Metabolism of n−3 polyunsaturated fatty acids by the isolated perfused rat liver

The hepatic metabolism of oleic acid and n−3 fatty acids (eicosapentaenoic acid, EPA and docosahexaenoic acid, DHA), and secretion of very low density lipoprotein (VLDL) were studied in isolated perfused rat livers from normal chow fed male rats. The basal perfusion medium contained 30% bovine erythrocytes, 6% bovine serum albumin (BSA), and 100 mg/dL glucose, in Krebs-Henseleit bicarbonate buffer (pH 7.4) which was recycled through the liver for 2 hr. Individual fatty acids (EPA, DHA or oleic acid), as complexes with 6% BSA, or albumin alone, were infused at a rate of 70 μmol/hr. When any of these fatty acids was infused at this rate, the ambient concentration in the medium was maintained at 0.3–0.4 μmol/mL, indicative of similar hepatic rates of uptake for each fatty acid (i.e., approximately 6 μmol/g liver/hr). When fatty acid was not infused, the ambient free fatty acid level was 0.16 μmol/mL. The concentrations of infused free fatty acids increased appropriately in the perfusion medium; however, with infusion of EPA, DHA, or oleate, the concentrations of perfusate palmitate and linoleate were the same as when fatty acid was not infused. Additionally, the perfusate concentration of oleate in the free fatty acid fraction was not affected by infusion of EPA and DHA. These data indicate a constant outflow of endogenous fatty acid unaffected by the presence of the exogenously supplied fatty acid. The net secretion rate of VLDL lipids and protein was stimulated by infusion of oleate, whereas when EPA was infused, secretion rates were lower and similar [except for VLDL cholesterol (C), which was greater] to those occuring when fatty acid was not provided. DHA stimulated the secretion of VLDL triacylglycerol (TG), phospholipid (PL) and C to a similar rate, as did oleate, but secretion of VLDL cholesteryl ester (CE) and protein was lower and similar to that with EPA. VLDL and hepatic TG and CE were enriched with the infused fatty acids, compared to experiments without fatty acids, as determined by gas chromatography. Enrichment of PL, however, was significant only in liver upon infusion of EPA. The formation of¹⁴CO₂ and perchloric acid soluble products from [1-¹⁴C]EPA, considered separately, did not differ statistically from that obtained with [1-¹⁴C]oleate, although the mean values were higher with [1-¹⁴C]EPA. However, the sum of oxidation products derived from EPA was significantly greater than that from oleate. Incorporation of [1-¹⁴C]EPA into TG and CE, but not into PL, was lower as compared to that from [1-¹⁴C]oleate. These lower rates of incorporation of [1-¹⁴C]EPA into VLDL lipids therefore paralleled the mass fatty acid enrichment-patterns. It may be concluded that EPA is used to a similar extent as oleate for synthesis of PL, but is a poorer substrate for synthesis of TG. The reduced output of newly synthesized (radioactive) PL reflected the lower hepatic output of VLDL. Since hepatic uptake of EPA, DHA or oleate was identical, utilization of EPA for TG synthesis was less than that of oleate or DHA. Further-more, utilization of endogenous fatty acids for TG synthesis and secretion of the VLDL was reduced in the presence of EPA. The decreased TG synthesis resulted in reduced formation of VLDL for transport of TG from the liver. These effects taken together with an apparently increased oxidation of EPA provide substantial evidence for a decrease in formation of VLDL and transport of TG, PL, C and CE into the circulation in response to EPA. DHA, however, appears to be an adequate substrate for TG synthesis and stimulates VLDL secretion. The reduced transport of CE may reflect lower selectivity of DHA by acyl-CoA; cholesterol acyltransferase for CE formation.     

