5-lipoxygenase-dependent biosynthesis of novel 20:4 n-3 metabolites with anti-inflammatory activity.

5-lipoxygenase (5-LO) catalyzes the conversion of arachidonic acid (AA) into pro-inflammatory leukotrienes. N-3 PUFA like eicosapentaenoic acid are subject to a similar metabolism and are precursors of pro-resolving mediators. Stearidonic acid (18:4 n-3, SDA) is a plant source of n-3 PUFA that is elongated to 20:4 n-3, an analogue of AA. However, no 5-LO metabolites of 20:4 n-3 have been reported. In this study, control and 5-LO-expressing HEK293 cells were stimulated in the presence of 20:4 n-3. Metabolites were characterized by LC-MS/MS and their anti-inflammatory properties assessed using AA-induced autocrine neutrophil stimulation and leukotriene B4-mediated chemotaxis. 8­hydroxy­9,11,14,17-eicosatetraenoic acid (Δ17-8-HETE) and 8,15-dihydroxy-9,11,13,17-eicosatetraenoic acid (Δ17-8,15-diHETE) were identified as novel metabolites. Δ17-8,15-diHETE production was inhibited by the leukotriene A4 hydrolase inhibitor SC 57461A. Autocrine neutrophil leukotriene stimulation and neutrophil chemotaxis, both BLT1-dependent processes, were inhibited by Δ17-8,15-diHETE at low nM concentrations. These data support an anti-inflammatory role for Δ17-8,15-diHETE, a novel 5-LO product.

Maternal iron deficiency and its effect on essential fatty acid and eicosanoid metabolism and spatial memory in the guinea pig offspring.

Iron deficiency is prevalent among infants and pregnant women in industrialized country. The goal of this study was to evaluate the impact of moderate maternal iron deficiency on the offspring's fatty acid and eicosanoid metabolism and spatial memory in guinea pigs. An iron-sufficient (IS) or iron-deficient (ID) diet was fed 14 days before mating and throughout pregnancy and lactation. The pups were tested for spatial memory on post-natal days 4-7. On post-natal day 9, the biochemical analysis included the pup's brain fatty acid profiles, prostaglandin (PGE(2) and PGF(2alpha)) concentrations and cyclooxygenase II protein levels. Spatial memory and indices of eicosanoid metabolism were comparable in both dietary groups. However, n-3 fatty acids were significantly higher (p<0.05) in brain of pups from the ID group. The data suggest that maternal iron deficiency results in a modification of the fatty acid profile of the offspring's brain that is not associated with any spatial memory deficits during early development.

Anti-CD3 and concanavalin A-induced human T cell proliferation is associated with an increased rate of arachidonate-phospholipid remodeling. Lack of involvement of group IV and group VI phospholipase A2 in remodeling and increased susceptibility of proliferating T cells to CoA-independent transacyclase inhibitor-induced apoptosis.

In this study arachidonate-phospholipid remodeling was investigated in resting and proliferating human T lymphocytes. Lymphocytes induced to proliferate with either the mitogen concanavalin A or with anti-CD3 (OKT3) in combination with interleukin 2 (OKT3/IL-2) showed a greatly accelerated rate of [3H]arachidonate-phospholipid remodeling compared with resting lymphocytes or with lymphocytes stimulated with OKT3 or IL-2 alone. The concanavalin A-stimulated cells showed a 2-fold increase in the specific activity of CoA-independent transacylase compared with unstimulated cells, indicating that this enzyme is inducible. Stimulation with OKT3 resulted in greatly increased quantities of the group VI calcium-independent phospholipase A2 but not of the quantity of group IV cytosolic phospholipase A2. However, group IV phospholipase A2 became phosphorylated in OKT3-stimulated cells, as determined by decreased electrophoretic mobility. Incubation of cells with the group VI phospholipase A2 inhibitor, bromoenol lactone, or the dual group IV/group VI phospholipase A2 inhibitor, methyl arachidonyl fluorophosphonate, did not block arachidonate-phospholipid remodeling resting or proliferating T cells, suggesting that these phospholipases A2 were not involved in arachidonate-phospholipid remodeling. The incubation of nonproliferating human lymphocytes with inhibitors of CoA-independent transacylase had little impact on cell survival. In contrast, OKT3/IL-2-stimulated T lymphocytes were very sensitive to apoptosis induced by CoA-independent transacylase inhibitors. Altogether these results indicate that increased arachidonate-phospholipid remodeling is associated with T cell proliferation and that CoA-independent transacylase may be a novel therapeutic target for proliferative disorders.

