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PubMed1997

Metabolism and Energetics of Lactococcus lactis during Growth in Complex or Synthetic Media.

When Lactococcus lactis was grown in various complex or synthetic media, the fermentation of glucose remained homolactic whatever the medium used, with a global carbon balance of about 87%. Moreover, the nitrogen balance was not equilibrated, indicating that some amino acids led to the production of unknown nitrogen-containing carbon compounds while part of the glucose might contribute to anabolic pathways. In minimal medium containing six amino acids, a high concentration of serine was deaminated to pyruvate. This did not occur in more complete media, suggesting the presence of a regulation of this phenomenon by an amino acid. Ammonia produced during serine consumption was partly reconsumed after serine exhaustion. The values for biomass yield and biomass yield relative to ATP (Y(infATP)), the maximal growth rate, the specific rate of glucose consumption, and the corresponding rate of ATP synthesis all increased with the complexity of the medium, amino acid composition having the most pronounced effect. The Y(infATP) values were shown to range from 6.6 to 17.6 g of biomass(middot)mol of ATP(sup-1) on minimal and complex media.

Journal Article↗
PubMed1976

Effect of haematocrit value and pO2 on the redox state and metabolism of the perfused liver.

1. The haematocrit value and pO2 of blood perfusing the isolated liver were varied. Provided O2 content of the blood was not rate-limiting, O2 consumption was related to haemotocrit value rather than O2 saturation or pO2. 2. Hypoxia caused the blood-glucose concentration and ketogenesis to increase and the output of very-low-density (d less than 1.006) lipoproteins to decrease. 3. A decrease in pO2 caused an increase in both the (lactate)/(pyruvate) and (3-hydroxybutyrate)/(acetoacetate) and a decrease in (ATP)/(ADP) ratios, independently of O2 consumption. 4. The more reduced redox state was associated with a shift in the balance between the oxidation and esterification of free fatty acids in favour of oxidation. 5. Acetoacetate may be an important hydrogen acceptor during hypoxia of the liver.

Acetoacetates↗
PubMed1981

Storage of platelet concentrates using ion exchange resin charged with dibasic phosphate..

Platelet concentrates were prepared at twice the normal concentration and stored at room temperature for 7 days in either standard bags (controls) or bags to which 1 or 2 g of Amberlite resin beads charged with dibasic phosphate had been added. The resin beads served as a buffer system by providing a "slow release" form of phosphate ions as well as by binding CO2 produced during platelet metabolism. Control platelets demonstrated rapid falls in pH, ATP content, morphology score, and thrombin-induced nucleotide release after 24 hr of storage with a fall in pH to less than 6.0 by day 3. Profound ultrastructural changes and a rise in pO2, suggesting loss of platelet viability, accompanied these changes. In contrast, the resin-stored platelets remained near normal after 24 hr of storage, with preservation of discoid morphology, 95% of ATP levels, excellent ultrastructural appearance, and evidence of continued oxygen consumption after 3 days of storage. Even after 7 days of storage, ATP levels remained greater than 50% of baseline and ultrastructurally intact platelets were seen. In the 1-g resin bags the pH remained at baseline levels (6.9-7.0), while there was a rise in pH in the 2-g resin bags. These results demonstrate the beneficial effects of maintaining a higher pH during platelet storage and provide a new approach to studying the metabolic changes that occur during longer term storage.

Adenine Nucleotides↗
PubMed1993

Inhibition by 2,3-butanedione-monoxime of mitochondrial ADP-dependent respiration and muscle contraction.

2,3-Butanedione-monoxime (BDM) inhibits tension development in skeletal, cardiac and smooth muscle. Experiments on isolated single fibres of frog-muscle have shown a use dependent intermittent failure of twitching during continued stimulation. Our analysis of the effect of BDM on rabbit liver mitochondria and submitochondrial particles revealed a reduction of oxygen consumption in coupled mitochondria stimulated by adding of ADP, while in uncoupled mitochondria the oxygen consumption was not inhibited by BDM. Furthermore the substance did not inhibit ATP hydrolysis by submitochondrial particles. Based on these findings we suggest that BDM inhibits the activity of the mitochondrial ATP/ADP translocase.

Adenosine Diphosphate↗
PubMed1996

Effects of the inhibitors of energy metabolism, lonidamine and levamisole, on 5-aminolevulinic-acid-induced photochemotherapy.

