Synthesis and structure elucidation of leukotrienes.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
E J Corey
Publications and source records attributed to E J Corey.
Characterization and biologic properties of 5,12-dihydroxy derivatives of eicosapentaenoic acid, including leukotriene B5 and the double lipoxygenase product.
Leukotriene B5 (LTB5) and three stereoisomers were prepared biosynthetically from eicosapentaenoic acid and compared with the analogous derivatives of arachidonic acid for their chemotactic and aggregating effects on human neutrophilic polymorphonuclear leukocytes. Leukotriene B4 (LTB4), LTB5, and the 6-trans-diastereoisomers of each were generated by activating polymorphonuclear leukocytes with the calcium ionophore A23187 in the presence of 14C-labeled and unlabeled arachidonic acid or 14C-labeled and unlabeled eicosapentaenoic acid, respectively. The double lipoxygenase products, (5S,12S)-6-trans-8-cis-LTB4 and (5S,12S)-6-trans-8-cis-LTB5, were generated from 5S-hydroxyeicosatetraenoic acid and racemic 5-hydroxyeicosapentaenoic acid intermediates by incubation with platelet sonicates. The products of each reaction were isolated by reverse-phase-high performance liquid chromatography and identified by their retention times relative to the appropriate totally synthetic standards, ultraviolet absorption spectra, immunoreactivity in a radioimmunoassay for LTB4, and, for all but the double lipoxygenase products, by incorporation of radiolabel from the specific polyunsaturated fatty acid source. When the concentration of LTB5 eliciting maximum chemotactic response of human polymorphonuclear leukocytes, 50 ng/ml (1.5 X 10(-7) M), and that eliciting a maximum aggregation response, 20 ng/ml (5.9 X 10(-8) M), were compared with the interpolated values of LTB4 eliciting comparable effects, the potency of LTB5 relative to LTB4 was approximately 1:8 as a chemotactic agent and about 1:20 as an aggregating agent. The double lipoxygenase products and the resolved 6-trans-diastereoisomers of the pentaene and tetraene series were about 2 logs less active as chemotactic factors than LTB4 and only (5S,12S)-6-trans-8-cis-LTB4 had even minimal aggregating activity.
Arachidonic Acid↗
Specific binding of leukotriene C4 to ileal segments and subcellular fractions of ileal smooth muscle cells.
A specific high-affinity receptor for leukotriene C4 (LTC4) has been identified on segments of longitudinal smooth muscle from guinea pig ileum, in disrupted cells obtained from the ileal segments, and in subcellular fractions enriched for mitochondrial membranes and plasma membranes, respectively. Specific [3H]LTC4 binding at a fixed input at 4 degrees C reached a plateau at 60 min with each of the four preparations and was greater than 80% reversible after binding reached equilibrium by the introduction of excess unlabeled homoligand. LIGAND analysis demonstrated a single high-affinity receptor on the smooth muscle segments, the disrupted cells, and the subcellular fractions enriched for mitochondrial membranes and for plasma membranes with respective Kd values of 7.6 nM, 1.3 nM, 13 nM, and 8.5 nM. These Kd values overlap with the concentration of LTC4 known to elicit a spasmogenic response in the ileal muscle, indicating that the radioligand recognizes a receptor that mediates the biological response. Competition analysis with disrupted ileal cells and subcellular fractions with a fixed input of LTC4 radioligand and incremental concentrations of the natural sulfidopeptide leukotrienes demonstrated leukotriene D4 (LTD4) to be 1-3 logarithms less active than LTC4 and leukotriene E4 (LTE4) to be essentially inactive. Thus, ileal longitudinal smooth muscle cells possess a high-affinity receptor that is selective for LTC4 and that receptor exhibits both a plasma membrane and subcellular distribution.
Animals↗
Leukotriene E4-induced airway hyperresponsiveness of guinea pig tracheal smooth muscle to histamine and evidence for three separate sulfidopeptide leukotriene receptors.
