15-hydroperoxy-5-8-11-13-eicosatetraenoic-acid and arachidonic-acid-5-hydroperoxide

This work was performed to elucidate further the main cellular events underlying the protective effect of ischaemic preconditioning in an in vivo rat liver model of 90 min ischaemia followed by 30 min reperfusion. A significant attenuation of the various aspects of post-ischaemic injury, namely necrosis and the levels of hydrogen peroxide and 5- and 15-hydroperoxyeicosatetraenoic acids, was afforded by the prior application of a short cycle of ischaemia/reperfusion (10 + 10 min) or when rats were previously treated with gadolinium chloride. However, when preconditioning was applied on Kupffer cell-depleted livers, no additional level of ischaemic tolerance was obtained. In terms of cellular pathology, this result could be suggestive of Kupffer cells as the target of the preconditioning phenomenon during the warm ischaemia/reperfusion injury. Accordingly, modulation of Kupffer cell activity was associated with a well-preserved hepatocyte integrity, together with low levels of pro-oxidant generation during reperfusion. As activated Kupffer cells can generate and release potentially toxic substances, their modulation by ischaemic preconditioning could help to provide new surgical and/or pharmacological strategies to protect the liver against reperfusion damage.

Topics: Animals; Arachidonate 5-Lipoxygenase; Gadolinium; Hydrogen Peroxide; Ischemic Preconditioning; Kupffer Cells; Leukotrienes; Lipid Peroxides; Liver; Male; Neutrophils; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Sodium Chloride; Transaminases

Electrospray ionization ion trap mass spectra of 5-, 12-, and 15-hydroperoxyeicosatetraenoic (HPETE), hydroxyeicosatetraenoic (HETE), and ketoeicosatetraenoic (KETE) acids were recorded. The HPETE were partly dehydrated to the corresponding KETE in the heated capillary of the mass spectrometer. 12-HPETE and 15-HPETE were also converted to KETE by collision-induced dissociation (CID) in the ion trap, whereas CID of 5-HPETE yielded little formation of 5-KETE. Subcellular fractions of bovine corneal epithelium were incubated with arachidonic acid (AA) and the metabolites were analyzed. 15-HETE and 12-HETE were consistently formed, whereas significant accumulation of HPETE and KETE was not detected. Biosynthesis of 12- and 15-HETE was quantified with octadeuterated 12-HETE and 15-HETE as internal standards. The average biosynthesis of 15-HETE and 12-HETE from 30 microM AA by the cytosol was 38 +/- 8 and below 3 ng/mg protein/30 min, respectively, which increased to 78 +/- 21 and 10 +/- 4 ng/mg protein/30 min in the presence of 1 mM free Ca2+. The microsomal biosynthesis was unaffected by Ca2+. The microsomes metabolized AA to 15-HETE as the main metabolite at a low protein concentration (0.3 mg/mL), whereas 12-HETE and 15-HETE were formed in a 2:1 ratio at a combined rate of 0.7 +/- 0.2 microg/mg protein/30 min at a high protein concentration (1.8 mg/mL). The level of 12-HETE in corneal epithelial cells was 50 +/- 13 pg/mg tissue, whereas the endogenous amount of 15-HETE was low or undetectable (<3 pg/mg tissue). Incubation of corneas for 20 min at 37 degrees C before processing selectively increased the amounts of 12-HETE in the epithelium fourfold to approximately 0.2 ng/mg tissue. We conclude that 12-HETE is the main endogenously formed lipoxygenase product of bovine corneal epithelium.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Cattle; Chromatography, Liquid; Epithelium, Corneal; Hydroxyeicosatetraenoic Acids; Leukotrienes; Lipid Peroxides; Lipoxygenase; Mass Spectrometry; Subcellular Fractions

Treatment of human natural killer (NK) cells with phospholipase A(2) (PLA(2)) inhibitors, mepacrine and 4-bromophenacyl bromide (BPB), diminished their ability to lyse K562 target cells by as much as 100%. The ability of NK cells to bind to K562 cells was significantly affected by BPB above 2 microM, but not by mepacrine at any concentration tested. This indicates that BPB is having effects on NK cells unrelated to its inhibition of PLA(2) activity at concentrations above 2 microM. The activation of phospholipase C in response to K562 cell binding (as measured by inositol phosphate turnover) was unaffected by inhibition of the PLA(2) activity. The products of PLA(2) catabolism are a fatty acid (often arachidonic acid) and a lysophospholipid. Inhibition of NK cytotoxicity by mepacrine or BPB is reversed significantly when lysophosphatidylcholine, but no other lysolipid, is added back to the NK cells before assaying for cytotoxicity. Arachidonic acid, but not linoleic acid, also significantly reverses inhibition of NK cytotoxicity. Finally, the 15-lipoxygenase product, 15S-hydroperoxyeicosatetraenoic acid (15S-HPETE), is also able to reverse mepacrine-induced inhibition of NK cytotoxicity. The 5-lipoxygenase product 5S-HPETE was not effective. These data indicate that PLA(2) activation is a necessary signal in human NK cytotoxicity and that it is not involved in protein tyrosine kinase and subsequent phospholipase C activation; these latter two enzymes are also required in the cytotoxic response. Thus PLA(2) activation is either a more distal signal, dependent on activation of some earlier signal, or an independent cosignal stimulated by tumor-target binding which generates lysophosphatidylcholine, arachidonic acid, and/or a lipoxygenase product(s).

