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.     

A simple steady state kinetic model for alcohol dehydrogenase. Use of reaction progress curves for parameters estimation at pH 7.4

A simple rate equation for alcohol dehydrogenase was obtained by assuming independent binding sites for ethanol and NAD+ and fully competitive inhibition by the products of the reaction, acetaldehyde and NADH. A random binding order was also assumed. The rate equation is described by six parameters: four association constants (two for the substrates and two for the products of the reaction), Vf for the forward direction, and the equilibrium constant of the reaction. The six parameters were determined at pH 7.4 by numerical analysis of progress curves of reactions started with different concentrations of ethanol and NAD+. The parameters for alcohol dehydrogenase partially purified from rat liver were: Km for ethanol = 0.746 mM, Km for NAD+ = 0.0563 mM, Km for acetaldehyde = 7.07 microM, Km for NADH = 4.77 microM and Keq = 2.36 X 10(-4). The computed values allowed a very good simulation of the experimental progress curves and little variation was observed in the kinetic parameters when the reactions were started in the presence of either NADH or acetaldehyde.     

Ethanol metabolism in guinea pig: In vivo ethanol elimination,alcohol dehydrogenase distribution,and subcellular localization of acetaldehyde dehydrogenase in liver

Guinea pig ethanol metabolism as well as distribution and activities of ethanol metabolizing enzymes were studied. Alcohol dehydrogenase (ADH; EC 1.1.1.1) is almost exclusively present in liver except for minor activities in the cecum. All other organ tissues tested (skeletal muscle, heart, brain, stomach, and testes) contained only negligible enzyme activities. In fed livers, ADH could only be demonstrated in the cytosolic fraction (2.94 μmol/g liver/min at 38 °C) and its apparent K_m value of 0.42 mm for ethanol as substrate is similar to the average K_m of the human enzymes. Acetaldehyde dehydrogenase (ALDH; EC 1.2.1.3) of guinea pig liver was measured at low (0.05 mm) and high (10 mm) acetaldehyde concentrations and its subcellular localization was found to be mainly mitochondrial. The total acetaldehyde activity in liver amounts to 3.56 μmol/g/ min. Fed and fasted animals showed similar zero-order alcohol elimination rates after intraperitoneal injection of 1.7 or 3.0 g ethanol/kg body wt. The ethanol elimination rate of fed animals after 1.7 g ethanol/kg body wt (2.59 μmol/g liver/min) was inhibited by 80% after intraperitoneal injection of 4-methylpyrazole. Average ethanol elimination rates in vivo after 1.7 g/kg ethanol commanded only 88% of the totally available ADH activity in fed guinea pig livers. Catalase (EC 1.11.1.6), an enzyme previously implicated in ethanol metabolism, is of 3.4-fold higher activity in guinea pig (10,400 U/g liver) than in rat livers (3,100 U/g liver), but 98% inhibition by 3-amino-1,2,4-triazole did not significantly alter ethanol elimination rates. After ethanol injection, fed and fasted guinea pigs reacted with prolonged hyperglycemia.     

Kinetics of inhibition of ethanol metabolism in rats and the rate-limiting role of alcohol dehydrogenase

If liver alcohol dehydrogenase were rate-limiting in ethanol metabolism, inhibitors of the enzyme should inhibit the metabolism with the same type of kinetics and the same kinetic constants in vitro and in vivo. Against varied concentrations of ethanol, 4-methylpyrazole is a competitive inhibitor of purified rat liver alcohol dehydrogenase (Kis = 0.11 microM, in 83 mM potassium phosphate and 40 mM KCl buffer, pH 7.3, 37 degrees C) and is competitive in rats (with Kis = 1.4 mumol/kg). Isobutyramide is essentially an uncompetitive inhibitor of purified enzyme (Kii = 0.33 mM) and of metabolism in vivo (Kii = 1.0 mmol/kg). Low concentrations of both inhibitors decreased the rate of metabolism as a direct function of their concentrations. Qualitatively, therefore, alcohol dehydrogenase activity appears to be a major rate-limiting factor in ethanol metabolism. Quantitatively, however, the constants may not agree because of distribution in the animal or metabolism of the inhibitors. At saturating concentrations of inhibitors, ethanol is eliminated by inhibitor-insensitive pathways, at about 10% of the total rate at a dose of ethanol of 10 mmol/kg. Uncompetitive inhibitors of alcohol dehydrogenase should be especially useful for inhibiting the metabolism of alcohols since they are effective even at saturating levels of alcohol, in contrast to competitive inhibitors, whose action is overcome by saturation with alcohol.     

