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PubMed1985

Synthesis of pyruvate-1-11C as a radiopharmaceutical for tumor imaging.

Pyruvate-1-11C was prepared enzymatically by the exchange reaction of 11CO2 with the carboxyl group of pyruvic acid using pyruvate-ferredoxin oxidoreductase from Clostridium butyricum. 11C-Labeled pyruvate was purified by sublimation in specially made glassware. The radiochemical yield of pure pyruvate-1-11C was 80% 35 min after the end of bombardment. The distribution of 11C in tumor-bearing rabbits after an i.v. injection of pyruvate-1-11C was observed using a gamma camera. In contrast to normal organs, the tumor was positively visualized. We also conducted a number of successful clinical studies. A case of brain tumor which exhibited a positive image on positron-emission tomography (PET) using pyruvate-1-11C is presented.

Animals↗
PubMed1980

Evidence for two genetic complementation groups in pyruvate carboxylase-deficient human fibroblast cell lines.

We have examined genetic complementation in pyruvate carboxylase deficiency by comparing the enzyme activity in polyethylene glycol-induced heterokaryons with that in unfused mixtures of fibroblasts from three affected children. Complementation, manifested as a three- to sevenfold increase in pyruvate carboxylase activity, was observed in fusions between a biotin-responsive multiple carboxylase (pyruvate carboxylase, propionyl CoA carboxylase, and beta-methylcrotonyl CoA carboxylase) deficient fibroblast line and two other lines deficient only in pyruvate carboxylase activity. Kinetic analysis of complementing pyruvate carboxylase deficient lines, measured by the rate of restoration of enzyme activity as a function of time, revealed that maximum restoration was achieved within 10-24 hr after fusion. This profile is similar to those oberved for fusions between the multiple carboxylase deficient line and two lines deficient in propionyl CoA carboxylase activity that are known to represent different gene mutations. Although the patients with pyruvate carboxylase deficiency had similar clinica findings, our studies indicate that pyruvate carboxylase deficiency is genetically heterogeneous, with at least two distinct, probably intergenic, complementation groups.

Biotin↗
PubMed1983

Paracatalytic inactivation of pig heart pyruvate dehydrogenase complex.

The phenomenon of paracatalytic inactivation has been demonstrated and characterized with the pig heart pyruvate dehydrogenase complex. The enzyme became progressively inactive when it was preincubated in the presence of pyruvate, thiamine pyrophosphate, and extrinsic oxidative agent, 2,6-dichloroindophenol. Not only the overall reaction of enzyme complex but the individual reactions catalyzed by enzyme components, pyruvate dehydrogenase, and dihydrolipoamide acetyltrasferase, were reduced after preincubation. The observed inactivation is due to two factors: (i) covalent incorporation of the pyruvate atoms, and (ii) formation of a thiamine pyrophosphate analog. (i) A covalent incorporation of radioactivity from [2-14C]pyruvate into the enzyme complex, which was proportional to the inhibition of overall reaction was observed. Seventy-eight percent of the radioactivity incorporated into the dihydrolipoamide transacetylase but not into the lipoic acid. Thus, modification of the dihydrolipoamide acetyltransferase component can be attributed to the covalent incorporation of pyruvate atoms. (ii) During inactivation, the enzyme-bound thiamine pyrophosphate underwent a modification resulting in the formation of a thiamine pyrophosphate analog, presumably thiamine thiazolone pyrophosphate, which inhibited exclusively the pyruvate dehydrogenase component.

2,6-Dichloroindophenol↗
PubMed1983

Regulation of fatty acid oxidation in heart muscle. Effects of pyruvate and dichloroacetate.

