Antiglycation effect of gliclazide on in vitro AGE formation from glucose and methylglyoxal

Gliclazide, a sulfonylurea widely used for treatment of diabetes mellitus, is known to scavenge reactive oxygen species. To clarify whether its antioxidative ability interferes with the glycation processes, we incubated bovine serum albumin (BSA) with 1 M glucose or 1 mM methylglyoxal, in the presence or absence of gliclazide, and observed the formation of advanced glycation end products (AGEs). AGE production was assessed by AGE-specific fluorescence, an enzyme-linked immunosorbent assay (ELISA), and Western blotting. The fluorescence at excitation/emission wavelengths of 320/383 nm and 335/385 nm was definitely increased by incubating BSA with 1 M glucose or 1 mM methylglyoxal, and 1 mM gliclazide significantly blunted the fluorescent augmentation, in both wavelengths, in a dose-dependent fashion. Gliclazide almost equaled to aminoguanidine, a putative antiglycation agent, in the inhibitory effect on the glucose-induced fluorescence, while the methylglyoxal-derived fluorescent formation was less suppressed by gliclazide than by aminoguanidine. The AGE concentrations determined by ELISA showed similar results. Incubation of BSA with 1 M glucose or 1 mM methylglyoxal yielded an apparent increase in carboxymethyllysine or argpyrimidine. Both AGEs were significantly lowered by 1 mM gliclazide and a reduction of glucose-derived carboxymethyllysine was comparable to that caused by aminoguanidine. The results of Western blotting supported the findings in ELISA. To our knowledge, the present study provides the first evidence of the antiglycation effect of gliclazide on in vitro AGE formation from glucose and methylglyoxal.     

The polyamines spermine and spermidine protect proteins from structural and functional damage by AGE precursors: a new role for old molecules?

Due to the importance of glycation in the genesis of diabetic complications, an intense search for synthetic new antiglycation agents is ongoing. However, a somewhat neglected avenue is the search for endogenous compounds that may inhibit the process and be a source of protodrugs. Based on their ubiquity, their polycationic nature, their essential role in growth, their relatively high concentrations in tissues, and their high concentrations in sperm, we hypothesized that polyamines inhibit glycation and that might be one of their so far elusive functions. In this study we demonstrate a potent antiglycation effect of physiological concentrations of the polyamines spermine and spermidine. We employed two approaches: in the first, we monitored structural changes on histones and ubiquitin in which polyamines inhibit glycation-induced dimer and polymer formation. In the second we monitored functional impairment of catalytic activity of antithrombin III and plasminogen. Protection is afforded against glycation by hexoses, trioses and dicarbonyls AGE precursors and is comparable to those of aminoguanidine and carnosine.     

Inhibition of human muscle-specific enolase by methylglyoxal and irreversible formation of advanced glycation end products

Methylglyoxal (MG) was studied as an inhibitor and effective glycating factor of human muscle-specific enolase. The inhibition was carried out by the use of a preincubation procedure in the absence of substrate. Experiments were performed in anionic and cationic buffers and showed that inhibition of enolase by methylglyoxal and formation of enolase-derived glycation products arose more effectively in slight alkaline conditions and in the presence of inorganic phosphate. Incubation of 15 micromolar solutions of the enzyme with 2 mM, 3.1 mM and 4.34 mM MG in 100 mM phosphate buffer pH 7.4 for 3 h caused the loss a 32%, 55% and 82% of initial specific activity, respectively. The effect of MG on catalytic properties of enolase was investigated. The enzyme changed the K(M) value for glycolytic substrate 2-phospho-D-glycerate (2-PGA) from 0.2 mM for native enzyme to 0.66 mM in the presence of MG. The affinity of enolase for gluconeogenic substrate phosphoenolpyruvate altered after preincubation with MG in the same manner, but less intensively. MG has no effect on V(max) and optimal pH values. Incubation of enolase with MG for 0-48 h generated high molecular weight protein derivatives. Advanced glycation end products (AGEs) were resistant to proteolytic degradation by trypsin. Magnesium ions enhanced the enzyme inactivation by MG and facilitated AGEs formation. However, the protection for this inhibition in the presence of 2-PGA as glycolytic substrate was observed and AGEs were less effectively formed under these conditions.     

