Conversion of Amadori product of the Maillard reaction to Nε-(carboxymethyl) lysine by short-term heating process

Abstract

The accumulation of advanced glycation endproducts (AGE) of the Maillard reaction is increased by aging process and pathogenesis of age-enhanced disease such as atherosclerosis and diabetic complications. Especially, immunohistochemical analyses have demonstrated accumulation of AGE in vivo such as diabetic nephropathy, atherosclerosis and Alzheimer's disease. In some issues of these reports, localization of AGE has been determined using formalin-fixed paraffin-embedded tissue sections after heat-induced epitope retrieval treatment. However, we speculated that AGE might be artificially formed from Amadori product, a crucial intermediate for AGE formation, by the heating process. To evaluate this possibility, we compared the reactivity of monoclonal antibody against Nε-(carboxymethyl) lysine (CML), a major AGE structure, to heat-treated and untreated tissue sections. Normal human skin, rat skin and liver samples were divided into two groups, one was rapidly frozen and the other was formalin-fixed paraffin-embedded, followed by heat-induced epitope retrieval treatment. In the heat-treated sections, the nuclei of the epidermis in the skin obtained from human and rat, and cytoplasm of rat liver were strongly stained by anti-CML antibody (6D12) while in the frozen sections, the staining of the same site was negligible. In order to analyze the mechanism, we conducted heat-treatment to glycated-human serum albumin (glycated-HSA), a model Amadori product, and generation of CML was determined by 6D12 and high performance liquid chromatography (HPLC). CML was generated from glycated-HSA by heat-treatment at over 80 °C, and it was increased in time dependent manner. In contrast, the generation of CML from glycated-HSA was significantly inhibited in the presence of NaBH4 (132 mM), diethylenetriamine pentaacetic acid (DTPA) (1 mM) or aminoguanidine (90 mM). Furthermore, reactive intermediates such as glucosone, 3-deoxyglucosone, methylglyoxal and glyoxal were detected by HPLC after heat treatment of glycated-HSA at 100 °C, indicating that these aldehydes generated from Amadori product by oxidative cleavage can contribute to further AGE formation. These results strongly demonstrated that Amadori product in tissues was able to convert into CML by the heating process.