MEASUREMENT OF HISTIDINOHYDROXYLYSINONORLEUCINE AND HYDROXYPROLINE INSKIN COLLAGEN BY REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY AFTER 9-FLUORENYLMETHYL CHLOROFORMATE LABELING

A novel, highly sensitive method to quantify histidinohydroxylysinonor leucine (HHL), a trifunctional type of cross-link in skin collagen, wa s developed. HHL in skin hydrolysates labelled with 9-fluorenylmethyl chloroformate (FMOC-Cl) was separated by reversed-phase high-performan ce liquid chromatography. Mass spectrometric analysis revealed that tw o FMOCs were bound to two primary amino acid residues, histidine and h ydroxylysine, but not to lysine residue in one HHL molecule. Hydroxypr oline was simultaneously measured to express the molar ratio of HHL to collagen. The detection range of HHL was from 1 to 10 pmol and that o f hydroxyproline from 1 to 50 pmol. A 6-mm punch-biopsied human skin s ample contained 0.40 to 0.69 mol of HHL per one molecule of collagen. This sensitive method is useful as it is rapid and can be used to exam ine the aging process or the change of HHL content in skin collagens o f various pathologic states. (C) 1997 Academic Press.