Regulation of ethanol metabolism in the rat

The purpose of these experiments was to examine the factors which regulate ethanol metabolism in vivo. Since the major pathway for ethanol removal requires flux through hepatic alcohol dehydrogenase, the activity of this enzyme was measured and found to be 2.9 mumol/(min X g liver). Ethanol disappearance was linear for over 120 min in vivo and the blood ethanol fell 0.1 mM/min; this is equivalent to removing 20 mumol ethanol/min and would require that flux through alcohol dehydrogenase be about 60% of its measured maximum velocity. To test whether ethanol metabolism was limited by the rate of removal of one of the end products (NADH) of alcohol dehydrogenase, fluoropyruvate was infused to reoxidize hepatic NADH and to prevent NADH generation via flux through pyruvate dehydrogenase. There was no change in the rate of ethanol clearance when fluoropyruvate was metabolized. Furthermore, enhancing endogenous hepatic NADH oxidation by increasing the rate of urea synthesis (converting ammonium bicarbonate to urea) did not augment the steady-state rate of ethanol oxidation. Hence, transport of cytoplasmic reducing power from NADH into the mitochondria was not rate limiting for ethanol oxidation. In contrast, ethanol oxidation at the earliest time periods could be augmented by increasing hepatic urea synthesis.     

Substituent effects in alkylated liver alcohol dehydrogenases

Alcohol dehydrogenase from horse liver was reductively alkylated with aldehydes having varied alkyl substituents. Kinetic studies of alkylated liver alcohol dehydrogenases which were modified in the absence and in the presence of NADH indicate that the alkylation of the specific lysine residues generally activates the enzyme by increasing Michaelis and inhibition constants for substrates and maximum velocities for the reactions. These kinetic parameters were analyzed in terms of electronic, steric, and hydrophobic effects of alkyl substituents. The hydrophilic character of the lysine residues is the most important factor which affects all kinetic parameters, particularly K_ia and V₂. In addition, the nucleophilic character of the lysine residues enhances the enzyme activity by increasing the maximum velocity of ethanol oxidation and the affinity of alcohol dehydrogenase for NADH and acetaldehyde. The steric interaction at the lysine residues favors the affinity of the enzyme for NADH and ethanol.     

Liver alcohol dehydrogenase activity in the female rat: Effects of ovariectomy and estradiol administration

The effects of ovariectomy and administration of estradiol on the activity of liver alcohol dehydrogenase and on the rate of ethanol elimination were determined in female Sprague-Dawley rats. The activity of the enzyme and the rates of ethanol elimination in the female sham-operated animals were higher than obtained previously in male rats of the same age. Ovariectomy had no effect on liver alcohol dehydrogenase and on rates of ethanol elimination. Estradiol administration resulted in an increase in liver weight and in total liver alcohol dehydrogenase activity per animal in sham-operated but not in ovariectomized animals. The increase in enzyme activity after estradiol administration in sham-operated animals was not associated with a significant increase in the rate of ethanol elimination, suggesting that the enzyme activity in female rats is not rate-limiting in in vivo ethanol oxidation.     

Characterization of a nonhepatic alcohol dehydrogenase from rat hepatocellular carcinoma and stomach.

