The identification and formation of 20-aldehyde leukotriene B4

Microsomes of human polymorphonuclear leukocytes (PMN) in the presence of 100 microM NADPH converted 0.6 microM leukotriene B4 (LTB4) to 20-OH-LTB4 (retention time = 18.0 min) and to two additional compounds designated I (retention time = 16.8 min) and II (retention time = 9.6 min) as analyzed by reverse-phase high performance liquid chromatography (HPLC). Compounds I and II were also formed from the reaction of 1.0 microM 20-OH-LTB4, PMN microsomes, and 100 microM NADPH; the identity of compound II was confirmed as 20-COOH-LTB4 by gas chromatography-mass spectrometry. Equine alcohol dehydrogenase in the presence of 100 microM NAD+ in 0.2 M glycine buffer (pH 10.0) converted 20-OH-LTB4 to 20-aldehyde (CHO) LTB4, which coeluted with compound I on reverse-phase HPLC. In the presence of 100 microM NADH in 50 mM potassium phosphate buffer (pH 6.5), equine alcohol dehydrogenase reduced both 20-CHO-LTB4 and compound I to 20-OH-LTB4, indicating the identity of compound I as 20-CHO-LTB4. Gas chromatography-mass spectrometry of trideuterated O-methyl-oxime trimethylsilyl ether methyl ester derivative of 3H-labeled compound I definitively established compound I as 20-CHO-LTB4. Addition of immune IgG to cytochrome P-450 reductase or 1.0 mM SKF-525A completely inhibited the formation of 20-CHO-LTB4 from 20-OH-LTB4, indicating that the reaction was catalyzed by a cytochrome P-450. LTB5 (3.0 microM), a known substrate for cytochrome P-450LTB and a competitive inhibitor of LTB4 omega-oxidation, completely inhibited the omega-oxidation of 1.5 microM 20-OH-LTB4 to 20-CHO-LTB4, indicating that the cytochrome P-450 was P-450LTB. Conversion of 1.0 microM 20-CHO-LTB4 to 20-COOH-LTB4 by PMN microsomes was also dependent on NADPH and inhibited by antibody to cytochrome P-450 reductase, 1.0 mM SKF-525A, or 5.0 microM LTB5, indicating that this reaction was also catalyzed by cytochrome P-450LTB. These results identify the novel metabolite 20-CHO-LTB4 and indicate that cytochrome P-450LTB catalyzes three sequential omega-oxidations of LTB4 leading to the formation of 20-COOH-LTB4 via 20-OH-LTB4 and 20-CHO-LTB4 intermediates.     

Properties of leukotriene B4 20-hydroxylase from polymorphonuclear leukocytes

Human polymorphonuclear leukocytes (PMNL) convert arachidonic acid (20:4) to a number of dihydroxy metabolites, including leukotriene B4 (LTB4) 5S,12R-dihydroxy-6,8,10,14-EEEZ-icosatetraenoic acid (isomer-1), 5S,12S-dihydroxy-6,8,10,14-EEEZ-icosatetraenoic acid, 5S,12S-dihydroxy-6,8,10,14-EZEZ-icosatetraenoic acid (5S,12S-dh-20:4), 5,6-dihydroxy-7,9,11,14-icosatetraenoic acid, and 5,15-dihydroxy-6,8,11,13-icosatetraenoic acid. LTB4 was synthesized rapidly after stimulation of PMNL with the divalent cation ionophore, A23187, but its concentration rapidly declined after about 4 min, in contrast to the other dihydroxy metabolites of 20:4 whose concentrations remained stable for at least 20 min. The amounts of polar metabolites (identified primarily as 20-hydroxy-LTB4) increased steadily with time up to 20 min. These results suggest that LTB4 may be specifically converted to its 20-hydroxy metabolite by PMNL. We prepared 3H- and 14C-labeled analogs of the dihydroxyicosatetraenoic acid metabolites described above by incubation of labeled 20:4 with PMNL. Although all of these substances were metabolized to some extent by human PMNL, LTB4 (apparent Km, 1.0 microM) was metabolized the most rapidly, followed by 5S,12S-dh-20:4 (apparent Km, 2.4 microM) and isomer-1 (apparent Km, 4.8 microM). All three substrates were shown by mass spectrometry to be converted to their 20-hydroxy metabolites. LTB4 was also metabolized to its omega-carboxy derivative. Human mononuclear leukocytes and rabbit PMNL metabolized LTB4 very slowly, whereas rat PMNL metabolized this substrate at about one-sixth the rate of human PMNL. These results demonstrate that human PMNL contain an omega-hydroxylase that specifically converts LTB4 to its 20-hydroxy metabolite. This enzyme may be important for the regulation of LTB4 levels in vivo.     