Topics: Acetophenones; Arachidonic Acid; Cytotoxicity, Immunologic; Enzyme Inhibitors; Humans; K562 Cells; Killer Cells, Natural; Leukotrienes; Linoleic Acid; Lipid Peroxides; Lysophosphatidylcholines; Phospholipases A; Quinacrine

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

The acute activation of adrenal glucocorticoid synthesis by ACTH has long been believed to be mediated by cAMP as the major second messenger, although increases in cellular cAMP concentration have not been observed at low concentrations of ACTH. We found that steroidogenesis in bovine adrenal fasciculata-reticularis cells was activated by the addition of arachidonic acid or its 15-lipoxygenase metabolite, 15-hydroperoxyeicosatetraenoic acid. The cellular 15-lipoxygenase pathway was significantly activated by 1 pM ACTH, at which concentration no increase in cellular cAMP synthesis was observed. The 1 pM ACTH-induced stimulation of steroidogenesis was completely suppressed by a lipoxygenase inhibitor, AA-861. The stimulation was independent of the increase in cellular cAMP. These results show that the action of 1 pM ACTH on steroidogenesis may be mediated by the 15-lipoxygenase metabolite(s) as a solo second messenger. The addition of ACTH at concentrations higher than 10 pM increased both the 15-lipoxygenase activity and cellular cAMP synthesis. Under these conditions, the 15-lipoxygenase metabolite(s) and cAMP were shown to mediate the activation of steroidogenesis synergistically. The presence of a dual second messenger system could explain the stimulation of steroidogenesis by ACTH at physiological concentrations.

Topics: Animals; Arachidonate 15-Lipoxygenase; Arachidonic Acids; Benzoquinones; Bucladesine; Cattle; Cells, Cultured; Cosyntropin; Cyclic AMP; Dihydrotestosterone; Enzyme Inhibitors; Indomethacin; Isoquinolines; Kinetics; Leukotrienes; Lipid Peroxides; Lipoxygenase Inhibitors; Masoprocol; Pregnenolone; Sulfonamides; Tetrahydronaphthalenes; Zona Fasciculata; Zona Reticularis

To understand the mechanism of the phototoxic effects of inperatorin, a psoralen derivative used as a pigmentation agent, we have investigated the photosensitized oxidation of arachidonic acid (ARA) by irradiation with visible light (> 400 nm) in the presence of inperatorin. HPLC and GC/MS analyses of the products showed the formation of many hydroperoxyeicosatetraenoic acids (HPETEs) including the products of lipoxygenase-catalyzed reactions such as 5- and 15-HPETEs, which are the precursors of chemical mediators such as leukotrienes and lipoxins, during the reaction. Active oxygen scavening agents such as D-mannitol, superoxide dismutase, and beta-carotene inhibited the formation of the oxidation products, indicating that the oxidation reaction was mediated by various active oxygen species. These results suggest that the phototoxic effects of inperatorin could also be induced by visible light and could be explained at least partially in terms of inflammation initiated by the biologically active HPETEs arising from photosensitized oxygenation reactions of ARA with the drug.

Topics: Arachidonic Acid; beta Carotene; Borohydrides; Carotenoids; Chromatography, High Pressure Liquid; Furocoumarins; Gas Chromatography-Mass Spectrometry; Leukotrienes; Light; Lipid Peroxides; Mannitol; Oxidation-Reduction; Photochemistry; Photosensitizing Agents; Reactive Oxygen Species; Superoxide Dismutase

Oxygen radical-induced genetic damage may be mediated by products of lipid peroxidation, in particular, arachidonic acid. Several isomeric hydroxy- and hydroperoxy-6,8,11,14-eicosatetraenoic acids (HETEs and HPETEs), intermediates of arachidonic acid metabolism, were evaluated for their ability to cause sister chromatid exchanges (SCEs) in Chinese hamster ovary (CHO) cells. Both HETEs and HPETEs induced SCEs in a dose-dependent fashion at concentrations of 5, 10, and 20 microM. At each concentration, HETEs were more effective in producing SCEs than the corresponding HPETEs. Each of the isomeric forms used were equally effective in producing genetic damage. Antioxidants (superoxide dismutase, catalase and mannitol) were protective suggesting an intermediate role for the hydroxyl radical. Iron chelation by desferrioxamine suppressed SCE induction by 45% and an additional 33% inhibition was observed upon the addition of the calcium chelator EGTA.

