Urinary excretion of hydroxylysine and its glycosides as an index of collagen degradation. S M Krane, … , S R Pinnell, F R Singer J Clin Invest. 1977;59(5):819-827. https://doi.org/10.1172/JCI108704. Research Article Urimary excretion of hydroxyprolin (Hyp) is one index of total collagen degradation, from all sources. Since some of the Hyp released from collagen may be further metabolized before it is excreted, other markers are necessary to measure collagen breakdown. Excretion of the glycosides of hydroxylysine (Hyl), glucosyl galactosyl hydroxylysine (Hy1[Gl)cGa1]), and galactosyl hydroxylysine (Hyl[Ga)]), more accurately reflects collagen metabolism since these products occur in specificratios in different tissue collagens and are themselves metabolized only to a minor degree. The ratios of total Hy1/Hyp and Hyl(GlcGal)/Hyl(Ga1) were measured in the urine of norma. subjects and of patients with Paget's disease of bone, hyperphosphatasia, and extensive thermal burns. In patients with extensive thermal burns the pattern of urinary Hy1 and its glycosides was consistent with degradation of collagen in dermis and fascia. When bone collagen degradation was dominant, the pattern of urinary metabolites reflected that source. Pagetic bone collagen has an amino acid composition similar to normal bone and Hy1(G1cGa1/Hyl(G1) of 0.396-0.743,vs. normal of 0.474+/-0.088. In untreated patients with severe Paget's disease of bone or hyperphosphatasia (urinary Hyp greater than 2.0 micronmol/mg creatinine) urinary Hyl/Hyp averaged 0.052+/-0.042 (0.042+/-0.009 in normal bone) and Hy1(G1cGa1)/Hy1(Ga1) 0.601+/- 0.017 (0.47+/-0.009 in normal bone). When bone resorption was decreased sufficiently with calcitonin or disodium etidronate in these patients, both the urinary ratios of Hy1/Hyp and Hy1(G1cGa1)/Hyl(Gal) rose. In […] Find the latest version: https://jci.me/108704/pdf Urinary Excretion of Hydroxylysine and its Glycosides as an Index of Collagen Degradation STEPHEN M. KRANE, FRED G. KANTROWITZ, MICHAEL BYRNE, SHELDON R. PINNELL, and FREDERICK R. SINGER From the Department of Medicine, Harvard Medical School and Medical Services (Arthritis and Endocrine Units), Massachusetts General Hospital, Boston, Massachuisetts 02114 A B S T R A C T Urinary excretion of hydroxyproline sorption was decreased stufficiently with calcitonin or (Hyp) is one index of total collagen degradation, disodiuim etidronate in these patients, both the urinary from all sources. Since some of the Hyp released ratios of Hyl/Hyp and Hyl(GlcGal)/Hyl(Gal) rose. In from collagen may be further metabolized before it is normal subjects treated with calcitonin and excreting excreted, other markers are necessary to measure col- relatively little Hyp, the ratio of Hyl/Hyp approached lagen breakdown. Excretion of the glycosides of 0.7 and Hyl(GlycGal)/Hyl(Gal) approached 3.5. These hydroxylysine (Hyl), glucosyl galactosyl hydroxylysine increased ratios reveal the existence of a source of (Hyl[GlcGal ]), and galactosyl hydroxylysine (Hyl- collagen breakdown other than skin or bone. The first [Gal]), more accurately reflects collagen metabolism subcomponent of complement, Cl(q, which has colla- since these products occur in specific ratios in dif- gen-like sequences, relatively high amounts of Hyl, ferent tissue collagens and are themselves metabolized and most of the glycosylated Hyl as Hyl(GlcGal), only to a minor degree. could be the source of these metabolites. The ratios of total Hyl/Hyp and Hyl(GlcGal)/Hyl- (Gal) were measured in the urine of normal subjects INTRODUCTION and of patients with Paget's disease of bone, hyper- phosphatasia, and extensive thermal burns. In patients Collagens contain uinique, modified amino acids, the with extensive thermal bums the pattern of urinary most abundant of which is 4-hydroxyproline (Hyp).2 Hyl and its glycosides was consistent with degrada- Measurement of urinary excretion of total Hyp (free tion of collagen in dermis and fascia. When bone Hyp plus Hyp-containing peptides) has been used collagen degradation was dominant, the pattern of extensively as an index of collagen degradation (1). urinary metabolites reflected that source. Pagetic bone It is not possible to obtain quantitative estimations collagen has an amino acid composition similar to of collagen breakdown by this measurement since most normal bone and Hyl(GlcGal)/Hyl(Gal) of 0.396-0.743, of the free Hyp is further metabolized to carbon dioxide vs. normal of 0.474+0.088. In untreated patients and other products not incluided in the Hyp determina- with severe Paget's disease of bone or hyperphos- tion and only a portion appears in the urine. Woessner phatasia (urinary Hyp > 2.0 umol/mg creatinine) found that less than 15% of the Hyp of degraded urinary Hyl/Hyp averaged 0.052±0.002 (0.042+0.009 collagen appears in the urine of the pospartum rat