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.     

The total synthesis and characterization of six methyl branched isomers of 8,11,14-eicosatrienoic acid

Six methyl branched isomers of 8,11,14-eicosatrienoic acid were prepared in which a methyl branch was located on carbons 5, 10, 13, 17, 18 and 19. The     

Identification of 15-hydroxy-11,12-epoxyeicosatrienoic acid as a vasoactive 15-lipoxygenase metabolite in rabbit aorta

Arachidonic acid (AA) causes endothelium-dependent smooth muscle hyperpolarizations and relaxations that are mediated by a 15-lipoxygenase-I (15-LO-I) metabolite, 11,12,15-trihydroxyeicosatrienoic acid (11,12,15-THETA). We propose that AA is metabolized sequentially by 15-LO-I and hydroperoxide isomerase to an unidentified hydroxyepoxyeicosatrienoic acid (HEETA), which is hydrolyzed by a soluble epoxide hydrolase (sEH) to 11,12,15-THETA. After incubation of aorta with 14C-labeled AA, metabolites were extracted and the HEETAs were resolved by performing HPLC. Mass spectrometric analyses identified 15-Hydroxy-11,12-epoxyeicosatrienoic acid (15-H-11,12-EETA). Incubation of aortic incubates with methanol and acetic acid trapped the acid-sensitive 15-H-11,12-EETA as methoxydihydroxyeicosatrienoic acids (MDHEs) (367 m/z, M-H). Pretreatment of the aortic tissue with the sEH inhibitor 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA; 10(-6) M) increased the formation of 15-H-11,12-EETA, measured as MDHEs. Thus 15-H-11,12-EETA is an acid- and sEH-sensitive precursor of 11,12,15-THETA. Aortic homogenates and endothelial cells contain a 57-kDa protein corresponding to the rabbit sEH. In preconstricted aortic rings, AA (10(-7)-10(-4) M) and acetylcholine (10(-9)-10(-6) M) caused concentration-related relaxations that were enhanced by pretreatment with AUDA. These enhanced relaxations were inhibited by increasing extracellular [K(+)] from 4.8 to 20 mM. AA (3 x 10(-6) M) induced cell membrane hyperpolarization (from -31.0 +/- 1 to -46.8 +/- 2 mV) in aortic strips with an intact endothelium, which was enhanced by AUDA. These results indicate that 15-H-11,12-EETA is produced by the aorta, hydrolyzed by sEH to 11,12,15-THETA, and mediates relaxations by membrane hyperpolarization. 15-H-11,12-EETA represents an endothelium-derived hyperpolarizing factor.     

Formation of murine macrophage-derived 5-oxo-7-glutathionyl-8,11,14-eicosatrienoic acid (FOG7) is catalyzed by leukotriene C4 synthase.

5-Oxo-7-glutathionyl-8,11,14-eicosatrienoic acid (FOG(7)), a biologically active glutathione (GSH) adduct of the eicosanoid 5-oxo-eicosatrienoic acid (5-oxoETE), is the major metabolite formed within the murine peritoneal macrophage. The conjugation of GSH to electrophilic 5-oxoETE in vitro was found to be catalyzed by both soluble glutathione S-transferase and membrane-bound leukotriene C(4) (LTC(4)) synthase. The cytosolic glutathione S-transferase-catalyzed products were not biologically active; however, the adduct formed from recombinant LTC(4) synthase had identical mass spectrometric properties and biological activity to the macrophage-derived FOG(7). The biosynthesis of FOG(7) in the macrophage was inhibited by MK-886, a known inhibitor of LTC(4) synthase, suggesting that this nuclear membrane-bound enzyme might be responsible for GSH conjugation to 5-oxoETE in the intact cell. Subcellular fractionation revealed that the microsomal fraction from the murine macrophage contained the enzyme responsible for FOG(7) biosynthesis. Western blot analysis confirmed the presence of LTC(4) synthase in the microsomal fraction that did not catalyze conjugation of GSH to 1-chloro-2,4-dinitrobenzene, indicating an absence of microsomal glutathione S- transferase activity. These results suggest that LTC(4) synthase, thought to be specific for the conjugation of GSH to LTA(4), can also recognize 5-oxoETE as an electrophilic substrate.     

Conversion of oleic acid to 10-hydroxystearic acid by Nocardia paraffinae

Resting cells of Nocardia paraffinaeCBS 255 58 anaerobically converted oleic acid to 10-hydroxystearic acid (optimum activity at 35°C and pH 7). Addition of oleic acid (0.22 mg/ml of medium), as an inducer, improved the hydroxylating activity. The biocatalyst was inactivated by sonication, lyophilization and heating, but kept 65% of its initial activity after five freeze-thawing treatments. This activity appeared to be located either in the cells or bound to the plasmic membrane.     