Inhibition of human muscle-specific enolase by methylglyoxal and irreversible formation of advanced glycation end products

Methylglyoxal (MG) was studied as an inhibitor and effective glycating factor of human muscle-specific enolase. The inhibition was carried out by the use of a preincubation procedure in the absence of substrate. Experiments were performed in anionic and cationic buffers and showed that inhibition of enolase by methylglyoxal and formation of enolase-derived glycation products arose more effectively in slight alkaline conditions and in the presence of inorganic phosphate. Incubation of 15 micromolar solutions of the enzyme with 2 mM, 3.1 mM and 4.34 mM MG in 100 mM phosphate buffer pH 7.4 for 3 h caused the loss a 32%, 55% and 82% of initial specific activity, respectively. The effect of MG on catalytic properties of enolase was investigated. The enzyme changed the K_M value for glycolytic substrate 2-phospho-D-glycerate (2-PGA) from 0.2 mM for native enzyme to 0.66 mM in the presence of MG. The affinity of enolase for gluconeogenic substrate phosphoenolpyruvate altered after preincubation with MG in the same manner, but less intensively. MG has no effect on V_max and optimal pH values. Incubation of enolase with MG for 0-48 h generated high molecular weight protein derivatives. Advanced glycation end products (AGEs) were resistant to proteolytic degradation by trypsin. Magnesium ions enhanced the enzyme inactivation by MG and facilitated AGEs formation. However, the protection for this inhibition in the presence of 2-PGA as glycolytic substrate was observed and AGEs were less effectively formed under these conditions.     

Isoferulic acid action against glycation-induced changes in structural and functional attributes of human high-density lipoprotein

Glycation-induced high-density lipoprotein (HDL) modification by aldehydes can result in loss of its antiinflammatory/antioxidative properties, contributing to diabetes-associated cardiovascular diseases. Isoferulic acid, a major active ingredient of Cimicifuga heracleifolia, shows antiinflammatory, antiviral, antioxidant, and antidiabetic properties. Thus, this study investigated the antiglycation effect of isoferulic acid against compositional modifications of HDL and loss of biological activity of HDL-paraoxonase induced on incubation with different aldehydes. Protective effect of isoferulic acid was assessed by subjecting purified HDL from human plasma to glycation with methylglyoxal, glyoxal, or glycolaldehyde and varying concentrations of isoferulic acid. The effect of isoferulic acid was analyzed by determining amino group number, tryptophan and advanced glycation end-product fluorescence, thermal denaturation studies, carboxymethyl lysine content, and activity of HDL-paraoxonase. Concentration-dependent inhibitory action of isoferulic acid was observed against extensive structural perturbations, decrease in amino group number, increase in carboxymethyl lysine content, and decrease in the activity of HDL-paraoxonase caused by aldehyde-associated glycation in the HDL molecule. Isoferulic acid, when taken in concentration equal to that of aldehydes, was most protective, as 82-88% of paraoxonase activity was retained for all studied aldehydes. Isoferulic acid shows antiglycation action against aldehyde-associated glycation in HDL, which indicates its therapeutic potential for diabetic patients, especially those with micro-/macrovascular complications.     

