Lactate metabolism in the perfused rat hindlimb.

A preparation of isolated rat hindleg was perfused with a medium consisting of bicarbonate buffer containing Ficoll and fluorocarbon, containing glucose and/or lactate. The leg was electrically prestimulated to deplete partially muscle glycogen. The glucose was labelled uniformly with 14C and with 3H in positions 2, 5 or 6, and lactate uniformly with 14C and with 3H in positions 2 or 3. Glucose carbon was predominantly recovered in glycogen, and to a lesser extent in lactate. The 3H/14C ration in glycogen from [5-3H,U-14C]- and [6-3H,U-14C]-glucose was the same as in glucose. Nearly all the utilized 3H from [2-3H]glucose was recovered as water. Insulin increased glucose uptake and glycogen synthesis 3-fold. When the muscle was perfused with a medium containing 10 mM-glucose and 2 mM-lactate, there was little change in lactate concentration. 14C from lactate was incorporated into glycogen. There was a marked exponential decrease in lactate specific radioactivity, much greater with [3H]- than with [14C]-lactate. The 'apparent turnover' of [U-14C]lactate was 0.28 mumol/min per g of muscle, and those of [2-3H]- and [3-3H]-lactate were both about 0.7 mumol/min per g. With 10 mM-lactate as sole substrate, there was a net uptake of lactate, at a rate of about 0.15 mumol/min per g, and the apparent turnover of [U-14C]lactate was 0.3 mumol/min per g. The apparent turnover of [3H]lactate was 3-5 times greater. When glycogen synthesis was low (no prestimulation, no insulin), the incorporation of lactate carbon into glycogen exceeded that from glucose, but at high rates of glycogen deposition the incorporation of lactate carbon was much less than that of glucose. Lactate incorporation into glycogen was similar in fast-twitch white and fast-twitch red muscle, but was very low in slow-twitch red fibres. We find that (a) pyruvate in muscle is incorporated into glycogen without randomization of carbon, and synthesis is not inhibited by mercaptopicolinate or cycloserine; (b) there is extensive lactate turnover in the absence of net lactate uptake, and there is a large dilution of 14C-labelled lactate from endogenous supply; (c) there is extensive detritiation of [2-3H]- and [3-3H]-lactate in excess of 14C utilization.     

Acidogenic fermentation of lactose

Cheese whey is the main component of waste streams from cheese manufacturing plants. Whey is a high biochemical oxygen demand (BOD) effluent that must be reduced before the streams are sent to the sewer. It is proposed in this article that the production of methane by anaerobic fermentation would be the best use of this stream, especially for small plants. Single-stage fermentation of lactose, the main component of whey, results in a very low pH and a stalled process. Two-phase fermentation will eliminate this problem. The acidogenic stage of fermentation has been studied at pH of between 4 and 6.5. The nature of the main products of the reaction have been found to be pH dependent. Below a pH of 4.5 a gas (CO(2) and H(2)) is produced along with ethanol, acetate, and butyrate. Above a pH of 4.5 no gas was produced, and the liquid products included less ethanol and butyrate and more acetate. A separate study on the conditions for gas formation showed that if the pH dropped for a short time below 4.5 gases were formed at all subsequent pH. This would indicate a change in population distribution due to the period at a low pH. By assuming that the desired products from the acidogenic stage were butyrate, acetate, and no gases, the optimum pH range was found to be between 6.0 and 6.5.     

Intrinsic fermentation kinetics of lactose in acidogenic biofilms

The intrinsic fermentation kinetics of lactose in acidogenic biofilms were investigated in situ in a continuous flow fermentor at 35 degrees C and pH 4.6. The external and internal mass transfer resistances to lactose molecules from bulk solution to inside the biofilms were experimentally minimized or eliminated in a thin biofilm and recycled medium. In a chemically defined culture medium, the immobilized acidogens converted lactose mainly to acetate and butyrate; the minor products included ethanol. propionate, lactate, and hydrogen. The utilization rate of lactose, as a function of lactose concentration in the fermentor, can be described by a Michaelis-Menten equation, as can the formation rates of acetate, butyrate, and ethanol. The production rates of propionate and lactate had a liner relationship with lactose concentration under the experimental conditions. The low pH (4.6) of culture medium could depress the formation of propionate, and intermediate which is most difficulty digested by acetogenic bacteria located in the second fermentor in a two-phase process. Production rate of acetate quickly reached a constant, and additional utilization of lactose produced more butyrate and other minor products. (c) 1993 John Wiley & Sons, Inc.     

