The role of tryptophan 2,3-dioxygenase in the hormonal control of tryptophan metabolism in isolated rat liver cells. Effects of glucocorticoids and experimental diabetes.

The metabolism of L-tryptophan by isolated liver cells prepared from control, adrenalectomized, glucocorticoid-treated, acute-diabetic, chronic-diabetic and insulin-treated chronic-diabetic rats was studied. Liver cells from adrenalectomized rats metabolized tryptophan at rates comparable with the minimum diurnal rates of controls, but different from rates determined for cells from control rats 4h later. Administration of dexamethasone phosphate increased the activity of tryptophan 2,3-dioxygenase (EC 1.13.11.11) 7-8-fold, and the flux through the kynurenine pathway 3-4-fold, in cells from both control and adrenalectomized rats. Increases in flux through kynureninase (EC 3.7.1.3) and to acetyl-CoA can be explained in terms of increased substrate supply from tryptophan 2,3-dioxygenase. The metabolism of tryptophan was increased 3-fold in liver cells isolated from acutely (3 days) diabetic rats, with a 7-8-fold increase in the maximal activity of tryptophan 2,3-dioxygenase. The oxidation of tryptophan to CO2 and metabolites of the glutarate pathway increased 4-5-fold, consistent with an increase in picolinate carboxylase (EC 4.1.1.45) activity. Liver cells isolated from chronic (10 days) diabetic rats metabolized tryptophan at rates comparable with those of cells from acutely diabetic rats, but with a 50% decrease in the activity of tryptophan 2,3-dioxygenase. The proportion of flux from tryptophan 2,3-dioxygenase to acetyl-CoA, however, was increased by 50%; this was indicative of further increases in the activity of picolinate carboxylase. Administration of insulin partially reversed the effects of chronic diabetes on the activity of tryptophan 2,3-dioxygenase and flux through the kynurenine pathway, but had no effect on the increased activity of picolinate carboxylase. The role of tryptophan 2,3-dioxygenase in regulating the blood tryptophan concentration is discussed with reference to its sensitivity to the above conditions.     

Differential effects of tryptophan on glucose synthesis in rats and guinea pigs.

1. Tryptophan inhibition of gluconeogenesis in isolated rat liver cells is characterized by a 20 min lag period before linear rates of glucose output are attained. 2. Half-maximal inhibition of gluconeogenesis in isolated rat hepatocytes is produced by approx. 0.1 mM-tryptophan. 3. Tryptophan inhibits gluconeogenesis from all substrates giving rise to oxaloacetate, but stimulates glycerol-fuelled glucose production. 4. Gluconeogenesis in guinea-pig hepatocytes is insensitive to tryptophan. 5. Changes in metabolite concentrations in rat liver cells are consistent with a locus of inhibition at the step catalysed by phosphoenolpyruvate carboxykinase. 6. Inhibition of gluconeogenesis persists in cells from rats pretreated with tryptophan in vivo. 7. Tryptophan has no effect on urea production from alanine, but decreases [1-14C]palmitate oxidation to 14CO2 and is associated with an increased [hydroxybutyrate]/[acetoacetate] ratio. 8. These results are discussed with reference to the control of gluconeogenesis in various species.     

The metabolism of L-tryptophan by liver cells prepared from adrenalectomized and streptozotocin-diabetic rats.

1. The metabolism of L-tryptophan by liver cells prepared from fed normal, adrenalectomized and streptozotocin-diabetic rats was studied. 2. At physiological concentrations (0.1 mM), the rate of oxidation of tryptophan by tryptophan 2,3-dioxygenase was 3-fold greater in liver cells from diabetic rats than in those from fed rats. In liver cells from diabetic rats, oxidation of tryptophan to CO2 and metabolites of the glutarate pathway was increased 7-fold. Quinolinate synthesis was decreased by 50%. These findings are consistent with an increase in picolinate carboxylase activity. 3. Rates of metabolism of 0.1 mM-tryptophan by hepatocytes from fed and adrenalectomized rats were similar. 4. In all three types of cell preparation, fluxes through tryptophan 2,3-dioxygenase with 2.5 mM-tryptophan were 7-fold greater than those obtained with 0.1 mM-tryptophan. Tryptophan 2,3-dioxygenase and kynureninase fluxes in hepatocytes from fed and adrenalectomized rats were comparable, whereas those in liver cells from diabetic rats were increased 2.5-fold and 3.3-fold respectively. Picolinate carboxylase activities of liver cells from diabetic rats were 15-fold greater than those of cells from fed rats, but rates of quinolinate synthesis were unchanged. 5. It is concluded that: (i) adrenal corticosteroids are not required for the maintenance of basal activities of the kynurenine pathway, whereas (ii) chronic insulin deficiency produces changes in both the rate of oxidation and metabolic fate of tryptophan carbon.     