Ethanol oxidation by the soluble fraction of a rat hepatoma was compared to that of the liver. Ethanol oxidation by the hepatoma was NAD⁺-dependent and sensitive to pyrazole, suggesting the presence of alcohol dehydrogenase. At low concentrations of ethanol (10.8 mm) the alcohol dehydrogenase activities of hepatoma and liver supernatant fractions were comparable. When the concentration of ethanol was raised to 108 mm, the activity of the liver enzyme decreased, whereas the activity in hepatoma supernatant fractions was strikingly elevated. m-Nitrobenzaldehyde-reducing activity was also conspicuously higher in hepatoma supernatant fractions. By contrast the ability to metabolize steroids and cyclohexanone was less than that in supernatant fractions of the liver.Electrophoresis of the liver supernatant fractions on ionagar at pH 7.0 revealed only one component that oxidized ethanol. On the other hand, hepatoma supernatant fractions contained two components with alcohol dehydrogenase activity; one with the same electrophoretic mobility as the liver enzyme, the other showing a slower rate of migration. The latter component, which is absent in the liver, is referred to as hepatoma alcohol dehydrogenase. By electrophoresis on starch gels at pH 8.5, it could be demonstrated that the liver and hepatoma enzymes moved in opposite directions.The liver and hepatoma enzymes differ in electrophoretic mobility, susceptibility to heat treatment, pH activity optimum and some catalytic properties. The substrate specificity of the hepatoma enzyme is narrower than that of liver alcohol dehydrogenase; cyclohexanone or 3β-hydroxysteroids of A/B cis configuration and the corresponding 3-ketones are not substrates for the hepatoma enzyme. The overall substrate specificity characteristics are, however, similar to those of the liver enzyme in that the effectiveness of substrates increases with an increase in chain length and introduction of unsaturation or an aromatic group. Both liver and hepatoma alcohol dehydrogenase cross-react with antibody to horse liver alcohol dehydrogenase EE. The Michaelis constant for ethanol with the hepatoma enzyme is 223 mm, compared to 0.3 mm for liver alcohol dehydrogenase; at 1.0 m ethanol the hepatoma enzyme is not fully saturated with substrate. The Michaelis constant for 2-hexene-1-ol is 0.3 mm, indicating that the hepatoma enzyme is better suited for dehydrogenation of longer chain alcohols. Stomach alcohol dehydrogenase has kinetic properties comparable to those of the hepatoma enzyme, as well as similar electrophoretic mobility. The hepatoma enzyme can be detected in the serum of rats bearing hepatomas.     

Ethanol metabolism in guinea pig: Ethanol oxidation and its effect on NADNADH ratios,oxygen consumption,and ketogenesis in isolated hepatocytes of fed and fasted animals

Ethanol metabolism was studied in isolated hepatocytes of fed and fasted guinea pigs. Alcohol dehydrogenase (EC 1.1.1.1) activities of fed or fasted liver cells were 2.04 and 1.88 μmol/g cells/min, respectively. Under a variety of in vitro conditions, alcohol dehydrogenase operates in fed hepatocytes at 34–74% and in fasted liver cells at 23–61% of its maximum velocity, respectively. Hepatocytes of fed animals, incubated in Krebs-Ringer bicarbonate buffer, oxidized ethanol at an average rate of 0.69 μmol/g wet weight cells/min, whereas cells of 48-h fasted animals consumed only 0.44 μmol/g/min under identical conditions. Various substrates and metabolites of intermediary metabolism significantly enhanced ethanol oxidation in fed liver cells. Maximum stimulatory effects were achieved with alanine (+138%) and pyruvate (+102%), followed in decreasing order by propionate, lactate, fructose, dihydroxyacetone, and galactose. In contrast to substrate couples such as lactate/pyruvate and glycerol/dihydroxyacetone, sorbitol with or without fructose significantly inhibited ethanol oxidation. The addition of hydrogen shuttle components such as malate, aspartate, or glutamate to fasted hepatocytes resulted in significantly higher stimulation of ethanol uptake than in fed hepatocytes. Also, the degree of inhibition of shuttle activity by n-butylmalonate was more pronounced in fasted liver cells (77% inhibition) than in fed cells (59% inhibition). These data as well as oxygen kinetic studies in intact guinea pig hepatocytes utilizing uncouplers (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone, dinitrophenol), electron-transport inhibitors (rotenone, antimycin), and malate-aspartate shuttle inhibitors (aminooxyacetate, n-butylmalonate) strongly suggested that the malate-aspartate shuttle is the predominant hydrogen transport system during ethanol oxidation in guinea pig liver.Comparison of the alcohol dehydrogenase-inhibitors 4-methylpyrazole and pyrazole on ethanol oxidation demonstrated that the alcohol dehydrogenase system is quantitatively the most important alcohol-metabolizing pathway in guinea pig liver. Supporting this conclusion, it was found that the H₂O₂-forming substrate glycolate slightly increased ethanol oxidation in liver cells of control animals (+26%), but prior inhibition of catalase by 3-amino-1,2,4-triazole resulted in a significant increase (+25%) instead of a decrease in alcohol oxidation. This finding does not support a quantitatively important role of peroxidatic oxidation of ethanol by catalase in liver.Cytosolic $\text{NAD}\text{NADH}$ ratios were greatly shifted toward reduction during ethanol oxidation. These reductive shifts were even more pronounced when cells were incubated in the presence of fatty acids (octanoate, oleate) plus ethanol. Inhibitor studies with 4-methylpyrazole demonstrated that the decrease of the cytosolic $\text{NAD}\text{NADH}$ ratio during fatty acid oxidation was due to an inhibition of hydrogen transport from cytosol to mitochondria and not the result of transfer of hydrogen, generated by fatty acid oxidation, from mitochondria to cytosol. Lactate plus pyruvate formation was slightly inhibited by ethanol in fed hepatocytes but greatly accelerated in fasted cells; this latter effect was mostly the result of increased lactate formation. Such regulation may represent a hepatic mechanism of alcoholic lactic acidosis as observed in human alcoholics. The ethanol-induced decrease of the mitochondrial $\text{NAD}\text{NADH}$ ratio was prevented by addition of 4-methylpyrazole. Endogenous ketogenesis was greatly increased (+80%) by ethanol in fed liver cells. This effect of ethanol was blunted in the presence of glucose. Propionate, by competing with fatty acid oxidation, was strongly antiketogenic. This effect was alleviated by ethanol. In 48-h fasted hepatocytes, endogenous ketogenesis was enhanced by 84%. Although ethanol did not further stimulate endogenous ketogenesis under these conditions, alcohol significantly increased ketogenesis in the presence of octanoate or oleate. This stimulatory effect of ethanol was almost completely prevented by 4-methylpyrazole. These findings demonstrate that the syndrome of alcoholic ketoacidosis may be due, at least partially, to the additional stimulation of ketogenesis by or from ethanol during fatty acid oxidation in the fasting state.     

