The metabolism of leukotriene B4 by peripheral blood polymorphonuclear leukocytes in psoriasis

The formation of leukotriene B4 and its omega-oxidised metabolites has been compared in calcium ionophore-stimulated polymorphonuclear leukocytes, in the absence of exogenous substrate, from fourteen psoriatic subjects and thirteen healthy controls. Although there was no significant difference in the levels of leukotriene B4, the psoriatic cells synthesised significantly greater amounts of omega-oxidation products than control cells. This difference was confirmed in an experiment comparing the time course of formation of the omega-oxidation products of leukotriene B4, under similar conditions, in polymorphonuclear leukocytes from four psoriatic subjects and three healthy controls. The kinetic constants for the metabolism of exogenous leukotriene B4 by 20-hydroxylase were determined by a radiochromatographic enzyme assay in polymorphonuclear leukocytes from three patients with psoriasis and three healthy controls. No significant differences were found in the apparent Km and Vmax values. It is concluded that the increased formation of omega-oxidation products in psoriatic cells may be secondary to increased synthesis of leukotriene B4 by these cells, with consequent increased metabolism, rather than to an inherent abnormality of the 20-hydroxylase system. Further work is needed to determine the kinetics of the enzymes involved in leukotriene B4 synthesis in the psoriatic polymorphonuclear leukocyte, and also to assess the contribution of the leukotriene B4 and omega-oxidation products from polymorphonuclear leukocytes infiltrating the skin to the pathogenesis of the psoriatic lesion.     

Characterization of leukotriene B4 synthesis in canine polymorphonuclear leukocytes.

The synthesis and release of leukotriene B4 (LTB4) from canine polymorphonuclear leukocytes (PMNs) was characterized in terms of incubation time, temperature and effects of calcium ionophore A23187 concentrations. Maximal LTB4 concentrations were determined when canine PMNs were incubated with 10 microM A23187. Increasing LTB4 concentrations were determined through 10 min incubation. The maximal LTB4 concentrations (310 +/- 30 pg LTB4/2.5 x 10(5) cells) determined at 10 min did not change through a 55 min incubation period. Greater LTB4 concentrations were synthesized by canine PMNs at 37 degrees C (268 +/- 12 pg LTB4/2.5 x 10(5) cells) than at 25 degrees C (206 +/- 11 pg LTB4/2.5 x 10(5) cells) or 5 degrees C (59 +/- 3 pg LTB4/2.5 x 10(5) cells). The synthesis of LTB4 in canine PMNs was inhibited by incubation of the cells with either of two known lipoxygenase inhibitors, BWA4C or BW755C. BWA4C inhibited LTB4 synthesis with an approximate IC50 = 0.1 microM, whereas BW755C inhibited LTB4 synthesis with an approximate IC50 = 10 microM. These results indicate canine PMNs have the capability to synthesize large quantities of LTB4 when stimulated with calcium ionophore A23187. Furthermore, the 5-lipoxygenase inhibitors BWA4C, an acetohydroxyamic acid, and BW755C, a phenyl pyrazoline, can readily inhibit LTB4 synthesis in canine PMNs.     

Catecholamines decrease leukotriene B4 and increase thromboxane B2 synthesis in A23187-stimulated human whole blood.

Catecholamines (adrenaline, dopamine, isoprenaline, noradrenaline) and caffeic acid (catecholic compound without adrenergic receptor activity) decreased leukotriene (LT)B4 synthesis in A23187-stimulated human whole blood. Salbutamol, a non-catecholic beta 2-adrenergic agonist, did not influence LTB4 synthesis. Catecholamines stimulated thromboxane (TX)B2 synthesis with a concomitant inhibition of LTB4 synthesis; caffeic acid and salbutamol did not stimulate TXB2 synthesis. These results, obtained in A23187-stimulated whole blood, which also takes into account the complex interaction between different cell types, are similar to our previous results with polymorphonuclear leukocytes. Catecholamines show an opposite effect on lipoxygenase and cyclooxygenase pathways, which may give rise to a marked change in LT/TX ratio in physiological or pathological conditions where sufficient concentrations of catecholamines are present.     