Eicosapentaenoic acid,but not docosahexaenoic acid,increases mitochondrial fatty acid oxidation and upregulates 2,4-dienoyl-CoA reductase gene expression in rats

The aim of the present study was to investigate whether eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) was responsible for the triglyceride-lowering effect of fish oil. In rats fed a single dose of EPA as ethyl ester (EPA-EE), the plasma concentration of triglycerides was decreased at 8 h after acute administration. This was accompanied by an increased hepatic fatty acid oxidation and mitochondrial 2,4-dienoyl-CoA reductase activity. The steady-state level of 2,4-dienoyl-CoA reductase mRNA increased in parallel with the enzyme activity. An increased hepatic long-chain acyl-CoA content, but a reduced amount of hepatic malonyl-CoA, was obtained at 8 h after acute EPA-EE treatment. On EPA-EE supplementation, both EPA (20:5n-3) and docosapentaenoic acid (DPA, 22:5n-3) increased in the liver, whereas the hepatic DHA (22:6n-3) concentration was unchanged. On DHA-EE supplementation retroconversion to EPA occurred. No statistically significant differences were found, however, for mitochondrial enzyme activities, malonyl-CoA, long-chain acyl-CoA, plasma lipid levels, and the amount of cellular fatty acids between DHA-EE treated rats and their controls at any time point studied. In cultured rat hepatocytes, the oxidation of [1-¹⁴C]palmitic acid was reduced by DHA, whereas it was stimulated by EPA. In thein vivo studies, the activities of phosphatidate phosphohydrolase and acetyl-CoA carboxylase were unaffected after acute EPA-EE and DHA-EE administration, but the fatty acyl-CoA oxidase, the rate-limiting enzyme in peroxisomal fatty acid oxidation, was increased after feeding these n-3 fatty acids. The hypocholesterolemic properties of EPA-EE may be due to decreased 3-hydroxy-3-methylglutaryl-CoA reductase activity. Furthermore, replacement of the ordinary fatty acids, i.e., the monoenes (16:1n-7, 18:1n-7, and 18:1n-9) with EPA and some conversion to DPA concomitant with increased fatty acid oxidation is probably the mechanism leading to changed fatty acid composition. In contrast, DHA does not stimulate fatty acid oxidation and, consequently, no such displacement mechanism operates. In conclusion, we have obtained evidence that EPA, and not DHA, is the fatty acid primarily responsible for the triglyceride-lowering effect of fish oil in rats.     

Effect of dietary fish oil on the rate of very low density lipoprotein triacylglycerol formation and on the metabolism of chylomicrons

The mechanism by which ω3 fatty acids lower plasma triacylglycerol levels was investigated. Rats were fed fish oil, olive oil (10% fat by weight) or a nonpurified diet 4% fat by weight) for 15 days. Lipoprotein lipase was inhibited by intra-arterial administration of Triton WR 1339 to estimate hepatic triacylglycerol output. Rats fed the olive oil diet showed a higher rate of triacylglycerol formation than rats fed the ω3 fatty acid diet or the low-fat diet. All three groups showed identical rates of removal from plasma of intraarterially administered artificial chylomicrons that had simultaneously been labeled with cholesteryl [1-¹⁴C]oleate and [9,10(n)-³H]triolein. Liver radioactivity and total fat content were lowest in rats fed the fish oil diet, indicating that ω3 fatty acids were preferentially metabolized in liver. Chylomicrons obtained from donor rats fed either fish oil containg [¹⁴C]cholesterol or olive oil containing [³H]cholesterol were removed at similar rates when infused together intraarterially into recipient animals. A slower formation of plasma very low density lipoprotein triacylglycerols in rats fed fish oil is probably due to a faster rate of oxidation of the fatty acid chains in the liver resulting in decreased plasma triacylglycerol concentrations.     

The hypotriglyceridemic effect of eicosapentaenoic acid in rats is reflected in increased mitochondrial fatty acid oxidation followed by diminished lipogenesis

