leukotriene-a4 and arachidonic-acid-5-hydroperoxide

The family of eicasanoids, biologically active metabolites of polyunsaturated C20 fatty acids such as arachidonic acid, has recently been enlarged by the recognition of a new biosynthetic pathway leading to the leukotrienes, including the compounds described two decades ago as 'slow reacting substances'. These biologically potent substances are involved in regulation of the immune response and also as mediators in various disease states. This account presents a brief history of this field, an overview of the biological relevance of leukotrienes, and a discussion of the investigations which led to the clarification of the molecular structures, pathway of biosynthesis and total chemical synthesis of the leukotrienes, including leukotrienes A, B, C, D and E (LTA-LTE). As a result of the synthetic work these rare substances are available for the first time in pure form and in quantities sufficient for biological and medical studies. Also reviewed are recent discoveries with regard to the development of inhibitors of leukotriene biosynthesis and anti-leukotrienes.

Topics: Animals; Arachidonic Acids; Asthma; Autacoids; Chemical Phenomena; Chemistry; Humans; Hydroxyeicosatetraenoic Acids; Hypersensitivity; Leukotriene A4; Leukotriene B4; Leukotriene E4; Leukotrienes; Lipoxygenase Inhibitors; Macrophages; Mast Cells; Molecular Conformation; Neutrophils; SRS-A; Stereoisomerism; Structure-Activity Relationship

5-Lipoxygenase (5-LOX) reacts with arachidonic acid (AA) to first generate 5(S)-hydroperoxy-6(E),8(Z),11(Z),14(Z)-eicosatetraenoic acid [5(S)-HpETE] and then an epoxide from 5(S)-HpETE to form leukotriene A4, from a single polyunsaturated fatty acid. This work investigates the kinetic mechanism of these two processes and the role of ATP in their activation. Specifically, it was determined that epoxidation of 5(S)-HpETE (dehydration of the hydroperoxide) has a rate of substrate capture (Vmax/Km) significantly lower than that of AA hydroperoxidation (oxidation of AA to form the hydroperoxide); however, hyperbolic kinetic parameters for ATP activation indicate a similar activation for AA and 5(S)-HpETE. Solvent isotope effect results for both hydroperoxidation and epoxidation indicate that a specific step in its molecular mechanism is changed, possibly because of a lowering of the dependence of the rate-limiting step on hydrogen atom abstraction and an increase in the dependency on hydrogen bond rearrangement. Therefore, changes in ATP concentration in the cell could affect the production of 5-LOX products, such as leukotrienes and lipoxins, and thus have wide implications for the regulation of cellular inflammation.

Topics: Adenosine Triphosphate; Allosteric Regulation; Arachidonate 5-Lipoxygenase; Arachidonic Acid; Calcium; Enzyme Activation; Epoxy Compounds; Humans; Leukotriene A4; Leukotrienes; Peroxides; Stereoisomerism; Viscosity

Leukotrienes are important mediators in a number of inflammatory diseases and therefore are a target of several therapeutic approaches. The first committed step in the synthesis of leukotrienes is the conversion of arachidonic acid to leukotriene A(4) (LTA(4)) in two successive reactions catalyzed by 5-lipoxygenase (5-LOX). Assays to measure 5-LOX activity typically have been low throughput and time consuming. In this article, we describe a fluorescence assay that is amenable to high-throughput screening in a 384-well microplate format. The fluorescent signal is measured during oxidation of 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) by human 5-LOX. The assay has been found to reliably identify small molecule inhibitors of human 5-LOX. The IC(50) values of several 5-LOX inhibitors in this new assay are comparable to those determined in a standard spectrophotometric assay that measures the formation of the 5(S)-hydroperoxyeicosatetraenoic acid (5-HpETE) product. In addition, we demonstrate the use of the assay in a high-throughput screen of the Pfizer compound collection to identify inhibitors of 5-LOX.