The possibility of a mutual regulation between carbohydrate and fatty acid oxidation was studied in isolated perfused rat hearts. Infusions of pyruvate and/or dichloroacetate were employed to convert fully the pyruvate dehydrogenase complex into its active form during [1-14C]octanoate oxidation, the rate of which was measured by the production of 14CO2. It was found that 5 mM dichloroacetate suppressed the oxidation of 0.1 mM octanoate by 58%, but 10 mM external pyruvate was without effect. Dichloroacetate reduced the tissue malate concentration by 41% and the citrate concentration by 76% at a 0.1 mM octanoate concentration, but had no effect on the metabolite concentrations or fatty acid oxidation rate in perfusions with 1 mM octanoate. Metabolite depletion was probably partly due to inhibition of the carboxylation of pyruvate, as verified by determination of the metabolite labelling kinetics from [1-14C]pyruvate. It is, therefore, possible that the dichloroacetate-induced inhibition of octanoate oxidation could also be partly due to inhibition of tricarboxylic acid cycle secondary to metabolite depletion. Since both dichloroacetate and pyruvate converted pyruvate dehydrogenase to its active form, but only dichloroacetate inhibited fatty acid oxidation, the latter effect could not be due to oxidation of a competing substrate, but instead may result from an inhibition of fatty acid uptake, activation or transport, as also indicated by the observed decrease in the acid-soluble acyl-CoA concentration. This interpretation is also supported by the mitochondrial redox effects of dichloroacetate observed during octanoate oxidation in the perfused heart.

Acetates↗
PubMed1992

Pyruvate transport across the plasma membrane of the bloodstream form of Trypanosoma brucei is mediated by a facilitated diffusion carrier.

The characteristics of pyruvate transport across the plasma membrane in the bloodstream form of Trypanosoma brucei were studied using [14C]pyruvate in combination with the silicone-oil centrifugation technique. We present evidence for the existence of a facilitated diffusion carrier in the plasma membrane of T. brucei which specifically mediates the translocation of pyruvate. The uptake of pyruvate followed saturation kinetics (Km 1.96 +/- 0.28 mM; Cmax 36.61 +/- 1.15 nmol pyruvate/30 sec.mg protein), after correction of the data for a nonsaturable diffusion component. The uptake of pyruvate was competitively inhibited by a number of (oxo)monocarboxylic acids, including pyruvate analogs and metabolically related substances, but not by L-lactate. The transport exhibited the phenomenon of transacceleration, indicative for the involvement of a facilitated diffusion carrier. The carrier is highly specific for pyruvate and differs from other known monocarboxylate carriers present in the mitochondrial and/or plasma membrane of other eukaryotic cells in that it does not transport L-lactate.

Animals↗
PubMed1983

H4-isozyme of lactate dehydrogenase in the solution of sodium chloride--3. The enzymatic activity and the pyruvate inhibition.

1. Low enzymatic activities in low pyruvate concentrations and high Km were observed in sodium chloride solutions. 2. The pyruvate inhibition shown by the % activity at 1 mM pyruvate was lower sodium chloride than in 0.1 M sodium phosphate. 3. At 40 degrees C, as compared with results at 20 degrees C, less pyruvate inhibition was observed in phosphate buffer and in sodium chloride solutions. 4. By using the equilibrium constants between dimer and tetramer, a theoretical explanation is proposed for the pyruvate inhibition. In this explanation, it is suggested that the quaternary complex which is composed of tetrameric enzyme, coenzyme and two kinds of pyruvates was the main cause of the pyruvate inhibition.

Isoenzymes↗
PubMed1985

Inhibition of palmitoylcarnitine oxidation by pyruvate in rat heart mitochondria.

The oxidation of 1-14C-palmitoylcarnitine by rat heart mitochondria has been measured by assessing both the disappearance of substrate and the appearance of labelled products. Pyruvate inhibited palmitoylcarnitine oxidation by about 40%. Fifty percent inhibition occurred at about 20 mumol/L pyruvate. The inhibitory effect of pyruvate required entry of pyruvate into mitochondria since it did not occur in the presence of alpha-cyano-4-hydroxycinnamic acid, an inhibitor of the mitochondrial pyruvate transporter. The inhibitory effect of pyruvate also required an active pyruvate dehydrogenase. These studies indicate a mechanism that may be important in the replacement of fatty acids by carbohydrate as fuels of respiration of the heart during the transition from the starved to the fed state.

