Rate Of Ethanol Oxidation Research Articles

Ethanol oxidation in isolated hepatocytes.

In isolated hepatocytes from fed and starved rats, rates of ethanol oxidation were 1.15 and 0.71 μmol x min−1 x (g wet wt)−1 respectively and were unchanged over the ethanol concentration range 8-96mM. The addition of the competitive inhibitors of alcohol dehydrogenase (ADH), pyrazole and particularly 4-methyl pyrazole (4-MP) abolished the oxidation of 8 mM ethanol and subsequently inhibited oxidation of 96 mM ethanol.In hepatocytes isolated from rats treated with ethanol, phenobarbitone or 3-amino-triazole, rates of ethanol oxidation were the same at ethanol concentrations of 8 and 96mM and the rates were similar to, and never exceeded the rate found in hepatocytes from normal fed rats. Pyrazole inhibited ethanol oxidation to the same extent in all preparations; 4-methyl pyrazole did likewise, but was more inhibitory than pyrazole.Pyruvate greatly stimulated cellular ethanol oxidation irrespective of the prior treatment of the donor animal. This stimulation together with the excess accumulation of lactate was totally abolished by 4-methyl pyrazole.Methylene blue, phenazine methosulphate and menadione stimulated both ethanol oxidation and respiration irrespective of prior treatment of the donor animal, but again the enhancement of ethanol oxidation and respiration was totally abolished by 4-methyl pyrazole. It is probable that these agents, like pyruvate, act by stimulating the oxidation of NADH generated in the cytoplasm by the alcohol dehydrogenase catalysed conversion of ethanol to acetaldehyde.These results indicate that under a wide variety of experimental conditions the contribution of the postulated microsomal ethanol oxidising system (MEOS) in isolated intact liver cells appears minimal. Thus they cast considerable doubt on its physiological role in vivo.KeywordsMethylene BlueEthanol OxidationDonor AnimalIsolate Liver CellArtificial Electron AcceptorThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

The effect of chronic ethanol consumption on pathways of ethanol metabolism.

The contribution of the known hepatic pathways for the disposal of ethanol was studied utilizing two different experimental designs. In vitro studies were carried out in hepatocytes isolated from rats fed Purina chow. Rates of ethanol oxidation in these preparations increased with increasing levels of ethanol (10-50 mM). After inhibition of alcohol dehydrogenase (ADH) by pyrazole (2 mM) and the catalase by azide (1 mM) approximately 25% of the ethanol oxidizing activity remained. The residual activity was also dependent upon ethanol concentration and the apparent Km was 13 mM. Hepatocytes from ethanol-fed rats exhibited rates of ethanol oxidation which were also dependent upon the concentration of ethanol. The rates were higher in the hepatocytes from the ethanol-fed rats than in the controls. The addition of inhibitors of ADH and catalase lowered the rates, but abolished neither the differences nor the concentration dependency. In the in vivo studies, ethanol elimination rates were measured in alcohol-fed and control baboons by using a constant ethanol infusion to maintain blood ethanol at three different levels: 5, 10 or 50 mM. Ethanol elimination rate was accelerated with increasing concentration, particularly in alcohol-fed baboons. These observations indicate that a pathway other than the low Km alcohol dehydrogenase participates in alcohol oxidation and is responsible in part for the adaptive increase in ethanol metabolism associated with chronic ethanol consumption.

Kinetics and control of alcohol oxidation in rats.

The rates of oxidation of ethanol and isopropanol by purified rat liver alcohol dehydrogenase were determined in vitro and compared to the rates of metabolism in vivo in order to estimate the extent to which alcohol dehydrogenase activity limits ethanol metabolism. The metabolism of isopropanol and isopropanol-d7 (CD3CDOHCD3) was examined by measuring blood alcohol and acetone levels at various times and apparently proceeds by an irreversible, enzyme-catalyzed pathway: isopropanol leads to acetone leads to an unidentified metabolite. The kinetic constants for the metabolism were computed from simultaneous fits to the appropriate differential equations using a nonlinear least-squares program. The relative rates of oxidation of the alcohols, ethanol:isopropanol:isopropanol-d7, at 25 mM were 9.6 : 2.3 : 1.0 in vitro and 4.1 : 2.4 : 1.0 in vivo. Since the ratio of rates for isopropanol is about the same in vitro and in vivo, it appears that alcohol dehydrogenase activity is the predominant rate-limiting factor in isopropanol metabolism. The relatively slower rate of ethanol oxidation in vivo as compared to in vitro suggests that liver alcohol dehydrogenase is partially (about 40%) limiting for ethanol metabolism.

