Methylglyoxal Formation Research Articles

Carbonyl stress phenomena during chronic infection with Opisthorchis felineus

Infection with the fish borne liver fluke Opisthorchis felineus is common in the Eastern Europe (Ukraine, European part of Russia), Northern Asia (Siberia) and Central Asia (Northern Kazakhstan). Better understanding of the molecular pathogenesis of the biliary tract and liver during chronic opisthorchiasis can be expected to improve protection against and management of complications of this disease. We hypothesize that infection with O. felineus associates with formation of methylglyoxal and carbonyl stress in the liver and hence here we investigated the glyoxalase system and the receptor for advanced glycated end products (RAGE) in the liver of hamsters infected with this liver fluke. Expression of mRNA encoding glyoxalase 1 decreased at 8weeks of the infection and catalytic activity as well decreased at 8 and 12weeks after infection, and the expression of the glyoxalase 2 decreased until 36week post-infection, which associated with the decreasing activity of the enzyme at 8 and 12weeks post-infection. Glutathione levels in infected livers had decreased at week 8, whereas up-regulation of RAGE at mRNA levels was seen for the extended duration of the experimental infection of the hamsters. This outcome supported the notion of hepatic dicarbonyl stress during chronic opisthorchiasis. The inhibition of the glyoxalase system and accumulation of methylglyoxal at the early stages of the infection may underpin development of insulin resistance during opisthorchiasis.

Influence of the presence of natural monosaccharides in the quantification of α-dicarbonyl compounds in high content sugar samples. A comparative study by ultra-high performance liquid chromatography–single quadrupole mass spectrometry using different derivatization reactions

The goal of this work is to evaluate the formation of side α-dicarbonyl compounds in high content sugar samples. These compounds may be originated from fructose and glucose, during different derivatization reactions. The formation of D-glucosone, 3-deoxyglucosone, glyoxal, and methylglyoxal, using three derivatization agents (5,6-diamino-2,4-hydroxypyrimidine, 2,4,5-triamine-6-hydroxypyrimidine, and o-phenylenediamine), and ultra-high pressure liquid chromatography in combination with MS detection, has been assessed in the presence of different levels of monosaccharides. 2,4,5-triamine-6-hydroxy-pyrimidine appears to be the most suitable for the analysis of α-dicarbonyl compounds, in this kind of food samples, and was selected as optimum reagent for the quantification of these compounds. The validation of the method was performed through the establishment of external standard calibration curves and analytical figures of merit, and it showed good linearity over a wide concentration range (r2>0.99), and limits of detection and quantification lower than 42μgL−1 and 142μgL−1, respectively. The validated method has been successfully applied to the determination of the target compounds in honey samples. The intraday and interday assay variability in the analysis of real samples was below 2.3 and 5.7%, respectively, for all analytes.