The effect of eicosapentaenoic acid (EPA) on fatty acid oxidation and on key enzymes of triglyceride metabolism and lipogenesis was investigated in the liver of rats. Repeated administration of EPA to normolipidemic rats resulted in a time-dependent decrease in plasma triglycerides, phospholipids and cholesterol. The triglyceride-lowering effect was observed after one day of feeding whereas lowering of plasma cholesterol and phospholipids was observed after five days of treatment. The triglyceride content of liver was reduced after two-day treatment. At that time, increased mitochondrial fatty acid oxidation occurred whereas mitochondrial and microsomal glycerophosphate acyltransferase was inhibited. The phosphatidate phosphohydrolase activity was unchanged. Adenosine triphosphate:citrate lyase, acetyl-CoA carboxylase, fatty acid synthetase and glucose-6-phosphate dehydrogenase were inhibited during the 15 d of EPA treatment whereas peroxisomal β-oxidation was increased. At one day of feeding, however, when the hypotriglyceridemic effect was established, the lipogenic enzyme activities were reduced to the same extent in palmitic acid-treated animals as in EPA-treated rats. In cultured rat hepatocytes, the oxidation of [¹⁴C]palmitic acid to carbon dioxide and acid-soluble products was stimulated in the presence of EPA. These results suggest that the instant hypolipidemia in rats given EPA could be explained at least in part by a sudden increase in mitochondrial fatty acid oxidation, thereby reducing the availability of fatty acids for lipid synthesis in the liver for export,e.g., in the form of very low density lipoproteins, even before EPA induced peroxisomal fatty acid oxidation, reduced triglyceride biosynthesis and diminished lipogenesis.     

Metabolism of trans fatty acids by hepatocytes

The present work was undertaken to study the metabolism of fatty acids with trans double bonds by rat hepatocytes. In liver mitochondria, elaidoyl-CoA was a poorer substrate for carnitine palmitoyltransferase I (CPT-I) than oleoyl-CoA. Likewise, incubation, of hepatocytes with oleic acid produced a more pronounced stimulation of CPT-I than incubation with trans fatty acids. This was not due to a differential effect of cis and trans fatty acids on acetyl-CoA carboxylase (ACC) activity and malonyl-CoA levels. Elaidic acid was metabolized by hepatocytes at a higher rate than oleic acid. Surprisingly, compared to oleic acid, elaidic acid was a better substrate for mitochondrial and, especially, peroxisomal oxidation, but a poorer substrate for cellular and very low density lipoprotein triacylglycerol synthesis. Results thus show that trans fatty acids are preferentially oxidized by hepatic peroxisomes, and that the ACC/malonyl-CoA/CPT-I system for coordinate control of fatty acid metabolism is not responsible for the distinct hepatic utilization of cis and trans fatty acids.     

Influence of dietary fish oil on the relative synthesis of triacylglycerol and phospholipids in rat liverin vivo

The influence of dietary fish oil containing n−3 polyunsaturated fatty acids on the biosynthesis of triacylglycerol relative to total individual phospholipids was studied in rat liverin vivo. The dietary lipid (10% by weight of diet) was either sunflower oil enriched in linoleic acid (SO group) or MaxEPA fish oil/sunflower oil, 9∶1 by weight (FO group) enriched in eicosapentaenoic acid (EPA, 20∶5n−3) plus docosahexaenoic acid (DHA, 22∶6n−3). After a 3-week feeding period, the triacylglycerol content (in μmmol/g liver) was 44% lower in the FO group relative to the SO animals. Thein vivo incorporation of [³H]glycerol into individual hepatic lipids resulted in triacyl-glycerol/total phospholipid radioactivity ratios of 2.1 and 0.9 for the SO and FO groups, respectively. These results indicate an inhibitory effect of dietary EPA/DHA on triacylglycerol relative to phospholipid synthesis from intermediary 1,2-diacylglycerol in rat liverin vivo. This metabolic alteration was accompanied by a substantially lower amount (in μmol/g liver) of arachidonic acid and higher levels of EPA plus DHA in the triacylglycerol, choline glycerophospholipid (CGP), and ethanolamine glycerophospholipid (EGP) of the FO group. A moderately higher labelling of the EGP from [³H]glycerol was observed in the FO as compared to the SO group (as evidenced by CGP/EGP radioactivity ratios of 1.3∶1 and 1.8∶1, respectively). The present study providesin vivo evidence for a dampening effect of dietary fish oil on the synthesis of liver triacylglycerol relative to phospholipid and a moderate alteration ofde novo synthesis of individual phospholipids. Presented in part at the 80th Annual Meeting of the AOCS in Cincinnati, Ohio (May, 1989).     

