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Nutrient Interactions and Toxicity

Dietary Saturated Fat Reduces Alcoholic Hepatotoxicity in Rats by Altering Fatty Acid Metabolism and Membrane Composition^1,2

Martin J. J. Ronis^*,,3, Soheila Korourian^**, Michelle Zipperman^*, Reza Hakkak and Thomas M. Badger^*,

^* Arkansas Children’s Nutrition Center and Department of Pharmacology/Toxicology, ^** Department of Pathology, Department of Dietetics & Nutrition, and Department of Physiology/Biophysics, University of Arkansas for Medical Sciences, Little Rock, AR 72205

³To whom correspondence should be addressed. E-mail: RonisMartinJ{at}uams.edu.

	ABSTRACT

TOP ABSTRACT METHODS RESULTS DISCUSSION LITERATURE CITED

 
Rats fed a saturated fat diet are protected from experimentally induced alcoholic liver disease, but the molecular mechanisms underlying this phenomenon remain in dispute. We fed male Sprague-Dawley rats intragastrically by total enteral nutrition using diets with or without ethanol. In 1 control and 1 ethanol group, the dietary fat was corn oil at a level of 45% of total energy. In other groups, saturated fat [18:82 ratio of beef tallow:medium-chain triglyceride (MCT) oil] was substituted for corn oil at levels of 10, 20, and 30% of total energy, while keeping the total energy from fat at 45%. After 70 d, liver pathology, serum alanine aminotransferase (ALT), biochemical markers of oxidative stress, liver fatty acid composition, cytochrome P450 2E1 (CYP2E1) expression and activity and cytochrome P450 4A (CYP4A) expression were assessed. In rats fed the corn oil plus ethanol diet, hepatotoxicity was accompanied by oxidative stress. As dietary saturated fat content increased, all measures of hepatic pathology and oxidative stress were progressively reduced, including steatosis (P < 0.05). Thus, saturated fat protected rats from alcoholic liver disease in a dose-responsive fashion. Changes in dietary fat composition did not alter ethanol metabolism or CYP2E1 induction, but hepatic CYP4A levels increased markedly in rats fed the saturated fat diet. Dietary saturated fat also decreased liver triglyceride, PUFA, and total FFA concentrations (P < 0.05). Increases in dietary saturated fat increased liver membrane resistance to oxidative stress. In addition, reduced alcoholic steatosis was associated with reduced fatty acid synthesis in combination with increased CYP4A-catalyzed fatty acid oxidation and effects on lipid export. These findings may be important in the nutritional management and treatment of alcoholic liver disease.

KEY WORDS: • ethanol • dietary fat • hepatotoxicity • oxidative stress • steatosis

Diet clearly plays an important role in determining the nature of liver pathology associated with chronic alcohol consumption (1–4). However, the delineation of dietary influences, identification of specific dietary factors, and the mechanisms of action remain controversial, especially given the variability in pathological outcomes reported by investigators using different rat models of alcohol-induced liver damage (ALD)⁴ involving orally fed and intragastrically infused liquid diets (3–7).

For example, chronic oral consumption of a liquid diet (such as the Lieber DeCarli diet) containing ethanol and high levels of carbohydrate causes hepatosteatosis, but few other indicators of pathology (5). However, studies using the Tsukamoto-French intragastric infusion model have reported that diets containing high levels of carbohydrate and as little as 5% total energy as dietary fat can produce steatohepatitis characterized by severe and progressive steatosis, focal necrosis, and centrilobular infiltration of mononuclear cells (6). In contrast, intragastric infusion of an ethanol-containing, high-carbohydrate diet (total energy consisting of 35% ethanol, 19% carbohydrate, 30% fat, and 16% protein) using the total enteral nutrition (TEN) model developed in this laboratory only produced hepatosteatosis similar to the pathology seen with the Lieber DeCarli liquid diet (4). When the carbohydrate content of this latter diet was reduced below 5% of total energy, steatohepatitis developed (4). In addition, when the Lieber DeCarli diet was modified to reduce carbohydrate to 5% of total energy as described by Lindros and Jarvelainen, additional liver pathology developed (8,9).

In addition to a role for carbohydrate content (or dietary carbohydrate to lipid ratio) in the development of ALD, it has been suggested that the type of dietary fat is an important factor affecting the pathological outcome. In particular, it has been suggested that dietary unsaturated fat is required in the development of ALD, with the severity of pathology correlated with the dietary linoleic acid content (1). Nanji et al. (10,11) reported that almost no liver damage occurred in rats fed ethanol and SFAs [medium-chain triglyceride (MCT) oil or beef tallow] and that dietary saturated fat reversed ethanol-induced pathology (12). More severe liver damage was reported by investigators using ethanol diets incorporating (n-3) or (n-6) polyunsaturated fats such as fish oil (13).