Role of Glucose Derived Reactive Metabolites in Diabetic Nephropathy

Discovery of differences in the formation of triose phosphate intermediates (TPint) and methylglyoxal (MG) by cell metabolism between mouse strains with identical levels of hyperglycemia suggested prospective importance to diabetes complications. To assess the possible relevance of triose phosphates [glyceraldehyde-3-phosphate and dihydroxyacetone phosphate] in modulating diabetic nephropathy (DN) in type 1 diabetes mellitus (T1DM) patients. A case–control study was conducted among 131 unrelated T1DM with (59 patients) or without nephropathy (72 patients) and 146 non-diabetic controls. Using red blood cells, metabolites of glyoxalase system and triose phosphates were analyzed in deproteinated hemolysate samples. Suitable descriptive statistics was used for different variables. Cross-sectional measures of TPint/mol of glucose in red blood cells were significantly increased in nephropathy cohort relative to diabetic patients with normal albumin excretion/healthy controls (p < 0.0001) and a concomitant increase in MG (R2: 0.3721; p < 0.0001) was observed. Decreased glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity correlated with increased MG levels (R2: 0.0841; p: 0.0259, Table 1) and TPint (R2: 0.0676; p: 0.0467; Table 1) in the DN cohort, but not in either diabetic group with normal albumin excretion or healthy controls. The concentration of d-lactate and activity of glyoxalase I in RBCs were increased markedly in the DN cohort as compared to other studied groups (p < 0.05). The surrogate markers for oxidative stress (thiobarbituric acid reactive substances and glutathione) in DN cohort were all within the range observed in the other studied groups. Diabetic patients who have an elevated levels of intracellular TPint and consequently the chronic exposure to high MG concentrations appears to be susceptible to the development of renal diabetic complications, despite good glycemic control and possibly related to modification of GAPDH by environmental factors or inherent differences. Table 1 Correlation coefficients for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with various biochemical measurements Healthy controls Diabetic patients with normal AER Diabetic nephropathy patients with macroalbuminuria R2 p R2 p R2 p HbA1c (%) 0.012 0.1783 0.0282 0.1584 0.017 0.319 Hemosylate glucose (nmol/mgHb) 0.0046 0.4148 0.0065 0.4988 0.0153 0.3494 TPint (nmol/mgHb) 0.0166 0.1207 0.0050 0.5534 0.0676 0.0467 MG (nmol/mgHb) 0.0021 0.5814 0.0037 0.6107 0.0841 0.0259 GSH 0.0059 0.3556 0.0077 0.4623 0.0065 0.542 TBARS 0.0028 0.5252 0.0019 0.7136 0.0088 0.4789 HbA1C glycated hemoglobin, TPint triose phosphate intermediates, MG methylglyoxal, GSH reduced glutathione, TBARS thiobarbituric acid reactive substances

The Role Formation of Methylglyoxal, Carbonyl Compound, Hydrogen Peroxide and Advance Oxidation Protein Product Induced Cadmium in Ovarian Rat

The Role Formation of Methylglyoxal, Carbonyl Compound, Hydrogen Peroxide and Advance Oxidation Protein Product Induced Cadmium in Ovarian Rat

The Role of Cadmium in Proteins Glycation by Glucose: Formation of Methylglyoxal and Hydrogen Peroxide in Vitro

The Role of Cadmium in Proteins Glycation by Glucose: Formation of Methylglyoxal and Hydrogen Peroxide in Vitro

The Critical Role of Methylglyoxal and Glyoxalase 1 in Diabetic Nephropathy

The discovery of increased formation of methylglyoxal (MG) by cell metabolism in high glucose concentration in vitro suggested possible relevance to diabetes and diabetes complications (1,2). MG is the precursor of quantitatively important advanced glycation end products (AGEs) of protein and DNA- and MG-derived AGEs increase in experimental and clinical diabetes (3,4). Increased MG and its metabolism by glyoxalase 1 (Glo1) was linked to clinical microvascular complications (nephropathy, retinopathy, and neuropathy) (5). Current clinical treatment decreasing MG and MG-derived AGEs, such as insulin lispro (6,7), has some clinical benefit in diabetic nephropathy (8), although the decrease in MG-derived AGE exposure is minor—∼17% (7). Greater benefits may be achieved with specific and effective anti-MG targeted therapy. An outstanding research problem is to gain unequivocal evidence that MG glycation is a key mediator of vascular complications and, if possible, provide some pointers as to how MG glycation could be effectively countered. In this issue, the study by Giacco et al. (9) provides key evidence by a functional genomic approach manipulating expression of Glo1 to increase or decrease endogenous MG glycation. The outcomes show that development of experimental diabetic nephropathy is driven by increased levels of MG glycation and increasing renal expression of Glo1 prevents this. Recent research has shown Glo1 expression may be increased …

A mitochondria-targeted mass spectrometry probe to detect glyoxals: implications for diabetes

