New biomarkers of Maillard reaction damage to proteins.

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.

Mechanism of autoxidative glycosylation: identification of glyoxal and arabinose as intermediates in the autoxidative modification of proteins by glucose.

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.

Formation of reactive intermediates from Amadori compounds under physiological conditions.

The Maillard or browning reaction between reducing sugars and proteins contributes to the chemical aging of tissue proteins in vivo and to the accelerated aging of proteins in diabetes. To identify reactive carbohydrate intermediates formed in the Maillard reaction under physiological conditions, we studied the decomposition of the model Amadori compound, N alpha-formyl-N epsilon-fructoselysine (fFL) and of Amadori compounds on glycated collagen at pH 7.4 and 37 degrees C. Because of effects of buffer and oxidative conditions on the decomposition of Amadori compounds, the kinetics and products of decomposition were studied in varying phosphate concentrations and in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes) buffer under both aerobic and anaerobic conditions. The half-life of fFL was significantly shorter in phosphate, compared to Hepes buffer, and under aerobic, compared to anaerobic, conditions. The decomposition of both fFL and Amadori adducts on glycated collagen was accelerated by increasing the phosphate concentration and/or pH. Glucose and mannose were identified as major products formed by reversal of the Amadori rearrangement, along with tetroses, pentoses, and 3-deoxyglucosone, formed by reverse aldol, rearrangement, and hydrolysis reactions. The tetrose and pentose products included both aldose and ketose sugars. These same products were also formed in similar yields on decomposition of Amadori adducts on glycated collagen in vitro. The spontaneous decomposition of Amadori compounds to more reactive sugars in vivo, including tetroses, pentoses, and 3-deoxyglucosone, provides a mechanism for generating reactive intermediates under physiological conditions and for propagating damage to protein as a result of glycation of proteins by glucose in vivo.

Chemistry of the fructosamine assay: D-glucosone is the product of oxidation of Amadori compounds.

The chemistry of the fructosamine assay was studied by using the Amadori compound, N alpha-formyl-N epsilon-fructose-lysine (fFL), an analog of glycated lysine residues in protein. Previously (Clin Chem 1993;39:2460-5), we reported that free lysine was formed from fFL at 70% yield during incubation with alkaline nitroblue tetrazolium (NBT) under the conditions routinely used for the fructosamine assay (sodium carbonate buffer, pH 10.35 at 37 degrees C). Here, we show that D-glucosone is the primary carbohydrate oxidation product formed from Amadori compounds in the fructosamine assay. Glucosone, which decomposes under alkaline assay conditions with a half-life of < 30 min, reaches a maximum concentration of approximately 50% of the initial fFL concentration after 10 min of incubation. Like fFL, glucosone reduces NBT to the purple monoformazan dye, but its decomposition is not accelerated by the presence of NBT. The dicarbonyl-trapping reagent, aminoguanidine, inhibits the fructosamine assay by approximately 25% when fFL is the substrate, but by nearly 100% with glucosone as substrate. Studies with serum samples from diabetics and nondiabetics indicate that glucosone formation does not have a significant effect on the clinical usefulness of the fructosamine assay; however, corrections for glucosone formation may be required when the assay is used for estimating the extent of glycation of proteins.

Mechanism of fructosamine assay: evidence against role of superoxide as intermediate in nitroblue tetrazolium reduction.

We studied the chemistry of the fructosamine assay for glycated serum proteins by using the model Amadori compound N alpha-formyl-N epsilon-fructoselysine (fFL), an analog of glycated lysine residues in protein. Free lysine was formed at approximately 70% yield during a standard 20-min incubation of fFL with alkaline nitroblue tetrazolium (NBT) at 37 degrees C. Although superoxide dismutase (SOD; EC 1.15.1.1) and catalase (EC 1.11.1.6) decreased the yield of the product, monoformazan dye (MF+), the yield of MF+ was slightly greater under anaerobic than aerobic conditions, excluding a role for superoxide as an intermediate in the reduction of NBT during the fructosamine assay. SOD added to diabetic patients' sera at physiological concentrations also caused a significant (approximately 50%) inhibition of MF+ formation. This inhibition was reduced by addition of nonionic detergents, which contain organic peroxide inhibitors of SOD, to the fructosamine reagent. Overall, these data indicate that the Amadori compound is the direct reductant of NBT in the fructosamine assay and that superoxide is not an intermediate in the reaction. The inhibitory effects of SOD and catalase are most likely the result of oxygen regeneration in the assay mixture.