CHAPTER 41 Target : Proteins 719

45. Bellows CG, Reimers SM, Heersche JN 1999 Expression of 61. Price PA, Williamson MK, Lothringer JW 1981 Origin of the mRNAs for type-I , bone sialoprotein, , vitamin K-dependent bone protein found in plasma and its and osteopontin at different stages of osteoblastic differentia- clearance by kidney and bone. J Biol Chem 256:12760Ð12766. tion and their regulation by 1,25-dihydroxyvitamin D3. Cell 62. Ducy P, Desbois C, Boyce B, Pinero G, Story B, Dunstan C, Tissue Res 297:249Ð259. Smith E, Bonadio J, Goldstein S, Gundberg C, Bradley A, 46. Broess M, Riva A, Gerstenfeld LC 1995 Inhibitory effects of Karsenty G 1996 Increased bone formation in osteocalcin- 1,25(OH)2 vitamin D3 on collagen type I, osteopontin, and deficient mice. Nature 382:448Ð452. osteocalcin expression in chicken osteoblasts. J Cell 63. Chenu C, Colucci S, Grano M, Zigrino P, Barattolo R, Biochem 57:440Ð451. Zambonin G, Baldini N, Vergnaud P, Delmas PD, Zallone AZ 47. Yoon K, Buenaga R, Rodan GA, Prince CW, Butler WT 1994 Osteocalcin induces chemotaxis, secretion of matrix pro- 1987 Tissue specificity and developmental expression of teins, and calcium-mediated intracellular signaling in human rat osteopontin. Biochem Biophys Res Commun 148: osteoclast-like cells. J Cell Biol 127:1149Ð1158. 1129Ð1136. 64. Watts NB 1999 Clinical utility of biochemical markers of bone 48. Beresford JN, Joyner CJ, Devlin C, Triffitt JT 1994 The effects remodeling. Clin Chem 45:1359Ð1368. of dexamethasone and 1,25-dihydroxyvitamin D3 on osteogenic 65. Ivaska KK, Hentunen TA, Vaaraniemi J, Ylipahkala H, differentiation of human marrow stromal cells in vitro. Arch Pettersson K, Vaananen HK 2004 Release of intact and frag- Oral Biol 39:941Ð947. mented osteocalcin molecules from bone matrix during bone 49. Prince CW, Butler WT 1987 1,25-Dihydroxyvitamin D3 regu- resorption in vitro. J Biol Chem 279:18361Ð18369. lates the biosynthesis of osteopontin, a bone-derived cell 66. Beresford JN, Gallagher JA, Poser JW, Russell RG 1984 attachment protein, in clonal osteoblast-like osteosarcoma Production of osteocalcin by human bone cells in vitro. cells. Coll Relat Res 7:305Ð313. Effects of 1,25(OH)2D3, 24,25(OH)2D3, parathyroid hormone, 50. Chen J, Thomas HF, Sodek J, Kim RH, Ogata Y 1996 Regulation and glucocorticoids. Metab Bone Dis Relat Res 5:229Ð234. of bone sialoprotein and osteopontin mRNA expression by 67. Lian J, Stewart C, Puchacz E, Mackowiak S, Shalhoub V, dexamethasone and 1,25-dihydroxyvitamin D3 in rat bone Collart D, Zambetti G, Stein G 1989 Structure of the rat osteo- organ cultures. Connect Tissue Res 34:41Ð51. calcin gene and regulation of vitamin D-dependent expression. 51. Matsue M, Kageyama R, Denhardt DT, Noda M 1997 HelixÐ Proc Natl Acad Sci USA 86:1143Ð1147. loopÐhelix-type transcription factor (HES-1) is expressed in 68. Lajeunesse D, Kiebzak GM, Frondoza C, Sacktor B 1991 osteoblastic cells, suppressed by 1,25(OH)2 vitamin D3, and Regulation of osteocalcin secretion by human primary bone modulates 1,25(OH)2 vitamin D3 enhancement of osteopontin cells and by the human osteosarcoma cell line MG-63. Bone gene expression. Bone 20:329Ð334. Miner 14:237Ð250. 52. Noda M, Rodan GA 1989 Transcriptional regulation of osteo- 69. Ingram RT, Bonde SK, Riggs BL, Fitzpatrick LA 1994 Effects pontin production in rat osteoblast-like cells by parathyroid of transforming growth factor beta (TGF beta) and 1,25-dihy- hormone. J Cell Biol 108:713Ð718. droxyvitamin D3 on the function, cytochemistry and morphol- 53. Staal A, Van Wijnen AJ, Desai RK, Pols HA, Birkenhager JC, ogy of normal human osteoblast-like cells. Differentiation Deluca HF, Denhardt DT, Stein JL, Van Leeuwen JP, Stein GS, 55:153Ð163. Lian JB 1996 Antagonistic effects of transforming growth 70. Bodine PV, Trailsmith M, Komm BS 1996 Development and factor-beta on vitamin D3 enhancement of osteocalcin and characterization of a conditionally transformed adult human osteopontin transcription: reduced interactions of vitamin D osteoblastic cell line. J Bone Miner Res 11:806Ð819. receptor/retinoid X receptor complexes with vitamin E response 71. Mosavin R, Mellon WS 1996 Posttranscriptional regulation of elements. Endocrinology 137:2001Ð2011. osteocalcin mRNA in clonal osteoblast cells by 1,25-dihydroxy- 54. Ganss B, Kim RH, Sodek J 1999 Bone sialoprotein. Crit Rev vitamin D3. 574.1905 A67 Arch Biochem Biophys 332:142Ð152. Oral Biol Med 10:79Ð98. 72. Hicok KC, Thomas T, Gori F, Rickard DJ, Spelsberg TC, 55. Oldberg A, Jirskog-Hed B, Axelsson S, Heinegard D 1989 Riggs BL 1998 Development and characterization of condi- Regulation of bone sialoprotein mRNA by steroid hormones. tionally immortalized osteoblast precursor cell lines from J Cell Biol 109:3183Ð3186. human bone marrow stroma. J Bone Miner Res 13:205Ð217. 56. Li JJ, Sodek J 1993 Cloning and characterization of the rat 73. Lajeunesse D, Frondoza C, Schoffield B, Sacktor B 1990 bone sialoprotein gene promoter. Biochem J 289:625Ð629. Osteocalcin secretion by the human osteosarcoma cell line 57. Kim RH, Li JJ, Ogata Y, Yamauchi M, Freedman LP 1996 MG-63. J Bone Miner Res 5:915Ð922. Identification of a vitamin D3-response element that overlaps 74. Hosoda K, Kanzaki S, Eguchi H, Kiyoki M, Yamaji T, a unique inverted TATA box in the rat bone sialoprotein gene. Koshihara Y, Shiraki M, Seino Y 1993 Secretion of osteocalcin Biochem J 318:219Ð226. and its propeptide from human osteoblastic cells: Dissociation 58. Sodek J, Kim RH, Ogata Y, Li J, Yamauchi M, Zhang Q, of the secretory patterns of osteocalcin and its propeptide. Freedman LP 1995 Regulation of bone sialoprotein gene J Bone Miner Res 8:553Ð565. transcription by steroid hormones. Connect Tissue Res 32: 75. Zhang R, Ducy P, Karsenty G 1997 1,25-Dihydroxyvitamin D3 209Ð217. inhibits osteocalcin expression in mouse through an indirect 59. Chang PL, Prince CW 1991 1α,25-Dihydroxyvitamin D3 stim- mechanism. J Biol Chem 272:110Ð116. ulates synthesis and secretion of nonphosphorylated osteopon- 76. Lian JB, Shalhoub V, Aslam F, Frenkel B, Green J, Hamrah M, tin (secreted phosphoprotein 1) in mouse JB6 epidermal cells. Stein GS, Stein JL 1997 Species-specific glucocorticoid and Cancer Res 51:2144Ð2150. 1,25-dihydroxyvitamin D responsiveness in mouse MC3T3-E1 60. Pockwinse SM, Stein JL, Lian JB, Stein GS 1995 Developmental osteoblasts: Dexamethasone inhibits osteoblast differentiation stage-specific cellular responses to vitamin D and glucocorti- and vitamin D down-regulates osteocalcin gene expression. coids during differentiation of the osteoblast phenotype: Endocrinology 138:2117Ð2127. Interrelationship of morphology and gene expression by in situ 77. Komori T, Yagi H, Nomura S, Yamaguchi A, Sasaki K, hybridization. Exp Cell Res 216:244Ð260. Deguchi K, Shimizu Y, Bronson RT, Gao YH, Inada M, Sato M, 720 PAUL H. ANDERSON ET AL.

Okamoto R, Kitamura Y, Yoshiki S, Kishimoto T 1997 86. Imai K, Neuman MW, Kawase T, Saito S 1992 Calcium in Targeted disruption of Cbfa1 results in a complete lack of bone osteoblast-enriched bone cells. Bone 13:217Ð223. formation owing to maturational arrest of osteoblasts. Cell 87. Halstead LR, Scott MJ, Rifas L, Avioli LV 1992 Characterization 89:755Ð764. of osteoblast-like cells from normal adult rat femoral trabecular 78. Otto F, Thornell AP, Crompton T, Denzel A, Gilmour KC, bone. Calcif Tissue Int 50:93Ð95. Rosewell IR, Stamp GW, Beddington RS, Mundlos S, Olsen BR, 88. El-Maadawy S, Kaartinen MT, Schinke T, Murshed M, Selby PB, Owen MJ 1997 Cbfa1, a candidate gene for Karsenty G, McKee MD 2003 Cartilage formation and calcifi- cleidocranial dysplasia syndrome, is essential for osteoblast cation in arteries of mice lacking matrix Gla protein. Connect differentiation and bone development. Cell 89:765Ð771. Tissue Res 44:272Ð278. 79. Javed A, Gutierrez S, Montecino M, van Wijnen AJ, Stein JL, 89. Fraser JD, Otawara Y, Price PA 1988 1,25-Dihydroxyvitamin D3 Stein GS, Lian JB 1999 Multiple Cbfa/AML sites in the rat stimulates the synthesis of matrix gamma-carboxyglutamic osteocalcin promoter are required for basal and vitamin DÐ acid protein by osteosarcoma cells. Mutually exclusive expression responsive transcription and contribute to chromatin organiza- of vitamin KÐdependent bone proteins by clonal osteoblastic cell tion. Mol Cell Biol 19:7491Ð7500. lines. J Biol Chem 263:911Ð916. 80. Aslam F, McCabe L, Frenkel B, van Wijnen AJ, Stein GS, 90. Fraser JD, Price PA 1990 Induction of matrix Gla protein Lian JB, Stein JL 1999 AP-1 and vitamin D receptor (VDR) synthesis during prolonged 1,25-dihydroxyvitamin D3 treatment signaling pathways converge at the rat osteocalcin VDR of osteosarcoma cells. Calcif Tissue Int 46:270Ð279. element: Requirement for the internal activating protein-1 site 91. Barone LM, Owen TA, Tassinari MS, Bortell R, Stein GS, for vitamin DÐmediated trans-activation. Endocrinology 140: Lian JB 1991 Developmental expression and hormonal regula- 63Ð70. tion of the rat matrix Gla protein (MGP) gene in chondrogenesis 81. Grigoriadis AE, Schellander K, Wang ZQ, Wagner EF 1993 and osteogenesis. J Cell Biochem 46:351Ð365. Osteoblasts are target cells for transformation in c-fos trans- 92. Hewison M, Zehnder D, Bland R, Stewart PM 2000 genic mice. J Cell Biol 122:685Ð701. 1α-Hydroxylase and the action of vitamin D. J Mol Endocrinol 82. McCabe LR, Banerjee C, Kundu R, Harrison RJ, Dobner PR 25:141Ð148. 1996 Developmental expression and activities of specific fos 93. Turner RT, Howard GA, Puzas JE, Baylink DJ, Knapp DR and jun proteins are functionally related to osteoblast maturation: 1983 Calvarial cells synthesize 1α,25-dihydroxyvitamin D3 Role of Fra-2 and Jun D during differentiation. Endocrinology from 25-hydroxyvitamin D3. Biochemistry 22:1073Ð1076. 137:4398Ð4408. 94. Atkins GJ, Anderson PH, Morris HA, Zannettino ACW, 83. Narisawa S, Frohlander N, Millan JL 1997 Inactivation of Kostakis P, Findlay DM 2003 The expression of 25-hydroxy- two mouse alkaline phosphatase genes and establishment of vitamin D 24-hydroxylase and 25-hydroxyvitamin D 1α- a model of infantile hypophosphatasia. Dev Dyn 208:432Ð446. hydroxylase in human osteoblasts. J Bone Miner Res 84. Noda M, Yoon K, Rodan GA, Koppel DE 1987 High lateral 18(Suppl. 2):M480. mobility of endogenous and transfected alkaline phosphatase: 95. Ke HZ, Qi H, Crawford DT, Xu G, Li M, Plum L, A phosphatidylinositol-anchored membrane protein. J Cell Clagett-Dame M, DeLuca HF, Thompson DD, Brown TA 2003 Biol 105:1671Ð1677. A novel vitamin D analogue restores bone mass and adds extra 85. Manolagas SC, Burton DW, Deftos LJ 1981 1,25- bone by markedly stimulating bone formation in ovariec- Dihydroxyvitamin D3 stimulates the alkaline phosphatase tomized rats with established osteopenia. J Bone Miner Res activity of osteoblast-like cells. J Biol Chem 256:7115Ð7117. 18(Suppl. 2):1153. CHAPTER 42

The Calbindins: -D9K and Calbindin-D28K

SYLVIA CHRISTAKOS, YAN LIU, PUNEET DHAWAN, AND XIAORONG PENG Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey

I. Introduction and General Considerations III. Regulation of Calbindin Gene Expression II. Localization and Proposed Functional IV. Conclusion Significance References

I. INTRODUCTION AND GENERAL 1,25(OH)2D3 target genes should result in novel insights CONSIDERATIONS related to tissue-specific molecular mechanisms involved in calcium homeostasis. In the two major target tissues of 1,25-dihydroxy- One of the most important findings in the vitamin D vitamin D3 [1,25(OH)2D3] action, intestine and kidney, field has been the discovery by Wasserman and Taylor one of the most pronounced effects of 1,25(OH)2D3 in 1966 of a 28,000 Mr vitamin DÐdependent calcium known is the induction of the calcium binding protein, binding protein in avian intestine [1]. Although calbindin, the first identified target of 1,25(OH)2D3 previously known as the vitamin DÐdependent calcium action. There are two major subclasses of calbindin: a binding protein (CaBP), in 1985 it became officially protein of approximately 28,000 molecular weight known as calbindin-D28K and calbindin-D9K for the (calbindin-D28K) and a protein of approximately 9,000 28,000 Mr and the 9000 Mr proteins, respectively [2]. molecular weight (calbindin-D9K). Calbindin-D28K is Initially identified in avian intestine [1], calbindin-D28K present in highest concentration in avian intestine and has since been reported in many other tissues including in avian and mammalian kidney, brain, and pancreas. kidney and bone, and in tissues that are not primary Calbindin-D28K has four functional high-affinity calcium regulators of serum calcium such as pancreas, testes, binding sites and is highly conserved in evolution. and brain and in a variety of species [3Ð9] (see Calbindin-D9K has two calcium binding domains, is Christakos et al. [9] for review). The importance of the present in highest concentration in mammalian intes- discovery of calbindin-D28K is that key advances in our tine, and, unlike calbindin-D28K, is not evolutionarily understanding of the diversity of the vitamin D conserved and has been observed only in mammals. endocrine system have been made through the study of There is no amino acid sequence similarity between its tissue distribution and its colocalization with the calbindin-D9K and calbindin-D28K. vitamin D receptor (VDR). In addition, the biosynthe- The discussion that follows reviews the chemistry, sis of calbindin has provided a model for studies that localization, proposed functional significance, and have resulted in an important basic understanding of regulation of these calcium binding proteins. In addi- the molecular mechanism of action of 1,25(OH)2D3 in tion, this chapter provides insight into the information major target tissues such as intestine and kidney. obtained by studying these proteins concerning the Chicken and mammalian calbindin-D28K proteins con- multiple actions of the vitamin D endocrine system and tain 261 amino acid residues, have a molecular weight of the basic molecular mechanism of 1,25(OH)2D3 action. approximately 28,000 (30,000 based on amino acid Findings indicating that calbindins can be regulated by sequence and 28,000 based on migration on sodium a number of different hormones and factors are also dodecyl sulfateÐpolyacrylamide gels), and are blocked at reviewed. The study of the molecular interactions of the amino terminus [9Ð12]. The mammalian calbindin- several members of the steroid hormoneÐretinoic acid D28K sequences are 98% similar to one another and 79% family as well as the role of signal transduction path- similar to chicken calbindin-D28K [9,11,12]. Calbindin- ways in the regulation of calbindin-D may be applicable D28K is highly conserved in evolution, suggesting an to the regulation of other targets of 1,25(OH)2D3 action. important, fundamental role for calbindin-D28K in medi- Elucidation of multiple factors and interactions regulating ating intracellular calcium-dependent processes [9]. VITAMIN D, 2ND EDITION Copyright © 2005, Elsevier, Inc. FELDMAN, PIKE, AND GLORIEUX All rights reserved. 722 SYLVIA CHRISTAKOS ET AL.

Unlike calbindin-D28K, calbindin-D9K is observed only in calbindin-D28K has yet to be elucidated by X-ray crys- mammals. It has no amino acid to tallography, circular dichroism experiments have calbindn-D28K. It is most abundant in mammalian duo- shown that calbindin-D28K contains approximately 30% denum, placenta, and uterus [13Ð16]. It is also present in α helix, 20.6% β sheet, and 51% random coil [31]. The mammalian yolk sac, lung, bone, and mouse kidney three-dimensional structure of calbindin-D9K has been [14,17Ð23]. Human calbindin-D9K has 79 amino acid elucidated [32]. Calbindin-D9K has been shown to residues and a calculated molecular weight of 9015 undergo limited conformational change in the presence [24,25]. It is 89% similar to the bovine and porcine or absence of calcium [33]. Both calbindins are heat- sequences and 78% and 77% similar to rat and mouse stable protein and acidic, having a pI value of approx- calbindin-D9K, respectively [24,25]. imately 5 [34,35]. The calbindins bind other cations in 2+ 2+ Calbindin-D28K and calbindin-D9K belong to a family addition to calcium with reduced affinity: Ca > Cd > of high-affinity calcium (Ca2+) binding proteins Sr2+ > Mn2+ > Zn2+ > Ba2+ > Co2+ > Mg2+ [36]. −8 −6 (Kd =10 Ð10 M) that contains more than 200 mem- bers and is characterized by the EF-hand structural motif [26] (Fig. 1). The EF-hand domain is an octahedral struc- II. LOCALIZATION AND PROPOSED ture consisting of two α helices separated by a 12-amino- FUNCTIONAL SIGNIFICANCE acid loop that contains side chain oxygens necessary for orienting the divalent calcium cation [26]. Calbindin- A. Intestine D28K contains six EF hands (Fig. 2); however, only four 2+ of these actively bind Ca [27,28]. Calbindin-D9K con- One of the most pronounced effects of 1,25(OH)2D3 is tains two calcium binding sites [29]. Other calcium increased synthesis of intestinal calbindin. Calbindin-D9K binding proteins belonging to this family include in mammalian intestine and calbindin-D28K in avian calmodulin, parvalbumin, , , cal- intestine have been localized primarily in the cyto- cineurin, calpain, Spec I, myosin light chains, S100, plasm of absorptive cells [37], which supports the and [26,30]. Although the structure of proposed role of calbindin in intestinal calcium

E helix

Ca2+

F helix

Ca2+

EF hand

FIGURE 1 EF-hand structural motif (helixÐloopÐhelix). The helices are represented by the extended forefinger and thumb. The clenched middle finger represents the loop that contains the oxygen ligands of the calcium ion. The EF hand is a recurring motif in calbindin and other calcium binding proteins. Reprinted with permission from Stryer L 1995 Biochemistry. Freeman, San Francisco, p. 1064. CHAPTER 42 The Calbindins: Calbindin-D9K and Calbindin-D28K 723

Ca2+ α-Helix Region Ca2+ Binding Binding Domains Domains

FIGURE 2 Position of intervening sequences within the structure of chicken calbindin-D28K. Locations of introns are indicated by circled numbers. Numbers above amino acids indicate codon positions. Invariant Glu/Leu and Gly amino acids are indicated by black circles. Calcium binding domains are separated from the α-helix region by vertical lines. Reprinted with permission from Minghetti et al. [121].

absorption [38Ð40]. Early studies in chicks established duodenum and distal tubule of the kidney), suggesting, a strong correlation between the level of calbindin and for the first time, a mechanism of calcium entry [44Ð46]. an increase in intestinal Ca2+ transport [41Ð43]. It is thought that calbindin acts to facilitate the diffusion In the intestine, 1,25(OH)2D3 effects the transfer of of calcium through the cell interior toward the basolat- Ca2+ across the luminal brush-border membrane, the eral membrane [40,42]. Supporting this hypothesis are transfer of calcium through the cell interior, and active findings observed in vitamin D receptor knockout calcium extrusion from the basolateral membrane. mice. In these mice, the major defect that results in A vitamin DÐinducible apical calcium channel has been rickets is in intestinal calcium absorption [47Ð49]. The identified in 1,25(OH)2D3 responsive epithelia (proximal defect in intestinal calcium absorption is accompanied by 724 SYLVIA CHRISTAKOS ET AL.

a 50% reduction in intestinal calbindin-D9K mRNA [50]. calcium and to stimulate ATP dependent extrusion of The 1,25(OH)2D3 regulation of intestinal calbindin-D9K calcium at the basolateral membrane [65]. The different is also evident in 25-hydroxyvitamin-D3 1α-hydroxy- functions of renal calbindin-D28K and calbindin-D9K lase knockout mice. In these mice, characterized by suggest different mechanisms that may be involved in hypocalcemia, hyperparathyroidism, and skeletal abnor- the enhancement by 1,25(OH)2D3 of calcium transport malities characteristic of rickets, intestinal calbindin-D9K in the distal nephron. mRNA is absent [51]. In addition to facilitated diffusion, Recent studies have provided additional evidence it has been suggested that intestinal calbindin may for a role of calbindin-D28K in distal tubular calcium also act as a cytosolic buffer to prevent toxic levels of reabsorption. Studies using immunosuppressant drugs calcium from accumulating in the intestinal cell during (CsA and FK-506) that result in nephrotoxicity have vitamin DÐmediated translocation of calcium [40]. noted decreases in calbindin-D28K in the rat coincident with increases in urinary calcium and intratubular calci- fication [66,67]. The authors suggested that calbindin- B. Kidney D28K has a role in calciuria and tubular calcification induced by the immunosuppressant drugs. In addition, Immunocytochemical studies have reported the calbindin-D28K knockout mice fed a high-calcium diet exclusive localization of calbindin-D28K in the distal were found to have significantly increased urinary nephron (distal convoluted tubule and connecting tubule) calcium/creatinine ratio compared to wild-type con- in a variety of species including mammals, chickens, trols [63,68]. The regulation of renal and intestinal and reptiles [3,9,52Ð54]. Renal calbindin-D28K is calbindin-D9K was found to be similar in wild-type and localized in the cytosol and the nucleus and is not knockout mice, indicating that changes in calbindin-D9K associated with membranes or filamentous elements. were not compensating for the lack of calbindin-D28K Both calbindin-D28K and calbindin-D9K are localized in and further suggesting different roles for these two mouse distal nephron and perinatal rat distal nephron vitamin DÐdependent calcium binding proteins [63]. [23]. Autoradiographic data indicated that the VDR is Serum calcium was not different in the wild-type and also predominantly localized in the distal nephron and calbindin-D28K knockout mice, suggesting compensatory both calbindins have been reported to be induced by changes in bone or in intestinal calcium absorption 1,25(OH)2D3 in the kidney [55,56]. Although micro- [63,68]. However, it should be noted that mechanisms puncture data [57] as well as studies using a mouse within the kidney, independent of calbindin-D28K, are distal convoluted tubule cell line [58] have indicated also associated with hypercalciuria [69]. In the future that vitamin D metabolites can enhance calcium trans- it will be of interest to develop calbindin-D9K knockout port in the distal nephron, little information is available mice as well as calbindin-D28K and calbindin-D9K double concerning the exact role of vitamin DÐinducible renal knockout mice in order to provide additional insight calbindins in this process. Transcellular calcium trans- into the role of each of these proteins in distal tubule port in the distal convoluted tubule, similar to transcel- calcium transport. lular intestinal calcium absorption, involves calcium entry through the apical plasma membrane, diffusion of calcium across the cell, and active extrusion of cal- C. Bone cium across the basolateral membrane mediated by a calcium-dependent ATPase [59]. Recent data using Calbindin-D28K and calbindin-D9K are both present apical membrane vesicles reconstituted with calbindin- in chondrocytes of growth plate cartilage in rats and D28K have indicated that this protein can increase the calbindin-D28K is present in the growth plate cartilage influx of calcium at the apical membrane [60]. of chicks [22,70,71]. Although it is not clear whether Whether calbindin-D28K affects the activity of the calbindin is vitamin D dependent in chondrocytes, recently identified vitamin DÐinducible epithelial cal- 1,25(OH)2D3 receptors have been reported in developing cium channel in the distal tubule or other calcium chick bone, specifically in dividing chondrocytes [72]. channels previously identified in the distal tubule is not It has been suggested that calbindin may be involved yet known [46,61,62]. It has also been suggested that in the movement of calcium in the process of calcifi- calbindin-D28K may act to ferry calcium across the cell as cation in the chondrocyte [70]. Calbindin-D9K and in the intestine as well as to buffer calcium, resulting in calbindin-D28K have also been localized to osteoblasts protection against calcium mediated cell death [63,64]. and ameloblasts of rodent teeth, and it has been Calbindin-D9K has been reported to have a different cel- reported that calbindin-D9K and calbindin-D28K mRNAs lular action. Calbindin-D9K has been reported to bind are induced by 1,25(OH)2D3 in these cells [20,21]. CHAPTER 42 The Calbindins: Calbindin-D9K and Calbindin-D28K 725

It has also been suggested that elevated expression of cal- and have important implications for the prevention of bindin may phenotypically characterize cells that are cellular degeneration in bone cells. involved in calcium handling during mineralization [20]. In addition to an association with mineralization, recent evidence has indicated that calbindin-D28K is D. Pancreas able to protect against apoptosis of bone cells. Calbindin-D28K was found to protect osteoblastic cells The pancreas was the first nonclassic target tissue in against tumor necrosis factor (TNF) induced apoptosis which receptors for 1,25(OH)2D3 were identified [75]. (Fig. 3) as well as to prevent glucocorticoid-induced Although 1,25(OH)2D3 has been reported to play a role apoptosis of osteoblastic and osteocytic cells [73,74]. in insulin secretion, the exact mechanisms remain The protection against both TNF and glucocorticoid unclear [76Ð78]. An early indication that the pancreas induced cell death was found to be at least partially may be a target for 1,25(OH)2D3 was the immunocyto- due to the ability of calbindin-D28K to inhibit endoge- chemical study of Morrissey et al. [3], which localized nous caspase 3, a key mediator of apoptosis in response calbindin-D28K to the islet. In the chick, calbindin-D28K to multiple signals. Calbindin-D28K was found to inhibit is detected exclusively in insulin-producing β cells [79] caspase 3 but was not cleaved by the caspase. In addition, and is responsive to vitamin D [80]. In the rat, however, the inhibition of caspase 3 by calbindin-D28K was calbindin-D28K has been reported to be localized in α as reported to be independent of its calcium binding ability. well as β cells of the pancreas [81]. Because auto- Besides the inhibitor of apoptotic proteins (IAPs), radiographic data have indicated that 1,25(OH)2D3 calbindin-D28K is the only other known natural, nononco- receptors are localized only in rat β cells [82], and genic inhibitor of caspase 3. These findings are novel because insulin but not glucagon secretion is affected

Empty Calbindin–D 28K vector cDNA

Empty vector Calbindin-D28K + TNFα cDNA + TNFα

FIGURE 3 Overexpression of calbindin-D28K suppresses nuclear fragmentation of osteoblastic cells induced by TNFα. Cells were transfected with the expression vector pREP4 alone (empty vector) or containing the cDNA for calbindin-D28K (calbindin-D28K) together with an expression vector containing the coding sequence of green fluorescent protein with a nuclear localization sequence. Forty-eight hours after transfection, cells were exposed to 1 nM TNFα for 16 hr. Cells were fixed, mounted, and examined with a Zeiss confocal laser scanning microscope. Note the presence of apoptotic nuclei in the TNFα-treated vector-transfected cells but not in the calbindin-transfected cells similarly treated. 726 SYLVIA CHRISTAKOS ET AL. in vitamin DÐdeficient animals [76], studies in the uterus, mouse oviduct epithelium and in primary folli- rat suggest that β-cell calbindin may be regulated cles of mouse ovary) [89]. 1,25(OH)2D3 has no effect by 1,25(OH)2D3 while non-β-cell calbindin may be on calbindin in these tissues. However, calbindin-D9K independent of vitamin D. Calbindin-D28K has also been and calbindin-D28K in rat and chick uterus, respectively, identified in human pancreatic islet cells [83]. Recent are under the positive control of estradiol [16,90]. studies using pancreatic beta cell lines as well as In the mouse, calbindin-D28K gene expression is down- calbindin-D28K knockout mice have suggested that regulated in the uterus but not in the ovaries and oviduct, calbindin-D28K, by regulating intracellular calcium, mod- suggesting tissue and species specific regulation of ulates depolarization-stimulated insulin release [84]. In calbindin-D28K by estradiol [89]. It has been suggested addition to modulating insulin release, more recent that transcellular calcium transport in epithelial cells studies have indicated that calbindin-D28K, by buffering of the uterus and oviduct is facilitated by calbindin [89]. calcium, can protect against destruction of beta cells by The presence of calbindin in the myometrium sug- cytokines by preventing calcium mediated mitochon- gests the involvement of calbindin in the modulation of drial damage and the resultant generation of free radi- intracellular calcium that may alter the frequency and cals [85]. These findings have important therapeutic strength of uterine contractions. implications for type 1 diabetes and the prevention of autoimmune destruction of pancreatic β cells. G. Nervous Tissue

E. Testes Calbindin-D28K is widely distributed throughout the brain of mammals, avians, reptiles, amphibians, fish, Calbindin-D28K has been reported in both chick and and mollusks [9]. It is present in most neuronal cell rat testes [5,6]. In chick and rat, immunocytochemical groups and fiber tracts and is localized in neuronal studies have revealed that calbindin-D28K is present in elements and some ependymal cells [8,90Ð92]. In brain, spermatogonia and spermatocytes of the seminiferous calbindin-D28K is not vitamin D dependent [9]. Neurons tubules and some interstitial Leydig cells [5,6]. It has containing calbindin-D28K are found in the cerebral cortex been reported that vitamin DÐdeficient chicks have in layers 2Ð4, primarily in pyramidal neurons [8,90Ð92]. significantly (threefold) lower testicular calbindin lev- In the hippocampus, both basket cells and pyramidal els than vitamin DÐreplete chicks [86]. As calbindin- neurons in CA1 stain positively for calbindin, as do D28K as well as VDR (which is also present in granule cells and fibers in the dentate gyrus [8,90Ð92]. seminiferous tubules) have been shown to correlate Purkinje cells of the cerebellum stain most intensely with testicular maturation [6,87], the involvement of for calbindin-D28K [8,90Ð92]. It is of interest that the calbindin-D28K and vitamin D in spermatogenesis and phenotype of the calbindin-D28K knockout mouse is steroidogenesis has been suggested [86]. impaired motor coordination [93]. It has been suggested that this phenotype may be the result of abnormal cere- bellar activity due to the alteration of synaptically evoked F. Placenta, Yolk Sac, Egg Shell Gland, calcium transients in the Purkinje cells in the absence and Uterus of calbindin [93]. Calbindin-D28K immunoreactive cells are also observed in the hypothalamus, amygdala, Calbindin-D9K is present in the placenta and yolk sac pyriform region, and thalamus [8,90Ð92]. of rats and mice [14,17]. Calbindin-D9K is also present In addition, specific neuronal sensory cells have been in rat endometrium and myometrium [16]. In pregnant shown to contain calbindin-D28K [94Ð100]. These cell rats calbindin-D9K is also expressed in the uterine populations include cochlear and vestibular hair cells in epithelium [16]. In placenta and yolk sac calbindin-D9K the inner ear [94,95], avian basilar papilla [96], cone but increases at the end of gestation, when there is increased not rod photoreceptor cells of avian and mammalian calcium need of the fetus, suggesting a role for calbindin- retina [97Ð99], and conelike, modified photoreceptor D9K in the transport of calcium to the fetus [14,17]. cells (pinealocytes) of pineal transducers [100]. The Unlike calbindin-D9K, calbindin-D28K is not present in presence of calbindin in specific cells of the sensory rat reproductive tissue. However, calbindin-D28K has pathway suggests the possible involvement of calbindin been localized in the tubular gland cells of the shell in mechanisms of signal transduction. gland in the chick [88], which are involved in calcium In the nervous system it has been suggested that secretion during egg-shell formation. Calbindin-D28K neuronal calbindin, by buffering calcium, can regulate is also found in the reproductive tissues of female mice intracellular calcium responses to physiological stimuli (endometrium and glandular epithelium of mouse and can protect neurons against calcium-mediated CHAPTER 42 The Calbindins: Calbindin-D9K and Calbindin-D28K 727

neurotoxicity [101,102]. It has been demonstrated Since calbindin-D28K can prevent neuronal damage in that introduction of exogenous calbindin into sensory neuropathies, these findings have important therapeutic neurons can modulate calcium signaling by decreasing implications. the rate of rise of intracellular calcium and by chang- ing the kinetics of decay of the calcium signal [103]. Using adenovirus as an expression vector, overexpres- H. Calbindin-D28K and Apoptosis sion of calbindin-D28K in hippocampal neurons was reported to suppress posttetanic potentiation, possibly Calcium is thought to play an important regulatory by restricting and destabilizing the evoked calcium function in apoptosis, but the precise mechanism(s) by signal [104]. Calbindin-D28K was also reported to play which calcium promotes cell death is unknown. The a role in the control of hypothalamic neuroendocrine first study suggesting that calbindin-D28K plays a pro- neuronal firing patterns [105]. The whole-cell patch- tective role in the process of apoptosis used subtraction clamp method was used to introduce calbindin into rat analysis between the cDNA libraries of two human 2+ supraoptic neurons. Calbindin-D28K suppressed Ca - prostate cell lines [115]. One of the cell lines was andro- dependent depolarization afterpotentials and converted gen independent and the other one androgen dependent, phasic into continuous firing [106]. As different firing and the results revealed that a hybrid calbindin-D28K patterns promote the release of different hypothalamic gene was specifically expressed in the hormone- hormones, it was suggested that calbindin-D28K, by independent cell line. Apoptosis has been observed in regulating firing patterns, may be involved in the prostate cells on androgen depletion. Androgen depri- control of hormone secretion from hypothalamic neu- vation of prostate cells triggers an influx of calcium roendocrine neurons. These studies [103Ð105] indicated ions into the cells, leading to an increase in intracellu- directly by introduction of exogenous calbindin-D28K lar calcium. It was suggested that calbindin-D28K might via the patch clamp method or by transfection and over- buffer intracellular calcium and contribute to protec- expression that calbindin is an important and effective tion against apoptosis and thus androgen independence regulator of calcium-dependent aspects of neuronal in the prostatic cell line. function. It has been reported that stable transfection and Correlative evidence between decreases in neuronal overexpression of calbindin-D28K in lymphocytes pro- calbindin-D28K and neurodegeneration in studies of tect against apoptosis induced by calcium ionophore, ischemic injury [106], seizure activity [107,108], and cAMP, and glucocorticoid [116]. A similar protective chronic neurodegeneration (Alzheimer’s, Huntington’s, role for calbindin-D28K has been observed in apoptosis- and Parkinson’s diseases) [109Ð111] have been reported. susceptible cells in the central nervous system [112Ð114] It has been suggested that decreased calbindin levels as well as in human embryonic kidney cells (HEK 293), may lead to a loss of calcium buffering or intracellular osteoblasts, and pancreatic β cells [64,73,74,85]. These calcium homeostasis, which leads to cytotoxic events findings indicate that calbindin-D28K has a major role associated with neuronal damage and cell death. Direct in different cell types in protecting against apoptotic evidence of a protective role of neuronal calbindin- cell death. A further understanding of the mechanisms D28K against a variety of insults including exposure involved will have important therapeutic implications to hypoglycemia and IgG from amyotrophic lateral for the prevention of a number of diseases including sclerosis patients has been shown in primary cultures osteoporosis and diabetes. of neuronal cells or in neuronal cell lines in which the calbindin-D28K gene has been transfected [112,113]. Expression of calbindin-D28K in neural cells was also I. Calbindin-D28K Enzyme Activation found to suppress the proapoptotic actions of mutant and Other Potential Targets presenilin 1 (PS-1), which is causally linked to about 50% of the cases of early-onset familial Alzheimer’s In addition to its role as an intracellular calcium disease [114]. Mutant PS-1 has been reported to sensi- buffer and in transepithelial calcium transport, there is tize cells to apoptosis induced by amyloid β peptide in vitro evidence that calbindin-D28K may modulate the (Aβ), the major component of plaques in Alzheimer’s activities of calmodulin-sensitive enzymes such as disease. Aβ has been reported to damage neurons by a calcium-dependent ATPase and phosphodiesterase [117]. mechanism involving oxidative stress and disruption There is also evidence for a calcium-dependent, specific of calcium homeostasis. Calbindin-D28K protected interaction of calbindin-D28K with intestinal alkaline against the proapoptotic action of mutant PS-1 by atten- phosphatase [118]. In addition recent studies using bac- uating the increase in intracellular calcium and prevent- teriophage display have suggested that myo-inositol ing the impairment of mitochondrial function [114]. monophosphatase, a key enzyme in the regulation of 728 SYLVIA CHRISTAKOS ET AL. the activity of the phosphatidylinositol-signaling path- knockout mice only a small reduction in basal levels of way, is an activated target of calbindin-D28K [119]. renal calbindin-D28K is observed. However, the response Although the physiological relevance of these findings of renal calbindin-D28K in the VDR knockout mouse to is not known, these studies suggest that calbindin-D28K 1,25(OH)2D3 is compromised [50]. Also in 25-hydroxy- may act as an enzyme regulator. In addition, studies vitamin D3 1α-hydroxylase knockout mice the expres- in opossum kidney cells indicated that expression sion of renal calbindin-D28K is reduced [51]. There of calbindin in these cells resulted in increased phos- is now increasing evidence that the large induction of phate transport that was associated with alterations calbindin-D28K mRNA long after 1,25(OH)2D3 treatment in the actin cytoskeleton. Pollock and Santiesteban may be due primarily to posttranscriptional mecha- suggested a possible role for calbindin in cytoskeletal nisms [124,129,134Ð136]. Exactly how this action is reorganization [120]. exerted is not known, but one report suggests that 1,25(OH)2D3 may regulate the expression of an inter- mediate protein that may be involved in calbindin-D28K III. REGULATION OF CALBINDIN mRNA accumulation [136]. These studies suggest that GENE EXPRESSION the mechanism of action of 1,25(OH)2D3 on calbindin- D28K regulation is more complicated than the conven- A. Calbindin-D28K tional hormone receptorÐtranscriptional activation model, and that this regulation may involve other factors 1. GENOMIC ORGANIZATION OF THE and is mostly posttranscriptional. CALBINDIN-D28K GENE The genomic organization of the chicken calbindin- 3. REGULATION BY OTHER STEROIDS AND FACTORS D28K gene has been elucidated [121,122] and a partial structure for the human gene was also reported [12,123]. Further studies in intestine and kidney have pro- The human gene is located on 8 [123,124] vided evidence that the calbindin-D28K gene is not and is believed to consist of 11 exons, analogous to that exclusively regulated by 1,25(OH)2D3 and that other demonstrated for the avian gene. Moreover, the total factors can modulate gene expression. It has been size of the gene is reported to be 18.5 kb in chicken. reported that glucocorticoids can inhibit the levels The protein coding region of the mouse gene shares of calbindin-D28K mRNA and protein in intestine of 77% sequence similarity with the chicken gene [125]. vitamin DÐtreated chicks, resulting in a comparable However, no obvious sequence similarity exists between decrease in intestinal calcium absorption [137,138]. the mammalian and avian promoters except in the These findings suggest the involvement of the inhibi- region of the TATA box [12,126]. tion of intestinal calbindin in the clinically important hypocalcemic action of glucocorticoids. Adding to 2. REGULATION BY 1,25(OH)2D3 the growing body of data suggesting that the regulation It is well known that calbindin-D28K in the avian of calbindin is more complex than previously thought intestine [1,127] and kidney [127] and in the mam- are studies describing the modulation by calcium of malian kidney [128,129] is induced by 1,25(OH)2D3. In intestinal and renal calbindin-D28K gene expression chicken, a putative calbindin-D28K vitamin D response [135,139,140]. element (VDRE) was suggested after computer analysis In other tissues where calbindin-D28K is present in of the promoter sequence [130], but only a twofold significant amounts, for example, in parts of the brain, response to 1,25(OH)2D3 was detected in primary kidney the regulation of calbindin-D28K appears to be very cells after transfection with a 2.1-kb segment of the different from that in the intestine and kidney. In the 5′ flanking region of the promoter [131]. A relatively central nervous system (CNS), 1,25(OH)2D3 has no inactive putative VDRE was also reported in the chicken apparent effect on the levels of calbindin-D28K [9]. calbindin-D28K promoter by others [132,133] and the Instead, a variety of different factors have been reported response of the mouse calbindin-D28K promoter to to be involved in regulating neuronal calbindin-D28K. 1,25(OH)2D3 is modest [five-fold maximal induction in Using rat hippocampal cultures, evidence has indicated chloramphenicol acetyltransferase (CAT) activity] [126]. that neurotropin 3 (NT-3) [141,142], brain-derived neu- The modest response reflects previous in vivo findings rotropic factor (BDNF) [141,142], fibroblast growth that indicated a small transcriptional response to factor (FGF) [141], and tumor necrosis factors (TNFs) 1,25(OH)2D3 [124,129]. Similar findings were reported [143,144] all can induce calbindin. It has been reported for the in vivo induction of the chick intestinal calbindin- that neurotropic factors may protect against excitotoxic D28K gene by 1,25(OH)2D3 [134]. In addition, in VDR neuronal damage [145]. The induction of calbindin by CHAPTER 42 The Calbindins: Calbindin-D9K and Calbindin-D28K 729

those factors suggests a role for calbindin-D28K in the The third exon codes for the second calcium binding process of protection against cytotoxicity. site and the 3′ untranslated region. In addition, corticosterone administration in vivo 2. REGULATION OF CALBINDIN-D9K BY 1,25(OH)2D3 has been reported to increase calbindin-D28K expres- sion in rat hippocampus [146,147]. The glucocorticoid Similar to the regulation of avian intestinal calbindin- response is specifically localized to the CA1 region [148]. D28K and mammalian renal calbindin-D28K, in vivo Retinoic acid has also been reported to induce calbindin- findings have indicated that intestinal calbindin-D9K is D28K protein and mRNA in medulloblastoma cells, which regulated by 1,25(OH)2D3 by a small, rapid transcrip- express a neuronal phenotype [148]. Furthermore, the tional stimulation followed by a posttranscriptional content of calbindin-D28K in cultured Purkinje cells can effect accounting for a sustained accumulation of mRNA be increased by insulin-like growth factor I (IGF-I) [149]. long after cessation of 1,25(OH)2D3 treatment [154]. Similarly IGF-I, as well as insulin, promoted the expres- Unlike renal calbindin-D28K, there is a marked decrease sion of calbindin-D28K protein in cultured rat embryonic of basal as well as 1,25(OH)2D3 induced levels of neuronal cells [150]. Thus, neuronal calbindin-D28K can intestinal calbindin-D9K mRNA in VDR knockout mice, be regulated by steroids as well as by factors that affect suggesting that intestinal calbindin-D9K is more sensi- signal transduction pathways. These different modes tive to control by VDR-mediated mechanisms than of activation may be important for cell-specific effects calbindin-D28K [50]. Recent studies using transgenic of calbindin-D28K. mice have shown that the proximal promoter of the The molecular basis for tissue-specific calbindin-D28K calbindin-D9K gene from Ð117 to +365 and distal ele- regulation is still not understood, but in vivo experi- ment at Ð3731 to Ð2894 together but not separately ments using transgenic mice suggest that tissue speci- confer the 1,25(OH)2D3 induced transcriptional response. ficity of calbindin-D28K expression to some degree is Since this region does not contain a classical VDRE, controlled by separate elements on the promoter [151]. these findings suggest that the 1,25(OH)2D3 regula- The transgenic mouse study demonstrated the impor- tion of calbindin-D9K may involve a nonconventional tance of an in vivo system to investigate the role of activation pathway. sequence elements needed for tissue-specific gene expression and regulation [151]. 3. REGULATION OF CALBINDIN-D9K BY OTHER STEROIDS AND FACTORS As discussed previously, calbindin-D28K is also present in the avian egg-shell gland and in the repro- In vitro footprinting and gel shift assays suggested ductive tissues of female mice. In the avian egg-shell that several trans-acting factors other than the VDR, gland, estradiol-17β induces calbindin-D28K in in vivo including an ubiquitous factor (NF1), liver-enriched experiments [90]. In the mouse, calbindin-D28K gene factors (HNF1, C/EBP alpha and beta, and HNF4), expression was found to be down-regulated by estradiol and the intestine-specific transcription factor caudal in the uterus and oviduct [89,152] but up-regulated in homeobox-2 (Cdx-2), may be important for intestine the ovaries [152]. Multiple imperfect half-palindromic specific calbindin-D9K gene expression [156]. Further estrogen responsive elements, which are likely to studies using transgenic mice showed that a mutation mediate the estrogen responsiveness of the calbindin- in the distal Cdx2-binding site of calbindin-D9K pro- D28K gene by estradiol-17β, were present in two regions moter dramatically decreased intestinal expression of (−1075/−702 and −175/−78) of the promoter [152]. the calbindin-D9K gene, directly demonstrating the cru- The complex regulation of calbindin-D28K highlights cial role of Cdx2 for the transcription of this gene in the fact that while discovered as a vitamin DÐregulated the intestine [157]. 2+ gene product, the protein play roles in Ca regulation With regard to other steroids, besides 1,25(OH)2D3 well beyond that involving vitamin D3. the expression of calbindin-D9K in the intestine is also regulated by glucocorticoids. Glucocorticoids have been reported to inhibit intestinal calbindin-D9K B. Calbindin-D9K expression [158,159]. It has been suggested that this decrease may be involved in the reported decrease by 1. GENOMIC ORGANIZATION OF THE glucocorticoids in intestinal calcium absorption. CALBINDIN-D9K GENE Whether the effect on intestinal calbindin-D9K expression The size of the calbindin-D9K gene is 2.5 kb, and is a primary or a secondary action of glucocorticoids is the gene consists of three exons and two introns [153]. not yet known. The first exon contains the 5′ untranslated region. The In the uterus, calbindin-D9K is under the control of second exon codes for the first calcium binding site. estrogen but is unaffected by 1,25(OH)2D3. An imperfect 730 SYLVIA CHRISTAKOS ET AL.

TABLE IDistribution of Calbindin

Calbindin-D9K Calbindin-D28K

Mammalian intestine [13] Avian intestine [3,34,37] Mouse and neonatal rat kidney [23] Avian, reptilian, amphibian, and mammalian kidney [3,52Ð54] Rat and mouse yolk sac [14,17] Hen egg-shell gland (uterus) [90] Rat uterus [15,16] Mouse reproductive tissues (uterus, oviduct, ovary) [89] Rat and mouse placenta [14] Avian and mammalian beta cells of the pancreas [79,83] Alpha cells of the rat pancreas [81] Rat growth cartilage [22] Rat and chick growth cartilage [22,70,71] Ameloblasts and osteoblasts of rodent teeth [20,21] Ameloblasts and osteoblasts of rodent teeth; mouse osteoblasts [20,21,73] Rat lung [18] Brain (avian, reptilian, amphibian, molluskan, fish, and mammalian brain) [9,91,92]

estrogen response element (ERE) that binds the estrogen 4. Christakos S, Norman AW 1978 A vitamin D-dependent receptor (ER) has been identified at the border of the calcium binding protein in bone tissue. Science 202:70Ð71. first exon and the first intron [160,161]. In vivo experi- 5. Inpanbutr N, Taylor AN 1992 Expression of calbindin-D28K in developing and growing chick testes. Histochemistry ments using transgenic mice suggest the functionality 97:335Ð339. of this imperfect ERE [162,163]. 6. Kagi U, Chafouleas JG, Norman AW, Heizmann CW 1988 Developmental appearance of the Ca2+-binding proteins parval- bumin, calbindin-D28K, S100 proteins and calmodulin during IV. CONCLUSION testicular development in the rat. Cell Tissue Res 252:359Ð365. 7. Taylor AN 1974 Chick brain calcium binding protein: Comparison with intestinal vitamin DÐinduced calcium bind- We once viewed calbindin-D28K and calbindin-D9K ing protein. Arch Biochem Biophys 161:100Ð108. as exclusively vitamin DÐdependent proteins. It is now 8. Jande SS, Maler L, Lawson DEM 1981 Immunohistochemical evident that the calbindins are not under the exclusive mapping of vitamin DÐinduced calcium binding protein in regulatory control of 1,25(OH) D . Calbindin-D and brain. Nature 294:765Ð767. 2 3 28K 9. Christakos S, Gabrielides C, Rhoten WB 1989 Vitamin DÐ calbindin-D9K are present in many different tissues dependent calcium binding proteins: Chemistry, distribution, (See Table I) and may serve many different functions. functional considerations, and molecular biology. Endocr Accordingly, the regulation of these calcium binding Rev 10:84Ð107. proteins is varied and quite complex. In future studies, 10. Fullmer CS, Wasserman RH 1987 Chicken intestinal the generation of calbindin-D knockout mice as well as 28-kilodalton calbindin-D: Complete amino acid sequence 9K and structural considerations. Proc Natl Acad Sci USA 84: studies using mice in which both calbindins are absent 4772Ð4776. will provide new insight into the mechanism of action of 11. Hunziker W, Schrickel S 1988 Rat brain calbindin-D28K: Six the calbindins, including the role of calbindin in intestinal domain structure and extensive amino acid homology with calcium absorption, in calcium reabsorption in the distal chicken calbindin-D28K. Mol Endocrinol 2:465Ð473. nephron, and in protection against cell death. 12. Parmentier M 1990 Structure of the human cDNAs and genes coding for calbindin-D28K and calretinin. Adv Exp Med Biol 140:21Ð25. 13. Thomasset M, Parkes CO, Cuisinier-Gleizes P 1982 Rat calcium-binding proteins: distribution, development, and References vitamin D dependence. Am J Physiol 243:E483ÐE488. 14. Bruns ME, Kleeman E, Mills SE, Bruns DE, Herr JC 1985 1. Wasserman RH, Taylor AN 1966 Vitamin D3 induced cal- Immunochemical localization of vitamin D-dependent cium binding protein in chick intestinal mucosa. Science calcium-binding protein in mouse placenta and yolk sac. 152:791Ð793. Anat Rec 213:514Ð517, 532Ð535. 2. Wasserman RH 1985 Nomenclature of the vitamin D induced 15. Warembourg M, Perret C, Thomasset M 1987 Analysis and calcium binding proteins. In: Norman AW, Schaefer K, in situ detection of cholecalcin messenger RNA (9000 Mr Grigoleit HG, Herrath DV (eds) Vitamin D: Chemical, CaBP) in the uterus of the pregnant rat. Cell Tissue Res Biochemical and Clinical Update, de Gruyter, Berlin, 247:51Ð57. pp. 321Ð322. 16. Bruns ME, Overpeck JG, Smith GC, Hirsch GN, Mills SE, 3. Morrissey RL, Bucci TJ, Richard B, Empson RN, Lufkin EG Bruns DE 1988 Vitamin DÐdependent calcium binding 1975 Calcium binding protein: Its cellular localization in protein in rat uterus: differential effects of estrogen, tamoxifen, jejunum, kidney and pancreas. Proc Soc Exp Biol Med progesterone, and pregnancy on accumulation and cellular 149:56Ð60. localization. Endocrinology 122:2371Ð2378. CHAPTER 42 The Calbindins: Calbindin-D9K and Calbindin-D28K 731

17. Delorme AV, Cassier P, Geny B, Mathieu H 1983 37. Taylor AN 1981 Immunohistochemical localization of Immunocytochemical localization of vitamin DÐdependent vitamin DÐinduced calcium binding protein: Relocation of calcium-binding protein in the yolk sac of the rat. Placenta antigen during frozen section processing. J Histochem 4:263Ð270. Cytochem 29:65Ð73. 18. Dupret JM, L’Horset F, Perret C, Bernaudin JF, Thomasset M 38. Feher JJ 1983 Facilitated calcium diffusion by intestinal 1992 Calbindin-D9K gene expression in the lung of the rat. calcium-binding protein. Am J Physiol 244:C303ÐC307. Absence of regulation by 1,25-dihydroxyvitamin D3 and 39. Bronner F, Pansu D, Stein WD 1986 An analysis of intestinal estrogen. Endocrinology 131:2643Ð2648. calcium transport across the rat intestine. Am J Physiol 150: 19. Balmain N 1991 Calbindin-D9K. A vitamin-D-dependent, G561ÐG569. calcium-binding protein in . Clin Orthop 40. Wasserman RH, Fullmer CS 1995 Vitamin D and intestinal 265:265Ð276. calcium transport: Facts, speculations and hypotheses. J Nutr 20. Berdal A, Hotton D, Saffar JL, Thomasset M, Nanci A 1996 125:1971SÐ1979S. Calbindin-D9K and calbindin-D28K expression in rat mineral- 41. Taylor AN, Wasserman RH 1969 Correlations between the ized tissues in vivo. J Bone Miner Res 11:768Ð779. vitamin DÐinduced calcium binding protein and intestinal 21. Berdal A, Hotton D, Pike JW, Mathieu H, Dupret JM 1993 absorption of calcium. Fed Proc 28:1834Ð1838. Cell- and stage-specific expression of vitamin D receptor and 42. Feher JJ, Wasserman RH 1979 Calcium absorption and calbindin genes in rat incisor: regulation by 1,25-dihydroxy- intestinal calcium-binding protein: Quantitative relationship. vitamin D3. Dev Biol 155:172Ð179. Am J Physiol 236:E556ÐE561. 22. Balmain N, Tisserand-Jochem E, Thomasset M, Cuisinier- 43. Corradino RA, Fullmer CS, Wasserman RH 1976 Embryonic Gleizes P, Mathieu H 1986 Vitamin-D-dependent calcium- chick intestine in organ culture: Stimulation of calcium binding protein (CaBP-9K) in rat growth cartilage. transport by exogenous vitamin DÐinduced calcium-binding Histochemistry 84:161Ð168. protein. Arch Biochem Biophys 174:738Ð743. 23. Rhoten WB, Bruns ME, Christakos S 1985 Presence and local- 44. Hoenderop JG, van der Kemp AW, Hartog A, van de Graaf SF, ization of two vitamin DÐdependent calcium binding proteins van Os CH, Willems PH, Bindels RJ 1999 Molecular identifi- in kidneys of higher vertebrates. Endocrinology 117:674Ð683. cation of the apical Ca2+ channel in 1,25-dihydroxyvitamin 24. Howard A, Legon S, Spurr NK, Walters JR 1992 Molecular D3Ðresponsive epithelia. J Biol Chem 274:8375Ð8378. cloning and chromosomal assignment of human calbindin- 45. Peng JB, Chen XZ, Berger UV, Vassilev PM, Tsukaguchi H, D9K. Biochem Biophys Res Commun 185:663Ð669. Brown EM, Hediger MA 1999 Molecular cloning and char- 25. Jeung EB, Krisinger J, Dann JL, Leung PC 1992 Molecular acterization of a channel-like transporter mediating intestinal cloning of the full-length cDNA encoding the human calbindin- calcium absorption. J Biol Chem 274:22739Ð22746. D9K. FEBS Lett 307:224Ð228. 46. Peng JB, Chen XZ, Berger UV, Vassilev PM, Brown EM, 26. Kawasaki H, Nakayama S, Kretsinger RH 1998 Classification Hediger MA 2000 A rat kidney-specific calcium transporter and evolution of EF-hand proteins. Biometals 11:277Ð295. in the distal nephron. J Biol Chem 275:28186Ð28194. 27. Hunziker W 1986 The 28-kDa vitamin D-dependent 47. Li YC, Pirro AE, Amling M, Delling G, Baron R, Bronson R, calcium-binding protein has a six-domain structure. Proc Demay MB 1997 Targeted ablation of the vitamin D recep- Natl Acad Sci USA 83:7578Ð8582. tor: an animal model of vitamin D-dependent rickets type II 28. Akerfeldt KS, Coyne AN, Wilk RR, Thulin E, Linse S 1996 with alopecia. Proc Natl Acad Sci USA 94:9831Ð9835. 2+ Ca -binding stoichiometry of calbindin-D28K as assessed by 48. Yoshizawa T, Handa Y, Uematsu Y, Takeda S, Sekine K, spectroscopic analyses of synthetic peptide fragments. Yoshihara Y, Kawakami T, Arioka K, Sato H, Uchiyama Y, Biochemistry 35:3662Ð3669. Masushige S, Fukamizu A, Matsumoto T, Kato S 1997 Mice 29. Nelson MR, Thulin E, Fagan PA, Forsen S, Chazin WJ 2002 lacking the vitamin D receptor exhibit impaired bone forma- The EF-hand domain: A globally cooperative structural unit. tion, uterine hypoplasia and growth retardation after wean- Protein Sci 11:198Ð205. ing. Nat Genet 16:391Ð396. 30. Heizmann CW, Braun K 1992 Changes in Ca2+-binding pro- 49. Amling M, Priemel M, Holzmann T, Chapin K, Rueger JM, teins in human neurodegenerative disorders. Trends Neurosci Baron R, Demay MB 1999 Rescue of the skeletal pheno- 15:259Ð264. type of vitamin D receptor-ablated mice in the setting 31. Gross MD, Nelsestuen GL, Kumar R 1987 Observations on the of normal mineral ion homeostasis: Formal histomorphome- binding of lanthanides and calcium to vitamin DÐdependent tric and biomechanical analyses. Endocrinology 140: chick intestinal calcium binding protein. J Biol Chem 262: 4982Ð4987. 6539Ð6545. 50. Li YC, Pirro AE, Demay MB 1998 Analysis of vitamin DÐ 32. Maler L, Blankenship J, Rance M, Chazin WJ 2000 Site-site dependent calcium-binding protein messenger ribonucleic communication in the EF-hand Ca2+-binding protein cal- acid expression in mice lacking the vitamin D receptor. bindin D9K. Nat Struct Biol 7:245Ð250. Endocrinology 139:847Ð851. 33. Skelton NJ, Kordel J, Akke M, Forsen S, Chazin WJ 1994 51. Panda DK, Miao D, Tremblay ML, Sirois J, Farookhi R, Signal transduction versus buffering activity in Ca2+-binding Hendy GN, Goltzman D 2001 Targeted ablation of the proteins. Nat Struct Biol 1:239Ð245. 25-hydroxyvitamin D 1α-hydroxylase enzyme: Evidence for 34. Wasserman RH, Corradino RA, Taylor AN 1968 Vitamin D skeletal, reproductive, and immune dysfunction. Proc Natl dependent calcium binding protein: Purification and some Acad Sci USA 98:7498Ð7503. properties. J Biol Chem 243:3978Ð3986. 52. Rhoten WB, Christakos S 1981 Immunocytochemical local- 35. Kesvatera T, Jonsson B, Thulin E, Linse S 1999 Ionization ization of vitamin DÐdependent calcium binding protein in behavior of acidic residues in calbindin D9K. Proteins mammalian nephron. Endocrinology 109:981Ð983. 37:106Ð115. 53. Roth J, Thorens B, Hunziker W, Norman AW, Orci L 1981 36. Ingersoll RJ, Wasserman RH 1971 Vitamin D3 induced cal- Vitamin DÐdependent calcium binding protein: Immuno- cium binding protein: Binding characteristics, conformational cytochemical localization in chick kidney. Science 214: effects and other properties. J Biol Chem 246:2808Ð2814. 197Ð200. 732 SYLVIA CHRISTAKOS ET AL.

54. Rhoten WB, Lubit B, Christakos S 1984 Avian and mam- 72. Suda S, Takahashi N, Shinki T, Horiuchi N, Yamaguchi A, malian vitamin DÐdependent calcium-binding protein in Yoshiki S, Enomato S, Suda T 1985 1,25-Dihydroxyvitamin D3 reptilian nephron. Gen Comp Endocrinol 55:96Ð103. receptors and their actions in embryonic chick. Calcif Tissue Int 55. Stumpf WE, Sar M, Reid FA, Tanaka Y, DeLuca HF 1979 37:82Ð90. Target cells for 1,25-dihydroxyvitamin D3 in intestinal tract, 73. Bellido T, Huening M, Raval-Pandya M, Manolagas SC, stomach, skin, pituitary and parathyroid. Science 209: Christakos S 2000 Calbindin-D28K is expressed in osteoblastic 1188Ð1190. cells and suppresses their apoptosis by inhibiting caspase-3 56. Stumpf WE, Sar M, Narbauitz R, Reid FA, DeLuca HF, activity. J Biol Chem 275:26328Ð26332. Tanaka Y 1980 Cellular and subcellular localization of 74. Liu Y, Porta A, Peng X, Gengaro K, Cunningham EB, Li H, 1,25(OH)2D3 in rat kidney: Comparison with localization of Dominguez LA, Bellido T, Christakos S 2004 Prevention of parathyroid hormone and estradiol. Proc Natl Acad Sci USA glucocorticoid induced apoptosis in osteocytes and osteoblasts 77:1149Ð1153. by calbindin-D28K. J Bone Miner Res 19:479Ð490. 57. Winaver J, Sylk DB, Robertson JS, Chen TC, Puschett JB 75. Christakos S, Norman AW 1979 Evidence for a specific 1980 Micropuncture study of the acute renal tubular trans- high affinity protein for 1,25-dihydroxyvitamin D3 in chick port effects of 25-hydroxyvitamin D3 in the dog. Miner kidney and pancreas. Biochem Biophys Res Commun 89: Electrolyte Metab 4:178Ð188. 56Ð63. 58. Friedman PA, Gesek FA 1993 Vitamin D accelerates PTH- 76. Norman AW, Frankel BJ, Heldt AM, Grodsky GM 1980 dependent calcium transport in distal convoluted tubule cells. Vitamin D deficiency inhibits pancreatic secretion of insulin. Am J Physiol 265:F300ÐF308. Science 209:823Ð825. 59. Friedman PA, Gesek FA 1993 Calcium transport in renal 77. Kadowaki S, Norman AW 1985 Demonstration that the epithelial cells. Am J Physiol 264:F181ÐF198. vitamin D metabolite 1,25(OH)2D3 and not 24R,25(OH)2- 60. Bouhtiauy I, Lajeunesse D, Christakos S, Brunette MG 1994 vitamin D3 is essential for normal insulin secretion in the Two vitamin D3Ðdependent calcium binding proteins perfused rat pancreas. Diabetes 34:315Ð320. increase calcium reabsorption by different mechanisms I: 78. Lee, S, Clark SA, Gill RK, Christakos S 1994 1,25-Dihydroxy- Effect of CaBP28K. Kidney Int 45:461Ð468. vitamin D3 and pancreatic β-cell function: Vitamin D recep- 61. Hoenderop JG, Muller D, Van Der Kemp AW, Hartog A, tors, gene expression and insulin secretion. Endocrinology Suzuki M, Ishibashi K, Imai M, Sweep F, Willems PH, 134:1602Ð1610. Van Os CH, Bindels RJ 2001 Calcitriol controls the epithelial 79. Roth J, Bonner-Weir S, Norman AW, Orci L 1982 calcium channel in kidney. J Am Soc Nephrol 12:1342Ð1349. Immunocytochemistry of vitamin DÐdependent calcium 62. Yu AS, Boim M, Hebert SC, Castellano A, Perez-Reyes E, binding protein in chick pancreas: Exclusive localization in Lytton J 1995 Molecular characterization of renal calcium β cells. Endocrinology 111:2216Ð2218. channel beta-subunit transcripts. Am J Physiol 268:F525ÐF531. 80. Kadowaki S, Norman AW 1984 Pancreatic vitamin DÐ 63. Sooy K, Kohut J, Christakos S 2000 The role of calbindin dependent calcium binding protein: Biochemical properties and and 1,25-dihydroxyvitamin D3 in the kidney. Curr Opin response to vitamin D. Arch Biochem Biophys 233:228Ð236. Nephrol Hypertens 9:341Ð347. 81. Pochet R, Pipeleers DG, Malaisse WJ 1987 Calbindin-D28K: 64. Turner PR, Mefford S, Christakos S, Nissenson RA 2000 preferential localization in non-β islet cells of the rat pancreas. Apoptosis mediated by activation of the G protein-coupled Biol Cell 61:155Ð161. receptor for parathyroid hormone (PTH)/PTH-related protein 82. Clark SA, Stumpf WE, Sar M, DeLuca HF, Tanaka Y 1980 (PTHrP). Mol Endocrinol 14:241Ð254. Target cells for 1,25-dihydroxyvitamin D3 in the pancreas. 65. Bouhtiauy I, Lajeunesse D, Christakos S, Brunette MG 1994 Cell Tissue Res 209:515Ð520. Two vitamin D3-dependent calcium binding proteins increase 83. Johnson JA, Grande JP, Roche PC, Kumar R 1994 Immuno- calcium reabsorption by different mechanisms. II. Effect of histochemical localization of the 1,25(OH)2D3 receptor and CaBP 9K. Kidney Int 45:469Ð474. calbindin-D28K in human and rat pancreas. Am J Physiol 66. Grenet O, Bobadilla M, Chibout SD, Steiner S 2000 267:E356ÐE360. Evidence for the impairment of the vitamin D activation path- 84. Sooy K, Schermerhorn T, Noda M, Surana M, Rhoten WB, way by cyclosporine A. Biochem Pharmacol 59:267Ð272. Meyer M, Fleischer N, Sharp GW, Christakos S 1999 2+ 67. Yang CW, Kim J, Kim YH, Cha JH, Mim SY, Kim YO, Shin YS, Calbindin-D28K controls [Ca ]i and insulin release. Evidence Kim YS, Bang BK 1998 Inhibition of calbindin-D28K expres- obtained from calbindin-D28K knockout mice and beta cell sion by cyclosporin A in rat kidney: The possible pathogenesis lines. J Biol Chem 274:34343Ð34349. of cyclosporin A-induced hypercalciuria. J Am Soc Nephrol 85. Rabinovitch A, Suarez-Pinzon WL, Sooy K, Strynadka K, 9:1416Ð1426. Christakos S 2001 Expression of calbindin-D28K in a pancre- 68. Lee CT, Huynh VM, Lai LW, Lien YH 2002 Cyclosporine atic islet beta-cell line protects against cytokine-induced AÐinduced hypercalciuria in calbindin-D28K knockout and apoptosis and necrosis. Endocrinology 142:3649Ð3655. wild-type mice. Kidney Int 62:2055Ð2061. 86. Inpanbutr N, Reiswig JD, Bacon WI, Siemens RD, 69. Caride AJ, Chini EN, Penniston JT, Dousa TP 1999 Selective Iacopino AM 1996 Effect of vitamin D on testicular CaBP28K decrease of mRNAs encoding plasma membrane calcium expression and serum testosterone in chickens. Biol Reprod pump isoforms 2 and 3 in rat kidney. Kidney Int 56:1818Ð1825. 54:242Ð248. 70. Balmain N, Brehier A, Cuisinier-Gleizes P, Mathieu H 1986 87. Walters MR 1984 1,25-Dihydroxyvitamin D3 receptors in Evidence for the presence of calbindin-D28K (CaBP28K) in the seminiferous tubules of the rat testes increase at puberty. tibial growth cartilages of rats. Cell Tissue Res 245:331Ð335. Endocrinology 114:2167Ð2173. 71. Zhou XY, Dempster DW, Marion SL, Pike JW, Haussler MR, 88. Lippiello L, Wasserman RH 1975 Fluorescent antibody Clemens TL 1986 Bone vitamin DÐdependent calcium binding localization of the vitamin D dependent calcium binding protein is localized in chondrocytes of growth plate cartilage. protein in the oviduct of the laying hen. J Histochem Cytochem Calcif Tissue Int 38:244Ð247. 23:111Ð116. CHAPTER 42 The Calbindins: Calbindin-D9K and Calbindin-D28K 733

89. Opperman LA, Saunders TJ, Bruns DE, Boyd JC, Mills SE, 107. Baimbridge KG, Mody I, Miller JJ 1985 Reduction of rat Bruns ME 1992 Estrogen inhibits calbindin-D28K expression hippocampal calcium binding protein following commis- in mouse uterus. Endocrinology 130:1728Ð1735. sural, amygdala, septal, perforant path and olfactory bulb 90. Corradino RA, Smith CA, Krook LP, Fullmer CS 1993 kindling. Epilepsia 26:460Ð465. Tissue-specific regulation of shell gland calbindin-D28K 108. Sonnenberg J, Frantz GD, Lee S, Heick A, Chu C, Tobin AJ, biosynthesis by estradiol in precociously matured, vitamin Christakos S 1991 Calcium binding protein (calbindin-D28K) D-depleted chicks. Endocrinology 132:193Ð198. and glutamate decarboxylase gene expression after kindling 91. Roth J, Baeten D, Norman AW, Garcia-Sergura LM 1981 induced seizures. Mol Brain Res 9:179Ð190. Specific neurons in chick central nervous system stain with 109. Iacopino AM, Christakos S 1990 Specific reduction of neu- an antibody against intestinal vitamin D-dependent calcium ronal calcium binding protein (calbindin-D28K) gene expres- binding protein. Brain Res 222:452Ð457. sion in aging and neurodegenerative diseases. Proc Natl Acad 92. Feldman SC, Christakos S 1983 Vitamin DÐdependent cal- Sci USA 87:4078Ð4082. cium binding protein in rat brain: Biochemical and immuno- 110. Ferrer I, Tunon T, Soriano E, Del Rio A, Iraizoz I, Fonseca M, cytochemical characterization. Endocrinology 112:290Ð302. Guionnet N 1993 Calbindin-D28K immunoreactivity in the 93. Barski JJ, Hartmann J, Rose CR, Hoebeek F, Morl K, Noll- temporal neocortex in patients with Alzheimer’s disease. Clin Hussong M, De Zeeuw CI, Konnerth A, Meyer M 2003 Neuropathol 12:53Ð58. Calbindin in cerebellar Purkinje cells is a critical determinant of 111. Geula C, Bu J, Nagykery N, Scinto LF, Chan J, Joseph J, the precision of motor coordination. J Neurosci 23:3469Ð3477. Parker R, Wu CK 2003 Loss of calbindin-D28K from aging 94. Rabie A, Thomasset M, Legrand C 1983 Immunocytochemical human cholinergic basal forebrain: relation to neuronal loss. detection of a calcium binding protein in the cochlear and J Comp Neurol 455:249Ð259. vestibular hair cells of the rat. Cell Tissue Res 232:691Ð696. 112. Meier TJ, Ho DY, Sapolsky RM 1997 Increased expression 95. Legrand C, Brehier A, Clavel MC, Thomasset M, Rabie A of calbindin D28K via herpes simplex virus amplicon vector 1988 Cholecalcin (28 kDa CaBP) in rat cochlea: Development decreases calcium ion mobilization and enhances neuronal in normal and hypothyroid animals: An immunocytochemical survival after hypoglycemic challenge. J Neurochem study. Brain Res 466:121Ð129. 69:1039Ð1047. 96. Oberholtzer JC, Buettger C, Summers MC, Matschinsky FM 113. Ho BK, Alexianu ME, Colom LV, Mohamed AH, Serrano F, 1988 The 28 kDa calbindin-D is a major calcium binding Appel SH 1996 Expression of calbindin-D28K in motoneuron protein in the basilar papilla of the chick. Proc Natl Acad Sci hybrid cells after retroviral infection with calbindin-D28K USA 85:3387Ð3390. cDNA prevents amyotrophic lateral sclerosis IgG-mediated 97. Rabie A, Thomasset M, Parkes CO, Clavel MC 1985 cytotoxicity. Proc Natl Acad Sci USA 93:6796Ð6801. Immunocytochemical detection of 28,000 MW calcium 114. Guo Q, Christakos S, Robinson N, Mattson MP 1998 binding protein in horizontal cells of the rat retina. Cell Calbindin-D28K blocks the proapoptotic actions of mutant Tissue Res 240:493Ð496. presenilin 1: reduced oxidative stress and preserved mito- 98. Schreiner DS, Jande SS, Lawson DEM 1985 Target cells of chondrial function. Proc Natl Acad Sci USA 95:3227Ð3232. vitamin D in the retina. Acta Anat 121:153Ð162. 115. Liu AY, Abraham BA 1991 Subtractive cloning of a hybrid 99. Haley TL, Pochet R, Baizer L, Burton MD, Crabb JW, human endogenous retrovirus and calbindin gene in the Parmentier M, Polans AS 1995 Calbindin-D28K immunoreac- prostate cell line PC3. Cancer Res 51:4104Ð4110. tivity of human cone cells varies with retinal position. Vis 116. Dowd DR, MacDonald PN, Komm BS, Haussler MR, Neurosci 12:301Ð307. Miesfeld RL 1992 Stable expression of the calbindin-D28K 100. Roman A, Brisson P, Pasteels B, Demol S, Pochet R, Collen JP complementary DNA interferes with the apoptotic pathway 1988 PinealÐretinal molecular relationships: Immunocyto- in lymphocytes. Endocrinology 6:1843Ð1848. chemical evidence of calbindin-D28K in pineal transducers. 117. Reisner PD, Christakos S, Vanaman TC 1992 In vitro enzyme Brain Res 442:33Ð42. activation with calbindin-D28K the vitamin D dependent 101. Blatow M, Caputi A, Burnashev N, Monyer H, Rozov A 2003 28kDa calcium binding protein. FASEB J 297:127Ð131. Ca2+ buffer saturation underlies paired pulse facilitation in 118. Norman AW, Leathers V 1982 Preparation of a photoaffinity calbindin-D28K-containing terminals. Neuron 38:79Ð88. probe for the vitamin D dependent intestinal calcium binding 102. Mattson MP, Rychlick B, Chu C, Christakos S 1991 protein: Evidence for a calcium-dependent, specific interac- Evidence for calcium reducing and excitoprotective roles tion with intestinal alkaline phosphatase. Biochem Biophys for the calcium binding protein calbindin-D28K in cultured Res Commun 108:220Ð226. hippocampal neurons. Neuron 6:41Ð51. 119. Berggard T, Szczepankiewicz O, Thulin E, Linse S 2002 103. Chard PS, Bleakman D, Christakos S, Fullmer CS, Miller RJ Myoinositol monophosphatase is an activated target of 1993 Calcium buffering properties of calbindin-D28K and calbindin-D28K. J Biol Chem 277:41954Ð41959. parvalbumin in rat sensory neurones. J Physiol 472:341Ð357. 120. Pollock AS, Santiesteban HL 1995 Calbindin expression 104. Chard PS, Jordan J, Marcuccilli CJ, Miller RJ, Leiden JM, in renal tubular epithelial cells: Altered sodium phosphate Roos RP, Ghadge GD 1995 Regulation of excitatory trans- cotransport in association with cytoskeletal rearrangement. mission at hippocampal synapses by calbindin-D28K. Proc J Biol Chem 270:16291Ð16301. Natl Acad Sci USA 92:5144Ð5148. 121. Minghetti PP, Cancela L, Fujisawa Y, Theofan G, Norman AW 105. Li Z, Decavel C, Hatton GI 1995 Calbindin-D28K: Role in 1988 Molecular structure of the chicken vitamin DÐinduced 2+ determining intrinsically generated firing patterns in rat calbindin-D28K gene reveals eleven exons, six Ca -binding supraoptic neurones. J Physiol 4883:601Ð608. domains, and numerous promoter regulatory elements. Mol 106. Burke RE, Baimbridge KG 1993 Relative loss of the Endocrinol 2:355Ð367. striatal striosome compartment, defined by calbindin-D28K 122. Wilson PW, Rogers J, Harding M, Pohl V, Pattyn G, immunostaining, following developmental hypoxic-ischemic Lawson DEM 1988 Structure of chick chromosomal genes injury. Neuroscience 56:305Ð315. for calbindin and calretinin. J Mol Biol 200:615Ð625. 734 SYLVIA CHRISTAKOS ET AL.

123. Parmentier M, De Vijlder JJM, Muir E, Szpirer C, Islam MQ, serum calcium and phosphate levels in vitamin D-replete Geurts van Kessel A, Lawson DEM, Vassart G 1989 The chick intestine. Mol Cell Endocrinol 54:135Ð140. human calbindin 27 kDa gene: Structure organization of the 140. Huang Y-C, Christakos S 1988 Modulation of rat calbindin- 5′ and 3′ regions, chromosomal assignment, and restriction D28K gene expression by 1,25-dihydroxyvitamin D3 and fragment length polymorphism. Genomics 4:309Ð319. dietary alteration. Mol Endocrinol 2:928Ð935. 124. Varghese S, Deaven LL, Huang Y-C, Gill RK, Iacopino AM, 141. Collazo D, Takahashi H, McKay RDG 1992 Cellular targets Christakos S 1989 Transcriptional regulation and chromoso- and trophic functions of neurotrophin-3 in the developing rat mal assignment of the mammalian calbindin-D28K gene. Mol hippocampus. Neuron 9:643Ð656. Endocrinol 3:495Ð502. 142. Ip NY, Li Y, Yancopoulos GD, Lindsay RM 1993 Cultured 125. Wood TL, Kobayashi Y, Franz G, Varghese S, Christakos S, hippocampal neurons show responses to BDNF, NT-3 and Tobin AJ 1988 Molecular cloning of mammalian 28,000 Mr NT-4 but not NGF. J Neurosci 13:3394Ð3405. vitamin D dependent calcium binding protein (calbindin-D28K) 143. Cheng B, Christakos S, Mattson MP 1994 Tumor necrosis in rodent kidney and brain. DNA 7:585Ð594. factors protect neurons against excitotoxic/metabolic insults 126. Gill RK, Christakos S 1993 Identification of sequence and promote maintenance of calcium homeostasis. Neuron elements in mouse calbindin-D28K gene that confer 1,25-dihy- 12:139Ð153. droxyvitamin D3- and butyrate-inducible responses. Proc Natl 144. Mattson MP, Cheng B, Baldwin SA, Smith-Swintosky VL, Acad Sci USA 90:2984Ð2988. Keller J, Geddes JW, Scheff SW, Christakos S 1995 Brain 127. Christakos S, Norman AW 1980 Vitamin DÐdependent injury and tumor necrosis factors induce calbindin-D28K calcium-binding protein synthesis by chick kidney and in astrocytes: Evidence for a cytoprotective response. duodenal polysomes. Arch Biochem Biophys 203:809Ð815. J Neurosci Res 42:357Ð370. 128. Pansini AR, Christakos S 1984 Vitamin DÐdependent calcium 145. Lindvall O, Kokaia Z, Bengzon J, Elmer E, Kokaia M 1994 binding protein in rat kidney: Purification and physico- Neurotrophins and brain insults. Trends Neurosci 17:490Ð496. chemical and immunological characterization. J Biol Chem 146. Iacopino AM, Christakos S 1990 Corticosterone regulates 259:9735Ð9741. calbindin-D28K mRNA and protein levels in rat hippocampus. 129. Varghese S, Lee S, Huang Y-C, Christakos S 1988 Analysis J Biol Chem 265:10177Ð10180. of rat vitamin DÐdependent calbindin-D28K gene expression. 147. Krugers HJ, Medema RM, Postema F, Korf J 1995 Region- J Biol Chem 263:9776Ð9784. specific alterations of calbindin-D28K immunoreactivity in 130. Minghetti PP, Gibbs PEM, Norman AW 1989 Computer the rat hippocampus following adrenalectomy and cortico- analysis of 1,25-dihydroxyvitamin D3-receptor regulated pro- sterone treatment. Brain Res 696:89Ð96. moters: Identification of a candidate D3-response element. 148. Wang Y-Z, Christakos S 1995 Retinoic acid regulates the Biochem Biophys Res Commun 162:869Ð875. expression of the calcium binding protein, calbindin-D28K. 131. Maiyar AC, Minghetti PP, Norman AW 1991 Transfection Mol Endocrinol 9:1510-1521. of avian vitamin DÐdependent calbindin-D28K 5′ flanking 149. Nieto-Bona MP, Busiguina S, Torres-Aleman I 1995 Insulin- promoter sequence in primary chick kidney cells. Mol Cell like growth factor I is an afferent trophic signal that modu- Endocrinol 78:127Ð135. lates calbindin-D28K in adult Purkinje cells. J Neurosci Res 132. Ferrari S, Battini R, Pike WJ 1990 Functional analysis of the 42:371Ð376. promoter region of the gene encoding chicken calbindin-D28K. 150. Yamaguchi T, Keino K, Fukuda J 1995 The effect of insulin Adv Exp Med Biol 140:21Ð25. and insulin-like growth factor-1 on the expression of cal- 133. MacDonald PN, Whitfield GK, Haussler CA, Hocker AM, retinin and calbindin-D28K in rat embryonic neurons in culture. Haussler MR 1992 Evaluation of a putative vitamin D response Neurochem Int 26:255Ð262. element in the avian calcium binding protein gene. DNA Cell 151. Pavlou O, Ehlenfeldt R, Horn S, Orr HT 1996 Isolation, char- Biol 11:377Ð383. acterization and in vivo analysis of the murine calbindin-D28K 134. Theofan G, Nguyen AP, Norman AW 1986 Regulation of upstream regulatory region. Mol Brain Res 36:268Ð279. calbindin-D28K gene expression by 1,25-dihydroxyvitamin D3 152. Gill RK, Christakos S 1995 Regulation by estrogen through is correlated to receptor occupancy. J Biol Chem 261: the 5′-flanking region of the mouse calbindin-D28K gene. Mol 16943Ð16947. Endocrinol 9:319Ð326. 135. Enomoto H, Hendy GN, Andrews GK, Clemens TL 1992 153. Perret C, Lomri N, Gouhier N, Auffray C, Thomasset M Regulation of avian calbindin-D28K gene expression in primary 1988 The rat vitamin-D-dependent calcium-binding protein chick kidney cells: Importance of posttranscriptional mecha- (9-kDa CaBP) gene. Complete nucleotide sequence and nisms and calcium ion concentration. Endocrinology 130: structural organization. Eur J Biochem 172:43Ð51. 3467Ð3474. 154. Dupret JM, Brun P, Perret C, Lomri N, Thomasset M, 136. Meyer J, Galligan MA, Jones G, Komm BS, Haussler CA, Cuisinier-Gleizes P 1987 Transcriptional and post-transcrip- Haussler MR 1995 l,25(OH)2D3-dependent regulation of tional regulation of vitamin DÐdependent calcium-binding calbindin-D28K mRNA requires ongoing protein synthesis in protein gene expression in the rat duodenum by 1,25-dihy- chick duodenal organ culture. J Cell Biochem 58:315Ð327. droxycholecalciferol. J Biol Chem 262:16553Ð16557. 137. Hall AK, Bishop JE, Norman AW 1987 Inhibitory and stimula- 155. Colnot S, Ovejero C, Romagnolo B, Porteu A, Lacourte P, tory effects of dexamethasone and 1,25-dihydroxyvitamin D3 Thomasset M, Perret C 2000 Transgenic analysis of the on chick intestinal calbindin-D28K and its mRNA. Mol Cell response of the rat calbindin-D9K gene to vitamin D. Endocrinol 51:25Ð31. Endocrinology 141:2301Ð2308. 138. Corradino RA, Fullmer C 1991 Positive cotranscriptional 156. Lambert M, Colnot S, Suh E, L’Horset F, Blin C, Calliot ME, regulation of intestinal calbindin-D28K gene expression by Raymondjean M, Thomasset M, Traber PG, Perret C 1996 1,25-dihydroxyvitamin D3 and glucocorticoids. Endocrinology Cis-acting elements and transcription factors involved in the 128:944Ð950. intestinal specific expression of the rat calbindin-D9K gene: 139. Theofan G, Kong MW, Hall AK, Norman AW 1987 Binding of the intestine-specific transcription factor Cdx-2 to Expression of calbindin-D28K mRNA as a function of altered the TATA box. Eur J Biochem 236:778Ð788. CHAPTER 42 The Calbindins: Calbindin-D9K and Calbindin-D28K 735

157. Colnot S, Romagnolo B, Lambert M, Cluzeaud F, Porteu A, the transcriptional regulation of calbindin-D9K gene in rat Vandewalle A, Thomasset M, Kahn A, Perret C 1998 uterus. J Biol Chem 266:551Ð558. Intestinal expression of the calbindin-D9K gene in transgenic 161. L’Horset F, Blin C, Colnot S, Lambert M, Thomasset M, mice. Requirement for a Cdx2-binding site in a distal activator Perret C 1994 Calbindin-D9K gene expression in the uterus: region. J Biol Chem 273:31939Ð31946. Study of the two messenger ribonucleic acid species and 158. Huang YC, Lee S, Stolz R, Gabrielides C, Pansini-Porta A, analysis of an imperfect estrogen-responsive element. Bruns ME, Bruns DE, Miffin TE, Pike JW, Christakos S Endocrinology 134:11Ð18. 1989 Effect of hormones and development on the expression 162. Romagnolo B, Cluzeaud F, Lambert M, Colnot S, Porteu A, of the rat 1,25-dihydroxyvitamin D3 receptor gene. Comparison Molina T, Tomasset M, Vandewalle A, Kahn A, Perret C 1996 with calbindin gene expression. J Biol Chem 264: Tissue-specific and hormonal regulation of calbindin-D9K 17454Ð17461. fusion genes in transgenic mice. J Biol Chem 271: 159. Li H, Christakos S 1991 Differential regulation by 1,25-dihy- 16820Ð16826. droxyvitamin D3 of calbindin-D9K and calbindin-D28K gene 163. Romagnolo B, Molina T, Leroy G, Blin C, Porteux A, expression in mouse kidney. Endocrinology 128:2844Ð2852. Thomasset M, Vandewalle A, Kahn A, Perret C 1996 160. Darwish H, Krisinger J, Furlow JD, Smith C, Murdoch FE, Estradiol-dependent uterine leiomyomas in transgenic mice. DeLuca HF 1991 An estrogen-responsive element mediates J Clin Invest 98:777Ð784. CHAPTER 43 Target Genes: PTHrP

DAVID GOLTZMAN AND RICHARD KREMER Department of Medicine, McGill University and McGill University Health Center, Montre«al, Que«bec, Canada

I. Introduction V. Therapeutic Strategies to Inhibit PTHrP Production II. PTHrP Gene and Its Products VI. Summary III. Mechanism of Action of PTHrP References IV. Regulation of PTHrP Production

I. INTRODUCTION 141 amino acid. The human gene is regulated by three promoters in the 5′ region [6Ð8] and potentially by In this chapter, we first discuss the structure of the mRNA stability in the 3′ AUUUA-rich untranslated parathyroid hormoneÐrelated peptide (PTHrP) gene region [9]. PTHrP gene expression is positively or and its products. We next examine the mechanism negatively regulated by several factors in its 5′ promoter of action of PTHrP through its endocrine, autocrine, region [10]. Several growth factors including epidermal and intracrine pathways. We then describe important growth factor and insulin-like growth factor 1 (IGF1) are biological properties of PTHrP relevant to the develop- stimulatory [10,11], whereas 1,25 dihydroxyvitamin D3 ment and progression of human cancer. Finally, based (1,25(OH)2D3) and dexamethasone down-regulate on the known biology of PTHrP, we outline the impor- PTHrP expression and secretion in vitro [10,12,13]. tance of using preclinical animal models to test potential The immature forms of PTHrP, the prepro forms, are strategies for blocking PTHrP production in cancer with extended at the amino terminus by 36 amino acids that particular emphasis on vitamin D and vitamin D analogs contains a signal sequence necessary to direct the as therapeutic agents. nascent peptide from the cytosol to the endoplasmic reticulum (ER). This signal sequence is likely cleaved in the ER by signal peptidases. The propeptide is then II. PTHRP GENE AND ITS PRODUCTS directed to the Golgi apparatus where it is cleaved and the mature form of PTHrP then stored in secretory The gene encoding human PTHrP is a complex granules. These three isoforms are identical up to transcriptional unit of approximately 15 kb encoding amino acid 139 but then diverge to encode a unique three potential isoforms through alternative splicing of C-terminal region [14]. The high homology of PTHrP 139, 141, and 173 amino acids [1] (Fig. 1A). At the and PTH at the amino-terminal end is responsible for its amino-terminal end, PTHrP shares a strong homology action on calcium homeostasis including bone resorp- with parathyroid hormone (PTH) within the first 13 tion, renal calcium reabsorption, and renal phosphate amino acids. Furthermore, human PTH and PTHrP excretion [15]. The first two amino acids are critical genes are located at similar positions of chromosome for adenylate cyclase stimulation [14]. Although not 11 and 12, respectively, suggesting that these two genes similar in their primary sequence to PTH, amino acids arose through duplication from a common ancestral 14 to 34 are also critical for binding to the PTH/PTHrP gene. There are at least seven exons with exon1 subdi- receptor [16], suggesting that the tertiary structure vided into Ia, Ib, and Ic [2]. Exon II encodes different 5′ contributes to hormone binding. The sequence beyond untranslated regions. Exon III and IV encode the prepro- amino acid 34 is not required for PTHrP activity on coding region and the mature peptide up to amino acid calcium homeostasis, but is believed to play a role in 139, respectively. Exons V, VI, and VII encode the other important functions such as growth and differenti- C-terminal amino acids, a stop codon, and 3′ noncod- ation. Indeed, amino acids 35Ð111 are highly conserved ing regions. In contrast, the rat, mouse, and chicken among species, an indication that this region plays a [3Ð5] PTHrP genes have a much simpler organization critical role in “other” functions of PTHrP. Beyond with four or five exons encoding a single isoform of amino acid 111, the sequences diverge among species,

VITAMIN D, 2ND EDITION Copyright © 2005, Elsevier, Inc. FELDMAN, PIKE, AND GLORIEUX All rights reserved. 738 DAVID GOLTZMAN AND RICHARD KREMER

A Organization of the human PTHrP gene Ia Ib Ic II III IV V VI VII

−36 1 139 173 141 B Structural function relationship Placental Ca++ Unknown Pre-Pro PTH-Liketransport NLS Osteostatin function

−36 1 34 37 86 87 106 107 139 141 173 FIGURE 1 Organization of the PTHrP gene and structureÐfunction relationship. (A) The boxes represent the exons with exon I subdivided into Ia, Ib, Ic. Hatched boxes represent 5′ untranslated (5′UTR) regions. Solid boxes represent coding sequences. The open boxes represent 3′ untranslated (3′UTR) regions. The solid lines represent alternative splicing. (B) The proposed biologically active domains of human PTHrP are represented. All of PTH-like bioactivity is contained in PTHrP1-34. PTHrP37-86 is thought to play a role in placental calcium transport. The PTHrP nuclear/nucleolar local- ization sequence (NLS) lies between amino acids 87 and 106. A sequence between amino acids 107 and 137 also called osteostatin is believed to be anabolic for osteoblasts and inhibitory for osteoclasts. and the human isoform contains a unique sequence receptor is able to activate the MAP kinase pathway in from amino acids 141 to 173 whose function has not yet a cell-specific fashion [28]. However, similarly to other been elucidated. peptides that bind to cell surface receptors that act Posttranslational processing also contributes to the through a classical endocrine/paracrine mechanism, it generation of additional PTHrP fragments (Fig. 1B) with has been proposed that PTHrP can also target the distinct functions. This processing yields an amino termi- nucleus of the cell using an intracrine signaling path- nal fragment PTHrP 1-36 which can bind the way [29]. Indeed, the sequence spanning amino acids PTH/PTHrP receptor, a mid region fragment (residues 88Ð106 has structural homology with previously 38Ð86), [17], which plays a role in placental calcium described nuclear and nucleolar localization sequences transport [18] and a carboxyterminal fragment also called (NLS) [30,31]. Removal of this putative nuclear/nucle- osteostatin (residues 107Ð139) which inhibits osteoclastic olar sequence abolishes intranuclear localization of the bone resorption [19] and stimulates osteoblastic bone for- prepro PTHrP cDNA transiently expressed in COS-7 mation [20]. In addition the human form of PTHrP can be cells [29]. Subsequently this nuclear/nucleolar local- O-glycosylated at the amino terminus [21] and cleaved ization has been reported in osteoblasts [29], ker- off at the carboxy terminus to produce fragments [22,23] atinocytes [32], vascular smooth muscle [33], that accumulate in renal failure. pancreatic adenocarcinoma [34], melanoma [35], and breast cancer [36]. PTHrP was indeed localized over the dense fibrillar component of the nucleolus [29], III. MECHANISM OF ACTION OF PTHRP suggesting that it could modulate specific nucleolar functions such as transcription/processing of rRNA or Both PTH and PTHrP bind to a common seven ribonucleoprotein complex formation prior to their transmembrane spanning receptor that is linked transport to the cytoplasm. In contrast to this specific through G proteins to both adenylate cyclase and localization in COS-7 cells and osteoblasts, PTHrP phospholipase-C signaling pathways [24]. In addition localization is more diffuse to the nuclear/nucleolar to the seven transmembrane domains, the PTH/PTHrP compartments in vascular smooth muscle cells [33] receptor contains an extracellular domain of around and could therefore regulate other nuclear actions in a 180 amino acids and an intracellular carboxyterminal tail cell-specific fashion. It is tempting to speculate that of approximately 120 amino acids [25]. The overall PTHrP localization to the nuclear/nucleolus has spe- organization of the receptor, although similar to those cific functions independent of the paracrine/autocrine of other G proteinÐcoupled receptors, has a unique effects that are secondary to the PTH/PTHrP receptor gene organization and no sequence homology to other activation. This hypothesis has been confirmed in at G proteinÐcoupled receptors [26,27]. In addition, the least one cell model [37]. The mechanism by which CHAPTER 43 Target Genes: PTHrP 739

PTHrP is directed to the nucleus/nucleolus is still functions such as DNA replication and/or gene tran- elusive and probably not unique. It is presumed that in scription when translocated to the nucleus. order to exert its endocrine/paracrine/autocrine func- tion, PTHrP should be internalized, as is the case for other peptides that have receptor-independent nuclear IV. REGULATION OF PTHRP actions such as insulin, prolactin, and epidermal PRODUCTION growth factor (EGF) [38]. This hypothesis is supported by studies demonstrating receptor-mediated internal- Traditionally, PTHrP has been regarded as a para- ization of PTHrP in cells harboring the osteoblast phe- neoplastic hypercalcemic mediator produced by many notype [39,40]. However, it has also been reported that types of cancer, especially solid tumors [45]. PTHrP is PTHrP can be internalized in cells that do not express now known to be produced not only by cancer cells but the classical PTH/PTHrP receptor [41], indicating that by a wide variety of normal cells (Table I). It has been an as-yet-unidentified PTHrP receptor recognizing the specific nuclear/nucleolar sequence could be responsible TABLE I PTHrP Expression in Normal and Cancer for PTHrP internalization and nuclear/nucleolar targeting. Cells and Tissues Other possible mechanisms of PTHrP targeting to the nucleus in PTHrP producing cells is the intracrine Normal cells/tissues Cancer cells/tissues route that does not utilize receptor/ligand internaliza- tion. In this case, PTHrP could be diverted from its secre- Adrenal cortex Breast tory pathway from the ER and back to the cytosol by a Amnion Bladder process called retrograde translocation [42]. Although Aortic smooth muscle Colon there is no direct evidence that PTHrP could utilize this Bladder Embryonal carcinoma pathway to enter the nucleus, indirect evidence indi- Bone HTLVI-infected T cells cates that PTHrP is a substrate of the ubiquitin and Brain Insulinoma proteasome-mediated proteolytic system [43] that Bronchial epithelium Lung diverts PTHrP from the ER to the cytosol. However, it Cardiac muscle Medullary carcinoma has not yet been established that PTHrP could then Cervical cells Melanoma escape ubiquitination and proteosomal degradation to Chondrocytes Multiple myeloma be redirected to the nucleus. As for other peptides, the Endometrium Oral nuclear uptake of PTHrP from the cytosol is likely regulated by the cell cycle [44], although only indirect Endothelium Osteosarcoma proof of this mechanism has so far been established Epidermis Ovary [32,33]. It has also been suggested that PTHrP can Gonads Prostate Kidney Renal serve as a substrate for CDK2-CDC2 kinase [29], since a consensus motif for CDK2-CDC2 is located immedi- Keratinocytes Squamous carcinoma ately upstream of the nuclear/nucleolar localization Liver Testicular sequence. Phosphorylation of the NLS can indeed Lung influence nuclear import of other peptides [30] and Mammary epithelium therefore could potentially modulate PTHrP nuclear Melanocytes α β import. Importins and , also known as kariopherins Ovaries α β and , play a major role in the nuclear import process Pancreatic islet cells of NLS-containing proteins [38]. In the case of PTHrP Parathyroid glands and in contrast to the majority of other NLS-containing proteins, importin β and not importin α is responsible for Placenta nuclear translocation [39]. Indeed, a sequence mapped Prostate to amino acids 89Ð93 in the PHTrP sequence was found Salivary ducts to be responsible for interacting with importin β, and Small intestine residues 380Ð643 of importin β mediated this interac- Spleen tion. Once translocated to the nucleus/nucleolus com- Stomach mucosa partment, the biological function of PTHrP at these sites Thyroid remains elusive, but its accumulation in the dense fibril- Thymus lar component of the nucleolus suggests that it would Urothelium modulate ribosomal gene transcription or ribosomal Uterus synthesis. Additionally, it could affect other nuclear 740 DAVID GOLTZMAN AND RICHARD KREMER proposed that PTHrP functions predominantly as an (Fig. 2) and end-stage disease in non-Hodgkin’s lym- autocrine/paracrine factor in normal tissues such as phoma [53]. One of the major features of PTHrP regu- keratinocytes [46]. Furthermore, PTHrP produced by lation is its positive regulation by mitogenic stimuli and cancer cells exhibits growth factorÐlike properties that cytokines such as epidermal growth factor (EGF), may enhance the growth of tumor cells locally in an insulin-like growth factor I (IGF-1), and TGFβ autocrine fashion [47Ð49] and subsequently favor [10,11,54Ð56] and its inhibition by 1,25-dihydroxy- tumor progression [50]. Indeed, indirect clinical vitamin D3 [10,57Ð61]. Additionally, studies have shown evidence indicates that PTHrP enhanced the capacity that the PTHrP promoter region contains both growth of cancer cells to invade bone [47,51] and that the pres- factor responsive sequences and a vitamin DÐresponsive ence of detectable PTHrP levels in hypercalcemic can- element (VDRE) [10,62Ð64]. A variety of other factors cer patients is associated with shorter survival [52] have also been shown to be either stimulatory or

A B 1.00 Pretreatment calcium and PTHrp 1.00 PTHrP 0, CA ≤ 12 10/25 PTHrP > 0 9/16 PTHrP 0, CA > 12 9/10 PTHrP > 0 22/25 0.75 0.75 Pretreament calcium, PTHrP for Age ≤ 65 PTHrP 0, CA ≤ 12 3/14 PTHrP > 0 6/10 PTHrP 0, CA > 12 5/6 0.50 0.50 PTHrP > 0 14/16

Proportion surviving 0.25 Proportion surviving 0.25

0.00 0.00 0123 0123 Years Years C 1.00

PTHrP and age group PTHrP 0, Age ≤ 65 8/20 PTHrP > 0 20/26 0.75 PTHrP 0, Age > 65 11/15 PTHrP > 0 11/15

0.50

Proportion surviving 0.25

0.00 0123 Years FIGURE 2 (A) Survival in 76 hypercalcemic cancer patients by parathyroid hormoneÐrelated peptide (PTHrP) status and pre- treatment calcium levels. Numbers shown in the inset are total number of deaths/number of patients at baseline. Number of patients at risk were 40 at 100 days, 22 at 1 year, and 3 at 3 years. (B) Survival in 46 hypercalcemic cancer patients ≤ 65 years old of PTHrP status and pretreatment calcium levels. Number of patients at risk were 26 at 100 days, 13 at 1 year, and 1 at 3 years. (C) Survival in hypercalcemic cancer patients by PTHrP status and age group. Number of patients at risk were 41 at 100 days, 22 at 1 year, and 3 at 3 years. CA ≤ 12, pretreatment serum calcium levels 10.3 to 12 mg/dl; CA > 12, pretreatment serum calcium levels > 12 mg/dl; PTHrP 0, PTHrP not elevated; PTHrP > 0, PTHrP elevated [52]. CHAPTER 43 Target Genes: PTHrP 741

TABLE II Stimulators and Inhibitors of PTHrP Expression/Production in Normal and Cancer Cells and Tissues

Stimulators Cell/tissue type Inhibitors Cell/tissue type

Serum Human keratinocytes [10] 1,25(OH)2D3 Human medullary carcinoma [57] Rat aortic smooth muscle [65] Human keratinocytes [10] Human squamous carcinoma [55] Human keratinocytes [58] Rat islet cell line [66] Human oral cancer cells [59] Human squamous carcinoma [54] Human squamous carcinoma [60] Human squamous carcinoma [60] Human T cells transfected with HTLV1 [61] Growth factors Human keratinocytes [10] Human melanocytes and melanoma cells [106] Human cervical epithelial cells [67] EB1089 Human squamous carcinoma [100]

Epidermal Human mammary epithelial [1,25(OH)2D3 analog] Human squamous carcinoma [60] growth factor cells [11] Human osteosarcoma cells [70] Human melanoma [106] Human squamous carcinoma [55] Human lung cancer cells [64]

Human keratinocyte cell line 22-oxa-1,25(OH)2D3 Human T cells transfected HaCaT [75] [1,25(OH)2D3 analog] with HTVL1 [61] IGFI Human mammary epithelial cells [11] Human lung cancer cells [64] Human lung carcinoma [76] 9-cis-retinoic acid Human oral cancer cells [59] TGFβ Human squamous carcinoma [55] Glucocorticoids Human medullary carcinoma [57] Human uterine cells [56] Human neuroendocrine cells [71] IL-6 Human lung carcinoma [76] Testosterone Rat testicular tumor [13] IL-1β Human lung carcinoma [76] Chromogranin A Human squamous carcinoma [72] TNFα Human lung carcinoma [76] Ras-signaling inhibitors Rat Leydig H500 tumor [95] (B1086 and lovastatin) Ras-Raf- Human vascular endothelial cells [73] MAP-Kinase Rat 3T3 fibroblasts transfected signaling with TRP-MET [78] Ras transformed human prostate epithelial cells [77] Calcium Human keratinocytes [10] Human squamous carcinoma [54] Estrogens Rat uterus [105] Rat kidney [68] MCF-7 breast cancer cells [74] Prolactin Rat mammary gland [107] Calcitonin Human squamous carcinoma [69] Human squamous carcinoma [72] All-trans- retinoic acid Human cervical epithelial cells [67] Phorbol ester Human osteosarcoma cells [70] Endothelin I Rat aortic smooth muscle [65]

inhibitory for PTHrP as indicated in Table II. Since expressed in cancer cells [77,78]. Additional mecha- growth factors are generally positive regulators for nisms have been proposed to explain PTHrP overex- PTHrP production, it should not be surprising that can- pression in cancer tissues and include silencing by cer cells may overexpress PTHrP following activation demethylation of specific regulatory sequences of the of the growth factor signaling pathways. The ras-raf- PTHrP gene [79], gene amplification [80], and resistance MAPKinase activation cascade is activated by EGF and to PTHrP-induced inhibition by 1,25(OH)2D3 [54]. This can lead to PTHrP overproduction when constitutively resistance to 1,25(OH)2D3 action was shown to be 742 DAVID GOLTZMAN AND RICHARD KREMER secondary to phosphorylation of the vitamin D receptor V. THERAPEUTIC STRATEGIES TO (VDR) heterodimeric partner retinoid X receptor (RXR) INHIBIT PTHRP PRODUCTION at a specific MAP kinase consensus site or SER 160 [81]. Overall, several mechanisms may in theory lead to over- Based on our current knowledge of PTHrP biology, production of PTHrP by tumor cells, allowing subse- several strategies may be devised to block PTHrP pro- quent release into the systemic circulation or enhanced duction or action by cancer cells. These include PTHrP local autocrine/paracrine action. Based on these obser- ablation by immunotherapy or antisense strategies, vations, a number of strategies could be devised to PTHrP inhibition by altering growth factor production/ block PTHrP production by cancer cells. A primary tar- signaling or vitamin D therapy (Fig. 3). get for such strategies is breast cancer, since over 60% of primary breast cancer and over 90% of metastatic breast cancer lesions have been reported to overexpress A. Immunotherapy PTHrP [82Ð87]. The higher percentage of metastatic lesions overexpressing PTHrP may be explained by a Neutralization of the PTHrP molecule using specific favorable bone microenvironment which produces antibodies is particularly attractive since it has the cytokines such as TGFβ, a known stimulator of PTHrP potential to work systemically as well as locally to block production [55,56]. Targeting PTHrP in breast cancer PTHrP production and action. Previous studies have has therefore the potential to inhibit mammary tumor shown that this strategy worked in an animal model of growth and its devastating skeletal complications. osteolytic metastases associated with breast cancer [88].

25-OHD3 1,25-(OH)2D3 and analogs

1α-hydroxylase

1,25(OH)2D3

RXR VDR RAS VDRE − PTHrP mRNA PTHrP + GFRE

GF MAPK PrePro PTHrP

PTHrP

Antibody

FIGURE 3 Regulation of PTHrP expression/production and potential targets for PTHrP inhibition. Positive regu- lators of PTHrP include growth factors that can utilize the ras-raf-mitogen-activated protein kinase (MAPK) path- way. Negative regulators include 1,25(OH)2D3 and its analogs. A reduction in PTHrP expression/production is possible through inactivation of the growth factor-MAPK pathway and/or through activation of the VDR/RXR com- plex by 1,25(OH)2D3, its analogs, or the inactive 1,25(OH)2D3 precursor 25OHD3. Additional strategies could include PTHrP antibody administration (immunotherapy) and gene expression knockout technology (antisense therapy). CHAPTER 43 Target Genes: PTHrP 743

Mice treated with a PTHrP antibody raised against undergo down-regulation of gene expression over PTHrP1-34 had fewer osteolytic lesions than control time [94]. Consequently, this approach for gene therapy animals. Furthermore, histomorphometric analysis of experiments may not yield sufficient effects on gene long revealed that this strategy reduced both expression over time to sustain the desired biological the number of active osteoclasts at the tumorÐbone effects. In another study, PTHrP inhibition was achieved interface and the tumor burden within bone. These using a nonretroviral antisense RNA approach and beneficial effects were observed even after the estab- resulted in normalization of serum calcium levels lishment of bone metastatic lesions [89]. In this animal and inhibition of tumor growth in an animal model of model, osteolytic lesions were not associated with malignancy-associated hypercalcemia, the H500 rat hypercalcemia or detectable PTHrP fragments in the Leydig cell tumor model [49]. Despite the potential dif- circulation, indicating that the antibody likely had a ficulties in future gene therapy experiments, the anti- neutralizing effect by preventing the binding of PTHrP sense RNA approach has the additional advantage over molecules to the PTH/PTHrP receptor within the bone immunotherapy to block both the secretory pathway microenvironment. Additional studies indicate that this and the intracrine PTHrP signaling pathway. strategy may also be effective in animal models of PTHrP-induced hypercalcemia demonstrating that sys- temic infusion of PTHrP antibodies can effectively C. Inhibition of PTHrP by Interfering with normalize serum calcium levels [90,91]. These authors Growth Factor Signaling used a humanized antibody constructed using the complementary-determining region grafting method. As indicated earlier, a number of growth factors and They showed that this humanized antibody inhibited cytokines positively regulate PTHrP production (see PTHrP-induced cAMP production in rat osteosarcoma Table II). Studies on the molecular mechanism of ROS17/28-5 cells in vitro and bone resorption markers PTHrP induction by growth factors indicate that ras in vivo. Furthermore, administration of a single dose of signaling is critical for this effect [77,78]. Constitutive this antibody was more effective than bisphosphonates expression of receptor tyrosine kinases and p21ras in correcting hypercalcemia and maintaining normal isoprenylation significantly enhanced PTHrP produc- serum calcium levels thereafter. In addition, adminis- tion indicating that targeting receptor tyrosine kinases tration of the humanized antibody but not of bisphos- and/or ras signaling is a possible strategy to block PTHrP phonates had anticachectic properties and prolonged production [78]. Interestingly, lovastatin, a commonly survival of these animals, suggesting that PTHrP used hypolipidemic agent, is able to block p21 isopreny- inhibition had additional properties independent of its lation at the inner surface of the plasma membrane and antihypercalcemic effect. consequently inactivate ras-signaling. In these studies, it However, the usefulness of this approach in cancer was demonstrated that lovastatin blocks PTHrP produc- patients remains to be established. tion [95] in cancer cells in vitro. It has not yet been demonstrated whether this strategy works in vivo.

B. Antisense RNA Technology D. Vitamin D Therapy Antisense RNA inhibits target mRNA sequences by hybridizing and consequently interfering through one of Since the discovery that 1,25(OH)2D3 inhibits several mechanisms, with the function of the targeted PTHrP production in normal human epidermal ker- mRNA. This ultimately results in reduced translation atinocytes [10], numerous studies have shown that of the gene product [92]. This strategy was applied pre- 1,25(OH)2D3 is a strong inhibitor of PTHrP production viously to block PTHrP production using two different in a wide variety of normal and cancer cells (Table II). approaches [46,49]. In one study, a PTHrP cDNA cloned It was further demonstrated that the mechanism of this in an antisense orientation in a replication defective inhibitory effect involves vitamin DÐresponsive elements retroviral vector [46] was used and resulted in the com- in the 5′ promoter region of both the rat [10,62,96] plete inhibition of PTHrP production in a human and human genes [63]. It is interesting to note that the keratinocyte cell line. The retrovector used contains putative 1,25(OH)2D3 mediated repression sequence the backbone of Moloney murine leukemia virus long of the rat PTHrP promoter overlapped with the growth terminal repeat promoter (MoMLV-LTR) [93]. However, factor/serum mediated stimulatory region localized the MoMLV-LTR and other MoMLV-based vectors that between 0.3 and 1.2 kb upstream of the transcriptional are the most widely used for retroviral-mediated gene start site [10]. Furthermore, although the rat PTHrP transfer experiments are not active in all cell types and VDRE interacts with the VDR/RXR complex like 744 DAVID GOLTZMAN AND RICHARD KREMER other VDREs [62], the human PTHrP VDRE appears be useful in targeting PTHrP-producing tumors with- not to interact. The human PTHrP VDRE was shown out hypercalcemia. Indeed, it has been suggested that to recognize VDR but not RXR [63], although studies PTHrP may play an important role in the establishment/ in human oral cancer cells seem to indicate that sup- development of osteolytic bone metastasis associated pression of PTHrP expression in this model is medi- with breast cancer [83,89]. A xenograft model of bone ated through the VDR/RXR complex activation [59]. metastasis in which human MDA-MB-231 breast can- 1,25(OH)2D3 therapy may therefore represent an alter- cer cells are implanted into the left ventricle of nude native option to block PTHrP production in cancer mice [89,103] was used to test the efficacy of EB1089. patients. However, since 1,25(OH)2D3 causes hyper- The analog was administered in a preventative fashion calcemia and hypercalciuria at relatively low doses, its at the time of tumor implantation [102]. The develop- applications in malignancy-associated hypercalcemia ment of osteolytic bone metastasis was evaluated radi- may be limited. These difficulties could in theory be ologically, histologically, and by histomorphometry. overcome by synthesizing new vitamin D analogs with Kaplan Meier analysis demonstrated that bone lesions lower calcemic activity compared to 1,25(OH)2D3 but detected by radiographs progressed more slowly in with potent anti-tumor effect. Interestingly a number of EB1089-treated animals and that animal survival time these analogs were found to have selective properties was increased. Furthermore, tumor burden in bone was on growth and differentiation [97Ð99] and in the hyper- significantly reduced in EB1089-treated animals. proliferative skin disorder psoriasis, due to their rapid Interestingly, PTHrP is expressed in this MDA-MB- metabolic degradation when applied locally to skin 231 xenograft model and released by these cancer cells lesions. Other analogs with strong antiproliferative and was previously shown to play a causal role in the prodifferentiative properties that are administered sys- development of osteolytic bone lesions [89]. Although temically have also been designed (see Chapters 80Ð88). not reported in the study of El Abdaimi et al. [102], it One such analog, EB1089, was found to have very low would be of major interest to know if EB1089 can calcemic potency relative to 1,25(OH)2D3 when infused reduce the production of PTHrP by metastatic tumors into control animals [100Ð102]. in bone in this model. An alternative strategy to block In vivo, EB1089 is 10Ð100 times more potent than PTHrP production would be to use an inactive vitamin 1,25(OH)2D3 in inhibiting PTHrP production in cancer D derivative that could subsequently be metabolized cells [60]. Subsequently this analog was used in two by tumor tissues to active vitamin D metabolites. preclinical animal models of malignancy-associated Such strategy has been used in a xenograft model of hypercalcemia including the rat Leydig tumor H500 ras-transformed keratinocytes transplanted in nude implanted into Fisher rats and a xenograft model in mice [104]. In this study, both alleles of the human which nude mice were implanted with PTHrP-producing 1α-hydroxylase gene were inactivated by homologous HPK1Aras cells. In these models, it was clearly estab- recombination (double knockout) in cancer cells and lished that EB1089 inhibited PTHrP production by the the effect of the inactive precursor 25-hydroxyvitamin tumors and its release in the circulation. These actions in D3 (25OHD3) was tested in both wild-type and double turn caused a reduction in hypercalcemia [100,101]. knockout cancer cells. In vivo tumor growth was Interestingly, in the H500 Leydig tumor model EB1089 significantly reduced by 25OHD3 in nude mice also prolonged survival, an added benefit that may be transplanted with wild-type cells but not in animals related to either its antihypercalcemic effect, its direct transplanted with knockout cells, indicating that antitumor effect, or its indirect antitumor effect through 1α-hydroxylase expression by cancer cells was neces- PTHrP inhibition. Furthermore, EB1089 demonstrated sary for the growth inhibitory effect of 25OHD3 to both preventative and therapeutic potential by either occur (Fig. 4). Furthermore, animals remained normo- preventing the development of hypercalcemia [100] or calcemic even when 25OHD3 was administered at by decreasing blood calcium levels once hypercal- doses 100 times higher than 1,25(OH)2D3. In a subse- cemia was achieved [101]. Both strategies represent quent study, 25OHD3 was found to be highly effective two distinct but clinically relevant situations. In the in reducing PTHrP production by human melanoma first scenario, the analog is administered to a patient cells following its conversion to 1,25(OH)2D3 [50]. with an established diagnosis of cancer but prior to the In vivo strategies using inactive 1,25(OH)2D3 precur- development of hypercalcemia. In the second scenario, sors may therefore be effective in controlling PTHrP the analog is administered to a cancer patient present- overproduction while avoiding the undesired hypercal- ing with hypercalcemia and usually an advanced stage cemic complications. A potential drawback common of cancer. to many cancer therapeutic strategies is the possibility In addition to being a potential therapy in malignancy- of drug resistance. Indeed it has been demonstrated associated hypercalcemia, vitamin D analogs may also that ras-transformed keratinocytes producing PTHrP CHAPTER 43 Target Genes: PTHrP 745

AB1600 1600 WT (+/+) DKO (−/−)

1400 Vehicle 1400 Vehicle

1200 1200 25 OHD3 25 OHD3 ) 3 1000 1000

800 800

600 600 Tumor volume (mm 400 400

200 200

0 0 0102030405060 0102030405060 Time (days)

CD 1.5 WT (+/+) 1.5 DKO (−/−)

1 1 Tumor weight (g) 0.5 0.5

0 0 Vehicle 25OHD3 Vehicle 25OHD3 FIGURE 4 In vivo tumor growth kinetics in SCID mice following s.c. injection of 3 × 106 cells in PBS mixed with matrigel (1:1). 25-OHD3 (2000 pM 24 hr) was administered by constant infusion using Alzet osmotic minipumps. SCID mice that received implants of WT control HPK1Aras/pcDNA3 cells (A) or DKO HPK1Aras cells (B). 25-OHD3 administration significantly inhibit tumor growth in animals that received implants of WT-HPK1Aras cells (A and C), but has no effect in animals that received implants of DKO HPK1Aras cells (B and D). Seven and a half weeks after tumor implantation and treatment with 25-OHD3 or vehicle, mice were killed and the weight of tumors in animals implanted with WT (C) or DKO (D) was measured. Data are expressed as means of 12 mice in each group. This experiment was repeated twice. *, significantly different from vehicle-treated animals at each time point; P < 0.05. (From Huang DC, Papavasiliou V, Rhim JS, Horst RL, Kremer R 2002 Mol Cancer Res 1:1Ð12.) are partially resistant to the inhibitory effect of analogs may be substantially reduced in the commonly 1,25(OH)2D3 [54]. It was subsequently demonstrated occurring ras-transformed tumors and strategies aimed that ras-induced resistance to vitamin D was a conse- at simultaneously inactivating the ras-raf-MAPKinase quence of the activation of the ras-raf-MAPKinase signaling pathways and administering the vitamin D pathway resulting in phosphorylation of the retinoid X analog may therefore be extremely useful. Similarly receptor on SER260 (a MAPKinase consensus strategies aimed at inhibiting growth factor signaling sequence) [81]. Consequently, the efficacy of vitamin D may also be used in combination with vitamin D 746 DAVID GOLTZMAN AND RICHARD KREMER analog therapy to target simultaneously the stimulatory 11. Sebag M, Henderson JE, Goltzman D, Kremer R 1994 and inhibitory pathways of PTHrP signaling. Regulation of parathyroid hormone-related peptide produc- tion in normal human mammary epithelial cells in vitro. Am J Physiol 267(3):C723Ð730. 12. Glatz JA, Heath JK, Southby J, L’Keefe LM, Kiriyama T, VI. SUMMARY Moseley JM, Martin TJ, Gillespie MT 1994 Dexamethasone regulation of PTHrP expression in a squamous cancer cell Our thorough understanding of PTHrP regulation line. Mol Cell Endocrinol 101:295Ð308. 13. Liu B, Goltzman D, Rabbani SA 1993 Regulation of parathy- by both positive and negative regulators has led to roid hormone related protein production in vitro by the the evaluation of new therapeutic options to control the rat hypercalcemic leydig tumor H500. Endocrinology 132: production of this peptide in cancer. Because of the 1658Ð1664. complex nature of PTHrP regulation, it is plausible 14. Orloff JJ, Reddy D, de Papp AE, Yang KH, Soifer NE, that combining strategies to target several steps of the Stewart AF 1994 Parathyroid hormoneÐrelated protein as a prohormone. Post-translational processing and receptor regulatory pathway may become a viable therapeutic interactions. Endocr Reviews 15:40Ð60. option in cancer therapy. Vitamin D signaling has 15. Kemp BE, Moseley JM, Rodda CP, Ebeling PR, Wettenhall RE, emerged as an important potential tool in these strate- Stapleton D, Diefenback-Jagger H, Ure F, Michelangeli VP, gies by using either more active tissue specific analogs Simmons HA, et al. 1987 Parathyroid hormone-related or inactive precursors that could be converted to active protein of malignancy: active synthetic fragments. Science 238: 1568Ð1570. forms within the target cancer cells. Additional efforts 16. Stewart AF, Broadus AE 1991 PTHrP: coming of age in the should be directed to find even more potent vitamin D 1990s. J Clin Endocrinol Metab 71:1410Ð1414. analogs or precursors that could be tested alone or in 17. Soifer NE, Dee KE, Insogna KL, Burtis WJ, Matovcik LM, combination with other strategies targeting a reduction Wu TL, Milstone LM, Broadus AE, Philbrick WM, Stewart AF of PTHrP production. 1992 PTHrP: Evidence of secretion of a novel mid region fragment by three different cell lines in culture. J Biol Chem 267:18236Ð18243. 18. Kovacs CS, Lanske B, Hunzelman JL, Guo J, Karaplis AC, References Kroninberg HM 1996 Parathyroid hormone related peptide (PTHrP) regulates fetalÐplacental calcium transport through 1. Martin TJ, Moseley J, Williams D 1997 Parathyroid hormone- a receptor distinct from the PTH/PTHrP receptor. Proc Natl related protein: hormone and cytokine. J Endocrinol 154: Acad Sci USA 93:15233Ð15238. 523Ð537. 19. Cornish J, Callon KE, Nicholson GC, Reid IR 1997 2. Martin TJ, Moseley J, Gillespie MT 1991 Parathyroid hormone- Parathyroid hormone-related protein (107Ð139) inhibits bone related protein: biochemistry and molecular biology. Crit Rev resorption in vivo. Endocrinology 138:1299Ð1304. Biochem Mol Biol 26:377Ð395. 20. Cornish J, Callon KE, Lin C, Xiao C, Moseley JM, Reid IR 3. Karaplis AC, Yasuda J, Hendy GN, Goltzman D, Banville D 1999 Stimulation of osteoblast proliferation by C-terminal 1990 Gene-encoding parathyroid hormone-like peptide: fragments of parathyroid hormone-related protein. J Bone nucleotide sequence of the rat gene and comparison with the Min Res 14:915Ð922. human homolog. Mol Endocrinol 4:441Ð446. 21. Wu TL, Soifer NE, Burtis WJ, Milstone LM, Stewart AF 4. Mangin M, Ikeda K, Broadus AE 1990 Structure of the 1991 Glycosylation of PTHrP secreted by human epidermal mouse gene encoding parathyroid hormone-related peptide. keratinocytes. J Clin Endocrinol Metab 73:1002Ð1007 Gene 95:195Ð202. 22. Burtis WJ, Brady TG, Orloff JJ, Ersbak BJ, Warrell RP Jr, 5. Thiede MA, Rutledge SJ 1990 Nucleotide sequence of a Olson BR, Wu TL, Mitnick ME, Broadus AE, Stewart AF parathyroid hormone-related peptide expressed by the 10-day 1990 Immunochemical characterization of circulating chicken embryo. Nucleic Acids Res 18:3062. parathyroid hormone-related protein in patients with humoral 6. Mangin M, Ikeda K, Dreyer BE, Broadus AE 1990 hypercalcemia. N Engl J Med 322:1106Ð1112. Identification of an upstream promoter of the human parathy- 23. Orloff JJ, Soifer NE, Fodero JP, Dann P, Burtis WJ 1993 roid hormone-related peptide gene. Mol Endocrinol 4:851Ð858. Accumulation of carboxyterminal fragments of PTHrP in 7. Yasuda T, Banville, D, Hendy GN, Goltzman D 1989 renal failure. Kidney Int 43:1371Ð1376. Characterization of the human parathyroid hormone-like 24. Chorev M, Rosenblatt M 1994 StructureÐfunction analysis peptide gene: Functional and evolutionary aspects. J Biol of parathyroid hormone and parathyroid hormone-related Chem 13:7720Ð7725. protein. In: Bilezekian JP, Marcus R, Levine MD (eds) The 8. Vasavada RC, Wysolmerski JJ, Broadus AE, Philbrick WM Parathyroids: Basic and Clinical Concepts. Raven Press, 1993 Identification and characterization of a GC-rich pro- New York, pp. 139Ð116. moter of the human PTHrP gene. Mol Endocrinol 7:273Ð282. 25. Jüppner H 1995 Functional properties of the PTH/PTHrP 9. Moseley JM, Gillespie MT 1995 Parathyroid hormone- receptor. Bone (Suppl. 17):39SÐ42S. related protein. Crit Rev Clin Lab Sciences 32:299Ð343. 26. Jüppner H 1994 Molecular cloning and characterization of a 10. Kremer R, Karaplis AC, Henderson J, Gulliver W, Banville D, PTH/PTHrP receptor: a member of an ancient family of G Hendy GN, Goiltzman D 1991 Regulation of parathyroid protein coupled receptors. Curr Opin Nephrol Hypertens hormone-like peptide in cultured normal human keratinocytes: 3:371Ð378. effect of growth factors and 1,25 dihydroxyvitamin D3 gene 27. Abou-Samra AB, Juppner H, Force T, Freeman MW, expression and secretion. J Clin Invest 87:884Ð893. Kong XF, Schipani E, Urena P, Richards J, Bonventre JV, CHAPTER 43 Target Genes: PTHrP 747

Potts JT Jr, et al. 1992 Expression cloning of a common 42. Kopito RR 1997 ER quality control: The cytoplasmic con- receptor for PTH and PTHrP from rat osteoblast-like cells: nection. Cell 88:427Ð430. A single receptor stimulates intracellular accumulation of both 43. Meerovitch K, Wing S, Goltzman D 1997 Pre-proparathyroid camp and inositol triphosphates and increases intracellular hormone-related protein, a secreted peptide is a substrate free calcium. Proc Natl Acad Sci USA 89:2732Ð2736. for the ubiquitin proteolytic system. J Biol Chem 272: 28. Verheijen MH, Defize LH 1997 Parathyroid hormone related 6707Ð6013. peptide activates mitogen-activated protein kinase via a 44. Fujihara SM, Nadler SG 1998 Modulation of nuclear protein cAMP-mediated pathway independent of Ras. J Biol Chem import: a novel means of regulating gene expression. Biochem 272:3423Ð3429. Pharmacol 56:157Ð161. 29. Henderson JE, Amizuka N, Warshawsky H, Biasotto D, 45. Stewart AF, Horst R, Deftos LJ, Camden EC, Lang R, Lanske BM, Goltzman D, Karaplis AC 1995 Nucleolar local- Broadus AE 1980 Biochemical evaluation of patients with ization of parathyroid hormoneÐrelated peptide enhances cancer-associated hypercalcemia. Evidence for humoral and survival of chondrocytes under conditions that promote non-humoral groups. N Engl J Med 303:1377Ð1383. apoptotic cell death. Mol Cell Biol 15:4064Ð4075. 46. Kaiser SM, Laneuville P, Bernier SM, Rhim JS, Kremer R, 30. Jans DA, Hubner S 1996 Regulation of protein transport to Goltzman D 1992 Enhanced growth of a human keratinocyte the nucleus: central role of phosphorylation. Physiol Rev cell line induced by antisense RNA for parathyroid hormone- 76:651Ð685. related peptide. J Biol Chem 267:13623Ð13628. 31. Jans DA, Xiao CY, Lam MH 2000 Nuclear targeting signal 47. Burton PB, Moniz C, Quirke P, Malik A, Bui TD, Juppner H, recognition: A key control point in nuclear transport? Segre GV, Knight DE 1992 Parathyroid hormone-related Bioessays 22:532Ð544. peptide: expression in fetal and neonatal development. 32. Lam MH, Olsen SL, Rankin WA, Ho PW, Martin TJ, J Pathol 167:291Ð296. Gillespie MT, Moseley JM 1997 PTHrP and cell division: 48. Benitezverguizas J, Esbrit P 1994 Proliferative effect of expression and localization of PTHrP in a keratinocyte cell parathyroid hormone-related protein on the hypercalcemic line (HaCaT) during the cell cycle. J Cell Physiol walker 256 carcinoma cell line. Biochem Biophys Res Commun 173:433Ð446. 198:1281Ð1289. 33. Massfelder T, Dann P, Wu TL, Vasavaba R, Helwig JJ, 49. Rabbani SA, Gladu J, Liu B, Goltzman D 1995 Regulation Stewart AF 1997 Opposing mitogenic and antimitogenic in vivo of the growth of Leydig cell tumors by antisense actions of parathyroid hormone related protein in vascular ribonucleic acid for parathyroid hormone related peptide. smooth muscle cells: a critical role for nuclear targeting. Proc Endocrinology 136:5416Ð5422. Natl Acad Sci USA 94:13630Ð13635. 50. Huang D, Kremer R 2002 Inhibition of metastasis in 34. Bouvet M, Nardin SR, Burton DW, Behling C, Carethers JM, melanoma following targeted disruption of the PTHrP gene: Moossa AR, Deftos LJ 2001 Human pancreatic adenocarci- Enhanced visualization of the invasive process with green nomas express parathyroid hormone-related protein. J Clin fluorescence protein. Proceedings of the 24th Annual Endocrinol Metab 86:310Ð316. Meeting of the ASBMR, San Antonio, TX. 35. Yeung SC, Eton O, Burton DW, Deftos LJ, Vassilopoulou- 51. Bouizar Z, Spyratos F, Deytieux S, de Vernejoul MC, Sellin R, Gagel RF 1998 Hypercalcemia due to parathyroid Jullienne A 1993 Polymerase chain reaction analysis of hormone-related protein secretion by melanoma. Horm Res parathyroid hormone-related protein gene expression in 49:288Ð291. breast cancer patients and occurrence of bone metastases. 36. Bucht E, Rong H, Pernow Y, Nordqvist AC, Eriksson E, Cancer Res 53:5076Ð5078. Rankin W, von Schoultz EW, Burtis WJ, Granberg B, 52. Truong NU, deB Edwardes MD, Papavasiliou V, Goltzman D, Falkmer UG, Burton DW, Deftos LJ 1998 Parathyroid Kremer R 2003 Parathyroid hormone-related peptide and hormone-related protein in patients with primary breast cancer survival of patients with cancer and hypercalcemia. Am J Med and eucalcemia. Cancer Res 58:4113Ð4116. 115:115Ð121. 37. Henderson JE, He B, Goltzman D, Karaplis AC 1996 53. Kremer R, Shustik C, Tabak T, Papavasiliou V, Goltzman D Constitutive expression of parathyroid hormoneÐrelated pep- 1996 Parathyroid-hormone-related peptide in hematologic tide (PTHrP) stimulates growth and inhibits differentiation of malignancies. Am J Med 100:406Ð411. CFK2 chondrocytes. J Cell Physiol 169:33Ð41. 54. Henderson JE, Sebag M, Rhim J, Goltzman D, Kremer R 38. Jans DA, Hassan G 1998 Nuclear targeting by growth factors, 1991 Dysregulation of parathyroid hormone-like peptide cytokines and their receptors: A role in signaling? Bioessays expression and secretion in a keratinocyte model of tumor 20:400Ð411. expression. Cancer Res 51:6521Ð6528. 39. Lam MH, Briggs LJ, Hu W, Martin TJ, Gillespie MT, Jans DA 55. Merryman JI, De Wille J, Wermeister JR, Capen CL, Rosol TJ 1999 Importin beta recognizes parathyroid hormone- 1994 Effects of transforming growth factors on parathyroid related protein with high affinity and mediates its nuclear hormoneÐrelated protein production and ribonucleic acid import in the absence of importin alpha. J Biol Chem 274: expression by a squamous carcinoma cell line in vitro. 7391Ð7398. Endocrinology 134:2424Ð2430. 40. Watson PH, Fraher LJ, Natale BV, Kisiel M, Hendy GN, 56. Casey ML, Mike M, Erk A, MacDonald PC 1992 Hodsman AB 2000 Nuclear localization of the type 1 parathy- Transforming growth factor β, stimulation of parathyroid roid hormone/parathyroid hormone-related peptide receptor hormone related protein expression in human uterine cells in in MC3T3-E1 cells: Association with serum-induced prolifer- culture; mRNA levels and protein secretion. J Clin ation. Bone 26:221Ð225. Endocrinol Metab 74:950Ð952. 41. Aarts MM, Rix A, Guo J, Bringhurst R, Henderson JE 1999 57. Ikeda K, Lu C, Weir EC, Mangin M, Broadus AE 1989 The nucleolar targeting signal of parathyroid hormoneÐrelated Transcriptional regulation of the parathyroid hormone related protein mediates endocytosis and nucleolar translocation. peptide gene by glucocorticoids and vitamin D in a human J Bone Min Res 14:1493Ð1503. C-cell line. J Biol Chem 264:15743Ð15746. 748 DAVID GOLTZMAN AND RICHARD KREMER

58. Sharpe GR, Dillon JP, Durham B, Gallagher JA, Fraser WD Roba K, Ouchi Y 1998 Cytokine-induced expression of 1998 Human keratinocytes express transcripts for three iso- parathyroid hormoneÐrelated peptide in cultured human forms of parathyroid hormoneÐrelated protein (PTHrP), but vascular endothelial cells. Biochem Biophys Res Commun not for the parathyroid hormone/PRTHrP receptor: Effects of 249:339Ð343. 1,25(OH)2D3. Brit J Dermatol 138:944Ð951. 74. Funk JL, Wei H 1998 Regulation of parathyroid hormoneÐ 59. Abe M, Abeno N, Oshida S, Horiuchi N 1998 Inhibitory related protein expression in MCF-7 breast carcinoma cells by effects of 1,25 dihydroxyvitamin D3 and 9-cis-retinoic acid estrogen and antiestrogens. Biochem Biophys Res Commun on parathyroid hormoneÐrelated protein expression by oral 251:849Ð854. cancer cells (NSC-3). J Endocrinol 156:349Ð357. 75. Heath JK, Southby J, Fukumoto S, O’Keefe LM, Martin TJ, 60. Yu J, Papavasiliou V, Rhim J, Goltzman D, Kremer R 1995 Gillespie MT 1995 Epidermal growth factor-stimulated Vitamin D analogs: New therapeutic agents for the treatment parathyroid hormone-related protein expression involves of squamous cancer and its associated hypercalcemia. increased gene transcription and mRNA stability. Biochem J Anticancer Drugs 6:101Ð108. 307:159Ð167. 61. Inoue D, Matsumoto T, Ogata E, Ikeda K 1993 22-Oxacalcitriol, 76. Rizzoli R, Feyen JHM, Grau G, Wohlwend A, Sappino AP, a noncalcemic analogue of calcitriol, suppresses both cell Bonjour JP 1994 Regulation of parathyroid hormone-related proliferation and parathyroid hormone-related peptide gene protein production in a human lung squamous cell carcinoma expression in human T cell lymphotrophic virus, type IÐ line. J Endocrinol 143:333Ð342. infected T cells. J Biol Chem 268:16730Ð16736. 77. Kremer R, Goltzman D, Amizuka N, Webber MM, Rhim JS 62. Kremer R, Sebag M, Champigny C, Meerovitch K, Hendy GN, 1997 ras Activation of human prostate epithelial cells induces White J, Goltzman D 1996 Identification and characterization overexpression of parathyroid hormoneÐrelated peptide. Clin of 1,25-dihydroxyvitamin D3-responsive repressor sequences in Cancer Res 3:855Ð859. the rat parathyroid hormone-related peptide gene. J Biol Chem 78. Aklilu F, Park M, Goltzman D, Rabbani SA 1996 Increased 271:16310Ð16316. PTHrP production by a tyrosine kinase oncogene, TRP-MET: 63. Nishishita T, Okazaki T, Ishikawa T, Igarashi T, Hata K, role of the ras signaling pathway. Am J Physiol 271:E277ÐE283. Ogata E, Fujita T 1998 A negative vitamin D response. DNA 79. Broadus AE, Stewart AF 1994 Parathyroid hormone related element in the human parathyroid hormoneÐrelated peptide protein. Structure, processing and physiological actions. In: gene binds to vitamin D receptor along with Ku antigen to Bilezikian JP, Levine MA, Marcus R (eds) The Parathyroids. mediate negative gene regulation by vitamin D. J Biol Chem Raven Press, New York, pp. 259Ð294. 273(18):10901Ð10907. 80. Sidler B, Alpert L, Henderson JE, Deckelbaum R, Amizuka N, 64. Falzon M, Zhong J 1998 The non-calcemic vitamin D Silva E, Goltzman D, Karaplis AC 1996 Amplification of the analogs EB1089 and 22-oxacalcitriol suppress serum-induced parathyroid hormoneÐrelated peptide (PTHrP) gene amplifica- parathyroid hormoneÐrelated peptide gene expression in a tion in a colonic carcinoma. J Clin Endocrinol 81:2841Ð2847. lung cancer cell line. Endocrinology 139:1046Ð1053. 81. Solomon C, White JH, Kremer R 1999 Mitogen-activated 65. Hongo T, Kupfer J, Enomoto H, Sharifi B, Giannella-Neto D, protein kinase inhibits 1,25-dihydroxyvitamin D3Ðdependent Forrester JS, Singer FR, Goltzman D, Hendy GN, Pirola C, signal transduction by phosphorylating human retinoid X et al. 1991 Abundant expression of parathyroid hormone receptor alpha. J Clin Invest 103:1729Ð1735. related protein in primary aortic smooth muscle cells accompa- 82. Powell GJ, Southby J, Danks JA, Stillwell RG, Hayman JA, nies serum-induced proliferation. J Clin Invest 88:1841Ð1847. Henderson MA, Bennett RC, Martin TJ 1991 Localization of 66. Streutker C, Drucker DJ 1991 Rapid induction of parathyroid parathyroid hormone-related protein in breast cancer metas- hormone-like peptide gene expression by sodium butyrate in tases: increased incidence in bone compared with other sites. a rat islet cell line. Mol Endocrinol 5:703Ð708. Cancer Res 51:3059Ð3061. 67. Kremer R, Woodworth CD, Goltzman D 1996 Expression 83. Southby J, Kissin MW, Danks JA, Hayman JA, Moseley JM, and action of parthyroid hormone-related peptide in human Henderson MA, Bennett RC, Martin TJ 1990 Immuno- cervical epithelial cells. Am J Physiol 271:C164ÐC171. histochemical localization of parathyroid hormone-related 68. Cros M, Silve C, Graulet AM, Morieux C, Urena P, protein in human breast cancer. Cancer Res 50:7710Ð7716. de Vernejoul MC, Bouizar Z 1998 Estrogen stimulates PTHrP 84. Vargas SJ, Gillespie MT, Powell GJ, Southby J, Danks JA, but not PTHPTHrP receptor gene expression in the kidney of Moseley JM, Martin TJ 1992 Localization of parathyroid ovariectomized rat. J Cell Biochem 70:84Ð93. hormone-related protein mRNA expression in breast cancer 69. Chilco PJ, Gerardi JM, Kaczmarczyk SJ, Chu S, Leopold V, and metastatic lesions by in situ hybridization. J Bone Miner Zajac JD 1993 Calcitonin invreases transcription of parathy- Res 7:971Ð979. roid hormone related protein via cAMP. Mol Cell Endocrinol 85. Bundred NJ, Walder RA, Ratcliffe WA, Wguuck J, 94:1Ð7. Morisson JM, Ratcliffe JG 1992 Parathyroid hormone related 70. Rodan SB, Wesolowski G, Ianacone J, Thiede MA, Rodan GA protein and skeletal morbidity in breast cancer. Eur J Cancer 1989 Production of parathyroid hormone-like peptide in a 28(213):690Ð692. human osteosarcoma cell line: Stimulation by phorbol esters 86. Kissin MW, Henderson MA, Danks JA, Hayman JA, and epidermal growth factor. J Endocrinol 122:219Ð227. Bennett RC, Martin TJ 1993 Parathyroid hormone related 71. Lu C, Ikeda K, Deftos LJ, Cazdar AF, Mangin M, Broadus AE protein in breast cancers of widely varying prognosis 1989 Glucocorticoid regulation of PTHrP gene transcription Eur J Surg Oncol 19:134Ð142. in a human neuroendocrine cell line. Mol Endocrinol 3: 87. Grill V, Ho P, Body JJ, Johanson N, Lee SC, Krukeja SC, 2034Ð2040. Moseley JM, Martin TJ 1991 Parathyroid hormone-related 72. Deftos LJ, Hogue-Angeletti R, Chalberg C, Su T 1989 PTHrP protein: elevated levels in both humoral hypercalcemia of secretion is stimulated by CT and inhibited by chromogranin malignancy and hypercalcemia complicating metastatic A peptides. Endocrinology 125:563Ð565. breast cancer. J Clin Endocrinol Metab 73:1309Ð1315. 73. Eto M, Akishita M, Ishikawa M, Kozaki K, Yoshizumi M, 88. Guise TA 1997 Parathyroid hormone-related protein and Hashimoto M, Ako J, Sugimoto N, Nagano K, Sudoh N, bone metastases. Cancer 80:1572Ð1580. CHAPTER 43 Target Genes: PTHrP 749

89. Guise TA, Yin JJ, Taylor SD, Kumagai Y, Dallas M, Boyce BF, 99. Kragballe K, Gjertsen BT, de Hoop D, Karlsmark T, Yoneda T, Mundy GR 1996 Evidence for a causal role of van de Kerkhof PC, Larko O, Nieboer C, Roed-Petersen J, parathyroid hormone-related protein in the pathogenesis Strand A, Tikjob G 1991 Double-blind, right/left comparison of human breast cancer mediated osteolysis. J Clin Invest 98: of calcipotriol and betamethasone valerate in treatment of 1544Ð1549. psoriasis vulgaris. Lancet 337:193Ð196. 90. Sato K 1999 Does PTHrP mediate cancer-associated 100. Haq M, Kremer R, Goltzman D, Rabbani SA 1993 A vitamin D cachexia as well as humoral hypercalcemia of malignancy? analogue (EB 1089) inhibits parathyroid hormone-related In: 7th Int Forum on Calcified Tissue and Bone Metabolism, peptide production and prevents the development of pp. 32Ð37. malignancy-associated hypercalcemia in vivo. J Clin Invest 91. Sato K, Yamakawa Y, Shimuze K, Satoh T, Mohojomi K, 91:2416Ð2422. Demura H, Akatsu T, Nagata N, Kasahara T, Ohkawa H, 101. El Abdaimi K, Papavasiliou V, Rabbani SA, Rhim JS, Ohsumi K 1993 Passive immunization with antiparathyroid Goltzman D, Kremer R 1999 Reversal of hypercalcemia with hormone-related protein antibody markedly prolongs survival the vitamin D analogue EB 1089 in a human model of squa- time of hypercalcemia nude mice bearing transplanted human mous cancer. Cancer Res 59:3325Ð3328. PTHrP-producing tumors. J Bone Min Res 8:849Ð860. 102. El Abdaimi K, Dion N, Papavasiliou V, Cardinal PE, 92. Stein CA, Cheng YC 1993 Antisense oligonucleotides as Binderup L, Goltzman D, Ste-Marie LG, Kremer R 2000 The therapeutic agents. Is the bullet really magical? Science vitamin D analogue EB 1089 prevents skeletal metastasis and 261:1004Ð1012. prolongs survival time in nude mice transplanted with human 93. Armentano D, Yu SF, Kantoff PW, Von Ruden T, Anderson breast cancer cells. Cancer Res 60:4412Ð4418. WF, Gilboa E 1987 Effects of internal viral sequences on the 103. Sasaki A, Boyce BF, Story B, Wright KR, Chapman M, utility of retroviral vectors. J Virol 61:1047Ð1050. Boyce R, Mundy GR, Yoneda T 1995 Bisphosphonate 94. Palmer JD, Rosman GJ, Osborne WRA, Miller AD 1991 residronate reduces metastatic human breast cancer burden in Genetically modified skin fibroblasts persist long after trans- bone in nude mice. Cancer Res 55:3551Ð3557. plantation but gradually inactivate introduced genes. Proc 104. Huang DC, Papavasiliou V, Rhim JS, Horst RL, Kremer R Natl Acad Sci USA 88:1330Ð1334. 2002 Targeted disruption of the 25-dihydroxyvitamin D3, 95. Aklilu F, Park M, Goltzman D, Rabbani SA 1997 Induction of 1α-hydroxylase gene in ras-transformed keratinocytes parathyroid hormoneÐrelated peptide by the Ras oncogene: demonstrates that locally produced 1α,25-dihydroxyvitamin role of Ras farnesylvation inhibitors as potential therapeutic D3 suppresses growth and induces differentiation in an agents for hypercalcemia of malignancy. Cancer Res 57: autocrine fashion. Mol Cancer Res 1:56Ð67. 4517Ð4522. 105. Thiede MA, Harm SC, Hasson DM, Gardner RM 1991 96. Falzon M 1996 DNA sequences in the rat parathyroid In vivo regulation of parathyroid hormone-related peptide hormone-related peptide gene responsible for 1,25-dihydroxy- messenger ribonucleic acid in the rat uterus by 17β-estradiol. vitamin D3Ðmediated transcriptional repression. Mol Endocrinology 128:2317Ð2323. Endocrinol 10:672Ð681. 106. El Abdaimi K, Papavasiliou V, Goltzman D, Kremer R 2000 97. Binderup L, Brammn E 1988 Effect of a novel vitamin D Expression and regulation of parathyroid hormone-related analog MC903 on cell proliferation and differentiation in vitro peptide in normal and malignancy melanocytes. Am J Physiol and on calcium metabolism in vivo. Biochem Pharmacol Cell Physiol 279:C1230ÐC1238. 37:889Ð895. 107. Thiede MA 1989 The mRNA encoding a parathyroid hormone- 98. Kissmeyer AM, Binderup L 1991 Calcipotriol (MC903): like peptide is produced in mammary tissue in response to pharmacokinetics in rats and biological activities of metabolites. elevations in serum prolactin. Mol Endocrinol 3:1443Ð1447. A comparative study with 1,25(OH)2D3. Biochem Pharmacol 108. Huang DC, Papavasiliou V, Rhim JS, Horst RL, Kremer R 2002 41:1601Ð1606. Mol Cancer Res 1:1Ð12. CHAPTER 44

Effects of 1,25-Dihydroxyvitamin D3 on Voltage-Sensitive Calcium Channels in the Vitamin D Endocrine System

MARY C. FARACH-CARSON AND JOEL J. BERGH Department of Biological Sciences, University of Delaware

I. Systemic and Intracellular Ca2+ Homeostasis V. Ca 2+ Induced Inactivation of VSCCs II. Voltage-Sensitive Calcium Channels VI. Calcium and Transcriptional Responses to 1,25(OH)2D3 III. 1,25-Dihydroxyvitamin D3 and Voltage-Sensitive VII. Cross-Talk between Membrane and Nuclear Actions Ca2+ Channels VIII. Summary and Conclusions 2+ IV. Membrane-Initiated Ca Responses to 1,25(OH)2D3 References

I. SYSTEMIC AND INTRACELLULAR stores in the endoplasmic reticulum through leak chan- Ca2+ HOMEOSTASIS nels and by influx of extracellular Ca2+ through plasma membrane channels that include voltage-sensitive All mammals must maintain plasma Ca2+ concentra- calcium channels (VSCCs), voltage-insensitive calcium tions within a narrow homeostatic set point to ensure channels (VICCs), receptor-operated calcium channels proper control of Ca2+-regulated cellular function and (ROCs), and mechanosensitive divalent cation channels phenotype. Ca2+ homeostasis involves hormonal regu- (MDCCs). Calcitropic hormones, including 1,25-dihy- lation by 1,25(OH)2D3 at three major sites, the kidney, droxyvitamin D3 (1,25(OH)2D3), and parathyroid hor- intestine, and bone. As serum Ca2+ levels decrease, mone (PTH), and mechanical load modulate the activity 2+ 1,25(OH)2D3 production increases, facilitating increased of these plasma membrane Ca channels. In osteoblasts, Ca2+ absorption in the intestines, decreased Ca2+ excre- VSCCs serve as key regulators of Ca2+ permeability tion in the urine, and a shift in bone remodeling to a and are the major Ca2+ channels present in the plasma state that favors resorption. The skeletal system is the membrane [3]. Additionally, Ca2+ influx through L-type location of roughly 99% of all Ca2+ found in the human VSCCs in response to membrane depolarization events body. The skeleton undergoes continuous remodeling, maintains the activity of the cAMP- and Ca2+-dependent generating a small pool of Ca2+ that is freely exchange- transcription factor CREB [4]. Evidence suggests that able with the extracellular fluid, and establishing a buffer transcriptional activation through CREB is more effi- system to aid in the maintenance of circulating Ca2+ cient when the Ca2+ signal is generated through the concentrations. L-type VSCC than by other channels that permit Ca2+ Ca2+ levels in cells are kept in a dynamic equilibrium entry [4,5]. by the activities of channels, pumps, and exchangers in both the plasma membrane and internal Ca2+ storage organelles, including the endoplasmic reticulum, mito- II. VOLTAGE-SENSITIVE chondria, and nucleus. While organelles and intracel- CALCIUM CHANNELS lular Ca2+-binding proteins can sequester intracellular Ca2+, they only transiently can buffer cytosolic increases. VSCCs are present in all excitable tissues and in Therefore, the overall maintenance of intracellular Ca2+ most nonexcitable cell types. VSCCs have been identi- levels is maintained by the plasma membrane, which fied in several tissues in the vitamin D endocrine extrudes Ca2+ into the extracellular space using the system, including the kidney [6], intestine [7], and energy of Ca2+-ATPases and collaborating Na+/Ca2+ bone [3,8]. VSCCs mediate the influx of Ca2+ in response exchangers [1,2]. Intracellular Ca2+ levels also are to membrane depolarization and regulate numerous buffered by the uptake of Ca2+ into internal stores, such intracellular functions, including contraction, secretion, as the mitochondria and the endoplasmic reticulum. Ca2+ gene transcription, neurotransmitter release, and cellular levels in the cytoplasm increase by release of Ca2+ from differentiation. Many of these responses are tuned to VITAMIN D, 2ND EDITION Copyright © 2005, Elsevier, Inc. FELDMAN, PIKE, AND GLORIEUX All rights reserved. 752 MARY C. FARACH-CARSON AND JOEL J. BERGH

2+ 2+ a specific temporal and spatial pattern of Ca entry [9,10] Ca α which must be balanced with the potential toxicity Extracellular 2 caused by high intracellular levels of Ca2+. The need for regulated patterns of Ca2+ influx is reflected by the δ α presence of multiple unique subtypes of VSCCs, each γ 1 differing in its kinetics, pharmacology, and tissue dis- tribution. VSCCs were first solubilized and purified from the transverse tubules of skeletal muscle [11]. The purifi- Intracellular β cation identified the α1, β, and γ subunits and showed that the α1 and β subunits contain consensus sites for FIGURE 1 Subunit structure of the L-type VSCC. The L-type cAMP-dependent phosphorylation [11]. Further bio- VSCC consists of four or five subunits depending on the cell type chemical analysis revealed the presence of the α /δ (figure adapted from [13,14]). The general structure consists of a 2 α 2+ subunit [12]. Analysis of the protein sequences, hydro- pore-forming 1 subunit through which Ca travels along the con- centration gradient. A glycosylated α2δ subunit is present on the pathicity, and glycosylation properties of these five extracellular face and is the result of posttranslational processing of subunits produced a model (Fig. 1) composed of the a single gene product. A γ subunit is sometimes present (dotted line). β transmembrane α1 subunit in association with the The subunits are located on the intracellular face and play an important role in modification of channel conductance behavior disulfide-linked α2/δ dimer, an intracellular β subunit, and a transmembrane γ subunit [13Ð15]. and in plasma membrane targeting of the VSCC. The α1 subunit has been the focus of many biologi- cal studies because it is the large pore-forming subunit, the site of Ca2+ translocation, and can generate a Ca2+ residues. With similar topology to Na+ and K+ channels, current in the absence of the other subunits [16]. With both the short amino-terminal and long carboxyl-terminal a mass of approximately 175 kDa, the α1 subunit is segments of the α1 are located intracellularly where they the receptor for three classes of organic Ca2+ channel can be modified during signal transduction [18Ð21]. blockers [17]. Figure 2 shows the general structure of Molecular cloning has revealed at least 10 distinct α1 all α1 subunits, consisting of four repeating domains, subunits [21,22], but only four are found associated with each containing six hydrophobic transmembrane seg- L-type channel currents [17]. Table I lists each known 2+ ments that are embedded in the plasma membrane. The α1 subunit, the classic nomenclature, the Ca current fourth segment of each domain is distinguished by a col- associated with the subunit, and the tissue where it is lection of repeating positively charged amino acid primarily expressed. Different gene products code for these four different subtypes of the α1, which are named the α , α , α , and the α subtypes. These four sub- α 1C 1D 1F 1S TABLE I 1 Subunit Types and Function types share approximately 80% sequence similarity. α 2+ 2+ When the 1 subunit alone is expressed in Xenopus Ca Common Ca Primary tissue + channel nomenclature current type localization oocytes, a Ca2 current is established [2]. Physiological and pharmacological studies demon- α CaV1.1 1S LSkeletal muscle strate functional similarities between various α1 subunits, CaV1.2 α1C L Cardiac muscle Neurons, Bone Ca α L Endocrine cells V1.3 1D Domain: I II III IV Neurons Extracellular CaV1.4 α1F L Retina

CaV2.1 α1A P/Q Nerve terminal Dendrites + CaV2.2 α1B NNerve terminal NH3 Dendrites − CO2 CaV2.3 α1E R Cell bodies Nerve terminals Ca α T Cardiac muscle V3.1 1G FIGURE 2 Transmembrane organization of the pore-forming α Skeletal muscle 1 subunit of the L-type VSCC. The L-type VSCC α subunit consists Neurons 1 of four domains linked by relatively large intracellular loops α CaV3.2 1H T Cardiac muscle (adapted from [13,14]). Both the amino and carboxyl termini also Neurons are located intracellularly. This orientation presents the cytosolic CaV3.3 α1I T Neurons face of the channel to interact with signaling cascades and permits it to interact with calmodulin (see text). CHAPTER 44 Effects of 1,25-Dihydroxyvitamin D3 on Voltage-Sensitive Calcium Channels 753 which allow for VSCCs to be classified into high-voltage of 35 kDa. In cardiac tissue, purification studies have activated (L-, P/Q-, N, and R-type) and low-voltage acti- not yet revealed the presence of a γ subunit, although vated(T-type) classes [23]. L-type Ca2+ currents are several protein bands in the range of 120Ð130 kDa mediated by VSCCs containing α1C, α1D, α1F, and α1S were found. These bands might be complexes of associ- subunits, which have about 80% amino acid identity ated auxiliary subunits because a γ subunit-like cDNA with one another. α1A, α1B, and α1E (P/Q-, N-type, and has been isolated from cardiac mRNA preparations by R-type VSCC, respectively) make up the remainder PCR cloning [2,17]. of the high-voltage activated VSCC families. The low- Expression of the α1 subunit is sufficient to produce voltage activated T-type VSCCs, composed of α1G, functional channels, but with lower expression and α1H, and α1I, are distantly related to the other known different kinetics and voltage dependence than native homologs, with less than 25% amino acid sequence channels [16]. Coexpression of the α2/δ subunit confers identity. more normal gating properties and enhances the expres- The β subunit is a 56-kDa protein that is highly sion of the channel [29]. Coexpression of β subunits phosphorylated in vitro. Hydrophobicity analysis generally increases channel expression, while shifting revealed the lack of a membrane-spanning segment, the voltage dependence of activation and inactivation to suggesting it is localized intracellularly. Pulse chase more negative membrane potentials and increasing the analysis shows that β subunits are posttranscriptionally rate of inactivation [13]. The γ subunit alters peak cur- modified by palmitoylation, which aids in membrane rents and reduces channel availability by a negative shift association [24]. In rabbit brain, four distinct β subunits of the voltage dependence of inactivation [37]. have been identified. When α1 subunits are expressed in Xenopus oocytes, the activation and inactivation kinetics of the Ca2+ channel are abnormally slow com- III. 1,25-DIHYDROXYVITAMIN D3 AND pared to measurements performed in native cell prepa- VOLTAGE-SENSITIVE Ca2+ CHANNELS rations [17]. Recombinant coexpression of the α1 subunit 2+ with β subunits almost fully restores normal channel 1,25(OH)2D3 is a critical hormonal modulator of Ca currents. This finding supports the idea that the β sub- homeostasis and plays an important role in osteoblast unit can regulate channel kinetics, voltage-dependent function during bone remodeling. Many of its calcemic gating properties, and channel density. The affiliation actions occur in concert with PTH and are sensitive to of different β subunits with the α1 subunit results in the cell cycle [38]. Regulation of bone resorption by calcium channels with different electrophysiological osteoclasts in response to 1,25(OH)2D3 is mediated properties [17,24Ð28]. through resorptive signals generated by osteoblastic The α2/δ subunits are disulfide linked and, together, cells. 1,25(OH)2D3 generates biological responses form a 155-kDa protein complex that, after reduction, through the regulation of gene transcription and by the produces a 125-kDa α2 and 30-kDa δ polypeptide. commencement of rapid, membrane-initiated events. These subunits are encoded by the same gene and Long-term (hours to days) treatment with 1,25(OH)2D3 formed by posttranslational processing [29]. The α2 can generate cellular responses by binding the nuclear subunit lies extracellularly, while the δ subunit, with a vitamin D receptor (nVDR) and altering nVDR sensi- single transmembrane-spanning segment, resides in tive gene transcription [39Ð44]. In mice, 1,25(OH)2D3 the plasma membrane. There are five separate mRNA stimulates the production of several noncollagenous species for these subunits, and more recent studies sug- matrix proteins, including osteopontin [41], and down- gest the existence of two more α2/δ genes. Osteoblastic regulates osteocalcin [42] and parathyroid hormone cells express the α2/δ1 and the α2/δ3 isoforms of the (PTH) [43,44]. α2/δ subunit [30]. The extracellular α2 subunit facili- The binding of 1,25(OH)2D3 to its receptor, translo- tates the assembly of α1 at the cell surface, and their cation to the nucleus, interaction with coactivators, and ability to modulate α1-induced current is more pro- modulation of gene expression is the best characterized nounced if they are coexpressed with the β subunit cellular response to 1,25(OH)2D3 treatment. However, [13,31Ð35]. This result shows that, like the β subunit, the initial response after exposure to secosteroid is the α2/δ is capable of regulating current amplitude in increasingly appreciated. Similar to observations that L-type Ca2+ channels [17,31,32,36]. other steroid hormones, including estrogen [45] and The last subunit involved in forming a native L-type glucocorticoids [46], have membrane-initiated actions, VSCC is the γ subunit, whose function is still largely the role of 1,25(OH)2D3 in membrane events warranted unknown. In skeletal muscle, this highly glycosylated study in bone. Rapid responses to 1,25(OH)2D3 are protein exhibits considerable hydrophobicity, suggest- proposed to be facilitated through the binding of the ing localization to the plasma membrane, and a mass ligand with a plasma membrane-associated receptor. 754 MARY C. FARACH-CARSON AND JOEL J. BERGH

The rapid actions of 1,25(OH)2D3 have been related Low nanomolar concentrations of 1,25(OH)2D3 can to the induction of protein kinase C [47], phospholi- elicit a transient local elevation in intracellular Ca2+ pase C [48], adenylyl cyclase [49], membrane sphin- through influx of extracellular Ca2+ through the plasma gomyelinases [50], phosphorylation of matrix proteins membrane, whereas supraphysiological levels of 2+ including OPN [51], and modulation of intracellular 1,25(OH)2D3 promote the release of Ca from internal Ca2+ levels [52]. Previous reports using microarray stores and yield measurable Ca2+ transients [62]. analysis demonstrated that treatment of osteoblasts Changes in osteoblast Ca2+ permeability in the pres- with 1,25(OH)2D3 alters gene expression through ence of 1,25(OH)2D3 are regulated by the activity of nVDR-dependent and independent pathways [53]. the plasma membrane vitamin D response system, Many changes in expression have been observed as although the role of the nVDR in this action remains 2+ early as 3 hr posttreatment for genes that include stress controversial. 1,25(OH)2D3 can trigger a Ca transient response proteins, transcription factors, and various in response to 1,25(OH)2D3 treatment; in some reports matrix proteins, which lack a vitamin D response this occurs even when the nVDR is missing [65]. Recent element in their promoters. These observations, along findings report a loss of rapid responses in knockout with 1,25(OH)2D3 responsiveness in nVDR-free mem- mice lacking the nVDR [66], and our laboratory has brane preparations, suggest the presence of separate found a strict correlation between nVDR positive status nuclear and membrane receptors for 1,25(OH)2D3 both and rapid responses in more than 30 cell lines tested of which can be linked to transcriptional change. with various ligand analogs [55,67]. Pharmacological A 64.5-kDa protein, called 1,25D3-MARRS studies provide further insights. The rapid elevation of (membrane-associated rapid response to steroids) has local intracellular Ca2+ can be mimicked using synthetic been identified as a potential membrane receptor for analogs of 1,25(OH)2D3. Analog 1,24-dihydroxy-22- 1,25(OH)2D3 [54Ð57]. The N-terminal sequence was ene-24-cyclopropyl D3 (termed analog BT) has a high used to generate a specific antibody that identified a binding affinity for the nVDR and activates genomic component of the membrane vitamin D response signaling pathways that lead to changes in gene tran- system in other tissues, including chick kidney, and scription of various bone matrix proteins, including brain [58] and in rat chondrocytes [59]. Using the anti- osteocalcin, osteopontin, and type-I collagen [67Ð69]. body in a function blocking role inhibited intracellular In contrast to 1,25(OH)2D3, analog BT does not stimu- 2+ 2+ Ca signaling caused by 1,25(OH)2D3 [55,58Ð60]. late rapid changes in Ca permeability. Other classes These data suggest that 1,25D3-MARRS is involved in of 1,25(OH)2D3 analogs that lack the 1-α-hydroxyl generating rapid signals in response to 1,25(OH)2D3. group, including 25-hydroxy-16-ene-23-yne-D3 (termed Interestingly, Ca2+ signals generated by activation of analog AT), do not bind to the nVDR, but application the plasma membrane vitamin D receptor have been of these analogs rapidly increases Ca2+ influx into associated with changes in gene expression, similar to osteoblastic cells [69]. The use of analogs that can selec- those seen in response to prostaglandins [47,53,61]. tively activate the plasma membrane vitamin D response Changes in gene transcription can be observed within system or the nVDR have provided powerful tools to 2+ 3 hr after addition of 1,25(OH)2D3 or Ca mobilizing help identify the specific attributes of short- and long- vitamin D3 analogs [53], indicating that the rapid tran- term cellular responses to 1,25(OH)2D3 treatment [68]. 2+ scriptional effects are related to Ca influx rather than 1,25(OH)2D3 binding to the membrane vitamin D to activation of the nVDR. Addition of Ca2+ channel response system activates several signaling pathways. blockers negates many of the rapid actions of In chick embryogenesis, 1,25(OH)2D3 stimulates VSCC 1,25(OH)2D3, including the ability to modify the phos- activity through the activation of adenylyl cyclase, phorylation state of OPN [51], suggesting that the leading to the production of cAMP and activation of membrane-initiated actions of 1,25(OH)2D3 require protein kinase A [70]. The channel activation can be 2+ the activity of plasma membrane Ca channels. mimicked in the absence of 1,25(OH)2D3 by the addi- tion of forskolin, an adenylyl cyclase activator, and by dibutyryl-cAMP. Electrophysiological studies have + IV. MEMBRANE-INITIATED Ca2 shown that the major mechanism for Ca2+ influx into RESPONSES TO 1,25(OH)2D3 the osteoblast cell is the L-type VSCC, which shows a prolonged open time in the presence of 1,25(OH)2D3 [3] Application of 1,25(OH)2D3 to target cells results (Fig. 3A). Furthermore, application of the L-type in a rapid and transient increase in intracellular Ca2+ VSCC agonist Bay K 8644 or analog AT increases in some cell types [62Ð64]. The elevation in intracellu- Ca2+ influx into the osteoblast and shifts the threshold lar Ca2+ is dependent on release from internal stores of activation towards the resting potential, an event and influx through the plasma membrane [64]. termed “left shift” [3,71] (Fig. 3B). “Left-shifting” of CHAPTER 44 Effects of 1,25-Dihydroxyvitamin D3 on Voltage-Sensitive Calcium Channels 755

Interactions of VSCCs and VICCs in Osteoblasts A Basal 1,25(OH)2D3 + Depolarization 1,25(OH)2D3 PTH = local response “left shift” depolarization

Change in single channel conductance B −40 −30 −20 −10 0204010 30

Left shift VSCC VICC VSCC Vm(mV) −2 Ca2+ Ca2+ Ca2+ DHP blockGd 3+block DHP block −4 FIGURE 4 Interactions of VSCCs and VICCs in osteoblasts. − Im(×10 10) Full depolarization of VSCCs occurs after osteoblasts are treated first with 1,25(OH)2D3 that “left shifts” the VSCC as shown in FIGURE 3 Effect of 1,25(OH)2D3 on behavior of the L-type Fig. 3, then with a local depolarizing stimulus such as PTH VSCC in osteoblasts. (A) The L-type α1c-containing VSCC in the interacting with a VICC. The left-shifted VSCC senses the local osteoblast demonstrates a basal activity seen in single-channel depolarization through the VICC and allows large amounts of Ca2+ recordings that supports basal Ca2+-dependent signaling. Addition to enter the cell through the pore (solid dark arrow). As reported of 1,25(OH)2D3 to the cell in continuous recording shows a change earlier [71], the left-shift and full depolarization response through in single-channel conductance marked by increased mean open the L-type VSCC can be inhibited by dihydropyridine channel time and long nulls. This occurs without an increase in whole cell blockers (DHP), whereas the VICC can be inhibited by Gd3+. Under inward current (B). The solid dots represent untreated cells; open the conditions shown, a Ca2+ transient would occur and involve fur- 2+ dots represent cells treated with 5.0 nM 1,25(OH)2D3. The open ther Ca release from intracellular stores as discussed in the text. squares show further increase in left-shift during washout to low nanomolar concentrations. The filled squares represent results when 1 µM BAY K8644, an L-type VSCC agonist, is added. The V. C a 2+-INDUCED INACTIVATION arrow indicates the direction of the “left shift” indicating a change in the threshold of channel activation toward the resting potential OF VSCCS (near −40 in these cells). Osteoblastic cells respond to 1,25(OH)2D3 and express significant amounts of the nVDR [74]. Exposure the membrane resting potential predicts that plasma to physiological levels of 1,25(OH)2D3 produces a rapid membrane VSCCs in osteoblastic cells are more sus- change in membrane permeability that precedes any ceptible to opening following stimulation with other changes in gene expression. In osteoblasts, alteration hormones acting through ROCs or in response to of membrane Ca2+ permeability is regulated primarily smaller membrane depolarizations such as may be by the activity of the L-type VSCC. Spontaneous and associated with activation of VICCs or MDCCs. hormonally regulated opening of Ca2+ channels has been The physiological implication for a “left shift” is observed in resting cells and leads to localized eleva- 2+ evident in the interactions between 1,25(OH)2D3 and tions of intracellular Ca , sometimes referred to as PTH. It has long been recognized that PTH can stimulate “sparks” [75], that directly control the release of secre- Ca2+ entry into the osteoblastic cell via influx through tory vesicles and the generation of nuclear-specific a gadolinium-sensitive Ca2+ channel that interacts Ca2+ signals [5,76]. with neighboring VSCCs [72] (Fig. 4). Pretreatment In cardiac cells, Ca2+ influx rapidly inactivates the of osteoblastic cell cultures with low nanomolar con- Ca2+ current, resulting in the channel current returning centrations of 1,25(OH)2D3 for 10 min results in an to its resting potential during long membrane depolar- enhancement of PTH-induced Ca2+ influx that is asso- izations [77,78]. Some time ago, our laboratory ciated with increased bone resorption rates [71,73]. demonstrated that inactivation of the L-type VSCC in 2+ This suggests that 1,25(OH)2D3 serves a priming func- ROS 17/2.8 cells was faster in 20 mM external Ca and tion to augment PTH-induced Ca2+ influx at the plasma that the steady-state inward current was smaller than in membrane (Fig. 4). Removal of extracellular Ca2+ or experiments that were performed in 5 mM external application of the L-type VSCC inhibitor nitrendipine, Ca2+ [3] (Fig. 5). Ca2+-dependent inactivation was a dihydropyridine (DHP), inhibits the elevation of observed when cardiac VSCCs were inserted into lipid 2+ 1,25(OH)2D3 and PTH induced Ca influx [71], indi- bilayers in the absence of cytosolic components and cating that the enhanced PTH-induced Ca2+ influx ATP [79], suggesting that Ca2+ is binding to the VSCC depends on the presence and influx of Ca2+ through the directly or to a protein that is coupled with the channel L-type VSCC [73]. complex. The detection of two exon splice variants of 756 MARY C. FARACH-CARSON AND JOEL J. BERGH

Ca2+-dependent inactivation to an up-regulation of gene transcription of osteoid −40 −20 0 20 40 60 80 proteins, mediated by the actions of the nuclear vita- min D receptor [88]. Activation of this nuclear receptor initiates a signaling cascade that results in alteration of target gene expression. Some genes that are transcrip- 1 tionally regulated by 1,25(OH)2D3 include alkaline phos- phatase, IL-3 receptor, osteopontin, osteocalcin, type I collagen, GM-colony stimulating factor, and PTH. 2 = 5 mM Ca2+ It has been established that depolarization and = 2+ 20 mM Ca hormonal stimulated Ca2+ influx into osteoblastic cells = 2+ 3 20 mM Ba is inhibited by the application of dihydropyridines [3], × −10 Im( 10 A) indicating that the L-type VSCCs are involved in this + FIGURE 5 Ca2+-dependent inactivation of the L-type VSCC in influx of Ca2 . Three of the four known L-type VSCCs, 2+ osteoblasts. When Ba is used as the permeant ion (solid dot) α1C, α1D, and α1S, were found to be expressed in human α 2+ a large inward current is seen through 1c. When Ca is placed into osteoblastic cells, although only α is always present the extracellular medium at two concentrations (solid triangle, 1C 5 mM; open circle, 20 mM) rapid channel inactivation occurs (arrow) [89]. The identification of these subclasses that are typi- (adapted from [3]). As discussed in the text, this Ca2+-dependent cally seen in excitable tissues, including neurons [90] inactivation involves calmodulin. and skeletal muscle [91], in osteoblastic cells is not surprising, given the fact that they are found in a variety of other nonexcitable tissues and cells including lung [92], kidney [93], pancreas [94], and fibroblasts [95]. Short- the L-type VSCC that have drastically different inacti- term application of 1,25(OH)2D3 increases the mean open vation rates led to the identification of Ca2+/calmodulin time of the L-type VSCC [3]. In primary osteoblast kinase as a regulator of the L-type VSCC [80Ð82]. The cultures, 1,25(OH)2D3 application leads to an increase C-terminal end of the rapid inactivating splice variant in Ca2+ permeability and, when this is accompanied by includes an IQ domain, a well-characterized calmodulin release from intracellular stores [62,63], to a subse- binding site [82]. Two calmodulin mutants, one unable quent elevation in intracellular Ca2+ levels, resulting in the to associate with the VSCC and the other incapable of stimulation of many Ca2+-dependent signaling pathways. 2+ 2+ binding Ca , lack the ability to generate the Ca - If unregulated, continued exposure to 1,25(OH)2D3 dependent inactivation of the L-type VSCC [82Ð84]. could result in sustained influx of intracellular Ca2+ This demonstrates that Ca2+-dependent inactivation of and lead to cell death [76]. It was found that long-term the L-type VSCC requires Ca2+ binding to calmodulin exposure of osteoblasts to low nanomolar concentra- and the subsequent complex must associate with the tions of 1,25(OH)2D3 resulted in down-regulation of 2+ C-terminal tail of the Ca channel. Calmodulin has α1C mRNA transcript levels, as well as a subsequent been reported to be tethered and localized to the decrease in α1C protein expression [96]. Using analog C-terminal tail of the L-type VSCC [85]. This supports BT, which binds the nuclear vitamin D receptor and the notion that Ca2+ influx across the plasma mem- does not elicit a plasma-membrane response, it was brane is a highly regulated localized event under further demonstrated that the down-regulation of the negative feedback regulation rather than a signal for α1C subunit is due to transcriptional changes mediated global change in intracellular Ca2+. through the nuclear receptor and not as a result of a membrane initiated signal [96]. Radioactive Ca2+ influx assays revealed that prolonged exposure to 1,25(OH)2D3 VI. CALCIUM AND TRANSCRIPTIONAL led to a decrease in the amount of Ca2+ that enters the RESPONSES TO 1,25(OH)2D3 cell through the L-type VSCC, presumably due to the decrease in expression of the α1C subunit. A potential The classic genomic signals generated by role for down-regulation of the α1C subunit in response 1,25(OH)2D3 are mediated through binding of the to long-term exposure to 1,25(OH)2D3 is to protect the secosteroidÐreceptor complex to vitamin D response cell from chronic elevations in intracellular Ca2+ that element sequences up-stream of target sequences and could lead to cell apoptosis. To that end, it has been the subsequent alteration of gene transcription [86,87] demonstrated in hippocampal neurons that neuronal (see Chapter 11Ð19). In osteoblasts, 48-hr treatment with vulnerability to excitotoxicity is mediated through Ca2+ 1,25(OH)2D3 results in the increase in deposition of influx through the L-type VSCC, and down-regulation of osteoid, an extracellular matrix specific to bone cells these channels with long-term exposure to 1,25(OH)2D3 (see Chapter 37, 41). The increase in deposition is due leads to increased neuroprotection [97]. Together, these CHAPTER 44 Effects of 1,25-Dihydroxyvitamin D3 on Voltage-Sensitive Calcium Channels 757

results suggest that 1,25(OH)2D3 exposure elicits a Finally, in myoblasts, phospholipase c redistribution and rapid cellular response, including the activation of var- activation occurs following rapid 1,25(OH)2D3-induced, ious protein kinases, phospholipases, and generation Ca2+-dependent signal transduction involving c-Src and of cAMP, by increasing the ability of Ca2+ to enter the PI3K [102]. Taken together, it is clear that the integrated osteoblast through the α1C subunit of the L-type VSCC. responses of cells to vitamin D hormone represent a Long-term exposure to the secosteroid results in a continuum of highly regulated responses that often 2+ down-regulation of the α1C subunit in a nuclear recep- begin with activation of VSCCs and Ca influx, and torÐmediated pathway, which results in a diminished ultimately lead to changes in gene transcription and cell ability of the cell to maintain elevated intracellular Ca2+ behavior and phenotype. levels, thus preventing apoptosis and a prolonged cel- lular response to 1,25(OH)2D3. VII. SUMMARY AND CONCLUSIONS

VII. CROSS-TALK BETWEEN Taken together, it is clear that the integrated MEMBRANE AND NUCLEAR ACTIONS responses of cells to vitamin D hormone represent a continuum of highly regulated responses that often The genomic and membrane-initiated actions of begin with activation of VSCCS and Ca2+ influx, and 1,25(OH)2D3 provide opportunities for cross-talk and ultimately lead to changes in gene transcription and feedback loops among the various pathways (recently cell behavior and phenotype. It is impossible to sepa- reviewed in [98]) (see Chapter 23). For example, rate the intracellular pathways that involve rapid immediately after 1,25(OH)2D3 treatment, the α1C sub- responses from those that modulate nuclear hormone unit if the VSCC increases its open time [3] and allows receptors, for the simple reason that elaborate cross- more Ca2+ to enter the cell, especially in sites proximal talk between these response systems is the norm rather to the pores. This happens without increasing current than the exception. Activation and inactivation of amplitude [3]. Local increases in Ca2+ activate VSCCs, both low and high voltage channels, as well as calmodulin and can lead to channel inactivation as dis- regulation of VSCC biosynthesis, assembly and mem- cussed earlier. Continued presence of the hormone brance insertion provide first tier controls that fine tune leads to activation of the nuclear receptor and eventual Ca2+-dependent hormone responses of target cells in decreases in mRNA levels encoding α1C [96]. The the vitamin D endocrine system. depression in VSCC mRNA and protein levels leads to decreased responsiveness to 1,25(OH)2D3 at the plasma membrane. This mechanism provides cells a Acknowledgments means to respond to and then attenuate the rapid response to secosteroid, preventing Ca2+ toxicity while The work in the authors’ laboratory was supported supporting Ca2+-dependent signaling. by grants from the NIH/NIDCR. We thank Dr. Errin 1,25(OH)2D3 also interacts with peptide hormones Lagow and Dr. Dan Carson for their many helpful dis- that include PTH, transforming growth factor β (TGFβ), cussions and proofreading. We acknowledge all of the and inflammatory cytokines to modulate cellular members of the Carson and Farach-Carson laboratories responses [98]. Gene expression in many cell types for their individual contributions to the development of changes in response to 1,25(OH)2D3 treatment alone and this work over the years, and to our many excellent as a consequence of cross-talk between 1,25(OH)2D3- collaborators. Some of this work was presented in the activated pathways and peptide hormone-activated path- doctoral thesis of J.J.B. ways. As an example, 1,25(OH)2D3 can cause a rapid phosphorylation of serine residues on IκBα in mono- cytes, which synergize with PKC-dependent signaling References pathways to regulate NFκB translocation and signaling [99]. In a similar fashion, Smad proteins conduct signals 1. Rasmussen H, Barrett P, Zawalich W, Isales C, Stein P, downstream of TGFβ that mediate cross-talk between Smallwood J, McCarthy R, Bollag W 1989 Cycling of + TGFβ and 1,25(OH)2D3 signaling in osteoblasts [100]. Ca2 across the plasma membrane as a mechanism for gener- As another example of cross-talk, PTH treatment of ating a Ca2+ signal for cell activation. Ann NY Acad Sci osteoblasts activates PKA, which through phosphoryla- 568:73Ð80. 2. Tsien RW, Tsien RY 1990 Calcium channels, stores, and tion modulates VSCC function, and in the presence of oscillations. Annu Rev Cell Biol 6:715Ð760. 2+ 1,25(OH)2D3-activated CaMK alters [Ca ]i, and regu- 3. Caffrey JM, Farach-Carson MC 1989 Vitamin D3 metabolites lates secretion of osteoclastic coupling factors [101]. modulate dihydropyridine-sensitive calcium currents in 758 MARY C. FARACH-CARSON AND JOEL J. BERGH

clonal rat osteosarcoma cells. J Biol Chem 264: 22. Randall A, Benham CD 1999 Recent advances in the molec- 20265Ð20274. ular understanding of voltage-gated Ca2+ channels. Mol Cell 4. Bading H, Ginty DD, Greenberg ME 1993 Regulation of Neurosci 14:255Ð272. gene expression in hippocampal neurons by distinct calcium 23. Reuter H 1996 Diversity and function of presynaptic calcium signaling pathways. Science 260:181Ð186. channels in the brain. Curr Opin Neurobiol 6:331Ð337. 5. Catterall WA 1998 Structure and function of neuronal Ca2+ 24. Catterall WA 1996 Molecular properties of sodium and cal- channels and their role in neurotransmitter release. Cell cium channels. J Bioenerg Biomembr 28:219Ð230. Calcium 24:307Ð323. 25. McEnery MW, Copeland TD, Vance CL 1998 Altered 6. Barry EL, Gesek FA, Yu AS, Lytton J, Friedman PA 1998 expression and assembly of N-type calcium channel α1B and Distinct calcium channel isoforms mediate parathyroid hor- β subunits in epileptic lethargic (lh/lh) mouse. J Biol Chem mone and chlorothiazide-stimulated calcium entry in trans- 273:21435Ð21438. porting epithelial cells. J Membr Biol 161:55Ð64. 26. Mermelstein PG, Foehring RC, Tkatch T, Song WJ, 7. Buffenstein R, Sergeev IN, Pettifor JM 1994 Absence of Baranauskas G, Surmeier DJ 1999 Properties of Q-type cal- calcitriol-mediated nongenomic actions in isolated intestinal cium channels in neostriatal and cortical neurons are corre- cells of the damara mole-rat (Cryptomys damarensis). Gen lated with β subunit expression. J Neurosci 19:7268Ð7277. Comp Endocrinol 95:25Ð30. 27. Gao T, Chien AJ, Hosey MM 1999 Complexes of the α1C and 8. Meszaros JG, Karin NJ, Farach-Carson MC 1996 Voltage- β subunits generate the necessary signal for membrane tar- sensitive calcium channels in osteoblasts: mediators of plasma geting of class C L-type calcium channels. J Biol Chem membrane signalling events. Connect Tissue Res 35:107Ð111. 274:2137Ð2144. 9. Dolmetsch RE, Lewis RS, Goodnow CC, Healy JI 1997 28. Walker D, Bichet D, Geib S, Mori E, Cornet V, Snutch TP, Differential activation of transcription factors induced by Mori Y, De Waard M 1999 A new β subtype-specific interac- 2+ 2+ Ca response amplitude and duration. Nature 386:855Ð858. tion in α1A subunit controls P/Q-type Ca channel activation. 10. Gu X, Spitzer NC 1995 Distinct aspects of neuronal differen- J Biol Chem 274:12383Ð12390. tiation encoded by frequency of spontaneous Ca2+ transients. 29. Singer D, Biel M, Lotan I, Flockerzi V, Hofmann F, Dascal N Nature 375:784Ð787. 1991 The roles of the subunits in the function of the calcium 11. Curtis BM, Catterall WA 1984 Purification of the calcium channel. Science 253:1553Ð1557. antagonist receptor of the voltage-sensitive calcium channel 30. Bergh JJ, Shao Y, Akanbi K, Farach-Carson MC 2003 Rodent from skeletal muscle transverse tubules. Biochemistry 23: osteoblastic cells express voltage-sensitive calcium channels 2113Ð2118. lacking a γ subunit. Calcif Tissue Int, 73:502Ð510. 12. Hosey MM, Barhanin J, Schmid A, Vandaele S, Ptasienski J, 31. Dolphin AC, Wyatt CN, Richards J, Beattie RE, Craig P, Lee O’Callahan C, Cooper C, Lazdunski M 1987 Photoaffinity RH, Cribbs LL, Volsen SG, Perez-Reyes E 1999 The effect labelling and phosphorylation of a 165 kilodalton peptide of α2-δ and other accessory subunits on expression and prop- associated with dihydropyridine and phenylalkylamine- erties of the calcium channel α1G. J Physiol 519:35Ð45. sensitive calcium channels. Biochem Biophys Res Commun 32. Klugbauer N, Lacinova L, Marais E, Hobom M, Hofmann F 147:1137Ð1145. 1999 Molecular diversity of the calcium channel α2δ subunit. 13. Catterall WA 2000 Structure and regulation of voltage-gated J Neurosci 19:684Ð691. Ca2+ channels. Annu Rev Cell Dev Biol 16:521Ð555. 33. Jay SD, Sharp AH, Kahl SD, Vedvick TS, Harpold MM, 14. Catterall WA 1995 Structure and function of voltage-gated Campbell KP 1991 Structural characterization of the ion channels. Annu Rev Biochem 64:493Ð531. dihydropyridine-sensitive calcium channel α2δ-subunit and 15. Takahashi M, Seagar MJ, Jones JF, Reber BF, Catterall WA the associated δ peptides. J Biol Chem 266:3287Ð3293. 1987 Subunit structure of dihydropyridine-sensitive calcium 34. Gurnett CA, De Waard M, Campbell KP 1996 Dual function 2+ channels from skeletal muscle. Proc Natl Acad Sci USA of the voltage-dependent Ca channel α2δ subunit in current 84:5478Ð5482. stimulation and subunit interaction. Neuron 16:431Ð440. 16. Perez-Reyes E, Kim HS, Lacerda AE, Horne W, Wei XY, 35. Felix R, Gurnett CA, De Waard M, Campbell KP 1997 Rampe D, Campbell KP, Brown AM, Birnbaumer L 1989 Dissection of functional domains of the voltage-dependent 2+ Induction of calcium currents by the expression of the Ca channel α2δ subunit. J Neurosci 17:6884Ð6891. α1-subunit of the dihydropyridine receptor from skeletal 36. De Jongh KS, Warner C, Catterall WA 1990 Subunits of puri- muscle. Nature 340:233Ð236. fied calcium channels α2 and δ are encoded by the same 17. Varadi G, Strobeck M, Koch S, Caglioti L, Zucchi C, Palyi G gene. J Biol Chem 265:14738Ð14741. 1999 Molecular elements of ion permeation and selectivity 37. Wei X, Pan S, Lang W, Kim H, Schneider T, Perez-Reyes E, within calcium channels. Crit Rev Biochem Mol Biol Birnbaumer L 1995 Molecular determinants of cardiac Ca2+ 34:181Ð214. channel pharmacology. Subunit requirement for the high 18. Chesnoy-Marchais D, Fritsch J 1988 Voltage-gated sodium affinity and allosteric regulation of dihydropyridine binding. and calcium currents in rat osteoblasts. J Physiol 398:291Ð311. J Biol Chem 270:27106Ð27111. 19. Chien AJ, Carr KM, Shirokov RE, Rios E, Hosey MM 1996 38. Bizzarri C, Civitelli R 1994 Activation of the Ca2+ message Identification of palmitoylation sites within the L-type system by parathyroid hormone is dependent on the cell calcium channel β2a subunit and effects on channel function. cycle. Endocrinology 134:133Ð140. J Biol Chem 271:26465Ð26468. 39. Yamamoto T, Ozono K, Shima M, Yamaoka K, Okada S 20. Mitterdorfer J, Froschmayr M, Grabner M, Moebius FF, 1998 24R,25-Dihydroxyvitamin D3 increases cyclic GMP Glossmann H, Striessnig J 1996 Identification of PK-A phos- contents, leading to an enhancement of osteocalcin synthesis phorylation sites in the carboxyl terminus of L-type calcium by 1,25-dihydroxyvitamin D3 in cultured human osteoblastic channel α1 subunits. Biochemistry 35:9400Ð9406. cells. Exp Cell Res 244:71Ð76. 21. Khan I, Ahmad S, Thomas N 1998 Differential translation of 40. Hannah SS, Norman AW 1994 1α,25(OH)2 vitamin D3- the α1 isoforms of L-type calcium channel in rat brain and regulated expression of the eukaryotic genome. Nutr Rev other tissues. Biochem Mol Biol Int 45:895Ð904. 52:376Ð382. CHAPTER 44 Effects of 1,25-Dihydroxyvitamin D3 on Voltage-Sensitive Calcium Channels 759

41. Noda M, Vogel RL, Craig AM, Prahl J, DeLuca HF, of a 1,25-dihydroxyvitamin D3 membrane-associated rapid- Denhardt DT 1990 Identification of a DNA sequence respon- response steroid binding protein during human tooth and sible for binding of the 1,25-dihydroxyvitamin D3 receptor and bone development and biomineralization. J Bone Miner Res 1,25-dihydroxyvitamin D3 enhancement of mouse secreted 17:1588Ð15896. phosphoprotein 1 (SPP-1 or osteopontin) gene expression. 58. Jia Z, Nemere I 1999 Immunochemical studies on the puta- Proc Natl Acad Sci USA 87:9995Ð9999. tive plasmalemmal receptor for 1,25-dihydroxyvitamin D3 II. 42. Zhang R, Ducy P, Karsenty G 1997 1,25-Dihydroxyvitamin D3 Chick kidney and brain. Steroids 64:541Ð550. inhibits osteocalcin expression in mouse through an indirect 59. Pedrozo HA, Schwartz Z, Rimes S, Sylvia VL, Nemere I, mechanism. J Biol Chem 272:110Ð116. Posner GH, Dean DD, Boyan BD 1999 Physiological impor- 43. Russell J, Lettieri D, Sherwood LM 1986 The effects of 1,25- tance of the 1,25(OH)2D3 membrane receptor and evidence dihydroxyvitamin D3 on the synthesis of pre-proparathyroid for a membrane receptor specific for 24,25(OH)2D3. J Bone hormone mRNA. Trans Assoc Am Physicians 99:189Ð196. Miner Res 14:856Ð867. 44. Russell J, Lettieri D, Sherwood LM 1986 Suppression by 60. Nemere I, Larsson D, Sundell K 2000 A specific binding 1,25(OH)2D3 of transcription of the pre-proparathyroid moiety for 1,25-dihydroxyvitamin D3 in basal lateral mem- hormone gene. Endocrinology 119:2864Ð2866. branes of carp enterocytes. Am J Physiol Endocrinol Metab 45. Pappas TC, Gametchu B, Watson CS 1995 Membrane estrogen 279:E614ÐE621. receptors identified by multiple antibody labeling and impeded- 61. Choi AM, Tucker RW, Carlson SG, Weigand G, Holbrook NJ ligand binding. FASEB J 9:404Ð410. 1994 Calcium mediates expression of stress-response genes 46. Hua SY, Chen YZ 1989 Membrane receptor-mediated elec- in prostaglandin A2-induced growth arrest. FASEB J trophysiological effects of glucocorticoid on mammalian 8:1048Ð1054. neurons. Endocrinology 124:687Ð691. 62. Civitelli R, Kim YS, Gunsten SL, Fujimori A, Huskey M, 47. van Leeuwen JP, Birkenhager JC, van den Bemd GJ, Avioli LV, Hruska KA 1990 Nongenomic activation of the Buurman CJ, Staal A, Bos MP, Pols HA 1992 Evidence for calcium message system by vitamin D metabolites in the functional involvement of protein kinase C in the action osteoblast-like cells. Endocrinology 127:2253Ð2262. of 1,25-dihydroxyvitamin D3 in bone. J Biol Chem 267: 63. Lieberherr M 1987 Effects of vitamin D3 metabolites on 12562Ð12569. cytosolic free calcium in confluent mouse osteoblasts. J Biol 48. Grosse B, Bourdeau A, Lieberherr M 1993 Oscillations in Chem 262:13168Ð13173. inositol 1,4,5-trisphosphate and diacylglycerol induced by 64. Vazquez G, Selles J, de Boland AR, Boland R 1999 Rapid vitamin D3 metabolites in confluent mouse osteoblasts. actions of calcitriol and its side chain analogues CB1093 and J Bone Miner Res 8:1059Ð1069. GS1500 on intracellular calcium levels in skeletal muscle 49. Zanello LP, Norman AW 1997 Stimulation by 1α,25(OH)2- cells: a comparative study. Br J Pharmacol 126:1815Ð1823. vitamin D3 of whole cell chloride currents in osteoblastic 65. Baran DT, Sorensen AM, Shalhoub V, Owen T, Oberdorf A, ROS 17/2.8 cells. A structureÐfunction study. J Biol Chem Stein G, Lian J 1991 1α,25-dihydroxyvitamin D3 rapidly 272:22617Ð22622. increases cytosolic calcium in clonal rat osteosarcoma cells 50. Liu R, Xu Y, Farach-Carson MC, Vogel JJ, Karin NJ 2000 lacking the vitamin D receptor. J Bone Miner Res 6:1269Ð1275. 1,25-Dihydroxyvitamin D3 activates sphingomyelin turnover 66. Erben RG, Soegiarto DW, Weber K, Zeitz U, Lieberherr M, in ROS17/2.8 osteosarcoma cells without sphingolipid- Gniadecki R, Moller G, Adamski J, Balling R 2002 Deletion induced changes in cytosolic Ca2+. Biochem Biophys Res of deoxyribonucleic acid binding domain of the vitamin D Commun 273: 95Ð100. receptor abrogates genomic and nongenomic functions of 51. Safran JB, Butler WT, Farach-Carson MC 1998 Modulation vitamin D. Mol Endocrinol 16:1524Ð1537. of osteopontin post-translational state by 1,25-dihydroxy- 67. Farach-Carson MC, Sergeev I, Norman AW 1991 Nongenomic vitamin D3. Dependence on calcium influx. J Biol Chem actions of 1,25-dihydroxyvitamin D3 in rat osteosarcoma cells: 273:29935Ð29941. structure-function studies using ligand analogs. Endocrinology 52. Long GJ, Rosen JF 1994 Lead perturbs 1,25 dihydroxy- 129:1876Ð1884. vitamin D3 modulation of intracellular calcium metabolism 68. Norman AW, Nemere I, Zhou LX, Bishop JE, Lowe KE, in clonal rat osteoblastic (ROS 17/2.8) cells. Life Sci Maiyar AC, Collins ED, Taoka T, Sergeev I, Farach-Carson MC 54:1395Ð1402. 1992 1,25(OH)2-vitamin D3, a steroid hormone that produces 53. Farach-Carson MC, Xu Y 2002 Microarray detection of gene biologic effects via both genomic and nongenomic pathways. expression changes induced by 1,25-dihydroxyvitamin D3 J Steroid Biochem Mol Biol 41:231Ð240. and a calcium influx-activated analog in ROS 17/2.8 cells. 69. Khoury R, Ridall AL, Norman AW, Farach-Carson MC 1994 Steroids 67:467Ð470. Target gene activation by 1,25-dihydroxyvitamin D3 in 54. Nemere I, Dormanen MC, Hammond MW, Okamura WH, osteosarcoma cells is independent of calcium influx. Norman AW 1994 Identification of a specific binding protein Endocrinology 135:2446Ð2453. for 1α,25-dihydroxyvitamin D3 in basal-lateral membranes 70. Norman AW, Wehling M 1999 Overview of the first interna- of chick intestinal epithelium and relationship to transcal- tional meeting on rapid responses to steroid hormones. tachia. J Biol Chem 269:23750Ð23756. Steroids 64:3Ð4. 55. Farach-Carson MC, Nemere I 2003 Membrane receptors 71. Li W, Duncan RL, Karin NJ, Farach-Carson MC 1997 1,25- for vitamin D steroid hormones: Potential new drug targets. Dihydroxyvitamin D3 enhances PTH-induced calcium tran- Curr Drug Targets 4:67Ð76 sients in preosteoblasts by activating L-type calcium 56. Norman AW, Song X, Zanello L, Bula C, Okamura WH 1999 channels. Am J Physiol 273:E599ÐE605. Rapid and genomic biological responses are mediated by dif- 72. Duncan RL, Hruska KA, Misler S 1992 Parathyroid hormone ferent shapes of the agonist steroid hormone, 1α,25(OH)2 activation of stretch-activated cation channels in osteosar- vitamin D3. Steroids 64:120Ð128. coma cells (UMR-106.01). FEBS Lett 307:219Ð223. 57. Mesbah M, Nemere I, Papagerakis P, Nefussi JR, 73. Li W, Farach-Carson MC 2001 Parathyroid hormone-stimulated Orestes-Cardoso S, Nessmann C, Berdal A 2002 Expression resorption in calvaria cultured in serum-free medium is 760 MARY C. FARACH-CARSON AND JOEL J. BERGH

enhanced by the calcium-mobilizing activity of 1,25-dihy- 90. Hell JW, Westenbroek RE, Warner C, Ahlijanian MK, droxyvitamin D3. Bone 29:231Ð235. Prystay W, Gilbert MM, Snutch TP, Catterall WA 1993 74. Dokoh S, Donaldson CA, Haussler MR 1984 Influence of Identification and differential subcellular localization of 1,25-dihydroxyvitamin D3 on cultured osteogenic sarcoma the neuronal class C and class D L-type calcium channel α1 cells: correlation with the 1,25-dihydroxyvitamin D3 receptor. subunits. J Cell Biol 123:949Ð962. Cancer Res 44:2103Ð2109. 91. Tanabe T, Takeshima H, Mikami A, Flockerzi V, Takahashi H, 75. Nelson MT, Cheng H, Rubart M, Santana LF, Bonev AD, Kangawa H, Kojima M, Matsuo H, Hirose T, Numa S 1987 Knot HJ, Lederer WJ 1995 Relaxation of arterial smooth Primary structure of the receptor for calcium channel blockers muscle by calcium sparks. Science 270:633Ð637. from skeletal muscle. Nature 328:313Ð318. 76. Berridge MJ, Bootman MD, Lipp P 1998 Calcium—a life 92. Biel M, Ruth P, Bosse E, Hullin R, Stuhmer W, Flockerzi V, and death signal. Nature 395:645Ð648. Hofmann F 1990 Primary structure and functional expression 77. Lee KS, Marban E, Tsien RW 1985 Inactivation of calcium of a high voltage activated calcium channel from rabbit lung. channels in mammalian heart cells: Joint dependence on FEBS Lett 269:409Ð412. membrane potential and intracellular calcium. J Physiol 93. Zhao PL, Wang XT, Zhang XM, Cebotaru V, Cebotaru L, 364:395Ð411. Guo G, Morales M, Guggino SE 2002 Tubular and cellular 78. Nilius B, Benndorf K 1986 Joint voltage- and calcium- localization of the cardiac L-type calcium channel in rat dependent inactivation of Ca channels in frog atrial kidney. Kidney Int 61:1393Ð1406. myocardium. Biomed Biochim Acta 45:795Ð811. 94. Seino S, Yamada Y, Espinosa R, 3rd, Le Beau MM, Bell GI 79. Haack JA, Rosenberg RL 1994 Calcium-dependent inactiva- 1992 Assignment of the gene encoding the α1 subunit of the tion of L-type calcium channels in planar lipid bilayers. neuroendocrine/brain-type calcium channel (CACNL1A2) Biophys J 66:1051Ð1060. to human chromosome 3, band p14.3. Genomics 13: 80. Soldatov NM, Oz M, O’Brien KA, Abernethy DR, Morad M 1375Ð1377. 1998 Molecular determinants of L-type Ca2+ channel inacti- 95. Soldatov NM 1992 Molecular diversity of L-type Ca2+ chan- vation. Segment exchange analysis of the carboxyl-terminal nel transcripts in human fibroblasts. Proc Natl Acad Sci USA cytoplasmic motif encoded by exons 40Ð42 of the human α1C 89:4628Ð4632. subunit gene. J Biol Chem 273:957Ð963. 96. Meszaros JG, Karin NJ, Akanbi KA, Farach-Carson MC 81. Soldatov NM, Zuhlke RD, Bouron A, Reuter H 1997 1996 Down-regulation of L-type Ca2+ channel transcript Molecular structures involved in L-type calcium channel levels by 1,25-dihyroxyvitamin D3. Osteoblastic cells express 2+ inactivation. Role of the carboxyl-terminal region encoded L-type α1C Ca channel isoforms. J Biol Chem 271: 2+ by exons 40Ð42 in α1C subunit in the kinetics and Ca depen- 32981Ð32985. dence of inactivation. J Biol Chem 272:3560Ð3566. 97. Brewer LD, Thibault V, Chen KC, Langub MC, Landfield PW, 82. Zuhlke RD, Bouron A, Soldatov NM, Reuter H 1998 Ca2+ Porter NM 2001 Vitamin D hormone confers neuroprotec- channel sensitivity towards the blocker isradipine is affected tion in parallel with downregulation of L-type calcium by alternative splicing of the human α1C subunit gene. FEBS channel expression in hippocampal neurons. J Neurosci Lett 427:220Ð224. 21:98Ð108. 83. Qin N, Olcese R, Bransby M, Lin T, Birnbaumer L 1999 98. Farach-Carson MC, Davis PJ (2003) Steroid hormone inter- Ca2+-induced inhibition of the cardiac Ca2+ channel depends actions with target cells: cross-talk between membrane and on calmodulin. Proc Natl Acad Sci USA 96:2435Ð2438. nuclear pathways. JPET, in press. 84. Zuhlke RD, Pitt GS, Deisseroth K, Tsien RW, Reuter H 1999 99. Berry DM, Clark CS, Meckling-Gill KA 2002 1α,25- Calmodulin supports both inactivation and facilitation of Dihydroxyvitamin D3 stimulates phosphorylation of IκBα L-type calcium channels. Nature 399:159Ð162. and synergizes with TPA to induce nuclear translocation of 85. Pitt GS, Zuhlke RD 2001 Molecular basis of calmodulin NFκB during monocytic differentiation of NB4 leukemia tethering and Ca2+-dependent inactivation of L-type Ca2+ cells. Exp Cell Res 272:176Ð184. channels. J Biol Chem 276:30794Ð30802. 100. Gurlek A, Kumar R 2001 Regulation of osteoblast growth by 86. Ozono K, Sone T, Pike JW 1991 The genomic mechanism of interactions between transforming growth factor-β and action of 1,25-dihydroxyvitamin D3. J Bone Miner Res 1α,25-dihydroxyvitamin D3. Crit Rev Eukaryot Gene Expr 6:1021Ð1027. 11:299Ð317. 87. Haussler MR 1986 Vitamin D receptors: Nature and function. 101. Bergh JJ, Xu Y, Farach-Carson MC 2004 Osteoprotegerin Annu Rev Nutr 6:527Ð562. expression and secretion are regulated by calcium influx 88. Pike JW 1991 Vitamin D3 receptors: Structure and function through the L-type voltage-sensitive calcium channel. in transcription. Annu Rev Nutr 11:189Ð216. Endocrinology 145:426Ð436. 89. Barry EL 2000 Expression of mRNAs for the α1 subunit of 102. Buitrago C, Gonzalez Pardo V, de Boland AR 2002 voltage-gated calcium channels in human osteoblast-like cell Nongenomic action of 1α,25(OH)2-vitamin D3. Activation of lines and in normal human osteoblasts. Calcif Tissue Int muscle cell PLC γ through the tyrosine kinase c-Src and 66:145Ð50. PtdIns 3-kinase. Eur J Biochem 269:2506Ð2515. CHAPTER 45 Vitamin D and the Cellular Response to Oxidative Stress

