REVIEW LIQUID DIET TECHNIQUE OF ETHANOL ADMINISTRATION: 1989 UPDATE

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1 Alcohol <«Alcoholism. Vol. 24. No. 3. pp /89 $ Printed in Great Britain Perganron Press pk 1989 Medical Council on Alcoholism REVIEW LIQUID DIET TECHNIQUE OF ETHANOL ADMINISTRATION: 1989 UPDATE CHARLES S. LIEBER* and LEONORE M. DECARLI Section of Liver Disease and Nutrition, Alcohol Research ai'.d Treatment Center, Bronx Veterans Administration Medical Center and Mount Sinai School of Medicine (CUNY), New York, U.S.A. (First received 4 October 1988; accepted for publication 8 November 1988) Abstract A technique of feeding alcohol as part of a liquid diet is reviewed that achieves an alcohol consumption of clinicaj relevance, while maintaining dietary control and providing adequate nutrition. With this procedure, blood alcohol levels are obtained which mimic clinical conditions and allow experimental duplications of many pathological complications caused by alcohol. In the rat, the liquid diet technique provides a model for the alcoholic fatty liver, various alcohol-induced metabolic, endocrine and central nervous system abnormalities (including tolerance and dependence) and the interaction of ethanol with industrial solvents, many commonly used drugs, analgesics, carcinogens and nutrients. This technique also resulted in the discovery of a new pathway of ethanol metabolism in the microsomes involving an ethanolspecific cytochrome P-450 (P450IIE1), which has now been confirmed in man. P450IIE1 contributes not only to the metabolic tolerance to ethanol, but also explains the enhanced susceptibility of the alcoholic to many ubiquitous xenobiotk agents. The liquid diet technique provides the flexibility to adjust to special experimental or physiological needs by allowing for various substitutions including changes in lipids, proteins or other dietary constituents. This procedure is thereby ideally suited for the study of the interactions of alcohol with deficiency or excess of various nutrients. The technique also facilitates the comparison with controls by simplifying pair feeding procedures. Although the flexibility of the liquid diet technique is one of its key advantages, a standard 'all purpose' liquid diet is described which is appropriate for most experimental applications. In addition, two other general formulae are given, namely a low fat diet (that allows the study of the effects of ethanol in the presence of minimal hepatic lipid accumulation) and a high protein diet (to meet increased needs, e.g. during pregnancy and lactation). The optimal amount of ethanol for the rat liquid diet was found to be 5 g/dl or 36% of total energy. With lesser amounts of alcohol, intake falls below a critical threshold; blood levels of alcohol then become negligible and the model becomes irrelevant to clinical conditions. In the rat, amounts of ethanol above 5 g/dl were not found to be associated with any further gain in alcohol ingestion. By contrast, in the baboon, the ethanol content could be raised profitably to 7 g/dl or 50% of total energy and resulted in the development of cirrhosis. This higher alcohol intake, together with species difference, may explain the greater severity of liver lesions produced by alcohol in the baboon. This first experimental model of alcoholic cirrhosis made it possible to clarify the pathogenesis of alcohol-induced fibrosis and has revealed precirrhotic lesions that have now found applicability to the human condition. Thus the alcohol-liquid diet feeding technique discovered 25 years ago and continuously improved since then has provided a thusfar unsurpassed tool for the experimental study of the effects of alcohol and the improvement of treatment and prevention. The success of this technique is due largely to the fact that it has resulted in an animal model with much greater ethanol intake than had heretofore been possible. As a consequence, many of the pathological disorders seen in patients, and which could not be reproduced before in animals, were now duplicated in rats and baboons. Address correspondence to: Charles S. Lieber, M.D., INTRODUCTION Alcohol Research and Treatment Center, Veterans Administration Medical Center, 130 West Kingsbridge Oie technique Of the feeding of ethanol as part Road, Bronx, New York 10468, U.S.A. of a totally liquid diet was devised over twenty- 197

2 198 CHARLES S. LIEBER and LEONORE M. DECARLI five years ago (Lieber et al., 1963) in response to the need to develop an animal model with an alcohol consumption of clinical relevance, while maintaining dietary control. Before the development of this technique of alcohol feeding, ethanol had been commonly administered to rats as part of their drinking water. With that technique, however, ethanol intake is insufficient to result in sustained appreciable levels in the blood and to cause significant liver damage when the diet is adequate (Best et al., 1949). The low intake results from a natural aversion of the animals to ethanol. However, when rats are given nothing to eat or to drink but the ethanol-containing liquid diet formula, their aversion to ethanol can be overcome and the intake is sufficient to sustain a high daily ethanol consumption of g/kg, two to three times more than can be achieved through the drinking water technique. Blood levels reached are also significantly higher. Although they fluctuate, in part due to the circadian rhythm, ethanol levels of mg/dl are not uncommon. In addition to achieving a significant ethanol intake, the liquid diet technique also has the advantage of allowing for an accurate recording of the nutrients consumed and for an easy change of the nutritional components according to specific experimental needs. Over the last two to three decades the liquid diet technique has been useful in characterizing the metabolism of ethanol, in assessing the interactions between ethanol and nutrition, other drugs, hepatotoxic agents and carcinogens as well as in elucidating the mechanisms of alcoholic liver injury, endocrine abnormalities, developmental problems, withdrawal states and other central nervous system changes, including a host of associated complications. IMPROVEMENTS OF THE LIQUID DIET Basic composition Whereas the first formula was based on an amino acid and sucrose mixture (Lieber et al., 1963), a more economical formula based on casein and dextrin-maltose was subsequently proposed (DeCarli and Lieber, 1967; Lieber and DeCarli, 1970a). Recommendations published in 1977 in a Report by the American Institute of Nutrition (AIN) and in 1978 by the National Research Council-National Academy of Science in terms of minerals and vitamins were subsequently taken into account in a