Formation of glyoxal, methylglyoxal and 3-deoxyglucosone in the glycation of proteins by glucose.

The glycation of proteins by glucose has been linked to the development of diabetic complications and other diseases. Early glycation is thought to involve the reaction of glucose with N-terminal and lysyl side chain amino groups to form Schiff's base and fructosamine adducts. The formation of the alpha-oxoaldehydes, glyoxal, methylglyoxal and 3-deoxyglucosone, in early glycation was investigated. Glucose (50 mM) degraded slowly at pH 7.4 and 37 degrees C to form glyoxal, methylglyoxal and 3-deoxyglucosone throughout a 3-week incubation period. Addition of t-BOC-lysine and human serum albumin increased the rate of formation of alpha-oxoaldehydes - except glyoxal and methylglyoxal concentrations were low with albumin, as expected from the high reactivity of glyoxal and methylglyoxal with arginine residues. The degradation of fructosyl-lysine also formed glyoxal, methylglyoxal and 3-deoxyglucosone. alpha-Oxoaldehyde formation was dependent on the concentration of phosphate buffer and availability of trace metal ions. This suggests that alpha-oxoaldehydes were formed in early glycation from the degradation of glucose and Schiff's base adduct. Since alpha-oxoaldehydes are important precursors of advanced glycation adducts, these adducts may be formed from early and advanced glycation processes. Short periods of hyperglycaemia, as occur in impaired glucose tolerance, may be sufficient to increase the concentrations of alpha-oxoaldehydes in vivo.

Accumulation of alpha-oxoaldehydes during oxidative stress: a role in cytotoxicity.

Glyoxal, methylglyoxal (MG), and 3-deoxyglucosone (3-DG) are physiological alpha-oxoaldehydes formed by lipid peroxidation, glycation, and degradation of glycolytic intermediates. They are enzymatically detoxified in cells by the cytosolic glutathione-dependent glyoxalase system (glyoxal and MG only) and by NADPH-dependent reductase and NAD(P)+-dependent dehydrogenase. In this study, the changes in the cellular and extracellular concentrations of these alpha-oxoaldehydes were investigated in murine P388D1 macrophages during necrotic cell death induced by median toxic concentrations of hydrogen peroxide and 1-chloro-2,4-dinitrobenzene (CDNB). Alpha-oxoaldehyde concentrations were determined by derivatization with 1,2-diamino-4,5-dimethoxybenzene. There were relatively small increases in cellular and extracellular glyoxal concentration, except that extracellular glyoxal was decreased with hydrogen peroxide. The cytosolic concentration of 3-DG and the cytosolic and extracellular concentrations of MG, however, increased markedly. Aminoguanidine inhibited alpha-oxoaldehyde accumulation and prevented cytotoxicity induced by hydrogen peroxide and CDNB. The accumulation of glyoxal and MG in toxicant-treated cells was a likely consequence of decreased in situ activity of glyoxalase 1. The effect was marked for MG but not for glyoxal, suggestive of a greater metabolic flux of MG formation than of glyoxal. The accumulation of 3-DG in toxicant-treated cells was probably due to the decreased availability of pyridine nucleotide cofactors for the detoxification of 3-DG. Impairment of alpha-oxoaldehyde detoxification is cytotoxic, and this may contribute to toxicity associated with GSH oxidation and S conjugation in oxidative stress and chemical toxicity, and to chronic pathogenesis associated with diabetes mellitus where there is oxidative stress and the formation of glyoxal, MG, and 3-DG is increased.

Rapid hydrolysis and slow alpha,beta-dicarbonyl cleavage of an agent proposed to cleave glucose-derived protein cross-links.

The putative protein glycation cross-link cleaving agent N-phenacylthiazolium bromide (PTB) underwent hydrolysis and cyclic hemithioacetal formation under physiological conditions to form two isomeric 2,3-dihydro-4-formyl-2-hydroxy-2-phenyl-1,4-thiazines: at pH 7.4 and 37 degrees, the rate constant k(Hydrolysis) was (2.6+/-0.1) x 10(-4) sec and the chemical half-life was ca. 44 min. The alpha,beta-dicarbonyl cleavage reaction only competed effectively with the hydrolysis when the alpha,beta-dicarbonyl substrate was at nonphysiological high levels. The high concentrations of PTB (10-30 mM) used previously to demonstrate chemical and biochemical activity also lead to acidification of incubation media. The mechanism of action of PTB now requires reappraisal.

Teratogenicity of 3-deoxyglucosone and diabetic embryopathy.