Identification and biological activity of dihydroleukotriene B4: a prominent metabolite of leukotriene B4 in the human lung

Exogenous [3H]leukotriene B4 (LTB4) was converted into several polar and non-polar metabolites in the chopped human lung. One of the major metabolites was identified as 5(S),12-dihydroxy-6,8,14-eicosatrienoic acid (10,11-dihydro-LTB4) by means of co-chromatography with authentic standards, ultraviolet spectrometry and gas chromatography-mass spectrometry. Analysis of chiral straight phase HPLC revealed the presence of both the 12(S) and 12(R) epimers of dihydro-LTB4. Dihydro-LTB4 was also formed from endogenously generated LTB4 in ionophore A23187 stimulated incubations. The dihydro metabolites were approximately 100 times less potent than LTB4 in causing guinea pig lung strip contraction and leukocyte-dependent inflammation in the hamster cheek pouch in vivo.     

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.     

Urinary metabolites of leukotriene B4 in the human subject

Leukotriene B4 (LTB4) is a potent chemoattractant for neutrophils and is thought to play a role in a variety of inflammatory responses in humans. The metabolism of LTB4 in vitro is complex with several competing pathways of biotransformation, but metabolism in vivo, especially for normal human subjects, is poorly understood. As part of a Phase I Clinical Trial of human tolerance to LTB4, four human subjects were injected with 150 nmol/kg LTB4 with one additional subject as placebo control. The urine of the subjects was collected in two separate pools (0-6 and 7-24 h), and aliquots from these urine collections were analyzed using high performance liquid chromatography, UV spectroscopy, and negative ion electrospray ionization tandem mass spectrometry for metabolites of LTB4. In the current investigation, 11 different metabolites of LTB4 were identified in the urine from those subjects injected with LTB4, and none were present in the urine from the placebo-injected subject. The unconjugated LTB4 metabolites found in urine were structurally characterized as 18-carboxy-LTB4, 10,11-dihydro-18-carboxy-LTB4, 20-carboxy-LTB4, and 10,11-dihydro-20-carboxy-LTB4. Several glucuronide-conjugated metabolites of LTB4 were characterized including 17-, 18-, 19-, and 20-hydroxy-LTB4, 10-hydroxy-4,6,12-octadecatrienoic acid, LTB4, and 10,11-dihydro-LTB4. The amount of LTB4 glucuronide (16.7-29.4 pmol/ml) and 20-carboxy-LTB4 (18.9-30.6 pmol/ml) present in the urine of subjects injected with LTB4 was determined using an isotope dilution mass spectrometric assay before and after treatment of the urine samples with beta-glucuronidase. The urinary metabolites of LTB4 identified in this investigation were excreted in low amounts, yet it is possible that one or more of these metabolites could be used to assess LTB4 biosynthesis following activation of the 5-lipoxygenase pathway in vivo.     

