Experimental alcoholic hepatitis: a new primate model.

Research into the pathogenesis of alcoholic cirrhosis has suffered from the lack of an animal model of alcoholic hepatitis, considered by many to be the link between alcoholic fatty liver and cirrhosis. The entire constellation of histologic features characteristic of alcoholic hepatitis has been produced for the first time in baboons by administration of ethanol with a nutritionally adequate diet. This includes fat, necrosis. inflammation, alcoholic hyaline, fibrosis, and central hyaline sclerosis.

Hepatic collagen metabolism: effect of alcohol consumption in rats and baboons.

Long-term ethanol feeding causes collagen accumulation in livers of rats and baboons. Activity of collagen proline hydroxylase in the liver is also stimulated, and incorporation of proline into collagen hydroxyproline in rat liver slices is significantly enhanced, a result indicating that increased synthesis is responsible, in part, for the collagen accumulation.

Hepatic microsomal enzymes in man and rat: induction and inhibition by ethanol.

The feeding of ethanol increased significantly the activities of hepatic pentobarbital and benzpyrene hydroxylases in rats, and, in human volunteers, doubled pentobarbital hydroxylase activity. In vitro ethanol inhibited aniline, pentobarbital, and benzpyrene hydroxylases. These data may explain, at least in part, the increased tolerance of alcoholics to sedatives when sober, and the enhanced sensitivity to sedatives when inebriated.

Plasma alpha amino-n-butyric acid to leucine ratio: an empirical biochemical marker of alcoholism.

The plasma ratio of alpha-amino-n-butyric acid to leucine was elevated in ambulatory and hospitalized alcoholics as well as in baboons fed alcohol along with an adequate diet. There was a statistically significant positive correlation between this ratio and the degree of alcoholism assessed by three separate medical and psychological criteria in patients maintained on methadone.

Acetaldehyde oxidation by hepatic mitochondria: decrease after chronic ethanol consumption.

Prolonged consumption of ethanol significantly reduces the capacity of rat liver mitochondria to oxidize acetaldehyde. This is associated with decreased mitochondrial respiration with acetaldehyde as substrate. The reduced ability of mitochondria to metabolize acetaldehyde may explain the high levels of acetaldehyde in the blood of alcoholics, which in turn could promote the perpetuation of liver injury.

Ethanol increases hepatic smooth endoplasmic reticulum and drug-metabolizing enzymes.

Rats were fed ethanol for 2 weeks along with diets either adequate or deficient in protein and choline, the latter intake being similar to that of many alcoholics. Hepatic lipids, smooth endoplasmic reticulum, and the activities of drug-metabolizing enzymes (aniline hydroxylase and nitroreductase) were increased with the adequate diet but more so with the deficient one. These results may explain the increased tolerance by alcoholics of drugs such as sedatives.

Ethanol oxidation by hepatic microsomes: adaptive increase after ethanol feeding.

Hepatic microsomes contain an ethanol-oxidizing system distinct from alcohol dehydrogenase. In vitro, it has characteristics comparable to those of microsomal drug-detoxifying enzymes and, in vivo, it is capable of adaptation to the administration of ethanol. The existence of this microsomal ethanol-oxidizing system may explain ultrastructural, pharmacological, and biochemical effects of ethanol.

Reduced nicotinamide-adenine dinucleotide phosphate oxidase: activity enhanced by ethanol consumption.

Prolonged consumption of ethanol enhances the activities of the hepatic microsomal ethanol oxidizing system and of reduced nicotinamide-adenine dinucleotide phosphate oxidase, but not of catalase. The oxidase-catalase system is not part of the microsomal ethanol oxidizing system since catalase inhibitors dissociate ethanol oxidation by the two pathways. Enhanced reduced nicotinamide-adenine dinucleotide phosphate oxidase activity may contribute to liver injury, possibly by favoring lipoperoxidation.

Lack of correlation between hepatic mitochondrial membrane structure and functions in ethanol-fed rats.

