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.     

Metabolism of arachidonic acid by isolated rat hepatocytes,renal cells and by some rabbit tissues: Detection of vicinal diols by mass fragmentography

Purified cytochromes P-450 (LM₂ and PB-B₂) in a reconstituted system and epoxide hydrolase were recently found to metabolize arachidonic (eicosatetraenoic) acid to four vicinal dihydroxyeicosatrienoic acids. These metabolites were chemically synthetized from octadeuterated arachidonic acid and employed as internal standards for mass fragmentography. Isolated rat hepatocytes and renal cells were incubated with arachidonic acid (0.1 mM; 37°C, 15 min) and, following extractive isolation and reversed-phase HPLC, formation of 11,12-dihydroxy-5,8,14-eicosatrienoic acid and 14,15-dihydroxy-5,8,11-eicosatrienoic acid was demonstrated by mass fragmentography using a capillary GC column. Furthermore, these diols were also detected in rabbit liver and renal cortex and they therefore appear to be formed endogenously. Formation of vicinal diols was also studied in cell free systems. Rabbit liver and renal cortical microsomes were incubated with NADPH (1 mM) and arachidonic acid (0.15 mM) for 15 min at 37°C and, besides 11,12-dihydroxy- and 14,15-dihydroxyeicosatrienoic acid, small amounts of 8,9-dihydroxy- and 5,6-dihydroxyeicosatrienoic acid could be detected by mass fragmentography. Renal as well as hepatic monooxygenases can thus epoxidize each of the four double bonds of arachidonic acid. In contrast, rabbit lung microsomes and NADPH metabolize arachidonic acid mainly to prostaglandins and 19-hydroxy- and 20-hydroxyarachidonic acid, while only small amounts of 11,12-dihydroxyeicosatrienoic acid could be found. Monooxygenase metabolism of arachidonic acid by epoxidation might therefore be a significant pathway for the metabolism of this essential fatty acid in isolated rat renal cells and hepatocytes but presumably not in the lung.     

Oxygenation of 5,8,11-eicosatrienoic acid by prostaglandin H synthase-2 of ovine placental cotyledons: isolation of 13-hydroxy-5,8,11-eicosatrienoic and 11-hydroxy-5,8,12-eicosatrienoic acids

Prostaglandin H synthase-1 of ram vesicular glands metabolises 5,8,11-eicosatrienoic (Mead) acid to 13R-hydroxy-5,8,11-eicosatrienoic and to 11R-hydroxy-5,8,12-eicosatrienoic in a 5:1 ration. We wanted to determine the metabolism of this fatty acid by prostaglandin H synthase-2. Western blot showed that microsomes of sheep and rabbit placental cotyledons contained prostaglandin H synthase-2, while prostaglandin H synthase-1 could not be detected. Microsomes of sheep cotyledons metabolised [1-¹⁴C]5,8,11-eicosatrienoic acid to many polar metabolites and diclofenac (0.05 mM) inhibited the biosynthesis. The two major metabolites were identified as 13-hydroxy-5,8,11-eicosatrienoic and 11-hydroxy-5,8,12-eicosatrienoic acids. They were formed in a ratio of 3:2, which was not changed by aspirin (2 mM). 5,8,11-Eicosatrienoic acid is likely oxygenated by removal of the pro-S hydrogen at C-13 and insertion of molecular oxygen at either C-13 or C-11, which is followed by reduction of the peroxy derivatives to 13-hydroxy-5,8,11-eicosatrienoic and 11-hydroxy-5,8,12-eicosatrienoic acids, respectively. Prostaglandin H synthase-1 and -2 oxygenate 5,8,11-eicosatrienoic acid only slowly compared with arachidonic acid.     

