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.     

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.     

Antibodies against rabbit omega-hydroxylases of prostaglandins and fatty acids cross-react with leukotriene B4 omega-hydroxylase in human neutrophils (cytochrome P-450LTB omega)

Leukotriene B4 (LTB4) omega-hydroxylase activity in human neutrophil microsomes was significantly inhibited by antisera against three rabbit omega-hydroxylase P-450s, lung prostaglandin omega-hydroxylase (P-450p-2), small intestine prostaglandin A omega-hydroxylase (P-450ia), and kidney fatty acid omega-hydroxylase (P-450kd). In contrast, the activity is not affected by antibodies raised against the phenobarbital-inducible forms of P-450s from both rabbits and rats. These findings suggest that the LTB4 omega-hydroxylase (P-450LTB omega) is structurally related to a group of rabbit omega-hydroxylase P-450s. The antiserum raised against P-450p-2 also inhibited the NADPH-dependent oxidation of 20-hydroxy LTB4 to 20-oxo LTB4 and 20-carboxy LTB4 by the microsomes, supporting that P-450LTB omega is able to catalyze the subsequent oxidation of 20-hydroxy LTB4 as well as the omega-hydroxylation of LTB4.     

Purification and characterization of 20-hydroxy-leukotriene B4 dehydrogenase in human neutrophils

Leukotriene B4 (LTB4) is converted to 20-hydroxy-LTB4 (20-OH-LTB4) which is subsequently oxidized to 20-carboxy-LTB4 (20-COOH-LTB4). The oxidation of the hydroxy LTB4 to the carboxy LTB4 by human neutrophils was associated with the reduction of NAD+ and required both cytosolic and microsomal fractions. We isolated a cytosolic protein which oxidized the hydroxy LTB4 in the presence of NAD+ and the microsomal fraction. It was homogeneous on SDS/PAGE, with a subunit molecular mass of 37 kDa, and may be a dimeric protein with two identical or similar subunits because its molecular mass, estimated by Sephadex G-100 column chromatography, was about 80 kDa. The protein was an alcohol dehydrogenase with high affinity for omega-hydroxy fatty acids, such as 12-hydroxylaurate and 16-hydroxypalmitate. We conclude that the cytosolic protein oxidizes 20-OH-LTB4 to 20-oxo-LTB4 and the microsomal fraction oxidizes the oxo-LTB4 to the carboxy-LTB4, based on the finding that the activity which oxidizes omega-hydroxy fatty acids is present only in the cytosol fraction, while that which oxidizes hydrophobic aldehydes is found only in the microsomal fraction and that the stoichiometry of the formation of 20-COOH-LTB4 to the reduction of NAD+ was 1:2. The affinity of the dehydrogenase for 20-OH-LTB4 may be higher than that for 12-hydroxylaurate (Km = 70 microM), because the latter inhibited the oxidation of the former by only 40%, at a concentration of 12-hydroxylaurate 10 times higher than that of 20-OH-LTB4. The enzyme activity was not affected by pyrazole and 4-methylpyrazole at millimolar concentrations. These characteristics indicate that the dehydrogenase is a unique type of alcohol dehydrogenase.     

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.     

Characterization of human neutrophil leukotriene B4 omega-hydroxylase as a system involving a unique cytochrome P-450 and NADPH-cytochrome P-450 reductase