Activation of leukotriene synthesis in human neutrophils by exogenous arachidonic acid: inhibition by adenosine A(2a) receptor agonists and crucial role of autocrine activation by leukotriene B(4).

We report here that the apparent inability of isolated human polymorphonuclear leukocytes (PMNs) to efficiently transform arachidonic acid (AA) is the consequence of A(2a) receptor engagement by endogenous adenosine accumulating in incubation media. Indeed, when adenosine is eliminated from PMN suspensions by the addition of adenosine deaminase, or when cells are incubated with adenosine A(2a) receptor antagonists, important quantities (40-80 pmol/10(6) cells) of 5-lipoxygenase products are synthesized by PMN incubated with 1 to 5 microM exogenous AA. The selective A(2a) receptor agonist CGS21680 was a very potent inhibitor of the AA-induced leukotriene (LT) synthesis, showing an IC(50) of approximately 1 nM. The mechanism of AA-induced stimulation of LT synthesis observed in the absence of extracellular adenosine was investigated. In adenosine deaminase-treated PMN, exogenous AA induced Ca(2+) mobilization and the translocation of 5-lipoxygenase to nuclear structures. A time lag of 20 to 60 s (variable between PMN preparations) was observed consistently between the addition of AA and the elevation of intracellular Ca(2+) concentration (and LT synthesis), indicating that AA itself did not trigger the Ca(2+) mobilization in PMN. This AA-induced Ca(2+) mobilization, as well as the corresponding 5-lipoxygenase translocation and stimulation of LT synthesis, was blocked efficiently by the LT synthesis inhibitor MK0591, the LTB(4) receptor antagonists CP105696 and LY223982, and the LTA(4) hydrolase inhibitor SC57461A. These data demonstrate that AA is a highly potent and effective activator of LT synthesis and acts through a mechanism that requires an autocrine stimulatory loop by LTB(4).

Perturbations in the control of cellular arachidonic acid levels block cell growth and induce apoptosis in HL-60 cells.

Our previous studies demonstrated that inhibitors of arachidonate-phospholipid remodeling [i.e. the enzyme CoA-independent transacylase (CoA-IT)] decrease cell proliferation and induce apoptosis in neoplastic cells. The goal of the current study was to elucidate the molecular events associated with arachidonate-phospholipid remodeling that influence cell proliferation and survival. Initial experiments revealed the essential nature of cellular arachidonate to the signaling process by demonstrating that HL-60 cells depleted of arachidonate were more resistant to apoptosis induced by CoA-IT inhibition. In cells treated with CoA-IT inhibitors a marked increase in free arachidonic acid and AA-containing triglycerides were measured. TG enrichment was likely due to acylation of arachidonic acid into diglycerides and triglycerides via de novo glycerolipid biosynthesis. To determine the potential of free fatty acids to affect cell proliferation, HL-60 cells were incubated with varying concentrations of free fatty acids; exogenously provided 20-carbon polyunsaturated fatty acids caused a dose-dependent inhibition of cell proliferation, whereas oleic acid was without effect. Blocking 5-lipoxygenase or cyclooxygenases had no effect on the inhibition of cell proliferation induced by arachidonic acid or CoA-IT inhibitors. An increase in cell-associated ceramides (mainly in the 16:0-ceramide fraction) was measured in cells exposed to free arachidonic acid or to CoA-IT inhibitors. This study, in conjunction with other recent studies, suggests that perturbations in the control of cellular arachidonic acid levels affect cell proliferation and survival.

