ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
175, 635-643 (1976)
Hepatic Ethanol Metabolism: Respective Roles of Alcohol Dehydrogenase, the Microsomal Ethanol-Oxidizing System, and Catalase’ ROLF TESCHKE,
YASUSHI
HASUMURA,
AND
CHARLES
S. LIEBER
Alcohol Research Laboratory of the Section of Liver Disease and Nutrition, Veterans Administration Hospital, Bronx, New York 10468, and Department of Medicine, Mount Sinai School of Medicine of the City University of New York, New York, New York 10029 Received
October 27, 1975
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 rn& 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 nn-alanine. Similarly, in vivo administration of 3-amino-1,2,4triazole, a compound which inhibits the activity of catalase but not that of the microsoma1 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 catalaseHZ02, significantly decreased the pyrazole-insensitive ethanol metabolism in liver slices. These results indicate that alcohol dehydrogenase is responsible for half or more of ethanol metabolism by liver slices and that the microsomal ethanol-oxidizing system rather than catalase-H,O, accounts for most if not all of the alcohol dehydrogenaseindependent pathway.
It was generally assumed that ethanol metabolism proceeds exclusively via alcohol dehydrogenase (ADH),2 an enzyme of the cell sap of the hepatocyte. Indeed, this concept is satisfactory at low ethanol concentrations since the oxidation of ethanol is almost completely abolished under these conditions by pyrazole, a potent inhibitor of alcohol dehydrogenase activity (1). At ’ This work was supported in part by USPHS Grants AA 00224 and AM 12511 and VA project No. 5251-02. Portions of this study were presented at the Spring Meeting of the American Society for Pharmacology and Experimental Therapeutics, Atlantic City, April 1975. Z Abbreviations used: ADH, alcohol dehydrogenase; MEOS, microsomal ethanol-oxidizing system; DEAE-, diethyl aminoethyl.
intermediate and higher concentrations, however, ethanol metabolism becomes less sensitive to pyrazole, a finding which suggested the operation of a non-ADH-mediated pathway for ethanol metabolism (l6). Since this ADH-independent pathway was considered to account for up to 75% of the overall rates of ethanol metabolism at high ethanol concentrations (1) the question of the biochemical nature of this pathway became relevant. Recent studies have shown that in addition to ADH, ethanol can also be metabolized by the microsomal fraction of the hepatocyte which comprises the endoplasmic reticulum. This microsomal ethanol-oxidizing system (MEOS) was extensively studied (2, 3, 7, 8) and it was separated by 635
Copyright 0 1976 by Academic F’rcss, Inc. All rights of reproduction in any form reserved.
636
TESCHKE,
HASUMURA
DEAE-cellulose column chromatography from both alcohol dehydrogenase and catalase (9-12). A variety of reports provided strong evidence for a significant role of the microsomal ethanol-oxidizing system in ethanol metabolism (1-6, 13). This concept, however, was challenged recently, and it has been claimed that the alcohol dehydrogenase-independent pathway of ethanol metabolism is due exclusively to catalase (14). In view of these conflicting interpretations, the present study was undertaken to clarify the respective roles of the microsoma1 ethanol-oxidizing system and catalase in ethanol metabolism. MATERIALS
AND
METHODS
Materials. The chemicals and enzymes were obtained from the following sources: NADP+ (yeast), P-NAD+ (grade III), m-isocitrate (type I), and isocitric dehydrogenase from Sigma Chemical Company, Na,-EDTA, sodium St. Louis, MO.; m.-alanine, azide, hydrogen peroxide (30%), semicarbazide hydrochloride, n-butanol, and n-glucose from Fisher Scientific Company, Fairlawn, N.J.; acetaldehyde and pyrazole from Eastman Kodak Company, Rochester, N.Y.; ethanol (dehydrated) from Publicker Industries Co., Linfeld, Pa.; 3-amino-1,2,4-triazole from Aldrich, Milwaukee, Wis.; glucose oxidase (type I) from Boehringer Mannheim Corp., New York, N.Y.; and the gas mixtures from Matheson Gas Products, East Rutherford, N.J. Animals. Male Sprague-Dawley rats (CD, Charles River Breeding Laboratories, North Wilmington, Mass.) of body weight 340-440 g were used; they were fed Purina laboratory chow ad Zibitum and had free access to tap water. When indicated, rats were pretreated with 3-amino-1,2,4-triazole (1 g/kg body weight ip in 0.9% saline) 1 h before sacrifice, whereas the corresponding littermate received 0.9% saline solution only. Procedure for the measurement of ethanol oxidation by liver slices. The animals were killed by decapitation, and liver slices of approximately 0.5-mm thickness with a wet weight of about 50-60 mg each were prepared by means of a Stadie-Riggs microtome (Arthur H. Thomas Company, Philadelphia, Pa.). Randomized liver slices of a total weight of about 500 mg were added to 50-ml Erlenmeyer flasks containing 4.5 ml of isotonic Krebs-Ringer bicarbonate buffer (pH 7.4) (151, and, when indicated, the following compounds: pyrazole, 2 mM, sodium azide, 1 mM, and nbalanine, 40 mM. Then 0.5 ml of ethanol in Krebs-Ringer bicarbonate buffer (pH 7.4) was added to achieve a final alcohol concentration of 50
