Arachidonic Acid Oxidation Products Research Articles

Thromboxane and prostacyclin generation by human umbilical veins after thrombin

Prostacyclin (PGI 2) and Thromboxane B 2 (TxB 2) production induced by thrombin in human umbilical veins (HUV) was studied. Successive stimulations of HUV segments were performed with and without restoration of arachidonic acid (AA). Thrombin consistently stimulated the production of both substances. The magnitude of the increment declined with progressive stimuli. The addition of exogenous AA could restore the prodcution of TXB 2 but not that of PGI 2. These results suggests that sustained stimulation of AA release may lead to an imbalance in the TXA 2/PGI 2 ratio perphaps through an effect of unkown products of AA oxidation on PGI 2 synthase.

Characterization of the activity of purified recombinant human 5-lipoxygenase in the absence and presence of leukocyte factors.

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.

CI-959, a New, Potential Antiallergic Drug, Inhibits Mediator Release from Lung and Contractions of Human Airways in vitro

Many symptoms of the immediate allergic response can be attributed to the synthesis/release and subsequent actions of histamine and metabolic products of arachidonic acid oxidation. CI-959 [5-methoxy-3-(1-methylethoxy)-N-1H-tetrazole-5-yl-benzo(b) thiophene-2-carboxamide], a new, potential antiallergic drug, inhibited the release of histamine, immunoreactive sulfidopeptide leukotrienes C4, D4 and E4 and immunoreactive thromboxane B2 from immunologically activated guinea-pig and human lung cells in vitro. The IC50s of CI-959 using guinea-pig lung were: histamine, 0.8 +/- 1.4 microM; leukotriene, 0.7 +/- 1.6 microM and thromboxane, 9.6 +/- 3.3 microM. Using human lung the IC50s were: 2.3 +/- 1.3 microM for histamine; 0.3 +/- 5.1 microM for leukotriene, and 0.3 +/- 2.6 microM for thromboxane. CI-959 caused a concentration-dependent inhibition of anti-IgE-induced contractions of human bronchial muscle. Mean percent inhibitions were 45, 65 and 96 at 1, 3 and 10 microM, respectively. Cromolyn, 10 microM, inhibited bronchial contractions only 42%. The ability of CI-959 to inhibit these immunologically induced contractions indicates that the release of all mediators responsible for bronchoconstriction was effectively inhibited. These data suggest that CI-959 may be effective in preventing the development of symptoms directly related to inflammatory mediator release in a variety of allergic and inflammatory states.

Cytochrome P-450-Dependent Omega-Oxidation of Leukotriene B4 in Rodent and Human Epidermis

Leukotriene B4 (LTB4,5-12-dihydroxy 6,8,10,14-eicosatetraenoic acid), an enzyme-catalyzed oxidation product of arachidonic acid, is a major inflammatory mediator. Human polymorphonuclear leukocytes and rodent hepatic microsomes catabolize LTB4 to 20-OH-LTB4 and 20-COOH-LTB4, which is mediated by a cytochrome P-450 catalyzed reaction termed the LTB4 omega-hydroxylase. In this study we investigated the catabolism of LTB4 in rat, guinea pig, and human epidermis. The incubation of 3H-LTB4 (9 microM) for 60 min in the presence of oxygen, NADPH, and epidermal microsomes prepared from neonatal fat (3.0 mg) or adult guinea pig (2.6 mg) resulted in the formation of 20-OH-LTB4 and 20-COOH-LTB4. Metabolite identification was based on co-chromatography on high pressure liquid chromatography with highly purified reference standards. The formation of 20-OH-LTB4 and 20-COOH-LTB4 was accompanied by the disappearance of LTB4. The rate of formation of 20-OH-LTB4 was 9-12-fold higher than that of 20-COOH-LTB4. Product formation was negligible with boiled microsomes, required NADPH and oxygen, was linear with respect to incubation time and protein, and was maximal at pH 7.4. LTB4-omega-hydroxylase activity was inhibited (greater than 90%) by carbon monoxide or 2-diethylaminoethyl-2,2-diphenylvalerate hydrochloride (SKF-525A) (1 mM), whereas alpha-naphthoflavone produced only moderate (13%) or no effects. Topical application of 3-methylcholanthrene and other conventional inducers of epidermal monooxygenase activities to neonatal rats (100 mg/kg, single treatment) did not result in an increase in epidermal LTB4-omega-hydroxylase activity. The addition of 3H-LTB4 (30 nmoles) to primary human keratinocytes followed by incubation at 37 degrees C resulted in time-dependent disappearance of LTB4 and appearance of 20-OH-LTB4 and 20-COOH-LTB4 in the medium. These results suggest that LTB4 is catabolized by the cytochrome P-450-dependent enzyme system in rodent and human skin and that this may participate in modulating the effects of this proinflammatory lipid in this tissue.

