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.     

The immediate and long term effects of clofibrate on the metabolism of the perfused rat liver.

A study was made of the immediate effects of CPIB (chlorophenoxy-isobutyrate) and of the effects of clofibrate (ethyl-CPIB) pretreatment on the metabolism of the perfused liver. Both treatments caused an increased hepatic uptake of lactate and free fatty acids. Pretreatment with clofibrate resulted in a decrease in perfusate glucose, an increase in ketogenesis and a decreased output of very low density lipoprotein triacylglycerol. A more oxidized redox state of both the cytosol and the mitochondria was indicated by decreased ratios of perfusate [lactate]/[pyruvate] and [3-hydroxybutyrate]/[acetoacetate] respectively. Increased hepatic O₂ consumption was associated with the increased liver weight of rats treated with the drug for 1 week. The fate of free fatty acids was followed by infusing [1—¹⁴Cloleate. The increased oxidation of oleate to both CO₂ and ketone bodies in livers from animals pretreated with clofibrate was accompanied by a corresponding decreased incorporation of ¹⁴C into very low density lipoprotein triacylglycerol. Lipogenesis was depressed upon addition of CPIB to the perfusate, but was increased after pretreatment with clofibrate. No changes in cholesterol synthesis were detected. A hypothesis to account for the hypolipidaemic and other effects of clofibrate pretreatment is advanced. This is based on a primary enhancement of fatty acid oxidation accompanied by a reciprocal decrease in hepatic triacylglycerol secretion. It is suggested that increased peroxisomal oxidation of fatty acids may be a cause of the decreased redox potential. A consequent activation of pyruvate dehydrogenase would explain both the changes in carbohydrate metabolism and the increase in lipogenesis.     

Effects of clofibrate on mitochondrial function

The effects of ethyl and sodium clofibrate on mitochondrial function were studied. Both compounds exerted similar effects, the ethyl derivative being more potent. State 3 respiration was inhibited; the order of inhibitory effectiveness was NAD⁺-dependent substrates > succinate > ascorbate. State 4 NAD⁺-linked oxidation was not significantly affected, but state 4 oxidations of succinate and ascorbate were stimulated by ethyl clofibrate. Energy production was inhibited, as evidenced by the decrease in the respiratory control ratio, the P/O ratio and the ATP-³²P exchange reaction. Energy utilization, assessed by substrate or ATP-supported energy-linked Ca²⁺ uptake, was also inhibited. By contrast, energy-independent Ca²⁺ uptake was not affected. Clofibrate interfered with the integrity of the mitochondrial membranes, since it stimulated ATPase activity and increased the normally low permeability of intact mitochondria toward NADH. The transfer of reducing equivalents into the mitochondria, catalyzed by the α-glycerophosphate, fatty acid or malate-aspartate shuttles, was inhibited by sodium clofibrate. These results may explain our previous finding that the reconstituted a-glycerophosphate shuttle was not stimulated in rats fed clofibrate, despite an increase in the activity of mitochondrial α-glycerophosphate dehydrogenase.     

Hypolipidemic effect of α-mono-p-myristyloxy-α'-methylcinnamoyl glycerol (LK-903) in rats

The hypolipidemic effect of an α-monoacylglycerol analog, α-monomyristyloxy-α-p -methyl-cinnamoyl glycerol (LK-903), was studied in rats. Its hypolipidemic activity in normal rats was about twice that of clofibrate. It did not produce hepatomegaly. Its free acid form, p-myristyloxy-α-methyl-cinnamic acid, was also active, although less active than the monoglyceride form. LK-903 was effective in fructose-, cholesterol-, and Triton-induced hyperlipidemias and phenobarbital-induced fatty liver. LK-903 differs from clofibrate in that it invariably depresses the triglyceride concentration of the liver. Its pharmacological effect seems to be ascribable to the depression of circulating free fatty acids.     