reformulation of the diet (Lieber and DeCarli, 1982). The latter diet also contained an increased amount of zinc, to adjust to the switch of rat cages from galvanized to stainless steel ones. New suspending agents (such as xanthan gum) also became available that facilitated preparation of the diet (Lieber and DeCarli, 1986). Recent independent reviews confirmed the usefulness of xanthan gum (Ramirez, 1987) and concluded that this or similar liquid diets provide adequate nutrients for the rat (Ramirez, 1987; Ward, 1987; Editorial, 1988). Preparation and stability The various liquid diets can be prepared in the laboratory using kitchen-type blenders. The standard nutrient mixtures can also be purchased from various commercial manufacturers that have marketed the 'Lieber- DeCarli' diet; their quality standards, however, vary widely. Furthermore, experience gained in the dairy industry as well as in the administration of liquid formulae in man has shown that stability of micronutrients must be a major concern in such preparations. For instance, there is a striking loss of vitamin A, particularly when subjected to fluorescent light, especially in low-fat products (Senyk and Shipe, 1981). Some commercial manufacturers of the liquid diet formulae have adopted the practice of selling complete dry diet mixes containing not only the vitamins but also the various minerals. Theoretically, the presence in the same mix of relatively high concentrations of metals and micronutrients may promote the degradation of the latter. Indeed, we found that feeding of some commercial preparations even soon after they were received from the manufacturer resulted in low hepatic vitamin A with levels only about a tenth to a fifth of those of rats fed diets freshly prepared in-house. When ethanol was given, hepatic vitamin A content of animals fed these diets dropped to negligible levels. Vitamin A deficiency is associated with growth retardation and might contribute to depressed growth

3 LIQUID DIET TECHNIQUE OF ETHANOL ADMINISTRATION: 1989 UPDATE 199 and conceivably decreased food consumption. Furthermore, low hepatic vitamin A levels cause lesions such as giant lysosomes (Leo et al., 1983) and other adverse effects. To avoid these problems, we recommend the following measures: susceptible nutrients such as vitamin A should be kept separate from the other dry ingredients and the two mixed together only when the diet is actually prepared in the liquid suspension; the latter should be kept refrigerated in the dark and used within 1 week of preparation (Lieber and DeCarli, 1986). Pair-feeding One of the major advantages of the liquid diet technique is to facilitate the pair-feeding process. Usually, the alcohol-fed animals, being rate limiting, are allowed dietary consumption ad libitum. Their dietary intake is monitored by determining the amount of liquid consumed. The control animals are then given the same amount of liquid control diet during the subsequent feeding period. This pairfeeding process can be repeated every 12 or 24 hr, or more often as necessary. However, when ethanol is fed as part of a totally liquid diet, the pair-fed control, being limited in food intake, has a tendency to ingest more rapidly the total daily ration than the corresponding ethanol-fed animals. The possibility that the pattern of food ingestion might affect the P450IIE1 induction was investigated by using a modification of the 'simultaneous pair feeding system' of Israel et al. (1984). By eliminating the jackets and administering the diet at room temperature, we facilitated the use of our standard liquid diets. No differences were found between animals fed ethanol with the special device as compared to the regular Richter drinking tubes in terms of body weight, liver weight, total hepatic lipids, total cytochrome P450, P450IIE1 and the activity of other microsomal enzymes (Lieber et al., 1988). It was concluded that for ordinary experimental purposes, there was no advantage in using the somewhat cumbersome special tubes of Israel et al. (1984). With both systems, blood ethanol levels were comparable and reached a clinically significant level varying between 100 and 200 mg/dl. Fibre and nutrient adjustments Another modification of the diet has been the incorporation of fibre. It has not been established whether inclusion of fibre is needed in the liquid diets, but, because of some evidence of beneficial effects in humans, it was incorporated in our 1982 formulation (Lieber and DeCarli, 1982). We have now established that, for the parameters studied, fibre inclusion does not alter either the normal morphology of the liver in controls nor the effects of ethanol in terms of fat accumulation and ultrastructural changes (Lieber and DeCarli, 1986). One of the advantages of the liquid diet formula is to allow for flexible adjustment of nutrients to meet specific experimental needs. Accordingly we have to date used over a hundred different formulations. One basic formula, however, meets the majority of experimental needs associated with alcohol research. This 'all purpose' normal-fat-containing diet will be discussed in detail, with some additional references to low-fat, highcarbohydrate and high-protein formulations {vide infra). Alcohol intake as a function of energy intake and oxidative capacity With all our liquid diets, a meaningful blood alcohol level above 20 mm or about 100 mg/dl can be achieved. It must be emphasized that a reasonable blood alcohol level is a sine qua non of such experimental models. Indeed, experimental models of alcohol consumption are useful to the extent that they mimick prevailing clinical conditions. These are characterized by significant blood alcohol levels and a consumption level of alcohol which, in alcoholics, averages 50% of total energy (Patek, 1979; Lieber, 1988a). The baboon liquid diet model with 50% of total calories as ethanol and high blood levels (Lieber and DeCarli, 1974) fulfils both criteria. The rat model (Lieber and DeCarli, 1982, 1986), with 36% of calories as ethanol, comes close. Attempts at increasing other energy-rich nutrients (i.e. carbohydrates: Rao et al., 1987ft) have failed, because they are inevitably associated with a relative decrease in alcohol content (i.e. 20% of total energy: Lieber era/., 1965; or 26%: Rao et al., 19876). Under those

