Metabolism of leukotriene E4 by rat tissues: formation of N-acetyl leukotriene E4.

Leukotriene E4 was incubated with subcellular fractions from rat liver homogenates. A product identified as 5-hydroxy-6-S-(2-acetamido-3-thiopropionyl)-7,9-trans-11,14- cis-eicosatetraenoic acid (N-acetyl leukotriene E4) was formed. Enzymes catalyzing the reaction were associated with particulate fractions sedimenting between 600 and 8500 g and 20,000 and 105,000 g. Acetyl coenzyme A served as the donor of the acetyl group. N-Acetyl leukotriene E4 was also formed by the 105,000g sediment fractions from kidney, spleen, skin, and lung. The myotropic activity of N-acetyl leukotriene E4 on isolated guinea pig ileum was reduced over 100-fold compared to that of leukotriene D4.

Metabolism of leukotriene D by porcine kidney.

An enzyme from porcine kidney converted leukotriene D4 into a less polar metabolite. The structure of this compound was 5-hydroxy-6-S-cysteinyl-7,9-trans-11,14-cis-eicosatetraenoic acid (leukotriene E4). Analogous products, viz. 5-hydroxy-6-S-cysteinyl-7,9,11-eicosatrienoic acid (leukotriene E3), 5-hydroxy-6-S-cysteinyl-7,9,11-trans-14-cis-eicosatetraenoic acid (11-trans-leukotriene E4), and 5-hydroxy-6-S-cysteinyl-7,9,11,14,17-eicosapentaenoic acid (leukotriene E5) were formed from leukotrienes D3, 11-trans-D4, and D5, respectively. Leukotriene E4 induced slow reacting substance-like contractions of guinea pig ileum but was less potent (8-12 times) than leukotriene C4. The biological potency of 11-trans-leukotriene E4 was similar to that of leukotriene E4.

Metabolism and excretion of cysteinyl-leukotrienes.

In vitro and in vivo experiments have demonstrated that a major pathway of metabolism of the glutathione containing leukotrienes involves modifications of the tripeptide substituent. The metabolic alterations are initiated by elimination of the N-terminal gamma-glutamyl residue, catalyzed by the enzyme gamma-glutamyl transferase. This reaction is followed by hydrolysis of the remaining peptide bond resulting in elimination of the C-terminal glycine residue. The enzyme catalyzing the latter reaction is a membrane bound dipeptidase which occurs in kidney and other tissues. The product formed by these reactions, leukotriene E4, has been tentatively identified as a urinary metabolite in man following intravenous administration of leukotriene C4. In rats, two major fecal metabolites of leukotriene C4 were characterized as having the structures N-acetyl leukotriene E4 and N-acetyl 11-trans leukotriene E4. These compounds are formed from leukotriene E4 and 11-trans leukotriene E4 in reactions with acetyl coenzyme A. A membrane bound enzyme, present in liver, kidney and other tissues, catalyzes these reactions.

Metabolism of leukotrienes.

The in vitro metabolism of leukotriene B4 is initiated by omega-hydroxylation. This reaction is followed by oxidation of the omega-hydroxyl group to a carboxyl group. In vivo extensive beta-oxidation occurs and the main excreted products after administration of leukotriene B4 are water and carbon dioxide. Experiments performed in vitro and in vivo have demonstrated that a major pathway of metabolism of the glutathione containing leukotrienes involves modifications of the tripeptide substituent. The metabolic alterations are initiated by enzymatic elimination of the N-terminal gamma-glutamyl residue, catalyzed by the enzyme gamma-glutamyl transferase. This reaction is followed by hydrolysis of the remaining peptide bond resulting in elimination of the C-terminal glycine residue. The enzyme catalyzing the latter reaction is a membrane bound dipeptidase which occurs in kidney and other tissues. The product formed by these reactions, leukotriene E4, has been tentatively identified as a urinary metabolite in man following intravenous administration of leukotriene C4. In rats, the two major fecal metabolities of leukotriene C4 were characterized as being N-acetyl leukotriene E4 and N-acetyl 11-trans leukotriene E4. These compounds are formed in reactions between leukotriene E4 or 11-trans leukotriene E4 and acetyl coenzyme A. The reactions are catalyzed by a membrane bound enzyme present in liver, kidney and other tissues.