Topics: Arachidonate 5-Lipoxygenase; Chromogenic Compounds; Cloning, Molecular; Drug Evaluation, Preclinical; Fluoresceins; Fluorescence; Fluorescent Dyes; Humans; Indicators and Reagents; Inhibitory Concentration 50; Leukotriene A4; Leukotrienes; Lipoxygenase Inhibitors; Sensitivity and Specificity; Spectrophotometry, Ultraviolet; Substrate Specificity

(-)-Epicatechin and its related oligomers, the procyanidins, are present in sizable amounts in some cocoas and chocolates. Intake of flavonoid-rich chocolate in humans has been reported to increase the plasma level of (-)-epicatechin and concomitantly to significantly decrease the plasma level of proinflammatory cysteinyl leukotrienes. Because leukotrienes are formed via the 5-lipoxygenase pathway of arachidonic acid metabolism, we examined whether 5-lipoxygenase is a possible target for the flavonoids of cocoa. Recombinant human 5-lipoxygenase was reacted with arachidonic acid and yielded a mixture of mainly 5-hydroperoxy-6E,8Z, 11Z,14Z-eicosatetraenoic acid (5-HpETE) and hydrolysis products of 5,6-leukotriene A(4) (LTA(4)). The formation of these products was significantly inhibited by (-)-epicatechin in a dose-dependent manner with 50% inhibitory concentrations (IC(50)) of 22 and 50 micromol/L, respectively. Among the procyanidin fractions isolated from the seeds of Theobroma cacao, only the dimer fraction and, to a lesser extent, the trimer through pentamer fractions exhibited comparable effects, whereas the larger procyanidins (hexamer through nonamer) were almost inactive. We conclude that (-)-epicatechin and its low-molecular procyanidins inhibit both dioxygenase and LTA(4) synthase activities of human 5-lipoxygenase and that this action may contribute to a putative anti-inflammatory effect of cocoa products.

Topics: Antioxidants; Biflavonoids; Cacao; Catechin; Dose-Response Relationship, Drug; Escherichia coli; Flavonoids; Humans; Hydrolysis; Leukotriene A4; Leukotriene Antagonists; Leukotrienes; Lipoxygenase Inhibitors; Molecular Weight; Proanthocyanidins; Protein Isoforms; Recombinant Proteins

Leukotriene A(4) hydrolase in mammals is a bifunctional zinc metalloenzyme that catalyzes the hydrolysis of leukotriene A(4) into the proinflammatory mediator leukotriene B(4), and also possesses an aminopeptidase activity. Recently we cloned and characterized an leukotriene A(4) hydrolase from Saccharomyces cerevisiae as a leucyl aminopeptidase with an epoxide hydrolase activity. Here we show that S. cerevisiae leukotriene A(4) hydrolase is a metalloenzyme containing one zinc atom complexed to His-340, His-344, and Glu-363. Mutagenetic analysis indicates that the aminopeptidase activity follows a general base mechanism with Glu-341 and Tyr-429 as the base and proton donor, respectively. Furthermore, the yeast enzyme hydrolyzes leukotriene A(4) into three compounds, viz., 5S,6S-dihydroxy-7,9-trans-11,14-cis-eicosatetraenoic acid, leukotriene B(4), and Delta(6)-trans-Delta(8)-cis-leukotriene B(4), with a relative formation of 1:0.2:0.1. In addition, exposure of S. cerevisiae leukotriene A(4) hydrolase to leukotriene A(4) selectively inactivates the epoxide hydrolase activity with a simultaneous stimulation of the aminopeptidase activity. Moreover, kinetic analyses of wild-type and mutated S. cerevisiae leukotriene A(4) hydrolase suggest that leukotriene A(4) binds in one catalytic mode and one tight-binding, regulatory mode. Exchange of a Phe-424 in S. cerevisiae leukotriene A(4) hydrolase for a Tyr, the corresponding residue in human leukotriene A(4) hydrolase, results in a protein that converts leukotriene A(4) into leukotriene B(4) with an improved efficiency and specificity. Hence, by a single point mutation, we could make the active site better suited to bind and turn over the substrate leukotriene A(4), thus mimicking a distinct step in the molecular evolution of S. cerevisiae leukotriene A(4) hydrolase toward its mammalian counterparts.

