Differences in kinetic characteristics and in sensitivity to inhibitors between human and rat liver alcohol dehydrogenase and aldehyde dehydrogenase

1. On the basis of kinetic properties and sensitivity to pyrazole inhibition, it is shown that liver alcohol dehydrogenase present in human mainly corresponded to class I and in rat to class ADH-3 which differed in a number of parameters. 2. Two different aldehyde dehydrogenase (ALDH) isoenzymes were detected in both human and rat liver. The human isoenzymes corresponded to the ALDH-I and ALDH-II type. 3. In the rat, one isoenzyme had low Km and showed similar activity than in human liver but differed in their sensitivity to both disulfiran and nitrofazole inhibition whereas the other presented high Km and showed greater activity than the human one. 4. Caution must be therefore paid when extrapolating to human subjects the data on ethanol metabolism obtained with rats.     

Effect of almond and anis oils on mouse liver alcohol dehydrogenase, aldehyde dehydrogenase and heart lactate dehydrogenase isoenzymes

The effects of short-term intraperitoneal injection of diluted almond or anis oil on heart lactate dehydrogenase isoenzymes, liver alcohol dehydrogenase and subcellular aldehyde dehydrogenase were studied in the female mouse. Hepatic alcohol dehydrogenase was induced from control by administration of almond oil 3.2 g/kg/d for 7 days, or anis oil 1.6 g/kg/d for 7 days. Treatment with almond but not anis oil inhibited both cytoplasmic and mitochondrial liver aldehyde dehydrogenase. The mitochondrial isoenzyme with an apparently low Km was also inhibited by the almond oil trial. No significant changes occurred in heart lactate dehydrogenase isoenzymes by the treatments used. The enzymatic inhibition kinetics were found to be non-competitive. The apparent Km for almond-treated mouse aldehyde dehydrogenase was greater than the controls. This indicates lower substrate affinity for almond oil than for acetaldehyde. The results suggest adverse hepatic metabolic interaction between almond oil and alcohol.     

Effect of carbidine on parameters of ethanol oxidation in experimental alcoholic intoxication

The effect of carbidine on enzymes of ethanol and acetaldehyde oxidation, the rate of ethanol elimination and the parameters of ethanol consumption were studied during long-term alcoholic intoxication. Carbidine administration was shown to increase the activity of alcohol dehydrogenase of the liver tissue, to decrease the activity of aldehyde dehydrogenase with a low Km to acetaldehyde. Also, the rate of ethanol elimination and a relative amount of consumed ethanol increase at the expense of an increase of the volume of consumed liquid.     

Action of metadoxine on isolated human and rat alcohol and aldehyde dehydrogenases. Effect on enzymes in chronic ethanol-fed rats

Metadoxine (pyridoxine-pyrrolidone carboxylate) has been reported to accelerate ethanol metabolism. In the present work we have investigated the effect of metadoxine on the activities of isolated alcohol and aldehyde dehydrogenases from rat and man, and on the activity of these enzymes in chronic ethanol-fed rats. Our results indicate that in vitro metadoxine does not activate any of the enzymatic forms of alcohol dehydrogenase (classes I and II) or aldehyde dehydrogenase (low-Km and high-Km, cytosolic and mitochondrial). At concentrations higher than 0.1 mM, metadoxine inhibits rat class II alcohol dehydrogenase, although this would probably not affect the physiological ethanol metabolism. Chronic ethanol intake for 5 weeks results in a 25% decrease of rat hepatic alcohol dehydrogenase (class I) activity as compared with the pair-fed controls. The simultaneous treatment with metadoxine prevents activity loss, suggesting that the positive effect of metadoxine on ethanol metabolism can be explained by the maintenance of normal levels of alcohol dehydrogenase during chronic ethanol intake. No specific effect of chronic exposure to ethanol or to metadoxine was detected on rat aldehyde dehydrogenase activity.     

