Eicosapentaenoic acid modulates arachidonic acid metabolism in rat alveolar macrophages activated by silica.

Eicosapentaenoic acid (EPA) was used to modulate the activation of alveolar macrophages, to examine its potential anti-inflammatory effect in addition to its anti-arteriosclerotic or anti-thrombotic effects. Wistar strain rat alveolar macrophages (2 x 10(6) cell) obtained by bronchoalveolar lavage were preincubated with EPA (0-20 microM), and further incubated with 1 mg of silica for 90 min. Leukotriene (LT) B4 and LTB5 of the supernatant were analyzed by reverse phase HPLC. EPA inhibited the production of LTB4 dose-dependently. The production of LTB5, a metabolite from EPA, was increased at low concentrations of EPA (0-10 microM) and decreased at high concentrations (>10 microM). These results suggest that EPA is competitive with arachidonic acid (AA) at low concentrations, and that EPA may inhibit AA metabolism via inhibition of 5-lipoxygenase or phospholipase A2 at high concentrations.     

Biosynthesis and biological activity of leukotriene B5

Several studies indicate that increased intake of eicosapentaenoic acid (EPA) in the diet may lead to decreased incidence of thrombotic events. Most investigators agree that this is achieved by competitively inhibiting the conversion of arachidonic acid (AA) to thromboxane A2 in the platelets. The effect of high EPA-intake on the formation of prostacyclin is less clear. However, EPA is a good substrate for lipoxygenase enzymes which results in formation of hydroperoxy- and hydroxy-acids, and, in some cases, leukotrienes. The biological activities of the leukotrienes derived from arachidonic acid suggest that they mediate or modulate some symptoms associated with inflammatory and hypersensitivity reactions. In order to clarify the possible effect of dietary manipulation on inflammatory processes, leukotriene B5 (LTB5) was prepared and its biological activities assessed. LTB5 was biosynthesized by incubation EPA with glycogen-elicited polymorphonuclear neutrophils (PMN) from rabbits in the presence of the divalent cation ionophore, A23187. The LTB5 was extracted from the incubate using mini-reverse phase extraction columns (Sep-pak) and purified by reverse-phase high pressure liquid chromatography (RP-HPLC). The purity of the product assessed by repeat RP-HPLC and straight phase (SP) HPLC was greater than 95%. Ultra-violet spectrophotometry of the product confirmed its purity and also provided assessment of the yield. The biological activity of LTB5 was assessed and compared with that of LTB4 in the following tests: aggregation of rat neutrophils, chemokinesis of human PMN, lysosomal enzyme release from human PMN and potentiation of bradykinin-induced plasma exudation. In all these tests, LTB5 was considerably less active (at least 30 times) than LTB4.     

Effect of dietary oils on the production of n-3 and n-6 metabolites of leukocyte 5-lipoxygenase in five rat strains

We examined the influence of various dietary oils, including linseed and fish oil on the relative rates of leukotriene B4 (LTB4) and LTB5 production by rat peritoneal exudate cells in five rat strains. While there was an association between the membrane phospholipid levels of the fatty acid precursors (arachidonic acid (AA) and eicosapentaenoic acid (EPA)) and the rate of synthesis of their respective 5-lipoxygenase products (LTB4 and LTB5), the rate of LTB4 synthesis was a combined function of both AA and EPA levels. We observed a strong linear relationship (correlation coefficient = 0.99) between the ratio of EPA/AA in the cell membrane phospholipids and the ratio of LTB5/LTB4 produced by these cells in vitro; this association was independent of genetic (strain) variability and was independent of the source of EPA (dietary EPA or EPA endogenously synthesized from dietary alpha-linolenic acid).     

