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Hippocampal infusions of pyruvate reverse the memory-impairing effects of septal muscimol infusions.

Hippocampal infusions of glucose reverse memory deficits in spontaneous alternation and in a continuous multiple trial inhibitory avoidance task. The current experiments tested whether glucose metabolism may participate in these effects of glucose. Specifically, these experiments determined whether the glycolytic metabolite pyruvate would mimic these effects of glucose. Male Sprague-Dawley rats were given septal infusions of vehicle or the gamma-aminobutyric acid (GABA) receptor agonist muscimol (0.15 nmol for spontaneous alternation or 5 nmol for continuous multiple trial inhibitory avoidance) combined with hippocampal infusions of vehicle or pyruvate (200 nmol) 15 min prior to assessing spontaneous alternation or training in a continuous multiple trial inhibitory avoidance task. The infusions of muscimol decreased percent alternation scores and continuous multiple trial inhibitory avoidance retention latencies tested 48 h after training. More importantly, hippocampal infusions of pyruvate reversed the deficits produced by septal infusions of muscimol on both tasks. These findings show for the first time that hippocampal infusions of pyruvate influence memory and suggest that glucose may affect memory via glycolytic metabolism.

Animals↗

Blockade of quinolinic acid-induced neurotoxicity by pyruvate is associated with inhibition of glial activation in a model of Huntington's disease.

In this study, we have examined the mechanisms involved in pyruvate-mediated neuroprotection against quinolinic acid (QA)-induced striatal damage. QA injection into the striatum caused widespread neuronal damage and extensive areas of lesions in core and penumbra. The involvement of oxidative-mediated striatal damage was suggested by increased expressions of peroxynitrite, marked lipid peroxidation, and formation of DNA oxidative damage products. Administration of pyruvate, a glycolysis end product with antioxidant activity, significantly reduced QA-mediated striatal lesions, neuronal degeneration, and oxidative damage, whereas another energy substrate, lactate, was ineffective against oxidative damage and only partially effective in reducing lesions and neuronal degeneration. Treatment with the iNOS inhibitor aminoguanidine attenuated QA-mediated striatal lesions and reduced oxidative damage, indicating that iNOS activation may be involved in the striatal oxidative damage induced by QA. A role for glial cells in mediating oxidative damage was suggested because pyruvate blocked the expression of iNOS and nitrotyrosine in activated microglia and astrocytes in QA-injected striatum. These data suggest that pyruvate reduces oxidative free radical damage in QA-injected striatum and could have clinical utility in the treatment of Huntington's disease (HD).

Animals↗

Pyruvate but not lactate prevents NADH-induced myoglobin oxidation.

In this work, we investigated the influence of NADH on the redox state of myoglobin and the roles of pyruvate and lactate in this process. NADH increased the autoxidation rate of myoglobin. Both a drop in pH and partial deoxygenation markedly stimulated the autoxidation process and the influence of NADH. A correlation between met-Mb formation rate and NADH oxidation rate was always observed. The increased rate of Mb autoxidation caused by NADH was inhibited by catalase and pyruvate but not by l-lactate. The antioxidant activity versus H2O2 of both pyruvate and lactate was evidenced by chemiluminescence experiments. The antioxidant activity of lactate disappeared completely in the presence of myoglobin or apo-myoglobin, whereas it was only reduced for pyruvate. These results could be of interest in preventing autoxidation of myoglobin that can contribute to ischemia-reperfusion injury during infarction or high-intensity exercise.

Animals↗

High-performance liquid chromatographic assay of lactic, pyruvic and acetic acids and lactic acid stereoisomers in calf feces, rumen fluid and urine.

To facilitate clinical investigation of metabolic acidosis, a high-performance liquid chromatographic method was adapted and validated for the chiral separation of D-(-) and L-(+)-lactic acid in calf feces, rumen fluid and urine. A non-chiral method was also adapted and validated for the separation of pyruvic, acetic and DL-(+/-)-lactic acids in calf feces and DL-(+/-)-lactic and pyruvic acids in rumen fluid. Separation and quantification were achieved using a reversed phase sulphonated polystyrenedivinylbenzene analytical column for pyruvic, acetic and racemic lactic acids and by a 3 microm octadecylsilane (ODS) packed analytical column coated with N,N-dioctyl-L-alanine as the chiral selector for the separation of lactic acid enantiomers with Cu(II)-containing eluents by stereoselective ligand exchange chromatography. Endogenous analytes were present in validation samples over a range of concentrations (0.2-14.8 mmol/l). For the stereoselective assay, mean intra-day accuracy ranged from 90.6 to 108.4% and intra-day precision from 0.3 to 13.8%. For the non-stereoselective assay, mean intra-day accuracy ranged from 90.4 to 108.8% and intra-day precision from 1.5 to 11.1%. The limit of quantitation was 1.0 mmol/l for D- and L-lactic acid, 0.06125 mmol/l for pyruvic acid, 1.0 mmol/l for DL-lactic acid and 1 mmol/l for acetic acid. These assays can be used to study the role of the gastrointestinal tract and kidney in metabolic acidosis.