Increased formation of methylglyoxal and protein glycation, oxidation and nitrosation in triosephosphate isomerase deficiency

Triosephosphate isomerase deficiency is associated with the accumulation of dihydroxyacetonephosphate (DHAP) to abnormally high levels, congenital haemolytic anaemia and a clinical syndrome of progressive neuromuscular degeneration leading to infant mortality. DHAP degrades spontaneously to methylglyoxal (MG)--a potent precursor of advanced glycation endproducts (AGEs). MG is detoxified to D-lactate intracellularly by the glyoxalase system. We investigated the changes in MG metabolism and markers of protein glycation, oxidation and nitrosation in a Hungarian family with two germline identical brothers, compound heterozygotes for triosephosphate isomerase deficiency, one with clinical manifestations of chronic neurodegeneration and the other neurologically intact. The concentration of MG and activity of glyoxalase I in red blood cells (RBCs) were increased, and the concentrations of D-lactate in blood plasma and D-lactate urinary excretion were also increased markedly in the propositus. There were concomitant increases in MG-derived AGEs and the oxidative marker dityrosine in hemoglobin. Smaller and nonsignificant increases were found in the neurologically unaffected brother and parents. There was a marked increase (15-fold) in urinary excretion of the nitrosative stress marker 3-nitrotyrosine in the propositus. The increased derangement of MG metabolism and associated glycation, oxidative and nitrosative stress in the propositus may be linked to neurodegenerative process in triosephosphate isomerase deficiency.     

Dual effect of methylglyoxal on the intracellular Ca2+ signaling and neurite outgrowth in mouse sensory neurons

The formation of advanced glycation end products is one of the major factors involved in diabetic neuropathy, aging, and neurodegenerative diseases. Reactive carbonyl compounds, such as methylglyoxal (MG), play a key role in cross-linking to various proteins in the extracellular matrix, especially in neurons, which have a high rate of oxidative metabolism. The MG effect was tested on dorsal root ganglia primary neurons in cultures from adult male Balb/c mice. Lower MG doses contribute to an increased adherence of neurons on their support and an increased glia proliferation, as proved by MTS assay and bright-field microscopy. Time-lapse fluorescence microscopy by Fura-2 was performed for monitoring the relative fluorescence ratio changes (ΔR/R(0)) upon depolarization and immunofluorescence staining for quantifying the degree of neurites extension. The relative change in fluorescence ratio modifies the amplitude and dispersion depending on the subtype of sensory neurons, the medium-sized neurons are more sensitive to MG treatment when compared to small ones. Low MG concentrations (0-150 μM) increase neuronal viability, excitability, and the capacity of neurite extension, while higher concentrations (250-750 μM) are cytotoxic in a dose-dependent manner. In our opinion, MG could be metabolized by the glyoxalase system inside sensory neurons up to a threshold concentration, afterwards disturbing the cell equilibrium. Our study points out that MG has a dual effect concentration dependent on the neuronal viability, excitability, and neurite outgrowth, but only the excitability changes are soma-sized dependent. In conclusion, our data may partially explain the distinct neuronal modifications in various neurodegenerative pathologies.     

Induction of sister-chromatid exchanges in Chinese hamster ovary cells by the biotic ketoaldehyde methylglyoxal

The number of sister-chromatid exchanges (SCEs) per metaphase was determined in Chinese hamster ovary cells after 16 h exposure to methylglyoxal (MG) concentrations ranging from 0.1 to 0.75 mM. MG produced an increase of SCE frequency that proved to be dose-dependent, and to reach a maximum of 2 X baseline at the highest nontoxic concentration (0.5 mM).     

