Effect of selected alcohol dehydrogenase inhibitors on human hepatic lactate dehydrogenase activity - an in vitro study

Metabolic acidosis severely complicates methanol and ethylene glycol intoxications. Acidosis is caused by acid metabolites and can be intensified by lactate elevation. Lactate concentration depends on the NADH(2)/NAD ratio. Lactate dehydrogenase (LDH, E.C.1.1.1.27.) supplies more lactate when the level of NADH(2) is elevated. The aim of the study was to evaluate the effect of alcohol dehydrogenase (ADH) inhibitors and substrates: cimetidine, EDTA, 4-methylpyrazole (4-MP), Ukrain and ethanol on LDH activity. The activity of LDH was determined spectrophotometrically in human liver homogenates incubated with cimetidine, EDTA, 4-MP and Ukrain at concentrations of 2 x 10(-6), 10(-5) and 5 x 10(-5) m as well as ethanol at concentrations of 12.50, 25.00, 50.00 mm. The LDH activity was significantly increased by 10(-5) and 5 x 10(-5) m concentrations of cimetidine and 4-MP, and by all concentrations of ethanol. The most effective change of LDH activity of about 26% (P<0.01) was observed at the highest concentration of ethanol. Ukrain inhibited LDH activity at both concentrations, i.e. 10(-5) and 5 x 10(-5) m (P<0.05). However, EDTA did not significantly influence LDH activity. The data showed that ethanol and 4-MP, the main antidotes in methanol or ethylene glycol poisoning, may increase liver LDH activity - an undesirable effect during the therapy of patients intoxicated with these alcohols. On the other hand, the decrease of LDH activity in the presence of Ukrain is a promising finding but definitely requires further investigation.     

In vitro studies of human alcohol dehydrogenase inhibition in the process of methanol and ethylene glycol oxidation

The liver is the major organ responsible for methanol and ethylene glycol oxidation, and alcohol dehydrogenase (alcohol: NAD⁺ oxidoreductase, EC 1.1.1.1.) is the main enzyme involved. In the present study, alcohol dehydrogenase (ADH) activity was measured spectrophotometrically in vitro at physiological pH 7.4 and 37 °C using human enzyme hepatic fraction. The percentage of residual activity was calculated for four inhibitors at concentrations of 10⁻³, 10⁻⁴, and 10⁻⁵ M (pyrazole, 4-methylpyrazole, cimetidine, theophylline) and methylene blue at concentrations 10⁻⁴ and 10⁻⁵ M. Our results have shown that the best inhibitor, cimetidine, decreased oxidation of 0.1 M and 0.05 M methanol to 24 and 29% respectively at a drug concentration of 1 mM. Reaction with 0.1 M ethylene glycol as the ADH substrate was blocked by the same substances and 4-methylpyrazole was found to be a highly effective inhibitor. Received: 3 November 1997 / Accepted: 5 May 1998     

The metabolite ratio as a function of chloral hydrate dose and intracellular redox state in the perfused rat liver

Chloral hydrate (CH), an intermediate metabolite of trichloroethylene, is reduced to trichloroethanol (TCE) by alcohol dehydrogenase and aldehyde reductase, and is also oxidized to trichloroacetic acid (TCA) by the nicotinamide adenine dinucleotide (NAD)-dependent enzyme, CH dehydrogenase. Alcohol dehydrogenase requires reduced NAD (NADH), aldehyde reductase requires reduced nicotinamide adenine dinucleotide phosphate (NADPH) and CH dehydrogenase requires NAD to complete the reaction. It is unclear which reaction is predominant at the physiological redox level in intact liver cells. To study this question, we perfused the livers of well-fed rats with Krebs-Ringer buffer solution containing 0.1 mM pyruvate/1.0 mM lactate. The levels of TCE and TCA in the effluent were measured by gas chromatography, and the fluorescence of reduced pyridine nucleotides was measured with a surface fluorometer. When a low concentration (below 0.25 mM) of CH was administered, more TCA than TCE was produced. When a high concentration of CH was administered (over 0.5 mM), TCE production was greater. Reduced pyridine nucleotides decreased inversely with the CH concentration. Even at low CH concentrations, pyridine nucleotides were not reduced. When 10 mM lactate was added to the perfusate in order to reduce the pyridine nucleotides in the liver cells, the TCE/TCA ratio increased. On the other hand, the TCE/TCA ratio tended to fall following the addition of 5.0 mM pyruvate. In conclusion, the TCE/TCA ratio was altered according to the concentration of CH, and to the redox level of pyridine nucleotides in the liver.     

