Role of oxidative stress in physiological albumin glycation: a neglected interaction.

Protein glycation is a key mechanism involved in chronic disease development in both diabetic and nondiabetic individuals. About 12-18% of circulating proteins are glycated in vivo in normoglycemic blood, but in vitro studies have hitherto failed to demonstrate glucose-driven glycation below a concentration of 30mM. Bovine serum albumin (BSA), reduced BSA (mercaptalbumin) (both 40g/L), and human plasma were incubated with glucose concentrations of 0-30mM for 4 weeks at 37°C. All were tested preoxidized for 8h before glycation with 10nM H2O2 or continuously exposed to 10nM H2O2 throughout the incubation period. Fructosamine was measured (nitroblue tetrazolium method) at 2 and 4 weeks. Oxidized BSA (both preoxidized and continuously exposed to H2O2) was more readily glycated than native BSA at all glucose concentrations (p = 0.03). Moreover, only oxidized BSA was glycated at physiological glucose concentration (5mM) compared to glucose-free control (glycation increased by 35% compared to native albumin, p < 0.05). Both 5 and 10mM glucose led to higher glycation when mercaptalbumin was oxidized than when unoxidized (p < 0.05). Fructosamine concentration in human plasma was also significantly higher when oxidized and exposed to 5mM glucose, compared to unoxidized plasma (p = 0.03). The interaction between glucose concentration and oxidation was significant in all protein models (p < 0.05). This study has for the first time demonstrated albumin glycation in vitro, using physiological concentrations of albumin, glucose, and hydrogen peroxide, identifying low-grade oxidative stress as a key element early in the glycation process.