LC-MS/MS analysis of epoxyalcohols and epoxides of arachidonic acid and their oxygenation by recombinant CYP4F8 and CYP4F22.

CYP4F22 and CYP4F8 are expressed in epidermis, and mutations of CYP4F22 are associated with lamellar ichthyosis. Epoxyalcohols (HEETs) and epoxides (EETs) of 20:4n-6 appear to be important for the water permeability barrier of skin. Our aim was to study the MS/MS spectra and fragmentation of these compounds and to determine whether they were oxidized by CYP4F22 or CYP4F8 expressed in yeast. HEETs were prepared from 15-hydroperoxyeicosatetraenoic acid (15-HPETE), 12-HPETE, and their [(2)H(8)]labeled isotopomers, and separated by normal phase-HPLC with MS/MS analysis. CYP4F22 oxygenated 20:4n-6 at C-18, whereas metabolites of HEETs could not be identified. CYP4F8 formed omega3 hydroxy metabolites of HEETs derived from 12R-HPETE with 11,12-epoxy-10-hydroxy configuration, but not HEETs derived from 15S-HPETE. 8,9-EET and 11,12-EET were also subject to omega3 hydroxylation by CYP4F8. We conclude that CYP4F8 and CYP4F22 oxidize 20:4n-6 and that CYP4F8 selectively oxidizes 8,9-EET, 11,12-EET, and 10,11R,12R-HEET at the omega3 position.

Oxidation of prostaglandin H(2) and prostaglandin H(2) analogues by human cytochromes P450: analysis of omega-side chain hydroxy metabolites and four steroisomers of 5-hydroxyprostaglandin I(1) by mass spectrometry.

The objective was to examine the NADPH-dependent oxygenation of prostaglandin H(2) (PGH(2)) and three PGH(2) analogues, 9,11-diazo-15-deoxy-PGH(2) (U51605), 9,11-epoxymethano-PGH(2) (U44069), and 11,9-epoxymethano-PGH(2) (U46619), by cytochromes P450, and to characterize the metabolites by mass spectrometry. CYP2C19, CYP4A11, CYP4F8, and liver and renal cortical microsomes oxidized the omega-side chain of U44069, U46619, and U51605, whereas only CYP4F8 oxidized the omega-side chain of PGH(2). PGH(2) was transformed to four stereoisomers of 5-hydroxy-PGI(1) by recombinant cytochromes P450. CYP4F8 formed the 5-hydroxy-PGI(1) isomers in small amounts compared to the 19-hydroxy metabolites of PGH(2). Isomers of 5-hydroxy-PGI(1) and 6-keto-PGF(1 alpha) were detectable when PGH(2) decomposed in the presence of hemin, hemoglobin, or heat-inactivated microsomes. 5-Hydroxy-PGI(1) is likely formed from PGH(2) in a pseudo-enzymatic reaction involving homolytic scission of the endoperoxide and formation of an ether between C-9 and C-6 and a carbon-centered radical at C-5, which reacts with molecular oxygen. CYP4F8 catalyzes 19-hydroxylation of PGH(2), but the absolute configuration of the 19-hydroxy group is unknown, whereas human seminal fluid contains (19R)-hydroxy-PGE(2). CYP4F8 was found to metabolize U51605 to 90% of the (19R)-hydroxy metabolite, providing further evidence in favor of a role of CYP4F8 in biosynthesis of (19R)-hydroxy PGE in human seminal vesicles. We conclude that omega-side chain hydroxylation of PGH(2) analogues may be catalyzed by many different cytochromes P450, but only CYP4F8 oxidizes the omega-side chain of PGH(2) efficiently.

Nimesulide aggravates kainic acid-induced seizures in the rat.

Treatment of rats with kainic acid (10 mg/kg, intraperitoneally) triggers limbic seizures. Cyclooxygenase-2 mRNA is expressed in the hippocampus and cortex after 8 hr and marked cell loss occurs after 72 hr in the CA1-CA3 areas of the hippocampus. We examined the effect of the cyclooxygenase-2 inhibitor, nimesulide (N-(4-nitro-2-phenoxyphenyl)-methanesulfonamide), on kainate-induced seizures and delayed neurotoxicity. Nimesulide (10 mg/kg, intraperitoneally) was well tolerated given alone or 6-8 hr after kainate. However, pretreatment with nimesulide augmented seizures and increased the mortality rate from approximately 10% to 69%. We examined the effect of nimesulide on delayed cell loss after 72 hr in the surviving animals with histological staining. Cell loss did not seem to be reduced in animals treated with nimesulide 6-8 hr after kainate, but in the surviving animals pretreated with nimesulide less cell loss occurred. We conclude that nimesulide should be used with caution as an antiinflammatory drug in patients with convulsive disorders.

