Microsomal Ethanol Oxidation Research Articles

Hepatic microsomal ethanol oxidation. Hydrogen peroxide formation and the role of catalase.

Microsomes from rat liver form hydrogen peroxide in the presence of an NADPH‐generating system in proportion to protein concentrations as determined by three independent methods: ferrithiocyanate, cytochrome c peroxidase, and scopoletin fluorescence. Maximal rates observed were about 15 μmol H2O2/g microsomal protein per minute. The oxygen concentration for half‐maximal rates was 50 μM. It is suggested that NADPH‐dependent hydrogen peroxide formation in microsomes is mainly due to NADPH oxidase; however, partial inhibition by carbon monoxide suggests that about one third arises from the autoxidation of cytochrome P‐450.Similarities exist between microsomal acetaldehyde production from ethanol (i.e. the microsomal ethanol‐oxidizing system of Lieber and DeCarli [4]) and hydrogen peroxide formation: viz. requirement for NADPH and oxygen, identical oxygen concentrations for halfmaximal rates, and sensitivity to carbon monoxide. Microsomal acetaldehyde production in the presence of either an NADPH‐ or an H2O2‐generating system exhibits identical characteristics as follows: (a) ethanol concentration for half‐maximal rates (i.e. 12 mM); (b) dependency of maximal rates on rates of hydrogen peroxide formation; (c) competitive inhibition by peroxidatic substrates for catalase, e.g. formate (half‐maximal effect: 150 μM); (d) inhibition by catalase inhibitors, e.g. azide (half‐maximal effect: 50 μM), with identical azide insensitive rates; (e) diminished acetaldehyde production in microsomes from rats pretreated with aminotriazole or pyrazole with identical residual rates. Moreover, NADPH‐dependent acetaldehyde production is suppressed in the presence of an active H2O2‐utilizing system.Thus, it is concluded that the NADPH‐dependent microsomal ethanol‐oxidizing system of Lieber and DeCarli [4] is due to a hydrogen peroxide formation from NADPH and a subsequent peroxidation of ethanol by contaminating catalase. The data indicate that the existence of a unique system in addition to the peroxidatic reaction of catalase as postulated recently [4] is highly doubtful.

Alcohol-drugs interaction in man: alcohol and tolbutamide.

Abstract. In man tolbutamide was shown to share certain properties with the drugs handled by the hepatic drug‐metabolizing system: its prolonged administration is capable of accelerating its own metabolism; the same effect is obtained by pretreatment with microsomal inducers such as phenobarbital, diphenylhydantoin and diazepam. — Chronic addiction to alcohol produces an enhanced rate of tolbutamide metabolism; on the other hand, prolonged treatment with tolbutamide is able to increase the rate of ethanol oxidation. The existence of a microsomal oxidation of ethanol supports the hypothesis that microsomes may be the site of the interference between alcohol and tolbutamide. — Additional evidence is provided by the observed inhibition of tolbutamide metabolism by ethanol infusion.

Effect of ethanol administration on hepatic ethanol and drug-metabolizing enzymes and on rates of ethanol degradation.

Abstract Ethanol administration was recently shown to enhance the activities of microsomal enzyme systems which metabolize ethanol and other drugs. The present study was designed to establish the time sequence and the mechanism that may account for the increases in enzyme levels, and to determine whether the increases in microsomal ethanol-oxidizing activity are accompanied by comparable changes in the rates of ethanol degradation in vivo. Male rats were fed ethanol for periods of 24, 48, and 72 hours, 7, and 14 days. Whereas liver weights and the cytosol enzymes alcohol dehydrogenase and lactate dehydrogenase were not changed, significant increases were obtained as early as 24 hours after ethanol administration for microsomal ethanol-oxidizing activity and at 48 hours for aniline hydroxylase and cytochrome P-450. Microsomal protein concentration was increased only at 14 days. Protein turnover studies demonstrated no changes in the rate of incorporation of 1- 14 C-l-leucine into microsomal protein, but significant decreases in the rate of degradation of 1- 14 C-l-leucine from microsomal protein were found at 14 days. The rates of ethanol disappearance from the blood were increased; however, the calculated mean increase of the microsomal ethanol-oxidizing activity accounted for only 13.8 per cent of the mean increase in the rate of in vivo ethanol disappearance at 7 days. The findings suggest that the mechanism of induction by ethanol is different from that of other known inducers, and that in vivo factors beside the absolute increase in microsomal enzyme activity must account for the observed changes in the rates of ethanol degradation.

Hepatic Microsomal Ethanol-oxidizing System: IN VITRO CHARACTERISTICS AND ADAPTIVE PROPERTIES IN VIVO

A hepatic microsomal ethanol-oxidizing system is described both in men and rats. It is distinguished from alcohol dehydrogenase by its subcellular localization (cytosol for alcohol dehydrogenase, microsomes for this system), its pH optimum (physiological pH versus pH 10 to 11 for alcohol dehydrogenase), and its cofactor requirements (NADPH versus NAD+ for alcohol dehydrogenase). It also requires oxygen and is inhibited by CO, properties commonly found among microsomal drug-detoxifying enzymes. That catalase is probably not involved was revealed by the partial or complete failure of cyanide, pyrazole, azide, or 3-amino-1,2,4-triazole to inhibit the NADPH-dependent microsomal ethanol-oxidizing system under conditions which diminished catalase activity. Moreover, a combination of administration in vivo of pyrazole and addition in vitro of azide virtually blocked catalase activity and abolished 95% of a H2O2-dependent microsomal ethanol oxidation, whereas two-thirds of the activity of the NADPH-dependent ethanol oxidation persisted. Ethanol feeding resulted in a striking rise of hepatic NADPH-dependent microsomal ethanol-oxidizing activity, whereas under the same conditions, activities of alcohol dehydrogenase in the cytosol and of microsomal as well as of total hepatic catalase did not increase. Furthermore, blood ethanol clearance was accelerated, which suggests that microsomal ethanol oxidation may play a role in vivo. Pyrazole, which inhibits alcohol dehydrogenase strongly (affecting also other hepatic functions, including microsomal enzymes) markedly reduced but did not block ethanol metabolism in vivo or in liver slices. Even after pyrazole, ethanol clearance rates remained significantly higher in ethanol-pretreated rats. The existence of a microsomal ethanol-oxidizing system, especially its capacity to increase in activity adaptively after ethanol feeding, may explain various effects of ethanol, including proliferation of hepatic smooth endoplasmic reticulum, induction of other hepatic microsomal drug-detoxifying enzymes, and the metabolic tolerance to ethanol which develops in alcoholics.