Influence of epinephrine on alcohol dehydrogenase activity in rat hepatocyte culture

The effects of epinephrine on alcohol dehydrogenase activity and on rates of ethanol elimination were determined in rat hepatocyte culture. Continuous exposure of the hepatocytes to epinephrine (10 microM) in combination with dexamethasone (0.1 microM) enhanced alcohol dehydrogenase activity on days 4-7 of culture, whereas neither hormone alone had an effect. The increased alcohol dehydrogenase activity was associated with an increased rate of ethanol elimination. Acute addition of 10 microM epinephrine to hepatocytes maintained in culture with 0.1 microM dexamethasone did not change alcohol dehydrogenase activity, but resulted in an immediate marked, but transitory, increase in ethanol elimination within the first 5 min after the addition of the hormone. Prazosin, an alpha 1-adrenergic blocker, and antimycin, an inhibitor of mitochondrial respiration, were powerful inhibitors of the transient increase in ethanol elimination, whereas 4-methylpyrazole was only partially inhibitory. These observations indicate that epinephrine has a chronic effect in increasing alcohol dehydrogenase activity and ethanol elimination and, also, an acute transient effect of increasing ethanol elimination which is not limited by alcohol dehydrogenase activity.     

Multiple retinoid dehydrogenases in testes cytosol from alcohol dehydrogenase negative or positive deermice.

Retinoic acid syntheses from retinol by cytosol from testes of alcohol dehydrogenase negative or positive deermice were similar in specific activity and in their insensitivity to 1 M ethanol or 100 mM 4-methylpyrazole. Anion-exchange followed by size-exclusion chromatography revealed multiple and similarly migrating peaks in each cytosol that had both retinol and retinal dehydrogenase activities. Thus, the effects of ethanol on testes cannot be caused by direct inhibition of cytosolic retinoic acid synthesis because retinoid dehydrogenases distinct from mouse class A2 alcohol dehydrogenases, which corresponds to human class I, occurred in testes and they were not inhibited by ethanol. These data also demonstrate the occurrence of multiple cytosolic retinoic acid synthesis activities and indicate that the two reactions of cytosolic retinoic acid synthesis, retinol and retinal dehydrogenation, may be catalyzed by enzymes that occur as complexes.     

Ethanol oxidizing enzyme activites in liver disease.

1 The activites of hepatic alcohol dehydrogenase, catalase and the reduced nicotinamide adenine dinucleotide phosphate (NADPH) dependent ethanol oxidizing system were determined in liver biopsies from nine patients with liver disease and seven control subjects with non evidence of liver disease. 2 Alcohol dehydrogenase and catalase activites were significantly lower in the patients with liver disease. 3 The activity of the NADPH dependent ethanol oxidizing system was significantly greater in the patients with liver disease, when its activity was expressed in terms of mg protein or g wet weight liver. 4 It is suggested that the greater activity of the NADPH dependent system may compensate for the low alcohol dehydrogenase activites found in patients with liver disease and maintain normal rates of ethanol metabolism.     

