Subacute toxicity of a Pseudomonas vaccine prepared from outer membrane fraction of Pseudomonas aeruginosa in beagle dogs.

CFC-101, a Pseudomonas vaccine, was administered to beagle dogs by intramuscular injection for 4 weeks (5 days/week) at 0.05, 0.15 and 0.45 mg/kg/d. Clinical signs considered to be related to treatment were restricted to swelling at the injection sites, being apparent 1-2 h after treatment. There was no effect on body weight, food consumption, ophthalmoscopy, electrocardiography, hematology, biochemical and urinary parameters. The histopathological examination revealed treatment-related changes at the injection sites at all dosages, particularly in the hindlimbs where both perivascular and intramuscular aggregations of inflammatory cells were seen. Thus, the only treatment-related changes seen in this study were local reactions to the test substance at the injection sites; furthermore these changes seem to represent a pharmacological response to the test material. Because no evidence of any systemic toxicity was observed at any dosage level, it is concluded that dosages of CFC-101 up to and including 0.45 mg/kg/d were well tolerated over a period of 4 weeks in the beagle dog.

The effect of di-isopropyl 1,3 dithiol-2-ylidenemalonate (malotilate) on the hepatic changes induced by ethanol administration in the rat.

The sulphur-containing drug, di-isopropyl-1,3-dithiol-2-ylidenemalonate (Malotilate) protects against the increase in hepatic triglyceride concentration after acute ethanol administration (either 6 g/kg p.o. or 2 g/kg i.p.) in rats. The compound had no influence on the increased hepatic NADH:NAD ratio (measured as the lactate:pyruvate and 3-hydroxybutyrate:acetoacetate ratios) after acute ethanol dosing (2 g/kg i.p.), but was found to lower hepatic acetaldehyde concentrations and prevent some of the disturbances in lipid metabolism observed in liver slices from ethanol-treated animals (e.g. decreased oxidation of [1-14C]palmitate to 14CO2) after this ethanol dose. The drug did not inhibit ethanol metabolism in this acute experiment. Administration of Malotilate to Wistar rats (100 mg/kg/day orally) during chronic feeding of ethanol as 36% of the total calorie intake in a liquid diet, resulted in a lower intake of the alcohol-containing diet by ethanol-fed animals and reduced body weight gain in rats which received the drug, without blood ethanol levels or the ethanol intake (expressed in g/kg body weight/day) being affected. In ethanol-fed animals, Malotilate prevented the production of fatty liver and the adaptive increase in the ethanol elimination rate (EER) normally seen in ethanol-fed animals, although the drug actually caused a slight increase in EER in glucose pair-fed controls. Malotilate did not significantly decrease the degree of induction of microsomal cytochrome P-450 by ethanol, but the increase in aniline hydroxylation was much less marked in animals receiving ethanol and Malotilate, suggesting that the activity of the inducible microsomal ethanol oxidising system (MEOS) may be reduced by the compound. Determination of hepatic acetaldehyde concentrations during ethanol feeding, and during an acute ethanol challenge test following long-term ethanol treatment showed that the compound significantly lowered the level of this ethanol metabolite in the liver under both circumstances. This reduction of hepatic acetaldehyde concentrations, probably resulting in part from the reduced EER as well as increased low-Km aldehyde dehydrogenase activities and glutathione contents seen in the livers of Malotilate-treated rats, are possible mechanisms by which the drug protects against triglyceride accumulation after ethanol administration.

Effects of cysteine and antioxidants on the hepatic redox-state, acetaldehyde and triglyceride levels after acute ethanol dosing.

Cysteine and the synthetic antioxidants butylated hydroxytoluene (BHT) and N,N'-diphenyl-phenylenediamine (DPPD) have been found to protect against the increase in hepatic triglycerides caused by acute ethanol administration (2 g/kg/i.p.) in rats. None of these agents affected the ethanol-induced increase in the hepatic redox-state, measured as lactate/pyruvate and 3-hydroxybutyrate/acetoacetate ratios, and there was no influence of any of the compounds on ethanol metabolism. Of the three agents tested, only cysteine was found to lower the liver acetaldehyde concentration after ethanol administration, confirming reports that trapping of acetaldehyde can protect against ethanol hepatotoxicity. The protective action of the anti-oxidants suggests that lipid peroxidation (probably initiated by acetaldehyde) is an important event in the pathogenesis of acute alcoholic fatty liver.

