0022-202X/ 8 1/ 770 1-005 1$02.00 / 0 TH E J OU RNAL Of' INVESTIGATIV E DERMATOLOGY, 76:5 1-58, 1981 Vol. 77 , No. 1 Copy righl © 1981 by The Williams & Wilkins Co. Prill ted ill U. .A. The Cutaneous Photosynthesis of Previtamin D3: A Unique Photoendocrine System

MICHAEL F. HOLICK, PH.D., M .D . Endocrine Unit, M a.ssachusetts General Hospita.l, a.nd Ha.rvard Medica.l School, BostOIl, Massa.chusetts, a.nd Department of Nutrition a.nd Food Sciences, Massa.clwsetts Institute of T ech.nology, Cambridge, Massa.chusetts, U.S.A.

The skin has been recognized as the site for the sun­ essentially absent in the rural districts [5]. A year later, Palm mediated photosynthesis of vitamin D ,, ; until recently, [6] reported on an epidemiologic survey of rickets in the British however, very little was known about either the se­ Isles and the Orient and observed that in Japan, China, and quence of events leading to the formation of vitamin D" India, where people were poorly nourished a nd lived in s qualor, in human skin or the factors that regulate the synthesis rickets was rare, whereas it was endemic in t he industrialized of this hormone. It is now established that, during ex­ districts of Great Britain, one of the wealthiest nations in t he posure to sunlight, the cutaneous reservoir of7-dehydro­ world. These clinical observations led him to urge t h e "sys­ cholesterol (principally in the stratum Malpighii) con­ tematic use of sunbaths as a preventive measure in rickets and verts to previtamin D;I. Once this thermally labile prev­ other diseases." However, despite these ma ny insightful obser­ itamin is formed, it undergoes a temperature-dependent vations, by the t um of the 20th century m ost physicia ns and isomerization to vitamin D" over a period of 3 days. The scientists could not accept such a simple explanation for the plasma vitamin-D binding protein preferentially trans­ etiology of this dreaded bone-deforming disease. In fact, by locates vitamin D;j from the skin into the circulation. 1900 the 3 most popular t heories of the etiology of this disease During prolonged exposure to the sun, the accumulation included (a) an infectious disease, ( b ) a heritable disorder, and of previtamin D" is limited to about 10 to 15% of the (c) a nutritional deficiency. original 7-dehydrocholesterol content because the pre­ In 1919, Huldschinsky [7] provided incontravertible evidence vitamin photoisomerizes to 2 biologically inert photo­ that exposure to radiation from a mercury-vapor quartz lamp products, lumisteroIa and tachysteroh. Increases in alone was sufficient to treat and cure rickets. Furthermore, he either latitude or the melanin concentration in the skin showed that this curative effect was not a local effect (because diminish the epidermal synthesis of previtamin D". A exposw'e of one arm had equal and dramatic curative effects in single total body exposure to 3 minimal erythemal doses the non exposed arm as well), prompting th e suggestion that a of ultraviolet radiation increased the vitamin-D" levels compound was photosynthesized in the skin and transported in the serum almost lO-fold within 2 days and doubled into the circulation to act at distant sites. Two years later, Hess the serum 25-hydroxyvitamin-D levels after 7 days. The and Unger [8] reported that infants with rickets were cured unique mechanism for the cutaneous synthesis, storage, after simple exposur e to sunlight. Hence, proof was finally and steady release of vitamin D" into the circulation demonstrated 3 decades after the first suggestion by Palm that prompted an investigation into the potential therapeutic exposure to sunlight alone had a curative effect on rickets. benefits of using the skin as the site for the synthesis and absorption of vitamin-D3 metabolites. CUTANEOUS PHOTOSYNTHESIS OF PREVITAMIN D ;l At the same time that Huldschinsky [7] a nd Hess and Unger Since the dawn of man, the sun has been worshipped and [8] were demonstrating that radiation from various a rtificial revered for its life-sustaining powers. However, the importance souJ'ces or the sun could cure rickets, other investigators were of exposure to sunlight for health was not appreciated until the developing rachitic animal models and s howing that rickets industrialization of northern Emope in the 17th century. Coin­ could also be cured by administration of cod liver oil [9] or by cident with the appearance of the pall of industrial smoke and ultraviolet irradiation of th e food that was subsequently eaten t h e congregation of people into densely populated towns, where [10,11]. The confusion over whether rickets was cured by direct the streets were narrow and poorly exposed to sunlight, was the exposure of the body to radiation from the sun (or a mercury­ recognition of a disease, known as rickets, associated with vapor quartz lamp) or by ingestion of a dietary factor was deformities of the skeleton in children [1-3]. As early as 1822, resolved when H ess [12] reported that rats fed ultraviolet (UV)­ t he Polish physician Sniadecki [4] suggested that the dreaded irradiated human cadaver skin did not develop rickets, and " English disease" (also known as rickets) was due to the lack of Powers et al [13] demonstrated in rachitic }'ats that the healing exposm e to sunlight. Further evidence that this disease was effects were t he same after treatment with e ither radiation from promoted by environmental factors was provided in a report, in a mercury-vapor quartz lamp or administration of cod liver oil. 1889, by the British Medical Association. A survey of the Collectively, these observations provided the basis for t he for­ incidence of rickets in the British Isles showed that rickets tification of dairy products and other food stuffs with vitamin abounded in thickly populated industrialized areas, but was D, the consequence of which was responsible for the eventual eradication of this endemic disease. Once it was established that the antirachitic factor could be This work was supported in part by NIH grant # AM -25506-01 and produced in vitro by ultraviolet irradiation of plant and animal NIH grant #AM-27334. tissues, numerous investigators became involved in the struc­ Reprint req uests to: Michael F. Holick, Ph.D., M.D., Endocrine Unit, tural characterization of the antirachitic factor and its precur­ Massachusetts General Hospital, Boston, Massachusetts 02114. sor. It was established that the antirachitic precursor was a 4- Abbreviations: member ring sterol with 2 conjugated double-bonds at carbons 7- DHC: 7-dehydrocholesterol 10',25-{OH h -7-DHC: 10',25-dihydroxy-7-dehydrocholesterol 5 and 7. Initial characterization of the antirachitic factor (also 10',25-{OH h-D,,: 10',25-dihydroxyvitamin 0:, known as vitamin D) established that ring B was opened at 25-0H-D,,: 25-hydroxyvitamin D" C9-ClO position (Fig 1) and that 3 double-bonds existed in a MED: minimal erythema dose 5,6-cis-configuration [14,15]. X-ray crystal analYSis of a heavy­ preD;j: prev itamin D" atom deri vative of vitamin D [1 6] provided evidence for its