AND
LIEBER
rnM in a final volume of 5 ml. With each incubation set, experiments were run in which boiled liver slices were incubated with 50 mM ethanol. The values thus obtained were used as evaporation controls and subtracted from the corresponding experimental values. The vessels were sealed with serum caps and flushed for 5 min with a gas mixture of 95% 0, and 5% CO,. The subsequent incubations were carried out at 37°C for a total of 150 min in a Dubnoff water bath shaking at 100 strokes/min. Aliquots of the incubation medium were harvested with a needle and syringe through the rubber top immediately before the start of the incubations and then at intervals of 30 min. One hundred microliters of the harvested incubation medium was added to 0.5 ml of 35% perchloric acid contained in a 25-ml glass flask designed for analysis by a Perkin-Elmer F-40 gasliquid chromatograph (16). The sample bottles were immediately closed and incubated for 20 min at 60°C in the water bath attached to the chromatograph. Aliquots of the head space gas of these flasks were then injected by an automatic electropneumatic dosing system (injection time 4 s) into the gas-liquid chromatograph, and a 2-m x 2-mm column packed with 15% polyethyleneglycol on 50-60 mesh Celite was used. Helium was employed as a carrier gas at a flow rate of 40 ml/min. The temperature was 75°C for the column and 145°C for the flash heater as well as for the hydrogen flame detector. Quantitative assessment of the ethanol remaining in the incubation medium following the incubation was achieved by the use of a Hewlett-Packard GC digital integrator (Model 3370A) connected to the gas-liquid chromatograph. Solutions with known amounts of ethanol served as standards. It was found that an excellent reproducibility could only be achieved with a gas-liquid chromatograph with an automatic electropneumatic dosing system which was equilibrated for at least 4 h before the start of the analysis. Subcellular fractionation. The rats were killed by decapitation and their livers were perfused with icecold 0.15 M KC1 through the portal vein, excised, chilled, and homogenized in three volumes of 0.15 M KC1 using a glass homogenizer with a Teflon pestle. The following steps were carried out at 0-4°C. The 25% homogenate was spun at 10,OOOgfor 30 min, and the supernatant was centrifuged at 105,OOOgfor another 30 min. The resulting supematant (cytosol) was used as enzyme source for alcohol dehydrogenase, whereas the pellet was resuspended in 0.15 M KCl, and washed microsomes were obtained by spinning this suspension at 105,OOOgfor another 30 min. Biochemical determinations. The activity of alcohol dehydrogenase was assayed in the hepatic cytosol following the method of Bonnichsen and Brink (17) with ethanol at a final concentration of 50 mM. Catalatic activity of catalase was determined by
HEPATIC
ETHANOL
measuring the disappearance at 240 nm of H,O, added to aliquots of the 25% liver homogenate and expressing the results in units according to Luck (18). The activity of the NADPH-dependent microsoma1 ethanol-oxidizing system (MEOS) was determined with washed microsomes (3 mg of protein/ flask) which were preincubated with ethanol (50 mM) and sodium azide (1 mM) for 5 min at 37°C. The reactions were initiated by addition of a NADPHgenerating system (0.4 mM NADP+, 8 mM sodium isocitrate, and 0.34 unit/ml of isocitric dehydrogenase). The incubation medium contained, in a final volume of 3.0 ml, 1.0 mM NaZ-EDTA and 5.0 mM MgCl, in 0.1 M phosphate buffer (pH 7.4). The incubations were performed in closed 50-ml Erlenmeyer flasks with center wells containing 0.6 ml of 0.015 M semicarbazide hydrochloride in 0.1 M phosphate buffer (pH 7.4), and the acetaldehyde bound to the semicarbazide after an overnight diffusion period was determined according to Lieber and DeCarli (2). When the effect of butanol was studied, this alcohol was preincubated at a final concentration of 10 mM with microsomes and ethanol (50 mM) for 5 min before the reaction was started by adding the cofactor. Under these conditions, acetaldehyde bound to the semicarbazide was determined by gas-liquid chromatography as described above for the determination of ethanol in the liver slice study (12). Aliquota (100 ~1) of the semicarbazone solution of the center wells were added to 0.5 ml of 