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Hepatic and Metabolic Effects of Alcohol
Vol. 50, No. I Printed in U S.A.
GASTROENTEROLOGY
Copyright © 1966 by The Williams & Wilkins Co
PROGRESS IN GASTROENTEROLOGY HEPATIC AND METABOLIC EFFECTS OF ALCOHOL CHARLES
S.
LIEBER,
M.D.
Liver Disease and Nutrition Unit, Second (Cornell) Medical Division, Bellevue Hospital, and the Department of Medicine, Cornell University ilJedical College, New York, New York
For many years the disorders observed in association with alcoholism, especially diseases of the liver, have been attributed to nutritional deficiencies accompanying alcoholism rather than to alcohol itself.! Recent studies, however, indicate that in addition to dietary deficiencies, alcohol per se exerts a number of direct effects on hepatic and intermediary metabolism. 2 - 4 These effects of alcohol have been linked, in part, to alterations in biochemical pathways produced by the metabolism of alcohol in the liver.3 The hepatic metabolism of alcohol and the associated changes in intermediary metabolism will therefore be briefly summarized prior to a review of the pathological changes which can be attributed to direct effects of alcohol on the liver. (Alcohol and ethanol are used synonymously in this paper.) A. Metabolism of Ethanol Ethanol, which is readily absorbed from the gastrointestinal tract, is oxidized, mainly in the liver, to the extent of 90 to 98%.5 Trace amounts of ethanol may also be synthesized endogenously.6 It is generally accepted that the metabolic pathway for disposal of alcohol inAddress requests for reprints to: Dr. Charles S. Lieber, Second (Cornell) Medical Division, Bellevue Hospital, New York, New York 10016. Preparation of this manuscript was supported in part by Research Grants AM-062S4 and AM-09536 from the United States Public Health Service, National Institute of Arthritis and Metabolic Diseases. Dr. Lieber is recipient of United States Public Health Service Research Career Development Award K3-AM-22,590 from the National Institute of Arthritis and Metabolic Diseases.
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volves oxidation to acetaldehyde (fig. 1), followed by conversion to acetyl coenzyme A (acetyl-CoA). The initial oxidation of ethanol to acetaldehyde is catalyzed by a zinc-containing enzyme,1 alcohol dehydrogenase (ADH) , which has been isolated in pure form from the soluble fraction of the liver cells ;s, 9 other tissues oxidize only small amounts of alcohol, although more recently it has been pointed out that the gastrointestinal tract may play a larger part than has been hitherto suspected. 10 Most studies on liver ADH have been conducted on the horse enzyme; human liver ADH, which has been recently isolated,1 has similar properties, but a wider substrate specificity, including methanol, which cannot be oxidized by the horse enzyme. 7 This offers a satisfactory explanation for the successful use of ethanol in the treatment of human methanol toxicity, which had remained unexplained on the basis of the horse enzyme data. Similarly, ethylene glycol is also a substrate for human ADH, and ethanol in high concentration prevents its oxidation and toxicity.7 The acetyl-CoA which results from the metabolism of alcohol is, to a large extent, oxidized to carbon dioxide through the citric acid cycle; ethanol-C14 can also be traced to fatty acids, cholesterol, glycerol, glycogen, amino acids, and protein. 3 , 11-16 Acetate, which results either from the breakdown of acetyl-CoA or originates directly from acetylaldehyde, has been found to increase in the blood after ethanol administrationP Although in vitro the liver can readily utilize acetate, in vivo acetate is largely metabolized in peripheral tissues.lS, 19
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HYPERURICEMIA SOLUBLE CYTOPLASM MITOCHONDRION ETHANOL
NAD
.............. -.--.--Ace"'[CoA
10 \. , l c~1~6c
~
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ACETALDEHYDE
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FIG. 1. Hepatic oxidation of ethyl alcohol and some of its effects, through increases (-) or decreases (--.. ) of metabolic pathways.
In addition to producing acetyl-GoA and acetate, the oxidation of ethanol also results in the transfer of hydrogen to nicotinamide adenine dinucleotide (NAD, previously called DPN), which is reduced to NADH2 (fig. 1) ,20,21 NADH2 is also generated by the oxidation of various other substrates such as glucose. In contrast to glucose, the extrahepatic metabolism of ethanol, although shown to occur,22,23 is smal1. 24 ,25 This "obligatory" hepatic metabolism results in a high rate of local NAD to NADH2 conversion, reflected by an increase in the NADH2/NAD ratio following ethanol administration. 26 , 27 These measurements of total hepatic NADH2/NAD, however, do not take into account cellular compartmentalization and give only an attenuated picture of the NADH2/ NAD change in the soluble cytoplasmic compartment, where the oxidation of ethanol takes place. 8, 9 At the present time, this ratio cannot be measured directly, but can be assessed indirectly by the ratio of metabolites whose reduction is coupled with oxidation of ethanol , such as pyruvate-lactate. NADH 2 , which results from the metabolism of ethanol, can be reoxidized by vari-
ous reactions (fig. 1). A major system for NADH2 oxidation (the flavoprotein-cytochrome system), however, is localized within the mitochondria,28 while ADH is part of the soluble cytoplasmic fraction of the hepatic cell. 8, 9 Furthermore, the mitochondrial membrane appears to be relatively impermeable to NADH2 .28,29 If the mitochondrial flavoprotein-cytochrome system has to take part in the reoxidation of NADH2 produced in the soluble compartment, it is likely that some intermediate carrier, for instance ,B-hydroxybutyrate30 or a-glycerophosphate,31.33 transports hydrogen from the NADH2 of the soluble cytoplasmic compartment to the mitochondrial flavoprotein- cytochrome system. Alcohol oxidation was indeed found to be accompanied by an increase in hepatic IXglycerophosphate,34 which could contribute to the accumulation of hepatic triglyceride through enhanced production of the glycerol moiety of triglyceride. Recent data indicate, however, that in this respect glycerol from extrahepatic sources may play a more important role than glycerol formed in the liver.35 Theoretically, increased fatty acid syn-
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thesis from acetyl-CoA could be considered as another means for disposing of an excess of hydrogen, and indeed, NADH 2-generating systems such as ethanol or sorbitol were found in liver slices to increase the incorporation of acetate-C14 into fatty acids, relative to the C 14 0 2 produced. These observations probably indicate both increased fatty acid synthesis and diminished citric acid cycle activity resulting from a change in hepatic NADH2/ NAD ratio on ethanol oxidation. 36 In these experiments, simultaneous measurement of C 14 0 2 was needed to assess fatty acid labeling independent of the dilution of the labeled precursor which varies with different substrates and substrate concentrations. When, in the absence of C 14 0 2 determinations, no correction was made for isotopic dilution, the ethanol effect was not observed. 37 Ethanol could affect activity of the citric acid cycle and fatty acid metabolism directly through enhanced production of NADH2 which, as well as NADPH 2 (reduced nicotinamide adenine dinucleotide phosphate, previously called TPNH) , stimulates fatty acid synthesis. 3S , 39 Ethanol may also act via NADPH 2 through transhydrogenation from NADH 2; it must be pointed out, however, that recent studies have indicated that NADH2 is as great or sometimes an even greater source of hydrogen for fatty acid synthesis than NADPH 2 .40, 41 Ethanol could also act via an increase of a-glycerophosphate which was found to accumulate on alcohol ingestion 34 and to stimulate fatty acid synthesis. 42 Reduced citric acid cycle activity may be responsible for decreased fatty acid oxidation produced by ethanol as evidenced by parallel reduction in C14 0 Z production from both C14-palmitate and C14-acetate.36 A diminution in citric acid cycle activity has been recently confirmed by the observation of a reduction in the absolute amount of CO 2 produced in liver perfused with ethanol, without change in oxygen consumption. 43 No reduction in hepatic oxygen consumption with ethanol has been observed in vitr0 43 - 45 and also in vivo in men undergoing hepatic vein catheterizationP Hepatic adenosine triphosphate (ATP) 46 was unaffected by ethanol, suggesting that de-
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spite decreased citric acid cycle activity, energy production continues unharmed with presumably ethanol providing the reducing equivalents normally arising from oxidation of other substrates primarily through the citric acid cycle (fig. 1). It has also been found recently that ethanol produces hepatic cholesterol accumulation and increases incorporation of acetate-C14 into cholesterol.47 Theoretically, increased cholesterogenesis could also be viewed as another means for disposing of excess hydrogen produced on ethanol oxidation. In addition to increased lipogenesis, other pathways can serve as hydrogen acceptors in the soluble cytoplasmic compartment. For instance, the oxidation of ethanol to acetaldehyde can be coupled with the reduction of pyruvate to lactate,21, 48 NADH2 acting as intermediate hydrogen carrier (fig. 1). The conversion of pyruvate to lactate leads to increased lactate formation in the liver and increased lactate/ pyruvate ratio in the blood 49 , 50 or, in vitro, in liver incubation media. 43 This channeling of pyruvate to lactate is associated with decreased gluconeogenesis with ethanol which appears to play a role in the hypoglycemia occurring when ethanol administration is superimposed upon glycogen depletion such as results from prolonged fasting. 45 • 51. 52 Furthermore, NADH2 has been shown to produce a dissociation of glutamic dehydrogenase into inactive subunits 53 which may also explain reduction of gluconeogenesis from amino acid metabolism. In addition to hypoglycemia, however, hyperglycemia has been described in asso ciation with alcoholism. The mechanism of the hyperglycemia is still largely not understood; pancreatitis and disturban ces in catecholamine metabolism presumably play a role.3 In addition to its possible role in alcoholic hypoglycemia, the increased hepat ic conversion of pyruvate to lactate leads to en hanced blood lactate concentration,49. [;0 , 54 which produces a diminution in urin ary uric acid output; this explains, at least in part, the observed hyperuricemia in subjects intoxicated with alcoholic beverages55 (fig. 1). The hyperuricemia produced by a lcohop5
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122
~
:.ill!!fffCHYLOM\~S 1)I)jJ1i)f
HE/PATIC LIPOGENESIS
"
)ljJJt!!I_FFA--+-FATTY LIVER
F
,/\,
'J/j
ADIPOSE TISSUE
'
,/
~' LIPOPROTEINS
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~ C02
FIG. 2. Schematic representation of the three major sources (-+) and the two principal modes of disposition (--7) of the fat accumulating in alcoholic fatty liver.