Metabolism of leukotriene B4 in isolated rat hepatocytes. Involvement of 2,4-dienoyl-coenzyme A reductase in leukotriene B4 metabolism

Radiolabeled leukotriene (LT) B4 was incubated with isolated rat hepatocytes in order to assess the metabolism of this chemotactic leukotriene by the liver. At least eight radioactive metabolites were observed, three of which were previously identified as 20-hydroxy-, 20-carboxy-, and 18-carboxy-19,20-dinor-LTB4. A less lipophilic major metabolite (designated HIV) was purified by two reverse phase high performance liquid chromatography separations and was found to exhibit maximal UV absorbance at 269 nm with shoulders at 260 and 280 indicating the presence of a conjugated triene chromophore. Negative ion electron capture gas chromatography/mass spectrometry analysis of the pentafluorobenzyl ester, trimethylsilyl ether derivative of HIV, and positive ion electron ionization mass spectra of the methyl ester trimethylsilyl derivative were consistent with a structure of this metabolite being 16-carboxy-14,15-dihydro-17,18,19,20-tetranor-LTB3. The appearance of this metabolite supports the concept of further beta-oxidation of LTB4 to the carbon 16 which requires the action of 2,4-dienoyl-CoA reductase to remove the 14,15-double bond located two carbon atoms removed from the CoA thioester moiety. One minor metabolite was analyzed by negative ion continuous flow fast atom bombardment mass spectrometry which revealed an ion at m/z 444 which by high resolution mass spectrometry was shown to contain both nitrogen and sulfur. Tandem mass spectrometry suggested the presence of SO3- as well as other fragments corresponding to the amino acid taurine. Incubation of isolated rat hepatocytes with [14C]taurine as well as [3H]LTB4 revealed the incorporation of both radioactive isotopes into this metabolite. The data supported the identification of this metabolite as tauro-18-carboxy-19,20-dinor-LTB4. Amino acid conjugation of leukotrienes has not been previously reported and suggests that such intermediates might participate in enterohepatic circulation of LTB4 metabolites in the intact animal and thus serve as an alternative metabolic route for LTB4 elimination.     

Metabolism of arachidonic acid by peripheral and elicited rat polymorphonuclear leukocytes. Formation of 18- and 19-oxygenated dihydro metabolites of leukotriene B4

Previous studies have shown that leukotriene B4 is metabolized by polymorphonuclear leukocytes (PMNL) by a 20-hydroxylase, a 19-hydroxylase, and a reductase. We have now identified for the first time LTB4 metabolites formed by a combination of the reductase and omega-oxidation pathways. We have also discovered that rat PMNL metabolize LTB4 by a novel pathway to 18-hydroxy products. Dihydro metabolites of LTB4 have formerly been reported only after incubation of exogenous LTB4 with PMNL, but we have now shown that they are formed to the same extent from endogenous arachidonic acid after stimulation of PMNL with the ionophore, A23187. The following metabolites have been identified after incubation of either LTB4 or arachidonic acid with rat PMNL: 10,11-dihydro-LTB4, 10,11-dihydro-12-epi-LTB4, 10,11-dihydro-12-oxo-LTB4, 19-hydroxy-LTB4, 19-hydroxy-10,11-dihydro-LTB4, 19-oxo-10,11-dihydro-LTB4, 18-hydroxy-LTB4, 18-hydroxy-10,11-dihydro-LTB4, and 18-hydroxy-10,11-dihydro-12-oxo-LTB4. Negligible amounts of 20-hydroxylated products were formed. Incubation of PMNL with 10,11-dihydro-LTB4 resulted in the formation of all of the above dihydro metabolites. However, none of the omega-oxidized metabolites of LTB4 was further metabolized to a significant extent when incubated with PMNL, possibly at least partially because they were not substrates for a specific LTB4 uptake mechanism. We found that the biosynthesis and metabolism of LTB4 is considerably enhanced in PMNL from an inflammatory site (carrageenan-induced pleurisy) compared with peripheral PMNL. When arachidonic acid was the substrate, the greatest increase was observed for products formed by the reductase pathway, which were about eight times higher in pleural PMNL. The rates of formation of both LTA hydrolase and omega-hydroxylase products were about three times higher, whereas the total amounts of 5-lipoxygenase products were about twice as high in pleural PMNL. The amounts of products formed by the above enzymatic pathways reached maximal levels about 4-6 h after injection of carrageenan and then declined.     