Formation of leukotrienes E3, E4 and E5 in rat basophilic leukemia cells.

Rat basophilic leukemia (RBL-1) cells incubated with ionophore A23187 and 5,8,11-eicosatrienoic acid produced three slow-reacting substances identified as leukotrienes C3, D3 and E3 by spectroscopic, chromatographic and enzymatic methods. 5,8,11,14,17-Eicosapentaenoic acid was similarly converted by RBL-1 cells to leukotrienes C5, D5. and E5. Leukotrienes C4, D4 and E4 were also formed in these experiments from endogenous arachidonic acid. Time-course studies, incubations with 3H-labeled leukotriene C3 and effects of acivicin [L-(alpha S, 5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid; a gamma-glutamyl transpeptidase inhibitor] indicated that leukotrienes C and D are intermediates in the formation of leukotrienes E. L-Cysteine enhanced the conversion of leukotriene C3 to leukotriene D3 and inhibited further degradation of leukotriene D3 to leukotriene E3.

Metabolism of leukotriene E4 to 5-hydroxy-6-mercapto7,9-trans-11,14-cis-eicosatetraenoic acid by microfloral cysteine-conjugate beta-lyase and rat cecum contents.

Leukotriene E4 was incubated with cysteine-conjugate beta-lyase isolated from the intestinal bacterium Eubacterium limosum. The reaction was terminated by addition of iodoacetic acid or dimethyl sulfate, and the products formed were isolated by reverse-phase high-performance liquid chromatography. The structures of two adducts of a metabolite were determined by uv spectroscopy, by gas-liquid radiochromatography, and by comparisons with chemically synthesized reference compounds. They were 5-hydroxy-6-S-carboxymethylthio-7,9-trans-11,14-cis-eicosatetraeno ic acid (iodoacetic acid adduct) and 5-hydroxy-6-S-methylthio-7,9-trans-11,14-cis-eicosatetraenoic acid (dimethyl sulfate adduct) indicating that the structure of the underivatized metabolite was 5-hydroxy-6-mercapto-7,9,11,14-eicosatetraenoic acid (5,6-HMETE). The latter product is formed by beta-lyase-catalyzed cleavage of the cysteine C-S bond in leukotriene E4. Leukotriene E4 was also metabolized to 5,6-HMETE by rat cecal contents. A product formed was trapped as the iodoacetic acid derivative and identified as 5-hydroxy-6-S-carboxy-methylthio-7,9,11,14-eicosatetraenoic acid. It is concluded that intestinal leukotriene E4, originating from biliary excretion of systemic cysteinyl leukotrienes or produced in the intestine, is converted by microfloral cysteine-conjugate beta-lyase to 5,6-HMETE.

Collisionally induced dissociation of epoxyeicosatrienoic acids and epoxyeicosatrienoic acid-phospholipid molecular species.