Leucine and tryptophan metabolism in rats.

The rate of tryptophan metabolism in isolated liver cells from animals fed on a high-leucine diet was greater than for cells from control animals. Leucine inhibited tryptophan metabolism and tryptophan uptake in isolated liver cells, probably by competing for membrane transport. Leucine had no effect on tryptophan 2,3-dioxygenase in vitro. 4-Methyl-2-oxovalerate increased tryptophan oxidation in incubations containing albumin, by displacing bound tryptophan and increasing the availability of the amino acid to the cell. The results suggest that, under extreme conditions, when the availability of tryptophan is low, leucine may be pellagragenic.     

Production of 4-hydroxypyrazole from the interaction of the alcohol dehydrogenase inhibitor pyrazole with hydroxyl radical

Pyrazole, an effective inhibitor of alcohol dehydrogenase, was previously shown to be a scavenger of the hydroxyl radical. 4-Hydroxypyrazole is a major metabolite in the urine of animals administered pyrazole in vivo. Experiments were conducted to show that 4-hydroxypyrazole was a product of the interaction of pyrazole with hydroxyl radical generated from three different systems. The systems utilized were the iron-catalyzed oxidation of ascorbate, the coupled oxidation of hypoxanthine by xanthine oxidase, and NADPH-dependent microsomal electron transfer. Ferric-EDTA was added to all the systems to catalyze the production of hydroxyl radicals. A HPLC procedure employing either uv detection or electrochemical detection was utilized to assay for the production of 4-hydroxypyrazole. The three systems all supported the oxidation of pyrazole to 4-hydroxypyrazole by a reaction which was sensitive to inhibition by competitive hydroxyl radical scavengers such as ethanol, mannitol, or dimethyl sulfoxide and to catalase. The sensitivity to catalase implicates H2O2 as the precursor of the hydroxyl radical by all three systems. Superoxide dismutase inhibited production of 4-hydroxypyrazole only in the xanthine oxidase reaction system. In the absence of ferric-EDTA (and azide), microsomes catalyzed the oxidation of pyrazole to 4-hydroxypyrazole by a cytochrome P-450-dependent reaction which was independent of hydroxyl radicals. This latter pathway may be primarily responsible for the in vivo metabolism of pyrazole to 4-hydroxypyrazole. The production of 4-hydroxypyrazole from the interaction of pyrazole with hydroxyl radicals may be a sensitive, rapid technique for the detection of these radicals in certain tissues or under certain conditions, e.g., increasing oxidative stress.     

The metabolism of L-tryptophan by isolated rat liver cells. Quantification of the relative importance of, and the effect of nutritional status on, the individual pathways of tryptophan metabolism.