Hydroxylation of leukotriene B4 in leukocytes from various species: identification of a metabolite to authentic 5S,12R,19-trihydroxy-6Z,8E,10E,14Z-eicosatetra enoic acid and relative importance of 19- and 20-hydroxylations

The major hydroxylated metabolite of leukotriene B4 in rat PMNL was found identical (UV spectrum and retention times in 3 different HPLC systems) to a synthetic compound of known stereochemistry, 19-hydroxy-LTB4. PMNL from various species exhibited 3 different types of behaviour for LTB4 hydroxylation. Human and monkey PMNL showed a high hydroxylating activity and a high regioselectivity with almost exclusive formation of products from 20-hydroxylation. Rat and mini-pig PMNL exhibited a very different regioselectivity with major formation of 19-OH-LTB4 (3:1 ratio). Finally, pig and beef PMNL were found almost devoid of any hydroxylating activity toward LTB4.     

Metabolism of leukotriene B4 in hepatic microsomes

Leukotriene B4 was metabolized in rat hepatic microsomes to two products. Mass spectral analysis of these two metabolites indicated that the major metabolite was the 20-hydroxy metabolite while the minor metabolite was the 19-hydroxy metabolite. The formation of these metabolites required NADPH and was linear with time (20 min) and protein (1.6 mg/ml). The Km apparent and Vmax for omega hydroxylation of LTB4 was 14 uM and 0.138 nmol/min/mg protein. In contrast, the km and Vmax for omega minus one hydroxylation was 54 uM and 0.093 nmol/min/mg protein. These results suggest that omega and omega minus one hydroxylations of LTB4 may be mediated by different isozymes of hepatic P-450.     

Human monocytes convert leukotriene B4 to two dihydro-leukotriene B4-metabolites

Human monocytes metabolize LTB4 by an additional pathway different from omega-oxidation. Reverse-phase high performance liquid chromatography showed four metabolites: 20-COOH-LTB4, 20-OH-LTB4 and two metabolites less polar than LTB4 with an UV maximum at 232 nm. Gas-chromatography mass-spectrometry showed nearly identical mass spectra for both metabolites. The main mass fragments of the two metabolites were increased by two mass units compared to LTB4. Our findings suggest that LTB4 had been reduced to a known and a new dihydro-metabolite of LTB4. Both metabolites together amounted to 85% of total metabolites. The remaining 15% were omega-oxidation products. Thus, the major pathway of LTB4 metabolism by human monocytes is reduction to dihydro-LTB4.     

Metabolism of leukotriene E4 in isolated rat hepatocytes. Identification of beta-oxidation products of sulfidopeptide leukotrienes

Little is known about the metabolic fate of the sulfidopeptide leukotrienes (LTC4/D4/E4). Earlier studies using radiolabeled leukotrienes have shown that these potent molecules are concentrated and metabolized in the liver when administered to mice and that isolated rat hepatocytes have a high affinity uptake system for LTE4. N-Acetyl-LTE4 has been identified as a metabolite of LTC4 in the bile of rats, but the majority of the metabolites in these studies were not characterized. Based on these earlier reports, incubation of LTE4 with isolated rat hepatocytes was chosen as a model for the study of sulfidopeptide leukotriene metabolism. [3H]LTE4 was incubated with isolated rat hepatocytes and the metabolites formed were purified extensively by ODS flash column chromatography, TLC, and reverse phase-high pressure liquid chromatography. Metabolites were identified by retention of the radiolabel and UV absorbance at 280 nm. Purified metabolites were characterized by UV spectroscopy, fast atom bombardment mass spectrometry, negative ion chemical ionization gas chromatography-mass spectrometry, and electron impact gas chromatography-mass spectrometry. Six LTE4 hepatocyte metabolites were characterized. Metabolite A was determined to be N-acetyl-LTE4. Metabolite B was determined to be the omega-oxidation product 20-carboxy-N-acetyl-LTE4. Metabolite C was characterized as the beta-oxidation product 18-carboxydinor-N-acetyl-LTE4. A further round of beta-oxidation with a concomitant double bond reduction produced Metabolite D, identified as 16-carboxytetranordihydro-N-acetyl-LTE4. The reduction of the 14-15 double bond was most likely the result of the action of 2,4-dienoyl-CoA reductase. The UV spectrum of Metabolite E indicated the presence of a conjugated tetraene, and this metabolite was determined to be 16-carboxytetranor-delta 13-N-acetyl-LTE4. Metabolite F was identified as 14-carboxyhexanor-N-acetyl-LTE4. The observed pathway of beta-oxidation of LTE4 proceeded entirely from the C-20 methyl terminus after omega-oxidation which is in contrast to the known metabolic fate of other eicosanoids. This may be due to the failure to generate the required thioester at C-1 in LTE4 through a strong interaction of the C-5 hydroxy group with the C-1 carboxyl.     

