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F Fitzpatrick

Publications and source records attributed to F Fitzpatrick.

30 records · Page 2Linked to original sources
PubMed1985

Enzymatic hydration of leukotriene A4. Purification and characterization of a novel epoxide hydrolase from human erythrocytes.

Human erythrocytes contained a soluble cytosolic epoxide hydrolase for stereospecific enzymatic hydration of leukotriene A4 into leukotriene B4. The enzyme was purified 1100-fold, to apparent electrophoretic homogeneity, by conventional DEAE-Sephacel fractionation followed by high performance anion exchange and chromatofocusing procedures. Its characteristics include a molecular weight of 54,000 +/- 1,000, an isoelectric point 4.9 +/- 0.2, a Km apparent from 7 to 36 microM for enzymatic hydration of leukotriene A4, and a pH optimum ranging from 7 to 8. The enzyme was partially inactivated by its initial exposure to leukotriene A4. There was slow but detectable enzymatic hydration (pmol/min/mg) of certain arachidonic acid epoxides including (+/-)-14,15-oxido-5,8-11-eicosatrienoic acid and (+/-)-11,12-oxido-5,8,14-eicosatrienoic acid, but not others, including 5,6-oxido-8,11,14-eicosatrienoic acid. Human erythrocyte epoxide hydrolase did not hydrate either styrene oxide or trans-stilbene oxide. In terms of its physical properties and substrate preference for leukotriene A4, the erythrocyte enzyme differs from previously described versions of epoxide hydrolase. Human erythrocytes represent a novel source for an extrahepatic, cytosolic epoxide hydrolase with a potential physiological role.

8,11,14-Eicosatrienoic Acid↗
PubMed1985

Cell damage unmasks 15-lipoxygenase activity in human neutrophils.

Metabolism of arachidonic acid (10 microM) into 15(S)-hydroxyl-5,8,11-cis-13-trans-eicosatetraenoic acid (15-HETE) was proportional to lactate dehydrogenase release from human neutrophils incubated with supratherapeutic concentrations of non-steroidal anti-inflammatory agents. In contrast to others (Vanderhoek, J., and Bailey, J. (1984) J. Biol. Chem. 259, 6752-6756), we report that increased 15-HETE formation was not uniquely attributable to 5 mM ibuprofen, and it did not originate from enzymatic activation. For instance, ibuprofen (1-5 mM) did not affect the isolated 15-lipoxygenase enzyme in the 100,000 X g supernatant from neutrophil lysates, and dose-dependent increases in 15-HETE biosynthesis, proportional to lactate dehydrogenase release, were evident with benoxaprofen, naproxen, flurbiprofen, or etodolac. At similar supratherapeutic concentrations (1-5 mM), aspirin and phenylbutazone did not influence lactate dehydrogenase release or 15-HETE production. In further contrast, neutrophils did not tolerate 1-5 mM ibuprofen. Biochemical, morphological, flow cytometric, and fluorochromatic analyses each indicated cytological damage. A correlation between lactate dehydrogenase release and increased 15-HETE formation was a dose-dependent property also exhibited by arachidonic acid alone (10-100 microM). We conclude that cytological damage, facilitating access of arachidonic acid to 15-lipoxygenase in a cytosolic compartment, accounts for this phenomenon.

Acetates↗
PubMed1984

Metabolism of leukotriene A4 by human erythrocytes. A novel cellular source of leukotriene B4.

Human erythrocytes transformed leukotriene A4 into leukotriene B4. Metabolism was proportional to the erythrocyte concentration, even at subphysiological levels (0.08-4 X 10(9) erythrocytes/ml). Comparative metabolic studies excluded the possibility that leukotriene B4 originated from trace amounts of polymorphonuclear leukocytes or platelets present in the purified erythrocyte suspensions. For example, suspensions of isolated platelets (100-500 X 10(6) cells/ml) failed to convert leukotriene A4 into leukotriene B4; and conversion by suspensions of isolated polymorphonuclear neutrophils was insufficient to account for the amounts of leukotriene B4 formed by erythrocytes. Leukotriene B4 formation was maximal within 2 min and substrate concentration dependent. Enzymatic activity originated from a 56 degrees C labile nondialyzable (Mr greater than 30,000) soluble component in the 100,000 X g supernatant obtained from lysed erythrocytes. In contrast to the contemporary view, our results indicate that human erythrocytes are not metabolically inert in terms of eicosanoid biosynthesis. The role of human erythrocytes during inflammatory or pulmonary disorders deserves re-examination in this context.

Arachidonic Acids↗
PubMed1984

Development of a gas chromatographic-mass spectrometric method using a stable isotope internal standard for quantitation of thromboxane B2.

Tetradeuterated 19, 19',20,20'-2H4-thromboxane B2 was synthesized and used as a stable isotope internal standard for the development of a gas chromatographic/mass spectrometric (GC/MS) method to quantitate thromboxane B2. Quantitation was based on the detection of fragment ions at m/z 301 for thromboxane B2 and m/z 305 for the corresponding tetradeuterated internal standard. At m/z 301/305 a response equivalent to a protium/deuterium ratio of 0.2% thromboxane B2 could be measured with a standard deviation of 15% when 100 nanograms of the tetradeuterated internal standard was analyzed. For measurement of cellular biosynthesis of thromboxane B2 in whole blood or platelet rich plasma, the internal standard and naturally occurring thromboxane B2 were i) isolated by solvent extraction, or sequestration to XAD-2 columns; ii) purified by reversed phase high performance liquid chromatography; and iii) converted to methoxime methyl ester trimethylsilyl ethers prior to analysis. A limited comparison was made using both radioimmunological and mass spectrometric quantitation of thromboxane B2.

