Functional alterations of liver mitochondria in chronic experimental alcoholism

EXPERIMENTAL

AND MOLECULAR

Functional

Alterations of Liver Mitochondria Experimental Alcoholism’

OSVALDO R. KOCH,~ GERMAN ANZOLA Centro

25,253-262 (1976)

PATHOLOGY

de Patologiu Experimental, Fact&ad de Medicina,

C&LOS D. MONTERO,~

BEDETTI,~ AND

in Chronic

MERCEDES

ANDI& 0. M.

GAMBONI, STOPPANI~

II Critedra de Patologia e lnstituto de Quimica BioZdgica, thkersidad de Buenos Aires, J. E. Uriburu 950, Buenos

Aires,

Argentina

Received May 28, 1975, and in retiised form May 11, 1976 Mitochondria obtained from nonfatty livers of male rats fed an alcoholic “super diet” for 4 months displayed enlargement and bizarre configurations. In vitro, the mitochondria from alcohol-treated rats showed reduced oxidation of succinate and of malate-glutamate, but the energy coupling remained apparently unchanged when compared with that of three different controls. With j%hydroxybutyrate as substrate, the maximal respiratory rate was affected in relation to those of control rats in whose regimens alcohol was isocalorically substituted by sucrose or fat, but not when the whole basal diet replaced alcohol. According to these results, it seems clear that the respiration of hepatic mitochondria is significantly altered when ethanol is supplied to rats at high levels over long periods and when a nutritional basal diet which does not induce fatty liver is used.

One of the most consistent changes associated with the chronic consumption of large amounts of alcohol, both in man and in experimental animals, is the distortion and enlargement of hepatocytic mitochondria (Porta et al., 1965a, b). Previous studies indicate that at least the mitochondrial enlargement (megamitochondria) was due to the direct effect of alcohol metabolism, although the quality of the diet may modify its development. In rats, enlarged mitochondria appeared earlier and were more numerous when the alcoholic regimens contained adequate levels of protein, vitamins, and lipotropes (“super diets”) (Porta et at., 1968; Koch et al., 1969) than when the regimens were nutritionally deficient and unbalanced (Porta et al., 1969). Thus, all of the nutritional factors that prevent the abnormal accumulation of hepatic triglycerides (fatty liver ) and other more severe hepatic changes promote, on the contrary, the development of mitochondrial enlargement. The functional significance of the alcohol-associated mitochondrial changes is still uncertain. The reported results of studies on certain in viva mitochondrial parameters are conflicting, probably reflecting differences in experimental conditions. While in some of these studies the livers of the rats were fatty (Rubin 1 Studies supported y Tknicas, Argentina. 2 Career Investigator, 3 Research Fellow, 4 Research Fellow,

by

a grant

from

the

Consejo

Consejo National de Investigaciones Consejo National de Investigaciones Organization of American States.

253 Copyright All rights

National

1976 by Academic Press, Inc. o9 reproduction in any form reserved

de Cientikas Cientificas

Investigaciones y TBcnicas, y Tkcnicas,

Cientificas Argentina. Argentina.

KOCH

254

AL.

ET

et al., 1970; Gordon, 1973; Cederbaurn et al., 1974) and/or the periods of consumption of high-alcohol regimens were relatively short (Rubin et al., 1970; Gordon, 1973; Videla et al., 1973; Cederbaum et al., 1974), in others the livers were apparently normal and the periods of consumption of low-alcohol regimens were substantially more prolonged (Kiessling and Tilander, 1961, 1963; Lundquist et al., 1966; K&sling, and Pilstrijm, 1967, 1968; Kiessling, 1968; Sardesai and Walt, 1969). To clarify the contradictory results reported, we have investigated in vitro some respiratory parameters of hepatic mitochondria in rats submitted to experimental conditions in which three capital requirements are fulfilled: (a) a high alcohol regimen; (b) a true chronic period of intake (4 months); and (c) the absence of fatty liver. With this experimental design it is possible to obtain the most dramatic ultrastructural alterations of hepatic mitochondria without any other morphological modification. MATERIALS