Evaluation of a screening method by liquid chromatography-tandem mass spectrometry for estimating effect of drugs on the activation and β-oxidation of fatty acids in mitochondria

Objectives Fatty acid metabolism is controlled not only by the acyl‐coenzyme A (CoA) synthetases but by some enzymes in the β‐oxidation cycle. Medium‐chain and long‐chain acyl‐CoA esters are key metabolites in fatty acid metabolism. We have developed an enzymatic assay method for determining chain shortening of the acyl‐CoAs via β‐oxidation from palmitic and octanoic acids in liver mitochondria. We have evaluated the assay method for detecting whether drugs influence the activation or the β‐oxidation of fatty acids. Methods Liver mitochondria were used for investigating the effect of drugs on fatty acid metabolism. The drugs selected were salicylic acid, diclofenac, valproic acid and paracetamol. Each acyl‐CoA formed was analysed by liquid chromatography–tandem mass spectrometry. Key findings After less than 5 min of incubation, the levels of acyl‐CoAs reflected the acyl‐CoA synthetase activity, whereas after 60‐min incubation they reflected the activity of some enzymes in the β‐oxidation cycle. Salicylic acid, diclofenac and valproic acid inhibited the medium‐chain acyl‐CoA synthetases, whereas valproic acid only exhibited a weak inhibitory activity toward the β‐oxidation of the medium‐chain fatty acids. In the case of long‐chain fatty acid metabolism, salicylic acid and diclofenac inhibited both the activation and β‐oxidation, whereas valproic acid was a weak inhibitor for only the β‐oxidation activity. Paracetamol showed hardly any influence on the metabolism of medium‐chain and long‐chain fatty acids. Conclusions These findings suggest that salicylic acid, diclofenac, valproic acid and paracetamol exert a different influence on fatty acid metabolism depending on the length of the acyl chain. This assay allows sensitive and selective analysis for predicting the pathways by which drugs exert a greater influence over fatty acid metabolism.     

Effect of clofibrate on the metabolism of oleate in the perfused rat liver

The effect of clofibrate on the metabolism of [1-¹⁴C]- and [U-¹⁴C]oleate was examined in the perfused rat liver. Clofibrate feeding severely reduced hepatic triglyceride secretion and enhanced ketone body production. The increase in the rate of incorporation of labeled tracers into perfusate oxidation products and ketone bodies due to the clofibrate treatment was demonstrated only with [U-¹⁴C]oleate. Clofibrate strongly reduced the rate of incorporation of oleate into perfusate triglyceride, whereas that into the phospholipid fraction of the post-perfused liver doubled. In consequence, the sum of the radioactivities in esterified lipids in the perfusate and the post-perfused liver was not altered by clofibrate. A clofibrate-dependent increase in phospholipid synthesis may restrict the amount of exogenous fatty acid which is available for the formation of triglyceride-rich lipoproteins.     

Disposition of clofibrate in the rat: Acute and chronic administration

The disposition of 1-[¹⁴C]clofibrate (0.4 $\text{mmole}\text{kg}$) was studied in rats after acute (single dose) and chronic (b.i.d., for 14 days) administration. With a single dose (orally or by intraperitoneal injection) of clofibrate, most (~90 per cent) of the ¹⁴C-dose appeared in the urine within 24 hr and the recovery of ¹⁴C from the urine and feces was nearly quantitative within 72 hr. Little fecal excretion of ¹⁴C (< 5 per cent) occurred after a single or chronic clofibrate administration. Clofibrate was readily absorbed and eliminated, as evidenced by a rapid increase in plasma ¹⁴C level within 90 min and a calculated biological half-life of 4.1 hr. The pharmacokinetic profile of ¹⁴C-elimination in rats was unaffected by pretreatment with cholestyramine. Clofibric acid [2-(4-chlorophenoxy)-2-methylpropionic acid] was identified as the major metabolite in plasma (~97 per cent) whereas the glucuronide of clofibric acid was the main urinary and biliary metabolite (~96 per cent). Clofibric acid, as the free acid and glucuronide form, accounted for 99 per cent of the total ¹⁴C-dose in rats, and unchanged clofibrate was not detected in any of the biological samples. Two unidentified, minor urinary metabolites were also detected. In cannulated bile duct studies, it was found that [¹⁴C]clofibrate, as clofibric acid, was rapidly and efficiently excreted in the bile. The biliary excretion rates of ¹⁴C and of the glucuronide of clofibric acid were also not altered by phenobarbital pretreatment. Chronic treatment with [¹⁴C]clofibrate did not alter the qualitative or quantitative nature of biotransformation in vivo. An increased rate of urinary ¹⁴C-elimination was observed following chronic 1-[¹⁴C]clofibrate treatment, with concomitant reductions in blood and heart ¹⁴C-content and an elevation in ¹⁴C-content of epididymal fat tissue. Subcellular fractionation of liver, from rats given [¹⁴C]clofibrate chronically, indicated an increased distribution of ¹⁴C into mitochondria and peroxisomes. Tissue ¹⁴C-levels, achieved in these in vivo studies, were an order of magnitude lower than those required for the pharmacological activities of clofibrate and clofibric acid in vitro.     

