A new rapid method to detect inhibition of Amadori product generated by delta-gluconolactone

Advanced glycation endproducts (AGEs) have been implicated as a major pathogenic process in leading to diabetic complications. An increasing number of drug candidates have recently been developed as potential inhibitors of AGEs. Aminoguanidine, a hydrazine-like molecule is the first drug that was extensively studied both in vitro and in vivo as an inhibitor of AGE formation. Several assay methods have been proposed to determine the inhibitory effect of glycation inhibitors, including assays based on inhibition of specific fluorescence generated in the course of glycation and AGE-formation; assays based on the inhibition of AGE-protein crosslinks, and dimer and trimer formation; and specific ELISA assays using anti-AGE antibodies for quantitative measurement of AGEs in the presence and absence of the inhibitor. However, none of these assays can accurately evaluate chemical intermediates and products generated during the early stages of glycation. We have devised a new rapid assay method for evaluation of the early stage of glycation (Amadori product). This assay is based on the interaction of delta-gluconolactone (delta-Glu), an oxidized (ketoaldehyde) analogue of glucose, with hemoglobin present in blood samples. The assay involves determination of the percentage of glycated hemoglobin (HbA1C) after incubation at 37 degrees for 16 h with delta-Glu (50 mmol/L) using a dedicated ion-exchange high-performance liquid chromatography (HPLC) system. The results using normal human red blood cells show HbA1C levels to be 180% higher than baseline controls. The effects of various inhibitors are determined by measuring the levels of HbA1C by the compound in question compared to delta-Glu-treated vehicle only blood samples. This new assay provides a relevant and physiological model to study glycation and potential inhibitors. Furthermore, it offers a means to differentiate between inhibitors of the early and late stages of glycation and provides a rapid method of screening large numbers of potential inhibitors of glycation. Contrary to the assay methods, which are based on the measurement of fluorescence of fluorophores generated during glycation, the proposed assay does not suffer from the possible problem of overlapping and interference of AGE-specific fluorescence with the intrinsic fluorescence of the inhibitor compound.