Advanced glycation end product (AGE): Characterization of the products from the reaction between D-glucose and serum albumin

We incubated bovine serum albumin (BSA) with glucose in an attempt to study how the advanced glycation end products (AGEs) are formed and what methods can be used for their identification and isolation. The reaction was monitored by boronated affinity gel, size exclusion and ion exchange chromatography, and chromatofocusing. Reaction products were also characterized by fluorescence measurement, fructosamine assay, and polyacrylamide gel electrophoresis (PAGE). Based on the measurement of AGE-associated fluorescence (excitation, 370 nm; emission, 440 nm) we found that the AGEs could be detected as early as after 3 days incubation. The fluorescence was always associated with the larger molecules of cross-linking product resulting from the reaction between BSA and glucose. The overall fluorescence intensity increased with incubation time and fluorescence of the highest intensity was found with the AGE product largest in size. As with the Amadori product, AGEs also bind to the boronated gel column but with an even higher affinity. Compared to the original albumin monomer AGE molecules are not only larger in size but also have lower isoelectric points and carry more negative charges. Both the size and the negative charges of AGEs continue to increase over time during incubation. This results in a group of cross-linking molecules heterogeneous in size and charge. These results will aid in both the isolation and selection of appropriate AGE molecules for the preparation of anti-AGE antibodies, calibrator, and control in the development of an AGE immunoassay.