RUTH KOREN AND AMIRAM RAVID Felsenstein Medical Research Center, Beilinson Campus, Rabin Medical Center, Petah Tivka, Israel

I. Reactive Oxygen Species and Redox Homeostasis IV. Discussion II. Vitamin D as a Prooxidant References III. Vitamin D as an Antioxidant

I. REACTIVE OXYGEN SPECIES Traditionally, ROS were considered the unwanted AND REDOX HOMEOSTASIS and toxic by-products of living in an aerobic environ- ment. Indeed, when present at vulnerable sites or in A. Introduction excess, ROS can damage the cell and initiate cellular “damage control systems” that arrest cell proliferation, Recent years have witnessed the accumulation of activate repair mechanisms, and induce programmed numerous reports on the effects of hormonally active cell death when the damage is irreparable. Cells vitamin D derivatives on cellular oxidationÐreduction exposed to even higher ROS levels cells may undergo balance. In fact, modulation of this balance may be necrotic cell death. The severity of cellular damage is one of the underlying mechanisms of long-recognized, determined by the extent of the imbalance between seemingly unrelated actions of the hormone. Vitamin D ROS production and antioxidant protection. Oxidative derivatives seem to have a prooxidant effect in some challenge elicits an adaptive response that results in systems but an antioxidant one in others. The purpose increased cellular repair and antioxidant capacities. of this chapter is to present the available evidence for Nondamaging smaller perturbations in ROS levels both modes of action and to discuss the possible under- appear to participate in cell signaling associated with lying mechanisms for this complex cross-talk, taking fundamental cellular processes such as cell prolifera- into account current knowledge regarding the regulation tion and differentiation and regulated exocytosis. It is of pro- and antioxidant cellular mechanisms. noteworthy that in addition to inducing cell death as a consequence of oxidative damage, ROS participate in signaling pathways that lead to programmed cell death B. The Janus Face of Reactive induced by other noxious agents [1Ð3]. Oxygen Species

Cells are continuously exposed to reactive oxygen C. The Cellular Redox State species (ROS) produced intracellularly by normal aerobic metabolism and occasionally to extracellular The major source of energy required for cellular sources of ROS such as immune cells, radiation, and metabolism in an aerobic environment is derived from xenobiotics. ROS are partially reduced compounds the movement of electrons from oxidizable donors to − of oxygen, which include superoxide (⋅O2), hydrogen oxygen. Redox couples in cells are responsive to the peroxide (H2O2), and hydroxyl (⋅OH), peroxyl (ROO⋅), electron flow. Some of these redox couples are linked and alkoxyl (RO⋅) radicals. The current state of knowl- and some are independent from other sets if activation edge regarding the effects of ROS on cellular energies are high and there are no enzyme systems that metabolism and cell fate and the various aspects of cel- can link them kinetically. Redox state is a term origi- lular ROS handling were reviewed extensively [1Ð13] nally coined to denote the ratio between the oxidized and the major insights pertinent to this chapter are and reduced forms of an interconvertible redox couple. outlined hereafter. In recent years, the use of this term was extended to

VITAMIN D, 2ND EDITION Copyright © 2005, Elsevier, Inc. FELDMAN, PIKE, AND GLORIEUX All rights reserved. 762 RUTH KOREN AND AMIRAM RAVID describe the overall redox environment of a cell. to form hydrogen peroxide and molecular oxygen. Schafer and Buettner [7] suggested a more general def- Two metal-containing SOD isoenzymes, mitochon- inition for the redox state: “The redox environment of drial Mn-SOD and the cytosolic Cu,Zn-SOD, are a linked set of redox couples as found in biological flu- active in eukaryotic cells. H2O2 reactivity is moderate. ids, organelle, cell or tissue is the summation of the It does not react spontaneously with carbon-centered products of the reduction potential and reducing capac- molecules or thiols, but reacts rapidly with thiolates ity of the linked redox couples present.” The reduction (R-S−) and transition metals. The highly toxic potential can be calculated by the Nernst equation and hydroxyl radical can be generated when H2O2 interacts the reducing capacity by the concentration of the with a reduced transition metal in a Fenton reaction. At reduced species of a redox couple. In practice, it may least three antioxidant enzyme systems are responsible be difficult or impossible to measure the potential and for the removal of H2O2 from the cellular milieu: cata- capacity of all the linked couples within a cell and a lase, glutathione peroxidases, and peroxiredoxins. representative redox couple is generally used as an Catalase catalyzes the dismutation of H2O2 molecules indicator of the redox environment. The most com- to water and molecular oxygen. This heme-containing monly employed indicator is the glutathione redox enzyme is predominantly localized in the peroxisomes couple (GSSG/2GSH), which provides a very large of mammalian cells. Glutathione peroxidases that con- pool of reducing equivalents and can be considered as tain a selenocysteine are cytosolic enzymes. They cat- the cellular redox buffer. It is now known that control alyze the reduction of H2O2 using reduced glutathione of the intracellular redox environment is vital for (GSH) as substrate. Glutathione disulfide (GSSG) proper cellular function. formed in the course of this reaction is reduced to For protection against the constant oxidative chal- regenerate GSH by the NADPH dependent flavoen- lenge, cells have developed defense mechanisms that zyme glutathione reductase. Peroxiredoxins are capa- insure the proper balance between the prooxidant and ble of directly reducing H2O2, and the oxidized antioxidant molecules. These antioxidant mechanisms enzymes formed during the catalytic cycle are reduced are indispensable for cellular defense against the dam- by thioredoxin. The ubiquitous thioredoxin system is age inflicted by ROS and play a crucial role in cellular composed of the antioxidant enzyme pair, thioredoxin redox homeostasis and regulation of redox-sensitive and thioredoxin reductase. Thioredoxin is a general metabolic processes. disulfide reductant and thioredoxin reductase catalyzes the reduction of the active site disulfide of thioredoxin utilizing NADPH as the source of reductive power. D. The Generation and Degradation of ROS Continuous availability of NADPH is needed to fuel the regeneration of reduced glutathione and thiore- ROS are generated in the cell both enzymatically, by doxin and the maintenance of the cellular redox state. oxidoreductases and nonenzymatically, as side prod- The reducing equivalents of NADPH are generated ucts of reactions utilizing electron transfer. by the flow of carbon through the pentose phosphate Mitochondria, the cytochrome P450s and their reduc- pathway while the regulatory enzyme glucose-6- tases, and nitric oxide synthases have been implicated phosphate dehydrogenase catalyzes the first and rate in ROS generation. There is no compelling evidence limiting step in this pathway. for regulation of ROS production through specific sig- naling pathways, although it is clearly subject to change according to substrate availability or energy E. ROS Signaling state. An exception to this notion is NADPH oxidase that generates superoxides. The mechanism of trigger- Various properties of ROS qualify them to act as ing and regulation of this enzyme was elucidated in second messengers in signaling cascades [6Ð13]: (1) macrophages but it is now recognized that it is also They are enzymatically generated (e.g., by NAD(P)H present in other cells. Moreover, it is activated by vari- oxireductases) in response to stimuli such as cytokines ous agonistic stimuli such as cytokines, growth factors and growth factors. (2) Peroxiredoxins maintain enzy- acting via receptor tyrosine kinases, and ligands of G- matically their basal concentration at a subthreshold protein coupled receptors. Superoxide anion is the main level. (3) Their stimulus-elicited rise decays rapidly ROS produced in the course of oxidationÐreduction by glutathione peroxidase and catalase. (4) Their − reactions in the presence of molecular oxygen. Two action is specific. ⋅O2 and H2O2 readily react with thio- molecules of superoxide rapidly dismutate in a reac- late (-S−), the ionized form of thiols, forming disulfide tion accelerated by superoxide dismutase (SOD) and sulfenic acid, but cannot react at a biologically CHAPTER 45 Vitamin D and the Cellular Response to Oxidative Stress 763 significant rate with thiols (-SH). Thus, only thiolate- Last, we will discuss the possible role of some containing proteins will be affected by ROS directly. 1,25(OH)2D3 target genes in mediating either the pro- However, ROS may mediate the oxidation of thiols in or antioxidant action of the hormone. proteins indirectly through disulfide exchange with glutathione disulfide, which is transiently increased during the reduction of hydroperoxides by glutathione II. VITAMIN D AS A PROOXIDANT peroxidases. (5) Oxidized thiols can be regenerated by GSH, thioredoxin and glutaredoxin, providing a mech- The first evidence for a prooxidant activity of anism for reversible inactivation of proteins by ROS. 1,25(OH)2D3 was reported in 1988 by Polla et al. [15]. ROS trigger and modulate various cellular signaling According to this study, pretreatment of human pathways and affect transcription mainly via the oxi- myelomonocytic leukemia cells with calcitriol increased dation of cysteine residues in redox-sensitive proteins. their susceptibility to the cytotoxic action of H2O2 This chemical modification may affect DNA binding administered in bolus. Twenty-five years later, this or enzymatic activities, the formation or release of finding was corroborated by a study reporting a simi- protein complexes, or the formation of multimers. ROS lar enhancing activity of calcitriol in the human breast were shown to modulate, among others, all the mitogen- cancer cell line MCF-7 [16]. The cellular effect of ROS activated protein kinases, the phosphatidyl inositol produced intracellularly in response to ROS-generating 3-kinase pathway, and the IKK/NFκB signaling path- agents may differ from that of ROS applied to cells in way. The molecular targets for this action include pro- bolus. In fact, it was demonstrated that the gene expres- tein phosphatases, protein kinases, small GTPases, sion profile and intensity were different in cells exposed and thioredoxin and glutathione S-transferase Pi (acting to H2O2 or to menadione [17], the latter simulating the as inhibitors of ASK-1 and c-Jun N-terminal kinases, endogenous cellular generation of ROS in terms of respectively). The modulation of transcription by ROS their nature and the site and rate of their production. is due to the activation of signaling systems, to the direct The quinone moiety of menadione may undergo a one- oxidation and inactivation of transcription factors, and electron reduction to the corresponding semiquinone to their ability to induce the expression of various tran- radical by various cellular flavin-centered reductases scription factors. Redox-sensitive transcription factors present in different compartments of the cell. In the include AP-1, NFκB, p53, Sp1, and nuclear receptors. presence of oxygen, this radical will form superoxide In this context, it is interesting to note that the vitamin D anions that will be dismutated by SOD to generate receptor (VDR) itself is subject to regulation by ROS H2O2. Despite the difference between the two agents, that oxidize the structural cysteines in its zinc fingers however, 1,25(OH)2D3 sensitized breast cancer cells to and inhibit its transcriptional activity [14]. the cytotoxic action of menadione similarly to its action The preceding discussion accentuates both the com- on exogenous H2O2 cytotoxicity [18]. plexity and the redundancy of the cellular networks To support the hypothesis that prooxidant action responsible for maintaining the essential balance of 1,25(OH)2D3 is the underlying mechanism for the between cellular ROS production and degradation. enhanced cytotoxicity, it is necessary to provide evidence In view of this scenario, it is not unexpected that the that its sensitizing action is specific to ROS-induced action of 1,25(OH)2D3 or other vitamin D3 metabolites cell damage and not to cell damage per se. ROS are is both cell and context dependent. The mode of action also involved in the cytotoxic activity of many natural of 1,25(OH)2D3 is expected to depend on the identity and pharmacological agents with anticancer activity. of the cellular system(s) involved in each case of ROS Pertinent examples are the immune cytokines tumor balance perturbation. It is thus not surprising that the necrosis factor (TNF) and interleukin 1 (IL-1), and dox- hormone can act as pro- or antioxidant in different orubicin, the widely used anticancer drug. 1,25(OH)2D3 cellular systems and even in the same cell under dif- and other active vitamin D derivatives enhance the ferent conditions. In the following paragraphs and cytotoxicity of these agents against breast cancer Table I we will summarize and discuss the experimental cells [18Ð21] and of TNF also against renal cell carci- evidence for both activities of vitamin D metabolites. noma cells [22]. In these experimental systems, cell We will first present some cases in which hormonally death is only partly due to ROS and other cytotoxic active vitamin D derivatives either enhance or attenuate mechanisms operate as well. By the use of antioxidants the impact of exposure to preformed ROS or ROS such as N-acetylcysteine, GSH, ascorbate, or lipoic acid generating agents. We will then describe the available it was demonstrated that 1,25(OH)2D3 preferentially direct evidence for vitamin D hormone-mediated mod- reinforced the ROS-dependent mechanisms of cell death ulation of the cellular ROS balance and redox state. [18,20,23]. A similar mechanism may also account for 764 RUTH KOREN AND AMIRAM RAVID

TABLE I Cellular Manifestations of the Prooxidant and Antioxidant Activities of Vitamin D

Species and Cell line or Outcome of vitamin D cell origin cell type action References

Prooxidant effects Human myelomonocytic leukemia U937 Increased H2O2 cytotoxicity [15]

Human breast cancer MCF-7 Increased H2O2 cytotoxicity [16] Human breast cancer MCF-7 Increased menadione cytotoxicity [18] Human breast cancer MCF-7 Increased doxorubicin cytotoxicity [18] Human breast cancer T-47D MDA-MB-231 Increased doxorubicin cytotoxicity [74] Human breast cancer MCF-7 Increased TNF and IL-1 cytotoxicity [19,20,23] Human renal cell carcinoma SK-RC-29 Increased TNF cytotoxicity [22] Human breast cancer MCF-7 Increased ionizing radiation cytotoxicity [25] Human renal tubular cells HK-2 Increased iron cytotoxicity [24] Antioxidant effects Rat brain substantia nigra Neurons (in vivo) Decreased 6-OHDA neurotoxicity [46] Rat brain Mesencephalic Decreased 6-OHDA, [46,47,48] neurons (in vitro) glutamate and MPP+ neurotoxicity

Rat brain Mesencephalic Decreased H2O2 neurotoxicity [46,47] neurons (in vitro) Human myelomonocytic U937 Decreased TNF cytotoxicity [53] leukemia Human promyelocytic HL-60 Increased differentiation by [51,52] leukemia antioxidants

Human keratinocytes HaCaT Decreased H2O2 cytotoxicity [60] Mouse skin Keratinocytes Decreased UV-induced sunburn [54,55,56] (in vivo) cell formation Rat epidermis Keratinocytes Decreased UV cytotoxicity [54] (in vitro) Human epidermis Keratinocytes Decreased UV cytotoxicity [56] (in vitro) Chick intestine Epithelium Calcium absorption [67,68,69]

the findings that 1,25(OH)2D3 sensitized renal tubular glutathione levels following TNF treatment, indicative cells to iron-mediated toxicity [24], and that both of a more oxidized cellular redox environment [20]. 1,25(OH)2D3 and other active vitamin D derivatives More impressively, it turned out that treatment with increased the rate of apoptosis of breast cancer cells 1,25(OH)2D3 alone significantly increased the GSSG/ exposed to ionizing radiation [25]. However, although GSH ratio [26]. In other words, the hormone itself can ROS are implicated in both scenarios of cell death, perturb the cellular redox environment and shift it whether ROS play a role in the enhancing effect of toward a more oxidized state. A shift in the redox state 1,25(OH)2D3 has not been unequivocally established. of the major cellular redox buffer, the glutathione sys- The notion that 1,25(OH)2D3 precipitates cell death by tem, should be reflected in the potential of other thiol a prooxidant mechanism is complemented by its lack redox couples like those in transcription factors and the of effect on ROS-independent modes of cell death, active site of enzymes. This surmise was substantiated such as those induced by etoposide, interferon α, and by the finding that the activity of the redox-sensitive cytotoxic lymphocytes [16,18,22]. glycolytic enzyme GAPDH was oxidatively inhibited The antioxidant system protects the cell from oxida- in breast cancer cells treated with 1,25(OH)2D3 in tive damage inflicted by exposure to excessive ROS association with an increase of the GSSG/GSH ratio [26]. levels and also participates in redox homeostasis. It is noteworthy that the observed increase in the glu- We have heretofore discussed the prooxidant role of tathione redox potential (3Ð8 mV) is close to the redox vitamin D metabolites, manifested as exacerbation change (15 mV) that was shown to abolish the DNA of oxidative stress-induced cell death. We will now binding capacity of the transcription factors AP-1 and proceed to describe the effects of the hormone on the NFκB [27]. Changes in the redox state could also cellular redox state. Pretreatment of breast cancer translate into reversible oxidation of cysteines in pro- cells with 1,25(OH)2D3 increased the drop in reduced teins that determine cell fate. Protein kinases, protein CHAPTER 45 Vitamin D and the Cellular Response to Oxidative Stress 765 tyrosine phosphatases, and key components of the consequently diminished intracellular ROS levels, apoptotic process, such as the mitochondrial permeabil- while up-regulation of the protein by calcitriol, increased ity transition pores and caspases, are all subject to redox ROSlevels. regulation [28,29]. The increase in the glutathione redox potential may be related to an independent observation of increased ROS levels in breast cancer III. VITAMIN D AS AN ANTIOXIDANT cells treated with 1,25(OH)2D3 alone under similar conditions [30]. Similar to the prooxidant action of 1,25(OH)2D3, The prooxidant activity of 1,25(OH)2D3 is attributed evidence for its antioxidant activity relies mainly on to its genomic action via the VDR. This may be the ability of the hormone to limit oxidative damage. It inferred from the structureÐfunction relationship of has long been recognized that besides the anticancer vitamin D metabolites and the time required for activity of the hormone, resulting in induction of apop- 1,25(OH)2D3 action [18,19,22,23]. It is thus expected tosis and inhibition of cell proliferation, it protects that vitamin DÐregulated proteins are involved in the some normal cells from death-inducing stimuli. In this mediation of the prooxidant effects of 1,25(OH)2D3. context, the neuroprotective action of the hormone has Two proteins that were shown to be affected by been a subject of numerous studies (see Chapter 100). 1,25(OH)2D3 and could be involved in this activity are In this chapter we will focus on the evidence that links Cu/Zn-SOD and vitamin D up-regulated protein 1 neuroprotective effects of 1,25(OH)2D3 to its antioxidant (VDUP1). As discussed earlier, the cytosolic enzyme properties. Cu/Zn-SOD is one of the major constituents of the cel- Parkinson’s disease is a chronic neurodegenerative lular defense system against ROS. Treatment of breast disorder characterized by the selective loss of dopami- cancer cells with 1,25(OH)2D3 was shown to decrease nergic neurons in the substantia nigra. Autopsy studies Cu/Zn-SOD gene expression, protein level, and backed by experimental models linked the mode of enzyme activity [18]. Decrease in SOD activity may dopaminergic neuron death with excessive production render these cells more vulnerable to oxidative chal- of ROS resulting from dopamine metabolism [39,40]. lenge as inferred from previous reports on the effect of Glutamate neurotoxicity may also play a critical role SOD overexpression [31,32]. Such a decrease would in dopaminergic neuron death. Glutamate serves as an . − cause a shift in the balance between O2 and H2O2. excitatory neurotransmitter, but excessive amounts of Increased levels of superoxides can, in turn, cause glutamate cause calcium overload and trigger exces- increased oxidative damage due to interaction with NO sive ROS and nitric oxide production that in turn brings to form the highly toxic peroxynitrite [33] or to release about cell death [41Ð43]. Parkinsonian symptoms of transition metal ions from intracellular stores, which may be induced in experimental animals by treatment supports hydroxyl radical formation via the Fenton with the neurotoxins 6-hydroxydopamine (6-OHDA) or reaction [34]. Decrease in SOD activity may also 1-methyl-4-phenylpyridine (MPP+). 6-OHDA is thought underlie the increased thiol oxidation in redox-sensitive to be formed endogenously in Parkinson’s disease . − proteins, since O2 was shown to react with thiolate patients through dopamine oxidation and to cause eight times faster than H2O2 [35]. dopaminergic cell death via a free radical mechanism Chen and DeLuca first described, cloned, and [44]. MPP+ accumulates within dopaminergic neurons sequenced a novel protein, VDUP1, induced in the where it induces a deleterious series of events starting promyelocytic leukemia cell line HL-60 in response to with inhibition of mitochondrial respiration, leading to 1,25(OH)2D3 treatment [36], but at the time (1994) its superoxide formation and energy failure. Again, ROS function and role remained obscure. Only later, after a are thought to be a major mediator of MPP+-induced series of unrelated studies concerned mainly with the cell death [40,45]. Pretreatment with 1,25(OH)2D3 regulation of the thioredoxin system, was it realized that reduced neurotoxicity of 6-OHDA in rats [46] and pro- VDUP1 is a thioredoxin binding protein that sequesters tected cultured neurons against cytotoxicity induced thioredoxin and limits its intracellular availability by glutamate [47], 6-OHDA [46], and MPP+ [47,48]. for the redox regulation and antioxidant systems [37]. Although suggestive, these findings alone do not Thioredoxin is now recognized to be as important for provide direct evidence for the antioxidant action of cellular redox homeostasis and redox regulation of 1,25(OH)2D3. A more direct evidence was obtained proteins as glutathione. Thus, changes in VDUP1 can by demonstrating a protective effect against cytotox- and do affect the cellular redox environment. Song icity induced by preformed, exogenous ROS such et al. have shown that 1,25(OH)2D3 up-regulated as H2O2 [46,47] or a superoxide generating system VDUP1 also in murine melanoma cells [38]. They (hypoxanthine/xanthine oxidase) [47]. The protective found that decreasing VDUP1 synthesis by antisense effect was detectable within hours of exposure to the cDNA raised the reducing capacity of thioredoxin and hormone and dependent on protein synthesis, indicating 766 RUTH KOREN AND AMIRAM RAVID

a genomic mode of action of calcitriol. Moreover, [15] of a 1,25(OH)2D3 dependent enhancement of 1,25(OH)2D3 reduced intracellular ROS levels formed H2O2 cytotoxicity in this same cell line. These opposite + in response to MPP [48] or exogenous H2O2 [47]. effects of calcitriol may be due to the different types of A clue to the mechanism of this antioxidant activity is ROS and antioxidant systems that participate in the the finding of a significant increase in total glutathione cytotoxic processes in these two scenarios. levels in mesencephalic neuron cultures following Vitamin D metabolites are known to be potent 1,25(OH)2D3 treatment [48]. inducers of keratinocyte differentiation. In recent years, Astrocytes play a major role in antioxidative and evidence has been brought forward for an additional detoxification processes in the brain. Administration of role of the hormone in the epidermis, the protection 1,25(OH)2D3 to rats increased the activity of γ-glutamyl of epidermal keratinocytes, both in vivo and in vitro, transpeptidase (γ-GT) in astrocytes and pericytes derived from UV radiation-induced apoptosis [54Ð57]. The from their brains [49]. γ-GT is a membrane-bound role of ROS, and particularly H2O2, in mediating the enzyme that hydrolyzes extracellular GSH, and thus effects of UV on cell fate is well established [58,59]. enables the cellular reutilization of its constituent amino The possibility that the protective effect of 1,25(OH)2D3 acids to generate intracellular GSH. These findings is related to its antioxidant capacity is born out by the were verified in primary astrocyte cultures treated finding that 1,25(OH)2D3 also protects keratinocytes with lipopolysaccharide in which 1,25(OH)2D3 treat- from H2O2 cytotoxicity [60]. These protective effects ment markedly increased γ-GT mRNA levels [50]. are associated with inhibition of the activation of the 1,25(OH)2D3 also brought about an increase in GSH stress-activated MAP kinase, c-Jun N-terminal kinase, levels in these cells, but this effect may not be entirely by H2O2 and UV [57,60]. This action could at least attributed to the up-regulation of γ-GT. γ-GT seems to partially account for the protective effect of the hor- play only a minor role in the increase of glutathione lev- mone as c-Jun N-terminal kinase is known to activate els in neuronal cultures treated with 1,25(OH)2D3 [48]. proapoptotic signaling pathways. Metallothioneins However, it is assumed that in vivo astrocytes protect (MTs) are ubiquitous sulfhydryl-rich proteins that neurons against ROS toxicity through the supply of are readily inducible by heavy metals. One-third of the glutathione precursors. Therefore, up-regulation of γ-GT 61 amino acids of MTs are cysteines and because in astrocytes may well reinforce this cross-talk and con- of this feature, they may serve as expendable targets tribute to the in vivo antioxidant action of vitamin D for oxidants. Although the antioxidant properties of metabolites. MTs derive mainly from sulfhydryl nucleophilicity, Another experimental system in which 1,25(OH)2D3 complexation of metals involved in the Fenton reaction acts as an antioxidant is the promyelocytic leukemia can also serve to reduce oxidative stress and ROS cell line HL-60. A prolonged, 96-hr treatment of these damage [61,62]. Indeed, overexpression of MT confers cells with 1,25(OH)2D3 decreased intracellular ROS protection of cells against Cu-dependent lipid peroxi- production both in resting cells and in response to dation and cytotoxicity [63] and reduces UV-induced oxidative challenge [51]. Although this finding is con- apoptosis in keratinocytes both in vivo and in vitro [64]. sistent with an antioxidant effect of the hormone, it Moreover, epidermis from MT-null mice is more must be borne in mind that 1,25(OH)2D3 induces HL-60 susceptible to the formation of sunburn cells following cells to differentiate and that the increased cellular exposure to UV [64]. In view of these findings, it antioxidant capacity may be one manifestation of the seems plausible that up-regulation of epidermal MT more differentiated state. It is noteworthy that well- gene expression can account, at least partially, for the characterized antioxidants such as vitamin E and antioxidant effect of 1,25(OH)2D3 in the skin [54,56]. carnosic acid also promote differentiation of HL-60 cells Treatment with 1,25(OH)2D3 increased MT mRNA and that this capacity is greatly enhanced in the presence levels in cultured keratinocytes but also in liver, kidney, of low concentrations of 1,25(OH)2D3 [51,52]. This syn- and skin when applied in vivo [65,66]. The effect of ergism may also be related to the antioxidant capacity of the hormone was apparent within 2 hr and maximal active vitamin D metabolites. after 24 hr and was not dependent on protein synthesis, In line with the foregoing antioxidant activity of indicating a direct genomic effect. These findings sug- 1,25(OH)2D3 is the report of an inhibitory effect exerted gest that 1,25(OH)2D3 may have antioxidant activity in by the hormone on cytotoxicty induced by the intracel- the liver and kidney under conditions when MT levels lular ROS-generating agent TNF in the myelomonocytic become rate limiting. leukemia cell line U937 [53]. This protective effect was It is intriguing that the most studied and the best- associated with increased TNF-dependent induction of characterized activity of 1,25(OH)2D3, namely, stimula- the antioxidant enzyme Mn-SOD. This finding is in tion of intestinal calcium absorption, may also be related apparent contradiction with the study of Polla et al. to its antioxidant properties. Depletion of glutathione CHAPTER 45 Vitamin D and the Cellular Response to Oxidative Stress 767 pools produced a rapid and reversible inhibition of have been attributed to its redox modulating activity. 1,25(OH)2D3-induced calcium transfer from lumen to Although a causal relationship between redox modula- plasma and of 1,25(OH)2D3-dependent enhancement tion by vitamin D and biological outcome was estab- of the activity of Ca-ATPase in the intestinal basolat- lished in some experimental systems [16,18,20,23], most eral membranes. It has been suggested that this inhibi- of the available evidence for such an association is cor- tion is due to reversible oxidation of SH groups in the relative in nature. Keeping in mind the genomic mode 2+ Ca transporter [67]. This surmise is supported by of action of 1,25(OH)2D3, it is not surprising that efforts finding that the number of reduced SH groups in brush have been made to discover vitamin D target genes, the border membrane preparations prepared from vitamin expression of which is up- or down-regulated by the D-deficient chicks increased twofold by a short-term hormone in a way that may account for its pro- or in vivo treatment with 1,25(OH)2D3 [68,69]. The rapid antioxidant actions. The current available knowledge action of 1,25(OH)2D3 suggests that its antioxidant regarding such putative redox regulators is described effect in this system is mediated via a nongenomic in the text and summarized in Table II. Some of these mechanism. genes are directly affected by calcitriol (metallothi- onein and VDUP1), while the modulation of others requires long incubation times suggesting an indirect IV. DISCUSSION action of the hormone. No indication of the existence of one master gene that mediates the redox action of The impressive body of experimental evidence laid 1,25(OH)2D3 emerges from the available data. However, out in this chapter attests to the ability of vitamin D taking into account the diversity of redox-related genes derivatives to modulate the cellular systems responsible modulated by 1,25(OH)2D3, it is plausible that the redox for redox homeostasis and the response to oxidative activity of the hormone is only one facet of a more stress. Since the cellular redox state and antioxidant general stress response. Transcriptome studies will systems have important roles in the regulation of cell probably help in answering this question. The func- metabolism and in the determination of cell fate, some tional association between the modulation of specific effects of 1,25(OH)2D3 such as modulation of cell gene expression and the biological outcome awaits the proliferation, differentiation, and programmed cell death use of specific inhibitors or gene knockout models.

TABLE II Effect of Vitamin D on ROS Levels and Redox-Associated Molecules

Target molecule Cell type or line Direction of effect Consequence References

Cu,Zn-SOD MCF-7 (breast cancer) Decrease Prooxidant [18] Mn-SOD U937 (myelomonocytic leukemia) Increase Antioxidant [53] Prostate cells (normal and tumor) Increase Antioxidant [70] γ-Glutamyl transpeptidase Astrocytes and pericytes Increase Antioxidant [49,50] Thioredoxin reductase 1 Prostate cells (normal and tumor) Increase Antioxidant [70] Metallothionein Epidermis, keratinocytes Increase Antioxidant [54,56,65] Prostate cells (normal) Increase Antioxidant [70] Liver, kidney Increase Antioxidant [65,66] Prostate cells (tumor) Decrease Prooxidant [70] VDUP1 B16F10 (mouse melanoma) Increase Prooxidant [38] Glutathione (total) Astrocytes Increase Antioxidant [50] Mesencephalic neurons Increase Antioxidant [48] MCF-7 (breast cancer) Decrease Prooxidant [20] HL-60 (promyelocytic leukemia) Increase Antioxidant [51] GSSG/GSH ratio MCF-7 (breast cancer) Increase Prooxidant [26] ROS levels HL-60 (promyelocytic leukemia) Decrease Antioxidant [51] Mesencephalic neurons Decrease Antioxidant [47,48] MCF-7 (breast cancer) Increase Prooxidant [30] B16F10 (mouse melanoma) Increase Prooxidant [38] 768 RUTH KOREN AND AMIRAM RAVID

An intriguing feature of 1,25(OH)2D3 is its ability 2. Jacobson MD 1996 Reactive oxygen species and programmed to exert both prooxidant and antioxidant effects in its cell death. Trends Biochem Sci 21:83Ð86. 3. Simon HU, Haj-Yehia A, Levi-Schaffer F 2000 Role of reactive target cells. This may occur even in the same cells as oxygen species (ROS) in apoptosis induction. Apoptosis exemplified by its prooxidant and antioxidant activities 5:415Ð418. in U937 cells treated with H2O2 or TNF, respectively 4. Mates M 2000 Effects of antioxidant enzymes in the molecu- [15,53]. Some possible explanations for the opposite lar control of reactive oxygen species toxicology. Toxicology effects of 1,25(OH) D are: (1) 1,25(OH) D can both 153:83Ð104. 2 3 2 3 5. Nordberg J, Arner ES 2001 Reactive oxygen species, antioxi- increase and decrease the level of different antioxidant dants, and the mammalian thioredoxin system. Free Radic Biol proteins in the same cell as was shown in malignant Med 31:1287Ð1312. prostate cells (decrease in MT and increase in thiore- 6. Forman HJ, Torres M 2001 Redox signaling in macrophages. doxin reductase 1 and Mn-SOD levels) [70]. The out- Mol Aspects Med 22:189Ð216. come of this complex hormonal effect will depend on 7. Schafer FQ, Buettner GR 2001 Redox environment of the cell as viewed through the redox state of the glutathione the role the different antioxidants have in various cellu- disulfide/glutathione couple. Free Radic Biol Med 30: lar scenarios. (2) 1,25(OH)2D3 can modulate the level of 1191Ð1212. the same antioxidant in opposite directions in different 8. Sen CK, Packer L 1996 Antioxidant and redox regulation of cell types (e.g., up-regulation of MT in normal prostate gene transcription. FASEB J 10:709Ð720. cells and down-regulation in malignant prostate cells 9. Sun Y, Oberley LW 1996 Redox regulation of transcriptional activators. Free Radic Biol Med 21:335Ð348. [70]). (3) The same effect of 1,25(OH)2D3 on a certain 10. Lander HM 1997 An essential role for free radicals and derived antioxidant may have opposite consequences, pro- or species in signal transduction. FASEB J 11:118Ð124. antioxidative, depending on the cellular context (e.g., 11. Nakamura H, Nakamura K, Yodoi J 1997 Redox regulation of decrease in SOD level may have either prooxidant or cellular activation. Annu Rev Immunol 15:351Ð369. antioxidant effects depending on the capacity of the 12. Finkel T 1998 Oxygen radicals and signaling. Curr Opin Cell γ Biol 10:248Ð253. cellular H2O2 degrading systems and increase in -GT 13. Kamata H, Hirata H 1999 Redox regulation of cellular activity may result in enhanced GSH synthesis on the signaling. Cell Signal 11:1Ð14. one hand and increase in H2O2 generation, on the other 14. Kroncke KD, Klotz LO, Suschek CV, Sies H 2002 Comparing hand [71]). (4) A primary prooxidant effect of nitrosative versus oxidative stress toward zinc finger-dependent 1,25(OH) D can induce an adaptive response that will transcription. Unique role for NO. J Biol Chem 277: 2 3 13294Ð13301. increase the antioxidant capacity of the cell. An exam- 15. Polla BS, Bonventre JV, Krane SM 1988 1,25-Dihydroxy- ple that may be relevant in this context is the increase vitamin D3 increases the toxicity of hydrogen peroxide in the in the cellular content of glucose-6-phosphate dehy- human monocytic line U937: The role of calcium and heat drogenase following treatment of breast cancer cells shock. J Cell Biol 107:373Ð380. with 1,25(OH) D [26,72]. Glucose-6-phosphate dehy- 16. Ravid A, Koren R 2003 The role of reactive oxygen species in 2 3 the anticancer activity of vitamin D. Recent Results Cancer drogenase is the rate-limiting enzyme in the pentose Res 164:357Ð367. phosphate pathway, which supplies the cell with 17. Chuang YY, Chen Y, Gadisetti, Chandramouli VR, Cook JA, NADPH, and its up-regulation is part of the adaptive Coffin D, Tsai MH, DeGraff W, Yan H, Zhao S, Russo A, Liu ET, cellular response to oxidative challenge [73]. Mitchell JB 2002 Gene expression after treatment with hydrogen The above discussion reflects the fact that the study peroxide, menadione, or t-butyl hydroperoxide in breast cancer cells. Cancer Res 62:6246Ð6254. of the effect of vitamin D derivatives on ROS balance 18. Ravid A, Rocker D, Machlenkin A, Rotem C, Hochman A, and handling and on redox homeostasis is a developing Kessler-Icekson G, Liberman UA, Koren R 1999 1,25- field and that many unexplored areas remain and many Dihydroxyvitamin D3 enhances the susceptibility of breast questions are still unanswered. cancer cells to doxorubicin-induced oxidative damage. Cancer Res 59:862Ð867. 19. Rocker D, Ravid A, LibermanUA, Garach-Jehoshua O, Koren R 1994 1,25-Dihydroxyvitamin D3 potentiates the cytotoxic effect Acknowledgments of TNF on human breast cancer cells. Mol Cell Endocrinol 106:157Ð162. We would like to acknowledge support of the Israel 20. Koren R, Rocker D, Kotestiano O, Liberman UA, Ravid A Science Foundation grants no. 684/931 and 601/99. 2000 Synergistic anticancer activity of 1,25-dihydroxyvitamin D3 and immune cytokines: The involvement of reactive oxygen species. J Steroid Biochem Mol Biol 73:105Ð112. 21. Colston KW, Hansen CM 2002 Mechanisms implicated in the growth regulatory effects of vitamin D in breast cancer. Endocr References Relat Cancer 9:45Ð59. 22. Yacobi R, Koren R, Liberman UA, Rotem C, Wasserman L, 1. Slater AF, Nobel CS, Orrenius S 1995 The role of intracellular Ravid A 1996 1,25-Dihydroxyvitamin D3 increases the sensi- oxidants in apoptosis. Biochim Biophys Acta 1271:59Ð62. tivity of human renal carcinoma cells to tumor necrosis factor CHAPTER 45 Vitamin D and the Cellular Response to Oxidative Stress 769

alpha but not to interferon alpha or lymphokine-activated killer 41. Sawada H, Shimohama S, Kawamura T, Akaike A, Kitamura Y cells. J Endocrinol 149:327Ð333. Taniguchi T, Kimura J 1996 Mechanism of resistance to 23. Weitsman GE, Ravid A, Liberman UA, Koren R 2003 Vitamin D NO-induced neurotoxicity in cultured rat dopaminergic neurons. enhances caspase-dependent and -independent TNFα-induced J Neurosci Res 46:509Ð518. breast cancer cell death: The role of reactive oxygen species 42. Sawada H, Kawamura T, Shimohama S, Akaike A, Kimura J and mitochondria. Int J Cancer 106:178Ð186. 1996 Different mechanisms of glutamate-induced neuronal 24. Zager RA 1999 Calcitriol directly sensitizes renal tubular death between dopaminergic and non-dopaminergic neurons in cells to ATP-depletion and iron-mediated attack. Am J Pathol rat mesencephalic culture. J Neurosci Res 43:503Ð510 154:1899Ð1909. 43. Sawada H, Shimohama S, Tamura Y, Kawamura T, Akaike A, 25. Sundaram S, Gewirtz DA 1999 The vitamin D3 analog EB Kimura J 1996 Methylphenylpyridium ion (MPP+) enhances 1089 enhances the response of human breast tumor cells to glutamate-induced cytotoxicity against dopaminergic neurons radiation. Radiat Res 152:479Ð486. in cultured rat mesencephalon. J Neurosci Res 43:55Ð62. 26. Koren R, Hadari-Naor I, Zuck E, Rotem C, Liberman UA, 44. Riobo NA, Schopfer FJ, Boveris AD, Cadenas E, Poderoso JJ Ravid A 2001 Vitamin D is a prooxidant in breast cancer cells. 2002 The reaction of nitric oxide with 6-hydroxydopamine: Cancer Res 61:1439Ð1444. implications for Parkinson’s disease. Free Radic Biol Med 27. Clive DR, Greene JJ 1996 Cooperation of protein disulfide 32:115Ð121. isomerase and redox environment in the regulation of NF-κB 45. Nakai M, Mori A, Watanabe A, Mitsumoto Y 2003 1-Methyl- and AP1 binding to DNA. Cell Biochem Funct 14:49Ð55. 4-phenylpyridinium (MPP+) decreases mitochondrial oxida- 28. Broekemeier KM, Klocek CK, Pfeiffer DR 1998 Proton selec- tionÐreduction (REDOX) activity and membrane potential tive substrate of the mitochondrial permeability transition pore: (Deltapsi(m)) in rat striatum. Exp Neurol 179:103Ð110. Regulation by the redox state of the electron transport chain. 46. Wang JY, Wu JN, Cherng TL, Hoffer BJ, Chen HH, Biochemistry 37:13059Ð13065. Borlongan CV, Wang Y 2001 Vitamin D3 attenuates 6-hydroxy- 29. Hampton MB, Fadeel B, Orrenius S 1998 Redox regulation of dopamine-induced neurotoxicity in rats. Brain Res 904: the caspases during apoptosis. Ann NY Acad Sci 854:328Ð335. 67Ð75. 30. Narvaez CJ, Welsh J 2001 Role of mitochondria and caspases 47. Ibi M, Sawada, H, Nakanishi M, Kume T, Katsuki H, Kaneko S, in vitamin D-mediated apoptosis of MCF-7 breast cancer cells. Shimohama S, Akaike A 2001 Protective effects of 1α,25- J Biol Chem 276:9101Ð9107. (OH)2D3 against the neurotoxicity of glutamate and reactive 31. Doroshow JH, Akman S, Esworthy S, Chu FF, Burke T 1991 oxygen species in mesencephalic culture. Neuropharmacology Doxorubicin resistance conferred by selective enhancement of 40:761Ð771. intracellular glutathione peroxidase or superoxide dismutase 48. Shinpo K, Kikuchi S, Sasaki H, Moriwaka F, Tashiro K 2000 content in human MCF-7 breast cancer cells. Free Radic Res Effect of 1,25-dihydroxyvitamin D3 on cultured mesencephalic Commun 12Ð13(Pt. 2):779Ð781. dopaminergic neurons to the combined toxicity caused by 32. Manna SK, Zhang HJ, Yan T, Oberley LW, Aggarwal BB 1998 L-buthionine sulfoximine and l-methyl-4-phenylpyridine. Overexpression of manganese superoxide dismutase sup- J Neurosci Res 62:374Ð382. presses tumor necrosis factor induced apoptosis and activation 49. Garcion E, Thanh XD, Bled F, Teissier E, Dehouck MP, of nuclear transcription factor-κB and activated protein-1. Rigault F, Brachet P, Girault A, Torpier G, and Darcy F 1996 J Biol Chem 273:13245Ð13254. 1,25-Dihydroxyvitamin D3 regulates γ1 transpeptidase activity 33. Squadrito GL, Pryor WA 1998 Oxidative chemistry of nitric in rat brain. Neurosci Lett 216:183Ð186. oxide: the roles of superoxide, peroxynitrite, and carbon diox- 50. Garcion E, Sindji L, Leblondel G, Brachet P, Darcy F 1999 ide. Free Radic Biol Med 25:392Ð403. 1,25-Dihydroxyvitamin D3 regulates the synthesis of gamma- 34. Minotti G 1993 Sources and role of iron in lipid peroxidation. glutamyl transpeptidase and glutathione levels in rat primary Chem Res Toxicol 6:134Ð146. astrocytes. J Neurochem 73:859Ð866. 35. Barrett WC, DeGnore JP, Konig S, Fales HM, Keng YF, 51. Danilenko M, Wang Q, Wang X, Levy J, Sharoni Y, Studzinski GP Zhang ZY, Yim MB, Chock PB 1999 Regulation of PTP1B via 2003 Carnosic acid potentiates the antioxidant and prodiffer- glutathionylation of the active site cysteine 215. Biochemistry entiation effects of 1α,25-dihydroxyvitamin D3 in leukemia cells 38:6699Ð6705. but does not promote elevation of basal levels of intracellular 36. Chen KS, DeLuca HF 1994 Isolation and characterization of calcium. Cancer Res 63:1325Ð1332. a novel cDNA from HL-60 cells treated with 1,25-dihydroxy- 52. Sokoloski JA, Hodnick WF, Mayne ST, Cinquina C, Kim CS, vitamin D3. Biochim Biophys Acta 1219:26Ð32. Sartorelli AC 1997 Induction of the differentiation of HL-60 37. Nishiyama A, Matsui M, Iwata S, Hirota K, Masutani H, promyelocytic leukemia cells by vitamin E and other antioxi- Nakamura H, Takagi Y, Sono H, Gon Y, Yodoi J 1999 dants in combination with low levels of vitamin D3: Possible Identification of thioredoxin-binding protein-2/vitamin D3 relationship to NF-kappaB. Leukemia 11:1546Ð1553. up-regulated protein 1 as a negative regulator of thioredoxin 53. Iwamoto S, Takeda K, Kamijo R, Konno K 1990 Induction of function and expression. J Biol Chem 274:21645Ð21650. resistance to TNF cytotoxicity and mitochondrial superoxide 38. Song H, Cho D, Jeon JH, Han SH, Hur DY, Kim YS, Choi I dismutase on U-937 cells by 1,25-dihydroxyvitamin D3. 2003 Vitamin D3 up-regulating protein 1 (VDUP1) antisense Biochem Biophys Res Commun 170:73Ð79. DNA regulates tumorigenicity and melanogenesis of murine 54. Hanada K, Sawamura D, Nakano H, Hashimoto I 1995 melanoma cells via regulating the expression of fas ligand and Possible role of 1,25-dihydroxyvitamin D3-induced metallo- reactive oxygen species. Immunol Lett 86:235Ð247. thionein in photoprotection against UVB injury in mouse skin 39. Perry TL, Yong VW 1986 Idiopathic Parkinson’s disease, and cultured rat keratinocytes. J Dermatol Sci 9:203Ð208. progressive supranuclear palsy and glutathione metabolism in 55. Youn JI, Park BS, Chung JH, Lee JH 1997 Photoprotective the substantia nigra of patients. Neurosci Lett 67:269Ð274. effect of calcipotriol upon skin photoreaction to UVA 40. Nakamura K, Wang W, Kang UJ 1997 The role of glutathione in and UVB. Photodermatol Photoimmunol Photomed 13: dopaminergic neuronal survival. J Neurochem 69:1850Ð1858. 109Ð114. 770 RUTH KOREN AND AMIRAM RAVID

56. Lee J, Youn JI 1998 The photoprotective effect of 1,25-dihydroxy- 66. Chou SY, Hannah SS, Lowe KE, Norman AW, Henry HL 1995 vitamin D3 on ultraviolet light B-induced damage in keratinocyte Tissue-specific regulation by vitamin D status of nuclear and and its mechanism of action. J Dermatol Sci 18:11Ð18. mitochondrial gene expression in kidney and intestine. 57. De Haes P, Garmyn M, Degreef H, Vantieghem K, Bouillon R, Endocrinology 136:5520Ð5526. Segaert S 2003 1,25-Dihydroxyvitamin D3 inhibits ultraviolet 67. Tolosa de Talamoni N, Marchionatti A, Baudino V, Alisio A B-induced apoptosis, Jun kinase activation, and interleukin-6 1996 Glutathione plays a role in the chick intestinal production in primary human keratinocytes. J Cell Biochem calcium absorption. Comp Biochem Physiol A. Physiol 115: 89:663Ð673. 127Ð132. 58. Tyrrell RM, Keyse SM 1990 New trends in photobiology. The 68. Mykkanen HM, Wasserman RH 1990 Relationship of interaction of UVA radiation with cultured cells. J Photochem membrane-bound sulfhydryl groups to vitamin DÐstimulated Photobiol B 4:349Ð361. uptake of [75Se]Selenite by the brush border membrane 59. Black HS 1987 Potential involvement of free radical reactions vesicles from chick duodenum. J Nutr 120:882Ð888. in ultraviolet light-mediated cutaneous damage. Photochem 69. Mykkanen HM, Wasserman RH 1990 Reactivity of sulfhydryl Photobiol 46:213Ð221. groups in the brush-border membranes of chick duodena is 60. Ravid A, Rubinstein E, Gamady A, Rotem C, Liberman UA, increased by 1,25-dihydroxycholecalciferol. Biochim Biophys Koren R 2002 Vitamin D inhibits the activation of stress- Acta 1033:282Ð286. activated protein kinases by physiological and environmental 70. Krishnan AV, Peehl DM, Feldman D 2003 Inhibition of stresses in keratinocytes. J Endocrinol 173:525Ð532. prostate cancer growth by vitamin D: Regulation of target gene 61. Lazo JS, Kuo SM, Woo ES, Pitt BR 1998 The protein thiol expression. J Cell Biochem 88:363Ð371. metallothionein as an antioxidant and protectant against anti- 71. Dominici S, Valentini M, Maellaro E, Del Bello, B, Paolicchi, A, neoplastic drugs. Chem Biol Interact 111Ð112:255Ð262. Lorenzini E, Tongiani R, Comporti M, Pompella A 1999 62. Viarengo A, Burlando B, Ceratto N, Panfoli I 2000 Antioxidant Redox modulation of cell surface protein thiols in U937 role of metallothioneins: a comparative overview. Cell Mol lymphoma cells: The role of gamma-glutamyl transpeptidase- Biol (Noisy-le-grand) 46:407Ð417. dependent H2O2 production and S-thiolation. Free Radic Biol 63. Fabisiak JP, Pearce LL, Borisenko GG, Tyhurina YY, Tyurin VA, Med 27:623Ð635. Razzack J, Lazo JS, Pitt BR, Kagan VE 1999 Bifunctional anti/ 72. Noun A, Garabedian M, Monet JD 1989 Stimulatory effect of prooxidant potential of metallothionein: redox signaling of 1,25-dihydroxyvitamin D3 on the glucose-6-phosphate dehy- copper binding and release. Antioxid Redox Signal 1:349Ð364. drogenase activity in the MCF-7 human breast cancer cell line. 64. Hanada K, Sawamura D, Tamai K, Baba T, Hashimoto I, Cell Biochem Funct 7:1Ð6. Muramatsu T, Miura N, Naganuma A 1998 Novel function of 73. Ursini MV, Parrella A, Rosa G, Salzano S, Martini G 1997 metallothionein in photoprotection: metallothionein-null Enhanced expression of glucose-6-phosphate dehydrogenase mouse exhibits reduced tolerance against ultraviolet B injury in human cells sustaining oxidative stress. Biochem J 323(Pt 3): in the skin. J Invest Dermatol 111:582Ð585. 801Ð806. 65. Karasawa M, Hosoi J, Hashiba H, Nose K, Tohyama C, Abe E, 74. Wang Q, Yang W, Uytingco MS, Christakos S, Wieder R 2000 Suda T, Kuroki T 1987 Regulation of metallothionein gene 1,25-Dihydroxyvitamin D3 and all-trans-retinoic acid sensitize expression by 1α,25-dihydroxyvitamin D3 in cultured cells breast cancer cells to chemotherapy-induced cell death. Cancer and in mice. Proc Natl Acad Sci USA 84:8810Ð8813. Res 60:2040Ð2048. CHAPTER 46 Vitamin D: Role in the Calcium Economy

ROBERT P. H EANEY Creighton University, Omaha, Nebraska

I. Introduction V. Optimal Vitamin D Status II. Overview of the Calcium Economy VI. Summary and Conclusions III. Calcium Absorptive Input References IV. Physiological Sources of Vitamin D Activity

I. INTRODUCTION apatite lattice with variable stoichiometry, and embedded in a dense protein matrix. Although cells (osteocytes) Vitamin D functions in many body systems, but ramify throughout bony tissue, the intercellular bony perhaps the best attested of the nutrient’s actions—and material itself lacks appreciable free water. As a result certainly the one most clearly associated with human there is very limited exchange of calcium ions between disease—is its role in transferring calcium (and phos- the bone and the circulating body fluids. Isotopic phorus) from ingested food into the body fluids. Calcium, exchange with tracers injected into the blood is con- like most divalent cations, is only partially absorbed fined to the surface layer of crystals in the bone situated from the chyme as it travels through the small intestine. along vascular channels and spaces, and to still incom- This situation creates an opportunity for regulation of pletely mineralized new forming sites. Taken all together absorption, with room both to increase and to decrease the exchangeable bone calcium moieties amount to only calcium extraction efficiency in response to physiologi- about 25 mmol (1000 mg), or ~0.1% of total skeletal cal controls. calcium [1]. Moreover, the insolubility of bone mineral Details of both the many cellular and tissue effects is such that, even when there is exchange, there is virtu- of vitamin D, and of the absorptive process itself, are ally never net transfer out of bone into the body fluids. covered in other chapters in this volume. Here I shall Net transfer normally requires formation or resorption attempt to summarize mainly the meaning and impor- of bone tissue.1 tance of the vitamin D-mediated transfer process from A second, biologically critical compartment is intra- gut to blood and to outline how it fits into the mainte- cellular calcium. Here calcium serves as a ubiquitous nance of the calcium economy. My frame of reference second messenger, linking signals from outside the cell will be the integrated functioning of the intact organism. to the mechanisms constituting the cell’s response. While free calcium ion concentrations in the cytosol

II. OVERVIEW OF THE CALCIUM ECONOMY 1One possible exception is the calcium carbonate of bone. A. Body Calcium Compartments Carbonate substitutes poorly for phosphate in the apatite lattice, and it is generally considered that, because of different valences Body calcium in an adult human amounts to about and ionic radii for the two ions, carbonate is confined to crystal sur- 15Ð20 g (0.375Ð0.5 mol)/kg body weight. This calcium faces. Generally it is assumed that the carbonate is more or less uniformly distributed throughout the bony material. However, bone exists in three quite distinct divisions (or compartments). carbonate is substantially more labile than is bone calcium gener- They are distinct because (1) movement of calcium ally, and it is likely therefore that the carbonate is situated even atoms between them is both limited and regulated; and more superficially than generally presumed, that is, primarily on (2) they can and do vary in magnitude independently of anatomic bone surfaces, rather than diffusely on crystal surfaces one another. generally. Calcium carbonate might, thus, be a kind of “icing” on the underlying mineralized matrix, sensitive to pH and pCO2 in the The first and most obvious compartment is the extracellular fluid. If this is the case, a limited amount of net move- calcium in the bones and teeth. Here calcium exists ment of calcium into and out of bone would be possible without as inorganic mineral crystals, arranged in an imperfect involving cell-mediated formation or resorption of bone tissue. VITAMIN D, 2ND EDITION Copyright © 2005, Elsevier, Inc. FELDMAN, PIKE, AND GLORIEUX All rights reserved. 774 ROBERT P. H EANEY are typically on the order of 1 × 10Ð5 mmol [2], total possibly fatal extremes of hypo- and hypercalcemia as cell calcium is on the order of 0.5Ð2 mmol/kg tissue [1]. the organism goes from fasting to feeding. Most of this quantity (~99.99%) is bound to specialized calcium storage proteins (e.g., sequestrin, parvalbumin, calbindin) and located in storage vesicles, typically B. Regulation of ECF [Ca2+] specialized units of the endoplasmic reticulum. Because the calcium ion is of just the right radius to fit neatly A detailed description of the processes involved in reg- into folds of the peptide chain, and because calcium is ulating ECF [Ca2+] is beyond the purpose of this chapter. capable of forming up to 12 (typically 6Ð8) coordina- To situate vitamin D in this system it is necessary only tion bonds with oxygen atoms in the side chains of to note that regulation occurs both by controlling the amino acids projecting off the peptide chain, calcium renal excretory threshold for calcium and by regulating stabilizes the tertiary structure of many catalytic calcium fluxes into and out of the ECF. Two of the molecules, thereby activating them. Cytosolic [Ca2+] transfers out of the ECF illustrated in Fig. 1 drive the must be kept very low to prevent constant, uncontrolled system and are essentially unregulated. The other activity of the many cell functions in which calcium transfers are at least partially responsive and constitute plays a messenger or activation role. On the other hand, the basis of the regulatory control of ECF [Ca2+]. The substantial intracellular stores of calcium are neces- unregulated, driving transfers are (1) mineralization of sary because the binding avidity of cytosolic proteins newly deposited bone matrix at bone forming sites and for calcium is so high that, typically, the free path of (2) daily obligatory loss out of the body. Vitamin D and a calcium ion in the cytosol is only a tiny fraction of its metabolites play critical roles in the two regulated a cell diameter [2]. If reservoirs were not diffusely transfers into the ECF—from ingested food and from distributed throughout the cytosol, and if extracellular bone resorption. calcium were the only source for this critical second messenger function, activation would be limited to the 1. DRIVING TRANSFERS zone immediately beneath the plasma membrane. a. Mineralization of Bone Mineralization of bone Because the cell membrane is relatively imperme- constitutes an unregulated drain because it is a passive able to calcium ions, and because the cytosolic [Ca2+] phenomenon, lagging several days, and even weeks, compartment is so tiny, tracer exchange between ECF behind the osteoblastic cellular activity that initiates calcium and cell stores of calcium is surprisingly slow— the process. The growing mineral deposits extract typically requiring hours or days to come into tracer calcium and phosphorus from blood flowing past the equilibrium [1]. Further, cell calcium levels are typi- mineralizing site at a decreasing exponential rate for cally unaffected by acute changes in calcium concen- up to 40 weeks after the matrix has been deposited. trations in the extracellular fluid (ECF). The only way, in theory, to stop the process is to shut The third and smallest division of body calcium is the down blood flow to bone. The magnitude of this miner- calcium present in the circulating blood and the ECF alization drain varies with skeletal remodeling activity bathing all the body tissues. This compartment contains and with body size. It amounts to about 0.15 mmol/d/kg typically about 0.4 mmol calcium per kg body weight. body weight in healthy mature adults. It is much higher Ionized calcium concentration in these fluids is ordi- during growth, of course, and rises once again after mid narily about 1.25 mmol/liter (5 mg/dl), a figure that is life [3,4]. regulated across the higher vertebrate orders with the b. Obligatory Loss Obligatory loss consists of a same exquisite precision as are, e.g., the concentrations combination of cutaneous loss and the fixed components of sodium and potassium. Departures from this level of urinary and endogenous fecal calcium excretion. produce well-studied, significant effects on interneu- Cutaneous losses consist not just of sweat calcium ronal signal transmission and on muscular excitability. but of the calcium contained in shed skin, hair, and ECF [Ca2+] is also important for supracellular protein nails. As has been noted above, all cells contain sub- activations such as those in the coagulation cascade. stantial amounts of calcium, and their loss from body Into and out of this compartment passes all the surfaces inexorably takes calcium with them. Cutaneous calcium entering and leaving the body from the out- losses have been hard to quantify but are estimated side, as well as entering and leaving bone. These fluxes to be at least 0.4 mmol/day and more likely closer to are summarized schematically in Fig. 1. Together they 1.5 mmol/day [5,6]. Much higher losses have been involve daily quantities amounting to 35Ð50% of the reported with vigorous physical activity [7]. size of the entire compartment in healthy adults, and to The fixed component of endogenous fecal and urinary several times that compartment size in infants. Without calcium losses is somewhat more complicated [8]. On tight regulation, ECF [Ca2+] would oscillate between average, 3.5Ð4.0 mmol calcium enters the gut each day CHAPTER 46 Vitamin D: Role in the Calcium Economy 775

Bone Diet Ca 20.0 Formation 9.0 Resorption 9.0

Skin 3.5 Extracellular 1.5 fluid 0.8

5.0 25 mmol

Filtered Reabsorbed 200 197.5

Fecal Ca 17.7 Urine Ca 3.0

FIGURE 1 Schematic representation of the principal inputs and outputs of the extracellular fluid compartment. Units are in Ca mmol for extracellular fluid and mmol Ca/day for all other entries. (Copyright Robert P. Heaney, 2004. Reproduced with permission.) from endogenous sources, principally as a component even on a very low calcium diet (see later discussion), of digestive secretions, but also as the calcium con- there is an irreducible minimum loss of endogenous tained in shed mucosal cells (which turn over once calcium through the gut averaging close to 2 mmol/day. every 4Ð5 days). The precise quantity varies directly If gross absorption is not greater than this figure, the with body size and with the amount of food consumed. gut becomes a net excretory organ for calcium. For reasons not understood, the amount of calcium enter- The third obligatory loss is through the kidney. It is ing the gut in the digestive secretions varies directly also generally held that renal calcium excretion is control- with phosphorus intake [8]. In any event, this endogenous lable, but this is only partly true. PTH certainly regu- calcium mixes with food calcium and much of it is lates tubular calcium reabsorption. However, there is a subject to absorption, just as is the calcium of food. fixed limit to what that mechanism can accomplish. However, as noted earlier, calcium absorption is always This limit is itself a function of other variables that are incomplete. Gross absorption averages about 25Ð30% outside the regulatory loop. Best studied of these in healthy adults, and net absorption about 10Ð11%. factors is the renal excretion of sodium, dietary intakes Hence much of the secreted calcium is lost in the feces. of protein and potassium, net endogenous acid produc- Moreover, some of the endogenous calcium secretion tion (NEAP), and absorbed dietary phosphorus. enters the intestinal stream effectively so low in the gut Sodium [9Ð11], protein [12,13], and NEAP [14] increase as to be essentially unreclaimable (e.g., the calcium in urinary calcium loss; phosphorus and potassium decrease colon mucus and in shed colon cells). On a normal diet it [15,16]. this distal component has been estimated to be about Because sodium and calcium compete for the same 0.6 mmol [8]. Typical endogenous fecal calcium values reabsorption mechanism in the proximal tubule, the two average in the range of 3 mmol/day. Since absorption ions influence one another’s excretion [17]. On average, efficiency in adults is essentially never above 60%, urine calcium increases by from 0.5 to 1.5 mmol for 776 ROBERT P. H EANEY

every 100 mmol of sodium excreted [9,10]. Similarly, levels of 1,25(OH)2D and augmenting absorption effi- urine calcium rises by about 0.25 mmol for every 10 g ciency for ingested food calcium. of protein ingested [12]. The result, for an adult woman These five effects reinforce one another in important ingesting the RDA for protein and the median sodium ways. The earliest effects, occurring within minutes, are intake for North Americans, is a level of obligatory a decrease in renal tubular phosphate reabsorption and urinary loss amounting to about 2 mmol/day. Reducing the resulting fall in serum phosphate. The latter immedi- sodium intake would certainly reduce this obligatory ately augments existing osteoclastic bone resorption [19] loss. Nevertheless such voluntary dietary change is and increases activity of the renal 1-α-hydroxylase [20]. clearly not a part of any physiological regulatory loop. The elevated production of 1,25(OH)2D leads to And thus, to the extent that sodium intake influences increased intestinal absorption, elevating ECF [Ca2+] obligatory calcium loss, it constitutes a demand to and thereby closing the feedback loop. [1,25(OH)2D which the calcium homeostatic system must respond. also suppresses parathyroid hormone release in its own Given typical adult diets in Europe and North right.] Finally, 1,25(OH)2D is necessary for efficient America, the sum of these obligatory losses through skin, osteoclast work. In this last role, it is not known gut, and kidney is about 5 ± 1 mmol/day, or about one- whether variations of 1,25(OH)2D in the physiologic fifth of the total calcium in the ECF. To offset these range produce corresponding alterations in bone losses (plus the demands of bone mineralization), the resorption, and it is a difficult question to study because organism regulates countervailing inputs into the ECF of the tight regulation of the various components of from food and bone. It is in these transfers that vitamin the system. Nevertheless, it is well established that D plays its role. The input from the first source is resorptive work is severely impaired in vitamin D defi- dependent both upon the presence of food in the upper ciency states (see Fig. 2 and later discussion), and that GI tract and the presence of sufficient calcium in 1,25(OH)2D in supraphysiologic doses is capable of that food. Because neither condition can be guaran- causing substantial increases in bone resorption. Finally, teed, the second source, bone, is the more reliable and all of the components of the intestinal calcium transport constitutes, in fact, the first line of defense against system, including vitamin D receptors and calbindins, hypocalcemia.2 are also found in the distal tubule of the nephron [21]. It is possible, though not yet firmly established, that 2. RESPONSE OF THE SYSTEM TO DEMAND 1,25(OH)2D may thus enhance recovery of filtered cal- a. PTH-Mediation of Response Briefly, a fall in cium and contribute to the PTH effect of elevating the ECF [Ca2+] evokes a prompt rise in parathyroid hor- renal calcium threshold. mone [PTH] release. PTH acts in a classical negative An example of the integrated response of the system feedback loop to raise the ECF [Ca2+], thereby closing to demand is afforded by what occurs during antler the loop and reducing PTH release. The mechanisms by formation in deer in the spring [22]. The calcium which PTH raises ECF [Ca2+] illustrate well the com- demands of antler formation exceed the calcium avail- plexities of the calcium economy. These mechanisms able in the nutrient-poor late winter foliage. Other include: (1) increasing renal phosphate clearance, things being equal, this drain would lower ECF [Ca2+], thereby lowering ECF phosphate levels; (2) increasing but the parathyroid glands, sensing minute reductions renal tubular reabsorption of calcium, thereby allowing in ECF [Ca2+], respond by increasing secretion of system inputs to elevate ECF [Ca2+]; (3) augmenting PTH, which in turn activates numerous remodeling osteoclast work at existing resorption loci; (4) activating loci throughout the deer’s skeleton. Since remodeling new bone resorption loci; and (5) increasing the activity is asynchronous, with resorption preceding formation of the renal 1-α-hydroxylase, thereby increasing serum at each remodeling locus, the extra burst of newly acti- vated remodeling sites provides a temporary surplus of calcium, which the animal promptly uses to mineralize matrix in the forming antler. A few weeks later, when the skeletal remodeling loci reach their own formation 2Because it is beyond the scope of this chapter, which is con- cerned mainly with calcium transfers, I shall not develop further phase, calcium content of the ingested foliage will be what is actually an even more fundamental mechanism by which higher, and the “mineral debt” created by antler for- PTH regulates ECF [Ca2+], that is, the control of the renal calcium mation will be repaid from ingested greens. threshold (which is covered in more detail elsewhere [18]). Suffice b. Vitamin D Deficiency As already noted, vitamin it to say here only that in the shift from a lower to higher PTH level, D is essential for the mobilization of calcium from the renal calcium losses are temporarily curtailed until calcium inputs from bone and gut succeed in elevating [Ca2+]. Then, as the filtered skeletal calcium reserves. This is well illustrated in the load at the glomerulus rises, calcium excretion returns to its previ- course of nutritional rickets observed in the children of ous level (although renal calcium clearance is now reduced). China and Northern Europe before the discovery of CHAPTER 46 Vitamin D: Role in the Calcium Economy 777

mineralization of the hypertrophic cartilage zone beneath the growth plate. Then, during the 8 or so months when solar vitamin D was lacking, the rachitic process resumed. The result, visible on X-ray, was a series of bands parallel with the growth plate, with densely mineralized bone tissue alternating with undermineralized layers (Fig. 2). One might have thought that, during the winter, PTH-mediated bone resorption would have attacked bone in the dense layers, making its calcium available to new bone-forming sites (as occurs with antler formation in deer). But in the absence of vitamin D, the bone resorptive process is severely impaired. As a result the rachitic child is unable adequately to mobilize its own calcium reserves. c. Feast and Famine The diets of hominids were high in calcium [24], just as are the diets of contempo- rary deer and other higher mammals. Foods available to contemporary hunter-gatherers exhibit an annual mean calcium nutrient density of 1.75Ð2 mmol (70Ð80 mg)/100 kCal. For individuals of contemporary body size, doing the work of a hunter-gatherer, that value translates to calcium intakes in the range of 50Ð75 mmol (2000Ð3000 mg)/day. But, as noted, the environment could not be relied upon to supply calcium-rich food continuously. Periods of fasting, famine, or drought would undoubtedly have threatened hypocalcemia. This fact underscores the importance both of bone as a calcium reserve and of the vitamin D-parathyroid hor- mone control system, with its ability to release calcium rapidly from bone.3 Contemporary humans in industrialized nations have the same paleolithic physiology as our hominid ancestors. We experience, however, a few crucial dif- ferences in external conditions. One is a generally lower exposure to sunlight. Rickets was endemic in Northern Europe in the 19th century, partly because of latitude, partly because of air pollution, and partly because of child labor. Routine use of vitamin D today has all but eliminated that problem in children, but FIGURE 2 X-ray of the knee in a child with vitamin D deficiency adults, and particularly the elderly, are often vitamin DÐ rickets. The alternating bands of high and low density reflect insufficient. (The consequences of this low vitamin D annual periods of greater and lesser vitamin D availability. (Reproduced from A Textbook of X-ray Diagnosis, Vol. 6, 4th edi- exposure are explored elsewhere in this volume.) tion, 1971, edited by SC Shanks, P Kerley. HK Lewis & Co., Ltd, Another difference is a much reduced nutrient density Toronto. Chapter XLIII, Metabolic and endocrine-induced bone for calcium in our ingested food. The importance of disease by CJ Hodson, p. 661, Fig. 774.) this latter departure from primitive conditions lies in

vitamin D, and now, unfortunately, occurring once 3Technically calcium is never actually “released” from bone. again in exclusively breast-fed children not given vita- (See, however, footnote 1. Rather, a volume of actual bone tissue min D supplements [23] (see Chapter 49). Prior to rou- is resorbed. In the process its calcium is released into the ECF. Thus transfer of calcium out of bone always means some removal tine prophylactic use of vitamin D in countries where of bone tissue. Bone, however, is a very rich source of calcium. rickets was endemic, summer sun exposure produced A single cubic centimeter of bone contains as much calcium as is some vitamin D—enough to allow reasonably normal contained in the entire circulating blood of an adult human. 778 ROBERT P. H EANEY the fact that it limits the efficacy of vitamin D in aug- these two metabolites to the calcium economy, and of menting input from the intestine. This limitation is not differences in their concentrations in ethnic groups, is widely recognized and rarely is its quantitative impact still unclear (see later discussion). adequately appreciated. Section III,B of this chapter develops this important component of the calcium economy in greater detail. III. CALCIUM ABSORPTIVE INPUT

3. INDEPENDENCE OF PTH EFFECTOR MECHANISMS Chapter 24 (by Wasserman) describes the mecha- The PTH regulatory system is unique in that the nisms of calcium transfer from the intestinal lumen. feedback loop operates through three independent Here I describe quantitative aspects of that transfer as effector mechanisms, already described (elevated renal a part of the integrated calcium economy, and will calcium threshold, elevated intestinal calcium absorp- focus as well on the factors determining the magnitude tion, and elevated bone resorption). This seeming of the input from the gut into the ECF. redundancy underscores the physiological importance of maintaining constant ECF [Ca2+]. The independence of response of the three PTH effector mechanisms con- A. Location and Timing of Absorption stitutes the substratum for still incompletely explored, in the Gut but interesting, differences in the way the organism adapts to deficiency and surfeit of calcium. As noted in Chapter 24, there is a gradient of con- The feedback loop of calcium regulation is typically centrations of vitamin D receptors and of calbindin in closed by some variable combination of all three mucosa along the gut, with highest levels in the duo- effects. For example, when calcium intake falls, there denum and lowest in the colon mucosa. Accordingly, is an obvious limit to what the absorptive mechanism the avidity (or rate) of active absorption is highest in can yield, forcing higher PTH levels and correspond- the duodenum. It is sometimes said that absorption ingly greater renal calcium retention and enhanced net itself is highest there, but this is not correct. That con- transfer out of bone. Relative differences in the intrin- clusion is based on studies of isolated loops or gut sic responsiveness of those effector organs have impor- sacs, where movement of the chyme along the intestine tant consequences for bone mass [25]. Slightly lower cannot occur. Absorbed quantity is the product of intrinsic responsiveness of osteoclastic resorption to absorption rate and residence time; and residence time PTH, other things being equal, leads to a slightly of the chyme in the duodenum is very brief. Only at higher PTH level, which, in turn, drives all three effec- very low calcium intakes (or test loads), and with max- tor mechanisms slightly harder. The result is that the imal 1,25(OH)2D-stimulated active transport, will it be intestinal absorptive and renal recovery mechanisms true that most of the calcium absorbed will be from the for calcium operate at higher efficiency, and bone duodenum. At more usual intakes, the much longer resorption, at lower efficiency. This leads to more bone residence time in the jejunum and ileum means that and is probably a large part of the explanation for the most of the quantity absorbed occurs from the lower higher bone mass in persons of black African ancestry. small intestine. The importance of length of exposure Although there has been some inconsistency in the to the absorptive surface is reflected in the finding that data reported to date in this regard, the bulk of the absorptive efficiency varies directly with mouth-to- evidence points to relative refractoriness to PTH- cecum transit time [34]. stimulated bone resorption in African Americans [26Ð32]. Absorption does not occur from the healthy stom- For example, Dawson-Hughes et al. [33] showed that ach, and thus the beginning of absorption is delayed blacks exhibited a greater response to dietary calcium until gastric emptying begins. This, in turn, is depen- reduction than did whites, with larger increments of both dent upon the character of the ingested meal or other PTH and 1,25(OH)2D, indicating a blackÐwhite differ- calcium source. Emptying tends to be most rapid with ence in bony response (see Chapter 47). Clear evidence small fluid ingestates and is slower with solid food and in this regard also comes from the calcium tracer studies with fat. In healthy individuals ingesting light meals of Abrams and his colleagues, showing higher calcium (such as would commonly be employed to test absorp- absorption and retention in adolescent black females tion efficiency), calcium absorption is nearly complete than in whites at the same pubertal stage [31]. There by 5 hours after ingestion [35]. Figure 3 presents data seem also to be differences in vitamin D metabolism in on the time course of absorption, using the ratio of the the two ethnic groups, with blacks showing typically time-dependent apparent absorption fraction to its ulti- lower serum 25OHD levels and higher 1,25(OH)2D mate value in the individual being tested. As the figure levels than whites [32]. The relative importance of shows, absorption has reached better than 80% of its CHAPTER 46 Vitamin D: Role in the Calcium Economy 779

105 0.8 * 100 * 0.7 * *

2 SEM) 95 * ± 0.6 90 Late phase absorption 0.5 85 (colonic) * 0.4 80

Calcium absorption 0.3 75 End of early phase absorption (small intestine) Absorption fraction 0.2 (percent completion *significantly diff. from 100% 0 010203040 0.1 Time (hrs) 0.0 FIGURE 3 Time course of absorption (derived from Barger-Lux 01020304050 et al. [35]). The data plot the percentage completion of absorption Calcium intake (mmol/d) (derived from expressing the double-isotope absorption fraction as FIGURE 4 Fractional absorption plotted as a function of usual a ratio of its value at any given time to its final value after 24 hr. calcium intake (in mmol/day) in 525 studies in healthy, middle- Completion of absorption is thus expressed as a value of 100%. aged women [36]. The solid line is the least squares regression line As the curve shows, absorption is about 94% complete by 5 hr derived from a log-log fit. (Copyright Robert P. Heaney, 1989. after oral ingestion. The remaining 6% occurs more slowly and Reproduced with permission.) may be presumed to reflect absorption from the colon and/or from ileo-cecal reflux. (Copyright Robert P. Heaney, 1966. Reproduced with permission.)

undoubtedly correct, it is also substantially incom- plete. This is shown by the data in Fig. 5, which plots nonadaptive absorption fraction as a function of a ultimate value by 3 hr after ingestion, and 96% by 7 hr. broad range of calcium load sizes. These studies were There is then only a very gradual approach to comple- performed in women, assigned randomly on any given tion over the next 20 hr. This last component probably morning to intake loads spanning a 30-fold range, from reflects a small amount of colonic absorption (or, alter- 0.4 to 12.5 mmol [37]. Clearly, an inverse relationship natively, cecal-ileal reflux, with delayed ileal absorp- tion). It should be stressed that the percentage values in Fig. 3 refer to the quantity absorbed, not the quantity ingested. Thus, with typically only 25Ð30% of a load absorbed (see later discussion), the 4Ð5% colonic com- 0.9 ponent represents absorption of only about 1% of the 0.8 ingested load. 0.7

0.6 B. Absorption as a Function of Intake 0.5

It has long been recognized that absorption effi- 0.4 ciency varies inversely with intake. Figure 4 illustrates 0.3 this relationship with data obtained from healthy, Absorption fraction middle-aged women in whom absorption fraction was 0.2 measured under controlled metabolic ward conditions 0.1 and plotted as a function of their ingested intakes [36]. The best fit regression line through the data shows the 0.0 expected rise in absorption fraction at low calcium 0.1 1 10 intakes. (Note, however, that even at the lowest intakes, Calcium load (mmol) predicted mean absorption efficiency is only ~45%.) FIGURE 5 Fractional absorption in nonadapted healthy women The higher efficiency at low intakes is traditionally for loads ranging from 0.4 to 12.5 mmol Ca. Error bars are ±2 SEM. The solid line is the least squares regression through the actual attributed to adaptation, specifically to higher produc- data (n = 75), derived from Heaney et al. [37]. The units of the hor- tion of 1,25(OH)2D, with a corresponding increase in izontal axis are natural logarithms of load size (in mmol Ca). active calcium absorption. While that explanation is (Copyright Robert P. Heaney, 1996. Reproduced with permission.) 780 ROBERT P. H EANEY

0.7 0.7 9 8 0.6 0.6 7 0.5 0.5 Adapted women 6 0.4 0.4 5 0.3 4 0.3 3 0.2

Non-adapted women Absorption fraction 0.2 2 Absorption fraction 0.1 1 Absorbed calcium (mmol/d) 0.1 0.0 0 01020304050 0.0 051015 20 25 Calcium intake (mmol/d) Calcium intake (mmol) FIGURE 7 Fractional absorption and mass absorption for the 525 FIGURE 6 Combination of the regression lines from Figs. 4 and studies of Fig. 4. The left axis and solid line represent 5, showing the extent of difference produced by adaptation to the fractional absorption and the right axis and dashed line, mass various intakes. (Copyright Robert P. Heaney, 1996. Reproduced absorption. (Copyright Robert P. Heaney, 1996. Reproduced with with permission.) permission.)

is present, just as in the data of Fig. 4. Equally clearly, It is, however, a necessary starting point because it is it cannot be due to adaptation, since the test load was the primary datum available from most studies of the first exposure these women had to the intake level absorptive physiology. Figure 7 presents the regression concerned. Figure 6 plots these two sets of data line from Fig. 4 and adds a second line representing the together and shows that, while both exhibit an inverse actual quantity of calcium absorbed in these same relationship between absorption and intake, there is in women (i.e., the product of absorption fraction and fact a difference between them, with the adapted intake). This variable is obviously the nutritionally rel- women absorbing more at low intakes than the non- evant one since, in offsetting obligatory losses (or spe- adapted (as would be predicted). The zone between the cial demands such as antler building or fetal skeletal two lines is a semiquantitative expression of the PTH- development), it is a quantity of calcium (not a fraction) vitamin D-mediated adaptation to the lower intake.4 that is needed to balance the drains created by calcium The most likely explanation for the inverse relation- leaving the ECF. ship observed under both sets of conditions is that Figure 7 also illustrates another important aspect of calcium transfer, whether active or passive, is a slow, this input to the calcium economy. At low intakes, inefficient process, with only a limited number of carrier absorption is quantitatively low, despite being rela- molecules or pores available at any given instant. In the tively more efficient. A moment’s reflection suffices to brief interval between exit of a bolus of food from the show that a large fraction of a small number is, of stomach and the time it reaches the colon, only so many necessity, a smaller number still. Thus, absorbing even calcium ions can use the available transport. If the num- a large fraction of a small intake cannot produce much ber of ions reaching the absorptive site is small, then calcium. The result is that, in the range of intakes com- by numerical necessity the fraction absorbed will be monly encountered among contemporary, industrial- larger than when the number of ions is large. ized humans, absorptive adaptation (via vitamin D) Absorption fraction (or efficiency) is thus a poten- mitigates the problem created by a low intake, but it tially misleading measure (at least if we stop there). does not counterbalance it. A concrete example, employing realistic numbers, will help illustrate this point, and will show additionally how optimal opera- tion of the vitamin D hormonal system is dependent 4As Fig. 6 shows, most of the difference occurs at intakes below upon—and in fact presumes—the kinds of high cal- 500 mg (12.5 mmol). However, the two sets of observations were cium intakes found among hunter-gatherer humans performed in different groups of women and even the non-adapted and high primates (in whom the system evolved). set must have had some basal level of 1,25(OH)2D-mediated adap- tive absorption. Hence the true extent of adaptive absorption is Contrast how two individuals are able to respond to undoubtedly somewhat greater than indicated solely by the differ- the increased obligatory loss occasioned by regular ence between the two lines. daily ingestion of an additional 100 mmol sodium CHAPTER 46 Vitamin D: Role in the Calcium Economy 781

(approximately the sodium contained in a single fast- calcium intakes are already low. That does not mean food chicken dinner). Assume that one individual is that ECF [Ca2+] regulation suffers. The bony calcium ingesting 5 mmol Ca (200 mg)/day (corresponding to the reserves are vast—essentially limitless. So long as lower quintile of calcium intakes in U.S. women [38]), vitamin D status is above rachitic levels, those reserves and the other, 40 mmol (1600 mg) (approximately the will readily be drawn upon to support ECF [Ca2+], NIH Consensus Conference recommendation [39] for using the well-studied mechanisms already described. estrogen-deprived, postmenopausal women). Using Naturally, if this drain continues, bone mass will data from the curve in Fig. 4, the individual with the inevitably decline. At high calcium intakes, such as lower intake absorbs at an efficiency of 44.5% prior to prevailed during hominid evolution, the vitamin D hor- the extra sodium load, and the individual with the monal system not only helps maintain ECF [Ca2+], but higher intake, at 17.8%. (The first, therefore, has a gross total body calcium as well; at low intakes, only the absorbed quantity from the diet of 2.2 mmol/day, and ECF is protected. the second, 7.1 mmol/day.) The increase in obligatory urinary loss occasioned by the increase in sodium intake will be about 1 mmol/day (see earlier discus- C. Partition of Absorption between Active sion). To offset this loss, the first individual, with the and Passive Mechanisms low intake, would have to increase the absorbed quan- tity to 3.2 mmol/day, which means increasing the As described in Chapter 24, absorption occurs already high absorption efficiency by a factor of nearly both by vitamin DÐmediated active transport across 1.5 (from 44.5 to 64.5%). By contrast, the individual the mucosal cells and by passive diffusion around the with the high intake needs to increase only from 17.8 to cells. Is it possible to partition absorption between the 20.3%. (These calculations are summarized in Fig. 8.) active, cellular process and the passive, paracellular The adjustment for the individual with a low calcium process? To a limited extent, the answer is yes. As intake is substantially more than most adults can should be clear from the foregoing, absorption fraction accomplish, while the second is easily accommodated. (both passive and active) exhibits an inverse relation to The first individual is forced, therefore, to get the needed intake or load. Absorption will be high by either mech- additional calcium from bone, while the second easily anism, even approaching 100%, if the load is suffi- gets it from her food. ciently small. But small loads are not nutritionally Thus, while vitamin D plays a critical role in relevant, no matter how well they are (or are not) increasing absorptive efficiency in response either to absorbed; so in this discussion I shall confine myself to increased losses from the body or to decreased intake, consideration of loads or intakes in the range of (or it must be stressed that there is little room in which the above) currently recommended intakes. PTHÐvitamin D endocrine system can operate when The right end of the regression line in Fig. 4 repre- sents an absorption fraction of approximately 0.15. In work published earlier from our laboratory [40], we extended intakes well above the 2 g (50 mmol) upper 0.8 limit of Fig. 4, to as high as 8.0 g (200 mmol) Ca/day. Added demand: 1 mmol/d 0.7 Absorption fraction in that study also averaged about 0.15 at these very high intakes, or approximately what 0.6 + 0.20 we observed at 50 mmol in Fig. 4. The essentially lin- 0.5 ear character of this absorption across such a broad 0.4 range of intakes probably reflects the fact that, if pas- sive absorption is due largely to solvent drag, the quan- 0.3 tity of calcium absorbed will be a linear function of + 0.025 Absorption fraction 0.2 luminal calcium concentration. 0.1 It is likely, at habitual intakes in the range of 40Ð50 mmol Ca/day, that active absorption would be 0.0 051015 20 25 30 35 40 45 50 minimal, and it is a virtual certainty that that would be Calcium intake (mmol/d) so at supraphysiological intakes of 200 mmol. Studies in patients with end-stage renal disease, with limited FIGURE 8 Changes in absorption efficiency required to offset ability to synthesize 1,25(OH) D, also report absorp- fully from diet an additional drain of 1 mmol Ca/d, at low and high 2 regions of the curve of Fig. 4. Numbers shown are the required tion fractions in the range of 10Ð20% [41,42]. Taken increases in absorption fraction at the respective intakes. together, these data indicate that passive absorption is able (Copyright Robert P. Heaney, 2003. Reproduced with permission.) to extract about 10Ð15% of the calcium in the ingested 782 ROBERT P. H EANEY food at nutritionally relevant loads. Variation around that to offset such losses at intakes in the range of current level will presumably relate to inter-individual variations recommendations (25Ð30 mmol/d). in mucosal mass and in intestinal transit time. What would an absorption efficiency of 15% mean for the calcium economy if passive absorption were the IV. PHYSIOLOGICAL SOURCES only means for extracting calcium from the diet, as, for OF VITAMIN D ACTIVITY example, must be the case with hereditary vitamin D resistant rickets (HVDRR) Type II? Assume an intake Thus far I have spoken of vitamin D only generically. of 20 mmol/day. As noted earlier, digestive secretions As discussed extensively elsewhere in this volume, add 3.5 mmol to the stream, 0.6 mmol of that total too native vitamin D (calciferol) has very little biological low to be absorbed. Calculation from these data easily activity in its own right. It is converted in the body into shows that, at a 15% absorption efficiency, the gut will a number of hydroxylated metabolites, the most be a net excretory organ (if only barely: approximately important of which for the purposes of this chapter are 0.1 mmol/day net loss). No calcium gain into the body 25OHD3 and 1,25(OH)2D3. It is well established that could occur at such an intake. Hence a background, cholecalciferol is 25-hydroxylated in the liver. The gross absorption of 15% without vitamin D seems reaction is loosely controlled by circulating levels of entirely compatible with development of severe rick- 1,25(OH)2D as well as by limited end-product inhibi- ets, either as found in HVDRR or in typical nutritional tion exerted by 25OHD itself. However, for the most vitamin D deficiency. part circulating 25OHD levels are driven mainly by the Figure 9 systematizes those calculations for varying circulating levels of the precursor, either ergo- or levels of active absorption and a broad range of calcium cholecalciferol. In studies performed in my laboratory, intakes, expressing the results in terms of net calcium serum 25OHD3 rises by approximately 0.7 nmol/L for absorption. It shows graphically, for example, that every µg of oral cholecalciferol after 16 to 20 weeks of without active transport it takes an intake of about daily oral administration [43]. This relationship is 26 mmol to ensure zero gut balance, and that an intake of consistent with published data from studies of high- 60 mmol would not suffice to offset extraintestinal losses dose vitamin D treatment and cases of vitamin D of 5 mmol through the kidney and skin. By contrast, intoxication [44,45]. active absorption of ~16% is sufficient in mature adults 1,25(OH)2D3 is produced in the kidney, as already described (Chapter 5), under control by serum PTH and serum inorganic phosphate concentrations. It is generally considered to be the biologically active form 35 of the vitamin, responsible for its full spectrum of 48 actions; it is also considered that the precursors (chole- 30 calciferol and 25OHD3) exert activity in their own 40 25 right only under conditions of intoxication. This con- 32 clusion is based mainly upon studies of both binding 20 Needed to offset 24 kinetics of the various metabolites with the vitamin D 15 5 mmol/d receptor and of consequent gene expression. If binding obligatory 16 affinity for 1,25(OH) D is taken as 1.0, reported values 10 loss 2 3 Ð3 Ð4 8 for 25OHD3 are in the range of 1×10 to 1×10 , and 5 Active absorption (percent) for native D, 1×10Ð6 or lower [46Ð48]. Accordingly, it

Net absorption (mmol/d) 0 0 is reasoned that normal serum levels of the precursor compounds are too low to exert significant action. −5 Zero balance across the gut However, since serum levels of 25OHD are typi- 0102030405060 Calcium intake (mmol/d) cally three orders of magnitude higher than those of 1,25(OH)2D, it is not clear, a priori, that 25OHD would FIGURE 9 Relationship of vitamin DÐmediated, active calcium be without effect under normal conditions. Protein absorption, calcium intake, and net calcium gain across the gut. Each of the contours represents a different level of active absorption binding in serum complicates interpretation of these above a baseline passive absorption of 12.5%. (The values along relationships. It is generally presumed that total serum each contour represent the sum total of passive and variable active levels are physiologically less meaningful than free absorption.) The horizontal dashed lines indicate 0 and 5 mmol/d serum levels of a metabolite. As discussed in Chapter 8, net absorption, respectively. The former is the value at which the the vitamin D family of compounds is carried in serum gut switches from a net excretory to a net absorptive mode, and the latter is the value needed to offset typical urinary and cutaneous losses complexed to a D-binding carrier protein [an alpha in mature adults. (Copyright Robert P. Heaney, 1999. Reproduced globulin designated DBP, with a gradient of affinities: with permission.) highest for 25OHD, lower for native vitamin D3, and CHAPTER 46 Vitamin D: Role in the Calcium Economy 783

lower still for 1,25(OH)2D]. However, binding to the produces a strong enhancement of absorption effi- carrier protein cannot be interpreted without reference ciency when given to intact humans [52Ð56] with non- to other factors, usually not readily determined in any deficient levels of serum 25OHD. Transcaltachia, given situation. These include the relative affinities of which is a rapid-response, nongenomic action of the D the binding protein and the receptor as well as the con- vitamin metabolites, requires occupancy of the nuclear centrations of both. Available data on binding at the receptor by 1,25(OH)2D [58]. This is shown, for exam- intact organism level are not sufficient to resolve this ple, in patients with HVDRR type II, who lack func- issue of hormone dynamics. But such theoretical con- tional vitamin D receptors, and who are not able to siderations aside, there is a substantial body of clinical absorb calcium efficiently despite normal to high cir- data indicating that 25OHD exerts appreciable biolog- culating levels of both 1,25(OH)2D and 25OHD ical activity at normal physiological concentrations. [59,60]. Thus vitamin DÐmediated absorption seems to Several studies show a surprisingly strong correla- require both a functioning receptor and some combi- tion between serum 25OHD and intestinal calcium nation of 1,25(OH)2D and 25OHD. Whatever the pre- absorption efficiency in intact humans [34,49Ð53]. If cise mechanism, the system operates as if there were a 25OHD were acting only as a precursor, one should floor or background level of absorption in normal indi- have expected no correlation at all. Where 1,25(OH)2D viduals that is determined in part by the long-half-life levels were measured in these studies, the correlation 25OHD, while 1,25(OH)2D produces a quick-acting of 1,25(OH)2D with absorption fraction was usually fine tuning of the system. weaker than for 25OHD, or nonsignificant altogether. Clinically, it is widely recognized that 1,25(OH)2D levels are commonly normal in sporadic nutritional V. OPTIMAL VITAMIN D STATUS rickets, while 25OHD levels are invariably low, and, more to the point, calcium absorption is low. Moreover, Optimal vitamin D status can be defined as the daily Colodro et al. [52], in a dose response study for both intake or production of the vitamin (and/or the serum metabolites, using calcium absorption in healthy human level of 25OHD) that is sufficient so that its availability adults as the endpoint, reported a molar potency for does not limit any of the metabolic functions depen- administered 25OHD relative to 1,25(OH)2D of 1:125, dent upon the vitamin. This notion of limit is illus- not the less than 1:2,000Ð1:4,000 figure predicted from trated in Fig. 10A. A large body of data indicates that in vitro receptor binding studies. Barger-Lux et al. [54], vitamin D-mediated absorption follows a curve such as in a similar study, found a nearly identical potency the one presented in Fig. 10A, rising with intake at lev- ratio (1:100). In this study the rise in absorption frac- els below the requirement, then flat through a range of tion produced by oral 25OHD occurred without a sufficiency, then rising again at pharmacologic (or detectable change in 1,25(OH)2D level. Taken together toxic) intakes. Figure 10B shows this behavior as these studies provide evidence that some fraction of related to actual serum 25OHD concentration, derived circulating vitamin DÐlike activity can be attributed to from two studies [61,62]. It is likely that the absorptive 25OHD.5 response, taken in isolation, would be a continuous The basis for the interaction of the two vitamin D smooth function of vitamin D dose, such as that indi- metabolites in intestinal calcium absorption is unclear. It cated by the dashed line in Fig. 10A. However, at the may be that mucosal cell expression of the 1α-hydroxy- whole-organism level, once calcium absorption is opti- lase converts circulating 25OHD to 1,25(OH)2D within mal, other factors alter the response to vitamin D expo- the cell, thereby augmenting the 1,25(OH)2D levels the sure. For example, at levels of calcium absorption receptor actually sees. Alternatively, the two metabo- higher than needed to offset daily losses, PTH levels lites may act synergistically—1,25(OH)2D in the would drop and 1,25(OH)2D synthesis would fall. canonical genomic manner, and 25OHD binding to Thus, despite a rising solar or oral dose of the precur- membrane receptors and opening calcium channels, a sor vitamin D, absorption plateaus. But when the dose process termed “transcaltachia” [57,58] (see Chapter 23). becomes sufficiently high (as in vitamin D intoxica- In any event, there can be no question about the potency tion), system controls are saturated and bypassed, and 6 of 1,25(OH)2D itself. This very active metabolite absorption begins to rise once again. Intakes below the plateau are clearly insufficient, since they limit

5Some of the apparent action of 25OHD in oral dosing studies may be due to a direct effect of the metabolite on the mucosal cell (a first-pass effect). During absorption of 25OHD, mucosal expo- 6This second rise in absorption has never been reported for sure to the agent would be substantially higher than would occur purely solar sources of vitamin D; however, it is well recognized as from exposure to plasma 25OHD. a component of vitamin D intoxication. 784 ROBERT P. H EANEY

A B 0.50 0.45 0.40 0.35 0.30 0.25 Absorption fraction Absorption fraction 0.20 0.00 0406080100 120 140 160 Vitamin D input Serum 25(OH)D (nmol/L)

FIGURE 10 (A) Schematic diagram of the likely relationship between absorptive performance and vitamin D input. The dashed line represents the curve in the absence of physiological controls (such as might be found in isolated gut loop studies), while the solid line represents the likely relationship in organisms with function- ing control systems. (B) Paired sets of measured absorption values in groups studied at differing values for serum 25OHD. The lines connecting the points denote the pairs of values within each study [61,63]. (Copyright Robert P. Heaney, 1996, 2003. Reproduced with permission.)

absorption anterior to any physiological controls. considered normal [~37.5 nmol/liter (15 ng/ml)]. Intake levels beyond the plateau, by contrast, represent (At the same time it is reassuring to note that a value toxicity, i.e., the overriding of physiological controls. of 80 nmol/liter is itself well below the mean for indi- (The subject of vitamin D toxicity is discussed in viduals with levels of sun exposure such as must have Chapter 78.) prevailed during hominid evolution [75]). Clinical con- Optimal status could thus be defined as an intake or firmation of the approximate correctness of this level is production of the vitamin sufficient to get an individ- found in a recent, large, randomized, controlled trial ual onto the absorptive plateau. Optimal position on in which raising serum 25OHD from 53 nmol/liter to the plateau would depend upon population-level rela- 76 nmol/liter resulted in a 33% decrease in typical tive risks of toxicity and deficiency. Defining such a osteoporotic fractures over 5 years of treatment [62]. level can be approached operationally in several ways. Note that the untreated level (53 nmol/liter) was itself One is by determining the level of vitamin D intake at well within the usual reference range and might there- which calcium absorption does not change further fore have been considered a “normal” value. The fact upon giving extra vitamin D at doses within the phys- that it permitted excess osteoporotic fractures shows iological range [61,63]. Since both PTH levels and clearly that the bottom end of the reference range can PTH-mediated bone resorption are known to rise in the no longer be used to determine normality. face of vitamin D insufficiency, a complementary Since vitamin D is not found in appreciable concen- approach would be to define the 25OHD level at which trations in most of the items in the food supply (either the parathyroid response evoked by inadequate primitive or modern), maintenance of optimal vitamin absorbed calcium intake is minimized [64,65]. D status requires either sun exposure or, in high north- Using the absorptive response to supplemental D ern or southern latitudes, some degree of supplementa- [49,61,63,66], such indices of vitamin D status as sea- tion/fortification. Such a conclusion has often been sonal variation in serum iPTH and bone remodeling uncongenial to traditional nutritionists, who have [67Ð72], or the point at which PTH concentration is min- maintained that humans ought to be able to get all of imized [64,65], available evidence converges on a serum the nutrients they need from a well-balanced diet. value of approximately 80 nmol/liter (32 ng/ml).7 This However, it must be stressed that vitamin D is an acci- value is well above the bottom end of the range currently dental nutrient, included with the other vitamins by mis- take at an early stage of the development of nutritional science. Whatever the merit of the traditional nutri- tionists’ position, it cannot apply to this essential com- 7The principal exception to this otherwise highly consistent body of data is found mainly in studies from the Netherlands, in pound. Now that we understand the situation, we must which PTH is reported to be minimized below 50 nmol/L [73,74]. see that it is certainly no more unnatural to sustain The reason for this discrepancy is unclear. vitamin D status in high latitudes by supplementation CHAPTER 46 Vitamin D: Role in the Calcium Economy 785 than it is to sustain body temperature there by clothing serum levels. Optimal vitamin D status is operationally or shelter. (See also Chapter 61.) defined as a level of D intake (or production) high enough Although more work needs to be done to define the to ensure that the D-mediated transfers are not limited by bottom end of the acceptable normal range with preci- D availability. Available data point to a value for serum sion, for now the prudent course would seem to be to 25OHD of about 32 ng/ml (80 nmol/liter) as the bottom aim for a vitamin D intake sufficient to produce a serum end of the optimal range. Confirmation of this estimate 25OHD level of at least 80 nmol/liter (32 ng/ml). As comes from the demonstration that calcium absorp- just noted, levels below that point result in calcium tion efficiency is suboptimal below ~80 nmol/liter and absorptive impairment [63] and carry a risk of bone that risk of fragility fractures rises as serum 25OHD loss and osteoporotic fractures [62]. The 80 nmol/liter concentration falls below 80 nmol/liter. level is certainly safe, since healthy college-age adults (with often generous sun exposure) commonly have levels twice that high. Oral cholecalciferol intakes References required to produce a level of 80 nmol/liter will depend upon degree of cutaneous synthesis and pretreatment 1. Heaney RP 1963 Evaluation and interpretation of calcium serum 25OHD concentration. As noted earlier, the kinetic data in man. Clin Orthop 31:153Ð183. 2. Clapham DE 1995 Calcium signaling. Cell 80:259Ð268. most direct evidence for dose response in midlife 3. Eastell R, Delmas PD, Hodgson SF, Eriksen EF, Mann KG, individuals points to a rise of 0.7 nmol/liter for each Riggs BL 1988 Bone formation rate in older normal women: microgram (40 IU) of daily oral cholecalciferol sup- concurrent assessment with bone histomorphometry, calcium plementation [43]. Less direct evidence mostly kinetics, and biochemical markers. J Clin Endocrinol Metab obtained in the elderly indicates that the rate of 67:741Ð748. 4. Chapuy MC, Schott AM, Garnero P, Hans D, Delmas PD, response from conditions of more extreme vitamin D Meunier PJ, and EPIDOS study group. 1996 Healthy elderly depletion could be as much as 1.6Ð2 nmol/liter per µg French women living at home have secondary hyperparathy- of continuous daily oral supplementation. Whatever roidism and high bone turnover in winter. J Clin Endocrinol the actual response rate, it is unlikely to be higher than Metab 81:1129Ð1133. 2 or much lower than 0.6 mmol/liter/µg/d. Hence a 5. Charles P 1989 Metabolic bone disease evaluated by a com- bined calcium balance and tracer kinetic study. Dan Med Bull patient with a baseline value of 40 nmol/liter can be 36:463Ð479. estimated to require a daily dose ranging from 800 to 6. Rianon N, Feeback D, Wood R, Driscoll T, Shackelford L, 2600 IU to achieve the desired level. It is worth noting LeBlanc A 2003 Monitoring sweat calcium using skin patches. that the entirety of this range of required intakes is Calcif Tissue Int 72:694Ð697. above the currently recommended intake. 7. Klesges RC, Ward KD, Shelton ML, Applegate WB, Cantler ED, Palmieri GMA, Harmon K, Davis J 1996 Changes in bone mineral content in male athletes. Mechanisms of action and intervention effects. JAMA 276:226Ð230. VI. SUMMARY AND CONCLUSIONS 8. Heaney RP, Recker RR 1994 Determinants of endogenous fecal calcium in healthy women. J Bone Miner Res 9:1621Ð1627. The best attested function of vitamin D is the facilita- 9. Nordin BEC, Need AG, Morris HA, Horowitz M 1993 The nature and significance of the relationship between urinary tion of transfer of calcium (and phosphorus) into the sodium and urinary calcium in women. J Nutr 123:1615Ð1622. extracellular fluid from ingested food and from bone. In 10. Itoh R, Suyama Y 1996 Sodium excretion in relation to this capacity, vitamin D functions as a part of a control calcium and hydroxyproline excretion in a healthy Japanese system that operates to maintain constancy of the calcium population. Am J Clin Nutr 63:735Ð740. ion concentrations in the extracellular fluid against the 11. Devine A, Criddle RA, Dick IM, Kerr DA, Prince RL 1995 A longitudinal study of the effect of sodium and calcium intakes demands of obligatory excretory losses and skeletal min- on regional bone density in postmenopausal women. Am J Clin eralization. In both transfers vitamin D works in concert Nutr 62:740Ð745. with parathyroid hormone. Quantitative analysis of the 12. Heaney RP, Recker RR 1982 Effects of nitrogen, phosphorus, inputs and drains of the calcium economy reveals that, at and caffeine on calcium balance in women. J Lab Clin Med contemporary calcium intakes, D-mediated absorptive 99:46Ð55. 13. Johnson NE, Alcantara EN, Linkswiler H 1970 Effect of level enhancement only partially mitigates the impact of low of protein intake on urinary and fecal calcium and calcium calcium intake or large calcium losses. However, at retention of young adult males. J Nutr 100:1425Ð1430. intakes closer to those prevailing during hominid evolu- 14. Sebastian A, Frassetto LA, Sellmeyer DE, Merriam RL, tion, minor shifts in vitamin D-mediated absorption Morris RC Jr 2002 Estimation of the net acid load of the diet are fully adequate to compensate for stresses on the cal- of ancestral preagricultural Homo sapiens and their hominid ancestors. Am J Clin Nutr 76:1308Ð1316. cium economy. While 1,25(OH)2D is clearly the most 15. Parfitt AM, Higgins BA, Nassim JR, Collins JA, Hilb A 1964 potent form of the vitamin, 25OHD exerts significant Metabolic studies in patients with hypercalciuria. Clinical vitamin DÐlike activity in its own right at physiological Science 27:463Ð482. 786 ROBERT P. H EANEY

16. New SA, Robins SP, Campbell MK, Martin JC, Garton MJ, the United States: Third National Health and Nutrition Bolton-Smith C, Grubb DA, Lee SJ, Reid DM 2000 Dietary Examination Survey, Phase 1, 1988Ð91. Advance data from influences on bone mass and bone metabolism: further vital and health statistics; no. 258. National Center for Health evidence of a positive link between fruit and vegetable con- Statistics, Hyattsville, MD. sumption and bone health. Am J Clin Nutr 71:142Ð151. 39. NIH Consensus Conference: Optimal Calcium Intake 1994 17. Walser M 1961 Calcium clearance as a function of sodium JAMA 272:1942Ð1948. clearance in the dog. Am J Physiol 200:769Ð773. 40. Heaney RP, Saville PD, Recker RR 1975 Calcium absorption 18. Heaney RP 2003 How does bone support calcium homeosta- as a function of calcium intake. J Lab Clin Med 85:881Ð890. sis? Bone 33:264Ð268. 41. Recker RR, Saville PD 1971 Calcium absorption in renal failure: 19. Raisz LG 1965 Bone resorption in tissue culture. Factors influ- its relationship to blood urea nitrogen, dietary calcium intake, encing the response of parathyroid hormone. J Clin Invest time on dialysis, and other variables. J Lab Clin Med 78:380Ð388. 44:103Ð116. 42. Coburn JW, Koppel MH, Brickman AS, Massry SG 1973 20. Portale AA, Halloran BP, Morris RC Jr 1987 Dietary intake of Study of intestinal absorption of calcium in patients with renal phosphorus modulates the circardian rhythm in serum concen- failure. Kidney International 3:264Ð272. tration of phosphorus. J Clin Invest 80:1147Ð1154. 43. Heaney RP, Davies KM, Chen TC, Holick MF, Barger-Lux MJ 21. Feldman D, Malloy PJ, Gross C: Vitamin D 1996 Metabolism 2003 Human serum 25-hydroxy-cholecalciferol response to and action. In: Marcus R, Feldman D, Kelsey J (eds) extended oral dosing with cholecalciferol. Am J Clin Nutr Osteoporosis. Academic Press, San Diego, California. pp. 77:204Ð210. 205Ð225. 44. Whyte MP, Haddad JG Jr, Walters DD, Stamp TCB 1979 22. Banks WJ Jr, Epling GP, Kainer RA, Davis RW 1968 Antler Vitamin D bioavailability: serum 25-hydroxyvitamin D levels in growth and osteoporosis. Anat Rec 162:387Ð398. man after oral, subcutaneous, intramuscular, and intravenous 23. Abrams SA 2002 Nutritional rickets: An old disease returns. vitamin D administration. J Clin Endocrinol Metab 48:906Ð911. Nutr Rev 60(4):111Ð115. 45. Byrne PM, Freaney R, McKenna MJ 1995 Vitamin D supple- 24. Eaton SB, Nelson DA 1991 Calcium in evolutionary perspec- mentation in the elderly: review of safety and effectiveness of tive. Am J Clin Nutr 54:281SÐ287S. different regimes. Calcif Tissue Int 56:518Ð520. 25. Heaney RP 1965 A unified concept of osteoporosis. Am J Med 46. Hughes MR, Baylink DJ, Jones PG, Haussler MR 1976 39:877Ð880. Radioligand receptor assay for 25-hydroxyvitamin D2/D3 and 26. Bell NH, Greene A, Epstein S, Oexmann MJ, Shaw S, Shary J 1-α,25-dihydroxyvitamin D2/D3. J Clin Invest 58:61Ð70. 1985 Evidence for alteration of the vitamin D-endocrine 47. Brumbaugh PF, Haussler MR 1973 1,25-dihydroxyvitamin D3 system in blacks. J Clin Invest 76:470Ð473. receptor: Competitive binding of vitamin D analogs. Life Sci 27. Bell NH, Stern PH, Paulson SK 1985 Tight regulation of cir- 13:1737Ð1746. culating 1-α,25-dihydroxyvitamin D in black children. N Engl 48. DeLuca HF 1983 The vitamin DÐcalcium axis. In: Rubin RP, J Med 313:1418. Weiss GB, Putney Jr JW (eds) Calcium in Biological Systems. 28. Weinstein RS, Bell NH 1988 Diminished rates of bone forma- Plenum Press, New York, pp. 491Ð511. tion in normal black adults. N Engl J Med 319:1698Ð1701. 49. Francis RM, Peacock M, Storer JH, Davies AEJ, Brown WB, 29. Cosman F, Morgan DC, Nieves JW, Shen V, Luckey MM, Nordin BEC 1983 Calcium malabsorption in the elderly: the Dempster DW, Lindsay R, Parisien M 1997 Resistance to bone effect of treatment with oral 25-hydroxyvitamin D3. Eur J Clin resorbing effects of PTH in black women. J Bone Miner Res Invest 3:391Ð396. 12(6):958Ð966. 50. Bell NH, Epstein S, Shary J, Greene V, Oexmann MJ, Shaw S 30. Aloia JF, Vaswani A, Yeh JK, Flaster E 1996 Risk for osteo- 1988 Evidence of probable role for 25-hydroxyvitamin D in porosis in black women. Calcif Tissue Int 59:415Ð423. the regulation of human calcium metabolism. J Bone Miner 31. Abrams SA, O’Brien KO, Liang LK, Stuff JE 1995 Res 3:489Ð495. Differences in calcium absorption and kinetics between black 51. Reasner CA II, Dunn JF, Fetchick D, Liel Y, Hollis BW, and white girls aged 5Ð16 years. J Bone Miner Res 10:829Ð833. Epstein S, Shary J, Mundy GR, Bell NH 1990 Alteration of 32. Heaney RP 2002 Ethnicity, bone status, and the calcium vitamin D metabolism in Mexican-Americans [Letter to the requirement. Nutr Res 22:(1Ð2):153Ð178. editor]. J Bone Miner Res 5:13Ð17. 33. Dawson-Hughes B, Harris S, Kramich C, Dallal G, Rasmussen 52. Colodro IH, Brickman AS, Coburn JW, Osborn TW, Norman HM 1993 Calcium retention and hormone levels in black and AW 1978 Effect of 25-hydroxyvitamin D3 on intestinal white women on high- and low-calcium diets. J Bone Miner absorption of calcium in normal man and patients with renal Res 8:779Ð787. failure. Metabolism 27:745Ð753. 34. Barger-Lux MJ, Heaney RP, Lanspa SJ, Healy JC, DeLuca HF 53. Devine A, Wilson SG, Dick IM, Prince RL 2002 Effects of 1995 An investigation of sources of variation in calcium vitamin D metabolites on intestinal calcium absorption and absorption efficiency. J Clin Endocrinol Metab 80:406Ð411. bone turnover in elderly women. Am J Clin Nutr 75:283Ð288. 35. BargerÐLux MJ, Heaney RP, Recker RR 1989 Time course of 54. Barger-Lux MJ, Heaney RP, Dowell S, Bierman J 1996 calcium absorption in humans: Evidence for a colonic compo- Relative molar potency of 25-hydroxyvitamin D indicates a nent. Calcif Tissue Int 44:308Ð311. major role in calcium absorption. J Bone Miner Res 11:S423. 36. Heaney RP, Recker RR, Stegman MR, Moy AJ 1989 Calcium 55. Gallagher JC, Jerpbak CM, Jee WSS, Johnson KA, DeLuca absorption in women: relationships to calcium intake, estrogen HF, Riggs BL 1982 1,25-Dihydroxyvitamin D3: short- and status, and age. J Bone Miner Res 4:469Ð475. long-term effects on bone and calcium metabolism in patients 37. Heaney RP, Weaver CM, Fitzsimmons ML 1990 The influence with postmenopausal osteoporosis. Proc Natl Acad Sci of calcium load on absorption fraction. J Bone Miner Res 79:3325Ð3329. 11:1135Ð1138. 56. Dawson-Hughes B, Harris SS, Finneran S, Rasmussen HM 38. Alaimo K, McDowell MA, Briefel RR, Bischof AM, 1995 Calcium absorption responses to calcitriol in black and Caughman CR, Loria CM, Johnson CL 1994 Dietary intake of white premenopausal women. J Clin Endocrinol Metab vitamins, minerals, and fiber of persons 2 months and over in 80:3068Ð3072. CHAPTER 46 Vitamin D: Role in the Calcium Economy 787

57. Norman AW 1990 Intestinal calcium absorption: a vitamin D- 66. Krall EA, DawsonÐHughes B 1991 Relation of fractional 47Ca hormone-mediated adaptive response. Am J Clin Nutr 51: retention to season and rates of bone loss in healthy post- 290Ð300. menopausal women. J Bone Miner Res 6:1323Ð1329. 58. Norman AW, Nemere I, Zhou L-X, Bishop JE, Lowe KE, 67. McKenna JM, Freaney R, Meade A, Muldowney FP 1985 Maiyar AC, Collins ED, Taoka T, Sergeev I, Farach-Carson Hypovitaminosis D and elevated serum alkaline phosphatase MC 1992 1,25(OH)2-vitamin D3, a steroid hormone that pro- in elderly Irish people. Am J Clin Nutr 41:101Ð109. duces biologic effects via both genomic and nongenomic path- 68. Rosen CJ, Morrison A, Zhou H, Storm D, Hunter SJ, ways. Steroid Biochem Molec Biol 41:231Ð240. Musgrave K, Chen T, Wei W, Holick MF 1994 Elderly women 59. al-Aqeel A, Ozand P, Sobki S, Sewairi W, Marx S 1993 The in northern New England exhibit seasonal changes in bone min- combined use of intravenous and oral calcium for the treatment eral density and calciotropic hormones. Bone Miner 25:83Ð92. of vitamin D dependent rickets type II (VDDRII). Clin 69. Dawson-Hughes B, Harris SS, Dallal GE 1997 Plasma calcid- Endocrinol Oxf 39:229Ð237. iol, season, and serum parathyroid hormone concentrations in 60. Simonin G, Chabrol B, Moulene E, Bollini G, Strouc S, healthy elderly men and women. Am J Clin Nutr 65:67Ð71. Mattei JF, Giraud F 1992 Vitamin D-resistant rickets type II: 70. Salamone LM, Dallal GE, Zantos D, Makrauer F, Dawson- apropos of 2 cases. Pediatrie-Bucur 47:817Ð820. Hughes B 1993 Contributions of vitamin D intake and seasonal 61. Barger-Lux MJ, Heaney RP: Effects of above average sunlight exposure to plasma 25-hydroxyvitamin D concentra- summer sun exposure on serum 25-hydroxyvitamin D tion in elderly women. Am J Clin Nutr 58:80Ð86. and calcium absorption. J Clin Endocrinol Metab 87(11): 71. Dawson-Hughes B, Dallal GE, Krall EA, Harris S, Sokoll LJ, 4952Ð4956. Falconer G 1991 Effect of vitamin D supplementation on 62. Trivedi DP, Doll R, Khaw KT 2003 Effect of four monthly wintertime and overall bone loss in healthy postmenopausal oral vitamin D3 (cholecalciferol) supplementation on fractures women. Ann Intern Med 115:505Ð512. and mortality in men and women living in the community: 72. Krall EA, Sahyoun N, Tannenbaum S, Dallal GE, randomised double blind controlled trial. Br Med J 326: Dawson-Hughes B 1989 Effect of vitamin D intake on seasonal 469Ð474. variations in parathyroid hormone secretion in postmenopausal 63. Heaney RP, Dowell MS, Hale CA, Bendich A 2003 Calcium women. N Engl J Med 321:1777Ð1783. absorption varies within the reference range for serum 73. Lips P 2001 Vitamin D deficiency and secondary hyper- 25-hydroxyvitamin D. J Am Coll Nutr 22(2):142Ð146. parathyroidism in the elderly: consequences for bone loss and 64. Chapuy M-C, Preziosi P, Maamer M, Arnaud S, Galan P, fractures and therapeutic implications. Endocr Rev 22:477Ð501. Hercberg S, Meunier PJ 1997 Prevalence of vitamin D 74. Lips P, Duong T, Oleksik A, Black D, Cummings S, Cox D, insufficiency in an adult normal population. Osteoporos Int Nickelsen T 2001 A global study of vitamin D status and 7:439Ð443. parathyroid function in postmenopausal women with osteoporo- 65. Thomas MK, Lloyd-Jones DM, Thadhani RI, Shaw AC, sis: baseline data from the multiple outcomes of raloxifene eval- Deraska DJ, Kitch BT, Vamvakas EC, Dick IM, Prince RL, uation clinical trial. J Clin Endocrinol Metab 86:1212Ð1221. Finkelstein JS 1998 Hypovitaminosis D in medical inpatients. 75. Matsuoka LY, Wortsman J, Hollis BW 1990 Suntanning and N Engl J Med 338:777Ð783. cutaneous synthesis of vitamin D3. J Lab Clin Med 116:87Ð90. CHAPTER 47 Effects of Race, Geography, Body Habitus, Diet, and Exercise on Vitamin D Metabolism

MEHRDAD RAHMANIYAN AND NORMAN H. BELL Department of Medicine, Medical University of South Carolina, Charleston, South Carolina

I. Introduction V. Effects of Exercise II. Effects of Race and Geography VI. Summary III. Effects of Diet References IV. Effects of Body Habitus

I. INTRODUCTION 24,25-dihydroxyvitamin D [24,25(OH)2D] by the mito- chondrial enzyme 25OHD-24-hydroxylase (24-hydroxy- Vitamin D metabolism is influenced by a number of lase) (CYP24A1), which is present in the kidney and factors. These include race, geographic location, diet, other organs [15Ð17], and less 1,25(OH)2D is produced. body habitus, and exercise. To understand how these Conversely, in states of vitamin D deficiency, less factors exert their influence, it is useful to briefly review 24,25(OH)2D and more 1,25(OH)2D is produced. The the metabolism of vitamin D and vitamin DÐendocrine two metabolites undergo additional hydroxylation to system. These subjects are discussed in greater detail in form 1,24,25-trihydroxyvitamin D, before being con- Chapter 2 of this book. A list of populations at risk for verted to calcitroic acid. This degradative pathway is developing rickets and osteomalacia and factors similar to the classic and alternative pathways that are involved is shown in Table I. involved in the transformation of cholesterol to bile Vitamin D metabolism can be summarized as acids. CYP24A1 is induced by 1,25(OH)2D by two vita- follows (see Chapter 3 for details). In skin, vitamin D3 is min D receptor (VDR) response elements in the gene synthesized from dermally produced 7-dehydrocholes- promoter. Thus, 1,25(OH)2D regulates not only its own terol. Previtamin D3 is converted from 7-dehydro- rate of degradation but that of 25OHD as well [18]. The cholesterol by absorption of one photon of ultraviolet regulation of degradation of 25OHD by 1,25(OH)2D sunlight, and the further conversion of previtamin D3 to is underscored by studies in rats which showed that vitamin D3 is regulated by body heat, a process that 1,25(OH)2D increases the metabolic clearance rate of takes place over a period of several days and is temper- 25OHD [19] and by studies in human subjects which ature dependent [1,2]. The vitamin is carried from cap- showed that the increase in serum 25OHD produced by illaries in skin by vitamin DÐbinding protein to the liver, pharmacologic doses of vitamin D was prevented by where it is converted to 25-hydroxyvitamin D (25OHD) the simultaneous administration of 1,25(OH)2D3 [20]. by vitamin D-25-hydroxylase (CYP2R1), the newly Calcium metabolism is modulated by a negative discovered hepatic microsomal enzyme [3,4]. 25OHD feedback control system that includes the parathyroids, is further converted to 1,25-dihydroxyvitamin D skeleton, kidneys, and intestine. Serum calcium is kept [1,25(OH)2D] in the proximal tubule of the kidney by within a very narrow range by parathyroid hormone the mitochondrial enzyme 25OHD-lα-hydroxylase (PTH) and 1,25(OH)2D by stimulating osteoclastic bone (lα-hydroxylase) (CYP27B1). The enzyme is stimu- resorption [21], the reabsorption of calcium by the renal lated directly by parathyroid hormone a reaction that is tubules [22], and the intestinal absorption of calcium, mediated by its messenger cyclic AMP [5Ð7], and indi- a biochemical event mediated by 1,25(OH)2D via the rectly by growth hormone, through stimulation of VDR [23,24]. Secretion of PTH by the parathyroid insulin-like growth factor-I [9,10] and is inhibited by glands is stimulated by phosphate [25] and is inhibited calcium [8,11,12] and inorganic phosphate [13,14]. In by both calcium and 1,25(OH)2D. Inhibition by calcium states of vitamin D excess, 25OHD is converted to is mediated via a calcium-sensing receptor [26] and VITAMIN D, 2ND EDITION Copyright © 2005, Elsevier, Inc. FELDMAN, PIKE, AND GLORIEUX All rights reserved. 790 MEHRDAD RAHMANIYAN AND NORMAN H. BELL

TABLE I Causes and Consequences of Vitamin D Deficiency in Various Races and Populations

Diminished Inadequate Increased exposure intake of Population skin pigment to sunshine vitamin D Rickets Osteomalacia

Asian Indians +++++ Blacks ++++− Caucasians −−++− Chinese −−++− Egyptians ++++− Hispanics +−−−− Jordanians ++++− Libyans ++++− Moroccans ++++− Pakistanis +++++ Polynesians +−−−− Saudi Arabians ++++− Lebanese −+++− Turkish ++++− Iranians ++++− Other Arabs ++++−

Newborn infants of any race or population are prone to develop rickets when breast-fed and kept indoors. Asians and Pakistanis are at risk to develop vitamin D deficiency and osteomalacia, particularly when they reside away from the equator. Despite knowledge of prevention of these diseases, they are widespread throughout the world.

inhibition by 1,25(OH)2D is mediated via the 7-dehydrocholesterol by absorption of photons of ultra- VDR [27,28]. The inhibition of PTH secretion by violet light is prevented by their absorption instead by 1,25(OH)2D in enhanced by up-regulation of the VDR the skin pigment melanin. Compared to Caucasians, in the parathyroids [29]. decreases in serum 25OHD in African-Americans of about 50% are found in most studies in the United States II. EFFECTS OF RACE AND GEOGRAPHY including newborns, children, and adults [32Ð41]. Compared to Caucasians, blacks show decreases in A. Blacks serum vitamin D and 25OHD, modest increases in serum PTH, serum 1,25(OH)2D, and urinary cyclic adenosine 1. VITAMIN D METABOLISM 3′,5′-monophosphate, and decreases in urinary calcium When blacks live in the Northern or Southern [32Ð42]. The changes are caused by low serum 25OHD Hemisphere at some distance from the equator, the as a consequence of vitamin D deficiency and are cor- vitamin D metabolic pathway is altered as a conse- rected by treatment with 25OHD3 [40]. Of course, they quence of diminished exposure to sunshine and inten- could also be corrected by treatment with vitamin D. sity of sunlight. For example, it was found that male The decrease in urinary calcium results from increased blacks in Zaire had a mean serum 25OHD value of secretion of PTH and increased renal tubular reabsorp- 29 ± 4 pg/ml (72.5 pmol/ml) (± SE) whereas blacks tion of calcium. In this regard, the incidence of calcium- in Belgium had a mean serum 25(OH)D value of containing kidney stones in blacks in South Africa is 9 ± 1 pg/ml (22.5 pmol/ml). A gradual decline in serum reduced because of a low urinary calcium [42]. 25OHD over a period of years occurred in black men There is a seasonal variation in vitamin D metabolism who moved from Zaire to Belgium [30]. Increased as a consequence of changes in duration of sunshine skin pigment and diminished dermal production of and intensity of sunlight in all individuals residing vitamin D3 accounts for the reduction in serum 25OHD in the Northern or Southern Hemisphere, regardless of in blacks [31]. The formation of previtamin D3 from race or skin color. In the Northern Hemisphere, serum CHAPTER 47 Effects of Race, Geography, Body Habitus, Diet, and Exercise on Vitamin D Metabolism 791

25OHD and 1,25(OH)2D are higher in summer than in premature and full-term healthy infants, 400 IU (10 µg) winter. However, serum 25OHD is lower and serum of vitamin D per day was found to be adequate [52,53]. 1,25(OH)2D is higher in black than in Caucasian men and A daily supplement of 1000 IU (25 µg) per day is rec- women regardless of season [37]. Studies of dynamics of ommended for preterm babies who are breast fed (see secretion of PTH show an exaggerated increased secretion Chapter 48). Apparently, there is no maturational delay of the hormone in response to induced hypocalcemia and in the postnatal development of hepatic 25-hydroxylase diminished suppression of secretion of the hormone in activity in well babies. response to induced hypercalcemia in black compared to white men and women [39]. The demonstration that 2. BONE MASS parathyroid glands are larger in black than in white A higher bone mineral density in blacks compared individuals in postmortem studies in the United States to whites dates from infancy and is present throughout indicates that moderate hypertrophy of the parathyroid life. In black compared to white prepubertal boys, glands is associated with long-term increased secretion bone mineral densities of the hip, spine, trochanter, of PTH [43]. and femoral neck were significantly greater but growth Blacks show a decreased skeletal sensitivity to PTH. hormone secretion and serum sex hormones were not This is evident not only by the alteration in vitamin D different [55]. A recent study found that total body metabolism system with secondary hyperparathy- bone mineral content is higher in black than in white roidism [32], but by the demonstration that the response children of the same age and Tanner stage [53]. Over of bone markers to infusion of hPTH(1-34) is blunted half of the difference could be accounted for by differ- in premenopausal black compared to premenopausal ences in body size and body composition. In contrast, white women [44]. Blacks retain more calcium than bone mineral content at the spine was found to be the whites through renal conservation and relative preser- same in black and white children. One study found vation of skeletal tissue. These findings may account no difference in intestinal absorption of calcium and for the higher bone mass and lower incidence of osteo- a lower urinary calcium in black compared to white porosis and fractures in black women [44]. children [37]. However, calcium kinetic studies showed In black and white men and women, 25OHD3 turnover that calcium accretion and intestinal absorption of cal- studies showed that reduction in serum 25OHD in blacks cium are higher and bone resorption and urinary calcium probably results from a diminished production rate and are lower in African-American compared to Caucasian not an increased metabolic rate of the metabolite since girls [42,54]. Whether these differences represent low values for serum 25OHD were associated with very heterogeneity in the populations studied or differences low values for serum vitamin D [45]. Thus, decreased in methodology is not known. Bone mineral density availability of substrate is the major factor that accounts at the lumbar spine, trochanter, and femoral neck are for low serum 25OHD values in blacks. There was an higher in black than in Caucasian women, both pre- 8- to 10-fold variation in the production rate of 25OHD menopausal and postmenopausal, and are higher in black in both black and Caucasian men and women. It is not than in white men [56Ð59]. clear whether genetic heterogeneity of CYP2R1 itself, In addition to growth during childhood and adoles- nonspecific hydroxylation by other enzymes, or other cence, the skeleton continuously remodels throughout factors account for the wide variation. life (see Chapter 28). Bone remodeling begins with The most recent NHANES (1988Ð1994) study found activation of osteoclasts and resorption of small areas that over 40% of African American women of reproduc- called Howship’s lacunae. This is followed by recruit- tive age had vitamin D deficiency as defined by serum ment of osteoblasts or bone-forming cells that repair values for 25OHD ≤ 15 ng/ml (37.5 nmol/liter) [38]. the resorption site. With aging, skeletal repair is not In the growing fetus, mothers are the sole source of complete, resulting in bone loss. Generally, the rate of vitamin D and 25OHD. Vitamin D and 25OHD in mater- skeletal remodeling is a major determinant of rate of nal milk alone are an inadequate source in newborns bone loss [60]. Histomorphometric studies showed that and infants, regardless of race. Indeed, concentrations the rate of skeletal remodeling in blacks is reduced. of vitamin D and 25OHD (39 and 310 pg/ml, respec- As a consequence, this difference could contribute to tively, 97.5 and 775 pmol/liter) are low even in milk or be responsible for the greater bone mass in black men from Caucasian women. Black infants in particular are at and women [60]. In one study, serum 17β-estradiol, risk for developing vitamin DÐdeficient rickets, espe- a major determinant of growth hormone secretion in cially when they are breast fed [46Ð50]. Thus, mater- both men and women, and growth hormone secretion nal milk provides only a modest portion of nutritional were 50% higher and bone mineral density of the total needs for infants and other sources are required [51]. body, forearm, trochanter, and femoral neck was signif- For maintenance of a normal serum 25OHD in both icantly higher in the black than in the white men [56]. 792 MEHRDAD RAHMANIYAN AND NORMAN H. BELL

Since estrogen diminishes skeletal remodeling and of bone resorption, is lower in black than in white growth hormone stimulates skeletal growth, racial dif- women both before and after menopause [72]. Urinary ferences in serum 17β-estradiol and growth hormone N-telopeptides of type I collagen, a highly specific secretion in men could be a contributing factor to marker of bone resorption, is lower in black than in white differences in bone mineral density [56]. In pre- men [73]. As noted, decreases in the rate of skeletal menopausal women, however, no racial difference in remodeling could contribute to the racial difference in serum 17β-estradiol or growth hormone secretion was bone mineral density. Findings in patients with thyroid found despite a higher bone mineral density of the total disease support this concept. Bone mineral density varies body and hip in the black women [59]. inversely with rates of skeletal remodeling. Remodeling Black men and women in the United States are less is increased in hyperthyroidism and decreased in likely to develop osteoporosis and atraumatic fractures hypothyroidism and bone density is decreased and than Caucasians. The rate of fracture for black women increased, respectively [74]. Inhibition of bone resorp- is 40% to 60% lower than that for Caucasian women tion with the bisphosphonate alendronate increases [61Ð64]. The racial difference in the incidence of frac- bone mineral density at both the lumbar spine and hip tures is attributed in part to the racial difference in as well in black as it does in Caucasian postmenopausal bone mass. In both black and white women, low body women [75,76]. weight, a previous stroke, use of aids in walking, and In subjects from South Africa, histomorphometric alcohol consumption are risk factors for hip fracture studies of bone biopsies of the iliac crest without double- [65,66]. In black women, a shorter hip axis length may tetracycline labeling demonstrated thicker trabecular protect against fractures [67]. Since the incidence of bone, greater osteoid volume, surface, and thickness, obesity is twice as high in black than in white women, and greater erosion surfaces in blacks compared to and since body weight is a major determinant of bone Caucasians, findings different from those in studies from mineral density, greater body weight is a contributing the United States [77]. The greater values for osteoid factor to higher bone mineral density in black women. volume and erosion in blacks were attributed to higher Greater body fat provides cushioning for falls and rates of bone turnover and to the fact that trabecular protects against fractures. bone in blacks was renewed more frequently and was Black men and women in South Africa also are less therefore less prone to fatigue failure and spontaneous likely to develop osteoporosis and atraumatic fractures fracture [77]. than Caucasians [68,69]. In children, no racial difference was found in bone mineral content of the forearm [70]. Bone mineral density of the femoral neck is higher in B. Asian Indians and Pakistanis black than in white women in South Africa, but bone mineral density of the forearm and lumbar spine are 1. VITAMIN D METABOLISM not different [71]. Thus, a greater bone mineral density Asian Indians living in South Carolina have a lower of the femoral neck may account for the lower inci- serum vitamin D, serum 25(OH)D, urinary calcium, and dence of femoral fractures in black women, but the urinary phosphorus compared with Caucasians, whereas racial difference in vertebral fractures must result from serum PTH and serum 1,25(OH)2D are significantly factors other than a difference in bone mass. higher in Asian Indians than in Caucasians [78]. Previous studies in Asian Indians residing in Great Britain showed 3. BONE HISTOMORPHOMETRY that, compared to values in Caucasians, serum 25OHD Bone formation rate, determined by histomorpho- is reduced [79Ð96], and vitamin D deficiency often leads metric analysis of biopsies of the iliac crest after to rickets in neonates [82,86] and infants [87], as well as double-tetracycline labeling, was found to be lower by in children and adolescents [79,88Ð94], and to osteo- about two-thirds in black compared to white men and malacia in adults [79,92Ð96]. Infants of mothers with women in the United States [60]. Static measurements severe osteomalacia develop rickets. Risk factors for in the two groups were not different. Since the error of rickets are inadequate exposure to sunlight, malabsorp- histomorphometric measurements is about 10% and tion of vitamin D, and renal and hepatic disorders [97,98]. racial differences in bone mineral density range from Rickets and osteomalacia caused by vitamin D deficiency 5% to 10%, these findings are consistent with differ- can be treated and prevented by vitamin D in daily doses ences in bone mineral density. Studies with markers of of 400 to 3000 IU [82,94,96,99,100]. bone turnover remodeling support the histomorphome- Factors that contribute to vitamin D deficiency in tric findings. For example, serum osteocalcin, a marker immigrant Asian Indians and Pakistanis include of bone formation, is lower in black than in Caucasian increased skin pigmentation and diminished dermal subjects [32,72], and urinary hydroxyproline, a marker production of vitamin D3 [31], diminished exposure to CHAPTER 47 Effects of Race, Geography, Body Habitus, Diet, and Exercise on Vitamin D Metabolism 793 sunlight in more northern climates [84Ð101], reduced or have had significant exposure to sunlight before blood intake of vitamin D [81,88,92], consumption of non- sampling. As expected, serum 25OHD was higher in fortified Chapputi flour that is not enriched with spring, summer, and fall than in winter, serum 25OHD vitamin D [81,88], and vegetarian diets [80,83,96]. was higher in maternal than cord blood, and serum Nevertheless, increases in serum vitamin D after expo- 25OHD in cord blood correlated with serum 25(OH)D sure to ultraviolet light are comparable in Indians, in maternal blood [109]. In a prospective study in two Pakistanis, and Caucasians [101]. In Pakistanis who northern and two southern Chinese cities, term infants live closer to the equator in Pakistan, serum 25OHD were treated with 100, 200, or 400 IU vitamin D daily values are comparable to those in Caucasians living in and cord blood at delivery as well as X-rays of the Britain [83,102]. Ingestion of vegetarian diets, particu- wrist and blood samples were obtained after 6 months larly those without meat, eggs, or fish, and not intake of of treatment [110]. Half were studied in the fall and Chapputi flour, contribute to vitamin D deficiency [103]. half in the spring. The results showed that none of the However, serum 25OHD also is lower in Asian adults infants had rickets. Serum 25OHD from cord blood and children who do not consume a vegetarian diet was lower in infants from the north than in those from than in Caucasians [80,84]. Because of maternal the south. Serum 25OHD was lower in infants at vitamin D deficiency and lack of supplementation of 6 months of age and was higher with increasing doses vitamin D, vitamin D deficiency and rickets were found of vitamin D. Ossification of the wrist was less com- in Pakistani infants, particularly those who were breast mon in northern than in southern infants and was more fed [95,104]. likely to be present in infants born in the fall who had a higher serum 25OHD. In breastfed infants, whereas 2. BONE MASS increased facial exposure to sunshine increased serum When body mass index and the area over which the 25OHD in some infants, supplementation with vitamin X-ray beam during measurement of BMD is projected D was required to prevent vitamin DÐdeficient rickets are taken into account, no difference in bone mass of in the general population [111]. Asian Indians compared to Caucasians can be demon- strated. Whereas earlier studies demonstrated lower bone 2. BONE MASS densities in Japanese and Chinese, who are smaller Chinese women in Beijing, China, have a lower bone compared to Caucasians, the studies did not take body mineral density and a modestly lower rate of vertebral mass index or size into consideration [105]. There is fracture than white women in the United States. As in no difference in the incidence of hip fracture in Asian other populations, low bone mineral density and a Indians and Caucasians living in England [106]. sedentary life style increase the risk of osteoporosis and fracture [112]. Compared to Caucasians, Asian boys 3. BONE HISTOMORPHOMETRY have greater bone mineral content of the lumbar spine In Asian Indians with clinical and biochemical at midpuberty and lower whole-body bone mineral changes of osteomalacia, biopsies of the iliac crest content at maturity and Asian girls have lower femoral showed increases in osteoid volume [92]. Since only neck bone mineral content through mid-puberty and static histomorphometric measurements were carried lower whole-body bone mineral content in pre-/early out, dynamic measurements after double-tetracycline puberty. Whole-body bone mineral density and whole- labeling also should be performed to demonstrate body bone mineral content/height values are signifi- impaired calcification, a sine-qua-non requirement for cantly lower in mature Asian versus Caucasian males. the diagnosis of osteomalacia. Differences in bone mineral density and apparent density between Asian and Caucasian subjects are due to differences in body weight and pubertal stage, and, C. Chinese at the femoral neck, to differences in weight-bearing activity [113]. 1. VITAMIN D METABOLISM In China, infantile rickets is common. The incidence is highest in breast-fed infants and in infants who are D. Hispanics 2 to 4 months of age [107,108]. Rickets sometimes occurs in infants who have a normal serum 25OHD, 1. VITAMIN D METABOLISM indicating that other factors may play a role in the Studies in prepubertal Mexican-American and pathogenesis of the bone disease in these infants [109]. Caucasian girls in southeastern Texas showed that Thus, normal serum 25OHD values may be found in calcium absorption, urinary calcium excretion, calcium rachitic patients who had received a dose of vitamin D kinetic values, and total body bone mineral content 794 MEHRDAD RAHMANIYAN AND NORMAN H. BELL were similar. However, serum PTH was greater and intake of vitamin D is the commonest cause of rickets in serum 25OHD was lower in Mexican-American girls Saudi Arabia. Rickets also occurs because of inadequate than in Caucasian girls and serum 1,25(OH)2D was not calcium intake and putative vitamin D-25-hydroxylase different in the two groups [114]. Mexican-American deficiency [123]. Saudi women are veiled, avoid sun- men and women have a lower serum vitamin D and light, and stay indoors. Consequently, serum 25OHD 25OHD and higher serum 1,25(OH)2D and serum PTH is often low [124,125]. Vitamin D deficiency is very than Caucasians [115]. In contrast to African Americans common in Saudi women who are pregnant, and serum [32], urinary calcium, serum osteocalcin, and urinary 25OHD is low in cord blood [125]. Vitamin D defi- cyclic adenosine 3′,5′-monophosphate are not different. ciency is common in Saudi men as well as in Jordanian, The low serum vitamin D and 25OHD in Mexican- Egyptian, and other men living in Saudi Arabia and is Americans is attributed to increased skin pigmentation. attributed to inadequate intake of vitamin D and avoid- ance of sunlight [126]. Vitamin D deficiency in elderly 2. BONE MASS Saudi men and women is associated with fractures of the Mexican-American women have higher bone mineral femoral neck because of avoidance of sunlight [127]. density at the proximal femur than Caucasian women, and this difference may account in part for their lower 2. BONE MASS rate of hip fracture. In the United States, Hispanics are Bone mineral density in healthy Saudi women is intermediate for risk of hip fracture between blacks lower than in American women. This is attributed to a and whites and Mexican-Americans are at higher risk higher number of pregnancies and longer duration of lac- than Puerto Ricans [115Ð119]. tation together with prevalent vitamin D deficiency [128]. One study showed a significant correlation between serum 25OHD and back pain. Treatment with vitamin D E. Polynesians increased serum 25OHD and alleviated the back pain [129]. Thus, vitamin D deficiency is very common 1. VITAMIN D METABOLISM in Saudis of all ages and is a cause of rickets and osteo- Serum 25OHD is lower in Polynesians compared porosis with fractures. to Caucasians. However, serum PTH, 1,25(OH)2D, osteocalcin, alkaline phosphatase, calcitonin, and urinary calcium/creatinine and urinary hydroxyproline/ G. Other Groups creatinine ratios are not different [120]. Reduction in serum 25OHD is attributed to increased skin In Arabs in the United Arab Emirates (UAE), a cor- pigmentation. relation between serum 25OHD and biosocial factors including age, education, and living accommodation 2. BONE MASS were found. Serum 25OHD in UAE Arabs and in non- Bone mineral density of the forearm, lumbar spine, Gulf Arabs was significantly lower than in Europeans, and femoral neck is higher in Polynesian than in whereas serum calcium, phosphorus, alkaline phos- Caucasian women even after adjustment for body mass phatase, and PTH among the groups were not different index [121,122]. It is known that a short femoral [130]. In Lebanese, the degree of vitamin D deficiency neck is associated with a low rate of hip fracture [67]. lies between that observed in Europe and the United However, the femoral neck is longer in Polynesians States [131]. A pilot study among Iranian women and than it is in Europeans so that the lower incidence of their neonates showed that 80% had a low maternal hip fracture cannot be attributed to a difference in serum 25OHD. Mean maternal plasma calcium and femoral neck length. Either a higher bone mineral alkaline phosphatase were in the normal range and density or other more subtle differences in proximal serum PTH was elevated. The mean cord serum 25OHD femoral geometry must account for the lower hip fracture was very low. Serum 25OHD in infants of mothers with incidence [122]. hypovitaminosis D was almost undetectable [132]. In Turkey, the extent to which clothing causes limited exposure to sunlight was shown to be a major factor F. Saudi Arabians in determination of serum 25OHD [133]. In the Netherlands, serum 25OHD was lower and serum PTH 1. VITAMIN D METABOLISM was higher in children of Turkish and Moroccan immi- Despite proximity to the equator, vitamin D deficiency grants who have dark skin [134]. In contrast, serum due to lack of exposure to sunlight and inadequate 25OHD in dark-skinned Bedouins of the Negev Desert CHAPTER 47 Effects of Race, Geography, Body Habitus, Diet, and Exercise on Vitamin D Metabolism 795 of Israel is not different compared to lighter-skinned vitamin D [143Ð147]. Serum vitamin D is low pre- Jewish men and women. Presumably a longer duration sumably because vitamin D is fat soluble and is stored in of exposure to sunshine and greater intensity of sunlight fat [148,149]. In obese men and women, compensatory prevented vitamin D deficiency [135]. Biochemical and increases in serum PTH, serum 1,25(OH)2D, and urinary clinical rickets occur in Libyan infants who are breast cyclic adenosine 3′,5′-monophosphate and decreases in fed. The rickets is attributed to inadequate maternal urinary calcium occur [145,147,150]. These changes are exposure to sunlight and inadequate maternal intake of reversible with weight loss [150,151]. Treatment with vitamin D [136]. In Alaska vitamin D deficiency among 25OHD3 also corrects these changes: serum 25OHD and children less than 2 years is common especially in urinary calcium are increased and serum 1,25(OH)2D breast-fed infants [137]. In Australia food is not forti- and urinary cyclic adenosine 3′,5′- monophosphate are fied with vitamin D, and the major source of vitamin D decreased [147]. After treatment, these changes return is casual exposure to the sunshine, leading to vitamin D to baseline values [147]. On the other hand, in nonobese deficiency in adults [138]. subjects, administration of 25OHD increases serum Again, serum 25OHD can vary as a consequence of 25OHD, and decreases serum 1,25(OH)2D, but does not geographical location. Serum values are lower in sub- alter urinary calcium [147]. As noted already, nonobese jects in southern Argentina (Ushuaia) than in subjects blacks living in the United States have changes in in northern Argentina (Buenos Aires) [139]. vitamin D metabolism similar to those of obese Caucasians [32]. These changes are not further altered III. EFFECTS OF DIET by obesity [150]. A. Vitamin D Metabolism B. Bone Mass Dietary intake of vitamin D was found to be signif- icantly lower in vegans and lactovegetarians compared Regardless of race, obesity is associated with with omnivores living in Finland. Throughout the year increases in bone mineral density of the lumbar spine serum 25OHD was lower and serum PTH was higher and hip [152,153]. Indeed, body weight is an important in vegans than in omnivores and lactovegetarians. determinant of bone mineral density of the lumbar BMD of the lumbar spine was lower in vegans than spine, trochanter, and femoral neck in postmenopausal in omnivores and tended to be lower than in lactoveg- women [154,155]. Radiographic measurements of the etarians. Bone mineral density of the neck of femur metacarpal cortical area showed a higher bone mass in tended to be lower in vegans than in omnivores and obese compared to nonobese individuals [156]. Also, lactovegetarians [140]. fat mass is a major determinant of total body bone mineral density in premenopausal and postmenopausal women [152,154,155]. B. Bone Mass

In the Netherlands, a high prevalence of rickets was C. Bone Histomorphometry found in infants on macrobiotic diets of unpolished rice, pulses, vegetables with high fiber content, seaweeds, fer- Whereas obesity is associated with vitamin D defi- mented foods, nuts, seeds, and fruits [141] and reduced ciency and a low serum 25(OH)D, bone histomorphom- bone mineral content of the whole body, spine, radius, etry of the iliac crest is almost always normal. In one and hip were found in adolescents who had been fed a study, one of 24 grossly obese individuals was found to macrobiotic diet in early life [142]. Thus, the vitamin D have mild osteomalacia and secondary hyperparathy- and calcium content of strict vegetarian and macrobiotic roidism and a second individual had changes associ- diets is inadequate, and the diets need to be fortified to ated with increased skeletal remodeling [157]. Morbid prevent vitamin D deficiency. obesity is sometimes treated with partial or total biliopancreatic bypass. When bone histomorphometric IV. EFFECTS OF BODY HABITUS analysis was performed 1 to 5 years after bypass in 41 patients, all of them had a normal serum 25OHD. A. Vitamin D Metabolism However, almost three quarters of them had abnormal bone mineralization, diminished bone formation, Obese individuals have a lower serum 25OHD and increased bone resorption [158]. Thus, following values than nonobese subjects as a result of low serum biliopancreatic bypass for treatment of obesity, 796 MEHRDAD RAHMANIYAN AND NORMAN H. BELL osteomalacia is common despite the absence of vita- sunlight occurs in Arabs of all ages and the populations min D deficiency. from the Middle East and leads to rickets in children and infants and osteoporosis and fractures in adults. Macrobiotic and vegetarian diets are deficient in V. EFFECTS OF EXERCISE vitamin D, and infants fed macrobiotic diets are prone to develop rickets. In obese men and women, serum Weight lifting and other weight-bearing exercises vitamin D is markedly reduced, probably because increase bone mass of the lumbar spine, trochanter, vitamin D is fat soluble and is stored in fat. Whereas femoral neck, and lower extremities in adult and serum 25OHD and urinary calcium are reduced and adolescent males [159Ð162]. Men who had undergone circulating PTH is increased in obesity, osteomalacia muscle-building exercise for at least 1 year showed is usually not a clinical problem. Weight-lifting and increases in serum osteocalcin, an index of bone for- muscle-building exercise apparently leads to increases in mation, serum 1,25(OH)2D, and urinary cyclic adeno- serum 1,25(OH)2D, and urinary calcium, and weight- sine 3′,5′-monophosphate compared to men who were bearing exercise increases bone mineral density and sedentary [169]. Serum 1,25(OH)2D and osteocalcin thus may help to prevent osteoporosis and fractures. did not correlate with each other. Thus, exercise may In summary, vitamin D deficiency is common in increase bone formation, serum 1,25(OH)2D, calcium peoples from many races throughout the world in both absorption and bone mineral density. In older indi- developed and undeveloped countries, even those near viduals, exercise increased total body calcium and the equator. Thus, it is widespread despite the ready bone mineral density of the lumbar spine [163Ð165]. availability of assays for serum 25(OH)D to ascertain In aging men, muscle strength of the back correlated vitamin D status and the known necessity for supple- with bone mineral density of the lumbar spine and mentary dietary vitamin D and adequate exposure to midradius [165]. sunshine, particularly during pregnancy, childhood, In postmenopausal women, aerobic, weight-bearing adolescence, and aging. and resistance exercises each increased bone mineral density of the spine and walking increased bone mineral density of the hip [166,167]. In college women, bone References mineral density of the lumbar spine was increased in tennis players but not in swimmers, indicating the 1. Holick MF, MacLaughlin JA, Doppelt SH 1981 Regulation necessity that the exercise be weight bearing [168]. of cutaneous previtamin D photosynthesis in man: Skin pigment is not an essential regulator. Science 211:590Ð593. 2. Holick MF 1981 The cutaneous synthesis of previtamin D3: A unique photo-endocrine system. J Invest Dermatol 77:51Ð58. VI. SUMMARY 3. Cheng JB, Motola DL, Mangelsdorf DJ, Russell DW 2003 De-orphanization of cytochrome P450 2R1: a microsomal A number of factors influence vitamin D metabolism. vitamin D-25-hydroxylase. J Biol Chem 278:38084Ð38093. Among these are race, geographic locus, sunlight expo- 4. Cheng JB, Levine MA, Bell NH, Mangelsdorf DJ, Russell DW sure, diet and cultural traditions, body habitus, and 2004 Genetic evidence that human CYP2R1 is a key vita- min D 25-hydroxylase. Proc Natl Acad Sci USA 101: exercise. Dark-skinned individuals who live in temperate 7711Ð7715. regions including blacks, Asian Indians, Pakistanis, 5. Garabedian M, Holick MF, DeLuca HF, Boyle IT 1972 Hispanics, and others have diminished production of Control of 25-hydroxycholecalciferol metabolism by vitamin D3 in the skin because photons of light energy parathyroid glands. Proc Natl Acad Sci USA 69:1673Ð1676. are absorbed by melanin instead of 7-dehydrocholes- 6. Henry HL 1989 Parathyroid hormone modulation of 25-hydrovitamin D3 metabolism by cultured chick kidney terol. Consequently, decreases in serum 25OHD and cells is mimicked and enhanced by forskolin. Endocrinology urinary calcium and increases in circulating PTH occur. 116:503Ð510. Further, vitamin DÐdeficiency rickets may occur in 7. Horiuchi N, Suda T, Takahashi H, Shimazawa E, Ogata E breast-fed infants of any race if they do not receive 1977 In vivo evidence for the intermediary role of 3′,5′- supplements of the vitamin and are kept out of sunlight cyclic AMP in parathyroid hormone-mediated stimulation of 1,25-dihydroxyvitamin D3 synthesis in rats. Endocrinology since the contents of vitamin D and 25(OH)D in human 101:969Ð974. milk are inadequate. Infants of dark-skinned mothers 8. Murayama A, Takeyama K, Kitanaka S, Kodera Y, are particularly susceptible in this regard. Kawaguchi Y, Hosoya T, Kato S 1999 Positive and negative α Vitamin DÐdeficient rickets and osteomalacia occur regulations of the renal 25-hydroxyvitamin D3 1 -hydroxy- in dark-skinned Asian Indians and Pakistanis who live lase gene by parathyroid hormone, calcitonin, and 1α,25 (OH)2D3 in intact animals. Endocrinology 140:2224Ð2231. in temperate regions. Vitamin D deficiency caused by 9. Caverzasio J, Montessuit C, Bonjour JP 1990 Stimulatory inadequate dietary vitamin D intake and avoidance of effect of insulin-like growth factor-I on renal Pi transport CHAPTER 47 Effects of Race, Geography, Body Habitus, Diet, and Exercise on Vitamin D Metabolism 797

and plasma 1,25-dihydroxyvitamin D3. Endocrinology 127: preproparathyroid hormone in isolated bovine parathyroid 453Ð459. cells. Proc Natl Acad Sci USA 82:4270Ð4273. 10. Nesbitt T, Drezner MK 1993 Insulin-like growth factor-I 29. Naveh-Many T, Marx R, Keshet E, Pike JW, Silver J 1990 regulation of renal 25-hydroxyvitamin D-1α-hydroxylase Regulation of 1,25-dihydroxyvitamin D3 receptor gene activity. Endocrinology 132:133Ð138. expression by 1,25-dihydroxyvitamin D3 in the parathyroid 11. Boyle IT, Graw RW, DeLuca HF 1971 Regulation by calcium in vivo. J Clin Invest 86:1968Ð1975. of in vitro synthesis of 1,25-dihydroxycholecalciferol. Proc 30. M’Buyamba-Kabangu JR, Fagard R, Lijnen P, Bouillon R, Natl Acad Sci USA 68:2131Ð2134. Lissens W, Amery A 1987 Calcium, vitamin D-endocrine 12. Bushinsky DA, Riera G, Mavus MJ, Coe FL 1985 Evidence system and parathyroid hormone in black and white males. that blood ionized calcium can regulate serum 1,25(OH)2D3 Calcif Tissue Int 41:70Ð74. independently of PTH and phosphorus in the rat. J Clin 31. Clemens TL, Henderson SL, Adams JS, Holick MF 1982 Invest 76:1599Ð1604. Increased skin pigment reduces the capacity of skin to 13. Tanaka Y, DeLuca HF 1973 The control of 25-hydroxy- synthesize vitamin D3. Lancet 1:74Ð76. vitamin D metabolism by inorganic phosphorus. Arch 32. Bell NH, Greene A, Epstein S, Oexmann MJ, Shaw S, Shary J Biochem Biophys 154:566Ð574. 1985 Evidence for alteration of the vitamin D-endocrine 14. Portale AA, Halloran BP, Murphy MM, Morris RC Jr 1986 system in blacks. J Clin Invest 76:470Ð473. Oral intake of phosphorus can determine the serum concentra- 33. Hollis BW, Pittard WB III 1984 Evaluation of the total feto- tion of 1,25-dihydroxyvitamin D by determining its produc- maternal vitamin D relationships at term: Evidence for racial tion rate in humans. J Clin Invest 77:7Ð12. differences. J Clin Endocrinol Metab 59:652Ð657. 15. Henry HL 1979 Regulation of the hydroxylation of 34. Luckey MM, Meier DE, Clemens TL 1986 PTH and vitamin D 25-hydroxyvitamin D3 in vivo and in primary cultures of metabolites in premenopausal white and black women. J Bone chick kidney cells. J Biol Chem 254:2722Ð2729. Miner Res 1(Suppl. 1):381. 16. Trechsel U, Bonjour J-P, Fleisch H 1978 Regulation of the 35. Dawson-Hughes B, Harris S, Kramich C, Dallal G, metabolism of 25-hydroxyvitamin D in primary cultures of Rasmussen HM 1993 Calcium retention and hormone levels chick kidney cells. J Clin Invest 64:206Ð217. in black and white women on high and low calcium diets. 17. Henry HL 1992 Vitamin D hydroxylases. J Cell Biochem J Bone Miner 8:779Ð787. 49:4Ð9. 36. Katz BS, Jackson GJ, Hollis BW, Bell NH 1992 Diagnostic 18. Henry HL 2001 The 25(OH)D3/1α,25(OH)(2)D3-24-hydroxy- criteria of vitamin D deficiency. Endocrinologist 3:248Ð253. lase: a catabolic biosynthetic enzyme? Steroids 66:391Ð398. 37. Bell NH, Yergey AL, Vieira NE, Oexmann MJ, Shary JR 19. Halloran BP, Bikle DD, Levens MJ, Castro ME, Globu RK, 1993 Demonstration of a difference in urinary calcium, not Holton E 1986 Chronic 1,25-dihydroxyvitamin D3 adminis- calcium absorption, in black and white adolescents. J Bone tration in the rat reduces the serum concentration of Miner Res 8:1111Ð1115. 25-hydroxyvitamin D by increasing metabolic clearance rate. 38. Nesby-O’Dell S, Scanlon KS, Cogswell ME, Gillespie C, J Clin Invest 78:622Ð628. Hollis BW, Looker CA, Allen C, Doughertly C, Gunter EW, 20. Bell NH, Shaw S, Turner RT 1984 Evidence that 1,25-dihy- Bowman BA 2002 Hypovitaminosis D prevalence and deter- droxyvitamin D3 inhibits the hepatic production of minants among African American and white women of repro- 25-hydroxyvitamin D in man. J Clin Invest 74:1540Ð1544. ductive age: Third National Health and Nutrition Examination 21. McSheely PM, Bibby NJ 1985 Osteoblastic cells mediate osteo- Survey, 1988Ð1994. Am J Clin Nutr 76:187Ð192. clastic responsiveness to parathyroid hormone. Endocrinology 39. Fuleihan GE-H, Gundberg CM, Gleason R, Brown EM, 118:824Ð828. Stromski ME, Grant FD, Conlin PR 1994 Racial differences 22. Agus ZS , Gardner LB, Beck LH, Goldberg M 1973 Effects of in parathyroid hormone dynamics. J Clin Endocrinol Metab parathyroid hormone on renal tubular reabsorption of calcium, 79:1642Ð1647. sodium and phosphate. Am J Physiol 224:1143Ð1148. 40. Bell NH 1995 25-Hydroxyvitamin D3 reverses alteration 23. Favus MJ 1985 Factors that influence absorption and secre- of the vitamin D-endocrine system in blacks. Am J Med 99: tion of calcium in the small intestine and colon. Am J Physiol 597Ð599. 248:G147ÐG157. 41. Abrams SA, O’Brien KO, Liang LK, Stuff JE 1995 Differences 24. Bikle DD, Zolock DT, Munson S 1984 Differential response in calcium absorption and kinetics between black and white of duodenal epithelial cells to 1,25-dihydroxyvitamin D3 girls aged 5Ð16 years. J Bone Miner Res 10:829Ð833. according to position in the villus: A comparison of calcium 42. Modlin M 1967 Urinary calcium in normal adults and in uptake, calcium-binding protein, and alkaline phosphatase patients with renal stones: An interracial study. Invest Urol activity. Endocrinology 115:2077Ð2084. 5:49Ð57. 25. Silver J, Yalcindag C, Sela-Brown A, Kilav R, Naveh-Many T 43. Ghandur-Mnaymneh L, Cassady J, Hajanpour MA, Reiss E 1999 Regulation of the parathyroid hormone gene by vitamin D, 1986 The parathyroid gland in health and disease. Am J Pathol calcium and phosphate. Kidney Int Suppl 73:S2ÐS7. 125:292Ð299. 26. Brown EM, Gamba G, Riccardi D, Lombardi M, Butters R, 44. Cosman F, Morgan DC, Nieves JW, Shen V, Luckey MM, Kifor O, Sun A, Hediger MA, Lytton J, Hebert SC 1993 Cloning Dempster DW, Lindsay R, Parisien M 1997 Resistance to and characterization of an extracellular Ca2+-sensing receptor bone resorbing effects of PTH in black women. J Bone Miner from bovine parathyroid. Nature 366:575Ð580. Res 12:958Ð966. 27. Naveh-Many T, Friedlaender MM, Mayer, Silver J 1989 45. Awumey E, Hollis BW, Bell NH 1996 Low serum Calcium regulates parathyroid hormone messenger ribo- 25-hydroxyvitamin D in blacks results from decreased nucleic acid (mRNA), but not calcitonin mRNA in vivo production rate and not increased metabolic clearance rate. in the rat. Dominant role of 1,25-dihydroxyvitamin D. J Bone Miner Res 11:8165 (abstract). Endocrinology 125:275Ð280. 46. Bachrach S, Fisher J, Parks JS 1980 An outbreak of vitamin D 28. Silver J, Russell J, Sherwood LM 1985 Regulation by deficiency rickets in a susceptible population. Pediatrics vitamin D metabolites of messenger ribonucleic acid for 64:277Ð283. 798 MEHRDAD RAHMANIYAN AND NORMAN H. BELL

47. Edidin DV, Levitsky LL, Schey W, Dumbovic M, Campos A 66. Grisso AJ, Kelsey JL, Strom BL, O’Brien LA, Maislin G, 1980 Resurgence of nutritional rickets associated with La Pann K, Samelson L, Hoffman S 1994 Risk factors for hip breast-feeding and special dietary practices. Pediatrics fracture in black women. The Northeast Hip Fracture Study 65:232Ð235. Group. N Engl J Med 330:1555Ð1559. 48. Kruger DM, Lyne ED, Kleerekoper M 1987 Vitamin D 67. Cummings SR, Cauley JA, Palermo L, Ross PD, Wasnich RD, deficiency rickets. A report of three cases. Clin Orthop 224: Black D, Faulkner KG 1994 Racial differences in hip axis 277Ð283. lengths might explain racial differences in rates of hip fracture. 49. Key LL 1992 Vitamin D deficiency rickets. Trends Endocrinol Osteoporosis Int 4:226Ð229. Metab 2:81Ð85. 68. Solomon L 1968 Osteoporosis and fracture of the femoral neck 50. Chang YT, Germain-Lee EL, Doran TF, Migeon CJ, in the South African Bantu. J Bone Joint Surg 50:2Ð5. Levine MA, Berkovitz GD 1992 Hypocalcemia in nonwhite 69. Solomon L 1979 Bone density in aging Caucasian and breast-fed infants. Clin Pediat 31:695Ð698. African populations. Lancet 3:1326Ð1329. 51. Hollis BW, Roos BA, Draper HH, Lambert PW 1981 Vitamin D 70. Patel DN, Pettifor JM, Becker PJ, Grieve C, Leschner K and its metabolites in human and bovine milk. J Nutr 111: 1993 The effect of ethnicity on appendicular bone mass in 1240Ð1248. white, coloured and Indian school children. S Afr Med J 83: 52. Pittard WB, Geddes KM, Husley TC, Hollis BW 1991 How 847Ð853. much vitamin D for neonates? Am J Dis Child 145:1147Ð1149. 71. Daniels ED, Pettifor JM, Schnitzler CM, Russel SW, Paten 53. Hui SL, Dimeglio LA, Longcope C, Peacock M, McClintock R, DN 1995 Ethnic differences in bone density in female South Perkins AJ, Johnston CC Jr 2003 Difference in bone mass African nurses. J Bone Miner Res 10:359Ð367. between black and white American children: Attributable to 72. Meier De, Luckey MM, Wallenstein S, Lapinski RH, body build, sex hormone levels, or bone turnover? J Clin Catherwood B 1992 Racial differences in pre- and post- Endocrinol Metab 88:642Ð649. menopausal bone homeostasis: Association with bone density. 54. Bryant RJ, Wastney ME, Martin BR, Wood O, McCabe GP, J Bone Miner Res 7:1181Ð1189. Morshidi M, Smith DL, Peacock M, Weaver CM 2003 Racial 73. Jackson G, Hollis BW, Eyre DR, Baylink DJ, Bell NH 1994 differences in bone turnover and calcium metabolism in Effects of race and calcium intake on bone markers and adolescent females. J Clin Endocrinol Metab 88:1043Ð1047. calcium metabolism in young adult men. J Bone Miner Res 55. Wright NM, Papadea N, Veldhuis JD, Bell NH 2002 Growth 9:S185 (abstract). hormone secretion and bone mineral density in prepubertal 74. Eriksen EF, Mosekilde L, Melsen F 1986 Kinetics of trabec- black and white boys. Calcif Tissue Int 70:146Ð152. ular bone resorption and formation in hypothyroidism: 56. Wright NM, Renault J, Willi S, Veldhuis JD, Pandey JP, Evidence for a positive balance per remodeling cycle. Bone Gordon L, Key LL, Bell NH 1995 Greater secretion of growth 7:101Ð108. hormone in black than in white men: Possible factor in greater 75. Chestnut CH, McClung MR, Ensrud KE, Bell NH, Genant bone mineral density—A clinical research center study. J Clin HK, Harris ST, Singer FR, Stock JL, Yood RA, Delmas PD, Endocrinol Metab 80:2291Ð2297. Pryor-Tillotson S, Santora AC 1995 Alendronate treatment of 57. Liel Y, Edwards J, Shary J, Spicer KM, Gordon L, Bell NH the postmenopausal osteoporotic woman: Effect of multiple 1988 The effects of race and body habitus on bone mineral dosages on bone mass and bone remodeling. Am J Med density of the radius, hip and spine in premenopausal 99:144Ð152. women. J Clin Endocrinol Metab 66:1247Ð1250. 76. Liberman UA, Weiss SR, Broil J, Minne HW, Quan H, 58. DeSimone DP, Stevens J, Edwards J, Shary J, Gordon L, Bell NH, Rodriquez-Portales J, Downs RW, Dequecker J, Bell NH 1989 Influence of body habitus and race on bone Favus M, Capizzi T, Santora II AC, Lombardi A, Shah RV, mineral density of the midradius, hip and spine in aging Hirsch LJ, Karpf DB 1995 Effect of three years treatment women. J Bone Miner Res 4:827Ð830. with oral alendronate on fracture incidence in women 59. Wright NM, Papadea N, Willi S, Veldhuis JD, Pandey JP, with postmenopausal osteoporosis. N Engl J Med 33: Key LL, Bell NH 1996 Demonstration of a lack of racial 1437Ð1443. difference in secretion of growth hormone despite a racial 77. Schnitzler CM, Pettifor JM, Mesqita JM, Bird MD, Schnaid E, difference in bone mineral density in premenopausal Smyth AE 1990 Histomorphometry of iliac crest bone in 346 women—A clinical research center study. J Clin Endocrinol normal black and white South African adults. Bone Miner Metab 81:1023Ð1026. 10:183Ð199. 60. Weinstein RS, Bell NH 1988 Diminished rates of bone 78. Awumey EM, Mitra DA, Hollis BW, Kumar R, Bell NH 1998 formation in normal black adults. N Engl J Med 319: Vitamin D metabolism is altered in Asian Indians in the 1698Ð1701. southern United States: A clinical research center study. 61. Gyepes M, Melliaz HZ, Katz I 1962 The low incidence of J Clin Endocrinol Metab 83:169Ð73. fracture of the hip in the Negro. JAMA 181:1073Ð1074. 79. Preece MA, Ford JA, Mclntosh WB, Dunnigan MG, 62. Silverman SL, Madison RE 1988 Decreased incidence of hip Tomlinson S, O’Riordan JLH 1973 Vitamin D deficiency fracture in hispanics, Asians and Blacks: California hospital among Asian immigrants to Britain. Lancet 1:907Ð910. discharge data. Am J Public Health 78:1482Ð1483. 80. Dent CE, Gupta MM 1975 Plasma 25-hydroxyvitamin-D 63. Kellie SE, Brody JA 1990 Sex-specific and race-specific hip levels during pregnancy in Caucasians and in vegetarian fracture rates. Am J Public Health 80:326Ð328. and non-vegetarian Asians. Lancet 2:1057Ð1060. 64. Griffin MR, Ray WA, Fought RL, Melton LJ III 1992 81. Hunt SP, O’Riordan JLH, Windo J, Truswell AS 1976 Black-white differences in fracture rates. Am J Epidemiol Vitamin D status in different sub-groups of British Asians. 136:1378Ð1385. Br Med J 2:1351Ð1354. 65. Pruzansky ME, Turano M, Luckey M, Senie R 1989 Low 82. Heckmatt JZ, Peacock M, Davies AEJ, McMurray J, body weight is a risk factor for hip fracture in both black and Isherwood DM 1979 Plasma 25-hydroxyvitamin D in white women. J Orthop Res 7:192Ð197. pregnant Asian women and their babies. Lancet 2:546Ð548. CHAPTER 47 Effects of Race, Geography, Body Habitus, Diet, and Exercise on Vitamin D Metabolism 799

83. Brooke OG, Brown IRF, Cleeve HJW, Sood A 1981 secondary hyperparathyroidism in middle-aged strict vege- Observations on the vitamin D state of pregnant Asian women tarians. Am J Clin Nutr 58:684Ð689. in London. Br J Obstet Gynaecol 88:18Ð26. 104. Ahmed I, Atiq M, Iqbal J, Khurshid M, Whittaker P 1995 84. Ellis G, Woodhead JS, Cooke WT 1977 Serum 25-hydroxy- Vitamin D deficiency rickets in breast-fed infants presenting vitamin-D concentrations in adolescent boys. Lancet with hypocalcemic seizures. Acta Paediatr 84:941Ð942. 1:825Ð828. 105. Cundy C, Cornish J, Evans MC, Gamble G, Stapleton J, Reid IR 85. O’Hare AE, Uttley WS, Belton NR, Westwood A, Levin SD, 1995 Sources of interracial variation in bone mineral density. Anderson F 1984 Persisting vitamin D deficiency in the J Bone Miner Res 10:368Ð373. Asian adolescent. Arch Dis Child 59:766Ð770. 106. Parker M, Anand JK, Myles JW, Lodwick R 1992 Proximal 86. Ford JA, Davidson DC, Mclntosh WB, Fyfe WM, femoral fractures: Prevalance in different racial groups. Dunnigan MG 1973 Neonatal rickets in Asian immigrant Eur J Epidemiol 8:730Ð732. population. Br Med J 3:211Ð212. 107. Fong JC, Huong YM, Shen KY, Dewen PL 1980 Prevention 87. Arneil GC, Crosbie JC 1963 Infantile rickets returns to and treatment of rickets (report). Zhengzhou, Henan, People’s Glasgow. Lancet 2:423Ð425. Republic of China: Henan College of Medicine publication 88. Goel KM, Sweet EM, Logan RW, Warren JM, Arneil GC, (Chinese), 1Ð10. Shanks RA 1976 Florid and subclinical rickets among 108. Xiao L 1982 Survey of infant rickets. J Chinese Pediatr immigrant children in Glasgow. Lancet 1:1141Ð1145. (Chinese) 20:63Ð64. 89. Henderson JB, Dunnigan MG, Mclntosh WB, 109. Danhui Z, Quingbing Z, Yan X 1990 Serum 25OHD levels in Abdul-Motaal AA, Gettinby G, Glekin BM 1987 The impor- maternal and cord blood in Beijing, China. Acta Paediatr tance of limited exposure to UV radiation and dietary factors Scand 79:1240Ð1241. in the aetiology of Asian rickets: A risk factor model. Q J Med 110. Specker BL, Ho ML, Oestreich A, Yin T-A, Shui Q-M, 63:413Ð425. Chen X-C, Tsang RC 1992 Prospective study of vitamin D 90. Dent CE, Rowe DJF, Round JM, Stamp TC B 1973 Effect of supplementation and rickets in China. J Pediatr 120: chapattis and ultraviolet irradiation on nutritional rickets in 733Ð739. an Indian immigrant. Lancet 1:1282Ð1284. 111. Specker BL, Valanis B, Hertzberg V, Edwards N, Tsang RC 91. Wills MR, Day RC, Phillips JB, Bateman EC 1972 Phytic 1985 Sunshine exposure and serum 25-hydroxyvitamin D con- acid and nutritional rickets in immigrants. Lancet 1:771Ð773. centrations in breast-fed infants in Beijing, China. J Pediatr 92. Holmes AM, Enoch BA, Taylor JL, Jones ME 1973 Occult 107:928Ð931. rickets and osteomalacia amongst the Asian immigrant 112. Ling X, Cummings SR, Mingwei Q, Xihe Z, Xioashu C, population. Q J Med 42:125Ð149. Nevitt M, Stone K 2000 Vertebral fractures in Beijing, China: 93. Ford JA, Colhoun EM, Mclntosh WB, Dunnigan MG 1972 the Beijing Osteoporosis Project. J Bone Miner Res 15: Rickets and osteomalacia in the Glasgow Pakistani community, 2019Ð2025. 1961Ð1971. Br Med J 1:677Ð679. 113. Bhudhikanok GS, Wang MC, Eckert K, Matkin C, Marcus R, 94. Preece MA, Tomlinson S, Ribot CA, Pietrek J, Korn HT, Bachrach LK 1996 Differences in bone mineral in young Davies DM, Ford JA, Dunnigan MG, O’Riordan JLH Asian and Caucasian Americans may reflect differences in 1975 Studies of vitamin D deficiency in man. Q J Med 44: bone size. J Bone Miner Res 11:1545Ð1556. 575Ð589. 114. Abrams SA, Copeland KC, Gunn SK, Stuff JE, Clarke LL, 95. Finch PJ, Ang L, Colston KW, Nisbet J, Maxwell JD 1992 Ellis KJ 1999 Calcium absorption and kinetics are similar Blunted seasonal variation in serum 25-hydroxyvitamin D in 7- and 8-year-old Mexican-American and Caucasian girls and increased risk of osteomalacia in vegetarian London despite hormonal differences. J Nutr 129:666Ð671. Asians. Eur J Clin Nutr 46:509Ð515. 115. Reasner III CA, Dunn JF, Fetchick DA, Leil Y, Hollis BW, 96. Henderson JB, Dunnigan MG, Mclntosh WB, Motaal AA, Epstein S, Shary J, Mundy GR, Bell NH 1990 Alteration of Hole D 1990 Asian osteomalacia is determined by dietary vitamin D metabolism in Mexican-Americans. J Bone Miner factors when exposure to ultraviolet radiation is restricted: Res 5:13Ð17. A risk factor model. Q J Med 76:923Ð933. 116. Bauer RL, Diehl AK, Barton SA, Brender JA, Deyo RA 1986 97. Teotia M, Teotia SP 1997 Nutritional and metabolic rickets. Risk of postmenopausal hip fracture in Mexican-American Indian J Pediatr 64:153Ð157. women. Am J Public Health 76:1020Ð1021. 98. Atiq M, Suria A, Nizami SQ, Ahmed I 1998 Vitamin D status 117. Bauer RL, Deyo RA 1987 Low risk vertebral fracture in of breastfed Pakistani infants. Acta Paediatr 87:737Ð740. Mexican-American women. Arch Intern Med 147:1437Ð1439. 99. Pietrek J, Preece MA, Windo J, O’Riordan J, Dunnigan MG, 118. Taaffe DR, Villa ML, Holloway L, Marcus R 2000 Bone Mclntosh WB, Ford JA 1976 Prevention of vitamin-D mineral density in older non-Hispanic Caucasian and deficiency in Asians. Lancet 1:1145Ð1148. Mexican-American women: Relationship to lean and fat 100. Dunnigan MG, Mclntosh WB, Sutherland GR, Gardee R, mass. Ann Hum Biol 27:331Ð344. Glekin B, Ford JA, Robertson I 1981 Policy for prevention of 119. Lauderdale DS, Jacobsen SJ, Furner SE, Levy PS, Brody JA, Asian rickets in Britain: A preliminary assessment of the Goldberg J 1998 Hip fracture incidence among elderly Glasgow rickets campaign. Br Med J 1:357Ð360. Hispanics. Am J Public Health 88:1245Ð1247. 101. Lo CW, Paris PW, Holick MF 1986 Indian and Pakistani 120. Reid IR, Cullen S, Schooler BA, Livingstone NE, Evans MC immigrants have the same capacity as Caucasians to produce 1990 Calciotropic hormone levels in Polynesians, evidence vitamin D in response to ultraviolet irradiation. Am J Clin against their role in interracial differences in bone mass. Nutr 44:683Ð685. J Clin Endocrinol Metab 70:1452Ð1456. 102. Rashid A, Mohammed T, Stephens WP, Warrington S, Berry JL, 121. Reid IR, Mackey M, Ibbertson HK 1986 Bone mineral con- Mawer EB 1983 Vitamin D state of Asians living in Pakistan. tent in Polynesian and white New Zealand women. Br Med J Br Med J 1:182Ð184. 2:1457Ð1458. 103. Lamberg B Allardt C, Karkkainen M, Seppanen R, Bistrom H 122. Chin K, Evans MC, Cornish J, Cundy T, Reid IR 1997 1993 Low serum 25-hydroxyvitamin D concentrations and Differences in hip axis and femoral neck length in 800 MEHRDAD RAHMANIYAN AND NORMAN H. BELL

premenopausal women of Polynesian, Asian and European 141. Dagnelie PC, Vergote FJVRA, van Staveren WA, origin. Osteoporos Int 7:344Ð347. van den Berg H, Dingjan PG, Hautvast JGAJ 1990 High 123. Abdullah MA, Salhi HS, Bakry LA, Okamoto E, prevalence of rickets in infants on macrobiotic diets. Am Abomelha AM, Stevens B, Mousa FM 2002 Adolescent J Clin Nutr 51:202Ð208. rickets in Saudi Arabia: A rich and sunny country. J Pediatr 142. Parsons TJ, van Dusseldorp M, van der Bliet M, van de Endocrinol Metab 15:1017Ð1025. Werken K, Schaafsma G, van Staveren WA 1997 Reduced 124. Fonseca V, Tongia R, El-Hazmi M, Abu-Aisha H 1984 bone mass in Dutch adolescents fed a macrobiotic diet in Exposure to sunlight and vitamin D deficiency in Saudi early life. J Bone Miner Res 12:1486Ð1494. Arabian women. Postgrad Med J 60:589Ð591. 143. Compston JE, Vedi S, Ledjer JE, Webb A, Gazet JC, 125. Serinius F, Elidrissy A, Dandona P 1984 Vitamin D nutrition Pilkington TR 1981 Vitamin D status and bone histomor- in pregnant women at term and in newly born babies in Saudi phometry. Am J Clin Nutr 34:2359Ð2363. Arabia. J Clin Pathol 37:444Ð447. 144. Rickers H, Christiansen C, Balslev I, Rodbro P 1984 Impairment 126. Sedrani SH 1984 Low 25-hydroxyvitamin D and normal of vitamin D metabolism and bone mineral content after serum calcium concentrations in Saudi Arabia: Riyadh intestinal bypass surgery. Scand J Gastroenterol 19:184Ð189. region. Ann Nutr Metab 28:181Ð185. 145. Bell NH, Epstein S, Greene A, Shary J, Oexmann MJ, Shaw S 127. Al-Arabi KM, Wahab A, Elidrissy TH, Sedrani SH 1984 Is 1985 Evidence for alteration of the vitamin D-endocrine avoidance of sunlight a cause of fractures of the femoral neck system in obese subjects. J Clin Invest 76:370Ð373. in elderly Saudis? Trop Geogr Med 36:273Ð279. 146. Liel Y, Ulmer E, Shary J, Hollis BW, Bell NH 1988 Low cir- 128. Ghannam NN, Hammami MM, Bakheet SM, Khan BA 1999 culating vitamin D in obesity. Calcif Tissue Int 43:199Ð201. Bone mineral density of the spine and femur in healthy Saudi 147 Bell NH, Epstein S, Shary J, Greene V, Oexmann MJ, Shaw S females: Relation to vitamin D status, pregnancy, and lacta- 1988 Evidence of a probable role for 25-hydroxyvitamin D tion. Calcif Tissue Int 65:23Ð28. in the regulation of calcium metabolism in man. J Bone Miner 129. Al Faraj S, Al Mutairi K 2003 Vitamin D deficiency and Res 3:489Ð495. chronic low back pain in Saudi Arabia. Spine 28:177Ð179. 148. Rosenstreich SJ, Rich C, Volwiler W 1971 Deposition in and 130. Dawodu A, Absood G, Patel M, Agarwal M, Ezimokhai M, release of vitamin D3 from body fat: Evidence for a storage Abdulrazzaq Y, Khalayli G 1998 Biosocial factors affecting site in the rat. J Clin Invest 50:679Ð687. vitamin D status of women of childbearing age in the United 149. Mawer EB, Backhouse J, Holman CA, Lumb GA, Stanbury SW Arab Emirates. J Biosoc Sci 30:431Ð437. 1972 The distribution and storage of vitamin D and its metabo- 131. Gannage-Yared MH, Brax H, Asmar A, Tohme A 1998 lites in human tissues. Clin Sci 43:414Ð431. [Vitamin D status in aged subjects. Study of a Lebanese 150. Epstein S, Bell NH, Shary J, Shaw S, Greene A, Oexmann MJ population]. Presse Med 27:900Ð904. 1986 Evidence that obesity does not influence the vitamin DÐ 132. Bassir M, Laborie S, Lapillonne A, Claris O, Chappuis MC, endocrine system in blacks. J Bone Miner Res 1:181Ð184. Salle BL 2001 Vitamin D deficiency in Iranian mothers and 151. Wortsman J, Matsuoka LY, Chen TC, Lu Z, Holick MF 2000 their neonates: A pilot study. Acta Paediatr 90:577Ð579. Decreased bioavailability of vitamin D in obesity. Am J Clin 133. Alagol F, Shihadeh Y, Boztepe H, Tanakol R, Yarman S, Nutr 72:690Ð693. Azizlerli H, Sandalci O 2000 Sunlight exposure and vitamin D 152. DeSimone DP, Stevens J, Edwards J, Shary J, Gordon L, deficiency in Turkish women. J Endocrinol Invest 23:173Ð177. Bell NH 1989 Influence of body habitus and race on bone 134. Meulmeester JF, van den Berg H, Wedel M, Boshuis PG, mineral density of the midradius, hip and spine in aging Hulshof KFAM, Luyken R 1990 Vitamin D status, parathy- women. J Bone Miner Res 4:827Ð830. roid hormone and sunlight in Turkish, Moroccan and Caucasian 153. Liel Y, Edwards J, Shary J, Spicer KM, Gordon L, Bell NH children in The Netherlands. Eur J Clin Nutr 44:461Ð470. 1988 The effects of race and body habitus on bone mineral 135. Shany S, Hirsh J, Berlyne GM 1976 25-Hydroxy- density of the radius, hip, and spine in premenopausal cholecalciferol levels in Bedouins in the Negev. Am J Clin Nutr women. J Clin Endocrinol Metab 66:1247Ð1250. 29:1104Ð1107. 154. Reid IR, Ames R, Evans MC, Sharpe S, Gamble F, France JT, 136. Elzouki AY, Markestad T, Elgarrah M, Elhoni N, Aksnes L Lim TMT, Cundy TF 1992 Determinants of total body and 1989 Serum concentrations of vitamin D metabolites in rachitic regional bone mineral density in normal postmenopausal Libyan children. J Pediatr Gastroenterol Nutr 9:507Ð512. women. A key role for fat mass. J Clin Endocrinol Metab 137. Gessner BD, Plotnik J Muth PT 2003 25-Hydroxyvitamin D 75:45Ð51. levels among healthy children in Alaska. J Pediatr 143: 155. Reid IR, Plank LD, Evans MC 1992 Fat mass is an important 434Ð437. determinant of whole body bone density in premenopausal 138. Pasco JA Henry MJ, Nicholson GC Sanders KM, Kotowicz MA women but not in men. J Clin Endocrinol Metab 75:779Ð782. 2001 Vitamin D status of women in the Geelong Osteoporosis 156. Dalen N, Hallberg D, Lamke B 1975 Bone mass in obese Study: Association with diet and casual exposure to sunlight. subjects. Acta Med Scand 197:353Ð355. Med J Aust 175:401Ð405. 157. Steiniche T, Vesterby A, Eriksen EF, Melsen F 1986 A histo- 139. Ladizesky M, Lu Z, Oliveri B, San Roman N, Diaz S, morphometric determination of iliac bone structure and Holick MF, Mautalen C 1995 Solar ultraviolet B radiation remodeling in obese subjects. Bone 7:77Ð82. and photoproduction of vitamin D3 in central and southern 158. Compston JE Verdi S, Gianetta E, Watson G, Civalleri D, parts of Argentina. J Bone Miner Res 10:545Ð548. Scopinaro N 1984 Bone histomorphometry and vitamin D 140. Outila TA, Karkkainen MU, Seppanen RH, Lamberg-Allardt CJ status after biliopancreatic bypass for obesity. Gastroenterology 2000 Dietary intake of vitamin D in premenopausal, healthy 87:350Ð356. vegans was insufficient to maintain concentrations of serum 159. Nilsson BE, Westlin NE 1971 Bone density in athletes. Clin 25-hydroxyvitamin D and intact parathyroid hormone within Orthop Related Res 77:179Ð182. normal ranges during the winter in Finland. J Am Diet Assoc 160. Granbed H, Jonson R, Hansson T 1987 The loads on the lumbar 100:434Ð441. spine during extreme weight lifting. Spine 12:146Ð149. CHAPTER 47 Effects of Race, Geography, Body Habitus, Diet, and Exercise on Vitamin D Metabolism 801

161. Colletti LA, Edwards J, Gordon L, Shary J, Bell NH 1989 aerobic capacity to bone density in older men and women. The effects of muscle-building exercise on bone mineral den- J Bone Miner Res 4:421Ð432. sity of the radius, spine, and hip in young men. Calcif Tissue 166. Bonaiuti D, Shea B, Iovine R, Negrini S, Robinson V, Int 45:12Ð14. Kemper HC, Wells G, Tugwell P, Cranney A 2002 Exercise 162. Conroy BP, Kraemer WJ, Maresh CM, Fleck SJ, Stone MH, for preventing and treating osteoporosis in postmenopausal Fry AC, Miller PD, Kalsky GP 1993 Bone mineral density in women. Cochrane Database Syst Rev 3:CD000333. elite junior Olympic weightlifters. Med Sci Sports Exercise 167. Kelley GA, Kelley KS, Tran ZV 2002 Exercise and lumbar 25:1103Ð1109. spine bone mineral density in postmenopausal women: A 163. Aloia JF, Cohn SH, Ostuni JA, Cane R, Ellis K 1978 Prevention meta-analysis of individual patient data. J Gerontol A Biol of involutional bone loss by exercise. Ann Intern Med 89: Sci Med Sci 57:M599ÐM604. 356Ð358. 168. Jacobson PC, Beaver W, Grubb SA, Taft TN, Talmadge RV 164. Krolner B, Toft B, Pors Nielson KS, Tondevold E 1983 1984 Bone density in women: College athletes and older Physical exercise as prophylaxis against involutional vertebral athletic women. J Orthop Res 2:328Ð332. bone loss: A controlled trial. Clin Sci 64:541Ð546. 169. Bell NH, Godsen RN, Henry DP, Shary J, Epstein S 1988 165. Bevier W, Wiswell R, Pyka G, Kozac K, Newhall K, Marcus R The effects of muscle building exercise on vitamin D and 1989 Relationship of body composition, muscle strength and mineral metabolism. J Bone Miner Res 3:369Ð373. CHAPTER 48 Perinatal Vitamin D Actions

NICHOLAS J. BISHOP Academic Department of Child Health, University of Sheffield, United Kingdom

I. Introduction VII. Late Neonatal Hypocalcemia II. The Last Trimester of Pregnancy VIII. Recommendations for Vitamin D Intake III. The Normal Term Infant in the Perinatal Period IV. The Term Growth-Retarded Infant IX. Summary V. The Premature Infant References VI. Infants of Diabetic Mothers

I. INTRODUCTION evidence for vitamin D deficiency causing osteomala- cia in the mother and abnormal skeletal metabolism in With the cutting of the umbilical cord, the provision the fetus and infant is strong [1Ð3]. Infants of severely of both mineral substrates and the placental factors that malnourished mothers may be born with rickets and had participated in the regulation of skeletal maturation suffer fractures in the neonatal period. Although much in utero ceases abruptly. The response of the neonate to of the literature relating to such infants was written early these sudden changes depends in part on the reserves in the 20th century, recent reports from European centres of calcium, phosphate, and vitamin D laid down during indicate the need for continued vigilance, particularly in pregnancy. The role of vitamin D in the pregnant mother immigrant or refugee populations. Four infants were born is dealt with elsewhere in this volume (see Chapter 51). with craniotabes to immigrant mothers with osteoma- Nevertheless, it is appropriate to include here a summary lacia in Berlin [2]. The infants exhibited typical bio- of the clinical consequences of deficiency or excess in chemical and radiological changes of rickets, which relation to the status of the infant at birth and during responded well to vitamin D therapy. Observational stud- the perinatal period. The events of the final trimester are ies suggest that radiographic bone density is reduced in important in establishing good nutritional reserves while both malnourished mothers and their infants [3], and maintaining rapid growth. Thus the period covered by this reduction can be ameliorated by calcium supple- this review spans the last 3 months of pregnancy and mentation during pregnancy. the first month of extrauterine life for infants born at term. For those born prematurely, the postnatal period covered is the first 3 months. In addition, because of B. Vitamin D Deficiency the potential for metabolic bone disease to develop as a result of inadequate calcium and phosphate intake in Vitamin D deficiency can of course occur in the preterm infants, vitamin D metabolism in this population absence of malnutrition. Vitamin D nutrition in pregnancy is considered in the light of mineral substrate provision. was investigated by Brooke and colleagues in 115 Asian (Pakistani, Hindu Indian, and East African Asian) women living in London, and in 50 of their newborn infants [4]. II. THE LAST TRIMESTER Maternal serum 25-hydroxyvitamin D (25OHD) con- OF PREGNANCY centration at the beginning of the last trimester was 20.2 nmol/liter (8.1 ng/ml), falling to 16.0 nmol/liter A. Malnutrition (6.4 ng/ml) after delivery. Postpartum, 36% of the women and 32% of the infants had undetectable 25OHD con- In countries where vitamin D supplementation of centrations (less than 3 nmol/liter or 1.2 ng/ml). Alkaline table milk is routine, vitamin D deficiency is unlikely phosphatase bone isoenzyme was elevated (compared to arise during pregnancy except in recent immigrants with appropriate well-nourished controls) in 20% of with chronic dietary insufficiency of calcium, vitamin the women postpartum, and in 50% of the infants. Five D, and other essential nutrients, in groups avoiding infants developed symptomatic hypocalcemia. dairy products for cultural or dietary reasons (e.g., Our own recent studies of vitamin D levels in the cord cow’s milk protein intolerance), and where sunlight blood of infants born to women pregnant during the exposure is negligible. In malnourished populations, spring and early summer months in Sheffield indicate VITAMIN D, 2ND EDITION Copyright © 2005, Elsevier, Inc. FELDMAN, PIKE, AND GLORIEUX All rights reserved. 804 NICHOLAS J. BISHOP widespread vitamin D insufficiency. More than groups, and the authors did not report any evidence of 60% of infants had cord blood levels of vitamin D active rickets in the infants. There were no differences below 20 nmol/liter. Of the subjects, 90% were white in birth weight or length between the groups. Caucasian (Am J Clin Nutr 2004). Further studies undertaken in France by Madelenat and colleagues examined the effect of 80,000 IU of vitamin D given in a single dose at around the end C. Vitamin D Supplementation of the second trimester. In this study, which recruited primarily white Caucasian women, 34% of women Maxwell and colleagues studied 126 Asian women had a serum 25-hydroxyvitamin D concentration in the whose mean serum 25OHD was 20 nmol/liter (8 ng/ml) osteomalacic range. Following supplementation only at the end of the second trimester. A double-blind study one woman continued to have a low 25-hydroxy- of supplementary vitamin D (1000 IU per day) versus vitamin D concentration at delivery. The study was placebo during the third trimester to Asian women undertaken during the winter months. There was no living in London was performed. There was increased evidence of vitamin D toxicity occurring in the women maternal weight gain (63 versus 46 g/day) and a 50% receiving this dose. reduction in the numbers of infants classified as Thus, maternal malnutrition with coexisting vitamin D “growth retarded” (born weighing less than 2500 g at deficiency can result in metabolic bone disease and term), which closely approached significance at the 5% disturbed calcium and vitamin D metabolism in the level, in the supplemented group [5]. Infants in the neonate. Vitamin D supplementation in the malnour- control group also had larger fontanelles, suggesting ished mother results in improved growth of the child delayed ossification. Further follow-up of the cohort both in terms of birth weight and also subsequent was reported to age 1 year, with the observers still linear growth during infancy. The neonatal metabolic blinded to the original randomization. There was bone disease resulting from maternal malnutrition is increasing divergence of the groups in terms of both amenable, at least in the short term, to standard treatment weight and length, so that by 1 year of age the infants with vitamin D and calcium supplements. There are no whose mothers had received the supplemental vitamin D long-term data on the outcome for infants treated in this during the third trimester were on average 0.4 kg heavier way. In women with better overall nutritional status, but and 1.6 cm longer than the control group [6]. not receiving routine vitamin D supplementation or Marya et al. gave vitamin D 600,000 IU twice (during consuming fortified foods, administration of vitamin D the seventh and eighth months of pregnancy), or a daily reduces the incidence of neonatal hypocalcaemia. supplement of 1200 IU vitamin D and 375 mg calcium per day throughout the third trimester, or placebo, to Hindu women living in India. The high-dose vitamin D III. THE NORMAL TERM INFANT supplement had a greater effect on infant birth weight and cord blood levels of calcium, inorganic phosphate, Stearns et al. in the 1930s showed that the rate of and alkaline phosphatase activity than the daily supple- linear growth and weight gain in normal infants was ment, which did not differ greatly in its effects from the related to vitamin D intake. Infants supplemented with placebo; however, there were no data regarding the com- 340 as opposed to 135 IU of vitamin D grew more pliance of the group taking the daily supplement [8]. rapidly in both weight and length [9]. Over the years In well-nourished mothers who do not receive there was a tendency to increase the amount of vitamin D vitamin D supplementation during pregnancy, the situ- given to infants so that by the 1950s some infants were ation is less clear. Delvin and colleagues studied the receiving over 2000 IU per day. Contemporaneously, effect of vitamin D supplementation from the end of a number of cases of “idiopathic hypercalcemia” were the first trimester in a group of French mothers [7]. reported. Hypervitaminosis D resulted in hypercalcemia They reported differences (comparing supplemented with polyuria leading to dehydration and its typical con- with unsupplemented controls) in vitamin D metabo- sequences, which were documented in some case lite levels at birth in both mothers and their infants. In reports. Of more concern, a recent report suggests that addition, the postnatal fall in plasma calcium in the some infants receiving intermittent high-dose vitamin D infants of unsupplemented mothers was more likely to prophylaxis may go on to develop nephrocalcinosis [10]. be associated with symptomatic hypocalcemia. In con- Normal term infants born to vitamin DÐsufficient trast with the studies of malnourished mothers detailed mothers have plasma levels of total vitamin D metabolites above, there were no differences in maternal blood cal- that correlate closely with those of their mothers [11]. cium or inorganic phosphate concentrations between A number of studies have reported that total levels of CHAPTER 48 Perinatal Vitamin D Actions 805