Leukotriene B4 (LTB4), a potent chemotactic agent, was catabolized to 20-hydroxyleukotriene B4 (20-OH-LTB4) by the 150,000 x g pellet (microsomal fraction) of human neutrophil sonicate. The reaction required molecular oxygen and NADPH, and was significantly inhibited by carbon monoxide, suggesting that a cytochrome P-450 is involved. The neutrophil microsomal fraction showed a carbon monoxide difference spectrum with a peak at 450 nm in the presence of NADPH or dithionite, indicating the presence of a cytochrome P-450. The addition of LTB4 to the microsomal fraction gave a type-I spectral change with a peak at around 390 nm and a trough at 422 nm, indicating a direct interaction of LTB4 with the cytochrome P-450. The dissociation constant of LTB4, determined from the difference spectra, is 0.40 microM, in agreement with the kinetically determined apparent Km value for LTB4 (0.30 microM). Such a spectral change was not observed with prostaglandins A1, E1 and F2 alpha or lauric acid, none of which inhibited the LTB4 omega-hydroxylation. The inhibition of the LTB4 omega-hydroxylation by carbon monoxide was effectively reversed by irradiation with monochromatic light of 450 nm wavelength. The photochemical action spectrum of the light reversal of the inhibition corresponded remarkably well with the carbon monoxide difference spectrum. These observations provide direct evidence that the oxygen-activating component of the LTB4 omega-hydroxylase system is a cytochrome P-450. Ferricytochrome c inhibited the hydroxylation of LTB4 and the inhibition was fortified by cytochrome oxidase. An antibody raised against rat liver NADPH-cytochrome-P-450 reductase inhibited both LTB4 omega-hydroxylase activity and the NADPH-cytochrome-c reductase activity of human neutrophil microsomal fraction. These observations indicate that NADPH-cytochrome-P-450 reductase acts as an electron carrier in LTB4 omega-hydroxylase. On the other hand, an antibody raised against rat liver microsomal cytochrome b5 inhibited the NADH-cytochrome-c reductase activity but not the LTB4 omega-hydroxylase activity of human neutrophil microsomal fraction, suggesting that cytochrome b5 does not participate in the LTB4-hydroxylating system. These characteristics indicate that the isoenzyme of cytochrome P-450 in human neutrophils, LTB4 omega-hydroxylase, is different from the ones reported to be involved in omega-hydroxylation reactions of prostaglandins and fatty acids.     

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.     

NAD+-dependent retinol dehydrogenase in liver microsomes

A microsomal NAD+-dependent retinol dehydrogenase is being described with optimal activity at physiological pH. The enzyme was present in liver microsomes of rats and also in a strain of deermice which lacks the cytosolic retinol dehydrogenase. Unlike the latter enzyme, the microsomal retinol dehydrogenase was not inhibited by either ethanol or 4-methylpyrazole; its activity was insensitive to CO and not oxygen dependent, in contradistinction with that of the microsomal cytochrome P-450 and NADPH-dependent retinol oxidase. Chronic ethanol consumption resulted in an increased activity of the microsomal retinol dehydrogenase which may contribute to hepatic retinol depletion, especially in view of the insensitivity of the enzyme to ethanol inhibition.     

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.     

Leukotriene B4 omega-hydroxylase in rat liver microsomes: identification as a cytochrome P-450 that catalyzes prostaglandin A1 omega-hydroxylation, and participation of cytochrome b5

The omega-hydroxylation of leukotriene B4 (LTB4) by rat liver microsomes requires NADPH and molecular oxygen, suggesting that the hydroxylation is catalyzed by a cytochrome P-450 (P-450)-linked monooxygenase system. The reaction is inhibited by CO, and the inhibition is reversed by irradiation of light at 450 nm in a light-intensity-dependent manner. The extent of the reversal is strongly dependent on the wavelength of the light used, the 450-nm light is most efficient. The finding provides direct evidence for the identification of the LTB4 omega-hydroxylase as a P-450. The P-450 seems to be also responsible for prostaglandin A1 (PGA1) omega-hydroxylation, but not for lauric acid omega-hydroxylation. The LTB4 omega-hydroxylation is competitively inhibited by PGA1, but not affected by lauric acid. The Ki value for PGA1 of 38 microM agrees with the Km value for PGA1 omega-hydroxylation of 40 microM. LTB4 inhibits the PGA1 omega-hydroxylation by rat liver microsomes in a competitive manner with the Ki of 43 microM, which is consistent with the Km for the LTB4 omega-hydroxylation of 42 microM. An antiserum raised against rabbit pulmonary PG omega-hydroxylase (P-450p-2) inhibits slightly the omega-hydroxylations of LTB4 and PGA1, while it has stronger inhibitory effect on lauric acid omega-hydroxylation. In addition to NADPH-cytochrome P-450 reductase, cytochrome b5 appears to participate in the LTB4 omega-hydroxylating system, since the reaction is inhibited by an antibody raised against the cytochrome b5 as well as one raised against the reductase.     

