leukotriene-b4 and 13-hydroxy-9-11-octadecadienoic-acid

We studied the effect of the linoleic acid metabolite 13-hydroxyoctadecadienoic acid (13-HODE) on the increase of the intracellular calcium concentration, [Ca2+]i, induced by platelet-activating factor (PAF), leukotriene B4 (LTB4), and formyl-Met-Leu-Phe (fMLP) in human polymorphonuclear leukocytes (PMNs). 13-HODE by itself did not change the [Ca2+]i of PMNs. However, when PMNs were preincubated with 13-HODE and subsequently stimulated with PAF, LTB4, or fMLP, an inhibition of the increase of the [Ca2+]i was observed. The PAF-induced calcium response was concentration-dependently inhibited between 0.1 and 5 microM 13-HODE. These results suggest that 13-HODE can affect the increase of the [Ca2+]i and hence can modulate activation of PMNs.

Topics: Amino Acid Sequence; Calcium; Humans; Intracellular Fluid; Leukotriene B4; Linoleic Acids; Molecular Sequence Data; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Platelet Activating Factor; Stimulation, Chemical

Intradermal 13-hydroxyoctadecadienoic acid (13-HODE; 10(-11)-10(-9) mol/site) inhibited oedema formation induced by the neutrophil-dependent mediator leukotriene B4 (LTB4) in the presence of calcitonin gene-related peptide (CGRP; 10(-11) mol/site) in rabbit skin. In contrast, the responses to the direct acting mediators bradykinin and histamine were unaffected by 13-HODE. 13-HODE failed to induce oedema formation in rabbit skin when injected alone or in the presence of the potent vasodilator CGRP. These results present a novel interaction between 13-HODE and LTB4 that could have important implications in the pathogenesis of inflammation.

Topics: Analysis of Variance; Animals; Calcitonin Gene-Related Peptide; Edema; Leukotriene B4; Linoleic Acids; Rabbits; Skin

Enzymatic transformation of the n-6 polyunsaturated fatty acid (PUFA) arachidonic acid (AA) by the 5-lipoxygenase (LO) enzyme results in the formation of leukotrienes (LTs) including leukotriene B4 (LTB4), which is a potent mediator of inflammation. The purpose of the present study was to determine the effect of other n-6 fatty acids on the formation of LTB4 by human neutrophils and to determine if these n-6 fatty acids themselves may be transformed into products with antiinflammatory capacity. Purified neutrophils isolated from heparinized human venous blood were incubated with A23187 (5 microM) and different concentrations (0-100 microM) of the n-6 fatty acids linoleic acid (LA) and dihomo-gamma-linolenic acid (DGLA). LO products were determined by use of quantitative reversed-phase high performance liquid chromatography (RP-HPLC) and mass spectrometry. The formation of LTB4 was dose dependently inhibited by both LA (IC50 = 45 microM) and DGLA (IC50 = 40 microM). This inhibition of LTB4 formation was associated with a dose dependent increase in the formation of the respective 15-LO products of LA (13-hydroxy-octadecadienoic acid; 13-HODE) and DGLA (15-hydroxy-eicosatrienoic acid; 15-HETrE). To determine whether these 15-LO products themselves might inhibit LTB4 formation, neutrophils were incubated with 13-HODE and 15-HETrE. Both 15-LO products lead to a dose-dependent inhibition of LTB4 formation (IC50 = 7.5 microM and IC50 = 0.2 microM). For comparison the 15-LO product of AA, 15-hydroxy-eicosatetraenoic acid (15-HETE), also inhibited LTB4 formation (IC50 = 0.75 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 8,11,14-Eicosatrienoic Acid; Anti-Inflammatory Agents, Non-Steroidal; Arachidonate 15-Lipoxygenase; Chromatography, High Pressure Liquid; Humans; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; L-Lactate Dehydrogenase; Leukotriene B4; Linoleic Acid; Linoleic Acids; Mass Spectrometry; Neutrophils

Fatty acid-derived inflammatory mediators are considered to play an important role in airway hyperresponsiveness of asthmatic patients. The pulmonary macrophage may be an important source for these mediators in airway tissue. We investigated the metabolism of arachidonic acid and linoleic acid by human bronchoalveolar lavage cells, mainly comprising pulmonary macrophages. Arachidonic was mainly metabolized by 5-lipoxygenase, giving rise to the formation of leukotriene B4 and 5-hydroxy-eicosatetraenoic acid (5-HETE). Linoleic acid was converted to 5 major metabolites, including the 9-hydroxy and 13-hydroxy derivatives, 9- and 13-hydroxy-octadecadienoic acid (9- and 13-HODE). The formation of HODEs could be inhibited by cyclooxygenase inhibitors as well as lipoxygenase inhibitors, indicating that both enzymic species play a role in the generation of HODEs.