Acetic Acid↗

Pyruvate ameliorates the defect in ureogenesis from ammonia in citrin-deficient mice.

BACKGROUND/AIMS: Mutations in SLC25A13, encoding the mitochondrial aspartate-glutamate carrier citrin, cause adult-onset type II citrullinemia (CTLN2) in humans. We have previously reported that although citrin-knockout (Ctrn-/-) mice fail to display symptoms of CTLN2, liver perfusion revealed a deficit in ureogenesis from ammonia accompanied by an increase in the perfusate lactate-to-pyruvate (L/P) ratio. The present study explores the effects of pyruvate, aspartate and citrate on improving the abnormalities observed in the Ctrn-/- liver. METHODS: We measured the rate of ureogenesis from ammonium chloride using the liver-perfusion system. RESULTS: Pyruvate infusion lowered the L/P ratio and corrected the deficit in ureogenesis in the Ctrn-/- liver. This effect was found to be dose-dependent in both instances. Phenazine methosulfate, a cytosolic oxidant, also improved the rate of ureogenesis in the Ctrn-/- liver and led to a fall in the L/P ratio. The addition of aspartate or citrate did not change either the rate of ureogenesis or the L/P ratio in the Ctrn-/- liver. CONCLUSIONS: Citrin deficiency disturbs urea synthesis primarily as a result of an elevated cytosolic NADH/NAD+ ratio owing to limited reoxidation of reducing equivalents. Clinically, pyruvate may have a therapeutic benefit for CTLN2 patients.

Amino Acids↗

Increased expression of hepatic pyruvate dehydrogenase kinases 2 and 4 in young and middle-aged Otsuka Long-Evans Tokushima Fatty rats: induction by elevated levels of free fatty acids.

The activity of the pyruvate dehydrogenase complex (PDC) is regulated by covalent modification of its E1 component, which is catalyzed by specific pyruvate dehydrogenase kinases (PDKs) and phosphatases. In the liver, PDK2 and PDK4 are the most abundant PDK isoforms, which are responsible for inactivation of PDC when glucose availability is scarce in the body. In the present study, regulatory mechanisms of hepatic PDC were examined before and after the onset of type 2 diabetes mellitus in Otsuka Long-Evans Tokushima Fatty (OLETF) rats, using Long-Evans Tokushima Otsuka (LETO) rats as controls. Plasma glucose and insulin concentrations were at normal levels in rats aged 8 weeks, but were significantly higher in OLETF than in LETO rats aged 25 weeks, indicating insulin resistance in OLETF rats. Plasma free fatty acids (FFAs) were 1.6-fold concentrated, and the liver PDC activity was significantly lower in OLETF than in LETO rats at both ages, suggesting suppression of pyruvate oxidative decarboxylation in OLETF rats before and after the onset of diabetes. Pyruvate dehydrogenase kinase activity and abundance of PDK2 and PDK4 proteins, as well as mRNAs, were greater in OLETF rats at both ages. These results suggest that persistently elevated levels of circulating free fatty acid in normal and diabetic OLETF rats play an important role in stimulating PDK2 and PDK4 expression in liver.

Age Factors↗

Pyruvate slows disease progression in a G93A SOD1 mutant transgenic mouse model.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease caused by selective motor neuron death, and currently no effective treatment is available for ALS. In this study, we investigated the neuroprotective effects of pyruvate, which acts as an anti-oxidant and as an energy source. We treated G93A SOD1 transgenic mice with pyruvate (from 70 days of age, i.p., at 1000 mg/kg/week), and found that it prolonged average lifespan by 12.3 days (10.5%), slowed disease progression, and improved motor performance, but did not delay disease onset. Pyruvate treatment was also associated with reduced nitrotyrosine immunoreactivity, gliosis, and increased Bcl-2 expression in the spinal cords of G93A SOD1 transgenic mice. These results suggest that pyruvate treatment may be a potential therapeutic strategy in ALS.

Amyotrophic Lateral Sclerosis↗

Measurement of gluconeogenesis and pyruvate recycling in the rat liver: a simple analysis of glucose and glutamate isotopomers during metabolism of [1,2,3-(13)C3]propionate.