Stereochemical requirements for substrate specificity of LTB4 20-hydroxylase

LTB4 20-hydroxylase (P-450LTB) is the cytochrome P-450 in the microsomes of human polymorphonuclear leukocytes that catalyzes the omega-oxidation of leukotriene B4 (LTB4) to 20-OH LTB4. The activity of P-450LTB for LTB4 compared to isomers and analogs of LTB4 at a concentration of 0.3 microM revealed a preference of P-450LTB for both the triene bond configuration of LTB4 and for the chirality of the 5S and 12R hydroxyl groups. 15S-Hydroxyeicosatetraenoic acid, 8(R/S), 15S-dihydroxy-5-cis-9,11,13-trans-eicosatetraenoic acid, 8R,15S-dihydroxy-5,13-cis-9,11-trans-eicosatetraenoic acid, and 5S,15S-dihydroxy-6,13-trans-8,11-cis-eicosatetraenoic acid were each not subject to omega-oxidation, indicating a negative effect of the presence of a 15-hydroxyl group on substrate recognition. At a concentration of 1.5 microM, 12R- and 12S-hydroxyeicosatetraenoic acid were converted to their respective 20-OH derivatives at rates that were 34.2 +/- 11.6% (mean +/- S.D., n = 3) and 3.5 +/- 4.3% (mean +/- S.D., n = 4), respectively, of that of LTB4 to 20-OH LTB4, further indicating that P-450LTB can distinguish the chirality of the 12-hydroxyl group. The lower Km of LTB4 (2.0 microM), as compared to those of its 6-trans-12-epi isomer (3.8 microM) and 5-epi-LTB4 (6.6 microM) confirmed the preference of P-450LTB for the specific triene bond structure of LTB4 and its preference for the chirality of the hydroxyl groups of LTB4 within this structurally related class of molecules. At equal 1.5-microM concentrations, LTB4 completely inhibited the omega-oxidation of all other substrates and partially suppressed that of leukotriene B5, consistent with the lower Km of LTB4 and indicating that P-450LTB catalyzed the omega-oxidation of all substrates. Thus, P-450LTB is a novel cytochrome P-450 of human polymorphonuclear leukocytes with substrate recognition determined by the triene bond configuration and the chirality of the hydroxyl groups.     

Immunochemical studies on the contribution of NADPH cytochrome P-450 reductase to the cytochrome P-450-dependent metabolism of arachidonic acid

We have studied the role of NADPH cytochrome P-450 reductase in the metabolism of arachidonic acid and in two other monooxygenase systems: aryl hydrocarbon hydroxylase and 7-ethoxyresorufin-o-deethylase. Human liver NADPH cytochrome P-450 reductase was purified to homogeneity as evidenced by its migration as a single band on SDS gel electrophoresis, having a molecular weight of 71,000 Da. Rabbits were immunized with the purified enzyme and the resulting antibodies were used to evaluate the involvement of the reductase in cytochrome P-450-dependent arachidonic acid metabolism by bovine corneal epithelial and rabbit renal cortical microsomes. A highly sensitive immunoblotting method was used to identify the presence of NADPH cytochrome P-450 reductase in both tissues. We used these antibodies to demonstrate for the first time the presence of cytochrome c reductase in the cornea. Anti-NADPH cytochrome P-450 reductase IgG, but not anti-heme oxygenase IgG, inhibited the NADPH-dependent arachidonic acid metabolism in both renal and corneal microsomes. The inhibition was dependent on the ratio of IgG to microsomal protein where 50% inhibition of arachidonic acid conversion by cortical microsomes was achieved with a ratio of 1:1. A higher concentration of IgG was needed to achieve the same degree of inhibition in the corneal microsomes. The antibody also inhibited rabbit renal cortical 7-ethoxyresorufin-o-deethylase activity, a cytochrome P-450-dependent enzyme. However, the anti-NADPH cytochrome P-450 reductase IgG was much less effective in inhibiting rabbit cortical aryl hydrocarbon hydroxylase. Thus, the degree of inhibition of monooxygenases by anti-NADPH cytochrome P-450 reductase IgG is variable. However, with respect to arachidonic acid, NADPH cytochrome P-450 reductase appears to be an integral component for the electron transfer to cytochrome P-450 in the oxidation of arachidonic acid.     