can be potentiated by fasting 56 which, in itself, is known to cause hyperuricemia. 57 Further effects possibly related to the hyperlactidemia include increased urinary magnesium excretion,58,59 and possibly increased splanchnic blood flOW,60 which has been described with large doses of ethanol,50. 60. 61 but not with small amounts. l7 • 62 Among the alterations produced by ethanol that can be attributed to the NADH 2/ NAD change, one should list decreased removal of galactose from the blood, resulting from the inhibition of uridinephosphategalactose-4-epimerase by NADH 2 63-65 and decreased urinary excretion of 5-hydroxyindole acetic acid (5-HIAA), presumably because of diminished oxidation of serotonin to 5-HIAA through competition for NAD.66 B. Pathogenesis of Alcohol Fatty Liver
The problem of whether the fatty liver observed after prolonged alcohol intake is due only to nutritional deficiencies associated with alcohol, or whether it is also caused by alcohol itself,!' 67-69 has been recently settled: subjects with a history of alcoholic fatty liver, but whose hepatic morphology had returned to normal, developed a fatty liver with the ingestion of ethanol, either in addition to, or as isocaloric substitution for, carbohydrate in an otherwise adequate diet. 70, 71 Similar effects were observed in rats given alcohol as part of a nutritionally adequate liquid diet. 71 When calories given as ethanol (or as carbohydrate in the control diet) were either
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omitted or isocalorically replaced with fat, no fatty liver was produced, indicating that the alcoholic fatty liver was not simply the consequence of a lack of carbohydrate or of available calories but resulted from alcohol per se, whose capacity for producing a fatty liver was found to be greater than that of fat itself.71 These studies demonstrate that in addition to nutritional deficiencies associated with alcoholism, alcohol itself has to be considered as a direct etiological agent in the pathogenesis of the fatty liver produced by prolonged alcohol intake. Lipids which accumulate in the liver can originate from three main sources: (a) dietary fat, which reaches the blood stream from the gut as chylomicrons; (b) adipose tissue fatty acids, which are transported to the liver as free fatty acids (FFA); and (c) lipids synthesized in the liver itself (fig. 2). Hepatic steatosis can result either from an excessive supply from one or more of these three sources, or from a disturbance of lipid disposition from the liver through reduced lipid oxidation, insufficient lipoprotein formation, or excretion into the blood (fig. 2). Dietary lipids. The importance of dietary fat in the pathogenesis of the alcoholic fatty liver has recently been brought into focus with the observation that both in man and in rat, fatty acids which accumulate in the liver on prolonged intake of alcohol and fat-containing diets are, to a large degree, dietary in origin. 72 -74 The role of dietary lipids has been further emphasized by the fact that prolonged ingestion of alcohol with low fat diets led to less hepatic steatosis than equal amounts of alcohol in isocaloric diets with a fat content equivalent to the average U. S. diet73 , 74 (fig. 3). There was no significant effect of alcohol on intestinal lipid absorption, hepatic chylomicron uptake, or hepatic lipoprotein formation and release, while decreased hepatic oxidation of dietary lipids appeared as the most likely explanation for the observed accumulation of dietary fat upon prolonged alcohol ingestion. n . 74 Adipose tissue lipids. After the administration of a very large single dose of alcohol (6 to 9 g per kg) by gastric tube to fast-
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~
HEPATIC LIPIDS IN 44 RATS GIVEN OVER 24 DAYS CONTROL SUCROSE DIET OR _ ISOCALORIC ETHANOL DIET TOTAL
LIPIDS
TRIGLYCERIDES
fat-conta i ning diets 143'4 col)
140
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FIG. 3. Comparison of hepatic steatosis produced with diets of varying fat but constant ethanol content (from C. S. Lieber et al. 1966. Role of dietary, adipose and endogenously synthesized fatty acids in the pathogenesis of the alcoholic fatty liver. J . Clin. Invest. 1,5: Jan.) .
ing rats, adipose tissue fatty acids were found t o accumulate in the liver. 73 - 77 This was considered to be due to increased peripheral fat mobilization, as under these experimental conditions, circulating FF A were found to be increased. 75 • 76. 78 Under apparently similar experimental conditions, however, other investigators found no rise in circulating FF A79 and no evidence of enhanced mobilization of fatty acids from labeled fat pads. so The latter experiments led to the conclusion that even after the administration of a single large dose of ethanol, decreased hepatic metabolism of
fatty acids (mobilized from adipose tissue at a normal rate) rather than increased peripheral fat mobilization is responsible for the hepatic steatosis. so Fat accumulation in the liver after a single large dose of ethanol was found to be moderate, and less in male than in female rats. Total lipids in male rats increased only about 20% 16 hr after the administration of 7.5 g per kg of ethanol by gastric tube. 74 The cause for this sex difference is not known; it could be related to the fact that female rats have more liver alcohol dehydrogenase than males. 9
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In man, large amounts of alcohol (400 g per day) were found to raise circulating FF A, but with more moderate quantities of ethanol, up to 300 g per day for as long as 18 days, circulating FFA remained unchanged (fig. 4).70, 81 Fatty liver was nevertheless observed with these moderate amounts of ethanoFo, 71 and it contained only a small fraction of adipose tissue fatty acids.72,73 In acute experiments in men (1 to 10 hr), ethanol decreased circulating FFA,82-84 with a reduction of FF A turnover.85 Thus, increased peripheral fatty acid mobilization has only been shown to play a role when very large amounts of ethanol are administered; even under these experimental conditions, the relative importance of enhanced peripheral mobilization versus decreased hepatic lipid metabolism has not been fully clarified. Lipids synthesized in the liver. In rats given low fat diets (2% of calories), the same amounts of alcohol produced less hepatic fat accumulation than in rats fed isocaloric diets containing 43% of calories as fat (fig. 3) the fatty liver had also a fatty acid composition very different from adipose tissue, with a large percent of endogenously synthesized fatty acids (such as palmitic acid), most likely produced in the liver itself.72-74 It is likely that the accumulation of endogenously synthesized fatty acids results from a combination of decreased oxidation and increased synthesis of hepatic fatty acids, effects which alcohol has been shown to produce in vivo and in vitro.36, 86, 87 The mechanism of this has been discussed above (section A). The major lipid fraction of the alcoholic fatty liver is triglyceride. The reason for the selective accumulation of triglyceride is not known. This could result from a specific stimulation of triglyceride production ,75, 77 with possibly a relative block in phospholipid synthesis. 75 Disturbances in lipoprotein metabolism and alcoholic hyperlipemia. Theoretically, a substantial interference with the hepatic mechanism for lipoprotein formation or release could lead to excessive accumulation of fat in the liver (fig. 2). Decreased hepatic lipoprotein release has been proposed as an explanation for the fatty liver,
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the decrease in circulating lipids, and the reduction in post-Triton hyperlipemia induced by carbon tetrachloride poisoning. s8 On the basis of hepatic perfusion studies, a similar mechanism has been proposed for the alcoholic fatty liver.89 In contrast to the carbon tetrachloride poisoning and the in vitro perfusion of liver with large amounts of ethanol, in vivo administration of alcohol was not accompanied by a fall, but rather produced an increase in blood lipids, especially triglycerides, both in man 70, 8], 83 and in experimental animals,2, 70, 90 with the concomitant development of a fatty liver. 70, 71 Furthermore, post-Triton hyperlipemia was found to be unaffected by ethanol. 91 ,92 These findings suggest that decreased transport of lipids from the liver is not the primary factor in the production of the alcoholic fatty liver, if one assumes no major change in peripheral fat utilization. The pathogenesis of alcoholic hyperlipemia, however, has not been fully clarified. Decreased lipolysis may playa role in the rare individuals suffering from marked hyperlipemia, the so-called "Zieve's Syndrome." 93-95 This is suggested by the observations, in these subjects, of low lipoprotein lipase activity,95 circulating inhibitor of lipoprotein lipase,96 and accumulation of a-chylomicrons. 97 Delayed clearing of postprandial hyperlipemia due to ethanol has also been reported in man98 and in rats. 99 Furthermore, it must be pointed out that, in addition to the production of lipoproteins, the liver is also responsible for the removal of an important fraction of the circulating lipoproteins; theoretically, alteration of this function by alcohol, possibly in association with steatosis, could also lead to hyperlipemia. Although the association of alcoholism and lactescent serum has been described by various authors, patients with alcoholic fatty liver do not necessarily present with hyperlipemia. On administration of alcohol, an increase in blood lipids was observed only with large doses, larger than necessary for the production of fatty liver. 7o ,71 In addition to its dependence on the amount of alcohol ingested, the hyperlipemia may also be caused or potentiated by factors other than alcohol, such as pan-
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PROGRESS IN GASTROENTEROLOGY 500 GLYCERIDES
400 300
~HOLIPIDS~
200
CHOLESTEROL
~
mg%
400 mg% 300
=~
mg%
200 100
o
FIG. 4. Effect of prolonged alcohol intake on serum lipids in seven chronic alcoholic individuals (average results ± SE of the mean) (from C. S. Lieber et al. 1963. Fatty liver, hyperlipemia and hyperuricemia produced by prolonged alcohol consumption, despite adequate dietary intake. Trans. Ass. Amer. Physicians 76: 289).
