Analogs of leukotriene B4: effects of modification of the hydroxyl groups on leukocyte aggregation and binding to leukocyte leukotriene B4 receptors

The syntheses and agonist and binding activities of 5(S)-hydroxy- 6(Z), 8(E), 10(E), 14(Z)-eicosatetraenoic acid (12-deoxy LTB4), 5(S), 12(S)-dihydroxy-6(Z), 8(E), 10(E), 14(Z)-eicosatetraenoic acid (12-epi LTB4), 12(R)-hydroxy-6(Z), 8(E), 10(E), 14(Z)-eicosatetraenoic acid (5-deoxy LTB4), 5(R), 12(S)-dihydroxy-6(Z), 8(E), 10(E), 14(Z)-eicosatetraenoic acid (5-epi LTB4), 6(Z), 8(E), 10(E), 14(Z)-eicosatetraenoic acid (5, 12-deoxy LTB4) are described. These leukotriene B4 analogs were all able to aggregate rat leukocytes and compete with [3H]-leukotriene B4 for binding to rat and human leukocyte leukotriene B4 receptors with varying efficacy. The analog in which the 12-hydroxyl group was removed was severely reduced both in agonist action (aggregation) and binding. The epimeric 12-hydroxyl analog demonstrated better agonist and binding properties than the analog without a hydroxyl at this position. In contrast, in the case of the 5-hydroxyl the epimeric hydroxyl analog had greatly reduced agonist and binding activities while the 5-deoxy analog demonstrated potency only several fold less than leukotriene B4 itself. The dideoxy leukotriene B4 analog was more than a thousand fold less active than leukotriene B4 as an agonist and in binding to the leukotriene B4 receptor. These results show that binding to the leukocyte leukotriene B4 receptor requires a hydroxyl group at the 12 position in either stereochemical orientation but that the presence of a hydroxyl at the 5 position is less important. However, the epimeric C5 leukotriene B4 analog clearly interacts unfavourably with the binding site of the leukotriene B4 receptor.     

Rearrangement of 5S, 12S-dihydroxy-6,8,10,14-(E,Z,E,Z)-eicosatetraenoic acid during gas chromatography: formation of a cyclohexadiene derivative

The 5S, 12S-dihydroxy-6,8,10,14-(E,Z,E,Z,)-eicosatetraenoic acid, a product of double dioxygenation of arachidonic acid by lipoxygenases, undergoes severe decomposition during gas chromatography-mass spectrometric (GC-MS) analysis of the trimethylsilyl ether methyl ester derivative. The decomposition product was studied by GC-MS and identified as a cyclohexadiene derivative of the parent compound formed by ring closure at C6 and C11. Under identical GC conditions, two stereoisomers, i.e. 5S,12R-dihydroxy-6,8,10,14-(Z,E,E,Z)-eicosatetraenoic acid (leukotriene B4), and 6-trans-leukotriene B4 showed excellent chromatographic properties. These data indicated that the 5,12-dihydroxy derivative of arachidonic acid carrying the trans-cis-trans triene unit selectively undergoes cyclization during GC. These studies also provided an explanation to the controversial GC-MS data reported for this lipoxygenase product.     

LTA(4)-derived 5-oxo-eicosatetraenoic acid: pH-dependent formation and interaction with the LTB(4) receptor of human polymorphonuclear leukocytes