Inhibition of hepatic gluconeogenesis by ethanol

1. Gluconeogenesis from 10mm-lactate in the perfused liver of starved rats is inhibited by ethanol. The degree of inhibition reached a maximum of 66% at 10mm-ethanol under the test conditions and decreased at higher ethanol concentrations. The concentration-dependence of the inhibition is paralleled by the concentration-dependence of the activity of alcohol dehydrogenase. The enzyme is also inhibited by ethanol concentrations above 10mm. 2. Gluconeogenesis from pyruvate is not inhibited by ethanol. 3. The degree of the inhibition of gluconeogenesis from lactate by ethanol depends on the concentration of lactate and other oxidizable substances, e.g. oleate, in the perfusion medium. 4. Ethanol also inhibits, to different degrees, gluconeogenesis from glycerol, dihydroxyacetone, proline, serine, alanine, fructose and galactose. 5. The inhibition of gluconeogenesis from lactate by ethanol is reversed by acetaldehyde. 6. Pyrazole, a specific inhibitor of alcohol dehydrogenase, also reverses the inhibition of gluconeogenesis by ethanol. 7. Gluconeogenesis in kidney cortex, where the activity of alcohol dehydrogenase is very low, is not inhibited by ethanol. 8. Kidney cortex, testis, ovary, uterus and certain tissues of the alimentary tract were the only rat tissues, apart from the liver, that showed measurable alcohol dehydrogenase activity. 9. The concentrations of pyruvate in the liver were decreased to about one-fifth by ethanol. 10. The concentration of lactate in the perfused liver was about 3mm below that of the perfusion medium 30min. after the addition of 10mm-lactate. 11. The great majority of the findings support the view that the inhibition of gluconeogensis by ethanol is caused by the alcohol dehydrogenase reaction, which decreases the [free NAD(+)]/[free NADH] ratio. The decrease lowers the concentration of pyruvate and this is the immediate cause of the inhibition of gluconeogenesis from lactate, alanine and serine: the fall in the concentration of pyruvate lowers the rate of the pyruvate carboxylase reaction, one of the rate-limiting reactions of gluconeogenesis. The cause of the inhibition of gluconeogenesis from other substrates is discussed.     

