Polyunsaturated fatty acids exert antiarrhythmic actions as free acids rather than in phospholipids

Previous studies have shown that exogenous free n-3 polyunsaturated fatty acids (PUFA) can prevent tachyarrhythmias caused by specific agents in isolated cardiac myocytes. However, the question as to whether incorporation of the n-3 PUFA into membrane phospholipids has the same immediate protective effects remained to be answered. To answer this question, we increased the content of n-3 PUFA in the phospholipids of cultured neonatal rat myocytes by growing them 2-3 d in a culture to which eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) in 15 microM concentration was added. Analysis of the fatty acid composition of membrane phospholipids revealed a significantly higher level of EPA and DHA (from 0.2 to 7.6% and from 1.2 to 6.5%) in cells supplemented with EPA or DHA, respectively. The responses of the myocytes grown in normal media or in media enriched with the PUFA to arrhythmogenic agents were examined after free fatty acids were removed from the medium and the cells. The arrhythmogenic agents used were the beta-adrenergic agonist isoproterenol or an elevated extracellular concentration of calcium. The results showed that there was no significant difference in the induction of tachyarrhythmias by isoproterenol or by elevated [Ca2+]o in cells grown in media enriched with PUFA, as compared with cells grown in normal media in the absence of the free PUFA. Under the conditions of this study, only the unesterified PUFA were able to protect the cardiomyocytes against induced arrhythmias. There was no antiarrhythmic effect due to an increased fraction of EPA or DHA in membrane phospholipids.     

Effect of medium-term supplementation with a moderate dose of n-3 polyunsaturated fatty acids on blood pressure in mild hypertensive patients

Several studies have shown that n-3 polyunsaturated fatty acids (n-3 PUFA) are able to lower blood pressure (BP) in humans, but large doses of fish oils have been often used. Moreover, most of the studies available in the literature were not able to evaluate the specific effects of n-3 PUFA because they employed fish oils which contain, together with n-3 PUFA, many other different components. The aim of this preliminary study was to evaluate if medium-term supplementation with a moderate dose of highly purified eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) ethyl esters is able to reduce BP in mild hypertensive patients. Sixteen mild essential hypertensive (diastolic BP: 95-104 mm Hg), non-diabetic, normolipidemic male outpatients and 16 normotensive male controls were recruited to participate in the study. Both hypertensive and control subjects were randomly assigned to receive either EPA and DHA ethyl esters (2.04 g EPA and 1.4 g DHA) as active treatment or olive oil (4 g/day) as a placebo for a period of 4 months. These subjects were followed up with 24-hour ambulatory BP monitoring and blood chemistry analyses at 2 and 4 months of treatment and 2 months after its discontinuation. The intake of n-3 PUFA was checked by red blood cell (RBC) phosphatidylcholine (PC) fatty acid composition. The effect of n-3 PUFA on BP in the active group was maximum after 2 months. Both systolic (-6 mm Hg, p<0.05) and diastolic (-5 mm Hg, p<0.05) BP significantly decreased during the n-3 PUFA ethyl ester supplementation. No further effect was observed at 4 months with a return to baseline values during the recovery period. These data indicate that 4 g/day of highly purified EPA + DHA ethyl esters are able to favorably affect BP in mild hypertensives.     

A survey of blood component use in a German university hospital

In view of the promising future for use of n-3 polyunsaturated fatty acids (PUFA) in the prevention of cancer and cardiovascular diseases, it is necessary to ensure that their consumption does not result in detrimental oxidative effects. The aim of the present work was to test a hypothesis that low doses of eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) do not induce harmful modifications of oxidative cell metabolism, as modifications of membrane fatty acid composition occur. Wistar rats received by gavage oleic acid, EPA, or DHA (360 mg/kg body weight/day) for a period of 1 or 4 wk. Fatty acid composition and alpha-tocopherol content were determined for plasma, red blood cell (RBC) membranes, and liver, kidney, lung, and heart microsomal membranes. Susceptibility to oxidative stress induced by tert-butylhydroperoxide was measured in RBC. EPA treatment increased EPA and docosapentaenoic acid (DPA) content in plasma and in all the membranes studied. DHA treatment mainly increased DHA content. Both treatments decreased arachidonic acid content and n-6/n-3 PUFA ratio in the membranes, without modifying the Unsaturation Index. No changes in tissue alpha-tocopherol content and in RBC susceptibility to oxidative stress were induced by either EPA or DHA treatment. The data suggest that EPA and DHA treatments can substantially modify membrane fatty acids, without increasing susceptibility to oxidative stress, when administered at low doses. This opens the possibility for use of low doses of n-3 PUFA for chemoprevention without risk of detrimental secondary effects.     