The effect of pargyline on the metabolism of ethanol and acetaldehyde by isolated rat liver cells

The effect of pargyline on the uptake of acetaldehyde (in the presence of pyrazole) by isolated rat liver cells was studied after incubating the liver cells for 0, 10, 30, 45, and 60 min with 0.40, 1.30, and 2.6 m m pargyline. Without any incubation period, pargyline had no effect on acetaldehyde uptake. With increasing time of incubation, there was a progressive increase in the extent of inhibition of acetaldehyde uptake by pargyline. This suggests the possibility that pargyline is metabolized to the effective inhibitor or the incubation period allows pargyline to reach its site(s) of action. Pargyline was also a more effective inhibitor of the uptake of lower concentrations of acetaldehyde, e.g., 0.167 m m, than of higher concentrations (1.0 m m) of acetaldehyde, especially after short incubation periods or when pyrazole was omitted from the reaction medium. After a 20- to 30-min incubation period, pargyline inhibited the control rate of ethanol oxidation by the liver cells, as well as the accelerated rate of ethanol oxidation found in the presence of pyruvate or an uncoupling agent. Pargyline had no effect on hepatic oxygen consumption. During ethanol oxidation, a time-dependent release of acetaldehyde into the medium was observed. Pyruvate, by increasing the rate of ethanol oxidation, increased the output of acetaldehyde five- to tenfold. Pargyline increased the output of acetaldehyde two- to threefold, despite decreasing the rate of ethanol metabolism by the liver cells. These data indicate that pargyline inhibits the low K m aldehyde dehydrogenase in intact rat liver cells and that this enzyme plays the major role in oxidizing the acetaldehyde which arises during the metabolism of ethanol. Although most of the acetaldehyde generated during the oxidation of ethanol is removed by the liver cells in an effective manner, changes in the activity of aldehyde dehydrogenase or the rate of acetaldehyde generation significantly alter the hepatic output of acetaldehyde.

Effect of natural amino acids on ethanol oxidation in isolated rat hepatocytes

The effects of the various naturally occurring amino acids on ethanol oxidation in hepatocytes from starved rats was systematically studied. In order to minimize the non ADH pathways, the ethanol concentration used was 4 mmol/litre, the amino acids being added at the same concentration. In hepatocytes from fasted rats, alanine, arginine, asparagine, aspartate, citrulline, cysteine, glutamate, glutamine, glycine, histidine, hydroxyproline, ornithine and serine increase significantly ethanol consumption. The stimulatory effect of glutamine being much less pronounced than the asparagine one and proline being devoid of action, the influence of ammonium chloride addition on ethanol consumption in the presence of these amino acids was studied. Ammonium chloride determines an enhancement of ethanol oxidation in these conditions, the results showing no apparent correlation between intracellular glutamate concentration and ethanol oxidation rate, contrarily to previous data. In hepatocytes from fed rats, only alanine, asparagine, cysteine, glycine, hydroxyproline, ornithine and serine increase ethanol oxidation, although to a lesser extent than in cells from starved rats.

Respective role of superoxide and hydroxyl radical in the activity of the reconstituted microsomal ethanol-oxidizing system

Superoxide dismutase, a scavenger of O − 2. does not affect the rate of ethanol oxidation in a reconstituted system containing purified cytochrome P-450, NADPH-cytochrome c reductase, and dilauroyl l-3-phosphatidyl choline. The same concentration of Superoxide dismutase (50 μg/ml) completely abolishes the oxidation of epinephrine in this reconstituted system and ethanol oxidation by the xanthine-xanthine oxidase. Ethanol is not oxidized by the reconstituted system when NADPH is replaced by H 2O 2 but the addition of H 2O 2 to this sytem containing NADPH accelerates ethanol oxidation. This increase is abolished by the addition of Superoxide dismutase. Hydroxyl radical scavengers (50 m m dimethylsulfoxide, 100 m m benzoate, 100 m m mannitol, 20 m m thiourea) diminish the oxidation of ethanol in the reconstituted system by 48 to 76%. Thus hydroxyl radical may participate in the activity of reconstituted ethanol-oxidizing system, whereas Superoxide is not involved.

Lactate-stimulated ethanol oxidation in isolated rat hepatocytes.

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.

