Potentiation of ethanol fatty liver in rats by chronic administration of nicotinic acid.

Two groups of rats were fed isocalorically on alcohol and control semi-liquid diet for 28 days; two other groups had the same diets except for supplementation with nicotinic acid at 50 mg/100 ml of diet, Blood ethanol levels were unaffected by nicotinic acid administration, even though nicotinic acid was well absorbed and stored in the liver. Lipid analyses of liver and plasma after 28 days revealed that nicotinic acid, per se, stimulated fatty infiltration of the liver and this effect was potentiated when given in conjunction with ethanol.

Paradoxical urinary excretion of D-glucaric acid in acute viral hepatitis.

The urinary excretion of D-glucaric acid and the plasma clearance of antipyrine were estimated during the acute phase of viral hepatitis and again during recovery. The plasma clearance of antipyrine was impaired during the acute stage of hepatitis, while the urinary excretion of D-glucaric acid was paradoxically high. Both parameters returned to normal during recovery. These findings suggest that the use of urinary D-glucaric acid excretion as an index of microsomal enzyme induction is unreliable when there is liver injury.

Assay for free and total choline activity in biological fluids and tissues of rats and man with Torulopsis pintolopessi.

The sensitive, specific growth response to choline activity of the thermophilic enteric yeast Torulopsis pintolopessi enables estimation of free and bound choline activity in rat and human fluids and tissues- as little as 10 ng/ml of choline is measurable. Unlike other microbial assays, estimation of unbound (free) choline activity is not influenced by methionine or phospholipids. The method also distinguishes differences in choline activity of fluids and tissues from choline-deficient and choline-replete rats. Free and bound choline activity in blood, red blood cells, plasma, and liver from choline-deficient rats were almost 2-fold lower than from choline-supplemented animals. Free and bound choline activity in whole brain from choline-deficient rats were signifigantly higher (more than 2-fold). The application of the T. pintolopessi method in studying choline status in man and reasons for high choline activity in brain of choline-deficient rats are suggested.

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, 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.

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.

Malignant hyperthermia. A potentially fatal syndrome in orthopaedic patients.

Malignant hyperthermia is an inherited muscular disorder and a cause of sudden death in afflicted patients. Stress arising from tramuma, surgery, and other causes can initiate the syndrome. We studied the types of orthopaedic problems commonly seen in patients with this condition as well as the clinical manifestations and pathophysiology of the syndrome. Recognition, management, and understanding of the pathophysiology of malignant hyperthermia can be aided by a study of its similarities to porcine stress syndrome. We performed experimental studies of muscle and prophylaxis in swine. We also demonstrated the extension of the prophylactic use of dantrolene sodium in a management protocol in eighty surgical procedures in humans.

A simplified procedure for the determination of betaine in liver.

A convenient procedure for the determination of hepatic betaine levels is described. The method takes advantage of ethanol precipitation to rid acidified tissue extracts of interfering substances. Betaine is reacted with potassium triiodide to form betaine periodide, which is selectively precipitated via pH adjustment. The precipitate of betaine periodide is dissolved in ethylene dichloride and measured spectrophotometrically. The method is specific, accurate, and simple and showed recoveries of from 97 to 103% at two different levels of added betaine. The applicability of the method was shown when it was demonstrated that diets containing different amounts of choline influenced levels of hepatic betaine.

Determination of free choline and phosphorylcholine in rat liver.

A simple procedure for determination of levels of free choline and phosphorylcholine in hepatic tissue is outlined. The method makes use of the enzyme acid phosphatase to liberate choline from phosphorylcholine and incorporates the ability of choline to react with potassium triiodide to yield choline periodide for the measurement of choline and phosphorylcholine in liver. The method is accurate for both entities (recovery of 97-100% for choline and 92-98% for phosphorylcholine). For phosphorylcholine, the method is markedly simpler than other methods previously described and the results for normally fed rats are of the same order of magnitude. The applicability of the method was shown when it was demonstrated that diets containing different amounts of choline influenced the level of choline and phosphorylcholine in liver.

Effect of serum lipoproteins of bile obstructed rats on 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in perfused rat liver.

Total lipoproteins as well as fractionated VLDL + LDL and HDL from fasted control rats and bile-ligated rats were tested in liver perfusion for their effect on 3-hydroxy-3-methylglutaryl CoA reductase activity in normal rat livers. The total lipoproteins of bile-obstructed rats had 3 times greater capacity to increase 3-hydroxy-3-methylglutaryl CoA reductase activity than that of the control total lipoproteins. When the fractionated lipoproteins were tested from fasted control rats, it was found that the major stimulating activity was in the HDL fraction with minor activity in the VLDL + LDL fraction. When these plasma components isolated from fasted bile-ligated rats were tested, it was found that the major activity had shifted to the VLDL + LDL fraction with the HDL having only a minor stimulatory role. The possible mechanism of action of the abnormal lipoproteins associated with bile obstruction in regulating 3-hydroxy-3-methylglutaryl CoA reductase activity is discussed.

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.

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.

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.

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."

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.