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 elcosapentaenoic 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 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.     

CHARGE association: an update and review for the primary pediatrician

A diet including 2-3 portions of fatty fish per week, which corresponds to the intake of 1.25 g EPA (20:5n-3) + DHA (22:6n-3) per day, has been officially recommended on the basis of epidemiological findings showing a beneficial role of these n-3 long-chain PUFA in the prevention of cardiovascular and inflammatory diseases. The parent fatty acid ALA (18:3n-3), found in vegetable oils such as flaxseed or rapeseed oil, is used by the human organism partly as a source of energy, partly as a precursor of the metabolites, but the degree of conversion appears to be unreliable and restricted. More specifically, most studies in humans have shown that whereas a certain, though restricted, conversion of high doses of ALA to EPA occurs, conversion to DHA is severely restricted. The use of ALA labelled with radioisotopes suggested that with a background diet high in saturated fat conversion to long-chain metabolites is approximately 6% for EPA and 3.8% for DHA. With a diet rich in n-6 PUFA, conversion is reduced by 40 to 50%. It is thus reasonable to observe an n-6/n-3 PUFA ratio not exceeding 4-6. Restricted conversion to DHA may be critical since evidence has been increasing that this long-chain metabolite has an autonomous function, e.g. in the brain, retina and spermatozoa where it is the most prominent fatty acid. In neonates deficiency is associated with visual impairment, abnormalities in the electroretinogram and delayed cognitive development. In adults the potential role of DHA in neurological function still needs to be investigated in depth. Regarding cardiovascular risk factors DHA has been shown to reduce triglyceride concentrations. These findings indicate that future attention will have to focus on the adequate provision of DHA which can reliably be achieved only with the supply of the preformed long-chain metabolite.     

Differential accumulation and release of long-chain n-3 fatty acids from liver, muscle, and adipose tissue triacylglycerols

Eicosapentaenoic acid (EPA, 20:5n-3) is less efficiently accumulated in tissue triacylglycerols (TAGs) during fish oil feeding than docosahexaneoic acid (DHA, 22:6n-3) or docosapentaenoic acid (DPA, 22:5n-3), and EPA is preferentially released from the TAG of isolated adipocytes in vitro and adipose tissue in vivo during fasting compared with DHA or DPA. It is not known if this preferential release occurs in vivo under nonfasting conditions or if it is limited to adipose tissue. Accordingly, we have carried out experiments to study the turnover of EPA, DHA, and DPA in the TAG of adipose tissue, liver, and skeletal muscle. Weanling rats were fed diets containing fish oil for 6 weeks and then switched to diets containing only corn oil as the dietary fat for 8 weeks. The fatty acid composition and mass in epididymal fat pads, omental fat, liver, and soleus muscle TAGs were determined weekly for the first 10 weeks and at weeks 12 and 14. Subsequent to the change to the corn oil diet, EPA (20:5n-3), DPA (22:5n-3), and DHA (22:6n-3), which had accumulated during fish oil feeding, were lost from the tissue TAG pools of each tissue examined. After 8 weeks on the corn oil diet, less than 10% of the accumulated EPA, DPA, and DHA remained in the liver and muscle. The loss of EPA, DPA, and DHA from epididymal fat pad was slower. In each tissue, EPA was lost more rapidly than DPA or DHA. This selective loss of EPA relative to DHA or DPA may explain the previously reported underrepresentation of EPA compared with DHA or DPA in tissue TAG.     