Inhibition of leukotriene B4 synthesis in human polymorphonuclear leukocytes after exposure to meningococcal lipopolysaccharide

Polymorphonuclear leukocytes (PMNL) were preincubated in the presence and absence of lipopolysaccharide (LPS) prior to stimulation of arachidonic acid (20:4) metabolism by addition of the divalent cation ionophore, A23187. Analysis of the products by high pressure liquid chromatography showed that LPS inhibited the formation of leukotriene B₄, 5-hydroxy-6,8,11,14- icosatetraenoic acid and 12-hydroxy-5,8,10,14-icosatetraenoic acid by about 70%. In the absence of ionophore, LPS had little effect on the basal synthesis of 20:4 metabolites. Preincubation with LPS also inhibited the formation of the above 3 products in the presence of an excess of exogenous 20:4, suggesting that its action was mediated by the inhibition of lipoxygenases rather than phospholipase.     

Specific binding of leukotriene B4 to receptors on human polymorphonuclear leukocytes

Leukotriene B4 (5(S),12(R)-di-hydroxy-eicosa-6,14-cis-8,10-trans-tetraenoic acid [LTB4]) is a product of the 5-lipoxygenation of arachidonic acid, which elicits human PMN leukocyte chemotactic responses in vitro that are 50% of the maximal level at concentrations of 3 X 10(-9) M to 10(-8) M and are maximal at 2 X 10(-8) M to 10(-7) M. The specific binding of highly purified [3H]LTB4 to human PMN leukocytes was assessed both by extracting the unbound and weakly bound [3H]LTB4 with acetone at -78 degrees C and by centrifuging the PMN leukocytes through cushions of phthalate oil to separate the unbound from bound [3H]LTB4. The levels of total binding of [3H]LTB4 and of nonspecific binding of [3H]LTB4, in the presence of a 1500-fold molar excess of nonradioactive LTB4, were approximately two times higher with the phthalate oil method. Scatchard plots of the concentration dependence of the specific binding (total - nonspecific binding) of [3H]LTB4 to PMN leukocytes were linear for the acetone extraction and phthalate oil methods and revealed dissociation constants of 10.8 X 10(-9) M and 13.9 X 10(-9) M, respectively, and mean of 2.6 X 10(4) and 4.0 X 10(4) receptors per PMN leukocyte. The 5(S),12(S)-all-trans-di-HETE analog of LTB4 and 5-HETE competitively inhibited by 50% the binding of [3H]LTB4 to PMN leukocytes at respective concentrations that evoked half-maximal chemotactic responses, whereas neither N-formyl-methionyl-leucyl-phenylalanine nor chemotactic fragments of C5 inhibited the binding. Human erythrocytes exhibited no specific binding sites for [3H]LTB4. Human PMN leukocytes possess a subset of receptors for LTB4 that are distinct from those specific for peptide chemotactic factors.     