Topics: Amino Acid Sequence; Animals; Binding Sites; Catalytic Domain; Enzyme Inhibitors; Epoxide Hydrolases; Escherichia coli; Glutamic Acid; Humans; Hydrolysis; Leukotriene A4; Leukotriene B4; Leukotrienes; Molecular Sequence Data; Mutagenesis, Site-Directed; Phenylalanine; Recombinant Proteins; Saccharomyces cerevisiae; Spodoptera; Tyrosine; Zinc

The positional specificity of arachidonic acid oxygenation is currently the decisive parameter for classification of lipoxygenases. Although the mechanistic basis of lipoxygenase specificity is not completely understood, sequence determinants for the positional specificity have been identified for various isoenzymes. In this study we altered the positional specificity of the human 5-lipoxygenase by multiple site-directed mutagenesis and assayed the leukotriene A(4) synthase activity of the mutant enzyme species with (5S,6E,8Z,11Z,14Z)-5-hydroperoxy-6,8,11,14-eicos atetraenoic acid (5S-HpETE) as substrate. The wild-type 5-lipoxygenase converts 5S-HpETE almost exclusively to leukotriene A(4) as indicated by the dominant formation of leukotriene A(4) hydrolysis products. Since leukotriene synthesis involves a hydrogen abstraction from C(10), it was anticipated that the 15-lipoxygenating quadruple mutant F359W + A424I + N425M + A603I might not exhibit a major leukotriene A(4) synthase activity. Surprisingly, we found that this quadruple mutant exhibited a similar leukotriene synthase activity as the wild-type enzyme in addition to its double oxygenation activity. The leukotriene synthase activity of the 8-lipoxygenating double mutant F359W + A424I was almost twice as high, and similar amounts of leukotriene A(4) hydrolysis products and double oxygenation derivatives were detected with this enzyme species. These data indicate that site-directed mutagenesis of the human 5-lipoxygenase that leads to alterations in the positional specificity favoring arachidonic acid 15-lipoxygenation does not suppress the leukotriene synthase activity of the enzyme. The residual 8-lipoxygease activity of the mutant enzyme and its augmented rate of 5-HpETE conversion may be discussed as major reasons for this unexpected result.

Topics: Animals; Arachidonate 12-Lipoxygenase; Arachidonate 15-Lipoxygenase; Arachidonate 5-Lipoxygenase; Catalysis; Chromatography, High Pressure Liquid; Enzyme Activation; Gas Chromatography-Mass Spectrometry; Humans; Leukotriene A4; Leukotrienes; Mutagenesis, Site-Directed; Rabbits; Recombinant Proteins; Substrate Specificity

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

Topics: 5-Lipoxygenase-Activating Proteins; Animals; Arachidonate 5-Lipoxygenase; Carrier Proteins; Cell Line; Electrophoresis, Polyacrylamide Gel; Humans; Kinetics; Leukotriene A4; Leukotrienes; Linoleic Acids; Lipid Peroxides; Membrane Proteins; Recombinant Proteins; Spodoptera; Substrate Specificity; Transfection

A Ca2+ and a phosphatidylcholine (PC) as stimulatory factors to human 5-lipoxygenase (5-LO) were assessed to examine aspects of the regulatory mechanism of 5-LO. In the presence of Ca2+ (1 microM or less), PC liposomes distinctly stimulated the dual activities of 5-LO for the production of 5-HPETE from arachidonate and for its subsequent conversion to LTA4. At the same concentration of Ca2+, 5-LO was found to bind to PC liposomes. As with 5-LO activities, the binding was dependent on the range of Ca2+ concentration. The conversion ratios of 5-HPETE to LTA4 were dependent on PC liposome concentration and reached a maximum of 50% conversion. Among the four cell membrane lipids examined, PC liposomes demonstrated the highest conversion ratio of 5-HPETE to LTA4 by 5-LO. Most of the arachidonate added to the reaction mixture localized in PC liposomes. These results confirm that the intracellular increase of Ca2+ concentration causes 5-LO to associate with the cell membrane and perform an interfacial reaction. They also suggest that this binding of 5-LO to the cell membrane enhances the subsequent conversion from 5-HPETE to LTA4.

Topics: Adenosine Triphosphate; Arachidonate 5-Lipoxygenase; Arachidonic Acid; Calcium; Enzyme Activation; Humans; Leukotriene A4; Leukotrienes; Liposomes; Phosphatidylcholines