Effect of triiodothyronine on alcohol dehydrogenase and aldehyde dehydrogenase activities in rat liver. Implications for the control of ethanol metabolism

Treatment of rats with 20 micrograms of 3,3',5-triiodo-L-thyronine (T3) per 100 g body wt for a period of 6 days led to a 45% decrease in total liver alcohol dehydrogenase and a 36% decrease in total liver aldehyde dehydrogenase. Most of the latter decrease was directly attributable to a 57% fall in the level of the physiologically-important low Km mitochondrial isoenzyme. The high Km isoenzyme of the postmitochondrial and soluble fractions was much less affected by T3-treatment. T3, at concentrations up to 0.1 mM, did not inhibit the activity of aldehyde dehydrogenase in vitro. Despite these large losses of the two enzymes most intimately involved in ethanol metabolism, the rate of ethanol elimination in vivo was the same in T3-treated and control animals. Moreover, there was no difference between the two groups in the susceptibility of ethanol elimination to inhibition by 4-methylpyrazole, making it unlikely that an alternative route of ethanol metabolism had been significantly induced by treatment with T3. As it had been suggested that T3 might create a "hypermetabolic state" in which constraints normally imposed on alcohol dehydrogenase and aldehyde dehydrogenase are removed thereby compensating for any loss in total enzymic activity, 2,4-dinitrophenol (0.1 mmoles/kg body wt) was administered to rats in order to raise the general metabolic rate. However, the uncoupler proved to be lethal to T3-treated animals and did not stimulate ethanol elimination in controls. The results do not support the notion that ethanol elimination in vivo is normally governed either by the level of alcohol dehydrogenase or by that of hepatic aldehyde dehydrogenase. However, the mode of control remains unclear.     

Interaction of ethanol oxidation with glucuronidation in isolated hepatocytes.

1. Glucuronidation of harmol, 2-naphthol, 4-methylumbelliferone and phenolphthalein in isolated hepatocytcs was inhibited up to 50 per cent in the presence of low concentrations of ethanol (10 mM). Sulphate conjugation was unaffected. The inhibitory effect of ethanol was reversed by 4-methylpyrazole, an inhibitor of alcohol dehydrogenase dependent ethanol oxidation. 2. The oxidation of harmine to harmol was not affected by 10 mM ethanol, but in hepatocytes isolated from phenobarbital-treated rats glucuronidation of the formed harmol was inhibited about 30 per cent in the presence of this amount of ethanol. 3. Ethanol increased the intracellular NADH/NAD⁺ ratio as did lactate and sorbitol. The latter two substances were also inhibitory to glucuronidation having no effect on the sulphate conjugation. 4. The synthesis of UDPglucuronic acid was inhibited by ethanol both in the presence and absence of a substrate undergoing glucuronidation. It is suggested that the inhibitory effect of ethanol on glucuronidation is due to a decreased UDPglucuronic acid synthesis caused by the increased NADH/NAD⁺ ratio resulting from the alcohol dehydrogenase dependent oxidation of ethanol.     

Human liver alcohol dehydrogenase--inhibition of methanol activity by pyrazole, 4-methylpyrazole, 4-hydroxymethylpyrazole and 4-carboxypyrazole.

With human liver alcohol dehydrogenase of high purity at pH 7.0 and 500 μM NAD the K_m for methanol is 7.0 mM (ten times greater than the K_m for ethanol) and the turnover number 1.4/active site/min (about one-tenth of the turnover with ethanol in the same conditions). From secondary kinetic plots it can be calculated that at saturating concentrations of both substrates, namely methanol and NAD, these constants do not change appreciably: the K_m for methanol is somewhat lower (5.2 mM) and the turnover number slightly higher (1.7/active site/min). The difference in turnover numbers with methanol and ethanol as substrates suggests that the kinetic mechanism for methanol is different from that for ethanol dehydrogenation. The dissociation constant between human alcohol dehydrogenase and NAD, determined kinetically with methanol as substrate, is 127 μM. The K_i values for pyrazole, 4-methylpyrazole and 4-hydroxymethylpyrazole are 0.54, 0.09 and 6.6 μM respectively; 4-carboxypyrazole (100 μM) at 3mM methanol does not inhibit human ADH. The inhibitory effect of 4-methylpyrazole is therefore not likely to be enhanced by a possible metabolic conversion to 4-hydroxymethylpyrazole and 4-carboxypyrazole.     

