AGEs in the Lens: Detection of Dideoxyosone Intermediates Using a Monoclonal Antibody

Abstract

Purpose: : Glycation or the Maillard reaction in lens proteins forms advanced glycation end products (AGEs) that contribute to protein crosslinking and pigmentation in the human lens. Dideoxyosones (DDO) are the newly discovered intermediates during AGE formation. They are formed by the long–range carbonyl shift of the Amadori product. They are likely the major precursors of AGEs in the human lens, Two AGEs derived from DDO, pentosidine and glucosepane accumulate in relatively large quantities in cataractous lenses

Methods: : DDO react with o–phenylenediamine (OPD) to produce quinoxalines. We developed a monoclonal antibody against 2–methylquinoxaline–6–carboxylate coupled to keyhole limpet hemocyanin and we characterized the antibody with glycated proteins and quinoxalines. HPLC was used to isolate major antibody reactive products from glycated lysine (+OPD).

Results: : The antibody reacted strongly with ribose and fructose (+ OPD)–modified RNase A and weakly with glucose and ascorbate (+ OPD)–modified RNase A. Reaction with substituted quinoxalines indicated that this antibody favored the 2–methyl group on the quinoxaline ring. Three antibody–reactive products were isolated from a reaction mixture of Nalpha–hippuryl–L–lysine + ribose + OPD. The two most reactive products were identified as diastereoisomers of N1–benzoylglycyl–N6– (2–hydroxy–3–quinoxalin–2–ylpropyl)lysine and the other less reactive product as N1–benzoylglycyl–N6–[2–hydroxy–2–(3–methylquinoxalin–2–yl)ethyl]lysine.

Conclusions: : Our study confirms that dideoxyosone intermediates form during glycation and offers a new tool for the study of this important pathway in lens aging and cataractogenesis.

Keywords: protein modifications-post translational • crystalline lens • diabetes