The reversibility of cytosolic dehydrogenase reactions in hepatocytes from starved and fed rats. Effect of fructose.

The metabolism of [2-3H]lactate was studied in isolated hepatocytes from fed and starved rats metabolizing ethanol and lactate in the absence and presence of fructose. The yields of 3H in ethanol, water, glucose and glycerol were determined. The rate of ethanol oxidation (3 mumol/min per g wet wt.) was the same for fed and starved rats with and without fructose. From the detritiation of labelled lactate and the labelling pattern of ethanol and glucose, we calculated the rate of reoxidation of NADH catalysed by lactate dehydrogenase, alcohol dehydrogenase and triosephosphate dehydrogenase. The calculated flux of reducing equivalents from NADH to pyruvate was of the same order of magnitude as previously found with [3H]ethanol or [3H]xylitol as the labelled substrate [Vind & Grunnet (1982) Biochim. Biophys. Acta 720, 295-302]. The results suggest that the cytoplasm can be regarded as a single compartment with respect to NAD(H). The rate of reduction of acetaldehyde and pyruvate was correlated with the concentration of these metabolites and NADH, and was highest in fed rats and during fructose metabolism. The rate of reoxidation of NADH catalysed by lactate dehydrogenase was only a few per cent of the maximal activity of the enzymes, but the rate of reoxidation of NADH catalysed by alcohol dehydrogenase was equal to or higher than the maximal activity as measured in vitro, suggesting that the dissociation of enzyme-bound NAD+ as well as NADH may be rate-limiting steps in the alcohol dehydrogenase reaction.     

Kinetic and stereochemical characterization of hamster liver 3 alpha-hydroxysteroid dehydrogenase and 3 alpha(17 beta)-hydroxysteroid dehydrogenase

The kinetic mechanism of NADP(+)-dependent 3 alpha-hydroxysteroid dehydrogenase and NAD(+)-dependent 3 alpha(17 beta)-hydroxysteroid dehydrogenase, purified from hamster liver cytosol, was studied in both directions. For 3 alpha-hydroxysteroid dehydrogenase, the initial velocity and product inhibition studies indicated that the enzyme reaction sequence is ordered with NADP+ binding to the free enzyme and NADPH being the last product to be released. Inhibition patterns by Cibacron blue and hexestrol, and binding studies of coenzyme and substrate are also consistent with an ordered bi bi mechanism. For 3 alpha(17 beta)-hydroxysteroid dehydrogenase, the steady-state kinetic measurements and substrate binding studies suggest a random binding pattern of the substrates and an ordered release of product; NADH is released last. However, the two enzymes transferred the pro-R-hydrogen atom of NAD(P)H to the carbonyl substrate.     

In vivo inhibition of liver alcohol dehydrogenase by ethanol administration

The activity of liver alcohol dehydrogenase (LADH) from rats sacrificed two hours after the administration of ethanol 3, 4 or 5 g/kg intraperitoneally was significantly inhibited compared to the activity of LADH from control rats. LADH activity was inversely related to the dose of ethanol administered, to the concentration of ethanol in the liver, and to the concentration of ethanol in the blood. The clearance of blood ethanol in rats was dose-dependent and was inversely related to the dose administered. The half-life of ethanol elimination increased as the dose of ethanol increased. These results suggest that ethanol-induced inhibition of LADH can occur in vivo and that the level of activity of this enzyme determines the rate of oxidation of ethanol.     

Human liver akdehyde dehydrogenase. Kinetics of aldehyde oxidation.

Steady state initial velocity studies were carried out to determine the kinetic mechanism of human liver aldehyde dehydrogenase. Intersecting double reciprocal plots obtained in the absence of inhibitors demonstrated that the dehydrogenase reaction proceeded by sequential addition of both substrates prior to release of products. Dead end inhibition patterns obtained with coenzyme and substrate analogues (e.g. thionicotinamide-AD+ and chloral hydrate) indicated that NAD+ and aldehyde can bind in random fashion. The patterns of inhibition by the product NADH and of substrate inhibition by glyceraldehyde were also consistent with this mechanism. However, comparisons between kinetic constants associated with the dehydrogenase and esterase activities of this enzyme suggested that most of the dehydrogenase reaction flux proceeds via formation of an initial binary NAD+-enzyme complex over a wide range of substrate and coenzyme concentrations.     