4 200 CHARLES S. LIEBER and LEONORE M. DECARLI conditions, and even with 29% of total energy as ethanol (Ward, 1987) only negligible blood alcohol levels were achieved. It appears that the capacity to oxidize alcohol is proportional to the total energy intake. Indeed, it has been known for a long time that the rate of ethanol metabolism is directly related to the basal metabolic rate with a linear relationship: the higher the metabolic rate is in a given species, the higher is its capacity to oxidize ethanol (Videla et al., 1975). Thus, the maximum capacity to oxidize ethanol is a function of total metabolism, which in turn is determined by the total energy intake. Thus, the maximum rate of ethanol oxidation in a given species under standard conditions should be expressed as a % of energy intake, and significant sustained blood ethanol levels will be achieved only if ethanol intake exceeds the threshold of this maximal oxidation capacity. As a consequence, when the energy intake is increased (e.g., by adding carbohydrate: Rao et al., 1987b) the contribution of alcohol to the total energy will decrease (i.e. from 36% to 26% of total calories). Furthermore, consumption of ethanol (expressed as g/kg body weight) decreases and falls below the maximal capacity to oxidize ethanol. As a result, blood levels will be negligible, although the total amount of alcohol ingested (expressed in g per animal, not per kg body weight or as % of energy) may not be significantly lower. This may explain why the various attempts by Rao et al. at modifying our liquid diet resulted in a lack of significant alcoholaemia and therefore failed to achieve a meaningful experimental model of excessive alcohol consumption of relevance to the human condition. The above mentioned difficulty does not pertain to nutrients with negligible caloric content. We took advantage of this and increased the mineral and vitamin content of our diet by 50% (Lieber and DeCarli, 1989): the overall effect of ethanol on the liver was unchanged. All liquid diets containing 36% of energy as ethanol allow greater intake than achieved by conventional feeding procedures (i.e. alcohol in drinking water). However, whereas the various Lieber-DeCarli diets assure intake of alcohol of g/kg per day, Miller et al. (1980) only reached a level of 9-11 g/kg per day. Reasons for this lower intake are not obvious; they could possibly be related to the fact that larger-sized animals were used at the start by Miller et al. (1980) than by us. We recommend for routine use that animals be started on the diet once they have reached a body weight of g, and that the ethanol be introduced progressively, with 3 g/dl of the liquid diet for 2 days, 4 g/dl for the subsequent 2 days followed by the final formula containing 5 g/dl. RECOMMENDED 'ALL PURPOSE' LIQUID DIET For the study of most effects of alcohol in the rat, the diet shown in Table 1 can be used. This diet contains an amount of fat (35%) comparable to that of the American diet and 18% of total energy as protein. The remainder of the energy is provided as carbohydrates which, in the ethanol formula, are isocalorically replaced by ethanol (to the extent of 36% of total Table 1. Composition of 'All Purpose' liquid diet Casein L-Cystine DL-Methionine Corn oil Olive oil Safflower oil Dextrin-maltose Choline bitartrate Fibre Xanthan gum Vitamin mix Mineral mix g/litre (1000 kcal) * Vitamins/1000 kcal: thiamin HC1 1.5 mg; riboflavin, 1.5 mg; pyridoxine HO, 1.75 mg; nicotinic acid, 7.5 mg; calcium pantothenate, 4.0 mg; folic acid, 0.5 mg, biotin, 0.05 mg; vitamin B )2, 25 ng; p-aminobenzoic acid, 12.5 mg; inositol, 25 mg, vitamin A, 6000 IU; vitamin D, 400 IU; vitamin E, 30 IU; vitamin K, 125 ug. Mineral mix (mg/1000 kcal): calcium, 1300; phosphorus, 1000; sodium, 255; potassium, 900; magnesium, 125; manganese, 13.5; iron, 8.8; copper, 1.5; zinc, 7.5; iodine, 0.05; selenium 0.025; chromium, 0.5; chloride, 390; sulphate, 250; fluoride, In the ethanol formula, replaced by 25.6 g of dextrinmaltose and 50 g of ethanol.

5 LIQUID DIET TECHNIQUE OF ETHANOL ADMINISTRATION: 1989 UPDATE 201 energy). The liquid diets were first created to address the view, prevailing at the time, that ethanol did not produce any changes in the liver different from those resulting from isocaloric carbohydrate (Best et al., 1949). To test this thesis, we replaced carbohydrate in the diet by isoenergetic amounts of ethanol and discovered that under those conditions, the ethanol-containing diet resulted in the development of striking liver changes, including marked ultrastructural alterations and fat deposition, whereas diets comprising isocaloric carbohydrate did not (Lieber et al., 1963, 1965; Iseri et al., 1964, 1966). These results were confirmed with our more recent version of the liquid diet (DeCarli and Lieber, 1967; Lieber and DeCarli, 1970a, 1982, 1986; Lieber et al., 1988). These studies therefore established the difference, in terms of hepatotoxicity, between ethanol and carbohydrates. ADEQUACY OF THE DIET IN TERMS OF OVERALL NUTRITION Carbohydrate, dietary restriction, growth rate, tnicronutrients and alcohol effects When ethanol is introduced in the diet, rodents decrease their overall food intake. Therefore, the question came up at that time (Lieber et al., 1965) as to whether the overall decreased food intake rather than the ethanol might be responsible for the liver changes. To control for this factor, we have always insisted that each control animal must be pair-fed with amounts of the liquid diet equal to those ingested by the corresponding alcohol-treated littermate. Under these conditions, the control animal does not develop any of the pathological changes caused by the introduction of alcohol in the diet. Therefore, the effects of ethanol can be attributed to alcohol itself rather than to insufficient nutrient intake, since the alcohol-fed and control animals ingested identical amounts of all nutrients save one, namely carbohydrate (isocalorically replaced by the ethanol). The question has been raised by Lieber et al. (1965) and more recently by Rao and Larkin (1984) whether the decreased carbohydrate supply might be responsible for the effects attributed to the alcohol-containing diet. This issue was answered by using ethanol not only as isocaloric replacement for carbohydrate but also a substitute for fat (Fig. 1). Under these conditions, despite equal intake of dietary carbohydrate, ethanol-fed animals developed lesions that were not present in pairfed controls, thereby clearly incriminating ethanol itself and not simply a lack of carbohydrate (Lieber et al., 1965). Similarly, when carbohydrate (36% of energy) was simply omitted from the control liquid diet, the effects that had been seen with alcohol were not reproduced (Lieber et al., 1965). The type of carbohydrate may also be of importance. Whereas in our 'all purpose' diet, we employ dextrin-maltose, some diets use, as primary ingredient, sucrose which has disadvantages in terms of favouring caries and having an unnaturally high fructose content. Furthermore, it can cause kidney lesions in some strains of rats on