creatitis,lOO or an unusually low lipoprotein lipase activity.95,96 Furthermore, when seven chronic alcoholic volunteers were given up to 300 g of alcohol per day for 18 days, hyperlipemia was found during the first 10 days only, with return toward normal thereafter, despite continuation, at least initially, of the same alcohol and dietary intake (fig. 4) .70, 81 This indicated that duration of alcohol ingestion may also be one of the crucial factors determining whether or not hyperlipemia will be seen in alcoholics. The reason for the disappearance of the hyperlipemia on continuous alcohol intake is not clear; this could be related to progressive deterioration of liver function. C. Prevention of Alcoholic Fatty Liver In experimental animals, the fatty liver produced by small amounts of alcohol in conjunction with diets deficient in lipotro-
pic agents can be prevented by simple correction of the dietary deficiency.1, 69 Lipotropic agents were found to be only partially effective 101 or even totally ineffective I02 , 103 in preventing fat accumulation resulting from the administration of a single large dose of alcohol by gastric tube. Similarly, fatty livers were produced in rats on prolonged alcohol intake despite diets containing adequate amounts of lipotropic agents (0.25 mg of choline chloride and 1.5 mg of DL-methionine per cal) ,70,71 even when supplemented with an additional 1.25 mg per cal of methionine,t04 an amount previously shown to protect against alcoholic fatty liver produced on deficient diets.I,69 Supplementation with larger amounts of methionine (2.5 mg per cal) or choline chloride (up to 5 mg per cal) resulted in a decrease of fat accumulation produced by alcohol; the protection was incomplete and variable, with some animals
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~howing no protection while others had a marked reduction in steatosis (C. S. Lieber and L. M. DeCarli, unpublished data). The fat which accumulates in the liver of rats after the administration of one large dose of alcohol was found to be reduced or even prevented by asparagine,t°5 or various antioxidants, such as vitamin E, N-N-diphenyl-p-phenylenediamine or G-50 (Griffith Laboratories, Chicago, Ill.) .106,107 These substances, as well as large amounts of vitamin K, methylene blue, cyanocobalamin, and inositol were found to be ineffective in the prevention of the fatty liver produced by prolonged alcohol intake, when the diet was otherwise adequate (C. S. Lieber and L. M. DeCarli, unpublished data). The injection of massive amounts of ATP has been reported to decrease hepatic fat accumulation after a single large dose of ethanol, possibly through hypothermia and shock. 108 The fatty liver produced by prolonged alcohol ingestion was reduced by decreasing the fat content of the diet, both in man and in experimental animals,73,74 underlining the important role of dietary lipids in the pathogenesis of the alcoholic fatty liver. A similar effect was obtained by replacing dietary fat with MCT (Drew Chemical Corp., Boonton, N. J.), a mixture of medium chain triglycerides (C. S. Lieber and L. M. DeCarli, unpublished data).
D. "Alcoholic Hepatitis" and Cirrhosis
In contrast to the problem of the fatty liver, not much progress has been made in unraveling the pathogenesis of alcoholic cirrhosis. It is still not clear which hepatic lesions have to be considered as precursors of the cirrhosis, for instance, whether alcoholic fatty liver per se leads to cirrhosis, such as has been described for experimental choline deficiency,l°9 or whether, independent of the fatty liver or in conjunction with it, other lesions are necessary. In addition to fat accumulation, alcoholic individuals may have a variety of hepatic lesions which have been grouped under the terms "alcoholic hepatitis," 110-115 "acute fatty metamorphosis of the liver," 116 or "acute hepatic insufficiency of the
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chronic alcoholic." 117 It is characterized by intensified drinking usually in a chronic alcoholic, leading to anorexia, nausea, vomiting, upper abdominal pain, hepatomegaly and, frequently, fever and jaundice. Biochemical tests are not characteristically altered, though they may suggest liver cell damage with sometimes a confusing obstructive element. Anemia is usually present and a severe leukocytosis may develop. In addition to marked fatty changes, the histological picture is characterized by local and massive cellular degeneration and necrosis, with acute inflammatory reaction. On electron microscopy, the mitochondria appear much enlarged, and clumped. Clumping of the mitochondria and giant forms has been proposed as a morphological basis for the "alcoholic hyaline bodies" of Mallory.109, 114, 118 In other electron microscopic studies, however, the hyaline material was considered to consist of altered endoplasmic reticulum.n9, 120 Among various nonspecific mitochondrial alterations described in alcoholic fatty liver, the most conspicuous was the presence of crystal-like inclusions resembling "myelin degeneration" often accompanied by marked mitochondrial enlargement.114.121 In rats, acute administration of a very large dose of alcohoF22 or prolonged intake of moderate amounts of alcohoF23. 124 also leads to mitochondrial swelling and deformation with a reduction in the capacity of the mitochondria to carry out oxidations. 125 The mitochondrial lesion is preceded by an alteration of the endoplasmic reticulum characterized by disappearance of the normal parallel arrays and their replacement by vesicular s1;ructures.123 Autoradiographic techniques, employing tritiated thymidine to evaluate hepatic deoxyribonucleic acid (DNA) synthesis, indicated that hepatic cell damage produced by ethanol is accompanied by an increase in DNA synthesis which is primarily found in mesenchymal and ductular cells, and appears to be correlated with hepatic necrosis and inflammation, rather than with fat accumulation. 126 Which of the morphological lesions described above has to be considered as "the
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precursor" to cirrhosis remains obscure; as for the fatty liver, it is possible that various factors may be involved, especially in view of the fact that alcoholic cirrhosis appears to comprise a heterogeneous group, with both portal and "postnecrotic" forms of cirrhosis. 127 It has been shown recently that, once cirrhosis was established, but provided an adequate protein ingestion was maintained, the intake of alcoholic beverages in amounts less than spontaneously consumed had no obvious adverse effect on the cirrhosis, though some fat infiltration was observed. 128 The significance of this observation is hard to assess in view of the difficulty of evaluating the progress of cirrhosis over relatively short periods of time in human biopsy material. It focuses our attention, however, on the importance of the dose of alcohol ingested and the possible role of factors other than alcohol, such as malnutrition. Furthermore, possible hepatotoxic roles of nonalcoholic components of alcoholic beverages must be considered. For instance, wine of certain vintages has been shown to contain large amounts of iron129-131 which was found to accumulate in liver and other organs of experimental animals given such wine over long periods of time ;129 this could also explain, at least in part, iron accumulation in various tissues (including the liver) of wine drinkers with cirrhosis of the liver.129-131 E. Effects of Alcohol on Varions Enzymes
In man, alcohol was found both to increase 132 or not to change 133 , 134 various serum enzymes, especially transaminases; in the former study alcohol was given orally, while in the latter studies it was administered intravenously. In rats, serum 135 and liver 136 catalase increased with alcohol administration. In patients with cirrhosis, the clearance of alcohol from the blood was found to be either normal 137 , 138 or reduced. 139 These results can presumably be reconciled on the basis of the severity of liver disease, the metabolism of alcohol being affected only by very advanced hepatic damage. 137 The rate of alcohol metab-
127
olism was found to be related to the ADH activity in the liver of rats,140 while no such correlation was found in human liver.138 In animals, ADH activity in the liver increased with alcohol ingestion,141-143 but on prolonged alcohol intake, after an initial rise, there was ultimately a decrease in ADH activity141, 142 potentiated by a low protein diet,144 In patients with alcoholic liver disease, hepatic ADH145, 146 and isocitric dehydrogenase 145 were found to be reduced. Hepatic glutamic-pyruvic transaminase was also found to be decreased in rats given ethanol.147 Despite these enzyme changes, hepatic protein synthesis, as assessed by amino acid incorporation, did not appear to be decreased.Hs In conclusion, a number of pathological effects of ethanol remain unexplained, especially the chain of events which leads to cirrhosis. Studies in recent years, however, have indicated that in addition to acting via nutritional deficiencies, ethanol per se has to be considered as a direct hepatotoxic agent, as evidenced by production of fatty liver, hyperlipemia, and various enzyme changes. A number of metabolic effects of ethanol (involving lipid, glucose, galactose, uric acid, and serotonin metabolism) have been given a satisfactory explanation on the basis of a single biochemical defect, namely, the increase of NADH2/ NAD ratio in the cytoplasmic compartment of the hepatic cell, which may represent the most characteristic change produced by ethanol. REFERENCES 1. Best, C. H ., W. S. Hartroft, C. C. Lucas, and J. H. Ridout. 1949. Liver damage produced by feeding alcohol or sugar and its prevention by choline. Brit. Med. J . 2: 4635 . 2. Klatskin, G. 1961. Alcohol and its relation to liver damage. Gastroenterology 41: 443. 3. Lieber, C . S., and C. S. Davidson. 1962. Some metabolic effects of ethyl alcohol. Amer. J. Med . 33: 319. 4. Isselbacher, K. J., and N. J. Greenberger. 1964. M etabolic effects of alcohol on the liver. N ew Eng. J. Med. 270: 351. 5. Thompson, G. N. 1956. Alcoholism. Charles C Thomas, Publisher, Springfield, Ill .