Cloning and characterization of CYP4F21: a prostaglandin E2 20-hydroxylase of ram seminal vesicles.

Ram semen contains high concentrations of PGE1, PGE2, 20-hydroxy-PGE1, and 20-hydroxy-PGE2, which mainly originate from the ram seminal vesicles. The 20-hydroxy-PGE compounds are formed by a tentatively identified cytochrome P450, designated PGE2 20-hydroxylase. Our aim was to clone the enzyme and express it in yeast. Total RNA was isolated from ram seminal vesicle. Reverse transcription-polymerase chain reaction (RT-PCR) with degenerate primers for the CYP4 family yielded a novel cDNA sequence of a cytochrome P450. The full coding region (1584 bp) was cloned by RT-PCR and designated CYP4F21. The deduced protein sequence of CYP4F21 contained 528 amino acids and showed 74% amino acid identity with CYP4F8 of human seminal vesicles. CYP4F21 was expressed in yeast, and its catalytic properties were studied by liquid chromatography-mass spectrometry. Recombinant CYP4F21 oxidizes three stable PGH2 analogs (U44069, U46619, and U51605) and PGE2 to their 20-hydroxy metabolites, whereas PGH1, PGH2, PGE1, and PGF2alpha appeared to be poor substrates. The apparent Km for hydroxylation of PGE2 was 0.05 mM. Microsomes of ram seminal vesicles and NADPH metabolized PGE2 and the three PGH2 analogs essentially in the same way as CYP4F21. Our results suggest that CYP4F21 might be a sheep homolog to CYP4F8 of human seminal vesicles. The reproductive function of CYP4F21 is likely to biosynthesize 20-hydroxy-PGE1 and 20-hydroxy-PGE2, which is excreted by the seminal vesicles.

The selective cyclooxygenase-2 inhibitor rofecoxib reduces kainate-induced cell death in the rat hippocampus.

Treatment of male Sprague-Dawley rats with kainic acid (10 mg/kg, i.p.) triggered limbic seizures in 60% of the animals starting within 30 min and lasting for about 6 h. Cyclooxygenase-2 (COX-2) mRNA was strongly induced in the pyramidal cells of the hippocampus, in the amygdala and the piriform cortex after 8 h, as shown by in situ hybridization, and returned to control levels after 72 h. At this time marked cell loss occurred in the CA1-CA3 areas of the hippocampus. We hypothesize that rofecoxib, a selective COX-2 inhibitor, might abbreviate the late neurotoxicity, possibly associated with COX-2 induction. Animals which developed seizures were treated for 3 days with rofecoxib (10 mg/kg, i.p., n = 12) starting 6 or 8 h after kainic acid injection. Histological staining of viable cells confirmed that rofecoxib treatment selectively diminished cell loss in the hippocampus. The TdT-mediated dUTP nick end labelling (TUNEL) technique was used to estimate delayed cell death. Abundant TUNEL-positive cells were detected in seizure rats 72 h after kainic acid injection in pyramidal cells of the hippocampus (CA1-CA3), in cells of the thalamus, the amygdala and the piriform cortex. Treatment with rofecoxib selectively and significantly (P < 0.05) attenuated the number of TUNEL-positive cells in the hippocampus, whereas the cells of the thalamus, amygdala and piriform cortex were not protected. Therefore we conclude that COX-2 might contribute to cell death of pyramidal cells of the hippocampus as a consequence of limbic seizures.

cDna cloning and expression of CYP4F12, a novel human cytochrome P450.

cDNA of a novel human cytochrome P450 was cloned from human liver by reverse transcription-polymerase chain reaction and designated CYP4F12. The open reading frame coded for 524 amino acids, and the sequence could be aligned with 78-83% amino acid identity to the four human CYP4F enzymes (CYP4F2, CYP4F3, CYP4F8 and CYP4F11). Northern blot analysis suggested three major transcripts of CYP4F12, which were detected in liver, kidney, colon, small intestine and heart. The CYP4F12 gene contained 13 exons and was located at chromosome 19p13.1. CYP4F12, expressed in yeast, oxidized arachidonic acid to 18-hydroxyarachidonic acid, and the omega-side chain of two stable prostaglandin (PG) H(2) analogs (11,9-epoxymethano-PGH(2) and 9,11-diazo-15-deoxy-PGH(2)). CYP4F12 oxidized the omega-side chain of leukotriene B(4), PGE(2), PGF(2 alpha), PGH(2), and 9,11-epoxymethano-PGH(2) poorly. Several CYP4F enzymes are important omega 1- and omega 2-hydroxylases of eicosanoids. The physiological function of CYP4F12 merits further investigation.

