Daniel K. Asiedu

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.

LONG-TERM EFFECT OF TETRADECYLTHIOACETIC ACID : A STUDY ON PLASMA LIPID PROFILE AND FATTY ACID COMPOSITION AND OXIDATION IN DIFFERENT RAT ORGANS

Administration of tetradecylthioacetic acid (a 3-thia fatty acid) increases mitochondrial and peroxisomal beta-oxidative capacity and carnitine palmitoyltransferase activity, but reduces free fatty acid and triacylglycerol levels in plasma compared to palmitic acid-treated rats and controls. The decrease in plasma triacylglycerol was accompanied by a reduction (56%) in VLDL-triacylglycerol. Prolonged supplementation of tetradecylthioacetic acid caused a significant increase in lipogenic enzyme activities (ATP-citrate lyase and acetyl-CoA carboxylase) and diacylglycerol acyltansferase, but did not affect phosphatidate phosphohydrolase. Plasma cholesterol, LDL- and HDL-cholesterol levels were reduced. 3-Hydroxy-3-methylglutaryl-coenzyme A reductase activity was, however, stimulated in 3-thia fatty acid-treated rats compared to controls. In addition. the mRNAs of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and LDL-receptor were increased. Tetradecylthioacetic acid administration affected the fatty acid composition in plasma and liver by increasing the amount of monoenes, especially 18:1(n-9), mostly at the expense of omega-3 fatty acids. Compared to liver a large amount of tetradecylthioacetic acid accumulated in the heart, and this accumulation was accompanied by an increase in omega-3 fatty acids, particularly 22:6(n-3) and a decrease in omega-6 fatty acids, mainly 20:4(n-6). The results show that the hypolipidemic effect of tetradecylthioacetic acid is sustained after prolonged administration and may, at least in part, be due to increased fatty acid oxidation and upregulated LDL-receptor gene expression. The increase in lipogenic enzyme activities as well as increased 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity, may be compensatory mechanisms to maintain cellular integrity. Decreased level of 20:4(n-6) combined with increased omega-3/omega-6 ratio in cardiac tissue after tetradecylthioacetic acid treatment may have influence on membrane dynamics and function.

Early effects on mitochondrial and peroxisomal β-oxidation by the hypolipidemic 3-thia fatty acids in rat livers

A single administration of 3-thiadicarboxylic and tetradecylthioacetic acids stimulates both mitochondrial and peroxisomal beta-oxidation and lowers plasma triacylglycerol levels. An increased rate of mitochondrial beta-oxidation and carnitine palmitoyl-transferase activity was established after 3 h and this was accompanied by a lowering of plasma triacylglycerol. Peroxisomal beta-oxidation, however, remained unchanged up to 8 h and was significantly increased after 12 h. These results suggest that after a single administration of 3-thia fatty acids mitochondrial beta-oxidation precedes peroxisomal beta-oxidation. Furthermore, they show that the observed tricylglycerol-lowering effect, which is established early (3-4 h) after the administration of 3-thia fatty acids, is initially due to an increased mitochondrial beta-oxidation.

Fatty acid metabolism in liver of rats treated with hypolipidemic sulphur-substituted fatty acid analogues

The purpose of this study was to investigate early biochemical changes and possible mechanisms via which alkyl(C12)thioacetic acid (CMTTD, blocked for beta-oxidation), alkyl(C12)thiopropionic acid (CETTD, undergo one cycle of beta-oxidation) and a 3-thiadicarboxylic acid (BCMTD, blocked for both omega- (and beta-oxidation) influence the peroxisomal beta-oxidation in liver of rats. Treatment of rats with CMTTD caused a stimulation of the palmitoyl-CoA synthetase activity accompanied with increased concentration of hepatic acid-insoluble CoA. This effect was already established during 12-24 h of feeding. From 2 days of feeding, the cellular level of acid-insoluble CoA began to decrease, whereas free CoASH content increased. Stimulation of [1-14C]palmitoyl-CoA oxidation in the presence of KCN, palmitoyl-CoA-dependent dehydrogenase (termed peroxisomal beta-oxidation) and palmitoyl-CoA hydrolase activities were revealed after 36-48 h of CMTTD-feeding. Administration of BCMTD affected the enzymatic activities and altered the distribution of CoA between acid-insoluble and free forms comparable to what was observed in CMTTD-treated rats. It is evident that treatment of peroxisome proliferators (BCMTD and CMTTD), the level of acyl-CoA esters and the enzyme activity involved in their formation precede the increase in peroxisomal and palmitoyl-CoA hydrolase activities. In CMTTD-fed animals the activity of cyanide-insensitive fatty acid oxidation remained unchanged when the mitochondrial beta-oxidation and carnitine palmitoyltransferase operated at maximum rates. The sequence and redistribution of CoA and enzyme changes were interpreted as support for the hypothesis that substrate supply is an important factor in the regulation of peroxisomal fatty acid metabolism, i.e., the fatty acyl-CoA species appear to be catabolized by peroxisomes at high rates only when uptake into mitochondria is saturated. Administration of CETTD led to an inhibition of mitochondrial fatty acid oxidation accompanied with a rise in the concentration of acyl-CoA esters in the liver. Consequently, fatty liver developed. The peroxisomal beta-oxidation was marginally affected. Whether inhibition of mitochondrial beta-oxidation may be involved in regulation of peroxisomal fatty acid metabolism and in development of fatty liver should be considered.

