Rate-limiting factors in the oxidation of ethanol by isolated rat liver cells.
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ABSTRACT:
SUBMITTER: Meijer AJ
PROVIDER: S-EPMC1165727 | biostudies-other | 1975 Aug
REPOSITORIES: biostudies-other
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Project description:Amino-oxyacetate (carboxymethoxylamine) was found to inhibit protein labelling in isolated liver cells. A similar degree of inhibition (about 70%) was observed of basal and substrate-stimulated rates of protein labelling, ruling out an action on the cellular energy state. Its effect does not seem to be related either to a perturbation of the reduction state of the NAD system or to rate changes in the gluconeogenic pathway. The following observations indicate that amino-oxyacetate inhibits protein labelling by limiting aspartate supply. Amino-oxyacetate was ineffective in a postmitochondrial supernatant under non-limiting amino acid supply conditions. The aspartate cellular content decreases in the presence of amino-oxyacetate, although most other amino acids tend to accumulate. L-Cycloserine was unable to decrease aspartate content and was ineffective in decreasing protein labelling. The inhibitory action of amino-oxyacetate was specifically reversed by incubating cells with amino acids that increase the cellular content of aspartate.
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Project description:1. Hepatocytes isolated from starved rats and incubated without other substrates oxidized ethanol at a rate of 0.8-0.9mumol/min per g wet wt. of cells. Addition of 10mm-lactate increased this rate 2-fold. 2. Quinolinate (5mm) or tryptophan (1mm) decreased the rate of gluconeogenesis with 10mm-lactate and 8mm-ethanol from 0.39 to 0.04-0.08mumol/min per g wet wt. of cells, but rates of ethanol oxidation were not decreased. From these results it appears that acceleration of ethanol oxidation by lactate is not dependent upon the stimulation of gluconeogenesis and the consequent increased demand for ATP. 3. As another test of the relationship between ethanol oxidation and gluconeogenesis, the initial lactate concentration was varied from 0.5mm to 10mm and pyruvate was added to give an initial [lactate]/[pyruvate] ratio of 10. This substrate combination gave a large stimulation of ethanol oxidation (from 0.8 to 2.6mumol/min per g wet wt. of cells) at low lactate concentrations (0.5-2.0mm), but rates remained nearly constant (2.6-3.0mumol/min per g wet wt. of cells) at higher lactate concentrations (2.0-10mm). 4. In contrast, owing to the presence of ethanol, the rate of glucose synthesis was only slightly increased (from 0.08 to 0.12mumol/min per g wet wt. of cells) between 0.5mm- and 2.0mm-lactate and continued to increase (from 0.12 to 0.65mumol/min per g wet wt. of cells) with lactate concentrations between 2 and 10mm. 5. In the presence of ethanol, O(2) uptake increased with increasing substrate concentration over the entire range. 6. Changes in concentrations of glutamate and 2-oxoglutarate closely paralleled changes in the rate of ethanol oxidation. 7. In isolated hepatocytes, rates of ethanol oxidation are lower than those in vivo apparently because of depletion of malate-aspartate shuttle intermediates during cell preparation. Rates are returned to those observed in vivo by substrates that increase the intracellular concentration of shuttle metabolites.
