Rates of Ethanol Clearance and Activities of the Ethanol-Oxidizing Enzymes in Chronic Alcoholic Patients

Vol. 61. No.5 Printed in U.S.A.

GASTIWENTEHOI.OGY

Copyright © 1971 by The Williams & Wi lkins Co.

LIVER PHYSIOLOGY AND DISEASE

RATES OF ETHANOL CLEARANCE AND ACTIVITIES OF THE ETHANOL-OXIDIZING ENZYMES IN CHRONIC ALCOHOLIC PATIENTS E STEBAN MEZEY , M.D., AN D F ABIO T OBON, M.D.

Alcoholism Research Unit and the Department of M edicine of the Baltimore City Hospitals, and The Johns Hopkins University School of Medicine, Baltimore, Maryland

The rates of ethanol disappearance from the blood and the activities of the hepatic ethanol-oxidizing enzymes were determined in 25 male chronic alcoholic patients during ethanol ingestion and in half of this group following withdrawal. During ingestion, the mean rate of ethanol disappearance from the serum and the activity of the reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent ethanol-oxidizing system were enhanced in the alcoholic patients when compared with control subjects. By contrast, there was no increase in the activity of alcohol dehydrogenase. In the repeat studies, performed in 12 patients (divided into three groups of 4 each) after 7, 14, and 21 days of withdrawal from alcohol, the mean rate of ethanol disappearance from the serum decreased to normal within 7 days, while the activity of the NADPH-dependent ethanol oxidizing system did not decrease significantly until 21 days after discontinuation of the ethanol. Alcohol dehydrogenase activity was increased 7 days after withdrawal from ethanol, returning to normal after 21 days . These observations suggest that increases in the activity of the NADPH-dependent ethanol-oxidizing system may contribute to, but do not fully explain, the enhanced rates of ethanol clearance in actively imbibing chronic alcoholics. Enhanced rates of ethanol disappearance from the blood have been shown in chronic alcoholic patients. 1 The rate-limiting step in the metabolism of ethanol is Received January 29, 1971. Accepted June 23, 1971. This work was presented in part at the Annual Meeting of the American Association for the Study of Liver Diseases, November 4, 1970. Address requests for reprin ts to: Dr. Esteban Mezey, Baltimore City Hospitals, 4940 Eastern Avenue, Baltimore, Maryland 21224 This work was supported by National Institute of Mental Health Grant 5 RO 1 MH 14251. The aut hors are greatly indebted to Dr. Richard E. Slavin for review of the liver biopsies, to Mrs. 707

its oxidation to acetaldehyde, principally catalyzed by alcohol d ehydrogenase 2 present in the soluble fraction of liver tissue homogenates, 3 and, recently, a reduced nicot inamide a denine dinucleotide phosphate (N ADPH)-dependent microsomal enzyme system was shown also to be capable of the oxidation of ethanol. • When ethanol was administered to rats, it was found to induce increases both in the microsomal ethanol-oxidizing system • and in the rates of in vivo ethanol disappearance Elaine Houston for her nursing supervision of the patients, and to Mrs. Jane Cooke, Mrs. Susan Swiderski, and Miss Carolyn Ochs for thei r techni cal help.

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from the blood, 5 leading to the suggestion that the increased rates of ethanol disappearance from the blood in acutely drinking alcoholics may be the result of induction of the microsomal ethanol-oxidizing system. 1• 6 In this study, the rates of ethanol disappearance from the blood and the activities of the ethanol-oxidizing enzymes were determined in chronic alcoholics during ethanol ingestion and following withdrawal to assess whether '6r not changes in the rates were accompanied by parallel changes in the activity of the enzymes. Methods Patients. Twenty-five male chronic alcoholic patients ranging in age from 25 to 53 years (mean, 38 years) without clinical evidence of chronic liver disease were studied. The patients, who had come to the hospital seeking help for their acute alcoholism, were admitted to the Metabolic Unit directly from the Emergency Room. Patients with jaundice, history, or clinical evidence of cirrhosis, gastrointestinal bleeding, abdominal surgery, or recent intake of medications were not considered for the study. All the patients included in the study acknowledged their alcoholism and agreed to remain in the Metabolic Unit for the length of the research protocol; however, only half of them volunteered for the follow-up studies. The history of alcohol intake in the patients varied from 9 to 35 years (mean, 20.4 years), all had been drinking recently in excess of 250. g of ethanol daily for 2 or more weeks and up to at least 24 hr prior to admission. Dietary intake had been poor (less than 20 g of protein per day) for a period of at least 10 days prior to TABLE

