Chronic ethanol consumption increases homocysteine accumulation in hepatocytes.

Results of previous studies have shown that chronic ethanol administration impairs methionine synthetase activity and decreases S-adenosylmethionine levels in the liver, indicating interference with homocysteine remethylation. The purpose of the present study was to investigate the effects of chronic ethanol feeding on the accumulation of homocysteine (Hcy), a potentially toxic agent. The research was divided into two experiments. In Experiment A, hepatocytes were isolated from pair-fed control and ethanol-fed rats after 2 weeks of feeding, and the release of Hcy into the medium was determined. Hepatocytes obtained from ethanol-fed rats released twice as much Hcy into the medium as did those obtained from controls. When hepatocytes were challenged by a methionine load, a marked increase in Hcy generation was observed, and the increase was further enhanced in hepatocytes obtained from ethanol-fed rats. In Experiment B, hepatocytes were isolated from pair-fed control and ethanol-fed rats after 4 weeks of feeding (the feeding time required for significant formation of alcoholic fatty liver in rats). In this experiment, similar results were obtained with Hcy generation as in Experiment A. In Experiment B, supplementation of the incubation medium with betaine prevented the increase in generation of Hcy by methionine-treated control cells as well as the generation of Hcy by cells of ethanol-treated rats. These results indicate that betaine may have the potential as a therapeutic agent against toxic Hcy formation.

Betaine reduces hepatic lipidosis induced by carbon tetrachloride in Sprague-Dawley rats.

Carbon tetrachloride-injected rats were given liquid diets with and without betaine for 7 d. Hepatic lipidosis was induced by 4 daily injections of carbon tetrachloride (CCl4). Animals were killed and their livers and blood taken for analysis of betaine, S-adenosylmethionine (SAM), betaine homocysteine methyltransferase (BHMT), triglyceride, alanine aminotransferase and aspartate aminotransferase. Liver samples were also processed and stained for histological examination. Supplemental betaine reduced triglyceride in the liver and centrilobular hepatic lipidosis induced by the CCl4 injections. In both the control and experimental groups receiving betaine, liver betaine, BHMT and SAM were significantly higher than in their respective groups not receiving betaine. This study provides evidence that betaine protects the liver against CCl4-induced lipidosis and may be a useful therapeutic and prophylactic agent in ameliorating the harmful effects of CCl4.

The effect of betaine in reversing alcoholic steatosis.

The feeding of ethanol to experimental animals results in fatty infiltration of the liver. Recent findings have shown that ethanol-induced steatosis is accompanied by a lowering in hepatic S-adenosylmethionine (SAM) levels. It is known that SAM provides substrates for reduced glutathione formation and offers the cell protection from toxic metabolic oxidants. A recent study in this laboratory demonstrated that dietary supplementation with betaine generated increased SAM in the liver and protected against ethanol-induced steatosis. The present study not only showed that betaine supplementation to rats protects the liver from alcoholic steatosis, but also demonstrated that once steatosis is established, treatment with betaine partially reversed the steatosis after cessation of ethanol feeding. Furthermore, this study indicated that betaine supplementation to the diet had the capacity to attenuate steatosis despite the continued feeding of ethanol.

Betaine effects on hepatic methionine metabolism elicited by short-term ethanol feeding.

Previous studies in this laboratory have shown that feeding of ethanol to rats produces prompt inhibition of methionine synthetase (MS) as well as a subsequent increase in activity of betaine homocysteine methyltransferase (BHMT). Further studies have shown that supplemental dietary betaine enhanced methionine metabolism and S-adenosylmethionine (SAM) generation in control and ethanol-fed rats. Because MS and BHMT are both involved in the formation of SAM, this study was conducted to determine early effects of ethanol on hepatic SAM levels and the influence of betaine supplementation on parameters of methionine metabolism during the early periods of MS inhibition and enhanced BHMT activity. Results showed that ethanol feeding produced a significant loss in SAM in the first week with a return to normal SAM levels in the second week. Betaine feeding enhanced hepatic betaine pools in control as well as ethanol-fed animals. This feeding attenuated the early loss of SAM in ethanol-fed animals, produced an early increase in BHMT activity, and generated increased levels of SAM in both control and ethanol-fed groups. Furthermore, betaine lowered significantly the accumulation of hepatic triglyceride produced by ethanol after 2 weeks of ingestion.

Betaine, ethanol, and the liver: a review.