Effect of salicylic acid and diclofenac on the medium‐chain and long‐chain acyl‐CoA formation in the liver and brain of mouse

Medium‐chain and long‐chain acyl‐CoA esters are key metabolites in fatty acid metabolism. Effects of salicylic acid on the in vivo formation of acyl‐CoAs in mouse liver and brain were investigated. Further, inhibition of the medium‐chain and long‐chain acyl‐CoA synthetases by salicylic acid and diclofenac was determined in mouse liver and brain mitochondria. Acyl‐CoA esters were analyzed by liquid chromatography–tandem mass spectrometry. The amounts of medium‐chain acyl‐CoAs (C₆, C₈ and C₁₀) were less than long‐chain acyl‐CoAs (C_16:0, C_18:0, C_18:1 and C_20:4) in both liver and brain. The administration of salicylic acid decreased the levels of both the medium‐chain (C₆, C₈ and C₁₀) and long‐chain acyl‐CoAs (C_16:0, C_18:0, C_18:1 and C_20:4) in liver. In brain, however, only long‐chain acyl‐CoAs were decreased. The level of salicylyl‐CoA detected in brain was about 12% of that in liver. Salicylic acid had a strong inhibitory activity (IC₅₀ = 0.1 mm ) for the liver mitochondrial formation of hexanoyl‐CoA from hexanoic acid, whereas diclofenac was weak (IC₅₀ = 4.4 mm ). In contrast, diclofenac (IC₅₀ = 1.4 mm ) inhibited the liver mitochondrial long‐chain acyl‐CoA synthetases more potently than salicylic acid (IC₅₀ = 25.5 mm ). Similar inhibitory activities for the acyl‐CoA synthetases were obtained in the case of the brain and liver mitochondria, except for the weak inhibition of brain medium‐chain acyl‐CoA synthetases by salicylic acid (IC₅₀ = 1.8 mm ). These findings suggest that salicylic acid and diclofenac exhibit different mechanisms of inhibition of fatty acid metabolism depending on the length of the acyl chain and tissues, and they may contribute to the further understanding of the toxic effects associated with these drugs. Copyright © 2009 John Wiley & Sons, Ltd.     

Raloxifene affects fatty acid oxidation in livers from ovariectomized rats by acting as a pro-oxidant agent

Estrogen deficiency accelerates the development of several disorders including visceral obesity and hepatic steatosis. The predisposing factors can be exacerbated by drugs that affect hepatic lipid metabolism. The aim of the present work was to determine if raloxifene, a selective estrogen receptor modulator (SERM) used extensively by postmenopausal women, affects hepatic fatty acid oxidation pathways. Fatty acids oxidation was measured in the livers, mitochondria and peroxisomes of ovariectomized (OVX) rats. Mitochondrial and peroxisomal β-oxidation was inhibited by raloxifene at a concentration range of 2.5–25 μM. In perfused livers, raloxifene reduced the ketogenesis from endogenous and exogenous fatty acids and increased the β-hydroxybutyrate/acetoacetate ratio. An increase in ¹⁴CO₂ production without a parallel increase in the oxygen consumption indicated that raloxifene caused a diversion of NADH from the mitochondrial respiratory chain to another oxidative reaction. It was found that raloxifene has a strong ability to react with H₂O₂ in the presence of peroxidase. It is likely that the generation of phenoxyl radical derivatives of raloxifene in intact livers led to the co-oxidation of NADH and a shift of the cellular redox state to an oxidised condition. This change can perturb other important liver metabolic processes dependent on cellular NADH/NAD⁺ ratio.     