Ethanol oxidation by isolated hepatocytes from ethanol-treated and control rats; factors contributing to the metabolic adaptation after chronic ethanol consumption

Chronic consumption of ethanol by rats produced a fatty liver and resulted in a pronounced increase in the rate of ethanol oxidation by isolated hepatocytes. Despite the increase in ethanol oxidation, oxygen consumption with several substrates was not enhanced after chronic ethanol treatment. Ouabain, an inhibitor of the (Na + + K + )-ATPase activity, did not abolish the increase in the rate of ethanol oxidation. About 40–50 per cent of the increase in ethanol oxidation persisted after inhibition of alcohol dehydrogenase, mitochondrial oxygen consumption or the malate-aspartate shuttle. The addition of substrates for the malate-aspartate shuttle slightly increased the rate of ethanol oxidation in hepatocytes from control and ethanol-treated animals. The increased rate of ethanol oxidation was not abolished by the uncoupling agent dinitrophenol. which by itself had little effect on ethanol oxidation. In the presence of aspartate or α-glycerophosphate, dinitrophenol augmented the rate of ethanol oxidation; in the presence of glutamate, the rate of ethanol oxidation was doubled by dinitrophenol. However, the higher rate of ethanol oxidation after ethanol consumption was still found in the presence of various combinations of substrate shuttles, with or without dinitrophenol. Pathways independent of alcohol dehydrogenase may contribute, at least in part, to the increase in ethanol oxidation found after chronic ethanol consumption. It is concluded that ethanol oxidation may be enhanced after chronic ethanol consumption without the estabishment of a hypermetabolic state of the liver.

Affinity Labelling of Alcohol Dehydrogenases

1. DL-alpha-Bromo-beta(5-imidazolyl)-propionic acid is a potential affinity labelling reagent for metallo-enzymes. It has been used with the alcohol dehydrogenases from liver and yeast. The liver enzyme is chemically modified and inactivated in a Michaelis-Menten-type reaction, where one molecule of the reagent is bound per subunit. The enzyme is protected from the inhibitor in a competitive manner by imidazole, 2,2'-dipyridyl, 1,10-phenanthroline and cyclohexanone, which all combine with the active-site zinc. The protection by chloride, acetate and NADH, which are considered to bind at the general anion binding site, is not strictly competitive. Inactivation has an optimum at pH 8.5. For the liver enzyme, the reagent was found to decrease the initial rate of ethanol oxidation. Prior to the irreversible alkylation of Cys-46, reversible binding is shown to occur at the active-site zinc atom. The yeast enzyme was extremely resistant to the reagent and no specific modification was found. 2. The potential affinity labelling and crosslinking reagent, symmetrical 1,3-dibromoacetone although unstable, has also been used for chemical modification. With the liver enzyme, concentrations below 5 mM gave a reaction of the Michaelis-Menten-type at pH 7.0. Several ligands known to complex with the active-site region protect the enzyme against the reagent. Dibromoacetone gave rapid inactivation of the yeast enzyme. Despite the fact that a pseudo-first-order reaction was observed with respect to enzyme as well as inhibitor, no saturating effect was found. In this work, dibromoacetone reacted like a monofunctional reagent.

Transfer and reoxidation of reducing equivalents as the rate-limiting steps in the oxidation of ethanol by liver cells isolated from fed and fasted rats

A study was made of factors regulating the oxidation of ethanol in liver cells isolated from fed and fasted rats. The rate of ethanol oxidation was greater in liver cells from fed rats than from fasted rats. Inhibitors of the malate-aspartate shuttle decreased the rate of ethanol oxidation, suggesting that this shuttle contributes to the reoxidation of cytosolic NADH produced during the oxidation of ethanol. The greater inhibition of ethanol oxidation by antimycin than by rotenone suggests that the α-glycerophosphate shuttle also plays an important role in transporting reducing equivalents. The components of the malate-aspartate and α-glycerophosphate shuttles stimulated ethanol oxidation to a greater extent in liver cells from fasted rats than those from fed rats, suggesting that in the fasted state, ethanol oxidation is regulated by the intracellular concentrations of substrate shuttle components which transfer reducing equivalents into the mitochondria. Therefore, uncoupling agents, which stimulate oxygen consumption, do not stimulate ethanol oxidation, and concentrations of antimycin which depress oxygen uptake are much less effective in decreasing ethanol oxidation. By contrast, in liver cells from fed rats, the rate of ethanol oxidation was increased by uncoupling agents. Such stimulation was not observed when cells were prepared in the absence of albumin, probably due to leakage of shuttle substrates which leads to abnormally low intracellular levels. Indeed, when the shuttle substrates were added back to these preparations, uncouplers were effective in stimulating the rate of ethanol oxidation beyond the stimulation produced by the shuttle substrates alone. Thus, under conditions of sufficient intracellular levels of the intermediates of the substrate shuttles, ethanol oxidation is regulated by the capacity of the mitochondrial respiratory chain to reoxidize reducing equivalents generated by the alcohol dehydrogenase reaction.

