Bone and Mineral, 22 (1993) 1-8 1 © 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved. 0169-6009/93/$06.00

BAM 00576

Urinary of bone

collagen crosslink excretion: a better index resorption than hydroxyproline in Paget's

disease of bone?

N.A.T. Hamdy a, S.E. Papapoulos a, A. Colwell b, R. Eastell b, R.G.G. Russell b

aClinical Investigation Unit, Department of Endocrinology and Metabolic Diseases, University Hospital Leiden, The Netherlands and bDepartment of Human Metabolism and Clinical Biochemistry, University of

Sheffield Medical School, UK

(Received 20 March 1992) (Accepted 2 February 1993)

Summary

The 24 h urinary excretion of the collagen degradation products pyridinoline (Pyr) and deoxypyridinoline (Dpyr) have been proposed as specific and quantitative indices of bone resorption. We compared the value of the urinary excretion of Pyr and Dpyr to that of hydroxyproline (OHP) in 11 patients with Paget's disease of bone before and during treatment with inhibitors of bone resorption, during admission to a metabolic ward and maintenance on a gelatin-free diet. Pyr and Dpyr excretion rates were significant- ly correlated with those ofOHP (r = 0.81 and 0.77, respectively, P < 0.001; n = 106). The rate and degree of suppression of bone resorption were monitored in 6 of the patients similarly treated with intravenous dimethyI-APD at a dose of 4 mg/day for 10 days, by daily measurements of the 24 h urinary excretion of Pyr, Dpyr and OHP. Treatment with dimethyl-APD resulted in a decrease in the three indices of bone resorption. The percentage change from baseline values was similar for the three indices, although changes in Dpyr appeared to follow more closely those of OHP. Our findings suggest that Pyr and Dpyr are useful and specific indices of bone resorption in Paget's disease of bone. They appear to confer no advantage, however, over the traditional determination of the urinary excretion of OHP, in the monitor- ing of response to treatment of such patients with inhibitors of bone resorption. Further studies are re- quired to establish the value of these new biochemical indices of bone resorption, possibly in more subtle disorders of bone metabolism such as osteoporosis.

Key words: Bisphosphonates; Indices bone resorption; Paget's disease of bone; Urinary hydroxyproline; Urinary crosslinks

Introduction

Over the past few years, there has been a growing interest in the development of new, specific biochemical indices of bone turnover, in the diagnosis and follow-up of met-

Correspondence to: Dr N.A.T. Hamdy, Clinical Investigation Unit, Department of Endocrinology and Metabolic Diseases, University Hospital Leiden, Rijnsburgerweg 10, 2333 AA, Leiden, The Netherlands.

abolic bone disease. The major collagen of bone, type I collagen, is synthesised in- tracellularly as a large procollagen molecule, which spontaneously assembles into fibrils following extracellular cleavage. These collagen fibrils only acquire their full tensile strength, however, when they acquire crosslinks [1]. Pyridinoline (Pyr) and deoxypyridinoline (Dpyr) are non-reducible intermolecular crosslinks between adja- cent mature collagen chains, present only in extracellular collagen fibrils and releas- ed in the circulation during the degradation of bone and cartilage [2-4]. Dpyr is located almost exclusively in type I collagen of bone, whilst Pyr is also a major crosslink in cartilage where it links type II and type IV collagen [4,5]. Unlike the case with hydroxyproline (OHP), Pyr and Dpyr are not found in immature or newly syn- thesised collagen [4]. The three products of collagen degradation are excreted in the urine where they can be measured [6-9]. OHP is the most widely used biochemical index of bone resorption in metabolic bone disease [10-12]. A main draw-back with its use, however, is that dietary restriction of gelatin is necessary for a more accurate interpretation of results, in contrast to the case with Pyr and Dpyr which are not affected by dietary gelatin [13].

It has been proposed that the urinary excretion of Pyr and Dpyr may be a more sensitive index of collagen degradation, and hence bone resorption, than hydroxy- proline in metabolic bone diseases, including Paget's disease of bone [14,15]. For this reason, we have undertaken the following study to assess the relationship between the urinary excretion of Dpyr and Pyr, and that of OHP, as biochemical indices of bone resorption, in patients with Paget's disease of bone. A second aim of the study was to assess the value of urinary Pyr and Dpyr, compared to that of OHP, for moni- toring the rate and degree of suppression of bone resorption during treatment of ac- tive Paget's disease of bone with bisphosphonates, which are known to suppress bone resorption [16].

