Exogenous Eicosapentaenoic Acid Research Articles

Compartmental analyses of 2H5-α-linolenic acid and C-U-eicosapentaenoic acid toward synthesis of plasma labeled 22:6n−3 in newborn term infants

During early postnatal development, the nervous system accretes docosahexaenoic acid (DHA; 22:6n-3), a highly unsaturated n-3 (omega-3) fatty acid (FA) used in the formation of neural cell membranes. DHA, which is present in human breast milk, may also be biosynthesized from n-3 FAs such as 18:3n-3 [alpha-linolenic acid (ALA)] or 20:5n-3 [eicosapentaenoic acid (EPA)]. An important concern is to what extent these precursors can supply DHA to the developing infant. We analyzed measurements of fractional percentages of plasma (2)H(5)-ALA and (13)C-U-EPA directed toward the synthesis of labeled 22:6n-3 in 11 newborn infants by using compartmental modeling procedures. One-week-old infants received doses of (2)H(5)-ALA and (13)C-U-EPA ethyl esters enterally. We drew blood from the infants periodically and analyzed the plasma for endogenous and labeled n-3 FAs. From the time-course concentrations of the labeled FAs, we determined rate constant coefficients, fractional synthetic rates, and plasma turnover rates of n-3 FAs. In infants, approximately 0.04% of the (2)H(5)-ALA dose converted to plasma (2)H(5)-EPA. Plasma (2)H(5)-EPA and (2)H(5)-22:5n-3 [docosapentaenoic acid (DPA)] efficiently converted to (2)H(5)-DPA and (2)H(5)-DHA, respectively. The percentage of plasma (13)C-U-EPA directed toward the synthesis of (13)C-DHA was lower than the percentage of plasma (2)H(5)-EPA that originated from (2)H(5)-ALA. Endogenously synthesized EPA was efficiently converted to DHA. In comparison, preformed EPA was less efficiently used for DHA biosynthesis, which suggests a differential metabolism of endogenous EPA compared with exogenous EPA. However, on a per mole basis, preformed EPA was 3.6 times more effective toward DHA synthesis than was ALA. Newborns required an intake of approximately 5 mg preformed DHA. kg(-1) x d(-1) to maintain plasma DHA homeostasis.

Effect of arachidonic and eicosapentaenoic acid metabolism on RAW 264.7 macrophage proliferation

Prostaglandins (PGs) and leukotrienes (LTs) derived from arachidonic acid (AA) are potent mediators of inflammation and cell proliferation. Dietary intake of eicosapentaenoic acid (EPA) appears beneficial to both inflammatory processes and cell proliferation. However, there is no clear mechanism explaining these effects. In this study, we investigated the effect of EPA on the AA incorporation in phospholipid membranes, on AA release and metabolism, and consequently, on PG synthesis. Our results showed not only that [(3)H]AA and [(14)C]EPA were similar incorporated into RAW 264.7 macrophage membranes, but also that the redistribution pattern between phospholipids was alike. [(3)H]AA or [(14)C]EPA release was induced by fetal bovine serum (FBS) in a similar fashion with AA metabolizing 3-fold more than EPA. In this way, we observed that AA could be metabolized by cyclooxygenase (COX)-1, COX-2 and 5-lipoxygenase (5-LOX) whereas EPA was metabolized by COX-2 and 5-LOX pathways. Moreover, both fatty acids were able to induce COX-2 expression. When we incubated [(3)H]AA labeled cells with exogenous EPA, we observed that EPA did not modify FBS-induced [(3)H]AA release but that the presence of EPA decreased [(3)H]AA metabolism and therefore PGE(2) synthesis. Moreover, we studied the effect of AA and EPA metabolites on macrophage proliferation. Our results showed that PGE(3) stimulated cell growth with a potency similar to that of PGE(2), whereas LTB(5) was less effective than LTB(4). These data suggest that the effects of EPA on cell growth might be attributable, at least in part, to the marked decrease of eicosanoid release.

