A collection method and high-sensitivity enzyme immunoassay for sweat pyridinoline and deoxypyridinoline cross-links

Abstract

Background: Collagen cross-link molecules such as pyridinoline (PYD), deoxypyridinoline (DPD), and N-terminal cross-linked peptides (NTX) have been measured in urine as indices of bone resorption. However, very little is known regarding the excretion of pyridinolines into other biological fluids. We report a collection device, normalizing analyte, and high-sensitivity immunoassay for quantitative analysis of free pyridinoline cross-links in sweat. Methods: Flame atomic emission and ion-selective electrode techniques were used to measure potassium as a sweat volume marker. The Pyrilinks immunoassay for urine free pyridinolines was optimized to increase sensitivity for measurements in sweat. The precision, accuracy, and detection limit of this assay were characterized. To assess values and variability of sweat pyridinolines in human subjects, a nonocclusive skin patch was used to collect sweat samples from a reference group and from a mixed group experiencing accelerated bone resorption, postmenopausal women and men receiving gonadotropin-releasing hormone for prostate cancer. Results: The immunoassay intra- and interassay variations were ≤10% and <16%, respectively, with a detection limit of 309 pmol/L. Linearity upon dilution and analytical recovery ranged from 93% to 109% and 85% to 122%, respectively. Sweat PYD values normalized to potassium output yielded a weekly intraindividual biological variability of 14.7%. The mean increase in the population experiencing increased bone resorption vs the reference group was 36% (P <0.05) for sweat PYD/K vs 23-40% (P <0.05) for urinary PYD/Cr, DPD/Cr, and NTX/Cr. Conclusion: We conclude that this new platform sweat collection technology and PYD immunoassay show potential as an indicator of bone resorption.