Effect of cyanamide on the metabolism of ethanol and acetaldehyde and on gluconeogenesis by isolated rat hepatocytes

Previous experiments demonstrated that acetaldehyde stimulated glucose production from pyruvate, whereas gluconeogenesis from glycerol, xylitol and sorbitol was inhibited [A.I. Cederbaum and E. Dicker, Archs Biochem. Biophys. 197, 415 (1979)]. To determine the mechanism whereby acetaldehyde affects glucose production from these precursors, and to evaluate the role of acetaldehyde in the actions of ethanol, experiments with cyanamide were carried out. The oxidation of acetaldehyde by isolated rat liver cells was inhibited by cyanamide after a brief incubation period. Associated with this inhibition of acetaldehyde oxidation was an inhibition of ethanol oxidation by cyanamide and an increase in the amount of acetaldehyde which arose during the oxidation of ethanol. Ethanol oxidation was decreased because of the ineffective removal of acetaldehyde in the presence of cyanamide. Cyanamide had no effect on hepatic oxygen uptake. The increase in the β-hydroxybutyrate/acetoacetate ratio produced by acetaldehyde was completely prevented by cyanamide, whereas the slight increase in the lactate/pyruvate ratio was not prevented by cyanamide. Cyanamide partially reversed the ethanol-induced increase in the lactate/pyruvate ratio, but it completely prevented the ethanol-induced increase in the β-hydroxybutyrate/acetoacetate ratio. The ethanol-induced change in the mitochondrial redox state may, therefore, be due primarily to the mitochondrial oxidation of the acetaldehyde which arises during the oxidation of ethanol. The inhibitory effects of acetaldehyde on gluconeogenesis from glycerol, xylitol and sorbitol, as well as the stimulation of acetaldehyde of glucose production from pyruvate, were completely prevented by cyanamide. These results indicate that the effects of acetaldehyde on gluconeogenesis represent metabolic effects, rather than direct effects of acetaldehyde. Changes in the cellular NADH/NAD^? ratio as a consequence of acetaldehyde metabolism are postulated to be responsible for these actions of acetaldehyde. Ethanol stimulated glucose production from pyruvate, while inhibiting gluconeogenesis from glycerol, xylitol and sorbitol. Cyanamide, which prevented the effects of acetaldehyde on gluconeogenesis, also prevented the effects of ethanol on gluconeogenesis. This prevention by cyanamide may be suggestive for a role for acetaldehyde in the actions of ethanol on gluconeogenesis. The possibility cannot be ruled out, however, that the prevention of the effects of ethanol by cyanamide may be due to the partial inhibition of ethanol oxidation by cyanamide. These results indicate that cyanamide is an effective inhibitor of acetaldehyde oxidation by isolated liver cells and therefore can be used to determine the mechanism whereby acetaldehyde affects metabolic function. Depending on the reaction under investigation, acetaldehyde can have direct or indirect effects on cellular metabolism.     

Contribution of non-ADH pathways to ethanol oxidation in hepatocytes from fed and hyperthyroid rats. Effect of fructose and xylitol

The metabolism of (1R)[1-3H]ethanol, [2-3H]lactate or [2-3H]xylitol was studied in hepatocytes from fed or T3-treated rats in the presence or absence of fructose or xylitol. The yields of tritium in ethanol, lactate, water, glycerol and glucose were determined. A simple model, describing the metabolic fate of tritium from these substrates is presented. The model allows estimation of the ethanol oxidation rate by the non-alcohol dehydrogenase pathways from the relative yield of tritium in water and glucose. The calculations are based on a comparison of the fate of the 1-proR-hydrogen of ethanol and the hydrogen bound to carbon 2 of lactate (or xylitol) under identical condition. In our calculations we have taken into account that the reactions catalyzed by lactate dehydrogenase and alcohol dehydrogenase are reversible and that lactate or ethanol labelled during the metabolism of the other tritiated substrates will contribute to the tritium found in water. The contribution of non-ADH pathways to ethanol oxidation varied from 10 to 50% and was correlated to changes in the lactate/pyruvate ratio from 80 to 500. In T3-treated rats the activity of non-ADH pathways were greater than in fed rats for the same lactate/pyruvate ratio.     

