The Relative Oxidation, Glycation and Crosslinking Activity of Glucose and Ascorbic Acid in vitro

Abstract

The protein crosslinking activities of glucose and ascorbic acid (ASA) were compared by the incorporation of Nα-formyl-[U-14C]lysine into lens proteins. On a molar basis, ASA was 60-fold more active than glucose and 100-fold more active than fructose. ASA, but not glucose, was rapidly oxidized by the metal ions present in 100 mM phosphate buffer, but the addition of a chelating agent or even lens proteins (0.5 mg/mL) almost completely prevented this oxidation. The generation of singlet oxygen (2.0 mM) by the UVA irradiation of human lens proteins oxidized 0.5 mM ASA in 1 hr, but 4 h of irradiation had no oxidative effect on 50 μM glucose. High levels of glutathione were unable to prevent completely the oxidation of ASA by singlet oxygen. This was due to the unique products of singlet oxygen-mediated oxidation of ASA, and by the aggregate nature of the lens proteins preventing GSH access to the sites of ASA oxidation. UVA irradiation of human lens proteins caused the incorporation of [U-14C]ASA into protein, even in the presence of physiological levels of glutathione. These data argue that ascorbic acid, rather than glucose, is responsible for the glycation-dependent crosslinking of lens proteins in vivo.