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6. Lester, D. 1961. Endogenous ethanol: A review. Quart. J . Stud. Alcohol. 22: 554. 7. von Wartburg, J. P., J. L. Bethune, and B. L. Vallee. 1964. Human liver-alcohol dehydrogenase. Kinetic and physicochemical properties. Biochemistry 3: 1775. 8. Nyberg, A., J. Schuberth, and L . Anggard. 1953. On the intracellular distribution of catalase and alcohol dehydrogenase. Acta Chern. Scand. 7: 1170. 9. Buttner, H. 1965. Aldehyd- und alkoholdehydrogenase-aktivitat in Leber und Niere der Ratte. Biochem. Z. 341: 300. 10. Spencer, R. P., K. R Brody, and B. M. Lutters. 1964. Some effects of ethanol on the gastrointestinal tract. Amer. J. Dig. Dis. 9: 599. 11. Forsander, O. A., and C. R. Riiihii. 1960. Metabolites produced in the liver during alcohol oxidation. J. BioI. Chern. 235: 34. 12. Schulman, M. P., R Zurek, and W. W. Westerfeld. 1957. The pathway of alcohol metabolism. In Alcoholism, publ. 47, p. 29. American Association for the Advancement of Science, Washington, D . C. 13. Curran, G. L., and D. Rittenberg. 1950. The role of ethyl alcohol in the biological synthesis of cholesterol. J. BioI. Chern. 190: 17. 14. Smith, M. E., and H. W. Newman. 1960. Ethanol-1-C" and acetate-1-C" incorporation into lipid fractions in the mouse. Proc. Soc. Exp. BioI. Med.l04: 282. 15. Russell, P. T., and J. T. Van Bruggen. 1964. Ethanol metabolism in the intact rat. J. BioI. Chern. 239: 719. 16. Schiller, D., N. Burbridge, V. C. Sutherland, and A. Simon. 1959. The conversion of labeled ethanol to glycerol and glycogen. Quart. J. Stud. Alcohol. 20: 432. 17. Lundquist, F., N. Tygstrup, K. Winkler, K. Mellemgaard, and S. Munck-Peterson. 1962. Ethanol metabolism and production of free acetate in the human liver. J. Clin. Invest. 41: 955. 18. Katz, J., and I. L. Chaikoff. 1955. Synthesis via the Krebs' cycle in the utilization of acetate by rat liver slices. Biochim. Biophys. Acta 18: 87. 19. Lindeneg, 0., K. Mellemgaard, J. Fabricius, and F. Lundquist. 1964. Myocardial utilization of acetate, lactate and free fatty acids after ingestion of ethanol. Clin. Sci. 27: 427. 20. Westheimer, F. H., H. F. Fisher, E. E. Conn, and B. Vennesland. 1951. The enzymatic transfer of hydrogen from alcohol to DPN. J. Amer. Chern. Soc. 73: 2403. 21. Lundquist , F., U . Fugmann, E. Kl aning, and
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PROGRESS IN GASTROENTEROLOGY
hepatic and plasma lipids and hepatic glycogen from glycerol-Cu. Life Sci. 2: 717. 36. Lieber, C. S., and R. Schmid. 1961. The effect of ethanol on fatty acid metabolism: stimulation of hepatic fatty acid synthesis in vitro. J. Clin. Invest. 40: 394. 37. Majchrowicz, E. 1964. Failure of ethanol to stimulate incorporation of acetate-l-C" into hepatic fatty acids in vitro. Proc. Soc. Exp. BioI. Med.115: 615. 38. Wakil, S. J. 1961. Mechanism of fatty acid synthesis. J. Lipid Res. 2: 1. 39. Harlan, W. R., Jr., and S. J. Wakii. 1962. The pathways of synthesis of fatty acids by mitochondria. Biochem. Biophys. Res. Commun. 8: 131. 40. Lamdin, E., W. W. Shreeve, N. Oji, and I. L. Schwartz. 1965. Transfer of tritium from succinate and other carbohydrates into liver fatty acids of mice in vivo. Fed. Proc. 24: 343. 41. Rognstadt, R., and J. Katz. 1965. Metabolism of tritium labeled glucose by adipose tissue. Fed. Proc. 24: 291. 42. Howard, C. F., and J. M. Lowenstein. 1964. The effect of a-glycerophosphate on the microsomal stimulation of fatty acid synthesis. Biochim. Biophys. Acta 84: 226. 43. Forsander, O. A., N. Raiha, M. Salaspuro, and P. Maenpaa. 1965. Influence of ethanol on the liver metabolism of fed and starved rats. Biochem. J. 94: 259. 44. Fondal, E., and C. D. Kochakian. 1951. In vitro effect of ethyl alcohol on respiration of rat liver and kidney slices. Proc. Soc. Exp. BioI. Med. 77: 823. 45. Freinkel, N., A. K. Cohen, R. A. Arky, and A. E. Foster. 1965. Alcohol hypoglycemia: II. A postulated mechanism of action based on experiments with rat liver slices. J. Clin. Endocr. 25: 76. 46. Buttner, H., F. Portwich, and K. Engelhardt. 1961. Der DPN+- und DPN-H-Gehalt der Rattenleber wahrend des Abbaues von Athanol und seine Beeinflussung durch Sulfonyl-harnstoff und Disulfiram. N aunyn Schmiedeberg Arch. Exp. Path. 240: 573. 47. Lieber, C. S., and L. M. DeCarli. 1964. Effect of ethanol on cholesterol metabolism. Clin. Res. 12: 274. 48. Westerfeld, W. W., E. Stolz, and R. L. Berg. 1943. The coupled oxidation-reduction of alcohol and pyruvate in vivo. J. BioI. Chern. 149: 237. 49. Seligson, D., S. S. Waldstein, B. Gige, W. H. Mekowey, and V. M. Sborov. 1953. Some metabolic effects of ethanol in humans. Clin. Res. 1: 86.
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activity in acute and chronic alcoholism. J. A. M. A. 168: 156. Galambos, J. T., M. Asada, and J. Z. Shanks. 1963. The effect of intravenous ethanol on serum enzymes in patients with normal or diseased liver. Gastroenterology 44: 267. Childs, A. W., R. M. Kivel, and A. Lieberman. 1963. Effect of ethyl alcohol on hepatic circulation, sulfobromophthalein clearance, and hepatic glutamic-oxalacetic transaminase production in man. Gastroenterology 45: 176. Tremolieres, J., and L. Carre. 1961. Production d'activites peroxydasiques dans Ie plasma de l'alcoolique par ingestion d'ethanol. C. R. Soc. BioI. (Paris) 155: 1022. Wartburg, J. P., and M. Roethlisberger. 1961. Enzymatische Veranderungen in der Leber nach langdauernder Belastung mit Aethanol und Methanol bei der Ratte. Helv. Physiol. Acta. 19: 30. Lieberman, F . L. 1963. The effect of liver disease on the rate of ethanol metabolism in man. Gastroenterology 44: 261. Asada, M., and J . T. Galambos. 1963. Liver disease, hepatic alcohol dehydrogenase activity, and alcohol metabolism in the human. Gastroenterology 45: 67. Pietz, D . G., B. D. Rosenak, and R. N. Harger. 1960. Alcohol metabolism in hepatic dysfunction. Amer. J. Gastroent. 34: 140. Mikata, A., A. A. Dimakulangan, and W. S. Hartroft. 1963. Metabolism of ethanol in rats with cirrhosis. Gastroenterology 44: 159. Figueroa, R: B., and A. P. Klotz. 1962. Alterations of alcohol dehydrogenase and other hepatic enzymes following oral alcohol intoxication. Amer. J. Clin. Nutr. 11: 235. Dajani, R. M., J. Danielski, and J. M. Orten. 1963. The utilization of ethanol. II. The alcohol-acetaldehyde dehydrogenase systems in the livers of alcohol-treated rats. J. Nutr. 80: 196. McClearn, G. E., E. L. Bennett, M . Hebert, R. Kakihana, and K. Schlesinger. 1964. Alcohol dehydrogenase activity and previous ethanol consumption in mice. N ature (London) :203: 793. Figueroa, R. B ., and A. P. Klotz. 1964. The effect of whiskey and low-protein diets on hepatic enzymes in rats. Amer. J. Dig. Dis. 9: 121. Figueroa, R. B., and A. P. Klotz. 1962. Alterations of liver alcohol dehydrogenase and
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other hepatic enzymes in alcoholic cirrhosis. Gastroenterology 43: 10. 146. Schwarz mann, V., C. Julien, P. Borenstein, J. Eteve, and N . Berthaux. 1962. L'alcooldeshydrogenase Mpatique chez les alcooliques. Rev. Franc. Etud. Clin. BioI. 7: 762. 147. Henley, K. S., H . S. Wiggins, B. 1. Hirschowitz, and H . M . Pollard. 1958. The effect
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GASTROENTEROLOGY
Copyright © 1966 by The Williams & Wilkins Co
PROGRESS IN GASTROENTEROLOGY HEPATIC AND METABOLIC EFFECTS OF ALCOHOL CHARLES
S.
