SINGLE-COLUMN HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC FLUORESCENCE DETECTION OF IMMATURE, MATURE, AND SENESCENT CROSSLINKS OF COLLAGEN

A high-performance liquid chromatographic-fluorescence detection metho d of reducible (immature) and nonreducible (mature and senescent) cros s-links of collagen was established without the use of a radioisotope and preliminary fractionation step. This method used a gradient elutio n procedure of sodium citrate buffer containing 7% ethanol. The reduci ble cross-links (dihydroxylysinonorleucine, hydroxylysinonorleucine, a nd lysinonorleucine) and nonreducible cross-link (histidinohydroxylysi nonorleucine) were detected by O-phthalaldehyde derivatization with th e postcolumn method, whereas other nonreducible cross-links (pyridinol ine, deoxypyridinoline, and pentosidine) were detected by natural fluo rescence. The linear ranges of contents of the O-phthalaldehyde deriva tive 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 mt of collag en was adequate for duplicate analyses of the reducible and nonreducib le cross-links. An equivalent of 0.25 mg of hydrolyzed collagen could be analyzed by this HPLC system. Using this system, age-related change s in the cross-links of collagen from human connective tissues were al so investigated. (C) 1997 Academic Press.