Effect of 4-(4′-chlorobenzyloxy)benzyl nicotinate (KCD-232) on triglyceride and fatty acid metabolism in rats

The effects of KCD-232, a new hypolipidemic agent with a structure of 4-(4'-chlorobenzyloxy) benzyl nicotinate, on triglyceride (TG) and fatty acid (FA) metabolism were studied in rats. KCD-232 dose-dependently reduced both liver and serum TG levels. From in vivo and in vitro studies, the hypotriglyceridemic action of KCD-232 was shown to be based on the inhibition of hepatic TG synthesis due to both decreased FA synthesis and increased FA oxidation in the liver. Of two metabolites of KCD-232, i.e. 4-(4'-chlorobenzyloxy)benzoic acid (MII) and nicotinic acid, MII was found to be responsible for the decreased synthesis and increased oxidation of FA in the liver, the latter apparently being due to increased mitochondrial oxidation activated by MII. MII was demonstrated to form a xenobiotic TG in which one fatty acid moiety was substituted by MII and to form a thioester with CoA by rat liver microsomes. This thioester, MII-CoA, inhibited fatty acid syntheses from [¹⁴C]acetate, [¹⁴C] acetyl-CoA and [¹⁴C]malonyl-CoA in cell-free enzyme systems from rat liver both with and without an NADPH-generating system, whereas MII as such showed no effect. MII-CoA was therefore considered to be a chemical entity for the inhibition of hepatic fatty acid synthesis by KCD-232 and was suggested to inhibit fatty acid synthetase directly.     

A subcellular study of the clofibratei-nduced increase in coenzyme a concentration in rat liver

When clofibrate [ethyl 2-(4-chlorophenoxv)-2-methylpropionatel was administered subcutaneously to rats (600 $\text{mg}\text{kg}$ per day for 3 days), the concentration of CoA and its acyl derivatives in the liver increased 2.5-fold. Forty-eight per cent of the total cellular CoA in the clofibrate-treated rat liver and 51 per cent in the control liver was found in the mitochondrial fraction. In order to study the intermediates of CoA synthesis, clofibrate-treated rats were injected with [³H]pantothenate intracardially and killed after 30 min, l or 2hr for determination of the incorporation of radioactivity into CoA and its precursors. The incorporation of pantothenate into CoA after 2 hr was 5.9-fold in the liver and 4.5-fold in the liver mitochondrial fraction as compared with the control values. Measurement of the pantothenate concentration and radioactivity in clofibrate-treated and control rat liver showed that the higher incorporation of [³H]pantothenate into CoA in clofibrate-treated rat liver cannot be the result of a higher specific radioactivity of pantothenate. It is therefore evident that clofibrate affects the CoA concentration by increasing the rate of synthesis, although the rate of CoA degradation is simultaneously decreased, as has been shown previously [9]. The present results indicate that clofibrate increases the total hepatic CoA concentration without affecting the intracellular compartmentation of CoA. The clofibrate-induced increase in the rate of CoA synthesis does not result in differences in the compartmentation of the intermediates of CoA synthesis.     

Further studies concerning the effects of clofibrate on respiration and oxidative phosphorylation of rat liver mitochondria

Clofibrate, administered in vitro, inhibited rat liver mitochondrial respiration at two sites within the respiratory chain. One site was between the interaction of NADH with NADH dehydrogenase and the point at which electrons from succinate oxidation enter the electron transport chain; another, less sensitive site, was between the interaction of succinate with succinate dehydrogenase and cytochrome c. In addition to these specific sites, clofibrate inhibited respiration by causing a depletion of pyridine nucleotides that was accompanied or followed by large-amplitude, non-energy-linked swelling. Clofibrate uncoupled oxidative phosphorylation at coupling sites II and III but not at site I. The concentrations required to cause loss of pyridine nucleotides were lower than those required to inhibit at the specific sites. p-Chlorophenoxyisobutyrate (CPIB) also inhibited succinate and β-hydroxybutyrate-linked respiration, and uncoupled oxidative phosphorylation, but at much higher concentrations (50 per cent inhibition of β-hydroxybutyrate oxidation at about 3·7 μmoles/mg of protein) than were required of clofibrate (50 per cent inhibition of β-hydroxybutyrate oxidation at about 0·17 μmole/mg of protein). Clofibrate administration to rats (100 and 300 mg/kg p.o. daily for 1 week) lowered serum lipid levels and increased the liver size, the amount of mitochondrial protein/g of liver, and the oxygen consumption of liver slices. However, mitochondria, isolated from livers of the treated rats, respired normally. A single administration of clofibrate (100 or 300 mg/kg, p.o.) did not affect liver slice respiration.     