1. The metabolism of L-tryptophan by liver cells prepared from fed and 48 h-starved rats was studied. Methods are described, with the use of L-[ring-2-(14)C], L-[carboxy-14C]-and L-[benzene-ring-U-14C]-tryptophan, for the simultaneous determination of tryptophan 2,3-dioxygenase and kynureninase activities and of the oxidation of tryptophan to CO2 and non-aromatic intermediates of the kynurenine-glutarate pathway. 2. At physiological concentrations (0.1 mM), tryptophan was oxidized by tryptophan 2,3-dioxygenase at comparable rates in liver cells from both fed and starved rats. Kynureninase activity of hepatocytes from starved rats was 50% greater than that of cells from fed rats. About 10% of the tryptophan metabolized by tryptophan 2,3-dioxygenase was degraded completely to CO2. 3. In the presence of 0.5 mM-L-tryptophan, tryptophan 2,3-dioxygenase and kynureninase activities increased 5--6-fold. Liver cells from starved rats oxidized tryptophan at about twice the rate of these from fed rats. Degradation of tryptophan to non-aromatic intermediates of the glutarate pathway and CO2 was increased only 3-fold, suggesting an accumulation of aromatic intermediates of the kynurenine pathway. 4. Rates of metabolism with 2.5 mM-L-tryptophan were not significantly different from those obtained with 0.5 mM-tryptophan. 5. Rates of synthesis of quinolinic acid from 0.5 mM-L-tryptophan, determined either by direct quantification or indirectly from rates of radioisotope release from L-[carboxy-(14)C]- and [benzene-ring-U-14C]tryptophan, were essentially similar. 6. At all three concentrations examined, tryptophan was degraded exclusively through kynurenine; there was no evidence of formation of either indol-3-ylacetic acid or 5-hydroxyindol-3-ylacetic acid.     

The metabolism of L-tryptophan by isolated rat liver cells. Effect of albumin binding and amino acid competition on oxidatin of tryptophan by tryptophan 2,3-dioxygenase.

1. Novel methods, using L-[ring-2-14C]tryptophan, are described for the measurement of tryptophan 2,3-dioxygenase activity and tryptophan accumulation in isolated rat liver cells. 2. The effects of bovine serum albumin, non-esterified fatty acids and neutral amino acids on tryptophan oxidation by hepatocytes and on the partition of tryptophan between free and albumin-bound forms were investigated. 3. Oxidation of physiological concentrations (0.1 mM) of tryptophan was inhibited by approx. 50% in the presence of 2% (w/v) bovine serum albumin; no effects were found at tryptophan concentrations of 0.5 mM and above. 4. Increases in free tryptophan concentrations produced by displacement of 0.1 mM-tryptophan from albumin-binding sites by palmitate resulted in increased flux through tryptophan dioxygenase. 5. Addition of a mixture of neutral amino acids, at plasma concentrations, to hepatocyte incubations had no effect on the rate of tryptophan oxidation. 6. It is concluded that alterations in free tryptophan concentrations consequent to changes in albumin binding may be an important factor in regulating tryptophan uptake and catabolism by the liver. The results are briefly discussed with reference to possible consequences on brain tryptophan metabolism.     

Enzyme activities involved in tryptophan metabolism along the kynurenine pathway in rabbits

The following enzyme activities of the tryptophan-nicotinic acid pathway were studied in male New Zealand rabbits: liver tryptophan 2,3-dioxygenase, intestine indole 2,3-dioxygenase, liver and kidney kynurenine 3-monooxygenase, kynureninase, kynurenine-oxoglutarate transaminase, 3-hydroxyanthranilate 3,4-dioxygenase, and aminocarboxymuconate-semialdehyde decarboxylase. Intestine superoxide dismutase and serum tryptophan were also determined. Liver tryptophan 2,3-dioxygenase exists only as holoenzyme, but intestine indole 2,3-dioxygenase is very active and can be considered the key enzyme which determines how much tryptophan enters the kynurenine pathway also under physiological conditions. The elevated activity of indole 2,3-dioxygenase in the rabbit intestine could be related to the low activity of superoxide dismutase found in intestine. Kynurenine 3-monooxygenase appeared more active than kynurenine-oxoglutarate transaminase and kynureninase, suggesting that perhaps a major portion of kynurenine available from tryptophan may be metabolized to give 3-hydroxyanthranilic acid, the precursor of nicotinic acid. In fact, 3-hydroxyanthranilate 3,4-dioxygenase is much more active than the other previous enzymes of the kynurenine pathway. In the rabbit liver 3-hydroxyanthranilate 3,4-dioxygenase and aminocarboxymuconate-semialdehyde decarboxylase show similar activities, but in the kidney 3-hydroxyanthranilate 3,4-dioxygenase activity is almost double. These data suggest that in rabbit tryptophan is mainly metabolized along the kynurenine pathway. Therefore, the rabbit can also be a suitable model for studying tryptophan metabolism in pathological conditions.     