The ability of endogenously synthesized protoporphyrin IX (PpIX) to damage Chinese hamster lung fibroblasts of the line V79 by exposure to light was examined. This treatment induced reduction of cellular ATP, GTP, of the NADH/NAD+ ratio and of oxygen consumption. The present results indicate a close relationship between inhibition of respiration of irradiated cells and their ability to survive, e.g. 1 min of light exposure induced 90% inhibition of oxygen consumption and inactivation of approximately 95% of the cells, while the cellular content of ATP was reduced by only 15%. This indicates that the mitochondria are one of the primary targets of 5-aminolevulinic acid (ALA)-mediated photochemotherapy (PCT). In the present study, ALA-PCT was combined with the modulators of the glycolysis and the respiration chain, levamisole (LEV) and lonidamine (LND). A synergistic effect of combining ALA-PCT with non-toxic concentrations of LND was observed when LND was given prior to light exposure. This synergism was observed despite a substantial LND-induced inhibition of PpIX formation. At increasing doses of LND (>0.15 mM) the combination treatment becomes less efficient. This is due to the inhibition of PpIX synthesis induced by LND. A synergistic effect of ALA-PDT and LEV was found when LEV was given prior to light exposure. This was at least partly due to an LEV-stimulated effect on ALA-induced PpIX formation. However, it is not clear from the present results whether LEV may perturb energy metabolism in V79 cells since LEV alone did not reduce the energy charge or the NADH/NAD+ ratio. When LEV or LND were given after ALA-PCT, these 2 treatment modalities acted in an additive or slightly synergistic manner.

Aminolevulinic Acid↗
PubMed1975

Systemic hypotension in neurosurgery.

The authors review the intraoperative use of elective hypotension to reduce the probability of hemorrhage, to increase pliability of the aneurysmal sac for ease of clip application, and to control hemorrhage. The optimum agent and techniques for lowering systemic blood pressure remain controversial, but trimethaphan, sodium nitroprusside, and halothane have been found most useful. When cerebral blood flow falls below the brain's capacity to autoregulate, distinct time-related alterations occur biochemically and histologically. The profile of prolonged reduced adenosine triphosphate (ATP), low phosphocreatine, low glucose, and elevated lactate and lactate/pyruvate ratio is associated with swelling of perivascular astrocytes and "blebbing" of vascular endothelial cells with subsequent cerebral damage. To prevent permanent alteration it is desirable to observe time constraints and to employ other means of protection such as hypothermia, although the authors believe the latter unnecessary for short hypotensive periods. It has been proposed, but not substantiated, that anesthetics which depress rate of cerebral oxygen consumption but do not affect cerebral ATP level protect the brain from hypotension. Several investigations suggest that halothane, a vasodiltor, satisfies the safety requirement. The most prominent contraindication to halothane, however, is elevation of intracranial pressure. At present hypotensive surgery for aneurysmorrhapy is usually performed when intracranial pressure has returned to normal. Experimentally the electroencephalogram has been observed to show alterations prior to biochemical parameters for following brain vulnerability, so that it conceivably could be an effective monitoring technique during prolonged profound hypotension.

Adenosine Triphosphate↗
PubMed2006

Metabolism of perfused pig intercostal muscles evaluated by 31P-magnetic resonance spectroscopy.

This study presents a perfused preparation for evaluation of metabolism in pig intercostal muscle in vitro. Preserved vessels and nerves to an intercostal segment including two adjacent ribs allowed for tissue perfusion and electrical stimulation with measurement of contraction force, oxygen consumption and 31P-magnetic resonance spectroscopy (31P-MRS). When perfused at rest with Krebs-Ringer buffer, the preparation maintained physiological levels of phosphocreatine (PCr), inorganic phosphate (Pi), ATP and pH at a stable oxygen consumption of 0.51 +/- 0.01 micromol min(-1) g(-1) for more than 2 h. Tonic stimulation of the nerve caused anaerobic energy consumption as PCr and pH decreased, and both variables recovered after the contraction with half-time values of approximately 7 min. Force increased to 0.040 N g(-1) (range, 0.031-0.103 N g(-1)) and it gradually decreased by about 70% during the subsequent 5 min of stimulation. The calculated free ADP concentration increased from 7.4 +/- 2.1 nmol g(-1) at rest to 28 +/- 12 nmol g(-1) (mean +/- s.d.) by the end of the stimulation. Thus anaerobic ATP turnover was zero at rest, 6.1 +/- 2 micromol min(-1) g(-1) during the first minute of stimulation and 3.5 +/- 0.5 micromol min(-1) g(-1) during the two last minutes, corresponding to the drop in force. When the preparation was left unperfused, anaerobic ATP turnover averaged 0.40 +/- 0.15 micromol min(-1) g(-1) for the first 10 min. The preparation can also be applied to human intercostal muscles, as demonstrated in one preliminary experiment. The results demonstrate a stable and functional in vitro preparation of intact perfused intercostal muscles in the pig.

