13-hydroxy-9-11-octadecadienoic-acid and 13-hydroperoxy-9-11-octadecadienoic-acid

To determine in vivo if L-4F differentially alters plasma levels of oxidized fatty acids resulting in more anti-inflammatory HDL. Injecting L-4F into apoE null mice resulted in a significant reduction in plasma levels of 15-HETE, 5-HETE, 13-HODE and 9-HODE. In contrast, plasma levels of 20-HETE were not reduced and plasma levels of 14,15-EET, which are derived from the cytochrome P450 pathway, were elevated after injection of L-4F. Injection of 13(S)-HPODE into wild-type C57BL/6J mice caused an increase in plasma levels of 13-HODE and 9-HODE and was accompanied by a significant loss in the anti-inflammatory properties of HDL. The response of atherosclerosis resistant C3H/HeJ mice to injection of 13(S)-HPODE was similar but much more blunted. Injection of L-4F at a site different from that at which the 13(S)-HPODE was injected resulted in significantly lower plasma levels of 13-HODE and 9-HODE and significantly less loss of HDL anti-inflammatory properties in both strains. i) L-4F differentially alters plasma levels of oxidized fatty acids in vivo. ii) The resistance of the C3H/HeJ strain to atherosclerosis may in part be mediated by a reduced reaction of this strain to these potent lipid oxidants.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Anti-Inflammatory Agents; Apolipoproteins E; Atherosclerosis; Chromatography, Liquid; Enzyme-Linked Immunosorbent Assay; Fatty Acids; Female; Hydroxyeicosatetraenoic Acids; Injections, Subcutaneous; Linoleic Acids; Linoleic Acids, Conjugated; Lipid Peroxides; Lipoproteins, HDL; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Knockout; Oxidation-Reduction; Peptides; Species Specificity; Tandem Mass Spectrometry; Time Factors; Up-Regulation

Human reticulocyte 15-lipoxygenase-1 (15-hLO-1) and human platelet 12-lipoxygenase (12-hLO) have been implicated in a number of diseases, with differences in their relative activity potentially playing a central role. In this work, we characterize the catalytic mechanism of these two enzymes with arachidonic acid (AA) as the substrate. Using variable-temperature kinetic isotope effects (KIE) and solvent isotope effects (SIE), we demonstrate that both k(cat)/K(M) and k(cat) for 15-hLO-1 and 12-hLO involve multiple rate-limiting steps that include a solvent-dependent step and hydrogen atom abstraction. A relatively low k(cat)/K(M) KIE of 8 was determined for 15-hLO-1, which increases to 18 upon the addition of the allosteric effector molecule, 12-hydroxyeicosatetraenoic acid (12-HETE), indicating a tunneling mechanism. Furthermore, the addition of 12-HETE lowers the observed k(cat)/K(M) SIE from 2.2 to 1.4, indicating that the rate-limiting contribution from a solvent sensitive step in the reaction mechanism of 15-hLO-1 has decreased, with a concomitant increase in the C-H bond abstraction contribution. Finally, the allosteric binding of 12-HETE to 15-hLO-1 decreases the K(M)[O(2)] for AA to 15 microM but increases the K(M)[O(2)] for linoleic acid (LA) to 22 microM, such that the k(cat)/K(M)[O(2)] values become similar for both substrates (approximately 0.3 s(-1) microM(-1)). Considering that the oxygen concentration in cancerous tissue can be less than 5 microM, this result may have cellular implications with respect to the substrate specificity of 15-hLO-1.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Allosteric Regulation; Arachidonate 12-Lipoxygenase; Arachidonate 15-Lipoxygenase; Arachidonic Acid; Biocatalysis; Blood Platelets; Carbon Isotopes; Humans; Kinetics; Leukotrienes; Linoleic Acid; Linoleic Acids; Lipid Peroxides; Models, Chemical; Oxygen; Recombinant Proteins; Reticulocytes; Solvents; Temperature