Selective inhibition of leukotriene C4 synthesis and natural killer activity by ethacrynic acid

We have investigated the role of arachidonic acid (AA) metabolism in natural killer (NK) cell activity. Human nonadherent (NA) peripheral blood lymphocytes were used as effector cells against 51Cr-labeled K562 target cells. Synthesis of leukotriene C4 (LTC4) is dependent on glutathione S-transferase (GST). We have chosen to study three putative GST inhibitors, namely, ethacrynic acid (ET), caffeic acid (CA), and ferulic acid (FA), with regard to NK activity and with regard to their effect on AA metabolism. The GST inhibitors inhibited NK lysis when added directly to the NK assay. The GST inhibitors inhibited LTC4 synthesis as induced by calcium ionophore A23187 in a dose-dependent manner similar to their inhibition of NK activity. However, only ET was selective, for it had little effect on LTB4, 5-hydroxyeicosatetraenoic acid, and prostaglandin E2 synthesis. LTC4 synthesis was associated with the NK-enriched fractions obtained from discontinuous Percoll gradients. NK-specific anti-Leu-11b antibody and C treatment could abrogate NK activity and LTC4 synthesis. ET was also inhibitory when NA cells were cultured at 37 degrees C for 18 hr. In this case, LTC4 could reverse the inhibitory effect of ET. Our data suggest that LTC4 plays an important role in NK activity.     

E. coli hemolysin-induced lipid mediator metabolism in alveolar macrophages: impact of eicosapentaenoic acid

Escherichia coli hemolysin (HlyA) is a prototype of a large family of pore-forming proteinaceous exotoxins that have been implicated in the pathogenetic sequelae of severe infection and sepsis, including development of acute lung injury. In the present study in rabbit alveolar macrophages (AMs), subcytolytic concentrations of purified HlyA evoked rapid synthesis of platelet-activating factor, with quantities approaching those in response to maximum calcium ionophore challenge. In parallel, large quantities of leukotriene (LT) B(4) and 5-, 8-, 9-, 12-, and 15-hydroxyeicosatetraenoic acid (HETE) were liberated from HlyA-exposed AMs depending on exogenous arachidonic acid (AA) supply. Coadministration of eicosapentaenoic acid (EPA) dose dependently suppressed generation of the proinflammatory lipoxygenase products LTB(4) and 5-, 8-, 9-, and 12-HETE in parallel with the appearance of the corresponding EPA-derived metabolites LTB(5) and 5-, 8-, 9-, and 12-hydroxyeicosapentaenoic acid (HEPE). At equimolar concentrations, EPA turned out to be the preferred substrate over AA for these AM lipoxygenase pathways, with the sum of LTB(5) and 5-, 8-, 9-, and 12-HEPE surpassing the sum of LTB(4) and 5-, 8-, 9-, and 12-HETE by >80-fold. In contrast, coadminstration of EPA did not significantly reduce HlyA-elicited generation of the anti-inflammatory AA lipoxygenase product 15-HETE. We conclude that AMs are sensitive target cells for HlyA attack, resulting in marked proinflammatory lipid mediator synthesis. In the presence of EPA, lipoxygenase product formation is shifted from a pro- to an anti-inflammatory profile.     

Arachidonic acid and 15(S)-hydroxy-5,8,11-cis-13-trans-eicosatetraenoic acid modulate human polymorphonuclear neutrophil activation by monocyte derived neutrophil activating factor

Exposure of human polymorphonuclear neutrophils (PMN) to human monocyte derived neutrophil activating factor(s) (NAF) resulted in a concentration-dependent extracellular release of granule constituents. NAF also induced the generation of 5(S),12(R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid [Leukotriene B4 (LTB4)] by PMNs which was enhanced in the presence of exogenous arachidonic acid (AA). In contrast to its enhancing effect on LTB4 production, AA inhibited NAF-stimulated PMN degranulation. 15(S)-hydroxy-5,8,11-cis-13-trans-eicosatetraenoic acid (15-HETE), a product of the 15-lipoxy-genation of AA in PMNS, caused a concentration-dependent suppression of degranulation and LTB4 generation by PMNs in contact with NAF. 15-HETE also inhibited the rise in cytosolic-free calcium [( Ca2+]i) observed in NAF activated PMNs. These data suggest that AA and a 15-lipoxygenase product modulate the NAF-associated activation pathway in human PMNs.     