Two of the most important biochemical hepatic pathways in the liver are those that synthesize methionine and S-adenosylmethionine (SAM) through the methylation of homocysteine. This article reviews some recent findings in this laboratory, which demonstrate that ethanol feeding to rats impairs one of these pathways involving the enzyme methionine synthetase (MS), but by way of compensation increases the activity of the enzyme betaine:homocysteine methyl transferase (BHMT), which catalyzes the second pathway in methionine and SAM biosynthesis. It has been shown that despite the inhibition of MS, the enhanced BHMT pathway utilizes hepatic betaine pools to maintain levels of SAM. Subsequent to the above findings, it has been shown that minimal supplemental dietary betaine at the 0.5% level generates SAM twofold in control animals and fivefold in ethanol-fed rats. Concomitant with the betaine-generated SAM, ethanol-induced hepatic fatty infiltration was ameliorated. In view of the fact that SAM has already been used successfully in the treatment of human maladies, including liver dysfunction, betaine, shown to protect against the early stages of alcoholic liver injury as well as being a SAM generator, may become a promising therapeutic agent and a possible alternative to expensive SAM in the treatment of liver disease and other human maladies.

S-adenosylmethionine generation and prevention of alcoholic fatty liver by betaine.

Earlier studies by other investigators have shown that S-adenosylmethionine (SAM) has the capacity to attenuate liver injury in experimental animals. In a recent study in this laboratory, it was shown that when supplemental dietary betaine was given to control and ethanol-fed rats at the level of 0.50% (W/V), SAM levels were doubled in the livers of control animals and increased fivefold in livers of ethanol-fed rats. The increased levels of SAM in the livers of ethanol-fed animals protected the livers from fatty infiltration due to ethanol feeding. In this study, an attempt was made to determine the minimum level of dietary betaine that protects against the fatty infiltration. Levels of betaine at 0.05%, 0.10%, 0.25%, and 0.50% in semiliquid control and alcohol diets were tested in rats for 30 days. When hepatic betaine, SAM, and triglyceride levels were determined, it was demonstrated that only the dietary level of betaine at 0.50% supplied enough hepatic betaine to generate the level of SAM that was required to protect against the alcoholic steatosis resulting from the dietary ethanol. These results suggest that betaine, when given in sufficient amounts, may be a promising therapeutic agent in the treatment of liver disease.

Effect of chronic alcohol ingestion on hepatic folate distribution in the rat.

The mechanism by which ethanol impairs folate metabolism remains uncertain. In the present study, we used our new technique (affinity/HPLC) for folate analysis to study the effect of chronic alcohol ingestion on the content and distribution of folates in livers. Twelve male Sprague-Dawley rats (180 g) were divided into two groups, and fed for 4 weeks with Lieber-DeCarli semi-liquid isocaloric diets, with and without 5% ethanol. Livers were extracted in boiling, pH 9.3 borate buffers containing ascorbate/dithioerythritol. Folates in the supernatant fractions were purified by affinity chromatography and analyzed using ion pair high performance liquid chromatography. The data obtained showed that hepatic folate distribution in alcohol-treated rats differed from that of control animals in two ways. Livers from the ethanol-fed rats, when compared with those from control rats, exhibited increases in the percent concentrations of methylated tetrahydrofolates (21.46 +/- 2.21 vs 14.8 +/- 1.23), decreases in the percent concentrations of formylated tetrahydrofolates (25.62 +/- 4.02 vs 46.18 +/- 2.65) and higher concentrations of unsubstituted tetrahydrofolates (52.91 +/- 3.84 vs 38.88 +/- 2.50). In addition, alcohol ingestion was associated with longer glutamate chains of the folate molecules, characterized by lower relative concentrations of pentaglutamyl folates (29 vs 48%), and higher relative concentrations of hexa- and heptaglutamyl folates (55 vs 46% and 15 vs 6%) when compared with controls. The data are discussed in relation to the possibility that alcohol exerts its effect through: (1) inhibition of B12-dependent methyl transfer from methyltetrahydrofolate to homocysteine; (2) diversion of formylated tetrahydrofolates toward serine synthesis; and (3) interaction of acetaldehyde with tetrahydrofolates, thereby interfering with folate coenzyme metabolism.

The relationship of ethanol feeding to the methyl folate trap.