Mechanisms of the priming effect of lipopolysaccharides on the biosynthesis of leukotriene B4 in chemotactic peptide-stimulated human neutrophils.

The goal of this study was to explain the priming effect of lipopolysaccharides (LPS) in human polymorphonuclear leukocytes on leukotriene B4 (LTB4) biosynthesis after stimulation with the receptor-mediated agonist formyl-methionyl-leucyl-phenylalanine (fMLP). This priming effect for LTB4 biosynthesis was maximal after a 30 min preincubation with LPS but was lost when incubations were extended to 90 min or longer. Priming with LPS resulted in an enhanced maximal activation of 5-lipoxygenase (5- to15-fold above unprimed cells) as well as a prolonged activation of the enzyme after stimulation with fMLP compared to that measured in unprimed cells. The activation of 5-lipoxygenase was associated with its translocation to the nuclear fraction of the cell after stimulation of LPS-primed cells but not of unprimed cells. Priming of cells with LPS also resulted in an enhanced capacity (fivefold increase) for arachidonic acid (AA) release after stimulation with fMLP compared to unprimed cells as measured by mass spectrometry. This release of AA was very efficiently blocked in a dose-dependent manner by the 85 kDa cytosolic phospholipase A2 (PLA2) inhibitor MAFP (IC50=10nM) but not by the 14 kDa secretory PLA2 inhibitor SB 203347 (up to 5 microM), indicating that the 85 kDa cPLA2 is the PLA2 responsible for AA release in response to receptor-mediated agonists. In accord with inhibitor studies, the LPS-mediated phosphorylation of cPLA2 followed the same kinetics as the priming for AA release, and a measurable fMLP-induced translocation of cPLA2 was observed only in primed cells. As with AA release and LTB4 biosynthesis, both the phosphorylation and capacity to translocate cPLA2 were reversed when the preincubation period with LPS was extended to 120 min. These results explain some of the cellular events responsible for the potentiation and subsequent decline of functional responses of human polymorphonuclear leukocytes recruited to inflammatory foci.

The distribution and metabolism of arachidonate-containing phospholipids in cellular nuclei.

The cell nucleus has been identified as a location to which several arachidonic acid-metabolizing enzymes are located in stimulated cells. However, little information exists describing the distribution of arachidonate-containing phospholipids associated with the nucleus or the control of their composition. In this study, nuclei isolated from human monocyte-like THP-1 cells were found to have a distribution of arachidonyl-phospholipids which is markedly different from that of other cellular membranes. THP-1 nuclei which contained 22% of total cellular arachidonate, showed a near equal distribution of arachidonate in 1-acyl-2-arachidonoyl-glycero-3-phosphocholine, 1-acyl-2-arachidonyl-glycero-3-phosphoethanolamine, 1-acyl-2-arachidonoyl-glycero-3-phosphoinositol and 1-alk-1-enyl-2-arachidonoyl-glycero-3-phosphoethanolamine molecular species. In contrast in non-nuclear membranes, arachidonate was located primarily in 1-alk-1-enyl-2-arachidonoyl-glycero-3-phosphoethanolamine molecular species which accounted for approximately half of the arachidonate in all non-nuclear phospholipids. Isolated nuclei were incapable of initially acylating arachidonic acid into their phospholipids in the absence of cellular cytosol. However, they were capable of efficiently remodelling existing arachidonate between phospholipid classes and subclasses. Isolated nuclei contained 25-30% of the cellular activity of CoA-independent transacylase, the key enzyme responsible for arachidonate-phospholipid remodelling. This enzyme is also critical in the control of arachidonate availability following cell stimulation. Given that the cellular distribution of arachidonate is such that nuclei are enriched in donor substrates for the CoA-independent transacylase reaction, that non-nuclear membranes are enriched in acceptor substrates and that nuclei have the enzymatic machinery to remodel arachidonate efficiently, these results suggest that CoA-independent transacylase may be responsible for the remodelling of arachidonate not only between different phospholipid species within the same organelles but also between different sub-cellular compartments.

