Dietary n-3 and n-6 PUFA Enhance DHA Incorporation in Retinal Phospholipids Without Affecting PGE<Subscript>1</Subscript> and PGE<Subscript>2</Subscript> Levels

The purpose of this study was to determine whether dietary n-3 and n-6 PUFA may affect retinal PUFA composition and PGE₁ and PGE₂ production. Male Wistar rats were fed for 3 months with diets containing: (1) 10% eicosapentaenoic acid (EPA) and 7% docosahexaenoic acid (DHA), or (2) 10% γ-linolenic acid (GLA), or (3) 10% EPA, 7% DHA and 10% GLA, or (4) a balanced diet deprived of EPA, DHA, and GLA. The fatty acid composition of retinal phospholipids was determined by gas chromatography. Prostaglandin production was measured by enzyme immunoassay. When compared to rats fed the control diet, the retinal levels of DHA were increased in rats fed both diets enriched with n-3 PUFA (EPA + DHA and EPA + DHA + GLA diets) and decreased in those supplemented with n-6 PUFA only (GLA diet). The diet enriched with both n-6 and n-3 PUFA resulted in the greatest increase in retinal DHA. The levels of PGE₁ and PGE₂ were significantly increased in retinal homogenates of rats fed with the GLA-rich diet when compared with those of animals fed the control diet. These higher PGE₁ and PGE₂ levels were not observed in animals fed with EPA + DHA + GLA. In summary, GLA added to EPA + DHA resulted in the highest retinal DHA content but without increasing retinal PGE₂ as seen in animals supplemented with GLA only.     

Platelet and aorta arachidonic and eicosapentaenoic acid levels andin vitro eicosanoid production in rats fed high-fat diets

There is a significant interest in the interrelationship between long-chain n-3, and n-6 fatty acids due to their ability to modulate eicosanoid production. In general, the intake of arachidonic acid (AA) results in enhanced eicosanoid production, whereas n-3 polyunsaturated fatty acids (PUFA) decrease the production of eicosanoids from AA. The purpose of this study was to investigate whether the effects of dietary AA on eicosanoid production in the rat were correlated with the AA and EPA levels in platelets and aorta (eicosanoid-producing tissues). Four groups of male Sprague-Dawley rats were fed a highfat diet enriched with eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (approximately 100 mg/day of EPA+DHA) for 24 d. During the last 10 d, the four groups were orally supplemented with 0,30,60, and 90 mg/day of ethyl arachidonate. A further group of rats was fed a control diet (without longchain n-3 PUFA) for 24 d.In vitro aorta prostacyclin (PGI₂) production, serum thromboxane A₂ (TxA₂) production and plasma, and platelet and aorta phospholipid (PL) fatty acids were measured. Enriching the diet with n-3 PUFA resulted in significant reductions in tissue AA levels and an increase in the n-3 PUFA, particularly EPA. On this diet, the AA to EPA ratio was 1:1 in platelet PL, and it was 2:1 in the aorta PL. There were significant decreases in thein vitro PGI₂ and TxA₂ production compared with the control animals. The inclusion of AA in the diet resulted in marked increases in AA levels in the platelet and aorta PL with corresponding decreases in EPA. The lowest dose of AA (30 mg/rat) reversed the effects of 100 mg/day of n-3 PUFA on AA levels in platelet and aortic PL and onin vitro aorta PGI₂ and serum TxA₂ production. The dietary AA caused a differential (twofold) increase in TxA₂ relative to PGI₂ for all three levels of AA supplementation. There were greater changes in the levels of AA and/or EPA in platelet PL compared with the aorta PL, which might have accounted for the differential effects of these PUFA on thromboxane production compared with PGI₂ production in this study.     