Patients and Methods

Eleven patients with biochemical, radiological, and scintigraphic evidence for Paget's disease of bone were studied. All patients were admitted to a metabolic ward and maintained on a constant gelatin-free diet.

Urine was collected for the determination of the 24 h urinary excretion of creatinine, OHP, Pyr, and Dpyr. OHP was measured using the Prockop and Uden- friend method [6] and Pyr and Dpyr were measured by fluorimetry and reverse phase HPLC using a previously described method [8,17]. The crosslinks Pyr and Dpyr were quantitated using an external standard prepared from canine femur using CF1 cellulose chromatography after demineralisation and acid hydrolysis. The standard was further purified using HPLC and exchange chromatography, and calibrated against a standard kindly provided by Dr S. Robins [8]. The intraassay coefficients of variation were 8% and 10.4% and the interassay variation were 8.5% and 13.4% for Pyr and Dpyr, respectively. To test the validity of the crosslinks assay at high urinary concentrations, high concentration samples (n = 9) were run through the assay at the usual concentration and at a 1:4 dilution. We found a mean undiluted to diluted ratio of 1:3.98 (SD 0.99), strongly indicating a linear response of the flu- orescence detector for both.

The relationship between the urinary excretion of OHP, and Pyr and Dpyr was

assessed in 106 twenty-four hour urine samples collected from the eleven patients studied, before and during treatment with inhibitors of bone resorption. Pamidro- hate (APD) was given intravenously at a dose of 20 mg/day for 10 days in one patient; the new bisphosphonate 3-dimethylamino-l-hydroxypropylidene-l,l-bis- phosphonate, dimethyl APD [18], was given intravenously at a dose of 4 and 8 mg/ day for 10 days in 6 and 3 patients, respectively; and the last patient was treated with human calcitonin given subcutaneously at a dose of 0.5 mg for 9 days.

Six patients treated uniformly with dimethyl-APD at a dose of 4 mg/day for 10 days had daily estimations of the 24 h urinary excretion of OHP, Pyr and Dpyr. The 24 h excretion of the three indices was expressed relative to urinary creatinine to correct for differences in excretion of the latter, and the rate and degree of sup- pression of the 24 h urinary excretion of the three indices was compared over the duration of treatment.

Results

The samples assessed before and during treatment of Paget's disease of bone (n = 106) represented a wide range of bone resorption with the 24 h excretion of OHP varying from 67 to 1657 t~mol/1. The 24 h urinary excretion of Pyr and Dpyr varied from 108.4 to 2408.7 nmol and from 19.5 to 680.8 nmol, respectively and correlated significantly with that of OHP excretion (r = 0.81 and 0.77, respectively, after log- transformation of the data, P < 0.001; Fig. 1).

During treatment with inhibitors of bone resorption, all three biochemical indices of bone resorption exhibited similar urinary excretion patterns. This is exemplified in two individual patients with similar activity of Paget's disease treated with intrave- nous dimethyl-APD (d-APD)and subcutaneous human calcitonin, respectively (Fig. 2). During the course of treatment with d-APD, effective suppression of bone resorption resulted in parallel decreases in the 24 h urinary excretion of OHP, Pyr and Dpyr. The decrease in Dpyr followed more closely the expected decrease in OHP than did that of Pyr (Fig. 2, upper panel). The patient treated with subcutaneous cal- citonin demonstrated a poor response as evidenced by the persistently raised urinary excretion of OHP, mirrored by the urinary excretion rates of both Pyr and Dpyr. It is of note that during the course of monitoring, little variability was demonstrated in the urinary excretion rates of the three indices of bone resorption (Fig. 2, lower panel).

In the group of 6 patients uniformly treated with dimethyl-APD, there was a signi- ficant decrease in the three indices of bone resorption, the rate and degree of sup- pression of which, as evidenced by percentage change from baseline values, were similar for the three indices studied. The changes in Dpyr appeared to follow more closely the changes in OHP than did those of Pyr (Fig. 3).

Discussion

We have chosen to study patients with Paget's disease of bone as this disease represents an ideal model to evaluate potential biochemical indices of bone metabo- lism. Thus, in this disorder, bone turnover is increased (with closely coupled resorp-

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Fig. 1. The 24 h urinary excretion of the collagen degradation products pyridinoline and deox- ypyridinoline correlated significantly with that of hydroxyproline excretion (r = 0.81 and 0.77, respective- ly, P < 0.001) in patients with Paget's disease of bone, before and during treatment with inhibitors of

bone resorption (n = 106). Data were log-transformed before statistical analysis.

