The omega-hydroxylation of arachidonic acid by human polymorphonuclear leukocytes

Incubations of [1-14C]arachidonic acid with unstimulated human polymorphonuclear leukocytes resulted in the formation of four new metabolites in a previously described reverse-phase HPLC system. Three of these metabolites were largely suppressed in a CO/O2 (80/20, by vol.) atmosphere indicating a cytochrome-P450-dependent monooxygenase reaction. In agreement with this assumption is their NADPH/O2-dependent formation in the microsomal fraction. One metabolite was identified by gas chromatography/mass spectrometry analysis as omega-hydroxy-arachidonic acid and the two others were secondary products identified as omega-carboxy-arachidonic acid and 5,20-dihydroxy-E,Z,Z,Z-6,8,11,14-eicosatetraenoic acid. Since the affinity for arachidonate of the omega-monooxygenase was quite low and the presence of LTB4 suppressed the omega-hydroxylation of arachidonate, we conclude that the known LTB4 omega-monooxygenase is responsible for the formation of omega-hydroxy-arachidonate. It is unlikely, however, that significant concentrations of these metabolites are formed by activated polymorphonuclear leukocytes in vivo. The fourth metabolite remains tightly associated with the leukocytes but has not been further characterized.     

Metabolism of platelet-activating factor by arachidonic acid-depleted rat polymorphonuclear leukocytes

1-O-[3H]Alkyl-2-acetyl-sn-glycero-3-phosphocholine ([3H]PAF) and 1-O-[3H]alkyl-2-lyso-sn-glycero-3-phosphocholine ([3H]lyso-PAF) when incubated with rat polymorphonuclear leukocytes (PMN) were rapidly metabolized to 1-O-[3H]alkyl-2-acyl-sn-glycero-3-phosphocholine ([3H]alkyl-acyl-GPC) containing long chain acyl groups in the sn-2 position. The specificity and the absolute requirements of arachidonate (20:4) for acylation into PAF and lyso-PAF were investigated by comparing the rate of [3H]PAF and [3H]lyso-PAF metabolism by control rat PMN with that by rat PMN depleted of 20:4. Comparable rates of metabolism of [3H]PAF and [3H]lyso-PAF by both control and 20:4-depleted PMN were observed at all the concentrations of PAF and lyso-PAF studied. The nature of the fatty acyl group incorporated into the sn-2 position of the [3H]alkyl-acyl-GPC formed was analyzed by argentation chromatography. Dienoic fatty acids were the major fatty acid incorporated into the alkyl-acyl-GPC by both control and 20:4-depleted PMN at all the incubation times studied. At 3 min of incubation with [3H]PAF and [3H]lyso-PAF, control PMN had small but significant amounts of [3H]alkyl-acyl-GPC containing tetraenoic fatty acids, the concentration of which gradually increased as the incubation time progressed. On the other hand, under similar conditions, 20:4-depleted PMN had only trace amounts of the [3H]alkyl-acyl-GPC with tetraenoic fatty acid and the concentration of which remained at the low level throughout the incubation time. At 3 min of incubation, the 20:4-depleted PMN had small but significant amounts of [3H]alkyl-acyl-GPC with saturated fatty acids, the amount of which declined by 10 min and remained at that level as the incubation time progressed. While the concentration of [3H]alkyl-acyl-GPC with dienoic fatty acids in the 20:4-depleted cells gradually increased with the progress of incubation time, these molecular species of GPC in the control PMN remained more or less constant. In spite of a very high concentration (equivalent to that of 20:4 in control PMN) of eicosatrienoic acid (20:3 delta 5,8,11) in the 20:4-depleted PMN, no significant amounts of [3H]alkyl-acyl-GPC with trienoic fatty acid were formed by these cells. The rate of metabolism of [3H]PAF and [3H]lyso-PAF by the resident macrophages isolated from control and 20:4-depleted rats was similar.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of fatty acid anilides on the generation of arachidonic acid by human polymorphonuclear leukocytes

The addition of oleoylanilide or linoleylanilide to human polymorphonuclear leukocytes induces a time- and dose-dependent generation of arachidonic acid. Half-maximal effect is caused by a dose of 0.2 mg linoleylanilide/ml. Fatty acid anilides also produce a time- and dose-dependent inhibition of the synthesis of triacylglycerol. Half-maximal effect is caused by 1 microgram linoleylanilide/ml. These results indicate that fatty acid anilides, which have been found in the illegal cooking oil which intoxicated thousands of Spaniards, alter lipid metabolism in human polymorphonuclear leukocytes.     