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

During the early stages of germination, a lipid-body lipoxygenase is expressed in the cotyledons of sunflowers (Helianthus annuus L.). In order to obtain evidence for the in vivo activity of this enzyme during germination, we analyzed the lipoxygenase-dependent metabolism of polyunsaturated fatty acids esterified in the storage lipids. For this purpose, lipid bodies were isolated from etiolated sunflower cotyledons at different stages of germination, and the storage triacylglycerols were analyzed for oxygenated derivatives. During the time course of germination the amount of oxygenated storage lipids was strongly augmented, and we detected triacylglycerols containing one, two or three residues of (9Z,11E,13S)-13-hydro(pero)xy-octadeca-9,11-dienoic acid. Glyoxysomes from etiolated sunflower cotyledons converted (9Z,11E,13S)-13-hydroxy-octadeca-9,11-dienoic acid to (9Z,11E)-13-oxo-octadeca-9,11-dienoic acid via an NADH-dependent dehydrogenase reaction. Both oxygenated fatty acid derivatives were activated to the corresponding CoA esters and subsequently metabolized to compounds of shorter chain length. Cofactor requirement and formation of acetyl-CoA indicate degradation via beta-oxidation. However, beta-oxidation only proceeded for two consecutive cycles, leading to accumulation of a medium-chain metabolite carrying an oxo group at C-9, equivalent to C-13 of the parent (9Z,11E,13S)-13-hydroxy-octadeca-9,11-dienoic acid. Short-chain beta-oxidation intermediates were not detected during incubation. Similar results were obtained when 13-hydroxy octadecanoic acid was used as beta-oxidation substrate. On the other hand, the degradation of (9Z,11E)-octadeca-9,11-dienoic acid was accompanied by the appearance of short-chain beta-oxidation intermediates in the reaction mixture. The results suggest that the hydroxyl/oxo group at C-13 of lipoxygenase-derived fatty acids forms a barrier to continuous beta-oxidation by glyoxysomes.

Topics: Chromatography, High Pressure Liquid; Cotyledon; Germination; Glyoxysomes; Helianthus; Linoleic Acids; Lipid Metabolism; Lipid Peroxides; Lipoxygenase; Oxidation-Reduction; Time Factors

Vegetables are generally recognized as rich sources of dietary antioxidants for inhibiting lipid peroxidation. Here we investigated lipid hydroperoxide (LOOH)-reducing activity of several vegetables to estimate their role on the prevention of lipid peroxidation in food and the digestive tract. By using HPLC analysis, we screened vegetables possessing the ability to convert 13-hydroperoxyoctadecadienoic acid (13-HPODE) to its reduced derivative, 13-hydroxyoctadecadienoic acid (13-HODE). Welsh onion (Allium fistulosum L.) was found to be highly active in the reduction of 13-HPODE among tested vegetables. There was no relationship between 13-HPODE reducing activity and GSH peroxidase (GPX) activity in the tested vegetables. 13-HPODE-reducing activity of welsh onion was enhanced by the addition of sulfhydryl compounds including glutathione (GSH). Neither GPX inhibitor nor heat treatment suppressed 13-HPODE-reducing activity effectively. These results suggest that welsh onion and other vegetables contain GPX mimics responsible for the reduction of LOOH. GPX mimics may be helpful in the attenuation of harmful effect of LOOH from food.

Topics: Antioxidants; Chromatography, High Pressure Liquid; Enzyme Inhibitors; Food; Glutathione Peroxidase; Hot Temperature; Linoleic Acids; Lipid Peroxidation; Lipid Peroxides; Onions; Oxidation-Reduction; Sulfhydryl Compounds; Vegetables

