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Abstract
PURPOSE: To determine the relative rate of oxidation of ascorbic acid (ASA) and glucose under conditions used for glycation reactions in vitro and by ultraviolet A (UVA)-generated oxygen free radicals using human lens sensitizers. METHODS: ASA and [14C]glucose were incubated in 0.1 M phosphate buffer, and the rate of oxidation was determined by absorbance at 265 nm and by thin-layer chromatography, respectively. Oxidation also was measured during the UVA irradiation of 2 mg/ml solutions of human lens water-insoluble proteins. The role of individual reactive oxygen species was determined by the protective effects of superoxide dismutase, catalase, and sodium azide. RESULTS: ASA was oxidized rapidly in 0.1 M phosphate buffer. This loss was prevented by the addition of a metal chelator, by previous chelex resin treatment of the buffer, or by the addition of lens proteins. Glucose was not oxidized under any of the above conditions. UVA irradiation with 2 mg/ml human lens protein as sensitizer oxidized 1 mM ASA after several hours but oxidized, at most, only 2 microM glucose even after 8 hours of irradiation. Superoxide anion was responsible for 24%, and singlet oxygen for 40%, of the ASA oxidized. UVA-generated H2O2 caused little or no oxidation of ASA. H2O2 did accelerate the oxidation of ASA in phosphate buffer, but this was almost completely prevented by the addition of either a chelating agent or lens proteins. CONCLUSIONS: The conditions used for glycation reactions in vitro rapidly oxidized ASA, but not glucose. The UVA-dependent generation of oxygen free radicals also oxidized ASA at a 10(3) faster rate than glucose. Superoxide anion and singlet oxygen were identified as the principal oxidants of ASA in this process. These data argue that ASA may be the primary glycating agent in aging normal lenses.