Bronchial hyperresponsiveness to contractile agonists and nonspecific irritants is a characteristic feature of bronchial asthma. The mechanisms causing this hyperirritability are unknown. The existence of separate receptors for leukotrienes C4 and D4 (LTC4 and LTD4) has been demonstrated previously by physiologic and radioligand binding studies. The rank order of potency of the sulfidopeptide leukotrienes for contracting tracheal spirals [leukotriene E4 (LTE4) greater than LTD4 = LTC4] is different from that for contracting parenchymal strips (LTD4 greater than LTE4 greater than LTC4), thereby suggesting the existence of a separate receptor for LTE4. We now report that LTE4, the most stable of the leukotrienes comprising slow reacting substance of anaphylaxis, enhances the contractile response of guinea pig tracheal spirals but not of parenchymal strips to histamine in a time- and dose-dependent fashion. The ability of LTE4 to increase histamine responsiveness occurred after removal of the free agonist and recovery of the tissues to baseline tensions and was not produced by leukotrienes C4 and D4, which elicited the same magnitude of contraction of tracheal smooth muscle as LTE4. These findings suggest that LTE4-induced airway hyperirritability is not mediated by the contractile response per se and may be mediated through a receptor distinct from those for leukotrienes C4 and D4.
Airway Resistance↗
Leukotriene B4 action on endothelium mediates augmented neutrophil/endothelial adhesion.
The attachment of human polymorphonuclear leukocytes (PMN) to bovine endothelial monolayers is increased by pretreatment of the endothelial cells with leukotriene B4 (LTB4). The response is dose dependent with a maximum at 10(-6) M, requires no more than 1 min of preincubation for the maximal effect, and decays linearly over 30 min once the LTB4 is removed. Preincubation of PMN with LTB4 does not augment subsequent adhesion to a plastic substratum or an untreated monolayer of endothelial cells and eliminates the augmented attachment to LTB4-pretreated endothelial cells. The simultaneous exposure of both cell types during the adhesion interaction gives a biphasic dose-related increment, suggesting that the inhibition of the neutrophil adherence is less marked than with a pretreatment interval. Thus, the LTB4-dependent adherence of PMN to endothelial cells is selectively mediated by endothelial cells, is dose related, is reversible upon removal of the agonist from the endothelial cells, and is opposed by the direct action of LTB4 on the PMN.
Animals↗
Studies on leukotriene B4 omega-oxidation in human leukocytes.
The kinetics of the transformation of arachidonic acid into 5-lipoxygenase products in human blood leukocytes stimulated with the ionophore A23187 (2 microM) was studied using high performance liquid chromatography. The levels of 5-hydroxyeicosatetraenoic acid (5-HETE), leukotriene B4 (LTB4), delta 6-trans-LTB4, and leukotriene C4 (LTC4) were maximum after 4-5 min of incubation; at longer incubation times, the levels of delta 6-trans-LTB4 and LTC4 remained unchanged, whereas those of 5-HETE and LTB4 decreased rapidly. The disappearance of LTB4 was concomitant with the formation of omega-hydroxy-LTB4 and omega-carboxy-LTB4. When synthetic LTB4 was added to a suspension of unstimulated human leukocytes, a similar pattern of omega-oxidation products was observed. This omega-oxidation process showed some specificity for LTB4, as indicated by the slower reaction of three stereoisomers of LTB4. Studies with subpopulations of human blood cells and human plasma clearly indicated that the polymorphonuclear leukocytes were the main source of enzymic activity for the omega-oxidation of LTB4. The liquid chromatographic behaviors of natural omega-hydroxy-LTB4 and omega-carboxy-LTB4 were studied in two systems.
Calcimycin↗
Renal and systemic hemodynamic responses to intravenous infusion of leukotriene C4 in the rat.
