Identification of glutathionyl-3-hydroxykynurenine glucoside as a novel fluorophore associated with aging of the human lens

A novel fluorophore was isolated from human lenses using high performance l iquid chromatography (HPLC), The new fluorophore was well separated from 3- hydroxykynurenine glucoside (3-OHKG) and its deaminated isoform, 4-(2-amino -3-hydroxyphenyl)-4-oxobutanoic acid O-glucoside, which are known UV filter compounds. The new compound exhibited UV absorbance maxima at 260 and 365 nm, was fluorescent (Ex(360 nm)/Em(500 nm)), and increased in concentration with age, Further analysis of the purified compound by microbore HPLC with in-line electrospray ionization mass spectrometry revealed a molecular mas s of 676 Da, This mass corresponds to that of an adduct of GSH with a deami nated form of 3-OHKG, This adduct was synthesized using 3-OHKG: and GSH as starting materials. The synthetic glutathionyl-3-hydroxykynurenine glucosid e (GSH-3-OHKG) adduct had the same HPLC elution time, thin-layer chromatogr aphy R-F value, UV absorbance maxima, fluorescence characteristics, and mas s spectrum as the lens-derived fluorophore. Furthermore, the H-1 and C-13 N MR spectra of the synthetic adduct were entirely consistent with the propos ed structure of GSH-3-OHKG. These data indicate that GSH-3-OHKG is present as a novel fluorophore in aged human lenses. The GSH-3-OHKG adduct was foun d to be less reactive with beta-glucosidase compared with 3-OHKG, and this could be due to a folded conformation of the adduct that was suggested by m olecular modeling.