Alexis Garras

Chronic administration of eicosapentaenoic acid and docosahexaenoic acid as ethyl esters reduced plasma cholesterol and changed the fatty acid composition in rat blood and organs

Fish oils rich in n-3 fatty acids have been shown to decrease plasma lipid levels, but the underlying mechanism has not yet been elucidated. This investigation was performed in order to further clarify the effects of purified ethyl esters of eicosapentaenoic acid (EPA-EE) and docosahexaenoic acid (DHA-EE) on lipid metabolism in rats. The animals were fed EPA-EE, DHA-EE, palmitic acid, or corn oil (1 g/kg/d) by orogastric intubation along with a chow background diet for three months. At the end the animals were sacrificed. Plasma and liver lipids were measured, as well as lipid-related enzyme activities and mRNA levels. The fatty acid composition of plasma and different tissues was also determined. This study shows that, compared to the corn oil control, EPA-EE and DHA-EE lowered plasma cholesterol level, whereas only EPA-EE lowered the amount of plasma triacylglycerol. In liver peroxisomes, both EE preparations increased fatty acyl-CoA oxidase FAO activities, and neither altered 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase activities. In liver microsomes, EPA-EE raised HMG-CoA reductase and acyl-CoAicholesterol acyltransferase activities, whereas DHA-EE lowered the former and did not affect the latter. Neither product altered mRNA levels for HMG-CoA reductase, low density lipoprotein-receptor, or low density lipoprotein-receptor related protein. EPA-EE lowered plasma triacylglycerol, reflecting lowered very low density lipoprotein secretion, thus the cholesterol lowering effect in EPA-EE-treated rats may be secondary to the hypotriacylglycerolemic effect. An inhibition of HMG-CoA reductase activity in DHA-EE treated rats may contribute to the hypocholesterolemic effect. The present study reports that 20∶5n-3, and not 22∶6n-3, is the fatty acid primarily responsible for the triacylglycerol lowering effect of fish oil. Finally, 20∶5n-3 was not converted to 22∶6n-3, whereas retroconversion of 22∶6n-3 to 20∶5n-3 was observed.

Rapid method for the separation and detection of tissue short-chain coenzyme A esters by reversed-phase high-performance liquid chromatography

A simple and rapid method for the separation and identification of tissue levels of short chain coenzyme A (CoA) esters by a reversed-phase high-performance liquid chromatography with ultraviolet-visible adsorbance detection is described. Samples of liver, heart and kidney tissues were homogenised in 5% sulfosalicylic acid containing 50 microM of dithioerythritol in 1:9 w/v proportion. Following centrifugation, 20 microliters of the supernatant were directly injected onto a 3-micron ODS C18 column (100 x 4.6 mm I.D.). The separation of acetyl-CoA, malonyl-CoA, methylmalonyl-CoA, succinyl-CoA, propionyl-CoA and free CoASH was achieved in less than 20 min using gradient elution with sodium phosphate, sodium acetate and methanol at a constant flow-rate of 1.5 ml/min. The lowest detection limit was 3 pmol.

Mitochondrial 3-hydroxy-3-methylglutaryl coenzyme A synthase and carnitine palmitoyltransferase II as potential control sites for ketogenesis during mitochondrion and peroxisome proliferation

3-Thia fatty acids are potent hypolipidemic fatty acid derivatives and mitochondrion and peroxisome proliferators. Administration of 3-thia fatty acids to rats was followed by significantly increased levels of plasma ketone bodies, whereas the levels of plasma non-esterified fatty acids decreased. The hepatic mRNA levels of fatty acid binding protein and formation of acid-soluble products, using both palmitoyl-CoA and palmitoyl-L-carnitine as substrates, were increased. Hepatic mitochondrial carnitine palmitoyltransferase (CPT) -II and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase activities, immunodetectable proteins, and mRNA levels increased in parallel. In contrast, the mitochondrial CPT-I mRNA levels were unchanged and CPT-I enzyme activity was slightly reduced in the liver. The CoA ester of the monocarboxylic 3-thia fatty acid, tetradecylthioacetic acid, which accumulates in the liver after administration, inhibited the CPT-I activity in vitro, but not that of CPT-II. Acetoacetyl-CoA thiolase and HMG-CoA lyase activities involved in ketogenesis were increased, whereas the citrate synthase activity was decreased. The present data suggest that 3-thia fatty acids increase both the transport of fatty acids into the mitochondria and the capacity of the beta-oxidation process. Under these conditions, the regulation of ketogenesis may be shifted to step(s) beyond CPT-I. This opens the possibility that mitochondrial HMG-CoA synthase and CPT-II retain some control of ketone body formation.