Topics: Animals; Catalase; Cell Line; Cricetinae; Deferoxamine; Egtazic Acid; Free Radicals; Hydroxyeicosatetraenoic Acids; Leukotrienes; Lipid Peroxidation; Lipid Peroxides; Mannitol; Sister Chromatid Exchange; Superoxide Dismutase

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

An improved direct chemical ionization (DCI) mass spectrometric technique, using a polyimide-coated fused silica fiber as an extended probe tip, was used to obtain molecular ions and diagnostic fragment ions of underivatized arachidonic acid, 5-hydroperoxyeicosatetraenoic acid, 15-hydroperoxyeicosatetraenoic acid, leukotriene B4 (LTB4) and, for the first time, of leukotriene A4 (LTA4)-free acid. In this technique, sample compounds are coated onto the fused silica fiber and vaporized in the plume of the reagent gas plasma of a chemical ionization source without external heating of the probe. Both ammonia and isobutane DCI spectra were obtained for each compound. A volatile alkaline eluent system was developed that allowed reversed-phase high-performance liquid chromatography of LTA4 to be followed rapidly by DCI mass spectrometry. With these techniques, the conversion of LTA4 to LTB4 during incubation with human liver microsomes was confirmed. Selected ion monitoring (SIM) of preselected ion fragments in the spectrum increases the selectivity of this technique and improves quantification in the range 100 ng to 10 pg.

Topics: Arachidonic Acids; Humans; In Vitro Techniques; Leukotriene A4; Leukotriene B4; Leukotrienes; Lipid Peroxides; Mass Spectrometry; Microsomes, Liver

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

Sulfhydryl reagents such as dithiothreitol stabilized human leukocyte 5-lipoxygenase (5-LO) during purification. During enzyme assay, however, these reagents led to irreproducible or unexpectedly low activity. This inconsistency in the assay was eliminated by inclusion of hydroperoxyeicosatetraenoic acids (1-5 microM) during the reaction which effected a 10-20-fold stimulation of 5-LO activity. Structural studies indicated that an intact hydroperoxy function, and a long-chain fatty acyl moiety were required for 5-LO stimulation. These data suggest that human leukocyte 5-LO is activated by hydroperoxy fatty acids, and that this results in a requirement for exogenous hydroperoxide in the presence of sulfhydryl reagents.

Topics: Arachidonate Lipoxygenases; Arachidonic Acids; Enzyme Activation; Humans; Leukocytes; Leukotrienes; Lipid Peroxides; Lipoxygenase; Sulfhydryl Reagents

The biosynthetic conversions of arachidonic acid to hydroperoxyeicosatetraenoic acids (HPETEs) and the further conversion of leukotriene epoxides are accompanied by stereoselective hydrogen abstraction from the reaction substrate. Furthermore, this hydrogen removal has always been found to occur in fixed stereochemical relationship to carbon-oxygen chiral center(s) in the substrate or product. We have used stereospecifically labeled 10-3H-substrates with 14C internal standard to investigate whether the same relationships bear in HPETE and leukotriene formation during autoxidation. After autoxidation of labeled arachidonate, both the 8(R)- and 8(S)-HPETE enantiomers (resolved as diastereomer derivatives) and the 12(RS)-HPETE were observed to retain 41-47% 3H relative to the starting material. In autoxidative formation of leukotrienes from labeled 15(S)-HPETE the four main leukotrienes, including two derived from 14,15-leukotriene A4 hydrolysis, were observed to have retained an average of 45% 3H. Primary and secondary isotope effects were found to accompany these reactions. The results prove that stereorandom hydrogen abstraction occurs in autoxidation and that the hydrogen loss bears no stereochemical relationship to chiral oxygen center(s) in the HPETE product, (8(R) or 8(S], or the 15(S)-hydroperoxy substrate. We conclude that the chiral features of the biosynthetic reactions are a reflection of enzymatic control of stereochemistry. Nonetheless, the findings of primary and secondary isotope effects in autoxidation which are similar to those observed in the analogous biosynthetic reactions suggests that, except for stereochemical control, the autoxidative and enzymatic reactions may be mechanistically similar.