Assignment of EPR Transitions in a Manganese-Containing Lipoxygenase and Prediction of Local Structure.

A new variant of lipoxygenases, one containing manganese instead of iron, is characterized by electron paramagnetic resonance (EPR) at two frequencies. In the manganous state (S(e) = 5/2), maganese lipoxygenase (MnLO) yields very broad X-band (9.2 GHz) EPR signals, extending over about 800 mT. In contrast, at W-band (94 GHz), the signal is much simplified, consisting of nested transitions centered near the free electron g-value. Computer simulation has been employed to derive estimates of the zero-field splittings for MnLO, with data from these two EPR frequencies. The general features of both X- and W-band spectra are fit, first, by simulations with S(e) = 5/2, but no nuclear hyperfine splitting. The simulations are then refined by inclusion of the hyperfine splitting. On the basis of the simulations, the ranges of zero-field splitting parameters are D = +0.07 to +0.10 cm(-1), and E/D = 0.13 to 0.23. Comparison of the value of D for MnLO with that of other manganese-containing proteins suggests that MnLO has three N-ligands to the metal center and O-ligands in the remainder of 6 coordination positions. The coordination environment of MnLO is similar to that in iron lipoxygenases.

Studies of lipoxygenases in the epithelium of cultured bovine cornea using an air interface model.

Epithelial lipoxygenases of bovine cornea were investigated in organ culture models. Subcellular fractions of the epithelium were incubated with(14)C-labelled arachidonate and the metabolites were analysed. Bovine corneal epithelial cells contain 15-lipoxygenase type 2 and 12-lipoxygenases of the leukocyte and the platelet types. The 15-lipoxygenase activity was prominent in the cytosolic fraction. Twelve- and 15-lipoxygenases occurred in the microsomal fraction, where the 15-lipoxygenase activity appeared to be favoured by low protein levels. The lipoxygenase activities strongly declined within 24 hr when the cornea was covered with cell culture medium, but were maintained with high activity in an air interface organ culture model for at least 72 hr. Cultured corneas were studied in pairs in the air interface model under influence of inflammatory stimuli. The epithelial 15- and 12-lipoxygenase activities were only slightly augmented by treatment with 12-O-tetradecanoyl-phorbol-13-acetate (10 microM, 8-72 hr), and remained unchanged after treatment with lipopolysaccharide (1-100 microgram ml(-1), 8-72 hr) or UV irradiation (301 nm, 0.17 J cm(-2); 8-24 hr). In some experiments, 5-lipoxygenase activity was detectable, as judged from liquid chromatography-mass spectrometry and chiral chromatography. Reverse transcription-polymerase chain reaction and Northern blot analysis were therefore used to identify mRNA of 5-lipoxygenase and related enzymes in bovine epithelium. 5-Lipoxygenase was detected as an amplicon of 695 bp, which had 91% nucleotide sequence identity with human 5-lipoxygenase and by Northern blot as a 3.0 kb mRNA. Leukotriene A(4)hydrolase was detected with the same techniques. The amino acid sequence of a 612 bp fragment was 90% identical with human leukotriene A(4)hydrolase and the size of the mRNA was 2.7 kb. The two enzymes were also detected in human corneal epithelium by reverse transcription-polymerase chain reaction.

Identification of CYP4F8 in human seminal vesicles as a prominent 19-hydroxylase of prostaglandin endoperoxides.