A current hypothesis suggests that alterations in the chemical composition and the subsequent changes in the structure of the membrane could account for the functional derangements observed in the hepatic mitochondria of animals fed ethanol for extended periods. An examination of this hypothesis reveals that the liver mitochondria of ethanol-fed rats show a dissociation between the respiratory functions and the lipid composition and microviscosity of the membranes.

Acetaldehyde production and transfer by the perfused human placental cotyledon.

Fetal injury associated with maternal ethanol ingestion is a major cause of congenital anomalies and mental retardation. Studies with animals suggest that acetaldehyde, the primary hepatic oxidative metabolite of ethanol, may contribute to fetal damage. It is not known, however, whether acetaldehyde reaches the human fetus, either by placental production or transfer. Studies utilizing the perfused human placental cotyledon show that the human placenta oxidizes ethanol to acetaldehyde, releasing it into the fetal perfusate. Moreover, when acetaldehyde is present in the maternal perfusate, it is transferred to the fetal side, reaching approximately 50 percent of the maternal level. These findings suggest that the human placenta may play a pivotal role in the pathophysiology of ethanol-associated fetal injury.

Prolonged ethanol consumption increases testosterone metabolism in the liver.

Male alcoholics often suffer from features of hypogonadism related to abnormal metabolism of sex steroids. Since the activity of testosterone reductases is rate limiting for testosterone metabolism in the liver, the effect of prolonged ethanol consumption by rats and human volunteers on the activities of these microsomal and cytosolic enzymes was studied. In rats, long-term ethanol ingestion doubled microsomal 5alpha-testosterone reductase activity, a major pathway for testosterone metabolism, while in human volunteers the activity was increased two- to fivefold. These changes may play a role in the altered androgenic activity of the chronic alcoholic.

Alcoholic hepatomegaly: accumulation of protein in the liver.

The hepatomegaly that appears after long-term feeding of ethanol results in accumulation of protein that is quantitatively as important as the increase in lipid. The bulk of protein accumulated in the soluble fraction of the cell. Hepatic albumin and transferrin concentrations increase and colchicine-binding protein decreases, thus suggesting an intrahepatic retention of export proteins.

Decrease in microviscosity and cholesterol content of rat liver plasma membranes after chronic ethanol feeding.

This investigation was performed to determine whether chronic ethanol feeding alters the lipid composition or the fluidity of liver plasma membranes. Male Sprague-Dawley rats were pair-fed nutritionally adequate liquid diets containing ethanol as 36% of energy or an isocaloric amount of carbohydrate for 4-5 wk. Contrasting with other membranes, chronic ethanol feeding resulted in an increase in hepatic plasma membrane fluidity as assessed by fluorescence anisotropy. This alteration was associated with a decrease in plasma membrane cholesterol content.

Normal testicular structure and reproductive function in deermice lacking retinol and alcohol dehydrogenase activity.

It was found that a strain of deermice (Peromyscus maniculatus), which genetically lacks liver alcohol dehydrogenase activity also displays no such activity in the testis and is devoid of the enzyme activity that converts retinol to retinal, both in liver and in the testis; nevertheless, these animals exhibit normal reproduction and testicular histology. Therefore, one must reconsider the theory that the testicular atrophy and aspermatogenesis commonly found in alcoholics is due, at least in part, to interaction of ethanol with these enzyme activities in the testis.

Effect of ethanol on ketone metabolism.