Screening for yeasts incorporating the exogenous eicosapentaenoic and docosahexaenoic acids from crude fish oil

The screening for yeasts incorporating exogenous eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) into their cellular lipids was conducted. Two percent of oil from fish scraps was added to a yeast isolation medium as a sole carbon source. From 143 soil samples, we isolated 23 yeast strains, nine of them were found to be capable of quickly assimilating the scrap fish oil. These nine strains, in addition to four previously isolated triglyceride-assimilating and lipase-producing strains, Yarrowia lipolytica and three Geotrichum species (FO274A, FO347-2 and FO401B), were cultured in a growth medium at 30°C. Strains FO726A, FO765A and FO347-2 were selected on the basis of dry cell weight production and ability to store EPA and DHA in their cells, and their performance was further compared by varying cultivation temperature and time. From 1 g of the scrap fish oil, FO726A yielded 620 mg of dry cells, containing 47.1% lipid, 38.1% triglyceride, 3.3% EPA, and 4.9% DHA, when cultured at 25°C for 36 h. Strain FO726A apparently has the highest ability to incorporate EPA and DHA into its cellular lipids. Results from further experiments showed that the incorporated EPA and DHA mainly existed in the form of triglyceride in the FO726A cell. These results suggest that FO726A is suitable for the production of cell mass rich in EPA and DHA for feed.

Differential effects of polyunsaturated fatty acids on chemosensitivity of NIH3T3 cells and its transformants

Polyunsaturated fatty acids (PUFAs) have been suggested, on the basis of animal-model studies, to be related not only to cancer development but also to chemotherapeutic effects. Controversy persists, however, as to which types of PUFAs are beneficial in terms of chemosensitivity. In this study, we used the NIH3T3 cell line and its SIC(sigmoid colon cancer)-oncogene transformants to investigate the effects of PUFAs on the chemosensitivity of non-malignant and malignant cells in terms of cell proliferation. We also determined the fatty-acid composition of cells by high-performance liquid chromatography (HPLC). The results revealed that the sensitivity of SIC transformants to mitomycin C (MC) was lower than that of NIH3T3 cells cultured in 10% calf-serum DMEM without PUFA supplementation. When cells were cultured in DMEM supplemented with eicosapentaenoic acid (EPA) at a concentration (2 μg/ml) that does not influence cell proliferation, the sensitivity of SIC transformants to MC increased, whereas that of NIH3T3 cells decreased in comparison with the sensitivity of cells cultured without PUFA supplementation (p < 0.05). There was no difference between the 2 cell lines in the chemosensitivity of cells cultured in medium supplemented with arachidonic acid (ARA). The SIC transformants contained more stearic acid (C:18) and less lauric acid (C:12) than NIH3T3 cells cultured without PUFA. Culturing the cells in medium supplemented with EPA or ARA modified the cellular fatty-acid composition. EPA caused the relative combined percentage of lauric acid and myristic acid (C:14) in SIC transformants to decrease significantly, and the SIC transformants tended to accumulate additional EPA, in contrast to the NIH3T3 cells. We conclude that the alterations in fatty-acid composition in malignant transformants caused by exogenous EPA differ from those in non-malignant cells, and that these changes account for the increased chemosensitivity of malignant transformants. Although preliminary, these findings imply that EPA specifically enhances the chemosensitivity of malignant cells. Int. J. Cancer, 70:357–361, 1997. © 1997 Wiley-Liss, Inc.

Differential effects of polyunsaturated fatty acids on chemosensitivity of NIH3T3 cells and its transformants.

Polyunsaturated fatty acids (PUFAs) have been suggested, on the basis of animal-model studies, to be related not only to cancer development but also to chemotherapeutic effects. Controversy persists, however, as to which types of PUFAs are beneficial in terms of chemosensitivity. In this study, we used the NIH3T3 cell line and its SIC(sigmoid colon cancer)-oncogene transformants to investigate the effects of PUFAs on the chemosensitivity of non-malignant and malignant cells in terms of cell proliferation. We also determined the fatty-acid composition of cells by high-performance liquid chromatography (HPLC). The results revealed that the sensitivity of SIC transformants to mitomycin C (MC) was lower than that of NIH3T3 cells cultured in 10% calf-serum DMEM without PUFA supplementation. When cells were cultured in DMEM supplemented with eicosapentaenoic acid (EPA) at a concentration (2 micrograms/ml) that does not influence cell proliferation, the sensitivity of SIC transformants to MC increased, whereas that of NIH3T3 cells decreased in comparison with the sensitivity of cells cultured without PUFA supplementation (p < 0.05). There was no difference between the 2 cell lines in the chemosensitivity of cells cultured in medium supplemented with arachidonic acid (ARA). The SIC transformants contained more stearic acid (C:18) and less lauric acid (C:12) than NIH3T3 cells cultured without PUFA. Culturing the cells in medium supplemented with EPA or ARA modified the cellular fatty-acid composition. EPA caused the relative combined percentage of lauric acid and myristic acid (C:14) in SIC transformants to decrease significantly, and the SIC transformants tended to accumulate additional EPA, in contrast to the NIH3T3 cells. We conclude that the alterations in fatty-acid composition in malignant transformants caused by exogenous EPA differ from those in non-malignant cells, and that these changes account for the increased chemosensitivity of malignant transformants. Although preliminary, these findings imply that EPA specifically enhances the chemosensitivity of malignant cells.