3-thia fatty acid treatment, in contrast to eicosapentaenoic acid and starvation, induces gene expression of carnitine palmitoyltransferase-II in rat liver

The aim of the present study was to investigate the hepatic regulation and β-oxidation of long-chain fatty acids in peroxisomes and mitochondria, after 3-thia- tetradecylthioacetic acid (C₁₄-S-acetic acid) treatment. When palmitoyl-CoA and palmitoyl-l-carnitine were used as substrates, hepatic formation of acid-soluble products was significantly increased in C₁₄-S-acetic acid treated rats. Administration of C₁₄-S-acetic acid resulted in increased enzyme activity and mRNA levels of hepatic mitochondrial carnitine palmitoyltransferase (CPT)-II. CPT-II activity correlated with both palmitoyl-CoA and palmitoyl-l-carnitine oxidation in rats treated with different chain-length 3-thia fatty acids. CPT-I activity and mRNA levels were, however, marginally affected. The hepatic CPT-II activity was mainly localized in the mitochondrial fraction, whereas the CPT-I activity was enriched in the mitochondrial, peroxisomal, and microsomal fractions. In C₁₄-S-acetic acid-treated rats, the specific activity of peroxisomal and microsomal CPT-I increased, whereas the mitochondrial activity tended to decrease. C₁₄-S-Acetyl-CoA inhibited CPT-I activity in vitro. The sensitivity of CPT-I to malonyl-CoA was unchanged, and the hepatic malonyl-CoA concentration increased after C₁₄-S-acetic acid treatment. The mRNA levels of acetyl-CoA carboxylase increased. In hepatocytes cultured from palmitic acid- and C₁₄-S-acetic acid-treated rats, the CPT-I inhibitor etomoxir inhibited the formation of acid-soluble products 91 and 21%, respectively. In contrast to 3-thia fatty acid treatment, eicosapentaenoic acid treatment and starvation increased the mitochondrial CPT-I activity and reduced its malonyl-CoA sensitivity. Palmitoyl-l-carnitine oxidation and CPT-II activity were, however, unchanged after either EPA treatment or starvation. The results from this study open the possibility that the rate control of mitochondrial β-oxidation under mitochondrion and peroxisome proliferation is distributed between an enzyme or enzymes of the pathway beyond the CPT-I site after 3-thia fatty acid treatment. It is suggested that fatty acids are partly oxidized in the peroxisomes before entering the mitochondria as acylcarnitines for further oxidation.     

Metabolism of erucic acid in adipocytes isolated from rat epididymal fat

The metabolism of [14-¹⁴C]erucic acid and [U-¹⁴C]palmitic acid has been investigated in adipocytes isolated from rat epididymal fat. The rate of acylation of [¹⁴C]erucic acid in cellular lipids and oxidation to CO₂ and acid-soluble activity was ca. 1/3 of the rate with [¹⁴C]palmitic acid as substrate. A maximal incorporation of fatty acids in triacylglycerol was found at a fatty acid concentration of 0.8 mM in the medium, both with [¹⁴C]erucic acid and [¹⁴C]palmitic acid as substrate. Glucose added to the medium increased the esterification and decreased the oxidation of both fatty acids. No significant chain-shortening of [¹⁴C]erucic acid to shorter monoenes was identified in the fat cells. Increasing concentrations of unlabeled palmitic acid in the incubation medium markedly inhibited the esterification of [¹⁴C]erucic acid, whereas unlabeled erucic acid had little effect on the rate of esterification of [¹⁴C]palmitic acid.     

Hepatic microsomal and peroxisomal docosahexaenoate biosynthesis during piglet development

The roles of peroxisomes and microsomes on the biosynthetic pathway for docosahexaenoic acid (DHA) from α-linolenic acid (ALA) were investigated. Microsomes and peroxisomes were prepared from livers of fetal and neonatal piglets by a combination of differential and gradient layer centrifugation. Microsomes, peroxisomes, and combined cell fractions were incubated with [¹³C-U]18∶3n−3. The [M] and [M+18] isotopomers of the fatty acids in the long-chain polyunsaturated fatty acid (LCPUFA) n−3 pathway were detected by gas chromatography-mass spectrometry. The quantity of each fatty acid was determined by gas chromatography, and synthesis of each fatty acid was calculated for a 30-min period. Synthesis of DHA was not detected in combined fetal liver fractions. The data suggest that DHA in the fetus is probably supplied from maternal sources through the placenta. In either singly incubated microsomal or peroxisomal preparations from neonatal livers, no DHA synthesis was detected. After combination of the microsomal and peroxisomal fractions, DHA synthesis was evident and increased rapidly between birth and 2 wk of age. This is the first demonstration of the entire biosynthetic LCPUFA n−3 pathway in subcellular organelles starting from isotopically labeled ALA to the final product, DHA, with all the intermediates present and isotopically labeled. The primary importance of the data is that it unequivocally demonstrates that peroxisomes are required for biosynthesis of DHA from ALA.     