Thermodynamic analysis of product formation in mesophilic acidogenesis of lactose

Thermodynamic analysis on the acidogenesis of lactose was performed to evaluate the different acidogenic patterns and mechanisms by using Gibbs free energy calculation. Batch acidogenesis of lactose was investigated by using an enriched culture at 37 degrees C, pH 5.5 and varied substrate levels. In addition to usual acidogenic products, i-butyrate, valerate, i-valerate, caproate, and propanol were also produced at a significant level. Thermodynamic analysis shows that valerate might be formed through the reaction requiring hydrogen as electron donor and consuming of propionate and carbon dioxide. Caproate was most likely produced directly from butyrate, hydrogen, and carbon dioxide. The minimum amount of Gibbs free energies needed to sustain isomerization of butyrate and valerate were approximately 5.7-5.8 and 4.5-4.6 kJ/mol, respectively. Propanol was produced from acetate, hydrogen, and carbon dioxide with a minimum amount of Gibbs free energy of 41.8-42.0 kJ/mol. Formation of butanol was controlled more by substrate level or population dynamics than by thermodynamics.     

Rates of production and utilization of lactate by microbial communities from the human colon

Lactate metabolism was studied in mixed bacterial communities using single-stage continuous flow fermentors inoculated with faecal slurries from four different volunteers and run for 6 days at pH 5.5 and 6.0, using carbohydrates, mainly starch, as substrates. A continuous infusion of [U-(13) C]starch and l-[3-(13) C]lactate was performed on day 5 and a bolus injection of l-[3-(13) C]lactate plus dl-lactate on day 6. Short-chain fatty acids and lactate concentrations plus enrichments and numbers of lactate-producing and -utilizing bacteria on day 5 were measured. Faecal samples were also collected weekly over a 3-month period to inoculate 24-h batch culture incubation at pH 5.9 and 6.5 with carbohydrates alone or with 35 mmol L(-1) lactate. In the fermentors, the potential lactate disposal rates were more than double the formation rates, and lactate concentrations usually remained below detection. Lactate formation was greater (P<0.05) at the lower pH, with a similar tendency for utilization. Up to 20% of butyrate production was derived from lactate. In batch cultures, lactate was also efficiently used at both pH values, especially at 6.5, although volunteer and temporal variability existed. Under healthy gut environmental conditions, bacterial lactate disposal seems to exceed production markedly.     

Citrate Fermentation by Lactococcus and Leuconostoc spp

Citrate and lactose fermentation are subject to the same metabolic regulation. In both processes, pyruvate is the key intermediate. Lactococcus lactis subsp. lactis biovar diacetylactis homofermentatively converted pyruvate to lactate at high dilution (growth) rates, low pH, and high lactose concentrations. Mixed-acid fermentation with formate, ethanol, and acetate as products was observed under conditions of lactose limitation in continuous culture at pH values above 6.0. An acetoin/butanediol fermentation with alpha-acetolactate as an intermediate was found upon mild aeration in continuous culture and under conditions of excess pyruvate production from citrate. Leuconostoc spp. showed a limited metabolic flexibility. A typical heterofermentative conversion of lactose was observed under all conditions in both continuous and batch cultures. The pyruvate produced from either lactose or citrate was converted to d-lactate. Citrate utilization was pH dependent in both L. lactis and Leuconostoc spp., with maximum rates observed between pH 5.5 and 6.0. The maximum specific growth rate was slightly stimulated by citrate, in L. lactis and greatly stimulated by citrate in Leuconostoc spp., and the conversion of citrate resulted in increased growth yields on lactose for both L. lactis and Leuconostoc spp. This indicates that energy is conserved during the metabolism of citrate.     