Simple equations that relate glucose and glutamate 13C-NMR multiplet areas to gluconeogenesis and pyruvate recycling during metabolism of [1,2,3-(13)C3]propionate are presented. In isolated rat livers, gluconeogenic flux was 1.2 times TCA cycle flux and about 40% of the oxaloacetate pool underwent recycling to pyruvate prior to formation of glucose. The 13C spectra of glucose collected from rats after gastric versus intravenous administration of [1,2,3-(13)C3]propionate indicated that pyruvate recycling was slightly higher in vivo (49%) while glucose production was unchanged. This indicates that a direct measure of gluconeogenesis and pyruvate recycling may be obtained from a single 13C-NMR spectrum of blood collected after oral administration of enriched propionate.

Animals↗

Regulation of mammalian pyruvate dehydrogenase kinase.

It is generally believed that mammalian pyruvate dehydrogenase kinase is a heterodimer consisting of catalytic and regulatory subunits. However, the contribution of the two subunits to the kinase-mediated signal transduction has remained undefined. In the present study recombinant components of mammalian pyruvate dehydrogenase complex were employed in order to characterize the role of the kinase catalytic subunit in the regulation of pyruvate dehydrogenase reaction. The results provide the first evidence strongly suggesting that the catalytic subunit of pyruvate dehydrogenase kinase is competent to respond to known effectors of kinase activity as well as to interact with the E2-core without assistance of a regulatory subunit.

Animals↗

Determination of pyruvate and lactate in primary liver cell culture medium during hypoxia by on-line microdialysis-liquid chromatography.

A microdialysis sampling device was constructed for the measurement of pyruvate and lactate in primary liver cell culture medium during hypoxia. It was composed of a Petri dish, a dialysis membrane and two transmission tubes within a hypoxia chamber. The dialysis membrane was located in the Petri dish such that it was immersed in the culture medium. Dialysates were collected and introduced by an on-line injector to a liquid chromatographic system for analysis of pyruvate and lactate. The detection limit of this assay was 0.2-2.0 microM with acceptable intra- and inter-assay reproducibilities. In order to validate the assay, primary liver cells were incubated in the Petri dish within a hypoxia chamber in an incubator. The baseline concentrations of pyruvate and lactate in primary liver cell culture medium were 10.6+/-5.6 and 607+/-143 microM, respectively. These levels drastically changed during hypoxia and reperfusion. In conclusion, the present assay provides a sensitive, direct measurement of pyruvate and lactate in culture medium while minimizing pretreatment procedures for sample preparation.

Animals↗

A new class of wine pigments generated by reaction between pyruvic acid and grape anthocyanins.

A new class of stable red pigments detected in grape pomace was analysed by electrospray ionisation mass spectrometry. They were shown to be pyruvic acid derivatives of genuine grape anthocyanins by synthesis experiments. The major product was identified by NMR (1H, NOE, HSQC, HMBC) experiments as the malvidin-3-monoglucoside pyruvic acid adduct. Its formation results from cyclisation between C-4 and the hydroxyl group at C-5 of the original flavylium moiety with the double bond of the enolic form of pyruvic acid, followed by dehydration and rearomatisation steps. This type of reaction leads to increased colour stability. Various yeast metabolites other than pyruvic acid were shown to react with grape anthocyanins following this mechanism, suggesting that it may be an important route of conversion into stable pigments during the maturation and ageing of wine.

Anthocyanins↗

Effect of differential addition of glycerol and pyruvate to extender on cryopreservation of Mediterranean buffalo (B.bubalis) spermatozoa.

