A. Colwell

Factors affecting the assay of urinary 3-hydroxy pyridinium crosslinks of collagen as markers of bone resorption

Abstract. The measurement of the 3–OH pyridinium compounds, pyridinoline (Pyr) and deoxypyridinoline (Dpyr), in urine by high performance liquid chromato‐graphy is potentially useful in clinical studies, since they are specific biochemical markers of bone resorption. The aims of the present study were to improve assay performance and optimize sample collection. An isocratic high performance liquid chromatogram (HPLC) separation with baseline resolution was accomplished within 4 min using heptafluorobutyric acid as an ion‐pair. The sample preparation for HPLC, using CFl cellulose, produced uncontami‐nated samples with a recovery higher than 90% for both crosslinks. An elastin‐derived material, tentatively identified as isodesmosine (Ides), was also tested and proved to be a suitable internal standard. Use of this standard improved assay precision. The effect of an oral gelatin load on the excretion of Pyr and Dyr was investigated. The creatinine corrected excretion of Pyr and Dpyr was unchanged over a 6 h period, in contrast to the 10–fold increase in the excretion of urinary hydroxyproline with a peak 2–4 h after ingestion. In 20 postmenopausal women, 2 h fasting morning urine results correlated with results from 24–h urine collections Dpyr/Cr (r = 0.70, n= 20). There was a day‐to‐day variation of 26% in adults studied for 10 days.

Biochemical markers of bone turnover in girls during puberty

OBJECTIVES Bone turnover and the rate of bone growth increase dramatically during puberty. A number of new assays for the estimation of bone resorption and formation rates have been developed over recent years, and puberty acts as a convenient model for evaluation of these measurements. The aim of this study was to explore the interrelationships between pubertal development, biochemical markers of bone turnover, Insulin‐like growth factor I and oestradiol in healthy pubertal girls.

Biochemical Markers of Bone Resorption Compared with Estimates of Bone Resorption from Radiotracer Kinetic Studies in Osteoporosis

The pyridinium cross‐links of collagen pyridinoline (Pyd) and deoxypyridinoline (Dpd) are released during bone resorption and are neither metabolized nor absorbed from the diet. The aim of this study was to validate their use in osteoporosis. We studied 19 women with osteoporosis and estimated the bone resorption rate from a combined calcium balance/kinetics technique without (R) and with partial (RH) and “complete” (Res) correction for long‐term exchange. The strongest correlation was observed between the bone‐specific marker (Dpd) and with complete correction for long‐term exchange (Res) (r = 0.71, p < 0.001). The intercept was not different from zero, suggesting that bone was the major source for Dpd. The crude ratio of Dpd to Res in the 19 women was 54.5; but the regression coefficient relating Dpd as the dependent variable to Res was 31.8 (95% CI 15.6–48.0), which was higher, but not significantly, than the ratio between Dpd and calcium (16.4) in 10 bone samples (cortical and trabecular bone). The weakest correlations between a biochemical marker and a kinetic index were those between hydroxyproline (a nonspecific marker of bone resorption) and R or RH. Treatment with hormone replacement therapy (HRT) or HRT and parathyroid hormone peptide 1–38 in seven women over 1 year resulted in similar percent changes in the biochemical markers and estimates of bone resorption. We conclude that the measurement of Dpd provides a reasonably accurate assessment of bone resorption in osteoporosis, and in the context of several repeat 24‐h collections of urine offers measurement precision that is similar to that obtainable with methods depending on the use of radioisotopic tracers and the assessment of metabolic calcium balance.

Effect of menopause and hormone replacement therapy on urinary excretion of pyridinium cross-links: a longitudinal and cross-sectional study.

OBJECTIVE To study longitudinally the effect of the menopause and hormone replacement therapy on the new markers of bone resorption: urinary excretion of pyridinoline and deoxypyridinoline.

Bone mineral density and bone turnover in spinal osteoarthrosis.

OBJECTIVES--To determine whether there was a generalised increase in bone mineral density (BMD) in spinal osteoarthrosis (OA), and to determine the mechanism of this possible protection against osteoporosis as assessed by biochemical markers of bone turnover. METHODS--We studied 375 women (ages 50 to 85) from a population based group. Spinal OA was defined from radiographs as the presence of degenerative changes affecting intervertebral or facet joints. BMD of the lumbar spine (LS), femoral neck (FN) and total body (TB) was measured by dual energy x ray absorptiometry (Lunar DPX). Bone turnover rates were estimated from measurement of biochemical markers of bone formation and resorption (urine deoxypyridinoline (Dpyr) and serum bone specific alkaline phosphatase (BAP)). RESULTS--BMD at each site was greater in the women with spinal OA (mean increase in LS-BMD 7.9%, 95% confidence interval (CI) 1.0 to 15.1; TB-BMD 8.4%, 95% CI 1.9 to 9.7; FN-BMD 6.4%, 95% CI 0.3 to 12.6). Twenty four hour urinary excretion of Dpyr, corrected for TB bone mineral content, and serum BAP were 19% lower in the women with spinal OA (95% CI for Dpyr 4.3 to 31.9%; for BAP 6.3 to 32.0%). CONCLUSIONS--Spinal OA is associated with a generalised increase in BMD and a decreased rate of bone turnover. This suggests that the protective effect of spinal OA against osteoporosis may be mediated by decreased bone turnover.