51 52 HOLICK Vol. 77, No.1

24 26 During the past 5 yr, my laboratory has been interested in the 25 sequential events OCCUlTing in the skin during exposure to 27 ultraviolet-radiation and culminating in the synthesis of vitamin D a. We chemically synthesized [3cx- :J H]7-DHC [24] and devel­ oped chromatographic techniques that would permit the direct HO analysis of skin lipids without any thermal isomerization. Ini­ tially, we topically applied [3cx- 3H]7-DHC to previously shaven 7-0EHYOROC HOLESTEROL vitamin-D-deficient rats. Then, 24 hI' after application, groups 2 (PROVITAM IN 0 3 ) of animals were exposed to 0.2 J /cm of UV radiation (obtained VITAM IN 03 from an F40-T12 Westinghouse sunlamp broad-band UV-radia­ tion 275-350 nm) and the irradiated skin was excised, extracted, and chromatographed. Lipid extracts from the skin of control nonirradiated animals contained 7-DHC and its reduction prod­ uct, «holesterol. Skin from exposed animals exhibited radiola­ beled 7-DHC, cholesterol, and a major photoproduct identified as preDa [24]. To be certain that this phenomenon was not due only to the photoconversion of radiolabeled 7-DHC on the stratum corneum surface but was also an occurrence within the epidermis, 40 vitamin-D-deficient rats were exposed to 0.2 J / HO cm2 of UV radiation, and the whole skin was immediately ERGOSTEROL excised and extracted in organic solvents. All purification steps were carried out at 4°C to ensure that any thermally labile (PROVITAMIN O ) 2 VITAMIN 02 photoproducts would not be altered during the extraction and chromatography procedures. After numerous chromatographic FIG 1. Structures for 7-dehydrocholesterol, ergosterol, vitamin Oa procedures, preDa was isolated in pure form and identified and vitamin O2• based upon (a) mass spectrum, m/e 384 (M+), 271 (M-side­ chain), 253 (271- H 20), 136 (ring A + (HC)6, (HCh, and (H2Ch9, 1. spatial orientation as shown in Fig The designations vitamin and 118 (136-H20), (6) UV-absorption spectrum Amox260 and "D/ ' and vitamin "D/' refer to differences in side-chain struc­ A01;,,230 nm, which is characteristic for the 6,7-cis-triene chro­ ture. Vitamin Da originates from the cutaneous sterol, 7-dehy­ mophore for pre vitamin Da, and (e) subsequent thermally con­ drocholesterol (7~DHC), whereas vitamin D 2, which has a C24 - trolled conversion to vitamin Da as demonstrated by spectral methyl and a double-bond between carbons 22 and 23, origi­ shiJt in the UV absorption from Amux260 to A01;n265 nm (Fig 2). nates from the fungal sterol, ergosterol (Fig 1). Furthermore, an analysis of the same lipid extract did not With the discovery that exposure of skin to radiation from demonstrate the presence of any vitamin Da, and thus ruled out the sun or artificial-light sources led .to the production of the possibility that vitamin Da was being made simultaneously vitamin Da, there was intensive investigation into which part of with the preDa (25). the sunlight spectrum caused this conversion and what steps led to the foxmation of vitamin Da. It was'demonstrated in vivo CUT ANEOUS SYNTHESIS OF VITAMIN Da and in vitro that, when human or animal skin was exposed to Once we established that, in vivo, when skin is exposed to the ultraviolet-B (UV -B) portion of natural sunlight (spectral UV radiation, cutaneous 7-DHC was photolyzed to preD , the range 290-320 nm), the stores of 7-DHC in the epidermis a intriguing question of whether there was any physiologic ad­ converted ultimately to vitamin D a, but the precise sequence of vantage to this process was explored. We examined some of the events was I10t known [17,18). It was well established by 1950 physiologic implications of this unique cutaneous photobiologic that, when 7-DHC is dissolved in an organic solvent in a quartz process, including (a) the temperature dependence of preD vessel and exposed to UV-B radiation, it (a) absorbs a photon 3 conversion to vitamin Da, (6) the transport of preD3 and/ or of radiation energy, which (b) causes cleavage of the C - C 9 IO vitamin Da from the skin into the circulation, and (c) the bond in the precursor, and (e) brings about the formation of distribution of sites of synthesis of preDa within the various the 9,lD-seeo-steroid, pre vitamin Da (preDa) [19]. Vitamin Da is layers of epidermis and dermis. The time course for the thermal not formed during this photochemical rel;lction, inasmuch as it isomerization of preDa to vitamin D a in vitro and in vivo at is derived solely from previtamin D a (20). PreD is thermally a various temperatures was first determined [26]. Incubation of labile and undergoes a temperature-dependent rearrangement preDa in methanol at O°C for 1 week caused less than 2% of its double-bonds to form the thermally stable 9,l0-seeo­ formation of vitamin Da, whereas, at 25° C, approximately 50% steroid, vitamin Da. A number of investigators have demonstrated that vi.tamin D a can be isolated from animal and human skin after exposure PREVITAMIN D3 to ultraviolet radiation [21-23]. There appeared to be at least ,QC . ,---,"'13"6c---::-:.4:---A-.- -=2-::6-::0---=------, 2 possible mechanisms resulting in cutaneous vitamin-Dol for­ 0 w u mation from the UV radiation: (a) 7-DHC in the skin converts z efficiently and quantitatively directly to vitamin Da, or (b) the 118 « ~02 process in vivo is identical to that seen in vitro, ttlat is, 7-DHC o absorbs a photon of radiation energy and is converted to pre­ en M' «CD 384 vitamin Da. Once formed, preDa would then undergo a thermal 40. isomerization to vitamin Da. In 1969, Rauschkolb et al [21] reported the isolation and identification of vitamin Da from o WAVELENGTH (nm) human cadaver skin. Okano et al (22) and Esvelt, Schnoes, and DeLuca [23] saponified whole rat skin that had been exposed to ultraviolet radiation and prepared lipid extracts in which, after several chromatographic procedures, they also demon­ M/E strated the presence of vitamin D ol . Because saponification was FIG 2. Mass spectrum of previtamin D, isolated fTom rat skin after used for the purification in each case, any thermally labile it was thermally converted to vitamin 0 ". Ultraviolet absorption spec­ intermediate photoproducts such as preDa were isomerized. trum of previtamin D" isolated from the rat skin (insert). July 1981 PHOTOSYNTHESIS OF PREVITAMIN D3 53 of the preD:) cO!1verted to vitamin Do in 48 h1" (Fig 3B). When SUN preD3 was incubated in methanol at 37°C, approximately 50% of preD3 converted to vitamin D3 by 28 hr, and equilibrium was - ~ tt! osb'."o~ "" - reached