35% perchloric acid contained in 25-ml sample bottles. The bottles were sealed and subsequently incubated for 20 min at 60°C before aliquots of the head space were injected into the gas-liquid chromatograph. Flasks to which known amounts of acetaldehyde were added were carried through the complete procedure and were used as standards. The amount of acetaldehyde produced upon incubation was measured by peak heights. Peroxidatic activity of catalase was determined in washed microsomes under assay conditions described for the NADPH-dependent microsomal ethanol-oxidizing system except that sodium azide was omitted from the incubation medium and the NADPH-generating system was replaced by a H202producing one consisting of glucose (10 mM) and glucose oxidase (1.0 wg/ml of incubation medium). Under these conditions, glucose was preincubated with microsomes and ethanol (50 mM), and the reaction was started by adding glucose oxidase. Protein concentration was determined according to the method of Lowry et al. (19). Statistical analysis. Each individual result was compared with the value of its corresponding control; the means (+SE) of individual differences were calculated and their significance was assessed by the paired Student’s t test.
637
METABOLISM RESULTS
Effect of Pyrazole, Sodium Azide, and DLAlanine on Ethanol Metabolism Ethanol was oxidized by liver slices at a rate of approximately 50-60 pmol h-’ (g of liver)-‘, when ethanol was employed at a final concentration of 50 mM. The amount of ethanol disappearance was measured 150 min following the start of the incubation at which time 2530% of the alcohol added to the incubation medium had been oxidized. To verify the linearity of the reaction, measurements were also performed during the incubation time at regular intervals of 30 min each. The rate of ethanol metabolism was linear for 150 min. To assess quantitatively the role of alcohol dehydrogenase in ethanol metabolism, liver slices were incubated with ethanol (50 mM) and, when indicated, with pyrazole added to the incubation medium at a final concentration 2 mM. At this concentration, pyrazole almost completely abolished the activity of alcohol dehydrogenase when measured in the cytosol of the hepatocyte with ethanol as substrate (Table I). Pyrazole also decreased catalatic activity in liver homogenate by 24% (Table I); under these conditions, MEOS activity was found before to be inhibited by 11% (2). In contrast to the almost complete abolition of the activity of cytosolic ADH (Table I), pyrazole decreased the rate of ethanol metabolism by only 60% (P < 0.001) in liver slices (Table II). This finding suggests that although ADH activity plays a major role in ethanol metabolism, a considerable fraction of ethanol metabolism in liver slices may be accounted for by a pathway not involving the activity of alcohol dehydrogenase. To determine to what extent the pyrazole-insensitive pathway of ethanol metabolism in liver slices is due to the activity of catalase, the catalase inhibitor sodium azide was included in the incubation medium to achieve a final concentration of 1 mM. At this concentration, sodium azide virtually abolished catalase activity in liver homogenates when assayed by its catalatic property to decompose added hy-
638
TESCHKE,
HASUMURA
AND
TABLE EFFECT
OF PYRAZOLE
AND SODIUM
AZIDE
LIEBER
I
ON THE ACTIVITY
OF ALCOHOL
DEHYDROGENASE
ADH P
Activity (rim01 min-* liver)-‘) Control Pyrazole Azide Pyrazole
1821 60 1710 55
+ azide
+ + k -t
(g of 61 5 99 4
AND CATALASE”
Catalase
(% of control) 100 3.3 93.9 3.0
Catalatic (units (g of liver)-‘)
OF
BIOCHEMISTRY
AND
BIOPHYSICS
175, 635-643 (1976)
Hepatic Ethanol Metabolism: Respective Roles of Alcohol Dehydrogenase, the Microsomal Ethanol-Oxidizing System, and Catalase’ ROLF TESCHKE,
YASUSHI
HASUMURA,
AND
CHARLES
S. LIEBER
Alcohol Research Laboratory of the Section of Liver Disease and Nutrition, Veterans Administration Hospital, Bronx, New York 10468, and Department of Medicine, Mount Sinai School of Medicine of the City University of New York, New York, New York 10029 Received
October 27, 1975
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 rn& 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 nn-alanine. Similarly, in vivo administration of 3-amino-1,2,4triazole, a compound which inhibits the activity of catalase but not that of the microsoma1 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 catalaseHZ02, significantly decreased the pyrazole-insensitive ethanol metabolism in liver slices. These results indicate that alcohol dehydrogenase is responsible for half or more of ethanol metabolism by liver slices and that the microsomal ethanol-oxidizing system rather than catalase-H,O, accounts for most if not all of the alcohol dehydrogenaseindependent pathway.