admission in 17 (68 %) of the patients . Sixteen patients (64%) had a history of prior hospital admissions for alcoholism, and 8 (32 %) had a history of one or more episodes of delirium tremens . On physical examination, hepatomegaly (liver edge palpated 1 to 6 em below the rib margin at the midclavicular line) was noted in 11 patients. Pertinent abnormal laboratory determinations present at admission are summarized in table 1. Upon admission , the patients were placed on a 2600-cal diet of which fat made up 355(, (100 g), protein 17% (112 g), and carbohydrate 48 % (312 g). This was supplemented by ethanol in a daily dose of 256 g, given as 95% ethanol diluted in noncaloric juice and administered in eight daily doses at 2-hr intervals from 8 AM to 10 PM . The same diet was continued throughout the hospitalization of the patient, but the ethanol was discontinued after the 10 PM dose on the day of the liver biopsy, performed 2 or 3 days after admission, and followed 1 day later by determination of the rate of ethanol disappearance from the blood after an intravenous ethanol load . Follow-up studies were obtained in 12 patients with initially elevated rates of ethanol clearance, 4 each after 7, 14, and 21 days of continuous hospitalization and withdrawal from ethanol. The pat ients received no medications during their hospitalization. Control subjects. Liver biopsies obtained for diagnostic purposes in 7 hospitalized nonalcoholic male patients ranging in age from 19 to 59 years (mean, 36 years) were assayed for ethanol-oxidizing enzyme activities . Histological examination of the liver biopsies revealed noncaseating granulomas in 3, nonspecific mild portal inflammation in 1, and normal liver in the remaining 3. The rate of ethanol disappearance from the

1. Abnormal laboratory determinations in the study group of 25 patients

Det erminati on

Hemoglobin (g/100 mg) ..... . . . . . . . . Serum bilirubin (total mg/100 ml) .. . .. .. SGOT (IU/Iiter) " ...................... . . Alkaline phosphatase (KA units/100 ml) ' Serum albumin (g/ 100 ml) Prothrombin concentration (?c ) . .. .. . . . ESP• (% retention after 45 min) .... . ····· ··

Ran ge of normal va lu es

No. of pat ients with abnormal va lu es

14.0- 17.0 < 1.0 < 40 4- 12 3.7- 5.7 80- 100 0- 4

SGOT, serum glutamic oxaloacetic transaminase; IU, international units. ' KA units, King-Armstrong units. c Corrected to normal with AquaMEPHYTON, 15 mg intramuscularly. • ESP, Eromsulphalein (Su lfobromophthalein).