Effect of alcohols on arachidonic acid metabolism in murine mastocytoma cells and human polymorphonuclear leukocytes

The effects of alcohols on the formation of leukotrienes, 5-HETE and prostaglandin D2 in mastocytoma cells and human neutrophils were studied. In murine mastocytoma cells, alcohols appear to have at least two different effects on the production of these arachidonic acid metabolites. At low levels of cellular arachidonic acid achieved after stimulation with calcium ionophore A23187 or addition of low levels of exogenous arachidonic acid, alcohols appear to have a general inhibitory effect on the production of lipoxygenase metabolites. In the presence of higher concentrations of cellular arachidonic acid, ethanol and methanol stimulated the production of lipoxygenase metabolites, but had no large stimulatory effect on the cyclo-oxygenase metabolite, prostaglandin D2. Under these conditions, n-propanol and t-butanol have inhibitory effects on leukotriene production. Human neutrophils are less sensitive to ethanol than mastocytoma cells, but stimulatory effects were still found at high ethanol concentrations (220-430 mM).     

The effect of dietary eicosapentaenoic acid on arachidonic acid incorporation and metabolism in rat leukocytes

Arachidonic Acid (AA) released from membrane phospholipids by phospholipase A2 during cell activation is the major polyunsaturated fatty acid precursor in mammals for the cyclooxygenase and lipoxygenase pathways. Eicosapentaenoic acid (EPA), a major polyunsaturated fatty acid in fish oils competes with AA for these enzymes. The resulting products from EPA are generally less potent than the corresponding AA metabolites which may explain the beneficial effects of this oil in reducing thrombotic and inflammatory responses. This study compares the incorporation of 14C-AA into leukocyte phospholipids and its release and metabolism by the cyclooxygenase and lipoxygenase pathways in rats fed a 'Max EPA' fish oil rich diet (EPA group) and a hydrogenated coconut/safflower oil control diet. More than 75% of radiolabel was incorporated into leukocytes with no difference seen between dietary groups. Upon stimulation with calcium ionophore, the EPA group released significantly more radiolabelled AA than the control group. The EPA diet showed a significant increase in the formation of 5-hydroxyeicosatetraenoic acid and 6-keto-prostaglandin F1 alpha but no difference was seen in leukotriene B4 formation. The majority of radiolabel released was free AA, this being significantly higher in the EPA group than in the control. The percentage of radiolabel remaining after stimulation in phosphatidylglycerol, phosphatidylethanolamine and neutral lipids was significantly less in EPA fed rats. As the release and metabolism of endogenous AA may not be the same as 14C-AA, these results do not necessarily indicate that the mass of AA available for eicosanoid biosynthesis has been altered by the EPA diet.     

Effects of endotoxin on oxidative metabolism of polymorphonuclear leukocytes

The influence of endotoxin on rat polymorphonuclear leucocytes (PMN) ability to generate oxygen free radicals (OFR) has been studied by chemiluminescence method. PMNs derived from intact animals were used as a control. PMNs derived from animals with 1.5 h endotoxemia increased OFR production after stimulation by latex. In contrast, PMNs derived from intact animals and preincubated with endotoxin for 1.5 h decreased OFR production after stimulation bw latex. It was proposed that stimulating effect of endotoxin on PMNs in vivo was mediated by plasma components.     