The current study assessed the differential incorporation of 12-hydroperoxyeicosatetraenoic acid (12-HPETE), arachidonic acid (AA), 12-hydroxyeicosatetraenoic acid (12-HETE) and the linoleic acid (LA) oxidation products, 13-hydroxyoctadecadienoic acid (13-HODE) and 13-hydroperoxyoctadecadienoic acid (13-HPODE), into human umbilical vein endothelial cells (HUVEC). Approximately 80-90% of AA (10(-8)-10(-5)M) and 80% of LA (10(-8)-10(-5)M) were incorporated into HUVEC within 12h, while less than 50% of the hydroxy metabolites (12-HETE, 12-HPETE, 13-HODE, 13-HPODE) were incorporated into HUVEC over 48h. Further, treatment of HUVEC with either 12-HPETE or 13-HPODE (concentrations of 10(-5)M) had no effect on cell number at a 48h time point when compared with control. These results demonstrate that exogeneous hydroxy metabolites are incorporated into HUVEC to a lesser degree than were endogenous fatty acids. Further, we speculate that 12-HPETE and 13-HPODE are rapidly metabolized to substances without significant cytotoxic effects.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonic Acid; Cells, Cultured; Endothelial Cells; Humans; Leukotrienes; Linoleic Acids; Lipid Peroxides; Umbilical Veins

The new route of the plant lipoxygenase pathway, directed specifically towards the ketodiene formation, was detected during in vitro experiments with Jerusalem artichoke (Helianthus tuberosus) tubers. Through this pathway (9Z,11E,13S)-13-hydroperoxy-9,11-octadecadienoic acid (13-HPOD) is reduced to corresponding 13-hydroxy acid (13-HOD), which is in turn dehydrogenated into ketodiene (9Z,11E,13S)-13-oxo-9,11-octadecadienoic acid (13-KOD). Dehydrogenation of 13-HOD into 13-KOD was not dependent on the presence of either NAD or NADP, but was strongly dependent on the presence of oxygen. Under anoxic conditions, 13-HOD dehydrogenation was blocked, but addition of 2,6-dichlorophenolindophenol restored it. Sulfite addition fully suppressed the aerobic dehydrogenation of 13-HOD. Hydrogen peroxide is a by-product formed by the enzyme along with 13-KOD. These data suggest that the ketodiene biosynthesis in H. tuberosus tubers is catalyzed by flavin dehydrogenase. (9S,10E,12Z)-9-Hydroxy-10,12-octadecadienoic acid (9-HOD) is dehydrogenated by this enzyme as effectively as 13-HOD, while alpha-ketol, (9Z)-12-oxo-13-hydroxy-9-octadecenoic acid, and ricinoleic acid did not act as substrates for dehydrogenase. The enzyme was soluble and possessed a pH optimum at pH 7.0-9.0. The only 13-HOD dehydrogenase known so far was detected in rat colon. However, unlike the H. tuberosus enzyme, the rat dehydrogenase is NAD-dependent.

Topics: Animals; Carbon Radioisotopes; Chromatography, High Pressure Liquid; Helianthus; Hydrogen-Ion Concentration; Hypoxia; Intracellular Space; Linoleic Acids; Linolenic Acids; Lipid Peroxides; Lipoxygenase; Molecular Structure; NAD; NADP; Nuclear Magnetic Resonance, Biomolecular; Oxidoreductases; Oxygen Radioisotopes; Plant Tubers; Rats; Spectrometry, Mass, Electrospray Ionization; Substrate Specificity

Various forms of oxidized low-density lipoproteins (Ox-LDL) are thought to play a major role in the development of atherosclerosis. The lipid components of Ox-LDL present a plethora of proatherogenic effects in in vitro cell culture systems, suggesting that oxidative stress could be an important risk factor for coronary artery disease. However, buried among these effects are those that could be interpreted as antiatherogenic. The present study demonstrates that various oxidants, including oxidized fatty acids and mildly oxidized forms of LDL (MO-LDL), are able to induce catalase (an antioxidant enzyme) expression in rabbit femoral arterial smooth muscle cells (RFASMC), RAW cells (macrophages), and human umbilical vein endothelial cells (HUVEC). In RFASMC, catalase protein, mRNA, and the enzyme activity are increased in response to oxidized linoleic acid (13-hydroperoxy-9,11-octadecadienoic acid [13-HPODE] and 13-hydroxy-9,11-octadecadienoic acid [13-HODE]), MO-LDL, or hydrogen peroxide (H(2)O(2)). Such an increase in catalase gene expression cannot totally be attributed to the cellular response to an intracellular generation of H(2)O(2) after the addition of 13-HPODE or 13-HODE because these agents induce a further increase of catalase as seen in catalase-transfected RFASMC. Taken together with the induction of heme oxygenase, NO synthase, manganese superoxide dismutase (Mn-SOD), and glutathione synthesis by oxidative stress, our results provide yet more evidence suggesting that a moderate oxidative stress can induce cellular antioxidant response in vascular cells, and thereby could be beneficial for preventing further oxidative stress.