A novel cytochrome P450, CYP4F8, was recently cloned from human seminal vesicles. CYP4F8 was expressed in yeast. Recombinant CYP4F8 oxygenated arachidonic acid to (18R)-hydroxyarachidonate, whereas prostaglandin (PG) D(2), PGE(1), PGE(2), PGF(2alpha), and leukotriene B(4) appeared to be poor substrates. Three stable PGH(2) analogues, 9,11-epoxymethano-PGH(2) (U-44069), 11, 9-epoxymethano-PGH(2) (U-46619), and 9,11-diazo-15-deoxy-PGH(2) (U-51605) were rapidly metabolized by omega2- and omega3-hydroxylation. U-44069 was oxygenated with a V(max) of approximately 260 pmol min(-)(1) pmol P450(-1) and a K(m) of approximately 7 micrometer. PGH(2) decomposes mainly to PGE(2) in buffer and to PGF(2alpha) by reduction with SnCl(2). CYP4F8 metabolized PGH(2) to 19-hydroxy-PGH(2), which decomposed to 19-hydroxy-PGE(2) in buffer and could be reduced to 19-hydroxy-PGF(2alpha) with SnCl(2). 18-Hydroxy metabolites were also formed (approximately 17%). PGH(1) was metabolized to 19- and 18-hydroxy-PGH(1) in the same way. Microsomes of human seminal vesicles oxygenated arachidonate, U-44069, U-46619, U-51605, and PGH(2), similar to CYP4F8. (19R)-Hydroxy-PGE(1) and (19R)-hydroxy-PGE(2) are the main prostaglandins of human seminal fluid. We propose that they are formed by CYP4F8-catalyzed omega2-hydroxylation of PGH(1) and PGH(2) in the seminal vesicles and isomerization to (19R)-hydroxy-PGE by PGE synthase. CYP4F8 is the first described hydroxylase with specificity and catalytic competence for prostaglandin endoperoxides.

Kinetics of manganese lipoxygenase with a catalytic mononuclear redox center.

Manganese lipoxygenase was isolated from the take-all fungus, Gaeumannomyces graminis, and the oxygenation mechanism was investigated. A kinetic isotope effect, k(H)/k(D) = 21-24, was observed with [U-(2)H]linoleic acid as a substrate. The relative biosynthesis of (11S)-hydroperoxylinoleate (11S-HPODE) and (13R)-hydroperoxylinoleate (13R-HPODE) was pH-dependent and changed by [U-(2)H]linoleic acid. Stopped-flow kinetic traces of linoleic and alpha-linolenic acids indicated catalytic lag times of approximately 45 ms, which were followed by bursts of enzyme activity for approximately 60 ms and then by steady state (k(cat) approximately 26 and approximately 47 s(-1), respectively). 11S-HPODE was isomerized by manganese lipoxygenase to 13R-HPODE and formed from linoleic acid at the same rates (k(cat) 7-9 s(-1)). Catalysis was accompanied by collisional quenching of the long wavelength fluorescence (640-685 nm) by fatty acid substrates and 13R-HPODE. Electron paramagnetic resonance (EPR) of native manganese lipoxygenase showed weak 6-fold hyperfine splitting superimposed on a broad resonance indicating two populations of Mn(II) bound to protein. The addition of linoleic acid decreased both components, and denaturation of the lipoxygenase liberated approximately 0.8 Mn(2+) atoms/lipoxygenase molecule. These observations are consistent with a mononuclear Mn(II) center in the native state, which is converted during catalysis to an EPR silent Mn(III) state. We propose that manganese lipoxygenase has kinetic and redox properties similar to iron lipoxygenases.

Cloning of linoleate diol synthase reveals homology with prostaglandin H synthases.

Linoleate diol synthase is a homotetrameric ferric hemeprotein, which catalyzes dioxygenation of linoleic acid to (8R)-hydroperoxylinoleate and isomerization of the hydroperoxide to (7S,8S)-dihydroxylinoleate. Ferryl intermediates and a tyrosyl radical are formed in the reaction. Linoleate diol synthase was digested with endoproteinase Lys-C, and internal peptides were sequenced. The sequence information was used for reverse transcription-polymerase chain reaction analysis, and a cDNA probe was obtained. Northern blot analysis of linoleate diol synthase suggested a 3.7-kilobase pair (kb) mRNA. A full-length clone of the linoleate diol synthase gene was obtained by screening of a genomic lambda-ZAP II library of the fungus Gaeumannomyces graminis. The 5'-untranslated region contained CAAT- and TATA-like boxes. The gene contained three short introns and spanned over 3.2-kb. The deduced open reading frame consisted of 2.9-kb, which corresponded to 978 amino acids and a molecular subunit mass of 108,000. Data base analysis with the gapped BLAST algorithm showed that 391 residues of linoleate diol synthase was 23-24% identical and 36-37% positive with the catalytic domain of mammalian prostaglandin H (PGH) synthase-2. Based on homology with PGH synthases, the proximal heme ligand of linoleate diol synthase was tentatively identified as His-379 and the important tyrosine for catalysis as residue 376 (apparent consensus EFNXXXYXWH). The distal heme ligand was tentatively identified as His-203 (apparent consensus THXXFXT). We conclude from catalytic and structural similarities that linoleate diol synthase and PGH synthases likely share common ancestry and may belong to a gene family of fatty acid heme dioxygenases.

