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.     

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.     

High-performance liquid chromatography of the triacylglycerols ofVernonia galamensis andCrepis alpina seed oils

The triacylglycerols ofVernonia galamensis andCrepis alpina seed oils were characterized because these oils have high concentrations of vernolic (cis-12,13-epoxy-cis-9-octadecenoic) and crepenynic (cis-9-octadecen-12-ynoic) acids, respectively. The triacylglycerols were separated from other components of crude oils by solid-phase extraction, followed by resolution and quantitation of the individual triacylglycerols by reversed-phase high-performance liquid chromatography with an acetonitrile/methylene chloride gradient and flame-ionization detection. Isolated triacylglycerols were characterized by proton and carbon nuclear magnetic resonance and by capillary gas chromatography of their fatty acid methyl esters. The locations of the fatty acids on the glycerol moieties in the oils were obtained by lipolysis. TheVernonia galamensis oil contained 50% trivernoloyl and 21% divernoloyllinoleoyl glycerols along with 20% triacylglycerols with one vernolic and two other fatty acids. TheCrepis alpina oil contained 36% tricrepenynoyl and 33% dicrepenynoyllinoleoyl glycerols, 17% triacylglycerols with two crepenynic and one other fatty acid and 7% triacylglycerols with one crepenynic acid and two other fatty acids. Vernolic acid was found at both the 1(3)- and 2-glycerol carbons but was more abundant at the 1,(3)-position in theVernonia galamensis oil. Crepenynic acid was found at both glycerol carbon positions but was more abundant at the 2-position in theCrepis alpina oil. Visiting scientist from Technical Research Institute, Snow Brand Milk Company, Ltd., Saitana, Japan.     

Tissue-selective inhibition of sterol synthesis in mice by pravastatin sodium after a single or repeated oral administrations

Pravastatin, an inhibitor of 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase, exhibits liver-selectivity in inhibiting sterol synthesis, when administered as a single oral dose to mice or rats, whereas lovastatin and simvastatin do not. This may be due to the fact that pravastatin is distributed intracellularly, to a large extent, in the liver and extracellularly in nonhepatic tissues. In the present study, we examined whether the difference in liver-selectivity among these three HMG-CoA reductase inhibitors observed in single-dose studies was preserved after repeated oral administrations of drugs to mice.De novo sterol synthesis in different tissues of mice was examinedin vivo three hours after the last dose of drug by measuring incorporation of intraperitoneally injected [¹⁴C]acetate into total sterols. Pravastatin administered orally for 11 consecutive days at 5 and 10 mg/kg exhibited a greater liver-selectivity than lovastatin and simvastatin: sterol synthesis was inhibited more than 60% in the liver by all three drugs, whereas that in nonhepatic tissues was inhibited less than 10% by pravastatin and more than 30% by lovastatin and simvastatinin in most of the nonhepatic tissues examined. Pravastatin administered orallyfor 11 consecutive days at 10 mg/kg caused more selective inhibition of sterol synthesis in liverex vivo than two other inhibitors at the same dose. Pravastatin inhibitedde novo sterol synthesis from [¹⁴C]acetate into sterol fraction in the liver slicesin vitro, but minimally in those of the spleen and testis, whereas lovastatin and simvastatin inhibited in those of all three tissues. Since the drug concentrations determined in the same tissue samples of the liver, spleen, and testis were almost comparable among the three drugs, it was suggested that the cellular distribution profiles of pravastatin observed in a single-dose study were preserved in the multiple-dose study. We conclude that the difference in tissue-selectivity between pravastatin and the other two inhibitors to inhibit sterol synthesis in mice is maintained, regardless of the duration of administration.     

Fatty Acid de Novo Synthesis in Adult Intrauterine Growth-Restricted Offspring,and Adult Male Response to a High Fat Diet

Intrauterine growth restriction (IUGR) with rapid catch‐up growth leads to adult obesity and insulin resistance. We have previously shown that IUGR male rats demonstrated increased de novo fatty acid synthesis in the subcutaneous (SC) fat, but not the visceral fat, during the nursing period prior to the onset of obesity. Young IUGR females do not exhibit the same increase. We further hypothesized that in male IUGR offspring, de novo synthesis is a programmed intrinsic effect that persists to adulthood and does not suppress in response to a high fat diet. We measured fatty acid de novo synthesis in IUGR adult males (6 months) using deuterium‐enriched drinking water as a stable isotope tracer, then further studied the response after consumption of an isocaloric high fat diet. Baseline de novo synthesis in adult females was also studied at age 9 months. Males demonstrated increased baseline de novo synthesis in both SC fat and visceral fat. Correspondingly, SC and visceral fat protein expression of lipogenic enzymes acetyl‐coA carboxylase‐α (ACCα) and fatty acid synthase were upregulated. After the isocaloric high fat diet, de novo synthesis was suppressed such that no differences remained between the two groups, although, IUGR SC fat demonstrated persistently increased lipogenic protein expression. In contrast, de novo synthesis among adult females is not impacted in IUGR. In conclusion, enhancement of male IUGR SC fat de novo synthesis appears to be an early consequence of metabolic programming, whereas enhancement in visceral fat appears to be a later consequence.     