Effect of propranolol on ethanol metabolism-evidence for the role of mitochondrial NADH oxidation.

Ethanol metabolism in the rat as measured in vivo by ¹⁴CO₂ production or in vitro by the removal of ethanol by liver slices was inhibited approximately 30 per cent by propranolol. There was no inhibitory effect of propranolol on rat liver alcohol dehydrogenase, catalase. NADPH-dependent microsomal ethanol oxidation or formate oxidation to ¹⁴CO₂. Propranolol inhibited fatty acid oxidation to ¹⁴CO₂in vivo as well as by liver slices and isolated hepatic mitochondria. NADH oxidation by hepatic mitochondria was also reduced by propranolol. 2,4-Dinitrophenol treatment or chronic ethanol feeding of rats stimulated alcohol metabolism as well as hepatic mitochondrial NADH oxidation. These increases were abolished by propranolol. The effect of propranolol in blocking the increase in ethanol oxidation after chronic alcohol feeding appears to be related to its action on the mitochondrial re-oxidation of NADH to NAD. Propranolol inhibits mitochondrial NADH oxidation, while 2,4-dinitrophenol or chronic ethanol feeding stimulates this process. The present studies support the concept that the rate of hepatic ethanol metabolism is limited, at least in part, by the mitochondrial oxidation of NADH.     

Disulfiram as a Tool in the Studies on the Metabolism of Acetaldehyde in Rats

Abstract The liver acetaldehyde dehydrogenases and the acetaldehyde level in the blood during ethanol metabolism were studied in rats 24 hrs after the administration of disulfiram. High doses of disulfiram (150–600 mg/kg) caused a threefold decrease in the activity of the mitochondrial low-K_m enzyme, whereas no significant effects were found on the activity of the high-K_m enzymes present in the mitochondrial, the microsomal and the cytosolic fractions. The concentration of acetaldehyde was threefold higher in the hepatic venous blood and fivefold higher in the peripheral blood in rats given disulfiram compared to rats given ethanol only. Low doses of disulfiram (25–50 mg/kg) decreased the activity of the low-K_m enzyme by 26 %, and caused a significant increase in the liver output of acetaldehyde. The rate of ethanol elimination decreased by 35 % at a high dose of disulfiram, whereas the alcohol dehydrogenase activity was not influenced. It is suggested that the mitochondrial low-K_m enzyme has a primary role in the regulation of the hepatic output of acetaldehyde, and the results will be discussed with special reference to the site and kinetics of acetaldehyde oxidation during ethanol metabolism in rat liver.     

Ethanol-induced alteration of dopamine metabolism in rat liver

Ethanol alters the metabolism of dopamine such that the final product is no longer predominantly the acid, 3,4-dihydroxyphenylacetic acid (DOPAC), but is a mixture of the acid and the alcohol derivative, 3,4-dihydroxyphenylethanol (DOPET). The ratio of DOPAC/DOPET produced in rat liver slices incubationed with [ethylamine-2-¹⁴C]dopamine hydrochloride in the absence of ethanol is ca. 10, while in the presence of ethanol it is 0.25. Addition of alcohol dehydrogenase (ADH) inhibitors prevents the alteration in metabolism. Changing the NAD/NADH ratio of the liver cytosol by adding lactate to the incubation medium does not cause an alteration in the metabolism of dopamine. Acetaldehyde addition in the presence or absence of ADH inhibitors does not enhance the production of the alcohol derivative, though there was a small decrease in DOPAC levels. Thus, neither the decreased liver cytosol NAD/NADH ratio nor the preferential oxidation of acetaldehyde over 3,4-dihydroxyphenyl acetaldehyde (DOPAL) can explain the ethanol-induced alteration in dopamine metabolism. 3-Etiocholan-3β-o1-17-one, an alternative substrate for ADH, whose product of oxidation is neither a substrate nor an inhibitor of aldehyde dehydrogenase, mimics the effect of ethanol such that in its presence the metabolism of dopamine to its alcohol derivative is enhanced. An increased reduction of DOPAL by the NADPH-dependent aldehyde reductase cannot explain the dramatic enhancement of DOPET formation observed in the presence of ethanol or the sterol because the NADPH/ NADP ratio is normally very high in the liver. Due to the unique enzyme mechanism of ADH, in which the rate-limiting step of the reaction is the release of NADH from the enzyme, a finite concentration of the enzyme-NADH complex will exist during alcohol metabolism. We propose that the biogenic aldehyde binds to this form of ADH and is reduced.     

