Preferential Generation of 15-HETE-PE Induced by IL-13 Regulates Goblet Cell Differentiation in Human Airway Epithelial Cells.

Type 2-associated goblet cell hyperplasia and mucus hypersecretion are well known features of asthma. 15-Lipoxygenase-1 (15LO1) is induced by the type 2 cytokine IL-13 in human airway epithelial cells (HAECs) in vitro and is increased in fresh asthmatic HAECs ex vivo. 15LO1 generates a variety of products, including 15-hydroxyeicosatetraenoic acid (15-HETE), 15-HETE-phosphatidylethanolamine (15-HETE-PE), and 13-hydroxyoctadecadienoic acid (13-HODE). In this study, we investigated the 15LO1 metabolite profile at baseline and after IL-13 treatment, as well as its influence on goblet cell differentiation in HAECs. Primary HAECs obtained from bronchial brushings of asthmatic and healthy subjects were cultured under air-liquid interface culture supplemented with arachidonic acid and linoleic acid (10 µM each) and exposed to IL-13 for 7 days. Short interfering RNA transfection and 15LO1 inhibition were applied to suppress 15LO1 expression and activity. IL-13 stimulation induced expression of 15LO1 and preferentially generated 15-HETE-PE in vitro, both of which persisted after removal of IL-13. 15LO1 inhibition (by short interfering RNA and chemical inhibitor) decreased IL-13-induced forkhead box protein A3 (FOXA3) expression and enhanced FOXA2 expression. These changes were associated with reductions in both mucin 5AC and periostin. Exogenous 15-HETE-PE stimulation (alone) recapitulated IL-13-induced FOXA3, mucin 5AC, and periostin expression. The results of this study confirm the central importance of 15LO1 and its primary product, 15-HETE-PE, for epithelial cell remodeling in HAECs.

10-oxo-12(Z)-octadecenoic acid, a linoleic acid metabolite produced by gut lactic acid bacteria, potently activates PPARγ and stimulates adipogenesis.

Our previous study has shown that gut lactic acid bacteria generate various kinds of fatty acids from polyunsaturated fatty acids such as linoleic acid (LA). In this study, we investigated the effects of LA and LA-derived fatty acids on the activation of peroxisome proliferator-activated receptors (PPARs) which regulate whole-body energy metabolism. None of the fatty acids activated PPARδ, whereas almost all activated PPARα in luciferase assays. Two fatty acids potently activated PPARγ, a master regulator of adipocyte differentiation, with 10-oxo-12(Z)-octadecenoic acid (KetoA) having the most potency. In 3T3-L1 cells, KetoA induced adipocyte differentiation via the activation of PPARγ, and increased adiponectin production and insulin-stimulated glucose uptake. These findings suggest that fatty acids, including KetoA, generated in gut by lactic acid bacteria may be involved in the regulation of host energy metabolism.

Lipoperoxidation affects ochratoxin A biosynthesis in Aspergillus ochraceus and its interaction with wheat seeds.

In Aspergillus nidulans, Aspergillus flavus, and Aspergillus parasiticus, lipoperoxidative signalling is crucial for the regulation of mycotoxin biosynthesis, conidiogenesis, and sclerotia formation. Resveratrol, which is a lipoxygenase (LOX) and cyclooxygenase inhibitor, downmodulates the biosynthesis of ochratoxin A (OTA) in Aspergillus ochraceus. In the genome of A. ochraceus, a lox-like sequence (AoloxA; National Center for Biotechnology Information (NCBI) accession number: DQ087531) for a lipoxygenase-like enzyme has been found, which presents high homology (100 identities, 100 positives %, score 555) with a lox gene of Aspergillus fumigatus (NCBI accession number: XM741370). To study how inhibition of oxylipins formation may affect the A. ochraceus metabolism, we have used a DeltaAoloxA strain. This mutant displays a different colony morphology, a delayed conidia formation, and a high sclerotia production. When compared to the wild type, the DeltaAoloxA strain showed a lower basal activity of LOX and diminished levels of 13-hydroperoxylinoleic acid (HPODE) and other oxylipins derived from linoleic acid. The limited oxylipins formation corresponded to a remarkable inhibition of OTA biosynthesis in the DeltaAoloxA strain. Also, wheat seeds (Triticum durum cv Ciccio) inoculated with the DeltaAoloxA mutant did not accumulate 9-HPODE, which is a crucial element in the host defence system. Similarly, the expression of the pathogenesis-related protein 1 (PR1) gene in wheat seeds was not enhanced. The results obtained contribute to the current knowledge on the role of lipid peroxidation governed by the AoloxA gene in the morphogenesis, OTA biosynthesis, and in host-pathogen interaction between wheat seeds and A. ochraceus.