5-oxo-(7E,9E,11Z,14Z)-eicosatetraenoic acid (5-oxo-ETE) has been identified as a non-enzymatic hydrolysis product of leukotriene A(4) (LTA(4)) in addition to 5,12-dihydroxy-(6E,8E,10E, 14Z)-eicosatetraenoic acids (5,12-diHETEs) and 5,6-dihydroxy-(7E,9E, 11Z,14Z)-eicosatetraenoic acids (5,6-diHETEs). The amount of 5-oxo-ETE detected in the mixture of the hydrolysis products of LTA(4) was found to be pH-dependent. After incubation of LTA(4) in aqueous medium, the ratio of 5-oxo-ETE to 5,12-diHETE was 1:6 at pH 7.5, and 1:1 at pH 9.5. 5-Oxo-ETE was isolated from the alkaline hydrolysis products of LTA(4) in order to evaluate its effects on human polymorphonuclear (PMN) leukocytes. 5-Oxo-ETE induced a rapid and dose-dependent mobilization of calcium in PMN leukocytes with an EC(50) of 250 nM, as compared to values of 3.5 nM for leukotriene B(4) (LTB(4)500 nM for 5(S)-hydroxy-(6E,8Z,11Z,14Z)-eicosatetraenoic acid (5-HETE). Pretreatment of the cells with LTB(4) totally abolished the calcium response induced by 5-oxo-ETE. In contrast, the preincubation with 5-oxo-ETE did not affect the calcium mobilization induced by LTB(4). The calcium response induced by 5-oxo-ETE was totally inhibited by the specific LTB(4) receptor antagonist LY223982. These data demonstrate that 5-oxo-ETE can induce calcium mobilization in PMN leukocyte via the LTB(4) receptor in contrast to the closely related analog 5-oxo-(6E,8Z,11Z, 14Z)-eicosatetraenoic acid which is known to activate human neutrophils by a mechanism independent of the receptor for LTB(4).     

Synthesis, structural identification and biological activity of 11,12-dihydroxyeicosatetraenoic acids formed in human platelets

An enantiospecific route for the synthesis of 11,12-dihydroxyeicosatetraenoic acids was developed and used to synthesize 11,12-dihydroxy-5(Z),7(E),9(E),14(Z)-eicosatetraenoic acids. The 11,12-DHETEs were synthesized with the stereochemistry of the hydroxyl group being 11(R),12(S) and 11(S),12(S). The synthetic compounds were used to elucidate the structure of 11,12-DHETEs formed in human platelets by comparison of the chromatographic retention time in HPLC and GC as well as their ion fragmentation pattern in GC-MS. The major 11,12-DHETE formed in human platelets was found to be identical with 11(R),12(S)-dihydroxy-5(Z),7(E),9(E),14(Z)-eicosatetraenoic acid. Two more compounds were tentatively identified as 11(S),12(S)-dihydroxy-5(Z),7(E),9(E),14(Z)-eicosatetraenoic acid and 11,12-dihydroxy-5(E),7(E),9(E),14(Z)-eicosatetraenoic acid. Furthermore, the 11(S),12(S)-dihydroxy-5(Z),7(E),9(E),14(Z)-eicosatetraenoic acid was found to possess biological activity on neutrophil functional responses. However, the major compound, 11(R),12(S)-dihydroxy-5(Z),7(E),9(E),14(Z)-eicosatetraenoic acid, formed in platelets lacks biological activity in the test systems used. The present data further support that 11,12-dihydroxy-eicosatetraenoic acids are formed in human platelets via a leukotriene like mechanism presumably by the 12-lipoxygenase. Furthermore, the biological effects of one of the compounds showed a unique activity profile compared to other lipoxygenase products.     

Identification of 5-oxo-15-hydroxy-6,8,11,13-eicosatetraenoic acid as a novel and potent human eosinophil chemotactic eicosanoid.

Incubation of human eosinophils with arachidonic acid led to the formation of a novel and potent eosinophil chemotactic lipid (ECL) (Morita, E., Schr?der, J.-M., and Christophers, E. (1990) J. Immunol. 144, 1893-1900). To test the working hypothesis of whether ECL could have been formed via eosinophil-arachidonic acid 15-lipoxygenase we investigated whether other arachidonic acid 15-lipoxygenases such as soybean lipoxygenase I catalyze formation of a similar ECL. In the presence of hemoproteins and soybean lipoxygenase I arachidonic acid is converted to an ECL, which has physicochemical properties similar to those found for the eosinophil-derived ECL. Purification of this ECL by high performance liquid chromatography revealed that ECL is structurally different from well known eosinophil chemotactic eicosanoids such as leukotriene B4, 5,15-(6E,8Z,11Z,13E)-dihydroxyeicosatetraenoic acid (5,15-diHETE), and (8S,15S)-(5Z,9E,11Z,13E)-dihydroxyeicosatetra eno ic acid ((8S,15S)-diHETE). UV spectra of this ECL with absorbance maxima at 230 and 278 nm revealed the presence of two independent chromophores such as a conjugated oxodiene and a conjugated diene. Catalytic hydrogenation of ECL methyl ester led to the formation of 5,15-dihydroxyarachidic acid methyl ester. Reduction of ECL with sodium borohydride produced a product which is identical with authentic (5S,15S)-(6E,8Z,11Z,13E)-diHETE. Formation of an ECL monomethoxime derivative supports the conclusion that this highly potent eosinophil chemotactic eicosanoid is structurally identical with 5-oxo-15-hydroxy-6,8,11,13-eicosatetraenoic acid.     