5-Lipoxygenase is the first committed enzyme in the leukotriene biosynthetic pathway and is known to catalyze not only the first oxygenation of arachidonate to form 5(S)-hydroperoxyeicosatetraenoic acid (5(S)-HPETE), but also dehydration of this intermediate into leukotriene A4 (LTA4) by an activity termed leukotriene A4 synthase. Inhibition of cytosolic 5-lipoxygenase prepared from human blood granulocytes with zileuton (100 microM) was virtually complete, but LTA4 synthase activity was only inhibited by 47%. Structural characterization of eicosanoids synthesized in these preparations revealed an abundance of 15-lipoxygenase metabolites including 15-HETE when arachidonate was used as substrate and 5(S),15(S)-dihydroxy-6,8,11,13(E,E,Z,Z)-eicosatetraenoic acid when 5(S)-HPETE was used as substrate. When neutrophils were prepared that contained less than 1% eosinophil contamination, zileuton was found to almost completely inhibit all 5-lipoxygenase, as well as LTA4 synthase products. Immunochemical analysis of the supernatants from purified neutrophils and eosinophils confirmed the previous observation that neutrophils do not express 15-lipoxygenase. Incubation of 5(S)-HPETE with recombinant mammalian 15-lipoxygenase resulted in the formation of 6-trans-LTB4 and 6-trans-12-epi-LTB4 as LTA4 products, as well as the 12-lipoxygenase product 5(S),12(S)-diHPETE. The mechanism of action of 15-lipoxygenase acting as an LTA4 synthase is proposed to involve removing the pro-R hydrogen atom at carbon-10 of 5(S)-HPETE, which is antarafacial to the hydroperoxy group to yield LTA4.

Topics: Arachidonate 15-Lipoxygenase; Arachidonate 5-Lipoxygenase; Arachidonic Acid; Cell-Free System; Cytosol; Dose-Response Relationship, Drug; Eosinophils; Granulocytes; Humans; Hydroxyeicosatetraenoic Acids; Hydroxyurea; Leukotriene A4; Leukotrienes; Lipoxygenase Inhibitors; Models, Chemical; Neutrophils; Recombinant Proteins

Low levels of 5-lipoxygenase (5-LO), the first committed enzyme in the synthesis of leukotrienes (LTs), have been reported in the porcine pancreas. We have quantitated 5-LO activity in subcellular fractions of pancreas samples from three human donors. 5-LO activity was detectable in all samples, although enzyme activity was lower than in human leukocytes. 5-LO in human pancreas samples displayed highest specific activity in membrane fractions, and did not require arachidonic acid (AA) addition for activity. These unusual characteristics of pancreatic 5-LO appear to be due, at least in part, to the presence of unesterified AA in the pancreas samples. Western blot analysis demonstrated that the human pancreas contains low levels of 5-lipoxygenase-activating protein (FLAP) in addition to 5-LO.

Topics: 5-Lipoxygenase-Activating Proteins; Arachidonate 5-Lipoxygenase; Arachidonic Acid; Blotting, Western; Carrier Proteins; Chromatography, High Pressure Liquid; Female; Humans; Leukotriene A4; Leukotriene B4; Leukotrienes; Male; Membrane Proteins; Middle Aged; Pancreas; Subcellular Fractions

Purified recombinant human 5-lipoxygenase was used to investigate the catalytic properties of the protein in the presence and absence of leukocyte stimulatory factors. Recombinant human 5-lipoxygenase was purified to apparent homogeneity (95-99%) from a high expression baculovirus system by chromatography on ATP-agarose with a yield of 0.6 mg of protein per 100 ml of culture (2 x 10(8) cells) and a specific activity of 3-6 mumol of 5-hydroperoxyeicosatetraenoic acid (5-HPETE) per mg of protein in the presence of ATP, Ca2+, and phosphatidylcholine as the only factors. In the absence of leukocyte factors, the reaction catalyzed by the purified recombinant enzyme showed a half-time of maximal 5-HPETE formation of 0.5-0.7 min and was sensitive to the selective 5-lipoxygenase inhibitors BW755C (IC50 = 13 microM) and L-656,224 (IC50 = 0.8 microM). The reaction products of arachidonic acid oxidation were 5-HPETE and 6-trans- and 12-epi-6-trans-leukotriene B4, the nonenzymatic hydrolysis products of leukotriene A4 (LTA4), indicating that the purified protein expressed both the 5-oxygenase and leukotriene A4 synthase activities (ratio 6:1). The microsomal fraction and the 60-90% ammonium sulfate precipitate fraction from sonicated human leukocytes did not increase product formation by the isolated enzyme when assayed in the presence of ATP, Ca2+, and phosphatidylcholine. These factors were found to stabilize 5-lipoxygenase during preincubation of the enzyme at 37 degrees C with the assay mixture but they failed to stimulate enzymatic activity when added at the end of the preincubation period. The results demonstrate that human 5-lipoxygenase can be isolated in a catalytically active form and that protein factors from leukocytes protect against enzyme inactivation but are not essential for enzyme activity.