Gene expression of a novel cytochrome P450 of the CYP4F subfamily in human seminal vesicles.

19R-Hydroxyprostaglandins are major components of human seminal fluid. They are apparently formed in the seminal vesicles by NADPH-dependent omega2-hydroxylation. The hydroxylase is likely a cytochrome P450 (CYP), which has not been identified. To address this issue we studied gene expression of CYPs in human seminal vesicles (n = 4) with reverse-transcription polymerase chain reaction (RT-PCR). CYP1B1, CYP2E1, CYP2J2, CYP3A5, CYP4B1, and CYP4B1 with insertion of three nucleotides (Ser207) were detected in all subjects. RT-PCR with degenerate primers for the CYP4 family yielded a novel cDNA sequence, which was derived from a previously reported genomic sequence on chromosome 19p13.1 and present in all subjects. cDNA cloning showed that the deduced amino acid sequence consisted of 520 amino acids. Northern blot analysis demonstrated mRNA transcripts of approximately 2.1 and approximately 2.3 kb. The deduced protein showed 81.2 and 76.7% amino acid identity with the human enzymes CYP4F2 and CYP4F3. The novel CYP was designated CYP4F8.

Analysis of cytochrome P450 metabolites of arachidonic and linoleic acids by liquid chromatography-mass spectrometry with ion trap MS.

We have used reversed phase-high performance liquid chromatography-mass spectrometry (RP-HPLC-MS) with an ion trap mass spectrometer to study the metabolism of arachidonic and linoleic acids by human recombinant cytochrome P450 (CYP) enzymes. We first recorded the MS2 spectra of the carboxylate anions of epoxides, diols, omega-side chain, and bisallylic hydroxy fatty acids of arachidonic, octadeuterated arachidonic, and linoleic acids. The metabolites formed by CYP2C9 and CYP2C19 were then studied. CYP2C9 converted arachidonic and linoleic acids to epoxides/diols and monohydroxy fatty acids. Some hydroxyeicosatetraenoic acids (HETEs) were studied in detail to investigate the oxygenation mechanism. Incubation of CYP2C9 under oxygen-18 gas showed that all HETEs had incorporated oxygen-18 to the same degree. Chiral HPLC showed that CYP2C9 formed 15R-HETE (72% of the R enantiomer), 13S-HETE (90%), and 11R-HETE (57%). RP-HPLC-MS analysis revealed that CYP2C19 oxygenated arachidonic acid to 19-HETE, 14,15-epoxyeicosatrienoic acid (EET), and 8,9-EET as main metabolites. The method was sufficiently sensitive to identify arachidonic acid metabolites formed by some other isozymes. RP-HPLC-MS with MS2 seems to be useful for rapid identification of fatty acid metabolites in complex mixtures formed by cytochrome P450.

Analysis of novel hydroperoxides and other metabolites of oleic, linoleic, and linolenic acids by liquid chromatography-mass spectrometry with ion trap MSn.

Linoleate is oxygenated by manganese-lipoxygenase (Mn-LO) to 11S-hydroperoxylinoleic acid and 13R-hydroperoxyoctadeca-9Z,11E-dienoic acid, whereas linoleate diol synthase (LDS) converts linoleate sequentially to 8R-hydroperoxylinoleate, through an 8-dioxygenase by insertion of molecular oxygen, and to 7S,8S-dihydroxylinoleate, through a hydroperoxide isomerase by intramolecular oxygen transfer. We have used liquid chromatography-mass spectrometry (LC-MS) with an ion trap mass spectrometer to study the MSn mass spectra of the main metabolites of oleic, linoleic, alpha-linolenic and gamma-linolenic acids, which are formed by Mn-LO and by LDS. The enzymes were purified from the culture broth (Mn-LO) and mycelium (LDS) of the fungus Gaeumannomyces graminis. MS3 analysis of hydroperoxides and MS2 analysis of dihydroxy- and monohydroxy metabolites yielded many fragments with information on the position of oxygenated carbons. Mn-LO oxygenated C-11 and C-13 of 18:2n-6, 18:3n-3, and 18:3n-6 in a ratio of approximately 1:1-3 at high substrate concentrations. 8-Hydroxy-9(10)epoxystearate was identified as a novel metabolite of LDS and oleic acid by LC-MS and by gas chromatography-MS. We conclude that LC-MS with MSn is a convenient tool for detection and identification of hydroperoxy fatty acids and other metabolites of these enzymes.