The glycation of protein and nucleic acids that occurs as a consequence of hyperglycemia disrupts cell function and contributes to many pathologies, including those associated with diabetes and aging. Intracellular glycation occurs after the generation of the reactive 1,2-dicarbonyls methylglyoxal and glyoxal, and disruption of mitochondrial function is associated with hyperglycemia. However, the contribution of these reactive dicarbonyls to mitochondrial damage in pathology is unclear owing to uncertainties about their levels within mitochondria in cells and in vivo. To address this we have developed a mitochondria-targeted reagent (MitoG) designed to assess the levels of mitochondrial dicarbonyls within cells. MitoG comprises a lipophilic triphenylphosphonium cationic function, which directs the molecules to mitochondria within cells, and an o-phenylenediamine moiety that reacts with dicarbonyls to give distinctive and stable products. The extent of accumulation of these diagnostic heterocyclic products can be readily and sensitively quantified by liquid chromatography–tandem mass spectrometry, enabling changes to be determined. Using the MitoG-based analysis we assessed the formation of methylglyoxal and glyoxal in response to hyperglycemia in cells in culture and in the Akita mouse model of diabetes in vivo. These findings indicated that the levels of methylglyoxal and glyoxal within mitochondria increase during hyperglycemia both in cells and in vivo, suggesting that they can contribute to the pathological mitochondrial dysfunction that occurs in diabetes and aging.

Changes in glyoxal and methylglyoxal content in the fried dough twist during frying and storage

Glyoxal (GO) and methylglyoxal (MGO) are important precursors for advanced glycation end products (AGEs) in foods and in vivo, which are also known as reactive carbonyls. Accumulation of AGEs may result in health risk. The effect of frying time and temperature on GO and MGO formation during fried dough twist processing was investigated. The changes in GO and MGO content of fried dough twist were also examined under the different storage condition. Sample extraction, derivatization, and cleanup conditions were evaluated to provide a sample preparation method for GO and MGO analysis by RP-HPLC–DAD. Quantification limits of GO and MGO were 0.01 and 0.02 mg/L for fried dough twist, respectively. The average recovery rate was 89 % with detection relative standard deviation of lower than 5.7 %. During frying and prolonged storage, GO and MGO contents in fried dough twist ranged from 6.5 to 11.8 mg/kg and from 5.1 to 12.0 mg/kg, respectively. The results indicated that content of GO and MGO in fried dough twist depended on the frying time and temperature. For GO, no significant differences (p < 0.05) among the all types of samples were found during the whole process of storage, with the exception of storage temperature after 60-day storage. Changes in MGO occurred during the 60-day storage, indicating storage temperature, oxygen content, lighting, and storage duration, exert a significant effect on the development of the MGO.

Edaravone Protected Human Brain Microvascular Endothelial Cells from Methylglyoxal-Induced Injury by Inhibiting AGEs/RAGE/Oxidative Stress

Subjects with diabetes experience an increased risk of cerebrovascular disease and stroke compared with nondiabetic age-matched individuals. Increased formation of reactive physiological dicarbonyl compound methylglyoxal (MGO) seems to be implicated in the development of diabetic vascular complication due to its protein glycation and oxidative stress effect. Edaravone, a novel radical scavenger, has been reported to display the advantageous effects on ischemic stroke both in animals and clinical trials; however, little is known about whether edaravone has protective effects on diabetic cerebrovascular injury. Using cultured human brain microvascular endothelial cells (HBMEC), protective effects of edaravone on MGO and MGO enhancing oxygen-glucose deprivation (OGD) induced injury were investigated. Cell injury was measured by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) formation, cell account, lactate dehydrogenase (LDH) release and Rhodamine 123 staining. Advanced glycation end-products (AGEs) formation and receptor for advanced glycation end-products (RAGE) expression were measured by western blotting. Cellular oxidative stress was measured by reactive oxygen species (ROS) release. Treatment of MGO for 24 h significantly induced HBMEC injury, which was inhibited by pretreatment of edaravone from 10–100 µmol/l. What’s more, treatment of MGO enhanced AGEs accumulation, RAGE expression and ROS release in the cultured HBMEC, which were inhibited by 100 µmol/l edaravone. Finally, treatment of MGO for 24 h and then followed by 3 h OGD insult significantly enhanced cell injury when compared with OGD insult only, which was also protected by 100 µmol/l edaravone. Thus, edaravone protected HBMEC from MGO and MGO enhancing OGD-induced injury by inhibiting AGEs/RAGE/oxidative stress.

