linoleic-acid and 13-oxo-9-11-octadecadienoic-acid

The ligand activated nuclear hormone receptor peroxisome proliferator-activated receptor gamma (PPARgamma) induces transcriptional repression of pro-inflammatory factors. Activation of PPARgamma is followed by amelioration of colitis in animal models of inflammatory bowel disease (IBD). A reduced expression of PPARgamma was found in epithelial cells of patients with ulcerative colitis. The eicosanoids 13-HODE and 15-HETE are products of 12/15-lipoxygenase (LOX) and endogenous ligands for PPARgamma. Dehydrogenation of 13-HODE by 13-HODE dehydrogenase results in formation of the 13-Oxo-ODE. Highest activity of 13-HODE dehydrogenase is found in colonic epithelial cells (CECs). We therefore investigated whether 13-Oxo-ODE is a new endogenous ligand of PPARgamma in CECs.. LOX activity and 13-HODE dehydrogenase in CECs were investigated after stimulation with arachidonic or linoleic acid. LOX metabolites were identified by RP-18 reversed-phase HPLC. Binding of (14)C-labelled 13-Oxo-ODE was demonstrated using a His-tagged PPARgamma.. Stimulation of HT-29 and primary CECs homogenates with and without Ca-ionophor was followed by the formation of high amounts of the linoleic acid metabolite 13-Oxo-ODE (155 and 85 ng/ml). The decrease of IL-8 secretion from IEC was more pronounced after pre-incubation with 13-Oxo-ODE compared to the PPARgamma agonist troglitazone and higher as with the known PPARgamma ligands 13-HODE and 15-HETE. Binding assays with (14)C-labelled 13-Oxo-ODE clearly demonstrated a direct interaction.. High amounts of 13-Oxo-ODE can be induced in CECs by stimulation of linoleic acid metabolism. 13-Oxo-ODE binds to PPARgamma and has anti-inflammatory effects. 13-HODE dehydrogenase might be a therapeutic target in IBD.

Topics: Animals; Arachidonic Acid; Blotting, Western; Carbon Radioisotopes; Cattle; Chromatography, High Pressure Liquid; Colon; Epithelial Cells; HT29 Cells; Humans; Hydroxyeicosatetraenoic Acids; Immunoprecipitation; Interleukin-8; Ligands; Linoleic Acid; Linoleic Acids; Linolenic Acids; Molecular Structure; PPAR gamma; Protein Binding; Reverse Transcriptase Polymerase Chain Reaction; Spectrometry, Mass, Electrospray Ionization; Transfection

The oxidation of linoleic acid produces several products with biological activity including the hydroperoxy fatty acid 13-hydroperoxyoctadecadienoic acid (13-HPODE), the hydroxy fatty acid 13-hydroxyoctadecadienoic acid (13-HODE), and the 2,4-dienone 13-oxooctadecadienoic acid (13-OXO). In the present work, the peroxidase activity of glutathione transferases (GST) A1-1, M1-1, M2-2, and P1-1(Val 105) toward 13-HPODE has been examined. The alpha class enzyme is the most efficient peroxidase while the two enzymes from the mu class exhibit weak peroxidase activity toward 13-HPODE. It was also determined that the conjugated diene 13-HODE is not a substrate for GST from the alpha and mu classes but that 13-HODE does inhibit the GST-catalyzed conjugation of CDNB by enzymes from the alpha, mu, and pi classes. Finally, both 13-HODE and 13-OXO were shown to be inducers of GST activity in HT-29 and HCT-116 colon tumor cells. These data help to clarify the role of GST in the metabolic disposition of linoleic acid oxidation products.

Topics: Acetonitriles; Cell Line, Tumor; Dinitrochlorobenzene; Dose-Response Relationship, Drug; Glutathione; Humans; Kinetics; Linoleic Acid; Linoleic Acids; Linolenic Acids; Lipid Peroxides; Models, Chemical; Oxygen; Peroxidase

The oxidation of linoleic acid leads to the generation of several products with biological activity, including 13-oxooctadeca-9,11-dienoic acid (13-OXO), a bioactive 2,4-dienone that has been linked to cell differentiation. In the current work, the conjugation of 13-OXO by human glutathione transferases (GSTs) of the alpha (A1-1, A4-4), mu (M1-1, M2-2) and pi (the allelic variants P1-1/ile, and P1-1/val) classes, and a rat theta (rT2-2) class enzyme has been evaluated. The kinetics and stereoselectivity of the production of the 13-OXO-glutathione conjugate (13-OXO-SG) have been examined. In contrast to many xenobiotic substrates, the endogenous substrate 13-OXO does not exhibit an appreciable non-enzymatic rate of conjugation under physiological conditions. Therefore, the GST-catalyzed conjugation takes on greater significance as it provides the only realistic means for formation of 13-OXO-SG in most biological systems. Alpha class enzymes are most efficient at catalyzing the formation of 13-OXO-SG with kcat/Km values of 8.9 mM(-1) s(-1) for GST A1-1 and 2.14 mM(-1) s(-1) for GST A4-4. In comparison, enzymes from the mu and pi classes exhibit specificity constants from 0.4 to 0.8 mM(-1) s(-1). Conjugation of 13-OXO with glutathione at C-9 of the substrate can yield a pair of diastereomers that can be resolved by chiral HPLC. GSTs from the mu and pi classes are the most stereoselective enzymes and there is no apparent relationship between catalytic efficiency and stereoselectivity. The role of GST in the metabolic disposition of the bioactive oxidation products of linoleic acid has implications for the regulation of normal cellular functions by these versatile enzymes.