Syntheses of enantiomerically pure 9(S)- and 9(R)-hete from D-mannitol. Use of a malic dialdehyde equivalent.

Starting from D-mannitol, the total syntheses of methyl 9(S)- and 9(R)-hydroxy 5(Z), 7(E), 11(Z) eicosatetraenoate are described by two different ways. One route uses a diepoxide opening by an acetylide while the second one involved the transformation of a diepoxide into an equivalent of malic aldehyde, an important intermediate in the synthesis of other oxidation products of arachidonic acid. Both methods are available for the synthesis of each enantiomer.

Synthesis of arachidonate oxidation products by synovial joint tissues during the development of chronic erosive arthritis

An animal model, antigen-induced arthritis in the rabbit, was used to investigate the synthesis of arachidonic acid oxidation products (eicosanoids) by tissues of the knee joints during the initiation and early phase of this developing chronic destructive lesion. High concentrations of immunoreactive prostaglandin E2 and immunoreactive leukotriene B4 were found to be present in the synovial fluid in the early lesion. These levels rapidly declined, as did the ability of the infiltrating cells of the arthritic joint fluids to synthesize leukotriene B4. Articular cartilage and synovial lining were maintained for 24 hours in nonproliferative organ culture, and the synthesis of eicosanoids was measured by assay of the culture fluids. The synovial lining was the major source of eicosanoids. Synthesis of prostaglandin E2, prostacyclin, thromboxane A2, and leukotriene B4 by the arthritic synovial lining was low during the first few days, but reached maximal values between day 5 and day 15 of disease duration. Maximal eicosanoid production occurred around the time that damage to articular cartilage and bone has been reported to occur, though it is not certain that these two events are linked. It was demonstrated that, in chronic lesions, the arthritic synovial lining was still producing elevated levels of eicosanoids compared with levels in the control tissue.

Reversible inhibition of lymphokine-activated killer cell activity by lipoxygenase-pathway inhibitors.

Natural killer (NK) cells and lymphokine-activated killer (LAK) cells are anomalous cytotoxic cells which are potentially important in host defense against cancer. Several studies have demonstrated that natural killer (NK) cell activity can be suppressed by chemical inhibitors of the lipoxygenase pathway through inhibition of the production of leukotriene B4 (LTB4). The present study investigated the effects of the lipoxygenase inhibitors BW755C and nordihydroguaiaretic acid (NDGA) on NK and LAK cell activity. NK cell function of fresh peripheral blood mononuclear cells (PBMC) was determined via a standard chromium release assay employing K562 as the tumor target. The LAK cell activity of PBMC which had been stimulated with 10 IU of interleukin-2 for 72 hr was determined against the NK-resistant cell line Daudi. Both BW755C and NDGA inhibited NK and LAK cell function at a variety of concentrations. Indomethacin, a prostaglandin synthesis inhibitor, did not bring about an appreciable diminution in NK or LAK cell activity. Inhibition of NK and LAK cell activities by BW755C and NDGA could be reversed by washing the effector cell suspensions prior to the cytotoxic assay or by adding LTB4 (10(-11)-10(-8) M) directly to the effector:target suspensions. These data indicate that certain arachidonic acid oxidation products of the lipoxygenase pathway are essential for the function of LAK cells.

Progress on the characterization and identification of endothelium‐derived relaxing factor(s)

AbstractSubstantial evidence indicates that there are novel factors released from the vascular endothelium that regulate vasodilatation. The weight of the present evidence is that endothelium‐derived relaxing factor(s) (EDRF) is an oxidation product of arachidonic acid. Whether the oxidative enzyme is a lipoxygenase or related to a cytochrome P450 monooxygenase remains under investigation. Future progress requires a reproducible source of EDRF to allow isolation, purification, and structural identification.