Enzymatic synthesis of a bicyclobutane fatty acid by a hemoprotein lipoxygenase fusion protein from the cyanobacterium Anabaena PCC 7120.

Biological transformations of polyunsaturated fatty acids often lead to chemically unstable products, such as the prostaglandin endoperoxides and leukotriene A(4) epoxide of mammalian biology and the allene epoxides of plants. Here, we report on the enzymatic production of a fatty acid containing a highly strained bicyclic four-carbon ring, a moiety known previously only as a model compound for mechanistic studies in chemistry. Starting from linolenic acid (C18.3omega3), a dual function protein from the cyanobacterium Anabaena PCC 7120 forms 9R-hydroperoxy-C18.3omega3 in a lipoxygenase domain, then a catalase-related domain converts the 9R-hydroperoxide to two unstable allylic epoxides. We isolated and identified the major product as 9R,10R-epoxy-11trans-C18.1 containing a bicyclo[1.1.0]butyl ring on carbons 13-16, and the minor product as 9R,10R-epoxy-11trans,13trans,15cis-C18.omega3, an epoxide of the leukotriene A type. Synthesis of both epoxides can be understood by initial transformation of the hydroperoxide to an epoxy allylic carbocation. Rearrangement to an intermediate bicyclobutonium ion followed by deprotonation gives the bicyclobutane fatty acid. This enzymatic reaction has no parallel in aqueous or organic solvent, where ring-opened cyclopropanes, cyclobutanes, and homoallyl products are formed. Given the capability shown here for enzymatic formation of the highly strained and unstable bicyclobutane, our findings suggest that other transformations involving carbocation rearrangement, in both chemistry and biology, should be examined for the production of the high energy bicyclobutanes.

15-Lipoxygenase-1 expression suppresses the invasive properties of colorectal carcinoma cell lines HCT-116 and HT-29.

Colorectal carcinoma (CRC) is often lethal when invasion and/or metastasis occur. 15-Lipoxygenase-1 (15-LO-1), a member of the inflammatory eicosanoid pathway, oxidatively metabolizes linoleic acid and its expression is repressed in CRC. In this study, we investigated the hypothesis that the lack of 15-LO-1 expression in CRC cells might contribute to tumorigenesis. Therefore we introduced 15-LO-1 into HCT-116 and HT-29 cells that do not have detectable levels of 15-LO-1. Our data indicate that expression of 15-LO-1 significantly decreased cell proliferation and increased apoptosis. In addition, we observed a reduction in adhesion to fibronectin, anchorage-independent growth on soft agar, cellular motility and ability to heal a scratch wound, and migratory and invasive capacity across Matrigel. 15-LO-1 expression also reduced the expression of metastasis associated protein-1, a part of the nucleosome remodeling and histone deacetylase silencing complex. We propose that 15-LO-1 expression in CRC might contribute to the inhibition of metastatic capacity in vitro and can be exploited for therapeutic purposes.

Lipoxygenase distribution in coffee (Coffea arabica L.) berries.

In this paper lipoxygenase (LOX) presence was investigated in coffee berries to determine its involvement in lipid degradative metabolism of plants grown in organic and conventional cultivations. An immunochemical analysis has evidenced a ca. 80 kDa protein, cross-reacting with an anti-LOX antibody, only in the pulp fraction of berries obtained from plants of both cultivations. LOX activity in this fraction could be monitored either as conjugated diene formation or reaction products (determined by HPLC) and was mainly associated with a heavy membrane fraction (HMF, enriched in tonoplast, endoplasmic reticulum, plasma membrane, and mitochondria) and a light membrane fraction (LMF, enriched in plasma membrane and endoplasmic reticulum, with low levels of tonoplast and mitochondria). The LOX activity of LMF from berries of both cultivations showed an optimum at pH 8.0. The HMF exhibited a different activity peak in samples from conventional (pH 8.0) and organic (pH 5.5) cultures, suggesting the presence of different isoenzymes. These findings were also confirmed by variation of the ratio of 9- and 13-hydroperoxides in organic (1:1) and conventional cultivations (1:10), indicating that the organic one was subjected to an oxidative stress in the coffee pulp fraction leading to the expression of an acidic LOX. Such de novo synthesized LOX activity could be responsible for the production of secondary metabolites, which may interfere with the organoleptic profile of coffee.