We studied the systemic and renal hemodynamic effects of leukotriene C4 (2 micrograms/kg per min for 5 minutes, iv) in the rat. During the period of its infusion, leukotriene C4 produced a significant elevation of mean arterial pressure and reductions in cardiac output and renal blood flow, as measured by electromagnetic flow probes. These effects were abolished by FPL55712 , a putative antagonist of sulfidopeptide leukotrienes, but not by saralasin or indomethacin. Leukotriene C4 also resulted in an average loss of 20% in plasma volume which, during the postinfusion period, perpetuated the low cardiac output state and thus provoked the release of angiotensin II. This vasoactive peptide sustained the elevation in systemic vascular resistance and the reduction in renal blood flow over a 70-minute postinfusion observation period. Consequently, glomerular filtration rate fell by approximately 50%. These angiotensin II-mediated effects were abolished by saralasin. Indomethacin prevented the leukotriene C4-induced loss of plasma volume and, thus, allowed for the significant recovery of cardiac output and renal blood flow during the post-infusion period, thereby preserving glomerular filtration rate. We conclude that leukotriene C4 exerts direct systemic and renal vasoconstrictor, as well as cardiodepressant effects, during the period of its infusion. By virtue of its vasopermeability enhancing effect, leukotriene C4 also results in an immediate loss of plasma volume, an effect which requires the presence of secondarily generated cyclooxygenase products and which perpetuates the hemodynamic abnormalities observed beyond the period of leukotriene C4 infusion.
Animals↗
Effects of exogenous arachidonic, eicosapentaenoic, and docosahexaenoic acids on the generation of 5-lipoxygenase pathway products by ionophore-activated human neutrophils.
Exogenous eicosapentaenoic acid (EPA) and docosahexaenoic acid (DCHA) have been compared with exogenous arachidonic acid for their capacity to modulate the oxidative metabolism of membrane-derived arachidonic acid by the 5-lipoxygenase pathway in ionophore-activated human neutrophils and for their suitability as parallel substrates in this pathway. The products from specific 14C- or 3H-labeled substrates were isolated by reverse phase high performance liquid chromatography (RP-HPLC) and were identified by elution of radiolabel at the retention times of the appropriate synthetic standards. Each product was also characterized by its ultraviolet (UV) absorption spectrum, and 7-hydroxy-DCHA was defined in addition by analysis of its mass spectrum. The metabolites, 5-hydroxyeicosatetraenoic acid, leukotriene B4 (LTB4), 6-trans-LTB4 diastereoisomers, 5-hydroxyeicosapentaenoic acid, 6-trans-leukotriene B5 diastereoisomers, leukotriene B5 (LTB5), and 7-hydroxy-DCHA were quantitated by integrated UV absorbance during resolution by RP-HPLC. LTB4 and LTB5 were also quantitated by radioimmunoassay of the eluate fractions, and leukotrienes C4 and C5 (LTC4 and LTC5, respectively) were quantitated by radioimmunoassay alone. None of the unlabeled exogenous fatty acids (5-40 micrograms/ml) altered the release of radioactivity from [14C]arachidonic acid-labeled, ionophore-activated neutrophils. The metabolism of 5 and 10 micrograms/ml of exogenous EPA by ionophore-activated, [14C]arachidonic acid-labeled neutrophils not only generated 5-hydroxyeicosapentaenoic acid, 6-trans-LTB5, LTB5, and LTC5, but also stimulated the formation of 5-hydroxyeicosatetraenoic acid, 6-trans-LTB4 diastereoisomers, and LTC4 from membrane-derived arachidonic acid. In contrast, LTB4 production was diminished throughout the EPA dose-response, beginning at 5 micrograms/ml EPA and reaching 50% suppression at 10 micrograms/ml and 84% suppression at 40 micrograms/ml. The selective decrease in extracellular LTB4 concentrations in the presence of EPA was not due to a change in the kinetic appearance of LTB4 or to an increase in conversion to its omega-oxidation metabolites. DCHA was metabolized to 7-hydroxy-DCHA, did not stimulate metabolism of membrane-derived arachidonic acid, did not appreciably inhibit LTB4 formation, and was not a substrate for leukotriene formation. Incremental doses of exogenous arachidonic acid resulted in increased production of 5-hydroxyeicosatetraenoic acid and 6-trans-LTB4 by ionophore-activated, [14C]arachidonic acid-labeled neutrophils without any change in LTB4 production. 5-hydroxyeicosapentaenoic acid and 7-hydroxy DCHA were inactive as chemotactic factors whereas 5-hydroxyeicosatetraenoic acid exhibited 2% of the potency of LBT4. Thus, exogenous DCHA does not appreciably interfere with the metabolism of membrane-derived arachidonic acid by ionophore-activated, [14C]arachidonic acid-labeled neutrophils and is converted only to a monohydroxy derivative. In contrast, exogenous EPA attenuates the generation of LTB4 and is converted to LTB5, which is a weak and partial agonist as compared with LTB4.