Incorporation of [3H]Arachidonic and [14C]Eicosapentaenoic Acids into Glycerophospholipids and Their Metabolism via Lipoxygenases in Isolated Brain Cells from Rainbow Trout Oncorhaynchus mykaiss

The incorporation of [3H]arachidonate [( 3H]AA) and [14C]eicosapentaenoate [( 14C]EPA) into glycerophospholipids was studied in isolated brain cells from rainbow trout, a teleost fish whose lipids are rich in (n-3) polyunsaturated fatty acids (PUFAs). EPA was incorporated into total lipid to a greater extent than AA, but the incorporation of both PUFAs into total glycerophospholipids was almost identical. The incorporation of both AA and EPA was greatest into phosphatidylethanolamine (PE). However, when expressed per milligram of individual phosphoglycerides, both AA and EPA were preferentially incorporated into phosphatidylinositol (PI), the preference being significantly greater with AA. On the same basis, significantly more EPA than AA was incorporated into phosphatidylcholine (PC). When double-labelled cells were challenged with calcium ionophore A23187, the 3H and 14C released from the cells closely paralleled each other, peaking at 10 min after addition of ionophore. The 12-monohydroxylated derivative was the pre-dominant lipoxygenase product from both AA and EPA with a rank order of 12-hydroxyeicosatetraenoic acid (12-HETE) greater than leukotriene B4 (LTB4) greater than 5-HETE greater than 15-HETE for the AA products and 12-hydroxyeicosapentaenoic acid (12-HEPE) greater than 5-HEPE greater than LTB5 greater than 15 HEPE for EPA products. The 3H/14C (dpm/dpm) ratios in the glycerophospholipids, total released radioactivity, and the lipoxygenase products suggested that PC rather than PI was the likely source of eicosanoid precursors in trout brain cells.     

The effect of dietary eicosapentaenoic acid on arachidonic acid incorporation and metabolism in rat leukocytes

Arachidonic Acid (AA) released from membrane phospholipids by phospholipase A2 during cell activation is the major polyunsaturated fatty acid precursor in mammals for the cyclooxygenase and lipoxygenase pathways. Eicosapentaenoic acid (EPA), a major polyunsaturated fatty acid in fish oils competes with AA for these enzymes. The resulting products from EPA are generally less potent than the corresponding AA metabolites which may explain the beneficial effects of this oil in reducing thrombotic and inflammatory responses. This study compares the incorporation of 14C-AA into leukocyte phospholipids and its release and metabolism by the cyclooxygenase and lipoxygenase pathways in rats fed a 'Max EPA' fish oil rich diet (EPA group) and a hydrogenated coconut/safflower oil control diet. More than 75% of radiolabel was incorporated into leukocytes with no difference seen between dietary groups. Upon stimulation with calcium ionophore, the EPA group released significantly more radiolabelled AA than the control group. The EPA diet showed a significant increase in the formation of 5-hydroxyeicosatetraenoic acid and 6-keto-prostaglandin F1 alpha but no difference was seen in leukotriene B4 formation. The majority of radiolabel released was free AA, this being significantly higher in the EPA group than in the control. The percentage of radiolabel remaining after stimulation in phosphatidylglycerol, phosphatidylethanolamine and neutral lipids was significantly less in EPA fed rats. As the release and metabolism of endogenous AA may not be the same as 14C-AA, these results do not necessarily indicate that the mass of AA available for eicosanoid biosynthesis has been altered by the EPA diet.     

Inhibition of natural killer cell activity of human lymphocytes by eicosapentaenoic acid

The effect of eicosapentaenoic acid (EPA) on natural killer (NK) cell activity of human lymphocytes was examined. The addition of an emulsion of trieicosapentaenoyl-glycerol (EPA-TG) emulsified with purified phosphatidylcholine from krill to a cytotoxicity assay system resulted in a marked depression of NK activity. The inhibition was proportional to the concentration of EPA-TG emulsion, and was observed as early as the first one hour of incubation at various effector to target cell ratios. Pretreatment of effector cells with EPA-TG emulsion resulted in significant suppression of their NK activity. Inhibition of cytotoxicity was not due to direct toxicity to effector cells or decreased target cell binding. These results indicate that EPA is a potent inhibitor of NK activity in vitro.     