The distribution and metabolism of acetaldehyde in rats during ethanol oxidation--II. Regulation of the hepatic acetaldehyde level.

The regulation of the hepatic acetaldehyde (AcH) level during ethanol oxidation was investigated in vivo in fed male and female Sprague-Dawley rats. Various doses of ethanol were administered orally, the livers were freeze-clamped during pentobarbital anaesthesia, and ethanol, AcH, lactate, pyruvate, acetoacetate, 3-hydroxybutyrate and the aldehyde dehydrogenase activity were measured. A positive correlation was found between the ethanol and AcH concentration, when the ethanol concentrations were between 5–30 μmole/g wet wt liver. A negative correlation was found within this ethanol range between the hepatic mitochondrial free NADH/free NAD⁺ ratio and the AcH concentration. A negative correlation was also obtained between the hepatic aldehyde dehydrogenase activity and the AcH concentration. A positive correlation was demonstrated between the hepatic mitochondrial free NADH/free NAD⁺ ratio and the AcH concentration at ethanol concentrations above 30 μmole/g. The results are discussed in relation to the regulation of the hepatic AcH level by the metabolism of ethanol and AcH within the liver.     

Ethanol and acetaldehyde metabolism in rat strains genetically selected for their ethanol preference

A study was made of ethanol and acetaldehyde metabolism in both sexes in rat strains genetically selected for their ethanol preference. The strains are denoted by ANA (Alko, Non-Alcohol), which prefers water to a 10% (v/v) ethanol solution, and AA (Alko, Alcohol), which prefers the ethanol solution. Peripheral blood and freeze-stopped livers were used for the in vivo studies. A once-through perfusion technique was applied so that in the same liver ethanol and acetaldehyde oxidation, the cytoplasmic redox state and oxygen consumption could be measured. In the female rats of the AA strain there was a higher rate of ethanol oxidation and oxygen consumption, compared with those of the ANA strain. A greater difference was found between the sexes, the female rats of both strains having a more rapid ethanol oxidation and oxygen consumption, compared with the respective males. The AA strain displayed a significantly lower level of acetaldehyde during ethanol oxidation than did the ANA strain. On comparison of the liver acetaldehyde concentrations with the mitochondrial NADH/NAD⁺ ratio, calculated from the 3-hydroxybutyrate/acetoacetate ratio, strain correlations were observed in both sexes, the ANA strain, with higher acetaldehyde, having a lower 3-hydroxybutyrate/acetoacetate ratio than the AA strain, with lower acetaldehyde. The results are discussed in relation to the regulation of the ethanol and acetaldehyde metabolism. The biochemical basis for ethanol preference is briefly discussed.     

Ethanol oxidation in systems containing soluble and mitochondrial fractions of rat liver: Regulation by acetaldehyde

Systems containing soluble fraction of rat liver, with or without mitochondrial fraction, oxidised [l-¹⁴C] ethanol to acetaldehyde, ¹⁴CO₂ and non-volatile ¹⁴C-products of which acetate was the principal, and possibly the only, component. Ethanol oxidation was stimulated by pyruvate which served as an electron sink thereby allowing rapid regeneration of NAD. When no mitochondria were present acetaldehyde accumulated, rapidly at first but eventually reaching a plateau. The rate of ethanol oxidation in these systems was much lower than the measured maximum activity of alcohol dehydrogenase (ADH) and it was concluded that ADH was inhibited by the accumulated acetaldehyde. Mitochondria, because of their relatively high aldehyde dehydrogenase (ALDH) activity, prevented the accumulation of acetaldehyde, or quickly removed acetaldehyde already accumulated. This action was accompanied by a sharp increase in the rate of ethanol oxidation, presumably due to the deinhibition of ADH. Cyanamide, an inhibitor of mitochondrial ALDH, blocked the stimulatory effect of mitochondria on ethanol oxidation. It was concluded that, in the reconstituted systems, acetaldehyde played a dominant role in controlling the rate of ethanol oxidation. The possible importance of acetaldehyde in governing ethanol oxidation in vivo is discussed.     

