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Previous studies have shown that immunoassay of urinary NTx (cross-linked N-telopeptides of type I collagen) provides a responsive index of human bone resorption. Here we report by a sensitive immunoassay that NTx is present in serum and is suppressed appropriately in patients with Paget disease of bone by bisphosphonate antiresorptive therapy. The monoclonal antibody (1H11) developed against urinary NTx was applied in a sensitive chemiluminescence format. Results for human serum and urine showed parallel inhibition curves. The NTx concentrations in paired serum and urine samples from individual patients correlated well when urinary concentrations were normalized to creatinine concentrations (in premenopausal and postmenopausal women and Paget disease patients, r = 0.90, n = 60). The percentage of NTx suppression from baseline values for Paget disease patients on bisphosphonate therapy was similar for serum and urine. Blood samples drawn from bone marrow at the site of Pagetic lesions in three patients with active disease had as much as 10-fold higher concentrations of NTx than did peripheral blood samples drawn at the same time. The latter finding is consistent with other evidence showing that immunoreactive NTx originates directly during the proteolytic cleavage of bone collagen by osteoclasts rather than, e.g., by degradative processes occurring later in the liver and kidney.

We have compared the analytical performance and biological variability of three commercially available bone resorption assays: Pyrilinks-D, Osteomark, and CrossLaps, for the measurement of urinary free deoxypyridinoline (Dpd), cross-linked N-telopeptides of type I collagen (NTx), and linear C-telopeptides of type I collagen (CTx), respectively. The intraassay and interassay CVs for precision of the Dpd and NTx assays were < 10% for analyte concentrations greater than the second calibrator (i.e., 3 nmol/L Dpd or 30 nmol bone collagen equivalents/L NTx). The CTx assay demonstrated poor precision for analyte concentration lower than the third calibrator (i.e., 200 micrograms/L). The NTx assay exhibited nonlinear recovery for sample dilutions prepared in buffer; however, this nonlinear recovery could be corrected for sample dilutions made in urine at a low analyte concentration. Supplement recoveries of each of the three assays were within 100% +/- 10% on average. All three analytes showed stability through five freeze-thaw cycles. The mean day-to-day variations were 16% for Dpd, and 23% for both NTx and CTx. Similar diurnal rhythm was observed for all three assays on average, with the peak in the early morning and the nadir in the afternoon. Mean amplitude of the diurnal variation was 37% for Dpd and NTx, and 57% for CTx. Variations within the reference intervals for a healthy premenopausal population were 28% for Dpd, 57% for NTx, and 56% for CTx. Pyrilinks-D has demonstrated analytical precision and accuracy equal or superior to Osteomark and CrossLaps in all areas. Dpd exhibits the least biological variability day-to-day, within individuals across the diurnal cycle, and within a healthy premenopausal population.

We present a coated-tube RIA that is useful for assessment of bone resorption. The assay uses a monoclonal antibody raised against a linear 8-amino-acid sequence (EKAHDGGR) derived from the C-telopeptides of type I collagen. Within-run and total CVs were 4.4% and 5.3-6.2%, respectively, at concentrations of 1-7 mg/L (n = 4-20). Analytical recovery was 98% +/- 8% and dilution 97% +/- 7%. Values obtained in a group of 36 premenopausal women were 227 +/- 89.6 mg/mol creatinine. In a group of 141 postmenopausal women, the values obtained were 429 +/- 225 mg/mol creatinine, a highly significant increase of 89% (P <0.001) over the premenopausal value. In a double-blind placebo-controlled clinical study of these postmenopausal women receiving five different doses of a bisphosphonate, a significant decrease of RIA-measured C-telopeptide values was seen in all bisphosphonate-treated groups, after just 3 months. Values in urine samples from postmenopausal women assayed with the RIA (gamma) and the CrossLaps(TM) ELISA (x) agreed well: slope = 0.98 (95% confidence interval, 0.94-1.01), intercept = 0.34 (0.25-0.43) mg/L, and Sylx = 0.93 mg/L (n = 678). We conclude that this RIA represents a valuable tool for assessing bone resorption.

Urinary excretion of the pyridinium crosslinks pyridinoline (Pyr) and deoxypyridinoline (Dpyr) is used as a biochemical marker of bone resorption. The present study was undertaken to determine the long-term stability of these compounds in stored urine, using the HPLC method. Systematic investigation of their chemical stability in urine demonstrated that both the free and conjugated forms of Pyr and Dpyr are extremely stable: No significant changes were observed after 6 weeks at -20 degrees C storage (e.g., free Pyr 9.6 +/- 1.2 mumol/mol creatinine (before) and 10.6 +/- 3.2 (after); free Dpyr 2.3 +/- 0.2 mumol/mol creatinine (before) and 2.5 +/- 1.2 (after)). These results predict stability of urines stored for 10-20 years at -20 degrees C in the dark. Also, freezing and thawing as many as 10 times had no effect on the concentrations of the crosslinks. Study of the stability of the excretion pattern in healthy women showed substantially higher variations in excretions of free and total Dpyr (18% and 13%, respectively) than of Pyr (10% for both forms).

An enzyme-linked immunosorbent assay (ELISA) for measuring type I collagen degradation products in urine < 3 h was evaluated. The measuring range was 0.5-10.5 mg/L with a detection limit of 0.2 mg/L. Within-run and total CVs were 5.3% and 6.6%, respectively. Analytical recovery averaged 100%. The mean (+/- SD) concentrations in urine samples from a healthy premenopausal population (n = 102) were 250 +/- 110 mg/mol creatinine (Cr). A group of healthy postmenopausal women (n = 410) gave a mean value of 416 +/- 189 mg/mol Cr. Values obtained in the ELISA correlated well (r = 0.83) to HPLC values for the established bone resorption marker deoxypyridinoline (n = 214), slightly better than the correlation to hydroxyproline measurements (r = 0.78, n = 421). We conclude that the assay described here presents a useful tool for further elucidating the importance of type I collagen degradation products in urine.