Inhibition of lipogenesis by vasopressin and angiotensin II in glycogen-depleted hepatocytes

Vasopressin and angiotensin II inhibited lipogenesis (measured with 3H2O) in hepatocytes from fed rats. Inhibition was also observed with hepatocytes from fed rats which had been depleted of glycogen in vitro and incubated with lactate + pyruvate (5 mM + 0.5 mM) as substrates. The inhibitory actions of the hormones are therefore independent of hormone-mediated changes in glycogenolytic or glycolytic flux from glycogen, and thus the site(s) of hormone action must be subsequent to the formation of lactate. (-)Hydroxycitrate, a specific inhibitor of ATP-citrate lyase, decreased lipogenesis in hepatocytes from fed rats incubated with lactate + pyruvate by approx. 51% but had little effect on lipogenesis in glycogen-depleted hepatocytes similarly incubated. There was parallel inhibition of incorporation of 14C from [U-14C]lactate into fatty acid and lipogenesis as measured with 3H2O in each case. Thus depletion of glycogen, or conceivably the process of glycogen-depletion (incubation with dibutyryl cyclic AMP) causes a change in the rate-determining step(s) for lipogenesis from lactate. Vasopressin and angiotensin II also decreased lipogenesis and incorporation of 14C into fatty acids in glycogen-depleted hepatocytes provided with [U-14C]proline as opposed to [U-14C]-lactate. However, proline-stimulated lipogenesis was inhibited by (-)hydroxycitrate, and proline-stimulated lipogenesis and incorporation of 14C from [U-14C]-proline were not decreased in parallel by this inhibitor (inhibition of 52% and 85% respectively). It is inferred that lactate and proline stimulate lipogenesis by different mechanisms and incorporation of 14C from [U-14C]proline and [U-14C]lactate into fatty acid occurs via different routes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proline and hepatic lipogenesis

The effects of proline on lipogenesis in isolated rat hepatocytes were determined and compared with those of lactate, an established lipogenic precursor. Proline or lactate plus pyruvate increased lipogenesis (measured with 3H2O) in hepatocytes from fed rats depleted of glycogen in vitro and in hepatocytes from starved rats. Lactate plus pyruvate but not proline increased lipogenesis in hepatocytes from starved rats. ( - )-Hydroxycitrate, an inhibitor of ATP-citrate lyase, partially inhibited incorporation into saponifiable fatty acid of 3H from 3H2O and 14C from [U-14C]lactate with hepatocytes from fed rats. Incorporation of 14C from [U-14C]proline was completely inhibited. Similar complete inhibition of incorporation of 14C from [U-14C]proline by ( - )-hydroxycitrate was observed with glycogen-depleted hepatocytes or hepatocytes from starved rats. Inhibition of phosphoenolpyruvate carboxykinase by 3-mercaptopicolinate did not inhibit the incorporation into saponifiable fatty acid of 3H from 3H2O or 14C from [U-14C]proline or [U-14C]lactate. Both 3-mercaptopicolinate and ( - )-hydroxycitrate increased lipogenesis (measured with 3H2O) in the absence or presence of lactate or proline with hepatocytes from starved rats. The results are discussed with reference to the roles of phosphoenolpyruvate carboxykinase, mitochondrial citrate efflux, ATP-citrate lyase and acetyl-CoA carboxylase in proline- or lactate-stimulated lipogenesis.     

The regulation of glyceride synthesis in isolated white-fat cells. The effects of acetate, pyruvate, lactate, palmitate, electron-acceptors, uncoupling agents and oligomycin