25OHD and 1,25-dihydroxyvitamin D [1,25(OH)2D], colleagues [19] demonstrated an increased incidence are decreased in cord blood compared with maternal of type I diabetes in young adults in Finland who as blood at the time of delivery [11Ð13]; unbound infants had not received the recommended dose of (free) metabolic levels of 25OHD, 24,25(OH)2D and vitamin D (2000 IU/day) [50]. The potential for early 25,26(OH)2D have been reported as higher in infants’ exposure to influence long-term outcomes such as blood, with free 1,25(OH)2D levels being equal [11]. diabetes and osteoporosis is of considerable interest as The first report of 1,25(OH)2D levels in infants born at the incidence of both these diseases is rising rapidly in term indicated that the initially low levels in cord all populations. blood rose to normal adult values by 24 hr of age [12]. Longitudinal measurement of vitamin D metabolites in the serum of breast-fed infants (not receiving vitamin D IV. THE TERM GROWTH- supplements) who were born to vitamin DÐreplete RETARDED INFANT mothers suggested that depletion of vitamin D stores occurs within 8 weeks of delivery in the majority [11]. There is no direct evidence that vitamin D metabolism In 1963 the American Academy of Pediatrics rec- is altered in the infant who is growth retarded (small for ommended that, for infants, daily intakes of vitamin D gestational age) due to uteroplacental factors rather be restricted to 400 IU from all sources [14]. These than maternal malnutrition. Reduced bone mineral recommendations are still regarded as appropriate by content and reduced serum 1,25(OH)2D and osteocalcin most pediatricians, although a recent report from the levels have been documented for growth retarded as Academy suggested that the recommended dietary opposed to term infants, but no difference in 25OHD allowance (RDA) should be 200 IU per day starting status was recorded [20]. The authors suggested that within the first 2 months of life and continuing through- reduced uteroplacental blood flow resulted in reduced out infancy, childhood, and adolescence [15]. The fetal production of 1,25(OH)2D and hence lower osteo- United Kingdom’s Department of Health recommends calcin and reduced bone mineral accretion, but reduced 340 IU vitamin D per day to age 6 months [16]. transfer of all nutrients including minerals is also likely Sufficient vitamin D is available in normal reconsti- to have contributed. tuted infant formulas to meet these recommendations. However, the vitamin D content of human milk is low. V. THE PREMATURE INFANT Unless the infant is exposed (face and hands) to sunlight for 10 min each day, there is a good case for A. Early Neonatal Hypocalcemia providing an oral supplement of vitamin D of up to 400 IU per day. The effect of vitamin D supplementa- Early neonatal hypocalcemia is a common event tion on bone mineralization in wholly breast-fed occurring in up to 75% preterm infants, chiefly those infants was investigated by Greer et al. [17]. Infants born with very low birth weight (under 1500 g) [21]. who received 400 IU per day of vitamin D had higher It is usually of short duration and does not express bone mineral content and serum 25OHD levels at age itself clinically in the majority of infants. Immaturity of 12 weeks than those not supplemented. The effect on the vitamin D activation pathway has been suggested as growth and bone mineralization beyond this period a major underlying factor either alone or in combina- remains unknown. tion with other abnormalities, particularly transient There is thus no evidence that increasing the level hypoparathyroidism, hypercalcitoninemia, and end-organ of vitamin D supplementation beyond 400 IU per day resistance to hormonal effects [22]. However, it has influences either linear growth or bone mass in the been clearly shown that there is an appropriate secre- immediate postnatal and infant period. Vitamin D tion of parathyroid hormone (PTH) in response to this supplementation in infancy may however have longer hypocalcemic stimulus [23,24]. This increase in serum term consequences. Zamora and colleagues have doc- immunoreactive PTH concentration appeared within umented a higher bone mineral mass of prepubertal the initial 24 hr after birth, with levels of both intact girls who were breast-fed as infants and who received PTH (1Ð84) and the carboxyl-terminal fragment vitamin D supplements [18]. This retrospective study (cPTH) following the same trend [25]. This physiolog- demonstrated increased areal bone mineral density ical response to a hypocalcemic stimulus is substanti- in the femoral neck in the supplemented group. The ated by the observation that the increment in increase in areal bone mineral density was thought to immunoreactive PTH levels was blunted when prema- arise primarily as a result in an increase in bone size. ture infants received calcium infusion; this calcium load A retrospective birth cohort study by Hyponnen and buffered the postnatal depression of serium calcium. 806 NICHOLAS J. BISHOP

By day 10 serum levels of PTH (1Ð84) and cPTH return D

to euparathyroid values [25,26]. 2 3.0

B. Vitamin D in the Neonate 2.0

In preterm as in full-term newborns, both total and (pmol/L) free 25OHD cord blood levels were lower than in 1.0 25OHD 1,25(OH) D maternal blood and were correlated to those of the 2 ree 25OHD or 1,25(OH) mothers [27Ð29]. Bouillon et al. [30] reported a positive F 0 correlation between maternal and cord serum concen- Cord 051015 20 25 30 trations of both total and free 1,25(OH)2D in premature Days of life babies; others found that only those of free 1,25(OH)2D FIGURE 2 Newborn serum free 25OHD and 1,25(OH)2D levels were correlated [27]. This discrepancy could be due to as a function of age. Values are means ± SEM. From Delvin et al. [23] the vitamin D depletion state of the subjects studied [25]. with permission. Cord and maternal blood vitamin D binding protein (DBP) levels were also positively related. Hirsfeld and Lunell [31] have excluded the possibility of a placental transfer of this protein by DBP polymorphism analysis; age, the plasma levels of 1,25(OH)2D were well above the most likely explanation for this fetomaternal rela- the range observed in reference adolescent groups tionship would therefore be common fetal and maternal [33Ð35]. This sharp elevation was probably linked to factors affecting its synthesis. hypocalcemia and the concomitant elevated PTH levels. Longitudinal trends in total and free 25OHD and Substrate concentration is a rate limiting factor in the 1,25(OH)2D estimates for preterm infants in the first synthesis of 1,25(OH)2D in the presence of hypocal- month of life are shown in Figs. 1 and 2. Many reports cemia, and thus a strong positive correlation between have clearly shown that in premature infants, after serum 25OHD and 1,25(OH)2D concentrations was 28 weeks of gestation, activation of vitamin D is opera- observed during the first 10 days of life over a wide tive as early as 24 hr after birth [23,32Ð35]. In European range of 25OHD levels [23,35]. This was well illustrated countries where dairy products are not enriched with by the report of Glorieux et al. [23] of twin preterm vitamin D, average levels of 25OHD in cord blood are babies in a controlled study of vitamin D supplementa- lower than those in North America [23]. Vitamin D tion. Their levels of 25OHD at birth were identical and supplementation (from 500 to 2000 IU/day) in French much higher than those measured in other preterm new- preterm infants just after birth improved vitamin D borns. In the protocol of early supplementation, one of nutritional status as evidenced by rising plasma the twins was assigned to the vitamin DÐsupplemented 25OHD levels. In addition, the administration of group. 1,25(OH)2D increased in both infants in a simi- vitamin D resulted in an increase in the circulating lar fashion and paralleled the average increase concentration of 1,25(OH)2D (Fig. 1). By 5 days of recorded in the supplemented group. This observation emphasizes the importance of maternal 25OHD ade- quacy during pregnancy and indicates its potential for limitation of 1,25(OH)2D production. 80 350 Serum bone Gla protein (BGP) values are high at birth 300 (15 + 3 ng/ml); maternal and cord serum BGP levels 60 250 were not correlated [36,37]. During the first month of life, serum BGP increases and parallels the changes in 200

40 D (pmol/L) ( ) 1,25(OH)2D but without sustained correlation. These 150 2 results indicate that serum BGP does not reflect changes 100 20 in serum 1,25(OH)2D but rather probably the overall

25OHD (nmol/L) ( ) 50

1,25(OH) rate of bone formation or growth at the tissue level. 0 0 Cord 0 5 10 15 20 25 30 Days of life C. Postnatal Vitamin D Supplementation

FIGURE 1 Newborn serum total 25OHD and 1,25(OH)2D levels as a function of age. Values are means ± SEM. From Delvin et al. [23] After the first week of life, in premature infants with permission. who received vitamin D, plasma 25OHD remained CHAPTER 48 Perinatal Vitamin D Actions 807

constant; 1,25(OH)2D concentration increased up to observations of ENH of premature infants [44]. day 30 with no further change until the end of the first Neither Salle et al. [44] nor Noguchi et al. [45] detected 3 months [35]. The levels of 1,25(OH)2D were more than any major impairment in PTH responsiveness in IDM. 2 or 3 times higher than those seen in older children. Thus, the pathogenesis of hypocalcemia of IDM During this time, there was no significant correlation remains unclear. Possibly, the increased fetal body size between vitamin D metabolite concentration and serum of these infants may be responsible for increased cal- calcium and phosphorus levels or calcium and phos- cium needs; the whole body bone mineral content of phorus intake. these babies measured by dual energy X-ray absorp- The high levels of plasma 1,25(OH)2D beyond the tiometry corresponds to that of a newborn baby of the neonatal period may represent a compensatory effect same weight [46]. to ensure calcium and phosphorus absorption from the A prospective controlled study has shown no evidence diet at a time where bone demineralization may occur. of hypocalcemia occurring in infants born to women Osteopenia is seen commonly in premature infants, with diet-controlled gestational diabetes [47]. particularly in those who have received prolonged peri- ods of parenteral feeding or who received a diet insuf- ficient in calcium and phosphate (European formula or VII. LATE NEONATAL HYPOCALCEMIA human milk). There is now a widespread agreement that deficiency of mineral substrate and not intake and Late neonatal hypocalcemia (LNH) is less frequent metabolism of vitamin D is the principal etiological than ENH and usually brought to attention by the clin- factor of osteopenia in low-birth-weight infants [38Ð40]. ical manifestations of tetany and convulsions [48,49]. Backstrom and colleagues conducted a randomized These are observed from the third or fourth day up to controlled trial of vitamin D supplementation on bone the end of the first month of life. LNH generally affects density and biochemical indices in preterm infants [41]. term infants, but it is also observed in premature infants Thirty-nine infants aged 32 weeks gestation at birth and IDM in the form of prolonged and severe ENH or less received either 200 IU/kilo of vitamin D (max- (see earlier discussion). A now-uncommon cause is the imum 400 IU/day) or 960 IU/day up to age 3 months. feeding of “doorstep” (unmodified) cow’s milk in Bone mass was determined by dual energy X-ray which vitamin D content is not controlled. Moreover, absorptiometry at the distal end shaft site of the left its large phosphate content suppresses production of forearm. At 3 and 6 months corrected age there was no 1,25(OH)2D. difference between the groups in bone mineral content Hypocalcemia resulting in convulsions can also or areal bone mineral density. The authors concluded occur as a result of osteopetrosis [50], maternal hyper- that a directly administered vitamin D for preterm infants parathyroidism [51], and maternal calcium carbonate of 200 IU/kg body weight per day up to a maximum of consumption during pregnancy [52]; in one case it was 400 IU provided biochemical evidence of vitamin D reported as a result of a phosphate enema administered sufficiency, and no functional differences were observed to an ex-premature infant at age 6 weeks [53]. Heart in the study period between this and the higher dose of failure can occur in some instances and may be misdi- 960 IU/day. agnosed as cardiomyopathy [54]. In late neonatal hypocalcemia, serum PTH levels remain inappropriately low (normal values). Relative VI. INFANTS OF DIABETIC MOTHERS hypoparathyroidism thus appears to be the main abnor- mality but is transient and not due to the absence or Hypocalcemia in infants of diabetic mothers (IDM) hypoplasia of the parathyroid glands as found in the Di has been the subject of a number of studies, and sev- George syndrome [55]. Serum 1,25(OH)2D remains eral pathogenic factors have been suggested in this normal to moderately elevated, corresponding to what metabolic disorder including hypoparathyroidism, one would expect in response to the low serum PTH hyperphosphatemia, hypomagnesemia, and defective levels. This contrasts with the sharp increase in serum vitamin D metabolism [42,43]. Hypocalcemia of IDM 1,25(OH)2D levels observed in infants with ENH [25]. shares some features with the early neonatal hypocal- Severe hypocalcemia occurs in perinatal asphyxia cemia (ENH) observed in premature babies. Indeed, it when hypoxemia and acidosis persist, despite raised appears during the very early hours of life and shows serum PTH levels, suggesting possible end-organ little further change after 24 hr of age. However, it resistance [56] in the context of generally sick or failing tends to be more severe than ENH and to persist for a cells. longer time. No consistent abnormality in vitamin D Mild LNH requires only a watchful eye, but metabolism has been observed in IDM, similar to the symptomatic and persistent LNH may require more 808 NICHOLAS J. BISHOP aggressive intervention. The therapeutic management 2 months of life, continuing until adulthood [59]. It of LNH based on the pathophysiological findings would should be noted that all the current recommendations are be to apply the treatment strategy of hypoparathy- based on biochemical measures of vitamin D sufficiency roidism. The active form of vitamin D [1,25(OH)2D] utilizing a variety of cutoff points for serum 25OHD may be effective. In most cases the treatment can be between 20 and 30 nmol/liter (8Ð12 ng/ml). Definitive discontinued after a few days without relapse of studies relating exposure to functional outcome are more hypocalcemia. In the case of symptomatic hypocal- difficult to conduct and are still awaited, in particular for cemia with convulsions, intravenous calcium is recom- vitamin D supplementation during pregnancy. mended (1Ð2 mmol/kg over 30Ð60 min, preferably by a central line), within the context of managing the underlying clinical situation. IX. SUMMARY

Maternal vitamin D intake during the last trimester of VIII. RECOMMENDATIONS FOR pregnancy significantly influences neonatal vitamin D VITAMIN D INTAKE IN THE stores and metabolism and may influence growth in PERINATAL PERIOD infancy. There is no impairment of neonatal vitamin D metabolism consequent on “immaturity,” whatever the Recommendations for vitamin D supplementation gestational age or birth weight of the infant. All mothers in pregnancy are currently hard to give since there have should receive an adequate vitamin D intake during the been no studies of functional outcomes across a range last trimester of pregnancy, and all infants should receive of populations encompassing both those to be per- vitamin D in their diet, either as a supplement when the ceived to be at risk, such as mothers with dark skins, infant is completely breast-fed or as part of a modified reduced sunlight exposure, and vitamin DÐdeficient cow’s milkÐderived formula. There is no place for the diets, and those traditionally perceived to be likely to use of active vitamin D metabolites in the routine care be vitamin D sufficient, such as white Caucasian mothers of healthy infants. with good sunlight exposure and taking foods fortified in some way with vitamin D. The available evidence suggests that 400 units a day during the whole of preg- nancy, 1000 units per day over the last 3 months of References pregnancy, or up to 100,000 units at monthly intervals during the last 3 months of pregnancy are all well 1. Coutinho M de L, Dormandy TL, Yudkin S 1968 Maternal malabsorption presenting as rickets. Lancet 1:1048Ð1052. tolerated, safe, and effective methods of delivering 2. Park W, Paust H, Kaufmann HJ, Offermann G 1987 vitamin D to pregnant women. Osteomalacia of the mother Ð rickets of the newborn. Eur J Pediat The vitamin D requirements of low-birth-weight 146:292Ð293. infants are influenced by the body stores at birth, which 3. Krishnamachari KAVR, Iyengar L 1975 Effect of maternal in turn are related to the length of gestation and maternal malnutrition on the bone density of neonates. Am J Clin Nutr 28:482Ð486. stores. These factors should be taken into considera- 4. Brooke OG, Brown IRF, Cleeve HJW, Sood A 1980 tion when deciding on the policy concerning vitamin D Observations on the vitamin D state of pregnant Asian women supplementation in each country. The American in London. Br J Obstet Gynaecol 88:18Ð26. Academy of Pediatrics recommended that daily intake 5. Maxwell JD, Ang L, Brooke OG, Brown IR 1981 Vitamin D should be at least 400 IU independently of the vitamin D supplements enhance weight gain and nutritional status in pregnant Asians. Br J Obstet Gynaecol 88:987Ð991. content of low-birth-weight formula [57]. The European 6. Brooke OG, Butters F, Wood C 1981 Intrauterine vitamin D Society of Pediatric Gastroenterology and Nutrition nutrition and postnatal growth in Asian infants. Br Med J Clin recommended that when low-birth-weight infants are fed Res 283:1024. human milk they should receive a vitamin D supplement 7. Delvin EE, Salle BL, Glorieux FH, Adeleine P, David LS 1986 of 1000 IU per day [58]. The work of Backstrom [41] Vitamin D supplementation during pregnancy: Effect on neonatal calcium homeostasis. J Pediatr 109:328Ð334. detailed earlier suggests that where maternal vitamin D 8. Marya RK, Rathee S, Lata V, Mudgil S 1981 Effects of stores are thought to be normal, 200 IU/kg is likely to vitamin D supplementation in pregnancy. Gynecol Obstet Invest be adequate. 12:155Ð161. Formula-fed infants should also be supplemented 9. Stearns G, Jeans PC, Vandecar V 1936 The effect of vitamin D with vitamin D in order to achieve the same intake as on linear growth in infancy. J Pediatr 9:1Ð10. 10. Misselwitz J, Hesse V, Markestad T 1990 Nephrocalcinosis, babies receiving breast milk. The American Academy hypercalciuria and elevated serum levels of 1,25-dihydroxy- of Pediatrics recently recommended that daily intake vitamin D in children. Possible link to vitamin D toxicity. Acta should be 200 IU/day, commencing within the first Paediatr Scand 79:637Ð643. CHAPTER 48 Perinatal Vitamin D Actions 809

11. Hoogenboezem T, Degenhart J, Munick Keizer-Schrama SM, 28. Delvin E, Glorieux F, Salle B, David L, Varenne J 1982 Control Bouillon R, Grose WF, Hackeng WH, Visser HK 1989 Vitamin D of vitamin D metabolism in preterm infants: Fetomaternal rela- metabolism in breast-fed infants and their mothers. Pediatr Res tionships. Arch Dis Child 57:754Ð757. 25:623Ð627. 29. Hillman LS, Haddad JG 1975 Perinatal vitamin D metabolism II. 12. Steichen JJ, Tsang RC, Gratton TL, Hamstra A, DeLuca HF Serial 25-hydroxyvitamin D concentrations in sera of term and 1980 Vitamin D homeostasis in the perinatal period: preterm infants. J Pediatr 86:928Ð935. 1,25-Dihydroxyvitamin D in maternal, cord, and neonatal 30. Bouillon R, van Assche FA, van Baelen H, Heyns W, De Moor P blood. N Engl J Med 302:315Ð319. 1981 Influence of the vitamin DÐbinding protein on the serum 13. Delvin EE, Salle BS, Glorieux FH 1991 Vitamin D and concentrations of 1,25-dihydroxyvitamin D3: Significance of calcium homeostasis in pregnancy: Feto-maternal relation- the free 1,25-dihydroxyvitamin D3 concentration. J Clin Invest ships. In: Glorieux FH (ed) Rickets. Vevey/Raven, New York, 67:589Ð596. pp. 91Ð105. 31. Hirsfeld J, Lunell O 1963 Serum protein synthesis in foetus: 14. American Academy of Pediatrics Committee on Nutrition Haptoglobins and group-specific components. Nature 196: 1963 The prophylactic requirements and the toxicity of 1220Ð1222. vitamin D. Pediatrics 31:512. 32. Salle BL, Glorieux FH, Delvin EE, David LS, Meunier G 1983 15. Gartner LM, Greer FR Section on Breastfeeding and Vitamin D metabolism in preterm infants. Serial serum cal- Committee on Nutrition 2003 Prevention of rickets and citriol values during the first four days of life. Acta Paediatr vitamin D deficiency: New guidelines for vitamin D intake. Scand 72:203Ð206. Pediatrics 111: 908Ð910. 33. Markestad T, Elzouki A, Legrain M, Ulstein M, Asknes L 1984 16. HMSO Report Number 41 1991 Dietary and nutrient reference Serum concentration of vitamin D metabolites in maternal values for food energy and nutrients for the United Kingdom. and umbilical cord blood of Libyan and Norwegian women. United Kingdom Department of Health, London. Hum Nutr Clin Nutr 38:55Ð62. 17. Greer FR, Searcy JE, Levin RS, Steichen JJ, Asch PS, Tsang RC 34. Schilling R, Haschke F, Schatten C, Schmid M, Woloszczuk W, 1981 Bone mineral content and serum 25-hydroxyvitamin D Steffan I, Schuster E 1990 High total and free 1,25-dihydroxy- concentrations in breast-fed infants with and without supple- vitamin D concentrations in serum of premature infants. Acta mented vitamin D. J Pediatr 98:696Ð701. Paediatr Scand 79:36Ð40. 18. Zamora SA, Rizzoli R, Belli DC, Slosman DO, Bonjour JP 35. Salle BL, Senterre J, Glorieux FH, Delvin EE, Putet G 1987 1999 Vitamin D supplementation during infancy is associated Vitamin D metabolism in preterm infants. Biol Neonate with higher bone mineral mass in prepubertal girls. J Clin 52:119Ð130. Endocrinol Metab 84:4541Ð4544. 36. Delmas PD, Glorieux FH, Delvin EE, Salle BL, Melki I 1987 19. Hypponen E, Laara E, Reunanen A, Jarvelin MR, Virtanen SM Perinatal serum bone Gla-protein and vitamin D metabolites in 2001 Intake of vitamin D and risk of type 1 diabetes: a birth- preterm and full term neonates. J Clin Endocrinol Metab cohort study. Lancet 358:1500Ð1503. 65:588Ð591. 20. Namgung R, Tsang RC, Specker BL, Sierra RI, Ho ML 1993 37. Pittard WBD, Geddes KM, Hulsey TC, Hollis BW 1992 Reduced serum osteocalcin and 1,25-dihydroxyvitamin D Osteocalcin, skeletal alkaline phosphatase, and bone mineral concentrations and low bone mineral content in small for content in very low birth weight infants: A longitudinal assess- gestational age infants: Evidence of decreased bone formation ment. Pediat Res 31:181Ð185. rates. J Pediatr 122:269Ð275. 38. Tsang RC, Demarini S 1995 Rickets and calcium and phos- 21. Rosli A, Fanconi A 1973 Neonatal hypocalcemia. “Early phorus requirements in very low birth weight infants. type” in low birth weight newborns. Helv Paediatr Acta 28: Monatsschr Kinderkeilkd 43:S125ÐS129. 443Ð457. 39. Shaw NJ, Bishop NJ 1995 Mineral accretion in growing bones— 22. Tsang RC, Light IJ, Sutherland JM, Kleinman LI 1973 A framework for the future? Arch Dis Child 72:177Ð179. Possible pathogenetic factors in neonatal hypocalcemia of pre- 40. Lapillonne A, Glorieux FH, Salle BL, Braillon PM, Chambon M, maturity. The role of gestation, hyperphosphatemia, hypomagne- Rigo J, Putet G, Senterre J 1994 Mineral balance and whole semia, urinary calcium loss, and parathormone responsiveness. body bone mineral content in very low-birth-weight infants. J Pediatr 82:423Ð429. Acta Paediatr 405(Suppl):117Ð122. 23. Glorieux FH, Salle BL, Delvin EE, David LS 1981 Vitamin D 41. Backstrom MC, Maki R, Kuusela AL, Sievanen H, Koivisto AM, metabolism in preterm infants: Serum calcitriol values during Ikonen RS, Kouri T, Maki M 1999 Randomised controlled trial the first five days of life. J Pediatr 99:640Ð643. of vitamin D supplementation on bone density and biochemi- 24. David L, Salle BL, Chopard JP, Frederich A 1976 Parathyroid cal indices in preterm infants.Arch Dis Child Fetal Neonatal function in low birth weight newborns during the first 48 hours Ed. 80:F161ÐF166. of life. In: Stern F-H (ed) Symposium on Intensive Care of the 42. Tsang RC, Chen IW, Friedman FA, Gigger M, Steichen J, Newborn. Masson, New York, pp. 107Ð117. Koffler H, Fenton L, Brown D, Pramanik A, Keenan W, Strub R, 25. Salle BL, Delvin EE, Lapillonne A, Bishop NJ, Glorieux FH. Joyce T 1975 Parathyroid function in infants of diabetic mothers. 2000 Perinatal metabolism of vitamin D. Am J Clin Nutr J Pediatr 86:399Ð404. 71(5 Suppl):1317SÐ1324S. 43. Bergman L, Kjellmer I, Seltam U 1974 Calcitonin and parathy- 26. David L, Salle BL, Putet G, Grafmeyer D 1981 Serum roid hormone. Relation to early neonatal hypocalcemia in immunoreactive calcitonin in low birth weight infants. infants of diabetic mothers. Biol Neonate 24:151Ð160. Description of early changes: Effect of intravenous calcium 44. Salle BL, David L, Glorieux FH, Delvin EE, Louis JJ, Troncy G infusion: relationships with early changes in serum calcium, 1982 Hypocalcemia in infants of diabetic mothers. Studies on phosphorus, magnesium, parathyroid hormone, and gastrin circulating calciotropic hormone concentrations. Acta Paediatr levels. Pediatr Res 15:803Ð808. Scand 71:573Ð577. 27. Delvin EE, Salle BL, Glorieux FH, David LS 1988 Vitamin D 45. Noguchi A, Eren M, Tsang R 1980 Parathyroid hormone in metabolism in preterm infants: Effect of a calcium load. Biol hypocalcemia and normocalcemic infants of diabetic mothers. Neonate 53:321Ð326. J Pediatr 97:112Ð114. 810 NICHOLAS J. BISHOP

46. Lapillonne A, Guerin S, Braillon P, Claris O, Delmas PD, 53. Walton DM, Thomas DC, Aly HZ, Short BL 2000 Morbid Salle BL 1997 Diabetes during pregnancy does not alter whole hypocalcemia associated with phosphate enema in a six-week-old body bone mineral content in infants. J Clin Endocrinol Metab infant. Pediatrics 106:E37. 82:3993Ð3997. 54. Gulati S, Bajpai A, Juneja R, Kabra M, Bagga A, Kalra V 2001 47. Sarkar S, Watman J, Seigel WM, Schaeffer HA 2003 A Hypocalcemic heart failure masquerading as dilated cardiomy- prospective controlled study of neonatal morbidities in infants opathy. Indian J Pediatr 68:287Ð90. born at 36 weeks or more gestation to women with diet- 55. Perez E, Sullivan KE 2002 Chromosome 22q11.2 deletion controlled gestational diabetes (GDM-class A1). J Perinatol syndrome (DiGeorge and velocardiofacial syndromes). Curr 23:223Ð228. Opin Pediatr 14:678Ð683. 48. Balsan S, Alizon M 1968 L’hypoparathyroidie transitoire 56. Schedewie HK, Odell WD, Fisher DA, Krutzik SR, Dodge M, idiopathique du nourrison. Arch Fr Pediatr 25:1151Ð1170. Cousins L, Fiser WP 1979 Parathormone and perinatal calcium 49. Fanconi A, Prader A 1967 Transient congenital idiopathic homeostasis. Pediatr Res 13:1Ð6. hypoparathyroidism. Helv Paediatr Acta 22:342Ð359. 57. American Academy of Pediatrics, Committee on Nutrition 50. Srinivasan M, Abinun M, Cant AJ, Tan K, Oakhill A, Steward 1977 Nutritional needs of low birth weight infants. Pediatrics CG 2000 Malignant infantile osteopetrosis presenting with 60:519Ð530. neonatal hypocalcaemia. Arch Dis Child Fetal Neonatal Ed 58. European Society of Paediatric Gastroenterology and 83:F21ÐF23. Nutrition, Committee on Nutrition of the Preterm Infant 1987 51. Ip P 2003 Neonatal convulsion revealing maternal hyper- Nutrition and feeding of preterm infants. Acta Paediatr Scand parathyroidism: An unusual case of late neonatal hypoparathy- 336(Suppl):6. roidism. Arch Gynecol Obstet. 268:227Ð229. 59. Gartner LM, Greer FR, Section on Breastfeeding and 52. Robertson WC Jr 2002 Calcium carbonate consumption Committee on Nutrition. American Academy of Pediatrics during pregnancy: An unusual cause of neonatal hypocalcemia. 2003 Prevention of rickets and vitamin D deficiency: new J Child Neurol 17:853Ð855. guidelines for vitamin D intake. Pediatrics 111:908Ð910. CHAPTER 49 Vitamin D Deficiency and Calcium Absorption during Infancy and Childhood

STEVEN A. ABRAMS USDA/ARS Children’s Nutrition Research Center, Houston, Texas

I. Introduction V. Calcium Absorption in Adolescents II. Premature Infants VI.Fortification of Foods with Calcium and Vitamin D for Children III. Full-Term Infants VII. Summary and Conclusions IV. Toddlers and Prepubertal Children References

I. INTRODUCTION intake of calcium and vitamin D needed to prevent severe bone loss or rickets, is not well described. Long- Although the importance of providing adequate term consequences of variations in calcium absorption calcium and vitamin D during childhood and adolescent and bone mineralization in early life are not known. growth is well known, there remain important gaps in Despite the evidence that most infant formulas provide our understanding regarding the process of calcium at least as much, if not more, absorbable calcium than absorption and utilization in childhood. Certain time human milk, some data suggest that, in later childhood, periods in development appear to be critical ones in the bone mass of infants who are breastfed may be the which calcium or vitamin D deficiency can pose very same or greater than that of formula-fed infants [4,5]. high risks. The first such time periods is in utero, or as An important issue then is to determine at what age commonly reflected in current medical care, the initial it becomes critical to maximize mineral intake and months of life of prematurely delivered infants (also absorption to lead to an optimal peak bone mass. see Chapter 48). These infants, especially those < 1.5 kg Some controlled trials have indicated that supplemen- at birth, are at very high risk for the development of tation of calcium before puberty may be important, clinical rickets or other manifestations of bone loss. whereas others have found benefit in pubertal child- This bone mineral deficiency is primarily related to ren [6]. Interpreting such studies is difficult, however, difficulties in providing, via parenteral or enteral sources, as prestudy calcium intakes are often poorly assessed adequate calcium and phosphorus for the extremely or controlled and supplemented intakes may exceed rapid bone growth that normally occurs via placental the absorptive threshold, leading to little benefit. transfer to the fetus [1]. In healthy full-term infants, Because it is the time of most rapid mineralization human milk is recommended as the sole nutritional of the skeleton, efforts to improve peak bone mass source for the first 6 months of life [2]. The vitamin D during childhood have focused on increasing absorbable content of human milk is very low and the photo- calcium intake in adolescents. Efforts in this regard conversion of vitamin D precursors to vitamin D is include advocacy campaigns to increase dairy products necessary to obtain adequate vitamin D levels. Since and considerable efforts to provide fortified foods and this may not be possible in many infants, it is recom- beverages to adolescents. Recent research efforts have mended that all infants receive supplemental vitamin D, looked at the effects of race, diet, gender, and other food either via infant drops or, for infant formula-fed infants, components on maximizing calcium absorption [6,7]. as provided in the formula [3]. As part of global efforts to develop nutritional plan- Although vitamin DÐdeficient rickets is a well- ning, consideration has been given to the role of calcium described problem for older infants and toddlers, and vitamin D supplements in food products designed extremely little information is available regarding for children in developing countries [8]. Although many vitamin D requirements and the relationship between of these are countries in which children are exposed to vitamin D status and calcium absorption in this age adequate sunshine, they may also have low calcium group. The optimal vitamin D intake or concentration and vitamin D status due to social or dietary causes. to maximize calcium absorption, as well as the lowest Extending research in calcium absorption to these VITAMIN D, 2ND EDITION Copyright © 2005, Elsevier, Inc. FELDMAN, PIKE, AND GLORIEUX All rights reserved. 812 STEVEN A. ABRAMS populations is important not only to prevent bone loss, The bioavailability of the calcium in these forti- but also to understand and prevent an increase in osteo- fiers may be a key aspect of their adequacy. Using a porosis in developing countries in the future. commercially available human milk fortifier, Schanler and Abrams [1] reported that net calcium retention was 104 ± 36 mg/kg/d in premature infants, a value approx- II. PREMATURE INFANTS imating the in utero accretion rate during the third trimester. These retention values are well above those Human milk is recognized as the ideal food for achieved using earlier human milk fortifiers [16] and virtually all infants [2]. The substantial health and those more recently reported using a human milk for- developmental benefits of human milk feeding have tifier not used in the United States [17]. That fortifier been well documented even in the smallest of prema- led to minimally positive calcium retention and there- ture infants. However, for many decades, it has been fore little if any benefit to the baby. It is not clear why known that one critical limitation is that the minerals, the bioavailability of the calcium in the fortified feed- especially the calcium and phosphorus, in human ing was low, but it is likely related either to the form of milk do not meet the needs of rapidly growing prema- the calcium or problems with the solubility of the cal- ture infants. This and other factors place premature cium in the fortified milk. infants at high risk for nutritional rickets (Table I). Of interest is that the calcium absorption percent- This can readily be seen in that the fetus accretes ages from both fortified human milk and from special- 100Ð120 mg/kg/d of calcium (about 50Ð60 mg/kg/d of ized preterm formula average 50Ð65% in many studies phosphorus) during the third trimester. Since human [13,14]. This percentage absorption is similar to that milk contains about 25 mg/dl of calcium (13 mg/dl of seen for unfortified human milk in both premature phosphorus) and is usually fed at 150Ð200 mg/kg/d, infants and full-term infants. However, in the case even if all of the calcium and phosphorus were to be of preterm formulas or fortified milk, the calcium completely absorbed and retained, it cannot provide intake is about 220 mg/kg/d. This intake vastly exceeds more than half of the minerals needed by a 1.0 kg the body-weight adjusted calcium intake from human infant to meet the in utero accretion rate [1,9Ð11]. In milk by full-term infants (10 mg/kg/d for a 10-kg reality, calcium absorption in unfortified human milk- infant) [5,12]. This constancy of absorptive fraction in fed infants is generally about 60%, leading to a net premature infants suggests that much of the calcium absorption of about 20Ð30 mg/kg/d or less than 1/3 of absorption by premature infants and newborn full-term the in utero rate [12Ð14]. infants is not vitamin DÐdependent. In a review of more The recent addition of various forms of mineral salts than 100 balance studies, Bronner et al. [13] showed and/or mineral fortifiers to human milk and the use of that the calcium absorption fraction varied little with specialized preterm infant formulas with very high cal- calcium intake in premature infants and thereby cium content levels have been shown to enhance the suggest that most if not virtually all calcium absorption amount of calcium and other minerals retained from is vitamin DÐindependent. the diet, to increase bone mineral content of the Unfortunately, there are no studies of calcium infants, and to decrease the incidence of osteopenia absorption in premature infants over a broad range of and frank rickets in preterm infants [1,15,16]. calcium intakes to directly determine the effects of calcium intake and vitamin D status on calcium absorp- tion. Such studies would be virtually impossible to do on a practical and ethical basis. Multiple studies have TABLE I High-Risk Criteria for Osteopenia in Premature Infants demonstrated that vitamin D intakes of 400 IU/day (or 200 IU/kg up to 400 IU/d) in premature infants • Born at < 27 weeks’ gestation leads to adequate vitamin D levels [18Ð20]. One study • Birth weight of <1000 g demonstrated adequate 25-hydroxyvitamin D concen- • Long-term parenteral nutrition trations and clinical outcomes with oral vitamin D • Severe bronchopulmonary dysplasia with use of diuretics intakes as low as 160 IU/d [21]. and fluid restriction In addition, studies have generally failed to show • Long-term steroid use any clinical benefit of increasing vitamin D intake • History of necrotizing enterocolitis above 400 IU/day in preterm infants. One study com- paring 500 IU to 1000 IU found no short-or long-term • High serum alkaline phosphatase activity (>900 IU/liter) and low serum phosphorus (<5.2 mg/dl) benefit (up to age 11) of higher amounts [22]. “There are no data to support the belief that preterm infants CHAPTER 49 Calcium Absorption in Children 813 need a disproportionately high vitamin D dose in rela- come from human milk, but there is some from solid tion to their weight” [22]. foods. The Adequate Intake for 7- to 12-month-old The effects of other formula components on mineral infants was therefore established as 270 mg/day, which absorption have also been considered. A study using is the sum of the usual intake of calcium from breast a triple lumen perfusion technique demonstrated that milk and solid foods. calcium absorption was greater using a solution that Infant formulas have a calcium concentration well included a glucose polymer than one with lactose [23]. above that usually found in human milk [28,29]. One As glucose polymers are widely used in preterm for- justification for providing more calcium in infant mulas, this effect may be clinically important. Altering formulas than human milk is the belief that calcium is the fat blend of infant formula to more closely resem- more poorly absorbed from infant formula than from ble that of human milk may also enhance mineral human milk. This perspective is based on studies in absorption in premature infants [24,25]. which calcium concentrations were much greater in formula than in human milk [5]. These high concentra- III. FULL-TERM INFANTS tions may lead to lower fractional calcium absorption in the infants. This inverse relationship was demonstrated A. Calcium by Devizia et al. [30] who reported decreasing frac- tional absorption in a very small group of infants as Infancy is a time of rapid body growth as infants formula concentration of calcium increased. may triple their birth weight in the first year of life. Several studies have suggested that in many cases, This rapid body growth is accompanied by comparably the fractional absorption of calcium from infant for- rapid bone growth [26]. Remarkably, however, the mulas [31Ð35] is similar to the value for human milk human milkÐfed baby readily mineralizes with all of (Table III). Studies of whole-body bone mineral con- the calcium coming from mother’s milk in the first tent using DXA support these findings. Calculations 6 months and most of it from human milk during the both from Fomon and Nelson [5] and from earlier data second 6 months of life [27]. This is possible because using metacarpal morphometry [36] suggest a mean of the withdrawal of bone from the maternal skeleton calcium accretion rate of approximately 80 mg/day to meet the infant’s needs and the continued high rates during the first year of life [28]. More recent bone min- of calcium absorption from human milk. eral content studies in breast-fed infants have shown Dietary recommendations for calcium intake in slightly higher rates of bone mineralization of approx- infancy are based on the knowledge that calcium- imately 100 mg/d during the first year of life [27]. deficiency rickets does not occur in healthy, vitamin DÐ Although there are few direct comparisons with sufficient, breast-fed infants. Therefore, the calcium formula-fed infants, recent studies suggest values of intake of the exclusively breast-fed infant, averaging about 150 mg/day in the first 6 months of life [26,37] 210 mg/day, was established by the National Academy for formula-fed infants. This rate appears not to change of Sciences [28], and set as the Adequate Intake for substantially in the second 6 months of life after more calcium in the first 6 months of life (Table II). In the solid foods are introduced [37,38]. More data are second 6 months of life, most calcium continues to needed though, especially in the second 6 months of life, to evaluate the relationship between rates of bone mineralization and diet and vitamin D status. With these high absorption fractions, it appears that TABLE II Calcium Recommendations for Infants and Children in the United States [28] it is possible to markedly increase total calcium absorption and net calcium retention in infants above Calcium (mg/d) that of primarily human milkÐfed infants. However, caution should be raised about the benefits of targeting Infants 0Ð6 mo high levels of calcium absorption in infancy, either Breast-fed 210 with high-mineral-containing formulas, or through the Formula-fed 315 use of highly calcium-fortified solid foods for older Infants 7Ð12 mo infants. Breast-fed 270 There are no data to support any long-term benefit Formula-fed 335 by exceeding the calcium absorption or bone mass of Children 1Ð3 yrs 500 breast-fed infants. A study by Jones et al. [4] found a Children 4Ð8 yrs 800 greater bone mineral density at the spine and whole body in 8-year-old children who had been breast-fed 814 STEVEN A. ABRAMS

TABLE III Calcium Absorption Fraction in Human Milk and Formula-Fed Infants Based on a Reference Intake of 780 ml/day

Ca concentration (mg/dl) Absorption (%) Net Ca absorption (mg/d)

Human milk [32] 25 61 ± 23 97 Standard Formula [33] 50 58 ± 13 205 Partially hydrolyzed [31] 46 66 ± 12 220 Human milk [5] 25 58 ± 17 113 Cow milk-based formula [35] 57 57 ± 15 255

Notes: An estimate was used for endogenous fecal calcium excretion for isotope studies and the net absorption for isotope-based studies was adjusted for this value.

compared to those bottle-fed as infants. This effect was Therefore, the American Academy of Pediatrics [3] only present for infants who had been breast-fed for has recommended universal vitamin D supplementa- at least 3 months. Studies in preterm infants have also tion for all infants. For breast-fed infants, the vitamin D failed to show any long-term benefit to greater mineral is provided as part of a supplement drop; for formula-fed intake during early infancy [39,40]. This view is sup- infants it is contained in the formula [Table IV]. Based ported by animal data in rabbits that do not show any on the recommendations of the Food and Nutrition benefit to increasing bone mineral content in early life Board in the Dietary Reference Intakes as revised in [41] and is consistent with similar classic data from 1997, the recommendation is to provide 200 IU/day to Gershoff et al. [42]. infants beginning by 2 months of age [3,28], Of note is that as vitamin DÐdeficient rickets is not usually seen in early infancy, it is not crucial to begin supplementation B. Vitamin D at birth, but as the policy allows, to delay beginning the vitamins for up to 2 months. Nutritional rickets in children is described in detail The choice of 200 IU/day is based on minimal data in Chapter 65. In this section, we will consider some primarily obtained in a very few infants regarding the specific issues related to the use of vitamin D supple- amount of vitamin D required to maintain adequate ments for infants, especially those in the United States. levels [3,28]. Many more data are needed on this topic, Human milk is a relatively small source of dietary however, and given the high level of safety of vitamin D vitamin D for most infants, usually providing an aver- in this dose range, it is possible that higher doses may age of 10Ð20 IU/day [43,44]. Increased vitamin D in be needed to optimize vitamin D status [46]. It is very human milk is related to increasing maternal vitamin D likely, however, that the dose of 200 IU/day is adequate status, but the level of supplementation required may for virtually all infants to prevent overt vitamin D be relatively high [3,44]. Infant formulas and cow’s deficiency. milk are fortified by statute in the United States, although cow’s milk is not fortified with vitamin D in many other countries. Therefore, those at greatest risk of vitamin D defi- TABLE IV Common Supplemental Vitamin D ciency in the United States are infants who are breast- Sources for Infants and Toddlers fed without adequate sunshine exposure, or those who Multivitamin dropsa 400 IU/ml are weaned to diets containing little vitamin D. In Vitamin D onlyb 8000 IU/ml addition to an increased frequency of breast-feeding c older infants and toddlers, social conditions and the D-vi-sol (Canada) 400 IU/ml widespread vigorous use of sun-block have made it Infant formulas 400 IU/liter more common for infants to receive little sunlight Whole milk/juices 100 IU/240 ml exposure. Because of the resurgence of rickets in the United aUsually combined with vitamins A and C. bConcentrated vitamin D available in the United States. Not recom- States [45], it has become clear that policies of selec- mended for routine use. tive vitamin D supplementation of high-risk infants are cSingle-source vitamin D (Mead-Johnson, Inc., Evansville, IN) available not adequately protective of the entire population at risk. (June 2003) in Canada but not the United States. CHAPTER 49 Calcium Absorption in Children 815

IV. TODDLERS AND PREPUBERTAL bone mass are uncertain. Further studies are needed to CHILDREN evaluate different levels of calcium intake in this age group. It is also not clear that benefits in bone mass There is a substantial gap in data regarding calcium achieved prepuberty will persist through puberty or absorption between infants and pubertal children. The once high intakes of calcium are stopped. calcium adequate intake (AI) of 500 mg/day for chil- dren from 12 to 48 months of age was developed based on extrapolation from desirable calcium retention for V. CALCIUM ABSORPTION IN 4- to 8-year-olds [28]. We have shown that increasing ADOLESCENTS calcium intake via fortified foods in small children leads to increased total calcium absorption [47]. These A. Effects of Puberty, Gender, Ethnicity, and similar data demonstrating increased calcium and Other Genetic Factors absorption or retention at intakes above 500 mg/day in toddlers imply that the AI for 1- to 3-year-old children Population- and age-related variability in calcium may be below the optimal intake level [47Ð49]. Higher absorption is largely related to factors that are not read- levels may significantly increase calcium absorption ily controlled such as pubertal status, ethnicity, and and retention without posing any risk to long-term bone other genetic factors [52Ð57]. Identification of these development. factors and understanding their relative contribution Few data are available regarding calcium require- to mineralization is an important area of ongoing ments in children prior to puberty. Most of this data is research. based on balance studies conducted over 50 years ago Calcium absorption efficiency increases substanti- on diets that are very different from those currently in ally during puberty and rapidly decreases postpuberty place [48]. An increase in net calcium absorption when (Fig. 1) [58]. In a longitudinal multiethnic study, we the intake of calcium in 3- to 5-year-old children was found a significant increase in the utilization of cal- increased from 500 to 1200 mg/day has been reported cium associated during early puberty (Fig. 2) com- [49]. The benefit was relatively modest, however, and pared with the year prior to the physical changes of intermediate intake levels, as might more readily be puberty. This change was evidenced by increased cal- achieved in preschool children, were not evaluated in cium absorption, and kinetically determined rates of this study. bone calcium deposition [60]. We found that increases We compared measures of calcium metabolism in in calcium absorption and deposition were associated 7- and 8-year-old Mexican-American and non-Hispanic with maturation of the hypothalamicÐpituitary axis as Caucasian girls living in southeastern Texas [50]. measured by a rise in the gonadotropin-releasing Fractional calcium absorption and total body bone hormone-simulated luteinizing hormone (LH) level. mineral content in the girls was not significantly Martin and co-workers [52] have reported the changes correlated to either PTH or vitamin D levels. We found lower serum 25-hydroxyvitamin D concentrations and higher PTH levels in the Mexican-American girls, but these were not significantly inversely correlated to each other. Seasonal variability was seen for 25-hydroxy- Age-related changes in calcium absorption in girls vitamin D concentrations in girls of both ethnic 600 groups, but values in all of the girls were >12 ng/ml. 500 Therefore it appears that differences in 25-hydroxy- vitamin D and PTH concentrations between Mexican- 400 American and Caucasian girls do not have a large effect 300 on calcium absorption in vitamin DÐsufficient prepu- bertal children. 200 The potential benefit to ultimate peak bone mass of 100 increasing calcium intake has been studied in groups Calcium absorption, mg/d 0 of prepubertal children. In one controlled calcium sup- 5 – 6 7 – 8 9 –10 11–12 13 –14 15 –16 plementation trial, an increase in bone mass was found Age groups, y when calcium supplements were given to children as FIGURE 1 The relationship between age and calcium absorp- young as 6 years of age [51]. However, relatively few tion in girls. Maximum rates of absorption are found from 9 to children this young were studied, and the duration of 14 years of age consistent with maximum rates of bone mass effect of this supplementation and its impact on peak accumulation [58]. 816 STEVEN A. ABRAMS

250 B. Effects of Inadequate Calcium Peak accretion rate and Vitamin D Intake 200 Extremely low calcium intakes at all ages have 150 been associated with fractures and rickets both in the United States and in other countries, especially Nigeria 100 and South Africa. Much more common in Western countries are intakes of calcium by adolescents far 50 below the recommendation of 1300 mg/day (AI) but Calcium accretion (mg/d) high enough to prevent overt clinical deficiency. For 0 example, in girls 14Ð18 years old, the 10th percentile Prepubertal Early pubertal of usual intakes is 413 mg/day and the 25th percentile FIGURE 2 Increased rates of calcium accretion are seen in girls is 541 mg/day. This means that nearly 25% of adoles- during early puberty. “Prepubertal” refers to the average rate of cent girls have a daily calcium intake of 40% or less calcium accumulation during a 1-year period before the onset of of the recommended amount [28]. Clearly, there is a physical changes of puberty and “early puberty” refers to the sub- considerable ability to adapt to these low intakes by sequent year during which pubertal changes were noted to have occurred [59]. increasing fractional absorption. This has been shown in earlier studies by Matkovic and Heaney [48], and in preliminary data from our group [63]. However, net calcium retention remains far below that achieved on more appropriate intakes. in total body bone mineral content in girls using DXA. There are very few data on the health consequences They found a maximum increment of 260 mg/day at or effects on calcium physiology of various levels of age 12Ð13 years. These more recent values for peak vitamin D intake or vitamin D status in children. One rates of calcium gain are very similar to the estimates recent study from Finland indicated that there was derived from rates of weight change by Leitch and markedly improved bone mineralization, especially Aitken in the 1950s [61]. at the lumbar spine, in girls who had high vitamin D There are marked differences in bone mass and the intakes. Those with the highest vitamin D levels showed incidence of osteoporosis between African-Americans the most increase longitudinally in bone mineral density and Caucasians (see Chapter 47). Several groups have at the lumbar spine [64]. This is consistent with findings found lower urinary calcium in African-American that vitamin D may be limiting in some populations of girls compared to Caucasians [54,56,62]. In addition, adolescents. For example, adolescent boys in France it appears that, at similar calcium intakes, African- had decreased vitamin D status in winter associated with American girls absorb more calcium than do Caucasians increased PTH levels [65]. Rickets has been reported in [9]. There are no data available for this comparison adolescents in Middle Eastern countries who have little among males. sunlight exposure for cultural reasons [66]. Numerous recent studies have focused on identifying Goulding [67] found lower bone mass in girls with the mechanisms of the relationship between genetics distal forearm fractures than in age-matched girls and osteoporosis by evaluating specific genetic markers without fractures. A lower calcium intake was reported and their relationship to bone mass (see Chapter 68). in the 11- to 15-year-old girls with fractures than in the One of the unknown aspects of this important interac- controls. Wyshak and Frisch [68] similarly reported tion is the relationship between these genetic differ- that high calcium intakes decreased the risk of bone ences and calcium absorption and excretion. Because fractures in adolescents. of the importance of puberty in bone mass, it is likely Epidemiological evidence regarding the conse- that an effect can be seen during pubertal development. quences of low calcium consumption in childhood and We reported [57] a significant relationship between adolescence and ultimate adult bone mass has gener- polymorphisms of the vitamin D receptor (VDR) Fok 1 ally shown the expected benefits [69]. However, such genotype and calcium absorption. Children with data are not conclusive nor do they demonstrate a the FF genotype absorbed on average 115 mg/day critical intake level of calcium at which long-term ben- more calcium than those with the ff genotype. efit or harm can be derived. Few long-term follow-up Furthermore, we found the FF genotype to be associ- studies have been done to evaluate the effects of ated with greater bone mineral density in the study calcium intake in childhood and adolescence on adult subjects. bone mass. CHAPTER 49 Calcium Absorption in Children 817

C. Effects of Other Factors Including lead levels (see below). This is in contrast to a 25th Soda Consumption percentile value of 599 mg/day for 1- to 3-year-olds (and 649 mg/day for 4- to 8-year-olds) in the United Wyshak also reported a positive relationship between States [28], suggesting a much greater prevalence of fractures and carbonated beverage intake [70]. However, very low calcium intakes in Mexico compared to the this link does not prove a direct cause-and-effect rela- United States. tionship. Some have suggested that the high phosphorus content of the sodas leads to increased calcium losses and lower bone mass. However, there are no prospec- VI. FORTIFICATION OF FOODS tive data relating bone mass to carbonated beverage WITH CALCIUM AND VITAMIN D intake and no prospective studies of the relationship. FOR CHILDREN Furthermore, it is likely that very high phosphorus intakes are needed to adversely affect calcium A. Rationale for Fortification metabolism [71]. The amount of phosphorus in sodas is small relative to the total daily intake of most ado- The potential for inadequate intake of calcium and lescents and therefore it is likely that reasonable soda vitamin D among children and adolescents throughout or carbonated beverage consumption is not a major the world is considerable. Among the strategies for cause of bone mass loss related to their phosphorus resolving this problem are (1) increasing the intake content. However, it remains of concern that excessive of foods that naturally contain calcium and vitamin D; intake of some beverage products places adolescents (2) increasing the absorption of calcium from foods by at risk for calcium deficiency probably primarily additional food components or genetic modification due to lowered intake of healthier calcium-containing (e.g., decreasing oxalate content) which might enhance beverages. Further data on the relationship between calcium absorption; (3) universal pill or liquid vitamin dietary factors and fractures and bone loss are needed, and mineral supplementation; or (4) increased manda- and at present this relationship remains highly con- tory or optional fortification of food sources [79]. troversial [72,73]. Each of these can and will be important in improving bone health in children and adults. Consideration of the use of prebiotics to enhance calcium absorption is pro- D. Developing Countries vided hereafter. Food fortification, however, is likely to be a key component of any strategy. In Mexican toddlers, Murphy et al. [74] found rela- In considering food fortification with calcium or tively high levels of calcium intake (mean 735 ± 199 vitamin D, one must evaluate both the potential bene- mg/day). This was far greater than the mean calcium fits of the fortification strategy and the risks related to intake of less than 220 mg/day in Egypt and Kenya. excess intake from overly zealous fortification. The higher intake in Mexico likely is due both to dairy To determine benefit, one must ensure that fortifica- products and lime-treated tortillas in parts of Latin tion is necessary based on a low intake by at least one America [74,75]. The bioavailability of the calcium in population subgroup, that the nutrient of importance this diet, especially from the treated tortillas, may be has an important public health need, and that the nutri- poor, however [76]. These calcium intakes may not ents being fortified are bioavailable. All of these crite- occur among poorer populations of Latin America. ria are easily met for both calcium and vitamin D Wyatt and Tejas [77] have reported large economic fortification of food and beverage sources for children differences in calcium intakes in 4- to 6-year-old chil- and adolescents. Good bioavailability of calcium- dren in Southern Mexico. In children in the poorest fortified orange juice has been shown [80]. Limited areas, mean calcium intake was only 272 mg/day, pri- data indicate relatively good bioavailability of calcium marily coming from corn tortillas. This increased in added to bread and grain products [81] and fortified medium-income families to more acceptable intake cereal [47]. levels of 625 mg/day due to the increased availability From a safety perspective, it is worth noting that of dairy products in families of higher socioeconomic the upper limit for calcium intake in children over status. 12 months of age is 2500 mg/day [28]. This intake is In Mexico City a mean calcium intake of 516 mg/day well above the 95th percentile of intakes for all popu- was reported for children 1 to 5 years of age [78]. In this lation groups and above the 99th percentile of intake group, 25% of the children had calcium intakes of less for all age and gender groups except adolescent males. than 361 mg/day that was associated with higher blood Therefore, reasonable calcium fortification of grain 818 STEVEN A. ABRAMS products and juices is unlikely to pose a problem for the diet would be unlikely to significantly increase cal- children and adolescents. cium absorption [85,86]. Traditionally in the United States, relatively few The mechanism by which oligosaccharides might foods and beverages have been vitamin D fortified increase calcium absorption is not known. NDOs resist except for milk. This situation is changing and it is digestion in the human gut, but are fermented to likely that increasing number of commercial products volatile fatty acids in the colon [84]. These fatty acids will have added vitamin D. Recently, juice products may have a local effect in the colon by reducing the pH including orange juice have been allowed to add vita- and increasing solubility of mineral in the aqueous min D. The safety margin for this is also likely to be phase of the colonic contents permitting higher absorp- very favorable, as the upper limit of 2000 IU/day is far tion of minerals in the colon, a site where little calcium above the intakes of almost anyone who is not taking absorption normally occurs. Alternatively, the NDO, high-dose medicinal supplements that are not recom- volatile fatty acids, or some other mediator may have a mended for children [82]. trophic effect on the gut [46], improving overall “gut The combination of substantial public need and a health.” Such an effect could increase calcium absorp- high safety margin make calcium and vitamin D tion throughout the length of the gastrointestinal tract. appropriate for food and beverage fortification efforts. Finally, NDOs may alter the composition of the However, continuing monitoring and education is colonic bacterial flora [83,84,87Ð89], and this might important to ensure that excess fortification does not lead, directly or indirectly, to a change in mineral occur and that high-dose supplements are not used at absorption or overall “gut health.” the same time as fortificants. Consideration should also be given to the effects of calcium and vitamin D fortification on the status of other minerals, including VII. SUMMARY AND CONCLUSIONS magnesium and zinc. Calcium and vitamin D are critical nutrients throughout infancy and childhood. Clinically apparent B. Enhancers of Calcium Absorption demineralization related to deficiencies can present as rickets, most commonly in premature infants or tod- An alternative dietary strategy to enhancing dlers. However, calcium intake is especially critical net absorbed calcium is to identify dietary strategies during adolescence. Inadequate intake and absorption that enhance the calcium bioavailability of the whole of calcium during puberty can lead to a lifelong risk of diet and which have other health benefits. For exam- osteoporosis due to failure to achieve peak bone mass. ple, functional foods including prebiotics such as non- Currently efforts to enhance the calcium status of chil- digestible oligosaccharides (NDO) may be of benefit dren have focused on improving intakes via food forti- [83,84]. fication strategies and increasing absorption using We have completed a study of the effect of 8 g/day specific enhancers of absorption. Inadequate calcium of Synergy1 (a NDO composed of a mixture of long and vitamin D status is a global problem with substan- and short chain-length molecules) on calcium absorp- tial health consequences for children throughout the tion in young girls (aged 11 to 13.9 years) [85]. Subjects world. received in random order 8 g/day of NDO (either Synergy1 or oligofructose) and placebo (sucrose), added to a diet providing approximately 1200Ð1300 mg/day Acknowledgment calcium. Calcium absorption was measured after 21 days of adaptation to the NDO or placebo using a This work is a publication of the U.S. Department stable isotope method. We found a significant increase of Agriculture (USDA)/Agricultural Research Service in calcium absorption while consuming NDOs. (ARS) Children’s Nutrition Research Center, Calcium absorption was significantly higher when Department of Pediatrics, Baylor College of Medicine subjects consumed 8 g/day Synergy1 than when con- and Texas Children’s Hospital, Houston, TX. This suming placebo, but no significant benefit was seen project has been funded in part with federal funds from from 8 g/day of oligofructose. The increase in calcium the USDA/ARS under Cooperative Agreement number absorption (32.3% to 38.2%) represents a relative 58-6250-6-001. Contents of this publication do not increase of more than 18%. A change of this magni- necessarily reflect the views or policies of the USDA, tude is clinically highly significant. Of importance, nor does mention of trade names, commercial prod- this effect was seen at a relatively high calcium intake, ucts, or organizations imply endorsement by the U.S. whereas simply increasing the amount of calcium in Government. CHAPTER 49 Calcium Absorption in Children 819

References indices in preterm infants. Arch Dis Child Fetal Neonatal Ed 80:F161ÐF166. 1. Schanler RJ, Abrams SA 1995 Postnatal attainment of 21. Koo WW, Krug-Wispe S, Neylan M, Succop P, Oestreich AE, intrauterine macromineral accretion rates in low birth weight Tsang RC 1995 Effect of three levels of vitamin D intake in infants fed fortified human milk. J Pediatr 126:441Ð447. preterm infants receiving high mineral-containing milk. 2. Work Group on Breastfeeding, American Academy of J Pediatr Gastroenterol Nutr 21:182Ð189. Pediatrics 1997 Breastfeeding and the use of human milk. 22. Backstrom MC, Maki R, Kuusela AL, Sievanen H, Koivisto AM, Pediatrics 100:1035Ð1039. Koskinen M, Ikonen RS, Maki M 1999 The long-term effect of 3. Gartner LM, Greer FR 2003 Prevention of rickets and vitamin D early mineral, vitamin D, and breast milk intake on bon deficiency: New guidelines for vitamin D intake. Pediatrics mineral status in 9- to 11-year-old children born prematurely. 111:908Ð910. J Pediatr Gastroenterol Nutr 29:575Ð582. 4. Jones G, Riley M, Dwyer T 2000 Breastfeeding in early life 23. Stathos TH, Shulman RJ, Schanler RJ, Abrams SA 1996 and bone mass in prepubertal children: A longitudinal study. Effects of carbohydrates on calcium absorption in premature Osteoporos Int 11:146Ð152. infants. Pediatr Res 39:666Ð670. 5. Fomon SJ, Nelson SE 1993 Calcium, phosphorus, magnesium, 24. Carnielli VP, Luijendijk IH, van Goudoever JB, Sulkers EJ, and sulfur. In: Nutrition of Normal Infants. Mosby-Year Book, Boerlage AA, Degenhart HJ, Sauer PJ 1995 Feeding premature St Louis, pp. 192Ð218. newborn infants palmitic acid in amounts and stereoisomeric 6. Heaney RP, Abrams SA, Dawson-Hughes B, Looker A, position similar to that of human milk: Effects on fat and mineral Marcus R, Matkovic V, Weaver C 2000 Peak bone mass. balance. Am J Clin Nutr 61:1037Ð1042. Osteoporos Int 11:985Ð1009. 25. Lucas A, Quinlan P, Abrams S, Ryan S, Lucas PJ 1997 7. Baker SS, Cochran WJ, Flores CA, Georgieff MK, Jacobson MS, Randomised controlled trial of a synthetic triglyceride milk Jaksic T, Krebs NF 1999 American Academy of Pediatrics. formula for preterm infants. Arch Dis Child 77:F178ÐF184. Committee on Nutrition. Calcium requirements of infants, 26. Koo WW, Hammami M, Margeson DP, Nwaesei C, children, and adolescents. Pediatrics 104:1152Ð1157. Montalto MB, Lasekan JB 2003 Reduced bone mineralization 8. Abrams SA, Atkinson S 2003 Calcium, magnesium, phosphorus, in infants fed palm olein-containing formula: A randomized, and vitamin D fortification of complementary foods. J Nutr double-blinded, prospective trial. Pediatrics 111:1017Ð1023. 133:29945Ð29995. 27. Butte NF, Wong WW, Hopkinson JM, Smith EO, Ellis KJ 2000 9. Schanler RJ 2001 The use of human milk for premature Infant feeding mode affects early growth and body composi- infants. Pediatr Clin North Am 48:207Ð219. tion. Pediatrics 106:1355Ð1366. 10. Greer FR, McCormick A 1988 Improved bone mineralization 28. Institute of Medicine Food and Nutrition Board’s Standing and growth in premature infants fed fortified own mother’s Committee on the Scientific Evaluation of Dietary Intervals milk. J Pediatr 112:961Ð969. 1997 Calcium. In: Dietary Reference Intervals for Calcium, 11. Lyon AJ, McIntosh N 1984 Calcium and phosphorus balance Phosphorus, Magnesium, Vitamin D and Fluoride. National in extremely low birthweight infants in the first six weeks of Academy Press, Washington, DC, pp. 71Ð146. life. Arch Dis Child 59:1145Ð1150. 29. Life Sciences Research Office (LSRO) report 1998 Assessment 12. Schanler RJ, Oh W 1985 Nitrogen and mineral balance in of nutrient requirements for infant formulas. J Nutr 128: preterm infants fed human milks or formula. J Pediatr 2140SÐ2143S. Gastroenterol Nutr 4:214Ð219. 30. DeVizia B, Fomon SJ, Nelson SE, Edwards BE, Ziegler EE 13. Bronner F, Salle BL, Putet G, Rigo J, Senterre J 1992 Net cal- 1985 Effect of dietary calcium on metabolic balance of normal cium absorption in premature infants: results of 103 metabolic infants. Pediatr Res 19:800Ð806. balance studies. Am J Clin Nutr 56:1037Ð1044. 31. Abrams SA, Griffin IJ, Davila PM 2002 Calcium and zinc 14. Abrams SA, Esteban NV, Vieira NE, Yergey AL 1991 Dual absorption from lactose-containing and lactose-free infant tracer stable isotopic assessment of calcium absorption and formulas. Am J Clin Nutr 76:442Ð446. endogenous fecal excretion in low birth weight infants. Pediatr 32. Abrams S A, Wen J, Stuff JE 1997 Absorption of calcium, zinc, Res 29:615Ð618. and iron from breast milk by five- to seven-month-old infants. 15. Schanler RJ 1998 The role of human milk fortification for Pediatr Res 41:384Ð390. premature infants. Clin Perinatol 25:645Ð657. 33. Lifschitz CL, Abrams SA 1998 Addition of rice cereal to 16. Schanler RJ, Abrams SA, Garza C 1988 Mineral balance studies formula does not impair mineral bioavailability. J Pediatr in very low birth weight infants fed human milk. J Pediatr 113: Gastroenterol Nutr 26:175Ð178. 230Ð238. 34. Carnielli VP, Luijendijk IH, Van Goudoever JB, Sulkers EJ, 17. Loui A, Raab A, Obladen M, Bratter P 2002 Calcium, phos- Boerlage AA, Degenhart HJ, Sauer PJ 1996 Structural position and phorus and magnesium balance: FM 85 fortification of human amount of palmitic acid in infant formulas: effects on fat, fatty acid, milk does not meet mineral needs of extremely low birth- and mineral balance. J Pediatr Gastroenterol Nutr 23:553Ð560. weight infants. Eur J Clin Nutr 56:228Ð235. 35. Nelson SE, Frantz JA, Ziegler EE 1998 Absorption of fat and 18. Cooke R, Hollis B, Conner C, Watson D, Werkman S, Chesney R calcium by infants fed a milk-based formula containing palm 1990 Vitamin D and mineral metabolism in the very low birth olein. J Am Coll Nutr 17:327Ð332. weight infant receiving 400 IU of vitamin D. J Pediatr 116: 36. Garn SM 1972 The course of bone gain and the phases of bone 423Ð428. loss. Ortho Clin North Am l3:503Ð520. 19. Pittard WB, Geddes KM, Hulsey TC, Hollis BW 1991 37. Specker BL, Beck A, Kalfwarf H, Ho M 1997 Randomized How much vitamin D for neonates? Am J Dis Child 145: trial of varying mineral intake on total body bone mineral 1147Ð1149. accretion during the first year of life. Pediatrics 99:E12. 20. Backstrom MC, Maki R, Kuusela AL, Sievanen H, Koivisto AM, 38. Koo WW, Bush AJ, Walters J, Carlson SE 1998 Postnatal Ikonen RS, Kouri T, Maki M 1999 Randomised controlled trial development of bone mineral status during infancy. J Am Coll of vitamin D supplementation on bone density and biochemical Nutr 17:65Ð70. 820 STEVEN A. ABRAMS

39. Schanler RJ, Burns PA, Abrams SA, Garza C 1992 Bone min- absorption and bone mineral density in children. J Bone Miner eralization outcomes in human milk-fed preterm infants. Res 14:740Ð746. Pediatr Res 31:583Ð586. 58. Bronner F, Abrams SA 1998 Development and regulation of 40. Fewtrell MS, Prentice A, Jones SC, Bishop NJ, Stirling D, calcium metabolism in healthy girls. J Nutr 128:1474Ð1480. Buffenstein R, Lunt M, Cole TJ, Lucas A 1999 Bone mineral- 60. Abrams SA, Copeland KC, Gunn SK, Gundberg CM, Klein KO, ization and turnover in preterm infants at 8Ð12 years of age: and Ellis KJ 2000 Calcium absorption, bone accretion and the effect of early diet. J Bone Miner Res 14:810Ð820. kinetics increase during early pubertal development in girls. 41. Gafni RI, McCarthy EF, Hatcher T, Meyers JL, Inoue N, J Clin Endocrinol Metab 85:1805Ð1808. Reddy C, Weise M, Barnes KM, Abad V, Baron J 2002 61. Leitch I, Aitken FC 1959 The estimation of calcium requirement: Recovery from osteoporosis through skeletal growth: early A re-examination. Nutr Abst Rev 29:393Ð409. bone mass acquisition has little effect on adult bone density. 62. Abrams SA, O’Brien KO, Liang LK, Stuff JE 1995 FASEB J 16:736Ð738. Differences in calcium absorption and kinetics between black 42. Gershoff SH, Legg, MA, Hegsted DM 1958 Adaptation to and white girls age 5Ð16 years. J Bone Miner Res 10:829Ð833. different calcium intakes in dogs. J Nutr 64:303Ð312. 63. Abrams SA, Griffin IJ, Davila P, Liang L, Powledge D 2001 43. Hollis BW, Roos BA, Draper HH, Lambert PW 1981 Vitamin D Effects of very low calcium intake on calcium metabolism in and its metabolites in human and bovine milk. J Nutr 111: pubertal girls. FASEB J 15:A1095. 1240Ð1248. 64. Lehtonen-Veromaa MK, Mottonen TT Nuotio IO, Irjala KM, 44. Greer FR, Hollis BW, Napoli JL 1984 High concentrations of Leino AE, Viikari JS 2002 Vitamin D and attainment of peak vitamin D2 in human milk associated with pharmacologic bone mass among peripubertal Finnish girls: A 3-y prospective doses of vitamin D2. J Pediatr 105:61Ð64. study. Am J Clin Nutr 76:1446Ð1453. 45. Abrams SA 2002 Nutritional rickets: An old disease returns. 65. Guillemant J, Le HT, Maria A, Allemandou A, Peres G, Nutr Rev 60:111Ð115. Guillemant S 2001 Wintertime vitamin D deficiency in male 46. Heaney RP, Weaver CM 2003 Calcium and vitamin D. adolescents: Effect on parathyroid function and response to Endocrinol Metab Clin North Am 32:181Ð194. vitamin D3 supplements. Osteoporos Int 12:875Ð879. 47. Abrams SA, Griffin IJ, Davila P, Liang L 2001 Calcium 66. Abdullah MA, Salhi HS, Bakry LA, Okamoto E, Abomelha AM, fortification of breakfast cereal enhances calcium absorption in Stevens B, Mousa FM 2002 Adolescent rickets in Saudi children without affecting iron absorption. J Pediatr 139: Arabia: A rich and sunny country. J Pediatr Endocrinol Metab 522Ð526. 15:1017Ð1025. 48. Matkovic V, Heaney RP 1992 Calcium balance during human 67. Goulding A, Jones IE, Taylor RW, Manning PJ, Williams SM growth: evidence for threshold behavior. Am J Clin Nutr 2000 More broken bones: A 4-year double cohort study of 55:992Ð996. young girls with and without distal forearm fractures. J Bone 49. Ames SK, Gorham BM, Abrams SA 1999 Effects of high vs Miner Res 15:2011Ð2018 low calcium intake on calcium absorption and red blood cell 68. Wyshak G, Frisch RE 1994 Carbonated beverages, dietary cal- iron incorporation by small children. Am J Clin Nutr cium, the dietary calcium/phosphorus ratio, and bone fractures 70:44Ð48. in girls and boys. J Adolesc Health 15:210Ð215. 50. Abrams SA, Copeland KC, Gunn SK, Stuff JE, Clark LL, 69. Teegarden D, Lyle RM, Proulx WR, Johnston CC, Weaver CM Ellis KJ 1999 Calcium absorption and kinetics are similar in 1999 Previous milk consumption is associated with greater 7- and 8-year-old Mexican-American and Caucasian girls despite bone density in young women. Am J Clin Nutr 69:1014Ð1017. hormonal differences. J Nutr 129:666Ð671. 70. Wyshak G 2000 Teenaged girls, carbonated beverage consump- 51. Johnston CC Jr, Miller JZ, Slemenda CW, Reister TK, Hui S, tion, and bone fractures. Arch Pediatr Adolesc Med 154:610Ð613. Christian JC, Peacock M 1992 Calcium supplementation and 71. Spencer H, Kramer L, Osis D 1988 Do protein and phosphorus increases in bone mineral density in children. N Engl J Med cause calcium loss? J Nutr 118:657Ð660. 327:82Ð87. 72. Calvo MS 2000 Dietary considerations to prevent loss of bone 52. Martin AD, Bailey DA, McKay HA, Whiting S 1997 Bone and renal function. Nutrition 16:564Ð566. mineral and calcium accretion during puberty. Am J Clin Nutr 73. Allison DB 2001 Hold the cola alarm. Arch Pediatr Adolesc 66:611Ð615. Med 155:201Ð202. 53. Matkovic V, Jelic T, Wardlaw GM, Ilich JZ, Goel PK, Wright JK, 74. Murphy SP, Beaton GH, Calloway DH 1992 Estimated mineral Andon MB, Smith KT, Heaney RP 1994 Timing of peak bone intakes of toddlers: Predicted prevalence of inadequacy in mass in Caucasian females and its implication for the preven- village populations in Egypt, Kenya, and Mexico. Am J Clin Nutr tion of osteoporosis. J Clin Invest 93:799Ð808. 56:565Ð572. 54. Bryant RJ Wastney ME, Martin BR, Wood O, McCabe GP, 75. Murphy SP, Calloway DH, Beaton GH 1995 Schoolchildren Morshidi M, Smith DL, Peacock M, Weaver CM 2003 Racial have similar predicted prevalences of inadequate intakes as differences in bone turnover and calcium metabolism in toddlers in village populations in Egypt, Kenya, and Mexico. adolescent females. J Clin Endocrinol Metab 88:1043Ð1047. Eur J Clin Nutr 49:647Ð657. 55. McKay HA, Bailey DA, Mirwald RL, Davison S, Faulkner RA 76. Wyatt CJ, Hernandez ME, Mendez RO 1996 Dialyzable cal- 1998 Peak bone mineral accrual and age at menarche in cium and phosphorus in Mexican diets high in insoluble fiber. adolescent girls: A 6-year longitudinal study. J Pediatrics J Agric Food Chem 46:4662Ð4666. 133:682Ð687. 77. Wyatt CJ, Tejas MAT 2000 Nutrient intake and growth of 56. Bell NH, Yergey AL, Vieira NE, Oexmann MJ, Shary JR 1993 preschool children from different socioeconomic regions in the Demonstration of a difference in urinary calcium, not calcium city of Oaxaca, Mexico. Ann Nutr Metab 44:4Ð20. absorption, in black and white adolescents. J Bone Miner Res 78. Lacasana M, Romiel I, Sanis LH, Palazuelos E, Hernandez- 8:1111Ð1115. Avila M 2000 Blood lead levels and calcium intake in Mexico 57. Ames SK, Ellis KJ, Gunn SK, Copeland KC, Abrams SA 1999 City children under five years of age. Int J Environ Health Res Vitamin D receptor gene Fok1 polymorphism predicts calcium 10:331Ð340. CHAPTER 49 Calcium Absorption in Children 821

79. Calvo MS, Whiting SJ 2003 Prevalence of vitamin D insuffi- 84. Roberfroid MB 1999 Concepts in functional foods: The case of ciency in Canada and the United States: Importance to health inulin and oligofructose. J Nutr 129:1398SÐ1401S. status and efficacy of current food fortification and dietary 85. Griffin IJ Davila PM, Abrams SA 2002 Non-digestible supplement use. Nutr Rev 61:107Ð113. oligosaccharides and calcium absorption in girls with adequate 80. Andon MB, Peacock M, Kanerva RL, De Castro JA 1996 calcium intakes. Br J Nutr 287:S187ÐS191. Calcium absorption from apple and orange juice fortified with 86. Griffin IJ, Hicks PMD, Heaney RP, Abrams SA 2003 Enhanced calcium citrate malate (CCM). J Am Coll Nutr 15:313Ð316. chicory inulin increases calcium absorption mainly in adoles- 81. Martin BR, Weaver CM, Heaney RP, Packard PT, Smith DL 2002 cents with lower calcium absorption. Nutr Res 23:901Ð909. Calcium absorption from three salts and CaSO4-fortified bread 87. Rémésy C, Levrat MA, Gamet L, Demigne C 1993 Cecal fer- in premenopausal women. J Agric Food Chem 50:3874Ð3876. mentations in rats fed oligosaccharides (inulin) are modulated 82. Heaney RP, Weaver CM 2003 Calcium and vitamin D. by dietary calcium level. Am J Physiol 264:G855ÐG862. Endocrinol Metab Clin North Am 32:181Ð194. 88. Brommage R, Binacua C, Antille S, Carrie AL 1993 Intestinal 83. Coudray C, Bellanger J, Castiglia-Delavaud C, Rémésy C, calcium absorption in rats is stimulated by dietary lactulose Vermorel M, Rayssignuier Y 1997 Effect of soluble or partly and other resistant sugars. J Nutr 123:2186Ð2194. soluble dietary fibres supplementation on absorption and bal- 89. Coudray C, Fairweather-Tait SJ 1998 Do oligosaccharides affect ance of calcium, magnesium, iron and zinc in healthy young the intestinal absorption of calcium in humans? Am J Clin Nutr men. Eur J Clin Nutr 51:375Ð380. 68:921Ð923. CHAPTER 50 Vitamin D Metabolism and Aging

BERNARD P. H ALLORAN AND ANTHONY A. PORTALE Departments of Medicine and Pediatrics, University of California, and Division of Endocrinology Veterans Affairs Medical Center, San Francisco, California

I. Introduction VI. Tissue Responsiveness and the Role of Vitamin D II. Cutaneous Production of Vitamin D in the Aging Process III. Dietary Vitamin D and Intestinal Absorption VII. Conclusions IV. Synthesis of 25-Hydroxyvitamin D References V. Synthesis and Metabolism of 1,25-Dihydroxyvitamin D

I. INTRODUCTION keratinocytes also changes with donor age [7,8]. Barrier function is compromised and total lipid content Aging in the context of this chapter refers to post- (including cholesterol) in the stratum corneum is maturational aging and not to growth and development. decreased in aged animals [9]. Skin blood flow decreases Postmaturational aging is a complex process beginning by nearly 40% between the ages of 20 and 70 years, with senescence at the cellular level. All cells gradually which reduces dermal clearance of vitamin D [10]. change phenotypically and eventually lose the ability Cutaneous production of vitamin D decreases to proliferate as they age. Tissues and organs also age with advancing age [11Ð13]. Approximately 80% of with consequent changes in metabolism and function. the vitamin D formed in the skin is produced in the And of course organisms age resulting in diminished epidermis, and the amount of precursor to vitamin D3, health and performance. Aging is heterogeneous. 7-dehydrocholesterol, is decreased in the epidermis Differences in genetic makeup and lifestyle (diet, activity of elderly subjects. Despite a decrease in the number level, environment) influence the progress of senescence. of melanocytes in the skin (normally melanin in the Some people appear to age more rapidly than others. melanocyte acts as a natural sunscreen to reduce produc- Disease confounds the aging process, and thus the tion of vitamin D), conversion of 7-dehydrocholesterol changes observed in an aging population might be a to previtamin D3 in human skin samples exposed to consequence of true aging or the cumulative effect of ultraviolet radiation is decreased as much as twofold chronic disease. The changes that occur during aging are in elderly subjects. Holick et al. [13,14] report that often subtle, but their compound effect can be dramatic. whole body exposure to one minimal erythemal dose Predictably, aging influences vitamin D metabolism, of ultraviolet B radiation can increase serum vitamin D and vitamin D influences the aging process. This chapter to a maximum of 78.1 nmol/liter-ng/ml in young sub- deals with how aging affects cutaneous production, jects but to a maximum of only 20.8 nmol/liter-ng/ml dietary availability, metabolism, and action of vitamin D, in elderly subjects 68Ð80 years of age (Fig. 1). The and how vitamin D influences the progression of aging. decrease in cutaneous vitamin D synthesis is presum- An attempt is made to separate the effects of common ably a consequence of the decrease in substrate age-related diseases from the effects of aging per se. (7-dehydrocholesterol) and reduced dermal clearance of vitamin D. The diminished ability to produce vitamin D in the II. CUTANEOUS PRODUCTION skin of older subjects is often aggravated by changes in OF VITAMIN D lifestyle and environmental factors. Many older people are homebound or hesitant to venture outdoors, and The production of vitamin D in the skin is described when exposed to the sun frequently wear potent sun- in Chapter 3. The structure and function of the skin screens to reduce the risk of skin cancer. Sunlight begin to deteriorate during the third decade of life [1]. that has passed through a glass windowpane will not Epidermal thinning begins around age 20 and tissue induce the synthesis of vitamin D because of the loss continues with advancing age [2]. Skin elasticity, UV absorbance by the glass [14]. Furthermore, chronic keratinocyte number, and cell turnover rate decrease with use of sunscreens can dramatically reduce serum vita- aging [3Ð6]. The pattern in gene expression in cultured min D levels [15]. The elderly, especially those that are

VITAMIN D, 2ND EDITION Copyright © 2005, Elsevier, Inc. FELDMAN, PIKE, AND GLORIEUX All rights reserved. 824 BERNARD P. H ALLORAN AND ANTHONY A. PORTALE

the phrase Adequate Intake (AI). The USDA has set 100 the AIs for young adults (< 50 years) at 200 IU/d, middle-aged adults (50Ð70 years) at 400 IU/d, and 80 elderly adults (> 70 years) at 600 IU/d [20]. The major dietary sources of vitamin D are milk, milk products, 60 butter, fortified margarine, eggs, and some fatty fish. Young Data from a survey conducted by the National Center 40 for Health Statistics, Centers for Disease Control (National Health and Nutrition Examination Survey — 20 Elderly NHANES III, 1988Ð1994), indicate that the usual adult (all ages) dietary intake of vitamin D in the United States 0 is approximately 5 µg/d and does not change with post- 0 1 2 3 4 5 6 7 Serum of vitamin D (nmol/L) concentration Day maturational aging [21]. More recent data will be available shortly [22]. Krall et al. [23] in a study of FIGURE 1 Effect of whole-body exposure to one minimal ery- 333 women observed vitamin D intakes ranging from themal dose of ultraviolet B radiation on the serum concentrations of vitamin D in young (22Ð30 years) and elderly volunteers. From 0.5 to more than 40 µg/d. With advancing age, protein, Holick M [14]. fat, and total calorie intake decrease [24,25]. Dietary calcium (excluding supplements but combining men and women) decreases from 909 mg/d between 20 and 39 years to 721 mg/d after 60 years [22] and vitamin D homebound or confined to a nursing home are, there- intake is tightly coupled to total dietary calcium. Thus, fore, at increased risk of becoming vitamin D deficient, one would predict that vitamin D intake might decrease as a result of both diminished efficiency in cutaneous with age. However, vitamin D supplementation of the production and reduced effective solar exposure. Gloth normal diet can account for as much as 50% of the total et al. [16] report that of 244 elderly men and women intake of the vitamin [26,27] and supplementation in at over the age of 65 who were deprived of direct sunlight least some people increases with age. Sowers et al. [27] exposure (homebound elderly and nursing home resi- report that in 373 women ranging in age from 20 to dents) 54% of the community dwellers and 38% of 80 years the intake of vitamin D from supplements nursing home residents have serum 25OHD levels increased from 104 ± 18 IU/d in 25- to 40-year-olds to below 25 nmol/liter (normal range = 25Ð137 nmol/liter). 202 ± 43 IU/d in 60- to 75-year-olds. Despite such Obesity is another factor that can contribute to vitamin D increases, Sharkey et al. [28], who studied a population deficiency in the elderly. Body mass index (BMI) of cognitively eligible homebound men and women frequently increases with age; vitamin D is stored in (age 60Ð85 years) whose meals were delivered to their fat tissue, and vitamin D levels have been shown to homes, found that 100% of the men and 99.6% of inversely correlate with BMI after irradiation or oral the women had intakes of vitamin D below the AIs. vitamin D administration [17]. The subject of vita- Although those elderly that are ill or confined indoors min D insufficiency in elderly subjects in relationship often have inadequate total vitamin D intakes [29], the to osteoporosis is discussed in Chapters 65 and 66, and predominance of evidence suggests that in most healthy, by Lips [18]. free-living elderly the total intake of vitamin D does not change with age [23,25,26,30]. Collectively, direct calculation of total vitamin D intake in elderly popula- III. DIETARY VITAMIN D tions suggests that overall intake decreases with age in AND INTESTINAL ABSORPTION some elderly, especially those that are chronically ill or confined indoors [21,23,27,29], and not in others. A. Dietary Intake Based on the latest Department of Agriculture AIs, which recommend successive increases in dietary vita- Dietary intake of vitamin D, because of differences min D with aging, this may still be inadequate to meet in dietary supplementation and food preferences, varies physiological needs. over a broad range in the elderly. The United States Department of Agriculture (USDA) previously estab- lished the adult recommended daily allowance (RDA) B. Intestinal Absorption of Vitamin D for vitamin D at 5.0 µg/d or 200 IU/d [19]. Because of the vagaries of cutaneous production of vitamin D, the Whether intestinal absorption of vitamin D changes USDA has abandoned the term RDA in favor of using with age is controversial. Vitamin D is absorbed in the CHAPTER 50 Vitamin D Metabolism and Aging 825 proximal intestine, and in the absence of disease range = 10Ð55 nmol/L). Clearly many older people are associated with intestinal malabsorption, some studies, vitamin D insufficient or frankly deficient. Disease and but not all [31,32], indicate that vitamin D absorption medication use frequently exacerbate the problem. remains normal in the elderly [33Ð35,36]. Clemens Lips [18] illustrates this by reporting a survey of serum et al. [33] compared serum vitamin D2 levels in young 25OHD in elderly subjects by health and residence adults and 25 chronically institutionalized but other- category. wise healthy elderly adults (mean age 72) after admin- Mild hyperparathyroidism, increased bone turnover, istration of 50,000 IU of vitamin D2. In the absence and diminished bone density are associated with mod- of gastrointestinal disease, they found no evidence of erate to severe vitamin D insufficiency in the elderly. impaired vitamin D absorption. In contrast, Harris and In elderly populations serum 25OHD has been observed Dawson-Hughes [36] studied the effect of administering to be inversely correlated with both the serum concen- 1800 IU/d of vitamin D2 on plasma concentrations in tration of parathyroid hormone (PTH), markers of young and elderly men. The increase in 25-hydroxy- bone turnover and bone loss [18,23,42,46Ð54]. Ooms vitamin D2 was greater in the young than in the elderly et al. [48], in a study of 330 healthy women over the men by nearly twofold. The data suggest that either age of 70, report that serum 25OHD in 65% of the sub- intestinal absorption or liver hydroxylation of vitamin D jects was less than 12 nmol/L. 25-Hydroxyvitamin D is impaired with aging. Importantly, studies to examine was negatively correlated to serum PTH but only at whether aging disrupts enterohepatic recirculation of levels of 25OHD below 10 nmol/L. Bone mineral den- vitamin D have not been performed. The effects of sity was positively correlated with serum 25OHD but gastrointestinal and hepatobiliary disease on vitamin D only below 12 nmol/L. Furthermore, in a prospective absorption are discussed in Chapter 75. study of 9704 elderly women, Stone et al. [53] report that lower levels of serum 25OHD are associated with increased bone loss in the hip. Severe dietary vitamin D C. Consequences of Vitamin D Insufficiency deficiency is associated with increased risk of hip fracture [54]. The consequences of diminished cutaneous produc- Supplementation with vitamin D increases serum tion (all elderly), insufficient intake (some elderly), 25OHD, in most cases decreases serum PTH, and nor- and impaired absorption (some elderly) in the elderly malizes bone turnover [47,55Ð57]. Brazier et al. [47] are inadequate vitamin D status. Serum concentrations compared vitamin DÐsufficient and insufficient elderly of vitamin D and 25-hydroxyvitamin D (25OHD) men and women before and after treatment of the are frequently inadequate to support normal mineral insufficient group with 800 IU of vitamin D/d. In the homeostasis [16,18,25,37Ð45]. The serum concentra- vitamin D insufficient subjects, serum PTH and markers tion of 25OHD in the elderly, a reflection of serum of bone turnover were increased prior to treatment but vitamin D levels (see Section IV, on synthesis of normalized to levels found in age-matched vitamin DÐ 25-hydroxyvitamin D) is related inversely to age and sufficient subjects after treatment. These data suggest directly to sun exposure. In the presence of disease, that dietary supplementation of elderly men and women malabsorption may contribute to poor vitamin D status. with poor or marginal vitamin D status may be beneficial In a study of 433 postmenopausal women, Need et al. in reducing bone loss associated with aging. Indeed, [42] showed that serum 25OHD decreased with age Dawson-Hughes et al. [58] report that vitamin D sup- and was positively correlated to hours of sunlight. plementation (10 µg/d) to postmenopausal women with Van der Wielen et al. [38], in a study of 824 elderly an otherwise normal mean intake of 5 µg/d can reduce people from 11 European countries, report that 36% of wintertime bone loss. Further studies by these investi- men and 47% of women had serum 25OHD levels below gators [59] in 247 healthy ambulatory postmenopausal 12 nmol/L (the lower range of normal). Serum 25OHD women consuming an average of 2.5 µg/d of vitamin D concentrations were directly related to hours of ultra- showed that supplementation with 17.5 µg/d could violet light exposure and factors of physical health status. reduce the rate of loss of bone mineral density in the A study of Fardellone et al. [46] further exemplifies femoral neck year-round. Interestingly, the anabolic the importance of sun exposure and health status, effects of vitamin D treatment, although associated with as well as demonstrates the heterogeneity of aging an increase in serum 25OHD, are not usually accom- populations. These investigators studied a group of panied by an increase in serum 1,25(OH)2D unless the chronically institutionalized elderly and observed a patients are frankly vitamin D deficient [49]. The report mean serum 25OHD of only 3.7 nmol/L. Eighty-five by Barger-Lux et al. [60] demonstrating a positive cor- percent of the subjects had serum 25OHD levels below relation between serum 25OHD (but not 1,25(OH)2D) 5 nmol/L and 98% had levels below 10 nmol/L (normal and calcium absorption in healthy premenopausal 826 BERNARD P. H ALLORAN AND ANTHONY A. PORTALE women treated with 25OHD is consistent with this V. SYNTHESIS AND METABOLISM idea. These data suggest that 25OHD may be acting OF 1,25-DIHYDROXYVITAMIN D directly on the intestine to stimulate calcium absorption (see Chapter 46). A consensus regarding the effects of aging on the Not all individuals benefit from vitamin D supple- synthesis and metabolism of 1,25(OH)2D is slowly mentation. Orwoll et al. [61] studied normal healthy emerging. The issue is complex because of the hetero- men ranging in age from 30 to 87 years. Administration geneity of the aging process and the independent effects of both calcium (1000 mg/d) and vitamin D (25 µg/d) of aging on renal function, acidÐbase balance, sex did not affect the normal rate of bone loss. Importantly, steroid levels, growth hormone status, and other factors, however, mean basal dietary calcium and vitamin D in each of which can affect the synthesis and metabolism this population was 1159 mg/d and approximately of 1,25(OH)2D (see Chapter 5). 9 µg/d, respectively. These data are consistent with the findings of Ooms et al. [55] and suggest that elderly populations with adequate dietary calcium and ade- A. Serum Concentration of 1,25(OH)2D quate vitamin D status are not likely to improve their mineral balance with additional vitamin D. The effect of aging on the serum concentration of 1,25(OH)2D is controversial. With advancing age in men, serum concentrations of 1,25(OH)2D are reported to IV. SYNTHESIS OF either decrease [64,65] or remain unchanged [66Ð68]. 25-HYDROXYVITAMIN D In women, serum 1,25(OH)2D is reported to decrease [64,69Ð71], remain unchanged [67], to increase and Synthesis of 25OHD does not appear to be influ- then decrease [65], or to decrease and then increase [72]; enced by aging with some exceptions [15,31,36]. The and in mixed populations to either decrease [73] or primary site for synthesis of 25OHD is the liver (see remain unchanged [33,74Ð76]. Findings in aging ani- Chapter 4). Severe liver disease can lead to a reduction mals are similar to those in humans. Using the rat as a in the synthesis of 25OHD and the serum concentra- model of human aging, serum 1,25(OH)2D levels are tions of 25OHD, vitamin D binding protein, and total reported to be decreased [77Ð80], unchanged [81,82], (but not free) 1,25(OH)2D [62,63], and elderly patients or increased [83] in aged animals compared to those in with inadequate hepatic function may have a reduction younger animals. These apparently discrepant findings in vitamin D 25-hydroxylase reserve. In the absence of result in part from differences in the definition of “old” hepatic disease, however, most studies suggest that the and differences in the gender, lifestyle, presence of dis- activity of the vitamin D-25-hydroxylase is normal in ease, and medication use in the populations studied. the elderly. Aknes et al. [37] examined the relation- Accounting for these differences and better defining ships among the serum concentrations of vitamin D, the populations studied, has permitted reconciliation of 25OHD, and vitamin D binding protein (DBP) in healthy earlier apparent discrepancies. adults ranging in age from 22 to 96 years. With advanc- In animal studies of aging, the definition of “old” ing age, they observed decreases in vitamin D, 25OHD, is critical to their interpretation. The most commonly and DBP but no change or a small increase in the used animal model for aging studies is the Fischer molar ratio of 25OHD to vitamin D. This suggests that 344 (F344) rat, which becomes sexually mature at hepatic hydroxylation at the 25-position of vitamin D, approximately 3 months of age and has a mean life- in the absence of disease, is not impaired by aging. span of 24 months. Serum levels of 1,25(OH)2D are Matsuoka et al. [15] studied the response of serum higher in young growing F344 rats when compared to 25OHD2 to an oral load of vitamin D2 in young and levels in mature “adult” animals 3–4 months of age. elderly subjects. The increase in serum 25OHD over In many “aging” studies, findings in growing or not yet time was similar between age groups, providing evi- mature animals have been compared to those in adult dence that both intestinal absorption and 25-hydroxy- animals. Such studies show that serum 1,25(OH)2D lation are normal in healthy elderly people. This levels are lower in adult than in growing animals. contrasts with the findings of Harris et al. [32]. These However, although such studies have been interpreted investigators found that the increase in serum 25OHD to indicate that serum 1,25(OH)2D decreases with age, in the elderly in response to a 3-week course of oral they do not address the question of whether serum vitamin D was roughly half that of young subjects, 1,25(OH)2D levels change during postmaturational implying either intestinal malabsorption or a defect in aging: that is, during the period between 3 and 24 months 25OHD synthesis. of age. CHAPTER 50 Vitamin D Metabolism and Aging 827

Nevertheless, even when findings in young adult 150 animals (4Ð6 months) are compared to those in aged animals (20Ð28 months) discrepancies remain. The heterogeneity of the aging process and the small number 100 of animals studied in aging studies can account, at least D (pmol/L)

in part, for differences in the findings. Loss of renal func- 2 tion is a major cause of death in the aging F344 rat. Since 50 1,25(OH)2D is synthesized in the kidney, it is predictable 1,25(OH) that serum 1,25(OH)2D will be reduced in aged animals Serum of concentration with renal insufficiency. However, in aged (24-month) male F344 rats with normal or near-normal glomerular 0 filtration rates, serum concentrations of 1,25(OH)2D are 0.9 )

not significantly different from those in young adult 1 − D (6-month) male F344 rats [82]. It is of interest that in 2 male F344 rats between the ages of 12 and 24 months 0.6 with normal renal function, the serum concentration of 70 kg IBW .

1,25(OH)2D tends to increase! The trend does not reach 1 − s statistical significance, but may explain why some inves- .

MCR of 1,25(OH) 0.3 tigators have observed an increase in serum 1,25(OH)2D (mL in the aging rat [83]. In humans, renal function deteriorates with post- maturational aging [84Ð87]. The rate of deterioration varies from subject to subject, and some older people 0.0 maintain normal or near-normal glomerular filtration 90 rates (GFR) well into their eighth decade of life. In ) healthy elderly men in whom renal function is normal 1 − D or near normal, serum 1,25(OH)2D levels are not signif- 2 icantly different from those in young men [68] (Fig. 2). 60 However, in elderly individuals with moderate to severe 70 kg IBW . 1

renal insufficiency (GFR < 70 ml/min), renal synthesis − s and serum concentrations of 1,25(OH)2D are reduced. . 30 Thus, the low serum concentrations of 1,25(OH) D PR of 1, 25(OH)

2 (fmol reported in many elderly populations reflect in large part the age-related fall in GFR [67,68,76]. Moreover, Young men Old men 0 serum vitamin D binding protein (DBP) levels in men do not change with age [73] suggesting that free levels FIGURE 2 Serum concentration, metabolic clearance rate of 1,25(OH) D do not change. (MCR), and production rate (PR) of 1,25(OH)2D in healthy young 2 and elderly men after ingesting a constant normal diet for 9 days. In women, comparison of 1,25(OH)2D concentra- The mean of each population is indicated by a dashed line. From tions in elderly and young women is confounded Halloran et al. [68]. by the potential effects of menopause in the elderly, and the observation that serum 1,25(OH)2D changes during the menstrual cycle in younger women. Most data indicate that serum total 1,25(OH)2D levels in increased [72] in postmenopausal women. Prince et al. healthy women remain constant or gradually increase [72] report that in a cross-sectional study of 655 women through the eighth decade [67,88,89]. Although ranging in age from 35 to 90 years, circulating DBP menopause appears to have little effect on the serum levels increase after the menopause, then decrease tran- concentration of 1,25(OH)2D [72,90,91], a transitory siently, and finally increase progressively with advanc- decrease in total serum 1,25(OH)2D has been observed ing age, resulting in a progressive decrease in the serum 5Ð15 years after the menopause, followed by a gradual free 1,25(OH)2D index beginning 10Ð15 years after the increase in serum 1,25(OH)2D that correlates with ris- menopause. Other studies, however, do not support ing serum PTH concentrations [72]. these findings [92], and thus it is not clear whether the The serum concentration of DBP is reported to serum concentration of free 1,25(OH)2D changes with be either decreased [73,92,93], normal [37,43], or age in women. 828 BERNARD P. H ALLORAN AND ANTHONY A. PORTALE

B. Kinetics of 1,25(OH)2D Metabolism Serum 1,25(OH)2D levels did not correlate with those of 25OHD, suggesting that substrate concentration was The serum concentration of 1,25(OH)2D is deter- not limiting for the production of 1,25(OH)2D in both mined by its production rate (PR) and its metabolic elderly and young men. clearance rate (MCR). The MCR of 1,25(OH)2D in the rat is increased by vitamin D deficiency [94] and chronic exogenous administration of 1,25(OH)2D [95], C. Synthesis of 1,25(OH)2D: Trophic Factors and in the rat [94] and pig [96], by depletion of dietary 1. SEX STEROIDS calcium. The MCR of 1,25(OH)2D is unaffected by changes in dietary phosphorus [96,97], PTH adminis- Serum total and free testosterone levels decrease tration [98], and glucocorticoid excess [99]. Loss of with advancing age in men [101,102], and administra- renal function decreases the MCR of 1,25(OH)2D in tion of testosterone to hypogonadal men has been the rat [100]. shown to increase modestly both serum total and free With advancing age in the F344 male rat with 1,25(OH)2D concentrations [103]. These data suggest normal or near normal renal function [82], the MCR that the decline in serum testosterone levels with aging of 1,25(OH)2D gradually increases (Table I). When may reduce tonic stimulation of the 1α-hydroxylase. compared to values in 6-month-old animals, the MCR Estrogen deficiency leads to a decrease in serum is 24% (p < 0.10) higher at 12 months, 30% (p < 0.05) 1,25(OH)2D levels regardless of age [75,104,105]. higher at 18 months and 57% higher (p < 0.01) at Estrogen replacement in postmenopausal women 24 months of age. The PR of 1,25(OH)2D also increases can increase both total and free serum 1,25(OH)2D with age in the F344 male rat, and by 24 months of age [92,104,106Ð110], suggesting that with menopause and is 91% (p < 0.01) higher than at 6 months. The increase the accompanying estrogen deficiency, an important in 1,25(OH)2D production correlates positively with trophic factor for the maintenance of serum 1,25(OH)2D an increase in serum PTH, presumably reflecting the is lost in aging women. normal stimulatory effect of PTH on 1α-hydroxylase activity. These data demonstrate that both production 2. CALCIUM AND PHOSPHORUS and clearance of 1,25(OH)D can increase during post- The morning fasting serum concentration of total maturational aging in the rat. They also suggest that calcium in elderly men and women is reported to be in animals with normal GFRs, the kidney remains either normal [67,69,111], decreased [43,69,111,112], sensitive to PTH. or increased [113,114] when compared with values in The kinetics of 1,25(OH)2D metabolism in healthy younger women. Serum total calcium concentrations men [68] and women [88] are unchanged with advanc- can directly influence the serum concentration of ing age. We have shown, in a group of healthy elderly 1,25(OH)2D [115], but studies to determine whether men (mean age 72 years) with normal or near-normal aging in humans affects the capacity of calcium to GFRs studied under strictly controlled metabolic con- modulate serum 1,25(OH)2D have not been reported. ditions, that the PR and MCR of 1,25(OH)2D, as well Armbrecht et al. [116] administered a low-calcium diet as its serum concentration, are not different from those to young (2-month) and adult (12-month) F344 rats in healthy young men (mean age 34 years) [68] (Fig. 2). and observed a severalfold increase in serum In both groups of men, the serum 1,25(OH)2D concen- 1,25(OH)2D concentrations and in renal 1α-hydroxy- trations correlated with values of PR but not of MCR. lase mRNA abundance in both groups of animals. However, the levels attained in the adult rats were only 37% and 10%, respectively, of those in the young rats, even though serum levels of PTH were stimulated by TABLE IEffect of Aging on the Serum Concentrations the low-calcium diet to a comparable extent in both of PTH and 1,25(OH)2D, and the Production and groups of animals. The effect of postmaturational Metabolic Clearance Rates of 1,25(OH)2D in the Rat aging on the response to low-calcium diet was not Age (months) 6 12 18 24 addressed in these studies. There is general agreement that the serum phospho- PTH (pg/ml) 15 ± 218± 322± 5a 36 ± 8a rus concentration decreases with advancing age in men ± ± ± ± 1,25(OH)2D (pg/ml) 29 4254313354 and remains unchanged in women [67,69]. We showed PR (pg/min) 1.1 ± 0.2 1.1 ± 0.2 1.4 ± 0.1 2.1 ± 0.2a in healthy young men that moderate and severe restric- MCR (µl/min) 37 ± 146± 448± 4a 58 ± 2a tion of dietary phosphorus can stimulate the serum con- centration of 1,25(OH)2D, independently of changes in Values are mean ± SEM, n = 6, ap < .05. From Wada et al. [82]. serum concentrations of either PTH or blood ionized CHAPTER 50 Vitamin D Metabolism and Aging 829

calcium, and these changes in serum 1,25(OH)2D are TABLE III Serum Concentrations of Phosphorus due to changes in its PR, since its MCR does not change (24 hr Mean) and 1,25(OH)2D in Healthy Young [97,117,118]. Since dietary and serum phosphorus (29 ± 2 yr, n = 9) and Elderly Men (71 ± 1 yr, n =7) can physiologically regulate 1,25(OH)2D production Consuming Constant Whole-Food Diets Containing in humans, it is of interest to determine whether such 625, 1500, 2300 mg/d/70 kg regulation is affected by aging. Villa et al. [119] report that in postmenopausal women given aluminum Diet P (mg/d/70 kg) hydroxide to reduce intestinal phosphorus absorption, 650 1500 2300 serum phosphorus concentrations decreased by 17% and Serum P (mg/dl) serum 1,25(OH)2D increased by 38%, without changes in serum total or ionized calcium or PTH concentrations. Young men 3.7 ± 0.1a 4.2 ± 0.1 4.3 ± 0.1 Although a control group of younger women was not Elderly men 3.2 ± 0.2 3.7 ± 0.2 3.8 ± 0.2 similarly studied, the investigators concluded that older Serum 1,25(OH)2D (pg/ml) women retain the capacity to increase 1,25(OH)2D con- Young men 43 ± 236± 229± 2 centrations in response to dietary phosphorus restriction. Elderly men 43 ± 233± 231± 2 We examined the effects of aging in healthy elderly men on the capacity of phosphorus restriction to stimu- aValues are mean ± SE. From Portale et al. [120]. late serum 1,25(OH)2D. We first studied healthy young and elderly men with normal or near-normal GFR while receiving a constant whole-food diet containing normal dietary intake of phosphorus, serum 1,25(OH)2D amounts of phosphorus and calcium [120,121]. Both levels in the young and elderly men were virtually iden- the morning fasting and the 24-hr mean blood concen- tical, but serum phosphorus levels in the elderly were trations of total and ionized calcium did not differ lower than those in the young men (Table III). Across between young and elderly men (Table II). In contrast, the range of phosphorus intakes, the 24-hr mean serum both the morning fasting and the 24-hr mean serum concentration of phosphorus varied inversely with phosphorus concentrations were significantly lower in serum 1,25(OH)2D concentrations in both groups of the elderly men than in the young men. Since a low subjects (r = −0.92, p < 0.0001) (Fig. 3); the slope of serum phosphorus concentration would be expected to stimulate 1,25(OH)2D production, mild hypophos- phatemia in the elderly would be expected to increase 50 serum levels of 1,25(OH)2D. Given our findings and Elderly those of others that serum 1,25(OH)2D is not increased Young in this population, this suggests that the capacity of phosphorus to regulate renal 1,25(OH)2D production is diminished with age. To investigate this possibility, we then varied dietary phosphorus within its normal range: 40 D (pg/ml)

With restriction of dietary phosphorus from 2300 to 2 625 mg/d, the magnitude of the increase induced in serum 1,25(OH)2D (47%) in the elderly was virtually the same as that induced in the young men. At each

Serum 1,25(OH) 30

TABLE II Whole Blood Ca2+ in Healthy Young (39 ±1 yr, n = 13) and Elderly (74 ± 2 yr, n =9) 2 Men (GFR > 70 ml/min/1.73 m ) after 9 Days on 3.0 3.5 4.0 4.5 a Constant Whole-Food Diet 24-hr Mean serum phosphorus (mg/dl) Young men Elderly men FIGURE 3 Relationship between concentrations of serum 1,25(OH)2D and 24-hr mean serum phosphorus when dietary phos- Whole blood Ca2+ (mg/dl) phorus was normal (squares), then supplemented (triangles), and Morning fasting 4.84 ± 0.03a 4.84 ± 0.04 then restricted (circles) in healthy elderly and young men. Each point represents the mean ± SE of serum 1,25(OH) D and 24-hr ± ± 2 24 hr mean 4.77 0.04 4.80 0.04 serum phosphorus at steady state. Multiple linear regression anal- ysis indicates that the intercept is significantly lower in elderly men aValues are mean ± SE. From Portale et al. [121]. (p < .001). From Portale et al. [120]. 830 BERNARD P. H ALLORAN AND ANTHONY A. PORTALE the relationship was not different between the two (NcAMP) (+56%), and fractional excretion of phos- groups, but the intercept was lower in the elderly men. phorus (FEPi) (+44%) were higher in the elderly. With These data suggest that the capacity of phosphorus PTH infusion, serum 1,25(OH)2D increased to virtually restriction to increase renal production and serum con- the same maximum concentration in the young and centration of 1,25(OH)2D is not impaired by aging in elderly men (Fig. 4). Urinary cAMP, NcAMP, and FEPi healthy men. However, the normal relationship between increased, and both the time course and increment the serum concentrations of phosphorus and 1,25(OH)2D were not significantly different between age groups is altered with advancing age, such that for at any given (Fig. 5). TmP/GFR decreased in response to PTH to the concentration of serum phosphorus, serum concentra- same extent in both age groups. These results demon- tions of 1,25(OH)2D are lower in the elderly. strate that whereas the time course of the increase in serum 1,25(OH)2D induced by PTH is delayed in the 3. PARATHYROID HORMONE elderly relative to that in the young, maximum renal The serum concentration of parathyroid hormone responsiveness to PTH, in terms of cAMP generation, (PTH) increases progressively with advancing age in 1,25(OH)2D production, and phosphaturia, is not men and women [65Ð68,87,88,111,121Ð124]. As early impaired in healthy elderly men. as the fifth decade [111], serum PTH concentrations These studies raise the question as to why the higher are increased in normal healthy men, and by age 70 are basal serum PTH concentrations in the elderly are two- to threefold higher than values in young men not associated with increased serum 1,25(OH)2D con- (30Ð40 years old) [68,111]. The age-related increase in centrations. Such an altered relationship between the serum PTH can be attributed, at least in part, to dimin- serum concentrations of 1,25(OH)2D and those of ishing GFR, but even in healthy elderly men in whom either PTH or phosphorus may reflect a disorder in the GFR is greater than 70 ml/min, serum PTH is higher growth hormone (GH) and insulin-like growth factor than values in young men, albeit within the normal (IGF) axis in the elderly. Both growth hormone and range [120]. IGF-I stimulate 1-hydroxylase activity, and an intact The observation that, in aging men, serum PTH concentrations increase but those of 1,25(OH)2D do not suggests that the capacity of PTH to stimulate 140 1,25(OH)2D production may decrease with advancing hPTH(1-34) Infusion (35U/70kg/hr) age [70,75]. Indeed, Slovik et al. [75] found that intra- venous infusion of human PTH(1-34) induced an increase in serum 1,25(OH)2D in healthy young sub- * jects but not in elderly patients with osteoporosis. 120 * Tsai et al. [70] reported that the increase in serum * 1,25(OH)2D induced by infusion of bovine PTH(1-34) * is blunted in elderly postmenopausal women with mild D (pmol/L) 2 to moderate renal insufficiency when compared to that * * in healthy young women. These studies, however, were 100 performed in elderly patients with osteoporosis or mild to moderate renal insufficiency, and thus they do not permit separation of the effects of aging from other Serum 1,25(OH) conditions that may impair the ability of the kidney to 80 respond to PTH. To determine whether aging in normal subjects without osteoporosis or renal insufficiency influences the ability of the kidney to respond to PTH, we admin- istered hPTH(1-34) by intravenous infusion for 24 hr 0 to healthy young and elderly men who were free of −4 0481216 20 24 conditions known to affect mineral metabolism and in Days Time (hr) whom the GFR was > 70 ml/min/1.73 m2 [125]. Before FIGURE 4 Effect of PTH infusion on the serum concentration ± administration of PTH, the concentrations of blood ion- of 1,25(OH)2D in young (39 1, n = 9) (solid line) and elderly ± ized calcium and serum 1,25(OH) D and urinary excre- (70 1, n = 7) (broken line) men. Values are given for the 2 days 2 before PTH administration and every 4 hr during a 24-hr infusion. tion of calcium and phosphorus were similar in both The asterisks denote significant differences (p < .05) from baseline age groups, but concentrations of serum PTH (+148%), (i.e., days 0, −1, −2), using two-way repeated measures ANOVA. plasma cAMP (+44%), nephrogenous cAMP excretion From Halloran et al. [125]. CHAPTER 50 Vitamin D Metabolism and Aging 831

Young men Elderly men Control PTH RecoveryControl PTH Recovery 30 * * ∆ ± ∆ = 12.7 ± 2.0 = 13.3 2.2 20

10 NcAMP (nmol/L GFR)

0

Control PTH RecoveryControl PTH Recovery 1.50

1.25 +

1.0 *

∆ = 0.46 ± 0.08

TmPi (mmol/L GFR) 0.75 * ∆ = 0.44 ± 0.06

0.5

FIGURE 5 Effect of PTH infusion and recovery on NcAMP and tubular reab- sorptive maximum for phosphorus (TmPi) in young and elderly men. Values repre- sent measurements taken between 0700Ð0900 hr. Differences are indicated between the control and PTH periods. Using two-way ANOVA, asterisks denote a significant (p < .05) difference from the control period; plus signs denote a signifi- cant difference from the control period in young men. From Halloran et al. [125].

GH/IGF-I axis is required for dietary phosphorus restric- renal 1α-hydroxylase activity failed to increase with tion to stimulate 1,25(OH)2D production [126Ð129]. restriction of either dietary calcium or phosphorus, but With advancing age, the serum concentrations of GH did increase when dietary restriction was combined and IGF-I decrease [130,131], and administration of with infusion of IGF-I, albeit to levels below those GH to elderly subjects induces an increase in serum achieved in young (4Ð6 weeks) rats fed the restricted 1,25(OH)2D [132,133]. These observations suggest diets. Whether age-related changes in factors other than that the age-related decrease in serum GH and IGF-I GH and IGF-I play a role, remains to be determined. may reduce tonic stimulation of the 1α-hydroxylase. If so, the serum concentration of 1,25(OH)2D would be predicted to decrease, resulting in decreased intestinal VI. TISSUE RESPONSIVENESS AND calcium absorption, hypocalcemia, and stimulation THE ROLE OF VITAMIN D IN THE of PTH release. The increase in serum PTH would be AGING PROCESS expected to stimulate renal synthesis of 1,25(OH)2D, thus restoring tonic stimulation otherwise provided by A. Intestinal Calcium Absorption GH/IGF-I. The mild hyperparathyroidism, consequent hypophosphatemia, and unchanged serum 1,25(OH)2D Active intestinal calcium absorption is an excellent levels observed in elderly men are consistent with marker of tissue responsiveness to vitamin D. Intestinal this formulation. Also consistent with this formula- calcium absorption is reported to decrease with post- tion is the observation by Wong et al. that in aging maturational aging suggesting that aging may be accom- (24 months) [134] and adult (3Ð4 months) [135] rats, panied by intestinal (and other end organ) resistance to 832 BERNARD P. H ALLORAN AND ANTHONY A. PORTALE

1,25(OH)2D [136Ð144]. In animal studies, evidence 44 healthy women between the ages of 20 and 87. strongly supports the contention that calcium absorption Over the age range of the population the serum con- decreases with age. Part of the reason for this is the age- centration of 1,25(OH)2D increased by approximately related decrease in circulating 1,25(OH)2D. However, 27%, VDR concentration decreased by 20% (affinity in addition to the contribution associated with the did not change) and intestinal calcium absorption did age-related fall in 1,25(OH)2D, most [140,141] but not not change, suggesting that intestinal responsiveness all [80] investigators find an age-related resistance to the to 1,25(OH)2D declines with age. Despite these obser- stimulatory effects of 1,25(OH)2D. Wood et al. [140], vations, Ebeling et al. [148], in a later report, indicate for example, report that the sensitivity of duodenal that the responses of serum 1,25(OH)2D and intestinal calcium transport to the plasma concentration of 1,25- calcium absorption to restriction of dietary calcium are (OH)2D is reduced by 54% in aged compared to young the same in young (30 ± 1 years) and elderly (74 ± 1 rats. These investigators also report that mucosal years) women. In a more recent study representing per- vitamin D receptor (VDR) levels do not change with haps the most thorough investigation of the age-related age, although other investigators [145Ð147] find some changes in intestinal calcium transport in response decrease in VDR number. Binding affinity appears to 1,25(OH)2D to date, Pattanaungkul et al. [143] unaffected by age. Other data show that there are also measured fractional calcium absorption (FCA), and age-related decrements in the intestinal absorptive cell the serum concentrations of 1,25(OH)2D and the vita- plasma membrane calcium pump [142], calbindin min D binding protein (to calculate the free 1,25(OH)2D binding protein concentration [140], and ability to index) in young and elderly women in whom they translocate protein kinase C to membrane fractions manipulated the circulating level of 1,25(OH)2D over [146] in response to 1,25(OH)2D in aged compared to a broad range. The results clearly demonstrate that young animals. the absorptive response to a steady-state level of free In humans, there is near-unanimous agreement that 1,25(OH)2D (index) is impaired in the elderly (Fig. 6). aging is associated with impaired intestinal calcium Vitamin D receptor number is reported to both be absorption and reduced sensitivity to 1,25(OH)2D decreased [88,89] and remain unchanged [139] with [88,89,139Ð142]. Eastell et al. [88] measured serum aging. 1,25(OH)2D and intestinal calcium absorption in Collectively the animal and human data suggest women 26 to 88 years of age. Calcium absorption did that the VDR concentration in the intestinal mucosa not change with age but serum 1,25(OH)2D gradually probably decreases with aging but by a small amount. increased. They concluded that true calcium absorp- Nevertheless, there is a marked decrease in intestinal tion in healthy elderly women must be resistant to responsiveness to 1,25(OH)2D with age. The mecha- 1,25(OH)2D. To assess the relationship among calcium nisms for this are not clear but appear to involve the absorption, serum 1,25(OH)2D and VDR concentration calcium transport machinery (Ca-pump, calbindin) of in the intestinal mucosa, Ebeling et al. [89] studied the absorptive cell.

Young Elderly 1.2 1.2 r = 0.63, p = 0.003 r = 0.35, p = 0.142

0.8 0.8 FCA FCA

0.4 0.4

0 0 024682 46 8 0 × 5 × 5 1,25(OH)2D/DBP 10 1,25(OH)2D/DBP 10

FIGURE 6 Effect of aging on fractional calcium absorption (FCA) in response to the free 1,25(OH)2D index. From Pattanaungkul et al. [143]. CHAPTER 50 Vitamin D Metabolism and Aging 833

B. Bone, Kidney, and Other Tissues modulation in brain neurons [161] have all been directly linked to vitamin D. Thus, aging changes vita- Parallel studies in bone comparing responsiveness min D metabolism, and in turn, alterations in tissue to 1,25(OH)2D in young and old rodents [147,149,150] sensitivity to vitamin D are likely to contribute to the and humans [151Ð155] also show distinct changes aging process. with age. Vitamin D receptor concentrations [147] and the number of osteoblasts expressing the VDR [150] both decrease with aging in the rat. Furthermore VII. CONCLUSIONS osteoblasts from old animals are less active and their response to 1,25(OH)2D is altered [149]. Studies with A complete and accurate description of the effects human cells are more extensive. Martinez et al. [154] of aging on vitamin D metabolism cannot yet be made. report lower VDR expression in osteoblasts from older Many aspects have not been adequately investigated subjects and osteoblasts from old donors are associated and many of the existing data remain controversial. The with reduced baseline, and impaired osteocalcin following is an attempt to integrate what we think we [151,152,154,155] and alkaline phosphatase [152,153] know combined with a bit of speculation. response to 1,25(OH)2D (Table IV). In the absence of disease and in a free-living popu- In the rat kidney the amount of unoccupied VDR lation consuming a normal Western diet vitamin D does not appear to change with age [156]. However, metabolism begins to change around mid-life. Dietary binding of the VDR to DNA-cellulose is reduced by calcium intake and cutaneous production of vitamin D nearly 50% in kidney preparations from aged rats. decline, serum growth hormone and IGF-I begin to Further, the concentration of the vitamin DÐdependent decrease, and renal function begins to deteriorate. As a protein, calbindin-D28K, is reported to decrease with consequence of these changes the serum concentration advancing age, and the decline has been linked to both of 25OHD decreases, calcium absorption in the intes- lower serum 1,25(OH)2D levels and to impaired tine diminishes, and calcium bioavailability to meet responsiveness to 1,25(OH)2D [114]. serum demands declines. This stimulates PTH secre- Since the VDR is found in numerous tissues, perhaps tion [162Ð164], but because of declining renal function all tissues, and has been shown to stimulate differentia- and loss of the normal trophic effects of testosterone, tion it is likely that the documented changes in intestinal, estrogen, and GH/IGF on 1-hydroxylase activity, pro- bone, and kidney VDR concentration and responsive- duction of 1,25(OH)2D decreases, remains the same, ness to 1,25(OH)2D reflect a much broader and more or increases modestly depending upon the individual. generalized change in all tissues. For example, ker- The mild hyperparathyroidism associated with aging atinocytes express the VDR and respond to 1,25(OH)2D stimulates bone turnover. Coupled with the decrease by altering metabolic activity and differentiation, and in serum 25OHD and decline in intestinal calcium mice containing the null allele of VDR (VDR−/−) are absorption this shifts the source of calcium for mainte- more sensitive to chemically induced skin carcinogen- nance of the serum pool away from the diet and toward esis [157]. Thus, age-related declines in skin VDR levels the bone. This shift in mineral balance aggravates the or cell responsiveness to vitamin D may predispose to gradual loss of bone associated with aging and con- neoplasia. Furthermore, hypertension [158], benign tributes to the eventual development of senile osteo- prostate hypertrophy and prostate cancer [159], age- porosis. The impact of disease and medication use related changes in the sex steroids [160], and calcium complicates this enormously. Each individual is different. As we age we become less alike. In addition to the changes in vitamin D bioavailability and metabolism, TABLE IV Cell Content of Osteocalcin (ng/mg Protein) in Human Osteoblastic Cells after aging is associated with alterations in tissue respon- Treatment with 1,25(OH) D siveness to vitamin D. These changes not only affect 2 mineral metabolism but also have broad effects on

1,25(OH)2D 1,25(OH)2D numerous tissues ranging from skin to brain. Thus, Age (yr) Basal (10−8 mol/liter) (10−6 mol/liter) aging causes changes in vitamin D metabolism and tissue responsiveness, and these changes in turn are <50 0.7 ± 0.2 8.0 ± 6.1a 12.8 ± 7.2a likely to contribute to the aging process. Further studies ± ± a,b ± a >50 0.3 0.2 2.8 1.5 5.0 2.3 are clearly needed to better define the effects of aging per se and the confounding effects of disease burden Values are mean ± SEM, n =6. ap < .05 vs baseline. on mineral homeostasis and vitamin D metabolism, and bp <.05 between 10−8 and 10−6 mol/liter. the role of age-related alterations in tissue vitamin D From Martinez et al. with permission [154]. responsiveness to aging. 834 BERNARD P. H ALLORAN AND ANTHONY A. PORTALE

References variations in parathyroid hormone secretion in post- menopausal women. N Engl J Med 321:1777Ð1783. 24. Schock NW 1972 Energy metabolism caloric intake and 1. Cerimele D, Celleno L, Serri F 1999 Physiological changes physical activity of the aging. In: LA Carson (ed) Nutrition in ageing skin. Brit J Dermatol 122(Suppl 35):13Ð20. in Old Age. Almqvist and Wiksell, Uppsala, Sweden. 2. Lavker RM 1979 Structural alterations in exposed and unex- 25. Elahi VK, Elahi D, Andres R, Tobin JD, Butler MG, Norris AH posed aged skin. J Invest Dermatol 73:59Ð66. 1983 A longitudinal study of nutritional intake in men. 3. Goodson WH, Hunt TK 1979 Wound healing and aging. J Gerontol 38:162Ð180. J Invest Dermatol 73:88Ð91. 26. Omdahl JL, Garry PJ, Hunsaker LA, Hunt WC, Goodwin JS 4. Daly CH, Odland GF 1979 Age-related changes in the 1982 Nutritional status in a healthy elderly population: mechanical properties of skin. J Invest Dermatol 73:84Ð87. Vitamin D. Am J Clin Nutr 36:1225Ð1233. 5. Grove GL, Kligman AM 1983 Age-associated changes in 27. Sowers MR, Wallace RB, Hollis BW, Lemke JH 1986 human epidermal cell renewal. J Gerontol 38:137Ð142. Parameters related to 25OHD levels in a population-based 6. Escoffier C, DeRegal J, Rochefort A 1989 Age-related mechan- study of women. Am J Clin Nutr 43:621Ð628. ical properties of human skin. J Invest Dermatol 93:353Ð360. 28. Sharkey JR, Branch LG, Zahoori N, Gueliani C, Bushy- 7. Gilchrest BA, Garmyn M, Yaar M 1994 Aging and photoaging Whitehead J, Hains PS 2002 Inadequate nutrient intakes affect gene expression in cultured human keratinocytes. Arch among homebound elderly and their correlation with indi- Dermatol 130:82Ð86. vidual characteristics and health-related factors. Am J Clin 8. Puschel HU, Chang J, Muller PK, Brinckman J 1995 Nutr 76:1435Ð1445. Attachment of intrinsically and extrinsically aged fibroblasts 29. Delvin EE, Imbach A, Copti M 1988 Vitamin D nutritional on collagen and fibronectin. J Photochem Photobiol 27:39Ð46. status and related biochemical indices in an autonomous 9. Ghadially R, Brown BE, Sequeira-Martin SM, Feingold KR, elderly population. Am J Clin Nutr 48:373Ð378. Elias PM 1995 The aged epidermal permeability barrier. 30. Payette H, Gray-Donald K 1991 Dietary intake and bio- J Clin Invest 95:2281Ð2290. chemical indices of nutritional status in an elderly population 10. Tsuchida Y 1993 The effect of aging and arteriosclerosis on with estimates of the precision of the 7-day food record. Am human skin blood flow. J Dermatol Sci 5:175Ð181. J Clin Nutr 54:478Ð488. 11. MacLaughlin J, Holich MF 1985 Aging decreases the 31. Barragry JM, France MW, Corless P, Gupta SP, Switala S, capacity of human skin to produce vitamin D. J Clin Invest Cohen RD 1978 Intestinal cholecalciferol absorption in 76:1536Ð1538. elderly and young adults. Clin Sci Mol Med 55:213Ð220. 12. Webb AR, Kline L, Holick MF 1988 Influence of season and 32. Harris SS, Dawson-Hughs B, Perrone GA 1999 Plasma latitude on the cutaneous synthesis of vitamin D3. J Clin 25-hydroxyvitmain D response of young and old men to Endocrinol Metab 67:373Ð378. three weeks of supplementation with 1800 IU/d of vitamin D. 13. Holick MF, Matsuoka LY, Wortsman J 1989 Age, vitamin D Am J Clin Nutr 18:470Ð474. and solar ultraviolet. Lancet 2:1104Ð1105. 33. Clemens TL, Zhou XY, Myles M, Endres D, Lindsay R 1986 14. Holick MF 1994 Vitamin D—new horizons for the 21st Serum vitamin D and vitamin D concentrations and absorp- century. Am J Clin Nutr 60:619Ð630. 3 2 tion of vitamin D2 in elderly subjects. J Clin Endocriol Metab 15. Matsuoka LY, Wortsman J, Hanifan N, Holick MF 1988 63:656Ð660. Chronic sunscreen use decreases circulating concentrations 34. Hollander D, Tarnawski H 1984 Influence of aging on of 25-hydroxvitamin D. Arch Dermatol 124:1802Ð1804. vitamin D absorption and unstirred water layer dimensions in 16. Gloth FM, Gundberg CM, Hollis BW, Haddad JG, Tobin JD the rat. J Lab Clin Med 103:462Ð469. 1995 Vitamin D deficiency in homebound elderly persons. 35. Fleming BB, Barrows CH 1982 The influence of aging on J Am Med Assoc 274:1683Ð1686. intestinal absorption of vitamins A and D by the rat. Exp 17. Wortsman J, Masuoka LY, Chen TC, Lu Z, Holick MF 2000 Gerontol 17:115Ð120. Decreased bioavailability of vitamin D in obesity. Am J Clin 36. Harris SS, Dawson-Hughes B 2002 Plasma vitamin D and Nutr 72:690Ð693. 25OHD responses of young and old men to supplementation 18. Lips P 2001 Vitamin D deficiency and secondary hyper- with vitamin D3. J Am Coll Nutr 21:357Ð362. parathyroidism in the elderly: consequences for bone loss and 37. Aksnes L, Rodland O, Aarskog D 1988 Serum levels of vita- fractures, and therapeutic implications. Endo Rev 22:477Ð501. min D3, 25-hydroxyvitamin D3 in elderly and young adults. 19. Recommended Daily Allowances, 10th ed. 1989 Food and Bone Miner 3:351Ð357. Nutrition Board, Commission of Life Sciences, National 38. Van der Wielen RP, Lowik MR, van den Berg H, de Groot LC, Research Council, National Academic Press, Washington, DC, Haller J, Moreiras O, van Staveren WA 1995 Serum vitamin D pp. 92Ð98. concentrations among elderly people in Europe. Lancet 20. Holick MF 1998 Vitamin requirements for humans of all 346:207Ð210. ages: New increased requirements for women and men 50 yr 39. McMurtry CT, Young SE, Downs RW, Adler RA 1992 Mild and older. Osteop Intern 8:24Ð29. vitamin D deficiency and secondary hyperparathyroidism in 21. NHANES III Series 11, No. 245 Dietary Intake of Macronu- nursing home patients receiving adequate dietary vitamin D. trients, Micronutrients, and Other Dietary Constituents: J Am Geriat Soc 40:343Ð347. United States, 1988Ð94: http://www.cdc.gov/nchs/data/series/ 40. Weisman Y, Schen RJ, Eisenberg Z, Edelstein S, Harell A sr11/sr11 245.pdf 1981 Inadequate status and impaired metabolism of vitamin D 22. Dietary Intake of Ten Key Nutrients for Public Health, in the elderly. Isr J Med Sci 17:19Ð21. 1999Ð2000. Advance data from vital and health statistics No. 41. Guillemant J, Oberlin F, Bourgeois P, Guillemant S 1984 334:http://www.cdc.gov/nchs/data/ad/ad334.pdf Age-related effect of a single oral dose of calcium on parathy- 23. Krall EA, Sahyoun N, Tannenbaum S, Dallal GE, roid function: relationship with vitamin status. Am J Clin Nutr Dawson-Hughs B 1989 Effect of vitamin D intake on seasonal 60:403Ð407. CHAPTER 50 Vitamin D Metabolism and Aging 835

42. Need AG, Morris HA, Horowitz M, Nordin C 1993 Effects women randomly assigned to one of two doses of vitamin D. of skin thickness, age, body fat and sunlight on serum Am J Clin Nutr 61:1140Ð1145. 25-hydroxyvitamin D. Am J Clin Nutr 58:882Ð885. 60. Barger-Lux MJ, Heaney RP, Lanspa SJ, Healy JC, DeLuca HF 43. Quesada JM, Coopmans W, Ruiz B, Aljama P, Jans I, 1995 An investigation of sources of variation in calcium Bouillon R 1992 Influence of vitamin D on parathyroid absorption efficiency. J Clin Endocrinol Metab 80:406Ð411. function in the elderly. J Clin Endocriol Metab 75:494Ð501. 61. Orwoll ES, Oviatt SK, McClung MR, Deftos LJ, Sexton G 44. McKenna MJ 1992 Differences in vitamin D status between 1990 The rate of bone mineral loss in normal men and the countries in young adults and the elderly. Am J Med 93:69Ð77. effects of calcium and cholecalciferol supplementation. Ann 45. Perry HM, Horowitz M, Morley JE, Patrick P, Vellas B, Intern Med 112:29Ð34. Baumgartner R, Garry R 1999 Longitudinal changes in 62. Bouillon R, Auwerx J, Dekeyser L, Fevery J, Lisssns W, serum 25OHD in older people. Metabolism 48:1028Ð1032. De Moor P 1984 Serum vitamin D metabolites and their bind- 46. Fardellone P, Sebert JL, Garabedian M, Bellony R, Maamer M, ing protein in patients with liver cirrhosis. J Clin Endocrinol Agbomson F, Brasier M 1995 Prevalence and biological con- Metab 59:86Ð89. sequences of vitamin D deficiency in elderly institutionalized 63. Bikle DD, Gee E, Halloran B, Haddad JG 1984 Free subjects. Rev Rhum Engl Ed 62:576Ð581. 1,25-dihydroxyvitamin D levels in serum from normal 47. Brazier M, Kamel S, Maamer M, Agbomson F, Elesper I, subjects, pregnant subjects and subjects with liver disease. Garabedian M, Desmet G, Sebert JL 1995 Markers of bone J Clin Invest 74:1966Ð1977. remodeling in the elderly subject: Effects of vitamin D insuffi- 64. Manolagas S, Culler FL, Howard JE, Brickman AS, Deftos LJ ciency and its correction. J Bone Miner Res 10:1753Ð1761. 1983 The cytoreceptor assay for 1,25(OH)2D and its applica- 48. Ooms ME, Lipps P, Roos JC, van der Vvijgh WJ, tion to clinical studies. J Clin Endocrinol Metab 56:751Ð760. Popp-Snijders C, Bezemer PD, Bouter LM 1995 Vitamin D 65. Epstein S, Bryce G, Hinman JW 1986 The influence of age status and sex hormone binding globulin: Determinants of on bone mineral regulating hormones. Bone 7:421Ð425. bone turnover and bone mineral density in elderly women. 66. Orwoll ES, Meier D 1986 Alterations in calcium, vitamin D J Bone Miner Res 10:1177Ð1184. and PTH physiology in normal men with aging. J Clin 49. Guillemant S, Guillemant J, Feteanu D, Sebag-Lanoe R 1989 Endocrinol Metab 63:1262Ð1269. Effect of vitamin D3 administration on serum 25-hydroxy- 67. Sherman SS, Hollis BW, Tobin JD 1990 Vitamin D status and vitamin D3, 1,25-dihydroxyvitamin D and osteocalcin in related parameters in a healthy population: the effects of age, vitamin D deficient elderly people. J Ster Biochem 33: sex and season. J Clin Endocrinol Metab 71:405Ð413. 1155Ð1159. 68. Halloran BP, Portale AA, Lonergan ET, Morris RC 50. Sakota O, Masud T, San P, Hosking DJ 1999 Vitamin D 1990 Production and metabolic clearance of 1,25(OH)2D in insufficiency increases bone turnover markers and enhances men: effect of advancing age. J Clin Endocrinol Metab 70: bone loss at the hip in patients with established vertebral 318Ð323. osteoporosis. Clin Endocrinol 51:217Ð221. 69. Gallagher C, Riggs BL, Eisman JA, Hamstra A, Arnaud SB, 51. Melin AL, Wilske J, Ringertz H, Saaf M 1999 Vitamin D DeLuca HF 1979 Intestinal calcium absorption and vitamin D status, parathyroid function and femoral bone density in an metabolites in normal subjects and osteoporotic patients. elderly Swedish population living at home. Aging 11:200. J Clin Invest 64:729Ð736. 52. Rapuri PB, Kinyamu HK, Gallagher JC, Haynatzka V 2002 70. Tsai KS, Heath H, Kumar R, Riggs BL 1984 Impaired vita- Seasonal changes in calcitropic hormones, bone markers and min D metabolism with aging in women. 73:1668Ð1672. bone mineral density in elderly women. J Clin Endocrinol 71. Buchanan JR, Myers CA, Greer RB 1988 Effect of declining Metab 87:2024Ð2032. renal function on bone density in aging women. Calcif Tissue 53. Stone K, Bauer DC, Black DM, Sklarin P, Ensrud KE, Int 43:1Ð6. Cummings SR 1998 Hormonal predictors of bone loss in elderly 72. Prince RL, Dick I, Garcia WP, Retallack RW 1990 The women: a prospective study. J Bone Miner Res 13:1167Ð1174. effects of the menopause on calcitriol and PTH: Responses 54. Myer HE, Hendriksen C, Falch JA, Pedersen JI, Tverdal A to a low dietary calcium stress test. J Clin Endocrinol Metab 1996 Risk factors for hip fracture in a high incidence area. 70:1119Ð1123. Osteop Int 5:239Ð246. 73. Fujisawa Y, Kida K, Matsuda H 1984 Role of change in vita- 55. Ooms ME, Roos JC, Bezemer PD, van der Vvijgh WJ, min D metabolism with age on calcium and phosphorus Bouter L, Lipps P 1995 Prevention of bone loss by vitamin D metabolism in normal human subjects. 59:719Ð726. supplementation in elderly women: a randomized double- 74. Dokoh S, Morita R, Fukunaga M, Yamamoto I, Torizuka K blind trial. J Clin Endocrinol Metab 80:1052Ð1058. 1978 Competitive protein binding assay for 1,25(OH)2D in 56. Himmelstein S, Clemens TL, Rubin A, Lindsay R 1990 human plasma. Endocrinol Jpn 25:431Ð436. Vitamin D supplementation in elderly nursing home resi- 75. Slovik SM, Adams JS, Neer RM, Holick MF, Potts JT 1981 dents increases 25OHD but not 1,25(OH)2D. Am J Clin Nutr Deficient production of 1,25(OH)2D in elderly osteoporotic 52:701Ð706. patients. N Engl J Med 305:372Ð374. 57. Lips P, Wiersinga A, van Ginkel FC 1988 The effect of vita- 76. Lund B. Sorensen OH, Lund B, Agner E 1982 Serum min D supplementation on vitamin D status and pararthyroid 1,25(OH)2D in normal subjects and in patients with post- function in elderly subjects. J Clin Endocrinol Metab menopausal osteopenia. Horm Metab Res 14:271Ð274. 67:644Ð650. 77. Armbrecht HJ, Strong R, Boltz M, Rocco D, Wood WG, 58. Dawson-Hughes B, Dallal GE, Krall EA, Harris S, Sokoll LJ, Richardson A 1988 A modulation of age-related changes in Falconor G 1991 Effect of vitamin D supplementation on serum 1,25(OH)2D and PTH by dietary restriction in F344 wintertime overall bone loss in healthy postmenopausal rats. J Nutr 118:1360Ð1365. women. Ann Intern Med 115:505Ð512. 78. Fox J, Mathew B 1991 Heterogeneous response to PTH in 59. Dawson-Hughes B, Harris SS, Krall EA, Dallal GE, Falconer G, aging rats: Evidence for skeletal PTH resistance. Am J Green CL 1995 Rates of bone loss in postmenopausal Physiol 260:E933ÐE937. 836 BERNARD P. H ALLORAN AND ANTHONY A. PORTALE

79. Gray RW, Gambert SR 1982 Effect of age on plasma concentration of 1,25(OH)D2 by determining its production 1,25(OH)2D in the rat. Age 5:54Ð56. rate in humans. J Clin Invest 77:7Ð12. 80. Liang CT, Barns J, Takamoto S, Sacktor B 1989 Effect of age 98. Young EW, Hsu CH, Patel S, Simpson RU, Komanicky P on calcium uptake in isolated duodenal cells: role of 1987 Metabolic degradation and synthesis of calcitriol in 1,25(OH)2D. Endocriology 124:2830Ð2836. spontaneously hypertensive rat. Am J Physiol 252:E778ÐE782. 81. Armbrecht HJ, Forte L, Halloran BP 1989 Effect of age 99. Seeman E, Kumar R, Hunder GG, Scott M, Heath H, and dietary calcium on renal 25(OH)D metabolism, serum Riggs BL 1980 Production, degradation and circulating levels 1,25(OH)2D and PTH. Am J Physiol 246:E266ÐE270. of 1,25(OH)2D in health and in chronic glucocorticoid excess. 82. Wada L, Daly R, Kern D, Halloran B 1992 Kinetics of J Clin Invest 66:664Ð669. 1,25-dihydroxyvitamin D metabolism in the aging rat. Am 100. Hsu CH, Patel S, Young EW, Simpson RU 1987 Production J Physiol 262:E906ÐE910. and degradation of calcitriol in renal failure rats. Am J Physiol 83. Theuns HM, van der Vijgh WJF, Hackeng WHL, Bekker H, 253:F1015ÐF1019. Barto R, Lips P, Roholl PJM, Netelenbos JC, Knook DL 101. Gray A, Feldman HA, McKinlay JB, Longcope C 1991 Age, 1991 Parathyroid hormone and vitamin D metabolites disease and changing sex hormone levels in middle-aged throughout life of two inbred rat strains. In: AW Norman, men: results of the Massachusettes male aging study. J Clin R Bouillon, M Thomasset (eds) Vitamin D, de Gruyter, Endocrinol Metab 73:1016Ð1025. New York, pp. 769Ð770. 102. Vermeulen A 1991 Androgens in the aging male. J Clin 84. Schock NW 1945 Inulin, diodrast and urea clearance studies Endocrinol Metab 73:221Ð224. on aged human subjects. Fed Proc 4:65Ð71. 103. Hagenfeldt Y, Linde K, Sjoberg HE, Zumkeller W, Arver S 85. Rowe JW, Anders R, Tobin JR, Norris AH, Schock NW. The 1992 Testosterone increases serum 1,25(OH)2D and IGF-I in effect of age on creatinine clearance in man: a cross-sectional hypogonadal men. Intern J Androl 15:93Ð102. and longitudinal study. J Geront 31:155Ð163. 104. Boucher A, D’Amour P, Hamel L 1989 Estrogen replacement 86. Lindeman RD, Tobin J, Schock NW 1985 Longitudinal studies decreases the set-point of PTH stimulation by calcium in on the rate of decline in renal function with age. J Am Geriatr normal postmenopausal women. J Clin Endocrinol Metab Soc 33:278Ð285. 68:831Ð836. 87. Marcus R, Madvig P, Young G 1984 Age related changes in 105. Scharla SH, Minne HW, Waibel TS 1990 Bone mass reduc- PTH and PTH action in normal humans. J Clin Endocrinol tion after estrogen deprivation by long-acting gonadotropin- Metab 58:223Ð230. releasing hormone agonists and its relation to pretreatment 88. Eastell R, Yergey AL, Vierira NE, Cedel SE, Kumar R, serum concentrations of 1,25(OH)2D. J Clin Endocrinol Metab Riggs BL 1991 Interrelationship among vitamin D metabolism, 70:1055Ð1061. true calcium absorption, parathyroid function and age in 106. Marcus R, Villa ML, Cheema M, Cheema C, Newhall K, women: Evidence of an age-related intestinal resistance to Holloway L 1992 Effects of conjugated estrogen on the cal- 1,25(OH)2D. J Bone Miner Res 6:125Ð132. citriol response to parathyroid hormone in postmenopausal 89. Ebeling PR, Sandgren ME, DiMagno EP, Lane AW, women. J Clin Endocrin Metab 74:413Ð418. DeLuca HF, Riggs BL 1992 Evidence of an age-related 107. Packer E, Holloway L, Newhall K, Kanwar G, Butterfield K, decrease in intestinal responsiveness to vitmain D: Relationship Marcus R 1990 Effects of estrogen on daylong circulating between serum 1,25-dihydroxyvitamin D and intestinal calcium,phosphorus,1,25-dihydroxyvitamin D and parathy- vitamin D receptor concentrations in normal women. J Clin roid hormone in postmenopausal women. J Bone Miner Res Endocrinol Metab 75:176Ð182. 5:877Ð884. 90. Hartwell D, Riis BJ, Christiansen C 1990 Changes in vitamin 108. Stock JL, Coderre JA, Mallette LE 1985 Effects of a short D metabolism during natural and medical menopause. J Clin course of estrogen on mineral metabolism in postmenopausal Endocrinol Metab 71:127Ð132. women. J Clin Endocrinol Metab 61:595Ð600. 91. Falch JA, Oftebro H, Haug E 1987 Early postmenopausal 109. Gallagher C, Riggs BL, DeLuca HF 1980 Effect of estrogen bone loss is not associated with a decrease in circulating on calcium absorption and vitamin D metabolites in post- 25OHD, 1,25(OH)2D or vitamin D binding protein. J Clin menopausal osteoporosis. J Clin Endocrinol Metab 51: Endocrinol Metab 64:836Ð841. 1359Ð1364. 92. Bikle DD, Halloran BP, Harris ST, Portale AA 1992 110. Selby PL, Peacock M, Barkworth SA, Brown WB, Progestin antagonism of estrogen stimulated 1,25(OH)2D Taylor GA 1985 Early effects of ethinyloestradiol and levels. J Clin Endocrinol Metab 75:519Ð524. norethisterone treatment in postmenopausal women. Clin Sci 93. Dick IM, Prince RL, Kelly JJ, Ho KK 1995 Oestrogen effects 69:265Ð271. on calcitriol levels in postmenopausal women: a comparison 111. Minisola S, Pacitti M, Scarda A 1993 Serum ionized calcium, of oral versus transdermal administration. Clin Endocrinol parathyroid hormone and related variables: Effect of age and 43:219Ð224. sex. J Bone Miner 23:183Ð193. 94. Jongen MJ, Bishop JE, Cade C, Norman AW 1987 Effect of 112. Wiske PS, Epstein S, Bell NH, Queener SF, Edmondson J, dietary calcium, phosphate and vitamin D deprivation on the Johnston CC 1979 Increases in immunoreactive PTH with pharmacokinetics of 1,25(OH)2D in the rat. Horm Metab Res age. N Engl J Med 300:1419Ð1421. 19:481Ð485. 113. Endres DB, Morgan CH, Garry PJ, Omdahl JL 1987 Age- 95. Halloran BP, Castro ME 1989 Vitamin D kinetics in vivo: Effect related changes in serum immunoreactive parathyroid of 1,25(OH)2D administration. Am J Physiol 256:E686ÐE691. hormone and its biological action in healthy men and women. 96. Fox J, Ross R 1985 Effect of low phosphorus and low J Clin Endocrinol Metab 65:724Ð731. calcium diets on the production and metabolic clearance 114. Chen ML, Boltz M, Christakos S, Armbrecht HJ 1992 Age- rates of 1,25(OH)2D in pigs. J Endocrinol 105:169Ð173. related alterations in calbindin-D28K induction by 1,25(OH)2D 97. Portale AA, Halloran BP, Murphy MM, Morris RC Jr in primary cultures of rat renal tubule cells. Endocrinology 1986 Oral intake of phosphorus can determine the serum 130:3295Ð3300. CHAPTER 50 Vitamin D Metabolism and Aging 837

115. Hulter HN, Halloran BP, Toto RD, Peterson JC 1985 Long 133. Lieberman SA, Holloway L, Marcus R, Hoffman AR 1999 term control of calcitriol concentration in dog and man: The Interactions of growth hormone and parathyroid hormone in dominant role of plasma calcium concentration in experi- renal phosphate, calcium and calcitriol metabolism and bone mental hyperparathyroidism. J Clin Invest 76:695Ð702. remodeling in postmenopausal women. J Bone Miner 116. Armbrecht HJ, Hodam TL, Boltz MA, Kumar VB 1999 Res:1723Ð1728. Capacity of a low calcium diet to induce the renal vitamin D 134. Wong MS, Tembe VA, Favus MJ 2002 Insulin-like growth 1α-hydroxylase is decreased in adult rats. Biochem Biophys factor-I stimulates renal 1,25-dihydroxycholecalciferol syn- Res Commun 18:731Ð734. thesis in old rats fed a low calcium diet. J Nutr 130:1147Ð1152. 117. Portale AA, Halloran BP, Morris RC Jr 1987 Dietary intake 135. Wong MS, Sriussadaporn S, Tembe VA, Favus MJ 1997 of phosphorus modulates the circadian rhythm in serum con- Insulin-like growth factor I increases renal 1,25(OH)2D3 centration of phosphorus: Implications for the renal produc- biosynthesis during low-P diet in adult rats. Am J Physiol tion of 1,25-dihydroxyvitamin D. J Clin Invest 8:1147Ð1154. 272:F698ÐF703. 118. Portale AA, Halloran BP, Morris RC Jr 1989 Physiologic 136. Francis RM, Peacock M, Taylor GA, Storer JH, Nordin BEC regulation of the serum concentration of 1,25-dihydroxy- 1984 Calcium malabsorption in elderly women with verte- vitamin D by phosphorus in normal men. J Clin Invest 83: bral fractures: evidence for resistance to the actions of 1494Ð1499. vitamin D metabolites on the bowl. Clin Sci 66:103Ð107. 119. Villa ML, Packer E, Cheema M, Holloway L, Marcus R 1991 137. Avioli LV, McDonald JE, Lee SW 1965 The influence of age Effects of aluminum hydroxide on the parathyroid-vitamin D on the intestinal absorption of Ca in women and its relation to axis of postmenopausal women. J Clin Endocrinol Metab Ca absorption in postmenopausal osteoporosis. J Clin Invest 73:1256Ð1261. 44:1960Ð1967. 120. Portale AA, Halloran BP, Morris RC, Lonergan ET 1996 138. Bullammore JR, Gallagher JC, Wilkinson JR, Nordin BEC Effect of aging on the metabolism of phosphorus and 1,25-dihy- 1970 Effect of age on calcium absorption. Lancet 2:535Ð537. droxyvitamin D in healthy men. Am J Physiol 270:E483ÐE490. 139. Heaney RP, Reckler RR, Stegman MR, Moy AJ 1989 Calcium 121. Portale AA, Lonergan ET, Halloran BP 1997 Aging alters absorption in women: relationships to calcium intake, estrogen calcium regulated parathyroid hormone release in healthy status, and age. J Bone Miner Res 4:469Ð475. men. Am J Physiol 272:E139ÐE146. 140. Kinyamu HK, Gallagher JC, Prahl JM, Deluca HF, 122. Young G, Marcus R, Minkoff JR, Kim LY, Segre GV 1987 Petranick KM, Lanspa SJ 1997 Association between intestinal Age-related rise in parathyroid hormone in man: the use of vitamin D receptor, calcium absorption, and serum 1,25- intact and midmolecule antisera to distinguish hormone dihydroxyvitamin D in normal young and elderly women. secretion from retention. J Bone Miner Res 2:367Ð373. J Bone Miner Res 12:922Ð928. 123. Forero MS, Klein RF, Nissenson RA, Nelson K, Heath III H, 141. Wood RJ, Fleet JC, Cashman K, Bruns ME, Deluca HF 1998 Arnaud CD, Riggs BL 1987 Effect of age on circulating Intestinal calcium absorption in the aged rat: Evidence immunoreactive and bioactive parathyroid hormone levels in of intestinal resistance to 1,25(OH)2D. Endocrinology 139: women. J Bone Miner Res 2:363Ð368. 3843Ð3848. 124. Ledger GA, Burritt MF, Kao PC, O’Fallon WM, Riggs BL, 142. Armbrecth HJ, Boltz MA, Kumar VB 1999 Intestinal plasma Khosla S 1994 Abnormalities of PTH secretion in elderly membrane calcium pump protein and its induction by women that are reversible by short term therapy with 1,25(OH)2D decrease with age. Am J Physiol 277:G41ÐG47. 1,25(OH)2D. J Clin Endocrinol Metab 79:211Ð216. 143. Pattanaungkul S, Riggs BL, Yergey AL, Vieira NE, 125. Halloran BP, Lonergan ET, Portale AA 1996 Aging and renal O’Fallon WM, Khosla S 2000 Relationship of intestinal responsiveness to parathyroid hormone in healthy men. calcium absorption to 1,25-dihydroxyvitamin D levels in J Clin Endocrinol Metab 81:2192Ð2197. young versus elderly women: Evidence for age-related 126. Gray RW, Garthwaite TL 1985 Activation of renal intestinal resistance to 1,25(OH)2D action. J Clin Endocrinol 1,25(OH)2D synthesis by phosphate deprivation: evidence Metab 85:4023Ð4027. for a role for growth hormone. Endocrinology 116:189Ð193. 144. Balogh G, Boland R, de Boland AR 2000 1,25(OH)2D affects 127. Gray RW 1987 Evidence that somatomedians mediate the the subcellular distribution of protein kinase C isoenzymes effect of hypophosphatemia to increase serum 1,25(OH)2D in rat duodenum: influence of aging. J Cell Biochem 79: levels in rats. Endocrinology 121:504Ð509. 686Ð694. 128. Halloran BP, Spencer EM 1988 Dietary phosphorus and 145. Takamoto S, Seino Y, Sacktor B, Liang CT 1990 Effect of 1,25-dihydroxyvitamin D metabolism: influence of insulin- age on duodenal 1,25-dihydroxyvitamin D receptors in Wistar like growth factor I. Endocrinology 123:1225Ð1230. rats. Biochim Biophys Acta 1034:22Ð28. 129. Menaa C, Vrtovsnik F, Friedlander G, Corvol M, Garabedian M 146. Liang CT, Barns J, Imanaka S, DeLuca HF 1994 Alterations 1995 Insulin-like growth factor I, a unique calcium-dependent in mRNA expression of duodenal 1,25-dihydroxyvitamin D stimulator of 1,25-dihydroxyvitamin D production. J Biol Chem receptor and vitamin D dependent calcium binding protein in 270:25461Ð25467. aged Wistar rats. Exp Gerontol 29:179Ð186. 130. Florini JR, Prinz PN, Vitiello MV, Hintz RL 1985 147. Horst RL, Goff JP, Reinhardt TA 1990 Advancing age results Somatomedin C levels in healthy young and old men: in the reduction of intestinal and bone 1,25-dihydroxy- Relationship to peak and 24-hour integrated levels of growth vitamin D receptor. Endocrinology 126:1053Ð1057. hormone. J Gerontol 40:2Ð7. 148. Ebeling PR, Yergey AL, Vierira NE, Burritt MF, O’Fallon WM, 131. Rudman D 1985 Growth hormone, body composition and Kumar R, Riggs BL 1994 Influence of age on effects of aging. J Am Geriatr Soc 33:800Ð806. endogenous 1,25(OH)2D on calcium absorption in normal 132. Marcus R, Butterfield G, Holloway L, Gilland L, Baylink D, women. Calcif Tissue Res 55:330Ð334. Hintz R, Sherman B 1990 Effects of short term administration 149. Wang X, Schwartz Z, Yaffe P, Ornoy A 1999 The expression of recombinant human growth hormone to elderly people. of TGF-beta and interleukin-1beta mRNA, and the response J Clin Endocrinol Metab 70:519Ð523. to 1,25(OH)2D′17 beta-estradiol and testosterone is age 838 BERNARD P. H ALLORAN AND ANTHONY A. PORTALE

dependent in primary cultures of mouse-derived osteoblasts 157. Zinser GM, Sundberg JP, Welsh J 2002 Vitamin D receptor in vitro. Endocrine 11:13Ð22. ablation sensitizes skin to chemically induced tumorigenesis. 150. Duque G, Abdaimi KE, Macoritto M, Miller MM, Kremer R Carcinogenesis 23:2103Ð2109. 2002 Estrogens regulate expression and response of 158. Pfeifer M, Bergerow B, Minne HW, Nachtigall D, Hansen C 1,25(OH)2D receptors in bone cells: Changes with aging and 2001 Effects of a short term vitamin D and calcium supple- hormone deprivation. Biochem Biophys Res Commun mentation on blood pressure and PTH levels in elderly 299:446Ð454. women. J Clin Endocrinol Metab 86:1633Ð1637. 151. Battmann A, Battmann A, Jungt G, Schulz A 1997 Endosteal 159. Crescioli C, Maggie M, Vannelli GB, Luconi M, Salerno R, human bone cells show age-related activity in vitro. Exp Clin Barni T, Gulisano M, Forti G, Serio M 2000 Effect of a Endocrinol Diabetes 105:98Ð102. vitamin D analogue on keratinocyte growth factor-induced 152. Martinez ME, Medina S, Sanchez M, Del Campo MT, Esbrit P, cell proliferation in benign prostate hyperplasia. J Clin Rodrigo A, Sanchez-Cabezudo MJ, Moreno I, Garces MV, Endocrinol Metab 85:2576Ð2583. Munuera L 1999 Influence of skeletal site of origin and 160. Kinuta K, Tanaka H, Moriwake T, Aya K, Kato S, Seino Y donor age on 1,25(OH)2D induced response of various 2000 Vitamin D is an important factor in estrogen biosynthesis osteoblastic markers in human osteoblastic cells. Bone of both female and male gonads. Endocrinology 141: 24:203Ð209. 1317Ð1324. 153. Katzberg S, Lieberherr M, Ornoy A, Klein BY, Hendel D, 161. Brewer LD, Thibault V, Chen KC, Langub MC, Landfield PW, Somjen D 1999 Isolation and hormonal responsiveness of Porter NM 2001 Vitamin D hormone confers neuro-protection primary cultures of human bone-derived cells: Gender and in parallel with down regulation of L-type calcium channel age differences. Bone 25:667Ð673. expression in hippocampal neurons. J Neurosci 21:98Ð108. 154. Martinez P, Moreno I, De Miguel F, Vila V, Esbrit P, 162. Dawson-Huges B, Harris SS, Dallal GE 1997 Plasma calcid- Martinez ME 2001 Changes in osteocalcin response to iol, season and serum PTH concentration in healthy elderly 1,25(OH)2D stimulation and basal vitamin D receptor men and women. Am J Clin Nutr 65:67Ð71. expression in human osteoblastic cells according to donor 163. Lips P, Duong T, Oleksik A, Black D, Cummings S, Cox D, age and skeletal origin. Bone 29:35Ð41. Nickelsen T 2001 A global study of vitamin D status and 155. Klein-Nuland J, Sterck JG, Semeins CM, Lips P, Joldersma M, parathyroid function in postmenopausal women with osteo- Baart JA, Burger EH 2002 Donor age and meachnosensitivity porosis: Baseline data from the multiple raloxifene evalua- of human bone cells. Osteopor Intern 13:137Ð146. tion clinical trial. J Clin Endocrinol Metab 86:1212Ð1221. 156. Koszewski NJ, Reinhardt TA, Beitz DC, Horst RL 1990 164. Need AG, Horowitz M, Morris HA, Nordin BC 2000 Vitamin Developmental changes in rat kidney 1,25(OH)2D receptor. D status: Effects on PTH and 1,25(OH)2D in postmenopausal Biochem Biophys Res Commun 170(1):65Ð72. women. Am J Clin Nutr 71:1577Ð1581. CHAPTER 51 Vitamin D Metabolism in Pregnancy and Lactation

HEIDI J. KALKWARF Division of General and Community Pediatrics, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio

BONNY L. SPECKER Martin Program in Human Nutrition, South Dakota State University, Brookings, South Dakota

I. Introduction IV. Adaptations in Vitamin D and Calcium Metabolism II. Adaptations in Vitamin D and Calcium Metabolism during Lactation and after Weaning during Pregnancy V. Effects of Low Maternal Vitamin D and Calcium Intakes III. Effects of Low Maternal Vitamin D on Breast Milk Vitamin D and Calcium Concentrations and Calcium Intake during Pregnancy VI. Conclusions on the Fetus and Neonate References

I. INTRODUCTION but 250 mg/d during the third trimester [1]. Maternal serum calcium concentrations decrease in the first half Significant changes in maternal calcium metabolism of pregnancy, reaching a nadir at mid-gestation due occur during pregnancy, lactation, and after weaning to to plasma volume expansion and decreased albumin provide the calcium needed for fetal bone mineral concentrations [2,3]. Serum concentrations of ionized accretion, for the synthesis of breast milk, and for the calcium or calcium adjusted for albumin concentra- restoration of the maternal skeleton. Multiple factors tions show less fluctuation and remain relatively stable are involved in regulating these processes so that mater- throughout pregnancy [2,4,5]. Serum concentrations of nal blood calcium concentrations are maintained within intact parathyroid hormone (PTH) have been reported a narrow range despite the large changes in calcium to decrease [2,4,6] or not change [3] over the course of fluxes that occur. Primary strategies include changes in pregnancy. In contrast, circulating concentrations of the efficiency of absorption of calcium from the intesti- the active form of vitamin D, 1,25-dihydroxyvitamin D nal tract, alterations in renal calcium reabsorption and (1,25(OH)2D), are increased during pregnancy. By the thus urinary calcium excretion, and the flux of calcium second trimester serum concentrations of 1,25(OH)2D in and out of bone. Reproductive hormones have impor- increase by 50Ð100% over pre-pregnant values, and in tant effects on calcium homeostasis and bone the third trimester they increase by 100% [3,7] (Fig. 1). metabolism, and during pregnancy and lactation their The signal to increase 1,25(OH)2D synthesis is not actions work in concert with the vitamin D endocrine clear, as PTH concentrations are not elevated. Vitamin D system to ensure that calcium needs are met for fetal binding protein (DBP) concentrations increase in bone mineral accretion, for breast milk production, and pregnancy possibly due to increased concentrations to maintain circulating maternal calcium concentrations. of estrogen. Although some of the increase in serum 1,25(OH)2D concentrations may be due to the increase in the amount bound to its binding protein, the amount II. ADAPTATIONS IN VITAMIN D of free 1,25(OH)2D is still elevated [3,8]. Some of the cir- AND CALCIUM METABOLISM culating 1,25(OH)2D may be of extrarenal origin as the DURING PREGNANCY decidua has been shown to synthesize 1,25(OH)2D [9] (see Chapter 79). Consistent with this is the fact that A. Vitamin D and Calcium Metabolism maternal 1,25(OH)2D concentrations rapidly decrease within a few days after delivery [10]. Approximately 25 to 30 g of calcium is transferred The increase in 1,25(OH)2D concentrations during to the fetal skeleton by the end of pregnancy, the most pregnancy is accompanied by an increase in intestinal of which is transferred during the last trimester. The calcium absorption (Fig. 1). Fractional calcium fetus accumulates 2 to 3 mg/d during the first trimester, absorption increases by 50Ð56% over prepregnant VITAMIN D, 2ND EDITION Copyright © 2005, Elsevier, Inc. FELDMAN, PIKE, AND GLORIEUX All rights reserved. 840 HEIDI J. KALKWARF AND BONNY L. SPECKER

80 Intestinal calcium absorption 180 some of the increase in bone turnover markers is due 1,25(OH)2D to increased turnover of soft tissue collagen of the uterus 65 140 and skin [6,12]. Increases in circulating concentrations of insulin-like growth factor-1 (IGF-1) and placental lactogen have 50 100 D (pmol/liter)

2 been suggested as the possible mechanisms behind increased bone turnover during pregnancy [2,6]. 35 60 Increases in IGF-1 concentrations precede the increase % Calcium absorption 1,25(OH) in bone formation markers, and IGF-1 concentrations 20 20 correlated more strongly with markers of bone forma- Prepregnant 1st trimester 2nd trimester 3rd trimester tion than bone resorption. A recent study investigated changes in calcium-regulating hormones and osteopro- FIGURE 1 Intestinal calcium absorption and serum 1,25(OH)2D concentrations before and during pregnancy. Data from Ritchie tegerin (OPG) during pregnancy and found that mater- et al. [3]. nal serum OPG concentrations steadily increased with gestational age [10]. Receptor activator of nuclear factor-κ B ligand (RANKL) is important in osteoclast differentiation [13], and OPG acts as a decoy receptor levels in the second trimester and by 54Ð62% in the for RANKL, thereby preventing the differentiation of third trimester [3,7]. Thus increased maternal intestinal osteoclast precursors into mature osteoclasts and absorption of calcium is an important physiologic decreasing bone resorption. The authors speculated that adaptation to secure sufficient amounts of this mineral higher OPG concentrations during pregnancy, possibly for the fetus. Despite the increased need for calcium, of placental origin, might play a role in the control of urinary calcium excretion increases by 40Ð50% over bone metabolism throughout gestation. the course of pregnancy. This is most likely due to the marked increase in glomerular filtration rate and increased absorptive load [2,3,11]. B. Changes in Bone Mineral Content Several studies report increased concentrations of and Density during Pregnancy biochemical markers of bone turnover during preg- nancy. Serum concentrations of biochemical markers Few studies have included bone mineral density of bone formation, namely bone specific alkaline measurements during pregnancy because of the poten- phosphatase and the propeptide of type 1 collagen tial risks to the fetus associated with radiation exposure. (PICP), are elevated in the third trimester with a steep There is conflicting evidence as to whether there is a peak in the last month of pregnancy [2,6]. It is not clear net change in bone density during pregnancy. Several whether there are changes in the first two trimesters of different approaches have been used to evaluate the pregnancy as an increase, decrease, or no change in the impact of pregnancy on maternal bone. Some longitudi- concentration of these bone formation markers have nal studies have measured bone density by dual energy been reported [2,6]. Osteocalcin concentrations have X-ray absorptiometry (DXA) before conception and been found to decrease [6], decrease then increase [3], shortly after delivery and have found no significant loss or not change during pregnancy [10]. Markers of bone of bone density [3,7,14,15]. However, other studies resorption, namely the breakdown products of colla- report losses of 2 to 2.6% at the ultradistal radius [16,17], gen such as pyridinoline, deoxypyridinoline, and NTx, 2 to 4% at the spine [2,6,18], and 2.4 to 3.6% at the increase throughout pregnancy reaching a peak at the hip [2,19]. In some of these studies bone density was end of pregnancy [2,3,6]. Although it appears that there measured as far as 6 weeks postpartum, and signifi- is a dissociation of bone resorption and bone formation cant losses of bone may occur within the immediate in the first two trimesters with elevated bone resorption postpartum period thereby making it hard to interpret predominating, it is difficult to predict whether there is these results. Naylor and co-workers found that the a net loss of maternal bone during pregnancy using changes in bone density during pregnancy varied accord- biochemical markers alone. Other factors confound ing to skeletal site. Bone density at trabecular-rich the interpretation of biochemical markers of bone sites (pelvis and spine) decreased by 3 to 4%, whereas turnover during pregnancy such as whether they are of bone density at cortical sites (arms and legs) increased maternal, placental, or fetal origin, and the effects of by 2% [6]. pregnancy on the metabolic clearance of these proteins Many investigators have investigated changes in by the liver and kidney. It also has been suggested that bone density by use of ultrasound as it does not involve CHAPTER 51 Vitamin D Metabolism in Pregnancy and Lactation 841 radiation exposure. Speed of sound (SOS) and bone due to increased glomerular filtration rate and an ultrasound attenuation (BUA) are strongly correlated increased absorptive calcium load. with bone density measured at the same skeletal site. Longitudinal studies over the course of pregnancy have documented a decrease in SOS and BUA mea- III. EFFECTS OF LOW MATERNAL sured at the os calcis or phalanges in the latter half of VITAMIN D AND CALCIUM INTAKE pregnancy, particularly in the third trimester [20Ð23]. DURING PREGNANCY ON THE FETUS These data are consistent with a loss of maternal bone AND NEONATE mineral toward the end of pregnancy when the fetus is accreting bone mineral most rapidly. Longitudinal A. Vitamin D studies of bone density in women who do not lactate after delivery show that bone density at the spine and Maternal vitamin D deficiency is more likely to occur hip increase by about 2% in the first year postpartum in winter months, in countries that do not routinely for- [24Ð26]. It is possible that this increase in bone may tify dairy or other food products with vitamin D, among compensate for bone lost during pregnancy. If so, this members of ethnic groups who cover most of their skin, may explain why studies comparing bone density of or among individuals with heavily pigmented skin. Few women with different pregnancy histories have found randomized nutritional vitamin D and calcium inter- no differences in bone density measured many years ventions have been conducted during pregnancy, and the later [27Ð30]. Henderson et al. found that even grand importance of maternal vitamin D and calcium intake is multiparous women having six or more pregnancies best illustrated from observational studies of women and long lactations did not have lower bone density of with poor calcium and/or vitamin D intake. These obser- the lumbar spine, femoral neck, or mid-radius than vational studies, along with the results of the few clinical nulliparous women later in life [31]. trials that have been conducted, indicate that maternal In summary, several adaptations in the maternal vitamin D and calcium status are important in neonatal calcium economy occur in order to provide sufficient handling of calcium, and possibly in fetal growth and calcium for fetal bone mineral accretion (Fig. 2). The bone maturation and mineralization. primary adaptive strategy is an increase in intestinal Maternal vitamin D deficiency during pregnancy can calcium absorption. Additional calcium may come affect neonatal calcium metabolism. Maternal vitamin D from demineralization of maternal bone. The increased deficiency is associated with secondary hyperparathy- concentrations of 1,25(OH)2D, estrogen, IGF-1, pla- roidism, and maternal hyperparathyroidism during cental lactogen, and OPG interact to facilitate these pregnancy may lead to neonatal hypocalcemia or changes. Despite the increased need for calcium dur- tetany [32,33]. In the early 1970s, Purvis and co-workers ing pregnancy, urinary calcium losses are increased noted that the occurrence of neonatal tetany among 112 infants was inversely related to the amount of sun- light exposure the mothers had during the last trimester of pregnancy [34]. The authors speculated that the Dietary calcium mothers developed hyperparathyroidism secondary to vitamin D deficiency leading to a transitory hypoparathy- roidism and hypocalcemia in the neonate. Several Intestinal calcium investigators subsequently reported that infants of absorption mothers with low vitamin D intake during pregnancy had low serum calcium concentrations in cord blood or during the first week of life [35Ð37]. Several random- ized trials of vitamin D supplementation during preg- Fetus Blood Urinary calcium nancy were later reported. Cockburn and co-workers randomized two obstetric wards, one with 506 women who received 400 IU vitamin D/day from the 12th week of gestation and another with 633 women who did not receive vitamin D Bone [38]. They reported higher maternal, cord, and infant FIGURE 2 Adaptations in the calcium economy during preg- 25-hydroxyvitamin D (25OHD) concentrations with nancy. Solid arrows indicate an increase with arrow thicknesses vitamin D supplementation. They also found that the representing the magnitude of the fluxes. incidence of neonatal hypocalcemia was less with 842 HEIDI J. KALKWARF AND BONNY L. SPECKER

18 Case reports of congenital rickets in newborn infants 16 of mothers with severe nutritional osteomalacia associ- 14 ated with vitamin D or calcium deficiency have been reported [43Ð45]. Reif and co-workers, in a case-control 12 study, reported an association between craniotabes, or 10 Human milk delayed ossification of the cranial vertex, and maternal Formula 8 and neonatal 25OHD concentrations. However, these findings have not been replicated in other observa- 6 tional studies or trials [36,39]. Although Brooke and 4 co-workers did not find an association between cran-

Incidence of hypocalcemia (%) 2 iotabes and vitamin D status, they did find that infants 0 of mothers who received placebo had larger fontanelles Control Vitamin D than infants of mothers supplemented with vitamin D, FIGURE 3 Incidence of neonatal hypocalcemia on day 6 by type which is consistent with impaired skull ossification [39]. of feeding (solid bars are formula-fed; open bars are human milk-fed) A study conducted in China also found possible evidence in infants whose mothers received either no vitamin D (Control) or for a relationship between maternal vitamin D deficiency 400 IU vitamin D/d from 12th week of gestation (Vitamin D). Data and impaired fetal bone ossification [46]. The presence from Cockburn et al. [38]. of wrist ossification centers in neonates was associated with cord serum 25OHD concentrations. A higher rate of ossification centers in newborn infants of mothers vitamin D supplementation, although this was modi- with adequate vitamin D status was apparent when fied by the infant’s feeding (hypocalcemia greater compared to infants of mothers with low vitamin D with formula feeding vs breast-feeding) (Fig. 3) [38]. status. Several randomized trials of vitamin D supplementation Few studies have investigated the role of maternal (1000 IU/d) during pregnancy subsequently found that vitamin D status on infant bone mineralization. infants of mothers receiving vitamin D had higher Congdon et al. measured forearm BMC using single serum calcium concentrations within the first week of photon absorptiometry and found that BMC did not life than infants of mothers receiving placebo [39]. differ by history of vitamin D supplementation during Brooke and co-workers conducted a randomized, dou- pregnancy and was not correlated with cord serum ble-blind trial of vitamin D supplementation (1000 IU/d 25OHD concentrations [36]. The majority of the vita- from 28 to 32 weeks gestation) and found that infants min D supplementation trials reported to date began of mothers receiving vitamin D had higher serum cal- supplements late in gestation. There are observational cium on days 3 and 6 and a lower incidence of symp- studies suggesting that maternal vitamin D status early tomatic hypocalcemia than infants of mothers receiving in gestation may be important in fetal bone develop- placebo [39Ð41]. These studies were completed in ment. Seasonal differences in adult bone density have populations that are at increased risk for vitamin D been reported by some [47,48] but not all investi- deficiency, and the results indicate that adequate mater- gators [49] and may be attributed to seasonal varia- nal vitamin D status during pregnancy may be necessary tions in vitamin D status. Studies that have examined to ensure appropriate neonatal calcium homeostasis. seasonal differences in newborn BMC have had Maternal vitamin D deficiency during pregnancy conflicting findings. Two studies conducted in the also may lead to impaired fetal growth and bone devel- United States found that infants born in the summer opment. The occurrence of vitamin D deficiency is have lower BMC compared to infants born in the winter high among Asians from the Indian subcontinent living months [50,51]. These findings are opposite to what is in Britain [33]. A trial of vitamin D supplementation seen in adults. However, Namgung and co-workers (1000 IU/d) among pregnant Asian women found that examined this association in infants born in Korea and a higher percent of the infants randomized to the also found that winter-born infants had lower BMC placebo group (28.6%) were small-for-gestational-age than summer-born infants in Korea [52]. One explana- compared to infants in the supplemented group (15.3%) tion for these contradictory findings is that many [39]. Some investigators [37,42], but not all [41], have United States women take prenatal vitamins contain- reported lower birth weights of infants born to mothers ing vitamin D beginning in the second trimester of with low vs adequate vitamin D status. Decreased pregnancy. Thus the observed seasonal effects on skeletal mineralization in utero may be manifested as infant BMC in the United States may reflect vitamin D rickets or osteopenia in the newborn infant. However, status in the first trimester of pregnancy. Because there fetal or congenital rickets of the newborn are rare. is minimal fetal calcium accretion in the first trimester, CHAPTER 51 Vitamin D Metabolism in Pregnancy and Lactation 843 this would indicate some other function of vitamin D IV. ADAPTATIONS IN VITAMIN D on fetal bone development. AND CALCIUM METABOLISM DURING In summary, maternal vitamin D status during LACTATION AND AFTER WEANING pregnancy has been shown to be associated with neona- tal calcium homeostasis. There are conflicting reports A. Vitamin D and Calcium Metabolism indicating a possible role of maternal vitamin D status during Lactation in fetal growth and bone development. Lactating women secrete approximately 200Ð240 mg of calcium daily in breast milk [55]. Over 6 months B. Calcium of lactation this is equivalent to approximately 6% of her total skeletal calcium reserve. Despite this large Few studies have evaluated the effects of maternal transfer of calcium from the maternal circulation, calcium intakes on fetal bone mineral accretion. A study maternal serum calcium concentrations are unchanged by Raman and co-workers in India found that under- [3,56] or slightly elevated [57,58]. There is no increase nourished pregnant mothers who were supplemented in PTH concentrations during lactation. In fact, serum with 300 or 600 mg calcium/d had similar maternal concentrations of PTH are lower in lactating as metacarpal bone density compared to mothers not compared to nonlactating women in the first 3 months supplemented, but the bone density of their newborns postpartum [56,59,60]. was greater [53]. Similar results have been reported in The lower PTH concentrations during lactation are a large randomized trial of maternal calcium supple- likely to be a consequence of the rapid bone resorption mentation for the prevention of preeclampsia [54]. that occurs especially early in lactation and the resultant A total of 256 women were enrolled in the randomized, increase in serum calcium concentrations. Two potential double-blind, placebo-controlled trial. Newborn infants causes of bone resorption are hypoestrogenemia and of mothers in the lowest quintile of calcium intake elevated circulating concentrations of parathyroid (<600 mg/d) who were randomized to calcium supple- hormone related peptide (PTHrP) [61Ð63]. Lactation mentation had higher BMC compared to newborns in results in prolonged postpartum amenorrhea and hypo- the lowest quintile whose mothers were randomized to estrogenemia due to suppression of the hypothalamicÐ placebo (Fig. 4). There was no difference in neonatal pituitaryÐgonadal axis. Hypoestrogenemia is known to BMC between placebo and supplemented maternal result in bone resorption in a variety of clinical and groups in the upper four quintiles of maternal calcium experimental situations. PTHrP also stimulates bone intake. These studies suggest that there is a lower limit resorption (see Chapter 43). PTHrP is made in the mam- to the mother’s calcium regulatory capacity to buffer mary gland and is present in very high concentrations in the fetus from variations in her calcium intake. This breast milk [64]. Presumably some of the PTHrP syn- intake of approximately 600 mg/d is below the current thesized by the mammary gland may gain access to the recommended calcium intakes for pregnant women. maternal circulation. Circulating PTHrP has actions similar to PTH and acts through the PTH receptor [65]. PTHrP is a potent stimulator of bone resorption, and 80 Placebo Calcium supplemented administration of PTHrP results in an immediate 70 ∗ increase in serum calcium concentrations [66]. In lactat- ing women, serum concentrations of calcium are more 60 highly correlated with PTHrP than with PTH [62], sug- 50 gesting that the decrease in PTH also may be secondary

40 to elevated PTHrP concentrations and subsequently increased serum calcium concentrations. 30 Whether or not there is a decrease in urinary calcium

Newborn BMC (g) 20 excretion during lactation is unclear. Some studies have found a 20 to 50% decrease in urinary calcium excre- 10 tion in lactating women [3,57,67Ð70]. However, some 0 of this decrease may be a postpartum phenomenon <561 562–775 776–969 970 –1374 >1374 and not just a result of lactation. Studies that compared Material dietary calcium intake (mg/d) urinary calcium excretion in lactating women to that FIGURE 4 Effects of maternal calcium intake and calcium sup- of nonlactating postpartum controls have not found plementation (2 g/d) on newborn total body bone mineral content. urinary calcium excretion to be lower in lactating Data from Koo et al. [54]. *p < 0.05 women [56,59,60,71]. 844 HEIDI J. KALKWARF AND BONNY L. SPECKER

Unlike during pregnancy, there is no increase in cir- a day [81]. Polatti et al. also found less of a deficit in culating concentrations of 1,25(OH)2D in lactating as bone density in lactating women who resumed menses compared to nonlactating postpartum women [56,59]. by 5 months postpartum as compared to those were Commensurate with this finding is that there is no dif- remained amenorrheaic (−3.0% vs −5.8%) [25]. These ference in intestinal calcium absorption in lactating as findings underscore the importance of ovarian hormones compared to nonlactating women [3,71Ð73]. Intestinal in regulating bone loss during lactation. calcium absorption is increased in lactating rats that Dietary calcium intake does not appear to affect the are suckling multiple pups, but this does not occur in amount of bone lost during lactation in women. Bone women nursing one infant. Greer et al. found that loss during lactation has been observed in women with women nursing twins had elevated concentrations of high calcium intakes (>1500 mg/d) [17,60,75], and PTH and 1,25(OH)2D [74]. Urinary calcium excretion dietary calcium intake has not been shown to be a sig- and intestinal calcium absorption were not measured, nificant predictor of bone loss during lactation but presumably elevations in PTH and 1,25(OH)2D [55,59,75,80]. Furthermore, three randomized calcium concentrations resulted in changes in urinary calcium supplementation trials have demonstrated that provi- excretion and intestinal calcium absorption. Although sion of supplemental calcium does not affect bone loss bone demineralization, and not improved efficiency of during lactation. Prentice et al. studied 60 lactating intestinal calcium absorption, is the primary compen- women in the Gambia who had a very low calcium satory response to secure calcium in lactating women, it intake (274 mg/d). Half of the women received an can be hypothesized that increased absorption efficiency average of 714 mg of supplemental calcium per day, may occur in situations of greater calcium demand such and half received a placebo. Overall there was a sig- as women nursing multiple infants. nificant loss (1.1%) of bone mineral at the radial shaft by 13 weeks postpartum, but there was no difference in the amount of bone lost between supplemented and B. Changes in Bone Mineral during Lactation unsupplemented lactating women [67]. Kalkwarf et al. randomized 83 lactating women and 81 nonlactating One of the primary changes in calcium homeostasis postpartum women whose dietary calcium intake aver- during lactation is the marked decrease in bone min- aged 735 mg/d, to receive a calcium supplement (1 g/d) eral content and density. Decreases of 3% to 9% at the or placebo for 6 months. There was a small effect lumbar spine and femoral neck have been reported (+1.2%) of calcium supplementation on bone density [3,17,24,25,60,75Ð78]. The decreases in bone density of the lumbar spine when considering all women [24]. of the spine and hip occur rapidly within the first 3 to However, bone loss did not differ between lactating 6 months of lactation, and bone density remains lower women who received the calcium supplement and with continued lactation [75,76]. The rate of bone loss those that received placebo (4.2% vs 4.9%) (Fig. 5). In at these sites in the first 6 months of lactation is sig- a supplementation trial conducted in 274 Italian women, nificant as it approaches 1% per month. In comparison, Polatti et al. found no difference in the amount of bone menopausal and early postmenopausal women lose lost at the lumbar spine (4.0% vs 4.4%) or the ultra- bone at the rate of 1% to 2% per year [79]. distal radius (2.0% vs 2.2%) between supplemented The amount of bone lost during lactation is variable among women. Women who breast-feed longer, or 8 who have a greater breast milk volume, have greater bone loss compared to women who breast-feed for 6 shorter periods of time [17,55,75,80]. In addition, the 4 length of postpartum amenorrhea is an important Nonlactating groups determinant of bone loss during lactation. Women who 2 Calcium resume menses early have less bone loss than women 0 Placebo who have longer periods of postpartum amenorrhea

Percent change −2 [17,25,76]. Although the length of postpartum amen- Lactating groups orrhea and length of lactation are related, some women −4 Calcium resume menses while still breastfeeding. Kalkwarf Placebo −6 et al. found that the net change in bone density at the 0.5 36 lumbar spine at 6 months postpartum was only Ð1.8% in Months since delivery women who had resumed menses whereas it was Ð4.4% FIGURE 5 Effects of lactation and calcium supplementation in women who had not resumed menses despite the (1 g/d) on percent change in bone density of the lumbar spine during fact that both groups were breast-feeding five times the first 6 months postpartum. Reproduced from Kalkwarf et al. [24]. CHAPTER 51 Vitamin D Metabolism in Pregnancy and Lactation 845

D. Changes in Bone Mineral after Weaning Dietary calcium Maternal bone density increases rapidly after weaning. Much of the bone density lost during lactation is Intestinal calcium recovered within the first 6 months after weaning. absorption Laskey and co-workers demonstrated that the resump- tion of menses was as good a predictor of bone changes as was the length of lactation [76]. Increases in bone density after weaning occur earlier for the Milk Blood Urinary calcium spine than for the femoral neck, which may be a con- sequence of the greater amount of trabecular bone at the spine [17,75,76,78]. Although most studies show a complete recovery of bone density at the spine after Bone weaning, it is not clear that the recovery of bone at the femoral neck is complete, as deficits in bone density FIGURE 6 Adaptations in the calcium economy during lactation. at this site were still evident at the end of the study Solid arrows indicate an increase with arrow thicknesses represent- follow-up in most of these studies [17,75,78]. It is possi- ing the magnitude of the fluxes. Dashed arrows indicate a decrease. ble that a complete recovery of bone may have occurred with a longer follow-up period. Consistent with this are the results of studies in postmenopausal women (1 g calcium/d) and unsupplemented women over that have found that lactation history is not a signifi- 6 months of lactation [25]. cant predictor of bone density [27Ð30] and is not asso- The primary adaptive strategy to secure calcium to ciated with increased risk of hip fracture [82Ð84]. support breast milk production is demineralization of Kalkwarf et al. conducted a calcium supplementa- maternal bone (Fig. 6). Lactation also may result in tion trial in 76 women who had lactated for 6 months urinary calcium conservation, although the results and then weaned their infants and 82 nonlactating from studies are conflicting as to whether this is a lac- postpartum controls to determine whether provision of tation effect or a postpartum effect. Bone loss during supplemental calcium could enhance bone recovery lactation is related to postpartum amenorrhea and after weaning [24]. By 6 months after the initiation of hypoestrogenemia. Elevated circulating concentrations weaning (4 months after complete weaning), lactating of PTHrP also may have a role in bone loss during women who had received 1 g/d of supplemental cal- lactation. cium had a significantly greater increase in spinal BMD compared to women who received the placebo (5.9% vs 4.4%) (Fig. 7). C. Vitamin D and Calcium Metabolism The restoration of bone mass after lactation has after Weaning ceased is important in maintaining maternal bone health.