Effect of degree of unsaturation in dietary fatty acids on arachidonic acid mobilization by peritoneal macrophages

Cells from rats fed with a tripalmitin diet showed a depletion of phospholipid arachidonate and n-3 fatty acids such as eicosapentaenoic and docosahexaenoic acids (EPA and DHA). In rats fed fish oil diet, a significant reduction in archidonic acid (AA) content was observed whereas EPA and DHA were incorporated into membranes lipids. These changes in lipid composition of membranes did not affect cellular adherence, phagocytic capability, or [³H]AA incorporation. However, both tripalmitin and fish oil diets induced a decrease in [³H]AA mobilization stimulated by 4β-phorbol-12-myristate 13-acetate, A23187, or opsonized-zymosan in rat peritoneal macrophages. These results demonstrate that the antiinflammatory effects of essential fatty acids deficiency or n-3 enrichment diets may be associated with a decreased AA mobilization in resident rat peritoneal macrophages treated with proinflammatory agents.     

Metabolism of [1-14C]docosahexaenoate (22∶6n−3), [1-14C]eicosapentaenoate (20∶5n−3) and [1-14C]linolenate (18∶3n−3) in brain cells from juvenile turbotScophthalmus maximus

The incorporation of [1-¹⁴C]18∶3n−3, (LNA) and [1-¹⁴C]-22∶6n−3 (DHA), and the metabolismvia the desaturase/elongase pathways of [1-¹⁴C]LNA, and [1-¹⁴C]20∶5n−3 (EPA) were studied in brain cells from newly-weaned (1-month-old) and 4-month-old turbot. The rank order of the extent of net incorporation of both LNA and DHA into glycerophospholipids was total diradyl glycerophosphocholines (CPL)> total diradyl glycerophosphoethanolamines (EPL)> phosphatidylserine (PS) and phosphatidylinositol (PI) and was independent of the polyunsaturated fatty acid added, the age of the fish and the time of incubation. However, the rate of incorporation of LNA into total lipid, CPL, EPL and PS was significantly greater than the rate of incorporation of DHA, and there was a significantly greater amount of DHA incorporated into EPL than LNA. There was no significant difference between the amounts of LNA and DHA incorporated into total lipid, CPL, PS and PI. Therefore, little preferential uptake and incorporation of DHA into brain cells was apparent. In 24-h incubations, on average 1.1% and 8.5% of radioactivity from [1-¹⁴C]LNA and [1-¹⁴C]EPA, respectively, were recovered in the DHA fraction. Therefore, LNA cannot contribute significantly to brain DHA levels in the turbot but EPA can. There were no significant differences between the amounts of radioactivity from either [1-¹⁴C]LNA or [1-¹⁴C]EPA recovered in the individual products/intermediates of the desaturase pathways in brain cells from 30-day-old and 120-day-old turbot.     

Effects of non-β-oxidizable sulfur-substituted fatty acid analogues on synthesis and secretion of triacylglycerol and cholesterol in cultured rat hapatocytes