Animals↗
PubMed1984

Hemoprotein catalysis of leukotriene formation.

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.

Arachidonic Acids↗
PubMed1983

Albumin stabilizes 14,15-leukotriene A4.

14,15-Leukotriene A4 is a pivotal biosynthetic intermediate in 15-lipoxygenase initiated leukotriene biosynthesis. This compound hydrolyzes instantaneously in phosphate buffer at pH 7.4. However, addition of human or bovine albumin to otherwise identical buffer solutions increases its stability. Intact 14,15-leukotriene A4 then decomposes by first-order kinetics with rate constants inversely proportional to the albumin concentration. Stabilization of 14,15-leukotriene A4 under certain conditions may influence its proportionate transformation by enzymatic vs non-enzymatic processes.

Animals↗
PubMed1983

Metabolism of leukotriene A4 by an enzyme in blood plasma: a possible leukotactic mechanism.

Cell-free mammalian plasma transformed leukotriene A4 into leukotriene B4. This conversion originated from a soluble enzymatic activity. Heating at 56 degrees C or digestion of plasma with a proteolytic enzyme eliminated formation of leukotriene B4 but not other diastereomeric dihydroxyicosatetraenoic acids formed by nonenzymatic hydrolysis of leukotriene A4. Plasma from several mammals, including guinea pigs, pigs, cows, sheep, rabbits, rats, dogs, and humans, exhibited a qualitatively similar activity. Maximal production of leukotriene B4 occurred in guinea pig plasma near a pH of 7.6; metabolic capacity approached saturation at a substrate concentration of 10 microM. Leukotriene A4 anion was transformed but not its methyl ester. The results suggest that conversion of leukotriene A4 by an enzyme in blood plasma of mammals may provide local gradients of the chemotactic substance leukotriene B4. High interfacial concentrations of leukotriene B4 between the vascular endothelium and leukocytes could facilitate their diapedesis and accumulation at inflammatory sites.

Animals↗
PubMed1979

9,11-Iminoepoxyprosta-5,13-dienoic acid is a selective thromboxane A2 synthetase inhibitor.

9,11-Iminoepoxyprosta-5,13-dienoic acid inhibits the thromboxane A2 synthetase in platelet and lung microsomal enzyme preparations and in intact platelets. It does not inhibit the protaglandin I2 synthetase in aorta or lung microsomes and intact Balb 3T3 fibroblasts. In lung microsomes, which contain both enzymes, 9,11-iminoepoxyprosta-5,13-dienoic acid inhibits only thromboxane A2 formation and augments prostaglandin I2 formation. This inhibitor is more selective than other reported prostaglandin endoperoxide analogs which inhibit the platelet thromboxane synthetase.

Animals↗
PubMed1979

Triene prostaglandins: prostaglandin D3 and icosapentaenoic acid as potential antithrombotic substances.

Addition of the 3-series fatty acid precursor (icosapentaenoic acid, IPA), its endoperoxide [prostaglandin (PG)H(3)], or thromboxane A(3) to human platelet-rich plasma (PRP) does not result in aggregation of the platelets. In fact, preincubation of human PRP with exogenous PGH(3) actually inhibited aggregation by increasing platelet cyclic AMP concentrations. PGH(3) undergoes rapid spontaneous degradation to PGD(3) in human PRP. The PGD(3) so formed is adequate to account for the increase of platelet cAMP and inhibition of aggregation. Furthermore, addition of PGD-specific antisera to human PRP blocked the platelet inhibitory activity of exogenous PGH(3). PGD(3) has considerable potential as a circulating antithrombotic agent. Pretreatment of human PRP with the adenylate cyclase inhibitor 2',5'-dideoxyadenosine blocked the increase of platelet cyclic AMP and the inhibition of aggregation normally produced by PGI(2), PGE(1), PGD(2), PGH(3), and PGD(3). Furthermore, the dideoxyadenosine unmasked a direct but moderate reversible aggregatory effect in response to the subsequent addition of PGH(3). Similarly, the dideoxyadenosine markedly enhanced the aggregation produced by exogenous PGH(2). IPA is readily incorporated into tissue lipids but proved to be a poor substrate for kidney, blood vessel, or heart cyclooxygenase. IPA was previously shown to be a poor substrate for platelet cyclooxygenase. IPA is readily deacylated from the renal phospholipid pool in response to bradykinin, a substance that also stimulates the release of arachidonic acid. A diet that relies primarily on cold-water fish, as in the case of the Greenland Eskimos, lowers endogenous arachidonic acid and markedly increases the IPA content of tissue lipids. Thus, because IPA has the potential to act as an antagonist with arachidonic acid for platelet cyclooxygenase and lipoxygenase, the simultaneous release of IPA could suppress any residual arachidonic acid conversion to its aggregatory metabolites.

Adenosine↗