Animals↗
PubMed1995

Triiodothyronine treatment increases substrate cycling between pyruvate carboxylase and malic enzyme in perfused rat liver.

The relative roles of pyruvate kinase and malic enzyme in substrate cycling between pyruvate and oxaloacetate were examined in perfused livers of 24-hour-fasted normal and triiodothyronine (T3)-treated rats using an inhibitor of malic enzyme (hydroxymalonate). Livers were perfused for 60 minutes in a recirculating system with [3-13C]alanine (10 mmol/L, 99% 13C-enriched). The combined flux through pyruvate kinase plus malic enzyme relative to pyruvate carboxylase flux was assessed by the 13C-enrichment ratio of alanine C2 to glucose C5 in the perfusate, determined with 13C and 1H nuclear magnetic resonance (NMR) spectroscopy. In normal rat livers, the relative carbon flux through pyruvate kinase plus malic enzyme to pyruvate carboxylase was 0.18 +/- 0.04, and increased to 0.44 +/- 0.08 (P < .05) in the T3-treated group. After addition of hydroxymalonate, this relative carbon flux was unchanged in normal rat livers, but decreased to 0.15 +/- 0.04 (P < .01) in the T3-treated group, suggesting that the increased carbon flux in T3-treated livers was caused by increased flux through malic enzyme. Malic enzyme activity increased from 0.36 +/- 0.05 U/g liver in normal livers to 2.51 +/- 0.50 U/g liver (P < .05) in the T3-treated group, whereas there was no effect of T3 treatment on pyruvate kinase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Alanine↗
PubMed1975

Activities of enzymes concerned with pyruvate and oxaloacetate metabolism in the heart and liver of developing sheep.

1. In order to assess whether the potential ability of heart ventricular muscle and liver to metabolise substrates such as alanine, aspartate and lactate varies as the sheep matures and its nutrition changes, the activities of the following enzymes were determined in tissues of lambs obtained at varying intervals between 50 days after conception to 16 weeks after birth and in livers from adult pregnant ewes: lactate dehydrogenase (EC 1.1.1.27), alanine aminotransferase (EC 2.6.1.2), pyruvate kinase (EC 2.7.1.40), pyruvate carboxylase (EC 6.4.1.1), phosphoenolpyruvate carboxykinase (GTP)(EC 4.1.1.32), malate dehydrogenase (EC 1.1.1.37), aspartate aminotransferase (EC 2.6.1.1) and citrate (si)-synthase (EC 4.1.3.7). 2. In the heart a most marked increase in alanine aminotransferase activity was found throughout development. During this period the activities of citrate (si)-synthase, lactate dehydrogenase and pyruvate carboxylase also increased. There were no substantial changes in the activities of aspartate aminotransferase, malate dehydrogenase or pyruvate kinase. Pyruvate kinase activities were five times greater in the heart compared with those found in the liver. No significant activity of phosphoenolpyruvate carboxykinase (GTP) was detected in heart muscle. 3. In the liver the activities of both alanine aminotransferase and aspartate aminotransferase increased immediately following birth although the activity of alanine aminotransferase was lower in livers of pregnant ewes than in any of the lambs. As with alanine aminotransferase the highest activities of lactate dehydrogenase were found during the period of postnatal growth. No marked changes were observed in malate dehydrogenase or citrate (si)-synthase activities during development. A small decline in pyruvate kinase activity occurred whilst the activities of pyruvate carboxylase and phosphoenolpyruvate carboxykinase (GTP) tended to rise during development.

Aging↗
PubMed2004

Effect of R(+)alpha-lipoic acid on pyruvate metabolism and fatty acid oxidation in rat hepatocytes.

