Release of prostaglandins and monohydroxy and trihydroxy metabolites of linoleic and arachidonic acids by adult and fetal aortae and ductus arteriosus

The conversion of arachidonic acid (20:4) to prostaglandins by vascular tissue is important in the adult because of the antithrombotic effect of prostacyclin and in the fetus because of the vasodilatory effect of prostaglandin (PG) E2 on the ductus arteriosus. We have shown that vascular tissue converts various polyunsaturated fatty acids to monohydroxy and trihydroxy metabolites derived from hydroperoxides, which may be involved in regulating prostaglandin synthesis. We have now measured the amounts of these hydroperoxide metabolites, as well as those of prostaglandins, released from slices of rat, rabbit and bovine aortae, as well as from fetal calf aorta and ductus arteriosus. The major oxygenated polyunsaturated fatty acid metabolite formed by rat and bovine blood vessels was 6-oxo-PGF1 alpha. Fetal calf aorta and ductus arteriosus produced about five times as much 6-oxo-PGF1 alpha as adult bovine aorta. Much smaller amounts of the cyclooxygenase products, PGE2, 12-hydroxy-5,8,10-heptadecatrienoic acid, 11-hydroxy-5,8,12,14-icosatetraenoic acid (11-hydroxy-20:4), and 15-hydroxy-20:4, were released by aortae. Small amounts of the lipoxygenase product, 12-hydroxy-20:4, were also detected. Substantial amounts of free and esterified monohydroxy and trihydroxy metabolites of linoleic acid (18:2) were detected, especially in rat and rabbit aortae. Rabbit aorta, which had low cyclooxygenase activity, formed more oxygenated 18:2 metabolites than 20:4 metabolites. Indomethacin did not inhibit the formation of the 18:2 metabolites, indicating that cyclooxygenase was not involved. Neither exogenous 13-hydroxy-18:2 nor trihydroxyoctadecenoic acid was incorporated to a large extent into lipids from vascular endothelial or smooth muscle cells, suggesting that the esterified 18:2 oxygenation products had arisen mainly via direct oxygenation of lipids.     

Metabolism of eicosapentaenoic acid by aorta: formation of a novel 13-hydroxylated prostaglandin

We have investigated the metabolism by fetal calf aorta of eicosapentaenoic acid (20:5) and docosahexaenoic acid (22:6), two polyunsaturated fatty acids found in high concentrations in marine oils. The major product formed from 20:5 by particulate fractions from fetal calf aorta is delta 17-6-oxoprostaglandin F1 alpha. In addition, we detected a novel isomer of delta 17-6-oxoprostaglandin F1 alpha, in which a hydroxyl group is present in the 13-position instead of the 15-position. Eicosapentaenoic acid is also converted to 12-hydroxy-5,8,10,14-heptadecatetraenoic acid as well as to five monohydroxy isomers with hydroxyl groups present in the 11, 12, 14, 15, and 18 positions. Although 20:5 was metabolized at about one-third the rate of arachidonic acid (20:4), greater amounts of monohydroxy fatty acids, the major one being the 11-hydroxy metabolite, were formed from 20:5. Unlike 20:5, 22:6 was not metabolized to any detectable products by fetal calf aorta, but both of these polyunsaturated fatty acids inhibited the oxygenation of 20:4 by cyclooxygenase from aorta with IC50 values of 4.1 microM (22:6) and 15 microM (20:5). These results suggest that 20:5 has a high affinity for cyclooxygenase, but that the intermediate 11-oxygenated intermediate has a lower affinity than the corresponding intermediate from 20:4, resulting in a greater loss of substrate after a single oxygenation. The formation of oxygenation products from both 20:4 and 20:5 was inhibited by 13-hydroperoxy-9,11-octadecadienoic acid (13hp-18:2). The IC50 values for inhibition of cyclooxygenase products by 13hp-18:2 were about twice as high as those for inhibition of prostacyclin synthase products. Consequently, there was little diversion of prostaglandin endoperoxides to other prostaglandins in the presence of 13hp-18:2.     