Interactions among triphenyltin degradation, phospholipid synthesis and membrane characteristics of Bacillus thuringiensis in the presence of d-malic acid.

Degradation pathway and surface biosorption of triphenyltin (TPT) by effective microbes have been investigated in the past. However, unclear interactions among membrane components and TPT binding and transport are still obstacles to understanding TPT biotransformation. To reveal the mechanism involved, the phospholipid expression, membrane potential, cellular mechanism and molecular dynamics between TPT and fatty acids (FAs) during the TPT degradation process in the presence of d-malic acid (DMA) were studied. The results show that the degradation efficiency of 1 mg L-1 TPT by Bacillus thuringiensis (1 g L-1) with 0.5 or 1 mg L-1 DMA reached values up to approximately 90% due to the promotion of element metabolism and cellular activity, and the depression of FA synthesis induced by DMA. The addition of DMA caused conversion of more linoleic acid into 10-oxo-12(Z)-octadecenoic acid, increased the membrane permeability, and alleviated the decrease in membrane potential, resulting in TPT transport and degradation. Fluorescence analysis reveals that the endospore of B. thuringiensis could act as an indicator for membrane potential and cellular activities. The current findings are advantageous for acceleration of biosorption, transport and removal of pollutants from natural environments.

Hepatic overproduction of 13-HODE due to ALOX15 upregulation contributes to alcohol-induced liver injury in mice.

Chronic alcohol feeding causes lipid accumulation and apoptosis in the liver. This study investigated the role of bioactive lipid metabolites in alcohol-induced liver damage and tested the potential of targeting arachidonate 15-lipoxygenase (ALOX15) in treating alcoholic liver disease (ALD). Results showed that chronic alcohol exposure induced hepatocyte apoptosis in association with increased hepatic 13-HODE. Exposure of 13-HODE to Hepa-1c1c7 cells induced oxidative stress, ER stress and apoptosis. 13-HODE also perturbed proteins related to lipid metabolism. HODE-generating ALOX15 was up-regulated by chronic alcohol exposure. Linoleic acid, but not ethanol or acetaldehyde, induced ALOX15 expression in Hepa-1c1c7 cells. ALOX15 knockout prevented alcohol-induced liver damage via attenuation of oxidative stress, ER stress, lipid metabolic disorder, and cell death signaling. ALOX15 inhibitor (PD146176) treatment also significantly alleviated alcohol-induced oxidative stress, lipid accumulation and liver damage. These results demonstrated that activation of ALOX15/13-HODE circuit critically mediates the pathogenesis of ALD. This study suggests that ALOX15 is a potential molecular target for treatment of ALD.

The histone deacetylase inhibitor suberoylanilide hydroxamic acid induces apoptosis via induction of 15-lipoxygenase-1 in colorectal cancer cells.

Histone deacetylases (HDACs) mediate changes in nucleosome conformation and are important in the regulation of gene expression. HDACs are involved in cell cycle progression and differentiation, and their deregulation is associated with several cancers. HDAC inhibitors have emerged recently as promising chemotherapeutic agents. One such agent, suberoylanilide hydroxamic acid, is a potent inhibitor of HDACs that causes growth arrest, differentiation, and/or apoptosis of many tumor types in vitro and in vivo. Because of its low toxicity, suberoylanilide hydroxamic acid is currently in clinical trials for the treatment of cancer. HDAC inhibitors induce the expression of <2% of genes in cultured cells. In this study, we show that low micromolar concentrations of suberoylanilide hydroxamic acid induce the expression of 15-lipoxygenase-1 in human colorectal cancer cells. The expression of 15-lipoxygenase-1 correlates with suberoylanilide hydroxamic acid-induced increase in 13-S-hydroxyoctadecadienoic acid levels, growth inhibition, differentiation, and apoptosis observed with these cells. Furthermore, specific inhibition of 15-lipoxygenase-1 significantly reduced the suberoylanilide hydroxamic acid-induced effects. These novel findings are the first demonstration of a mechanistic link between the induction of 15-lipoxygenase-1 by a HDAC inhibitor and apoptosis in cancer cells. This result has important implications for the study of suberoylanilide hydroxamic acid and other HDAC inhibitors in the prevention and therapy of colorectal cancer and supports future investigations of the mechanisms by which HDAC inhibitors up-regulate 15-lipoxygenase-1.

