Priscilla I. Spach

Effect of ethanol consumption on the phospholipid composition of rat liver microsomes and mitochondria

Male Sprague-Dawley rats were maintained for 31 days on a liquid diet containing 36% of calories as ethanol. Pair-fed controls were administered a similar diet, but with maltose-dextrin isocalorically substituted for ethanol. A phospholipid analysis has been carried out in liver microsomes and mitochondria isolated from the two groups of animals. The phospholipid phosphorus/protein ratio was not significantly different in the organelles of the ethanol-fed animals as compared to the same organelles of liquid diet controls, which indicates that ethanol feeding did not influence the total phospholipid content of microsomes and mitochondria. The phospholipid distribution within organelles was not changed, except for a significant increase in the phosphatidylinositol content of microsomes from ethanol-fed animals. The fatty acid compositions of both microsomal and mitochondrial phospholipids were significantly altered by ethanol feeding. In microsomes from ethanol-fed rats, palmitic acid levels were lowered in the total phospholipid fraction, phosphatidylcholine and phosphatidylethanolamine; oleic acid levels were elevated in microsomal phosphatidylethanolamine. In mitochondria from ethanol-fed animals, palmitic and arachidonic acid were lowered in phosphatidylcholine and phosphatidylethanolamine. Oleic and linoleic acid were elevated in the same phospholipids. In contrast, linoleic acid levels in cardiolipin were depressed significantly. These alterations in the fatty acid composition are suggestive of ethanol-induced changes in fatty acid desaturation activities.

Control of state 3 respiration in liver mitochondria from rats subjected to chronic ethanol consumption

Male Sprague-Dawley rats were pair-fed a liquid diet containing 36% of calories as ethanol for at least 31 days. Mitochondria were isolated from the livers and assayed for state 3, state 4 and uncoupled respiration at all three coupling sites. Assay conditions were established that maximized state 3 respiration with each substrate while maintaining a high respiratory control ratio. In mitochondria from ethanol-fed animals, state 3 respiratory rates were decreased at all three coupling sites. The decreased state 3 rate observed at site III was still significantly higher than the state 3 rates observed at site II in mitochondria from either ethanol-fed or control animals. Moreover, the maximal (FCCP-uncoupled) rates with succinate and alpha-ketoglutarate were the same in mitochondria from ethanol-fed and control animals, whereas with glutamate-malate as substrate it was lowered 23% by chronic ethanol consumption. To investigate the role of cytochrome oxidase in modulating the respiratory rate with site I and site II substrates, the effects of cyanide on state 3 and FCCP-uncoupled respiration were determined. When the mitochondria were uncoupled there was no decrease in the rate of succinate oxidation until the rates of ascorbate and succinate oxidation became equivalent. Conversely, parallel inhibition of ascorbate, succinate and glutamate-malate state 3 respiratory rates were observed at all concentrations (1-50 microM) of cyanide utilized. These observations suggest strongly that in coupled mitochondria ethanol-elicited decreases in cytochrome oxidase activity depress the state 3 respiratory rates with site I and II substrates.

Effect of chronic ethanol consumption on energy-linked processes associated with oxidative phosphorylation: Proton translocation and ATP-Pi exchange

Abstract Male rats were administered an ethanol-containing diet for 31 days during which time they demonstrated fatty liver. Mitochondria and submitochondrial particles were prepared from their livers (ethanol mitochondria, ethanol submitochondrial particles) and from their pair-fed partners (control mitochondria, control submitochondrial particles). The H+/coupling site ratio was not significantly different in ethanol and control mitochondria with succinate as electron donor. A 13% decrease in the H+/coupling site ratio was observed in ethanol mitochondria, however, when β-hydroxybutyrate was used as substrate. The rate of ATP-Pi exchange was decreased significantly in both ethanol mitochondria and submitochondrial particles as compared to control preparations. These observations demonstrate ethanol-elicited decreases in energy conservation in the site I region of the electron transport chain and in the activity of the ATP synthetase complex.

The effect of chronic ethanol consumption on the lipids in liver mitochondria.