R-(+)-alpha-lipoic acid (R-LA) is the naturally occurring enantiomer of LA. It is a strong antioxidant and cofactor of key metabolic enzyme complexes catalyzing the decarboxylation of alpha-keto acids. Racemic LA (rac-LA) has shown promise in treating diabetic polyneuropathy, and some studies suggest that it improves glucose homeostasis in patients with type 2 diabetes. We examined the effects of R-LA on pyruvate metabolism and free fatty acid (FFA) oxidation in primary cultured hepatocytes isolated from 24-hour fasted rats. After overnight culture in serum-free medium, cells were pre-exposed to R-LA for 3 hours before assays. R-LA (25 to 200 micromol/L) significantly increased pyruvate oxidation ( approximately 2-fold at the highest dose tested) measured as (14)CO(2) production from [1-(14)C]pyruvate by the cells over 1 hour post-treatment. These effects correlated with proportional, significant increases in the activation state of the pyruvate dehydrogenase (PDH) complex. R-LA treatment inhibited glucose production from pyruvate by approximately 50% at 50 micromol/L R-LA and approximately 90% at 200 micromol/L. Palmitate oxidation was measured in hepatocytes cultured in the presence of albumin and physiological (0.1 mmol/L) or high (1.5 mmol/L) concentrations of FFA. The latter markedly enhanced FFA oxidation. R-LA treatment significantly inhibited FFA oxidation in both media, but was more effective in high FFA, where it reduced FFA oxidation by 48% to 82% at 25 to 200 micromol/L, respectively. Identical doses of R-LA did not affect FFA oxidation by L6 myotubes (a cell culture model for skeletal muscle) in either high or low FFA medium, but enhanced pyruvate oxidation. In conclusion, 3-hour exposure of primary cultured rat hepatocytes to R-LA at therapeutically relevant concentrations increased pyruvate oxidation, apparently by activation of the PDH complex, and decreased gluconeogenesis and FFA oxidation. These features may prove useful in the control of type 2 diabetes.

Animals↗
PubMed1985

Regulation of pyruvate dehydrogenase during infusion of fatty acids of varying chain lengths in the perfused rat heart.

The effects of a homologous series of fatty acids with a chain length of two to eight on the rate of pyruvate oxidation and covalent interconversions of the pyruvate dehydrogenase complex (PDH) were studied in isolated perfused rat hearts. In the Langendorff-perfused heart beating at 5 Hz against an aortic pressure of 59 mmHg (7.85 kPa), a positive linear correlation was found between the fraction of PDH existing in the active non-phosphorylated form of pyruvate dehydrogenase complex (PDHa) and the pyruvate oxidation rate until the PDHa fraction increased to 48%. This value resulted in a saturation of the citric acid cycle and further activation did not increase the metabolic flux. The PDHa content of the tissue was higher during infusion of odd carbon number fatty acids than during infusion of even carbon number fatty acids. Propionate caused an almost maximal (93%) activation of PDH. A negative correlation was found between the mitochondrial NADH/NAD+ ratio and the PDHa content. A negative correlation was also found between the acetyl-CoA/CoA ratio and the tissue PDHa content. The rate of labelled CO2 production, the specific radioactivity of tissue alanine and the metabolic balance sheet demonstrated that the alanine aminotransferase reaction in the total tissue does not reach equilibrium with the mitochondrial pyruvate pool during propionate oxidation, but the equilibrium is reached during the oxidation of even-number carbon fatty acids. This suggests that pyruvate is formed from propionate-derived metabolites also in the cytosol, although the primary metabolism of propionate occurs in the mitochondria. The results indicate that the rate of pyruvate oxidation in the myocardium is mainly regulated by covalent interconversion of PDH. During propionate oxidation the PDHa content in the tissue can increase beyond the point of saturation of the citric acid cycle and this indicates that feedback inhibition of the enzyme is rate-determining under these conditions.

Acetyl Coenzyme A↗
PubMed1998

The pyruvate dehydrogenase complex from thermophilic organisms: thermal stability and re-association from the enzyme components.