AND METHODS

Animals ad diets. A total of 36 Wistar male rats (98 to 114 gm, initial hod, weight) was allotted to four groups (A, B, C, and D) and the rats were fed as follows: Members of Group A were offered ad I&. the diet shown in Table I and, apart from this, a mixture of 25% sucrose32s ethanol (w/v) in Richter drinking tubes, also an lib. The amounts of vitamins and lipotropes of the solid diet were almost twice and 10 times as high, respectively, as the usually recommended allowances (Warner, 1962). The total caloric intake consumed by rats of Group A, derived from the solid food as well as from the alcohol-sucrose mixture, was recorded daily. All the control groups were offered water ad lib. as the only drinking fluid and their solid diets were based on the lineal regimen consumed by Group A in which, however, alcohol was isocalorically replaced by sucrose (Group B ), by fat ( Group C ), or by the basal diet (Group D). The control rats were isocaIorically pair-fed (individually) with those of the alcoho1 group. All animals were housed in wire-bottomed individual cages in air-conditioned rooms. Body weights were recorded weekly. Hours of light and darkness were controlled for all groups. The animals from all groups were killed during a period between the fourth or the fifth month, and their livers were removed and processed for histological and biochemical studies. TABI&: Composit*ion

I of t.he I )ief,

Componenk Laboratory chowcL Partially hydrogennt’ed Corn oil Vitamin mixture’ Choline chloride

(Gram

shortening”

percent, 93.0 2.0 2.0 2.0 1.0

” Labina. Purina de Argent.ina Sociedad An6nima, Buenos Aires, Argentina. * La Favorita. Molinos Rio de la Plats, Buenos Aires, Argent.ina. r Vitamin diet. foltifieation mixture, Nut.rit.ional Biochemicsls Corporation,

Cleveland,

Ohio.

tIVER

MITOCHONDRIA

IN CHRONIC

ALCOHOLISM

255

Light and electron microscopy. The material for these studies was always obtained from the median hepatic lobes, For light microscopy, sections were fixed in Baker’s solution. Paraffin sections were stained with hematoxylin and eosin, Masson’s trichrome stain, and aniline blue-chromotrope 2 R for the detection of megamitochondria. Fat in frozen sections was stained with oil red 0. For electron microscopy, small blocks were fixed in glutaraldehyde, postfixed in Caulfield’s solution, and embedded in Epon 812. Ultrathin sections were doublestained with uranyl acetate and lead, using Reynolds’ method, and were photographed in a Siemens Elmiskop I A electron microscope. Mitochondrial respiration. Mitochondria were prepared by the method of Schneider ( 1948). The homogenization medium was 0.25 M sucrose, 1 mM Tris-HCl ( pH 7.4). The homogenates were fractioned according to Myers and Slater ( 1957). Pellets of the mitochondrial and nuclear fractions were studied by electron microscopy by the same methods as described above. The mitochondria were suspended in the homogenization medium and the respiratory activity was measured no later than 3 hr after the end of the preparation. Mitochondrial respiration was determined polarographically at 30°C with a vibrating platinum electrode (Model K Oxigraph, Gilson Med. Elec.). The basic reaction medium contained 0.24 M sucrose, 34 mM KCl, 5 mM MgC12, 1 mM EDTA, 9 mM Tris-HCl (pH 7.4), 5 mM K,HPO, (pH 7.4), and substrates as stated in Table II. When malate-glutamate was used as substrate, 2.5 mM malonate was added. The mitochondrial protein (l-2 mgm/ml) and ADP (0.2-0.3 mM) were added successively. The final volume of the reaction mixture was 2 ml. The oxygen concentration in the reaction media was taken as 0.22 mM. The ADP:O ratio was determined according to Estabrook (1967) (corrected for the small quantity of hydrolized ADP, about 80/r, and is expressed as the P: 0 ratio in Table II ), The protein of mitochondrial preparations was determined by a biuret method (Gornall et al., 1949). Statistical analysis. In order to test the differences between group means (one of the groups was considered as “control”), a one-way analysis of variance was performed first; then, each one of the three “treated” group means was compared with the control group by means of a t test, using as estimator of variance the “pooled” estimate of the four individual variance estimates (Steel and Torrie, 1960) . RESULTS Solid, fluid, and caloric intake. The average amounts of daily solid food and fluid (expressed in kilocalories and in grams ) spontaneously consumed by rats of Group A -throughout the entire experiment are presented in Fig. 1. The animals consumed relatively low amounts of solid food (about 31 kcal/day) and almost the same amounts of calories from the sucrose-ethanol solution. The final caloric intake and the percentages of caloric ingredients in this final regimen are also shown in Fig. 1. The lipotropic value is expressed in milligrams of choline per 100 kcal (Porta and Gomez Dumm, 1968). Animals of the pair-fed control groups consumed the same amounts of calories as those consumed by members of Group A. Body and liver weights. Animals of the alcohol group A grew during the entire experiment, attaining an average daily growth rate of 1.14 gm. Control animals of Groups B, C, and D grew at a higher rate than the animals in Group A (B,,