Topics: Arachidonate 5-Lipoxygenase; Baculoviridae; Blood Proteins; Blotting, Western; Catalysis; Chromatography, Gel; Electrophoresis, Polyacrylamide Gel; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Viral; Genes, Viral; Humans; Kinetics; Leukocytes; Leukotriene A4; Leukotrienes; Recombinant Proteins

A method for the simultaneous single-step organic extraction from biological matrices of peptido- and dihydroxyleukotrienes as well as 5-hydroperoxy- and 5-hydroxyeicosatetraenoic acid followed by separation and quantitation in a single run on reversed-phase high-performance liquid chromatography was evaluated. Using an extraction system comprising 400/1200/4800 (v/v/v) aqueous phase/isopropanol/dichloromethane, pH 3.0, absolute recoveries of 82.3 +/- 2.0, 89.7 +/- 1.0, 93.7 +/- 1.4, 92.8 +/- 1.4, 90 +/- 4, and 90 +/- 4% for prostaglandin B1 (PGB1), leukotriene C4 (LTC4), leukotriene B4 (LTB4), leukotriene D4 (LTD4), 5-hydroperoxyeicosatetraenoic acid (5-HETE), respectively, were achieved. Separation and quantitation of products were performed on a Nucleosil 100 C18 column (5 microns, 4.6 X 250 mm) using, at pH 6.0, a gradient system comprising 72/28/0.02 (v/v/v) methanol/water/glacial acetic acid from 0 to 15 min, followed by a convex gradient to 76/24/0.02 (v/v/v) methanol/water/glacial acetic acid, followed by a 10-min hold at this methanol concentration. The method was used to investigate the profile of leukotrienes synthesized by rat hepatocyte homogenates from 5-HPETE or leukotriene A4 in absence or presence of glutathione (GSH). During a 5-min incubation with 100 microM 5-HPETE, 9.6 ng LTB4/mg protein and 2.2 micrograms 5-HETE/mg protein were formed in the absence of GSH. In the presence of 0.4 mM GSH, 3.7 ng LTB4/mg protein and 11.0 micrograms 5-HETE/mg protein were formed. Using 20 microM LTA4 as a substrate, 17.3 and 324.0 ng LTC4/mg protein X min and 14.3 and 19.3 ng LTB4/mg protein X min were formed in the presence of 0.4 and 10 mM GSH, respectively.

Topics: 1-Propanol; Animals; Chromatography, High Pressure Liquid; Glutathione; Hydrogen-Ion Concentration; Hydroxyeicosatetraenoic Acids; Leukotriene A4; Leukotriene B4; Leukotrienes; Liver; Male; Methylene Chloride; Prostaglandins B; Rats; Rats, Inbred Strains; SRS-A

Rat hepatocyte homogenates convert 5-hydroperoxyeicosatetraenoic acid (5-HPETE) into biologically active leukotriene B4 (LTB4) as well as less active all-trans-LTB4 (i.e., 6-trans-LTB4 and 6-trans-12-epi-LTB4). Here, we present a hypothesis of the reaction mechanism and the minimal structural requirements of the active enzyme based on the following experimental evidence: The ED50 of the inhibitors 5,8,11,14-eicosatetraynoic acid (ETYA) and 5,6-dehydro-eicosatetraenoic acid was approximately 100-fold higher than for 5-lipoxygenase. Propanethiol and O2 were strong inhibitors of LTB4 formation, whereas butylated hydroxytoluene, nordihydroguaiaretic acid, metyrapone, Desferal and CO had no effect. Cytochrome c, catalase, hematin, and a Fe3+/Fe2+ couple, but not iron-free protoporphyrin IX, catalyzed the formation of only all-trans-LTB4. LTB4 formation in hepatocyte homogenates was heat- and trypsin-sensitive whereas all-trans-LTB4 formation was not. We propose that a ferric heme iron forms a ferryl-hydroxo complex upon homolytic scission of the oxygen-oxygen bond in 5-HPETE and the resulting 5,6-trans-epoxide radical is oxidized by the ferryl-hydroxo complex to yield LTA4. A mechanism for hydrolysis of LTA4 is described that results in formation of LTB4 (less than 1% yield) rather than all-trans-LTB4.