after 4 days with approximately 80% of preD:J convert­ ing to vitamin D 3. In contrast, in vivo (the sill'face temperature / HO ~ ~ of rat skin was determined with a thermal couple to be 36.5 ± 7- DEHYOROCHOI..ESTEROI.. Hf PREVITAMIN 0 0.5°C, and the dermis, 37.5 ± 0.5°C), 50% of the 1"adiolabeled 3 preD3 that was topically applied 01" generated by exposing [3a- JrSK/N TEMP 3H]7-DHC to UV-B radiation converted to vitamin Da in 18 hr, SKIN and, by 3 days, there was a 95:5 ratio of vitamin D3 to preD3 remaining in the skin (Fig 3B). We next examined the possibility that there was a transport system that would preferentially remove vitamin D3 from the skin, leaving behind preD3 to continue its thermal isomerization to vitamin D j . The binding affinity of the plasma vitamin-D binding protein (an a,-globulin important for the transport of vitamin D :) and its metabolites in the circulation) for preD3 and vitamin D 3 was determined [26]. Figure 3A illustrates that the vitamin-D binding protein has essentially no affinity for preD 3 ~ compared with the affinity for vitamin D:1• The potential phys­ LIVER iologic advantage of this selective binding is summarized in Fig Flc 4. Diagrammatic representation of the formation of previtamin 4. Only after thermal conversion of preDa to vitamin D3 is this D" in the skin during sun exposure and its subsequent thermal conver­ seeo-steroid removed from the skin by binding to the vitamin­ sion to vitamin D", which in turn binds to the plasma vitamin-D binding D binding protein in the capillary bed of the dermis. protein from transport into the circulation. To investigate the location in the skin where preDa is syn­ thesized during sunlight exposure, we used 2 separate tech­ niques that separate layers of epidermis without destroying the LOCATION OF PREVITAMIN ~ integrity of the cells [26]. Surgically obtained hypopigmented A Caucasian skin (from the lower limb), with subcutaneous fat removed, was cut into samples 6.25 cm 2 and separated by either incubation with 20 mg/ml of staphylococcal exfoliatin (a sub­ stance that specifically cleaves the epidermis at the stratum ~S C granulosum/stratum spinosum interface) or immersion in a ~q~SG - HPLC 60°C water bath for 30 sec (this technique cleaves epidermis at the stratum-spinosum/stratum-basale interface) (Fig 5). After SM s B 7 - Dehydrocholesterol ' .. ' .. , _ HPLC A PreD3 . . 0z , J . ". DERMIS 6 40 - " 0'- 0- CD ..., 0 I 020 Staph To)":n 'I" ,- . .,. _.,.- ...... "'N 'I' .!2... 1.0 10 100 1000 8 * NANOGRAMS 100 B / 0 -- '-0 in'l.i.l£ll 36.5-37.5°C

0 / / 80 I / l / .. ~ 0"" 60 / ~ ...... in I'i1ro 25°C z •...... 'A" " " "" ~ / ~ 40 / ~...... 5 ;,-'1 FIC 5. A , Diagrammatic illustration of the separation of the stratum .: corneum and stratum granulosum from the rest of the epidermis and 20 dermis by toxin treatment. B, Diagrammatic illustration of the sepa­

!D Vit ro O°C ration of the skin · into the epidermis minus stratum basale; and the o ___ _t::. ______t::.. ______6 stratum basale and dermis by a heat separation technique. 01234567 DAYS either treatment, but before physical separation of the skil) FIG 3. A, Thermal conversion of preDa to Da as a function of time layers, the stratum-corneum side of the skin samples was ex­ in vitro at O°C (-6 -), 25°C ( . . ·6 · · .), 37°C (-e-), and in vivo at posed to 0.5 J / cm 2 ofUV-radiation with a narrow band centered 36.5- 37.5°C (-0 -) (Each time point represents 2 experiments that at 295 nm with a lO-nm half-band width. Control skin samples were each determined in triplicate. There was exceUent agreement for were kept in an UV -free environment. Immediately after irra­ each of the data points with less than 2% variation). B, Displacement diation, the toxin-treated skin was separated into a top layer of ["H]25-hyd.roxyvitamin D" (["H]25-0H-D:,) from rat D-binding pro­ (stratum corneum + stratum granulosum) a nd the bottom layer tein by vitamin D:.I (Da), previtamin D:, (preD:,), and 7-dehyd.rocholes­ terol (7-DHC) (Each data point represents an average of 2 sepa.rate (stratum spinosum, stratum basale, and dermis), whereas the determinations). Reproduced from Holick et al [26] with permission. heat-treated skin was sepal'ated into a top layer (stratum cor­ (Copyright 1980, American Association for the Advancement of Sci­ neum, stratum granulosum, stratum spinosum) and the bottom ence.) layer (stratum basale and dermis). The basal cells of the stra- 54 HOLICK Vol. 77, No.1 tum basale were collected by mechanically scraping them off amount of vitamin D3 synthesized in the skin. He speculated the dermis (Fig 5) . Contents of all skin layers were confIrmed that heavily pigmented individuals living at or near the equator by histologic examination. The separated layers of skin were were protected from vitamin-D toxicity principally because the extracted with 8% ethyl acetate in n-hexane for 24 hr at -20°C, skin pigment absorbed UV-B photons. He further speculated dried under nitrogen and weighed, and a portion of each sample that as mankind moved north and south of the equator, hypo­ was chromatographed by high-pressure liquid chromatography pigmentation evolved to permit adequate quantities of UV-B in duplicate to determine 7-DHC and preD 3 concentrations. As photons to penetrate the epidermis to permit the synthesis of demonstrated in Table I, even though all of the epidermal adequate amounts of vitamin D 3. strata and the dermis contain 7-DHC, the highest concentration For our investigation of the role of pigmentation, latitude, of 7-DHC per mg of lipid was in the stratum basale. The largest and other factors in the cutaneous synthesis of preD:J, we amount of this sterol per unit surface of skin, however, was exposed hypopigmented and hyperpigm~nted human skin for present in the stratum spinosum. When human hypopigmented various times to simulated solar UV radiation. We then deter­ Caucasian skin was exposed to UV radiation, photosynthesis of mined the photoproducts that resulted from 7-DHC in order to preDa occurred throughout the entire epidermis and to a small study the sequential photochemical events quantitatively, and extent in the dermis, with the highest concentration of preD" in examined the regulator processes, if any, and the role of melanin the stratum spinosum ano. stratum basale. and latitude as modifiers of the production of preD3 in the skin Collectively, these data indicate that the photosynthesis of [30]. Surgically obtained hypopigmented (type III) and hyper­ preD" in the epidermis during exposW'e to the sun is advanta­ pigmented (types V and VI) human skin specimens obtained geous. When hypopigmented Caucasian skin is exposed to sunlight, UV-B radiation penetrates the skin and causes the photochemical conversion of 7-DHC to preD:! throughout the entire epidermis and in the dermis. The regions of the skin that A B potentially have the highest capacity for preD3 formation per unit area are the stratum spinosum and the stratum basale. 