Mechanism for the antitumor and anticachectic effects of n-3 fatty acids.

Dietary intake of the n-6 fatty acid (FA) linoleic acid (LA) has a strong growth-promoting effect on many rodent tumors and human tumor xenografts grown in immunodeficient rodents. n-3 FAs such as alpha-linolenic and eicosapentaenoic acids (EPAs), which differ from LA and arachidonic acid, respectively, by only a single double bond in the n-3 position, are recognized cancer chemopreventive and anticachectic agents. Understanding how this seemingly small structural difference leads to such remarkable functional differences has been a challenge. In a previous study, we showed that LA uptake, [3H]thymidine incorporation into DNA, and total DNA content were decreased in tissue-isolated hepatoma 7288CTC perfused in situ with arterial blood containing alpha-linolenic acid, EPA, or docosahexaenoic acids. The Ki for the inhibition of LA uptake and [3H]thymidine incorporation by alpha-linolenic acid was 0.18 and 0.25 mM, respectively. Here we show that the addition of alpha-linolenic acid or EPA to arterial blood inhibits tumor FA uptake, including LA, and the subsequent conversion of LA to the mitogen 13-hydroxyoctadecadienoic acid (13-HODE) in vivo and during perfusion in situ. [3H]Thymidine incorporation during perfusion in situ was also inhibited. Addition of 13-HODE to the arterial blood reversed the inhibition of [3H]thymidine incorporation but had no effect on FA uptake. These two n-3 FAs also inhibited FA transport in inguinal fat pads in vivo and during perfusion in situ in fed (FA uptake) and fasted (FA release) rats. The effects of EPA and talinolenic acid on transport of saturated, monounsaturated, and n-6 polyunsaturated FAs in hepatoma 7288CTC and inguinal fat pads during perfusion in situ were reversed by the addition of forskolin (1 microM), pertussis toxin (0.5 microg/ml), or 8-bromo-cyclic AMP (10 microM) to the arterial blood. We conclude that the antitumor and anticachectic effects of n-3 FAs on hepatoma 7288CTC and inguinal fat pads in vivo result from an inhibition of FA transport. These inhibitions are mediated by a putative n-3 FA receptor via a Gi protein-coupled signal transduction pathway that decreases intracellular cyclic AMP. A specific decrease in LA uptake and its conversion to the mitogen 13-HODE causes the tumor growth inhibition.

Biosynthesis of 8R-hydroperoxylinoleic acid by the fungus Laetisaria arvalis.

8-Hydroxylinoleic acid is known to be a fungicidal metabolite formed by the fungus Laetisaria arvalis (Bowers, W.S. et al. (1986) Science 232, 105-106). In the present report, the mechanism of formation of 8-hydroxylinoleic acid was investigated. L. arvalis metabolized [14C]linoleic acid to 8-hydroperoxylinoleic acid and 8-hydroxylinoleic acid as major metabolites. The identification is based on the reduction of the hydroperoxide to an alcohol with stannous chloride and gas chromatography-mass spectrometry. The absolute configuration of the hydroxyl was determined to be R by ozonolysis of the (-)-menthoxycarbonyl derivative of 8-hydroxylinoleic acid. Linoleic acid 8R-dioxygenase activity was present in the 100,000 x g supernatant of the cell lysate. In summary, the 8R-linoleic acid dioxygenase of L. arvalis shows many similarities with the 8R-dioxygenase recently described in the fungus Gaeumannomyces graminis.

Stereochemistry in the formation of 9-hydroxy-10,12-octadecadienoic acid and 13-hydroxy-9,11-octadecadienoic acid from linoleic acid by fatty acid cyclooxygenase.

