IN DEVELOPMENT OF ALCOHOLIC LIVER DISEASE IN ... - CiteSeerX

TRANSACTIONS OF THE AMERICAN CLINICAL AND CLIMATOLOGICAL ASSOCIATION, VOL. 113, 2002

INTERACTIONS OF ETHANOL AND FOLATE DEFICIENCY IN DEVELOPMENT OF ALCOHOLIC LIVER DISEASE IN THE MICROPIG CHARLES H. HALSTED and (by invitation) JESUS A. VILLANUEVA and ANGELA M. DEVLIN DAVIS, CA

S. JILL JAMES JEFFERSON, AR

ABSTRACT Folate deficiency is present in most patients with alcoholic liver disease (ALD), whereas folate regulates and alcoholism perturbs intrahepatic methionine metabolism, and S-adenosyl-methionine prevents the development of experimental ALD. Our studies explored the hypothesis that abnormal methionine metabolism is exacerbated by folate deficiency and promotes the development of ALD in the setting of chronic ethanol exposure. Using the micropig animal model, dietary combinations of folate deficiency and a diet containing 40% of kcal as ethanol were followed by measurements of hepatic methionine metabolism and indices of ALD. Alcoholic liver injury, expressed as steatohepatitis in terminal 14 week liver specimens, was evident in micropigs fed the combined ethanol containing and folate deficient diet but not in micropigs fed each diet separately. Perturbations of methionine metabolism included decreased hepatic S-adenosylmethionine and glutathione with increased products of DNA and lipid oxidation. Thus, the development of ALD is linked to abnormal methionine metabolism and is accelerated in the presence of folate deficiency.

INTRODUCTION Alcohol is a frequent component of the American diet that provides 7.1 kcal/g, is essentially devoid of micronutrients, and, when consumed in excess, influences the availability and hepatic metabolism of many From the University of California, Davis, California and National Toxicological Research Center, Jefferson Arkansas. Address for reprints: Charles H. Halsted, MD, Department of Internal Medicine, TB156, School of Medicine, One Shields Avenue, University of California Davis, Davis, CA 95616. Phone: 530-752-6778; Fax: 530-752-3470. 151

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nutrients (1). At least 5% of the population consume alcohol in excess and are at risk for alcoholic liver disease (ALD), the 11th cause of mortality in the United States (2). It has been increasingly recognized that one or many features of malnutrition are present in virtually all patients who develop ALD. For example, varied degrees of protein calorie malnutrition that correlated with the severity of ALD were found universally in more than 500 ALD patients enrolled in two multicenter U.S. Veterans Administration studies (3). Among micronutrients, folate deficiency is the most common finding in chronic alcoholics, occurring in up to 80% of those at risk for developing ALD (4-6). In clinical studies of alcoholics and in animal models of chronic alcohol exposure, folate deficiency was shown to result from poor diet and combinations of decreased folate absorption, hepatic uptake, and increased renal excretion (7-13). The frequent finding of folate deficiency in chronic alcoholism suggests a role for this vitamin in the pathogenesis of ALD. According to extensive studies in animal models, current concepts on the pathogenesis ofALD involve ethanol and cytokine induced production of reactive oxygen species (ROS), which, together with reduced antioxidant defense, results in hepatocyte necrosis, inflammation, and eventual collagen production and cirrhosis (14-16). The metabolism of ethanol in the liver by the microsomal enzyme CYP2E1 generates oxidative injury through formation of the hydroxyethyl radical (17,18). At the same time, gut-derived lipopolysaccharide (LPS) enterotoxin stimulates the production in Kupffer cells of tumor necrosis factor a (TNFa), which, in turn, invokes a signal transduction cascade in hepatocytes that involves production of ceramide from membrane sphingomyelin and enhances the mitochondrial generation of ROS (19,20). Oxidative liver injury is the net result of enhanced generation of ROS and depletion of antioxidants such as glutathione (GSH). At the same time, apoptosis or cell death is promoted by stimulation ofintracellular caspases and enhanced DNA strand breakage (15,21). Over the past decade, it has become apparent that abnormal hepatic methionine metabolism is integral to ALD and perhaps central to its pathogenesis. As depicted in Figure 1, folate in the form of 5-methyltetrahydrofolate (5-MTHF) and homocysteine are substrates with vitamin B12 co-factor methionine synthase for the production of endogenous methionine that, in turn, is substrate for methionine adenosyl transferase (MAT1) in the production of 6-8 g/d of S-adenosylmethionine (SAM) in the human liver. SAM is the principal methyl donor in a multitude of reactions and regulates a number of methionine cycle pathways, including the generation of GSH from homocysteine (16,22).

