15-hydroperoxy-5-8-11-13-eicosatetraenoic-acid and 5-hydroxy-6-8-11-14-eicosatetraenoic-acid

This report describes the application of direct chemical ionization mass spectrometry (DCIMS) to the identification and quantification of 5- and 15-HPETEs. A unique feature of the method is use of a polyimide-coated fused silica fiber that allows vaporization of the hydroperoxides, with very low excess energy, into the plume of the chemical ionization reagent gas plasma. Mass spectra are obtained that allow identification of the nonreduced and nonderivatized free acid forms of 5- and 15-HPETE as well as their quantification from 1 microgram to 100 picograms.

Topics: Animals; Arachidonic Acids; Humans; Hydroxyeicosatetraenoic Acids; Leukotrienes; Lipid Peroxides; Magnetic Resonance Spectroscopy; Mass Spectrometry; Spectrophotometry, Infrared

Capsaicin, a pungent ingredient of hot peppers, causes excitation of small sensory neurons, and thereby produces severe pain. A nonselective cation channel activated by capsaicin has been identified in sensory neurons and a cDNA encoding the channel has been cloned recently. However, an endogenous activator of the receptor has not yet been found. In this study, we show that several products of lipoxygenases directly activate the capsaicin-activated channel in isolated membrane patches of sensory neurons. Among them, 12- and 15-(S)-hydroperoxyeicosatetraenoic acids, 5- and 15-(S)-hydroxyeicosatetraenoic acids, and leukotriene B(4) possessed the highest potency. The eicosanoids also activated the cloned capsaicin receptor (VR1) expressed in HEK cells. Prostaglandins and unsaturated fatty acids failed to activate the channel. These results suggest a novel signaling mechanism underlying the pain sensory transduction.

Topics: Animals; Capsaicin; Cell Line; Cells, Cultured; Dinoprostone; Eicosanoids; Ganglia, Spinal; Humans; Hydroxyeicosatetraenoic Acids; Inflammation; Ion Channel Gating; Leukotriene B4; Leukotrienes; Ligands; Lipid Peroxides; Lipoxygenase; Molecular Structure; Neurons, Afferent; Prostaglandin D2; Prostaglandin H2; Prostaglandins H; Rats; Receptors, Drug; Structure-Activity Relationship

The effects of 1 microM concentrations of arachidonic acid hydroperoxide (HPETES) products of 5-, 12- and 15-lipoxygenase on Na+, K(+)-ATPase activity were investigated in synaptosomal membrane preparations from rat cerebral cortex. 5-HPETE inhibited Na+, K(+)-ATPase activity by up to 67 %. In contrast, 12-HPETE and 15-HPETE did not inhibit Na+, K(+)-ATPase activity. In addition, neither 5-HETE or LTA4 inhibited Na+, K(+)-ATPase activity. Dose-response studies indicated that 5-HPETE was a potent (IC25 = 10(-8) M) inhibitor of Na+, K(+)-ATPase activity. These findings indicate that 5-HPETE inhibits Na+, K(+)-ATPase activity by a mechanism that is dependent on the hydroperoxide position and independent of further metabolism by 5-lipoxygenase. It is proposed that 5-HPETE production by 5-lipoxygenase and subsequent inhibition of neuronal Na+, K(+)-ATPase activity may be a mechansim for modulating synaptic transmission.

Topics: Animals; Arachidonate Lipoxygenases; Cerebral Cortex; Enzyme Inhibitors; Hydroxyeicosatetraenoic Acids; Leukotriene A4; Leukotrienes; Lipid Peroxides; Male; Neurons; Rats; Rats, Sprague-Dawley; Sodium-Potassium-Exchanging ATPase; Synaptic Transmission; Synaptosomes

1. Prostaglandin endoperoxide synthetase (PES) and lipoxygenase (Lox) activities were compared in the cerebella and cerebra of vitamin E-sufficient young chicks and in chicks in which nutritional encephalomalacia (NE) was induced by a diet deficient in vitamin E. 2. Eicosanoid production patterns were qualitatively similar in the brains of both groups of chicks, but prostaglandin production was 50-60% less in cerebella of ataxic chicks, compared to control cerebella, while the opposite trend was observed in the cerebellar Lox pathway, as measured by radioimmunoassay of 15-HETE. 3. Cerebellar phospholipase A2 activity was twice that of the cerebrum but was not affected by NE. 4. Purification of Lox activity from the cerebellar homogenates produced a lower yield and enrichment when the starting material was taken from ataxic chicks, compared to the controls. 5. In addition there were qualitative differences in the purified fractions from both groups, as seen by pH optima and kinetics. 6. The results are consistent with the view that the cerebellum has less antioxidant protection than the cerebrum and that its higher phospholipase A2 activity and greater propensity to oxygenate arachidonic acid via the Lox pathway at the expense of the PES pathway may render this region of the brain particularly vulnerable to oxidative damage in NE.