The mechanisms behind the hypolipidemic effect of two sulfur-substituted fatty acid analogues, 3-thiadicarboxylic acid and tetradecylthioacetic acid, have been investigated in cultured hepatocytes. There was a dose-dependent reduction in incorporation of [³H]water into triacylglycerol and diacylglycerol when tetradecylthioacetic acid was added to rat hepatocytes cultured in the presence of 200 μM oleic acid. Tetradecylthioacetic acid also increased the oxidation of [¹⁴C]palmitic acid compared to oleic acid, inhibited the incorporation of radiolabeled precursors into diacylglycerol to a greater extent than into triacylglycerol, and reduced the secretion of triacylglycerol more than its synthesis. A stimulation, rather than a reduction, in glycerolipid synthesis and secretion by tetradecylthioacetic acid was observed when oleic acid was omitted from the culture medium. When 3-thiadicarboxylic acid was added to cultured hepatocytes, the effects on glycerolipid synthesis were generally similar to those observed with tetradecylthioacetic acid, but 3-thiadicarboxylic acid did not increase the oxidation of [¹⁴C]palmitic acid. The two fatty acid analogues also had different effects on the synthesis and secretion of cholesterol and cholesteryl esters—3-thiadicarboxylic acid reduced the incorporation of [³H]water into synthesized and secreted cholesterol and cholesteryl esters, whereas tetradecylthioacetic acid only reduced the secretion of cholesteryl esters without affecting its synthesis. It is concluded that tetradecylthioacetic acid increases the oxidation of fatty acids and reduces the synthesis and secretion of glycerolipids. 3-Thiadicarboxylic acid reduces the synthesis and secretion of both glycerolipids and cholesterol to approximately the same extent without a concomitant increase in the oxidation of fatty acids.     

The differential effect of eicosapentaenoic acid and oleic acid on lipid synthesis and VLDL secretion in rabbit hepatocytes

The suppression of plasma very low density lipoprotein (VLDL) triglyceride levels by dietary fish oils rich in polyunsaturated n−3 fatty acids has been attributed to decreased hepatic VLDL secretion. To investigate the effect of n−3 fatty acids on lipid metabolism and VLDL secretion in a tissue culture system, we incubated rabbit hepatocytes with oleic acid and eicosapentaenoic acid (EPA) and examined [³H]glycerol and [¹⁴C]fatty acid incorporation into hepatocyte triglyceride and phospholipid and into media VLDL. Glycerol incorporation studies showed that EPA failed to stimulate VLDL triglyceride secretion from hepatocytes as occurred with oleic acid (P<0.05). Oleic acid preferentially enhanced hepatocyte triglyceride synthesis while EPA stimulated significantly phospholipid synthesis (P<0.01). Varying the relative concentrations of oleic acid and EPA at a constant total fatty acid concentration corroborated preferential triglyceride synthesis from oleic acid. Synthesis shifted predominantly to phospholipids with increasing concentrations of EPA and lower levels of oleic acid. Incorporation of the [¹⁴C]fatty acids (800 μM) followed similar patterns: 87% of [¹⁴C]oleic acid was incorporated into hepatocyte triglyceride and 44% of [¹⁴C]EPA was assimilated in hepatocyte phospholipid (p<0.001). Fatty acids at trace concentrations (53 nM) showed a more divergent pattern of lipid incorporation: 60% of [¹⁴C]oleic acid was incorporated into triglyceride while 91% of [¹⁴CEPA was incorporated into phospholipid (p<0.001). We conclude that in primary rabbit hepatocyte culture, which appears to be a useful model to study lipid metabolism and VLDL secretion, EPA is avidly incorporated into phospholipid while oleic acid predominantly becomes esterified in triglyceride. In addition, EPA, unlike oleic acid, fails to stimulate hepatocyte VLDL secretion. These divergent effects on hepatocyte lipid metabolism are, at least in part, likely to be responsible for fish oil induced suppression of plasma triglycerides.     

Total and peroxisomal oxidation of various saturated and unsaturated fatty acids in rat liver,heart and m. quadriceps

Rates of total and peroxisomal fatty acid oxidation were estimated from the production of¹⁴C-labeled CO₂ and acid-soluble products from differently labeled [¹⁴C]fatty acids, in the absence and presence of antimycinrotenone, in homogenates of liver, heart and m. quadriceps. Total and peroxisomal oxidation rates of palmitic, oleic and linoleic acid were 3–4 times higher than those of arachidonic and adrenic acid which had higher oxidation rates than those of lignoceric and erucic acid. The peroxisomal contribution to the oxidation of the last fatty acids was similar to or higher than that of palmitic acid. For all fatty acids tested in these tissues, the mitochondrial contribution to β-oxidation was higher than the peroxisomal contribution. Production of¹⁴CO₂ and¹⁴C-labeled, acid-soluble metabolites from [13-¹⁴]arachidonic acid indicated that polyunsaturated fatty acids can be chain-shortened beyond their double bonds in m. quadriceps and heart as well as in liver. Although 2,4-dienoyl-CoA reductase requires NADPH, addition of this coenzyme did not influence arachidonic acid oxidation. Arachidonic acid oxidation was inhibited by palmitic acid in mitochondria and peroxisomes, but arachidonic acid had only a slight effect on palmitic acid oxidation.     