METHIONINE CYCLE, FOLATE DEFICIENCY AND ALD

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cystttlonlne l glutathione CY(SAH -t FIG. 1. Folate and methionine metabolism in the liver. 5-methyltetrahydrofolate (5-MTHF), is substrate for the methionine synthase (MS) reaction that generates methionine from homocysteine. In an alternate salvage pathway, betaine, a product of choline metabolism, is the substrate for betaine homocysteine methyltransferase (BHMT). Methionine is converted to S-adenosylmethionine (SAM) by methionine adenosyl transferase (MAT). Through reactions that include DNA methylation and the synthesis of phosphatidylcholine (PC) from phophatidylalcoholamine (PE) by PE methyl transferase (PEMT), SAM is converted to S-adenosylhomocysteine (SAH), which is also up-regulated by synthesis from homocysteine through the reversible SAH hydrolase (SAHh) reaction. SAM regulates the synthesis of glutathione (GSH) by up-regulation of cystathionine dsynthase and the homocysteine transsulfuration pathway. SAM provides negative regulatory feedback to the methylenetetrahydrofolate reductase (MTHFR) reaction that converts 5,10 methenyltetrahydrofolate (5,10-MTHF) to 5-MTHF.

The ratio of SAM to its product S-adenosylhomocysteine (SAH) is considered a comprehensive measure of inhibition of functional SAM activity, in particular as related to methylation (23). Early clinical studies in this field demonstrated decreased levels of MATi activity and SAM in liver biopsies from ALD patients (24,25). The administration of SAM attenuated the decrease in GSH and the experimental development of ALD in ethanol fed baboons, and improved the clinical status of patients with varied degrees of ALD (26,27). Recently it was demonstrated in ethanol fed rats that SAM promotes the fluidity of the mitochondrial membrane and the transport of intracellular GSH to its mitochondrial site of activity (28). We developed an animal model ofALD in the micropig, a species that consumes ethanol voluntarily in the diet. Initially, castrated male Yucatan micropigs were fed 40% of kCal as ethanol or cornstarch

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control and all essential nutrients including an excess of folate. Among the ethanol-fed micropigs we observed typical features of alcoholic liver injury after 12 months of feeding and cirrhosis at 21 months, together with the accumulation of protein adducts of the alcohol metabolite acetaldehyde and the lipid oxidant product malondialdehyde (29,30). There were no histological changes in a subsequent study of intact and uncastrated micropigs fed the same diets for 12 mo, which we ascribed to changes in testosterone secretion (31,32). On the bases of the high frequency of folate deficiency in human ALD, the essential role of folate in hepatic methionine metabolism, and prior evidence for disturbed methionine metabolism in ALD, the present study tests the hypothesis that folate deficiency promotes both abnormal hepatic methionine metabolism and the development of ALD. MATERIALS AND METHODS Twenty-four intact male micropigs each weighing -20 kg were purchased at 6 mo age (Sinclair, Columbia, MO) and were grouped to receive four different diets for 14 weeks. Each diet provided 90 kCal/kg body wt/d as polyunsaturated corn oil at 33% of kCal, protein as vitamin-free casein at 2 g/lkg body wt/d, and cornstarch as carbohydrate, or the same diet substituting ethanol for cornstarch at 40% of kCal, equivalent to 5 glkg body wt/d. Methionine, choline, and all essential minerals and vitamins were provided with or without folic acid in accord with the established requirements of growing swine (33). The four diets were folate-sufficient (FS, or control) with excess folate at 14.5 ggfkg body wt, folate deficient containing no added folate (FD), folate sufficient with ethanol (FSE), and folate deficient with ethanol (FDE). The animals were weighed and group-paired weekly to ingest the same mean amount as ingested by the FDE group. The U.C. Davis Animal Welfare Committee approved the feeding protocol and all experimental studies. Animals were housed in individual kennels at the University of California Davis Animal Resources Center facilities, which are approved by the National Institutes of Health and animal care followed the standards and procedures outlined in the National Academy of Sciences "Guide for the Care and Use of Laboratory Animals." Blood samples were obtained for measurements of serum homocysteine, aspartyl aminotransferase (AST), alanine aminotransferase (ALT), and malondialdehyde (MDA) (34,35) as an index of lipid peroxidation. Terminal urine was obtained by bladder puncture and was