Conversion of linoleic acid hydroperoxide to hydroxy, keto, epoxyhydroxy, and trihydroxy fatty acids by hematin

We have carried out a study of the reaction of 13-hydroperoxy-9-cis,11-trans-octadecadienoic acid (linoleic acid hydroperoxide) with hematin. The major products are erythro-11-hydroxy-12,13-epoxy-9-octadecenoic acid, threo-11-hydroxy-12,13-epoxy-9-octadecenoic acid, 9,12,13-trihydroxy-10-octadecenoic acid, 13-keto-9,11-octadecadienoic acid, and 13-hydroxy-9,11-octadecadienoic acid. Several minor products have also been identified, including 9-hydroxy-12,13-epoxyoctadecenoic acid, 11-hydroxy-9,10-epoxy-12-octadecenoic acid, 9-hydroxy-10,12-octadecadienoic acid, and 9-keto-10,12-octadecadienoic acid. Oxygen labeling studies indicate that the observed products arise by at least two pathways. In the major pathway, hematin reduces 13-hydroperoxy-9,11-octadecadienoic acid by one electron to an alkoxyl radical that cyclizes to an adjacent double bond to form an epoxy allylic radical. The allylic radical either couples to the hydroxyl radical coordinated to hematin or diffuses from the solvent cage and couples to O2, forming a peroxyl radical. In the minor pathway, the hydroperoxide is oxidized by one electron to a 13-peroxyl radical that undergoes beta-scission to a pentadienyl radical and O2. Exchange of hydroperoxide-derived O2 for dissolved O2 occurs at this stage followed by coupling of O2 to either terminus of the pentadienyl radical. Both pathways of hydroperoxide metabolism generate significant quantities of peroxyl radicals that epoxidize the isolated double bonds of dihydroaromatic molecules. The products of hydroperoxide reaction with hematin and the oxygen labeling patterns are very similar to the products of unsaturated fatty acid hydroperoxide metabolism by platelets, aorta, and lung. Our results not only provide a mechanism for the formation of a series of mammalian metabolites of linoleic and arachidonic acids but also offer an estimate of the yield of peroxyl radicals generated during the process.     

Identification of diacylglycerol and triacylglycerol containing 11,12,13-trihydroxy-9,14-octadecadienoic acid in castor oil

Castor oil has many industrial uses. Molecular species of acylglycerols containing monohydroxy, dihydroxy and trihydroxy fatty acids in castor oil have been reported. We report here the identification of acylglycerols containing a triOH18:2 fatty acid in castor oil. The structure of this novel fatty acid was proposed as 11,12,13-trihydroxy-9,14-octadecadienoic acid by the mass spectrometry of the lithiated adducts of acylglycerols in the HPLC fractions of castor oil. The fragmentation pathways of the lithiated adduct of 11,12,13-trihydroxy-9,14-octadecadienoic acid were proposed. We also proposed the biosynthetic pathways of polyhydroxy fatty acids in castor.     

Metabolism of linoleic and arachidonic acids in VX2 carcinoma tissue: Identification of monohydroxy octadecadienoic acids and monohydroxy eicosatetraenoic acids

The metabolism of arachidonic and linoleic acids by VX₂ carcinoma tissue was determined. Prostaglandin E₂ was the major metabolic product of arachidonic acid in the neoplastic tissue. Minor products accounting for 3– 8% of arachidonic acid metabolism were 11-hydroxy-5, 8, 12, 14-eicosatetraenoic acid (11-HETE) and 15-hydroxy-5, 8, 11, 13-eicosatetraenoic acid (15-HETE). Linoleic acid was converted to a mixture of 9-hydroxy-10, 12-octadecadienoic acid (9-HODD) and 13-hydroxy-9, 11-octadecadienoic acid (13-HODD). The conversion of linoleic acid to monohydroxy C-18 fatty acids varied from 40–80% 9-HODD and 20–60% 13-HODD in tumor tissue harvested from different animals. The quantity of monohydroxy C-18 fatty acids biosynthesized by VX₂ carcinoma tissue from endogenous linoleic acid equals or exceeds that of prostaglandin E₂ biosynthesis from endogenous arachidonic acid. The presence of a hydroxyl group adjacent to a conjugated diene suggest that the monohydroxy C-18 and monohydroxy C-20 fatty acids were formed via the action of lipoxygenase-like enzymes. These lipoxygenase-like reactions are inhibited by indomethacin in a concentration-dependent fashion similar to the inhibition of prostaglandin E₂ biosynthesis. The enzymes catalyzing the lipoxygenase-like reactions of linoleic and arachidonic acids are localized in the microsomal fraction of VX₂ carcinoma tissue. These data suggest that the lipoxygenase-like reactions are catalyzed by fatty acid cyclooxygenase and that there are two major pathways of fatty acid cyclooxygenase metabolism of polyenoic fatty acids in the neoplastic tissue. One pathway involves the formation of prostaglandin E₂ via cyclic endoperoxy intermediates. The second pathway involves the formation of monohydroxy C-18 fatty acids from linoleic acid via lipoxygenase-like reactions.     

