Effect of glucose concentration on formation of AGEs in erythrocytes in vitro

Posttranslational modifications, such as advanced glycoxidation and lipoxidation end products (AGE/ALES), are implicated in the pathogenesis of diabetic complications and atherosclerosis. Recent studies have demonstrated that AGE/ALEs are generated not only in extracellular matrix proteins, but also in intracellular proteins from metabolic intermediates. In this study we investigate the effect of glucose concentration on the formation of the AGE/ALEs, N-epsilon(carboxymethyl)lysine (CML), N-epsilon-(carboxyethyl)lysine (CEL), S-(carboxymethyl)cysteine (CMC), and S-(2-succinyl)cysteine (2SC) in erythrocytes as a function of glucose concentration. Human erythrocytes (10% bematocrit) were incubated in Dulbecco's modified Eagle's medium (DMEM) containing 5 mM or 30 mM glucose for 5 days at 37 degrees C. Globin was recovered by precipitation with 0.25 M HCl in acetone. Following acid hydrolysis, amino acids were converted to their trifluoroacetyl methyl ester derivatives and analyzed by GC/MS/MS. The CML and CEL content of globin increased in a time- and glucose-dependent manner and also increased 1.3- and 1.8-fold, respectively, in incubations containing 30 mM glucose; whereas CNIC and 2SC content did not change during the five-day incubations. Furthermore, CEL content of globin in erythrocytes incubated with 30 mM was the highest in the other AGES, indicating that methylglyoxal may play a major role in AGE formation in erythrocytes. The erythrocyte system should be useful for cellular screening of the efficacy of inhibitors of AGE/ALE formation.