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analyzed for 8-oxo-2'-deoxyguanosine (Oxo8dG) as an index of DNA oxidation (36). Terminal liver samples were homogenized and analyzed for folate (37), methionine and choline metabolites (38,39), activities of MS (40) and BHMT (41), and histology. Data were analyzed by repeated measures or 2-way analysis of variance, where the independent variables were folate status (sufflcient or deficient), ethanol treatment, and time. When interactions were significant, separate sub-group analyses were performed; otherwise analyses were done with both variable groups pooled. Correlations between variables were determined by linear regressions using all points. RESULTS During the 14 weeks of the experiment, micropigs in the FS control group gained more than twice the weight of each of the other three groups. Mean plasma homocysteine levels were increased in all three experimental groups from week 6 onward. The greatest effect occurred in the FDE group, where levels were increased 3-fold over those in FS control and were greater than those each of the other experimental groups. Table 1 describes the significant effects of each dietary regimen on terminal hepatic methionine enzymes and metabolites, urinary Oxo8dG, plasma MDA, AST, and ALT. Terminal hepatic folate levels were decreased by one half in groups FD and FDE, homocysteine was elevated in FSE and FDE, and methionine was reduced in FD, FSE, and FDE. Consistent with reduced methionine, MS activity was reduced in FSE and FDE, whereas the compensatory BHMT activity pathway was increased in FD and FDE. Choline levels were unchanged, while betaine was reduced in FD and FDE consistent with its role as substrate for BHMT. At the same time, hepatic PC was reduced in all three experimental groups due to an interaction between ethanol and folate deficiency. Consistent with the effects of feeding on reductions of liver methionine levels in all three experimental groups, hepatic SAM was reduced in FSE and FDE, hepatic SAH was increased and the SAM to SAH ratio was decreased in all three experimental groups due to an interaction between ethanol and folate deficiency. Hepatic GSH, the principal mitochondrial antioxidant, was reduced in FSE and FDE, correlating with levels of SAM. Urinary Oxo8dG, a measure of DNA oxidation, was increased in FD, FSE, and FDE. Plasma MDA, a measure of lipid peroxidation, was increased in FSE and FDE. Levels of plasma AST and ALT, each a measure of liver

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TABLE 1 Effects of Experimental Diets on Hepatic Methionine Enzymes and Metabolites, DNA and Lipid Peroxidation, and Liver Injury Enzymes Variable FD FSE FDE Folate (-) * Homocysteine (+) t t Methionine (-) * t t MS (-) t t BHMT(±) * * Choline Betaine (-) * * PC (-) * t SAM (-) t SAH(+) * .t *t SAM/SAH (-) * t GSH (-) t t Oxo'dG (urine) (+) * t t MDA (plasma) (+) t t AST (plasma) (+) t tt ALT (plasma) (+) t t (+) Positive effect, (-) negative effect, * significant effect of folate deficiency, t significant effect of ethanol feeding, t significant interaction of folate deficiency and ethanol feeding. A significant positive correlation was found by linear regressions between individual values of hepatic SAM and GSH. Significant negative correlations were found between values of hepatic GSH or the SAM to SAH ratio and levels of urine Oxo'dG and plasma MDA, and between the SAM to SAH ratio and values of plasma ALT.

injury, were increased in FSE and FDE. Levels of urinary Oxo8dG and plasma MDA were each correlated negatively to the SAM to SAH ratio and to levels of hepatic GSH. The level of plasma ALT was correlated negatively to the SAM to SAH ratio. While there were no changes in liver histology in FS control, FD and FSE micropigs, livers from 5 of the 6 animals in group FDE demonstrated lesions characteristic of alcoholic liver injury, specifically diffuse intralobular hepatocytes steatosis and necrosis with infiltration of inflammatory cells.

DISCUSSION The significance of the present findings relate to the potential importance of perturbations of methionine metabolism in chronic alcoholism during development of ALD. In addition to rodent ethanol feeding studies describing reduction in MS activity and SAM production with compensatory increase in BHMT (42-44), a clinical study demonstrated decreased transcripts of MATl1A, BHMT, C,3S and MS in liver biopsies from ALD patients (45). Using the intragastric ethanol-