Metabolism of 5(6)Oxidoeicosatrienoic acid by ram seminal vesicles. Formation of two stereoisomers of 5-hydroxyprostaglandin I1

Cytochrome P-450 can metabolize arachidonic (5,8,11,14-eicosatetraenoic) acid to four epoxides. One of them, cis-5(6)oxido-8,11,14-eicosatrienoic acid, has been reported to possess biological activity. To ascertain whether this epoxide could be a substrate for the enzyme fatty acid cyclooxygenase, synthetic 3H-labeled cis-5(6)-oxido-8,11,14-eicosatrienoic acid was incubated with microsomes of ram seminal vesicles and incubated with microsomes of ram seminal vesicles and the products were separated by reversed phase high performance liquid chromatography. The substrate was enzymatically transformed into products, which were more polar than 5,6-dihydroxy-8,11,14-eicosatrienoic acid. The biosynthesis was strongly inhibited by indomethacin or diclofenac sodium, two inhibitors of fatty acid cyclooxygenase. Two of the major metabolites could be identified by capillary gas chromatography-mass spectrometry as two stereoisomers of 5-hydroxyprostaglandin I1, viz. (5R,6R)-5-hydroxyprostaglandin I1 and (5S,6S)-5-hydroxyprostaglandin I1. The structures were established by comparison with the mass spectra of authentic material and by the retention time on capillary gas chromatography using deuterated internal standards. The two stereoisomers were presumably formed nonenzymatically from the intermediate 5(6)oxidoprostaglandin endoperoxides or from 5(6)oxidoprostaglandin F1 alpha during the isolation procedure.     

Double bond requirement for the 5-lipoxygenase pathway

We showed previously that polyenoic fatty acids with double bonds at carbon 5,8,11 are good substrates for the 5-lipoxygenase and also can be converted to LTC and dihydroxy acids. In order to determine whether all three double bonds are necessary for the 5-lipoxygenase-leukotriene pathway we studied 5,8,14-eicosatrienoic and 5,11,14-eicosatrienoic acid. C14-labeled fatty acids were incubated with 10,000 X g supernatant of homogenate of rat basophilic leukemia (RBL-1) cells in the presence of Ca++ at 37 degrees C. 5,11,14-Eicosatrienoic acid was not converted by the 5-lipoxygenase pathway and 5,8,14-eicosatrienoic acid was mainly converted to 5-hydroxy-6,8,14-eicosatrienoic acid (5-HETE). This monohydroxy was identified by UV spectrometry (UV max 235 nm) and GC-mass spectrometry. Incubations with whole homogenate analyzed by HPLC and bioassay showed that no detectable LTC, LTD or LTE was formed. These data indicate that fatty acids which have double bonds at carbon 5 and carbon 8 are readily converted to the 5-hydroperoxide. However double bonds at carbon 5,8 and 11 are necessary for LTA biosynthesis. This study therefore extends the characterization of the double bond requirement of the 5-lipoxygenase-leukotriene pathway. The number of double bonds necessary at each step varies and increases with each step in the pathway.     

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.     

Oxygenation of linolenic and linoleic acid to novel vicinal dihydroxy acids by hepatic microsomes of the rabbit

[14C] Linolenic acid (18: omega 3) and [14C] linoleic acid (18:2 omega 6) were incubated with hepatic microsomes of the rabbit in the presence of NADPH (1 Mm) for 15 min at 37 degrees C. The products were extracted and purified by high performance liquid chromatography. The major metabolites of linolenic and linoleic acid were identified by capillary gas chromatography mass spectrometry as 15,16-dihydroxy-9,12-octadecadienoic acid, 12,13-dihydroxy-9,15-octadecadienoic acid and 9,10-dihydroxy-12,15-octadecadienoic acid and as 12,13-dihydroxy-9-octadecaenoic acid and 9,10-dihydroxy-12-octadecaenoic acid, respectively. The results were confirmed by comparison with mass spectra of the authentic compounds. These metabolites are presumably formed by cytochrome P-450 catalyzed epoxidation of the omega 3, omega 6 and omega 9 double bonds, followed by enzymatic hydrolysis to 1,2-diols. The ratio of omega 3, omega 6 and omega 9 oxygenated metabolites of linolenic acid was approximately 2:1:1 and the ratio of the omega 6 and omega 9 metabolites of linoleic acid was 2:1, indicating that the double bond closest the omega end is most easily oxygenated.     

Beta-oxidation of 12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid by MOLT-4 lymphocytes.