Effects of selenium-deficient diets on the production of prostaglandins and other oxygenated metabolites of arachidonic acid and linoleic acid by rat and rabbit aortae

Selenium is an essential component of glutathione peroxidase, which reduces free and esterified hydroperoxides of polyunsaturated fatty acids. Adequate glutathione peroxidase activity could be important for the maintenance of prostacyclin synthesis by blood vessels, since hydroperoxides can inhibit the formation of this substance. We have investigated the effects of dietary selenium deficiency on glutathione peroxidase activity and the synthesis of 6-oxoprostaglandin F1 alpha and monohydroxy and trihydroxy metabolites of polyunsaturated fatty acids by aorta. The latter products can be formed either by the actions of cyclooxygenase or lipoxygenase or by lipid peroxidation. Aortic glutathione peroxidase activity was reduced by over 80% by feeding rats a selenium-deficient diet for 4 weeks, and to undetectable levels after 6 weeks. There were no appreciable differences in the levels of free and esterified oxygenated metabolites of linoleic acid or arachidonic acid between the control and treated groups after 4 weeks. However, after 6 weeks, there were modest, but statistically significant reductions in the formation of 6-oxoprostaglandin F1 alpha and monohydroxy products formed by cyclooxygenase. On the other hand, the amounts of esterified 18:2 metabolites appeared to be higher in aortae from animals on the selenium-deficient diet, although only the increase in esterified 9-hydroxy-10,12-octadecadienoic acid was statistically significant. These results suggest that selenium deficiency can affect the formation of prostacyclin and other oxygenated metabolites of polyunsaturated fatty acids by aorta, possibly by increasing lipid peroxidation. However, the differences between control and selenium-deficient rats after 6 weeks were not very dramatic, in spite of the fact that glutathione peroxidase activity was undetectable. It would therefore appear that additional mechanisms are also involved in controlling the levels of lipid hydroperoxides in aorta.     

Biotransformation of linoleic acid by porcine leukocytes

Linoleic acid is an important essential fatty acids of leukocyte cell membrane phospholipids from some animals, e.g. from pigs and rabbits, and is a known substrate for lipoxygenase(s), especially in plant systems. Lipoxygenase activity has also been well documented in leukocytes using arachidonic acid as a substrate. These findings and our own interest in the fate of linoleic acid have prompted us to investigate the biotransformation of this essential fatty acids in leukocytes.Porcine leukocytes were isolated from whole blood by dextrane precipitation of the erythrocytes and by centrifugation. Broken cells were incubated with exogenous linoleic acid and four major biotransformation products, X₁, X₂, X₃ and X₄, were formed. Following isolation by silicagel column chromatography and thin layer chromatography, the products were derivatized and characterized by GC/MS. Derivatization included hydrogenation, methyl ester formation, n-butyl boronate formation and trimethylsilylation, and various types of derivatives were made in order to facilitate the structure elucidation. The major product X₁, which represented 60.5% of the total metabolites formed, was identified as 13-hydroxy-9,11-octadecadienoic acid. Product X₂ (16.2%) was shown to be 11-hydroxy-12,13-epoxy-9-octadecenoic acid. Products X₃ and X₄ (respectively 5.2 and 7.5%) resulted in identical thermore, each of the products X₃ and X₄ was shown to be a mixture of two positional isomers, i.e. of 9,12,13-trihydroxy-10-octadecenoic acid (70%) and 9,10,13-trihydroxy-12-octadecenoic acid (30%). With regard to the structure elucidation of the latter isomers, the mixed hydrogenated, n-butylboronate, methyl ester, TMS-ether derivatives were shown to be of particular value for the determination of the vicinal diol position.The metabolism of linoleic acid in porcine leukocytes is analogous to that by cereal lipoxygenases. A major difference however is that porcine leukocyte lipoxygenase predominantly yields products, which arise through 13-lipoxygenation, whereas, in cereals, transformation products of 9-hydroperoxy-10,12-octadecadienoic acid are formed to the same extent as metabolites of 13-hydroperoxy-9,11-octadecadienoic acid.     