Regulation of B-lymphocyte proliferative responses by arachidonate metabolites: Effects on membrane-directed versus intracellular activators

Immunoregulatory effects of the oxidative metabolites of arachidonic acid (AA) on proliferation of B-lymphocytes were assessed in a serum-free culture system. Activation of B cells by membrane-directed ligands and intracellular activators was regulated by AA metabolites in very distinct fashions. Thus exogenous cyclooxygenase products (particularly prostaglandins E 1 and E 2 amplified the response to anti-immunoglobulin antibodies, whereas lipoxygenase products damped this response. In contrast, B cell activation with 8-mercaptoguanosine (an intracellular activator) was inhibited by cyclooxygenase products and remained relatively unaffected by several lipoxygenase products tested. This pattern of results was confirmed in studies with pathway inhibitors. Moreover, when liberation of endogenous AA was induced by stimulation of phospholipase A 2 activity with melittin, inhibition of the response to each class of activator was counteracted with the appropriate pathway inhibitor. Results suggest that the two major groups of AA oxidation products function as a system of counterbalancing regulatory influences, serving to modulate B cell activation at the plasma membrane and to downregulate B cell activation at the intracellular level.

The Leukotrienes, Highly Biologically Active Substance Involved in Allergy and Inflammation

AbstractArachidonic acid, a fourfold unsaturated C20‐fatty acid, has attracted interest over the last 15 years far beyond the bounds of nutritional research. It forms an essential acyl component of the phospholopids in all animal cells. The long know fact that certain unsaturated fatty acids must be contained in the diet in order to ensure normal function of the organism has been explained by the discovery of a broad palette of biologically highly active oxidation products of arachidonic acid, a cascade, as it is often referred to. Arachidonic acid is formed in organisms from linoleic and linolenic acids, which belong to the so‐called essential fatty acids. As early as 1935 Ulf von Euler was able to detect a substance in semen which caused smooth muscles to contract. This factor, which was later isolated in pure form, was termed prostaglandin. The number of prostaglandins, including the synthetically obtained analogues, then rapidly increased. Prostaglandins are biosynthesized from arachidonic acid and similar C20‐fatty acids by enzymatic oxidation with a cyclooxygenase. Its activity is inhibited by acetylsalicylic acid, indomethazine, and other substances, because these relieve or cure inflammations arising from the action of certain prostaglandins. After the prostaglandins, the thromboxanes, which promote aggregation of blood platelets and thus cause thrombosis, were discoverd as further metabolic products of arachidonic acid. In a well‐balanced equilibrium the thromboxanes have a biological effect completely opposite to that of prostacyclin, discovered soon after this, which inhibits platelet aggregation. Both of these biologically active substances are metabolites of arachidonic acid, and are formed in the course of the cyclizing oxidation. Aspirin and substances having a similar mode of action, therefore have an influence on thrombosis. There are, however, contraction processes and inflammation symptoms which do not respond to these substances, but to steroid hormones (cortisone). For symptoms of this type, to which asthma also belongs, the most recently discovered metabolic products of arachidonic acid are probably highly decisive; they are formed by another enzymatic route via a hydroperoxide of arachidonic acid. These compounds are formed in leukocytes and contain the triply unsaturated structural element of a triene and were, therefore, termed leukotrienes. Their astonishingly high histamine‐like activity in producing contraction of lung capillaries, their marked property of making capillary vessels permeable to blood plasma, i.e. of generating oedema, the ability of leukotriene LTB4 to cause adherence of human leukocytes and their adhesion to the endothelium, make the leukotrienes chemical mediators in allergies and inflammations, and produce biological effects when present at minute concentrations. In the following article one of the three scientists who were awarded this years Nobel prize for medicine and physiology, and who provided an explanation for this extraordinarily interesting class of natural products, gives an account of the conversion of arachidonic acid into the labile epoxide LTA4 and further into the dihydroxy compound LTB4, or the glutathione cysteinylglycine and cysteine conjugates LTC4. LTD4, and LTE4, respectively, as well as of the physiological‐pathological implications. The two other prize winners in this area of biologically active compounds, which are often referred to as eicosanoids, are Samuelsson's teacher Sune Bergstöm and the Britain John Vane.

Modulation of lymphocyte activation: II. Alteration of intracellular cyclic nucleotide concentrations by an oxidation product of arachidonic acid