Biotransformation of oleic acid into 10-hydroxy-8E-octadecenoic acid by Pseudomonas sp. 42A2

Pseudomonas sp. 42A2 when incubated for 36 h with oleic acid (20 g l^–1) in a stirred bioreactor, accumulated 10-hydroxy-8E-octadecenoic acid. Production in a 2 l bioreactor with 1.4 l of working volume, was increased from 0.65 g l^–1 to 7.4 g l^–1 with K _L a values ranging between 15 and 200 h^–1. A linear relationship was found between volumetric productivity and oxygen transfer rates and an exponential relation between the specific rate of product formation and specific growth rate.     

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

The enzyme prostaglandin H leads to 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.     

Conversion of oleic acid to 10-hydroxystearic acid by two species of ruminal bacteria

Bacteria able to convert oleic acid to 10-hydroxystearic acid were isolated from the ovine rumen. The solid hydroxy fatty acid produced from bacterial fermentations containing oleic acid was recovered by filtration, extraction into ether and crystallisation. The identity of the product was confirmed by HPLC and gas chromatography/mass spectrometry. One 10-hydroxystearic-acid-producing bacterial group was represented by two strains of an anaerobic gram-negative curved rod with tufts of flagella on the concave surface of the cell. The morphology and other characteristics enabled the strains to be tentatively identified as Selenomonas ruminantium. Another bacterium capable of the same transformation, represented by two strains of a facultatively anaerobic gram positive chain-forming coccus, was identified as Enterococcus faecalis. Since unsaturated fatty acids entering the rumen are normally hydrogenated, hydration of oleic acid represents an alternative fate of unknown significance in vivo.     

Steric analysis of 8-hydroxy- and 10-hydroxyoctadecadienoic acids and dihydroxyoctadecadienoic acids formed from 8R-hydroperoxyoctadecadienoic acid by hydroperoxide isomerases

8-Hydroxyoctadeca-9Z,12Z-dienoic acid (8-HODE) and 10-hydroxyoctadeca-8E,12Z-octadecadienoic acid (10-HODE) are produced by fungi, e.g., 8R-HODE by Gaeumannomyces graminis (take-all of wheat) and Aspergillus nidulans, 10S-HODE by Lentinula edodes, and 10R-HODE by Epichloe typhina. Racemic [8-(2)H]8-HODE and [10-(2)H]10-HODE were prepared by oxidation of 8- and 10-HODE to keto fatty acids by Dess-Martin periodinane followed by reduction to hydroxy fatty acids with NaB(2)H(4). The hydroxy fatty acids were analyzed by chiral phase high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) with 8R-HODE and 10S-HODE as standards. 8R-HODE eluted after 8S-HODE on silica with cellulose tribenzoate (Chiralcel OB-H), and 10S-HODE eluted before 10R-HODE on silica with an aromatic chiral selector (Reprosil Chiral-NR). 5S,8R-Dihydroxyoctadeca-9Z,12Z-dienoic acid (5S,8R-DiHODE) is formed from 18:2n-6 by A. nidulans and 8R,11S-dihydroxyoctadeca-9Z,12Z-dienoic acid (8R,11S-DiHODE) by Agaricus bisporus. 8R-Hydroperoxylinoleic acid (8R-HPODE) can be transformed to 5S,8R-DiHODE and 8R,11-DiHODE by Aspergillus spp., and 8R,13-dihydroxy-9Z,11E-dienoic acid (8R,13-DiHODE) can also be detected. We prepared racemic [5,8-(2)H(2)]5,8- and [8,11-(2)H(2)]8,11-DiHODE by oxidation and reduction as above and 8R,13S- and 8R,13R-DiHODE by oxidation of 8R-HODE by S and R lipoxygenases. The diastereoisomers were separated and identified by normal phase HPLC-MS/MS analysis. We used the methods for steric analysis of fungal oxylipins. Aspergillus spp. produced 8R-HODE (>95% R), 10R-HODE (>70% R), and 5S,8R- and 8R,11S-DiHODE with high stereoselectivity (>95%), whereas 8R,13-DiHODE was likely formed by nonenzymatic hydrolysis of 8R,11S-DiHODE.     

Total synthesis of the novel bacterial fatty acid 16-methyl-8(Z)-heptadecenoic acid.

The recently discovered bacterial fatty acid 16-methyl-8(Z)-heptadecenoic acid was synthesized for the first time in four steps (22% overall yield) starting from commercially available 8-methylnonanoic acid. The synthetic approach provided enough material to corroborate the structure and stereochemistry of the acid, which was recently identified in a Micrococcus bacterium from Lake Pomorie in Bulgaria. Reference equivalent-chain length values in nonpolar capillary gas chromatography for methyl 16-methyl-8(Z)-heptadecenoate and methyl 16-methyl-8(E)-heptadecenoate are also reported. This information will be helpful in subsequent characterizations of these fatty acids, as well as in the total identification of the fatty acid profile of bacteria producing these compounds.     