The effect of highly purified eicosapentaenoic and docosahexaenoic acids on monocyte phagocytosis in man

The n-3 fatty acids (FA) from marine sources are known to exert antiinflammatory effects on monocyte function. There is still controversy whether n-3 FA may increase the susceptibility to infections. The present study was designed to assess the effect of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on monocyte phagocytosis and respiratory burst activity. Fifty-eight healthy men were randomized to take a daily supplement of 3.8 g highly purified EPA (n = 20), 3.6 g DHA (n = 19), or corn oil (n = 19) for 7 wk. Mononuclear leukocytes were collected, isolated, and cryopreserved prior to and after dietary supplementation. Paired samples were analyzed in the presence of autologous serum in a crossover design. Monocyte phagocytosis and respiratory burst activity were measured by flow cytometry after ingestion of Escherichia coli. Monocytes retained their phagocytic ability and respiratory burst activity after supplementation. No reduction in internalization of bacteria was registered. Dietary n-3 FA and particularly EPA improved bacterial adherence to the monocyte surface. In the crossover experiments, there was an adverse effect of serum enriched with n-3 FA on bacterial adherence. We conclude that monocytes retain their phagocytic potential after supplementation with purified EPA and DHA.     

High-throughput screening: advances in assay technologies

The expression and up-regulation of cell adhesion molecules on a human colonic epithelial cell line HT-29, and the peripheral blood T lymphocyte proliferation responses to bacterial superantigens presented by this cell line were investigated, compared with peripheral blood monocytes. In HT-29 cells, there was constitutive expression of intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function-associated antigen-3 (LFA-3) at a low level, but no constitutive expression of HLA-DR, LFA-1, B7-1 and B7-2 molecules. After stimulation with the supernatants of staphylococcal enterotoxin B (SEB)-stimulated peripheral blood mononuclear cells for 48 h, there was significant up-regulation of HLA-DR and ICAM-1 molecules (both > 90% positive). However, this stimulation had no effect on the expression of LFA-1, B7-1, B7-2 and LFA-3 molecules. In the presence of all tested superantigens SEB, toxic shock syndrome toxin-1, and streptococcal pyogenic exotoxin A, stimulated HT-29 cells caused significant T cell proliferation. When monocytes were used as antigen-presenting cells (APC), the MoAbs against HLA-DR, B7-2 and LFA-3 showed a significant inhibition of SEB-induced T cell proliferation. Anti-ICAM-1 MoAb had no effect on this response. On the other hand, when stimulated HT-29 cells were used as APC, the MoAbs against HLA-DR and ICAM-1 significantly inhibited SEB-induced T cell proliferation. In contrast to monocytes, anti-B7-2 and anti-LFA-3 had no effect on this response. SEB could not induce HT-29 cells to produce IL-8 directly; however, SEB significantly induced the stimulated HT-29 cells to produce IL-8 in the presence of T cells. Thus these data demonstrate that the products of superantigen-stimulated T cell activation can increase the expression of HLA-DR and ICAM-1 molecules on HT-29 cells significantly. Stimulated HT-29 cells can serve as APC to bacterial superantigens. This response is an HLA-DR- and ICAM-1-dependent, but B7-2- and LFA-3-independent process, which was different from professional APC monocytes.     

Arachidonic acid enhances the tissue factor expression of mononuclear cells by the cyclo-oxygenase-1 pathway: beneficial effect of n-3 fatty acids

Monocytes express tissue factor (TF) upon stimulation by inflammatory agents. Dietary administration of fish oil rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) results in an impairment of TF expression by monocytes. EPA and DHA are metabolized differently from arachidonic acid (AA), the major fatty acid present in cell membranes. We examined the effects of AA on the TF expression of isolated human PBMC, and we determined whether EPA and DHA modulated this phenomenon differently. Nonstimulated PBMC had a low TF-dependent procoagulant activity. When PBMC were incubated with increasing concentrations of AA, the TF-dependent procoagulant activity increased in a dose-dependent manner to 190% at 7.5 microM. Indomethacin, a cyclo-oxygenase inhibitor, totally abolished the stimulating effect of AA, whereas specific pharmacologic inhibitors of cyclo-oxygenase-2 or of 5-lipoxygenase had no inhibitory effect. A thromboxane (TX)A2/endoperoxides receptor antagonist and a TX synthase inhibitor blocked the potentiating effect of AA. Purified PGG2 and carbocyclic TXA2, a TXA2 agonist, enhanced the procoagulant activity of PBMC in a dose-dependent manner whereas, in contrast, PGE2 inhibited it. Finally, contrary to AA, EPA or DHA did not increase TXB2 production or TF expression by PBMC. The TF-dependent procoagulant activity of isolated PBMC was increased by AA through the production of cyclo-oxygenase-1 metabolites; the combined action of PGG2 and TXA2, which potentiated it, was greater than that of PGE2, which inhibited it. Dietary n-3 fatty acids exert part of their beneficial effect by modulating this procoagulant activity differently from AA.     