prolonged administration (Report of the American Institute of Nutrition Ad Hoc Committee on Standards for Nutritional Studies, 1977). These are some of the reasons why sucrose was replaced in our original diet (Lieber et al., 1963) with dextrinmaltose (DeCarli and Lieber, 1967). It has been established that energy restriction achieved through partial reduction of dietary carbohydrate has no apparent ill effects in terms of liver morphology or fat content (Lieber et al., 1965). We now have extended this observation to the administration of control diets with an across-the-board restriction of nutrients (Lieber et al., 1988). These control animals ended up with weights comparable to those of the alcohol-fed rats; however, their liver morphology and fat content remained normal and identical with those of controls strictly pair-fed with the alcohol-fed animals. By contrast, the alcohol-fed littermates developed the classic chemical and structural hepatic abnormalities that we have previously described (Lieber et al., 1965). The slower weight gain of rats fed alcoholcontaining liquid diets (compared to rats fed ad libitum Purina Rat Chow or other nonalcohol-containing diets) is not due to a deficiency in the diet but rather to the fact that alcohol decreases food intake and depresses growth. Indeed, when our liquid diets were

6 202 CHARLES S. LIEBER and LEONORE M. DECARLI TOTAL 100 HEPATIC LIPIDS mg/g (wet wt.) 0 CONTROL DIETARY COMPOSITION % of total calories MP HIOH FAT Hi * 1i [ «* HYPOCALORIC 36* HYPOCALORIC I ETHANOL if m * l if ij SUCROSE l.l w1 - i 1ill lilll AMINO-ACIOS given ad libitum without alcohol, growth rates were fully comparable to or exceeded those achieved with Purina Rat Chow or other diets (Lieber et al., 1965; Saville and Lieber, 1969). Despite the fact that ethanol-fed animals have an overall food intake lower than that of animals fed the control diet ad libitum, the intake of nutrients by the former remains adequate. Indeed, in our hands, intake of the ethanol-containing diets, though variable (depending on animal size), averages 60 kcal/day, significantly more than the figure of 41 kcal/ day used by Rao et al. (1988) in their calculations. Actually, this consumption figure of 60 kcal/day is the same as the intake reported by Rao and co-workers themselves in two of their previous papers (Rao et al., 1986, 1987a) with the Lieber-DeCarli alcohol diet. A daily consumption of 60 kcal falls within the recommended intake for other diets and provides adequate amounts of nutrients, including protein, Ca, Cu, I, Fe, Mg, Mn, P, K, Se, vitamin D, choline and other minerals and vitamins (National Research Council, 1978). Conclusions to the contrary (Rao et al., 1988; Rao and Larkin, 1985) were based not only on an unrepresentative low intake, but also on the use of outdated nutritional tables. Despite dietary adequacy, liver injury developed with ethanol (Lieber et al., 1965; Lieber and DeCarli, 1982, 1986). Although the nutrient intake of our alcohol-fed animals meets accepted standards, the question may be asked whether the overall lower intake (compared to ad libitum fed controls) might, in some way, potentiate the alcohol effects. To address this issue, we have now treated a group of animals with our standard diet in which the vitamins and minerals were all increased by 50% (Lieber and DeCarli, 1989). Total lipid accumulation after ethanol was similar with or without the supplement (133.0 ± 15.8 vs ± 7.4 mg/g liver, with 53.1 ± 2.9 vs 58.5 ± 3.5 in the corresponding controls). Induction of the microsomal ethanol oxidizing system (MEOS) was also the same as that achieved Fig. 1. Effect on total hepatic lipids of five types of liquid diets fed to rats for 24 days. Isocaloric substitution of carbohydrate by ethanol produced an increase in hepatic lipids. Ethanol rather than the reduction in carbohydrate must be incriminated, since isocaloric substitution of carbohydrate with fat ('high fat') or decreases in carbohydrate (18 or 36% of energy) had no similar effect. (From Lieber, C. S. (1968) Metabolic effects produced by alcohol in the liver and other tissues. In Advances in Internal Medicine, Vol. XIV, pp , Snapper, I., and Strollerman, G. H. eds. Year Book Medical Publishers, Chicago; data from Lieber el al., 1965.)

7 LIQUID DIET TECHNIQUE OF ETHANOL ADMINISTRATION: 1989 UPDATE 203 with our regular diet (Lieber and DeCarli, 1989). Thus, there is no evidence that the hepatic effects observed with our 'all purpose' ethanol-containing diet is in any way due to a deficiency in minerals, vitamins or carbohydrates, or that the moderate limitation in food intake inherent to the technique is detrimental. In fact, it should be noted that in terms of bone characteristics (Saville and Lieber, 1969), longevity (Barrows, 1972) and susceptibility to diseases (Yu et al., 1982), moderate undernutrition without malnutrition may be more favourable than ad libitum nutrition. Dietary restriction is also beneficial regarding spontaneous cancer incidence (Weindruch and Walford, 1982), radiation-induced tumors in rats (Gross and Dreyfuss, 1984) and hepatic protein synthesis; the latter was significantly higher in rats fed a diet restricted to 60% of the amount consumed by the control animals fed ad libitum (Birchnall-Sparks et al., 1985). Thus, under ordinary circumstances, there is no need to supplement our 'all purpose' liquid diet, and we found addition of vitamins and minerals to be without consequence on the alcohol-effect (Lieber and DeCarli, 1989). Other experiments with vitamin supplementation have resulted in equivocal results: Rao et al. (1987b) observed in one group of rats that a supplement of extra calcium, phosphorus, iron, copper, zinc, manganese, pyridoxine, and choline improved the growth rate of rats approximately 25% over a 4-week period, but the liver triglyceride (triacylglycerol) level was unchanged. Furthermore, in another group of animals, the growth rate was hardly changed, while triglyceride levels appeared decreased. It is questionable, therefore, whether any reproducible effect was obtained with this supplementation. Since the publication of the first alcoholcontaining diet (Lieber et al., 1963), several other variations of the original diet formula have appeared. Some of these fulfil specific experimental needs, others, however, introduce unnecessarily minor variations in an otherwise satisfactory formulation. It is hoped that with time, investigators can agree on an overall useful basic formula that can serve for most alcohol-related studies and that changes are only introduced to satisfy particular experimental goals. Such uniformity, coupled with the use of standardized animal strains, would greatly facilitate comparison of