It was generally assumed that ethanol metabolism proceeds exclusively via alcohol dehydrogenase (ADH),2 an enzyme of the cell sap of the hepatocyte. Indeed, this concept is satisfactory at low ethanol concentrations since the oxidation of ethanol is almost completely abolished under these conditions by pyrazole, a potent inhibitor of alcohol dehydrogenase activity (1). At ’ This work was supported in part by USPHS Grants AA 00224 and AM 12511 and VA project No. 5251-02. Portions of this study were presented at the Spring Meeting of the American Society for Pharmacology and Experimental Therapeutics, Atlantic City, April 1975. Z Abbreviations used: ADH, alcohol dehydrogenase; MEOS, microsomal ethanol-oxidizing system; DEAE-, diethyl aminoethyl.
intermediate and higher concentrations, however, ethanol metabolism becomes less sensitive to pyrazole, a finding which suggested the operation of a non-ADH-mediated pathway for ethanol metabolism (l6). Since this ADH-independent pathway was considered to account for up to 75% of the overall rates of ethanol metabolism at high ethanol concentrations (1) the question of the biochemical nature of this pathway became relevant. Recent studies have shown that in addition to ADH, ethanol can also be metabolized by the microsomal fraction of the hepatocyte which comprises the endoplasmic reticulum. This microsomal ethanol-oxidizing system (MEOS) was extensively studied (2, 3, 7, 8) and it was separated by 635
Copyright 0 1976 by Academic F’rcss, Inc. All rights of reproduction in any form reserved.
636
TESCHKE,
HASUMURA
DEAE-cellulose column chromatography from both alcohol dehydrogenase and catalase (9-12). A variety of reports provided strong evidence for a significant role of the microsomal ethanol-oxidizing system in ethanol metabolism (1-6, 13). This concept, however, was challenged recently, and it has been claimed that the alcohol dehydrogenase-independent pathway of ethanol metabolism is due exclusively to catalase (14). In view of these conflicting interpretations, the present study was undertaken to clarify the respective roles of the microsoma1 ethanol-oxidizing system and catalase in ethanol metabolism. MATERIALS
AND
METHODS
Materials. The chemicals and enzymes were obtained from the following sources: NADP+ (yeast), P-NAD+ (grade III), m-isocitrate (type I), and isocitric dehydrogenase from Sigma Chemical Company, Na,-EDTA, sodium St. Louis, MO.; m.-alanine, azide, hydrogen peroxide (30%), semicarbazide hydrochloride, n-butanol, and n-glucose from Fisher Scientific Company, Fairlawn, N.J.; acetaldehyde and pyrazole from Eastman Kodak Company, Rochester, N.Y.; ethanol (dehydrated) from Publicker Industries Co., Linfeld, Pa.; 3-amino-1,2,4-triazole from Aldrich, Milwaukee, Wis.; glucose oxidase (type I) from Boehringer Mannheim Corp., New York, N.Y.; and the gas mixtures from Matheson Gas Products, East Rutherford, N.J. Animals. Male Sprague-Dawley rats (CD, Charles River Breeding Laboratories, North Wilmington, Mass.) of body weight 340-440 g were used; they were fed Purina laboratory chow ad Zibitum and had free access to tap water. When indicated, rats were pretreated with 3-amino-1,2,4-triazole (1 g/kg body weight ip in 0.9% saline) 1 h before sacrifice, whereas the corresponding littermate received 0.9% saline solution only. Procedure for the measurement of ethanol oxidation by liver slices. The animals were killed by decapitation, and liver slices of approximately 0.5-mm thickness with a wet weight of about 50-60 mg each were prepared by means of a Stadie-Riggs microtome (Arthur H. Thomas Company, Philadelphia, Pa.). Randomized liver slices of a total weight of about 500 mg were added to 50-ml Erlenmeyer flasks containing 4.5 ml of isotonic Krebs-Ringer bicarbonate buffer (pH 7.4) (151, and, when indicated, the following compounds: pyrazole, 2 mM, sodium azide, 1 mM, and nbalanine, 40 mM. Then 0.5 ml of ethanol in Krebs-Ringer bicarbonate buffer (pH 7.4) was added to achieve a final alcohol concentration of 50