Differential utilization of eicosapentaenoic acid and docosahexaenoic acid in human plasma

It has recently been shown that the ω3 fatty acid status in humans can be predicted by the concentration of eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids in plasma phospholipids [Bjerve, K.S., Brubakk, A.M., Fougner, K.J., Johnsen, H., Midjthell, K., and Vik, T. (1993)Am. J. Clin. Nutr., in press]. In countries with low intake of ω3 fatty acids, the level of EPA in plasma phospholipids is often only about one-fifth the concentration of DHA. The purpose of this study was to investigate whether this difference in the concentration of these two fatty acids was due to a selective loss of EPA relative to DHA or to a lower dietary intake of EPA. Seven female volunteers ingested four grams of MaxEPA daily for 2 wk and in the following 4 wk they ate a diet almost completely devoid of the long-chain ω3 fatty acids. The concentrations of the ω3 fatty acids in the plasma cholesteryl esters, triglycerides and phospholipids and the high density lipoprotein phospholipids were examined at weekly intervals throughout the study. There was a more rapid rise in the concentration of EPA than in DHA levels in the supplementation period in all lipid fractions, but there was a disproportionate rise in DHA relative to EPA in the plasma lipids compared with the ratio in the supplement. In the depletion phase there was a rapid disappearance of EPA from all fractions, such that pre-trial levels were reached by one week post-supplementation. The disappearance of DHA was slower, particularly for the plasma phospholipids: at 4 wk post-supplementation, the DHA concentration in this fraction was still 40% above the pre-trial value. It is suggested that the low plasma EPA values relative to DHA are the result of increased β-oxidation of EPA and/or low dietary intake, rather than a rapid conversion of EPA to DHA. One practical result of this experiment is that, compared with DHA, the maintenance of increased EPA levels in plasma (and therefore tissues) would require constant inputs of EPA due to its more rapid loss from the plasma.     

Preferential oxidation of linolenic acid compared to linoleic acid in the liver of catfish (Heteropneustes fossilis andClarias batrachus)

The fate of [1-¹⁴C] linoleic acid and [1-¹⁴C] linolenic acid in the liver slices and also in the liver tissues of live carnivorous catfish,Heteropneustes fossilis andClarias batrachus, was studied. Incorporation of the fatty acids into different lipid classes in the live fish differed greatly from the tissue slices, indicating certain physiological control operative in vivo. The extent of desaturation and chain elongation of linoleic and linolenic acids into long-chain polyunsaturated fatty acids was low. Linolenic acid was oxidized (thus labeling the saturated fatty acid with liberated¹⁴C-acetyl-CoA) in preference to linoleic acid, and this oxidation also seemed to be under physiological control since both of the fatty acids were poorly oxidized in the tissue slices and in the killed fish. These fish can therefore recognize the difference in the acyl chain structures of linoleate and linolenate. The higher oxidation of liolenic acid and poor capacity for its conversion to longer chain, highly unsaturated derivatives indicates a higher demand for the dietary supply of these essential fatty acids in these two species.     

The effects of clofibrate feeding on the metabolism of palmitate and erucate in isolated hepatocytes

The metabolism of palmitate and erucate has been investigated in hepatocytes isolated from control rats and from rats fed 0.3% clofibrate. Clofibrate increased the oxidation of [1-¹⁴C]palmitate 1.5 to 2-fold while the esterification was decreased. At a high concentration of palmitate (1.5 mM), the total rate of fatty acid metabolism was stimulated. Clofibrate stimulated both the oxidation (3.5 to 5-fold) and the esterification (1.7-fold) of [14-¹⁴C]erucate. Erucate undergoes chain-shortening in isolated liver cells. This chain-shortening was stimulated at least 2-fold by clofibrate feedings. The isolated mitochondrial fraction from clofibrate-fed rats showed an increased capacity for oxidation of short-chain acylcarnitines (including acetylcarnitine), while the oxidation of palmitoyl- and erucoylcarnitine showed little change. It is suggested that erucate is shortened by the recently detected β-oxidation system of peroxisomes.     