results obtained in various laboratories. Taking advantage of almost three decades of experience with alcohol liquid diets, we would like to propose the formula described in Table 1. With this diet, the results are essentially the same as those obtained with our and 1982 (Lieber and DeCarli, 1982) formulae. For those who are using the formulae and house animals in galvanized cages, there is actually no need for change. When animals are kept in stainless steel cages, the greater zinc content of our new formula is appropriate. The other proposed changes are minor and could be considered optional. This regular Lieber- DeCarli formula is intended to mimick the clinical situation in which heavy alcohol consumption is associated with fatty liver development, even in the presence of an adequate diet (Lieber et al., 1965). The fat content of this diet is similar to that prevailing in Western human diets (see Handbook of Clinical Dietetics, 1981). Whereas this all purpose diet will meet most requirements for experiments carried out with alcohol in rats, two important variants are being proposed namely a 'low fat' formula intended for those who wish to study the effects of ethanol while minimizing hepatic fat accumulation, and a high protein formula useful for conditions (such as gestation and lactation) that require increased protein consumption {vide infra). Role of dietary fat and 'low fat' formula When alcohol is given as part of a liquid diet containing 35% of energy as fat, the bulk of the fat which accumulates in the liver is derived from dietary lipids (Lieber etal., 1966). Consequently, the quantity of fat in the diet has a striking effect on the overall amounts which accumulates in the liver (Fig. 2). To minimize the fatty liver change, the optimal fat content of the diet is about 5-15% of energy. Therefore we have formulated 'low fat diets' with 5 or 12% of total energy as fat, and a reciprocal increase of carbohydrates (Table 2). Such diets are particularly recommended for those who wish to study the effects of ethanol (such as various pharmacological actions, including

8 204 CHARLES S. LIEBER and LEONORE M. DECARLI o 1 2* 5* 10* 15% 25* 35* DIETARY FAT (*ol total calories) 43* Fig. 2. Effect of varying amounts of dietary fat on hepatic fat accumulation produced by ethanol. Hepatic triglycerides were measured in seven groups of rats given ethanol (36% of calories) with a diet of normal protein (18% of calories) content. Average hepatic triglycerides concentration in the control animals is indicated by a dotted line. (From Lieber and DeCarli (1970a.) Table 2. Overall composition of rat liquid diets (% of calories) Protein Fat Carbohydrate All purpose Low fat /18 5/12 77/70 High protein In the ethanol-containing diets, carbohydrates (36% of energy) are isocalorically replaced by ethanol. The composition of the 'all purpose' liquid diet is given in Table 1. The composition of the 'low fat' diet is identical except that the amount of fat is decreased to 5 or 12% and the amount of carbohydrates correspondingly increased. In the 'high protein' diet, the composition is identical to that of the low fat diet, except that the amount of protein is increased and the carbohydrates correspondingly decreased. brain effects) in the absence of significant liver changes. It is noteworthy that some of the 'inductive' properties of ethanol on microsomal systems, including the microsomal ethanol oxidizing system, can be achieved even with a low fat diet (Lieber et al., 1988). Not only the amount, but also type, of dietary fat may affect the degree of steatosis. When long-chain fatty acids are replaced by medium-chain fatty acids introduced as medium-chain triglycerides (MCT), fat accumulation in the liver decreases, presumably because of the propensity of MCT for oxidation rather than esterification (Lieber et al., 1967). Various mechanisms have been proposed to explain the alcohol-induced fatty liver, as discussed in detail elsewhere (Lieber and Savolainen, 1984). One mechanism involves dietary essential fatty acids. Ethanol has been shown in vitro to inhibit the hepatic desaturase system that converts linoleate (18:2) to arachidonate (20:4) (Nervi et al., 1980; Wang and Reitz, 1983). This would lead to reduced amounts of arachidonate and an elevated ratio of linoleate to arachidonate in the membranes of the hepatocytes. It was reported that when arachidonic acid is incorporated into the ethanol-containing diet, rats increase their food consumption, gain weight at rates comparable to those of rats fed the control diet ad libitum and accumulate less fat in their livers (Goheen et al., 1980). It is noteworthy that a subsequent study from the same laboratory (Goheen et al., 1981) found no protective effect of arachidonic acid in pair-fed rats, nor could such a protective effect be confirmed by our own laboratory. In fact arachidonate was found to promote hepatic total lipids and triacylglycerol deposition. Dietary protein When the experimental design calls for a nutritionally adequate diet, the protein content of our 'all purpose' diet, namely about 18% of total energy is fully adequate; in fact it exceeds the 13% recommended by the National Research Council (1978). Under special circumstances (e.g. pregnancy and lactation) a higher amount of protein may be required in which case diets containing 25% of total energy as protein can be used. The increase in protein should be achieved as isocaloric substitution for either carbohydrate (Table 2) or fat, but not by merely adding the excess protein to the basic formula, as has been done by others (Weiner et al., 1981). The latter results in a dilution of all remaining dietary constituents,

9 LIQUID DIET TECHNIQUE OF ETHANOL ADMINISTRATION: 1989 UPDATE 205 including the ethanol. The decrease in the relative contribution of ethanol to the total energy content to a level below the original 36% results in a significant decrease in blood ethanol levels (vide supra), thereby defeating one of the original goals of the liquid diet technique. A high protein diet containing 36% of energy as ethanol has been used successfully in studies of pregnancy and lactation in rats (Gordon et al., 1985). Our pregnant animals consume about 75 ml/day of this diet, an intake that provides adequate nutrition for gestation. Should the experimental design result in an excessive lowering of food intake, the amount of minerals and vitamins can be raised by 50%. Such a supplement can be readily achieved without altering the basic effects of ethanol. INFUSION OF LIQUID DIETS Liquid diets have also been administered by continuous infusion (Tsukamoto et al., 1985). This mode of administration allows for better control of the diet intake in the