Interaction between ethanol metabolism and mixed-function oxidation in alcohol dehydrogenase positive and negative deermice

To assess the effect of non alcohol dehydrogenase (ADH) ethanol metabolism on mixed-function oxidation, aminopyrine demethylation was studied in vivo and in vitro in deermice having normal liver ADH (ADH+) or lacking it (ADH-), in the presence and absence of ethanol. When injected 15 min prior to administration of [14C]aminopyrine, ethanol reduced the 14CO2 exhalation rate in both ADH- and ADH+ deermice. The inhibitory effect of ethanol was dose dependent in both strains, and there was no significant difference between strains. Chronic ethanol feeding increased 14CO2 production from [14C]aminopyrine in both animal strains (ADH- alcohol 5.9 +/- 1.3 vs ADH- control 2.9 +/- 0.03, P less than 0.025; ADH+ alcohol 5.9 +/- 0.3 vs ADH+ control 2.7 +/- 1.3 nmoles aminopyrine/100 g body wt/min, P less than 0.001). Alcohol feeding also induced aminopyrine N-demethylase activity measured in vitro. This induction was more pronounced in ADH- deermice. Ethanol also inhibited aminopyrine demethylation in liver homogenates from ADH- and ADH+ animals in a dose-dependent manner and to a comparable degree in both strains. The kinetics of aminopyrine N-demethylase inhibition by ethanol was competitive in the microsomal fraction from ADH- as well as ADH+ animals. These results suggest that inhibition of mixed-function oxidation by ethanol may be due to an effect of ethanol on the hepatic microsomes rather than to redox changes produced by ADH-mediated ethanol oxidation. Further, chronic ethanol feeding increased microsomal aminopyrine demethylation independently of the presence of ADH.     

The effects of substrates of mixed function oxidase on ethanol oxidation in rat liver microsomes

Summary The effects of mixed function oxidase substrates, aminopyrine and ethylmorphine, on the NADPH and O₂ dependent rate of ethanol oxidation have been examined.Aminopyrine like ethylmorphine exerts different effects on the rate of acetaldehyde formation depending upon the ethanol concentration used. At saturating ethanol concentration V _max increases. Inhibition is observed at low concentrations of ethanol. Plots of acetaldehyde formation versus ethanol concentration reveal, in the presence of aminopyrine, curves which indicate the simultaneous action of two enzymes functional in ethanol oxidation.These data provide support for the existence of a microsomal ethanol oxidizing enzyme system (Orme-Johnson et al., 1965; Lieber et al., 1970), in addition to the well documented azide sensitive and H₂O₂ dependent pathway for ethanol oxidation (Thurman et al., 1972; Feytmans et al., 1973).Supported by Deutsche Forschungsgemeinschaft; Schwerpunktprogramm: Biologische Grundlagen der Arzneimittel- und Fremdstoffwirkungen.     

The roles of the hepatocellular redox state and the hepatic acetaldehyde concentration in determining the ethanol elimination rate in fasted rats

Ethanol administration (2 g/kg i.p.) to fasted male Wistar rats caused, on average, a 64% decrease in the cytosolic free NAD+:NADH ratio and a 41% decrease in the mitochondrial free NAD+:NADH ratio measured 90 min after ethanol was injected. Treatment of animals with either Naloxone (2 mg/kg i.p.) 1 hr after ethanol or 3-palmitoyl-(+)-catechin (100 mg/kg p.o. 1 hr before ethanol) prevented these ethanol induced redox state changes, without affecting the ethanol elimination rate or the hepatic acetaldehyde concentration measured at 90 min after ethanol administration. The thiol compounds cysteine and malotilate (diisopropyl-1,3-dithiol-2-ylidene malonic acid) significantly lowered the hepatic acetaldehyde concentrations measured at 0.75, 1.5 and 6.0 hr after ethanol, and caused a 29% and 12% increase respectively in the ethanol elimination rate, without affecting the ethanol induced alterations in the NAD+:NADH ratio. Pretreatment of animals with the aldehyde dehydrogenase inhibitor, cyanamide (1 mg/kg or 15 mg/kg p.o. one hour before ethanol), caused increases of up to 23-fold in the hepatic acetaldehyde level, without influencing the cytosolic NAD+:NADH ratio in ethanol dosed rats, while significantly reducing the ethanol elimination rate by up to 44%, compared with controls. These results suggest that ethanol oxidation by cytosolic alcohol dehydrogenase may be regulated in part by the hepatic acetaldehyde concentration achieved during ethanol metabolism rather than NADH reoxidation, either to supply NAD for the dehydrogenase, or to reduce inhibition of the enzyme by NADH, being a rate-limiting factor in ethanol metabolism in fasted rats.     