The oxidation of external NADH by an intermembrane electron transfer in mitochondria from the ubiquinone-deficient mutant E3-24 of Saccharomyces cerevisiae

Cells of the E3-24 mutant of the strain D273-10B of Saccharomyces cerevisiae, grown in a fermentable substrate not showing catabolite repression of respiration (2% galactose), are able to respire, in spite of their ubiquinone deficiency in mitochondrial membranes. Mitochondria isolated from these mutant cells oxidize exogenous NADH through a pathway insensitive to antimycin A but inhibited by cyanide. Addition of methanolic solutions of ubiquinone homologs stimulates the oxidation rate and restores antimycin A sensitivity in both isolated mitochondria and whole cells. Mersalyl preincubation of isolated mitochondria inhibits both NADH oxidation and NADH-cytochrome c oxido-reductase activity (assayed in the presence of cyanide) with the same pattern. Electrons resulting from the oxidation of exogenous NADH reduce both cytochrome b5 and endogenous cytochrome c. The increase in ionic strength stimulates NADH oxidation, which is also coupled to the ATP synthesis with an ATP/O ratio similar to that obtained with ascorbate plus N,N,N',N'-tetramethyl-p-phenylendiamine (TMPD) as substrate. The effect of cyanide on these activities and on NADH-induced endogenous cytochrome c reduction is also comparable. These results support the existence in vivo and in isolated mitochondria of a energy-conserving pathway for the oxidation of cytoplasmatic NADH not related to the dehydrogenases of the inner membrane, the ubiquinone, and the b-c1 complex, but involving a cytochrome c shuttle between the NADH-cytochrome c reductase of the outer membrane and cytochrome oxidase in the inner membrane.     

Substrate Oxidation by Indoleamine 2,3-Dioxygenase: EVIDENCE FOR A COMMON REACTION MECHANISM*

The kynurenine pathway is the major route of l-tryptophan (l-Trp) catabolism in biology, leading ultimately to the formation of NAD⁺. The initial and rate-limiting step of the kynurenine pathway involves oxidation of l-Trp to N-formylkynurenine. This is an O₂-dependent process and catalyzed by indoleamine 2,3-dioxygenase and tryptophan 2,3-dioxygenase. More than 60 years after these dioxygenase enzymes were first isolated (Kotake, Y., and Masayama, I. (1936) Z. Physiol. Chem. 243, 237–244), the mechanism of the reaction is not established. We examined the mechanism of substrate oxidation for a series of substituted tryptophan analogues by indoleamine 2,3-dioxygenase. We observed formation of a transient intermediate, assigned as a Compound II (ferryl) species, during oxidation of l-Trp, 1-methyl-l-Trp, and a number of other substrate analogues. The data are consistent with a common reaction mechanism for indoleamine 2,3-dioxygenase-catalyzed oxidation of tryptophan and other tryptophan analogues.     

Fluidizing effect of endogenous ubiquinone in bovine heart mitochondrial membranes

Exposure of isolated rat liver cells to glucagon or dibutyryl cyclic AMP leads to a prompt decrease in the rate of cellular peroxide generation as evidenced by (i) a reduced rate of [14C]formate oxidation and (ii) a lowered steady-state concentration of catalase Compound I.     

Oxidation of pyrazole by reconstituted systems containing cytochrome P-450 IIE1

Rat liver microsomes oxidize pyrazole to 4-hydroxypyrazole and this oxidation is increased in microsomes isolated from rats treated with inducers of cytochrome P-450 IIE1, such as pyrazole or ethanol. A reconstituted system containing the P-450 IIE1, purified from pyrazole-treated rats, oxidized pyrazole to 4-hydroxypyrazole in a time- and P-450-dependent manner. Oxidation of pyrazole was dependent on the concentration of pyrazole over the range of 0.15 mM to 1.0 mM. In isolated microsomes, glycerol inhibited pyrazole oxidation by about 50% under concentration conditions which occur in the reconstituted system; hence, the values for pyrazole oxidation by the reconstituted systems are underestimated because of the presence of glycerol. Oxidation of pyrazole was inhibited by competitive substrates for P-450 IIE1, such as 4-methylpyrazole, aniline and ethanol, as well as by an antibody raised against the pyrazole-induced P-450 IIE1. Thus, pyrazole is an effective substrate for oxidation by purified P-450 IIE1, extending the substrate specificity of this isozyme to potent inhibitors of alcohol dehydrogenase.     