Palmitoyl-catechin for alcoholic liver disease: results of a three-month clinical trial.

A prospective randomized double-blind trial of 3-palmitoyl-(+)-catechin at a dose of 1500 mg daily (500 mg t.d.s.) for 3 months vs placebo has failed to demonstrate statistically significant clinical, biochemical or histological benefit in patients with biopsy-proven alcoholic liver disease. Nevertheless, this trial has confirmed the beneficial effect of a reduction in the rate of alcohol consumption on alcoholic liver disease. Apart from clinical evidence of a higher rate of alcohol consumption by patients receiving the active drug during the trial, no adverse side-effects were identified and for this reason, it is suggested that a further trial should be considered with the daily dosage so far used in man (20 mg/kg) increased toward that (100 mg/kg) employed with benefit in animal experiments.

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.

Inhibition of ethanol induced hepatic vitamin A depletion by administration of N,N'-diphenyl-p-phenylene-diamine (DPPD).

The administration of ethanol as 36% of the total calories in a nutritionally adequate liquid diet for three weeks to male Wistar rats caused a 36% decrease in hepatic vitamin A levels (P less than 0.001) when compared with glucose pair-fed control rats, without affecting serum levels of the vitamin. Simultaneous administration of a synthetic antioxidant, DPPD (N,N'-diphenyl-p-phenylene-diamine) to ethanol-fed rats caused a 73% decrease in the extent of the ethanol induced hepatic vitamin A depletion (P less than 0.001). DPPD administration did not affect weight gain, dietary (and hence ethanol) intake or serum ethanol and vitamin A levels in ethanol-fed rats, nor did it affect hepatic or serum vitamin A levels in pair-fed controls. Increased hepatic catabolism of retinoic acid due to induction of cytochrome P450 by ethanol has been suggested as a mechanism of depletion. In the current study, DPPD administration to ethanol-fed rats did not reverse the ethanol induced increase in microsomal cytochrome P450 concentrations or aniline hydroxylase activity. These findings indicate that the ethanol induced hepatic vitamin A depletion can be largely dissociated from the induction of cytochrome P450. In view of the potent free radical scavenging activity of vitamin A, and the protective effect of DPPD against ethanol induced hepatic loss of the vitamin, this study suggests that increased free radical generation and direct peroxidation of vitamin A may be an important mechanism by which ethanol induced hepatic vitamin A depletion occurs in the rat.

The effect of methylene blue on the hepatocellular redox state and liver lipid content during chronic ethanol feeding in the rat.

Feeding of ethanol in a liquid diet to male Wistar rats caused decreases in the hepatic cytosolic and mitochondrial [NAD+]/[NADH] ratios. This redox-state change was attenuated after 16 days of feeding ethanol as 36% of the total energy intake. Supplementation of the ethanol-containing liquid diet with Methylene Blue largely prevented the ethanol-induced redox state changes, but did not significantly decrease the severity of the hepatic lipid accumulation that resulted from ethanol ingestion. Methylene Blue did not affect body-weight gain, ethanol intake or serum ethanol concentrations in ethanol-fed rats, nor did the compound influence the hepatic redox state or liver lipid content of appropriate pair-fed control animals. These findings suggest that the altered hepatic redox state that results from ethanol oxidation is not primarily responsible for the production of fatty liver after long-term ethanol feeding in the rat.

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.

Alcoholic malnutrition and the small intestine.