AND
LIEBER
rnM in a final volume of 5 ml. With each incubation set, experiments were run in which boiled liver slices were incubated with 50 mM ethanol. The values thus obtained were used as evaporation controls and subtracted from the corresponding experimental values. The vessels were sealed with serum caps and flushed for 5 min with a gas mixture of 95% 0, and 5% CO,. The subsequent incubations were carried out at 37°C for a total of 150 min in a Dubnoff water bath shaking at 100 strokes/min. Aliquots of the incubation medium were harvested with a needle and syringe through the rubber top immediately before the start of the incubations and then at intervals of 30 min. One hundred microliters of the harvested incubation medium was added to 0.5 ml of 35% perchloric acid contained in a 25-ml glass flask designed for analysis by a Perkin-Elmer F-40 gasliquid chromatograph (16). The sample bottles were immediately closed and incubated for 20 min at 60°C in the water bath attached to the chromatograph. Aliquots of the head space gas of these flasks were then injected by an automatic electropneumatic dosing system (injection time 4 s) into the gas-liquid chromatograph, and a 2-m x 2-mm column packed with 15% polyethyleneglycol on 50-60 mesh Celite was used. Helium was employed as a carrier gas at a flow rate of 40 ml/min. The temperature was 75°C for the column and 145°C for the flash heater as well as for the hydrogen flame detector. Quantitative assessment of the ethanol remaining in the incubation medium following the incubation was achieved by the use of a Hewlett-Packard GC digital integrator (Model 3370A) connected to the gas-liquid chromatograph. Solutions with known amounts of ethanol served as standards. It was found that an excellent reproducibility could only be achieved with a gas-liquid chromatograph with an automatic electropneumatic dosing system which was equilibrated for at least 4 h before the start of the analysis. Subcellular fractionation. The rats were killed by decapitation and their livers were perfused with icecold 0.15 M KC1 through the portal vein, excised, chilled, and homogenized in three volumes of 0.15 M KC1 using a glass homogenizer with a Teflon pestle. The following steps were carried out at 0-4°C. The 25% homogenate was spun at 10,OOOgfor 30 min, and the supernatant was centrifuged at 105,OOOgfor another 30 min. The resulting supematant (cytosol) was used as enzyme source for alcohol dehydrogenase, whereas the pellet was resuspended in 0.15 M KCl, and washed microsomes were obtained by spinning this suspension at 105,OOOgfor another 30 min. Biochemical determinations. The activity of alcohol dehydrogenase was assayed in the hepatic cytosol following the method of Bonnichsen and Brink (17) with ethanol at a final concentration of 50 mM. Catalatic activity of catalase was determined by
HEPATIC
ETHANOL
measuring the disappearance at 240 nm of H,O, added to aliquots of the 25% liver homogenate and expressing the results in units according to Luck (18). The activity of the NADPH-dependent microsoma1 ethanol-oxidizing system (MEOS) was determined with washed microsomes (3 mg of protein/ flask) which were preincubated with ethanol (50 mM) and sodium azide (1 mM) for 5 min at 37°C. The reactions were initiated by addition of a NADPHgenerating system (0.4 mM NADP+, 8 mM sodium isocitrate, and 0.34 unit/ml of isocitric dehydrogenase). The incubation medium contained, in a final volume of 3.0 ml, 1.0 mM NaZ-EDTA and 5.0 mM MgCl, in 0.1 M phosphate buffer (pH 7.4). The incubations were performed in closed 50-ml Erlenmeyer flasks with center wells containing 0.6 ml of 0.015 M semicarbazide hydrochloride in 0.1 M phosphate buffer (pH 7.4), and the acetaldehyde bound to the semicarbazide after an overnight diffusion period was determined according to Lieber and DeCarli (2). When the effect of butanol was studied, this alcohol was preincubated at a final concentration of 10 