Effect of alcohol and aldehyde dehydrogenase inhibitors on the toxicity of 3-nitropropanol in rats

The nitrocompounds 3-nitropropanol (NPOH) and 3-nitropropionic acid (NPA) were shown to be equally toxic when injected intraperitoneally into male Wistar rats. The LD50 for NPOH was 0.58 mmol/kg and for NPA it was 0.56 mmol/kg. NPOH was rapidly metabolized to NPA but this conversion was suppressed by prior administration of ethanol or 4-methylpyrazole to inhibit alcohol dehydrogenase. Administration of ethanol or 4-methylpyrazole before NPOH protected rats from intoxication. However, if the alcohol dehydrogenase inhibitors were given after the nitroalcohol, toxicity still occurred. Administration of the aldehyde dehydrogenase inhibitor diethyldithiocarbamic acid had little effect on the conversion of NPOH to NPA and did not alter the toxicity of NPOH. It was concluded that NPOH and NPA are equally toxic to rats but that NPOH is toxic due to its being rapidly converted to NPA.     

Comparison of rat pulmonary and hepatic cytosolic alcohol dehydrogenase activities.

The liver is the major organ responsible for ethanol oxidation, and alcohol dehydrogenase (ADH) is the main enzyme involved. There is limited evidence suggesting the involvement of the lung in ethanol metabolism. To determine the degree to which pulmonary ADH plays a role in ethanol metabolism, ADH activity was measured spectrophotometrically using hepatic and pulmonary cytosolic fractions prepared by differential centrifugation and Sephadex G-50 column chromatography. Apparent Km values for hepatic and pulmonary ADHs were determined. Inhibition constants were calculated using 4-methylpyrazole. The ADHs were characterized by examining the influence of pH on enzyme activity. Pulmonary ADH activity was much lower at near neutral pH than at pH 9.0 or 10, whereas hepatic ADH activity was also pH dependent but was significantly higher. Pulmonary ADH is less sensitive to inhibition by 4-methylpyrazole than is hepatic ADH, as evidenced by a 1000-fold higher Ki. Pulmonary ADH would be expected to make only a minor contribution to ethanol metabolism in vivo.     

Severe Lactic Acidosis in a Diabetic Patient after Ethanol Abuse and Floor Cleaner Intake

An intoxication with drugs, ethanol or cleaning solvents may cause a complex clinical scenario if multiple agents have been ingested simultaneously. The situation can become even more complex in patients with (multiple) co‐morbidities. A 59‐year‐old man with type 2 diabetes mellitus (without treatment two weeks before the intoxication) intentionally ingested a substantial amount of ethanol along with ~750 mL of laminate floor cleaner containing citric acid. The patient was admitted with severe metabolic acidosis (both ketoacidosis and lactic acidosis, with serum lactate levels of 22 mM). He was treated with sodium bicarbonate, insulin and thiamine after which he recovered within two days. Diabetic ketoacidosis and lactic acidosis aggravated due to ethanol intoxication, thiamine deficiency and citrate. The high lactate levels were explained by excessive lactate formation caused by the combination of untreated diabetes mellitus, thiamine deficiency and ethanol abuse. Metabolic acidosis in diabetes is multi‐factorial, and the clinical situation may be further complicated, when ingestion of ethanol and toxic agents are involved. Here, we reported a patient in whom diabetic ketoacidosis was accompanied by severe lactic acidosis as a result of citric acid and mainly ethanol ingestion and a possible thiamine deficiency. In the presence of lactic acidosis in diabetic ketoacidosis, physicians need to consider thiamine deficiency and ingestion of ethanol or other toxins.     

Current management of ethylene glycol poisoning.