Examples of pyruvate dehydrogenase complexes, and of its probable precursors, the pyruvate ferredoxin oxidoreductases, both isolated from thermophilic organisms, are described. The pyruvate ferredoxin oxidoreductases are mostly characterized from thermophilic archaea like Sulfolobus solfataricus and Pyrococcus furiosus. They retain their catalytic activity up to 60 and 90 degreesC, respectively. Characteristic for the thermophilic nature is a biphasic temperature behavior, reflecting a more stable low temperature and a metastable high temperature form. Another feature is the strong binding of the cofactor thiamin diphosphate. Detailed analysis of thermostable pyruvate dehydrogenase complexes so far only exist for the enzymes from Bacillus stearothermophilus and Thermus flavus. In most respects, especially in the structural features, the enzyme complex from B. stearothermophilus resembles its mesophilic counterparts and only an elevated temperature maximum for the catalytic activity reveals the thermophilic nature. In contrast to this, the more thermostable enzyme complex from T. flavus shows a quite distinct behavior. One single protein chain (Mr=100 kDa) instead of an alpha2beta2 aggregate was found for the pyruvate dehydrogenase (E1) subunits of this enzyme complex. Its catalytic activity is controlled by allosteric regulation, while the enzyme complex from B. stearothermophilus shows no such regulation. Reversible phosphorylation as a regulatory principle of pyruvate dehydrogenase complexes from higher organisms does not take place in the thermophilic enzyme complexes. The overall activity of the enzyme complex from B. stearothermophilus remains stable at 60 degreesC for 50 min while that from T. flavus is active up to 83 degreesC. Thermophilic pyruvate dehydrogenase complexes do not spontaneously renature from their separated enzyme components. However, chaperonins from Thermus thermophilus stimulate the reactivation of the enzyme complex from T. flavus.

Archaea↗
PubMed1987

Purification and properties of Escherichia coli 4'-phosphopantothenoylcysteine decarboxylase: presence of covalently bound pyruvate.

4'-Phosphopantothenoylcysteine decarboxylase was purified 900-fold from Escherichia coli B with an overall yield of 6%. The enzyme migrates as a single band with a molecular weight of 35,000 +/- 3000 in 10% polyacrylamide gel electrophoresis under denaturing conditions. The native enzyme has an apparent molecular weight of 146,000 +/- 9000 as determined by a gel exclusion column. At pH 7.6 and 25 degrees C, Km = 0.9 mM and Vmax = 600 nmol/(min X mg of protein). The pH optimum for Vmax is between 7.5 and 7.7. Hydroxylamine, phenylhydrazine, potassium cyanide, and sodium borohydride as well as pyridoxal phosphate and pyridoxal inactivated the enzyme. The enzyme contains covalently bound pyruvate as suggested by the isolation of [3H]lactate and pyruvate from [3H]NaBH4-reduced enzyme and native enzyme, respectively. One mole of [3H]lactate was isolated per 39,000 g of [3H]NaBH4-reduced and completely inactivated enzyme, and 1 mol of pyruvate was isolated per 31,000 +/- 4000 g of native enzyme. Mild base treatment released lactate and pyruvate from the reduced and the native enzymes, respectively, suggesting the pyruvate is attached to the enzyme by an ester bond. These findings are in accord with similar results obtained with the horse liver enzyme (R. Scandurra, personal communication). The presence of covalently bound pyruvate in the bacterial and mammalian enzymes suggests that pyruvate plays a major role in the mechanism of action.

Binding Sites↗
PubMed1997

Molecular mechanism of regulation of the pyruvate dehydrogenase complex from E. coli.

The pyruvate dehydrogenase multienzyme complex from E. coli shows a sigmoidal dependency of the reaction rate on the substrate concentration when product formation is followed in the presence of physiological concentrations of the cofactor thiamin diphosphate. To elucidate the molecular mechanism of this regulation, the influence of the substrate pyruvate on the coenzyme-protein interaction has been investigated using several coenzyme analogues. The observed binding constants of all coenzymatically active analogues are increased in the presence of the substrate pyruvate, whereas those of all coenzymatically inactive analogues are not altered in the presence of pyruvate. This points to an increased binding affinity of a reaction-intermediate-coenzyme complex to the protein. Since cofactor binding and dissociation at physiological concentrations of thiamin diphosphate are slow compared to the catalytic reaction, a slow transition to the active state of the enzyme occurs. After lowering the pyruvate concentration, the opposite effect, a dissociation of the thiamin diphosphate from the enzyme is observed. This slow substrate dependent enhancement of cofactor binding enables efficient regulation of the pyruvate dehydrogenase complex by its substrate pyruvate.