1. The incorporation of 5mm-[U-(14)C]glucose into glyceride fatty acids by fat cells from normal rats incubated in the presence of 20munits of insulin/ml was increased by acetate, pyruvate, palmitate, NNN'N'-tetramethyl-p-phenylenediamine, phenazine methosulphate, dinitrophenol, tetrachlorotrifluoromethyl benzimidazole and oligomycin. Lactate did not stimulate glucose incorporation into fatty acids. The effects of these agents were concentration-dependent. 2. In the presence of 5mm-glucose+insulin, [U-(14)C]acetate, [U-(14)C]pyruvate and [U-(14)C]lactate were incorporated into fatty acids in a concentration-dependent manner, thereby further increasing the total rate of fatty acid synthesis. 3. NNN'N'-tetramethyl-p-phenylenediamine decreased the incorporation of [U-(14)C]pyruvate into fatty acids in normal cells and increased the incorporation of [U-(14)C]lactate into fatty acids. 4. In fact cells from 72h-starved rats the stimulatory effects of NNN'N'-tetramethyl-p-phenylenediamine upon glucose and lactate incorporation into fatty acids were totally and partially abolished respectively whereas the stimulatory effects of acetate upon glucose incorporation were retained. 5. Combinations of the optimum concentrations of the substances that stimulate glucose incorporation into fatty acids were tested and compared. The effects of acetate+NNN'N'-tetramethyl-p-phenylenediamine and acetate+palmitate upon normal cells were additive. The effects of NNN'N'-tetramethyl-p-phenylenediamine+palmitate were not additive. It was found that total fatty acid synthesis in the presence of glucose was most effectively increased by raising the concentration of pyruvate in the incubation system. 6. The significance of these results in supporting the proposal that fatty acid synthesis from glucose in adipose tissue is a ;self-limiting process' is discussed.     

Carbon Balance Studies of Glucose Metabolism in Rat Cerebral Cortical Synaptosomes

Synaptosomes were isolated from rat cerebral cortex and incubated with [U-14C]-, [1-14C]- or [6-14C]glucose. Glucose utilization and the metabolic partitioning of glucose carbon in products were determined by isotopic methods. From the data obtained a carbon balance was constructed, showing lactate to be the main product of glucose metabolism, followed by CO2, amino acids and pyruvate. Measuring the release of 14CO2 from glucose labelled in three different positions allowed the construction of a flow diagram of glucose carbon atoms in synaptosomes, which provides information about the contribution of the various pathways of glucose metabolism. Some 2% of glucose utilized was calculated to be degraded via the pentose phosphate pathway. Addition of chlorpromazine, imipramine or haloperidol at concentrations of 10(-5) M reduced glucose utilisation by 30% without changing the distribution pattern of radioactivity in the various products.     

Amino acid oxidation and alanine production in rat hemidiaphragm in vitro. Effects of dichloroacetate.

Dichloroacetate (an activator of pyruvate dehydrogenase) stimulates 14CO2 production from [U-14C]glucose, but not from [U-14C]glutamate, [U-14C]aspartate, [U-14C]- and [1-14C]-valine and [U-14C]- and [1-14C]-leucine. It is concluded (1) that pyruvate dehydrogenase is not rate-limiting in the oxidation to CO2 of amino acids that are metabolized to tricarboxylic acid-cycle intermediates, and (2) that carbohydrate (and not amino acids) is the main carbon precursor in alanine formation in muscle.     

Impact of pH on lactate formation and utilization by human fecal microbial communities

The human intestine harbors both lactate-producing and lactate-utilizing bacteria. Lactate is normally present at <3 mmol liter(-1) in stool samples from healthy adults, but concentrations up to 100 mmol liter(-1) have been reported in gut disorders such as ulcerative colitis. The effect of different initial pH values (5.2, 5.9, and 6.4) upon lactate metabolism was studied with fecal inocula from healthy volunteers, in incubations performed with the addition of dl-lactate, a mixture of polysaccharides (mainly starch), or both. Propionate and butyrate formation occurred at pH 6.4; both were curtailed at pH 5.2, while propionate but not butyrate formation was inhibited at pH 5.9. With the polysaccharide mix, lactate accumulation occurred only at pH 5.2, but lactate production, estimated using l-[U-(13)C]lactate, occurred at all three pH values. Lactate was completely utilized within 24 h at pH 5.9 and 6.4 but not at pH 5.2. At pH 5.9, more butyrate than propionate was formed from l-[U-(13)C]lactate in the presence of polysaccharides, but propionate, formed mostly by the acrylate pathway, was the predominant product with lactate alone. Fluorescent in situ hybridization demonstrated that populations of Bifidobacterium spp., major lactate producers, increased approximately 10-fold in incubations with polysaccharides. Populations of Eubacterium hallii, a lactate-utilizing butyrate-producing bacterium, increased 100-fold at pH 5.9 and 6.4. These experiments suggest that lactate is rapidly converted to acetate, butyrate, and propionate by the human intestinal microbiota at pH values as low as 5.9, but at pH 5.2 reduced utilization occurs while production is maintained, resulting in lactate accumulation.     