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fed rat model, others recently demonstrated decreased hepatic levels of methionine and SAM, together with decreased MAT1 but increased MAT2 activity and increased DNA strand breaks in the ethanol group (46). In a previous study of uncastrated male micropigs fed control or ethanol containing diets identical to the present FS and FSE groups for 12 months, we found that ethanol feeding significantly reduced MS and the SAM to SAH ratio, while causing DNA nucleotide imbalance and increasing hepatocellular apoptosis (31). In the present studies, we tested the hypothesis that the development of ALD involves perturbations in hepatic folate-regulated methionine metabolism that result from chronic ethanol ingestion and are enhanced in the presence of folate deficiency in the micropig. The experimental design permitted analysis of the separate, additive, and synergistic effects of folate deficiency and chronic ethanol exposure on methionine and choline metabolism. By deleting folate from the diet, we achieved -50% lower liver folate levels in animals fed folate deficient diets with or without ethanol (Table 1). Ethanol feeding acted alone in elevating liver homocysteine and plasma MDA and ALT and in reducing MS activity, SAM, and GSH. Folate deficiency acted alone in increasing BHMT activity and reducing liver folate and betaine. Ethanol feeding and folate deficiency were additive in elevating serum homocysteine levels and liver SAH and Oxo8dG levels. On the other hand, ethanol feeding and folate deficiency were interactive and synergistic in reducing the liver SAM to SAH ratio and PC levels, in elevating serum AST levels, and in production of the histopathological features of steatonecrosis. The finding of a positive correlation of SAM to GSH is explained by the known regulatory role of SAM in the transsulfuration pathway (47), and links methionine metabolism to the anti-oxidative GSH response. The finding of a negative correlation of the SAM to SAH ratio and of GSH to plasma MDA levels links increased lipid peroxidation to SAM through its regulation of GSH production. Similarly, the finding of a negative correlation of the SAM to SAH ratio and of GSH to urinary Oxo8dG levels links increased DNA oxidation to SAM through its regulation of the anti-oxidant GSH. At the same time, the negative correlation of the hepatic SAM to SAH ratio to the plasma ALT level links hepatic liver injury to aberrations in the methionine cycle. By demonstrating the presence of steatonecrosis only in the FDE group and its absence in the FSE group, we determined that folate deficiency promotes while folate sufficiency attenuates the development ofALD. Further underscoring the significance of folate deficiency in presence of chronic ethanol feeding, the finding of abnormal histo-

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pathology in the FDE group micropigs after 14 weeks of feeding contrasts with absence of overt histopathology in our previous 12 mo study of micropigs fed the identical folate sufficient control or ethanol containing diets (31). How might the observed perturbations of methionine metabolism triggered by ethanol and accentuated by folate deficiency be related to the pathogenesis of ALD? A prior study showed induction of apoptosis of hepatocytes in rats fed diets deficient in folate, methionine, and choline, in association with nucleotide imbalance, as described by elevation in dUMP and reduction of dTMP levels (48). Since we achieved the same findings in ethanol fed but folate replete micropigs (31), we can presume that this apoptotic mechanism is magnified in pigs fed a combination of folate deficient and ethanol containing diets. Our present findings linking decreased SAM to GSH are relevant in view of the known effects of chronic alcoholism and ALD on reduction of liver GSH and its correction by SAM (26,27), and are consistent with concepts on the production of ethanol induced oxidative liver injury and the important role of GSH antioxidant defense (15,16). Lastly, a prior finding of SAM attenuation of plasma TNFa and LPS-stimulated steatonecrosis in choline deficient rats suggests that SAM deficiency accentuates TNFa mediated liver injury (49). The present study has potential implications for the prevention and treatment of ALD. As demonstrated previously in the baboon model (26), the provision of SAM would be expected to correct the observed decrease in SAM levels and increase levels of the antioxidant GSH in the ethanol fed groups, thereby countering the process of oxidative liver injury. Through its positive effect in maintaining 5,10-MTHF for the TS reaction, SAM would also predictably maintain DNA nucleotide balance and prevent DNA strand breakage and hepatocellular apoptosis. Although mechanisms were not provided, a multicenter clinical trial demonstrated the efficacy of SAM treatment of patients with established alcoholic hepatitis, an intermediate form of ALD (27). Further, provision of PC has been shown to prevent the development of ALD in the baboon model by uncertain mechanism (50). Based on our finding of diminished PC in all experimental groups and choline pathways, the salutary effect of correction of PC deficiency could be explained by enhancement of betaine as substrate for BHMT and methionine and SAM replenishment, or by enhancing sphingomyelin synthesis and thereby decreasing the availability of ceramide for the induction of oxidative hepatocellular necrosis (15,51). Finally, the present finding that steatonecrosis was limited to animals fed the combined folate deficient and ethanol FDE diet suggests that mainte-


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nance of normal folate stores through provision of supplemental folic acid or 5-MTHF may delay the onset or mitigate the severity of ALD. The present model of ALD in micropigs fed ethanol and folate deficient diets lends itself to further exploration of these potential mechanisms of liver injury and their potential correction by various modalities targeted at methionine metabolism in the liver.