The ethanol-related alterations in hepatic mitochondrial phospholipids are primarily changes in acyl chain composition. There are no alterations in the unesterified cholesterol content in the mitochondrion, as measured by the cholesterol-phospholipid ratio. Moreover, the distribution of mitochondrial phospholipids are not changed as a result of chronic ethanol consumption. There was a significant ethanol-related decrease (18%) in the phospholipid-protein ratio in mitochondria from rats maintained on a low-fat diet, which was not observed in studies where animals were fed diets containing a higher proportion of lipid. This effect of dietary composition on the phospholipid-protein ratio was also paralleled by the interaction between diet and ethanol in influencing the phospholipid acyl composition. The alterations in acyl chain distribution indicated that ethanol consumption stimulated elongation of palmitic acid, and depressed the delta 5 desaturation step required for the formation of arachidonic acid. Elongation of palmitic acid was stimulated in studies where animals were fed diets with moderate amounts of fat, whereas depressed synthesis of arachidonate occurred more frequently, but not exclusively, in studies where low-fat diets were employed. These results indicate that there is a significant interaction between diet and ethanol in eliciting changes in hepatic mitochondrial phospholipids. The significant decrease in the linoleic acid content of cardiolipin and the more prominent ethanol-associated alterations in mitochondrial phospholipids suggest that ethanol consumption depresses the phospholipid reacylation activities associated with the mitochondrion. The above observations indicate, therefore, that the alterations occurring in mitochondrial phospholipids are influenced by ethanol-related changes in mitochondrial enzymes involved in phospholipid metabolism. In addition, alterations in the availability of fatty acids due to ethanol-related changes in microsomal elongation and desaturation activities also appear to affect the fatty acid composition of phospholipids in mitochondria from ethanol-fed animals.

Identification and quantitation of electron-transport components in human polymorphonuclear neutrophils

Using dithionite difference spectra we have detected cytochrome b in highly purified human neutrophils at a concentration of 0.08 nmol/mg protein. The presence of quinone was identified in lipid extracts at a concentration of approx. 0.06 nmol/mg protein. It was identified as ubiquinone-10 by mass spectrographic analysis. Simultaneous measurements of cytochrome oxidase indicated that these compounds could not be attributed to mitochondrial contamination. These results are compatible with the hypothesis that initiation of the respiratory burst in human neutrophils involves a multicomponent electron-transport system.

The interaction between chronic ethanol consumption and oxygen tension in influencing the energy state of rat liver

Hepatocytes were isolated from chow-fed and liquid-diet control rats, and animals fed ethanol chronically for 31 days. These preparations were analyzed for adenine nucleotide and inorganic phosphate concentrations after being maintained under various conditions of oxygenation and nutrient availability. Hepatocytes from ethanol-fed animals resuspended at high cell density (oxygen tensions near zero) demonstrated a greater depression in cellular energy state as indicated by decreases in phosphorylation potential and energy charge. If, however, these hepatocytes were restored to high oxygen tension their energy state was equivalent to that observed with preparations from liquid-diet control animals. Moreover, their rate of oxygen consumption was equivalent to that of control hepatocytes. Analyses of livers from chow-fed, liquid diet control, and ethanol-fed rats which were freeze-clamped while being perfused by the animals blood revealed that there were no significant differences in the energy states of the hepatic tissue from these three animal groups. These results indicate that (1) the hepatic energy state in rats fed ethanol chronically is maintained under conditions of normal oxygen tension and (2) that hepatic tissue from these animals experiences a much more dramatic depression in energy state than tissue from control rats when subjected to oxygen deprivation.

Characteristics of a succinate-di chlorophenolindophenol reductase reconstituted with bovine heart electron transport components☆

Abstract The reconstituted succinate-dichlorophenolindophenol reductase complex described previously (1) has been further characterized. The complex is very sensitive to 4,4,4 trifluoro-1-(2-thienyl)-1,3 butanedione (TTB), being inhibited 50% by a concentration of 2.5 μM. Combinations of complex III (reduced ubiquinone-cytochrome c reductase) and the DCPIP reductase catalyze a TTB and antimycin A sensitive succinate-cytoohrome c reductase. These observations suggest that the reconstructed succinate-DCPIP reductase is very similar in properties to isolated complex II (succinate-ubiquinone reductase).