a

8 6 18 11 1 3< 8

Ra nge of abnormal va lu es

11.0- 13.9 1.2- 2.1 48- 232 12.4- 21.4 3.0 56- 68 5- 26

November 1971

709

LIVER PHYSIOLOGY AND DISEASE

blood after an intravenous ethanol load was determined in 6 male control subjects ranging in age from 28 to 44 years (mean , 34 .0 years). None of the control subjects had received any recent medications, and , while most of them were occasional drinkers of alcohol, none had ingested ethanol for at least 5 weeks prior to the determinations. Enzyme determinations. Liver biopsies were performed with a Menghini needle. In a few cases, two biopsies were necessary to obtain sufficient tissue. Approximately one-third of the tissue was fixed in 109;, formaldehyde for histological examination and the remainder was immediately placed in a petri dish over ice, weighed, and homogenized. The samples weighing 25 to 50 mg were homogenized at 0 C in a Potter-Elvehjem apparatus with 5 ml of 0.1 M K 2HP0 4 -NaH 2PO. buffer, pH 7.4. Alcohol dehydrogenase activity was assayed on 5 11liters of the tissue homogenate by the spectrofluorometric method of Morrison and Brock. 7 Fluorescence was read in an Aminco Bowman spectrofluorometer (catalog no. A-8202); wavelength of the activating light equalled 365 m11, and recording (fluorescing) light equalled 470 m11. The activity of alcohol dehydrogenase was expressed in micromoles per mg of protein per hr. The NADPH-dependent ethanol-oxidizing activity was determined by the method of Lieber and DeCarli, • modified as follows. (a) One milliliter of homogenate (corresponding to 5 to 10 mg of fresh liver tissue) was added to 2 ml of reaction mixture in the main chamber of an Erlenmeyer flask. The final composition of the incubation mixture was: nicotinamide adenine dinucleotide phosphate (NADP +), 0.3 mM; magnesium chloride, 5.0 mM; nicotinamide, 5.0 mM ; isocitric dehydrogenase (Sigma type IV), 100 11liters; ethanol, 115 mM; and K 2HP0 4 NaH2P04 buffer, 0.1 M, pH 7.4. The pH of the buffer was increased from 7.0 to 7.4 because the pH optimum for the reaction was found to be between 7.2 and 7.6. The concentration of ethanol and that of the other reagents was shown to be optimal. (b) Only 0.3 ml of 0.015 M semicarbazide hydrochloride solution was added to the center well of the flask. (c) The reaction was stopped after 10 min of incubation with shaking by adding 0.5 ml of 709;, trichloroacetic acid, and the concentration of acetaldehyde bound to semicarbazide was determined after 24 hr by diluting a 0.25-ml aliquot of the center well to 1 ml and reading the absorbance at 224 m11 against a blank containing the semicarbazide hydrochloride solution. • The small amount of liver tissue obtained on needle biopsy necessitated a single timed determina-

tion of enzyme activity. The 10-min incubation period was chosen after the enzyme reaction with homogenates of normal human liver acquired at autopsy in quantities and with activities similar to those obtained on liver biopsy was shown to be linear with respect to time for up to 15 min after the addition of ethanol. The absorption spectrum of the solution recovered from the center well after incubation of liver homogenates with the reaction mixture and ethanol was identical to that obtained after the incubation of microsomes from human autopsy liver or rat liver and that of a standard acetaldehyde-semicarbazone solution . The activity of the NADPH ethanol-oxidizing system was expressed in millimicromoles per mg of protein per min. Protein concentration was determined by the Folin-Ciocalteau method of Lowry eta!. , 9 with bovine serum albumin used as a standard. Rates of et!w.nol disappearance. The rates of ethanol disappearance from the blood were determined 1 day after the liver biopsy, following a 12-hr period of fasting. An intravenous load of 0.8 g of ethanol per kg of body weight, given as a 20% solution in normal saline, was administered over 45 to 60 min. Five milliliters of blood were withdrawn at the end of the infusion, and hourly thereafter for 6 hr. The blood was centrifuged at 2000 g for 10 min and the separated serum was frozen . Ethanol concentrations were determined in all serum samples by gas-liquid chromatography.'" A Packard dual column oven gas-liquid chromatograph, model 803, with a hydrogen flame detector was used. The serum was diluted 10 times with distilled water . Ten microliters of this diluted sample were injected into a Poropak Q (Waters Associates, Framingham, Mass.) chromatographic column maintained at 185 C. Ethanol standards were injected before and after the injection of each sample. Ethanol concentration in the serum when plotted against time followed a linear function. The rate of ethanol disappearance from the serum was obtained from the slope of the regression line calculated by the method of least squares 11 and expressed in milligrams of ethanol cleared per 100 ml of serum per hr. The results are expressed as means ± 1 sn .

Results Histological examination of the liver biopsies revealed varying degrees of fatty infiltration in all 25 chronic alcoholic patients. The percentage of fatty infiltration of the liver parenchyma was minimal (0 to

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LIVER PHYSIO LOG Y AND DISEASE

710

10'() in 7, mild (10 to 30~() in 9, and moderate (30 to 70~() in 9 patients. Focal liver cell necrosis and Mallory bodies were present in two of the biopsies, while increased fibrous tissue was noted in only one of them. The mean rate of ethanol disappearance from the serum in the alcoholic patients was 19.7 ± 3.4 mg per 100 ml per hr (fig. 1) , which is significantly higher than the rate of 14.9 ± 2.5 mg per 100 ml per hr obtained in 6 control subjects (P < 0.001). The mean activity of the NADPH-dependent ethanol-oxidizing enzyme system was likewise increased in the alcoholic patients as compared with control patients: 7.7 ± 2.6 mJ.!moles per mg of protein per min , or 902.4 ± 380.0 mJ.!moles per g of liver (wet weight) per min, as compared with 4.2 ± 1.0 mJ.!moles per mg of protein per min, or 500.3 ± 203.2 mJ.!ffioles per g of liver (wet weight) per min (P < 0.001). RATE OF ETHANOL CLEARANCE