1-QHC 7-DHC However, the a mount of preD" that is formed in the various layers will be dependent on the amount of UV -radiation reach­ ing each layer. Immediately upon its formation, the previtamin begins to isomerize by a nonphotochemical rearrangement of its triene system, the rate of which is dictated by the tempera­ w uz ture of the skin at or near the stratum basale, to vitamin 0,1. « CD This process permits the skin to continually synthesize (from a:: o preD;I ) and release vitamin Da into the circulation for up to 2 to Ul 3 days after a single exposure to sunlight. Once vitamin D;l is «CD formed, the vitamin-D binding protein translocates it prefer­ entiall y into the circulation and assures the efficient convel'sion of small quantities .of preDa to vitamin D :l by shifting the previtamin D :J ~ vitamin D:l equilibrium to the right at the time when equilibrium is being approached (Fig 4). RE TENTION TIME (min) FACTORS THAT LIMIT THE CUTANEOUS PHOTOPRODUCTION OF PREVITAMIN Da 100 E In 1922, Hess [27] showed that skin pigmentation in rats \ influenced tRe antirachitic potency of sunlight. This observation 90 i '. Tropical Solar Simulation correlated with the clinical observation that the darker the \ skin, the greater the susceptibility to rickets in childhood, BO \ especially during seasons or in environments in which sunlight was reduced [28]. In 1967, Loomis [29] popularized the theory ~ 70 I u \ ~ that melanin pigmentation not only limited cutaneous vitamin- '0 \ 0 0,1 formation but that it was most imporfant for controlling the 60 \ .... 1iL ~ \ 0 .0""" '6. \ 50 \ .0 ~ . . '1 TABLE 1. The concentration of 7-dehydrocholesterol (7-DHC) and 0 " u 4 0 pre vitamin D:, in the various strata of a. hypopigmented Caucasian I " 2 0 whole shin sample after exposure to 0.5 J/cm of UV radiation" ,..:. 30 p' 7-DHC 7-DHC previ- Skin Strata (ng/rng (ng/6.25 tamin 0 ;, (ng/6.25 "" lipid) em' ) 'X 7 - 0HC ern' ) ~ PreDo Stratum corneum + stratum 180 360 20 granulosum {; T Stratum spit10sum 630 2459 114 Stratum basale 1720 1892 113 Time (J10ur s) Dermis 88 1670 3 FIG 6. High-performance liquid chromatographic profiles of a lipid " The concentrations represent the average of 2 determinations from extr act from the basal cells of sUl'gically obt.ained hypopigmented skin the same thigh skin sample. Analysis of the 7-DHG concentration in that was previously shielded from (A) or exposed to tropical simulated the various 'skin strata from skin samples obtained from different areas solar UV radiation that reaches the Earth at sea level at noon for (B) of the body or from different individuals suggests that the absolute 10 min; (C) ] hI'; and (D) 8 hr. E represents an analysis of the photolysis amount of 7-DHC may vary but that the stratum basale contains the of 7 -dehydrocholesterol (7- DHC) (_ ..... -) in the basal-cell layer and highest concentration of 7-DHC pel' mg of lipid and the stratum the appearance of the photoproducts previtamin 0 " (preD,,) (-e-), spinosum has the highest content of 7-DHC per unit surface area. lumisterol:, (L),j--O -- ), and tachysterol:, (T) (-6 -) with increasing Reproduced with the kind permission of the publisher [26]. (Copyright exposure time. 6 Represents the SEM from 3 determinations. Repro­ 1980, American Association for the Advancement of Science) duced with the kind permission of the publisher [30]. July 1981 PHOTOSYNTHESIS OF PREVITAMIN D.1 55 from differe nt areas of the body with subcutaneous fat removed lumisterol and tachysterol. Exposure times necessar y to maxi­ 2 were cut into samples (6.25 cm ) and immersed in a hot-water mize preD:\ formation in hypopigmented type-III skin when it bath at 60°C for 30 sec. The skin was blotted dry, and the was exposed to simulated solar UV radiation at the equator or stratum-corneum side was exposed at various times to simu­ in Boston are shown in Table II and indicate the effect of lated solar UV-radiation. Conditions were adjusted to approxi­ latitude on preD3 formation. The decrease in UV radiation from mate UV-radiation that reaches the Earth in June at sea level the equator to Boston (due to the increase in the zenith angle at either the equator (0° Latitude) or in Boston (42°,20' North) of the sun) increased t he exposure time required to maximize at noontime. Immediately after irradiation, the skin was sepa­ preD3 formation [3~). rated into its component parts (see Fig 5). Control skin samples Our data suggest that the photochemical conversion ofpreD3 maintained in an UV-free enviJ"Onment for the same period of to lumisterol and tachysterol is the major limiting factor in time were similarly separated. Figure 6 illustrates the repre­ preventing vitamin-D:I intoxication due to a single prolonged sentative chromatographic proflles of lipid extracts of the stra­ expOSUl'e to the sun. Our theory is supported by the fact that tum basale, separated from surgically obtained hypopigmented there aTe no documented cases of vitamin-D toxicity due to human skin samples of type III, that were exposed for various prolonged exposure to the sun. For example, if a hypopigmented times to simulated tropical solar UV-radiation. After 15 min of person and a heavily pigmented person aloe exposed to 3 hr of exposure, the principal photoproduct in the stratum basa le was UV radiation at the equator (Fig 6 and Table II), the amount previtamin D :j • Skin samples subjected to longer exposme time of preD:\ formed is essentially the same, i.e., about 15% of the showed the appearance of 2 additional photoproducts (lumis­ initial 7-DHC content in the epidermis. The major difference is terol and tachysterol), but, as seen in Fig 6-c, -d, -e, tachysterol that the photoproduction of the biologicall y inert photoisomer, formation reached the maximum at 5% of the original 7-DHC lumisterol, in hypopigmented skin is significantly greater than concentration by 1 hI', whereas lumisteJ"OI formation steadily it is in heavily pigmented skin [3D). Melanin competes with 7- increased to 50% by 8 hI". Correlated with the increase in DHC for UV -B photons, and therefore an increase in melanin photoproduct concentration was the decline in 7-DHC to 30% in human skin also increases the time of exposure to UV of its original concentration by 8 hL We next compared t he radiation t hat is needed to maximize preD:1 formation (Table exposure time necessary to maximize preD:I formation in hy­ II). popigmented Type-III and hyperpigmented types-V and VI Therefore, when we aJ°e exposed to minimum amounts of skin. As pigmentation increased 6'om type III to types V and sunlight, our cutaneous stores of 7-DHC convert to preD".