Enhanced 5-lipoxygenase activity in lung macrophages compared to monocytes from normal subjects

We compared lipoxygenase activities of lung macrophages obtained from bronchoalveolar lavage to activities of blood monocytes purified by using discontinuous plasma/Percoll density gradients and adherence to tissue culture plastic in five normal subjects. Cells were incubated with ionophore A23187 (10(-9) to 10(-5) M) or arachidonic acid (0.12 to 80 microM) for 1 to 60 min at 37 degrees C to construct dose-response and time-dependence curves of lipoxygenase product generation. Products were identified and were quantified by using high-pressure liquid chromatography and ultraviolet spectroscopy. Under all conditions of product generation, both macrophages and monocytes generated predominantly (5S,12R)-dihydroxy-(6Z, 8E, 10E, 14Z)-eicosatetraenoic acid (leukotriene B4 (LTB4] and (5S)-hydroxy-(6E, 8Z, 11Z, 14Z) - eicosatetraenoic acid (5 - HETE), but, in each subject, macrophages invariably released greater amounts of LTB4 and 5-HETE than monocytes. In response to A23187, macrophages released a maximum of 183 +/- 96 pmol of LTB4 and 168 +/- 108 pmol of 5-HETE per 10(6) cells (mean +/- SEM), whereas monocytes released only 16 +/- 1 and 18 +/- 8 pmol per 10(6) cells of LTB4 and 5-HETE, respectively. After adding arachidonic acid, macrophages released a maximum of 52 +/- 21 pmol of LTB4 and 223 +/- 66 pmol of 5-HETE, whereas monocytes released no detectable products. The results suggest that mononuclear phagocyte maturation in the lung may be accompanied by an enhanced ability to generate 5-lipoxygenase products.     

Relationship of cyclic-AMP levels in leukotriene B4-stimulated leukocytes to lysosomal enzyme release and the generation of superoxide anions

Leukotriene B4 stimulated the formation of cyclic AMP, the release of lysosomal enzyme and generation of superoxide anions by human leukocytes. Dose-response curves have shown that the enzyme release proceeded in parallel with increments in cyclic AMP, suggesting a linkage between cyclic AMP and leukotriene B4-induced leukocyte activation. However, preincubation of the cells with (5S,12S)-dihydroxy-6,8,10,14-eicosatetraenoic acid or leukotriene B4 resulted in a dose-dependent inhibition of leukotriene B4-induced degranulation, without causing parallel changes in the levels of cyclic AMP. Both dihydroxy acids also blocked leukotriene B4-induced superoxide anion generation. These results suggest that the leukocyte responses to leukotriene B4 and the concomitant cyclic-AMP increments may be merely coincidental. In addition, the present study further supports the suggestion that (5S,12S)-dihydroxy-6,8,10,14-eicosatetraenoic acid may modulate the action of leukotriene B4 in the leukocyte.     

The 6R-oxygenase activity of arachidonate 5-lipoxygenase purified from porcine leukocytes

Arachidonate 5-lipoxygenase purified from porcine leukocytes was incubated with (5S)-hydroperoxy-6,8,11,14-eicosatetraenoic acid. In addition to degradation products of leukotriene A4 (6-trans-leukotriene B4 and its 12-epimer and others), (5S,6R)-dihydroperoxy-7,9,11,14-eicosatetraenoic acid was produced as a major product especially when the incubation was performed on ice rather than at room temperature. The amount of the (5S,6R)-dihydroperoxy acid was close to the total amount of leukotriene A4 degradation products. Under the anaerobic condition, production of the (5S,6R)-dihydroperoxy acid was markedly reduced. 5-Hydroxy-6,8,11,14-eicosatetraenoic acid could be a substrate of the enzyme and was transformed predominantly to a compound identified as (5S)-hydroxy-(6R)-hydroperoxy-7,9-trans-11,14-cis-eicosatetraenoic acid at about 1-2% rate of arachidonate 5-oxygenation. These findings indicated that the purified 5-lipoxygenase exhibited a 6R-oxygenase activity with (5S)-hydroxy and (5S)-hydroperoxy acids as substrates. The 6R-oxygenase activity, like the leukotriene A synthase activity, was presumed to be an integral part of 5-lipoxygenase because it required calcium and ATP and was affected by selective 5-lipoxygenase inhibitors.     