Chemotactic LTB4 metabolites produced by hepatocytes in the presence of ethanol.

Ethanol in low concentrations significantly alters the hepatocyte metabolism of the neutrophil chemotactic lipid leukotriene B4 (LTB4). Two novel metabolites of LTB4 which are encountered only when ethanol is present, retained significant biological activity. One metabolite, 3-hydroxy-LTB4 increased intracellular free calcium in the human neutrophil at concentrations as low as 3 x 10(-10) M as well as induced shape change and adherence to albumin-coated latex beads at 10 nM. The 3-hydroxy-LTB4 and 3,20-hydroxy-LTB4 metabolites were also potent chemotactic agonists with an ED50 at 3.0 and 9.0 nM, respectively. These results suggest that the presence of ethanol can substantially alter inactivation of LTB4 by the liver and may mediate neutrophil accumulation into the liver, thereby contributing to the pathogenesis of alcoholic hepatitis even when LTB4 biosynthesis occurs at some site distant to the liver.     

Metabolism of leukotriene B4 to dihydro and dihydro-oxo products by porcine leukocytes

Porcine leukocytes contain a novel pathway for the metabolism of leukotriene B4 (LTB4) which results in reduction of the conjugated triene chromophore to a conjugated diene. These cells converted LTB4 to two major metabolites, both of which exhibited maximal absorbance at 230 nm in their UV spectra. These products were purified by high pressure liquid chromatography and identified as 10, 11-dihydro-LTB4 and 10,11-dihydro-12-oxo-LTB4 on the basis of the mass spectra of various derivatives. The position of the double bond of LTB4 which had been reduced was established by cleaving the remaining double bonds of 10, 11-dihydro-LTB4 with ozone followed by oxidation or reduction of the resulting ozonide and analysis of the products by mass spectrometry. Experiments with deuterium-labeled substrate indicated that LTB4 could be directly converted to 10, 11-dihydro-LTB4 without the prior oxidation of either of its hydroxyl groups, as is required for the formation of dihydro metabolites of prostaglandins. Incubation of porcine leukocytes with 10, 11-dihydro-LTB4 and 10, 11-dihydro-12-oxo-LTB4 indicated that these two products can be interconverted and are in equilibrium with one another. The dihydro-oxo metabolite can therefore be formed from 10, 11-dihydro-LTB4, although we have not ruled out the possibility that it is also produced via 12-oxo-LTB4, which could be a transitory intermediate. These results indicate that porcine leukocytes contain a novel reductase/dehydrogenase pathway distinct from the pathway responsible for the metabolism of prostaglandins. This pathway is also different from the pathway in human polymorphonuclear leukocytes which converts 6-trans-isomers of LTB4 to dihydro products, since the latter pathway involves 5-oxo intermediates and results in a shift in the positions of the remaining double bonds.     