Topics: Animals; Glutathione Transferase; Isoenzymes; Linoleic Acid; Linolenic Acids; Models, Chemical; Oxidation-Reduction; Stereoisomerism; Substrate Specificity

Cytochrome c oxidized linoleic acid, with lipoxygenase-like activity, to 9-hydroperoxy-10,12-octadecadienoic acid (9-HpODE), 13-hydroperoxy-9,11-octadecadienoic acid (13-HpODE), 13-keto-9,11-octadecadienoic acid (13-KODE), and hydroxy-epoxy-octadecenoic acids (HEPOs) in the presence of calcium chloride at the physiological pH of 7.5. In the absence of calcium, the reaction was remarkably reduced. Statistically significant increase of 13-KODE was observed 1 min after the reaction was started by adding calcium. Calcium chloride at concentrations as low as 30 microM caused statistically significant increase of 13-KODE production in the presence of 1 microM cytochrome c, and cytochrome c at concentrations as low as 10 nM caused statistically significant increase of 13-KODE production in the presence of 2 mM calcium. The addition of bleomycin enhanced the reaction, and the addition of adriamycin attenuated the reaction. The same oxidized linoleic acid products were found in the submitochondrial particles which were hydrolyzed by phospholipase-A2 in the presence of calcium. Lipoxygenase activity potentiated by calcium may be involved in the lipid peroxidation in the submitochondrial particles hydrolyzed by phospholipase-A2.

Topics: Animals; Calcium; Cattle; Cytochrome c Group; Enzyme Inhibitors; Hydrogen-Ion Concentration; Linoleic Acid; Linolenic Acids; Lipid Peroxidation; Lipoxygenase; Mitochondria, Heart; Peroxides; Phospholipases A; Phospholipases A2; Stearic Acids

To observe lipid peroxidation of additive-free submitochondrial particles, we incubated submitochondrial particles in the absence of exogenous irons and t-butyl hydroperoxide. After the incubation, the phospholipids were hydrolyzed by phopholipase A2, and the fatty acid constituents were analyzed by high-performance liquid chromatography, gas chromatography-mass spectrometry, and liquid chromatography-mass spectrometry. Contrary to a commonly accepted theory, lipid peroxidation in the submitochondrial particles did not need the addition of NADH. In the phospholipid constituent fatty acids of the oxidized submitochondrial particles, derivatives of hydroperoxides of linoleic acid such as keto, hydroxy, trihydroxy, and hydroxyepoxy compounds were generated. Lipid peroxidation in the submitochondrial particles was not inhibited by the addition of catalase, superoxide dismutase, hydroxyl radical scavengers, or ethylenediaminetetraacetic acid, but was inhibited by the addition of KCN, antimycin-A, NADH, ubiquinol, deferoxamine mesylate, ascorbic acid, and alpha-tocopherol. The cardiolipin-cytochrome c lipid peroxidation system could mimic the lipid peroxidation of the submitochondrial particles, in terms of linoleic acid products and the inhibitory patterns of radical scavengers and electron transfer chain inhibitors. Thus, lipid peroxidation in the submitochondrial particles seems to be due to phospholipid-hemoprotein lipid peroxidation systems such as the cardiolipin-cytochrome c system.

Topics: Animals; Cattle; Chromatography, High Pressure Liquid; Electron Transport; Free Radical Scavengers; Free Radicals; Gas Chromatography-Mass Spectrometry; Iron; Linoleic Acid; Linolenic Acids; Lipid Peroxidation; Mitochondria, Heart; NAD; Submitochondrial Particles; Time Factors

Oxidation products of linoleic acid, including hydroperoxy- and hydroxyoctadecadienoic acids have been shown to possess biological activities in a number of different systems. In this work we describe an enzymatic activity which catalyzes the conversion of 13-hydroxyoctadecadienoic acid to a 2,4-dienone product, 13-oxooctadecadienoic acid. The enzyme activity is widely distributed, with the highest activity in the colon and the liver. The distribution of activity among various tissues is distinct from other dehydrogenases known to use oxygenated unsaturated fatty acids as substrates. This enzyme may play a key role in the metabolism of 13-hydroxyoctadecadienoic acid in epithelial tissues.

Topics: Alcohol Oxidoreductases; Animals; Colon; Linoleic Acid; Linoleic Acids; Linolenic Acids; Liver; Male; Organ Specificity; Oxidation-Reduction; Rats; Rats, Inbred Strains; Subcellular Fractions