In light of the above differences between rat models in the effects of diet on the development of ALD, the current study examined the effects of increasing substitution of saturated for unsaturated fat on in vivo ethanol metabolism, liver pathology, cytochrome P450 2E1 (CYP2E1) and cytochrome P450 4A1 (CYP4A1) content and activity, lipid peroxidation, and hepatic fat composition, using isocaloric low-carbohydrate and high-ethanol TEN model diets.

	METHODS

TOP ABSTRACT METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Animals. The experiments were approved by the Institutional Animal Care and Use Committee at the University of Arkansas for Medical Sciences. Male Sprague-Dawley rats were purchased from Harlan Industries. All rats were housed in an Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) approved animal facility with a 12-h light–dark cycle and constant humidity. Rats were killed by decapitation after administration of 90 mg/kg body weight of phenobarbital.

    Experiments. Groups of male Sprague-Dawley rats (body weight 250 to 300 g, n = 8/group) were surgically implanted with intagastric cannulae at the Arkansas Children’s Nutrition Center. After surgery, the rats were allowed to recover for 14 d until their presurgical weight was attained. Thereafter, the rats were fed by TEN as previously described (4). Diets were isocaloric (782 kJ · kg^-0.75 · d^-1), met NRC nutrient requirements for normal growth, and were infused at 3 mL/h for 23 h/d (14). There were 8 treatment groups, each infused with a different diet (Table 1). In the control group diets, the levels of protein, carbohydrate, and fat were held constant at 16, 39, and 45% of total energy, respectively. In the alcohol diets, ethanol was substituted for carbohydrate energy, beginning at 10 g · kg^-1 · d^-1 and increased by 0.5 g · kg^-1 · wk^-1 to attain a final concentration of 12.5 g · kg^-1 · d^-1 (39% of total energy; Table 1). The levels of saturated fat were 0, 10, 20, and 30% of total energy. To achieve the dietary fat levels, saturated fat (an 18:82 ratio of beef tallow:MCT oil) was substituted for corn oil (Table 2). Diets were infused for 70 d.

Liver FFA composition was analyzed as follows. Liver tissue (0.5 g) was weighed and homogenized in 4 mL/g of distilled water. To extract the lipid fraction, the homogenate was first mixed with 20 mL/g of chloroform:methanol (2:1, v:v) and stored for 2 h at room temperature. After centrifugation at 1800 x g for 20 min, the upper phase of methanol was removed and discarded. Approximately 5 mL of the lower chloroform layer was analyzed. An internal standard of 20 µL of 17:0 fatty acid (100 g/L) was added for quantification. To assay total fatty acid recovery, the internal standard was also added to marked samples before phase separation and extraction instead of to the chloroform layer. The liquid was evaporated under N₂ to limit lipid peroxidation, and the sample was redissolved in 200 µL of chloroform. The FFAs were separated and extracted as described by Morimoto et al. (17). Aliquots (10 µL) were spotted on 3 lanes of 200-µm TLC plates (LHPKDF Silica Gel 60 nm; Whatman). A solvent of petroleum ether:diethylether:acetic acid (90:9:1, by vol) was used to separate bands of triglyceride, cholesterol ester, and fatty acid from top to bottom; a lower phospholipid band was visible in some samples. The bands were detected with a short-wave UV light source. The fatty acids (R_f= 0.53) were scraped and methylated directly by adding 1 mL of boron trifluoride (12% in methanol), and heating at 80°C for 12 to 15 min at a moderate boil. Large 10- x 75-mm glass test tubes were used to prevent reflux loss. The (FAMEs were extracted with petroleum ether 3 times, removed in the upper layer, and dried under N₂. Samples were redissolved in 100 µL of carbon disulfide, and 1 µL was analyzed using a QP5000 GC/MS with a SP-2340 fused silica capillary column, 30 m x 25 mm i.d. (Supelco). A time program maintained an isothermal column temperature of 50°C for 2 min, then increased the temperature 15°C/min to 100°C and held for 4 min, then increased 5°C/min to 240°C and held for 4.67 min. The injector and interface temperatures were 250 and 260°C, respectively. Retention times were compared to authentic FAME standards, and quantification was calculated based on an internal standard curve of 17:0. Liver microsomes were prepared by differential ultracentrifugation (18), and microsomal carbon tetrachloride–dependent lipid peroxidation was assessed as described by Johansson and Ingelman-Sundberg (19). p-Nitrophenol hydroxylation was measured spectrophotometrically as described by Koop et al. (20). Lauric acid 12-hydroxylation was measured by TLC, using ¹⁴C-lauric acid, and CYP2E1 and CYP4A1 apoprotein expression were quantitated by Western immunoblotting as described previously (21).