Effects of exogenous eicosapentaenoic acid on generation of leukotriene C4 and leukotriene C5 by calcium ionophore-activated human eosinophils in vitro.

Exogenous eicosapentaenoic acid (EPA) has been compared with exogenous arachidonic acid (AA) for its ability to modulate the oxidative metabolism of membrane-derived arachidonic acid by the 5-lipoxygenase pathway in ionophore-activated human eosinophils, and for its suitability as a parallel substrate in this pathway. Products were quantitated by specific RIA and tetraene and pentaene leukotrienes (LT) were separated by reverse-phase HPLC. Eosinophils were preincubated with control buffer, exogenous EPA or AA and stimulated optimally with 10 microM calcium ionophore (A23187) for 15 min. Mean generation of LTC4 in the absence of fatty acid was 6.0 = 1.1 ng/10(6) eosinophils (mean = SEM, n = 5). In the presence of EPA, the amount of LTC4 generated rose to peak at 16.5 +/- 1.9 ng/10(6) eosinophils at 10 micrograms/ml EPA and then fell to 8.3 +/- 3.1 ng/10(6) cells at 40 micrograms/ml EPA. The EPA derivative, LTC5 was first detectable at 5 micrograms/ml EPA with 4.8 +/- 1.2 ng/10(6) cells and gradually rose with increasing dose of EPA to be maximal at 40 micrograms/ml with 12.7 +/- 2.2 ng/10(6) cells. Identity of the LTC5 was confirmed by an identical retention time to synthetic LTC5 standard, immunoreactivity to a specific antibody against LTC4 and LTC5 and a typical UV absorbance spectrum. When eosinophils were preincubated with AA and similarly stimulated, LTC4 generation gradually increased from a baseline of 6.7 +/- 0.7 ng/10(6) cells in the absence of fatty acid to reach a maximum of 12.9 +/- 0.8 ng/10(6) cells at 40 micrograms/ml of AA. Total LTC generation was nearly twofold more with cells incubated with EPA than with cells incubated with AA (p < 0.05). Thus, EPA does not suppress LTC generation from eosinophils but stimulates it at lower doses and is a substrate for LTC5 generation.

Subhemolytic doses of Escherichia coli hemolysin evoke large quantities of lipoxygenase products in human neutrophils

Polymorphonuclear leukocytes (PMN) have been identified as preferred target cells for Escherichia coli hemolysin in human blood (Bhakdi, S., Greulich, S., Muhly, M., Eberspächer, B., Becker, H., Thiele, A., and Hugo, F. (1989) J. Exp. Med. 169, 737-754). Leukotriene and 5-hydroxyeicosatetraenoic acid generation was investigated in human PMN challenged with E. coli hemolysin in the absence or presence of free arachidonic acid or eicosapentaenoic acid (EPA). In the absence of exogenous free fatty acids, E. coli hemolysin (0.01-10 hemolytic units/ml) induced moderate generation of leukotriene B4 (LTB4) and its omega-oxidation products. The presence of free arachidonic acid (10 microM) during E. coli hemolysin (0.1 hemolytic unit/ml) challenge evoked the generation of large quantities of these products (greater than 100 pmol/1.5 x 10(7) PMN). In parallel, large amounts of 5-hydroxyeicosatetraenoic acid and nonenzymatic LTA4 hydrolysis products appeared. Product release peaked or plateaued 5-10 min after E. coli hemolysin challenge. The presence of exogenous EPA upon E. coli hemolysin challenge resulted in the exclusive generation of LTB5 and metabolites, LTA5 decay products and 5-hydroxyeicosapentaenoic acid. Dose and time dependences corresponded to those with arachidonic acid provision, and the total of EPA-derived products surpassed that of arachidonic acid metabolites in corresponding experiments approximately 2-fold. Increasing the time between free fatty acid provision and E. coli hemolysin challenge resulted in a rapid decline in the generation of arachidonic acid or EPA metabolites. Thus, subhemolytic doses of E. coli hemolysin evoke marked PMN eicosanoid generation that is dependent on exogenous free fatty acid supply, with total amounts approximating those found in calcium ionophore-stimulated neutrophils.