The pro-oxidant role of methylglyoxal in mesenteric artery smooth muscle cells

Methylglyoxal (MG), a highly reactive metabolite of glucose, causes non-enzymatic glycation of proteins to form irreversible advanced glycation endproducts (AGEs). The present study investigated whether methylglyoxal induced oxidative stress and activated nuclear factor kappa B (NF-kappaB) in freshly isolated and cultured smooth muscle cells (SMCs) from rat mesenteric artery. The treatment of cells with MG (50 or 100 micromol/L) induced a significant increase in AGE formation and oxidation of DCF. MG-enhanced generation of AGEs and the oxidation of DCF was markedly inhibited by antioxidant n-acetylcysteine (NAC, 600 micromol/L). MG at a concentration of 100 micromol/L increased the heme-oxygenase-1 expression in these cells. Moreover, MG activated NF-kappaB p65, indicated by an increased immuno cytochemistry stain for NF-kappaB p65 located in the nucleus after the treatment of mesenteric artery SMCs with MG. MG-induced activation of NF-kappaB p65 was inhibited by NAC. In summary, MG significantly increases oxidative stress and activates NF-kappaB p65 in mesenteric artery SMCs. The pro-oxidant role of methylglyoxal may contribute to various pathological changes of SMCs from resistance arteries.     

Monitoring nonenzymatic glycation of human immunoglobulin G by methylglyoxal and glyoxal: A spectroscopic study

The accumulation of dicarbonyl compounds, methylglyoxal (MG) and glyoxal (G), has been observed in diabetic conditions. They are formed from nonoxidative mechanisms in anaerobic glycolysis and lipid peroxidation, and they act as advanced glycation endproduct (AGE) precursors. The objective of this study was to monitor and characterize the AGE formation of human immunoglobulin G (hIgG) by MG and G using ultraviolet (UV) and fluorescence spectroscopy, circular dichroism (CD), and matrix-assisted laser desorption/ionization–mass spectrometry (MALDI–MS). hIgG was incubated over time with MG and G at different concentrations. Formation of AGE was monitored by UV and fluorescence spectroscopy. The effect of AGE formation on secondary structure of hIgG was studied by CD. Comparison of AGE profile for MG and G was performed by MALDI–MS. Both MG and G formed AGE, with MG being nearly twice as reactive as G. The combination of these techniques is a convenient method for evaluating and characterizing the AGE proteins.     

Site-selective modifications of arginine residues in human hemoglobin induced by methylglyoxal

Methylglyoxal (MG) is an important glycating agent produced under physiological conditions. MG could react with DNA and proteins to generate advanced glycation end products. Human hemoglobin, the most abundant protein in blood cells, has not been systematically investigated as the target protein for methylglyoxal modification. Here we examined carefully, by using HPLC coupled with tandem mass spectrometry (LC-MS/MS), the covalent modifications of human hemoglobin induced by methylglyoxal. Our results revealed that hemoglobin could be modified by methylglyoxal, and the major form of modification was found to be the hydroimidazolone derivative of arginine residues. In addition, Arg-92 and Arg-141 in the alpha chain as well as Arg-40 and Arg-104 in the beta chain were modified, whereas two other arginine residues, that is, Arg-31 in the alpha chain and Arg-30 in the beta chain, were not modified. Semiquantitative measurement for adduct formation, together with the analysis of the X-ray structure of hemoglobin, showed that the extents of arginine modification were highly correlated with the solvent accessibilities of these residues. The facile formation of hydroimidazolone derivatives of arginine residues in hemoglobin by methylglyoxal at physiologically relevant concentrations suggested that this type of modification might occur in vivo. The unambiguous determination of the sites and extents of methylglyoxal modifications of arginines in hemoglobin provided a basis for understanding the implications of these modifications and for employing this type of hemoglobin modification as molecular biomarkers for clinical applications.     

Modulation of plasminogen activation and plasmin activity by methylglyoxal modification of the zymogen

The effect of methylglyoxal on the plasminogen-plasmin system is studied. Treatment of plasminogen with methylglyoxal at a 20-fold molar excess results in covalent modification of the molecule as evidenced by the decreased number of NH(2) side chains, arginine side chain residues and the new band in the non-tryptophan dependent fluorescent spectrum. This structural modification is associated with profound functional alterations: the rate of activation by streptokinase, tissue-type plasminogen activator, urokinase-type plasminogen activator and trypsin decreases and the amidolytic activity of the generated plasmin is impaired. Plasmin treatment with methylglyoxal on the other hand does not alter its steady-state kinetic parameters on a peptidyl-anilide synthetic substrate, indicating that modification susceptible side chains are sensitive to methylglyoxal only in the zymogen. Our data suggest that in vivo fibrinolysis could be impaired under pathological conditions, e.g. increased methylglyoxal formation in diabetes mellitus.     

