Rate of formation of AGEs during ascorbate glycation and during aging in human lens tissue

Abstract

The similarity of the yellow chromophores isolated from human cataracts with those from ascorbic acid modified calf lens proteins was recently published [Biochim. Biophys. Acta 1537 (2001) 14]. The data presented here additionally quantify age-dependent increases in individual yellow chromophores and fluorophores in the water-insoluble fraction of normal human lens. The water-insoluble fraction of individual normal human lens was isolated, solubilized by sonication and digested with a battery of proteolytic enzymes under argon to prevent oxidation. The level of A330-absorbing yellow chromophores, 350/450 nm fluorophores and total water-insoluble (WI) protein were quantified in each lens. The total yellow chromophores and fluorophores accumulated in parallel with the increase in the water-insoluble protein fraction during aging. The digest from each single human lens was then subjected to Bio-Gel P-2 size-exclusion chromatography. The fractions obtained were further separated by a semi-preparative prodigy C-18 high-performance liquid chromatography (RP-HPLC). Bio-Gel P-2 chromatography showed four major fractions, each of which increased with age. RP-HPLC of the amino acid peak resolved five major A330-absorbing peaks and eight fluorescent peaks, and each peak increased coordinately with age. A late-eluting peak, which contained hydrophobic amino acids increased significantly after age 60. Aliquots from an in vitro glycation of calf lens proteins by ascorbic acid were removed and subjected to the same enzymatic digestion. Ascorbic acid-modified calf lens protein digests showed an almost identical profile of chromophores, which also increased in a time-dependent manner. The late-eluting peak, however, did not increase with the time of glycation and may not be an advanced glycation endproduct (AGE) product. The data indicate that the total water-insoluble proteins, individual yellow chromophores and fluorophores increased equally both with aging in normal human lens and during ascorbate glycation in vitro. The major protein modifications, which accumulate during aging, therefore, appear to be AGEs. Whereas the late-eluting peak, which showed poor correlation to ascorbylation, may represent UV filter compounds bound to lens proteins. © 2002 Elsevier Science B.V. All rights reserved.