Effect of pyrazole on ethanol metabolism in ethanol-tolerant rats.

Adult male rats were pair-fed liquid diets, providing 37% of calories as ethanol or sucrose, for 1 month. Alcohol dehydrogenase (ADH) activity in the cytosol fractions of liver homogenates from the two groups did not differ with respect to total activity per 100 g body weight, Km for ethanol, or Ki for pyrazole. Other rats, fed in the same way, were fasted for 18-24 H, then given an intraperitoneal injection of pyrazole followed 1 h later by an injection of ethanol, 3g/kg. Blood alcohol curves showed an unexplained slower rise to maximum level in the chronic alcohol group. Both groups showed a period of several hours in which the blood alcohol stayed at the respective maximum concentrations, which were higher in the control group. After 7-8h the alcohol concentration began to fall in both groups, significantly more rapidly in the chronic alcohol-fed animals. A kinetic analysis shows that the results are adequately explained by the known effects of pyrazole on the ADH-mitochondrial system. The results are interpreted as evidence against the function of any microsomal ethanol oxidizing system in vivo.     

De novo design and utilization of photolabile caged substrates as probes of hydrogen tunneling with horse liver alcohol dehydrogenase at sub-zero temperatures: a cautionary note

In order to understand the influence of protein dynamics on enzyme catalysis and hydrogen tunneling, the horse liver alcohol dehydrogenase (HLADH) catalyzed oxidation of benzyl alcohol was studied at sub-zero temperatures. Previous work showed that wild type HLADH has significant kinetic complexity down to -50 degrees C due to slow binding and loss of substrate [S.-C. Tsai, J.P. Klinman, Biochemistry, 40 (2001) 2303]. A strategy was therefore undertaken to reduce kinetic complexity at sub-zero temperatures, using a photolabile (caged) benzyl alcohol that prebinds to the enzyme and yields the active substrate upon photolysis. By computer modeling, a series of caged alcohols were designed de novo, synthesized, and characterized with regard to photolysis and binding properties. The o-nitrobenzyl ether 15, with a unique long tail, was found to be most ideal. At sub-zero temperatures in 50% MeOH, a two-phase kinetic trace and a rate enhancement by the use of 15 were observed. Despite the elimination of substrate binding as a rate-limiting step, the use of caged benzyl alcohol does not produce a measurable H/D kinetic isotope effect. Unexpectedly, the observed fast phase corresponds to multiple enzyme turnovers, based on the stoichiometry of the substrate to enzyme. Possible side reactions and their effects, such as the re-oxidation of bound NADH and the dissipation of photo-excitation energy, may offer an explanation for the observed multiple-turnovers. The lack of observable deuterium isotope effects offers a cautionary note for the application of caged substrates to isolate and study chemical steps of enzyme reactions, particularly when NADH is involved in the reaction pathway.     

In vivo ethanol elimination in man, monkey and rat: a lack of relationship between the ethanol metabolism and the hepatic activities of alcohol and aldehyde dehydrogenases

The in vivo ethanol elimination in human subjects, monkeys and rats was investigated after an oral ethanol dosage. After 0.4 g. ethanol/kg of body weight, ethanol elimination was much slower in human subjects than in monkeys. In order to detect a rise in monkey plasma ethanol concentrations as early as observed in human subjects, ethanol had to be administered at a dose of 3 g/kg body weight. Ethanol metabolism in rats was also much faster than in human subjects. However, human liver showed higher alcohol dehydrogenase activity and higher low Km aldehyde dehydrogenase activity than rat liver. Thus, our data suggest a lack of relationship between hepatic ethanol-metabolizing activities and the in vivo ethanol elimination rate.     

Hydrogen transfer between ethanol molecules during oxidoreduction in vivo.