Ethylene glycol, a common antifreeze, coolant and industrial solvent, is responsible for many instances of accidental and intentional poisoning annually. Following ingestion, ethylene glycol is first hepatically metabolised to glycoaldehyde by alcohol dehydrogenase. Glycoaldehyde is then oxidised to glycolic acid, glyoxylic acid and finally oxalic acid. While ethylene glycol itself causes intoxication, the accumulation of toxic metabolites is responsible for the potentially fatal acidosis and renal failure, which characterises ethylene glycol poisoning. Treatment of ethylene glycol poisoning consists of emergent stabilisation, correction of metabolic acidosis, inhibition of further metabolism and enhancing elimination of both unmetabolised parent compound and its metabolites. The prevention of ethylene glycol metabolism is accomplished by the use of antidotes that inhibit alcohol dehydrogenase. Historically, this has been done with intoxicating doses of ethanol. At a sufficiently high concentration, ethanol saturates alcohol dehydrogenase, preventing it from acting on ethylene glycol, thus allowing the latter to be excreted unchanged by the kidneys. However, ethanol therapy is complicated by its own inherent toxicity, and the need to carefully monitor serum ethanol concentrations and adjust the rate of administration. A recent alternative to ethanol therapy is fomepizole, or 4-methylpyrazole. Like ethanol, fomepizole inhibits alcohol dehydrogenase; however it does so without producing serious adverse effects. Unlike ethanol, fomepizole is metabolised in a predictable manner, allowing for the use of a standard, validated administration regimen. Fomepizole therapy eliminates the need for the haemodialysis that is required in selected patients who are non-acidotic and have adequate renal function.     

Assessment of the role of non-ADH ethanol oxidation in vivo and in hepatocytes from deermice

Deermice genetically lacking alcohol dehydrogenase (ADH-) were used to quantitate the effect of 4-methylpyrazole (4-MP) on non-ADH pathways in hepatocytes and in vivo. Although primarily an inhibitor of ADH, 4-methylpyrazole was also found to inhibit competitively the activity of the microsomal ethanol-oxidizing system (MEOS) in deermouse liver microsomes. The degree of 4-MP inhibition in ADH- deermice then served to correct for the effect of 4-MP on non-ADH pathways in deermice having ADH (ADH+). In ADH+ hepatocytes, the percent contributions of non-ADH pathways were calculated to be 28% at 10 mM and 52% at 50 mM ethanol. When a similar correction was applied to in vivo ethanol clearance rates in ADH+ deermice, non-ADH pathways were found to contribute 42% below 10 mM and 63% at 40-70 mM blood ethanol. The catalase inhibitor 3-amino-1,2,4-triazole, while reducing catalase-mediated peroxidation of ethanol by 83-94%, had only a slight effect on blood ethanol clearance at ethanol concentrations below 10 mM, and no effect at all at 40-70 mM ethanol. These results indicate that non-ADH pathways (primarily MEOS) play a significant role in ethanol oxidation in vivo and in hepatocytes in vitro.     

Buformin suppresses the expression of glyceraldehyde 3-phosphate dehydrogenase

The biguanides metformin and buformin, which are clinically used for diabetes mellitus, are known to improve resistance to insulin in patients. Biguanides were reported to cause lactic acidosis as a side effect. Since the mechanism of the side effect still remains obscure, we have examined genes whose expression changes by treating HepG2 cells with buformin in order to elucidate the mechanisms of the side effect. A subtraction cDNA library was constructed by the method of suppressive subtractive hybridization and the screening of the library was performed with cDNA probes prepared from HepG2 cells treated with or without buformin for 12 h. The expression of the gene and the protein obtained by the screening was monitored by real-time RT-PCR with specific primers and Western blotting with specific antibody. The amounts of ATP and NAD+ were determined with luciferase and alcohol dehydrogenase, respectively. We found that expression of the glyceraldehyde 3-phosphate dehydrogenase (GAPD) gene was suppressed by treating HepG2 cells with 0.25 mM buformin for 12 h as a result of the library screening. The decrease in the expression depended on the treatment period. The amount of GAPD protein also decreased simultaneously with the suppression of the gene expression by the treatment with buformin. The amount of ATP and NAD+ in the HepG2 cells treated with buformin decreased to 10 and 20% of the control, respectively. These observations imply that the biguanide causes deactivation of the glycolytic pathway and subsequently the accumulation of pyruvate and NADH and a decrease in NAD+. Therefore, the reaction equilibrium catalyzed by lactate dehydrogenase leans towards lactate production and this may result in lactic acidosis.     