Formation of 6-oxoprostaglandin F1 alpha, 6,15-dioxoprostaglandin F1 alpha, and monohydroxyicosatetraenoic acids from arachidonic acid by fetal calf aorta and ductus arteriosus

Particulate fractions and slices from fetal calf aorta convert arachidonic acid to 6-oxoprostaglandin F1 alpha (6-oxoPGF1 alpha), 6,15-dioxoPGF1 alpha, 12-hydroxy-5,8,10-heptadecatrienoic acid, 11-hydroxy-5,8,12,14-icosatetraenoic acid (11h-20:4), and 15-hydroxy-5,8,11,13-icosatetraenoic acid (15h-20:4). In some cases, small amounts of 12-hydroxy-5,8,10,14-icosatetraenoic acid (12h-20:4) were also detected. The products were all identified by gas chromatography-mass spectrometry after purification by normal phase and argentation high pressure liquid chromatography. Both 11h-20:4 and 15h-20:4 appeared to be formed by prostaglandin endoperoxide synthetase rather than by lipoxygenases, since their formation was inhibited by indomethacin but not by nordihydroguaiaretic acid. The formation of 12h-20:4, on the other hand, was stimulated by indomethacin, probably due to increased substrate availability. The formation of hydroxyicosatetraenoic acids was markedly stimulated by adrenaline. Substantial amounts of 6,15-dioxoPGF1 alpha were formed from arachidonic acid by particulate fractions from fetal calf blood vessels, especially in the presence of relatively high substrate concentrations. The formation of this product was stimulated by methemoglobin and inhibited by adrenaline, glutathione, and tryptophan. It would appear that particulate fractions from fetal calf aorta convert arachidonic acid to 15-hydroperoxyPGI2, which can either be reduced in the presence of various cofactors to form PGI2 or dehydrated to give 15-oxoPGI2. The formation of hydroperoxides from arachidonic acid could be an important factor in regulating PGI2 synthesis in aorta, since PGI2 synthetase is strongly inhibited by such intermediates.     

Acid-catalyzed transformation of 13(S)-hydroperoxylinoleic acid into epoxyhydroxyoctadecenoic and trihydroxyoctadecenoic acids

Acid treatment of (13S)-(9Z,11E)-13-hydroperoxy-9,11-octadecadienoic acid in tetrahydrofuran-water solvent afforded mainly (11R,12R,13S)-(Z)-12,13-epoxy-11-hydroxy-9-octadecenoic acid, diastereomeric (Z)-11,12,13-trihydroxy-9-octadecenoic acids and four isomers of (E)-9,12,13(9,10,13)-trihydroxy-10(11)-octadecenoic acid. Other minor products were oxooctadecadienoic, (E)-9(13)-hydroxy-13(9)-oxo-10(11)-octadecenoic and (E)-12-oxo-10-dodecenoic acids. A heterolytic mechanism for acid catalysis was indicated, even though most of the products characterized also have been observed as a result of homolytic decomposition of the hydroperoxide via an oxy radical. Most of the products found in this study have been observed as metabolites of (13S)-(9Z,11E)-13-hydroperoxy-9,11-octadecadenoic acid in biological systems, and analogous compounds have been reported as metabolites of (12S)-(5Z,8Z,10E, 14Z)-12-hydroperoxy-5,8,10,14-hydroperoxy-5,8,10,14-eicosatetraenoic acid in either blood platelets or lung tissue.     