LIEBER,
M.D.
Liver Disease and Nutrition Unit, Second (Cornell) Medical Division, Bellevue Hospital, and the Department of Medicine, Cornell University ilJedical College, New York, New York
For many years the disorders observed in association with alcoholism, especially diseases of the liver, have been attributed to nutritional deficiencies accompanying alcoholism rather than to alcohol itself.! Recent studies, however, indicate that in addition to dietary deficiencies, alcohol per se exerts a number of direct effects on hepatic and intermediary metabolism. 2 - 4 These effects of alcohol have been linked, in part, to alterations in biochemical pathways produced by the metabolism of alcohol in the liver.3 The hepatic metabolism of alcohol and the associated changes in intermediary metabolism will therefore be briefly summarized prior to a review of the pathological changes which can be attributed to direct effects of alcohol on the liver. (Alcohol and ethanol are used synonymously in this paper.) A. Metabolism of Ethanol Ethanol, which is readily absorbed from the gastrointestinal tract, is oxidized, mainly in the liver, to the extent of 90 to 98%.5 Trace amounts of ethanol may also be synthesized endogenously.6 It is generally accepted that the metabolic pathway for disposal of alcohol inAddress requests for reprints to: Dr. Charles S. Lieber, Second (Cornell) Medical Division, Bellevue Hospital, New York, New York 10016. Preparation of this manuscript was supported in part by Research Grants AM-062S4 and AM-09536 from the United States Public Health Service, National Institute of Arthritis and Metabolic Diseases. Dr. Lieber is recipient of United States Public Health Service Research Career Development Award K3-AM-22,590 from the National Institute of Arthritis and Metabolic Diseases.
119
volves oxidation to acetaldehyde (fig. 1), followed by conversion to acetyl coenzyme A (acetyl-CoA). The initial oxidation of ethanol to acetaldehyde is catalyzed by a zinc-containing enzyme,1 alcohol dehydrogenase (ADH) , which has been isolated in pure form from the soluble fraction of the liver cells ;s, 9 other tissues oxidize only small amounts of alcohol, although more recently it has been pointed out that the gastrointestinal tract may play a larger part than has been hitherto suspected. 10 Most studies on liver ADH have been conducted on the horse enzyme; human liver ADH, which has been recently isolated,1 has similar properties, but a wider substrate specificity, including methanol, which cannot be oxidized by the horse enzyme. 7 This offers a satisfactory explanation for the successful use of ethanol in the treatment of human methanol toxicity, which had remained unexplained on the basis of the horse enzyme data. Similarly, ethylene glycol is also a substrate for human ADH, and ethanol in high concentration prevents its oxidation and toxicity.7 The acetyl-CoA which results from the metabolism of alcohol is, to a large extent, oxidized to carbon dioxide through the citric acid cycle; ethanol-C14 can also be traced to fatty acids, cholesterol, glycerol, glycogen, amino acids, and protein. 3 , 11-16 Acetate, which results either from the breakdown of acetyl-CoA or originates directly from acetylaldehyde, has been found to increase in the blood after ethanol administrationP Although in vitro the liver can readily utilize acetate, in vivo acetate is largely metabolized in peripheral tissues.lS, 19
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HYPERURICEMIA SOLUBLE CYTOPLASM MITOCHONDRION ETHANOL
NAD
.............. -.--.--Ace"'[CoA
10 \. , l c~1~6c
~
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\
I ~ ~.
'i:'"
ACETALDEHYDE
CYCLE
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ELECTRON
TRANSPORT
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-----
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.1
.... -.:'------------------- ... ---------------.,...-----------------~,'
FIG. 1. Hepatic oxidation of ethyl alcohol and some of its effects, through increases (-) or decreases (--.. ) of metabolic pathways.
In addition to producing acetyl-GoA and acetate, the oxidation of ethanol also results in the transfer of hydrogen to nicotinamide adenine dinucleotide (NAD, previously called DPN), which is reduced to NADH2 (fig. 1) ,20,21 NADH2 is also generated by the oxidation of various other substrates such as glucose. In contrast to glucose, the extrahepatic metabolism of ethanol, although shown to occur,22,23 is smal1. 24 ,25 This "obligatory" hepatic metabolism results in a high rate of local NAD to NADH2 conversion, reflected by an increase in the NADH2/NAD ratio following ethanol administration. 26 , 27 These measurements of total hepatic NADH2/NAD, however, do not take into account cellular compartmentalization and give only an attenuated picture of the NADH2/ NAD change in the soluble cytoplasmic compartment, where the oxidation of ethanol takes place. 8, 9 At the present time, this ratio cannot be measured directly, but can be assessed indirectly by the ratio of metabolites whose reduction is coupled with oxidation of ethanol , such as pyruvate-lactate. NADH 2 , which results from the metabolism of ethanol, can be reoxidized by vari-
ous reactions (fig. 1). A major system for NADH2 oxidation (the flavoprotein-cytochrome system), however, is localized within the mitochondria,28 while ADH is part of the soluble cytoplasmic fraction of the hepatic cell. 8, 9 Furthermore, the mitochondrial membrane appears to be relatively impermeable to NADH2 .28,29 If the mitochondrial flavoprotein-cytochrome system has to take part in the reoxidation of NADH2 produced in the soluble compartment, it is likely that some intermediate carrier, for instance ,B-hydroxybutyrate30 or a-glycerophosphate,31.33 transports hydrogen from the NADH2 of the soluble cytoplasmic compartment to the mitochondrial flavoprotein- cytochrome system. Alcohol oxidation was indeed found to be accompanied by an increase in hepatic IXglycerophosphate,34 which could contribute to the accumulation of hepatic triglyceride through enhanced production of the glycerol moiety of triglyceride. Recent data indicate, however, that in this respect glycerol from extrahepatic sources may play a more important role than glycerol formed in the liver.35 Theoretically, increased fatty acid syn-
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PROGRESS IN GASTROENTEROLOGY
thesis from acetyl-CoA could be considered as another means for disposing of an excess of hydrogen, and indeed, NADH 2-generating systems such as ethanol or sorbitol were found in liver slices to increase the incorporation of acetate-C14 into fatty acids, relative to the C 14 0 2 produced. These observations probably indicate both increased fatty acid synthesis and diminished citric acid cycle activity resulting from a change in hepatic NADH2/ NAD ratio on ethanol oxidation. 36 In these experiments, simultaneous measurement of C 14 0 2 was needed to assess fatty acid labeling independent of the dilution of the labeled precursor which varies with different substrates and substrate concentrations. When, in the absence of C 14 0 2 determinations, no correction was made for isotopic dilution, the ethanol effect was not observed. 37 Ethanol could affect activity of the citric acid cycle and fatty acid metabolism directly through enhanced production of NADH2 which, as well as NADPH 2 (reduced nicotinamide adenine dinucleotide phosphate, previously called TPNH) , stimulates fatty acid synthesis. 3S , 39 Ethanol may also act via NADPH 2 through transhydrogenation from NADH 2; it must be pointed out, however, that recent studies have indicated that NADH2 is as great or sometimes an even greater source of hydrogen for fatty acid synthesis than NADPH 2 .40, 41 Ethanol could also act via an increase of a-glycerophosphate which was found to accumulate on alcohol ingestion 34 and to stimulate fatty acid synthesis. 42 Reduced citric acid cycle activity may be responsible for decreased fatty acid oxidation produced by ethanol as evidenced by parallel reduction in C14 0 Z production from both C14-palmitate and C14-acetate.36 A diminution in citric acid cycle activity has been recently confirmed by the observation of a reduction in the absolute amount of CO 2 produced in liver perfused with ethanol, without change in oxygen consumption. 43 No reduction in hepatic oxygen consumption with ethanol has been observed in vitr0 43 - 45 and also in vivo in men undergoing hepatic vein catheterizationP Hepatic adenosine triphosphate (ATP) 46 was unaffected by ethanol, suggesting that de-
121
spite decreased citric acid cycle activity, energy production continues unharmed with presumably ethanol providing the reducing equivalents normally arising from oxidation of other substrates primarily through the citric acid cycle (fig. 1). It has also been found recently that ethanol produces hepatic cholesterol accumulation and increases incorporation of acetate-C14 into cholesterol.47 Theoretically, increased cholesterogenesis could also be viewed as another means for disposing of excess hydrogen produced on ethanol oxidation. In addition to increased lipogenesis, other pathways can serve as hydrogen acceptors in the soluble cytoplasmic compartment. For instance, the oxidation of ethanol to acetaldehyde can be coupled with the reduction of pyruvate to lactate,21, 48 NADH2 acting as intermediate hydrogen carrier (fig. 1). The conversion of pyruvate to lactate leads to increased lactate formation in the liver and increased lactate/ pyruvate ratio in the blood 49 , 50 or, in vitro, in liver incubation media. 43 This channeling of pyruvate to lactate is associated with decreased gluconeogenesis with ethanol which appears to play a role in the hypoglycemia occurring when ethanol administration is superimposed upon glycogen depletion such as results from prolonged fasting. 45 • 51. 52 Furthermore, NADH2 has been shown to produce a dissociation of glutamic dehydrogenase into inactive subunits 53 which may also explain reduction of gluconeogenesis from amino acid metabolism. In addition to hypoglycemia, however, hyperglycemia has been described in asso ciation with alcoholism. The mechanism of the hyperglycemia is still largely not understood; pancreatitis and disturban ces in catecholamine metabolism presumably play a role.3 In addition to its possible role in alcoholic hypoglycemia, the increased hepat ic conversion of pyruvate to lactate leads to en hanced blood lactate concentration,49. [;0 , 54 which produces a diminution in urin ary uric acid output; this explains, at least in part, the observed hyperuricemia in subjects intoxicated with alcoholic beverages55 (fig. 1). The hyperuricemia produced by a lcohop5
PRCGRESSDVGASTROENTEROLOGY
122
~
:.ill!!fffCHYLOM\~S 1)I)jJ1i)f
HE/PATIC LIPOGENESIS
"
)ljJJt!!I_FFA--+-FATTY LIVER
F
,/\,
'J/j
ADIPOSE TISSUE
'
,/
~' LIPOPROTEINS
"
'"
~ C02
FIG. 2. Schematic representation of the three major sources (-+) and the two principal modes of disposition (--7) of the fat accumulating in alcoholic fatty liver.