MOLT-4 lymphocytes metabolize 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12(S)-HETE via beta-oxidation with retention of the hydroxyl group at the omega 9 carbon atom. The isolation of 6-hydroxy-4,8-tetradecadienoic acid documents that these cells have the capacity to catabolize the conjugated diene system. 12(S)-HETE was also metabolized to 3,12-dihydroxy-8,10,14-eicosatrienoic acid and 1,9-dihydroxy-5,7,11-heptadecatriene as well as to 17- and 19-carbon aldehydes. When MOLT-4 cells were incubated with the beta-oxidation product, 10-hydroxy-6,8,12-octadecatrienoic acid, it was in part further catabolized but in addition it served as an anabolic precursor as defined by the accumulation 3,12-dihydroxy-8,10,14-eicosatrienoic acid as well as 1,11-dihydroxy-7,9,13-nonadecatriene. Neither 10-hydroxy-6,8,12-octadecatrienoic acid nor 13-hydroxy-5,8,11-octadecatrienic acid was as potent in inhibiting phytohemagglutin-induced lymphocyte mitogenesis as were their parent compounds--i.e., 12(S)- and 15(S)-HETE. These findings argue against the hypothesis that beta-oxidation products of 12(S)- and 15(S)-HETE are the potential modulators of lymphocyte function. However, neither the pathway for synthesis, nor the role of odd chain aldehydes and diols as potential lipid mediators was determined in this study.     

Formation of reactive 1-nitropyrene metabolites by lung microsomes and isolated lung cells

The metabolism and activation of 1-nitropyrene (1-NP) to reactive intermediates by lung microsomes and isolated lung cells was studied. Mutagenicity of 1-NP metabolites was assayed in Salmonella typhimurium TA98NR, a strain lacking a major component of nitroreductase activity. In the presence of NADPH, microsomes from rabbit, rat and hamster lung metabolized 1-NP to mutagenic products to a similar degree. Pretreatment with a mixture of polychlorinated biphenyls (PCB) decreased the formation of mutagenic metabolites by rabbit lung microsomes, but did not affect the production of mutagens by rat or hamster lung microsomes. ³H-1-NP was metabolized to covalently bound protein products at a rate of 82 and 10 pmol/mg by rabbit and hamster lung microsomes, respectively, whereas no binding was detected in rat lung microsomes. PCB-pretreatment increased covalent protein binding of ³ H-1-NP in lung microsomes from hamster and rat, but decreased the binding in rabbit lung microsomes. High performance liquid chromatography analysis indicated that ³H-1-NP was readily converted to ring-hydroxylated products by rabbit and hamster lung microsomes; the rate was much lower with rat lung microsomes. ³H-1-NP was activated to metabolites that covalently bound to protein in isolated rabbit lung cells, with the following rates being observed: Clara cells > lung digest > type II cells. In contrast, covalent protein binding in cells isolated from rat lung was very low. 1-NP was not activated to products mutagenic for S. typhimurium TA 98 N R when co-incubated with cells isolated either from rabbit or rat lung.Abbreviations 1-AP 1-aminopyrene - DMSO dimethyl sulfoxide - EGTA ethylene glycol-bis(ß-aminoethyl ether) - EM electron microscopy - HEPES N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid - HPBS HEPES-phosphate-buffered-saline - HPLC high performance liquid chromatography - NBT nitroblue tetrazolium - 1-NP 1-nitropyrene - 1-NP-4,5-diol trans-4,5-dihydro-4,5-dihydroxy-1-nitropyrene - 1-NP-9,10-diol trans-9,10-dihydro-9,10-dihydroxy-1-nitropyrene - 1-NP-4,5-oxide 1-nitropyrene-4,5-oxide - 1-NP-9,10-oxide 1-nitropyrene-9,10-oxide - 3-OH-1-NP 3-hydroxy-1-nitropyrene - 6-/8-OH-1-NP a mixture of 6- and 8-hydroxy-1-nitropyrene - PBS phosphate-buffered saline - PCB a mixture of polychlorinated biphenyls (Aroclor 1254) - TLC thin layer chromatography     

12(R)-hydroxyeicosatrienoic acid: a vasodilator cytochrome P-450-dependent arachidonate metabolite from the bovine corneal epithelium

