Immunological evidence that non-carboxymethyllysine advanced glycation end-products are produced from short chain sugars and dicarbonyl compounds in vivo

Background: The Maillard reaction that leads to the formation of advanced g lycation end-products (AGE) plays an important role in the pathogenesis of angiopathy in diabetic patients and in the aging process. Recently, it was proposed that AGE were not only created by glucose, but also by dicarbonyl compounds derived from the Maillard reaction, autoxidation of sugars and ot her metabolic pathways of glucose. In this study, we developed four types o f non- carboxymethyllysine (CML) anti-AGE antibodies that recognized protei ns modified by incubation with short chain sugars and dicarbonyl compounds. Materials and Methods: AGE-modified serum albumins were prepared by incubat ion of rabbit serum albumin with glyceraldehyde, glycolaldehyde, methylglyo xal or glyoxal. After immunization of rabbits, four types of AGE-specific a ntisera were obtained that were specific for the AGE modification. To separ ate non-CML AGE antibodies (Ab) (non-CML AGE-Ab-2, -3, -4, and -5), these a nti-AGE antisera were subjected to affinity chromatography on a matrix coup led with four kinds of AGE bovine serum albumin (BSA) or CML-BSA. These non -CML AGE antibodies were used to investigate the AGE content of serum obtai ned from diabetic patients on hemodialysis. Results: Characterization of the four types of non-CML AGE antibodies obtai ned by immunoaffinity chromatography was performed by competitive ELISA and immunoblot analysis. Non-CML AGE-Ab-2 crossreacted with the protein modifi ed by glyceraldehyde or glycolaldehyde. Non-CML AGE-Ab-3 and -Ab-4 specific ally cross-reacted with protein modified by glycolaldehyde and methylglyoxa l, respectively. Non-CML AGE-Ab-5 cross-reacted with protein modified with glyoxal as well as methylglyoxal and glycolaldehyde. Three kinds of non-CML AGE (AGE-2, -4, and -5) were detected in diabetic serum as three peaks wit h apparent molecular weights of 200, 1.15, and 0.85 kD; whereas, AGE-3 was detected as two peaks with apparent molecular weights of 200 and 0.85 kD. Conclusion: We propose that various types of non-CML AGE are formed by the Maillard reaction, sugar autoxidation and sugar metabolism. These antibodie s enable us to identify such compounds created by the Maillard reaction in vivo.