Increased Methylglyoxal Formation with Upregulation of Renin Angiotensin System in Fructose Fed Sprague Dawley Rats

The current epidemic of obesity and type 2 diabetes is attributed to a high carbohydrate diet, containing mainly high fructose corn syrup and sucrose. More than two thirds of diabetic patients have hypertension. Methylglyoxal is a highly reactive dicarbonyl generated during glucose and fructose metabolism, and a major precursor of advanced glycation end products (AGEs). Plasma methylglyoxal levels are increased in hypertensive rats and diabetic patients. Our aim was to examine the levels of methylglyoxal, mediators of the renin angiotensin system and blood pressure in male Sprague-Dawley rats treated with a high fructose diet (60% of total calories) for 4 months. The thoracic aorta and kidney were used for molecular studies, along with cultured vascular smooth muscle cells (VSMCs). HPLC, Western blotting and Q-PCR were used to measure methylglyoxal and reduced glutathione (GSH), proteins and mRNA, respectively. Fructose treated rats developed a significant increase in blood pressure. Methylglyoxal level and protein and mRNA for angiotensin II, AT1 receptor, adrenergic α1D receptor and renin were significantly increased, whereas GSH levels were decreased, in the aorta and/or kidney of fructose fed rats. The protein expression of the receptor for AGEs (RAGE) and NF-κB were also significantly increased in the aorta of fructose fed rats. MG treated VSMCs showed increased protein for angiotensin II, AT1 receptor, and α1D receptor. The effects of methylglyoxal were attenuated by metformin, a methylglyoxal scavenger and AGEs inhibitor. In conclusion, we report a strong association between elevated levels of methylglyoxal, RAGE, NF-κB, mediators of the renin angiotensin system and blood pressure in high fructose diet fed rats.

PL10 Hydrogen sulfide, glucose, and methylglyoxal formation

Hydrogen sulfide, an important gasotransmitter, has been implicated in the pathogenesis of diabetes. Increased methylglyoxal (MG) level is linked to the development of type 2 diabetes and hypertension. As a member of the reactive carbonyl species, MG is formed mainly through the nonenzymatic conversion of triosephosphates, such as dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (GA3P). The triosephosphate pool, in turn, is regulated by cellular levels of glucose and fructose. The present study investigated whether MG level was altered in mice with cystathionine r-lyase knockout (CSE-KO) and its underlying mechanisms. We measured plasma and renal MG levels in both CSE-KO and wild type (WT) mice at different age groups (6–22 weeks). We also evaluated the role of fructose-1,6-bisphosphatase (FBPase) and related signaling pathway in the regulation of MG formation. We observed a significant decrease in plasma glucose levels along with a significant increase in plasma MG levels in all three age groups (6–8, 14–16, and 20–22 week-old) of the CSE-KO mice. Renal MG, DHAP and GA3P were increased, whereas renal FBPase activity and its mRNA levels were decreased in CSE-KO mice. Decreased FBPase activity was accompanied by lower levels of its product, fructose-6-phosphate, and higher levels of its substrate, fructose-1,6-bisphosphate, in renal extracts from CSE-KO mice. These data indicate an important role of hydrogen sulfide in the regulation of glucose metabolism and MG generation.