Human neutrophils convert the sebum-derived polyunsaturated fatty acid Sebaleic acid to a potent granulocyte chemoattractant

Sebaleic acid (5,8-octadecadienoic acid) is the major polyunsaturated fatty acid in human sebum and skin surface lipids. The objective of the present study was to investigate the metabolism of this fatty acid by human neutrophils and to determine whether its metabolites are biologically active. Neutrophils converted sebaleic acid to four major products, which were identified by their chromatographic properties, UV absorbance, and mass spectra as 5-hydroxy-(6E,8Z)-octadecadienoic acid (5-HODE), 5-oxo-(6E,8Z)-octadecadienoic acid (5-oxo-ODE), 5S,18-dihydroxy-(6E,8Z)-octadecadienoic acid, and 5-oxo-18-hydroxy-(6E,8Z)-octadecadienoic acid. The identities of these metabolites were confirmed by comparison of their properties with those of authentic chemically synthesized standards. Both neutrophils and human keratinocytes converted 5-HODE to 5-oxo-ODE. This reaction was stimulated in neutrophils by phorbol myristate acetate and in keratinocytes by oxidative stress (t-butyl-hydroperoxide). Both treatments dramatically elevated intracellular levels of NADP(+), the cofactor required by 5-hydroxyeicosanoid dehydrogenase. In keratinocytes, this was accompanied by a rapid increase in intracellular GSSG levels, consistent with the involvement of glutathione peroxidase. 5-Oxo-ODE stimulated calcium mobilization in human neutrophils and induced desensitization to 5-oxo-6,8,11,14-eicosatetraenoic acid but not leukotriene B(4), indicating that this effect was mediated by the OXE receptor. 5-Oxo-ODE and its 8-trans isomer were equipotent with 5-oxo-6,8,11,14-eicosatetraenoic acid in stimulating actin polymerization and chemotaxis in human neutrophils, whereas 5-HODE, 5-oxo-18-hydroxy-(6E,8Z)-octadecadienoic acid, and 5S,18-dihydroxy-(6E,8Z)-octadecadienoic acid were much less active. We conclude that neutrophil 5-lipoxygenase converts sebaleic acid to 5-HODE, which can be further metabolized to 5-oxo-ODE by 5-hydroxyeicosanoid dehydrogenase in neutrophils and keratinocytes. Because of its chemoattractant properties, sebum-derived 5-oxo-ODE could be involved in neutrophil infiltration in inflammatory skin diseases.     

Arachidonate 12-lipoxygenase purified from porcine leukocytes by immunoaffinity chromatography and its reactivity with hydroperoxyeicosatetraenoic acids

Arachidonate 12-lipoxygenase was purified to near homogeneity from the cytosol fraction of porcine leukocytes by ammonium sulfate fractionation, DEAE-cellulose chromatography, and immunoaffinity chromatography using a monoclonal antibody against the enzyme. The purified enzyme was unstable (half-life of about 24 h at 4 degrees C) but was markedly protected from the inactivation by storage in the presence of ferrous ion or in the absence of air. The lag phase which was observed before the start of the enzyme reaction was abolished by the presence of 12-hydroperoxy-5,8,10,14-eicosatetraenoic acid. An apparent substrate inhibition was observed with arachidonic acid and other active substrates; however, the substrate concentration curve was normalized by the presence of 0.03% Tween 20. Arachidonic acid was transformed to the omega-9 oxygenation product 12-hydroperoxy-5Z,8Z,10Z,14Z-eicosatetraenoic acid. C-12 oxygenation also occurred with 5-hydroxy- and 5-hydroperoxyeicosatetraenoic acids; the respective maximal velocities were 60 and 150% of the rate with arachidonic acid. Octadecaenoic acids were also good substrates. gamma-Linolenic acid was oxygenated in the omega-9 position (C-10), while linoleic and alpha-linolenic acids were subject to omega-6 oxygenation (C-13). A far more complex reaction was observed using 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid as substrate. Reaction occurred at 70% of the rate with arachidonic acid. The dihydroperoxy and dihydroxy products were identified by their UV absorption spectra, high performance liquid chromatography, and gas chromatography-mass spectrometry. Among these products, (8S,15S)-dihydroperoxy-5Z,9E,11Z,13E-eicos atetraenoic acid and (14R,15S)-erythro-dihydroperoxy-5Z,8Z,10E, 12E-eicosatetraenoic acid were produced in larger amounts than the (8R)- and (14S,15S)-threo isomers, respectively; these products were attributed to 8- and 14-oxygenation of the 15-hydroperoxy acid. Furthermore, formation of 14,15-leukotriene A4 was inferred from the characteristic pattern of its hydrolysis products comprised of equal amounts of (8R,15S)- and (8S,15S)-dihydroxy-5Z,9E,11E,13E-eicosatetraenoi c acids together with smaller amounts of (14R,15S)-erythro- and (14S,15S)-threo-dihydroxy-5Z,8Z,10E,12E-eicosate traenoic acids. Thus, both lipoxygenase and leukotriene synthase activities were demonstrated with the homogeneous preparation of porcine leukocyte 12-lipoxygenase.     