KOCH

256

ET AL.

70 r

ColBCal

64.05-

60, 50’

A

34

c

44

40, et < Y

33

67,

Col%Col

“‘%

’ 20 v 10

-F

’

9 P

13

-!

0) 6 779/.iar Lx

A24

6 100

/day

t.v.

0

Lv.

201

FIG. 1. Diets. Average daily amounts of solid food and fluid consumed by rats of Group A throughout the experiment. A, alcohol; C, carbohydrate; F, fat; P, protein; Lv, lipotropic value (expressed in milligrams of choline per 100 kcal).

1.31 gm; C, 1.62 gm; D, 1.27 gm), but only the rate of the “fat” control group C was statistically different from that of the alcohol group A. No significant differences were found in the weight ‘of the livers between alcohol and control groups. The values expressed in grams per 100 gm of body weight were as follows: Group A, 3.22; Group B, 3.25; Group C, 3.16; Group D, 3.49. Light

microscopy.

Livers

from

rats of Group

A showed

a few

fatty

changes

in

the form of tiny droplets located predominantly in centrilobular hepatocytes. In sections stained with aniline blue-chromotrope 2 R, numerous megamitochondria (some larger than the nuclei) were observed. Their preferential hepatocytic lobular location was in midzonal areas, although they were also found in centrilobular hepatocytes. Sections from livers of control groups B, C, and D showed fatty changes simi-

FIG. 2. Electron micrograph of a portion of a hepatocyte from alcohol group A. Enlargement and distortion of mitochondria with scanty number of cristae. Uranyl acetate-lead stain. x 16,000.

LIVER

MITOCHONDRIA

IN CHRONIC

ALCOHOLISM

257

FIG. 3. Electron micrograph of mitochondrial pellet from alcohol group A. The fraction was composed of organelles of different sizes. Uranyl acetate stain. X14,000.

lar to those of alcohol group A, but the hepatocytes predominantly affected were those of periportal areas. No megamitochondria were observed in these control groups. Electron microscopy. The most conspicuous ultrastructural changes in hepatocytes of rats from Group A were found in the mitochondria. The organelles were enlarged and displayed bizarre shapes. Their cristae were diminished in number but their matrical densities were normal (Fig. 2). Helical filaments were occasionally observed in dilated cristae and/or in the outer mitochondrial space. The ultrastructural configuration of hepatocytes from the control groups B, C, and D was normal. The study by electron microscopy of the samples obtained from the mitochondrial pellets of Group A showed almost the same ultrastructural appearance as that observed in the liver sections. The mitochondrial population was composed of organelles with sizes ranging from normal to a two- or threefold increase (Fig. 3). In this fraction we did not observe the remarkably large mitochondria present in the tissue sections. However, they were to be seen in the nuclear fraction. Mitochondriul respiration. The results of mitochondrial respiratory activities from rats fed according to the different regimens are presented in Table II.

KOCH

2.58 ML-GLU n

ET

AL.