Topics: Animals; Arachidonate 5-Lipoxygenase; Arachidonic Acid; Arachidonic Acids; Binding Sites; Chemical Phenomena; Chemistry; Chromatography, High Pressure Liquid; Endoplasmic Reticulum; Free Radicals; Leukotriene A4; Leukotriene B4; Leukotrienes; Lipoxygenase Inhibitors; Liver; Male; Models, Biological; Molecular Structure; Oxygen; Rats; Rats, Inbred F344

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

Rat hepatocyte homogenates converted 5-hydroperoxyeicosatetraenoic acid into leukotriene B4 (LTB4). The reaction was dependent on time and protein and substrate concentration, did not require NADPH or oxygen, and was not supported by heat-inactivated hepatocyte homogenates. The authenticity of the biologically generated LTB4 that eluted at the position of synthetic LTB4 during high performance liquid chromatography was established by UV spectrophotometry, mass spectral analysis, radioimmunoassay, and a LTB4 receptor displacement assay. In addition, a leukotriene bioassay is described in which transient increases in cytosolic Ca2+ within human neutrophils are measured by means of fura-2 fluorescence. Biologically generated LTB4 was 40, 40, and 33% as active as synthetic LTB4 in the radioimmunoassay, receptor displacement assay, and cytosolic calcium bioassay, respectively. This activity is consistent with the biologically derived LTB4 being an epimeric mixture of (5S),(12R)-LTB4 and the much less active (5S),(12S)-LTB4. The formation of LTB4 was inhibited by 5,8,11,14-eicosatetraynoic acid (1 mM), 5,6-dehydro-arachidonic acid (50 microM), propanethiol (1 mM), and O2 (100%) to the extent of 53, 42, 48, and 66%, respectively. No inhibition was observed in the presence of diethylcarbamazine (1 mM) and desferal (1 mM). A possible contribution towards LTB4 formation by contaminating Kupffer cells was excluded (less than 0.2%). These results suggest that hepatocytes can convert lipid peroxides into potent chemoattractants that may alter the homeostasis of immunomediators within the liver.

Topics: Animals; Arachidonic Acids; Diethylcarbamazine; In Vitro Techniques; Leukotriene A4; Leukotriene B4; Leukotrienes; Lipoxygenase Inhibitors; Liver; Male; Mass Spectrometry; Rats; Rats, Inbred Strains; Receptors, Immunologic; Receptors, Leukotriene B4

Topics: Arachidonic Acids; Blood Platelets; Glutathione; Humans; In Vitro Techniques; Leukotriene A4; Leukotrienes; SRS-A

Topics: Animals; Arachidonic Acids; Epoxide Hydrolases; Glutathione Transferase; Guinea Pigs; Leukotriene A4; Leukotriene B4; Leukotrienes; Tissue Distribution

When arachidonic acid is added to lysates of rat polymorphonuclear leukocytes, it is oxidized to (5S)-hydroperoxy-6(E),8(Z),11(Z),14(Z)-eicosatetraenoic acid (5-HPETE). The 5-HPETE then partitions between reduction to the 5-hydroxyeicosanoid and conversion to leukotriene A4 (LTA4). Both steps in the formation of LTA4 are catalyzed by the enzyme 5-lipoxygenase. When [3H]arachidonic acid and unlabeled 5-HPETE were incubated together with 5-lipoxygenase, approximately 20% of the arachidonic acid oxidized at low enzyme concentrations was converted to LTA4 without reduction of the specific radioactivity of the LTA4 by the unlabeled 5-HPETE. A significant fraction of the [3H]-5-HPETE intermediate that is formed from arachidonic acid must therefore be converted directly to LTA4 without dissociation of the intermediate from the enzyme. This result predicts that even in the presence of high levels of peroxidase activity, which will trap any free 5-HPETE by reduction, the minimum efficiency of conversion of 5-HPETE to LTA4 will be approximately 20%, and this prediction was confirmed. 5-HPETE was found to be a competitive substrate relative to arachidonic acid, so that it is likely that the two substrates share a common active site.