Lipid-protein interactions: enhancement of enzyme activity of L-glutamic acid dehydrogenase by nonionic detergents.

Five nonionic detergents enhanced the activity of L-glutamic acid dehydrogenase [L-glutamate:nicotinamide adenine dinucleotide phosphate oxidoreductase (deaminating) (EC 1.4.1.3)]. These detergents activated the enzyme toward alpha-ketoglutaric acid reduction, causing a decrease in the sensitivity of the enzyme to allosteric regulation by guanosine 5-triphosphate. There was also a diminution of the enhancing effect of the modifier adenosine 5-diphosphate on the enzyme's L-glutamic acid dehydrogenase activity. These detergents may cause a conformational change in the enzyme, and this change could lead to an increase in the binding of the substrates for the alpha-ketoglutaric acid reduction. Accompanied with this conformational change would be a decrease in the binding of the modifier guanosine 5'-triphosphate, with no concomitant change in the binding of the adenosine 5'-diphosphate modifier.     

Dehydrogenase binding by tiazofurin anabolites

Thiazole-4-carboxamide adenine dinucleotide (TAD) is the active anabolite of the new antitumor agent tiazofurin (NSC 286193). TAD is an analogue of NAD in which the nicotinamide ring has been replaced by a thiazole-4-carboxamide heterocycle. TAD putatively acts by inhibition of inosine monophosphate dehydrogenase (IMPd). In this study it is shown that TAD is a competitive inhibitor, with respect to NAD, of mammalian glutamate, alcohol, lactate, and malate dehydrogenases. TAD binds to these enzymes with 1-2 orders of magnitude less affinity than it binds to IMPd. Computer modeling studies suggest that dehydrogenase binding by TAD occurs at the regular cofactor site, the thiazole-4-carboxamide group mimicking the steric and hydrogen-bonding properties of the nicotinamide ring. Noncompetitive kinetics of TAD inhibition of the target enzyme IMPd are potentially due to a reverse order of addition of substrate and cofactor from that observed in the dehydrogenases studied here. The weaker binding of TAD to these dehydrogenases may be due to their inability to preserve a close sulfur-oxygen contact in the bound inhibitor.     

WIF-B cells as a model for alcohol-induced hepatocyte injury

A potential in vitro model for studying the mechanisms of alcohol-induced hepatocyte injury is the WIF-B cell line. It has many hepatocyte-like features, including a differentiated, polarized phenotype resulting in formation of bile canaliculi. The aim of this study was to examine the effects of ethanol treatment on this cell line. WIF-B cells were cultured up to 96 h in the absence or presence of 25 mM ethanol and subsequently were analyzed for ethanol-induced physiological and morphological changes. Initial studies revealed WIF-B cells exhibited alcohol dehydrogenase (ADH) activity, expressed cytochrome p4502E1 (CYP2E1), and efficiently metabolized ethanol in culture. This cell line also produced the ethanol metabolite acetaldehyde and exhibited low K(m) aldehyde dehydrogenase (ALDH) activity, comparable to hepatocytes. Ethanol treatment of the WIF-B cells for 48 h led to significant increases in the lactate/pyruvate redox ratio and cellular triglyceride levels. Ethanol treatment also significantly altered WIF-B morphology, decreasing the number of bile canaliculi, increasing the number of cells exhibiting finger-like projections, and increasing cell diameter. The ethanol-induced changes occurring in this cell line were negated by addition of the ADH inhibitor, 4-methylpyrazole (4-MP), indicating the effects were due to ethanol metabolism. In summary, the WIF-B cell line metabolizes ethanol and exhibits many ethanol-induced changes similar to those found in hepatocytes. Because of these similarities, WIF-B cells appear to be a suitable model for studying ethanol-induced hepatocyte injury.     