Dietary supplementation with gamma-linolenic acid alters fatty acid content and eicosanoid production in healthy humans.

To understand the in vivo metabolism of dietary gamma-linolenic acid (GLA), we supplemented the diets of 29 volunteers with GLA in doses of 1.5-6.0 g/d. Twenty-four subjects ate controlled eucaloric diets consisting of 25% fat; the remaining subjects maintained their typical Western diets. GLA and dihomo-gamma-linolenic acid (DGLA) increased in serum lipids of subjects supplemented with 3.0 and 6.0 g/d; serum arachidonic acid increased in all subjects. GLA supplementation with 3.0 and 6.0 g/d also resulted in an enrichment of DGLA in neutrophil phospholipids but no change in GLA or AA levels. Before supplementation, DGLA was associated primarily with phosphatidylethanolamine (PE) of neutrophil glycerolipids, and DGLA increased significantly in PE and neutral lipids after GLA supplementation. Extending the supplementation to 12 wk did not consistently change the magnitude of increase in either serum or neutrophil lipids in subjects receiving 3.0 g/d. After GLA supplementation, A23187-stimulated neutrophils released significantly more DGLA, but AA release did not change. Neutrophils obtained from subjects after 3 wk of supplementation with 3.0 g/d GLA synthesized less leukotriene B4 (P < 0.05) and platelet-activating factor. Together, these data reveal that DGLA, the elongase product of GLA, but not AA accumulates in neutrophil glycerolipids after GLA supplementation. The increase in DGLA relative to AA within inflammatory cells such as the neutrophil may attenuate the biosynthesis of AA metabolites and may represent a mechanism by which dietary GLA exerts an anti-inflammatory effect.

Inhibitors of coenzyme A-independent transacylase induce apoptosis in human HL-60 cells.

ET-18-O-CH3 (1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine) is an antiproliferative agent, blocking the growth of cancer cells both in vitro and in vivo. However, there is controversy regarding the mechanism leading to its antiproliferative effects. CoA-independent transacylase (CoA-IT) is an enzyme that remodels arachidonate between specific phospholipid donor and acceptor molecules in a variety of mammalian cells. ET-18-O-CH3 was found to be a potent inhibitor of CoA-IT (IC50, 0.5 microM), and kinetic analysis revealed that its inhibition was competitive with the lyso-phospholipid substrate. The goal of the current study was to explore the connection between inhibition of CoA-IT and antiproliferative effects using several structurally distinct inhibitors of CoA-IT. ET-18-O-CH3 and other inhibitors of CoA-IT were found to inhibit cell proliferation and thymidine incorporation into the DNA, as well as to induce apoptosis in human HL-60 monocytic leukemia cells. The mechanism of apoptosis induced by ET-18-O-CH3 appeared to be different from that induced by tumor necrosis factor; the former failed to activate NF-kappa B, whereas tumor necrosis factor did. Closer examination of the pharmacology of apoptosis in this model revealed that compounds that were structurally related to CoA-IT inhibitors, but lacked CoA-IT inhibitory activity, also failed to induce apoptosis. In addition, compounds that inhibited other enzymes that participate in arachidonic acid metabolism, cyclooxygenase, 5-lipoxygenase and phospholipase A2, did not induce apoptosis. Taken together, these results demonstrate that inhibition of CoA-IT can be linked to blockade of proliferation and the induction of apoptosis in HL-60 cells.

Relationship between arachidonate--phospholipid remodeling and apoptosis.