Effect of various lipoxygenase metabolites of arachidonic acid on degranulation of polymorphonuclear leukocytes

Lipoxygenase metabolites of guinea pig peritoneal polymorphonuclear leukocytes stimulated with 10 microM A23187 plus arachidonic acid were isolated and identified. These metabolites were compared with each other and to chemically synthesized arachidonate metabolites for their ability to stimulate leukocyte degranulation. 5(S),12(R)-Dihydroxy-6,8,10-(cis/trans/trans)14-cis-eicosatetraenoic acid (leukotriene B4) produced a significant release of lysozyme, but not beta-glucuronidase or beta-N-acetylglucosaminidase at low concentrations (EC50 = 6.5 x 10(-9) M), while the leukocyte nonenzymatically generated 5,12-or 5,6-dihydroxyeicosatetraenoic acids had no effect at these concentrations. Higher concentrations (1--10 microM) of all the dihydroxyeicosatetraenoic acids, 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) and its hydroperoxy precursor stimulated significant lysozyme release which was greater than that produced by 15-hydroxy-5,8,11-13-eicosatetraenoic acid, arachidonic acid, or its acetylene analogue, 5,8,11,14-eicosatetraynoic acid. Micromolar concentrations of leukotriene B4 and 5-HETE also stimulated significant release of beta-N-acetylglucosaminidase above controls, but not beta-glucuronidase. These results suggest that leukotriene B4 may play a role in regulating the release of certain granule-bound enzymes from polymorphonuclear leukocytes.     

Methanol is a potent inhibitor of the metabolism of endogenous arachidonic acid by elicited rat peritoneal leukocytes

The effect of methanol on the ability of elicited rat peritoneal leukocytes to metabolise endogenous and exogenous arachidonic acid was studied using 2H8-arachidonic acid as the source of exogenous arachidonic acid and calcium ionophore A23187 as the lipoxygenase stimulus. As the methanol concentration increased from 0 to 992 mM there was a slight decrease in the total amount of LTB4 and related compounds formed, however examination of the ratio of undeuterated to deuterated LTB4 formed revealed that as the methanol concentration increased from 0 to 992 mM, the percentage of undeuterated LTB4 present decreased significantly from 57 +/- 9% to 2 +/- 1%. Methanol interferes with the ability of these cells to utilise endogenous arachidonic acid even in the presence of the powerful stimulus calcium ionophore A23187 thus allowing the facile biosynthesis of a range of deuterium labelled arachidonic acid metabolites.     

Transformation of arachidonic acid by rabbit polymorphonuclear leukocytes. Formation of a novel dihydroxyeicosatetraenoic acid.

A new metabolite of arachidonic acid, 5-D-(S),12-D-(R)-dihydroxy-6,8,10,14-eicosatetraenoic acid, was found upon incubation of the fatty acid with a suspension of rabbit peritoneal polymorphonuclear leukocytes collected 4 h after injection of glycogen into the peritoneal cavity. The yield of the dihydroxy acid was 0.5 to 2%. The compound possesses three conjugated double bonds and was found to be stereochemically pure at C-5 and C-12. Incubation of the cells with 8,11,14-eicosatrienoic acid did not lead to the formation of the analogous triunsaturated dihydroxy acid.     

Methanol is a potent inhibitor of the metabolism of endogenous arachidonic acid by elicited rat peritoneal leukocytes

The effect of methanol on the ability of elicited rat peritoneal leukocytes to metabolise endogenous and exogenous arachidonic acid was studied using ²H₈-arachidonic acid as the source of exogenous arachidonic acid and calcium ionophore A23187 as the lipoxygenase stimulus. As the methanol concentration increased from 0 to 992 mM there was a slight decrease in the total amount of LTB₄ and related compounds formed, however examination of the ratio of undeuterated to deuterated LTB₄ formed revealed that as the methanol concentration increased fro 0 to 992 mM, the percentage of undeuterated LTB₄ present decreased significantly from 57 ± 9% to 2 ± 1 %. Methanol interfers with the ability of these cells to utilise endogenous arachidonic acid even in the presence of the powerful stimulus calcium ionophore A23187 thus allowing the facile biosynthesis of a range of deuterium labelled arachidonic acid metabolites.     

