Conversion of Amadori products of the Maillard reaction to Nε-(carboxymethyl) lysine by short-term heating:: Possible detection of artifacts by immunohistochemistry

Accumulation of advanced glycation end products (AGE) of the Maillard reaction increases by aging and in age-enhanced diseases such as atherosclerosis and diabetic complications. Immunohistochemical analysis has been used to demonstrate AGE in vivo. In immunochemistry, the heat-induced epitope retrieval technique is extensively used with formalin-fixed, paraffin-embedded tissue sections. Here we examined whether AGE could be formed artificially through the heating process. Normal rat skin and liver samples were divided into two groups, one rapidly frozen, the other formalin-fixed, paraffin-embedded and submitted to heat-induced epitope retrieval treatment. In heat-treated sections, the cytoplasm of rat epidermal cells and hepatocytes were strongly stained by monoclonal antibody against N-6-(carboxymethyl)lysine (CML), while the staining was negligible in either frozen sections or in paraffin-embedded but heat-untreated sections. To clarify the mechanism, we conducted heat treatment to glycated human serum albumin (HSA), a model Amadori protein, and generation of CML was determined by immunochemical and HPLC analysis. CML was generated from glycated HSA by heat treatment (above 80degrees C) and increased in a time-dependent manner. In contrast, generation of CML from glycated HSA was significantly inhibited in the presence of NaBH4, a reducing agent, diethylenetriamine pentaacetic acid, a chelator of transition metal ion, or aminoguanidine, a trapping reagent for alpha-oxoaldehydes. Furthermore, heat-induced CML formation in rat liver samples determined by HPLC was markedly reduced by pretreatment with NaBH4. Reactive intermediates such as glucosone, 3-deoxyglucosone, methylglyoxal, and glyoxal were formed upon heat treatment of glycated HSA at 100degrees C, indicating that these aldehydes generated from Amadori products by oxidative cleavage can contribute to further CML formation. CML generated by heating, directly from Amadori products or via these aldehydes, might serve as an artifact upon immunohistochemistry.