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.     

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)     

Metabolism of arachidonic acid through the 5-lipoxygenase pathway in normal human peritoneal macrophages

Peritoneal macrophages (PM), obtained from 39 healthy women with normal laparoscopy findings, were stimulated with the ionophore A23187 or/and arachidonic acid (AA) both in adherence and in suspension. AA lipoxygenase metabolites were determined by reversed-phase HPLC. The major metabolites identified were 5-hydroxyeicosatetraenoic acid (5-HETE), leukotriene (LT)B4 and LTC4. The 20-hydroxy-LTB4, 20-carboxy-LTB4, and 15-HETE were not detected. Incubations of adherent PM with 2 microM A23187 induced the formation of LTB4, 110 +/- 19 pmol/10(6) cells, 5-HETE, 264 +/- 53 pmol/10(6) cells and LTC4, 192 +/- 37 pmol/10(6) cells. When incubated with 30 microM exogenous AA, adherent PM released similar amounts of 5-HETE (217 +/- 67 pmol/10(6) cells), but sevenfold less LTC4 (27 +/- 12 pmol/10(6) cells) (p less than 0.01). In these conditions LTB4 was not detectable. These results indicate that efficient LT synthesis in PM requires activation of the 5-lipoxygenase/LTA4 synthase, as demonstrated previously for blood phagocytes. When stimulated with ionophore, suspensions of Ficoll-Paque-purified PM produced the same lipoxygenase metabolites. The kinetics of accumulation of the 5-lipoxygenase/LTA4 synthase products in A23187-stimulated adherent cells varied for the various metabolites. LTB4 reached a plateau by 5 min, whereas LTC4 levels increased up to 60 min, the longest incubation time studied. Levels of 5-HETE were maximal at 5 min, and then slowly decreased with time. Thus, normal PM, in suspension or adherence, have the capacity to produce significant amounts of 5-HETE, LTB4, and LTC4. The profile of lipoxygenase products formed by the PM and the reactivity of this cell to AA and ionophore A23187 are similar to those of the human blood monocyte, but different from those of the human alveolar macrophage.     

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.     

Mechanism for the formation of dihydro metabolites of 12-hydroxyeicosanoids. Conversion of leukotriene B4 and 12-hydroxy-5,8,10,14-eicosatetraenoic acid to 12-oxo intermediates.

Eicosanoids containing a 12-hydroxyl group preceded by at least two conjugated double bonds are metabolized to 10,11-dihydro and 10,11-dihydro-12-oxo products by porcine polymorphonuclear leukocytes (PMNL) (Wainwright, S. L., Falck, J. R., Yadagiri, P., and Powell, W. S. (1990) Biochemistry 29, 10126-10135). These 10,11-dihydro metabolites could either have been formed by the direct reduction of the 10,11-double bond of the substrate, as previous evidence suggested, or via an initially formed 12-oxo intermediate. To gain some insight into the mechanism for the formation of dihydro products by this pathway, we investigated the metabolism of leukotriene B4 (LTB4), 12(S)-hydroxy-5,8,10,14-eicosatetraenoicacid(12(S)-HETE), and 12(R)-HETE by subcellular fractions from porcine PMNL. In the presence of NAD+ and a microsomal fraction from PMNL, each of the above 12-hydroxyeicosanoids was converted to a single product with a lambda max approximately 40 nm higher than that of the substrate, indicating that the conjugated diene or triene chromophore had been extended by one double bond, presumably by oxidation of the 12-hydroxyl group to an oxo group. In the case of LTB4, this was confirmed by mass spectrometry, which indicated that the product was identical to 12-oxo-LTB4. LTB4 was not converted to any products by a cytosolic fraction from PMNL, but was converted to both 10,11-dihydro-LTB4 and 10,11-dihydro-12-oxo-LTB4 by the 1500 x g supernatant in the presence of NAD+. Negligible amounts of dihydro products were formed in the presence of NADH or NADPH, suggesting that initial oxidation of the 12-hydroxyl group is a requirement for reduction of the 10,11-double bond. Consistent with this hypothesis, 12-oxo-LTB4 was rapidly metabolized to 10,11-dihydro-12-oxo-LTB4 by the cytosolic fraction in the presence of NADH. Only small amounts of this product, along with some LTB4, were formed by the microsomal fraction. These results indicate that the initial step in the formation of 10,11-dihydro products from 12-hydroxyeicosanoids is oxidation of the 12-hydroxyl group by a microsomal 12-hydroxyeicosanoid dehydrogenase in the presence of NAD+, which is followed by reduction of the olefinic double bond by a cytosolic delta 10-reductase in the presence of NADH.     