Protective Effects of Vescalagin from Pink Wax Apple [Syzygium samarangense (Blume) Merrill and Perry] Fruit against Methylglyoxal-Induced Inflammation and Carbohydrate Metabolic Disorder in Rats

The unbalance of glucose metabolism in humans may cause the excessive formation of methylglyoxal (MG), which can react with various biomolecules to form the precursor of advanced glycation end products (AGEs). Vescalagin (VES) is an ellagitannin that alleviates insulin resistance in cell study. Results showed that VES reduced the value of oral glucose tolerance test, cardiovascular risk index, AGEs, and tumor necrosis factor-α contents while increasing C-peptide and d-lactate contents significantly in rats orally administered MG and VES together. The preventive effect of VES on MG-induced inflammation and carbohydrate metabolic disorder in rats was thus proved. On the basis of the experiment data, a mechanism, which involves the increase in d-lactate to retard AGE formation and the decrease in cytokine release to prevent β-cell damage, is proposed to explain the bioactivities of VES in antiglycation and in the alleviation of MG-induced carbohydrate metabolic disorder in rats.

Formation of carbonyls and hydroperoxyenals (HPALDs) from the OH radical reaction of isoprene for low-NOx conditions: influence of temperature and water vapour content

The formation of gas-phase products from the reaction of OH radicals with isoprene for low-NOx conditions ([NOx] ≤ 1010 molecule cm−3) has been studied in an atmospheric pressure flow tube (Institute for Tropospheric Research-Laminar Flow Tube, IfT-LFT) operating in the temperature range of 293–343 K and a relative humidity of < 0.5 % up to 50 %. The photolysis of H2O2 or ozone photolysis in the presence of water vapour served as the NOx-free OH radical sources. For dry conditions at 293 K, the measured yields of methyl vinyl ketone (MVK), 0.07 ± 0.02, and methacrolein (MACR), 0.12 ± 0.04, were in reasonable agreement with literature data. Beside the C4-carbonyls, further product signals have been attributed tentatively to glycolaldehyde, methylglyoxal, hydroxyacetone, 3-methylfuran, C5-hydroperoxyenals (HPALDs) and C5-hydroxy-hydroperoxides. A simplified, “classical” reaction mechanism without efficient HPALD production describes well the observed yield for MVK and MACR. Unexpected high MVK and MACR yields of up to 0.65 in total were measured under conditions of a relative humidity of 50 % using both OH radical sources and two different measurement techniques for organics (proton transfer reaction mass spectrometry and gas chromatography with flame ionization detector). The reaction mechanism applied is not able to describe the strong increase of MVK and MACR yields with increasing water vapour content. The signal attributed to the HPALDs showed a distinct rise of about one order of magnitude increasing the temperature from 293 K to 343 K. A rough estimate leads to a HPALD yield of 0.32 at 343 K with an uncertainty of a factor of two. The results of this study do not support a predominant formation of HPALDs under atmospheric conditions in low-NOx areas. The surprisingly high MVK and MACR yields measured for a relative humidity of 50 % and the formation of glycolaldehyde, methylglyoxal and hydroxyacetone necessitate further research.

Studies on the formation of methylglyoxal from dihydroxyacetone in Manuka (Leptospermum scoparium) honey

Dihydroxyacetone (DHA) and methylglyoxal (MGO) are unique carbohydrate metabolites of manuka honey. A method for the reliable quantification of DHA in honey samples was established, based on derivatization with o-phenylenediamine (OPD) and subsequent RP-HPLC with UV detection. The previously unknown reaction product of DHA and OPD was identified as 2-hydroxymethylquinoxaline by spectroscopic means. DHA was exclusively determined in 6 fresh manuka honeys originating directly from the beehive as well as 18 commercial manuka honey samples, ranging from 600 to 2700mg/kg and 130 to 1600mg/kg, respectively. The corresponding MGO contents varied from 50 to 250mg/kg in fresh and 70 to 700mg/kg in commercial manuka honey samples. A good linear correlation between DHA and MGO values in commercial manuka honeys was observed, resulting in a mean ratio of DHA to MGO of 2:1. In contrast to this, the DHA-to-MGO relation was much higher in fresh manuka honeys but approximated to a ratio of 2:1 while honey ripening. Heating experiments revealed that MGO formation based on thermal treatment as a consequence, for example, of caramelization in honey does not occur. DHA and MGO can serve as suitable unique quality parameter for manuka honey.