The mechanism of leukotriene B4 export from human polymorphonuclear leukocytes

Recently, we characterized the export of leukotriene (LT) C4 from human eosinophils as a carrier-mediated process (Lam, B. K., Owen, W. F., Jr., Austen, K. F., and Soberman, R. J. (1989) J. Biol. Chem. 264, 12885-12889). To determine whether a similar mechanism regulates the release of leukotriene B4 (LTB4), human polymorphonuclear leukocytes (PMN) were preloaded with LTB4 by incubation with 25 microM leukotriene A4 (LTA4) at 0 degrees C for 60 min. PMN converted LTA4 to LTB4 in a time-dependent manner as determined by resolution of products by reverse-phase high performance liquid chromatography and quantitation by integrated optical density. When PMN preloaded with LTB4 were resuspended in buffer at 37 degrees C for 0-90 s, there occurred a time-dependent release of LTB4 but little formation or release of 20-hydroxy-LTB4 and 20-carboxy-LTB4. When PMN were preloaded with increasing amounts of intracellular LTB4 by incubation with 3.1-50.0 microM LTA4 and were then resuspended in buffer at 37 degrees C for 20 s, there occurred a concentration-dependent and saturable release of LTB4 with a Km of 798 pmol/10(7) cells and a Vmax of 383 pmol/10(7) cells/20 s. The release of LTB4 was temperature-sensitive with a Q10 of 3.0 and an energy of activation of 19.9 kcal/mol. The rate of LTB4 release at 37 degrees C is about 50 times the rate of 20-carboxy-LTB4 release. PMN preloaded with LTB4 and resuspended at 0 degree C for 1-60 min in the presence of 30 microM LTA5 progressively converted LTA5 to LTB5. The rate of LTB4 release at 0 degree C was inhibited over the entire time period, peaking at about 50% at 30 min. These results indicate that the release of LTB4 from PMN is a carrier-mediated process that is distinct from its biosynthesis.     

Superoxide production of human polymorphonuclear leukocytes stimulated by leukotriene B4

Leukotriene B4 stimulated a transient production of superoxide anions (O2-) by human polymorphonuclear leukocytes which continued for only about 1 min. The production was dependent on Ca2+ in the suspending medium and no production was observed without the addition of calcium. The concentrations of leukotriene B4 and calcium for the half-maximal production were about 1 microM and 200 microM, respectively. 8-(N,N,-Diethylamino)-octyl-3,4,5-trimethoxybenzoate (TMB-8), an intracellular calcium antagonist, did not inhibit the O2- production stimulated by leukotriene B4 in the presence of calcium, while N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a calmodulin inhibitor, did. When leukotriene B4 was added to the cells treated with cytochalasin B, the production of O2- was biphasic: an initial rapid phase, followed by a slow one. The slow phase was also dependent on Ca2+ concentrations but it could be induced even without the addition of Ca2+ to the medium. The cells treated with both cytochalasin B and TMB-8 in Ca2+-free medium showed a negligible production of superoxide on addition of leukotriene B4, but the production appeared upon addition of CaCl2. These findings suggest that the superoxide production stimulated by leukotriene B4 is associated with the influx of Ca2+.     

Serum-coated zymosan stimulates the synthesis of leukotriene B4 in human polymorphonuclear leukocytes. Inhibition by cyclic AMP

Addition of serum-treated zymosan particles to a suspension of human peripheral blood polymorphonuclear leukocytes led to the formation of leukotriene B₄. Prostaglandin I₂ and RO20-1724 (an inhibitor of cyclic 3′:5′-nucleotide phosphodiesterase) decreased the synthesis of this compound, indicating that cyclic AMP exerts an inhibitory effect on the formation of leukotriene B₄.     

Investigations of the biological activity of leukotriene A4 in human polymorphonuclear leukocytes

An investigation was undertaken to compare the responses of human neutrophils to the epoxide leukotriene A4 with those elicited by its stable product leukotriene B4 under identical IN VITRO conditions. LTA4 evokes neutrophil responses similar in nature to those induced by LTB4 but at much higher concentrations. Evidence suggests that LTA4 is important primarily for its role as an intermediate rather than for inherent activity.     