Metabolism of leukotriene B4 in isolated rat hepatocytes. Identification of a novel 18-carboxy-19,20-dinor leukotriene B4 metabolite

Isolated rat heptocytes were found to metabolize leukotriene B4 (LTB4) to a number of products which could be separated by reverse phase high performance liquid chromatography (HPLC). After incubation of LTB4 with hepatocytes for 15 min, the known omega-oxidized metabolites, 20-hydroxy- and 20-carboxy-LTB4, were identified by HPLC retention time and gas chromatography-mass spectrometry. An early fraction corresponding to 15% of the initial LTB4 was structurally characterized as a novel metabolite, 18-carboxy-19,20-dinor-LTB4, by ultraviolet spectroscopy and gas chromatography-mass spectrometry of the derivatized and derivatized, reduced metabolite. The short HPLC retention time of this metabolite was consistent with its reduced lipophilicity. An additional minor metabolite was tentatively identified as 3-hydroxy-LTB4. These two novel metabolites provide evidence for beta-oxidation as an important route of hepatic biotransformation of LTB4 and 20-hydroxy-LTB4.     

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.     

Effect of structural modification at carbon atom 1 of leukotriene B4 on the chemotactic and metabolic response of human neutrophils

Human neutrophils biosynthesize the chemoattractant leukotriene B4 (LTB4) and metabolize LTB4 to omega oxidative products 20-hydroxy-LTB4 (20-OH-LTB4) and 20-carboxy-LTB4 (20-COOH-LTB4). In this study, we prepared the C-1 methyl ester and N-methyl amide of LTB4 and then examined neutrophil chemotaxis and metabolism of these derivatives of LTB4. The results show that chemical modification of LTB4 at carbon atom 1 dramatically affects metabolism of the lipid molecule. The free acid form of LTB4 was taken up and metabolized by human neutrophils, while the methyl ester and N-methyl amide derivatives were poor substrates for omega oxidation. Although human neutrophils were poorly attracted to the methyl ester of LTB4, the amide derivative was a complete agonist of the neutrophil chemotactic response and displayed an ED50 for chemotaxis identical to that of LTB4. Therefore, we concluded that omega oxidation is not a requirement for the neutrophil chemotactic response induced by LTB4. These results also indicate that the N-methyl amide of LTB4 may be a useful ligand for the elucidation of molecular mechanisms operative in neutrophil chemotaxis to LTB4, since the C-1 derivative is not further metabolized. Two separate responses of human neutrophils are elicited by LTB4, resulting in both cellular activation and generation of omega oxidation products. It appears that putative receptors on the neutrophils can distinguish between LTB4 and certain derivatives that are structurally identical except for modification at the C-1 position (i.e., the methyl ester). LTB4 derivatives modified at the C-1 position do not undergo conversion to omega oxidation products by the neutrophil.     

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.     

Solubilization and characterization of leukotriene B4 receptor-GTP binding protein complex from porcine spleen

A high amount of leukotriene B4 (LTB4) binding protein was observed in the porcine spleen. It was solubilized and partially purified from spleen membrane with 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS). Scatchard analysis indicated the presence of a single class of receptor with Kd and Bmax values of 0.26 nM and 120 fmol/mg protein, respectively. The receptor was specific for LTB4, and Ki values for 20-hydroxy- and 20-carboxy-LTB4, both inactive metabolites of LTB4, were 1.7 nM and over 1,000 nM, respectively. By the addition of 10 microM GTP gamma S, a low affinity binding site appeared with a Kd value of 390 nM. A pretreatment of the receptor-GTP binding protein complex with islet-activating protein (IAP) increased the inhibitory effect of GTP gamma S on LTB4 binding, indicating that the LTB4 receptor is coupled with an IAP-sensitive GTP-binding protein in the porcine spleen.     