Aldolase B Knockdown Prevents High Glucose-Induced Methylglyoxal Overproduction and Cellular Dysfunction in Endothelial Cells

We used cultured endothelial cells as a model to examine whether up-regulation of aldolase B and enhanced methylglyoxal (MG) formation play an important role in high glucose-induced overproduction of advanced glycosylation endproducts (AGEs), oxidative stress and cellular dysfunction. High glucose (25 mM) incubation up-regulated mRNA levels of aldose reductase (an enzyme converting glucose to fructose) and aldolase B (a key enzyme that catalyzes MG formation from fructose) and enhanced MG formation in human umbilical vein endothelial cells (HUVECs) and HUVEC-derived EA. hy926 cells. High glucose-increased MG production in EA. hy926 cells was completely prevented by siRNA knockdown of aldolase B, but unaffected by siRNA knockdown of aldolase A, an enzyme responsible for MG formation during glycolysis. In addition, inhibition of cytochrome P450 2E1 or semicarbazide-sensitive amine oxidase which produces MG during the metabolism of lipid and proteins, respectively, did not alter MG production. Both high glucose (25 mM) and MG (30, 100 µM) increased the formation of N(ε)-carboxyethyl-lysine (CEL, a MG-induced AGE), oxidative stress (determined by the generation of oxidized DCF, H2O2, protein carbonyls and 8-oxo-dG), O-GlcNAc modification (product of the hexosamine pathway), membrane protein kinase C activity and nuclear translocation of NF-κB in EA. hy926 cells. However, the above metabolic and signaling alterations induced by high glucose were completely prevented by knockdown of aldolase B and partially by application of aminoguanidine (a MG scavenger) or alagebrium (an AGEs breaker). In conclusion, efficient inhibition of aldolase B can prevent high glucose-induced overproduction of MG and related cellular dysfunction in endothelial cells.

Laboratory Kinetic and Mechanistic Studies on the OH-Initiated Oxidation of Acetone in Aqueous Solution

The OH-initiated oxidation of acetone in aqueous solution is investigated because of its potential implications in atmospheric chemistry. The UV-spectrum of the transient acetonylperoxy radical was measured. Two characteristic absorption bands of the acetonylperoxy radical spectrum are found in the 220-400 nm wavelength region. The rate constant for the recombination reaction of the acetonylperoxy radical was determined as a function of temperature for the first time in aqueous solution with k(rec,298K) = (7.3 ± 1.3) × 10(8) M(-1) s(-1), E(A) = 4.5 ± 3.3 kJ mol(-1), and A = (4.7 ± 2.7) × 10(9) M(-1) s(-1). Furthermore, kinetic investigations of the OH-initiated oxidation of methylglyoxal and pyruvic acid were performed with the following results: for methylglyoxal, k(second) = (6.2 ± 0.2) × 10(8) M(-1) s(-1), E(A) = 12 ± 2 kJ mol(-1), and A = (7.8 ± 0.2) × 10(9) M(-1) s(-1); for pyruvic acid (pH = 0), k(second) = (3.2 ± 0.6) × 10(8) M(-1) s(-1), E(A) = 15 ± 5 kJ mol(-1), and A= (1.1 ± 0.1) × 10(11) M(-1) s(-1); for pyruvate (pH = 6), k(second) = (7.1 ± 2.4) × 10(8) M(-1) s(-1), E(A) = 25 ± 19 kJ mol(-1), and A = (1.5 ± 0.4) × 10(13) M(-1) s(-1). Quantitative product studies were done as a function of the number of laser photolysis pulses for acetone and its oxidation products methylglyoxal, hydroxyacetone, pyruvic acid, acetic acid, and oxalic acid. After the recombination reaction of acetonylperoxy radicals, there are two possible decomposition reactions where the primary products methylglyoxal and hydroxyacetone are formed. From product analysis after a single photolysis laser shot, the ratio of the main product-forming reactions was determined as (A) 30% and (B) 56% for the methylglyoxal formation via channel A to yield two molecules of methylglyoxal and channel B to yield one molecule of methylglyoxal and one molecule of hydroxyacetone. The remaining product can be ascribed to channel C, the radical-retaining channel forming alkoxy radicals with a yield of 14%. Pyruvic acid and acetic acid were found to be the major intermediates estimated with concentrations in the same order of magnitude and a similar time profile, indicating that acetic acid is also a possible oxidation product of methylglyoxal.