    Pathological evaluation. Liver pathology was assessed in hematoxilin-eosine–stained liver sections and evaluated by a board-certified pathologist (S.K.) using blind scoring procedures. For statistical comparisons, the levels of steatosis (macro- and microvesicular), leukocyte infiltration, and necrosis were scored on a scale of 1 to 5 (1 = no pathology, 5 = maximal pathology), and total pathology was defined as the sum of the steatosis, inflammation, and necrosis scores.

    Statistical analysis. The data are presented as means ± SEM. All data were analyzed by two-way ANOVA followed by the Student-Newman-Keuls test for specific comparisons between means, using the SigmaStat for Windows program (Jandal Scientific Software). Where data were not normally distributed, a two-way ANOVA of ranks followed by Dunn’s test was used to determine whether group medians differed, but means ± SEM were listed in the corresponding data tables for clarity. Differences between means or medians were considered significant at P < 0.05. The specific statistical comparisons between means or medians were as follows: within each level of saturated fat between rats fed the control and ethanol diets, respectively; within all levels of saturated fat for rats fed the control diets relative to those fed the 0% saturated fat control diet; and within all levels of saturated fat for rats fed the ethanol diets relative to those fed the 0% ethanol diet.

	RESULTS

TOP ABSTRACT METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Liver pathology. Interestingly, body weight gain decreased as the saturated fat content of the diet increased (P < 0.05), despite the isocaloric intake of energy. The significant ethanol-associated growth reduction of rats in the lower–saturated fat groups was eliminated at the highest concentration of dietary saturated fat. In contrast, the liver of rats fed ethanol and low-carbohydrate diets had significant hyperplasia at all levels of saturated fat (Table 3). There were no pathological changes in the liver in any control group (Figs. 1and 2; Table 3). Pathological evaluation revealed the presence of micro- and macrovasicular steatosis and inflammatory infiltrates of monocytes, polymorphonucleated granulocytes, and lymphocytes accompanied by foci or confluent foci of necrosis and increased serum ALT values in rats fed ethanol and low-carbohydrate, high–unsaturated fat diets (P < 0.05) (Figs. 1and 2; Table 3). As the dietary saturated fat content increased, liver pathology scores and ALT values decreased (P < 0.05). At 30% dietary saturated fat, ethanol-induced hepatic necrosis was eliminated, and micro- and macrovesicular steatosis and inflammation were markedly reduced, even though the carbohydrate and total fat content of the diets and the ethanol dose were identical to those administered to rats fed the low-carbohydrate, unsaturated fat diets.

View larger version (87K): [in this window] [in a new window]   FIGURE 1 Alcohol-induced liver pathology in male rats fed control or ethanol TEN diets containing various levels of saturated fat: liver from rat fed control TEN diet with 0% saturated fat (A); liver from rat fed ethanol TEN diet with 0% saturated fat (B); liver from rat fed ethanol TEN diet with 30% saturated fat (C); liver from rat fed ethanol TEN diet with 0% saturated fat at higher magnification, showing development of micro- and macrovesicular steatosis accompanied by foci of macrophage infiltration and necrosis (D); and liver from rat fed ethanol TEN diet with 30% saturated fat at higher magnification, showing dramatically reduced pathology (E).

View larger version (15K): [in this window] [in a new window]   FIGURE 2 Total pathology score (steatosis, inflammation, and necrosis) of livers from male rats fed control or ethanol (EtOH) TEN diets containing various levels of saturated fat. Values are means ± SEM, n = 4–8. Significant differences (P 0.05) are marked as follows: adifference between 0% control diet and control diet with higher percentage of saturated fat, bdifference between 0% EtOH diet and EtOH diet with higher percentage of saturated fat, and cdifference between EtOH and control diets with the same level of saturated fat.