Metabolism of arachidonic acid in polymorphonuclear leukocytes. Structural analysis of novel hydroxylated compounds.

Arachidonic acid was incubated with rabbit peritoneal polymorphonuclear leukocytes (glycogen-induced) and compounds obtained from ether extractions were fractionated by silicic acid column chromatography. A fraction containing several unidentified metabolites of arachidonic acid was analyzed by reversed phase-high pressure liquid chromatography. The metabolites were esterified and further purified by silicic acid high pressure liquid chromatography. The structures of the pure compounds were elucidated by infrared and ultraviolet spectrometry, ozonolysis, and gas chromatography-mass spectrometry. The following novel compounds were identified: Compound 1, 5S, 12R-dihydroxy-(E,E,E,Z)-6,8,10,14-eicosatetraenoic acid; Compound 2, 5S, 12S-dihydroxy-(E,E,E,Z)-6,8,10,14-eicosatetraenoic acid; Compound 3, 5, 6-dihydroxy-7,9,11,14-eicosatetraenoic acid; Compound 4, a diastereoisomer of the latter. Evidence for the occurrence of the delta-lactone forms of the 5,12-dihydroxy acids is also presented.     

Specificity of the effect of lipoxygenase metabolites of arachidonic acid on calcium homeostasis in neutrophils. Correlation with functional activity

The ability of the major neutrophil-derived lipoxygenase metabolites of arachidonic acid to increase the rate of 45Ca influx in rabbit neutrophils was examined. The results obtained demonstrate that (5S),(12R)-dihydroxy-6,8,11,14-(cis,trans,trans,cis)-eicosatetraenoic acid (leukotriene B4) is the most active of the arachidonic acid metabolites. The activity of leukotriene B4 is highly stereospecific in that its three nonenzymatically derived isomers are essentially inactive. The omega-hydroxylation of leukotriene B4 results in a compound that is nearly as active as leukotriene B4 as far as its ability to stimulate calcium influx and neutrophil aggregation while being a much weaker secretagogue. The further conversion of leukotriene B4 into a dicarboxylic acid removes all detectable biological activity. 5,6-Oxido-7,9,11,14-eicosatetraenoic acid (leukotriene A4) methyl ester was also found to increase the rate of calcium influx, while the degradation products of native leukotriene A4 were essentially inactive. These results demonstrate that a close correlation exists between the ability of the various lipoxygenase products to alter calcium homeostasis in rabbit neutrophils and their biological activities.     

Stimulation of human myelopoiesis by leukotriene B4

Cultivation of human mononuclear bone marrow cells for 10 days in the presence of leukotriene B4 (8 X 10(-8) - 3 X 10(-6)M) led to an increase in the formation of granulocyte-macrophage colonies. The increase varied between 19 and 122% when compared to control cells. 5S, 12S-Dihydroxy-6, 8, 10, 14-eicosatetraenoic acid (5S, 12S-DHETE), an isomer of leukotriene B4, did not stimulate colony formation. Preincubation of the cells with 5S, 12S-DHETE inhibited the stimulatory action of leukotriene B4 on the proliferation of bone marrow cells. The present study indicates that leukotriene B4 amplifies the stimulation caused by the colony stimulating factor(s) and may play a role in modulating granulocyte and macrophage poiesis by a positive feedback mechanism.     

