Multiple molecular forms of pyridinolines cross‐links excreted in human urine evaluated by chromatographic and immunoassay methods

The measurement of the collagen cross-links, hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP), excreted in urine either in free or peptide-bound forms represents the most extensively investigated biochemical marker of bone collagen degradation. We studied the urinary molecular forms of pyridinolines after separation in free and peptide-linked fractions by chromatography and serial dialysis. The pyridinoline amounts of molecular species (free, 10,000 D) were evaluated by high performance liquid chromatography (HPLC) as well as with the two newly introduced enzyme-linked immunosorbent assay (ELISA) methods for determination of free pyridinolines (collagen Pyrilinks™ and collagen Pyrilinks™ -D). The variability of urinary pyridinoline forms were studied in healthy adult control subjects (n = 10, 38.4 ± 7.5) years), in adolescents (n = 10, 16 ± 3.3 years), and in elderly subjects with vitamin D insufficiency (n = 10,87.3 ± 4.3 years). Free and peptide-conjugated pyridinolines with MW 10,000 D) represent a low percentage of total pyridinolines (about 5%), in the elderly a significantly greater amount (15–20%, p < 0.01) is measured. Performance characteristics of the new Pyrilinks-D kit, which uses a monoclonal antibody able to recognize only free LP, were also studied. This immunoassay is convenient in terms of reproducibility, accuracy, precision, and sensitivity. The correlation between HPLC and immunoassay yielded a high r value (0.90, p < 0.0001). Additionally, the antibody bound preferentially to free LP, and the other molecular species reacted very poorly. The specific free LP measurement with this antibody can be used to assess the higher bone resorption in elderly patients with vitamin D insufficiency (8.2 ± 3.0 nmol/mmol Cr) than in healthy adults (2.4 ± 2.1 nmol/mmol Cr, p = 0.0001) as well as by HPLC (16.2 ± 7.8 nmol/mmol Cr in elderly and 4.5 ± 4 nmol/mmol Cr in healthy adults, p = 0.0001). However, the significant changes in excretion of molecular forms of pyridinolines must be taken into consideration when bone resorption is measured by different techniques.