Coenzymes↗
PubMed1969

Pyruvate kinase variants of the Alaskan king-crab. Evidence for a temperature-dependent interconversion between two forms having distinct and adaptive kinetic properties.

1. Pyruvate kinase of Alaskan king-crab leg muscle exists in two kinetically distinct forms, each of which displays a different temperature-dependence in the K(m) for phosphoenolpyruvate. 2. A ;cold' variant of the enzyme has hyperbolic kinetics and exhibits a minimal K(m) for substrate at 5 degrees . At physiological concentrations of phosphoenolpyruvate the ;cold' enzyme is active only below 10 degrees . A ;warm' pyruvate kinase has a minimal K(m) for substrate at about 12 degrees . This enzyme displays sigmoidal kinetics and is likely to be inactive, at physiological substrate concentrations, at temperatures below 9 degrees . 3. The combined activities of these two pyruvate kinases yield highly temperature-independent rates of catalysis, at physiological substrate concentrations, over the range of habitat temperatures encountered by the organism, namely 4-12 degrees . 4. The two variants of pyruvate kinase do not appear to be isoenzymes in the conventional sense. Electrophoretic and electrofocus analyses revealed only single peaks of activity. 5. The results suggest that the ;warm' pyruvate kinase and the ;cold' pyruvate kinase are formed by a temperature-dependent interconversion of one protein species. This interconversion has major adaptive significance: as the temperature is lowered the ;warm' enzyme is converted into the ;cold' enzyme; the opposite situation obtains when the temperature is raised. Temperature changes thus mimic the effects noted for fructose 1,6-diphosphate on certain mammalian pyruvate kinases.

Animals↗
PubMed1976

Selective modification of rabbit muscle pyruvate kinase by 5-chloro-4-oxopentanoic acid.

Rabbit muscle pyruvate kinase was irreverisbly inactivated by 5-chloro-4-oxopentanoic acid with a pKa of 9.2. The inhibition was time-dependent and was related to the 5-chloro-4-oxopentanoic acid concentration. Analysis of the kinetics of inhibition showed that the binding of the inhibitor showed positive co-operativity (n = 1.5 +/- 0.2). Inhibition of pyruvate kinase by 5-chloro-4-oxopentanoic acid was prevented by ligands which bind to the active site. Their effectiveness was placed in the order Mg2+ greater than phosphoenolpyruvate greater than ATP greater than ADP greater than pyruvate. Inhibitor-modified pyruvate kinase was unable to catalyse the detritiation of [3-(3)H]pyruvate in the ATP-promoted reaction, but it did retain 5-10% of the activity with either phosphate or arsenate as promoters. 5-Chlor-4-oxo-[3,5-(3)H]pentanoic acid was covalently bound to pyruvate kinase and demonstrated a stoicheiometry of 1 mol of inhibitor bound per mol of pyruvate kinase protomer. The incorporation of the inhibitor and the loss of enzyme was proportional. These results are discussed in terms of 5-chloro-4-oxopentanoic acid alkylating a functional group in the phosphoryl overlap region of the active site, and a model is presented in which this compound alkylates an active-site thiol in a reaction that is controlled by a more basic group at the active site.

Alkylation↗
PubMed1988

The kinetics of transport of lactate and pyruvate into rat hepatocytes. Evidence for the presence of a specific carrier similar to that in erythrocytes.