When corneal microsomes were incubated with arachidonic acid in the presence of an NADPH-generating system, two biologically active metabolites of arachidonic acid were formed. The structure of one of the metabolites, compound C, was previously reported to be 12(R)-hydroxy-5,8,10,14-eicosatetraenoic acid and was found to be a potent inhibitor of the Na+/K+-ATPase in the cornea. The second metabolite, compound D, was found to be a potent vasodilator as well as having the property of stimulating protein influx into the aqueous humor of the eye. Following purification of compound D by thin layer chromatography and high pressure liquid chromatography, it was found to lack a UV chromophore in contrast to the previously reported cytochrome P-450-dependent metabolite. Mass spectrometric analysis using positive and negative ionization modes was carried out on derivatized compound D that had been synthesized from a mixture of labeled [( 5,6,8,9,11,12,14,15-2H8]) and unlabeled arachidonic acid incubated with corneal microsomes. The novel arachidonate metabolite had abundant fragment ions consistent with compound D being a monooxygenated derivative of arachidonic acid with a hydroxyl substituent at carbon 12 of the eicosanoid backbone; only seven deuterium atoms from [2H8]arachidonate were retained in the structure. Oxidative ozonolysis yielded a product indicating that the double bonds in metabolite D resided between carbons at positions 8 and 9 and positions 14 and 15 of the 20-carbon chain. Compound D was therefore characterized as 12-hydroxy-5,8,14-eicosatrienoic acid. Model compounds were synthesized from dimethyl malate with the hydroxy at the 12 position with both the R and S absolute configuration and with all double bonds of the cis configuration. Only the 12(R) isomer was found to be a potent vasodilator and to increase aqueous humor protein concentration, suggesting that the biologically active compound D was 12(R)-hydroxy-5,8,14-(Z,Z,Z)-eicosatrienoic acid. As this compound possesses proinflammatory properties, it may play a role in the wound-healing processes of corneal injury.     

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.     

Calcium-dependent oxidation of 5,8,11-icosatrienoic acid by the cyclooxygenase enzyme system

Mead (5,8,11-icosatrienoic) acid was found to be metabolized by the cyclooxygenase enzyme system of ram seminal vesicle microsomes in a calcium-dependent manner. Although the enzyme converted Mead acid to products more slowly and less completely than the isomeric 8,11,14-icosatrienoic acid, both oxidations were inhibitable by indomethacin. Experiments using purified cyclooxygenase confirmed the participation of this enzyme system in the calcium-dependent oxidation. The products of the oxidation were separated by high performance liquid chromatography and analyzed by ultraviolet and gas chromatography-mass spectrometry. The spectra obtained were consistent with the products having the structures 13-hydroxy-5,8,11-icosatrienoate (the major product), 11-hydroxy-5,8,12-icosatrienoate, 9-hydroxy-5,7,11-icosatrienoate, and two isomeric 8,11-dihydroxy-5,9,12-icosatrienoates. No prostaglandin-like, cyclized products could be identified. This report is only the second to illustrate a calcium-dependent oxidation of a polyunsaturated fatty acid by a cyclooxygenase enzyme system and further extends the metabolic potential of Mead acid.     

Synthesis and structural identification of four dihydroxy acids and 11,12-leukotriene C4 derived from 11,12-leukotriene A4

Using a partially purified 12-lipoxygenase from porcine leukocytes, (5Z,8Z,10E,14Z)-12-hydroperoxy-5,8,10,14-icosate traenoic acid was synthesized from arachidonic acid with a yield of over 35%. The absolute configuration of C-12 was determined as S by chiral-phase column chromatography. It was chemically converted to at least three epoxides with the conjugated triene structure. Two were identified by proton NMR and mass spectrometry to be (5Z,7E,9E,14Z)-(11S,12S)-11,12-oxido-5,7,9,14-ic osatetraenoic acid (11,12-leukotriene A4) and (5Z,7Z,9E,14Z)-(11S,12S)-11,12-oxido-5,7,9,14-ic osatetraenoic acid (7-cis-11,12-leukotriene A4). 11,12-Leukotriene A4 underwent acid hydrolysis to yield two diastereomers of (6E,8E,10E,14Z)-(12S)-5,12-dihydroxy-6,8,10,14-i cosatetraenoic acid and two isomers of (14Z)-(12S)-11,12-dihydroxy-5,7,9,14-icosatetraenoic acid. Upon incubation with rat liver glutathione S-transferase, 11,12-leukotriene A4 was converted to 11,12-leukotriene C4, a spasmogenic compound.     