Topics: Arachidonic Acids; Chemical Phenomena; Chemistry, Physical; Chromatography, High Pressure Liquid; Hydrogen; Leukotriene A4; Leukotrienes; Lipid Peroxides; Stereoisomerism

A series of stereospecifically labeled polyunsaturated fatty acids were prepared by biosynthesis from [8-DR-3H]- and [8-LS-3H]stearic acids. The labeled stearic acids were synthesized by a novel scheme employing readily available alkyne and aldehyde starting materials. The stereochemical purity of the prochiral tritium labels was judged to be greater than 99%, as determined by analysis of the octadec-1-yn-8(R)- and 8(S)-ol intermediates in the synthesis. Previously, the labeled arachidonic acids were used to investigate the stereoselectivity of hydrogen abstraction in the biosynthesis of leukotriene epoxides. We have now investigated the selectivity of hydrogen abstraction in a chemical synthesis of 14,15-leukotriene (LT) A4 from mixtures of [3-14C]- and either [10-DR-3H]- or [10-LS-3H]15(S)-HPETE methyl esters. Reaction with either chirally labeled precursor led to 70-95% retention of 3H relative to 14C in the 14,15-LTA4 and 10-Z-14,15-LTA4 products after purification by high performance liquid chromatography. The 15-dienone obtained from this reaction was consistently enriched in 3H relative to 14C after isolation and purification. Evidence was obtained to indicate that the majority of the 3H in the products was retained in its original location and configuration. These results indicate that the biomimetic chemical reaction is stereo-random with respect to hydrogen loss from carbon 10 and that, in contrast to the reaction as it occurs in leukocytes and platelets, in the chemical model the reaction begins by decomposition of the hydroperoxide group, with hydrogen loss from carbon 10 occurring as a late or final step.

Topics: Arachidonic Acid; Arachidonic Acids; Chemical Phenomena; Chemistry, Physical; Chromatography, High Pressure Liquid; Hydrogen; Leukotriene A4; Leukotrienes; Lipid Peroxides; Magnetic Resonance Spectroscopy; Spectrophotometry, Ultraviolet; Stereoisomerism

The authors are studying the molecular details of the process that begins with hepatic metabolism of halogenated inhalation anesthetics and ends with hepatic necrosis. In previous studies they have shown that the halothane-free radical produced by UV-irradiation is identical to that produced during reductive metabolism of halothane by hepatic cytochrome P-450. In the present study, the authors have examined a mechanism by which free radicals may propagate damage in the endoplasmic reticulum of liver cells. The 1-chloro-2,2,2-trifluoroethyl free radical produced by UV-irradiation of halothane can abstract a hydrogen radical from arachidonic acid to yield 2-chloro-1,1,1-trifluoroethane and an arachidonic acid-free radical. The arachidonic acid-free radical reacts with molecular oxygen to form 5- and 15-hydroperoxyeicosatetraenoic acid. There is considerable evidence that the peroxidation process that we studied in the model system will be similar when the arachidonic acid is an acyl chain on a membrane phospholipid and the free radicals are generated metabolically. The authors suggest that these hydroperoxides may be toxic by acting as intermediates in the pathway of leukotriene production as well as by direct oxidation of membrane components.

Topics: Arachidonic Acids; Chemical Phenomena; Chemistry; Free Radicals; Halothane; Leukotrienes; Lipid Peroxides; Mass Spectrometry; Ultraviolet Rays

Incubation of various hemoproteins with 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid or 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid resulted in formation of epimeric 5(S),12-dihydroxy-6,8,10,14 -eicosatetraenoic acids and epimeric 8,15(S)-dihydroxy-5,9,11,13 -eicosatetraenoic acids, respectively. These dihydroxy acids were earlier recognized as nonenzymatic hydrolysis products of 5(S),6-oxido-7,9,11,14-eicosatetraenoic acid (leukotriene A4) and 14,15(S)-oxido-5,8,10,12-eicosatetraenoic acid (14,15-leukotriene A4). These allylic epoxides could be isolated as such from the hemoprotein incubations, and most probably they are intermediates in formation of the dihydroxy acids.

Topics: Arachidonic Acids; Chromatography, High Pressure Liquid; Cytochromes; Ferrous Compounds; Gas Chromatography-Mass Spectrometry; Horseradish Peroxidase; Iron; Leukotrienes; Lipid Peroxides; Peroxidases; SRS-A

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Chromatography, High Pressure Liquid; Eosinophils; Humans; Leukotrienes; Lipid Peroxides; Mice; Oxidation-Reduction; Species Specificity; Swine

Topics: Animals; Arachidonic Acids; Chromatography, High Pressure Liquid; Eicosapentaenoic Acid; Gas Chromatography-Mass Spectrometry; Humans; Leukemia, Experimental; Leukocytes; Leukotriene A4; Leukotrienes; Lipid Peroxides; Lipoxygenase; Peroxides; Rats; SRS-A