Production of 10(S)-hydroxy-8(E)-octadecenoic acid mono-estolides by lipases in non-aqueous media

In this study, Novozym 435, a lipase B from Candida antarctica, was used for fatty acid polymerization. For the first time, an apolar reaction media, n-hexane, was used to synthesize in vitro estolides from trans-hydroxy-fatty acids derived from the biotransformation of oleic acid by Pseudomonas aeruginosa 42A2 NCIMB 40045. We studied the effects of the substrate, the enzyme ratio, the enzyme stability and the reusability of the biocatalyst. To determine the structure of the oligomers formed, both liquid chromatography mass spectrometry and MALDI-TOF mass spectrometry, with a DHB matrix neutralized with lithium hydroxide, were used to obtain simpler mass spectra. Estolides composed of two units of (10S)-HOME were synthesized with a reaction yield of 30%. Finally, various lipases were screened, and another apolar organic solvent, iso-octane, was assayed to try to increase the reaction yield.     

Transformations of 12,13-epoxy-11-hydroxy-9-octadecenoic acid and 4,5-epoxy-N-acetylsphingosine by incubation with liver homogenate and liver microsomes

Transformation of 12,13-epoxy-11-hydroxy-9-octadecenoic acid and 4,5-epoxy-N-acetylsphingosine by addition of porcine liver homogenate and human liver microsomes, respectively was investigated. Both epoxides were converted to corresponding dioles by porcine liver homogenate, but not by human liver microsomes, suggesting location of the hydrolyzing enzymes not in the microsomes, but within the cell wall.     

Hydroxylation of 10-deoxoartemisinin to 15-hydroxy-10-deoxoartemisinin by Aspergillus niger

The microbial hydroxylation of 10-deoxoartemisinin was investigated with the aim of obtaining preparative yields of hydroxy derivatives. During 14 d at 28°C and pH 6.5 Aspergillus niger transformed 10-deoxoartemisinin (500 mg l^–1) to 15-hydroxy-10-deoxoartemisinin (26%) and 7-hydroxy-10-deoxoartemisinin (69%).     

Inhibitory effect of 5,8,11-eicosatrienoic acid on angiogenesis

IntroductionCartilage contains high levels of n-9 eicosatrienoic acid (20:3n-9) but no blood vessels. 20:3n-9 might inhibit angiogenesis.Materials and methodsAngiogenesis was measured in human umbilical vein endothelial cells and diploid fibroblasts. Co-culture was performed with vascular endothelial growth factor-A (VEGF-A, 10 ng/mL) and fatty acids (0.1–10 μmol/L). After 10 days of incubation and immunostaining for endothelial cells, vessel areas were calculated with image analyser software.ResultsAddition of 20:3n-9 and n-3 eicosatrienoic acid (20:3n-3) dose dependently inhibited VEGF-A-stimulated angiogenesis (more than the positive control suramin). Arachidonic, eicosapentaenoic, dihomo-γ-linolenic (20:3n-6) and oleic acids did not affect VEGF-A-stimulated angiogenesis even at 10 μmol/L. Arachidonic and dihomo-γ-linolenic acids enhanced angiogenesis without VEGF-A.Discussion and conclusionsWe suggest that the presence of 20:3n-9 in cartilage may be related to its vessel-free status and that 20:3n-9 may be useful for the treatment of disorders with excessive vasculature.AcknowledgementsThis work was partly supported by Polyene Project, Inc.     

Specificity and mode of action of the antifungal fatty acid cis-9-heptadecenoic acid produced by Pseudozyma flocculosa

cis-9-Heptadecenoic acid (CHDA), an antifungal fatty acid produced by the biocontrol agent Pseudozyma flocculosa, was studied for its effects on growth and/or spore germination in fungi. Inhibition of growth and/or germination varied considerably and revealed CHDA sensitivity groups within tested fungi. Analysis of lipid composition in these fungi demonstrated that sensitivity was related primarily to a low intrinsic sterol content and that a high level of unsaturation of phospholipid fatty acids was not as involved as hypothesized previously. Our data indicate that CHDA does not act directly with membrane sterols, nor is it utilized or otherwise modified in fungi. A structural mechanism of CHDA, consistent with the other related antifungal fatty acids produced by P. flocculosa, is proposed in light of its activity and specificity. The probable molecular events implicated in the sensitivity of fungi to CHDA are (i) partitioning of CHDA into fungal membranes; (ii) a variable elevation in fluidity dependent on the buffering capability (sterol content) in fungi; and (iii) higher membrane disorder causing conformational changes in membrane proteins, increased membrane permeability and, eventually, cytoplasmic disintegration.     