Stereochemistry of leukotriene B4 metabolites formed by the reductase pathway in porcine polymorphonuclear leukocytes: inversion of stereochemistry of the 12-hydroxyl group

Leukotriene B4 (LTB4), a potent proinflammatory agent, is a major metabolite of arachidonic acid in polymorphonuclear leukocytes (PMNL). When porcine PMNL were incubated with LTB4 and the products purified by reversed-phase high-pressure liquid chromatography (HPLC), we previously identified two metabolites: 10,11-dihydro-LTB4 and 10,11-dihydro-12-oxo-LTB4 [Powell, W. S., & Gravelle, F. (1989) J. Biol. Chem. 264, 5364-5369]. Further analysis of the reaction products by normal-phase HPLC has now revealed the presence of a third major metabolite of LTB4. This product is not formed in detectable amounts in the first 5 min of the reaction but accounts for about 20-30% of the reaction products after 60 min, when LTB4 has been completely metabolized. The mass spectrum and gas chromatographic properties of the new metabolite are identical with those of 10,11-dihydro-LTB4, suggesting that it is a stereoisomer of this compound. This product was identified as 10,11-dihydro-12-epi-LTB4 [i.e., 5(S),12(R)-dihydroxy-6,8,14-eicosatrienoic acid] by comparison of its chromatographic properties with those of the authentic chemically synthesized compound. Both 10,11-dihydro-LTB4 and 10,11-dihydro-12-oxo-LTB4 were enzymatically converted to 10,11-dihydro-12-epi-LTB4 by porcine PMNL, the former compound being the better substrate. The reaction was reversible, since both 10,11-dihydro-12-epi-LTB4 and 10,11-dihydro-12-oxo-LTB4 could be converted to 10,11-dihydro-LTB4.(ABSTRACT TRUNCATED AT 250 WORDS)     

The development of a sensitive and specific radioreceptor assay for leukotriene B4

To establish a simple and sensitive quantitation of leukotriene B4 (LTB4), we developed a radioreceptor assay (RRA) using a highly specific [3H]leukotriene B4[( 3H]LTB4) binding to a guinea pig spleen homogenate. The assay detected LTB4 levels as low as 0.12 pmol per tube. Fifty percent inhibition of bound [3H]LTB4 was obtained by 2.5 nM of unlabeled LTB4. [3H]LTB4 competition studies indicated that 20-hydroxy-LTB4 was 8 times, 6-trans-LTB4 was 640 times and 20-carboxy-LTB4 was 1000 times less effective than LTB4. The peptide leukotrienes C4, D4 and E4 showed no effect on [3H]LTB4 binding. Recovery rates averaged 97% after ethanol extraction and evaporation of known amounts of LTB4. The intra-assay coefficients of variation for three samples were 2.4%, 7.2% and 8.4%, respectively. This assay was validated by measuring LTB4 released from human granulocytes stimulated with calcium ionophore A23187. The LTB4 level was maximal at 10 min (156.8 +/- 36.2 pmol/3 x 10(6) cells) and decreased rapidly after 15 min. This radioreceptor assay for leukotriene B4 is highly sensitive and is comparable to the reported sensitivity by radioimmunoassay. The method is simpler and less expensive than other methods such as high pressure liquid chromatography and is suitable for routine measurement of leukotriene B4.     

A novel hepatointestinal leukotriene B4 receptor. Cloning and functional characterization

Leukotriene B(4) (LTB(4)) is a product of eicosanoid metabolism and acts as an extremely potent chemotactic mediator for inflammation. LTB(4) exerts positive effects on the immigration and activation of leukocytes. These effects suggest an involvement of LTB(4) in several diseases: inflammatory bowel disease, psoriasis, arthritis, and asthma. LTB(4) elicits actions through interaction with one or more cell surface receptors that lead to chemotaxis and inflammation. One leukotriene B(4) receptor has been recently identified (LTB(4)-R1). In this report we describe cloning of a cDNA encoding a novel 358-amino acid receptor (LTB(4)-R2) that possesses seven membrane-spanning domains and is homologous (42%) and genetically linked to LTB(4)-R1. Expression of LTB(4)-R2 is broad but highest in liver, intestine, spleen, and kidney. In radioligand binding assays, membranes prepared from COS-7 cells transfected with LTB(4)-R2 cDNA displayed high affinity (K(d) = 0.17 nm) for [(3)H]LTB(4). Radioligand competition assays revealed high affinities of the receptor for LTB(4) and LTB(5), and 20-hydroxy-LTB(4), and intermediate affinities for 15(S)-HETE and 12-oxo-ETE. Three LTB(4) receptor antagonists, 14,15-dehydro-LTB(4), LTB(4)-3-aminopropylamide, and U-75302, had high affinity for LTB(4)-R1 but not for LTB(4)-R2. No apparent affinity binding for the receptors was detected for the CysLT1-selective antagonists montelukast and zafirlukast. LTB(4) functionally mobilized intracellular calcium and inhibited forskolin-stimulated cAMP production in 293 cells. The discovery of this new receptor should aid in further understanding the roles of LTB(4) in pathologies in these tissues and may provide a tool in identification of specific antagonists/agonists for potential therapeutic treatments.     