Inhibition of mitochondrial fatty acid oxidation in pentenoic acid-induced fatty liver: A possible model for Reye's syndrome

Rats treated with six to eight doses (80 mg/kg, i.p.) of 4-pentenoic acid, an inhibitor of mitochondrial fatty arid oxidation in vitro, during a 48-hr starvation period developed microvesicular fatty infiltration of the liver similar to that observed in Reye's Syndrome. Hepatic triglycorides were elevated an average of 5-fold, although considerable variability was found between individual rats. Fed rats did not develop fatty liver upon similar treatment with pentenoic acid. Liver mitochondria isolated from rats with pentenoic acid-induced fatty liver showed a persistent inhibition of fatty acid oxidation. Rates of oxidation of pahnitoylcamitine and decanoylcamitine were decreased about 70%, while that of octanoylcamitine was decreased 50%. Camitine-independent oxidation of octanoate was also inhibited. Oxidation rates for substrates other than fatty acids, including glutamate, succinate, pyruvate, and α-ketoglutarate, were unaffected. Measurements of flavoprotein reduction in intact mitochondria indicated that neither palmitoylcamitine nor palmitoyl CoA plus l-carnitine could elicit reduction of acyl-CoA dehydrogenase and electron transferring flavoprotein in mitochondria from rats with pentenoic acid-induced fatty liver. These results support a site of inhibition of mitochondrial β-oxidation at the level of acyl-CoA dehydrogenase for pentenoic add treatment in vivo, and they suggest a role for nutritional or hormonal factors in the metabolic disposition of pentenoic add _vivo and in the development of fatty liver.     

The effects of 2{5(4-chlorophenyl) pentyl}oxirane-2-carbonyl-CoA on mitochondrial oxidations

2{5(4-Chlorophenyl)pentyl}oxirane-2-carbonyl-CoA (POCA-CoA) was prepared from 2{5(4-chlorophenyl)pentyl}oxirane-2-carboxylate (POCA) and characterised chromatographically. POCA-CoA does not inhibit citrate cycle oxidations or effect oxidative phosphorylation by rat liver mitochondria. POCA-CoA at low (μM) concentrations, but not free POCA^?, specifically inhibits palmitoyl-CoA oxidation at the stage of carnitine palmitoyltransferase I (CPT I) situated on the outer face of the inner mitochondria membrane. Palmitoyl-carnitine oxidation was not inhibited by POCA-CoA. POCA-CoA inhibits palmitoyl-CoA oxidation in liver mitochondria from fed rats more strongly than it does in mitochondria from fasted rats, similarly to the inhibition by malonyl-CoA [E. D. Saggerson and C. A. Carpenter, FEBS Lett. 129, 225 (1981)]. Palmitoyl-CoA, by contrast with palmitoylcarnitine, is not quantitatively oxidised to acetoacetate by liver mitochondrial fractions, presumably due to competing palmitoyl-CoA hydrolase activity. In the presence of POCA-CoA the amount oxidised is decreased further because the slower rate of oxidation allows more palmitoyl-CoA to be hydrolysed to palmitate. The oxidation of palmitoyl-CoA, but not that of palmitoyl-carnitine, was strongly decreased in washed liver and muscle mitochondrial fractions from POCA-fed animals. POCA^? inhibited the oxidation of {U-¹⁴C}palmitate in cultured human fibroblasts, and caused small increases in ¹⁴CO₂ production from {1-¹⁴C}pyruvate and {U-¹⁴C}glucose. Inhibition of β-oxidation at the stage of CPT I by POCA-CoA can explain the powerful hypoketonaemic and hypoglycaemic effects of POCA in fasted normal and fasted diabetic animals [H. P. O. Wolf, K. Eistetter and G. Ludwig, Diabetologia22, 456 (1982)].     