Comparison between dietary monounsaturated and polyunsaturated fatty acids as regards diet-related diseases

We have studied the effects of dietary fatty acid (FA) composition on lipids and lipoproteins, platelet function and other hemostatic variables as well as on the endogenous formation of DNA adducts of malonaldehyde (MA) in healthy subjects in controlled dietary experiments. The FAs studied were monounsaturated oleic acid (OA, 18:1 n-9), n-6-polyunsaturated linoleic acid (LA, 18:2 n-6), n-3 polyunsaturated alpha-linolenic acid (ALA, 18:3 n-3), and two long-chain, n-3 polyunsaturated FAs, eicosapentaenoic acid (EPA, 20:5 n-3) and docosahexaenoic acid (DHA, 22:6 n-3). The results indicated that a high OA and high LA diet had comparable effects on lipids and lipoproteins when they replaced saturated FAs in a diet. Furthermore, the effect of ALA did not differ from that of LA in this respect. Both diets also similarly increased in vitro platelet aggregation when compared with high saturated FA baseline diet. In another study the effect of LA and ALA on platelet function was studied. In this study ALA decreased in vitro platelet aggregation when compared with LA. When ALA was compared with EPA + DHA it was found that platelet function and some coagulation and fibrinolysis parameters were mainly affected in a similar manner by ALA and EPA + DHA treatments. The high LA diet increased the levels of DNA adducts of MA when compared with the effect of the high OA diet. Our findings indicate that the interpretation of the effect of diet, dietary fat or a specific FA on the development of chronic disease is extremely complex.     

Dietary modulation of phase 1 and phase 2 activities with benzo(a)pyrene and related compounds in the intestine but not the liver of the channel catfish, Ictalurus punctatus

EPA, DHA, C15SCH2COOH (n-3), C15SCH2COOH (n-6) and C18SCH2COOH (n-3) are extensively incorporated into phospholipids and triacylglycerol in rat hepatocytes after 24 h incubation with 80 microM fatty acid/derivative. Only traces of polyunsaturated 3-oxa fatty acids (C15OCH2COOH, C18OCH2COOH) were incorporated. C15-S-butyric acid (n-3) is a stronger inhibitor of delta6-desaturase in rat liver-microsomes than C15SCH2COOH (n-3), C15-S-propionic acid (n-3), EPA and DHA. It inhibits delta5-desaturase in a similar manner to EPA and DHA. Arachidonic acid and C15SCH2COOH, (n-6) are better substrates for PGH-synthase than EPA and C15SCH2COOH, (n-3), showing the inhibitory effect of the n-3 bond. The n-3 polyunsaturated fatty acids, including the sulfur-substituted fatty acid derivatives, are poor substrates for PGH-synthase. However, they inactivate the PGH-synthase activity at least as efficiently as arachidonic acid. C15SCH2COOH (n-3), C15S(CH2)2COOH (n-3) and C18SCH2COOH (n-3) induce peroxisomal beta-oxidation more than EPA and DHA.     

Effects of dietary docosahexaenoic acid on surface molecules involved in T cell proliferation

It is known that n-3 polyunsaturated fatty acids (PUFA) such as docosahexaenoic acid (DHA) suppress immunity as compared with n-6 PUFA such as linoleic acid (LA), but the mechanism involved in this phenomenon is still unclear. The present study was designed to assess the effect of dietary DHA on the surface molecules involved in T cell proliferation. Weanling male C57BL/6 mice were divided into four dietary groups that were fed a 10% fat diet for 4 weeks varying in amounts of DHA and LA. As the dietary DHA concentration increased, the surface expression of CD4 and CD8 on splenic T cells decreased, while that of CD28 increased. The surface expression of CD3, however, was invariable in all dietary groups. DNA synthesis of splenic T cells, induced by CD3 crosslinkage with anti-CD3 epsilon monoclonal antibody in the presence of CD28-mediated costimulation, increased as the DHA concentration was elevated. These observations suggest that diets rich in DHA exert some of their immunomodulatory effects by a downregulation of surface expression of CD4 and CD8 and by an upregulation of CD28-mediated costimulatory signal.     