Modulation of plasma and hepatic oxidative status and changes in plasma lipid profile by n-3 (EPA and DHA), n-6 (corn oil) and a 3-thia fatty acids in rats

This manuscript describes changes in plasma lipid profiles and parameters of oxidative status in the plasma and liver of rats fed 5 different fatty acids: 95% eicosapentaenoic acid, 92% docosahexaenoic acid (DHA), corn oil (n-6), 1-mono-(carboxymethylthio)-tetradecane (CMTTD) and palmitic acid (controls) for 3 months. At the given doses both EPA and the 3-thia fatty acid, CMTTD, caused a significant decrease in plasma triglycerides, phospholipids, free fatty acids and cholesterol. DHA decreased plasma free fatty acids and cholesterol, while corn oil feeding reduced only plasma free fatty acids. Plasma and hepatic vitamin E levels were significantly decreased in EPA, DHA and CMTTD fed rats, but remained unchanged in corn oil fed rats. Plasma glutathione was noted to decrease after EPA and DHA feeding but remained unchanged in other groups. However, hepatic glutathione content was increased in EPA, DHA and CMTTD fed rats, whereas cysteine levels were noted to decrease. As hepatic levels of cysteinylglycine remained unchanged, increased rate of cellular glutathione synthesis rather than its decreased degradation is likely to contribute to the increased hepatic glutathione content in EPA, DHA and CMTTD fed rats. Except for reduction in the levels of plasma lipid peroxidation caused by CMTTD, no significant changes were noted between the different treatment groups. Hepatic lipid peroxidation was elevated only in rats given DHA. Furthermore, our results show that EPA and DHA cause minimal imbalance of the peroxisomal H2O2 metabolising enzymes as compared to CMTTD. In addition, contrary to the potent peroxisome proliferator compound CMTTD which decreased the activities of glutathione transferase and glutathione peroxidase, EPA and DHA increased the activities of these detoxification enzymes.

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.

Gas chromatographic measurement of 3- and 4-thia fatty acids incorporated into various classes of rat liver lipids during feeding experiments.

A practical procedure is described for the quantitative measurement of the amount of acyl units derived from tetradecylthioacetic acid (effecting hypolipemia in rats) and tetradecylthiopropionic acid (effecting hyperlipidemia). The procedure involves three main successive steps: (1) extraction; (2) solid-phase lipid class separation yielding free fatty acids, phospholipids, triacylglycerides, cholesterol esters, and diacylglycerides without crosscontamination; and (3) gas chromatography of hydrolyzed lipids derivatized to picolinyl esters, combined with unambiguous identification by gas chromatography-mass spectrometry. The overall recoveries of heptadecanoyl lipids added as internal standards during extraction were 94-96%, except for cholesteryl heptadecanoate where the recovery was 60% owing to incomplete hydrolysis. Recoveries of thia fatty acids from samples spiked with these compounds were 95%. Flame-ionization response factors were found to be 0.92 and 0.81 for the tetradecylthioacetic acid and tetradecylthiopropionic acid picolinyl esters, respectively, compared to that of heptadecanoic acid. The lower limit of quantitation was 25 pmol as injected. Measurement of the amount of thia fatty acyl units in rat plasma and in liver lipids 4 h after administration of single doses by gastric intubation indicated efficient absorbtion and rapid incorporation into liver lipids, particularly in the phospholipid fraction. Both plasma clearance and channelling into lipids was slower for the 4-thia fatty acid.

Effect of local processing methods (cooking, frying and smoking) on three fish species from Ghana: Part 2—Amino acids and protein quality

Abstract The effect of processing methods (cooking, frying and smoking) on the amino acid composition of Sardinella sp., Dentex sp. and Tilapia sp. was studied. Protein quality, as expressed by AD, TD, NPU and Bal% was also investigated. All the processed fish had good quality protein; as expressed by their high AD, TD and NPU. The good quality of the processed fish was further supported by the amino acid composition. Cooked fish was better absorbed than fried and smoked.

Acute modulation of rat hepatic lipid metabolism by sulphur-substituted fatty acid analogues

A single oral dose of two 3-thia (3-thiadicarboxylic and tetradecylthioacetic acids) and of 4-thia (tetradecylthiopropionic acid) fatty acids were administered to normolipidemic rats and their effects on lipid metabolism over a 24 hr period were studied. All three thia fatty acids could be detected in plasma 2 hr after treatment. Tetradecylthioacetic and tetradecylthiopropionic acids were detected in different hepatic lipid fractions but were incorporated mainly into hepatic phospholipids. Two hours after administration hepatic mitochondrial beta-oxidation and the total liver level of long-chain fatty acyl-CoA increased with a concomitant decrease in saturated fatty acids, total hepatic malonyl-CoA and plasma triacylglycerol levels in the 3-thia fatty acid groups. Tetradecylthiopropionic acid administration caused a decrease in mitochondrial beta-oxidation and an increase in plasma triacylglycerol at 24 hr. The activities of key lipogenic enzymes were unaffected in all treatment groups. Plasma cholesterol level was reduced only at 8 hr in 3-thiadicarboxylic acid treated rats although 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase was suppressed already at 2, 4, 8 and 12 hr. The results show that thia fatty acids are rapidly absorbed and are systemically available after oral administration but the 3-thia fatty acids reached systemic circulation more slowly and less completely than the 4-thia fatty acid. Very low levels of the thia fatty acids are detected in plasma 24 hr after a single administration. They are incorporated into all hepatic lipid classes, especially phospholipids. Rapid incorporation of a non beta-oxidizable thia fatty acid into hepatic lipids may cause a diversion of other fatty acids from glycerolipid biosynthesis to mitochondrial beta-oxidation. Stimulation of mitochondrial beta-oxidation and suppression of HMG-CoA reductase are primary events, occurring within hours, after 3-thia fatty acid administration. The hypotriglyceridemic effect of the 3-thia fatty acids observed at 2-4 hr is independent of the activities of key lipogenic and triacylglycerol synthesising enzymes.