Enhanced superoxide anion generation but reduced leukotriene B4 productivity in thioglycollate-elicited peritoneal macrophages

Lipoxygenase metabolism of arachidonic acid was compared between peritoneal macrophages from untreated rats and those from rats on day 7 after intraperitoneal injection of thioglycollate broth (TG). Resident macrophages (M phi) from untreated rats produced mainly LTB4 (303 +/- 25 pmol/5 x 10(6) cells) and 5-HETE (431 +/- 56 pmol/5 x 10(6) cells) when stimulated with 5 micrograms/ml calcium ionophore A23187 for 20 min at 37 degrees C. On the other hand, TG-elicited M phi generated less amounts of lipoxygenase metabolites (157 +/- 10 pmol LTB4 and 319 +/- 19 pmol 5-HETE/5 x 10(6) cells) with the same stimulus. Then, leukotriene productivity was examined by using subcellular fractions of each M phi lysate and an unstable epoxide intermediate, leukotriene A4. LTA4 hydrolase activity was mainly contained in soluble fractions from the both groups of M phi. The cytosol fraction from the resident M phi exhibited the following specific and total activity; 2.2 +/- 0.1 nmol LTB4/mg protein/5 min and 12.2 +/- 0.5 nmol LTB4/5 min per 10(8) cells. On the contrary, the cytosol fraction from the TG-elicited M phi showed 1.9 +/- 0.1 nmol LTB4/mg protein/5 min and 9.6 +/- 0.3 nmol LTB4/5 min per 10(8) cells. The resident M phi, however, generated 0.14 +/- 0.04 nmol O2-/min/4 x 10(5) cells whereas the TG-elicited M phi did 0.49 +/- 0.13 nmol O2-/min/4 x 10(5) cells when stimulated with wheat germ lectin. These results suggest that the TG-elicited macrophages show enhanced superoxide production but generate less lipoxygenase metabolites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conversion of stereoisomers of leukotriene B4 to dihydro and tetrahydro metabolites by porcine leukocytes

We have previously shown that porcine leukocytes convert leukotriene B4 (LTB4) to two major products, 10,11-dihydro-LTB4 and 10,11-dihydro-12-oxo-LTB4. Although we did not detect these products after incubation of LTB4 with human polymorphonuclear leukocytes, these cells converted 12-epi-6-trans-LTB4 to the corresponding 6,11-dihydro metabolite (i.e., there appeared to be a shift in the positions of the remaining double bonds). The objective of the present investigation was to determine whether 6-trans isomers of LTB4 are metabolized by porcine leukocytes by a pathway similar to LTB4, or whether they are metabolized by a pathway analogous to that in human leukocytes. We found that 6-trans-LTB4 and 12-epi-6-trans-LTB4 are metabolized more much extensively than LTB4 by porcine leukocytes. 6-trans-LTB4 appears to be converted by two different reductase pathways to two dihydro products differing in the positions of the two remaining double bonds between carbons 5 and 12. Dihydro-12-oxo and dihydro-5-oxo metabolites are also formed from this substrate. Porcine leukocytes also convert 6-trans-LTB4, presumably by a combination of the above two pathways, to tetrahydro, tetrahydro-12-oxo and tetrahydro-5-oxo metabolites, none of which possesses any conjugated double bonds. 12-epi-6-trans-LTB4 is also converted to tetrahydro metabolites by these cells. Experiments with deuterium-labeled 6-trans-LTB4 indicated that the deuterium in the 5-position was almost completely lost during the formation of tetrahydro-6-trans-LTB4, whereas about 80-85% of the deuterium in the 12-position was lost, suggesting a requirement for a 5-oxo intermediate. As with LTB4, 12-epi-8-cis-6-trans-LTB4, the product of the combined actions of 5-lipoxygenase and 12-lipoxygenase, was converted principally to dihydro and dihydro-12-oxo metabolites. Only a relatively small amount of the tetrahydro metabolite was detected.     