The composition of the extender in which semen is diluted before freezing plays a major role in successful cryopreservation of spermatozoa. Substances of high osmolarity, like glycerol, protect sperm cells during the freezing process and energy-rich compounds, like pyruvate provide extra energy during capacitation and fertilization. Since cryopreservation procedures for Buffalo spermatozoa have not been adequately defined, the aim of the study was to improve the survival rate of buffalo (Bubalus bubalis) spermatozoa after cryopreservation by optimizing the timing for adding glycerol and by enriching the cryoprotectant extender with an energy source substrate. Semen was collected with an artificial vagina from 5 bulls and the ejaculates were immediately evaluated for motility, forward progressive motility and for viability, pooled and held at room temperature (28 degrees C) for 1 h. Then aliquots of pooled semen were subjected to dilution and equilibration in triplicate as follows: Experiment 1. Glycerol (3%) in a commercial extender was added to the semen at 28 degrees C and cooled to 5 degrees C for 1 h; then extender with 11% glycerol was added before further equilibration (initial glycerol addition; IGA) and the samples held at 5 degrees C for 1, 3 or 5 additional hours (IGA 1, n = 24; IGA 3, n = 24; IGA 5, n = 24) before freezing. Experiment 2. Glycerol (3%) was added and the mixture brought to 5 degrees C as described above. Then extender with 11% glycerol was added (late glycerol addition; LGA) and after equilibration for 1, 3 and 5 h (LGA 1, n= 24; LGA 3, n = 24; LGA 5, n = 24) the samples were frozen. In Experiments 3 and 4 Na pyruvate (1.25 mM) was added to the extender as described for IGA and LGA above (IPA and LPA samples). The effect of addition time (initial vs late) of glycerol and pyruvate was evaluated by measuring sperm motility, progressively forward motility and viability. After freezing-thawing the percentage of motile spermatozoa was significantly higher (0.001<P<0.01) after a late addition of glycerol and pyruvate (LGA 5 and LPA 5). The optimizing of the timing of the glycerol addition and the presence in the extender of an energy source rendered a higher efficiency in thawed spermatozoa.

Animals↗

Rat-brain pyruvate kinase: purification and effects of lithium.

Purified pyruvate kinase was prepared from pooled brains obtained from untreated rats. Its properties suggest that it is similar to type 'M' pyruvate kinase. Lithium inhibition was demonstrated at pharmacologically significant lithium concentrations (7%-12% at 2 mmol 1-1 Li) and this was similar in character to that previously seen in rabbit muscle pyruvate kinase, namely noncompetitive with respect to phosphoenol pyruvate, K+, and Mg2+ but competitive with ADP.

Animals↗

Purification and characterization of recombinant pyruvate phosphate dikinase from Giardia.

The gene encoding pyruvate phosphate dikinase (PPDK) from Giardia duodenalis was expressed using a baculovirus system. The recombinant enzyme was purified to homogeneity and its enzymological and solution structure properties characterized. The catalytic constant for the pyruvate-producing reaction was about twice as high (1560 min(-1) at 30 degrees C) as that for the reverse reaction (700 min(-1)) and the k(cat)/Km for PPi was about two orders of magnitude higher than k(cat)/Km for Pi, indicating that the pyruvate-forming reaction is much more efficient than the reverse, phosphoenolpyruvate (PEP)-forming process. The endogenous substrate levels found for PEP (0.5 mM) and pyruvate (< 80 microM) support the assumption that, under physiological conditions, the enzyme primarily performs a catabolic function. The molecular mass of the purified recombinant PPDK was analyzed by analytical ultracentrifugation and size exclusion chromatography using different assay conditions that have been reported to affect the quaternary structure of PPDKs in other organisms. Both methods clearly indicated a dimeric structure for giardial PPDK with a molecular mass of about 197 kDa (monomer mass 97.6 kDa). Several compounds, primarily structural analogs of PPi, were tested for their ability to inhibit PPDK activity. Most of the bisphosphonates examined showed either no, or only a moderate, inhibitory effect on the enzyme. Imidodiphosphate was the only competitive inhibitor with respect to PPi (Kic = 0.55 mM), whereas the bisphosphonates produced a mixed type of inhibition. The most active compound in inhibiting PPDK activity was oxalate, with a Kic value of less than 1 microM with respect to PEP.

Animals↗

Practical implications of lactate and pyruvate metabolism by lactic acid bacteria in food and beverage fermentations.

This article reviews the metabolism of pyruvate and lactate by lactic acid bacteria (LAB) involved in food and beverage fermentations with an emphasis on practical implications. First, the formation of pyruvate and lactate from a range of substrates, including carbohydrates, organic acids and amino acids, is briefly described. The catabolism of pyruvate and lactate by LAB is then reviewed. This is followed by a discussion of lactate degradation and racemisation by LAB from specific fermented foods and beverages. Finally, the impact of environmental factors and metabolic engineering on pyruvate and lactate metabolism by LAB is evaluated with regard to practical significance.

Beverages↗

Decarboxylation and carboxylation of pyruvate in the living mice.

Mice received intravenously [1- or 2-14C]acetate, [1-, 2- or 3-14C] or [2-14C]pyruvate and were killed 1, 3, 5 or 15 min later. The radioactivity of CO2 or HCO3- of liver or carcass as well as the radioactivity of blood glucose were measured. The ratio of the radioactivity found in these compounds after [3-14C] or [2-14C-A1pyruvate injection suggests that in the fed aminals: 1. the decarboxylation of the pyruvate was more rapid than its carboxylation, 2. most of the neosynthesized glucose was derived from pyruvate molecules which had undergone a decarboxylation followed by a condensation to citrate, 3. 1/4 to 1/3 of the pyruvate was carboxylated and 2/3 to 3/4 was decarboxylated in animals receiving a diet poor in fats.