Rates of exchange catalysed by alcohol dehydrogenase were determined in vivo in order to find rate-limiting steps in ethanol metabolism. Mixtures of [1,1-2H2]- and [2,2,2-2H3]ethanol were injected in rats with bile fistulas. The concentrations in bile of ethanols having different numbers of 2H atoms were determined by g.l.c.-m.s. after the addition of [2H6]ethanol as internal standard and formation of the 3,5-dinitrobenzoates. Extensive formation of [2H4]ethanol indicated that acetaldehyde formed from [2,2,2-2H3]ethanol was reduced to ethanol and that NADH used in this reduction was partly derived from oxidation of [1,1-2H2]ethanol. The rate of acetaldehyde reduction, the degree of labelling of bound NADH and the isotope effect on ethanol oxidation were calculated by fitting models to the found concentrations of ethanols labelled with 1-42H atoms. Control experiments with only [2,2,2-2H3]ethanol showed that there was no loss of the C-2 hydrogens by exchange. The isotope effect on ethanol oxidation appeared to be about 3. Experiments with (1S)-[1-2H]- and [2,2,2-2H3]ethanol indicated that the isotope effect on acetaldehyde oxidation was much smaller. The results indicated that both the rate of reduction of acetaldehyde and the rate of association of NADH with alcohol dehydrogenase were nearly as high as or higher than the net ethanol oxidation. Thus, the rate of ethanol oxidation in vivo is determined by the rates of acetaldehyde oxidation, the rate of dissociation of NADH from alcohol dehydrogenase, and by the rate of reoxidation of cytosolic NADH. In cyanamide-treated rats, the elimination of ethanol was slow but the rates in the oxidoreduction were high, indicating more complete rate-limitation by the oxidation of acetaldehyde.     

An enzymatic model for rat liver alcohol dehydrogenase in the presence of low-Km aldehyde dehydrogenase

Four isoenzymes of aldehyde dehydrogenase were partially purified from rat liver mitochondria by hydroxylapatite chromatography and gel filtration. While three forms display low affinity for acetaldehyde, the fourth is active at extremely low aldehyde concentrations (Km less than or equal to 2 microM) and allows the oxidation of the acetaldehyde formed by catalysis of alcohol dehydrogenase at pH 7.4. Different models of alcohol dehydrogenase have been examined by analysis of progress curves of ethanol oxidation obtained in the presence of low-km aldehyde dehydrogenase. According to the only acceptable model, when the acetaldehyde concentration is kept low by the action of aldehyde dehydrogenase, NADH no longer binds to alcohol dehydrogenase, but acetaldehyde still competes with ethanol for the active site of the enzyme. The seven kinetic parameters of the two enzymes (four for alcohol dehydrogenase and three for aldehyde dehydrogenase) and the equilibrium constant of the reaction catalyzed by alcohol dehydrogenase have been determined by applying a new fitting procedure here described.     

Estimation of rate dissociation constants involving ternary complexes in reactions catalysed by yeast alcohol dehydrogenase

Stopped-flow studies of oxidation of butan-1-ol and propan-2-ol by NAD(+) in the presence of Phenol Red and large concentrations of yeast alcohol dehydrogenase give no evidence for the participation of a group of pK(a) approx. 7.6 in alcohol binding. Such a group has been implicated in ethanol binding to horse liver alcohol dehydrogenase [Shore, Gutfreund, Brooks, Santiago & Santiago (1974) Biochemistry13, 4185-4190]. The present result supports previous findings based on steady-state kinetic studies with the yeast enzyme. Stopped-flow studies of the yeast alcohol dehydrogenase-catalysed reduction of acetaldehyde by NADH in the presence of ethanol as product inhibitor indicate that the rate-limiting step is NAD(+) release from the enzyme-NAD(+)-ethanol product complex. This finding permits calculation of K(3), the dissociation constant for ethanol from the enzyme-NAD(+)-ethanol complex, by using the product-inhibition data of Dickenson & Dickinson (1978) (Biochem. J.171, 613-627). The calculations show that K(3) varies very little with pH in the range 5.95-8.9, and this agrees with the findings of the stopped-flow experiments described above. Absorption and fluorescence measurements on mixtures of substrates and coenzymes in the presence of high concentrations of alcohol dehydrogenase have been used to estimate values for the ratio [enzyme-NADH-acetaldehyde]/ [enzyme-NAD(+)-ethanol] at equilibrium. The values obtained were in the range 0.11+/-0.04, and this value together with estimates of K(3) was used to provide estimates of values for rate constants and dissociation constants for steps within the catalytic mechanism.