Additional adjustments in calcium metabolism occur shortly after lactation has stopped as the maternal phys- 8 Lactating groups iologic system switches from mobilizing calcium from Weaning 6 Calcium the skeleton and secreting calcium into breast milk to Placebo restoring maternal calcium reserves. Some studies have 4 found an increase in serum concentrations of PTH Calcium shortly after weaning and decreased urinary calcium 2 Placebo excretion, presumably in response to increased PTH 0 Nonlactating groups concentrations [7,57]. However, these changes have not Percent change − been found in all studies [56]. Kalkwarf et al. found 2 that serum concentrations of 1,25(OH)2D were higher −4 in women shortly after weaning [72], and this was −6 accompanied by a higher intestinal calcium absorption 6 9 12 (37% vs 31%). Ritchie et al. did not find a significant Months since delivery increase in intestinal calcium absorption after weaning, FIGURE 7 Effects of weaning and calcium supplementation (1 g/d) but the smaller sample size in that study may have on percent change in bone density of the lumbar spine during the limited their ability to detect a small increase [3]. second 6 months postpartum. Reproduced from Kalkwarf et al. [24]. 846 HEIDI J. KALKWARF AND BONNY L. SPECKER

infant is dependent upon endogenous synthesis or other Dietary calcium dietary sources for vitamin D. Most reported cases of rickets have been of black infants, supporting the premise that persons with dark skin have difficulty synthesizing adequate amounts of vitamin D due to the relative inability of sunlight Intestinal calcium to penetrate heavily pigmented skin. In addition, the absorption diet of mothers of rachitic infants appears to be low in vitamin D and the mothers may be vitamin D deficient themselves. Although some investigators have found breast milk vitamin D or 25OHD concentrations to be correlated Blood Urinary calcium with maternal intake of vitamin D, mothers who con- sume 600Ð700 IU vitamin D/d still have low concen- trations of vitamin D in breast milk ranging from only 5 to 136 IU/liter. The biological activity of vitamin D in human milk averages 13 IU/liter, while the 25OHD concentrations represent 38 IU/liter. The average biological activity of vitamin D in human milk is Bone less than 50 IU per day, assuming an average intake of 0.75 liter/d [87]. FIGURE 8 Adaptations in the calcium economy after weaning. Solid arrows indicate an increase, with arrow thicknesses repre- Investigators recently found that consumption of senting the magnitude of the fluxes. Dashed arrows indicate cod liver oil supplements among Icelandic women a decrease. increased milk vitamin D concentrations, but the milk concentrations were still below the current Nordic recommendations [88]. Ala-Houhala and co-workers from Finland, in a series of vitamin D supplementation Bone mass and density increase in parallel with the trials, found that supplementing lactating mothers with return of menstruation and presumably normalization up to 1000 IU vitamin D/d in northern latitudes during of circulating estrogen concentrations. The recovery of winter months increased maternal serum 25OHD con- bone mass may be facilitated by an increase in intestinal centrations, but did not stabilize infant serum 25OHD calcium absorption efficiency and a decrease in urinary concentrations (Fig. 9) [89]. Maternal supplementation calcium excretion, but these alterations have not been with 2000 IU/d was found to normalize infant serum found consistently across studies (Fig. 8). 25OHD concentrations [90]. There were no differences in infant serum calcium or alkaline phosphatase con- centrations when mothers were supplemented with V. EFFECTS OF LOW MATERNAL VITAMIN D AND CALCIUM INTAKES ON BREAST MILK VITAMIN D AND CALCIUM CONCENTRATIONS 50

A. Vitamin D 40

30 Vitamin D deficiency leads to rickets in children. Ala-Houhala, 1985 Infant formula is routinely fortified with vitamin D, Ala-Houhala, 1986

25-OHD 20 but very low vitamin D concentrations have been Infant serum found in human milk [85]. Infant serum 25OHD is 10 correlated with maternal vitamin D status early in the 0 neonatal period and is probably a result of placental Infant: 0 0 0 400 1,000 IU/d vitamin D transfer and fetal stores. Beyond the neonatal Maternal 0 1,000 2,000 0 0 IU/d period, the breast-fed infant’s serum 25OHD concentra- FIGURE 9 Infant serum 25OHD concentrations (ng/ml) after tions are correlated with neither breast milk vitamin D 20 weeks of either infant or maternal vitamin D supplementation. nor maternal serum 25OHD concentrations [86], and the Data from Ala-Houhala et al. [89,90]. CHAPTER 51 Vitamin D Metabolism in Pregnancy and Lactation 847 either 1000 or 2000 IU vitamin D/d or when infants were These results are consistent with older observational supplemented with 400 IU/d. studies showing that milk calcium concentrations are Specker and co-workers found based on conservative not associated with maternal calcium intake [92,93]. estimates, that exclusively breast-fed infants residing in Cincinnati could maintain serum 25OHD concentra- tions above the lower limit of normal (11 ng/ml) with VI. CONCLUSIONS 2 hr of sunshine exposure per week if fully clothed except for the face [86]. The cutoff for defining low Multiple changes in the maternal calcium economy 25OHD is based on the concentration at which nutri- occur during pregnancy, lactation, and after weaning to tional rickets has been observed. Other factors such as protect maternal calcium concentrations while provid- latitude, season, weather conditions, and use of sun- ing sufficient calcium for fetal bone mineral accretion, screens may affect vitamin D status. Large seasonal breast milk production and maternal bone recovery. The differences in sunlight exposure and serum 25OHD strategies to secure calcium during these physiologic concentrations over the first year of life have been states differ. During pregnancy, the primary strategy to observed in infants followed longitudinally [91]. These secure additional calcium is by increases in serum con- findings indicate that the infant’s sunlight exposure centrations of 1,25(OH)2D and intestinal calcium plays a more dominant role in determining his or her absorption. During lactation, maternal bone is deminer- vitamin D status than the mother’s vitamin D status or alized, possibly because of the lactation-induced amen- milk vitamin D concentrations. orrhea, in order to secure adequate availability of calcium for breast milk production. After weaning and the return of menses, maternal bone density increases. B. Calcium Lactation does not appear to have a long-term negative effect on maternal bone density nor does it increase Two of the randomized trials described earlier that osteoporotic fracture risk. The maternal calcium regula- investigated the effect of supplemental calcium on tory system is able to provide sufficient calcium to the maternal bone changes during lactation also measured fetus and for breast milk even when calcium intake is milk calcium concentrations. Kalkwarf and co-workers low. However, there is some evidence that neonatal found that women with habitually low calcium intake calcium homeostasis and fetal bone mineral accretion (<800 mg/d) who were supplemented with calcium may be compromised when maternal vitamin D status is (1 g/d) had breast milk calcium concentrations similar low or calcium intake is below 600 mg/d. to that of women who received the placebo [24] (Fig. 10). Prentice and co-workers also conducted a randomized calcium supplementation trial among References Gambian women and found no effect of calcium intake on breast milk calcium concentrations [67]. 1. Widdowson EM 1981 Changes in body composition during growth. In: Davis JA, Dobbings J (eds) Scientific Foundations of Paediatrics. William Heinemann Medical Books, London, 35 pp. 330Ð342. Calcium supplemented 2. Black A, Topping J, Durham B, Farquharson R, Fraser W 2000 30 Placebo A detailed assessment of alterations in bone turnover, calcium homeostasis, and bone density in normal pregnancy. J Bone 25 Miner Res 15:557Ð564. 3. Ritchie LD, Fung EB, Halloran BP, Turnlund JR, Van Loan MD, 20 Cann CE, King JC 1998 A longitudinal study of calcium homeostasis during human pregnancy and lactation and after 15 resumption of menses. Am J Clin Nutr 67:693Ð701. 4. Gallacher SJ, Fraser WD, Owens OJ, Dryburgh FJ, Logue FC, 10 Jenkins A, Kennedy J, Boyle IT 1994 Changes in calciotrophic hormones and biochemical markers of bone turnover in normal Breast milk calcium (mg/dL) 5 human pregnancy. Eur J Endocrinol 131:369Ð374. 5. Pitkin RM, Reynolds WA, Williams GA, Hargis GK 1979 0 0.5 3 6 Calcium metabolism in normal pregnancy: A longitudinal Months postpartum study. Am J Obstet Gynecol 133:781Ð790. 6. Naylor KE, Iqbal P, Fledelius C, Fraser RB, Eastell R 2000 FIGURE 10 Effects of calcium supplementation (1 g/d) on breast The effect of pregnancy on bone density and bone turnover. milk calcium concentrations. Data from Kalkwarf et al. [24]. J Bone Miner Res 15:129Ð137. 848 HEIDI J. KALKWARF AND BONNY L. SPECKER

7. Cross NA, Hillman LS, Allen SH, Krause GF, Vieira NE 1995 26. Hopkinson JM, Butte NF, Ellis K, Smith EO 2000 Lactation Calcium homeostasis and bone metabolism during pregnancy, delays postpartum bone mineral accretion and temporarily lactation, and postweaning: A longitudinal study. Am J Clin Nutr alters its regional distribution in women [In Process Citation]. 61:514Ð523. J Nutr 130:777Ð783. 8. Bikle DD, Gee E, Halloran B, Haddad JG 1984 Free 27. Kritz-Silverstein D, Barrett-Connor E, Hollenbach KA 1992 1,25-Dihydroxyvitamin D levels in serum from normal subjects, Pregnancy and lactation as determinants of bone mineral density pregnant subjects, and subjects with liver disease. J Clin Invest in postmenopausal women. Am J Epidemiol 136:1052Ð1059. 74:1966Ð1971. 28. Melton LJ, III, Bryant SC, Wahner HW, O’Fallon WM, 9. Delvin EE, Arabian A, Glorieux FH, Mamer OA 1985 In vitro Malkasian GD, Judd HL, Riggs BL 1993 Influence of breast- metabolism of 25-OH-hydroxycholecalciferol by isolated cells feeding and other reproductive factors on bone mass later in life. from human decidua. J Clin Endocrinol Metab 60:880Ð885. Osteoporos Int 3:76Ð83. 10. Uemura H, Yasui T, Kiyokawa A, Kuwahara A, Ikawa H, 29. Fox KM, Magaziner J, Sherwin R, Scott JC, Plato CC, Nevitt M, Matsuzaki T, Maegawa M, Furumoto H, Irahara M 2002 Cummings S 1993 Reproductive correlates of bone mass in Serum osteoprotegerin/osteoclastogenesis-inhibitory factor dur- elderly women. J Bone Miner Res 8:901Ð908. ing pregnancy and lactation and the relationship with calcium- 30. Grainge MJ, Coupland CAC, Cliffe SJ, Chilvers CED, regulating hormones and bone turnover markers. J Endocrinol Hosking DJ 2001 Reproductive, menstrual and menopausal 174:353Ð359. factors: which are associated with bone mineral density in 11. Gertner JM, Coustan DR, Kliger AS, Mallette LE, Ravin N, early postmenopausal women? Osteopor Int 12:777Ð787. Broadus AE 1986 Pregnancy as state of physiologic absorptive 31. Henderson PH, 3rd, Sowers M, Kutzko KE, Jannausch ML hypercalciuria. Am J Med 81:451Ð456. 2000 Bone mineral density in grand multiparous women with 12. Kent GN, Price RI, Gutteridge DH, Allen JR, Rosman KJ, extended lactation. Am J Obstet Gynecol 182:1371Ð1377. Smith M, Bhagat CI, Wilson SG, Tetallack RW 1993 Effect of 32. Daaboul J, Sanderson S, Kristensen K, Kitson H 1997 Vitamin D pregnancy and lactation on maternal bone mass and calcium deficiency in pregnant and breast-feeding women and their metabolism. Osteoporos Int (Suppl. 1):S44ÐS47. infants. J Perinatol 17:10Ð14. 13. Martin TJ, Gillespie MT 2001 Receptor activator of nuclear 33. Okonofua F, Menon RK, Houlder S, Thomas M, Robinson D, factor kappa B ligand (RANKL): Another link between breast O’Brien S, Dandona P 1986 Parathyroid hormone and neonatal and bone. Trends Endocrinol Metab 12:2Ð4. calcium homeostasis: Evidence for secondary hyperparathy- 14. Matsumoto I, Kosha S, Noguchi S, Kojima N, Oki T, Douchi T, roidism in the Asian neonate. Metabolism 35:803Ð806. Nagata Y 1995 Changes of bone mineral density in pregnant 34. Purvis RJ, MacKay GS, Cockburn F, Barrie WJM, and postpartum women. J Obstet Gynaecol 21:419Ð425. Wilkinson EM, Belton NR, Forfar JO 1973 Enamel hypoplasia 15. Sowers M, Crutchfield M, Jannausch M, Updike S, Corton G of the teeth associated with neonatal tetany: A manifestation of 1991 A prospective evaluation of bone mineral change in preg- maternal vitamin-D deficiency. Lancet (October 13):811Ð814. nancy. Obstet Gynecol 77:841Ð845. 35. Paunier L, Lacourt G, Pilloud P, Schlaeppi P, Sizonenko PC 16. Bjorklund K, Naessen T, Nordstrom ML, Bergstrom S 1999 1978 25-Hydroxyvitamin D and calcium levels in maternal, Pregnancy-related back and pelvic pain and changes in bone cord and infant serum in relation to maternal vitamin D intake. density. Acta Obstet Gynecol Scand 78:681Ð685. Helvetica Paediatr Acta 33:95Ð103. 17. Kolthoff N, Eiken P, Kristensen B, Nielsen SP 1998 Bone min- 36. Congdon P, Horsman A, Kirby PA, Dibble J, Bashir T 1983 eral changes during pregnancy and lactation: A longitudinal Mineral content of the forearms of babies born to Asian and cohort study. Clin Sci 94:405Ð412. white mothers. Br Med J 286:1234Ð1235. 18. Shefras J, Farquharson R 1996 Bone density studies in preg- 37. Marya RK, Rathee S, Lata V, Mudgil S 1981 Effects of nant women receiving heparin. Eur J Obste Gynecol Reprod vitamin D supplementation in pregnancy. Gynecol Obstet Biol 65:171Ð174. Inves 12:155Ð161. 19. Drinkwater BL, Chesnut CH, III 1991 Bone density changes 38. Cockburn F, Belton NR, Purvis RJ, Giles MM, Brown JK, during pregnancy and lactation in active women: A longitudi- Turner TL, Wilkinson EM, Forfar JO, Barrie WJM, McKay GS, nal study. Bone Miner 14:153Ð160. Pocock SJ 1980 Maternal vitamin D intake and mineral 20. Paparella P, Giorgino R, Maglione A, Lorusso D, Scirpa P, metabolism in mothers and their newborn infants. Br Med J Del Bosco A, Mancuso S 1995 Maternal ultrasound bone den- 281:11Ð14. sity in normal pregnancy. Clin Exp Obstet Gynecol 22:268Ð278. 39. Brooke OG, Brown IRF, Bone CDM, Carter ND, Cleeve HJW, 21. Gambacciani M, Spinetti A, Gallo R, Cappagli B, Teti GC, Maxwell JD, Robinson VP, Winder SM 1980 Vitamin D sup- Facchini V 1995 Ultrasonographic bone characteristics during plements in pregnant Asian women: Effects on calcium status normal pregnancy: Longitudinal and cross-sectional evalua- and fetal growth. Br Med J 280:751Ð754. tion. Am J Obstet Gynecol 173:890Ð893. 40. Delvin EE, Salle BL, Glorieux FH, Adeleine P, David LS 1986 22. Yamaga A, Taga M, Minaguchi H, Sato K 1996 Changes in Vitamin D supplementation during pregnancy: Effect on bone mass as determined by ultrasound and biochemical mark- neonatal calcium homeostasis. J Pediatr 109:328Ð334. ers of bone turnover during pregnancy and puerperium: A lon- 41. Mallet E, Gugi B, Brunelle P, Henocq A, Basuyua JP, Lemeur H gitudinal study. J Clin Endocrinol Metab 81:752Ð756. 1986 Vitamin D supplementation in pregnancy: A controlled 23. Aguado F, Revilla M, Hernandez ER, Menendez M, trial of two methods. Obstet Gynecol 68:300Ð304. Cortes-Prieto J, Villa LF, Rico H 1998 Ultrasonographic bone 42. Marya RK, Rathee S, Dua V, Sangwan K 1988 Effect of velocity in pregnancy: A longitudinal study. Am J Obstet Gynecol vitamin D supplementation during pregnancy on foetal growth. 178:1016Ð1021. Indian J Med Res 88:488Ð492. 24. Kalkwarf HJ, Specker BL, Bianchi DC, Ranz J, Ho M 1997 43. Zhou H 1991 Rickets in China. In: Glorieux FH (ed) Raven The effect of calcium supplementation on bone density during Press, New York, p. 253. lactation and after weaning. N Engl J Med 337:523Ð528. 44. Russell JGB, Hill LF 1974 True fetal rickets. Br Radiol 25. Polatti F, Capuzzo E, Viazzo F, Colleoni R, Klersy C 1999 Bone 47:732. mineral changes during and after lactation. Obstet Gynecol 45. Moncrieff M, Fadahunsi TO 1974 Congenital rickets due to 94:52Ð56. maternal vitamin D deficiency. Arch Dis Child 49:810Ð811. CHAPTER 51 Vitamin D Metabolism in Pregnancy and Lactation 849

46. Specker B, Ho M, Oestreich A, Yin T, Shui Q, Chen X, Tsang R gestation: relationship to changes in bone and mineral 1992 Prospective study of vitamin D supplementation and metabolism. J Clin Endocrinol Metab 80:3699Ð3707. rickets in China. J Pediatr 120:733Ð739. 63. Lippuner K, Zehnder H-J, Casez J-P, Takkinen R, Jaeger P 47. Dawson-Hughes B, Harris S 1991 Regional changes in body 1996 PTH-related protein is released into the mother’s blood- composition by time of year in healthy postmenopausal stream during lactation: Evidence for beneficial effects on women. Am J Clin Nutr 56:307Ð313. maternal calcium-phosphate metabolism. J Bone Miner Res 48. Chapuy MC, Schott AM, Garnero P, Hans D, Delmas PD, 11:1394Ð1399. Meunier PJ 1996 Healthy elderly French women living at 64. Seki K, Kato T, Sekiya S, Makimura N, Kudoh K, Furuya K, home have secondary hyperparathyroidism and high bone Nagata I 1997 Parathyroid-hormone-related protein in human turnover in winter. J Clin Endocrinol Metab 81:1129Ð1133. milk and its relation to milk calcium. Gynecol Obstet Invest 49. Melin A, Wilske J, Ringertz H, Saaf M 2001 Seasonal varia- 44:102Ð106. tions in serum levels of 25-hydroxyvitamin D and parathyroid 65. Fraher LJ, Klein K, Marier R, Freeman D, Hendy GN, hormone but no detectable change in femoral neck bone Goltzman D, Hodsman AB 1995 Comparison of the pharma- density in an older population with regular outdoor exposure. cokinetics of parenteral parathyroid hormone-(1-34) J Am Geriatr Soc 49:1190Ð1196. [PTH-(1-34)] and PTH-related peptide-(1-34) in healthy 50. Namgung R, Mimouni F, Campaigne BN, Ho ML, Tsang RC young humans. J Clin Endocrinol Metab 80:60Ð64. 1992 Low bone mineral content in summer compared with 66. Fraher LJ, Hodsman AB, Jonas K, Saunders D, Rose CI, winter-born infants. J Pediatr Gastroenterol Nutr 15:285Ð288. Henderson JE, Hendy GN, Goltzman D 1992 A comparison of 51. Namgung R, Tsang RC, Specker BL, Sierra RI, Ho ML 1994 the in vivo biochemical responses to exogenous parathyroid Low bone mineral content and high serum ostocalcin and 1,25- hormone-(1-34) [PTH-(1-34)] and PTH-related peptide-(1-34) dihydroxyvitamin D in summer- versus winter-born newborn in man. J Clin Endocrinol Metab 75:417Ð423. infants: An early fetal effect? J Pediatr Gastroenterol Nutr 67. Prentice A, Jarjou LMA, Cole TJ, Stirling DM, Dibba B, 19:220Ð227. Fairweather-Tait S 1995 Calcium requirements of lactating 52. Namgung R, Tsang RC, Lee C, Han D-G, Ho ML, Sierra RI Gambian mothers: Effects of a calcium supplement on 1998 Low total body bone mineral content and high bone breast-milk calcium concentration, maternal bone mineral resorption in Korean winter-born versus summer-born content, and urinary calcium excretion. Am J Clin Nutr newborn infants. J Pediatr Gastro Nutr 132:421Ð425. 62:58Ð67. 53. Raman L, Rajalakshmi K, Krishnamachari KAVR, Gowrinath 68. Klein CJ, Moser-Veillon PB, Douglass LW, Ruben KA, Trocki O Sastry J 1978 Effect of calcium supplementation to undernour- 1995 A longitudinal study of urinary calcium, magnesium, and ished mothers during pregnancy on the bone density of the zinc excretion in lactating and nonlactating postpartum women. neonates. Am J Clin Nutr 31:466Ð469. Am J Clin Nutr 61:779Ð786. 54. Koo W, Walters J, Esterlitz J, Levine R, Bush A, Sibai B 1999 69. Specker BL, Vieira NE, O’Brien KO, Ho ML, Heubi JE, Maternal calcium supplementation and fetal bone mineraliza- Abrams SA, Yergey AL 1994 Calcium kinetics in lactating tion. Obstet Gynecol 94:577Ð582. women with low and high calcium intakes. Am J Clin Nutr 55. Laskey MA, Prentice A, Hanratty LA, Jarjou LMA, Dibba B, 59:593Ð599. Beavan SR, Cole TJ 1998 Bone changes after 3 mo of 70. Donangelo CM, Trugo NMF, Melo GJO, Gomes DD, lactation: Influence of calcium intake, breast-milk output, and Henriques C 1996 Calcium homeostasis during pregnancy and vitamin D-receptor genotype. Am J Clin Nutr 67:685Ð692. lactation in primiparous and multiparous women with sub- 56. Kalkwarf HJ, Specker BL, Ho M 1999 Effects of calcium adequate calcium intakes. Nutr Res 16:1631Ð1640. supplementation on calcium homeostatis and bone turnover in 71. Moser-Veillon PB, Mangels AR, Vieira NE, Yergey AL, lactating women. J Clin Endocrinol Metab 84:464Ð470. Patterson KY, Hill AD, Veillon C 2001 Calcium fractional 57. Kent GN, Price RI, Gutteridge DH, Smith M, Allen JR, absorption and metabolism assessed using stable isotopes Bhagat BH, Barnes MP, Hickling CJ, Retallack RW, Wilson SG, differ between postpartum and never pregnant women. J Nutr Devlin RD, Davies C, St. John A 1990 Human lactation: 131:2295Ð2299. Forearm trabecular bone loss, increased bone turnover, and 72. Kalkwarf HJ, Specker BL, Heubi JE, Vieira NE, Yergey AL renal conservation of calcium and inorganic phosphate with 1996 Intestinal calcium absorption of women during lactation recovery of bone mass following weaning. J Bone Miner Res 5: and after weaning. Am J Clin Nutr 63:526Ð531. 361Ð369. 73. Fairweather-Tait S, Prentice A, Heumann KG, Jarjou LMA, 58. Specker BL, Tsang RC, Ho ML 1991 Changes in calcium Stirling DM, Wharf SG, Turnlund JR 1995 Effect of calcium homeostasis over the first year postpartum: Effect of lactation supplements and stage of lactation on the calcium absorption and weaning. Obstet Gynecol 78:56Ð62. efficiency of lactating women accustomed to low calcium 59. Krebs NF, Reidinger CJ, Robertson AD, Brenner M 1997 Bone intakes. Am J Clin Nutr 62:1188Ð1192. mineral density changes during lactation: Maternal, dietary, 74. Greer FR, Lane J, Ho M 1984 Elevated serum parathyroid and biochemical correlates. Am J Clin Nutr 65:1738Ð1746. hormone, calcitonin, and 1,25-dihydroxyvitamin D in lactating 60. Affinito P, Tommaselli GA, Di Carlo C, Guida F, Nappi C. 1996 women nursing twins. Am J Clin Nutr 40:562Ð568. Changes in bone mineral density and calcium metabolism in 75. Sowers MF, Corton G, Shapiro B, Jannausch ML, Crutchfield M, breastfeeding women: A one year follow-up study. J Clin Smith ML, Randolph JF, Hollis B 1993 Changes in bone density Endocrinol Metab 81:2314Ð2318. with lactation. JAMA 269:3130Ð3135. 61. Sowers MF, Hollis BW, Shapiro B, Randolph J, Janney CA, 76. Laskey M, Prentice A 1999 Bone mineral changes during and Zhang D, Schork MA, Crutchfield M, Stanczyk F, after lactation. Obstet Gynecol 94:608Ð615. Russell-Aulet M 1996 Elevated parathyroid hormone-related 77. Honda A, Kurabayashi T, Yahata T, Tomita M, Takakuwa K, peptide associated with lactation and bone density loss. JAMA Tanaka K 1998 Lumbar bone mineral density changes during 276:549Ð554. pregnancy and lactation. Int J Gynecol Obstet 63:253Ð258. 62. Dobnig H, Kainer F, Stepan V, Winter R, Lipp R, Schaffer M, 78. Karlsson C, Obrant KJ, Karlsson M 2001 Pregnancy and Kahr A, Nocnik S, Patterer G, Leb G 1995 Elevated lactation confer reversible bone loss in humans. Osteoporosis parathyroid hormone-related peptide levels after human Int 12:828Ð834. 850 HEIDI J. KALKWARF AND BONNY L. SPECKER

79. Nordin BEC, Need AG, Chatterton BE, Horowitz M, Morris HA 87. Specker BL, Tsang RC, Hollis BW 1985 Effect of race and diet 1990 The relative contributions of age and years since menopause on human milk vitamin D and 25-hydroxyvitamin D. Am J Dis to postmenopausal bone loss. J Clin Endocrinol Metab 70:83Ð88. Child 139:1134Ð1137. 80. Little KD, Clapp JF 1998 Self-selected recreational exercise 88. Olafsdottir AS, Wagner KH, Thorsdottir I, Elmadfa I 2001 has no impact on early postpartum lactation-induced bone loss. Fat-soluble vitamins in the maternal diet, influence of cod liver Med Sci Sports Exerc 30:831Ð836. oil supplementation and impact of maternal diet on human 81. Kalkwarf HJ, Specker BL 1995 Bone mineral loss during lac- milk composition. Ann Nutr Metab 45:265Ð272. tation and recovery after weaning. Obstet Gynecol 86:26Ð32. 89. Ala-Houhala M 1985 25-Hydroxyvitamin D levels during 82. Michaelsson K, Baron JA, Farahmand BY, Ljunghall S 2001 breast-feeding with or without maternal or infantile Influence of parity and lactation on hip fracture risk. Am J supplementation of vitamin D. J Pediatr Gastro Nutr 4: Epidemiol 153:1166Ð1172. 220Ð226. 83. Cumming RG, Klineberg RJ 1993 Breastfeeding and other 90. Ala-Houhala M, Koskinen T, Terho A, Koivula T, Visakorpi J reproductive factors and the risk of hip fractures in elderly 1986 Maternal compared with vitamin D supplementation. women. Int J Epidemiol 22:684Ð691. Arch Dis Child 61:1159Ð1163. 84. Hoffman S, Grisso JA, Kelsey JL, Gammon MD, O’Brien LA 91. Specker BL, Tsang RC 1987 Cyclical serum 25-hydroxy- 1993 Parity, lactation and hip fracture. Osteoporosis Int vitamin D concentrations paralleling sunshine exposure in 3:171Ð176. exclusively breast-fed infants. J Pediatr 110:744Ð747. 85. Hollis BW, Roos BA, Draper HH, Lambert PW 1981 Vitamin D 92. Moser PB, Reynolds RD, Acharya S, Howard MP, Andon MB and its metabolites in human and bovine milk. J Nutr 1988 Calcium and magnesium dietary intakes and plasma and 111:1240Ð1248. milk concentrations of Nepalese lactating women. Am J Clin 86. Specker BL, Valanis B, Hertzberg V, Edwards N, Tsang RC Nutr 47:735Ð739. 1985 Sunshine exposure and serum 25-hydroxyvitamin D 93. Vaughn LA, Weber CW, Kemberling SR 1979 Longitudinal concentrations in exclusively breast-fed infants. J Pediatr changes in the mineral content of human milk. Am J Clin Nutr 107:372Ð376. 32:2301Ð2306. CHAPTER 52 Vitamin D and Reproductive Organs

KEIICHI OZONO, SHIGEO NAKAJIMA Department of Developmental Medicine (Pediatrics), Osaka University Graduate School of Medicine, Osaka, Japan

TOSHIMI MICHIGAMI Department of Environmental Medicine, Osaka Medical Center and Institute for Maternal and Child Health, Osaka, Japan

I. Historical View VIII. Ovary II. Chick Embryonic Development and Egg Hatchability IX. Uterus III. Mouse Models for a Lack of Vitamin D Function X. Mammary Gland IV. Fetal Development and Vitamin D Synthesis XI. Aromatase V. Calcium Homeostasis in the Fetus and Its Mother XII. Placenta VI. Fertility XIII. Concluding Remarks VII. Testis References

I. HISTORICAL VIEW Before hatching, calcium is mobilized from the eggshell to the embryo. There it supports skeletal development, Vitamin D is an essential nutrient in human that and the resulting weakening of the eggshell allows the prevents rickets, and its classical actions are targeted to chick to hatch. Immunological analyses demonstrated bone, intestine, and kidney to maintain mineral and the presence of the vitamin D receptor (VDR) in the shell bone homeostasis [1]. Besides these classical actions, gland, suggesting that vitamin D is involved in the con- the natural vitamin D metabolites including 1α,25- trol of calcium deposition onto the eggshell [6]. It is dihydroxycholecalciferol [1,25(OH)2D3] and 24R,25- also known that the chorioallantoic membrane just dihydroxycholecalciferol [24R,25(OH)2D3] are reported inside the eggshell is rich in VDR, especially just to be necessary for embryonic development and normal before hatching [7]. When a hen is deficient in vitamin egg hatchability in white leghorn hens [2]. These inter- D, the eggshell becomes soft due to deficiency of cal- esting reports represent among the first to shed light on cium, reducing egg hatchability. nonclassical action of vitamin D such as cell differen- Norman et al. reported that both 1,25(OH)2D3 and tiation, proliferation, insulin secretion, and fertility. In 24R,25(OH)2D3 were necessary for embryonic devel- mammals, it has been argued that vitamin D itself plays opment and normal egg hatchability in the hen and in a role in reproductive organs. However, mice in which the Japanese quail Coturnix coturnix japonica [2,8]. In the vitamin D receptor (VDR) gene has been deleted their experiments, hatchability equivalent to that of D3- (VDR-null) show infertility in at least one strain [3]. repleted hens was obtained only when hens received Moreover, another mouse model for vitamin D resis- a combination of 1,25(OH)2D3 and 24R,25(OH)2D3. tance in which 1α-hydroxylase was ablated exhibited These results suggest a specific biological role for infertility as well [4]. In humans, osteomalacia may cause 24R,25(OH)2D3 in hatchability. In further studies using distortion of the maternal pelvis and be a risk factor for the two stereoisomers of the vitamin D3 metabolite, cephalopelvic disproportion, although no association namely the naturally occurring 24R,25(OH)2D3 and its of vitamin D deficiency with obstructed labor was unnatural epimer 24S,25(OH)2D3, it was concluded found in the Karachi study [5]. These findings revealed that the former is essential for hatching in vitamin DÐ the significance of the role of vitamin D in reproductive deficient hens and Japanese quail [8]. organs in vivo. In this chapter, the roles of vitamin D in In general, 24R,25(OH)2D3 is believed to be an inac- reproductive organs are reviewed. tive metabolite of vitamin D3. The compound has weak binding affinity for the VDR and increases serum cal- cium levels only when administered in large amounts in II. CHICK EMBRYONIC DEVELOPMENT vitamin DÐdeficient animals. However, some reports AND EGG HATCHABILITY describe specific functions of 24R,25(OH)2D3 in chon- drocyte differentiation and bone formation as well as Hen’s eggs have calcium stored in the shell, and in turn hatchability [9,10]. 1,25(OH)2D3 is able to induce the calcium precipitation provides the eggshell with strength. activity of 24-hydroxylase (this enzyme also converts VITAMIN D, 2ND EDITION Copyright © 2005, Elsevier, Inc. FELDMAN, PIKE, AND GLORIEUX All rights reserved. 852 KEIICHI OZONO, SHIGEO NAKAJIMA AND TOSHIMI MICHIGAMI

1,25(OH)2D3) after binding to VDR. In fact, two vita- IV. FETAL DEVELOPMENT AND min DÐresponsive elements (VDREs) have been VITAMIN D SYNTHESIS identified in the rat and human 24-hydroxylase gene promoter [11]. Because of a reduction in the levels of As shown in the vitamin DÐresistance models 24R,25(OH)2D3 in mouse models for vitamin D resis- described earlier, fetal development is not generally tance, the resulting phenotype of these mice does not altered under conditions of vitamin D deficiency. appear to rule out a specific role for 24R,25(OH)2D3 However, a subtle abnormality in the development of in vivo. The mechanism of the specific action of several organs, including reproductive organs, was 24R,25(OH)2D3, if any, remains to be clarified. reported. Kidney is an important organ for vitamin D activation, and its development is related to some extent to the genitals. Immunohistochemical techniques were III. MOUSE MODELS FOR A LACK OF used to examine the distribution of VDR in developing VITAMIN D FUNCTION rat and mouse kidneys and murine metanephric organ culture [14]. This study showed that VDR was present To fully understand the in vivo function of VDR, a in cells of branching ureteral buds and in the sur- member of the nuclear hormone receptor superfamily, rounding mesenchyme from gestational day 15, and at VDR-null mutant mice were generated using established later developmental stages in glomerular visceral and gene engineering technology [3,12] (see Chapter 7). In parietal epithelial cells and proximal and distal the mice deficient in VDR, no defects in development tubules. Expression of the 28-kDa calcium-binding and growth were observed before weaning, whereas in protein (calbindin D28k) gene, a target of vitamin D, the neonate and adult, the mutation resulted in reduced was found exclusively in distal tubules from gesta- expression of vitamin D target genes. Accordingly, after tional day 19. Hence, calbindin D28k appears later in weaning, the mutant mice failed to thrive and showed developing rat and mouse kidney and was distributed alopecia, hypocalcemia, and impaired bone formation. differently from that of VDR [14]. These findings are consistent with typical features of We also detected mRNA for VDR in kidneys vitamin DÐdependent rickets type II in humans. While from gestational day 13.5 using reverse transcriptionÐ the lethality among VDR-null mice varies, perhaps due polymerase chain reaction (RT-PCR) analysis [15]. to the difference in calcium contents in water, infertility RT-PCR and whole-mount in situ hybridization were then in the mutant mice was reported in at least one strain [3]. performed to examine the expression of 1α-hydroxylase Uterine hypoplasia and impaired folliculogenesis were and 24-hydroxylase, to investigate the role of VDR in found in the reproductive organs. These effects will be vitamin D metabolism. In the absence of stimulants, the reviewed later in this chapter. expression of 1α-hydroxylase and 24-hydroxylase was As another form for vitamin D resistance, a mouse detected from day 13.5 of gestation. Forskolin and deficient in 1α-hydroxylase was developed through 1α,25(OH)2D3 induced the expression of 1α-hydroxy- targeted ablation of the hormone-binding and heme- lase and 24-hydroxylase, respectively, in a dose- and binding domains of the 1α-hydroxylase gene [4,13] (see time-dependent manner. Signals for the expression of Chapter 7). These mice also developed normally in the either 1α-hydroxylase (Fig. 1) or 24-hydroxylase were womb and as neonates, and only showed hypocalcemia, detected in kidney explants taken from embryos at secondary hyperparathyroidism, retarded growth, and 15.5 days of gestation after the appropriate stimula- the skeletal abnormalities characteristic of rickets after tion, but the localization of signals differed between weaning. These abnormalities are similar to those the two enzymes. The expression of both hydroxylases described in humans with the heritable disease vitamin was restricted to the epithelium of developing renal DÐdependent rickets type I [also known as pseudovita- tubules. A similar pattern of the expression of megalin, min DÐdeficiency rickets (PDDR)] (see Chapter 7). an endocytotic receptor, to that of 1α-hydroxylase was Again, female mutant mice were infertile and exhibited shown by whole-mount in situ hybridization. These uterine hypoplasia and absent corpora lutea. results suggest that the expression of 1α-hydroxylase In the mice where vitamin D did not function well, is induced in epithelium of renal tubules distinct from infertility was observed, though not in all mice and not that of 24-hydroxylase even at the early stage of kid- completely, indicating an essential role for vitamin D ney development and prior to glomerulogenesis. This in fertility. It is not clear whether these effects of vita- result is consistent with the idea that the roles of both min D are direct or involve the maintenance of calcium enzymes in terms of vitamin D metabolism are sepa- homeostasis. A discussion on the direct or indirect rate. Megalin was shown to play an essential role in the effect of vitamin D on fertility will be described in the activation of vitamin D in renal tubular cells by the next section. uptake of 25-hydroxyvitamin D bound to vitamin D CHAPTER 52 Vitamin D and Reproductive Organs 853

with pregnant women even in developed countries and leads to a congenital vitamin D deficiency in the baby. In pregnant women, renal 1,25(OH)2D3 production is stimulated, and there is evidence of 1,25(OH)2D3 production by the decidua/placenta and fetal kidney in vitro [20]. The renal 1α-hydroxylase activity is pos- sibly induced by estrogens and PTH. It is likely that increased serum 1,25(OH)2D3 concentrations increase intestinal calcium absorption during pregnancy. PTH and 1,25(OH)2D3 levels decrease after delivery in the mother, but are increased when lactation is prolonged. The lα-hydroxylase activity may be stimulated by PTH and prolactin during lactation. What is responsible for the active transport of cal- cium in the placenta is not fully understood. Parathyroid hormoneÐrelated protein (PTHrP) is at least one of the factors needed for transport. PTHrP was originally FIGURE 1 Expression of 1α-hydroxylase during early develop- cloned in 1987 as a causative factor of humoral hyper- ment. Whole-mount in situ hybridization for 1α-hydroxylase mRNA. calcemia of malignancy, and named based on its struc- Kidney explants obtained from mouse embryo at 15.5 days of ges- tural similarity to PTH [21]. In fact, PTHrP shares with tation were incubated with 10−4 M forskolin for 6 hr and hybridized PTH a membrane-bound receptor (PTH1R) [22]. When α with the antisense probes for 1 -hydroxylase mRNA. See the secreted in excess, PTHrP causes hypercalcemia. detailed materials and methods described in [15]. However, PTHrP functions physiologically in the development of cartilage, mammary gland, heart, skin, binding protein based on findings in megalin-knockout hair follicles, tooth, pancreas, and kidney as a paracrine mice [16]. Therefore, developmental analyses of the factor expressed throughout the body [23,24]. During expression sites in kidneys suggest that the function the course of pregnancy, the serum concentration of of megalin may couple with that of the 1α-hydroxylase PTHrP is elevated, and the elevation is maintained and that the function of VDR may couple with that of postpartum for several weeks [25]. Previous studies the 24-hydroxylase. have shown that mice lacking either the PTHrP or the PTH1R gene exhibit severe chondrodysplasia [26]. In addition, in most genetic backgrounds, the PTH1R- V. CALCIUM HOMEOSTASIS IN THE null mice die at midgestation in utero. The cause of FETUS AND ITS MOTHER death has been reported to be maldevelopment of the heart [27]. No abnormalities were observed in the yolk Before focusing on the direct or indirect effect of sac or placenta, implicating the degeneration of the vitamin D on fertility, the characteristic state of cal- heart as the primary cause of death. cium and calciotrophic hormones in fetal development, In PTH1R-null mice, an increase in placental cal- pregnancy, and lactation in the mother will be briefly cium transfer was observed [27]. Because PTHrP lev- reviewed [17,18]. During pregnancy and lactation, the els are increased in PTH1R-null fetuses, it is presumed level of calcium is dynamically regulated to ensure that elevated levels of PTHrP contribute to the increased appropriate development of the skeleton in both the transfer of calcium in the placenta. With its midregion fetus and neonate. The fetus has a relatively high level sequence, PTHrP is able to promote active calcium of serum calcium, which decreases sharply at birth to transport in the placenta in the absence of PTH1R [28]. below normal in infancy, because the active transport Hence the lack of PTHrP action may indirectly impair of calcium in the placenta is stopped. This decrease in the mineralization of the skeleton in the fetus due both serum calcium induces the secretion of parathyroid to a failure to actively transport calcium in the placenta hormone (PTH), and PTH in turn increases the serum and to the disruption to chondrocyte differentiation. calcium concentration to the normal level. PTHrP has also been shown to be necessary for the Active vitamin D is also necessary to maintain calcium normal development of the mammary gland, and large and bone metabolism in the fetus and neonate at least in amounts of PTHrP are found in human milk. humans. Vitamin D deficiency causes hypocalcemia As another participant in the active transport of cal- and rickets even early in the neonatal period in humans cium in the placenta, the calcium-sensing receptor [19]. Hypovitaminosis D is sometimes associated (CaSR) has an essential role in monitoring calcium 854 KEIICHI OZONO, SHIGEO NAKAJIMA AND TOSHIMI MICHIGAMI concentrations [29]. CaSR is expressed in both villous homozygous (−/−) disruption of CaSR caused a further and extravillous regions of the human placenta [30]. increase in the fetal calcium level [34]. This increase CaSR expression was detected in both first-trimester was modestly blunted or not blunted by concomitant and term placentas. In the villous region of the pla- disruption of the PTHrP gene and completely reversed centa, CaSR was detected in syncytiotrophoblasts and by disruption of the PTH1R gene. These results sug- at lower levels in cytotrophoblasts. Local expression of gest that PTH is important to the increase in serum cal- CaSR in the brush border of syncytiotrophoblasts sug- cium levels in these mutant mice. Actually, serum gests a role for the maternal Ca concentration in the con- levels of PTH and 1,25(OH)2D3 were substantially trol of the active transport of calcium between the increased above the normal low fetal levels by disrup- mother and fetus. In the extravillous region of the pla- tion of CaSR. On the other hand, placental calcium centa, CaSR was detected in cells forming trophoblast transfer was reduced and renal calcium excretion was columns in anchoring villi, in close proximity to maternal increased by disruption of CaSR. Thus, increased lev- blood vessels, and in transitional cytotrophoblasts, sug- els of PTH and 1,25(OH)2D3 did not compensate for gesting an important role in controlling calcium levels of placental calcium transfer, suggesting a specific role of both mother and fetus in the development of the pla- PTHrP in the transfer. These in vivo studies indicate centa and fetus [31]. CaSR may be a key molecule pro- that CaSR normally suppresses PTH secretion in the moting the formation of the placenta because it may presence of the normal raised (and PTHrP-dependent) be involved in the control of the growth of placental fetal calcium level. Disruption of CaSR causes fetal trophoblasts. In addition, PTHrP is responsible for the hyperparathyroidism and hypercalcemia, with addi- active calcium transport in the placenta, suggesting that tional effects on placental calcium transfer [33,35]. the interaction of PTHrP with CaSR plays an important These findings indicate that CaSR plays an important role in regulating calcium homeostasis in the fetus. role in fetal and neonatal calcium homeostasis by CaSR was found originally to regulate the secretion conducting the secretion of PTH. of PTH to control extracellular calcium concentrations in adults, but its role in fetal life remains to be eluci- dated. CaSR is expressed in the fetal period, but a VI. FERTILITY postnatal increase in the expression of the CaSR gene has been observed in renal tubular cells [32]. A study As described previously, fertility was reduced in on CaSR gene knockout mice revealed a role for CaSR in vitamin DÐdeficient animals (Table I). Whether this regulating fetal calcium metabolism [33]. Because of results from direct or indirect effect of vitamin D the active transport of calcium in the placenta, normal remains controversial, however. This issue needs to be calcium concentrations in fetal blood are increased above considered relative to the difference in species and sex. the maternal level. The increase in fetal calcium levels Initially, several experiments concerning vitamin D depends upon PTHrP as described earlier. However, PTH deficiency and fertility were done in chickens. For and 1,25(OH)2D3 also play a role in maintaining serum example, chickens were raised from 1 day of age to calcium levels in the fetus. Heterozygous (+/−) and 8 weeks of age on a vitamin DÐdeficient diet to induce

TABLE I Infertility in Several Subjects with Defect of Vitamin D Actions

Subject Infertility Ca supplementation Reference

Vitamin DÐdeficient male chicken Infertile N.D. [36] Vitamin DÐdeficient female rat Infertile Fertile [37] Vitamin DÐdeficient female chicken Infertile N.D. [38] Vitamin DÐdeficient male rat Reduced N.D. [39] VDR-null mouse Died within 15 weeks N.D. [3] Uterine hypoplasia VDR-null mouse Not mentioned N.D. [12] VDR-null mouse Infertile Fertile [41] 1α-Hydroxylase-null mouse Infertile (female) N.D. [4] Uterine hypoplasia 1α-Hydroxylase-null mouse Not mentioned N.D. [13]

N.D., not done. CHAPTER 52 Vitamin D and Reproductive Organs 855 a vitamin DÐdeficient state [36]. After 8 weeks, serum in new mouse models of vitamin D resistance. As vitamin D levels were significantly lower in the chickens described previously, infertility has been reported in fed a vitamin DÐdeficient diet than those fed a normal VDR-null mutant mice. Hypoplasia of uterus and diet. In male chickens, calbindin-D28K (CaBP28K) decreased sperm count associated with sperm motility and testosterone levels were reduced in association with are observed in female and male mutant mice, respec- the deficiency. The morphology of the seminiferous tively. In addition, low activity of aromatase was tubules did not differ between the vitamin DÐreplete and also shown in these mice. Serum estradiol levels and vitamin DÐdeficient chickens. Concerning hens with sperm motility were partially reversed with calcium vitamin D deficiency, they may lay fertile eggs but the supplementation [40]. embryos fail to hatch as described earlier. In another study, VDR-null mutant mice fed a normal In vitamin DÐdeficient female rats, a reduction of diet were hypocalcemic and were found to be largely overall reproductive capacity was observed. Vitamin infertile (14% fertility), whereas the fertility level of DÐdeficient rats fed a high-calcium, high-phosphorus, normocalcemic VDR-null mutant mice and wild-type 20% lactose diet had normal serum calcium, slightly mice was between 86% and 100%. In these reports, lower phosphorus, and undetectable 25-hydroxy- a high-calcium diet led to 100% fertility in the VDR- vitamin D levels [37]. Johnson and DeLuca reported null mice [41]. Thus, high dietary calcium levels are that the decrease in reproductive capacity, previously required for normal reproduction in VDR-null mutant seen in vitamin DÐdeficient rats, as indicated by the female mice. These results suggest that the defect in fertility ratio and pup number per litter, was completely reproduction reported previously for VDR-null mutant corrected when serum calcium and phosphorus levels mice is not the lack of a direct effect of 1,25(OH)2D3 were normalized relative to vitamin DÐreplete rats. on reproductive function but is the result of hypocal- Based on these results, it seems likely that the dimin- cemia. These results support the idea of an indirect role ished reproductive performance attributed to vitamin D of vitamin D for reproduction. deficiency is the result of hypocalcemia and/or Similar to observations in VDR-null mice, uterine hypophosphatemia caused by the deficiency [37]. hypoplasia and decreased ovarian size were also In contrast, some studies suggest a specific and found in a mouse model for the genetic disorder vita- direct role of vitamin D in fertility. In one study, both min DÐdependent rickets type I in humans where the fertility and reproductive capacity of female rats fed on 1α-hydroxylase activity is impaired [13,42]. Panda et al. vitamin DÐdeplete diet were decreased even when reported that folliculogenesis was also abnormal in the serum levels of calcium were not reduced [38]. These mutant mice [4]. Female mutant mice were infertile and results support an essential role of vitamin D in repro- exhibited uterine hypoplasia and absent corpora lutea. duction via mechanisms other than hypocalcemia. In addition, the null mutant mice were acyclic and Thus, the mechanism of action of 1,25(OH)2D3 sepa- did not ovulate. In a different study, the ovary and rate from an increase in serum calcium levels remains uterus of female 1α-hydroxylase-null mutant mice to be determined, although the direct role of vitamin D were smaller [4]. Histological analysis of female in fertility remains controversial. 1α-hydroxylase-null mutant mice showed a poorly Reduced fertility associated with vitamin D defi- developed endometrium in the hypoplastic uterine at ciency was also reported in male rats. Male weanling rats 7 weeks. Ovaries of these mice were smaller than in were fed a vitamin DÐdeficient or vitamin D-replete diet wild-type mice, ovarian follicles were immature, inter- until maturity, and mated to age-matched, vitamin D- stitial tissue was increased, and there were no corpora replete females. Vitamin DÐdeficient males were found lutea. This study supports the idea that 1,25(OH)2D3 is to be capable of reproduction. However, successful mat- important for reproductive organ development and its ings, i.e., the presence of sperm in the vaginal tract of the deficiency results in infertility. However, male repro- female, by vitamin DÐdeficient males were reduced by ductive organs were grossly normal in 1α-hydroxylase- 45% when compared to matings by vitamin DÐreplete null mutant mice. In addition, the abnormality of males [39]. Fertility (successful pregnancies in sperm- reproductive organs was more severe in females than positive females) was reduced by 73% in litters from males. The reduced activity of aromatase with vitamin vitamin DÐdeficient male inseminations when com- D deficiency, which is described in this chapter, may pared to litters from females inseminated by vitamin account for the female abnormality in fertility. DÐreplete males [39]. These results supported the hypothesis that vitamin D and its metabolites are nec- essary for normal spermatogenesis and sperm function VII. TESTIS in the male rat. In addition to data based on vitamin D deficiency, the Since vitamin DÐdeficient male rats are infer- effect of vitamin D on infertility was also investigated tile, vitamin D may exert its effect on the testis and 856 KEIICHI OZONO, SHIGEO NAKAJIMA AND TOSHIMI MICHIGAMI involve spermatogenesis. In VDR-null mice, decreased immunoreactivity, while the remaining 16.7% of the nor- sperm count and motility associated with histological mal surface ovarian epithelium was VDR negative [45]. abnormalities consisting of dilated lumen of seminiferous Ovarian calcinoma cells also expressed VDR, and the tubules and thinner layer of epithelial cells were intensity of VDR immunostaining was significantly observed in the male [40]. With respect to the existence increased in ovarian carcinomas as compared to nor- of VDR in testis, evidence from autoradiographic stud- mal ovarian tissue [45]. Concerning the distribution of ies with 1,25(OH)2D3 labeled with tritium and immuno- VDR in ovary, immunostaining for VDR was seen in histochemistry using monoclonal and polyclonal ovarian follicles, specifically in granulosa cells. antibodies against VDR shows a broad distribution of Weaker immunostaining of VDR was observed in fol- VDR in the testes of animals [43]. In rat testes, VDR epi- licular thecal cells and in the ovarian stroma and ger- topes were observed in seminiferous tubules, specifically minal epithelium. Corpus luteal cells stained intensely in spermatogonia, Sertoli cells, and spermatocytes, but for VDR. Epithelium of fallopian tubes and the uterus spermatozoa were faintly stained. Epithelial cells of also expressed VDR. Hence, both nuclear and cytoplas- the epididymis, seminal vesicles, and prostate also mic VDR immunostaining was observed in female rat expressed VDR epitopes. Biochemical data also reproductive tissues as detected in testis in male rats. revealed the VDR in testis, and its binding affinity for These results suggest that vitamin D may function in the −10 1,25(OH)2D3 was 1.8 × 10 M [44]. These results maturation of ovocytes, ovulation, and reproduction. suggest a nonclassical role for vitamin D in testis other The relationship between ovarian cancer and VDR than maintaining calcium and bone homeostasis. The is another issue to be considered. On analyzing the coex- role of vitamin D in testis is also supported by the find- pression of VDR with the proliferation marker Ki-67 or ing of infertility in vitamin DÐdeficient animals and with estrogen and progesterone receptors, no significant VDR or 1α-hydroxylase-deficient mice. correlation was found with ovarian cancer cells [45]. The The intracellular protein calbindin D28K is a primary expression of VDR with ovarian carcinomas seemed to target gene for vitamin D. The regulation of calbindin be regulated independently from that of the estrogen D28K expression by vitamin D has been intensively receptor or progesterone receptor. VDR expression is investigated in chick and rat intestine. Calbindin D28K generally increased in ovarian carcinomas as com- exists in testis, and the effects of vitamin D deficiency pared to normal ovarian tissue, suggesting a role for on calbindin D28K and testosterone levels have been vitamin D in ovarian cell proliferation [45]. investigated in male chickens [36]. The study demon- strated that vitamin D deficiency exerted an effect on calbindin D28K expression in chicken testes. The IX. UTERUS morphology of the seminiferous tubules did not differ between the vitamin DÐreplete and vitamin DÐdeficient During decidualization, endometrial cells prolifer- chickens. Immunohistochemical analysis revealed that ate rapidly, resulting in an increase in uterine weight. calbindin D28K was present in spermatogonia and sper- At the same time, endometrial cells differentiate into matocytes of the seminiferous tubules. A few interstitial decidual cells. This process is called a decidual reac- Leydig cells were positive for calbindin D28K. The tion and is usually associated with ovum implantation. amount of calbindin D28K was qualified by radio- Because endometrial cells and decidual cells possess immunoassay in the testes and was found to be three- VDR and respond to 1,25(OH)2D3 (induction of fold higher in chickens raised on a normal diet than in 24-hydroxylase), the decidual process may be affected those raised on a vitamin DÐdeficient diet. These results by 1,25(OH)2D3. However, the characteristics of indicate that the decrease in the testicular calbindin endometrial and decidual cells are distinct. Halhali and D28K concentration might be attributable to vitamin D colleagues reported that intraluminal injection of female deficiency despite normal serum testosterone and rats with 10Ð500 ng of 1,25(OH)2D3 on day 5 of pseu- calcium levels in 8-week-old chickens. dopregnancy significantly increased uterine weight and induced a decidual reaction [46]. This effect was observed as early as the third day after the injection. VIII. OVARY These results suggest that 1,25(OH)2D3 plays a physi- ological role in the differentiation of endometrial cell Immunohistochemical studies using monoclonal into decidual cells, a crucial step in pregnancy. antibody against VDR demonstrated that VDR was Indeed, although renal tubular cells are the main site of expressed in both normal and carcinomatous ovarian expression of 1alpha-hydroxylase, human decidual cells tissues. Villena-Heisen and colleagues reported that a are also reported to produce 1,25(OH)2D3, particularly total of 83.3% exhibited weak to moderate VDR at the end of pregnancy. A study using stromal decidual CHAPTER 52 Vitamin D and Reproductive Organs 857 cells showed that human uterine cells were capable of syn- mammary cells in culture, but little was known about thesizing 1,25(OH)2D3 even during early pregnancy [47]. the physiological relevance of the vitamin D endocrine On the other hand, the synthesis of 1,25(OH)2D3 by system in the developing mammary gland in vivo. It endometrial cells is controversial. was reported that VDR was expressed in epithelial As the main thesis in the chapter, poor reproductive cells of the terminal end bud and subtending ducts, in performance was observed in rats deficient in vitamin D. stromal cells, and in a subset of lymphocytes within On the other hand, vitamin D toxicity was also asso- the lymph node [50]. In the terminal end bud, a distinct ciated with poor reproductive activity reflected in a gradient of VDR expression has been observed, with decrease in the number of matings, implantations, and weak staining in proliferative populations and strong live births [48]. These changes were reversible, and staining in differentiated populations. recovery was observed after treatment with active The role of VDR in ductal morphogenesis was exam- vitamin D was discontinued. In order to clarify the ined in VDR-null mice fed high dietary calcium, which mechanism of these reversible toxicities, female rats normalizes fertility, serum estrogen levels, and neonatal treated with the vitamin D3 metabolite were compared growth [51]. The results of the study indicate that mam- with untreated rats with respect to several reproductive mary glands from virgin VDR-null mice are heavier and parameters [48]. The estrous cycle was disturbed in vita- exhibit accelerated growth, as evidenced by higher min DÐtreated rats. Also, hypofunctional changes in the numbers of terminal end buds, greater ductal outgrowth, corpus luteum in the ovary and the epithelium, and enhanced secondary branch points, compared with endometrium, and uterine gland in the uterus with a glands from age- and weight-matched wild-type mice decrease in the serum progesterone level were also both in vivo and in organ culture. In addition, glands from observed. In addition, vitamin DÐinduced hypercalcemia VDR-null mice exhibit enhanced growth in response to decreased calcitonin or PTH levels in serum with mor- exogenous estrogen and progesterone compared with phological changes including atrophy and cyst forma- glands from wild-type mice. These results provided tion in the parathyroid. However, these changes were in vivo evidence that 1,25(OH)2D3 and VDR impact reversible, and a recovery was observed after adminis- ductal elongation and branching morphogenesis during tration of the compound was discontinued. These results pubertal development of the mammary gland [51]. indicate that the hypercalcemia caused by 1,25(OH)2D3 Given these results, the vitamin D signaling pathway disrupts endocrinological homeostasis, which in turn may participate in negative growth regulation of the temporarily disrupts the female reproductive system. mammary gland. Cervical cells in the uterus are another possible tar- 1,25(OH)2D3 interacts with VDR to modulate pro- get of vitamin D, and cervical cell carcinoma is one of liferation and apoptosis in a variety of cell types, the most common cancers in women. The proliferation including breast cancer cells. It is interesting that mam- and differentiation of cervical epithelial cells are mary glands from VDR-null mice exhibit accelerated mainly regulated by estrogens and progestins. growth and branching during puberty, pregnancy, and lac- 1,25(OH)2D3 is reported to be an important determi- tation as compared to wild-type mice [51]. In addition, nant of the responsiveness to these hormones [49]. involution after weaning, a process driven by epithelial 1,25(OH)2D3 induced the expression of insulin-like cell apoptosis, proceeds at a slower rate in VDR-null mice growth factor-binding protein 3 and inhibited the compared to wild-type mice. These in vivo findings were growth of human ectocervical epithelial cells [49]. further evaluated using cells isolated from VDR-null and wild-type mice. In these experiments, the growth of both normal and transformed mammary cells derived X. MAMMARY GLAND from wild-type mice was inhibited by 1,25(OH)2D3. In contrast, cells derived from VDR-null mice did Development of the mammary gland occurs predom- not respond to 1,25(OH)2D3, indicating VDR was inantly postnatally, and the morphogenesis of mammary responsible for the growth inhibition by 1,25(OH)2D3. gland is achieved through the coordination of signaling In addition to normal development of mammary networks in both epithelial and stromal cells [50]. While gland and proliferation of mammary cells, 1,25(OH)2D3 the major proliferative hormones driving pubertal mam- and its various analogs have been shown to inhibit pro- mary gland development are estrogen and proges- liferation in human breast cancer cells and in experi- terone, studies in transgenic and knockout mice have mental mammary tumors in vivo and in vitro. successfully identified other steroid and peptide hor- Epidemiological studies have suggested an association mones that affect development. VDR with an active between 1,25(OH)2D3 deficiency and increased risk of vitamin D metabolite has been implicated in the con- various malignancies including cancer of the colon and trol of the differentiation, cell cycle, and apoptosis of breast [52]. 1,25(OH)2D3-mediated growth inhibition 858 KEIICHI OZONO, SHIGEO NAKAJIMA AND TOSHIMI MICHIGAMI was enhanced by the addition of a variety of agents, tissue is a significant source of estrogen in men and including steroid hormones, phytoestrogens, and postmenopausal women. The CYP19 gene was cloned growth factors that up-regulate VDR expression [51]. in 1989 and found to consist of 10 exons with a variety These results support a role for 1,25(OH)2D3 and VDR of tissue-specific promoters [54]. in the negative growth regulation of both normal mam- In aromatase-deficient patients, an estrogen defi- mary gland and breast cancer cells. Loss of VDR in ciency appeared prenatally. The phenotype of these breast cancer cells, however, does not always lead to patients indicates the significant role of aromatase for increased aggressiveness. Nonetheless, 1,25(OH)2D3 the development and functions of reproductive organs inhibits the growth of most breast cancer cells both in humans. In men with an aromatase deficiency, male in vivo and in vitro. reproductive functions are impaired. Because estrogens Concerning the mechanisms of growth inhibition play a pivotal role in the control of serum gonadotropin of mammary cells and breast cancer cells by concentrations in the male, male rodents with an aro- 1,25(OH)2D3, an assessment of 1,25(OH)2D3 increased matase defect actually show impaired sexual behavior growth arrest and apoptosis was made using cell lines and fertility as a consequence of this estrogen defect [56]. established from DMBA-induced mammary tumors In contrast, the patients who had gain-of-function muta- derived from VDR-null and wild-type mice [53]. tions in the CYP19 gene have been described [57]. Zinser and colleagues obtained two VDR-null and two The patients showed gynecomastia and relatively low wild-type cell lines, and characterized the growth of levels of testosterone and follicle-stimulating hormone these cells in response to 1,25(OH)2D3 [53]. Both wild- caused by severe estrogen excess. type cell lines expressed the VDR protein and were In a 46,XX female with aromatase-deficiency due to sensitive to growth inhibition by 1,25(OH)2D3 at doses molecular defects in the CYP19 (P450arom) gene, a as low as 1 nM. 1,25(OH)2D3 induced G(0)/G(1) arrest nonadrenal form of pseudohermaphrodism appeared at and apoptosis in the wild-type cell lines. In contrast, birth because of the excess of androgen and deficiency both cell lines from VDR-null mice were completely of estrogen [58]. The fetal masculinization in this syn- resistant to 1,25(OH)2D3-mediated growth arrest and drome is likely to be caused by exposure of the female apoptosis even when the high concentration of fetus to excessive amounts of testosterone as a result of 1,25(OH)2D3 was used. It was confirmed that both the defective placental conversion of C19 steroids to cells established from tumors that developed in VDR- estrogens. According to the case report of a female null mice lacked VDR mRNA and protein. Therefore, patient with aromatase deficiency, bone aging was the induction of cell cycle arrest and apoptosis in breast delayed. This finding is consistent with the idea that cancer cells by 1,25(OH)2D3 is dependent on the nuclear estrogens, in contrast to androgens, are the major sex VDR. Cells lacking VDR remain sensitive to growth steroid driving skeletal maturation during puberty. arrest mediated by 9-cis-retinoic acid, a ligand for the Moreover, estrogen replacement therapy was effective retinoid X receptor (RXR) that can heterodimerize in the growth spurt, breast development, menarche, with VDR. Sensitivity to apoptosis induced by the suppression of gonadotropin levels, and resolution of DNA-damaging agent etoposide was not altered in cysts [58]. Pubertal failure, mild virilization, multicystic VDR-null cells, indicating that VDR ablation did not ovaries, and hyperstimulation of the ovaries by FSH and impair apoptotic pathways in general. LH all result from the inability of the ovary to convert testosterone and androstenedione into estrogens. As described in the first paragraph of this section, the XI. AROMATASE CYP19 gene employs several different promoters (I.1, I.2, I.3, I.4, I.5, I.6, 2a, 1f, and PII) to control expres- Aromatase, the product of the CYP19 gene, is a P-450 sion in a tissue-specific fashion. Although the regulation containing enzyme that is involved in estrogen bio- of aromatase gene expression is complex, one of the synthesis [54]. It is a key enzyme that confers both main regulators, especially in the ovary, is protein endocrine and paracrine actions of estrogen. In con- kinase A. It is suggested that a nuclear receptor system trast to estrogen, the production of which is limited to comprising the RAR-RXR heterodimer is also involved the gonads and brain in most vertebrate species, aro- in the regulation of aromatase activity in MCF-7 breast matase is expressed in various tissues and cells includ- cancer cells [59]. On the other hand, the PPARgamma ing adipose stromal cells and syncytiotrophoblasts [55]. ligand troglitazone or RXR ligand LG100268 alone In men, estrogen is produced from androgens in decreased aromatase activity in cultured human ovarian Leydig cells of the testis. In women the primary syn- granulosa cells, while combined treatment with troglita- thetic sites are granulose and luteal cells of the ovary. zone and LG100268 caused an even greater reduction in At the same time, peripheral conversion in adipose this activity. This suggests that the PPARgammaÐRXR CHAPTER 52 Vitamin D and Reproductive Organs 859 heterodimer system is involved in the regulation of the gene expression. Gel retardation analysis using nuclear aromatase gene expression in human granulosa cells. extracts of JEG3 cells and the imperfect palindromic Therefore, RXR is involved in the regulation of the aro- sequence as a probe showed the formation of a het- matase gene expression in a unique manner in different erodimer of RXR alpha and VDR. These results suggest tissues, i.e., as an RARÐRXR heterodimer in human that the imperfect palindromic sequence upstream of breast cancer cells, and as a PPARgammaÐRXR exon I.1 represents a novel VDRE. heterodimer in ovarian granulosa cells. The role of vitamin D in the regulation of estrogen synthesis in gonads was also investigated in a mouse XII. PLACENTA model [40]. VDR-null mutant mice showed gonadal insufficiencies: Uterine hypoplasia and impaired fol- Maternal calcium regulation is adapted to allow liculogenesis were observed in the female, and a fetal growth through enhanced intestinal calcium decreased sperm count and decreased motility with absorption rather than through the mobilization of histological abnormality of the testis were observed in maternal skeletal reserves [17]. This adaptive process the male [40]. Aromatase activity in these mice was depends mainly on the two major calciotrophic hor- also reported to be weak in the ovary, testis, and epi- mones, PTH and 1,25(OH)2D3, which show quantitative didymis at 24%, 58%, and 35% of the wild-type val- changes from the nonpregnant state. Maternal plasma ues, respectively. The expression of aromatase itself levels of 1,25(OH)2D3 are elevated during pregnancy in was also reduced in these organs. Elevated serum lev- humans and experimental animals [17]. Placenta is els of LH and FSH revealed hypergonadotropic hypo- thought to be a site for the production of 1,25(OH)2D3 gonadism in these mice. However, in response to and some reports described 1α-hydroxylase activity supplementation with estradiol, histological abnormal- here. The expression of the 1α-hydroxylase gene is ities were normalized in both male and female gonads. controversial, however [60]. Nevertheless, in patients Interestingly, calcium supplementation increased aro- with a lack of renal 1α-hydroxylase activity, convert- matase activity and partially corrected the hypogo- ing activity was also absent in cells isolated from the nadism, but LH and FSH levels remained high. Hence, decidua of patients with vitamin D-dependency type 1, the action of vitamin D on estrogen biosynthesis was suggesting that the decidual and renal enzymes are partially explained by maintaining calcium homeostasis, encoded by the same gene [61]. although a direct effect of active vitamin D on the expres- Characterization of VDR in placenta was performed; sion of the aromatase gene was also apparent [40]. the molecular weight of the receptor was estimated to be These results are consistent with the idea that vitamin 55 kDa based on gel filtration [62]. The binding affinity −10 D is essential for full gonadal function in both sexes. of the receptor for 1,25(OH)2D3 was 3.0 × 10 M. These As described previously, the tissue-specific expres- results indicate that the properties of the 1,25(OH)2D3 sion of the CYP19 gene is regulated by means of receptor in human placenta are similar to those of the tissue-specific promoters through the use of alternative chicken intestinal and human osteoblastic VDR. splicing mechanisms. Thus, transcripts containing var- In contrast to maternal mineral homeostasis, the ious 5′-untranslated termini are present in ovary, brain, maintenance of fetal calcium homeostasis depends adipose stromal cells, and placenta [55]. Sun and largely on PTHrP, which regulates active placental cal- co-workers reported that VDR is involved in the con- cium transfer and the calcium fluxes across the kidney trol of the promoter I.1 of the CYP19 gene [55]. This pro- and bone. The major source of PTHrP is the fetal moter drives expression of the gene in human placenta parathyroid gland and placenta, although some organs and choriocarcinoma cells. Various deletion mutations including bone and cartilage are able to synthesize of the upstream flanking region of exon I.1 were con- PTHrP. The mechanism of active calcium transport by structed and transfected into human choriocarcinoma PTHrP remains to be elucidated, although PTHR1 (JEG3) cells to examine the region regulated by VDR or exists in placenta. The relationship between intracellu- retinoic acid receptor (RAR) activity. The longest con- lar cAMP and calcium transport needs to be elucidated. struct, −924/+10 bp, exhibited the highest level of luciferase reporter gene activity [55]. Interestingly, this activity was induced by vitamin D as well as by LG69 XIII. CONCLUDING REMARKS and TTNPB, ligands specific for RXR and RAR respec- tively. The imperfect palindromic sequence (AGGT- For complete reproduction, a complex and har- CATGCCCC) located at −183 to −172 bp upstream of monic regulation of the expression of entire sets of the transcriptional start site of exon I.1 was found to be genes is necessary. A copy of the genome is reserved important for both basal and retinoid-induced reporter in the gamete, and development begins after the male 860 KEIICHI OZONO, SHIGEO NAKAJIMA AND TOSHIMI MICHIGAMI and female germ cells are joined. During the process of 12. Li YC, Pirro AE, Amling M, Delling G, Baron R, Bronson R, fetal development, calcium is dynamically mobilized Demay MB 1997 Targeted ablation of the vitamin D receptor: an animal model of vitamin D-dependent rickets type II with from mother to fetus, and there appears to be a specific alopecia. Proc Natl Acad Sci USA 94:9831Ð9835. system that is involved, for example, in active transport 13. Dardenne O, Prud’homme J, Arabian A, Glorieux FH, St- of calcium in the placenta. Vitamin D clearly has a role Arnaud R 2001 Targeted inactivation of the 25-hydroxyvita- α in reproduction because vitamin D deficiency leads to min D3-1 -hydroxylase gene (CYP27B1) creates an animal infertility in chickens and mammals. An indirect effect model of pseudovitamin DÐdeficiency rickets. Endocrinology 142:3135Ð3141. of vitamin D that is mediated by serum calcium levels 14. Johnson JA, Grande JP, Roche PC, Sweeney WE Jr, Avner ED, appears to play a major part, but any direct effect is still Kumar R 1995 1α,25-Dihydroxyvitamin D3 receptor ontogen- to be considered. The establishment of mouse models esis in fetal renal development. Am J Physiol 269:F419ÐF428. for certain types of vitamin D resistance will contribute 15. Yamagata M, Kimoto A, Michigami T, Nakayama M, Ozono K to the elucidation of the mechanism of infertility under 2001 Hydroxylases involved in vitamin D metabolism are dif- ferentially expressed in murine embryonic kidney: application conditions of defective vitamin D action. of whole mount in situ hybridization. Endocrinology 142: 3223Ð3230. 16. Nykjaer A, Dragun D, Walther D, Vorum H, Jacobsen C, Herz J, References Melsen F, Christensen EI, Willnow TE 1999 An endocytic pathway essential for renal uptake and activation of the steroid 25-(OH) vitamin D3. Cell 96:507Ð515. 1. Jones G, Strugnell SA, DeLuca HF 1998 Current understand- 17. Hosking DJ 1996 Calcium homeostasis in pregnancy. Clin ing of the molecular actions of vitamin D. Physiol Rev Endocrinol (Oxf) 45:1Ð6. 78:1193Ð1231. 18. Verhaeghe J, Bouillon R 1992 Calciotropic hormones during 2. Henry HL, Norman AW 1978 Vitamin D: two dihydroxylated reproduction. J Steroid Biochem Mol Biol 41:469Ð477. metabolites are required for normal chicken egg hatchability. 19. Dawodu A, Agarwal M, Hossain M, Kochiyil J, Zayed R 2003 Science 201:835Ð837. Hypovitaminosis D and vitamin D deficiency in exclusively 3. Yoshizawa T, Handa Y, Uematsu Y, Takeda S, Sekine K, breast-feeding infants and their mothers in summer: a justifi- Yoshihara Y, Kawakami T, Arioka K, Sato H, Uchiyama Y, cation for vitamin D supplementation of breast-feeding infants. Masushige S, Fukamizu A, Matsumoto T, Kato S 1997 Mice J Pediatr 142:169Ð173. lacking the vitamin D receptor exhibit impaired bone forma- 20. Stumpf WE, Denny ME 1989 Vitamin D (soltriol), light, and tion, uterine hypoplasia and growth retardation after weaning. reproduction. Am J Obstet Gynecol 161:1375Ð1384. Nat Genet 16:391Ð396. 21. Suva LJ, Winslow GA, Wettenhall RE, Hammonds RG, 4. Panda DK, Miao D, Tremblay ML, Sirois J, Farookhi R, Moseley JM, Diefenbach-Jagger H, Rodda CP, Kemp BE, Hendy GN, Goltzman D 2001 Targeted ablation of the 25- Rodriguez H, Chen EY, Hudson PJ, Martin TJ, Wood WI 1987 hydroxyvitamin D 1α-hydroxylase enzyme: evidence for A parathyroid hormone-related protein implicated in malig- skeletal, reproductive, and immune dysfunction. Proc Natl nant hypercalcemia: cloning and expression. Science Acad Sci USA 98:7498Ð7503. 237:893Ð896. 5. Brunvand L, Shah SS, Bergstrom S, Haug E 1998 Vitamin D 22. Abou-Samra AB, Juppner H, Force T, Freeman MW, Kong XF, deficiency in pregnancy is not associated with obstructed labor. Schipani E, Urena P, Richards J, Bonventre JV, Potts JT Jr, A study among Pakistani women in Karachi. Acta Obstet Kronenberg HM, Segre GV 1992 Expression cloning of a Gynecol Scand 77:303Ð306. common receptor for parathyroid hormone and parathyroid 6. Yoshimura Y, Ohira H, Tamura T 1997 Immunocytochemical hormone-related peptide from rat osteoblast-like cells: a single localization of vitamin D receptors in the shell gland of immature, receptor stimulates intracellular accumulation of both cAMP laying, and molting hens. Gen Comp Endocrinol 108:282Ð289. and inositol trisphosphates and increases intracellular free 7. Elaroussi MA, Uhland-Smith A, Hellwig W, DeLuca HF 1994 calcium. Proc Natl Acad Sci USA 89:2732Ð2736. The role of vitamin D in chorioallantoic membrane calcium 23. Orloff JJ, Reddy D, de Papp AE, Yang KH, Soifer NE, transport. Biochim Biophys Acta 1192:1Ð6. Stewart AF 1994 Parathyroid hormone-related protein as a 8. Norman AW, Leathers V, Bishop JE 1983 Normal egg hatch- prohormone: posttranslational processing and receptor interac- ability requires the simultaneous administration to the hen of tions. Endocr Rev 15:40Ð60. 1α,25-dihydroxycholecalciferol and 24R,25-dihydroxychole- 24. Karaplis AC 2001 PTHrP: novel roles in skeletal biology. Curr calciferol. J Nutr 113:2505Ð2515. Pharm Des 7:655Ð670. 9. Boyan BD, Sylvia VL, Curry D, Chang Z, Dean DD, Schwartz Z 25. Ardawi MSM, Nasrat HA, BA’Aqueel HS 1997 Calcium- 1998 Arachidonic acid is an autocoid mediator of the differen- regulating hormones and parathyroid hormone-related peptide tial action of 1,25-(OH)2D3 and 24,25-(OH)2D3 on growth in normal human pregnancy and postpartum: a longitudinal plate chondrocytes. J Cell Physiol 176:516Ð524. study. Eur J Endocrinol 137:402Ð409. 10. Nakamura T, Suzuki K, Hirai T, Kurokawa T, Orimo H 1992 26. Escande B, Lindner V, Massfelder T, Helwig JJ, Simeoni U Increased bone volume and reduced bone turnover in vitamin 2001 Developmental aspects of parathyroid hormone-related DÐreplete rabbits by the administration of 24R,25-dihydroxy- protein biology. Semin Perinatol 25:76Ð84. vitamin D3. Bone 13:229Ð236. 27. Qian J, Colbert MC, Witte D, Kuan CY, Gruenstein E, 11. Ohyama Y, Ozono K, Uchida M, Shinki T, Kato S, Suda T, Osinska H, Lanske B, Kronenberg HM, Clemens TL 2003 Yamamoto O, Noshiro M, Kato Y 1994 Identification of a Midgestational lethality in mice lacking the parathyroid vitamin D-responsive element in the 5′-flanking region of the hormone (PTH)/PTH-related peptide receptor is associated rat 25-hydroxyvitamin D3 24-hydroxylase gene. J Biol Chem with abrupt cardiomyocyte death. Endocrinology 144: 269:10545Ð10550. 1053Ð1061. CHAPTER 52 Vitamin D and Reproductive Organs 861

28. Kovacs CS, Lanske B, Hunzelman JL, Guo J, Karaplis AC, of 1,25-dihydroxyvitamin-D3-receptors, estrogen and proges- Kronenberg HM 1996 Parathyroid hormone-related peptide terone receptors and Ki-67 in ovarian carcinoma. Anticancer (PTHrP) regulates fetal-placental calcium transport through a Res 22:2261Ð2267. receptor distinct from the PTH/PTHrP receptor. Proc Natl 46. Halhali A, Acker GM, Garabedian M 1991 1,25- Acad Sci USA 93:15233Ð15238. Dihydroxyvitamin D3 induces in vivo the decidualization of rat 29. Brown EM, MacLeod RJ 2001 Extracellular calcium sensing endometrial cells. J Reprod Fertil 91:59Ð64. and extracellular calcium signaling. Physiol Rev 81:239Ð297. 47. Kachkache M, Rebut-Bonneton C, Demignon J, Cynober E, 30. Bradbury RA, Cropley J, Kifor O, Lovicu FJ, de Iongh RU, Garabedian M 1993 Uterine cells other than stromal decidual Kable E, Brown EM, Seely EW, Peat BB, Conigrave AD 2002 cells are required for 1,25-dihydroxyvitamin D3 production Localization of the extracellular Ca2+-sensing receptor in the during early human pregnancy. FEBS Lett 333:83Ð88. human placenta. Placenta 23:192Ð200. 48. Horii I, Takizawa S, Fujii T 1992 Effect of 1,25-dihydroxy- 31. Kovacs CS, Ho-Pao CL, Hunzelman JL, Lanske B, Fox J, vitamin D3 on the female reproductive system in rats. J Toxicol Seidman JG, Seidman CE, Kronenberg HM 1998 Regulation Sci 17:91Ð105. of murine fetal-placental calcium metabolism by the calcium- 49. Agarwal C, Lambert A, Chandraratna RA, Rorke EA, Eckert RL sensing receptor. J Clin Invest 101:2812Ð2820. 1999 Vitamin D regulates human ectocervical epithelial 32. Riccardi D, Hall AE, Chattopadhyay N, Xu JZ, Brown EM, cell proliferation and insulin-like growth factor-binding Hebert SC 1998 Localization of the extracellular Ca2+/poly- protein-3 level. Biol Reprod 60:567Ð572. valent cation-sensing protein in rat kidney. Am J Physiol 50. Zinser G, Packman K, Welsh J 2002 Vitamin D3 receptor abla- 274:F611ÐF622. tion alters mammary gland morphogenesis. Development 129: 33. Ho C, Conner DA, Pollak MR, Ladd DJ, Kifor O, Warren HB, 3067Ð3076. Brown EM, Seidman JG, Seidman CE 1995 A mouse model of 51. Welsh J, Wietzke JA, Zinser GM, Smyczek S, Romu S, Tribble E, human familial hypocalciuric hypercalcemia and neonatal Welsh JC, Byrne B, Narvaez CJ 2002 Impact of the Vitamin D3 severe hyperparathyroidism. Nat Genet 11:389Ð394. receptor on growth-regulatory pathways in mammary gland 34. Kovacs CS, Manley NR, Moseley JM, Martin TJ, Kronenberg and breast cancer. J Steroid Biochem Mol Biol 83:85Ð92. HM 2001 Fetal parathyroids are not required to maintain 52. Garland FC, Garland CF, Gorham ED, Young JF 1990 placental calcium transport. J Clin Invest 107:1007Ð1015. Geographic variation in breast cancer mortality in the United 35. Clemens TL, Cormier S, Eichinger A, Endlich K, Fiaschi- States: A hypothesis involving exposure to solar radiation. Taesch N, Fischer E, Friedman PA, Karaplis AC, Massfelder T, Prev Med 19:614Ð622. Rossert J, Schluter KD, Silve C, Stewart AF, Takane K, Helwig JJ 53. Zinser GM, McEleney K, Welsh J 2003 Characterization of 2001 Parathyroid hormone-related protein and its receptors: mammary tumor cell lines from wild type and vitamin D3 nuclear functions and roles in the renal and cardiovascular sys- receptor knockout mice. Mol Cell Endocrinol 28(200):67Ð80. tems, the placental trophoblasts and the pancreatic islets. Br 54. Meinhardt U, Mullis PE 2002 The aromatase cytochrome J Pharmacol 134:1113Ð1136. P-450 and its clinical impact. Horm Res 57:145Ð152. 36. Inpanbutr N, Reiswig JD, Bacon WL, Slemons RD, Iacopino AM 55. Sun T, Zhao Y, Mangelsdorf DJ, Simpson ER 1998 1996 Effect of vitamin D on testicular CaBP28K expression and Characterization of a region upstream of exon I.1 of the serum testosterone in chickens. Biol Reprod 54:242Ð248. human CYP19 (aromatase) gene that mediates regulation by 37. Johnson LE, DeLuca HF 2002 Reproductive defects are cor- retinoids in human choriocarcinoma cells. Endocrinology rected in vitamin D-deficient female rats fed a high calcium, 139:1684Ð1691. phosphorus and lactose diet. J Nutr 132:2270Ð2273. 56. Rochira V, Balestrieri A, Madeo B, Baraldi E, Faustini-Fustini M, 38. Hickie JP, Lavigne DM, Woodward WD 1983 Reduced fecun- Granata AR, Carani C 2001 Congenital estrogen deficiency: dity of vitamin D deficient rats. Comp Biochem Physiol in search of the estrogen role in human male reproduction. A 74:923Ð925. Mol Cell Endocrinol 178:107Ð115. 39. Kwiecinski GG, Petrie GI, DeLuca HF 1989 Vitamin D is nec- 57. Shozu M, Sebastian S, Takayama K, Hsu WT, Schultz RA, essary for reproductive functions of the male rat. J Nutr Neely K, Bryant M, Bulun SE 2003 Estrogen excess associ- 119:741Ð744. ated with novel gain-of-function mutations affecting the 40. Kinuta K, Tanaka H, Moriwake T, Aya K, Kato S, Seino Y 2000 aromatase gene. N Engl J Med 348:1855Ð1865. Vitamin D is an important factor in estrogen biosynthesis of both 58. Conte FA, Grumbach MM, Ito Y, Fisher CR, Simpson ER 1994 female and male gonads. Endocrinology 141:1317Ð1324. A syndrome of female pseudohermaphrodism, hyper- 41. Johnson LE, DeLuca HF 2001 Vitamin D receptor null mutant gonadotropic hypogonadism, and multicystic ovaries associated mice fed high levels of calcium are fertile. J Nutr 131:1787Ð1791. with missense mutations in the gene encoding aromatase 42. Dardenne O, Prud’homme J, Hacking SA, Glorieux FH, (P450arom). J Clin Endocrinol Metab 78:1287Ð1292. St-Arnaud R 2003 Correction of the abnormal mineral ion 59. Yanase T, Mu YM, Nishi Y, Goto K, Nomura M, Okabe T, homeostasis with a high-calcium, high-phosphorus, high- Takayanagi R, Nawata H 2001 Regulation of aromatase by lactose diet rescues the PDDR phenotype of mice deficient for nuclear receptors. J Steroid Biochem Mol Biol 79:187Ð192. the 25-hydroxyvitamin D-1α-hydroxylase (CYP27B1). Bone 60. Fu GK, Lin D, Zhang MY, Bikle DD, Shackleton CH, 32:332Ð340. Miller WL, Portale AA 1997 Cloning of human 25-hydroxy- 43. Johnson JA, Grande JP, Roche PC, Kumar R 1996 vitamin D-1α-hydroxylase and mutations causing vitamin Immunohistochemical detection and distribution of the 1,25- DÐdependent rickets type 1. Mol Endocrinol 11:1961Ð1970. dihydroxyvitamin D3 receptor in rat reproductive tissues. 61. Glorieux FH, Arabian A, Delvin EE 1995 Pseudo-vitamin D Histochem Cell Biol 105:7Ð15. deficiency: Absence of 25-hydroxyvitamin D 1α-hydroxylase 44. Habib FK, Maddy SQ, Gelly KJ 1990 Characterisation of activity in human placenta decidual cells. J Clin Endocrinol receptors for 1,25-dihydroxyvitamin D3 in the human testis. Metab 80:2255Ð2258. J Steroid Biochem 35:195Ð199. 62. Ross R, Florer J, Halbert K, McIntyre L 1989 Characterization 45. Villena-Heinsen C, Meyberg R, Axt-Fliedner R, Reitnauer K, of 1,25-dihydroxyvitamin D3 receptors and in vivo targeting of 3 Reichrath J, Friedrich M 2002 Immunohistochemical analysis [ H]-1,25(OH)2D3 in the sheep placenta. Placenta 10:553Ð567. CHAPTER 53 Vitamin D Receptor as a Sensor for Toxic Bile Acids

DAVID J. MANGELSDORF AND DANIEL L. MOTOLA Howard Hughes Medical Institute, Department of Pharmacology, University of Texas Southwestern Medical Center at Dallas, Texas

I. Introduction V. Bile Acids, Vitamin D Receptor, and Colon Cancer II. Bile Acids: Physiologic Roles in Lipid Digestion VI. A Model of Lithocholic Acid Detoxification and Absorption in the Intestine III. The Fate of Bile Acids VII. Perspectives IV. Nuclear Receptors: Key Regulators of Bile Acid Metabolism References

I. INTRODUCTION The rate-limiting step catalyzing this process is modu- lated by a feedforward loop that controls the conversion In this chapter we highlight recent findings that of cholesterol into bile acids and a feedback loop that build on our current understanding of the function of limits bile acid production. Both the feedforward and the vitamin D receptor (VDR) within the enterohepatic feedback loops are governed by a unique sensing appara- system. These findings show that in the intestine, VDR tus that involves several nuclear hormone receptors [3,4] acts as a sensor of carcinogenic bile acids and induces (see section IV). their catabolism by up-regulating expression of key After synthesis by the liver, the majority of bile acids detoxifying enzymes [1]. Specifically, VDR binds a toxic are conjugated to either glycine or taurine before being secondary bile acid, lithocholic acid (LCA), a bioactive secreted through the bile canuliculi of the liver for stor- metabolite believed to play a role in the development of age in the gallbladder and secretion into the duodenum. colon cancer. Binding of either vitamin D or LCA to Conjugation converts bile acids into a stronger acid and VDR leads to receptor activation and induces the detox- thus at a physiological pH, conjugated bile acids become ification of LCA in the intestine through up-regulating fully ionized, membrane impermeable, and more water the expression of a cytochrome P450, CYP3A [1]. These soluble [5,6]. As they reach high concentrations within findings provide a new understanding of the ability of the the intestine they begin to solubilize dietary lipids enteric system to protect itself against carcinogenic bile through the formation of mixed micelles [5,6]. The cen- acids and underscore the importance of developing new ter of the mixed micelle is essentially a fat globule pharmacological therapies that target VDR for the pre- while the outside is charged because of the ionized vention and treatment of colon cancer. A brief review of sterol nucleus of bile acids. Thus, the mixed micelles bile acid physiology along with the details and impor- are free to associate with the water layer adjacent to the tant implications of this discovery are discussed next. surface mucosa of the intestine to allow for the absorp- tion of dietary lipids, cholesterol, and lipid-soluble vitamins. II. BILE ACIDS: PHYSIOLOGIC ROLES IN LIPID DIGESTION AND ABSORPTION III. THE FATE OF BILE ACIDS Bile acids are produced by the liver and are secreted A. Enterohepatic Circulation into the small intestine where they serve several impor- tant functions. Because of their amphipathic properties, Approximately 95% of bile acids synthesized in the bile acids are essential as detergents that solubilize and liver are reclaimed through a process called enterohep- facilitate absorption of dietary fats. The primary bile atic circulation [5,6]. A small portion of bile acids acids, cholic acid (CA) and chenodeoxycholic acid undergoes passive diffusion and uptake by the proximal (CDCA), are produced in the liver from cholesterol intestine. However, the majority of conjugated bile acids, through a series of more than 17 enzymatic steps [2]. which are hydrophilic, are actively absorbed by the VITAMIN D, 2ND EDITION Copyright © 2005, Elsevier, Inc. FELDMAN, PIKE, AND GLORIEUX All rights reserved. 864 DAVID J. MANGELSDORF AND DANIEL L. MOTOLA terminal ileum through the help of an apical membrane allowed to accumulate because of the danger it poses to sodium/bile salt transporter (ABST) [7]. From the peripheral tissues, most notably the circulatory system. enterocytes of the terminal ileum, the bile acids enter Thus, conversion of cholesterol into bile acids is a the portal venous circulation and return to their site of major pathway through which the body excretes excess production in the liver to be secreted once again. This cholesterol and maintains homeostasis, especially when enterohepatic circulation of bile acids helps to main- faced with a large dietary cholesterol load [5,6]. tain a pool of bile acids that can continuously function throughout the day to participate in digestion and absorption of dietary lipids. B. Production of Secondary Bile Acids During the process of circulating between the liver and the intestine, 5% of the bile acid pool is lost with As the bile acid pool continually circulates, enteric each round. This 5% loss is made up for by the conver- flora located within the intestine metabolize primary sion of approximately 500 mg of cholesterol into bile bile acids into the secondary bile acids, deoxycholic acids each day [2]. Although cholesterol serves several acid (DCA) and lithocholic acid (LCA) (Fig. 1) [5,6]. important functions in our bodies, it must not be These secondary bile acids result from bacterial

HO Cholesterol CYP7A1 CYP8B1 CYP7A1 CYP27A1 CYP27A1 Primary bile acids O OH O OH OH

Liver synthesis HO OH HO OH and conjugation Cholic acid Chenodeoxycholic acid

O OH O O O N S OH N S OH H H O O HO OH HO OH Taurocholic acid Taurochenodeoxycholic acid

O OH O Secondary bile acids Bacterial OH OH modifications Lithocholic acid HO Deoxycholic acid HO

Hydroxylation Sulfation

Detoxification O O OH OH _ O SO HO 3 OH Sulfolithocholic acid Hyodeoxycholic acid

FIGURE 1 Classical pathway of bile acid synthesis including the production and detoxification of the secondary bile acid lithocholic acid (LCA). Conversion of cholesterol to bile acids occurs exclusively in the liver through a classical and acidic pathway. For a comprehensive review of these two pathways refer to [2]. In the classical pathway, illustrated here, CYP7A1 hydroxylates cholesterol at position 7. Further modifications to the ring structure occur followed by modifications to the side chain, which are carried out by CYP27A1. The primary bile acids, cholic acid (CA) and chenodeoxycholic acid (CDCA), are produced as a result Before being released into the intestine, these bile acids are conjugated to amino acids taurine or glycine. Bacteria within the colon deconjugate and dehydroxylate CA and CDCA into the less polar and more toxic secondary bile acids, deoxycholic acid (DCA) and LCA, respectively. Sulfation or hydroxylation of secondary bile acids, such as LCA, aids in their detoxification and elimination from the body. CHAPTER 53 Vitamin D Receptor and Bile Acids 865 deconjugation and 7α-dehydroxylation of CA and rate-limiting enzyme in the conversion of cholesterol CDCA, respectively. Both DCA and LCA are much into bile acids [2,3]. In both humans and rodents, more hydrophobic than their primary bile acid coun- LXRs also up-regulate the expression of several ATP- terparts and therefore more readily concentrate within binding cassette transporters, which pump cholesterol cells. The detergent like properties and hydrophobicity out of the liver and into the intestine [3]. In the intestine, of DCA and LCA pose a threat to the cell integrity and these same proteins are also regulated by the LXRs to are believed to cause damage directly to DNA through reverse-transport cholesterol out of the enterocytes the formation of adducts and strand breaks and indirectly and thus limit its absorption [3]. When challenged through the inhibition of DNA repair enzymes [8Ð10]. with cholesterol, mice lacking either LXRα alone or These properties alone make bile acids potential both isoforms fail to turn on these pathways and rapidly carcinogens. Additionally, one of these bile acids, LCA, accumulate toxic levels of cholesterol within the is associated with liver damage seen in cases of liver [14]. cholestatic liver disease. In addition, administration of LCA has been shown to lead to cholestasis in rodents and high levels of LCA have been observed in patients B. Farnesoid X Receptor (FXR): suffering from chronic cholestatic liver disease [11Ð13]. A Bile Acid Receptor Thus, although bile acids serve several important func- tions, they are also inherently toxic to cells at high In the case of bile acids, feedback mechanisms exist levels and therefore must also be maintained at constant to control their production as well as maintain a nor- homeostatic levels. mal bile acid pool size and composition. The farnesoid X receptor, FXR, is expressed in the liver and intestine and acts as a receptor for bile acids [15Ð17]. Bile acid IV. NUCLEAR RECEPTORS: activation of FXR represses the expression of CYP7A1, KEY REGULATORS OF BILE ACID the rate-limiting enzyme for bile acid synthesis. FXR METABOLISM reduces CYP7A1 gene expression by regulating the expression of a potent repressor called short heterodimer It should be apparent that very tightly regulated partner (SHP), an unusual orphan receptor that does not mechanisms of control must exist to maintain both possess a DNA binding domain [18,19]. As a result, cholesterol and bile acid levels. The molecular mecha- SHP dimerizes with other nuclear receptors that are nisms that govern this regulation have been elucidated bound to the CYP7A1 promoter and dominantly within the past few years and have uncovered an impor- represses transcription, thereby turning off bile acid tant role for nuclear receptors in this process. The fol- synthesis. FXR also helps maintain the normal flow of lowing section will briefly describe the role of nuclear bile acids by modulating the expression of various bile receptors in this process and will end with the discus- acid transporters and binding proteins in the liver and sion of how VDR participates in these mechanisms of intestine [20]. regulation. C. Pregnane X Receptor (PXR): A. Liver-X-Receptor (LXR): A Xenobiotic Receptor An Oxysterol Receptor The pregnane X receptor (PXR) functions primarily The nuclear receptors play a major role in the con- in the liver to protect the body against the accumula- trol of cholesterol and bile acid levels. The feed- tion of toxic exogenous (xenobiotics) or endogenous forward control of cholesterol’s conversion into bile (endobiotic) chemicals [21Ð24]. The large ligand-binding acids as well as its excretion into bile is now known to pocket of PXR allows it to bind a structurally diverse be mediated through the action of two nuclear hor- group of chemicals that include many prescription drugs, mone receptors, the liver X receptors (called LXRα and steroids, and over-the-counter herbal remedies [24]. PXR LXRβ). LXRα is expressed predominately in the liver controls the levels of these compounds by up-regulating where it senses the levels of cholesterol through bind- the gene expression of the enzymes responsible for ing of oxysterols (oxidized cholesterol) and in turn their metabolism and clearance. These enzymes include modulates the expression of gene networks involved in the cytochrome p450s, in particular the CYP3A sub- the catabolism, transport, storage, and absorption of family members, and the sulfotransferases. cholesterol [3]. One target of LXRα in mice but not in In cholestatic liver disease, a failure to secrete humans is the cytochrome p450 enzyme CYP7A1, the bile results in the progressive accumulation of bile 866 DAVID J. MANGELSDORF AND DANIEL L. MOTOLA constituents in the liver causing liver damage and even- age-adjusted colon cancer mortality rates are highest in tual cirrhosis. LCA, as mentioned above, is a highly the northern parts of the country, especially in the toxic secondary bile acid metabolite that is elevated in northeast United States where a combination of lati- the liver and serum of patients under cholestatic condi- tude, climate, and pollution results in a 5-month period tions [13]. Recent investigations have now demonstrated without any vitamin D synthesis [30] (see Chapter 90). that PXR functions as an LCA sensor in the liver so that The geographical differences in fat intake are not it can protect the liver against LCA-induced hepato- likely to account for the differences in colon cancer toxicity [21Ð24]. At high concentrations (∼100 µM), mortality as the southern United States typically con- LCA binds PXR, which is then activated to induce the sumes more fat than the northern United States [30]. expression of two genes, N-sulfotransferase (SULT-N), Furthermore, clinical and laboratory studies have a member of the sulfotransferase gene family, and shown that calcium and/or vitamin D supplementation CYP3A, both of which convert LCA into a more polar can decrease the incidence of colon cancer associated and less toxic bile acid that is more easily eliminated with a high-fat diet, in high-risk patients, or as a result from the body [21Ð24]. of LCA administration in rodents [30Ð32]. These epidemiological and experimental findings suggest that calcium or vitamin D may help protect against the V. BILE ACIDS, VITAMIN D RECEPTOR, mortality and/or development of colon cancer (also see AND COLON CANCER Chapters 90 and 91). The ability of vitamin D to inhibit growth and The most recent finding involving nuclear receptors induce differentiation and apoptosis in malignant cells and the regulation of bile acid metabolism is the is well established [33]. However, the mechanism by surprising role of VDR as an enteric sensor of carcino- which vitamin D prevents colon cancer remains an genic bile acids. The finding may help explain the area of active investigation. A possible explanation for protective effects of vitamin D and VDR in preventing the protective effects of vitamin D comes from recent colon cancer. investigations involving nuclear receptors in the regu- lation of bile acid metabolism [1]. As was previously mentioned, under conditions of cholestasis the nuclear A. High-Fat Diets Increase Risk for hormone receptor PXR may be responsible for the Colon Cancer detoxification of LCA in the liver through up-regulating the gene expression of CYP3A. However, an additional In the United States, colon cancer is the third most PXR-independent pathway for LCA detoxification must common cancer in men and women [25]. Besides exist, since PXR-null animals still showed LCA-induced genetic predisposition and other environmental risk expression of CYP3A [22]. The only other receptor factors, epidemiological and animal studies have given known to interact with primary or secondary bile acids rise to the prevailing theory that a high dietary intake is FXR [15Ð17] (Section IV, B); however, CYP3A is of fat is a primary contributing factor in the develop- not known to be an FXR target gene, nor is vitamin D ment of colon cancer [26] (see Chapter 91). Associated a ligand for FXR or PXR. Interestingly, two reports with this high-fat diet is an increase in the secretion of suggested that CYP3A is a VDR target gene [34,35]. fecal bile acids, specifically the toxic secondary bile This led to the exploration of the potential role of VDR acid, LCA [27]. In animal studies, LCA administration in bile acid metabolism, specifically of LCA within the has been shown to induce colon cancer [28,29] and its intestine [1]. concentrations have been reported to be elevated in patients with colon cancer [27]. C. VDR as a Bile Acid Receptor

B. A Protective Role for Vitamin D Strong correlative evidence supports the idea that VDR may have evolved the ability to sense bile acids, In contrast to the positive correlations between in particular LCA. Phylogenetic analysis of VDR shows high-fat diets, LCA, and colon cancer, the levels of that its primary amino acid sequence is most similar to another dietary component, vitamin D, is negatively that of PXR and FXR. The highest identity is found correlated with the incidence of colon cancer [30]. between the DNA-binding and ligand-binding domains Epidemiological studies have shown that strikingly of VDR and PXR [36,37]. Interestingly, both PXR and similar global geographical patterns exist between colon VDR bind to DNA response elements configured cancer and rickets. For example, in the United States, as everted repeats spaced by six nucleotides (ER-6) CHAPTER 53 Vitamin D Receptor and Bile Acids 867

or direct repeats spaced by three nucleotides (DR-3) LCA and 3-keto-LCA to VDR (inhibition constant Ki = [33,38] (see Chapter 18). Several experiments were 29 ± 6 µM and 8 ± 3 µM, respectively) [1]. Furthermore, carried out to determine whether or not VDR indeed the binding affinity of LCA and 3-keto-LCA for VDR mediates the PXR-independent LCA-induced expres- matched closely the dose response data from transactiva- sion of CYP3A. The results of these experiments are tion experiments and provided convincing evidence that detailed next. LCA and its metabolites are bona fide ligands for VDR. In order to identify the LCA receptor, a panel of enterohepatic nuclear receptors was screened for their ability to bind LCA and induce the expression of D. VDR Regulates CYP3A-Dependent CYP3A [1]. The ability of a receptor to bind a ligand Detoxification of LCA and activate gene expression can be measured in vitro using cell-based, ligand-induced reporter activation The ability of LCA to induce the expression of assays. These results can also be confirmed by using CYP3A through VDR was then confirmed using competitive binding assays, which also assess speci- both in vitro and in vivo experiments [1]. Response ficity and kinetics of ligand binding. To identify the elements to which VDR binds in its known target genes LCA receptor, an approach was taken based on ligand- were found in the promoters of the human, rat, and induced interaction of a nuclear receptor with its coac- mouse CYP3A genes. These CYP3A response elements tivator. The coactivator protein SRC-1 was fused to the were shown to be bound by VDR/RXR heterodimers DNA binding domain of the yeast transcription factor, using electrophoretic mobility shift assays (EMSA) and GAL4. Each receptor within the panel was also fused to were also able to confer VDR-dependent LCA induction the activation domain of the herpes virus protein,VP16. of a reporter gene in cell-based transfection assays. Human embryonic kidney cells (HEK 293) were trans- These results were confirmed in vivo, as administration fected with the appropriate plasmids, and each nuclear of either vitamin D or LCA induced the expression of receptor was tested for its ability to bind LCA, recruit CYP3A within the intestine and livers of wild-type mice coactivator protein, and activate the expression of a as well as in mice lacking PXR. These experiments pro- GAL4 responsive reporter gene. At the concentration vided further evidence confirming the existence of a of LCA tested, 30 µM, only VDR and FXR were acti- PXR-independent pathway for CYP3A induction and vated. Interestingly, a much higher concentration of supported the idea that VDR is a sensitive receptor for LCA (≥100 µM) was required for activation of PXR. LCA and can induce LCA catabolism within the intes- Further analysis of the ligand specificity of VDR and tine by up-regulating the expression of CYP3A. In FXR revealed that each receptor has a distinct speci- addition to CYP3A, other genes involved in LCA ficity profile. VDR was activated by 1,25(OH)2D3, metabolism are also induced by VDR (B. Chatterjee, while FXR was not. Similarly, FXR was activated by personal communication). One such example is SULT-N, the primary bile acids, CA and CDCA, while VDR was which is a PXR target gene and is important for the not. Both receptors bound LCA and its 3-keto metabolite, clearance of LCA from the liver [23]. but only VDR was activated by the 6-keto metabolite of LCA. Dose response curves revealed that VDR was activated by LCA and 3-keto LCA with a median effec- VI. A MODEL OF LITHOCHOLIC ACID tive concentration (EC50) of 8 µM and 3 µM, respec- DETOXIFICATION IN THE INTESTINE tively. However, FXR required two to three times greater concentrations of LCA and its 3-keto metabo- The experiments just described provided new lite for activation, while PXR required a concentration insights into the mechanism by which vitamin D might at least 10 times greater, when compared to activation protect against colon cancer and revealed an unexpected of VDR. Taken together, the results from this work role for VDR as a sensor of carcinogenic bile acids. The suggested that VDR is a more sensitive receptor for epidemiological findings of similar geographical distri- LCA and its metabolites than are FXR and PXR, and butions of both rickets and colon cancer may therefore that the ligand specificity profile of each receptor has be explained under the following model (Fig. 2). Under allowed each receptor to perform distinct functions. normal nutritional status, VDR utilizes 1,25(OH)2D3 To further confirm the direct interaction of LCA and or LCA to maintain increased levels of CYP3A gene 3-keto-LCA with VDR, competitive binding experiments expression. CYP3A converts LCA or its metabolites were performed using purified recombinant VDR, into less toxic bile acids, thus preventing injury to the radiolabeled 1,25(OH)2D3,and increasing concentrations intestinal epithelium. However, the risk for developing of unlabeled LCA or 3-keto-LCA as competitors. These colon cancer rises significantly when LCA levels are experiments revealed a direct and specific binding of increased as a result of either a sustained high-fat diet 868 DAVID J. MANGELSDORF AND DANIEL L. MOTOLA

AB

Low fat diet Vitamin D High fat diet normal vitamin D LCA and/or low vitamin D Catabolized LCA Excessive level

Normal level

CYP3A CYP3A

N

FIGURE 2 A model for the detoxification of LCA in the intestine. (A) On a low-fat diet, low levels of LCA (open square) and high levels of vitamin D (filled triangle) tip the balance away from colon can- cer and toward a normal state. Vitamin D levels are important for maintaining this normal physiologi- cal state as 1,25(OH)2D3 can also bind to VDR and activate CYP3A catabolism of LCA. (B). Certain pathological conditions such as sustained high-fat diets or vitamin D deficiency tip the balance toward colon cancer. A sustained high-fat diet overwhelms the capacity of CYP3A to metabolize LCA and excessive levels of LCA “spill over” and cause injury to the colonic epithelium. Alternatively, in condi- tions of low vitamin D, such as rickets, the balance is similarly shifted toward colon cancer. or vitamin D deficiencies. Since both of these conditions dietary lipid excess. These receptors (including the (i.e., sustained high-fat diets and vitamin D deficiency) LXRs, FXR, and PXR) are all expressed primarily in are pathophysiologic and beyond what the body has the enterohepatic system, where they are ideally posi- evolved to cope with, the detoxification system cannot tioned to sense toxic dietary constituents and eliminate keep up with the elevated levels of LCA even though them by activating expression of a metabolic gene net- the VDR sensing mechanism is still in place. This work [3]. Further up the evolutionary ladder, a second results in LCA-induced toxicity to the colon. As pre- group of RXR heterodimers has evolved as endocrine dicted by this model, mice lacking VDR have been receptors (e.g., thyroid hormone and retinoic acid shown to display enhanced preneoplastic cellular pro- receptors). The fact that VDR has elements of both liferations in the colon [39]. the endocrine and lipid sensing receptor class lends credence to the hypothesis that the primordial VDR ancestor was likely a bile acid sensor. It is tempting to VII. PERSPECTIVES speculate that from this ancestral receptor, the family expanded to become PXR, FXR, and the LXRs, all of In this chapter we have focused on the recent, and which are phylogenetically related to VDR and share indeed unexpected, finding that VDR can function as similar sterol-derived ligands. The evolution of VDR both an endocrine receptor (for vitamin D) and a lipid from this group of lipid-sensing receptors into an sensing receptor (for LCA). This finding has both evo- endocrine receptor that regulates calcium homeostasis lutionary and pharmacological implications. From an may not be that surprising if one considers that such a evolutionary perspective, it is interesting to note that receptor must have some of the same properties as other VDR exists as one of a number of RXR heterodimer receptors in this class, such as an abundant enterohep- partners that evolved to protect the body against atic expression. The fact that VDR kept its vestigial CHAPTER 53 Vitamin D Receptor and Bile Acids 869 bile acid receptor qualities suggests that there was a 10. Ogawa A, Murate T, Suzuki M, Nimura Y, Yoshida S 1998 strong evolutionary drive to keep LCA concentration Lithocholic acid, a putative tumor promoter, inhibits mam- malian DNA polymerase beta. Jpn J Cancer Res 89:1154Ð1159. in check. It will be interesting to test this prediction 11. Fisher MM, Magnusson R, Miyai K 1971 Bile acid further by investigating the ligand binding properties metabolism in mammals. I. Bile acid-induced intrahepatic of VDR in more primitive species. cholestasis. Lab Invest 25:88Ð91. This work also suggests the importance of this 12. Javitt NB 1966 Cholestasis in rats induced by taurolitho- group of receptors as potential therapeutic targets for cholate. Nature 210:1262Ð1263. 13. Fischer S, Beuers U, Spengler U, Zwiebel FM, Koebe HG novel drug discovery. In particular, the work high- 1996 Hepatic levels of bile acids in end-stage chronic lighted here should give rise to future studies and cholestatic liver disease. Clin Chim Acta 251:173Ð186. efforts aimed at the development of new pharmacolog- 14. Peet DJ, Turley SD, Ma W, Janowski BA, Lobaccaro JM, ical strategies for the prevention of colon cancer. With Hammer RE, Mangelsdorf DJ 1998 Cholesterol and bile acid respect to VDR, the ideal candidate would be a drug metabolism are impaired in mice lacking the nuclear oxysterol receptor LXR alpha. Cell 93:693Ð704. that does not carry the unwanted side effects such as 15. Makishima M, Okamoto AY, Repa JJ, Tu H, Learned RM, hypercalcemia, which are currently seen with many of Luk A, Hull MV, Lustig KD, Mangelsdorf DJ, Shan B 1999 the VDR agonists currently used to treat psoriasis and Identification of a nuclear receptor for bile acids. Science osteoporosis. The notion that such selective VDR mod- 284:1362Ð1365. ulators may be found is strengthened by recent find- 16. Parks DJ, Blanchard SG, Bledsoe RK, Chandra G, Consler TG, Kliewer SA, Stimmel JB, Willson TM, Zavacki AM, Moore DD, ings showing that most vitamin D analogs bind the Lehmann JM 1999 Bile acids: Natural ligands for an orphan ligand-binding pocket of VDR differently than LCA [40]. nuclear receptor. Science 284:1365Ð1368. Consequently, it may be possible to design novel VDR 17. Wang H, Chen J, Hollister K, Sowers LC, Forman BM 1999 agonists that induce multiple conformations of the Endogenous bile acids are ligands for the nuclear receptor VDR’s ligand binding domain, thereby resulting in dif- FXR/BAR. Mol Cell 3:543Ð553. 18. Lu TT, Makishima M, Repa JJ, Schoonjans K, Kerr TA, ferential therapeutic actions. The discovery of selective Auwerx J, Mangelsdorf DJ 2000 Molecular basis for feedback estrogen receptor modulators (SERMs) and elucidation regulation of bile acid synthesis by nuclear receptors. Mol Cell of their mechanisms of action provides an important 6:507Ð515. proof of principle as to how this might actually be 19. Goodwin B, Jones SA, Price RR, Watson MA, McKee DD, accomplished with VDR [41,42]. Moore LB, Galardi C, Wilson JG, Lewis MC, Roth ME, Maloney PR, Willson TM, Kliewer SA 2000 A regulator cascade of the nuclear receptors FXR, SHP-1, and LRH-1 represses bile acid biosynthesis. Mol Cell 6:517Ð526. References 20. Sinal CJ, Tohkin M, Miyata M, Ward JM, Lambert G, Gonzalez FJ 2000 Targeted disruption of the nuclear receptor 1. Makishima M, Lu TT, Xie W, Whitfield GK, Domoto H, FXR/BAR impairs bile acid and lipid homeostasis. Cell Evans RM, Haussler MR, Mangelsdorf DJ 2002 Vitamin D 102:731Ð744. receptor as an intestinal bile acid sensor. Science 296:1313Ð1316. 21. Staudinger JL, Goodwin B, Jones SA, Hawkins-Brown D, 2. Russell DW 2003 The Enzymes, regulation, and genetics of MacKenzie KI, LaTour A, Liu Y, Klaassen CD, Brown KK, bile acid synthesis. Annu Rev Biochem. Reinhard J, Willson TM, Koller BH, Kliewer SA 2001 The 3. Chawla A, Repa JJ, Evans RM, Mangelsdorf DJ 2001 Nuclear nuclear receptor PXR is a lithocholic acid sensor that protects receptors and lipid physiology: opening the X-files. Science against liver toxicity. Proc Natl Acad Sci USA 98:3369Ð3374. 294:1866Ð1870. 22. Xie W, Radominska-Pandya A, Shi Y, Simon CM, Nelson MC, 4. Lu TT, Repa JJ, Mangelsdorf DJ 2001 Orphan nuclear recep- Ong ES, Waxman DJ, Evans RM 2001 An essential role for tors as eLiXiRs and FiXeRs of sterol metabolism. J Biol Chem nuclear receptors SXR/PXR in detoxification of cholestatic 276:37735Ð37738. bile acids. Proc Natl Acad Sci USA 98:3375Ð3380. 5. Hofmann AF 1999 The continuing importance of bile acids 23. Sonoda J, Xie W, Rosenfeld JM, Barwick JL, Guzelian PS, in liver and intestinal disease. Arch Intern Med 159: Evans RM 2002 Regulation of a xenobiotic sulfonation 2647Ð2658. cascade by nuclear pregnane X receptor (PXR). Proc Natl 6. Carey MaD, WC 1994 Enterohepatic circulation. In: Arias IM BJ, Acad Sci USA 99:13801Ð13806. Fausto N, Jakoby WB, Schachter DA, Sharfritz DA (eds) The 24. Kliewer SA, Goodwin B, Willson TM 2002 The nuclear preg- Liver: Biology and Pathobiology, 3rd ed. Raven Press, New York, nane X receptor: A key regulator of xenobiotic metabolism. pp. 719Ð768. Endocr Rev 23:687Ð702. 7. Craddock AL, Love MW, Daniel RW, Kirby LC, Walters HC, 25. Cancer Facts and Figures: American Cancer Society; 2003 Wong MH, Dawson PA 1998 Expression and transport proper- [Postscript Document] URL: http://www.cancer.org/docroot/ ties of the human ileal and renal sodium-dependent bile acid STT/stt_0.asp transporter. Am J Physiol 274:G157ÐG169. 26. Lipkin M, Reddy B, Newmark H, Lamprecht SA 1999 Dietary 8. Nagengast FM, Grubben MJ, van Munster IP 1995 Role of factors in human colorectal cancer. Annu Rev Nutr bile acids in colorectal carcinogenesis. Eur J Cancer 31A: 19:545Ð586. 1067Ð1070. 27. Owen RW, Dodo M, Thompson MH, Hill MJ 1987 Fecal 9. Hamada K, Umemoto A, Kajikawa A, Seraj MJ, Monden Y steroids and colorectal cancer. Nutr Cancer 9:73Ð80. 1994 In vitro formation of DNA adducts with bile acids. 28. Narisawa T, Magadia NE, Weisburger JH, Wynder EL 1974 Carcinogenesis 15:1911Ð1915. Promoting effect of bile acids on colon carcinogenesis after 870 DAVID J. MANGELSDORF AND DANIEL L. MOTOLA

intrarectal instillation of N-methyl-N′-nitro-N-nitrosoguani- 36. Rochel N, Wurtz JM, Mitschler A, Klaholz B, Moras D 2000 dine in rats. J Natl Cancer Inst 53:1093Ð1097. The crystal structure of the nuclear receptor for vitamin D 29. Reddy BS, Watanabe K 1979 Effect of cholesterol metabolites bound to its natural ligand. Mol Cell 5:173Ð179. and promoting effect of lithocholic acid in colon carcinogenesis in 37. Watkins RE, Wisely GB, Moore LB, Collins JL, Lambert MH, germ-free and conventional F344 rats. Cancer Res 39:1521Ð1524. Williams SP, Willson TM, Kliewer SA, Redinbo MR 2001 The 30. Garland CF, Garland FC, Gorham ED 1999 Calcium and vita- human nuclear xenobiotic receptor PXR: structural determi- min D. Their potential roles in colon and breast cancer preven- nants of directed promiscuity. Science 292:2329Ð2333. tion. Ann NY Acad Sci 889:107Ð119. 38. Kliewer SA, Moore JT, Wade L, Staudinger JL, Watson MA, 31. Pence BC, Buddingh F 1988 Inhibition of dietary fat-promoted Jones SA, McKee DD, Oliver BB, Willson TM, Zetterstrom RH, colon carcinogenesis in rats by supplemental calcium or vita- Perlmann T, Lehmann JM 1998 An orphan nuclear receptor acti- min D3. Carcinogenesis 9:187Ð190. vated by pregnanes defines a novel steroid signaling pathway. 32. Kawaura A, Tanida N, Sawada K, Oda M, Shimoyama T 1989 Cell 92:73Ð82. Supplemental administration of 1α-hydroxyvitamin D3 39. Kallay E, Pietschmann P, Toyokuni S, Bajna E, Hahn P, inhibits promotion by intrarectal instillation of lithocholic acid Mazzucco K, Bieglmayer C, Kato S, Cross HS 2001 in N-methyl-N-nitrosourea-induced colonic tumorigenesis in Characterization of a vitamin D receptor knockout mouse as a rats. Carcinogenesis 10:647Ð649. model of colorectal hyperproliferation and DNA damage. 33. Haussler MR, Whitfield GK, Haussler CA, Hsieh JC, Carcinogenesis 22:1429Ð1435. Thompson PD, Selznick SH, Dominguez CE, Jurutka PW 40. Choi M, Yamamoto K, Itoh T, Makishima M, Mangelsdorf DJ, 1998 The nuclear vitamin D receptor: biological and molecular Moras D, DeLuca HF, Yamada S 2003 Interaction regulatory properties revealed. J Bone Miner Res 13:325Ð349. between vitamin D receptor and vitamin D ligands. Two- 34. Thummel KE, Brimer C, Yasuda K, Thottassery J, Senn T, Lin Y, dimensional alanine scanning mutational analysis. Chem Biol Ishizuka H, Kharasch E, Schuetz J, Schuetz E 2001 10:261Ð270. Transcriptional control of intestinal cytochrome P-4503A by 41. Brzozowski AM, Pike AC, Dauter Z, Hubbard RE, Bonn T, 1α,25-dihydroxyvitamin D3. Mol Pharmacol 60:1399Ð1406. Engstrom O, Ohman L, Greene GL, Gustafsson JA, Carlquist M 35. Schmiedlin-Ren P, Thummel KE, Fisher JM, Paine MF, 1997 Molecular basis of agonism and antagonism in the Watkins PB 2001 Induction of CYP3A4 by 1α,25-dihydroxy- oestrogen receptor. Nature 389:753Ð758. vitamin D3 is human cell line-specific and is unlikely to involve 42. Shang Y, Brown M 2002 Molecular determinants for the tissue pregnane X receptor. Drug Metab Dispos 29:1446Ð1453. specificity of SERMs. Science 295:2465Ð2468. CHAPTER 54 Vitamin D and the ReninÐAngiotensin System

YAN CHUN LI Department of Medicine, University of Chicago, Chicago, Illinois

I. Introduction V. 1,25-Dihydroxyvitamin D3 as a Negative Endocrine II. The ReninÐAngiotensin System Regulator of the ReninÐAngiotensin System III. Sunlight, Vitamin D, and Blood Pressure: VI. Vitamin D Analogs as Potential Antihypertensive Agents Epidemiological and Clinical Observations VII. Conclusion IV. Vitamin D and Cardiovascular Functions References

I. INTRODUCTION cells are highly granulated smooth muscle cells located in the media of the afferent arteriole at the vascular The reninÐangiotensin system (RAS) plays a central pole of the glomerulus, where the afferent arteriole role in the regulation of blood pressure, extracellular enters, and the efferent arteriole exits, the renal cor- volume, and electrolyte homeostasis. Inappropriate puscle (Fig. 2). The main function of renin is to cleave activation of the RAS may lead to hypertension, which angiotensin I (Ang I), a 10-amino-acid peptide, from is one of the major risk factors for stroke, myocardial angiotensinogen, which is predominantly produced in infarction, congestive heart failure, progressive the liver. Ang I is then converted to an 8-amino-acid atherosclerosis, and renal failure. These are major dis- peptide, angiotensin II (Ang II), by the angiotensin- eases with high mortality rates in both developing and converting enzyme (ACE), which primarily resides in industrialized countries. However, prevention and ther- the endothelial cells in blood vessels. Further processing apeutic intervention of hypertension remain a major of Ang II by animopeptidase A and N produces Ang III medical challenge at present, and hypertension preva- and Ang IV. Ang II is the central biological effector lence in the United States has increased in the past of the RAS. Through binding to its receptors, which are decade [1]. Our understanding of the renocardiovascular G protein-coupled receptors widely distributed and system, including the RAS, will have a direct impact, at expressed by many cell types [5], Ang II exerts diverse least to some degree, on how well we face this chal- physiological actions that regulate the homeostasis of lenge. Recent advance has placed the vitamin D electrolytes, extracellular volume and blood pressure endocrine system at an important position in the regu- [6,7]. For instance, Ang II acts on blood vessel smooth lation of the RAS, and this chapter will focus on the relationship between the vitamin D endocrine system and the RAS. Prorenin

Renin II. THE RENINÐANGIOTENSIN SYSTEM Angiotensinogen Angiotensin I ACE A. An Overview Angiotensin II

The RAS is a systemic endocrine regulatory cascade Angiotensin II receptors (Fig. 1) that involves multiple organs [2], but components of the RAS have also been found inside many tissues Thirst Intestinal Na ADH Aldosterone such as the brain, the heart, and the kidney, suggesting Vasoconstriction that it may also function in a paracrine fashion [3]. The H2O intake Na intake H2O retention Na retention exact physiological role of the tissue RAS remains unclear. The first and rate-limiting component of the Extracellular volume Blood pressure RAS cascade is renin, an aspartyl protease synthesized FIGURE 1 The reninÐangiotensin system. ADH, antidiuretic and secreted predominantly by the juxtaglomerular hormone, also known as vasopressin; ACE, angiotensin-converting (JG) cells in the JG apparatus of the kidney [4]. The JG enzyme. VITAMIN D, 2ND EDITION Copyright © 2005, Elsevier, Inc. FELDMAN, PIKE, AND GLORIEUX All rights reserved. 872 YAN CHUN LI muscle cells to cause vasoconstriction; stimulates B. Control of Renin Synthesis and Secretion antidiuretic hormone (ADH, also called vasopressin) production by the central nervous system, which Because of its central role in the reninÐangiotensin increases water retention in the kidney; stimulates cascade, the biosynthesis and secretion of renin is aldosterone synthesis and secretion in the adrenal tightly regulated. Renin is synthesized in the JG cortex, which enhances renal sodium reabsorption in cells as a prepropolypeptide precursor during trans- the distal tubules; and stimulates the sensation of thirst lation in the endoplasmic reticulum. The targeting in the central nervous system and thus increases water signal sequence is cleaved during translocation in intake. Therefore, activation of the reninÐangiotensin the endoplasmic reticulum, yielding the inactive cascade generally leads to an increase in extracellular prorenin. Prorenin is glycosylated and activated during volume and blood pressure (Fig. 1), and inappropriate intracellular transport through the Golgi apparatus, stimulation of the RAS thus induces detrimental effects. and eventually stored in secretory granules. The Adding a new level of complexity to the RAS, recently prosequence is removed in this process. Renin is a renin receptor has been reported in the heart, kidney, secreted from these granules through exocytosis upon brain, placenta, and liver, which may function to increase stimulation [4,12]. the catalytic activity of renin [8], and an ACE homolog, It is well known that renin secretion in the kidney is ACE2, has been isolated [9,10] and found to play an commonly regulated by a variety of physiological essential role in heart functions [11]. factors, including renal perfusion pressure, renal

Proximal convoluted tubule

Basement membrane

Glomerular Erythrocytes capillaries

Glomerular basement Mesangial membrane cells

Endothelium Capsular Glomerular (parietal (visceral epithelium) epithelium)

Lacis cells JG cells

Efferent Afferent arteriole Macula arteriole densa

Distal tubule Smooth muscle cells in media FIGURE 2 Diagram of the renal corpuscle structure, showing the juxtaglomerular (JG) apparatus. The tubular pole, which is where the proximal tubule leaves the corpuscle, is at the top, and the vascular pole, which is where the afferent arteriole enters and the efferent arteriole leaves the corpuscle, is at the bottom. The granu- lated renin-producing JG cells are located in the afferent arteriole at the vascular pole and are in close vicinity to the macula densa. (Adapted from Cormack 1984, Introduction to Histology, Fig. 15-5, p. 344, with permission.) CHAPTER 54 Vitamin D and the ReninÐAngiotensin System 873 sympathetic nerve activity, and tubular sodium chlo- renin secretion, and several cAMP response elements ride load [4,6]. The perfusion pressure in the renal (CRE) have been identified in both murine and human artery is the most profound parameter to influence renin gene promoters [12]. However, both CREB- renin secretion: When renal perfusion pressure falls, dependent and -independent mechanisms may be renin secretion rises; when renal perfusion pressure rises, involved in the cAMP-PKA pathway in human renin renin secretion falls. This effect is mediated by a promoter activation [20]. Transgenic studies have baroreceptor or stretch receptor mechanism in the demonstrated that sequences required for the tissue- JG cells [13]. The JG apparatus and the glomerulus specific and development stageÐspecific expression of have rich sympathetic nerve endings, which express the renin gene, as well as for the response to a variety β-adrenergic receptors. Stimulation of renin synthesis of physiological stimuli, are located within 5 kb of the and release by renal innervation has been well docu- 5′-flanking region of the murine renin gene [21Ð23]. mented, which is mediated by β-adrenergic receptors In recent years, studies of the renin gene regulation and intracellular cyclic AMP [14]. This pathway may have been greatly facilitated by the establishment of a JG exert a tonic stimulatory influence on renin production cell-like cell line, namely As4.1, from kidney tumors [15]. Renin secretion is also tightly regulated by the of SV40 T antigen transgenic mice [23]. The As4.1 tubular sodium chloride load [16]. There is an inverse cells express a high level of endogenous renin. In the relationship between dietary sodium chloride intake 4.1 kb 5′-flanking region of murine Ren-1c gene, a and renin secretion. Tubular control of renin release is 223 bp minimal promoter (−117 to +6) and a 242 bp mediated by the macula densa, which is part of the distal enhancer (−2866 to Ð2625) at about Ð2.6 kb upstream tubule and anatomically in close association with the of the transcriptional start site have been found to be renin-producing JG cells (Fig. 2). The macula densa essential for high-level expression of the renin gene in senses the sodium chloride load and transduces the As4.1 cells [24]. Recent studies have identified an signal, possibly via adenosine and ATP, to the JG cells array of transcriptional factors involved in the tran- to influence renin production and secretion [12]. At the scriptional regulation of renin gene expression. These local level, renin synthesis and release are influenced factors include positive regulators such as LXRα, by a large variety of bioactive molecules. For instance, RAR/RXR, CREB/CREM, USF1/USF2, HOX genes, prostaglandins, nitric oxide, and adrenomedullin are NFI, and SP1/SP3 [25Ð29], and negative regulators known to stimulate renin secretion, whereas Ang II (as a such as NF-Y and Ear-2 [30,31]. Thus, the production feedback regulator), endothelin, vasopressin, and adeno- of renin is determined by a combined interplay of mul- sine are inhibitors of renin release [4,6,12]. tiple transcriptional regulators available or activated under a specific physiological condition. C. Transcriptional Regulation of Renin Gene Expression III. SUNLIGHT, VITAMIN D, AND BLOOD PRESSURE: EPIDEMIOLOGICAL Renin is encoded by a single gene in humans. In mice, AND CLINICAL OBSERVATIONS some strains (e.g., C57BL/6) have one renin gene (Ren-1c), whereas others (e.g., DBA/2, J129) contain Evidence from epidemiological and clinical studies two renin genes (Ren-1d and Ren-2), which are closely in recent decades has suggested a connection between linked and probably resulted from a duplication of the vitamin D endocrine system and blood pressure. the 21 kb Ren-1c-like ancestral gene [17]. The human As ultraviolet (UV) irradiation is essential for the cuta- renin gene and the three mouse renin genes all share neous production of vitamin D, circulating vitamin D the same overall genomic organization (e.g., nine exons levels are greatly influenced by geographic locations, and eight introns) and encode highly homologous pro- seasonal changes, and skin pigmentations. Data teins. For instance, Ren-1 and Ren-2 share 97% amino obtained from the INTERSALT study centers reveal a acid identity [18]. It is believed that the Ren-1 protein linear correlation between the rise in blood pressure or is the major source of circulating renin and thus is the prevalence of hypertension and the latitudes north the major systemic regulator of the reninÐangiotensin or south of the equator [32]. Similarly, data from a cascade. Recent studies in transgenic mice demonstrate national survey in China also show a high-to-low gra- that the Ren-1d and Ren-2 genes cooperate to preserve dient from the north to the south of the country in the the homeostasis of the RAS [19]. prevalence of hypertension and stroke incidence [33]. Recent studies suggest that expression of the renin Seasonal variations in blood pressure have been gene is regulated by a complex network of transcrip- reported in temperate climates, with blood pressure tional factors. Cyclic AMP is a major mediator for higher in the winter (low UV irradiation) than in the 874 YAN CHUN LI summer (high UV irradiation) [34,35]. Dark skin pig- mentation, which affects efficient UV light penetra- 8 tion [36], has also been reported to be associated with higher blood pressure [37,38]. Indeed, UV irradiation has been reported to lower blood pressure in patients 6 with mild essential hypertension [39]. Numerous studies have shown that the serum level of 1,25(OH)2D3 is inversely associated with blood 4 pressure in normotensive and hypertensive subjects (Fig. 3) [40Ð42]. More interestingly, such inverse rela- tionship has also been reported between circulating 2 Plasma renin activity (ng/ml/h) 1,25(OH)2D3 levels and plasma renin activity in patients with essential hypertension (Fig. 4) [43]. Vitamin D supplement has been shown to be beneficial to the heart. 20 40 60 80 100 120 For instance, in double-blinded, placebo-controlled 1,25(OH) D (pg/ml) clinical trials, long-term treatment with 1α-hydroxy- 2 3 vitamin D3 results in a reduction in blood pressure FIGURE 4 Inverse relationship between circulating 1,25(OH)2D3 in patients with essential hypertension [44], and levels and the plasma renin activity in patients with essential hypertension. (Adapted from Resnick et al. 1986, Fig. 2, p. 652, with short-term vitamin D3 and calcium supplementation permission.) reduces blood pressure in elderly women [45]. In clinical cases, it has been reported that 1,25(OH)2D3

supplementation reduces the blood pressure and plasma renin activity in a patient with pseudohyper- 130 parathyroidism and high plasma renin activity [46], and intravenous 1,25(OH)2D3 treatment of hemodialy- 127 126.12 sis patients with secondary hyperparathyroidism sig- nificantly reduces the plasma renin and Ang II levels 124 and concomitantly regresses the myocardial hypertro- 120.55 120.51 phy [47]. These observations are consistent with a role 121 of the vitamin D endocrine system in the regulation of SBP (mm Hg) renocardiovascular functions and blood pressure. 118 115.44 115 <=60 61–70 71–80 >80 IV. VITAMIN D AND CARDIOVASCULAR FUNCTIONS 82 The heart is believed to be a vitamin D target organ, 80 79.13 as the vitamin D receptor (VDR) is found expressed in 78 skeletal and cardiac myocytes [48,49]. Early studies, 76.43 76.38 76.61 mostly using vitamin DÐdeficient rats, have showed 76 that 1,25(OH)2D3 may play some roles in the regulation 74 of cardiovascular functions. In rats rendered vitamin DÐ DBP (mm Hg) deficient for 9 weeks, systolic blood pressure as well 72 as cardiac and vascular muscle contractile response 70 are increased [50]. The ratio of heart weight to body <=60 61–70 71–80 >80 weight is also significantly increased in the deficient animals [51]. Simpson and colleagues suggested that Serum 1,25(OH) D (pmol/L) 2 3 the rise in blood pressure and the change in vascular FIGURE 3 Inverse relationship between circulating 1,25(OH)2D3 muscle contractile function may be due to hypocal- levels and blood pressure in humans. The graphs show blood cemia induced by vitamin D-deficiency and thus repre- pressure levels at different quartiles of serum 1,25(OH)2D3 con- centrations. SBP, systolic blood pressure; DBP, diastolic blood sent an indirect response to vitamin D, whereas the pressure. (Adapted from Kristal-Boneh et al. 1997, Fig. 2, p. 1291, cardiac hypertrophy and cardiac contractile function with permission.) may be directly affected by vitamin D, as they cannot be CHAPTER 54 Vitamin D and the ReninÐAngiotensin System 875 prevented by preventing the hypocalcemia with a high- this hypothesis. By inference from the hypothesis, calcium diet in vitamin D-deficient rats [50,52,53]. VDR(−/−) mice are expected to display elevated renin Further studies demonstrate a significant increase in expression. Indeed, we have demonstrated that both myocardial collagen in the extracellular space and in V1 renin mRNA and protein levels in the kidney are dras- myosin isozyme expression in the heart of vitamin DÐ tically increased in VDR(−/−) mice. The plasma level deficient rats, which might be the basis for abnormal of Ang II, which is a downstream product of renin, is cardiac contractility [51,54]. Interestingly, these vita- also markedly increased in the mutant mice, whereas min DÐdeficient rats also show a marked increase in the expression of angiotensinogen, the substrate of plasma renin activity in both hypocalcemic and normocal- renin, in the liver is the same as in wild-type mice cemic states [52]. Therefore, the activation of the RAS may (Fig. 5) [57]. Therefore, the increase in plasma Ang II also contribute to the aberrant blood pressure and cardio- production appears to be mainly due to an increase in vascular functions associated with vitamin D deficiency. renin activity. As a consequence of the aberrant RAS In primary cultures of neonatal rat cardiac myocytes, overstimulation, VDR(−/−) mice develop high blood 1,25(OH)2D3 has been shown to inhibit ventricular pressure, cardiac hypertrophy, and an overdrinking myocyte proliferation [55], and antagonize endothelin- behavior, mostly due to the potent vasoconstricting and induced myocyte hypertrophy [56]. These studies thirst-inducing effect of Ang II [6,60]. As expected, demonstrate that genes associated with myocyte pro- plasma and urinary aldosterone levels are also liferation and hypertrophy, including c-myc, PCNA, markedly increased in VDR(−/−) mice [59]. Urinary and ANP, are suppressed by 1,25(OH)2D3 treatment, volume and urinary salt excretion are also higher, suggesting a direct effect of this secosteroid hormone whereas plasma sodium and potassium concentrations on the cardiac myocytes. However, whether the mech- remain normal in the mutant mice. Cardiac hypertrophy, anism of direct regulation of cardiac myocytes by likely induced by elevated Ang II production, is reflected 1,25(OH)2D3 can be applied in intact animals remains by a higher heart-weight-to-body-weight ratio and to be established. increased cardiac myocyte size in the left ventricle revealed by histological analyses. Accompanying the cardiac hypertrophy, both cardiac ANP mRNA expres- V. 1,25-DIHYDROXYVITAMIN D3 AS A sion and plasma ANP concentration are increased in NEGATIVE ENDOCRINE REGULATOR VDR(−/−) mice, as a compensatory response of the OF THE RENINÐANGIOTENSIN SYSTEM body. In fact, an increase in ANP expression is the most common feature of cardiac hypertrophy. A. A Hypothesis VDR inactivation alters many physiological param- eters; thus it is important to directly correlate the car- Based mostly on the epidemiological and clinical diovascular phenotype with the changes in the RAS. In evidence regarding a possible connection between the this regard, we have shown that the high blood pres- vitamin D endocrine system and blood pressure, partic- sure, cardiac hypertrophy, and increased water intake ularly the inverse relationship between the circulating seen in VDR(−/−) mice can be corrected by treatment 1,25(OH)2D3 level and plasma renin activity (Fig. 4) [43], with captopril, an ACE inhibitor, or losartan, an Ang II we speculate that vitamin D may control blood pressure AT 1 receptor antagonist. These results confirm that through regulating the RAS. Specifically, we hypothe- overstimulation of the RAS is mostly responsible for size that 1,25(OH)2D3 may function as a negative these abnormalities. endocrine regulator of renin gene expression in vivo [57]. Because of the critical role of the vitamin D Thus, if the hypothesis is correct, disruption of the endocrine system in the regulation of calcium home- vitamin D signaling pathway should lead to deregulated ostasis, inactivation of VDR leads to development of elevation of renin expression and consequent stimulation hypocalcemia and secondary hyperparathyroidism of the RAS, whereas increasing the circulating level [61], which may influence renin production and secre- of 1,25(OH)2D3 should lead to a suppression of renin tion. To address the contribution of serum calcium or production. This hypothesis is strongly supported by parathyroid hormone (PTH) to renin up-regulation a number of in vivo and in vitro studies [57Ð59]. seen in VDR(−/−) mice, we have examined renin expression in 20-day-old VDR(−/−) mice, adult VDR(−/−) mice whose blood calcium levels are nor- B. Animal Studies malized by a high calciumÐhigh lactose diet treatment [62], and Gcm2(−/−) mice that lack the parathyroid VDR-null mutant mice, which lack VDR-mediated glands [63]. The 20-day-old VDR(−/−) mice are still vitamin D signaling, are an ideal animal model to test normocalcemic, as intestinal calcium absorption is 876 YAN CHUN LI

A B +/+−/− 6 5 Renin 4 3 36B4 2 Renin mRNA 1 0 +/+−/− C

+/+ −/−

DE +/+ −/− 600 500 Angiotensinogen 400 300 36B4 200 Ang II (pg/ml) 100 0 +/+ −/− FIGURE 5 VDR knockout mice display elevated renin expression and plasma Ang II production. (A) Renin mRNA expression in the kidney. (B) Quantitative results of the Northern blot analyses shown in A. *, P < 0.001 vs +/+ mice. (C) Immunohistochemical staining of the kidney cortex from wild-type and VDR knockout mice with anti-renin antiserum. Arrows indicate the afferent glomerular arterioles in the juxtaglomerular region. (D) Plasma Ang II concentrations in wild- type and VDR knockout mice. *, P < 0.001 vs +/+ mice. (E) Liver angiotensinogen mRNA expression in wild-type and VDR knockout mice. +/+, wild-type mice; −/−, VDR knockout mice; Ang II, angiotensin II. (From Li et al. 2002, with permission.) independent of vitamin D before weaning. Renin or alopecia. This conclusion is consistent with previ- up-regulation is already evident in these young mice, ous observations in humans that the inverse relation- apparently before hypocalcemia develops. Furthermore, ship between serum 1,25(OH)2D3 levels and plasma in the normocalcemic adult VDR(−/−) mice, renin renin activity or blood pressure appears independent of mRNA level remains elevated, as is the plasma Ang II the serum calcium level [40,43]. However, the contri- level. On the other hand, renin expression is normal in bution of PTH to the renin up-regulation in VDR(−/−) the Gcm2(−/−) mice, even though these mutant mice are is less certain yet, because serum PTH starts to rise as hypocalcemic as VDR(−/−) mice [63]. In addition, early in life before hypocalcemia develops and cannot renin expression remains up-regulated in VDR(−/−) be completely normalized by the dietary intervention, mice whose alopecia is rescued by targeted expression due to the lack of the VDR-mediated vitamin D inhibi- of human VDR in the skin [64]. These data strongly tion of PTH biosynthesis [65]. suggest that regulation of renin expression by The hypothesis has been further tested in wild-type 1,25(OH)2D3 is independent of calcium metabolism mice and in mice lacking 25-hydroxyvitamin D3 CHAPTER 54 Vitamin D and the ReninÐAngiotensin System 877

1α-hydroxylase (1αOHase). In wild-type mice rendered can then be derived from the in vitro studies. In As4.1 vitamin DÐdeficient by dietary strontium treatment, cells transiently or stably transfected with human VDR which inhibits 1,25(OH)2D3 biosynthesis [66], or in cDNA, treatment with 1,25(OH)2D3 drastically 1αOHase(−/−) mice, renin expression in the kidney is reduces renin mRNA expression in 24 hr in a dose- markedly up-regulated, as seen in VDR(−/−) mice. On dependent manner [57]. To elucidate the molecular the other hand, in wild-type mice that have received mechanism whereby 1,25(OH)2D3 suppresses renin several doses of 1,25(OH)2D3 injection, renin expres- gene expression, stable hVDR-As4.1 cells are used to sion is significantly suppressed. Thus, the inhibitory analyze the renin gene promoter by luciferase reporter role of 1,25(OH)2D3 in renin biosynthesis is confirmed assays. When the cells are transfected with a luciferase in other mouse models. reporter plasmid containing the 4.1kb 5′-flanking DNA c sequence of the murine Ren-1 gene, 1,25(OH)2D3 treatment markedly reduces the promoter activity, con- C. Vitamin D Suppression vs Other firming that 1,25(OH)2D3 directly and negatively regu- Regulatory Mechanisms in Renin Regulation lates renin gene transcription by a VDR-mediated mechanism. Through deletion analysis two critical As renin biosynthesis is regulated by multiple phys- DNA fragments in the Ren-1c gene promoter have iological factors and pathways, it is important to deter- been identified. One fragment contains the base pairs mine whether 1,25(OH)2D3 regulates renin expression from Ð2720 to Ð2642, and the other from Ð117 to +1. by altering other regulatory mechanisms. To this end, The later is the minimal promoter of the Ren-1c gene. we have examined the response of VDR(−/−) mice to These DNA fragments are sufficient to mediate the high-salt diet and water deprivation [57]. As in wild- 1,25(OH)2D3 repression of the promoter activity and type mice, renin expression in VDR(−/−) mice is are the focus of current investigations aiming at com- markedly suppressed by a diet containing 8% sodium pletely elucidating the molecular mechanism under- chloride, and is dramatically stimulated by 24-hr dehy- lying the transcriptional repression of renin gene dration. The same is true for plasma Ang II production. expression by 1,25(OH)2D3. Thus, the mechanisms to sense changes in salt intake and extracellular volume are still functionally intact in VDR(−/−) mice. Moreover, treatment with captopril or E. Physiological Implications losartan, which blocks Ang II signaling, leads to a drastic up-regulation of renin expression in wild-type The finding that 1,25(OH)2D3 regulates the RAS is as well as in VDR(−/−) mice [59], suggesting that the consistent with the view that the vitamin D endocrine regulatory loop of Ang II feedback inhibition of renin system plays multiple physiological roles. Figure 6 out- production is also functionally intact in VDR(−/−) lines the interaction between the vitamin D endocrine mice. In all the above cases VDR(−/−) mice always system and the RAS in the regulation of calcium, maintain a significantly higher renin expression than volume, and blood pressure homeostasis. 1,25(OH)2D3 wild-type mice in any circumstance. These observa- and PTH are known to be the principal regulators tions indicate that, despite a high basal renin synthesis, for maintaining the blood calcium concentration. the basic regulatory mechanisms that control renin 1,25(OH)2D3 also functions as a negative regulator of production, including the Ang II feedback inhibition renin production and thus of the RAS, which helps and the salt- and volume-sensing mechanisms, are nor- explain the inverse relationship between the circulating mal in VDR(−/−) mice. Therefore, the sustained renin 1,25(OH)2D3 level and blood pressure reported previ- up-regulation is not mediated by these mechanisms. In ously. As some low 1,25(OH)2D3/high renin subjects another words, vitamin D regulation of renin synthesis are able to maintain normal serum calcium levels, the is an independent mechanism. threshold of the circulating 1,25(OH)2D3 level to induce hypocalcemia and hyperreninemia might be different. Under what physiological conditions 1,25(OH)2D3 D. Mechanism of Vitamin D Suppression of exerts its inhibitory action on the renin gene remains a Renin Gene Expression matter of speculation. 1,25(OH)2D3 may be one of the general “gate-keepers” to maintain an appropriate We have also tested the hypothesis using cell level of renin in the body, and/or may act as a counter- cultures. As4.1 cells maintain a high level of endoge- balance regulator to antagonize other renin-stimulating nous renin synthesis [23] and thus are very suitable for factors and prevent the detrimental overstimulation studying the effect of 1,25(OH)2D3 on renin gene of the RAS. Generally speaking, long-term vitamin D expression. Mechanistic knowledge of the regulation deficiency may increase the risk of high blood pressure 878 YAN CHUN LI

UV light

Renal perfusion pressure ++ Tubular sodium chloride load Ca PTH 1,25(OH) D Renin 2 3 Sympathetic nerve activity

Ca++ Cardiovascular Angiotensin II functions

Blood Extracellular pressure volume

FIGURE 6 Interaction between the vitamin D endocrine system and the reninÐ angiotensin system. 1,25(OH)2D3 feedback regulates the production of parathyroid hor- mone and suppresses renin biosynthesis. Renin is also feedback-suppressed by Ang II. Ultraviolet light influences blood pressure via 1,25(OH)2D3. 1,25(OH)2D3, PTH, and calcium may also directly affect the cardiovascular functions (dashed lines), as suggested by other studies. PTH, parathyroid hormone. (Adapted from Li 2003, with permission.)

and hypertension, whereas vitamin D supplement may with equal or better potency but less calcemic effects be beneficial to the cardiovascular system. than 1,25(OH)2D3 are good candidates. To search for such candidates, we have set to screen vitamin D analog compounds using the stable hVDR- VI. VITAMIN D ANALOGS AS POTENTIAL As4.1 cells by Northern blot and renin promoter ANTIHYPERTENSIVE AGENTS luciferase reporter assays. Interestingly, of the nine compounds we initially screened, only the two Gemini As a major pathogenic contributor to hypertension, the compounds, which have double side chains at the RAS has been an important drug target for therapeutic carbon 20 position (see Chapter 85) [75], display intervention of hypertension, with ACE inhibitors and renin-suppressing activity equal to or better than that Ang II receptor antagonists being among the most of 1,25(OH)2D3, whereas all other vitamin D analogs popular anti-hypertensive drugs [67]. As high-renin had little or much less inhibitory activity. Continued hypertension accounts for 10Ð20% of the patient pop- screening of 11 more Gemini compounds identified six ulation with essential hypertension, specific inhibitors more candidates. The reason why the double-side-chain for renin production are of significant therapeutic values. Gemini compounds possess more potent activity in Such inhibitors, in theory, can be used alone or in renin inhibition than other vitamin D analogs or even combination with ACE inhibitors or Ang II receptor 1,25(OH)2D3 itself remains to be explored. The in vivo antagonists. Patients with high-renin hypertension efficacy of these Gemini compounds is being tested in generally have higher blood pressure [68] and tend to animals. Preliminary data show that some analogs can have a more active sympathetic nervous system [69]; indeed significantly inhibit renin mRNA expression in thus renin inhibitors may also be used with sympa- mouse kidneys. Whether the Gemini compounds can tholytic agents such as the β-blockers. Great efforts reduce blood pressure needs to be further tested in have been made in the past to develop angiotensin sub- high-renin hypertensive animal models before clinical strate analogs as renin inhibitors [70Ð72]. Unfortunately, trials are considered. these peptide renin inhibitors are toxic, and thus are of little use for administration to humans. The finding that 1,25(OH)2D3 suppresses renin VII. CONCLUSION biosynthesis has raised the possibility to develop vita- min D analogs into renin inhibitors for therapeutic pur- The discovery that 1,25(OH)2D3 functions as a poses. With a large number of low calcemic vitamin D negative endocrine regulator of the RAS reveals a novel analogs synthesized, and some of them already physiological function of the vitamin D endocrine approved for clinical applications [73,74], such a pos- system. This finding has great physiological and phar- sibility is not unrealistic. In theory, vitamin D analogs macological implications for the vitamin D hormone CHAPTER 54 Vitamin D and the ReninÐAngiotensin System 879 and its analogs. Given the importance of this subject, 13. Carey RM, McGrath HE, Pentz ES, Gomez RA, Barrett PQ the role of vitamin D in the renocardiovascular system 1997 Biomechanical coupling in renin-releasing cells. J Clin Invest 100:1566Ð1574. is worth further exploration in future studies. It is also 14. Holmer SR, Kaissling B, Putnik K, Pfeifer M, Kramer BK, important to elucidate the molecular mechanism under- Riegger GA, Kurtz A 1997 Beta-adrenergic stimulation of lying vitamin D suppression of renin gene expression. renin expression in vivo. J Hypertens 15:1471Ð1479. Finally, low-calcemic vitamin D analogs may open a 15. Wagner C, Hinder M, Kramer BK, Kurtz A 1999 Role of renal new era for the long-sought therapeutic renin inhibitors nerves in the stimulation of the renin system by reduced renal arterial pressure. Hypertension 34:1101Ð1105. and potentially offer a new class of antihypertensive 16. Skott O, Briggs JP 1987 Direct demonstration of macula drugs. For diseases such as chronic renal failure and densaÐmediated renin secretion. Science 237:1618Ð1620. end-stage renal disease, which are commonly associ- 17. Abel KJ, Gross KW 1990 Physical characterization of genetic ated with cardiovascular problems and secondary rearrangements at the mouse renin loci. Genetics 124:937Ð947. hyperparathyroidism, vitamin D analogs with activities 18. Sigmund CD, Gross KW 1991 Structure, expression, and regulation of the murine renin genes. Hypertension 18: to suppress renin and PTH production might offer mul- 446Ð457. tiple clinical benefits [74,76,77]. 19. Pentz ES, Lopez ML, Kim HS, Carretero O, Smithies O, Gomez RA 2001 Ren1d and Ren2 cooperate to preserve home- ostasis: evidence from mice expressing GFP in place of Ren1d. Physiol Genomics 6:45Ð55. References 20. Ying L, Morris BJ, Sigmund CD 1997 Transactivation of the human renin promoter by the cyclic AMP/protein kinase A path- 1. Hajjar I, Kotchen TA 2003 Trends in prevalence, awareness, way is mediated by both cAMP-responsive element binding treatment, and control of hypertension in the United States, protein-1 (CREB)-dependent and CREB-independent mecha- 1988Ð2000. JAMA 290:199Ð206. nisms in Calu-6 cells. J Biol Chem 272:2412Ð2420. 2. Moffett DF, Moffett SB, Schauf CL 1993 Human Physiology. 21. Jones CA, Sigmund CD, McGowan RA, Kane-Haas CM, Foundation and Frontiers, 2nd ed. Wm. C. Brown, Dubuque, IA. Gross KW 1990 Expression of murine renin genes during fetal 3. Lavoie JL, Sigmund CD 2003 Minireview: overview of the development. Mol Endocrinol 4:375Ð383. renin–angiotensin system—an endocrine and paracrine system. 22. Jones CA, Hurley MI, Black TA, Kane CM, Pan L, Pruitt SC, Endocrinology 144:2179Ð2183. Gross KW 2000 Expression of a renin/GFP transgene in 4. Hackenthal E, Paul M, Ganten D, Taugner R 1990 Morphology, mouse embryonic, extra-embryonic, and adult tissues. Physiol physiology, and molecular biology of renin secretion. Physiol Genomics 4:75Ð81. Rev 70:1067Ð1116. 23. Sigmund CD, Okuyama K, Ingelfinger J, Jones CA, Mullins JJ, 5. Berry C, Touyz R, Dominiczak AF, Webb RC, Johns DG 2001 Kane C, Kim U, Wu CZ, Kenny L, Rustum Y, et al. 1990 Angiotensin receptors: Signaling, vascular pathophysiology, and Isolation and characterization of renin-expressing cell lines from interactions with ceramide. Am J Physiol Heart Circ Physiol transgenic mice containing a renin-promoter viral oncogene 281:H2337ÐH2365. fusion construct. J Biol Chem 265:19916Ð19922. 6. Ballermann BJ, Zeidel ML, Gunning ME, Brenner BM 1991 24. Petrovic N, Black TA, Fabian JR, Kane C, Jones CA, Loudon Vasoactive peptides and the kidney. In: Brenner BM, Rector JA, Abonia JP, Sigmund CD, Gross KW 1996 Role of proxi- FC (eds) The Kidney, (4th ed). W.B. Saunders, Philadelphia, mal promoter elements in regulation of renin gene transcrip- pp. 510Ð583. tion. J Biol Chem 271:22499Ð22505. 7. Inagami T 1994 The reninÐangiotensin system. Essays 25. Tamura K, Chen YE, Horiuchi M, Chen Q, Daviet L, Yang Z, Biochem 28:147Ð164. Lopez-Ilasaca M, Mu H, Pratt RE, Dzau VJ 2000 LXRalpha 8. Nguyen G, Delarue F, Burckle C, Bouzhir L, Giller T, Sraer JD functions as a cAMP-responsive transcriptional regulator of 2002 Pivotal role of the renin/prorenin receptor in angiotensin gene expression. Proc Natl Acad Sci USA 97:8513Ð8518. II production and cellular responses to renin. J Clin Invest 26. Shi Q, Gross KW, Sigmund CD 2001 Retinoic acid-mediated 109:1417Ð1427. activation of the mouse renin enhancer. J Biol Chem 9. Donoghue M, Hsieh F, Baronas E, Godbout K, Gosselin M, 276:3597Ð3603. Stagliano N, Donovan M, Woolf B, Robison K, Jeyaseelan R, 27. Pan L, Black TA, Shi Q, Jones CA, Petrovic N, Loudon J, Breitbart RE, Acton S 2000 A novel angiotensin-converting Kane C, Sigmund CD, Gross KW 2001 Critical roles of enzyme-related carboxypeptidase (ACE2) converts a cyclic AMP responsive element and an E-box in regulation of angiotensin I to angiotensin 1-9. Circ Res 87:E1ÐE9. mouse renin gene expression. J Biol Chem 276:45530Ð45538. 10. Tipnis SR, Hooper NM, Hyde R, Karran E, Christie G, Turner AJ 28. Pan L, Xie Y, Black TA, Jones CA, Pruitt SC, Gross KW 2001 2000 A human homolog of angiotensin-converting enzyme. An Abd-B class HOX.PBX recognition sequence is required Cloning and functional expression as a captopril-insensitive for expression from the mouse Ren-1c gene. J Biol Chem carboxypeptidase. J Biol Chem 275:33238Ð33243. 276:32489Ð32494. 11. Crackower MA, Sarao R, Oudit GY, Yagil C, Kozieradzki I, 29. Pan L, Glenn ST, Jones CA, Gronostajski RM, Gross KW Scanga SE, Oliveira-dos-Santos AJ, da Costa J, Zhang L, 2003 Regulation of renin enhancer activity by nuclear factor I Pei Y, Scholey J, Ferrario CM, Manoukian AS, Chappell MC, and Sp1/Sp3. Biochim Biophys Acta 1625:280Ð290. Backx PH, Yagil Y, Penninger JM 2002 Angiotensin-converting 30. Shi Q, Gross KW, Sigmund CD 2001 NF-Y antagonizes renin enzyme 2 is an essential regulator of heart function. Nature enhancer function by blocking stimulatory transcription fac- 417:822Ð828. tors. Hypertension 38:332Ð336. 12. Bader M, Ganten D 2000 Regulation of renin: New evidence 31. Liu X, Huang X, Sigmund CD 2003 Identification of a nuclear from cultured cells and genetically modified mice. J Mol Med orphan receptor (Ear2) as a negative regulator of renin gene 78:130Ð139. transcription. Circ Res 92:1033Ð1040. 880 YAN CHUN LI

32. Rostand SG 1997 Ultraviolet light may contribute to geo- 51. Weishaar RE, Kim SN, Saunders DE, Simpson RU 1990 graphic and racial blood pressure differences. Hypertension Involvement of vitamin D3 with cardiovascular function. III. 30:150Ð156. Effects on physical and morphological properties. Am J Physiol 33. He J, Klag MJ, Wu Z, Whelton PK 1995 Stroke in the People’s 258:E134ÐE142. Republic of China. I. Geographic variations in incidence and 52. Weishaar RE, Simpson RU 1989 The involvement of the risk factors. Stroke 26:2222Ð2227. endocrine system in regulating cardiovascular function: 34. Kunes J, Tremblay J, Bellavance F, Hamet P 1991 Influence of emphasis on vitamin D3. Endocr Rev 10:351Ð365. environmental temperature on the blood pressure of hyperten- 53. Weishaar RE, Simpson RU 1987 Involvement of vitamin D3 sive patients in Montreal. Am J Hypertens 4:422Ð426. with cardiovascular function. II. Direct and indirect effects. 35. Woodhouse PR, Khaw KT, Plummer M 1993 Seasonal varia- Am J Physiol 253:E675ÐE683. tion of blood pressure and its relationship to ambient tempera- 54. O’Connell TD, Weishaar RE, Simpson RU 1994 Regulation of ture in an elderly population. J Hypertens 11:1267Ð1274. myosin isozyme expression by vitamin D3 deficiency and 36. Holick MF 1987 Photosynthesis of vitamin D in the skin: 1,25-dihydroxyvitamin D3 in the rat heart. Endocrinology Effect of environmental and life-style variables. Fed Proc 134:899Ð905. 46:1876Ð1882. 55. O’Connell TD, Berry JE, Jarvis AK, Somerman MJ, Simpson RU 37. Harburg E, Gleibermann L, Roeper P, Schork MA, Schull WJ 1997 1,25-Dihydroxyvitamin D3 regulation of cardiac myocyte 1978 Skin color, ethnicity, and blood pressure I: Detroit blacks. proliferation and hypertrophy. Am J Physiol 272:H1751ÐH1758. Am J Public Health 68:1177Ð1183. 56. Wu J, Garami M, Cheng T, Gardner DG 1996 1,25(OH)2 vita- 38. Klag MJ, Whelton PK, Coresh J, Grim CE, Kuller LH 1991 min D3, and retinoic acid antagonize endothelin-stimulated The association of skin color with blood pressure in US blacks hypertrophy of neonatal rat cardiac myocytes. J Clin Invest with low socioeconomic status. JAMA 265:599Ð602. 97:1577Ð1588. 39. Krause R, Buhring M, Hopfenmuller W, Holick MF, 57. Li YC, Kong J, Wei M, Chen ZF, Liu SQ, Cao LP 2002 1,25- Sharma AM 1998 Ultraviolet B and blood pressure. Lancet Dihydroxyvitamin D3 is a negative endocrine regulator of the 352:709Ð710. renin-angiotensin system. J Clin Invest 110:229Ð238. 40. Kristal-Boneh E, Froom P, Harari G, Ribak J 1997 Association 58. Li YC 2003 Vitamin D regulation of the renin-angiotensin of calcitriol and blood pressure in normotensive men. system. J Cell Biochem 88:327Ð331. Hypertension 30:1289Ð1294. 59. Kong J, Li YC 2003 Effect of angiotensin II type I receptor 41. Lind L, Hanni A, Lithell H, Hvarfner A, Sorensen OH, antagonist and angiotensin-converting enzyme inhibitor on Ljunghall S 1995 Vitamin D is related to blood pressure and vitamin D receptor null mice. Am J Physiol Regul Integr Comp other cardiovascular risk factors in middle-aged men. Am J Physiol 285:R255ÐR261. Hypertens 8:894Ð901. 60. Fitzsimons JT 1980 Angiotensin stimulation of the central 42. Burgess ED, Hawkins RG, Watanabe M 1990 Interaction of nervous system. Rev Physiol Biochem Pharmacol 87:117Ð167. 1,25-dihydroxyvitamin D and plasma renin activity in high 61. Li YC, Pirro AE, Amling M, Delling G, Baron R, Bronson R, renin essential hypertension. Am J Hypertens 3:903Ð905. Demay MB 1997 Targeted ablation of the vitamin D receptor: 43. Resnick LM, Muller FB, Laragh JH 1986 Calcium-regulating an animal model of vitamin DÐdependent rickets type II with hormones in essential hypertension. Relation to plasma alopecia. Proc Natl Acad Sci USA 94:9831Ð9835. renin activity and sodium metabolism. Ann Intern Med 62. Li YC, Amling M, Pirro AE, Priemel M, Meuse J, Baron R, 105:649Ð654. Delling G, Demay MB 1998 Normalization of mineral ion 44. Lind L, Wengle B, Wide L, Ljunghall S 1989 Reduction of homeostasis by dietary means prevents hyperparathyroidism, blood pressure during long-term treatment with active vitamin D rickets, and osteomalacia, but not alopecia in vitamin D receptor- (alphacalcidol) is dependent on plasma renin activity and calcium ablated mice. Endocrinology 139:4391Ð4396. status. A double-blind, placebo-controlled study. Am J Hypertens 63. Gunther T, Chen ZF, Kim J, Priemel M, Rueger JM, Amling M, 2:20Ð25. Moseley JM, Martin TJ, Anderson DJ, Karsenty G 2000 45. Pfeifer M, Begerow B, Minne HW, Nachtigall D, Hansen C Genetic ablation of parathyroid glands reveals another source 2001 Effects of a short-term vitamin D(3) and calcium supple- of parathyroid hormone. Nature 406:199Ð203. mentation on blood pressure and parathyroid hormone levels in 64. Kong J, Li XJ, Gavin D, Jiang Y, Li YC 2002 Targeted expression elderly women. J Clin Endocrinol Metab 86:1633Ð1637. of human vitamin D receptor in the skin promotes the 46. Kimura Y, Kawamura M, Owada M, Oshima T, Murooka M, initiation of postnatal hair follicular cycle and rescue the alopecia Fujiwara T, Hiramori K 1999 Effectiveness of 1,25-dihydroxy- in vitamin D receptor null mice. J Invest Dermatol 118:631Ð638. vitamin D supplementation on blood pressure reduction in a 65. Silver J, Naveh-many T, Mayer H, Schmeizer HJ, Popovtzer MM pseudohypoparathyroidism patient with high renin activity. 1986 Regulation by vitamin D metabolites of parathyroid Intern Med 38:31Ð35. hormone gene transcription in vivo in the rat. J Clin Invest 47. Park CW, Oh YS, Shin YS, Kim CM, Kim YS, Kim SY, Choi EJ, 78:1296Ð1301. Chang YS, Bang BK 1999 Intravenous calcitriol regresses 66. Omdahl JL, DeLuca HF 1971 Strontium induced rickets: myocardial hypertrophy in hemodialysis patients with secondary Metabolic basis. Science 174:949Ð951. hyperparathyroidism. Am J Kidney Dis 33:73Ð81. 67. August P 2003 Initial treatment of hypertension. N Engl J Med 48. Simpson RU, Thomas GA, Arnold AJ 1985 Identification of 348:610Ð617. 1,25-dihydroxyvitamin D3 receptors and activities in muscle. 68. Brunner HR, Laragh JH, Baer L, Newton MA, Goodwin FT, J Biol Chem 260:8882Ð8891. Krakoff LR, Bard RH, Buhler FR 1972 Essential hypertension: 49. Fraga C, Blanco M, Vigo E, Segura C, Garcia-Caballero T, renin and aldosterone, heart attack and stroke. N Engl J Med Perez-Fernandez R 2002 Ontogenesis of the vitamin D receptor 286:441Ð449. in rat heart. Histochem Cell Biol 117:547Ð550. 69. Esler M, Julius S, Zweifler A, Randall O, Harburg E, Gardiner H, 50. Weishaar RE, Simpson RU 1987 Vitamin D3 and cardiovascular DeQuattro V 1997 Mild high-renin essential hypertension. function in rats. J Clin Invest 79:1706Ð1712. Neurogenic human hypertension? N Engl J Med 296:405Ð411. CHAPTER 54 Vitamin D and the ReninÐAngiotensin System 881

70. Kokubu T, Ueda E, Fujimoto S, Hiwada K, Kato A 1968 Peptide 75. Uskokovic MR, Manchand PS, Peleg S, Norman AW inhibitors of the renin-angiotensin system. Nature 217:456Ð457. 1997 Synthesis and preliminary evaluation of the biological 71. Poulsen K, Burton J, Haber E 1975 Purification of hog renin by properties of a 1α,25-dihydroxyvitamin D3 analog with two affinity chromatography using the synthetic competitive inhibitor side chains. In: Norman AW, Bouillon R, Thomasset M (eds) (D-Leu6)octapeptide. Biochim Biophys Acta 400:258Ð262. Vitamin D: Chemistry, Biology and Clinical Applications 72. Burton J, Cody RJ Jr, Herd JA, Haber E 1980 Specific inhibi- of the Steroid Hormone. University of California, Riverside, tion of renin by an angiotensinogen analog: studies in sodium pp. 19Ð21. depletion and renin-dependent hypertension. Proc Natl Acad 76. Park CW, Oh YS, Shin YS, Kim CM, Kim YS, Kim SY, Sci USA 77:5476Ð5479. Choi EJ, Chang YS, Bang BK 1999 Intravenous calcitriol 73. Brown AJ, Dusso AS, Slatopolsky E 2002 Vitamin D ana- regresses myocardial hypertrophy in hemodialysis patients logues for secondary hyperparathyroidism. Nephrol Dial with secondary hyperparathyroidism. Am J Kidney Dis Transplant 17(Suppl 10):10Ð19. 33:73Ð81. 74. Malluche HH, Mawad H, Koszewski NJ 2002 Update 77. Rostand SG, Drueke TB 1999 Parathyroid hormone, vitamin D, on vitamin D and its newer analogues: actions and rationale and cardiovascular disease in chronic renal failure. Kidney for treatment in chronic renal failure. Kidney Int 62:367Ð374. Int 56:383Ð392. CHAPTER 55 Vitamin D and Muscle

RICARDO L. BOLAND Departamento de Biología, Bioquímica y Farmacia, Universidad Nacional del Sur, (8000), Bahía Blanca, Argentina

I. Introduction VI. Effect of 1α,25(OH)2D3 on Muscle Cell Proliferation II. Vitamin DÐDependent Myopathies and Differentiation III. Muscle Vitamin D Receptor VII. Mechanism of Action of 1α,25(OH)2D3 in Muscle IV. 1α,25(OH)2D3 Regulation of Calcium Homeostasis VIII. Summary in Muscle Cells References V. Modulation of Muscle Cell Phosphate Uptake by Vitamin D3 Metabolites

I. INTRODUCTION the clinical issues of vitamin D action on muscle the reader is referred to Chapter 102 of this book. Vitamin D plays a major role in the regulation of vertebrate calcium and phosphorus metabolism by acting II. VITAMIN DÐDEPENDENT at the level of intestine, bone, and kidney [1,2]. As dis- MYOPATHIES cussed in detail in Chapter 2 of this book, vitamin D3 from either dietary sources or endogenous synthesis by A. Clinical Background ultraviolet irradiation of the skin is transformed into the most active hormonal form, 1α,25-dihydroxyvitamin D3 Early clinical observations suggested a relationship (1α,25(OH)2D3), by 25-hydroxylation in the liver fol- between vitamin D and muscle. These studies have lowed by 1α-hydroxylation of 25-hydroxyvitamin D3 shown that a myopathy characterized by muscle weak- (25(OH)D3) in the kidney. 1α,25(OH)2D3 elicits its ness, followed by atrophy, is a common symptom in effects through two different mechanisms. In addition to vitamin D deficiency states of various origins [10Ð13]. regulating gene transcription via its specific intracellular The main clinical feature of the myopathy associated receptor (VDR) like other steroid hormones [3,4], 1α,25- with osteomalacia consists of predominantly proximal (OH)2D3 induces rapid, nontranscriptional responses muscle weakness, which often gives rise to a waddling involving activation of transmembrane signal trans- gait. Muscle wasting without fasciculation or depres- duction pathways such as growth factors and peptide sion of the tendon reflexes may be observed. Plasma hormones [5,6]. muscle enzyme profiles are generally unaltered and In the past few years, 1α,25(OH)2D3 receptors have only slight nonspecific histopathological abnormalities been identified in a wide range of tissues and cell lines, may be detected [13,14]. Electromyographic evaluation implying the hormone in effects not directly related to of patients reveals a myopathic pattern as evidenced by mineral metabolism. Thus, 1α,25(OH)2D3, among vari- a significant reduction in motor unit potential duration ous nonclassical actions, may influence the proliferation and amplitude, and an increased percentage of polypha- and differentiation of various cell types, has immunoreg- sicity as compared to controls [11,12]. A similar myopa- ulatory properties and modulates insulin secretion and thy has been observed in patients with postgastrectomy prolactin synthesis [7,8]. vitamin D deficiency, those with idiopathic hypophos- Several lines of evidence have demonstrated that phatemic osteomalacia, and in gluten-sensitive enteropa- skeletal muscle is also a target tissue for vitamin D [9]. thy [13]. Muscle weakness responds to the treatment This chapter reviews and updates relevant experimental with vitamin D3 suggesting that the sterol plays an etio- research performed to characterize cellular processes logical role. Moreover, it has been shown that osteoma- and mechanisms involved in the effects of vitamin D on lacic myopathy and neuropathy are not interrelated [12]. muscle. These issues will be preceded by a description Patients with end-stage renal failure also develop proxi- of essential clinical aspects of vitamin DÐdependent mal muscle weakness. The myopathy is demonstrable by myopathies in order to provide a background that will quantitative electromyography and its histological char- allow to point out the clinical relevance of the basic acterization reveals selective atrophy of type II muscle scientific concepts discussed. For a detailed account of fibers. Electron microscopy may show in addition VITAMIN D, 2ND EDITION Copyright © 2005, Elsevier, Inc. FELDMAN, PIKE, AND GLORIEUX All rights reserved. 884 RICARDO L. BOLAND nonspecific degenerative changes [14]. The myopathy In addition, there is evidence which suggests that may be considerably improved by the administration impairment of the structure and functional properties of small amounts of 1α,25(OH)2D3 [15,16] and renal of the actomyosin contractile complex may also con- transplantation [14], whereas treatment with large tribute to muscle dysfunction [9]. doses of vitamin D3 causes only moderate improve- The contractionÐrelaxation cycle is regulated by the ment [14]. This suggests that impaired synthesis of concentration of free Ca2+ ions in the sarcoplasm 1α,25(OH)2D3 may play a role in the development of [22Ð24]. Several data obtained in vivo indicate that muscle weakness in renal diseases. In disagreement vitamin D acts on muscle calcium transport. Ca uptake with this interpretation, prior work reported a high inci- by the sarcoplasmic reticulum (SR) is diminished dence of a similar myopathy in patients with in vitamin DÐdepleted rabbits [25] and chicks [21]. primary hyperparathyroidism [17]. However, this 1α,25(OH)2D3 may be the active metabolite on SR observation could not be confirmed in two subsequent as nephrectomy or strontium feeding (inhibits 1α,25- investigations [10,11]. At that early stage of the (OH)2D3 synthesis in kidney) in rabbits and chicks, research it was proposed that hypophosphatemia was respectively, decreased Ca uptake. Dosage of animals the basic disorder underlying these myopathies as it is with 1α,25(OH)2D3 reversed these changes [26Ð29]. a common feature of primary hyperparathyroidism, 1α,25(OH)2D3 may affect calcium transport across osteomalacia, and nutritional vitamin D deficiency [18]. sarcoplasmic reticulum membranes by increasing the Contrary to this proposal it was noted that the myopathy number and kinetics of formation of active transport of hypophosphatemic osteomalacia may not resolve sites of the SR Ca-ATPase. Steady-state levels of phos- with increased phosphate intake, and vitamin D intake phoderivative (EP, enzyme phosphate) and phosphory- is required for recovery. Moreover, the fact that muscle lation velocity of the transport enzyme are decreased in weakness is absent in X-linked hypophosphatemic experimental uremia. Administration of 1α,25(OH)2D3 rickets has been taken as indication that mere restores both parameters to normal [28,29]. It has hypophosphatemia need not be the etiological factor been reported that vitamin D3 also affects Ca fluxes involved [13]. In addition, 1α,25(OH)2D3 levels are through mitochondrial membranes from skeletal muscle low [19], and it has been pointed out that intracellular in vivo [21]. In addition, an action of vitamin D3 on Ca phosphate level may remain normal or even higher in transport across muscle plasma cell membranes has spite of severe hypophosphatemia [18]. been shown. Administration of the sterol to vitamin DÐ deficient chicks markedly increased Ca uptake in sub- sequently isolated sarcolemma vesicles. This change B. Animal Model Studies was accompanied by an increase in Ca-ATPase activity that could in turn be related to increased Vmax and The myopathy associated with vitamin D deficiency decreased Km [30]. Sarcolemmal Ca transport probably has been experimentally characterized. First, in elec- reflects the Ca-ATPase activity of vesicles with inside- trophysiological studies using an in situ neuromuscu- out membranes as the physiological function of the lar preparation of the soleus, Rodman and Baker [20] pump is to extrude Ca2+ from the cells [30]. Desaturation found diminished peak tension and prolonged time for analysis of rachitic chick soleus muscle Ca pools prela- 45 recovery halfway to resting tension for single twitches beled with Ca suggested that 1α,25(OH)2D3 stimu- and prolonged relaxation half-life after tetanic contrac- lates Ca efflux from mitochondria and Ca transport tion in vitamin DÐdeficient rats. Impaired tension (influx and efflux) across plasma cell membranes [31]. development and slow relaxation after muscle contrac- The action of 1α,25(OH)2D3 on muscle Ca fluxes tion in response to repetitive electrical simulation were may contribute to extracellular calcium homeostasis in also observed in vitamin DÐdepleted chicks [21]. conditions of vitamin D deficiency. It has been Interestingly enough, both studies clearly showed that reported that vitamin D depletion of chicks leads to Ca these changes were not related to modifications in accumulation in muscle tissue. Treatment with a single blood Ca and P levels and could only be reversed to dose of 1α,25(OH)2D3 quickly reversed this change. normal by administration of vitamin D3 [20,21]. These The prompt increase in blood Ca that followed admin- data indicate that vitamin DÐdependent muscle weak- istration of the steroid correlated better with the fall in ness may be ascribed to a primary disorder of skeletal Ca content of muscle tissue than with stimulation of muscle function. intestinal Ca absorption [32]. Increased Ca efflux from Studies with animal models have provided information mitochondria and cytoplasm as revealed by the studies pointing to alterations in muscle calcium metabolism, and with soleus muscle may provide a mechanism for the to a lesser extent in phosphate metabolism, as etiological 1α,25(OH)2D3 action on Ca mobilization from skeletal factors responsible for vitamin DÐdependent myopathies. muscle of rachitic chicks. CHAPTER 55 Vitamin D and Muscle 885

An action of vitamin D on muscle phosphate fluxes totally excluded, preventing an accurate description of was first suggested by Birge and Haddad [33], who by the mechanisms involved and the identification of administration of vitamin D3 or 25(OH)D3 to vitamin DÐ active vitamin D3 metabolite(s). deficient, phosphate-depleted rats, detected first a fall However, more recent experimental studies using in serum P and an increase in 32P uptake by muscle, fol- skeletal muscle cell culture systems (myoblasts/ lowed by an increase in muscle ATP and protein syn- myotubes) have contributed substantially to identify thesis. Nephrectomy did not obliterate these responses, 1α,25(OH)2D3 as the main biologically active form of indicating that further conversion of the sterols to vitamin D3 acting in muscle, and to elucidate the 1α,25(OH)2D3 was not required. On the basis of this mechanisms by which this hormonal metabolite regu- evidence it was proposed that a reduction in muscle lates muscle cell Ca2+ and phosphate, proliferation, and intracellular phosphate may contribute to osteomalacic differentiation, providing thereby a new basis for the myopathy [18]. In agreement with these observations, abnormalities in skeletal muscle contractility and measurement of 32P specific activities in serum and growth that occur in states of vitamin D deficit. The skeletal muscle sarcoplasm and intracellular mem- following sections will deal with these aspects. branes after in vivo 32P-labeling of vitamin DÐdeficient and acutely vitamin D3Ðtreated chicks suggested that the sterol stimulates phosphate transport across muscle III. MUSCLE VITAMIN D RECEPTOR plasma membranes. Furthermore, prior treatment of vitamin DÐdepleted chicks with vitamin D3 resulted There is evidence for the presence in muscle cells of + in a stimulation of Na gradientÐmediated phosphate an intracellular receptor for 1α,25(OH)2D3, analogous transport in isolated sarcolemma vesicles [30]. to that found in classical tissues (VDR). Density gradient 3 Studies in vivo have implied vitamin D3 in the syn- analysis of [ H]1α,25(OH)2D3 binding by cytosol from thesis of skeletal muscle contractile proteins. A signif- primary-cultured chick myoblasts revealed that the icant reduction in actomyosin levels has been noted in hormone specifically binds to a 3.7 S macromolecule, rats fed a vitamin DÐdeficient diet [34]. Moreover, and saturation analysis of the binding showed that the treatment of vitamin DÐdepleted chicks with vitamin D3 receptor proteins binds 1α,25(OH)2D3 with high affinity −10 caused an increase in actin and troponin C levels [35]. (KD 2.46 × 10 M) and low capacity (74 fmol/mg pro- Pointon et al. [36] also observed a reduction in tro- tein) [38]. The presence of a specific 1α,25(OH)2D3 ponin C in rachitic rabbits. The changes observed may binding protein with similar characteristics in murine also contribute to impaired tension development in and human growing myoblasts and myotubes has been vitamin D deficiency. Ca binding to troponin C initi- reported [39,40]. Additional data on the expression of ates the mechanism of muscle contraction in vertebrate the VDR in avian muscle cells has been obtained by striated muscle [37]. detection of VDR-mRNA by RT-PCR, Western blot, and Vitamin D3 induces, in addition, changes in skeletal immunocytochemical analysis using highly specific muscle phospholipids, which may represent part of the antibodies [41,42]. Receptor levels are low in undiffer- mechanism involved in the effects of the sterol on the entiated myoblasts and significantly increase during the transport properties of muscle membranes. Thus, an differentiation process to myotubes [42]. increase in phosphatidylcholine content at the expense of Notably, it has been shown that treatment of chick a proportional decrease in phosphatidylethanolamine muscle cells with 1α,25(OH)2D3 promotes tyrosine levels is observed in sarcolemmal membranes after phosphorylation of the VDR, in agreement with the administration of vitamin D3 to vitamin DÐdepleted database for protein consensus motifs indicating the animals [30]. Vitamin D3 also increases in vivo the presence within the primary sequence of the chick total phospholipid content of sarcoplasmic reticulum VDR of a putative tyrosine phosphorylation site corre- and mitochondria without affecting the relative pro- sponding to amino acids 164Ð170 (KTFDTTY), a portions of phospholipid classes [35]. This may reflect region located near the C-terminal end of the receptor increased availability of sarcoplasmic phosphate due DNA binding domain [43]. Similar putative tyrosine to the action of vitamin D3 metabolites on sarcolemmal phosphorylation sites are also found in human, rat, and phosphate transport [30,33]. mouse VDR sequences (Boland, unpublished observa- The preceding information on vitamin D3 regulation tions). All steroid hormone receptors, including the of skeletal muscle ion fluxes, and protein and lipid VDR, are phosphorylated and undergo ligand-induced composition, obtained with animal models, does not hyperphosphorylation [44]. However, most of the necessarily imply a direct action of the sterol on muscle phosphorylated residues identified to date are serines tissue, since the contribution of systemic effects, e.g., in the N-terminal motif, and tyrosine phosphorylation changes in blood Ca, P, and PTH levels, cannot be has only been documented for the estrogen receptor [45]. 886 RICARDO L. BOLAND

Phosphorylation of serine residues in the human VDR be mimicked by the VDCC agonist BAY K8644, has been involved in 1α,25(OH)2D3-dependent tran- and plasma membrane depolarization induced by a scriptional activation [46,47]. Recent evidence sug- high K+ medium. The effects of the combined treat- + gests that phosphorylation of tyrosine residues in the ment with 1α,25(OH)2D3 and BAY K8644 or K depo- VDR may play a role in hormone modulation of mus- larization were not additive. Moreover, the action cle mitogenic tyrosine phosphorylation cascades of the hormone was dependent on extracellular Ca2+ as (Section VI) and Ca2+ influx through store operated they were reversibly inhibited by the Ca2+ chelator channels (Section VII,B). EGTA. Furthermore, on the basis of their sensitivity to nifedipine and verapamil, the voltage-dependent Ca2+ channels activated in skeletal muscle cells by IV. 1α,25(OH)2D3 REGULATION 1α,25(OH)2D3 were pharmacologically identified as OF CALCIUM HOMEOSTASIS from the L-type [49,50]. Various lines of evidence IN MUSCLE CELLS described in Section VII,B have demonstrated that the hormone rapidly regulates muscle cell Ca2+ influx by It is possible that alterations in the mechanisms by G proteinÐmediated activation of both phospholipase C which intracellular Ca2+ is regulated in the muscle cell and adenylyl cyclase, leading to the stimulation of PKC play a major role in the muscle weakness associated and PKA and activation of VDCC. PKA and PKC mod- with vitamin D deficiency and renal diseases as ulate the activity of VDCC by phosphorylation, thus suggested by the animal model studies described in increasing the probability of channel opening [51]. Section II,B. Cultured avian embryonic skeletal muscle 1α,25(OH)2D3 stimulates the phosphorylation of sev- cells have proven to be an adequate model to further eral proteins in muscle cells [50,52]. Identification of characterize 1α,25(OH)2D3 regulation of muscle intra- these proteins may help to further understand the reg- cellular Ca2+ homeostasis. Chick myoblasts differentiate ulatory action of the hormone on VDCC. Interestingly, in vitro into functionally competent myotubes that are both in myoblasts and soleus muscle it has been shown endowed with a VDR and the molecular machinery to that in response to 1α,25(OH)2D3, calmodulin rapidly respond to 1α,25(OH)2D3 through both the genomic translocates from the cytosol to the membrane where it and nongenomic mechanisms that mediate the actions binds a major hormone-dependent phosphoprotein of of the hormone (Sections III, VI, and VII). 28 kDa. There is evidence suggesting that this event 2+ Long-term effects of 1α,25(OH)2D3 on Ca trans- mediates the 1α,25(OH)2D3 fast increase in calcium port in chick skeletal muscle cells have been scarcely entry through the dihydropyridine-sensitive pathway studied. Thus, it has been only reported that physiolog- [50, and references therein]. 45 2+ ical levels of 1α,25(OH)2D3 increase myoblast Ca Spectrofluorometric studies with Fura-2-loaded uptake after 8Ð24 hr treatment of cultures [9], probably muscle cells have confirmed the foregoing observa- in line with the operation of a nuclear mechanism that tions and revealed additional key information related involves sterol-induced de novo synthesis in chick mus- to the regulation of muscle intracellular Ca2+ homeo- 2+ cle of calbindin-D9K (instead of calbindin-D28K as in stasis by 1α,25(OH)2D3. The cytosolic Ca response other avian tissues) and enzymes that convert phos- to the hormone involves an initial rapid sterol-induced 2+ phatidylethanolamine to phosphatidylcholine, according Ca mobilization from IP3/thapsigargin-sensitive stores to the information provided in Section VII,A. followed by cation influx from the extracellular milieu, The nongenomic regulation of myoblast/myotube accounting for a sustained Ca2+ phase that does not 2+ intracellular Ca by 1α,25(OH)2D3 has also been well return to baseline as long as the cells are exposed to the characterized. Various aspects of this mechanism have sterol. This Ca2+ influx was shown to be contributed not been reproduced using intact differentiated skeletal only by the well established L-type VDCC-mediated muscle in vitro. Indeed, studies with isolated chick Ca2+ entry but also by a store-operated Ca2+ (SOC; soleus muscle demonstrated for the first time that capacitative Ca2+ entry, CCE) channel, therefore intro- 45 2+ 1α,25(OH)2D3 exerts acute effects (1Ð15 min) on Ca ducing a novel aspect into the mechanism of 1α,25- 2+ uptake, which are not blocked by inhibitors of RNA (OH)2D3-induced Ca influx across the plasma and protein synthesis but are suppressed by blockers of membrane of muscle cells [53,54]. The effects of the voltage-dependent calcium channels (VDCC) [48]. hormone on the profile of changes in intracellular Ca2+ Interestingly, this rapid stimulation of skeletal muscle levels are clearly detectable within the 10−12 MÐ10−8 M calcium fluxes in response to 1α,25(OH)2D3 has also concentration range [55]. The SOC influx activated been observed in vivo [32]. In myoblasts the operation by 1α,25(OH)2D3 was identified by being insensitive of this mechanism has been firmly established by the to L-type Ca2+ channel antagonists but was fully 2+ fact that 1α,25(OH)2D3Ðdependent Ca influx could inhibitable by low micromolar concentrations of CHAPTER 55 Vitamin D and Muscle 887

3+ 2+ + La (3 µM) and Ni . PI(polyphosphoinositide)-specific evidence that 1α,25(OH)2D3 affects the Na -linked PLC blockade prior to 1α,25(OH)2D3 stimulation sup- component of myoblast phosphate uptake involving pressed both the cytosolic Ca2+ transient and SOC influx. de novo RNA and protein synthesis, suggesting that 2+ Accordingly, depletion of intracellular Ca stores by 1α,25(OH)2D3 acts on embryonic muscle phosphate 2+ thapsigargin reproduced 1α,25(OH)2D3-induced Ca uptake through a receptor-mediated mechanism. influx, inhibiting any further response to the hormone. Information is lacking on postuptake phosphate Furthermore, 1α,25(OH)2D3 increased the rate of metabolism and its possible regulation by vitamin D3 quenching of Fura-2 fluorescence by Mn2+, indicating metabolites in muscle cells. activation of Mn2+ influx that specifically permeates SOC channels [53,54]. α There is evidence on the involvement of protein VI. EFFECT OF 1 ,25(OH)2D3 kinases in the regulation of capacitative calcium influx ON MUSCLE CELL PROLIFERATION in muscle cells by 1α,25(OH)2D3. It has been shown AND DIFFERENTIATION that the hormone stimulation of CCE is prevented by inhibitors of PKC (calphostin C, bisindolylmaleimide) 1α,25(OH)2D3 regulates the proliferation and differ- and tyrosine kinase (genistein) but unaffected by entiation of several cell types, including myeloid blockade of the PKA pathway [54]. Of relevance, leukemia and hemopoietic cells [60,61], chondrocytes SOC influx stimulated by 1α,25(OH)2D3 is insensitive [62], and keratinocytes [63]. It was proposed that the to both calmodulin (CAM) antagonists and CAM- 1α,25(OH)2D3 receptor complex activates or represses dependent protein kinase II (CAMKII) inhibitors genes related to the cellular cycle [64], for example the 2+ when added after the IP3-mediated Ca transient but protooncogenes c-myc, c-fos, and c-jun [65,66]. In completely abolished when added before it. Moreover, addition, data were obtained indicating that transcrip- in cells microinjected with antisense oligonucleotides tional regulation of protein kinase C is secondarily directed against the CAM mRNA the sterol-stimulated involved in the control of c-myc expression by SOC influx is reduced up to 60% with respect to 1α,25(OH)2D3 [66]. uninjected cells [56]. These results suggest that the There is experimental evidence demonstrating that 2+ 1α,25(OH)2D3-induced (IP3-mediated) cytosolic Ca 1α,25(OH)2D3 also regulates muscle cell proliferation transient is required for CAM activation, which in turn and differentiation, in keeping with the clinical obser- mediates SOC influx in a mechanism that seems to vation of muscle atrophy in states of 1α,25(OH)2D3 include CAMKII. deficit (Section II). The action of the hormone on myogenesis has been characterized in chick myoblast cultures. Myoblasts are mononucleated cells that pro- V. MODULATION OF MUSCLE CELL liferate actively in culture followed by differentiation PHOSPHATE UPTAKE BY VITAMIN D3 into multinucleated myotubes expressing phenotypic METABOLITES characteristics of mature muscle fibers [67,68]. Specifically, the morphological profiles of myoblasts [32P]Phosphate uptake by cultured chick myoblasts is cultured for 1Ð6 days indicate that undifferentiated to a great extent Na+-dependent, saturable with respect muscle cells elongate, become aligned, and fuse to to phosphate, energy-dependent, and inhibited by form differentiated myotubes as the culture period ouabain and arsenate, in agreement with the operation progresses. Accordingly, in parallel to these changes of a Na+Ðphosphate cotransport system in the muscle the rate of DNA synthesis decreases 10-fold during the cell plasma membrane as described for intestine and 1- to 6-day interval and creatine kinase activity augments kidney [57]. Treatment of myoblast cultures with 10- to 11-fold. In addition, myosin levels markedly 1α,25(OH)2D3 for 4Ð24 hr causes a significant dose- increase from 2 to 6 days of culture during myogene- dependent (10−10Ð10−7 M) stimulation of phosphate sis [69]. Protein kinase C (PKC) is involved in myoge- accumulation by the cells, increasing the velocity of nesis, the isoform α playing a pivotal role. Thus, high phosphate uptake to a greater extent than the total levels of PKCα are detected in the proliferative stage capacity. 25(OH)D3 increases phosphate accumula- that markedly diminish as myoblasts differentiate. In tion by myoblasts roughly to the same extent as addition, down-regulation of PKCα with a phorbol ester 1α,25(OH)2D3, whereas 24,25(OH)2D3 and vitamin D3 inhibits DNA synthesis in dividing myoblasts [69]. are devoid of activity [58,59]. These results are in par- Furthermore, specific blockage of the expression of tial agreement with previous work of Birge [18] show- PKCα in myoblasts by using antisense oligodeoxy- ing 25(OH)D3 stimulation of phosphate influx by rat nucleotides (ODNs) results in a significant decrease epitrochlear muscle cultures. Our studies also provided of culture cell density and DNA synthesis, clearly 888 RICARDO L. BOLAND showing that this isoenzyme is involved in signaling interact with Src through the SH2 domain of the latter. pathways that promote muscle cell proliferation [70]. Preincubation of myoblasts with a pool of different Using as criteria the just-described changes in mor- antisense ODNs against the VDR mRNA (AS-VDR phology and specific biochemical markers of myogen- ODNs) significantly reduces Src stimulation, further esis, it has been clearly shown that physiological levels implying the VDR in hormone activation of Src [78]. of 1α,25(OH)2D3 stimulate muscle cell proliferation However, MAP kinase tyrosine phosphorylation by and differentiation [31,71Ð73]. In accord with the 1α,25(OH)2D3 is affected to a lesser extent by trans- involvement of PKC in the regulation of myogenesis, fection with AS-VDR ODNs implying that both VDR- increased activity is observed during 1α,25(OH)2D3 dependent and VDR-independent signaling mediate stimulation of myoblast proliferation, whereas inhibi- hormone stimulation of MAPK [78,79]. tion of PKC activity accompanied the effects of the In agreement with this interpretation, using intracel- hormone on myoblast differentiation; the specific PKC lular and extracellular Ca2+ mobilizing agents and chela- inhibitor calphostin suppressed hormone potentiation tors, as well as specific PKC activators and inhibitors, it of DNA synthesis in proliferating myoblasts [73,74]. has been shown that calcium and protein kinase C are Moreover, the early stimulation of myoblast prolifera- also involved in the stimulation of MAP kinase by tion mainly correlated to PKCα expression, whereas 1α,25(OH)2D3 [80]. Recent investigations [81] have decreased PKCα levels were observed during the sub- established the role of PKC and uncovered other sequent activation of myoblast differentiation [73]. metabolic steps that participate in hormone up-regulation Recent studies have revealed that activation of tyro- of the MAPK cascade. Thus, 1α,25(OH)2D3 causes a sine phosphorylation pathways plays an important role fast significant increase of Raf-1-serine phosphoryla- in the mechanism that mediates the effects of tion, indicating activation of Raf-1 by the hormone. 1α,25(OH)2D3 on muscle growth. Tyrosine phospho- The PKC inhibitors calphostin C, bisindolylmaleimide I, rylation is a crucial event in signal transduction linked and Ro 318220 blocked 1α,25(OH)2D3-induced Raf-1 to the mitogen-activated protein kinase (MAPK). serine phosphorylation, revealing that PKC partici- Stimulation of the MAPK cascade may occur through pates in hormone stimulation of MAPK at the level of activation of receptor tyrosine kinases or G protein- Raf-1. Moreover, application of antisense oligonu- coupled receptors by stimulation of nonreceptor Src cleotide technology revealed that PKCα specifically kinases or by direct signaling to Raf via PKC. Upon mediates this action. In addition, by using a specific phosphorylation by mitogens, MAPK is translocated Ras peptide inhibitor, Ras has also been involved in from the cytoplasm into the nucleus, which results in 1α,25(OH)2D3 activation of Raf-1. The hormone the activation or induction of transcription factors lead- rapidly induced tyrosine dephosphorylation of Ras- ing to the expression of genes involved in the control GTPase-activating protein, suggesting that inhibition of cellular growth [75]. of Ras-GTP hydrolysis is part of the mechanism by 1α,25(OH)2D3 rapidly (within 1 min) promotes which 1α,25(OH)2D3 activates Ras in myoblasts [81]. tyrosine phosphorylation of MAPK in cultured Stimulation of tyrosine phosphorylation cascades myoblasts. Other major tyrosine-phosphorylated by 1α,25(OH)2D3 through the just-described mecha- targets of the hormone in muscle cells are PLCγ, and nisms causes translocation of MAPK from the cyto- surprisingly, the c-myc oncoprotein, a novel finding plasm to the nucleus in an active phosphorylated form for which there is no information on the signaling and induces the expression of the growth-related pro- component(s) leading to phosphorylation of its tyro- tein c-myc, as the MAPK kinase (MEK) inhibitor sine residues [76]. Fast 1α,25(OH)2D3-dependent PD98059 abolishes stimulation of c-myc synthesis by increased Src kinase activity has been observed in 1α,25(OH)2D3 [77]. Early studies had already shown myoblasts [43]. Preincubation of muscle cells with that the mitogenic effects of the hormone in myoblasts specific Src inhibitors or their transfection with an are correlated to increased c-myc mRNA levels [71]. antisense ODN against Src mRNA inhibits There is also information available on the signaling 1α,25(OH)2D3 activation of MAPK, involving Src as cascade leading to PLCγ activation, a relevant event an upstream element that leads to hormone signaling that may account for the activation of PKCα (followed through this cascade [77]. Coimmunoprecipitation by that of Raf-1) via release of DAG and IP3-mediated 2+ analysis have provided evidence that 1α,25(OH)2D3 Ca . Investigations based on the utilization of specific promotes the formation of complexes between Src and inhibitors and antisense technology have involved the VDR, and between Src and c-myc, which can be Src and phosphatidylinositol 3-kinase (PtdIns3K) in explained by the fact that both the VDR and c-myc 1α,25(OH)2D3 stimulation of PLCγ tyrosine phospho- behave as 1α,25(OH)2D3-dependent tyrosine phospho- rylation and its translocation to the cell membrane. rylated proteins (see Section III for the VDR) and may Evidence has been obtained indicating that the CHAPTER 55 Vitamin D and Muscle 889

1α,25(OH) D 2 3 Plasma membrane

γ PLC y y Raf–1 Ras GTP GDP GAP S − PI–3K Ras α y PKC Shc c-Src Sos GTP GDP Grb2 S MEK y VDR T c-myc

Y MAPK T Nuclear membrane

Cellular proliferation c-myc expression

FIGURE 1 Tyrosine phosphorylation cascades involved in the mitogenic effects of 1α,25(OH)2D3 in skeletal muscle cells. PI-3K, phosphatidylinositol 3-kinase; PLCγ, phospholipase Cγ; Ras-GAP, Ras-GTPase-activating protein; MAPK, mitogen activated protein kinase; MEK, MAPK kinase.

hormone increases the physical association of Src and abolished by actinomycin D and cycloheximide, PtdIns3K with PLCγ and induces a Src-dependent suggesting that it is dependent on de novo RNA and tyrosine phosphorylation of the p85 regulatory subunit protein synthesis [83]. Temporally correlated to the 2+ of PtdIns3K [82]. 1α,25(OH)2D3-induced changes in Ca transport, an Altogether, 1α,25(OH)2D3 stimulates in muscle increase in phosphatidylcholine (PC) at the expense of cells an intrincate network of signaling components phosphatidylethanolamine (PE) levels is observed in and interacting pathways (Fig. 1), which provide a response to the hormone, which is mainly accounted mechanism underlying the regulation of muscle cellu- for by up-regulation of PE-N-methyltransferases lar growth by the hormone. [83,84]. The effects of 1α,25(OH)2D3 on phospholipid metabolism are also abolished by both actinomycin D VII. MECHANISMS OF ACTION and cycloheximide, reflecting the participation of a OF 1α,25(OH) D IN MUSCLE nuclear mechanism [85,86]. 2 3 In agreement with these observations, treatment of A. Genomic Mechanism myoblasts with 1α,25(OH)2D3 for 24 hr results in a marked increase in the incorporation of [3H]leucine Congruent with the presence of the VDR in into total cell proteins, which is abolished by the addition myoblasts and myotubes (Section III), various lines of of cycloheximide or actinomycin D [87]. SDS-PAGE 14 3 evidence have shown that 1α,25(OH)2D3 modulates the coelectrophoresis of [ C]leucine- and [ H]leucine- expression of genes related to the regulation of muscle labeled proteins from control and 1α,25(OH)2D3-treated calcium transport and phospholipid metabolism. The chick embryo skeletal muscle myoblasts, respectively, increase in cell 45Ca2+ uptake elicited by the incubation has shown that the hormone preferentially stimulates of cultured chick myoblasts/myotubes with the hor- the synthesis of seven proteins. These myoblast proteins mone for long treatment intervals (Section III) can be have been partially characterized on the basis of their 890 RICARDO L. BOLAND molecular weight (9Ð100 kDa), isoelectric points, Ca2+ Subcloning and sequencing of a partial 160-bp cDNA binding properties, carbohydrate content, and subcel- PCR product has shown that the cDNA corresponds to lular localization [87]. Of interest, four of the proteins calbindin D9K-cDNA [89,90], documenting for the 2+ induced by 1α,25(OH)2D3 bind Ca , one of them char- first time the expression of the calbindin-D9K gene in acterized as a cytosolic 9-kDa macromolecule with an an avian species. Calbindin D-28K was thought hereto- acidic isoelectric point. The 9-kDa myoblast calcium- fore to be the only calcium-binding protein expressed 125 binding protein (CaBP) comigrates with I-labeled rat in the chick in response to 1α,25(OH)2D3 [2,7]. calbindin-D9K in SDS-PAGE gels, and Western blot An unresolved issue is the identification of the analysis with a specific rat intestine calbinding-D9K remaining gene products regulated by 1α,25(OH)2D3 in antibody revealed the presence of an immunoreactive muscle cells, e.g., other Ca2+ binding proteins (17, 40, and protein of 9 kDa in chick embryo myoblasts treated with 100 kDa; [87]), enzymes of phospholipid metabolism. 1α,25(OH)2D3 as well as in chick skeletal muscle [88]. 1α,25(OH)2D3 induces a two- to fourfold increase in 9 kDa CaBP mRNA levels after 20Ð24 hr treatment, B. Nongenomic Signal Transduction coincident with maximum Ca uptake responses generated Pathways by the hormone. Northern hybridization analysis using a specific rat calbindin-D9K probe showed that the There is a wealth of information showing that increase was related to a single 550-nucleotide mRNA 1α,25(OH)2D3 also exerts nongenomic actions at the species, in accordance with the reported size for rat level of transmembrane second messenger systems, calbinding-D9K mRNA [88]. However, very low con- which mediate the fast effects of the hormone on mus- centrations of mRNA are usually detected, suggesting cle intracellular Ca2+ regulation (Fig. 2). that the 9-kDa myoblast protein is the stable product of It has been shown that 1α,25(OH)2D3 within the translation of a transient rare mRNA. Further molecu- physiological concentration range modifies the activity lar evidence on the expression of calbindin-D9K of myoblast phospholipases in a mode independent of mRNA in muscle cells as well as in other tissues from the nucleus. Within seconds to minutes the hormone the chick has been recently obtained by combined activates phospholipase C (PLC) generating the second 2+ reverse transcription and polymerase chain reaction. messengers inositol trisphosphate (IP3, a Ca mobilizer)

1,25(OH)2D3

mVDR? mVDR? mVDR? PIP2 PC AC PLC PLA2 PLD DAG Gi IP3 Arachidonic acid cAMP ER PKA Ca2+ Ca2+ VDCC Nucleus VDCC

SOC

FIGURE 2 Nongenomic signal transduction pathways activated by 1α,25(OH)2D3 in skeletal muscle cells and their relationship to intracellular Ca2+ regulation. mVDR, novel membrane 1α,25(OH)2D3 receptor; PIP2, phosphatidylinositol bisphosphate; PLD, PLC, and PLA2, phos- pholipases D, C, and A2; AC, adenylyl cyclase; Gi, inhibitory G protein; PC, phosphatidyl- choline; ER, endoplasmic reticulum; VDCC, voltage-dependent Ca channel; SOC, store-operated Ca channel. CHAPTER 55 Vitamin D and Muscle 891 and diacylglycerol (DAG, a PKC activator) from mem- Physiological concentrations of the hormone elicit brane phosphoinositides [91]. The formation of DAG very fast (within 30 sec) increases in AC, cAMP levels is biphasic, with the second phase independent of IP3 and PKA activity in both intact differentiated muscle production and peaking at 5 min. 1α,25(OH)2D3 also and cultured myoblasts/myotubes. 1α,25(OH)2D3 stimulates the rapid hydrolysis of phosphatidylcholine stimulation of dihydropyridine-sensitive Ca2+ influx is (PC) in myoblasts by a phospholipase D (PLD)- abolished by specific inhibitors of AC and PKA and catalyzed mechanism. PLD activity generates choline mimicked by forskolin and dibutyryl cAMP, involving and phosphatidic acid, which in turn can be converted the AC/cAMP/PKA pathway in the hormone non- to diacylglycerol by the action of a phosphohydrolase, genomic modulation of VDCC [100,101]. accounting for the second peak of DAG observed in Studies with muscle cells and tissue on the effects response to the hormone [92]. In addition, 1α,25(OH)2D3 of G protein modulators, e.g., fluoride, GTPγS, activates phospholipase A2 (PLA2) and the subsequent GDPβS, cholera, and Bordetella pertussis toxins, on 2+ release of arachidonic acid. The response is rapid 1α,25(OH)2D3-mediated Ca uptake, as well as the (within 1 min) and dose-dependent (10−11Ð10−7 M) [93]. observation of hormone-induced decrease of [35S]GTPγS Rapid modulation of myoblast PLC, PLD, and PLA2 is binding to membranes and increased ADP ribosylation 1α,25(OH)2D3 specific, as 25(OH)D3 and 24,25(OH)2D3 of the pertussis toxinÐsensitive 41-kDa substrate, led do not influence enzyme activities. to the proposal that negative regulation of an inhibitory The rapid activation of phospholipases by 1α,25- protein coupled to AC is part of the mechanism by 2+ (OH)2D3 involves the participation of guanine nucleotide which 1α,25(OH)2D3 increases Ca influx through the − binding (G) proteins. (AlF4) and the stable analog cAMP-dependent pathway [101,102] (Fig. 2, right side). GTPγS, which activate G proteins, mimic hormone stim- Direct evidence on the involvement of G proteins in the ulation of PLA2-mediated arachidonic acid (AA) release fast activation of adenylyl cyclase by 1α,25(OH)2D3 from myoblasts prelabeled with [3H]AA, whereas has been obtained in experiments in which the effect of GDPβS and Bordetella pertussis toxin pretreatment the hormone on AC, GTPase, and PKA activities as abolish 1α,25(OH)2D3-dependent AA release [94]. By well as on the phosphorylation of Gαi was studied in using similar experimental approaches, it has been membranes from chick skeletal muscle cells [103]. shown that like PLA2, hormone modulation of PLC 1α,25(OH)2D3 stimulates AC activity in a dose (0.1Ð10 and PLD is mediated by a pertussis toxinÐsensitive nM)- and time (1Ð5 min)-dependent fashion provided GTP-binding protein [95]. GTP is present in the assay. High-affinity GTPase, In agreement with the 1α,25(OH)2D3-induced gen- related to Gs, is unaffected by the hormone. In the eration of DAG via PLC and PLD, it has been reported absence of GTP or in the presence of a high concentra- that the hormone rapidly translocates protein kinase C tion of Mn2+, a condition that provides information on into the cell membrane and increases its activity in adenylyl cyclase activity devoid of G-protein regula- chick muscle soleus muscle in vitro as well as in cul- tion, 1α,25(OH)2D3 effects on AC are abolished. PKA tured embryonic muscle cells. In addition, the partici- activity is increased in cells pretreated with the hormone. 32 pation of protein kinase C in the fast 1α,25(OH)2D3 Moreover, immunoprecipitation of Gαi from [ P]-labeled 45 2+ stimulation of Ca influx through VDCC channels myoblast membranes shows that 1α,25(OH)2D3 is supported by experimental evidence obtained with increases the phosphorylation of its α subunit. Therefore, phorbol esters and DAG analogs, which mimic the these data altogether indicate that in muscle cells action of the hormone, as well as PKC inhibitors, 1α,25(OH)2D3 activates adenylyl cyclase by a GTP- which reduce its effects [96,97]. Recent studies have dependent action implying, as suggested before, revealed that PKCα is the only PKC isoform activated amelioration of Gi function by hormone-induced αi and translocated from cytosol to the membrane upon phosphorylation [103]. 1α,25(OH)2D3 stimulation of muscle cells [98] (Fig. 2, The rapidity with which 1α,25(OH)2D3 activates left side). Moreover, transfection of specific anti- second messenger systems and Ca2+ channels in muscle PKCα antibodies or intranuclear microinjection of cells and the hormone specificity at the physiological antisense oligonucleotides against PKCα mRNA cou- vitamin D3 metabolite concentrations suggest that a pled to spectrofluorimetric analysis of changes in intra- plasma membrane-bound receptor (mVDR; Fig. 2) cellular Ca2+ in Fura-2-loaded myoblasts/myotubes may be responsible for the initiation of its effects. The shows a marked reduction of hormone-dependent Ca2+ existence of a putative novel cell-surface receptor for influx [98,99]. 1α,25(OH)2D3 that mediates the nongenomic actions There are also data implying the participation of of the hormone has been reported for other target cells. the adenylyl cyclase (AC)/cAMP pathway in the non- Thus, a 1α,25(OH)2D3 binding protein of 65Ð66 kDa, genomic mode of action of 1α,25(OH)2D3 in muscle. different from the nuclear VDR, detected in intestine, 892 RICARDO L. BOLAND cartilage, kidney, and brain cells, has been functionally (TRPC1ÐTRPC7), which are homologs of the inverte- related to transcaltachia and rapid activation of PKC brate counterparts [108]. The involvement of TRPC [104Ð106]. It has also been suggested that annexin II may proteins in SOC entry induced by 1α,25(OH)2D3 in be the membrane receptor that mediates 1α,25(OH)2D3- muscle cells has been investigated. Two fragments induced rapid increases in cytosolic Ca2+ in rat have been amplified from avian myoblasts by RT-PCR, osteoblast-like cells ROS 24/1, which have very few or exhibiting >85% sequence homology with human undetectable VDR [107]. TRPC3. In agreement with these observations, Northern However, recent observations support the hypothesis and Western blots employing TRPC3-probes and anti- that the classic nuclear VDR may be the receptor that TRPC3 antibodies, respectively, confirmed endogenous mediates, at least in part, the nongenomic effects of expression of a TRPC3-like protein in muscle cells. As 2+ 1α,25(OH)2D3 on store-operated Ca (SOC; CCE) shown in Fig. 3A, transfection of myoblasts with anti- influx in muscle. The structural components of SOC TRPC3 antisense ODNs shows reduced CCE induced channels are the TRP (transient receptor potential; by 1α,25(OH)2D3. Anti-VDR antisense ODNs also Drosophila melanogaster) proteins, designated as the inhibit hormone-dependent SOC Ca2+ influx (Fig. 3A), TRP-canonical (TRPC) subfamily of the larger TRP and coimmunoprecipitation of TRPC3 and VDR is superfamily of gene products, which function as Ca2+- observed (Fig. 3B), suggesting an association between permeable channels mainly regulated by store depletion both proteins and a functional role of the receptor in when expressed in heterologous systems. At present, 1α,25(OH)2D3 activation of CCE. Accordingly, it has seven mammalian TRPC proteins are at least known been shown that short treatment with 1α,25(OH)2D3

A Ca2+ Control

) 1,25

M (50 n i ] 2+ anti-TRP [Ca anti-VDR

1 min B 1 234 1234

140 kDa

60 kDa

WB: anti-TRP WB: anti-VDR

FIGURE 3 1α,25(OH)2D3-dependent muscle SOC influx is mediated by TRPC3 proteins and the VDR. (A) Effects of anti-TRPC3 and anti-VDR antisense ODNs on 1α,25(OH)2D3-induced SOC influx, fluorimetrically measured after hormone-elicited store depletion by the Ca2+ readdition protocol [54]. (B) Coimmunoprecipitation of TRPC3 and VDR. Immunoprecipitation was carried out with anti-TRPC3 antibody (right) or with anti-VDR antibody (left) followed by Western blotting with anti-TRPC3 antibody (left) or anti-VDR antibody (right). Lane 1, anti-TRPC3 antibody; lane 2, donkey anti-goat antibody; lane 3, without primary and secondary antibodies; lane 4, cell lysate. CHAPTER 55 Vitamin D and Muscle 893

AB

3 2,5 1,25 EGTA 2,5 + Ca2 1,25 2 EGTA Ca2+ 2

1,5 1,5 1 Ratio (340/390) Ratio (340/390) 1 0,5 1 min 1 min 0 0,5

FIGURE 4 Effect of an anti-INAD antibody on 1α,25(OH)2D3-dependent SOC influx in muscle cells. Cultured chick embryo skeletal muscle cells were permeabilized with saponin (50 µg/ml buffer) for 5 min at room temperature (as in [98]), in the presence of either normal rabbit Ig G (A) or an antibody against INAD from Caliphora vicinia (B). After washing, the cells −9 were loaded with Fura-2 and the SOC influx dependent upon store depletion by 1α,25(OH)2D3 [10 M] was fluorimetrically measured by the Ca2+ readdition protocol [54].

induces translocation of the VDR from the nucleus to and relaxation, are observed in vitamin DÐdeficiency plasma membranes in chick myoblasts/myotubes. This states. These myopathies are independent of changes reverse translocation is blocked by colchicine, genistein, in blood mineral composition or PTH levels and or herbimycin, suggesting the involvement of micro- respond only to vitamin D3 or its metabolites. Various tubular transport and tyrosine kinase/s in the relocation lines of experimental evidence described in this chapter of the receptor [42]. obtained with animal models and cultured embryonic TRP channels have been shown to be modulated by muscle cells (myoblasts), which differentiate in vitro association of macromolecules integrating signaling to functionally competent muscle fibers (myotubes), supramolecular complexes. The scaffold protein INAD have demonstrated that the hormonal metabolite clusters these macromolecules through its PDZ 1α,25(OH)2D3 is essential for normal homeostasis of domains [109]. A functional role for a INAD-like pro- intracellular calcium and growth in skeletal muscle tein in hormone activation of CCE is implied by the and thereby plays an important role in contractility and 2+ reduction of 1α,25(OH)2D3-induced Ca influx upon myogenesis. Other lines of research have implied an transfection of muscle cells with an anti-INAD anti- action of 25(OH)D3 in muscle phosphate uptake and body (Fig. 4) or microinjection with anti-INAD anti- contractile protein synthesis. 2+ sense ODNs (not shown). In addition to TRPC3 and 1α,25(OH)2D3 regulates muscle intracellular Ca VDR, other components of the putative signaling com- through both genomic and nongenomic mechanisms. plex may be calmodulin/CaMKII and Src, which have Congruent with the presence of the VDR in myoblasts been shown to interact with TRP proteins (recent and myotubes, the hormone induces transcriptionally unpublished observations) and the VDR [78], respec- the synthesis of various Ca2+ binding proteins, one of tively, in keeping with the fact that CaM antagonists them identified as calbindin-D9K, and enzymes related and inhibitors of CaMKII as well as of tyrosine kinases to the synthesis of phosphatidylcholine, whose changes 2+ block 1α,25(OH)2D3-induced SOC influx [54,56]. correlate with those in 1α,25(OH)2D3-dependent Ca Figure 5 illustrates the proposed mechanism by which transport. INAD-based signaling complexes with the interven- Fast actions of 1α,25(OH)2D3 also exert rapid tion of the VDR participate in modulation of SOC effects in skeletal muscle initiated in the cell surface influx by 1α,25(OH)2D3. by interacting with an as-yet-unidentified hormone plasma membrane receptor, followed by the stimula- tion of second messenger systems that transmit the VIII. SUMMARY signal to the cytoplasm. This mode of action involves the activation of Ca2+ entry through voltage-dependent Muscle weakness and atrophy, and electrophysiolog- Ca2+ channels by G-protein-mediated modulation of ically demonstrable abnormalities in muscle contraction the adenylyl cyclase/cAMP/PKA and phospholipase 894 RICARDO L. BOLAND

P PLCβ

P SERCA pump

P

Nucleus

FIGURE 5 Proposed mechanism of action involved in 1α,25(OH)2D3 regulation of SOC influx with participation of the VDR and INAD-based signaling complexes. PM, plasma membrane; PLCβ, phospholipase C β; IP3, inositol trisphosphate; DAG, diacylglycerol; IP3R, IP3 receptor; SERCA, sarcoplasmicÐendoplasmic reticulum Ca ATPase; PMCA, plasma membrane Ca ATPase; SOC/CCE, store-operated Ca/capacitative Ca entry channel; VDCC, voltage-dependent Ca channel; CaM, calmodulin; INAD, scaffold protein, TRPC3, C3 isoform of TRP protein.