Modulation of fatty acid incorporation and desaturation by trifluoperazine in fungi

The effects of trifluoperazine (TFP) on [1-¹⁴C]fatty acid incorporation into the lipids ofMortierella ramanniana var.angulispora were studied. TFP decreased [1-¹⁴C]-fatty acid incorporation into phosphatidylcholine, phosphatidylethanolamine and triacylglycerol, but greatly increased¹⁴C-labeling in phosphatidic acid. These changes in [1-¹⁴C]fatty acid incorporation induced by TFP were accompanied by a decrease in desaturation of some [1-¹⁴C]fatty acids taken up by the fungal cells. When [1-¹⁴C]lioleic acid (LA) was incubated with the fungal cells, total γ-linolenic acid (GLA) formation from incorporated [1-¹⁴C]LA decreased, but the¹⁴C-labeled GLA conent in individual lipid classes was essentially unchanged. This suggests that the site of the TFP effect on GLA formation from [1-¹⁴C]LA taken up from the medium is not the desaturase acting on LA linked to complex lipids. On the other hand, GLA formation from [1-¹⁴C]oleic acid was much less susceptile to TFP, which suggests that in this fungus Δ6 desaturation to GLA has at least two different pathways with different degrees of susceptibility to TFP.     

Detection of [U-13C]eicosapentaenoic acid in rat liver lipids using13C nuclear magnetic resonance spectroscopy

Fatty acid carbons are well-resolved in¹³C nuclear magnetic resonance (NMR) spectra of lipid extracts, but application of this methodology to the metabolism of¹³C-labelled fatty acids has not yet been reported. In the present study,¹³C NMR was used to monitor the presence of 98% [U-¹³C]eicosapentaenoic acid (EPA) in liver and carcass lipids 24 h after it had been injected into the stomach of a rat. Natural abundance¹³C NMR spectra of liver total fatty acid extracts were obtained from four control rats for comparison. At 24 h post-injection, quantitative high resolution¹³C NMR showed¹³C enrichment in liver fatty acid extracts was present mainly at olefinic and at the n−1 to n−4 carbons, but¹³C signal intensities for C−1 to C−4 of [U-¹³C]EPA were markedly reduced or absent. Small¹³C resonances, possibly indicative of some¹³C incorporation into docosahexaenoic acid and saturated or monounsaturated fatty acids, were present in spectra of liver fatty acids. Liver and carcass fatty acid composition was similar in both the controls and the EPA-injected rat, suggesting little accumulation of the injected [U-¹³C]EPA after 24 h. We conclude that the carbon-specific data provided by¹³C NMR of lipid extracts may be useful in monitoring the fate of individual carbons during tracer studies using¹³C-labelled fatty acids.     

The preparation of concentrates of eicosapentaenoic acid and docosahexaenoic acid by lipase-catalyzed transesterification of fish oil with ethanol

The objective of this study was to investigate the use of lipases as catalysts for producing concentrates of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) from fish oil as an alternative to conventional chemical procedures. Transesterification of fish oil with ethanol was conducted under anhydrous solvent-free conditions with a stoichiometric amount of ethanol. Among the 17 lipases tested, the results showed that Pseudomonas lipases had the highest activity toward the saturated and monounsaturated fatty acids in the fish oil, much lower activity toward EPA and DHA and, at the same time, good tolerance toward the anhydrous alcoholic conditions. With 10 wt% of lipase, based on weight of the fish oil triacylglycerol substrate (15% EPA and 9% DHA initial content), a 50% conversion into ethyl esters was obtained in 24 h at 20°C, in which time the bulk of the saturated and monounsaturated fatty acids reacted, leaving the long-chain n-3 polyunsaturated fatty acids unreacted in the residual mixture as mono-, di-, and triacylglycerols. This mixture comprised approximately 50% EPA+DHA. Total recovery of DHA and EPA was high, over 80% for DHA and more than 90% for EPA. The observed fatty acid selectivity, favoring DHA as a substrate, was most unusual because most lipases favor EPA.