Identification of oxylipins with antifungal activity by LC–MS/MS from the supernatant of Pseudomonas 42A2

In microorganisms hydroxy fatty acids are produced from the biotransformation of unsaturated fatty acids. Such compounds belong to a class of oxylipins which are reported to perform a variety of biological functions such as anti-inflammatory or cytotoxic activity. These compounds have been found in rice and timothy plants after being infected by specific fungus. When grown in submerged culture with linoleic acid, Pseudomonas 42A2 accumulated in the supernatant several hydroxy fatty acids. In this work LC–MS/MS has been used to elucidate the structure of the components form the organic extract: 9-hydroxy-10,12-octadecadienoic acid; 13-hydroxy-9,11-octadecadienoic acid; 7,10-dihydroxy-8E-octadecenoic acid; 9,10,13-trihydroxy-11-octadecenoic acid and 9,12,13-trihydroxy-10-octadecenoic acid. Antimicrobial activity against several pathogenic fungal strains is presented: MIC (μg/mL) Verticillium dhaliae, 32; Macrophonia phaesolina, 32; Arthroderma uncinatum, 32; Trycophyton mentagrophytes, 64.     

Hydroperoxide-dependent epoxidation of unsaturated fatty acids in the broad bean (Vicia faba L.)

Incubation of linoleic acid with the 105,000g particle fraction of the homogenate of the broad bean (Vicia faba L.) led to the formation of the following products: 13(S)-hydroxy-9(Z),11(E)-octadecadienoic acid, 9,10-epoxy-12(Z)-octadecenoic acid (9(R),10(S)/9(S)/10(R), 80/20), 12,13-epoxy-9(Z)-octadecenoic acid (12(S),13(R)/12(R)/13(S), 64/36), and 9,10-epoxy-13(S)-hydroxy-11(E)-octadecenoic acid (9(S),10(R)/9(R),10(S), 91/9). Oleic acid incubated with the enzyme preparation in the presence of 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoic acid or cumene hydroperoxide was converted into 9,10-epoxyoctadecanoic acid (9(R),10(S)/9(S),10(R), 79/21). Two enzyme activities were involved in the formation of the products, an omega 6-lipoxygenase and a hydroperoxide-dependent epoxygenase. The lipoxygenase, but not the epoxygenase, was inhibited by low concentrations of 5,8,11,14-eicosatetraynoic acid and nordihydroguaiaretic acid. In contrast, the epoxygenase, but not the lipoxygenase, was readily inactivated in the presence of 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoic acid. Studies with 18O2-labeled 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoic acid showed that the epoxide oxygens of 9,10-epoxyoctadecanoic acid and of 9,10-epoxy-13(S)-hydroxy-11(E)-octadecenoic acid were derived from hydroperoxide and not from molecular oxygen.     

Peroxygenase-Catalyzed Fatty Acid Epoxidation in Cereal Seeds (Sequential Oxidation of Linoleic Acid into 9(S),12(S),13(S)-Trihydroxy-10(E)-Octadecenoic Acid)

Peroxygenase-catalyzed epoxidation of oleic acid in preparations of cereal seeds was investigated. The 105,000g particle fraction of oat (Avena sativa) seed homogenate showed high peroxygenase activity, i.e. 3034 [plus or minus] 288 and 2441 [plus or minus] 168 nmol (10 min)-1 mg-1 protein in two cultivars, whereas the corresponding fraction obtained from barley (Hordeum vulgare and Hordeum distichum), rye (Secale cereale), and wheat (Triticum aestivum) showed only weak activity, i.e. 13 to 138 nmol (10 min)-1 mg-1 protein. In subcellular fractions of oat seed homogenate, peroxygenase specific activity was highest in the 105,000g particle fraction, whereas lipoxygenase activity was more evenly distributed and highest in the 105,000g supernatant fraction. Incubation of [1-14C]linoleic acid with the 105,000g supernatant of oat seed homogenate led to the formation of several metabolites, i.e. in order of decreasing abundance, 9(S)-hydroxy-10(E),12(Z)-octadecadienoic acid, 9(S),12(S),13(S)-trihydroxy-10(E)-octadecenoic acid, cis-9,10-epoxy-12(Z)-octadecenoic acid [mainly the 9(R),10(S) enantiomer], cis-12,13-epoxy-9(Z)-octadecenoic acid [mainly the 12(R),13(S) enantiomer], threo-12,13-dihydroxy-9(Z)-octadecenoic acid, and 12(R),13(S)-epoxy-9(S)-hydroxy-10(E)-octadecenoic acid. Incubation of linoleic acid with the 105,000g particle fraction gave a similar, but not identical, pattern of metabolites. Conversion of linoleic acid into 9(S),12(S),13(S)-trihydroxy-10(E)-octadecenoic acid, a naturally occurring oxylipin with antifungal properties, took place by a pathway involving sequential catalysis by lipoxygenase, peroxygenase, and epoxide hydrolase.     