various groups of animals and possibly could also enhance the overall dose of ethanol administered, although the latter is questionable since with the standard liquid diet technique, the amount of alcohol ingested seems to be limited by the overall capacity of the rat to metabolize ethanol. In any event, using this technique, lesions more severe than simple fatty liver have been produced (Tsukamoto et al., 1985). APPLICATION OF THE LIQUID DIET TECHNIQUE TO OTHER ANIMAL SPECIES The liquid diet technique has been adapted to a number of animal species other than the rat. One of the most successful applications has been the baboon liquid diet (Lieber and DeCarli, 1974). The composition of the liquid diet was adjusted to meet the primate's needs. The energy is derived from protein (18% of total calories), carbohydrate (61%), and fat (21%). In the alcohol-containing diet, ethanol replaces isocalorically carbohydrate up to 50% of total energy. Thus, the alcohol content of the baboon liquid diet is significantly higher than that in the rat because of a lesser aversion to ethanol in the former species. The higher alcohol intake, coupled with the longer periods of administration may, in addition to species difference, be responsible for the fact that the baboon not only develops fatty liver, but progresses to more severe stages of alcoholic liver disease, including cirrhosis in one-third of the animals (Lieber et al., 1975; Popper and Lieber, 1980). Supplementation with choline failed to prevent the effect, although very large and even toxic amounts of choline were used (Lieber et al., 1985). Recently, Ainley et al. (1988) failed to reproduce alcohol-induced cirrhosis in baboons. However, only two animals received the regular ethanol-containing diet for more than 18 months. Since only onethird of our animals (Popper and Lieber, 1980) developed cirrhosis (with an average ethanol treatment of three years), the negative result of Ainley et al. (1988) is not surprising. Moreover, the amount of alcohol consumed by the baboons of Ainsley et al. (1988) is not clear. A daily administration of 25 g/kg is reported; if the baboons had consumed that amount, they would not have survived, since it is a lethal dose. Actually, the reported postprandial blood levels were, if anything, lower than the one we observed with a five fold lower ethanol administration (Lieber et al., 1975)! The liquid diet technique has also been successfully applied to the feeding of alcohol to mice (Peterson and Atkinson, 1980), rabbits (M. Rothschild, unpublished results) and deermice (Shigeta et al., 1984). ACHIEVEMENTS OBTAINED WITH THE LIQUID DIET TECHNIQUE The introduction of the technique of the feeding of alcohol as part of totally liquid diets has provided a useful tool for the study of many of the pathological disorders associated with alcoholism. The success of this technique is due largely to the fact that it has resulted in an animal model with much greater ethanol intake than had heretofore been possible. As a consequence, many of the pathological disorders seen in patients and which could not be reproduced before in animals were now duplicated in this experimental model. In addition, the liquid diet technique provided an easy

10 206 CHARLES S. LIEBER and LEONORE M. DECARLI method to control and measure the intake of various nutrients, thereby allowing for the assessment of their contribution to the ethanol-induced pathology. Furthermore, the liquid diet technique allows for relatively easy changes in dietary constituents. For instance, in studies on the interaction of ethanol with thiamine (Shaw et al., 1981) and vitamin A (Sato and Lieber, 1981; Leo etal., 1982, 1983), the content of these vitamins was varied widely from extreme deficiency to excess. Similarly the liquid diet technique has allowed for the study of the effects of protein deficiency on alcohol induced fat accumulation in the liver (Lieber and DeCarli, 1970a), ethanol metabolism (Wilson et al., 1986a) and pancreas (Wilson et al., 1986b, 1988). One major accomplishment has been the demonstration of the hepatotoxicity of ethanol, whether given as isocaloric replacement for carbohydrate (Lieber et al., 1963, 1965; DeCarli and Lieber, 1967) or for fat (Lieber et al., 1965). Fatty liver was produced in the rat (Lieber et al., 1963, 1965) and cirrhosis in the baboon (Lieber and DeCarli, 1974). These experimental models in turn have allowed the detailed studies of the interaction of ethanol with lipid metabolism and with fibrosis, as reviewed elsewhere (Lieber and Savolainen, 1984; Lieber and Leo, 1986). The same technique has also been used to study the effects of ethanol and acetaldehyde on liver organelles (Lieber, 1988b), other tissues (including the gastro-intestinal tract and the endocrine system) and developmental effects of ethanol (e.g. the fetal alcohol syndrome) and central nervous system changes such as ethanol withdrawal (Lieber and De- Carli, 1973). A detailed discussion of the several hundred papers contributed by many laboratories would clearly exceed the scope of this article. The clinically relevant results have been summarized in a monograph (Lieber, 1982). One observation that should perhaps be singled out is the demonstration that chronic ethanol consumption results in increased activity of a microsomal ethanol-oxidizing system (MEOS) (Lieber and DeCarli, 1968, 1970b). It was the observation in rats (Iseri et al., 1964) confirmed in man (Lane and Lieber, 1966) that chronic ethanol consumption is associated with proliferation of hepatic microsomal membranes that had prompted the suggestion that liver microsomes could be the site for a distinct and adaptive system of ethanol oxidation. This MEOS was differentiated from alcohol dehydrogenase and catalase, purified, and its composition demonstrated in reconstituted systems (Ohnishi and Lieber, 1977). The ethanolinducible form of P-450 (P450IIE1) was purified from rabbit liver microsomes (Koop etal., 1982) and ethanol- or isoniazid-treated rats (Ryan et al., 1985, 1986). A similar purified human protein has been obtained in a catalytically active form, with a high turnover rate for ethanol (Lasker et al., 1987a). Inasmuch as compounds other than ethanol (e.g. acetone) can also serve as P450IIE1 inducers, and since the administration of ethanol (together with fat-containing diets) can be associated with ketonaemia (Lefevre etal., 1970), the question has been raised as to whether the induction of P450IIE1 is mediated specifically by ethanol. Recent studies, however, showed that induction of the activity of P450IIE1 occurs already after short-term and relatively modest ethanol consumption, in the absence of increased acetonaemia or hepatic steatosis (Lieber