Immunological detection of fructose-derived advanced glycation end-products

Abstract

The advanced stage of non-enzymatic glycation (also called the Maillard reaction) that leads to the formation of advanced glycation end-products (AGEs) has an important function in the pathogenesis of angiopathy in diabetic patients. So far, most studies have been focused on the Maillard reaction by glucose. Although an elevated level of glucose had been thought to have a primary function in the Maillard reaction, on a molecular basis, glucose is among the least reactive sugars within biological systems. In addition to the extracellular formation of AGEs, rapid intracellular AGEs formation by various intracellular precursors (fructose, trioses, and dicarbonyl compounds) has recently attached attention. In this study, we considered the Maillard reaction with particular attention to the potential function of fructose. Fructose AGE-modified serum albumins were prepared by incubation of rabbit or bovine serum albumin (RSA or BSA) with D-fructose. After immunization of rabbits, fructose-derived AGEs (Fru-AGE) antiserum was subjected to affinity chromatography on a Sepharose 4B column coupled with Fru-AGE-BSA. Characterization of the novel anti-Fru-AGE antibody was performed with a competitive enzyme-linked immunosorbent assay and immunoblot analysis. The assay of Fru-AGE was established using the immunoaffinity-purified-specific antibody, and the presence of Fru-AGE in healthy and diabetic serum was shown (7.04±4.47 vs 29.13±18.08 U/ml). We also investigated whether high glucose treatment could stimulate intracellular Fru-AGE production in cultured pericytes, and we analyzed the amount of Fru-AGE contained in some common commercial beverages and condiments. It is possible that Fru-AGE formation by these endogenous and exogenous routes contributes importantly to the tissue pathology of diabetes and aging. This paper provides novel and clinically relevant information on the detection of Fru-AGE between fructose and proteins.