Effects of eicosapentaenoic acid and arachidonic acid on incorporation and metabolism of radioactive linoleic acid in cultured human fibroblasts

Effects of exogenous eicosapentaenoic acid, arachidonic acid and oleic acid on incorporation and metabolism of [ 14C] linoleic acid were examined in cultured human fibroblasts obtained from three donors of different ages. Eicosapentaenoic acid treatment (40 μM) inhibited incorporation of radioactive linoleic acid and actively reduced radioactivity of desaturation-elongation metabolites in phospholipids, predominantly in the phosphatidylethanolamine fraction. In contrast, radioactivities of the metabolites in triacylglycerols were significantly increased with arachidonic acid treatment (40 μM): eicosapentaenoic acid had a smaller effect or none. Oleic acid had virtually no effect. These effects were consistent in the three cell lines, but responses to treatment with the acids differed considerably among individual cells. The pool of linoleic acid metabolites in triacylglycerols may not be negligible. The exogenous fatty acids may influence both the transfer of lipids between the major lipid pools as well as the activities of the desaturation-elongation system.

Catabolism of leukotriene B5 in humans

Human neutrophils, enriched by dietary supplementation with eicosapentaenoic acid, form leukotriene (LT)B5 in addition to LTB4 upon stimulation. LTB5 is one order of magnitude less biologically active than the potent chemokinetic and chemoattractant LTB4. Catabolites of LTB5 have not yet been characterized in vitro and ex vivo. It is unknown whether catabolism of LTB5 interferes with catabolism of LTB4. This report describes catabolism of LTB5 to 20-OH-LTB5, which in turn is catabolized to 20-COOH-LTB5. The structures of the two catabolites were established by UV-absorbance, behavior on reverse-phase high-performance liquid chromatography, enzymatic analysis of human neutrophils, and gas chromatography-mass spectrometry. In vitro, formation of LTB4 was delayed and formation of its catabolites was depressed by exogenous eicosapentaenoic acid. By supplementing the diet of six volunteers with 5 g eicosapentaenoic acid/day for 7 days, eicosapentaenoic acid quadrupled in neutrophil phospholipid fatty acids. Consequently, LTB5, 20-OH-LTB5, and 20-COOH-LTB5 were detected ex vivo. In contrast to the findings in vitro, however, levels of LTB4, 20-OH-LTB4, and 20-COOH-LTB4 were unaltered by the dietary intervention. Thus, in vitro, but not ex vivo, addition of eicosapentaenoic acid, and subsequent formation of LTB5, impeded catabolism of proinflammatory LTB4.

ヒト単球におけるエイコサペンタエン酸の代謝 ５‐リポキシゲナーゼを中心に

Metabolism of eicosapentaenoic acid (EPA) and effect of EPA on arachidonic acid (AA) metabolism in human blood monocytes in vitro were examined. Highly purified monocytes were preparated by an improved method in which hypertonic treatment was employed. Metabolism of EPA in monocytes stimulated with calcium ionophore A 23187 was studied compared with that of AA. 5-lipoxygenase (5-LO) metabolites of AA or EPA were separated and quantitated by reverse phase-HPLC (RP-HPLC) . Production of leukotriene B4 (LTB4) and 5-hydroxyeicosatetraenoic acid (5-HETE), AA derived metabolites, were inhibited and formation of LTB5 and 5-hydroxyeicosapentaenoic acid (5-HEPE), EPA derived metabolites, were increased dose dependently by the addition of exogenous EPA (1.0-5.0μg/ml) in A 23187 stimulated monocytes.The current data indicate that EPA is a good substrate for 5-LO and is metabolized to LTB5 by A 23187 stimulation and also inhibits production of LTB4 in monocytes. It is well known that monocytes play an important role in inflammation and immune response, so that EPA might exert its antiinflammatory or immunomodulatory effect through the modulation of production of 5-LO metabolites especially LTB4 and LTB5 in monocytes.

Modulation of the contractile activity of the guinea-pig lung parenchymal strip by exogenous 5,8,11,14,17-eicosapentaenoic acid.