Analogs of arachidonic acid methylated at C-7 and C-10 as inhibitors of leukotriene biosynthesis

The syntheses and biological activity of (all )-7,7-dimethyl-5-8,- 11,14-eicosatetraenoic acid, (all )-7,7,-dimethyl-5,8,11-eicosatrienoic acid, ( , -7,7-dimethyl-5,8-eicosadienoic acid, (all )-10,10-dimetyl- 5,8,11,14-eicosatetraenoic acid, (all -10,10-dimethyl-5,8,11-eicosatrienoic acid, and .-( , -15-hydroxy-7,7-dimethyl-5,8-eicosadienoic acid are described. These arachidonic acid analogs are all inhibitors of ionophore-induced SRS-A biosynthesis in rat peritoneal cells. Their mode of action may involve inhibition of phospholipase A₂ rather than Δ⁵-lipoxygenase. These compounds failed to exhibit significant activity in an model designed to detect inhibitors of antigen-induced, leukotriene-mediated bronchoconstriction is sensitized guinea pigs.     

Inhibition of prostaglandin biosynthesis by c−5, c−8, c−11-eicosatrienoic acid

The enzyme prostaglandin H → E-isomerase (EC 5.3.99.3), which is present in sheep vesicular gland and needs glutathione as cofactor, is inhibited by c−5, c−8, c−11-eicosatrienoic acid, the fatty acid accumulating during essential fatty acid deficiency. The EFA-deficiency syndrome can partly be explained from a prostaglandin deficiency caused by lack of precursors. The present finding indicates that 5,8,11-eicosatrienoic acid could well be an additional factor in modifying the symptoms of EFA-deficiency.     

Enzymatic hydration of leukotriene A4. Purification and characterization of a novel epoxide hydrolase from human erythrocytes

Human erythrocytes contained a soluble cytosolic epoxide hydrolase for stereospecific enzymatic hydration of leukotriene A4 into leukotriene B4. The enzyme was purified 1100-fold, to apparent electrophoretic homogeneity, by conventional DEAE-Sephacel fractionation followed by high performance anion exchange and chromatofocusing procedures. Its characteristics include a molecular weight of 54,000 +/- 1,000, an isoelectric point 4.9 +/- 0.2, a Km apparent from 7 to 36 microM for enzymatic hydration of leukotriene A4, and a pH optimum ranging from 7 to 8. The enzyme was partially inactivated by its initial exposure to leukotriene A4. There was slow but detectable enzymatic hydration (pmol/min/mg) of certain arachidonic acid epoxides including (+/-)-14,15-oxido-5,8-11-eicosatrienoic acid and (+/-)-11,12-oxido-5,8,14-eicosatrienoic acid, but not others, including 5,6-oxido-8,11,14-eicosatrienoic acid. Human erythrocyte epoxide hydrolase did not hydrate either styrene oxide or trans-stilbene oxide. In terms of its physical properties and substrate preference for leukotriene A4, the erythrocyte enzyme differs from previously described versions of epoxide hydrolase. Human erythrocytes represent a novel source for an extrahepatic, cytosolic epoxide hydrolase with a potential physiological role.     

Rabbit aorta converts 15-HPETE to trihydroxyeicosatrienoic acids: potential role of cytochrome P450

Previous work showed that rabbit aorta metabolizes arachidonic acid via 15-lipoxygenase to 15-hydroperoxyeicosatetraenoic acid (15-HPETE), which undergoes an enzymatic rearrangement to 11-hydroxy-14,15-epoxyeicosatrienoic acid (11-H-14,15-EETA) and 15-hydroxy-11,12-epoxyeicosatrienoic acid (15-H-11,12-EETA). Hydrolysis of the epoxy group results in the formation of 11,14,15- and 11,12,15-trihydroxyeicosatrienoic acids (THETAs). Endothelial cells have several heme-containing enzymes including cytochromes P450 (CYP), nitric oxide synthase (eNOS), and prostacyclin (PGI(2)) synthase that catalyze the rearrangement of 15-HPETE to HEETAs. Incubation of arachidonic acid and 15-lipoxygenase, or 15-HPETE with rabbit aortic microsomes or rat liver microsomes, a rich source of CYP, resulted in the formation of a product that comigrated with THETAs and HEETAs on HPLC. Immunoblot analysis showed the presence of CYP2C8 and CYP2J2 in aortic tissue and when CYP2J2 or CYP2C8 was incubated with arachidonic acid and 15-lipoxygenase, the major products were 11,12,15- and 11,14,15-THETAs. Incubation of purified hematin, CYP2C11, eNOS or PGI(2) synthase enzymes with arachidonic acid and 15-lipoxygenase produced a different pattern of metabolites from rabbit aortic microsomes. Clotrimazole, a non-specific CYP inhibitor, and ebastine and terfenadone, specific CYP2J2 inhibitors, blocked the ability of aortic microsomes to produce THETAs while specific inhibitors of PGI(2) synthase, eNOS or CYP2C8/2C9 had no effect on THETA production. We suggest that a CYP, possibly CYP2J2, may function as the hydroperoxide isomerase converting 15-HPETE to HEETAs in rabbit vascular tissue. Further hydrolysis of the epoxy group of the HEETAs results in the formation of 11,12,15- and 11,14,15-THETAs. The HEETAs and THETAs are both vasodilators and may function as important regulators of vascular tone.     