Ketonuria has been observed in alcoholics. To study the mechanism of this effect, healthy, volunteers were given adequate diets (36% of calories as lipid and 15% as protein) for 18 days, with isocaloric replacement of carbohydrate (46% of calories) by either ethanol or additional fat. The latter resulted in a high fat diet, with 82% of calories as lipid. After about 1 wk of alcohol, massive and persistent ketonuria developed. Compared with the control period, there was a 30-fold increase in fasting blood acetoacetate and beta-hydroxybutyrate (P < 0.001). With the high fat diet, acetoacetate and beta-hydroxybutyrate increased 8- to 10-fold (P < 0.001). In the postprandial state, ethanol also induced hyperketonemia, but less markedly than when ethanol followed an overnight fast. With low fat diets (5% of calories), alcohol (46% of total calories) did not induce ketonuria or hyperketonemia, suggesting that a combination of alcohol and dietary fat is necessary. The addition of alcohol to rat liver slices did not affect ketogenesis. In rats pretreated with alcohol for 3 days, however, ketonemia developed, hepatic glycogen was decreased, and liver slices (incubated with palmitate-(14)C and glucose) had a significant increase in acetoacetate production, when compared to carbohydrate pretreated controls. Alcohol pretreatment or addition of alcohol in vitro had no effect on acetoacetate utilization by rat diaphragms, and decreased only slightly the conversion of beta-hydroxybutyrate-(14)C to (14)CO(2). Thus, the hyperketonemia and ketonuria observed after alcohol consumption cannot be attributed to an immediate effect of alcohol, but is the consequence of a delayed change in intermediary metabolism characterized by increased hepatic ketone production from fatty acids, possibly linked to ethanol-induced glycogen depletion and depression of citric acid cycle activity.

Efcts of chronic ethanol feeding on serum lipoprotein metabolism in the rat.

In rats, chronic ethanol feeding was found to enhance the postprandial hyperlipemia and to increase the incorporation of dietary palmitic acid-(3)H and intravenously injected L-lysine-(14)C into serum lipoproteins. The main increases of total amount, labeling, and specific activity of lipid and protein occurred in the d < 1.019 lipoprotein fraction. Fat absorption and the clearance of injected chylomicrons were not affected by ethanol feeding. Blocking of lipoprotein and chylomicron removal with Triton did not prevent the action of ethanol on serum lipids, indicating that the ethanol effect is not likely due to defective removal of lipids from the circulation. Ethanol enhanced the incorporation of chylomicron fatty acids into newly synthetized very low density lipoproteins, as shown by an increased reappearance of the fatty acid label into the lipids of this fraction after injection of palmitate-(14)C/glycerol-(3)H doubly labeled chylomicrons. These results indicate that alcoholic hyperlipemia is due, at least in part, to an increase in newly synthetized lipoproteins. The hyperlipemia produced by ethanol was accompanied by hepatic steatosis. The simultaneous production of fatty liver and hyperlipemia makes it unlikely that defective lipoprotein synthesis or secretion is a primary mechanism for the pathogenesis of the alcoholic fatty liver.

Pathogenesis of postprandial hyperlipemia in rats fed ethanol-containing diets.

To study the mechanism of the increase in serum lipoproteins which occurs in rats fed alcohol chronically, and especially to assess the role of the intestine, the effects of acute and chronic ethanol administration on lymph and plasma lipids were compared in rats with and without intestinal lymph fistulae. In rats not previously given alcohol, the administration of one dose of a diet containing ethanol (3 g/kg) produced a significant increase in lymph flow, lipid output, and incorporation of dietary fat into lymph lipids when compared with the effects of a control diet containing isocaloric carbohydrate. However, no hyperlipemia developed after ethanol. By contrast, previous feeding of ethanol for several weeks modified the acute effects of ethanol on both lymph and serum lipids. Compared with control animals pair-fed with isocaloric carbohydrate-containing diets, rats which had been fed a diet with 36% of total calories as ethanol for 3-4 wk developed postprandial hyperlipemia when given a single dose of the ethanol-containing or even the ethanol-free diet. This was associated with an increased incorporation of labeled dietary fat and of intravenously injected [(3)H]lysine into plasma lipoproteins of d < 1.006. However, postprandial lymph flow and lipid output were not higher in rats fed alcohol chronically than in their pair-fed controls. Moreover, when rats with lymph fistulae were given intravenous (i.v.) infusions of lymph lipids (to substitute for the diverted intestinal lymph), the ethanol-fed animals still developed hyperlipemia. Incorporation of i.v. lysine into d < 1.006 plasma lipoproteins also remained significantly increased. Thus, under these conditions, alcoholic hyperlipemia does not result from changes in intestinal lymph lipids. Two main factors appear to be involved; the acute effects of ethanol on hepatic lipid metabolism and the development of an increased capacity for lipoprotein synthesis during chronic ethanol feeding. The latter most likely occurs in the liver and it is postulated that it is linked to the associated changes in the hepatic endoplasmic reticulum.