The cerebral oxidative metabolic response to acute ethanol administration in rats and cats

Ethanol effects on mitochondrial respiratory chain components in intact cerebral cortex of cats and rats are reported. Changes in redox levels were monitored by non-invasive optical techniques (microfluorometry for intramitochondrial NADH: dual wavelength reflection spectrophotometry for cytochrome a,a₃). Acute ethanol injections were accompanied by progressive increases in the level of reduced cytochrome a,a₃, a response consistent with the cytochrome response to other depressants such as phenobarbital and chlorpromazine. Low ethanol doses (below approx. 1 g/kg) were accompanied by increased levels of reduced NAD and it appeared that the ethanol effect in vivo is due to a decrease in tissue activity common to central depressants. Higher ethanol doses (1–3 g/kg) produced increased levels of oxidized NAD whereas phenobarbital and chlorpromazine each resulted in increased levels of reduced NAD. Since many of the effects of ethanol have been attributed to the activity of its primary metabolic product, acetaldehyde. the latter's effects on energy metabolism were also considered. Acetaldehyde at all effective doses produced similar changes in NAD (i.e. oxidation) and cytochrome a,a₃ (reduction) to those of the higher ethanol doses. The oxidation of NAD at higher ethanol doses and at all doses of acetaldehyde was interpreted as a unique effect of ethanol and due to acetaldehyde.     

Metabolism of ethanol and acetaldehyde in parenchymal and non-parenchymal rat liver cells

Suspensions of isolated parenchymal (P) and non-parenchymal (NP) cells were prepared by collagenase perfusion followed by centrifugation of the primary cell suspension. Suspensions of P cells were able to metabolize ethanol (8–16 nmoles/min/10⁶ viable cells) while NP cells did not metabolize ethanol at all. Acetaldehyde was metabolized in P-cell suspensions at rates ranging from 14 to 20 nmoles/min/10⁶ viable cells. Some acetaldehyde metabolism also occurred in NP-cell suspensions (0.18–0.33 nmoles/min/10⁶ viable cells). In accordance with these studies on ethanol and acetaldehyde metabolism we found alcohol dehydrogenase activity only in homogenates of P cells, and aldehyde dehydrogenase activity in homogenates of P cells was 20 times higher per cell than in homogenates of NP cells. It was concluded that the P cells of rat liver are responsible for ethanol metabolism and probably also responsible for most, if not all metabolism of acetaldehyde arising from ethanol oxidation. Biochemical effects which are consequences of ethanol metabolism are probably not found in NP cells.     

Free radicals as mediators of alcohol toxicity

In this article we have reviewed recent evidence in support of the hypothesis that acute/chronic alcohol toxicity is mediated primarily via the generation of damaging free radical species in various tissues. Studies in man, animal model or in vitro experimental systems have shown: (1) the demonstration of alcohol-induced free radical species directly via esr spectroscopic analysis; (2) increases in indirect markers of ethanol-induced free radical damage in tissues, such as lipid peroxides and protein carbonyl; (3) ethanol-induced alterations in the levels of endogenous tissue antioxidants. These data show the induction of free radicals by ethanol to be a complex interactive process. The classical pathway for ethanol metabolism, catalysed by alcohol dehydrogenase to form acetaldehyde, results in the formation of free radicals, resulting from concomitant changes in NADH levels and NADH/NAD+ redox ratios, which in turn modulate the activity of the free radical generating enzyme xanthine oxidase. The induction of CYP 2E1 in the microsomes results in the generation of HER, another major route by which ethanol induces free radical formation. In addition to the above, ethanol may also induce free radical formation via the reaction of aldehyde oxidase with acetaldehyde or NADH to generate oxyradicals via disturbance in the metabolism of the pro-oxidant iron, or via increased efflux from mitochondria following altered mitochondrial oxidative metabolism.     

