5-hydroxyeicosapentaenoic Acid Research Articles

Long-Term Administration of Highly Purified Eicosapentaenoic Acid Provides Improvement of Psoriasis

Nine patients with chronic stable psoriasis (4 males and 5 females) were entered in this trial. Eicosapentaenoic acid (EPA) ethylester (90% pure) without docosahexaenoic acid (DHA) in gelatin-coated capsules at a daily dose of 3.6 g was administered to 9 patients for 3 months, 7 patients for 6 months and 6 patients for 12 months. The clinical changes of skin lesions of the patients with 12 months of treatment were as follows: marked improvement 1, improvement 3, relative improvement 1, no change 1. A clinical improvement of skin lesions was first observed 2-3 months after EPA treatment. The supplementation of highly purified EPA caused a significant increase in the content of plasma EPA and docosapentaenoic acid without affecting that of arachidonic acid (AA) and DHA. EPA decreased the production of leukotriene B4 (LTB4) and increased the formation of leukotriene B5 (LTB5) and 5-hydroxyeicosapentaenoic acid significantly in A23187-stimulated neutrophils. The LTB5/LTB4 ratio positively correlated with the plasma EPA/AA ratio and was directionally related to the clinical score, although the directional data were not statistically significant. We could not observe any side effects of EPA over 1 year. Although its effects are modest, it is nontoxic and its favorable effect appears to continue for the duration of its usage, indicating that EPA could be beneficial for the long-term treatment of psoriasis.

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.

Production of lipoxygenase metabolites of eicosapentaenoic acid by bovine alveolar macrophages in vitro.

Lipoxygenase metabolites of arachidonic acid (AA), the leukotrienes (LTs), and hydroxyeicosatetraenoic acids (HETEs) are potent proinflammatory mediators. Release of LTs and HETEs by bovine alveolar macrophages (BAMs) was measured by reverse-phase high performance liquid chromatography. LTB4 (1.1 +/- 0.2 ng/10(6) cells) and 5-HETE (2.2 +/- 0.2 ng/10(6) cells) were the major metabolites calcium ionophore A23187-stimulated BAMs produced from endogenous AA. The tritiated forms of these compounds and their precursor fatty acids were produced following incorporation of [3H]AA into the cells and stimulation by calcium ionophore A23187. Incorporation of an alternative substrate, [3H]eicosapentaenoic acid [( 3H]EPA) into BAMs incubated in parallel resulted in production of [3H]LTB5 and [3H]5-hydroxyeicosapentaenoic acid (5-HEPE). Equivalent amounts of [3H]AA and [3H]EPA and of [3H]LTB4 and homologous [3H]LTB5 were released. BAM produced significantly greater amounts of [3H]5-HEPE than [3H]5-HETE, however. These findings indicate that the BAM 5-lipoxygenase is capable of metabolizing EPA to LTB5 and 5-HEPE, with the production of 5-HEPE preferred over 5-HETE.

Arachidonic and eicosapentaenoic acid metabolism in bovine neutrophils and platelets: effect of calcium ionophore.

Substitution of dietary fatty acids has potential for altering the inflammatory response. The purpose of the present study was to define the metabolites of arachidonic acid (AA) and eicosapentaenoic acid (EPA) secreted by bovine peripheral blood neutrophils and platelets. High performance liquid chromatography was used to characterize cyclooxygenase and lipoxygenase metabolites secreted in response to the calcium ionophore A23187. Cells were prelabelled with 3H-AA or 3H-EPA prior to challenge with the calcium ionophore. Bovine neutrophils secreted leukotriene B4 (LTB4) and 5-hydroxyeicosatetraenoic acid (5-HETE) as the major metabolites of AA, as well as the corresponding leukotriene B5 (LTB5) and 5-hydroxyeicosapentaenoic acid (5-HEPE) metabolites of EPA. Peptidoleukotrienes derived from 3H-AA or 3H-EPA were not detected under these conditions. The major tritiated metabolites secreted from bovine platelets were: thromboxane A2, measured as the stable metabolite thromboxane B2 (TXB2); hydroxyheptadecatrienoic acid (HHT) and 12-HETE derived from 3H-AA; and the omega-3 analogs TXB3 and 12-HEPE, derived from 3H-EPA. Preferred substrate specificities existed amongst the AA- and EPA-derived metabolites for the intermediary enzymes involved in the arachidonic acid cascade. These findings support the hypothesis that substitution of membrane-bound AA by EPA has potential for modulation of the host inflammatory response following cellular phospholipid mobilization.

Eggs of the sea urchin, Strongylocentrotus purpuratus, contain a prominent (11R) and (12R) lipoxygenase activity.