Screening of the inhibitory effect of xenobiotics on alcohol metabolism using S9 rat liver fractions

The purpose of this work was to develop and optimize a simple and suitable method to detect the potential inhibitory effect of drugs and medicines on alcohol dehydrogenase (ADH) activity in order to evaluate the possible interactions between medicines and alcohol metabolism. Commonly used medicines that are often involved in court litigations related with driving under the influence of alcohol were selected. Alprazolam, flunitrazepam and tramadol were tested as drugs with no known effect on ADH activity. Cimetidine, reported previously as having inhibitory effect on ADH, and 4-methylpyrazole (4-MP), a well known ADH inhibitor, were tested as positive controls. Apart from 4-MP, tramadol was identified as having the higher inhibitory effect with an IC50 of 44.7 × 10^?3 mM, followed by cimetidine (IC50 of 122.9 × 10^?3 mM). Alprazolam and flunitrazepam also reduced liver ADH activity but to a smaller extent (inhibition of 11.8 ± 5.0% for alprazolam 1.0 mM and 34.5 ± 7.1% for flunitrazepam 0.04 mM). Apart from cimetidine, this is the first report describing the inhibitory effect of these drugs on ethanol metabolism. The results also show the suitability of the method to screen for inhibitory effect of drugs on ethanol metabolism helping to identify drugs for which further study is justified.     

Effects of pyrazole and 3-amino-1,2,4-triazole on methanol and ethanol metabolism by the rat

The metabolism of methanol-¹⁴C and ethanol-1-¹⁴C in rats was evaluated from the rates of ¹⁴CO₂ production. 3-Amino,1,2,4-triazole, a known catalase inhibitor, decreased by 10 and 35 per cent the rates of oxidation of ethanol and methanol, whereas pyrazole, an alcohol dehydrogenase inhibitor, decreased the rates 85 and 50 per cent respectively. However, the simultaneous use of both inhibitors gave the same effects produced by pyrazole alone. Thus the relative contributions in vivo to alcohol metabolism of rat liver alcohol dehydrogenase and catalase-mediated peroxidation, cannot be estimated only in this way. Rat liver alcohol dehydrogenase was purified 14·7 times. At pH 7·0 and 30°, the K_m for methanol was 340 mM and for ethanol 0·26 mM. The V_max/e was 2·36 nM for methanol and 22·3 nM for ethanol (NADH × U^?1 × 1^?1 × sec^?1). 3-Amino-1,2,4-triazole inhibited the purified enzyme with a K_i of 55 mM for methanol and 33 mM for ethanol. The K_i of pyrazole was 2·3 mM for methanol and 2·2 mM for ethanol. The amount of alcohol dehydrogenase present in rat liver, with the found kinetic constants, can account for the ethanol oxidation in vivo, but fails to account, as methanol dehydrogenase, for the observed pyrazole-sensitive methanol oxidation. A mechanism for the complete oxidation of methanol to CO₂ and water through the concerted action of catalase and alcohol dehydrogenase is suggested. 3-Amino-1,2,4-triazole in a dose of 1 g/kg decreases more than 90 per cent of the catalatic activity of catalase, but under certain conditions in vitro, only about 50 per cent of the peroxidative activity of catalase towards methanol and ethanol. Consequently, the degree of catalase-mediated peroxidation should not be controlled or estimated from the residual catalatic activity when using catalase inhibitors. Pyrazole, at a dose of 0·3 g/kg, does not affect catalase activity 1 hr after administration, but decreases it more than 90 per cent after 24 hr. This effect is completely prevented in the presence of alcohol.