VI, the exposw'e time necessary to maximize preD:1 formation PreD" can either thermally isomerize in the skin to vitamin D :l increased from 30 min to 1 hr and 3 hr, respectively (Table II). 01' photochemically isomerize to lumisterol a nd tachysterol (Fig However, regardless of skin type, once preD:1 maximized and 7). During prolonged exposure to the sun, the synthesis ofpreD.l plateaued at about 15% of the original 7-DHC concentration, reaches a plateau at a concentration of about 10-15% of t he further expOSUl'e to UV radiation produced only increases in original cutaneous 7-DHC concentration, and preD:1 photoiso­ merizes to 2 biologically iner t products, lumisterol and tachys­ terol. Tachysterol formation also plateaus at a concentration of TABLE II. The effect o[ melanin concentration and latitude on the about 5%, whereas lumisterol formation continues to increase [ormation o[ preuitamin D" and lumisterol3 in hum.an shin a[ter with increased exposure to solar UV radiation. An analysis of irradiation with simulated solar UV radiation. the binding affinity of the vitamin-D binding protein for lumis­ % Lumis­ terol and tachysterol has demonstrated that t here is essentiall y Time (h]') of expo­ t.erob fO rilla­ Simulated geographi c sure to simulated so­ tion after 3- Skin type(s) location IaI' UV radiaL io n to hr exposure Ilulximize preD;1 for- of simulated mation solar UV ra- diation Type III" Equator" 0.50-0.75" 40 Type V" Equator 1.00-1.50 20 Type VI" Equator 3.00-3.50 14 Type III" Equator 0.25-0.50 48 HOR Type m" Boston" 0.50-1.00 30 7- DEHYDROCHIXESTERIX " Surgically obtained foresk in specimens from healthy males (26-36 yr old). SKIN " Surgically obtained thigh skin from a 38-yr-old male. c A #WG-305-C Schott filter (Scho tt, Optical Glass Inc., York Ave­ nue, Duryea, PA 18642) was used to simulate noon sunlight at the equator at sea level. Based on the United States Gov ernment Standard (USGS) that skin exposed to sunlight on a cloudless day for an 8- h1' period will be irradiated with approx imately 175 J /cm2 of UV-A and 4 J/cm2 UV-B. BLOW " A #WG-320-B Schott filter was used to simulate Boston sunlight at noon in June at sea level at a ze nith angle of 20° . Based on the USGS DBP report that skin exposed to sunlight on a cloudless day for 8-hr period FIG 7. This is a sc hematic represe ntation of the formation of pre­ will be irradiated with approximately 140 J /cm2 of UV-A and 2.4 J /cm2 vitamin D3 in the skin during ex posure to th e sun, its thermal isomer­ UV-B. ization to vitamin D", wh ich is specifically translocated by the vit;all'lin ­ • Skin samples were exposed to simulated solar UV radiation using D-binding protein (DBP) in to the circulation. During continual elC po­ a 2.5-kw xenon arc lamp with a dichroic mirror and an approp riate sure to the sun, previtamin Do also photoiso merizes to lumisterob and filter. Skin was kept on ice during irradiation to avoid overheating. tachysterola, which are photopl'Oducts that al'e biologically inert (i.e., Because of the high intensity of this ligh t system, ex posure times to they do not stimulate intestinal calcium absorption). Becau e the DBP simulated solar UV radiation were .significa ntly decreased compared has no affinity fOl- lumisterob but has minimal affi ni ty fOJ'ta chystet'ol;" with natural conditions. For example, the amount of time it took to the translocation of these photoisomers into the circulation is negligible deliver 3-3.5 hI' of simulated equatorial solar UV radiation was approx­ and these photoproducts are sloughed off during the natural turnover imately 10 min. Each value represents an average of 3 sepal'ate deter­ of the skin. Because these photoisomers are in a state of quasiphotoe­ minations. This was excellent agreement for each value, with less than quilibrium as soon as previtamin D3sto res al'e depleted, (due to thertnal 10% variation. Reproduced with the kind permission of the publisher iso merization to D,,), exposure of lumisterol and tachysterol to UV [30]. (Copyright 1981, American Association for the Advancement of radiation will promote these isomers to photoisomerize to preD". Re­ Science.) produced with the kind permission of the publisher [30]. 56 HOLICK Vol. 77, No.1 no affinity for lumisterol and minimum affinity for tachysterol, tamin Da in a group of J apanese who went on vacation to the thus making unlikely the translocation of these photoisomers South Sea Islands for 2 weeks. After 2 weeks of intermittent into the circulation (Fig 7) . Therefore, these inert photoprod­ sunbathing, the vitamin-D values had increased by 92%, and ucts would be sloughed off during the natural turnover of the the 25-0H-D values had increased by 45%, whereas the serum skin. Furthermore, because these photoisomers are in a qua­ 1,25-(OHb-D values did not change significantly. Recently, we sistationary state with preD:J, as soon as preDa stores are exposed both vitamin-D-sufficient and vitamin-D-deficient in­ depleted due to their thermal isomerization to vitamin D 3 , dividuals to a quantitative amount of ultraviolet radiation and exposure of cutaneous lumisterol and tachysterol to solar UV determined sequential changes in the serum vitamin-D, 25-0H­ radiation could promote the photo isomerization of these prod­ D, and 1,25-(OHb-D concentrations (J. S. Adams, T. L. Cle­ ucts to preD ~ (Fig 7). Therefore, in order of importance, the mens, and M. F. Holick, unpublished results). In healthy young significant determinants limiting cutaneous previtamin-Da pro­ Caucasian adults whose entire bodies w e~e exposed to 3 minimal duction appeared to be (a) photochemical regulation, (b) pig­ erythema doses (MED) of broad-band UV radiation, the serum mentation, and (c) latitude [30]. vitamin-D values increased by 7- to lO-fold within 24 ill" and then declined to basal values by one week after the initial VITAMIN-D METABOLISM exposure (Fig 