Intracellular utilization of superoxide anion by indoleamine 2,3-dioxygenase of rabbit enterocytes.

The participation of superoxide anion (O2-) in the intracellular indoleamine 2,3-dioxygenase activity was studied using the dispersed cell suspension of the rabbit small intestine. The dioxygenase activity was assayed by measuring [14C]formate released from DL-[ring-2-14C]tryptophan. The addition of diethyldiethiocarbamate, a superoxide dismutase inhibitor, markedly accelerated the intracellular dioxygenase activity while the superoxide dismutase activity decreased concomitantly. Furthermore, substrates of xanthine oxidase such as inosine, adenosine, and hypoxanthine also increased the dioxygenase activity in the cells, particularly in the presence of methylene blue. This increase was completely abolished by the addition of allopurinol, a specific inhibitor of xanthine oxidase. These results, taken together, indicate that the intracellular accumulation of O2- results in acceleration of the in situ dioxygenase activity, and that indoleamine 2,3-dioxygenase utilizes O2- in the isolated intestinal cells.     

Insulin-stimulated intracellular hydrogen peroxide production in rat epididymal fat cells.

Insulin stimulation of hydrogen peroxide production by rat epididymal fat cells was investigated by studying the oxidation of formate to CO2 by endogenous catalase. Under optimal concentrations of formate (0.1 to 1 mM) and glucose (0.275 mM), insulin stimulated formate oxidation 1.5- to 2.0-fold. Inhibitors of catalase activity, including nitrite and azide, inhibited both basal and insulin-stimulated formate oxidation at concentrations that did not interfere with insulin effects on glucose C-1 oxidation or glucose H-3 incorporation into lipids. The addition of exogenous catalase increased formate oxidation only slightly, while exogenous H2O2 (0.5 mM) stimulated formate oxidation by endogenous catalase strongly. These data indicate that the insulin-stimulated H2O2 production was intracellular. Insulin dose-response curves for formate oxidation were identical with those for glucose H-3 incorporation into lipids. The dependence of relative insulin effects on the logarithm of the glucose concentration was bell-shaped for formate oxidation and correlated highly with the coresponding dependences of glucose C-1 oxidation and glucose H-3 incorporation into lipids. This suggests that insulin stimulation of intracellular H2O2 production is linked to glucose metabolism. Since it is known that extracellular H2O2 can mimic insulin in several respects, these observations suggest that H2O2 may act as a "second messenger" for the observed effects of insulin.     

Acclimatization to hypoxia modulates the tryptophan 2,3-dioxygenase activity in rats exposed to simulated high altitude.

1. Exposure of rats to an 8000 m altitude increased the hepatic tryptophan 2,3-dioxygenase (EC 1.13.1.12) activity. 2. Acclimatization to hypoxia by a repeated exposure to an altitude of 5000 m induced a marked decrease in liver tryptophan dioxygenase activity after the rats were subjected to an 8000 m altitude, but a pre-exposure to 4000 m altitude showed no effect on the enzyme activity. 3. Plasma tryptophan was rapidly decreased by exposure to 8000 m altitude to the same extent in the acclimatized and non-acclimatized rats. 4. Plasma tryptophan may be utilized as the substrate for tryptophan dioxygenase in liver of the non-acclimatized rats under highly hypoxic conditions; however, acclimatized rats can reserve tryptophan as the substrate for the alternative metabolism other than the degradation pathway in liver.     

The influence of starvation and tryptophan administration on the metabolism of phenylalanine, tyrosine and tryptophan in isolated rat liver cells.

