Measurement of histidinohydroxylysinonorleucine and hydroxyproline in skin collagen by reversed-phase high-performance liquid chromatography after 9-fluorenylmethyl chloroformate labeling

A novel, highly sensitive method to quantify histidinohydroxylysinonorleucine (HHL), a trifunctional type of cross-link in skin collagen, was developed. HHL in skin hydrolysates labelled with 9-fluorenylmethyl chloroformate (FMOC-Cl) was separated by reversed-phase high-performance liquid chromatography. Mass spectrometric analysis revealed that two FMOCs were bound to two primary amino acid residues, histidine and hydroxylysine, but not to lysine residue in one HHL molecule. Hydroxyproline 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 of hydroxyproline from 1 to 50 pmol. A 6-mm punch-biopsied human skin sample 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 examine the aging process or the change of HHL content in skin collagens of various pathologic states. (C) 1997 Academic Press.