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.     

Catabolism of leukotriene B5 in humans

Human neutrophils, enriched by dietary supplementation with eicosapentaenoic acid, form leukotriene (LT)B5 in addition to LTB4 upon stimulation. LTB5 is one order of magnitude less biologically active than the potent chemokinetic and chemoattractant LTB4. Catabolites of LTB5 have not yet been characterized in vitro and ex vivo. It is unknown whether catabolism of LTB5 interferes with catabolism of LTB4. This report describes catabolism of LTB5 to 20-OH-LTB5, which in turn is catabolized to 20-COOH-LTB5. The structures of the two catabolites were established by UV-absorbance, behavior on reverse-phase high-performance liquid chromatography, enzymatic analysis of human neutrophils, and gas chromatography-mass spectrometry. In vitro, formation of LTB4 was delayed and formation of its catabolites was depressed by exogenous eicosapentaenoic acid. By supplementing the diet of six volunteers with 5 g eicosapentaenoic acid/day for 7 days, eicosapentaenoic acid quadrupled in neutrophil phospholipid fatty acids. Consequently, LTB5, 20-OH-LTB5, and 20-COOH-LTB5 were detected ex vivo. In contrast to the findings in vitro, however, levels of LTB4, 20-OH-LTB4, and 20-COOH-LTB4 were unaltered by the dietary intervention. Thus, in vitro, but not ex vivo, addition of eicosapentaenoic acid, and subsequent formation of LTB5, impeded catabolism of proinflammatory LTB4.     

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.     

Oxidation of 20-hydroxyleukotriene B4 to 20-carboxyleukotriene B4 by human neutrophil microsomes. Role of aldehyde dehydrogenase and leukotriene B4 omega-hydroxylase (cytochrome P-450LTB omega) in leukotriene B4 omega-oxidation

Leukotriene B4 (LTB4), a potent chemoattractant for leukocytes, is catabolized by human neutrophils via omega-oxidation. Neutrophil microsomes are known to oxidize 20-hydroxy-LTB4 (20-OH-LTB4) to its 20-oxo and 20-carboxy derivatives in the presence of NADPH. This activity has been ascribed to LTB4 omega-hydroxylase (cytochrome P-450LTB omega), a conclusion supported by our finding of the reversal of carbon monoxide inhibition by 450 nm light and by competitive inhibition studies. The oxidation of 20-oxo-LTB4 to 20-carboxy-LTB4 is also catalyzed by microsomes fortified with 1 mM NAD+, and this activity is not affected by cytochrome P-450LTB omega inhibitors. The evidence is compatible with involvement of a disulfiram-insensitive aldehyde dehydrogenase in this second oxidation pathway. Interaction of the two pathways is evidenced by facilitation of NADPH-dependent oxidation of 20-OH-LTB4 by the addition of NAD+. This synergism may be explained by removal of the aldehyde intermediate by the NAD(+)-dependent aldehyde dehydrogenase. Taken together with the finding that the NAD(+)-dependent activity is severalfold higher than the NADPH-dependent one, the dehydrogenase may be important in the oxidation of 20-OH-LTB4 to 20-carboxy-LTB4.