Effect of various lipoxygenase metabolites of arachidonic acid on degranulation of polymorphonuclear leukocytes

Lipoxygenase metabolites of guinea pig peritoneal polymorphonuclear leukocytes stimulated with 10 microM A23187 plus arachidonic acid were isolated and identified. These metabolites were compared with each other and to chemically synthesized arachidonate metabolites for their ability to stimulate leukocyte degranulation. 5(S),12(R)-Dihydroxy-6,8,10-(cis/trans/trans)14-cis-eicosatetraenoic acid (leukotriene B4) produced a significant release of lysozyme, but not beta-glucuronidase or beta-N-acetylglucosaminidase at low concentrations (EC50 = 6.5 x 10(-9) M), while the leukocyte nonenzymatically generated 5,12-or 5,6-dihydroxyeicosatetraenoic acids had no effect at these concentrations. Higher concentrations (1--10 microM) of all the dihydroxyeicosatetraenoic acids, 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) and its hydroperoxy precursor stimulated significant lysozyme release which was greater than that produced by 15-hydroxy-5,8,11-13-eicosatetraenoic acid, arachidonic acid, or its acetylene analogue, 5,8,11,14-eicosatetraynoic acid. Micromolar concentrations of leukotriene B4 and 5-HETE also stimulated significant release of beta-N-acetylglucosaminidase above controls, but not beta-glucuronidase. These results suggest that leukotriene B4 may play a role in regulating the release of certain granule-bound enzymes from polymorphonuclear leukocytes.     

A novel leukotriene produced by stimulation of leukocytes with formylmethionylleucylphenylalanine

Stimulation of human polymorphonuclear leukocytes with the chemotactic peptide formylmethionylleucylphenylalanine led to the formation of a novel leukotriene: 5(S),12(R)-dihydroxy-6,8,10,14-eicosatetraen-1,20-dioic acid. This dihydroxydicarboxylic acid is derived from omega-oxidation of 5(S),12(R),dihydroxy-6,8,10,14-eicosatetradienoic acid (leukotriene B4). The intermediate 5(S),12(R),20-trihydroxy-6,8,10,14-eicosatetraenoic acid was also isolated from these incubations. The two metabolites of leukotriene B4 exhibit chemotactic properties for human polymorphonuclear leukocytes but are less active in this respect than the parent compound.     

Effect of Bryonia cucurbitacins on the biosynthesis of eicosanoids in human leukocytes

2 beta,25-di (beta-D-glucopyranosyl)-16 alpha,20-dihydroxy-3,11,22- trioxocucurbit-5-en and 2 beta-(beta-D-glucopyranosyl)-16 alpha,20,25-trihydroxy-3,11,22-trioxocucurbit-5-en isolated from bryonia (Bryonia alba L.) roots have been demonstrated to inhibit in vitro the [1-14C]arachidonic acid release from neutrophils. Aglicon 2 beta,16 alpha,20,25-tetrahydroxy-3,11,22-trioxocucurbit-5-en is much less active. When the cells are stimulated by calcium ionophore A23187, the aglycon potentiates the release of arachidonic acid. In these conditions the glucosides show little activity. Both the glucosides and their aglycon suppress the biosynthesis of 5S,12R-dihydroxy-6,8,10,14(Z, E, E, Z)-eicosatetraenoic acid (LTB4) and 5S,12S-dihydroxy-6,8,10,14(E, Z, E, Z)-eicosatetraenoic acid (5S,12S-DHETE). Inhibition of the biosynthesis of these compounds by 2 beta,16 alpha,20,25-tetrahydroxy-3,11,22-trioxocucurbit-5-en also takes place on incubation of human neutrophils with exogenous arachidonic acid. The formation of other products of cycloxygenase and lipoxygenase oxidation pathways remains practically unchanged.     