By contrast, there was no significant difference in alcohol dehydrogenase activity between chronic alcoholic and control patients: 9.4 ± 3.0 J.!moles per mg of protein per hr, or 1.07 ± 0.36 mmoles per g of liver (wet weight) per hr, as compared with 7.9 ± 2.6 J.!moles per mg of protein per hr , or 0.95 ± 0.38 mmoles. per g of liver (wet weight) per hr (P > 0.05). The concentration of protein per g of liver (wet weight) was similar in alcoholic patients and control subjects: 121.9 ± 22.2 mg per g as compared with 123.2 ± 44.6 mg per g (P > 0.05). When the enzyme values were plotted on scatter diagrams, no correlation was found between the rates of ethanol disappearance from the serum and the activities of either the NADPH-dependent ethanol-oxidizing enzyme system or al cohol dehydrogenase . Follow-up liver biopsies in 12 patients after varying intervals of withdrawal from

ALCOHOL DEHYDROGENASE

NADPH DEPENDENT ETHANOL OXIDIZING SYSTEM

32

16

c:

.c 21. E ::>

0

.E 12

:Sc: 12

c:

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0

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0;
E

16

a. "'

a.

-;;;

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S! ~ 16

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0

8

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8

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.

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CONTROL

ALCOHOLIC

8

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CONTROL

ALCOHOLIC

.I

CONTROL

A LCOHOL!C

FIG . 1. Rates of ethanol disappearance from the serum and activities of the ethanol-oxidizing enzymes in alcoholic patients an d control subjects. The bars indicate the mean values ± · 1 so. The differences between alcoholic patients and control subjects are significant for the rate of ethanol clearance and the activity of the reduced nicotinamide adenine dinucleotide phosphate (NADPH)-depe~dent ethanol-oxidizing system (P < 0.001). Differences in the activity of alcohol dehydrogenase are not statistically significant .

November 1971

LIVER PHYSIOLOGY AND DISEASE

alcohol revealed decreases in the degree of fatty infiltration in all of them (table 2). Focal necrosis and Mallory bodies, present in the initial biopsy of 1 of the patients, was not observed on repeat biopsy 21 days after withdrawal from ethanol. The mean rate of ethanol disappearance from the serum decreased to normal as early as 7 days after withdrawal from alcohol (P < 0.01), while the mean NADPH-dependent ethanol-oxidizing activity was decreased significantly only after 21 days (P < 0.02). By contrast, alcohol dehydrogenase was increased significantly at 7 days (P < 0.05), returning to normal 21 days after withdrawal from alcohol. The concentration of protein per g of liver (wet weight) was not significantly changed following ethanol withdrawal in any of the groups. The expression of enzyme activities per g of wet liver weight followed by statistical analysis yielded the same results as when expressed per mg of homogenate protein. Discussion The development of behavioral and pharmacological tolerance to ethanol by chronic alcoholics as well as by nonalcoholic subjects is a well known phenomenon. 12 • 13 A possible cause of the increased tolerance is an enhanced rate of degradation of ethanol. The chronic administration of ethanol to rats, for example, results in increases both in the rates of ethanol disappearance from the blood 6 • 14 and in the metabolism of ethanol by liver slices. 15 Although determinations of the rates of ethanol disappearance from the blood in chronic alcoholics have yielded conflicting results (Kater et al. 1 demonstrated increases in the rates of ethanol disappearance in chronic alcoholics studied soon after hospitalization following a drinking episode, while Clark and Senior 16 found no enhanced rates in chronic alcoholics studied after 2 weeks of abstinence from alcohol), this study clarifies the differences by showing that chronic alcoholics studied immediately after the cessation of alcohol intake do indeed have faster rates of ethanol degradation, which, however, return to normal as early as 7 days after withdrawal from alcohol and institution of