C/DAG + IP3/PKCα pathways. 1α,25(OH)2D3 also of action of the hormone in skeletal muscle, where it stimulates the release of Ca2+ from intracellular stores appears to play an important role both in the regulation and the capacitative influx of the cation through store- of cellular Ca2+ and in growth and development. operated calcium (SOC; TRP) channels. Tyrosine kinases (TKs) and the VDR participate in hormone reg- α ulation of SOC channels. Accordingly, 1 ,25(OH)2D3 Acknowledgments induces rapid translocation of the VDR from the nucleus to the plasma membrane, a process that is The author is indebted to current and previous blocked by TK inhibitors. Of mechanistic relevance, laboratory colleagues who have contributed during molecular and immunochemical studies and the appli- various phases of the investigations reported, in particu- cation of oligonucleotide antisense technology, in con- lar Ana R. De Boland, Teresita Bellido, Susana Morelli, junction with microspectrofluorimetric analysis, have Guillermo Vazquez, Maria Julia Marinissen, Claudia provided evidence on the participation of signaling Buitrago, Graciela Santillàn, and Daniela Capiati. supramolecular structures integrated by TRP proteins, Work in our laboratory described in this chapter calmodulin, Src, VDR, and the scaffold protein INAD. has been supported by the Consejo Nacional de This is an emerging novel concept within the field Investigaciones Cientìficas (CONICET), Agencia of nongenomic actions of vitamin D3 actions, which Nacional de Promociòn Cientìfica y Tecnològica, and deserve further study. Fundaciòn Antorchas, Argentina. 1α,25(OH)2D3 stimulates proliferation and growth of muscle cells by promoting tyrosine phosphorylation of the MAP kinase cascade, mainly upstream at the level of Src and Raf-1, which requires PKCα, Ca2+, and, References in part, the VDR itself. Clearly, significant advances in the characterization 1. DeLuca HF 1988 The vitamin D story: a collaborative effort α of basic science and clinical medicine. FASEB J 2:224Ð236. of the effects of 1 ,25(OH)2D3 on muscle calcium 2. Reichel H, Koeffler H, Norman AW 1989 The role of the metabolism and growth have been achieved in recent vitamin D endocrine system in health and disease. New Engl years, which may provide useful insights into the mode J Med 320:980Ð991. CHAPTER 55 Vitamin D and Muscle 895

3. Minghetti P, Norman A 1988 1,25(OH)2-vitamin D3 26. Heimberg KW, Matthews C, Ritz E, Augustin J, Hasselbach W receptors: Gene regulation and genetic circuitry. FASEB J 1976 Active Ca transport of sarcoplasmic reticulum during 2:3043Ð3053. experimental uremia: changes in kinetics and lipid composi- 4. Perlman KA, Kutnerei A, Prahl J, Smith C, Inaba M, Schnoes H, tion. Eur J Biochem 61:207Ð213. DeLuca HF 1990 24-Homologated 1,25-dihydroxyvitamin D3 27. Matthews C, Heimberg KW, Ritz E, Agostini B, Fritzsche J, compounds: Separation of calcium and differentiation activities. Hasselbach W 1977 Effect of 1,25-dihydroxy-cholecalciferol Biochemistry 29:190Ð196. on impaired calcium transport by the sarcoplasmic reticulum 5. De Boland A, Nemere I 1992 Rapid actions of vitamin D in experimental uremia. Kidney Int 11:227Ð235. compounds. J Cell Biochem 49:32Ð36. 28. Boland R, Matthews C, De Boland AR, Ritz E, Hasselbach W 6. Nemere I, Farach-Carson MC 1998 Membrane receptors for 1983 Reversal of decreased phosphorylation of sarcoplasmic steroid hormones: A case for specific cell surface binding reticulum calcium transport ATPase by 1,25-dihydroxy- sites for vitamin D metabolites and estrogens. Biochim cholecalciferol in experimental uremia. Calcif Tissue Int Biophys Res Commun 248:443Ð449. 35:195Ð201. 7. Walters M 1992 Newly identified functions of the vitamin D 29. Boland R, De Boland AR, Ritz E, Hasselbach W 1983 Effect endocrine system. Endocr Rev 13:719Ð764. of 1,25-dihydroxy-cholecalciferol on sarcoplasmic reticulum 8. Walters M 1995 Newly identified functions of the vitamin D calcium transport in strontium-fed chicks. Calcif Tissue Int endocrine system: Update. Endocr Rev Monogr 4:47Ð56. 35:190Ð194. 9. Boland R 1986 Role of vitamin D in skeletal muscle func- 30. De Boland AR, Gallego S, Boland R 1983 Effects of vitamin D3 tion. Endocr Rev 7:434Ð448. on phosphate and calcium transport across and composition 10. Smith R, Stern G 1967 Myophathy, osteomalacia and hyper- of skeletal muscle plasma cell membranes. Biochim Biophys parathyroidism. Brain 90:593Ð602. Acta 733:264Ð273. 11. Smith R, Stern G 1969 Muscular weakness in osteomalacia 31. Giuliani D, Boland R 1984 Effects of vitamin D metabolites and hyperparathyroidism. J Neurol Sci 8:511Ð520. on calcium fluxes in intact chicken skeletal muscle and 12. Skaria J, Katiyar BC, Srivastava TP, Dube B 1975 Myopathy myoblasts cultured in vitro. Calcif Tissue Int 36:200Ð205. and neuropathy associated with osteomalacia. Acta Neurol 32. Bauman VK, Valinietse MY, Babarykin DA 1984 Vitamin D3 Scandinav 51:37Ð58. and 1,25-dihydroxy-vitamin D3 stimulate the skeletal muscle- 13. Schott GD, Wills MR 1976 Muscle weakness in osteomala- calcium mobilization in rachitic chicks. Arch Biochem cia. Lancet 1:626Ð629. Biophys 231:211Ð216. 14. Floyd M, Ayyar DR, Barwick DD, Hudgson P, Weightman D 33. Birge SJ, Haddad JC 1975 25-Hydroxy-cholecalciferol stim- 1974 Myopathy in chronic renal failure. Q J Med 43:509Ð523. ulation of muscle metabolism. J Clin Invest 56:1100Ð1107. 15. Henderson RG, Ledingham JGG, Oliver DO, Small DJ, 34. Ströder J, Arensmeyer E 1965 Der Actomyosin-Gehalt der Russel RGG, Smith R, Walton RJ, Preston C 1974 Effects of Skeletmuskulatur bei experimenteller Rachitis. Klin 1,25-dihydroxy-cholecalciferol on calcium absorption, mus- Wochenschr 43:1201Ð1202. cle weakness and bone disease in chronic renal failure. 35. De Boland AR, Albornoz LE, Boland R 1983 The effect of Lancet 1:379Ð384. cholecalciferol in vivo on proteins and lipids of skeletal 16. Brickman AS, Coburn JW, Massry SG, Norman AW 1974 muscle from rachitic chicks. Calcif Tissue Int 35:798Ð805. 1,25-Dihydroxy-vitamin D3 in normal man and patients with 36. Pointon JJ, Francis MJO, Smith R 1979 Effects of vita- renal failure. Ann Int Med 80:161Ð168. min D deficieny on sarcoplasmic reticulum function and 17. Vicale CT 1949 Diagnostic features of muscular syndrome troponin c concentration of rabbit skeletal muscle. Clin Sci resulting from hyperparathyroidism osteomalacia owing to 57:257Ð263. renal tubular acidosis and perhaps to related disorders of 37. Zot HB, Potter JD 1984 The role of calcium in the regulation calcium metabolism. Trans Am Neurol Assoc 74:143Ð147. of the sekeletal muscle contraction-relaxation cycle. In: Sigel H 18. Birge SJ 1978 Vitamin D, muscle and phosphate homeosta- (ed) Metal Ions in Biological Systems. Marcel Dekker, sis. Mineral Electrolyte Metab 1:57Ð64. New York, pp. 381Ð402. 19. Haussler MR, Baylink DJ, Hughes MR, Brumbraugh PF, 38. Boland RL, Norman AW, Ritz E, Hasselbach W 1985 Presence Vergedal JE, Shen FH, Nielsen RL, Counts SJ, Bursak KM, of a 1,25-dihxdroxy-vitamin D3 Receptor in chick skeletal mus- McCain TA 1976 The assay of 1,25-dihydroxy-vitamin D3. cle myoblasts. Biochem Biophys Res Commun 128:305Ð311. Physiologic and pathological modulation of circulating hor- 39. Simpson RU, Thomas GA, Arnold AJ 1985 Identification of mone levels. Clin Endocrinol (Oxf) 5(Suppl):151SÐ165S. 1,25-dihydroxyvitamin D3 receptors and activities in muscle. 20. Rodman JS, Baker T 1978 Changes in the kinetics of muscle J Biol Chem 260:8882Ð8891. contraction in vitamin D-depleted rats. Kidney Int 13:189Ð193. 40. Costa EM, Blau HM, Feldman D 1986 1,25-Dihydroxy- 21. Pleasure D, Wyszynski B, Summer A, Schotland D, vitamin D3 receptors and hormonal responses in human Feldmann B, Hitz K, Goodman DB, Nugent N 1979 Skeletal skeletal muscle cells. Endocrinology 119:2214Ð2220. muscle calcium metabolism and contractile force in vitamin D- 41. Zanello SB, Collins ED, Marinissen MJ, Norman AW, deficient chicks. J Clin Invest 64:1157Ð1167. Boland R 1997 Vitamin D receptor expression in chicken 22. Weber A, Herz R, Reiss I 1964 Role of calcium in contrac- muscle tissue and cultured myoblasts. Horm Metab Res tion and relaxation of muscle. Fed Proc 23:896Ð200. 29:231Ð236. 23. Ebashi S, Endo M 1968 Calcium ion and muscle contraction. 42. Capiati D, Benassati S, Boland R 2002 1,25(OH)2-Vitamin D3 Prog Biophys Mol Biol 18:123Ð183. induces translocation of the vitamin D receptor (VDR) to the 24. Carafoli E, Crompton M 1978 The regulation of intracellular plasma membrane in skeletal muscle cells. J Cell Biochem calcium. Curr Top Membr Transp 10:151Ð216. 86:128Ð135. 25. Curry OB, Bastein JF, Francis MJA, Smith R 1974 Calcium 43. Buitrago C, Vazquez G, De Boland AR, Boland R 2000 uptake by sarcoplasmic reticulum of muscle from vitamin DÐ Activation of Src kinase in skeletal muscle cells by 1,25(OH)2- deficient rabbits. Nature 249:83Ð84. vitamin D3 correlates with tyrosine phosphorylation of the 896 RICARDO L. BOLAND

vitamin D receptor (VDR), and VDRÐSrc interaction. J Cell 60. Abe E, Miyaura C, Sakagami H, Takeda M, Kormo K, Biochem 79:274Ð281. Yamazaki T, Yoshiki S, Suda T 1981 Differentiation of mouse 44. Bodwell JE, Webster JC, Jewell CM, Cidlowski JA, Hu JM, myeloid cells induced by 1alpha,25-dihydroxy-vitamin D3. Munck A 1998 Glucocorticoid receptor phosphorylation: Proc Natl Acad Sci USA 78:4990Ð4994. Overview, function and cell cycle-dependence. J Steroid 61. Suda T 1989 The role of 1,25-dihydroxyvitamin D3 in myeloid Biochem Mol Biol 65:91Ð99. cell differentiation. Proc Soc Exp Biol Med 191:214Ð220. 45. Kuiper GG, Brinkmann AO 1994 Steroid hormone receptor 62. Fahrquahrson C, Whitehead C, Rennie J, Loverigde N 1993 phosphorylation: Is there a physiological role? Mol Cell In vivo effects of 1,25-dihydroxycholecalciferol on the Endocrinol 100:103Ð107. proliferation and differentiation of avian chondrocytes. J Bone 46. Hsieh JC, Jurutka PW, Galligan MA, Terpening CM, Miner Res 8:1081Ð1088. Haussler CA, Samuels DS, Shimizu Y, Shimizu N, Haussler 63. Bikle D, Pillai S 1993 Vitamin D, calcium, and epidermal MR 1991 Human Vitamin D receptor is selectively phospho- differentiation. Endocr Rev 14:3Ð19. rylated by protein kinase C on serine 51, a residue crucial to 64. Simpson R, Hsu T, Begley D, Mitchell B, Alizadeh J 1987 its trans-activation function. Proc Natl Acad Sci USA Transcriptional regulation of the c-myc protooncogene by 88:9315Ð9319. 1,25-dihydroxyvitamin D3 in HL-60 promyelocytic cells. 47. Jurutka PW, Hsieh JC, MacDonald PN, Haussler CA, J Biol Chem 262:4104Ð4108. Haussler MR, Whitfield GK 1993 Phosphorylation of serine 65. Tu-Yu A, Curtiss Morris R, Ives H 1993 Differential modu- 208 in the human Vitamin D receptor. The predominant lation of fos and jun gene expression by 1,25-dihydroxy- amino acid phosphorylated by casein kinase II, in vitro, and vitamin D3. Biochem Biophys Res Commun 193:161Ð166. identification as a significant phosphorylation site in intact 66. Simpson R, Hsu T, Wendt M, Taylor J 1989 1,25-dihydroxy- cells. J Biol Chem 268:6791Ð6799. vitamin D3 regulation of c-myc protooncogene transcription. 48. De Boland AR, Boland R 1987 Rapid changes in skeletal Possible involvement of protein kinase C. J Biol Chem muscle calcium uptake induced in vitro by 1,25-dihydroxy- 264:19710Ð19715. vitamin D3 are suppressed by calcium channel blockers. 67. Wakelam MJO 1985 The fusion of myoblasts. Biochem J Endocrinology 120:1858Ð1864. 228:1Ð12. 49. Vazquez G, De Boland AR 1993 Stimulation of dihydropyri- 68. O’Neill MC, Stockdale FE 1972 Kinetic analysis of myo- dine-sensitive Ca2+ influx in cultured myoblasts by genesis in vitro. J Cell Biol 52:52Ð65. 1,25(OH)2-vitamin D3. Biochem Mol Biol Int 31:677Ð684. 69. Capiati DA, Limbozzi F, Tellez-Inon MT, Boland R 1999 50. De Boland AR, Boland R 1994 Non-genomic signal trans- Evidence on the participation of protein kinase C α in the duction pathway of vitamin D in muscle. Cell Signal proliferation of cultured myoblasts. J Cell Biochem 6:717Ð724. 74:292Ð300. 51. McDonald TF, Pelzer S, Trautwein W, Pelzer DJ 1994 70. Capiati DA, Vazquez G, Tellez-Inon MT, Boland R 2000 Regulation and modulation of calcium channels in cardiac, Antisense oligonucleotides targeted against protein kinase C skeletal, and smooth muscle cells. Physiol Rev 74:365Ð507. α inhibit proliferation of cultured avian myoblasts. Cell 52. Boland R, De Boland AR, Marinissen MJ, Santillan G, Prolif 33:307Ð315. Vazquez G, Zanello S 1995 Avian muscle cells as targets for 71. Drittanti L, De Boland AR, Boland R 1989 Modulation of the secosteroid hormone 1,25-dihydroxy-vitamin D3. Mol DNA synthesis in cultured muscle cells by 1,25-dihydroxy- Cell Endocrinol 114:1Ð8. vitamin D3. Biochim Biophys Acta 1014:112Ð119. 53. Vazquez G, De Boland AR, Boland R 1997 Stimulation of 72. Drittanti L, De Boland AR, Boland R 1990 Stimulation of Ca2+ release-activated Ca2+ channels as a potential mecha- calmodulin synthesis in proliferating myoblasts by 1,25- nism involved in non-genomic 1,25(OH)2-vitamin D3- dihydroxy-vitamin D3. Mol Cell Endocrinol 74:143Ð153. induced Ca2+ entry in skeletal muscle cells. Biochem 73. Capiati DA, Tellez-Inon MT, Boland R 1999 Participation of Biophys Res Commun 239:562Ð565. protein kinase C α in 1,25-dihydroxy-vitamin D3 regulation 54. Vazquez G, De Boland AR, Boland R 1998 1α,25-dihydroxy- of chick myoblast proliferation and differentiation. Mol Cell 2+ vitamin D3-induced store-operated Ca influx in skeletal Endocrinol 153:39Ð45. muscle cells. Modulation by phospholipase C, protein kinase C, 74. Bellido T, Morelli S, Fernandez LM, Boland R 1993 and tyrosine kinases. J Biol Chem 273:33954Ð33960. Evidence for the participation of protein kinase C and 55. Vazquez G, Selles J, De Boland A, Boland R 1999 Rapid 3′,5′-cyclic AMP-dependent protein kinase in the stimulation actions of calcitriol and its side chain analogues CB1093 and of muscle cell proliferation by 1,25-dihydroxy-vitamin D3. GS1500 on intracellular calcium levels in skeletal muscle cells: Mol Cell Endocrinol 90:231Ð238. a comparative study. British J Pharmacol 126:1815Ð1823. 75. Neary JI 1997 MAPK cascades in cell growth and death. 56. Vazquez G, De Boland AR, Boland R 2000 Involvement of News Physiol Sci 12:286Ð293. calmodulin in 1α,25-dihydroxyvitamin D3 stimulation of 76. Morelli S, Buitrago C, Vazquez G, De Boland AR, Boland R 2+ store-operated Ca influx in skeletal muscle cells. J Biol 2000 Involvement of tyrosine kinase activity in 1,25(OH)2- Chem 275:16134Ð16138. vitamin D3 signal transduction in skeletal muscle cells. J Biol 57. Bellido T, Boland R 1987 Phosphate accumulation by mus- Chem 275:3626Ð3628. cle in vitro and the influence of vitamin D3 metabolites. 77. Buitrago C, Boland R, De Boland AR 2001 The tyrosine Z Naturforsch 42c:237Ð244. kinase c-Src is required for 1,25(OH)2-vitamin D3 signaling 58. Bellido T, Boland R 1988 Stimulation of myoblast membrane to the nucleus in muscle cells. Biochim Biophys Acta protein synthesis by 25-hydroxy-vitamin D3. Z Naturforsch 1541:179Ð187. 44c:807Ð812. 78. Buitrago C, Vazquez G, De Boland AR, Boland R 2001 The 59. Bellido T, Boland R 1991 Effects of 1,25-dihydroxy-vitamin vitamin D receptor mediates rapid changes in muscle protein D3 on phosphate accumulation by myoblasts. Horm Metab tyrosine phosphorylation induced by 1,25(OH)2D3. Biochem Res 23:113Ð116. Biophys Res Commun 289:1150Ð1156. CHAPTER 55 Vitamin D and Muscle 897

79. Boland R, De Boland, Buitrago C, Morelli S, Santillan G, 95. Morelli S, Boland R, De Boland AR 1996 1,25(OH)2-vitamin Vazquez G, Capiati D, Baldi C 2002 Non-genomic stimula- D3 stimulation of phospholipases C and D in muscle cells tion of tyrosine phosphorylation cascades by 1,25(OH)2D3 by involves extracellular calcium and a pertussis-sensitive G VDR-dependent and -independent mechanisms in muscle protein. Mol Cell Endocrinol 122:207Ð211. cells. Steroids 67:477Ð482. 96. Massheimer V, De Boland AR 1992 Modulation of 1,25- 2+ 80. Morelli S, Buitrago C, Boland R, De Boland AR 2001 The dihydroxyvitamin D3Ðdependent Ca uptake in skeletal stimulation of MAP kinase by 1,25(OH)2-vitamin D3 in muscle by protein kinase C. Biochem J 281:349Ð352. skeletal muscle cells is mediated by protein kinase C and cal- 97. Vazquez G, De Boland AR 1996 Involvement of protein cium. Mol Cell Endocrinol 173:41Ð52. kinase C in the modulation of 1α,25-dihydroxy-vitamin D3- 81. Buitrago C, Gonzalez Pardo V, De Boland AR, Boland R induced 45Ca2+ uptake in rat and chick cultured myoblasts. 2003 Activation of Raf-1 through Ras and protein kinase Cα Biochim Biophys Acta 1310:157Ð162. mediates 1α,25(OH)2-vitamin D3 regulation of the mitogen- 98. Capiati DA, Vazquez G, Tellez-Inon MT, Boland R 2001 activated protein kinase pathway in muscle cells. J Biol Role of PKC in 1,25(OH)2-vitamin D3 regulation of intracel- Chem 278:2199Ð2205. lular calcium levels during development of skeletal muscle 82. Buitrago C, Gonzalez Pardo V, De Boland AR 2002 cells in culture. J Cell Biochem 77:200Ð212. Nongenomic action of 1α,25(OH)2-vitamin D3. Activation of 99. Capiati DA, Vazquez G, Boland R 2001 Protein kinase C muscle cell PLCγ through the tyrosine kinase c-Src and modulates the Ca2+ influx phase of the Ca2+ response to PtdIns 3-kinase. Eur J Biochem 269:2506Ð2515. 1α,25-dihydroxy-vitamin D3 in skeletal muscle cells. Horm 83. De Boland AR, Boland R 1985 Suppression of 1,25-dihy- Metab Res 33:201Ð206. droxy-vitamin D3 calcium transport by protein synthesis 100. Fernandez LM, Massheimer V, De Boland AR 1990 Cyclic inhibitors and changes in phospholipids in skeletal muscle. AMP-dependent membrane protein phosphorylation and Biochim Biophys Acta 845:237Ð241. calmodulin binding are involved in the rapid stimulation of 84. Bellido T, Drittanti L, De Boland AR, Boland R 1987 The muscle calcium uptake by 1,25-dihydroxyvitamin D3. Calcif phospholipid and fatty acid composition of skeletal muscle Tissue Int 47:314Ð319. cells during culture in the presence of Vitamin D metabolites. 101. Vazquez G, Boland R, De Boland AR 1995 Modulation by Biochim Biophys Acta 922:162Ð169. 1,25(OH)2-vitamin D3 of the adenylyl cyclase/cyclic AMP 85. Drittanti L, De Boland AR, Boland R 1987 Changes in pathway in rat and chick myoblasts. Biochim Biophys Acta muscle lipid metabolism induced in vitro by 1,25-dihydroxy- 1269:91Ð97. vitamin D3. Biochim Biophys Acta 918:83Ð92. 102. Boland AR, Flawia M, Coso O, Boland R 1991 A guanine- 86. Drittanti L, De Boland AR, Boland R 1988 Effects of 1,25- nucleotide protein mediates 1,25-dihydroxy-vitamin D3Ð 2+ dihydroxyvitamin D3 on phospholipid metabolism in chick dependent rapid stimulation of Ca uptake in skeletal mus- myoblasts. Biochim Biophys Acta 962:1Ð7. cle. Biochim Biophys Acta 1094:238Ð242. 87. Drittanti L, Boland R, De Boland AR 1989 Induction 103. Vazquez G, De Boland AR, Boland R 1997 1α,25-(OH)2- of specific proteins in cultured skeletal muscle cells by vitamin D3 stimulates the adenylyl cyclase pathway in mus- 1,25-dihydroxyvitamin D3. Biochim Biophys Acta 1012: cle cells by a GTP-dependent mechanism which presumably 16Ð23. involves phosphorylation of Gαi. Biochem Biophys Res 88. Drittanti L, Zanello S, Boland R 1994 Induction of a Commun 234:125Ð128. calbindin-D9K-like protein in avian muscle cells by 1,25- 104. Nemere I, Dormanen MC, Hammond MW, Okamura WH, dihydroxy-vitamin D3. Biochem Mol Biol 5:859Ð868. Norman AW 1994 Identification of a specific binding protein 89. Zanello S, Drittanti L, Norman A, Boland R 1994 for 1α,25-dihydroxyvitamin D3 in basal-lateral membranes Identification of a 9 kDa calcium-binding protein in the chick of chick intestinal epithelium and relationship to transcal- as calbindin D-9K. In: Norman A, Bouillon R, Thomasset M tachia. J Biol Chem 269:23750Ð23576. (eds) Vitamin D. A Pluripotent Steroid Hormone: Structural 105. Nemere I, Schwartz S, Pedrozo H, Sylvia VL, Dean DD, Studies, Molecular Endocrinology and Clinical Applications. Boyan BD 1998 Identification of a membrane receptor for Walter de Gruyter, pp. 418Ð419. 1,25-dihydroxyvitamin D3 which mediates rapid activation of 90. Zanello S, Boland R, Norman A 1995 cDNA sequence protein kinase C. J Bone Min Res 13:1353Ð1359. identity of a vitamin-dependent calcium-binding protein 106. Jia Z, Nemere I 1999 Immunochemical studies on the putative in the chick to calbindin D-9K. Endocrinology 136:2784Ð2787. plasmalemmal receptor for 1,25-dihydroxyvitamin D3. II. 91. Morelli S, De Boland AR, Boland R 1993 Generation Chick kidney and brain. Steroids 64:541Ð550. of inositol phosphates, diacylglycerol and calcium fluxes 107. Baran DT, Quail JM, Ray R, Leszyk J, Honeyman T 2000 in myoblasts treated with 1,25-dihydroxyvitamin D3. Annexin II is the membrane receptor that mediates the rapid Biochem J 289:675Ð679. actions of 1α,25-dihydroxyvitamin D3. J Cell Biochem 92. De Boland AR, Morelli S, Boland R 1994 1,25(OH)2- 78:34Ð46. vitamin D3 signal transduction in chick myoblasts involves 108. Birnbaumer L, Zhu X, Jiang M, Boulay G, Peyton M, phosphatidylcholine hydrolysis. J Biol Chem 269:8675Ð8679. Vannier B, Brown D, Platano D, Sadeghi H, Stefani E, 93. De Boland AR, Boland R 1993 1,25-Dihydroxyvitamin D3 Birnbaumer M 1996 On the molecular basis and regulation induces arachidonate mobilization in embryonic chick of cellular capacitative calcium entry: roles for Trp proteins. myoblasts. Biochim Biophys Acta 1179:98Ð104. Proc Natl Acad Sci USA 93:15195Ð15202. 2+ 94. De Boland AR, Morelli S, Boland R 1995 1,25(OH)2- 109. Huber A, Sander P, Bahner M, Paulsen R 1998 The TRP Ca vitamin D3 stimulates phospholipase A2 activity via a guanine channel assembled in a signaling complex by the PDZ domain nucleotide-binding protein in chick myoblasts. Biochim protein INAD is phosphorylated through the interaction with Biophys Acta 1257:274Ð278. protein kinase C (εPKC). FEBS Lett 425:317Ð322. CHAPTER 56 Vitamin D and Cardiovascular Medicine

DWIGHT A. TOWLER Department of Medicine, Division of Bone and Mineral Diseases, Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, MO 63110

THOMAS L. CLEMENS Department of Cell Biology and Physiology, Department of Internal Medicine, University of Cincinnati School of Medicine, Cincinnati, OH

I. Introduction V. Vascular Calcification and Calcitropic Hormones: II. Clinical Evidence for Vitamin D Signaling in Cardiovascular Toxicology of Vitamin D Cardiovascular Health VI. Summary and Conclusions III. Indirect Cardiovascular Actions of Vitamin D References IV. Direct Actions of Vitamin D in the vasculature

I. INTRODUCTION overview of our current knowledge of vitamin D sig- naling in cardiovascular physiology and toxicology. It is The role of vitamin D in skeletal physiology meant to highlight the need for additional contributions and calcium/phosphate homeostasis has been well- by biologists, nutritionists, epidemiologists, and clini- recognized for over 50 years; rachitic musculoskeletal cians in order to develop a better understanding of vas- frailty arising from vitamin D deficiency and the cular biology and vitamin D signaling—relevant to clinical response to vitamin D repletion were defining preserving cardiovascular health and preventing frailty characteristics of this novel fat-soluble agent [1,2]. in our aging population. In recent years, the important roles for vitamin D in epithelial cell cycle physiology and terminal differen- tiation have become apparent, most saliently in skin II. CLINICAL EVIDENCE FOR [3] and in colonic and prostatic epithelia [4,5]. Perhaps VITAMIN D SIGNALING IN foreseeable from the actions of vitamin D on monocyte CARDIOVASCULAR HEALTH proliferation and development ex vivo, in vivo modula- tion of cell-mediated immunity by vitamin D has been A. Epidemiology demonstrated via clinical studies of psoriasis, animal models of autoimmune diabetes, and organ transplan- As detailed elsewhere in this text (Chapters 2 and 3), tation [6Ð8]. Clinically, concern of cardiovascular tox- outside of dietary supplementation, the major source of icity has dominated our thinking of vitamin D in vitamin D arises from photoconversion of 7-dehydro- vascular biology; indeed, several well-studied rat mod- cholesterol in skin in response to ultraviolet light. els utilize vitamin D toxicity in order to study the phar- Given the tremendous variation in human cutaneous macological inhibition of vascular calcification and pigment content, several groups have noted the differ- calciphylaxis [9Ð13]. Only recently, however, has car- ential effects of diminished sunlight exposure on vita- diovascular physiology been tied to vitamin D bioactiv- min D levels in African Americans vs. Caucasians [19] ity. A woefully small number of key patient-oriented (Chapter 47). Because of the increased risk for hyper- research studies [14,15] and preclinical investigations tension in the former population, Rostand studied the [16] has adequately addressed the role of vitamin D sig- potential correlation of light exposure and vitamin D naling in cardiovascular health—emphasizing the reg- levels to racial and geographic variations in blood ulation of hypertension, vascular inflammation, and pressure [19]; he found that vitamin D deficiency is diabetes risk as immediately germane to atherosclero- indeed a risk factor for hypertension. No intervention sis [17,18]. The goal of this chapter is to provide an study has been performed to date that directly assesses

VITAMIN D, 2ND EDITION Copyright © 2005, Elsevier, Inc. FELDMAN, PIKE, AND GLORIEUX All rights reserved. 900 DWIGHT A. TOWLER AND THOMAS L. CLEMENS the effects of vitamin D nutritional supplementation on B. Vitamin D Receptor Clinical Genetics blood pressure in African Americans. However, Pfeifer and Cardiovascular Disease and colleagues recently published a seminal clinical study of calcium with or without vitamin D supple- The vitamin D receptor (VDR) is a prototypic mem- mentation on systolic blood pressure (SBP) and diastolic ber of the large family of nuclear receptor transcription blood pressure (DBP) in elderly, vitamin DÐdeficient factors [25,26]. Like other nuclear receptors, this zinc Caucasian women—a population at high risk for osteo- finger transcription factor regulates gene transcription porosis and concomitant cardiovascular disease [14]. in heterodimeric VDR complexes via protein-protein While calcium (1200 mg) had little, if any, effect on and protein-DNA interactions directed by lipophilic blood pressure, two months of 800 IU of vitamin D ligands (e.g., calcitriol) and Ser/Thr phosphorylation with 1200 mg of calcium resulted in an average 9% [25,26]. Both genomic and nongenomic responses to (13 mm Hg) decrease in SBP (p = 0.02; n = 148). A vitamin D require the ligand binding domain and zinc similar, borderline trend (P = 0.10) was observed for fingers of the VDR [27]. A decade ago, a polymorphism DBP as well, and a modest decrease in basal heart rate in a BsmI cognate was identified in the 3′-UTR of the was noted, suggesting globally reduced sympathetic VDR gene. As compared to transcripts containing this tone (not directly assessed). Most importantly, 80% BSM sequence (denoted “b”), transcripts lacking this of subjects treated with vitamin D plus calcium expe- BSM sequence (denoted “B”) result in higher levels of rienced a 5 mm Hg decrement in SBP, compared with VDR mRNA accumulation, although this finding is 40% of calcium treated subjects. The mechanism controversial (see Chapter 68) [28,29]. Small studies was unexamined, but may be related to suppressive have provided evidence that the B VDR allele is effects of vitamin D on renin production [16] (vide infra, enriched in patients with calcific aortic stenosis [30]. section III). Moreover, the B allele is associated with acute onset This past year, Farhleitner and colleagues showed type I diabetes [31], and the VDR BB genotype is that peripheral arterial vascular disease (PAD) is associ- associated with a threefold increased risk for type II ated with a high incidence of vitamin D deficiency [15]. diabetes with coronary artery disease (CAD) [32]. In a They found that a remarkable 71% of individuals with larger study of 3,441 patients referred for angiography, PAD had 25-hydroxyvitamin D levels below 9 ng/ml. once adjustments are made for traditional risk factors Of greater significance, they demonstrated that 40% had such as diabetes and hypertension, no independent secondary hyperparathyroidism, and approximately association exists between the VDR BsmI genotype 10% had frank hypocalcemia—biochemical indices and cardiovascular risk or extent [33]. Thus, genetic that independently confirmed the high incidence of contributions of VDR polymorphisms to CAD may be reduced vitamin D tone in patients with PAD [15]. The more closely related to modifying the risk for these data are consistent with vitamin D deficiency arising well-known cardiovascular risk factors. Consistent secondary to the immobilization and isolation that can with this notion, vitamin D supplementation has been be associated with severe PAD. However, other studies shown to decrease the risk for type I diabetes in have shown inverse relationships between 25-hydroxy Finnish children [34] (see Chapter 99 for additional vitamin D levels and the risk for myocardial infarction discussion of vitamin D and diabetes). [20,21], suggesting a cardiovascular benefit to adequate The VDR BB allele is also associated with lower vitamin D nutrition—or detriment with deficiency. serum calcium concentrations and higher serum To date, no adequately powered study evaluating the phosphate concentrations in patients with chronic contribution of vitamin D supplementation on primary renal failure [35]—a population at high risk for car- prevention of either coronary artery disease or PAD diovascular disease and calcific vasculopathy. This has been performed [15]. Of note, a key placebo- subset of our population—the hemodialysis patient— controlled arm of the Women’s Health Initiative (WHI) has severely perturbed calcium phosphate homeosta- clinical study will assess contributions of vitamin D sis. It is tempting to speculate that VDR genetic (400 IU daily) and calcium (1000 mg daily as calcium polymorphisms exert a greater impact on cardiovascu- carbonate) supplementation to bone health and colo- lar health in this metabolically stressed milieu, as rectal cancer outcomes in post-menopausal women. reflected in PTH responses to calcitriol pharmacother- Since cardiovascular health endpoints modifiable by apy (bb genotype relatively resistant to calcitriol sup- hormone replacement therapy are primary endpoints in pression of PTH) [36,37] and cardiovascular mortality other arms of this study [22], potential cardiovascular risk [38]. benefits of dietary vitamin D + calcium supplementa- The hypophosphatemia and hypocalcemia of severe tion may also emerge from analysis of the WHI rickets is an uncommon but reversible cause of con- [23,24] (minimum of 7 years planned follow-up). gestive heart failure (CHF) [39Ð41]. Little data exist CHAPTER 56 Vitamin D and Cardiovascular Medicine 901 on VDR genotype and CHF, particularly in adults. via the activation of RANKL production from susten- Recently in a small study (n = 75), Katagiri and col- tacular bone marrow stromal cells, calcitriol enhances leagues examined relationships between the VDR FokI RANKL-dependent differentiation of the bone resorb- genotype in women with mild to moderate CHF [42]. ing osteoclast that also releases calcium and phosphate While no connection to severity or prevalence of CHF from skeletal stores (Chapter 38). Via its suppression of was identified, the fractional excretion of calcium was PTH induction, vitamin D also enhances renal tubular found to be elevated twofold in CHF patients with the resorption of phosphate, reducing phosphate loss in the VDR FF allele [42]. Loss of spinal bone mineral den- urine [45]. Thus, a major consequence of vitamin D sity was also greater in VDR FF patients. Calciuresis is action is the long-term regulation (days to months) of coupled with natriuresis; the VDR FF allele correlates the serum calcium-phosphate product. This has several positively with furosemide dose and elevated atrial important indirect cardiovascular consequences rele- natriuretic peptide concentrations—pharmacological vant to severe vitamin D deficiency and vitamin D tox- and physiological factors that regulate natriuresis in icity. The phosphate depletion of severe rickets is an the CHF patient [42]. While genotype correlation with uncommon but reversible cause of congestive heart furosemide dose administered suggests correlation failure (CHF) [39Ð41]; this highlights the deleterious, with severity of cardiovascular compromise [42], no indirect consequences of extreme vitamin D malnutri- direct evidence exists. However, these data do high- tion via reduced myocardial phosphate stores (ATP, light that CHF patients with the VDR FF genotype are creatine phosphate) necessary for normal cardiac con- at greater risk for negative calcium balance, bone loss, tractility. However, in addition to providing adequate and perhaps fracture. mineral substrates for bone deposition at sites of skeletal ossification, an elevated calcium-phosphate product enhances mineral deposition at nonosseous sites [46Ð48]; III. INDIRECT CARDIOVASCULAR a key feature of vitamin D toxicity arises from the dys- ACTIONS OF VITAMIN D trophic vascular calcification (vide infra, section V). Thus, at the extremes of vitamin D exposure, perturba- A. Vitamin D Regulation tions in serum calcium and phosphate indirectly con- of the ReninÐAngiotensin-Aldosterone tribute to cardiovascular pathophysiology. (RAA) Axis It has been recognized for almost 20 years that the PTH gene is a direct target of vitamin D action [49]. As mentioned above, serum vitamin D levels are As detailed elsewhere in the text (Chapter 30), the inversely related to blood pressure, and vitamin D sup- production of PTH is under negative regulation by cal- plementation in borderline deficient women reduces citriol [50], and the VDR bb allele has been associated blood pressure. Recently, Li and colleagues (Chapter with an increased incidence of primary hyperparathy- 54) provided important insights into the mechanism of roidism [51] and impaired PTH suppression by cal- this physiological response [16]. They demonstrated citriol [37]. Thus, a subset of vitamin D cardiovascular that activated VDR signaling suppresses the renin gene actions are thus potentially elicited via changes in cir- via ligand-dependent VDR interactions with a negative culating PTH. Vitamin D deficiency is known to result response element in the renin promoter. Activation of in secondary hyperparathyroidism, and chronic hyper- the RAA axis not only increases blood pressure via parathyroidism is associated with hypertension, partic- angiotensin II signaling, but concomitantly drives a ularly salient in chronic renal insufficiency [52]. myocardial fibrosis response via aldosterone [43]. However, the vascular response in X-linked hypophos- Given the well-documented cardiovascular benefits of phatemic rickets—a hereditary disorder characterized reducing blood pressure and RAA axis activity [43], by secondary hyperparathyroidism in the absence of reduced renin expression may explain, in part, the vitamin D deficiency (Chapter 69)—provides additional emerging and desirable cardiovascular actions of ade- evidence for a role for hyperparathyroidism in vascular quate vitamin D nutrition [14,19,44]. tone [53]. Children afflicted with XLH who subse- quently develop hypertension have marked secondary hyperparathyroidism and hypertension [53]. Of note, B. Calcium Phosphate Homeostasis since the latter is often associated with nephrocalci- and PTH Secretion nosis and elevated renin levels, hypertension may occur in response to renal injury [53]. Jorde and As detailed elsewhere in this volume, calcitriol colleagues demonstrated that reduced calcium intake enhances gastrointestinal absorption of calcium and at any given level of vitamin D intake is associated phosphate from dietary sources (Chapter 24). In addition, with elevated systolic blood pressure, diastolic blood 902 DWIGHT A. TOWLER AND THOMAS L. CLEMENS pressure, and circulating PTH [54,55]; however, cal- cardiovascular disease [65]. The nutritional and phar- cium supplementation in patients with secondary macological anti-inflammatory actions of vitamin D— hyperparathyroidism does not appear to reduce blood while predicted to reduce cardiovascular risk in part pressure even though PTH levels are reduced [56]. via these mechanisms—have not been adequately Thus, the relative contributions of vitamin D defi- studied to either demonstrate or discount potential con- ciency to hypertension and ventricular function via tributors to cardiovascular health. the RAA axis (Section III.A), PTH secretion, and direct actions described (Section IV.B) remain to be determined. IV. DIRECT ACTIONS OF VITAMIN D IN THE VASCULATURE C. Vitamin D Regulation of the A. Calcitriol D Actions in Vascular Smooth Coagulation Cascade Muscle Cells (VSMCs)

Relatively little data are available concerning the Immunoreactive vitamin D receptors are present in actions of vitamin D on the coagulation cascade. In VSMCs [66Ð68], cardiomyocytes [69Ð71], and circulating human monocytes and the U937 monocytic endothelial cells [72]. The direct actions of calcitriol cell line [57], calcitriol upregulates thrombomodulin on mesenchymal myofibroblasts and VSMCs are well (coactivator of protein C-mediated anticoagulation), and described. Calcitriol exerts a profound suppressive down-regulates the production of tissue factor, which is effect on VSMC and myofibroblast proliferation in a key procoagulant that facilitates intrinsic pathway acti- response to thrombin [73], but can increase DNA syn- vation during injury and sepsis. Importantly, monocyte thesis in serum-free conditions [73] and enhances procoagulant responses to TNF-alpha were abrogated VSMC migration [66]. Both proliferative and inhibitory by calcitriol and synthetic analogs [58]. Consistent activities suggest a potential role for vitamin D in with this, calcitriol inhibits disseminated intravascular modulating vascular injury responses and neointima coagulation (DIC) in response to lipopolysaccharide, formation, but this has not been systematically exam- but not tissue factor, in a rat model of DIC [59]. ined. However, evidence for a role of VSMC vitamin D Whether vitamin D signaling is of any clinical rele- signaling in calcific vasculopathy of vitamin D toxicity vance in the setting of gram-negative sepsis or acute is strong [74]. Vitamin D intoxication induces medial coronary thrombosis is as yet unexamined. degeneration in both humans and animal models [13,75]. Calcitriol also upregulates the production of alkaline phosphatase in cultured VSMCs [76,77]. This has D. Regulation of Inflammation and tremendous functional importance, since alkaline Cell-Mediated Immunity by Vitamin D phosphatase—in addition to being an early marker of bone-forming osteoblast differentiation—degrades Freedman and colleagues using the U937 myelomono- inorganic pyrophosphate and dephosphorylates osteo- cytic cell line highlighted an important role for calcitriol pontin (OPN) [78]Ðmajor physiological inhibitors of in limiting the proliferative expansion of hematopoi- heterotopic calcium deposition [79,80]. This is likely etic progenitors [60,61]. Calcitriol enhances expression secondary to the actions of vitamin D on vascular of p21, a negative regulator of G1 cell-cycle transit. expression of PTHrP, an autocrine inhibitor of VSMC This activity of calcitriol and its analogs has been mineralization, proliferation, and OPN gene expres- proved clinically useful in the treatment of psoriasis, a sion [76]. Mechanistically, VDR targets a nuclear memory T-cellÐdriven hyperproliferative disorder of matrix Ku antigen complex, enhancing stable associa- skin and joints [62]. Calcitriol inhibits the production of tion of the DNA-dependent protein kinase to the IL-12 [63], an important macrophage-derived inflam- PTHrP promoter with loss of Ku subunit binding and matory Th1 cytokine that plays an important role in resultant transcriptional suppression [81,82]. The atherosclerotic progression [64]. The immunomod- physiologically important roles of adequate vitamin D ulatory actions of vitamin D are detailed thoroughly nutrition in controlling blood pressure, diabetes risk, in Chapter 36. Of note, a compelling view of cardio- and inflammatory responses relevant to atherosclerosis vascular disease in the setting of diabetes, dyslipi- are just emerging (Section III). While the consequences demia, and heart transplantation emphasizes the role of toxic vitamin D exposure of VSMC are well estab- of vascular inflammation in disease progression [65]; lished and being studied, much more research is circulating markers of inflammation robustly portend required to determine the physiological/nutritional CHAPTER 56 Vitamin D and Cardiovascular Medicine 903 contribution of VSMC VDR signaling in cardiovascular complex via protein-protein interaction with RXRα health. [104]. Whether similar complexes assemble on other Of note, the matrix cytokine OPN [83] is produced MyHC genes is unknown. Of note, similar multiprotein by vascular smooth muscle cells and is a direct target of VDR repressor complexes have been identified in the phosphate and glucose induction [84]. OPN enhances nVDRE of the PTHrP promoter [81,82]. the proliferation of VSMCs [85], regulates the size and The heart is an endocrine organ [105]; atrial car- extent of atheroma formation in animal models of diomyocytes produce natriuretic peptides that control atherosclerosis [86,87], and has the strong potential to cardiac development and renal sodium excretion [106]. promote intimal-medial thickening and calcification in Gardner and colleagues [107,108] demonstrated that the clinical settings of diabetes, atherosclerosis, and calcitriol suppresses transcription from the atrial natri- renal insufficiency [85,88Ð92]. As a Th1-type cytokine uretic peptide (ANP) promoter via ligand-dependent [93], OPN plays an important role in enhancing assembly of an unusual repressor complex containing macrophage activation and migration [94] in the coactivator GRIP1 [109]. Consistent with this, in atherosclerosis [86,87], in part by suppression of the the clinical setting of CHF, calcitriol and 25-hydroxy- anti-inflammatory cytokine IL-10 and upregulation of vitamin D are inversely correlated with pro-ANP lev- IL-12 [86]. However, OPN also can limit VSMC calcifi- els (pro-ANP levels determined by rates of production cation [87] in a phosphorylation-dependent manner [78]. and section) [110]. Potential benefits of manipulating Of note, OPN is directly upregulated by VDR signal- VDR signaling in the setting of acute and chronic CHF ing in osteoblasts [95] and in monocyte/macrophages have not been systematically examined, but would be [96,97], but apparently not in vascular smooth muscle predicted to have actions on hypertrophic responses, cells [76]. The net consequences and relative contribu- afterload reduction, cardiac output, and sodium/fluid tions of OPN cytokine induction vs. the direct, antipro- retention. liferative actions of vitamin D in leukocytes and VSMCs In addition to the genomic actions of VDR described have yet to be fully explored. above in cardiomyocytes, nongenomic actions of vita- min D also occur [111,112], including the rapid acti- vation of protein kinase A, tyrosine phosphorylation, B. Vitamin D Signaling in Cardiomyocytes and potentially store-operant calcium mobilization [113]. Boland’s expert contributions are most complete As mentioned previously, profound vitamin D defi- (Chapter 55) [114,115]; while ligand specificity is ciency is a recognized reversible cause of congestive relaxed, signaling still requires the presence of VDR heart failure in children, and allelic variations in the [114,115]. Drawing upon parallels that exist for non- VDR gene portend CHF severity. Simpson and col- genomic signaling by other “nuclear” receptors [116], leagues were among the first to identify VDR expres- a subpopulation of VDR tethered to a caveolar signal- sion in the cardiomyocytes, and detailed the evolution ing complex may be responsible for inducing this of cardiac hypertrophy in the vitamin D deficient response [117]. The reader is referred elsewhere in this rat [69,98,99]. A combination of in vivo and in vitro text for additional detail (Chapters 23 and 55). studies indicated that indirect actions (vide supra, section III A) likely contribute. Vitamin D participates in chamber-specific regulation of cardiac gene expres- C. Endothelial Responses to Vitamin D sion during development [100], and globally inhibits ventricular cardiomyocte myosin heavy chain gene As compared with actions of vitamin D in other cell expression (MyHC1, MyHC2, MyHC3) [101,102]. types, relatively little information exists concerning the Consistent with this, VDR −/− mice and vitamin D defi- mechanisms whereby VDR ligands regulate endothelial cient mice exhibit cardiac hypertrophy [16]. Moreover, cell (EC) functions. Calcitriol, via its down-regulation calcitriol antagonizes cardiomyocyte maturation of tissue factor (vide supra) in monocytes in response to [101,102] and endothelin mediated hypertrophy in inflammatory cytokines, inhibits coagulation cascade culture [103]. Genomic mechanisms appear to be activation during inflammation. Although actions in important for suppression of MyHC3 in ventricular umbilical vein ECs appear minimal [57], other ECs myocardium. A VDR:RXRα heterodimer binds to a have not been examined. In the pulmonary vasculature, negative vitamin D response element (nVDRE) about ICAM and ELAM expression and neutrophil margina- 780 bp upstream from the transcription initiation site tion is inhibited by calcitriol [118]. Indeed, evidence has of MyHC3 [100]. Irx4, a cardiac homeodomain protein, emerged that ECs may express a 1-alpha hydroxylase is recruited as a negative coregulator to the VDR:RXRα activity that produces calcitriol as paracrine/intracrine 904 DWIGHT A. TOWLER AND THOMAS L. CLEMENS regulator of EC activation to inflammation (Chapter 79) and/or chronic renal insufficiency [89,147]. Unlike the [119]. Additionally, Canfield and colleagues were calcification associated with initiation of the atheroscle- among the first to demonstrate that calcitriol inhibits rotic plaque, medial artery calcification initiates via an bovine aortic EC proliferation in response to vascular active osteogenic process that mimics intramembranous endothelial growth factor (VEGF) stimulation [120]. bone formation. There is matrix vesicle-dependent Moreover, calcitriol induced apoptosis of EC under- accumulation of calcium phosphate [148,149] occurring taking capillary sprouting in culture [120]. Consistent in immediate juxtaposition to a subset of medial VSMCs with this observation, in models of tumor angiogene- called CVCs (calcifying vascular cells) by Demer and sis, calcitriol and its derivatives function as anti-angio- colleagues [132,133]. The dysmetabolic state of dia- genic agents, suppressing EC proliferation and betes and hyperlipidemia upregulates a BMP2ÐMsx2 differentiation [121,122]. Such actions might be pre- regulated osteogenic potential in the aorta [91,132,150]. dicted to exert deleterious actions during collateral Data from several labs that pleuripotent microvascular vessel development in ischemic heart disease. However, smooth muscle cells—pericytic myofibroblasts— angiogenic responses may be tissue specific; calcitriol represent the wide-spread vascular osteoprogenitors that can also exert pro-angiogenic actions via parenchymal can be diverted to the osteogenic lineage [151Ð154]. cell-type specific induction of VEGF [123,124]. For example, calcitriol promotes vascularization of the chondro-osseous junction during endochondral bone B. Regulation of Vascular Calcification formation [125]. Calcitriol also upregulates VEGF pro- by Vitamin D duction in hypertrophic chondrocytes and osteoblasts [126Ð129] and increases VEGF release in an aortic The mechanisms controlling the active and passive VSMC cell culture model [130]. Thus, a great deal components of vascular calcification are poorly under- more is to be learned on the net effects of vitamin D on stood. Price and colleagues have extensively imple- vascular health as programmed by endothelial compo- mented a vitamin D intoxication/calciphylaxis model nents of this complex organ system. to study the pharmacological regulation of vascular calcification in the rat [155Ð158]. Their data strongly suggest that vascular calcium accumulation is inversely V. VASCULAR CALCIFICATION related to net calcium influx into bone [156]. Morii and AND CALCITROPIC HORMONES: colleagues identified an important role for paracrine CARDIOVASCULAR TOXICOLOGY PTHrP signaling in a culture model of vascular calcifi- OF VITAMIN D cation [76]. They demonstrated that calcitriol promotes bovine VSMC alkaline phosphatase expression and A. Overview of Vascular Calcification calcification in part by suppression of endogenous VSMC PTHrP [76]. Moreover, calcitriol-induced alka- Vascular calcification is a heterogeneous disease line phosphatase expression and VSMC calcification is that can be usefully categorized into a minimum of inhibited in a dose-dependent fashion by PTHrP [76]. three histopathological variants—atherosclerotic inti- Thus, an additional contribution of vitamin D excess mal calcification, valvular calcification, and medial to arterial calcification may occur via the down- artery calcification. Calcification of atherosclerotic regulation of a paracrine PTHrP signal. Of note, in the plaques has been well-known for over a century, first LDL receptor −/− mouse, intermittent administration recognized and denoted by Virchow [131]. Initial cal- of PTH suppresses vascular calcification while promot- cium deposition is probably dystrophic, with calcium ing orthotopic mineral deposition (Towler et al, unpub- phosphate deposition visualized in extracellular matrix lished), consistent with the in vitro results of Morii fibrils of the intima, fibrinous aggregates, and necrotic [76]. Given the results outlined above, it is tempting to cellular debris. However, with progression, active ossi- speculate that the vasculature has a protective surveil- fication processes resembling bone formation are lance system for preventing medial calcification pro- rapidly recruited [132Ð134]. Calcification of cardiac ceeding via VSMC PTH/PTHrP receptor signaling— valves—most notably the aortic and mitral valves— recruited by either pulsatile PTH(1-34) administration has been shown to be histologically similar to the process (Towler, unpublished observations) or paracrine of endochondral and intramembranous ossification PTHrP production [76]. In the setting of vitamin D [135,136]. In one key study, Kaplan and colleagues toxicosis [155Ð158] calcitriol may enhance vascular identified that 13% of calcified valves have histologi- mineral deposition by suppression of protective PTH cal evidence of lamellar bone formation [136]. Medial and PTHrP signals [76]—in addition to elevating the calcification occurs in response to diabetes [137Ð146] pro-calcific calcium phosphate product [46] (vide supra). CHAPTER 56 Vitamin D and Cardiovascular Medicine 905

Demer and colleagues once again published a sem- working model of vitamin D nutrition in cardiovascu- inal study demonstrating an inverse relationship lar health has yet to be established. Mechanism-based between calcitriol levels and coronary calcification as preclinical studies that dissect cell-type specific contri- quantified by electron beam computed tomography [159] butions of VDR signaling to cardiovascular biology are suggesting a protective effect of calcitriol signaling required—approachable in genetically manipulatable within the physiological range. This relationship held mouse models (Chapter 20). Such basic research in both individuals at high risk for coronary disease investigations will greatly complement the ongoing (patients heterozygous for the LDL receptor deficiency clinical studies that explore the utility of VDR ligands of familial hypercholesterolemia) and in normal indi- in cardiovascular medicine. Additional contributions viduals [15,159]. Unfortunately, levels of 25-hydroxy- by biologists, endocrinologists, nutritionists, epidemi- vitamin D—the longer lived metabolite that accurately ologists, and clinicians are sorely needed in order to reflects vitamin D nutrition—were not assessed in this develop a better understanding of vascular biology and study [15,159]. Nevertheless, these data provide tanta- vitamin D nutrition—immediately relevant to promoting lizing evidence that arterial vascular disease—whether the health of our aging population. coronary [159] or peripheral [15]—may be beneficially modulated by vitamin D metabolites. References

VI. SUMMARY AND CONCLUSIONS 1. DeLuca HF 1998 The vitamin D story: a collaborative effort of basic science and clinical medicine. Faseb J 2(3):224Ð236. Without a doubt, vitamin D exerts important physio- 2. Holick MF 2003 Vitamin D: A millenium perspective. J Cell logical and pathophysiological actions in the vascula- Biochem 88(2):296Ð307. 3. Smith EL, Pincus SH, Donovan L, Holick MF 1988 A novel ture. A biphasic cardiovascular “dose-response” curve approach for the evaluation and treatment of psoriasis. Oral is emerging that highlights the benefits of adequate or topical use of 1,25-dihydroxyvitamin D3 can be a safe vitamin D nutrition and the deleterious consequences and effective therapy for psoriasis. J Am Acad Dermatol of vitamin D excess or deficiency (Fig. 1). Both direct 19(3):516Ð528. and indirect mechanisms contribute to the net vascular 4. Fletcher RH, Fairfield KM 2002 Vitamins for chronic disease prevention in adults: clinical applications. Jama 287(23): responses to vitamin D signaling. Whereas most ani- 3127Ð3129. mal studies have emphasized the role of excessive vita- 5. Doherty TM, Tang W, Dascalos S, Watson KE, Demer LL, min D in vascular toxicity, studies in humans have Shavelle RM, Detrano RC 1997 Ethnic origin and serum studied consequences of nutritional deficiency on car- levels of 1alpha,25-dihydroxyvitamin D3 are independent diovascular health. Because of the biphasic response predictors of coronary calcium mass measured by electron- beam computed tomography. Circulation 96(5):1477Ð1481. curve, the added complexity of both direct vs. indirect 6. DeLuca HF, Zierold C 1998 Mechanisms and functions of mechanisms (for example secondary to changes in vitamin D. Nutr Rev 56(2 Pt 2):S4Ð10; discussion S54ÐS75. calcium homeostasis), and the paucity of “bench-to- 7. Becker BN, Hullett DA, O’Herrin JK, Malin G, Sollinger bedside” research programs in the area, a detailed HW, DeLuca H 2002 Vitamin D as immunomodulatory therapy for kidney transplantation. Transplantation 74(8): 1204Ð1206. 8. Adorini L, Penna G, Giarratana N, Uskokovic M 2003 Tolerogenic dendritic cells induced by vitamin D receptor ligands enhance regulatory T cells inhibiting allograft Suppresses VSMC PTHrP, Increases Alkaline Phosphatase rejection and autoimmune diseases. J Cell Biochem 88(2): Promotes VascularIncreases Calcification, Serum Calcium Myocardial x Phosphate Calcinosis Product 227Ð233. 9. Rosenblum IY, Black HE, Ferrell JF 1977 The effects of var- ious diphosphonates on a rat model of cardiac calciphylaxis. Calcif Tissue Res 23(2):151Ð159. 10. Block GA 2000 Prevalence and clinical consequences of ele- vated Ca × P product in hemodialysis patients. Clin Nephrol 54(4):318Ð324. 11. Cannon EP, Williams BJ 1990 Raised vascular calcium in an Suppresses Cardiovascular Hypertrophy animal model: effects on aortic function. Cardiovasc Res 24(1):47Ð52. Reduces Blood Pressure & Diabetes Risk Cardiovascular Health 12. Kingma JG Jr., Roy PE 1988 Ultrastructural study of hyper- Decreases Renin-Angiotensin-AldosteroneNutrition Axis Activity Toxicity vitaminosis D induced arterial calcification in Wistar rats. Vitamin D Dosage Artery 16(1):51Ð61. 13. Niederhoffer N, Bobryshev YV, Lartaud-Idjouadiene I, FIGURE 1 A biphasic vitamin D dose-response curve for car- Giummelly P, Atkinson J 1997 Aortic calcification produced diovascular health. by vitamin D3 plus nicotine. J Vasc Res 34(5):386Ð398. 906 DWIGHT A. TOWLER AND THOMAS L. CLEMENS

14. Pfeifer M, Begerow B, Minne HW, Nachtigall D, Hansen C 30. Ortlepp JR, Hoffmann R, Ohme F, Lauscher J, Bleckmann F, 2001 Effects of a short-term vitamin D3 and calcium Hanrath P 2001 The vitamin D receptor genotype predis- supplementation on blood pressure and parathyroid hormone poses to the development of calcific aortic valve stenosis. levels in elderly women. J Clin Endocrinol Metab 86(4): Heart 85(6):635Ð638. 1633Ð1637. 31. Motohashi Y, Yamada S, Yanagawa T, Maruyama T, Suzuki R, 15. Fahrleitner A, Dobnig H, Obernosterer A, Pilger E, Leb G, Niino M, Fukazawa T, Kasuga A, Hirose H, Matsubara K, Weber K, Kudlacek S, Obermayer-Pietsch BM 2002 Vitamin D and others 2003 Vitamin D receptor gene polymorphism affects deficiency and secondary hyperparathyroidism are common onset pattern of type 1 diabetes. J Clin Endocrinol Metab complications in patients with peripheral arterial disease. 88(7):3137Ð3140. J Gen Intern Med 17(9):663Ð669. 32. Ortlepp JR, Lauscher J, Hoffmann R, Hanrath P, Joost HG 16. Li YC, Kong J, Wei M, Chen ZF, Liu SQ, Cao LP 2002 1,25- 2001 The vitamin D receptor gene variant is associated with Dihydroxyvitamin D3 is a negative endocrine regulator of the the prevalence of type 2 diabetes mellitus and coronary artery renin-angiotensin system. J Clin Invest 110(2):229Ð238. disease. Diabet Med 18(10):842Ð845. 17. Raisanen-Sokolowski AK, Pakkala IS, Samila SP, Binderup L, 33. Ortlepp JR, von Korff A, Hanrath P, Zerres K, Hoffmann R Hayry PJ, Pakkala ST 1997 A vitamin D analog, MC1288, 2003 Vitamin D receptor gene polymorphism BsmI is not inhibits adventitial inflammation and suppresses intimal associated with the prevalence and severity of CAD in a lesions in rat aortic allografts. Transplantation 63(7):936Ð941. large-scale angiographic cohort of 3441 patients. Eur J Clin 18. Moreno PR, Purushothaman KR, Fuster V, O’Connor WN Invest 33(2):106Ð109. 2002 Intimomedial interface damage and adventitial inflam- 34. Hypponen E, Laara E, Reunanen A, Jarvelin MR, Virtanen SM mation is increased beneath disrupted atherosclerosis in the 2001 Intake of vitamin D and risk of type 1 diabetes: a birth- aorta: implications for plaque vulnerability. Circulation cohort study. Lancet 358(9292):1500Ð1503. 105(21):2504Ð2511. 35. Chudek J, Karkoszka H, Schmidt-Gayk H, Ritz E, Kokot F 19. Rostand SG 1997 Ultraviolet light may contribute to geo- 2000 Plasma parathyroid hormone, phosphatemia and graphic and racial blood pressure differences. Hypertension vitamin D receptor genotype: are they interrelated? J Nephrol 30(2 Pt 1):150Ð156. 13(1):54Ð58. 20. Scragg R, Holdaway I, Jackson R, Lim T 1992 Plasma 36. Nagaba Y, Heishi M, Tazawa H, Tsukamoto Y, Kobayashi Y 25-hydroxyvitamin D3 and its relation to physical activity 1998 Vitamin D receptor gene polymorphisms affect sec- and other heart disease risk factors in the general population. ondary hyperparathyroidism in hemodialyzed patients. Am J Ann Epidemiol 2(5):697Ð703. Kidney Dis 32(3):464Ð469. 21. Scragg R, Jackson R, Holdaway IM, Lim T, Beaglehole R 37. Marco MP, Martinez I, Betriu A, Craver L, Fibla MJ, 1990 Myocardial infarction is inversely associated with Fernandez E 2001 Influence of Bsml vitamin D receptor gene plasma 25-hydroxyvitamin D3 levels: a community-based polymorphism on the response to a single bolus of calcitrol study. Int J Epidemiol 19(3):559Ð563. in hemodialysis patients. Clin Nephrol 56(2):111Ð116. 22. Wassertheil-Smoller S, Hendrix SL, Limacher M, Heiss G, 38. Marco MP, Craver L, Betriu A, Fibla J, Fernandez E 2001 Kooperberg C, Baird A, Kotchen T, Curb JD, Black H, Influence of vitamin D receptor gene polymorphisms on Rossouw JE, and others 2003 Effect of estrogen plus progestin mortality risk in hemodialysis patients. Am J Kidney Dis on stroke in postmenopausal women: the Women’s Health 38(5):965Ð974. Initiative: a randomized trial. JAMA 289(20):2673Ð2684. 39. Abdullah M, Bigras JL, McCrindle BW, Mustafa A 23. McGowan JA, Pottern L 2000 Commentary on the Women’s 1999 Dilated cardiomyopathy as a first sign of nutritional Health Initiative. Maturitas 34(2):109Ð112. vitamin D deficiency rickets in infancy. Can J Cardiol 15(6): 24. Design of the Women’s Health Initiative clinical trial and 699Ð701. observational study. The Women’s Health Initiative Study 40. Gulati S, Bajpai A, Juneja R, Kabra M, Bagga A, Kalra V Group 1998 Control Clin Trials 19(1):61Ð109. 2001 Hypocalcemic heart failure masquerading as dilated 25. MacDonald PN, Baudino TA, Tokumaru H, Dowd DR, cardiomyopathy. Indian J Pediatr 68(3):287Ð290. Zhang C 2001 Vitamin D receptor and nuclear receptor coac- 41. Uysal S, Kalayci AG, Baysal K 1999 Cardiac functions in tivators: crucial interactions in vitamin D-mediated transcrip- children with vitamin D deficiency rickets. Pediatr Cardiol tion. Steroids 66(3Ð5):171Ð176. 20(4):283Ð286. 26. Pike JW, Yamamoto H, Shevde NK 2002 Vitamin D receptor- 42. Nishio K, Mukae S, Aoki S, Itoh S, Konno N, Ozawa K, mediated gene regulation mechanisms and current concepts Satoh R, Katagiri T 2003 Congestive heart failure is associ- of vitamin D analog selectivity. Adv Ren Replace Ther ated with the rate of bone loss. J Intern Med 253(4):439Ð446. 9(3):168Ð174. 43. Lavoie JL, Sigmund CD 2003 Minireview: overview of the 27. Erben RG, Soegiarto DW, Weber K, Zeitz U, Lieberherr M, Renin-Angiotensin system—an endocrine and paracrine Gniadecki R, Moller G, Adamski J, Balling R 2002 Deletion system. Endocrinology 144(6):2179Ð2183. of deoxyribonucleic acid binding domain of the vitamin D 44. Holick MF 2002 Sunlight and vitamin D: both good for car- receptor abrogates genomic and nongenomic functions of diovascular health. J Gen Intern Med 17(9):733Ð735. vitamin D. Mol Endocrinol 16(7):1524Ð1537. 45. Silver J, Yalcindag C, Sela-Brown A, Kilav R, Naveh-Many T 28. Morrison NA, Qi JC, Tokita A, Kelly PJ, Crofts L, Nguyen TV, 1999 Regulation of the parathyroid hormone gene by vitamin D, Sambrook PN, Eisman JA 1994 Prediction of bone density calcium, and phosphate. Kidney Int Suppl 73:S2ÐS7. from vitamin D receptor alleles. Nature 367(6460):284Ð287. 46. Cozzolino M, Dusso AS, Slatopolsky E 2001 Role of 29. Riggs BL, Nguyen TV, Melton LJ, 3rd, Morrison NA, calcium-phosphate product and bone-associated proteins on O’Fallon WM, Kelly PJ, Egan KS, Sambrook PN, Muhs JM, vascular calcification in renal failure. J Am Soc Nephrol Eisman JA 1995 The contribution of vitamin D receptor gene 12(11):2511Ð2516. alleles to the determination of bone mineral density in normal 47. Kistler A, Galli B, Horst R, Truitt GA, Uskokovic MR 1989 and osteoporotic women. J Bone Miner Res 10(6):991Ð996. Effects of vitamin D derivatives on soft tissue calcification in CHAPTER 56 Vitamin D and Cardiovascular Medicine 907

neonatal and calcium mobilization in adult rats. Arch Toxicol Involvement of NF-kappaB downregulation in transcriptional 63(5):394Ð400. repression of the p40 gene. J Clin Invest 101(1):252Ð262. 48. Safadi FF, Thornton P, Magiera H, Hollis BW, Gentile M, 64. Uyemura K, Demer LL, Castle SC, Jullien D, Berliner JA, Haddad JG, Liebhaber SA, Cooke NE 1999 Osteopathy and Gately MK, Warrier RR, Pham N, Fogelman AM, Modlin RL resistance to vitamin D toxicity in mice null for vitamin D 1996 Cross-regulatory roles of interleukin (IL)-12 and IL-10 binding protein. J Clin Invest 103(2):239Ð251. in atherosclerosis. J Clin Invest 97(9):2130Ð2138. 49. Russell J, Lettieri D, Sherwood LM 1986 Suppression by 65. Libby P 2003 Vascular biology of atherosclerosis: overview 1,25(OH)2D3 of transcription of the preproparathyroid hor- and state of the art. Am J Cardiol 91(3A):3AÐ6A. mone gene. Endocrinology 119(6):2864Ð2866. 66. Rebsamen MC, Sun J, Norman AW, Liao JK 2002 1α,25- 50. Russell J, Ashok S, Koszewski NJ 1999 Vitamin D receptor dihydroxyvitamin D3 induces vascular smooth muscle cell interactions with the rat parathyroid hormone gene: synergistic migration via activation of phosphatidylinositol 3-kinase. effects between two negative vitamin D response elements. Circ Res 91(1):17Ð24. J Bone Miner Res 14(11):1828Ð1837. 67. Rajasree S, Umashankar PR, Lal AV, Sarma PS, Kartha CC 51. Carling T, Kindmark A, Hellman P, Lundgren E, Ljunghall S, 2002 1,25-dihydroxyvitamin D3 receptor is upregulated in Rastad J, Akerstrom G, Melhus H 1995 Vitamin D receptor aortic smooth muscle cells during hypervitaminosis D. Life genotypes in primary hyperparathyroidism. Nat Med 1(12): Sci 70(15):1777Ð1788. 1309Ð1311. 68. Ishibashi K, Evans A, Shingu T, Bian K, Bukoski RD 1995 52. Rostand SG, Drueke TB 1999 Parathyroid hormone, vitamin D, Differential expression and effect of 1,25-dihydroxyvitamin D3 and cardiovascular disease in chronic renal failure. Kidney Int on myosin in arterial tree of rats. Am J Physiol 269(2 Pt 1): 56(2):383Ð392. C443ÐC450. 53. Alon US, Monzavi R, Lilien M, Rasoulpour M, Geffner ME, 69. Weishaar RE, Simpson RU 1987 Vitamin D3 and cardiovas- Yadin O. Hypertension in hypophosphatemic rickets-role cular function in rats. J Clin Invest 79(6):1706Ð1712. of secondary hyperparathyroidism. Pediatr Nephrol 18(2): 70. Fraga C, Blanco M, Vigo E, Segura C, Garcia-Caballero T, 155Ð158. Perez-Fernandez R 2002 Ontogenesis of the vitamin D 54. Jorde R, Sundsfjord J, Haug E, Bonaa KH 2000 Relation receptor in rat heart. Histochem Cell Biol 117(6):547Ð550. between low calcium intake, parathyroid hormone, and blood 71. O’Connell TD, Simpson RU 1996 Immunochemical identifi- pressure. Hypertension 35(5):1154Ð1159. cation of the 1,25-dihydroxyvitamin D3 receptor protein in 55. Jorde R, Bonaa KH 2000 Calcium from dairy products, vita- human heart. Cell Biol Int 20(9):621Ð624. min D intake, and blood pressure: the Tromso Study. Am J 72. Merke J, Milde P, Lewicka S, Hugel U, Klaus G, Clin Nutr 71(6):1530Ð1535. Mangelsdorf DJ, Haussler MR, Rauterberg EW, Ritz E 1989 56. Saleh F, Jorde R, Sundsfjord J 2003 Effect of calcium sup- Identification and regulation of 1,25-dihydroxyvitamin D3 plementation on blood pressure in patients with secondary receptor activity and biosynthesis of 1,25-dihydroxyvitamin D3. hyperparathyroidism. J Endocrinol Invest 26(1):35Ð41. Studies in cultured bovine aortic endothelial cells and human 57. Koyama T, Shibakura M, Ohsawa M, Kamiyama R, dermal capillaries. J Clin Invest 83(6):1903Ð1915. Hirosawa S 1998 Anticoagulant effects of 1alpha,25-dihy- 73. Mitsuhashi T, Morris RC Jr., Ives HE 1991 1,25-dihydroxy- droxyvitamin D3 on human myelogenous leukemia cells and vitamin D3 modulates growth of vascular smooth muscle monocytes. Blood 92(1):160Ð167. cells. J Clin Invest 87(6):1889Ð1895. 58. Ohsawa M, Koyama T, Yamamoto K, Hirosawa S, Kamei S, 74. Fleckenstein-Grun G, Thimm F, Frey M, Matyas S 1995 Kamiyama R 2000 1alpha,25-dihydroxyvitamin D3 and its Progression and regression by verapamil of vitamin D3Ð potent synthetic analogs downregulate tissue factor and induced calcific medial degeneration in coronary arteries of upregulate thrombomodulin expression in monocytic cells, rats. J Cardiovasc Pharmacol 26(2):207Ð213. counteracting the effects of tumor necrosis factor and oxi- 75. Dahl PR, Winkelmann RK, Connolly SM 1995 The vascular dized LDL. Circulation 102(23):2867Ð2872. calcification-cutaneous necrosis syndrome. J Am Acad 59. Asakura H, Aoshima K, Suga Y, Yamazaki M, Morishita E, Dermatol 33(1):53Ð58. Saito M, Miyamoto K, Nakao S 2001 Beneficial effect of the 76. Jono S, Nishizawa Y, Shioi A, Morii H 1998 1,25- active form of vitamin D3 against LPS-induced DIC but not Dihydroxyvitamin D3 increases in vitro vascular calcification against tissue-factor-induced DIC in rat models. Thromb by modulating secretion of endogenous parathyroid hormone- Haemost 85(2):287Ð290. related peptide. Circulation 98(13):1302Ð1306. 60. Liu M, Lee MH, Cohen M, Bommakanti M, Freedman LP 77. Shioi A, Katagi M, Okuno Y, Mori K, Jono S, Koyama H, 1996 Transcriptional activation of the Cdk inhibitor p21 Nishizawa Y 2002 Induction of bone-type alkaline phosphatase by vitamin D3 leads to the induced differentiation of in human vascular smooth muscle cells: roles of tumor necro- the myelomonocytic cell line U937. Genes Dev 10(2): sis factor-alpha and oncostatin M derived from macrophages. 142Ð153. Circ Res 91(1):9Ð16. 61. Rots NY, Iavarone A, Bromleigh V, Freedman LP 1999 78. Jono S, Peinado C, Giachelli CM 2000 Phosphoryla- Induced differentiation of U937 cells by 1,25-dihydroxy- tion of osteopontin is required for inhibition of vascular vitamin D3 involves cell cycle arrest in G1 that is preceded by smooth muscle cell calcification. J Biol Chem 275(26): a transient proliferative burst and an increase in cyclin 20197Ð20203. expression. Blood 93(8):2721Ð2729. 79. Hessle L, Johnson KA, Anderson HC, Narisawa S, Sali A, 62. Scott LJ, Dunn CJ, Goa KL 2001 Calcipotriol ointment. A Goding JW, Terkeltaub R, Millan JL 2002 Tissue-nonspecific review of its use in the management of psoriasis. Am J Clin alkaline phosphatase and plasma cell membrane glycoprotein-1 Dermatol 2(2):95Ð120. are central antagonistic regulators of bone mineralization. 63. D’Ambrosio D, Cippitelli M, Cocciolo MG, Mazzeo D, Proc Natl Acad Sci USA 99(14):9445Ð9449. Di Lucia P, Lang R, Sinigaglia F, Panina-Bordignon P 1998 80. Steitz SA, Speer MY, McKee MD, Liaw L, Almeida M, Yang H, Inhibition of IL-12 production by 1,25-dihydroxyvitamin D3. Giachelli CM 2002 Osteopontin inhibits mineral deposition 908 DWIGHT A. TOWLER AND THOMAS L. CLEMENS

and promotes regression of ectopic calcification. Am J Pathol and 1,25-dihydroxyvitamin D3 enhancement of mouse 161(6):2035Ð2046. secreted phosphoprotein 1 (SPP-1 or osteopontin) gene 81. Okazaki T, Nishimori S, Ogata E, Fujita T 2003 Vitamin DÐ expression. Proc Natl Acad Sci USA 87(24):9995Ð9999. dependent recruitment of DNA-PK to the chromatinized 96. Andersson G, Johansson EK 1996 Adhesion of human negative vitamin D response element in the PTHrP gene is myelomonocytic (HL-60) cells induced by 1,25-dihydroxy- required for gene repression by vitamin D. Biochem Biophys vitamin D3 and phorbol myristate acetate is dependent on Res Commun 304(4):632Ð637. osteopontin synthesis and the alpha v beta 3 integrin. Connect 82. Nishishita T, Okazaki T, Ishikawa T, Igarashi T, Hata K, Tissue Res 35(1Ð4):163Ð171. Ogata E, Fujita T 1998 A negative vitamin D response DNA 97. Miyazaki Y, Setoguchi M, Yoshida S, Higuchi Y, Akizuki S, element in the human parathyroid hormone-related peptide Yamamoto S 1990 The mouse osteopontin gene. Expression gene binds to vitamin D receptor along with Ku antigen to in monocytic lineages and complete nucleotide sequence. mediate negative gene regulation by vitamin D. J Biol Chem J Biol Chem 265(24):14432Ð14438. 273(18):10901Ð10907. 98. Weishaar RE, Simpson RU 1987 Involvement of vitamin D3 83. Denhardt DT, Noda M, O’Regan AW, Pavlin D, Berman JS. with cardiovascular function. II. Direct and indirect effects. Osteopontin as a means to cope with environmental insults: Am J Physiol 253(6 Pt 1):E675Ð83. regulation of inflammation, tissue remodeling, and cell 99. Weishaar RE, Kim SN, Saunders DE, Simpson RU 1990 survival. J Clin Invest 107(9):1055Ð1061. Involvement of vitamin D3 with cardiovascular function. III. 84. Beck GR, Jr., Zerler B, Moran E 2000 Phosphate is a specific Effects on physical and morphological properties. Am J Physiol signal for induction of osteopontin gene expression. Proc 258(1 Pt 1):E134ÐE142. Natl Acad Sci USA 97(15):8352Ð8357. 100. Wang GF, Nikovits W, Jr., Schleinitz M, Stockdale FE 1998 85. Isoda K, Nishikawa K, Kamezawa Y, Yoshida M, Kusuhara M, A positive GATA element and a negative vitamin D receptor- Moroi M, Tada N, Ohsuzu F 2002 Osteopontin plays an like element control atrial chamber-specific expression of a important role in the development of medial thickening and slow myosin heavy-chain gene during cardiac morphogenesis. neointimal formation. Circ Res 91(1):77Ð82. Mol Cell Biol 18(10):6023Ð6034. 86. Isoda K, Kamezawa Y, Ayaori M, Kusuhara M, Tada N, 101. O’Connell TD, Weishaar RE, Simpson RU 1994 Regulation Ohsuzu F 2003 Osteopontin transgenic mice fed a high- of myosin isozyme expression by vitamin D3 deficiency and cholesterol diet develop early fatty-streak lesions. Circulation 1,25-dihydroxyvitamin D3 in the rat heart. Endocrinology 107(5):679Ð681. 134(2):899Ð905. 87. Matsui Y, Rittling SR, Okamoto H, Inobe M, Jia N, Shimizu T, 102. O’Connell TD, Giacherio DA, Jarvis AK, Simpson RU 1995 Akino M, Sugawara T, Morimoto J, Kimura C, and others Inhibition of cardiac myocyte maturation by 1,25-dihydroxy- 2003 Osteopontin deficiency attenuates atherosclerosis in vitamin D3. Endocrinology 136(2):482Ð488. female apolipoprotein e-deficient mice. Arterioscler Thromb 103. Wu J, Garami M, Cheng T, Gardner DG 1996 1,25(OH)2 Vasc Biol 23(6):1029Ð1034. vitamin D3, and retinoic acid antagonize endothelinÐstimulated 88. Nitta K, Ishizuka T, Horita S, Hayashi T, Ajiro A, Uchida K, hypertrophy of neonatal rat cardiac myocytes. J Clin Invest Honda K, Oba T, Kawashima A, Yumura W, and others 2001 97(7):1577Ð1588. Soluble osteopontin and vascular calcification in hemodialysis 104. Wang GF, Nikovits W, Jr., Bao ZZ, Stockdale FE 2001 Irx4 patients. Nephron 89(4):455Ð458. forms an inhibitory complex with the vitamin D and retinoic 89. Moe SM, O’Neill KD, Duan D, Ahmed S, Chen NX, X receptors to regulate cardiac chamber-specific slow MyHC3 Leapman SB, Fineberg N, Kopecky K 2002 Medial artery expression. J Biol Chem 276(31):28835Ð28841. calcification in ESRD patients is associated with deposition 105. Needleman P, Greenwald JE 1986 Atriopeptin: a cardiac of bone matrix proteins. Kidney Int 61(2):638Ð647. hormone intimately involved in fluid, electrolyte, and blood- 90. Chen NX, Moe SM 2003 Arterial calcification in diabetes. pressure homeostasis. N Engl J Med 314(13):828Ð834. Curr Diab Rep 3(1):28Ð32. 106. Cameron VA, Ellmers LJ 2003 Minireview: Natriuretic Peptides 91. Towler DA, Bidder M, Latifi T, Coleman T, Semenkovich CF during Development of the Fetal Heart and Circulation. 1998 Diet-induced diabetes activates an osteogenic gene Endocrinology 144(6):2191Ð2194. regulatory program in the aortas of low density lipoprotein 107. Wu J, Garami M, Cao L, Li Q, Gardner DG 1995 1,25(OH)2D3 receptor-deficient mice. J Biol Chem 273(46):30427Ð30434. suppresses expression and secretion of atrial natriuretic 92. Bidder M, Shao JS, Charlton-Kachigian N, Loewy AP, peptide from cardiac myocytes. Am J Physiol 268(6 Pt 1): Semenkovich CF, Towler DA 2002 Osteopontin transcription E1108ÐE1113. in aortic vascular smooth muscle cells is controlled by glucose- 108. Li Q, Gardner DG 1994 Negative regulation of the human regulated upstream stimulatory factor and activator protein-1 atrial natriuretic peptide gene by 1,25-dihydroxyvitamin D3. activities. J Biol Chem 277(46):44485Ð44496. J Biol Chem 269(7):4934Ð4939. 93. Ashkar S, Weber GF, Panoutsakopoulou V, Sanchirico ME, 109. Chen S, Cui J, Nakamura K, Ribeiro RC, West BL, Gardner DG Jansson M, Zawaideh S, Rittling SR, Denhardt DT, 2000 Coactivator-vitamin D receptor interactions mediate Glimcher MJ, Cantor H 2001 Eta-1 (osteopontin): an early inhibition of the atrial natriuretic peptide promoter. J Biol component of type-1 (cell-mediated) immunity. Science Chem 275(20):15039Ð15048. 287(5454):860Ð864. 110. Zittermann A, Schleithoff SS, Tenderich G, Berthold HK, 94. Weber GF, Zawaideh S, Hikita S, Kumar VA, Cantor H, Korfer R, Stehle P 2003 Low vitamin D status: a contributing Ashkar S 2002 PhosphorylationÐdependent interaction of factor in the pathogenesis of congestive heart failure? J Am osteopontin with its receptors regulates macrophage migra- Coll Cardiol 41(1):105Ð112. tion and activation. J Leukoc Biol 72(4):752Ð761. 111. Selles J, Boland R 1991 Evidence on the participation of the 95. Noda M, Vogel RL, Craig AM, Prahl J, DeLuca HF, 3′,5′-cyclic AMP pathway in the nongenomic action of 1,25- Denhardt DT 1990 Identification of a DNA sequence respon- dihydroxy-vitamin D3 in cardiac muscle. Mol Cell Endocrinol sible for binding of the 1,25-dihydroxyvitamin D3 receptor 82(2Ð3):229Ð235. CHAPTER 56 Vitamin D and Cardiovascular Medicine 909

112. Santillan GE, Boland RL 1998 Studies suggesting the participa- endothelial growth factor mRNA expression by 1,25-dihy- tion of protein kinase A in 1,25(OH)2-vitamin D3Ðdependent droxyvitamin D3 in human osteoblast-like cells. J Bone protein phosphorylation in cardiac muscle. J Mol Cell Cardiol Miner Res 11(4):472Ð479. 30(2):225Ð233. 128. Schlaeppi JM, Gutzwiller S, Finkenzeller G, Fournier B 1997 113. Vazquez G, de Boland AR, Boland RL 2000 Involvement of 1,25-Dihydroxyvitamin D3 induces the expression of vascular calmodulin in 1alpha,25-dihydroxyvitamin D3 stimulation of endothelial growth factor in osteoblastic cells. Endocr Res store-operated Ca2+ influx in skeletal muscle cells. J Biol 23(3):213Ð229. Chem 275(21):16134Ð16138. 129. Wang DS, Miura M, Demura H, Sato K 1997 Anabolic 114. Boland R, De Boland AR, Buitrago C, Morelli S, Santillan G, effects of 1,25-dihydroxyvitamin D3 on osteoblasts are Vazquez G, Capiati D, Baldi C 2002 Nongenomic stimulation enhanced by vascular endothelial growth factor produced by of tyrosine phosphorylation cascades by 1,25(OH)2D3 by osteoblasts and by growth factors produced by endothelial VDRÐdependent and Ðindependent mechanisms in muscle cells. Endocrinology 138(7):2953Ð2962. cells. Steroids 67(6):477Ð482. 130. Yamamoto T, Kozawa O, Tanabe K, Akamatsu S, Matsuno H, 115. Capiati D, Benassati S, Boland RL 2002 1,25(OH)2-vitamin D3 Dohi S, Hirose H, Uematsu T 2002 1,25-dihydroxyvitamin D3 induces translocation of the vitamin D receptor (VDR) to the stimulates vascular endothelial growth factor release in aortic plasma membrane in skeletal muscle cells. J Cell Biochem smooth muscle cells: role of p38 mitogen-activated protein 86(1):128Ð35. kinase. Arch Biochem Biophys 398(1):1Ð6. 116. Chambliss KL, Yuhanna IS, Mineo C, Liu P, German Z, 131. Virchow R 1989 Cellular pathology. As based upon physiolog- Sherman TS, Mendelsohn ME, Anderson RG, Shaul PW ical and pathological histology. Lecture XVIÐAtheromatous 2000 Estrogen receptor alpha and endothelial nitric oxide affection of arteries. 1858. Nutr Rev 47(1):23Ð25. synthase are organized into a functional signaling module in 132. Bostrom K, Watson KE, Horn S, Wortham C, Herman IM, caveolae. Circ Res 87(11):E44ÐE52. Demer LL 1993 Bone morphogenetic protein expression in 117. Norman AW, Olivera CJ, Barreto Silva FR, Bishop JE 2002 human atherosclerotic lesions. J Clin Invest 91(4):1800Ð1809. A specific binding protein/receptor for 1alpha,25-dihydroxy- 133. Bostrom K, Demer LL 2000 Regulatory mechanisms in vitamin D3 is present in an intestinal caveolae membrane vascular calcification. Crit Rev Eukaryot Gene Expr 10(2): fraction. Biochem Biophys Res Commun 298(3):414Ð419. 151Ð158. 118. Chen SF 1995 1alpha,25-Dihydroxyvitamin D3 decreased 134. Fitzpatrick LA, Turner RT, Ritman ER 2003 Endochondral ICAM-1 and ELAM-1 expressions on pulmonary microvas- bone formation in the heart: a possible mechanism of coro- cular endothelial cells and neutrophil motivation. J Steroid nary calcification. Endocrinology 144(6):2214Ð2219. Biochem Mol Biol 52(1):67Ð70. 135. Srivatsa SS, Harrity PJ, Maercklein PB, Kleppe L, Veinot J, 119. Zehnder D, Bland R, Chana RS, Wheeler DC, Howie AJ, Edwards WD, Johnson CM, Fitzpatrick LA 1997 Increased Williams MC, Stewart PM, Hewison M 2002 Synthesis of cellular expression of matrix proteins that regulate mineral- 1,25-dihydroxyvitamin D3 by human endothelial cells is regu- ization is associated with calcification of native human and lated by inflammatory cytokines: a novel autocrine determinant porcine xenograft bioprosthetic heart valves. J Clin Invest of vascular cell adhesion. J Am Soc Nephrol 13(3):621Ð629. 99(5):996Ð1009. 120. Mantell DJ, Owens PE, Bundred NJ, Mawer EB, Canfield AE 136. Mohler ER, 3rd, Gannon F, Reynolds C, Zimmerman R, 2000 1 alpha,25-dihydroxyvitamin D3 inhibits angiogenesis Keane MG, Kaplan FS 2001 Bone formation and inflamma- in vitro and in vivo. Circ Res 87(3):214Ð220. tion in cardiac valves. Circulation 103(11):1522Ð1528. 121. Bernardi RJ, Johnson CS, Modzelewski RA, Trump DL 2002 137. Mozes G, Keresztury G, Kadar A, Magyar J, Sipos B, Antiproliferative effects of 1alpha,25-dihydroxyvitamin D3 Dzsinich S, Gloviczki P 1998 Atherosclerosis in amputated and vitamin D analogs on tumorÐderived endothelial cells. legs of patients with and without diabetes mellitus. Int Angiol Endocrinology 143(7):2508Ð2514. 17(4):282Ð286. 122. Iseki K, Tatsuta M, Uehara H, Iishi H, Yano H, Sakai N, 138. Chantelau E, Lee KM, Jungblut R 1997 Distal arterial occlu- Ishiguro S 1999 Inhibition of angiogenesis as a mechanism sive disease in diabetes is related to medial arterial calcifica- for inhibition by 1alpha-hydroxyvitamin D3 and 1,25-dihy- tion. Exp Clin Endocrinol Diabetes 105(Suppl 2):11-13. droxyvitamin D3 of colon carcinogenesis induced by 139. Lehto S, Niskanen L, Suhonen M, Ronnemaa T, Laakso M azoxymethane in Wistar rats. Int J Cancer 81(5):730Ð733. 1996 Medial artery calcification. A neglected harbinger of car- 123. Ferrara N 2002 Role of vascular endothelial growth factor in diovascular complications in non-insulin-dependent diabetes physiologic and pathologic angiogenesis: therapeutic impli- mellitus. Arterioscler Thromb Vasc Biol 16(8):978Ð983. cations. Semin Oncol 29(6 Suppl 16):10Ð14. 140. Chantelau E, Lee KM, Jungblut R 1995 Association of 124. Ferrara N, Davis-Smyth T 1997 The biology of vascular below-knee atherosclerosis to medial arterial calcification in endothelial growth factor. Endocr Rev 18(1):4Ð25. diabetes mellitus. Diabetes Res Clin Pract 29(3):169Ð172. 125. Lin R, Amizuka N, Sasaki T, Aarts MM, Ozawa H, Goltzman D, 141. Niskanen L, Siitonen O, Suhonen M, Uusitupa MI 1994 Medial Henderson JE, White JH 2002 1α,25-dihydroxyvitamin D3 artery calcification predicts cardiovascular mortality in promotes vascularization of the chondro-osseous junction by patients with NIDDM. Diabetes Care 17(11):1252Ð1256. stimulating expression of vascular endothelial growth factor 142. Young MJ, Adams JE, Anderson GF, Boulton AJ, Cavanagh PR and matrix metalloproteinase 9. J Bone Miner Res 17(9): 1993 Medial arterial calcification in the feet of diabetic patients 1604Ð1612. and matched non-diabetic control subjects. Diabetologia 126. Martinez P, Esbrit P, Rodrigo A, Alvarez-Arroyo MV, 36(7):615Ð621. Martinez ME 2002 Age-related changes in parathyroid 143. Niskanen LK, Suhonen M, Siitonen O, Lehtinen JM, hormone-related protein and vascular endothelial growth factor Uusitupa MI 1990 Aortic and lower limb artery calcification in human osteoblastic cells. Osteoporos Int 13(11):874Ð881. in type 2 (non insulinÐdependent) diabetic patients and non 127. Wang DS, Yamazaki K, Nohtomi K, Shizume K, Ohsumi K, diabetic control subjects. A five year follow-up study. Shibuya M, Demura H, Sato K 1996 Increase of vascular Atherosclerosis 84(1):61Ð71. 910 DWIGHT A. TOWLER AND THOMAS L. CLEMENS

144. Everhart JE, Pettitt DJ, Knowler WC, Rose FA, Bennett PH 152. Diaz-Flores L, Gutierrez R, Lopez-Alonso A, Gonzalez R, 1988 Medial arterial calcification and its association with Varela H 1992 Pericytes as a supplementary source of mortality and complications of diabetes. Diabetologia osteoblasts in periosteal osteogenesis. Clin Orthop 275: 31(1):16Ð23. 280Ð286. 145. Nelson RG, Gohdes DM, Everhart JE, Hartner JA, Zwemer 153. Brighton CT, Lorich DG, Kupcha R, Reilly TM, Jones AR, FL, Pettitt DJ, Knowler WC 1988 Lower-extremity amputa- Woodbury RA, 2nd 1992 The pericyte as a possible osteoblast tions in NIDDM. 12-yr follow-up study in Pima Indians. progenitor cell. Clin Orthop 275:287Ð299. Diabetes Care 11(1):8Ð16. 154. Zuk PA, Zhu M, Mizuno H, Huang J, Futrell JW, Katz AJ, 146. Lithner F, Hietala SO, Steen L 1984 Skeletal lesions and arte- Benhaim P, Lorenz HP, Hedrick MH 2001 Multilineage rial calcifications of the feet in diabetics. Acta Med Scand cells from human adipose tissue: implications for cellÐbased Suppl 687:47Ð54. therapies. Tissue Eng 7(2):211Ð228. 147. Shioi A, Taniwaki H, Jono S, Okuno Y, Koyama H, Mori K, 155. Price PA, June HH, Buckley JR, Williamson MK 2001 Nishizawa Y 2001 Monckeberg’s medial sclerosis and Osteoprotegerin inhibits artery calcification induced by inorganic phosphate in uremia. Am J Kidney Dis 38(4 Suppl 1): warfarin and by vitamin D. Arterioscler Thromb Vasc Biol S47ÐS49. 21(10):1610Ð1616. 148. Tanimura A, McGregor DH, Anderson HC 1986 Calcification 156. Price PA, Faus SA, Williamson MK 2001 Bisphosphonates in atherosclerosis. I. Human studies. J Exp Pathol 2(4): alendronate and ibandronate inhibit artery calcification at 261Ð273. doses comparable to those that inhibit bone resorption. 149. Tanimura A, McGregor DH, Anderson HC 1986 Matrix vesi- Arterioscler Thromb Vasc Biol 21(5):817Ð824. cles in atherosclerotic calcification. Proc Soc Exp Biol Med 157. Price PA, Buckley JR, Williamson MK 2001 The amino 172(2):173Ð177. bisphosphonate ibandronate prevents vitamin D toxicity 150. Tyson KL, Reynolds JL, McNair R, Zhang Q, Weissberg PL, and inhibits vitamin DÐinduced calcification of arteries, car- Shanahan CM 2003 Osteo/chondrocytic transcription factors tilage, lungs, and kidneys in rats. J Nutr 131(11):2910Ð2915. and their target genes exhibit distinct patterns of expression 158. Price PA, June HH, Buckley JR, Williamson MK 2002 SB in human arterial calcification. Arterioscler Thromb Vasc 242784, a selective inhibitor of the osteoclastic V-H+ATPase, Biol 23(3):489Ð494. inhibits arterial calcification in the rat. Circ Res 91(6):547Ð552. 151. Doherty MJ, Ashton BA, Walsh S, Beresford JN, Grant ME, 159. Watson KE, Abrolat ML, Malone LL, Hoeg JM, Doherty T, Canfield AE 1998 Vascular pericytes express osteogenic Detrano R, Demer LL 1997 Active serum vitamin D levels are potential in vitro and in vivo. J Bone Miner Res 13(5): inversely correlated with coronary calcification. Circulation 828Ð838. 96(6):1755Ð1760. CHAPTER 57 Approach to the Patient with Metabolic Bone Disease

MICHAEL P. W HYTE Center for Metabolic Bone Disease and Molecular Research, Shriners Hospitals for Children, and Division of Bone and Mineral Diseases, Washington University School of Medicine at Barnes-Jewish Hospital; St. Louis, Missouri

I. Introduction IV. Summary II. Diagnostic Evaluation References III. Treatment

I. INTRODUCTION the skeleton. Significantly, a variety of potent hormones and drugs that regulate mineral homeostasis and alter Metabolic bone disease traditionally encompasses a bone remodeling are now available to clinicians [3]. considerable number and variety of conditions [1Ð3]. This pharmaceutical armamentarium must be used In fact, the list is rapidly growing as the molecular knowledgeably for efficacy, yet safety. bases of inherited skeletal syndromes and dysplasias This chapter emphasizes a number of considerations are elucidated using DNA technology [4,5]. Although for the approach to the patient with metabolic bone dis- these disorders are often rare, several are epidemic ease, particularly those with disturbances in vitamin D in various regions of the world (e.g., postmenopausal homeostasis. Subsequent chapters in this section of the osteoporosis, vitamin D deficiency rickets). Some can book discuss in detail the radiology of rickets and be life-threatening (e.g., severe forms of osteopetrosis osteomalacia (Chapter 60), bone histomorphometry and osteogenesis imperfecta); others can be incidental (Chapter 59), measurement of the vitamin D metabolites findings (e.g., Paget bone disease and fibrous dysplasia). (Chapter 58), and the pharmacology and therapeutic use Patients reflect all ages. Cumulatively, the number of of vitamin D preparations (Chapter 61). individuals with clinically important metabolic bone disease is significant [1Ð3]. Diagnosis and treatment of metabolic bone disease II. DIAGNOSTIC EVALUATION can be both intriguing and satisfying, but there are numerous challenges. We now have at our disposal a Patients with metabolic bone disease are often chal- variety of techniques to image the skeleton and to mea- lenging to physicians because many nutritional, envi- sure bone mass, assays for many of the factors that ronmental, genetic, pharmacological, and toxic factors condition mineral and skeletal homeostasis as well as can disturb mineral metabolism and impact the skele- biochemical markers of bone turnover, and qualitative ton [2,3]. This book is testimony to the number and and quantitative histopathological methods to directly diversity of internal and external perturbations that can examine osseous tissue [6]. Furthermore, molecular impair the biosynthesis, bioactivation, and actions of tests for genetic disorders of the skeleton are becoming vitamin D. increasingly available in commercial and research lab- Patient age is yet another challenge for several reasons. oratories [4]. Initial and follow-up patient evaluation In infants, children, and adolescents, complications of benefit greatly from skilled use of these tools. metabolic bone disease can be especially severe and Nevertheless, clinical acumen is more important than complex because bone growth and modeling are ever. Judicious selection from among these advances occurring in addition to skeletal remodeling. All three in technology and circumspect interpretation of the physiological processes can be disturbed in pediatric information they provide comes from experience with patients, leading to impairment of growth and alter- patients. In fact, successful treatment of metabolic ations in the shape of bones. The outcome is novel phys- bone disease often requires multidisciplinary medical ical and radiological findings in children compared to approaches that may need to be especially broad-based adults. Patient age conditions the pathogenesis and clin- when there is deformity or other structural problems of ical manifestations of these disorders and also provides VITAMIN D, 2ND EDITION Copyright © 2005, Elsevier, Inc. FELDMAN, PIKE, AND GLORIEUX All rights reserved. 914 MICHAEL P. W HYTE a guide to the etiology. However, diagnoses can be For the physician skilled in medical history acquisition missed or delayed if the diagnostician is unaware that concerning metabolic bone disease, it is not unusual the reference ranges for some of the biochemical for the physical examination and laboratory testing to parameters of mineral homeostasis (e.g., serum inor- uphold a diagnosis. ganic phosphate, alkaline phosphatase) and all of the Physical examination of the patient with metabolic markers of skeletal turnover are different for infants disease may show findings for or against the diagnosis and children compared to adults [2,3]. Elderly patients suspected from the medical history, but the informa- are also uniquely challenging, because they are espe- tion is also important for revealing deformity or other cially likely to have metabolic bone disease with mul- structural problems of the skeleton requiring attention. tifactorial etiology and pathogenesis [1Ð3,7]. Radiological investigation of metabolic bone dis- Broad-based medical knowledge is necessary to ease may appropriately be minimal or extensive, but it fully understand the relationships between mineral and should be directed by the complete medical history and skeletal homeostasis and the consequences of specific physical examination. Not uncommonly, the diagnosis or complex disturbances [8]. Clinicians who encounter is established from characteristic radiographic findings patients with metabolic bone disease routinely need (e.g., Paget bone disease, fibrous dysplasia) [10Ð12]. If some of the skills of the endocrinologist, nutritionist, not, x-ray studies often provide an important basis for nephrologist, geneticist, and often the pediatrician or differential diagnosis (e.g., osteopenia, osteosclerosis, gerontologist. Familiarity with skeletal radiology and rickets, etc.) or support the diagnostic impression that pathology is required. Furthermore, if there is significant must instead be confirmed by additional tests (e.g., bony deformity, the expertise of orthopedics, rheuma- pseudohypoparathyroidism, mastocytosis, hyperparathy- tology, and rehabilitation medicine will be helpful. roidism). However, radiological studies are also useful Metabolic bone disease is remarkable for the many because they may help to assess the severity and evo- subspecialties that can contribute to the comprehensive lution of the disorder. In addition, they can reveal evaluation and effective care of patients. skeletal complications not detected by physical exam- Diagnosis of metabolic bone disease must begin ination. Some changes (e.g., physeal widening in rickets, with the acquisition of all of the important information osteolytic lesions in Paget bone disease, etc.) can then extractable from the medical history and obtainable be followed to precisely assess responses to medical from a thorough physical examination. This founda- treatment. tion should be supplemented with only the helpful and Biochemical testing is crucial to characterize any cost-effective choices from the ever-expanding menu disturbance of mineral homeostasis. Furthermore, of biochemical tests, as well as the available radiolog- biochemical testing provides quantitative information ical and histological studies. Most situations will to help guide the intensity of medical treatment and require some biochemical and radiological investiga- to monitor the patient’s response. Proper selection of tion [2,3]. For the majority of these patients, however, laboratory studies at the time of diagnosis is important histological assessment before therapy or during to completely establish baseline data. Most metabolic follow-up is not necessary. But, when it is, nondecal- bone diseases will be treated medically. If laboratory cified bone processing, staining, and interpretation investigation is incomplete when pharmacological or following tetracycline labeling are often crucial. nutritional intervention begins, an opportunity for The importance of the medical history for evalua- diagnosis or for evaluating therapy may be lost. In fact, tion of metabolic bone disease cannot be overempha- freezing away some patient serum and an aliquot of sized. Foremost in diagnosing these disorders is this urine before medication is prescribed is sometimes orderly accumulation of pertinent information directly worthwhile in case retrospective diagnosis may become from patients [9]. The revelations help to guide the necessary. Furthermore, this can help provide espe- physical examination and subsequent laboratory studies cially precise pre- and posttreatment comparisons. and to disclose potentially important medical records. Additionally, in select circumstances where a thera- Examples are endless. For deficiency of vitamin D, peutic challenge is given, preserving pretreatment historical details alone often explain how the patient specimens can be a tactic for saving money. Expensive has come to require supplementation. Prolonged breast and low-yield testing can await results of a brief trial feeding, lack of sunlight exposure together with failure of therapy (e.g., suspected environmental vitamin D to consume foods fortified with vitamin D, use of cer- deficiency rickets). Among the biochemical tests for tain anticonvulsants, or a family history suggestive of metabolic bone disease, there is now considerable a similar heritable disorder exemplify critical informa- redundancy (e.g., markers of skeletal turnover) [2,3,6]. tion that will be obtained by talking with the patient. Furthermore, some of these assays are useful primarily CHAPTER 57 Approach to the Patient with Metabolic Bone Disease 915 for research purposes where groups of patients are TABLE I Some Potentially Adverse Influences on studied. The utility of a single measurement of some of Mineral and Skeletal Homeostasis the markers of bone turnover for an individual patient can be limited because of technical or physiological Genetic Medical disorders variability, leading to just modest correlation with Ethnic background Acromegaly pathological processes. Heritable disorders Anorexia nervosa/Bulimia Histological study of the skeleton is essential in Lifestyle Celiac disease relatively few clinical circumstances, although it often Inactivity Cushing syndrome has considerable research importance. Here too, (immobilization) Cystic fibrosis however, an opportunity for diagnosis and/or baseline Smoking Early menopause assessment may be lost once pharmacologic therapy Alcohol abuse Fanconi syndrome begins [13]. Nutritional Fibrous dysplasia (polyostotic) Excessive tea drinking Gastrointestinal disease (fluorosis) Glycogen storage disease A. Medical History High protein intake Hemochromatosis Low dietary The complete and accurate medical history and calcium intake Hemolytic anemia thorough physical examination is the cornerstone for Milk intolerance Hepatitis C infection diagnosis, effective and safe treatment, and sound clin- Vegetarian diet Hepatobiliary disease ∗ ical investigation of metabolic bone disease [9]. Drugs Homocystinuria A detailed medical history helps assure that the Anticonvulsants Hyperparathyroidism many adverse external or heritable factors that can Bisphosphonate excess Hypogonadism affect the patient with metabolic bone disease will be Chemotherapy Lymphoproliferative disease uncovered (Table I). Cyclosporin A McCune-Albright syndrome A questionnaire completed by the patient may serve Diuretics (producing Mastocytosis as a beginning, but is hardly a satisfactory substitute. calciuria) Multiple myeloma Only by talking with his/her patient will the physician Fluoride Osteogenesis imperfecta sense how knowledgeable this individual might be, and Pancreatic insufficiency therefore be able to judge the value of any historical Glucocorticoids information. Only by talking with the patient will the Gonadotropin-releasing Postmenopausal osteoporosis hormone (GnRH) Prolactinoma character of the major signs and symptoms be revealed. agonists or antagonists Renal failure (transplantation) Subsequently, the medical history should be reported as Heparin a narrative description of the clinical problem. Rheumatologic disorders Lithium As discussed below, all of the principal elements of Secondary amenorrhea Protease inhibitors the medical history are potentially important for Thyrotoxicosis Thyroid replacement patients with metabolic bone disease. Turner syndrome therapy Type I diabetes 1. CHIEF COMPLAINT Vitamin A or D The chief complaint (or CC) may readily lead to the diagnosis; for example, pain from hip fracture due to osteoporosis or increasing head size due to Paget bone disease. Often, however, the patient’s major concern is more subtle. The above conditions may manifest with chronic back discomfort or progressive leg deformity, respectively. Such less dramatic difficulties must be recognized not only because they may be pointing to a diagnosis, but because they are objectives of treatment and can help monitor the efficacy of treatment. For * The more resources we have, and the more complex they are, example, when vitamin D deficiency causing weak- the greater are the demands upon our clinical skill. These resources are calls upon judgment and not substitutes for it. Do not, there- ness from myopathy or bone pain from osteomalacia is fore, scorn clinical examination; learn it sufficiently to get from it effectively treated, these difficulties should resolve. all it holds, and gain in it the confidence it merits. Furthermore, no matter how mild or severe the CC, Sir F.M.R. Walshe (1881Ð1973) this is the worry for which the patient with metabolic Canadian Medical Association Journal 67:395, 1952. bone disease seeks help. 916 MICHAEL P. W HYTE

TABLE II Vitamin D Deficiency: Age-Dependent Signs and Symptoms

Metabolic Skeletal Lax ligaments Hypocalcemia (see Table IV) Bone tenderness Limb deformity Muscle Cranial sutures widened Listlessness Asthenia Craniotabes (skull flattening or asymmetry) Low back pain Potbelly with lumbar lordosis Dystocia Pneumonia Proximal myopathy Flared wrists and ankles Rachitic rosary Waddling gait Fracture Rib deformity → respiratory Denial Frontal bossing compromise Caries Harrison’s groove Short stature Delayed eruption Hypotonia Sternal indention or protrusion Enamel defects Kyphosis “String-of-pearls” deformity in hands

With disturbances in vitamin D homeostasis, there especially useful way. Tactfully diverting them from may be one or more major complaints that can be excessive or extraneous details usually becomes easy metabolic or skeletal in origin (Table II). This aspect of because they now appreciate that the physician is truly the medical history is sometimes particularly challeng- concerned and wishes to know, understand, and help ing, because one problem can be emphasized from them to organize this important information. among this myriad collection of important symptoms Nutritional factors could be considered in the HPI. or signs, or because complaints may seem vague or Both mineral metabolism and vitamin D homeostasis excessive. are influenced by diet in countries that fortify foods with vitamin D. Strict vegetarians (vegans), who avoid 2. HISTORY OF PRESENT ILLNESS all foods derived from animals, will not benefit from Most metabolic bone diseases (including many the safety net of vitamin D supplementation of milk caused by disturbances in vitamin D homeostasis) are products in the United States. Avoidance of dairy foods chronic conditions. The detailed history of present ill- may also lead to a calcium-deficient diet (Table III). ness (or HPI) may therefore be lengthy. Nevertheless, Ovolactovegetarians, however, do drink milk. the time invested is crucial because this effort provides When the HPI is reported completely, not only will the most important historical information. Attention critical clues to disease etiology and pathogenesis to the details also demonstrates to the patient that the emerge, but the physician may also gain important physician understands and cares about his/her illness. insight for treatment, and here may be a glimpse of the Hence, this effort helps secure the patient’s confidence patient’s prognosis. Has this individual been compliant needed for effective treatment. with regard to recommendations for diagnostic studies From the HPI, the physician should gather an under- or medical care; if not, will pharmacologic therapy standing of the temporal evolution of the disorder— be safe? Have the manifestations of the disorder been likely essential for accurate and complete diagnosis lifelong (suggesting a congenital or genetic problem), and effective treatment. Clues predating the symptoms or has there been the development of recent symptoma- of vitamin D deficiency would be necessary to fully tology that should prompt very different diagnostic uncover the pathogenesis and etiology. Appreciation of considerations and interventions? Have complications past therapeutic attempts may reveal factors masking a been substantial, and likely to remain so if medical diagnosis. Indeed, the outcome of previous therapy therapy cannot effect a cure? (successful or unsuccessful) may guide diagnosis and Only by inquiring about the patient’s illness in detail future treatment. is the physician likely to learn that previous medical I find it worthwhile at the outset (for the sake of records, radiographs, etc., are available to help avoid time and effort, organization, and completeness) to expensive duplication of effort, and perhaps to help alert the patient that information will be especially address important diagnostic and prognostic issues. valuable if obtained in historical sequence. Patients Finally, by carefully documenting this aspect of a often require some guidance as this effort begins, but metabolic bone disease, the physician is providing the most will then help present their medical history in this basis for sound clinical research. CHAPTER 57 Approach to the Patient with Metabolic Bone Disease 917

TABLE III Vegetarians Furthermore, some medications confound interpreta- tion of biochemical studies aimed at metabolic bone Vegans (strict vegetarians who do not consume dairy disease because they alter mineral homeostasis (e.g., products or eggs) diuretics that increase or decrease urine calcium levels), Ovolactovegetarians (vegetarians who will eat dairy products and eggs) elevate serum alkaline phosphatase activity from the liver, etc. Fortunately, in the United States, pharmaco- Buddhists (some sects) logical doses of vitamin D require a prescription. Zen (Chinese) Nevertheless, vitamin D intoxication has occurred from ′ Ch an (Japanese) excesses inadvertently added to dairy milk (and other Muslims (some Islamic sects) errors) that would have gone unrecognized were it not Ethnic groups of African, Hispanic, American Indian, Jewish, for detailed medical histories. or Oriental descent (who may have lactose intolerance) The PMH may also help to predict the nature and Yoga frequency of recurrent illness and therefore complica- Seventh Day Adventists tions from pharmaceutical treatments. Depending on International Society for Krishna Consciousness the patient’s prior medical problems and compliance, Zen Macrobiotic Movement will use of vitamin D2 (with its prolonged biological effects) be safer or more risky than a shorter-acting, but more potent, vitamin D metabolite? Such information might help to prevent the abrupt development of hypocalcemia if therapy will be suddenly compromised, 3. PAST MEDICAL HISTORY or prevent prolonged hypercalcemia if dosing might A considerable number and variety of perturbations become excessive. can cause metabolic bone disease, or can influence the outcome of medical treatment, by impacting on min- 4. SOCIAL HISTORY eral and skeletal homeostasis (Table I). The detailed Patient compliance for medical treatment, especially past medical history (or PMH) will help to disclose these for chronic disorders, is often imperfect. Recognition disturbances or factors that can influence or obscure that an individual will be uncooperative or has health a diagnosis. insurance problems or financial difficulties that could In the PMH, previous diagnostic studies may be affect his/her ability or willingness to undergo diagnostic revealed that could prove useful for assessing the testing or remain compliant for therapy or follow-up metabolic bone disease. Radiographs (e.g., chest x-rays, may come in the social history (or SH). Such informa- intravenous pyelogram) taken years ago for other rea- tion can be necessary to formulate not only an effective sons could show whether osteopenia, osteosclerosis, or but also a safe treatment plan, particularly when the rachitic change is old or new. Routine biochemical disease is severe and/or requires potent medication. testing reports might help to date the onset of vitamin D The various pharmaceutical preparations of vitamin D, deficiency by documenting when serum alkaline such as 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], phosphatase activity began to rise. 1α-hydroxyvitamin D3 (lαOHD3), dihydrotachysterol Drug history must be carefully assessed here and, if (DHT), 25OHD3, and vitamin D2, have very different relevant, perhaps detailed instead in the HPI. Many potencies, biological half-lives, and price (Chapter 61). pharmaceuticals can adversely affect the skeleton and What will the issues of long-term treatment and drug cost disturb mineral or vitamin D homeostasis (e.g., gluco- mean for patient compliance, safety, and/or follow-up? corticoids, certain diuretics, anticonvulsants, etc.) The medical literature contains enough case reports of (Chapter 61). A seemingly incidental problem like acne, renal failure from vitamin D intoxication when patient if overlooked, might fail to reveal tetracycline or retinoid monitoring was inadequate. exposure. In fact, these concerns also apply to some Because vitamin D3 is produced naturally by exposure over-the-counter preparations (e.g., vitamin A, calcium to ultraviolet light in sunshine, and because nutrition also supplements, antacids). In large amounts, each of these importantly influences mineral homeostasis, apprecia- nonprescription items can have important effects on tion of climate, clothing, diet, and skin pigmentation is mineral or skeletal homeostasis and cause illness. important. Several ethnic, religious, and other groups Nevertheless, they may inadvertently be dismissed dur- have vegetarian members who will not consume dairy ing elicitation of a drug history (“Pardon me, products for health, spiritual, or ethical reasons doctor. You asked me what medications I was taking.”). (Table III). Recognition in the SH that the patient belongs Many patients will not consider vitamins, mineral sup- to one of these populations may disclose a significant plements, or antacids to be in this category (Table I). factor contributing to his/her metabolic bone disease. 918 MICHAEL P. W HYTE

Physical factors (e.g., exercise and work activities) To report that the FH is “negative” without first often impact patients with metabolic bone disease. establishing the value of this information is misleading. Indeed, as regimens continue to improve for increasing If the patient is adopted, he/she is unlikely to give low skeletal mass, much of what the clinician can do relevant data. An understanding of the size of a family for a child or adult with osteoporosis still comes from is essential before dismissing transmission of a herita- cautioning them against potentially traumatic pursuits at ble disorder. The patient who is the only child of only play or work. Prevention of falls and proscription against children, or from a disrupted family, is not as likely to heavy lifting for pediatric or adult forms of osteoporosis disclose a heritable disorder as will be the patient from are important for minimizing fracture and spinal defor- a large, cohesive kindred. This effort can facilitate a mity. In fact, this advice, often guided by history-taking, diagnosis. Medical records from similarly affected can sometimes correct vertebral crush deformities in living or deceased family members may establish the children with brittle bone disease who (unlike adults) diagnosis, and may be an important guide not only can naturally reconstitute their spinal anatomy (Fig. 1). concerning prognosis, but also for treatment.

5. FAMILY HISTORY 6. REVIEW OF SYSTEMS The family history (or FH) is often revealing for Metabolic bone diseases can cause a considerable metabolic bone disease, because many of these condi- variety of symptoms. This is especially true for disor- tions are heritable [1Ð5]. In fact, a correct diagnosis ders that disturb vitamin D homeostasis and lower may be disclosed by study of kindred members— extracellular calcium and phosphate levels (Table II). familial benign (hypocalciuric) hypercalcemia or Therefore, a careful “review of systems” may uncover a osteogenesis imperfecta are good examples. Inborn sufficient number of these problems so that a diagnosis errors of vitamin D bioactivation or resistance are rare, becomes apparent, or a new or additional condition is but they may be uncovered in the FH. Furthermore, suspected. Furthermore, this effort provides a baseline significant benefit may come from screening studies to from which to judge the impact of subsequent medical identify, and then to treat or to counsel, other affected therapy. Symptoms that persist after a course of other- relatives who may also represent important clues to the wise effective treatment would need further investiga- patient’s future complications and prognosis. tion if they were expected to respond.

FIGURE 1 Considerable reconstitution of vertebrae (here, L3ÐL5) has occurred between ages 14 (left) and 16 (right) years in a boy with idiopathic osteoporosis. He was counseled against lifting and to avoid falls and stopped participating in traumatic exercises in physical education class. No pharmacological intervention had been attempted. CHAPTER 57 Approach to the Patient with Metabolic Bone Disease 919

B. Physical Examination diaphragmatic pull producing a horizontal depression along the lower border of the chest at costal insertions Many clinical signs as well as significant skeletal of the diaphragm). Although weight bearing typically deformities can accompany or result from metabolic bows the lower limbs, especially during the adolescent bone disease—especially in children. Furthermore, not growth spurt a knock-knee deformity may occur instead. all of these disorders manifest themselves with overt The patient can also have myopathy with reduced mus- disturbances of hormone or mineral homeostasis (e.g., cle strength and tone causing a waddling gait, lax liga- postmenopausal osteoporosis). Accordingly, physical ments, indentation of the sternum from forces exerted examination is especially important. Discovery as well by the diaphragm and intercostal muscles, delayed as comprehensive treatment of metabolic bone disease eruption of permanent teeth, and enamel defects. In often depends on this skill. infants, floppiness and hypotonia are characteristic. Occasionally, diagnosis of a metabolic bone disease Rachitic infants and young children commonly are list- starts with the identification of one physical finding; less and irritable. Bone pain can also occur from fracture for example, blue or gray sclerae (osteogenesis imper- and deformity. fecta), cafe-au-lait spots (McCune-Albright syndrome), Hypocalcemia (Chapter 64) can also result from tumor (oncogenic rickets/osteomalacia), premature vitamin D deficiency (Chapters 65 and 66), pseudode- loss of a deciduous tooth (hypophosphatasia), hallux ficiency (Chapter 71), or resistance (Chapter 72) [14]. valgus (fibrodysplasia ossificans progressiva), or brachy- Hence, it is important that symptoms of hypocalcemia dactyly (pseudohypoparathyroidism, type IA). For some are elicited during the medical history and physical signs metabolic bone diseases, a constellation of physical of latent or overt tetany are recognized during the phys- findings leads to the diagnosis. Paget disease of bone, ical examination (Table IV). Hypocalcemia enhances when advanced, can feature an enlarging calvarium neuromuscular excitability. Consequently, there may with bulging temporal arteries, deafness, asymmetrical be varying degrees (latent or overt) of tetany. Overt bowing of the limbs, and localized areas of skeletal tetany usually presents with numbness and tingling warmth. Postmenopausal osteoporosis causes loss of vertebral height with reduced stature leading to a “short- waisted” appearance, kyphosis or a gibbus (dowager’s hump), a protuberant abdomen (that the patient may TABLE IV Signs and Symptoms of Hypocalcemia mistakenly attribute to weight gain), ribs lowered toward (or in) the pelvic brim, paravertebral muscle Nervous system spasm, and thin skin (McConkey’s sign) [7]. Unless Increased irritability with latent or overt tetany such physical abnormalities individually or in combina- Mental status changes tion are correctly identified, a diagnosis may be missed. Seizures Furthermore, these findings should focus attention Basal ganglia calcification on anatomical structures of concern, perhaps requiring Mental retardation treatment (e.g., vertebroplasty for osteoporosis, leg- Cardiovascular bracing for rickets, etc.). With disturbances in vitamin D homeostasis, a Prolonged ST-interval with arrhythmia plethora of physical findings can develop (Table II). Cardiomyopathy with congestive heart failure Patient age determines which are likely to be encoun- Hypotension tered. Low levels or ineffective action of vitamin D can Other be especially harmful for infants and for children. As Papilledema discussed below, distinctive physical findings occur in Lenticular cataracts the pediatric age group. Intestinal malabsorption Rickets disturbs the most actively growing bones. Dysplastic teeth Because the skull is enlarging especially quickly at birth, Rickets/osteomalacia craniotabes (flattened posterior skull) is characteristic Integument changes of essentially congenital disease. A rachitic rosary Joint contractures (enlargement of the costochondral junctions) can appear Vertebral ligament calcification during the first year of life, when the rib cage forms rapidly. During infancy or childhood, there may be flared Reprinted with permission from MP Whyte 1993 Hypocalcemia. In: BEC wrists and ankles from metaphyseal widening, bony Nordin, AG Need, HA Morris (eds) Metabolic Bone and Stone Disease, 3rd tenderness, and Harrison’s groove (rib cage ridging from edn. Churchill Livingstone, Edinburgh, pp. 147Ð162. 920 MICHAEL P. W HYTE around the mouth and in the fingertips, and can be fol- physes close at the end of puberty. Height or length is lowed by muscle spasm in the extremities, face, larynx determined with a stadiometer or a tape measure with (causing stridor), and elsewhere, and mental status the patient in bare feet standing or supine, respectively. changes including epileptic seizures. Symptoms and Weight should also be carefully assessed (and con- signs may be particularly striking when reductions in trolled, if excessive). Obesity or inordinate weight gain extracellular levels of ionized calcium are severe, or in girls during late childhood may transiently improve when hypocalcemia occurs rapidly. Typically, there is stature, not necessarily reflecting a favorable response carpopedal spasm manifesting as adduction of the thumb, to medical therapy. Instead, the physician can mistake metacarpophalangeal joint flexion, and interphalangeal the influence of excess weight on accelerating pubertal joint extension. Latent tetany can be unmasked by elic- growth for efficacy of treatment. Here, the growth spurt iting Chvostek’s sign or Trousseau’s sign. Chvostek’s has merely occurred early, but physes will fuse soon sign is a spasm of the ipsilateral muscles of the face on after menarche, negating any improvement in final tapping the facial nerve near its exit from the skull in adult stature. the region of the parotid gland (just anterior to the ear Skeletal deformation can cause much of the mor- lobe, below the zygomatic arch). A positive Chvostek’s bidity of metabolic bone disease. Bowing of the lower sign ranges from twitching of the lip at the corner of limbs predisposes to osteoarthritis especially in the the mouth, to spasm of all of the facial muscles on the knees. Prevention, control, or correction of deformities stimulated side. However, slightly positive responses is an important goal of patient care. Without a complete occur in as many as 10 to 15% of healthy adults. physical examination, such important clinical prob- Trousseau’s sign is provoked when a sphygmomanome- lems may go unnoticed. Something as inexpensive as a ter is inflated on the arm to above the systolic blood shoe lift can be of considerable benefit, but the correct pressure for up to 3 minutes [9]. Positive responses consist size and placement must come from accurate evalua- of carpal spasm with resolution occurring 5 to 10 seconds tion of leg-length inequality if iatrogenic problems are after the cuff is deflated (i.e., relaxation is not immedi- to be avoided. ate). Both Chvostek’s and Trousseau’s signs can be For children with rickets, determination of height absent, however, even in severe hypocalcemia. They and arm span will help to quantitate skeletal deformity seem to reflect the rapidity of change in serum calcium as will measurements of the upper and lower segment levels. Chronic hypocalcemia causes cataracts, der- lengths and calculation of their ratio. Simple measure- mopathy (Fig. 2), and basal ganglia calcification [14]. ments—including finger breadth separation of knees or Growth rate is an important parameter to follow in ankles with bowed legs or knock knees, respectively— infants and children with metabolic bone disease, espe- are useful. With rickets (e.g., X-linked hypophos- cially rickets. Improvement or correction of short stature phatemia), some time may pass before the metabolic can derive from effective treatment. With it should also bone disease is controlled medically with active come reduction or resolution of skeletal deformity if metabolites of vitamin D3 and phosphate supplementa- there is sufficient time for longitudinal growth before tion. Accordingly, photography, gait analysis, and even videotaping of skeletal deformity may also help assess progression or document response to therapy. A “metabolic myopathy” is a prominent clinical fea- ture of vitamin D deficiency and tumor-induced rickets or osteomalacia. Proximal muscle weakness of the limbs is suspected from a history of difficulty rising from sitting position, negotiating stairs, or combing hair, and can be confirmed by physical examination. Gower’s sign is a simple and excellent way to detect this problem in children who are observed getting up from a seated position on the floor—if they must push up with their hands on their thighs to achieve upright posture, this is a positive test. Other routine assessments of muscle strength should be recorded [9]. In infants and children with rachitic disease, skull FIGURE 2 Hyperkeratotic dermatosis (shown here, posterior shape and calvarial growth should be followed— neck) recurs in a 19-year-old black man with pseudohypoparathy- roidism, who is poorly compliant with medical therapy. When he including recordings of head circumference using stops treatment with calcium and vitamin D, he becomes markedly standard charts. Early closure of cranial sutures is not hypocalcemic, and the hyperkeratotic lesions reappear. uncommon in these disorders [15]. Premature union of CHAPTER 57 Approach to the Patient with Metabolic Bone Disease 921 sagittal sutures commonly causes a dolichocephalic TABLE V Biochemical Markers of Bone Remodeling skull in X-linked hypophosphatemia, but this is usually a cosmetic concern only. In hypophosphatasia, however, Resorption (osteoclast products) there can be either functional or true premature fusion of Tartrate-resistant acid phosphatase (serum) multiple cranial sutures leading to a scafalocephalic Hydroxyproline (urine) skull, sometimes with raised intracranial pressure. Hydroxylysine glycosides (urine) Dystocia from too narrow a birth canal due to pelvic Collagen cross-links (urine and serum) deformity from vitamin D deficiency during childhood Total pyridinolines (Pyr and/or Dpy)* was a major cause of puerperal mortality in mothers in Free pyridinolines (Pyr and/or Dpy)* the early twentieth century. This deformity should be Cross-linked N- and C-telopeptides (NTX, CTX) searched for in pregnant women with a history of rickets. Alopecia is a distinctive clinical finding in some Formation (osteoblast products) patients with hereditary vitamin D-resistant rickets Propeptides of type I collagen (serum) (vitamin D-dependent rickets, type II) (Chapter 72). C-propeptide However, alopecia or hypotrichosis is also a manifesta- N-propeptide tion of far more prevalent vitamin D-deficiency rickets Osteocalcin (serum) accompanying malnutrition. Additionally, hypotrichosis Alkaline phosphatase (serum) occurs in some forms of metaphyseal dysplasia that Total activity can be confused clinically and radiographically with Bone-specific enzyme rickets [5]. The benign tumors that cause oncogenic (tumor- *Pyr = pyridinoline; Dpy = deoxypyridinoline. induced) rickets or osteomalacia are often at least pal- pable if not visible, but they may be no more than pea-size and are sometimes hidden (Chapter 70). They are frequently found subcutaneously, but can occur anywhere on the body. Some have been discovered usually all that are needed. Radiological procedures intravaginally or in the nasopharynx, and some lie used for skeletal disease are relatively limited but often within the skeleton. Because extirpation of these give critical information. However, it is wasteful to lesions is curative, especially thorough physical exam- cover the diagnostic “waterfront” by ordering a “bone ination is essential when this disorder is suspected. If battery” of biochemical tests, or a series of low-yield the physician cannot find the tumor on imaging studies radiological studies. Histopathological assessments are (including bone and octreotide scanning), patients indicated in fewer situations, but may provide definitive should be instructed concerning periodic searches for or insightful findings. subcutaneous masses or symptoms from tumor else- where. Physical examination yearly is warranted in hopes that previously undetectable lesions have appeared. D. Radiological Examination

1. X-RAY IMAGES C. Laboratory Testing Radiographs of the skeleton chosen selectively are often crucial for diagnosis and follow-up of patients The medical history and physical examination guide with metabolic bone disease (Chapter 60) [10Ð12,16]. further assessment of the patient with metabolic bone However, the “skeletal survey” that examines all bones disease by laboratory methods. Beginning the testing is an expensive, relatively insensitive, and laborious pro- without this fundamental information, but instead with cedure, causing a not trivial exposure to X-irradiation. biochemical or radiological studies, is not appropriate. Visualization of the shape of the entire skeleton (but Of consternation for some physicians who only using films of just one upper and one lower extremity) occasionally encounter patients with metabolic bone is usually indicated to assess a bone dysplasia, but disease is the often bewildering array of expensive is rarely necessary for evaluation of metabolic bone assays for factors that condition mineral or skeletal disease. Instead, the “metabolic bone survey” generally homeostasis as well as the plethora of markers of skele- gives the necessary radiographic information for diag- tal apposition or resorption (Table V). Although some nosis and follow-up. Here, one studies the appendicular, biochemical testing is necessary for effective diagnosis as well as the axial skeleton, and therefore delineates or treatment of metabolic bone disease, especially dis- both cortical and trabecular bone as well as “yellow” turbances of vitamin D homeostasis, a few studies are and “red” marrow spaces. The necessary films are 922 MICHAEL P. W HYTE a lateral view of the skull and thoracolumbar spine, does not establish a diagnosis [17]. Enhanced radioiso- posteroanterior view of a hand and wrist as well as tope uptake occurs in areas of increased blood flow the chest, and an anteroposterior view of the pelvis to the skeleton, excess osteoid, and accelerated bone and a knee. Chapter 60, as well as several comprehen- formation. Cost-effective assessment starts with the sive texts [10Ð12,16], and other resources (London bone scan followed by x-rays of the “hot spots” to guide Dysmorphology Database, Oxford University Press further study. Bone scanning is generally unnecessary and POSSUM—Pictures Of Standard Syndromes in children with rachitic disease, unless a search for And Undiagnosed Malformations—The Murdoch a skeletal source of tumoral rickets is needed when Institute for Research Into Birth Defects, Melbourne, physical examination fails to yield an obvious lesion. Australia), describe the radiographic findings of the In adults, bone scanning can also disclose complica- metabolic bone diseases and help to distinguish the tions of osteomalacia, including “true” as well as “false” skeletal dysplasias. (pseudo) fractures. When there is rickets, posteroanterior radiographs of the hands and anteroposterior radiographs of the 3. BONE DENSITOMETRY knees will precisely document the presence and degree Bone mass quantitation is now widely available for of physeal and metaphyseal change. Long cassette clinicians [6]. Dual energy x-ray absorptiometry films of the lower extremities, taken while the patient (DEXA) and quantitative computed tomography (QCT) is standing, help quantitate bowing or knock-knee can give helpful assessments of skeletal mass desig- deformity. Radiographic studies can also provide clues nated “bone mineral density” (BMD). DEXA or QCT to the particular etiology or pathogenesis of rickets. densitometry, however, does not provide a diagnosis. For those disorders that cause rickets by disturbing In fact, each of the principal categories of metabolic vitamin D homeostasis and result in secondary hyper- bone disease (see below)—namely, osteoporosis, parathyroidism, subperiosteal bone resorption and osteomalacia, and osteitis fibrosa cystica—can mani- osteopenia may be noted in addition to growth plate fest with low BMD. The presence of worrisome BMD widening and irregular metaphyses (Fig. 3). These revealed by these techniques is merely a point of findings contrast to most cases of X-linked hypophos- departure for differential diagnosis. For those rare con- phatemia where the skeleton typically has normal or ditions associated with increased BMD, these tools are sometimes increased radiodensity and evidence of similarly helpful. hyperparathyroidism is generally absent. In hypophos- Densitometry has some important technical caveats. phatasia, there are characteristically peculiar “tongues” When using DEXA for children, it is crucial to under- of radiolucency that project from the physes into the stand that the technique generates a so-called areal metaphyses. Rachitic disease of recent onset will sym- (two-dimensional) rather than volumetric (three- metrically widen growth plates, whereas long-standing dimensional) assessment of BMD. BMD is measured as rachitic disease with bony deformity alters the mechan- gm/cm2 by DEXA and as gm/cm3 by QCT. Accordingly, ical forces acting on the physes, which in turn become values for BMD from DEXA are importantly depen- asymmetrically widened. Accordingly, chronic rachitic dent on body size. Children have lower BMD on DEXA disease sometimes seems more difficult to diagnose compared to adults, but this is largely an artifact of with x-rays (Fig. 4). their body size. Small stature individuals, who have The rapidity of resolution of rachitic changes on otherwise normal skeletons, will seem to have low x-ray images also may be of diagnostic significance. BMD compared to taller subjects. Hence, the pediatric With vitamin D deficiency rickets from lack of sunlight age group (or individuals who are small for reasons exposure, radiographic improvement can occur rapidly other than metabolic bone disease) may be incorrectly (within a few weeks) following a single pharmacologi- diagnosed with “osteopenia” or “osteoporosis” if this cal dose of vitamin D. Other forms of rickets, especially technical phenomenon with DEXA goes unrecognized those due to renal phosphate wasting, generally take or uncorrected. QCT can measure BMD with the advan- longer (several months or more) to improve or correct tage that it provides a volumetric measurement, and can with appropriate medical therapy. Sequential radio- focus on either cortical or trabecular bone, but with graphs are essential to evaluate the response to therapy higher exposure to X-irradiation. QCT can also pre- for rickets. cisely evaluate the anatomy of the skeleton and readily detect extracellular calcification [17]. Quantitation of 2. BONE SCINTIGRAPHY low BMD in children uses Z-scores (standard devia- Bone scintigraphy is an excellent tool for uncover- tions from mean values) matched for age and gender ing a variety of abnormalities of the skeleton, but it and ideally for ethnicity, whereas T-scores (which refer CHAPTER 57 Approach to the Patient with Metabolic Bone Disease 923

ABC

DEF

FIGURE 3 Characteristic changes of rickets (growth plate widening and frayed ends of metaphyses) occur in anteroposterior radiographs of the knees of five boys each newly diagnosed with a different type of rachitic disease (AÐE). However, as shown, addi- tional information is apparent concerning the pathogenesis and/or etiology. (A) A 1-year-old boy with osteopenia due to secondary hyperparathyroidism from “nutritional” rickets. (B) Similar findings in a 5-year-old boy with osteopenia consistent with documented secondary hyperparathyroidism from anticonvulsant-induced rickets. (C) A 3-year-old boy has normal bone mass and no secondary hyperparathyroidism, consistent with X-linked hypophosphatemia (XLH). (D) A 10-year-old boy has symmetrical widening of the growth plates (seen best in the proximal tibia) but no long bone deformity. Together, these changes suggest recent-onset disease in keeping with suspected tumor-induced rickets. (E) A 10-year-old boy has “tongues” of radiolucency (arrows), that project from physes into metaphyses, characteristic of the childhood form of hypophosphatasia. (F) However, not all disorders that cause metaphyseal irregularity are forms of rickets. This 4-year-old boy, with unremarkable biochemical studies and iliac crest histology following “tetracycline labeling,” has metaphyseal dysplasia. 924 MICHAEL P. W HYTE

TABLE VI Causes of Chronic Hypophosphatemia

Decreased intestinal absorption Alcohol abuse Antacid abuse Vitamin D deficiency Malabsorption Starvation

Increased urinary losses Renal tubular defects X-linked hypophosphatemia (XLH) Oncogenic rickets or osteomalacia (tumor-induced osteomalacia) Abnormalities of vitamin D metabolism Vitamin D deficiency Vitamin D-dependent rickets Hyperparathyroidism Renal transplantation Alcohol abuse FIGURE 4 A 9-year-old girl, who is poorly compliant for medi- Poorly controlled diabetes mellitus cal therapy for X-linked hypophosphatemia (XLH), has lower limb Metabolic or respiratory acidosis bowing that asymmetrically deforms the growth plates in her knees. Drugs: calcitonin, diuretics, glucocorticoids, bicarbonate Respiratory alkalosis to peak bone mass) are used to assess low bone mass Extracellular fluid volume expansion in adults. Software may be FDA-approved for displaying Severe burns a child’s lumbar spine BMD value, but not for other skeletal regions (e.g., hip). A Web site (http://www-stat- class.stanford.edu/pediatric-bones/) provides DEXA- based BMD reference ranges for various skeletal sites important when there is defective skeletal mineralization. in children, but cautions that the information reflects Chronic hypophosphatemia occurs in a number of condi- research experience (“not for diagnosis”) and warns tions associated with rickets or osteomalacia (Table VI). about the caveats (see above) for interpreting areal Accordingly, hypophosphatemia is an especially BMD values in children. Interpretation of high BMD important finding. Clinicians must recognize that the values, uses Z-scores for either children or adults [18]. pediatric age group normally has higher fasting blood phosphate levels compared to adults. Serum phosphate 4. OTHER RADIOLOGICAL PROCEDURES levels should be assayed with fasting blood specimens, Magnetic resonance imaging (MRI) is particularly because food (depending on content) can acutely raise useful for marrow space examination, including delin- or lower the level. Assay of phosphate levels in a urine eation of ischemic necrosis of bone. Fat-suppressed specimen can help to distinguish whether hypophos- imaging showing bone edema may disclose a cause for phatemia is due to renal phosphate wasting or to unexplained bone pain. New applications of MRI include dietary deficiency. Detailed tests of renal phosphate assessment of trabecular bone microanatomy [17]. handling using timed collections [e.g., tubular Ultrasound studies of the skeleton may provide reabsorptive maximum for phosphorus/glomerular qualitative as well as quantitative information [6]. filtration rate (TmP/GFR) ratios provide a definitive assessment [2,3]. Disturbances in vitamin D stores or bioactivation E. Biochemical Investigation commonly result in hypocalcemia, secondary hyper- parathyroidism and, consequently, hypophosphatemia. Understandably, circulating calcium levels are closely Assay of the serum levels of the major active vitamin D scrutinized in patients with metabolic bone disease, metabolites—25OHD and 1,25(OH)2D—is essential to especially those suspected of having rickets or osteo- detect disturbances in vitamin D stores or in vitamin D malacia (Chapters 65 and 66). However, extracellular bioactivation, respectively. The important effects (serum) phosphate levels may be equally (perhaps more) of season on serum 25OHD levels should be considered. CHAPTER 57 Approach to the Patient with Metabolic Bone Disease 925

Furthermore, 25OHD is transported in the circulation number of disorders cause rickets or osteomalacia bound to vitamin D-binding protein (DBP). Accordingly, where serum 25OHD levels are low (Table VII). Assay of hypoproteinemia must be recognized before interpreting serum 1,25(OH)2D concentration is most helpful in exploring a serum 25OHD concentration. hypercalcemia, but is also necessary to characterize Low levels of serum 25OHD usually indicate vita- inborn errors of vitamin D bioactivation. Finding a min D deficiency, but this biochemical finding is merely low serum 1,25(OH)2D level also helps in the inves- a starting point for differential diagnosis. A considerable tigation of oncogenic osteomalacia. However, serum

TABLE VII Causes of Rickets or Osteomalacia

I. Vitamin D deficiency B. Acquired A. Deficient endogenous synthesis 1. Ureterosigmoidostomy 1. Inadequate sunshine 2. Drug-induced 2. Other factors, e.g., genetic, aging, pigmentation a. Chronic acetazolamide use B. Dietary b. Chronic ammonium chloride use 1. Classic “nutritional” V. Chronic renal failure 2. Fat-phobic VI. Phosphate depletion II. Gastrointestinal A. Dietary A. Intestinal 1. Low phosphate intake 1. Small-bowel diseases with malabsorption, 2. Total parenteral nutrition e.g., celiac disease (gluten-sensitive enteropathy) 3. Aluminum hydroxide antacid abuse B. Hepatobiliary (or other nonabsorbable hydroxides) 1. Cirrhosis B. Impaired renal tubular (? intestinal) 2. Biliary fistula phosphate reabsorption 3. Biliary atresia 1. Hereditary C. Pancreatic a. X-linked hypophosphatemic rickets 1. Chronic pancreatic insufficiency b. Adult-onset vitamin DÐresistant hypophosphatemic osteomalacia III. Disorders of vitamin D bioactivation 2. Acquired A. Hereditary a. Sporadic hypophosphatemic osteomalacia 1. Vitamin D dependency, type I (pseudovitamin D (phosphate diabetes) deficiency) b. Tumor-associated rickets and osteomalacia 2. Vitamin D dependency, type II (hereditary vitamin D-resistant rickets) c. Neurofibromatosis B. Acquired d. McCune-Albright syndrome 1. Anticonvulsant therapy VII. General renal tubular disorders (Fanconi syndrome) 2. Renal insufficiency (see below) A. Primary renal IV. Acidosis 1. Idiopathic A. Distal renal tubular acidosis (classic, type I) a. Sporadic 1. Primary (specific etiology not determined) b. Familial a. Sporadic 2. Associated with systemic metabolic process b. Familial a. Cystinosis 2. Secondary b. Glycogenosis a. Galactosemia c. Lowe’s syndrome b. Hereditary fructose intolerance with nephrocalcinosis B. Systemic disorder with associated renal disease c. Fabry’s disease 1. Hereditary 3. Hypergammaglobulinemic states a. Inborn errors 4. Medullary sponge kidney (i) Wilson’s disease 5. Post renal transplantation (ii) Tyrosinemia

Continued 926 MICHAEL P. W HYTE

TABLE VII Causes of Rickets or Osteomalacia—Cont’d 2. Acquired IX. States of rapid bone formation with or without a. Multiple myeloma a relative defect in bone resorption b. Nephrotic syndrome A. Postoperative hypoparathyroidism with osteitis fibrosa cystica c. Transplanted kidney B. Osteopetrosis (“osteopetrorickets”) 3. Intoxications X. Defective matrix synthesis a. Cadmium A. Fibrogenesis imperfecta ossium b. Lead XI. Miscellaneous c. Outdated tetracycline A. Magnesium-dependent VIII. Primary mineralization defects B. Steroid-sensitive A. Hereditary C. Axial osteomalacia 1. Hypophosphatasia B. Acquired 1. Bisphosphonate intoxication 2. Fluorosis

1,25(OH)2D levels should be interpreted mindful that as well as for judging the efficacy of treatment. Thus, pediatric and adult reference ranges differ, and that low bone biopsy may be more helpful in adult patients. serum phosphate levels are expected to increase 1,25(OH)2D levels in healthy individuals. Hypophos- phatemia without physiological elevation in 1,25(OH)2D III. TREATMENT levels points to a disturbance in renal 1,25(OH)2D biosynthesis. Treatment of metabolic bone disease can range from simple (e.g., exposure to sunlight for environmental vitamin D deficiency rickets) to complex (e.g., bone F. Histopathological Assessment marrow transplantation for the malignant form of osteopetrosis). When there is a disturbance in vitamin D Throughout the past century, clinicians diagnosed homeostasis, pharmaceuticals are usually needed. and dealt with three major types of metabolic bone However, additional treatment approaches may be disease based on the principal histopathological fea- required when there is skeletal deformity. Patients tures and the mineral-to-protein ratios—osteoporosis, who require vitamin D sterols can benefit greatly; but osteomalacia, and hyperparathyroidism (osteitis fibro- medical care often must be especially skilled. sis cystica). Biopsy of the iliac crest samples cortical as Rickets or osteomalacia can occur from deficiency well as trabecular bone and distinguishes especially or impaired bioactivation of vitamin D. Either distur- well these types of metabolic bone disease [13]. bance will decrease calcium absorption from the Histological examination of the iliac crest must, gastrointestinal tract, leading to variable degrees of however, follow in vivo “labeling” of the patient’s hypocalcemia, secondary hyperparathyroidism, and skeleton by ingestion of two 3-day courses of hypophosphatemia. The hypocalcemia and hypophos- tetracycline [6,13] and nondecalcified sectioning and phatemia together engender the defective skeletal histomorphometric analysis of specimens. However, mineralization (Chapters 65 and 66). which of these types of bone disease is present actu- Fortunately, it is possible to prevent, cure, or control ally provides another starting point for differential most aberrations in vitamin D homeostasis. Vitamin D diagnosis [2,3,13]. deficiency stemming from socioeconomic factors has Bone biopsy is not routinely needed to diagnose relatively uniform etiology and pathogenesis, consider- rachitic disease, which is easily detected by radiologi- able prevalence, and a long history for mankind that cal studies of the physes of the wrist and knees make prevention or treatment well understood and together with biochemical testing. In adults with osteo- theoretically straightforward. Inborn errors of vita- malacia, however, growth plate changes on x-ray min D bioactivation can be relatively easy to control, imaging have been “lost” as guideposts for diagnosis e.g., merely by providing a “replacement” dose of CHAPTER 57 Approach to the Patient with Metabolic Bone Disease 927

1,25(OH)2D3 for pseudovitamin D deficiency rickets biochemical and skeletal dynamics change in response (vitamin DÐdependent rickets, type I), or extremely to treatment, but there is increasing body size to con- difficult to treat, as in some patients with hereditary tend with. Alterations in weight, a growth spurt, or vitamin D resistant rickets (vitamin D-dependent healing of rachitic disease will all likely require rickets, type II). changes in dosage. Greater weight means higher doses Here, resistance to 1,25(OH)2D is so great in some of vitamin D sterols and/or mineral supplements to cases that it must be “bypassed” therapeutically by control an otherwise static metabolic disturbance. A providing calcium intravenously in order to heal the growth spurt can undo successful therapy if treatment rickets. For other clinical situations (especially hepato- does not increase to keep up with the greater skeletal biliary, gastrointestinal, or pancreatic disease) leading demands. Conversely, healing of rickets or osteomala- to vitamin D deficiency, the precise regimen and dura- cia may require a reduction in dosage, because the tion of treatment will vary greatly from patient to skeletal “sump” is now satisfactorily mineralized. patient and depend on measures to control the primary It should be clear that individualized follow-up, disorder. Hence, an accurate diagnosis, understanding especially for pediatric patients, is crucial. of pathogenesis, and familiarity with the pharmacolog- When managing chronic forms of rickets, help from ical armamentarium is essential for successful therapy other medical and surgical (e.g., orthopedic) subspe- which may require adjustment from time to time. cialties is often necessary, and is optimal when there is Now, a considerable number of pharmaceuticals are significant interdisciplinary exchange. Children with available for the treatment of metabolic bone disease. rickets who do not respond to pharmacologic therapy In the United States, the three major biological forms may benefit from bracing, epiphysiodesis (physeal sta- of vitamin D [vitamin D, 25OHD, and 1,25(OH)2D] pling), or osteotomy to improve lower limb deformity. can be prescribed. Also still available is dihydro- However, these procedures can reflect suboptimal tachysterol. There are also several preparations that medical therapy and are usually avoidable if dosing, provide inorganic phosphate, and innumerable types compliance, and follow-up are satisfactory. For some of calcium supplementation. Hence, both repletion patients, particularly children and adolescents, pill of vitamin D reserves as well as bypassing steps in counts to assess compliance can provide important vitamin D bioactivation are treatment options. information. Cooperation and monitoring can be Furthermore, direct (intravenous) administration of cal- improved using pillboxes marked by days of the week. cium is possible when there might be insufficient or School nurses may help administer patient medica- delayed action of a vitamin D sterol on the gastroin- tions when family life is disrupted. If deformities are testinal tract. Importantly, these drugs have different not obviously correcting, at least yearly orthopedic durations of fat and muscle storage and potency. evaluation (perhaps biannually during the growth Additionally, the lag times for onset of action and spurt) is advisable. longevity of biological effects vary considerably for Not all types of rickets or osteomalacia manifest these preparations. Finally, the cost of these pharmaceu- with hypocalcemia or low serum levels of 25OHD ticals differ greatly (Chapter 61). An accurate diagnosis or 1,25(OH)2D. Hypophosphatemia, with or without from among the many disorders that cause rickets or hypocalcemia, can lead to defective skeletal mineral- osteomalacia is essential for therapeutic efficacy, safety, ization. Nevertheless, many of these disorders respond and economy. to treatment with 1,25(OH)2D3 and mineral supple- An understanding of the pathophysiology of the ments [2,3]. Here again, the etiology and pathogenesis aberration in vitamin D homeostasis is required for of the specific condition must be understood for effective and safe therapy. Use of 1,25(OH)2D3, when successful medical management. there is depletion of body stores of vitamin D, merely The most common form of heritable rickets, circumvents but does not correct the basic problem. X-linked hypophosphatemia (XLH), is transmitted as Because many disorders disturb vitamin D homeostasis an X-linked dominant trait. The pathogenesis of XLH and their severities can differ markedly, each patient includes a renal tubular defect engendering loss of and condition must be treated individually. phosphate by the kidney [3]. Despite hypophos- From the above, it is apparent that follow-up of med- phatemia, serum 1,25(OH)2D levels are paradoxically ical treatment for metabolic bone disease—especially normal instead of elevated. More complex proximal rickets and osteomalacia—is essential for many rea- renal tubular defects that also feature renal phosphate sons. Only some generalities can be discussed here. For wasting (Fanconi syndrome) can reflect other inborn infants, children, and adolescents with rickets, close errors of metabolism, heritable disorders, or exposure monitoring of drug therapy is especially important to certain drugs or toxins including heavy metal poi- because these individuals are growing. Not only will soning (Table VI). Acquired hypophosphatemic rickets 928 MICHAEL P. W HYTE

IV. SUMMARY

The complex interaction of the many exogenous and endogenous factors that impact vitamin D homeo- stasis, discussed throughout this book, explain why patients with disturbances in this endocrine process are especially challenging. Nevertheless, these individuals typically benefit greatly from the efforts of physicians who understand such disorders. Demonstration of concern for, and commitment to, the patient begins with a complete medical history and thorough physical examination that wins their confidence and trust. Such rapport will likely be essential for effective manage- ment of what will often prove to be a chronic disorder. Physical examination is crucial not only for diagnosis, but to uncover structural skeletal problems. Information gathered by the medical history and physical examina- tion will be the guide to the myriad of biochemical, radiological, and other technologies that can help to establish the etiology and pathogenesis and to set the stage for subsequent treatment and follow-up. Effective therapies are available for derangements of vitamin D homeostasis, but the pharmaceuticals vary significantly in potency, duration of effect, and cost. Once the proper clinical foundation is in place, the physician will usually be gratified by a patient that FIGURE 5 In X-linked hypophosphatemia (XLH), the extremes he/she has greatly helped. of lower limb deformity in children that can be corrected by phar- macological therapy alone are not widely appreciated. Accordingly, close cooperation between the medical and orthopedic disciplines Acknowledgments is essential. At age 3 years, a girl with XLH has severe bowing deformity of the lower extremities that can be quantitated with Supported by Shriners Hospitals for Children and standing, long-cassette radiographs. Compliance for 1,25(OH)2D3 the Clark and Mildred Cox Inherited Metabolic Bone and inorganic phosphate supplementation therapy was excellent, and her deformity is remarkably improved by age 10 years without Disease Research Fund. osteotomy, epiphysiodesis, or lower extremity bracing. References

1. Avioli LV, Krane SM 1998 Metabolic bone disease and clini- cally related disorders. Academic Press, San Diego, CA. is also a common complication in McCune-Albright 2. Coe FL, Favus MJ 2002 Disorders of bone and mineral syndrome and is characteristic of oncogenic rickets. metabolism. Lippincott, Williams & Wilkins, Philadelphia, PA. Treatment with 1,25(OH) D and phosphate is used for 3. Favus MJ 2003 Primer on the Metabolic Bone Diseases and 2 3 Disorders of Mineral Metabolism. American Society for Bone the rickets complicating these disorders. Normalization and Mineral Research, Washington, DC. of blood phosphate levels occurs only transiently with 4. Scriver CR, Beaudet AL, Sly WS, Valle D 2001 The meta- oral phosphate supplementation in conditions charac- bolic and molecular bases of inherited disease. McGraw-Hill, terized by renal phosphate wasting. Nevertheless, clin- New York, NY. ical improvement can be substantial (Fig. 5), without a 5. McKusick V. Online Mendelian Inheritance in Man, OMIM (TM). McKusick-Nathans Institute for Genetic Medicine, likely hazardous push to correct phosphate levels mea- Johns Hopkins University (Baltimore, MD) and National sured with fasting blood specimens. Correction of Center for Biotechnology Information, National Library of hypophosphatemia should not be considered the objec- Medicine (Bethesda, MD). (http://www.ncbi.nlm.nih.gov/ tive of therapy, but rather correction of deformity and entrez/query.fcgi?db=OMIM). restoration of an adequate growth rate. Radiographic 6. Tovey FI, Stamp TCB 1995 The Measurement of Metabolic Bone Disease. Parthenon Publishing Group, New York, NY. and other biochemical studies are, instead, useful for 7. Marcus R, Feldman D, Kelsey JL 1996 Osteoporosis. judging adequacy and safety or therapy. Academic Press, San Diego, CA. CHAPTER 57 Approach to the Patient with Metabolic Bone Disease 929

8. Bilezikian JP, Raisz LG, Rodan GA 2002 Principles of bone 14. Whyte MP 1993 Hypocalcemia, In: BEC Nordin, AG Need, biology. Academic Press, San Diego, CA. HA Morris (eds) Metabolic Bone and Stone Disease. Churchill 9. Degowen, Degowen. Diagnostic Examination. 1999 McGraw- Livingstone, Edinburgh, UK, pp. 147Ð162. Hill Health Professionals Division New York, NY. 15. Reilly BJ, Leeming JM, Fraser D 1964 Craniosynostosis in the 10. Edeiken J, Dalinka MK, Karasick D 1990 Edeiken’s roentgen diag- rachitic spectrum. J Pediatr 64:396Ð405. nosis of diseases of bone. Williams & Wilkins, Baltimore, MD. 16. Greenfield GB 1990 Radiology of Bone Diseases. Lippincott, 11. Taybi H, Lachman RS 1996 Radiology of syndromes, metabolic Williams & Wilkins, Philadelphia, PA. disorders, and skeletal dysplasias. Mosby, St. Louis, MO. 17. Resnick D 1996 Bone and Joint Imaging. Saunders, 12. Ravell PA 1985 Pathology of Bone. Springer-Verlag, Berlin, Philadelphia. Germany. 18. Whyte MP Misinterpretation of osteodensitometry in high bone 13. Resnick D 2002 Diagnosis of bone and joint disorders. mass disease (it isn’t all osteoporasis). Journal of Clinical Saunders, Philadelphia. Densitometry (submitted for publication). CHAPTER 58 Detection of Vitamin D and Its Major Metabolites*

BRUCE W. HOLLIS Departments of Pediatrics, Biochemistry, and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina

I. Introduction V. Detection of 1,25(OH)2D II. Detection of Vitamin D VI. Clinical Interpretation and Relevance of Antirachitic III. Detection of 25OHD Sterol Measurements IV. Detection of 24,25(OH)2D References

I. INTRODUCTION quantitated, and only two of those, namely, 25OHD and 1,25(OH)2D, provide any clinically relevant information. Vitamin D is a 9,10-seco steroid and is treated as However, the quantitation of vitamin D and 24,25(OH)2D such in the numbering of its carbon skeleton (Fig. 1). can provide important information in a research environ- Vitamin D occurs in two distinct forms: vitamin D2 and ment. Thus, this chapter addresses the quantitation of vitamin D3. As shown in Fig. 1, vitamin D3 is a 27-carbon these four important vitamin D compounds. Further, it derivative of cholesterol; vitamin D2 is a 28-carbon is not the intent of this chapter to address the detailed molecule derived from the plant sterol ergosterol. Besides history of vitamin D metabolite analysis, as this can be containing an extra methyl group, vitamin D2 differs obtained from previous reviews [1Ð3]. Rather, the intent from vitamin D3 in that it contains a double bond of this chapter is to describe how we currently measure between carbons 22 and 23. The most important aspects vitamin D and its major metabolites in our laboratory, of vitamin D chemistry center on its cis-triene struc- as well as to discuss the appropriate clinical judgments ture. This unique cis-triene structure makes vitamin D in the selection of a given compound for analysis. and related metabolites susceptible to oxidation, ultra- The first semiquantitative assay for vitamin D was a violet (UV) light-induced conformational changes, heat- bioassay based on the rat-line test [4]. This assay was induced conformational changes, and attack by free cumbersome, expensive, and relatively inaccurate. radicals. As a rule, the majority of these transformation Real progress in vitamin D analysis was not achieved products have lower biological activity than vitamin D. It until the advent of high specific activity 3H-labeled is important to note that, in humans, vitamin D2 and D3 vitamin D3 compounds [5]. The introduction of these provide similar potency, (although some controversy exists as discussed in Chapter 61), and in this chapter the 28 CH term vitamin D refers to both compounds. 3 Metabolic activation of vitamin D is achieved through 21 22 24 26 18 25 hydroxylation reactions at both carbon 25 of the CH 20 23 3 CH3 side chain and, subsequently, carbon 1 of the A ring. 12 17 27 Metabolic inactivation of vitamin D takes place primarily 11 13 16 9 14 15 through a series of oxidative reactions at carbons 23, 8 24, and 26 of the side chain of the molecule. These metabolic activations and inactivations are well char- 7 acterized and result in a plethora of vitamin D metabo- 6 CH CH lites (Fig. 2). Of the compounds shown in Fig. 2, only 5 2 2 four, vitamin D, 25-hydroxyvitamin D (25OHD), 24,25- 4 10 3 1 dihydroxyvitamin D [24,25(OH)2D], and 1,25-dihy- 2 HO droxyvitamin D [1,25(OH)2D] have been extensively HO

Vitamin D3 Vitamin D2 *In the interest of full disclosure, the author wishes to inform the readers that he has been a paid consultant to the DiaSorin Company. FIGURE 1 Molecular structures of vitamins D2 and D3. VITAMIN D, 2ND EDITION Copyright © 2005, Elsevier, Inc. FELDMAN, PIKE, AND GLORIEUX All rights reserved. 932 BRUCE W. HOLLIS

DIET OH 7-dehydrocholesterol hν 5(E)-10-nor-10-oxo-D 24OHD OH O 3 3 HO HO Vitamin D3 OH HO α 1 ,24R(OH)2D3 OH OH α 8 ,25(OH)2-9, 10-seco- 5(E)-25(OH)- HO OH OH -4,6,10(19)-choleste 25OHD -19-nor-10-oxo-D3 trien-3-one 3 OH O HO OH OH 5(E)-25OHD3 25(OH)-23-oxo-D3 OH OH OH OH O 24R,25(OH)2D3 OH α 1 ,25(OH)2D3 OH HO HO HO OH 5(E)-24R,25(OH)2- O -19-nor-10-oxo-D3 OH OH O OH 25(OH)-23- OH OH OH OH OH -dehydro-D OH OH OH OH 3 α 25S,26(OH) D 1 ,24R- 2 3 24S,25(OH) D 1α,238,25(OH) D HO 23S,25(OH) D 2 3 -26(OH) D 3 3 2 3 25R,26(OH)2D3 2 3 HO (mixture1:1) OH HO HO HO OH HO OH HO OH α OH 1 ,26R,26(OH) D OH OH 3 3 OH O O 23,24,25(OH) O 3 3 OH OH OH OH OH OH O OH OH OH OH HO H α 1α,23S,25R,26(OH) D 24,25,26(OH) D 25(OH)-24-oxo-D 1 ,25(OH)2 α 3 3 23,25R- 3 3 3 1 ,24S,25(OH)3D3 -24-oxo-D3 -26(OH)3D3 HO HO HO HO OHHO OH O HO OH O O C O OH OH C OH OH O OH O O O OH OH 1α,25R(OH) -26,23S- 25R(OH)-26- 23S,25(OH) - 1α,23,25(OH) 2 2 3 -lactol-D -23S-lacto-D3 -24-oxo-D -24-oxo-D 3 HO 2 3 HO OH 25R(OH)-26,23S- OH HO HO OH -peroxylactone D3 O OH O CCH O COH CH2OH CH2OH O O

HO HO HO HO HO OH OH 25R(OH)-26,23S- 23(OH)-24-25,26,27- 1,23(OH) -24,2- α 25,26,27-trinor- 2 1 ,25R(OH)2-26,23S- -lactone-D -tetranor-D 26,27,-tetranor-D lactonel-D 3 -24-COOH-D3 3 3 3

O O C C OH OH

HO HO OH 24,25,26,27-tetranor 1α,(OH)-24,25,26,27- -23-COOH-D3 tetranor-23-COOH-D3 (Calcitroic acid)

FIGURE 2 Summary of metabolic transformations of vitamin D3. From Bouillon R, Okamura WH, Norman AW 1995 Structure- function relationships in the vitamin D endocrine system. Endocrine Review 16:200Ð257. © The Endocrine Society. tracers led to the development of competitive protein coupled with 125I-labeled tracers that require little or binding assays (CPBA) for vitamin D and 25OHD no chromatographic treatment of the sample [14,15] [6,7]. A short time later CPBA for 24,25(OH)2D and and the instrument automation for the direct detection radioreceptor assays (RRA) for 1,25(OH)2D were of circulating 25OHD. introduced [8,9]. In the late 1970s, high-performance The assays for vitamin D and its major metabolites liquid chromatographic (HPLC) analytical procedures that we currently utilize in our laboratory are described for vitamin D and 25OHD were described [10,11]. in this chapter. These assays are all standalone types of Subsequently, radioimmunoassay (RIA) techniques assays as opposed to the multiple-metabolite assays began to appear as a means to quantitate 25OHD and described in years past [16,17]. We chose to do this 1,25(OH)2D [12,13]. Finally, recent advances in because seldom in a clinical situation does one require antirachitic sterol analysis have included RIA a battery of vitamin D metabolite values. Further, many CHAPTER 58 Detection of Vitamin D and Its Major Metabolites 933

assays, especially for 25OHD and 1,25(OH)2D, have UV quantitation of vitamins D2 and D3 following non- been optimized as single metabolite procedures [14,15]. aqueous reversed phase HPLC provides an accurate, convenient method to measure circulating vitamin D. II. DETECTION OF VITAMIN D This method is described here in detail. A. Background B. Methodology Vitamin D, the parent compound, is by far the most lipophilic of the antirachitic sterols, and for this reason 1. SAMPLE EXTRACTION it is the most difficult to quantitate (Table I). The first A 0.5- to 1-ml of sample serum or plasma is placed serious attempt to quantitate vitamin D was performed into a 13 × 100 mm borosilicate glass culture tube con- 3 in 1971 utilizing CPBA [7]. This initial study grossly taining 1000 cpm of H-vitamin D3 in 25 µl of ethanol overestimated the actual amount of circulating vitamin D to monitor recovery of the endogenous compound because of insufficient sample prepurification prior to through the extraction and chromatographic proce- CPBA. It was later shown that vitamin D could be dures. Following a 15-min incubation with the tracer, assessed by CPBA, but only following extensive chro- 2 plasma volumes of HPLC-grade methanol are added matographic purification of the organic extract, includ- to each sample. The sample is then vortex-mixed for ing HPLC [18,19]. The first valid determination of 1 min followed by the addition of 3 plasma volumes of circulating vitamin D was achieved in 1978 by utiliz- HPLC-grade hexane. Each tube is capped and vortex- ing direct UV detection following a two-step HPLC mixed for an additional 1 min followed by centrifuga- purification procedure [10]. A short time later, valid tion at 1000 g for 10 min. The hexane layer is removed CPBA were introduced for the quantitation of circulat- into another 13 × 100 mm culture tube, and the aqueous ing vitamin D [18,19]. However, these procedures layer is reextracted in the same fashion. The hexane were cumbersome, as they required extensive sample layers are combined and dried in a heated water bath, prepurification prior to CPBA, including HPLC. 55¡C, under N2. The lipid residue is then resuspended Vitamin D is also difficult to quantitate because it is in 1 ml of HPLC-grade methylene chloride and capped. the only antirachitic sterol that cannot be extracted from aqueous media utilizing solid-phase extraction 2. SILICA CARTRIDGE CHROMATOGRAPHY techniques [20]. Therefore, unlike its more polar Silica Bond-Elut cartridges (500 mg) and a Vac-Elut metabolites, vitamin D must be extracted from serum cartridge rack were obtained from Varian Instruments or plasma using liquid-liquid organic extraction tech- (Harbor City, CA). The silica cartridges are washed in niques. Many of the initial studies used Bligh and order with 5 ml HPLC-grade methanol, 5 ml HPLC- Dyer-type total lipid extraction to extract vitamin D grade isopropanol, and 10 ml HPLC-grade methylene from serum samples [7,10,18]. However, these types chloride. The sample, in 1 ml of methylene chloride, is of extractions remove an extraordinary amount of lipid then applied to the cartridge and eluted through the from the plasma sample. We therefore utilized a more cartridge under vacuum into waste. This initial step is selective organic extraction procedure incorporating followed by 3 ml of 0.2% isopropanol in methylene chlo- methanol-hexane [21]. This extraction method coupled ride (discard) and 8 ml of the same solvent (vitamin D). with open cartridge silica chromatography and direct The 8-ml fraction contains vitamins D2 and D3 and is

TABLE I Significant Methods for the Estimation of Vitamin D in Human Seruma

Detection method Extraction Preliminary chromatography Ref. Normal circulating levelsb

CPBA Methanol-chloroform Silicic acid Belsey et al. [7] 24Ð40 ng/ml HPLC Methanol-chloroform Preparative HPLC Jones [10] 2.2 ± 1.1 ng/ml CPBA Ether-methylene chloride Sephadex LH-20, preparative HPLC Horst et al. [19] Ð CPBA Methanol-methylene chloride Lipidex-5000, preparative HPLC Hollis et al. [18] 2.3 ± 1.1 ng/ml HPLC Methanol-hexane Silica cartridges, Preparation HPLC Liel et al. [21] 9.1 ± 1.0 ng/ml (normal), 1.3 ± 0.1 ng/ml (obese)

aAs noted in the text, the Belsey method overestimated the circulating vitamin D levels because of insufficient prepurification prior to competitive protein binding assay (CPBA). bng/ml × 2.6 = nmol/liter. 934 BRUCE W. HOLLIS subsequently dried in a heated water bath, 55¡C, under 4. QUANTITATIVE REVERSED-PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY N2. The elution profile of vitamin D from the silica car- tridge is displayed in Fig. 3. The silica cartridges can be The final quantitative step is performed using non- cleaned and regenerated by washing with methanol, aqueous reversed-phase HPLC. The column is a Vydac isopropanol, and methylene chloride and reused many TP-201-54, 5 µm, wide pore, nonendcapped octadecyl- times. silane (ODS) silica material, 0.4 × 25 cm. This particu- lar column must be used for this procedure to work. The 3. PREPARATIVE NORMAL-PHASE HIGH-PERFORMANCE mobile phase comprises acetonitrile-methylene chloride LIQUID CHROMATOGRAPHY (65:35, v/v) utilizing a flow rate of 1.2 ml/min. This sys- High-performance liquid chromatography can be per- tem provides clear resolution of vitamins D2 and D3 formed on any available HPLC system. This normal-phase (Fig. 4B). This system is calibrated with varying HPLC step is performed in our laboratory using a 0.4 × amounts of vitamin D2 and D3 (1Ð100 ng). The sample 25 cm Zorbax-Sil column packed with 5 µm silica, but residue from normal-phase HPLC is dissolved in 15 µl any equivalent column could be utilized. The mobile methylene chloride followed by the addition of 135 µl of phase comprises hexane-methylene chloride-isopropanol acetonitrile and injected onto the HPLC. After elution, (49.5 : 49.5 : 0.5, v/v) at a flow rate of 2 ml/min. The sam- final quantitation of vitamin D2 and D3 is by direct UV ple residue from the silica cartridge is dissolved in 150 µl monitoring of 265 nm. The vitamin D3 portion is col- of the mobile phase and injected onto the HPLC column lected, dried under N2, and subjected to liquid scintilla- that had been previously calibrated with 10 ng of stan- tion counting in order to determine the final recovery of dard vitamin D3. The elution of vitamins D2 and D3 endogenous vitamin D3 from the sample. Calculations (they coelute on this system) can be seen in Fig. 4A. are then performed, and the results are reported in as The vitamin D fraction is collected in a 12 × 75 mm glass nanograms vitamin D2 and/or D3 per milliliter. A flow culture tube and dried under N2 at 55¡C. diagram of the entire procedure is displayed in Fig. 5.

0.004 CH Cl :ISP Hexane: ISP A + 2 2 D2 D3 99.8:0.2 95:5 92:8 85:15 500 98.5:1.5 3 0.003 H-1,25-(OH)2-D3 300

0.002 100

0.001 500 3 H-24,25-(OH)2-D3

300 0

100 B 0.004 D2 D3 Optical density 265 nm

500 3 H-25-OH-D3 0.003

Radioactivity (cpm) 300

100 0.002

500 3 0.001 H-Vitamin D3

300 0 24681012 14 100 Elution time (min)

04 812162024 28 32 36 FIGURE 4 High-performance liquid chromatographic profiles of Elution volume (ml) standard vitamins D2 and D3 on normal-phase (A) and reversed- phase (B) systems. Column calibration was achieved by injecting FIGURE 3 Elution profiles of radioactive vitamin D3 and its metabo- 10 ng of each compound and monitoring optical density at 265 nm. lites chromatographed on a silica Bond-Elut (500 mg) cartridge. Column conditions are described in the text. CHAPTER 58 Detection of Vitamin D and Its Major Metabolites 935

0.5 ml Sample or control + 3H-Vitamin D protein (DBP) from rat serum as a specific binding 3 agent. These assays all contained some type of organic Incubate 15 min room temp extraction coupled with sample prepurification by 3 column chromatography, all used H-25OHD3 as a Extract with methanol : hexane tracer, and all required individual sample recovery estimates to account for endogenous losses of 25OHD during the extraction and purification procedures. Centrifuge and remove hexane layer A nonchromatographic assay for circulating 25OHD was introduced in 1974 [22], but it was never widely Dry hexane under N2 and resuspend in methylene chloride accepted because of its nonspecificity and susceptibility to serum lipid interference. Apply to silica cartridge and collect vitamin D-containing fraction Various CPBAs for 25OHD dominated the literature until 1977 when the first valid direct UV quantitative Dry fraction under N2 and resuspend HPLC assay was introduced [11]. 25OHD circulates in in normal-phase HPLC mobile phase the nanogram per milliliter (nanomole/liter) range and Apply to normal-phase silica HPLC and thus could be directly quantitated by UV detection collect vitamin D-containing fraction following its separation by normal-phase HPLC. Also, HPLC detection provided the advantage of being able Dry fraction under N2 and resuspend to individually quantitate 25OHD and 25OHD . The in methylene chloride:acetonitrile 2 3 disadvantages of HPLC quantitation methods are their Apply to nonaqueous reversed-phase Vydac ODS HPLC and requirements for expensive equipment and large sample quantitate vitamins D2 and D3 by direct UV absorption. Collect 3 size, cumbersome nature, and the technical expertise to vitamin D3-containing fraction and monitor for H-content recovery correction perform this type of analysis. However, HPLC analysis for 25OHD is frequently used in research environments, FIGURE 5 Flow diagram of the HPLC-UV assay for the quanti- including our own, and has provided a great deal of tation of vitamins D2 and D3. significant information. As the clinical demand for circulating 25OHD analysis increased, it was clear that simpler, rapid yet III. DETECTION OF 25OHD valid assay procedures would be required. To this point, all valid assays required liquid-liquid organic extraction, A. Background some sort of chromatographic prepurification, and evap- oration of the organic solvents, hardly practical for a One of the major factors responsible for the explo- clinical chemistry laboratory. Thus, in 1985, the first sion of knowledge related to vitamin D metabolism valid RIA for assessing circulating 25OHD was intro- was the introduction of valid CPBA for 25OHD in the duced [13]. This RIA eliminated the need for sample early 1970s [6,7] (Table II). One of these assays in prepurification prior to assay and had no requirement particular gained widespread use owing to its relative for organic solvent evaporation. However, the method 3 simplicity and, as a result, has been cited nearly 1000 was still based on the use of H-25OHD3 as a tracer. times [6]. The first assays utilized the vitamin D-binding This final shortcoming was solved in 1993 when an

TABLE II Significant Methods for the Estimation of 25OHD in Human Serum

Detection Preliminary method Extraction chromatography Ref. Normal circulating levelsa

CPBA Methanol-chloroform Silicic acid Belsey et al. [7] 18Ð36 ng/ml CPBA Ether Silicic acid Hadad and Chyu [6] 27.3 ± 11.8 ng/ml CPBA Ethanol None Belsey et al. [22] 20Ð100 ng/ml HPLC Methanol-chloroform Sephadex LH-20 Eisman et al. [11] 31.9 ± 1.7 ng/ml RIA Acetonitrile None Hollis and Napoli [13] 25.5 ± 11.8 ng/ml RIA Acetonitrile None Hollis et al. [14] 9.9Ð41.5 ng/ml CLIA None None DiaSorin Corp. 9.5Ð52.0 ng/ml

ang/ml × 2.4 = nmol/liter. 936 BRUCE W. HOLLIS

125 I-labeled tracer was developed and incorporated into a heated water bath, 55¡C, under N2. The elution profile the RIA for 25OHD [14]. This assay has become the of 25OHD3 from the silica cartridge is displayed in method of choice for assessing 25OHD status and has Fig. 3. The silica cartridges can be cleaned and reused become the first test for vitamin D approved for clinical many times. diagnosis by the U.S. Food and Drug Administration (FDA). Most recently, DiaSorin Corporation (Stillwater, 4. QUANTITATIVE NORMAL-PHASE MN) has introduced an automated, nonextracted HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY chemiluminescent immunoassay (CLIA) for the direct The final quantitative step is performed using normal- determination of circulating 25(OH)D. phase HPLC with a 0.4 × 25 cm Zorbax-Sil column packed with 5 µm spherical silica. The mobile phase is composed of hexane-methylene chloride-isopropanol B. HPLC Methodology (50 : 50 : 2.5, v/v) at a flow rate of 2 ml/min. The sample residue from the silica cartridge is dissolved in 150 µl 1. SAMPLE EXTRACTION of mobile phase and injected onto the HPLC column A 0.5-ml sample of serum or plasma is placed into previously calibrated with varying amounts of 25OHD2 a 12 × 75 mm borosilicate glass culture tube containing and 25OHD3 (1Ð100 ng). This HPLC system provides 3 1000 cpm of H-25OHD3 in 25 µ of ethanol to monitor clear resolution of 25OHD2 and 25OHD3 (Fig. 6A). recovery of endogenous compound through the extrac- After elution, final quantitation of 25OHD2 and 25OHD3 tion and chromatographic procedures. Following a is by direct UV monitoring at 265 nm. The 25OHD3 15-min incubation with the tracer, 1 plasma volume of portion is collected, dried under N2, and subjected to HPLC-grade acetonitrile is added to each sample. The liquid scintillation counting in order to determine the sample is then vortex-mixed for 1 min followed by final endogenous recovery of 25OHD2 and 25OHD3 centrifugation at 1000 g for 10 min. The supernatant from the sample. Calculations are then performed, and is removed into another 12 × 75 mm culture tube, and the results reported as nanograms 25OHD2 and/or 1 plasma volume of 0.4M K2HPO4, pH 10.4, is added. 25OHD3 per milliliter. A flow diagram of the entire procedure is displayed in Fig. 7. 2. SOLID-PHASE EXTRACTION CHROMATOGRAPHY

C18 silica Sep-Pak cartridges (500 mg) and a Sep- Pak rack were obtained from Waters Associates C. RIA Methodology (Milford, MA). The C18 cartridges are washed in sequence with 5 ml HPLC-grade isopropanol and 5 ml 1. PREPARATION OF ASSAY CALIBRATORS HPLC-grade methanol. The sample is applied to the One of the goals of the RIA procedure for 25OHD cartridge and eluted through the cartridge under vacuum was to eliminate the need for individual sample recov- into waste. This initial step is followed by 5 ml of 30% ery. Another goal was to obtain FDA approval for water in methanol (discard) and 3 ml of acetonitrile clinical use of this procedure in the United States. Both (25OHD). The acetonitrile fraction is dried in a heated of these goals place 25OHD3 in a human serum-based water bath, 55¡C, under N2. The lipid residue is then set of assay calibrators. To prepare these calibrators, suspended in 1 ml of 1.5% isopropanol in hexane and human serum was “stripped” free of vitamin D metabo- capped. The C18 cartridges can be cleaned and regen- lites by treatment with activated charcoal. Absence of erated by washing with 2 ml of methanol and reused endogenous 25OHD in the stripped sera was confirmed many times. by direct UV detection of 25OHD in serum following HPLC as described in Section III,B. Subsequently, 3. SILICA CARTRIDGE CHROMATOGRAPHY crystalline 25OHD3 dissolved in absolute ethanol was Silica Bond-Elut cartridges (500 mg) and a Vac-Elut added to the stripped sera to yield calibrators at con- cartridge rack were obtained from Varian Instruments. centrations of 0, 5, 12, 40, 100 ng/ml. The silica cartridges are washed in order with 5 ml HPLC-grade methanol, 5 ml HPLC-grade isopropanol, 2. SAMPLE AND CALIBRATOR EXTRACTION and 5 ml HPLC-grade hexane. The sample, in 1 ml of To extract 25OHD from calibrators and samples, 0.5 1.5% isopropanol in hexane, is then applied to the car- ml of acetonitrile is placed into a 12 × 75 mm borosil- tridge and eluted through the cartridge under vacuum icate glass tube after which 50 µl of sample or calibra- into waste. This initial elution is followed by 4 ml of tor is dropped through the acetonitrile. After 1.5% isopropanol in hexane (discard) and 6 ml of 5% vortex-mixing, the tubes are centrifuged (1000 g, 4¡C, isopropanol in hexane (25OHD). The 6-ml fraction con- 5 min) and 25 µl of supernatant transferred to 12 × 75 tains 25OHD2 and 25OHD3 and is subsequently dried in mm borosilicate glass tubes and placed on ice. CHAPTER 58 Detection of Vitamin D and Its Major Metabolites 937

A 0.004 25OHD2 25OHD3 + 3 0.5 ml Sample or control H-25OHD3

0.003 Incubate 15 min room temp

0.002

Solid-phase extract using C18 Sep-Pak 0.001

Dry acetonitrile under N2 and resuspend in 0 methylene chloride B 0.004

Apply to silica cartridge and collect 25OHD-containing fraction 0.003 24,25(OH)2D2 24,25(OH)2D2

Dry fraction under N and 0.002 2 resuspend in normal-phase HPLC mobile phase

0.001 Optical density 265 nm Apply to normal-phase silica HPLC and quantitate 25OHD2 and 25OHD3 by direct UV absorption. Collect 25OHD3 0 containing fraction and monitor for 3H-content for recovery correction C 0.004

FIGURE 7 Flow diagram of the HPLC-UV assay for the quanti- 0.003 1,25(OH)2D2 1,25(OH)2D3 tation of 25OHD2 and 25OHD3.

0.002 buffer (50 mM, pH 7.4, containing 0.1% swine skin gelatin). Nonspecific binding is estimated using the 0.001 above buffer minus the antibody. Vortex-mix the contents of the tubes and incubate them for 90 min at 20Ð25¡C. Following this period, add 0.5 ml of a second antibody 0 precipitating complex to each tube, vortex-mix, incubate 24681012 14 Elution time (min) at 20Ð25¡C for 20 min, and centrifuge (20¡C, 2000 g, 20 min). Discard the supernatant and bound the tubes FIGURE 6 High-performance liquid chromatographic profiles of in a gamma well counting system. 25OHD values are standard vitamin D metabolites. (A) 25OHD2 and 25OHD3; calculated directly from the standard curve by the (B) 24,25(OH)2D2 and 24,25(OH)2D3; (C) 1,25(OH)2D2 and 1,25- counting system using a smooth-spline method of (OH)2D3. The HPLC was performed on a normal-phase Zorbax-Sil calculation. The entire 25OHD RIA procedure is dis- column, and column calibration was achieved by injecting 10 ng of each compound and monitoring optical density at 265 nm. Column played in Fig. 8. conditions are described in the text. 4. COMMENTS ON THE 25OHD RIA This 125I-based RIA is similar to an RIA we intro- 3. RADIOIMMUNOASSAY 3 duced several years ago that used H-25OHD3 as a The assay tubes are 12 × 75 mm borosilicate glass tracer [13]. In both cases, antisera were raised against tubes containing 25 µl of acetonitrile-extracted calibrators the synthetic vitamin D analog 23,24,25,26,27-pentanor or samples. To each tube add 125I-25OHD derivative vitamin D-C(22)-carboxylic acid. The syntheses of this (50,000 cpm in 50 µl 1:1 ethanol-10 mM phosphate analog and its 125I-labeled counterpart have been buffer, pH 7.4) that was synthesized as previously described in detail [13,14]. Coupling this compound to described [14]. Then added to each tube 1.0 ml of pri- bovine serum albumin allowed us to generate antibodies mary antibody diluted 1:15,000 in sodium phosphate that cross-reacted equally with most vitamin D2 and D3 938 BRUCE W. HOLLIS

TABLE III Cross-reactivity of Various 50 µl Sample, standard or control Vitamin D Compounds with 25OHD Antiserum and 125I-labeled Vitamin D Derivativea

Steroid Cross-reactivity (%)b 500 µl Acetonitrile, 10 min spin Vitamin D2 0.8

Vitamin D3 0.8 DHT < 0.1 25(OH)D 100 25 µl Extract + 50 µl tracer + 1.0 ml 2 primary antibody 25(OH)D3 100

25(OH)D3-26,23-lactone 100

24,25(OH)2D2 100

24,25(OH)2D3 100 90 min Incubation 25,26(OH)2D2 100 at room temperature 25,26(OH)2D3 100

1,25(OH)2D2 2.5

1,25(OH)2D3 2.5

+ 0.5 ml Precipitating complex aFrom Hollis et al. Clin Chem 39:529-533. bAbility to displace 50% of the 125I tracer from the 25(OH)D antiserum diluted 15,000-fold.

20 min Incubation at room temperature - a variety of human serum samples (Fig. 9). Further, the 20 min spin present 125I-based RIA was shown to identify vitamin D deficiency in biliary atresia patients as well as vitamin D toxicity in hypoparathyroid patients who were receiving massive vitamin D therapy for the maintenance of Decant and count plasma calcium (Table IV).

FIGURE 8 Flow diagram of the direct RIA or the quantitation of 25OHD. 200 RIA = 0.98(HPLC) + 0.01 r2 = 0.98 n = 63 metabolites (Table III). The structures for vitamins D2 and D differ only with respect to their side chains 3 150 (Fig. 1). Because the analog retained the intact structure of vitamin D only up to carbon 22, the structural differ- ences between vitamins D2 and D3 were not involved in the antibody recognition, and antibodies directed 100 against this analog could not discriminate with respect to side chain metabolism of vitamin D. The antibody, however, was specific for the open B-ring cis-triene β structure containing a 3 -hydroxyl group that is inherent 50 in all vitamin D compounds. RIA 25OHD determination (ng/ml) Many vitamin D metabolites other than 25OHD are present in the circulation; however, they contribute only a small percentage (6Ð7%) to the overall assess- 0 ment of nutritional vitamin D status as compared with 050100 150 200 25OHD [23]. This fact is supported by the comparison HPLC 25OHD determination (ng/ml) of the 25OHD RIA with the UV quantitative HPLC FIGURE 9 25OHD values obtained by the 25OHD RIA (y axis) assay for 25OHD described earlier in Section III,B on and by direct UV quantitation of 25OHD following HPLC (x axis). CHAPTER 58 Detection of Vitamin D and Its Major Metabolites 939

TABLE IV Concentration of 25OHD as Determined 160.0 125 a by I-RIA in Various Physiological States 140.0 y = 0.98x − 1.5 ) ng/ml

 r = 0.77 Subject type n Mean (ng/ml)b Range (ng/ml)b 120.0 100.0 c Normal 36 25.7 9.9Ð41.5 80.0 Biliary atresia 12 6.3 4.3Ð8.3 60.0 Vitamin D therapyd 8 145 92Ð202 40.0 20.0 aFrom Hollis et al. Clin Chem 39:529Ð533. b × = ng/ml 2.496 nmol/liter. Vitamin D (Liaison 25 OH 0.0 cSamples from subjects in Minnesota in October. 0.0 20.0 40.0 60.0 80.0 100.0 dSamples from subjects with hypoparathyroidism or pseudohypo- 25 OH Vitamin D (RIA) ng/ml parathyroidism receiving pharmacological doses of vitamin D . 2 FIGURE 10 Linear regression analysis of 200 normal clinical samples by 125I-RIA and LIAISON¨

D. Automated Instrumentation CLIA Methodology protein results in the Nichols assay being less com- pliant when compared with the standard RIA (Fig. 12). DiaSorin Corporation (Stillwater, MN) has introduced This result is not unexpected since the vitamin DÐ a method for the direct (no extraction) quantitative deter- binding protein is susceptible to nonspecific interferences mination of 25OHD in serum or plasma utilizing com- from unknown substances in serum or plasma [24]. petitive chemiluminescence immunoassay (CLIA). The original version of this method encountered the This assay is offered in an automated format using the same problems 30 years ago and was discarded as an companies’ LIAISON¨ platform. Much of the method- invalid procedure [22]. Further, the Nichols system is ology is proprietary, although the assay utilizes a spe- less sensitive and much less efficient than the compara- cific antibody to 25OHD that is coated onto magnetic ble DiaSorin system. A direct comparison of the systems particles (solid phase). The tracer vitamin D is linked is displayed on Table V. to an isoluminol derivative. During the incubation of the sample, 25OHD is dissociated from its binding IV. ADDENDUM protein, and competes with the labeled vitamin D for binding sites on the antibody. After the incubation, the Recently, two articles have appeared that demon- unbound material is removed with a wash cycle. strate disturbing deficiencies in the Nichols Advantage Subsequently, the starter reagents are added and a flash 25(OH)D assay system. Binkley, et al. [24a] has shown chemiluminescent reaction is initiated. The light signal the Advantage assay to perform poorly on clinical samples is measured by a photomultiplier as relative light units and advised against its use in clinical settings. A more (RLU) and is inversely proportional to the concentration disturbing deficiency in the Advantage assay is its total of 25OHD present in calibrators, controls, and samples. inability to detect 25(OH)2 in clinical samples [24b]. This procedure will assay up to 180 samples/hr with This is in direct conflict with the manufacturer’s claim excellent linearity, precision, and sensitivity. Further, this automated assay is in excellent agreement with the widely used RIA (14) in both normal and chronic renal failure patients (Figs. 10 and 11). This assay format 40.0 would be the method of choice for large throughput y = 0.91x + 0.6 clinical laboratories. 30.0 r = 0.82 A similar assay platform has also been introduced by Nichols Institute Diagnostics (San Clemente, CA). 20.0 ¨ This platform is the Nichols ADVANTAGE system. 10.0 This instrument is similar to the DiaSorin LIAISON Liaison (ng/ml) system; however, the assay is very different. The 0.0 Nichols ADVANTAGE 25OHD assay utilizes the 0.0 10.0 20.0 30.0 40.0 RIA (ng/ml) human serum vitamin DÐbinding protein as a compet- itive binder instead of an antibody such as that used FIGURE 11 Linear regression analysis of 118 chronic renal fail- in the DiaSorin system. The use of the serum binding ure samples by 125I-RIA and LIAISON¨ 940 BRUCE W. HOLLIS l 160.0 V. DETECTION OF 24,25(OH2)D y = 1.07x + 11.0

) ng/m 140.0

 r = 0.59 120.0 A. Background 100.0 Next to 25OHD, 24,25(OH)2D is quantitatively 80.0 the most abundant circulating vitamin D metabolite, 60.0 and, as a result, interest in its circulating levels has 40.0 persisted. However, to this day, the biological func- 20.0 tion(s) of 24,25(OH)2D3, if any, remains unresolved. 0.0 The first assay for 24,25(OH)2D was first reported 0.0 20.0 40.0 60.0 80.0 100.0 25 OH Vitamin D (Advantage 25 OH in 1977 and used CPBA in conjunction with sample 25 OH Vitamin D (RIA) ng/ml prepurification on Sephadex LH-20 [8] (Table VI). FIGURE 12 Linear regression analysis of 200 normal clinical However, it was soon discovered that more exten- samples by 125I-RIA and Advantage¨ sive sample prepurification was required prior to 24,25(OH)2D quantitation owing to substances that interfered in the 24,25(OH)2D CPBA [25]. To further complicate matters, the quantitation of 24,25(OH)2D (Table V) that the assay can identify 25(OH)D2 at is especially difficult when both the vitamin D2 and D3 100% efficiency. The seriousness of this problem cannot forms are present in the circulation [16]. When both be overstated in that the Advantage assay system will forms of 24,25(OH)2D are present, it is extremely fail to monitor vitamin D therapy involving Drisdol hard to remove other vitamin D metabolites that coelute (vitamin D2) supplementation. with 24,25(OH)2D2 and 24,25(OH)2D3 on HPLC

TABLE V Direct Comparison of Liaison¨ and Advantage¨ 25OHD Automated Assay Systems

Parameter LIAISON¨ ADVANTAGE¨

Time to First Result 35 minutes 75 minutes Maximum Throughput 180 tests/hr 75 tests/hr Lowest Reportable Value 2.6 ng/ml (6.5 nmol/L) 7.0 ng/ml (17.5 nmol/l) Sensitivity (Analytical) < 2.0 ng/ml < 2.0 ng/ml Mean % Recovery 105% ± 14% 94% ± 6% Intra-Assay Precision 13.6 ng/ml 10% 13.3 ng/ml 7% (% CV at 3 concentrations) 24.4 ng/ml 8% 26.6 ng/ml 4% 50.7 ng/ml 6% 55.5 ng/ml 2% Inter-Assay Precision 13.6 ng/ml 19% 13.3 ng/ml 20% (% CV at 3 concentrations) 24.4 ng/ml 17% 26.6 ng/ml 16% 50.7 ng/ml 13% 55.5 ng/ml 14% Dilution Linearity Observed = Expected (1.04) − 0.4; r = 0.95 Observed = Expected (0.95) + 3.7; r = 0.99 Sample Equivalence (Serum vs. Plasma) Plasma = Serum (1.1) Ð 2.4; r = 0.93 Plasma = Serum (0.96) + 1.3; r = 0.95

Cross-Reactivity D2 0.0% D2 0.0%

D3 0.0% D3 4.5%

25D2 100.0% 25D2 100.0%

25D3 100.0% 25D3 100.0%

1,25D2 7.1% 1,25D2 0.0%

1,25D3 21.7% 1,25D3 2.2% Carry-Over None None Reference Range Median = 25.5 ng/ml Median = 45.4 ng/ml (2.5th to 97.5th %) Range = 8.6Ð54.8 ng/ml Range = 20.4Ð90.2 ng/ml CHAPTER 58 Detection of Vitamin D and Its Major Metabolites 941

TABLE VI Significant Methods for the Estimation of 24,25(OH)2D in Human Serum Detection Preliminary Normal method Extraction chromatography Ref. circulating levelsb

CPBA Methanol-methylene chloride Sephadex LH-20 Haddad et al. [8] 3.7 ± 0.2 ng/ml CPBA Methanol-methylene chloride Sephadex LH-20, preparative HPLC Shepard et al. [25] 3.5 ± 1.4 ng/ml HPLC Methanol-methylene chloride Sephadex LH-20, preparative HPLC Dreyer and Goodman [26] 2.4 ± 1.1 ng/ml RIA Methanol-methylene chloride Sephadex LH-20, preparative HPLC Hummer and 0.1Ð4.0 ng/ml Christiansen [27]

CPBA Solid phase C18OH Silica cartridges Wei et al. [28] 3.1 ± 0.7 ng/ml

ang/ml × 2.4 = nmol/liter.

separation [16]. Further, once 24,25(OH)2D2 and and eluted through the cartridge under vacuum into 24,25(OH)2D3 are adequately separated and ready for waste. This initial step is followed by 5 ml of 40% CPBA, varying affinities of the two metabolites for water in methanol (discard), 5 ml of 1% methylene the DBP require standard curves to be constructed chloride in hexane (discard), and 5 ml of 5% iso- for final quantitation [29]. propanol in hexane [24,25(OH)2D]. The final fraction is A report published in 1994 questions the require- dried in a heated water bath, 55¡C, under N2. The lipid ment of HPLC prepurification of the serum sample residue is then resuspended in 150 µl 5% isopropanol in prior to CPBA [28]. However, we are firm believers hexane and capped. that in order to perform a valid assay for 24,25(OH)2D, one has to incorporate HPLC prepurification into the 3. PREPARATIVE NORMAL-PHASE HIGH-PERFORMANCE assay protocol. We have also chosen to do the final LIQUID CHROMATOGRAPHY quantitation of 24,25(OH)2D by RIA instead of CPBA. The normal-phase HPLC step is performed using a The RIA was chosen because the antibody used is 0.4 × 25 cm Zorbax-Sil column packed with 5 µm silica. cospecific for the vitamin D2 and D3 forms, and thus The mobile phase is composed of hexane-methylene only one compound, 24,25(OH)2D3, is required to con- chloride-isopropanol (80:15:3.5, v/v) at a flow rate struct the standard curve (Table III). This procedure is of 2 ml/min. The sample residue from the C18 silica car- described here in detail. tridge is injected onto the HPLC column that had been previously calibrated with 10 ng of 24,25(OH)2D2 and 24,25(OH)2D3. The elution of these metabolites can be B. Methodology seen in Fig. 6B. The fractions containing 24,25(OH)2D2 and 24,25(OH)2D3 are collected individually in 12 × AMPLE XTRACTION 1. S E 75 mm glass tubes and dried under N2 at 55¡C. The A 0.5-ml sample of serum or plasma is placed into residue is then redissolved in 500 µl absolute ethanol a 12 × 75 mm borosilicate glass culture tube containing and capped. 3 1000 cpm of H-24,25(OH)2D3 in 25 µl of ethanol to monitor recovery of endogenous compound through the 4. RADIOIMMUNOASSAY extraction and chromatographic procedures. Following The assay tubes are 12 × 75 mm borosilicate glass a 15-min incubation with the tracer, 1 plasma volume of tubes containing 25 µl of the HPLC-purified extracts in HPLC-grade acetonitrile is added to each sample. The ethanol. The standards for this RIA, which are sample is then vortex-mixed for 1 min followed by 24,25(OH)2D3 are placed in 12 × 75 mm tubes in 25 µl centrifugation at 1000 g for 10 min. The supernatant is ethanol at concentrations between 0 and 200 pg/tube. removed into another 12 × 75 mm culture tube, and 1 To each tube add 125I-25OHD derivative (50,000 cpm plasma volume of distilled water is added. in 50 µl 1:1 ethanol-10 mM phosphate buffer, pH 7.4) 3 or H-25OHD3 (5000 cpm in 25 µl ethanol). Then add 2. SOLID-PHASE EXTRACTION CHROMATOGRAPHY to each tube 1.0 ml of primary antibody diluted C18 silica Bond-Elut cartridges (500 mg) and a 1:15,000 in sodium phosphate buffer (50 mM, pH 7.4, Vac-Elut cartridge rack were obtained from Varian containing 0.1% swine skin gelatin). Nonspecific bind- Instruments. The C18 cartridges are washed in order with ing is estimated using the above buffer minus the anti- 5 ml HPLC-grade isopropanol and 5 ml HPLC-grade body. Vortex-mix the contents of the tubes and methanol. The sample is then applied to the cartridge incubate them for 90 min at 20Ð25¡C. Following this 942 BRUCE W. HOLLIS

with respect to quantitation. 1,25(OH) D circulates at 0.5 ml Sample or control 2 + 3 low picogram per milliliter concentrations (too low for H-24,25(OH)2D3 direct UV quantitation), is highly lipophilic, and is rela- Incubate 15 min room temp tively unstable, and its precursor, 25OHD, circulates at concentrations in excess of 103 to 104 times that of 1,25(OH)2D. The first RRA for 1,25(OH)2D was intro- Isolate 24,25(OH)2D by simultaneous duced in 1974 [9] (Table VII). Although this initial assay extraction and purification using C18- support in "phase-switching" mode was extremely cumbersome, it did provide invaluable information with respect to vitamin D homeostasis.