OHB

lmm

FIG. 4. Oxygen uptake of liver mitochondria isolated animals. Experimental conditions were as described in near the traces indicate oxygen utilization in ngm-atoms group; C, control group D.

from alcohol and control (Group D) Materials and Methods. The numbers of O/min/mgm of protein. A, alcohal

Figure 4 shows typical curves with the different substrates employed. With succinate as substrate, the mitochondrial respiratory rate of rats of Group A was significantly lower than the rates of the control groups. Diminution of the maximal respiratory rate was the cause of the lower respiratory control values, as may be calculated from the data presented in Table II. The P:O ratio was unaffected. Similar results were obtained using malate-glutamate as substrate. With ,&hydroxybutyrate, the maximal respiratory rate in Group A was significantly lower than the rates of the control groups B and C but not significantly different from that of Group D. The respiratory control values, although lower in rats exposed to alcohol, were not statistically different from those of the control groups, probably because the small values obtained camlot be positively reflected in the respiratory control values. The P : 0 ratio in Group A was signficantly lower than in the control groups R and C but not significantly different from that of Group D. DISCUSSION When rats are fed for prolonged periods on “super diets” plus the sweetened alcohol solution originally proposed by Porta and Gomez-Dumm (1968), they do not develop fatty livers or any other significant hepatic alteration other than the mitochondrial structural changes reported by Koch et al. (1968) and Porta et al. (1969). In order to extend these observations in the present study, some biochemical parameters strictly related to the functioning of the imler mitochondrial membrane have been investigated. Selection of “proper” controls in

LIVER

MITOCHONDRIA

IN TABLE

Phosphorylation

Group

Substrate A B C D

(alcohol) (sucrose) (fat) (complete

Substrate: A B C D

(alcohol) (sucrose) (fat) (complete

Substrate: A B C D

‘I Mean b Ratio c Ratio of ADP.

dP eP fP @P ?‘P

< < < < <

1.34 1.48 1.46 1.37

(6 mM)-rrglutamate

diet)

diet)

P:O

10 7 7 4

8 6 6 4

fi-hydroxybutyrate

(alcohol) (sucrose) (fat) (comp1et.e

II

Maximal r&e of mitochondrial respiration (ngm-atoms of O/min/mgm of protein)

from

Respiratory

control -__ Af t,erc

Beforeb

(10 mM)

diet,)

n-malate

259

ALCOHOLISM

and Substrate Oxidation in Mitoehondria Alcohol and Control Rats”

Number of animals

: succinate

CHRONIC

i zk zk i

0.18 0.13 0.14 0.12

83 176 163 159

f f zk f

27 16d 2gd 315

3.91 3.35 5.21 4.64

f f f f

1.08 1.25” 1.27” 1.10

3.30 4.96 5.15 4.47

f f f f

0.55 0.98d 0.78d 1.35d

61 118 108 117

f f f f

14 12d 17d 24d

4.29 7.24 7.18 7.27

f f f f

1.26 1.83’ 1.73’ 2.48’

3.64 5.76 6.03 5.75

f 0.68 f.1.12s f 1.140 f 1.89u

45& Sli 59 f 57i

9 9” 12’ 15

3.61 f 4.28 f 4.60 f 4.13 i

1.25 1.12 1.49 1.32

2.79 3.47 3.78 3.40

* f f zk

(5 mM) 2.22 2.32 2.34 2.28

* 0.14 f 0.11 f 0.10 f 0.13

(5 mM) 8 6 6 4

1.89 2.28 2.24 2.05

f 0.24 f 0.13* f 0.17h f 0.42

-

values are followed by the standard deviation. of respiratory rate in the presence of ADP vs the respiratory of respiratory rat,e in t,he presence of ADP vs the respiratory

0.45 0.90 0.69 1.26

rate before ADP addition. rate after t,he expenditure

0.001. 0.05. 0.01.

0.003. 0.02.

studies on experimental chronic alcoholism has always been a difficult problem to solve and, therefore, in the investigation here described we have included three different control groups, consisting of rats receiving (a) a large amount of fat, (b) a large amount of sucrose, and (c) the complete basal diet. According to the observations described under Results, it is clear that the respiration of hepatic mitochondria is significantly altered when alcohol, plus a nutritional basal diet not conducive to fatty liver, is supplied to rats at high levels, over a prolonged period of time ( 4 months). Under these conditions and when succinate was the substrate, the mitochondria of alcohol-treated rats showed a striking depression of respiratory activity (more than 50% ) in both state 3 and state 4, as compared with the mitochondria from the three different controls. A depression of succinate respiration of lower magnitude was observed by Rubin et al. (1970), Gordon (1973), and Cederbaum et at. (1974) in rats treated with an alcohol-liquid diet; but it must be noted that these rats showed