Topics: Animal Nutritional Physiological Phenomena; Animals; Arachidonic Acid; Brain; Cerebellum; Chickens; Encephalomalacia; Hydroxyeicosatetraenoic Acids; Kinetics; Leukotrienes; Lipid Peroxides; Lipoxygenase; Male; Phospholipases A; Phospholipases A2; Poultry Diseases; Prostaglandin-Endoperoxide Synthases; Prostaglandins

The role of several lipoxygenase metabolites of arachidonic acid in the action of luteinizing hormone-releasing hormone (LHRH) on ovarian hormone production was investigated. Like LHRH, treatment of rat granulosa cells with 5-HETE, 5-HPETE, 12-HETE, 15-HETE or 15-HPETE stimulated progesterone (P) and prostaglandin E2 (PGE2) production. 12-HEPE was most potent and stimulated P and PGE2 equally well. By contrast, 5-HETE stimulated P better than PGE2, while 15-HETE was a potent stimulator of PGE2 but not of P. Stimulation of P and PGE2 by LHRH or 12-O-tetradecanoylphorbol 13-acetate (TPA) was further augmented by several HETEs and HPETEs. Like protein kinase C, arachidonic acid metabolites appear to mediate the multiple actions of LHRH in the ovary.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acid; Arachidonic Acids; Dinoprostone; Drug Interactions; Female; Gonadotropin-Releasing Hormone; Granulosa Cells; Hydroxyeicosatetraenoic Acids; Leukotrienes; Lipid Peroxides; Lipoxygenase; Progesterone; Rats; Rats, Inbred Strains; Tetradecanoylphorbol Acetate

Oxygen radical-induced genetic damage may be mediated by products of lipid peroxidation, in particular, arachidonic acid. Hydroxy- and hydroperoxyeicosatetraenoic acids (HETEs and HPETEs) are intermediates in the metabolism of arachidonic acid to the leukotrienes. Several isomeric hydroxy- and hydroperoxy-6,8,11,14-eicosatetraenoic acids were evaluated for their ability to cause DNA single-strand breaks in human lymphocytes. Both HETEs and HPETEs induced strand breaks in a dose-dependent fashion at concentrations of 5, 10 and 20 microM. At each concentration, HETEs were more effective in producing breakage than the corresponding HPETEs. Each of the isomeric forms used were equally effective in producing strand breaks. Antioxidants (superoxide dismutase, catalase and mannitol) were protective. Iron chelation by desferrioxamine suppressed strand breakage by 45% and an additional 33% inhibition was observed upon the addition of the calcium chelator EGTA.

Topics: Antioxidants; Cells, Cultured; Deferoxamine; DNA; DNA Damage; DNA, Single-Stranded; Egtazic Acid; Humans; Hydroxyeicosatetraenoic Acids; Leukotrienes; Lipid Peroxides; Lymphocytes

Liver fatty acid binding protein (L-FABP) binds avidly the arachidonic acid metabolites, hydroperoxyeicosatetraenoic acids (HPETEs) and hydroxyeicosatetraenoic acids (HETEs). Binding of 15-[3H]HPETE was specific, saturable, reversible, and rapid. Protein specificity was indicated by the following order: L-FABP greater than bovine serum albumin greater than ovalbumin = beta-lactoglobulin greater than ribonuclease. Ligand specificity was evidenced by the following order of apparent competition: 15-HPETE greater than or equal to 5-HETE greater than or equal to 5-HPETE = oleic acid greater than 12-HETE greater than 12-HPETE greater than or equal to 15-HETE greater than prostaglandin E1 much greater than leukotriene C4 greater than prostaglandin E2 much greater than thromboxane B2 = leukotriene B4. Once bound, 15-HPETE was reversibly displaced. Ligand was recovered from the protein complex and confirmed to be 15-[3H]HPETE by TLC. L-FABP bound HPETE with a dissociation constant of 76 nM,5-HETE at 175 nM, and 15-HETE at 1.8 microM, and the reference fatty acids oleic acid at 1.2 microM and arachidonic acid at 1.7 microM. Thus, the affinity was approximately 16-fold greater for 15-HPETE, and 7-fold higher for 5-HETE, than for oleic acid. The need exists for studies of complexes of L-FABP with the HPETEs and HETEs in hepatocytes, especially since L-FABP has previously been associated with mitosis in normal hepatocytes, and shown to be the target protein of two liver carcinogens, and these arachidonic acid metabolites have been found to be able to modulate activities related to cell growth.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Binding, Competitive; Carrier Proteins; Fatty Acid-Binding Protein 7; Fatty Acid-Binding Proteins; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Kinetics; Leukotrienes; Ligands; Lipid Peroxides; Lipoxygenase; Liver; Neoplasm Proteins; Nerve Tissue Proteins; Oleic Acid; Oleic Acids; Protein Binding; Rats; Structure-Activity Relationship