Arachidonate Lipoxygenases↗
Immunologically induced generation of tetraene and pentaene leukotrienes in the peritoneal cavities of menhaden-fed rats.
The generation of sulfidopeptide leukotrienes and leukotriene B (LTB) in response to an IgG-mediated immune complex reaction in the peritoneal cavities of rats fed either a menhaden oil-supplemented diet or a beef tallow-supplemented diet for 9 to 10 wk was determined with the combined techniques of radioimmunoassay (RIA) and reverse-phase high performance liquid chromatography. Rats on the fish fat diet (FFD) incorporated eicosapentaenoic acid (EPA) into pulmonary and splenic tissues with an EPA:arachidonic acid ratio of approximately 2:1, whereas rats on the beef fat diet (BFD) showed no detectable EPA. The estimated total quantities of immunoreactive sulfidopeptide leukotrienes generated by each group of rats were similar, ranging from 70 to 99 ng/ rat in the FFD groups and 65 to 109 ng/rat in the BFD groups; for rats on the FFD this total included the pentaene products LTC5, LTD5, and LTE5 in quantities ranging from 24 to 39 ng/rat. The total quantities of immunoreactive LTB generated in the two groups of rats were similar, being 6 to 29 ng LTB4/rat for the BFD groups and the sum of LTB4 and LTB5 of 8 to 36 ng/rat for the FFD groups. There was a two- to seven-fold preferential generation of immunoreactive LTB5 over LTB4 in the FFD rats. LTC5 was equipotent with LTC4 in contracting guinea pig pulmonary parenchymal strips and ileal tissues. In contrast, LTB5 was 1/30 to 1/60 as potent and did not reach the same maximum as LTB4 in eliciting neutrophil chemotaxis. The finding that FFD favors the immunologic generation of LTB5, which has attenuated biologic activity when compared to LTB4, suggests that EPA-enriched tissues may produce less pro-inflammatory activity than tissues that are EPA-poor.
Animals↗
An analysis of the relationship between 5-lipoxygenase product generation and the secretion of preformed mediators from mouse bone marrow-derived mast cells.