Lipogenesis in rat brown adipocytes. Effects of insulin and noradrenaline, contributions from glucose and lactate as precursors and comparisons with white adipocytes.

1. Brown adipocytes were isolated from the interscapular depot of male rats maintained at approx. 21 degrees C. In some experiments parallel studies were made with white adipocytes from the epididymal depot. 2. Insulin increased and noradrenaline decreased [U-14C]glucose incorporation into fatty acids by brown adipocytes. Brown adipocytes differed from white adipocytes in that exogenous fatty acid (palmitate) substantially decreased fatty acid synthesis from glucose. Both noradrenaline and insulin increased lactate + pyruvate formation by brown adipocytes. Brown adipocytes converted a greater proportion of metabolized glucose into lactate + pyruvate and a smaller proportion into fatty acids than did white adipocytes. 3. In brown adipocytes, when fatty acid synthesis from [U-14C]glucose was decreased by noradrenaline or palmitate, incorporation of 3H2O into fatty acids was also decreased to an extent which would not support proposals for extensive recycling into fatty acid synthesis of acetyl-CoA derived from fatty acid oxidation. 4. In the absence of glucose, [U-14C]lactate was a poor substrate for lipogenesis in brown adipocytes, but its use was facilitated by glucose. When brown adipocytes were incubated with 1 mM-lactate + 5 mM-glucose, lactate-derived carbon generally provided at least 50% of the precursor for fatty acid synthesis. 5. Both insulin and noradrenaline increased [U-14C]glucose conversion into CO2 by brown adipocytes (incubated in the presence of lactate) and, in combination, stimulation of glucose oxidation by these two agents showed synergism. Rates of 14CO2 formation from glucose by brown adipocytes were relatively small compared with maximum rates of oxygen consumption by these cells, suggesting that glucose is unlikely to be a major substrate for thermogenesis. 6. Brown adipocytes from 6-week-old rats had considerably lower maximum rates of fatty acid synthesis, relative to cell DNA content, than white adipocytes. By contrast, rates of fatty acid synthesis from 3H2O in vivo were similar in the interscapular and epididymal fat depots. Expressed relative to activities of fatty acid synthase or ATP citrate lyase, however, brown adipocytes synthesized fatty acids as effectively as did white adipocytes. It is suggested that the cells most active in fatty acid synthesis in the brown adipose tissue are not recovered fully in the adipocyte fraction during cell isolation. Differences in rates of fatty acid synthesis between brown and white adipocytes were less apparent at 10 weeks of age.     

The role of phosphoenolpyruvate carboxykinase in muscle alanine synthesis.

3-Mercaptopicolinic acid (3-MPA) is reportedly a specific inhibitor of phosphoenolpyruvate (PEP) carboxykinase and has hitherto been used accordingly to elucidate the metabolic role of PEP carboxykinase in vitro and in vivo. We show that 3-MPA has multiple effects on intermediary metabolism in hemidiaphragms from 40 h-starved rats. It decreases the release of lactate + pyruvate and alanine in hemidiaphragms provided with no added substrate or with valine, leucine or isoleucine. Moreover, irrespective of the substrate provided (none, valine, leucine, isoleucine, glucose, acetate, oleate), 3-MPA decreases the [lactate]/[pyruvate] ratio. 3-MPA is without effect on 14CO2 production from [U-14C]valine, [1-14C]valine, [1-14C]leucine, [U-14C]isoleucine or [1-14C]oleate, but stimulates 14CO2 production from [U-14C]glucose and [1-14C]pyruvate and inhibits 14CO2 production from [1-14C]acetate. Glycolytic flux (measured as 3H2O formation from [5-3H]glucose) is stimulated by 3-MPA. It is concluded that 3-MPA has site(s) of actions other than PEP carboxykinase and that the putative role of PEP carboxykinase in alanine synthesis de novo in skeletal muscle from tricarboxylic acid-cycle intermediates and related amino acids requires reappraisal.     