The metabolism of leukotriene B4 by peripheral blood polymorphonuclear leukocytes in psoriasis

The formation of leukotriene B₄ and its ω-oxidised metabolites has been compared in calcium ionophore-stimulated polymorphonuclear leukocytes, in the absence of exogenous substrate, from fourteen psoriatic subjects and thirteen healthy controls. Although there was no significant difference in the levels of leukotriene B₄, the psoriatic cells synthesised significantly greater amounts of ω-oxidation products than control cells. This difference was confirmed in an experiment comparing the time course of formation of the ω-oxidation products of leukotriene B₄, under similar conditions, in polymorphonuclear leukocytes from four psoriatic subjects and three healthy controls. The kinetic constants for the metabolism of exogenous leukotriene B₄ by 20-hydroxylase were determined by a radiochromatographic enzyme assay in polymorphonuclear leukocytes from three patients with psoriasis and three healthy controls. No significant differences were found in the apparent K_m and V_max values. It is concluded that the increased formation of ω-oxidation products in psoriatic cells may be secondary to increased synthesis of leukotriene B₄ by these cells, with consequent increased metabolism, rather than to an inherent abnormality of the 20-hydroxylase system. Further work is needed to determine the kinetics of the enzymes involved in leukotriene B₄ synthesis in the psoriatic polymorphonuclear leukocyte, and also to assess the contribution of the leukotriene B₄ and ω-oxidation products from polymorphonuclear leukocytes infiltrating the skin to the pathogenesis of the psoriatic lesion.     

Transduction by leukotriene B4 receptors of increases in cytosolic calcium in human polymorphonuclear leukocytes

The uptake of Quin-2 by human polymorphonuclear (PMN) leukocytes permitted accurate fluorimetric quantification of the cytosolic concentration of intracellular calcium [( Ca+2]in), without altering the expression of the two subsets of leukotriene B4 (LTB4) receptors, as assessed by the binding of [3H]LTB4. Chemotactic concentrations of LTB4 elicited a rapid increase in [Ca+2]in, which reached a peak within 0.6 to 1 min and then decayed back to baseline levels by 6 to 10 min. The maximal increase and the half-maximal increase in [Ca+2]in were achieved by LTB4 at mean concentrations of 5 X 10(-10) M and 2 X 10(-10) M, respectively, where the binding of LTB4 to high-affinity receptors predominates. A rank order of potency of LTB4 greater than 5(S),12(S)-6-trans-LTB4 greater than 12(S)-LTB4 was established for the elicitation of increases in [Ca+2]in, which reflects the binding of the isomers to low-affinity receptors. PMN leukocytes were preincubated with 10(-8) M LTB4 to induce chemotactic deactivation, which eliminates the expression of high-affinity receptors without altering the expression of the low-affinity receptors for LTB4. LTB4 elicited an increase in [Ca+2]in in the deactivated PMN leukocytes with an EC50 of 3 X 10(-8) M, which is similar to the Kd for LTB4 binding to the low-affinity receptors. Two lines of cultured human leukemic cells, IM-9 and HL-60, did not bind LTB4 specifically and did not show any change in [Ca+2]in upon the addition of 3 X 10(-8) M LTB4. The HL-60 human promyelocytic leukemia cell line was induced to differentiate in 1% dimethyl sulfoxide to leukocytes with more mature myelocytic characteristics. Differentiated HL-60 cells expressed an average of 54,000 low-affinity receptors for LTB4 per cell with an average dissociation constant of 7.3 X 10(-8) M and concurrently developed the capacity to respond to LTB4 with an increase in [Ca+2]in. The binding of LTB4 to either high-affinity or low-affinity receptors appears to be sufficient to initiate an increase in [Ca+2]in in human PMN leukocytes and differentiated HL-60 cells. The specificity of LTB4 receptors in transducing maximum increases in [Ca+2]in is determined by the subset of receptors that predominate as a result of the concentration of LTB4 and the state of the responding cells.     