Recent work has shown that oocytes of the starfish synthesize (8R)-hydroxyeicosatetraenoic acid and that this eicosanoid has a potent and highly specific action in induction of oocyte maturation. These striking results prompted us to examine the lipoxygenase activity of eggs of the sea urchin Strongylocentrotus purpuratus. Four hydroxyeicosanoids were formed in homogenates of sea urchin eggs; their structures and stereochemistry were characterized by high pressure liquid chromatography, UV spectroscopy, and gas chromatography-mass spectrometry. The compounds were identified as (11R)-hydroxy-5,8,12,14-ZZEZ-eicosatetraenoic acid and (12R)-hydroxy-5,8,10,14-ZZEZ-eicosatetraenoic acid (from arachidonic acid) and the corresponding (11R)- and (12R)-hydroxy analogs of eicosapentaenoic acid. The formation of these egg products was not blocked by a cyclooxygenase inhibitor, indomethacin (10 microM), and their precise structures are consistent with their formation by a lipoxygenase reaction. Eicosapentaenoic acids with a prochiral tritium label in the 10-D or 10-L position were used to investigate the mechanism of biosynthesis. The formation of (12R)-hydroxyeicosapentaenoic acid proceeded with the stereoselective abstraction of the 10-D hydrogen from the substrate. This reaction was shown to be opposite to the (12S) oxygenation catalyzed by porcine leukocyte 12-lipoxygenase. These results with S. purpuratus eggs constitute the first demonstration of (11R)- or (12R)-lipoxygenase activity in any cell type or tissue.

Proinflammatory properties of unsaturated fatty acids and their monohydroxy metabolites

The proinflammatory effects of unsaturated fatty acids and, where appropriate, their monohydroxy derivatives, have been investigated both by application to human skin and with respect to human polymorphonuclear leukocyte (PMN) migration. Of the fatty acids applied to the skin only eicosapentaenoic and arachidonic acids (EPA;AA) produced consistent, measurable erythema. The monohydroxy derivatives of the two fatty acids also caused erythema. the 12-hydroxy isomers being the most potent. Chemokinetic activity towards PMNs was observed in the presence of AA, EPA and α-linolenic acid using an agarose microdroplet chemokinesis assay. In contrast to their in vivo properties, the 5-hydroxy isomers of AA and EPA were the most potent, being approximately 10 times more chemokinetically active than the other isomers. Quantification of the hydroxyeicosatetraenoic and hydroxyeicosapentaenoic acids (HETEs;HEPEs) in the lesional skin of psoriatic patients demonstrated that, of the metabolites measured, 12-HETE was present in the greatest amounts. Twenty five times more 12-HETE than 12- or 15-HEPE was detected, these being the most abundant of teh HEPEs formed. The monohydroxy derivatives of AA and EPA may contribute to the inflammatory changes observed in psoriasis. The HETEs appear to be of greater importance than the HEPEs in view of the relative amounts present.

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.

Characterization and biologic properties of 5,12-dihydroxy derivatives of eicosapentaenoic acid, including leukotriene B5 and the double lipoxygenase product.

Leukotriene B5 (LTB5) and three stereoisomers were prepared biosynthetically from eicosapentaenoic acid and compared with the analogous derivatives of arachidonic acid for their chemotactic and aggregating effects on human neutrophilic polymorphonuclear leukocytes. Leukotriene B4 (LTB4), LTB5, and the 6-trans-diastereoisomers of each were generated by activating polymorphonuclear leukocytes with the calcium ionophore A23187 in the presence of 14C-labeled and unlabeled arachidonic acid or 14C-labeled and unlabeled eicosapentaenoic acid, respectively. The double lipoxygenase products, (5S,12S)-6-trans-8-cis-LTB4 and (5S,12S)-6-trans-8-cis-LTB5, were generated from 5S-hydroxyeicosatetraenoic acid and racemic 5-hydroxyeicosapentaenoic acid intermediates by incubation with platelet sonicates. The products of each reaction were isolated by reverse-phase-high performance liquid chromatography and identified by their retention times relative to the appropriate totally synthetic standards, ultraviolet absorption spectra, immunoreactivity in a radioimmunoassay for LTB4, and, for all but the double lipoxygenase products, by incorporation of radiolabel from the specific polyunsaturated fatty acid source. When the concentration of LTB5 eliciting maximum chemotactic response of human polymorphonuclear leukocytes, 50 ng/ml (1.5 X 10(-7) M), and that eliciting a maximum aggregation response, 20 ng/ml (5.9 X 10(-8) M), were compared with the interpolated values of LTB4 eliciting comparable effects, the potency of LTB5 relative to LTB4 was approximately 1:8 as a chemotactic agent and about 1:20 as an aggregating agent. The double lipoxygenase products and the resolved 6-trans-diastereoisomers of the pentaene and tetraene series were about 2 logs less active as chemotactic factors than LTB4 and only (5S,12S)-6-trans-8-cis-LTB4 had even minimal aggregating activity.