Four isomers of epoxyeicosatrienoic acid (EET) can be formed by cytochrome P-450 oxidation of arachidonic acid: 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acid. The collision-induced dissociation of the [M-H]- anion at m/z 319 from each of these isomers, using negative-ion fast atom bombardment ionization and a triple quadrupole mass spectrometer, resulted in a series of common ions as well as ions characteristic of each isomer. The common ions were m/z 301 [M-H2O]- and 257 [M-(H2O + CO2)]-. Unique ions resulted from cleavages alpha to the epoxide moiety to form either conjugated carbanions or aldehydes. Mechanisms involving charge site transfer are suggested for the origin of these ions. A distonic ion series that may involve a charge-remote fragmentation mechanism was also observed. The epoxyeicosatrienoic acids were also incorporated into cellular phospholipids following incubation of the free acid with murine mast cells in culture. Negative fast atom bombardment mass spectrometry of purified glycerophosphoethanolamine-EET species and glycerophosphocholine-EET species yielded abundant [M-H]- and [M-CH3]- ions, respectively. The collision-induced dissociation of these specific high-mass ions revealed fragment ions characteristic of the epoxyeicosatrienoic acids incorporated (m/z 319, 301, and 257) and the same unique ions as those seen with each isomeric epoxyeicosatrienoic acid. With this direct method of analysis, phospholipids containing the four positional isomers of EET, including the highly labile (5,6-EET), could be identified as unique molecular species in mast cells incubated with EET.

Release of small amounts of free fatty acids from human adipocytes as determined by chemiluminescence.

A semiautomatic luminometric method for determination of small amounts of free fatty acids (FFA) released from human adipocytes in vitro is described. Bovine serum albumin (BSA) is used as acceptor of free fatty acids in the incubation medium of isolated fat cells. The assay involves pretreatment with the detergent sodium dodecyl sulfate (SDS) to liberate the free fatty acids from the bovine serum albumin before activation by acyl-CoA synthetase (ACS) (EC 6.2.1.3). This is followed by oxidation of the resulting thioesters by acyl-CoA oxidase (ACO). The H2O2 formed is subsequently measured in a horseradish peroxidase (HRP) (EC 1.11.1.7)-catalyzed luminol reaction. The assay is linear in the interval of 0.01-1 nmol in the cuvette corresponding to 2-200 microM in the sample, and 25 samples are automatically assayed in the luminometer within 75 min. FFA release could easily be studied in a small incubation volume (200 microliters) of very diluted (10(4) cells/ml) human adipocyte suspensions. Samples (25 microliters) containing 0.25% BSA from incubates of adipose tissue cells did not interfere with the standard curve. The analytical interference from different factors that could be used in studies of lipolysis was investigated. No interference was observed up to the following concentrations: 5 microM epinephrine, 5 microM norepinephrine, 80 microM isoproterenol, 1 mM insulin, 2.5 mM propranolol, 5 mM phentolamine, and 5 microM ascorbate. Results obtained with the present assay were highly correlated (r = 0.997) with those obtained by a 260-times less sensitive spectrophotometric kit method.(ABSTRACT TRUNCATED AT 250 WORDS)

Free fatty acid determination by peroxidase catalysed luminol chemiluminescence.

A sensitive, specific, and partly automatic method for the analysis of free fatty acids is described. The assay involves activation of free fatty acids by acyl-CoA synthetase (EC 6.2.1.3) followed by oxidation of the thioesters by acyl-CoA oxidase. The H2O2 formed is determined in a reaction catalysed by horseradish peroxidase (EC 1.11.1.7) using luminol as electron donor. The assay has a linear range of 0.05 to 5 nmol of different free fatty acids (C10-C18) in the original sample. The efficiency of the method toward capric, lauric, myristic, palmitic, palmitoleic, stearic, oleic, and linoleic acid measured as recovery of light emission compared to that of H2O2 standards, was over 90%. AffiGel 501 was used to covalently bind the free thiol group in CoASH eliminating interference of this substance in the peroxidase-luminol reaction.

Endogenous leukotriene D4 formation during anaphylactic shock in the guinea pig.