Inhibition by dietary tea polyphenols of chemical mediator release from rat peritoneal exudate cells

Male Wistar rats were given purified diets containing safflower (SAF), perilla (PER), or palm (PAL) oils with or without 1% tea polyphenols (TP) for 3 weeks, and chemical mediator releasing activity from rat peritoneal exudate cells (PEC) was measured. Histamine releasing activity was not influenced by TP, while histamine release and intracellular histamine content were significantly increased in the PAL-fed group. On the contrary, leukotriene B4 (LTB4) release was significantly lower in rats fed PER than in those fed SAF and PAL, and TP significantly decreased the release in all fat groups. TP also significantly inhibited the release of LTB5, which was generated only in rats fed PER. TP significantly decreased the proportion of arachidonic acid (AA) in PEC in the SAF-fed group and that of eicosapentaenoic acid (EPA), the precursor of LTB5 in the PER-fed group, but did not influence that of AA in the PAL- and PER-fed group. These results suggest that ingestion of TP improves type I allergic symptom through the inhibition of LT release though the inhibition by TP could not be totally explained by the reduction of substrate fatty acid.     

Products of the lipoxygenase pathway in human natural killer cell cytotoxicity

As earlier data suggested the importance of lipoxygenase activation for expression of human NK cell cytotoxicity, four different lipoxygenase inhibitors were tested for suppression of natural killer (NK) cell lysis. All inhibitors were found active at nontoxic concentrations with 50% inhibition at approximately 15 microM for nordihydroguaiaretic acid (NDGA). NK cell lysis could be reconstituted to NDGA-suppressed cells with leukotriene B4 (LTB4), the all-trans isomers 6-trans-LTB4 and 12-epi-6-trans-LTB4, and 20-COOH-LTB4. LTB4 reconstitution was best in the concentration range 1-100 pM and near control levels at both higher and lower concentrations. Herpesvirus Ateles-transformed killer T cells could also be inhibited by NDGA. These data indicate that lipoxygenase activity is required for human NK cell lysis and that several different LTB4-related products can restore NK activity in inhibited cells; they also suggest that the lipoxygenase pathway is present in the killer cell population.     

The effect of dietary eicosapentaenoic acid on arachidonic acid incorporation and metabolism in rat leukocytes

Arachidonic Acid (AA) released from membrane phospholipids by phospholipase A₂ during cell activation is the major polyunsaturated fatty acid precursor in mammals for the cyclooxygenase and lipoxygenase pathways. Eicosaspentaenoic acid (EPA), a major polyunsaturated fatty acid in fish oils competes with AA for these enzymes. The resulting products from EPa are generally less potent than the corresponding AA metabolites which may explain the beneficial effects of this oil in reducing thrombotic and inflammatory responses. This study compares the incorporation of ¹⁴C-AA into leukocyte phospholipids and its release and metabolism by the cyclooxygenase and lipoxygenase pathways in rats fed a ‘Max EPA’ fish oil rich diet (EPA group) and a hydrogenated coconut/safflower oil control diet. More than 75% of radiolabel was incorporated into leukocytes with no difference seen between dietary groups. Upon stimulation with calcium ionophore, the EPA group released significantly more radiolabelled AA than the control group. The EPA diet showed a significant increase in the formation of 5-hydroxyeicosatetraenoic acid and 6-keto-prostaglandin F_1α but no difference was seen in leukotriene B₄ formation. The majority of radiolabel released was free AA, this being significantly higher in the EPA grou than in the control. The percentage of radiolabel remaining after stimulation in phosphatidylglycerol, phosphatidylethanolamine and neutral lipids was significantly less in EPA fed rats. As the release and metabolism of endogenous AA may not be the same as ¹⁴C-AA, these results do not necessarily indicate that the mass of AA available for eicosanoid biosynthesis has been altered by the EPA diet.     

Stimulation of eicosapentaenoic acid metabolism in washed human platelets by 12-hydroperoxyeicosatetraenoic acid

Washed human platelets were not able to convert eicosapentaenoic acid (EPA) to thromboxane B3 (TXB3) and 12-hydroxyeicosapentaenoic acid (AA) to washed human platelets induced conversion of EPA to TXB3 and 12-HEPE. Esculetin, a specific inhibitor of 12-lipoxygenase, prevented the effect of AA, but cyclooxygenase inhibitor did not. The conversion of AA to TXB2 was not affected by the same dose of esculetin. These data suggest that products of AA formed by 12-lipoxygenase in human platelets have stimulatory effects on EPA metabolism. When AA was preincubated with washed human platelets, its effect on EPA conversion was reduced, suggesting that a labile product of AA formed by 12-lipoxygenase is involved in the facilitation of EPA metabolism. Addition of 12-hydroperoxyeicosatetraenoic acid directly to washed human platelets caused dose-dependent synthesis of TXB3 and 12-HEPE, while addition of 12-hydroxyeicosatetraenoic acid had no effect. Thus, 12-hydroperoxyeicosatetraenoic acid formed from AA promotes the metabolism of EPA in washed human platelets.     