Inhibition of the metabolism of ethyl carbamate by acetaldehyde

Ethyl carbamate is an animal carcinogen when administered in large doses; it is naturally present in minute concentrations in fermented foods and beverages. Previous studies from this laboratory have demonstrated that ethanol, in vivo, inhibits the metabolism of ethyl carbamate in mice, but the enzyme system has not been identified. In an effort to further characterize the enzyme system responsible, the metabolic products of ethanol metabolism were studied to determine whether ethanol or either of its metabolites is inhibitory. Acetaldehyde (400 mg/kg) is a potent inhibitor of ethyl carbamate metabolism for about 2 hr in vitro, but sodium acetate is not. Paraldehyde (250 mg/kg) has a slower onset and longer duration of inhibition, suggesting that its conversion to acetaldehyde produces the inhibitory molecule. Disulfiram (200 mg/kg) has a prolonged inhibitory effect; this effect is enhanced and extended when the disulfiram is combined with acetaldehyde (400 mg/kg). D-Penicillamine, given in a regimen of 1.2 g/kg 0.5 hr before and 0.6 g/kg 1.5 and 3.5 hr after ethyl carbamate, is not inhibitory; however, it abolishes the inhibitory effect of acetaldehyde, presumably from sequestration of acetaldehyde. These studies demonstrate that acetaldehyde is an inhibitor of the metabolism of ethyl carbamate and suggest that acetaldehyde is one, and perhaps the only, molecule responsible for the inhibition seen when ethanol is administered to mice. In vitro incubation studies determined that ethyl carbamate was not metabolized by human plasma.     

Deuterium D(V/K) isotope effects on ethanol oxidation in hepatocytes: importance of the reverse ADH-reaction

The kinetic deuterium isotope effect, D(V/K), on ethanol oxidation was measured on hepatocytes from rat and pig by the radiometric competitive method using 14C-labelled ethanol containing deuterium in the (1-R)-position. The corrected D(V/K) values of 2.68 and 2.80 for rat and pig hepatocytes respectively were significantly different, suggesting differences in the amount of non-ADH ethanol oxidizing activity. The apparent isotope effects declined rapidly with time when acetaldehyde was present in the medium as a result of the reduction to ethanol of the [14C]-acetaldehyde formed from the double labelled ethanol by alcohol dehydrogenase (ADH). Fructose and cyanamide caused the acetaldehyde concentration during ethanol oxidation to increase by entirely different mechanisms, and the isotope effect to decrease with time, as did also the addition of acetaldehyde. The apparent first order rate constant for the reverse ADH reaction, assuming the reactants to be acetaldehyde and the ADH-NADH complex, was determined by two methods giving comparable results. In the presence of semicarbazide, which removes acetaldehyde, the isotope effect was nearly constant. This was the case also when the acetaldehyde concentration was very low (less than 1 microM) for other reasons, as in hepatocytes from starved animals. A mathematical formula describing the expected decrease of the apparent isotope effect with time was derived. The different response of pig and rat hepatocytes to addition of fructose (the 'fructose effect') is suggested to be caused by differences in activity of aldehyde dehydrogenases in the two species.     

Metabolism of ethanol and acetaldehyde in intact rats during pregnancy

Ethanol and acetaldehyde contents in the peripheral blood of intact non-pregnant and pregnant rats have been determined after an intraperitoneal injection of ethanol. Determinations have also been made of the ethanol and acetaldehyde contents and the lactate/pyruvate ratios in frozen, clamped livers of intact pregnant and non-pregnant rats with or without a prior injection of ethanol. The in vitro activities of the liver alcohol and acetal-dehyde dehydrogenase have also been measured. Elimination rate of ethanol in vivo was found to be equal in pregnant and non-pregnant rats, but the acetaldehyde content of the peripheral blood after ethanol administration was higher in pregnant than in non-pregnant animals. Because the ethanol and acetaldehyde contents of frozen, clamped livers were similar in magnitude in pregnant and non-pregnant rats and no differences were found in the in vitro activities of the liver alcohol and acetaldehyde dehydrogenase between the two animal groups a difference in the extrahepatic metabolism of acetaldehyde is suggested to explain the high acetaldehyde content in the peripheral blood of pregnant rats after ethanol administration. The lactate and pyruvate contents of frozen, clamped livers of pregnant rats without a prior close of ethanol were higher than those of non-pregnant animals indicating a high rate of glycolysis during pregnancy, but the lactate/pyruvate ratios of the livers were equal in the two animal groups both with and without previous ethanol loading.     