tion and formation), the response to treatment with bisphosphonates is well described, and the value of known indices of bone metabolism well recognised [11,12,19]. The urinary excretion of hydroxyproline is a widely used, practical, and in our hands, most reliable index of bone collagen degradation and hence bone resorption in patients with Paget's disease of bone [11,19-21]. Our aims were to study the value of urinary collagen crosslinks as indices of bone turnover over a wide

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Fig. 2. The 24 h urinary excretion of hydroxyproline (OHP: 0), and the collagen degradation products pyridinoline (Pyr: D), and deoxypyridinoline (Dpyr: O) are shown in two patients with active Paget's disease of bone and similar initial increased bone resorption, during treatment with intravenous dimethyl- APD (upper panel), or with subcutaneous calcitonin (lower panel). Parallel decreases in the urinary excre- tion of the three indices are demonstrated with effective suppression of bone resorption. Decreases in Dpyr followed more closely those of OHP than did those of Pyr (upper panel). In the patient poorly responding to treatment, little variability is noted in the excretion rates of the three indices over the period

of monitoring (lower panel). Data are shown on a log scale.

range of bone turnover states, and to establish whether these new biochemical in- dices offered any advantage over the traditionally used OHP in monitoring the re- sponse to treatment of Paget's disease of bone with inhibitors of bone resorption.

In keeping with published data, our findings suggest that the urinary excretion of the collagen degradation products Pyr and Dpyr are indeed useful and specific in- dices of bone resorption in Paget's disease of bone. In this disorder, they were also useful in monitoring the rate and degree of suppression of bone resorption as a re- sponse to treatment with bisphosphonates, with deoxypyridinoline appearing to be

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Fig. 3. Rate and degree of suppression of the 24 h urinary excretion of hydroxyproline (OHP), pyridinoline (Pyr) and deoxypyridinoline (Dpyr), corrected for creatinine excretion and expressed as per- centage change from a basal value (time 0), in 6 patients with active Paget's disease of bone uniformly treated with intravenous dimethyl-APD at a dose of 4 mg/day for 10 days. During treatment, changes in

Dpyr appeared to be more closely related to changes in OHP than did those of Pyr.

more sensitive than pyridinoline in that latter respect, probably as the former is almost exclusively found in bone collagen.

Our data are at odds, however, with a recent publication suggesting that crosslinks are more sensitive than OHP in the monitoring of Paget's disease activity during treatment with inhibitors of bone resorption [14]. The reason for this discrepancy in findings is not clear, although we used a different method for measuring OHP in our study [6].

In the light of our findings, we feel that the determination of the urinary excretion of the collagen degradation products Pyr and Dpyr in the monitoring of the response to treatment with inhibitors of bone resorption in patients with Paget's disease of bone, does not offer any advantage over the traditional determination of the urinary excretion of hydroxyproline.

There is no doubt however, that Pyr and Dpyr are specific biochemical indices of bone resorption which have the advantage of not being affected by diet. The mea- surement of urinary crosslinks may prove to be of greater value as biochemical in- dices of bone resorption, when the interpretation of OHP measurements is more controversial such as in rheumatological disorders (specially Dpyr), or in cases with subtle, albeit significant changes in bone turnover as in osteoporosis [9,17,22-25]. Several studies are in progress to establish whether Pyr and Dpyr are of value in the diagnosis and management of disorders of bone metabolism other than Paget's disease of bone.

Acknowledgements

We are grateful to the Dutch Organisation for Scientific Research (NWO 900-541- 191) and to the Arthritis and Rheumatism Council, UK, for support of this work.

References

1 Prockop DJ, Kivirikko KI, Tuderman L, Guzman NA. The biosynthesis of collagen and its dis- orders. N Engl J Med 1979;301:13-23.

2 Fujimoto D, Moriguchi T, Ishida T, Hayashi H. The structure of pyridinoline, a collagen crosslink. Biochem Biophys Res Commun 1978;84:52-57.

3 Robins SP. Crosslinking of collagen: isolation, structural characterisation and glycosylation of pyridinoline. Biochem J 1983;215:167-173.

4 Eyre DR, Koob T J, Van Ness KP. Quantitation of hydroxypyridinium crosslinks in collagen by high-performance liquid chromatography. Anal Biochem 1984;137:380-388.

5 Robins SP, Duncan A. Pyridinium crosslinks of bone collagen and their location in peptides isolated from rat femur. Biochim Biophys Acta 1987;914:233-239.

6 Prockop DJ, Udenfriend S. A specific method for the analysis of hydroxyproline in tissues and urine. Anal Biochem 1960;1:228-239.