Topics: Animals; Arachidonate 5-Lipoxygenase; Arachidonate Lipoxygenases; Arachidonic Acid; Arachidonic Acids; Kinetics; Leukotriene A4; Leukotrienes; Neutrophils; Rats; Tritium

Bovine polymorphonuclear leukocytes exhibit a 12-lipoxygenase activity upon sonication. In contrast to bovine platelet 12-lipoxygenase and other 12-lipoxygenases, this enzyme is unable to convert 5(S)-HETE (5(S)-hydroxy,6-trans-8,11,14-cis-eicosatetraenoic acid) or 5(S)-HPETE (5(S)-hydroperoxy,6-trans-8,11,14-cis-eicosatetraenoic acid) into 5(S),12(S)-dihydroxy-6,10-trans,8,14-cis-eicosatetraenoic acid. Surprisingly, the formation of leukotriene A4-derived products namely leukotriene B4 and the leukotriene B4-isomers 12-epi,6-trans- leukotriene B4 and 6-trans-leukotriene B4, was observed upon incubation of this enzyme with 5(S)-HPETE. Hence, the 12-lipoxygenase from bovine polymorphonuclear leukocytes possesses leukotriene A4-synthase activity.

Topics: Animals; Arachidonate 12-Lipoxygenase; Arachidonate Lipoxygenases; Arachidonic Acids; Cattle; Cell-Free System; Hydroxyeicosatetraenoic Acids; Leukotriene A4; Leukotriene B4; Leukotrienes; Neutrophils

A single protein from human leukocytes possesses both 5-lipoxygenase and leukotriene A4 (LTA4) synthase activities. It has been reported that LTA4 production is more efficient when the enzyme utilizes arachidonic acid, than when 5-HPETE is exogenously supplied as substrate. In the present study, human leukocyte homogenate 100,000 X g supernatant was incubated with 100 microM octadeuterated arachidonic acid and exogenous 5-HPETE (0-80 microM), and the isotopic composition of LTA4 hydrolysis products was determined by gas chromatography-mass spectrometry. Even though 100 microM deuterated arachidonic acid results in 20-30 microM deuterated 5-HPETE, 80 microM exogenous 5-HPETE in the incubation could reduce the amount of deuterated LTA4 by only approx. 20%. The present study would thus indicate that the arachidonic acid moiety is preferentially converted to LTA4 in a concerted reaction without dissociation of a 5-HPETE intermediate.

Topics: Arachidonate 5-Lipoxygenase; Arachidonate Lipoxygenases; Arachidonic Acid; Arachidonic Acids; Humans; Leukocytes; Leukotriene A4; Leukotrienes; Substrate Specificity

Tritium-labelled leukotriene A4 is converted by a suspension of human platelets into leukotriene C4. The conversion is stimulated by reduced glutathione and is dependent on the platelet concentration. Formation of leukotriene C4 is temperature and time dependent and is destroyed by heating the platelets at 100 degrees C for 5 min. Verification of leukotriene C4 formation was obtained by conversion into leukotriene D4 during reaction of the HPLC-purified platelet-derived leukotriene C4 with commercial gamma-glutamyl transpeptidase. In separate experiments we incubated authentic tritiated leukotriene C4 with human platelets and we showed the formation of tritiated leukotriene D4, demonstrating the presence of gamma-glutamyl transpeptidase activity in these cells. This activity could be blocked by the presence of reduced glutathione in the incubation mixture. In contrast, erythrocytes converted tritiated leukotriene A4 almost exclusively into leukotriene B4. Although platelets have been reported to lack 5-lipoxygenase activity, our study demonstrates that platelets possess the necessary machinery to transform leukotriene A4 into leukotrienes C4 and D4. Our results suggest that an intracellular interaction between platelets and leukotriene A4-forming cells, e.g., polymorphonuclear leukocytes, could lead to the formation of these potent peptidolipids in the circulation.