Leukotriene B4 metabolism by hepatic cytochrome P-450

Leukotriene B4 (LTB) was found to be metabolized by suspensions of rat liver microsomes in the presence of NADPH and oxygen. The rate of LTB metabolism was also measured in reconstituted systems of both micelles and phospholipid vesicles containing cytochrome P-450-LM2, NADPH cytochrome P-450 reductase, and cytochrome b5. A 1 microM concentration of LTB was metabolized by rat hepatic microsomes at a rate of 4 pmol LTB/min/nmole P-450, and by vesicle and micelle reconstituted systems at 3 pmole/min/nmole P-450-LM2. At this rate a 10 g rat liver exposed to 1 microM LTB can metabolize 30 micrograms per hour. In that the leukotrienes are pharmacologically active at nanomolar concentrations, hepatic metabolism may be an important pathway of leukotriene inactivation.     

The effect of alpha-ecdysone and phenobarbital on the alpha-ecdysone 20-monooxygenase of house fly larva

The NADPH-dependent cytochrome P-450 20-monooxygenation of alpha-ecdysone is catalyzed both by mitochondria and microsomes isolated from Musca domestica, L. larvae, but about 50% of the activity is associated with mitochondria and 37% with microsomes. The mitochondrial activity is increased by pretreatment with alpha-ecdysone with a concomitant decrease in Km values. This effect is not observed in microsomes. Induction with phenobarbital represses the mitochondrial 20-monooxygenase but does not change the microsomal activity, although a large increase in cytochrome P-450 is observed in the latter fraction. It is concluded that only the mitochondrial 20-monooxygenase appears to be regulated by alpha-ecdysone which suggests that mitochondrial cytochrome P-450 forms are involved in the moulting phenomenon; whereas, microsomal cytochrome P-450 activity may be of a nonspecific nature and not relevant to development.     

Styrene metabolism in Exophiala jeanselmei and involvement of a cytochrome P-450-dependent styrene monooxygenase.

The yeast-like fungus Exophiala jeanselmei degrades styrene via initial oxidation of the vinyl side chain to phenylacetic acid, which is subsequently hydroxylated to homogentisic acid. The initial reactions are catalyzed by a NADPH- and flavin adenine dinucleotide-dependent styrene monooxygenase, a styrene oxide isomerase, and a NAD(+)-dependent phenylacetaldehyde dehydrogenase. The reduced CO-difference spectrum of microsomal preparations of styrene-grown cells shows a characteristic absorption maximum at 450 nm, which strongly suggests the involvement of a cytochrome P-450-dependent styrene monooxygenase. Inhibition of styrene monooxygenase activity in cell extracts by cytochrome P-450 inhibitors SKF-525-A, metyrapone, and CO confirms this assumption.     

Formation of 20-oxoleukotriene B4 by an alcohol dehydrogenase isolated from human neutrophils

When 20-hydroxyleukotriene B4 (20-OH-LTB4) is incubated at pH 10.5 in the presence of NAD+ with an alcohol dehydrogenase isolated from human neutrophils, a polar product is formed as detected on reverse-phase high-performance liquid chromatography (RP-HPLC). The product is identified as 20-oxo-LTB4 (20-CHO-LTB4) on the basis of its co-elution with the authentic compound on HPLC, ultraviolet spectrometry and gas chromatography-mass spectrometry. The 20-CHO-LTB4-forming activity requires NAD+, but NADP+ scarcely replaces NAD+. The apparent Km for 20-OH-LTB4 is 83 microM and the Vmax is 2.04 mumol/min per mg of protein. The activity is inhibited by omega-hydroxy fatty acids such as 12-hydroxylauric acid, 16-hydroxypalmitic acid and 12(S), 20-dihydroxyeicosatetraenoic acid, but not by 4-methylpyrazole. At pH 7.0 with NADH, the purified dehydrogenase catalyzes the reverse reaction, the reduction of 20-CHO-LTB4 to 20-OH-LTB4.     

NAD+-dependent oxidation of 20-hydroxyleukotriene B4 to 20-carboxyleukotriene B4 by rat liver cytosol

20-Hydroxyleukotriene B4 was converted by rat liver homogenates in the presence of NAD+ to a more polar product on reverse-phase high-performance liquid chromatography. The product was identified as 20-carboxyleukotriene B4 by straight-phase high performance liquid chromatography, ultraviolet spectrophotometry and gas chromatography-mass spectrometry. The oxidative activity of the homogenates was located in the cytosol with an optimal pH of 8.0. The activity was dependent on NAD+, and NADP+ could not substitute for NAD+. 1 mol of 20-carboxyleukotriene B4 was formed with the reduction of 2 mol of NAD+. The reaction was inhibited by pyrazole and 4-methylpyrazole, inhibitors of alcohol dehydrogenase, and by various alcohols, such as ethanol, 12-hydroxylaurate, and 20-hydroxyprostaglandin E1. Disulfiram, an inhibitor of aldehyde dehydrogenase, also inhibited the activity. These results suggest that two discrete steps catalyzed by different enzymes, alcohol dehydrogenase and aldehyde dehydrogenase, are involved in the oxidation of 20-hydroxyleukotriene B4 in rat liver cytosol. The enzyme system seems to be different from that of human neutrophils.     