Effect of Micronutrients on Methylglyoxal-Mediated In Vitro Glycation of Albumin

Nonenzymatic glycation of long-lived proteins has been implicated in several complications related to age and diabetes. Dicarbonyl compounds such as methylglyoxal (MGO) have been identified as the predominant source for the formation of advanced glycation end-products (AGEs) in various tissues. We investigated the effect of 13 micronutrients on MGO-mediated in vitro glycation of bovine serum albumin (BSA), as formation of AGEs and protein carbonyls. BSA (10 mg/ml) was incubated at 37°C with 100 mM MGO for 24 hours, in presence of ascorbic acid, Trolox (water-soluble α-tocopherol analog), β-carotene, retinol, riboflavin, thiamin, folic acid, niacin, pyridoxine, zinc, iron, manganese, and selenium. Fluorescence was measured at the wavelength pair of 370 and 440 nm as an index of the formation of AGEs and spectra were recorded for promising interactions at λex = 280 nm and λex = 370 nm. Within four standard antiglycating agents, aminoguanidine showed highest inhibitory response for BSA glycation followed by quercetin, gallic acid, and tannic acid. Promising antiglycation potential was seen for Trolox, riboflavin, Zn, and Mn as evidenced by decrease in the formation of AGEs and protein carbonyls.     

Increased glyoxalase I levels inhibit accumulation of oxidative stress and an advanced glycation end product in mouse mesangial cells cultured in high glucose

Chronic high glucose levels lead to the formation of advanced glycation end-products (AGEs) as well as AGE precursors, such as methylglyoxal (MG) and glyoxal, via non-enzymatic glycation reactions in patients with diabetic mellitus. Glyoxalase 1 (GLO-1) detoxifies reactive dicarbonyls that form AGEs. To investigate the interaction between AGEs and GLO-1 in mesangial cells (MCs) under diabetic conditions, AGE levels and markers of oxidative stress were measured in GLO-1-overexpressing MCs (GLO-1-MCs) cultured in high glucose. Furthermore, we also examined levels of high glucose-induced apoptosis in GLO-1-MCs. In glomerular MCs, high glucose levels increased the formation of both MG and argpyrimidine (an MG-derived adduct) as well as GLO-1 expression. GLO-1-MCs had lower intracellular levels of MG accumulation, 8-hydroxy-deoxyguanosine (an oxidative DNA damage marker), 4-hydroxyl-2-nonenal (a lipid peroxidation product), and nitrosylated protein (a marker of oxidative-nitrosative stress) compared to control cells. Expression of mitochondrial oxidative phosphorylation complexes I, II, and III was also decreased in GLO-1-MCs. Furthermore, fewer GLO-1-MCs showed evidence of apoptosis as determined by terminal deoxynucleotidyl transferase-mediated dUTP nick labeling assay, and activation of both poly (ADP-ribose) polymerase 1 cleavage and caspase-3 was lower in GLO-1-MCs than in control cells cultured in high glucose. These results suggest that GLO-1 plays a role in high glucose-mediated signaling by reducing MG accumulation and oxidative stress in diabetes mellitus.     