The effect of dimethylsulfoxide and other hydroxyl radical scavengers on the oxidation of ethanol by rat liver microsomes

The NADPH-dependent oxidation of ethanol by rat liver microsome preparations was studied in the presence of azide to inhibit the peroxidatic activity of catalase. Dimethylsulfoxide, benzoate, mannitol and thiourea, four compounds that react rapidly with hydroxyl radicals, each inhibited the oxidation rate of ethanol. Inhibition was competitive with respect to ethanol. In contrast, urea, a compound that reacts poorly with hydroxyl radicals, was essentially without effect. Dimethylsulfoxide at concentrations that inhibited the oxidation of ethanol had no effect on the xanthine oxidase-mediated oxidation of ethanol or on aniline hydroxylase or aminopyrine demethylase activity of microsomes. These results suggest that ethanol oxidation by microsomes can be dissociated from drug metabolism and that the mechanism of ethanol oxidation may involve, in part, the interaction of ethanol with hydroxyl radicals that are generated by microsomes during the oxidation of NADPH.

Isolation of II-alcohol dehydrogenase of human liver: Is it a determinant of alcoholism?

HUMAN LIVER ALCOHOL DEHYDROGENASE (ALCOHOL: NAD(+) oxidoreductase, EC 1.1.1.1), homogeneous by physicochemical criteria, has been available in quantity only recently [Lange, L. G. & Vallee, B. L. (1976) Biochemistry 15, 4681-4686]. Until now, the biochemical basis of human alcohol metabolism had to be extrapolated from the properties and behavior of enzymes from other species, primarily horses and yeast. The biological determinants of human alcoholism have remained obscure, although recent evidence indicates a genetic predisposition, requiring delineation. A functionally distinct form of human liver alcohol dehydrogenase (ADH), which we have designated II-ADH, is provocative since, thus far, it seems to be unique to human beings. It has a high K(m) for ethanol and is remarkably insensitive (apparent K(I), 500 muM) to pyrazole and its derivatives, which are usually potent ADH inhibitors (K(I), 1 muM), a property that is the basis for the isolation of II-ADH. The affinity resin 4-[3-(N-6-aminocaproyl)aminopropyl]pyrazole-Sepharose binds all other known forms of ADH but not II-ADH, thereby separating it selectively by affinity chromatography. In turn, this has led to the establishment of its identity with that enzyme form which was previously known as the anodic band and characterized by a high K(m) for ethanol (20 mM at pH 7.5). The remarkable insensitivity of II-ADH to pyrazole inhibition has also permitted quantitation of its role in hepatic ethanol oxidation. At 5 mM ethanol, a saturating concentration for virtually all other forms of ADH, II-ADH contributes less than 15% to total ethanol oxidation. However, at intoxicating concentrations, e.g., 60 mM, it can account for as much as 40% of the total ethanol oxidation rate of liver, indicating a seemingly unique role for this enzyme form in ethanol elimination. Thus far, we have found the amount of II-ADH varies from liver to liver of individuals and is considerably more labile than the other molecular forms, phenomena whose inter- or independence requires further study. The isolation of human II-ADH advances efforts to recognize and understand biochemical mechanisms that may be biological determinants of alcoholism and alcohol-related disease states, now generally approached and managed largely as psychosocial disorders.

Effect of Acute Murine Hepatitis (MHV-A-59) on Ethanol Oxidation in Vivo

Studies were performed to examine the effect of mild, moderate, and severe hepatitis infection induced by murine A-59 virus on ethanol metabolism in vivo. Female inbred mice were injected intraperitoneally with 8, 12, and 16 complement fixation units of virus, which produced liver damage ranging from mild (three to four foci of necrosis and inflammation per hepatic lobule) to severe hepatitis (destruction of 70 to 90% of hepatocytes). Ethanol oxidation was measured 48 hr after infection when maximal cell damage was present as determined by light microscopy and SGOT values. Ethanol (2.4 g per kg) was injected intraperitoneally and the rate of ethanol oxidation was measured by (1) cumulative production of I4CO2 from [14C]ethanol, (2) sequential serum ethanol levels, and (3) ethanol clearance using a subcutaneous air bubble technique. Using these methods there was no difference in ethanol oxidation between infected and control animals (P