Differential effects of eicosapentaenoic acid and docosahexaenoic acid on human skin fibroblasts

To better understand the mode of action of ω3 fatty acids in cell membranes, human foreskin fibroblasts were grown in serum-free medium supplemented with 50 μM oleic acid linoleic acid, eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA), and the effects on membrane composition, fluorescence polarization and enzyme activities were followed. The cells were enriched with EPA and DHA up to 7 and 13% of total lipids, respectively, of which >95% was associated with phospholipids. In addition, the concentration of 22∶5n−3 increased with both EPA and DHA to 7.5, and 2.1% of the total fatty acids, respectively. When compared to controls (oleic acid), cells treated with DHA showed a decrease in cholesterol, phospholipids, arachidonic acid (AA) and free cholesterol/phospholipid ratio (P<0.05). In the presence of EPA, only decreases in AA and cholesterol were significant (P<0.05). Membrane fluidity, assessed by fluorescence anisotropy, was increased 16% in cells enriched with DHA (P<0.05), but showed no change with EPA or linoleic acid. There was an increase in membrane-associated 5′-nucleotidase (+27%) and adenylate cyclase (+19%) activities (P<0.05), in DHA-enriched, but not in EPA-enriched cells, when compared with oleate controls. The studies show that incorporation of DHA, but not EPA, into cell membranes of fibroblasts alters membrane biophysical characteristics and function. We suggest that these two major n−3 fatty acids of fish oils have differential effects on cell membranes, and this may be related to the known differences in their physiological effects.     

Concentration and purification of stearidonic,eicosapentaenoic, and docosahexaenoic acids from cod liver oil and the marine microalgaIsochrysis galbana

n-3 Polyunsaturated fatty acids (n-3 PUFA) from the marine microalgaIsochrysis galbana were concentrated and purified by a two-step process—formation of urea inclusion compounds followed by preparative high-performance liquid chromatography. These methods had been developed previously with fatty acids from cod liver oil. By the urea inclusion compounds method, a mixture that contained 94% (w/w) stearidonic (SA), eicosapentaenoic (EPA), plus docosahexaenoic (DHA) acids (4:1 urea/fatty acid ratio and 4°C crystallization final temperature) was obtained from cod liver oil fatty acids. Further purification of SA, EPA, and DHA was achieved with reverse-phase C₁₈ columns. These isolations were scaled up to a semi-preparative column. A PUFA concentrate was isolated fromI. galbana with methanol/water (80:20, w/w) or ethanol/water (70:30, w/w). With methanol/water, a 96% EPA fraction with 100% yield was obtained, as well as a 94% pure DHA fraction with a 94% yield. With ethanol/water as the mobile phase, EPA and DHA fractions obtained were 92% pure with yields of 84 and 88%, respectively.     

Relationship between dietary supply of long-chain fatty acids and membrane composition of long- and very long chain essential fatty acids in developing rat photoreceptors

The present study was designed to determine if dietary supply of long-chain fatty acid (LCFA, C20∶4n-6, and/or C22∶6n-3), reflecting levels that might be incorporated into infant formulas, influences the fatty acid composition of the visual cell membrane. The rod outer segment (ROS) of the retina was analyzed from rats fed diets varying in the ratio of 18∶2n-6 to 18∶3n-3 with or without 20∶4n-6 [arachidonic acid (AA)] and 22∶6n-3 (docosahexaenoic acid) from birth to six weeks of age. The level of very long chain fatty acids (VLCFA, C₂₄−C₃₆) was identified using gas chromatography and gas chromatography-mass spectrometry. In the ROS, the highest relative percent of AA was attained in phosphatidylcholine (PC) and phosphatidylethanolamine (PE) of animals fed 1% AA diet, whereas feeding 0.7% docosahexaenoic acid (DHA) diet significantly increased the DHA level in PC, phosphatidylserine, and phosphatidylinositol compared to feeding diets containing AA. VLCFA of n-6 and n-3 up to C₃₆ were found in PC, with the most abundant fatty acids being C₃₂ and C₃₄. In PC, phosphatidylserine and PE, the n-6 tetraenoic VLCFA level was highly increased in animals fed 1% AA compared to other dietary groups. This study suggests that dietary fat containing small amounts of AA or DHA is an important factor influencing membrane fatty acid composition of the visual cell during development. Based on a presentation at the AOCS Annual Meeting & Expo in San Antonio, Texas, May 7–11, 1995.     