Dry organics under N2 and This initial RRA required a 20-ml serum sample, resuspend in normal- which was extracted using Bligh-Dyer organic extrac- phase HPLC mobile phase tion. The extract had to be purified by three successive laborious chromatographic systems (there was no Apply to normal-phase silica HPLC and individually collect HPLC at the time), and chickens had to be sacrificed 24,25(OH)2D3 and 24,25(OH)2D2 and vitamin D receptor (VDR) harvested from their intestines at the time of the RRA. By 1976, the volume requirement for this RRA had been reduced to a 5-ml Remove a portion Dry organics under N2, for recovery and resuspend each sample and sample prepurification had been modified estimation fraction in ethanol to include HPLC [30]. However, the sample still had to be extracted using a modified Bligh-Dyer extraction, and then prepurified on Sephadex LH-20, and chicken Quantitate 24,25(OH)2D2 and intestinal VDR was still utilized as a binding agent. 24,25(OH)2D3 individually by RIA using antibody cospecific for each In 1978, the first RIA for 1,25(OH)2D was intro- compound. 3H or 125I tracers duced [12]. Although it was an advantage not to have are available for use to isolate the intestinal VDR as a binding agent, this RIA was relatively nonspecific, so the cumbersome sample preparative steps were still required. Because FIGURE 13 Flow diagram of the HPLC-RIA assay for the quan- titation of 24,25(OH) D and 24,25(OH) D . of the extreme technical nature of these assays, and the 2 2 2 3 cost of HPLC systems, few laboratories could afford to measure circulating 1,25(OH)2D. Further, because period, add 0.5 ml of a second antibody precipitating these early techniques were so cumbersome, commer- 125 complex to each tube if I tracer was used, 0.2 ml of cial laboratories did not offer 1,25(OH)2D determina- 0.1 M borate buffer containing 1.0% norit A charcoal tions as a clinical service. 3 and 0.1% dextran T-70 if H-25(OH)D3 was used, This all changed in 1984 with the introduction of a vortex-mix, incubate at 20Ð25¡C for 20 min and cen- radically new concept for the determination of 125 trifuge (20¡C, 2000 g, 20 min). In the case of I tracer, circulating 1,25(OH)2D [31]. This new RRA utilized discard the supernatant and count the tubes in a gamma solid-phase extraction of 1,25(OH)2D from serum 3 well counting system. In the case of H-25OHD3, remove along with silica cartridge purification of 1,25(OH)2D. the supernatant into vials, add scintillation fluid, and As a result, the need for HPLC sample prepurification monitor for radioactive content in a scintillation was eliminated. Also, this assay utilized VDR isolated counter. 24,25(OH)2D values are calculated from the from calf thymus, which proved to be quite stable and standard curve in picograms per tube. To convert this thus had to be prepared only periodically. Further, the value to nanograms per milliliter, correct for dilution volume requirement was reduced to 1 ml of serum or 3 used as well as final recovery of H-24,25(OH)2D3 plasma. This assay opened the way for any laboratory added at the beginning of the sample extraction proce- to measure circulating 1,25(OH)2D. This procedure dure. The entire 24,25(OH)2D RIA procedure is also resulted in the production of the first commercial displayed in Fig. 13. kit for 1,25(OH)2D measurement. This RRA was fur- ther simplified in 1986 by decreasing the required VI. DETECTION OF 1,25(OH) D chromatographic purification steps [32]. Through the 2 mid-1990s, no new advances were reported with A. Background respect to the quantitation of circulating 1,25(OH)2D. As good as the calf thymus RRA for 1,25(OH)2D Of all the steroid hormones, 1,25(OH)2D represented was, it still possessed two serious shortcomings. First, the most difficult challenge to the analytical biochemist VDR had to be isolated from thymus glands, which CHAPTER 58 Detection of Vitamin D and Its Major Metabolites 943

TABLE VII Significant Methods for the Estimation of 1,25(OH)2D in Human Serum Detection Preliminary Normal method Extraction chromatography Ref. circulating levelsb

RRA Methanol-chloroform Silicic acid, Sephadex LH-20, celite Brumbaugh et al. [9] 39 ± 8 pg/ml RRA Methanol-methylene chloride Sephadex LH-20, preparative HPLC Eisman et al. [30] 29 ± 2 pg/ml RIA Methanol-chloroform Sephadex LH-20, preparative HPLC Clemens et al. [12] 35 pg/ml

RRA Solid phase C18OH Silica cartridge Reinhardt et al. [31] 37.4 ± 2.2 pg/ml

RRA Solid phase C18OH None Hollis [32] 28.2 ± 11.3 pg/ml

RIA Solid phase C18OH/silica None Hollis et al. [15] 32.2 ± 8.5 pg/ml

apg/ml × 2.4 = pmol/liter

was still a difficult technique. Second, because the 2. SAMPLE EXTRACTION 3 VDR is so specific for its ligand, only H-1,25(OH)2D3 A 1.0 ml sample of serum or plasma is placed into a could be used as a tracer, eliminating the possibility of 12 × 75 mm borosilicate glass culture tube containing 125 3 using a I-based tracer. This is a major handicap, 700 cpm of H-1,25(OH)2D3 in 25 µl of ethanol to mon- especially for the commercial laboratory. As a result, itor recovery of endogenous compound through the we have developed and reported in 1996 the first extraction and chromatographic procedures. Following a significant advance in 1,25(OH)2D quantification in a 15-min incubation with the tracer, 1 ml of HPLC-grade decade [15]. This new RIA incorporates an 125I-tracer, acetonitrile is added to each sample. The sample is then as well as standards in an equivalent serum matrix, so vortex-mixed for 1 min, followed by centrifugation at individual sample recoveries are no longer required. 1000 g for 10 min. The supernatant is removed into We describe this new RIA for 1,25(OH)2D along with another 12 × 75 mm culture tube, and 1 vol of 0.4 M the standard RRA. K2HPO4, pH 10.4, is added followed by vortex-mixing.

3. SOLID-PHASE EXTRACTION AND PURIFICATION B. RRA Methodology CHROMATOGRAPHY C18OH silica Bond-Elut cartridges (500 mg) and a 1. PREPARATION OF CALF THYMUS VDR Vac-Elut cartridge rack were obtained from Varian Frozen or fresh tissue is processed for VDR as fol- Instruments. The C18OH cartridges are washed in order lows (all steps are carried out at 4¡C). Thymus tissue is with 5 ml HPLC-grade methylene chloride, 5 ml HPLC- minced with a meat grinder and homogenized (20% w/v) grade isopropanol, and 5 ml HPLC-grade methanol. in a buffer containing 50 mM K2HPO4, 5 mM dithio- The sample is applied to the cartridge and eluted through threitol, 1 mM EDTA, and 400 mM KCl, pH 7.5. The the cartridge under vacuum into waste. This initial step tissue is homogenized using five, 30-sec bursts of a is followed by 5 ml of 30% water in methanol (discard), Polytron PT-20 tissue disrupter at a maximum power 5 ml of 10% methylene chloride in hexane (discard), setting. The homogenate is then centrifuged for 15 min 5 ml of 1% isopropanol in hexane (discard), and 5 ml at 20,000 g to remove large particles. The resulting of 3% isopropanol in hexane [1,25(OH)2D] (Fig. 14). supernatant is centrifuged at 100,000 g for 1 hr, and the This final fraction is dried in a heated water bath, “cytosol” (actually a high salt extract that includes 55¡C, under N2. The residue is then suspended in 200 µl nucleus VDR) is collected minus the floating lipid layer. absolute ethanol and capped. The VDR is then precipitated by the slow addition of 4. RADIORECEPTOR ASSAY solid (NH4)2SO4 to 35% saturation. The cytosol- (NH4)2SO4 mixture is stirred for 30 min while main- Prior to assay, the VDR-containing pellet is reconsti- taining the temperature at 4¡C. The mixture is then tuted to its original volume with assay buffer. The assay divided into 15-ml centrifuge tubes and centrifuged at buffer contains 50 mM K2HPO4, 5 mM dithiothreitol, 20,000 g for 20 min. The supernatant is discarded, and 1.0 mM EDTA, and 150 mM KCl at pH 7.5. The recep- tubes are allowed to drain for 5 min. The precipitated tor pellet is dissolved by gentle stirring on ice using a VDR is lyophilized and stored under inert gas at −70¡C. magnetic stir bar. The receptor solution is allowed to mix VDR prepared in this manner is stable for up to 60 hr for 20Ð30 min. Typically, a small portion of the pellet at room temperature. resists solubilization and is removed by centrifugation 944 BRUCE W. HOLLIS

H O CH OH:H O Hexane: CH CI 2 3 2 2 2 Hexane: Isopropanol 100% 70:30 90:10 99:1 97:3 100 3 H-25(OH)D3 80 60 40 20

3 100 H-24,25(OH)2D3 80 60 40 20

ercent of total radioactivity 100 3 P H-1,25(OH)2D3 80 60 40 20

0 2468101214 16 18 20 22 24 Elution volume (ml)

3 FIGURE 14 Elution of H-vitamin D3 and its metabolites from a C18OH Bond-Elut cartridge. From Hollis BW, Clin Chem 31:1815Ð1819.

at 3000 g for 10 min. The receptor solution is then diluted + 3 1:3Ð1:9 with assay buffer and kept on ice until use. The 1.0 ml Sample or control H-1,25(OH)2D3 correct dilution of receptor used in the assay is deter- 1.0 ml Acetonitrile, mined for each new batch of receptor. At the appropriate 10 min spin dilution for assay use, specific binding in the absence of Combine supernatant with unlabeled 1,25(OH)2D is 1600Ð2000 cpm; nonspecific 1 vol K HPO , pH 10.4 binding is 200Ð300 cpm. These results assume a spe- 2 4 3 cific activity of 130 Ci/mmol for H-1,25(OH)2D3 and a 40% counting efficiency for tritium. The assay tubes are 12 × 75 mm borosilicate glass Isolate 1,25(OH)2D by simultaneous extraction and purification tubes containing 50 µl of C18OH-purified extracts in ethanol. The standards for the assay, 1,25(OH) D , are Dry organics under N2 and 2 3 resuspend in ethanol placed in 12 × 75 mm tubes in 50 µl ethanol at concen- trations between 1 and 15 pg/tube. Nonspecific bind- 50 µl Extract + 500 µl thymus receptor preparation ing is estimated by adding 1 ng/tube of 1,25(OH)2D3. To each tube add 0.5 ml of reconstituted thymus 50 µl for recovery cytosol, vortex-mix, and incubate for 1 hr at 15Ð20¡C. 1 hr Incubation at 15–20°C estimation Following this initial incubation, each tube receives 3H-1,25(OH) D (5000 cpm in 50 µl ethanol) and the + µ 3 2 3 50 l H-1,25(OH)2D3 incubation proceeds for an additional 1 hr at 15Ð20¡C. Finally, place the assay tubes in an ice bath and add 1 hr Incubation at 15–20°C M 0.2 ml of 0.1 borate buffer containing 1.0% norit A + 200 µl Dextran-charcoal solution charcoal and 0.1% dextran T-70, vortex-mix, incubate 20 min, and centrifuge (4¡C, 2000 g, 10 min). Remove 20 min Incubation at room temp + 10 min spin the supernatant into vials, add scintillation fluid, and Decant and count monitor for radioactive content in a scintillation counter. 1,25(OH)2D values are calculated from the FIGURE 15 Flow diagram of the RRA for the quantitation of standard curve in picograms per tube. To convert this 1,25(OH)2D. CHAPTER 58 Detection of Vitamin D and Its Major Metabolites 945 value to picograms per milliliter, correct for dilution µ 3 500 l Sample, standard or control used, as well as final recovery of H-1,25(OH)2D3 added at the beginning of the sample extraction proce- µ dure. The 1,25(OH)2D RRA procedure is displayed 500 l Acetonitrile, in Fig. 15. 10 min spin

C. RIA Methodology Combine supernatant with 1 vol K2PO4, pH 10.4 1. PREPARATION OF ASSAY CALIBRATORS As was described for the 25OHD RIA (Section III,C), one of the goals of this RIA procedure was to eliminate the need for individual sample recovery. To prepare the assay calibrators, human serum was stripped free Isolate 1,25(OH)2D by simultaneous of vitamin D metabolites. The absence of endogenous extraction and purification 1,25(OH)2D in the stripped sera was confirmed by RRA for 1,25(OH)2D as previously described in Section V,B. Subsequently, crystalline 1,25(OH)2D3 dissolved in Dry organics under N2 and absolute ethanol was added to the stripped sera to resuspend in ethanol yield calibrators at concentrations of 0, 5, 15, 30, 75, and 200 pg/ml. 75 µl Extract/tracer 2. SAMPLE AND CALIBRATOR EXTRACTION + 300 µl primary antibody AND PRETREATMENT

The 1,25(OH)2D is extracted from calibrators and samples as follows. First, 0.5 ml of serum of plasma is 2 hr Incubation at room temp placed into a 12 × 75 mm borosilicate glass culture tube; 0.5 ml of HPLC-grade acetonitrile is added and vortex-mixed for 1 min followed by centrifugation at + 500 µl Precipitating complex 1000 g for 10 min. The supernatant is removed into another 12 × 75 mm culture tube, and 1 vol of 0.4 M K2HPO4, pH 10.4, is added followed by vortex-mixing. 20 min Incubation at room temp + 20 min spin 3. SOLID-PHASE EXTRACTION AND SILICA PURIFICATION CHROMATOGRAPHY Decant and count C18OH silica “Extra Clean” cartridges (500 mg) and a Vac-Elut cartridge rack were obtained from DiaSorin FIGURE 16 Flow diagram of the RIA for the quantitation of Corp and Varian Instruments, respectively. The 1,25(OH)2D. C18OHEC cartridges are washed in order with 5 ml HPCL-grade methylene chloride, 5 ml HPLC-grade isopropanol, and 5 ml HPLC-grade methanol. The sam- ple is applied to the cartridge and eluted through the 4. RADIOIMMUNOASSAY cartridge under vacuum into waste. This initial step is The assay tubes are 12 × 75 mm borosilicate glass followed by 5 ml of 30% water in methanol (discard), tubes containing 75 µl of the ethanol/tracer-reconstituted 5 ml of 10% methylene chloride in hexane (discard), extracted calibrators or samples. Then add to each tube 3 ml of 1% isopropanol in hexane (discard), and 3 ml 0.35 ml of primary antibody diluted 1:40,000 in sodium of 8% isopropanol in hexane [1,25(OH)2D]. This final phosphate buffer [50 mM, pH 6.2, containing 0.1% fraction is dried in a heated water bath, 55°C under N2 swine-skin gelatin and 0.35% polyvinyl alcohol or in a rapid vacuum device such as a Labconco Rapid- (Mr 13,000Ð23,000)]. Nonspecific binding is estimated Vap. The residue is first reconstituted with 50 µl of 95% by using the above buffer without the antibody. Vortex- ethanol with vortex-mixing. Each tube now receives 125 µl mix the contents of the tubes, incubate them for 2 hr at of I125-tracer solution with additional vortex-mixing. 20Ð25¡C, add 0.5 ml of second antibody precipitating The sample may be capped and stored at Ð20°C or one complex, incubate at 20Ð25¡C for 20 minutes, and may proceed to finish the assay at this point. then centrifuge (20¡C, 2000 g, 20 min). Discard the 946 BRUCE W. HOLLIS supernatant and count the tubes in a gamma well count- TABLE VIII Cross-reactivity of Various Vitamin D ing system. 1,25(OH)2D values are calculated directly Compounds with 1,25(OH)2D Antiserum from standard curve by the counting system using a and 125I-labeled 1-Hydroxylated Tracer smooth-spline method of calculation. The entire Steroid Cross-reactivity (%)a 1,25(OH)2D RIA procedure is displayed in Fig. 16.

Vitamin D3 < 0.001 5. COMMENTS ON THE 1,25(OH)2D RIA 25(OH)D3 0.002 Of the procedures developed for determining 24,25(OH)2D3 0.012 1,25(OH)2D status in humans, only a few RRA [31,32] 25,26(OH)2D3 0.003 have been able to quantify circulating 1,25(OH)2D without using HPLC for sample prepurification. Many 1,25(OH)2D2 100 RIA have been published and validated for the quan- 1,25(OH)2D3 100 tification of 1,25(OH) D, but all have included HPLC 2 a 125 Ability to displace 50% of the I tracer from the 1,25(OH)2D antiserum steps for sample prepurification [12,33,34]. Development diluted 1:40,000. of an RIA for quantification of circulating 1,25(OH)2D has been hampered from the beginning by the relatively 125 poor specificity of the antibodies that have been gener- I-based RIA for 1,25(OH)2D that involves the ated. To date, the best antibodies toward 1,25(OH)2D immunoextraction of 1,25(OH)2D from serum samples have, at best, a cross-reactivity with the non-1-hydroxy- and is marketed by IDS Ltd. (Tyne and Wear, UK) [35]. lated metabolites of vitamin D of approximately 1%. The basis of this kit is selective immunoextraction In comparison, the VDR used in the RRA has a cross- of 1,25(OH)2D from serum or plasma with a specific reactivity of approximately 0.01% with these more monoclonal antibody bound to a solid support. This abundant metabolites [31]. Given that the non-1- antibody is directed toward the lα-hydroxylated A ring hydroxylated metabolites circulate at concentrations of 1,25(OH)2D [36]. We concluded that this immuno- over 1000 times greater than that of 1,25(OH)2D, the extraction procedure was highly specific for the magnitude of the problem becomes clear. However, the lα-hydroxylated forms of vitamin D [35]. However, VDR is so specific that any attempt to introduce a there was a serious flaw in the assumptions made when 125 radionuclide such as I into 1,25(OH)2D3 erodes the this kit was designed: 1,25(OH)2D was the only signifi- binding between this steroid hormone and the VDR. cant lα-hydroxylated vitamin D metabolite that circulates. Therefore, if one wishes to develop 125I-based assays Many other lα-hydroxylated metabolites exist in the to quantify 1,25(OH)2D, RIA is the only choice. circulation, including 1,24,25(OH)3D3, 1,25,26(OH)3D3, Since the original paper on this assay was published l,25(OH)2D3-26,23-Lactone, 1,25(OH)2-24-oxo-D3, cal- [15], several improvements have been implemented. citroic acid, and probably various water-soluble, side- The major improvement involved the generation of a chain conjugates. Some of these compounds are new antibody. This new antibody has greater sensitivity, specificity as compared to the original antibody [15]. Further, the new antibody expresses 100% cross- 90 RIA reactivity between 1,25(OH)2D2 and 1,25(OH)2D3 80 (Table VIII). This new antibody has allowed the RIA to RRA return to a single column format for sample purifica- 70

tion along with the removal of a sample pretreatment D (pg/ml) 60 2 step involving NaIO4. This RIA recently received FDA approval for clinical diagnosis in humans and 50 thus far is the only test for 1,25(OH)2D to achieve this 40 status. 30 The concentrations of 1,25(OH)2D as determined in serum from various groups of healthy and pathological 20 Circulating 1,25(OH) subjects (Fig. 17) agree well with values reported in 10 previous studies [31,32]. It is very important to include 0 pathological samples such as those from subjects with Normal Chronic Hypopara- Biliary Pregnant biliary atresia and vitamin D toxicity in any assay subjects renal thyroid atresia subjects validation procedure for circulating 1,25(OH)2D. This failure importance was underlined in our previous report on an FIGURE 17 Comparison of circulating 1,25(OH)2D measured unpublished, unvalidated, but commercially available by RIA and RRA. From Hollis et al. Clin Chem 42:586Ð592. CHAPTER 58 Detection of Vitamin D and Its Major Metabolites 947

120

Immobilized 1-OH-specific 100 MAb 80

Measured Removed 60 D assayed (pg/ml) D assayed 2 40

1) 100% 1,25(OH)2D 1) All non-1-hydroxylated metabolites 1,25(OH) 2) 100% 1,24,25(OH)3D 20 3) 100% 1,25,26(OH)3D 4) 100% 1,23,25(OH)3D 0 0510 15 20 50 5) 100% 1,25(OH)2D3-26,23-lactone 1,25(OH) D -26,23-lactone added (pg/ml) 6) 100% Calcitroic acid 2 3

7) 100% Water-soluble conjugates of FIGURE 20 Effect of exogenously added l,25(OH)2D-26, calcitroic acid 23-lactone on the “apparent” serum concentration of 1,25(OH)2D. Concentrations were assessed by RRA (squares), RIA as described FIGURE 18 Graphic description of the vitamin D metabolites in the text (triangles), and IDS immunoextraction RIA (diamonds). assayed as “apparent” circulating 1,25(OH)2D by utilizing immu- From Hollis BW. Clin Chem 41:1313Ð1314. noextraction with a 1-hydroxy-specific monoclonal antibody (MAb) in conjunction with RIA.

l,25(OH)2D3-26,23-lactone is a significant in vivo bioactive, but most are not, and this assay cannot distin- metabolite in a variety of clinical samples, and we guish among them (Fig. 18). Further, compare how the find its concentration to be 0Ð30% of the respective IDS RIA for 1,25(OH)2D performs outside of normal 1,25(OH)2D concentration. Further, using the RIA or chronic renal failure human samples (Fig. 19). This based on immunoextraction, we have found “apparent” assay appears to be inadequate when presented with levels of 1,25(OH)2D to be grossly higher than the selected pathological samples. actual concentration in vitamin D-intoxicated subjects, We have specifically investigated the effects of l,25- hypoparathyroid subjects receiving vitamin D therapy, (OH)2D3-26,23-lactone on the “apparent” 1,25(OH)2D and biliary atresia patients (Fig. 19). We have also levels using the immunoextraction technique and observed some normal samples that displayed 100% found it to interfere on an equal molar basis compared elevation from the actual levels. What this assay is rec- with 1,25(OH)2D3 (Fig. 20). We also know that ognizing in these samples remains unknown, but it is undoubtedly some lα-hydroxylated metabolite, proba- bly a catabolic product. It is important to note that the 400 RIA described in this chapter, which is based on classic RRA IDS-RIA separation procedures, appears to escape this problem of detecting inactive lα-hydroxylated vitamin D metabo- 300 lites (Figs. 17). D (pg/ml) 2

200 VII. CLINICAL INTERPRETATION AND RELEVANCE OF ANTIRACHITIC 100 STEROL MEASUREMENTS

Circulating 1,25(OH) A. Vitamin D 0 Normal Chronic Hypo-para- Biliary Calcium Vitamin D subjects renal thyroid atresia deficient deficient The quantitation of circulating vitamin D is essen- failure rat rat tially of no clinical importance. The parent compound is a poor indicator of nutritional status because of its FIGURE 19 Circulating 1,25(OH)2D as determined by the RRA (squares) or IDS immunoextraction RIA (triangles) on a variety of short circulating half-life. The circulating levels of clinical samples. The same samples were compared in each assay. vitamin D are also difficult to interpret because the Horizontal lines denote means. levels are greatly affected by short-term sun exposure TABLE IX Relative Circulating Concentrations of Vitamin D, 25OHD, 24,25(OH)2 and 1,25(OH)2 in Various Disease States

a b c d Condition Vitamin D 25(OH)D 24,25(OH)2D 1,25(OH)2D

Nutritional deficiency Decreased Decreased Normal Increased followed by decrease Hypoparathyroidism Normal Normal Normal Decreased Pseudohypoparathyroidism Normal Normal Normal Decreased Hyperparathyroidism Normal Normal Normal Decreased Tumor-induced osteomalacia Normal Normal Normal Decreased Vitamin D-dependent rickets, type I Normal Normal Normal Decreased Vitamin D-dependent rickets, type II Normal Normal Normal Increased Sarcoidosis Normal Normal Normal Increased during hypercalcemia Renal failure Normal or decreased Normal or decreased Decreased Decreased Nephrotic syndrome Decreased Decreased Decreased Decreased Hypervitaminosis D Increased Increased Increased Normal or decreased Cirrhosis Normal or decreased Normal or decreased Normal or decreased Normal or decreased Tuberculosis Normal Normal Normal Increased during hypercalcemia Hodgkin’s disease Normal Normal Normal Increased during hypercalcemia Lymphoma Normal Normal Normal Increased during hypercalcemia Wegener’s granulomatosis Normal Normal Normal Increased during hypercalcemia X-linked hypophosphatemic rickets Normal Normal Normal Decreased or normal

aNormal range is 0Ð30 ng/ml (0Ð78 nmol/liter) and is extremely variable with respect to sunlight exposure and dietary intake. bNormal range is 15Ð80 ng/ml (37Ð192 nmol/liter) and is related to season, latitude, and diet. cNormal range is 0.5Ð4 ng/ml (1.2Ð9.6 nmol/liter) and is directly related to circulating 25OHD. dNormal range is 20Ð60 pg/ml (48Ð144 pmol/liter). CHAPTER 58 Detection of Vitamin D and Its Major Metabolites 949 and dietary intake of vitamin D [38,39]. Vitamin D has endocrine system, including hypoparathyroidism, proved to be useful in assessing intestinal lipid absorp- hyperparathyroidism, and chronic renal failure, the tive capacity associated with fat malabsorption assay of 1,25(OH)2D is a confirmatory test. It is also syndromes [40,41]. However, this use is more of a important to remember that circulating 1,25(OH)2D research application as opposed to an application used in provides essentially no information with respect to the a clinical diagnosis. Table IX lists a variety of clinical patient’s nutritional vitamin D status. Thus, circulating conditions for which the circulating levels of vitamin D 1,25(OH)2D should not be used as an indicator for have been defined. hypo- or hypervitaminosis D when nutritional factors are suspected (Table IX).

B. 25OHD References Nutritional vitamin D status is defined by the amount of circulating 25OHD [42]. The assessment of 1. Seamark DA, Trafford DHJ, Makin HLJ 1981 The estimation circulating 25OHD is thus an important measurement of vitamin D and its metabolites in human plasma. J Steroid to the clinician. Subnormal circulating levels of Biochem 14:111Ð123. 2. Porteous CE, Coldwell RD, Trafford DJH, Makin HLJ 1987 25OHD usually result from inadequate vitamin D Recent developments in the measurement of vitamin D and its intake and/or insufficient sunlight exposure. This com- metabolites in human body fluids. J Steroid Biochem bination of events usually puts elderly patients at 28:785Ð801. risk of developing vitamin D deficiency and ensuing 3. Jones G, Trafford DJH, Makin HLJ, Hollis BW 1992 Vitamin D: secondary hyperparathyroidism, especially if they are Cholecalciferol, ergocalciferol, and hydroxylated metabolites. In: DeLeenheer AP, Lambert WE, Nelis HJ (eds) Modem homebound [43]. This, in turn, has been shown to Chromato-graphic Analysis of Vitamins. Dekker, New York, result in an increased incidence of hip fractures in the pp. 73Ð151. elderly [44]. Other conditions that contribute to nutri- 4. McCollum EV, Simmonds N, Shipley PG, Park EA 1922 tional vitamin D deficiency include nephrotic syndrome, Studies on experimental rickets. XVI. A delicate biological test chronic renal disease, cirrhosis, and malabsorption syn- for calciumÐdepositing substances. J Biol Chem 51:41Ð49. 5. Suda T, DeLuca HF, Hallick RB 1971 Synthesis of [26 > 27- dromes such as biliary atresia (Table IX). Vitamin D 3Hj-25-hydroxycholecalciferol. Anal Biochem 43:139Ð146. intoxication, though rare, still occurs and is most accu- 6. Haddad JG, Chyu KJ 1971 Competitive protein-binding rately diagnosed by determining circulating 25OHD. radioassay for 25-hydroxycholecalciferol. J Clin Endocrinol Thus, from a clinical standpoint, the determination of Metab 33:992Ð995. circulating 25OHD is the most frequently requested 7. Belsey R, DeLuca HF, Potts JT 1971 Competitive binding assay for vitamin D and 25-OH vitamin D. J Clin Endocrinol antirachitic sterol measurement. Metab 33:554Ð557. 8. Haddad JG, Min C, Mendelsohn M, Slatopolsky E, Hahn TJ 1977 Competitive protein-binding radioassay of 24,25-dihy- droxyvitamin D in sera from normal and anephric subjects. C. 24,25(OH)2D Arch Biochem Biophys 182:390Ð395. 9. Brumbaugh PF, Haussler DH, Bursac DM, Haussler MR 1974 At the present time, there does not appear to be a Filter assay for 1,25-dihydroxyvitamin D3. Utilization of the compelling reason to measure circulating 24,25(OH)2D hormones target tissue chromatin receptor. Biochemistry in a clinical setting. Even in a research setting, the 13:4091Ð4097. 10. Jones G 1978 Assay of vitamins D and D , and 25-hydroxy- determination of 24,25(OH)2D is of questionable value 2 3 as evidenced by the decreased usage of this assay in vitamins D2 and D3 in human plasma by high-performance liquid chromatography. Clin Chem 24:287Ð298. the literature since the early 1990s. 11. Eisman JA, Shepard RM, DeLuca HF 1977 Determination of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 in human plasma using high-pressure liquid chromatography. Anal Biochem 80:298Ð305. D. 1,25(OH)2D 12. Clemens TL, Hendy GN, Graham RF, Baggiolini EG, Uskokovic MR, O’Riordan JLH 1978 A radioimmunoassay for Circulating 1,25(OH)2D is diagnostic for several 1,25-dihydroxyholecaliferol. Clin Sci Mol Med 54:329Ð332. clinical conditions, including vitamin D-dependent 13. Hollis BW, Napoli JL 1985 Improved radioimmunoassay for rickets types I and II, hypercalcemia associated with vitamin D and its use in assessing vitamin D status. Clin Chem sarcoidosis, and other hypercalcemic disorders causing 31:1815Ð1819. increased 1,25(OH) D levels. These other disorders 14. Hollis BW, Kamerud JQ, Selvaag SR, Lorenz JD, Napoli JL 2 1993 Determination of vitamin D status by radioimmunoassay include tuberculosis, fungal infections, Hodgkin’s with an 125I-labeled tracer. Clin Chem 39:529Ð533. disease, lymphoma, and Wegener’s granulomatosis. 15. Hollis BW, Kamerud JQ, Kurkowski A, Beaulieu J, Napoli JL In all other clinical conditions involving the vitamin D 1996 Quantification of circulating 1,25-dihydroxyvitamin D 950 BRUCE W. HOLLIS

125 by radioimmunoassay with an I-labeled tracer. Clin Chem protein-binding assays for 25-hydroxyvitamin D3, 24,25-dihy- 42:586Ð592. droxyvitamin D3 and 1,25-dihydroxyvitamin D3. J Clin 16. Horst RL, Littledike ET, Riley JL, Napoli JL 1981 Endocrinol Metab 50:773Ð775. Quantitation of vitamin D and its metabolites and their 30. Eisman JA, Hamstra AJ, Kream BE, DeLuca HF 1976 A sen- plasma concentrations in five species of animals. Anal sitive, precise, and convenient method for determination of Biochem 116:189Ð203. 1,25-dihydroxyvitamin D in human plasma. Arch Biochem 17. Lambert PW, DeOreo PB, Hollis BW, Fu IY, Ginsberg DJ, Biophys 176:235Ð243. Roos BA 1981 Concurrent measurement of plasma levels of 31. Reinhardt TA, Horst RL, Orf JW, Hollis BW 1984 A microas- vitamin D3 and five of its metabolites in normal humans, say for 1,25-dihydroxyvitamin D not requiring high perfor- chronic renal failure patients, and anephric subjects. J Lab mance liquid chromatography: Application to clinical Clin Med 98:536Ð548. studies. J Clin Endocrinol Metab 58:91Ð98. 18. Hollis BW, Roos BA, Lambert PW 1981 Vitamin D in 32. Hollis BW 1986 Assay of circulating 1,25-dihydroxyvitamin plasma: Quantitation by a nonequilibrium ligand binding Dinvolving a novel single-cartridge extraction and purifica- assay. Steroids 37:609Ð613. tion procedure. Clin Chem 32:2060Ð2063. 19. Horst RL, Reinhardt TA, Beitz DC, Littledike ET 1981 A 33. Bouillon R, De Moor P, Baggiolini EG, Uskokovic MR 1980 sensitive competitive protein binding assay for vitamin D in A radioimmunoassay for 1,25-dihydroxycholecalciferoI. plasma. Steroids 37:581Ð591. Clin Chem 26:562Ð567. 20. Rhodes CJ, Claridge PA, Traffold DJH, Makin KLJ 1983 An 34. Gray TK, McAdoo T, Pool D, Lester GE, Williams ME, Jones evaluation of the use of Sep-Pak Qg cartridges for the extrac- G 1981 A modified radioimmunoassay for 1,25-dihydroxyc- tion of vitamin D3 and some of its metabolites from plasma hole-calciferol. Clin Chem 27:458Ð463. and urine. J Steroid Biochem 19:1349Ð1354. 35. Hollis BW 1995 1,25-Dihydroxyvitamin D3-26,23-lactone 21. Liel Y, Ulmer E, Shary J, Hollis BW, Bell NH 1988 Low interferes in determination of 1,25-dihydroxyvitamin D by circulating vitamin D in obesity. Calcif Tissue Int RIA after immunoextraction. Clin Chem 41:1313Ð1314. 43:199Ð201. 36. Mawer EB, Berry JL, Bessone J, Shany S, White A 1985. 22. Belsey RE, DeLuca HF, Potts JT 1974 A rapid assay for Selection of high-affinity and high specificity monoclonal 25-OH-vitamin D3 without preparative chromatography. J antibodies for 1,25-dihydroxyvitamin D. Steroids Clin Endocrinol Metab 38:1046Ð1051. 46:741Ð754. 23. Hollis BW, Pittard WB 1984 Evaluation of the total fetoma- 37. Bouillon R, Okamura WH, Norman AW 1995 Structure- ternal vitamin D relationships at term: Evidence for racial function relationships in the vitamin D endocrine system. differences. J Clin Endocrinol Metab 59:652Ð657. Endocr Rev 16:200Ð257. 24. Dorantes LM, Arnaud SB, Arnaud CD 1978 Importance of 38. Adams JA, Clemens TL, Parrish JA, Holick MF 1982 Vita- the isolation of 25-hydroxyvitamin D before assay. J Lab min D synthesis and metabolism after ultraviolet irradiation Clin Med 91:791Ð796. of normal and vitamin DÐdeficient subjects. N Engl J Med 24a. Binkley N, Krueger D, Cowgill CS, Plum L, Lake E, 306:722Ð725. Hansen KE, DeLuca HF, Drezner MK 2004 Assay variation 39. Hollis BW, Lowery JW, Pittard WB, Guy DG, Hansen JW confounds the diagnosis of hypovitaminosis D: A cell for 1996 Effect of age on the intestinal absorption of vitamin D3- standardization. J Clin Endocrinol Metab 89:3152Ð3157. palmitate and nonesterified vitamin D2 in the term human 24b. Hollis BW 2004 Editorial: The determination of circulating infant. 81:1385Ð1388. 25-hydroxyvitamin D: No easy task. J. Clin Endocrinol Metab 40. Heubi JE, Hollis BW, Tsang RC 1990 Bone disease in 89:3149Ð3151. chronic childhood cholestasis II. Better absorption of 25. Shepard RM, Horst RL, Hamstra AJ, DeLuca HF 1979 25(OH)D than vitamin D in extrahepatic biliary atresia. Determination of vitamin D and its metabolites in plasma Pediatr Res 27:26Ð31. from normal and anephric man. Biochem J 182:55Ð69. 41. Argao EA, Heubi JE, Hollis BW, Tsang RC 1992 d-alpha- 26. Dreyer BE, Goodman DBP 1981 A simple direct spec- tocopheryl polyethylene glycol-1000 succinate enhances the trophoto-metric assay for 24,25-dihydroxyvitamin D3. Anal absorption of vitamin D in chronic cholestatic liver disease of Biochem 114:37Ð41. infancy and childhood. Pediatr Res 31:146Ð150. 27. Hummer L, Christiansen C 1984 A sensitive and selective 42. Haddad JG, Stamp TCB 1974 Circulating 25-hydroxyvitamin radioimmunoassay for serum 24,25-dihydroxycholecaliferol D in man. Am J Med 57:57Ð62. in man. Clin Endocrinol 21:71Ð79. 43. Gloth MF, Gundberg CM, Hollis BW, Haddad JG, Tobin JD 28. Wei S, Tanaka H, Kubo T, Ichikawa M, Seino Y 1994 A multi- 1995 Vitamin D deficiency in homebound elderly persons. ple assay for vitamin D metabolites without high-performance JAMA 274:1683Ð1686. liquid chromatography. Anal Biochem 22:59Ð365. 44. Chapuy MC, Arlot ME, DuBoeuf F, Brun J, Crouzet B, Arnaud S, 29. Jones G, Byrnes B, Palma F, Segev D, Mazur Y 1980 Meunier P 1992 Vitamin D3 and calcium to prevent hip fractures Displacement potency of vitamin D2 analogs in competitive in elderly women. N Engl J Med 327:1637Ð1642. CHAPTER 59 Bone Histomorphometry

JULIET E. COMPSTON University of Cambridge School of Clinical Medicine, Cambridge, United Kingdom

I. Introduction VI. Assessment of Bone Turnover II. Bone Biopsy VII. Assessment of Remodeling Balance III. Histomorphometry VIII. Assessment of Bone Structure IV. Assessment of Mineralization IX. Conclusions and Future Developments V. Histological Diagnosis of Osteomalacia References

I. INTRODUCTION in the supine position, and the specimen is obtained approximately 2 cm below and behind the anterior supe- Bone histomorphometry describes the quantitative rior iliac spine. Most operators use a mild sedative, assessment of bone remodeling, modeling, and structure. such as midazolam, and some also routinely administer It provides information that is not currently available an analgesic by mouth or injection before the proce- from other investigative approaches, for example, bone dure. However, if care is taken to ensure adequate local densitometry and biochemical markers of bone turnover. anesthesia during the biopsy, the latter measure is not Bone histomorphometry also enables a more precise generally necessary. The biopsy should be performed characterization of disease states and their response to under sterile conditions. treatment than can be obtained from qualitative exami- The area around the anterior superior iliac spine is nation of bone histology. In the last two decades, there infiltrated with local anesthetic, and the inner and outer have been significant advances in histomorphometric periosteum are then anesthetized. The author uses a techniques, most notably the use of computerized rather small trocar and stilette, which is driven with a weighted than manual techniques and the development of sophis- instrument until it lies just in the outer cortex; local ticated approaches to the assessment of bone microar- anesthetic is infiltrated under the periosteum, and the chitecture. The application of these techniques has been trocar and stilette are then advanced through the bone to particularly valuable in determining the cellular patho- the inner cortex, where the procedure is repeated. Other physiology of different forms of bone diseases and in investigators anesthetize the inner cortex by introducing defining the mechanisms by which drugs affect bone. a needle through the skin from opposite the biopsy site. A small skin incision is then made, and a hollow II. BONE BIOPSY cannula with a serrated edge is introduced and placed firmly on the outer periosteum. A smaller, hollow can- A. Procedure nula is then inserted through the larger cannula, and the

The iliac crest is the preferred site for bone biopsy in patients with metabolic bone disease. Most investi- gators favor the transverse approach, in which a biopsy containing two cortices and intervening cancellous bone is obtained (Fig. 1), in contrast to vertical biopsies, which contain only one cortical plate. In growing indi- viduals, only the transverse approach should be consid- ered, because of the presence of the growth plate along the top of the crest. A number of specially designed trephines are commercially available; ideally for bone histomorphometry, the internal diameter of the specimen should be at least 6 mm. FIGURE 1 Section of transiliac biopsy obtained with an 8mm In most cases, the biopsy is performed as an outpa- internal diameter trephine. The biopsy contains inner and outer tient procedure. For a transiliac biopsy, the patient lies cortical plates and intervening cancellous bone. VITAMIN D, 2ND EDITION Copyright © 2005, Elsevier, Inc. FELDMAN, PIKE, AND GLORIEUX All rights reserved. 952 JULIET E. COMPSTON biopsy is obtained by advancing the serrated edge of this III. HISTOMORPHOMETRY cannula through the iliac crest until it has reached the outer surface of the inner cortex. The cannula is then A. Theoretical Considerations withdrawn after rotation through 360¡ (to ensure that the core of bone has been freed from adjoining tissues), and Histological sections of bone provide a two- the biopsy is removed from the cannula using a metal dimensional representation of a three-dimensional struc- rod. The incision is then sutured and dressed, and the ture; extrapolation from one to the other requires the patient is instructed to lie on the side of the biopsy to application of stereological formulae that are based on apply pressure to the site and reduce the risk of bruising. the assumptions that sampling is random and unbiased Ideally, this position should be maintained for 2 hr, and and, for most applications, that the structure is isotropic thereafter the patient should be advised to rest for 24 hr. (i.e., evenly dispersed and randomly orientated in space) [2]. Although these conditions cannot be strictly fulfilled in the case of bone histomorphometry, conver- B. Adverse Effects sion of histomorphometric indices to three-dimensional units is used by some investigators, whereas others Bone biopsy is safe and generally well tolerated, express their data as two-dimensional values. The abso- although there is often some discomfort after the lute values generated by these different approaches procedure, usually lasting between 24 and 48 hr. clearly differ, but in practical terms either is acceptable The morbidity is low and mainly due to hematoma, provided that consistency is maintained. which may occasionally be extensive; this is most The requirement, for stereological purposes, for likely to occur in obese subjects or in patients with bone to be isotropic is clearly not fulfilled in bone in bleeding diatheses. Other reported complications which the macro- and microarchitecture are primarily include infection, transient femoral nerve palsy, determined by mechanical forces. However, random avulsion of the superior ramus of the iliac crest, frac- and unbiased sampling with respect to isotropy can be ture of the iliac crest, and osteomyelitis. It should achieved using vertical sections, as first described by be stressed, however, that these are extremely rare, Baddeley et al. [3] and subsequently applied to bone and, overall, the incidence of all complications is less by Vesterby et al. [4]; the vertical axis of the sections than 1% [1]. is kept parallel to the axis of the cycloid test system, in which the test lines are defined in relation to the axis (sine-weighted). C. Indications for Bone Biopsy

In clinical practice, bone biopsy is most often B. Methodology performed to exclude or confirm a diagnosis of osteo- malacia and to characterize the different forms of renal The use of manual techniques using grids and grati- osteodystrophy. In patients with suspected osteomala- cules has now been almost entirely replaced by inter- cia, the diagnosis may be evident from biochemical active computerized systems. These are faster, more and/or radiological abnormalities, but in the absence of operator-friendly, and possess the ability to perform these, bone biopsy is required. Chronic renal failure is complex measurements that could not be achieved associated with several types of bone disease, as dis- manually. A number of systems are now available, both cussed in Chapter 76; accurate diagnosis is essential to commercial and in-house [5,6]. establish the correct treatment. Bone biopsy is also helpful in a number of other, rarer forms of metabolic bone disease, for example, fibrogenesis imperfecta C. Terminology ossium and hypophosphatemic osteomalacia. For diag- nostic purposes, qualitative assessment of bone by an The nomenclature applied to bone histomorphometry experienced histopathologist is sufficient, and histo- has been standardized [7] in an attempt to clarify the morphometry is not required. cumbersome and sometimes unintelligible terminol- Although bone biopsy is a valuable research tool in ogy used previously. The revised system expresses all osteoporosis, it is of little value diagnostically, mainly data in terms of the source (the structure on which the because of the heterogeneity of bone loss and the rela- measurement is made), the measurement, and the refer- tively weak correlations between bone mass in iliac ent. The recommended format (including punctuation) crest biopsies and clinically relevant sites such as the is source-measurement/referent, although because spine and femoral neck. only one source is usually used in a particular study, it CHAPTER 59 Bone Histomorphometry 953

TABLE I Referents Commonly Used in Bone (two-dimensional nomenclature) or volume, surface, Histomorphometry and thickness (three-dimensional nomenclature) (Table II). It should be noted that absolute area and Referent (3D/2D) Abbreviation (3D/2D) perimeter measured on conventional histological sec- Bone surface/perimeter BS/B.Pm tions have no three-dimensional equivalent but, if Bone volume/area BV/B.Ar three-dimensional nomenclature is adopted, these are Tissue volume/area TV/T.Ar referred to as volume and surface although the abso- lute values are identical. Cortical width and thickness Core volume/area CV/C.Ar are also numerically equal; however, in other situa- Osteoid surface/perimeter OS/O.Pm tions, for example, trabecular width, conversion to Eroded surface/perimeter ES/E.Pm thickness requires division of width by 4/π (1.273) for Mineralized surface/perimeter Md.S/Md.Pm isotropic structures or 1.2 for human iliac cancellous Osteoblast surface/perimeter Ob.S/Ob.Pm bone [2,8]. The units recommended for the revised Osteoclast surface/perimeter Oc.S/Oc.Pm nomenclature are micrometer and millimeter for length and day and year for time; surface/surface and 3D, three dimensions; 2D, two dimensions. Adapted from Parfitt et al. [7], volume/volume ratios are expressed as percentages, with permission. whereas volume/surface ratios are expressed in mm3/mm2. Some of the more commonly used derived is unnecessary to specify this once it has been defined. histomorphometric indices are shown in Table III. Area or perimeter (or volume and surface) are used as Most histomorphometric studies have been confined referents for most measurements (Table I). to cancellous bone which, because of its high surface Histomorphometric data may be described in two- to volume ratio, exhibits greater remodeling activity dimensional or three-dimensional terms; the system than cortical bone. Although cortical bone predomi- used should be consistent within studies. Primary mea- nates throughout the skeleton and is a major determi- surements are referred to as area, perimeter, and width nant of bone strength and fracture risk, it is largely ignored by histomorphometrists. The application of histomorphometric techniques to cortical bone was TABLE II Primary Histomorphometric Indices first described by Frost [9] in the rib and more recently of Bone Remodeling

Name Abbreviation Units TABLE III Derived Histomorphometric Indices Bone area B.Ar/T.Ar % of Bone Remodeling Osteoid area O.Ar/T.Ar or % Name Abbreviation Units O.Ar/B.Ar Osteoid perimeter O.Pm/B.Pm % Adjusted apposition rate Aj.AR µm/day Osteoblast perimeter Ob.Pm/B.Pm % Bone formation rate BFR/B.Pm µm2/µm/day Osteoid width O.Wi µm BFR/B.Ar %/year Interstitial width It.Wi µm Erosion rate ER µm/day Trabecular width Tb.Wi µm Mineralization lag time Mlt day(s) Eroded perimeter E.Pm/B.Pm % Osteoid maturation time Omt day(s) Osteoclast perimeter Oc.Pm/B.Pm % Formation period FP day(s) Mineralizing surface Md.Pm/B.Pm % Active formation period FP(a+) day(s) Mineral apposition rate MAR µm/day Erosion period EP day(s) Wall width W.Wi µm Reversal period Rv.P day(s) Erosion depth E.De µm Quiescent period QP day(s) Erosion length E.Le µm Remodelling period Rm.P day(s) Erosion area E.Ar µm2 Total period Tt.P day(s) Cavity number N.Cv./B.Pm or no./mm or Activation frequency Ac.f /year 2 N.Cv./T.Ar no./mm Trabecular separationa Tb.Sp µm or mm Quiescent perimeter Q.Pm % Trabecular numbera Tb.N /mm Reversal perimeter Rv.Pm % aMay also be measured directly. Adapted from Parfitt et al. [7], with Adapted from Parfitt et al. [7], with permission. permission. 954 JULIET E. COMPSTON a detailed analysis of the bone remodeling cycle in osteoporosis and have generally been consistent with human cortical bone [10] and studies of cortical bone the observed changes in bone mass at sites such as the structure in the femoral neck and iliac crest [11Ð14] spine and hip. Finally, current histomorphometric tech- have been discribed. niques are seriously limited by the lack of reliable markers for activation and resorption. Dynamic indices related to these processes are at present calculated D. Limitations of Bone Histomorphometry from bone formation rates, based on the assumptions that bone resorption and formation are coupled tempo- Certain limitations of bone histomorphometry rally and spatially and that bone remodeling is in a should be recognized. Some of these are inherent in the steady state; however, these are unlikely to be tenable restrictions imposed by a single biopsy site and disease in many cases of untreated or treated osteoporosis [20]. heterogeneity, whereas others reflect imperfections in measurement techniques and difficulties in identification IV. ASSESSMENT OF MINERALIZATION of some of the key processes in remodeling; at least some of those in the latter categories may eventually be over- A. In vivo Tetracycline Labeling come by methodological improvements in the future. A number of studies have documented the large mea- The administration of two time-spaced doses of a surement variance associated with bone histomorphom- tetracycline derivative prior to bone biopsy enables etry, which arises from a number of sources including measurement and calculation of dynamic indices of bone intra- and interobserver variation, sampling variation, formation and, by extrapolation, bone resorption [21]. and methodological factors [15Ð17]. Intra- and Such information is not currently obtainable by other interobserver variation reflect the subjective approach means, and tetracycline labeling should therefore be to identification of many of the histological features performed whenever possible. Various regimens have assessed, for example, resorption cavities and osteoid been described; most involve a 10Ð12 day gap between seams. Methodological factors include the criteria used the two doses, bone biopsy being performed 3Ð5 days for corticomedullary differentiation, which are often after the last dose. The regimen used by the author is arbitrary, the staining method used, and the magnifica- as follows: tion at which measurements are made. The technique used for quantitation may also affect the values obtained, Days 1 and 2 150 mg demeclocycline twice daily as a result of different sampling procedures and varia- 10 days No demeclocycline tions in the number of sampling points utilized. Many Days 13 and 14 150 mg demeclocycline twice daily of these sources of variance can be minimized by the standardization of staining, corticomedullary delin- The bone biopsy is performed 3Ð5 days after day 14. eation, and magnification and the employment of criteria for identification of osteoid seams, resorption cavities, Adverse effects of demeclocycline and related com- and newly formed bone structural units. pounds are rare but include diarrhea and other gastroin- The important issue of how closely bone remodeling testinal symptoms. Occasionally, skin rashes occur; these in the iliac crest resembles that at other skeletal sites are sometimes severe and may exhibit photosensitivity. has not been fully resolved. Some metabolic bone There is evidence that different tetracycline com- disorders, for example, osteomalacia and most forms of pounds differ with respect to their uptake by mineraliz- renal osteodystrophy, appear to affect the whole skele- ing bone. Parfitt et al. [22] reported that demeclocycline ton, and in such cases an iliac crest biopsy is represen- labeling resulted in a greater surface extent of fluores- tative. In osteoporosis, however, there is clear evidence cence than oxytetracycline, significantly affecting val- of disease heterogeneity and it is well documented that ues for dynamic indices of bone formation. These bone volume in iliac crest biopsies is a poor indicator of differences should be borne in mind when comparing bone loss elsewhere in the skeleton [18]. There is also data between centers and, in particular, when using con- some evidence for variations in bone turnover at different trol data obtained from other sources. skeletal sites [19]; however, the demonstration of clear abnormalities of bone remodeling in iliac crest bone obtained from patients with osteoporosis indicates that B. Measurement of Osteoid changes responsible for the disease process are reflected, at least to some extent, in bone from this site. Osteoid may be distinguished from mineralized bone Similarly, changes in bone turnover and remodeling by several staining procedures, including von Kossa, tolu- balance have been shown in patients with treated idine blue, Goldner’s trichrome, and solochrome cyanin. CHAPTER 59 Bone Histomorphometry 955

of osteoid, as osteoid amount tends to be greater in the corticoendosteal region than in pure cancellous bone.

C. Dynamic Indices of Mineralization

The administration of time-spaced tetracycline labels prior to biopsy enables calculation of the dynamic indices of matrix formation and mineralization that are central to the histomorphometric diagnosis of osteomalacia. Tetracycline binds to calcium and becomes perma- nently incorporated into the mineralization fronts at sites of active mineralization (Fig. 3; see color insert). FIGURE 2 Toluidine blue-stained section of iliac crest cancel- In this respect, the timing of the biopsy in relation to lous bone showing mineralized bone (purple/blue) and osteoid the labeling regime is crucial, with a period of 3 to (pale blue). The calcification front can be seen as a dark blue line at 5 days after the last label enabling deposition of a suf- the interface of the osteoid and mineralized bone (See color plate). ficiently thick layer of new mineral to retain the tetra- cycline label [25]. The primary histomorphometric Primary measurements of osteoid include area, indices of bone remodeling are listed in Table II and perimeter, and seam width; assessment of dynamic the derived indices in Table III. Normative histomor- indices of mineralization, for example, mineral apposi- phometric data from the author’s laboratory are shown tion rate, mineralization lag time, and osteoid matura- for females (Table IV) and males (Table V). Other tion rate, requires double tetracycline labeling prior to groups have also published normative data and in par- biopsy [7]. Calcification fronts along osteoid seams ticular normative data for the pediatric age group are can be demonstrated by staining with toluidine blue now available [25a]. (Fig. 2; see color insert), Sudan black B, or thionin, but the surface extent assessed in this way does not always 1. MINERAL APPOSITION RATE correlate well with the mineralizing perimeter as mea- The mineral apposition rate (MAR) is calculated as sured using tetracycline uptake [23]. the distance between two time-spaced tetracycline labels Osteoid seam width may be measured directly or divided by the time between the administration of the calculated from osteoid area and perimeter. However, labels. Measurements are made from the midpoint of when relatively small amounts of osteoid are present, each label at approximately equidistant points along the latter approach is inaccurate because the value for the labeled surface, and the interlabel period is calcu- osteoid area, expressed as a percentage of bone area, is lated as the number of days between the midpoints of influenced not only by the seam width but also by the the two labeling periods [26]. MAR is used in the mineralized bone area [24]. Direct assessment can be calculation of many derived indices of bone formation, made using an eyepiece micrometer and calculating the mean of several equidistant measurements of width along each seam. Measurements of osteoid, in particular its perimeter, are strongly influenced by the magnification used. At high magnifications, it becomes difficult to distinguish osteoid seams from the thin endosteal membrane cov- ering the quiescent bone surface, and for this reason it is preferable to use defined criteria, for example, all seams less than one lamella (3 µm) are excluded. Osteoid mea- surements may also be affected by the staining procedure used, and poor differentiation of osteoid from mineral- ized bone in images used for semiautomatic techniques may reduce the accuracy of measurements; thus, values obtained using image analysis are generally lower than those generated by manual measurements [16]. FIGURE 3 Unstained section of iliac crest cancellous bone Finally, the delineation of the corticomedullary junction viewed by fluorescence microscopy. The tetracycline labels are may affect the values obtained for primary measurements seen as double yellow fluorescent bands. (See color plate.) 956 JULIET E. COMPSTON

TABLE IV Normative Histomorphometric Data in Femalesa

Age range

19Ð30 years 31Ð40 years 41Ð50 years 51Ð60 years 61Ð80 years Parameter (n = 5) (n = 6)b (n = 6)c (n = 10)d (n = 6)

BV/TV (%) 25.9(3.1) 27.7(5.5) 29.6(2.1) 23.9(4.5) 19.8(3.9) OV/BV (%) 2.4(1.4) 2.7(2.4) 2.3(1.3) 3.1(1.9) 4.7(1.9) OS/BS (%) 13.1(5.9) 17.7(11.5) 14.5(7.8) 21.2(11.3) 35.0(12.1) ES/BS (%) 2.15(0.36) 1.84(0.92) 1.78(1.03) 1.76(0.83) 1.66(0.66) Md.S/BS (%) 9.4(1.8) 8.1(3.4) 8.1(4.2) 13.0(6.7) 14.8(8.1) O.Th (µm) 5.4(1.9) 3.9(1.6) 5.8(1.1) 5.8(1.6) 5.8(2.3) W.Th (µm) 45.7(4.9) 51.2(6.6) 47.5(4.9) 36.1(2.9) 32.5(3.6) MAR (µm/day) 0.59(0.06) 0.60(0.12) 0.61(0.09) 0.61(0.10) 0.54(0.07) Mlt (days) 12.2(6.2) 16.5(11.8) 20.7(8.7) 21.2(19.6) 29.6(13.5) BFR (µm3/µm2/day) 0.056(0.013) 0.060(0.022) 0.051(0.031) 0.084(0.045) 0.081(0.043)

aResults are expressed as means, with SD values in parentheses. Data from Vedi et al. [70,71]. Md.S/BS was calculated as the double plus half the single tetracycline-labelled surface. bn = 4 for dynamic variables. cn = 5 for dynamic variables. dn = 9 for dynamic variables.

and accurate measurement is thus of key importance. 2. ADJUSTABLE APPOSITION RATE In the absence of tetracycline uptake, mineral apposition The adjusted apposition rate (Aj.AR) represents rate and derived indices should be treated as missing the mineral apposition rate averaged over the osteoid data; in biopsies in which only single labels can be surface. In the absence of a mineralization defect, the detected, the finite lower limit of 0.3 µm/day for min- apposition of matrix and mineral, whilst not syn- eral apposition rate should be used for the calculation chronous, can be assumed to occur at the same rate, of derived indices [27]. and under such circumstances, the adjusted apposition

TABLE V Normative Histomorphometric Data in Malesa

Age range

19Ð30 years 31Ð40 years 41Ð50 years 51Ð60 years 61Ð80 years Parameter (n = 3) (n = 6) (n = 3) (n = 6) (n = 6)

BV/TV (%) 31.3(6.4) 22.2(3.9) 26.9(7.1) 23.0(5.5) 21.4(2.6) OV/BV (%) 1.6(0.8) 4.1(1.6) 2.8(0.9) 3.1(0.9) 5.6(3.6) OS/BS (%) 10.0(5.3) 28.3(7.8) 26.3(6.0) 20.0(7.0) 34.8(15.7) ES/BS (%) 2.84(1.27) 1.69(0.62) 1.68(0.32) 1.77(0.68) 1.91(0.42) Md.S/BS (%) 9.9(4.0) 13.5(8.4) 8.7(7.0) 8.8(1.5) 9.2(5.1) O.Th (µm) 8.3(3.0) 6.1(1.3) 6.9(2.9) 6.2(2.0) 6.6(2.8) W.Th (µm) 49.7(9.6) 45.9(4.4) 42.8(4.0) 36.9(2.0) 33.4(3.3) MAR (µm/day) 0.67(0.07) 0.60(0.10) 0.55(0.10) 0.57(0.13) 0.53(0.05) Mlt (days) 10.5(1.9) 28.5(14.6) 36.1(6.2) 34.7(40.8) 49.4(28.1) BFR (µm3/µm2/day) 0.066(0.006) 0.079(0.049) 0.047(0.005) 0.051(0.015) 0.045(0.023)

aResults are expressed as means with SD values in parentheses. Data from Vedi et al. [70,71] Md.S/BS was calculated as the double plus half the single tetracycline-labeled surface. CHAPTER 59 Bone Histomorphometry 957 rate is equivalent to the osteoid or matrix apposition rate. It is calculated as follows:

Aj.AR = MAR × Md.Pm/O.Pm.

From the above formula, it is clear that Aj.AR is usually less than MAR and cannot exceed it.

3. MINERALIZATION LAG TIME AND OSTEOID MATURATION TIME The mineralization lag time (Mlt) is the interval be- tween deposition and mineralization of a given amount of osteoid, averaged over the life span of the osteoid seam. It is calculated as

Mlt = O.Wi/Aj.AR.

The osteoid maturation time (Omt) is the period between the deposition and onset of mineralization of a given amount of osteoid and results from processes such as collagen cross-linking that are necessary before mineralization can proceed. In humans it is usually shorter than Mlt and never exceeds it. It is calculated as follows:

Omt = O.Wi/MAR. FIGURE 4 (Top) Section of iliac crest stained by the von Kossa technique to demonstrate mineralized bone (black) and osteoid (pink) in a normal subject. (Bottom) Section of iliac crest stained V. HISTOLOGICAL DIAGNOSIS OF by the von Kossa technique to show osteoid accumulation in a OSTEOMALACIA woman with severe privational osteomalacia. (See color plate.) A. Generalized Osteomalacia

Osteomalacia is essentially a histological diagnosis, although biochemical and radiological abnormalities therefore, inaccessible to osteoclastic resorption. In may enable a firm diagnosis to be made without such cases, tunneling resorption may be apparent, and the necessity for histological examination of bone. the irregular outline of mineralized bone beneath the Nonetheless, osteomalacia may exist in the absence of thick osteoid seams provides evidence of previous biochemical and radiological abnormalities [28], and resorption. Para-trabecular fibrosis is also seen in severe in such cases bone biopsy is the only certain means by cases. In contrast, histological evidence of secondary which the diagnosis can be established. hyperparathyroidism is absent in untreated hypophos- The cardinal feature of osteomalacia is defective phatemic osteomalacia. mineralization, which results in accumulation of osteoid In histomorphometric terms, osteomalacia is with an increase in the width of osteoid seams (Fig. 4; defined as an increased osteoid seam width and a pro- see color insert) and a reduction in the surface extent longed mineralization lag time [25]. The criteria for of osteoid showing tetracycline labeling; there is often abnormality in these indices depend on the source of an increase in the width of individual tetracycline the reference data, which may vary as a result of both labels, and the distance between double labels is geographical and methodological factors, but in most reduced or undetectable. In severe cases, tetracycline centers a mean osteoid seam width greater than 12.5 um labeling may be absent. Increased bone turnover and and a mineralization lag time in excess of 100 days erosion depth, due to secondary hyperparathyroidism, would be regarded as abnormal. Although the mineral are present in the earlier stages of osteomalacia but apposition rate is also reduced in osteomalacia, this become less apparent as the mineralized bone surface is not a specific feature because low mineral apposi- becomes covered with thick osteoid seams and, tion rates may also result from a reduction in matrix 958 JULIET E. COMPSTON apposition rate as occurs, for example, in post- termination between administration of the two labels menopausal osteoporosis, osteogenesis imperfecta, and [31], and probably also the switch from an active to some forms of secondary osteoporosis. Similarly, the resting state in a minority of osteoid seams (the so- mineralization lag time will be increased in the pres- called on/off phenomenon) [2]. Because of the former, ence of reduced matrix apposition rate and is therefore the extent of double-labeled surface underestimates the not, by itself, pathognomonic of osteomalacia. The actively mineralizing surface, and the double plus half distinguishing feature of osteomalacia is that the min- the single label is therefore used to estimate the miner- eralization lag time is prolonged relative to the ad- alizing perimeter. In cases where only single labels can justed apposition rate, whereas in osteoporosis the be detected, it has been suggested that the mineralizing reverse is true; this phenomenon accounts for the perimeter should be expressed as half the single- increase in osteoid seam width that is specific to osteo- labeled perimeter [27]. malacia. In the absence of tetracycline administration prior to biopsy, the osteoid perimeter may provide some indication of bone turnover, with increased osteoid B. Focal Osteomalacia perimeter being characteristic of high turnover states. An increase in the extent of perimeter occupied by Focal osteomalacia has been described in patients resorption cavities does not necessarily imply taking bisphosphonate therapy and is characterized by increased bone turnover, however, as these may not the focal distribution of abnormally thick osteoid represent active resorption but rather reflect failure of seams with impaired mineralization [29,30]. In such formation to occur in previously resorbed cavities. cases, the osteoid area and perimeter may be normal, and, because some osteoid seams are of normal width and exhibit normal mineralization, the abnormality may B. Bone Formation Rates only be detected by examination of the distribution, within single biopsies, of values for osteoid seam width. Bone formation rates are usually expressed in terms The significance of these histological changes in terms of of the bone perimeter or area. In the former case, either fracture risk has not been established; they do not appear, the osteoid perimeter may be used as the referent however, to be associated with clinical symptoms or (adjusted apposition rate) or the total bone perimeter biochemical abnormalities. (tissue-based bone formation rate). They are calculated as follows:

VI. ASSESSMENT OF BONE TURNOVER Adjusted apposition rate = MAR × Md.Pm/O.Pm, Bone formation rate (tissue-based) (BFR/BS) Bone turnover describes the tissue level of bone re- = MAR × Md.Pm/B.Pm. sorption and formation, a key determinant of which is activation frequency, that is, the probability that a new The bone formation rate/bone area (BFR/B.Ar) is remodeling cycle will be initiated at any point of the calculated as bone surface. The uptake of tetracycline derivatives at sites of actively forming bone enables the rate of BFR/B.Ar = MAR × Md.Pm × (B.Pm/B.Ar) × 100. mineralization of osteoid seams and the surface extent of bone formation to be assessed and a number of derived indices, including activation frequency, to be C. Bone Resorption Rates calculated. Tetracycline labeling in bone is detected by fluorescence microscopy under blue light. Because of the lack of markers of active resorption analogous to the use of tetracycline to identify actively forming bone, bone resorption rates can only be calcu- A. Mineralizing Perimeter or Surface lated indirectly, from bone formation rates, based on the assumptions discussed earlier. Erosion rate (ER) is The extent of bone perimeter or surface that exhibits expressed in micrometers per day and calculated as tetracycline fluorescence is an important primary follows: measurement from which many dynamic indices are derived. When a double tetracycline label has been ER = E.De/EP. administered, both double and single labels will be seen; this reflects the labeling escape error, caused by Calculation of the erosion period (EP) is shown initiation of mineralization before the first label or its below. CHAPTER 59 Bone Histomorphometry 959

D. Remodeling Periods previously activated units. Because activation may occur at any site on the bone surface, however, there The average duration of a single remodeling cycle is seems no a priori reason why this should be so, par- described as the remodeling period, which can be fur- ticularly in nonsteady states [20]. In addition, the use ther divided into quiescent, erosion, reversal, and for- of indices of bone formation to calculate activation fre- mation remodeling periods [2]. The formation period quency relies on assumptions about the coupling of (FP) can be further divided into the active [FP(a+)] and bone resorption and formation that are unlikely to be inactive formation period [FP(a−)] [32]; the latter is a tenable in many disease states. measure of the “off-time,” which accounts for the dis- crepancy between osteoid and mineralizing perimeter after correction for label escape [31]. Formation peri- VII. ASSESSMENT OF ods are calculated as follows: REMODELING BALANCE FP = W.Wi/Aj.AR FP(a+) = W.Wi/M AR, A. Bone Formation FP(a−) = FP − FP(a+), Within individual remodeling units, the amount of where W.Wi (wall width) is the mean width of com- bone formed is termed the wall width [34]; this is mea- pleted bone structural units. Quiescent, erosion, and sured as the mean width of completed bone structural reversal periods are calculated as follows: units that are identified under polarized light (Fig. 5; see color insert) or by stains such as toluidine blue or QP = Q.Pm/B.Pm × FP, thionin [35], which demonstrate the cement line. Com- EP = E.Pm/B.Pm × FP, pleted structural units are identified by the absence of Rv.P = Rv.Pm/B.Pm × FP. resorption lacunae or osteoid. There is a large variation in reported values for wall width in both normal and osteoporotic subjects [20], reflecting differences in where Rv.Pm = E.Pm − osteoclastic perimeter; sampling procedures and difficulties in accurate Q.Pm = B.Pm − (O.Pm + E.Pm). identification of the cement line, which forms the base of the original resorption cavity. The mean time between initiation of two successive Investigation of the effect of a disease or its treat- remodeling cycles at the same site is defined as the ment on wall width (and calculated dynamic indices total period and calculated as follows: for which wall width is required) necessitates differen- tiation of those units formed during the period of Tt.P = Rm.P + QP. observation from those formed prior to this time. This can only be achieved by identification of uncompleted bone structural units, which have a covering of osteoid and hence can be presumed to represent current or E. Activation Frequency recent remodeling activity. Reconstruction of these

Activation frequency (Ac.f) is a key determinant of bone mass in the adult skeleton, and increased activa- tion frequency, resulting in high bone turnover, forms quantitatively the most important mechanism of bone loss in osteoporosis [33]. At present, however, there are no in situ markers of activation, and hence direct assess- ment of activation frequency cannot be made. Rather, it is calculated as the frequency with which a given site on the bone surface undergoes new remodeling, as follows:

Ac.f = 1/Tt.P or (BFR/B.Pm)/W.Wi.

These formulae define activation frequency as the reciprocal of the time taken from the initiation of FIGURE 5 Section of iliac crest biopsy viewed under polarized one remodeling cycle to initiation of a new one at that light to show a completed bone structural unit bounded by the site, thus implying its dependence on the life span of cement line and mineralized bone surface. 960 JULIET E. COMPSTON forming sites can then be achieved [36]; however, the number of such units that can be identified in any one biopsy is likely to be extremely small and variance correspondingly high.

B. Bone Resorption

There are several problems associated with accurate assessment of the amount of bone resorbed during each remodeling cycle. The identification of resorption cavities is often difficult and always to some extent subjective; in addition, it is difficult to identify those cavities in which resorption has been completed. The use of polarized light microscopy to demonstrate cut off lamellae at the edges of the cavity assists recogni- tion [37] (Fig. 6; see color insert), as does the presence of osteoclastlike cells within the cavity. More precise identification of osteoclasts can be achieved by histo- chemical techniques that demonstrate the presence of tartrate-resistant acid phosphatase, although this is not specific to osteoclasts [38,39]. Finally, it is not usually possible to identify those cavities that have resulted in trabecular perforation. Indirect assessment of erosion depth was first re- ported by Courpron et al. [40], based on the postulate that the interstitial width, (i.e., the distance between FIGURE 6 (Top) Resorption cavity in cancellous iliac crest bone two bone structural units on opposite sides of a trabec- stained by toluidine blue. (Bottom) Same resorption cavity viewed ula) is inversely proportional to erosion depth. In this under polarized light. Note the cutoff collagen lamellae at the edges of the cavity. (See color plate.) model, interstitial width is calculated as the difference between the trabecular width and twice the wall width. However, the relationship between interstitial width and erosion depth is not a simple inverse one, as it is influenced by concomitant changes in wall width and cavities by cell type [44]. The latter technique provides trabecular width [41,42]. an estimate of the mean depth of cavities in all stages Another approach to the direct measurement of ero- of completion and thus underestimates the final resorp- sion depth was reported by Eriksen et al. [43]. In this tion depth. method, the number of lamellae eroded beneath the The computerized method developed by Garrahan bone surface is counted, and cavities are characterized et al. [45] involves reconstruction of the eroded bone according to the presence of osteoclasts, mononuclear surface by a curve-fitting technique (cubic spline) and cells, and preosteoblastic cells, these being specifically provides measurements of mean and maximum ero- associated with increasing stages of completion of the sion depth together with the area, surface extent, and resorptive phase. This approach depends critically number of cavities (Fig. 8). Because all resorption upon the accurate identification, on morphological cavities are included in the measurements, the mean grounds, of these different cell types within resorption values for mean and maximum depth and for area con- cavities; even in high quality histological sections siderably underestimate the final resorption depth and this can be difficult, and in the author’s hands 24% of area. This method may also be performed using inter- resorption cavities were excluded from measurement active reconstruction of the eroded bone surface [46]. because of failure to classify by cell type or inability to Another modification is to include for measurement define and count the eroded lamellae. This method has only those cavities that contain a thin layer of osteoid, not been widely adopted by other groups, although ensuring that the final resorption depth has been some have used a simplified approach in which the achieved [46]; however, the number of such cavities eroded lamellae are counted without subdivision of that can be identified in a single biopsy is usually CHAPTER 59 Bone Histomorphometry 961 extremely small. Interestingly, the values reported length. Calculation of trabecular separation and num- using this approach in normal human biopsies were ber from trabecular width and bone area is based on a approximately 17% lower than those obtained in the parallel plate model [55] that may often be inappropri- same biopsies by the technique of counting eroded ate in cancellous bone. Nevertheless, these approaches lamellae and were more consistent with reported age- have generated useful information and have stimulated related changes in trabecular width [47,48]. the development of more sophisticated techniques for Further work is thus required to improve existing analysis of bone structure. techniques for the measurement of erosion depth. The Because all of the structural determinants of cancel- approaches most widely used at present measure the lous bone strength are three-dimensional characteris- depth of all resorption cavities and therefore underesti- tics, their assessment on conventional histological mate the completed erosion depth, whereas measure- sections provides only indirect information about these ments made using Eriksen’s method probably qualities, and three-dimensional images are required overestimate the true value [49]. Although these limita- for direct measurement of connectivity, anisotropy, and tions preclude accurate assessment of remodeling bal- trabecular size and shape [56]. Although further studies ance at present, measurement of erosion depth has are required to examine the relationship between struc- generated valuable information about the pathophysiol- tural indices obtained using two- and three-dimensional ogy of bone loss in untreated and treated disease states. approaches, there are several lines of evidence to support the contention that measurements from two-dimensional sections are representative of three-dimensional VIII. ASSESSMENT OF structure [57,58]. BONE STRUCTURE

The importance of cortical and cancellous bone B. Two-dimensional Approaches structure as a determinant of bone strength is well established, and there has been increasing interest in 1. STRUT ANALYSIS the quantitative assessment of bone microarchitecture. Strut analysis is based on the definition of nodes and Changes in bone structure have important implications termini and the topological classification of trabeculae not only for bone strength and fracture risk but also for and struts. Garrahan et al. [5] have described a semi- the timing and efficacy of treatment in osteoporosis. In automated procedure in which the binary image of a particular, the potential for anabolic agents to restore section is skeletonized and the different strut types are cancellous and cortical bone architecture in patients classified as shown in Fig. 7 (see color insert). The with advanced bone loss is of considerable interest. total length of each strut type may be expressed as a percentage of the total strut length or in absolute terms. Node-to-node and node-to-loop strut lengths are posi- A. Structural Determinants of Bone Strength tively related to connectivity, whereas node-to-terminus and terminus-to-terminus strut lengths are inversely Structural determinants of the mechanical strength related. Because of the edge effect, termini may be of bone include cortical width and porosity and, in created artefactually, whereas node-to-node and node- cancellous bone, trabecular size, shape, connectivity, to-loop struts are true indices of connectedness, although and anisotropy. Early approaches to the quantitative their number may be underestimated. Termini may assessment of cancellous bone structure were based on also result from sectioning through a trabecular window direct or indirect measurements of trabecular width, in a connected structure. separation, and number [50Ð54]. Direct measurements of trabecular width can be made using an eyepiece 2. STAR VOLUME graticule or grid, but nowadays are most commonly The star volume is defined as the mean volume of performed by using computerized techniques [53,54]. solid material or empty space that can be seen unob- These measurements provide information not only scured from a point of measurement chosen at random about the mean trabecular width but also about the dis- inside the material [59]. Its assessment in histological tributions of trabecular width within individual biop- sections of bone was first reported by Vesterby [60] sies. Calculation of trabecular width from area and using the vertical section technique [3] and a cycloid perimeter measurements may also be performed [55], test system. The method may be applied to measure- this approach being based on the assumption that the ment both of trabecular width (trabecular star volume) width of measured structures is small relative to their and trabecular separation (marrow space star volume), 962 JULIET E. COMPSTON

which a large proportion of the measured intercepts Strut analysis may hit the boundary rather than bone, resulting in underestimation of star volume [61].

3. TRABECULAR BONE PATTERN FACTOR Nd.Nd Another method is based on the concept that patterns or structures can be defined by the relationship between

Nd.Tm convex and concave surfaces [62], with convexity indi- cating poor connectivity and concavity reflecting Nd.Lp structural integrity. Using a computer-based system, convexity and concavity are assessed by measurement Tm.Tm of the bone perimeter before and after computer-based dilatation of the trabecular surface; whereas thickening of convex structures increases their perimeter, the reverse FIGURE 7 Diagrammatic representation of different strut types applies to concave structures. The values obtained for in strut analysis. Cancellous bone is shown in gray. Lines represent trabecular bone pattern factor, which is calculated as the skeletonized axis of the original bone profile. Squares represent nodes (Nd) and termini (Tm) Terminus-to-terminus (Tm.Tm), the difference between perimeter measurements before node-to-loop (Nd.Lp), and node-to-terminus (Nd.Tm) strut types and after dilatation divided by the corresponding dif- are illustrated. Reprinted from Croucher et al. [61] with permission. ference in area, may be significantly influenced by the (See color plate.) computer-based smoothing technique used, the degree of dilatation employed, and the magnification at which the measurements are performed. and theoretically provides an unbiased stereological approach to these indices. The method involves the 4. FRACTAL ANALYSIS generation of intercepts from random sampling points, Fractal objects are characterized by scale invariance or with the cubed length of the intercepts being used in self-similarity over a wide range of magnifications so that the calculation of star volume (Fig. 8; see color insert). any one piece of a fractal, if magnified sufficiently, The values generated by marrow star volume measure- resembles the intact object [63]. Fractal analysis has been ments are significantly influenced by biopsy size, par- described in a number of biological systems, including ticularly in poorly interconnected cancellous bone in the bronchial tree and vascular networks; in bone, it has been applied to radiological images and histological sections of bone [64]. The value obtained for the fractal dimension is critically dependent on the magnification Marrow space star volume used for measurement, and the relationship between fractal dimension and connectivity in cancellous bone has not been established. Multidirectional fractal analysis can be used to assess structural anisotropy [65].

C. Three-dimensional Approaches

A number of techniques have been used to generate three-dimensional images of bone. These include recon- struction of serial sections, scanning and stereo micros- copy, volumetric, high resolution, and microcomputed × π √ V*m.space = 4 /3 3/n tomography, and magnetic resonance imaging [66,67]. The potential for imaging techniques such as magnetic FIGURE 8 Binary image used in the assessment of marrow space resonance imaging and computed tomography to star volume. The upper active region is defined by the green and provide information about bone structure in vivo is an blue lines and the lower region by red and blue lines. Blue and important and active area of current research. At present, yellow squares represent grid points hitting the marrow space. Blue such application of these approaches is restricted by and yellow lines represent grid lines intercepting trabeculae and the edge of the active region. The white arrow shows the direction of the limited resolution, partial volume effects, and noise. vertical axis. Reprinted from Croucher et al. [61] with permission. Nevertheless, such approaches enable direct assessment (See color plate.) of connectivity using the Euler number, a topological CHAPTER 59 Bone Histomorphometry 963 property based on the number of holes and number of Standardization of nomenclature, symbols, and units. J Bone connected components in an object [68]. This measure- Miner Res 2:595Ð610. 8. Schwartz MP, Recker RR 1981 Comparison of surface den- ment can also be obtained using the ConnEuler method sity and volume of human iliac trabecular bone measured in which projections are made through parallel thin directly and by applied sterology. Calcif Tissue Int section pairs, or dissectors, spaced approximately 10Ð40 33:561Ð565. apart [69]. 9. Frost HM 1963 Mean formation time of human osteons. Can J Biochem Physiol 41:1307Ð1310. 10. Agerbaek MO, Eriksen EF, Kragstrup J, Mosekilde LE, Melsen F 1991 A reconstruction of the remodelling cycle IX. CONCLUSIONS AND FUTURE in normal human cortical iliac bone. Bone Miner 12: DEVELOPMENTS 101Ð112. 11. Bell NL, Loveridge N, Reeve J, Thomas CD, Feik SA, Bone histomorphometry is a valuable tool in the Clement JG 2001. Super-osteons (remodeling clusters) in the cortex of the femoral shaft: influence of age and gender. Anat assessment of metabolic bone diseases, in terms both Rec 264:378Ð386. of their diagnosis and pathophysiology. It also pro- 12. Jordan GR, Loveridge N, Bell KL, Power J, Rushton N, Reeve J vides information about the safety and mechanisms of 2000 Spatial clustering of remodeling osteons in the femoral action of pharmacological interventions used in the neck cortex: a cause of weakness in hip fracture? Bone 26: treatment of bone diseases. In the future, the advances 305Ð313. 13. Power J, Noble BS, Loveridge N, Bell KL, Rushton N, in molecular biological techniques and our knowledge Reeve J 2001. Osteocyte lacunar occupancy in the femoral of bone cell biology should enable more accurate iden- neck cortex: an association with cortical remodeling in tification of the processes of activation and resorption hip fracture cases and controls. Calcif Tissue Int 69: in situ, leading to a better understanding of mechanisms 13Ð19. of bone loss and bone gain. In addition, advances in the 14. Vedi S, Bell KL, Loveridge N, Garrahan N, Purdie DW, Compston JE 2003 The effects of hormone replacement in vivo assessment of cortical and cancellous bone therapy on cortical bone in postmenopausal women: a histo- architecture will provide new insights into structural morphometric study. Bone 33:330Ð334. determinants of bone strength and the ability of 15. de Vernejoul MC, Kuntz D, Miravet L, Goutalier D, anabolic skeletal agents to restore bone architecture in Ryckewaert A 1981 Histomorphometric reproducibility in patients with advanced bone loss. Finally, the importance normal patients. Calcif Tissue Int 33:369Ð374. 16. Chavassieux PM, Arlot ME, Meunier PJ 1985 Intermethod of changes in cortical bone, both in untreated and treated variation in bone histomorphometry: Comparison between disease, is increasingly recognized and this previously manual and computerized methods applied to iliac bone neglected area of bone histomorphometry is likely to be biopsies. Bone 6:211Ð219. more intensively studied in forthcoming years. 17. Wright CDP, Vedi S, Garrahan NJ, Stanton M, Duffy SW, Compston JE 1992 Combined inter-observer and inter-method variation in bone histomorphometry. Bone 13:205Ð208. 18. Compston JE, Crowe JP, Wells IP, Horton LWL, Hirst D, Merrett AL, Woodhead JS, Williams R 1980 Vitamin D References prophylaxis and osteomalacia in chronic cholestatic liver disease. Dig Dis 25:28Ð32. 1. Rao DS 1983 Practical approach to bone biopsy. In Recker R 19. Eventov I, Frisch B, Cohen Z, Hammel I 1991 Osteopenia, (ed) Bone Histomorphometry: Techniques and Interpretation. hematopoiesis, and bone remodelling in iliac crest and CRC Press, Boca Raton, Florida, pp. 3Ð11. femoral biopsies: A prospective study of 102 cases of 2. Parfitt AM 1983 The physiological and clinical significance femoral neck fractures. Bone 12:1Ð6. of bone histomorphometric data, In Recker R (ed) Bone 20. Compston JE, Croucher PI 1991 Histomorphometric assess- Histomorphometry: Techniques and Interpretations. CRC ment of trabecular bone remodeling in osteoporosis. Bone Press, Boca Raton, Florida, pp. 143Ð224. Miner 14:91Ð102. 3. Baddeley AJ, Gundersen HJG, Cruz Drive LM 1986 21. Frost HM 1969 Tetracycline-based histological analysis of Estimation of surface area from vertical sections. J Microsc bone remodelling. Calcif Tissue Int 3:211Ð237. 142:259Ð276. 22. Parfitt AM, Foldes J, Villanueva AR, Shih MS 1991 4. Vesterby A, Kragstrup J, Gundersen HJG, Melsen F 1987 Difference in length between demethylchlortetracycline Unbiased stereologic estimation of surface density in bone and oxytetracycline: Implications for the interpretation using vertical sections. Bone 8:13Ð17. of bone histomorphometric data. Calcif Tissue Int 48: 5. Garrahan NJ, Mellish RWE, Compston JE 1986 A new 74Ð77. method for the analysis of two-dimensional trabecular bone 23. Compston JE, Vedi S, Webb A 1985 Relationship between structure in human iliac crest biopsies. J Microsc 142:341Ð349. toluidine blue-stained calcification fronts and tetracycline 6. Compston JE, Garrahan NJ, Croucher PI, Yamaguchi K 1993 labeled surfaces in normal human iliac crest biopsies. Calcif Quantitative analysis of trabecular bone structure. Bone Tissue Int 37:32Ð35. 14:187Ð192. 24. Vedi S, Compston JE 1984 Direct and indirect measurements 7. Parfitt AM, Drezner MK, Glorieux FH, Kanis JA, Malluche H, of osteoid seam width in human iliac crest biopsies. Metab Meunier PJ, Ott SM, Recker RR 1987 Bone histomorphometry. Bone Dis Related Res 5:269Ð274. 964 JULIET E. COMPSTON

25. Parfitt AM 1990 Osteomalacia and related disorders. In: 43. Eriksen EF, Gunderson HJG, Melsen F, Mosekilde L 1984 Krane SM (ed) Metabolic Bone Disease, 2 Ed., Gnine & Reconstruction of the resorptive site in iliac trabecular bone; Stratton, New York. a kinetic model for bone resorption in 20 normal individuals. 25a. Glorieux FH, Travers R, Taylor A, Bowen JR. Rauch F, Metab Bone Dis Related Res 5:235Ð242. Norman M, Parfitt AM 2000 Normative data for iliac bone 44. Palle S, Chappard D, Vico L, Riffat G, Alexandre C 1989 histomorphometry in growing children. Bone 26:103Ð109. Evaluation of the osteoclastic population in iliac crest biopsies 26. Frost HM 1983 Bone histomorphometry: Analysis of trabecu- from 36 normal subjects: A histoenzymologic and histomor- lar bone dynamics. In Recker R (ed) Bone Histomorphometry: phometric study. J Bone Miner Res 4:501Ð506. Techniques and Interpretations. CRC Press, Boca Raton, 45. Garrahan NJ, Croucher PI, Compston JE 1990 A comput- Florida, pp. 109Ð131. erised technique for the quantitative assessment of resorption 27. Foldes J, Shih M-S, Parfitt AM 1990 Frequency distribu- cavities in trabecular bone. Bone 11:241Ð246. tions of tetracycline-based measurements: Implications for 46. Cohen-Solal ME, Shih M-S, Lundy MW, Parfitt AM 1991 the interpretation of bone formation indices in the absence A new method for measuring cancellous bone erosion of double-labeled surfaces. J Bone Miner Res 5:1063Ð1067. depth: Application to the cellular mechanisms of bone loss in 28. Peach H, Compston JE, Vedi S, Horton LWL 1982 The value postmenopausal osteoporosis. J Bone Miner Res 6: of plasma calcium, phosphate and alkaline phosphatase in 1331Ð1338. the diagnosis of histological osteomalacia. J Clin Pathol 47. Weinstein RS, Hutson MS 1987 Decreased trabecular width 35:625Ð630. and increased trabecular spacing contribute to bone loss with 29. Boyce BF, Fogelman I, Ralston S, Johnston E, Ralston S, ageing. Bone 8:137Ð142. Boyle IT 1984 Focal osteomalacia due to low-dose diphos- 48. Mellish RWE, Garrahan NJ, Compston JE 1989 Age-related phonate therapy in Paget’s disease. Lancet 1:821Ð824. changes in trabecular width and spacing in human iliac crest 30. Adamson BB, Gallacher SJ, Byars J, Ralston SH, Boyle IT, biopsies. Bone Miner 6:331Ð338. Boyce BF 1993 Mineralization defects with pamidronate 49. Parfitt AM 1991 Bone remodeling in type 1 osteoporosis therapy for Paget’s disease. Lancet 342:1459Ð1460. (letter). J Bone Miner Res 6:95Ð97. 31. Frost HM 1983 Bone histomorphometry: Choice of marking 50. Wakamatsu E, Sissons HA 1969 The cancellous bone of the agent and labeling schedule. In Recker R (ed) Bone iliac crest. Calcif Tissue Res 4:147Ð161. Histomorphometry: Techniques and Interpretations. CRC 51. Whitehouse WJ 1974 The quantitative morphology of Press, Boca Raton, Florida, pp. 37Ð51. anisotropic trabecular bone. J Microsc 101:153Ð168. 32. Arlot M, Edouard C, Meunier PJ, Neer RM, Reeve J 1984 52. Aaron JE, Makins NB, Sagreiya K 1987 The microanatomy Impaired osteoblast function in osteoporosis: Comparison of trabecular bone loss in normal aging men and women. between calcium balance and dynamic histomorphometry. Clin Orthop Related Res 215:260Ð271. Br Med J 289:517Ð520. 53. Clermonts ECGM, Birkenhager-Frenkel DH 1985 Software 33. Frost HM 1985 The pathomechanics of osteoporosis. Clin for bone histomorphometry by means of a digitizer. Comput Orthop Related Res 200:198Ð225. Math Prog Biomed 21:185Ð194. 34. Lips P, Courpron P, Meunier PJ 1978 Mean wall thickness of 54. Garrahan NJ, Mellish RWE, Vedi S, Compston JE 1987 trabecular bone packets in the human iliac crest: Changes Measurement of mean trabecular plate thickness by a new with age. Calcif Tissue Res 26:13Ð17. computerized method. Bone 8:227Ð230. 35. Derkz P, Birkenhager-Frenkel DH 1995 A thionin stain for 55. Parfitt AM, Mathews CHE, Villanueva AR, Kleerekoper M, visualizing bone cells, mineralizing fronts and cement lines in Frame B, Rao DS 1983 Relationship between surface, vol- undecalcified bone sections. Biotech Histochem 70:70Ð74. ume and thickness of iliac trabecular bone in aging and in 36. Steiniche T, Eriksen EF, Kudsk H, Mosekilde L, Melsen F osteoporosis: Implications for the microanatomic and cellu- 1992 Reconstruction of the formative site in trabecular bone lar mechanism of bone loss. J Clin Invest 72:1396Ð1409. by a new, quick, and easy method. Bone 13:147Ð152. 56. Compston JE 1994 Connectivity of cancellous bone: 37. Vedi S, Tighe JR, Compston JE 1984 Measurement of total Assessment and mechanical implications. Bone 15:463Ð466. resorption surface in iliac crest trabecular bone in man. 57. Feldkamp LA, Goldstein SA, Parfitt AM, Jesion G, Metab Bone Dis Related Res 5:275Ð280. Kleerekoper M 1989 The direct examination of bone archi- 38. Burstone MS 1959 Histochemical demonstration of acid tecture in vitro by computed tomography. Bone 4:3Ð11. phosphatase activity in osteoclasts. J Histochem Cytochem 58. Odgaard A, Gundersen HJG 1993 Quantification of connec- 7:39Ð41. tivity in cancellous bone with special emphasis on 3-D recon- 39. Evans RA, Dunstan CR, Baylink DJ 1979 Histochemical struction. Bone 14:173Ð182. identification of osteoclasts in undecalcified sections of 59. Serra J 1982 Image Analysis and Mathematical Morphology. human bone. Miner Electrolyte Metab 2:179Ð185. Academic Press, London. 40. Courpron P, Lepine P, Arlot M, Lips P, Meunier PJ 1980 60. Vesterby A 1990 Star volume of marrow space and trabecu- Mechanisms underlying the reduction with age of the mean lae in iliac crest: Sampling procedure and correlation to star wall thickness of trabecular basic structure unit (BSU) in volume of first lumbar vertebra. Bone 11:149Ð155. human iliac bone. In Jee WSS, Parfitt AM (eds) Bone histo- 61. Croucher PI, Garrahan NJ, Compston JE 1996 Assessment of morphometry, 3rd International Workshop. Armour cancellous bone structure: Comparison of strut analysis, Montagu, Paris, pp. 323Ð329. trabecular bone pattern factor and marrow space star volume. 41. Croucher PI, Mellish RWE, Vedi S, Garrahan NJ, Compston J Bone Miner Res 11:955Ð961. JE 1989 The relationship between resorption depth and 62. Hahn M, Vogel M, Pompesius-Kempa M, Delling G 1992 mean interstitial bone thickness: Age-related changes in Trabecular bone pattern factor—A new parameter for man. Calcif Tissue Int 45:15Ð19. simple quantification of bone microarchitecture. Bone 13: 42. Parfitt AM, Foldes J 1991 The ambiguity of interstitial bone 327Ð330. thickness: A new approach to the mechanism of trabecular 63. Mandelbrot BB 1977 Fractals: Form, chance, and dimension. thinning. Bone 12:119Ð122. Freeman, San Francisco. CHAPTER 59 Bone Histomorphometry 965

64. Weinstein RS, Majumdar S, Genant HK 1992 Fractal geom- 68. De Hoff RT, Aigeltinger EH, Craig KR 1972 Experimental etry applied to the architecture of cancellous bone biopsy determination of the topological properties of three-dimen- specimens. Bone 13:A38. sional micro-structures. J Microsc 95:69Ð91. 65. Jacquet G, Ohley WJ, Mont MA, Siffert R, Schmukler R 69. Gundersen HJG, Boyce RW, Nyengaard JR, Odgaard A 1993 1990 Measurement of bone structure by fractal dimension. The ConnEulor: Unbiased estimation of connectivity using Proc Ann Conf IEEE/EMBS 12:1402Ð1403. physical dissectors under projection. Bone 14:217Ð222. 66. Majumdar S, Genant HK 1995 A review of the recent 70. Vedi S, Compston JE, Webb A, Tighe JR 1982 Histomorphome- advances in magnetic resonance imaging in the assessment of tric analysis of bone biopsies from the iliac crest of normal osteoporosis. Osteoporosis Int 5:79Ð92. British subjects. Metab Bone Dis Related Res 4:231Ð236. 67. Genant HK, Engelke K, Fuerst T, Gliier C-C, Grampp S, 71. Vedi S, Compston JE, Webb A, Tighe JR 1983 Harris ST, Jergas M, Lang T, Lu Y, Majumdar S, Mathur A, Histomorphometric analysis of dynamic parameters of Takada M 1996 Noninvasive assessment of bone mineral and trabecular bone formation in the iliac crest of normal British structure: State of the art. J Bone Miner Res 11:707Ð730. subjects. Metab Bone Related Res 5:69Ð74. CHAPTER 60 Radiology of Rickets and Osteomalacia

JUDITH E. ADAMS Clinical Radiology, Imaging Science and Biomedical Engineering, Stopford Building, Oxford Road, The University, Manchester, England, United Kingdom

I. Introduction and Historical Aspects VI. Differential Diagnoses II. Vitamin D Deficiency VII. Vitamin D Intoxication III. Renal Osteodystrophy VIII. Technical Aspects of Imaging IV. Renal Tubular Defects and Hypophosphatemia IX. Conclusions V. Acidemia References

I. INTRODUCTION AND Disease of English Children which is popularly termed HISTORICAL ASPECTS Rickets” for his doctorate at the University of Leyden [4]. Glisson in 1650 wrote a treatise for the Royal College of Bone resorption is a one-stage process, with osteo- Physicians on, “De Rachitide sive morbo puerili qui clasts resorbing mineral and osteoid together. In contrast, vulgo the Rickets dicitur” [5,6]. In 1666 a postmortem bone formation occurs in two stages: osteoblasts lay examination was reported by John Locke of a child with down osteoid, which subsequently becomes mineralized. rickets who died of pneumonia [7]. The disease was well The mineralization of bone matrix depends on the pres- known in Europe at this time and was regarded as “the ence of adequate supplies of not only vitamin D, in the English disease.” The origin of the term “rickets” is still form of its active metabolite 1,25-dihydroxyvitamin D debated [8,9]. [1,25(OH)2D], but also calcium, phosphorus, and alka- Fish oil was recognized as a popular cure for line phosphatase, and on a normal pH prevailing in the “chronic rheumatism” (osteomalacia) and limb deformi- body environment. If there is a deficiency of these sub- ties (rickets) in children [10]. Robert Darbey, a house stances for any reason, or if there is severe systemic aci- surgeon and apothecary to the Manchester Infirmary, dosis, then mineralization of bone will be defective. was quoted by Percival in 1783 [11] as saying that There will be a qualitative abnormality of bone (in con- 50Ð60 gallons of cod-liver oil were issued every year to trast to osteoporosis, which is a quantitative abnormal- patients, usually for the treatment of chronic rheuma- ity of bone), with reduction in the mineral/osteoid ratio, tism [6]. In 1890 Palm [12] published an essay on the resulting in rickets in children and osteomalacia in distribution of rickets around the world and noted that it adults. In the immature skeleton, the radiographic was common in cities where people were deprived of abnormalities predominate at the growing ends of the sunlight. He suggested that rickets could be treated with bones, where endochondral ossification is taking place, sunlight, but did not proceed to perform the experiment. giving the classic appearances of rickets. When the Others observed that the disease is more common in skeleton reaches maturity and the process of endochon- cities [13]. dral ossification has ceased, the defective mineralization There was much confusion between conditions that of osteoid is evident radiographically as Looser’s zones had similar clinical features but different courses of pro- (pseudofractures, Milkman’s fractures), which are gression and responses to therapies of the day [14,15]. It pathognomonic of osteomalacia. Rickets and osteoma- was the microscopic studies of Pommer in 1885 [16] lacia are therefore synonymous and represent the same which distinguished, for the first time, between rickets, disease process, but are the manifestation in either the achondroplasia, and osteogenesis imperfecta. In 1895 growing or the mature skeleton. A large number of dif- Roentgen discovered X-rays, and it then became possi- ferent diseases can cause the same radiological abnor- ble to display the radiographic features of rickets and malities of rickets and osteomalacia [1Ð3]. osteomalacia. Rickets appeared when people began to live in cities The unraveling of the structure and function of vita- during the industrial revolution. The first descriptions of min D and its metabolites during the twentieth century the condition are attributed to Daniel Whistler, who in has elucidated the causes for confusion that existed in 1692, while a student at Merton College Oxford, wrote a the past as to the etiology of rickets and the variable thesis entitled, “Inaugural Medical Disputation in the response to treatment (see Chapter 1). VITAMIN D, 2ND EDITION Copyright © 2005, Elsevier, Inc. FELDMAN, PIKE, AND GLORIEUX All rights reserved. 968 JUDITH E. ADAMS

Vitamin D deficiency may occur as a consequence of As the changes become more severe, there is simple nutritional deficiency (diet, lack of sunlight; see “cupping” of the metaphysis with irregular and poor Chapters 65 and 66), due to malabsorption states, mineralization (Figs. 1Ð 4). There is some expansion chronic liver disease which affects hydroxylation at the in width of the metaphysis which results in the appar- 25 position (see Chapter 75), and chronic renal disease in ent soft tissue swelling around the ends of the long which the active metabolite 1,25(OH)2D is not produced bones affected. This produces the expansion at the ante- (see Chapter 76). Consequently, a large variety of dis- rior ends of the ribs referred to as a “rachitic rosary” eases may result in vitamin D deficiency [17Ð19] (see (Fig. 1C) [29]. There is often a thin “ghost-like” rim of Chapter 46). The radiological features of all will be sim- mineralization at the periphery of the metaphysis, ilar, being those of rickets and osteomalacia. This simi- since this mineralization occurs by membranous ossi- larity of radiological features but variations in response fication at the periosteum. The margin of outline of the to treatment contributed to some of the early confusion epiphysis also appears indistinct as endochondral ossifi- [2,20,21]. Rickets due to nutritional deprivation was cation at this site is also defective. A method for scoring cured by ultraviolet light or physiological doses of vita- the severity of rickets has been described (30). min D (400 IU per day), but rickets associated with The changes are most pronounced at the sites of chronic renal disease was not cured, except if very large bone that are growing most actively. These sites, in pharmacological doses (up to 300,000 IU per day) were sequence, are around the knee, the wrist (particularly used. This lead to the terms “refractory rickets” and the ulna, Fig. 3A), the anterior ends of the middle ribs, “vitamin D resistant rickets” being used for these condi- the proximal femur, and the distal tibia. It is these ana- tions. In these groups were included the diseases that tomical sites that should be radiographed if rickets is caused the clinical and radiological features of rickets suspected. As rachitic bone is soft and bends, addi- but were related to phosphate, not vitamin D, deficiency, tional features that develop, once the child begins to such as X-linked hypophosphatemia (see Chapter 70) walk, are bowing of the legs (genu valgum) or knock- and genetic diseases involving defects in 1-hydroxylase knees (genu varum), deformity of the hips (coxa valga (Chapter 71) and the vitamin D receptor (Chapter 72). or, more usually, coxa vara), in-drawing of the ribs at the insertion of the diaphragm (Harrison’s sulcus), and protrusio acetabulae and tri-radiate deformity of the II. VITAMIN D DEFICIENCY pelvis. The latter can result in problems with parturi- tion at subsequent pregnancies. Involvement of the The hormone 1,25(OH)2D plays an important role in bones of the thorax and respiratory tract (larynx and calcium homeostasis by its actions principally on the trachea) can result in stridor and respiratory distress bone and intestine, but also on the kidney and parathyroid [31,32]. Paradoxically, in very severe rickets, where glands. It promotes the intestinal absorption of calcium little growth is taking place (i.e., owing to nutritional and phosphorus from the intestine. On the bone it has two deprivation or chronic ill health), the features of rickets actions; one is to mobilize calcium and phosphorus from may not be evident at the pubertal growth plate [33]. In the skeleton as required, and the other is to promote mat- mild vitamin D deficiency, the radiographic features of uration and mineralization of the osteoid matrix. rickets may only become apparent during the pubertal Deficiency of vitamin D results in rickets in children and growth spurt associated with puberty, and then the osteomalacia in adults [22]. There are known to be sea- changes are most prominent at the knee. sonal variations in vitamin D status, with plasma levels The radiographic changes may be quite subtle and not being lower in the winter months [23] (see also Chapters involve the entire metaphysis (Fig. 2). The causes, pre- 3 and 47). The pathophysiology, clinical descriptions and sentation, and features of rickets may vary according to treatments are discussed elsewhere in this volume. the age of onset [26]. In the rickets of prematurity, there may be little abnormality at the metaphysis, as no skele- tal growth is taking place in the premature neonate. A. Rickets However, the bones are osteopenic and prone to fracture. Periosteal reaction is probably due to the accumulation In the growing skeleton, the effect of vitamin D defi- of unmineralised osteoid on the periosteal surface [34]. ciency and consequent defective mineralization of The cause in preterm infants is an inadequate supply of osteoid is seen at the growing ends of the bones [24Ð26]. phosphorus and calcium during periods in the hospital, In the early phase, there is widening of the growth plate, or those receiving unsupplemented human milk. In which is the radiolucent (unmineralized) gap between young infants, vitamin D levels are closely related to the mineralized metaphysis and epiphysis [27,28]. maternal vitamin D status. Although there has been a CHAPTER 60 Radiology of Rickets and Osteomalacia 969

A B C

FIGURE 1 Nutritional vitamin D deficiency rickets in young children. Radiograph of the wrist in a child of approximately 2 years (A) and 3.5 years (B) showing widening of the growth plate with cupping and expansion of the metaphyses, which are poorly mineralized. The expansion results in soft tissue swelling around the ends of the bones. The cortical “tunneling” (subcortical erosion) and hazy trabec- ular pattern indicate secondary hyperparathyroidism. (C) Chest radiograph in an infant showing the bulbous expansion of the anterior ends of the ribs (arrows) known as a “rachitic rosary.”

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FIGURE 2 Nutritional rickets in Asian adolescents. (A) Antero-posterior (AP) radiograph of the knee showing widening of the radiolu- cent growth plate due to poor mineralization and some splaying and cupping of the metaphyses. In other individuals in whom the vitamin D deficiency was mild, the features of rickets only became apparent clinically and radiologically during the growth spurt of puberty in the knee (B) and ankle (C). The changes may be quite subtle and not involve the entire metaphysis, as illustrated. The widened growth plate is evident only at the medial aspect of the metaphyses of the distal femur and proximal tibia (B), and in the lateral aspect of the distal tibia, and in the fibula (C). There is not the cupping, splaying, and irregular and poor mineralization of the metaphyses, which is evident in more florid rickets. 970 JUDITH E. ADAMS

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FIGURE 3 Rickets and the effect of treatment. (A) Wrist before treatment. The radiolucent growth plate is increased in width, and there is some cupping of the metaphyses of the distal radius and ulna with irregular and deficient mineralization. Note that the ulnar plate is more severely affected than the radial plate. This is the consequence of the ulna growing in length exclusively from its distal end, the proximal end forming the olecranon. This is the most sensitive site for assessing for the radiographic presence of rickets. (B) Wrist 4 months after treatment with vitamin D. There is healing of the rickets, although the distal segments of the radius and ulna are still reduced in density. This indicates the stage of bone development at which the rickets occurred and the amount of growth that has taken place since then. With time and remodeling, the appearances will become normal. (C) A series of radiographs of the wrist taken over a period of 4 months following treatment with vitamin D; left image: there are the characteristic features of rickets with widening of the growth plates of the radius and ulna and poor mineralization of the metaphyses. Middle image at approximately 6Ð8 weeks: the radiolucent growth plate is less wide and there is increased mineralization of the metaphyses, but minor abnormalities are still present. Right image: the abnormalities previously present have now disappeared almost completely with healing of the rachitic changes. CHAPTER 60 Radiology of Rickets and Osteomalacia 971

infant, softening of skull bones may result in craniotabes [35] and frontal bossing. Depending on the age of onset, there will be effects on the teeth (delay in dental eruption and enamel hypoplasia). If the vitamin D deficiency is treated appropriately, then the radiographic abnormalities of rickets will heal over about 2Ð3 months (Fig. 3). The radiographic features of healing will lag behind the improvement in biochemical parameters (2 weeks) and clinical symptoms. With treatment, the unmineralized osteoid of the growth plate of the metaphysis and epiphysis will mineralize. This section of abnormal bone may be visible for a period of time and gives some indication as to the age of onset and duration of the period of rickets (Fig. 3). Eventually, this zone becomes indis- tinguishable from the normal bone with time and remodeling. The zone of provisional calcification that was present at the onset of the disturbance to endo- chondral ossification may remain (Harris growth arrest line) (Figs. 6 and 12A) as a marker of the age of skele- tal maturation at which the rickets occurred [36]. However, this is not specific for rickets and can result from any condition (i.e., a period of ill health, lead poi- soning) that inhibits normal endochondral ossification. There is evidence of retarded growth and development in rickets, but this is more marked when the vitamin D deficiency is associated with chronic diseases that reduce calorie intake, general well-being, and activity (i.e., malabsorption, chronic renal disease) than with simple nutritional vitamin D deficiency (J. E. Adams, personal observation). With vitamin D deficiency, there is associated FIGURE 4 AP radiograph of the legs of a young child with severe rickets. The radiolucent growth plate is increased in width, the hypocalcemia. To maintain calcium homeostasis, the metaphyses are splayed and poorly mineralized. There are also parathyroid glands are stimulated to secrete parathy- Looser’s zones (pseudo fractures) evident as radiolucent lines roid hormone (PTH). This results in another important though the distal fibula bilaterally (arrows). These have some radio- feature of vitamin D deficiency rickets [37]. Evidence dense callus formation, indicating that vitamin D therapy has of secondary hyperparathyroidism, with increased already commenced. There is also some periosteal reaction evident. This is thought to be caused by the accumulation of unmineralized osteoclastic resorption (erosions, bone cysts), is always osteoid on the periosteal surface of bone which lifts the periosteum, evident histologically (see Chapter 59), although radio- stimulating it to mineralize. graphically evident features are uncommon [38Ð40], and cystic lesions of bone (brown tumors) are rare [41]. decline in the incidence of rickets, with improved social and environmental awareness, vitamin D deficiency B. Osteomalacia remains a significant public health problem in the UK among children of Southeast Asian and Middle Eastern At skeletal maturity, the epiphysis fuses to the meta- immigrants as a result of a number of factors. These physis, and longitudinal bone growth ceases. However, include diminished cutaneous vitamin D synthesis due to bone turnover continues throughout life to maintain the migration and residency in more northern latitudes where tensile integrity of the skeleton. Vitamin D deficiency in the sun is lower in the sky, and to voluntary avoidance of the adult skeleton results in osteomalacia, the pathog- sunshine due to religious and cultural practices. There nomonic feature of which is the Looser’s zone (pseudo- are the additional factors of the rachitic role of vegetar- fracture, Milkman’s fracture) [42–47]. Looser’s zones ian diets and prolonged breastfeeding without vitamin D are radiolucent areas in the bone that are composed of supplementation [24Ð26]. In the newborn and young unmineralized osteoid. They appear as radiolucent lines 972 JUDITH E. ADAMS

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E F G CHAPTER 60 Radiology of Rickets and Osteomalacia 973 in the bone that are perpendicular to the bone cortex, do deficiency has persisted since childhood and has been not extend across the entire bone shaft, and characteristi- inadequately treated or untreated. cally have a sclerotic margin (Fig. 5) [48]. They can As in rickets, secondary hyperparathyroidism is occur in any bone, but typically are found in the medial present and can be manifested radiographically as portion of the femoral neck, the pubic rami, the lateral subperiosteal erosions, particularly in the phalanges, border of the scapula, and the ribs. They may involve the but at other sites also (sacroiliac joints, symphysis first and second ribs, in which traumatic fractures are pubis, proximal tibiae, outer ends of the clavicle, and uncommon and are usually associated with severe “pepper-pot” skull), depending on the intensity of the trauma. Other less common sites are the metatarsals and hyperparathyroidism (Fig. 7). There can also be corti- metacarpals, the base of the acromium, and the ilium cal tunneling and a “hazy” trabecular pattern (Fig. 7B). (Figs. 5D, E, G). Again, traumatic fractures of the ilium There may be generalized osteopenia, and vertebral require very severe trauma, and a lack of history of such bodies may have concave end plates. This is due to severe injury should alert one to the fact that a “fracture” softening of the malacic bone, which is deformed by of the ilium may be related to vitamin D deficiency. the cartilagenous intervertebral disc (“codfish” defor- The etiology of why Looser’s zones occur in the mity). The etiology of this deformation is different anatomical sites that they do has been much debated from that which results in endplate irregularity in [49,50]. At one time, it was thought that they were in osteoporosis, in which microfractures occur owing to the sites of vascular channels, but this theory has been the bone being brittle rather than soft. discarded. Their position is most likely to be related to sites of stress in the skeleton. Looser’s zones must be differentiated from insufficiency fractures that occur in C. Secondary Hyperparathyroidism osteoporotic bone, particularly in the pubic rami, sacral ala, and calcaneum [51Ð53]. Insufficiency frac- The most sensitive site for the radiographic features tures consist of multiple microfractures and often have of hyperparathyroidism is the radial sides of the middle florid callus formation, which differentiates them from phalanges of the second and third fingers. There are Looser’s zones [54]. Incremental fractures occur in characteristic erosions along the cortical surface of these Paget’s disease of bone and resemble Looser’s zones in bones (Figs. 7AÐC). If there is florid hyperparathyroid- appearance (translucent zone, suggesting incomplete ism, then cortical erosions may be seen more widely fracture, with sclerotic margin), but these tend to occur and involve the distal phalanges (Fig. 7A), the outer on the outer (convex) cortex of the bone involved. The ends of the clavicle (Fig. 7D), the symphysis pubis, the typical features of Paget’s disease (sclerotic, disor- sacroiliac joints, the upper medial cortex of the tibiae, dered trabecular pattern, enlarged bone) serve as dis- and the skull vault (“pepper-pot” skull) (Figs. 7E–G). tinguishing radiographic features [55]. The erosions of hyperparathyroidism in the sacroiliac Complete fractures can occur through Looser’s joints tend to involve the iliac margin of the joint, in zones. As in rickets, osteomalacic bone is soft and contrast to the involvement of both joint surfaces in bends. This is evident by protrusio acetabulae, in inflammatory and erosive arthritides (Figs. 7F and 11D). which the femoral head deforms the acetabular margin The erosions of hyperparathyroidism in the skull vault so that the normal “teardrop” outline of the acetabulum (pepper-pot skull) must be differentiated from the is lost (Figs. 5A and B). There may be bowing of the “granularity” of the parietal region of the skull vault, long bones of the legs and tri-radiate deformity of the which may be a variant of normal. In the former, the pelvis (Fig. 6), particularly if the cause of the vitamin D margins of the erosions are indistinct; in the latter they

FIGURE 5 Osteomalacia. (A) Nutritional osteomalacia in an Asian. AP radiograph of the right hip. This shows a classic Looser's zone in the medial cortex of the femoral neck. Looser’s zones are generally radiolucent lines that are perpendicular to the bone cortex, do not extend fully across the bone (unless a fracture has occurred through the site), and have sclerotic margins as illustrated. There is also some protrusio acetabulae due to softening of the bone. (B) Pelvis in untreated celiac disease. Bilateral protrusio acetabulae and tri-radiate defor- mity of the pelvis are apparent. There are Looser's zones through both superior and inferior pubic rami (arrows), with complete fracture through that in the left superior pubic ramus. (C) Nutritional osteomalacia. Chest radiograph left apical area: there is a Looser's zone through the entire width of the left first rib (arrow). Traumatic fractures through the first and second rib normally occur only with severe trauma. (D) Malabsorption osteomalacia. In the pelvis there is an extensive Looser's zone through the left ilium and the right pubic ramus (arrows). There is evidence of pinning of a previous left hip fracture. Severe trauma is required to fracture the ilium. Malabsorption osteo- malacia (celiac disease): (E) Chest radiograph with Looser's zones in the lateral border of the right scapula and at the base of the acromium (arrows). (F) Forearm with Looser’s zones with sclerotic margins in both the radius and ulna. Nutritional vitamin D deficiency (G) hand radiograph with Loozer’s zone in the mid shaft of the third metacarpal. 974 JUDITH E. ADAMS

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FIGURE 6 Stigmata of rickets in childhood. (A) Pelvis showing stigmata of past rickets with a tri-radiate deformity of the pelvis and varus deformity of the femoral necks. (B) Bowing of tibia and fibula with evidence of Harris growth arrest lines in the distal shaft of the left tibia (arrows).

are distinct and corticated. Loss of the lamina dura (azo-temic) osteodystrophy, is complex and multifac- around the teeth does occur but is not specific to hyper- torial [57Ð60], and has changed in both clinical and parathyroidism, occurring also in other conditions such imaging features over the past three decades [60]. as Paget’s disease of bone and dental infection. Previously they occurred a combination of vitamin D With intense hyperparathyroidism, there is an in- deficiency, which resulted in rickets and osteomalacia, crease in cortical “tunneling” due to bone resorption and and hypocalcemia [61,62], the latter inducing severe an indistinct, “hazy” trabecular pattern. Erosions may secondary hyperparathyroidism that stimulated osteo- occur along the growth plate and result in displacement clastic resorption of bone [63Ð65]. This resulted radio- of the epiphysis from the metaphysis of the shaft of the graphically in subperiosteal erosions, most frequently bone. This is most likely to occur in association with identified along the radial aspect of the middle phalanx chronic renal disease (see Section III), since the inten- of the second and third fingers (Figs. 7A and C). sity of the hyperparathyroidism secondary to vitamin D Because the stimulus to secondary hyperparathyroidism deficiency is related to the duration and severity of the in chronic renal disease was intense and long standing, hypocalcemia which acts as the stimulant. the skeletal manifestations were often extensive and Although the changes of rickets occur predominantly manifest radiographically not only as sub-periosteal at the growth plates, Looser’s zones may also, but rarely, erosions in the hands, but other features also (e.g “pep- be present in the juvenile skeleton (Fig. 4) [56]. per pot” skull, brown “tumors” causing bone cysts, osteosclerosis and metastatic calcification). However, these features are now rarely evident on radiographs. III. RENAL OSTEODYSTROPHY This has occurred through the better understanding of vitamin D metabolism and improvements in therapeutic The bone disease that occurs in chronic renal management of patients with chronic renal impair- impairment, namely, renal osteodystrophy or uremic ment (calcitriol, 1α vitamin D, renal transplantation, B C

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FIGURE 7 Erosions of hyperparathyroidism. (A) Acro-osteolysis and resorption of the distal phalanges that results in pseudo-clubbing. There are also subperiosteal erosions in the radial cortex of the middle phalanges of the second, third, and fourth fingers (renal osteodys- trophy). (B) Intracortical tunneling of the cortex of the phalanges, with subperiosteal erosions in the radial cortex of the middle phalanx of the index finger. (C) Extensive subperiosteal erosions along the radial border of the middle phalanx of the third finger. There is adjacent metastatic vascular calcification in the digital artery, indicating phosphate retention and azotemic osteodystrophy. (D) Erosions in the outer end of the clavicle. (E) Lateral skull showing erosions in skull vault giving pepper-pot appearance. (F) Erosions along the iliac margin of the left sacro-iliac joint. (G) Erosions in the syphysis pubis (coronal tomogram). 976 JUDITH E. ADAMS and dialysis). Metastatic calcification in soft tissues and clavicle (Fig. 7D), the medial aspect of the proximal “adynamic” bone disease (related to aluminum) portion of the tibia, humerus, and femur, and the supe- remain problematic, and new complications have devel- rior and inferior borders of the ribs [67]. oped as a consequence of treatments (dialysis and Erosions may also occur adjacent to joints, and transplantation), including amyloid deposition, noninfec- consequent damage to the articular subchondral tive sponyloarthropathy, osteonecrosis and osteopenia/ bone can cause symptomatic arthritis [68]. These osteoporosis, all of which may have characteristic erosions may simulate those that occur in rheumatoid imaging features [60,66]. arthritis (RA) but tend to be a little further from the joint margin and are not generally associated with joint narrowing, periarticular osteopenia, or soft tissue A. Hyperparathyroidism swelling, which are early features of RA [69]. Joints that can be affected include the acromioclavicular, 1. SUBPERIOSTEAL EROSIONS sternoclavicular, sacroiliac, and the symphysis pubis Subperiosteal erosions occur most commonly in (Figs. 7F and G). In the hand, the distal interphalangeal the middle phalanges but are also present in the distal joints and ulnar aspect of the metacarpophalangeal phalangeal tufts, causing acro-osteolysis and the joints can be involved [70]. Subperiosteal erosions clinical sign of “pseudo-clubbing” (Figs. 7A and C). of the phalanges are diagnostic of hyperparathy- Other sites of erosions include the outer end of the roidism. In children, erosions can occur in the region

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FIGURE 8 Renal osteodystrophy in an adolescent in the past. (A) Pelvis showing rickets (widened growth plate at femoral metaphyses; varus deformity of femoral necks) and hyperparathryoidism (bone sclerosis and erosions in sacroiliac joints and along proximal femoral metaphyses), resulting in slipped upper femoral epiphysis at right. (B) Anteroposterior view of ankle showing extensive erosions of the metaphyses due to severe secondary hyperparathyroidism. With improved treatment of patients with chronic renal insufficiency (calcitriol, 1α vitamin D, renal transplantation, and dialysis), one should no longer see such cases of rickets and intense secondary hyperparathyroidism related to azotemic osteodystrophy. CHAPTER 60 Radiology of Rickets and Osteomalacia 977 of the growth plate, causing radiographic abnor- 2. INTRACORTICAL BONE RESORPTION malities, which may be mistaken for rickets and which can result in slipped epiphysis and deformity [71,72] Intracortical bone resorption is caused by osteoclastic (Fig. 8). resorption of Haversian canals within the cortex of the If the hyperparathyroidism is treated successfully, bones. Radiographically this causes linear translucencies erosions will fill in, and the cortex will revert to its within the cortex (Fig. 7B). This feature is not specific normal appearance. However, if there has been severe for hyperparathyroidism and can be found in other resorption of the distal phalangeal tufts, these cannot disorders in which bone turnover is increased (e.g., reconstitute to their normal shape and may remain Paget’s disease of bone). shortened and stubby (Fig. 7A). Bone resorption can occur in the regions of insertion 3. OSTEOSCLEROSIS of tendons and ligaments, particularly the trochanters, Osteosclerosis occurs uncommonly in primary hyper- the ischial and humeral tuberosities, the inferior aspect parathyroidism, but was a common feature of disease of the calcaneum, and around the elbow. Excessive bone secondary to chronic renal disease. Radiographically, the resorption in the skull vault causes the mottled texture of bones appeared increased in density (Fig. 9). This alternating areas of lucency and sclerosis, referred to as affected particularly the axial skeleton. In the vertebral a “salt and pepper” or “pepper-pot” skull (Fig. 7E). bodies, the endplates were preferentially involved, As the intensity of hyperparathyroidism is now much giving bands of dense bone adjacent to the end plates less intense and longstanding than previously, through with a central band of lower, normal bone density. the introduction of effective treatments (calcitriol, 1α These alternating bands of normal and sclerotic bone vitamin D, renal transplantation and dialysis), there may gave a striped pattern described as a “rugger jersey” be no features present radiographically, or only subper- spine (Figs. 9A and B). The osteosclerosis may be iosteal erosions in the phalanges. If there are no erosions more generalized. It may result from excessive accu- in this site, then it is unlikely that they will be found in mulation of poorly mineralized osteoid, which would other sites; skeletal surveys previously performed in appear more dense radiographically than normal patients with chronic renal impairment are now inappro- bone. It was also suggested that it results from an priate, particularly as parathyroid hormone levels can be exaggerated osteoblastic response following bone measured directly; a hand radiograph would suffice. resorption [73].

A B C

FIGURE 9 Renal osteodystrophy: bone sclerosis of the lateral lumbar spine showing end plate sclerosis, giving a “rugger jersey” appear- ance of alternating bands of stripes in the lumbar (A) and thoracic (B) spine. (C) Hand radiograph showing Looser's zone at the base of the first metacarpal, indicating osteomalacia, and generalized osteosclerosis due to secondary hyperparathyroidism. With improved management of chronic renal disease, the intensity of secondary hyperparathyroidism is less than in previous decades. 978 JUDITH E. ADAMS

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FIGURE 10 Brown “tumors” (osteitis fibrosa cystica) in hyperparathyroidism. (A) Oblique radiograph of right lower ribs showing a lytic area expanding the rib (arrow). In renal osteodystrophy: right knee radiograph lateral (B) and antero-posterior (C) projections showing well-defined lytic areas in the distal femur and the proximal fibula. The lesions are causing expansion of the bone with cortical destruction and soft tissue masses, radiological features of aggressive bone lesions. (D) Following parathryoidectomy and regression of the secondary hyperparathyroidism, the destructive lesions have filled in with woven bone and so have increased in density, and the cortex has reconstituted, although there is still expansion of the fibula. Such florid features of hyperparathyroidism are now infrequently seen with improvement in management. CHAPTER 60 Radiology of Rickets and Osteomalacia 979

4. BROWN TUMORS deposits of calcium will cause swelling and may be Brown tumors represent cavities within the bone in painful. They may increase rapidly in size and are which there has been excessive osteoclastic resorption. sometimes erroneously diagnosed as “tumors” on ini- Histologically, they are filled with fibrous tissue and tial clinical examination. Whereas rickets, osteomala- osteoclasts, and may undergo necrosis and liquifac- cia, and secondary hyperparathyroidism have become tion. Radiographically, they appear as radiolucent much less evident radiographically in patients with cysts within the bone. They can occur anywhere in the chronic renal disease over the past three decades, the skeleton and may cause expansion of bones (Fig. 10). prevalence of metastatic calcification has perhaps They constitute the osteitis fibrosa cystica of hyper- become more frequent in recent years [79]. This may parathyroidism first described by Von Recklinghausen. be due to a combination of excess phosphate not being When appropriate treatment was given, these bone removed effectively by dialysis, the increased use of cysts would will fill with woven bone and increase in calcium carbonate as a phosphate binder, and the occur- density (Fig. 10D). rence of “adynamic” renal bone disease [80]. This may result in the skeleton being a less effective reservoir for 5. OSTEOPOROSIS calcium than normal, so that the calcium remains in the With excessive bone resorption, combined with extracellular fluid. defective mineralization, the bones can appear These calcific masses can regress with appropriate osteopenic (reduced in radiographic density) in some treatment (phosphate binders such as aluminum hydrox- patients. ide or calcium carbonate) (Figs. 11B, C, and E). Initially, the masses may liquify, and apparent fluid levels are 6. PERIOSTEAL REACTION seen on radiographs taken with the patient in the erect Periosteal new bone formation (radiodense line paral- position. These fluid levels are the result of the inter- lel to the petriosteal cortex of a bone) used to be observed face between serum and serum plus mineral. There can in up to 25% of patients with renal osteodystrophy. It oc- be complete regression of periarticular calcific masses curred most frequently in those with severe bone disease with appropriate treatment; vascular calcification rarely and is thought to be a manifestation of intense hyper- regresses [81]. parathyroidism. It occurs in the metatarsals, femur, and The metastatic calcification in end stage renal disease pelvis and less commonly in the humerus, radius, ulna, can involve the intimal layer of the coronary arteries. tibia, metacarpals, and phalanges [74,75]. This is common, severe, and significantly associated with ischemic cardiovascular disease. The latter is the etiology of death in half the patients on dialysis. One B. Metastatic Calcification of the exciting recent developments is the quantitative imaging by electron-beam computed tomography With the reduced glomerular function of chronic (EBT) of the calcification in coronary arteries and renal failure, there is phosphate retention [76]. This cardiac valves. The calcium in coronary arteries is results in an increase in the calcium X phosphate highly correlated to myocardial infarction and angina product, and, as a consequence, amorphous calcium in patients on dialysis. Electron beam tomography has phosphate is precipitated in organs and soft tissues [77]. the potential to identify those patients at highest risk of This metastatic calcification can occur in the eyes cardiovascular morbidity and mortality, and to monitor and skin, causing symptoms of “gritty,” sore eyes and the change in cardiac calcification with time and the itching, and, in severe cases, calciphylaxis (ischemic effectiveness of treatment [82,83]. necrosis of soft tissue) [78]. Other organs in which cal- cium is deposited include the heart, stomach, kidneys, lung, and skeletal muscle, where it is rarely detected C. Aluminum Toxicity radiographically. However, radiographic evidence of metastatic calcification is seen in arteries and around Aluminum toxicity may occur in patients with the joints (Fig. 11). chronic renal disease due to excessive ingestion of The periarticular calcification is more common aluminum hydroxide taken to reduce serum phosphate. around the large joints (hip, shoulder) (Figs. 11B and C), It also occurs in those patients on hemodialysis in but can also occur around small joints. The calcifica- which the dialysate water contains excessive amounts tion can involve the joint capsule or tendons but more of aluminum [84Ð86]. The control of aluminum con- usually lies in the bursae adjacent to joints and bony tent in dialysate water in recent years has reduced the protuberances (ischial tuberosity). These metastatic prevalence of this disorder [87]. Aluminum accumulates 980 JUDITH E. ADAMS

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D E

FIGURE 11 Metastatic calcification in renal osteodystrophy. (A) Extensive vascular calcification in vessels of the foot. (B) Extensive “tumor-like” calcification around the right shoulder. There is also bowing of the proximal humerus due to osteomalacic bone softening. (C) Following treatment with a phosphate binder (aluminium hydroxide or calcium carbonate), these calcified masses can liquefy and regress, as illustrated. If the radiographs are taken with the patient in the erect position, there will be seen fluid/fluid mineral interface levels (arrows). (D) Pelvis showing extensive soft tissue calcification around the ischia and left hip. Widening of the sacroiliac joints indicates erosions of hyperparathyroidism. (E) Fingers showing (left) extensive metastatic calcification around the distal phalanx and subperiosteal erosions along the radial cortex of the middle phalanx. (Right) Following therapy with oral phosphate binder, there is reduction in the extent of the metastatic soft tissue calcification. Note the acro-osteolysis due to hyperparathyroidism. Although the features of osteomalacia and sec- ondary hyperparathyroidism are now infrequently seen radiographically with improvement in management of chronic renal impairment, metastatic calcification is still prevalent. This may be due to a combination of ineffective removal of phosphate through dialysis, the use of calcium carbonate as a phosphate binder and “adynamic” bone disease. at the bone/osteoid interface and inhibits mineraliza- These fractures can occur in unusual sites (second tion. This results in rickets and osteomalacia, but the to fourth ribs, odontoid) or have an atypical appear- bone also becomes “adynamic,” with reduced osteoid ance in long bones [88]. In patients with azotaemic production and turnover. Radiographically this results osteodystrophy and aluminium toxicity, there is less in reduced bone density and easy fracture [88]. osteosclerosis, fewer periosteal erosions and increase CHAPTER 60 Radiology of Rickets and Osteomalacia 981 in the rate of osteonecrosis following transplantation, and rickets and osteomalacia. Rickets becomes clini- than occurs in those individuals with chronic renal failure cally evident around 12 to 18 months of age. The radio- but without excess aluminium [85]. logical features of XLH are characteristic. Although in the past affected patients were often short in stature with quite marked deformities (bow-legs), improvements in IV. RENAL TUBULAR DEFECTS management have reduced these consequences of the AND HYPOPHOSPHATEMIA disorder.

Inorganic phosphate, glucose, and amino acids are 1. RICKETS absorbed in the proximal renal tubule; concentration There is widening of the growth plate and defective and acidification of the urine in exchange for a fixed base mineralization of the metaphysis; however the metaphy- occur at the distal renal tubule. Disorders of the renal seal margin tends to be less indistinct than in nutritional tubules may involve either the proximal or distal tubule, rickets, and there is less expansion in the width of the or both. They will result in a spectrum of biochemical metaphysis (Fig. 12). Changes are most marked at the disturbances that may result in loss of phosphate, knee, wrist, ankle, and proximal femur. Healing does glucose, or amino acids alone, or in combination, with occur with appropriate treatment. The growth plates fuse additional defects in urine acidification and concen- normally at skeletal maturation. The bones are often tration. These defects may be inherited and present short and undertubulated (shaft wide in relation to bone from birth (de Toni-Fanconi syndrome, cystinosis, length), with bowing of the femur and tibia (Fig. 12D). X-linked hypophosphatemia or XLH) [89] or acquired later in life (e.g., Wilson’s disease, hereditary tyrosine- 2. OSTEOMALACIA mia, neurofibromatosis, mesenychymal tumors, cad- After skeletal maturation, Looser’s zones persist in mium poisoning, drug induced [ifosfamide]) [90Ð92], patients with XLH. These tend to occur in sites different by tubular function being interfered with either by from those in nutritional osteomalacia, and are often crystal deposition (e.g., copper, in Wilson’s disease) or present in the outer cortex of the bowed femur (Fig. 12B), a humoral substance, such as is produced by tumors in although they occur along the medial cortex of the shaft tumor-induced osteomalacia (TIO) also known as also [101]. Looser’s zones in the ribs and pelvis are rare. “oncogenic” rickets [93]. It is the renal tubular disorders They may heal with appropriate treatment, but those that that cause phosphaturia, which results in rickets and have been present for many years will persist and are osteomalacia [94]. As the serum calcium is generally presumably filled with fibrous tissue (Figs. 14B and E). normal in these diseases, secondary hyperparathyroidism In the untreated patient, there is no evidence of does not occur (see Chapter 69). Hypophosphatemic hyperparathyroidism as there is no hypocalcemia, in rickets has also been described in association with the contrast to vitamin D deficiency osteomalacia. However, epidermal naevus syndrome [95]. treatment with large doses of oral phosphate for long periods may induce secondary hyperparathyroidism, which may be exceptionally evident by subperiosteal A. X-linked Hypophosphatemia erosions in the hand [102].

The genetic disorder XLH is transmitted as an 3. OSTEOSCLEROSIS X-linked dominant trait. Sporadic cases through Although there is defective mineralization of osteoid spontaneous mutations also occur. An autosomal in XLH, the bones are commonly increased in density, dominant variety with variable penetrance has also with a coarse and prominent trabecular pattern [103] been described [96]. The incidence is approximately (Fig. 12). This is a characteristic feature of the disease 1 in 25,000, and XLH is now the most common of and is not related to treatment with vitamin D and phos- genetically induced rickets [97Ð99]. The pathophysiol- phate supplements, as it can be present in those who ogy of XLH and its mode of treatment are discussed have not received treatment. This bone sclerosis has in detail in Chapter 69. been shown to involve the petrous bone and structures The disease results through mutations in the PHEX of the inner ear and may be responsible for the hydropic gene (phosphate regulating gene with homologies to cochlear pattern of deafness that these patients can endopeptidases on the X chromosome, previously known develop in later life [104]. Deformity and thickening as PEX), which codes for a type 2 membrane glyco- of the skull and involvement of the skull base can rarely protein that activates the putative phosphate-regulating be associated with stenosis of the foramen magnum hormone known as “phosphatonin” [19,100] and is char- with a Type 1 Chiari malformation and syringomyelia acterized by lifelong phosphaturia, hypophosphatemia, [105,106]. 982 JUDITH E. ADAMS

AB

C D

FIGURE 12 Familial, X-linked hypophosphatemic rickets (XLH). (A) Anteroposterior view of ankle showing widened growth plate and Harris growth arrest lines. The appearance of the metaphysis is different from that in nutritional rickets (see Fig. 1). (B) Pelvis showing rachitic changes at the proximal femoral metaphyses, dense bones with a coarse trabecular pattern, and bowing of the femoral shafts with bilateral Looser's zones (through medial cortex right femoral shaft and outer cortex of left femoral shaft [arrows]). (C) Pelvis showing rickets at the proximal femoral metaphyses, dense bones with a coarse trabecular pattern and chronic Looser’s zones in the medial aspect of the proximal shafts of both femora. (D) Femora showing rickets at the proximal and diskal femoral metaphyses, and the femora are bowed and undertubulated with broad shafts. There are Looser's zones through the medial cortex in the proximal shaft of each femur. The bones are also increased in density.

4. ABNORMALITIES OF BONE MODELING in many affected patients [108,109]. This results in new In XLH the bones are often short, with widening of bone formation around the pelvis and spine, with the the shaft. The ribs are broad and tend to slope down- changes resembling ankylosing spondylitis (Fig. 14). ward more than normal, causing a bell-shaped chest There can be complete ankylosis of the spine, which (Fig. 13A). There can be broadening of the distal end limits movements. As there is no inflammatory arthritis, of the ulna (Fig. 13B), and often marked bowing of the the sacro-iliac joints are normal, an important radio- femur and tibia [107]. graphic feature that serves to differentiate this condition from ankylosing spondylitis (Figs. 14A, 15A and B). 5. EXTRASKELETAL OSSIFICATION Ossification can occur in the interosseous membrane X-linked hypophosphatemia is characterized by an of the forearm, forming a synchondrosis between the enthesopathy (inflammation in the junctional area be- radius and ulna, and in the leg between the tibia and tween bone and tendon insertion) that heals by fibula (Figs. 14D and E). Separate small ossicles can ossification of ligament and tendon insertions to bone occur around the joints of the hands (Fig. 14C); there CHAPTER 60 Radiology of Rickets and Osteomalacia 983

A B

FIGURE 13 Modeling deformities in familial X-linked hypophosphatemic rickets (XLH). (A) Chest radiograph showing that the ribs are broad and slope downward more than normal, giving a rather bell-shaped chest. (B) Wrist showing some bulbous expansion of the distal shaft of the ulna. is also ossification at tendon insertions in the hands, The extent to which the radiographic abnormalities causing “whiskering” of bone margins. discussed in Section IV,A,1Ð6 are present varies between Rarely, spinal cord compression may be caused by a affected individuals [113]. In some, all the features are combination of ossification of the ligamentum flavum, present and so are diagnostic of XLH. In others, there thickening of the laminae, and hyperostosis around the may only be minor abnormalities, and the condition apophyseal joints [110] (Fig. 15). It is the ossification may be overlooked [114]. of the ligamentum flavum that causes the most signifi- cant narrowing of the spinal canal [111,112]. This occurs most commonly in the thoracic spine and gener- B. Tumor-Induced (“Oncogenic”) ally involves two or three adjacent vertebral segments. Rickets/Osteomalacia Patients may be asymptomatic even with severe spinal canal narrowing, and acute cord compression can be Tumor induced osteomalcia (TIO) or “oncogenic” precipitated by quite minor trauma. It is important to be rickets and osteomalacia were first reported in 1947 aware of this rare, but recognized, complication of the [93,115]. There is hypophosphatemia due to excessive disease since surgical decompression by laminectomy urinary phosphate loss, and serum concentrations of is curative. The extent of ossification cannot be pre- 1,25(OH)2D are low or undetectable. The clinical and dicted by the degree of paraspinal or extraskeletal ossi- radiographic features of rickets or osteomalacia can pre- fication at other sites. Computerized tomography (CT), cede identification of the causative tumor by long periods with its cross-sectional depiction of anatomy, is a (1–16 years). Bilateral Looser’s zones in the tibia have useful imaging technique for demonstrating the extent been described, and can mimic radiographically stress of intraspinal ossification (Figs. 15CÐF). Extraskeletal fractures that might occur in athletes in this site [116]. ossification is uncommon in patients with XLH under The tumors are usually small, benign, and vascular in 40 years of age. origin (hemangiopericytoma) [117,118], but some may be malignant [119] (Fig. 16). Rickets and osteomalacia 6. OSTEOARTHRITIS will heal with surgical removal of the tumor [120,121]. It The deformity and bowing of the long bones in XLH has now been established that circulating concentrations cause altered stress through joints, predisposing to of fibroblast growth factor 23 (FGF23) are high in tumor degenerative joint disease. This is particularly promi- induced osteomalacia, and the levels fall after the tumor nent in the knee joint. is removed [122,123]. Often, the tumors are extremely 984 JUDITH E. ADAMS

A B

C D E

FIGURE 14 Enthesopathy and extraskeletal ossification in XLH. (A) Anteroposterior view of pelvis and left hip (B) showing a tri-radiate deformity of the pelvis indicative of rickets in childhood. There is new bone formation around the hip joints and at lesser trochanters. There is a chronic Looser’s zone in the outer cortex of the proximal shaft of the left femur. Note the normal sacroil- iac joints which help in distinguishing the etiology of paravertebral ossification, which occurs in XLH, from that which occurs in ankylosing spondylitis. (C) Hands showing ossicles and bony outgrowths related to heads of the metacarpals and joints. (D) Forearm showing synostosis between the radius and ulna due to ossification of the interosseous ligament. (E) Lower leg showing deformity due to bowing and chronic Looser's zones with ossification of the interosseous membrane. Such severe deformity reflects inadequate treatment during childhood. small and elude detection for many years (Fig. 19). systemic acidosis then the features of rickets or osteo- It is important that the patient is vigilant about self- malacia may be evident radiographically. This may examination and reports any small palpable lump or skin occur with ileal replacement of the ureters [130] and lesion. More sophisticated imaging (CT, magnetic reso- with chronic and excessive antacid ingestion [131,132]. nance imaging [MRI] and radionuclide scanning [ocreotide]) may be helpful in localizing more deep-seated lesions [124Ð128]. The condition is discussed in detail in VI. DIFFERENTIAL DIAGNOSES Chapter 70. If no causal lesion comes to light with thor- ough imaging and spontaneous remission occurs, the Other etiologies may cause radiographic abnormal- condition described as “pseudo-(tumor-induced) rickets” ities of the metaphyses that have to be distinguished should be considered, to avoid prolonged medical treat- from those of rickets. These are the syndromes associ- ment and futile searches for a neoplasm [129]. ated with metaphyseal dysostoses (chondrodysplasias) (e.g. types of Jansen, Schmidt, etc.) (Fig. 17). The meta- physeal changes can vary in severity from mild, such as V. ACIDEMIA occur in the Shwachman-Diamond syndrome (Figs. 17A and B), in which there is associated neutropenia and The mineralization of osteoid also requires a normal pancreatic insufficiency, to more significant fragmenta- pH to prevail in the body environment. If there is a tion of the metaphyses [133Ð135] (Figs. 17C and D). A B

C D

E

F

FIGURE 15 Changes in XLH. (A) Lateral lumbar spine showing ossification of the paraspinal ligaments and at apophyseal joints. The appearances resemble those of ankylosing spondylitis but can be differentiated from it by the sacroiliac joints being normal and not eroded, as would occur in seronegative arthropathies. (B) Anteroposterior view of lumbar spine showing ossification of paraspinal ligaments result- ing in ankylosis but normal sacroiliac joints (arrows). (CÐF) Narrowing of the spinal canal may be caused by various factors. (C) Computerized tomography (CT) scan through thoracic spine showing thickening of the laminae and hypertrophy of the apophyseal joints causing narrowing and trefoil deformity of the spinal canal. (D) CT scan through the lower thoracic spine showing severe narrowing of the spinal canal by posterior ossification of the ligamentum flavum. (E) CT thoracic spine of a different patient showing narrowing of the spinal canal by new bone formation lying anterior to the laminae (arrow), severely narrowing the thoracic spinal canal (CT scan). (F) Sagittal reformation of thin (3 mm) contiguous CT sections showing ossification of the ligamentum flavum (arrows), severely reducing the antero- posterior diameter of the spinal canal. D, E, and F reprinted from Adams and Davies [111] with permission. 986 JUDITH E. ADAMS

vomiting due to hypercalcemia (see Chapter 78). The hypercalcemia results in hypercalciuria, nephrocalci- nosis, renal impairment, and hypertension. Metastatic calcification and bone sclerosis also occur [141,142] (Fig. 18).

VIII. TECHNICAL ASPECTS OF IMAGING A. Plain Radiographs

Despite tremendous developments and expansion in the imaging techniques available since the 1970s, plain radiographs remain the principal imaging method in the radiographic diagnosis of metabolic bone disorders, including those involving vitamin D deficiency, rickets, and osteomalacia. When radiographing the hands and FIGURE 16 Tumor-induced or oncogenic osteomalacia in a 46- year-old woman who 18 months previously had presented with feet, image quality must be optimized by using fine hypophosphatemic osteomalacia. There is a lytic lesion in the grain, single-sided emulsion film and a fine X-ray focal proximal fibula. This was a fibrosarcoma. The osteomalacia healed spot (0.6 mm or less). Meticulous attention to detail following surgical excision of the tumor. of the radiographic technique used will enhance the diagnostic features present in the hand radiograph, such as the subperiosteal erosions and intracortical tunneling In contrast to rickets of vitamin D deficiency, although of hyperparathyroidism. Magnification techniques, the metaphyses are fragmented and splayed, they do either optical or radiographic, can further enhance iden- not exhibit the same cupping and widening of the radi- tification of such diagnostic features of metabolic bone olucent growth plate [136]. disease. High-resolution radiographs of the torso regions Deficiency in alkaline phosphatase will also result of the body are generally precluded because of the high in rickets and osteomalacia. This occurs in hypophos- radiation doses required. phatasia, which is an inborn error of metabolism first reported in 1948 by Rathburn [137]. There is an accu- mulation of the enzyme substrates, including phospho- B. Nuclear Medicine ethanolamine and of inorganic phosphate, which promotes the development of articular chondrocalci- In the imaging technique of the skeleton referred to as nosis. There are several forms of the disorder, which nuclear medicine, 99mTc-labeled phosphate compounds varies in its severity and age of onset [138]. are administered intravenously [143,144]. They are incor- porated into the skeleton, particularly in sites that have either increased vascularity or increased new bone forma- VII. VITAMIN D INTOXICATION tion. Such areas of increased uptake are evident as “hot spots” on the scan, which is performed, using a gamma In the past, vitamin D was advocated in the treat- camera, 2 hours after administration of the radionuclide. ment of a great variety of conditions, including tuber- This bone scanning technique is very sensitive to disease culosis (especially lupus vulgaris), sarcoidosis, in bone, but not specific, in that numerous pathologies rheumatoid arthritis, hay fever, chilblains, and asthma. may cause hot spots, including infection, Paget’s disease This treatment had no beneficial effect in these condi- of bone, metastases, and degenerative joint disease tions, and its use was eventually abandoned, but not (hyperostosis). Radiographs of the relevant anatomical before many cases of vitamin D intoxication had been site will help to differentiate these various pathologies. described [139,140]. Although vitamin D intoxication The radionuclide bone scan is sensitive to detecting has become less common with the advent of Looser’s zones that may not be evident radiographi- 1,25(OH)2D3 and other active metabolites, the new era cally [145Ð149] (Fig. 19). The areas of increased of vitamin D usage to treat cancer, psoriasis, and uptake of label may be bilateral and symmetrical and be immunological disease (see Section X of this volume) present in anatomical sites typical for Looser’s zones may see a resurgence of interest in vitamin D intoxica- (femoral necks, ribs, pubic rami) [150] (Figs. 19B and C). tion. Clinically, the symptoms are of fatigue, malaise, If there is associated secondary hyperparathyroidism, weakness, thirst and polyuria, anorexia, nausea, and there will be generalized increase in uptake of the CHAPTER 60 Radiology of Rickets and Osteomalacia 987

AB

C

D E F

FIGURE 17 Differential diagnoses. Metaphyseal dysostosis (chondrodysplasia). The metaphyseal abnormalities may be mild and resemble rickets as evident in the knees (A) and proximal femora (B) in this patient with Shwachman-Diamond syndrome, in which there is associated neutropenia and pancreatic insufficiency. In more severe types, there is greater fragmented miner- alization of the metaphyses in the wrists (C) and the hips (D), which do not resemble the radiographic abnormalities of rick- ets. Hypophosphatasia, which is an inborn error of metabolism in which alkaline phosphatase levels are reduced, also results in rickets, as is evident in the knee (E) of this affected child, and osteomalacia in affected adults (F). As there are no specific therapeutic options, Looser’s zones are chronic, as seen in the neck and shaft of the left femur, and require repeated orthopaedic interventions, such as the intramedullary nailing, which has been undertaken in this case. Chondrocalcinosis may also be evident. 988 JUDITH E. ADAMS

A B

FIGURE 18 Vitamin D intoxication with associated renal impairment. (A) Pelvis showing bone sclerosis, calcification of vessels in the pelvis, and periosteal reaction around the pelvic brim. There is also calcification of the ilio-lumbar ligaments. (B) Lateral foot showing bone sclerosis and calcification of the ligaments in the foot. There was calcification of ligaments in other sites and of the falx and tentorium in the head. radionuclide by the skeleton (“super scan”), with ele- management of rickets and osteomalacia. The excep- vation of the bone/soft tissue ratio. There will be poor tion to this is their application to the identification and renal uptake of the radionuclide if the cause of the localization of tumors that cause oncogenic rickets. osteomalacia and vitamin D deficiency is chronic renal Such tumors are often very small and may be deep disease. With appropriate treatment of the osteomala- seated, so they can be extremely difficult to identify cia, the Looser’s zones will heal, and the hot spots will (see Chapter 70). Whole body MR scanning and CT not be present on subsequent radionuclide scanning. have proved useful in such identification and localiza- Radionuclide scanning offers a method of monitoring tion [126,156]. These imaging methods may be partic- response of osteomalacia to therapy [151,152]; how- ularly applicable after RNS with Indium-111 labeled ever, clinical symptoms, biochemical parameters, and ocreotide scanning has localized an area of increased plain radiographs often suffice for this purpose. uptake, since they can then be targeted at the abnormal As mesenchymal tumors have somatostatin receptors, site, and will provide superior anatomical localization and may be associated with tumor-induced osteomalacia, and spatial resolution than RNS. Indium-111 labeled pentetreotide has become an impor- tant imaging technique to identify the presence and con- firm the site of such tumors, which may otherwise prove D. Bone Mineral Densitometry elusive to clinical localization [153Ð155] (Fig. 19E). If the radionuclide scan demonstrates an abnormal area of Methods of bone mineral densitometry (BMD) high uptake, then it may additionally be helpful to per- play an important role in diagnosis of patients with form CT of MR targeted at the area of abnormality, since osteoporosis and monitoring the efficacy of treatment these imaging methods offer higher spatial resolution [157,158]. The methods available include single energy and superior anatomical detail of the site and size of the X-ray absorptiometry (SXA) for forearm (and calca- lesion (Fig. 19G). neus) measurements, dual energy X-ray absorptiometry Radionuclide scanning in children is of limited (DXA) for measurements in the lumbar spine (L1Ð4), value in metabolic bone disorders, because there is proximal femur (total hip, femoral neck, trochanteric, high uptake in the normal metaphysis of the growing and Ward’s area) and whole body, quantitative com- skeleton. In addition, the examination carries a signif- puterized tomography (QCT) for measuring cortical and icant radiation dose to the bone marrow. trabecular bone separately in the lumbar spine and fore- arm, and broadband ultrasound attenuation (BUA), used to make measurements in the calcaneum. In disease, and C. Other Imaging Techniques treatment, these techniques can provide complementary information because they measure different types of Ultrasonography (US), computerized tomography bone in different sites of the skeleton. Axial DXA is the (CT), magnetic resonance imaging (MRI), and angiog- most widely used method in clinical practice [159,160]. raphy generally play little part in the diagnosis and The World Health Organization (WHO) has defined CHAPTER 60 Radiology of Rickets and Osteomalacia 989

A B

C D

E

F G

FIGURE 19 Acquired hypophosphatemic osteomalacia. A 40-year-old man presented with low back and buttock pain. He was thought to have ankylosing spondylitis. (A) Pelvic radiograph, which was considered to be normal at the original hospital of referral. (B) Radionuclide scan of pelvis showing, increased uptake in both femoral necks, which are sites of Looser’s zones. (C) Radionuclide scan of upper torso showing multi- ple “hot spots” in ribs. The distribution of the hot spots suggests Looser's zones. A radionuclide scan is more sensitive than a radiograph for iden- tifying Looser’s zones. The patient subsequently had a road traffic accident in which he suffered bilateral femoral neck fractures through the Looser’s zones, which in retrospect are subtly present in the original radiograph (A). The patient was found to have hypophosphatemia presumed to be tumor-induced, but no tumor was identified after 15 years despite careful clinical examination and use of other imaging techniques. However, recently he had a radionuclide ocreotide scan, which showed a hot spot in the pelvis (E). The sagittal reformatted crude CT image (F) performed on the gamma camera at the end of the scan indicates that the lesion lies anterior to the bladder. (G) Conventional CT scan through the pelvis con- firms a sclerotic lesion in the left superior pubic ramus, adjacent to the symphysis. The etiology of the lesion and whether this is the cause of the oncogenic hypophosphatemic osteomalacia cannot yet be confirmed, as the patient does not wish to have the lesion removed. Figures B, C, E, and F courtesy of Dr Mary Prescott, Consultant in Nuclear Medicine, and Figure G courtesy of Dr Richard Whitehouse, Consultant Radiologist, both at The Royal Infirmary, Manchester, UK. Figure E case report reference 155 (Moran and Paul Int Orthop 2002:26:61Ð62 with permission). 990 JUDITH E. ADAMS osteoporosis in terms of bone densitometry as a T score this acts as a stimulus to secondary hyperparathy- (standard deviation below the mean of peak bone mass roidism, features of which can be identified radiograph- in an appropriate sex- and race-match reference group) ically (bone erosions). The bone disease of chronic renal below Ð2.5. DXA has advantages (rapid scanning impairment (renal osteodystrophy) is complex, being a approximately 5 min per site, extremely low radiation combination of rickets and osteomalacia [1,25(OH)2D doses 2Ð6 µSv [similar to one day’s natural background deficiency], hyperparathyroidism (bone erosions, radiation] and good reproducibility (CV 1%), but there sclerosis), and metastatic calcification (due to phos- are also some limitations (artefacts in the lumbar spine phate retention). However, the pattern of bone disease from degenerative and hyperostotic changes, particularly in chronic renal impairment has changed over the past in the elderly; size dependency as it is an ‘areal’ density 30 years with improved knowledge of vitamin D in g/cm2, a particular problem in growing children and metabolism, treatments to prevent vitamin D deficiency patients who are small through disease. QCT is unique (calcitriol, 1α vitamin D), transplantation, and dialysis. amongst methods in providing a true volumetric den- It is therefore now rare to see florid rickets and osteo- sity (mg/cc), so is not size dependent, and can measure malacia radiographically with the associated features cortical and trabecular bone density separately [161]. of severe and longstanding secondary hyperparathy- SXA measures integral (cortical and trabecular) bone roidism that was evident in the past. However, in the forearm, and DXA measures integral bone in the metastatic calcification in soft tissues is still witnessed proximal femur, the lumbar spine, and the whole body. and remains problematic. In vitamin D deficiency and rickets or osteomalacia, Plain radiographs remain the most important imaging there may be osteopenia; bone desitometry techniques technique for the diagnosis of metabolic bone disease; cannot distinguish between reduced BMD being due radionuclide bone scanning is more sensitive for identi- to osteoporosis (reduced bone mass) and osteomalacia fying Looser’s zones. Radionuclide scanning using (reduced mineral/osteoid ratio) [162]. If there is sec- Indium-111 labeled ocreotide can be useful in localizing ondary hyperparathyroidism, the forearm cortical bone tumors associated with tumoral (oncogenic) osteomala- mineral density (BMD) (e.g. distal SXA/DXA) mea- cia, as may other cross-sectional imaging methods surement might show the most marked reduction [163]. (ultrasound, computed tomography, magnetic resonance Where vitamin D deficiency osteomalcia is treated imaging). CT is particularly well suited to demonstrate appropriately, there is very rapid increase (+25% or the intra-spinal ossification which is a rare, but recog- more) in BMD (2Ð4 weeks) on serial bone densitometry nized, complication of the enthesopathy associated with (see Chapter 71). X-linked hypophosphatemic osteomalacia.

IX. CONCLUSIONS References

Mineralization of bone matrix depends on the pres- 1. Harrison HE, Harrison HC 1975 Rickets then and now. J Pediatr ence of adequate supplies of not only vitamin D, in the 87:1144. 2. Pitt MJ 1991 Rickets and osteomalacia are still around. form of its active metabolite 1,25(OH)2D, but also cal- Radiol Clin North Am 29:97Ð118. cium and phosphorus and the presence of alkaline 3. Turner ML, Dalinka MK 1979 Osteomalacia: Uncommon phosphatase and a normal pH. If there is deficiency of causes. A J Roentgenol 133:539Ð540. these substances for any reason, or if there is severe 4. Smerdon GT 1950 Daniel Whistler and the English Disease. acidosis, then mineralization of bone will be defective. A translation and biographical note. J Hist Med 5:397Ð415. 5. Glendening L 1942 Source Book of Medical History. Dover, This results in rickets in childhood and osteomalacia in New York, pp. 268Ð269. adults. Radiographically, rickets is evident by bone 6. Fourman P, Royer 1960 Vitamin D. In: Calcium Metabolism deformity caused by softening and metaphyseal abnor- and the Bone. Blackwell, Oxford, and Edinburgh, pp. 104Ð129. malities where endochondral ossification is defective. 7. Dewhurst K 1962 Postmortem examination in case of rickets The pathognomonic feature of osteomalacia is the performed by John Locke. Br Med J 2:1466. 8. Hunter R 1972 Rickets, ruckets, rekets, or rackets? Lancet Looser’s zone (pseudofracture). 1:1176Ð1177. Many different diseases that result in rickets and 9. LeVay D 1975 On the derivation of the name “rickets.” Proc R osteomalacia (vitamin D deficiency, calcium deficiency, Soc Med 68:46Ð50. hypophosphatemia, hypophosphatasia, and acidemia) 10. Schutte D 1824 Beobachtungen uber den Nutzen des Berger may therefore have similar radiographic appearances. Leberthrans. Arch Med Erfahrung 2:79Ð92. 11. Percival T 1783 Observations on the medicinal uses of There may be distinguishing features, such as bone scle- the oleum jecoris aselli or cod liver oil, in the chronic rosis and extraskeletal ossification in XLH. In conditions rheumatism and other painful disorders. Lond Clin Med J in which there is hypocalcemia (vitamin D deficiency), 3:392Ð401. CHAPTER 60 Radiology of Rickets and Osteomalacia 991

12. Palm TA 1890 The geographical distribution and aetiology of 41. Bereket A, Casur Y, Firat P, Yordam N 2000 Brown tumor as a rickets. Practitioner 45:270Ð279. complication of secondary hyperparathyroidism in severe long- 13. Morse JL 1900 The frequency of rickets in infancy in Boston lasting vitamin DÐdeficiency rickets. Eur J Paediatr 159:70Ð73. and vicinity. JAMA 34:724. 42. Looser E 1908 Uber spatrachitis und die Beziehungen zwischen 14. Parsons LG 1927 The bone changes occurring in renal and rachitis und osteomalacie. Mitt Grenzgeb Med Chir 18: coeliac infantilism, and their relationship to rickets. Part I. 678Ð744. Renal rickets. Arch Dis Child 2:1Ð25. 43. Looser E 1920 Uber spatrachitis und osteomalacie 15. Parsons LG 1927 The bone changes occurring in renal and Klinishe ront-genologische und pathologischanatomische coeliac infantilism and their relationship to rickets. Part II. Untersuchungen. Drsch Z Chir 152:210Ð357. Coeliac rickets. Arch Dis Child 2:198Ð211. 44. Looser E 1920 Uber pathologische Formen von Infraktionen 16. Pommer G 1885 Untersuchungen ober osteomalacie und und Callusbildungen bei Rachitis und osteomalakie und rachitis. Leipzig. anderen knocken Erkrankungen. Zbl Chir 47:1470Ð1474. 17. Dent CE 1970 Rickets (and osteomalacia), nutritional, and 45. Milkman LA 1930 Pseudofractures (hunger osteopathy, late metabolic (1919Ð1969). Proc R Soc Med 63:401Ð408. rickets, osteomalacia). Am J Roentgenol 24:29Ð37. 18. Hutchison FN, Bell NH 1992 Osteomalacia and rickets. 46. Milkman LA 1934 Multiple spontaneous idiopathic symmet- Semin Nephrol 12:127Ð145. rical fracture. Am J Roentgenol 32:622Ð634. 19. Berry JL, Davies M, Mee AP 2002 Vitamin D metabolism, 47. Reginato AJ, Falasca GF, Pappu R, McKnight B, Agha A rickets, and osteomalacia. Sem Musculoskel Radiol 6: 1999 Musculoskeletal manifestations of osteomalacia: Report 173Ð181. of 26 cases and literature review. Semin Arthritis Rheum 20. Pitt MJ 1981 Rachitic and osteomalacia syndromes. Radiol 28:287Ð303. Clin North Am 19:581Ð598. 48. Camp JD, McCullough JAL 1941 Pseudofractures in diseases 21. Smith R 1972 The pathophysiology and management of affecting the skeletal system. Radiology 36:651Ð661. rickets. Orthop Clin North Am 3:601Ð621. 49. LeMay M, Blunt JW 1949 A factor determining the location 22. Stamp TCB, Exton-Smith AN, Richens A 1976 Classical of pseudofractures in osteomalacia. J Clin Invest 28:521Ð525. rickets and osteomalacia in Britain. Lancet 2:308. 50. Steinbach HL, Kolb FO, Gilfillan R 1954 A mechanism of the 23. Stamp TCB, Round JM 1974 Seasonal changes in human production of pseudofractures in osteomalacia (Milkman’s plasma levels of 25-hydroxyvitamin D. Nature 247:563Ð565. syndrome). Radiology 62:388Ð394. 24. Mughal Z 2002 Rickets in childhood. Semin Musculoskelet 51. Cooper KL 1994 Insufficiency stress fractures. Curr Prob Radiol. 6:183Ð190. Diagn Radiol 23:29Ð68. 25. Pettifor JM 2002 Rickets Calcif Tissue Int 70:398Ð399. 52. McKenna MJ, Kleerekoper M, Ellis BI, Rao DS, Parfitt AM, 26. Wharton B, Bishop N 2003 Rickets. Lancet 362:1389Ð1400. Frame B 1987 Atypical insufficiency fractures confused with 27. Oestreich AE, Ahmad BS 1993 The periphysis and its effect Looser zones of osteomalacia. Bone 8:71Ð78. on the metaphysis. II. Application to rickets and other abnor- 53. North KAK 1966 Multiple stress fractures simulating osteo- malities. Skel Radiol 22:115Ð119. malacia. Am J Roentgenol 97:672Ð675. 28. Steinbach HL, Noetzli M 1964 Roentgen appearance of 54. Quek ST, Peh WCG 2002 Radiology of osteoporosis. Semin the skeleton in osteomalacia and rickets. Am J Roentgenol Musculoskelet Radiol 6:197Ð206. 91:955Ð972. 55. Whitehouse RW 2002 Paget’s disease of bone. Semin 29. Ontell FK, Moore EH, Shephard JA, Shelton DK 1997 The Musculoskeletal Radiol 6:313Ð322. costal cartilages in health and disease. Radiographics 56. Bulloch B, Schubert CJ, Brophy PD, Johnson N, Reed MH, 17:571Ð577. Shapiro RA 2000 Cause and clinical characteristics of rib 30. Thatcher TD, Fischer PR, Pettifor JM, Lawson JO, Manaster BJ fractures in infants. Pediatrics 105:E48. 2000 Radiographic scoring method for the assessment of the 57. Pitt MJ 1995 Rickets and osteomalacia. In: Resnick D severity of nutritional rickets. J Trop Pediatr 46:132Ð139. (ed) Diagnosis of Bone and Joint Disorders. Saunders, 31. Frankel A, Gruber B, Schey WL 1994 Rickets presenting as Philadelphia, Pennsylvania, pp. 1885Ð1920. stridor and apnea. Ann Otol Rhinol Laryngol 103:905Ð972. 58. Shapiro R 1972 Radiologic aspects of renal osteodystrophy. 32. Glasgow JFT, Thomas PS 1977 Rachitic respiratory distress Radiol Clin North Am 10:557Ð568. in small preterm infants. Arch Dis Child 52:268Ð273. 59. Sundaram M 1989 Renal osteodystrophy. Skeletal Radiol 33. Park EA 1954 The influence of severe illness on rickets. Arch 18:415Ð426. Dis Child 29:369Ð380. 60. Adams JE 2002 Dialysis bone disease. Semin Dial 15:277Ð289. 34. Eek S, Gabrielsen LH, Halvosen S 1957 Prematurity and 61. McCarthy JT, Kumar R 1986 Behavior of the vitamin D rickets Pediatrics 20:63Ð77. endocrine system in the development of renal osteodystro- 35. Pettifor JM, Pentopoulos M, Moodley GP, Isdale JM, Ross FP phy. Semin Nephrol 6:21Ð30. 1984 Is craniotabes a pathognomonic sign of rickets in 62. Peacock M 1978 Renal bone disease. Practitioner 220:913Ð918. 3-month-old infants? S Afr Med J 65:549Ð551. 63. Massry SG, Ritz E 1978 The pathogenesis of secondary 36. Harris HA 1933 Rickets. In: Bone Growth in Health and hyperparathyroidism of renal failure: Is there a controversy? Disease. Oxford Medical Publications, Oxford University Arch Intern Med 138:853Ð856. Press, London, p. 87. 64. Norfray J, Calenoff L, DelGreco F, Krumlovsky F 1975 Renal 37. Davies DR, Dent CE, Willcox A 1956 Hyperparathyroidism osteodystrophy in patients on hemodialysis as reflected in the and steatorrhoea. B Med J 2:1133Ð1137. bony pelvis. Am J Roentgenol 125:352Ð358. 38. Mankin HJ 1974 Rickets, osteomalacia, and renal 65. Parfitt AM 1972 Renal osteodystrophy. Orthop Clin North osteodystrophy—Part I. J Bone Joint Surg (Am) 56:101Ð128. Am 3:681Ð698. 39. Mankin HJ 1974 Rickets, osteomalacia, and renal 66. Theodorou DJ, Theodorou SJ, Resnick D 2002 Imaging in osteodystrophy—Part II. J Bone Joint Surg (Am) 56:352Ð386. dialysis sponyloarthropathy. Semin Dial 15:290Ð296. 40. Mankin HJ 1990 Rickets, osteomalacia, and renal osteodys- 67. Resnick D, Niwayama G 1976 Subchondral resorption of trophy. Orthop Clin North Am 21:81Ð96. bone in renal osteodystrophy. Radiology 118:315Ð321. 992 JUDITH E. ADAMS

68. Andresen J, Nielsen HE 1981 Juxta-articular erosions and 88. Sundaram M, Dessner D, Ballal S 1991 Solitary spontaneous calcifications in patients with chronic renal failure. Acta cervical and large bone fracture in aluminum osteodystrophy. Radiol Diagn 22:709Ð713. Skel Radiol 20:91Ð94. 69. Tigges S, Nance EP, Carpenter WA, Erb R 1995 Renal 89. Schulman JD, Schneider JA 1976 Cystinosis and the Fanconi osteodystrophy: imaging findings that mimic those of other syndrome. Pediatr Clin North Am 23:779Ð793. diseases. Am J Roentgenol 165:143Ð148. 90. Dubois J, Garel L, Patriquin H, Paradis K, Forget S, 70. Resnick DL 1974 Erosive arthritis of the hand and wrist in Filiatrault D, Grignon A, Russo P, St-Vil D 1996 Imaging hyperparathyroidism. Radiology 110:263Ð269. features of Type 1 hereditary tyrosinemia: A review of 30 71. Goldman AB, Lane JM, Salvati E 1978 Slipped capital patients. Pediatr Radiol 26:845Ð851. femoral epiphyses complicating renal osteodystrophy: A report 91. Takebayashi S, Jimi S, Segawa M, Kiyoshi Y 2000 Cadmium of three cases. Radiology 126:333Ð339. induces osteomalacia mediated by proximal tubular atrophy 72. Mehls O, Ritz E, Krempien B, Gilli G, Link K, Willich E, and disturbance of phosphate reabsorption. A study of 11 Scharer K 1975 Slipped epiphyses in renal osteodystrophy. autopsies. Pathol Res Pract 196:653Ð663. Arch Dis Child 50:545Ð554. 92. Lawson J 2002 Drug-induced metabolic bone disorders. 73. Wolf HL, Deubo JV 1958 Osteosclerosis in chronic renal dis- Semin Musculoskel Radiol 6:285Ð297. ease. Am J Med Sci 235:33Ð42. 93. Ryan EA, Reiss E 1984 Oncogenous osteomalacia: Review 74. Meema HE, Oreopoulos DO, Rabinovich S, Husdan H, of the world literature of 42 cases. Am J Med 77:501Ð512. Rapoport A 1974 Periosteal new bone formation (periosteal 94. Clarke BL, Wynne AG, Wilson DM, Fitzpatrick LA 1995 neostosis) in renal osteodystrophy. Radiology 110:513Ð522. Osteomalacia associated with adult Fanconi’s syndrome: 75. Ritchie WGM, Winney RJ, Davison AM, Robson JS 1975 Clinical and diagnostic features. Clin Endocrinol 43: Periosteal new bone formation developing during haemodial- 479Ð490. ysis for chronic renal failure. Br J Radiol 48:656Ð661. 95. Olivares JL, Ramos FJ, Carapeto FJ, Bueno M 1999 76. Slatopolsky E, Rutherford WE, Hruska K, Martin K, Klahr S Epidermal naevus syndrome and hypophosphatemic rickets: 1978 How important is phosphate in the pathogenesis of Description of a patient with central nervous system anoma- renal osteodystrophy? Arch Intern Med 138:848Ð852. lies and a review of the literature. Eur J Pediatr 158:103Ð107. 77. Parfitt AM 1969 Soft-tissue calcification in uremia. Arch 96. Econs MJ, McEnery PT 1997 Autosomal dominant hypophos- Intern Med 124:544Ð556. phatemic rickets/osteomalacia: Clinical characterization of 78. Gipstein RM, Coburn JW, Adams DA, Lee DB, Parsa KP, a novel renal phosphate-wasting disorder. J Clin Endocrinol Sellars A, Suki WN, Massry SG 1976 Calciphylaxix in man: Metab 82:674Ð681. a syndrome of tissue necrosis and vascular calcification in 11 97. Hanna JD, Niimi K, Chan JCM 1991 X-linked hypophos- patients with chronic renal failure. Arch Intern Med phatemia. Genetic and clinical correlates. Am J Dis Child 136:1273Ð1280. 145:865Ð870. 79. Weinreich T 1998 Prevention of renal osteodystrophy in peri- 98. Walton J 1976 Familial hypophosphatemic rickets: A delin- toneal dialysis. Kidney Int 54:2226Ð2233. eation of its subdivisions and pathogenesis. Clin Pediatr 80. Musci I, Hercz G 1977 Adynamic bone disease: pathogenesis, 15:1007Ð1012. diagnosis, and clinical relevance. Curr Opin Nephrol 99. Weisman Y, Hochberg Z 1994 Genetic rickets and osteoma- Hyperten 6:356Ð361. lacia. Curr Ther Endocrinol Metab 5:492Ð495. 81. Verberckmoes R, Bouillon R, Krempien B 1975 Disappearance 100. Rowe PS 1998 The role of the PHEX (PEX) gene in families of vascular calcification during treatment of renal with X-linked hypophosphatemic rickets. Curr Opin Nephrol osteodystrophy—two patients treated with high doses of Hypertens 7:367Ð376. vitamin D and aluminum hydroxide. Ann Intern Med 101. Milgram JW, Compere CL 1981 Hypophosphatemic vitamin D 82:529Ð533. refractory osteomalacia with bilateral femoral pseudofractures. 82. Raggi P 2002 Effects of excess calcium load on the cardio- Clin Orthop 160:78Ð85. vascular system measured with electron beam tomography in 102. Rivkees SA, el-Hajj-Fuleihan G, Brown EM, Crawford JD end-stage renal failure. Nephrol Dial Transplant 17:332Ð335. 1992 Tertiary hyperparathyroidism during high phosphate 83. Raggi P, Boulay A, Chasan-Taber S, Amin N, Dillon M, therapy of familial hypophosphatemic rickets. J Clin Burke SK, Chertow GM 2002 Cardiac calcification in Endocrinol Metab 76:1514Ð1518. adult hemodialysis patients. A link between end-stage renal 103. Steinbach HL, Kolb FO, Crane JT 1959 Unusual roentgen disease and cardiovascular diseases. J Am Coll Cardio manifestations of osteomalacia. Am Roentgenol 82:875Ð886. 20:695Ð701. 104. O’Malley SP, Adams JE, Davies M, Ramsden RT 1988 The 84. Andress DL, Maloney NA, Coburn JW, Endres DB, Sherrard DJ petrous temporal bone and deafness in X-linked hypophos- 1987 Osteomalacia and aplastic bone disease in aluminum- phatemic osteomalacia. Clin Radiol 39:528Ð530. related osteodystrophy. J Clin Endocrinol Metab 65:11Ð16. 105. Kuether TA, Piatt JH 1998 Chiari malformation associated 85. Kriegshauser JS, Swee RG, McCarthy JT, Hauser MF 1987 with vitamin DÐresistant rickets: Case report. Neurosurgery Aluminum toxicity in patients undergoing dialysis: 42:1168Ð1171. Radiographic findings and prediction of bone biopsy results. 106. Caldermeyer KS, Boaz JC, Wappner RS, Moran CC, Smith Radiology 164:399Ð403. RR, Quets JP 1995 Chiari 1 malformation: Association with 86. Ward MK, Feest TG, Ellis HA, Parkinson IS, Kerr DNS 1978 hypophosphatemic rickets and MR imaging appearances. Osteomalacic dialysis osteodystrophy: Evidence for a water- Radiology 195:733Ð738. borne aetiological agent, probably aluminum. Lancet 107. McAlister WH, Kim GS, Whyte MP 1987 Tibial bowing 4:841Ð845. exacerbated by partial premature epiphyseal closure in sex- 87. Smith GD, Winney RJ, McLean A, Robson JS 1987 linked hypophosphatemic rickets. Radiology 162:461Ð463. Aluminum-related osteomalacia: Response to reverse osmosis 108. Burnstein MI, Lawson JP, Kottamasu SR, Ellis BI, Micho J water treatment. Kidney Int 32:96Ð101. 1989 The enthesopathic changes of hypophosphatemic CHAPTER 60 Radiology of Rickets and Osteomalacia 993

osteomalacia in adults: Radiologic findings. Am J Roentgenol 128. Avila NA, Skarulois M, Rubino DM, Doppman JL 1996 153:785Ð790. Oncogenic osteomalacia: Lesion detected by MR skeletal 109. Polisson RP, Martinez S, Khoury M, Harrell RM, Lyles KW, survey. Am J Roentgenol 167:343Ð345. Friedman N, Harrelson JM, Reisner E, Drezner MK 1985 129. Whyte MP, Podgornik MN, Wollberg VA, Eddy MC, Calcification of entheses associated with X-linked hypophos- McAlister WH 2001 Pseudo-(tumor-induced) rickets. J Bone phatemic osteomalacia. N Engl J Med 313:1Ð6. Miner Res 16:1564Ð1571. 110. Cartwright DW, Latham SC, Masel JP, Yelland JDN 1979 130. Salahudeen AK, Elliott RW, Ellis HA 1984 Osteomalacia Spinal canal stenosis in adults with hypophosphatemic due to ileal replacement of ureters: A report of 2 cases. J Urol vitamin DÐresistant rickets. Aust New Zealand J Med 131:335Ð337. 9:705Ð708. 131. Boutsen Y, Devogelaer JP, Malghem J, Noel H, 111. Adams JE, Davies M 1986 Intra-spinal new bone formation Nagant de Deuxchais C 1996 Antacid-induced osteomalacia. and spinal cord compression in familial hypophosphataemic Clin Rheumatol 15:75Ð80. vitamin DÐresistant osteomalacia. Q J Med 61:1117Ð1129. 132. Kassem M, Eriksen EF, Melsen F, Mosekilde L 1999 112. Dunlop DJ, Stirling AJ 1996 Thoracic cord compression Antacid-induced osteomalacia: A case report with a histo- caused by hypophosphatemic rickets: a case report and a morphometric analysis. J Intern Med 229:275Ð279. review of the world literature. Eur Spine J 5:272Ð274. 133. Berrocal T, Simon MJ, al-Assir I, Prieto C, Pastor I, de Pablo L, 113. Hardy DC, Murphy WA, Siegel BA, Reid IR, Whyte MP Lama R 1995 Shwachman-Diamond syndrome: Clinical, radio- 1989 X-linked hypophosphatemia in adults: Prevalence of logical, and sonographic aspects. Pediatr Radiol 25:289Ð292. skeletal radiographic and scintigraphic features. Radiology 134. Cohen MM 2002 Some chondrodysplasias with short limbs: 171:403Ð414. Molecular perspectives. Am J Med Genet 112:304Ð313. 114. Econs MJ, Samsa GP, Monger M, Drezner ML, Fuessner JR 135. Cohen MM 2000 Merging the old skeletal biology with the 1994 X-linked hypophosphatemic rickets: A disease often new. II. Molecular aspects of bone formation and growth. unknown to affected patients. Bone Miner 24:17Ð24. J Craniofac Genet Dev Biol 20:94Ð106. 115. Prader A, Illig R, Uehlinger RE 1959 Rachitis infolge 136. Vohra P 1995 Metaphyseal chondrodysplasia: A differential Knochen-tumors. Helv Paediatr Acta 14:554Ð565. diagnosis of rickets. Indian J Pediatr 62:131Ð132. 116. Ohashi K, Ohnishi T, Ishikawa T, Tani H, Uesugi K, Takagi M 137. Rathbun JC, MacDonald JW, Robinson HMC, Wanklin JM 1999 Oncogenic osteomalacia presenting as bilateral stress 1961 Hypophosphatasia: Genetic study. Arch Dis Child fractures of the tibia Skelet Radiol 28:46Ð48. 36:540Ð542. 117. Renton P, Shaw DG 1976 Hypophosphatemic osteomalacia 138. Wedling D, Jeannin-Louys L, Kremer P, Fellmann F, secondary to vascular tumors of bone and soft tissue. Skeletal Toussirot E, Mornet E. Adult hypophosphatasia. Current Radiol 1:21Ð24. concepts. Joint Bone Spine 68:120Ð124. 118. Choi JJ, Murphey MD 2000 Angiomatous skeletal lesions. 139. Davies M, Adams PH 1978 The continuing risk of vitamin-D Semin Musculoskel Radiol 4:103Ð112. intoxication. Lancet 16:621Ð623. 119. Taylor HC, Fallen MD, Velasco ME 1984 Oncogenic 140. Anning ST, Dawson J, Dolby DE, Ingram JT 1948 The toxic osteomalacia and inappropriate antidiuretic hormone secre- effects of calciferol. Q J Med 17:203Ð228. tion due to oat-cell carcinoma. Ann Intern Med 101:786Ð788. 141. Harris PWR 1969 An unusual case of calcinosis due to 120. Linovitz RJ, Resnick D, Keissling P, Kondon JJ, Sehler B, vitamin D intoxication. Guy’s Hospital Rev 118:553Ð541. Nejdl RJ, Rowe JH, Deftos LJ 1976 Tumor-induced osteo- 142. Irnell L 1969 Metastatic calcification of soft tissue on malacia and rickets: A surgically curable syndrome, report of overdosage of vitamin D. Acta Med Scand 185:147Ð152. two cases. J Bone Joint Surg (Am) 58:419Ð423. 143. Alazraki N 1995 Radionuclide Techniques. In: Resnjck D 121. Pollack JA, Schiller AL, Crawford JD 1973 Rickets and (Ed) Diagnosis of Bone and Joint Disorders. Saunders, myopathy cured by removal of a nonossifying fibroma of Philadelphia, Pennsylvania, pp. 430Ð474. bone. Pediatrics 52:363Ð371. 144. Murray IPC 1994 Nuclear medicine in disorders of bone 122. Wilkins GE, Granleese S, Hegele RG, Holden J, Anderson DW, and joints: Growth and Metabolic Disorders. In: Murray IPC Bond GP 1995 Oncogenic osteomalacia: Evidence for a humeral and Ell PJ (eds). Nuclear Medicine in Clinical Diagnosis and phosphaturic factor. J Clin Endocrinol Metab 80:1628Ð1634. Treatment. Churchill Livingstone, London, pp. 1035Ð1040. 123. John MR, Wickert H, Zaar K, Jonsson KB, Grauer A, 145. Lee HK, Sung WW, Solodnik P, Shimshi M 1995 Bone scan Puppersberger P, Schmidt-Gayk H, Murer H, Zeigler R, in tumor-induced osteomalacia. J Nucl Med 36:247Ð249. Blind E 2001 A case of neuroendocrine oncogenic osteomala- 146. Rosenthall L, Kaye M 1976 Observations in the mechanism cia associated with a PHEX and fibroblast growth factor Ð23 of 99mTc-labeled phosphate complex uptake in metabolic expressing sinusoidal malignant schwannoma. Bone bone disease. Semin Nucl Med 6:59Ð67. 29:393Ð402. 147. Hain SF, Fogelman I 2002 Nuclear medicine studies in 124. Leicht E, Kramann B, Seitz G, Trentz O, Remberger K 1993 metabolic bone disease. Semin Musculoskel Radiol 6:323Ð329. Oncogenic osteomalacia: Imaging studies. Bildgebung 148. Mari C, Catafau A, Carrio I 1999 Bone scintigraphy and 60:13Ð17. metabolic disorders. Q J Nucl Med 43:259Ð267. 125. Shapiro D, Ben Izhak O, Nachtigal A, Burstein A, Shalom RB, 149. McAfee JG 1987 Radionuclide imaging in metabolic and Shagrawi I, Best LA 1995 Tumor, induced osteomalacia. systemic skeletal diseases. Semin Nucl Med 17:334Ð349. Semin Arthritis Rheum 25:35Ð46. 150. Singh BN, Spies SM, Mehta SP, Kesala BA, Quinn JL 1978 126. Edminster KA, Sundaram M 2002 Oncogenic osteomalacia. Unusual bone scan presentation of osteomalacia: symmetrical Semin Musculoskel Radiol 6:191Ð196. uptake—a suggestive sign. Clin Nucl Med 3:292Ð295. 127. Rhee Y, Lee JD, Shin KH, Lee HC, Huh KB, Lim SK 2001 151. Wu YW, Seto H, Shimizu M, Kageyama M, Watanabe N, Oncogenic osteomalacia associated with mesenchymal Kakishita M 1995 Postgastrectomy osteomalacia with pseu- tumour detected by indium-111 ocreotide scintigraphy. Clin dofractures assessed by repeated bone scintigraphy. Ann Endocrinol 54:551Ð554. Nucl Med 9:29Ð32. 994 JUDITH E. ADAMS

152. Meindok H, Rapoport A, Oreopoulos DG, Rabinovich S, 158. Adams JE 1995 Quantitative measurements in osteoporosis. Meema HE, Meema S 1985 Quantitative radionuclide scanning In: Tovey FI and Stamp TCB (eds). The measurement in metabolic bone disease. Nucl Med Commun 6:141Ð148. of metabolic bone disease. Parthenon Publishing Group, 153. Nguyen BD, Wang EA 1999 Indium-111 pentetreotide pp. 107Ð142. scintigraphy of mesenchymal tumour with oncogenic osteo- 159. Blake GM, Fogelman I 2002 Dual energy X-ray absorptiom- malacia. Clin Nucl Med 24:130Ð131. etry and its clinical applications. Semin Musculoskel Radiol 154. Jan de Beur SM, Streeten EA, Civelek AC, McCarthy EF, 6:207Ð217. Uribe L, Marx SJ, Onobrakpeya O, Raisz LG, Watts NB, 160. Adams JE 2002 Dual-energy X-ray absorptiometry. In: Sharon M, Levine M 2002 Localization of mesenchymal Radiology of Osteoporosis. Grammp S (Ed). Medical tumors by somatostatin receptor imaging. Lancet 359: Radiology. Springer-Verlag Berlin Heidelberg, pp. 87Ð100. 761Ð761. 161. Guglielmi G, Lang TF 2002 Quantitative tomography. 155. Moran M, Paul A 2002 Ocreotide scanning in the detection Semin Musculoskel Radiol 6:219Ð227. of a mesenchymal tumor in the pubic symphysis causing 162. Wahner HW 1987 Single- and dual-photon absorptiometry hypophosphatemic osteomalacia. Int Orthop 26:61Ð62. in osteoporosis and osteomalacia. Semin Nucl Med 17: 156. Fukumoto S, Takeuchi Y, Nagano A, Fujita T 1999 Diagnostic 305Ð315. utility of magnetic resonance imaging skeletal survey in a 163. Wishart J, Horowitz M, Need A, Nordin BE 1990 patient with oncogenic osteomalacia. Bone 25:375Ð377. Relationship between forearm and vertebral mineral density 157. Adams JE 1992 Osteoporosis and bone mineral densitometry. in postmenopausal women with primary hyperparathy- Curr Opin Radiol 4:11Ð19. roidism. Arch Intern Med 150:1329Ð1331. CHAPTER 61 The Pharmacology of Vitamin D, Including Fortification Strategies

REINHOLD VIETH Department of Laboratory Medicine and Pathobiology, University of Toronto, and Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Canada

I. Introduction V. Pharmacokinetic Principles, Volume II. Indications and Clinical Use: of Distribution, Turnover and Half-life as It Potential Health Effects of Vitamin D Pertains to Vitamin D III. Overview of the System of Vitamin D Metabolism, VI. Vitamin D Toxicity and Safety Issues and Its Regulation VII. Summary IV. Dosage Considerations References

I. INTRODUCTION that became our species, Homo sapiens, in our natural, tropical environment. The living conditions of modern Lack of cholecalciferol (vitamin D) in the diet humans differ from those that affected our evolution— can cause disease; therefore, vitamin D is a true nutri- we avoid exposure of skin to the vitamin DÐforming ent in the full sense of the word. The notion that UVB rays of sunshine, and even if we do spend time true nutrients may be available only from foods is a outdoors, many of us live at latitudes with compara- misconception; vitamin D, like niacin, is a vitamin tively little UVB. I contend that we, modern humans, that can be acquired without eating it [1]. Vitamin D is might benefit if we could compensate for the biological readily metabolized to calcidiol [25(OH)D], whose consequences of modern life. One such consequence level is the accepted measure of vitamin D nutritional may be an endemic lack of vitamin D that can be status [2]. 25(OH)D is a prehormone in the same sense corrected by appropriate supplementation. that testosterone and T4 are, because like them, it is My usual perspective about vitamin D is the circulating, immediate precursor of a signaling North American, where vitamin D is primarily regarded molecule [1]. as a nutrient. However, in Europe and in most of the The aim of this chapter is to offer the reader a fresh rest of the world, the daily use of even 20 mcg (800 IU) look at an antiquated nutrient, vitamin D, to address it of vitamin D is treated as if it were a prescription drug. from the perspective of pharmacology, as if it were a This drug-oriented perspective has the advantage of new drug that we need to understand fully so that we imposing a higher expectation on our understanding of can exploit its potential for clinical medicine and to the use of vitamin D. Before approving any new drug, optimize health. Vitamin D differs from other nutrients government regulators expect to see the answers to rela- because we have never had dietary intakes of vitamin D tively standard questions. Pharmaceutical firms need to as a reasonable reference point for deciding on how anticipate these issues as they plan the research neces- much of this nutrient that people should be consuming. sary for implementation of new products. If vitamin D Compared to the 250 mcg (10,000 IU) of vitamin D were a new drug, these questions would include, but that adults can obtain by exposing their full skin sur- are not limited to, the following: face to the sunshine [3], foods contain small amounts of vitamin D (Table I). 1a. What is the disease indication for the drug? Our biology was designed by evolution for life in 1b. What kind of clinical or health effects should we equatorial Africa. It is clear from Table I that foods be looking for, based on preclinical animal and containing a meaningful amount of vitamin D were laboratory research? not readily available to primates or to early humans. 2a. What are the most useful approaches to delivering Thus, diet could not have played a role in determining the drug to people: the vehicle? human vitamin D requirements. Requirements for 2b. What is the appropriate dosage, route of adminis- vitamin D were satisfied by the life of the naked ape tration, and interval between doses? VITAMIN D, 2ND EDITION Copyright © 2005, Elsevier, Inc. FELDMAN, PIKE, AND GLORIEUX All rights reserved. 996 REINHOLD VIETH

TABLE IVitamin D Content of Selected Food Sources*

Amount of Vitamin D per 100 g food, unless specified otherwise under Food

International Food Microgram (µg) units (IU)

According to NIH, USA [139] Cod Liver Oil, per 100 mL 219 9067 1 Salmon, cooked, 100 g or 3 /2 oz 9 360 1 Mackerel, cooked, 100 g or 3 /2 oz 9 345 1 Sardines, canned in oil, drained,100 g or 3 /2 oz 7 270 1 Eel, cooked, 100 g; 3 /2 oz 5 200 Milk, fortified with vitamin D, (250 mL) 1 cup 2 98 Margarine, fortified, 1 tablespoon 2 60 Dry breakfast cereal, if optionally fortified with vitamin D as permitted in the USA, 1 50 one serving, 190 mL 3/4 cup 1 Liver, beef, cooked, 100 g; 3 /2 oz 1 30 Egg, 1 whole (vitamin D is present in the yolk) 1 25

According to A. Takeuchi et al., Japan [140] Fishes Anglerfish (liver only) 110 4400 Skijack (viscera perserves) 120 4800 Skipjack (whole meat) 10 400 Indo-Pacific marlin 35 1400 Chum salmon 33 1300 Herring 28 1100 Flatfish 23 920 Bastard halibut (cultured) 18 720 Bluefin tuna (fatty meat) 18 720 Grunt 15 600 Rainbow trout 15 600 Eel 14 560 Red sea bream (cultured) 13 520 Mackerel 11 440

Meats Beef (lean separated) 00 Beef (total edible) 00 Beef, liver 00 Pork (lean separated) Pork (total edible) 1 28 Pork, liver 150 Chicken (breast) 00 Chicken liver 08 Turkey 04

Milk products (non-fortified) Cow’s milk 013 Human milk 013 Yogurt 00

*Amounts of Vitamin D shown in this table are per 100 g of the food, unless specified otherwise under food. (Continued) CHAPTER 61 The Pharmacology of Vitamin D, Including Fortification Strategies 997

TABLE IVitamin D Content of Selected Food Sources*—Cont’d

Amount of Vitamin D per 100 g food, unless specified otherwise under Food

International Food Microgram (µg) units (IU)

Butter 124 Cheese (cheddar) 00

Eggs Duck egg 18 720 Japanese quail egg 3 100 Chicken egg (whole) 3 120 Chicken egg (yolk) 6 230 Chicken egg (white) 00

Fungi Woody ear fungus (dried) 400 16000 Silver ear fungus (dried) 400 16000 Shitake (dried) 16 640 Shimeji 4 160 Matsutake 4 140 Commom mushroom 3 100 Winter fungus 150 Nameko 016

*Amounts of Vitamin D shown in this table are per 100 g of the food, unless specified otherwise under food.

3. What is the desirable target for the plasma and that I advocate higher requirements of vitamin D concentration; what dose would be needed to than most other investigators. attain or ensure this? 4. What, if any, are the biological markers to monitor toxicity, and what are our criteria for determining II. INDICATIONS AND CLINICAL USE: therapeutic effectiveness? What is the “therapeutic POTENTIAL HEALTH EFFECTS index”, the ratio between toxic vs beneficial dose OF VITAMIN D levels?