The quantitative relationships between the secretion of a granule-associated mediator, beta-hexosaminidase, and the oxidative metabolism of arachidonic acid by the 5-lipoxygenase pathway were analyzed for a homogeneous population of T cell-dependent, bone marrow-derived, murine mast cells. The mast cells were either sensitized with a monoclonal IgE and challenged with specific antigen, or to bypass a transmembrane signal, were stimulated with calcium ionophore A23187. The released products of the 5-lipoxygenase pathway were quantitated by integrated ultraviolet absorbance after resolution by reverse phase-high performance liquid chromatography in the case of 5-hydroxy-eicosatetraenoic acid (5-HETE), and by separate radioimmunoassays for leukotriene C4 (LTC4) and leukotriene B4 (LTB4). The activation-release response of the cells was perturbated by the introduction of three pharmacologic agents, each directed to different steps in the 5-lipoxygenase pathway of arachidonic acid metabolism, and the action of each agent was determined for separate cell samples while present and after its removal by washing. 5,6-Dehydroarachidonic acid (5,6-DHA), an irreversible inhibitor of 5-lipoxygenase, prevented formation of 5-HETE from exogenous [14C]arachidonic acid and from membrane-derived arachidonic acid in a dose-related fashion when sensitized mast cells, preincubated with drug, were washed before antigen activation. Release of 5-HETE, LTC4, and LTB4 was inhibited by 5,6-DHA in a corresponding dose-related fashion, with a minimal preincubation period of 1 to 5 min before the cells were washed and subjected to antigen-dependent activation. In contrast, the inhibitory effect of 5,6-DHA on beta-hexosaminidase release was lost after three washes and was not evident after one wash unless the preincubation period was extended to 15 min. The capacity of 5,6-DHA to prevent leukotriene generation without altering beta-hexosaminidase release was also observed with ionophore-activated mast cells. Preincubation of sensitized cells with diethylcarbamazine (DEC), followed by a wash before antigen-dependent activation, produced inhibition of leukotriene generation, no effect on beta-hexosaminidase release, and augmentation of 5-HETE release at the maximum dose studied; thus, DEC interrupts the pathway distal to the formation of 5-hydroperoxy-eicosatetraenoic acid (5-HPETE) from arachidonic acid by 5-lipoxygenase. Preincubation of sensitized cells with incremental amounts of the prostacyclin analog U-60,257, followed by washing and antigen challenge, inhibited LTC4 release without altering the release of LTB4 or beta-hexosaminidase.(ABSTRACT TRUNCATED AT 400 WORDS)
8,11,14-Eicosatrienoic Acid↗
The myeloperoxidase-dependent metabolism of leukotrienes C4, D4, and E4 to 6-trans-leukotriene B4 diastereoisomers and the subclass-specific S-diastereoisomeric sulfoxides.
The mechanism of the metabolic inactivation of leukotrienes C4, D4, and E4 by human polymorphonuclear leukocytes activated by phorbol myristate acetate has been analyzed with the use of activated cells, their supernatants, the isolated components of the myeloperoxidase system, and chemically synthesized hypochlorous acid (HOC1). The metabolic products were resolved by reverse-phase high performance liquid chromatography in one or more solvent systems, and each product was characterized relative to chemically synthesized standards by its reverse-phase high performance liquid chromatography retention time, UV absorption spectrum, nonvascular smooth muscle spasmogenic activity, and immunoreactivity. The phorbol myristate acetate-activated human polymorphonuclear leukocytes converted each of the sulfidopeptide leukotrienes to products identical with synthetic diastereoisomers of 6-trans-leukotriene B4 by the following criteria: retention times in two solvent systems, lambda max of 269 nm with shoulders at 259 and 279 nm, and mass spectral analysis. Each of the sulfidopeptide leukotrienes was simultaneously converted to subclass-specific S-diastereoisomeric sulfoxides which co-chromatographed with synthetic standards, exhibited a bathochromic shift to a lambda max at 284 nm, were fully immunoreactive, and possessed less than 5% spasmogenic activity of the corresponding sulfidopeptide leukotrienes. When the sulfidopeptide leukotrienes were incubated with supernatants of phorbol myristate acetate-activated cells in the presence of 0.1 mM H2O2, with a mixture of 100 milliunits of myeloperoxidase, 0.1 mM H2O2, and 10 mM Cl-, or with chemically prepared HOCl, the metabolic products formed were the same as those obtained with the activated cells, indicating that extracellular inactivation was due to HOCl produced by the action of released myeloperoxidase.
Cell-Free System↗
Airway constriction in normal humans produced by inhalation of leukotriene D. Potency, time course, and effect of aspirin therapy.