Topics: Arachidonate 5-Lipoxygenase; Arachidonic Acid; Bronchoalveolar Lavage Fluid; Chromatography, High Pressure Liquid; Cyclooxygenase Inhibitors; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Linoleic Acid; Linoleic Acids; Linoleic Acids, Conjugated; Lipoxygenase Inhibitors; Macrophages

Locally administered 9Ds-hydroxy-10,12(E,Z)-octadecadienoic acid (9-HODE) caused a drastic inflammatory response in the experimental model of granulation tissue formation of the rat according to RUDAS (Arzneimittelforsch. 10,226-229, 1960). Three days after implantation of the polyvinyl chloride rings the granulation tissue became inhomogeneous with proliferation islets surrounded by edematous regions containing a diminished number of cells. The number of polymorphonuclear leukocytes and of macrophages was greatly enhanced in the whole tissue, whereas the number of lymphocytes was reduced. After seven days the whole granulation tissue was loosened, and its mass was twice as high as in the control animals. The number of fibroblasts per area unit and the hydroxyproline content were diminished. Linoleic acid and 13Ls-hydroxy-9,11(Z,E)-octadecadienoic acid (13-HODE) caused also some changes in the formation of granulation tissue, but in a different manner, in particular, without accumulation of polymorphonuclear leukocytes and macrophages, indicating the specificity of the effect of 9-HODE. The recruitment of leukocytes was not due to a direct chemotactic action of 9-HODE as shown in an agarose diffusion test comparing the effects of 9-HODE and leukotriene B4. The possible biological importance of the proinflammatory effect of 9-HODE is discussed.

Topics: Animals; Chemotaxis, Leukocyte; Fibroblasts; Foreign-Body Reaction; Granulation Tissue; Inflammation; Leukotriene B4; Linoleic Acid; Linoleic Acids; Linoleic Acids, Conjugated; Macrophages; Male; Neutrophils; Prostheses and Implants; Rats; Wound Healing

We have previously shown that porcine polymorphonuclear leukocytes (PMNL) reduce leukotriene B4 (LTB4) to 10,11-dihydro-LTB4, 10,11-dihydro-12-epi-LTB4, and 10,11-dihydro-12-oxo-LTB4 [Wainwright et al. (1990) Biochemistry 29, 1180-1185]. We have now demonstrated that 12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid [12(S)-HETE] is metabolized by a similar pathway in porcine PMNL. 12(S)-HETE was metabolized to two products that were identified by gas chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy as 12-hydroxy-5,8,14-eicosatrienoic acid (10,11-dihydro-12-HETE) and 12-oxo-5,8,14-eicosatrienoic acid (10,11-dihydro-12-oxo-ETE). Derivatization of 12-hydroxy-5,8,14-eicosatrienoic acid with (R)-(+)-alpha-methoxy-alpha-(trifluoromethyl)phenylacetic acid, followed by chromatography on a silicic acid column, enabled the resolution of 12R and 12S stereoisomers, which were identified by cochromatography with synthetic standards. Incubation of 12(S)-HETE with PMNL for various times revealed that the stereochemistry of the 12-hydroxyl group of 12-hydroxy-5,8,14-eicosatrienoic acid was initially the same as that of 12(S)-HETE. However, after 40 min, 30% of the 12-hydroxy-5,8,14-eicosatrienoic acid had the opposite configuration at C12. 13-Hydroxy-9,11-octadecadienoic acid (13-HODE) was metabolized in a similar fashion by porcine PMNL to 13-hydroxy-9-octadecenoic acid (11,12-dihydro-13-HODE) and 13-oxooctadecenoic acid (11,12-dihydro-13-oxo-ODE). The apparent Km values for the reduction of 12-HETE, LTB4, and 13-HODE were 0.21, 0.28, and 2.22 microM, respectively. All three substrates had the same apparent Vmax [0.029 pmol min-1 (10(6) cells)-1]. Competition experiments between LTB4 and 12-HETE indicated that they were metabolized by the same pathway. Various structurally related compounds were metabolized by porcine PMNL in the order LTB4 = 6-trans-LTB4 greater than 12-epi-6-trans,8-cis-LTB4 greater than 12-epi-6-trans-LTB4 greater than 12-HETE greater than LTB5 greater than 15-HETE = 13-HODE much greater than 5-HETE greater than 9-HODE greater than 20-hydroxy-LTB4 greater than 12-hydroxy-5,8,10-heptadecatrienoic acid. Prostaglandins E2 and F2 alpha were not metabolized to any detectable products by porcine PMNL.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; 5,8,11,14-Eicosatetraynoic Acid; Aldehyde Oxidoreductases; Animals; Fatty Acids; Fatty Acids, Unsaturated; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Linoleic Acids; Molecular Conformation; Neutrophils; Oxidation-Reduction; Substrate Specificity; Swine

Topics: Animals; In Vitro Techniques; Leukotriene B4; Linoleic Acids; Lipopolysaccharides; Lipoxygenase; Macrophage Activation; Macrophages; Mice; Phagocytosis; SRS-A

A non-suppurative chronic arthritis was induced in the juvenile dog knee by intra-articular instillations with Carrageenan. Lipoxygenase products of arachidonic acid were separated from synovial fluid by reversed-phase high-performance liquid chromatography (RP-HPLC). After ten weeks we observed an accumulation of leukotriene B4 (LTB4) in synovial fluid in five out of six experimental knees (0.94 to 5.5 ng/ml), as judged by integrated optical density. Biological activity of LTB4 was confirmed by chemokinesis. LTB4 was not detected in control knees. The 15-lipoxygenase products, 15-hydroxyeicosatetraenoic acid (15-HETE) and 13-hydroxy-9,11-octadecadienoic acid (13-HODD), being inhibitors of 5-lipoxygenase, were found in relatively high levels in the control knees compared to the arthritic knees. The results denote LTB4 as a pro-inflammatory local mediator during carrageenan-induced arthritis; possibly, the decreased levels of 15-HETE and 13-HODD in the arthritic knees may have a regulatory function, thus facilitating LTB4 generation.