et al., 1988). Mechanisms involved in P450IIE1 regulation are under active investigation. Research with a P450IIE1 cdna probe indicated that P450IIE1 protein induction by 'ethanol-like' agents may be regulated by post-translational events (Song et al., 1986), whereas other studies showed an increase in translatable P450IIE1 mrna following treatment with ethanol (Kubota etal., 1988). In any event, the demonstration of MEOS allowed for better understanding of the metabolic tolerance produced by ethanol (Lieber and DeCarli, 1972). Since in addition to P450IIE1, other microsomal cytochrome P-450 isozymes can also contribute to ethanol oxidation (Lasker et al., 19876), the term MEOS should be maintained when one refers to the overall capacity of the microsomes to oxidize ethanol, rather than to that fraction of the activity which is specifically catalyzed by P450IIE1. The ethanol inducible form of cytochrome P450 (P450IIE1) has a high affinity not only for ethanol but also for a variety of other potentially toxic substrates. Accordingly, alcohol pretreatment remarkably enhances CCI 4 (Hasumura et al., 1974) and

11 LIQUID DIET TECHNIQUE OF ETHANOL ADMINISTRATION: 1989 UPDATE 207 acetaminophen or paracetamol (Sato et al., 1981) hepatotoxicity. Alcohol abuse is also associated with an increased incidence of upper alimentary and respiratory tract cancers; many factors have been incriminated in the cocarcinogenic effect of ethanol (Lieber et al., 1986). One of the mechanisms is the effect of ethanol on cytochrome /M50 dependent carcinogen activation. This has been demonstrated by using microsomes derived from a variety of tissues including the liver (Garro et al., 1981; Seitz et al., 1981; Farinati et al., 1985), lungs (Seitz et al., 1981), intestine (Seitz et al., 1978) and esophagus (Farinati et al., 1985). Ethanol has a unique effect on the chemical carcinogen N-nitrosodimethylamine (NDMA): it induces an NDMA demethylase which functions at low NDMA concentrations (Garro etal., 1981) and which has now been identified as P450IIE1. The induction of MEOS activity after chronic alcohol consumption 'spills over' to various other drug-metabolizing systems in liver microsomes, thereby accelerating drug metabolism: increased rates of blood clearance of meprobamate, pentobarbital, warfarin, diphenylhydantoin (phenytoin), tolbutamide, propranolol and rifampicin have been described (Kater et al., 1969; Misra et al., 1971; Pritchard and Schneck, 1977), and steroid degradation is enhanced (Gordon et al., 1979). Contrasting with the chronic administration, the main effect of the presence of ethanol is the inhibition of hepatic drug metabolism (Rubin et al, 1970; Sato C. and Lieber, 1981; Lieber, 1985). Alcoholics commonly have very low hepatic vitamin A concentration (Leo and Lieber, 1982), possibly because of accelerated microsomal degradation of vitamin A (Sato and Lieber, 1982). In reconstituted systems with purified forms of cytochrome P-450, retinoic acid and retinol can serve as substrates for microsomal oxidation (Leo et al., 1984; Leo and Lieber, 1985). Induction of microsomes may also result in energy wastage. As discussed before, it has been known for a long time that despite strict isocaloric pair feeding, ethanol-fed animals do not gain as much weight as their pair-fed controls although the latter receive diets with the same energy content. Similarly, alcoholics given ethanol (in addition to a normal diet) failed to gain weight (Lieber et al., 1965; Pirola and Lieber, 1972) and isocaloric substitution of ethanol for carbohydrate, up to 50% of total energy in a balanced diet, resulted in a decline in body weight (Pirola and Lieber, 1972). One postulated mechanism for this apparent energy wastage is oxidation without phosphorylation by the MEOS. Indeed, whereas ethanol oxidation to acetaldehyde via the ADH pathway is associated with the generation of NADH, a high energy compound, this is not the case when ethanol is oxidized via MEOS. In the latter pathway, a high energy compound (NADPH) is in fact utilized and no high energy compound is formed; the reaction only generates heat. To the extent that this calorigenesis exceeds the needs for thermoregulation, it can be considered as energy wastage. Other mechanisms may also contribute to such a hypermetabolic state (Lieber, 1982). In any event, as mentioned before, administration of a hypocaloric diet (by reduction of dietary carbohydrate or an across the board restriction of nutrients) did not result in apparent deleterious effects. Thus, although some of the alcohol-derived calories do not 'fully count', it does not appear that the resulting deficit in weight gain can account for the commonly assessed alcohol effects on the liver. Induction of MEOS leads to increased production of acetaldehyde, a very reactive compound. Acetaldehyde, in turn, causes injury through the formation of adducts with proteins (Nomura and Lieber, 1981; Behrens et al., 1988), resulting in antibody formation (Hoerner et al., 1986, 1988), enzyme inactivation, decreased DNA repair (Espina et al., 1988), and alterations in microtubules (Matsuda et al., 1979), mitochondria and plasma membranes, as reviewed elsewhere (Lieber, 1982, 19886). Acetaldehyde also promotes glutathione depletion, free-radical mediated toxicity, lipid peroxidation and hepatic collagen synthesis (Savolainen et al., 1984). The feeding of ethanol as part of the Lieber-DeCarli liquid diet also results in marked changes in skeletal muscle (Preedy et al., 1988). The latter observations were similar to those of skeletal myopathy in man and it was concluded that the use of the Lieber-DeCarli

12 208 CHARLES S. LIEBER and LEONORE M. DECARLI feeding regime in experimental animals provides a suitable model for investigating the mechanism of this important complication of alcoholism (Preedy et al., 1988). GENERAL CONCLUSIONS Whereas traditionally (Best et al., 1949) the disorders affecting the liver had been attributed mainly to the nutritional deficiencies that accompany alcoholism (Lieber, 1988a), studies carried out over the last three decades indicate that in addition to the role of dietary deficiencies, alcohol per se can be incriminated as a direct aetiologic factor in the production of alcoholic liver disease (Lieber, 1982; 19886). Indeed, even in the absence of dietary deficiencies, alcohol can lead to the development of fatty liver in humans. An experimental model for this toxic effect was created by overcoming the natural aversion of the rodent for alcohol by incorporating the ethanol in a totally liquid diet. Application of this liquid diet-feeding technique to the baboon resulted in the demonstration of a direct causal role of ethanol in the pathogenesis