Influence of epinephrine on alcohol dehydrogenase activity in rat hepatocyte culture

The effects of epinephrine on alcohol dehydrogenase activity and on rates of ethanol elimination were determined in rat hepatocyte culture. Continuous exposure of the hepatocytes to epinephrine (10 microM) in combination with dexamethasone (0.1 microM) enhanced alcohol dehydrogenase activity on days 4-7 of culture, whereas neither hormone alone had an effect. The increased alcohol dehydrogenase activity was associated with an increased rate of ethanol elimination. Acute addition of 10 microM epinephrine to hepatocytes maintained in culture with 0.1 microM dexamethasone did not change alcohol dehydrogenase activity, but resulted in an immediate marked, but transitory, increase in ethanol elimination within the first 5 min after the addition of the hormone. Prazosin, an alpha 1-adrenergic blocker, and antimycin, an inhibitor of mitochondrial respiration, were powerful inhibitors of the transient increase in ethanol elimination, whereas 4-methylpyrazole was only partially inhibitory. These observations indicate that epinephrine has a chronic effect in increasing alcohol dehydrogenase activity and ethanol elimination and, also, an acute transient effect of increasing ethanol elimination which is not limited by alcohol dehydrogenase activity.     

Cholinergic, anticholinergic agents and ethanol interaction

The effect of cholinergics and an anticholinergic agent on hepatic ethanol metabolizing enzymes was studied. Short-term administration of the cholinomimetic arecoline or the anticholinergic scopolamine induced rat liver mitochondrial aldehyde dehydrogenase (L-ALDH) isoenzyme with the apparent high and low Km, respectively. In addition, scopolamine inhibited cytoplasmic L-ALDH. This suggests differential sensitivity of the L-ALDH isoenzymes to these agents. Scopolamine and the cholinomimetic pilocarpine enhanced rat and mouse liver alcohol dehydrogenase (L-ADH) in vitro, respectively. This indicates species-dependent effect of these agents on L-ADH. The results suggest interaction of the cholinergic system with ethanol metabolizing enzymes which may contribute to the peripheral action of alcohol.     

Preferential inhibition of the low Km aldehyde dehydrogenase activity by pargyline

Pargyline has previously been shown to increase ethanol-induced sleep time, decrease ethanol elimination rate and greatly increase acetaldehyde levels after ethanol administration in mice. In rats pargyline treatment did not significantly alter blood ethanol levels but increased blood acetaldehyde levels in a dose-dependent manner. Using 5 mM propionaldehyde to assay aldehyde dehydrogenase activity only about 40 per cent inhibition of the mitochondrial aldehyde oxidizing capacity was seen with the highest pargyline dose (100 mg/kg). Almost total inhibition of the low K_m mitochondrial aldehyde dehydrogenase activity was observed with 50 μM propionaldehyde or 1 mM formaldehyde.     

Effect of a low-protein diet on acetaldehyde metabolism in rats

The effect of dietary changes on liver alcohol and aldehyde dehydrogenase activities as related to effects on ethanol and acetaldehyde metabolism was investigated. Feeding rats for 8 weeks on diets rich in carbohydrate or fat, but with normal protein content, induced minor changes relative to giving a balanced diet. A low-protein, high-carbohydrate diet (5 per cent and 80 per cent of calory content, respectively) caused a significant reduction of both alcohol and aldehyde dehydrogenase activities in the liver. The activity of the high-Km aldehyde dehydrogenase in the microsomal and soluble fractions appeared to be more reduced than that of the low-Km enzyme in the mitochondrial fraction. The tail blood acetaldehyde was significantly higher in rats on the protein deficient diet in spite of their reduced ethanol elimination rates. The results suggest that protein deficiency deranges acetaldehyde metabolism and may thus increase the possible contribution of acetaldehyde to the effects caused by ethanol metabolism.     

Effects of long-term ethanol treatment on aldehyde and alcohol dehydrogenase activities in rat liver.