Liver cells from fed Sprague-Dawley rats metabolized phenylalanine, tyrosine and tryptophan at rates consistent with the known kinetic properties of the first enzymes of each pathway. Starvation of rats for 48 h did not increase the maximal activities of phenylalanine hydroxylase, tryptophan 2,3-dioxygenase and tyrosine aminotransferase in liver cell extracts, when results were expressed in terms of cellular DNA. Catabolic flux through the first two enzymes was unchanged; that through the aminotransferase was elevated relatively to enzyme activity. This is interpreted in terms of changes in the concentrations of 2-oxoglutarate and glutamate. Cells from tryptophan-treated animals exhibited significant increases in the catabolism of tyrosine and tryptophan, but not of phenylalanine. The activities of tyrosine aminotransferase and tryptophan 2,3-dioxygenase were also increased, although the changes in flux and enzyme activity did not correspond exactly. These results are discussed with reference to the control of aromatic amino acid catabolism in liver; the role of substrate concentration is emphasized.     

Enzyme activities along the tryptophan-nicotinic acid pathway in alloxan diabetic rabbits

Recent data from our laboratory have indicated that the rabbit is a suitable animal model for the study of enzyme activities of the tryptophan-nicotinic acid pathway. We report here the pattern of tryptophan metabolism in rabbits made diabetic with alloxan treatment, and hypercholesterolemic with a high-cholesterol diet. A group of rabbits with only hypercholesterolemia was also considered. The enzymes assayed were: liver tryptophan 2,3-dioxygenase (TDO), intestine indoleamine 2,3-dioxygenase (IDO), liver and kidney kynurenine 3-monooxygenase, kynurenine-oxoglutarate transaminase, kynureninase, 3-hydroxyanthranilate 3,4-dioxygenase and aminocarboxymuconate-semialdehyde decarboxylase.TDO showed a reduction of specific activity in liver of diabetic-hyperlipidemic and hyperlipidemic rabbits compared to controls. Intestine IDO activities and liver and kidney kynurenine monooxygenase were unchanged with respect to controls.Kynurenine-oxoglutarate transaminase and kynureninase activities were reduced in the kidneys, but not in the liver, of diabetic-hyperlipidemic rabbits.The main finding was the reduction of 3-hydroxyanthranilate 3,4-dioxygenase activity (expressed as activity per g of fresh tissue) in the liver and kidneys of diabetic-hypercholesterolemic and hyperlipidemic rabbits compared to controls. Conversely, aminocarboxymuconate-semialdehyde decarboxylase activity was significantly higher in diabetic hypercholesterolemic rabbits in comparison with control and hypercholesterolemic rabbits.These data demonstrate that also in diabetic rabbits there is an alteration of tryptophan metabolism at the level of 3-hydroxyanthranilic acid-->nicotinic acid step. Also dyslipidemia seems to be involved in enzyme activity variations of the tryptophan metabolism along the kynurenine pathway.     

Mode of inhibition of mitochondrial energy transduction by chlorophenoxyisobutyrate

1. alpha-p-Chlorophenoxyisobutyric acid, the ethyl ester of which is widely used as an antihypercholesterolaemic drug, is an inhibitor of energy-transfer reactions in isolated rat liver mitochondria. 2. The compound at lower concentrations (<4.0mumol/mg of mitochondrial protein) inhibits state 3 oxidation, stimulates state 4 oxidation, abolishes respiratory control and stimulates the latent adenosine triphosphatase activity of mitochondria. The inhibition imposed on state 3 oxidation is relieved by dinitrophenol. 3. At higher concentrations it inhibits coupled phosphorylation as well as dinitrophenol-stimulated adenosine triphosphatase activity. The inhibition of state 3 oxidation under these conditions is not reversed by uncouplers. 4. The three coupling sites of phosphorylation exhibit differential susceptibility to inactivation by this compound. Coupled phosphorylation at the first site is abolished at a drug concentration of 3.0mumol/mg of protein. The third site is inactivated when the concentration of the drug reaches 5.0mumol/mg of protein. The second site is the most refractory and drug concentrations of the order of 10.0mumol/mg of protein are required effectively to inhibit phosphorylation at this site. 5. The compound also inhibits ATP-dependent reversal of electron transport as well as the adenosine triphosphatase activity in submitochondrial particles. 6. The oxidation of NADH and succinate in these particles is not inhibited. 7. These properties indicate that the compound acts as an ;inhibitory uncoupler' of energy-transfer reactions in isolated mitochondria.     

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 mm. Pyrazole (2 mm) 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 mm). 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-H₂O₂, 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-H₂O₂ accounts for most if not all of the alcohol dehydrogenase-independent pathway.