Dietary effects of eicosapentaenoic and docosahexaenoic acid esters on lipid metabolism and immune parameters in Sprague-Dawley rats

Sprague-Dawley rats were fed eicosapentaenoic (EPA) and docosahexaenoic acid (DHA) ethyl esters at the 2% level for 3 weeks to clarify their effects on immune functions. In the rats fed EPA or DHA, serum cholesterol, triglyceride, and phospholipid (PL) levels were significantly lower than those in the rats fed safflower oil. In PL fractions of serum, liver, lung, splenocytes, and peritoneal exudate cells (PEC), increases in linoleic and dihomo-gamma-linolenic acid contents and a decrease in arachidonic acid (AA) content were observed in the rats fed EPA or DHA. In addition, the EPA content increased in the rats fed EPA and DHA. In the rats fed EPA or DHA, a decrease of LTB4 productivity and an increase of LTBs productivity were observed in the PEC, in response to the treatment with 5 microM calcium ionophore A23187 for 20 min. The changes in leukotriene production were more marked in EPA-fed rats than in DHA-fed rats. These results suggest that dietary EPA affects lipid metabolism and leukotriene synthesis more strongly than DHA.     

Dietary effect of EPA-rich and DHA-rich fish oils on the immune function of Sprague-Dawley rats

The dietary effect of fish oils (FOs) rich in eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) on the immune function of Sprague-Dawley rats was compared with that of safflower oil. After 3 weeks of feeding at the 10% level of a dietary fat, the IgG and IgM production by splenocytes and IgG production by mesenteric lymph node (MLN) lymphocytes were significantly higher in the FO-fed rats, while no significant difference was found in IgA or IgE productivity by both the spleen and MLN lymphocytes. In the FO-fed rats, peritoneal exudate cells released a lower amount of LTB4, reflecting their lower arachidonic acid level, and a higher amount of LTB5, reflecting their higher EPA level in phospholipids. On these EPA-rich FO exerted a stronger effect than DHA-rich FO immune functions.     

Effect of supplementation of arachidonic acid (AA) or a combination of AA plus docosahexaenoic acid on breastmilk fatty acid composition

We investigated whether supplementation with arachidonic acid (20:4 omega 6; AA), or a combination of AA and docosahexaenoic acid (22:6 omega 3; DHA) would affect human milk polyunsaturated fatty acid (PUFA) composition. Ten women were daily supplemented with 300 mg AA, eight with 300 mg AA, 110 mg eicosapentaenoic acid (20:5 omega 3; EPA) and 400 mg DHA, for one week and eight women served as unsupplemented controls. Milk samples were collected on days 0, 1 and 7. The fatty acid composition of the milk was analyzed by capillary gas chromatography with flame ionisation detection. Supplementation with AA alone had no effect on breastmilk AA, but tended to reduce EPA and DHA levels. Administration of a combination of AA, EPA and DHA tended to increase both milk AA and long chain PUFA (LCPUFA)omega 3 content. A larger simultaneous increase of milk AA, DHA and EPA than observed in the present study can probably be accomplished by the use of a combination of a lower LCPUFA omega 6/LCPUFA omega 3 ratio and higher AA, EPA and DHA dosages.     

The effect of calcium ionophore A23187 on the metabolism of arachidonic and eicosapentaenoic acids in neutrophils from a marine teleost fish rich in (n-3) polyunsaturated fatty acids

1. [1-14C]AA and [1-14C]EPA were incorporated equally into plaice neutrophil phospholipids. 2. Incubation with A23187 resulted in the loss of label from total phospholipids and increased label released from the neutrophils. 3. Labelled LTB4 and LTB5 production was increased 2.5- and 3-fold, respectively, by A23187 treatment. 4. However, the incorporated AA was generally more metabolically active than the incorporated EPA with approx. 3-4 times as much LTB4 produced than LTB5 by cells in the presence or absence of A23187, respectively. 5. The results obtained in this (n-3) PUFA-rich species were discussed in comparison with current knowledge of AA and EPA metabolism in mammalian neutrophils.     