Immunoregulatory and anti-inflammatory effects of n-3 polyunsaturated fatty acids

1. Fish oils are rich in the long-chain n-3 polyunsaturated fatty acids (PUFAs), eicosapentaenoic (20:5n-3) and docosahexaenoic (22:6n-3) acids. Linseed oil and green plant tissues are rich in the precursor fatty acid, alpha-linolenic acid (18:3n-3). Most vegetable oils are rich in the n-6 PUFA linoleic acid (18:2n-6), the precursor of arachidonic acid (20:4n-6). 2. Arachidonic acid-derived eicosanoids such as prostaglandin E2 are pro-inflammatory and regulate the functions of cells of the immune system. Consumption of fish oils leads to replacement of arachidonic acid in cell membranes by eicosapentaenoic acid. This changes the amount and alters the balance of eicosanoids produced. 3. Consumption of fish oils diminishes lymphocyte proliferation, T-cell-mediated cytotoxicity, natural killer cell activity, macrophage-mediated cytotoxicity, monocyte and neutrophil chemotaxis, major histocompatibility class II expression and antigen presentation, production of pro-inflammatory cytokines (interleukins 1 and 6, tumour necrosis factor) and adhesion molecule expression. 4. Feeding laboratory animals fish oil reduces acute and chronic inflammatory responses, improves survival to endotoxin and in models of autoimmunity and prolongs the survival of grafted organs. 5. Feeding fish oil reduces cell-mediated immune responses. 6. Fish oil supplementation may be clinically useful in acute and chronic inflammatory conditions and following transplantation. 7. n-3 PUFAs may exert their effects by modulating signal transduction and/or gene expression within inflammatory and immune cells.     

Deficit in prepulse inhibition in mice caused by dietary n-3 fatty acid deficiency.

Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) may be biosynthesized from a precursor α-linolenic acid (LNA) or obtained preformed in the diet. Dams were fed four diets with different levels of the various n-3 fatty acids during pregnancy and lactation, and their offspring were weaned to the same diets: “n-3 Deficient,” containing (as % total fatty acids) 0.07% of LNA; “Low LNA” (0.4%); “High LNA” (4.8%); and a “DHA + EPA” diet, containing 0.4% of LNA, 2% DHA, and 2% EPA. Sensorimotor gating was measured by prepulse inhibition (PPI) of the acoustic startle response in C57Bl6 mice. The n-3 Deficient and Low LNA diets caused a substantial deficit in PPI compared to the DHA + EPA diet, whereas the High LNA diet induced a less pronounced, but significant reduction of PPI. These are the first data that demonstrate a deficit in sensorimotor gating in rodents caused by an inadequate amount of the n-3 fatty acids in the diet. Our results differentiate the effects of a High LNA diet from one with added EPA and DHA even though the difference in brain DHA content is only 12% between these dietary groups. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Docosahexaenoic and eicosapentaenoic acids inhibit human lymphoproliferative responses in vitro but not the expression of T cell surface activation markers

The effects of polyunsaturated fatty acids (PUFAs: docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids) on induced lymphocyte proliferation and expression of CD25alpha chain of interleukin-2 receptor, CD71 and HLA-DR were investigated. PUFAs had no effect on phytohaemagglutinin (PHA)-induced lymphocyte agglutination, but they strongly inhibited the lymphoproliferative response to PHA. This inhibitory effect is PUFA dose-dependent and seems to be more potent with DHA than EPA, Pre-incubation experiments showed that lymphocytes cultured with PUFAs for 6 h then washed and exposed to PHA, still inhibited lymphocyte proliferation. The authors also showed that this inhibitory activity was time dependent but became nonsignificant when PUFAs were added after 48 h lymphocyte culture. The addition of excess exogenous human recombinant rIL-2 partly restored PHA-lymphocyte proliferation inhibited by EPA but not by DHA. On the other hand, the authors showed that PUFAS did not inhibit IL-2 stimulated lymphocyte proliferation. The addition of PUFAs to cell culture medium had no inhibitory action on the PHA-induced lymphocyte expression of CD25, CD71 and HLA-DR. Furthermore, this effect appeared independent of eicosanoid synthesis or peroxide formation. Indeed, the inclusion of aspirin and vitamin E in the culture medium did not prevent the inhibitory effects of PUFAs on lymphocyte proliferation. Regardless of the mechanism of action, the inhibitory effect of PUFAs on activated lymphocytes may explain why some clinical trials of fish oil supplemented diets containing high amounts of DHA and EPA have been successful in improving the health status of patients suffering from inflammatory and autoimmune disorders.     