Metabolism of 12(R)-hydroxy-5,8,10,14-eicosatetraenoic acid (12(R)-HETE) in corneal tissues: formation of novel metabolites

12(R)-Hydroxy-5,8,10,14-eicosatetraenoic acid [12(R)-HETE], a cytochrome P450 arachidonate metabolite, is metabolized by corneal tissues via three distinct metabolic pathways: beta-oxidation, omega-hydroxylation, and keto-reduction. The major metabolite released from the intact rabbit corneal epithelium or cultured cells was identified by mass spectrometric analysis as 8-hydroxy-4,6,10-hexadecatrienoic acid, the tetranor metabolite derived following two steps of beta-oxidation from the carboxy terminus. The beta-oxidation pathway was expressed in both microsomes and mitochondria isolated from bovine corneal epithelium and was dependent on the addition of oxidizing equivalents. The major metabolite of 12(R)-HETE in subcellular fractions of bovine corneal epithelial cells was a dihydro compound, 12-hydroxy-5,8,14-eicosatrienoic acid (12-HETrE). This derivative is presumably formed by an oxidation of the hydroxyl group followed by two keto-reduction steps, since its formation was accompanied by the appearance of a keto metabolite identified as 12-oxo-5,8,14-eicosatrienoic acid. The omega-hydroxylation, in contrast to other cell types, was a minor route for 12(R)-HETE metabolism in these tissues. Since 12(R)-HETE has been implicated as a modulator of Na(+)-K(+)-ATPase activity and its related functions in ocular tissues, these findings raise the possibility that the newly described metabolites may be involved in regulating corneal functions. In addition, the presence of a keto reductase in the cornea may be of great importance following injury since 12(R)-HETrE resulting from 12(R)-HETE by this activity is a potent ocular proinflammatory compound.     

Thyroid autoregulation. Inhibitory effects of iodinated derivatives of arachidonic acid on iodine metabolism

Thyroid autoregulation has been linked to an organified iodocompound. Since several iodolipids are produced by the gland their possible role in thyroid autoregulation was examined. The following pure synthetic compounds were prepared: 1) 14-iodo-15-hydroxy-5,8,11-eicosatrienoic acid (I-OH-A); 2) its omega lactone (IL-ω);3)5-hydroxy-6-iodo-8,11,14-eicosatrienoic acid delta lactone (IL-δ). Their action on iodine metabolism was studied. Iodine uptake was measured in calf thyroid slices. At 10-⁴M I-OH-A caused a 64% decrease in the T/M ratio while IL-ω inhibited it by 36% and IL-δ was without effect. At 10⁻⁵M the inhibition was 44% for I-OH-A and 19% for IL-ω, while T₃ was without action. A possible isotopic dilution effect was excluded, and no change in iodine efflux was observed. The inhibition by I-OH-A of iodine uptake was observed after only 15 min preincubation. This compound alse decreased ¹²⁵I accumulation in rats.In calf thyroid slice, I-OH-A at 10⁻⁴M, inhibited PB¹²⁵I formation by 80%, IL-ω 62% and IL-ω by 37% and arachidonic acid were without action. I-OH-A also caused a dose-dependent inhibition of TSH-stimulated iodide organification.The present results demonstrate, for the first time, that iodinated derivatives of arachidonic acid inhibition thyroid function and mimic the effect of iodine on thyroid autoregulation.