Correlation of ATP/citrate lyase activity with lipid accumulation in developing seeds of Brassica napus L.

The temporal distribution of ATP/citrate lyase (ACL) activity in developing seeds of Brassica napus L. closely paralleled both that of acetyl-CoA carboxylase and the overall rate of lipid biosynthesis. Maximum ACL activities (250 nmol acetyl-CoA formed min⁻¹·g fresh seed) were recorded between 35 to 42 d after pollination and, if the in vitro data could be extrapolated to the situation in vivo, could account for half of the acetyl-CoA required for the measured rate of fatty acid biosynthesis during seed development. The enzyme appeared to be localized in a subcellular compartment, which was clearly separated from mitochondria on a sucrose gradient and by differential centrifugation, and which corresponded to the chloroplast organelle.     

Effects of perfluorodecanoic acid onde novo fatty acid and cholesterol synthesis in the rat

Perfluorodecanoic acid (PFDA) is a peroxisome proliferator that causes a dose-dependent (20–80 mg/kg) increase in hepatic triacylglycerol and cholesteryl ester levels in the rat. We hypothesized that PFDA may cause an increase in thede novo synthesis of fatty acids and cholesterol in this species, which would explain observed effects. The incorporation of³H₂O into tissue lipids was examined 7 days after rats received vehicle or 20 or 80 mg/kg of PFDA. PFDA treatment decreased the rate of synthesis of cholesterol and fatty acids in the liver and in epididymal fat pad. At a PFDA dose (20 mg/kg) that decreasedde novo synthesis of fatty acids and cholesterol, there was no effect on the concentration of fatty acids and cholesterol in the liver, epididymal fat pads, and plasma. We conclude that PFDA induced fatty liver is due to either a decrease in the oxidation of fatty acids in the liver, or an impairment of triacylglycerol catabolism and/or export from the liver, and is not the result of an increase inde novo synthesis of fatty acids and cholesterol.     

Sesamol as an inhibitor of growth and lipid metabolism in Mucor circinelloides via its action on malic enzyme

Sesamol, a nonoil component of sesame seed oil, inhibited growth, fatty acid synthesis, and desaturation by Mucor circinelloides in vivo. Although sesamol also inhibited the growth of other fungi and yeasts, its effect on the lipid metabolism of M. circinelloides was exceptional. An enzymological study demonstrated that sesamol affected lipid synthesis primarily by the inhibition of malic enzyme activity, thereby limiting the NADPH supply for fatty acid synthesis and desaturation. Sesamol itself had no inhibitory effect on malic enzyme activity in vitro. A metabolite of sesamol is therefore probably responsible for the in vivo effects of sesamol on lipid metabolism.     

Positional distribution of fatty acids in the triacylglycerols of developing oil palm fruit

The pattern of accumulation of triacylglycerols, their fatty acid compositions and the positional distribution of the fatty acids at thesn-2- andsn-1,3-positions of the triacylglycerol molecules at progressive stages of oil palm fruit development were determined. There was an exponential rate of increase of triacylglycerols and their fatty acids toward the end of fruit development. The fatty acid composition of the triacylglycerols in the early stages of development, prior to active accumulation, was more or less similar, but differed appreciably from the later stages, and the transition of fatty acid composition toward that of normal palm oil occurred at around 16 wk after anthesis (WAA) and stabilized at 20 WAA. All fatty acids increased in terms of absolute quantity. There was an overall consistency in fatty acid positional distribution, irrespective of development stage. More saturated fatty acids were found to be esterified at thesn-1,3-positions and more unsaturated fatty acids at thesn-2-position of triacylglycerol. Higher rate of incorporation of 16:0 at the 1,3-positions during the active phase of triacylglycerol synthesis was observed, while 18:1 acid exhibited a reverse trend.     