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

To better understand the mode of action of omega 3 fatty acids in cell membranes, human foreskin fibroblasts were grown in serum-free medium supplemented with 50 microM 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.     

Low levels of eicosapentaenoic and docosahexaenoic acids mimic the effects of fish oil upon rat lymphocytes

Fish oil is rich in the long chain n-3 polyunsaturated fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA); typically these fatty acids constitute 20 to 25 g/100 g total fatty acids in fish oil. Feeding rodents diets rich in fish oil has been shown to decrease lymphocyte proliferation and natural killer cell activity. It is not known what level of EPA + DHA is required in the diet to exert these effects. This question was addressed in the current study. Weanling rats were fed on high fat (178 g/kg) diets which contained 4.4 g alpha-linolenic acid (control) or 4.4 g EPA + DHA (4.4 EPA + DHA) or 6.6 g EPA + DHA (6.6 EPA + DHA)/100 g total fatty acids. The n-6 to n-3 polyunsaturated fatty acid ratio was maintained at approximately 7. The fatty acid compositions of the serum and of spleen leukocytes were markedly influenced by that of the diet. Spleen lymphocyte proliferation in response to concanavalin A, spleen natural killer cell activity and PGE2 production by spleen leukocytes were reduced by feeding the EPA + DHA diets compared with feeding the control diet; the 4.4 and 6.6 EPA + DHA diets caused very similar reductions. The 4.4 EPA + DHA diet reduced popliteal lymph node weight following a localised graft versus host response; this response was not investigated in rats fed the 6.6 EPA + DHA diet. The reductions in lymphocyte functions and in the in vivo graft versus host response caused by the EPA + DHA diets were similar to those previously reported following the feeding of diets rich in fish oil. Thus, this study shows that diets containing relatively low levels of EPA + DHA (20 to 25% of the level found in fish oil) exert immunomodulatory effects. Furthermore, this study suggests that the maximal effect of EPA + DHA is exerted when these fatty acids constitute a level of less than or equal to 4.4 g/100 g total dietary fatty acids.     

Development of an in vitro model of essential fatty acid deficiency in fish cells

Levels of eicosapentaenoic acid (EPA; 20:5, n-3) greatly exceed those of arachidonic acid (AA; 20:4, n-6) in the tissue phospholipids of most fish species. Despite this, it is 20:4, n-6-derived eicosanoids that are produced predominantly in fish cells. The development of an essential fatty acid (EFA)-deficient fish cell line would greatly assist the study of this selectivity and so several fish cell lines were cultured in EFA-deficient (EFAD) media. All n-3 and n-6 polyunsaturated fatty acids (PUFA) and total PUFA were considerably reduced in all lines, except turbot fin (TF) in which total n-9 PUFA doubled from 13.8% to 27.5% of total fatty acids. In the topminnow hepatocarcinoma cell line (PLHC-1), there was almost complete depletion of both n-3 and n-6 PUFA and in TF cells, no n-3 PUFA were detected. In the carp epithelial papilloma cell line (EPC), both n-6 and n-3 PUFA were reduced by approximately 70%. The reduced PUFA in cells cultured in EFAD media was compensated to a large extent in most cell lines by significantly increased percentages of monounsaturated fatty acids, particularly 18:1, n-9. Total n-9 PUFA were significantly increased in all cell lines by culture in EFAD media, with 20:2, n-9 significantly increased in all cell lines. There were relatively small increases, but often significant, in 20:3, n-9 in all cell lines. Of the cell lines investigated, only EPC and PLHC-1 showed proliferation after four passages in EFAD medium, although the growth rates were reduced in comparison with media supplemented with serum, but EPC was the only cell line able to survive and proliferate in long-term culture on EFAD medium. The EFAD-EPC line is a potentially useful model system for the study of the effects of EFA deficiency on cell structure and function and eicosanoid metabolism in fish.