Glycation and oxidation reactions contribute to protein modification in aging and diabetes. Formation of dicarbonyl sugars during autoxidation of glucose is the hypothetical first step in the autoxidative glycosylation and subsequent browning of proteins by glucose [Wolff, S. P., & Dean, R. T. (1987) Biochem. J. 245, 243-250]. In order to identify the dicarbonyl sugar(s) formed during autoxidation of glucose under physiological conditions, glucose was incubated in phosphate buffer (pH 7.4) at 37 degrees C under air (oxidative conditions) or nitrogen with transition metal chelators (antioxidative conditions). Dicarbonyl compounds were analyzed spectrophotometrically and by HPLC after reaction with Girard-T reagent. Carbohydrates were analyzed by gas chromatography-mass spectrometry. Both dicarbonyl sugar and arabinose concentrations increased with time and glucose concentration in incubations conducted under oxidative conditions; only trace amounts of these products were detected in glucose incubated under antioxidative conditions. HPLC analysis of adducts formed with Girard-T reagent indicated that glyoxal was the only alpha-dicarbonyl sugar formed on autoxidation of glucose. Glyoxal and arabinose accounted for > or = 50% of the glucose lost during a 21 day incubation. Neither glucosone nor its degradation product, ribulose, was detectable. Reaction of glyoxal with RNase yielded the glycoxidation product, N epsilon-(carboxymethyl)lysine, while arabinose is a source of pentosidine. Our results implicate glyoxal and arabinose as intermediates in the browning and crosslinking of proteins by glucose under oxidative conditions. They also provide a mechanism by which antioxidants and dicarbonyl trapping reagents, such as aminoguanidine, limit glycoxidation reactions and support further evaluation of these types of compounds for inhibition of chemical modification and crosslinking of proteins during aging and diabetes.

Advanced glycation end products (AGEs) and glycoxidation products are formed during Maillard or browning reactions between sugars and proteins and are implicated in the pathophysiology of aging and the complications of diabetes. To determine the structure of AGEs, antibodies were prepared to protein browned by incubation with glucose and used in ELISA assays to measure AGEs formed in model reactions between bovine serum albumin (BSA) or N alpha-acetyllysine and glucose, fructose, or glyoxal. AGEs were formed from glucose and fructose only under oxidative conditions, but from glyoxal under both oxidative and antioxidative conditions. Gel permeation chromatographic analysis indicated that a similar AGE was formed in reactions of N alpha-acetyllysine with glucose, fructose, and glyoxal and that this AGE co-eluted with authentic N alpha-acetyl-N epsilon-(carboxymethyl)lysine. Amino acid analysis of AGE proteins revealed a significant content of N epsilon-(carboxymethyl)lysine (CML). In ELISA assays using polyclonal antibodies against AGE proteins, CML-BSA (approximately 25 mol of CML/mol of BSA), prepared by chemical modification of BSA, was a potent inhibitor of the recognition of AGE proteins and of AGEs in human lens proteins. We conclude that AGEs are largely glycoxidation products and that CML is a major AGE recognized in tissue proteins by polyclonal antibodies to AGE proteins.

The Maillard or browning reaction between sugar and protein contributes to the increased chemical modification and cross-linking of long-lived tissue proteins in diabetes. To evaluate the role of glycation and oxidation in these reactions, we have studied the effects of oxidative and antioxidative conditions and various types of inhibitors on the reaction of glucose with rat tail tendon collagen in phosphate buffer at physiological pH and temperature. The chemical modifications of collagen that were measured included fructoselysine, the glycoxidation products N epsilon-(carboxymethyl)lysine and pentosidine and fluorescence. Collagen cross-linking was evaluated by analysis of cyanogen bromide peptides using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by changes in collagen solubilization on treatment with pepsin or sodium dodecylsulfate. Although glycation was unaffected, formation of glycoxidation products and cross-linking of collagen were inhibited by antioxidative conditions. The kinetics of formation of glycoxidation products proceeded with a short lag phase and were independent of the amount of Amadori adduct on the protein, suggesting that autoxidative degradation of glucose was a major contributor to glycoxidation and cross-linking reactions. Chelators, sulfhydryl compounds, antioxidants, and aminoguanidine also inhibited formation of glycoxidation products, generation of fluorescence, and cross-linking of collagen without significant effect on the extent of glycation of the protein. We conclude that autoxidation of glucose or Amadori compounds on protein plays a major role in the formation of glycoxidation products and cross-liking of collagen by glucose in vitro and that chelators, sulfhydryl compounds, antioxidants, and aminoguanidine act as uncouplers of glycation from subsequent glycoxidation and cross-linking reactions.

The amount of advanced glycation end-products (AGE) in tissue proteins increases in diabetes mellitus, and the concentration of a subclass of AGEs, known as glycoxidation products, also increases with chronological age in proteins. The rate of accumulation of glycoxidation products is accelerated in diabetes and age-adjusted concentrations of two glycoxidation products, N epsilon-(carboxymethyl)lysine (CML) and pentosidine, correlate with the severity of complication in diabetic patients. Although AGEs and glycoxidation products are implicated in the development of diabetic complications, these compounds are present at only trace concentrations in tissue proteins and account for only a fraction of the chemical modifications in AGE proteins prepared in vitro. The future of the AGE hypothesis depends on the chemical characterization of a significant fraction of the total AGEs in tissue proteins, a quantitative assessment of their effects on protein structure and function, and an assessment of their role as mediators of biological responses. In this manuscript we describe recent work leading to characterization of new AGEs and glycoxidation products. These compounds include: (1) the imidazolone adduct formed by reaction of 3-deoxyglucosone with arginine residues in protein; (2) N epsilon-(carboxyethyl)lysine, an analogue of CML formed on reaction of methylglyoxal with lysine; (3) glyoxal-lysine dimer; and (4) methyl-glyoxal-lysine dimer, which are imidazolium crosslinks formed by reaction of glyoxal or methylglyoxal with lysine residues in protein. The presence of 3-deoxyglucosone, methylglyoxal and glyoxal in vivo and the formation of the above AGEs in model carbonyl-amine reaction systems suggests that these AGEs are also formed in vivo and contribute to tissue damage resulting from the Maillard reaction.

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