Linoleate 9R-dioxygenase and allene oxide synthase activities of Aspergillus terreus

Oxygenation of linoleic acid by Aspergillus terreus was studied with LC-MS/MS. 9(R)-Hydroperoxy-10(E),12(Z)-octadecadienoic acid (9R-HpODE) was identified along with 10(R)-hydroxy-8(E),12(Z)-octadecadienoic acid and variable amounts of 8(R)-hydroxy-9(Z),12(Z)-octadecadienoic acid. 9R-HpODE was formed from [11S-²H]18:2n − 6 with loss of the deuterium label, suggesting antarafacial hydrogen abstraction and oxygenation. Two polar metabolites were identified as 9-hydroxy-10-oxo-12(Z)-octadecenoic acid (α-ketol) and 13-hydroxy-10-oxo-11(E)-octadecenoic acid (γ-ketol), likely formed by spontaneous hydrolysis of an unstable allene oxide, 9(R),10-epoxy-10,12(Z)-octadecadienoic acid. α-Linolenic acid and 20:2n − 6 were oxidized to hydroperoxy fatty acids at C-9 and C-11, respectively, but α- and γ-ketols of these fatty acids could not be detected. The genome of A. terreus lacks lipoxygenases, but contains genes homologous to 5,8-linoleate diol synthases and linoleate 10R-dioxygenases of aspergilli. Our results demonstrate that linoleate 9R-dioxygenase linked to allene oxide synthase activities can be expressed in fungi.     

Oxygenation of monounsaturated fatty acids by soybean lipoxygenase-1: evidence for transient hydroperoxide formation

Soybean lipoxygenase-1 (SBLO-1) catalyzes the oxygenation of polyunsaturated fatty acids to produce conjugated diene hydroperoxides. Previous work from our laboratories has demonstrated that SBLO-1 will also catalyze the oxygenation of monounsaturated acids (Clapp, C. H., Senchak, S. E., Stover, T. J., Potter, T. C., Findeis, P. M., and Novak, M. J. (2001) Soybean Lipoxygenase-Mediated Oxygenation of Monounsaturated Fatty Acids to Enones, J. Am. Chem. Soc. 123, 747-748). Interestingly, the products are alpha,beta-unsaturated ketones rather than the expected allylic hydroperoxides. In the present work, we provide evidence that the monoolefin substrates are initially converted to allylic hydroperoxides, which are subsequently converted to the enone products. The hydroperoxide intermediates can be trapped by reduction to the corresponding allylic alcohols with glutathione peroxidase plus glutathione or with SnCl2. Under some conditions, the hydroperoxide intermediates accumulate and can be detected by HPLC and peroxide assays. Kinetics measurements at low concentrations of [1-14C]-9(Z)-octadecenoic acid indicate that oxygenation of this substrate at 25 degrees C, pH 9.0 occurs with kcat/Km = 1.6 (+/-0.1) x 10(2) M-1 s-1, which is about 105 lower than kcat/Km for oxygenation of 9(Z),12(Z)-octadecadienoic acid (linoleic acid). Comparison of the activities of 9(Z)-octadecenoic acid and 12(Z)-octadecenoic acid implies that the two double bonds of linoleic acid contribute almost equally to the C-H bond-breaking step in the normal lipoxygenase reaction. The results are consistent with the notion that SBLO-1 functionalizes substrates by a radical mechanism.     