711

an adequate diet. Of note is that the rates of ethanol disappearance from the blood in both our control subjects and alcoholic patients were significantly lower than those reported by Kater eta!.; however, the rates in our control subjects were similar to those reported by Clark and Senior 16 and Lieberman. 17 The higher rates of ethanol disappearance from the blood reported by Kater et a!. 1 may be related to their finding that the rate of fall in ethanol concentration from the blood in both their control and alcoholic patients appeared to lie between a linear and an exponential clearance. 1 • 18 The rates obtained in our study always fitted a straight line, with correlat:on values (r) ranging between - 0.88 and - 0.99. That there were no significant differences in the activity of liver alcohol dehydrogenase between the chronic alcoholics studied immediately following cessation of drinking and the nonalcoholic control subjects is at variance with the findings of Ugarte et a!. 19 who found decreased liver alcohol dehydrogenase activity in chronic alcoholics when compared with moderate drinkers regardless of whether or not they had liver disease. Previous studies had shown decreased liver alcohol dehydrogenase activity in chronic alcoholics with cirrhosis of the liver. 20 Animal studies on the effect of ethanol administration on liver alcohol dehydrogenase activity have shown increases, 1 4 • 21 no changes, 4 • 22 or even decreases. 22 • 23 Decreased alcohol dehydrogenase, however, always occurred in association with severe fatty infiltration of the liver and was particularly marked in rats given ethanol while on a protein-deficient diet. 24 The transient increases in alcohol dehydrogenase activity found in this study 7 days after abstinence from ethanol and institution of a normal diet may reflect increases in protein enzyme synthesis and liver cell repair. The finding that the NADPH-dependent ethanol .. oxidizing enzyme system was increased in the chronic alcoholic patients is in agreement with the studies of Lieber and DeCarli 4 showing induction of this enzyme system by the administration of ethanol in rats. Besides the stimulation

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LIVER PHYSIOLOGY AND DISEA S E

712 TABLE

2. Rates of ethanol disappearance from the serum, activities of the ethanol-oxidizm g enzym es, and liver histology in 12 alcoholic patients before and after varying tim e p eriods of w ithdra ~al fr om alcohol Ra te of eth anol

Tim e period between initia l nnd fi na l stud.v

Patient no.

disappearance from the serum

In itia l

Fina l

NA DP H-d epend ent Alcohol dehydrogenase

Initia l

7 Days

1 2 3 4 Mean

± p 14 Days

SD

5 6 7 8 Mean

± p 21 Days

SD

9 10 11

12 Mean

± p

SD

Final

J.J.moles /m!-!lhr··

mp / 100 ml/l>r

ethanol·oxidi zing enz.vme syrstem" Initial

Liver histoloJ,.ryr>

Final

Initia l

Fina l

+3" +2 + 1 +3

+ 1d + 1 0 +1

+3 +2 +3 +3

+ 1 +1 +1 +1

+2 +1 + 2' +2

0 0 +1 0

m ~J, moles/mp/mi~·:.

21.3 21.6 18.9 17.6 19.9 1.9

14.7 14.5 14.9 15.6 14.9 0.48 < 0.01 '

12.9 10.4 9. 3 8. 6 10.3 1. 9

15.8 21.6 13.1 25.5 19.0 5.6 <0.05'

5.9 9. 5 7. 5 11. 7 8.7 2.5

6:9 6.:2 3.8 7.7 6.2 1. 7 >0 .05

20.0 19.0 21.8 19.8 20.2 1. 2

10.7 16.2 17.6 17.8 15.6 3.3 < 0.05'

15.3 12.5 5.5 10.9 11.1 4.1

11.1 16.3 13. 7 13.9 13.8 2. 1 > 0.05

5.9 8.9 13.3 4.8 8.2 3.8

7·.9 9.3 8,5· . 6.5 ·'

25 .8 25 .5 22.4 18.5 23 .1 3.4

15.9 17.4 13.5 14.2 15.3 1.8 < 0.01'

10.1 8.7 12.5 8. 1 9.9 2.0

9.4 7.5 8.4 6.6 8.0 1.2 > 0.05

6.8 7.5 10. 2 8.8 8.3 1. 5

..