Effects of pyrazole and 3-amino-1,2,4-triazole on methanol and ethanol metabolism by the rat

The metabolism of methanol-¹⁴C and ethanol-1-¹⁴C in rats was evaluated from the rates of ¹⁴CO₂ production. 3-Amino,1,2,4-triazole, a known catalase inhibitor, decreased by 10 and 35 per cent the rates of oxidation of ethanol and methanol, whereas pyrazole, an alcohol dehydrogenase inhibitor, decreased the rates 85 and 50 per cent respectively. However, the simultaneous use of both inhibitors gave the same effects produced by pyrazole alone. Thus the relative contributions in vivo to alcohol metabolism of rat liver alcohol dehydrogenase and catalase-mediated peroxidation, cannot be estimated only in this way. Rat liver alcohol dehydrogenase was purified 14·7 times. At pH 7·0 and 30°, the K_m for methanol was 340 mM and for ethanol 0·26 mM. The V_max/e was 2·36 nM for methanol and 22·3 nM for ethanol (NADH × U^?1 × 1^?1 × sec^?1). 3-Amino-1,2,4-triazole inhibited the purified enzyme with a K_i of 55 mM for methanol and 33 mM for ethanol. The K_i of pyrazole was 2·3 mM for methanol and 2·2 mM for ethanol. The amount of alcohol dehydrogenase present in rat liver, with the found kinetic constants, can account for the ethanol oxidation in vivo, but fails to account, as methanol dehydrogenase, for the observed pyrazole-sensitive methanol oxidation. A mechanism for the complete oxidation of methanol to CO₂ and water through the concerted action of catalase and alcohol dehydrogenase is suggested. 3-Amino-1,2,4-triazole in a dose of 1 g/kg decreases more than 90 per cent of the catalatic activity of catalase, but under certain conditions in vitro, only about 50 per cent of the peroxidative activity of catalase towards methanol and ethanol. Consequently, the degree of catalase-mediated peroxidation should not be controlled or estimated from the residual catalatic activity when using catalase inhibitors. Pyrazole, at a dose of 0·3 g/kg, does not affect catalase activity 1 hr after administration, but decreases it more than 90 per cent after 24 hr. This effect is completely prevented in the presence of alcohol.     

ATP metabolism in an ethanol induced fatty liver.

A marked decrease in the hepatic concentration of ATP lowers the phosphate potential of the liver of rats fed ethanol for prolonged periods of time. Either a decreased synthesis or an increased demand could account for this change in ATP. The experiments presented here were designed to investigate both possibilities. Changes in the utilization of ATP were assessed by measuring the activity of the [Na⁺ + K⁺]-activated ATPase system, and changes in the synthesis of ATP by the activity of the adenine nucleotide translocase system. Since the level of long-chain CoA derivatives of fatty acids regulate the translocation of ADP into mitochondria, their total cellular content and mitochondrial level were also determined. The experiments were conducted on male Sprague-Dawley rats (275–300 g) maintained on a liquid diet having 36 per cent of the caloric intake as ethanol. After 2 weeks, the experimental animals had a fatty liver. This change was not associated with any significant alteration in the ATP content of the liver or in the activity of the adenine nucleotide translocase system. After 4 weeks, the excess of neutral lipid in the liver still persisted, but was associated with a marked increase in the level of long-chain CoA derivatives of fatty acids. The level of ATP in the liver was only 50 per cent of normal, and the rate of translocation of ADP into the mitochondria was decreased. The activity of the adenine nucleotide translocase system could be restored to normal values if the long-chain CoA derivatives of fatty acids were removed from the surface of the mitochondrial membrane by treatment of the preparation with defatted albumin. At this time there was only a slight (15%) enhancement of the [Na⁺ + K⁺]-activated ATPase system. With the removal of ethanol from the diet the ATP level returned to normal rather quickly. Changes in the level of hepatic ATP correlated well with the activity of the adenine nucleotide translocase system but did not parallel changes in the activity of the Na⁺ pump system. These findings indicate that the dominant feature leading to the decrease in the ($\text{ATP}\text{ADP}\text{× P}{}_{\mathrm{i}}$) ratio is decreased synthesis of ATP.     

The Hypolipidemic Activity of Furoic Acid and Furylacrylic Acid Derivatives in Rodents

2-Furoic acid, 3-furoic acid, 3,4-furan dicarboxylic acid and furyl-acrylic acid were evaluated for hypolipidemic activity in mice and rats. 2-Furoic acid was the most potent agent of the four tested, lowering serum cholesterol levels 41 % and serum triglyceride levels 56 % at 20 mg/kg/day in mice and serum cholesterol 50 % and serum triglyceride levels 42 % in rats. 2-Furoic acid effectively suppressed liver mitochondrial citrate exchange, ATP dependent citrate lyase, acetyl CoA synthetase, acyl CoA cholesterol acyl transferase, sn-glycerol-3-phosphate acyl transferase, phosphatidate phosphohydrolase and hepatic lipoprotein lipase enzymatic activities. Lipid levels after 16 days in mice were reduced in the liver. In the rat cholesterol content of the HDL fraction was elevated and lowered in the chylomicron fraction. 2-Furoic acid administration for 14 days resulted in a large portion of ³H-cholesterol being excreted by the biliary route. The furoic acid derivatives appear to have promise as hypolipidemic agents and further studies on their ability to lower lipids are warranted.     