Time courses of L-lactate and pyruvate uptake into isolated rat hepatocytes were measured in a citrate-based medium to generate a pH gradient (alkaline inside), by using the silicone-oil-filtration technique at 0 degrees C to minimize metabolism. At low concentrations of lactate and pyruvate (0.5 mM), transport was inhibited by over 95% by 5 mM-alpha-cyano-4-hydroxycinnamate, whereas at higher concentrations (greater than 10 mM) a significant proportion of transport could not be inhibited. The rate of this non-inhibitable transport was linearly related to the substrate concentration, was less with pyruvate than with L-lactate, and appeared to be due to diffusion of undissociated acid. Uptake of D-lactate was not inhibited by alpha-cyano-4-hydroxycinnamate and occurred only by diffusion. Kinetic parameters for the carrier-mediated transport process were obtained after correction of the initial rates of uptake of lactate and pyruvate in the absence of 5 mM-alpha-cyano-4-hydroxycinnamate by that in the presence of inhibitor. Under the conditions used, the Km values for L-lactate and pyruvate were 2.4 and 0.6 mM respectively and the Ki for alpha-cyano-4-hydroxycinnamate as a competitive inhibitor was 0.11 mM. Km values for the transport of L-lactate and pyruvate into rat erythrocytes under similar conditions were 3.0 and 0.96 mM. The Vmax. of lactate and pyruvate transport into hepatocytes at 0 degrees C was 3 nmol/min per mg of protein. Carrier-mediated transport of 0.5 mM-L-lactate was inhibited by 0.2 mM-p-chloromercuribenzenesulphonate (greater than 90%), 0.5 mM-quercetin (80%), 0.6 mM-isobutylcarbonyl-lactyl anhydride (70%) and 0.5 mM-4,4'-di-isothiocyanostilbene-2,2'-disulphonate (50%). A similar pattern of inhibition of lactate transport is seen in erythrocytes. It is suggested that the same or a similar carrier protein exists in both tissues. The results also show that L-lactate transport into rat hepatocytes is very rapid at physiological temperatures and is unlikely to restrict the rate of its metabolism. Differences between our results and those of Fafournoux, Demigne & Remesy [(1985) J. Biol. Chem. 260, 292-299] are discussed.

Animals↗
PubMed1995

Metabolism of [3-13C]pyruvate and [3-13C]propionate in normal and ischaemic rat heart in vivo: 1H- and 13C-NMR studies.

The oxidation of [3-13C]pyruvate and [3-13C]propionate was studied in vivo in infused rats. The infused [3-13C]pyruvate was quickly converted to [3-13C]lactate in the blood, and the [3-13C]lactate formed was well metabolized in both normoxic and ischaemic hearts. Large differences (200-600%) in the 13C enrichment of alanine (C-3) and acetyl-CoA (C-2) compared with lactate (C-3) were found in both normoxic and ischaemic hearts, suggesting that the extracellular [3-13C]lactate preferentially entered a region of the cytoplasm which specifically transfers the labelled pyruvate (formed from [3-13C]lactate) to the mitochondria. The highly enriched mitochondrial pyruvate gave high enrichment in alanine and acetyl-CoA, which was detected by 1H- and 13C-NMR spectroscopy. Ischaemia increased 13C incorporation into the main cytoplasmic lactate pool and decreased 13C incorporation into citric acid cycle intermediates, mainly decreasing the pyruvate anaplerosis. Isoprenaline-induced ischaemia of the heart caused only a slight decrease in pyruvate oxidation. In contrast to the decreased anaplerosis of pyruvate, the anaplerosis of propionate (and propionyl-carnitine) increased significantly in ischaemic hearts, which may contribute to the protective effect of propionyl-carnitine seen in ischaemia. In addition, we found that [3-13C]propionate preferentially labelled aspartate C-3 in rat heart, suggesting incomplete randomization of label in the succinyl-CoA-malate span of the citric acid cycle. These data show that proton observed 13C edited spectroscopic methods, i.e. heteronuclear spin-echo and the one-dimensional heteronuclear multiple quantum coherence sequence, can be successfully used to study heart metabolism in vivo.

Acetyl Coenzyme A↗