Murine spleen cells incubated with arachidonic acid oxidized with lipoxidase (AA/L) do not respond to mitogenic stimulation. Analysis of the components of AA/L separated by high-pressure liquid chromatography has previously demonstrated that purified 15-hydroperoxyarachidonic acid, the predominant product generated, is the seat of the inhibitory activity observed. Correlative studies were undertaken to determine the relationship between the pervasive inhibitory effect of AA/L and the modulation of intracellular cyclic nucleotide concentration. AA/L elevated intracellular levels of cGMP in murine spleen cells cultured under serum-free conditions and caused a similar (two-fold) increment in intracellular levels of cyclic AMP. These are the first cellular activities observed in this laboratory that are not subject to inhibition by treatment of cells with AA/L. Therefore, studies were conducted to determine if AA/L-mediated inhibition of lymphocyte mitogenesis could be mimicked by elevation of intracellular nucleotide levels by pharmacologic agents. Attempts to recreate this inhibitory phenomenon by use of such agents indicated that inhibition could not be induced by modulation of cyclic AMP alone, cyclic GMP alone, or of both together. Similarly, when the same stimuli of cyclic nucleotide accumulation were employed to determine if modulation of intracellular cyclic nucleotide content could counteract the inhibitory effect of AA/L, these agents did not interfere with AA/L-mediated inhibition. The capability of the cyclic nucleotide agonists to elevate levels of cAMP and cGMP was confirmed by direct assay. Thus, although cyclic nucleotides may play an essential, adjunctive role in inhibition of lymphocyte mitogenesis, their modulation is not of itself sufficient either to evoke or to interfere with the establishment of a state of profound inhibition.

Oxidation products of arachidonic acid. IV. Model studies on the attachaient of the C(13)—C(20) side-chain in the synthesis of tetrahydrofurans and pyrans 1,2, and 3

The synthesis of methyl 15-hydroxy-8(R)-methoxy-11(R)-p-nitrobenzoyloxy-9(S),12(R)-oxy-eicosa-13-ynoate, an arachidonic acid metabolite derivative, is described. As well, methods for the attachment of the C(13)—C(20) prostaglandin side-chain to furanosides were studied and the results are described. The reduction of the C-15 keto group on a model compound was performed and was found to have the same behavior as in the prostaglandins.A partial structure proof of Pace-Asciak's compound, 9,12-oxy-8,11,15-trihydroxy-13-enoic acid is given.

Oxidation products of arachidonic acid II. The synthesis of methyl 8R,9S,11R-trihydroxy-5Z,12E,14Z-eicosatrienoate

A stereospecific total synthesis of the title compound, 37, starting from diacetone glucose (9), is described. Key intermediates in the synthesis are 3-deoxy-5,6-anhydro-1,2-O-isopropylidene-glucofuranose (15), obtained in 55% yield from 9, and methyl 8-tert-butyldiphenylsilyl-9,11-O-isopropylidene-8R,9S,11R-trihydroxy-12-oxo-dodeca-5Z-enoate (33), an important intermediate in the synthesis of other oxidation products of arachidonic acid.

Modulation of lymphocyte activation. I. Inhibition by an oxidation product of arachidonic acid.

Modulation of lymphocyte activation. I. Inhibition by an oxidation product of arachidonic acid.

Oxidation products of arachidonic acid I. The synthesis of methyl 8R, 11R, 15-trihydroxy-9S, 12-oxyeicosa-5Z, 13E-dienoate (19)

Diacetone glucose was converted in high yield to 3-deoxy-5-6-anhydro-1,2-0-isopropylidene-D-glucofuranose( 3 ), which was transformed to title compound. 3 Its mass spectrum confirms the structure of a naturally occurring oxidation product of arachidonic acid first described by wolfe and Pace-Asciak. 4

The role of iron in prostaglandin synthesis: Ferrous iron mediated oxidation of arachidonic acid

Arachidonic acid (AA) is the essential substrate for production of platelet endoperoxides and thromboxanes. Iron or heme is an essential cofactor for the peroxidase, lipoxygenase and cyclo-oxygenase enzymes involved in formation of these products. The present study has examined the direct interactions between iron and arachidonic acid. Iron caused the oxidation of AA into more polar products which could be detected by UV absorbtion at 232 nM or the thiobarbituric acid (TBA) reaction. High pressure liquid chromatography, chem-ionization and electron-impact mass spectrometry and nuclear magnetic resonance spectroscopy suggest that the major product was a hydroperoxide of AA. Ferrous iron (Fe ++) and oxygen were absolute requirements. Fe ++ was converted to the ferric iron (Fe +++) state during oxidation of AA, but Fe +++ could not substitute for Fe ++. No other enzymes, cofactors or ions were involved. Conversion of AA to a hydroperoxide by Fe ++ was inhibited by the antioxidant, 2, (3)-Tert-butyl-4-hydroxyanisole, the radical scavenger, nitroblue tetrazolium, and iron chelating agents, including EDTA, imidazole and dihydroxybenzoic acid. The reaction was not affected by superoxide dismutase, catalase or aspirin. These findings and preliminary studies of the Fe ++ induced oxidation product of AA as a substrate for prostaglandin synthesis and inhibitor of prostacyclin production indicate the critical role of Fe ++ in AA activation.