Incorporation of isotopic carbon into cerebral glycogen from non-glucose substrates.

1. Measurable incorporation of radioactive carbon from [U-14C]pyruvate, [U-14C]-glutamate and [14C]bicarbonate into the glycogen synthesized by brain slices in vitro was demonstrated. 2. The fructose diphosphatase activity of guinea-pig brain was determined and found to be about 0.03 mumol of substrate degraded/min per g of fresh tissue. 3. The specific radioactivity of the glucose carbon from glycogen relative to that of the precursor added to the incubation medium gave approximate values of 0.195 for glucose, 0.006 for pyruvate, 0.039 for glutamate and 0.001 for bicarbonate.     

Lactate and pyruvate transport is dominated by a pH gradient-sensitive carrier in rat skeletal muscle sarcolemmal vesicles

The mechanisms of lactate and pyruvate transport across the plasma membrane of rat skeletal muscle under various pH and ionic conditions were studied in skeletal muscle sarcolemmal (SL) membrane vesicles purified from 22 female Sprague-Dawley rats. Transport by SL vesicles was measured as uptake of L(+)-[U-14C] lactate and [U-14C] pyruvate. Lactate (La-) transport is pH-sensitive; stimulations to fivefold overshoot above equilibrium values were observed both directly by a proton gradient directed inward, and indirectly by a monensin- or nigericin-stimulated exchange of Na+ or K+ for H+ across the SL. Isotopic pyruvate could utilize the transporter, and demonstrated pH gradient-stimulated overshoot and cis-inhibition characteristics similar to those of lactate. Overshoot kinetics were also demonstrated by pH gradient formed by manipulation of external media at pH 5.9, 6.6, and 7.4 and intravesicular media at 6.6, 7.4, and 8.0, respectively. Carbonyl cyanide m-chlorophenylhydrazone, an H+ ionophore, was used as a "pH clamp" to return all stimulated uptake courses back to equilibrium values. Lactate uptake was depressed when internal pH was lower than external pH. These data strongly suggest that La- and H+ are either cotransported by the carrier, or transported as the undissociated HLa, and can account for the majority of the lactate uptake at pH 7.4. The mechanism does not require cotransport of either K+ or Na+. However, an inwardly directed Na+ gradient without ionophore in the absence of a pH gradient doubled La- transport; treatment with amiloride, an inhibitor of the Na+/H+ exchanger, abolished this stimulation, suggesting that this transporter may be an important coregulator of intracellular pH, and could disrupt 1:1 H+ and La- efflux stoichiometry in vivo. We conclude that the majority of La- crosses the skeletal muscle SL by a specific carrier-mediated process that is saturable at high La- concentrations, but flux is passively augmented at low intracellular pH by undissociated lactic acid. In addition, a Na+/H+ exchange mechanism was confirmed in skeletal muscle SL, does affect both lactate and proton flux, and is potentially an important coregulator of intracellular pH and thus, cellular metabolism.     

The effect of glucagon on the carbon flux from palmitate into glucose, lactate and ketone bodies, studied with isolated hepatocytes

Glucagon induced a rapid (within 3 min) increase in glucose radioactivity and a decrease in the labeling of ketone bodies when isolated hepatocytes were incubated in the presence of [1-14C]palmitate. Simultaneously, the hormone induced a decrease in the levels of pyruvate and Krebs cycle intermediates and an increase in the level of phosphoenolpyruvate (PEP). The glucagon-induced increase in glucose radioactivity was much larger than the simultaneous decrease in lactate labeling. A comparison of the incorporation of labeled carbon from [1-14C]palmitate and [U-14C]palmitate into glucose and CO2 indicates a selective stimulatory action of glucagon on the flux through the phosphoenolpyruvate carboxykinase (PEPCK) reaction.     

Performance of anaerobic granules for degradation of pentachlorophenol.