The role of methylglyoxal metabolism in type-2 diabetes and its complications

Transient or chronic hyperglycaemia increases the formation of intracellular reactive oxygen species and aldehydes. The accumulation of reactive aldehydes is implicated in the development of diabetic complications. Methylglyoxal, a glucose dependent α-dicarbonyl might be the most important reactive aldehyde in diabetes and its complications. Diabetes was the first disease in which evidence emerged for the increased formation of methylglyoxal in the cells and in the serum. Methylglyoxal has a toxic effect on insulin secretion from pancreatic beta-cells, and on modifications of proteins and nucleic acids. Moreover, methylglyoxal is one of the major precursors of advanced glycation end-products. The glyoxalase enzyme system that exists in all mammalian cells is catalyzing the detoxification of methylglyoxal. This review summarizes the methylglyoxal metabolism in normoglycaemic and hyperglycamic conditions and the role of methylglyoxal in the development of late diabetic microvascular complications.

Unusually Fast 1,6-H Shifts of Enolic Hydrogens in Peroxy Radicals: Formation of the First-Generation C2 and C3 Carbonyls in the Oxidation of Isoprene

In a theoretical investigation using the CBS-QB3//UB3LYP/6-31+G** method supported by higher-level computations such as CBS-QB3//UQCISD/6-31+G**, the 1,6-H shifts of the enolic hydrogen in peroxy radicals of the type Z-HO-CH═CH-CH(2)-OO(•) were found to face exceptionally low energy barriers of only about 11 kcal mol(-1)--i.e., 6-9 kcal mol(-1) lower than the barriers for similar shifts of alkane hydrogens--such that they can proceed at unequaled rates of order 10(5) to 10(6) s(-1) at ambient temperatures. The unusually low barriers for enolic 1,6-H shifts in peroxy radicals, characterized here for the first time to our knowledge, are rationalized. As cases in point, the secondary peroxy radicals Z-HO-CH═C(CH(3))-CH(OO(•))-CH(2)OH (case A) and Z-HO-CH═CH-C(CH(3))(OO(•))-CH(2)OH (case B) derived from the primary Z-δ-hydroxy-peroxy radicals in the oxidation of isoprene, are predicted to undergo 1,6-H shifts of their enolic hydrogens at TST-calculated rates in the range 270-320 K of k(T)(A) = 5.4 × 10(-4) × T(5.04) × exp(-1990/T) s(-1) and k(T)(B) = 109 × T(3.13) × exp(-3420/T) s(-1), respectively, i.e., 2.0 × 10(6) and 6.2 × 10(4) s(-1), respectively, at 298 K, far outrunning in all relevant atmospheric and laboratory conditions their reactions with NO proposed earlier as their dominant pathways (Dibble J. Phys. Chem. A 2004, 108, 2199). These fast enolic-H shifts are shown to provide the explanation for the first-generation formation of methylglyoxal + glycolaldehyde, and glyoxal + hydroxyacetone in the oxidation of isoprene under high-NO conditions, recently determined by several groups. However, under moderate- and low-NO atmospheric conditions, the fast interconversion and equilibration of the various thermally labile, initial peroxy conformers/isomers from isoprene and the isomerization of the initial Z-δ-hydroxy-peroxy radicals, both recently proposed by us (Peeters et al. Phys. Chem. Chem. Phys. 2009, 11, 5935), are expected to substantially reduce the yields of the small carbonyls at issue.