Omega-oxidation is the major pathway for the catabolism of leukotriene B4 in human polymorphonuclear leukocytes

Leukotriene B4 (LTB4), formed by the 5-lipoxygenase pathway in human polymorphonuclear leukocytes (PMN), may be an important mediator of inflammation. Recent studies suggest that human leukocytes can convert LTB4 to products that are less biologically active. To examine the catabolism of LTB4, we developed (using high performance liquid chromatography) a sensitive, reproducible assay for this mediator and its omega-oxidation products (20-OH- and 20-COOH-LTB4). With this assay, we have found that human PMN (but not human monocytes, lymphocytes, or platelets) convert exogenous LTB4 almost exclusively to 20-OH- and 20-COOH-LTB4 (identified by gas chromatography-mass spectrometry). Catabolism of exogenous LTB4 by omega-oxidation is rapid (t1/2 approximately 4 min at 37 degrees C in reaction mixtures containing 1.0 microM LTB4 and 20 X 10(6) PMN/ml), temperature-dependent (negligible at 0 degrees C), and varies with cell number as well as with initial substrate concentration. The pathway for omega-oxidation in PMN is specific for LTB4 and 5(S),12(S)-dihydroxy-6,8,10,14-eicosatetraenoic acid (only small amounts of other dihydroxylated-derivatives of arachidonic acid are converted to omega-oxidation products). Even PMN that are stimulated by phorbol myristate acetate to produce large amounts of superoxide anion radicals catabolize exogenous leukotriene B4 primarily by omega-oxidation. Finally, LTB4 that is generated when PMN are stimulated with the calcium ionophore, A23187, is rapidly catabolized by omega-oxidation. Thus, human PMN not only generate and respond to LTB4, but also rapidly and specifically catabolize this mediator by omega-oxidation.     

Ligand-induced formation of the leukotriene B4 receptor-G protein complex of human polymorphonuclear leukocytes.

The components of the polymorphonuclear leukocyte (PMNL) receptor for leukotriene B4 (LTB4) were examined by Sephacryl S-300 exclusion chromatography of PMNL membrane proteins, which were solubilized before and after the binding of [3H] LTB4. When the PMNL membranes were solubilized in 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) and filtered on Sephacryl S-300 prior to addition of [3H] LTB4, the binding activity was associated with a 65 kD protein. In contrast, the radioactivity of [3H] LTB4 bound to PMNL membranes prior to solubilization was recovered predominantly with a 140 kD protein. When PMNL membranes had been pretreated with pertussis toxin, but not cholera toxin, before the addition of LTB4 and subsequent solubilization, radioactivity was recovered predominantly with the 65 kD protein. The addition of guanylylimidodiphosphate (GMP-PNP), a nonhydrolyzable derivative of guanosine triphosphate (GTP), to PMNL membrane receptors bearing [3H] LTB4 either prior to or after CHAPS solubilization reduced the yield of the 140 kD presumed LTB4 receptor protein-G protein complex. That the maximum specific binding of [35S] guanosine-5'-0-3-thiotriphosphate (GTP-gammaS) to LTB4-binding proteins in the Sephacryl S-300 effluent corresponded to the 140 kD protein supported the presence of a G protein in the LTB4 receptor complex.     

Kinetics of leukotriene A4 synthesis by 5-lipoxygenase from rat polymorphonuclear leukocytes

When arachidonic acid is added to lysates of rat polymorphonuclear leukocytes, it is oxidized to (5S)-hydroperoxy-6(E),8(Z),11(Z),14(Z)-eicosatetraenoic acid (5-HPETE). The 5-HPETE then partitions between reduction to the 5-hydroxyeicosanoid and conversion to leukotriene A4 (LTA4). Both steps in the formation of LTA4 are catalyzed by the enzyme 5-lipoxygenase. When [3H]arachidonic acid and unlabeled 5-HPETE were incubated together with 5-lipoxygenase, approximately 20% of the arachidonic acid oxidized at low enzyme concentrations was converted to LTA4 without reduction of the specific radioactivity of the LTA4 by the unlabeled 5-HPETE. A significant fraction of the [3H]-5-HPETE intermediate that is formed from arachidonic acid must therefore be converted directly to LTA4 without dissociation of the intermediate from the enzyme. This result predicts that even in the presence of high levels of peroxidase activity, which will trap any free 5-HPETE by reduction, the minimum efficiency of conversion of 5-HPETE to LTA4 will be approximately 20%, and this prediction was confirmed. 5-HPETE was found to be a competitive substrate relative to arachidonic acid, so that it is likely that the two substrates share a common active site.     

