Harriet C. Beckenhauer

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.

Methionine synthase: a possible prime site of the ethanolic lesion in liver

Among the most important pathways for liver integrity in the body are the two that synthesize methionine and S-adenosylmethionine (SAM) through methylation of homocysteine. Results of studies in this laboratory have demonstrated ethanolic inhibition of one of these pathways catalyzed by methionine synthetase. It has been shown elsewhere that alcohol per se does not inhibit the enzyme, but that the metabolite of ethanol, acetaldehyde, is responsible through the formation of an inhibiting covalent adduct. Because hepatic SAM has been shown to be essential in the transport of fat from the liver, avoiding steatosis and further liver damage, it is entirely feasible that this repression of methionine synthase is an important site of the injurious action of alcohol metabolism in the liver. This loss of activity is particularly important in human beings who cannot produce methionine and SAM by means of the alternate pathway mediated by betaine:homocysteine:transmethylase, because of the lack of production of the betaine substrate for this enzyme.

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.

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.

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.

Effect of ethanol on uptake of choline by the isolated perfused rat liver.

Abstract 1. 1. Insofar as most studies on the effects of ethanol on the organism have involved chronic ingestion, this study was designed to measure the acute influence of this alcohol on choline uptake in the liver. 2. 2. Using the isolated perfused liver technique it was found that ethanol circulated in the perfusate increased the uptake of exogenous choline by the perfused liver. Acetate supplements had no effect on the uptake. 3. 3. It was found that increased uptake was not unique to ethanol and that two other alcohols that are NADH generators, methanol and sorbitol, also increased choline uptake. 4. 4. A hydrogen acceptor, 2,3,5-triphenyltetrazolium chloride, was shown to depress choline uptake. This finding tends to implicate NADH in the mechanism responsible for increased choline uptake seen in this study. 5. 5. It is concluded from this study that of the acetate and NADH resulting from ethanol metabolism, NADH is possibly the most potent stimulator of choline uptake.

Use of S-adenosylmethionine as an index of methionine recycling in rat liver slices

Abstract A simplified method for the measurement of methionine recycling in liver slices was developed. The method, a double-labeling procedure, utilizes S-adenosylmethionine as an index of recycling and offers several advantages over methionine, which has been used in other laboratories to determine methionine recycling in plants and intact animals. These advantages include (i) ease of isolation of index substance from tissue samples; (ii) elimination of oxidation problems prevalent with use of methionine; (iii) need for less initial concentrations of [methyl-3H] methionine and [35S]methionine used as tracers in the measurement of methionine recycling.

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.

Studies of isolated perfused rat livers—I: Effect of arginine and glutamate on ammonia metabolism in normal, fatty, and precancerous livers

Abstract Arginine or arginine-glutamate was shown to assist isolated perfused normal rat livers and experimentally produced pathological livers in detoxifying administered ammonia. This detoxification was reflected both in removal of ammonia from the perfusate and in the stimulation of urea production. In general, fatty livers and azo dye-fed livers were not as efficient as normal livers in producing urea from ammonium salts, amino acids, or combinations of these supplements placed in the blood perfusate. Glutamate when added to the perfusate of fatty livers did not elevate urea as in normal and precancerous livers.