Increased Renal Methylglyoxal Formation with Down-Regulation of PGC-1α-FBPase Pathway in Cystathionine γ-Lyase Knockout Mice

We have previously reported that hydrogen sulfide (H2S), a gasotransmitter and vasodilator has cytoprotective properties against methylglyoxal (MG), a reactive glucose metabolite associated with diabetes and hypertension. Recently, H2S was shown to up-regulate peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α, a key gluconeogenic regulator that enhances the gene expression of the rate-limiting gluconeogenic enzyme, fructose-1,6-bisphosphatase (FBPase). Thus, we sought to determine whether MG levels and gluconeogenic enzymes are altered in kidneys of 6–22 week-old cystathionine γ-lyase knockout (CSE-/-; H2S-producing enzyme) male mice. MG levels were determined by HPLC. Plasma glucose levels were measured by an assay kit. Q-PCR was used to measure mRNA levels of PGC-1α and FBPase-1 and -2. Coupled-enzymatic assays were used to determine FBPase activity, or triosephosphate levels. Experimental controls were either age-matched wild type mice or untreated rat A-10 cells. Interestingly, we observed a significant decrease in plasma glucose levels along with a significant increase in plasma MG levels in all three age groups (6–8, 14–16, and 20–22 week-old) of the CSE-/- mice. Indeed, renal MG and triosephosphates were increased, whereas renal FBPase activity, along with its mRNA levels, were decreased in the CSE-/- mice. The decreased FBPase activity was accompanied by lower levels of its product, fructose-6-phosphate, and higher levels of its substrate, fructose-1,6-bisphosphate in renal extracts from the CSE-/- mice. In agreement, PGC-1α mRNA levels were also significantly down-regulated in 6-22 week-old CSE-/- mice. Furthermore, FBPase-1 and -2 mRNA levels were reduced in aorta tissues from CSE-/- mice. Administration of NaHS, a H2S donor, increased the gene expression of PGC-1α and FBPase-1 and -2 in cultured rat A-10 cells. In conclusion, overproduction of MG in CSE-/- mice is due to a H2S-mediated down-regulation of the PGC-1α-FBPase pathway, further suggesting the important role of H2S in the regulation of glucose metabolism and MG generation.

Crh, the paralogue of the phosphocarrier protein HPr, controls the methylglyoxal bypass of glycolysis in Bacillus subtilis

The histidine protein HPr has a key role in regulation of carbohydrate utilization in low-GC Gram-positive bacteria. Bacilli possess the paralogue Crh. Like HPr, Crh becomes phosphorylated by kinase HPrK/P in response to high fructose-1,6-bisphosphate concentrations. However, Crh can only partially substitute for the regulatory functions of HPr leaving its role mysterious. Using protein co-purification, we identified enzyme methylglyoxal synthase MgsA as interaction partner of Crh in Bacillus subtilis. MgsA converts dihydroxyacetone-phosphate to methylglyoxal and thereby initiates a glycolytic bypass that prevents the deleterious accumulation of phospho-sugars under carbon overflow conditions. However, methylgyloxal is toxic and its production requires control. We show here that exclusively the non-phosphorylated form of Crh interacts with MgsA in vivo and inhibits MgsA activity in vitro. Accordingly, Crh inhibits methylglyoxal formation in vivo under nutritional famine conditions that favour a low HPr kinase activity. Thus, Crh senses the metabolic state of the cell, as reflected by its phosphorylation state, and accordingly controls flux through the harmful methylglyoxal pathway. Interestingly, HPr is unable to bind and regulate MgsA, making this a bona fide function of Crh. Four residues that differ in the interaction surfaces of HPr and Crh may account for this difference.