9-Hydroxy-10,12-octadecadienoic acid and 13-hydroxy-9,11-octadecadienoic acid are formed from linoleic acid upon incubation with the microsomal fraction of homogenates of the sheep vesicular gland (Hamberg, M. and Samuelsson, B. (1967) J. Biol. Chem. 242, 5344-5354. This communication is concerned with the stereochemical aspects of the conversion. The ratio between the 9- and 13-hydroxy isomers was 77:23. Steric analysis of the individual isomers showed that the hydroxyl group of both isomers had mainly the L configuration, i.e. 9L:9D, 79:21 and 13L:13D, 9- and 13-hydroxyoctadecadienoates which had largely lost the tritium label (6% and 7% retention of tritium relative to precursor, respectively) showing that the hydrogen which is removed from C-11 during the conversion has the L (pro-S) configuration.

A novel transformation of 13-LS-hydroperoxy-9,11-octadecadienoic acid.

13-LS-Hydroperoxy-9,11-octadecadienoic acid (13-HPOD) was incubated with human hemoglobin. Apart from monoketo-, monohydroxy- as well as a number of hydroxy-epoxy acid derivatives earlier identified (Hamberg, M. (1975) Lipids 10, 87-92) a small conversion (1-2%) of 13-HPOD into 8-LR, 13-LS- and 8-DS, 13-LS-dihydroxy-9trans,11trans-octadecadienoic acids was demonstrated. An unstable intermediate, i.e., 12,13-epoxy-8,10-octadecadienoic acid, was detected in the conversion of 13-HPOD into the two dihydroxy-octadecadienoates by trapping experiments. It was concluded that compounds structurally related to the leukotrienes may be formed nonenzymatically from 13-HPOD.

bis-Allylic hydroxylation of linoleic acid and arachidonic acid by human hepatic monooxygenases.

[14C]Linoleic acid was incubated with human liver microsomes and NADPH and biosynthesis of allylic hydroxy fatty acids was investigated. 11-Hydroxy-9Z,12Z-octadecadienoic acid (11-HODE), 9-hydroxy-10E,12Z-octadecadienoic acid (9-HODE), 13-hydroxy-9Z,11E-octadecadienoic acid (13-HODE) and 14-hydroxy-9Z,12Z-octadecadienoic acid were identified. 9-HODE and 13-HODE were formed with stereoselectivity (80% R) provided that 11-HODE was prevented from decomposing to 9(R,S)-HODE and 13(R,S)-HODE by extractive isolation at pH 5-6. Human hepatic microsomes metabolized [14C]arachidonic acid to many products, including 13-HETE and small amounts of 15-HETE (> 90% R), 11-HETE (59% R) and 12-HETE (> 90% R). Hepatic microsomes of untreated rats metabolized [14C]linoleic acid to 11-HODE as a major product, but significant formation of 11-HODE by purified cytochrome P-450 (P450) (CYP1A1, CYP2B1, CYP2B4, CYP2E1, CYP3A6 and CYP4A1) in a reconstituted system could not be detected, indicating that 11-HODE might be formed by other and constitutive P450 isozymes.

Formation of 9-hydroxy linoleic acid as a product of phospholipid peroxidation in diabetic erythrocyte membranes.

The increased production of oxygen-derived free radicals (OFR) and lipid peroxidation may contribute to vascular complications in diabetes. Some lipid peroxidation products have already been reported to be formed via glucose-induced oxidative stress. We have identified 9-hydroxy linoleic acid (9-OH-C18:2) in the red cell membrane phospholipid of diabetic subjects. We hypothesized that 9-OH-C18:2 would be formed in hydroxyl radical reactions to linoleic acid (C18:2) during glucose-induced oxidative stress, and confirmed that the formation of 9-OH-C18:2 was induced by ultraviolet (UV)-C irradiation to the synthetic C18:2. UV-C light generates highly reactive hydroxy radicals. C18:2 is confirmed to be the precursor of 9-OH-C18:2. To estimate the degree of oxidative damage to red cell membrane phospholipids, we developed a selective ion monitoring gas chromatography-mass spectrometric measurement for C18:2 and 9-OH-C18:2, following methanolysis of red cell membrane phospholipids. The relative peak height ratio of C18:2 to 9-OH-C18:2 (9-OH-C18:2/C18:2) was measured in phospholipid extracts of red cell membranes from healthy (n=29, 3.1+/-1.9%) and diabetic (n=27, 20. 9+/-16.1%) subjects. It was confirmed that 9-OH-C18:2/C18:2 is significantly (P<0.001) elevated in patients with diabetes. The measurement of 9-OH-C18:2/C18:2 in red cell membranes should be useful for assessing oxidative damage to membrane phospholipids in diabetes.