Our previous studies reveal that three structurally distinct inhibitors of the enzyme CoA-independent transacylase, including the antiproliferative alkyllysophospholipid ET-18-O-CH3, induce programmed cell death (apoptosis) in the promyelocytic cell line HL-60. The objective of the current study was to better elucidate the mechanism responsible for apoptosis. CoA-IT is an enzyme believed to be responsible for the remodeling of long chain polyunsaturated fatty acids like arachidonate between the phospholipids of mammalian cells. The chronic (24-48 h) treatment of HL-60 cells with all three CoA-IT inhibitors resulted in the inhibition of the remodeling of labeled arachidonate from choline- into ethanolamine-containing phospholipid molecular species. GC-MS analysis of the fatty acids in phospholipids revealed that CoA-IT inhibitor treatment induced a marked loss of arachidonate-containing phosphatidylethanolamine and an increase in arachidonate-containing phosphatidylcholine. This redistribution was specific to arachidonate since the mass distribution of linoleic acid in glycerolipids was not affected. In spite of the dramatic redistribution of arachidonate, the total cellular arachidonate content was not altered nor was the relative distribution of total phospholipid classes. The increase of arachidonate in phosphatidylcholine was specifically due to an increase in 1-acyl-2-arachidonoyl-sn-glycero-3-phosphocholine species, whereas the loss of arachidonate in PE was from both 1-acyl- and 1-alk-1-enyl-2-arachidonoyl-sn-glycero-3-phosphoethanolamine species. The incubation of cells with exogenous arachidonic acid or ethanolamine did not reverse the inhibition of proliferation induced by CoA-IT inhibitor treatment. Incubation with CoA-IT inhibitors also induced the characteristic cytoplasmic and nuclear changes associated with apoptosis as assessed by transmission electron microscopy and DNA fragmentation as determined by flow cytometry. Taken together, these data show that apoptosis in HL-60 cells, induced by blocking arachidonate-phospholipid remodeling, is correlated with a redistribution of arachidonate in membrane phospholipids and suggest that such alterations represent a signal which controls the capacity of cells to proliferate.

Priming of human peripheral blood mononuclear cells with lipopolysaccharides for enhanced arachidonic acid release and leukotriene synthesis.

In a previous study, we have shown that the ability of lipopolysaccharides (LPS) to prime isolated neutrophils for enhanced leukotriene B4 (LTB4) synthesis was dependent on the presence of plasma and involved the CD 14 antigen. In the present study, we have investigated the priming of human peripheral blood mononuclear cells (PBMC) with LPS for the subsequent release and metabolism of arachidonic acid. When PBMC were incubated with LPS for up to 2 h or when freshly isolated PBMC were stimulated with N-formyl-methionyl-leucyl-phenylalanine (fMLP) or with LPS alone, little or no synthesis of 5-lipoxygenase products nor arachidonic acid liberation were detected. However, the preincubation of PBMC with LPS for as little as 5 min primed cells for the subsequent synthesis of LTB4 upon stimulation with fMLP. Maximal priming was observed following a 15-min preincubation period and the priming effect was transient as cells preincubated with LPS for 90 min or more were no longer primed for leukotriene synthesis. Monocytes were found to be responsible for the enhanced response to fMLP since purified lymphocytes did not produce LTB4 nor LTC4 in contrast to monocyte-enriched suspensions. The priming for leukotriene synthesis coincided with an increased capacity for the release of free arachidonic acid as measured by mass spectrometry; LPS-primed cells released 8-15 times more arachidonic acid than unprimed cells within 1 min of stimulation with fMLP. Priming was observed with as little as 0.001-0.01 microg LPS/mL when cells were incubated in the presence of 10% autologous plasma. Interestingly, in the absence of plasma, priming was only observed at LPS concentrations of 0.1 microg/mL or greater. Pretreatment of cells with anti-CD14 antibodies significantly decreased the priming effect observed with 0.01 microg/mL LPS but did not affect priming with 1 microg/mL LPS. These results indicate that the priming of human PBMC with LPS for the subsequent synthesis of arachidonic acid metabolites via the 5-lipoxygenase pathway is dependent on plasma and CD14 at lower concentrations of LPS (0.001-0.01 microg/mL) but not at LPS concentrations of 0.1 microg/mL or greater.

Metabolism of gammalinolenic acid in human neutrophils.