Hemoprotein catalysis of leukotriene formation

Incubation of various hemoproteins with 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid or 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid resulted in formation of epimeric 5(S),12-dihydroxy-6,8,10,14 -eicosatetraenoic acids and epimeric 8,15(S)-dihydroxy-5,9,11,13 -eicosatetraenoic acids, respectively. These dihydroxy acids were earlier recognized as nonenzymatic hydrolysis products of 5(S),6-oxido-7,9,11,14-eicosatetraenoic acid (leukotriene A4) and 14,15(S)-oxido-5,8,10,12-eicosatetraenoic acid (14,15-leukotriene A4). These allylic epoxides could be isolated as such from the hemoprotein incubations, and most probably they are intermediates in formation of the dihydroxy acids.     

5-Lipoxygenase-derived oxylipins from the red alga Rhodymenia pertusa

The lipid extract of the temperate red alga Rhodymenia pertusa has yielded four eicosanoid metabolites, three of which are new natural products. Using principally NMR and MS techniques, their structures were deduced as 5R,6S-dihydroxy-7(E),9(E),11(Z),14(Z)-eicosatetraenoic acid (5R,6S-diHETE), 5R*,6S*-dihydroxy-7(E),9(E),11(Z),14(Z),17(Z)-eicosapentaenoic acid (5R*,6S*-diHEPE), 5-hydroxy-6(E),8(Z),11(Z),14(Z)-eicosatetraenoic acid (5-HETE), 5-hydroxy-6(E),8(Z),11(Z),14(Z),17(Z)-eicosapentaenoic acid (5-HEPE). The co-occurrence of these metabolites strongly suggests that R. pertusa contains a unique 5R-lipoxygenase system acting on both arachidonic and eicosapentaenoic acids.     

Kinetics of leukotriene A4 synthesis by 5-lipoxygenase from rat polymorphonuclear leukocytes

When arachidonic acid is added to lysates of rat polymorphonuclear leukocytes, it is oxidized to (5S)-hydroperoxy-6(E),8(Z),11(Z),14(Z)-eicosatetraenoic acid (5-HPETE). The 5-HPETE then partitions between reduction to the 5-hydroxyeicosanoid and conversion to leukotriene A4 (LTA4). Both steps in the formation of LTA4 are catalyzed by the enzyme 5-lipoxygenase. When [3H]arachidonic acid and unlabeled 5-HPETE were incubated together with 5-lipoxygenase, approximately 20% of the arachidonic acid oxidized at low enzyme concentrations was converted to LTA4 without reduction of the specific radioactivity of the LTA4 by the unlabeled 5-HPETE. A significant fraction of the [3H]-5-HPETE intermediate that is formed from arachidonic acid must therefore be converted directly to LTA4 without dissociation of the intermediate from the enzyme. This result predicts that even in the presence of high levels of peroxidase activity, which will trap any free 5-HPETE by reduction, the minimum efficiency of conversion of 5-HPETE to LTA4 will be approximately 20%, and this prediction was confirmed. 5-HPETE was found to be a competitive substrate relative to arachidonic acid, so that it is likely that the two substrates share a common active site.     

Calcium mobilization and right-angle light scatter responses to 12-oxo-derivatives of arachidonic acid in neutrophils: evidence for the involvement of the leukotriene B4 receptor.

The biological activities of two carbonyl compounds derived from arachidonic acid, (5Z,8Z,10E,14Z)-12-keto-5,8,10,14-eicosatetraeno ic acid (12-OxoETE) and (5Z,8Z,10E)-12-oxo-5,8,10-dodecatrienoic acid (12-OxoDTrE) were investigated. The ability of these compounds to induce a mobilization of calcium and to trigger a right-angle scatter response in isolated peripheral blood human neutrophils was determined. The two compounds induced a rapid and dose-dependent increase in the concentration of cytoplasmic free calcium; these effects were clearly detectable at concentrations greater than or equal to 10(-8) M. Pre-exposure of neutrophils to leukotriene B4 completely abolished the calcium mobilization induced by 12-OxoDTre and 12-OxoETE, while pre-exposure of the cells to the carbonyl compounds only slightly reduced the response to subsequent stimulation of neutrophils by leukotriene B4. The carbonyl compounds also induced a decrease in right-angle light scatter and these effects were abolished by pretreatment of neutrophils with leukotriene B4. These data demonstrate that 12-OxoETE and 12-OxoDTrE show significant agonist activities towards human neutrophils and strongly suggest that their mechanisms of action involve the leukotriene B4 binding sites or a common activation sequence.