Enzyme activity and AGE formation in a model of AST glycoxidation by D-fructose in vitro

Non-enzymatic glycation as the chain reaction between reducing sugars and free amino groups of proteins has been shown to correlate with physiological ageing and severity of diabetes. The process involves oxidative steps (glycoxidation). In this paper, the effect of D-fructose as a reactive sugar on aspartate aminotransferase (AST) as a model protein was monitored by measurements of the enzyme activity and formation of fluorescent advanced glycation end products (AGEs). Change in the AST activity was considered as a measure of the overall protein damage caused by glycation, and total AGEs and pentosidine represent, at least partly, the formation of glycoxidation products. Catalytic activity of AST in an incubation mixture containing D-fructose (50 mmol L(-1)), decreased compared to control values to 42% (p < 0.05) and to 11% (p < 0.05) on the 5th and on 21st day of incubation, respectively. In the presence of fructose, total fluorescent AGEs concentration was significantly higher since 5th day of incubation (110%, p < 0.05) and the fluorescent pentosidine concentration from 15th day of incubation (117%, p < 0.05) compared to control values, respectively. Catalytic activity of AST clearly and quantitatively demonstrated functional changes in the enzyme molecule caused by structural modifications initiated by fructose, while the evaluation of AGE formation and especially that of pentosidine by fluorescence measurement was less reliable.