260

KOCH

ET AL.

fatty livers, which complicates the interpretation of the modification of mitochondrial respiration. Rubin et al. (1970) have also suggested that the respiratory depressions may be related to a reduced succinate dehydrogenase activity and a lower level of cytochromes a and b in these mitochondria. Unpublished results from our group confirm these findings. On the other hand, in rats fed on normal diets plus 15-200/o ethanol in the drinking fluid for periods ranging from a few weeks to several months, and apparently having nonfatty livers, the succinatelinked mitochondrial respiration was found to be normal (Sardesai and Walt, 1969; Kiessling, 1968), reduced (Kiessling and Pilstriim, 1967; Kiessling, 1968), or the state 3 was depressed while the state 4 was increased (Kiessling and Tilander, 1961). These contradictory observations may be explained by the different experimental models used by these authors, who failed to induce an alcohol consumption of over 25% of the total caloric intake. Moreover, Videla et al. ( 1973), using for 3-4 weeks an ethanol-liquid diet that did not produce fatty liver, also failed to observe a reduction in succinate respiration. The difference between these results and those reported in Table II may be due to the relatively short period of the alcohol regimen in the Videla et al. (1973) study. A similar cause could account for the lack of effect of alcohol feeding on the respiratory control values, as described by Videla et al. (1973), Rubin et al. (197O), and Cederbaum et al. (1974). The results with NAD+-dependent substrates (mnlate-glutamate) showed an effect of alcohol similar to that when succinate is used as substrate. In this connection, our observations are in good agreement with other reports in the literature using glutamate as substrate (Kiessling and Pilstrijm, 1966; Cederbaum et al., 1974). Our studies with P-hydroxybutyrate as substrate showed a 25% depression of the maximal rate of respiration in alcohol-treated rats when compared with animals of the three control groups. The respiratory control values, although lower in Group A, were not significantly different from those of the other groups. These results may be related to the relatively small rates of respiration in states 3 and 4. The reduced values obtained with ,&hydrosybutyrate are at variance with those of other authors, who found that in rats consuming relatively low alcohol diets during several months, the respiration was either normal (Lundquist et al., 1966; Sardesai and Walt, 1969) or increased (Kiessling and PilstrBm, 1968; Kiessling, 1968). Using liquid diets for 3-4 weeks, Videla et cd. (1973) were also unable to detect changes in the mitochondrial respiration with P-hydroxybutyrate as substrate. On the other hand, Cederbaum et al. (1974) found a decreased rate of ,&hydroxybutyrate oxidation (a 38% decrease in state 3) with liver mitochondria from rats fed for 3-4 weeks with an alcoholic liquid diet, but here again the rats showed fatty livers. With succinate or malate-glutamate as substrate, the coupling of oxidative phosphorylation in the alcohol-treated rats was similar to that of controls, but with p-hydroxybutyrate a small reduction was noted in relation to the sucrose and fat controls. However, the P:O values did not differ from that of controls consuming the complete basal diet. The results are in agreement with the information available. In fact, with succinate as substrate, the reported values of the energy coupling of mitochondria remained unchanged in rats fed alcohol under different experimental models (Kiessling and Pilstram, 1968; Sardesai and Walt,