A protein radical and ferryl intermediates are generated by linoleate diol synthase, a ferric hemeprotein with dioxygenase and hydroperoxide isomerase activities.

Linoleate diol synthase (LDS) was isolated as a hemeprotein from the fungus Gaeumannomyces graminis. LDS converts linoleate sequentially to 8R-hydroperoxylinoleate (8-HPODE) through an 8-dioxygenase by insertion of molecular oxygen and to 7S,8S-dihydroxylinoleate through a hydroperoxide isomerase by intramolecular oxygen transfer. Light absorption and EPR spectra of LDS indicated that the heme iron was ferric and mainly high spin. Oxygen consumption during catalysis started after a short time lag which was reduced by 8-HPODE. Catalysis declined due to suicide inactivation. Stopped flow studies with LDS and 8-HPODE at 13 degreesC showed a rapid decrease in light absorption at 406 nm within 35 ms with a first order rate constant of 90-120 s-1. Light absorption at 406 nm then increased at a rate of approximately 4 s-1, whereas the absorption at 421 nm increased after a lag time of approximately 5 ms at a rate of approximately 70 s-1. EPR spectra at 77 K of LDS both with linoleic acid and 8-HPODE showed a transient doublet when quenched after incubation on ice for 3 s (major hyperfine splitting 2.3 millitesla; g = 2.005), indicating a protein radical. The relaxation properties of the protein radical suggested interaction with a metal center. 8-HPODE generated about twice as much radical as linoleic acid, and the 8-HPODE-induced radical appeared to be stable. Our results suggest that LDS may form, in analogy with prostaglandin H synthases, ferryl intermediates and a protein radical during catalysis.

Manganese lipoxygenase. Purification and characterization.

A linoleic acid (13R)-lipoxygenase was purified to homogeneity from the culture medium of Gäumannomyces graminis, the take-all fungus, by hydrophobic interaction, cation exchange, lectin affinity, and size-exclusion chromatography. The purified dioxygenase lacked light absorption between 300 and 700 nm. Gel filtration indicated an apparent molecular mass of approximately 135 kDa in 6 M urea and approximately 160 kDa in buffer. SDS-polyacrylamide gel electrophoresis (PAGE) showed that the enzyme was heterogeneous in size and consisted of diffuse protein bands of 100-140 kDa. Treatment with glycosidases for N- and O-linked oligosaccharides yielded a distinct protein of approximately 73 kDa on SDS-PAGE. Atomic emission spectroscopy indicated 0.5-1.0 manganese atom/enzyme molecule. The isoelectric point was approximately 9.7, and the enzyme was active between pH 5 and 11 with optimum activity at pH 7. 0. For molecular oxygen, Km was 30 microM and Vmax 10 micromol mg-1min-1; for linoleic acid, Km was 4.4 micromol, Vmax 8.2 micromol mg-1min-1, and the turnover number 1100 min-1. The enzyme oxidized linolenic acid twice as fast as linoleic acid. The main products were identified by mass spectrometry as 13-hydroperoxy-(9Z,11E, 15Z)-octadecatrienoic and 13-hydroperoxy-(9Z,11E)-octadecadienoic acids, respectively. After reduction of the hydroperoxide, steric analysis of methyl 13-hydroxyoctadecadienoate by chiral high performance liquid chromatography yielded one enantiomer (>95%), which co-eluted with the R-stereoisomer of methyl (13R, 13S)-hydroxyoctadecadienoate. Arachidonic and dihomogammalinolenic acids were not substrates, while oxygen consumption, UV analysis, and mass spectrometric analysis indicated that gamma-linolenic acid was oxygenated both at C-11 and C-13. The enzyme was active at 60 degreesC and after treatment with 6 M urea. It was strongly inhibited by 10-50 microM concentrations of eicosatetraynoic acid and a lipoxygenase inhibitor (N-(3-phenoxycinnamyl)acetohydroxamic acid), but many other lipoxygenase inhibitors (100 microM) were without effect. We conclude that, after deglycosylation, the enzyme has the same size on SDS-PAGE as mammalian and marine lipoxygenases, but it differs from all previously described lipoxygenases in three ways. It is secreted, it forms (13R)-hydroperoxy-(9Z, 11E)-octadecadienoic acid, and it contains manganese.