Separation of carbamazepine and five metabolites,and analysis in human plasma by micellar electrokinetic capillary chromatography

A rapid and feasible method was developed for the analysis of carbamazepine and its five metabolites (10,11-dihydro-10,11-epoxycarbamazepine, 10,11-dihydro-10,11-dihydroxycarbamazepine, 10,11-dihydro-10-hydroxycarbamazepine, 2-hydroxycarbamazepine and 3-hydroxycarbamazepine) in human plasma. Separation of the analytes is based on micellar electrokinetic chromatography, in untreated fused-silica capillary (48.5/40.0 cm length, 50 microm I.D.) with phosphate buffer (30 mM, pH 8.00) as background electrolyte, containing 50 mM sodium dodecylsulfate, and methanol (15%, v/v) as organic modifier. Clean up of human plasma samples was carried out by means of a solid-phase extraction procedure, which gave a high extraction yield for all six carbamazepines (>88%). The overall precision of the method gives a mean RSD of about 1.8%. The limit of quantitation for all analytes is < or = 0.30 microg ml(-1), the limit of detection < or = 0.12 microg ml(-1).     

Biochemical characterization of hepatic microsomal leukotriene B4 hydroxylases

omega-Hydroxylation of leukotriene B4 (LTB4) has been reported in human and rodent polymorphonuclear leukocytes; preliminary information indicates that this metabolism is cytochrome P-450 dependent. Therefore, these studies were initiated to characterize the cytochrome P-450-dependent metabolism of LTB4 in other tissues. LTB4 was metabolized by rat hepatic microsomes to two products, 20-hydroxy(omega)-LTB4 and 19-hydroxy(omega-1)-LTB4. The formation of these metabolites was both oxygen and NADPH dependent indicating that a monooxygenase(s) was responsible for these reactions. The apparent Km and Vmax for LTB4 omega-hydroxylase were 40.28 microM and 1202 pmol/min/mg of protein, respectively. In contrast, the apparent Km and Vmax for LTB4 (omega-1)-hydroxylase were 61.52 microM and 73.50 pmol/min/mg of protein, respectively. Both LTB4 omega- and (omega-1)-hydroxylases were inhibited by metyrapone in a concentration-dependent fashion. However, SK&F 525A inhibited LTB4 (omega-1)- but not omega-hydroxylase. In contrast, alpha-naphthoflavone decreased LTB4 omega- but not (omega-1)-hydroxylase activities. The differences in the Km apparent for substrate as well as the differential inhibition by inhibitors of cytochrome P-450 suggest that the omega- and (omega-1)-hydroxylations of LTB4 in hepatic microsomes are mediated by different isozymes of P-450. Furthermore, several additional characteristics of LTB4 hydroxylases indicate that these isozymes of P-450 may be different from those which catalyze similar reactions on medium-chain fatty acids, such as laurate and prostaglandins.     

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.     

Regio- and stereo-selective metabolism of 4-methylbenz[a]anthracene by the fungus Cunninghamella elegans.

Metabolism of 4-methylbenz[a]anthracene by the fungus Cunninghamella elegans was studied. C. elegans metabolized 4-methylbenz[a]anthracene primarily at the methyl group, this being followed by further metabolism at the 8,9- and 10,11-positions to form trans-8,9-dihydro-8,9-dihydroxy-4-hydroxymethylbenz[a]anthracene and trans-10,11-dihydro-10,11-dihydroxy-4-hydroxymethylbenz[a]anthracene. There was no detectable trans-dihydrodiol formed at the methyl-substituted double bond (3,4-positions) or at the 'K' region (5,6-positions). The metabolites were isolated by reversed-phase high-pressure liquid chromatography and characterized by the application of u.v.-visible-absorption-, 1H-n.m.r.- and mass-spectral techniques. The 4-hydroxymethylbenz[a]anthracene trans-8,9- and -10,11-dihydrodiols were optically active. Comparison of the c.d. spectra of the trans-dihydrodiols formed from 4-methylbenz[a]anthracene by C. elegans with those of the corresponding benz[a]anthracene trans-dihydrodiols formed by rat liver microsomal fraction indicated that the major enantiomers of the 4-hydroxymethylbenz[a]anthracene trans-8,9-dihydrodiol and trans- 10,11-dihydrodiol formed by C. elegans have S,S absolute stereochemistries, which are opposite to those of the predominantly 8R,9R- and 10R,11R-dihydrodiols formed by the microsomal fraction. Incubation of C. elegans with 4-methylbenz[a]anthracene under 18O2 and subsequent mass-spectral analysis of the metabolites indicated that hydroxylation of the methyl group and the formation of trans-dihydrodiols are catalysed by cytochrome P-450 mono-oxygenase and epoxide hydrolase enzyme systems. The results indicate that the fungal mono-oxygenase-epoxide hydrolase enzyme systems are highly stereo- and regio-selective in the metabolism of 4-methylbenz[a]anthracene.     