can be potentiated by fasting 56 which, in itself, is known to cause hyperuricemia. 57 Further effects possibly related to the hyperlactidemia include increased urinary magnesium excretion,58,59 and possibly increased splanchnic blood flOW,60 which has been described with large doses of ethanol,50. 60. 61 but not with small amounts. l7 • 62 Among the alterations produced by ethanol that can be attributed to the NADH 2/ NAD change, one should list decreased removal of galactose from the blood, resulting from the inhibition of uridinephosphategalactose-4-epimerase by NADH 2 63-65 and decreased urinary excretion of 5-hydroxyindole acetic acid (5-HIAA), presumably because of diminished oxidation of serotonin to 5-HIAA through competition for NAD.66 B. Pathogenesis of Alcohol Fatty Liver
The problem of whether the fatty liver observed after prolonged alcohol intake is due only to nutritional deficiencies associated with alcohol, or whether it is also caused by alcohol itself,!' 67-69 has been recently settled: subjects with a history of alcoholic fatty liver, but whose hepatic morphology had returned to normal, developed a fatty liver with the ingestion of ethanol, either in addition to, or as isocaloric substitution for, carbohydrate in an otherwise adequate diet. 70, 71 Similar effects were observed in rats given alcohol as part of a nutritionally adequate liquid diet. 71 When calories given as ethanol (or as carbohydrate in the control diet) were either
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omitted or isocalorically replaced with fat, no fatty liver was produced, indicating that the alcoholic fatty liver was not simply the consequence of a lack of carbohydrate or of available calories but resulted from alcohol per se, whose capacity for producing a fatty liver was found to be greater than that of fat itself.71 These studies demonstrate that in addition to nutritional deficiencies associated with alcoholism, alcohol itself has to be considered as a direct etiological agent in the pathogenesis of the fatty liver produced by prolonged alcohol intake. Lipids which accumulate in the liver can originate from three main sources: (a) dietary fat, which reaches the blood stream from the gut as chylomicrons; (b) adipose tissue fatty acids, which are transported to the liver as free fatty acids (FFA); and (c) lipids synthesized in the liver itself (fig. 2). Hepatic steatosis can result either from an excessive supply from one or more of these three sources, or from a disturbance of lipid disposition from the liver through reduced lipid oxidation, insufficient lipoprotein formation, or excretion into the blood (fig. 2). Dietary lipids. The importance of dietary fat in the pathogenesis of the alcoholic fatty liver has recently been brought into focus with the observation that both in man and in rat, fatty acids which accumulate in the liver on prolonged intake of alcohol and fat-containing diets are, to a large degree, dietary in origin. 72 -74 The role of dietary lipids has been further emphasized by the fact that prolonged ingestion of alcohol with low fat diets led to less hepatic steatosis than equal amounts of alcohol in isocaloric diets with a fat content equivalent to the average U. S. diet73 , 74 (fig. 3). There was no significant effect of alcohol on intestinal lipid absorption, hepatic chylomicron uptake, or hepatic lipoprotein formation and release, while decreased hepatic oxidation of dietary lipids appeared as the most likely explanation for the observed accumulation of dietary fat upon prolonged alcohol ingestion. n . 74 Adipose tissue lipids. After the administration of a very large single dose of alcohol (6 to 9 g per kg) by gastric tube to fast-
123
PROGRESS I N GASTROENTEROLOGY
January 1966
~
HEPATIC LIPIDS IN 44 RATS GIVEN OVER 24 DAYS CONTROL SUCROSE DIET OR _ ISOCALORIC ETHANOL DIET TOTAL
LIPIDS
TRIGLYCERIDES
fat-conta i ning diets 143'4 col)
140
120
low fat diets
W 100
12'4 col)
fat-containing diets
~
.,
.~
:J
(43% col)
80
0;
.! E
0'
"-
low fat diets 12'4 co I )
60
0'
E
40
20
o
p
p
p
p
FIG. 3. Comparison of hepatic steatosis produced with diets of varying fat but constant ethanol content (from C. S. Lieber et al. 1966. Role of dietary, adipose and endogenously synthesized fatty acids in the pathogenesis of the alcoholic fatty liver. J . Clin. Invest. 1,5: Jan.) .
ing rats, adipose tissue fatty acids were found t o accumulate in the liver. 73 - 77 This was considered to be due to increased peripheral fat mobilization, as under these experimental conditions, circulating FF A were found to be increased. 75 • 76. 78 Under apparently similar experimental conditions, however, other investigators found no rise in circulating FF A79 and no evidence of enhanced mobilization of fatty acids from labeled fat pads. so The latter experiments led to the conclusion that even after the administration of a single large dose of ethanol, decreased hepatic metabolism of
fatty acids (mobilized from adipose tissue at a normal rate) rather than increased peripheral fat mobilization is responsible for the hepatic steatosis. so Fat accumulation in the liver after a single large dose of ethanol was found to be moderate, and less in male than in female rats. Total lipids in male rats increased only about 20% 16 hr after the administration of 7.5 g per kg of ethanol by gastric tube. 74 The cause for this sex difference is not known; it could be related to the fact that female rats have more liver alcohol dehydrogenase than males. 9
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In man, large amounts of alcohol (400 g per day) were found to raise circulating FF A, but with more moderate quantities of ethanol, up to 300 g per day for as long as 18 days, circulating FFA remained unchanged (fig. 4).70, 81 Fatty liver was nevertheless observed with these moderate amounts of ethanoFo, 71 and it contained only a small fraction of adipose tissue fatty acids.72,73 In acute experiments in men (1 to 10 hr), ethanol decreased circulating FFA,82-84 with a reduction of FF A turnover.85 Thus, increased peripheral fatty acid mobilization has only been shown to play a role when very large amounts of ethanol are administered; even under these experimental conditions, the relative importance of enhanced peripheral mobilization versus decreased hepatic lipid metabolism has not been fully clarified. Lipids synthesized in the liver. In rats given low fat diets (2% of calories), the same amounts of alcohol produced less hepatic fat accumulation than in rats fed isocaloric diets containing 43% of calories as fat (fig. 3) the fatty liver had also a fatty acid composition very different from adipose tissue, with a large percent of endogenously synthesized fatty acids (such as palmitic acid), most likely produced in the liver itself.72-74 It is likely that the accumulation of endogenously synthesized fatty acids results from a combination of decreased oxidation and increased synthesis of hepatic fatty acids, effects which alcohol has been shown to produce in vivo and in vitro.36, 86, 87 The mechanism of this has been discussed above (section A). The major lipid fraction of the alcoholic fatty liver is triglyceride. The reason for the selective accumulation of triglyceride is not known. This could result from a specific stimulation of triglyceride production ,75, 77 with possibly a relative block in phospholipid synthesis. 75 Disturbances in lipoprotein metabolism and alcoholic hyperlipemia. Theoretically, a substantial interference with the hepatic mechanism for lipoprotein formation or release could lead to excessive accumulation of fat in the liver (fig. 2). Decreased hepatic lipoprotein release has been proposed as an explanation for the fatty liver,
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the decrease in circulating lipids, and the reduction in post-Triton hyperlipemia induced by carbon tetrachloride poisoning. s8 On the basis of hepatic perfusion studies, a similar mechanism has been proposed for the alcoholic fatty liver.89 In contrast to the carbon tetrachloride poisoning and the in vitro perfusion of liver with large amounts of ethanol, in vivo administration of alcohol was not accompanied by a fall, but rather produced an increase in blood lipids, especially triglycerides, both in man 70, 8], 83 and in experimental animals,2, 70, 90 with the concomitant development of a fatty liver. 70, 71 Furthermore, post-Triton hyperlipemia was found to be unaffected by ethanol. 91 ,92 These findings suggest that decreased transport of lipids from the liver is not the primary factor in the production of the alcoholic fatty liver, if one assumes no major change in peripheral fat utilization. The pathogenesis of alcoholic hyperlipemia, however, has not been fully clarified. Decreased lipolysis may playa role in the rare individuals suffering from marked hyperlipemia, the so-called "Zieve's Syndrome." 93-95 This is suggested by the observations, in these subjects, of low lipoprotein lipase activity,95 circulating inhibitor of lipoprotein lipase,96 and accumulation of a-chylomicrons. 97 Delayed clearing of postprandial hyperlipemia due to ethanol has also been reported in man98 and in rats. 99 Furthermore, it must be pointed out that, in addition to the production of lipoproteins, the liver is also responsible for the removal of an important fraction of the circulating lipoproteins; theoretically, alteration of this function by alcohol, possibly in association with steatosis, could also lead to hyperlipemia. Although the association of alcoholism and lactescent serum has been described by various authors, patients with alcoholic fatty liver do not necessarily present with hyperlipemia. On administration of alcohol, an increase in blood lipids was observed only with large doses, larger than necessary for the production of fatty liver. 7o ,71 In addition to its dependence on the amount of alcohol ingested, the hyperlipemia may also be caused or potentiated by factors other than alcohol, such as pan-
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125
PROGRESS IN GASTROENTEROLOGY 500 GLYCERIDES
400 300
~HOLIPIDS~
200
CHOLESTEROL
~
mg%
400 mg% 300
=~
mg%
200 100
o
FIG. 4. Effect of prolonged alcohol intake on serum lipids in seven chronic alcoholic individuals (average results ± SE of the mean) (from C. S. Lieber et al. 1963. Fatty liver, hyperlipemia and hyperuricemia produced by prolonged alcohol consumption, despite adequate dietary intake. Trans. Ass. Amer. Physicians 76: 289).
