New products in the hepoxilin pathway: isolation of 11-glutathionyl hepoxilin A3 through reaction of hepoxilin A3 with glutathione S-transferase.

We describe herein the metabolism of hepoxilin A3 (HxA3) by glutathione S-transferase (GST) into a glutathione conjugate. The reaction was carried out with HxA3 (unlabelled and 14C-labelled) and glutathione (unlabelled and tritium labelled). When two isomers of HxA3 were reacted with GST, two products were formed. Only one product was formed when a single isomer of HxA3 was used. The isomeric product HxB3 was marginally active indicating considerable specificity in the reaction with GST. The products were characterized by retention of tritium from glutathione and by comparison of their migration on high performance liquid chromatography with authentic reference compounds. The products bear the structure, 11-glutathionyl HxA3.

Formation and action of 8-hydroxy-11,12-epoxy-5,9,14-icosatrienoic acid in Aplysia: a possible second messenger in neurons.

In Aplysia neural tissue, the release and metabolism of arachidonic acid are stimulated by histamine or by activation of the identified L32 nerve cell circuit of the abdominal ganglion. Previously we found that histamine and intracellular stimulation of L32 cells, which are putatively histaminergic neurons, cause the production of 12-hydroxy-5,8,10,14-icosatetraenoic acid (12-HETE), a product of the 12-lipoxygenase pathway formed through 12-hydroperoxy-5,8,10,14-icosatetraenoic acid (12-HPETE). 12-HPETE, but not 12(S)-HETE, mimics the dual-action response of L14 ink motor neurons to histamine and stimulation of L32. 12-HPETE can also be further metabolized to 8-hydroxy-11,12-epoxy-5,9,14-icosatrienoic acid (8-HEpETE) which was identified by HPLC, enzymatic hydrolysis, and GC/MS. Production of 8-HEpETE is specific, as its positional isomer 10-hydroxy-11,12-epoxy-5,8,14-icosatrienoic acid is not formed after physiologic stimulation. 8-HEpETE can elicit the late component (hyperpolarization) of the dual-action response in L14 cells, suggesting that it may be a second messenger in Aplysia.

Dietary manipulation of macrophage phospholipid classes: selective increase of dihomogammalinolenic acid.

Because alterations in the dietary content of fatty acids are an important method for modulating macrophage eicosanoid production, we have quantitated the levels of n-6 and n-3 polyunsaturated fatty acids in peritoneal macrophage individual phospholipids from mice fed diets (3 wk) with either safflower oil (SAF), predominantly containing 18:2n-6, borage (BOR) containing 18:2n-6 and 18:3n-6, fish (MFO) containing 20:5n-3 and 22:6n-3, and borage/fish mixture (MIX) containing 18:2n-6, 18:3n-6, 20:5n-3 and 22:6n-3. Dietary n-3 fatty acids were readily incorporated into macrophage phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylinositol (PI). The increase in n-3 fatty acid levels was accompanied by a decrease in the absolute levels of 18:2n-6, 20:4n-6 and 22:4n-6 in PC, PE and PS. Interestingly, PI 20:4n-6 levels were not significantly lowered (P greater than 0.05) in MIX and MFO macrophages relative to SAF and BOR. These data demonstrate the unique ability of this phospholipid to selectively maintain its 20:4n-6 levels. In BOR and MIX animals, 20:3n-6 levels were significantly increased (P less than 0.05) in all phospholipids relative to SAF and MFO. The combination of borage and fish oils (MIX diet) produced the highest 20:3n-6/20:4n-6 ratio in all phospholipids. These data show that the macrophage eicosanoid precursor levels of 20:3n-6, 20:4n-6 and n-3 acids can be selectively manipulated through the use of specific dietary regimens. This is noteworthy because an increase in phospholipid levels of 20:3n-6 and 20:5n-3, while concomitantly reducing 20:4n-6, may have therapeutic potential in treating inflammatory disorders.

Formation of cis-14,15-oxido-5,8,11-icosatrienoic acid from phosphatidylinositol in human platelets.

Human platelets contain a soluble enzyme or enzyme system that catalyzes the formation of lysophosphatidylinositol and a compound more polar than arachidonic acid (compound A) from 2-arachidonoyl sn-phosphatidylinositol. Arachidonic acid, 2-arachidonoyl sn-phosphatidylcholine, or 2-arachidonoyl sn-phosphatidylethanolamine did not serve as substrate for the production of compound A. The reaction required Ca2+ and was not affected by aspirin, indomethacin, or mepacrin. Enzyme activity was not enhanced in the presence of NADPH, but it was inhibited greater than 90% by CO or N2; inhibition was readily reversible by exposure to atmospheric air. Neither metapyrone (SKF 525A) nor cyanide, inhibitors of cytochrome P-450, inhibited compound A formation, suggesting that a cytochrome P-450 system was not involved. Thrombin stimulated the formation of compound A in whole platelets; ionophore A23187 did so much less effectively; and other agonists such as collagen, ADP, and epinephrine were ineffective. Compound A exhibited a fragmentation pattern by GC/MS identical to that of authentic cis-14,15-oxido-5,8,11-icosatrienoic acid. Collectively, these data indicate that human platelets may contain an enzyme system that catalyzes the epoxidation of the arachidonic acid moiety of phosphatidylinositol and its hydrolysis to liberate cis-14,15-oxido-5,8,11-icosatrienoic acid.