of liver cirrhosis. Although alcohol depresses appetite, the liquid diets still meet nutritional requirements. Thus, it is now obvious that, although some of the alcohol effects are due to nutritional consequences, others can be attributed to the toxicity of ethanol itself, irrespective of malnutrition. By raising the alcohol intake to clinically meaningful levels while facilitating the manipulation of dietary constituents one by one, and by maintaining the same nutrient intake in control animals, the original liquid diet feeding technique has allowed for the definition not only of the various toxic effects of ethanol, but also of the interactions of ethanol with nutritional factors. In fact, at the biochemical cellular level, both 'toxic' and 'nutritional' effects are often intertwined. A better understanding of the biochemical alterations produced by ethanol in the body now provides insight into processes whereby ethanol alters both the activation and the degradation of key nutrients. One of the major achievements of the ethanol-liquid diet technique has been to delineate some nutritional effects of ethanol as well as the toxicity of the drug and to establish a biochemical link between the two types of actions. Thus, our liquid diet model has allowed for the elucidation of many alcoholrelated pathologies in the liver and other tissues and provides a uniquely suited model for the experimental production and the study of the multiple adverse effects of alcohol. Acknowledgements The authors thank Ms. R. Cabell for expert typing of the manuscript. Original studies reviewed here were supported, in part, by DHHS grant AA03508 and the Veterans Administration. REFERENCES Ainley, C. C, Senapati, A., Brown, I. M. H., lies, C. A., Slavin, B. M., Mitchell, W. D., Davies, D. R., Keeling, P. W. N. and Thompson, R. P. H. (1988) Is alcohol hepatotoxic in the baboon? Journal of Hepatology 7, Barrows, C. H. (1972) Nutrition, aging, and genetic program. American Journal of Clinical Nutrition 25, Behrens, U. H., Hoerner, M., Lasker, J. M. and Lieber, C. S. (1988) Formation of acetaldehyde adducts with ethanol-inducible P450IIE1 in vivo. Biochemical and Biophysical Research Communications 154, Best, C. H., Hartroft, W. S., Lucas, C. C. and Ridout, J. H. (1949) Liver damage produced by feeding alcohol or sugar and its prevention by choline. British Medical Journal 2, Birchnall-Sparks, M. C, Roberts, M. S., Staecker, J., Hardwick, J. P. and Richardson, A. (1985) Effect of dietary restriction on liver protein synthesis in rats. Journal of Nutrition 115, DeCarli, L. M. and Lieber, C. S. (1967) Fatty liver in the rat after prolonged intake of ethanol with a nutritionally adequate new liquid diet. Journal of Nutrition 91, Editorial (1988) Nutrition and alcoholism in rats. Nutrition Reviews 46, Espina, N., Lima, V., Lieber, C. S. and Garro, A. J. (1988) In vitro and in vivo inhibitory effect of ethanol and acetaldehyde on D-methylguanine transferase. Carcinogenesis 9, Farinati, F., Zhou, Z. C, Bellah, J., Lieber, C. S. and Garro, A. J. (1985) Effect of chronic ethanol consumption on activation of nitrosopyrrolidine to a mutagen by rat upper alimentary tract, lung and hepatic tissue. Drug Metabolism and Disposition 13, Garro, A. J., Seitz, H. K. and Lieber, C. S. (1981) Enhancement of dimethylnitrosamine metabolism and activation to a mutagen following chronic ethanol consumption. Cancer Research 41, Goheen, S. C, Larkin, E. C, Manix, M. and Rao, G. A. (1980) Dietary arachidonic acid reduces fatty liver, increases diet consumption and weight gain in ethanolfed rats. Upids IS, Goheen, S. C, Pearson, E. E., Larkin, E. C. and Rao,

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14 210 CHARLES S. LIEBER and LEONORE M. DECARLI Lieber, C. S. and DeCarii, L. M. (1986) The feeding of istration in man and in rats. American Journal of ethanol in liquid diets: 1986 update. Alcoholism: Clinical Medicine 51, and Experimental Research 10, National Research Council (1978) Nutrient requirements Lieber, C. S. and DeCarii, L. M. (1989) Effects of of the laboratory rat. In Nutrient Requirements of mineral and vitamin supplementation on the alcoholinduced fatty liver and mictosomal induction. Alcohol- National Academy of Sciences, Washington, DC. Laboratory Animals, No. 10, 3rd rev. ed., pp. 7-37, ism: Clinical and Experimental Research 13, Nervi, A. M., Peluffo, R. O., Brenner, R. R. and Leikin, Lieber, C. S., DeCarii, L. M. and Rubin, E. (1975) A. I. (1980) Effect of ethanol administration on fatty Sequential production of fatty liver, hepatitis and cirrhosis in sub-human primates fed ethanol with adequate Nomura, F. and Lieber, C. S. (1981) Binding of acetal- acid desaturation. Lipids 15, diets. Proceedings of the National Academy of Science of dehyde to rat liver microsomes: enhancement after the U.S.A. 72, chronic alcohol consumption. Biochemical and Biophysical Research Communications 100, Lieber, C. S., Garro, A., Leo, M. A., Mak, K. M. and Worner, T. (1986) Alcohol and cancer. Hepatology 6, Ohnishi, K. and Lieber, C. S. (1977) Reconstitution of the microsomal ethanol oxidizing system (MEOS): Qualitative and quantitative changes of cytochrome f-450 after Lieber, C. S., Jones, D. P., Mendelson, J. and DeCarii, L. M. (1963) Fatty liver, hyperlipemia and hyperuricemia produced by prolonged alcohol consumption, Chemistry 252, chronic ethanol consumption. Journal of Biological despite adequate dietary intake. Transactions of the Patek, A. J. (1979) Alcohol, malnutrition, and alcoholic Association of American Physicians 76, cirrhosis. American Journal of Clinical Nutrition 32, Lieber, C. S., Jones, D. 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American of long and medium-chain fatty acids: The role of fatty Journal of Pathology 98, acid chain length in the production of the alcoholic fatty Preedy, V. R., Duane, P. and Peters, T. J. (1988) liver. Journal of Clinical Investigation 46, Biological effects of chronic ethanol consumption: a Lieber, C. S. and Leo, M. A. (1986) Interaction of alcohol reappraisal of the Lieber-DeCarli liquid-diet model with and nutritional factors with hepatic fibrosis. In Progress reference to skeletal muscle. Alcohol and Alcoholism in Liver Disease, Vol. Ill, Popper, H. and Schaffner, F., 23, eds, Chap. 14, pp , Grune and Stratton, Pritchard, J. F. and Schneck, D. W. (1977) Effects of New York. ethanol and phenobarbital on the metabolism of propranolol by 9000 g rat liver supernatant. Biochemical Lieber, C. S., Leo, M. A., Mak, K. M., DeCarii, L. M. and Sato, S. (1985) Choline fails to prevent liver fibrosis Pharmacology 26, in ethanol-fed baboons but causes toxicity. Hepatology Ramirez, I. 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