Exogenous eicosapentaenoic acid (EPA, 16.5 mumol/l or 33 mumol/l) inhibited dose-dependently the anaphylactic contractile response of guinea-pig lung parenchymal strips suspended in an organ bath. As determined by radioimmunoassay, EPA inhibited in a dose-dependent manner the anaphylactic release of the cyclooxygenase products thromboxane (TX) B2 and 6-keto-prostaglandin (PG) F1 alpha but simultaneously enhanced the release of sulfidopeptide (SP)-leukotrienes (LT). Indomethacin (2.8 mumol/l) abolished the release of cyclooxygenase products but potentiated the release of SP-LT. However, indomethacin treatment did not affect the inhibitory action of EPA on the contractile response of the anaphylactic lung strips. The lipoxygenase inhibitor, esculetin (50 mumol/l), inhibited the release of SP-LT and also that of cyclooxygenase products of polyunsaturated fatty acid metabolism. The combination of esculetin and EPA resulted in enhanced inhibition of the anaphylactic contractile response as compared to EPA alone. By reversed phase high pressure liquid chromatography (HPLC), SP-LT from anaphylactic lung parenchymal strips was shown to consist of LTD4 and LTE4. EPA-pretreated lung strips released upon immunologic challenge additional immunoreactivity comigrating with authentic LTC4, LTC5, LTD5 and LTE5. While anaphylactic control strips also released LTB4, in the bath fluid of EPA-treated strips, an additional immunoreactive compound migrating with the retention time of LTB5 was observed. In non-sensitized guinea-pig lung parenchymal strips EPA inhibited the myotropic activity of exogenous mediators such as histamine (9 mumol/l), LTC4 (16 nmol/l) and the TX mimetic U 46619 (28.4 nmol/l), an effect which was neither affected by indomethacin (2.8 mumol/l) nor by the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA, 10 mumol/l).(ABSTRACT TRUNCATED AT 250 WORDS)

PGI 3 production from eicosapentaenoic acid in 3T3 fibroblast cells and cultured bovine pulmonary artery endothelial cells

Production of PGI 3 from eicosapentaenoic acid (EPA) in 3T3 fibroblast cells and in cultured bovine pulmonary artery endothelial cells (CPAE) which had an ability to produce PGI 2 from arachidonic acid (AA), was investigated by bioassay, gas chromatography-mass spectrometry (GC-MS) and thin layer chromatography (TLC). Inhibition of platelet aggregation was observed in the supernatant obtained from a culture medium of 3T3 fibroblast cells after incubation with EPA. This active substance in the supernatant could be identified as PGI 3, since the inhibitory effect of the supernatant was blocked by tranylcypromine, a potent inhibitor of prostacyclin synthetase. The inhibitory effect on platelet aggregation by the supernatant from culture medium with EPA was low compared with that with AA. More Δ 17-6-keto PGF 1α from EPA was produced than 6-keto PGF 1α from AA at the same concentration of substrates, when the formation of prostacyclins from EPA and AA was measured as their stable metabolites, Δ 17-6-keto PGF 1α and 6-keto PGF 1α by GC-MS respectively. It is suggested that the anti-aggregatory ability of PGI 3 is lower than that of PGI 2. On the contrary, the amount of PGI 3 produced from EPA was significantly less than that of PGI 2 produced from AA in CPAE. The production of 6-keto PGF 1α from exogenous AA was decreased by increasing the ratio of EPA to AA in the culture medium and the production of Δ 17-6-keto PGF 1α from EPA also decreased by an addition of AA to the culture medium. This result suggests that AA and EPA compete each other at the site of action of cyclooxygenase. In aged CPAE (passage number 40), 6-keto PGF 1α production from exogenous AA decreased to 60%, while Δ 17-6-keto PGF 1α production from exogenous EPA increased to 170% of the levels produced with CPAE of passage number 18. There is a significant difference in the ability of PGI 3 production from EPA between in 3T3 fibrobrast cells in CPAE. Further studies on PGI 3 formation via the cyclooxygenase pathway are needed.

Effect of exogenous 5,8,11,14,17-eicosapentaenoic acid on cardiac anaphylaxis.