Evidence for lipoxin formation by bovine polymorphonuclear leukocytes via triple dioxygenation of arachidonic acid

Incubation of bovine polymorphonuclear leukocytes (PMNs) with arachidonic acid leads to the formation of four lipoxins. The same lipoxins are also formed upon incubation of bovine PMNs with 5(S)-hydroperoxy-6-trans-8,11,14-cis-eicosatetraenoic acid, 5-hydroxy-6-trans-8,11,14-cis-eicosatetraenoic acid, 5(S)-hydroperoxy, 15(S)-hydroxy-6,13-trans-8,11-cis-eicosatetraenoic acid or 5(S),15(S)-dihydroxy-6,13-trans-8,11-cis-eicosatetraenoic acid. A 5,6-epoxide as intermediate in lipoxin formation in the bovine PMN is highly improbable because the 5-hydroxy compounds are as good substrates as the 5-hydroperoxy compounds. Moreover, the two main lipoxins were found to coelute with the two lipoxins produced via a triple dioxygenation of arachidonic acid by soybean lipoxygenase-1. Hence the bovine PMN is the first cell for which evidence is presented that the formation of lipoxins proceeds mainly via triple dioxygenation and not via 15-hydroxy-leukotriene A4 as is proposed for human and porcine PMNs.     

Differences in arachidonic acid release, metabolism and leukotriene B4 synthesis in human polymorphonuclear leukocytes activated by different stimuli

Products of the 5-lipoxygenase pathway were analyzed after different stimuli in human polymorphonuclear leukocytes prelabeled with 3H-arachidonic acid. Upon stimulation with the Ca2+ ionophore, A23187, polymorphonuclear leukocytes generate 118.2 +/- 18 pg [3H]dihydroxyeicosatetraenoic acids (diHETEs, including 3H-leukotriene B4 and its 6-trans-stereoisomers), after exposure to serum coated zymosan (35.8 +/- 9 pg) and N-fMet-Leu-Phe (39.5 +/- 9 pg). Conversion of 3H-arachidonic acid paralleled its release after A23187 and fMet-Leu-Phe exposure leaving only 13.8 +/- 7% and 13.6 +/- 3% of the released 3H-arachidonic acid unmetabolized, respectively. In contrast, after stimulation with serum-coated zymosan only a small fraction of the released 3H-arachidonate was converted to 5-lipoxygenase products leaving 73.0 +/- 5% of the released 3H-arachidonic acid unmetabolized. In parallel, leukotriene B4 synthesis was studied in unlabeled polymorphonuclear leukocytes, resulting in 40 +/- 15 ng upon A23187 stimulation, 4 +/- 0.9 ng upon stimulation with fMet-Leu-Phe and 1.8 +/- 0.9 ng after serum-coated zymosan, showing a different ratio of leukotriene B4 to 3H-diHETE for A23187 in contrast to serum-coated zymosan and fMet-Leu-Phe. These results indicate that the coupling between the release of the precursor fatty acid and the metabolism via the 5-lipoxygenase pathway differs greatly between different stimuli.     

Inhibition of the formation of 5-hydroxy-6,8,11,14-eicosatetraenoic acid from arachidonic acid in polymorphonuclear leukocytes by various coumarins

The effects of various coumarins (i.e. esculetin, daphnetin and fraxetin) on the formation of the 5-lipoxygenase product, 5-HETE, and the cyclooxygenase product, HHT, were studied. Esculetin (6,7-dihydroxycoumarin) was found to inhibit the formation of 5-HETE more strongly than HHT; its concentrations for 50% inhibition (IC50) were 1.46 +/- 1.02 microM for the formation 5-HETE and 57.3 +/- 17.3 microM for the formation of HHT. Daphnetin (7,8-dihydroxycoumarin) and fraxetin (6-methoxy-7,8-dihydroxycoumarin) also inhibited the formation of the 5-lipoxygenase product, 5-HETE, and the cyclooxygenase product, HHT; their IC50 values were, respectively, 6.90 +/- 2.07 microM and 2.57 +/- 0.088 microM for the formation of 5-HETE and 139.0 +/- 30.0 microM and 532.5 +/- 33.0 microM for the formation of HHT. The monohydroxy coumarin derivatives umbelliferone (7-hydroxycoumarin) and scopoletin (6-methoxy-7-hydroxycoumarin) and the coumarin glucosides fraxin (6-methoxy-7,8-dihydroxycoumarin 8-O-D-glucoside) and esculin (6,7-dihydroxycoumarin 6-O-D-glucoside) also inhibited the formation of 5-HETE, though less strongly. 4-Hydroxycoumarin and coumarin had no effect on either 5-lipoxygenase or cyclooxygenase at concentrations of up to 1 mM. Esculetin inhibited the formation of 5-HETE noncompetitively. In contrast, the dimethoxycoumarin fraxidin (6,8-dimethoxy-7-hydroxycoumarin) inhibited the formation of HHT more strongly than the formation of 5-HETE at a concentration of 1 mM.     