S. i 1.2 > 0:05 4:8 4.2 5,.2 6.5 5,2 ' 1. 0 < 0.02' ' '

" NADPH, reduced nicotinamide adenine dinucleotide phosphat e. • Fat. See text for histological gra ding. ' Milligrams of liver homogenate protein . • Mild fibrosis. • Significant. 1 Focal necrosis, alcoholic hya line.

of the microsomal NADPH-dependent ethanol-oxidizing system, ethanol has been reported to induce in rats other microsomal enzymes; namely, N-hydroxylation of aniline, aliphatic hydroxylation of pentobarbital, aromatic hydroxylation of 3 , 4- · benzpyrene, 2 5 and nitro group reduction of p-nitrobenzoic acid as well as cytochrome P-450. 26 In man, aliphatic hydroxylation of pentobarbital 25 and glucuronidation of bilirubin 27 have been shown to be stimulated by the administration of ethanol. The increases in microsomal en zyme activity were associated with hypertrophy of the hepatic smooth endoplasmic reticulum in both rats and man. The duration of enha nced enzyme activity after discontinuation of the inducin g agent has

been . shown : to vaw. in different species. While ih. rats increased . microsomal enzyme activities r'e tu'm · to normal within 1 week after discontinuation of the inducing agerit,'28. 29 -in , dogs arid man the enhanced enzyme activities have been shown to persist for 2 or more weeks. 30 • 3 1 In this study , the increased NADPH-dependent ethanoloxidizing activity returned to normal after 21 days of withdrawal" from ethanol and institution of a normal diet. The in vitro activities of drug-metabolizirrg enzymes duri~g :drug induction 32 • 33 and . after withdrawal29 in animals have been --shown to parallel changes in the in vivo rates of drug . metabolism. In acute a lcoholics there were increases both in the rate.s of ethanol clearance from the blood

November 1971

LIVER PHYSIOLOGY AND DISEASE

and in the NADPH ethanol-oxidizing enzyme, but no increase in alcohol dehydrogenase. Upon withdrawal from ethanol, the rapid fall in the rate of ethanol clearance to normal was associated with a paradoxical rise in alcohol dehydrogenase activity and no significant decrease in the NADPH-dependent ethanol-oxidizing enzyme system. It should be noted, however, that calculation of total enzyme activities would have to take into account liver size, which decreased following withdrawal from ethanol in conjunction with decreases in histologically demonstrable fatty infiltration of the liver. The decrease in liver size would indicate a greater fall in the total activity of the NADPH-dependent ethanol-oxidizing system following withdrawal from ethanol and this might correlate better with the early fall in the rate of ethanol clearance from the blood. Comparison of the roles of the two ethanol-oxidizing enzymes suggests that alcohol dehydrogenase, because of its higher activity, probably accounts for a greater portion of the ethanol clearance in normal subjects, while the NADPH-dependent ethanol-oxidizing enzyme is more likely to contribute to the increases in ethanol clearance following ethanol consumption. Recent studies showing a lack of effect of the administration of the microsomal enzyme inhibitor SKF 525-A on in vivo rates of ethanol metabolism have been presented as evidence against a role of the NADPH-dependent ethanol-oxidizing enzyme activity in ethanol oxidation. 34 However, in vitro studies have shown that this enzyme 5 as well as the oxidation by microsomal enzymes of some other lipidsoluble compounds 35 are not inhibited by SKF 525-A. Our conclusions remain tentative because different sets of only 4 patients were studied per time interval and the historical information on dietary intake and amount of alcohol consumed by the patients prior to the study is of questionable reliability. The results indicate a need for sequential studies of a larger number of patients both during prolonged ethanol feeding and following withdrawal. It is likely that factors in addition to the absolute total enzyme activities play an