Biosynthesis of acyl groups on molecular species of biliary phosphatidylcholines during metabolism of [2,2,2-2H3]ethanol.

Incorporation of deuterium into different positions of individual molecular species of biliary phosphatidylcholines was determined in bile fistula rats given [2,2,2-2H3]ethanol under conditions ensuring maximal rate of oxidation for 24 h. The deuterium-labelling of the glycerol moiety of the major molecular species was about 6-8 atom% at the end of ethanol administration. The deuterium excess at each of the different positions of the glycerol moiety of 1-palmitoyl-2-linoleoyl phosphatidylcholine was less than 3 atom%. From the isotopic composition of the palmitoyl residues of the phosphatidylcholines, it was calculated that [2,2,2-2H3]ethanol supplied about 35-40% of the acetyl-CoA forming the terminal methyl group and about 25-30% of the other C2 units of the palmitic acid chain. This difference in deuterium incorporation was interpreted as being due to an isotope effect, probably in the rate-limiting carboxylation step of acetyl-CoA. Most or perhaps all of the acetyl groups derived from ethanol were introduced into the terminal methyl group without loss of deuterium. This indicates that citrate is not an important carrier of acetyl-CoA in the biosynthesis of fatty acids from ethanol.

Metabolic profile of linoleic acid in porcine leukocytes through the lipoxygenase pathway.

Porcine neutrophilic leukocytes were found to contain a lipoxygenase which converted linoleic acid into 13-hydroxy-9,11-octadecadienoic acid (n-6 specificity), arachidonic acid into 12-hydroxy-5,8,10,14-eicosatetraenoic acid (n - 9 specificity) and 5-hydroxy-6,8,11,14-eicosatetraenoic acid into 5,12-dihydroxy-6,8,10,14-eicosatetraenoic acid. This lipoxygenase was partially purified and it appeared that its substrate specificity and other properties were quite different from the 12-lipoxygenase of blood platelets. Incubations of intact or broken porcine leukocytes with added linoleic acid revealed the formation of not only 13-hydroxy-9,11-octadecadienoic acid but also of substantial amounts of epoxyhydroxy and trihydroxy isomers. These products from linoleate, collectively described by the name 'octadecanoids' were characterized in detail by a combination of chemical, chromatographic and mass spectrometric techniques. The phospholipids of porcine leukocytes contain more than twice as much linoleate than arachidonate (22 vs. 8%). In accordance with this fatty acid composition we found that in the stimulated neutrophil the endogenous production of octadecanoids often surpassed that of the eicosanoids. Lipoxygenation of endogenously liberated linoleic acid was especially pronounced when a suspension of leukocytes in citrated plasma was recalcified and allowed to clot.

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.

Differential biosynthesis of polyunsaturated fatty acids by Tetrahymena supplemented with ergosterol.

Tetrahymena grown with foreign sterols such as ergosterol incorporate them into cellular membranes at the expense of the native compound, tetrahymanol. It is shown that cells grown with ergosterol have a lessened capacity to produce the polyunsaturated linoleic and gamma-linolenic acids from [14C]oleic acid. However, the same cells have normal capacities to introduce double bonds at C-6 into linoleate, alpha-linolenate, or cis-vaccenate. Thus, a presumed 12-desaturase is inhibited in the presence of ergosterol, while desaturation at C-6 is unaffected.

Characterization of monohydroxylated lipoxygenase metabolites of arachidonic and linoleic acid in rabbit peritoneal tissue.

Rabbit peritoneal tissue contains a lipoxygenase which converts arachidonic acid preferentially into 15-hydroxy-5,8,11,13-eicosatetraenoic acid. Stereochemical analysis of the menthyloxycarbonyl derivative of this metabolite by means of a high-pressure liquid chromatography method, involving the use of a Ag+ -loaded cation-exchange column, indicated that it has mainly the 15-Ls-hydroxy configuration. The biosynthesis of 15-hydroxy-5,8,11,13-eicosatetraenoic acid could be confirmed during examination of the monohydroxy acids obtained without addition of fatty acids, thus formed from endogenously released substrate. However, the 9-and 13-hydroxy derivatives of linoleic acid were also formed and in quantities exceeding those of 15-hydroxy-5,8,11,13-eicosatetraenoic acid.