A novel source of methylglyoxal and glyoxal in retina: implications for age-related macular degeneration

Aging of retinal pigment epithelial (RPE) cells of the eye is marked by accumulations of bisretinoid fluorophores; two of the compounds within this lipofuscin mixture are A2E and all-trans-retinal dimer. These pigments are implicated in pathological mechanisms involved in some vision-threatening disorders including age-related macular degeneration (AMD). Studies have shown that bisretinoids are photosensitive compounds that undergo photooxidation and photodegradation when irradiated with short wavelength visible light. Utilizing ultra performance liquid chromatography (UPLC) with electrospray ionization mass spectrometry (ESI-MS) we demonstrate that photodegradation of A2E and all-trans-retinal dimer generates the dicarbonyls glyoxal (GO) and methylglyoxal (MG), that are known to modify proteins by advanced glycation endproduct (AGE) formation. By extracellular trapping with aminoguanidine, we established that these oxo-aldehydes are released from irradiated A2E-containing RPE cells. Enzyme-linked immunosorbant assays (ELISA) revealed that the substrate underlying A2E-containing RPE was AGE-modified after irradiation. This AGE deposition was suppressed by prior treatment of the cells with aminoguanidine. AGE-modification causes structural and functional impairment of proteins. In chronic diseases such as diabetes and atherosclerosis, MG and GO modify proteins by non-enzymatic glycation and oxidation reactions. AGE-modified proteins are also components of drusen, the sub-RPE deposits that confer increased risk of AMD onset. These results indicate that photodegraded RPE bisretinoid is likely to be a previously unknown source of MG and GO in the eye.     

The proglycation effect of caffeic acid leads to the elevation of oxidative stress and inflammation in monocytes, macrophages and vascular endothelial cells

In this study, the effects of phenolic acids [caffeic acid (CA), ferulic acid, m-coumaric acid, and chlorogenic acid] on methylglyoxal (MG)-induced protein glycation were investigated in vitro. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and advanced glycation end products (AGEs)-specific fluorescence showed that MG-mediated protein modification was enhanced dose-dependently by CA (P<.05), whereas α-lipoic acid, glutathione and EDTA inhibited these changes. Electron paramagnetic resonance spectra showed that CA increased reactive oxygen species (ROS) production during glycation, suggesting the proglycation mechanism of CA is associated with its pro-oxidative properties. Additionally, fetal bovine serum (FBS) was utilized as the source of target proteins for evaluating the effects of CA in cells. Differential glycation of FBS samples was performed by incubating FBS with MG, CA or aminoguanidine (AG, an AGE inhibitor). FBS incubated with MG and CA (MG/CA-FBS) evoked the greatest deleterious responses, as follows: (1) inducing proinflammatory tumor necrosis factor (TNF)-α and interleukin-1β expression and ROS production in monocytic THP-1 cells, (2) stimulating TNF-α secretion in RAW 264.7 macrophages and (3) causing oxidative DNA damage and inducing the expression of receptor for AGEs (RAGE), intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 in human umbilical vein endothelial cells. Furthermore, adhesion and transendothelial migration of monocytes were also significantly increased by MG/CA-FBS treatment compared to MG-FBS (P<.05). In conclusion, our data show that CA exhibits pro-oxidative and pro-glycative effects during the glycation process, suggesting a detrimental role for CA under high-glycotoxin conditions.     

Dimerumic acid attenuates receptor for advanced glycation endproducts signal to inhibit inflammation and diabetes mediated by Nrf2 activation and promotes methylglyoxal metabolism into d-lactic acid