Factors Contributing to the Adaptive Increase in Ethanol Metabolism due to Chronic Consumption of Ethanol

SUMMARYChronic ethanol consumption, in a diet which leads to a fatty liver, results in an increase in the rate of ethanol oxidation in isolated rat hepatocytes. The percent increase (30%‐40%) is similar to that previously found in vivo, in the same model system.1,11 This increase does not correlate with the activity of alcohol dehydrogenase, which was slightly decreased after chronic ethanol consumption.1,16 The increase in ethanol oxidation is not associated with a hypermetabolic state of the liver since oxygen consumption with a variety of substrates was not enhanced after chronic ethanol consumption. Approximately 40%‐50% of this increase persists in the presence of an inhibitor of alcohol dehydrogenase, inhibitors of mitochondrial oxygen consumption and inhibitors of the malate‐aspartate shuttle. The increase persists in the presence of ouabain, dinitrophenol and various shuttle metabolites (in the presence and absence of dinitrophenol). Chronic consumption of ethanol results in hypertrophy of the smooth endoplasmic reticulum and an adaptive increase in the ability of microsomal preparations to oxidize ethanol to acetaldehyde.1,11 The fact that part of the increase in ethanol oxidation which occurs after chronic ethanol administration persists in the presence of the various additives discussed above, indicates that pathways that are independent of alcohol dehydrogenase contribute to this metabolic adaptation. These data extend the observation that the metabolic adaptation found after chronic ethanol consumption was not abolished by 2mM pyrazole.1,26

Regulation of acetaldehyde metabolism during ethanol oxidation in perfused rat liver.

Acetaldehyde (AcH) metabolism during ethanol oxidation has been studied in once-through perfused rat livers. The interest was focused upon the interrelations between the hepatic AcH concentration, AcH oxidation rate, ethanol metabolism, cytosolic and mitochondrial redox state, and aldehyde dehydrogenase (ALDH) activity. Correlational analyses between the possible regulative factors and regulated parameters were made and the following main conclusions drawn. The AcH concentration leaving the liver during the ethanol metabolism was regulated by both the ethanol and AcH oxidation rates, which in turn were regulated by the cytosolic redox state and the ALDH activity, respectively.

Hepatic ethanol metabolism: Respective roles of alcohol dehydrogenase, the microsomal ethanol-oxidizing system, and catalase

The respective role of alcohol dehydrogenase, of the microsomal ethanol-oxidizing system, and of catalase in ethanol metabolism was assessed quantitatively in liver slices using various inhibitors and ethanol at a final concentration of 50 m m. Pyrazole (2 m m) virtually abolished cytosolic alcohol dehydrogenase activity but inhibited ethanol metabolism in liver slices by only 50–60%. The residual pyrazole-insensitive ethanol oxidation in liver slices remained unaffected by in vitro addition of the catalase inhibitor sodium azide (1 m m). At this concentration, sodium azide completely abolished catalatic activity of catalase in liver homogenate as well as peroxidatic activity of catalase in liver slices in the presence of dl-alanine. Similarly, in vivo administration of 3-amino-1,2,4-triazole, a compound which inhibits the activity of catalase but not that of the microsomal ethanol-oxidizing system, failed to decrease both the overall rates of ethanol oxidation and the activity of the pyrazole-insensitive pathway. Finally, butanol, a substrate and inhibitor of the microsomal ethanol-oxidizing system but not of catalase-H 2O 2, significantly decreased the pyrazole-insensitive ethanol metabolism in liver slices. These results indicate that alcohol dehydrogenase is responsible for half or more of ethanol metabolism by liver slices and that the microsomal ethanol-oxidizing system rather than catalase-H 2O 2 accounts for most if not all of the alcohol dehydrogenase-independent pathway.

Mechanism of the stimulatory effect of fructose on ethanol oxidation in perfused rat liver.