When it comes to plain and simple, nutritional vita- The only officially recognized indications for use of min D (cholecalciferol), the answer to each of these vitamin D in adults are the treatment and prevention of questions is that we have just started to address it in the osteomalacia, bone loss, and fractures. Figure 1 sum- past decade. Any opinion about vitamin D here is con- marizes randomized control trials looking at whether troversial. Lips presents a more conservative perspec- vitamin D, with or without calcium, affects risk of non- tive on vitamin D requirements elsewhere [4,5] and vertebral fracture. A recent, thorough literature review also see Chapter 62. In an effort to provide some of vitamin D, 1,25(OH)2D and its analogs is also avail- answers to the preceding questions, I will present my able, addressing the issue of osteoporosis prevention personal perspective about the vitamin D system that and treatment [6]. The purpose of Fig. 1 is to show that relates to pharmacological aspects of vitamin D in the there has been no evidence that doses of vitamin D less adult context. My opinion about vitamin D require- than 800 IU/day are effective in preventing osteoporotic ments is not shared by all workers in the field, but I fractures. will provide data to support my views. However, the Although it makes sense conceptually, it is difficult reader is cautioned that differences of opinion exist, to tell whether or not additional calcium is needed in 998 REINHOLD VIETH

Fracture-prevention studies with 3.5 mcg/day vitamin D [16]. The authors failed to detect vitamin D3 100 any effect of calcium intake, but they suggested that in this cross-sectional study, women with a family history 90 800 IU/d of osteoporosis would have been more likely to take sup-

RR)) 80 ? plemental calcium, confounding a calcium effect. (1 − 70 I have a concern that 1,25(OH)2D may be used too Dawson-Hughes 1997 NEJM 337:670 60 aggressively as an alternative to improved vitamin D (100% × nutrition in the prevention or treatment of osteoporosis. 50 The point that 1,25(OH) D has a narrower margin of 40 Chapuy 1992 NEJM 327:1637 2 Chapuy 2002 Osteoporosis Int 13:257 safety (therapeutic index) than vitamin D has never 30 Trivedi 2003 BMJ 326:469 been raised in analyses comparing them [6,17]. If one 20 cause of osteoporosis is that the vitamin D system is % Fewer fractures 10 somehow deficient or defective [18], it makes little Lips 1996 Ann Intern Med 124:400 0 sense to resort to the use of 1,25(OH)2D. Rickets and Meyer 2002 J Bone Min Res 17:709 osteomalacia may exist despite normal 1,25(OH)2D 0 2010 90807060504030 100 concentrations. Increases in vitamin D will not increase Vitamin D3 mcg/day 1,25(OH)2D levels further [19Ð22]. As kidney function deteriorates, its endocrine capability also declines, and FIGURE 1 Summary of randomized-control clinical trials of frac- ture-prevention using vitamin D, with or without calcium. None of thus a low serum 1,25(OH)2D level reflects impaired the studies using doses of vitamin D3 providing less than 20 mcg/day renal function, not poor nutrition [22,23]. Despite many was effective in reducing fracture risk [90,137,137]. However, all the studies looking into the use of 1,25(OH)2D and its studies in which there was a reduction in fracture risk used approxi- analogs to prevent or treat osteoporosis, the review of mately 20 mcg/day of vitamin D3 [7,10Ð12,135,137]. This dose includes the background intake; for the work by Dawson-Hughes, background this topic by Papadimitropoulos concludes that there no intake was 5 mcg/day [11]. reason for anyone to resort to any metabolite other than nutritional vitamin D [6]. I would add that this should be vitamin D3, and at a dose of at least 20 mcg/d. concert with vitamin D, because most studies have combined calcium and vitamin D for comparison to a placebo group receiving neither. There are now two A. Non-Bone Effects of Vitamin D randomized-controlled studies which show that vita- min D3 given by itself in doses of 100,000 IU (2500 µg) Vitamin D nutrition probably affects health beyond orally every 4 months [7], or 150,000 IU (3750 µg) just bone. The mechanisms involved in mediating by annual injection [8] reduces the occurrence of the non-classic (i.e. non-bone) effects of vitamin D are fractures. probably through 1,25(OH)2D produced locally, using Bone density declines more quickly during winter circulating 25(OH)D as the substrate (see Chapter 79). than during summer [9]. Vitamin D supplements Many tissues possess 25(OH)D-1-alpha-hydroxylase (about 20 mcg (800 IU) per day) combined with cal- activity, including the skin (basal keratinocytes and hair cium, eliminate the faster fall in bone density during follicles), lymph nodes (granulomata), pancreas (islets), winter [9]. Furthermore, three studies show that the adrenal medulla, brain, pancreas, and colon [24]. An even combination of calcium and 20 mcg vitamin D daily wider range of tissues possess receptors for 1,25(OH)2D lower fracture risk in adults older than age 65 [10Ð12]. (VDR) [25]. All of this reveals a system for autocrine or Occurrence of fractures is reduced by about a third, paracrine regulation of tissue processes that involves even within the first year of these studies, when bone the local production of 1,25(OH)2D [26]. Sufficient density is not increased by enough to account for the vitamin D nutrition, and hence, appropriate 25(OH)D fewer fractures [11]. The explanation for this may be concentration is essential to this local, paracrine role of that vitamin D improves muscle strength and balance. 1,25(OH)2D that is not generally reflected in the circu- This reduces the occurrence of the falls that cause lating level of 1,25(OH)2D. The paracrine components fractures [13Ð15]. of the vitamin D endocrine/paracrine systems account In people younger than age 65, risk of osteoporotic for the many effects of vitamin D nutrition and/or UVB fracture has been difficult to assess. Data from the light on health and disease prevention. Nurses Health Study suggest a 37% lower risk of osteo- Vitamin D has been implicated in a wide array of porotic fracture in postmenopausal women younger diseases (Table II). While all of the relationships with than 65, if they consume vitamin D in amounts of at least vitamin D in Table II are statistically significant, most of 12.5 mcg/day, compared to women consuming less than the evidence for a role of vitamin D is circumstantial. CHAPTER 61 The Pharmacology of Vitamin D, Including Fortification Strategies 999

TABLE II Diseases and Conditions Known to Be, or Implicated as Being Prevented by Greater Vitamin D Nutrition or Skin UV Exposure

Disease Type of evidence supporting the association Reference

Rickets Long established, curative Osteomalacia Long established, curative Osteoporosis Placebo-controlled, randomized studies that vitamin D [7,9Ð11] prevents loss of bone density, and lessens fracture risk Poor calcium absorption Modest increase in Vitamin D nutrition increases this [161] Blood-pressure regulation Epidemiological and randomized interventional data [13,14,41,42] Risk of diabetes Epidemiological and case-control data [46,141,142] Progression osteoarthritis Epidemiological, cross-sectional studies [39,40] Diminished intra-uterine growth Presumed effect [143] Effects on brain development Rat experiments [144] Resistance to pneumonia Epidemiological association with rickets [45] Multiple sclerosis, occurrence and progression Epidemiological data, and lab effects on tissue [38,145,146] Prevention of tuberculosis Epidemiological data, and lab effects on tissue [147,148] Prevent depression or SAD or improve mood Small RCT’s 400 IU/day or 100000 in winter [149,150] No mood effect of 400 IU/day [151] Lessen risk/severity of fibromyalgia Cross-sectional study [152] Protection against cancers Breast Epidemiological data, and lab effects on tissue [153,154] Prostate Epidemiological, and lab effects on tissue [155] [64,154,156,157] Large bowel epidemiological and cross-sectional data, based [153,154,158] on latitude and serum 25(OH)D

Epidemiological studies show that higher serum Chapter by Li. Vitamin D deficiency may impair immune 25(OH)D, and/or environmental ultraviolet exposure is function in animals [44]. In children there is a strong associated with lower rates of breast, ovarian, prostate, association between pneumonia and nutritional rickets and colorectal cancers [27Ð34] (see Chapter 90). More [45]; however, patients with rickets generally have soft recent statistical analyses also show significant rela- ribs, so that pneumonia could have resulted from a tionships including non-Hodgkin’s lymphoma and mechanical respiratory inadequacy, not related to cancer of the bladder, esophagus, kidney, lung, pancreas, immune function. The concept that there is a connection rectum, stomach and corpus uteri [35]. Multiple sclerosis between vitamin D nutrition and immune function is is more prevalent in populations having lower levels of further supported by the apparent protective effect of vitamin D nutrition or ultraviolet exposure [32,36Ð38], improved vitamin D nutrition during infancy and child- and it has been proposed that vitamin D intake, ranging hood against type I diabetes mellitus [46]. The role of from 33Ð95 mcg (1,300 to 3,800 IU) per day, helps vitamin D on the immune system and immune-mediated prevent the disease [38]. Established osteoarthritis pro- disease is discussed in Chapter 36, 98, and 99. If any of gresses more slowly (is less severe) in adults with higher these nontraditional effects of vitamin D were taken into vitamin D nutritional status, with serum 25(OH)D that account, they would result in a substantial upward revi- exceeds 75 nmol/L [39,40]. The prevalence of hyper- sion of official recommendations for vitamin D beyond tension increases with population distance, north or the current Adequate Intake (AI) values [47]. south, from the equator [41]. Blood pressure is lowered The level of evidence needed to make a health claim in mildly hypertensive patients whose 25(OH)D levels that can be sanctioned officially involves more than are raised to over 100 nmol/L by tanning [42]. One the circumstantial evidence of laboratory experiments randomized intervention study showing that vitamin D and epidemiology. It requires direct intervention, the supplementation at 20 µg/d (800 IU/d) lowers blood controlled administration of the agent to many healthy pressure in elderly women [43]. The role of vitamin D people, and showing an effect that stands up to in regulating the renin-angiotensin system is discussed in statistical analysis. We need randomized intervention 1000 REINHOLD VIETH

trials to take this field beyond preclinical basic research vitamin D2 supplementation might prevent bone loss in and epidemiological evidence. There are ongoing ran- steroid-treated patients [48,49]; the effects of vitamin D domized trials involving vitamin D that relate to cancer, were marginal, but since plain and simple vitamin D3 multiple sclerosis, and osteoporosis, but for the most part, was never part of the experimental protocol, the issue they deal with analogs of 1,25(OH)2D, not the nutrient. remains unresolved. Another example of the unfortunate The nutrient has been very much overlooked for all focus on vitamin D2 instead of the D3 form is the recent purposes except rickets, osteomalacia, and osteoporosis. Australian study using vitamin D2 at a substantial dose There are three reasons for this. First, the financial of 250 µg (10,000 IU) weekly, yet producing no signif- incentive lies with the proprietary analogs, driven by icant effect on bone density preservation, and showing private funding that diverts the focus of investigators essentially no effect on serum 25(OH)D either [50]. who are able to do such studies. Second, an optimized dose of vitamin D has never been established for adults. Therefore, “plain” vitamin D sometimes compares III. OVERVIEW OF THE SYSTEM poorly with 1,25(OH)2D and its analogs, whose dose is more thoroughly optimized [17], and whose dose is OF VITAMIN D METABOLISM, usually designed to be very close to the point where it AND ITS REGULATION could cause hypercalcemia. Optimal doses of vitamin D probably vary, depending on the indication, so that one Administration of vitamin D is unusual in pharma- dose may not always be optimal. Third, the official mis- cology or in endocrinology, because this molecule is representation that vitamin D2 and vitamin D3 are equal two metabolic steps away from the biologically active has resulted in efficacy studies at higher doses that agent, 1,25(OH)2D. Furthermore, the laboratory test to usually involve vitamin D2 because high-dose com- monitor dose is the concentration of a metabolite, mercial preparations of vitamin D are comprised of 25(OH)D, and not the compound administered. As a way this. One example of this is worth looking at whether to provide a conceptual model for metabolic regulation in

A B Low input of High/normal input of Cholecalciferol from diet or UVB Legend Cholecalciferol from diet or UVB Legend Metabolite Liver mitochondrial vit D- 1 Liver mitochondrial vit D- “compartment” 25-hydroxylase Metabolite 1 25-hydroxylase 2 Liver microsomal vit D-25- “compartment” Vitamin D -> hydroxylase 2 Liver microsomal vit D-25- 3 12 Vitamin D - > 3 12 hydroxylase 3 Renal 25(OH)D-1- Renal 25(OH)D-1- 25(OH)D -> hydroxylase 3 4 5 4 Tissue (non-renal) hydroxylase 3 25(OH)D- > 25(OH)D-1-hydroxylase 4 5 4 Tissue (non-renal) 5 Renal mitochondrial 3 25(OH)D-1-hydroxylase Within Tissues 25(OH)D-24-hydroxylase 5 Renal mitochondrial 1,25(OH) D -> 2 Possessing In 25(OH)D-24-hydroxylase 1-OHase Plasma 6 Non-renal 1,25(OH)2D-24- 6 hydroxylase 1,25(OH) D -> Within 2 Tissues In 6 Non-renal 1,25(OH)2D-24- A “unregulated” step in the Possessing Plasma hydroxylase flow of metabolism 1-OHase 6 24,25(OH)2D An regulated step in the flow of An “unregulated” step in the and catabolism- > metabolism flow of metabolism 7 Catabolism and excretion Regulated step in the flow of 24,25(OH) D metabolism 2 -> and catabolism 7 Catabolism and excretion

FIGURE 2 (A) Metabolism of vitamin D under conditions of low vitamin D supply. The vessels represent metabolic compartments, stages in the metabolism of vitamin D. The height of the shaded portion of each vessel represents the relative concentration of each metabolite indicated in the figure. This figure illustrates the concept that vitamin D metabolism in vivo functions below its Km, i.e., the system behaves according to the first-order reaction kinetics. Just as the flow of water through a hole in a pail reflects the height of water in that pail, the rates of metabolism in the vitamin D system reflect the concentration of precursor at each step. Open pas- sages represent steps in metabolism in which the pertinent enzymes are relatively unregulated. Valves represent steps in metabolism in which there is regulation of flow at the enzyme (this regulation is usually through changes in the amount of enzyme protein in spe- cific tissues, and not allosteric). When vitamin D supplies are low, the flow of 25(OH)D through other potential pathways is com- promised to maintain the circulating concentration of 1,25(OH)2D at the level determined by the priority requirements of bone and mineral metabolism. (B) Metabolism of vitamin D under conditions of adequate vitamin D supply. When vitamin D supplies are ade- quate, the flow of 25(OH)D through other potential pathways, including its utilization by peripheral tissues for paracrine regulation, is no longer compromised. Higher 25(OH)D concentration makes available routes of metabolism other than the one path needed for bone and mineral metabolism. Furthermore, a higher supply of vitamin D leads to an upregulation of 24-hydroxylase and the catabolic path- ways associated with it; this accelerates rate of metabolic clearance and metabolite turnover in each compartment. CHAPTER 61 The Pharmacology of Vitamin D, Including Fortification Strategies 1001 this system, Fig. 2 illustrates the metabolite “compart- The vitamin D system entails endocrine, paracrine, ments” occupied by vitamin D after ingestion or expo- and autocrine functions that could have never been sure to sunshine. I will show later in this chapter that optimized to cope with the lack of vitamin D created less than 25 percent of vitamin D that enters the body by our modern culture of clothing and sun-avoidance. actually becomes 25(OH)D. More than 75 percent of Inadequate supplies of vitamin D limit the local con- vitamin D entering the circulation bypasses what we trol that many tissues need so that they can function recognize as the vitamin D endocrine system. Instead, properly. The question is now being raised, whether most vitamin D entering the circulation is excreted the modern, “normal” prevalence of some of the dis- and/or metabolized by other routes not shown here, and eases listed in Table II could be reduced substantially if most likely, excreted into the bile. we were to increase our intakes of vitamin D [56Ð59]. Figure 2 consists of two panels to illustrate the Control of metabolism in the vitamin D endocrine metabolic adaptations that exist so that the vitamin D system is very different from the way other steroid endocrine system can accommodate to a wide range in hormones are regulated. For conventional steroid the substrate concentration. The vitamin D system is opti- hormones, the concentration of substrate (cholesterol) mized to maintain plasma 1,25(OH)2D levels according is far higher than the substrate in the vitamin D system. to the requirements of calcium homeostasis. The earliest Figure 3 illustrates the effective in-vivo Km of 1-hydroxy- compromise to progressive restriction in vitamin D lase, in relation to the physiological concentration range supply is probably a diminished capacity of nonrenal tis- of its substrate. Our circulating cholesterol concentra- sues to produce 1,25(OH)2D. Kidney possesses megalin, tion is in the order of 5 million nmol/liter; in contrast, which facilitates substrate access [51] (see Chapter 10). 25(OH)D typically circulates at less than 200 nmol/liter. Since few nonrenal tissues possess megalin, their activ- Cholesterol concentration is not a rate-limiting aspect ity of existing 1-hydroxylase depends primarily on the of the body’s capacity to generate steroid hormones; 25(OH)D concentration [52Ð55]. This results in a com- however, 25(OH)D concentration is absolutely rate- promise at nonrenal tissues when 25(OH)D levels limiting for 1,25(OH)2D production. are low. This is illustrated in Figure 2 by the greater height of one of the valves (number 4 in the figure) rep- resenting nonrenal 1-hydroxylase on the “pail” (Fig. 2A Two ways to increase production of hormone (1,25(OH)2D versus Steroid hormones) vs 2B) that represents the 25(OH)D compartment. If one looks at the system of vitamin D metabolism (1) More 25(OH)D [= D nutrition] or (2) More enzyme (Vmax) in Fig. 2 from the perspective of a system designed to Vmax adapt to various inputs of the nutrient, it becomes clear

that this is a system better designed to cope with an D abundance of supply, not a lack of it. First, the flow of 2 (2) vitamin D toward 25(OH)D is remarkably ineffi- Physiological More cient—most vitamin D entering the body never range of Km enzyme 25(OH)D (Vmax) becomes 25(OH)D. Second, there is no way to correct synthesis for deficiency of vitamin D, other than to redirect uti-

Rate of 1,25(OH) Concentration lization of 25(OH)D toward 1,25(OH)2D production, 0 300 nmol/L 5,000,000 which is the pathway most acutely important for life. (1) More (Physiological cholesterol substrate That is, when supplies of vitamin D are severely concentration) restricted, its metabolism is directed only toward the Mass action maintenance of calcium homeostasis. To expand on the FIGURE 3 The difference in enzyme kinetics between the vitamin point that the system of vitamin D metabolism is effec- D endocrine system and the substrate supply for conventional steroid tively designed for adjusting for higher inputs, not lower hormone systems based on cholesterol. The purpose of this figure is inputs, I offer the example of an air-conditioner system. to emphasize that the range of physiologic concentration of 25(OH)D Air conditioners are designed to compensate for exces- in mammals is less than the Michaelis-Menten constant (Km) of 1-hydroxylase that has been characterized in vitro [138] and in vivo sive heat, but they are a useless way to compensate for [55]. There are two ways to improve capacity for 1,25(OH)2D pro- a cold environment. Human vitamin D metabolism duction at kidney, and at peripheral tissues: provide more substrate, or was effectively designed through evolution and natural increase 1-hydroxylase content of the tissue. This is fundamentally selection for people in an environment without clothing, different from the situation relevant to every other part of the and living at equatorial latitudes where UVB intensity endocrine system. No other hormone is so dependent on the arbitrary, external supply of its structural raw material. The concept of a mass- is always enough to produce a relative abundance of action relationship for 1,25(OH)2D production is the basis of the vitamin D. In contrast, most modern humans cover argument that operation of paracrine control systems dependent on close to 95 percent of skin surface and avoid sunshine. vitamin D supply can be improved by improving vitamin D nutrition. 1002 REINHOLD VIETH

In the acute situation, before adjustments can be carrier proteins. Sex-steroid binding globulin, and gluco- made to 24-hydroxylase and catabolic pathways (before corticoid binding globulin each circulate at concen- the “valves” in Figure 2 can be adjusted), the in vivo trations of about 150 nmol/L, in the same order of production of 1,25(OH)2D is directly proportional to magnitude as their ligands [66]; in contrast, the concen- circulating 25(OH)D concentration. In rats, the acute tration of vitamin D binding protein is 4700 nmol/L [67]; injection of 25(OH)D into the circulation produces a this represents a 50-fold excess over its vitamin D- rapid, transient increase in 1,25(OH)2D, directly propor- derived ligands (see Chapters 8Ð9). tional to the percentage increase in 25(OH)D [55,60]. The dynamics of 25(OH)D in tissues are remark- Since in vivo concentrations of 25(OH)D change able. Its carrier protein, DBP, is cleared from plasma slowly, over many months, this first-order relationship with a half-life of 1.7 d, which is shorter than the 5-day between 25(OH)D and 1,25(OH)2D is not normally half life of albumin [68]. Within one hour after injec- seen in adults [22]. However, in situations where tion of radiolabeled DBP, the radiolabel is present in a 1-hydroxylase is tonically stimulated, either because greater concentration than in plasma, within kidney, of primary hyperparathyroidism [61] or in granuloma- liver, skeletal muscle, heart, lung, intestine, testis, and tous disease [62,63], modest increases in vitamin D bone [68]. In contrast to DBP, its ligand, 25(OH)D, is supply will raise plasma 1,25(OH)2D concentration cleared slowly from the body, with a half-life of about and aggravate hypercalcemia. 10 days in both rabbit [68] and human [69]. The pool The model of regulation represented by Figure 2 may of DBP outside plasma is double the size of the help to explain the U-shaped risk curve for prostate can- intravascular DBP pool, and the molar replacement rate cer vs 25(OH)D for men in Nordic countries. Tuohimaa of DBP is reported to be 1,350-fold higher than that of et al. reported the fascinating observation that in north- 25(OH)D. The binding of 25(OH)D to DBP does not ern countries, the narrow range of 25(OH)D levels affect the turnover or tissue uptake of DBP [68]. between 40Ð60 nmol/L coincides with the lowest risk As a short summary of the preceding, the DBP of prostate cancer [64]. Although this topic is covered and/or DBP-25(OH)D complex is removed from in other chapters of this book (Schwartz and Chen, plasma by a variety of tissues. The DBP is degraded Chapter 88; Feldman et al., Chapter 93), I propose during this process, and most 25(OH)D released a hypothesis that a falling 25(OH)D concentration is within those tissues is recycled. The molar excess of a nonÐsteady-state situation during which it may not DBP to 25(OH)D in plasma and the relatively rapid be possible to sustain 1,25(OH)2D at its long-term set- turnover of DBP indicate that a high capacity, high point at tissues like the prostate that produce autocrine affinity, and dynamic transport mechanism for vitamin 1,25(OH)2D. High 25(OH)D concentrations may not D sterols exists in plasma. Although 25(OH)D released be problematic per se, but those Nordic men with the into cells because of the metabolic clearance of DBP is highest summertime 25(OH)D levels should be recycled, the clearance of DBP provides ready access expected to suffer the greatest decline in 25(OH)D dur- to vitamin D, 25(OH)D and its metabolites to the liver ing their long winters. This is based on a longitudinal, and kidney. These are the two organs most involved in seasonal study of men in the USA [65]. During winter, the clearance of DBP, and the two organs central to the steadily falling 25(OH)D concentrations create a need endocrine function of the vitamin D system. for prostate tissue to continuously increase the ratio Recent new knowledge of the megalin/cubulin of 1-hydroxylase and 24-hydroxylase so that optimal system has shed light on the mechanisms of DBP- setpoint concentrations of tissue 1,25(OH)2D can be tissue interactions and tissue-specific uptake of DBP sustained. This hypothesis of a suboptimal setpoint (chapters 8Ð10) [70]. Megalin and cubulin are cell- during winter is not unlike what is experienced by a per- surface, endocytic receptors, members of the low-density son taking a shower as the supply of hot water is running lipoprotein receptor gene family. These help to regu- out; he or she must keep adjusting faucets to maintain late the concentration of ligands in the extracellular water temperature—not a comfortable process. The fluids and deliver metabolites to cells in need of these hypothesis predicts that the U-shaped risk curve for metabolites [71]. Differences in tissue distribution of prostate cancer is distinct to high latitudes where winters these cell-surface proteins will affect the accessibility produce prolonged, gradual declines in 25(OH)D levels. of different tissues to circulating 25(OH)D.

IV. DOSAGE CONSIDERATIONS A. Role of Vitamin D Binding Protein (DBP) A. Infants Differences between steroid hormones and the vitamin D system are amplified further by the large Cholecalciferol, or vitamin D3, given in the form of differences in concentrations of their respective plasma cod liver oil, has been a folk remedy in northern CHAPTER 61 The Pharmacology of Vitamin D, Including Fortification Strategies 1003

Europe since the 1700s [72]. Empirically, a small tea- prevent rickets and osteomalacia would have been the spoonful daily was thought to help infants thrive. This vast majority in any region. Survival depended on arbitrary dose of cod-liver oil has turned out to be a adequacy of vitamin D nutrition, and at latitudes away good guess, so far as infants are concerned. The 375 IU from the equator, natural selection for lighter skin color (9 µg) of vitamin D3 contained in that teaspoon [73] helped to ensure adequacy for the quality of pelvis was confirmed relatively recently as being appropriate needed for vaginal birth. for infants [74,75]. A French study utilizing vitamin D2 In the 1960s, an expert committee on vitamin D could concluded that neonates might need somewhat more, provide only anecdotal support for “the hypothesis of 1000 IU [76]. If safety of vitamin D during infancy is a small requirement” for vitamin D in adults and a concern, it should be kept in mind, that until the late recommended one-half the infant dose, to ensure that 1960s, the recommended amount of vitamin D for adults obtain some from the diet [79]. Despite the new infants in Finland was 2000 IU/day (50 µg/day). A large knowledge uncovered since that time, dietary vitamin D epidemiologic study suggests that this higher dose recommendations for adults have remained very lowered risk of juvenile diabetes before age 30 years, conservative, and still derive from amounts established by 85% compared to people not receiving vitamin D as for neonates. In contrast to the way decisions are made infants [46]. about the dose of any new drug, recommendations for Compared to the adult, vitamin D nutrition in the vitamin D have been arbitrary, because there was no infant and child has been well characterized, and it is firm evidence on which to base decisions. However, even the focus of Chapters 48,49,65. There is also an excellent though the evidence about the effects of vitamin D review of the field available by Chesney [77]. The pres- dosages on adult health have become characterized sci- ent chapter focuses on the pharmacology of vitamin D in entifically, those with the final say in setting official the adult. nutrient guidelines (not the experts they consulted) con- tinued to focus on lower doses of vitamin D than had been shown effective in the fracture prevention trials dis- B. Adults cussed previously. The revised recommendations were referred to as the “adequate intake” (AI), because there Until it became clear that vitamin D was important was no published evidence of efficacy for them [2,47]. to the health of adults, there was very little thought The objective measure of vitamin D nutritional sta- directed at how much vitamin D adults might need to tus is the 25-hydroxyvitamin D (25(OH)D) concentra- consume. Until recently, there has been no consensus tion in serum or plasma [2]. The consensus on this point about what the objective criteria should be for appro- has made it possible for researchers to focus on a mea- priate vitamin D nutrition. In England, an adult recom- surable target when it comes to vitamin D nutrition. mendation of 2.5 µg (100 IU)/day was established Table III summarizes two views of the relationships simply because 7 women with severe nutritional osteo- between long-term vitamin D intakes and the anticipated malacia showed a response to this amount [78]. range of 25(OH)D concentration associated with them. Interestingly, the oils of different fish contain differ- Figure 4 is a dose-response curve to showing the ent amounts of vitamin D. For example, a teaspoon full final average 25(OH)D concentrations attained in stud- of halibut liver oil contains twice as much vitamin D3 ies reported in the literature [3,56]. Table IV summa- as does cod liver oil. If it had been halibut liver oil used rizes incremental responses to different treatment in the past, recommendations for vitamin D supple- strategies to raise 25(OH)D to steady-state concentra- mentation could well have been double what they have tions. Responsiveness to vitamin D administration, been through most of the last century. as measured by the nmol per liter increase per mcg con- Into the 1960s, the absence of overt rickets or osteo- sumption per day, increases with: a) lower vitamin D malacia was the only criterion that vitamin D nutrition dosage, b) lower initial 25(OH)D concentration; c) longer was adequate [79]. By that same criterion of bone duration of supplementation, suggesting a long half-life deformity, anthropologists consider vitamin D nutrition and time to plateau. to have been a relatively minor problem for ancient The conventional approach to improving vitamin D populations. The lack of evidence of bone deformity in nutritional status has been to give either vitamin D3 ancient populations is now explained by the new concept or vitamin D2 (ergocalciferol). Until recently, avail- that the lack of vitamin D resulted in a natural selection ability of 25(OH)D was another option (supply of for white skin color to prevent rickets and osteomalacia this product has been discontinued by Organon, NJ, within defined environments [80]. Women with osteo- USA). The company’s discontinuation of 25(OH)D malacia would have produced few offspring because may have made sense, because the objective of increas- rickets and osteomalacia produce a misshapen pelvis. ing plasma 25(OH)D concentrations can be almost Those women able to produce enough vitamin D to as easily achieved by providing enough vitamin D3. 1004 REINHOLD VIETH

TABLE III The Clinical Interpretation of Serum 25(OH)D Levels and the Estimated Intakes of Vitamin D Needed to Ensure These Levels (1 µg = 40 IU)

Desirable Insufficiency (suppress PTH, Toxic/Therapeutic Deficiency (increased PTH optimize (might increase (rickets and secretion, calcium urine and osteomalacia) osteoporosis) Sufficiency absorption) serum calcium)

Serum 25(OH)D nmol/L 0Ð25 25Ð40 40Ð100 75Ð160 >220 Serum 25(OH)D ng/mL 0Ð10 10Ð16 16Ð40 30Ð64 >88

Daily intake of Vitamin D3 per day needed to reach the 25(OH)D above: Food and Nutrition Boarda 0 mcg 5Ð10 mcg 5Ð15 mcg not stated ≥95 mcg (200Ð400 IU) (200Ð600 IU) (3800 IU) From the literature reviewedb 0Ð5 µg 10Ð15 25Ð100 µg 100Ð250 µg >1000 mcg (200 IU) (400Ð600 IU) (1000Ð4000 IU) (4000Ð10,000 IU) (>40000 IU)

aImplications drawn from current National Academy of Sciences nutritional guidelines that the stated intake will deliver the level of adequacy, i.e. 25(OH)D concentration indicated [2]. The “adequate intake” recommendations for vitamin D vary according to age: adults < 50, 5 mcg/day; 50Ð70 years, 10 mcg/day; > 70 Years, 15 mcg/day. There is no RDA for vitamin D. bBased on literature [3,5,118,135].

Nonetheless, useful perspectives can be gained from However, when vitamin D3 is used, the increment in previous experience with 25(OH)D. Barger-Lux and 25(OH)D per mcg per day of vitamin D3 decreases as Heaney et al. have shown that as 25(OH)D dosage the dose increases. increases, there is effectively a linear increase in the Since the increase in plasma 25(OH)D concentration average 25(OH)D concentration achieved (Table IV). per mcg dose is at least four times higher for 25(OH)D

TABLE IV Strategies to Increase Circulating 25(OH)D Concentration in Adults: Effects of Compound, Dose, and Duration1

25(OH)D nmol/L Duration Absolute increase increase DOSE of dose in 25(OH)D Compound per µg/day µg/day wks nmol/L Reference

25(OH)D3 4.1 50 4 206.4 [119]

25(OH)D3 4.0 10 4 40 [119]

25(OH)D3 3.8 20 4 76.1 [119] Cholecalciferol 1.5 15 52 22 [159] Cholecalciferol 1.4 20 8 27 [160] Cholecalciferol 1.1 25 8 28.6 [119] Cholecalciferol 1.1 21 20 23.4 [135] Cholecalciferol 0.8 100 52 81 [159] Cholecalciferol 0.8 25 20 19 [118] Cholecalciferol 0.7 138 20 102.7 [135] Cholecalciferol 0.6 275 20 169.8 [135] Cholecalciferol 0.6 250 8 146 [119] Cholecalciferol 0.5 100 20 51.8 [118] Cholecalciferol 0.5 1250 8 643 [119] Ergocalciferol 0.3 36 104 [50]

1The results in this table represent recent work not included in Figure 4. These data were assembled to permit comparison of efficacy dose of different strategies for increasing 25(OH)D concentration. The results are sorted in order of decreasing response to the dose, based on the nmol/L increase in 25(OH)D per mcg/day of oral doses used in these studies. These are studies done during winter and/or comparisons versus parallel control groups. CHAPTER 61 The Pharmacology of Vitamin D, Including Fortification Strategies 1005

administration than for vitamin D3 administration, we Nonetheless, vitamin D2 continues to be used clinically can conclude that fewer than 25 percent of vitamin D as if it is equivalent, since official guidelines [2] and molecules ever become 25(OH)D. At least three quarters pharmacopeas respond slowly to new evidence. of the molecules of vitamin D that enter the body are The presumption of equivalence is based on 60-year removed by some other fate. old studies of rickets prevention in infants—evidence recognized as weak, even at the time [73,83]. The older rat data suggesting that vitamin D2 and vitamin D3 were C. The Case Against Ergocalciferol, Vitamin D2 equivalent lose their meaning when it is noted that the rat-line tests last done over 50 years ago were bioassays Vitamin D is available in two forms for nutritional to establish units for the quantity of vitamin D not readily supplementation, ergocalciferol (vitamin D2) and measured in any other way [84]. For a bioassay yielding cholecalciferol (vitamin D3). Vitamin D2 is manufactured “units,” equivalence is not the same thing as equivalence by exposing a fat extract of yeast to UV light. Since no per milligram or per mole. Furthermore, all species tested metabolite of vitamin D2 is normally detectable in the show differences between the vitamin D2 and vitamin D3 blood of humans or primates [81,82], I contend that [85,86]. Despite these obvious problems about units, this should be regarded as a drug, and not a physiolog- the very conservative approach used by those who ical compound. The present discussion focuses on frame official statements about nutrients has remained vitamin D3 cholecalciferol, the form of vitamin D unchanged, that one international unit of vitamin D naturally present in mammals. Vitamin D3 (from here is equivalent to 25 nanograms of either vitamin D2 or on, vitamin D) is the more potent form of vitamin D vitamin D3 [2,84]. In Australia, vitamin D3 has never in all primate species and in man [81,82]. Comparisons been licensed for use, and the only nutritional form between the two versions of vitamin D [82], and of vitamin D available is vitamin D2. the meta-analysis of effects on 25(OH)D (Fig. 4) indi- I have summarized the differences between vitamin D2 cate that vitamin D3 is about 4 times as potent as and vitamin D3 in Table V. Based on the many major vitamin D2, i.e., 1 µg of D3 = approximately 4 µg of D2. differences between the two, it is clear that unless there is some well-characterized reason to favor vitamin D2 (I am not aware of any), all use of vitamin D for nutri- Vitamin D intake IU/day tional and clinical purposes should in my opinion specify 400 4000 40000 400000 cholecalciferol, vitamin D3. Study group mean data

Vit D2-Treated group mean data Individuals, Vit D Hypercalcemia 1000 D. UVB Light on Human Skin as a Dose of Vitamin D

In any discussion of vitamin D pharmacology or dosage, it would be a major oversight to ignore the role of sunshine, particularly its UVB component. As 100 described elsewhere in this book (Chapter 3), the syn- thesis of vitamin D is a self-limiting reaction, reaching

Serum or plasma 25(OH)D nmol/L an equilibrium after 20Ð25 min of summer UVB expo- sure for people with white skin, and producing no net increase in vitamin D production after that [87]. Darker 10 100 1000 1E4 Vitamin D intake µg/day skin requires longer exposure, but the potential yield of vitamin D is the same. Exposure of full skin surface to FIGURE 4 Dose-response relationship between daily vitamin D UVB light, in an amount less than erythemal, is equiv- intake and mean 25(OH)D concentration, based on data published alent to a vitamin D consumption of about 250 µg in the literature. The solid points show mean results for groups of adults consuming the indicated doses of vitamin D. Results for (10,000 IU)/day [88Ð91]. Lifeguards in the United groups of adults that are unambiguously consuming vitamin D2 are States and in Israel, as well as farmers in the Caribbean, shown by the circled points. Vitamin D3 is about 4 times as potent all exhibit serum 25(OH)D concentrations greater as vitamin D2, based on tracing the circled points for subjects con- than 100 nmol/L [92Ð94]. Furthermore, even regular suming vitamin D2 back to the trend-line based on vitamin D3. short periods in sun-tan parlors consistently raise Both axes are log scale. The results, represented by Xs, are for indi- viduals showing the classic hypercalcemic response to toxic levels serum 25(OH)D well beyond 80 nmol/L [42,95Ð100]. of prolonged vitamin D consumption. The data used to generate The highest 25(OH)D concentrations in the groups this graph were compiled and published previously [3,56]. of adults acquiring vitamin D physiologically (via UV 1006 REINHOLD VIETH

TABLE V The Case Against Vitamin D2, Compared to Vitamin D3

Vitamin D2 Vitamin D3 Ref.

Not detectable in humans or primates unless administered from The natural metabolite generated within [162] an external source skin and the oils of fur

Vitamin D binding protein has lower affinity for vitamin D2 than [163] for vitamin D3 and its metabolites

Generates metabolites for which there is no vitamin D3 equivalent [164] Microsomal 25-hydroxylase does not act on it Substrate for both microsomal and [165,166] mitochondrial 25-hydroxylases

Per mole of dose, 25(OH)D increases by less than with vitamin D3 [82]

The 25(OH)D response to vitamin D2 is less in the elderly than in 25(OH)D response to vitamin D3 is the [160,167] younger adults same for young vs older adults [159] All known cases of iatrogenic toxicity with vitamin D involved the All known adult cases of toxicity with [3,110] vitamin D2 form (albeit, formulations > 25 µg (1000 IU) vitamin D3 have been unintentional, [121] have usually been vitamin D2) “industrial” accidents [109] Dose preparations are less stable [71,82]

exposure) range up to 235 nmol/L [42,92], and none of because of the technical issue of measuring the these studies imply that such 25(OH)D levels have nanomolar quantities of vitamin D potentially embed- caused hypercalcemia. Since humans evolved as naked ded within tissues or excreted in catabolized forms. It apes, whose native habitat was within 30 degrees lati- is extremely difficult to detect or to measure vitamin D tude of the equator, I contend that our genome was and its metabolites when they exist among great selected under conditions of abundant vitamin D supply excesses of other lipids. Perhaps the most careful study [3]. As such, it is reasonable to think that the substan- into the fate of physiological amounts of cholecalciferol tially lower levels of 25(OH)D prevalent among mod- was reported by Lawson et al. [101,102]. They exposed ern humans have been accompanied by biological rats with shaved skin to ultraviolet light (UVB), and compromises, such as increased PTH secretion [22] measured vitamin D and 25(OH)D in tissues at various and altered cellular metabolism [26]. By now, these times afterwards. Although adipose tissue concentra- compromises may have been detrimental to the health tion of vitamin D was never greater than the plasma of modern humans for so long, that we are no longer in concentration, it contained the largest exchangeable a position to realize them. pool of vitamin D. Recovery of vitamin D3 in adipose Barger-Lux and Heaney studied the effect of sun- was less than 5 percent of the amount produced within shine on healthy outdoor workers in the US Midwest, the skin [101], and this low recovery was attributed to relating it to the vitamin D intakes needed to bring vitamin D excretion into the bile. Lawson et al esti- about the 25(OH)D levels observed [65]. They con- mated that the volume of distribution of unmetabolized cluded that for these men, the summertime supply of vitamin D3 was approximately four liters per kg (based vitamin D from sunshine was approximately 70 µg on concentration decay curves from plasma and total (2800 IU)/day. This supply during summer did not amounts recovered from tissues). Vitamin D is not ensure sufficiency through the winter, when 25(OH)D detectable in the adipose tissue of normal rats [102,103], fell to less than 50 nmol/liter in 3 of 26 subjects and but with administration of pharmacologic doses [104], or less than 75 nmol/liter in 15 of 26 subjects. shaving of fur to increase yield fivefold [101], vitamin D is detectable. Brouwer et al estimate the half-life of vitamin D in rat adipose tissue to be 96 days, which is V. PHARMACOKINETIC PRINCIPLES, plausible because it compares with the functional half- life of 25(OH)D in humans [3]. In contrast, Lawson VOLUME OF DISTRIBUTION, et al estimated the vitamin D in rat adipose tissue to TURNOVER AND HALF-LIFE AS have a half-life of 13.8 days [101]. The more rapid IT PERTAINS TO VITAMIN D half-life reported by Lawson et al was likely due to the younger age of the rats. A complete understanding of the pharmacokinetics Pharmacokinetic studies are extremely difficult with of the vitamin D system has eluded researchers. This is vitamin D, since unlike a drug, vitamin D is present in CHAPTER 61 The Pharmacology of Vitamin D, Including Fortification Strategies 1007 the body naturally. It is impossible to start with com- adipose tissue [116,117]. Two studies showed that fol- pletely deprived individuals to do appropriate studies lowing a defined dose of vitamin D or sunshine, the of pharmacokinetics, and the half-life is exceptionally rise in 25(OH)D was less for obese individuals than for long. Furthermore, the component of nutritional interest people who weighed less. These studies did not show is 25(OH)D, a metabolite of either vitamin D2 or vita- that adipose tissue concentrated vitamin D, and they min D3. Studies using isotopic techniques show that in failed to account for the obvious effect of a larger body humans, molecules of 25(OH)D have a plasma half-life compartment size, which should produce a lower con- of about 10 days [69,105]. However, a more practical centration of anything, regardless of whether adipose measure of the half-life of 25(OH)D is reflected in the plays a role or not. In our study using vitamin D3 doses rate at which 25(OH)D concentrations decline upon of 100 µg/day in adults, we found no correlation between the sudden elimination of sources of vitamin D (acute weight and serum 25(OH)D [118]. At physiological deprivation of ultraviolet light). Two studies show that doses, cholecalciferol (unmetabolized vitamin D3) dis- when sailors embark upon two-month-long missions tributes widely into tissues, not just to adipose, but to in submarines, the 25(OH)D concentration decreases by skeletal muscle and other organs as well [101,115]. As approximately 50 percent [3,106,107]. Follow-up of stated above, turnover of vitamin D stored in tissues 25(OH)D concentrations in adults who have been intox- produces a long half-life of about two months. icated with vitamin D3 suggest that the functional in vivo The amounts of vitamin D recoverable from tissue half-life is of the order of several months [108Ð110]. stores account for only a fraction of the dose adminis- During summer, we can accumulate and store vita- tered [101]. The animal data indicate that more than min D well enough so that supplies for vitamin D do 75% of the molecules of vitamin D that enter the body not become completely depleted during the winter are catabolized and excreted without ever being stored months. Within three days of a dose of vitamin D, very in tissues, and without ever becoming 25(OH)D. The little of the original vitamin D is detectable in the human data also support this. In humans, when vitamin D plasma of rats [111] or humans [112]. Most vitamin D or 25(OH)D are given over the long-term, to achieve initially entering the circulation appears to be excreted an equilibrium concentration of 25(OH)D, it takes more into the bile. The highest total concentrations of vita- than 4 times as much vitamin D to produce the same min D and its metabolites occur in plasma. However, 25(OH)D plateau [119]. By definition, at that plateau since plasma represents only 2.5% of body mass, in 25(OH)D, exchange of vitamin D with tissues is at larger pools of vitamin D and 25(OH)D exist in fat and equilibrium where release of stored vitamin D equals muscle [113Ð115]. storage of new vitamin D. Still the fourfold difference When there is a continuous supply of vitamin D, like in efficacy at sustaining 25(OH)D exists when com- the situation for people who regularly expose a large paring effects of doses of 25(OH)D and vitamin D. proportion of their skin surface to tropical sunshine, the That is, the difference in efficacy between 25(OH)D equilibrium state maintains a balance between vitamin and vitamin D at sustaining plasma 25(OH)D concen- D stored within body compartments and the removal trations cannot be explained by deposition of vitamin from tissue stores for metabolism and clearance. Under D into storage sites. The difference in efficacy at these physiologic, sun-derived circumstances, 25(OH)D sustaining 25(OH)D can only be explained by the loss concentrations in plasma sustain levels of more than of vitamin D entering the circulation to fates other than 200 nmol/L [3]. At these levels of vitamin D nutrition, 25-hydroxylation or storage. there has never been a concern raised that sudden loss of adipose tissue would either raise 25(OH)D or predispose to vitamin D toxicity. Likewise, despite 70 years of VI. VITAMIN D TOXICITY experience with the oral use of vitamin D in amounts AND SAFETY ISSUES that exceed the amounts tenable through sun exposure, there has never been a report of an eventual, sudden Amounts of vitamin D substantially greater than excess of vitamin D caused by release from adipose physiologic amounts >250 µg/day (>10,000 IU/day) stores because of weight loss. are toxic because they saturate circulating vitamin D binding protein (DBP), and they force the percent of vitamin D that is free and unbound to increase [3,120]. A. Body Storage of Vitamin D and Inefficient At toxic doses, the freely circulating vitamin D, along Conversion to 25(OH)D with its metabolites, accumulate in adipose [104] and muscle [115]. The 100 µg (4000 IU)/day of vitamin D It is thought that since vitamin D is a fat-soluble we have used in adults is physiologic and far below vitamin, it must show preferential accumulation in what would be needed to change the free fraction of 1008 REINHOLD VIETH vitamin D or its circulating metabolites [67]. The average even if modestly, to the vitamin D receptor to trigger a capacity of human plasma DBP to bind vitamin D and response as part of the mechanisms for toxicity [123]. its metabolites is 4700 nmol/L [67], and this exceeds We recently reported a safety evaluation of vitamin D3 by 20 times the physiologic total concentration of its supplementation of normal adults, involving daily con- vitamin DÐderived ligands. sumption of 100 µg (4,000 IU). Contrary to the hyper- The vast majority of cases of vitamin D intoxication calcemia in normal adults reported by Narang [124], have involved vitamin D2 [3]. The situations involving and which was used by the Food and Nutrition Board vitamin D3 to date, have been industrial accidents to establish the 50 µg/d (2,000 IU/day) upper limit for [109,120,121] or poisonings from an unknown source vitamin D intake, 100 µg (4000 IU)/day produced no [110]. In our case, we assayed blood levels of vitamin D detectable change in serum or urine calcium in more and its metabolites by chromatography and found that rigorous studies [118,125]. despite record-high 25(OH)D concentrations in humans The official safety limit for vitamin D intake with- (2,400 nmol/liter), they were still small in comparison out supervision by a physician is referred to as the to a large excess of vitamin D3 (17,000 nmol/liter), sug- “upper limit” (UL) [125,126]. This is the amount of gesting that the capacity of the liver to hydroxylate vitamin D that the general public can take safely on vitamin D is limited [110]. a long-term basis with no anticipation of harm. Like anything that has an effect on living things, Guidelines in both North America [2] and Europe [127] vitamin D can be harmful if taken in excess. I contend have established the UL as 50 µg (2000 IU)/day. This that the ratio of the physiologically effective dose vs. is a very conservative value that seems to remain con- the toxic level for vitamin D is similar to the safety stant, despite accumulating evidence to show that higher margin of many other nutrients (including even water). intakes are safe. The value of 50 µg (2000 IU)/day has The reason vitamin D has been perceived as toxic was remained unchanged since it was mentioned in the probably because daily ingestion in the milligram 1968 Recommended Dietary Allowance publication as (>1000 µg) range has caused harm. In contrast, mil- a dose approaching a harmful level [128]. To sustain ligram amounts of other nutrients are benign. Toxicity the very conservative approach of making minimal in normal adults requires intake of more than 1,000 µg changes to past recommendations, the only thing to (40,000 IU)/day, which reflects amounts of vitamin D change over the years has been the safety margin applied that are four times more than the 250 µg (10,000 IU)/day to the evidence. For example, when the “no observed that can be acquired naturally by sunshine [3]. In what adverse effect level” (the highest dose shown to have no I see as an overreaction to the potential for toxicity harmful effect) was 2400 IU/day, based on the Narang with vitamin D, the current recommendation of 5 µg study [124], the safety factor applied by the United (200 IU)/day (called an “Adequate Intake” in North States food and nutrition Board was 1.2. When subse- America) for adults under age 50 represents what can quent data were published indicating that 4000 IU/day only be regarded as a homeopathic dose—about 2% of was safe, the safety margin was increased by The what adults with white skin can make within 20 min of European Commission to a value of 2.0 [127]. Recent summer sun. In other words, the fear of vast excess has evidence in men shows that eight weeks of supple- resulted in physiologically miniscule intake recom- mentation with 275 µg (12,500 IU]/day of vitamin D mendations for adults. does not affect circulating calcium concentration (urine Concentrations of 1,25(OH)2D are not increased results were not reported) [129]. That is, the dose is much by vitamin D intoxication. This reflects the high non-hypercalcemic, and safe by the safety criterion level of regulation of this hormone via both its synthesis applied to drug studies of vitamin D analogs [130Ð132]. and catabolism. Nonetheless, vitamin D toxicity is Even with the application of a safety factor of 2.75, probably manifest by the excessive levels of “free” this would suggest that 100 µg (4000 IU) of vitamin D 1,25(OH)2D, displaced from its carrier protein, DBP, could be a safe adult UL for vitamin D. I predict that by the vast excess of other vitamin D metabolites [122]. past conservative patterns will remain for officially This excess was confirmed by studies looking into “free” mandated nutrition guidelines, and that the response to 1,25(OH)2D concentrations in vitamin D intoxicated evidence of greater safety of vitamin D will be to individuals [120]. This excess of metabolite over binding adjust safety factor for deriving the UL, so that the UL capacity was also confirmed by the high total of vita- can stay unchanged at 50 µg (2000 IU)/day until there min D and 25(OH)D concentrations (19,500 nmol/L) is solid research showing evidence of a need for higher in a patient intoxicated after consuming over a million intakes. units (>25000 µg) daily for many months [110]. It is The weight of published evidence on toxicity shows also likely that very high levels of 25(OH)D can bind, that the lowest dose of vitamin D proven to cause CHAPTER 61 The Pharmacology of Vitamin D, Including Fortification Strategies 1009 hypercalcemia in some healthy adults is 1,000 µg hypercalciuria, which would be a more sensitive indi- (40,000 IU)/day of the vitamin D2 form [3] (Fig. 4). cator of toxicity [118]. This translates to 1,000 micrograms, or 1 milligram, Ten years ago, a dairy in the Boston area, servicing taken daily for many months. If a consumer wanted to 10,000 households, made prolonged, gross errors in achieve this toxic dose, he or she would need to take 40 fortifying milk with several milligrams per quart. The of the 1000 unit pills (the highest dose available in case was published quickly [133] and covered by the North America without a prescription) every day for media. The more rigorous epidemiological follow-up many months. One must bear in mind that few studies was published later. That showed that the situation con- have looked at effects of high doses of vitamin D on tributed to two deaths of susceptible elderly people [121].

TABLE VI Opinions and Best Guesses at the Answers to Pharmacologic Questions that Need to be Addressed in Relation to Vitamin D Nutrition

Question Answer

1a. What is the indication for use of the compound? Prevention and cure of rickets and osteomalacia. Fracture prevention; preservation of bone mineral density; normalization of PTH levels 1b. What kind of clinical or health effects should Disease prevention: cancer, autoimmune conditions, diabetes, multiple we be looking for, based on preclinical animal sclerosis, high blood pressure, fibromyalgia, muscle strength, mood. and laboratory research? 2a. What are the most useful approaches to Encouragement to expose a large percent of skin surface to summertime increasing vitamin D status? sunshine, 10 min daily for white skin, up to 6 times longer for black skin. Fortification of foods to physiologically meaningful levels of vitamin D, consumption of supplement preparations with vitamin D. 2b. The dosage, route of administration, and Dosage depends on the target concentration of 25(OH)D desired. We can assume a rule of thumb, that a dose of 1 mcg/day vitamin D increases 25(OH)D by 1 nmol/L, after 8 months of use (See Table IV). In non-SI units, this is the equivalent to saying that 100 IU/day increases 25(OH)D by one ng/mL. Oral vitamin D is probably more effective than injection. 2c. Interval between doses? Since the half-life for decline in 25(OH)D is effectively 2 months, doses of vitamin D could be given monthly (we use weekly in our studies). Less frequent dosing than once every 2 months will generate large fluctuations in 25(OH)D concentrations that may not be desirable, because the enzymes involved in the regulation of 25(OH)D metabolism are functioning in a first-order relationship with substrate. 3. What is the desirable target for the plasma Current consensus points to a goal of ensuring that 25(OH)D levels concentration; what dose would be needed be higher than 70Ð100 nmol/L (28Ð40 ng/mL) [5]. This range reflects to attain or ensure this? average 25(OH)D levels seen in adults taking 25 µg (1,000 IU)/day vitamin D, and who are getting sunshine. To ensure this level for those of the normal population with the weakest response to vitamin D, we need to aim for an average 25(OH)D concentration of about 120 nmol/L (48 ng/mL). This objective requires an intake of 100 µg (4,000 IU)/day for all adults.1 4. What, if any, are the biological markers to Hypercalcemia is the classic criterion for toxicity of vitamin D, its monitor toxicity, and what are our criteria for metabolites, and their analogues. “Non-calcemic” doses are those that determining therapeutic effectiveness? do not cause hypercalcemia, and are considered “safe” by conventional What is the “therapeutic index”, the ratio criteria. However, the most sensitive clinical index of safety is urine between toxic vs beneficial dose levels? calcium. This is easily monitored by measuring a morning urine calcium/creatinine ratio (normal for this would be mmol/mmol < 1; or in non-SI units, mg/mg < 0.35) [118]. Of greater concern for the long-term use of vitamin D, its metabolites, or analogs, should be the effects on soft-tissue calcification, within aorta, kidney, or other tissues. These effects may be seen radiologically in humans, or by direct measure of tissue calcium in preclinical animal studies.

1Please note that this is not an official recommendation, but rather a scientific opinion offered by the author for research purposes. The dosage of vitamin D required for humans remains a controversial issue. 1010 REINHOLD VIETH

While hypercalcemia did occur, it was not widespread. one Kg of adipose tissue that had been primed by prior By far the most susceptible group to the excess vitamin D vitamin D intoxication, with daily adipose catabolism was women over age 65 years of age, suggesting that to continue for several weeks. When toxic doses of diminished renal function may play a role. The average vitamin D are administered, the effect will be manifest 25(OH)D concentration of the confirmed cases of during the period of administration. There is no evidence vitamin D toxicity was 900 nmol/L (214 ng/mL) [121]; that enough residual vitamin D can be stored in adipose in comparison, physiologically attained 25(OH)D con- tissue that vitamin D toxicity could possibly arise at centrations, obtained through sunshine exposure, can some later time, because of weight loss. reach 235 nmol/L safely, without hypercalcemia or hypercalciuria. When physiologically higher vitamin D nutrition is VII. SUMMARY associated with hypercalcemia, this reflects aberrant control of 25(OH)D-1-hydroxylase. This would reflect To conclude, I return to the pharmacological questions either primary hyperparathyroidism, where PTH con- posed at the start of this chapter, and offer Table VI as a tinuously stimulates the enzyme in the kidney [61], or way to address the issues, based on the material in this granulomatous disease, where peripheral tissue may chapter. not have the ability to regulate the 1-hydroxylase that normally serves autocrine/paracrine roles [3,134]. > In people with abundant sun exposure (25(OH)D References 150 nmol/L), the presupplement supply of vitamin D could be equivalent to about 100 µg (4000 IU)/day 1. Vieth R 2004 Why “Vitamin D” is not a hormone, and not a [65]. If such people were to take an additional dosage synonym for 1,25-dihydroxy-vitamin D, its analogs or by mouth of 100 µg/day of vitamin D, this would deltanoids. J Steroid Biochem Mol Biol 89Ð90:571Ð3 still be less than the dose of vitamin D shown to be safe 2. Standing Committee on the Scientific Evaluation of Dietary in a recent study [135]. Since long-term vitamin D Reference Intakes 1997 Dietary reference intakes: calcium, consumption of at least 1000 mcg/d would be needed to phosphorus, magnesium, vitamin D, and fluoride. National Academy Press. cause hypercalcemia, there is a large margin of safety 3. Vieth R 1999 Vitamin D supplementation, 25-hydroxy- with 100 µg (4000 IU)/day. vitamin D concentrations, and safety. Am J Clin Nutr 69(5): One concern sometimes expressed, is that if adipose 842Ð856. tissue were to break down, a sudden influx of vitamin D 4. Lips P 2004 Which circulating level of 25-hydroxyvitamin D from adipose might be toxic [136]. In both rats and cattle, is appropriate? J Steroid Biochem Mol Biol 89Ð90: 611Ð4. high doses of vitamin D are needed before vitamin D 5. Dawson-Hughes B, Heaney R, Lips P, Meunier P, Vieth R ends up as detectable in adipose tissue [104,115]. 2004 Vitamin D Round Table. In: Dawson-Hughes B, Despite being present in “significant” amounts in tissues, Heaney R, Burckhardt P eds. Nutritional Aspects of storage in tissues in not efficient. As a proportion of what Osteoporosis. Academic Press, New York. enters the body via the skin or the diet, the amounts of 6. Papadimitropoulos E, Wells G, Shea B et al 2002 Meta- analyses of therapies for postmenopausal osteoporosis. VIII: vitamin D stored in adipose are a fraction of the total. Meta-analysis of the efficacy of vitamin D treatment in pre- In normal humans, adipose tissue content of vitamin D venting osteoporosis in postmenopausal women. Endocr Rev has been reported to be as high as 116 ng/g (approx 23(4):560Ð569. 5 IU/g adipose) [102]. In cattle intoxicated with 7. Trivedi DP, Doll R, Khaw KT 2003 Effect of four monthly 7.5 million IU vitamin D (to cause hypercalcemia, in oral vitamin D3 (cholecalciferol) supplementation on frac- tures and mortality in men and women living in the commu- an experimental process to activate proteases to make nity: randomized double blind controlled trial. BMJ beef more tender after slaughter), muscle levels of 326:469Ð475. vitamin D reached 91 ng/g tissue (4 IU/g). The highest 8. Heikinheimo RJ, Inkovaara JA, Harju EJ et al. 1992 Annual tissue level reported in those animals was in the liver, injection of vitamin D and fractures of aged bones. Calcif which contained vitamin D at 979 ng/g (39 IU/g) [115]. Tissue Int 51(2):105Ð110. 9. Dawson-Hughes B, Dallal GE, Krall EA, Harris S, Sokoll LJ, The point is that while there is “meaningful” storage of Falconer G 1991 Effect of vitamin D supplementation on vitamin D in tissues, all the evidence indicates that wintertime and overall bone loss in healthy postmenopausal only a fraction of any vitamin D dose ends up in tissues women [see comments]. Annals of Internal Medicine to be withdrawn at later times. 115:505Ð512. If there were a sudden breakdown of 1 Kg of adipose 10. Chapuy MC, Arlot ME, Duboeuf F et al 2002 Vitamin D3 and µ calcium to prevent hip fractures in the elderly women. tissue, or liver, this would release as much as 979 g N Engl J Med 327(23):1637Ð1642. (39,000 IU) of vitamin D into the body. A toxic excess 11. Dawson-Hughes B, Harris SS, Krall EA, Dallal GE 1997 of vitamin D would require the breakdown daily of Effect of calcium and vitamin D supplementation on bone CHAPTER 61 The Pharmacology of Vitamin D, Including Fortification Strategies 1011

density in men and women 65 years of age or older. N Engl 29. Tangrea J, Helzlsouer K, Pietinen P et al 1997 Serum levels J Med 337(10):670Ð676. of vitamin D metabolites and the subsequent risk of colon and 12. Chapuy MC, Pamphile R, Paris E et al 2002 Combined cal- rectal cancer in Finnish men. Cancer Causes Control 8(4): cium and vitamin D3 supplementation in elderly women: 615Ð625. confirmation of reversal of secondary hyperparathyroidism 30. Garland CF, Garland FC, Gorham ED 1991 Can colon cancer and hip fracture risk: the Decalyos II study. Osteoporos Int incidence and death rates be reduced with calcium and 13(3):257Ð264. vitamin D? Am J Clin Nutr 54(1 Suppl):193S-201S. 13. Pfeifer M, Begerow B, Minne HW, Abrams C, Nachtigall D, 31. Emerson JC, Weiss NS 1992 Colorectal cancer and solar Hansen C 2000 Effects of a short-term vitamin D and radiation. Cancer Causes & Control 3:95Ð99. calcium supplementation on body sway and secondary 32. Schwartz GG 1992 Multiple sclerosis and prostate cancer: hyperparathyroidism in elderly women. J Bone Miner Res what do their similar geographies suggest? Neuroepidemiology 15(6):1113Ð1118. 11:244Ð254. 14. Bischoff HA, Stahelin HB, Dick W et al 2003 Effects of 33. Hanchette CL, Schwartz GG 1992 Geographic patterns of vitamin D and calcium supplementation on falls: a randomized prostate cancer mortality. Evidence for a protective effect of controlled trial. J Bone Miner Res 18(2):343Ð351. ultraviolet radiation. Cancer 70:2861Ð2869. 15. Janssen HC, Samson MM, Verhaar HJ 2002 Vitamin D defi- 34. Ainsleigh HG 1993 Beneficial effects of sun exposure ciency, muscle function, and falls in elderly people. Am J Clin on cancer mortality. [Review]. Preventive Medicine 22: Nutr 75(4):611Ð615. 132Ð140. 16. Feskanich D, Willett WC, Colditz GA 2003 Calcium, 35. Grant WB 2002 An estimate of premature cancer mortality in vitamin D, milk consumption, and hip fractures: a prospec- the U.S. due to inadequate doses of solar ultraviolet-B radia- tive study among postmenopausal women. Am J Clin Nutr tion. Cancer 94(6):1867Ð1875. 77(2):504Ð511. 36. Hayes CE 2000 Vitamin D: a natural inhibitor of multiple 17. Lau KW, Baylink DJ 1999 Vitamin D therapy of osteoporosis: sclerosis. Proc Nutr Soc 59(4):531Ð535. plain vitamin D therapy versus active vitamin D analog 37. McGrath J 2001 Does “imprinting” with low prenatal (D-hormone) therapy. Calcif Tissue Int 65(4):295Ð306. vitamin D contribute to the risk of various adult disorders? 18. Heaney RP 1999 Lessons for nutritional science from Med Hypotheses 56(3):367Ð371. vitamin D [editorial; comment]. Am J Clin Nutr 69(5):825Ð826. 38. Hayes CE, Cantorna MT, DeLuca HF 1997 Vitamin D and 19. Bouillon RA, Auwerx JH, Lissens WD, Pelemans WK 1987 multiple sclerosis. Proc Soc Exp Biol Med 216(1):21Ð27. Vitamin D status in the elderly: seasonal substrate deficiency 39. McAlindon TE, Felson DT, Zhang Y et al 1996 Relation of causes 1,25-dihydroxycholecalciferol deficiency. Am J Clin dietary intake and serum levels of vitamin D to progression Nutr 45(4):755Ð763. of osteoarthritis of the knee among participants in the 20. Himmelstein S, Clemens TL, Rubin A, Lindsay R 1990 Framingham Study. Ann Intern Med 125(5):353Ð359. Vitamin D supplementation in elderly nursing home residents 40. Lane NE, Gore LR, Cummings SR et al 1999 Serum vitamin D increases 25(OH)D but not 1,25(OH)2D. Am J Clin Nutr levels and incident changes of radiographic hip osteoarthritis: 52:701Ð706. a longitudinal study. Study of Osteoporotic Fractures 21. Landin-Wilhelmsen K, Wilhelmsen L, Wilske J et al 1995 Research Group. Arthritis Rheum 42(5):854Ð860. Sunlight increases serum 25(OH) vitamin D concentration 41. Rostand SG 1997 Ultraviolet light may contribute to geo- whereas 1,25(OH)2D3 is unaffected. Results from a general graphic and racial blood pressure differences. Hypertension population study in Goteborg, Sweden (The WHO MONICA 30(2 Pt 1):150Ð156. Project). Eur J Clin Nutr 49(6):400Ð407. 42. Krause R, Buhring M, Hopfenmuller W, Holick MF, 22. Vieth R, Ladak Y, Walfish PG 2003 Age-related changes in Sharma AM 1998 Ultraviolet B and blood pressure. Lancet the 25-hydroxyvitamin D versus parathyroid hormone rela- 352(9129):709Ð710. tionship suggest a different reason why older adults require 43. Pfeifer M, Begerow B, Minne HW, Nachtigall D, Hansen C more vitamin D. J Clin Endocrinol Metab 88(1):185Ð191. 2001 Effects of a short-term vitamin D3 and calcium 23. Ishimura E, Nishizawa Y, Inaba M et al 1999 Serum levels of supplementation on blood pressure and parathyroid hormone 1,25-dihydroxyvitamin D, 24,25-dihydroxyvitamin D, and levels in elderly women. J Clin Endocrinol Metab 86(4): 25-hydroxyvitamin D in nondialyzed patients with chronic 1633Ð1637. renal failure. Kidney Int 55(3):1019Ð1027. 44. McMurray DN, Bartow RA, Mintzer CL, Hernandez- 24. Zehnder D, Bland R, Williams MC et al 2001 Extrarenal Frontera E 1990 Micronutrient status and immune function in Expression of 25-Hydroxyvitamin D3-1alpha-Hydroxylase. J tuberculosis. Annals of the New York Academy of Sciences Clin Endocrinol Metab 86(2):888Ð894. 587:59Ð69. 25. Zineb R, Zhor B, Odile W, Marthe RR 1998 Distinct, tissue- 45. Muhe L, Lulseged S, Mason KE, Simoes EA 1997 Case- specific regulation of vitamin D receptor in the intestine, kidney, control study of the role of nutritional rickets in the risk of and skin by dietary calcium and vitamin D [In Process Citation]. developing pneumonia in Ethiopian children. Lancet Endocrinology 139(4):1844Ð1852. 349(9068):1801Ð1804. 26. Chen TC, Holick MF 2003 Vitamin D and prostate cancer 46. Hypponen E, Laara E, Reunanen A, Jarvelin MR, Virtanen SM prevention and treatment. Trends Endocrinol Metab 14(9): 2001 Intake of vitamin D and risk of type 1 diabetes: 423Ð430. a birth-cohort study. Lancet 358(9292):1500Ð1503. 27. Lefkowitz ES, Garland CF 1994 Sunlight, vitamin D, and 47. Vieth R, Fraser D 2002 Vitamin D insufficiency: no recom- ovarian cancer mortality rates in US women. Int J Epidemiol mended dietary allowance exists for this nutrient. CMAJ 23(6):1133Ð1136. 166(12):1541Ð1542. 28. Martinez ME, Giovannucci EL, Colditz GA et al 1996 48. Adachi JD, Bensen WG, Bianchi F et al 1996 Vitamin D and Calcium, vitamin D, and the occurrence of colorectal cancer calcium in the prevention of corticosteroid-induced osteo- among women. J Natl Cancer Inst 88(19):1375Ð1382. porosis: a 3-year followup. J Rheumatol 23(6):995Ð1000. 1012 REINHOLD VIETH

49. Adachi JD, Ioannidis G 1999 Calcium and vitamin D therapy 67. Vieth R 1994 Simple method for determining specific binding in corticosteroid-induced bone loss: what is the evidence? capacity of vitamin DÐbinding protein and its use to calculate Calcif Tissue Int 65(4):332Ð336. the concentration of “free” 1,25-dihydroxyvitamin D. Clin 50. Cooper L, Clifton-Bligh PB, Nery ML et al 2003 Vitamin D Chem 40:435Ð441. supplementation and bone mineral density in early post- 68. Haddad JG, Fraser DR, Lawson DE 1981 Vitamin D plasma menopausal women. Am J Clin Nutr 77(5):1324Ð1329. binding protein. Turnover and fate in the rabbit. J Clin Invest 51. Moestrup SK, Verroust PJ 2001 Megalin- and cubilin- 67(5):1550Ð1560. mediated endocytosis of protein-bound vitamins, lipids, and 69. Vicchio D, Yergey A, O’Brien K, Allen L, Ray R, Holick M hormones in polarized epithelia. Annu Rev Nutr 21:407Ð428. 1993 Quantification and kinetics of 25-hydroxyvitamin D3 52. Chen TC, Schwartz GG, Burnstein KL, Lokeshwar BL, by isotope dilution liquid chromatography/thermospray mass Holick MF 2000 The in vitro evaluation of 25-hydroxy- spectrometry. Biol Mass Spectrom 22(1):53Ð58. vitamin D3 and 19-nor-1alpha,25-dihydroxyvitamin D2 as 70. Nykjaer AWTE 2002 The low-density lipoprotein receptor therapeutic agents for prostate cancer [In Process Citation]. gene family: a cellular Swiss army knife? TRENDS in Cell Clin Cancer Res 6(3):901Ð908. Biology 12:273Ð280 53. Reichel H, Bishop JE, Koeffler HP, Norman AW 1991 71. Nykjaer A, Dragun D, Walther D et al 1999 An endocytic Evidence for 1,25-dihydroxyvitamin D3 production by cul- pathway essential for renal uptake and activation of the tured porcine alveolar macrophages. Mol Cell Endocrinol steroid 25-(OH) vitamin D3. Cell 96(4):507Ð515. 75(2):163Ð167. 72. Rajakumar K 2003 Vitamin D, cod-liver oil, sunlight, and 54. Reichel H, Koeffler HP, Norman AW 1987 25-Hydroxy- rickets: a historical perspective. Pediatrics 112(2):e132-e135. vitamin D3 metabolism by human T-lymphotropic virus- 73. Park EA 1940 The therapy of rickets. The J American Medical transformed lymphocytes. J Clin Endocrinol Metab 65(3): Association (JAMA) 115(5):370Ð379. 519Ð526. 74. Cooke R, Hollis B, Conner C, Watson D, Werkman S, 55. Vieth R, McCarten K, Norwich KH 1990 Role of 25-hydroxy- Chesney R 1990 Vitamin D and mineral metabolism in the vitamin D3 dose in determining rat 1,25-dihydroxyvitamin D3 very low birth weight infant receiving 400 IU of vitamin D. production. American Journal of Physiology 258(5 Pt 1): Journal of Pediatrics 116:423Ð428. E780Ð9. 75. Pittard WB, Geddes KM, Hulsey TC, Hollis BW 1991 How 56. Vieth R 2001 Vitamin D nutrition and its potential health much vitamin D for neonates? Am J Dis Child 145(10): benefits for bone, cancer, and other conditions. Journal of 1147Ð1149. Nutrition and Environmental Medicine 11:275Ð291. 76. Zeghoud F, Vervel C, Guillozo H, Walrant-Debray O, 57. Barthel HR, Scharla SH 2003 Benefits beyond the bones— Boutignon H, Garabedian M 1997 Subclinical vitamin D vitamin D against falls, cancer, hypertension and autoimmune deficiency in neonates: definition and response to vitamin D diseases. Dtsch Med Wochenschr 128(9):440Ð446. supplements. Am J Clin Nutr 65(3):771Ð778. 58. Holick MF 2003 Vitamin D: A millenium perspective. J Cell 77. Chesney RW 2001 Vitamin D deficiency and rickets. Biochem 88(2):296Ð307. Rev Endocr Metab Disord 2(2):145Ð151. 59. Zittermann A, Schleithoff SS, Tenderich G, Berthold HK, 78. Dent CE, Smith R 1969 Nutritional osteomalacia. Q J Med Korfer R, Stehle P 2003 Low vitamin D status: a contributing 38(150):195Ð209. factor in the pathogenesis of congestive heart failure? J Am 79. Blumberg RW, Forbes GB, Fraser D et al 1963 The prophy- Coll Cardiol 41(1):105Ð112. lactic requirement and the toxicity of vitamin D. Pediatrics 60. Vieth R, Milojevic S 1995 Moderate vitamin D3 supplemen- 31:512Ð525. tation lowers serum 1,25-dihydroxy-vitamin D3 in rats. 80. Jablonski NG, Chaplin G 2000 The evolution of human skin Nutrition Research 15(5):725Ð731. coloration. J Hum Evol 39(1):57Ð106. 61. Vieth R, Bayley TA, Walfish PG, Rosen IB, Pollard A 1991 81. Marx SJ, Jones G, Weinstein RS, Chrousos GP, Renquist DM Relevance of vitamin D metabolite concentrations in sup- 1989 Differences in mineral metabolism among non- porting the diagnosis of primary hyperparathyroidism. human primates receiving diets with only vitamin D3 or Surgery 110(6):1043Ð6; discussion 1046Ð7. only vitamin D2. J Clin Endocrinol Metab 69(1282): 62. Bell NH 1991 Endocrine complications of sarcoidosis. 1282Ð1289. Endocrinology & Metabolism Clinics of North America 82. Trang H, Cole DE, Rubin LA, Pierratos A, Siu S, Vieth R 20:645Ð654. 1998 Evidence that vitamin D3 increases serum 25-hydroxy- 63. Morita R, Yamamoto I, Takada M, Ohnaka Y, Yuu I 1993 vitamin D more efficiently than does vitamin D2. The Hypervitaminosis D. [Review] [Japanese]. Nippon Rinsho— American Journal of Clinical Nutrition 68:854Ð848. Japanese Journal of Clinical Medicine 51:984Ð988. 83. Bicknell F, Prescott F 1946 Vitamin D. The antirachitic or 64. Tuohimaa P, Tenkanen L, Ahonen M et al 2004 Both high calcifying vitamin. In: Bicknell F, Prescott F (eds) Vitamins and low levels of blood vitamin D are associated with in Medicine. Whitefriars Press, London, pp. 630Ð707. a higher prostate cancer risk: a longitudinal, nested case- 84. Norman AW 1972 Problems relating to the definition of control study in the Nordic countries. Int J Cancer 108(1): an international unit for Vitamin D and its metabolites. 104Ð108. J Nutrition 102:1243Ð1246. 65. Barger-Lux MJ, Heaney RP 2002 Effects of above average 85. Horst RL, Napoli JL, Littledike ET 1982 Discrimination summer sun exposure on serum 25-hydroxyvitamin D in the metabolism of orally dosed ergocalciferol and chole- and calcium absorption. J Clin Endocrinol Metab 87(11): calciferol by the pig, rat, and chick. Biochem J 204(1): 4952Ð4956. 185Ð189. 66. Dunn JF, Nisula BC, Rodbard D 1981 Transport of steroid 86. Marx SJ, Jones G, Weinstein RS, Chrousos GP, Renquist DM hormones: binding of 21 endogenous steroids to both 1989 Differences in mineral metabolism among nonhuman testosterone-binding globulin and corticosteroid-binding glob- primates receiving diets with only vitamin D3 or only vita- ulin in human plasma. J Clin Endocrinol Metab 53(1):58Ð68. min D2. J Clin Endocrinol Metab 69(6):1282Ð1290. CHAPTER 61 The Pharmacology of Vitamin D, Including Fortification Strategies 1013

87. Webb AR, DeCosta BR, Holick MF 1989 Sunlight regulates 106. Preece MA, Tomlinson S, Ribot CA et al 1975 Studies of the cutaneous production of vitamin D3 by causing its vitamin D deficiency in man. Q J Med 44(176):575Ð589. photodegradation. J Clin Endocrinol Metab 68:882Ð887. 107. Dlugos DJ, Perrotta PL, Horn WG 1995 Effects of the sub- 88. Stamp TC 1975 Factors in human vitamin D nutrition and in marine environment on renal-stone risk factors and vitamin the production and cure of classical rickets. Proc Nutr Soc D metabolism. Undersea Hyperb Med 22(2):145Ð152. 34(2):119Ð130. 108. Adams JS, Lee G 1997 Gains in bone mineral density with 89. Davie MW, Lawson DE, Emberson C, Barnes JL, Roberts GE, resolution of vitamin D intoxication. Ann Intern Med Barnes ND 1982 Vitamin D from skin: contribution to 127(3):203Ð206. vitamin D status compared with oral vitamin D in normal 109. Koutkia P, Chen TC, Holick MF 2002 Vitamin D intoxication and anticonvulsant-treated subjects. Clin Sci 63(5):461Ð472. associated with an over-the-counter supplement. N Engl J Med 90. Chel VG, Ooms ME, Popp-Snijders C et al 1998 Ultraviolet 345(1):66Ð67. irradiation corrects vitamin D deficiency and suppresses 110. Vieth R, Pinto T, Reen BS, Wong MM 2002 Vitamin D secondary hyperparathyroidism in the elderly. J Bone Miner Res poisoning by table sugar. Lancet 359:672. 13(8):1238Ð1242. 111. Vieth R, Chan A, Pollard A 1995 125I-RIA kit cannot distin- 91. Holick MF 1995 Environmental factors that influence guish vitamin D deficiency as well as a more specific assay the cutaneous production of vitamin D. Am J Clin Nutr for 25-hydroxyvitamin D. Clinical Biochemistry 28:175Ð179. 61(3 Suppl):638S-645S. 112. Stanbury SW, Mawer EB 1980 The metabolism of a physio- 92. Haddock L, Corcino J, Vazquez Md 1982 25(OH)D serum logical dose of radioactive cholecalciferol (vitamin D3) to levels in the normal Puerto Rican population and in subjects its hydroxylated metabolites in man. Clin Sci (Colch) 58(6): with tropical sprue and parathyroid disease. Puerto Rico 523Ð535. Health Sciences Journal 1:85Ð91. 113. Mawer EB, Backhouse J, Holman CA, Lumb GA, Stanbury SW 93. Haddad JG, Kyung JC 1971 Competitive protein-binding 1972 The distribution and storage of vitamin D and its radioassay for 25-hydroxycholecalciferol. Journal of Clinical metabolites in human tissues. Clin Sci 43(3):413Ð431. Endocrinology 33:992Ð995. 114. Rosenstreich SJ, Rich C, Volwiler W 1971 Deposition in and 94. Better OS, Shabtai M, Kedar S, Melamud A, Berenheim J, release of vitamin D3 from body fat: evidence for a storage Chaimovitz C 1980 Increased incidence of nephrolithiasis in site in the rat. J Clin Invest 50(3):679Ð687. lifeguards in Israel. In: Massry SG, Ritz E, Jahreis G (eds) 115. Montgomery JL, Parrish FC, Jr., Beitz DC, Horst RL, Phosphate and Minerals in Health and Disease. Plenum Huff-Lonergan EJ, Trenkle AH 2000 The use of vitamin D3 Press, New York, pp. 467Ð472. to improve beef tenderness. J Anim Sci 78(10):2615Ð2621. 95. Matsuoka LY, Wortsman J, Hollis BW 1990 Suntanning 116. Arunabh S, Pollack S, Yeh J, Aloia JF 2003 Body fat content and cutaneous synthesis of vitamin D3. J Lab Clin Med and 25-hydroxyvitamin D levels in healthy women. J Clin 116(1):87Ð90. Endocrinol Metab 88(1):157Ð161. 96. Mawer EB, Berry JL, Sommer-Tsilenis E, Beykirch W, 117. Wortsman J, Matsuoka LY, Chen TC, Lu Z, Holick MF 2000 Kuhlwein A, Rohde BT 1984 Ultraviolet irradiation increases Decreased bioavailability of vitamin D in obesity. Am J Clin serum 1,25-dihydroxyvitamin D in vitamin-DÐreplete adults. Nutr 72(3):690Ð693. Miner Electrolyte Metab 10(2):117Ð121. 118. Vieth R, Chan PC, MacFarlane GD 2001 Efficacy and safety 97. Stamp TC, Haddad JG, Twigg CA 1977 Comparison of oral of vitamin D3 intake exceeding the lowest observed adverse 25-hydroxycholecalciferol, vitamin D, and ultraviolet light effect level. Am J Clin Nutr 73(2):288Ð294. as determinants of circulating 25-hydroxyvitamin D. Lancet 119. Barger-Lux MJ, Heaney RP, Dowell S, Chen TC, Holick MF 1(8026):1341Ð1343. 1998 Vitamin D and its major metabolites: serum levels 98. Dent CE, Round JM, Rowe DJ, Stamp TC 1973 Effect of after graded oral dosing in healthy men. Osteoporos Int chapattis and ultraviolet irradiation on nutritional rickets in 8(3):222Ð230. an Indian immigrant. Lancet 1(7815):1282Ð1284. 120. Pettifor JM, Bikle DD, Cavaleros M, Zachen D, Kamdar MC, 99. Varghese M, Rodman JS, Williams JJ et al. The effect of Ross FP 1995 Serum levels of free 1,25-dihydroxyvitamin D ultraviolet B radiation treatments on calcium excretion and in vitamin D toxicity. Ann Intern Med 122(7):511Ð513. vitamin D metabolites in kidney stone formers. Clin Nephrol 121. Blank S, Scanlon KS, Sinks TH, Lett S, Falk H 1995 An 31(5):225Ð231. outbreak of hypervitaminosis D associated with the over- 100. Falkenbach A, Unkelbach U, Boehm BO et al. 1993 Bone fortification of milk from a home-delivery dairy. Am J Public metabolism before and after irradiation with ultraviolet light. Health 85(5):656Ð659. Eur J Appl Physiol 66(1):55Ð59. 122. Vieth R 1990 The mechanisms of vitamin D toxicity. Bone & 101. Lawson DE, Sedrani SH, Douglas J 1986 Interrelationships Mineral 11:267Ð272. in rats of tissue pools of cholecalciferol and 25-hydroxy- 123. Lou YR, Laaksi I, Syvala H et al 2003 25-Hydroxyvitamin D3 cholecalciferol formed in u.v. light. Biochem J 233(2): 535Ð540. is an active hormone in human primary prostatic stromal 102. Lawson DE, Douglas J, Lean M, Sedrani S 1986 Estimation cells. FASEB J, pp. of vitamin D3 and 25-hydroxyvitamin D3 in muscle and adi- 124. Narang NK, Gupta RC, Jain MK, Aaronson K 1984 Role of pose tissue of rats and man. Clin Chim Acta 157(2):175Ð181. vitamin D in pulmonary tuberculosis. Journal of Association 103. Vieth R. Reply to FAJ Muskiet et al 2001 Am J Clin Nutr of Physicians of India 32(2):185Ð186. 74(6):863Ð864. 125. Munro I 2001 Derivation of tolerable upper intake levels of 104. Brouwer DA, van Beek J, Ferwerda H et al 1998 Rat adipose nutrients. Am J Clin Nutr 74(6):865Ð867. tissue rapidly accumulates and slowly releases an orally- 126. Hathcock JN 2001 Tolerable upper intake level of vitamin D. administered high vitamin D dose. Br J Nutr 79(6):527Ð532. Am J Clin Nutr 74(6):865Ð867. 105. Clements MR, Davies M, Hayes ME et al 1992 The role 127. Health & Consumer Protection Directorate-General. Opinion of 1,25-dihydroxyvitamin D in the mechanism of acquired of the Scientific Committee on Food on the Tolerable Upper vitamin D deficiency. Clin Endocrinol (Oxf) 37(1):17Ð27. Intake Level of Vitamin D. European Commission, editor. 1014 REINHOLD VIETH

http://europa.eu.int/comm/food/fs/sc/scf/out157_en.pdf-Accessed 147. Chan TY 2000 Vitamin D deficiency and susceptibility to August 11, 2003. 2002. Brussels, Belgium. 1Ð10Ð0030. tuberculosis. Calcif Tissue Int 66(6):476Ð478. 128. Recommended Dietary Allowances 1968 Seventh Revised 148. Douglas AS, Ali S, Bakhshi SS 1998 Does vitamin D Edition ed. Washington, D.C.: National Academy Press. deficiency account for ethnic differences in tuberculosis 129. Heaney RP 2003 Quantifying human calcium absorption seasonality in the UK? Ethn Health 3(4):247Ð253. using pharmacokinetic methods. J Nutr 133(4):1224Ð1226. 149. Lansdowne AT, Provost SC 1998 Vitamin D3 enhances mood 130. Bouillon R, Verstuyf A, Verlinden L, Eelen G, Mathieu C in healthy subjects during winter. Psychopharmacology (Berl) 2003 Prospects for vitamin D receptor modulators as candidate 135(4):319Ð323. drugs for cancer and (auto)immune diseases. Recent Results 150. Gloth FM, III, Alam W, Hollis B 1999 Vitamin D vs broad Cancer Res 164:353Ð356. spectrum phototherapy in the treatment of seasonal affective 131. Posner GH 2002 Low-calcemic vitamin D analogs disorder. J Nutr Health Aging 3(1):5Ð7. (deltanoids) for human cancer prevention. J Nutr 132(12): 151. Harris S, Dawson-Hughes B 1993 Seasonal mood changes in 3802SÐ3803S. 250 normal women. Psychiatry Res 49(1):77Ð87. 132. van den Bemd GJ, Chang GT 2002 Vitamin D and vitamin D 152. Al Allaf AW, Mole PA, Paterson CR, Pullar T 2003 Bone analogs in cancer treatment. Curr Drug Targets 3(1):85Ð94. health in patients with fibromyalgia. Rheumatology (Oxford) 133. Jacobus CH, Holick MF, Shao Q et al 1992 Hypervitaminosis D 42:1Ð5. associated with drinking milk. New England Journal of 153. Garland CF, Garland FC, Gorham ED 1999 Calcium and Medicine 326:1173Ð1177. vitamin D. Their potential roles in colon and breast cancer 134. Bell NH 1998 Renal and nonrenal 25-hydroxyvitamin D- prevention. Ann N Y Acad Sci 889:107Ð119. 1alpha-hydroxylases and their clinical significance. J Bone 154. Grant WB 2001 An estimate of excess cancer mortality in Miner Res 13(3):350Ð353. the US due to inadequate exposure to solar RV-B radiation 135. Heaney RP, Davies KM, Chen TC, Holick MF, Barger-Lux MJ (abstract). Photodermatol Photoimmunol Photomed 17:142. 2003 Human serum 25-hydroxycholecalciferol response to 155. Schwartz GG, Wang MH, Zang M, Singh RK, Siegal GP extended oral dosing with cholecalciferol. Am J Clin Nutr 1997 1α,25-Dihydroxyvitamin D (calcitriol) inhibits the 77(1):204Ð210. invasiveness of human prostate cancer cells. Cancer 136. Muskiet FA, Dijck-Brouwer DJ, van d, V, Schaafsma A 2001 Epidemiol Biomarkers Prev 6(9):727Ð732. Do we really need 100 mcg vitamin D/day, and is it safe for 156. Schwartz GG, Whitlatch LW, Chen TC, Lokeshwar BL, all of us? Am J Clin Nutr 74(6):862Ð863. Holick MF 1998 Human prostate cells synthesize 1,25-dihy- 137. Lips P, Graafmans WC, Ooms ME, Bezemer PD, Bouter LM droxyvitamin D3 from 25-hydroxyvitamin D3 [In Process 1996 Vitamin D supplementation and fracture incidence in Citation]. Cancer Epidemiol Biomarkers Prev 7(5):391Ð395. elderly persons. A randomized, placebo-controlled clinical 157. Hsu JY, Feldman D, McNeal JE, Peehl DM 2001 Reduced trial. Ann Intern Med 124(4):400Ð406. 1alpha-hydroxylase activity in human prostate cancer cells 138. Vieth R, Fraser D 1979 Kinetic behavior of 25-hydroxy- correlates with decreased susceptibility to 25-hydroxy- vitamin D-1-hydroxylase and -24-hydroxylase in rat kidney vitamin D3Ðinduced growth inhibition. Cancer Res 61(7): mitochondria. J Biol Chem 254(24):12455Ð12460. 2852Ð2856. 139. NIH 2003 Facts about dietary supplements: Vitamin D. 158. Grau MV, Baron JA, Sandler RS et al 2003 Vitamin D, Clinical Nutrition Service,Warren Grant Magnuson Clinical calcium supplementation, and colorectal adenomas: results Center, National Institute of Health (NIH), Bethesda, MD, in of a randomized trial. J Natl Cancer Inst 95(23):1765Ð1771. conjunction with the Office of Dietary Supplements (ODS) 159. Vieth R, Dogan M, Cole DEC et al 2003 Vitamin D3 at in the Office of the Director of NIH. National Institute of 90 or 700 mcg weekly for 1 year: responses of 25(OH)D, Health. 12-12-0030. PTH, urine and plasma calcium. European Calcified Tissue 140. Takeuchi A, Okano T, Ishida Y, Kobayashi T 1995 Effects of Society, Rome, May, 2003. Calcified Tissue Int 72(4): dietary vitamin D intake on plasma levels of parathyroid hor- 377Ð378 mone and vitamin D metabolites in healthy Japanese. Miner 160. Harris S 2002 Can vitamin D supplementation in infancy Electrolyte Metab 21(1-3):217Ð222. prevent type 1 diabetes? Nutr Rev 60(4):118Ð121. 141. Stene LC, Ulriksen J, Magnus P, Joner G 2000 Use of cod 161. Heaney RP, Dowell MS, Hale CA, Bendich A 2003 Calcium liver oil during pregnancy associated with lower risk of Type I absorption varies within the reference range for serum diabetes in the offspring. Diabetologia 43(9):1093Ð1098. 25-hydroxyvitamin D. J Am Coll Nutr 22:142Ð6. 142. Eva JK 1999 Vitamin D supplement in early childhood and 162. Marx SJ, Jones G, Weinstein RS, Chrousos GP, Renquist DM risk for Type I (insulin-dependent) diabetes mellitus. The 1989 Differences in mineral metabolism among nonhuman EURODIAB Substudy 2 Study Group. Diabetologia primates receiving diets with only vitamin D3 or only vita- 42(1):51Ð54. min D2. J Clin Endocrinol Metab 69(6):1282Ð1290. 143. Fuller K 2000 Lactose, rickets, and the coevolution of genes 163. Jones G, Byrnes B, Palma F, Segev D, Mazur Y 1980 and culture. Human Ecology 28(3):471Ð477. Displacement potency of vitamin D2 analogs in competitive 144. Eyles D, Brown J, Mackay-Sim A, McGrath J, Feron F 2003 protein-binding assays for 25-hydroxyvitamin D3, 24,25- Vitamin D3 and brain development. Neuroscience 118(3): dihydroxyvitamin D3, and 1,25-dihydroxyvitamin D3. J Clin 641Ð653. Endocrinol Metab 50(4):773Ð775. 145. Mahon BD, Bemiss C, Cantorna MT 2001 Altered cytokine 164. Mawer EB, Jones G, Davies M et al 1998 Unique 24-hydroxy- profile in patients with multiple sclerosis following vitamin D lated metabolites represent a significant pathway of supplementation. FASEB J 837:4. metabolism of vitamin D2 in humans: 24-hydroxyvitamin D2 146. Embry AF, Snowdon LR, Vieth R 2000 Vitamin D and and 1,24-dihydroxyvitamin D2 detectable in human serum. seasonal fluctuations of gadolinium-enhancing magnetic J Clin Endocrinol Metab 83(6):2156Ð2166. resonance imaging lesions in multiple sclerosis. Ann Neurol 165. Holmberg I, Berlin T, Ewerth S, Bjorkhem I 1986 48(2):271Ð272. 25-Hydroxylase activity in subcellular fractions from CHAPTER 61 The Pharmacology of Vitamin D, Including Fortification Strategies 1015

human liver. Evidence for different rates of mitochondrial National Academy of Sciences of the United States of hydroxylation of vitamin D2 and D3. Scand J Clin Lab Invest America 90:8668Ð8672. 46(8):785Ð790. 167. Harris SS, Dawson-Hughes B, Perrone GA 1999 Plasma 166. Guo YD, Strugnell S, Back DW, Jones G 1993 Transfected 25-hydroxyvitamin D responses of younger and older men to human liver cytochrome P-450 hydroxylates vitamin D three weeks of supplementation with 1800 IU/day of vitamin analogs at different side-chain positions. Proceedings of the D. J Am Coll Nutr 18(5):470Ð474. CHAPTER 62 How to Define Normal Values for Serum Concentrations of 25-Hydroxyvitamin D? An Overview

PAUL LIPS Department of Endocrinology, VU University Medical Center, Amsterdam, The Netherlands

I. Introduction IV. Classification of Vitamin D Replete and Deficient States II. How to Define Normal Values V. Dietary Vitamin D Intake and Recommended Daily Allowances III. Variables Influencing Normal Values of Serum 25(OH)D References

I. INTRODUCTION as in many developing countries [14], or on the high side when a large proportion of the population takes sup- Vitamin D deficiency may cause rickets and osteo- plements, when food is fortified with vitamin D, or when malacia in the long term [1]. Mild or moderate vitamin sunshine is abundant and sunshine exposure is common D deficiency causes secondary hyperparathyroidism, [15,16]. Traditionally, reference values for serum bone loss, and osteoporosis and has been associated 25(OH)D have been higher in the U.S. and Australia than with fractures [2,3]. It has also been related to muscle in most European countries. Alternatively, reference val- weakness, colon cancer, and auto-immune diseases, ues will be low in China or Russia [17]. This is caused by such as diabetes mellitus and multiple sclerosis [4,5]. differences in sunshine exposure, food fortification with Vitamin D status is assessed by serum 25(OH)D, which vitamin D, and the use of the vitamin supplements. is not the active metabolite [6]. Normal “reference” val- Another way of defining normal values is by assess- ues have traditionally been derived from population data. ing biological or clinical outcomes of vitamin D status. However, these depend on sunshine exposure, latitude, These outcomes include the serum concentration of and other factors, such as skin pigmentation [7]. The 1,25(OH)2D, the serum concentration of parathyroid assessment is subject to interlaboratory variation, which hormone (PTH), bone mineral density (BMD), and the may be considerable [8]. The dietary calcium intake influ- occurrence of bone disease, e.g. osteoporotic fractures ences the consequences of vitamin D deficiency, which or rickets and osteomalacia [3]. These outcomes can be become visible earlier when calcium intake is low [9]. assessed cross-sectionally, as in case-control studies, During the last decade, it has become general practice to or longitudinally in prospective epidemiological define normal values of serum 25(OH)D with respect to studies or intervention studies. These methods are biological endpoints, for example, parathyroid function summarized in Table I. or bone mineral density [10,11]. A. Substrate-dependent Synthesis II. HOW TO DEFINE NORMAL VALUES of 1,25(OH)2D

Serum 25(OH)D has been used to assess vitamin D In the case of vitamin D deficiency, the synthesis of status for more than 20 years, as it is the main circu- the active metabolite 1,25(OH)2D becomes substrate- lating metabolite. Reference values have been obtained dependent, i.e. dependent on the serum 25(OH)D con- from population studies, or from presumably healthy centration. This was observed in a Belgian study in subjects, e.g. blood donors [10,12,13]. The problem is nursing home residents. The low serum 25(OH)D in win- that such values may be on the low side when the ter caused relatively low serum 1,25(OH)2D in winter greater part of the population is vitamin DÐdeficient, and spring (Fig. 1). When the amount of substrate VITAMIN D, 2ND EDITION Copyright © 2005, Elsevier, Inc. FELDMAN, PIKE, AND GLORIEUX All rights reserved. 1020 PAUL LIPS

TABLE I Biological Static and Dynamic Methods A nmol/l µg/l to Define the Threshold Between Deficient and 25-Hydroxycholecalciferol 75 Normal Values of Serum 25(OH)D 30 p < 0.001

Static methods 25 Positive correlation between serum 1,25(OH)2D and 25(OH)D below threshold serum 25(OH)D. 50 20 Negative correlation between serum PTH and 25(OH)D below p < 0.05 threshold serum 25(OH)D. 15 Positive correlation between bone mineral density (BMD) and serum 25(OH)D below threshold. 25 10 Association of fractures with low serum 25(OH)D in epidemiological studies. 5 Dynamic methods

Substrate dependent increase of serum 1,25(OH)2D following vitamin D supplementation when serum 25(OH)D is below B pmol/l1,25-Dihydroxycholecalciferol ng/l threshold. 175 70

Decrease of serum PTH after vitamin D supplementation NS when baseline serum 25(OH)D is below threshold. 125 50 Increase of BMD after vitamin D supplementation when serum 25(OH)D is below threshold. Decrease of fracture incidence after vitamin D supplementation 75 30 when baseline serum 25(OH)D is below threshold. p < 0.01 25 10

[serum 25(OH)D] increased in spring and summer 0246810 12 to 30Ð40 nmol/l serum, 1,25(OH)2D increased and Month approached normal values [18]. This substrate- FIGURE 1 Seasonal low serum 25(OH)D causes low serum dependent synthesis of 1,25(OH) D is also apparent 2 1,25(OH)2D. When serum 25(OH)D increases in summer, serum from the positive correlation between serum 25(OH)D 1,25(OH)2D approaches normal values. Reproduced from and serum 1,25(OH)2D, which was observed in patients Bouillon R et al. 1987 Am J Clin Nutr 45:755Ð763. With permis- with hip fracture and geriatric patients with low serum sion of the American Society for Clinical Nutrition. levels of 25(OH)D. A vitamin D supplementation study (vitamin D3 400 or 800 IU/day) in residents of a home absorption and serum calcium within normal limits [3]. for the elderly and a nursing home showed that serum In the end, serum 1,25(OH)2D is maintained within 1,25(OH)2D increased significantly when baseline normal limits at the expense of an increase of serum serum 25(OH)D was lower than 30 nmol/l, while there PTH [22]. The increase of serum PTH usually is within was no increase with higher baseline serum 25(OH)D normal limits, so the secondary hyperparathyroidism [20]. On the other hand, a study in nursing home resi- only can be observed at a group level and not in most dents in the U.S. did not show an increase of serum individuals. As the maintenance of serum 1,25(OH)2D 1,25(OH)2D, probably because baseline serum and serum calcium is the outcome of a homeostatic 25(OH)D was 45 nmol/l [21]. These studies suggest control system, the negative relationship is visible that serum 25(OH)D should be above 30Ð40 nmol/l in between serum PTH and serum 25(OH)D and not serum order to achieve a normal serum 1,25(OH)2D. 1,25(OH)2D [3]. This was clearly observed in a study of patients with hip fracture and elderly control sub- jects. While serum 25(OH)D is maximal in summer B. Secondary Hyperparathyroidism and reaches its lowest point in late winter or early spring, serum PTH shows an inverse seasonal pattern When serum 25(OH)D falls below a certain level, with maximal levels in winter or early spring and serum 1,25(OH)2D will decrease as its synthesis minimal levels in summer or autumn. However, serum becomes substrate-dependent. Calcium absorption from 1,25(OH)2D stayed at constant levels throughout the the gut will decrease, leading to an increase of PTH year [22]. A negative correlation has been observed secretion. The increase of serum PTH will stimulate in observational or epidemiological studies in adults, the synthesis of 1,25(OH)2D in order to keep calcium especially elderly people (Fig. 2). The Amsterdam CHAPTER 62 How to Define Normal Values for Serum Concentrations of 25-Hydroxyvitamin D? An Overview 1021

A PTH(1–84), pmol/l (log-scale)B iPTH (pg/ml) 15 16 12 130 p < 0.01 8 110

90 4 70 55 2 50 36 30 1 10 0 11.6 40 60 78 100 120 140 160 180 200 25(OH) D (nmol/l) 0102030 40 50 60 70 80 90 25(OH) vitamin D (nmol/l)

C 110

100

90

80

70

60

50 Parathyroid hormone (pg/ml)

40

30

20 0–5 >30 6–10 11–15 16–20 21–25 26–30 25-Hydroxyvitamin D (ng/ml)

FIGURE 2 Negative relationship between serum PTH and serum 25(OH)D with different threshold levels where serum PTH starts to increase. (A) Amsterdam Vitamin D Study, reproduced from Ooms ME 1994 PhD Thesis, Vrije Universiteit, Amsterdam. (B) SUVIMAX study, reproduced with permission from Chapuy MC et al. 1997 Osteoporosis Int 7:439Ð443. (C) A study in hospital in-patients, repro- duced from Thomas MK et al. 1998 N Engl J Med 338:777Ð783. With permission © Massachusetts Medical Society.

Vitamin D Study, a randomized placebo-controlled 25 nmol/l the correlation was no longer significant. study of the effect of vitamin D supplementation on However, the SUVIMAX study, a study in French post- the incidence of hip fractures, showed a negative cor- menopausal women, showed an increase of serum PTH relation between serum PTH and serum 25(OH)D when serum 25(OH)D was lower than 78 nmol/l [10] when serum 25(OH)D was lower than 25 nmol/l [23] (Fig. 2B). A study in adult patients admitted to a (Fig. 2A). When serum 25(OH)D was higher than general ward in a U.S. hospital showed an increase of 1022 PAUL LIPS serum PTH when serum 25(OH) was lower than BMD left femoral neck, g/cm2 (log-scale) 75 nmol/l [24] (Fig. 2C). These differences may be partially caused by interlaboratory differences in the 0.9 assays for 25(OH)D [8,25]. Other important determi- nants are population characteristics such as age, sex, 0.8 genetic difference, and dietary calcium intake. Another 0.7 point is whether a small increase in serum PTH within the normal range should be considered pathological or just a physiological compensatory phenomenon. 0.6

0.5 C. Relationship with Bone Mineral Density

0.4 Vitamin D deficiency is associated with a lower 0102030 40 50 60 70 80 intestinal absorption of calcium. The availability of cal- 25 (OH) vitamin D (nmol/l) cium for mineralization of newly formed bone matrix, the osteoid, may be too low. This causes insufficient FIGURE 3 Relationship between BMD of the femoral neck and serum 25(OH)D. Significant correlation (p < 0.001) when serum secondary mineralization and bone with a low mineral 25(OH)D < 30 nmol/l. When serum 25(OH)D > 30 nmol/l the content [26]. When vitamin D deficiency is severe, correlation was not significant. Reproduced from Ooms ME et al. primary mineralization is also hampered, leading to 1995 J Bone Miner Res 10:1177Ð1184. With permission of the accumulation of osteoid tissue and rickets or osteoma- American Society for Bone and Mineral Research. lacia. The secondary hyperparathyroidism causes high bone turnover. Even in mild or moderate vitamin D the BMD increase in the femoral neck was 6% in the deficiency, high turnover is associated with relatively group treated with vitamin D3 800 IU/day and calcium young osteons in which secondary mineralization is 1200 mg/day in comparison with the control group that not yet completed. The accumulation of osteoid and received double placebo [30]. However, the treatment young incompletely mineralized bone is associated effect in the former group may be due to calcium as well with low bone mineral density (BMD) when measured as vitamin D. The increase in BMD was not linked to a by densitometric techniques such as DXA [26]. low baseline serum 25(OH)D in these studies. In some cross-sectional studies, a relationship was observed between BMD and serum 25(OH)D. In 330 elderly women participating in the Amsterdam D. Relationship with Fractures Vitamin D Study, a positive relationship was observed between baseline serum 25(OH)D and BMD of the The question of whether vitamin D deficiency is a femoral neck [11]. This relationship was significant risk factor for fractures has been assessed in large when serum 25(OH)D was lower than 30 nmol/l (Fig. 3). prospective epidemiological studies, such as the Study The regression indicated that BMD could be 5 or 10% on Osteoporotic Fractures (SOF). In this study of 9704 lower when serum 25(OH)D was 20 or 10 nmol/l, elderly women, vitamin D deficiency, defined as serum respectively. A positive correlation between serum 25(OH)D lower than 47 nmol/l, was not associated 25(OH)D and BMD of the hip was also observed in with an increased risk for hip or vertebral fracture [31]. studies from the U.K. and from New Zealand [27,28]. However, a low serum 1,25(OH)2D (≤57 pmol/l) Baseline data of the MORE study, a placebo-controlled increased the risk for hip fracture (RR 2.1 adjusted for study in more than 6000 postmenopausal women, age and weight). A high serum PTH was not a risk fac- showed a relationship between serum 25(OH)D and tor in this study. The Longitudinal Aging Study BMD of the trochanter [16]. Subjects with a serum Amsterdam also assessed the effect of a low serum 25(OH)D lower than 25 nmol/l had a BMD 4% lower 25(OH)D on fracture risk [32]. A low serum 25(OH)D than those with serum 25(OH)D higher than 25 nmol/l. (<30 nmol/l) was associated with an increased fracture There was no difference between the groups with risk (RR 1.6 adjusted for age and sex). Fracture risk regard to BMD of the femoral neck or lumbar spine. according to fracture type was not studied because of Data on the effect of vitamin D treatment on BMD are limited follow-up. scarce. In the Amsterdam Vitamin D Study, treatment A Norwegian study has assessed the effect of with vitamin D3 400 IU/day increased the BMD of the vitamin D intake on hip fracture risk. A low intake femoral neck by 2.2% after 2 years in comparison with (<100 IU/day) was associated with an increased hip the placebo group [29]. In the French Decalyos Study, fracture risk, but serum 25(OH)D was not measured in CHAPTER 62 How to Define Normal Values for Serum Concentrations of 25-Hydroxyvitamin D? An Overview 1023 this study [33]. The incidence of hip fractures is much 25(OH)D (nmol/l) higher in Northern Europe than in Western or Southern 100 Europe [34]. This suggests a possible involvement of r=0.69 vitamin D deficiency as a risk factor. However, the 80 Euronut Seneca Study and the MORE Study showed that serum 25(OH)D was higher in Northern European countries than in Southern Europe [13,16]. This may 60 be due to sun-seeking behavior in northern climates and sun-avoiding behavior in sunny countries. The 40 consumption of fatty fish and the use of vitamin D sup- r=0.72 r=0.84 plements may also explain this gradient of higher serum 25(OH)D with more northern latitudes. 20

0 III. VARIABLES INFLUENCING NORMAL CPB RIA HPLC VALUES OF SERUM 25(OH)D Lyon Lyon Amsterdam A. Comparability of Assays for FIGURE 4 Interlaboratory comparison between assays for serum 25(OH)D from Lyon and Amsterdam. Reproduced with permission Serum 25(OH)D from Lips P et al. 1999 Osteoporosis Int 9:394Ð397. The observed differences in threshold values for serum 25(OH)D may be explained in part by differ- ences in assays for serum 25(OH)D (see Chapter 58). B. Influence of Calcium Intake Interlaboratory comparison studies have demonstrated a great variability in assay results [8,25]. Most assays A low calcium intake is associated with a higher are able to discriminate between low and average serum PTH than a high calcium intake. Serum PTH serum 25(OH)D values. However, it is difficult to com- decreases within one hour after oral calcium load [36]. pare studies from different countries and to establish When the mean calcium intake was increased from internationally validated threshold levels for discrimi- 800 mg to 2400 mg per day in postmenopausal nation of vitamin DÐdeficient states. Most assays use an women, serum PTH decreased about 30% during extraction step followed by competitive protein binding 24 hours [37]. A low calcium intake may also influ- assay (CPB) or radioimmunoassay (RIA). The gold ence vitamin D metabolism because PTH stimulates standard may be purification by high performance liquid the renal hydroxylation of 25(OH)D into 1,25(OH)2D. chromatography (HPLC) followed by CPB or RIA [8]. A recent interlaboratory comparison showed large differences in serum 25(OH)D between Lyon using CPB and Amsterdam using HPLC followed by CPB [25]. TABLE II Mean Serum 25(OH)D in Lyon The results on the same serum samples were 85% higher (Decalyos Study) and Amsterdam (Amsterdam in Lyon than in Amsterdam (Fig. 4, Table II). This Vitamin D Study) Before and After Supplementation 1 cross-calibration was used to correct serum values in with Placebo or Vitamin D order to compare the results of two large prospective Lyon Lyon intervention studies [30,35]. Following correction, it before after could be concluded that the elderly in Lyon were more correction correction Amsterdam vitamin DÐdeficient than those in Amsterdam and that Placebo groups the values of the vitamin D treatment were very similar in both studies. An interlaboratory comparison of four Baseline 32.5 17.9 26.0 nmol/l laboratories using a CPB and one laboratory using a 1 year 25.0 13.7 23.0 nmol/l RIA for serum 25(OH)D showed that the highest labo- Vitamin D groups ratory produced 38% higher serum 25(OH)D values Baseline 40 21.6 27.0 nmol/l than the lowest laboratory [25]. This is an important 1 year 105 56.7 62.0 nmol/l observation when considering a threshold for vitamin D insufficiency of 37.5 nmol/l (15 ng/ml) or 50 nmol/l 1The values for Lyon were corrected to those of Amsterdam using a (20 ng/ml), which may be similar or very different correction factor of 0.54 derived from the study in ref. 25 (Fig. 4). Other according to the assay used. data are derived from ref 29 and 30. 1024 PAUL LIPS

Rats on a low calcium intake had a higher serum PTH against the consequences of vitamin D deficiency and and serum 1,25(OH)2D than rats with a high calcium secondary hyperparathyroidism. intake [38]. This was associated with an increased metabolic clearance rate of 25(OH)D. This was con- firmed by a clinical study of patients with primary IV. CLASSIFICATION OF VITAMIN D hyperparathyroidism or secondary hyperparathyroidism REPLETE AND DEFICIENT STATES following gastrectomy [9]. A high serum 1,25(OH)2D was associated with a low half life of 25(OH)D. Thus, It may be clear from the above, that vitamin D status a low calcium intake may aggravate vitamin D deficiency is the complex result of sunshine exposure, latitude, while a high calcium intake may have a vitamin D sunscreen use, clothing, skin pigmentation, vitamin D sparing effect. Calcium intake may influence the degree intake with fatty fish, dairy products, fortified foods, of secondary hyperparathyroidism associated with vita- and vitamin D supplement use [3,7]. In addition, the con- min D deficiency and thus influence the threshold sequences of vitamin D deficiency depend on calcium serum 25(OH)D where serum PTH starts to increase. intake and on other variables that influence parathyroid This may partially explain why this threshold is lower function, such as the decrease of renal function with in countries where calcium intake is high such as The age, low estrogen status, and the use of loop diuretics Netherlands, than in countries such as France where such as furosemide [3]. In addition, the reported serum calcium intake is low. 25(OH)D levels for sufficiency or deficiency depend on interlaboratory variation, which may explain unex- pected findings [25]. C. Other Variables Influencing Serum PTH As discussed above, the serum PTH concentration starts to rise when serum 25(OH)D falls below Serum PTH is also influenced by renal function, the 25 nmol/l, 50 nmol/l or higher up to 78 nmol/l in various use of diuretics, and estrogen deficiency. The glomerular surveys [10,11,16]. The consequences of vitamin D filtration rate slowly decreases with age from about deficiency may be arranged in three stages (Table III). 125 ml/min at age 20 to 60 ml/min at age 80. This is Early, mild vitamin D deficiency or insufficiency causes accompanied by a gradual increase of serum PTH, which secondary hyperparathyroidism, high turnover, and bone is positively correlated with serum creatinine [39,40]. loss. Moderate vitamin D deficiency is associated with The increase of serum PTH may be caused by slight secondary hyperparathyroidism and slower secondary phosphate retention and lower synthesis of 1,25(OH)2D. mineralization, leading to bone with a lower degree of The loop diuretic furosemide increases calcium excre- mineralization. The third stage, severe vitamin D defi- tion, decreases ionized calcium by inducing alkalosis ciency causes a disturbance of primary mineralization, and thereby increases serum PTH [41,42]. The thiazide leading to the accumulation of osteoid tissue, the hall- diuretics increase calcium reabsorption, but paradoxi- mark of osteomalacia in adults, and accumulation of non- cally may also increase serum PTH [43]. mineralized hypertrophic cartilage at the epiphysial zones The effect of estrogen on serum PTH is complex. characterizing rickets in children (see also Chapter 63). The age-related increase in serum PTH did not occur in postmenopausal women on estrogen replacement ther- apy [44]. Estrogen also interacts with the secondary A. Mild Vitamin D Deficiency Worldwide hyperparathyroidism following vitamin D deficiency. In the Amsterdam Vitamin D Study, the serum concen- During the last decade, it has been suggested that tration of sex hormone binding globulin (SHBG) inter- the required normal range of serum 25(OH)D level acted with the negative relationship between serum PTH should be raised because it became clear that the point and serum 25(OH)D. Serum SHBG correlates negatively where serum PTH starts to rise is not as low as previ- with the free estrogen concentration. Mean serum PTH ously assumed [3]. In the same period, investigators was high in vitamin DÐdeficient elderly with high serum started to realize that mild vitamin D deficiency or SHBG, and it was normal with a low serum SHBG [11]. insufficiency or inadequacy was more common than This suggests that estrogen protects against secondary anticipated. In this chapter, mild vitamin D deficiency hyperparathyroidism caused by vitamin D deficiency. is defined as a serum 25(OH)D between 25 and Treatment with vitamin D had a greater effect on BMD 50 nmol/l [3]. While the consequences on an individ- of the femoral neck when serum SHBG was high than ual basis may be mild, a somewhat greater bone loss when serum SHBG was low [29]. This supports the and a slightly increased fracture risk, the consequences hypothesis that a higher free estrogen level protects on a population scale may be more important because CHAPTER 62 How to Define Normal Values for Serum Concentrations of 25-Hydroxyvitamin D? An Overview 1025

Table III Classification of Vitamin D Deficient States

Serum 25(OH)D Serum 25(OH)D Serum PTH Vitamin D status (nmol/l) ng/ml increase Bone histology Consequences

Vitamin D replete >50 >20 0% Normal — Mild deficiency 25Ð50 10Ð20 5Ð15% Normal/high turnover Increased fracture risk (insufficiency) Moderate deficiency 12.5Ð25 5Ð10 15Ð30% High turnover Increased fracture risk Severe deficiency <12.5 <5 >30 % Incipient or overt Bone pain, fractures, osteomalacia Looser zones

of the large number of people who are vitamin D insuf- adults (Fig. 5). A limitation of these reviews is the ficient. In addition, other diseases that might be due to probably large interlaboratory variation in the assays for vitamin D insufficiency or disturbances in vitamin D serum 25(OH)D. The Euronut Seneca study in Europe metabolism become more important [3]. and the MORE study, which was done globally, used a Several investigators have attempted to define the central laboratory facility [13,16]. Serum 25(OH)D var- problem on a worldwide scale. McKenna and col- ied in the Seneca Study between 22 nmol/l in Greece leagues identified 117 studies on vitamin D status in and 46 nmol/l in Scandinavia. There was a very signif- young adults and/or elderly [15]. These studies mainly icant positive relationship between serum 25(OH)D came from North America, Scandinavia, and Europe. and latitude with the highest levels in Northern and the It appeared from these studies that mild vitamin D lowest levels in Southern Europe. Similar observations deficiency was more common in Western and Southern were made in the MORE study, a double blind study on Europe than in Scandinavia or North America. While the effect of raloxifene in women with postmenopausal vitamin D deficiency was very common in the elderly osteoporosis [16]. In this study, a similar positive rela- in winter and spring in Europe and North America, tionship was observed between serum 25(OH)D and vitamin D deficiency was not rare in young adults northern latitude in Europe with levels ranging from in Europe during winter. A similar trend was observed 50Ð60 nmol/l in Southern Europe to 70Ð90 nmol/l in in another review with higher serum 25(OH)D levels in Northern Europe. There are several explanations for North America and Australia than in Europe and this south-north gradient, the inverse of what should be Asia. Serum 25(OH)D was lower in institutionalized expected. An explanation might be that people in elderly than in independent living young and older northern countries like direct sunshine and sunbathing,

A B 100 100 90 90 80 80 70 70 60 60 50 50 40 40

Serum 25(OH)D (nmol/l) 30 30

Serum 25(OH)D (nmol/l) 20 20 10 10 0 0 NW Europe Middle & Middle Asia Australia USA, Adults, Independent Outpatients, Inpatients, Hip fracture S. Europe East N.Zealand Canada postmenop. elderly home for geriatric p., patients women elderly nursing home

FIGURE 5 Mean values of serum 25(OH)D from 43 studies according to geographical region (A) or to subject/patient/residence category (B). Reproduced from Lips P 2001 Endocr Rev 22:477Ð501. With permission of Endocrine Society. 1026 PAUL LIPS whereas people living in sunny countries tend to avoid exposure and for a small part from dietary intake. the sun. The light skin in Northern Europe favors vita- Dietary intake becomes more important when sunshine min D synthesis, while the synthesis is less in a more exposure is low, as is common in elderly people and pigmented skin in Mediterranean countries. In addi- non-western immigrants [19,51]. However, most diets tion, consumption of fatty fish and the use of vitamin only contain small amounts of vitamin D3 in dairy supplements may be more widespread in Northern products and eggs. Only fatty fish such as herring, Europe. In the Seneca Study, vitamin D deficiency mackerel, and halibut contain considerable amounts (defined as serum 25(OH)D < 30 nmol/l) was observed of vitamin D [19]. An important dietary source may in 47% of the participants. In the MORE Study, a serum be vitamin DÐfortified products. Milk fortified with 25(OH)D < 50 nmol/l was found in 39.3% of the par- 400 IU of vitamin D3 per quart or liter is common in ticipants in Southern Europe and in 12.6% of those in the U.S. In many European countries, however, only Northern Europe. A sunny climate is no guarantee for margarine is fortified with vitamin D3 3 IU/g. Typical an adequate vitamin D status as follows from recent dietary intakes of vitamin D are more than 200 IU/day studies in adults from Lebanon [45], Ethiopia [14], and in the U.S. and about 100 IU/day in many European Italy [46]. countries [19,52]. A dietary vitamin D intake of 400 IU/day may be attained in the U.S. when sufficient for- tified dairy products are used. In most Europeans, high B. Risk Groups dietary intakes of vitamin D can only be obtained with regular consumption of fatty fish. Practicing physicians should be aware of risk Currently, dietary recommendations are given as factors for vitamin D insufficiency or deficiency. Risk adequate intakes. These intakes have been defined in factors relating to vitamin D production include low the U.S. to be 200 IU/day in adults until age 50 years, sunshine exposure, sunscreen use, highly pigmented 400 IU/day in adults from 51 to 70 years, and 600 IU/day skin, and clothes covering most parts of the body [7] in persons older than 70 years [53]. The European (see Chapter 47). Risk factors relating to vitamin D Community has recommended a vitamin D3 intake of metabolism are low calcium intake and medication 0 to 400 IU/day for adults from 18 to 44 years, depend- such as antiepileptics, that increase vitamin D ing on sunshine exposure, being high in active people catabolism [47] (see Chapter 74). Using these risk and low in the housebound [54]. The recommended factors, risk groups can be defined. Older people often intake for older persons (65 years and older) has been are immobile and do not go outside. Persons with defined as 400 IU/day. It is difficult to assure these skin conditions, e.g. skin cancer, should not stay out- intakes with a normal diet unless a considerable amount side in direct sunshine. People with a dark skin need of fatty fish or vitamin D3 fortified products is consumed. much more sunshine exposure to synthesize a similar Therefore, most risk groups have to rely on vitamin D quantity of vitamin D than people with a light skin supplements. (see Chapter 3). Cultural and religious customs may determine clothing habits and may restrict sunshine exposure. Very low serum 25(OH)D levels have been A. Consequences for Public Health reported from Saudi Arabia, Lebanon, and Ethiopia [14,45,48]. Immigrants from North Africa, the Middle The above mentioned risk groups, older persons, East, and India often have very low serum 25(OH)D immigrants, and persons with dark skin, are of consid- levels [49,50]. Calcium intake is low in people with erable size and comprise a large part of the population intolerance for dairy products due to lactase deficiency for which vitamin D supplementation may be consid- or following gastrectomy. Patients who are on chronic ered. A daily supplement of 400Ð600 IU is very effec- anti-epileptic medication may also carry a high risk tive but impractical. A larger supplement once per week, for vitamin D deficiency [47]. These groups need once per month, or once per three months may be special attention from general practitioners and public equally effective and easier to distribute [55,56]. health care. Dietary advice may be effective when fatty fish is recommended three times per week, but this is not fea- sible for many people. Fortification of dairy products V. DIETARY VITAMIN D INTAKE AND with vitamin D3 is more attractive [57] as these RECOMMENDED DAILY ALLOWANCES products are widely used and these products usually contain a lot of calcium. Milk is an excellent carrier for The circulating 25(OH)D originates for the greater vitamin D fortification, and it also is an important part from cutaneous synthesis following sunshine source of protein. CHAPTER 62 How to Define Normal Values for Serum Concentrations of 25-Hydroxyvitamin D? An Overview 1027

References 19. Lips P, van Ginkel FC, Jongen MJM, Rubertus A, van der Vijgh WJF and Netelenbos JC 1987 Determinants of 1. Frame B, Parfitt AM 1978 Osteomalacia: current concepts. vitamin D status in patients with hip fracture and elderly Ann Intern Med 89:966Ð982. control subjects. Am J Clin Nutr 46:1005Ð1010. 2. Chalmers J, Barclay A, Davison AM, Macleod DAD, 20. Lips P, Wiersinga A, van Ginkel FC, Jongen MJM, Netelenbos JC, Williams DA 1969 Quantitative measurements of osteoid in Hackeng WHL, Delmas PD and van der Vijgh WJF 1988 The health and disease. Clin Orthop 63:196Ð209. effect of vitamin D supplementation on vitamin D status and 3. Lips P 2001 Vitamin D deficiency and secondary hyper- parathyroid function in elderly subjects. J Clin Endocrinol Metab parathyroidism in the elderly: consequences for bone loss and 67:644Ð650. fractures and therapeutic implications. Endocrine Rev 21. Himmelstein S, Clemens TL, Rubin A, Lindsay R 1990 22:477Ð501. Vitamin D supplementation in elderly nursing home residents 4. Walters MR 1992 Newly identified actions of the vitamin D increases 25-OHD but not 1,25(OH)2D. Am J Clin Nutr endocrine system. Endocr Rev 13:719Ð764. 52:701Ð706. 5. Bouillon R, Okamura WH, Norman AW 1995 Structure-function 22. Lips P, Hackeng WHL, Jongen MJM, van Ginkel FC, relationships in the vitamin D endocrine system. Endocr Rev Netelenbos JC 1983 Seasonal variation in serum concentra- 16:200Ð257. tions of parathyroid hormone in elderly people. J Clin 6. Reichel H, Koeffler HP, Norman AW 1989 The role of the vita- Endocrinol Metab 57:204Ð206. min D endocrine system in health and disease. N Engl J Med 23. Ooms ME 1994 Osteoporosis in elderly women: vitamin D 20:980Ð991. deficiency and other risk factors. PhD Thesis, Vrije Universiteit, 7. Holick MF 1995 Environmental factors that influence the Amsterdam. cutaneous production of vitamin D. Am J Clin Nutr 1(Suppl 3): 24. Thomas MK, Lloyd-Jones DM, Thadhani RI, Shaw AC, 638SÐ645S. Deraska DJ, Kitch BT, Vamvakas EC, Dick IM, Prince RL, 8. Jongen MJM, Ginkel v FC, Vijgh vd WJF, Kuipers S, Finkelstein JS 1998 Hypovitaminosis D in medical inpatients. Netelenbos JC, Lips P 1984 An international comparison of N Engl J Med 338:777Ð783. vitamin D metabolite measurements. Clin Chem 30:399Ð403. 25. Lips P, Chapuy MC, Dawson-Hughes B, Pols HAP, Holick MF 9. Davies M, Heys SE, Selby PL, Berry JL, Mawer EB 1997 1999 An international comparison of serum 25-hydroxy- Increased catabolism of 25-hydroxyvitamin D in patients with vitamin D measurements. Osteoporos Int 9:394Ð397. partial gastrectomy and elevated 1,25-dihydroxyvitamin D levels. 26. Parfitt AM 1980 Morphologic basis of bone mineral measure- Implications for metabolic bone disease. J Clin Endocrinol ments: transient and steady state effects of treatment in osteo- Metab 82:209Ð212. porosis. Miner Electrolyte Metab 4:273Ð287. 10. Chapuy MC, Preziosi P, Maamer M, Arnaud S, Galan P, 27. Khaw KT, Sneyd MJ, Compston J 1992 Bone density, parathy- Hercberg S, Meunier PJ 1997 Prevalence of vitamin D insuffi- roid hormone and 25-hydroxyvitamin D concentrations in ciency in an adult normal population. Osteoporos Int middle-aged women. BMJ 305:273Ð277. 7:439Ð443. 28. McAuley KA, Jones S, Lewis-Barned NJ, Manning P, 11. Ooms ME, Lips P, Roos JC, van der Vijgh WJF, Popp-Snijders C, Goulding A 1997 Low vitamin D status is common among Bezemer PD, Bouter LM 1995 Vitamin D status and sex elderly Dunedin women. N Z Med J 110:275Ð277. hormone binding globulin: determinants of bone turnover and 29. Ooms ME, Roos JC, Bezemer PD, van der Vijgh WJF, bone mineral density in elderly women. J Bone Miner Res Bouter LM, Lips P 1995 Prevention of bone loss by vitamin D 10:1177Ð1184. supplementation in elderly women: a randomized double-blind 12. Netelenbos JC, Jongen MJM, van der Vijgh WJF, Lips P, trial. J Clin Endocrinol Metab 80:1052Ð1058. van Ginkel FC 1985 Vitamin D status in urinary calcium stone 30. Chapuy MC, Arlot ME, Duboeuf F, Brun J, Crouzet B, Arnaud S, formation. Arch Intern Med 145:681Ð685. Delmas PD, Meunier PJ 1992 Vitamin D3 and calcium to 13. Wielen vd RPJ, Lowik MRH, Berg vd H, Groot de LCPGM, prevent hip fractures in elderly women. N Engl J Med 327: Haller J, Moreiras O, Staveren v WA 1995 Serum vitamin D 1637Ð1642. concentrations among elderly people in Europe. Lancet 31. Cummings SR, Browner WS, Bauer D, Stone K, Ensrud K, 346:207Ð210. Jamal S, Ettinger B 1998 Endogenous hormones and the risk of 14. Feleke Y, Abdulkadir J, Mshana R, Mekbib TA, Brunvand L, hip and vertebral fractures among older women. N Engl J Med Berg JP, Falch JA 1994 Low levels of serum calcidiol in an 339:733Ð738. African population compared to a North European population. 32. Lips P, Pluijm SMF, Popp-Snijders C, Smit JH 2001 Vitamin D Eur J Endocrinol 141:358Ð360. status, sex hormone binding globulin, IGF-1 and markers of 15. McKenna M 1992 Differences in vitamin D status between bone turnover as determinants of bone mass and fractures in countries in young adults and the elderly. Am J Med 93:69Ð77. the Longitudinal Aging Study Amsterdam. J Bone Miner Res 16. Lips P, Duong T, Oleksik A, Black D, Cummings S, Cox D, 16(Suppl 1):S166. Nickelsen T 2001 A global study of vitamin D status and 33. Meyer HE, Henriksen C, Falch JA, Pedersen JI, Tverdal A parathyroid function in postmenopausal women with osteo- 1995 Risk factors for hip fracture in a high incidence area: porosis: Baseline data from the Multiple Outcomes of A caseÐcontrol study from Oslo, Norway. Osteoporos Int Raloxifene Evaluation Clinical Trial. J Clin Endocrinol Metab 5:239Ð246. 86:1212Ð1221. 34. Johnell O, Gullberg B, Allender E, Kanis JA 1992 The apparent 17. Zhao XH 1992 Nutritional situation of Beijing residents. incidence of hip fracture in Europe: a study of national register Southeast Asian J Trop Med Pub Health 23(Suppl 3):65Ð68. sources. MEDOS Study Group. Osteoporosis Int 2:298Ð302. 18. Bouillon RA, Auwerx JH, Lissens WD, Pelemans WK 1987 35. Lips P, Graafmans WC, Ooms ME, Bezemer PD, Bouter LM Vitamin D status in the elderly: seasonal substrate deficiency 1996 Vitamin D supplementation and fracture incidence in causes 1,25-dihydroxycholecalciferol deficiency. Am J Clin Nutr elderly persons. A randomized, placebo-controlled clinical 45:755Ð763. trial. Ann Intern Med 124:400Ð406. 1028 PAUL LIPS

36. Lips P, Netelenbos JC, van Doorn L, Hackeng WH, Lips CJM women referred to an osteoporosis outpatient clinic in 1991 Stimulation and suppression of intact parathyroid hor- Northern Italy for initial screening. Osteoporos Int 9:226Ð229. mone (PTH1-84) in normal subjects and hyperparathyroid 47. Hahn TJ, Birge SJ, Scharp CR, Avioli LV 1972 Phenobarbital- patients. Clin Endocrinol 35:35Ð40. induced alterations in vitamin D metabolism. J Clin Invest 37. McKane R, Khosla S, Egan KS, Robins SP, Burritt MF, 51:741Ð748. Riggs BL 1996 Role of calcium intake in modulating age- 48. Sedrani SH, Elidrissy AWTH, El Arabi KM 1983 Sunlight and related increases in parathyroid function and bone resorption. vitamin D status in normal Saudi subjects. Am J Clin Nutr J Clin Endocrinol Metab 81:1699Ð1703. 38:129Ð132. 38. Clements MR, Johnson L, Fraser DR A new mechanism for 49. Preece MA, Ford JA, McIntosh WB, Dunnigan MG, induced vitamin D deficiency in calcium deprivation. Nature Tomlinson S, O’Riordan JLH 1973 Vitamin D deficiency 325:62Ð65. among Asian immigrants to Britain. Lancet 1: 907Ð910. 39. Wiske PS, Epstein S, Bell NH, Queener SF, Edmondson J, 50. Grootjans-Geerts I 2001 Hypovitaminosis D: een versluierde Johnston C 1997 Increases in immunoreactive parathyroid hor- diagnose. Ned Tijdschr Geneeskd 145:2057Ð2060. mone with age. N Engl J Med 300:1419Ð1421. 51. Glerup H, Mikkelsen K, Poulsen L, Hass E, Overbeek S, 40. Marcus R, Madvig P, Young G 1984 Age-related changes in Andersen H 2000 Hypovitaminosis D myopathy without bio- parathyroid hormone and parathyroid hormone action in chemical signs of osteomalacic bone involvement. Calcif normal humans. J Clin Endocrinol Metab 58:223Ð230. Tissue Int 66:419Ð424. 41. Gabow PA, Hanson TJ, Popovtzer MM, Schrier RW 1977 52. Omdahl JL, Garry PJ, Hunsaker LA, Hunt WC, Goodwin JS Furosemide-induced reduction in ionized calcium in 1982 Nutritional status in a healthy elderly population: vitamin D. hypoparathyroid patients. Ann Intern Med 86:579Ð581. Am J Clin Nutr 36:1225Ð1233. 42. Stein MS, Scherer SC, Walton SL, Gilbert RE, Ebeling PR, 53. Holick MF 1998 Vitamin D requirements for humans of all Flicker L, Wark JD 1996 Risk factors for secondary hyper- ages: new increased requirements for women and men 50 years parathyroidism in a nursing home population. Clin Endocrinol and older. Osteoporos Int 8(Suppl 2):S24ÐS29. (Oxf) 44:375Ð383. 54. European Commission. Report on osteoporosis in the European 43. Rejnmark L, Vestergaard P, Heickendorff L, Andreasen F, Community: action for prevention. European Commission DG Mosekilde L 2001 Effects of thiazide- and loop-diuretics, alone V Directorate for Public Health, Luxembourg 1998. or in combination, on calcitropic hormones and biochemical 55. Weisman Y, Schen RJ, Eisenberg Z, Amarilio N, Graff E, bone markers: a randomized controlled study. J Intern Med Edelstein-Singer M, Goldray D, Harell A 1986 Single oral 250:144Ð53. high-dose vitamin D3 prophylaxis in the elderly. J Am Geriatr Soc 44. Khosla S, Atkinson EJ, Melton SLJ, Riggs BL 1997 Effects 34:515Ð518. of age and estrogen status on serum parathyroid hormone 56. Khaw KT, Scragg R, Murphy S 1994 Single-dose cholecalcif- levels and biochemical markers of bone turnover in women: a erol suppresses the winter increase in parathyroid hormone populationÐbased study. J Clin Endocrinol Metab 82:1522Ð1527. concentrations in healthy older men and women. Am J Clin 45. Gannage-Yared MH, Chemali R, Yaacoub N, Halaby G 2000 Nutr 59:1040Ð1044. Hypovitaminosis D in a sunny country: relation to lifestyle and 57. Keane EM, Rochfort A, Cox J, McGovern D, Coakley D, biochemical markers. J Bone Miner Res 15:1856Ð1862. Walsh JB 1992 Vitamin-DÐfortified liquid milk—a highly 46. Bettica P, Bevilacqua M, Vago T, Norbiato G 1999 High preva- effective method of vitamin D administration for house-bound lence of hypovitaminosis D among free-living postmenopausal and institutionalized elderly. Gerontology 38:280Ð284. CHAPTER 63 Vitamin D and the Pathogenesis of Rickets and Osteomalacia

A. MICHAEL PARFITT Division of Endocrinology and Center for Osteoporosis and Metabolic Bone Disease, University of Arkansas for Medical Sciences, Little Rock, Arkansas

I. Introduction V. Aspects of Vitamin D Metabolism Relevant II. Morphologic and Biochemical Aspects to Rickets and Osteomalacia of Mineralization VI. Vitamin D and the Pathogenesis of Impaired III. Processes Leading to Accumulation of Unmineralized Mineralization Tissue References IV. Evolution of Vitamin D Related Bone Disease

I. INTRODUCTION II. MORPHOLOGIC AND BIOCHEMICAL ASPECTS OF MINERALIZATION The vitamin D field has become so diverse and so complex that many forget how it all started—it was the The process whereby ions in solution are trans- study of rickets that led to the discovery of vitamin D. formed into a solid phase falls within the domain of Despite the multiplicity of effects on nontraditional physical chemistry, but skeletal mineralization is also a target tissues, the principal function of vitamin D and biological process that is controlled with regard to its its derivatives, in humans and most other mammals, is location, timing, rate, and relationship to cells and to still to facilitate the processes and mechanisms that are extracellular connective tissue matrices. A comprehen- necessary to prevent rickets and its adult counterpart sive theory of mineralization must be consistent with the osteomalacia. These diseases are both consequences of laws of chemistry and physics but must also account for defective mineralization, but within different tissues; its morphologic features. Disregard of these features the mineralization of growth plate cartilage and of led early students of bone, such as Franklin McLean, to bone have many features in common, but there are also believe that the matrix became mineralized as soon as important differences. Vitamin D deficiency may be it was formed, and that the presence of any unmineral- broadly classified as extrinsic, due to some combina- ized matrix was pathological [1]. This belief matched tion of nutritional deficiency and inadequate exposure the notion that biological mineralization was nothing to sunlight, and intrinsic, due to some combination more than the precipitation, within the appropriate of impaired absorption and accelerated catabolism of matrix, of crystals from a supersaturated solution, and vitamin D metabolites. The relative importance of these that only the composition of the solution determined mechanisms may be different in different parts of whether mineralization occurred [2]. The invariable the world, and different in children and adults. In both existence of a significant amount of unmineralized bone rickets and osteomalacia, there may be hypophos- matrix, or osteoid, in mammalian bone was first demon- phatemia, hypocalcemia, and secondary hyperparathy- strated by Lacroix and students in dogs and cats [3], and roidism, but their temporal relationships to one another soon after confirmed in human subjects by Frost and and to the events in bone may be different. A major Villanueva [4]. unsolved problem is whether changes in the composi- Microscopic examination of undecalcified sections tion of the blood are sufficient to account for the of bone obtained after double tetracycline labeling effects of vitamin D deficiency on cartilage and bone, (Chapter 59) allows the process of mineralization to be or whether one or more of the metabolites of vitamin D observed in situ, with preservation of its spatial rela- has actions on skeletal cells that promote mineral tionships to the bone and the cells and introduction of deposition. the dimension of time [5,6]. Tetracycline labeling has

VITAMIN D, 2ND EDITION Copyright © 2005, Elsevier, Inc. FELDMAN, PIKE, AND GLORIEUX All rights reserved. 1030 A. MICHAEL PARFITT been applied much less frequently to the study of min- present in ECF, and simultaneous removal of protons, eralization in cartilage than in bone, because the rate of generates a series of compounds beginning with sec- advance of the mineralization front and the consequent ondary calcium phosphate or brushite (CaHPO42H2O), distance between the two labels is driven mainly by the the first solid phase to be formed, and ending with rate of longitudinal growth, which reflects the rate of [Ca10(PO4)6(OH)2] [6]. The relevant chondrocyte proliferation [7]. However, two important activity products in ECF are in the region of metasta- features apply to mineralization in both tissues: spatial bility with respect to bone mineral, being undersatu- localization and a measurable time delay between the rated with respect to brushite but supersaturated with synthesis of matrix and the deposition of mineral within respect to hydroxyapatite [16]. Mechanisms to accom- it. In cartilage, the label is an aggregate of discrete plish initial mineral deposition include concentration patches, each corresponding to a single longitudinal sep- gradients between mineralizing and nonmineralizing tum, that form a band about 50 µm in width, extending sites maintained by cells and by the ion binding and all the way across the growth plate [8], and the average releasing properties of a variety of macromolecules time delay is about 24 hr. In bone, the label is contin- synthesized by cells [13,15,16,18], sequestration and uous, more sharply demarcated, and only 2 to 3 µmin subsequent release of calcium by mitochondria [20], width, and the average time delay is about 2 weeks. In and heterogeneous nucleation by outside agents or cartilage, the delay may reflect changes in gene substances [17]. Alkaline phosphatase is essential for expression in the chondrocytes [9], but in bone the normal mineralization [21], but its function remains delay reflects the need for extracellular changes in unknown. Mechanisms to restrain the growth of hydroxy- the matrix, collectively referred to as maturation, to apatite crystals include the precise spatial relationships occur before mineralization can begin. These changes between mineral and matrix [13], the presence at critical include completion of cross-linking between collagen locations of chelators of calcium [13] and inhibitors of fibrils [10,11] and the development of precise orienta- mineralization such as pyrophosphate [22], albumin [23], tion, conformation, and aggregation of a variety of non- and decorin [24], and the cellular and biochemical collagenous proteins and proteoglycans [12,13]. In characteristics of the quiescent bone surface, which is culture, maturation does not occur in the absence of the site of reversible mineral exchange with systemic viable cells [14]. ECF [19]. 2+ 3− − 2− Bone mineral consists of Ca , PO4 , OH , and CO Within this general framework, two types of miner- ions, arranged in space in accordance with the crystal alization can be recognized [25] (Chapter 27). In growth lattice structure of hydroxyapatite [15Ð18] (Chapter 27). plate cartilage and woven bone, which are temporary The composition is indeterminate because some of the structures destined soon to be removed, the matrix constituent ions can be replaced by other ions of similar is loosely textured and the collagen fibrils are small, radius, and at some lattice points calcium ions can be immature, and disordered. Mineral is deposited in the missing altogether [19]. The mineralizing potential of form of approximately spherical clusters of randomly extracellular fluid (ECF) depends on the free ionic oriented crystals of varying size. The clusters, termed activity, or effective concentration (denoted a), of Ca2+, calcospherulites, are spatially associated with matrix 2− + HPO4 , and H ions. The activity coefficients relating vesicles, which are small membrane-bound particles effective to actual ionic concentrations depend mainly that are derived by an unknown mechanism from on pH, temperature, and total ionic strength, which are chondrocytes. The vesicles are abundant and appear to all fairly constant in ECF, but the coefficients are lower be the only structures available for nucleation [26]. and more variable for divalent than for univalent Additional, more active roles in promoting mineraliza- ions [15]. For both Ca and P, ionic concentrations dif- tion that have been proposed [26Ð28] must be recon- fer from the total concentrations normally measured ciled with their distribution, with highest density in the because of protein binding and ion complexing, which resting and hypertrophic zones and lowest density in are also affected by pH. The often calculated total the proliferative and calcifying zones [29]. In lamellar plasma calcium × phosphate product, although mean- bone, which is invariably formed in apposition to an ingless in terms of physical chemistry, bears a rough existing surface, the matrix is compact in texture, empirical relationship to the true thermodynamic matrix vesicles are infrequent or absent, and the colla- 2+ 2− activity product [aCa ] [aHPO4 ]. gen fibrils are long and highly ordered. The mineral Mineralization is a phase transformation, not a crystals are aligned with their long axis parallel to the chemical reaction [17], but it is more likely that the collagen fibrils and are initially deposited within the complex structural order of hydroxyapatite is attained in hole zones by heterogeneous nucleation, but longer steps rather than all at once [16]. At sites of mineraliza- and wider crystals are subsequently formed on and 2+ 2− tion, the successive addition of Ca and HPO4 ions between the fibrils [18,25]. The differences between CHAPTER 63 Vitamin D and the Pathogenesis of Rickets and Osteomalacia 1031 these two types of mineralization explain why rickets bone tissue occupies all the available space enclosed and osteomalacia can under some circumstances by the cortices and does not undergo removal, but is vary independently in their severity and response to remodeled in a manner similar to the adult skeleton. treatment [30]. During both intramembranous ossification [35] and long bone growth, osteoid is formed beneath the perios- teum and mineralized bone is removed on the inner sur- III. PROCESSES LEADING face. When mineralization is defective, the accumulation TO ACCUMULATION OF of osteoid at sites other than the growing metaphysis UNMINERALIZED TISSUE should be referred to as osteomalacia, not as rickets. Thus, impaired mineralization leads to both rickets A. Growth Plate Cartilage and Osteoid and osteomalacia in the growing skeleton but only to in the Growing Skeleton osteomalacia in the mature skeleton. The kinetics of osteoid production and mineraliza- The process of endochondral ossification is tion in the growing skeleton have been studied most described in Chapters 27 and 33. The width of the epi- thoroughly in the rat tibia; several important observa- physeal growth plate depends on the rate of longitudi- tions were made [36]. First, although osteoid seams are nal growth, which is determined by the rate of new generally thinner in rats than in larger animals, they are chondrocyte production [31] and the life span for com- invariably present at sites of bone formation; because pletion of maturation prior to initial calcification. For of the temporal separation between matrix apposition example, in 5-week-old rats a width of about 600 µm and mineralization, there is also spatial separation. corresponds to a growth rate of 330 µm/day and a life Second, in 3-week-old rats, osteoid is found underneath span of about 1.8 days [26], and in 10-week-old rats the entire circumference of the periosteum; because its a width of 350 µm corresponds to a growth rate of extent cannot change except as a result of growth, a 180 µm/day and a life span of about 1.9 days [32]. significant increase in osteoid accumulation can occur These and other data indicate that although longitudinal only if the thickness of the seam increases. This con- growth slows progressively with increasing age, growth trasts with the mature skeleton, in which osteoid almost plate life span remains approximately constant. In always increases in surface extent before it increases in experimental rickets in rats, even though longitudinal thickness. Third, analogous to the regulation of growth growth is reduced threefold, growth plate width plate width, osteoid thickness depends on the rate of increases about fourfold in 6 weeks or by about matrix apposition, which corresponds to the rate of 27 µm/day because of an increase in life span of at least lateral (and hence longitudinal) growth [37], and on twelvefold [32]; in severe rickets, in the absence of the delay before the onset of mineralization that is treatment, growth plate life span is limited only by the imposed by osteoid maturation, known as the mineral- age of the animal. Evidently, the characteristic increase ization lag time (Mlt), which corresponds to the growth in growth plate width occurs despite a reduction in the plate life span. Like growth plate width, osteoid thick- rate of growth and is due entirely to a profound delay ness declines with increasing age because of a decline in mineralization, the pathogenesis of which is dis- in matrix apposition rate with a relatively constant cussed later. There is also structural disorganization of lag time. the metaphysis, partly due to mechanical effects [33] and partly to a profoundly altered pattern of vascular invasion [34]. B. Life History of Individual Osteoid During the transformation of calcified cartilage to Seams in the Adult Skeleton primary and then secondary spongiosa there is exten- sive deposition of osteoid. The kinetics of its produc- The formation of each new bone structural unit tion, life span, and mineralization have never been (osteon or hemiosteon) begins at the cement surface, studied by tetracycline labeling, but accumulation of a thin layer of lowly mineralized collagen-poor but osteoid contributes to the microscopic characteristics glycoprotein-rich connective tissue [38] that is laid down of the rachitic metaphysis. A possible source of confu- on the floor of the resorption cavity at the end of the sion must be addressed at this point. The term “rickets” reversal phase of each remodeling cycle, represented is commonly applied to the totality of skeletal abnor- in two-dimensional histological sections by the cement malities associated with defective mineralization in line, which remains in the same location, separating the growing skeleton, but it is more accurate to restrict new bone from old. The boundary between mineralized the term to changes in the growth plate and adjacent and unmineralized bone, referred to as the osteoid- metaphysis. In the vertebral bodies and ilium, cancellous bone interface, is the location of the mineralization 1032 A. MICHAEL PARFITT front, which normally moves away from the cement the osteoblast, and is presumably governed entirely by line during bone formation. Its rate of advance must be physicochemical factors [39]. distinguished from the rate at which mineralization A team of osteoblasts assembles on the cement proceeds after it is initiated [39]. In an individual moi- surface and begins to deposit a layer of bone matrix ety of bone matrix, mineral accumulation as a function referred to as an osteoid seam, which in standard of time is a continuous process that is conveniently histological sections appears in cortical bone as a ring, subdivided into two stages. There is an early rapid and in cancellous bone as a crescent tapering at each increase to about 75Ð80% of maximum within the end. Each seam has a measurable life span, during which first few days, referred to as primary mineralization. characteristic changes occur in the morphological It involves multiplication in the number of crystals, features and function of the osteoblasts, and in the occurs close to the osteoblast, and may be influenced thickness of the seam (Fig. 1). Matrix apposition is by its function. A much slower increase to about 95% most rapid (2.0 to 3.0 µm/day) at the outset, and the of maximum or more, over many months or even years, seam reaches a maximum thickness of approximately is referred to as secondary mineralization. It involves 15 to 20 µm after about 10Ð15 days, just before miner- slow growth in the size of crystals, with displacement alization begins. Mineral apposition is also most rapid of water, occurs remote in both time and space from at the outset (1.0 to 1.5 µm/day), and thereafter is always

FP

50

40

m) Mlt µ

Mx-AR 30 O.Th W.Th

20

MAR

Distance from cement line ( 10 MB.Th

0 0 20 40 60 80 100 120 Time from onset of matrix synthesis (days)

FIGURE 1 Model of bone formation, with growth curves for matrix apposition above and min- eral apposition below, showing distances from the cement line as functions of time at a single cross-sectional location of a representative basic metabolic unit (BMU). At any distance from the cement line, the horizontal distance between the lines is the instantaneous mineralization lag time (Mlt) at that distance. At any time, the vertical distance between the lines is the instantaneous osteoid thickness (O.Th) at that time. At any point the slopes of the lines (tangents) represent instantaneous apposition rates for matrix (Mx.AR) or mineral (MAR). For example, at t = 30 days, the instantaneous values are 20 µm for mineralized bone thickness (MB.Th), 16 µm for osteoid thickness, 0.5 µm/day for matrix osteoid apposition rate, and 0.8 µm/day for mineral apposition rate; the matrix deposited at that time will have a mineralization lag time of 26 days. Formation period (FP) is counted from the onset of matrix synthesis to the completion of mineralization, and in this example is 120 days, at which time completed wall thickness (W.Th) equals 50 µm. It is evident that the total area between the curves is given by FP × mean O.Th and by W.Th × mean Mlt, so that these expressions are equal. Furthermore, it follows that O.Th = Mlt × Mx.AR. Reprinted from Parfitt [5], in Chemistry and Biology of Mineralized Tissues, 1992, pp. 465Ð474, with kind permission from Elsevier Science. CHAPTER 63 Vitamin D and the Pathogenesis of Rickets and Osteomalacia 1033 more rapid than matrix apposition so that osteoid seam O.Th is osteoid thickness). In humans, bone formation thickness declines. Both matrix and mineral apposition is cyclical (Chapter 28), tetracycline fixation does not progressively slow with time, as the osteoblasts become occur during terminal mineralization, and it is impor- flatter and more extended in shape. About 80 days after tant to distinguish between instantaneous and mean the onset of mineralization when the bone surface has values. Osteoid thickness at any distance from the returned to its previous location about 50 µm from cement line is the product of the instantaneous matrix the cement line, matrix synthesis stops. The osteoid apposition rate (Mx.AR) and the instantaneous Mlt seam thickness has by now fallen to about 6 µm, and (Fig. 1). Instantaneous values are provided only by for a further 30 days mineral apposition continues at complete remodeling sequence reconstruction [41], and a progressively declining rate that is too slow for tetra- in practice only mean values are obtained. The best esti- cycline fixation to occur [40]. Eventually, the osteoid mate of the mean matrix apposition rate is the mineral seam disappears, because all the new matrix has become apposition rate averaged over the entire osteoid surface, mineralized. The osteoblasts have now completed their referred to as the adjusted apposition rate (Aj.AR), so histological transformation into lining cells, and con- that mean Mlt = mean O.Th/Aj.AR [39]. struction of the new BSU at that cross-sectional location In the rat, Mlt is identical with the osteoid matura- is finished. tion time (Omt), but in humans this may be true only A key quantity in understanding the mechanisms for the initial Mlt, which is usually about 10 days of osteoid accumulation and the pathogenesis of osteo- (Fig. 1). The cause of the subsequent increase in Mlt to malacia is the mineralization lag time (Mlt); this was about 30 days, which has no counterpart in the rat, is defined earlier for the rat, but its method of calculation unknown. One possibility is that the time required and significance are somewhat different in the adult for matrix maturation increases with the age of the human skeleton. In the rat, periosteal bone formation is osteoblast, and changes in matrix apposition rate continuous, the entire bone-forming surface is labeled and lag time together determine the progress of miner- with tetracycline, there is no need to distinguish alization (Fig. 2A). In this case, lag time would be an between instantaneous and mean values, and the best independent variable that remained identical with mat- estimate of the matrix apposition rate is the mineral uration time, and the changes in mineral apposition rate apposition rate (MAR) so that Mlt = O.Th/MAR (where would follow automatically. Alternatively, the rates of

AB m) µ Distance (

0 40 80 120 04080120 Time (days)

FIGURE 2 Two models of the relationship between matrix apposition, mineral apposition, and mineralization lag time. (A) Matrix apposition rate and mineralization lag time are separately and independently regulated as functions of osteoblast age, and the rate of mineral apposition changes as an automatic consequence. (B) Rates of matrix and mineral apposition are separately and independently regulated as functions of time, and the lag time changes as an automatic consequence. In both cases, the genuine independent variables are depicted by solid lines and the auto- matically determined variables by dashed lines. Reprinted from Parfitt [5], in Chemistry and Biology of Mineralized Tissues, 1992, pp. 465Ð474, with kind permission from Elsevier Science. 1034 A. MICHAEL PARFITT matrix and mineral apposition could be separately the mean life span of an individual moiety of osteoid, and independently regulated as functions of osteoblast which is the same as the mineralization lag time: age (Fig. 2B). For example, there could be a decline in the supply of mineral, since the net inward calcium OV/BV (%) = BFR/BV (%/year) × Mlt (years) (5) flux characteristic of osteoblasts must at some point change to the outward calcium gradient without net Because in the steady state bone turnover is determined flux characteristic of lining cells [19]. In this case, lag entirely by the frequency of remodeling activation and time would not be an independent variable but would the surface to volume ratio, and because FP is inversely progressively exceed maturation time. Fortunately, this proportional to Aj.AR [39], each of the three static uncertainty does not detract from the usefulness of Mlt indices of osteoid accumulation is determined by in the understanding of histomorphometric data and of a different pair of the same three kinetic indices [39]. the mechanisms of osteoid accumulation [5,36]. To Notably, osteoid volume is independent of matrix (or avoid the inconvenience of an infinite value (MAR = 0), mineral) apposition rate, which in the steady state affects it is sometimes useful to calculate the reciprocal of Mlt surface and thickness equally in opposite directions. as the osteoid mineralization rate (OMR): Although a reduction in mineral apposition rate is frequently taken to indicate defective mineralization, it OMR (%/d) = 100/Mlt (d) (1) is evident from Fig. 1 that matrix and mineral apposi- tion are closely coupled and that the mean mineral apposition rate can never exceed the mean matrix apposition rate. Consequently, a reduction in the mean C. Osteoid Indices and the Recognition rate of matrix apposition inevitably leads to, and is much of Impaired Mineralization the most common cause of, a reduction in the mean rate of mineral apposition. Both in normal subjects and Osteoid accumulation is assessed by three indepen- in patients with any metabolic bone disease except dent measurements (Chapter 59) that are related as fol- osteomalacia, there is a significant positive correlation lows [42] and defined below and in Fig. 1: between mean osteoid thickness and mean adjusted apposition rate, with broadly similar slopes (b) and OV/BV (%) = O.Th (mm) × OS/BS (%) intercepts (a) of the regression lines [42]. Although × BS/BV (mm2/mm3) (2) such a relationship is to be expected, it has an unantic- ipated consequence for the interpretation of the miner- A fall in trabecular thickness, as occurs to a modest alization lag time, as we can write extent in aging and in osteoporosis, will increase BS/BV and so increase OV/BV even if surface and O.Th = b(Aj.AR) + a. (6) width are unchanged; for the most accurate interpreta- tion, OV/BV should be corrected to the expected trabec- If this is combined with Eq. (3), we obtain ular thickness for age and sex. Each of the three indices is related differently to the underlying kinetic determi- Mlt = b + a/Aj.AR (7) nants. Osteoid thickness has already been discussed and is given by Because of this relationship, which defines a rectangu- lar hyperbola, when the matrix apposition rate falls, O.Th (µm) = Aj.AR (µm/day) × Mlt (days) (3) the mineralization lag time increases [6]. Another way of arriving at the same conclusion is to consider the Osteoid surface per unit of bone surface (OS/BS) is effect of prolongation of FP, which is also an inevitable determined entirely by the mean osteoid seam life span consequence of a reduction in matrix apposition rate. or formation period (FP) and by the average frequency From Fig. 1 it is clear that with which new osteoid appears at any point on the bone surface, which in the steady state is the same as FP × mean O.Th = W.Th × mean Mlt (8) the frequency of remodeling activation (Ac.f): Because O.Th has a minimum value [the intercept in OS/BS (%) = FP (years) × Ac.f (year−1) × 100 (4) Eq. (6)] and W.Th (the mean thickness of a completed BSU) is effectively constant in the short term, an Osteoid volume is determined entirely by the frac- increase in FP must be accompanied by an increase tional rate of bone turnover, which is the same as the in Mlt. It follows from this reasoning that neither a volume-based bone formation rate (BFR/BV), and by reduction in Aj.AR nor an increase in Mlt indicate that CHAPTER 63 Vitamin D and the Pathogenesis of Rickets and Osteomalacia 1035 mineralization is defective unless they are accompa- (Aj.AR; Fig. 3B). When Aj.AR is above 0.1 µm/day, nied by an increase in O.Th. there is the usual positive relationship between these variables; below 0.1 µm/day further decrements in Aj.AR are associated not with a fall in osteoid thick- IV. EVOLUTION OF VITAMIN D ness as in all other situations, but with a progressive RELATED BONE DISEASE increase, limited only by the normal thickness of new matrix or W.Th [44]. This reversal is the cardinal A. Histological Evolution and the Kinetic kinetic characteristic of defective mineralization; a Definition of Osteomalacia similar hyperbolic relationship is found between osteoid thickness and the fraction of osteoid surface In most patients, osteomalacia is preceded for many undergoing mineralization [45]. years by clinically silent secondary hyperparathy- On the basis of these relationships, the author defines roidism that accelerates the irreversible age-related osteomalacia by a combination of mean mineralization loss of cortical bone [43]. Exposition of this concept is lag time more than 100 days (OMR < 1/d) and mean aided by using the term “hypovitaminosis D osteopathy” osteoid thickness above the upper 95% confidence limit (HVO) to encompass the totality of osseous complica- predicted by the regression of osteoid thickness on tions of deficiency or altered metabolism of vitamin D Aj.AR in normal and osteoporotic subjects (Fig. 3B), or, [6]. There is usually no relationship between osteoid more simply, above an absolute value of 12.5 µm thickness and osteoid surface, but in HVO there is a (corrected for section obliquity). To distinguish general- hyperbolic relationship between these variables [6,42] ized osteomalacia from focal and atypical variant forms (Fig. 3A). This indicates that osteoid surface increases (6) needs an additional criterion of OV/BV greater than first and that osteoid thickness increases only slightly 10%. Diagnosis can be simplified by combining the dif- until OS/BS exceeds 70%, after which further increases ferent measurements into a single mineralization index in osteoid volume are due mainly to increasing thick- (MI, 46), which in arbitrary units is 0 to 15 in normal ness. In the same patients, osteoid thickness shows a subjects and greater than 30 in patients fulfilling the more complex relationship to adjusted apposition rate other criteria for osteomalacia. Patients with HVO who

60 AB

50

= 100d 40 Mlt

O.Th 30 (µm)

20

10

0 0 20 40 60 80 100 0 0.1 0.2 0.3 0.4 0.5 OS/BS (%) Aj.AR (µm/d)

FIGURE 3 Relationship of osteoid thickness (O.Th) to osteoid surface (OS/BS) (A) and adjusted appo- sition rate (Aj.AR) (B). In normal subjects and in patients with osteoporosis, there is no relationship between osteoid thickness and surface (A), but there is a significant positive relationship between osteoid thickness and adjusted apposition rate (B). During the development of osteomalacia there is a direct hyperbolic relationship between osteoid thickness and surface (A) and an inverse hyperbolic relationship between osteoid thickness and adjusted apposition rate (B); the usual ranges of values in established osteomalacia are shaded. The oblique line through the origin in (B) corresponds to a mineralization lag time of 100 days. Reprinted with permission from Parfitt AM. The physiologic and pathogenetic signif- icance of bone histomorphometric data. In: Coe FL, Favus MJ (eds.), Disorders of Bone and Mineral Metabolism. 2nd Edition. 2002. Lippincott Williams and Wilkins, Philadelphia, pp. 469Ð485. 1036 A. MICHAEL PARFITT do not meet these criteria have increased volume and sur- that the earliest formed matrix at each forming site face but not thickness of osteoid, increased bone forma- becomes mineralized but the later formed matrix does tion rate (BFR), the normal positive relationship between not, and HVOiii, in which mineralization never starts, so O.Th and Aj.AR, increased osteoclast indices [6], and that none of the matrix formed becomes mineralized. As low forearm bone density due to cortical thinning [47], all patients with HVOiii at the time of biopsy have likely resembling in every respect the histologic and densito- been through the stage of HVOii, they show a mixture metric features of primary hyperparathyroidism. of the two types of osteoid seam depicted in Fig. 4. This analysis identifies the earliest stage of HVO Both thickness and volume of osteoid are significantly (HVOi), when osteoid accumulation is due mainly to greater in HVOiii than in HVOii [Table I], but even in the increased frequency of remodeling activation and bone most severe cases individual values for mean osteoid turnover, before the emergence of a significant miner- thickness fall within the reference range for mean wall alization defect, as being due to secondary hyper- thickness [44]. parathyroidism [47] (Chapter 30). This concept has been sharpened by the MI [46], which permits HVOi to be subdivided into HVOia in which MI is normal (0Ð15), B. Biochemical Evolution of Rickets and HVOib, in which MI is moderately increased and Osteomalacia [15Ð30], but BFR is still high. Patients with HVO who meet the criteria for defective mineralization are further The cardinal metabolic consequence of vitamin D subdivided into those who retain some tetracycline deficiency is reduced net intestinal absorption of cal- double labels (HVOii) and those with no double labels cium [6,45] (see also Chapter 24). Fecal calcium (HVOiii). Pertinent histologic data in the four subgroups excretion is close to and can even exceed dietary of HVO are given in Table I. intake, but urinary calcium is low and calcium balance A different perspective on the fundamental nature of rarely more negative than −100 mg/day [48]. There is osteomalacia can be gained from the model of osteoid an equimolar deficit in net absorption of inorganic seam life span (Fig. 4). In every other condition, all phosphate, but the relative change is much smaller. matrix formed eventually mineralizes; the slopes of the According to the usual interpretation, calcium malab- curves representing matrix and mineral apposition, sorption leads in sequence to a fall in plasma calcium, although initially divergent, eventually converge, and the secondary hyperparathyroidism, reduced renal tubular loop formed by these curves ultimately closes. In con- reabsorption of phosphate, hypophosphatemia, and trast, in untreated osteomalacia some matrix remains reduction in calcium × phosphate product, which falls permanently unmineralized, the slopes of matrix and even further with the advent of more severe hypocal- mineral apposition remain divergent, and the loop never cemia. Eventually, deposition of mineral in osteoid is closes. The model also illuminates the difference between impaired because the supply of the relevant ions is HVOii, in which mineralization ceases prematurely, so reduced, and the alkaline phosphatase activity then

TABLE I Histology of Bone Mineralization

Variable Normal HVOia HVOib HVOii HVOiii n 143 18 8 13 29 OS/BS (%) 18.0 (9.5) 37.4 (16.7) 57.5(18.8) 87.0(5.9) 90.3(8.4) O.Th (µm)a 9.0 (1.7) 8.4(2.4) 10.6(1.9) 20.6(8.1) 26.5(10.8) OV/BV (%) 1.25 (0.74) 6.2(3.4) 10.8(3.8) 28.8(8.6) 40.8(17.0) BFR/BSb 13.8 (9.7) 34.2(26.3) 26.7(22.3) 12.4(8.4) 0 Mlt (d)c 36.8 (1.73) 39.8(1.62) 113(2.10) 355(2.2) 8 OMR (%/d) 3.06 (1.23) 2.89(1.41) 1.24(0.59) 0.213(0.169) 0 MId 8.0 (3.3) 9.3(3.8) 20.0(3.0) 54.8(18.1) 75.1(28.5)

Histologic indices of osteoid accumulation and their determinants in normal subjects and in patients with different degrees of hypovitaminosis D osteopathy (HVO), as defined in the text. Effects of sex, race, and age are small in relation to disease-related changes. Data expressed as mean (SD) but for Mlt expressed as geometric mean and multiplicative SD. aCorrected for section obliquity. bUnits are µm3/µm2/y. cCalculated after log transformation. dArbitrary units [46]. CHAPTER 63 Vitamin D and the Pathogenesis of Rickets and Osteomalacia 1037

Matrix

Mlt Mineral Distance (µm) 60

40 O. Th

Normal 20 Normal W. Th 0

60

40 (Mild) 20

0

60 Osteomalacia 40

20 (Severe)

0 100 200 300 400 500 600 Time (days) FIGURE 4 Kinetics of matrix and mineral apposition in osteomalacia. Evolution of bone formation at a single cross-sectional location. Each graph is constructed in a man- ner similar to Fig. 1, but with altered time scale. Mlt, Mineralization lag time; O.Th, osteoid thickness; W.Th, wall thickness. Note that in mild osteomalacia (HVOii) min- eralization is delayed in onset, retarded in rate, and premature in termination, whereas in severe osteomalacia (HVOiii) no mineralization occurs at all. Reprinted from Parfitt AM 1990 Bone-forming cells in clinical conditions. In: Hall BK (ed.), Bone: A Treatise, Vol. 1, The Osteoblast and Osteocyte, 1990, p. 395. Copyright CRC Press, Boca Raton, FL. rises. This traditional scheme requires considerable growth, but in infants the abnormality is associated modification with regard to the differences related to with a delayed increase in serum parathyroid hormone age of onset, the order in which the changes occur, their (PTH) [54], normal skeletal and renal tubular respon- pathophysiology, and their diagnostic significance. siveness to exogenous PTH [49], and spontaneous Many years ago, three stages were recognized in improvement; in adolescents and adults there is simple vitamin D deficiency in infants [49]. In Stage I an appropriate increase in PTH levels but impaired there was hypocalcemia and normal plasma phosphate, renal tubular as well as skeletal responsiveness to PTH in Stage II plasma calcium rose to normal, plasma [52,55], and the abnormality persists in the absence phosphate fell below normal, and alkaline phosphatase of treatment. Further discussion of this acquired form increased modestly, and in Stage III plasma calcium of pseudohypoparathyroidism is beyond the scope of and phosphate were both reduced, with no further this chapter, except for two points pertinent to subse- change in alkaline phosphatase. However, in current quent discussion. First, plasma calcium is determined practice there are many exceptions to this sequence by the homeostatic system at quiescent bone surfaces [50] (Chapter 65). Early hypocalcemia is rarely [19]. This system is independent of remodeling and is observed in older children [50,51] but is quite common regulated jointly by PTH and one or more metabolites during adolescence [52], although very uncommon in of vitamin D [15]. Second, deficiency of vitamin D adults [53] (Table I). The ability to release calcium from causes hypocalcemia mainly by loss of its effects bone may be compromised during periods of rapid on bone. 1038 A. MICHAEL PARFITT

TABLE II Biochemical Evolution of Hypovitaminosis D Osteopathy (HVO)a

Normalb (n = 23) HVOi (n = 26) HVOii (n = 11) HVOiii (n = 28)

Age (years) 60.3 ± 1.3 57.2 ±2.1 50.5 ± 6.3 58.1 ± 2.4 Plasma calcidiol (ng/ml) 23.7 ± 3.0 6.0 ± 0.5 6.8 ± 0.9(10) 4.1 ± 0.6(18)* Plasma calcitriol (pg/ml) 40.8 ± 6.9 46.0 ± 4.7(11) 39.1 ± 3.7(8) 21.7 ± 3.2(10)+ Plasma calciumc (md/dl) 9.64 ± 0.08 9.12 ± 0.11+ 7.95 ± 0.39+ 8.02 ± 0.17 Plasma phosphate (mg/dl) 3.47 ± 0.08 3.36 ± 0.11 2.91 ± 0.23+ 2.64 ± 0.13 Plasma Ca × P [(mg/dl)2] 33.5 ± 0.7 30.7 ± 1.1* 23.3 ± 1.6‡ 21.2 ± 1.2 Alkaline phosphatase (IU) 82.8 ± 3.9 132 ± 7.2‡ 201 ± 31.1 284 ± 24.2* NcAMPd (nmol/dl GF) 2.04 ± 0.26 4.01 + 0.34 6.62 ± 0.79 5.94 ± 0.61

a Stages are defined in the text. Number of analyses are shown in parentheses when less than number of subjects. Data are means ± SE. Significance levels are shown for differences in mean values from the column immediately to the left: *, p < 0.05; +, p < 0.01; ‡ p < 0.001. Data reprinted from Parfitt [6]. bVolunteers for bone biopsy. c Corrected for albumin. dN cAMP, Nephrogenous cyclic AMP, an in vivo bioassay for circulating PTH, which is given in units of nanomoles per deciliter of glomerular filtrate.

In most adults with HVOi, the mean plasma calcium Plasma and urinary calcium, TmPi/GFR, and plasma is slightly reduced, but the individual values are almost phosphate levels become lower, and PTH, NcAMP, always normal (Table II). PTH secretion is increased and alkaline phosphatase levels higher (Table II), but as shown both by radioimmunoassay [54,56] and by there are many individual exceptions. As in rickets, excretion of nephrogenous cyclic AMP (NcAMP) patients with severe hyperparathyroidism may suffer (Table II). Although mean tubular reabsorptive maxi- impaired tubular reabsorption of bicarbonate and mum for phosphorus divided by the glomerular filtra- amino acids as well as phosphate, resembling proximal tion rate (TmPi/GFR) and plasma phosphate are both renal tubular acidosis or the Fanconi syndrome [6,45], slightly reduced, individual values are usually normal. except for increased rather than decreased tubular Twenty-four hour urinary calcium excretion and fasting reabsorption of calcium. Hypophosphatemia is ade- urinary calcium/creatinine are often but not invariably quately explained by increased PTH secretion without reduced, and a moderate elevation of alkaline phos- the need to postulate an additional effect of vitamin D phatase is the most consistent abnormality. In extrinsic metabolite deficiency. Indeed, for the same increase in vitamin D depletion, the plasma calcidiol level at which NcAMP, TmPi/GFR is higher in secondary than in pri- abnormal mineral metabolism can first be detected in mary hyperparathyroidism because of the independent an individual is usually below 5 ng/ml [45,52], but in effect of plasma calcium to decrease phosphate reab- subjects with values between 5 and 10 ng/ml there is a sorption [53,57]. The mean calcidiol level is not sig- slight but statistically significant depression of mean nificantly lower in HVOii than in HVOi, but it does fall plasma calcium and phosphate and urinary calcium further in HVOiii (Table II). In contrast, the calcitriol and elevation of PTH [56], and most patients with his- levels can be normal in Stage II and do not become tologically verified HVOi have calcidiol values in this consistently subnormal until Stage III. Others have range (Table II). In intrinsic vitamin D depletion, the also found both normal and subnormal calcitriol levels complete biochemical, histological, and bone densito- in osteomalacia, although not classifying their cases in metric syndrome of HVOi can occur at plasma calcidiol the same manner [6]. levels between 10 and 20 ng/ml [47,53], presumably In summary, in HVOi there are characteristically no because there is an independent mechanism for calcium symptoms until a fracture occurs, which is why the malabsorption and consequent secondary hyper- existence of this intermediary stage was unrecognized parathyroidism that is unrelated to vitamin D but for so long. The only biochemical abnormality that accounts for the normal mean level of plasma calcitriol. would be revealed by routine screening is a raised The progression of HVO through Stages II and III is plasma alkaline phosphatase. Both fasting and 24-hr similar to the progression of infantile rickets through urinary calcium excretion are usually reduced. Skeletal Stages II and III, but it occurs over a much longer time radiographs are either normal or show only nonspecific scale and differs somewhat in detail. In general all osteopenia, but bone densitometry reveals that age-related the biochemical abnormalities become more severe. loss of bone is accelerated, especially appendicular CHAPTER 63 Vitamin D and the Pathogenesis of Rickets and Osteomalacia 1039 cortical bone but also axial trabecular bone, with a cor- Metabolic pathways leading from calciferol to cal- responding increase in fracture risk [47,53]. Plasma citriol have been extensively investigated but are pref- calcidiol is usually but not invariably low. There is both erentially followed only when body stores are greatly biochemical and histological evidence of secondary depleted. Normally, about 70% of the daily supply of hyperparathyroidism and of increased bone turnover. both calciferol and calcidiol is converted to more polar Defective mineralization is either absent (HVOia) or metabolites of low or absent biological activity that no more severe than in primary hyperparathyroidism undergo biliary and eventually fecal excretion [52], so (HVOib). Paradoxically, the deficit in forearm bone that only about 10% of available calciferol is used for density is greater in HVOi than in HVOii and HVOiii calcitriol production. The proportion following the alter- despite less severe hyperparathyroidism. This can only nate pathways can decrease to very low levels when be explained by slower progression and consequently necessary, but it increases to 90% for calciferol and 99% longer duration of accelerated cortical bone loss and for calcidiol in vitamin D treated hypoparathyroidism increased fracture risk [6]. It is likely that some patients [61]. Despite their quantitative importance in overall remain arrested indefinitely at this stage, a few progress vitamin D economy, not much is known about either to more severe hyperparathyroidism with radiographic the metabolites formed or their mechanism of production, osteitis fibrosa, but others eventually develop the com- and even less about how distribution between different plete clinical, biochemical, radiographic, and histologi- pathways is regulated (Chapter 2). cal syndrome of osteomalacia. However, it may Because of the usual wide margin of safety, malab- reasonably be assumed that all patients in Stages II or III sorption of dietary vitamin D is rarely of sufficient at the time of diagnosis traveled earlier through Stage I. severity to be the sole mechanism responsible for vitamin D depletion. The first additional mechanism to be proposed was interruption of a conservative entero- V. ASPECTS OF VITAMIN D hepatic circulation of calcidiol [6]. This proposal METABOLISM RELEVANT TO accounted for depletion of vitamin D of dermal as well RICKETS AND OSTEOMALACIA as dietary origin, but the magnitude of this pathway in human subjects, if it occurs at all, is much too small to Vitamin D metabolism can be affected at one of six fulfill its postulated role [52,62] (Chapter 75). It now levels [6]. Identification of the level is important in seems much more likely that the additional mechanism planning treatment, although the summation of inde- is accelerated catabolism of calcidiol in the liver initi- pendent factors at several levels may be needed to pro- ated by calcium deprivation [63], whether due to dietary duce clinical effects, and some diseases affect more than deficiency or intestinal malabsorption. It is because of one level. Each level is associated with a characteristic calcium deprivation that plasma calcitriol levels are profile of vitamin D metabolite concentrations in blood, increased in the earlier stages of intrinsic HVOi [64], but these must be interpreted with caution because as they are also in patients treated with anticonvulsants changes in vitamin D binding protein (DBP) can alter [52], although this is not evident from Table II, which total concentrations without altering free concentra- reports only data from patients who had bone biopsy. tions [6] (Chapter 8). Body stores of vitamin D, located Accelerated calcidiol catabolism is mediated by mainly in fat and to a lesser extent in muscle, are derived secondary hyperparathyroidism, either as a direct effect either from the photochemical production in skin of of increased circulating PTH [59,63] or (more likely) cholecalciferol or from dietary intake and intestinal as an indirect effect due to increased serum levels of cal- absorption of either chole- or ergocalciferol. Although citriol [59,65,66]. This mechanism explains the occur- the former source is more physiological [58], with rence of vitamin D deficiency in geographic regions current lifestyles the latter is equally important. The with high sun exposure but low calcium intake [59], and distinction was made earlier between extrinsic vitamin D it contributes to vitamin D deficiency in a variety of depletion, due to some combination of reduced skin gastrointestinal disorders [60,66] (Chapter 75). synthesis and reduced intake, and intrinsic depletion, The hepatic 25-hydroxylation of calciferol to calcid- due to intestinal malabsorption of vitamin D, augmented iol provides the principal transport form of vitamin D by an additional mechanism for increased fecal loss of and an additional component of body stores, located vitamin D [6]. mainly in muscle [6]. This process is impaired in Two infrequently emphasized features of normal cirrhosis of the liver, but rarely to a level that causes vitamin D metabolism are relevant to the pathogenesis osteomalacia (unless there is also malabsorption, as in of rickets and osteomalacia: first, its wastefulness in biliary cirrhosis) because the liver has such a large normal circumstances [52] and second, its suscepti- reserve capacity [52]. Significant calcidiol deficiency bility to disruption by calcium deficiency [59,60]. that is not due to depletion of its precursor is most 1040 A. MICHAEL PARFITT commonly the result of increased catabolism to biolog- Claims to the contrary [69,74] reflect the inability of ically inactive metabolites from drug-induced enzyme intermittent oral administration to sustain an adequate induction (Chapter 74) or from stimulation of existing blood level [72]. No other metabolite is essential; the enzymes by calcitriol or PTH [65] (Chapter 75). Loss impairment of mineralization during intramembranous of calcidiol bound to protein (both DBP and albumin) ossification that was previously attributed to deficiency occurs in the nephrotic syndrome and leads to sec- of 24 hydroxy-calcidiol [75] was shown to be the result ondary hyperparathyroidism and osteoid accumulation of very high circulating levels of calcitriol [70]. This in the absence of impaired renal function [6]. A similar counterintuitive effect has been known for some time mechanism operates during peritoneal dialysis, and [77], but its mechanism is still unknown. Nevertheless, it urinary loss of calcidiol is also increased in patients is possible that 24-hydroxy calcidiol contributes to pro- with biliary cirrhosis (Chapter 75). cesses such as bone embryonic development and matu- Calcitriol deficiency with normal body stores of ration of growth plate chondrocytes [75] (Chapter 33). vitamin D is most commonly the result of chronic Concerning the second controversy, there is evidence renal failure (Chapter 76) but can also be due to a that the effects of vitamin D deficiency in humans can genetic defect in renal 1α-hydroxylation, referred to as be corrected by giving enough calcium and phosphate pseudo vitamin D deficiency rickets (PVDR), heredi- intravenously [78], and that defects in calcitriol recep- tary hypocalcemia or vitamin D dependency type I tor binding can also be bypassed by providing sufficient (Chapter 71). Plasma calcitriol levels are reduced by mineral substrate [79Ð81]. The strength of the evidence magnesium depletion [6], but osteomalacia as a conse- is examined later in Section VI,B. In completely vita- quence has not been demonstrated. Calcitriol synthesis min DÐdeficient rats, maintenance of normal plasma is impaired by deficiency of PTH (Chapter 64), but it calcium and phosphate levels maintained normal is doubtful whether this causes osteomalacia, possibly growth, as well as normal growth plate and bone min- because bone turnover is so low. However, there is one eralization [82Ð83]. In clinical studies, the determina- adequately documented case of osteomalacia due to tion that radiographic and histological abnormalities are pseudohypoparathyroidism with secondary hyper- due to defective mineralization rather than to secondary parathyroidism [67]. Very low plasma calcitriol levels hyperparathyroidism is not as straightforward as is often are found during prolonged total parenteral nutrition, assumed, but if the conclusions of these studies are but they have not been clearly related to the presence taken at face value [78Ð81], then vitamin D is clearly or type of metabolic bone disease [6]. Finally, cal- not essential for mineralization. Nevertheless, one or citriol may be ineffective because of one of several more metabolites of vitamin D could have direct actions kinds of defect in its receptors (VDR), referred to as on those cells that contribute to the process in normal hereditary vitamin D-resistant rickets (HVDRR) or circumstances [6,52,74]. vitamin D dependency rickets type II (Chapter 72). A. The Effects of Vitamin D Are Not VI. VITAMIN D AND THE PATHOGENESIS Mediated Solely by Circulating Calcitriol OF IMPAIRED MINERALIZATION In patients with histologically verified osteomalacia The role of vitamin D in sustaining normal mineral- or with radiographically unambiguous rickets, plasma ization has given rise to two related controversies. First, calcitriol concentrations can be within the appropriate is calcitriol the only metabolite of physiological impor- reference ranges [45,84Ð87] (Table II). The levels are tance, other than as a precursor [68], or must some indeed inappropriately low for the degrees of PTH other metabolite also be considered [69]? Second, is hypersecretion and hypophosphatemia [86], as is indi- the action of vitamin D on bone mediated solely by cated by the very high levels attained during the early changes in the calcium and phosphate concentrations stages of treatment with vitamin D [71,87,88], but the in ECF [68], or does it influence mineralization more lack of target cell responses to a concentration of cal- directly [48,52]? In both cases the contestants have citriol that is normally adequate requires explanation. often failed to recognize the difference between an In adults with osteomalacia, both biochemical and histo- essential function that confers an absolute requirement logical indices of vitamin D depletion appear to corre- and a contributory function that confers only a relative late better with either the sum of calcidiol (in ng/ml) requirement. Concerning the first controversy, calcitriol and calcitriol (in pg/ml) concentrations, or with calcidiol alone can correct the clinical, biochemical, radio- alone, than with calcitriol alone [45,53]. This suggests graphic, and histological effects of vitamin D defi- that calcidiol, or some other metabolite for which ciency both in humans [70,71] and in the rat [72,73]. calcidiol is a precursor, such as 24-hydroxycalcidiol, CHAPTER 63 Vitamin D and the Pathogenesis of Rickets and Osteomalacia 1041 might function as an agonist for calcitriol, or might disease (Chapter 75) or anticonvulsant administration have its own effects mediated by a different receptor. [89]. Theories that ascribe all manifestations of rickets In infants with untreated rickets, the plasma Ca × P and osteomalacia to deficiency of circulating calcitriol product correlated with the plasma concentration of cal- alone may be able to account for its persistence, but citriol and not calcidiol, although a higher than normal they have much greater difficulty accounting for its ini- calcitriol level was needed to maintain a normal prod- tiation. At the onset of HVO in patients with extrinsic uct [88]. In this study the plasma level of 24-hydroxy- vitamin D deficiency, what causes calcium malabsorp- calcidiol was often undetectable; this compound might tion and a small but significant fall in plasma calcium function in a permissive manner, so that a fall in its when plasma calcitriol is maintained at a normal (or concentration below a critical level would increase the even increased) level by secondary hyperparathyroidism need for calcitriol, but without dose-related effects [90,91]? A similar argument applies to the increased above the critical level. vitamin D requirement of primary hyperparathy- Higher than normal calcitriol levels could be needed roidism, due to accelerated calcidiol catabolism in the to correct hypocalcemia and to restore normal miner- presence of increased plasma calcitriol levels [92]. alization when the calcitriol responsive cells are sepa- During the evolution of HVO, there is an early rated from the mineralized bone by a much wider than fall in plasma concentrations of both calcidiol normal layer of uncalcified osteoid through which the [47,53,84,91,93] (Table I) and 24-hydroxycalcidiol mineral ions must travel [5,6,52] (Fig. 5). However, no [74,85,88]. Calcium absorption and retention in bone similar reason is evident for the failure of intestinal are increased in humans by pharmacological doses of mucosal cells to accomplish normal calcium transport, 24-hydroxycalcidiol [69], but it is not known whether at least in patients who do not have an independent cause such effects occur at physiological blood levels. for impaired calcium absorption, such as intestinal Calcidiol binds to intestinal receptors for calcitriol, but

Mineralization: Systemic or local control? + + 2− H Ca HPO4 25HCC 1,25DHCC Ca P Capillary

Osteoblast

1,25

DHCC Osteoid

Osteocyte

M. front interface

Mineralized bone

FIGURE 5 Biochemical and morphological approaches to mineralization. At left are shown the directional movements of ions between a blood vessel above and the bone below without reference to intervening structures. These structures are depicted diagrammatically at right. 25HCC, 25-Hydroxycholecalciferol (calcidiol); 1,25DHCC, 1,25-dihydroxychole- calciferol (calcitriol); M., mineralization; interface, boundary between osteoid and mineral- ized bone. The osteoblasts and osteocytes can be influenced by circulating levels of calcium, phosphate, and calcitriol, and also by locally produced calcitriol, either autocrine or paracrine. Reprinted from Parfitt [5] 1992 in Chemistry and Biology of Mineralized Tissues, pp. 465Ð474, with kind permission from Elsevier Science. 1042 A. MICHAEL PARFITT with approximately 500- to 1000-fold lower affinity (less than 3.0 mg/dl) and low total plasma Ca × P prod- (Chapter 11), although calcidiol is only 100 times less uct [less than 30 (mg/dl)2] and healing rickets with effective than calcitriol in promoting bone resorption increases in these values [106,107]. Freshly harvested in vitro [94]. Seemingly, these differences in activity rachitic rat growth plate cartilage will mineralize in could be offset by the much higher total plasma con- normal rat serum and in aqueous solutions with the centration of calcidiol, but there is only a tenfold dif- same pH and Ca × P product [108,109]. Calcification ference in free concentrations, based on estimates of the occurs in the same region as the cartilage as in vivo, association constants for binding to the same circulating and fails to occur if the viability of the tissue is protein [95]. Consequently, a fall in plasma calcidiol compromised. The relationship of rickets to the total level below normal could not significantly modify total plasma Ca × P product has been confirmed many receptor occupancy in the target cells that respond to times [6,45], and the relationship of the product to the circulating calcitriol, although some more complex effect thermodynamic activity product for various solid on receptor function remains possible [96]. phases has been analyzed [109]. The same relationship A more promising approach to the clinical paradox is holds in rickets complicating osteopetrosis, in which the possibility that one or more dihydroxylated metabo- low plasma levels of calcium and/or phosphate are lites are produced locally in target tissues, as is strongly due to increased mineral sequestration in bone [110]. suggested by studies with isolated bone (97,98) and The relationship is disturbed in patients with renal intestinal cells [99,100], by the in vivo intestinal response failure [111], possibly due to the presence of mineral- to a pharmacological oral dose of calcidiol [101], and by ization inhibitors, such as magnesium [112], or the the in vitro effects of calcidiol to increase bone resorp- effect of metabolic acidosis on the activity coefficient 3− tion [102] and intestinal calcium transport [103] in of PO4 [109]. The data clearly established the impor- isolated tissues. These studies were carried out when cal- tance of plasma composition, which is disturbed indi- cidiol was believed to be the active form of vitamin D, rectly by vitamin D deficiency [109], but do not rule and were forgotten (or suppressed) when this belief was out additional direct local effects. Such effects of cal- superseded, but the results can only be explained by the citriol have been demonstrated in matrix vesicles, includ- local conversion of calcidiol to calcitriol. If bone cell and ing increases in the activity of alkaline phosphatase and intestinal cell 1a-hydroxylases were less influenced by metalloproteinase [113]. PTH and phosphate than is the renal 1a-hydroxylase, as In older children and adults, the relationship is generally the case for extrarenal calcitriol production between plasma composition and the state of mineral- [104, Chapter 5], local production of calcitriol would be ization is less consistent [6,48,74], being clearly evident more substrate dependent than circulating calcitriol and in some series of patients [114Ð116] but not in others would be impaired sooner by a fall in plasma calcidiol [117Ð119]. There are significant correlations between concentration below normal. This mechanism would plasma phosphate and adjusted apposition rate, between also account for the improvement in bone mineralization plasma calcium and mean osteoid thickness [120] and and intestinal calcium absorption brought about by between plasma Ca × P product and MI [46], but their calcidiol administration in patients with chronic renal magnitude is too low for useful prediction in individual failure [105], and for the much greater relative therapeu- patients. Also the high unexplained variance leaves plenty tic potency of dihydrotachysterol in hypoparathyroidism of room for other factors [46]. Calculation of an ion prod- than in osteomalacia [61]. Similar considerations would uct more clearly related to the physical chemistry of bone apply to local production of 24-hydroxycalcidiol [99]. It mineral may remove some discrepancies [109], but many seems reasonable to speculate that circulating calcitriol is remain. A more serious flaw in this line of reasoning is most important for the regulation of calcium homeostasis, that single measurements in the fasting state, as in normal locally produced calcitriol (and possibly 24-hydroxy- clinical practice, do not adequately represent body fluid calcidiol) are most important for the regulation of bone composition because of the substantial circadian varia- remodeling and mineralization, and both circulating and tion [19,121]. Nevertheless, it seems unlikely that such locally produced metabolites are important for the regu- variation could account for the absence of osteomalacia lation of calcium absorption. in some patients with a degree of persistent hypophos- phatemia that in other patients would be regarded as a sufficient explanation for their osteomalacia [120,122]. B. Evidence for Direct as Well as Indirect Even in the rat, a species in which mineralization proba- Effects of Vitamin D on Bone bly depends more closely on plasma composition than in humans, healing of rickets can be detected radiograph- It has been known since the early 1920s that active ically in response to vitamin D administration while infantile rickets is associated with low plasma phosphate the Ca × P product is still subnormal [73]. CHAPTER 63 Vitamin D and the Pathogenesis of Rickets and Osteomalacia 1043

It was previously demonstrated that the early effects been higher in the infused than in the vitamin DÐ of vitamin D deficiency in adults are due entirely to sec- replete animals. Osteoid surface and volume were sig- ondary hyperparathyroidism in the presence of normal nificantly lower in the infused rats [83], consistent bone mineralization; the correction of such effects by with oversuppression of PTH secretion. Consequently, calcium has no bearing on the issue in question. Neither it remains possible that higher mean plasma levels of does the cure by calcium administration of clinical and calcium and phosphate are needed to sustain normal radiographic appearances that resemble rickets but mineralization in the absence than in the presence of are due to severe dietary calcium deficiency [123]. vitamin D. Radiographic evidence for impaired mineralization The persistence in early osteomalacia of some dou- is often ambiguous. Looser’s zones can occur in the bly labeled surfaces with normal or only moderately absence of osteomalacia and heal spontaneously [124]. reduced rates of mineral apposition [6] indicates that Metaphyseal erosions in the absence of increased mineralization can proceed at the beginning of the growth plate width are due to osteitis fibrosa, but they osteoid seam life span, although it ceases prematurely have often been attributed erroneously to rickets [125]. (Fig. 4). Mineralizing and nonmineralizing osteoid Problems in the histological recognition of defective seams are often close together, sometimes even in mineralization were outlined earlier and are discussed direct continuity, and they are exposed to the same in more detail elsewhere [6]. In the previously cited microcirculation, so that the difference between them clinical studies [78Ð81], the authors demonstrated lit- cannot be explained in terms of chemical change tle awareness of the difficulties just mentioned, and the alone. However, at doubly labeled seams, a higher pro- evidence that the lesions corrected by mineral admin- portion of the surface is lined by osteoblasts [44,128], istration alone were the result of defective mineraliza- suggesting that these cells, possibly in conjunction tion rather than of secondary hyperparathyroidism was with the osteocytes derived from them lying within the inconclusive. Of six cases included in the four reports, osteoid [129], are able to promote mineralization in the three had metaphyseal erosions with normal growth face of a moderate reduction in plasma ion product, but plate width, wrongly reported as rickets [79,80], three do so for a shorter time than normal in vitamin D had qualitative bone histology only [78,79], and two depletion. When this function is lost, mineralization had no bone biopsy at all [79,80]. Only in one case was ceases even though matrix apposition continues slowly there convincing histological evidence of osteomalacia, and the osteoid seam gets progressively thicker. In more which healed with prolonged calcium infusion [81]. severe osteomalacia, osteoblasts are fewer or absent Studies in the rat have provided stronger evidence altogether [130], mineralization never begins, and dou- that mineral alone can be effective in vivo in the ble labels are not found (Fig. 4). A similar relationship absence of vitamin D. Restoration of normocalcemia is observed during treatment: the recovery of mineral- in vitamin DÐdeficient rats by a high calcium diet cor- ization in response to calcitriol administration, indicated rected both abnormal bone enzyme activity [126] and by double labeling, occurs preferentially at surfaces defective osteoid maturation [127]. Complete vitamin D where new osteoblasts have appeared [131,132]. deficiency was not demonstrated in these experiments, The bone histological data in patients with osteo- but in 25-day-old rats weaned from vitamin DÐdeficient malacia strongly suggest that deficiency of calcitriol mothers, and maintained without access to ultraviolet (and possibly also other metabolites) impairs some func- light or dietary vitamin D, plasma levels of calcitriol tion of the osteoblast that favors mineralization. This were undetectable [82]. Calcium chloride and buffered proposal is consistent with the presence in osteoblasts of sodium phosphate infused into separate jugular veins, calcitriol receptors (VDR) [133], the autoradiographic in amounts sufficient to maintain the same plasma cal- localization of labeled calcitriol in osteoblast nuclei [134], cium and phosphate levels as in vitamin DÐreplete rats, the in vitro stimulation by calcitriol of several actions of maintained normal growth plate width and normal osteoblasts including production of alkaline phosphatase tetracycline based indices of mineralization [83]. This [135] and osteocalcin [136], and sodium independent experiment demonstrated conclusively that vitamin D phosphate transport [137], the in vivo enhancement was not essential for mineralization but did not exclude by calcitriol of mineral apposition rate in young mice a contributory role for vitamin D under normal condi- [138], and the morphological changes induced by cal- tions. First, as previously discussed, there is only a citriol in the cells lining quiescent bone surfaces [139] very approximate relationship between total plasma that are of osteoblast lineage [19]. A local cellular levels and thermodynamic activity products. More effect of one or more vitamin D metabolites would also importantly, the plasma levels were measured only account for the abnormalities in collagen cross-linking once every 3 days at an unspecified time; because of and other changes in bone matrix maturation and com- circadian variation [121] the mean levels could have position [36,140,141], and the changes in intermediary 1044 A. MICHAEL PARFITT metabolism in cartilage cells [142,143], that have been could plausibly be accompanied by similar defects in found in vitamin D deficiency, although it is less clear transport across the quasi-epithelium that covers all that these are the result of a direct rather than an indi- bone surfaces [5,146]. rect effect of vitamin D on osteoblast and chondrocyte function. Finally, the proposal also accounts for the greater ability of vitamin D than calcium carbonate to References improve bone mineralization in chronic renal failure, despite equivalent changes in total Ca × P product 1. McLean FC, Urist MR 1955 Bone: An Introduction to the [144], on the assumption that bone cells can make Physiology of Skeletal Tissue. Univ. of Chicago Press, calcitriol from its precursor. Chicago. The usual approach to mineralization has been to 2. Robison R 1932 The Significance of Phosphoric Esters in Metabolism. New York Univ. Press, New York. study the physical chemistry of the solution in which 3. Lacroix P 1960 45Ca autoradiography in the study of bone the ions originate, and the events taking place in bone, tissue. In: Rodahl K, Nicholson JT, Brown EM (eds) Bone as and to largely ignore what happens in between [5]. But a Tissue. McGraw-Hill, New York, pp. 262Ð279. the circulating ions have to traverse a rather complex 4. Frost HM, Villanueva AR 1960 Observations on osteoid pathway before they arrive at the site of mineralization seams. Henry Ford Hospital Med Bull 8:212Ð219. 5. Parfitt AM 1992 Human bone mineralization studied by (Fig. 5). Having left the capillary and diffused through in vivo tetracycline labeling: Application to the pathophysi- marrow connective tissue, they must pass through a ology of osteomalacia. In Slavkin M, Price P (eds) Chemistry layer of osteoblasts and a layer of osteoid before they and Biology of Mineralized Tissues. Excerpta Medica, can reach the site of mineral disposition. Osteoblasts on Amsterdam, pp. 465Ð474. the surface, osteocytes within the osteoid, and osteocytes 6. Parfitt AM 1997 Osteomalacia and related disorders. In: Avioli LV, Krane SM (eds) Metabolic Bone Disease and within mineralized bone are joined by a communicating Clinically Related Disorders, 3rd Ed. Academic Press Inc., network of cellular processes within the canaliculi. Very San Diego, pp. 645Ð662. little is known about how mineral ions actually travel 7. Hansson LI, Menander-Sellman K, Stenstrom A, Thorngren KG through this complex structure, but it would be sur- 1972 Rate of normal longitudinal bone growth in the rat. prising if cellular transport mechanisms of some kind Calcif Tissue Res 10:238Ð251. 8. Thorngren KG, Hansson LI, Menander-Sellman K, were not involved in the movement of ions from the Stenstrom A 1973 Effect of hypophysectomy on longitudinal extracellular fluid to the site of mineral deposition. bone growth in the rat. Calcif Tissue Res 11:281Ð300. Indeed, it seems likely that calcitriol could stimulate 9. Caplan AI, Boyan BD 1994 Endochondral bone formation: the inward transport of calcium and/or phosphate ions The lineage cascade. In Hall BK (ed) Bone: A Treatise, through or between cells at sites of mineralization [5], Volume 8, Mechanisms of Bone Development and Growth. CRC Press, Boca Raton, Florida, pp. 1Ð46. consistent with its known effects on the cells of the 10. Yamauchi M, Chandler GS, Katz EP 1992 Collagen cross- intestinal mucosa (Chapter 24) and the renal tubule linking and mineralization. In: Slavkin M, Price P (eds) (Chapter 29). Chemistry and Biology of Mineralized Tissues. Excerpta The osteoblast thus influences mineralization in two Medica, Amsterdam, pp. 39Ð46. ways, by its effects on matrix maturation [14] and by 11. Gerstenfeld LC, Riva A, Hodgens K, Eyre DR, Landis WJ 1993 Post-translational control of collagen fibrillogenesis in its effects on mineral transport [52]. Furthermore, cal- mineralizing cultures of chick osteoblasts. J Bone Miner Res cium and phosphate ions must be regarded not just as 8:1031Ð1043. substrates for apatite formation but as part of the envi- 12. Boskey AL 1990 Mineral-matrix interactions in bone and ronment of the cell that is involved in their transport, cartilage. Clin Orthop Related Res 281:244Ð274. as osteoblast function is influenced by the circulating 13. Gorkale JA, Robey PG, Boskey AL 2001 The biochemistry of bone. In: Marcus R, Feldman D, Kelsey J (eds) Osteoporosis. and presumably also local levels of calcium [145] and 2nd Ed. Academic Press, San Diego, pp. 107Ð188. phosphate [52,120]. The concept that mineralization 14. Marsh ME, Munne AM, Vogel JJ, Cui Y, Franceschi RT 1995 normally depends both on the availability of substrate Mineralization of Bone-like Extracellular Matrix in the ions via the circulation and on the activity of Absence of Functional Osteoblasts. J Bone Min Res osteoblasts and chondrocytes, and that vitamin D influ- 10:1635Ð1643. 15. Parfitt AM, Kleerekoper M 1980 The divalent ion homeo- ences both of these processes, although by no means static system: Physiology and metabolism of calcium, phos- rigorously established, enables all the apparently con- phorus, magnesium, and bone. In: Maxwell M, Kleeman CR flicting data, laboratory and clinical, to be reconciled. (eds) Clinical Disorders of Fluid and Electrolyte Metabolism, The concept has the additional merit of providing a 3rd Ed. McGraw-Hill, New York, pp. 269Ð398. basis for unifying the pathogenesis of all major forms 16. Neuman WF 1980 Bone material and calcification mechanisms. In: Urist MR (ed) Fundamental and Clinical Bone Physiology. of osteomalacia, as hereditary or acquired defects in Lippincott, Philadelphia, Pennsylvania, pp. 83Ð107. phosphate transport across the renal tubular epithe- 17. Glimcher MJ 1992 The nature of the mineral component lium, whether intrinsic or due to humoral factors, of bone and the mechanism of calcification. In: Coe FL, CHAPTER 63 Vitamin D and the Pathogenesis of Rickets and Osteomalacia 1045