Topics: Animals; Arthritis; Carrageenan; Chemotaxis, Leukocyte; Chromatography, High Pressure Liquid; Disease Models, Animal; Dogs; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Linoleic Acids; Lipoxygenase; Synovial Fluid

Keratomed epidermal tissue from normal individuals and from the lesional and non-lesional skin of psoriasis patients served as source materials for the extraction, separation, and quantitation of eicosanoids that may be important to cutaneous function and pathophysiology. The eicosanoids were extracted in ethanol and buffer, partially purified on SEP-PAKs, separated by reverse phase microbore high-performance liquid chromatography, and quantitated by radioimmunoassay or integration of absorbency peaks obtained by high performance liquid chromatography. The involved areas from psoriasis patients contained a statistically significant seven- to 11-fold increase in the levels of leukotriene B4, 13-hydroxyoctadecadienoic acid-like compound, 15-hydroxyeicosatetraenoic acid compound and 12-hydroxyeicosatetraenoic acid in comparison to normal samples and a three- to sevenfold increase in comparison to uninvolved tissue. The uninvolved areas contained 40% to 100% increases in the levels of these compounds in comparison to normal tissue; these increases were statistically significant except for 13-hydroxyoctadecadienoic acid-like compound. From a single keratome biopsy, multiple eicosanoids can be separated and quantitated; in addition, levels before, during, and after therapy for psoriasis may be compared.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonate 15-Lipoxygenase; Arachidonate Lipoxygenases; Biopsy; Chromatography, High Pressure Liquid; Epidermis; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Linoleic Acids; Radioimmunoassay

Leukotriene B4 (LTB4) release by calcium ionophore-stimulated human leucocytes was measured by use of selective solvent partition of reaction mixtures and an agarose microdroplet chemokinesis assay, and the inhibitory effects of four monohydroxy fatty acids were determined. 15-Hydroxy-eicosatetraenoic acid (15-HETE) was the most effective inhibitor of LTB4 production with an approximate IC50 value of 6 microM and 99% inhibition at 50 microM, whereas 13-hydroxy-octadecadienoic acid (13-HODD) and 12-HETE were weaker inhibitors with approximate IC50 values of 32 microM and 23 microM, and 59% and 68% inhibition at 50 microM, respectively. We suggest that 13-HODD and 12-HETE, which are present in large amounts in the lesions of the skin disease psoriasis, may act as endogenous modulators of 5-lipoxygenase activity in skin.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonate Lipoxygenases; Calcimycin; Chromatography, High Pressure Liquid; Humans; Hydroxy Acids; Hydroxyeicosatetraenoic Acids; Leukocytes; Leukotriene B4; Linoleic Acids; Linoleic Acids, Conjugated; Lipoxygenase; Skin; Time Factors

Porcine neutrophilic leukocytes were found to contain a lipoxygenase which converted linoleic acid into 13-hydroxy-9,11-octadecadienoic acid (n-6 specificity), arachidonic acid into 12-hydroxy-5,8,10,14-eicosatetraenoic acid (n - 9 specificity) and 5-hydroxy-6,8,11,14-eicosatetraenoic acid into 5,12-dihydroxy-6,8,10,14-eicosatetraenoic acid. This lipoxygenase was partially purified and it appeared that its substrate specificity and other properties were quite different from the 12-lipoxygenase of blood platelets. Incubations of intact or broken porcine leukocytes with added linoleic acid revealed the formation of not only 13-hydroxy-9,11-octadecadienoic acid but also of substantial amounts of epoxyhydroxy and trihydroxy isomers. These products from linoleate, collectively described by the name 'octadecanoids' were characterized in detail by a combination of chemical, chromatographic and mass spectrometric techniques. The phospholipids of porcine leukocytes contain more than twice as much linoleate than arachidonate (22 vs. 8%). In accordance with this fatty acid composition we found that in the stimulated neutrophil the endogenous production of octadecanoids often surpassed that of the eicosanoids. Lipoxygenation of endogenously liberated linoleic acid was especially pronounced when a suspension of leukocytes in citrated plasma was recalcified and allowed to clot.

Topics: Animals; Arachidonic Acids; Chromatography, High Pressure Liquid; Gas Chromatography-Mass Spectrometry; Leukotriene B4; Linoleic Acid; Linoleic Acids; Lipoxygenase; Neutrophils; Phospholipids; Substrate Specificity; Swine