Adenosine Diphosphate↗
PubMed1993

Anaerobic glycolysis and postanoxic recovery of respiration of rat cortical synaptosomes are reduced by synaptosomal sodium load.

Synaptosomes of rat cerebral cortex were used to study the effect of veratridine-induced Na+ load on postanoxic recovery of respiration and on aerobic and anaerobic ATP turnover, calculated from rates of oxygen consumption and lactate production. Non-stimulated synaptosomes: after onset of anoxia lactate synthesis of synaptosomes rose immediately from 0.8 to 17.7 nmol lactate/min/mg protein indicating an anaerobic ATP turnover of 17.7 nmol ATP/min/mg protein. This value accounts for 80% of ATP synthesized during oxygenated conditions and seems to cover the energetic demand of anoxic synaptosomes. This assumption was supported by linearity of lactate production throughout anoxia (90 min), by unaffected synaptosomal integrity and by complete recovery of postanoxic respiration after 90 min of anoxia. Stimulated synaptosomes: stimulation of oxygenated synaptosomes with 10(-5) mol/l veratridine enhanced ATP turnover 5-fold, due to activation of Na+/K+ ATPase, as a result of veratridine-induced Na+ influx. Consequently, if not limited in capacity, anaerobic ATP synthesis should be enhanced after addition of veratridine during anoxia. However, the opposite effect was observed. Veratridine reduced anaerobic glycolysis in a concentration-dependent manner. This inhibitory effect could be prevented by tetrodotoxin applied 5 min prior to veratridine. Inhibition of anaerobic glycolysis was independent of extrasynaptosomal glucose (1-30 mmol/l) and Ca2+ concentration (Ca(2+)-free and 1.2 mmol/l Ca2+). Veratridine stimulation of anoxic synaptosomes reduced also the recovery of postanoxic respiration. The data indicate that Na+ load inhibits anaerobic ATP synthesis, the only energy source during anaerobic conditions. To our knowledge, inhibition of anaerobic glycolysis due to increased Na+ influx has not been shown so far.

Anaerobiosis↗
PubMed1993

L-propionylcarnitine does not affect myocardial metabolic or functional response to chronotropic and inotropic stimulation after repetitive ischemia in anesthetized pigs.