Lipids of selected molds grown for production of n−3 and n−6 polyunsaturated fatty acids

The lipid classes and component fatty acids of seven fungi were examined. Three marine fungi,Thraustochytrium aureum, Thraustochytrium roseum andSchizochytrium aggregatum (grown at 30, 25 and 25°C, respectively), produced less than 10% lipid but contained docosahexaenoic acid (DHA) up to 30% and eicosapentaenoic acid (EPA) up to 11% of the total fatty acids.Mortierella alpinapeyron produced 38% oil containing solely n-6 polyunsaturated fatty acids (PUFA) with arachidonic acid (AA) at 11% of the total fatty acids.Conidiobolus nanodes andEntomorphthora exitalis produced 25% oil and contained both n−3 and n−6 PUFA, with AA at 16% and 18%, respectively.Saprolegnia parasitica produced 10% oil and contained AA and EPA, respectively, at 19% and 18%. The triacylglycerol fraction always represented the major component at between 44% and 68% of the total lipid. Each fungus, exceptT. aureum, had the greatest degree of fatty acid unsaturation in the phospholipid fraction. The triacylglycerol fraction ofT. aureum was the most unsaturated with DHA representing 29% (w/w) of all fatty acids present. The presence of the enzyme ATP:citrate lyase correlated with the ability of molds to accumulate more than 10% (w/w) lipid when the fungi were grown in nitrogen-limiting media. In those molds that failed to accumulate more than 10% lipid, the enzyme was absent.     

Modulation of arachidonate and docosahexaenoate in Morone chrysops larval tissues and the effect on growth and survival

The extent to which extreme dietary levels of arachidonate (AA) and/pr docosahexaenoate (DHA) modulate lipid composition in the body tissues and consequently affect growth and survival in freshwater Morone larvae species was examined. White bass, M. chrysops, larvae (day 24–46) were fed Artemia nauplii enriched with algal oils containing varying proportions of AA and DHA (from 0 to over 20% the total fatty acids). Growth was significantly reduced (P<0.05) in larvae fed a DHA-deficient Artemia diet. Increases in dietary levels of AA also were associated with a significant growth reduction. However, the inhibitory effect of AA on larvae growth could be suppressed by the dietary addition of DHA (at a level of 21.6% of the total fatty acids in enrichment lipids). Larval brain+eyes tissue accumulated over 10 times more DHA than AA in its structural lipids (phosphatidylcholine, phosphatidylethanolamine) at any dietary ratio. In contrast, DHA accumulation, as compared to AA, in gill lipids declined considerably at higher than 10∶1 DHA/AA tissue ratios. DHA and eicosapentaenoic acid (EPA) contents in brain+eyes tissue were most sensitive to competition from dietary AA, being displaced from the tissue at rates of 0.36±0.07 mg DHA and 0.46±0.11 mg EPA per mg increase in tissue AA, and 0.55±0.14 mg AA per mg increase in tissue DHA. On the other hand, AA and EPA levels in gill tissue were most sensitive to dietary changes in DHA levels; AA was displaced at rates of 0.37±0.11 mg, whereas EPA increased at rates of 0.68±0.28 mg per mg increase in tissue DHA. Results suggest that balanced dietary DHA/AA ratios (that allow DHA/AA ratios of 2.5∶1 in brain+eyes tissue) promote a high larval growth rate, which also correlates with maximal regulatory response in tissue essential fatty acids.     