LIVER

MITOCHONDRIA

IN

CHRONIC

261

ALCOHOLISM

1969; Rubin et al., 1970; Videla et al., 1973; Cederbaum et al., 1974). However, with P-hydroxybutyrate as well as with other NAD+-linked substrates, Videla et al. (1973) and Cederbaum et al. (1974) found a significant decrease in the ADP:O ratio. With rats showing fatty livers, in studies such as those of Cederbaum et al. ( 1974), the decreased P: 0 ratio observed may be due to release of fatty acids and lysoderivatives during mitochondrial isolation. In fact, mitochondria isolated from fatty livers are frequently uncoupled (Vester and Stadie, 1957; Hall et al., 1960) since, after cell disruption, endogenous phospholipase A2 (Waite and Sisson, 1971) releases lysoderivatives and fatty acid from phospholipids, the former being active uncoupler agents ( Vasquez-Colon et al., 1966). In conclusion, whatever substrate is employed for testing the intactness of the mitochondrial energy-conserving mechanism, the reduction of respiratory activity in the present study largely exceeds those previously described by other authors. The longer feeding time (4 months) and the relatively high alcoholic content of our diets may account for the significant ultrastructural and biochemical lesions reported in this paper. The fact that only the alcohol-fed rats showed alterations of the mitochondria1 respiratory activity points to the alcohol, products of alcohol oxidation, or both as being responsible for the observed mitochondrial changes. The mitochondrial preparations employed in the present study do not include megamitochondria (over 5 pm in diameter). As a matter of fact, megamitochondria constitute but a small fraction of the mitochondrial population altered by chronic alcoholism (2% ), as described in the present study and in previous studies (Porta et al., 1965b, 1968, 1969; Koch et al., 1968, 1969; Porta and Gomez Dumm, 1968). Most of the liver mitochondria from rats treated with an alcoholsupplemented diet show an enlargement not greater than two or three times the normal size and are, therefore, not megamitochondria. Nevertheless, these mitochondria are abnormal, as shown by the reduced number and smaller length of the cristae and by the corresponding expansion of the matrix. Since the mitochondrial samples employed by us for the respiratory measurements systematically showed these structural features, we feel entitled to assume that these were representative of the liver mitochondrion, despite the fact that megamitochondria were practically absent from the samples investigated. Hence, the observed biochemical alterations, observed in vitro, are the counterpart of the ultrastructural alterations observed in the liver of the alcoholic rats in vivo. ACKNOWLEDGMENTS The authors wish Mrs. Silvia Hartman

to thank Professor Eduardo A. Porta for valuable assistance in the statistical

for reviewing analysis.

the

manuscript

and

REFERENCES CEDERBAUM, A. I., LIEBER, C. S., and RUBIN, E. ( 1974). Effects of chronic ethanol treatment on mitochondrial functions damage to coupling site I. Arch. Rio&em. Biophys. 165, 560-569. ESTABROOK, R. W. ( 1967). Mitochondrial respiratory control and the polarographic measurement of ADP:O ratios. In “Methods in Enzymology” (R. W. Estabrook and M. E. Pullman, Eds.), Vol. 10, pp. 41-47. Academic Press, New York. GORDON, E. R. (1973). Mitochondrial functions in an ethanol-induced fatty liver. J. Biol. Chem. 248, 8271-8280. GORNALL, A. G., BARDAWILL, C, J., and DAVLD, M. M. (1949). Determination of serum proteins by means of the biuret reaction. .I. Biol. Chem. 177, 751-766.

262

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ET AL.

HALL, J. C., SORDAHL, L. A., and STEFKO, P. 0. (1960). The effect of insulin on oxidative phosphorylation in normal and diabetic mitochondria. J. Biol. Chem. 235, 1536-1539. KIESSLING, K. H. ( 1968). Effect of ethanol on rat liver. VI. A possible correlation between alpha-glicerophosphate oxidase activity and mitochondrial size in male and female rats fed ethanol. Acta Pharmacol. Toxicol. 26, 245-252. KIESSLING, K. H., and PrrsrnBq L. (1967). Effect of ethanol on rat liver. IV. The influence of vitamins, electrolyte, and amino acids on the structure and function of mitochondria from rats receiving ethanol. Brit. J. Nutr. 21, 547-556. KIESSLING, K. H., and Pnsrnijhq L. (1968). Effect of ethanol on rat liver. V. Morphological and functional changes after prolonged consumption of various alcoholic beverages. Quart. J. Stud. Alcoh. 29, 819-827. KESSLING, K. H., and TILANDER, K. ( 1961). Biochemical changes in rat tissues due to prolonged alcohol consumption. Quart. I. Stud. Alcoh. 22, 535-543. KIESSLING, K. H., and TILANDER, K. (1963). Effects of prolonged alcohol treatment on respiration of liver and brain mitochondria from male and female rats. Exp. Cell Res. 30, 476-