Elongation of (n−9) and (n−7)cis-monounsaturated and saturated fatty acids in seeds ofsinapis alba

Lipids in developing seeds ofSinapis alba contain appreciable proportions of (n−7)octadecenoic (vaccenic) acid besides its (n−9) isomer (oleic acid), whereas the constituent very long chain (>C₁₈) monounsaturated fatty acids of these lipids are overwhelmingly composed of the (n−9) isomers. Cotyledons of developingSinapis alba seed use [1-¹⁴C]acetate, [1-¹⁴C]malonate or [1,3-¹⁴C]malonyl-CoA for de novo synthesis of palmitic, stearic and oleic acids and for elongation of preformed oleic, vaccenic and stearic acids to their higher (n−9), (n−7) and saturated homologs, respectively. Moreover, elongation of preformed (n−7)palmitoleic acid to vaccenic acid is observed. Stepwise C₂-additions to preformed oleoyl-CoA by acetyl-CoA or malonyl-CoA yielding (n−9)icosenoyl-CoA, (n−9)docosenoyl-CoA and (n−9)tetracosenoyl-CoA are by far the most predominant reactions catalyzed by the elongase system, which seems to have a preference for oleoyl-CoA over vaccenoyl-CoA as the primer. The pattern of¹⁴C-labeling of the very long chain fatty acids formed from either acetate or malonate shows a close analogy in the mode of elongation of monounsaturated and saturated 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).     

Hypolipidemic activity of 6-alkoxycarbonyl-3-aryl-1,3,5-triazabicyclo[3.1.0]hexane-2,4-diones and 2-alkoxycarbonyl-5-aryl-1,3,5-triazine-4,6(1H,5H)-diones in rodents

Significant hypolipidemic activity was demonstrated by 6-ethoxycarbonyl-3-phenyl-1,3,5-triazabicyclo[3.1.0]hexane-2,4-dione, 2-ethoxycarbonyl-5-phenyl-1,3,5-triazine-4,6(1H,5H)-dione and 2-ethoxycarbonyl-5-(4-chlorophenyl)-1,3,5-trizine-4,6(1H,5H)-dione in rodents at 20 mg/kg/day. These agents lowered serum cholesterol and triglyceride levels by approximately 40% in mice after 16 d. Tissue lipids in rat liver, small intestinal mucosa, aortic wall and feces were reduced by treatment with the agents. Very low density lipoprotein (VLDL) and low density lipoprotein (LDL) cholesterol levels were reduced in the rat; high density lipoprotein (HDL) cholesterol levels were elevated after 14 d of treatment. The activities of regulatory enzymes,e.g., acetyl-CoA synthetase, acyl-CoA:cholesterol acyltransferase, cholesterol 7α-hydroxylase,sn-glycerol-3-phosphate acyltransferase, phosphatidylate phosphohydrolase and heparin-induced lipoprotein lipase, involved inde novo synthesis of hepatic lipids were affected by the agents. The new compounds may represent another class of potentially useful hypolipidemic agents for the treatment of atherosclerosis since HDL cholesterol levels were increased and VLDL and LDL cholesterol levels were lowered by some of the agents.     

Isolation and identification of acetyl-CoA carboxylase from rainbow trout (Salmo gairdneri) liver

Acetyl-CoA carboxylase is the pivotal enzyme in the de novo synthesis of fatty acids and is the only carboxylase with a biotin-containing subunit greater than 200,000 daltons. The biotin moiety is covalently linked to the active site and has a high affinity (K_d=10⁻¹⁵ M) for the protein avidin. This relationship has been used in previous studies to identify acetyl-CoA carboxylase isolated from mammalian species. However, acetyl-CoA carboxylase has not been isolated and characterized in a poikilothermic species such as the rainbow trout. The present study describes the isolation and identification of acetyl-CoA carboxylase in the cytosol of rainbow trout (Salmo gairdneri) liver. The enzyme was isolated using two distinct procedures—polyethylene glycol precipitation and avidin-Sepharose affinity chromatography. Identification of the isolated protein as acetyl-CoA carboxylase was made by the following: (1) sodium dodecyl sulfate-polyacrylamide gel electrophoresis; (2) avidin binding; (3) in vivo labeling with [¹⁴C]biotin; and (4) acetyl-CoA carboxylase-specific activity. The subunit molecular weight of the major protein was 230,000 daltons ±3.3%. This protein was shown to bind avidin (M_r=16,600) prior to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating the presence of biotin. In addition, protein isolated from fish that had previously received intraperitoneal injections of [¹⁴C]biotin, showed the majority of radioactivity associated with the 230,000 dalton protein. The polyethylene glycol precipitation yielded 200 μg protein (4.4 μg/g liver), with a specific activity of 5 nmol malonyl-CoA/min/mg protein, whereas avidin affinity chromatography yielded 1.75±1.1 mg protein (9.0 μg/g liver), with a specific activity of 1.37±0.18 μmol malonyl-CoA/min/mg protein. The enzyme was citrate dependent showing maximum activity between 10 and 20 mM. Acetyl-CoA carboxylase-specific activity decreased by 50% in the presence of 0.2 M NaCl. These findings suggest that the major protein (M_r=230,000) purified from rainbow trout liver is acetyl-CoA carboxylase with enzyme characteristics comparable to mammalian acetyl-CoA carboxylase.     