The effects of infusions of eicosapentaenoic acid (EPA) (6 X 10(-8) mol min-1 and 15 X 10(-8) mol min-1) on the coronary constriction and the release of immunoreactive sulphidopeptide-leukotrienes (SP-LT), thromboxane B2(TXB2) and 6-keto-prostaglandin F1 alpha (PGF1 alpha) from perfused anaphylactic guinea-pig hearts were investigated. EPA dose-dependently inhibited the profound early coronary flow reduction after antigen injection. The less pronounced late phase of anaphylactic coronary flow reduction was, however, not significantly affected. EPA (15 X 10(-8) mol min-1) significantly shortened the average duration of antigen-induced arrhythmias. EPA dose-dependently decreased release of immunoreactive TXB2 and 6-keto-PGF1 alpha from anaphylactic guinea-pig hearts. Release of immunoreactive SP-LT was dose-dependently increased after antigen challenge in the presence of EPA. Inhibiton of the release of SP-LT by the lipoxygenase inhibitor esculetin (1 X 10(-7) mol min-1) was accompanied by a significant attenuation of flow reduction during the late phase of anaphylactic vasoconstriction. Reversed phase h.p.l.c. of perfusates from anaphylactic guinea-pig hearts revealed immunoreactivity comigrating with authentic leukotriene C4 (LTC4), LTD4, and LTE4. In perfusates from hearts treated with EPA infusions, additional immunoreactivity was detected comigrating with LTC5, LTD5 and LTE5. In addition to immunoreactivity migrating with LTB4, as observed in control heart perfusates, in perfusates from EPA-treated hearts, a second peak was observed, which coincides with the retention time described for LTB5. Exogenous LTC5 (1 X 10(-12) mol min-1 and 20 X 10(-12) mol min-1) induced dose-dependent reductions of coronary flow and was found to be a slightly weaker constrictor than LTC4, but no significant differences were observed. Coronary vasoconstriction elicited by infusion of exogenous LTC4 (20 X 10(-12) mol min-1) was dose-dependently inhibited by infusions of EPA. However, the negative inotropic effect of LTC4 remained unaffected. Thus, in the isolated anaphylactic heart of the guinea-pig exogenous EPA was effectively metabolized via the 5-lipoxygenase pathway whereas the cyclo-oxygenase pathway of polyunsaturated fatty acid metabolism was found to be inhibited. The results are in agreement with the suggestion that cyclo-oxygenase products are mediators of the early phase of the anaphylactic coronary constriction, while vasoconstrictor SP-LT are involved in the later phase. However, in spite of enhanced release of SP-LT, EPA infusion did not result in increased coronary constriction. Considering the fact that EPA antagonizes LTC4-induced coronary constriction, it seems possible, that EPA might act as a functional antagonist of vasoconstrictor eicosanoids including EPA-derived SP-LT.

Effects of fatty acid modification of ascites tumor cells on pulmonary metastasis in rat.

Yoshida sarcoma cells were incubated with each of 4 different saturated and 17 different unsaturated fatty acid methyl and ethyl esters in order to modify the fatty acid composition of the cell membrane, and a possible correlation between the lipid fluidity of the cell membrane and the metastatic efficiency was studied. Almost all the unsaturated fatty acids used were incorporated into the tumor cell membrane, resulting in an increase of fluidity. In contrast, the incorporation of saturated fatty acids brought no change in fluidity. Exogenous treatment of the cells with palmitoleic acid (16:1, cis) induced a remarkable increase of membrane lipid fluidity resulting in easier passage of modified cells through capillary vessels, which was detected in an in vitro perfusion test of the lung. Intravenous injection of the modified cells resulted in fewer metastatic foci in the lung as compared with that of unmodified cells. A similar effect on lung metastasis was also observed in exogenous modification of the cells by linoleic acid (18:2, cis) or linolenic acid (18:3, n-3). On the other hand, although exogenous eicosapentaenoic acid (20:5, n-3) and docosahexaenoic acid (22:6, n-3) treatments induced a slight increase in the lipid fluidity of the cells, intravenous injection of the cells produced a significant increase in their metastatic potential in the lung. Interestingly, the treatment with 20:5 and 22:6 produced increased stickiness of the cells to a glass surface and reduced cellular passage through the vessels as detected by the lung perfusion test. Thus, the present results suggested that selective modification of membrane fatty acid may be a useful method for artificial regulation of the ability of circulating tumor cells to pass through vessels.

Effects of exogenous arachidonic, eicosapentaenoic, and docosahexaenoic acids on the generation of 5-lipoxygenase pathway products by ionophore-activated human neutrophils.

