Urinary collagen crosslink excretion: a better index of bone resorption than hydroxyproline in...
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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.
Transcript of Urinary collagen crosslink excretion: a better index of bone resorption than hydroxyproline in...
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.
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