Platelet-activating factor and leukotriene biosynthesis is inhibited in polymorphonuclear leukocytes depleted of arachidonic acid

Rat peripheral or elicited polymorphonuclear leukocytes 90% deficient in arachidonic acid incorporate, after stimulation with the calcium ionophore A23187, 86% less acetate into platelet-activating factor than control. The total amount of platelet-activating factor in the ionophore stimulated elicited polymorphonuclear leukocytes deficient in arachidonate, measured by gas chromatography-negative ion chemical ionization mass spectrometry, was 84% less than that of control. The mass spectrometry also revealed the presence of various molecular species of platelet-activating factor ranging from 1-O-tetradecyl to 1-O-nonadecyl forms in both the deficient and control cells. However, the 1-O-hexadecyl was the predominate molecular species representing 79 and 96% of the total platelet-activating factor in the respective deficient and control cells. Other molecular species were less than 1.5 and 8.5% of the total for control and deficient polymorphonuclear leukocytes, respectively. Leukotriene B4 formation was also inhibited by 90% in the deficient cells. Both platelet-activating factor and leukotriene B4 biosynthesis could be partially restored in arachidonic acid-deficient cells by prelabeling the cells with arachidonate. This represents the first dietary link with platelet-activating factor biosynthesis.     

Characterization and separation of the arachidonic acid 5-lipoxygenase and linoleic acid omega-6 lipoxygenase (arachidonic acid 15-lipoxygenase) of human polymorphonuclear leukocytes

The cytosolic fraction of human polymorphonuclear leukocytes precipitated with 60% ammonium sulfate produced 5-lipoxygenase products from [14C]arachidonic acid and omega-6 lipoxygenase products from both [14C]linoleic acid and, to a lesser extent, [14C]- and [3H]arachidonic acid. The arachidonyl 5-lipoxygenase products 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HPETE) and 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) derived from [14C]arachidonic acid, and the omega-6 lipoxygenase products 13-hydroperoxy-9,11-octadecadienoic acid (13-OOH linoleic acid) and 13-hydroxy-9,11-octadecadienoic acid (13-OH linoleic acid) derived from [14C]linoleic acid and 15-hydroxyperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE), and 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE) derived from [14C]- and [3H]arachidonic acid were identified by TLC-autoradiography and by reverse-phase high-performance liquid chromatography (RP-HPLC). Products were quantitated by counting samples that had been scraped from replicate TLC plates and by determination of the integrated optical density during RP-HPLC. The arachidonyl 5-lipoxygenase had a pH optimum of 7.5 and was 50% maximally active at a Ca2+ concentration of 0.05 mM; the Km for production of 5-HPETE/5-HETE from arachidonic acid was 12.2 +/- 4.5 microM (mean +/- S.D., n = 3), and the Vmax was 2.8 +/- 0.9 nmol/min X mg protein (mean +/- S.D., n = 3). The omega-6 linoleic lipoxygenase had a pH optimum of 6.5 and was 50% maximally active at a Ca2+ concentration of 0.1 mM in the presence of 5 mM EGTA. When the arachidonyl 5-lipoxygenase and the omega-6 lipoxygenase were separated by DEAE-Sephadex ion exchange chromatography, the omega-6 lipoxygenase exhibited a Km of 77.2 microM and a Vmax of 9.5 nmol/min X mg protein (mean, n = 2) for conversion of linoleic acid to 13-OOH/13-OH linoleic acid and a Km of 63.1 microM and a Vmax of 5.3 nmol/min X mg protein (mean, n = 2) for formation of 15-HPETE/15-HETE from arachidonic acid.     

Metabolism of arachidonic acid in polymorphonuclear leukocytes. Structural analysis of novel hydroxylated compounds.