This study was designed to evaluate the effects of dimerumic acid (DMA) on receptor for advanced glycation endproducts (RAGE) signal activation and THP-1 monocyte inflammation treated with S100b, a specific ligand of RAGE. We found that DMA inhibited inflammatory cytokine production via upregulation of nuclear factor-erythroid 2-related factor 2 (Nrf2) and alleviated oxidative stress through attenuation of p47phox translocation to the membrane of S100b-treated THP-1 monocytes. We found that DMA activated Nrf2 mediated by the p38 kinase pathway in THP-1 monocytes. However, anti-inflammatory activity of DMA was attenuated by Nrf2 siRNA treatment. In an animal model, methylglyoxal (MG; 200 mg/kg bw) was chosen to induce diabetes in Balb/C mice (6 weeks) in this work. The in vivo verification of anti-inflammation in peripheral blood mononuclear cells by DMA treatment was confirmed by tumor necrosis factor-α and interleukin-1β measurements. Oral glucose tolerance test, insulin tolerance test, hyperinsulinemia, and hyperglycemia were improved in MG-treated mice by DMA treatment and these effects were greater than those of silymarin and N-acetylcysteine. Furthermore, DMA increased hepatic glyoxalase mRNA and glutathione mediated by Nrf2 activation to metabolize MG into d-lactic acid, thereby reducing serum and hepatic AGE levels and suppressing inflammatory factor generation in MG-treated mice. However, DMA did not exert the antiglycation activity in MG–bovine serum albumin incubation. Taken together, the results indicate that DMA is a novel antioxidant and Nrf2 activator that lowers AGE levels and may prove to be an effective treatment for diabetes.     

Fisetin lowers methylglyoxal dependent protein glycation and limits the complications of diabetes

The elevated glycation of macromolecules by the reactive dicarbonyl and α-oxoaldehyde methylglyoxal (MG) has been associated with diabetes and its complications. We have identified a rare flavone, fisetin, which increases the level and activity of glyoxalase 1, the enzyme required for the removal of MG, as well as the synthesis of its essential co-factor, glutathione. It is shown that fisetin reduces two major complications of diabetes in Akita mice, a model of type 1 diabetes. Although fisetin had no effect on the elevation of blood sugar, it reduced kidney hypertrophy and albuminuria and maintained normal levels of locomotion in the open field test. This correlated with a reduction in proteins glycated by MG in the blood, kidney and brain of fisetin-treated animals along with an increase in glyoxalase 1 enzyme activity and an elevation in the expression of the rate-limiting enzyme for the synthesis of glutathione, a co-factor for glyoxalase 1. The expression of the receptor for advanced glycation end products (RAGE), serum amyloid A and serum C-reactive protein, markers of protein oxidation, glycation and inflammation, were also increased in diabetic Akita mice and reduced by fisetin. It is concluded that fisetin lowers the elevation of MG-protein glycation that is associated with diabetes and ameliorates multiple complications of the disease. Therefore, fisetin or a synthetic derivative may have potential therapeutic use for the treatment of diabetic complications.     

Effect of pyridoxamine on chemical modification of proteins by carbonyls in diabetic rats: characterization of a major product from the reaction of pyridoxamine and methylglyoxal

Advanced glycation end products (AGEs) from the Maillard reaction contribute to protein aging and the pathogenesis of age- and diabetes-associated complications. The alpha-dicarbonyl compound methylglyoxal (MG) is an important intermediate in AGE synthesis. Recent studies suggest that pyridoxamine inhibits formation of advanced glycation and lipoxidation products. We wanted to determine if pyridoxamine could inhibit MG-mediated Maillard reactions and thereby prevent AGE formation. When lens proteins were incubated with MG at 37 degrees C, pH 7.4, we found that pyridoxamine inhibits formation of methylglyoxal-derived AGEs concentration dependently. Pyridoxamine reduces MG levels in red blood cells and plasma and blocks formation of methylglyoxal-lysine dimer in plasma proteins from diabetic rats and it prevents pentosidine (an AGE derived from sugars) from forming in plasma proteins. Pyridoxamine also decreases formation of protein carbonyls and thiobarbituric-acid-reactive substances in plasma proteins from diabetic rats. Pyridoxamine treatment did not restore erythrocyte glutathione (which was reduced by almost half) in diabetic animals, but it enhanced erythrocyte glyoxalase I activity. We isolated a major product of the reaction between MG and pyridoxamine and identified it as methylglyoxal-pyridoxamine dimer. Our studies show that pyridoxamine reduces oxidative stress and AGE formation. We suspect that a direct interaction of pyridoxamine with MG partly accounts for AGE inhibition.