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.     

Cytochrome P-450 and oxygen toxicity. Oxygen-dependent induction of ethanol-inducible cytochrome P-450 (IIE1) in rat liver and lung

The ethanol-inducible form of cytochrome P-450 (P-450IIE1) has previously been shown to exhibit an unusually high rate of oxidase activity with the subsequent formation of reactive oxygen species, e.g., hydrogen peroxide, and to be the main contributor of microsomal oxidase activity in liver microsomes from acetone-treated rats [Ekstr?m & Ingelman-Sundberg (1989) Biochem. Pharmacol. (in press)]. The results here presented indicate that oxygen exposure of rats causes an about 4-fold induction of P-450IIE1 in rat liver and lung microsomes. The induction in liver was not accompanied by any measurable increase in the P-450IIE1 mRNA levels, but the enhanced amount of P-450IIE1 accounted for 60% of the net 50% increase in the level of hepatic P-450 as determined spectrophotometrically. The induction of P-450IIE1 was maximal after 60 h of O2 exposure, and concomitant increases in the rates of liver microsomal CCl4-dependent lipid peroxidation, O2 consumption, NADPH oxidation, O2- formation, H2O2 production, and NADPH-dependent microsomal lipid peroxidation were seen. Liver microsomes from oxygen-treated rats had very similar properties to those of microsomes isolated from acetone-treated rats with respect to the P-450IIE1 content and catalytic properties, but different from those of thyroxine-treated animals. Treatment of rats with the P-450IIE1 inducer acetone in combination with oxygen exposure caused a potentiation of the NADPH-dependent liver and lung microsomal lipid peroxidation and decreased the survival time of the rats. The results reached indicate a role for cytochrome P-450 and, in particular, for cytochrome P-450IIE1 in oxygen-mediated tissue toxicity.     

On the intracellular localization of the ecdysone 20-monooxygenase in Musca domestica. L

The cytochrome P-450-dependent 20-monooxygenation of ecdysone is catalyzed both by mitochondria and microsomes isolated from Musca domestica (L.) larvae; however, about 50% of the activity is associated with mitochondria, and 37% is associated with microsomes. Pretreatment of larvae with ecdysone results in an increase in Vmax and a decrease in Km values in mitochondria but not in microsomes. Phenobarbital, a known cytochrome P-450 inducer, increases the cytochrome P-450 levels in microsomes without affecting the 20-monooxygenase activity, but both the cytochrome P-450 levels and monooxygenase activity are depressed in mitochondria from phenobarbital-pretreated larvae. The ecdysone 20-monooxygenase activity is equally distributed between mitochondria and microsomes in adult insects. Pretreatment of the insects with ecdysone does not significantly modify the 20-monooxygenase activity of either mitochondrial or microsomal fractions, but the cytochrome P-450 levels are reduced in mitochondria. Phenobarbital also depresses the mitochondrial cytochrome P-450 levels while markedly increasing the microsomal cytochrome P-450 levels. However, no significant changes in ecdysone 20-monooxygenase activity are produced by phenobarbital pretreatment. The effects of ecdysone on adult cytochrome P-450 are mostly evidenced in mitochondria isolated from females, whereas in males the changes are not statistically significant. It is concluded that the mitochondrial ecdysone 20-monooxygenase is under regulatory control by ecdysone in the larval stage, which suggests that only the mitochondrial activity has a physiological role during insect development in M. domestica. In adults, both the mitochondrial and microsomal ecdysone 20-monooxygenase activities are not responsive to ecdysone, which, coupled to their high Km values, indicates that the reaction may not be of physiological importance in adult insects and that the mitochondrial cytochrome P-450 species being depressed by ecdysone in females are possibly not involved in ecdysone metabolism.