Formation pathways of Nε-(carboxymethyl)lysine and dicarbonyl compounds by peroxynitrite

Abstract

In our previous paper, hydroxyl radical was found to be a major reactive oxygen species for Nε-(carboxymethyl)lysine (CML) formation through Amadori products. Peroxynitrite (PN) is also known as one of the reactive oxygen species. However, the contribution of PN to the formation of advanced glycation end products (AGE) such as CML has been poorly understood so far. In this work, the effect of PN on CML formation was discussed. First, glycated human serum albumin (glycated HSA) was incubated with 70 μM PN at 37 °C for 1 h. Second, d-glucose was incubated with 1 mM PN at 37 °C for 30 min in advance, followed by further incubation in the presence of HSA at 37 °C for 24 h. The amount of CML in these samples was determined by ELISA. After incubation of glycated HSA with PN for 5 min, CML formation was observed to a significant extent. Furthermore, the reaction of PN-treated glucose with HSA resulted in the production of CML. In contrast, CML was not formed in the parallel incubation without PN. To determine aldehydes produced during PN-induced CML formation, aliquots of the reaction mixtures were mixed with 2,3-diaminonaphthalene (DAN) and then quantified by HPLC. This experiment demonstrated that glucosone is an important intermediate on the formation of CML.