Sequential enzymes of linoleic acid oxidation in corn germ: lipoxygenase and linoleate hydroperoxide isomerase

Linoleic acid oxidation catalyzed by lipoxygenase (lipoxidase) activity in extracts of defatted corn germ does not terminate in the product, linoleic acid hydroperoxide, unless the lipoxygenase is first partially purified. If purification is not attempted, the hydroperoxide product exists only as a barely detectable intermediate in the synthesis of three products. One of these was identified as 9-hydroxy-10-oxo-cis-12-octadecenoic acid formed from the hydroperoxide by the enzyme, linoleate hydroperoxide isomerase. Another product, 13-hydroxy-10-oxo-trans-11-octadecenoic acid, is believed to be formed by an isomerase also. The third product was the linoleate ester of one of the hydroxy-oxo-fatty acids, 9-(cis-9,cis-12-octadecadienoyl)-10-oxo-cis-12-octadecenoic acid. It is not known if the synthesis of the ester is enzyme-catalyzed. When a mixture of 13-hydroperoxy-cis-9,trans-11-octa-decadienoic acid and 9-hydroperoxy-trans-10,cis-12-octa-decadienoic acid from soybean lipoxygenase oxidation of linoleic acid was used as a substrate, 13-hydroxy-12-oxo-cis-9-octadecenoic acid and 9-hydroxy-12-oxo-trans-10-octadecenoic acid were formed as the major products of catalysis by linoleate hydroperoxide isomerase(s) from corn. Smaller quantities of 9-hydroxy-10-oxo-cis-12-octadecenoic acid and 13-hydroxy-10-oxo-trans-11-octadecenoic acid were also formed.     

Antifungal Hydroxy Fatty Acids Produced during Sourdough Fermentation: Microbial and Enzymatic Pathways,and Antifungal Activity in Bread

Lactobacilli convert linoleic acid to hydroxy fatty acids; however, this conversion has not been demonstrated in food fermentations and it remains unknown whether hydroxy fatty acids produced by lactobacilli have antifungal activity. This study aimed to determine whether lactobacilli convert linoleic acid to metabolites with antifungal activity and to assess whether this conversion can be employed to delay fungal growth on bread. Aqueous and organic extracts from seven strains of lactobacilli grown in modified De Man Rogosa Sharpe medium or sourdough were assayed for antifungal activity. Lactobacillus hammesii exhibited increased antifungal activity upon the addition of linoleic acid as a substrate. Bioassay-guided fractionation attributed the antifungal activity of L. hammesii to a monohydroxy C_18:1 fatty acid. Comparison of its antifungal activity to those of other hydroxy fatty acids revealed that the monohydroxy fraction from L. hammesii and coriolic (13-hydroxy-9,11-octadecadienoic) acid were the most active, with MICs of 0.1 to 0.7 g liter⁻¹. Ricinoleic (12-hydroxy-9-octadecenoic) acid was active at a MIC of 2.4 g liter⁻¹. L. hammesii accumulated the monohydroxy C_18:1 fatty acid in sourdough to a concentration of 0.73 ± 0.03 g liter⁻¹ (mean ± standard deviation). Generation of hydroxy fatty acids in sourdough also occurred through enzymatic oxidation of linoleic acid to coriolic acid. The use of 20% sourdough fermented with L. hammesii or the use of 0.15% coriolic acid in bread making increased the mold-free shelf life by 2 to 3 days or from 2 to more than 6 days, respectively. In conclusion, L. hammesii converts linoleic acid in sourdough and the resulting monohydroxy octadecenoic acid exerts antifungal activity in bread.     

Effect of abscisic Acid on the linoleic Acid metabolism in developing maize embryos

Partially purified protein extracts from maize (Zea mays L.) embryos, whether treated or not with abscisic acid (ABA), were incubated with linoleic acid (LA) and 1-[¹⁴C]LA. The resulting LA metabolites were monitored by high performance liquid chromatography with a radioactivity detector and identified by gas chromatography-mass spectrometry. α- and γ-ketol metabolites arising from 9-lipoxygenase activity were the more abundant compounds detected in the incubates, although the corresponding metabolites produced by 13-lipoxygenase were also present in the samples. In addition, a group of stereoisomers originating from two isomeric trihydroxy acids (9, 12, 13-trihydroxy-10-octadecenoic and 9, 10, 13-trihydroxy-11-octadecenoic acids) are described. Important variations in the relative proportions of the LA metabolites were observed depending on the embryo developmental stage and on ABA treatment. Two new ABA-induced compounds have been detected. These compounds are present in embryos at all developmental stages, being more abundant in old (60 days) embryos. Furthermore, ABA induction of these compounds is maximum at very young developmental stages, decreasing as maturation progresses. A tentative structure for these compounds (10-oxo-9, 13-dihydroxy-11-octadecenoic acid and 12-oxo-9, 13-dihydroxy-10-octadecenoic acid) is also provided. This study revealed an early stage in maize embryogenesis characterized by a higher relative sensitivity to ABA. The physiological importance of ABA on LA metabolism is discussed.     