Feeding rats a semiliquid ethanol diet for a period of four weeks produced a hepatic accumulation of the methylating agent N5-methyltetrahydrofolate (N5CH3THF). When the ethanol-containing diet was supplemented with 0.5% betaine, an agent known to promote the generation of methionine and S-adenosylmethionine (SAM) in ethanol-fed animals, the accumulation of N5CH3THF was prevented. One index that the methyl folate trap exists is the hepatic accumulation of N5CH3THF, and a second index is that the N5CH3THF accumulation can be relieved by methionine administration. Since ethanol is shown to produce N5CH3THF accumulation in this study, and since betaine (a generator of methionine and SAM) acts to eliminate this accumulation, it is suggestive that ethanol can contribute to the impairing hepatic condition referred to as the "methyl folate trap."

Dietary betaine promotes generation of hepatic S-adenosylmethionine and protects the liver from ethanol-induced fatty infiltration.

Previous studies have shown that ethanol feeding to rats alters methionine metabolism by decreasing the activity of methionine synthetase. This is the enzyme that converts homocysteine in the presence of vitamin B12 and N5-methyltetrahydrofolate to methionine. The action of the ethanol results in an increase in the hepatic level of the substrate N5-methyltetrahydrofolate but as an adaptive mechanism, betaine homocysteine methyltransferase, is induced in order to maintain hepatic S-adenosylmethionine at normal levels. Continued ethanol feeding, beyond 2 months, however, produces depressed levels of hepatic S-adenosylmethionine. Because betaine homocysteine methyltransferase is induced in the livers of ethanol-fed rats, this study was conducted to determine what effect the feeding of betaine, a substrate of betaine homocysteine methyltransferase, has on methionine metabolism in control and ethanol-fed animals. Control and ethanol-fed rats were given both betaine-lacking and betaine-containing liquid diets for 4 weeks, and parameters of methionine metabolism were measured. These measurements demonstrated that betaine administration doubled the hepatic levels of S-adenosylmethionine in control animals and increased by 4-fold the levels of hepatic S-adenosylmethionine in the ethanol-fed rats. The ethanol-induced infiltration of triglycerides in the liver was also reduced by the feeding of betaine to the ethanol-fed animals. These results indicate that betaine administration has the capacity to elevate hepatic S-adenosylmethionine and to prevent the ethanol-induced fatty liver.

Hepatic transmethylation and blood alcohol levels.

Golden Syrian hamsters that have elevated hepatic alcohol dehydrogenase activity were divided into four groups and group-fed on four different liquid diets for five weeks. Group I was fed a control diet formulated for hamsters. Group II was fed the control diet containing 20 micrograms of 4 methylpyrazole per litre. Group III was fed the hamster ethanol liquid diet (ethanol amounting to 36% of total calories). Group IV was fed the ethanol diet to which 4-methylpyrazole (20 micrograms/litre) was added. Groups I, II and III were group-fed the amount consumed by Group IV on a daily basis. Upon killing the animals, blood alcohol levels were found to be elevated in Group IV but not in Group III. Hepatic methionine synthetase (MS) was inhibited in Group IV. Betaine-homocysteine methyltransferase was induced in this group to compensate for the MS inhibition and liver betaine was lowered reflecting this induction. None of these changes were seen in Group III. Since none of the animals showed an aversion to their respective diets and gained weight normally, these data indicate that it was the elevated blood levels of ethanol rather than nutritional factors that were related to the changes in methionine metabolism.

Methionine metabolism in the Buffalo rat following ethanol feeding.

Earlier studies have demonstrated that the Buffalo strain rat may have a defect in the process of methylneogenesis or lack the methyl-transfer enzyme system in the liver involved in methionine biosynthesis. The results of this study demonstrate that the enzyme systems responsible for methionine formation are present in the Buffalo rat and that these systems respond to ethanol feeding in a manner similar to that already shown in the Sprague-Dawley animal. These findings suggest that the previously described defect in methionine metabolism of the Buffalo rat may lie within the mechanism of methylneogenesis.

The influence of ethanol on hepatic transmethylation.