Topics: Animals; Catalase; Cells, Cultured; Endothelium, Vascular; Fatty Acids; Femoral Artery; Gene Expression; Humans; Hydrogen Peroxide; Linoleic Acid; Linoleic Acids; Lipid Peroxides; Lipoproteins, LDL; Muscle, Smooth, Vascular; Oxidants; Oxidation-Reduction; Rabbits; RNA, Messenger; Transfection; Umbilical Veins

Expression of cellular adhesion molecules (CAMs) at endothelial surfaces represents a physiological response to vascular damage and mediates the initiation of inflammation and possibly of atherogenesis. The cytokines TNF alpha and IL-1 are potent inducers of CAMs in endothelial cells. Reactive oxygen species comprising lipid oxidation products have been implicated in the signaling pathways of both TNF alpha and IL-1 and accordingly could modulate atherogenic events. We, therefore, investigated the potential role of the lipoxygenase product, 13-hydroperoxyoctadecadienoic acid (13-HPODE), which has also been identified in oxidized low density lipoproteins on CAM expression in HUVEC. 13-HPODE induced the expression of ICAM-1 in a concentration dependent manner up to 75 microM. Higher concentrations were toxic. Similar effects were observed with H2O2 and phosphatidylcholine hydroperoxide. VCAM-1 and E-selectin were not induced by 13-HPODE. 13-HPODE administered simultaneously with IL-1 or TNF alpha induced ICAM-1 additively, suggesting that hydroperoxides and cytokines act on the same signaling pathways. In contrast, pretreatment of cells with 50 microM 13-HPODE for 1 hour rather inhibited subsequent cytokine-induced ICAM-1 and E-selectin expression. Surprisingly, the reduction product of 13-HPODE, 13-hydroxyoctadecadienoic acid (13-HODE) proved to be an even better inducer of ICAM-1 than 13-HPODE. Pretreatment with 13-HODE did not show any inhibitory effect on ICAM-1 expression. Our data show that lipoxygenase products differentially affect CAM expression. 13-HPODE is stimulatory by itself and can positively or negatively affect cytokine signaling depending on time of exposure. 13-HODE induces CAM expression by itself but does not inhibit cytokine signaling. Thus, the interplay of lipoxygenase products with proinflammatory cytokines can not simply be explained by an oxidant-mediated facilitation of cytokine signaling.

Topics: Cell Adhesion Molecules; Cells, Cultured; Cytokines; Dose-Response Relationship, Drug; E-Selectin; Endothelium, Vascular; Humans; Hydrogen Peroxide; Intercellular Adhesion Molecule-1; Linoleic Acids; Lipid Peroxides; Phosphatidylcholines; Reducing Agents; Signal Transduction; Time Factors; Tumor Necrosis Factor-alpha; Umbilical Cord; Up-Regulation; Vascular Cell Adhesion Molecule-1