in normal bone) and Hyl(GlcGal)/Hyl(Gal) 0.601 (2). Weiss and Klein (3) determined that when collagen +0.017 (0.47+0.009 in normal bone). When bone re- peptides were administered to rats, 75% were metab- olized to respiratory carbon dioxide and 25% excreted. This is publication no. 724 of the Robert W. Lovett In their studies of a patient with deficiency in Hyp Memorial Group for the Study of Diseases Causing Deformi- oxidase (hydroxyprolinemia), Efron et al. (4) found that ties. only 20% of the urinary Hyp was peptide bound Dr. Kautrowitz' present address is Department of Medicine, whereas 80% was free. An additional deficiency in Beth Israel Hospital, Boston, Mass. 02215. Dr. Pinnell's present address is Division of Dermatology, Department of the use of measurement of Hyp excretion to assess Medicine, Duke University Medical Center, Durham, N. C. 27710. Dr. Singer's present address is Department of Medi- I Abbreviations used in this paper: EHDP, disoditum cine, University of Southem Califomia School of Medicine, etidronate; Gal, galactosyl; Hyl, hydroxylysine; Hyp, 4- Los Angeles, Calif. 90033. hydroxyproline; Hyl(Gal),,8- 1-galactosyl-0-hydroxylysine; Received for publication 4 June 1976 and in revised form (Hyl[GlcGal]),a-1-2-glucosyl-galactosyl-0-hydroxylysine; MRC, 29 December 1976. Medical Research Council; SCT, salmon calcitonin. The Journal of Clinical Investigation Volume 59 May 1977 819-827 819 collagen degradation is that its estimation provides vals. 24-h urine samples were collected from four patients no information concerning the source of collagen since with bums covering an estimated 50-88% of body surface. Multiple urine samples from two subjects with hereditary its content varies little from one tissue collagen to hyperphosphatasia were supplied through the courtesy of another. Dr. Melvin Horwith (New York Hospital-Cornell Medical Hydroxylysine (Hyl) is another amino acid unique to Center, New York). 12-h specimens were collected before and collagens and proteins containing collagen-like during human calcitonin therapy in one of these. Sam- is not ples of bone from pagetic and normal subjects were ob- sequences. Like Hyp, Hyl reutilized for collagen tained at time of hip surgery for pagetic coxopathy or at biosynthesis and although it is considerably less necropsy. Fascia and skin were obtained at necropsy from abundant than Hyp, it is a potential marker for col- two additional subjects who did not have obvious disease lagen metabolism. A variable proportion of Hyl resi- of collagenous tissues. dues in collagen are glycosylated as 38-1-galactosyl- 0-hydroxylysine (Hyl[Gal]) and some of these residues Preparation of samples may be glucosylated as a-1-2-glucosyl-galactosyl-0- Bone. Fragments ofnormal and pagetic bone were cleaned hydroxylysine (Hyl[GlcGal]) (5, 6). The relative pro- and stripped of periosteum. Specimens were demineralized portion and total content of glycosylated Hyl are dif- with three changes of 0.5 M disodium EDTA in 0.1 M Tris- HCI, pH 8.0, 20 ml/g of tissue, over a period of 3 days, at 50C ferent for different collagens. For example the ratio with constant agitation by means of a magnetic stirrer (7). The Hyl(GlcGal)/Hyl(Gal) is 0.47±0.09 in human bone, but samples were rinsed with distilled water and freeze-dried. No 2.06±0.47 in human skin (7). Cunningham et al. (5) Hyl or Hyp was found on amino acid analysis of acid and Kakimoto and Akazawa (8) identified these Hyl hydrolysates of the supematant solutions. glycosides in normal human urine and Segrest and Skin. Fat and hair were stripped by dissection and the Cunningham (9) that skin placed in 2 M NaBr at 20°C for 12 h, allowing the suggested since they were not epidermis to be peeled away from the dermis (12, 13). metabolized to the same extent as Hyp, they could The dermis was rinsed with distilled water and freeze- serve as more quantitative markers for collagen break- dried. down as well as provide information as to the source Fascia. Any muscle was stripped off by dissection and the of the collagen metabolites. fascia rinsed with distilled water and freeze-dried. Analysis of total Hyl and Hyp. Samples of bone, skin, In the present study we have measured the urinary and fascia (approximately 8 mg dry weight) and 1-5 ml excretion of Hyp, Hyl, and Hyl glycosides in normal equivalents of freeze-dried urine were hydrolyzed under subjects and patients with accelerated collagen break- nitrogen in sealed tubes in constant boiling HCI at 108°C down, particularly in disorders of the skeleton, to for 20 h. The samples were then dried in a desiccator containing solid NaOH under vacuum. Dried hydrolysates determine relative contributions of different col- were quantitatively transferred with 0.1 N HCI to 5-ml lagenous tissues to the urinary pool of collagen degra- volumetric flasks and brought to final volume.