Five normal human subjects inhaled aerosols generated from solutions of leukotriene D (LTD) to determine the bronchoconstrictor potency and the time course of airway obstruction produced by this constituent of slow-reacting substance of anaphylaxis. The dose-effect and time-effect curves were compared with curves similarly generated for the well-characterized airway constrictor histamine. Leukotriene D was, on average, 5,900 times more potent than histamine on a molar basis in producing an identical decrement in maximal expiratory flow rate at 30% of control vital capacity above residual volume. In addition, although LTD had a rapid onset of effect, similar to that of histamine, the airway obstruction produced by LTD was longer lasting, thereby reflecting more closely the response of asthmatic allergic individuals to antigen inhalation. The response of these subjects to inhalation of LTD was not altered by ingestion of aspirin, suggesting that the airway obstruction was not mediated by cyclooxygenase products of arachidonic acid.
Adult↗
Effects of leukotriene D on the airways in asthma.
Explore the source record for details and available documents.
Asthma↗
Identification and quantitation of arachidonic-acid metabolic products in rabbit, rat and human saliva.
Arachidonic-acid metabolites were identified in salivary secretions of rabbits, rats and man and in homogenates of rabbit parotid and submandibular glands. The following levels were determined in mixed saliva of healthy volunteers: PGE2, 0.65 +/- 0.8 ng/ml; PGF2 alpha, 0.41 +/- 0.06 ng/ml; serologically active hydroxyeicosatetraenoic acids (HETE) 319.6 +/- 36.6 ng/ml; serologically active 6-sulphidopeptide-containing leukotrienes 1.72 +/- 0.21 ng/ml (all values mean +/- SEM). In all instances, HETE markedly predominated over all other arachidonic-acid metabolites.
12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
Effects of leukotrienes C4 and D4 on glycoprotein and lysozyme secretion by human bronchial mucosa.
The effects of leukotrienes C4 (LTC4) and D4 (LTD4) on the secretion by human bronchial mucosa of [14C]glucosamine-labeled, trichloro-acetic acid/phosphotungstic acid-precipitable glycoprotein and lysozyme were evaluated in vitro. LTC4 and LTD4, in the concentration range of 0.16 to 1600 nM, induced a dose-related increase in the release of radiolabeled glycoprotein, but not of lysozyme. This secretagogue effect was selective for high molecular weight glycoproteins of about 2-5 x 10(6) daltons, and the median effective concentrations (EC50) of LTC4 of 9.4 x 10(-9) M and of LTD4 of 2.44 x 10(-8) M, indicate that these leukotrienes are approximately 100-fold more potent than the cholinergic agonist methacholine. Incubation of [14C]glucosamine-labeled bronchial mucosal explants with LTC4 or LTD4 for six sequential 15-min periods revealed a rapid, progressive decrement in glycoprotein release, compatible with stimulatory action on secretion rather than augmentation of the rate of glycoprotein synthesis. This interpretation is also consistent with the finding that the specific activity (ratio of bound radiolabel: protein content) of the macromolecular glycoprotein secreted by the explants is not changed with stimulation of release by the leukotrienes. Based upon the activity of synthetic leukotriene analogs, the specific C-6 chirality of the sulfidopeptide of LTD4, the presence of a hydroxyl at C-5 and the presence of eicosanoid carbons 9-20 were of no importance for secretagogue activity. These findings contrast with the stereochemical requirements for the spasmogenic response to sulfidopeptide leukotrienes and suggest that leukotriene-induced secretion is not likely to be mediated via a specific receptor.
Bronchi↗
Effects of indomethacin on the guinea-pig pulmonary response to intravenous leukotrienes C4 and D4.