Malnutrition is common in chronic alcoholics, although its severity may depend on the social characteristics of the patient group under study and their severity of alcohol dependence. General malnutrition is often reflected in body weight loss, mainly of adipose and muscle tissue. This loss of nutritional reserves is partly due to inadequate protein intake in the face of continued alcohol ingestion. However, there is also evidence that ethanol is relatively ineffective as a source of calories, in spite of its high theoretical calorific value. An increased metabolic rate and tissue oxygen consumption following alcohol ingestion, without parallel increases in phosphate bond energy production or anabolic processes demonstrate the poor value of ethanol as an alternative calorie source to carbohydrate, fat or protein. This situation of nutritional imbalance is often compounded in chronic alcoholics by the effects that ethanol has on gastrointestinal function. These include increased mucosal permeability which may lead to 'leakage' of nutrients from the blood to the gut lumen, increased gut motility with increased transit times, and impaired salt and water absorption. Alcohol inhibits absorption of vitamins and nutrients by active transport processes, an effect that may be crucial in precipitating specific nutrient deficiencies (e.g. thiamine) in the alcoholic, in addition to the role of reduced dietary intake of vitamins and minerals in alcoholics that also contributes to such deficiency states. The end result may be severe functional impairment and tissue damage in other organs, notably the liver and the brain, as a consequence of specific vitamin and nutrient deficiencies arising in chronic alcoholics by these mechanisms.

The effect of naloxone on the hepatocellular redox state and serum ethanol concentrations following acute ethanol administration.

Naloxone hydrochloride (2.0 mg/kg) has been found to reverse the significant decreases in the hepatic cytosolic and mitochondrial [NAD+]/[NADH] ratios observed after acute ethanol administration in rats. This correction of the ethanol-induced changes in the hepatocellular redox state by naloxone was, however, not associated with any lowering of serum ethanol concentrations or an observable reduction in the extent of intoxication. This lack of antagonism of alcohol intoxication by naloxone was not affected by the feeding status of the animals, the time point after naloxone administration at which serum ethanol concentration was determined or the method used for ethanol analysis. Thus this study has failed to confirm that naloxone antagonises acute alcohol intoxication, in spite of its potent ability to reverse the ethanol-induced changes in the hepatic redox state.

Alloxan-induced diabetes in the rat - protective action of (-) epicatechin?

Administration of alloxan (150 mg/kg body weight, i.p.) to male Wistar rats induced a reproducible and persistent diabetes mellitus as evidenced by elevated serum glucose and low serum insulin concentrations. Administration of either (-)epicatechin or (+)catechin (250 mg/kg, i.p. on each occasion) at 36, 24, 12 and 1 hour before and at 12 and 24 hours after alloxan administration did not prevent the induction of the diabetes. Similarly, treatment of animals with (-)epicatechin or (+)catechin (125 mg/kg i.p. twice daily) for 21 days commencing 24 hours after alloxan administration did not reverse the persisting elevated serum glucose and low serum insulin concentrations. Moreover, administration of these compounds did not relieve any of the symptoms of the alloxan-induced diabetes such as poor weight gain, polyuria or polydipsia. Thus, this study does not confirm previous reports of prophylactic and curative effects of (-)epicatechin against alloxan-induced diabetes in rats, in spite of the high dosages of the compounds used in these experiments.

Nutrition and vitamins in alcoholism.

Chronic alcoholics frequently have evidence of nutritional deficiency due to decreased intake, reduced uptake and impaired utilisation of nutrients. The alcoholic has increased nutrient requirements due to greater metabolic demands and the need for tissue repair. Chronic alcohol-related brain damage can often be a direct result of nutrient depletion, particularly of the vitamins thiamine, B12, nicotinamide and pyridoxine. Lesser degrees of brain damage are frequently unrecognised, and by the time a vitamin deficiency syndrome has developed and been diagnosed, irreversible damage has often occurred. The development of suitable computerised psychometric tests may allow earlier detection of brain malfunction associated with malnutrition, which can be reversed by nutrient repletion before permanent damage occurs. Circulating levels of vitamins can be a valuable guide to nutritional status, although care is needed when interpreting the results of such tests in the alcoholic. Sensitive microbiological and biochemical tests for assessing vitamin status in man have been available for some years, and in addition, new biochemical methods are constantly being developed. It is important that such methods are evaluated, and possibly adapted for clinical use where appropriate. Newer methods may have significant advantages over older, more established techniques. For thiamine and pyridoxine, for example, methods now exist to determine accurately circulating levels of the active forms of these vitamins, which could give more direct assessment of vitamin status than earlier methodology that uses indirect measurements, such as red cell enzyme activities. On the other hand, in the case of folate and B12, there has been a tendency to opt for the easy-to-perform radioassay techniques, when in fact the earlier microbiological methods offer greater sensitivity and probably also better accuracy. Technically difficult assays should not be disregarded if they can give information which is of greater clinical use than a simpler assay technique. Clinical laboratories should always bear in mind what their vitamin methods are actually measuring, particular consideration being given to whether metabolically inactive forms or analogues are determined in the assay. This can be of importance to the interpretation of vitamin data in the alcoholic, who often has problems forming active vitamins from their precursors.(ABSTRACT TRUNCATED AT 400 WORDS)

Heterogeneity of human erythrocyte transketolase: a preliminary report.