View larger version (56K): [in this window] [in a new window]   FIGURE 3 Western blots of CYP apoprotein expression in hepatic microsomes from male rats fed control (C) or ethanol (E) TEN diets containing various levels of saturated fat. The ethanol induction of CYP2E1 is independent of dietary saturated fat content (panel A), and the induction of CYP4A1 occurs with both ethanol and saturated fat (panel B).

    Fatty acid -hydroxylation and CYP4A1 expression.

    Liver antioxidant status and oxidative stress. The hepatic GSH concentration decreased (P < 0.05) and the GSSG:GSH ratio increased (P < 0.05) in rats fed ethanol and unsaturated fat diets, suggesting impaired antioxidant status in the livers with the greatest pathology (Table 5). This was accompanied by increased (P < 0.05) lipid peroxidation (TBARS), indicative of oxidative stress (Table 5). As dietary saturated fat content increased, these effects of ethanol decreased markedly. In addition, CYP2E1-mediated, carbon tetrachloride–dependent lipid peroxidation decreased (P < 0.05) in a dose-dependent manner as the percentage of dietary saturated fat increased (Fig. 4).

View larger version (17K): [in this window] [in a new window]   FIGURE 4 Carbon tetrachloride–dependent lipid peroxidation in in vitro preparations of liver microsomes from male rats fed control or ethanol (EtOH) TEN diets containing various levels of saturated fat. Thiobarbituric acid (TBA)-reactive metabolites are lipid peroxidation products such as malondialdehyde and other short-chain aldehydes. Values are means ± SEM, n = 4–8. Significant differences (P 0.05) are marked as follows: adifference between 0% control diet and control diet with higher percentage of saturated fat, bdifference between 0% EtOH diet and EtOH diet with higher percentage of saturated fat, and cdifference between EtOH and control diets with the same level of saturated fat.

	DISCUSSION

TOP ABSTRACT METHODS RESULTS DISCUSSION LITERATURE CITED

The reduction in liver pathology was accompanied by significant reductions in indexes of oxidative stress, such as the GSSG:GSH ratio and markers of lipid peroxidation (TBARS). However, this was not the result of changes in either ethanol clearance (as indicated by characteristics of the UEC pulses) or the induction of CYP2E1, because neither CYP2E1 apoprotein expression nor a direct measure of CYP2E1-dependent activity (p-nitrophenol hydroxylation) differed in any of the ethanol-treated groups. This finding differs from reports by some investigators using intragastric infusion models who found marked increases in CYP2E1 expression as the degree of unsaturation of the dietary fat source increased (13,24). Reduced lipid peroxidation may be due to lower unsaturated fatty acid concentrations in the cellular membranes, because carbon tetrachloride–dependent lipid peroxidation in liver microsomes isolated from ethanol-treated rats in the present study decreased as the dietary saturated fat content increased (P < 0.05). In vitro lipid peroxidation decreased even though CYP2E1, which initiates lipid peroxidation in this system as a result of reductive dechlorination of carbon tetrachloride to the trichloroethyl radical (20), was expressed at similar levels in all ethanol-treated groups. Because saturated and monounsaturated fats are not substrates for lipid radical propagation, and polyunsaturated fats are, a reduction in unsaturated fat content would explain the observed effect. In the present study, total liver concentrations of free PUFAs, such as linoleic acid and arachidonic acid, decreased markedly in ethanol-treated rats as the dietary saturated fat content increased. Thus, the levels of fatty acid substrates for lipid peroxidation were decreased. Nanji et al. (25) reported similar results, with a decrease in microsomal membrane linoleic acid concentrations of almost 50%, compared with those of rats fed ethanol and corn oil diets after feeding diets containing saturated fat and ethanol for 4 wk.