Pathogenesis of alcohol-induced accumulation of protein in the liver.

Alcohol feeding to rats produced hepatomegaly, associated with enlargement of the hepatocytes. The increase in liver dry weight was accounted for not only by fat but also by protein accumulation, primarily in microsomes and cytosol, with a selective increase in export proteins: concentrations of both immunoreactive albumin and transferrin were augmented in liver microsomes and cytosol of ethanol-fed rats. To investigate the mechanism of this protein accumulation, [14C]leucine was injected intravenously and its incorporation into both liver and serum proteins was measured after various time intervals. Rates of synthesis and export were assessed from protein labeling and specific activities of leucyl-tRNA. Synthesis of liver protein and proalbumin were enhanced by chronic ethanol feeding, but this was not associated with a corresponding rise in serum albumin output. Actually, there was a significant retention of the label in liver albumin and transferrin with delayed appearance in the serum of ethanol-fed rats. This indicated that, regardless of the changes in synthesis, the export of protein from the liver into the plasma was impaired. This alteration in export was associated with a decreased amount of polymerized tubulin in the liver of ethanol-treated animals. Thus, both enhanced protein synthesis and defective export contribute to the ethanol-induced accumulation of liver protein, and the decrease in liver microtubules represents a possible site for impairment of protein export.

Effects of chronic ethanol intake on mobilization and excretion of cholesterol in baboons.

To investigate the effects of chronic ethanol administration on the mobilization and excretion of cholesterol, turnover and balance studies were carried out in baboons pair-fed cholesterol-free diets containing 50% of energy either as ethanol or as additional carbohydrate for several years. Ethanol feeding increased free cholesterol in all plasma lipoprotein fractions, and esterified cholesterol in very low density lipoprotein, intermediate density lipoprotein, and high density lipoprotein (HDL). The major increase occurred in HDL, mainly as esterified cholesterol. The latter was associated with decreased transfer of esterified cholesterol from HDL to low density lipoprotein. By contrast, the smaller increase in HDL-free cholesterol was associated with increased turnover in the plasma, increased splanchnic uptake, and increased fecal excretion of plasma cholesterol, mainly as neutral steroids. Cholesterol extraction predominated over release in the splanchnic vascular bed, suggesting that the excess of cholesterol excreted in the feces originated in extrasplanchnic tissues. Thus, these findings indicate that alcohol consumption favors mobilization of tissue free cholesterol for hepatic removal and excretion. By contrast the increase in HDL-cholesterol (mainly esterified) appears to be a poor indicator of cholesterol mobilization.

Difference in hepatic metabolism of long- and medium-chain fatty acids: the role of fatty acid chain length in the production of the alcoholic fatty liver.

Replacement of dietary triglycerides containing long-chain fatty acids (LCFA) by triglycerides containing medium-chain fatty acids (MCFA) markedly reduced the capacity of alcohol to produce fatty liver in rats. After 24 days of ethanol and MCFA, the increase in hepatic triglycerides was only 3 times that of controls, whereas an 8-fold rise was observed after ethanol and LCFA. The triglyceride fatty acids that accumulated in the liver after feeding of ethanol with MCFA contained only a small percentage of the MCFA; their composition also differed strikingly from that of adipose lipids. To study the mechanism of the reduction in steatosis, we compared oxidation to CO(2) and incorporation into esterified lipids of (14)C-labeled chylomicrons or palmitate-(14)C (representing LCFA), and of octanoate-(14)C (as MCFA) in liver slices and isolated perfused livers, in the presence or absence of ethanol. Ethanol depressed the oxidation of all substrates to CO(2); MCFA, however, was much more oxidized and reciprocally much less esterified than LCFA, with a 100-fold difference in the ratio of esterified lipid-(14)C to (14)CO(2). Furthermore, in hepatic microsomal fractions incubated with alpha-glycerophosphate, octanoate was much less esterified than palmitate. This propensity of MCFA to oxidation rather than esterification represents a likely explanation for the reduction in alcoholic steatosis upon replacement of dietary LCFA by MCFA.