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.     

Sources of triacylglycerol accumulation in livers of rats fed a cholesterol-supplemented diet

The source of free fatty acids (FFA) and the pathways contributing to the accumulation of neutral fats in livers of rats fed a cholesterol-enriched diet were investigated in this report. Supplementation with 1% cholesterol in the diet for four weeks resulted in hepatomegaly in the rats. The contents of cholesterol and triacylglycerols (TG) per gram liver measured in rats fasted overnight increased by 48 mg (∼tenfold) and 66 mg (∼fourfold), respectively. The activities of glycerophosphate acyltransferase and diacylglycerol acyltransferase, the two key enzymes for TG synthesis in liver microsomes, were found to increase by 23 and 19%, respectively, in the cholesterol-fed rats. The secretion of plasma TG present predominantly in very low density lipoprotein was found to decrease by ∼30%. The incorporation of tritium from tritiated water in liver FFA increased by twofold in rats fed the cholesterol-supplemented diet, whereas the activity of CPT I in liver mitochondria decreased by 23%. The uptake of plasma FFAin vivo in livers of fasted rats maintained on the cholesterol-supplemented diet decreased by 60%. Our data thus indicate that the excess TG accumulated in livers of rats fed the cholesterol-enriched diet resulted from increased synthesis and decreased secretion of TG. To meet the demand of fatty acids for this purpose,de novo lipogenesis increased, whereas β-oxidation decreased. Although difference in the uptake of extrahepatic FFA may be discounted, a difference in the uptake of chylomicron remnants between the control and cholesterol-fed rats may not be ruled out.     

Eicosapentaenoic and docosahexaenoic acid affect mitochondrial and peroxisomal fatty acid oxidation in relation to substrate preference

Decreased triacylglycerol synthesis within hepatocytes due to decreased diacylglycerol acyltransferase (DGAT) activity has been suggested to be an important mechanism by which diets rich in fish oil lower plasma triacylglycerol levels. New findings suggest that eicosapentaenoic acid (EPA), and not docosahexaenoic acid (DHA), lowers plasma triacylglycerol by increased mitochondrial fatty acid oxidation and decreased availability of fatty acids for triacylglycerol synthesis. To contribute to the understanding of the triacylglycerol-lowering mechanism of fish oil, the different metabolic properties of EPA and DHA were studied in rat liver parenchymal cells and isolated rat liver organelles. EPA-CoA was a poorer substrate than DHA-CoA for DGAT in isolated rat liver microsomes, and in the presence of EPA, a markedly lower value for the triacyl[³H]glycerol/diacyl[³H]glycerol ratio was observed. The distribution of [1-¹⁴C]palmitic acid was shifted from incorporation into secreted glycerolipids toward oxidation in the presence of EPA (but not DHA) in rat liver parenchymal cells. [1-¹⁴C]EPA was oxidized to a much greater extent than [1-¹⁴C]DHA in rat liver parenchymal cells, isolated peroxisomes, and especially in purified mitochondria. As the oxidation of EPA was more effective and sensitive to the CPT-I inhibitor, etomoxir, when measured in a combination of both mitochondria and peroxisomes, we hypothesized that both are involved in EPA oxidation, whereas DHA mainly is oxidized in peroxisomes. In rats, EPA treatment lowered plasma triacylglycerol and increased hepatic mitochondrial fatty acid oxidation and carnitine palmitoyltransferase (CPT)-I activity in both the presence and absence of malonyl-CoA. Whereas only EPA treatment increased the mRNA levels of CPT-I, DHA treatment increased the mRNA levels of peroxisomal fatty acyl-CoA oxidase and fatty acid binding protein more effectively than EPA treatment. In conclusion, EPA and DHA affect cellular organelles in relation to their substrate preference. The present study strongly supports the hypothesis that EPA, and not DHA, lowers plasma triacylglycerol by increased mitochondrial fatty acid oxidation.     