We measured pulmonary conductance (GL), dynamic compliance (Cdyn.), and arterial pressure in chloralose-urethane anaesthetized guinea pigs for 30 min after intravenous infusion of either leukotriene (LT) C4 (1 microgram/kg) or LTD4 (0.5 microgram/kg) to control or indomethacin-pretreated (30 mg/kg) animals. LT infusion to control animals caused decrements in GL, which were maximal at 30 s and persisted for 7.5 min (LTD4) and 15 min (LTC4), decrements in Cdyn., which were maximal at 30 s and persisted for 30 min, and hypotension. Indomethacin treatment before LT infusion significantly altered the changes in GL by reducing the early (less than 1 min) response and potentiating the later (3-5 min) response, significantly reduced the fall in Cdyn. at all time points, resulting in a change in the GL/Cdyn. ratio to 1.0 and prolonged the hypotensive response. We conclude that cyclo-oxygenase products are responsible for the early (less than 1 min) pulmonary response to LT infusion and modulate the later (3-5 min) response but do not cause hypotension. With cyclo-oxygenase inhibition, the significant primary activity of LT is possibly less preferentially directed to contractile tissues associated with small airways.
Airway Resistance↗
Docosahexaenoic acid is a strong inhibitor of prostaglandin but not leukotriene biosynthesis.
Docosahexaenoic acid (DCHA), a major polyunsaturated acid component of fish lipid, is not a substrate for prostaglandin synthetase from ram seminal vesicles but is a strong competitive inhibitor (Ki, 0.36 microM) of the conversion by this enzyme of arachidonate (Km, 5.9 microM) to prostaglandins. In contrast, DCHA exhibits little interference to the conversion of arachidonate to metabolites on the leukotriene pathway. DCHA is a very poor substrate for the leukotriene-synthesizing system from RBL-1 cells and no formation of the C22 analog of leukotriene B could be detected from it. The C22 analog of leukotriene C4 was produced by chemical synthesis from DCHA and found to be less than 1/10,000th as active as leukotriene C4 in contracting guinea pig ileum. The DCHA used in this work was obtained in greater than 99.8% purity by a practical process developed for its separation from fish lipid. The cardiovascular protective effects ascribed to dietary intake of fish lipid by certain populations may be due in part to the biological action of DCHA, including the inhibition of prostaglandin biosynthesis.
Animals↗
Generation and metabolism of 5-lipoxygenase pathway leukotrienes by human eosinophils: predominant production of leukotriene C4.
5-Lipoxygenase pathway-derived products of arachidonic acid released by human eosinophils activated in vitro have been measured by using radioimmunoassays specific for leukotriene B4 (LTB4) and for sulfidopeptide leukotrienes including leukotriene C4 (LTC4). Eosinophil-enriched leukocytes (mean, 85% eosinophils) from five hypereosinophilic donors activated with 5.0 microM ionophore A23187 for 15 min at 37 degrees C in the presence of 50 mM L-serine released 69 +/- 28 and 1.5 +/- 0.8 (mean +/- SEM) ng of LTC4 and LTB4, respectively, per 10(6) cells; ratios of LTC4 to LTB4 ranged from 16 to 149. Eosinophils stimulated with ionophore (2.5 microM) or phorbol myristate acetate (1 microgram per ml) metabolized exogenously added LTC4 to products that coeluted on reverse-phase high-performance liquid chromatography with synthetic S-diastereoisomeric LTC4 sulfoxides and 6-trans-LTB4 diastereoisomers, and this metabolic inactivation was inhibited by L-serine or catalase. Ionophore-activated eosinophils purified from three normal donors also preferentially generated LTC4 (38 +/- 3 ng per 10(6) cells) relative to LTB4 (6.0 +/- 3.1 ng per 10(6) cells), whereas neutrophils from the same donors released LTB4 (48 +/- 21 ng per 10(6) cells) in a greater than 7-fold excess to LTC4. The predominant production by human eosinophils of LTC4 with its potent smooth muscle spasmogenic and vasoactive properties may contribute to the pathobiology of allergic and other diseases associated with eosinophilia.
Arachidonate Lipoxygenases↗