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Project description:1. Urate synthesis and other metabolic characteristics of isolated chicken hepatocytes were studied. 2. The distinction is made between immediate precursors of the purine ring (glycine, glutamine, aspartate, formyltetrahydrofolate, bicarbonate) and ultimate precursors from which the immediate precursors are formed in the liver. 3. In hepatocytes from well-fed chickens the rate of urate synthesis was not greatly increased by the addition of amino acids or NH(4)Cl, but in hepatocytes from 72h-starved chickens the rate was much increased when alanine or asparagine was added as the only substrate. Other amino acids, when added alone, did not affect the rate. The exceptional effect of alanine and asparagine is due to the ready formation of the immediate precursors. 4. Conditions are described under which glutamine, serine, glycine plus formate, ribose and glucose increased the rate of urate synthesis. 5. At 1mm-NH(4)Cl (a concentration not much higher than that of blood plasma) the rate of urate synthesis in the presence of lactate was increased, but higher concentrations inhibited urate synthesis in the presence of lactate or alanine; with alanine even 1mm-NH(4)Cl was inhibitory. 6. Glucose synthesis from lactate, alanine or dihydroxyacetone was also inhibited by 1mm-NH(4)Cl. 7. NH(4)Cl inhibition of urate and glucose synthesis was paralleled by an increased rate of glutamine synthesis. Thus in the presence of NH(4)Cl the gluconeogenic precursors are diverted from the pathway of gluconeogenesis to that of glutamate and glutamine synthesis. This implies that the synthesis of these amino acids is the primary process in the detoxication of ammonia in the avian liver. 8. Urate synthesis, like urea synthesis, can be looked on as a cyclic process with either phosphoribosyl pyrophosphate or ribose acting as the carrier on which the purine ring is assembled. 9. The energy requirements of urate synthesis depend on whether phosphoribosyl pyrophosphate is regenerated from IMP by pyrophosphorylase or by phosphorylation and pyrophosphorylation of ribose. It is 6 or 9 pyrophosphate bonds of ATP respectively.
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Project description:In order to distinguish between the mechanism of microsomal ethanol oxidation and hydroxyl-radical formation, the rate of cytochrome P-450 (P-450)-dependent oxidation of dimethyl sulphoxide (Me2SO) was determined in the presence and in the absence of iron-chelating compounds, in liver microsomes from control, ethanol- and phenobarbital-treated rats. Ethanol treatment resulted in a specific increase (3-fold) of the microsomal ethanol oxidation and NADPH consumption per nmol of P-450. A form of P-450 was purified to apparent homogeneity from the ethanol-treated rats and characterized with respect of amino acid composition and N-terminal amino acid sequence. Specific ethanol induction of a cytochrome P-450 species having a catalytic-centre activity of 20/min for ethanol and consuming 30 nmol of NADPH/min could account for the results observed with microsomes. Phenobarbital treatment caused 50% decrease in the rate of ethanol oxidation and NADPH oxidation per nmol of P-450. The rate of oxidation of the hydroxyl-radical scavenger Me2SO was increased 3-fold by ethanol or phenobarbital treatment when expressed on a per-mg-of-microsomal-protein basis, but the rate of Me2SO oxidation expressed on a per-nmol-of-P-450 basis was unchanged. Addition of iron-chelating agents to the three different types of microsomal preparations caused an 'uncoupling' of the electron-transport chain accompanied by a 4-fold increase of the rate of Me2SO oxidation. It is concluded that ethanol treatment results in the induction of P-450 forms specifically effective in ethanol oxidation and NADPH oxidation, but not in hydroxyl-radical production, as detected by the oxidation of Me2SO.
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Project description:It has been reported that chronic and acute alcohol exposure decreases cerebral glucose metabolism and increases acetate oxidation. However, it remains unknown how much ethanol the living brain can oxidize directly and whether such a process would be affected by alcohol exposure. The questions have implications for reward, oxidative damage, and long-term adaptation to drinking. One group of adult male Sprague-Dawley rats was treated with ethanol vapor and the other given room air. After 3 wk the rats received i.v. [2-(13)C]ethanol and [1, 2-(13)C2]acetate for 2 h, and then the brain was fixed, removed, and divided into neocortex and subcortical tissues for measurement of (13)C isotopic labeling of glutamate and glutamine by magnetic resonance spectroscopy. Ethanol oxidation was seen to occur both in the cortex and the subcortex. In ethanol-naïve rats, cortical oxidation of ethanol occurred at rates of 0.017 ± 0.002 µmol/min/g in astroglia and 0.014 ± 0.003 µmol/min/g in neurons, and chronic alcohol exposure increased the astroglial ethanol oxidation to 0.028 ± 0.002 µmol/min/g (P = 0.001) with an insignificant effect on neuronal ethanol oxidation. Compared with published rates of overall oxidative metabolism in astroglia and neurons, ethanol provided 12.3 ± 1.4% of cortical astroglial oxidation in ethanol-naïve rats and 20.2 ± 1.5% in ethanol-treated rats. For cortical astroglia and neurons combined, the ethanol oxidation for naïve and treated rats was 3.2 ± 0.3% and 3.8 ± 0.2% of total oxidation, respectively. (13)C labeling from subcortical oxidation of ethanol was similar to that seen in cortex but was not affected by chronic ethanol exposure.