Severe cerebral vasospasm as confirmed by angiography was induced in dogs by injection of autologous blood into the cisterna magna, and the resultant leukotriene formation in the isolated basilar artery was examined. When stimulated with calcium ionophore (A 23187), the arteries of the treated animals produced a significant amount of leukotrienes B4 (85 +/- 12 pmol/mg protein, n = 3) and C4 (72 +/- 14 pmol/mg), in addition to 5(S)-hydroxy-6,8,11,14-eicosatetraenoic acid. Structural elucidations of these metabolites were performed by radioimmunoassays or gas chromatography-mass spectrometry, following purification with HPLC. The artery of the untreated dog produced none of these compounds from either exogenous or endogenous arachidonic acid, under stimulation with the calcium ionophore. However, the homogenates from both animals converted exogenous leukotriene A4 to leukotrienes B4 and C4. These observations suggest that the normal basilar artery contains no detectable amount of 5-lipoxygenase, and that a prominent activation of this enzyme occurred (2.1 nmol 5-HETE/5 min/mg of protein) after subarachnoidal hemorrhage. The observation that fatty acid hydroperoxides stimulated the 5-lipoxygenase activity indicates a possible role of lipid peroxides in the development of vasospasm.

Topics: Animals; Arachidonate 5-Lipoxygenase; Arachidonate Lipoxygenases; Arachidonic Acid; Arachidonic Acids; Basilar Artery; Chromatography, High Pressure Liquid; Dogs; Enzyme Activation; Gas Chromatography-Mass Spectrometry; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Leukotrienes; Lipid Peroxides; Radioimmunoassay; SRS-A; Subarachnoid Hemorrhage

Arachidonate 5-lipoxygenase purified from porcine leukocytes was incubated with (5S)-hydroperoxy-6,8,11,14-eicosatetraenoic acid. In addition to degradation products of leukotriene A4 (6-trans-leukotriene B4 and its 12-epimer and others), (5S,6R)-dihydroperoxy-7,9,11,14-eicosatetraenoic acid was produced as a major product especially when the incubation was performed on ice rather than at room temperature. The amount of the (5S,6R)-dihydroperoxy acid was close to the total amount of leukotriene A4 degradation products. Under the anaerobic condition, production of the (5S,6R)-dihydroperoxy acid was markedly reduced. 5-Hydroxy-6,8,11,14-eicosatetraenoic acid could be a substrate of the enzyme and was transformed predominantly to a compound identified as (5S)-hydroxy-(6R)-hydroperoxy-7,9-trans-11,14-cis-eicosatetraenoic acid at about 1-2% rate of arachidonate 5-oxygenation. These findings indicated that the purified 5-lipoxygenase exhibited a 6R-oxygenase activity with (5S)-hydroxy and (5S)-hydroperoxy acids as substrates. The 6R-oxygenase activity, like the leukotriene A synthase activity, was presumed to be an integral part of 5-lipoxygenase because it required calcium and ATP and was affected by selective 5-lipoxygenase inhibitors.

Topics: Animals; Arachidonate 5-Lipoxygenase; Arachidonate Lipoxygenases; Arachidonic Acids; Chromatography, High Pressure Liquid; Hydroxyeicosatetraenoic Acids; Leukocytes; Leukotrienes; Lipid Peroxides; Oxygenases; Spectrophotometry, Ultraviolet; Swine

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonate 12-Lipoxygenase; Arachidonate 5-Lipoxygenase; Arachidonate Lipoxygenases; Arachidonic Acids; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Leukocytes; Leukotrienes; Lipid Peroxides; Substrate Specificity; Swine

It was shown that 15-hydroxyeicosatetraenoic acid (15-HETE) but not 15-H(P)ETE or 5-HETE is a potent agonist for secretion of glycoprotein-containing mucus from the in vivo canine trachea. Given by aerosol into the lungs or by intra-arterial injection into the trachea, 15 HETE also caused the chemotaxis of inflammatory cells into the lumen of the airways. Accompanying this inflammatory cell infiltrate was an increase (183%, p less than 0.05) of expiration of fluid in the partially saturated air coming from the lung. The levels of 15-HETE extracted from tracheal mucus correlated well with hillocks and weight of secreted mucus found in the mucus after hypoxia or after arachidonic acid loading. Indomethacin and atropine blocked the mucus secretagogue effect of 15-HETE in the trachea. Indomethacin and U-52, 412 (a 15-lipoxygenase inhibitor) pretreatment abolished a portion of the 15-HETE-induced enhancement of mucus weight and 15-HETE level in the secretion.