Incorporation of n-3 fatty acids into plasma lipid fractions, and erythrocyte membranes and platelets during dietary supplementation with fish, fish oil, and docosahexaenoic acid-rich oil among healthy young men

The effects of n-3 fatty acid supplementation in the form of fresh fish, fish oil, and docosahexaenoic acid (DHA) oil on the fatty acid composition of plasma lipid fractions, and platelets and erythrocyte membranes of young healthy male students were examined. Altogether 59 subjects (aged 19–32 yr, body mass index 16.8–31.3 kg/m²) were randomized into the following diet groups: (i) control group; (ii) fish diet group eating fish meals five times per week [0.38±0.04 g eicosapentaenoic acid (EPA) and 0.67±0.09 g DHA per day]; (iii) DHA oil group taking algae-derived DHA oil capsules (1.68 g/d DHA oil group taking algae-derived DHA oil capsules (1.68 g/d DHA in triglyceride form); and (iv) fish oil group (1.33 g EPA and 0.95 g DHA/d as free fatty acids) for 14 wk. The fatty acid composition of plasma lipids, platelets, and erythrocyte membranes was analyzed by gas chromatography. The subjects kept 4-d food records four times during the study to estimate the intake of nutrients. In the fish diet, in DHA oil, and in fish oil groups, the amounts of n-3 fatty acids increased and those of n-6 fatty acids decreased significantly in plasma lipid fractions and in platelets and erythrocyte membranes. A positive relationship was shown between the total n-3 polyunsaturated fatty acids (PUFA) and EPA and DHA intake and the increase in total n-3 PUFA and EPA and DHA in all lipid fractions analyzed. DHA was preferentially incorporated into phospholipid (PL) and triglyceride (TG) and there was very little uptake in cholesterol ester (CE), while EPA was preferentially incorporated into PL and CE. The proportion of EPA in plasma lipids and platelets and erythrocyte membranes increased also by DHA supplementation, and the proportion of linoleic acid increased in platelets and erythrocyte membranes in the DHA oil group as well. These results suggest retroconversion of DHA to EPA and that DHA also interferes with linoleic acid metabolism.     

Incorporation of polyunsaturated fatty acids into CT-26, a transplantable murine colonic adenocarcinoma

Previous studies in our laboratory have shown that marine oils, with high levels of eicosapentaenoic (EPA, 20∶5n−3) and docosahexaenoic acids (DHA, 22∶6n−3), inhibit the growth of CT-26, a murine colon carcinoma cell line, when implanted into the colons of male BALB/c mice. Anin vitro model was developed to study the incorporation of polyunsaturated fatty acids (PUFA) into CT-26 cells in culture. PUFA-induced changes in the phospholipid fatty acid composition and the affinity with which different fatty acids enter the various phospholipid species and subspecies were examined. We found that supplementation of cultured CT-26 cells with either 50 μM linoleic acid (LIN, 18∶2n−6), arachidonic acid (AA, 20∶4n−6), EPA, or DHA significantly alters the fatty acid composition of CT-26 cells. Incorporation of these fatty acids resulted in decreased levels of monounsaturated fatty acids, while EPA and DHA also resulted in lower levels of AA. While significant elongation of both AA and EPA occurred, LIN remained relatively unmodified. Incorporation of radiolabeled fatty acids into different phospholipid species varied significantly. LIN was incorporated predominantly into phosphatidylcholine and had a much lower affinity for the ethanolamine phospholipids. DHA had a higher affinity for plasmenylethanolamine (1-O-alk-1′-enyl-2-acyl-sn-glycero-3-phosphoethanolamine) than the other fatty acids, while EPA had the highest affinity for phosphatidylethanol-amine (1,2-diacyl-sn-glycero-3-phosphoethanolamine). These results demonstrate that,in vitro, significant differences are seen between the various PUFA in CT-26 cells with respect to metabolism and distribution, and these may help to explain differences observed with respect to their effects on tumor growth and metastasis in the transplantable model.     