Linoleic acid, a polyunsaturated C18 fatty acid, is one of the major fatty acids in the coronary arterial wall. Although diets rich in linoleic acid reduce blood pressure and prevent coronary artery disease in both humans and animals, very little is known about its mechanism of action. We believed that its beneficial effects might be mediated by changes in vascular tone. We investigated whether linoleic acid induces relaxation of porcine coronary artery rings and the mechanism involved in this process. Linoleic acid and two of its metabolites, 13-hydroxyoctadecadienoic acid (13-HODE) and 13-hydroperoxyoctadecadienoic acid (13-HPODE), induced dose-dependent relaxation of prostaglandin (PG) F2alpha-precontracted rings that was not affected by indomethacin (10[-5] mol/L), a cyclooxygenase inhibitor, or cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate (CDC; 10[-5] mol/L), a lipoxygenase inhibitor. Removal of endothelial cells had no effect on vasorelaxation, suggesting a direct effect on the vascular smooth muscle cells (VSMC). When rings were contracted with KCl, linoleic acid failed to induce relaxation. Although tetrabutylammonium (5 x 10[-3] mol/L), a nonselective K+ channel blocker, slightly inhibited the relaxation caused by linoleic acid, glibenclamide (10[-6] mol/L), an ATP-sensitive K+ channel blocker, and charybdotoxin (7.5x10[-8] mol/L) or tetraethylammonium (5x10[-3] mol/L), two different Ca2+-activated K+ channel blockers, had no effect. However, relaxation was completely blocked by ouabain (5x10[-7] mol/L), a Na+/K+-ATPase inhibitor, or by a K+-free solution. In addition, linoleic acid (10[-6] mol/L) caused sustained hyperpolarization of porcine coronary VSMC (from -49.5+/-2.0 to -60.7+/-4.2 mV), which was also abolished by ouabain. We concluded that linoleic acid induces relaxation and hyperpolarization of porcine coronary VSMC via a mechanism that involves activation of the Na+/K+-ATPase pump.

Topics: Animals; Coronary Vessels; Electrophysiology; Fatty Acids; Linoleic Acid; Linoleic Acids; Lipid Peroxides; Membrane Potentials; Swine; Vasodilation

The effect of dopamine-melanin (DA-melanin), synthetic model of neuromelanin, on the stability of 13-hydroperoxyoctadecadienoic acid (13-HPODE) has been investigated using a reverse-phase high-performance liquid chromatography (HPLC) with UV detection. It was found that DA-melanin effectively accelerated the decomposition of 13-HPODE, both in the absence and in the presence of ferrous ions. The disappearance of 13-HPODE was accompanied by the formation of 13-hydroxyoctadecadienoic acid (13-HODE). The results obtained indicate that DA-melanin is able to reduce linoleic acid hydroperoxide to its stable hydroxyl derivative. The fatty acid hydroperoxide-reducing ability of DA-melanin appears to play an important role in the antioxidative activity of neuromelanin in the process of lipid peroxidation.

Topics: Antioxidants; Chromatography, High Pressure Liquid; Linoleic Acids; Lipid Peroxides; Melanins; Models, Chemical; Oxidation-Reduction

The effect of 13-hydroperoxyoctadecadienoic acid (13-HPODE) on the activity of glucosamine synthetase, the rate-limiting enzyme of mucus synthesis, in rabbit gastric corporal mucosa was examined. 13-HPODE inhibited the glucosamine synthetase activity at concentrations ranging from 10 to 100 microM. The effect was concentration-dependent, and the concentration required for 50% inhibition was approximately 20 microM. Experiments utilizing Fe2+ and 13-hydroxyoctadecadienoic acid revealed that the inhibitory effect of 13-HPODE on the glucosamine synthetase activity is not due to the alcohol adduct and hydroxyl radical which are expected to be formed from 13-HPODE, and that the hydroperoxyl functional group is a prerequisite. The fact that tert-butyl hydroperoxide exhibited about 50 times weaker inhibition than 13-HPODE indicates the relative specificity of fatty acyl hydroperoxides in the modulation of the glucosamine synthetase activity. These results suggest that 13-HPODE has the potential to modulate the synthesis of gastric mucus by affecting the glucosamine synthetase activity.