482. KOCH, 0. R., POHTA, E. A., and HARTHOFT, W. S. (1968). A new experimental approach in the study of chronic alcoholism. III. Role of alcohol versus sucroseor fat-derived calories in hepatic damage. Lab. Inuest. 18, 379-386. KOCH, 0. R., PORTA, E. A., and HARTROFT, W. S. ( 1969). A new experimental approach in the study of chronic alcoholism. V. Super diet. Lab. Invest. 21, 298-303. LUNDQUIST, C. G., KIESSLING, K. H., and PILSTR~M, L. ( 1966). Effect of ethanol on rat livers. III. Lipid compositions of liver mitochondria from rats after prolonged alcohol consumption. Acta Chem. Stand. 20, 2751-2754. MYERS, D. K., and SLATER, E. C. (1957). Th e enzymic hydrolysis of adenosine triphosphate by liver mitochondria. Biochem. J. 67, 558-572. PORTA, E. A., BERGMAN, B. J., and STEIN, A. A. ( 1965a). Acute alcoholic hepatitis. Amer. J.

Pathol. 46, 657-689. PORTA, E. A., and Gohmz Duhlhr, C. L. A. (1968). A new experimental approach in the study of chronic alcoholism. I. Effects of high alcohol intake in rats fed a commercial laboratory diet. Lab. Invest. 18, 352-364. PORTA, E. A., HARTROFT, W. S., and L)E LA IGLESIA, F. A. (1965b). Hepatic changes associated with chronic alcoholism in rats. Lab. Znuest. 14, 1437-1455. PORTA, E. A., KOCH, 0. R., GO~IEZ DUMM, C. L. A., and HARTROFT, W. S. (1968). Effect of dietary protein on the liver of rats in experimental chronic alcoholism. J. Nutr. 94, 437-

446. PORTA, E. A., KOC:H, 0. R., and HARTHOFT, W. S. ( 1969 ). A new experimental approach in the study of chronic alcoholism. IV. Reproduction of alcoholic cirrhosis in rats and the role of lipotropes versus vitamins. Lob. Incest. 20, 562-572. RUBIN, E., BEATTIE, D. S., and LIEBER, C. S. (1970). Effects of ethanol on the biogenesis of mitochondrial membranes and associated mitochondrial functions. Lab. Incest. 23, 620-627. SARDESAL, V. M., and WALT, A. J. (1969). Effect of ethanol on tissue carbohydrate and energy metabolism. In “Biochemical and Clinical Aspects of Alcohol Metabolism” (V. M. Sardesai, Ed.), pp. 117-122. Charles C Thomas, Springfield, Illinois. SCHNEIDER, W. C. ( 1948). Intracellular distribution of enzymes. III. The oxidation of octanoic acid by rat liver fractions. J. Biol. Chem. 176, 259-266. STEEL, R., and TOHRIE, J. (1960). Analysis of variance. I. The one-way classification. Irl “Principles and Procedures of Statistics” (R. Steel and J. Torrie, Eds.), pp. 99-131. McGraw-Hill, New York. V~SQIJEZ-COLON, L., ZIEGLER, F. D., and ELLIOT, W. B. (1966). On the mechanism of fatty acid inhibition of mitochondrial metabolism. Biochemistry 5, 1134-1139. VESTER, J. W., and STADIE, W. C. ( 1957 ). St II d ies of oxidative phosphorylation by hepatic mitochondria from the diabetic cat. J. Biol. Chem. 227, 669-676. VIDELA, L., BERNSTEIN, J., and ISRAEL, Y. (1973). Metabolic alterations produced in the liver by chronic ethanol administration. Biochem. J. 134, 507-514. WAITE, M., and SISSON, P. (1972). Partial purification and characterization of the phosphoIipase A, from rat liver mitochondria. Biochemistry 10, 2377-2383. WARNER, R. G. (1962). Nutrient requirements of the laboratory rat. In “Nutrient Requirements of Domestic Animals” (National Academy of Sciences, Ed.), Vol. 10, pp. 51-95. National Research Council, Washington, DC.