Anaerobic granules degrading pentachlorophenol (PCP) with specific PCP removal activity up to 14.6 mg/g of volatile suspended solids per day were developed in a laboratory-scale anaerobic upflow sludge blanket reactor at 28 degrees C, by using a mixture of acetate, propionate, butyrate, and methanol as the carbon source. The reactor was able to treat synthetic wastewater containing 40 to 60 mg of PCP per liter at a volumetric loading rate of up to 90 mg/liter of reactor volume per day, with a hydraulic retention time of 10.8 to 15 h. PCP removal of more than 99% was achieved. Results of adsorption of PCP by granular biomass indicated that the PCP removal by the granules was due to biodegradation rather than adsorption. A radiotracer assay demonstrated that the PCP-degrading granules mineralized [14C]PCP to 14CH4 and 14CO2. Toxicity test results indicated that syntrophic propionate degraders and acetate-utilizing methanogens were more sensitive to PCP than syntrophic butyrate degraders. The PCP-degrading granules also exhibited a higher tolerance to the inhibition caused by PCP for methane production and degradation of acetate, propionate, and butyrate, compared with anaerobic granules unadapted to PCP.     

Metabolic compartmentation of pyruvate in the isolated perfused rat heart.

1. Prompted by the finding of markedly differing specific radioactivities of tissue alanine and lactate in isolated rat hearts perfused with [1-14C]pyruvate, a more detailed study on the cytosolic subcompartmentalization of pyruvate was undertaken. Isolated rat hearts were perfused by the once-through Langendorff technique under metabolic and isotopic steady-state conditions but with various routes of radioactive label influx, and the specific radioactivities of pyruvate, lactate and alanine were determined. An enzymic method was devised to determine the specific radioactivity of C-1 of pyruvate. 2. Label introduction as [1-14C]pyruvate resulted in a higher specific radioactivity of tissue alanine and mitochondrial pyruvate than of lactate, and a higher specific radioactivity of perfusate lactate than of tissue lactate. Label introduction as [1-14C]lactate resulted in a roughly similar isotope dilution into the tissue and perfusate pyruvate and the tissue alanine. Label introduction as [3,4-14C]glucose resulted in the same specific radioactivity of tissue lactate and alanine and a roughly similar specific radioactivity of mitochondrial pyruvate. 3. The results can be reconciled with a metabolic model containing two cytosolic functional pyruvate pools. One pool (I) communicates more closely with the glycolytic system, whereas the other (II) communicates with extracellular pyruvate and intracellular alanine. Pool II is in close connection with intramitochondrial pyruvate. The physical identity of the cytosolic subcompartments of pyruvate is discussed.     

Synthesis of fluoroacetate from fluoride, glycerol, and beta-hydroxypyruvate by Streptomyces cattleya.

Streptomyces cattleya produces fluoroacetate and 4-fluorothreonine from inorganic fluoride added to the culture broth. We have shown by 19F nuclear magnetic resonance (NMR) spectrometry that fluoroacetate is accumulated first in the culture broth and that accumulation of 4-fluorothreonine is next. To show precursors of the carbon skeleton of fluoroacetate, we carried out tracer experiments with various 14C- and 13C-labeled compounds. Radioactivity of [U-14C]glucose, [U-14C]glycerol, [U-14C]serine, and [U-14C]beta-hydroxypyruvate was incorporated into fluoroacetate to an extent of 0.2 to 0.4%, whereas [3-14C]pyruvate, [2,3-14C]succinate, and [U-14C]aspartate were less efficiently incorporated (0.04 to 0.08%). The addition of [2-13C]glycerol to the mycelium suspension of Streptomyces cattleya caused exclusive enrichment of the carboxyl carbon of fluoroacetate with 13C; about 40% of carboxyl carbon of fluoroacetate was labeled with 13C. We studied the radioactivity incorporation of [3-14C]-, [U-14C]-, and [1-14C]beta-hydroxypyruvates to show that C-2 and C-3 of beta-hydroxypyruvate are exclusively converted to the carbon skeleton of fluoroacetate. These results suggest that the carbon skeleton of fluoroacetate derives from C-1 and C-2 of glycerol through beta-hydroxypyruvate, whose hydroxyl group is eventually replaced by fluoride.