Topics: Animals; Antithrombins; Dose-Response Relationship, Drug; Gastric Mucosa; Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing); Linoleic Acids; Lipid Peroxidation; Lipid Peroxides; Male; Rabbits

Topics: Animals; Calcium-Calmodulin-Dependent Protein Kinases; Cell Division; Cell Line, Transformed; Cell Transformation, Neoplastic; Cricetinae; Embryo, Mammalian; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Fibroblasts; Hybrid Cells; Linoleic Acid; Linoleic Acids; Lipid Peroxides; Mesocricetus; Phosphorylation; Protein Processing, Post-Translational; Signal Transduction; Stimulation, Chemical

Arachidonate 12-lipoxygenase, which oxygenates positions 12 and 13 of arachidonic and linoleic acids, is present in porcine anterior pituitary cells. Colocalization of the 12-lipoxygenase with various pituitary hormones was examined by immunohistochemical double-staining using antibodies against 12-lipoxygenase and various anterior pituitary hormones. Under light microscopy, approximately 7% of the cells producing luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were positive for 12-lipoxygenase, whereas the enzyme was detected in less than 2% of the cells producing thyrotrophin, prolactin, growth hormone (GH), and adrenocorticotrophin. In an attempt to examine the participation of 12-lipoxygenase metabolites in pituitary hormone release, we incubated the primary culture of porcine anterior pituitary cells with 12-hydroperoxy-arachidonic acid or 13-hydroperoxy-linoleic acid. Significant stimulation of LH and FSH release by these hydroperoxides was observed at 10 microM in a time-dependent manner. At doses around 10 microM these compounds produced responses of similar magnitude to 1 nM gonadotrophin-releasing hormone (GnRH), but higher concentrations (30 microM) of the compounds were required for GH release. In contrast, 12-hydroxy-arachidonic and 13-hydroxy-linoleic acids were almost ineffective. Furthermore, the gonadotrophin release by 1 nM GnRH was inhibited by nordihydroguaiaretic acid (a lipoxygenase inhibitor) with an IC50 of about 5 microM. Thus, the hydroperoxy (but not hydroxy) products of 12-lipoxygenase may be involved in the release of pituitary hormones especially LH and FSH.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Antithrombins; Arachidonate 12-Lipoxygenase; Cells, Cultured; Chromatography, High Pressure Liquid; Fatty Acids, Unsaturated; Follicle Stimulating Hormone; Gonadotropin-Releasing Hormone; Growth Hormone; Hydroxyeicosatetraenoic Acids; Immunohistochemistry; Leukotrienes; Linoleic Acids; Lipid Peroxides; Luteinizing Hormone; Pituitary Gland, Anterior; Swine; Time Factors

Low density lipoprotein that was oxidized by activated human monocytes was analyzed to determine the identity of oxidized fatty acids present and the conditions required for their formation. The oxidized lipids were also analyzed under conditions allowing preservation of their oxidation state. Using reversed-phase high performance liquid chromatography (HPLC) analysis of native and saponified lipid extracts of oxidized low density lipoprotein (LDL), we found that the major fatty acid oxidation product was esterified hydroperoxyoctadecadienoic acid (HPODE), the oxidized product of the most abundant polyunsaturated fatty acid in human LDL, linoleic acid. Although some esterified hydroxyoctadecadienoic acid (HODE) was also detected, the reduction of HPODE to HODE did not appear to be monocyte-dependent. Essentially all of the HPODE was found to be esterified with the majority being esterified to cholesterol followed by phospholipids and generally following the abundance of esterified linoleic acid within the lipid classes. The percent of cholesteryl linoleate converted to cholesteryl HPODE and cholesteryl HODE at the end of the 24-h incubation was determined to be approximately 13.5%. The formation of oxidized esterified linoleic acid in the LDL was shown to require immunological activation of the human monocytes, a previously observed requirement for general LDL oxidation in this culture system. The oxidized esterified linoleic acid was present in the supernatant with the LDL and was not cell-associated. HPODE formation on LDL was prevented by including superoxide dismutase (SOD) or eicosatetraynoic acid (ETYA) during the 24-h coincubation of activated monocytes with LDL whereas indomethacin was without effect. The analysis of the lipid oxidation products in oxidized LDL can provide insight into the mechanisms involved in oxidation of LDL by activated human monocytes.

