Pre-Fractionation with Cation Exchanger for Determination of Intermolecular Crosslinks, Pyridinoline, and Pentosidine, in Hydrolysate

Abstract

A 3-hydroxypyridinium crosslink of mature collagen, pyridinoline (Pyr), is an essential substance in connective tissue except skin. A previous study of ours showed that the amount of Pyr in a hydrolysate of human articular cartilage could be used as an endogenous standard for the fluorometric analysis of pentosidine (Pen) [J. Biochem. 110, 714-718, 1991]. In the HPLC analysis of Pyr, partition chromatography utilizing CF-1 cellulose was usually employed to improve the resolution. However, this treatment seemed to be useless for Pen analysis. In this study, the usefulness of a cation exchanger, SP-Sephadex C-25 in the prefractionation of a hydrolysate is shown. The recovery of standards (mean ± SD) using CF-1 cellulose (n = 6) was 75.3 ± 3.7 % for Pyr and 11.5 ± 1.5 % for Pen, while that of standards using SP-Sephadex C-25 (n = 6) was 75.4 ± 2.3 % for Pyr and 71.1 ± 5.8 % for Pen. Using this method, age-related changes in the content of Pen in urinary hydrolysates of normal humans (n = 60) utilizing fluorometry on HPLC under isocratic conditions were determined. This method is easy and useful for the study of intermolecular crosslinks, including Pyr and Pen in hydrolysates of tissues and humors.