Acute and chronic effects of eicosapentaenoic acid on voltage-gated sodium channel expressed in cultured human bronchial smooth muscle cells

This study investigated acute and chronic effects of eicosapentaenoic acid (EPA) on voltage-gated Na+ current (I(Na)) expressed in cultured human bronchial smooth muscle cells (hBSMCs). The whole-cell voltage clamp technique and quantitative real-time RT-PCR analysis were applied. The alterations in the fatty acid composition of phospholipids after treatment with EPA were also examined. Extracellular application of EPA produced a rapid and concentration-dependent suppression of tetrodotoxin-sensitive I(Na) with the half-maximal inhibitory concentration of 2 microM. After washing out EPA with albumin, I(Na) returned to the control level. Similar inhibitory effects were observed regarding other fatty acids (docosahexaenoic, arachidonic, stearic, and oleic acids), but EPA was the most potent inhibitor. The effect of EPA on I(Na) was not blocked by nordihydroguaiaretic acid and indometacin, and was accompanied by a significant shift of the steady-state inactivation curve to more negative potentials. In cells chronically treated with EPA, the EPA content of the cell lipid fraction (mol%) increased time-dependently, while arachidonic acid (AA) decreased, resulting in an increase of EPA to AA ratio. Then, the level of mRNA (SCN9A) encoding I(Na) decreased significantly. These results provide novel evidence that EPA not only rapidly inhibits I(Na), but also reduces the mRNA levels of the Na+ channel after cellular incorporation of EPA in cultured hBSMCs.     

Arachidonic acid strongly stimulates prostaglandin I3 (PGI3) production from eicosapentaenoic acid in human endothelial cells

Eicosapentaenoic acid (EPA) is a prominent polyunsaturated fatty acid in fish oil which inhibits blood platelet aggregation and thromboxane A2 formation but not prostacyclin-like material generation from vascular endothelium. In this study we investigated interaction between EPA and arachidonic acid (AA) during their oxygenation by cultured endothelial cells. As measured by gas chromatography-mass spectrometry (GC-MS), AA increased markedly prostaglandin I3 (PGI3) production from EPA while that of PGI2 from AA was decreased by EPA. However, increasing the ratio AA/EPA over one almost suppressed the inhibition of PGI2 formation by EPA, and the stimulation of PGI3 production by AA was even higher. The effect of AA on EPA conversion to minor prostaglandins like PGE3 and PGF3 alpha was similar then confirming the stimulating effect and suggesting it is occurring at the cyclooxygenase instead of the prostacyclin synthase level. Altogether these data indicate that, in certain nutritional states where the liberation of EPA from endothelial cells will be accompanied with that of endogenous AA, substantial amounts of PGI3 could contribute to the prostacyclin-like activity of the vessel wall in addition to PGI2.     

Effects of lipoxygenase metabolites of arachidonic acid on the growth of human mononuclear marrow cells and marrow stromal cell cultures.

The effects of various lipoxygenase metabolites of arachidonic acid (AA) were investigated on the growth of freshly isolated human bone marrow mononuclear cells and marrow stromal cell cultures. LTB4, LXA4, LXB4, 12-HETE and 15-HETE (1 microM) decreased [3H]-thymidine incorporation on marrow stromal cell cultures without affecting cell number. Only 12-HETE showed a dose-response effect on [3H]-thymidine incorporation. While LTB4 (1 microM) decreased thymidine incorporation on marrow mononuclear cells, LTC4, LXA4, LXB4, 12-HETE and 15-HETE had no effect. The lipoxygenase inhibitor NDGA had no effect on both cell types suggesting no role of endogenous lipoxygenase metabolites on cell growth. These results suggest no important role of lipoxygenase metabolites of AA on the proliferation of human marrow mononuclear cells and marrow stromal cell cultures.