Glutathione inhibits the glycation and crosslinking of lens proteins by ascorbic acid.
The incubation of crude extracts of bovine lens with 20 mM ascorbic acid leads to the formation of covalent adducts even in the presence of saturating levels of a metal chelator. When dialysed lens extracts were used both ASA-protein adducts and highly crosslinked lens proteins were observed which are similar to those found in the water insoluble fraction from cataractous lenses. Both adduct formation and protein crosslinking, however, were markedly inhibited if undialysed lens extracts were used or if increasing concentrations of glutathione were added to the incubation mixture. Similar inhibition was seen with cysteine, dithiothreitol and sodium bisulfite, but little effect was observed with the glutathione analog ophthalmic acid or with free radical quenchers. Glutathione was readily oxidized during the incubation and no oxidation of ascorbic acid was observed until all the reduced glutathione was exhausted. No loss of ascorbic acid and no protein crosslinking were observed when oxygen was completely removed from the reaction mixture. These data strongly suggest that the glycating species was an oxidized form of ascorbic acid. Ascorbic acid solutions displayed a rapid oxidation in vitro, which was decreased 80-fold upon the addition of 1 mM chelator and was completely inhibited by both glutathione and chelator. A rapid decrease in the level of dissolved oxygen was seen in the presence of ascorbic acid or ascorbic acid and glutathione, but not with glutathione alone. These data argue that glutathione inhibits glycation by rapidly reducing dehydroascorbic acid back to ascorbic acid, which is not active in protein glycation
Ortwerth
B J
BJ
Mason Institute of Ophthalmology, University of Missouri, Columbia 65212.
Olesen
P R
PR
D000818:Animals; D001205:Ascorbic Acid / Q000037:antagonists & inhibitors*; D002417:Cattle; D003432:Cross-Linking Reagents; D003459:Crystallins / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005978:Glutathione / Q000494:pharmacology*; D015416:Maillard Reaction; D010084:Oxidation-Reduction
D003432:Cross-Linking Reagents; D003459:Crystallins; D005978:Glutathione; D001205:Ascorbic Acid; D004229:Dithiothreitol