The role of the hepatocellular redox state in the hepatic triglyceride accumulation following acute ethanol administration

The role of the increased hepatocellular redox-state [( NADH]/[NAD+] ratio) as a mechanism underlying hepatic triglyceride deposition after acute ethanol dosing has been investigated in the rat. Following a single dose of ethanol (2 g/kg i.p.) in fasted rats, increases were observed at 1.5 hr in the hepatic [lactate]/[pyruvate] (133%), [3-hydroxybutyrate]/[acetoacetate] (69%) ratios, and the liver triglyceride concentration (129%). At the same time point, ethanol increased radioactivity incorporated into hepatic total lipid and triglyceride, after an injection of [U-14C] palmitic acid, by 76% and 158% respectively. Treatment of animals with Naloxone hydrochloride (2 mg/kg i.p.) at 1.0 hr and 2.5 hr after ethanol abolished these ethanol-mediated redox-state changes, without inhibiting ethanol oxidation or affecting hepatic acetaldehyde levels. This, however, did not prevent completely the triglyceride accumulation in the liver or reverse the enhanced uptake of radio-labelled palmitate caused by ethanol. Administration of sorbitol (3.5 g/kg i.p.) caused 109%, 57% and 200% increases in the hepatic [lactate]/[pyruvate], [3-hydroxybutyrate]/[acetoacetate] ratios and glycerol-3-phosphate concentrations respectively. However, the hepatic triglyceride concentration and the incorporation of [U-14C] palmitic acid into hepatic lipids were not influenced by this treatment. In vitro studies in which rat liver slices were incubated with [1-14C] palmitic acid also indicated that the altered [NADH]/[NAD+] ratio was not responsible for the decreased rate of fatty acid oxidation seen after ethanol administration or after the addition of ethanol to the incubation medium. In conclusion, these experiments indicate that increases in the hepatic [NADH]/[NAD+] ratio resulting from ethanol oxidation may not be directly implicated in the altered hepatic fatty acid utilisation and triglyceride deposition observed after acute ethanol administration in rats.     

Metabolic reactions in vitro of psoralens with liver and epidermis

In recent years, psoralens have been widely-used clinically, but until now their in vitro and in vivo metabolic reactions have not been studied. The photochemotherapeutic agent 4,5',8-trimethyl-psoralen (TMeP) is readily metabolized in vitro by mouse liver under mixed-function oxidase conditions, with formation of three products. One of the products is 4,8-dimethyl, 5'-carboxypsoralen which has been identified previously as an in vivo product of TMeP metabolism in the urine of mice and human subjects receiving oral TMeP. A second product, with a molecular weight of 244, is apparently 4,8-dimethyl, 5'-hydroxymethylpsoralen. A metabolic pathway that integrates in vivo and in vitro TMeP reactions is proposed. By contrast, 8-methoxypsoralen (8-MOP) apparently is minimally metabolized in vitro by mouse liver. Guinea pig epidermis did not show any _vitro reactions with either 8-MOP or TMeP. The difference in liver biotransformations of 8-MOP and TMeP may help explain why orally administered TMeP is less photosensitizing and less effective than 8-MOP in phototherapy of skin diseases such as psoriasis.     

Effects on Rat Liver Acetaldehyde Dehydrogenases in Vitro and in Vivo by Coprine,the Disulfiram-like Constituent of Coprinus atramentarius

Abstract Coprine, the disulfiram-like constituent of the mushroom Coprinus atramentarius was found to inhibit the low-K_m acetaldehyde dehydrogenase in rat liver and to increase the acetaldehyde level in blood during ethanol metabolism in vivo. Coprine did not inhibit the low-K_m enzyme in vitro, but the hydrolytic product of coprine, 1 -aminocyclo-propanol, was a potent inhibitor both in vitro and in vivo. A rapid onset of inhibition was observed after administration of coprine and the inhibition was long-lasting. It is suggested that 1-aminocyclo-propanol is responsible for the inhibition caused by coprine in vivo.