mM with microsomes and ethanol (50 mM) for 5 min before the reaction was started by adding the cofactor. Under these conditions, acetaldehyde bound to the semicarbazide was determined by gas-liquid chromatography as described above for the determination of ethanol in the liver slice study (12). Aliquota (100 ~1) of the semicarbazone solution of the center wells were added to 0.5 ml of 35% perchloric acid contained in 25-ml sample bottles. The bottles were sealed and subsequently incubated for 20 min at 60°C before aliquots of the head space were injected into the gas-liquid chromatograph. Flasks to which known amounts of acetaldehyde were added were carried through the complete procedure and were used as standards. The amount of acetaldehyde produced upon incubation was measured by peak heights. Peroxidatic activity of catalase was determined in washed microsomes under assay conditions described for the NADPH-dependent microsomal ethanol-oxidizing system except that sodium azide was omitted from the incubation medium and the NADPH-generating system was replaced by a H202producing one consisting of glucose (10 mM) and glucose oxidase (1.0 wg/ml of incubation medium). Under these conditions, glucose was preincubated with microsomes and ethanol (50 mM), and the reaction was started by adding glucose oxidase. Protein concentration was determined according to the method of Lowry et al. (19). Statistical analysis. Each individual result was compared with the value of its corresponding control; the means (+SE) of individual differences were calculated and their significance was assessed by the paired Student’s t test.
637
METABOLISM RESULTS
Effect of Pyrazole, Sodium Azide, and DLAlanine on Ethanol Metabolism Ethanol was oxidized by liver slices at a rate of approximately 50-60 pmol h-’ (g of liver)-‘, when ethanol was employed at a final concentration of 50 mM. The amount of ethanol disappearance was measured 150 min following the start of the incubation at which time 2530% of the alcohol added to the incubation medium had been oxidized. To verify the linearity of the reaction, measurements were also performed during the incubation time at regular intervals of 30 min each. The rate of ethanol metabolism was linear for 150 min. To assess quantitatively the role of alcohol dehydrogenase in ethanol metabolism, liver slices were incubated with ethanol (50 mM) and, when indicated, with pyrazole added to the incubation medium at a final concentration 2 mM. At this concentration, pyrazole almost completely abolished the activity of alcohol dehydrogenase when measured in the cytosol of the hepatocyte with ethanol as substrate (Table I). Pyrazole also decreased catalatic activity in liver homogenate by 24% (Table I); under these conditions, MEOS activity was found before to be inhibited by 11% (2). In contrast to the almost complete abolition of the activity of cytosolic ADH (Table I), pyrazole decreased the rate of ethanol metabolism by only 60% (P < 0.001) in liver slices (Table II). This finding suggests that although ADH activity plays a major role in ethanol metabolism, a considerable fraction of ethanol metabolism in liver slices may be accounted for by a pathway not involving the activity of alcohol dehydrogenase. To determine to what extent the pyrazole-insensitive pathway of ethanol metabolism in liver slices is due to the activity of catalase, the catalase inhibitor sodium azide was included in the incubation medium to achieve a final concentration of 1 mM. At this concentration, sodium azide virtually abolished catalase activity in liver homogenates when assayed by its catalatic property to decompose added hy-
638
TESCHKE,
HASUMURA
AND
TABLE EFFECT
OF PYRAZOLE
AND SODIUM
AZIDE
LIEBER
I
ON THE ACTIVITY
OF ALCOHOL
DEHYDROGENASE
ADH P
Activity (rim01 min-* liver)-‘) Control Pyrazole Azide Pyrazole
1821 60 1710 55
+ azide
+ + k -t
(g of 61 5 99 4
AND CATALASE”
Catalase
(% of control) 100 3.3 93.9 3.0
Catalatic (units (g of liver)-‘)