In vivo formation of hepoxilin A3 in the rat.

Bolus intravenous injection of arachidonic acid (10 mg/kg) in the rat led to the appearance of hepoxilin A3 in the circulation. The product was assayed as the Me t-BDMSi derivative of its stable trihydroxy product trioxilin A3, by capillary gas chromatography-electron impact mass spectrometry using the stable deuterium isotope dilution technique. Hepoxilin A3, was undetected in blood samples taken prior to the injection of arachidonic acid, but rapidly appeared (4.62 +/- 1.3 ng/ml blood, n = 3) within 1 minute after injection of arachidonic acid. The plasma concentration of insulin increased by 36% over the same period after injection of arachidonic acid. These experiments demonstrate for the first time the formation of this new class of insulin secretagogues in vivo and their temporal correlation with plasma insulin concentrations in vivo.

Arachidonic acid epoxides. Isolation and structure of two hydroxy epoxide intermediates in the formation of 8,11,12- and 10,11,12-trihydroxyeicosatrienoic acids.

Arachidonic acid and 12-hydroperoxyeicosa-5,8, 10, 14-tetraenoic acid are converted by a 0-30% ammonium sulfate fraction (Fraction A) of the high speed supernatant of rat lung into two hydroxy epoxides (EH-1 and EH-2) which have been purified by high performance liquid chromatography. These hydroxy epoxides are converted quantitatively into two triols (10,11,12- from EH-1 and 8,11,12- from EH-2) by a 30-50% ammonium sulfate fraction (Fraction B) of the high speed supernatant. We propose the structures, 8-hydroxy-11,12-epoxyeicosa-5,9,14-trienoic acid (EH-2) and 10-hydroxy-11,12-epoxyeicosa-5,8,14-trienoic acids (EH-1) for these intermediates on the basis of mass spectral interpretation of several derivatives including the lithium aluminum hydride reduction product of both natural and 18Oxygenated derivatives.

Resolution by DEAE-cellulose chromatography of the enzymatic steps in the transformation of arachidonic acid into 8, 11, 12- and 10, 11, 12-trihydroxy-eicosatrienoic acid by the rat lung.

The 30-50% ammonium sulfate fraction of the high speed supernatant (100,000 xg) of a rat lung homogenate is capable of catalysing the conversion of arachidonic acid into 8,11,12- and 10,11, 12-trihydroxyeicosatrienoic acids. This enzyme preparation was resolved through DEAE cellulose chromatography into three stages which were assayed with precursors specific for each stage. Thus in the first stage arachidonic acid is converted by 12-lipoxygenase into 12-hydroperoxy-5,8,10,14-eicosatetraenoic acid (12-HPETE) detected as the corresponding 12-hydroxy product (12-HETE). 12-HPETE in turn is converted into 8-hydroxy-11,12-epoxy-5,9,14-eicosatrienoic acid and 10-hydroxy-11,12-epoxy-5,8,14-eicosatrienoic acid. These epoxides are in turn selectively converted through an epoxide hydrase into the respective triols. While the first and third stages were carried out by distinct fractions from the DEAE columns, the second i.e. conversion of 12-HPETE into epoxides, was detected in all fractions as was the reduction of 12-HPETE into 12-HETE.

The enzymatic conversion of arachidonic acid into 8,11,12-trihydroxyeicosatrienoic acid. Resolution of rat lung enzyme into two active fractions.

The conversion of arachidonic acid into 8,11,12-trihydroxyeicosatrienoic acid by rat lung high-speed supernatant has been resolved into two separate stages through ammonium sulfate precipitation. The first stage is catalysed by 0-30% ammonium sulfate fraction and converts arachidonic acid and 12-hydroperoxyeicosatetraenoic acid into an intermediate, X. X is subsequently utilized in the second stage by the fraction sedimented at 30-50% saturation in ammonium sulfate to form two isomeric 8,11,12-trihydroxyeicosatrienoic acids.

Isolation of an enzyme system in rat lung cytosol which converts arachidonic acid into 8,11,12-trihydroxyeicosatrienoic acid.

An enzyme system in rat lung is described which transforms arachidonic acid into 8,11,12-trihydroxyeicosatrienoic acid. The enzyme system is present in the high-speed supernatant fraction of a rat lung homogenate and can be precipitated with ammonium sulfate (30 to 50% saturation) and concentrated. Preliminary experiments indicate that the enzyme activity can be eluted from Sephadex G-200 and Sepharose 6B gel chromatography, whereas all activity is lost on DEAE cellulose chromatography. Enzyme activity is heat-liable, being lost after 10 min at 50 degrees C.

Oxygenation of arachidonic acid by hepatic microsomes of the rabbit. Mechanism of biosynthesis of two vicinal dihydroxyeicosatrienoic acids.

