histidinohydroxylysinonorleucine and pentosidine

A high-performance liquid chromatographic-fluorescence detection method of reducible (immature) and nonreducible (mature and senescent) cross-links of collagen was established without the use of a radioisotope and preliminary fractionation step. This method used a gradient elution procedure of sodium citrate buffer containing 7% ethanol. The reducible cross-links (dihydroxylysinonorleucine, hydroxylysinonorleucine, and lysinonorleucine) and nonreducible cross-link (histidinohydroxylysinonorleucine) were detected by O-phthalaldehyde derivatization with the postcolumn method, whereas other nonreducible cross-links (pyridinoline, deoxypyridinoline, and pentosidine) were detected by natural fluorescence. The linear ranges of contents of the O-phthalaldehyde derivative cross-links and the natural fluorescent nonreducible cross-links were 20-600, 5-500 (pyridinoline, deoxypyridinoline), and 0.2-20 pmol (pentosidine), respectively. Tissue containing 1-2 mg dry wt of collagen was adequate for duplicate analyses of the reducible and nonreducible cross-links. An equivalent of 0.25 mg of hydrolyzed collagen could be analyzed by this HPLC system. Using this system, age-related changes in the cross-links of collagen from human connective tissues were also investigated.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Aging; Amino Acids; Arginine; Cartilage, Articular; Child; Child, Preschool; Chromatography, High Pressure Liquid; Collagen; Cross-Linking Reagents; Diaphyses; Dipeptides; Femur; Histidine; Humans; Indicators and Reagents; Infant; Lysine; Male; Middle Aged; Sensitivity and Specificity; Skin; Spectrometry, Fluorescence