Conversion of calciferols to isotachysterols by trifluoroacetic acid

The conversion of the calciferols to isotachysterols using trifluoroacetic acid is described. The yield of isotachysterol formed is greater by about 10% than that obtained with either antimony trichloride or acetyl chloride. Trifluoroacetic acid also produces a more intense absorption at 500 nm with calciferols than does antimony trichloride, however, the color is less stable.     

Conjugated linoleic acid accumulation via 10-hydroxy-12-octadecaenoic acid during microaerobic transformation of linoleic acid by Lactobacillus acidophilus

Specific isomers of conjugated linoleic acid (CLA), a fatty acid with potentially beneficial physiological and anticarcinogenic effects, were efficiently produced from linoleic acid by washed cells of Lactobacillus acidophilus AKU 1137 under microaerobic conditions, and the metabolic pathway of CLA production from linoleic acid is explained for the first time. The CLA isomers produced were identified as cis-9, trans-11- or trans-9, cis-11-octadecadienoic acid and trans-9, trans-11-octadecadienoic acid. Preceding the production of CLA, hydroxy fatty acids identified as 10-hydroxy-cis-12-octadecaenoic acid and 10-hydroxy-trans-12-octadecaenoic acid had accumulated. The isolated 10-hydroxy-cis-12-octadecaenoic acid was transformed into CLA during incubation with washed cells of L. acidophilus, suggesting that this hydroxy fatty acid is one of the intermediates of CLA production from linoleic acid. The washed cells of L. acidophilus producing high levels of CLA were obtained by cultivation in a medium containing linoleic acid, indicating that the enzyme system for CLA production is induced by linoleic acid. After 4 days of reaction with these washed cells, more than 95% of the added linoleic acid (5 mg/ml) was transformed into CLA, and the CLA content in total fatty acids recovered exceeded 80% (wt/wt). Almost all of the CLA produced was in the cells or was associated with the cells as free fatty acid.     

Activation of rat mesenteric arterial KATP channels by 11,12-epoxyeicosatrienoic acid

Epoxyeicosatrienoic acids (EETs), the cytochrome P-450 epoxygenase metabolites of arachidonic acid, are candidates of endothelium-derived hyperpolarizing factors. We have previously reported that EETs are potent activators of cardiac ATP-sensitive K(+) (K(ATP)) channels, but their effects on the vascular K(ATP) channels are unknown. With the use of whole cell patch-clamp techniques with 0.1 mM ATP in the pipette and holding at -60 mV, freshly isolated smooth muscle cells from rat mesenteric arteries had small glibenclamide-sensitive currents at baseline (13.1 +/- 3.9 pA, n = 5) that showed a 7.2-fold activation by 10 microM pinacidil (94.1 +/- 21.9 pA, n = 7, P < 0.05). 11,12-EET dose dependently activated the K(ATP) current with an apparent EC(50) of 87 nM. Activation of the K(ATP) channels by 500 nM 11,12-EET was inhibited by inclusion of the PKA inhibitor peptide (5 microM) but not by the inclusion of the PKC inhibitor peptide (100 microM) in the pipette solution. These results were corroborated by vasoreactivity studies. 11,12-EET produced dose-dependent vasorelaxation in isolated small mesenteric arteries, and this effect was reduced by 50% with glibenclamide (1 microM) preincubation. The 11,12-EET effects on vasorelaxation were also significantly attenuated by preincubation with cell-permeant PKA inhibitor myristoylated PKI(14-22), and, in the presence of PKA inhibitor, glibenclamide had no additional effects. These results suggest that 11,12-EET is a potent activator of the vascular K(ATP) channels, and its effects are dependent on PKA activities.     

Conversion of beta-methylbutyric acid to beta-hydroxy-beta-methylbutyric acid by Galactomyces reessii.

beta-Hydroxy-beta-methylbutyric acid (HMB) has been shown to increase strength and lean mass gains in humans undergoing resistance-exercise training. HMB is currently marketed as a calcium salt of HMB, and thus, environmentally sound and inexpensive methods of manufacture are being sought. This study investigates the microbial conversion of beta-methylbutyric acid (MBA) to HMB by cultures of Galactomyces reessii. Optimal concentrations of MBA were in the range of 5 to 20 g/liter for HMB production. Preliminary shake flask experiments indicated that HMB yields were sensitive to dissolved oxygen levels and that cell growth decreased significantly as MBA concentrations increased. Degradation of HMB was faster at acidic pH, and pH 7.0 was optimal for HMB production. Resting cells obtained from media without MBA could efficiently convert MBA to HMB. Thus, a two-step, fed-batch fermentation procedure in which biomass was first produced, followed by coaddition of MBA and glucose, while dissolved oxygen was maintained at 20% of saturation, was designed. A maximum HMB concentration of 38 g/liter was obtained after 136 h, and the molar conversion yield was more than 0.50 mol of HMB/mol of MBA during the fermentation.