creatitis,lOO or an unusually low lipoprotein lipase activity.95,96 Furthermore, when seven chronic alcoholic volunteers were given up to 300 g of alcohol per day for 18 days, hyperlipemia was found during the first 10 days only, with return toward normal thereafter, despite continuation, at least initially, of the same alcohol and dietary intake (fig. 4) .70, 81 This indicated that duration of alcohol ingestion may also be one of the crucial factors determining whether or not hyperlipemia will be seen in alcoholics. The reason for the disappearance of the hyperlipemia on continuous alcohol intake is not clear; this could be related to progressive deterioration of liver function. C. Prevention of Alcoholic Fatty Liver In experimental animals, the fatty liver produced by small amounts of alcohol in conjunction with diets deficient in lipotro-
pic agents can be prevented by simple correction of the dietary deficiency.1, 69 Lipotropic agents were found to be only partially effective 101 or even totally ineffective I02 , 103 in preventing fat accumulation resulting from the administration of a single large dose of alcohol by gastric tube. Similarly, fatty livers were produced in rats on prolonged alcohol intake despite diets containing adequate amounts of lipotropic agents (0.25 mg of choline chloride and 1.5 mg of DL-methionine per cal) ,70,71 even when supplemented with an additional 1.25 mg per cal of methionine,t04 an amount previously shown to protect against alcoholic fatty liver produced on deficient diets.I,69 Supplementation with larger amounts of methionine (2.5 mg per cal) or choline chloride (up to 5 mg per cal) resulted in a decrease of fat accumulation produced by alcohol; the protection was incomplete and variable, with some animals
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PROGRESS IN GASTROENTEROLOGY
~howing no protection while others had a marked reduction in steatosis (C. S. Lieber and L. M. DeCarli, unpublished data). The fat which accumulates in the liver of rats after the administration of one large dose of alcohol was found to be reduced or even prevented by asparagine,t°5 or various antioxidants, such as vitamin E, N-N-diphenyl-p-phenylenediamine or G-50 (Griffith Laboratories, Chicago, Ill.) .106,107 These substances, as well as large amounts of vitamin K, methylene blue, cyanocobalamin, and inositol were found to be ineffective in the prevention of the fatty liver produced by prolonged alcohol intake, when the diet was otherwise adequate (C. S. Lieber and L. M. DeCarli, unpublished data). The injection of massive amounts of ATP has been reported to decrease hepatic fat accumulation after a single large dose of ethanol, possibly through hypothermia and shock. 108 The fatty liver produced by prolonged alcohol ingestion was reduced by decreasing the fat content of the diet, both in man and in experimental animals,73,74 underlining the important role of dietary lipids in the pathogenesis of the alcoholic fatty liver. A similar effect was obtained by replacing dietary fat with MCT (Drew Chemical Corp., Boonton, N. J.), a mixture of medium chain triglycerides (C. S. Lieber and L. M. DeCarli, unpublished data).
D. "Alcoholic Hepatitis" and Cirrhosis
In contrast to the problem of the fatty liver, not much progress has been made in unraveling the pathogenesis of alcoholic cirrhosis. It is still not clear which hepatic lesions have to be considered as precursors of the cirrhosis, for instance, whether alcoholic fatty liver per se leads to cirrhosis, such as has been described for experimental choline deficiency,l°9 or whether, independent of the fatty liver or in conjunction with it, other lesions are necessary. In addition to fat accumulation, alcoholic individuals may have a variety of hepatic lesions which have been grouped under the terms "alcoholic hepatitis," 110-115 "acute fatty metamorphosis of the liver," 116 or "acute hepatic insufficiency of the
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chronic alcoholic." 117 It is characterized by intensified drinking usually in a chronic alcoholic, leading to anorexia, nausea, vomiting, upper abdominal pain, hepatomegaly and, frequently, fever and jaundice. Biochemical tests are not characteristically altered, though they may suggest liver cell damage with sometimes a confusing obstructive element. Anemia is usually present and a severe leukocytosis may develop. In addition to marked fatty changes, the histological picture is characterized by local and massive cellular degeneration and necrosis, with acute inflammatory reaction. On electron microscopy, the mitochondria appear much enlarged, and clumped. Clumping of the mitochondria and giant forms has been proposed as a morphological basis for the "alcoholic hyaline bodies" of Mallory.109, 114, 118 In other electron microscopic studies, however, the hyaline material was considered to consist of altered endoplasmic reticulum.n9, 120 Among various nonspecific mitochondrial alterations described in alcoholic fatty liver, the most conspicuous was the presence of crystal-like inclusions resembling "myelin degeneration" often accompanied by marked mitochondrial enlargement.114.121 In rats, acute administration of a very large dose of alcohoF22 or prolonged intake of moderate amounts of alcohoF23. 124 also leads to mitochondrial swelling and deformation with a reduction in the capacity of the mitochondria to carry out oxidations. 125 The mitochondrial lesion is preceded by an alteration of the endoplasmic reticulum characterized by disappearance of the normal parallel arrays and their replacement by vesicular s1;ructures.123 Autoradiographic techniques, employing tritiated thymidine to evaluate hepatic deoxyribonucleic acid (DNA) synthesis, indicated that hepatic cell damage produced by ethanol is accompanied by an increase in DNA synthesis which is primarily found in mesenchymal and ductular cells, and appears to be correlated with hepatic necrosis and inflammation, rather than with fat accumulation. 126 Which of the morphological lesions described above has to be considered as "the
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PROGRESS IN GASTROENTEROLOGY
precursor" to cirrhosis remains obscure; as for the fatty liver, it is possible that various factors may be involved, especially in view of the fact that alcoholic cirrhosis appears to comprise a heterogeneous group, with both portal and "postnecrotic" forms of cirrhosis. 127 It has been shown recently that, once cirrhosis was established, but provided an adequate protein ingestion was maintained, the intake of alcoholic beverages in amounts less than spontaneously consumed had no obvious adverse effect on the cirrhosis, though some fat infiltration was observed. 128 The significance of this observation is hard to assess in view of the difficulty of evaluating the progress of cirrhosis over relatively short periods of time in human biopsy material. It focuses our attention, however, on the importance of the dose of alcohol ingested and the possible role of factors other than alcohol, such as malnutrition. Furthermore, possible hepatotoxic roles of nonalcoholic components of alcoholic beverages must be considered. For instance, wine of certain vintages has been shown to contain large amounts of iron129-131 which was found to accumulate in liver and other organs of experimental animals given such wine over long periods of time ;129 this could also explain, at least in part, iron accumulation in various tissues (including the liver) of wine drinkers with cirrhosis of the liver.129-131 E. Effects of Alcohol on Varions Enzymes
In man, alcohol was found both to increase 132 or not to change 133 , 134 various serum enzymes, especially transaminases; in the former study alcohol was given orally, while in the latter studies it was administered intravenously. In rats, serum 135 and liver 136 catalase increased with alcohol administration. In patients with cirrhosis, the clearance of alcohol from the blood was found to be either normal 137 , 138 or reduced. 139 These results can presumably be reconciled on the basis of the severity of liver disease, the metabolism of alcohol being affected only by very advanced hepatic damage. 137 The rate of alcohol metab-
127
olism was found to be related to the ADH activity in the liver of rats,140 while no such correlation was found in human liver.138 In animals, ADH activity in the liver increased with alcohol ingestion,141-143 but on prolonged alcohol intake, after an initial rise, there was ultimately a decrease in ADH activity141, 142 potentiated by a low protein diet,144 In patients with alcoholic liver disease, hepatic ADH145, 146 and isocitric dehydrogenase 145 were found to be reduced. Hepatic glutamic-pyruvic transaminase was also found to be decreased in rats given ethanol.147 Despite these enzyme changes, hepatic protein synthesis, as assessed by amino acid incorporation, did not appear to be decreased.Hs In conclusion, a number of pathological effects of ethanol remain unexplained, especially the chain of events which leads to cirrhosis. Studies in recent years, however, have indicated that in addition to acting via nutritional deficiencies, ethanol per se has to be considered as a direct hepatotoxic agent, as evidenced by production of fatty liver, hyperlipemia, and various enzyme changes. A number of metabolic effects of ethanol (involving lipid, glucose, galactose, uric acid, and serotonin metabolism) have been given a satisfactory explanation on the basis of a single biochemical defect, namely, the increase of NADH2/ NAD ratio in the cytoplasmic compartment of the hepatic cell, which may represent the most characteristic change produced by ethanol. REFERENCES 1. Best, C. H ., W. S. Hartroft, C. C. Lucas, and J. H. Ridout. 1949. Liver damage produced by feeding alcohol or sugar and its prevention by choline. Brit. Med. J . 2: 4635 . 2. Klatskin, G. 1961. Alcohol and its relation to liver damage. Gastroenterology 41: 443. 3. Lieber, C . S., and C. S. Davidson. 1962. Some metabolic effects of ethyl alcohol. Amer. J. Med . 33: 319. 4. Isselbacher, K. J., and N. J. Greenberger. 1964. M etabolic effects of alcohol on the liver. N ew Eng. J. Med. 270: 351. 5. Thompson, G. N. 1956. Alcoholism. Charles C Thomas, Publisher, Springfield, Ill .