The development of alcoholic liver disease is clearly a multifactoral process in which many possible mechanisms may operate in parallel to produce a similar pathological outcome. Some investigators suggest that ALD is initiated by the CYP2E1-mediated production of reactive oxygen species (ROS) as the result of cycling in the absence of substrate, loosely coupled cycling to produce superoxide and H₂O₂, metabolism of ethanol to produce reactive hydroxyethyl radicals, and peroxidative metabolism of unsaturated fatty acids (26). The hypothesis is that oxidative stress produced by ROS generation causes lipid peroxidation and inflammatory changes resulting from Kupffer cell activation, which in turn cause necrosis and the activation of Stellate cells to produce fibrosis (26,27). The relative importance of CYP2E1 and other pathways of alcohol metabolism in the production of ROS and the development of ALD remain controversial. Some investigators report that chemical inhibitors of CYP2E1 can significantly reduce pathology in rat models of ALD (28,29). Others report no differences in pathological outcome with either CYP2E1 inhibitors or pan-P450 inhibitors or in CYP2E1 knockout mice (30–32). Most recently, increases in ethanol-induced ALT values and inflammation were reported in CYP2E1-overexpressing transgenic mice (33). However, Kono et al. (34) report that the Kupffer cell enzyme NADPH-oxidase is the major source of hepatic ROS following ethanol treatment and that ALD does not occur in p47-knockout mice lacking a critical subunit of this enzyme. These investigators suggest that Kupffer cell activation and ROS production resulting from ethanol-induced endotoxemia cause inflammation and necrosis independent of CYP2E1 or ethanol metabolism (32,34,35). It is clear from the current study that other factors, potentially downstream of CYP2E1 or alcohol metabolism to acetaldehyde, are important in pathological outcome, because dietary saturated fatty acids protected against ALD in the absence of important differences in either CYP2E1 expression or activity or in ethanol metabolism as indicated by urine alcohol levels. However, in the rat TEN model of ALD utilized in these studies, ethanol treatment did not appreciably increase plasma endotoxin concentrations (9).

Oxidative stress has been proposed as both a cause and a consequence of ALD (11,36) Some investigators report that indicators of oxidative stress, such as increased lipid peroxidation, increase with ethanol-induced pathology rather than precede it (36). Other laboratories report markedly increased oxidative stress in the liver of rats fed the Lieber DeCarli diet even though these rats did not develop substantial inflammation, necrosis, or fibrosis in addition to steatosis (2,5). In addition, a study reports that the abolition of lipid peroxidation by feeding antioxidants such as vitamin E does not affect the development of steatosis (2). The current study supports a role for oxidative stress in the development of ALD because reduction of the pathology score correlated with reduced oxidative stress markers. It is of interest that in a recent study, Nanji et al. (12) were able to reverse inflammatory and fibrotic changes in the liver of ethanol-fed rats by replacing diets containing only polyunsaturated fat (fish oil) with diets containing only saturated fatty acids (palm oil or MCT oil) despite continued ethanol administration. The protection afforded by saturated fat was accompanied by decreased levels of lipid peroxidation.

    Molecular mechanisms underlying alcohol-induced steatosis. Hepatic triglyceride and FFA concentrations both decreased markedly in rats in the 30% saturated fat diet groups relative to those in the corn oil–only groups. In this regard it is interesting that none of the medium-chain fatty acids found in the dietary MCT oil were present in the fatty acid pool in the liver. The MCT oil oxidizes rapidly in the liver and increases energy expenditure (24). Reductions in the size of the fatty acid precursor pool might partly explain the decrease in liver triglyceride concentration. The increase of CYP4A1 expression in the 30% saturated fat group would also decrease FFA concentration through the induction of fatty acid degradation via oxidation at the terminal carbon (-hydroxylation). Because the hepatosteatosis is primarily caused by triglyceride accumulation, decreased fatty acid synthesis and increased degradation could both contribute to the decrease in steatosis in the saturated fat diet groups. However, the decreases in both micro- and macrosteatosis in the liver in the ethanol-treated 30% saturated fat group relative to the ethanol-treated corn oil group were much greater than the decrease in total liver triglyceride concentrations. It appears that most of the triglycerides in the ethanol-treated corn oil group were present as intracellular and extracellular lipid droplets, whereas the triglycerides in the saturated fat group may have been packaged differently, perhaps as lipoprotein complexes with apolipoprotein B-100 (apoB100). It is also interesting that despite enormous differences in steatosis, all livers from rats in the ethanol-treated groups had a similar increase in weight. It appears that ethanol-induced liver hyperplasia is not related to fat accumulation.

Among the few points on which researchers investigating ALD agree is that although the development of steatosis does not in itself necessarily lead to further pathological changes (4), steatosis appears to be an essential prerequisite for the development of inflammation, necrosis, and fibrosis following ethanol treatment, and the severity of additional liver damage correlates with the degree of steatosis (6). The current study found a dose-dependent decrease in the severity of alcohol-induced micro- and macrovesicular steatosis as dietary saturated fat content increased from 0 to 30% of total energy, even though the total dietary fat content, fat:carbohydrate ratio, ethanol dose, and rates of CYP2E1-dependent ethanol metabolism and overall alcohol clearance did not change. In addition, ethanol-induced steatosis was almost completely eliminated at 30% of total energy as saturated fat, even though the diet still included 15% of total energy as corn oil and liver triglyceride concentrations were markedly increased.