In postischemic myocardium, fatty acid oxidation may be deficient owing to depletion of carnitine and citric acid cycle intermediates and fatty acylCoA-induced inhibition of adenine nucleotide translocase. During postischemic stress, the impairment of the fatty acid oxidation may become more apparent. We therefore investigated in open-chest anesthetized pigs the effect of L-propionylcarnitine [100 mg/kg per day orally (p.o.) for 3 days and 50 mg/kg intravenously (i.v.) 2 h before the first occlusion; n = 13] on myocardial function and metabolism of postischemic (two cycles of 10-min occlusion each followed by 30-min reperfusion) myocardium under resting conditions and during chronotropic and inotropic stimulation with dobutamine. Myocardial levels of free carnitine were higher after pretreatment (5.7 +/- 1.4 vs. 4.0 +/- 1.3 mumol/g protein, p < 0.05). The ischemia-reperfusion-induced decreases in free carnitine were similar for both the untreated and treated animals, but in the latter free carnitine was not different from the baseline levels in the control animals. In untreated animals (n = 15), regional systolic segment shortening (SS) was 18.5 +/- 5.5% (means +/- SD) at baseline, but was reduced to 5.1 +/- 5.5% (p < 0.05) at the end of the second reperfusion period. Myocardial ATP levels had decreased by 30% (p < 0.05) in the presence of a maintained energy charge, while myocardial oxygen and lactate consumption had decreased to 61% and 9% of baseline, respectively. During subsequent i.v. infusion of dobutamine (2 micrograms/kg/min), SS and myocardial oxygen consumption per beat increased to 75 and 65% of baseline, respectively, whereas lactate consumption per beat increased to only 25% of baseline. Decreases in myocardial ATP and oxygen and lactate consumption were not different between treated and untreated animals. L-Propionylcarnitine-treated animals displayed slightly better postischemic recovery of systolic SS than did control animals; to 39 and 28% (p = 0.056) of baseline, respectively, probably owing to a reduction in arterial blood pressure (BP), because L-propionylcarnitine prevented the increase in systemic vascular resistance produced by ischemia-reperfusion. L-Propionylcarnitine did not affect myocardial metabolic and contractile functional responses to chronotropic and inotropic stimulation. In a model of repetitive myocardial ischemia, L-propionylcarnitine prevents systemic vasoconstriction in response to ischemia and reperfusion and, probably as a result of the lower afterload, slightly ameliorates postischemic hypofunction, but loss of carnitine apparently does not play a role in myocardial hypofunction after brief repetitive ischemia and reperfusion in pigs.

Adenosine Diphosphate↗
PubMed1993

Enhanced antibody production associated with altered amino acid metabolism in a hybridoma high-density perfusion culture established by gravity separation.

A high density hybridoma perfusion culture was established by separating and recycling cells from the product stream to the reactor using a simple external sedimentation-based separator-an inclined modified Erlenmeyer flask. After 3 weeks, when the optimal perfusion rate of 1.0 day-1 had been reached, viable cell density stabilized at around 10 x 10(6) cells ml-1, a level five times that obtained by simple batch culture. The efficiency of the separator was enhanced by cell flocculation. Specific antibody productivity, which was initially 0.4 micrograms 1 x 10(6) cells-1 h-1, decreased to half that value while cell density was increasing, but recovered to the initial level when the culture finally stabilized at a high cell density. During the final phase, when viable cell density and specific antibody production were high, there was a marked shift in metabolism. Consumption of the two most important substrates for energy generation, glucose and glutamine, caused their broth concentrations to decrease to 1.5 mM and 1 mM, respectively, from input medium concentrations of 25 mM and 10 mM, respectively. At the same time there was an increase in the specific production of glycine and aspartate, their broth concentrations reaching 1.5 mM and 0.02 mM, respectively. We suggest that this shift in metabolism results in enhanced production of ATP from glutamine. The specific glucose consumption and lactate production also indicate that there is a shift to more energy efficient metabolism. The mechanism whereby this leads to enhanced specific antibody production remains to be elucidated. Nevertheless, the combination of high cell density and enhanced productivity obtained with the present perfusion culture resulted in a high monoclonal antibody production-100 mg 1-1 d-1.

Amino Acids↗
PubMed1978

Kinetics of oxygen consumption after a single isometric tetanus of frog sartorius muscle at 20 degrees C.

The time-course of the rate of oxygen consumption (QO2) has been measured in the excised frog sartorius muscle after single isometric tetani of 0.1-1.0 s at 20 degrees C. To measure deltaQO2(t), the change in QO2 from its basal level, a novel method was devised, based on the validity in this tissue of the one-dimensional diffusion equation for oxygen, established in the preceding paper. After a tetanus, deltaQO2 reached a peak within 45-90 s, then declined exponentially, and could be well fit by deltaQO2(t) = QO + Q1(epsilon -k1t - epsilon-k2t). tau2 (= 1/k2), which characterized the rise of deltaQO2, was a decreasing function of tetanus duration (range: from 1.1 +/- 0.28 min [nu = 5] for a 0.1-s tetanus, to 0.34 +/- 0.05 min [nu = 8] for a 1.0-sec tetanus). tau1 (= 1/k1), which characterized the decline of deltaQO2, was not dependent on tetanus duration, with mean 3.68 +/- -.24 min (nu = 46). A forthcoming paper in this series shows that these kinetics of deltaQO2 are the responses to impulse-like changes in the rate of ATP hydrolysis. The variation of tau2 with tetanus duration thus indicates the involvement of a nonlinear process in the coupling of O2 consumption to ATP hydrolysis. However, the monoexponential decline of deltaQO2(t), with time constant independent of tetanus duration, suggests that during this phase, the coupling is rate-limited by a single reaction with apparent first order kinetics.

