Ilmo Hassinen

Ethanol metabolism in rats treated with ethyl-α-p-chlorophenoxyiso-butyrate (Clofibrate)

Clofibrate treatment (60 mg/day/100 g body wt im. for nine days) increased the liver to body weight ratio of the rats by 71 %. The activity of mitochondrial α-glycerophosphate dehydrogenase in the liver of clofibrate-treated rats was five times greater than in normal rat liver. The amounts of cytochrome a and mitochondrial protein per gram of liver wet weight were not changed. Clofibrate treatment increased the oxygen consumption of liver slices by 16 %. Ethanol increased the lactate/pyruvate ratio (an index of the redox state in the cytosolic compartment of the liver cell) in perfused normal livers and only slightly in the livers from clofibrate-treated rats. This effect of clofibrate is similar to that of thyroxine and seems to be due to enzyme induction, as diethylaminoclofibrate hydrochloride added in vitro did not prevent ethanol-induced increase in hepatic lactate/pyruvate ratio. Clofibrate treatment in rats significantly increased the disappearance rate of ethanol. The activity of hepatic alcohol dehydrogenase per gram of liver wet weight of per milligram of soluble protein was not changed by clofibrate treatment.

Absorption spectrophotometry of perfused rat liver applied to fructose-induced inhibition of respiration

Abstract In a hemoglobin-free perfusion of rat liver, a single addition of 5 mM fructose caused inhibition of respiration. Simultaneously, the intramitochondrial NAD, fluorescent flavoproteins and cytochrome c were reduced and cytochrome b oxidized. In a rotenone-blocked liver ethanol did not cause any reduction of cytochrome b , indicating that the hydrogen transfer mechanisms between cytosolic NADH and the mitochondrial respiratory chain do not operate above the level of the first phosphorylation site. The relative significance of the respiratory chain and regulation at substrate level in the control of respiration are discussed.

Effect of disulfiram (tetraethylthiuram disulfide) on mitochondrial oxidations

Abstract Disulfiram inhibits the oxidation of NAD-linked substrates by mitochondria but does not affect their succinoxidase activity. The oxidation of β-hydroxybutyrate was totally blocked by 0·27 mM disulfiram. Disulfiram did not change the P/O ratios. Uncoupled mitochondria were less sensitive to disulfiram than phosphorylating mitochondria. Disulfiram inhibited the dinitrophenol-activated ATPase of mitochondria by about 60 per cent whilst having little effect on Mg 2+ -activated ATPase. The ATP-P i exchange reaction was approximately 50 per cent inhibited by disulfiram. The inhibitory effect of disulfiram on mitochondria is irreversible. 2-Mercaptoethanol protected the mitochondria if present before the addition of disulfiram. The mechanism of action of disulfiram and the implications of the results for the understanding of the disulfiram-alcohol reaction and the mechanism of oxidative phosphorylation are discussed.

Regulation of cellular respiration by thyroid hormone. Spectroscopic evidence of mitochondrial control in intact rat liver

Abstract In order to elucidate the role of the mitochondrial control (coupling) mechanism in the regulation of respiration in intact liver, we investigated perfused rat livers using direct spectrophotometry. The acute effects of fructose on hepatic concentrations of adenine nucleotides, inorganic phosphate, and citrate were also studied. In hypothyroid rat livers fructose caused a small stimulation of respiration, followed by marked inhibition lasting about 10 min. During the respiratory inhibition the redox state of the flavoproteins was reduced but that of cytochrome b was markedly oxidized. In euthyroid rats fructose first stimulated hepatic oxygen consumption, but the subsequent inhibition of respiration was much smaller than that in hypothyroid rats. During the inhibitory phase flavoproteins were reduced, but the oxidation of cytochrome b was significantly less than in hypothyroid rats. In hyperthyroid rat liver, fructose stimulated respiration without any subsequent inhibition. Flavoproteins and all cytochromes were reduced, and no transient oxidation was observed in contrast to hypo- and euthyroid rats. In all groups of rats fructose induced a prompt decrease in hepatic concentrations of ATP and inorganic phosphate. Fructose did not induce any acute changes in the concentrations of citrate, but in hyperthyroid rat liver the citrate concentration was three times greater than in hypo- or euthryoid liver. These results suggest that, in intact tissue also, mitochondrial control is important in the regulation of energy metabolism during the metabolism of fructose. Thyroxine treatment seems to loosen this coupling, perhaps by increasing the concentration and oxidation of fatty acids in the liver.

Metabolic interactions of fructose and ethanol in perfused liver of normal and thyroxine-treated rats.

Abstract Combined effects of thyroxine treatment and fructose on the rate of ethanol oxidation, the hepatic redox state and the hepatic oxygen consumption were studied in perfused rat liver. Neither thyroxine treatment nor fructose was found to influence the rate of ethanol elimination by the liver. An ethanol-induced increase in lactate/pyruvate concentration ratio in the perfusion medium was augmented by the simultaneous addition of fructose. Thyroxine treatment markedly inhibited this redox change. In perfused livers of thyroxine-treated rats, fructose and glucose were utilized significantly more rapidly than in normal livers. In both kinds of liver, fructose was metabolized about three times as fast as glucose. Lactate and pyruvate production from fructose was ten times that of glucose. Ethanol had no effect on the rate of fructose or glucose elimination in either group of rats. The glucose production from fructose was greater in normal rat livers than in thyroxine-treated ones and was inhibited by ethanol in normal livers. In perfused livers of normal rats, fructose had a biphasic effect on the respiration. During the first 2 min after the addition of fructose, a stimulation of respiration occurred. The oxygen consumption was then inhibited for 8 to 10 min. In normal rats, after an injection of fructose, the hepatic adenosine triphosphate (ATP) and inorganic phosphate (P i ) concentrations diminished coincidently with the respiratory inhibition observed in the perfusions. In perfused livers of hyperthyroid rats, fructose only stimulated the oxygen consumption, but in contrast to normal livers, no inhibition was observed. Hepatic ATP and P i concentrations in these rats were decreased by fructose but not as much as in normal livers. The fructose-induced changes in hepatic oxygen consumption and ATP and P i content are interpreted as an example of mutual control between glycolysis and oxidative metabolism. The mechanism of this phenomenon and its control by thyroid hormones are discussed.