Δ6 Desaturase mRNA Abundance in HepG2 Cells Is Suppressed by Unsaturated Fatty Acids

The effect of unsaturated fatty acids on the abundance of Δ6 desaturase (D6D) mRNA and the fatty acid composition of HepG2 cell membranes was examined. Supplementation of HepG2 cells with oleic acid (18:1n-9, OA), linoleic acid (18:2n-6, LA), α-linolenic acid (18:3n-3, ALA), arachidonic acid (20:4n-6, AA) or eicosapentaenoic acid (20:5n-3, EPA) reduced D6D mRNA abundance by 39 ± 6.6, 40 ± 2.2, 31 ± 5.2, 55 ± 4.8, and 52 ± 5.0%, respectively, compared with control cells. Despite the reduction in D6D mRNA abundance, the level of D6D conversion products (20:3n-9, EPA and AA) in OA, ALA and LA supplemented cells, respectively, was elevated above that in control cells. Our results suggest that although unsaturated fatty acids decrease the abundance of D6D mRNA by as much as 50%, the conversion of polyunsaturated fatty acids and accumulation of long chain polyunsaturated fatty acids (LCPUFA) in HepG2 cell phospholipids continues to occur.     

Effects of an eicosapentaenoic and docosahexaenoic acid concentrate on a human lung carcinoma grown in nude mice

The effects of the n−3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), on the growth of a human lung mucoepidermoid carcinoma (HLMC) in athymic mice were studied. The mice were divided into three groups which were given either a control chow diet (C), a chow diet supplemented with EPA/DHA (P) (25 or 50 mg of free n−3 fatty acids/g of pellet/day), or chow diet supplemented with palmitic acid (S) (isocaloric with P). Two independent experimental schedules were followed: i) host mice bearing either tumors that were allowed to reach 4000 mm³, or only 35 mm³, were fed C, P or S for 21 or 41 days; ii) animals were fed C, P and S for 9 days before tumor implant and were maintained on these diets throughout tumor growth. Food consumption, mouse weight and liver/body weight ratio showed no significant differences between supplemented diets and chow. Tumor growth was markedly inhibited (45%) in both experiments by the EPA/DHA supplemented diet. In Experiment 2, only 60% of mice fed diet P had tumors. The fatty acid composition of neutral and polar lipids of host liver and tumor reflected the dietary intake of n−3 fatty acids; the content of arachidonic acid was reduced by 50%, and EPA/DHA was increased 3-to 5-fold. Tumor prostaglandin E₂ levels were reduced 7.4-fold in the P group. The reduced PGE₂ content may be a factor in tumor growth inhibition. Preliminary data were presented at the Annual Meeting of the American Oil Chemists' Society, (1990), Baltimore, MD, (de Antueno, R.J., Bravo, M.G. de, Toledo, J., De Tomas, M.E., Mercuri, O., Quintans, C. (1990)INFORM 1, 328 (Abstract HH1).     

A rapid method for determining the oxidation of n-3 fatty acids

The stability of unsaturated fatty acids to oxidation was monitored by following gas chromatographic (GC) analyses of headspace volatiles in comparison to changes in polyunsaturated fatty acids (PUFA) and increases in malonaldehydevia the 2-thiobarbituric (TBA) assay. Pure standards of linoleic acid (Lo) and n-3 fatty acids [eicosapentaenoic (EPA) and docosahexaenoic acid (DHA)] were added to headspace vials, equilibrated in air for 10 min, followed by heating at 80°C in teflon-capped vials for different time intervals. Headspace analysis showed increases in acetaldehyde, propenal, and propanal, corresponding to the oxidation of n-3 fatty acids, whereas hexanal production corresponded to losses of linoleic acid. The analysis of propanal by GC-headspace after only five minutes of heating appeared to be the most effective method of monitoring the oxidation of n-3 fatty acids, as indicated by correlations between TBA values and loss of PUFA. The oxidation of Lo, EPA and DHA appeared to be a function of the number of double bonds. Correlations between PUFA depletion, TBA values and volatile formation indicate that under the prescribed conditions of this experiment, GC-headspace analysis of propanal and pentane/hexanal is an excellent method for following the oxidation of selected n-3 fatty acids and linoleic acid.     