8) . In contrast, the serum 25-0H-D concentra­ During the past decade-it has been clearly demonstrated that tions, after a gradual rise, increased to a maximum of approxi­ vitamin Da either ingested in the diet or photosynthesized in mately two-fold by 2 to 3-weeks after exposure (Fig 8). The the skin is biologically inert. It must undergo 2 successive serum 1,25-(OHh -D values also increased by about 20%, al­ hydroxylations, first in the liver to 25-hydroxyvitamin D3 (25- though the values remained within normal range. Surprisingly OH-Da), and then in the kidney to 1a,25-dihydroxyvitamin Da enough, in the patient with vitamin-D defi ciency who had (la,25-(OHb-D3) before it is able to carry out its 2 main phys­ received a total-body exposure to 1 MED of UV radiation, the iologic functions: The stimulation of intestinal calcium absorp­ serum 1,25-(OH)2-D concentrations markedly increased from tion and the mobilization of calcium from the bone [31-34]. 25 pg/ml before irradiation to 260 pg/ ml 6 days after the T herefore, based upon the facts that (a) vitamin D3 is not an exposure, and returned to baseline 3 weeks later. absolute nutritional requirement, inasmuch as it can be synthe­ sized in the epidermis when skin is exposed to adequate PHOTOPRODUCTION OF VITAMIN-D3 METABOLITES amounts of sunlight, and, furthermore, that (b) vitamin D3 must IN THE SKIN be transported to 2 separate organs for metabolism before it The detection of specific hereditary or acquired defects in can carry out its biological effects at distant organs, vitamin Da vitamin-D metabolism has provided new insights into the path­ is considered a' hormone rather than a vitamin. Very little ophysiology of certain disorders of calcium and phosphorus information has been available on the blood levels of vitamin homeostasis and has focused interest on the clinical use of D3 as a result of exposure to sunlight. However, because the chemically synthesized vitamin-D metabolites and analogs for concentration of 25-0H-D 3 (which is the major circulating the treatment of these disorders [31-34]' Although administra­ metaboli te of vitamin D 3 ) in the serum .correlates with dietary tion of the active form of vitamin D 3, 1a,25- (OHh-D3, has intake of vitamin D 3 , many investigators have measured the proved effective in th e treatment of many vitamin-D-resistant circulating levels of this metabolite during a val'iety of environ­ syndromes, such as chronic renal failure, hypoparathyroidism, mental conditions and demonstrated that serum 25-0H-D and vitamin-D-dependent rickets, Type I, occasional hypercal­ levels reflect directly upon the cutaneous syn thesis of vitamin cemia is a problem perhaps related to the high transient local D 3 . Preece et al [35] and Fairney, Fry, and Lipscomb [36] concentrations of the biologically active metabolite in the small reported a marked lowering of 25-0H-D levels in the serum of intestine. Recent studies on the unique features of the skin for submariners and of one Antarctic explorer during sunless pe­ riods, wher~as Haddad and Chyu [37] reported that there is almost a doubling of 25-0H-D levels in the serum of lifeguards 3 med on the beach. There is now strong evidence in both children , 60 and adults that serum 25-0H-D levels vary with the season, E with peak levels occurring in the summer when exposure to the "-co sun and the intensity of UV radiation are maximized, whereas .5- .0 the lowest levels occur in the midwinter when exposure to the Cl sun and the intensity of the UV radiation are minimized. The 20 contribution of the cutaneous photoproduction of vitamin Da in preventing osteomalacia and rickets relative to the dietary intake of vitamin D2 or vitamin D3 h as been a subject of much E 60 examination. In the British Isles, where supplementation of "-co food with vitamin D is not practiced, the cutaneous photosyn­ .5- thesis of preD is quite important. In the United States, how­ Cl '0 3 :x: ever, dietary supplementation is widely practiced, and the role 0 0{) 20 of cutaneous preD3 synthesis for the maintenance of calcium N homeostasis is less clear. Recently, Neer et al [38] reported on the. concentrations of 25-0H-D in the serum of groups of adult E males who either worked primarily indoors or primarily out­ "-co doors in 6 cities across the United States, determined at inter­ 2 vals of 6 weeks after the summer and winter solstic'es and soon Cl -!:' after the vernal and autumnal equinoxes. Although all the :x: 2 serum levels of 25-0H-D were within the normal range of I 20 between 15 and 80 ng/ml at every season, the blood 25-0H-D 0{) N_ levels were 10 to 15 ng/ ml lower in the indoor workers. The conclusion from this study was that the significant determinants o 2 4 6 8 10 12 14 21 of blood 25-0H-D levels in normal adult men are: geography TIME (days) > individual variation> occupational exposure to sun> season FIG 8. Serum vitamin D, 25-0H-D, and la,25-(OHh-D levels in a of the year. 24 -yr-old Caucasian fe male before and after receiving a total body Morita et al [39] reported the serum concentrations of vi- exposure to 3 minimal erythemal doses of UV radiation. July 1981 PHOTOSYNTHESIS OF PREVITAMIN Da 57 controlling t he synthesis of vitamin D3 suggested that the skin UV-8 may also serve as a site for the application or synthesis, 01' both, of vitamin-D metabolites and would have pharmacologic ad­ vantages a nd be preferable to the effects achieved by the oral administration of vitamin-D3 metabolites. To test this hypoth­ esis, we chemically synthesized raruolabeled 10:,25-dihydroxy-7- dehydrocholesterol (l0:,25-(OHh-7-DHC) and topically applied 1•. 2 5 - (OH)2 -7 - DHC 1•• 25-(OH)2- PRE - D3 it to vitamin-D-deficient rats to determine whether expOSUl"e to UV radiation would convert t his hydroxylated 7-DHC precur­ jfSKIN TEMP sor to the preD:J derivative (Fig 9). Lipid extracts from the skin SKIN "'-' of rats that received a topical application of radiolabeled 10:,25- OH (OHh-7-DHC either with 01' without UV radiation demon­ strated that only the animals that received UV radiation 1• . 25- (OH)2 - D3 showed t he conversion of 10:,25-(OHh-7-DHC to 10:,25-dihy- I p~