Animals↗
PubMed1990

Response time of mitochondrial oxygen consumption following stepwise changes in cardiac energy demand.

We determined the speed with which mitochondrial oxygen consumption and therefore the mitochondrial ATP-synthesis adapted to changes in metabolic demand in the rabbit heart. This was done by measuring the oxygen uptake of the whole heart during a stepwise change in heart rate and correcting for the time taken by diffusion and by convective transport in the blood vessels. Data for the correction for transport time were obtained from the response of venous oxygen concentration to a stepwise change of arterial oxygen concentration. The time constant of the response of mitochondrial oxygen consumption to a step change in heart rate was found to be 4-8 s.

Adenosine Triphosphate↗
PubMed1983

Metabolic characteristics of cells cultured from human umbilical blood vessels: comparison with 3T3 fibroblasts.

Little metabolic information is available for cells cultured from umbilical vascular tissue. These studies were undertaken to compare the metabolism of cells isolated from human umbilical arteries and veins with that of umbilical vascular segments. These studies also compared umbilical vascular cells to standard fibroblast preparations. Oxygen consumption by umbilical venous cells or tissues was greater than that for either arterial cells or tissues. Cellular adenosine triphosphate (ATP) content was greater in umbilical venous than arterial cells. However, an oxidation-phosphorylation ratio (R) was constant for either arterial or venous cells. Oxygen consumption by vascular cells was greater than that by nonvascular cells, as was cellular ATP content. R for nonvascular cells was much greater than that for vascular cells, indicating loose coupling between oxygen consumption and cellular ATP content. Finally, cellular oxygen consumption was dependent upon cell density, and upon media serum content in vascular endothelial cells. We conclude therefore that the metabolism of umbilical vascular cells in culture reflects that of the parent tissue but is different from that of either vascular or nonvascular fibroblasts.

Adenosine Triphosphate↗
PubMed1982

Ketone-body utilization by homogenates of adult rat brain.

The regulation of ketone-body metabolism and the quantitative importance of ketone bodies as lipid precursors in adult rat brain has been studied in vitro. Utilization of ketone bodies and of pyruvate by homogenates of adult rat brain was measured and the distribution of 14C from [3-14C]ketone bodies among the metabolic products was analysed. The rate of ketone-body utilization was maximal in the presence of added Krebs-cycle intermediates and uncouplers of oxidative phosphorylation. The consumption of acetoacetate was faster than that of D-3-hydroxybutyrate, whereas, pyruvate produced twice as much acetyl-CoA as acetoacetate under optimal conditions. Millimolar concentrations of ATP in the presence of uncoupler lowered the consumption of ketone bodies but not of pyruvate. Indirect evidence is presented suggesting that ATP interferes specifically with the mitochondrial uptake of ketone bodies. Interconversion of ketone bodies and the accumulation of acid-soluble intermediates (mainly citrate and glutamate) accounted for the major part of ketone-body utilization, whereas only a small part was oxidized to CO2. Ketone bodies were not incorporated into lipids or protein. We conclude that adult rat-brain homogenates use ketone bodies exclusively for oxidative purposes.

2,4-Dinitrophenol↗
PubMed1989

The effect of fructose on the cellular content of adenine nucleotides in the perfused rat liver.

In order to evaluate the validity of using fructose in total parenteral nutrition, the mechanism of the resultant decrease in hepatic ATP content on fructose loading was examined using perfused rat livers. The ATP content of the livers perfused with fructose at a concentration of 2 mM or less, was about 2.8 mumol/g liver. Perfusion with 3 mM and 5 mM fructose decreased the ATP level to 1.65 and 1.50 mumol/g liver, respectively. Transient suppression of respiration occurred after the liver was perfused with fructose at a concentration of 3 mM or more, but not after 2 mM or less. Perfusion of the liver with fructose caused an accumulation of fructose 1-phosphate and a decrease in Pi. At higher concentrations of fructose, the accumulation of fructose 1-phosphate and the depletion of Pi were greater. The Pi depletion, caused by the accumulation of fructose 1-phosphate, suppressed O2 consumption and subsequently decreased the hepatic ATP content. A stoichiometric relationship was found between the total loss of adenine nucleotides in the liver and the amount of purine catabolites in the perfusate. After the administration of fructose at a concentration of 2 mM or less, however, the amount of remaining intracellular Pi was sufficient to maintain respiration and no decrease in the hepatic ATP content was observed.