Administration of intoxicating doses of ethanol by gavage for 3 weeks caused weight loss and reduced hepatic aldehyde dehydrogenase activity in the soluble, mitochondrial and microsomal fractions. Rats receiving equivalent amounts of ethanol as a constituent of a liquid diet for 5 weeks gained weight and showed no changes in aldehyde dehydrogenase activity. Alcohol dehydrogenase activity was decreased in the rats treated by gavage and unchanged in those given ethanol in the diet, but in spite of this the rate of ethanol elimination was accelerated in both groups. In the livers of two strains of rats genetically selected for their difference in voluntary alcohol consumption, the mitochondrial and microsomal aldehyde dehydrogenase activities had previously been shown to be significantly higher in the alcohol-consuming (AA) than in the alcohol-avoiding (ANA) rats. Similar differences were now found after long-term intragastric ethanol administration, although in both strains the absolute levels of aldehyde dehydrogenase were reduced. Profound reduction of mitochondrial low-K_m aldehyde dehydrogenase activity and high blood acetaldehyde were observed, especially in the ANA rats. This suggests a possible connection between the low activity of this enzyme and the increased acetaldehyde level.     

Role of alcohol dehydrogenase in the swift increase in alcohol metabolism (SIAM). Studies with deer mice deficient in alcohol dehydrogenase

Previous studies have shown that rates of ethanol metabolism increase markedly 2-4 hr after the administration of ethanol in rats and in four inbred strains of mice. This phenomenon, called the swift increase in alcohol metabolism (SIAM), also exists in humans. To determine whether alcohol dehydrogenase (ADH) is necessary for the SIAM response, we compared ethanol metabolism in two strains of the deer mouse, Peromyscus maniculatus. One strain lacks alcohol dehydrogenase (ADH-negative), whereas the other strain has normal ADH levels (ADH-positive). Rates of ethanol elimination were determined after a single intraperitoneal injection of ethanol at different doses (0.5 to 3.0 g/kg) and also after both strains were exposed to various levels of ethanol vapor for 4 hr. The ADH-positive strain exhibited up to a 72% increase in the rate of ethanol elimination after exposure to ethanol vapor compared to the ethanol-injected controls. In contrast, treatment with ethanol vapor did not alter rates of ethanol elimination in the ADH-negative strain. These data demonstrate clearly that ADH is required for SIAM in the deer mouse. In addition, in both the ADH-positive and the ADH-negative strain, rates of ethanol elimination increased in both the ethanol-injected and vapor-treated groups 2- to 3-fold as the dose of ethanol was increased from 100 to 500 mg/100 ml. Thus, it is concluded that this "concentration effect" of ethanol on rates of ethanol metabolism does not involve ADH in the . deer mouse.     

The oxidation of acrolein by rat liver aldehyde dehydrogenases. Relation to allyl alcohol hepatotoxicity

The oxidation of acrolein by aldehyde dehydrogenase was studied in several subcellular fractions of rat liver by measuring acrolein-dependent production of NADH from NAD+. Mitochondrial and cytosolic fractions each contained two aldehyde dehydrogenase activities with Km values for acrolein of 0.4-0.7 mM and 0.015-0.025 mM. Microsomes demonstrated only a high Km (1.5 mM) activity. The low Km activities of mitochondria and cytosol differed in their sensitivity to inhibition by chloral hydrate and in their response to 1 mM MgCl2 (activation vs. inhibition). The metabolism of acrolein by low Km aldehyde dehydrogenase activities was markedly depressed in mitochondrial or cytosolic fractions from rats pretreated with cyanamide (2 mg/kg for 1 hr) or disulfiram (100 mg/kg for 24 hr). The effect of aldehyde dehydrogenase inhibition on allyl alcohol toxicity was determined by pretreating rats with cyanamide or disulfiram prior to treatment with allyl alcohol. Hepatotoxicity was assessed on the basis of elevated serum alanine aminotransferase and sorbitol dehydrogenase activities and the loss of microsomal cytochrome P-450. Pretreatment with the aldehyde dehydrogenase inhibitors enhanced the hepatotoxicity of allyl alcohol in both male and female rats. The results suggest that acrolein metabolism by rat liver aldehyde dehydrogenase isozymes is important for the inactivation of allyl alcohol-derived acrolein.     

Comparison of rat pulmonary and hepatic cytosolic alcohol dehydrogenase activities.