Acetates↗

Oxidative damage to mouse lens in culture. Protective effect of pyruvate.

Studies have been conducted to examine the feasibility of preventing oxyradical-dependent oxidative stress to mouse lens in culture, using pyruvate as an antioxidant. The extent of oxidative damage to the tissue was assessed by measurement of the status of Na(+)-K(+) ATPase dependent active transport of rubidium 86Rb(+). The tissue levels of adenosine triphosphate (ATP), glutathione (GSH), malonaldehyde (MDA) and catalase were also determined. While the measurement of 86Rb(+) uptake provides an assessment of the integrity of the primary active transport system, measurement of the other components reflects the status of intracellular oxidative stress. ATP measurement also reflected on the overall status of metabolic integrity. Incubation of the lens with xanthine (XA)/xanthine oxidase (XO) system had an adverse effect on all these parameters. Incorporation of pyruvate was strikingly protective. The protective effect of pyruvate is apparently due to its ability to scavenge ROS generated in the medium with the possibility of its action on tissue metabolism as well. The findings are hence considered useful for further studies on the prevention of oxidative stress to tissues by exogenous supplementation with pyruvate, specially the human lens where the biochemistry of its antioxidant mechanisms is similar to the mouse lens, contrary to the rat lens.

Adenosine Triphosphate↗

Stereoselective biosynthesis of chloroarylpropane diols by the basidiomycete Bjerkandera adusta: exploring the roles of amino acids, pyruvate, glycerol and phenyl acetyl carbinol.

Bjerkandera adusta produces many chlorometabolites including chlorinated anisyl metabolites (CAMs) and 1-arylpropane-1,2-diols (1, 2, 3, 4) as idiophasic metabolic products of L-phenylalanine. These diols are stereoselectively biosynthesized from a C7-unit (benzylic, from L-phenylalanine) and a C2-unit, of unknown origin, as predominantly erythro (1R,2S) enantiomers. Of the labeled amino acids tested as possible C2-units, at the 4-10 mM level, none were found to efficiently label the 2,3-propane carbons of the diols. However, glycine (2-13C), L-serine (2,3,3-d3) and L-methionine (methyl-d3) entered the biomethylation pathway. Neither pyruvate (2,3-13C2), acetate (1,2-13C2), acetaldehyde (d4) nor ethanol (ethyl-d5) labeled the 2,3-propane carbons of the diols at the 4-10 mM level. Pyruvate (2,3-13C2) and L-serine (2,3,3-d3) (which also entered the biomethylation pathway) did, however, effectively label the 2,3-propane carbons of the alpha-ketols and diols at the 40 mM level as evidenced by mass spectrometry. Glycerol (1,1,2,3,3-d5) also appeared to label one of the 2,3-propane carbons (ca. 5% as 2H2 in the C3 side chain) as suggested by mass spectrometric data and also entered the biomethylation pathway, likely via amino acid synthesis. Glycerol (through pyruvate), therefore, likely supplies C2 and C3 of the propane side chain with arylpropane diol biosynthesis. Incubation of B. adusta with synthetic [2-2H1, 2-18O]-glycerol showed that neither 2H nor 18O were incorporated in the alpha-ketols or diols. The oxygen atom on the C2 of the ketols/diols, therefore, does not appear to come from the oxygen atom on the C2 of glycerol. Glycerol, however, can readily form L-serine (which can then form pyruvate via PLP/serine dehydratase and involve transamination washing out the 18O label and providing the oxygen from water), and can then go on to label the C2-unit. Labeled alpha-ketol, phenyl acetyl carbinol (5) (PAC; ring-d(5), 2,3-13C2 propane) cultured with B. adusta leads to stereospecific reduction to the (1R,2S)-diol (6) (ring-d5 and 2,3-13C2); in all other metabolites produced, the 2,3-13C2) label is washed out. Incubation of the fungus with 4-fluorobenzaldehyde (13) produces a pooling of predominantly erythro (1R,2S) 1-(4'-fluorophenyl)-1,2-propane diol (18 as diacetate) (through the corresponding alpha-ketols 16, 17). Blocking the para-position with fluorine thus appears to prevent ring oxygenation and also chlorination, forcing the conclusion that para-ring oxygenation precedes meta-chlorination.

Acetone↗