Eicosapentaenoic acid and docosahexaenoic acid selectively attenuate U46619-induced smooth muscle cell proliferation

It is well known that vascular smooth muscle cell (SMC) proliferation is a key step in atheromatous plaque formation. Thromboxane A₂ (T×A₂), released from aggregating platelets and an injured vessel wall, may play an important role in the development of atheromatous plaque. Many animal studies have suggested that n−3 polyunsaturated fatty acids eicosapentaenoic acid (EPA, 20∶5n−3) and docosahexaenoic acid (DHA, 22∶6n−3) present in the fish oils have antiatherosclerotic effects. In the present study, we investigated the effect of EPA and DHA on I×A₂-induced SMC proliferation. To determine the functional selectivity of n−3 fatty acids, we also tested the effect of arachidonic acid (AA, 20∶4n−6), γ-linolenic acid) (INA, 18∶3n−6), and oleic acid (OA, 18∶1n−9) on T×A₂-induced SMC proliferation. Only LPA and DHA prevented the SMC proliferation induced by the T×A₂ mimetic U46619. When EPA and DHA were added together in the ratio in which they are present in menhaden oil, EPA and DHA acted synergistically to block the SMC proliferation induced by the TXA₂-mimetic. These findings suggest that the n−3 polyunsaturated fatty acids in fish oils may exert antiatherosclerotic effects by blocking the mitogenstimulated proliferation of SMC.     

Daily Intake of Cod or Salmon for 2?Weeks Decreases the 18:1n-9/18:0 Ratio and Serum Triacylglycerols in Healthy Subjects

Intake of fish and omega-3 (n-3) fatty acids is associated with a reduced concentration of plasma triacylglycerols (TAG) but the mechanisms are not fully clarified. Stearoyl-CoA desaturase-1 (SCD1) activity, governing TAG synthesis, is affected by n-3 fatty acids. Peripheral blood mononuclear cells (PBMC) display expression of genes involved in lipid metabolism. The aim of the present study was to estimate whether intake of lean and fatty fish would influence n-3 fatty acids composition in plasma phospholipids (PL), serum TAG, 18:1n-9/18:0 ratio in plasma PL, as well as PBMC gene expression of SCD1 and fatty acid synthase (FAS). Healthy males and females (n = 30), aged 20–40, consumed either 150 g of cod, salmon, or potato (control) daily for 15 days. During intervention docosahexaenoic acid (DHA, 22:6n-3) increased in the cod group (P < 0.05), while TAG concentration decreased (P < 0.05). In the salmon group both eicosapentaenoic acid (EPA, 20:5n-3) and DHA increased (P < 0.05) whereas TAG concentration and the 18:1n-9/18:0 ratio decreased (P < 0.05). Reduction of the 18:1n-9/18:0 ratio was associated with a corresponding lowering of TAG (P < 0.05) and an increase in EPA and DHA (P < 0.05). The mRNA levels of SCD1 and FAS in PBMC were not significantly altered after intake of cod or salmon when compared with the control group. In conclusion, both lean and fatty fish may lower TAG, possibly by reducing the 18:1n-9/18:0 ratio related to allosteric inhibition of SCD1 activity, rather than by influencing the synthesis of enzyme protein.     