The stimulatory effect of fructose on ethanol oxidation was studied in livers from fasted rats perfused with Krebs-Henseleit-bicarbonate buffer in a non-recirculating system. Two series of experiments were performed: (A) ethanol was infused with stepwise increasing concentrations (0.1-20 mM) in the presence of 4 mM fructose; (B) fructose was infused with stepwise increasing concentrations (0.5-10 mM) in the presence of 2 mM ethanol. From measured metabolic rates the following parameters were calculated: energy-rich phosphates consumed for fructose metabolism which were provided from oxidative phosphorylation (delta approximately P); reducing equivalents derived from stimulated ethanol utilization which were disposed by mitochondrial oxidation (delta2H). Under the various conditions studied a linear relationship between these parameters was observed. The ratio delta approximately P/delta2H was about 2.0. It is suggested that fructose stimulates ethanol oxidation indirectly by increasing the energy consumption of the liver due to the production of glucose from fructose. Consequetnly, the rate of oxidative phosphorylation is increased and, therefore, the capacity of the respiratory chain for oxidizing reducing equivalents derived from ethanol is enhanced. The data support the more general hypothesis that the rate of ethanol oxidation depend upon the rate of hepatic energy consumption in a given metabolic state.

Some properties of an alcohol dehydrogenase partially purified from baker's yeast grown without added zinc

Alcohol dehydrogenase was partially purified from yeast (Saccharomyces cerevisiae) grown in the presence of 20 muM-MnSO4 without added Zn2+ and from yeast grown in the presence of 1.8 muM-MnSO4. The enzyme from yeast grown with added Zn2+ has the same properties as the crystalline enzyme from commercial supplies of baker's yeast. The enzyme from yeast grown without added An2+ has quite different properties. It has a mol.wt. in the region of 72000 and an S 20 w of 5.8S. The values can be compared with a mol.wt. of 141000 and an S 20 w of 7.6S for the crystalline enzyme. ADP-ribose, a common impurity in commercial samples of NAD+, is a potent competitive inhibitor of the new enzyme (K1 = 0.5 muM), but is not so for the crystalline enzyme. The observed maximum rate of ethanol oxidation at pH 7.05 and 25 degrees C was decreased 12-fold by the presence of 0.06 mol of inhibitor/mol of NAD+ when using the enzyme from Zn2+-deficient yeast, but with crystalline enzyme the maximum rate was essentially unchanged by this concentration of inhibitor. The kinetic characteristics for the two enzymes with ethanol, butan-1-ol, acetaldehyde and butyraldehyde as substrates are markedly different. These kinetic differences are discussed in relation to the mechanism of catalysis for the enzyme from Zn2+-deficient yeast.

Influence of Ethanol Oxidation Rate on the Lactate/Pyruvate Ratio and Phosphorylation State of the Liver in Fed Rats.

The effect of the ethanol oxidation rate on the interaction between the phosphorylation state (the [ATP]/[ADP] X [HPO4]2- ratio) and the redox state (the free [NAD+]/[NADH] ratio) of the liver cytosol was studied in intact fed rats. The rate of ethanol oxidation was inhibited to different degrees with pyrazole. The ethanol oxidation rate had no influence on the liver lactate level but correlated significantly with the pyruvate level. Accordingly, a significant correlation was also found between the ethanol oxidation rate and the lactate/pyruvate ratio. The rate of ethanol oxidation correlated significantly with the liver 3-phosphoglycerate level. No change in the glyceraldehyde-3-phosphate level was found. No correlation was found between the ethanol oxidation rate and the glyceraldehyde-3-phosphate/3-phosphoglycerate redox couple. Ethanol administration slightly increased the liver ATP level, but the simultaneous administration of pyrazole eliminated this effect. Other adenine nucleotides and HPO4 2- were not changed. The changes in the rate of ethanol oxidation had no effect on the phosphorylation state in the fed liver. It is assumed that in the fed liver the phosphorylation state is so well stabilized that the redox level has no influence.

Effect of ethanol on utilization of plasma free fatty acids for liver triacylglycerol synthesis and its relation to hepatic triacylglycerol accumulation in rats.

The effect of 3 different single doses of ethanol on the liver triacylglycerol concentration and on the metabolism of intravenously injected 14C-oleic acid in fasted rats was studied. All 3 doses (2, 3.75, and 6 g ethanol/kg body wt) caused a rapid increase in the liver triacylglycerol concentration during the first 5-6 hr after the ethanol was given. Until the plasma ethanol concentration had fallen to low values, the high liver triacylglycerol levels were raised and were independent of the ethanol dose given. The incorporation of radioactivity from intravenously injected 14C-oleic acid into liver triacylglycerols was increased over control values to the same extent in all rats given ethanol as long as the plasma ethanol concentration was above a low level. High rates of ethanol oxidation and increased utilization of plasma free fatty acids for liver triacylglycerol synthesis were closely correlated with the development and maintenance of the ethanol induced liver triacylglycerol accumulation.