A modified procedure for the preparation of transketolase from erythrocytes by dialysis and column ion-exchange resin chromatography is described. Changes have been made in the resin used, the column dimensions and the elution procedure so as to separate the enzyme with improved resolution, prepare the apoenzyme free of thiamine pyrophosphate and study the kinetics of its activation or reactivation by the coenzyme. On the basis of the elution profile of the enzyme activity from the chromatographic column, two different samples of the transketolase have been isolated, which differ not only in their isoelectric properties, but also in the proportion of the transketolase present in the apoenzyme form. Not only do the apoenzymes isolated from each of the two fractions differ in the way in which they recombine with thiamine pyrophosphate but kinetic analysis of the results shows that each fraction contains at least two variants of transketolase differing in their affinity for thiamine pyrophosphate. Three, probably four, separate variants have been identified which differ in their affinities for thiamine pyrophosphate over a range greater than 10(4). It is concluded that these two fractions of the enzyme must contain different subsets of the eight isoenzymes of transketolase of differing isoelectric points and that some of these isoenzymes must differ also in their affinity for the coenzyme. The implications of these findings for the Blass and Gibson hypothesis about the pathogenesis of the Wernicke-Korsakoff syndrome are considered.

Differential effect of chronic alcohol intake and poor nutrition on body weight and fat stores.

A six-months out-patient study of chronic alcoholics with undecompensated liver disease has shown a statistically significant inverse correlation between the change in mean corpuscular volume and the change in body weight (r = -0.4, P less than 0.01). A fall in body weight over this period was the best clinical indicator of apparently continuing alcohol abuse. Previous anthropometric studies have indicated that reduced adipose tissue is one cause of lower body weights in such patients. To determine whether this is due to the effects of alcohol or of poor nutrition, the epididymal fat pad weights of rats following 28 days administration of alcohol (36% of total calories) as part of a nutritionally adequate liquid diet were compared with those of pair-fed controls initially matched for body weight. At the end of the experiment, body weight gain was the same in both groups but the mean weight of the fat pads of alcohol-fed animals (371.7 mg +/- 60.0 mg SD) all of which developed hepatic steatosis was 29% greater than that of pair-fed controls (288.7 mg +/- 42.4 mg). This difference was statistically significant (P less than 0.025). This study shows that alcohol intake per se does not prevent an increase in body weight or fat even if hepatic steatosis is induced and that loss of adipose tissue in chronic alcoholics who continue to drink is probably due to simultaneous inadequate nutritional intake.

Biochemical mode of action of a hepatoprotective drug: observations on (+)-catechin.

(+)-Catechin inhibits the hepatic lipid accumulation resulting from chronic ethanol ingestion in the rat. Experiments have been carried out to determine the mechanisms underlying this effect. Ethanol was administered (2.0 g/kg intraperitoneally) to Wistar rats and 90 min later 1 microCi [U-14C] palmitic acid injected intraperitoneally. Animals were sacrificed 10 min after injection of palmitate and the liver freeze-clamped. Ethanol caused a 250% increase in the hepatic lactate:pyruvate (L:P) ratio and a 100% increase in the amount of [U-14C] palmitate incorporated into the hepatic lipids when compared with controls. Pretreatment of animals with (+)-catechin (200 mg/kg orally) at 24 and 0 hr before ethanol caused significant reductions in the L:P ratio and amount of radioactivity incorporated into hepatic lipids, when compared with animals receiving ethanol alone. (+)-Catechin also stimulated 14CO2 production from [1-14C] palmitate by liver slices taken from rats 90 min after ethanol administration. Thus, (+)-catechin appears to mediate its effect on fat accumulation partly by correcting the ethanol-induced alterations in hepatic redox state as there is no evidence of the drug inhibiting ethanol metabolism.