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Project description:One weakness of batteries is the rapid falloff in charge-storage capacity with increasing charge/discharge rate. Rate performance is related to the timescales associated with charge/ionic motion in both electrode and electrolyte. However, no general fittable model exists to link capacity-rate data to electrode/electrolyte properties. Here we demonstrate an equation which can fit capacity versus rate data, outputting three parameters which fully describe rate performance. Most important is the characteristic time associated with charge/discharge which can be linked by a second equation to physical electrode/electrolyte parameters via various rate-limiting processes. We fit these equations to ~200 data sets, deriving parameters such as diffusion coefficients or electrolyte conductivities. It is possible to show which rate-limiting processes are dominant in a given situation, facilitating rational design and cell optimisation. In addition, this model predicts the upper speed limit for lithium/sodium ion batteries, yielding a value that is consistent with the fastest electrodes in the literature.
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Project description:1. A spectrophotometric direct-reading assay for measurements of beta-oxidation by intact mitochondria is described. The procedure relies on the ability of ferricyanide to trap reducing equivalents generated by the acyl-CoA dehydrogenases (EC 1.3.99.3). The reduction of ferricyanide was recorded by using a dual-wavelength spectrophotometer. 2. Oxaloacetate or acetoacetate was used to stimulate the rate of beta-oxidation by rotenone-blocked mitochondria. Although both were effective with rat liver mitochondria, oxaloacetate gave about 75% more stimulation. With heart or kidney mitochondria, only oxaloacetate gave marked stimulation. Acetoacetate had no stimulatory effect with heart mitochondria, but a small stimulatory effect on the rate of beta-oxidation by kidney mitochondria. 3. The stoicheiometry of beta-oxidation-dependent reduction of ferricyanide was examined, and good correlations were found between experimental and theoretical amounts of ferricyanide reduced. 4. Ferricyanide appears as efficient a final electron acceptor as O2. With ferricyanide the rate of beta-oxidation by heart mitochondria can be measured without interference from the oxidation of tricarboxylic acid-cycle intermediates.
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2020-07-01 | GSE137147 | GEO
Project description:Glutathione (GSH) is transported into renal mitochondria by the dicarboxylate (DIC; Slc25a10) and 2-oxoglutarate carriers (OGC; Slc25a11). To determine whether these carriers function similarly in liver mitochondria, we assessed the effect of competition with specific substrates or inhibitors on GSH uptake in isolated rat liver mitochondria. GSH uptake was uniphasic, independent of ATP hydrolysis, and exhibited K(m) and V(max) values of 4.08 mM and 3.06 nmol/min per mg protein, respectively. Incubation with butylmalonate and phenylsuccinate inhibited GSH uptake by 45-50%, although the individual inhibitors had no effect, suggesting in rat liver mitochondria, the DIC and OGC are only partially responsible for GSH uptake. H4IIE cells, a rat hepatoma cell line, were stably transfected with the cDNA for the OGC, and exhibited increased uptake of GSH and 2-oxoglutarate and were protected from cytotoxicity induced by H(2)O(2), methyl vinyl ketone, or cisplatin, demonstrating the protective function of increased mitochondrial GSH transport in the liver.
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