Arachidonic acid was incubated with rabbit peritoneal polymorphonuclear leukocytes (glycogen-induced) and compounds obtained from ether extractions were fractionated by silicic acid column chromatography. A fraction containing several unidentified metabolites of arachidonic acid was analyzed by reversed phase-high pressure liquid chromatography. The metabolites were esterified and further purified by silicic acid high pressure liquid chromatography. The structures of the pure compounds were elucidated by infrared and ultraviolet spectrometry, ozonolysis, and gas chromatography-mass spectrometry. The following novel compounds were identified: Compound 1, 5S, 12R-dihydroxy-(E,E,E,Z)-6,8,10,14-eicosatetraenoic acid; Compound 2, 5S, 12S-dihydroxy-(E,E,E,Z)-6,8,10,14-eicosatetraenoic acid; Compound 3, 5, 6-dihydroxy-7,9,11,14-eicosatetraenoic acid; Compound 4, a diastereoisomer of the latter. Evidence for the occurrence of the delta-lactone forms of the 5,12-dihydroxy acids is also presented.     

Effect of single oral administrations of non steroidal antiinflammatory drugs to healthy volunteers on arachidonic acid metabolism in peripheral polymorphonuclear and mononuclear leukocytes

The effects of a single oral administration of acetylsalicylic acid (500 mg), indomethacin (50 mg) and piroxicam (40 mg) to healthy volunteers on functional and biochemical parameters of platelets, polymorphonuclear (PMN) and mononuclear (MNL) leukocytes were evaluated. Blood was collected before and two hours after the drug intake and blood cells separated according to conventional techniques. The considered drugs almost completely suppressed the aggregation of platelets, whereas they did not affect either PMN and MNL aggregation. Superoxide anion generation by leukocytes was (PMN), or no effect (MNL) was observed after piroxicam and indomethacin respectively. The formation of arachidonate metabolites via the 5-lipoxygenase pathway by PMN and MNL challenged with 10 microM A23187 was unchanged following aspirin and indomethacin. In this respect a selective increase of 5-HETE and LTC4 synthesis by MNL only was detected after piroxicam administration. The three drugs similarly reduced TXB2 synthesis by platelets and PMN (-80% for aspirin and indomethacin, and -40% for piroxicam). As far as MNL is concerned, aspirin inhibited this metabolite by 80%, while indomethacin reduced it by 40% only. In contrast piroxicam increased TXB2 synthesis by stimulated MNL. It can be concluded that the considered antiinflammatory drugs 1) differently affect the cyclooxygenase enzyme in platelets and leukocytes; 2) at variance with the situation in platelets, the inhibition of thromboxane formation by leukocytes is not related to modifications of cellular function; 3) the formation of arachidonate metabolites via the 5-lipoxygenase pathway is affected by piroxicam only.     

A novel dioxygenation product of arachidonic acid possesses potent chemotactic activity for human polymorphonuclear leukocytes

We have found that a novel dioxygenation product of arachidonic acid, 8(S),15(S)-dihydroxy-5,11-cis-9,13-trans-eicosatetraenoic acid (8,15-diHETE), possesses chemotactic activity for human polymorphonuclear leukocytes comparable to that of leukotriene B4. Authentic 8,15-diHETE, identified by gas chromatography-mass spectrometry, was prepared by treating arachidonic acid with soybean lipoxygenase and was purified by reverse-phase high performance liquid chromatography. Using a "leading front" assay, 8,15-diHETE exhibited significant chemotactic activity at a concentration of 5.0 ng/ml. Maximum chemotactic activity was observed at a concentration of 30 ng/ml. The 8,15-diHETE generated by mixed human leukocytes after stimulation with arachidonic acid and the calcium ionophore, A23187, exhibited quantitatively similar chemotactic activity. Two synthetic all-trans conjugated isomers of 8,15-diHETE, however, were not chemotactic at concentrations up to 500 ng/ml. In contrast to its potent chemotactic activity, 8,15-diHETE (at concentrations up to 10 micrograms/ml) was relatively inactive with respect to its ability to provoke either degranulation or generation of superoxide anion radicals by cytochalasin B-treated leukocytes. Both leukotriene B4 and 8,15-diHETE may be important mediators of inflammation.