6 A SMALL C SKIN INTESTINE ~-.. 1o<,25-(OH1 -7-0HC 5 :i ~ NO UV , 2 NO UV DBP Ia. 25- (OH)2D3 tf'Q 4 4

:>< 3 3 / "'- :::< SMALL INTESTINE BONE a. 2 2 £:) F IG 11. D iagrammatic representation of the photochemical conver­ sion of l a,25-(OHh-7-DHC to la,25- (OH)"-preD, in the skin and t he subsequent thermal conversion of the previtamin to 1. (t,25-(OHh-D,,; once formed, lLf,25-(OH)z-D:J is bound to the plasma vitamin-D-binding 6 B SMALL D protein (DBP) for transport through the circulation to its tru'get tissues. SKIN 5 INTESTINE Reproduced with the kind permission of the publisher [40]. ~:i A + + UV '"'Q 4 uv 4 1 ~ . 25-(0~ 1 2 - PRE 03 droxyprevitamin D3 (10:,25-(OH)2-preD3). We further demon­ :>< 3 lo:.25;(OH12 -03 3 strated that cutaneous 10:,25-(OHh-preD:J thermally isomerized :::< 1o<.25-(OHl -7-DHC to 10:,25-(OH)2-D:J, which t hen entered the circulation and a. 2 t 2 2 £:) localized in its target tissues: the small intestine and bone [40). We next measUl"ed intestinal calcium transport and serum calcium responses in vitamin-D-deficient anephric rats that o 20 40 60 80 100 120 140 0 20 40 60 80 100 120 140 received a topical dose of 10:,25-(OHb-7-DHC and were either FRACTION NUMBER (2 ml) FRACTION NUMBER (2 ml) UV -irradiated or shielded. Rats t hat received either a topical F IG 9. Sephadex LH-20 chl"omatographic profiles of lipid extracts of application of l a,25-(OHh-7-DHC without radiation therapy or tissu es from rats that received a topical application of [24-"H]-lLf,25- a topical application of 50 Ilg of vitamin D:] showed no elevation (OHh-7-DHC. (A) Skin and (C ) small-intestine extracts from rats that in intestinal calcium transport or serum calcium when com­ were exposed to ultraviolet radiation, and (B) skin and (D) sma U­ pared with the control group that received only a topica l intestine extracts from rats that were exposed to ultraviolet photother­ apy. Reproduced with the kind permission of the publisher [40]. application of 95% ethanol. However, the anephric rats t hat received a topical dose of la,25-(OHh-7-DHC and UV-photo­ therapy demonstrated a marked stimulation in intestinal cal­ A cium transport and a subsequent parallel elevation in serum calcium [40]. To study t he long-term biological consequences .of topical application of l a,25- (OHh-7-DHC combined with UV-photo­ therapy, vitamin-D-deficient rats were given a topical applica­

tion of 1 Ilg of lcr,25-(OH)2-preD3 (the photoproduct of l a,25- (OHh-7-DHC) to determine serum calcium concentration and intestinal calcium transport a nd to compare the values in sim­ il ar rats given (a) topical application of a vehicle (95% EtOH), (b) topical application of 1 Ilg of lcr,25-(OH)2-D:] , or (c) an oral dose of 1 Ilg of 10:,25-(OHh-D". Animals that had received an oral dose of la,25-(OHh-D:J had a very rapid elevation in 80 intestinal calcium absorption and a parallel rise in serum cal­ o'! cium (Fig 10) that reached a maximum at 12 hr and decreased ~ 7.0 rapidly to control values by 2 weeks. Rats that received a ~ 60 topical dose of 1 Ilg of 10:,25-(OH)2-D;] or 1lY.,25 -(OHh-preD" ~ showed gradual increases in intestinal calcium absorption that ("j 50 were sustained for 14 to 21 days, respectively, before returning :2 to control values. Therefore, the proven historical success of ii! 40 w en phototherapy for the treatment of rickets along with our exper­ T iments that demonstrate the fe asibility of using t he skin as the o 14 21 28 35 site for the synthesis and absorption of vitamin D metabolites DAYS AFTER DOSING (Fig 11) offers a potentially attractive a nd easily- ma naged al­ FIG 10. Intestinal calcium transport response (A) and serum calcium ternative for t he treatment of vitamin-D-resistant disorders. levels (B) in vitamin-D-defi cient rats that received either a topical application of 20 fll of 95% EtOH (-e-), topical appli cation of 1 flg of CONCLUSION la,25-(OHh-preD" (-6-), topical application of I /lg of l Lf,25- (OHh­ D a (-.&-), or an ora l dose of 1 flg of lLt,25-(OHh-D" (-0 -). Repro­ R ickets evolved into a devastating human affli ction because duced with the kind permission of the publisher [40]. most of the scientists a nd physicians in the 19th century were 58 HOLICK Vol. 77, No.1 unwilling to appreciate the healthful effects of exposure to 20. Schl\ltmann JLM, Pot J, Havinga E: An investigation into the natural sunlight. Finally, in the 1920's, it was clearly establish ed interconversion of precalciferol and calciferol and analogous co m­ pounds. Rec Trav Chim Pays-Bas 83:1173-1184,1964 that vitamin D a was photosynthesized in the skin a nd that this 21. Rauschkolb EW, Winston D, Fenimore DC, Bilack HS, Fabre LF: h ormone was responsible for the maintenance of normal skele­ Identification of vitamin D:J in human skin. J Invest Dermatol tal growth a nd developme nt. U ntil recently, h owever, little was 53:289-293, 1969 known about t h e sequential events involved in t h e cutaneous 22. Okano T, Yasumura M, Mizuno K, Kobayashi T: Photochemical conversion of 7-dehydrocholesterol into vitamin D3 in rat skins. synth esis of vita min D :] and about t h e factors t h at r egulated J NutI' Sci Vitaminol 23:165-168, 1977 this process. It is now establish ed t h at when skin is initially 23. Esvelt RP, Schnoes HK, DeLuca HF: Vitamin D ;] from rat skins exposed to solar r a diation, the stores of epidermal 7-DHC irradiated in vitro with ultraviolet light. Arch Biochem Biophys 188:282-286, 1978 convert to preD3. PreDa can eith er t h ermally isom erize to 24. Holick MF, Frommer JE, McNeill SC, Richtand NM, Henley JF, vitamin D 3 or undergo a photochemical isomerization