Eicosapentaenoic acid is primarily responsible for hypotriglyceridemic effect of fish oil in humans

The aim of this study was to determine whether eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA), or both, were responsible for the triglyceride (TG)-lowering effects of fish oil. EPA (91% pure) and DHA (83% pure), a fish oil concentrate (FOC; 41% EPA and 23% DHA) and an olive oil (OO) placebo (all ethyl esters) were tested. A total of 49 normolipidemic subjects participated. Each subject was given placebo for 2–3 wk and one of the n-3 supplements for 3 wk in randomized, blinded trials. The target n-3 fatty acid (FA) intake was 3 g/day in all studies. Blood samples were drawn twice at the end of each supplementation phase and analyzed for lipids, lipoproteins, and phospholipid FA composition. In all groups, the phospholipid FA composition changed to reflect the n-3 FA given. On DHA supplementation, EPA levels increased to a small but significant extent, suggesting that some retroconversion may have occurred. EPA supplementation did not raise DHA levels, however, FOC and EPA produced significant decreases in both TG and very low density lipoprotein (VLDL) cholesterol (C) levels (P<0.01) and increases in low density lipoprotein (LDL) cholesterol levels (P<0.05). DHA supplementation did not affect cholesterol, triglyceride, VLDL, LDL, or high density lipoprotein (HDL) levels, but it did cause a significant increase in the HDL₂/HDL₃ cholesterol ratio. We conclude that EPA appears to be primarily responsible for TG-lowering (and LDL-C raising) effects of fish oil.     

Docosahexaenoic acid and other dietary polyunsaturated fatty acids suppress leukotriene synthesis by mouse peritoneal macrophages

The efficacy of individual ω-t-3 polyunsaturated fatty acids (PUFA) in altering eicosanoid synthesis in peritoneal macrophages was studied by feeding mice for 10 days a diet containing 2 wt% fat, which included 0.5 wt% ethyl esters of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) or linolenic acid (LNA). Upon stimulation with calcium ionophore A23187, macrophages from these animals produced significantly lower amounts of leukotriene C₄, leukotriene B₄ and 12-hydroxyeicosatetraenoic acid, prostaglandin E₂ and 6-keto prostaglandin F_1α compared with those obtained from animals on the diets containing olive oil or safflower oil. The decrease in leukotriene synthesis was similar in the animals fed DHA, EPA or LNA diets. This depression of eicosanoids by DHA and EPA was associated with decreased levels of arachidonic acid (AA); however, LA that altered eicosanoids did not have the same effect on AA levels.     

Eicosapentaenoic and arachidonic acid: Comparison of metabolism and activity in murine epidermal cells

The biological activity, including metabolism and modulation of ornithine decarboxylase activity and DNA synthesis, of arachidonic acid (AA) and eicosapentaenoic acid (EPA) were compared in epidermal cells from SENCAR mice. Radiolabelled AA and EPA were found to be similarly incorporated into and released from membrane phospholipids of unstimulated cultures. However, when cells were stimulated with the tumor promoter 12-0-tetradecanoylphorbol-13-acetate (TPA), the release of AA was significantly higher than the release of EPA. The extent of metabolism of AA and EPA to prostaglandins was determined in both freeze-thawed cell preparations and in viable cultured cells. In the freeze-thawed preparations, use of AA as a substrate resulted in significantly more PGF than when EPA was used as the substrate. However, more PGE₃ was formed than PGE₂. PGD levels were the same for either fatty acid precursor. Prostaglandin production was also determined in viable cultured cells since other influences such as phospholipase A₂ activity can modify prostaglandin production. Control cultures prelabelled with either AA or EPA produced similar amounts of the respective PGF, PGE, and PGD. However, TPA-stimulated cultures produced significantly higher amounts of each prostaglandin in cultures prelabelled with AA compared to cells prelabelled with EPA. HETE or HEPE production was the same both for cultured cells prelabelled with AA or EPA and for homogenates from uncultured cells incubated directly with the radiolabelled fatty acids. TPA-induced ornithine decarboxylase (ODC) was significantly higher in AA-treated cultures compared to EPA-treated cultures. AA supports DNA synthesis to a greater extent than EPA, either alone or in the presence of TPA. These findings suggest that AA and EPA do not have equivalent biological activity in mouse epidermal cells.     