[1-14C] Arachidonic (eicosatetraenoic) acid was incubated at 37 degrees C for 15 min with rabbit liver microsomes fortified with NADPH (1 mM). The products were purified by high-pressure liquid chromatography (HPLC) and analyzed by gas chromatography-mass spectrometry. Based on polarity on reversed phase HPLC, the metabolites could be divided into three groups. The major metabolites of lowest polarity were 19- and 20-hydroxyarachidonic acid and 19-oxoarachidonic acid. The major metabolites of medium polarity were two diols, 14,15-dihydroxy-5,-8,11-eicosatrienoic acid and 11,12-dihydroxy-5,8,14-eicosatrienoic acid. Microsomal incubation under atmospheric isotopic oxygen led to incorporation of only one 18O molecule in each diol, indicating that the diols could originate from breakdown of 14(15)-oxido-5,8,11-eicosatrienoic acid and 11(12)-oxido-5,8,14-eicosatrienoic acid, respectively. Major metabolites in the most polar group were 14,15,19- and 14,15,20-trihydroxy-5,8,11-eicosatrienoic acid. 11,12,19- and 11,12,20-trihydroxy-5,8,14-eicosatrienoic acid and 11,12-dihydroxy-19-oxo-5,8,-14-eicosatrienonic acid. About 0.5% of exogenous radioactively labelled arachidonic was covalently bound to microsomal proteins. The metabolites and the protein-bound products were formed in considerably smaller amounts by non-fortified microsomes. Carbon monoxide inhibited this pathway of arachidonic acid metabolism, indicating that these reactions might be catalyzed by the cytochrome P-450-linked monooxygenase systems.

Arachidonic acid metabolism in rabbit renal cortex. Formation of two novel dihydroxyeicosatrienoic acids.

[1-14C]Eicosatetraenoic (arachidonic) acid was incubated with a low speed (17,000 X g) rabbit renal cortical supernatant or with a cortical microsomal suspension fortified with NADPH for 15 min at 37 degrees C. The products which were less polar than prostaglandins on reversed phase high performance liquid chromatography were identified by gas chromatography-mass spectrometry. Both the fortified microsomes and the low speed supernatant formed significant amounts of two novel metabolites, 11,12-dihydroxy-5,8,14-eicosatrienoic acid and 14,15-dihydroxy-5,8,11-eicosatrienoic acid. Other identified products were 19- and 20-hydroxyeicosatetraenoic acid, 19-oxoeicosatetraenoic acid, and in the low speed supernatant, eicosatetraen-1,20-dioic acid. The metabolites were not formed in significant amounts by high speed cortical supernatant or by nonfortified cortical microsomes. Carbon monoxide inhibited formation of these compounds, indicating that they may be formed by the cytochrome P-450-linked renal monooxygenase systems.

Formation of 8,11,12-trihydroxyeicosatrienoic acid by a rat lung high-speed supernatant fraction.

An enzyme was found in the high-speed (100 000 x g) supernatant fraction of a rat lung homogenate which catalysed the conversion of arachidonic acid into 8,11,12-trihydroxyeicosatrienoic acid. The isomeric 8,9,12-triol was not detected. The structure of the isolated product was confirmed by mass spectrometric analysis of the methyl ester t-butyldimethylsilyl ether derivative. These results indicate that formation of both positional isomers is carried out by separate enzymes, the distributions of which are not restricted to platelets.

Conversion of 5,8,11-eicosatrienoic acid to leukotrienes C3 and D3.

5,8,11-Eicosatrienoic acid was converted by mouse mastocytoma cells stimulated with ionophore A23187 to two slow reacting substances. These were characterized by spectroscopy and by chemical and enzymatic degradations as two geometrical isomers of 5-hydroxy-6-S-glutathionyl-7,9,11-eicosatrienoic acid (E,E,Z; leukotriene C3 and E,E,E; 11-trans-leukotriene C3). Corresponding cysteinylglycine compounds (leukotriene D3 and 11-trans leukotriene D3) were obtained from the leukotriene C3 isomers by treatment with kidney gamma-glutamyl transpeptidase. The biological effects of leukotrienes C3 and D3, on the isolated guinea pig ileum, were approximately the same as of leukotrienes derived from arachidonic acid.

Isolation of a new lipoxygenase metabolite of arachidonic acid, 8. 11, 12-trihydroxy-5,9,14-eicosatrienoic acid from human platelets.

Washed human platelets incubated with 1-14C-arachidonic acid (1mM) produced a new metabolite which migrated on thin layer chromatography close to thromboxane B2, but which was identified by mass spectrometry as a trihydroxy fatty acid. The mass spectrum was consistent with the assigned structure, 8,11,12-trihydroxy-5,9,14-eicosatrienoic acid (THETE). Platelet THETE synthesis from arachidonate was not inhibited by preincubation with asprin or indomethacin but was blocked by 5,8,11,14-eicosatetraynoic acid. Therefore, THETE appears to arise via the platelet lipoxygenase pathway rather than via the prostaglandin cyclooxygenase. Two proposed structures, including a novel dihydro-hydroxy-pyran cyclic intermediate, which could give rise to THETE are presented.