Metabolic effects of acetaldehyde in the intact rat brain cortex and its subcellular fractions

Summary The effect of acetaldehyde on the redox state of the nerve cell was investigated at 4 levels of organization: intact brain cortex in vivo , cortex slices, isolated nerve endings and the conventional subcellular fractions of the nerve cell. Aldehyde dehydrogenase activity was determined fluorometrically in the nuclear, mitochondrial, cytosolic and synaptosomal fractons from rat brain cortex. The activities for acetadehyde in these fractions were 0.331, 1.80, 0.219 a nd 0.759 nmole · mg protein −1 · min −1 , respectively. Two apparent Km values were observed in frozen and thawed mitochondrial preparations,1.0 × 10 −4 M and 1.3 × 10 −6 M. The higher value was probably due to the presence of intact mitochondrial membranes. The oxygen consumption of the synaptosomes was not sensitive to uncouplers but was inhibited by acetaldehyde (0.02–5m M ). The redox state was studied by measuring the NAD(P)H and flavoprotein fluorescence of brain mitochondria and synaptosomes. In these preparations the nicotinamide nucleotides were largely oxidized and the redox state was not affected by acetaldehyde. However, in cerebral cortex slices acetaldehyde caused a reduction of NAD(P) ** , detectible by measuring the surface fluorescence of the tissue slices at appropriate wavelengths. In vivo , acetaldehyde also caused reductionof NAD(P) in the cerebral cortex. The reduction could be demonstrated by measurements of the surface fluorescence of the cerebral cortex in vivo and by chemical measurements of the nicotinamide nucleotides in tissue samples obtained by the freeze-stop echnique.

Mixed Function Oxidase and Ethanol Metabolism in Perfused Rat Liver

Oxidation-reduction changes of cytochrome P-450 and oxygen consumption were measured in isolated perfused livers from normal and phenobarbital-treated rats. Phenobarbital treatment markedly increased the aminopyrine-induced reduction of cytochrome P-450, but ethanol did not cause any redox changes of this cytochrome. It was concluded that the microsomal ethanol-oxidizing system has an insignificant role in the metabolism of ethanol in intact liver.

Studies on the mechanism on inhibition of acute alcoholic fatty liver by clofibrate

Abstract The effect of ethanol on hepatic α-glycerophosphate (α-GP), dihydroxyacetone phosphate (DAP) and triglyceride (TG) concentrations, and on serum levels of triglycerides (TG) and free fatty acids (FFA) was studied 4 and 12 hr after administration of ethanol in normal and ethyl-α-p-chlorophenoxyisobutyrate (clofibrate)-treated rats. In the normal rats, ethanol increased liver α-GP and TG concentrations, α- GP DAP ratio and serum FFA concentration. In the clofibrate-treated rats there was no change in α- GP DAP ratio or α-GP concentration after ethanol, nor was there a significant rise in liver TG concentration. The initial serum TG concentration in the clofibrate-treated rats was about 30% of that in the normal animals. In the normal rats, ethanol administration produced a higher serum TG concentration than glucose, but in the clofibrate-treated rats it did not cause a significant rise in serum TG level. Thus, it appears that clofibrate treatment can inhibit the ethanol-induced change in the hepatic redox state in vivo as in the isolated liver. These results strengthen the view that clofibrate inhibits the production of acute ethanolic fatty liver at least in part by abolishing the ethanol-induced change in the hepatic redox state.

Effect of a transaminase inhibitor on the transport of cytosolic reducing equivalents into mitochondria.

Summary The effects of a transaminase inhibitor, cycloserine (CS), on extra and intramitochondrial redox state were studied in perfused rat liver. The addition of CS induced a rapid increase in perfusate lactate/pyruvate (L/P) ratio indicating a shift towards reduction in the cytosolic NAD(H) pool. The flavoprotein absorbance, measured from a lobe of liver, increased simultaneously indicating an oxidation of the intramitochondrial redox state. CS potentiated the effects of ethanol on the cytosolic redox state (L/P ratio), but decreased the ethanol-induced reduction of intramitochondrial flavo-proteins. The results can be interpreted to mean that transamination is of importance in the transport of reducing equivalents across the mitochondrial membrane.

Comparison of the effects of disulfiram and dimercaptopropanol arsenite on mitochondrial structure and function

Abstract The effects of disulfiram and arsenite + BAL∗ on the respiratory chainlinked NADH2 dehydrogenase and oxidase activities, reduction of mitochondrial flavoprotein and cytochrome b, effects on mitochondrial volume and mitochondrial ATPases were examined. Disulfiram inhibited NADH2 oxidase activity without affecting the dehydrogenase moiety of the enzyme. NADH2 oxidase was inhibited by BAL + arsenite. Disulfiram inhibited the reduction of cytochrome b by NADH2 without affecting reduction by succinate. BAL + arsenite inhibited the reduction of cytochrome b by NADH2 and succinate. Large-amplitude swelling of the mitochondria was induced by disulfiram, whereas rapid small-amplitude swelling was observed in the presence of BAL + arsenite. Both disulfiram and BAL + arsenite inhibited the DNP-activated ATPase of mitochondria in a closely similar manner. The role of structural alterations of mitochondria on the effects of disulfiram are discussed.