Experiments on the metabolism and excretion of i.v. administered selectively labeled [3H8]leukotriene C4 in bile duct-cannulated guinea pigs indicated predominantly biliary excretion of tritium. The major leukotriene metabolite in bile was identified as leukotriene D4. By monitoring leukotriene excretion radioimmunochromatographically, it was shown that guinea pigs suffering from anaphylactic shock produce leukotriene D4 endogenously. Immunological challenge of animals sensitized to ovalbumin was accompanied by an increase of biliary leukotriene D4 concentrations from 10 +/- 1 to 86 +/- 10 nM (mean +/- SEM, n = 5, P less than 0.001). When considering that bile flow was decreased to about half after challenge, the excretion rate of leukotriene D4 in bile increased from 0.88 +/- 0.16 before to 3.18 +/- 0.38 pmol X min-1 X kg-1 after challenge (mean +/- SEM, n = 5, P less than 0.002). It is concluded that systemic anaphylaxis in the guinea pig is associated with endogenous generation of leukotriene C4 (up to 1 nmol/kg during a 30-min period after the challenge.

Incorporation and distribution of epoxyeicosatrienoic acids into cellular phospholipids.

The different regioisomers of epoxyeicosatrienoic acids derived from cytochrome P-450 monooxygenase are readily esterified into phospholipids of mastocytoma cells. Incorporation of 14,15-epoxyeicosatrienoic acid was concentration-dependent, with Km = 1.1 microM and Vmax = 36 pmol/min/10(7) cells. Half-maximal incorporation occurred in 30 min, reaching a steady-state concentration of 470 pmol/10(6) cells. This was slightly lower than the values for arachidonic acid (665 pmol/10(6) cells) or 5-hydroxyeicosatetraenoic acid (554 pmol/10(6) cells). The distribution of 14,15-epoxyeicosatrienoic acid was preferential in the order phosphatidylethanolamine greater than phosphatidylcholine greater than phosphatidylinositol greater than phosphatidyl serine much greater than neutral lipids plus fatty acids. This contrasted with 5(S)-hydroxyeicosatetraenoic acid, which was distributed primarily into phosphatidylcholine. Fast atom bombardment/tandem mass spectrometry facilitated identification of molecular species containing epoxyeicosatrienoic acids without relying on radioisotopes. Phosphatidylethanolamine plasmalogens with 16:1 or 18:2 at the sn-1 position, or an 18:0 acyl group, and phosphatidylcholine with 16:0 alkyl ether or an acyl group at the sn-1 position incorporated all possible epoxyeicosatrienoic acid regioisomers. Under basal conditions, cells eliminated 14,15-cis-epoxyeicosatrienoic acid slowly with a half-life of 34.9 +/- 7 h. Cells stimulated with calcium ionophore A23187 eliminated 14,15-epoxyeicosatrienoic acid rapidly. It was notable that its rate of release from phosphatidylcholine and phosphatidylinositol exceeded that for arachidonic acid. A coenzyme A-independent transacylase also catalyzed the transfer of epoxyeicosatrienoic acids from mastocytoma cell membranes into 1-palmitoyl-2-lysophosphatidylcholine. The cellular incorporation, release, and distribution of epoxyeicosatrienoic acids is distinctive and contrasts with most other eicosanoids, suggesting that these compounds may have both autocoid and nonautocoid functions.

14, 15-cis-episulfide-eicosatrienoic acid, an 'epoxygenase' eicosanoid analog, inhibits ionophore- but not thrombin-induced platelet aggregation.

An 'epoxygenase' eicosanoid analog, 14, 15-cis-episulfide-eicosatrienoic acid, has several unique pharmacological effects on platelets. These include (i) inhibition of ionophore A23187- but not thrombin-induced activation, (ii) inhibition of thromboxane B2 biosynthesis derived from endogenous but not exogenous arachidonic acid, and (iii) attenuation of ionophore-mediated increases in cytosolic Ca2+ when extracellular or membrane Ca2+ is available but not when these pools are excluded. Neither elevation of cyclic AMP levels, a potent inhibitory process, nor direct antagonism of the prostaglandin H2/thromboxane A2 receptor is responsible for the actions of 14, 15-cis-episulfide-eicosatrienoic acid. These properties distinguish 14, 15-cis-episulfide-eicosatrienoic acid from other antiaggregatory substances.