Gammalinolenic acid (GLA), when provided as a dietary supplement, has been reported to improve clinical symptoms of several inflammatory disorders. The goal of the current study was to examine the metabolism of GLA and its relationship to arachidonic acid (AA) in the human neutrophil. Initial studies indicated that neutrophils provided GLA in vitro rapidly elongate it (by two carbons) to dihomogammalinolenic acid (DGLA). The bulk of this newly formed DGLA is incorporated into neutral lipids and specifically triacylglycerides. Neutrophils from volunteers supplemented with GLA as borage oil also had elevated quantities of DGLA but not GLA, when compared with neutrophils from volunteers not consuming the GLA supplement. To determine whether DGLA could be mobilized from cellular glycerolipids, neutrophils were stimulated with ionophore A23187 and fatty acid levels were determined. DGLA and AA were both released during stimulation, and the quantities of DGLA mobilized increased threefold after in vitro GLA supplementation. Exogenously provided DGLA was converted to one major metabolite during cell stimulation; this product migrated on reverse-phase HPLC with the 15-lipoxygenase product, 15-hydroxy-eicosa-trienoic acid (15-HETre). Both 15-HETre and DGLA (provided exogenously) inhibited the formation of leukotriene B4, (LTB4) and 20-hydroxy-leukotriene B4 (20-OH-LTB4). The IC50 for 15-HETre inhibition of both LTR, and 20-OH-LTB4 in A23187-stimulated neutrophils was 5 microM. This inhibition could be reversed by removing the compounds from the cells. Taken together, these data reveal that there are enzymes within the human neutrophil that metabolize GLA or its elongation product DGLA, and that the metabolism of GLA and AA may interact at a number of critical junctures.

Evidence that dietary arachidonic acid increases circulating triglycerides.

Male Syrian hamsters and male CD-1 mice were fed diets supplemented with ethyl esters of oleic, linoleic, arachidonic, and eicosapentaenoic acids (1.1-5%, w/w) for 3-4 wk. Plasma and serum triglycerides were significantly higher in the arachidonic acid-supplemented animals compared to those in the other supplementation groups. Changes in serum insulin and glucose levels did not appear to be related to the changes in circulating triglycerides observed in the arachidonic acid-supplemented group. These data indicate that dietary arachidonic acid elevates circulating triglyceride levels compared to other unsaturated fatty acids in hamsters and mice by unknown mechanisms.

Dietary n - 3 polyunsaturated fatty acids modify Syrian hamster platelet and macrophage phospholipid fatty acyl composition and eicosanoid synthesis: a controlled study.

The aim of this study was to determine the effects of varying intakes of dietary n - 3 polyunsaturated fatty acids (PUFA) on the fatty acyl composition and arachidonic acid metabolite synthesis of platelets and macrophages in Syrian hamsters consuming diets that were strictly controlled for n - 6 PUFA content. Animals consumed highly controlled diets which were not supplemented with n - 3 PUFA (control) or supplemented with 0.4%, 0.8% or 2% (w/w) n - 3 fatty acids. The content of n - 3 PUFA in cellular phospholipids increased progressively with the intake of n - 3 PUFA, while n - 6 PUFA, including arachidonic acid, decreased despite the constant intake of 18:2(n - 6); this latter effect was more substantial in macrophages than in platelets. The synthesis by stimulated macrophages of prostaglandin E2, 6-keto-prostaglandin F1 alpha, thromboxane B2 and 11- and 15-hydroxyeicosatetraenoic acids decreased with the intake of 0.8% n - 3 PUFA to 30-50% of the control values. Little effect of diets on platelet aggregation and eicosanoid synthesis was observed reflecting the limited effect on platelet arachidonic acid content. The synthesis of 12-hydroxyeicosapentaenoic acid by stimulated platelets increased with n - 3 PUFA consumption in a dose-dependent fashion. Circulating triacylglycerols and HDL-cholesterol were decreased only in animals consuming 2% n - 3 PUFA. The strict control of n - 6 PUFA intake allows the determination of the effects of n - 3 PUFA intake on the measured parameters without confounding effects of other dietary lipids.

Priming for the synthesis of 5-lipoxygenase products in human blood ex vivo by human granulocyte-macrophage colony-stimulating factor and tumor necrosis factor-alpha.