Liver microsomes from the yellow rat snake (Elaphe obsoleta) and American bullfrog (Rana catesbeiana) oxidize polyunsaturated fatty acids by NADPH-dependent hydroxylation and epoxidation.

Polyunsaturated fatty acids (PUFAs) can be oxygenated by mammalian hepatic P450s to a series of metabolites. The most prominent of these are formed by omega- and (omega-1)-hydroxylation, epoxidation of the double bonds or bisallylic hydroxylation. The object of the present investigation was to determine whether similar oxygenations are catalyzed by liver microsomes of the yellow rat snake (Elaphe obsoleta) and the American bullfrog (Rana catesbeiana). Liver microsomes were incubated with [1-14C]-labeled arachidonic (AA), eicosapentaenoic (EPA), and linoleic acids (LA) in the presence or absence of 1 mM NADPH, and the major metabolites were analyzed by reverse-phase and straight-phase high performance liquid chromatography and capillary gas chromatography-mass spectroscopy. No metabolites were produced in the absence of NADPH. Profiles of metabolites were different depending on the organism and the acclimation state. In all incubations, EPA was the most effective substrate utilized and LA the least effective. The major products from EPA were 19-HEPE, 13-HEPE, and 20-HEPE from cold-acclimated (5 degrees C), warm-acclimated (22 degrees C) frogs, and snakes (22 degrees C), respectively. In contrast, 20-HETE production from AA was greater than 19-HETE in all three. Cold-acclimated frog liver microsomes produced significantly more of all metabolites when compared with microsomes from warm-acclimated frogs. We conclude that amphibian and snake liver can catalyze epoxidation and hydroxylation of PUFAs and that products are species-specific and acclimation-state dependent.

Cytochromes P450 with bisallylic hydroxylation activity on arachidonic and linoleic acids studied with human recombinant enzymes and with human and rat liver microsomes.

Bisallylic carbons of polyunsaturated fatty acids can be hydroxylated in NADPH-dependent reactions in liver microsomes. Human recombinant cytochromes P450 and human and rat liver microsomes were assayed for bisallylic hydroxylation activity. CYP1A2, CYP2C8, CYP2C9, CYP2C19 and CYP3A4 converted [14C]linoleic acid to 14C-labeled 11-hydroxyoctadecadienoic acid (11-HODE), whereas [14C]arachidonic acid was oxygenated by CYP1A2 and CYP3A4 to 14C-labeled 13-hydroxyeicosatrienoic acid (13-HETE), 10-HETE and 7-HETE as determined by HPLC. Both substrates were also converted to many other metabolites. CYP2C9 appeared to form 12R-HETE and 13-HETE, whereas CYP2C8 formed 13-HETE, 11-HETE and 15-HETE as main monohydroxy metabolites. Fetal human liver microsomes metabolized linoleic acid to 11-HODE as a major hydroxy metabolite, whereas arachidonic acid appeared to be hydroxylated at C13, C20 and, to some extent, at C10, C19 and C7. Fetal liver microsomes mainly formed 13R-HETE, whereas adult human liver microsomes and CYP1A2 mainly formed 13S-HETE. 7,8-Benzoflavone (5 microM) and furafylline (20 microM), two inhibitors of CYP1A2, reduced the bisallylic hydroxylation activity of adult human liver microsomes. Treatment of rats with erythromycin or dexamethasone induced bisallylic hydroxylation of linoleic acid to 11-HODE in liver microsomes by 2- and 10-fold, respectively. The biosynthesis of 11-HODE by microsomes of dexamethasone-treated rats was inhibited by troleandomycin (ED50 = 1 microM) and by polyclonal antibodies against CYP3A1, suggesting that CYP3A1 could catalyze bisallylic hydroxylations in the dexamethasone-treated rat. We conclude from steric analysis of 13-HETE and the effects of CYP inhibitors on adult human liver microsomes that CYP1A2 might contribute to its bisallylic hydroxylation activity.

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 ratio. 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.