The liver is the major organ responsible for ethanol oxidation, and alcohol dehydrogenase (ADH) is the main enzyme involved. There is limited evidence suggesting the involvement of the lung in ethanol metabolism. To determine the degree to which pulmonary ADH plays a role in ethanol metabolism, ADH activity was measured spectrophotometrically using hepatic and pulmonary cytosolic fractions prepared by differential centrifugation and Sephadex G-50 column chromatography. Apparent Km values for hepatic and pulmonary ADHs were determined. Inhibition constants were calculated using 4-methylpyrazole. The ADHs were characterized by examining the influence of pH on enzyme activity. Pulmonary ADH activity was much lower at near neutral pH than at pH 9.0 or 10, whereas hepatic ADH activity was also pH dependent but was significantly higher. Pulmonary ADH is less sensitive to inhibition by 4-methylpyrazole than is hepatic ADH, as evidenced by a 1000-fold higher Ki. Pulmonary ADH would be expected to make only a minor contribution to ethanol metabolism in vivo.     

The metabolism of acetaldehyde and not acetaldehyde itself is responsible for in vivo ethanol-induced lipid peroxidation in rats

A single oral administration of ethanol (5 g/kg) to rats induced a marked increase in lipid peroxidation, in the liver and kidney within 9 hr, as assessed by malondialdehyde accumulation. The pretreatment with alcohol dehydrogenase (ADH) inhibitor, 4-methylpyrazole (1 mmol/kg) caused approximately 50% inhibition of the hepatic ADH activity and abolished this ethanol-induced lipid peroxidation. The disulfiram treatment (100 mg/kg) significantly inhibited 63% of the hepatic low Km aldehyde dehydrogenase (ALDH) but not the high Km ALDH. The cyanamide treatment (15 mg/kg) effectively decreased 83% of the low Km and 70% of the high Km ALDH in the liver. Although there was more than a 20-fold elevation of acetaldehyde levels by the inhibition of acetaldehyde metabolism with disulfiram or cyanamide, the ethanol-induced lipid peroxidation was significantly suppressed by pretreatment with these drugs. More than 90% inhibition of xanthine oxidase and dehydrogenase by the pretreatment with allopurinol (100 mg/kg), with no effect on the hepatic ADH and ALDH activities, did not alter the enhancement of lipid peroxidation following ethanol administration. We propose that the metabolism of acetaldehyde (probably via the low Km ALDH) and not acetaldehyde itself is responsible for the ethanol-induced lipid peroxidation in vivo and that the contribution of xanthine oxidase, as an initiator of lipid peroxidation through acetaldehyde oxidation is minute during acute intoxication.     

Blockade of hepatic aldehyde dehydrogenase in mice on chronic ingestion of 4-bromopyrazole and 4-iodopyrazole

4-Halopyrazoles acutely administered decreased the alcohol dehydrogenase activity of livers of treated mice but exerted little or no effect on the activity of aldehyde dehydrogenase. Ethanol administered to mice pretreated in this manner with 4-bromopyrazole disappeared slowly from blood as expected and gave no accumulation of acetaldehyde. In contrast, 4-bromopyrazole and 4-iodopyrazole, administered chronically via the drinking fluid, diminished the aldehyde dehydrogenase activity of livers of imbibing mice and elevated somewhat the alcohol dehydrogenase activity. In agreement with the blockade of aldehyde dehydrogenase observed in vivo, ethanol given to mice continually ingesting 4-bromopyrazole or 4-iodopyrazole resulted in the accumulation of acetaldehyde in blood. Moreover, chronic ingestion of 4-bromopyrazole caused a decrease in the natural ethanol preference of C57BL mice, a finding consistent with aldehyde dehydrogenase inhibition and the production of acetaldehyde from the interaction of 4-bromopyrazole and ethanol.     

Role of propiolaldehyde and other metabolites in the pargyline inhibition of rat liver aldehyde dehydrogenase