713

important role in the regulation of rates of ethanol oxidation. Changes in hepatic blood flow in our patients, for example, could have affected the rates of ethanol clearance from the blood, but the direction of these changes is unknown, since the administration of ethanol can result in increases36· 37 or decreases 38 of hepatic blood flow depending upon the dose administered, while fatty infiltration of the liver is associated with decreases in hepatic blood flow. 39 Furthermore, the availability of coenzymes could have been an important regulating factor. This is suggested by in vitro studies showing that the dissociation of the alcohol dehydrogenase reduced nicotinamide adenine dinucleotide complex is a rate-limiting step in ethanol oxidation by this enzyme. 40 Also, in vivo studies show that stimuli to increased reoxidation of reduced nicotinamide adenine dinucleotide such as elevated pyruvate concentrations in children with type I glycogen storage disease, 41 or infusion of fructose in normal subjects, 42 · 43 enhance the rates of ethanol disappearance from the blood. REFERENCES 1. Kater RMH, Carulli N , Iber FL: Differences in the rate of ethanol metabolism in recently drinking alcoho lic and nondrinking subjects. Amer J Clin Nutr 22:1608-1617, 1969 2. Isselbacher KJ , Greenberger NJ: Metabolic effects of alcohol on the liver. New Eng J Med 270: 351-356, 402-410, 1964 3. von Wartburg JP, Bethune JL, Vallee BL: Human liver alcohol dehydrogenase. Kinetics and physicochemical properties. Biochemistry (Wash) 3:1775-1782, 1964 4. Lieber CS, DeCarli LM: Ethanol oxidation by hepatic microsomes: adaptive increase after ethanol feeding. Science 162:917-918, 1968 5. Lieber CS , DeCarli LM: Hepatic microsomal ethanol-oxidizing system . In vitro characteristics and adaptive properties in vivo. J Bioi Chern 245: 2505- 2512, 1970 6. Lieber CS: New pathway of ethanol metabolism in the liver. Gastroenterology 59:930-937, 1970 7. Morrison GR, Brock FE: Quantitative measurement of alcohol dehydrogenase in the lobule of normal livers. J Lab Clin Med 70:116- 120, 1967 8. Gupta NK, Robinson WG: Coupled oxidationreduction activity of liver alcohol dehydrogenase. Biochim Biophys Acta 118:431-434, 1966 9. Lowry OH, Rosebrough NJ , Farr AL, et al: Pro-

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LIVER PHYSIOLOGY AND DISEASE

tein measurement with the Folin phenol reagent. J Bioi Chern 193:265- 275, 1951 10. Payne JP, Foster DV, Hill DW, et al: Observations on interpretation of blood alcohol levels derived from analysis of urine. Brit Med ,J 3:819823, 1967 11. Bancroft H: Introduction to Biostatistics. New York, Hoeber Medical Division, Harper and Row Inc, 1957, p 161 1:2. Mendelson .JH, La Dou J: Experimentally induced chronic intoxication and withdrawal in alcoholics. Quart ,J Stud Alcohol (suppl 2):14-39, 1964 13. Isbell H, Fraser HF, Wikler A, et al: An experimental study of the etiology of "rum fits" and delirium tremens. Quart J Stud Alcohol 16:1-33, 1955 14. Hawkins RD, Kalant H, Khanna JM: Effects of chronic intake of ethanol on rate of ethanol metabolism. Canad J Physiol Pharmacal 44:241- 257, 1966 15. Videla L, Israel Y: Factors that modify the metabolism of ethanol in rat liver and adaptive changes produced by its chronic administration. Biochem J 118:275-281, 1970 16. Clark CG, Senior JR: Ethanol clearance and oxidation of ethanol to carbon dioxide in persons with and without liver disease. Gastroenterology 55:670-676, 1968 17. Lieberman FL: The effect of liver disease on the rate of ethanol metabolism in man. Gastroenterology 44:261-266, 1963 18. Iber FL, Carulli N, Kater RMH: The kinetics of alcohol removal from the blood of man: comparison in recently drinking alcoholics and non-alcoholics. Fed Proc 28:626, 1969 19. Ugarte G, Pino ME, Insunza I: Hepatic alcohol dehydrogenase in alcoholic addicts with and without hepatic damage. Amer J Dig Dis 12:589592, 1967 20. Figueroa RB, Klotz AP: Alterations of liver alcohol dehydrogenase and other hepatic enzymes in alcoholic cirrhosis. Gastroenterology 43:10-12, 1962 21. Mistilis SP, Garske A: Induction of alcohol dehydrogenase in liver and gastroi ntestinal tract. Aust Ann Med 18:227-231, 1969 22. Greenberger NJ, Cohen RB, Isselbacher KJ: The effect of chronic ethanol administration on liver alcohol dehydrogenase activity in the rat. Lab Invest 14:264-271, 1965 23. Morrison GR, Brock FE: Quantitative measurement of alcohol dehydrogenase activity within the liver lobule of rats after prolonged ethanol ingestion. J Nutr 92:286-292, 1967 24. Figueroa RB, Klotz AP: The effect of whiskey and low-protein diets on hepatic enzymes in rats. Amer ,J Dig Dis 9:121-127, 1964