To determine the effects of ultraviolet light, natural light and ionizing radiation on pyridinium cross-links in bone and urine using high-performance liquid chromatography.

The aims of the study were to characterize the denaturation of urinary free and conjugated pyridinoline (Pyr) and deoxypyridinoline (Dpyr) on exposure to ultraviolet (UV) and natural light at different pH levels and to study the effects of X‐ and γ‐irradiation on Pyr and Dpyr in urine and in the mineralized and non‐mineralized compartments of human bone. Urine samples from six normal subjects, adjusted to pH 3.0, 7.0 and 9.0, were exposed to UV light for up to 3 days. Urine collections (2 mL and 24 h) from three subjects, pH adjusted to 1.0, 2.0, 3.0, 4.0 and 5.0, were exposed to natural light for up to 1 day. Urine samples and bone slices from seven human cadaveric femurs were irradiated with increasing doses of X‐rays (0–100 Gy) and high‐dose γ‐radiation (28kGy). Mineralized and non‐mineralized bone were separated using a modification of a published method employing heat denaturation followed by trypsin hydrolysis and analysed for Pyr, Dpyr and hydroxyproline (Hypro). The rate of UV photolysis of urinary Pyr and Dpyr increased with pH and was faster in the free fraction (after 3 days’ exposure: free Pyr and Dpyr at pH 7.0 vs. 9.0, P<0.05, conjugated pH 3.0 vs. 9.0, P<0.05). Exposure to natural light for 3 h did not significantly decrease urinary Pyr and Dpyr in either sample collections, but levels were reduced in the 2‐mL aliquots after exposure for 1 day (P<0.05). X‐irradiation of urine and bone did not affect Pyr and Dpyr. Pyr content was similar in both bone compartments (Pyr/Hypro=0.12±0.004), but Dpyr was higher in the non‐mineralized compartment (Dpyr/Hypro=0.047±0.002 vs. 0.038±0.002, P<0.001). UV light and γ‐irradiation result in denaturation of pyridinium cross‐links in urine. These cross‐links are present in both the mineralized and non‐mineralized bone compartments but are not affected by the doses of γ‐irradiation that denature these cross‐links in urine.

Urinary collagen crosslink excretion: a better index of bone resorption than hydroxyproline in Paget's disease of bone?

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 Pagets 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 significantly correlated with those of OHP (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 dimethyl-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 Pagets disease of bone. They appear to confer no advantage, however, over the traditional determination of the urinary excretion of OHP, in the monitoring of response to treatment of such patients with inhibitors of bone resorption. Further studies are required to establish the value of these new biochemical indices of bone resorption, possibly in more subtle disorders of bone metabolism such as osteoporosis.

The effect of rat parathyroid hormone (1-34) infusion on urinary 3-hydroxypyridinium cross-link excretion in the rat.

The utility of measurement of the urinary excretion of the 3-hydroxypyridinium cross-links, pyridinoline (Pyr) and deoxypyridinoline (Dpyr) as indices of bone resorption in rats was investigated. Total Pyr and Dpyr excretion were measured in young rats treated by s.c. infusion with rat parathyroid hormone (1-34) (PTH) at 22-30 micrograms/kg/day or with diluent (controls) for 14 days. During infusion, average urinary excretion of both cross-links was significantly higher in PTH rats (Pyr: 11.77 +/- 0.44 nmol/day), Dpyr: 15.81 +/- 0.95 nmol/day) than in controls (Pyr: 10.17 +/- 0.35 nmol/day, Dpyr: 12.03 +/- 0.67 nmol/day). These results were consistent with the magnitude of the expected increase in bone resorption rate with this dose of PTH. The method appears to provide a sensitive measure of bone resorption for in vivo bone studies in rats.

Parathyroid gland hormones in the skeletal development of the ovine foetus: the effect of parathyroidectomy with calcium and phosphate infusion

It has been confirmed that the foetal parathyroid glands are important in development and that thyroparathyroidectomy (TXPTX) of the ovine foetus with thyroxine (T4) replacement leads to hypocalcaemia, retarded skeletal development, depressed calcification and rickets, relative to thyroidectomy plus T4 replacement. Histomorphometric and biochemical (urinary excretion of deoxypyridinoline) indices of bone resorption are also reduced. However, skeletal calcification can be restored to normal by long-term infusion of the TXPTX foetuses with phosphate and calcium sufficient to normalise the plasma Ca2+ x Pi ion product. Nevertheless, depressed resorption, reduced osteoblast numbers and delayed development persisted. The evidence suggests that the abnormally low number of resorption cavities and osteoclasts may result from the reduction in circulatory parathyroid-hormone-related protein consequent upon the removal of the foetal parathyroid glands and that this hypercalcaemic factor has a direct effect upon the process of resorption and primary trabecular remodelling of the foetal skeleton.

The effect of age on bone collagen turnover as assessed by pyridinium crosslinks and procollagen I C-terminal peptide

ConclusionBone collagen turnover increases with age up to the ninth decade in women. This increase in bone turnover may result from secondary hyperparathyroidism and it could play a role in the pathogenesis of age-related bone loss.