Adenine Nucleotides↗
PubMed1987

Actions of extracellular UTP and ATP in perfused rat liver. A comparative study.

1. In perfused rat liver infusion of UTP and ATP in micromolar concentrations increased the portal pressure, with UTP being three times more effective than ATP at concentrations below 50 microM. Whereas ATP (up to 100 microM) increased oxygen consumption, there was a dose-dependent inhibition of oxygen uptake by UTP. 2. Both nucleotides stimulated hepatic glucose output; however, the time-courses were different. Withdrawal of UTP, but not of ATP (up to 100 microM) caused a further transient, but substantial stimulation of glucose output. 3. ATP led to a transient net K+ uptake by the liver being followed by a K+-release phase. Similar changes were observed with UTP; however, the initial K+ uptake was prolonged compared to ATP (1.9 min versus 3.5 min) and withdrawal of UTP, but not of ATP, stimulated hepatic K+ release markedly. 4. Metabolic and hemodynamic effects comparable to those induced by ATP were obtained with beta- and gamma-thio substituted ATP, whereas beta,gamma-methylene-substituted ATP was much less effective. The characteristic effects of UTP on glucose output, portal pressure and K+ fluxes were preserved during constant infusion of ATP or its beta,gamma-methylene derivative, pointing to additive effects. 5. ATP (20 microM) led to a net Ca2+ release (50-60 nmol/g liver) within 2-3 min. When the extracellular Ca2+ concentration was lowered from 1.25 mM to 0.3 mM, this Ca2+ release was increased to about 110 nmol/g liver whereby its time course remained largely unchanged. With 1.25 mM Ca2+, UTP induced Ca2+ movements only near the detection level (i.e. below 10-20 nmol/g liver); however, with 0.3 mM Ca2+ in influent perfusate, there was a slow Ca2+ release (not completed within 5-6 min). The maximal rates of Ca2+ efflux following ATP and UTP (20 microM each) were 70 nmol and 30 nmol g-1 min-1. Withdrawal of UTP led to a short Ca2+ release superimposing a phase of net Ca2+ uptake. 6. The data show that extracellular UTP is a potential and effective regulator of hepatic metabolism, ion fluxes across the hepatocyte membrane and hemodynamics. Compared to ATP, UTP seems to be more effective and the responses to both nucleotides are different. The data suggest that the action of UTP could involve a receptor distinct from the purinergic P2 receptor, whereas the ATP action involves predominantly the P2Y purinoceptor subtype.

Adenosine Triphosphate↗
PubMed2000

Exogenous Mg-ATP induces a large inhibition of pyruvate kinase in intact rat hepatocytes.

Mg-ATP infusion in vivo has been reported to be beneficial both to organ function and survival rate in various models of shock. Moreover, a large variety of metabolic effects has been shown to occur in several tissues due to purinergic receptor activation. In the present work we studied the effects of exogenous Mg-ATP in rat liver cells perifused with dihydroxyacetone to investigate simultaneously gluconeogenetic and glycolytic pathways. We found a significant effect on oxidative phosphorylation as characterized by a decrease in oxygen consumption rate and in the cellular ATP-to-ADP ratio associated with an increase in lactate-to-pyruvate ratio. In addition, exogenous Mg-ATP induced rapid and reversible inhibition of both gluconeogenesis and glycolysis. The main effect on gluconeogenesis was located at the level of the fructose cycle, whereas the decrease in glycolysis was due to a strong inhibition of pyruvate kinase. Although pyruvate kinase inhibition induced by exogenous Mg-ATP was allosteric when assessed in vitro after enzyme extraction, we found a large decrease in the apparent maximal velocity when kinetics were assessed in vivo in intact perifused hepatocytes. This newly described short-term regulation of pyruvate kinase occurs only in the intact cell and may open new potentials for the pharmacological regulation of pyruvate kinase in vivo.

Adenosine Diphosphate↗