Topics: Cholesterol Esters; Esterification; Humans; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Kinetics; Linoleic Acids; Linoleic Acids, Conjugated; Lipid Peroxides; Lipoproteins, LDL; Monocytes; Oxidation-Reduction; Thiobarbituric Acid Reactive Substances

1. One of the major fatty acids in the arterial wall is linoleic acid. It has been shown that its 13-hydroxy metabolite (13-HODE) is generated in significant amounts by cultured endothelial cells. The aim of the present study was to investigate the relaxations to 13-HODE and its hydroperoxyprecursor (13-HPODE) and to examine the role of the endothelial cells. 2. Ring segments of canine circumflex and splenic artery were mounted in organ chambers for isometric tension recording. During contractions induced by prostaglandin F2 alpha or noradrenaline, 13-HODE and 13-HPODE evoked dose-dependent relaxations. Removal of the endothelial cells reduced the relaxations to 13-HODE, but had no effect on those elicited by 13-HPODE. 3. Indomethacin and meclofenamate (0.3 microM to 30 microM) blocked the relaxations evoked by 13-HODE and 13-HPODE in endothelium-denuded rings. In segments with endothelium, both cyclo-oxygenase inhibitors again abolished the relaxations to 13-HODE, but only diminished those to 13-HPODE. 4. Prostacyclin biosynthesis, as measured by radioimmunoassay, increased upon incubation with 13-HODE and 13-HPODE (10 microM). Bioassay of the release of nitric oxide (NO) indicated that NO was not involved in the relaxations elicited by either metabolite. Moreover, L-NG-nitroarginine (100 microM), a specific inhibitor of NO synthesis, did not influence the relaxations to 13-HODE and 13-HPODE. The responses to 13-HPODE were also not altered by superoxide dismutase. 5. In the splenic artery 13-HPODE and 13-HODE induced contractions above 3 microM which were blocked by the thromboxane receptor antagonist, daltroban.In the circumflex artery contractile responses to high concentrations of 13-HODE could be observed only after inhibition of cyclo-oxygenase.6. We conclude that the vasodilatation induced by 13-HODE and 13-HPODE was due to stimulation of prostacyclin biosynthesis both in the endothelium and smooth muscle cells or other subendothelial structures. An additional, unidentified intermediate, which was neither NO nor a cyclo-oxygenase product nor superoxide anion, contributed to the relaxations to 13-HPODE in arteries with endothelium.

Topics: Animals; Dogs; Dose-Response Relationship, Drug; Endothelium, Vascular; Epoprostenol; Female; Linoleic Acids; Lipid Peroxides; Male; Muscle, Smooth, Vascular; Nitric Oxide; Radioimmunoassay; Splenic Artery; Vasodilation

The breakage of double-strand (ds) DNA by 13-L-hydroperoxy-cis-9,trans-11-octadecadienoic acid (LAHPO) was investigated by agarose gel electrophoresis of supercoiled pBR322 DNA and the site of cleavage on the DNA molecule was determined by the method of DNA sequence analysis using 3'-end and 5'-end-labeled DNA fragments as substrates. LAHPO caused cleavage at the position of guanine nucleotide in dsDNA. LAHPO caused dsDNA breaks at specific sites, but linoleic acid (LA) and 13-L-hydroxy-cis-9,trans-11-octadecadienoic acid (LAHO) have no such effects on dsDNA. The active oxygen atom of the hydroperoxy group of LAHPO was perhaps responsible for the site-specific cleavage of dsDNA.

Topics: Animals; DNA, Superhelical; Drosophila melanogaster; Linoleic Acid; Linoleic Acids; Lipid Peroxides; Piperidines; Plasmids