Synthesis and biological activity of hydroxylated derivatives of linoleic acid and conjugated linoleic acids

Allylic hydroxylated derivatives of the C18 unsaturated fatty acids were prepared from linoleic acid (LA) and conjugated linoleic acids (CLAs). The reaction of LA methyl ester with selenium dioxide (SeO₂) gave mono-hydroxylated derivatives, 13-hydroxy-9Z,11E-octadecadienoic acid, 13-hydroxy-9E,11E-octadecadienoic acid, 9-hydroxy-10E,12Z-octadecadienoic acid and 9-hydroxy-10E,12E-octadecadienoic acid methyl esters. In contrast, the reaction of CLA methyl ester with SeO₂ gave di-hydroxylated derivatives as novel products including, erythro-12,13-dihydroxy-10E-octadecenoic acid, erythro-11,12-dihydroxy-9E-octadecenoic acid, erythro-10,11-dihydroxy-12E-octadecenoic acid and erythro-9,10-dihydroxy-11E-octadecenoic acid methyl esters. These products were purified by normal-phase short column vacuum chromatography followed by high-performance liquid chromatography (HPLC). Their chemical structures were characterized by liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance spectroscopy (NMR). The allylic hydroxylated derivatives of LA and CLA exhibited moderate in vitro cytotoxicity against a panel of human cancer cell lines including chronic myelogenous leukemia K562, myeloma RPMI8226, hepatocellular carcinoma HepG2 and breast adenocarcinoma MCF-7 cells (IC₅₀ 10-75 μM). The allylic hydroxylated derivatives of LA and CLA also showed toxicity to brine shrimp with LD₅₀ values in the range of 2.30-13.8 μM. However these compounds showed insignificant toxicity to honeybee at doses up to 100 μg/bee.     

Isolation and identification of two isomeric trihydroxy octadecenoic acids with prostaglandin E-like activity from onion bulbs (Allium cepa)

Two fractions with prostaglandin E-like activity were isolated from onion (Allium cepa) by using XAD-2 adsorption, silicic acid column chromatography and thin layer chromatography. The fractions were analyzed by gas chromatography/mass spectrometry and were characterized as isomeric mixtures of 9,10,13-trihydroxy-11-octadecenoic and 9,12,13-trihydroxy-10-octadecenoic acid, which are lipoxygenase metabolites of linoleic acid. Bio-assay, for which cascade superfusion was used and the rabbit coeliac and mesenteric arteries and the rat fundus strip were employed as assay organs, was utilized to monitor the bio-active profile throughout the isolation procedures. The activity of 1 microgram of the pharmacologically active fractions T1 and T2 was found to be equivalent to that of respectively 1.33 and 0.63 ng of prostaglandin E2.     

Isolation and identification of two isomeric trihydroxy octadecenoic acids with prostaglandin E-like activity from onion bulbs ( )

Two fractions with prostaglandin E-like activity were isolated from onion ( ) by using XAD-2 adsorption, silicic acid column chromatography and thin layer chromatography. The fractions were analyzed by gas chromatography/mass spectrometry and were characterized as isomeric mixtures of 9,10,13-trihydroxy-11-octadecenoic and 9,12,13-trihydroxy-10-octadecenoic acid, which are lipoxygenase metabolites of linoleic acid. Bio-assay, for which cascade superfusion was used and the rabbit coeliac and mesenteric arteries and the rat fundus strip were employed as assay organs, was utilized to monitor the bio-active profile throughout the isolation procedures. The activity of 1 μg of the pharmacologically active fractions T1 and T2 was found to be equivalent to that of respectively 1.33 and 0.63 ng of prostaglandin E₂.