The metabolism of pargyline proceeds by way of three separate cytochrome P-450 catalyzed N-dealkylation reactions: N-depropargylation, N-demethylation and N-debenzylation. Propiolaldehyde, a product of N-depropargylation, is a potent inhibitor of aldehyde dehydrogenase (AlDH). The formation of pargyline-derived propiolaldehyde by isolated rat liver microsomes in vitro was confirmed using gas chromatographic/mass spectrometric techniques. The measured rates of propiolaldehyde formation for uninduced and phenobarbital-induced microsomes in vitro were 0.2 +/- 0.03 and 0.9 +/- 0.2 mumole/30 min/g wet weight liver respectively. However, these rates may have been artificially low due to competition between semicarbazide, the trapping agent, and microsomal proteins for the generated propiolaldehyde. CO significantly inhibited the microsome-catalyzed N-depropargylation reaction in vitro, whereas CoCl2 pretreatment of rats partially blocked the pargyline-induced rise in blood acetaldehyde after ethanol. Inhibition of the low Km liver mitochondrial AlDH by propiolaldehyde in vitro exhibited first-order kinetics, which is consistent with irreversible inhibition. Acetaldehyde did not attenuate the inhibition of AlDH by propiolaldehyde in vitro or by pargyline in vivo. Propargyl alcohol, a substance which is metabolized to propiolaldehyde by alcohol dehydrogenase, also inhibited AlDH in vivo and caused a quantitatively similar rise in blood acetaldehyde after ethanol as pargyline. Other putative metabolites of pargyline, namely benzylamine and propargylamine, inhibited AlDH in vivo, albeit to a lesser degree than pargyline, but neither of these amines inhibited AlDH directly. Monoamine oxidase was implicated in the conversion of benzylamine to an active inhibitory species, possibly an imine. From these studies, we conclude that propiolaldehyde was the primary metabolite responsible for the pargyline inhibition of AlDH in vivo; however, certain amine metabolites may have contributed to a lesser degree by conversion to yet unknown inhibitory forms.     

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.     

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.     

Effects of castration and testosterone administration on rat liver alcohol dehydrogenase activity

The effects of castration and testosterone administration on the activity of liver alcohol dehydrogenase and on the rate of ethanol elimination were determined in male Sprague-Dawley rats. Castration increased liver alcohol dehydrogenase activity. The total liver activity in castrated animals was 2.37 ± 0.229 (S.E.) $\text{mmoles}\text{hr}$ as compared with a value of 1.39 ± 0.125 $\text{mmoles}\text{hr}$ in sham-operated controls (P < 0.01). Testosterone administration partially suppressed the enhanced activity of liver alcohol dehydrogenase produced by castration. By contrast, in control animals testosterone administration resulted in a small paradoxical increase in liver alcohol dehydrogenase. The increase in the enzyme activity in castrated animals was associated with a parallel increase in the rate of ethanol elimination. Castrated and control animals showed decreases in free cytosolic and mitochondrial NAD⁺/NADH ratios after ethanol administration. These observations suggest that testosterone (and probably other as yet unknown factors modified by castration) affects liver alcohol dehydrogenase activity, and that the total enzyme activity can be a principal limiting factor in ethanol elimination.     

Quantitative correlation of ethanol elimination rates in vivo with liver alcohol dehydrogenase activities in fed, fasted and food-restricted rats.

The effects of nutritional states upon liver alcohol dehydrogenase (ADH) activity and ethanol elimination rate in vivo have been examined in the rat. Male Sprague-Dawley rats, 250–280 g, were studied in the fed state, after fasting for 24, 48 and 72 hr, and after 9 days of food restriction (5g food/day). Total ADH activity per liver or per animal (2.20 m-moles/hr in fed rats) decreased after a 24-hr fast and was 1.32 and 0.94 m-moles/hr after a 48-hr fast and food restriction respectively. Cytosolic protein and liver wet weight decreased in parallel with total ADH activity, but DNA content exhibited only a 10% decrease with fasting and a 20% decrease with food restriction. Ethanol elimination rate in vivo per animal after intraperitoneal injection of 2g ethanol/kg was 1.92, 1.14 and 0.84 m-moles/hr in the fed, 48 hr-fasted and food-restricted rats, respectively. These data indicate that the decrease in the ethanol elimination rate with fasting and food restriction may be caused by decreasing ADH activity, since the cytosolic free NAD⁺/ NADH in liver after acute administration of alcohol in vivo has been reported to be nearly identical in the fed and 48 hr-fasted rats. The close agreement between liver ADH activity and ethanol elimination rate in vivo suggests that the total enzymatic activity of liver ADH is an important rate-limiting factor in ethanol metabolism under the nutritional conditions examined.     

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.