Topics: Aerosols; Animals; Arachidonic Acid; Arachidonic Acids; Body Fluids; Cell Movement; Chemotaxis; Dogs; Hydroxyeicosatetraenoic Acids; Hypoxia; Inflammation; Leukotrienes; Lipid Peroxides; Lipoxygenase Inhibitors; Mucus; Respiration; Respiratory System; Respiratory Tract Diseases; Trachea

The leukotrienes, prominent mediators of immediate hypersensitivity and inflammation, are derived from the major product of leukocyte lipoxygenase activity, 5-HPETE. An alternative pathway for 5-HPETE metabolism is reduction to 5-HETE. I hypothesize that, in analogy to its established role in platelet lipoxygenase metabolism, the selenium-dependent glutathione peroxidase mediates this reduction. If so, supplementary selenium may decrease leukotriene production, and thus have value as a nutritional adjuvant in the management of asthma and various other inflammatory disorders.

Topics: Animals; Arachidonic Acids; Blood Platelets; Glutathione Peroxidase; Hydroxyeicosatetraenoic Acids; Leukocytes; Leukotriene B4; Leukotrienes; Lipid Peroxides; Lipoxygenase; Oxidation-Reduction; Rats; Selenium; SRS-A

The lipoxygenase products of arachidonic acid, 5-HPETE, 5-HETE, LTB4, LTC4 and LTD4, were examined for their capacity to enhance the expression of complement (C3b) receptors and to evoke chemotaxis of human neutrophils and eosinophils. With the exception of LTD4 all gave enhancement of C3b receptors. LTB4 and LTC4 enhanced over the concentration range 10(-7) to 10(-11) moles/l, and 5-HETE and 5-HPETE from 5 X 10(-6) to 5 X 10(-10) moles/l. The rank order of activity, as assessed by the magnitude of enhancement, was LTB4 (concentration for maximal effect = 10(-7) moles/l) greater than 5-HETE (5 X 10(-7)) greater than 5-HPETE (5 X 10(-6)) greater than LTC4 (10(-9)). High dose inhibition was observed with LTC4 and 5-HETE. Chemotaxis experiments performed in parallel over the same concentration ranges indicated that neither neutrophils nor eosinophils migrated towards LTC4 or LTD4. However, LTB4 evoked chemotaxis with a linear dose response from 10(-9) to 10(-7) moles/l and 5-HPETE and 5-HETE from 5 X 10(-8) to 5 X 10(-6) moles/l. At 10(-7) moles/l LTB4 was approximately 6 and 8 X more potent in chemotaxis than 5-HPETE and 5-HETE respectively. In general, complement receptor enhancement and chemotaxis of eosinophils were similar to that observed with neutrophils and did not vary with the patient source.

Topics: Arachidonate Lipoxygenases; Arachidonic Acids; Chemotaxis, Leukocyte; Eosinophils; Humans; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Leukotriene B4; Leukotrienes; Lipid Peroxides; Lipoxygenase; Neutrophils; Peroxides; Receptors, Complement; SRS-A

During allergic and inflammatory reactions, arachidonic acid is oxidized by lipoxygenases to a variety of biologically active products, including leukotrienes. The mechanisms for regulation of the different lipoxygenase activities are not well defined. We report here that [14C]arachidonic acid metabolism by the 5- and 15-lipoxygenase activities in rabbit leukocytes and the 12-lipoxygenase in rabbit platelets is inhibited by various hydroxyeicosatetraenoic acids (HETEs). 15-HETE was the most effective inhibitor of the 5- and 12-lipoxygenases, whereas similar inhibitory potencies were observed for 5-HETE and 12-HETE acting on the 15-lipoxygenase. These three enzyme pathways were all least sensitive to their own products HETEs. To determine which structural characteristics of 15-HETE are essential for inhibition of the 5-lipoxygenase, various derivatives were prepared and purified by high pressure liquid chromatography, and their structures were confirmed by gas chromatography-mass spectrometry. The inhibitory potencies of 15-HETE analogs with different degrees of unsaturation were in the order of three double bonds greater than 4 greater than 2 greater than 0. 15-Hydroperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE) was four times more potent than 15-HETE. The 15-acetoxy, 15-keto and methyl ester derivatives were of comparable activity to 15-HETE, and the 15-acetoxy methyl ester derivative was less potent. Based upon the observed patterns of inhibition, we postulate that complex interregulatory relationships exist between the various lipoxygenases, and that cells containing these lipoxygenases may interact with each other via their lipoxygenase metabolites.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acid; Arachidonic Acids; Blood Platelets; Humans; Hydroxyeicosatetraenoic Acids; Leukotrienes; Lipid Peroxides; Lipoxygenase; Lipoxygenase Inhibitors; Neutrophils; Rabbits; Species Specificity; Structure-Activity Relationship