Endothelial cell leukotriene C4 synthesis results from intercellular transfer of leukotriene A4 synthesized by polymorphonuclear leukocytes

Leukotriene (LT) synthesis and metabolism were studied in porcine aortic endothelial cells. Leukotrienes were identified by combinations of guinea pig lung parenchymal strip bioassay, radioimmunoassay, and UV spectrophotometry with high performance liquid chromatography. Endothelial cells stimulated with the calcium ionophore, A23187, were unable to convert arachidonic acid to detectable levels of LTA4-derived products including the biologically active metabolites, LTB4 or LTC4. However, these cells readily converted exogenous LTA4 to the potent slow-reacting substance, LTC4. Smaller quantities of 11-trans-LTC4 and LTD4 were also observed. LTB4 was not detectable in these incubations nor was LTB4 metabolism observed. The possible intercellular transfer of LTA4 between polymorphonuclear leukocytes (PMNL) and endothelial cells was tested since PMNL release LTA4 when stimulated and have significant contact with endothelium. When A23187-stimulated neutrophils were coincubated with endothelial cells, a significant increase in LTC4 levels was detected over PMNL alone. LTC4 is formed by the enzymatic conjugation of glutathione (GSH) with LTA4. Therefore in some experiments, endothelial cells were prelabeled with [35S]cysteine to allow intracellular synthesis of [35S]GSH. When unlabeled PMNL were added, as a source of LTA4 to the prelabeled endothelial cells, substantial levels of [35S] LTC4 were recovered. The data indicate that endothelial cells synthesize LTC4 from LTA4. They also demonstrate a specific PMNL-endothelial cell interaction in which endothelial cell LTC4 synthesis results from the intercellular transfer of LTA4 produced by PMNL.     

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.     

Inhibitory effect of stobadine on FMLP-induced chemiluminescence in human whole blood and isolated polymorphonuclear leukocytes.

The chemiluminescence (CL) technique with luminol and isoluminol was used to characterize the effect of stobadine on reactive oxygen metabolites (ROM) generation in human whole blood and in isolated polymorphonuclear leukocytes (PMNL) stimulated with N-formyl-methionyl-leucyl-phenyl-alanine (FMLP). In whole blood and in isolated PMNL, stobadine in the concentrations of 1, 10 and 100 micromol/L significantly inhibited the CL signal after FMLP, which activated predominantly extracellular generation of ROM. The same concentrations of stobadine were effective on CL in a cell-free system. On the other hand, myeloperoxidase (MPO) liberation was decreased by stobadine only in the concentration of 100 micromol/L. The results showed stobadine to act as a potent inhibitor/scavenger of extracellularly produced ROM in human PMNL and indicated interference of stobadine with ROM as well as with signalling events resulting in NADPH-oxidase activation and MPO liberation.     

Metabolism of 6-trans isomers of leukotriene B4 to dihydro products by human polymorphonuclear leukocytes