The mechanisms underlying the ethanol-induced development of fatty liver appear complex. It was originally suggested that the generation of NADH through ethanol oxidation might inhibit NAD⁺-dependent steps of the Krebs cycle and fatty acid ß-oxidation (37). However, reductive stress resulting from the apparent excess of NADH dissipates following chronic feeding of ethanol to baboons, and yet steatosis persists (38). Alternative explanations for the development of steatosis include suggestions of impaired lipoprotein transport (39), reduced fatty acid oxidation through inhibition of peroxisome proliferator-activated receptor (PPAR) pathways (40), and enhanced lipogenesis (41). Of these, inhibition of PPAR is perhaps the least likely, because the current study found increased expression of CYP4A1, a classic PPAR regulated gene, in steatotic livers from rats in the ethanol-treated 0% saturated fat diet group and previous studies have also found increased hepatic CYP4A expression following ethanol treatment (42,43).

In a recent study, You et al. (41) suggested that ethanol-induced steatosis is caused by the acetaldehyde-mediated activation of sterol regulatory element-binding protein 1. This in turn could activate a battery of lipogenic enzymes, including fatty acid synthease, stearoyl-CoA desaturase, citrate lyase, and malic enzyme, and could be inhibited by sterols such as cholesterol. In the current study, ethanol treatment caused a marked 33 to 50% increase in liver FFA concentration in all groups, consistent with increased fatty acid synthesis. However, the severity of steatosis varied widely depending on the dietary saturated fat content, even though rates of in vivo ethanol metabolism, and thus presumably acetaldehyde concentrations, were identical. In addition, liver FFA concentrations doubled in rats in the 0% saturated fat control groups, compared to those in the 30% saturated fat control groups, yet none of the rats in these control groups developed steatosis.

Dietary saturated fat composition may significantly alter fatty acid synthesis. It certainly alters fatty acid degradation via oxidation at the terminal carbon, because the CYP4A1 concentration was dramatically higher in rats fed the 30% saturated fat diet. Although the level of fatty acid precursors could affect the degree to which triglycerides are synthesized after ethanol treatment, ethanol must also induce micro- and macrovesicular triglyceride accumulation by mechanisms downstream from fat synthesis, possibly through effects on triglyceride transport (39). The effects of ethanol on these pathways might be inhibited by saturated fatty acids.

A recent report of marked deceases in the synthesis of apoB100, a rate-determining step in hepatic lipid export in patients with nonalcoholic steatohepatitis, a condition with a pathology very similar to that of ALD, suggests that decreased synthesis of this protein may also be involved in the development of alcohol-induced fatty liver (44). In addition, it was recently reported that ethanol treatment markedly reduced levels of microsomal triglyceride transport protein, another enzyme with an important role in the transfer of triglycerides between membranes and lipoproteins such as apoB100 (45). This effect, together with ethanol-induced steatosis, was reversed by treatment with human recombinant hepatocyte growth factor (45). We are continuing to investigate these pathways in rats fed ethanol with various diets.

	ACKNOWLEDGMENTS

 
The authors thank Terry Fletcher, Matt Ferguson, Kim Hale, Jamie Badeaux, Shanda Ferguson, Drew Holder, Carolyn Cimino, Misty Reeves, Chris Dahl, Greg Heird, James M. Robinette, and Michele Perry for technical assistance on this project.

	FOOTNOTES

 
¹ Previously presented, in part, as an abstract at the Society of Toxicology Meeting, San Francisco, CA, March 25–29, 2001. Hakkak, R., Korourian, S., Fletcher, T., Mercado, C., Holder, D., Ronis, M.J.J. & Badger, T. M. (2001) Effects of dietary saturated fat on growth and ethanol-induced hepatotoxicity in male rats. Toxicologist 60: 347 (abs.).

² Supported in part by R01 AA088645 (T.M.B.).

⁴ Abbreviations used: ALD, alcohol-induced liver damage; ALT, alanine aminotransferase; apoB100, apolipoprotein B-100; CYP2E1, cytochrome P450 2E1; CYP4A1, cytochrome P450 4A1; GSH, glutathione; GSSG, oxidized glutathione; MCT, medium-chain triglyceride; PPAR, peroxisome proliferator-activated receptor ; ROS, reactive oxygen species; TEN, total enteral nutrition; UEC, urine ethanol concentration.

Manuscript received 16 October 2003. Initial review completed 1 December 2003. Revision accepted 12 January 2004.

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