Collagen reduction by sodium borohydride: Effects of reconstitution, maturation and lathyrism

Abstract

This chapter emphasizes on the methods for detecting and quantifying the mature hydroxypyridinium cross-links, but also describes the procedures for quantifying them together with the borohydride-reducible cross-linking residues in the same sample of collagen. To measure cross-linking amino acids in a collagen hydrolyzate using a conventional amino acid analyzer with ninhydrin detection, the bulk amino acids must be largely removed to avoid plugging the reaction coil. Tissue is thoroughly reacted with sodium borohydride to convert all intermediate cross-linking residues to their reduced, acid-stable forms. More vigorous reduction conditions may be used than when preparing profiles of tritium-labeled cross-links using NaB3H4 and direct amino acid analysis. This chapter presents the direct quantitation of hydroxypyridinium cross-links in tissue hydrolyzates by HPLC and fluorescence detection. The following method based on reverse-phase high-performance liquid chromatography can resolve and quantify the HP and LP forms of hydroxypyridinium residue down to 1 pmol directly in hydrolyzates of whole connective tissue, without any preliminary clean-up or enrichment step.