Detection of 3-deoxyglucosone-derived AGE structures in vitro

In the Maillard reaction, 3-deoxyglucosone (3-DG) was known to generate from Amadori product and contribute to further AGE formation such as Íå-(carboxymethyl)lysine (CML), 3-DG-derived imidazolone (3-DG-imidazolone) and pyrraline. However, main AGE structures generated from 3-DG modification have not yet been elucidated. To solve this issue, we set up the detection system for AGE by immunochemical analysis as well as high-performance liquid chromatography (HPLC) and determined them in 3-DG-modified bovine serum albumin (BSA). When BSA was incubated at 37 °C for up to 4 weeks with 3-DG, the reactivity of antibodies against CML and 3-DG-imidazolone increased steeply with incubation time, whereas the reactivity of antibodies against pyrraline was increased slightly. In contrast, in addition to CML and 3-DG-imidazolone, significant amount of pyrraline was observed when AGE formation was enhanced by high temperature at 60 °C. In HPLC analysis, CML (1.10 mol/mol of BSA), 3-DG-imidazolone (0.84 mol/mol of BSA) and pyrraline (0.33 mol/mol of BSA) were detected in BSA incubated with 3-DG at 37 °C for 4 weeks. Taken together, our analyses clarified that formation of CML and 3-DG-imidazolone is higher than that of pyrraline during the incubation of protein with 3-DG.