Effect of tetracycline on the metabolism of [1-14C]oleate by the liver.

The effects of tetracycline on the synthesis, outward transport, and accumulation of triglyglycerides from [1-¹⁴C]oleate were investigated using the isolated perfused rat liver. The effects of the antibiotic on production of CO₂, and ketone bodies were studied also. Tetracycline either was added to the medium perfusing livers from normal fed male rats, or alternatively, was administered to rats intravenously (pure tetracycline HCl, or Achromycin, 100 mg/kg body weight) 3 hr prior to removal of the livers for perfusion. The quantity ofoleate infused was sufficient to yield approximately half-maximal rates of output of triglycerides. Under these conditions, on the basis of the isotopic data, the hepatic accumulation of triglycerides derived from the exogenous sources (perfusate ¹⁴C free fatty acid) was balanced by a decrease in outward transport triglycerides, whether tetracycline was added directly to the medium perfusing the livers in vitro or was administered to the animals in vivo. Decreases in hepatic oxidation of oleate induced by tetracycline did not appear to be an important mechanism for induction of steatosis. These data suggest that the fatty liver resulting from intoxication by tetracycline results primarily from inhibition of outward transport of triglycerides.     

The effect of chlorhexidine on the anaerobic fermentation of Saccharomyces cerevisiae related to the release of protein.

The relationship between the inhibitory effect of chlorhexidine (CHX) on the anaerobic fermentation of baker's yeast and the release of protein from the cells has been studied. The experiments were based on the ability of Ca²⁺ to protect the cells against the effect of CHX. Maximal depression of the anaerobic fermentation was produced by a concentration of 21 nmoles per mg of wet cells (29 μM) without the addition of Ca²⁺. This amount of CHX had no effect when 41 mM Ca²⁺ was added prior to or concomitantly with CHX. The inhibitory effect of CHX increased proportionally to the time of incubation up to about 75 sec. Fifty per cent inhibition of the CO₂ production was achieved after 41 sec of incubation without Ca²⁺, while maximal inhibition (90 per cent) was obtained after 100 sec of incubation. The yeast could lose up to 5 μg of protein per mg of cells corresponding to 4 per cent of their total content without any impairment of CO₂ production. The release of protein at the maximal inhibition of the CO₂ production amounted to 14 μg per mg of cells corresponding to 11 per cent of the total content. CHX exerted a concentration dependent protein releasing effect on the yeast in total concentrations up to 836 nmoles per mg of cells (1153 μM). The maximal release of protein was 50 μg per mg of cells corresponding to 38 per cent of the total content. Higher concentrations of CHX exerted a precipitating effect on the released proteins.     

Effects of clofibrate on ethanol-induced modifications in liver and adipose tissue metabolism: role of hepatic redox state and hormonal mechanisms.

Mechanisms by which clofibrate decreases the development of acute alcoholic fatty liver were studied, especially in regard to hepatic redox state and hormonal regulation of carbohydrate and lipid metabolism. A partial inhibition of the ethanol-induced increase in cytosolic NADH/NAD ratio was observed. As a result, in clofibrate-treated rats hepatic α-GP concentration during ethanol oxidation also remained at the same level as in normal control rats. Clofibrate treatment prevented the ethanol-induced increase in the adipose tissue cAMP and plasma FFA concentrations. Hepatic concentrations of CoA-SH, acetyl-CoA and long chain acyl-CoA were markedly increased by clofibrate treatment. Plasma insulin concentration was decreased in clofibrate-treated rats, which also showed an impaired glucose tolerance. The results show that clofibrate is able to restrict the availability of substrates (α-GP and fatty acids) for hepatic triglyceride synthesis in vivo. In addition, it was concluded that the partial inhibition of ethanol-induced fatty liver by clofibrate may result from the enhancement of the oxidation pathway of fatty acid metabolism as suggested by the enormous increase in hepatic content of CoA-SH and its derivatives.