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.     

Enhanced 5-lipoxygenase activity in lung macrophages compared to monocytes from normal subjects

We compared lipoxygenase activities of lung macrophages obtained from bronchoalveolar lavage to activities of blood monocytes purified by using discontinuous plasma/Percoll density gradients and adherence to tissue culture plastic in five normal subjects. Cells were incubated with ionophore A23187 (10(-9) to 10(-5) M) or arachidonic acid (0.12 to 80 microM) for 1 to 60 min at 37 degrees C to construct dose-response and time-dependence curves of lipoxygenase product generation. Products were identified and were quantified by using high-pressure liquid chromatography and ultraviolet spectroscopy. Under all conditions of product generation, both macrophages and monocytes generated predominantly (5S,12R)-dihydroxy-(6Z, 8E, 10E, 14Z)-eicosatetraenoic acid (leukotriene B4 (LTB4] and (5S)-hydroxy-(6E, 8Z, 11Z, 14Z) - eicosatetraenoic acid (5 - HETE), but, in each subject, macrophages invariably released greater amounts of LTB4 and 5-HETE than monocytes. In response to A23187, macrophages released a maximum of 183 +/- 96 pmol of LTB4 and 168 +/- 108 pmol of 5-HETE per 10(6) cells (mean +/- SEM), whereas monocytes released only 16 +/- 1 and 18 +/- 8 pmol per 10(6) cells of LTB4 and 5-HETE, respectively. After adding arachidonic acid, macrophages released a maximum of 52 +/- 21 pmol of LTB4 and 223 +/- 66 pmol of 5-HETE, whereas monocytes released no detectable products. The results suggest that mononuclear phagocyte maturation in the lung may be accompanied by an enhanced ability to generate 5-lipoxygenase products.     

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 [14C]methamphetamine in man, the guinea pig and the rat

1. The metabolites of (+/-)-2-methylamino-1-phenyl[1-(14)C]propane ([(14)C]methamphetamine) in urine were examined in man, rat and guinea pig. 2. In two male human subjects receiving the drug orally (20mg per person) about 90% of the (14)C was excreted in the urine in 4 days. The urine of the first day was examined for metabolites, and the main metabolites were the unchanged drug (22% of the dose) and 4-hydroxymethamphetamine (15%). Minor metabolites were hippuric acid, norephedrine, 4-hydroxyamphetamine, 4-hydroxynorephedrine and an acid-labile precursor of benzyl methyl ketone. 3. In the rat some 82% of the dose of (14)C (45mg/kg) was excreted in the urine and 2-3% in the faeces in 3-4 days. In 2 days the main metabolites in the urine were 4-hydroxymethamphetamine (31% of dose), 4-hydroxynorephedrine (16%) and unchanged drug (11%). Minor metabolites were amphetamine, 4-hydroxyamphetamine and benzoic acid. 4. The guinea pig was injected intraperitoneally with the drug at two doses, 10 and 45mg/kg. In both cases nearly 90% of the (14)C was excreted, mainly in the urine after the lower dose, but in the urine (69%) and faeces (18%) after the higher dose. The main metabolites in the guinea pig were benzoic acid and its conjugates. Minor metabolites were unchanged drug, amphetamine, norephedrine, an acid-labile precursor of benzyl methyl ketone and an unknown weakly acidic metabolite. The output of norephedrine was dose-dependent, being about 19% on the higher dose and about 1% on the lower dose. 5. Marked species differences in the metabolism of methamphetamine were observed. The main reaction in the rat was aromatic hydroxylation, in the guinea pig demethylation and deamination, whereas in man much of the drug, possibly one-half, was excreted unchanged.