Exogenous eicosapentaenoic acid (EPA) and docosahexaenoic acid (DCHA) have been compared with exogenous arachidonic acid for their capacity to modulate the oxidative metabolism of membrane-derived arachidonic acid by the 5-lipoxygenase pathway in ionophore-activated human neutrophils and for their suitability as parallel substrates in this pathway. The products from specific 14C- or 3H-labeled substrates were isolated by reverse phase high performance liquid chromatography (RP-HPLC) and were identified by elution of radiolabel at the retention times of the appropriate synthetic standards. Each product was also characterized by its ultraviolet (UV) absorption spectrum, and 7-hydroxy-DCHA was defined in addition by analysis of its mass spectrum. The metabolites, 5-hydroxyeicosatetraenoic acid, leukotriene B4 (LTB4), 6-trans-LTB4 diastereoisomers, 5-hydroxyeicosapentaenoic acid, 6-trans-leukotriene B5 diastereoisomers, leukotriene B5 (LTB5), and 7-hydroxy-DCHA were quantitated by integrated UV absorbance during resolution by RP-HPLC. LTB4 and LTB5 were also quantitated by radioimmunoassay of the eluate fractions, and leukotrienes C4 and C5 (LTC4 and LTC5, respectively) were quantitated by radioimmunoassay alone. None of the unlabeled exogenous fatty acids (5-40 micrograms/ml) altered the release of radioactivity from [14C]arachidonic acid-labeled, ionophore-activated neutrophils. The metabolism of 5 and 10 micrograms/ml of exogenous EPA by ionophore-activated, [14C]arachidonic acid-labeled neutrophils not only generated 5-hydroxyeicosapentaenoic acid, 6-trans-LTB5, LTB5, and LTC5, but also stimulated the formation of 5-hydroxyeicosatetraenoic acid, 6-trans-LTB4 diastereoisomers, and LTC4 from membrane-derived arachidonic acid. In contrast, LTB4 production was diminished throughout the EPA dose-response, beginning at 5 micrograms/ml EPA and reaching 50% suppression at 10 micrograms/ml and 84% suppression at 40 micrograms/ml. The selective decrease in extracellular LTB4 concentrations in the presence of EPA was not due to a change in the kinetic appearance of LTB4 or to an increase in conversion to its omega-oxidation metabolites. DCHA was metabolized to 7-hydroxy-DCHA, did not stimulate metabolism of membrane-derived arachidonic acid, did not appreciably inhibit LTB4 formation, and was not a substrate for leukotriene formation. Incremental doses of exogenous arachidonic acid resulted in increased production of 5-hydroxyeicosatetraenoic acid and 6-trans-LTB4 by ionophore-activated, [14C]arachidonic acid-labeled neutrophils without any change in LTB4 production. 5-hydroxyeicosapentaenoic acid and 7-hydroxy DCHA were inactive as chemotactic factors whereas 5-hydroxyeicosatetraenoic acid exhibited 2% of the potency of LBT4. Thus, exogenous DCHA does not appreciably interfere with the metabolism of membrane-derived arachidonic acid by ionophore-activated, [14C]arachidonic acid-labeled neutrophils and is converted only to a monohydroxy derivative. In contrast, exogenous EPA attenuates the generation of LTB4 and is converted to LTB5, which is a weak and partial agonist as compared with LTB4.

Effects of the ratio of exogenous eicosapentaenoic acid to arachidonic acid on platelet aggregation and serotonin release

We added arachidonic acid (AA) and eicosapentaenoic acid (EPA) to washed platelet suspensions in the absence of albumin, holding the total amount of the fatty acids constant at 2 μM, and changing the ratio of EPA to AA. Platelet aggregation, serotonin release and the amount of thromboxane (TX) B 2, a cyclooxygenase product synthesized from exogenous AA, decreased as the ratio was increased. The decreases were greater than the expected ones from the diminution of the amount of exogenous AA. On the other hand, 12-hydroxyeico-satetraenoic acid (HETE), a lipoxygenase product synthesized from exogenous AA,increased in the presence of EPA. Although EPA was reported to be a poor substrate for platelet cyclooxygenase, the amount of TXB3 synthesized from exogenous EPA increased markedly by the simultaneous addition of AA. These results suggest that the EPA / AA ratio-dependent decrease in platelet aggregation and serotonin release is caused at least by both the decrease in the absolute amount of AA and the inhibitory effect of EPA on AA-metabolism via the cyclooxygenase pathway. Further studies on effects of EPA-metabolites via the cyclooxygenase pathway on platelet responses will be needed.