Topics: Arachidonate Lipoxygenases; Arachidonic Acids; Blood Platelets; Chromatography, High Pressure Liquid; Erythrocytes; gamma-Glutamyltransferase; Glutathione; Humans; Leukotriene A4; Leukotrienes; Lipoxygenase; SRS-A

The activity of leukotriene A4 (LTA4) synthase in crude human leukocyte homogenates was found to have a similar requirement for Ca2+ and ATP as had been noted previously for 5-lipoxygenase activity. Purification of the 5-lipoxygenase using ammonium sulfate fractionation, AcA 44 gel-filtration chromatography, and HPLC on anion-exchange and hydroxyapatite columns demonstrated that LTA4 synthase activity copurified with the 5-lipoxygenase with similar recoveries and increases in specific activity. Furthermore, the two enzymatic activities coeluted exactly on three different HPLC systems. Maximal activity of purified LTA4 synthase required the addition of three nondialyzable stimulatory factors, two of which were cytosolic and one of which was membrane-bound. These findings were identical for 5-lipoxygenase activity. When incubated with arachidonic acid, the purified 5-lipoxygenase converted approximately equal to 15% of its endogenously generated 5-hydroperoxyicosatetraenoic acid (5-HPETE) to LTA4. LTA4 production was more efficient when the enzyme utilized 5-HPETE generated from arachidonic acid than when 5-HPETE was exogenously supplied as substrate. These findings suggest that a single protein from human leukocytes possesses 5-lipoxygenase and LTA4 synthase activities and that the synthesis of LTA4 from 5-HPETE is controlled by the same complex multicomponent system that regulates the 5-lipoxygenase reaction.

Topics: Adenosine Triphosphate; Arachidonate Lipoxygenases; Arachidonic Acid; Arachidonic Acids; Calcium; Chromatography, Affinity; Chromatography, Gel; Chromatography, High Pressure Liquid; Humans; Leukocytes; Leukotriene A4; Leukotrienes; Lipoxygenase

Topics: Animals; Anti-Inflammatory Agents; Arachidonic Acids; Azoles; Blood Platelets; Humans; In Vitro Techniques; Isoindoles; Leukocytes; Leukotriene A4; Leukotriene B4; Leukotrienes; Organoselenium Compounds; Selenium; Swine

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

Polymorphonuclear neutrophils, when stimulated, rapidly form platelet-activating factor (PAF) and metabolize their arachidonate into 5-hydroperoxyeicosatetraenoate (5-HPETE), 5-hydroxyeicosatetraenoate (5-HETE), leukotriene (LT)A4, and LTB4. PAF and LTB4 degranulate neutrophils; 5-HETE, while lacking intrinsic degranulating actions, potentiates these responses. We now find that: a) 5-HPETE similarly potentiates the two lipids and has weak degranulating actions, b) LTA4 and LTB4 degranulate neutrophils by a common pathway, c) PAF degranulates neutrophils by a distinctly different pathway, d) the actions of either LT are additive to those of PAF, e) 5-HETE is particularly effective in potentiating response to combinations of PAF and LTB4, and f) combinations of the lipids partially circumvent requirements for cytochalasin B in these degranulation responses. Thus, the five lipids can be classified into potentiators (i.e., 5-HPETE and 5-HETE) and two types of independently acting agonists (i.e., LT's are one type, PAF a second type). At low concentrations, potentiator, LT, and PAF can all interact to produce prominent responses. They may similarly interact to promote function within their cells of origin.

Topics: Arachidonic Acids; Cytoplasmic Granules; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene A4; Leukotriene B4; Leukotrienes; Neutrophils; Platelet Activating Factor; Stimulation, Chemical

Topics: Animals; Arachidonic Acids; Guinea Pigs; Indicators and Reagents; Leukotriene A4; Leukotrienes; Lipoxygenase; Prostaglandin-Endoperoxide Synthases; Rabbits; Structure-Activity Relationship

Hydrolysis of 5-hydroperoxyeicosatetraenoic acid catalyzed by cytochrome P-450 in the absence of NADPH formed a product with spectral and HPLC characteristics identical with those of leukotriene B4, 5(S),12(R)-dihydroxy-6-cis,8,10-trans,14-cis-eicosatetraenoic acid (LTB4). The product mixture suggests that trans-5,6-oxido-7,9-trans,11,14-cis-eicosatetraenoic acid, leukotriene A4 (LTA4) is an intermediate in the reaction. We suggest that the formation of LTB4 with the essential 6-cis stereochemistry is due to tight binding of the 5-HPETE to the cytochrome P-450 at a site that does not allow it to isomerize to the kinetically favored 6,8,10-trans configuration during attack of hydroxyl ion at C-12.

Topics: Animals; Arachidonic Acids; Chromatography, High Pressure Liquid; Cytochrome P-450 Enzyme System; Isomerism; Kinetics; Leukotriene A4; Leukotriene B4; Leukotrienes; Microsomes, Liver; Phenobarbital; Rabbits

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