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25. Rubin E, Lieber CS: Hepatic microsomal enzymes in man and rat. Induction and inhibition by ethanol. Science 162:690-691, 1968 26. Rubin E, Hutterer F, Lieber CS: Ethanol increases hepatic smooth endoplasmic reticulum and drug-metabolizing enzymes. Science 159: 1469-1470, 1968 27. Waltman R, Bonura F, Nigrin G, et al: Ethanol and neonatal bilirubin levels. Lancet 2:108, 1969 28. Wilson JT, Fouts JR: The effect of chronic phenobarbital administration on some hepatic drugmetabolizing enzyme activities in the rat. Biochem Pharmacol15:1238-1241, 1966 29. Stevenson IH , Turnbull MJ: Hepatic drug-metaholising enzyme activity and duration of hexobarbitone anaesthesia in barbitone-dependent and withdrawn rats. Biochem Pharmacol17:22972305, 1968 30. Remmer H, Siegert M, Liebenschutz HW : Die Beschleunigung der Oxydation von Arzneimitteln wahrend und nach qer Behandlung mit Luminal. Klin Wschr 39:490-491 , 1966 31. Schmid K, Cornu F, Imhof P, et al: Die biochemische Deutung der Gewohnung an Schlafmittel. Schweiz Med Wschr 94 :235-240, 1964 32. Rem mer H : Drugs' as activators of drug enzymes, vol 6, Metabolic factors controlling duration of drug action, Proceedings of the First International Pharmacological Meeting. Edited by B Uvnas. New York, Macmillan Co, 1962, p 235256 33. Conney AH, Davison C, Gastel R, et al: Adaptive increases in drug-m etabolizing enzymes induced by phenobarbital and other drugs. J Pharmacol Exp Ther 130:1-8, 1960 34. Tephly TR, Tinelli · F, Watkins WD: Alcohol metabolism: role of · microsomal oxidation in vivo. Science 166:627-628, 1969 35. Anders MW, Mannering GJ: Inhibition of drug metabolism. 1. Kin'etics of the inhibition of the N-demethylation of ethylmorphine by 2-diethylaminoethyl 2, 2-diphenylvalerate HCl (SKF 525-A) and related compounds. Molec Pharmacal 2:319-327, 1966. 36. Mendeloff AI: Effect of intravenous infusions of ethanol upon estimated hepatic blood flow in man . J Clin Invest 33:1298-1302, 1954 37. Stein SW, Lieber CS, Leevy CM, et al: The effect of ethanol upon systemic and hepatic blood flow in man. Amer J Clin Nutr 13:68-74, 1963 38. Lundquist F, Tygstrup N, Winkler K, et a!: Ethanol metabolism and production of free acetate in the human liver. J Clin Invest 41:955-961, 1962 39. Leevy CM: Fatty liver: a study of 270 patients with biopsy proven fatty liver and a review of the literature. Medicine (Bait) 41:249-278, 1962 40. Theorell H, Chance B: Studies on liver alcohol

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LIVER PHYSIOLOGY AND DISEASE

dehydrogenase. II. The kinetics of the compound of horse liver alcohol dehydrogenase and reduced diphosphopyridine nucleotide. Acta Chern Scand 5:ll27-ll44, 1951 41. Zuppinger K, Papenberg J, Schurch P, et a!: Vermehrte Alkoholoxydation bei der Glykogenose Typ I. Schweiz Med Wschr 97:ll10-1ll7, 1967 42. Pletscher A, Bernstein A, Staub H: Zur

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Beeinflussung der Umsatzgeschwindigkeit des Alkohols. 1. Die Beschleunigung des oxydativen Alkoholabbaus durch Fructose. Helv Physiol Pharmacal Acta 10:74-83, 1952 43. Pawan GLS: Vitamins, sugars and ethanol metabolism in man. Nature (London) 220:374-376, 1968