Similarity of the yellow chromophores isolated from human cataracts with those from ascorbic acid-modified calf lens proteins: evidence for ascorbic acid glycation during cataract formation

Chromatographic evidence supporting the similarity of the yellow chromophor es isolated from aged human and brunescent cataract lenses and calf lens pr oteins ascorbylated in vitro is presented. The water-insoluble fraction fro m early stage brunescent cataract lenses was solubilized by sonication (WIS S) and digested with a battery of proteolytic enzymes under argon to preven t oxidation, Also, calf lens proteins were incubated with ascorbic acid for 4 weeks in air and submitted to the same digestion. The percent hydrolysis of the proteins to amino acids was approximately 90% in every case. The co ntent of yellow chromophores was 90, 130 and 250 A(330) units/g protein for normal human WISS, cataract WISS and ascorbate-modified bovine lens protei ns respectively. Aliquots equivalent to 2.0 g of digested protein were subj ected to size-exclusion chromatography on a Bio-Gel P-2 column. Six peaks w ere obtained for both preparations and pooled. Side by side thin-laver chro matography (TLC) of each peak showed very similar R-f values for the long w avelength-absorbing fluorophores. Glycation with [U-C-14]ascorbic acid, fol lowed by digestion and Bio-Gel P-2 chromatography, showed that the incorpor ated radioactivity co-eluted with the A(330)-absorbing peaks, and that most of the fluorescent bands were labeled after TLC. Peaks 2 and 3 from the P- 2 were further fractionated by preparative Prodigy C-18 reversed-phase high -performance liquid chromatography. Two major A(330)-absorbing peaks were s een in peak 2 isolated from human cataract lenses and 5 peaks in fraction 3 , all of which eluted at the same retention times as those from ascorbic ac id glycated calf lens proteins. HPLC fractionation of P-2 peaks 4. 5 and 6 showed many A(330)-absorbing peaks from the cataract WISS, only some of whi ch were identical to the asorbylated proteins. The major fluorophores, howe ver, were present in both preparations. These data provide new evidence to support the hypothesis that the yellow chromophores in brunescent lenses re present advanced glycation endproducts (AGEs) probably due to ascorbic acid glycation in vivo. (C) 2001 Elsevier Science B.V. All rights reserved.