BACKGROUND: Previous studies reported the priming effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor-alpha (TNF alpha) on leukotriene synthesis by isolated polymorphonuclear leukocytes; however, little is known as of yet of these biologic effects of the two cytokines in a physiologic environment. EXPERIMENTAL DESIGN: In this study, we investigate the effects of GM-CSF and TNF alpha on the synthesis of 5-lipoxygenase (5-LO) products in heparinized blood stimulated ex vivo, using reverse phase high performance liquid chromatography analysis of deproteinized plasma samples. RESULTS: Stimulation of blood with f-Met-Leu-Phe resulted in the accumulation of up to 30 pmol of 5-LO products/ml of plasma. Preincubation of blood with 100 pM GM-CSF or 1.2 nM (200 units/ml) TNF alpha for 30 minutes at 37 degrees C before stimulation with f-Met-Leu-Phe resulted in a marked enhancement (> 5-fold) of the synthesis of leukotriene B4 and 5(S)-hydroxyeicosatetraenoic acid, which were formed in equivalent amounts. GM-CSF and TNF alpha priming activities were detectable at concentrations as low as 3 pM and 6 pM (1 unit/ml), respectively. The preincubation times required for optimal priming by GM-CSF and TNF alpha were different (40 and 10 minutes, respectively), and the effects of the two cytokines on leukotriene B4 and 5(S)-hydroxyeicosatetraenoic acid synthesis were additive, suggesting different priming mechanisms. The synthesis of 5-LO products in primed blood was also induced by platelet-activating factor, the complement fragment C5a, the particulate stimulus zymosan, and the ionophore A23187, but not by interleukin-8. Polymorphonuclear leukocytes and mononuclear cells accounted for 80% and 20% of the synthesis of 5-LO products, respectively. CONCLUSIONS: These data demonstrate that GM-CSF and TNF alpha exert very potent priming effects on the biosynthesis of 5-LO products in whole blood stimulated by various stimuli and strongly support that these cytokines could be important modulators of lipid mediator synthesis in physiologic and pathophysiologic conditions.

Reverse-phase high-performance liquid chromatography analysis of arachidonic acid metabolites in plasma after stimulation of whole blood ex vivo.

Following the stimulation of heparinized blood ex vivo, aliquots of plasma were denatured with organic solvents containing the internal standards prostaglandin (PG) B2 and 19-hydroxy-PGB2. Precipitated material was removed by centrifugation and the supernatants were directly analyzed by reverse-phase HPLC with on-line extraction and uv detection. Stimulation of blood with A23187 lead to the formation of both leukocyte and platelet arachidonic acid metabolites as the 5-lipoxygenase products leukotriene (LT) B4, 5-hydroxy-eicosatetraenoic acid (5-HETE), 20-hydroxy-LTB4 and 20-carboxy-LTB4, the 12-lipoxygenase product 12-HETE, and the cyclooxygenase product 12-hydroxy-heptadecatrienoic acid (HHTrE) were detected in plasma; in some plasma samples LTC4 and/or LTE4 were also detected. Stimulation of blood with zymosan lead to the synthesis of LTB4 and 5-HETE as major products and of 12-HETE. Recoveries of 20-hydroxy-LTB4, LTB4, 5-HETE, 12-HETE, and HHTrE added to plasma were high (> or = 90%) while those of LTC4 and LTE4 were lower (50-70%); however, washing of the precipitated protein pellet resulted in > 90% recoveries for all metabolites including the cysteinyl-leukotrienes. Levels of arachidonic acid metabolites in native plasma samples stored at -20 degrees C were stable for at least 28 days, while significant loss of material was observed over the same period of time in denatured plasma samples. Finally, we made the critical observation that the capacity for A23187- (but not zymosan-, ionomycin-, or LPS and FMLP-) induced arachidonic acid metabolite synthesis in blood decreased by 80% within 1 h of blood collection.(ABSTRACT TRUNCATED AT 250 WORDS)