Subcellular localisation and induction of NADH-sensitive acetyl-CoA hydrolase and propionyl-CoA hydrolase activities in rat liver under lipogenic conditions after treatment with sulfur-substituted fatty acids

The effects of sulfur-substituted fatty acid analogues on the subcellular distribution and activities of acetyl-CoA and propionyl-CoA hydrolases in rats fed a high carbohydrate diet were studied. Among subcellular fractions of liver homogenates from rats fed a high carbohydrate diet (20%), the acetyl-CoA and propionyl-CoA hydrolase activities are found in the mitochondrial, peroxisome-enriched and cytosolic fractions. We have shown that the subcellular distribution of acetyl-CoA hydrolase appears to be different from the distribution propionyl-CoA hydrolase activity. Thus, the highest specific activity of acetyl-CoA hydrolase was found in the mitochondrial fraction, whereas the highest specific activity of propionyl-CoA hydrolase was found in the peroxisome-enriched fraction. Rats treated with sulfur-substituted fatty acids, i.e., 3-thiadicarboxylic acid (400 mg/day per kg body weight), showed a significant increase in acetyl-CoA hydrolase activity where the peroxisomal and cytosolic hydrolases were increased 3.9- and 2.7-fold, respectively, compared to palmitic acid treated rats. Similar results were obtained with tetradecylthioacetic acid treated rats. Propionyl-CoA hydrolase activities, in rats treated with these two peroxisome proliferating fatty acid analogues showed increased activity mainly in the mitochondrial and the cytosolic subcellular fractions. Acetyl-CoA hydrolase activity was sensitive to NADH, whereas no stimulation of the propionyl-CoA hydrolase activity was observed in the presence of NADH. The hepatic amounts of acetyl-CoA, propionyl-CoA, and free CoASH were elevated after sulfur-substituted fatty acid treatment. Sulfur-substituted fatty acids also elevated the specific acetyl-CoA hydrolase activity in the mitochondrial fraction and the propionyl-CoA hydrolase activity in the light-mitochondrial fraction. These results, therefore, suggest that acetyl-CoA hydrolase and propionyl-CoA hydrolase are two distinct proteins and that these two enzymes have a multiorganelle localisation.

A nonradioactive assay fo N5-methyltetrahydrofolate-homocysteine methyltransferase (methionine synthase) based on o-phthaldialdehyde derivatization of methionine and fluorescence detection

Abstract The enzyme N 5 -methyltetrahydrofolate-homocysteine methyltransferase (methionine synthase, EC 2.1.1.13) catalyzes the conversion of homocysteine to methionine in the presence of a reducing system. N 5 -Methyltetrahydrofolate serves as a methyl donor in this reaction. An assay for the enzyme is described, which is based on methionine quantitation by o -phthaldialdehyde (OPA) derivatization and reversed-phase liquid chromatography. The enzymatic reaction is linear for at least 120 min under reducing conditions (125 m m 2-mercaptoethanol) and running the assay below an oil layer. This reducing system does not interfere with formation of the methionine-OPA adduct, which is separated from interfering compounds and an internal standard (norvaline) by a mobile phase adjusted to pH 5.0. The inclusion of internal standard increases the precision of the assay and corrects for the variable fluorescence yield due to occasional inaccurate pH adjustment before the derivatization step. Norvaline was suitable for this purpose because it elutes close to methionine and is not a natural amino acid present in biological extracts. This nonradioactive assay for methionine synthase was evaluated by comparison with a conventional method based on isolation of radioactive methionine by anion-exchange chromatography and by determination of enzyme activity in extract from cultured cells and liver.

Mitochondrial 3-hydroxy-3-methylglutaryl CoA synthase and carnitine palmitoyltransferase II are potential control sites of hepatic ketogenesis under conditions of peroxisome proliferation

3-Thia fatty acids are potent hypolipidemic fatty acid derivatives and mitochondrion and peroxisome proliferators. They increase the transport of fatty acids into the mitochondria and increase the capacity of the B-oxidation process. Administration of 3-thia fatty acids to rats was followed by a significantly increased production of acid-soluble products and ketone bodies. The levels of nonesterified fatty acids in plasma were decreased, whereas the hepatic mRNA levels of fatty acid-binding protein were increased. Increased mitochondrial carnitine palmitoyltransferase (CPT)-II and 3-hydroxy-3methylglutaryl (HMG)-CoA synthase activities, immunodetectable proteins, and mRNA levels were also observed in the liver. In contrast, the mitochondrial CPT-I mRNA levels were unchanged, and CPT-I enzyme activity was slightly reduced. The immunodetectable CPT-I protein, however, was increased. An increase in immunoreactive protein, which is not essential for catalytic function or sensitivity to malonyl-CoA, is possible due to formation of a 3-thia fatty acid CoA-ester which inhibits the CPT-I activity. Acetoacetyl-CoA thiolase and HMG-CoA lyase activities involved in ketogenesis were increased. The citrate synthase activity was decreased. We conclude that under conditions of peroxisome proliferation by 3-thia fatty acids, HMG-CoA synthase and CPT-II may be regulatory sites of ketone bodies formation.