Carnitine palmitoyltransferase I, carnitine palmitoyltransferase II, and Acyl-CoA oxidase activities in atlantic salmon (Salmo salar)

Salmon farmers are currently using high-energy feeds containing up to 35% fat; the fish's capability of fully utilizing these high-energy feeds has received little attention. Carnitine is an essential component in the process of mitochondrial fatty acid oxidation and, with the cooperation of two carnitine palmitoyltransferases (CPT-I and CPT-II) and a carnitine acylcarnitine transporter across the inner mitochondrial membrane, acts as a carrier for acyl groups into the mitochondrial matrix where β-oxidation occurs. However, no reports are available differentiating between CPT-I and CPT-II activities in fish. In order to investigate the potential for fatty acid catabolism, the activities of key enzymes involved in fatty acid oxidation were determined in different tissues from farmed Atlantic salmon (Salmo salar), i.e., acyl-CoA oxidase (ACO) and CPT-I and CPT-II. Malonyl-CoA was a potent inhibitor of CPT-I activity not only in red muscle but also in liver, white muscle, and heart. By expressing the enzyme activities per wet tissue, the CPT-I activity of white muscle equaled that of the red muscle, both being>> liver. CPT-II dominated in red muscle whereas the liver and white muscle activities were comparable. ACO activity was high in the liver regardless of how the data were calculated. Based on the CPT-II activity and total palmitoyl-l-carnitine oxidation in white muscle, the white muscle might have a profound role in the overall fatty acid oxidation capacity in fish.     

The origin of palmitic acid in brain of the developing rat

A rat milk substitute containing lower amounts of palmitic and oleic acid in the triacylglycerols in comparison to natural rat milk was fed to artificially reared rat pups from day 7 after birth to day 14. Pups reared by their mother served as controls. Free trideuterated (D₃) palmitic acid [(C²H₃)(CH₂)₁₄COOH, 98 atom % D] and free perdeuterated (D₃₁) palmitic acid [C₁₅ ²H₃₁COOH, 99 atom % D] in equal quantity were mixed into the triacylglycerols of the milk substitute in an amount equal to 100% of the palmitic acid in the triacylglycerols. A control milk substitute contained unlabeled free palmitic acid in an amount equal to 100% of the palmitic acid in the triacylglycerols of the milk substitute. The objective was to determine if palmitic acid in the diet contributed significantly to the palmitic acid content of developing brain and other organs. The methyl esters of the fatty acids were analyzed by gas chromatography and the palmitic acid methyl ester was examined by fast atom bombardment mass spectrometry. The proportion of deuterated methyl palmitate as a percentage of total palmitate was determined; 32% of the palmitic acid in liver and 12% of the palmitic acid in lung were trideuterated and perdeuterated palmitic acid in approximately equal amounts. The brain, by contrast, did not contain the deuterated palmitic acid moiety. Quantitation of palmitic acid and total fatty acids revealed a significant accumulation in organs in the interval from 7 to 14 days of age. Under our experimental conditions, labeled palmitic acid does not enter the brain. Consequently, we conclude that the developing brain produces all required palmitic acid byde novo synthesis.     

Stereospecific analysis of triacylglycerols rich in long-chain polyunsaturated fatty acids

Six oils of marine, algal, and microbial origin were analyzed for stereospecific distribution of component fatty acids. The general procedure involved preparation ofsn-1,2-(2,3)-diacylglycerols by partial deacylation with ethylmagnesium bromide or pancreatic lipase, separation of X-1,3- andsn-1,2(2,3)-diacylglycerols by borate thin-layer chromatography, resolution of thesn-1,2- andsn-2,3-enantiomers by chiral phase high-performance liquid chromatography following preparation of dinitrophenylurethane derivatives, and determination of the fatty acid composition by gas chromatography. Unexpected complications arose during a stereospecific analysis of triacylglycerols containing over 33% of either 20∶4 or 22∶6 fatty acids. Thesn-1,2(2,3)-diacylglycerols made up of two long-chain polyunsaturated acids migrated with the X-1,3-diacylglycerols and required separate chiral phase resolution. Furthermore, the enzymatic method yieldedsn-1,2(2,3)-diacylglycerols, overrepresenting the polyenoic species due to their relative resistance to lipolysis, but prolonged digestion yielded correct composition for the 2-monoacylglycerols. The final positional distribution of the fatty acids was established by pooling and normalizing the data from subfractions obtained by norman- and chiral-phase separation of diacylglycerols. The molecular species of X-1,3-,sn-1,2- andsn-2,3-diacylglycerol dinitrophenylurethanes were identified by chiral-phase liquid chromatography/mass spectrometry with electrospray ionization, which demonstrated a preferential association of the paired long-chain acids with thesn-1,2- andsn-2,3-diacylglycerol isomers.