The increased rate of embryonic dysmorphogenesis in diabetic pregnancy is correlated with the severity and duration of the concurrent hyperglycemia during early gestation. Whole embryo culture was used to investigate a possible association of hyperglycemia-induced disturbances of embryo development with tissue levels of the three alpha-oxoaldehydes: glyoxal, methylglyoxal, and 3-deoxyglucosone (3-DG). Rat embryos exposed to high glucose levels in vitro showed severe dysmorphogenesis and a 17-fold increased concentration of 3-DG compared with control embryos cultured in a low glucose concentration. Exogenous 3-DG (100 micromol/l) added to the medium of control cultures yielded an increased embryonic malformation rate and a 3-DG concentration similar to that of embryos cultured in high glucose. Addition of superoxide dismutase (SOD) to the culture medium decreased the malformation rates of embryos exposed to either high glucose or high 3-DG levels, but it did not decrease the high embryonic 3-DG concentrations caused by either agent. Our results implicate the potent glycating agent 3-DG as a teratogenic factor in diabetic embryopathy. In addition, the anti-teratogenic effect of SOD administration appears to occur downstream of 3-DG formation, suggesting that 3-DG accumulation leads to superoxide-mediated embryopathy.

Reduced glutathione esters--antidotes to toxicity. Cytotoxicity induced by hydrogen peroxide, 1-chloro-2,4-dinitrobenzene, and menadione in murine P388D1 macrophages in vitro.

Repletion of depleted cellular reduced glutathione (GSH) levels in oxidative stress and exposure to arylating agents is a strategy for the development of antidotes to chemical toxicity. The effect of GSH, reduced glutathione ethyl monoester (GSHEt), and reduced glutathione ethyl diester (GSHEt2) on the cytotoxicity of hydrogen peroxide, 1-chloro-2,4-dinitrobenzene (CDNB), and menadione to P388D1 macrophages in vitro was investigated. The median toxic concentration TC50 values of the toxicants were hydrogen peroxide 24 +/- 2 mM (N = 19), CDNB 63 +/- 6 microM (N = 18), and menadione 30 +/- 4 microM (N = 22). Reduced glutathione, GSHEt, and GSHEt2 were poor antidotes to hydrogen peroxide toxicity. Indeed, the observed antidote effects were attributed to the nonenzymatic reaction of the GSH derivatives with hydrogen peroxide in the extracellular medium. Reduced glutathione ethyl diester was a more potent antidote of CDNB- and menadione-mediated toxicity than GSHEt and GSH. For cell incubations with the approximate median toxic concentration TC50 values of hydrogen peroxide, CDNB, and menadione, the respective median effective antidote concentration EC50 values were GSHEt 23.8 +/- 4.1 mM (N = 9), 3.6 +/- 0.6 mM (N = 11), and 226 +/- 93 microM (N = 12); and GSHEt2 20.4 +/- 1.9 mM (N = 6), 603 +/- 2 microM (N = 9), and 7.6 +/- 2.3 microM (N = 12). Reduced glutathione ethyl diester was a potent antidote to CDNB- and menadione-induced toxicities but not to hydrogen peroxide-induced toxicity under acute intoxication conditions.

Comparison of the delivery of reduced glutathione into P388D1 cells by reduced glutathione and its mono- and diethyl ester derivatives.

The effect of reduced glutathione, reduced glutathione monoethyl ester and reduced glutathione diethyl ester on the cellular concentration of reduced glutathione and cysteine in P388D1 macrophages in vitro, and the cellular and extracellular de-esterification of reduced glutathione esters, was investigated. At 1 mM reduced glutathione derivative, only reduced glutathione diester markedly increased the cellular concentration of reduced glutathione. There was little delivery of reduced glutathione monoethyl ester into the cells. Reduced glutathione, and monoethyl and diethyl ester derivatives all increased the cellular concentration of cysteine; reduced glutathione diethyl ester also increased the cellular concentration of gamma-glutamylcysteine. Reduced glutathione diethyl ester also increased the cellular concentration of gamma-glutamylcysteine. Reduced glutathione esters were de-esterified intracellularly where the diester was rapidly converted to the monoester. The diester was also converted to the monoester extracellularly by interaction with cell surface esterases and by a much slower spontaneous hydrolysis. This indicates that the diester of reduced glutathione was a much more effective vehicle for delivery of reduced glutathione into cells than the monoester. Reduced glutathione diester also increased the cellular concentrations of cysteine and gamma-glutamylcysteine, suggesting that de novo synthesis of reduced glutathione was also stimulated.