One of the most important biochemical pathways in the organism is the biosynthesis of methionine from the methylation of homocysteine. Two different reactions are responsible for this methylation, one utilizing N5-methyltetra-hydrofolate as a methylating agent and the other using betaine as the methyl donor. This paper reviews some recent findings in this laboratory, which demonstrate that ethanol-feeding to rats impairs the folate-induced reaction. Our findings also show that this impairment is compensated for through the adaptive increase in the enzyme using betaine in the biosynthesis of methionine. Further studies indicate that the mechanism of action in the impairment may occur through the formation of individual adducts between the folate-induced enzyme (methionine synthetase), its essential cofactors and acetaldehyde, a metabolic product of ethanol. These findings suggest a basis for why rats are more resistant to alcoholic liver injury than humans and may offer a means of protecting against alcoholic liver injury in man.

Effects of prolonged ethanol feeding on methionine metabolism in rat liver.

Pairs of rats were fed control and alcohol liquid diets for periods of 1, 2, 3, and 4 months. The animals were then killed, and their livers analyzed for betaine, S-adenosylmethionine (SAM), methionine synthetase activity, and betaine--homocysteine methyltransferase (BHMT) activity. The results of this time-course study showed that chronic ethanol feeding inhibited the activity of the methionine synthetase throughout the study, but increased the activity of BHMT and lowered betaine levels. These data suggest that the rat, because of its ability to produce betaine from choline, has the capacity to compensate for the ethanol-induced impairment of methionine synthetase and maintain vital tissue levels of SAM over prolonged periods of time via an adaptive increase in BHMT activity.

Ethanol, the choline requirement, methylation and liver injury.

The findings obtained in this laboratory and others over the past decade are discussed in order to formulate a thesis, indicating the adverse action of ethanol on a vital methylation process in the liver. Evidence is shown that the rat may have a means of compensating for this impairment in methylation whereas humans do not have this same ability to protect against this action of ethanol. These considerations may offer a basis of why rats are apparently more resistant to alcoholic liver injury than humans.

Methotrexate hepatotoxicity.

An inhibitor of folate metabolism, amethopterin (methotrexate) has been successfully used in the treatment of psoriasis and neoplastic disease. This drug produces several dangerous side effects of both an acute and chronic nature which have widely curtailed its use. A serious chronic side effect of the drug is its hepatotoxicity, which may culminate in hepatic cirrhosis and death. To date the underlying mechanism of methotrexate in producing liver damage is unknown. Results of three studies conducted in this laboratory on the nutritional effects of methotrexate offer some evidence that the hepatotoxicity may possibly be incurred through the effect of the drug on methionine biosynthesis and methylation processes. This thesis is discussed in the light of methylating agents vital to the synthesis of methionine.

Betaine, metabolic by-product or vital methylating agent?

The possible physiological role of betaine, the oxidative product of choline, is considered. It is proposed that betaine, instead of merely being a metabolic by-product of choline oxidation, may serve as an important methylating agent when normal methylating pathways are impaired by ethanol ingestion, drugs or nutritional imbalances. Furthermore, betaine may prove to have therapeutic application in cases of altered folate, vitamin B12 or methionine metabolism.

Methotrexate effects on hepatic betaine levels in choline-supplemented and choline-deficient rats.

Groups of rats fed both choline-supplemented and choline-deficient diets were injected with methotrexate (MTX) at dose levels of 0.1 mg/kg/day and 0.2 mg/kg/day. Both doses produced lowered hepatic betaine levels in the choline-supplemented animals when compared with nontreated, pair-fed controls. Since betaine levels were very low in the livers of choline-deficient rats, MTX had no further effect on hepatic betaine in these animals. These data suggest that the betaine lowering seen in livers of choline-fed rats may be due to utilization as a means of compensating for the MTX-induced loss of N5 methyltetrahydrofolate for the vital methylation of homocysteine in methionine biosynthesis.

The effect of methotrexate on homocysteine methylating agents in rat liver.

Methotrexate (MTX) was administered to rats in a time course study throughout a 4-month period. Despite an inhibition of the hepatic dihydrofolate reductase by 88 to 90% in these animals, the body weights and liver weights of the treated animals did not vary from those of the pair-fed controls during this time course, and the livers showed no fatty infiltration as demonstrated by triglyceride analyses. The prime methylating agent of homocysteine, N5 methyltetrahydrofolate, progressively increased in control livers throughout the experimental period; however, MTX impaired the increases at the 2- and 4-month periods in the treated animals. Hepatic betaine, the secondary methylator of homocysteine in the liver, also increased in the controls during the experimental period. These increases were also impaired in the MTX-treated animals. These data suggest that betaine may compensate for N5 methyltetrahydrofolate as a methylating substance when folate metabolism is antagonized in rat liver.