th at Potts JT Jr: Photometabolism of 7-dehydrocholesterol to previ­ limits the cutaneous synth esis of preD" during prolonged ex­ tamin D" in skin. Biochem Biophys Res Commun 76:107-114, posure to the sun. The development of (a) n ew chromato­ 1977 graphic procedures t h at permit a direct a nalysis of cutaneous 25. Holick MF, Richtand NM, McNeill SC, Holick SA, Frommer JE, Henley JW, Potts JT Jr: Isolation and identification of previ­ 7-DHC and its photoproducts, (b) sensitive assays for vitamin tamin D" from the skin of rats exposed to ultraviolet irradiation. D 3 and its metabolites, a nd (c) new a pproach es for treating Biochemistry 18: 1003-1008, 1979 vitamin-D-resistan t syndrom es with the cuta neous synth esis 26. Holick MF, MacLaughlin JA, Clark MB, Holick SA, Potts JT Jr., a nd absorption of vitamin-D3 metabolites should stimulate new Anderson RR, Blank IH, Parrish JA: Photosynthesis of previ­ interest in th e basic a nd practical aspects of the cutaneous tamin D3 in human skin and the physiologic consequences. Sci­ ence 210:203-205, 1980 photobiology of vitamin D a. 27. Hess AF: New aspects of the rickets problem. JAMA 78: 1177-1183, ~~ . REFERENCES 28. Levinsohn S: Rickets in the Negro: Effect of treatment with ultra­ violet rays. Am J Dis Child 34:955- 966, 1927 1. Foote JA: Evidence of rickets prior to 1650. Am J Dis Child 34:443- 29. Loomis F: Skin-pigment regulation of vitamin D biosynthesis in 452, 1927 man. Science 157:501-506, 1967 2. Griffenhagen G: A brief history of nutritional diseases. II. Rickets. 30. Holick MF, MacLaughlin JA, Doppelt SH: Factors that influence Bull Nat! Inst NutI' 2:9, 1952 the cutaneous photosynthesis of previta min Do. Science, 211:590- 3. Guthrie D: In, A History of Medicine. Lippincott, Philadelphia, 593, 1981 1964, p 69 31. Holick MF, Potts JT Jr: Vitamin D, chapt 351, Harrison's Principles 4. Sniadecki J: Cited by W Mozo lowski : Jedrzej Sniadecki (1768-1883) of Internal Medicine. KJ Isselbacher, RD Adams, E Braunwald, on the cure of rickets. Nature 143:121, 1939 RG Petersdorf, JD Wilson. New York, McGraw-Hill Book Com­ 5. Owen I: Geogj'aphical distribution of rickets, acute and subacute pany, 9th ed, 1980, pp 1843-1849 rheumatism, chorea, cancer, and urinary calculus in the British 32. Haussler MR, McCain T A: Basic and clinical concepts related to Islands. Br Med J 1:113-116,1889 vitamin D metabolism and action. N E ngl J Med 297:974-983, 6. Palm T A: T he geographic distribution and etiology of rickets. 1041-1050, 1977 Practitioner 45:270-279,321-342, 1890 33. DeLuca HF, Schnoes HK: Metabolism and mechanism of action of 7. Huldschinsky K: Heilung von Rachitis durch Kunstliche H ohen­ vitamin D. Annu Rev Biochem 45:631-642, 1976 sonne. Deutsche Med WochenschT xiv:712- 713, 1919 34 . Norman AW, Henry H: 1,25-Dihydroxycholecalciferol-A hormon­ 8. Hess AF, Unger LJ: CUTe of infantile rickets by sunlight. JAMA 77: ally active form of vitamin D3. Recent Prog Horm Res 30:431- 39, 1921 480, 1974 9. Mellanby E. An experimental in vestigation on rickets. Lancet 1: 35. Preece MA, Tomlinson S, Ribot CA, Pietrek J, Korn HT, Davies 407-412, 1919 DM, Ford JA, Dunnigan MG, O'Riordan JLH: Studies of vitamin 10. Hess AF, Weinstock M: Antirachitic properties imparted to inert D deficiency in man. Quart J Med 44:575-589, 1975 fluids and green vegetables by ultraviolet irradiation. J Bioi 36. Fairney A, Fry J, Lipscomb A: The effect of darkness on vitamin D Chem 62:301-313, 1924 in adults. Postgrad Med J 55:248- 250, 1979 11. Steenbock H: The induction of growth-promoting and calcifying 37. Haddad JG, Chyu KJ: Competitive protein binding radioassay for properties in a ration exposed to light. Science 60:224- 225, 1924 25-hydroxycholecalciferol. J Clin Endocrinol Metab 33:992-996, 12. Hess AF:"I'he antirachitic activation of foods and of cholesterol by 1971 ultraviolet irradiation. JAMS 84:1910-1913, 1925 38. NeeI' R, Clark M, Friedman V, Belsey R, Sweeney M, Buonchris­ 13. Powers GF, Park EA, Shipley PG, McCollum EV, Simmonds N: tiani J , Potts JT Jr: Environmental and nutritional influences on T he prevention of rickets in the rat by means of radiation with plasma 25- hydroxyvitamin D concentration and calcium metab­ the mercury vapor quartz lamp. Proc Soc Exp Bioi Med 19:120- olism in man, Vitamin D: Biochemical, Chemical and Clinical 121, 192 L Aspects Related to Calcium Metabolism (Proceedings of the 14. Fieser LD, Fieser M: Steroids, chap 4, Vitamin D. Reinhold, NY, Third Workshop on Vitamin D). Edited by A W Norman, K 1959, pp 90-168 . Schaefer, JW Coburn, HF DeLuca, D Fraser, HG Grigoleit, Dv 15. Havinga E: Vitamin D, example and challenge. Experientia 29: Herrath. New York, Berlin, Walter de Gruyter, 1977, pp 595-606 1181-1193, 1973 39. Morita R, Dokoh S, Fukunaga M, Yamamoto I, Roizuka 1: Effects 16. Crawfoot D, Dunitz JD: Structure of calciferol. Nature 162:608- of sunlight exposure on blood concentration of vitamin D deriv­ 610, 1948 atives in healthy Japanese adults, Vitamin D: Basic Research 17. Steenbock H, Black A: The production of growth-promoting and and its Clinical Application (Proceedings of the Fourth Workshop calcifying properties in a ration by exposure to ul traviolet light. on Vitamin D). Edited by A W Norman, K Schaefer, Dv Herrath, J Bioi Chem 61:408-422, 1924 . HG Grigoleit, JW Coburn, HF DeLuca, EB Mawer, T Suda. New 18. Hess AF, Weinstock M, Helman DF: The antirachi tic value of York, Berlin, Walter de Gruyter, 1979, pp 165-168 . irradiated phytosterol and cholesterol. J Bioi Chem 63:305- 308, 40. Holick MF, Uskokovic M, Henley JW, MacLaughlin J, Holick SA, 1925 Potts JT JR: The photoproduction in skin of lex,25-hydroxyvi­ L9. Velluz L, Amiard G, Petit A: Le precalciferol-ses relations tamin Do: An approach to therapy of vitamin-D-resistant syn­ 'd'equilibre avec Ie calciferol. Bull Soc Chim Fr 16:501-508, 1949 dromes. N E ngl J Med 303:349-354, 1980