ACKNOWLEDGMENTS This work was supported by USPHS Grants DK 45301 and DK 35747 to CHH. The authors are indebted to the following individuals for expert technical assistance: Tim Garrow, University of Illinois, Urbana, IL; Steven Zeisel, University of North Carolina, Chapel Hill, NC; and Lynn Wallock and Mark Shigenaga, Children's Hospital of Oakland Research Institute, Oakland, CA.

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methionine deficiency and TNF-alpha in lipopolysaccharide-induced hepatic injury. Am J Physiol 1998;275:G125-G129. 50. Lieber CS, Robins SJ, Li J, et al. Phosphatidylcholine protects against fibrosis and cirrhosis in the baboon [see comments]. Gastroenterology 1994;106:152-159. 51. Yen CL, Mar MH, Zeisel SH. Choline deficiency-induced apoptosis in PC12 cells is associated with diminished membrane phosphatidylcholine and sphingomyelin, accumulation of ceramide and diacylglycerol, and activation of a caspase. Faseb J 1999;13:135-142.

DISCUSSION Oates, Nashville: I wonder if you could comment on the cardiac toxicity of ethanol and how it's altered with these interventions? Halsted, Davis: We did not do cardiac studies on our micropigs. In general, there are two dichotomous views about alcohol and the heart. One is that alcoholic consumption promotes elevated circulating HDL cholesterol levels and therefore alcoholics are protected against coronary artery disease. On the other hand, the entity of alcoholic cardiomyopathy, considered a direct toxic effect of alcohol, is probably much more common than we think, occurs in heavy drinkers, and results in congestive heart failure. Thiamin deficiency, also common in heavily drinking alcoholics is a nutritional cause of high output cardiac failure, or "wet beri-beri." Fallon, Charleston: It has been some time since I delved into this field, and I'm glad you are continuing in doing so well. I had a couple of questions. I think I heard you that the folate deficient animals alone had no abnormalities in liver histology. Is that correct? Halsted: That's correct. Fallon: And that the development of the alcoholic lesion also required the addition of folate deficiency. Halsted: That is correct in this model of early changes after 14 weeks' feeding. Fallon: Well the problem that has always been in this field, and I know that you are well aware of this is that not all humans who develop alcoholic liver disease are folate deficient. It depends a little bit on socio-economic class and whether there are bankers who tipple or whether they are down and out and malnourished. So I wonder how you put this together back into the framework of the original problem which we've all struggled with is; why do 20% of alcoholic humans develop alcoholic liver disease? Halsted: The question why some people who drink a lot get liver disease and some do not is still unanswered. As you know, an old study of well-nourished German executives found a direct correlation between the amount of ethanol consumed and the likelihood of developing cirrhosis according to their liver biopsies. I can't tell you whether some of them had folate deficiency, but suspect that our model is more reflective of derelict malnourished alcoholics who are typically folate-deficient. At the same time, our micropig model suggests that people who are folate-sufficient, i.e., well-nourished, may be at less risk, or may take longer to develop alcoholic liver disease than folate-deficient derelict alcoholics. Billings, Baton Rouge: You may have a slide that you are about to show us, which presents the data for the group that were treated with folate to a more than sufficient level. We here at the Homestead, are, of course, going to have more than enough folate and, maybe more than enough alcohol. When we return home should we go on additional folate to reduce our potential alcohol induced hepatoxicity? Halsted: I knew someone was going to ask that. First of all we are all getting an adequate amount of folate in our diets thanks to the US national food folate fortification


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program. Whether that's going to make a difference in the incidence of alcoholic liver disease in this country is an interesting question. It is noteworthy that many physicians don't determine whether their patients drink too much and are at risk for liver disease. Since alcohol consumption is conducive of folate deficiency for a variety of reasons, I would recommend folic acid supplementation for any man consuming more than 2-3 drinks daily, and since women are more susceptible to alcoholic liver disease, I would recommend folic acid supplementation for any woman consuming more than 1 drink daily. There are retrospective epidemiological studies suggesting that 3-5 drinks per day may be the cutoff point before an individual is at risk for alcoholic liver disease. I doubt that many of us at this meeting have reached that point.