Arachidonic acid to eicosapentaenoic acid ratio in blood correlates positively with clinical symptoms of depression

In this study of 20 moderately to severely depressed patients, diagnosed using current research diagnostic criteria and excluding known bipolar affective disorder and reactive depression, we investigated relationships between severity of depression and levels and ratios of n-3 and n-6 long-chain polyunsaturated fatty acids (PUFA) in plasma and erythrocyte phospholipids (PL). Severity of depression was measured using the 21-item Hamilton depression rating scale (HRS) and a second linear rating scale (LRS) of severity of depressive symptoms that omitted anxiety symptoms. There was a significant correlation between the ratio of erythrocyte PL arachidonic acid (AA) to eicosapentaenoic acid (EPA) and severity of depression as rated by the HRS (P<0.05) and the LRS for depression (P< 0.01). There was also a significant negative correlation between erythrocyte EPA and the LRS (P<0.05). The AA/EPA ratio in plasma PL and the ratio of erythrocyte long-chain (C₂₀ and C₂₂ carbon) n-6 to long-chain n-3 PUFA were also significantly correlated with the LRS (P<0.05). These findings do not appear to be simply explained by differences in dietary intake of EPA. We cannot determine whether the high ratios of AA/EPA in both plasma and erythrocyte PL are the result of depression or whether tissue PUFA change predate the depressive symptoms. We suggest, however, that our findings provide a basis for studying the effect of the nutritional supplementation of depressed subjects, aimed at reducing the AA/EPA ratio in tissues and severity of depression.     

Lipase-assisted concentration of n-3 polyunsaturated fatty acids in acylglycerols from marine oils

Preparation of n-3 polyunsaturated fatty acid (PUFA) concentrates from seal blubber oil (SBO) and menhaden oil (MHO) in the form of acylglycerols was carried out by hydrolysis with a number of commercial microbial lipases. The lipases tested were Aspergillus niger, Candida cylindracea (CC), Chromobacterium viscosum, Geotrichum candidum, Mucor miehei, Pseudomonas sp., Rhizopus oryzae, and Rhizopus niveus. After lipase-assisted hydrolysis of oils, free fatty acids were removed, and fatty acid composition of the mixture containing mono-, di-, and triacylglycerols was determined. All lipases were effective in increasing the n-3 PUFA content of the remaining acylglycerols of both SBO and MHO. The highest concentration of n-3 PUFA was provided by CC lipase; 43.5% in SBO [9.75% eicosapentaenoic acid (EPA), 8.61% docosapentaenoic acid (DPA), and 24.0% docosahexaenoic acid (DHA)] and 44.1% in MHO (18.5% EPA, 3.62% DPA, and 17.3% DHA) after 40 h of hydrolysis. Thus, CC lipase appears to be most suitable for preparation of n-3 PUFA in the acylglycerol form from marine oils.     

Regulation by long-chain fatty acids of the expression of cholesteryl ester transfer protein in HepG2 cells

Cholesteryl ester transfer protein (CETP) is an important determinant of lipoprotein function, especially high density lipoprotein (HDL) metabolism, and contributes to the regulation of plasma HDL levels. Since saturated and polyunsaturated fatty acids (FA) appear to influence the CETP activity differently, we decided to investigate the effects of FA on the expression of CETP mRNA in HepG2 cells using an RNA blot hybridization analysis. Long-chain FA (>18 carbons) at a 0.5 mM concentration were added to the medium and incubated with cells for 48 h at 37°C under 5% CO₂. After treatment with 0.5 mM arachidonic (AA), eicosapentaenoic (EPA), and docosahexaenoic acid (DHA), the levels of CETP mRNA were less than 50% of the control levels (AA, P=0.0005; EPA, P<0.01; DHA, P<0.0001), with a corresponding significant decrease in the CETP mass. These results suggest that FA regulate the gene expression of CETP in HepG2 and this effect is dependent upon the degree of unsaturation of the acyl carbon chain in FA.