Favus MJ (eds) Disorders of Bone and Mineral Metabolism. 36. Baylink D, Stauffer M, Wergedal J, Rich C 1970 Formation, Raven, New York, pp. 265Ð286. mineralization, and resorption of bone in vitamin DÐdeficient 18. Glowacki J 2002 Mechanisms of Biomineralization. In: rats. J Clin Invest 49:1122Ð1134. Disorders of Bone and Mineral Metabolism 2nd Ed. FL Coe, 37. Parfitt AM 1997 Genetic effects on bone mass and turnover- MJ Favus (eds) Lippincott Williams & Wilkins, pp. 227Ð234. relevance to black/white differences. J Amer Coll Nutr 19. Parfitt AM 1993 Calcium homeostasis. In: Mundy GR, 16:325Ð333. Martin TJ (eds) Handbook of Experimental Pharmacology, 38. Schaffler MB, Burr DB, Frederickson RG 1987 Morphology Volume 107, Physiology and Pharmacology of Bone. Springer- of the osteonal cement line in human bone. Anat Rec Verlag, Heidelberg, pp. 1Ð65. 217:223Ð228. 20. Brighton CT, Hunt RM 1978 The role of mitochondria in 39. Parfitt AM 1992 The physiologic and pathogenetic signifi- growth plate calcification as demonstrated in a rachitic cance of bone histomorphometric data. In: FL Coe, MJ Favus, model. J Bone Joint Surg 60-A:630Ð639. (eds) Disorders of Bone and Mineral Metabolism. 2nd 21. Fedde KN, Blair L, Silverstein J, Coburn SP, Ryan LM, Edition. 2002. Lippincott Williams and Wilkins. Philadelphia, Weinstein RS, Waymire K, Narisawa S, Millan JL, pp. 469Ð485. MacGregor GR, Whyte MP 1999 Alkaline phosphatase 40. Parfitt AM, Foldes J, Villanueva AR, Shih MS 1991 The dif- knock-out mice recapitulate the metabolic and skeletal ference in label length between demethylchlortetracycline defects of infantile hypophosphatasia. J Bone Min Res. and oxytetracycline: Implications for the interpretation of 14:2015Ð2026. bone histomorphometry. Calcif Tissue Int 48:74Ð77. 22. Alcock NW, Shils ME 1968 Association of inorganic 41. Eriksen EF 1986 Normal and pathological remodeling of pyrophosphatase activity with normal calcification of rat human trabecular bone: Three-dimensional reconstruction of costal cartilage in vivo. Biochem J 112:505Ð510. the remodeling sequence in normals and in metabolic bone 23. Meyer JL, Fleisch H 1984 Determination of calcium phos- disease. Endocr Rev 4:379Ð408. phate inhibitor activity; critical assessment of the methodology. 42. Parfitt AM 1984 The cellular mechanisms of osteoid accu- Miner Electrolyte Metab 10:249Ð258. mulation in metabolic bone disease. In: Mineral Metabolism 24. Hoshi K, Kemmotsu S, Takeuchi Y, Amizuka N, Ozawa H Research in Italy, Vol. 4. Wichtig Editore, Milano, pp. 3Ð9. 1999 The Primary Calcification in Bones Follows Removal 43. Parfitt AM 1986 Accelerated cortical bone loss: Primary and of Decorin and Fusion of Collagen Fibrils. J Bone Min Res secondary hyperparathyroidism. In: Uhthoff H (ed) Current 14:273Ð280. Concepts of Bone Fragility. Springer-Verlag, New York, 25. Christoffersen J, Landis WJ 1991 A contribution with review pp. 279Ð285. to the description of mineralization of bone and other calci- 44. Meunier PJ, Van Linthoudt D, Edouard C, Charhon S, Arlot M fied tissues in vivo. Anat Rec 230:435Ð450. 1981 Histological analysis of the mechanisms underlying 26. Schenk RK, Hunziker EB 1991 Growth plate: Histophysiology, pathogenesis and healing of osteomalacia. Proceedings, cell, and matrix turnover. In: Glorieux FH (ed) Rickets, Sixteenth European Calcified Tissue Symposium. Abstracts Nestle Nutrition Workshop Series, Vol. 21. Raven, New York, 33:771Ð774. pp. 63Ð78. 45. Peacock M 1993 Osteomalacia and rickets. In: Nordin BEC, 27. Anderson HC, Morris DC 1993 Mineralization. In: Mundy GR, Need AG, Morris HA (eds), Metabolic Bone and Stone Martin TJ (eds) Handbook of Experimental Pharmacology, Disease, 3rd Ed. Churchill Livingstone, London, pp. 83Ð118. Volume 107, Physiology and Pharmacology of Bone. Springer- 46. Parfitt AM, Qiu S, Rao DS. The Mineralization Index: A new Verlag, Heidelberg, pp. 267Ð298. approach to the histomorphometric appraisal of osteomalacia. 28. Boskey AL, Boyan BD, Schwartz Z 1997 Matrix Vesicles (in press). Promote Mineralization in a Gelatin Gel. Calcif Tissue Int, 47. Parfitt AM 2001 The Evolution of Vitamin D related Bone 60:309Ð315. Disease: The Importance of an Early Stage of Increased Bone 29. Reinholt, Engfeldt B, Hjerpe A, Jansson K 1982 Turnover Without Impaired Mineralization. In: Nutritional Stereological studies on the epiphyseal growth plate with Aspects of Osteoporosis, P Burkhardt, R Heaney, B Dawson- special reference to the distribution of matrix vesicles. Hughes (eds). Academic Press, San Diego, pp. 197Ð208. J Ultrastruct Res 80:270Ð279. 48. Stanbury SW 1980 Vitamin D and calcium metabolism. In: 30. Glorieux FH, Marie PJ, Pettifor JM, Delvin EE 1980 Bone Norman AW (ed) Vitamin D. Molecular Biology and Clinical response to phosphate salts, ergocalciferol, and calcitriol in Nutrition. Dekker, New York, pp. 251Ð319. hypophosphatemic vitamin D resistant rickets. N Engl J Med 49. Fraser D, Kooh SW, Scriver CR 1967 Hyperparathyroidism 303:1023Ð1031. as the cause of hyperaminoaciduria and phosphaturia in 31. Kember NF, Kirkwood JK 1991 Cell kinetics and the study human vitamin D deficiency. Pediatr Res 1:425Ð435. of longitudinal bone growth: A perspective. In: Dixon AD, 50. David L 1991 Common vitamin DÐdeficiency rickets. In: Sarnat BG, Hoyte DA (eds) Fundamentals of Bone Growth: Glorieux FH (ed) Rickets, Nestle Nutrition Workshop Series, Methodology and Applications. CRC Press, Boca Raton, Vol 21. Raven, New York, pp. 107Ð122. Florida, pp. 153Ð162. 51. Vainsel M, Manderlier T, Corvilain J, Vis HL 1974 Study of 32. Dodds GS, Cameron HC 1934 Studies on experimental rickets the secondary hyperparathyroidism in vitamin DÐdeficiency in rats. I. Structural modifications of the epiphyseal cartilages rickets. I. Aspects of mineral metabolism. Biomedicine in the tibia and other bones. Am J Anat 55:135Ð165. 21:368Ð371. 33. Park EA 1939 Observations on the pathology of rickets with 52. Stanbury SW, Mawer EB 1990 Metabolic Disturbances in particular reference to the changes at the cartilage-shaft junc- Acquired Osteomalacia. In: Cohen RD, Lewis B, tions of the growing bones. Bull NY Acad Med 15:495Ð543. Alberti KGMM, Denman AM (eds) The Metabolic and 34. Bicknell F, Prescott F 1953 The Vitamins in Medicine. Molecular Basis of Acquired Disease. Bailliere Tindall, Heinemann, London, pp. 529Ð533. London, pp. 1717Ð1782. 35. Bailie JM, Irving JT 1955 Development and healing of rickets 53. Rao DS, Villanueva A, Mathews M, Pumo B, Frame B, in intramembranous bone. Acta Med Sci 152(Suppl. 306):1Ð14. Kleerekoper M, Parfitt AM 1983 Histologic evolution of 1046 A. MICHAEL PARFITT

vitamin depletion in patients with intestinal malabsorption or 70. Nagant de Deuxchaisnes C, Rombouts-Lindemans C, Huaux JP, dietary deficiency. In: Frame B, Potts JT Jr (eds) Clinical Withofs H, Meersseman F 1979 Healing of vitamin DÐ Disorders of Bone and Mineral Metabolism. Excerpta deficient osteomalacia by the administration of 1,25(OH)2D3. Medica, Amsterdam, pp. 224Ð226. In: MacIntyre I, Szelke M (eds) Molecular Endocrinology, 54. Arnaud CD 1991 Parathyroid hormone and its role in the pp. 375Ð404. pathophysiology of the common forms of rickets and osteo- 71. Papapoulos SE, Clements TL, Fraher LJ, Gleed J, malacia. In: Glorieux FH (ed) Rickets, Nestle Nutrition O’Riordan JLH 1980 Metabolites of vitamin D3 in human Workshop Series, Vol. 21. Raven, New York, pp. 47Ð61. vitamin deficiency: Effect of vitamin D3 or 1,25-dihydroxy- 55. Rao DS, Parfitt AM, Kleerekoper M, Pumo BS, Frame B cholecalciferol. Lancet 2:612Ð615. 1985 Dissociation between the effects of endogenous 72. Parfitt AM, Mathews CHE, Brommage R, Jarnagin K, parathyroid hormone on cAMP generation and on phosphate DeLuca HF 1984 Calcitriol but no other metabolite of vita- reabsorption in hypocalcemia due to vitamin D depletion: An min D is essential for normal bone growth and development acquired disorder resembling pseudohypoparathyroidism in the rat. J Clin Invest 73:576Ð586. type II. J Clin Endocrinol Metab 61:285Ð290. 73. Lund B, Charles P, Egsmose C, Lund BJ, Melson F, 56. Brazier M, Kamel S, Maamer M, Agbomson F, Elesper I, Mosekilde L, Storm T, Sondergard H, Thode J, Sorensen OH Garabedian M, Desmet G, Sebert JL 1995 Markers of bone 1985 Changes in vitamin D metabolites and bone histology remodeling in the elderly subject: Effects of vitamin D insuf- in rats during recovery from rickets. Calcif Tissue Int ficiency and its correction. J Bone Miner Res 10:1753Ð1761. 37:478Ð483. 57. Parfitt AM, Kleerekoper M, Cruz C 1986 Reduced phosphate 74. Rasmussen H, Baron R, Broadus A, DeFronzo R, Lang R, reabsorption unrelated to parathyroid hormone after renal Horst R 1980 1,25(OH)2D3 is not the only D metabolite transplantation: Implications for the pathogenesis of hyper- involved in the pathogenesis of osteomalacia. Am J Med parathyroidism in chronic renal failure. Miner Electrolyte 69:360Ð368. Metab 12:356Ð362. 75. St Amaud R, Glorieux FH 1998 Editorial: 24, 25-dihydroxy- 58. Fraser DR 1983 The physiological economy of vitamin D. vitamin D-Active Metabolite or Inactive Catabolite? Lancet 1:969Ð972. Endocrinol 39:3371Ð3374. 59. Fraser DR 1991 Physiology of vitamin D and calcium home- 76. St. Amaud R, Arabian A, Travers R, Barletta F, Raval-Pandya M, ostasis. In: Glorieux FH (ed) Rickets, Nestle Nutrition Chapin K, Depovere J, Mathieu C, Christakos S, Demay MB, Workshop Series, Vol 21. Raven, New York, pp. 23Ð34. Glorieux FH 2000 Deficient mineralization of intramem- 60. Mawer EF, Davies M 2001 Vitamin D nutrition and bone dis- branous bone in vitamin D-24-hydroxylaseÐablated mice is ease in adults. Reviews Endocrine & Metabolic Disorders. due to elevated 1,25-dihydroxyvitamin D and not to the 2:153Ð164. absence of 24, 25-dihydroxyvitamin D. Endocrinology 141: 61. Parfitt AM 1978 Adult hypoparathyroidism: Treatment with 2658Ð2666. calcifediol. Arch Intern Med 138:874Ð881. 77. Wronski TJ, Halloran BP, Bikle DD, Globus RK, 62. Clements MR, Chalmers TM, Fraser DR 1984 Enterohepatic Morey-Holton ER 1986 Chronic administration of 1,25- circulation of vitamin D: A reappraisal of the hypothesis. dihydroxyvitamin D3: increased bone but impaired mineral- Lancet 1:1376Ð1379. ization. Endocrinology 119:2580Ð2585. 63. Clements MR, Johnson L, Fraser DR 1987 A new mecha- 78. Popovtzer MM, Mathay R, Alfrey AC, Block M, Beck P, nism for induced vitamin D deficiency in calcium depriva- Miles J, Reeve EB 1973 Vitamin D deficiency osteomalacia— tion. Nature 325:62Ð65. Healing of the bone disease in the absence of vitamin D with 64. Bisballe S, Eriksen EF, Melsen F, Mosekilde L, Sorensen OH, intravenous calcium and phosphorus infusions. In: Frame B, Hessov I 1991 Osteopenia and osteomalacia after gastrec- Parfitt AM, Duncan H (eds) Clinical Aspects of Metabolic tomy: Interrelations between biochemical markers of bone Bone Disease. Excerpta Medica, Amsterdam, pp. 382Ð387. remodeling, vitamin D metabolites, and bone histomorphom- 79. Sakati N, Woodhouse NJY, Niles N, Harfl H, de Grange DA, etry. Gut 32:1303Ð1307. Marx S 1986 Hereditary resistance to 1,25-dihydroxyvitamin D: 65. Clements MR, Davies M, Hayes ME, Hickey CD, Lumb GA, Clinical and radiological improvement during high-dose oral Mawer EB, Adams PH 1992 The role of 1,25-dihydroxyvita- calcium therapy. Horm Res 24:280Ð287. min D in the mechanism of acquired vitamin D deficiency. 80. Weisman Y, Bab I, Gazit D, Spirer Z, Jaffe M, Hochberg Z Clin Endocrinol 37:17Ð27. 1987 Long-term intracaval calcium infusion therapy in end- 66. Davies M, Heys SE, Selby PL, Berry JL, Mawer EB 1997 organ resistance to 1,25-dihydroxyvitamin D. Am J Med Increased catabolism of 25-hydroxyvitamin D in patients 83:984Ð990. with partial gastrectomy and elevated 1,25-dihydroxyvitamin 81. Balsan S, Garabedian M, Larchet M, Gorski AM, Cournot G, D levels. Implications for metabolic bone disease. J Clin Tau C, Bourdeau A, Silve C, Ricour C 1986 Long-term Endocrinol 82:209Ð212. nocturnal calcium infusions can cure rickets and promote 67. Epstein S, Meunier PJ, Lambert PW, Stern PH, Bell NH 1993 normal mineralization in hereditary resistance to 1,25-dihy- 1,25-Dihydroxyvitamin D3 corrects osteomalacia in droxyvitamin D. J Clin Invest 77:1661Ð1667. hypoparathyroidism and pseudohypoparathyroidism. Acata 82. Underwood JR, DeLuca H 1984 Vitamin D is not directly Endocrinol 103:241Ð247. necessary for bone growth and mineralization. Am J Physiol 68. Brommage R, DeLuca HF 1985 Evidence that 1,25-dihy- 246:493Ð498. droxyvitamin D3 is the physiologically active metabolite of 83. Weinstein RS, Underwood JL, Hutson MS, DeLuca HF vitamin D3. Endocr Rev 6:491Ð511. 1984 Bone histomorphometry in vitamin D-deficient rats 69. Norman AW, Roth J, Orci L 1982 The vitamin D endocrine infused with calcium and phosphorus. Am J Physiol 246: system: Steroid metabolism, hormone receptors, and biolog- E499ÐE505. ical response (calcium binding proteins). Endocr Rev 84. Compston JE, Vedi S, Merrett AL, Clemens TL, O’Riordan 3:331Ð366. JLH, Woodhead JS 1981 Privational and malabsorption CHAPTER 63 Vitamin D and the Pathogenesis of Rickets and Osteomalacia 1047

metabolic bone disease: Plasma vitamin D metabolite 1,25(OH)2D3, 25(OH)D3, and vitamin D3 in the rat. Am concentrations and their relationship to quantitative bone J Physiol 248:G633ÐG638. histology. Metab Bone Dis Related Res 3:165Ð170. 102. Trummel CL, Raisz LG, Blunt JW, DeLuca HF 1969 85. Kashiwa H, Nishi Y, Usui T, Seino Y 1981 A case of rickets 25-Hydroxycholecalciferol: Stimulation of bone resorption with normal serum level of 1,25-(OH)2D and low 25-OHD. in tissue culture. Science 163:1450Ð1451. Hiroshima J Med Sci 30:61Ð63. 103. Olson EB, DeLuca HF 1969 25-Hydroxycholecalciferol: 86. Chesney RW, Zimmerman J, Hamstra A, DeLuca MF, Direct effect on calcium transport. Science 165:405Ð407. Mazess RB 1981 Vitamin D metabolite concentrations in 104. Parfitt AM, Gallagher JC, Heaney RP, Johnston CC, Neer R, vitamin D deficiency. Are calcitriol levels normal? Am J Dis Whedon GD 1982 Vitamin D and bone health in the elderly. Children 135:1025Ð1028. Am J Clin Nutr 36:1014Ð1031. 87. Markestad T, Halvorsen S, Halvorsen KS, et al. 1984 Plasma 105. Eastwood JB, Stamp TCB, DeWardener HE, Bordier PJ, concentrations of vitamin D metabolites before and during Arnaud CD 1976 The effect of 25-hydroxy vitamin D3 in the treatment of vitamin D deficiency rickets in children. Acta osteomalacia of chronic renal failure. Clin Sci Mol Med Paediatr Scand 73:225Ð231. 52:499Ð508. 88. Stanbury SW, Taylor CM, Lumb GA, Mawer B, Berry J, 106. Howland J, Kramer B 1921 Calcium and phosphorus in the Hann J, Wallace J 1981 Formation of vitamin D metabolites serum in relation to rickets. Am J Dis Chldren 22:105Ð119. following correction of human vitamin D deficiency. Miner 107. Howland J, Kramer B 1923 A study of the calcium and inor- Electrolyte Metab 5:212Ð227. ganic phosphorus of the serum in relation to rickets and 89. Wahl TO, Gobuty AH, Lukert BP 1981 Long-term anticon- tetany. Monatschrift fur Kinderheilkd 25:279Ð293. vulsant therapy and intestinal calcium absorption. Clin 108. Shipley PG, Kramer B, Howland J 1926 Studies upon calci- Pharmacol Ther 30:506Ð516. fication in vitro. Biochem J 20:379Ð387. 90. Demiaux B, Arlot ME, Chapuy M-C, Meunier PJ, 109. Nordin BEC, Smith DA 1967 Pathogenesis and treatment of Delmas PD 1992 Serum osteocalcin is increased in patients osteomalacia. In: Hioco DJ (ed) L’Osteomalacie. Masson & Cie, with osteomalacia: Correlations with biochemical and Paris, pp. 374Ð399. histomorphometric findings. J Clin Endocrinol Metab 74: 110. Kaplan FS, August CS, Fallon MD, Gannon F, Haddad JG 1993 1146Ð1151. Osteopetrorickets—The paradox of plenty. Pathophysiology 91. Stanbury SW 1981 Vitamin D and hyperparathyroidism. J R and treatment. Clin Orthop Related Res 294:64Ð78. Coll Phys Lond 15:205Ð217. 111. Stanbury SW 1962 Osteomalacia, Schweiz Med Wochenschr 92. Clements MR, Davies M, Fraser DR, Lumb GA, Mawer EB, 29:883Ð892. Adams PH 1987 Metabolic inactivation of vitamin D is 112. Yendt ER, Connor TB, Howard JE 1955 In vitro calcification enhanced in primary hyperparathyroidism. Clin Sci of rachitic rat cartilage in normal and pathological human 73:659Ð664. sera with some observations on the pathogenesis of renal 93. Stamp TCB, Walker PG, Perry W, Jenkins MW 1980 rickets. Bull Johns Hopkins Hospital 96/97:1Ð19. Nutritional osteomalacia and late rickets in greater London 113. Dean DD, Boyan BD, Muniz OE, Howell DS, Schwartz Z 1974Ð1979: Clinical and metabolic studies in 45 patients. 1996 Vitamin D Metabolites Regulate Matrix Vesicle Clin Endocrinol Metab 9:81Ð105. Metalloproteinase content in a Cell Maturation-Dependent 94. Raisz LG, Trummel CL, Holick MF, DeLuca HF 1972 1,25- Manner. Calci Tiss Internat. 59:109Ð116. Dihydroxycholecalciferol: A potent stimulator of bone 114. Bordier PH, Hioco D, Roquier, Hepner GW, Thompson GR resorption in tissue culture. Science 175:768Ð869. 1969 Effects of intravenous vitamin D on bone and phos- 95. Bouillon R, Van Baelen H 1981 Transport of vitamin D: phate metabolism in osteomalacia. Calcif Tissue Res Significance of free and total concentrations of the vitamin D 4:78Ð83. metabolites. Calcif Tissue Int 33:451Ð453. 115. Bordier P, Pechet MM, Hesse R, Marie P, Rasmussen H 1974 96. Wilhelm F, Norman AW 1984 Cooperativity in the binding of Response of adult patients with osteomalacia to treatment with 1,25-dihydroxyvitamin D3 to the chick intestinal receptor. crystalline 1α-hydroxy vitamin D3. N Engl J Med 291:866Ð871. FEBS Lett 170:239Ð242. 116. Bordier P, Rasmussen H, Marie P, Miravet L, Gueris J, 97. Howard GA, Turner RT, Sherrard DJ, Baylink DJ 1981 Human Ryckwaert A 1978 Vitamin D metabolites and bone mineral- bone cells in culture metabolize 25-hydroxyvitamin D3 ization in man. J Clin Endocrinol Metab 46:284Ð294. to 1,25-dihydroxyvitamin D3 and 24,25-dihydroxyvitamin D3. 117. Compston JE, Horton LWL, Thompson RPH 1979 Treatment J Biol Chem 256:7738Ð7740. of osteomalacia associated with primary biliary cirrhosis 98. Ichikawa F, Sato K, Nanjo M, Nishii Y, Shinki T, Takahashi N, with parenteral vitamin D2 or oral 25-hydroxyvitamin D3. Suda T 1995 Mouse primary osteoblasts express vitamin D3 Gut 20:133Ð136. 25-hydroxylase mRNA and convert 1 α-hydroxyvitamin D3 118. Compston JE, Crowe JP, Horton LWL 1979 Treatment of into 1α,25-dihydroxyvitamin D3. Bone. 16:129Ð135. osteomalacia associated with primary biliary cirrhosis with 99. Puzas JE, Turner RT, Howard GA, Baylink DJ 1983 Cells oral 1α-hydroxy vitamin D3. Br Med J 309Ð312. isolated from embryonic intestine synthesize 1,25-dihy- 119. Compston JE, Horton LWL, Laker MF, Merrett AL, droxyvitamin-D3 and 24,25-dihydroxyvitamin-D3 in culture. Woodhead JS, Gazet J-C, Pilkington TRE 1980 Treatment of Endocrinology 112:378Ð380. bone disease after jejunoileal bypass for obesity with oral 100. Takezawa K, Moorthy B, Mandel ML, Garancis JC, 1α-hydroxyvitamin D3. Gut 21:669Ð674. Ghazarian JG 1990 Antigenic and catalytic disparity in the 120. Parfitt AM, Villanueva AR 1982 Hypophosphatemia and distribution of cytochrome P-450-dependent 25-hydroxy- osteoblast function in human bone disease. In: Massry SG, vitamin D3-1 α- and 24-hydroxylases. Histochemistry 95: Letteri JM, Ritz E (eds) Proceedings, 5th International 37Ð42. Workshop on Phosphate and Other Minerals. Regulation of 101. McDonald GB, Lau K-HW, Schy AL, Wergedal JE, Baylink DJ Phosphate and Mineral Metabolism. Adv Exp Med Biol 1985 Intestinal metabolism and portal venous transport of 151:209Ð216. 1048 A. MICHAEL PARFITT

121. Markowitz ME, Rosen JF, Laxminarayan S, Mizruchi M 133. Manolagas SC, Haussler MR, Deftos LJ 1980 1,25- 1984 Circadian rhythms of blood minerals during adoles- Dihydroxyvitamin D3 receptor-like macromolecule in rat cence. Pediatr Res 18:456Ð462. osteogenic sarcoma cell lines. J Biol Chem 255:4414Ð4417. 122. De Vernejoul MC, Marie PJ, Miravet L, Ryckewaert A 1983 134. Stumpf WE, Ssar M, DeLuca HF 1981 Sites of action of Chronic hypophosphatemia without osteomalacia. In: Frame B, 1,25(OH)2 vitamin D3 identified by thaw-mount autoradiog- Potts JT Jr (eds) Clinical Disorders of Bone and Mineral raphy. In: Cohn DV, Talmage RV, Matthews JL (eds) Metabolism. Excerpta Medica, Amsterdam, pp. 232Ð236. Hormonal Control of Calcium Metabolism. Excerpta Medica, 123. Oginni LM, Sharp CA, Worsfold M, Badru OS, Davie JWJ Amsterdam, pp. 222Ð229. 1999 Healing of rickets after calcium supplementation. 135. Manolagas SC, Burton DW, Deftos LJ 1981 1,25- Lancet 353:296. Dihydroxyvitamin D3 stimulates the alkaline phosphatase 124. McKenna MJ, Kleerekoper M, Ellis BI, Dao BS, Parfitt AM, activity of osteoblastlike cells. J Biol Chem 256:7115Ð7117. Frame B 1987 Atypical insufficiency fractures confused with 136. Lian JB, Coutts M, Canalis E 1985 Studies of hormonal Looser zones of osteomalacia. Bone 8:71Ð78. regulation of osteocalcin synthesis in cultured fetal rat cal- 125. Parfitt AM 1977 The clinical and radiographic manifestations variae. J Biol Chem 260:8706Ð8710. of renal osteodystrophy. In: David DG (ed) Perspectives 137. Veldman CM, Schläpper I, Schmid Ch 1997 1α,25-Dihy- in Hypertension and Nephrology: Calcium Metabolism in droxyvitamin D3 stimulates sodium-dependent phosphate Renal Failure and Nephrolithiasis. Wiley, New York, transport in osteoblast-like cells. Bone 21:41Ð47. pp. 145Ð195. 138. Marie PJ, Hott M, Garba M-T 1985 Contrasting effects of 126. Wergedal JE, Baylink JE 1971 Factors affecting bone enzy- 1,25-dihydroxyvitamin D3 on bone matrix and mineral appo- matic activity in vitamin DÐdeficient rats. Am J Physiol sitional rates in the mouse. Metabolism 34:777Ð783. 220:406Ð409. 139. Krempien B, Klimpel F 1980 Action of 1,25-dihydroxy- 127. Howard GA, Baylink DJ 1980 Matrix formation and osteoid cholecalciferol on cartilage mineralization and on endosteal maturation in vitamin DÐdeficient rats made normocalcemic lining cells of bone. Virch Arch [A] 388:335Ð347. by dietary means. Miner Electrolyte Metab 3:44Ð50. 140. Stern PH 1980 The D vitamins and bone. Pharmacol Rev 128. Sebert JL, Meunier PJ 1984 Role physiopathologique de la 32:47Ð80. vitamine D et de ses metabolites dans l’osteomalacie. In: 141. Dickson IR, Roughley PJ 1993 The effects of vitamin D defi- Bouillon R, Boudailliez B, Marie A, et al. (eds) Vitamine D ciency on proteoglycan and hyaluronate constituents of chick et Maladies des Os et du Metabolisme Mineral. Masson, bone. Biochim Biophys Acta 1181:15Ð22. Paris, pp. 109Ð145. 142. Tulpule PG, Patwardhan VN 1954 Mode of action of vitamin D. 129. Bordier PJ, Marie P, Miravet L, et al. 1976 Morphological The effect of vitamin D deficiency on the rate of anaerobic and morphometrical characteristics of the mineralization glycolysis and pyruvate oxidation by epiphyseal cartilage. front. A vitamin D regulated sequence of the bone remodeling. Biochem J 58:61Ð65. In: Meunier PJ (ed) Bone Histomorphometry. Second 143. Klein GL, Simmons DJ 1993 Nutritional rickets: Thoughts International Workshop. Armour Montagu, Paris, pp. 335Ð354. about pathogenesis. Ann Med 25:379Ð384. 130. Weinstein RS 2002 Clinical use of bone biopsy. In: Coe FL, 144. Eastwood JB, Bordier PJ, Clarkson EM, Tun Chot S, Favus MJ (eds) Disorders of Bone and Mineral Metabolism. De Wardener HE 1973 The contrasting effects on bone 2nd Ed. Lippincott, Williams & Wilkins. Philadelphia, pp. histology of vitamin D and of calcium carbonate in the osteo- 449Ð468. malacia of chronic renal failure. Clin Sci Mol Med 47:23Ð42. 131. Meunier PJ, Edouard C, Arlot M, et al. 1979 Effects of 1,25- 145. Stauffer M, Baylink D, Wergedal J, Rich C 1975 Decreased dihydroxyvitamin D on bone mineralization. In: Maclntyre I, bone formation, mineralization, and enhanced resorption in Szelke M (eds) Molecular Endocrinology. Elsevier/North- calcium-deficient rats. Am J Physiol 225:269Ð276. Holland, Amsterdam, pp. 283Ð292. 146. Ecarot B, Glorieux FH, Desbarats M, Travers R, Labelle L 132. Marie PJ, Glorieux FH 1981 Histomorphometric study of 1992 Defective bone formation by Hyp mouse bone cells bone remodeling in hypophosphatemic vitamin DÐresistant transplanted into normal mice: Evidence in favor of an intrin- rickets. Metab Bone Dis Related Res 3:31Ð38. sic osteoblast defect. J Bone Miner Res 7:215Ð220. CHAPTER 64 The Hypocalcemic Disorders: Differential Diagnosis and Therapeutic Use of Vitamin D

THOMAS O. CARPENTER Department of Pediatrics, Yale University School of Medicine, New Haven, Connecticut

KARL L. INSOGNA Department of Medicine, Yale University School of Medicine, New Haven, Connecticut

I. Physiology III. Therapy for Hypocalcemia II. Differential Diagnosis of Hypocalcemia References

I. PHYSIOLOGY is normally less than 0.40 ± 0.04 sec. This abnormality is not always present during hypocalcemia, and it may A. Hypocalcemia and Its Manifestations also be seen in hypokalemia. Cardiac failure may occur in the setting of hypocalcemia [3]. Papilledema has also Hypocalcemia refers to an abnormally low concentra- been attributed to hypocalcemia [4]. tion of ionized calcium in extracellular fluid, almost Chronic hypocalcemia caused by deficient calcium invariably sampled from the bloodstream. Manifestations intake during periods of significant skeletal growth may of hypocalcemia are related to increased neuromuscular result in rickets and osteomalacia (see Chapter 63). irritability [1]. Tetany is the classic sign of hypocalcemia, Severe osteoporosis and dental abnormalities have also yet it is variable in presentation. Paresthesias often occur been reported in long-standing untreated hypoparathy- first around the mouth or in the fingertips and may roidism [5,6]. A mineralization defect has been described progress to overt spasm of the muscles of the face and in hypoparathyroidism; however, these skeletal conse- extremities, the latter typified by carpopedal spasm. quences appear to be more prevalent in the setting of More subtle presentations have included complaints of endemic calcium deficiency, where secondary hyper- writer’s cramp or generalized stiffness. Children with parathyroidism is evident. Basal ganglia calcifications tetanic laryngospasm due to hypocalcemia have been are typical findings in long-standing hypoparathyroidism mistakenly diagnosed with croup [2]. Infants are more as well [7]. Abnormalities in the integument including likely than adults to present with jitteriness or twitching, dry skin, coarse hair, and a form of psoriasis that responds which can progress to overt tonic-clonic seizure activity. to normalization of the serum calcium concentration [8] Lethargy and cyanosis have also been described in this have all been described in states of long-standing age group. The term latent tetany refers to signs elicitable hypocalcemia. with provocative stimuli such as ischemia (Trousseau Regulatory mechanisms maintain the concentration test) or percussion (e.g., of the facial nerve to elicit of ionized calcium within a remarkably narrow range Chvostek’s sign). Neither the degree of hypocalcemia of 4.48 to 5.28 mg/dl in whole blood [9]. The ionized nor the rapidity with which it develops necessarily fraction of total serum calcium is generally estimated correlate with clinical manifestations. to be approximately 50%, with the remainder of the total Hypomagnesemia or hyperkalemia may present serum calcium being bound to serum proteins, most with similar findings, which can be exacerbated in the notably albumin, and to a lesser extent complexed with setting of hypocalcemia. Conversely, hypermagnesemia anions, such as citrate or sulfate. Only the ionized frac- or hypokalemia can mask symptoms in a hypocalcemic tion of total serum calcium is physiologically important, individual. Abnormalities of repolarization of cardiac and it is this component that is regulated on a minute- musculature may result in a prolonged Q-T interval on the to-minute basis. electrocardiogram (EKG). The Q-T interval corrected for Although it is possible to measure ionized calcium 1/2 heart rate [Q-TC, which equals Q-T/(R-R interval) ], routinely in large clinical laboratories, the specimen VITAMIN D, 2ND EDITION Copyright © 2005, Elsevier, Inc. FELDMAN, PIKE, AND GLORIEUX All rights reserved. 1050 THOMAS O. CARPENTER AND KARL L. INSOGNA must usually be obtained anaerobically and analyzed bone and thereby increasing the ionized calcium concen- promptly. Therefore, total serum calcium is often used tration of the extracellular fluid [9]. Details of the cellu- as an indirect assessment of the ionized calcium frac- lar mechanism by which this occurs are incompletely tion. A decrease in serum protein concentrations understood. The principal target cell for PTH in bone (particularly albumin) often results in reduced total appears to be the osteoblast or osteoblast-like stromal serum calcium concentrations, with preservation of a cell, rather than the resorbing cell itself, the osteoclast normal concentration of ionized calcium. Patients in [16,17]. In response to PTH, osteoblasts or osteoblast- whom this occurs will be asymptomatic, displaying like stromal cells release locally active cytokines that none of the signs or symptoms of hypocalcemia. These appear to increase the number and activity of osteo- findings are often present in patients with nephrotic clasts [18]. As the resorptive response to PTH is quite syndrome, chronic illness, malnutrition, cirrhosis, and rapid, the early effects of the hormone are likely medi- volume over-expansion. ated by activation of existing osteoclasts. A miscible pool A number of clinical guidelines have been suggested of incompletely mineralized calcium at the endosteal that correct for the effects of decreased serum albumin surface of bone may be the most readily accessible on total serum calcium concentration. One commonly source of calcium liberated in response to this action. It cited rule of thumb is to add 0.8 mg/dl to the total has been speculated that osteocytes may mediate serum calcium for every 1 g/dl decline in serum albumin release of calcium from this pool [19]. These effects below 4.0 g/dl. However, these estimates have been are evident in 6Ð12 hr [20]. shown to be somewhat inaccurate under many circum- The renal effects of PTH to defend serum calcium stances, and it is preferable to directly determine the occur within minutes (see Chapter 76). PTH increases ionized calcium concentration in the setting where the calcium reabsorption in the distal tubule. This effect is total serum calcium measure is not representative of greatest in the distal convoluted tubule, where a the ionized calcium measure [10]. sodium/calcium exchanger is regulated by PTH [21]. In the proximal renal tubule, PTH acts via a cAMP- dependent mechanism to decrease phosphate reabsorp- B. Role of Parathyroid Hormone tion, resulting in increased phosphaturia. PTH effects this in the Acute Defense of Ionized change by prompting the removal of sodium/phosphate Serum Calcium Concentration cotransporters from the renal tubular apical membrane [22]. These two effects both serve to acutely increase Parathyroid hormone (PTH) is secreted by the para- serum calcium; one by causing less calcium to be thyroid glands in response to a fall in ionized serum excreted by the kidney, the other by lowering circulating calcium concentration [9]. The relationship between concentrations of phosphate which favors an increase decrements in ionized calcium within the physiologi- in ionized calcium. cal range and increments in PTH secretion is quite The third site of action of PTH in the defense of steep, permitting rapid and substantial changes in PTH serum calcium is at the intestine. This is an indirect secretion in response to minor fluctuations in ionized effect, described in detail below, and is a consequence calcium [9]. The details of this response have been elu- of the ability of PTH to stimulate the renal production cidated with the cloning of the seven transmembrane of 1,25-dihydroxyvitamin D [1,25(OH)2D]. domain, GÐprotein-coupled calcium sensor, which is expressed in the parathyroid glands as well as in a variety of other tissues [11] (see Chapter 31). A rise in ionized C. Vitamin D in the Long-term calcium suppresses PTH secretion by activating this Maintenance of Eucalcemia receptor [11]. Parathyroid hormone acts to regulate ionized calcium Long-term eucalcemia is maintained, in large part, through its effects in three principal target tissues, bone, via the vitamin D endocrine system. This system oper- kidney, and intestine (see Chapter 30). The cellular mech- ates in the classic manner of a steroid hormone, resulting anisms of action of PTH have been clarified by the in de novo protein synthesis directed by vitamin D cloning of the PTH receptor, also a seven transmembrane- responsive genes [23] as discussed in detail in Section II domain, GÐprotein-coupled receptor [12]. Downstream of this volume. As noted above, acute changes in serum signaling from the PTH receptor involves activation of ionized calcium levels are sensed by GÐprotein-coupled both protein kinase A- and protein kinase C-dependent calcium-sensing receptors located within the parathy- pathways [13Ð15]. roid cell membrane [11]. PTH acts rapidly to correct Parathyroid hormone acts to increase bone resorp- a fall in serum calcium, and a sustained increase in tion, liberating calcium from the mineralized matrix of PTH also stimulates production of 1,25(OH)2D, which CHAPTER 64 The Hypocalcemic Disorders: Differential Diagnosis and Therapeutic Use of Vitamin D 1051 enhances intestinal calcium absorption. PTH mediates The vitamin D system has further complexities that this change by increasing the activity of the renal are currently not well understood. For example, some 25-hydroxyvitamin D (25OHD) 1-hydroxylase enzyme children with vitamin D deficiency (defined by low complex, located in the inner mitochondrial membrane 25OHD levels) may manifest hypocalcemia even when of renal tubular cells. A number of physiological studies circulating 1,25(OH)2D levels are actually elevated. have demonstrated increased enzyme activity in animals Possible explanations for this phenomenon include that were administered PTH [24,25] and decreased decrements in expression of calbindin during hypo- activity following parathyroidectomy [26]. The mech- calcemia, or a requirement for other circulating anism is discussed in Chapters 5 and 29. PTH also vitamin D metabolites which are not present. This acutely regulates the 1-hydroxylase enzyme complex by situation is discussed further in Chapter 31. Whether altering the phosphorylation state of the associated ferre- changes in the concentration of VDR levels play a role doxin molecule [27]. Direct stimulation of 1-hydroxy- in this “resistant” state is not clear, as receptor levels lase activity in the absence of PTH can also occur have been reported to increase, decrease, or not change because a low serum calcium level enhances enzyme with manipulation of the ambient calcium concentration activity in parathyroidectomized rats [28,29] and in [33Ð35] (see Chapters 12 and 78). Finally, the system hypoparathyroid humans [5]. must return to basal levels of function after calcium Increased synthesis of 1,25(OH)2D results in greater availability is restored. The absence of this self- circulating levels of the metabolite, which gain access to regulating feature would result in hypercalcemia. specific vitamin D receptors (VDR). The hormone- receptor complex then binds to vitamin D response elements (VDRE) in the regulatory regions of target D. Biochemical Changes Induced genes (Chapters 11Ð14). Of importance to long-term by Hypocalcemia control of calcium homeostasis is the induction by 1,25(OH)2D of the intestinal 9-kDa calcium binding As noted above, the immediate consequence of protein, calbindin-D9k (Chapter 42), which is thought to hypocalcemia is secretion of PTH. In addition to play a role in vitamin DÐmediated increases in calcium increasing serum calcium levels, PTH stimulates renal absorption in the jejunum and duodenum [30] (see also phosphate (Pi) excretion. The fall in serum phosphate Chapters 24 and 25). Induction of calbindin takes hours may, however, be compensated by sufficient mobiliza- to days and is more sustained than the acute compen- tion of phosphate (as well as calcium) from bone, so satory changes that occur with the initial rise in PTH in that circulating phosphate remains largely unchanged. response to hypocalcemia. These features define a clas- The principle of mass action is thought to maintain the sic feedback loop suitable for long-term calcium homeo- stability of the Ca × Pi ion product in the blood. As a stasis. More recently rapid, nongenomic actions of consequence, local concentrations of the two major 1,25(OH)2D mediating calcium transport across intesti- mineral components (Ca and Pi) of hydroxyapatite nal mucosa have been described [31] (see Chapter 23). (HA) are able to influence the rate of movement in and The importance of this system is emphasized by out of the mineral phase of bone: clinical observations in patients with severe vitamin D deficiency (see below). During vitamin D deprivation, [Ca] + [Pi] [HA]. the initial decline in ionized serum calcium results in secondary hyperparathyroidism, which maximizes Thus, a fall in ionized Ca would favor an increase 1,25(OH)2D production and allows for maintenance in serum phosphate concentration. Given all of these of eucalcemia in the early stages. Eventually, this influences, sustained hypocalcemia usually results compensatory mechanism fails, and intestinal calcium in a biochemical picture of secondary hyperparathy- absorption is sufficiently compromised that frank roidism, elevated 1,25(OH)2D levels, and variable hypocalcemia develops. This may be compounded by changes in serum phosphate. If hypocalcemia develops an induced resistance to PTH seen in severe hypocal- in the setting of diminished or absent PTH function, cemic or vitamin DÐdeficient states [1]. serum phosphate is usually elevated, owing to increased In children with hereditary resistance to vitamin D renal phosphate reabsorption. In this instance, treatment (HVDRR; see Chapter 72) caused by mutations in the with 1,25(OH)2D would also increase serum phosphate, VDR, the compensatory changes described previously since this metabolite enhances intestinal phosphate are interrupted by defective VDR and the inability of absorption. The effect of hypocalcemia on circulating 1,25(OH)2D to signal to the nucleus [32]. Such vitamin D metabolites is complex. An increase in the patients can have severe hypocalcemia leading to con- biosynthesis of 1,25(OH)2D occurs, as reviewed above. vulsions, coma, and death. This is largely secondary to the induced increase in 1052 THOMAS O. CARPENTER AND KARL L. INSOGNA

Vit D Milk

25(OH)D

1,25(OH)2D

1,25(OH)2D Ca Absorption

Low Ca Intake [Ca]i

PTH

[PO4]

Bone mineralization

FIGURE 1Mechanisms of the development of osteomalacia and rickets. Deficiency of vitamin D intake and/or limited ultraviolet light exposure lead to limited vitamin D stores as reflected by a decreased circulating 25OH D level. Reduced availability of this substrate is presumed to limit 1,25(OH)2D produc- tion, resulting in impaired intestinal calcium absorption. Calcium availability for skeletal mineralization is subsequently compromised, and secondary hyperparathyroidism, with concomitant hypophosphatemia occur. Restricted dietary calcium intake can also result in a similar pathophysiology. Increased turnover of vitamin D in the calcium deficient state may result in a greater risk of vitamin D insufficiency. The paradox of elevated levels of 1,25 dihydroxyvitamin D in these disorders is well recognized.

circulating PTH, but can be a direct consequence of the PTH resistance as a consequence of PTH/PTH-related fall in calcium ion concentration. It has been deter- peptide (PTHrP) receptor or postreceptor defects, or mined that calcium deprivation results in a general (3) in the setting of normal or increased PTH activity increase in turnover of the parent vitamin D metabo- and normal PTH receptor function. Thus, although lite, 25OHD, such that vitamin D stores are depleted at many of the etiologies relate to abnormalities in PTH, a more rapid rate than normal [36]. The clinical impli- they are very relevant to this book because vitamin D cation of this finding is that susceptibility to vitamin D metabolism is always involved and vitamin D is the deficiency may be greater when concomitant calcium cornerstone of therapy. deprivation is present (see Fig. 1). 1. HYPOCALCEMIA DUE TO ABNORMALITIES OF PTH AVAILABILITY II. DIFFERENTIAL DIAGNOSIS A variety of congenital or acquired disorders can OF HYPOCALCEMIA lead to developmental failure of the parathyroid glands, failure of functional hormone production, or A. Classification destruction of the glands. These are all present as hypocalcemia, usually with attendant hyperphos- A rapid increase in PTH serves as the major defense phatemia and undetectable or inappropriately low against acute hypocalcemia. We therefore classify levels of circulating PTH. these disorders as those which manifest hypocalcemia a. Failure of Organogenesis: DiGeorge Sequence (1) due to abnormalities of PTH availability, (2) due to DiGeorge sequence is an uncommon developmental CHAPTER 64 The Hypocalcemic Disorders: Differential Diagnosis and Therapeutic Use of Vitamin D 1053 disorder that affects the third and fourth branchial known to be represented by mutations in a gene encod- clefts and results in dysgenesis of the thymus and the ing (AIRE) an autoimmune regulatory protein contain- parathyroid glands [37]. Tetany and seizures are common ing a zinc finger motif, and is a candidate transcription features of the early course of infants with DiGeorge factor [47]. sequence. However, abnormalities in T-cell function with f. Postsurgery Reduction in PTH Given the close subsequent increased risk for infection often become anatomic relationship of the parathyroid glands to the a major feature of this disorder later in life [38]. thyroid, complete or near complete extirpation of the The gene(s) responsible for the cardiac, thymic, and thyroid gland as part of the management of either parathyroid features of the DiGeorge phenotype is Graves’ disease or thyroid cancer can be complicated by TBX1 [39]. Microdeletions of chromosome 22 are found destruction or vascular compromise of parathyroid tissue in many patients with this disorder. and varying degrees of hypoparathyroidism. This should b. Idiopathic Hypoparathyroidism As discussed be a rare complication of thyroid surgery, and with expe- in Sections II.A.1.c-e, a variety of causes of inherited rienced surgeons occurs with a frequency less than 10%. idiopathic hypoparathyroidism have now been delin- Even when destruction of the parathyroid glands does eated, including one family where the abnormality maps not occur following neck surgery, so-called stunned to the X chromosome. Linkage analysis has identified parathyroids with transient declines of approximately the Xq26-X27 region as the probable site for the 1 mg/dl in total serum calcium are often observed in molecular abnormality, which presents with failure of the first 24 to 48 hr postoperatively. This is presumably parathyroid gland development [40]. due to transient vascular or mechanical damage to the c. Molecular Abnormalities in the PTH Gene glands. Considerable variability in the degree of hypo- Parathyroid hormone is secreted and synthesized by a parathyroidism following neck surgery occurs, ranging classic secretory pathway. The initial translation product from asymptomatic reduction in parathyroid reserve to is a prepropeptide, which requires cleavage of the frank tetany, requiring chronic therapy with vitamin D amino-terminal pre and pro sequences before secretion. (calcitriol) and calcium. A family with autosomal dominant inheritance of g. Infiltrative Diseases and Deposition of Heavy hypoparathyroidism has been reported in which a mis- Metals Although uncommon, malignant metastasis sense mutation (Cys-18 → Arg) results in an abnormal to the parathyroid glands with hypoparathyroidism has signal sequence and diminished uptake of preproPTH been reported, usually with breast cancer [48]. It has into the endoplasmic reticulum [41]. Another family been postulated that granulomatous involvement of with recessively inherited hypoparathyroidism has the parathyroids in sarcoidosis can lead to hypopara- been reported in which the prepro sequence is deleted thyroidism [49]. Patients with transfusion-dependent by a splicing mutation [42]. thalassemia can develop hypoparathyroidism due to d. Molecular Abnormalities in the Calcium-Sensing hemochromatosis secondary to deposition of iron in the Receptor Gene The gene for the calcium-sensing glands [50]. In Wilson’s disease hypoparathyroidism receptor has been mapped to chromosome 3 [43]. As can occur, presumably because of copper deposition [51]. mentioned in Section I.B, ionized calcium is a ligand Finally, impaired parathyroid reserve has been reported for this receptor, and receptor occupancy suppresses in diabetic patients with uremia [52]. PTH secretion (see Chapter 31). Numerous individuals h. Radiation Although the parathyroid glands are and families with a variety of activating mutations in quite resistant to radiation, hypoparathyroidism follow- this receptor have now been reported; the associated ing radioactive iodine treatment for hyperthyroidism condition is referred to as autosomal dominant familial has been described [53]. hypocalcemia [44]. One such kindred with a peculiar i. Functional Defects in PTH Secretion Magnesium predisposition to nephrocalcinosis and renal insuffi- is an important cofactor for parathyroid hormone ciency has been described [45]. secretion, apparently required for release of the stored e. Autoimmune Polyglandular Syndrome Type 1 An hormone from secretory granules. In severe cases of autoimmune disorder termed autoimmune polyglandular hypomagnesemia, usually with circulating levels below syndrome type 1 is characterized by early development 1 mg/dl, suppressed parathyroid secretion can occur [54]. of hypoparathyroidism in association with Addison’s This can be seen in the settings of chronic gastrointestinal disease and mucocutaneous candidiasis. The majority disease, nutritional deficiency especially in alcoholics, of affected individuals will manifest hypocalcemia by or therapy with cis-platinum. Resistance to the action the age of 10 [46]. In addition to Addison’s disease, of PTH at the level of bone and kidney may also one-third of the patients will develop other endocrine contribute to the hypocalcemia seen in the setting of disorders, diabetes mellitus, pernicious anemia, or magnesium deficiency. Replenishment of magnesium premature ovarian failure [46]. This disorder is now stores promptly restores parathyroid function to normal. 1054 THOMAS O. CARPENTER AND KARL L. INSOGNA

Transient hypocalcemia in neonates has been reported that this phenotype may be due to tissue specific to be associated with maternal hyperparathyroidism. imprinting of the Gs alpha gene, or tissue specific splice variants that variably alter the function of the protein in 2. HYPOCALCEMIA DUE TO RESISTANCE different tissues [59]. TO THE ACTIONS OF PTH A diagnosis of type II PHP appears to describe a Several disorders of PTH action have hypocalcemia variety of defects distal to cAMP generation in the cas- as their principal manifestation. cade of hormone action. There is no distinct pheno- a. Pseudohypoparathyroidism Peripheral tissue type, although various autoimmune findings have been insensitivity or resistance to PTH is classically described in some patients. Finally, others have sug- termed pseudohypoparathyroidism (PHP) [55]. The gested that a circulating PTH inhibitor may play a role characteristic biochemical manifestations of PHP in the pathogenesis of PHP and have suggested that are hypocalcemia and hyperphosphatemia, as in this inhibitor may be generated by parathyroid tissue hypoparathyroidism; however, circulating levels of itself [60]. Resistance to PTH has also been described PTH are elevated, rather than low or undetectable. The in hypomagnesemia, as described below. renal tubule is the primary site of PTH resistance, b. Hypomagnesemia Magnesium deficiency can although variable degrees of skeletal resistance, depend- interfere with parathyroid secretion and function [54]. ing on treatment status, have also been reported [56]. Serum magnesium levels are usually moderately to However, if the skeletal response is unimpaired, lesions severely depressed (below the range of 1.0Ð1.4 mg/dl) characteristic of hyperparathyroidism, including osteitis before this occurs. Despite hypocalcemia, PTH levels fibrosa cystica, can develop. PTH stimulates renal may be inappropriately low or only modestly elevated, cAMP production, and levels of cAMP increase in and tetany refractory to calcium supplementation can the urine following administration of the hormone. ensue. Hypomagnesemia, per se, may cause tetanic A direct correlation has been demonstrated between symptoms, although concomitant hypocalcemia is the degree of PTH resistance (as assessed by the mag- more common. Insufficient PTH secretion is the most nitude of the change in cAMP excretion or renal widely accepted cause of refractory hypocalcemia in phosphate threshold) and the ambient circulating PTH magnesium deficiency [54], although it has been sug- level [57]. gested that resistance to the calcemic actions of PTH The renal cAMP response is the basis of a diagnos- and vitamin D may play a role as well [61]. Impairment tic test that allows partial classification of this hetero- of vitamin D synthesis may also be at work [62]. As geneous group of disorders. Individuals with PHP that PTH stimulates conversion of 25OHD to 1,25(OH)2D, demonstrate a blunted urinary cAMP response have PHP hypoparathyroidism may result in low circulating type I. Those that generate a normal cAMP response 1,25(OH)2D levels, further compromising the body’s have PHP type II. defense against hypocalcemia [63]. Whether target tis- PHP type I has been further characterized into types sue resistance to infused PTH occurs in magnesium Ia and Ib. Type Ia describes those individuals with the deficiency remains controversial, and it has been sug- Albright’s hereditary osteodystrophy (AHO) pheno- gested that this apparent resistance may simply reflect type, which includes short stature and large frame, differences in the basal levels of circulating PTH [64]. broad facies, and shortened fourth metacarpals. Soft tis- To further complicate matters, generalized malnutri- sue calcifications and multiple endocrine abnormalities tion including vitamin D deficiency is often present are often present. These individuals often have a muta- in hypomagnesemic patients [65]. Hypomagnesemia tion in the α subunit of the stimulatory guanine has been associated with alcohol abuse and may nucleotide binding regulatory protein, Gs [58]. This reg- result from inherited disorders of magnesium excre- ulatory protein couples membrane receptors to adeny- tion and/or absorption [66]. It can also be induced by late cyclase, thereby regulating receptor-dependent the renal tubular effects of several drugs, including cAMP production. The presence of Gs in various cell amphotericin B, aminoglycoside antibiotics, chemother- types accounts for the generalized hormone resistance apeutic agents (particularly cis-platinum), diuretics, and that may occur. For example, affected patients often have cyclosporin. elevated thyrotropin (TSH) levels with a compensated euthyroid state. Variable degrees of gonadotropin, anti- 3. HYPOCALCEMIA IN THE SETTING OF NORMAL diuretic hormone (ADH), adrenocorticotropin (ACTH), OR INCREASED PTH ACTIVITY AND NORMAL PTH and glucagon resistance have been described. Type Ib RECEPTOR FUNCTION PHP is manifest primarily by PTH resistance, and the Despite normal PTH function and downstream sig- AHO phenotype is not present. It has been speculated naling from its receptor, hypocalcemia can still occur CHAPTER 64 The Hypocalcemic Disorders: Differential Diagnosis and Therapeutic Use of Vitamin D 1055 due to disturbances in skeletal homeostasis, vitamin D of UV light required to penetrate melanin in the pig- metabolism, and a variety of medical illnesses. mented dermis and induce previtamin D formation [68]. a. Neonatal Hypocalcemia The newborn infant Breast milk contains only small amounts of vitamin D, undergoes an acute transition to independently regulated even when the mother is receiving pharmacological mineral homeostasis at parturition (see also Chapter 48). doses of the vitamin (Chapter 51). Others have pointed The maternal source of calcium is eliminated, and the out that dietary practices, including vegetarianism and infant’s circulating calcium level transiently decreases, high grain intake, may place infants at greater risk for with recovery occurring by the third day post-partum. the development of this condition [69] (Chapter 47). Infants of diabetic or preeclamptic mothers and infants Another group at high risk for development of vita- who suffer perinatal asphyxia or other fetal complications min D deficiency is found at the other extreme of may experience an exaggerated fall in serum calcium life, the elderly, because of general nutritional compro- with a delayed recovery phase. Management with mise and limited sunlight exposure (see Chapters 50 intravenous calcium supplementation is required in the and 66). event of symptoms or severely low serum calcium levels. Biochemical findings in these conditions vary with This condition is referred to as early neonatal hypocal- the severity or duration of deficiency. Most agree that cemia, and is usually transient. It may be associated with serum calcium levels in moderate to severe vitamin D transient hypomagnesemia. deficiency are often normal, compensated by secondary Hypocalcemia presenting at 5Ð10 days of life is re- elevations in PTH [70]. In severe vitamin D deficiency, ferred to as late neonatal hypocalcemia. This presen- however, overt hypocalcemia is usually manifest, tation is more typical of term infants after enteral despite elevated circulating PTH. Serum phosphate feeding has been established. Infants of hyperparathy- levels tend to be slightly low or normal. Circulating roid mothers may present with symptomatic hypocal- alkaline phosphatase activity of bone origin is usually cemia within this period, but have also been reported markedly elevated in children and can be elevated in to present as late as 1 year of life. Familial forms adults. The best available test to assess total body of hypoparathyroidism may present as either “early” or vitamin D status is the level of circulating 25OHD “late” hypocalcemia. Mild to moderate neonatal (see Chapter 58). Levels of this metabolite are low in hypocalcemia commonly occurs in patients with con- vitamin D deficiency but may rise to normal with genital heart disease (apart from those defects common recent ingestion of vitamin D or significant sunlight in the DiGeorge sequence) [67] and in some cases can exposure, whereas bone symptoms such as pain, and be attributed to transient impairment of parathyroid signs such as leg bowing, persist. In children, radio- function. graphs of rachitic extremities at the time of sampling b. Hypocalcemia Due to Vitamin D Malnutrition may reveal hyperdense lines of remineralization at the (see Fig. 1). Vitamin D synthesis in the skin requires physes, consistent with recent exposure to vitamin D, adequate exposure to ultraviolet light. Thus, vitamin D despite the presence of overt physical findings (see deficiency is uncommon in settings where sunlight Chapter 60). Circulating 1,25(OH)2D levels may be exposure is abundant. In extremes of latitude (e.g., low, normal, or elevated during vitamin D deficiency. northern climates in North America), and where indus- This may appear paradoxical, but it should be trial pollution can interfere with transmission of UV recognized that 1,25(OH)2D circulates in 1000-fold light, normal vitamin D status is dependent on adequate lower concentrations than 25OHD. Furthermore, in dietary vitamin D intake. Supplementation of milk the setting of vitamin D deficiency, production of products with vitamin D has significantly reduced the 1,25(OH)2D is maximized. Thus, efficient conversion of incidence of vitamin D deficiency in North America. small amounts of newly ingested or synthesized 25OHD Despite these measures, certain populations are at risk may markedly increase the circulating 1,25(OH)2D for development of vitamin D deficiency, and severe concentration. Perhaps a more intriguing paradox in hypocalcemia may be a presenting manifestation of the this setting is the continued malabsorption of calcium disorder (see also Chapters 47, 61, 62, and 65). despite normal concentrations of 1,25(OH)2D. A convergence of various risk factors for development The skeletal consequence of isolated severe vitamin D of vitamin D deficiency occurs in breast-fed infants in deficiency in children is rickets, a disorder of the the first 18 months of life. Presentation appears to be epiphyseal growth plate. The defective mineralization most common during the winter or early spring in processes ultimately result in malalignment deformi- northern U.S. cities. The limited direct sunlight expo- ties of the long bones. In adult bone, vitamin D defi- sure during the winter season is a major factor. Black ciency causes osteomalacia, which is characterized children are at greater risk due to the greater quanta histomorphometrically by excess undermineralized 1056 THOMAS O. CARPENTER AND KARL L. INSOGNA osteoid and a markedly delayed mineralization rate most resistant cases, however, long-term parenteral (see Chapter 63). Adults with osteomalacia may suffer calcium infusions can normalize the serum chemistries painful pseudofractures, particularly in weight-bearing and cure the skeletal lesions [74]. The positive thera- long bones. peutic response to parenteral calcium suggests that c. Hypocalcemia Due to Vitamin D Malabsorption mediation of calcium absorption at the intestine is the Because vitamin D is a fat-soluble vitamin, gener- critical systemic action for 1,25(OH)2D. alized fat malabsorption may result in vitamin D defi- Several defects in the coding region of the vitamin D ciency. Gastrointestinal diseases such as Crohn’s receptor (VDR) that impair or prevent either hormone disease, celiac sprue, and pancreatic insufficiency can or DNA binding have been described in these patients. be accompanied by hypocalcemia due to vitamin D Reduced expression of the VDR has also been malabsorption [71] (see also Chapter 75). We have also described. This condition is quite rare but serves as an encountered children presenting with vitamin D defi- interesting experiment of nature in which the receptor- ciency rickets who have ultimately been diagnosed mediated function of 1,25(OH)2D3 is specifically with cystic fibrosis and fat malabsorption. In addition, ablated (see also Chapter 72). interruption of the enterohepatic circulation of both f. Hypocalcemia Due to Dietary Calcium Deficiency 25OHD and 1,25(OH)2D may lower body vitamin D Although uncommon, extremely low calcium intakes stores. It is also possible that the diseased bowel may have been reported to be associated with mild hypocal- not be able to respond to 1,25(OH)2D. Mild hypocal- cemia. Nigerian and South African children with cal- cemia and secondary hyperparathyroidism is also seen cium intakes of 150 mg/day or less were found to have in cholestatic liver diseases such as primary biliary cir- decreased serum calcium values, secondary hyperpara- rhosis [71]. Circulating levels of 25OHD are reduced thyroidism, and rickets [75Ð77]. The children were not in this setting owing to impaired hydroxylation of vita- vitamin DÐdeficient, and their biochemical abnormali- min D in the liver and also because of intestinal mal- ties and bone disease responded to treatment with cal- absorption of vitamin D. cium alone. Our group has recently demonstrated that d. Hypocalcemia Due to 1-Hydroxylase Deficiency this disorder may be more common in North American Impaired metabolism of 25OHD to 1,25(OH)2D is an infants than previously expected. A review of nutritional autosomal recessive condition, in which hypocalcemia rickets in New Haven, Connecticut revealed that 50% of and severe rachitic abnormalities occur [72] (see also cases had normal circulating values of 25-OHD, and Chapter 71). The disorder (also termed pseudo-vitamin some were even on vitamin supplementation. Increasing D-deficiency rickets or vitamin DÐdependent rickets, dietary calcium resulted in radiographic and biochemi- type 1) is inherited in an autosomal recessive manner cal improvement. This phenomenon appears to occur and is characterized by biochemical features similar to after children have been weaned to diets with little to no those of vitamin DÐdeficiency rickets, with the excep- dairy product content, with fluids consisting mostly of tions that circulating 25OHD levels are normal and cir- juices and soft drinks [78]. culating 1,25(OH)2D levels are low. Mutations in the g. Hypocalcemia Induced by Hyperphosphatemia gene encoding the ferrodoxin-binding component of Since the 1930s, it has been appreciated that oral or par- the mitochondrial P450 enzyme, 25-hydroxyvitamin D enteral phosphate can induce a decline in serum calcium 1 alpha hydroxylase (CYP27B1) have been shown to concentrations. Herbert et al. have demonstrated that cause this condition [73]. Restoration of eucalcemia phosphate infusions lower serum calcium in both the and correction of rickets is attainable with physiologi- presence and absence of parathyroid glands [79]. cal doses of 1,25(OH)2D3. Moreover, they reported that the changes in peak urinary e. Hypocalcemia Due to Hereditary Resistance to calcium excretion during phosphate administration are 1,25(OH)2D A defect in target tissue responsivity to not sufficient to account for the fall in the serum calcium. 1,25(OH)2D was clinically described shortly after the The theory they advanced remains the best explanation capacity to measure circulating 1,25(OH)2D became available for this phenomenon and centers on the hypoth- available [32]. Patients with hypocalcemia caused by esis that the calcium × phosphate molar product, when hereditary resistance to 1,25(OH)2D have severe mani- exceeded, leads to spontaneous precipitation of calcium festations of vitamin DÐdeficiency rickets; however, salts in soft tissues. The Ca × P product, when estimated serum 25OHD concentrations are normal, and from total serum ion concentrations (as mg/dl), is nor- 1,25(OH)2D levels are usually elevated. This disorder mally taken to be <60 in adults or <80 in small children. is inherited in an autosomal recessive manner. Additional Hyperphosphatemia sufficient to cause hypocalcemia features in many patients include alopecia totalis and is usually abrupt in onset and severe in magnitude. oligodontia. The disease is variably responsive to large Typical clinical settings include (1) excessive enteral or doses of 1,25(OH)2D3 and oral calcium therapy. In the parental phosphate administration, (2) the tumor lysis CHAPTER 64 The Hypocalcemic Disorders: Differential Diagnosis and Therapeutic Use of Vitamin D 1057 syndrome, and (3) rhabdomyolysis-induced acute renal although distinguishing it from permanent postopera- failure. Hypocalcemia induced by either oral or parental tive hypoparathyroidism can be difficult and requires phosphate administration is often associated with soft gradual discontinuation of supportive therapy with care- tissue calcification. Such ectopic calcification has been ful monitoring. Hypocalcemia also may occur in patients observed during the treatment of hypophosphatemia with bony metastases that induce bone formation, as due to either diabetic ketoacidosis or acute alcoholism. with prostatic and breast cancer [86]. Adults receiving phosphate-containing enemas and Finally, institution of therapy for vitamin D defi- infants fed “humanized” cow milk rich in phosphate ciency osteomalacia or rickets can sometimes lead to a may also become hypocalcemic [80,81]. Under most fall in serum calcium associated with rapid mineraliza- circumstances discontinuation of exogenous phosphate tion of previously unmineralized osteoid [87]. This is intake leads to prompt return of the serum calcium level self-limited and can usually be prevented with supple- to normal. mental calcium. Hypocalcemia in the setting of massive tumor lysis i. Medical Illness Hypocalcemia in the setting of results from the release of intracellular phosphate as a renal failure results from hyperphosphatemia due to consequence of chemotherapy-induced cell death, usu- reduced renal phosphate clearance by the failing kid- ally during the treatment of rapidly proliferating neo- ney and is complicated by impaired biosynthesis of plasms [82]. The hypocalcemia may continue beyond 1,25(OH)2D [88] (see Chapter 76). the period of hyperphosphatemia and appears to be Hypocalcemia and tetany were first reported in aggravated by suppressed 1,25(OH)2D levels [83]. The patients with pancreatitis in the early 1940s [89]. use of phosphate binding antacids, oral calcium, and, Pancreatic lipase released from the damaged gland is in severe cases, 1,25(OH)2D3 may help to correct the believed to liberate free fatty acids that chelate calcium, serum calcium level. thereby removing it from the extracellular fluid [90]. Rhabdomyolysis-induced acute renal failure occurs Hypomagnesemia resulting from poor oral intake, with trauma and drug or alcohol abuse. Marked hypo- alcohol use, or vomiting may contribute to the hypocal- calcemia can occur in the early oliguric phase, and cemia. Hypocalcemia in the setting of pancreatitis often moderate to severe hypercalcemia in the subsequent suggests a poor clinical course. Treatment consists of polyuric phase. Llach et al. have described hyperphos- parental calcium and magnesium when indicated. phatemia and suppressed serum 1,25(OH)2D levels Hypocalcemia may also occur in patients with acute during the initial hypocalcemia, suggesting a mecha- sepsis. In one series, 20% of such patients evidenced nism similar to that seen in the tumor lysis syndrome reductions in ionized serum calcium [91]. Hypocalcemia [84]. The appearance of hypercalcemia and high serum in this series was associated with a poor prognosis (50% 1,25(OH)2D levels during the diuretic phase may result mortality, compared to 30% in eucalcemic patients). from rapid development of secondary hyperpara- This phenomenon is most often reported with gram- thyroidism during the initial hypocalcemic period. negative sepsis but has occurred in toxic shock syn- Treatment includes restriction of phosphate intake and drome caused by staphylococcal infection [92]. The efforts to prevent hypocalcemia during the early stages pathophysiology of hypocalcemia in these two settings of the disease. is unknown. Finally, it has been suggested that h. Hypocalcemia Due to Accelerated Skeletal parathyroid gland reserve is subnormal in patients with Mineralization Bone remodeling is a controlled pro- AIDS, although hypocalcemia is not a prominent fea- cess of tissue renewal which, in healthy individuals, ture of that disorder [93]. results in closely matched rates of bone resorption and j. Medications A variety of medications have been formation (see Chapter 28). If skeletal mineralization reported to decrease serum ionized calcium concentra- exceeds the rate of bone resorption, hypocalcemia can tion. Many of these drugs are used to treat hypercalce- occur. One setting in which this can be observed is fol- mia and/or excessive bone resorption, and hypocalcemia lowing surgical correction of primary or tertiary hyper- results from their overzealous use. Thus, mithramycin, parathyroidism. The abrupt cessation of PTH-mediated calcitonin, and the bisphosphonates can all cause osteoclastic bone resorption with concomitant rapid hypocalcemia. In susceptible individuals, prolonged remineralization of an undermineralized skeleton can therapy with diphenylhydantoin or phenobarbital can lead to “hungry bone syndrome” with severe, even life- lead to hypocalcemia, owing in part to enhanced threatening hypocalcemia [85]. Postoperative treatment catabolism of vitamin D metabolites [94] (Chapter 74). should be instituted when the serum calcium level falls Citrated blood products, particularly when used for below 8.0 mg/dl, using oral or parenteral calcium sup- large volume transfusions or plasma plasmapheresis, plements and if necessary 1,25(OH)2D3. In general, can cause hypocalcemia [95]. Radiocontrast agents that this condition resolves over the course of several days, contain EDTA (ethylenediaminetetraacetic acid) can 1058 THOMAS O. CARPENTER AND KARL L. INSOGNA also induce falls in serum ionized calcium levels [96]. per 8 hr, as necessary, until the underlying intestinal dis- Finally, foscarnet (trisodium phosphonoformate), used turbance has resolved. in the treatment of patients with AIDS, has been reported to cause a decline in ionized serum calcium, 3. ROLE OF MAGNESIUM SUPPLEMENTATION perhaps through complexing extracellular calcium [97]. In the setting of hypomagnesemia, magnesium therapy may be required to restore PTH secretion and III. THERAPY FOR HYPOCALCEMIA peripheral activity. Prior to administration of magne- sium salts, assessment of renal function and urinary A. Acute Management output should be performed. Magnesium treatment in infancy consists of 5Ð10 mg 1. NEWBORNS of elemental magnesium (Mg) per kilogram body weight. It may be necessary to treat early neonatal hypocal- Although magnesium may be given intramuscularly, cemia when the circulating concentration of total the intravenous route is preferred. Magnesium sulfate serum is less than 5Ð6 mg/dl in premature infants, and septahydrate (MgSO4 ¥ 7H2O) is available as a 50% less than 6Ð7 mg/dl in term infants. Appropriate emer- solution, containing 48 Mg/ml of elemental magne- gency therapy of acute symptomatic hypocalcemia sium. These small volumes may be further diluted, but consists of a slow intravenous infusion (<1 ml/min) of they should be infused slowly; the dose may be repeated calcium gluconate in a 10% (w/v) solution. The cal- every 12Ð24 hr. In older individuals, up to 2.4 mg of cium gluconate salt consists of 9% elemental calcium. elemental Mg per kilogram body weight can be given A well-functioning indwelling intravascular catheter over a 10Ðmin period (to a maximum of 180 mg). should be used, to avoid extravasation. Calcium should Others prefer a continuous infusion of 576 mg of never be administered intramuscularly because of local elemental magnesium over 24 hr. The length of therapy tissue toxicity. It is important to perform cardiac mon- must be individualized, and maintenance with oral itoring and careful observation during acute infusions. magnesium salts should be implemented in cases where A total infusion of 1Ð3 ml will usually arrest convul- ongoing hypomagnesemia is anticipated. sions, and no more than 2 mg of elemental calcium per Magnesium levels should be monitored to avoid kilogram body weight should be given as a single dose. toxicity. Deep tendon reflexes can be examined, and Such bolus infusions may be repeated up to 4 times in therapy should be halted if they diminish. As with cal- a 24-hr period. If severe hypocalcemia persists, how- cium therapy, cardiac monitoring should be performed ever, it is generally more effective to use a long-term and therapy stopped if EKG changes occur. Intravenous calcium gluconate infusion, such that 20Ð50 mg of calcium gluconate is a useful antidote for magnesium elemental calcium per kilogram body weight is infused intoxication, and should be available at the bedside. over an entire 24-hr period. Calcium chloride is more irritating than calcium gluconate and is not the pre- ferred salt for infusion. Neither bicarbonate nor phos- B. Long-term Treatment phate should be coinfused with calcium in order to prevent precipitation of their respective calcium salts, Many of the causes of hypocalcemia discussed either in the infusion line or in the vein. previously are corrected by treating the underlying dis- order (e.g., vitamin D deficiency, tumor lysis syndrome). 2. ADULTS Relatively few of these disorders require maintenance In adults, emergency management consists of therapy for hypocalcemia, and of those, the most impor- 10Ð20 ml of 10% calcium gluconate infused over a tant are hypoparathyroidism and pseudohypoparathy- 10- to 15-min period. In the longer term one can dilute roidism. Vitamin DÐresistant states, although rare, 10 ampules of calcium gluconate in 1 liter of 5% dex- comprise a third group of patients that require long-term trose and, beginning at a rate of 50 ml/hr, titrate the treatment. rate to maintain the serum calcium in the low normal Hypoparathyroidism, whether primary or secondary range. Finally, in the setting of acute exacerbations to trauma or surgery, is the most frequently encountered of calcium malabsorption, as may typically occur in condition that requires chronic therapy to maintain patients with autoimmune hypoparathyroidism with eucalcemia. In these individuals, the goal is to maintain associated gastrointestinal disorders, nocturnal naso- serum calcium in the low normal range (8.5 to 9.2 mg/dl gastric supplementation with calcium carbonate or as measured by atomic absorption spectrophotometry). calcium gluconate has been employed, providing up to This will reduce the likelihood of symptoms such as 20 mg of elemental calcium per kilogram body weight circumoral tingling, signs such as carpopedal spasm, as CHAPTER 64 The Hypocalcemic Disorders: Differential Diagnosis and Therapeutic Use of Vitamin D 1059 well as more long-term complications such as cataracts. our patients. The dose of calcitriol can range from as Long-term treatment with PTH is not yet practical little as 0.25 up to 2.0 ug/day [99,100]. We have esti- although short-term therapy has been successful [98]. mated the biological half-life of the drug as 12Ð14 hr. There is no single best way to achieve stable Hypercalcemia, when it develops during therapy eucalcemia, although the combination of a vitamin D with calcitriol, usually resolves within 3Ð4 days after metabolite with calcium supplements is generally pre- discontinuing the drug, although we have had patients ferred. A wide variety of preparations of both are avail- in whom it has taken over 1 week for serum calcium able (see Tables I and II). Because of the prolonged to normalize. In addition to a high calcium diet, cal- toxicity that occurs with excessive ingestion of either cium supplements are important for the treatment ergocalciferol (vitamin D2) or 25OHD, we generally of hypoparathyroidism. Doses of 1000Ð2000 mg/day prefer to use rapid acting preparations of vitamin D for of calcium may be necessary. Preparations including the treatment of this disorder. Toxicity, when it occurs the carbonate, citrate, lactate, gluconate, and gluco- with these preparations, corrects more rapidly with bionate salts are suitable for this purpose (Table I). discontinuation of the drug. Calcitriol [1,25(OH)2D3] We prefer calcium carbonate because it is inexpen- and dihydrotachysterol (DHT) are two preparations sive, well-tolerated, and easily acquired. In some cases suited to this purpose. Both are fully active in vivo. In of hypoparathyroidism, a thiazide diuretic may be general, it is best to aim for a stable dose of one of useful in augmenting serum calcium levels and these agents and to further regulate the serum calcium reducing the hypercalciuria that can occur with the by adjusting the intake of supplemental calcium, rather institution of treatment. Several vitamin D analogs than by making repeated changes in vitamin D have been developed for the treatment of secondary metabolite therapy. We use calcitriol in the majority of hyperparathyroidism in the setting of renal failure

TABLE I Oral Calcium Preparationsa

Cost per 1000 mg of Drug Dosage form Elemental calcium (mg/tablet) elemental calcium*

Calcium Carbonate Calcium carbonate (available Various formulations, including 100 mg/ml (susp), 260 mg (tab), Cost will vary by manufacturer in generic brands) suspension, tablets, chewable 500 mg (tab) (depends on (generally lower than name tablets (depending on manufacturer) brands) manufacturer) Os-Cal 500¨ Tablet 500 mg $0.18 Caltrate 600¨ Tablet 600 mg $0.16 Tums¨ (Regular, EX, Ultra) Chewable tablet 200 mg, 300 mg, 400 mg $0.30, $0.12, $0.18 Alka-Mints¨ Chewable tablet 340 mg $0.12 Viactiv¨ Chewable 500 mg $0.26

Calcium citrate Calcium citrate (available Various formulations including 260 mg, 500 mg (depends Cost will vary by manufacturer in generic brands) tablets, effervescent tablets, on manufacturer) (generally lower than name oral suspension (depending brands) on manufacturer) Citracal¨ Tablet 200 mg $0.13

Calcium glubionate Neo-Calglucon¨ Syrup 115 mg/5 ml $1.74

*Retail cost will vary between retail pharmacies. References ÐLacy CF, Armstrong LL, Goldman MP, Lance LL. Drug Information Handbook. Lexi-Comp. 2003;11th ed. 220Ð23. ÐKastrup EK ed. Drug Facts and Comparisons. St. Louis: Facts & Comparisons; August 2003. ÐWalgreens Pharmacy. www.walgreens.com. Accessed on July 31, 2003. aAdapted with permission from Carpenter T 1996 Rickets. In: Berg F, Inglefinger J, Wald E (eds) Gellis and Kagan’s Current Pediatric Therapy, 15th ed. Saunders, Philadelphia, pp. 363Ð367. 1060 THOMAS O. CARPENTER AND KARL L. INSOGNA

TABLE II Vitamin D and Related Agentsa,b

Name Formulation Typical dosec

Vitamin D (calciferol) Drisdol¨ Solution: 8000 IU/ml 2000 IU/day Tablet: 25,000 IU 1 tablet/day 50,000 IU 1 tablet/day Dihydrotachysterol (DHT) (Hytakerol¨) Solution: 0.2 mg/5ml 0.5 mg/day Tablets: 0.125 mg 0.5 mg/day 0.2 mg 0.5 mg/day 0.4 mg 0.4 mg/day 1,25 dihydroxyvitamin D (calcitriol) Rocaltrol¨ 0.25 µg capsule 0.5 µg/day 0.50 µg capsule 0.5 µg/day 1.0 µg/ml (oral solution) 0.5 µg/day Calcijex¨ solution: ampules for IV use containing solutions with 1 or 2 µg/ml of drug 1 µg vitamin D = 40 IU

aAdapted with permission from Carpenter T 1996 Rickets. In: Berg F, Ingelfinger J, Wald E (eds) Gellis and Kagan’s Current Pediatric Therapy, 15th ed. Saunders, Philadelphia, pp. 363Ð367. b Daily dose may vary significantly, depending on condition.

(see Chapter 76) [101]. These analogs are relatively of calcium have resulted in improvement of the rickets more selective inhibitors of parathyroid proliferation and normalization of all serum biochemical parameters than calcitriol. The limited calcemic activity of these (see Chapter 72) [74]. compounds renders them less useful in the management Although as discussed above, recently developed of hypocalcemia. analogs of vitamin D have not been generally recom- Magnesium deficiency can occur in patients with mended for therapy of hypocalcemia, two novel hypoparathyroidism, most often secondary to steator- analogs (20-Epi-1,25(OH)2D3 and JK-1626-2) have rhea, which is seen in the autoimmune forms of this dis- specifically been found to be efficacious in the treat- order [102,103]. This may render a patient relatively ment of cases of hereditary resistance to vitamin D resistant to therapy, and therefore magnesium deficiency caused by mutations in the ligand-binding domain of should be considered in individuals whose therapeutic VDR [104]. requirements unexpectedly increase. A variety of stresses such as trauma, infection, and In pseudohypoparathyroidism, the therapeutic pregnancy can increase the therapeutic requirements of approach is similar to that in primary hypoparathy- patients with chronic hypocalcemia, and the clinician roidism, the principal difference being that hypercalci- should be alert to this possibility. uria is less of an issue, and it is generally easier to maintain eucalcemia in these individuals. Untreated patients with pseudohypoparathyroidism may have Acknowledgments variable defects in mineralization and initially may require high dose therapy to achieve eucalcemia as Dr. Carpenter is supported by a grant from their bones remineralize. Requirements will drop as the National Institutes of Health (HD1288). Dr. Insogna the bone lesion heals, often heralded by a fall in serum is supported by grants from the NIH (AR39571) alkaline phosphatase and a rise in serum calcium and both are supported an NIH Core Center Grant levels. (AR 46032). Individuals with 1-hydroxylase deficiency have a defect in the ability to generate 1,25(OH)2D from the precursor metabolite 25OHD. In this disorder, eucal- References cemia can be achieved by supplying 1,25(OH)2D3 in physiological dosages [72]. In contrast, hereditary resis- 1. Harrison H, Harrison H 1979 Hypocalcemia states. In: tance to 1,25(OH)2D represents a spectrum of resistance Disorders of Calcium and Phosphate Metabolism in Childhood to therapy, with some individuals responding to doses and Adolescence. Saunders, Philadelphia, Pennsylvania, pp. 47Ð99. of calcitriol in the usual therapeutic range and others 2. Sharief N, Matthew DJ, Dillon MJ 1991 Hypocalcaemic resistant to even massive doses of the drug [32]. As stridor in children. How often is it missed? Clin Pediatr noted above, chronic therapy with parenteral infusions 30:51Ð52. CHAPTER 64 The Hypocalcemic Disorders: Differential Diagnosis and Therapeutic Use of Vitamin D 1061

3. Wong C, Lau C, Cheng C, Leung W, Freedman B 1990 20. King G, Holtrop M, Raisz L 1978 The relation of ultrastruc- Hypocalcemic myocardial dysfunction: Short- and long-term tural changes in osteoclasts to resorption in bone cultures improvement with calcium replacement. Am Heart J stimulated with parathyroid hormone. Metab Bone Dis 120:381Ð386. Related Res 1:67Ð74. 4. Alpan G, Glick B, Peleg O, Eyal F 1991 Pseudotumor 21. Friedman PA, Gesek FA 1993 Calcium transport in renal cerebri and coma in vitamin DÐdependent rickets. Clin Pediatr epithelial cells. Am J Physiol (Renal, Fluid Electrolyte Physiol) 20:254Ð256. 33:F181ÐF198. 5. Carpenter TO, Insogna KL, Boulware SD, Mitnick MA 1990 22. Letscher M, Kaissling B, Biber J, Murer H, Kempson ST, Vitamin D metabolism in chronic childhood hypoparathy- Levi M 1996 Regulation of rat renal Na/Pi cotransporter by roidism: Evidence for a direct regulatory effect of calcium. parathyroid hormone: Immunohistochemistry. Kidney Int J Pediatr 116:252Ð257. 49:1010Ð1018. 6. Nikiforuk G, Fraser D 1979 Etiology of enamel hypoplasia 23. Minghetti PP, Norman AW 1988 l,25(OH)2-Vitamin D3 and interglobular dentin: The roles of hypocalcemia and receptors: Gene regulation and genetic circuitry. FASEB J hypophosphatemia. Metab Bone Dis Related Res 2:17Ð23. 2:3043Ð3053. 7. Ilium F, Dupont E 1985 Prevalence of CT-detected calcifica- 24. Armbrecht J, Wongsurawat N, Zenser TV, Davis BB 1982 tion in the basal ganglia in idiopathic hypoparathyroidism Differential effects of parathyroid hormone on the renal and pseudo-hypoparathyroidism. Neuroradiology 27:32Ð37. 25-dihydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 8. Stewart AF, Battaglini-Sabetta J, Milstone LM 1984 production of young and adult rats. Endocrinology 111: Hypocalcemia-induced psoriasis of von Zumbusch: New expe- 1339Ð1344. rience with an old syndrome. Ann Intern Med 100:677Ð680. 25. Walker AT, Stewart AF, Korn EA, Shiratori T, Mitnick MA, 9. Brown EM 1991 Extracellular Ca2+ sensing, regulation of Carpenter TO 1989 Effect of parathyroid hormone-like parathyroid cell function, and role of Ca2+ and other ions as peptides on 25-hydroxyvitamin D-1a-hydroxylase activity in extracellular (first) messengers. Physiol Rev 71:371Ð411. rodents. Am J Physiol 258:E297ÐE303. 10. Ladenson J, Lewis J, Boyd J 1978 Failure of total calcium 26. Booth BE, Tsai HC, Morris C 1977 Parathyroidectomy corrected for protein, albumin, and pH to correctly assess reduces 25-hydroxyvitamin D3 1a-hydroxylase activity in the free calcium status. J Clin Endocrinol Metab 46:986Ð993. hypocalcemic vitamin DÐdeficient chick. J Clin Invest 11. Chattopadhyay N, Mithal A, Brown EM 1996 The calcium- 60:1314Ð1320. sensing receptor: A window into the physiology and patho- 27. Siegel N, Wongsurawat N, Armbrecht J 1986 Parathyroid physiology of mineral ion metabolism. Endocr Rev hormone stimulates dephosphorylation of the renoredoxin 17:289Ð307. component of the 25-hydroxyvitamin D3-1a-hydroxylase from 12. Juppner H, Abou-Samra AB, Freeman MV, Kong X, rat renal cortex. J Biol Chem 261:16998Ð17003. Schipani E, Richards J, Kolakawski L, Hock J, Kronenberg H, 28. Matsumoto T, Ikeda K, Morita K, Fukumoto S, Takahashi H, Serge G 1991 G protein-linked receptor for parathyroid hor- Ogata E 1987 Blood Ca2+ modulates responsiveness of renal mone and parathyroid hormone-related peptide. Science 25(OH)D3-1a-hydroxylase to PTH in rats. Am J Physiol 254:1024Ð1026. (Endocrinol Metab 16) 253:E503ÐE507. 13. Livesey SA, Kemp BE, Re C, Partridge NC, Martin T 1982 29. Bushinsky DA, Riera GS, Favus MJ, Coe FL 1985 Evidence Selective hormonal activation of cyclic AMP-dependent that blood ionized calcium can regulate serum 1,25(OH)2D3 protein-kinase isoenzymes in normal and malignant osteoblasts. independently of parathyroid hormone and phosphorus in the J Biol Chem 257:14983Ð14987. rat. J Clin Invest 76:1599Ð1604. 14. Hruska KA, Moskowitz D, Esbrit P, Civitelli R, Westbrook S, 30. Kumar R 1991 Vitamin D and calcium transport. Kidney Int Huskey M 1987 Stimulation of inositol triphosphate and 40:1177Ð1189. diacyl-glycerol production in renal tubular cells by parathy- 31. Norman AW, Song X, Zanello L, Bula C, Okamura WH roid hormone. J Clin Invest 79:230Ð239. 1999 Rapid and genomic biological responses are mediated 15. Abou-Samra A, Juppner H, Force T, Freeman M, Knog X, by different shapes of the agonist steroid hormone, Schipani E, Urena P, Richards J, Bonventre J, Potts J, 1alpha,25(OH)2vitamin D3. Steroids 64:120Ð128. Kronenberg H, Segre G 1992 Expression cloning of a 32. Liberman UA, Marx SJ 1996 Vitamin DÐdependent rickets. common receptor for parathyroid hormone and parathyroid In: Favus M (ed) Primer on the Metabolic Bone Diseases and hormone-related peptide from rat osteoblast-like cells: A single Disorders of Mineral Metabolism, 3rd Ed. Raven, New York, receptor stimulates intracellular accumulation of both cAMP pp. 311Ð316. and inositol triphosphates and increases intracellular free 33. Favus MJ, Mangelsdorf DJ, Tembe V, Coe BJ, Haussler MR calcium. Proc Natl Acad Sci USA 89:2732Ð2736. 1988 Evidence for in vivo upregulation of the intestinal 16. Lee K, Deeds D, Bond A, Juppner H, Abou-Samra A-B, vitamin D receptor during dietary calcium restriction in the Segre G 1993 In situ localization of PTH/PTHrP receptor rat. J Clin Invest 82:218Ð224. mRNA in the bone of fetal and young rats. Bone 14:341Ð345. 34. van Leeuwen JPTM, Birkenhager JC, Buurman CJ, 17. Rouleau M, Mitchell J, Goltzman D 1990 Characterization of Semite JP, Pols HAP 1990 Functional involvement of calcium the major parathyroid hormone target cell in the endosteal in the homologous up-regulation of the 1,25-dihydroxy- metaphysis of rat long bones. J Bone Miner Res 5:1043Ð1053. vitamin D3 receptor in osteoblast-like cells. FEBS Lett 270: 18. McSheehy P, Chambers T 1986 Osteoblast-like cells in the 165Ð167. presence of parathyroid hormone release soluble factor that 35. Sandgren ME, DeLuca HF 1990 Serum calcium and stimulates osteoclastic bone resorption. Endocrinology vitamin D regulate 1,25-dihydroxyvitamin D3 receptor con- 119:1654Ð1659. centration in rat kidney in vivo. Proc Natl Acad Sci USA 87: 19. Talmage R, Doppelt S, Fondren F 1976 An interpretation of 4312Ð4314. acute changes in plasma 45Ca following parathyroid hormone 36. Clements MR, Johnson L, Fraser DR 1987 A new mechanism administration to thyroparathyroidectomized rats. Calcif for induced vitamin D deficiency in calcium deprivation. Tissue Res 22:117Ð128. Nature 325:62Ð65. 1062 THOMAS O. CARPENTER AND KARL L. INSOGNA

37. Goltzman D, Cole D 1996 Hypoparathyroidism. In: Favus M 56. Kruse K, Kracht U, Wohlfart K, Kruse U 1989 Biochemical (ed) Primer on the Metabolic Bone Diseases and Disorders of markers of bone turnover, intact serum parathyroid hormone Mineral Metabolism, 3rd Ed. Raven, New York, pp. 220Ð223. and renal calcium excretion in patients with pseudo- 38. Conley M, Beckwith J, Mancer J, Tenckhoff L 1979 The hypoparathyroidism and hypoparathyroidism before and spectrum of DiGeorge syndrome. J Pediatr 94:883Ð890. during vitamin D treatment. Eur J Pediatr 148:535Ð539. 39. Yagi H, Furutani Y, Hamada, Sasaki T, Asakawa S, 57. Stone M, Hosking D, Garcia-Himmelstine C, White D, Minoshima S, Ichida F, Joo K, Kimura M, Imamura S, Rosenblum D, Worth H 1993 The renal response to exoge- Kamatani N, Momma K, Takao A, Nakazawa M, Shimizu N, nous parathyroid hormone in treated pseudohypoparathy- Matsuoka R 2003 Role of TBX1 in human del22q11.2 roidism Bone 14:727Ð735. syndrome. Lancet 362:1366Ð1373. 58. Patten JL, Johns DR, Valle D, Eil C, Gruppuso P, Steele G, 40. Thakker R, Davies K, Whyte M, Wooding C, O’Riordan J Smallwood PM, Levine MA 1990 Mutation in the gene 1990 Mapping the gene causing X-linked recessive idio- encoding the stimulatory G protein of adenylate cyclase pathic hypoparathyroidism to Xq26-Xq27 by linkage studies. in Albright’s hereditary osteodystrophy. N Engl J Med J Clin Invest 86:40Ð45. 322:1412Ð1419. 41. Arnold A, Horst S, Gardella T, Baba H, Levine M, 59. Weinstein LS, Yu S, Warner DR, Liu J 2001 Endocrine mani- Kronenberg H 1990 Mutation of the signal peptide-encoding festations of stimulatory G protein alpha-subunit mutations region of the preproparathyroid hormone gene in familial and the role of genomic imprinting. Endocrine Reviews hypoparathyroidism. J Clin Invest 86:1084Ð1087. 22:675Ð705. 42. Parkinson D, Thakker R 1992 A donor splice site mutation in 60. Loveridge N, Tschopp FT, Born W, Devogelaer JP, the parathyroid gene is associated with autosomal recessive de Deux-chaisnes CN, Fischer JA 1986 Separation of hypoparathyroidism. Nature Genet 1:149Ð152. inhibitory activity from biologically active parathyroid 43. Chou Y, Brown E, Levi T, Crowe G, Atkinson A, Arnqvist H, hormone in patients with pseudohypoparathyroidism type I. Toss G, Fuleihan G, Seidman J, Seidman C 1992 The gene Biochim Biophys Acta 889:117Ð122. responsible for familial hypocalciuric hypercalcemia maps to 61. Rude RK, Oldham SB, Singer FR 1976 Functional chromosome 3 in four unrelated families. Nature Genet hypoparathyroidism and parathyroid hormone end-organ 1:295Ð300. resistance in human magnesium deficiency. Clin Endocrinol 44. Pollak MR, Brown EM, Step L, McLaine PN, Kifor O, Park J, 5:209Ð224. Hebert SC, Seidman CE, Seidman JG 1994 Autosomal dom- 62. Carpenter T 1988 Disturbances of vitamin D metabolism and inant hypocalcaemia caused by a Ca2+-sensing receptor gene action during clinical and experimental magnesium defi- mutation. Nature Genet 8:303Ð307. ciency. Magnesium Res 1:131Ð139. 45. Pearce S, Williamson C, Kifor O, Bai M, Coulthard M, 63. Rude RK, Adams JS, Ryzen E, Endres DB, Miimi H, Horst RI, Davies M, Lewis-Earned N, McCredie D, Powell H, Kendall- Haddad JG, Singer FR 1985 Low serum concentrations of Taylor P, Brown E, Thakker R 1996 A familial syndrome of 1,25-dihydroxyvitamin D in human magnesium deficiency. hypocalcemia with hypercalciuria due to mutations in the J Clin Endocrinol Metab 61:933Ð940. calcium-sensing receptor. N Engl J Med 335:1115Ð1122. 64. Allgrove J, Adami S, Fraher L, Reuben A, O’Riordan JLH 46. Neufeld M, MacLaren N, Blizzard R 1981 Two types of autoim- 1984 Hypomagnesaemia: Studies of parathyroid hormone mune Addison’s disease associated with different polyglandular secretion and function. Clin Endocrinol 21:435Ð449. autoimmune (PGA) syndromes. Medicine 60:355Ð362. 65. Fuss M, Bergmann P, Bergans A, Bagon J, Cogan E, 47. Nagamine K, Peterson P, Scott HS, Kudoh J, Minoshima S, Pepersack T, van Gossum M, Corvilain J 1989 Correction Heino M, Krohn KJ, Lalioti MD, Mullis PE, Antonarakis SE, of low circulating levels of 1,25-dihydroxyvitamin D by Kawasaki K, Asakawa S, Ito F, Shimizu N 1997 Positional 25-hydroxyvitamin D during reversal of hypomagnesaemia. cloning of the APECED gene. Nature Genetics. 17:393Ð398. Clin Endocrinol 31:31Ð38. 48. Horwitz C, Myers W, Foote F 1972 Secondary malignant 66. Anast CS, Gardner DW 1981 Magnesium metabolism. In tumors of the parathyroid glands. Report of two cases with Bronner F, Coburn J (eds) Disorders of Mineral Metabolism, associated hypoparathyroidism. Am J Med 52:797Ð808. Vol. 3. Academic Press, New York, pp. 423Ð506. 49. Dill J 1983 Hypoparathyroidism in sarcoidosis. South Med J 67. Robertie PG, Butterworth JF, Prielipp RC, Tucker WY, 76:414. Zaloga GP1992 Parathyroid hormone responses to marked 50. Brezis M, Shalev O, Leibel B, Bernheim J, Ben-Ishay D hypocalcemia in infants and young children undergoing repair 1980 Phosphorus retention and hypoparathyroidism associ- of congenital heart disease. J Am Collect Cardiol 20:672Ð677. ated with transfusional iron overload in thalassaemia. Miner 68. Clemens TL, Henderson SL, Adams JS, Holick MF 1982 Electrolyte Metab 4:57. Increased skin pigment reduces the capacity of skin to 51. Carpenter TO, Carnes DL Jr, Anast CS 1983 Hypoparathy- synthesize vitamin D3. Lancet 1:74Ð76. roidism in Wilson’s disease. N Engl J Med 309:873Ð877. 69. Dagnelie PC, Vergote FJVRA, van Staveren WA, van den 52. Heidbreder E, Gotz R, Schafferhans K, Heidland A 1986 Berg H, Dingjan PG, Hautvast JGAJ 1990 High prevalence Diminished parathyroid gland responsiveness to hypocal- of rickets in infants on macrobiotic diets. Am J Clin Nutr cemia in diabetic patients with uremia. Nephron 42:285Ð289. 51:202Ð208. 53. Burch WM, Posillico JT 1983 Hypoparathyroidism after 131I 70. Kruse K 1995 Pathophysiology of calcium metabolism in therapy with subsequent return of parathyroid function. children with vitamin DÐdeficiency rickets. J Pediatr J Clin Endocrinol Metab 57:398Ð401. 126:736Ð741. 54. Anast C, Mohs J, Kalpan S, Burns P 1972 Evidence for parathy- 71. Kumar R 1983 Hepatic and intestinal osteodystrophy and the roid failure in magnesium deficiency. Science 177:606Ð608. hepatobiliary metabolism of vitamin D. Ann Intern Med 55. Albright F, Burnett CH, Smith PH, Parson W 1942 98:662Ð663. Pseudohypoparathyroidism. An example of “Seabright-Bantam 72. Balsan S 1991 Hereditary pseudo-deficiency rickets or syndrome.” Endocrinology 30:922Ð932. vitamin DÐdependency type I. In: Glorieux FH (ed) Rickets CHAPTER 64 The Hypocalcemic Disorders: Differential Diagnosis and Therapeutic Use of Vitamin D 1063

(Nestle Nutrition Workshop Series, Vol. 21). Raven, New York, 91. Zaloga GP, Chernow B 1987 The multifactorial basis for pp. 55Ð165. hypocalcemia during sepsis. Studies of the parathyroid 73. Wang X, Zhang MYH, Miller WL, Portale AA 2002 Novel hormone-vitamin D axis. Ann Intern Med 107:36Ð41. gene mutations in patients with 1α-hydroxylase deficiency 92. Chesney RW, McCarron DM, Haddad JG, Hawker CD, that confer partial enzyme activity in vitro. J Clin Endocrinol DiBella FP, Chesney PJ, Davis JP 1983 Pathogenic mecha- Metab 87:2424Ð2430. nisms of the hypocalcemia of the staphylococcal toxic-shock 74. Balsan S, Garabedian M, Larchet M, Gorski A, Coumot G, syndrome. J Lab Clin Med 101:576Ð585. Tau C, Bourdeau A, Silve C, Ricour C 1986 Long-term noc- 93. Jaeger P, Otto S, Speck RF, Villiger L, Horber FF, Casez J-P, turnal calcium infusions can cure rickets and promote normal Takkinen R 1994 Altered parathyroid gland function mineralization in hereditary resistance to 1,25-dihydroxy- in severely immunocompromised patients infected with vitamin D. J Clin Invest 77:1661Ð1667. human immunodeficiency virus. J Clin Endocrinol Metab 75. Pettifor JM, Ross FP, Travers R, Glorieux FH, DeLuca HF 79:1701Ð1705. 1981 Dietary calcium deficiency: A syndrome associated 94. Weinstein R, Bryce G, Sappington L, King K, Gallagher B with bone deformities and elevated serum 1,25-dihydroxy- 1984 Decreased serum ionized calcium and normal vitamin vitamin D concentration. Metab Bone Dis Related Res D metabolite levels with anticonvulsant drug treatment. 2:301Ð305. J Clin Endocrinol Metab 58:1003Ð1009. 76. Pettifor JM, Ross P, Wang J, Moodley GP, Couper-Smith J 95. Tofalletti J, Nissenson RA, Endres D, McGarry E, 1978 Rickets in children of rural origin in South Africa: Is Mogollon G 1985 Influence of continuous infusion of low dietary calcium a factor? J Pediatr 92:320Ð324. citrate on responses of immunoreactive PTH, calcium, 77. Marie P, Pettifor J, Ross F, Glorieux F 1982 Histological magnesium components, and other electrolytes in normal osteomalacia due to dietary calcium deficiency in children. adults during plasmapheresis. J Clin Endocrinol Metab 60: N Engl J Med 307:584Ð588. 874Ð879. 78. DeLucia MC, Mitnick ME, Carpenter TO 2003 Nutritional 96. Mallette LE, Gomez LS1982 Systemic hypocalcemia after rickets with normal circulating 25-hydroxyvitamin D: a call clinical injection of radiographic contrast media: for re-examining the role of dietary calcium intake in North Amelioration by omission of calcium chelating agents. American children. J Clin Endocrinol Metab 88:3539Ð3545. Radiology 147:677Ð679. 79. Herbert L, Lemann J, Petersen J, Lennon E 1966 Studies of 97. Jacobson MA, Gambertoglio JG, Aweeka FT, Causey DM, the mechanism by which phosphate infusion lowers serum Portale AA 1991 Foscarnet-induced hypocalcemia and effects calcium concentration. J Clin Invest 45:1886Ð1894. of foscarnet on calcium metabolism. J Clin Endocrinol Metab 80. Chernow B, Rainey T, Georges L, O’Brian J 1981 Iatrogenic 72:1130Ð1135. hyperphosphatemia: A metabolic consideration in critical 98. Winer K, Yanovski J, Cutler G 1996 Synthetic human care medicine. Crit Care Med 9:772Ð774. parathyroid hormone 1-34 vs calcitriol and calcium in the 81. Venkataraman P, Tsang R, Greer F, Noguchi A, Laskarzewski P, treatment of hypoparathyroidism: Results of a short-term Steichen J 1985 Late infantile tetany and secondary hyper- randomized crossover trial. JAMA 276:631Ð636. parathyroidism in infants fed humanized cow milk formula. 99. Russell R, Smith R, Walton R, Preston C, Basson R, Am J Dis Children 139:664Ð668. Henderson R 1976 1,25-Dihydroxycholecalciferol and 82. Arrambide K, Toto R 1993 Tumor lysis syndrome. Semin 1a-hydroxycholecalciferol in hypoparathyroidism. Lancet Nephrol 13:273Ð280. 2:14Ð17 (July 6). 83. Dunlay R, Camp M, Allon M, Fanti P, Malluche H, Llach F 100. Neer R, Holick M, DeLuca H, Potts J 1975 Effects of 1989 Calcitriol in prolonged hypocalcemia due to the tumor 1a-hydroxyvitamin D3 on calcium and phosphorus metabolism lysis syndrome. Ann Intern Med 110:162Ð164. in hypoparathyroidism. Metabolism 24:1403Ð1413. 84. Llach F, Felsenfeld A, Haussler M 1981 The pathophysiol- 101. Slatopolsky E, Dusso A, Brown AJ 2002 Control of uremic bone ogy of altered calcium metabolism in rhabdomyolysis- disease: role of vitamin D analogs. Kidney Int 61(Suppl 80): induced acute renal failure. N Engl J Med 305:117Ð123. S143ÐS148. 85. Brasier A, Nussbaum S 1988 Hungry bone syndrome: 102. Rosier A, Rabinowitz D 1973 Magnesium-induced reversal Clinical and biochemical predictors of its occurrence after of vitamin DÐresistance in hypoparathyroidism. Lancet parathyroid surgery. Am J Med 84:654Ð660. 1:803Ð804 (April 4). 86. Abramson EC, Gajardo H, Kukreja SC 1990 Hypocalcemia 103. Ahonen P, Myllarniemi S, Sipila I, Perheentupa J 1990 in cancer. Bone Miner 10:161Ð169. Clinical variation of autoimmune polyendocrinopathy- 87. Anast C, Carpenter T, Key L 1990 Metabolic bone disorders candidiasis-ectodermal dystrophy (APECED) in a series of in children. In: Avioli L, Krane S (eds) Metabolic Bone 68 patients. N Engl J Med 322:1829Ð1836. Disease and Related Research. Saunders, Philadelphia, 104. Gardezi SA, Nguyen C, Malloy PJ, Posner GH, Feldman D, Pennsylvania, pp. 850Ð887. Peleg S 2001 A rationale for treatment of hereditary vitamin DÐ 88. Llach F, Bover J 1996 Renal Osteodystrophy. In: Brenner B resistant rickets with analogs of 1 alpha,25-dihydroxy- (ed) The Kidney, 5th Ed. Saunders, Philadelphia, Pennsylvania, vitamin D3. J Biol Chem 276:29148Ð29156. pp. 2187Ð2273. 105. Lacy CF, Armstrong LL, Goldman MP, Lance LL. Drug 89. Edmondson H, Berne C 1944 Calcium changes in acute Information Handbook. Lexi-Comp. 2003;11th ed. 220Ð23. pancreatic necrosis. Surg Gynecol Obstet 79:240Ð244. 106. Kastrup EK ed. Drug Facts and Comparisons. St. Louis: 90. Stewart AF, Longo W, Kreutter D, Jacob R, Burtis WJ 1986 Facts & Comparisons; August 2003. Hypocalcemia due to calcium soap formation in a patient 107. Walgreens Pharmacy. www.walgreens.com. Accessed on with a pancreatic fistula. N Engl J Med 315:496Ð498. July 31, 2003. CHAPTER 65 Vitamin D Deficiency and Nutritional Rickets in Children

JOHN M. PETTIFOR MRC Mineral Metabolism Research Unit, Department of Pediatrics, University of the Witwatersrand and Chris Hani Baragwanath Hospital, P O Bertsham 2013, South Africa

I. Introduction VI. Radiologic Changes II. Historical Perspective VII. Treatment and Prevention III. The Epidemiology of Vitamin D Deficiency and Nutritional VIII. Dietary Calcium Deficiency Rickets IX. The Pathogenetic Spectrum of Nutritional Rickets IV. Clinical Presentation X. Conclusions V. Biochemical Abnormalities References

I. INTRODUCTION was known in Europe as “the English disease,” was more common in the cities than in rural areas. Prior to Rickets is a clinical syndrome that presents in chil- the industrial revolution, it was associated with afflu- dren as a result of a failure of or delay in mineraliza- ence, as the children of well-to-do families were often tion of the growth plate of growing bones. There are completely covered by clothing and were kept indoors. numerous different causes, the majority of which can With the migration of large numbers of people from be grouped into three major categories: those which rural to urban areas at the time of the industrial revolu- primarily result in a failure to maintain normal calcium tion, the disease became associated with poverty and homeostasis; those which primarily affect phosphate overcrowding in the developing urban slums. homeostasis; and those which directly inhibit the min- A number of studies in the late 19th and early 20th eralization process. Globally, rickets due to nutritional centuries documented the almost universal prevalence causes (which fall into the calciopenic group) remains of rickets in young children in cities in northern the most frequent form of the disease seen. However, Europe (for example in Glasgow [1] and Vienna [2]). in a number of industrialized countries, such as the However, with the realization of the importance of U.S., the genetic forms of hypophosphatemic rickets ultraviolet light in preventing nutritional rickets and are now probably more prevalent than the nutritional the discovery and isolation of vitamin D in the first causes outside the neonatal period, as a result of the quarter of the 20th century [3] (see Chapter 1), pro- fortification of foods with vitamin D and the use of gram were introduced to prevent vitamin D deficiency. vitamin D supplements in at-risk groups. Nevertheless, In the United Kingdom, a number of foods were for- the last decade has seen a resurgence of nutritional rick- tified with vitamin D during the World War II. This led ets in minority communities in a number of developed to a rapid reduction in the number of children diag- countries. nosed with rickets, but in the following years the inci- dence of idiopathic hypercalcemia rose in infants, which at the time was thought to be due to uncon- II. HISTORICAL PERSPECTIVE trolled fortification of various foods (especially milk and cereals) leading to daily intakes of 100 µg or more Although nutritional rickets is often considered to [4]. As a result, the fortification of foods and the use of be a disease of industrialization, descriptions of rickets vitamin D supplements fell into disrepute, and the have been attributed to both Homer (900 BC) and prevalence of vitamin D deficiency and nutritional Soranus Ephesius (130 AD). More recently, attention rickets has increased, particularly among the immigrant was drawn to rickets by Daniel Whistler in 1645, and Asian population. five years later Francis Glisson (1650) provided a clas- In the United States, the universal fortification of sic description of the disease. It was described as a dis- milk with vitamin D at 400 IU/quart from the 1930s has ease that occurred in young children, produced severe almost eradicated nutritional rickets except in families deformities, and was often fatal. The condition, which who exclude milk from their diets [5]. However, as had VITAMIN D, 2ND EDITION Copyright © 2005, Elsevier, Inc. FELDMAN, PIKE, AND GLORIEUX All rights reserved. 1066 JOHN M. PETTIFOR occurred in the United Kingdom, a few cases of acids was increased in one-third of the infants a long vitamin D toxicity have been reported as a result of time after the rickets had healed, many of the parents the lack of monitoring of the fortification process [6]. had increased amino acid and phosphorus excretion, In central Europe, rickets has been effectively pre- and a good correlation was found between the excre- vented in infants and young children by the intermit- tion of individual amino acids by an infant and its par- tent administration (every three to five months for the ents. The authors suggested that these findings indicate first two years of life) of high doses of vitamin D a genetic factor playing a role in predisposing a child (“stosstherapie”) [7]. to rickets; however, the mode of inheritance is unclear. In the early literature, breast feeding was reported to be protective against rickets [26]. More recently III. THE EPIDEMIOLOGY OF VITAMIN D however, it has been described as a risk factor for the DEFICIENCY AND NUTRITIONAL development of rickets [5,27Ð29]. In recent years, RICKETS specifically designed breast milk substitutes have replaced natural cow’s milk as the major source of nutri- Vitamin D deficiency is a prerequisite for the devel- ents for the non breast-fed infant. This alteration in feed- opment of nutritional rickets in the majority of chil- ing patterns may account for the apparent change in risk dren. Thus, the disease is typically associated with a associated with breast-feeding for several reasons; first, lack of ultraviolet light exposure or dietary vitamin D. breast milk substitutes are fortified with vitamin D at As commonly ingested foods are generally deficient in 400 IU/liter while natural cow’s milk contains little vita- vitamin D (the exceptions being oily fishes or fortified min D [30]; second, the calcium:phosphorus ratio in foods), the normal diet contributes little to the vitamin breast milk substitutes (ratio ~2:1) is more appropriate D status of an individual [8], so adequate skin expo- than that in cow’s milk (ratio ~1:1) for optimizing sure to ultraviolet radiation is essential for the preven- intestinal calcium absorption; and third, breast milk usu- tion of rickets in most situations [9] (see Chapter 3). ally contains only small quantities of vitamin D or its Consequently, rickets occurs most frequently in infants metabolites (between 20Ð65 IU/liter) [30,31]. However, before they are able to walk and get outside, in children there is evidence that vitamin D metabolites may cross living in countries at the extremes of latitude, or in into breast milk from the mother in sufficient quantities communities in which social custom prevents adequate to maintain normal serum concentrations of 25OHD sunlight exposure through excessive skin coverage by in the suckling infant if the mother receives vitamin D clothes or through the practice of purdah. supplements in high doses (~2000 IU/day) [15,32]. Vitamin D deficiency rickets is most prevalent in In the breast-fed infant not receiving vitamin D sup- children under two years of age, with a peak incidence plements, the maintenance of an adequate vitamin D between 3 and 18 months [10,11]. The disease is status is dependent mainly on the infant’s exposure to uncommon in infants under three months of age ultraviolet light [33,34]. Specker and coworkers because 25OHD readily crosses the placenta [12,13], [33,34] have shown a marked seasonal variation in thus providing the newborn infant with some protection serum 25OHD concentrations in breast-fed infants, against vitamin D deficiency [14] (see Chapter 51). which is dependent on the time spent outdoors and on Because 25OHD is not the major storage form of the extent of skin exposed to sunlight (see Chapter 51). vitamin D and has a turnover time of three to four They have estimated that an infant in Cincinnati (latitude weeks, serum levels fall rapidly after birth unless 39° 09′ N) needs to be outdoors for either 20 minutes additional sources of vitamin D are obtained by the a week in a diaper only or for two hours a week fully young infant [15]. Neonatal or congenital rickets has clothed but without a hat to maintain normal circulating been described in infants born to mothers who are concentrations of 25OHD [33]. themselves vitamin D deficient [16Ð21], and hypocal- Seasonal variations in serum 25OHD concentrations cemia is a common finding in neonates born to vitamin have also been documented in a number of countries in DÐdeficient mothers. In a number of studies vitamin D older children and adults [35Ð37], and these variations deficiency rickets has been noted to occur more com- appear to correlate with the amount of ultraviolet light monly in boys than girls [22Ð24], however the mecha- reaching the earth [38]. These observations highlight the nism for this remains unclear. It has been suggested importance of the photo-biosynthesis of vitamin D3 in that vitamin D deficiency rickets might be an heredi- the skin to prevent vitamin D deficiency and thus rick- tary disease, which manifests itself only under adverse ets in many populations in the world. In a number of circumstances [23,25]. In a study of infants with rick- countries such as Turkey [39], Saudi Arabia [29], India ets and their parents [25], urinary excretion of α-amino [40], China [21] (including Tibet [41]), Algeria [37], CHAPTER 65 Nutritional Rickets in Children 1067

Iran [10], Kuwait [42], Nigeria [43], Ethiopia [44] and IV. CLINICAL PRESENTATION in others in the tropics and subtropics [45Ð47] rickets remains a problem despite generally good daily hours The majority of clinical signs in children with rick- of sunshine. A number of factors contribute to the per- ets results from the effects of vitamin D deficiency on sistence of the problem in these areas; these include the mineralization process at the growth plate or on overcrowding and poverty, atmospheric pollution [48], calcium homeostasis. Fraser and coworkers [24] have purdah, lack of access to sunlight, a lack of vitamin D described three stages in the progression of vitamin D fortified foods or regular vitamin D supplements, and deficiency. Stage I is characterised by hypocalcemia diets that are low in calcium and high in inhibitors of with clinical signs related to the presence of hypocal- calcium absorption. This subject is dealt with in more cemia, in stage II the clinical features of impaired bone detail in Chapters 47 and 62. mineralization become apparent, and in stage III signs In the U.S., despite the almost complete eradication of both hypocalcemia and severe rickets are present. of vitamin D deficiency among Caucasian children, This division of the progression of vitamin D deficiency several studies have highlighted the resurgence of the rickets is conceptually useful, but there is considerable problem in specific groups [5,27,28,49Ð53], namely clinical overlap between the various stages. vegans and children on macrobiotic diets, children The early clinical manifestations of vitamin D who are breast-fed for prolonged periods, and black deficiency (stage I) are related to hypocalcemia and children [54]. It is suggested that the combination of are more commonly seen in young infants (less than decreased vitamin D3 formation in the dark skin, 6 months of age). They may present with convulsions extensive skin coverage by clothing, low dietary vita- [71,72], apnoeic episodes [73] or tetany with no clini- min D intakes because of the lack of dairy products, cal signs of rickets. Few children present clinically in and the generally low dietary calcium intakes associ- stage I as the majority who later present with rickets ated with vegetarian diets all contribute to an increased pass through this phase without developing symp- risk for vitamin D deficiency in these groups. tomatic hypocalcemia. Pseudotumor cerebri [74] and A similar pattern has also been documented in a cataracts, probably due to hypocalcemia, have been number of European countries [55Ð59], and in reported in a young infant with rickets [75]. It has been Australia and New Zealand [60Ð62]. Perhaps the most suggested that symptomatic hypocalcemia in infants intensively investigated community has been the Asian with vitamin D deficiency might be precipitated by population in Great Britain because of the high preva- an acute illness [76], in which there is a release of lence of vitamin D deficiency and “Asian rickets” in intracellular phosphate [77]. children of all ages [63] and adults [64,65]. The age As the deficiency progresses, the classical features distribution of Asian children with rickets is described of rickets become apparent. Typically, the infant or as being biphasic, with one peak in the classical age young child presents with a delay in motor milestones, group of vitamin D deficiency (9Ð36 months) and the hypotonia, and progressive deformities of the long other related to the pubertal growth spurt [1,66]. Since bones. The deformities are most noticeable at the dis- the initial descriptions of the resurgence of rickets in tal forearm with enlargement of the wrist and bowing the United Kingdom in the early 1960s, numerous of the distal radius and ulna, and in the legs with pro- studies have been undertaken to determine why Asians gressive lateral bowing of the femur and tibia. The site are predisposed to the problem when other immigrants and type of deformity are dependent on the age of the such as West Indians are not. Among the hypotheses child and the weight bearing patterns in the limbs. put forward are simple vitamin D deficiency due to the Thus, in the small infant, deformities of the forearms dark skin and lack of skin surface exposed to sunlight and anterior bowing of the distal tibias are more com- [67,68], low calcium diets associated with vegetarian- mon, while in the toddler who has started to walk an ism [69], and impaired intestinal calcium absorption exaggeration of the normal physiological bowing of associated with high phytate diets [70]. A unifying the legs (genu varum) is characteristic. In the older hypothesis, proposed by Clements [55], suggests that child, valgus deformities of the legs or a windswept in a situation of relative vitamin D insufficiency, the deformity (valgus deformity of one leg and varus defor- low dietary calcium and high phytate content of the mity of the other) may be apparent. The characteristic typical Asian vegetarian diet leads to mild secondary feature in the ribs is enlargement of the costochondral hyperparathyroidism and a resultant increase in the junctions leading to visible beading along the antero- catabolism of vitamin D. The progressive decline in lateral aspects of the chest (the rachitic rosary). In the vitamin D status culminates in the development of infant or young child with severe rickets, the muscular rickets (see Section VIII of this chapter). pull of the diaphragmatic attachments to the lower ribs 1068 JOHN M. PETTIFOR

is often considered to be pathognomonic of rickets, it may be a normal finding in normal young infants [80,81]. Craniosynostosis, involving the coronal or mul- tiple sutures, has been described in approximately 25% of patients, who were followed up after having suffered from vitamin D deficiency rickets [82]. The development of craniosynostosis appears to be related to the degree of severity of the rickets and thus to the severity of the mineralization defect, and inversely to the age of onset of the rickets. A delay in tooth eruption is a feature of rickets in the young child and enamel hypoplasia of teeth may occur if rickets develops prior to the completion of enamel deposition. The latter has been reported in the primary dentition of infants born to mothers who are vitamin D deficient [83], and is seen in the secondary dentition of children who have suffered from rickets during early childhood. Hypotonia, decreased activity, and a protuberant abdomen are characteristic features of advanced vita- min D deficiency rickets in the infant and young child. These signs are probably analogous to the proximal mus- cle weakness described in vitamin D deficient adoles- cents and adults [84]. In this situation, deep tendon reflexes are retained and may be brisk. The pathogenesis of the myopathy is thought to be due primarily to vita- min D deficiency, rather than hypophosphatemia [85] (see Chapters 55 and 102). Dilated cardiomyopathy and cardiac failure [86,87] have also been described in young infants with vitamin D deficiency. The mechanism is thought to be due to FIGURE 1 A young infant with vitamin D deficiency rickets, the effect of hypocalcemia on cardiac muscle function, presenting with respiratory distress. The child shows the character- rather than a direct effect of hypovitaminosis D [88]. istic deformities of the chest associated with severe rickets. The lateral diameter of the chest is reduced and bilateral Harrison’s Infants and young children with rickets are prone to sulci are present. The abdomen has a protuberant appearance. an increased number and severity of infections [39]. Although the increase in respiratory infections may be explained on the thoracic cage abnormalities (soften- ing of the ribs, the enlarged costochondral junctions, and the decreased thoracic movement due to muscle results in the development of Harrison’s sulcus (Fig. 1). weakness), other reasons for the increase in diarrheal The negative intrapleural pressure associated with disease must be sought. The now well-documented role breathing may result in narrowing of the lateral diam- of 1,25(OH)2D in modulating immune function [89,90] eter of the chest (the violin case deformity) with con- may contribute to the observed increase in infections sequent severe respiratory embarrassment. Increased (see Chapter 35). Impaired phagocytosis [91] and neu- sweating has also been described in young infants and trophil motility [92] have been described in children probably relates to the increased work of breathing due with vitamin D deficiency rickets. to the decreased compliance associated with the exces- A possibly associated abnormality is anemia, sively malleable ribs. In premature infants with rickets thrombocytopenia, leucocytosis, myelocytosis, erythro- (which may also be due to dietary phosphorus defi- blastosis, myelofibrosis [93], myeloid metaplasia, and ciency), fractures of the ribs may be the first clinical hepatosplenomegaly (von Jacksch-Luzet syndrome) [94], sign to draw attention to the problem [78]. which has been described in infants with rickets [95,96]. Other skeletal abnormalities include a delay in the clo- Although the exact pathogenetic mechanisms for this sure of the fontanelles, parietal and frontal bossing, and syndrome are unclear, vitamin D deficiency has been the presence of craniotabes [79]. Although craniotabes implicated based on the clinical observation that CHAPTER 65 Nutritional Rickets in Children 1069 vitamin D therapy cures the condition and on experi- be found. As the disease progresses, secondary hyper- mental evidence showing that 1,25(OH)2D has antipro- parathyroidism in response to the hypocalcemia induces liferative activity on myeloid leukaemia cell lines [97]. a partial correction of the low serum calcium concentra- tion, which may return to levels within the normal range, and increases phosphate excretion by the kidney result- V. BIOCHEMICAL ABNORMALITIES ing in hypophosphatemia (stage II) [109]. At this stage, serum alkaline phosphatase concentrations are usually The hallmark of vitamin D deficiency is a low cir- elevated and other renal manifestations of secondary culating level of 25OHD. In children, a normal range hyperparathyroidism, such as increased cyclic AMP of approximately 12 to 50 ng/ml (30Ð125 nmol/liter) excretion, generalized aminoaciduria, impaired acid has been found in the majority of studies [33,98,99] excretion, and decreased urinary calcium excretion, conducted in communities in which vitamin D defi- are found [110]. In stage III of the disease, the radio- ciency rickets is uncommon. However, the normal logical features are more severe, hypocalcemia once range is dependent on the vitamin D and calcium con- again becomes apparent, and alkaline phosphatase tents of the diet and on the ultraviolet light exposure of concentrations rise further [24]. the skin. In a number of studies a marked seasonal The elegant studies conducted by Fraser and cowork- variation in levels has been recorded [34,35,100], ers [24] before the availability of immunoassays for the reflecting in part the seasonal changes in the amount of measurement of serum parathyroid hormone (PTH) ultraviolet light reaching the earth. In countries at high concentrations, suggested that in stage I vitamin D defi- latitude where foods are not vitamin D fortified, serum ciency serum concentrations of PTH are normal as 25OHD concentrations in some “normal” children serum phosphorus values and urinary amino acid excre- may be in the range documented in symptomatic chil- tion are within the normal range. Their patients only had dren with vitamin D deficiency [35,63]. Thus, the radiologic evidence of calvarial demineralization with- development of symptoms depends on the duration out other bone changes of rickets. More recent data sup- and severity of low 25OHD concentrations and on the port this conclusion as normal PTH concentrations have ability of the kidney to achieve adequate 1,25(OH)2D been reported in the early hypocalcemic phase of symp- concentrations in the face of decreased substrate for tomatic vitamin D deficiency [71]. However, Kruse the gastrointestinal tract to maintain calcium absorp- [111] found elevated PTH values and increased urinary tion at a level appropriate to meet the demands of the cyclic AMP excretion in children with stage I rickets. growing child. In children with 25OHD concentrations This discrepancy can possibly be explained by the fact within the normal reference range, there is no correlation that the patients in the latter study might represent a between serum 25OHD and 1,25(OH)2D concentrations. slightly later stage of vitamin D deficiency than those in However, once 25OHD levels fall below ∼12 ng/ml the other studies as the children were selected on the (30 nmol/liter), 1,25(OH)2D concentrations correlate presence of radiologic changes. with those of 25OHD [101,102]. In the majority of Evidence of end-organ resistance to PTH has been studies in which 25OHD values have been measured in found in the young children with both mild and more children with rickets, concentrations have been found severe radiological rickets [111,112]. In the study by to be less than 4Ð5 ng/ml (10Ð12.5 nmol/liter) in most Kruse [111] the children with mild rickets remained patients [63,103,104], although other workers have normophosphatemic and had normal renal handling of found higher values [105Ð107]. phosphate (TmP/GFR) despite elevated PTH concentra- The classical biochemical changes in vitamin DÐ tions and increased urinary cyclic AMP excretion. Similar deficient children who have radiological changes of indirect evidence of PTH resistance (hypocalcemia, nor- rickets are a combination of hypocalcemia, hypophos- mophosphatemia, and a decrease in the phosphate excre- phatemia, and elevated alkaline phosphatase and para- tion index) was noted by Taitz and de Lacy [112] in thyroid hormone concentrations. In the early phase of infants with more severe radiologic rickets. Resistance to vitamin D deficiency before the development of radio- PTH has also been described in hypocalcemic adoles- logical signs (stage I), hypocalcemia may be the only cents with mild rickets [113]. Usually, however, as the biochemical abnormality [24]. Acute illness may precip- severity of the rickets increases (stages II and III), so itate hypocalcemia in the vitamin DÐdepleted infant PTH values rise further and renal hyporesponsiveness is through the sudden increase in serum phosphorus overcome [111]. Thus, hypophosphatemia and a decrease concentrations [77]. The biochemical picture in stage I in TmP/GFR become hallmarks of the disease. rickets may be confused with that of pseudohy- Markers of bone turnover are typically elevated in poparathyroidism [108], as serum hypocalcemia, hyper- nutritional rickets in response to the development of phosphatemia, and normal alkaline phosphatase may secondary hyperparathyroidism. Urinary hydroxyproline 1070 JOHN M. PETTIFOR excretion may be within the normal range in stage I the maintenance of normal calcium homeostasis rickets, but is elevated in patients with radiologic [123,126,127]. Others have suggested that although rickets [111], and an increase in serum concentrations concentrations are within the normal range, they are of bone resorption markers have been reported in chil- inappropriately low for the degree of hyperparathy- dren with untreated rickets [114,115]. Similarly, serum roidism [111,124]. As discussed later, the latter alkaline phosphatase values may be normal in stage I hypothesis is more likely. of vitamin D deficiency, but rise with the degree of A possible pathophysiological progression of vita- severity of the radiologic changes. Bone turnover mark- min D deficiency rickets in children may be described ers (especially those of bone resorption) rise in the first as follows [111,128]. As the child becomes progres- 2Ð3 weeks of treatment, and then fall progressively to sively vitamin DÐdepleted, a stage is reached when the normal values over a period of 4Ð6 weeks [115]. Of all serum 25OHD concentration falls below that required the readily available biochemical tests that might be to maintain a serum 1,25(OH)2D level necessary for deranged in nutritional rickets, alkaline phosphatase normal calcium homeostasis. The resultant hypocal- has been used most frequently as a screening test. cemia (stage I rickets) leads to secondary hyper- However, although alkaline phosphatase is elevated in parathyroidism, which through the stimulation of the vast majority of children with radiological changes, 1α-hydroxlase, increases 1,25(OH)2D production it lacks specificity [45,81,116]. Further, the degree of despite falling 25OHD concentrations. In concert with elevation of serum concentrations does not necessarily PTH, 1,25(OH)2D increases bone resorption and correlate with the radiological severity of the bone dis- intestinal calcium absorption, thus returning serum ease [45]. Whether or not the measurement of bone calcium concentrations towards normal (stage II rick- specific alkaline phosphatase in patients with sus- ets). The presence of hypophosphatemia at this stage is pected rickets will be of greater sensitivity and speci- probably responsible for the mineralization defect and ficity is unclear at present [117]. Recently, in a small the development of radiologic rickets. study of rachitic subjects, it has been suggested that It is during this phase that serum 1,25(OH)2D con- the measurement of deoxypyridinoline in a first morn- centrations may be elevated [111]. A possible explana- ing void urine sample might be a useful indicator of tion for the failure of the elevated 1,25(OH)2D levels to rickets, values being significantly higher in patients reduce the hyperparathyroidism and heal the bone dis- than in age matched controls [118]. ease at this stage is that they are not high enough to Osteocalcin is a noncollagenous bone matrix pro- meet the increased calcium requirements associated tein that binds to hydroxyapatite and is secreted by with the generalized mineralization defect and osteoblasts during mineralization [119]. Serum con- increased bone turnover. Support for this hypothesis centrations are higher in children than adults and peak come from data which show that 1,25(OH)2D concen- during the pubertal growth spurt [120]. In the few chil- trations rise to considerably higher levels (3Ð5 times dren with untreated vitamin D deficiency rickets, in normal) during the healing process even when only whom serum osteocalcin concentrations have been small doses of vitamin D are provided [101,111] and measured, values have been reported to be low [115] or that intestinal calcium absorption may reach ~80% of normal [121], and may rise rapidly on therapy to supra- dietary calcium intake during this phase [101]. normal concentrations [122]. A Nigerian study [114] of As 25OHD concentrations fall further, 1,25(OH)2D 12 rachitic children found slightly elevated serum levels once again fall, despite persistent hyperparathy- osteocalcin concentrations compared to values in age- roidism, because of the lack of substrate. Hypocalcemia matched controls, however it was suggested that the again becomes apparent as intestinal calcium absorp- children might have suffered from dietary calcium tion falls and calcium mobilization from bone deficiency rather than vitamin D deficiency. decreases due to the lack of 1,25(OH)2D, which has a In patients with vitamin D deficiency, serum permissive action on bone resorption by PTH 1,25(OH)2D concentrations have been reported to be low, [129,130]. The combination of both hypocalcemia and normal, or even elevated [101,104,107,111,123,124], hypophosphatemia increases the severity of the bone while 24,25(OH)2D values are low or undetectable disease (stage III). [101,104,107,124,125]. Kruse [111] found that 1,25(OH)2D values were higher in children with stage II rickets than in those with either stage I or stage III VI. RADIOLOGIC CHANGES rickets. The finding of normal or elevated levels of 1,25(OH)2D in vitamin D deficiencyÐrickets has led The typical radiologic changes associated with vitamin some researchers to conclude that other vitamin D D deficiency rickets have been well described and are dis- metabolites, such as 24,25(OH)2D, are necessary for cussed in Chapter 60. Stage I rickets characteristically CHAPTER 65 Nutritional Rickets in Children 1071 shows few radiologic signs, although demineralization of are uncommon. However, loss of the lamina dura the calvarium and loss of definition of the skull sutures round the teeth is frequently seen. have been described [24], but these signs are difficult to Enlargement and splaying of the costochondral quantify. The changes of rickets are best visualized at the junctions on the lateral radiographs of the chest have growth plate of rapidly growing bones. In the upper been used as a sign of rickets; however, in one study, limbs, the distal ulna is the site that may show best the mild changes were found to be unreliable as their pres- early signs of impaired mineralization. In the older child, ence did not correlate with serum 25OHD concentra- the metaphyses around the knees become more useful. tions or with other features of rickets at the distal The early signs of rickets include widening of the epi- radius and ulna [133]. physeal plate and a loss of definition of the provisional Rickets during adolescence may be difficult to zone of calcification at the metaphysis [131]. As the dis- detect using the conventional radiographic sites of the ease progresses, the disorganization of the growth plate wrist and knees as the epiphyseal plates narrow and becomes more apparent with cupping, splaying, spur for- epiphyses fuse. A radiograph of the pelvis may be use- mation, and stippling [77,132] (Fig. 2). The appearance of ful in this situation as the secondary iliac and ischial epiphyses may be delayed or they appear small, ossification centres may be abnormally wide [134]. osteopenic and ill-defined. These centers appear at puberty and normally unite with The shafts of the long bones show features of both the rest of the bone between the 15th and 25th years hyperparathyroidism and osteomalacia. Osteopenia is of age. a characteristic feature which in the so called The sign of early healing of rickets is described as “atrophic” form of the disease may be very severe [45]. broadened bands of increased density replacing the The cortices become thin and may show periosteal new normal sharp metaphyseal lines (Fig. 2). The demarca- bone formation, although this is more frequently seen tion of the broad bands on the diaphyseal side of the during healing. The trabecular pattern is reduced and shaft may be poorly defined [131]. Healing in more appears coarse. Deformities of the shafts of the long severe cases of rickets may first appear as bands of bones are typically present and in severe rickets, mineralization occurring distal to and separated from pathological fractures and Looser’s zones may be the irregular and frayed metaphyses. There is then noted. In vitamin D deficiency rickets, features of gradual filling in of the demineralized area proximal to hyperparathyroidism, such as subperiosteal erosions, the initial band of mineralization with remodelling and

FIGURE 2 The radiographic features of vitamin D deficiency rickets at the wrist. Left panel: untreated vitamin D deficiency showing underdevelopment of the epiphyses, widening of the epiphyseal plates, splaying and irregularity of the metaphyses and loss of the provisional zones of calcification. The shafts show coarsening of the trabecular pattern and loss of the normal cor- tical definition. Middle panel: Response after three months of vitamin D therapy. The metaphyses show clear signs of healing with dense bands of calcification at the distal ends of the metaphyses, narrowing of the epiphyseal plates, and more clearly defined epiphyses. The trabecular pattern still appears coarse but shows improvement. Right panel: Six months after starting vitamin D therapy. The radiographic changes of rickets have disappeared. The epiphyses, epiphyseal plates, metaphyses, and trabecular structure are normal. (Reproduced with permission [132].) 1072 JOHN M. PETTIFOR the development of a normal trabecular pattern. Periosteal dose [137]. These authors suggest that a dose of new bone formation may be seen which gradually 150,000 IU is equally effective as the larger dose in the becomes incorporated into the cortices of the long bones. management of the disease without running the risk of hypercalcemia. VII. TREATMENT AND PREVENTION Besides ensuring an adequate vitamin D intake, the calcium content of the diet should be optimized (between A. Treatment 600 and 1000 mg/day) during the initial stages of management. This is particularly true for children who Vitamin D deficiency rickets can be effectively are on vegetarian or low calcium containing diets [138] treated by the oral administration of small doses of and for those who are severely hypocalcemic. In symp- either vitamin D2 or D3, provided there is no evidence tomatic patients, a single dose of calcium gluconate of gastrointestinal malabsorption. Stanbury et al. [101] (1Ð2 ml/kg of a 10% solution) may be given slowly intra- showed that an oral vitamin D dose of between 200 to venously and the diet supplemented with 10% calcium 450 IU/day produced a rise in serum 1,25(OH)2D con- gluconate (5 ml/kg/day in divided doses). centrations to normal values within 1Ð3 days. The latter climbed to reach a peak some five times the normal mean after one to three weeks, despite serum 25OHD values B. Prevention remaining less than 10 ng/ml (25 nmol/l). Spontaneous improvement in the biochemical features of rickets has As discussed in section III of this chapter, vitamin D been reported to occur in children with biochemical deficiency rickets remains a problem in a number of at- abnormalities during the summer months, associated risk groups despite readily available methods of pre- with a rise in serum 25OHD values due presumably to venting the disease. A number of studies in several increased ultraviolet light exposure [103]. countries have been conducted prospectively in breast- More generally, however, doses of vitamin D between fed infants to assess vitamin D status. Several have 5,000 and 15,000 IU/day for three to four weeks are shown a fall in serum 25OHD concentrations in those used in the management of rickets. Normalization of infants who were not vitamin DÐsupplemented, to lev- serum calcium and phosphorus concentrations occur els in the vitamin DÐdeficient range [15,139,140], within 1 and 3 weeks [111], although serum alkaline although this is not a universal finding [141,142]. phosphatase concentrations and urinary hydroxypro- Further, a number of studies have highlighted the line excretion remain elevated for several months. high prevalence of vitamin D deficiency in mothers Despite the return to normal of serum PTH, calcium, during pregnancy and lactation, which exacerbates and phosphorus values within three weeks, serum the severity and onset of vitamin D deficiency in their 1,25(OH)2D concentrations may remain elevated for offspring [14,65,143,144]. up to 10 weeks [107,111]. Serum 24,24(OH)2D values, Preventive strategies should be directed not only at which are often undetectable in the untreated patient, breast-fed infants but also at pregnant and breast-feeding rise with the progressive increase in serum 25OHD women [145]. Both North America [146] and the concentrations during treatment [107]. Lower doses of United Kingdom [147] recommend dietary intakes of vitamin D (1000Ð2000 IU/day) do produce healing but vitamin D of between 200 and 400 IU/day for pregnant the response is less rapid. and lactating women to ensure adequate circulating In Central Europe, a single dose of 600,000 IU vita- 25OHD levels. Although at normal circulating mater- min D (either orally or intramuscularly) has been found nal 25OHD concentrations, the vitamin D content of to be effective, resulting in a rapid improvement in bio- breast milk is limited (see Section III), there is evi- chemical abnormalities within a few days and radiologic dence that maternal supplementation with vitamin D evidence of healing within two weeks [77,135]. A sus- at 2000 IU/day may increase breast-milk vitamin D tained drop in serum alkaline phosphatase is seen within concentrations sufficiently to maintain the infant’s 6 to 12 weeks [135]. Single dose therapy has an advan- 25OHD within the normal range. tage over smaller daily doses as it avoids the problem The North American and United Kingdom groups of compliance, which was thought to be responsible for recommend dietary intakes of between 200 and 350 IU the lack of response in 40% of children with vitamin DÐ vitamin D for the breast-fed infant [146,147]. The deficiency rickets in a study conducted in Kuwait [136]. American Academy of Pediatrics in its latest recom- A recent article has raised concern about the use of mendations suggest that infants less than 6 months of 600,000 IU vitamin D in the treatment of rickets as age should be kept out of direct sunlight, that children’s hypercalcemia was reported in a small number of activities should minimize sunlight exposure, and that infants a month after having received the vitamin D sunscreens should be used because of the indirect CHAPTER 65 Nutritional Rickets in Children 1073 evidence that early exposure to sunlight might deter- 50% of the infants who had received 15 mg at birth, mine the risk of skin cancer in later life [148]. These still had elevated 25OHD concentrations, while in the recommendations make it imperative that if the above 5 mg group none had elevated levels. In the group guidelines are followed, supplemental vitamin D receiving 2.5 mg every three months, serum 25OHD (200Ð400 IU/day) should be provided to all breast-fed values were in the normal range on each occasion prior and weaned infants ingesting less than 500 ml of infant to receiving the next dose. Although hypercalcemia milk formula/day [148Ð151]. In a prospective study was not detected in any of the infants, serum calcium conducted in China on infants from birth to 6 months concentrations were higher in the 15 mg group two of age, it was concluded that supplemental vitamin D weeks after receiving the dose than in the other two at a dose of 400 IU/day produced more normal circu- groups. The authors concluded that intermittent doses lating 25OHD concentrations than did either 100 or of 15 mg vitamin D during the first year of life are 200 IU/day [152], however, in a small number of excessive, and that 5 mg every six months or even better infants even 400 IU/day did not maintain 25OHD lev- 2.5 mg every three months are more suitable for the els above 11 ng/ml (27.5 nmol/liter). Nevertheless, no prevention of vitamin D deficiency in at-risk infants. radiologic evidence of rickets was found in any of the Vitamin D supplementation should be considered for infants in the three groups at six months of age. The all breast-fed infants living in temperate climates until use of 400 IU vitamin D daily to prevent vitamin D they are ambulatory and are able to play outside [154]. deficiency in at-risk infants is supported by a study Even in countries closer to the equator, where sunlight from Turkey, in which it was found that no rickets exposure should not be a problem, social customs may occurred in infants receiving the supplement compared place the mother and infant at risk from vitamin D to a prevalence of 3.8% in those that did not [39]. deficiency. In such situations (e.g., the Middle East and in Infants fed milk formulas or cow’s milk fortified Muslim communities in North Africa) vitamin D supple- with vitamin D do not require vitamin D supplements, mentation may also be necessary to reduce the high as their intake of milk generally provides sufficient prevalence of vitamin D deficiency [144,155]. vitamin D to prevent deficiency [141]. In a number of countries, vitamin D deficiency is As discussed earlier, high single dose therapy not just a disease of breast-fed infants and their moth- (stosstherapie) has been used with success in the treat- ers. Rickets has been described in adolescents of ment of vitamin D deficiency rickets in a number of Indian and Pakistani descent in the United Kingdom countries. A similar dose has also been used on a regu- and in the Middle East [156,157], while hypovita- lar intermittent basis of every 3 to 5 months for the first minosis D has been reported in adolescents in a num- 18 months of life as a means of prevention of vitamin ber of European countries [158Ð161], India [162,163] D deficiency. Little data is available on the efficacy of and China [164]. Furthermore there is an increasing such prophylaxis. However, in a study to assess the awareness of the high prevalence of what is considered effect of these high doses of vitamin D (600,000 IU) on to be vitamin D insufficiency in many elderly subjects in calcium and vitamin D metabolism in infants [7], it Europe and North America [165]. With the widespread was found that serum 25OHD concentrations reached nature of vitamin D deficiency in many countries, vita- very high levels two weeks after each administration, min D supplementation is unlikely to be an effective but that these had returned to normal prior to the next means of combating the disease on a community basis, dose. 1,25(OH)2D generally remained within the nor- so food fortification should be considered as a possible mal range, but 34% of infants were hypercalcemic at solution. some stage during the study. These results led the Although the untargeted fortification of foods other authors to conclude that the dosage regimen as used than milk and infant milk formulas has been used in during the study was excessive and unsafe [7]. the past as a means of addressing the high prevalence Following these results, a study to assess the of vitamin D deficiency in countries where the risk of efficacy of a single dose of vitamin D (600,000 IU or vitamin D deficiency is high, the problems experi- 15 mg) at 15 days of life, compared to 200,000 IU enced in the United Kingdom after World War II have (5 mg) at birth or 100,000 IU (2.5 mg) at birth and led to it falling into disfavor (see Section II of this three monthly for nine months was undertaken [153]. chapter). More recently, the use of targeted food fortifi- Two weeks after the initial administration, 28 of 30 cation has been studied in the Asian community in Great infants in the 15 mg group had serum 25OHD concen- Britain as a means of reducing the high prevalence of trations above the upper limit of normal (mean ± SD vitamin D deficiency in both adults and children in that for the group; 307 ± 160 nmol/liter) compared to 58% community [166]. In a small pilot study, it was found that (150 ± 55 nmol/liter) in the 5 mg group and 23% (92 ± the fortification of chapatti flour at a level of 6000 IU/kg 42 nmol/liter) in the 2.5 mg group. At 6 months of age, produced a sustained and significant rise in serum 1074 JOHN M. PETTIFOR

25OHD concentrations to values within the normal cereals (maize [corn], cassava, yam, rice, and plantain) range over a six-month period comparable to that [42,179Ð181]. In the South African children, dietary achieved by a weekly dose of 3000 IU vitamin D. Over calcium intakes have been estimated to be between 90 the six-month period, serum calcium and phosphorus and 300 mg/day in those children suffering from rick- values rose, and the number of subjects with biochem- ets compared to between 200 and 500 mg/day in age ical abnormalities suggestive of rickets fell. The matched controls [179], while in the Nigerian children, authors conclude that fortification of chapatti flour is a both patients and controls had similar but very low cheap and effective method of preventing vitamin D calcium intakes (200 mg/day) [182]. deficiency in the Asian community in Britain, and has In South Africa, the children typically come from the advantage over daily or intermittent vitamin D sup- rural areas and present with signs and symptoms of plementation as the compliance utilizing the latter rickets between the ages of 4 and 15 years [181], while form of prevention is often poor. Nevertheless, food in Nigeria they present younger, between 1 and 9 years fortification remains an emotional public issue. In the of age [42]. In the South African series, half the chil- U.S. not only have there been isolated reports of vita- dren presented with knock-knees, while the others min D toxicity related to inadequate monitoring of the presented with either bow-legs or wind-swept defor- fortification process, but underfortification is also a mities (Fig. 3). Bow-legs were more common in the problem. Holick [9] reports that in his study fewer than Nigerian children, probably reflecting their earlier age 30% of milk samples from all sections of the U.S. and of presentation [182]. Unlike vitamin D deficiency, British Columbia contained the specified amount, and symptoms of muscle weakness are characteristically that 14% to 21% of skim milk samples contained no absent in older children with dietary calcium deficiency. detectable vitamin D. Radiologically, the features are typical of cal- ciopenic rickets with osteopenia and features of hyper- parathyroidism being frequent findings (Fig. 4). The VIII. DIETARY CALCIUM DEFICIENCY severity of the metaphyseal changes is variable. Older children (teenagers) may have no radiologic changes Conventional wisdom has been that nutritional rick- of rickets, despite features of osteomalacia on the iliac ets is primarily due to vitamin D deficiency, although crest bone biopsy [183]. Younger children may show dietary calcium intake modulates the severity and evidence of only minor degrees of impaired endochon- rapidity of onset of the disease [167,168]. However, dral calcification, while in others the metaphyseal over the last three decades evidence has been accumu- changes may be quite marked. In a Nigerian study, the lating that implicates low dietary calcium intakes as a degree of severity of radiologic rickets was correlated cause of rickets in the face of serum 25OHD concen- with serum alkaline phosphatase values [184]. trations within the normal reference range. The subject The biochemical features are similar to those of is also discussed in Chapter 64. other causes of calciopenic rickets. Hypocalcemia, low Isolated case reports of rickets developing in infants urinary calcium excretion, and elevated serum PTH and toddlers, who were placed on very low calcium and alkaline phosphatase concentrations are character- diets, have been published [169Ð171]. Their clinical and istic, while serum phosphorus values are variable and biochemical presentations were very similar to those of often within the reference range for age [42,180,181]. infants with vitamin D deficiency, however in three of Serum 25OHD values are normal (mean 16.4 ng/ml the five infants, serum 25OHD and 1,25(OH)2D values and 14.4 ng/ml in the South African and Nigerian chil- were reported to be greater than 9 ng/ml (22.5 nmol/liter) dren respectively) and 1,25(OH)2D concentrations are and 118 pg/ml (295 pmol/liter), respectively. In none elevated [175,176,185]. In a Nigerian study [42] and in of the five infants was a therapeutic trial of calcium the South African children [121], serum osteocalcin supplementation of the diet alone tried; however, the levels are similar to those of nonrachitic controls in the clinical and biochemical presentation suggested to the majority of patients, although another report from authors that dietary calcium deficiency was the primary Nigeria found slightly higher levels in rachitic patients factor responsible for the development of rickets. than controls [114]. The finding of normophosphatemia More convincing evidence of dietary calcium defi- and normal renal handling of phosphorus (TmP/GFR) ciency as a cause for rickets in children comes from suggests that a peripheral resistance to PTH might be studies in South Africa [172,173], Nigeria [174Ð177] prevalent in this form of rickets. and India [162], and possibly Bangladesh [178], where Iliac crest bone biopsies reveal evidence of osteo- the staple diets of children are characteristically low in malacia and hyperparathyroidism in those children calcium because of the lack of readily available dairy who have radiologic features of rickets [173], while in products and the low/extra calcium content of the the teenagers without radiologic changes but lower CHAPTER 65 Nutritional Rickets in Children 1075

FIGURE 4 The radiographic features of dietary calcium defi- ciency rickets in the lower limbs of a child. The long bones are osteopenic with deformities characteristic of long-standing rickets. The metaphyses show evidence of impaired mineralization and growth arrest lines. FIGURE 3 The clinical presentation of children with dietary cal- cium deficiency. The deformities are typically more severe in the legs with a predominance of knock-knees or windswept deformi- ties. Upper limb deformities are usually mild if present at all. (Reproduced with permission [181]) More recently, a study using a calcium supplement of only 350 mg/day, reported complete healing within six months [177]. In a randomised controlled trial, cal- limb deformities, the histologic picture varies from cium supplements alone or calcium and vitamin D that of decreased bone volume, through features of together were equally effective in healing the bone dis- hyperparathyroidism, to frank osteomalacia and hyper- ease and were significantly better than vitamin D ther- parathyroidism [183]. apy alone [175]. In the majority of the South African In both the Nigerian and South African studies, clin- children, orthopedic corrective surgery has been nec- ical, biochemical, and radiologic healing has been essary to correct the deformities of the legs once bio- achieved through increasing the calcium intake of the chemical and radiologic healing has occurred. This has children to between 800 and 1500 mg/day without the not been the pattern in the younger Nigerian children administration of vitamin D supplements [42,172]. with rickets, who have shown remarkable remodelling 1076 JOHN M. PETTIFOR and straightening of deformities without orthopedic deficiency rickets in dogs. More recently, studies in surgical intervention [177]. baboons have confirmed these findings [170]. The data available from epidemiologic studies con- The resurgence of rickets and osteomalacia in the ducted in a rural area in South Africa in which a number Asian community in Great Britain has provided the of the affected children live, suggest that asymptomatic impetus for detailed studies into the pathogenesis of dietary calcium deficiency is prevalent in school- vitamin D deficiency, and bone disease in that com- children living in the area. Some 13% of children munity. Although vitamin D deficiency as assessed by between the ages of 7 and 12 years were hypocalcemic, circulating 25OHD concentrations, is the hallmark of 41.5% had elevated alkaline phosphatase concentra- the disease in Asians [63,191,192], the mechanisms for tions, and 76% had low urinary calcium excretion the low vitamin D status and the high prevalence of [186]. It is unclear whether these children have long rickets were unclear. It is apparent that the majority of term sequelae as a result of the poor calcium intakes. Asians in Britain do not spend less time outdoors than However, studies do indicate that asymptomatic chil- their Caucasian counterparts [193]. Further, although dren with biochemical abnormalities living in the rural they have darker skins than Caucasians, which might community have lower appendicular bone mass than reduce the amount of vitamin D formed in response to those with normal biochemistries [179] and that chil- sunlight exposure, West Indians living in Britain have dren in the community as a whole have lower appen- even darker skins, yet very few cases of rickets have dicular bone mass than their urban peers [187]. been described in this ethnic group [55]. Within the Although dietary calcium intakes in children with Asian community, studies have highlighted the find- biochemical changes suggestive of dietary calcium ings that risk factors for the disease include: living at deficiency are very low, it is unclear what role the high high latitude, Hindu religion, immigration from East phytate or oxalate contents of the diet play in aggravat- Africa, vegetarianism, high fiber diets, and the consump- ing the symptoms. Nevertheless, biochemical improve- tion of chapatti [64,69,70]. The association with vegetar- ment can be achieved by supplementing the children ianism, high fiber diets and cereals of high extraction with 500 mg calcium daily [188]. The finding of suggests that dietary factors play a role. Support for this similarly low dietary calcium intakes in patients with comes from two studies that have documented healing of rickets and age matched controls in Nigeria is intrigu- rickets on removing chapattis from the diet [194,195], ing [182], as it suggests that other factors besides low although this is not a universal finding [68]. dietary calcium intakes might influence the develop- Over the past fifteen years, research has shown that ment of rickets in affected children. Such factors might both high fiber diets and intestinal malabsorption include differing amounts of inhibitors of calcium reduce the serum half-life of 25OHD by approximately absorption in the diet, differing growth rates and there- one-third [196,197]. Further, experiments in rats have fore calcium requirements in the children, or genetic dif- demonstrated that an elevation in serum 1,25(OH)2D ferences that make the rachitic children less able to adapt concentrations, either by exogenous administration or to low dietary calcium intakes than control subjects. A endogenously through a low calcium diet, increases number of these factors are currently under investigation. the metabolic clearance rate of 25OHD without alter- A small study has found that there are significant differ- ing its rate of production [198Ð200]. The fall in serum ences in the frequency of vitamin D polymorphisms 25OHD levels could be accounted for by an increase in between affected and control children, but the signifi- polar metabolites appearing in the feces. Similar find- cance of these findings is unclear at present [189]. ings have been reported from studies in man [201,202]. Conversely, increasing the calcium content of the diet has been shown to increase serum 25OHD and decrease IX. THE PATHOGENETIC SPECTRUM serum 1,25(OH)2D concentrations [203]. These studies OF NUTRITIONAL RICKETS convincingly show that dietary calcium and phytate content influence the catabolism of 25OHD through Nutritional rickets has been viewed for some time as altering serum 1,25(OH)2D concentrations. being due to an inadequate supply of vitamin D In the light of the above studies, Clements [55] has through either an inadequate dietary intake or insuffi- proposed that the low dietary calcium and high phytate cient skin exposure to ultraviolet radiation, or more diet of the Asian population in Britain increases vitamin recently due to low dietary calcium intake in the face D catabolism and vitamin D requirements. In the face of of a normal vitamin D status. However, these patho- a marginal vitamin D status due to living at high latitude genetic concepts are too simplistic. Early studies by and the low dietary vitamin D content of the diet, the Mellanby [190] had shown the effect of cereals in increased catabolism is sufficient to precipitate vitamin exacerbating the clinical development of vitamin D D deficiency and clinical rickets and osteomalacia. CHAPTER 65 Nutritional Rickets in Children 1077

Thus, nutritional rickets has a spectrum of patho- dietary calcium intakes were responsible for rickets genetic mechanisms ranging from pure vitamin D defi- in young children while vitamin D deficiency played ciency associated with adequate calcium intakes, as a major role in adolescents [162]. might occur in the breast-fed infant, at one end of the spectrum, to pure dietary calcium deficiency with an adequate vitamin D status, as documented in Nigerian X. CONCLUSIONS and South African rural children, at the other end of the spectrum [204]. In between these two extremes lies the Despite readily accessible and effective means to situation exemplified by the Asian community in eradicate rickets globally, the disease remains a major Britain, where both poor calcium intakes or absorption public health problem in many countries, not only in and marginal vitamin D status combine to lead to frank temperate regions of the world but also in tropical and vitamin D deficiency and rickets (Fig. 5). It is likely subtropical countries. In many developed countries, the that the high prevalence of rickets in vegetarian or promotion of exclusive breast-feeding during the first immigrant children reported from the U.S. [27,28], six months of life and the concerns about the long-term Norway [57], Holland [58,59], and a number of tropi- effect of sunlight exposure during this period have exac- cal and subtropical countries [45] might be due to a erbated the risks of vitamin D deficiency in the young mechanism similar to that in the Asian community, infant. In some subtropical countries, social customs while osteomalacia in Bedouin adults in the Middle play an important role in preventing adequate vitamin D East reflects mainly dietary calcium deficiency [205]. status not only in the young infant but also in the preg- A number of recent studies have highlighted the nant and lactating mother. In a number of developing complex interaction between vitamin D and calcium countries, low dietary calcium intakes appear to play a intakes in the pathogenesis of nutritional rickets in major role in the pathogenesis of rickets in older chil- children. A review of 43 patients diagnosed as having dren. Recent studies have helped to provide an all nutritional rickets in New Haven, Connecticut, found embracing concept of the interaction of vitamin D and low 25OHD levels in only 22%, and the majority of calcium intakes in the pathogenesis of rickets. infants had been weaned onto diets with minimal dairy There remains a need for international agencies to content [51]. The authors concluded that low dietary place the eradication of vitamin D deficiency among calcium intakes probably played a major role in the young children in many parts of the world as a priority. pathogenesis of the disease. Similar findings are Nutritional rickets not only leads to an increase in infant reported from India, where it is suggested that low mortality but also has serious long-term health sequelae.

Lack of UV Light Inadequate dietary vitamin D

Low 25OHD 25OHD catabolism

Low 1,25(OH)2D 1,25(OH)2D

Impaired calcium absorption High dietary phytate/ Dietary calcium low dietary calcium deficiency Indequate calcium absorption for requirement of growing child

Serum ionized calcium PTH

Serum phosphate

Impaired mineralization

RICKETS

FIGURE 5 The spectrum of nutritional rickets. At either ends of the pathogenetic spec- trum are vitamin D deficiency and dietary calcium deficiency. In between lie combinations in varying degrees of relative vitamin D insufficiency and decreased dietary calcium content or bioavailability. 1078 JOHN M. PETTIFOR

24. Fraser D, Kooh SW, Scriver CR 1967 Hyperparathyroidism References as the cause of hyperaminoaciduria and phosphaturia in human vitamin D deficiency. Pediat Res 1:425Ð435. 1. Arneil GC 1975 Nutritional rickets in children in Glasgow. 25. Doxiadis S, Angelis C, Karatzas P, Vrettos C, Lapatsanis P Proc Nutr Soc 34:101Ð109. 1976 Genetic aspects of nutritional rickets. Arch Dis Child 2. Chick DH 1976 Study of rickets in Vienna 1919Ð1922. Med 51:83Ð90. Hist 20:41Ð51. 26. Dancaster CP, Jackson WPU 1961 Studies in rickets in the 3. Rafter GW 1987 Elmer McCollum and the disappearance of Cape Peninsula II. Aetiology. S Afr Med J 35:890Ð894. rickets. Perspect Biol Med 30:527Ð534. 27. Edidin DV, Levitsky LL, Schey W, Dumbuvic N, Campos A 4. Stephen JML 1975 Epidemiological and dietary aspects of 1980 Resurgence of nutritional rickets associated with breast- rickets and osteomalacia. Proc Nutr Soc 34:131Ð138. feeding and special dietary practices. Pediatrics 65:232Ð235. 5. Rudolf M, Arulanantham K, Greenstein RM 1980 28. Bachrach S, Fisher J, Parks JS 1979 An outbreak of vitamin D Unsuspected nutritional rickets. Pediatrics 66:72Ð76. deficiency rickets in a susceptible population. Pediatrics 64: 6. Jacobus CH, Holick MF, Shao Q, Chen TC, Holm IA, 871Ð877. Kolodny JM, Fuleihan GE-H, Seeley EW 1992 29. Elidrissy ATH, Sedrani SH, Lawson DEM 1984 Vitamin D Hypervitaminosis D associated with drinking milk. N Engl J deficiency in mothers of rachitic infants. Calcif Tissue Int Med 326:1173Ð1177. 36:266Ð268. 7. Markestad T, Hesse V, Siebenhuner M, Jahreis G, Aksnes L, 30. Hollis BW, Roos BA, Draper HH, Lambert PW 1981 Vitamin D Plenert W, Aarskog D 1987 Intermittent high-dose vitamin D and its metabolites in human and bovine milk. J Nutr 111: prophylaxis during infancy: effect on vitamin D metabolites, 1240Ð1248. calcium, and phosphorus. Am J Clin Nutr 46:652Ð658. 31. Specker BL, Tsang RC, Hollis BW 1985 Effect of race and 8. Portale AA, Halloran BP, Harris ST, Bikle DD, Morris RC diet on human-milk vitamin D and 25-hydroxyvitiamin D. 1992 Metabolic acidosis reverses the increase in serum 1,25- Am J Dis Child 139:1134Ð1137. (OH)2D in phosphorus-restricted normal men. Am J Physiol 32. Ala-Houhala M, Koskinen T, Terho A, Kiovula T, Visakorpi J 263:E1164ÐE1170. 1986 Maternal compared with infant vitamin D supplemen- 9. Holick MF 1995 Environmental factors that influence tation. Arch Dis Child 61:1159Ð1163. the cutaneous production of vitamin D. Am J Clin Nutr 61: 33. Specker BL, Valanis B, Hertzberg V, Edwards N, Tsang RC 638SÐ645S. 1985 Sunshine exposure and serum 25-hydroxyvitamin D 10. Salimpour R 1975 Rickets in Tehran. Arch Dis Child 50: concentrations in exclusively breast-fed infants. J Pediatr 63Ð66. 107:372Ð376. 11. Al Hag AI, Karrar ZA 1995 Nutritional vitamin D defi- 34. Specker BL, Tsang RC 1987 Cyclical serum 25-hydroxy- ciency rickets in Sudanese children. Ann Trop Paediatr 15: vitamin D concentrations paralleling sunshine exposure in 69Ð76. exclusively breast-fed infants. J Pediatr 110:744Ð747. 12. Hillman LS, Haddad JG 1974 Human perinatal vitamin D 35. Olivieri MB, Ladizesky M, Mautalen CA, Alonso A, metabolism 1:25-hydroxyvitamin D in maternal and cord Martinez L 1993 Seasonal variations of 25 hydroxyvitamin D blood. J Pediatr 84:742Ð749. and parathyroid hormone in Ushuaia (Argentina), the south- 13. Gertner JM, Glassman MS, Coutsan DR, Goodman DBP ernmost city of the world. Bone Miner 20:99Ð108. 1980 Fetomaternal vitamin D relationships at term. J Pediatr 36. Harris SS, Dawson-Hughes B 1998 Seasonal changes in 97:637Ð640. plasma 25-hydroxyvitamin D concentrations of young 14. Andiran N, Yordam N, Ozon A 2002 Risk factors for vitamin American black and white women. Am J Clin Nutr 67: D deficiency in breast-fed newborns and their mothers. 1232Ð1236. Nutrition 18:47Ð50. 37. Garabedian M, Ben-Mekhbi H 1991 Is vitamin DÐdeficiency 15. Rothberg AD, Pettifor JM, Cohen DF, Sonnendecker EWW, rickets a public health problem in France and Algeria? Ross FP 1982 Maternal-infant vitamin D relationships during In: Glorieux FH (ed) Rickets. Nestec, Vevey; Raven Press, breast-feeding. J Pediatr 101:500Ð503. New York, pp. 215Ð221. 16. Maxwell JP 1934 Further studies in adult rickets (osteomala- 38. Ladizesky M, Lu Z, Oliveri B, Roman NS, Diaz S, Holick cia) and fetal rickets. Proc Roy Soc Med 28:265Ð300. MF, Mautalen C 1995 Solar ultraviolet B radiation and pho- 17. Anatoliotaki M, Tsilimigaki A, Tsekoura T, Schinaki A, toproduction of vitamin D3 in central and southern areas of Stefanaki S, Nikolaidou P 2003 Congenital rickets due to Argentina. J Bone Miner Res 10:545Ð549. maternal vitamin D deficiency in a sunny island of Greece. 39. Beser E, Cakmakci T 1994 Factors affecting the morbidity of Acta Paediatr 92:389Ð391. vitamin D deficiency rickets and primary protection. East Afr 18. Mohapatra A, Sankaranarayanan K, Kadam SS, Binoy S, Med J 71:358Ð362. Kanbur WA, Mondkar JA 2003 Congenital rickets. J Trop 40. Ghai OP, Koul PB 1991 Rickets in India. In: Glorieux FH Pediatr 49:126Ð127. (ed) Rickets. Nestec, Vevey: Raven Press, New York, 19. Maiyegun SO, Malek AH, Devarajan LV, Dahniya MH 2002 pp. 247Ð252. Severe congenital rickets secondary to maternal hypovita- 41. Harris NS, Crawford PB, Yangzom Y, Pinzo L, Gyaltsen P, minosis D: a case report. Ann Trop Paediatr 22:191Ð195. Hudes M 2001 Nutritional and health status of Tibetan chil- 20. Moncrieff M, Fadahunsi TO 1974 Congenital rickets due to dren living at high altitudes. N Engl J Med 344:341Ð347. maternal vitamin D deficiency. Arch Dis Child 49:810Ð811. 42. Okonofua F, Gill DS, Alabi ZO, Thomas M, Bell JL, 21. Zhou H 1991 Rickets in China. In: Glorieux FH (ed), Dandona P 1991 Rickets in Nigerian children: a consequence Rickets. Nestec, Vevey: Raven Press, New York, pp. 253Ð261. of calcium malnutrition. Metabolism 40:209Ð213. 22. Chen Y 1994 Prematurity as a predictor of rickets in 43. Laditan AAO, Adeniyi A 1975 Rickets in Nigerian children: Shanghai infants. Public Health 108:333Ð339. Response to vitamin D. J Trop Med Hyg 78:206Ð209. 23. Lapatsanis P, Deliyanni V, Doxiadis S 1968 Vitamin D defi- 44. Lulseged S, Fitwi G 1999 Vitamin D deficiency rickets: ciency rickets in Greece. J Pediatr 73:195Ð202. socio-demographic and clinical risk factors in children seen CHAPTER 65 Nutritional Rickets in Children 1079

at a referral hospital in Addis Ababa. East Afr Med J 66. Moncrieff MW, Lunt HRW, Arthur LJH 1973 Nutritional 76:457Ð461. rickets at puberty. Arch Dis Child 48:221Ð224. 45. Bhattacharyya AK 1992 Nutritional rickets in the tropics. 67. Hodgkin P, Kay GH, Hine PM, Lumb GA, Stanbury SW In: Simopoulos AP (ed) Nutritional triggers for health and in 1973 Vitamin-D deficiency in Asians at home and in Britain. disease. Karger, Basel, pp. 140Ð197. Lancet ii:167Ð172. 46. Karrar ZA 1998 Vitamin D deficiency rickets in developing 68. Dent CE, Round JM, Rowe DJF, Stamp TCB 1973 Effect of countries. Ann Trop Paediatr 18(Suppl):S89ÐS92. chapattis and ultraviolet irradiation on nutritional rickets in 47. Bereket A 2003 Rickets in developing countries. Endocr Dev an Indian immigrant. Lancet i:1282Ð1284. 6:220Ð232. 69. Henderson JB, Dunnigan MG, McIntosh WB, Abdul-Motaal A, 48. Agarwal KS, Mughal MZ, Upadhyay P, Berry JL, Mawer EB, Hole D 1990 Asian osteomalacia is determined by dietary Puliyel JM 2002 The impact of atmospheric pollution on factors when exposure to ultraviolet radiation is restricted: vitamin D status of infants and toddlers in Delhi, India. Arch a risk factor model. Quart J Med 76:923Ð933. Dis Child 87:111Ð113. 70. Henderson JB, Dunnigan MG, McIntosh WB, Abdul-Motaal 49. Finberg L 1979 Human choice, vegetable deficiencies, and AA, Gettinby G, Glekin BM 1987 The importance of limited vegetarian rickets. Am J Dis Child 133:129. exposure to ultraviolet radiation and dietary factors in the 50. Chesney RW 2002 Rickets: the third wave. Clin Pediatr aetiology of Asian rickets: a risk-factor model. Quart J Med (Phila) 41:137Ð139. 63:413Ð425. 51. DeLucia MC, Mitnick ME, Carpenter TO 2003 Nutritional 71. Bonnici F 1978 Functional hypoparathyroidism in infantile rickets with normal circulating 25-hydroxyvitamin D: a call hypocalcaemic stage I vitamin D deficiency rickets. S Afr for reexamining the role of dietary calcium intake in North Med J 54:611Ð612. American infants. J Clin Endocrinol Metab 88:3539Ð3545. 72. Johnson GH, Willis F 2003 Seizures as the present- 52. McCaffree J 2001 Rickets on the rise. J Am Diet Assoc ing feature of rickets in an infant. Med J Aust 178: 101:16Ð17. 467Ð468. 53. Dwyer JT, Dietz WH, Hass G, Suskind R 1979 Risk of nutri- 73. Buchanan N, Pettifor JM, Cane RD, Bill PLA 1978 Infantile tional rickets among vegetarian children. Am J Dis Child apnoea due to profound hypocalcaemia associated with 133:134Ð140. vitamin D deficiency. S Afr Med J 53:766Ð767. 54. Kreiter SR, Schwartz RP, Kirkman HN Jr, Charlton PA, 74. Salaria M, Poddar B, Parmar V 2001 Rickets presenting Calikoglu AS, Davenport ML 2000 Nutritional rickets as pseudotumour cerebri and seizures. Indian J Pediatr in African American breast-fed infants. J Pediatr 137: 68:181. 153Ð157. 75. Hochman HI, Mejlszenkier JD 1977 Cataracts and pseudo- 55. Clements MR 1989 The problem of rickets in UK Asians. tumor cerebri in an infant with vitamin DÐdeficiency rickets. J Hum Nutr Diet 2:105Ð116. J Pediatr 90:252Ð254. 56. Pedersen P, Michaelsen KF, Molgaard C 2003 Children with 76. Park EA 1954 The influence of severe illness on rickets. nutritional rickets referred to hospitals in Copenhagen during Arch Dis Child 29:369Ð380. a 10-year period. Acta Paediatr 92:87Ð90. 77. Harrison HE, Harrison HC 1979 Rickets and osteomalacia. 57. Hellebostad M, Markestad T, Halvorsen KS 1985 Vitamin D In: Disorders of calcium and phosphate metabolism in deficiency rickets and vitamin B12 deficiency in vegetarian childhood and adolescence. W.B. Saunders, Philadelphia, children. Acta Paediatr Scand 74:191Ð195. pp. 141Ð256. 58. Meulmeester JF, van den Berg H, Wedel M, Boshuis PG, 78. Geggel RL, Pereira GR, Spackman TJ 1978 Fractured ribs: Hulshof KFAM, Luyken R 1990 Vitamin D status, parathy- Unusual presentation of rickets in premature infants. roid hormone, and sunlight in Turkish, Moroccan, and J Pediatr 93:680Ð682. Caucasian children in The Netherlands. Eur J Clin Nutr 79. Opie WH, Muller CJB, Kamfer H 1975 The diagnosis of 44:461Ð470. vitamin D deficiency rickets. Pediat Radiol 3:105Ð110. 59. Dagnelie PC, Vergote FJVRA, van Staveren WA, van den 80. Pettifor JM, Pentopoulos M, Moodley GP, Isdale JM, Berg H, Dingjan PG, Hautvast JGAJ 1990 High prevalence Ross FP 1984 Is craniotabes a pathognomonic sign of rickets of rickets in infants on macrobiotic diets. Am J Clin Nutr in 3-month-old infants? S Afr Med J 65:549Ð551. 51:202Ð208. 81. Dancaster CP, Jackson WPU 1960 Studies in rickets in the 60. Nozza JM, Rodda CP 2001 Vitamin D deficiency in mothers Cape Peninsula 1. Cranial softening in a colored population of infants with rickets. Med J Aust 175:253Ð255. and its relationship to the radiological and biochemical 61. Mason RS, Diamond TH 2001 Vitamin D deficiency and changes of rickets. S Afr Med J 34:776Ð780. multicultural Australia. Med J Aust 175:236Ð237. 82. Reilly BJ, Leeming JM, Fraser D 1964 Craniosynostosis in 62. Blok BH, Grant CC, McNeil AR, Reid IR 2000 Characteristics the rachitic spectrum. J Pediatr 64:396Ð405. of children with florid vitamin D deficient rickets in the 83. Purvis RJ, Barrie MWJ, MacKay GS, Wilkinson EM, Auckland region in 1998. NZ Med J 113:374Ð376. Cockburn F, Belton NR, Forfar JO 1973 Enamel hypoplasia 63. Ford JA, McIntosh WB, Butterfield R, Preece MA, Pietrek J, of the teeth associated with neonatal tetany: a manifestation Arrow-Smith WA, Arthurton MW, Turner W, O’Riordan of maternal vitaminÐD deficiency. Lancet ii:811Ð814. JLH, Dunnigan MG 1976 Clinical and subclinical vitamin D 84. Schott GD, Wills MR 1976 Muscle weakness in osteomalacia. deficiency in Bradford children. Arch Dis Child 51:939Ð943. Lancet i:626Ð629. 64. Finch PJ, Ang L, Eastwood JB, Maxwell JD 1992 Clinical 85. Glerup H, Mikkelsen K, Poulsen L, Hass E, Overbeck S, and histological spectrum of osteomalacia among Asians in Andersen H, Charles P, Eriksen EF 2000 Hypovitaminosis D South London. Quart J Med 83:439Ð448. myopathy without biochemical signs of osteomalacic bone 65. Datta S, Alfaham M, Davies DP, Dunstan F, Woodhead S, involvement. Calcif Tissue Int 66:419Ð424. Evans J, Richards B 2002 Vitamin D deficiency in pregnant 86. Price DI, Stanford LC, Braden DS, Ebeid MR, Smith JC women from a non-European ethnic minority population— 2003 Hypocalcemic rickets: An unusual cause of dilated an interventional study. Brit J Obstet Gynaec 109:905Ð908. cardiomyopathy. Pediatr Cardiol 24:510Ð512. 1080 JOHN M. PETTIFOR

87. Olgun H, Ceviz N, Ozkan B 2003 A case of dilated car- 107. Markestad T, Halvorsen S, Seeger Halvorsen K, Aksnes L, diomyopathy due to nutritional vitamin D deficiency rickets. Aarskog D 1984 Plasma concentrations of vitamin D metabo- Turk J Pediatr 45:152Ð154. lites before and during treatment of vitamin D deficiency rickets 88. Uysal S, Kalayci AG, Baysal K 1999 Cardiac functions in in children. Acta Paediatr Scand 73:225Ð231. children with vitamin D deficiency rickets. Pediatr Cardiol 108. Srivastava T, Alon US 2002 Stage I vitamin D-deficiency 20:283Ð286. rickets mimicking pseudohypoparathyroidism type II. Clin 89. Manolagas SC, Yu XP, Girasole G, Bellido T 1994 Vitamin D Pediatr (Phila) 41:263Ð268. and the hematolymphopoietic tissue: a 1994 update. Semin 109. Taitz LS and de Lacy CD 1962 Parathyroid function in vita- Nephrol 14:129Ð143. min D deficiency rickets 1. Phosphorus excretion index in 90. DeLuca HF, Cantorna MT 2001 Vitamin D: its role and uses vitamin D deficiency rickets in South African bantu infants. in immunology. FASEB J 15:2579Ð2585. Pediatrics 30:875Ð883. 91. Stroder J, Kasal P 1970 Evaluation of phagocytosis in rickets. 110. Muldowney FP, Freaney R, McGeeney D 1968 Renal tubular Acta Paediatr Scand 59:288Ð292. acidosis and amino-aciduria in osteomalacia of dietary or 92. Lorente F, Fontan G, Jara P, Casas C, Garcia-Rodriguez MC, intestinal origin. Quart J Med 37:517Ð539. Ojeda JA 1976 Defective neutrophil motility in hypovita- 111. Kruse K 1995 Pathophysiology of calcium metabolism in minosis D rickets. Acta Paediatr Scand 65:695Ð699. children with vitamin D-deficiency rickets. J Pediatr 126: 93. Atiq M, Fadoo Z, Naz F, Khurshid M 1999 Myelofibrosis in 736Ð741. severe vitamin D deficiency rickets. J Pak Med Assoc 49: 112. Taitz LS, de Lacy CD 1962 Parathyroid function in vitamin D 174Ð177. deficiency rickets II. The relationship of parathyroid function 94. Gruner BA, DeNapoli TS, Elshihabi S, Britton HA, to bone changes and incidence of tetany in vitamin D defi- Langevin AM, Thomas PJ, Weitman SD 2003 Anemia and ciency rickets in South African bantu infants. Pediatrics hepatosplenomegaly as presenting features in a child with 30:884Ð892. rickets and secondary myelofibrosis. J Pediatr Hematol Oncol 113. Stanbury SW, Torkington P, Lumb GA, Adams PH, De Silva 25:813Ð815. P, Taylor CM 1975 Asian rickets and osteomalacia: patterns 95. Yetgin S, Ozoylu S 1982 Myeloid metaplasia in vitamin D of parathyroid response in vitamin D deficiency. Proc Nutr deficiency rickets. Scand J Haematol 28:180Ð185. Soc 34:111Ð117. 96. David L 1991 Common vitamin D-deficiency rickets. 114. Scariano JK, Walter EA, Glew RH, Hollis BW, Henry A, In: Glorieux FH (ed) Rickets. Nestec, Vevey: Raven Press, Ocheke I, Isichei CO 1995 Serum levels of the pyridinoline New York, pp. 107Ð122. crosslinked carboxyterminal telopeptide of type I collagen 97. Suda T 1987 Cellular mechanisms of fusion of hemopoietic (ICTP) and osteocalcin in rachitic children in Nigeria. Clin cells induced by 1α,25 dihydroxyvitamin D3. In: Cohn, DV, Biochem 28:541Ð545. Martin TJ, Meunier P J (eds) Calcium regulation and bone 115. Baroncelli GI, Bertelloni S, Ceccarelli C, Amato V, Saggese G metabolism: basic and clinical aspects. Excerpta Medica, 2000 Bone turnover in children with vitamin D deficiency Amsterdam, pp. 363Ð370. rickets before and during treatment. Acta Paediatrica 89: 98. Haddad JG, Chyu KJ 1971 Competitive protein binding 513Ð518. radioassay for 25-hydroxycholecalciferol. J Clin Endocrinol 116. Editorial 1971 Diagnosis of nutritional rickets. Lancet 33:992Ð995. ii:28Ð29. 99. Pettifor JM, Ross FP, Moodley GP, Margo G 1978 Serum 117. Nawawi H, Girgis SI 2002 Serum levels of bone-specific calcium, magnesium, phosphorus, alkaline phosphatase and alkaline phosphatase and procollagen type I carboxyterminal 25-hydroxyvitamin D concentrations in a paediatric popula- peptide in vitamin D deficiency. Southeast Asian J Trop Med tion. S Afr Med J 53:751Ð754. Public Health 33(Suppl 2):124Ð130. 100. McLaughlin M, Raggatt PR, Fairney A, Brown DJ, Lester E, 118. Soylu H, Aras S, Kutlu NO, Egri M, Sazak S 2001 Urinary Wills MR 1974 Seasonal variation in serum 25-hydroxy- free deoxypyridinoline assessment in recognition of rickets. cholecalciferol in healthy people. Lancet i:536Ð538. J Trop Pediatr 47:186Ð187. 101. Stanbury SW, Taylor CM, Lumb GA, Mawer EB, Berry J, 119. Calvo S, Eyre DR, Gundberg CM 1996 Molecular basis and Hann J, Wallace J 1981 Formation of vitamin D metabolites clinical application of biological markers of bone turnover. following correction of human vitamin D deficiency: obser- Endocr Rev 17:333Ð368. vations in patients with nutritional osteomalacia. Miner 120. Cole DEC, Carpenter TO, Gundberg CM 1985 Serum Electrolyte Metab 5:212Ð227. osteocalcin concentrations in children with metabolic bone 102. Mawer EB, Backhouse J, Hill LF, Lumb GA, De Silva P, disease. J Pediatr 106:770Ð776. Taylor CM, Stanbury SW 1975 Vitamin D metabolism and 121. Daniels ED, Pettifor JM, Moodley GP 2000 Serum osteocal- parathyroid function in man. Clin Sci Mol Med 48:349Ð365. cin has limited usefulness as a diagnostic marker for rickets. 103. Gupta MM, Round JM, Stamp TCB 1974 Spontaneous cure Eur J Pediatr 159:730Ð733. of vitamin-D deficency in Asians during summer in Britain. 122. Greig F, Casas J, Castells S 1989 Changes in plasma osteo- Lancet i:586Ð588. calcin concentrations during treatment of rickets. J Pediatr 104. Garabedian M, Vainsel M, Mallet E, Guillozo H, Toppet M, 114:820Ð823. Grimberg R, NGuyen TM, Balsan S 1983 Circulating vita- 123. Eastwood JB, de Wardener HE, Gray RW, Lemann JR Jr min D metabolite concentrations in children with nutritional 1979 Normal plasma-1,25-(OH)2-vitaminÐD concentrations rickets. J Pediatr 103:381Ð386. in nutritional osteomalacia. Lancet i:1377Ð1378. 105. Arnaud SB, Stickler GB, Haworth JC 1976 Serum 25-hydroxy- 124. Chesney RW, Zimmerman J, Hamstra A, DeLuca HF, vitamin D in infantile rickets. Pediatrics 57:221Ð225. Mazess RB 1981 Vitamin D metabolite concentrations in 106. Goel KM, Sweet EM, Logan RW, Warren JM, Arneil GC, vitamin D deficiency. Am J Dis Child 135:1025Ð1028. Shanks RA 1976 Florid and subclinical rickets among immi- 125. NGuyen TM, Guillozo H, Garabedian M, Mallet E, Balsan S grant children in Glasgow. Lancet i:1141Ð1145. 1979 Serum concentrations of 24,25-dihydroxyvitamin D in CHAPTER 65 Nutritional Rickets in Children 1081

normal children and in children with rickets. Pediat Res summer: a justification for vitamin D supplementation of 13:973Ð976. breast-feeding infants. J Pediatr 142:169Ð173. 126. Rosen JF, Chesney RW 1983 Circulating calcitriol concen- 145. Wharton B, Bishop N 2003 Rickets. Lancet 362:1389Ð1400. trations in health and disease. J Pediatr 103:1Ð17. 146. Standing Committee on the Scientific Evaluation of Dietary 127. Rasmussen H, Baron R, Broadus A, DeFronzo R, Lang R, Reference Intakes 1997 Dietary reference intakes for cal- Horst R 1980 1,25(OH)2D3 is not the only D metabolite cium, phosphorus, magnesium, vitamin D, fluoride. National involved in the pathogenesis of osteomalacia. Am J Med Academy Press, Washington. 69:360Ð368. 147. Department of Health 1998 Nutrition and bone health: with 128. Arnaud CD 1991 Parathyroid hormone and its role in the particular reference to calcium and vitamin D. Her Majesty’s pathophysiology of the common forms of rickets and osteo- Stationery Office, London, p. 115. malacia. In: Glorieux FH (ed) Rickets. Nestec, Vevey: Raven 148. Gartner LM, Greer FR 2003 Prevention of rickets and Press, New York, pp. 47Ð61. vitamin D deficiency: new guidelines for vitamin D intake. 129. Rasmussen H, De Luca H, Arnaud CD, Hawker C, Von Pediatrics 111:908Ð910. Stedingk M 1963 The relationship between vitamin D and 149. Finberg L 1981 Human milk feeding and vitamin D supple- parathyroid hormone. J Clin Invest 42:1940Ð1946. mentation. J Pediatr 99:228Ð229. 130. Arnaud CD, Rasmussen H, Anast C 1966 Further studies on 150. Ozsoylu S, Hansaoglu A 1981 25-hydroxycholecalciferol the interrelationship between parathyroid hormone and vita- levels in breast-fed infants. Arch Dis Child 56:318. min D. J Clin Invest 45:1955Ð1964. 151. Editorial 1973 The need for vitamin-D supplements. Lancet 131. Richards IDG, Sweet EM, Arneil GC 1968 Infantile rickets i:1097Ð1098. persists in Glasgow. Lancet i:803Ð805. 152. Specker BL, Ho ML, Oestreich A, Yin T, Shui Q, Chen X, 132. Pettifor JM 1991 Calcium, phosphorus, and vitamin D. Tsang RC 1992 Prospective study of vitamin D supplemen- In: Ballabriga A, Brusner O, Dobbing J, Gracey M, Senterre J tation and rickets in China. J Pediatr 120:733Ð739. (eds) Clinical nutrition of the young child. Nestec, Vevey: 153. Zeghoud F, Ben-Mekhbi H, Djeghri N, Garabedian M 1994 Raven Press, New York, pp. 497Ð516. Vitamin D prophylaxis during infancy: comparison of the 133. Pettifor JM, Isdale JM, Sahakian J, Hansen JDL 1980 long-term effects of three intermittent doses (15, 5, or 2.5 mg) Diagnosis of subclinical rickets. Arch Dis Child 55:155Ð157. on 25-hydroxyvitamin D concentrations. Am J Clin Nutr 134. Hunter GJ, Schneidau A, Hunter JV, Chapman M 1984 60:393Ð396. Rickets in adolescence. Clin Radiol 35:419Ð421. 154. Chesney RW 2003 Rickets: An old form for a new century. 135. Shah BR and Finberg L 1994 Single-day therapy for nutri- Pediatr Int 45:509Ð511. tional vitamin DÐdeficiency rickets: a preferred method. 155. Atiq M, Suria A, Nizami SQ, Ahmed I 1998 Vitamin D sta- J Pediatr 125:487Ð490. tus of breastfed Pakistani infants. Acta Paediatr 87:737Ð740. 136. Lubani MM, Al-Shab TS, Al-Saleh QA, Sharda DC, 156. Al Jurayyan NA, El Desouki ME, Al Herbish AS, Al Mazyad Quattawi SA, Ahmed SAH, Moussa MA, Reavey PC 1989 AS, Al Qhtani MM 2002 Nutritional rickets and osteomalacia Vitamin DÐdeficiency rickets in Kuwait: the prevalence of a in school children and adolescents. Saudi Med J 23:182Ð185. preventable disease. Ann Trop Paediatr 3:134Ð139. 157. Narchi H 2000 Case-control study of diet and sun exposure 137. Cesur Y, Caksen H, Gundem A, Kirimi E, Odabas D 2003 in adolescents with symptomatic rickets. Ann Trop Paediatr Comparison of low and high dose of vitamin D treatment in 20:217Ð221. nutritional vitamin D deficiency rickets. J Pediatr Endocrinol 158. Scharla SH 1998 Prevalence of subclinical vitamin D defi- Metab 16:1105Ð1109. ciency in different European countries. Osteoporos Int 138. Kutluk G, Cetinkaya F, Basak M 2002 Comparisons of oral (Suppl 8):S7ÐS12. calcium, high dose vitamin D, and a combination of these in 159. Cheng S, Tylavsky F, Kroger H, Karkkainen M, Lyytikainen A, the treatment of nutritional rickets in children. J Trop Pediatr Koistinen A, Mahonen A, Alen M, Halleen J, Vaananen K, 48:351Ð353. Lamberg-Allardt C 2003 Association of low 25-hydroxyvita- 139. Greer FR, Searcy JE, Levin RS, Steichen JJ, Steichen-Asche min D concentrations with elevated parathyroid hormone PS, Tsang RC 1982 Bone mineral content and serum concentrations and low cortical bone density in early 25-hydroxyvitamin D concentrations in breast-fed infants pubertal and prepubertal Finnish girls. Am J Clin Nutr 78: with or without supplemental vitamin D: One-year follow-up. 485Ð492. J Pediatr 100:919Ð922. 160. Outila TA, Karkkainen MU, Lamberg-Allardt CJ 2001 140. Ala-Houhala M 1985 25-hydroxyvitamin D levels during Vitamin D status affects serum parathyroid hormone concen- breast-feeding with or without maternal or infantile supple- trations during winter in female adolescents: associations mentation of vitamin D. J Pediatr Gastroenterol Nutr 4: with forearm bone mineral density. Am J Clin Nutr 220Ð226. 74:206Ð210. 141. Roberts CC, Chan GM, Folland D, Rayburn C, Jackson R 161. Guillemant J, Le HT, Maria A, Allemandou A, Peres G, 1981 Adequate bone mineralization in breast-fed infants. Guillemant S 2001 Wintertime vitamin D deficiency in male J Pediatr 99:192Ð196. adolescents: effect on parathyroid function and response to 142. Birkbeck JA, Scott HF 1980 25-hydroxycholecalciferol vitamin D3 supplements. Osteoporos Int 12:875Ð879. serum levels in breast-fed infants. Arch Dis Child 162. Balasubramanian K, Rajeswari J, Gulab, Govil YC, Agarwal 50:691Ð695. AK, Kumar A, Bhatia V 2003 Varying role of vitamin D defi- 143. Bassir M, Laborie S, Lapillonne A, Claris O, Chappuis MC, ciency in the etiology of rickets in young children vs. ado- Salle BL 2001 Vitamin D deficiency in Iranian mothers and lescents in northern India. J Trop Pediatr 49:201Ð206. their neonates: a pilot study. Acta Paediatr 90:577Ð579. 163. Rajeswari J, Balasubramanian K, Bhatia V, Sharma VP, 144. Dawodu A, Agarwal M, Hossain M, Kochiyil J, Zayed R Agarwal AK 2003 Aetiology and clinical profile of osteoma- 2003 Hypovitaminosis D and vitamin D deficiency in lacia in adolescent girls in northern India. Natl Med J India exclusively breast-feeding infants and their mothers in 16:139Ð142. 1082 JOHN M. PETTIFOR

164. Du X, Greenfield H, Fraser DR, Ge K, Trube A, Wang Y 183. Schnitzler CM, Pettifor JM, Patel D, Mesquita JM, Moodley 2001 Vitamin D deficiency and associated factors in adoles- GP, Zachen D 1994 Metabolic bone disease in black cent girls in Beijing. Am J Clin Nutr 74:494Ð500. teenagers with genu valgum or varum without radiologic 165. Calvo MS, Whiting SJ 2003 Prevalence of vitamin D insuffi- rickets: a bone histomorphometric study. J Bone Miner Res ciency in Canada and the United States: importance to health 9:479Ð486. status and efficacy of current food fortification and dietary 184. Thacher TD, Fischer PR, Pettifor JM, Lawson JO, Manaster supplement use. Nutr Rev 61:107Ð113. BJ, Reading JC 2000 Radiographic scoring method for the 166. Pietrek J, Preece MA, Windo J, O’Riordan JLH, Dunnigan assessment of the severity of nutritional rickets. Journal of MG, McIntosh WB, Ford JA 1976 Prevention of vitamin-D Tropical Pediatrics 46:132Ð139. deficiency in Asians. Lancet i:1145Ð1148. 185. Pettifor JM, Ross FP, Travers R, Glorieux FH, DeLuca HF 167. Walker ARP 1953 Does a low intake of calcium cause 1981 Dietary calcium deficiency: a syndrome associated with or promote the development of rickets? Am J Clin Nutr bone deformities and elevated serum 1,25-dihydroxyvitamin 3:114Ð120. D concentrations. Metab Bone Rel Res 2:301Ð305. 168. Irwin MI, Kienholz EW 1973 A conspectus of research on 186. Pettifor JM, Ross FP, Moodley GP, Shuenyane E 1979 calcium requirements of man. J Nutr 103:1020Ð1095. Calcium deficiency in rural black children in South Africa— 169. Maltz HE, Fish MB, Holliday MA 1970 Calcium deficiency a comparison between rural and urban communities. Am J rickets and the renal response to calcium infusion. Pediatrics Clin Nutr 32:2477Ð2483. 46:865Ð870. 187. Pettifor JM, Moodley GP 1997 Appendicular bone mass in 170. Sly MR, van der Walt WH, Du Bruyn D, Pettifor JM, Marie children with a high prevalence of low dietary calcium PJ 1984 Exacerbation of rickets and osteomalacia by maize: intakes. J Bone Miner Res, 12:1824Ð1832. a study of bone histomorphometry and composition in young 188. Pettifor JM, Ross FP, Moodley GP, Shuenyane E 1981 baboons. Calcif Tissue Int 36:370Ð379. The effect of dietary calcium supplementation on serum 171. Proesman W, Legius E, Eggermont E 1988 Rickets due to calcium, phosphorus, and alkaline phosphatase concen- calcium deficiency. Mary Ann Liebert, New York, p. 15. trations in a rural black population. Am J Clin Nutr 172. Pettifor JM, Ross P, Wang J, Moodley G, Couper-Smith J 34:2187Ð2191. 1978 Rickets in children of rural origin in South Africa: is 189. Fischer PR, Thacher TD, Pettifor JM, Jorde LB, Eccleshall low dietary calcium a factor? J Pediatr 92:320Ð324. TR, Feldman D 2000 Vitamin D receptor polymorphisms and 173. Marie PJ, Pettifor JM, Ross FP, Glorieux FH 1982 nutritional rickets in Nigerian children. J Bone Miner Res Histological osteomalacia due to dietary calcium deficiency 15:2206Ð2210. in children. N Engl J Med 307:584Ð588. 190. Mellanby E 1919 An experimental investigation on rickets. 174. Thacher TD, Ighogboja SI, Fischer PR 1997 Rickets without Lancet i:407Ð412. vitamin D deficiency in Nigerian children. Ambulatory Child 191. Iqbal SJ, Kaddam I, Wassif W, Nichol F, Walls J 1994 Health 3:56Ð64. Continuing clinically severe vitamin D deficiency in Asians 175. Thacher TD, Fischer PR, Pettifor JM, Lawson JO, Isichei in the UK (Leicester). Postgrad Med J 70:708Ð714. CO, Reading JC, Chan GM 1999 A comparison of calcium, 192. Preece MA, Ford JA, McIntosh WB, Dunnigan MG, vitamin D, or both for nutritional rickets in Nigerian chil- Tomlinson S, O’Riordan JL H 1973 Vitamin-D deficiency dren. N Engl J Med 341:563Ð568. among Asian immigrants to Britain. Lancet i:907Ð910. 176. Oginni LM, Worsfold M, Oyelami OA, Sharp CA, Powell 193. Dunnigan MG, McIntosh WB, Ford JA 1976 Rickets in DE, Davie MW 1996 Etiology of rickets in Nigerian chil- Asian immigrants. Lancet i:1346. dren. J Pediatr 128:692Ð694. 194. Wills MR, Day RC, Phillips JB, Bateman EC 1972 177. Oginni LM, Sharp CA, Badru OS, Risteli J, Davie MW, Phytic acid and nutritional rickets in immigrants. Lancet Worsfold M 2003 Radiological and biochemical resolution i:771Ð773. of nutritional rickets with calcium. Arch Dis Child 195. Ford JA, Colhoun EM, McIntosh WB, Dunnigan MG 1972 88:812Ð817. Biochemical response of late rickets and osteomalacia to a 178. Fischer PR, Rahman A, Cimma JP, Kyaw-Myint TO, Kabir chupatty-free diet. Br Med J 3:446Ð447. AR, Talukder K, Hassan N, Manaster BJ, Staab DB, Duxbury 196. Batchelor AJ, Compston JE 1983 Reduced plasma half-life JM, Welch RM, Meisner CA, Haque S, Combs GF Jr 1999 of radio-labelled 25-hydroxyvitamin D3 in subjects receiving Nutritional rickets without vitamin D deficiency in a high-fiber diet. Br J Nutr 49:213Ð216. Bangladesh. J Trop Pediatr 45:291Ð293. 197. Batchelor AJ, Watson G, Compston JE 1982 Changes 179. Eyberg C, Pettifor JM, Moodley G 1986 Dietary calcium in plasma half-life and clearance of 3H-25-hydroxyvitamin intake in rural black South African children. The relationship D3 in patients with intestinal malabsorption. Gut 23: between calcium intake and calcium nutritional status. Hum 1068Ð1071. Nutr Clin Nutr 40C:69Ð74. 198. Clements MR, Johnson L, Fraser DR 1987 A new mecha- 180. Bhimma R, Pettifor JM, Coovadia HM, Moodley M, nism for induced vitamin D deficiency in calcium deprivation. Adhikari M 1995 Rickets in black children beyond infancy in Nature 325:62Ð65. Natal. S Afr Med J 85:668Ð672. 199. Halloran BP, Castro ME 1989 Vitamin D kinetics in vivo: 181. Pettifor JM 1991 Dietary calcium deficiency. In: effect of 1,25-dihydroxyvitamin D administration. Am J Glorieux FH (ed) Rickets. Nestec, Vevey: Raven Press, Physiol 256:E686ÐE691. New York, pp. 123Ð143. 200. Halloran BP, Bikle DD, Levens MJ, Castro ME, Globus RK, 182. Thacher TD, Fischer PR, Pettifor JM, Lawson JO, Isichei C, Holton E 1986 Chronic 1,25-dihydroxyvitamin D3 adminis- Chan GM 2000 Case-control study of factors associated tration in the rat reduces serum concentration of 25-hydroxy- with nutritional rickets in Nigerian children. J Pediatr 137: vitamin D by increasing metabolic clearance rate. J Clin 367Ð373. Invest 78:622Ð628. CHAPTER 65 Nutritional Rickets in Children 1083

201. Clements MR, Davies M, Fraser DR, Lumb GA, Mawer B, 203. Berlin T, Bjorkhem I 1988 Effect of calcium intake on Adams PH 1987 Metabolic inactivation of vitamin D is serum levels of 25-hydroxyvitamin D3. Eur J Clin Invest 18: enhanced in primary hyperparathyroidism. Clin Sci 73: 52Ð55. 659Ð664. 204. Pettifor JM 1994 Privational rickets: a modern perspective. 202. Clements MR, Davies M, Hayes ME, Hickey CD, Lumb GA, J Roy Soc Med 87:723Ð725. Mawer EB, Adams PH 1992 The role of 1,25-dihydroxyvita- 205. Shany S, Hirsh J, Berlyne GM 1976 25-Hydroxy- min D in the mechanism of acquired vitamin D deficiency. cholecalciferol levels in bedouins in the Negev. Am J Clin Clin Endocrinol 37:17Ð27. Nutr 29:1104Ð1107. CHAPTER 66 Vitamin D Insufficiency in Adults and the Elderly

PIERRE J. MEUNIER AND MARIE-CLAIRE CHAPUY INSERM Unit 403, Faculty Laennec and Department of Rheumatology and Bone Disease, Edouard Herriot Hospital, Lyon, France

I. Introduction V. Prevalence of Vitamin D Insufficiency II. Definition of Vitamin D Deficiency and Insufficiency VI. Preventive Measures: Correction of Low Vitamin D Status III. Determinants of Vitamin D Insufficiency VII. Conclusions IV. Consequences of Low Vitamin D Status References

I. INTRODUCTION II. DEFINITION OF VITAMIN D DEFICIENCY AND INSUFFICIENCY In contrast to the extensive attention paid to vitamin D as a component of nutritional health in infants At the outset, it is essential to clarify the definition of and children, for whom vitamin D deficiency was vitamin D “insufficiency.” Some confusion exists in the synonymous with rickets, the relationship of vitamin D literature between the terms vitamin D “insufficiency” to the nutritional health of adults or elderly subjects and “deficiency” or depletion. The term “insufficient” had been largely ignored for a long time. Studies is defined as lacking in something necessary for on the vitamin D status of the elderly began only in the completeness, and “deplete” is defined as empty [2]. mid-1970s when assays for serum 25-hydroxyvitamin D “Repletion,” or “sufficiency” on the other hand, con- (25OHD), the best barometer of vitamin D status, note fullness. In this chapter, we define a subject with became available. The initial studies were carried reduced vitamin D as vitamin D insufficient, and a out in Europe, and it was not until 1982 that studies on subject severely lacking in vitamin D as vitamin D the nutritional status of the elderly living in the United deplete or deficient. States included data on the vitamin D status of the aged Peacock et al., in 1985, have been among the first population [1]. authors to propose definitions of vitamin D deficiency With the major changes in demography that and insufficiency based on serum 25OHD concentra- occurred since the 1960s leading to increased life tions [3]. According to these authors, vitamin D defi- expectancy of the population, vitamin D insufficiency ciency occurs with serum 25OHD levels ranging from represents a timely and common health problem in 0 to 4 ng/ml (0Ð10 nmol/liter) with evident secondary older people. Vitamin D insufficiency is frequently hyperparathyroidism and malabsorption of calcium, associated with abnormal bone metabolism including leading to the histological evidence of frank osteoma- secondary hyperparathyroidism, which induces an lacia. In vitamin D insufficiency, that is, 25OHD levels increase in bone turnover and bone loss, particularly ranging from 4 to 20 ng/ml (10Ð50 nmol/liter), there is in cortical bone. This leads to an increased risk of mild hyperparathyroidism, suboptimal calcium absorp- fracture, especially of hip fracture, in those subjects tion, high bone turnover, and reduced bone density. In with vitamin D insufficiency. The purpose of this vitamin D sufficiency the 25OHD levels range from chapter is to define this state of vitamin D insuffi- 20 to 80 ng/ml (50 to 200 nmol/liter), and there is no dis- ciency, discuss its prevalence among adults and older turbance in calcium homeostasis and bone metabolism. subjects, and analyze its causes and its consequences Peacock et al. have further defined vitamin D suffi- on parathyroid function, bone, and muscle. At the end ciency by examining the serum 1,25-dihydroxyvitamin D of the chapter, the utility of increasing vitamin D [1,25(OH)2D] response to treatment with 25OHD3 in var- intake, not only in the elderly but also in adults, is ious groups of patients and healthy subjects. Vitamin D discussed. sufficiency [i.e., no change in serum 1,25(OH)2Dlevels]

VITAMIN D, 2ND EDITION Copyright © 2005, Elsevier, Inc. FELDMAN, PIKE, AND GLORIEUX All rights reserved. 1086 PIERRE J. MEUNIER AND MARIE-CLAIRE CHAPUY occurred at a serum 25OHD level in excess of 20 ng/ml On the other hand, because the major biological (50 nmol/liter), which is above the lower limit of the effects of vitamin D are mediated by 1,25(OH)2D, many classical normal range for 25OHD values. researchers would probably agree that vitamin D defi- Other investigators have used the parathyroid hor- ciency should entail 1,25(OH)2D deficiency. However, mone (PTH) level as an index of vitamin D repletion, low 1,25(OH)2D may not always be associated with low on the basis of the rationale that vitamin D insuffi- 25OHD, and normal values of 1,25(OH)2D can exist in ciency results in decreased calcium absorption, a subtle the presence of low 25OHD levels combined with an decline in blood ionized calcium, and consequently an increased PTH concentration. The determination of increase in PTH values [4Ð6]. For Gloth and coworkers circulating 1,25(OH)2D concentrations has very limited [7,8] and Webb et al. [9], the lowest acceptable limit clinical utility except in the diagnosis of some condi- for 25OHD levels was 37 nmol/liter (15 ng/ml). Later, tions involving the vitamin D endocrine system such in 1998, Lips et al. pointed out that the 25(OH)D level as vitamin DÐdependent rickets types I (PDDR) and associated with maximal suppression of PTH was II (HVDRR) and some hypercalcemic states caused by 30 nmol/L (12 ng/ml) [5], but more recently the results increased levels of 1,25(OH)2D. Even if 1,25(OH)2D is of several studies have shown that the serum 25(OH)D the active form of vitamin D, its measurement provides level corresponding to the lower limit of vitamin D little information in many disorders and no information sufficiency was much higher than this “classical” at all on the nutritional status of vitamin D [18]. threshold of 30 nmol/L. These studies have placed the Returning to the question of terminology, it appears estimate at 75 to 80 nmol/L (Chapuy et al., 1997 [10]), that the term “deficiency” used in many studies or 65 to 75 nmol/L (Thomas et al., 1998 [11]), 50 nmol/L reports is heterogeneous and includes several different (Malabanan et al., 1998 [12]), 82 nmol/L (Krall et al., states. We believe that the term “insufficiency” is well- 1989 [6]), 99 nmol/L (Dawson-Hughes et al., 1997 [13]), suited to define the state of hypovitaminosis D that 75 nmol/L (Tangpricha et al., 2002 [14]) and 100 nmol/L induces other abnormalities of bone metabolism and (Vieth et al., 2003 [15]). Thus, the estimates of 25 (OH)D changes in biochemical indices, but this is not yet required for maximal PTH suppression vary widely accepted by all investigators. from 30 to 100 nmol/L, and there is a cluster of estimates The reappraisal at a higher level of the definition in the 75 to 80 nmol/L range. However, serum 25OHD threshold for vitamin D insufficiency has two practical concentration varies with country, season, and sunshine consequences: first, vitamin D insufficiency is much exposure. The mean values of serum 25OHD are higher more common than formerly believed and this is rele- in the United States than in northwestern European coun- vant to larger possibilities of vitamin D supplements tries. This may result in different thresholds for diagnos- for preventing bone loss and fractures, particularly in ing insufficiency versus sufficiency. elderly people; second, it is important to ensure that As noted above, substrate-dependent synthesis is the serum 25 OHD level obtained after vitamin D sup- another way to diagnose a vitamin DÐdeficient state; plementation reaches this new threshold. Most studies when vitamin D therapy results in an increase in the have shown that an intake of 800 IU/day (20 µg) is serum 1,25(OH)2D concentration, vitamin D insuffi- needed to attain the desired 25 OHD level (also see ciency is probable [3,16]. This approach might be Chapters 61 and 62). complicated by an increase in PTH values secondary to low 25OHD values. Also, the comparability of the cutoffs proposed by III. DETERMINANTS OF VITAMIN D different authors may vary because different methods INSUFFICIENCY have been used for 25OHD assays (see Chapter 58). As the diagnosis of hypervitaminosis D is based on The vitamin D status of a subject is derived mainly a blood test, whether very low or “undetectable” levels from cutaneous synthesis initiated by solar irradiation of vitamin D represent deficiency or depletion of the skin and also from dietary intake. A reduction of will depend in part on the sensitivity of the assay one or both sources unavoidably leads to vitamin D method. Assays using extraction and purification give insufficiency. Some of these issues are also discussed lower results than those without a preparative chro- in Chapter 46. matographic step. In the future, new radioimmuno- assays, which give a higher correlation with the high-performance liquid chromatography (HPLC) A. Sunlight Deprivation method [17], certainly will be used more frequently and will allow better comparison of the data from The body stores of vitamin D are mainly dependent different studies. on the cutaneous synthesis of vitamin D3, but this CHAPTER 66 Vitamin D Insufficiency in Adults and the Elderly 1087 synthesis is in turn dependent on several factors such 10 as the duration of sunlight exposure, the latitude of the Young adults Recommended intake country, the season, the time of day, and atmospheric Elderly conditions (see Chapter 3). Holick found that in Boston (42° N), exposure to sunlight on cloudless days 8 between the months of November and February for up to 5 hr did not result in any significant production of /day) vitamin D [19]. At high latitudes, the stores of vitamin D µ are mainly generated during the summer months. 6 However, the penetration of effective ultraviolet rays (285 to 310 nm) into the cutaneous layers is modified by the type of clothing, the blockage of effective rays 4 by window glass, and the capacity of skin to produce vitamin D. Generally, on going outdoors, healthy elderly sub- ( vitamin D intake Oral jects take protective action to reduce sunlight exposure 2 either by use of clothing and sunscreens or simply by just avoiding direct sun. Even in a sunny country, Lebanon, a severe hypovitaminosis D has been shown in 31% of a population of 316 volunteers, more preva- 0 lent in women (41.5%), particularly in the veiled ones N Amer Scand Europe (62%) [20]. Recommendations for reducing the risk of FIGURE 1 Mean of the average vitamin D intake in young adults skin cancer have heightened the situation. Aging has a and elderly from studies collated according to geographic region. dramatic impact on the skin: after the age of 20, skin The recommended intake is 10 µg/day. From McKenna [24] with thickness decreases linearly with age, and the capacity permission. to produce previtamin D3 is reduced. The increase in 25OHD values after the same amount of simulated sunlight was 3 times higher in younger subjects aged that upward of 70% of all milk samples tested did 22Ð30 years than in elderly subjects aged 62Ð80 years not contain this amount. In Scandinavia (as in Japan), [21]. This is due in part to the age-related decline in people have a substantial amount of fish (salmon, skin thickness [22]. However, it has been shown that mackerel, herring) as a part of their diet. In the review irradiation with UV-B in the very elderly for a few by McKenna [24], the mean vitamin D intake was minutes per day leads to adequate improvement of the found to be 2.5 ± 1.3 µg/day in Europe, 6.2 ± 2.4 µg/day vitamin D status. It is as effective as oral vitamin D3 in North America, and 5.2 ± 2.0 µg/day in Scandinavia. (400 IU/day) in increasing serum 25 OHD and sup- In all studies combined, no differences were found pressing secondary hyperparathyroidism [23]. Even in between the vitamin D intake of young adults and the young adults, the seasonal variation of 25OHD con- elderly (Fig. 1). centrations might be explained by sun deprivation dur- The primacy of oral intake over sunlight exposure in ing winter months, when hypovitaminosis D is more maintaining vitamin D stores during the wintertime, frequent. Elderly institutionalized people often are especially in elderly subjects residing in Western unable to go outdoors because many are infirm or sick; Europe, and even in North American healthy post- thus, their vitamin D status depends exclusively on menopausal women, has been demonstrated [6]. The vitamin D supplied by food. current U.S. recommended dietary allowance of vita- min D for adults is 200 IU or 5 µg. From many studies, it appears that the need is greater and may approach B. Low Vitamin D Intake 400 IU or 10 µg per day in order to maintain adequate 25OHD concentrations in adults living in high latitude The natural sources of dietary vitamin D are fatty countries. fishes, fish liver oil, and to a lesser extent eggs. Regional It is likely that the true vitamin D requirement, in differences in vitamin D intake are important. Mean the absence of any exposure to sunlight, is closer to vitamin D intake per day is lower in Europe compared 600 IU or 15 µg/day [19]. In elderly people who are with both North America and Scandinavia. In North confined indoors and who get no direct sunlight expo- America, milk is fortified with 400 lU/quart of either sure, the requirement is increased to 800 IU/day. In vitamin D2 or vitamin D3, but studies have revealed addition, some studies, but not all [25,26], found that