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6. Lester, D. 1961. Endogenous ethanol: A review. Quart. J . Stud. Alcohol. 22: 554. 7. von Wartburg, J. P., J. L. Bethune, and B. L. Vallee. 1964. Human liver-alcohol dehydrogenase. Kinetic and physicochemical properties. Biochemistry 3: 1775. 8. Nyberg, A., J. Schuberth, and L . Anggard. 1953. On the intracellular distribution of catalase and alcohol dehydrogenase. Acta Chern. Scand. 7: 1170. 9. Buttner, H. 1965. Aldehyd- und alkoholdehydrogenase-aktivitat in Leber und Niere der Ratte. Biochem. Z. 341: 300. 10. Spencer, R. P., K. R Brody, and B. M. Lutters. 1964. Some effects of ethanol on the gastrointestinal tract. Amer. J. Dig. Dis. 9: 599. 11. Forsander, O. A., and C. R. Riiihii. 1960. Metabolites produced in the liver during alcohol oxidation. J. BioI. Chern. 235: 34. 12. Schulman, M. P., R Zurek, and W. W. Westerfeld. 1957. The pathway of alcohol metabolism. In Alcoholism, publ. 47, p. 29. American Association for the Advancement of Science, Washington, D . C. 13. Curran, G. L., and D. Rittenberg. 1950. The role of ethyl alcohol in the biological synthesis of cholesterol. J. BioI. Chern. 190: 17. 14. Smith, M. E., and H. W. Newman. 1960. Ethanol-1-C" and acetate-1-C" incorporation into lipid fractions in the mouse. Proc. Soc. Exp. BioI. Med.l04: 282. 15. Russell, P. T., and J. T. Van Bruggen. 1964. Ethanol metabolism in the intact rat. J. BioI. Chern. 239: 719. 16. Schiller, D., N. Burbridge, V. C. Sutherland, and A. Simon. 1959. The conversion of labeled ethanol to glycerol and glycogen. Quart. J. Stud. Alcohol. 20: 432. 17. Lundquist, F., N. Tygstrup, K. Winkler, K. Mellemgaard, and S. Munck-Peterson. 1962. Ethanol metabolism and production of free acetate in the human liver. J. Clin. Invest. 41: 955. 18. Katz, J., and I. L. Chaikoff. 1955. Synthesis via the Krebs' cycle in the utilization of acetate by rat liver slices. Biochim. Biophys. Acta 18: 87. 19. Lindeneg, 0., K. Mellemgaard, J. Fabricius, and F. Lundquist. 1964. Myocardial utilization of acetate, lactate and free fatty acids after ingestion of ethanol. Clin. Sci. 27: 427. 20. Westheimer, F. H., H. F. Fisher, E. E. Conn, and B. Vennesland. 1951. The enzymatic transfer of hydrogen from alcohol to DPN. J. Amer. Chern. Soc. 73: 2403. 21. Lundquist , F., U . Fugmann, E. Kl aning, and
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H. Rasmussen. 1959. The metabolism of acetaldehyde in mammalian tissues. Biochern. J. 713: 409. Larsen, J. A. 1959. Extrahepatic metabolism of ethanol in man. Nature (London) 184: 1236. Forsander, 0., N. Raihii, and H. Suomalainen. 1960. Oxydation des Athylakohols in isolierter Leber und isoliertem Hinterkorper der Ratte. Hoppe Seyler Z. Physiol. Chern. 318: 1. Lundsgaard, E. 1938. Alcohol oxidation as a function of the liver. C. R. Lab. Carlsberg 22: 333. Bartlett, G. R, and H. N. Barnet. 1949. Some observations on alcohol metabolism with radioactive ethyl alcohol. Quart. J. Stud. Alcohol. 10: 381. Smith, M . E., and H . W. Newman. 1959. The rate of ethanol metabolism in fed and fasting animals. J. BioI. Chern. 234: 1544. Forsander, 0 ., N. Riiihii, and H. Suomalainen. 1958. Alkoholoxydation und bildung von Acetoacetat in normaler und glykogenarmer intakter Rattenleber. Hoppe Seyler Z. Physiol. Chern. 312: 243. Green, D. E., and F. L. Crane. 1958. Structure of the mitochondrial electron transport system, p. 275. In Proceedings of the international symposium on enzyme chemistry. Maruzen Co., Ltd., Tokyo. Lehninger, A. L. 1951. Phosphorylation coupled to oxidation of dihydrodiphosphopyridine nucleotide. J. BioI. Chern . 190: 345. Devlin, T. M" and B. Bedell. 1960. Effect of acetoacetate on the oxidation of reduced diphosphopyridine nucleotide by intact rat liver mitochondria. J. BioI. Chern. 235: 2134. Chefurk a, W. 1954. Oxidative metabolism of carbohydrates in insects. 1. Glycolysis in the housefly Musca Domestica L. Enzymologia 17: 73. Estabrook, R W., and B. Sacktor. 1958. Alpha-glycerophosphate oxidase of flight muscle mitochondria. J . BioI. Chern. 233: 1014. Bucher, T., and M. Klingenberg. 1958. Wege des Wasserstoffs in der lebendigen Organisation. Angew. Chern. 70: 552 . Nikkila, E. A., and K. Ojala. 1963. Role of hepatic L-cx-glycerophosphate and triglyceride synthesis in production of fatty liver by ethanol. Proc. Soc. Exp. BioI. Med. 113: 814. Nikkila, E. A., and K Ojala. 1963. Ethanolinduced alterations in the synthesis of
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hepatic and plasma lipids and hepatic glycogen from glycerol-Cu. Life Sci. 2: 717. 36. Lieber, C. S., and R. Schmid. 1961. The effect of ethanol on fatty acid metabolism: stimulation of hepatic fatty acid synthesis in vitro. J. Clin. Invest. 40: 394. 37. Majchrowicz, E. 1964. Failure of ethanol to stimulate incorporation of acetate-l-C" into hepatic fatty acids in vitro. Proc. Soc. Exp. BioI. Med.115: 615. 38. Wakil, S. J. 1961. Mechanism of fatty acid synthesis. J. Lipid Res. 2: 1. 39. Harlan, W. R., Jr., and S. J. Wakii. 1962. The pathways of synthesis of fatty acids by mitochondria. Biochem. Biophys. Res. Commun. 8: 131. 40. Lamdin, E., W. W. Shreeve, N. Oji, and I. L. Schwartz. 1965. Transfer of tritium from succinate and other carbohydrates into liver fatty acids of mice in vivo. Fed. Proc. 24: 343. 41. Rognstadt, R., and J. Katz. 1965. Metabolism of tritium labeled glucose by adipose tissue. Fed. Proc. 24: 291. 42. Howard, C. F., and J. M. Lowenstein. 1964. The effect of a-glycerophosphate on the microsomal stimulation of fatty acid synthesis. Biochim. Biophys. Acta 84: 226. 43. Forsander, O. A., N. Raiha, M. Salaspuro, and P. Maenpaa. 1965. Influence of ethanol on the liver metabolism of fed and starved rats. Biochem. J. 94: 259. 44. Fondal, E., and C. D. Kochakian. 1951. In vitro effect of ethyl alcohol on respiration of rat liver and kidney slices. Proc. Soc. Exp. BioI. Med. 77: 823. 45. Freinkel, N., A. K. Cohen, R. A. Arky, and A. E. Foster. 1965. Alcohol hypoglycemia: II. A postulated mechanism of action based on experiments with rat liver slices. J. Clin. Endocr. 25: 76. 46. Buttner, H., F. Portwich, and K. Engelhardt. 1961. Der DPN+- und DPN-H-Gehalt der Rattenleber wahrend des Abbaues von Athanol und seine Beeinflussung durch Sulfonyl-harnstoff und Disulfiram. N aunyn Schmiedeberg Arch. Exp. Path. 240: 573. 47. Lieber, C. S., and L. M. DeCarli. 1964. Effect of ethanol on cholesterol metabolism. Clin. Res. 12: 274. 48. Westerfeld, W. W., E. Stolz, and R. L. Berg. 1943. The coupled oxidation-reduction of alcohol and pyruvate in vivo. J. BioI. Chern. 149: 237. 49. Seligson, D., S. S. Waldstein, B. Gige, W. H. Mekowey, and V. M. Sborov. 1953. Some metabolic effects of ethanol in humans. Clin. Res. 1: 86.
129
50. Mendeloff, A. I. 1954. Effects of intravenous infusions of ethanol upon estimated hepatic blood flow in man. J. Clin. Invest. 33: 1298. 51. Field, J. B., H. E. Williams, and G. E. Mortimore. 1963. Studies on the mechanism of
52.
53.
54.
55.
56.
57.
58.
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