The major dihydroxy metabolites of arachidonic acid formed by human polymorphonuclear leukocytes (PMNL) are leukotriene B4 (LTB4), 6-trans-LTB4, and 12-epi-6-trans-LTB4. LTB4, and to a lesser extent its 6-trans isomers, are metabolized to 20-hydroxy products by a hydroxylase in PMNL. We have recently reported the existence of a second pathway involving a reductase which, combined with the hydroxylase, results in the conversion of 6-trans-LTB4 to dihydro-6-trans-LTB4. We have now investigated some of the characteristics of this novel triene reductase pathway in human PMNL and have characterized some of the products and their mechanism of formation. At low substrate concentrations, the major pathway for the initial metabolism of both 6-trans-LTB4 and 12-epi-6-trans-LTB4 is reduction of the conjugated triene chromophore to give dihydro products with single absorption maxima at about 230 nm. Dihydro-6-trans-LTB4 is rapidly converted to its 20-hydroxy metabolite by LTB4 20-hydroxylase. However, 20-hydroxy-6-trans-LTB4 is not a substrate for the reductase. Neither 12-epi-6-trans-LTB4 nor its dihydro metabolite, 5,12-dihydroxy-7,9,14-eicosatrienoic acid, which was identified by gas chromatography-mass spectrometry, were very good substrates for the hydroxylase. The dihydro metabolites of 6-trans-LTB4 and 12-epi-6-trans-LTB4 were formed rapidly during the initial phase of the reaction, whereas the corresponding dihydro-20-hydroxy metabolites were formed only after a lag phase. Experiments utilizing deuterium-labeled 12-epi-6-trans-LTB4 indicated that a hydrogen atom is lost from the 5-position of the substrate, suggesting that the initial step in the formation of the dihydro products is the formation of a 5-oxo intermediate. LTB4 is metabolized very rapidly by LTB4 20-hydroxylase in PMNL, and we have not yet identified dihydro products derived from this substance. However, LTB4 strongly inhibits the conversion of 12-epi-6-trans-LTB4 to dihydro products, suggesting that it may also interact with the reductase.     

Identification of an aldehyde dehydrogenase in the microsomes of human polymorphonuclear leukocytes that metabolizes 20-aldehyde leukotriene B4

We have previously reported that cytochrome P-450LTB in the microsomes of human polymorphonuclear leukocytes (PMN) catalyzes three omega-oxidations of leukotriene B4 (LTB4), leading to the sequential formation of 20-OH-LTB4, 20-CHO-LTB4, and 20-COOH-LTB4 (Soberman, R.J., Sutyak, J.P., Okita, R.T., Wendelborn, D.F., Roberts, L.J., II, and Austen, K. F. (1988) J. Biol. Chem. 263, 7996-8002). The identification of the novel final intermediate, 20-CHO-LTB4, allowed direct analysis of its metabolism by PMN microsomes in the presence of adenine nucleotide cofactors. Microsomes in the presence of 100 microM NAD+ or 100 microM NADP+ converted 1.0 microM 20-CHO-LTB4 to 20-COOH-LTB4 with a Km of 2.4 +/- 0.8 microM (mean +/- S.E., n = 4) and a Vmax of 813.9 +/- 136.6 pmol.min-1.mg-1, for NAD+, as compared to 0.12 microM and 5.0 pmol.min-1.mg-1 (n = 2) for NADPH as a cofactor. The conversion of 1.0 microM of 20-CHO-LTB4 to 20-COOH-LTB4 in the presence of saturating concentrations (1.0 mM) of both NAD+ and NADP+ was not greater than the reaction in the presence of 1.0 mM of each cofactor separately, indicating that NAD+ and NADP+ were cofactors for the same enzyme. Antibody to cytochrome P-450 reductase did not inhibit the conversion of 20-CHO-LTB4 to 20-COOH-LTB4. When 1.0 microM 20-OH-LTB4 was added to microsomes in the presence of NADPH, approximately three-fourths of the product formed (63.7 +/- 5.1 pmol; mean +/- S.E., n = 3) was 20-CHO-LTB4 and approximately one-fourth (21.3 +/- 3.9 pmol; mean +/- S.E., n = 3) was 20-COOH-LTB4. In the presence of both NADPH and NAD+, only 20-COOH-LTB4 (85.5 +/- 9.9 pmol; mean +/- S.E., n = 3) was formed. PMN microsomes also contain an NADH-dependent aldehyde reductase which converts 20-CHO-LTB4 to 20-OH-LTB4, a member of the LTB4 family of molecules with biological activity. Based upon kinetic, cofactor and inhibition data, microsomal aldehyde dehydrogenase preferentially regulates the final and irreversible inactivation step in the LTB4 metabolic sequence.