7 Gunja-Smith Z, Boucek RJ. Collagen crosslink components in human urine. Biochem J 1981; 197:759-762.

8 Black D, Duncan A, Robins SP. Quantitative analysis of the pyridinium crosslinks of collagen in urine using ion-paired reversed-phased high-performance liquid chromatography. Anal Biocbem 1988;169:197-203.

9 Black D, Maharani M, Sturrock RD, Robins SP. Urinary excretion of hydroxypyridinium crosslinks of collagen in patients with rheumatoid arthritis. Ann Rheum Dis 1989;48:641-644.

10 Kivirikko KI. Urinary excretion of hydroxyproline in health and disease. Int Rev Connect Tissue Res 1970;5:93-163.

11 Russell RGG, Beard DJ, Cameron EC, Douglas DL, Forrest ARW, Guilland-Cumming D, Pater- son AD, Poser J, Preston C J, Milford-Ward A, Woodhead S, Kanis JA. Biochemical markers of bone turnover in Paget's disease. Bone 1981;3:255-263.

12 Harinck HI J, Bijvoet OLM, Vellenga CJLR, Blanksma H J, Frijlink WB. Relation between signs and symptoms in Paget's disease of bone. Q J Med 1986;226:133-151.

13 Colwell A, Eastell R, Assiri AMA, Russell RGG. Effect of diet on deoxypyridinoline excretion. In: Christianssen C, Overgaard K, eds. Osteoporosis. Aalborg, Denmark: Osteopress APS, 1990; 590-591.

14 Uebelhart D, Gineyts EC, Chapuy MC, Delmas PD. Urinary excretion of pyridinium crosslinks: a new marker of bone resorption in metabolic bone disease. Bone Miner 1990;8:87-96.

15 Robins SP, Black D, Paterson CR, Reid DM, Duncan A, Seibel MJ. Evaluation of urinary hydrox- ypyridinium crosslink measurements as resorption markers in metabolic bone diseases. Eur J Clin Invest 1991;21:310-315.

16 Fleisch H. Bisphosphonates: Mechanism of action and clinical applications. In: Peck WA, ed. Bone and mineral research, Amsterdam: Excerpta Medica, 1983;1:319-357.

17 Hassager C, Colwell A, Assiri AMA, Eastell R, Russell RGG, Christiansen C. Effect of menopause and hormone replacement therapy on urinary excretion of pyridinium crosslinks: a longitudinal and cross-sectional study. Clin Endocrinol 1992;37:45-50.

18 Papapoulos SE, Hoekman K, Lowik CWGM, Vermeij P, Bijvoet OLM. Application of an in vitro model and a clinical protocol in the assessment of the potency of a new bisphosphonate. J Bone Miner Res 1989;4:775-781.

19 Kanis JA. Pathophysiology and treatment of Paget's disease of bone. London: Martin Dunitz, 1991. 20 Harinck HI J, Bijvoet OLM, Blanksma H J, Dahlinghaus-Nienhuys PJ. Efficaceous management

with aminobisphosphonate (APD) in Paget's disease of bone. Clin Orthop 1987;217:79-98. 21 Harinck HIJ, Papapoulos SE, Blanksma HJ, Moolenaar AJ, Vermeij P, Bijvoet OLM. Paget's

disease of bone: early and late responses to three different modes of treatment with aminohydroxy- propylidene bisphosphonate (APD). Br Med J 295:1301-1305.

22 Black D, Marabani M, Sturrock RD, Robins SP, Urinary excretion of the hydroxypyridinium crosslinks of collagen in patients with rheumatoid arthritis, Ann Rheum Dis 1989;48:641-644.

23 Seibel M J, Duncan A, Robins SP. Urinary hydroxy-pyridinium crosslinks provide indices of carti- lage and bone involvement in arthritic diseases. J Rheumatol 1989;16:964-970.

24 Eastell R, Colwell A, Assiri AMA, Lufkin EG, Russell RGG, Riggs BL. Prediction of response to estrogen therapy using urine deoxypyridinoline. J Bone Miner Res 1990;5:s275.

25 Delmas PD, Schlemmer A, Gineyts E, Riis B, Christiansen C. Urinary excretion of pyridinoline crosslinks correlates with bone turnover measured on iliac crest biopsy in patients with vertebral os- teoporosis. J Bone Miner Res 1991;6:639-644.

26 Deacon AD, Hulme P, Hesp R, Green JR, Tellez M, Reeve J. Estimation of whole body bone re- sorption rate: a comparison of urinary total hydroxyproline excretion with two radioisotopic tracer methods in osteoporosis. Clin Chim Acta 1987;166:297-306.