the of chemistry vol. no. 5, pp. by for in u. s. a ... · mation of dihydro products by this...

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1991 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 266, No. 31, Issue of November 5, pp. 20899-20906,1991 Printed in U. S. A.

Mechanism for the Formation of Dihydro Metabolites of 12-Hydroxyeicosanoids CONVERSION OF LEUKOTRIENE B4 AND 12-HYDROXY-5,8,10,14-EICOSATETRAENOIC ACID T O 12-OXO INTERMEDIATES*

(Received for publication, June 21, 1991)

Sandra L. Wainwright and William S. Powell$ From the Meakins-Christie Laboratories, Respiratory Health Network of Centers of Excellence, Dept. of Medicine, McGill University, Montreal, Quebec, Canada, H2X 2P2, and the Endocrine Laboratory, Royal Victoria Hospital, Montreal, Quebec, Canada H3A l A 1

Eicosanoids containing a 12-hydroxyl group preceded by at least two conjugated double bonds are metabolized to l0,ll-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 l0,ll-dihydro metabolites could either have been formed by the direct reduction of the 10,ll- 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 (LTB,), 12(S)-hydroxy-5,8,10,14-eicosatetraenoicacid(l2(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 X,,, 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 LTB,, this was confirmed by mass spectrometry, which indicated that the product was identical to 12-oxo-LTB,. LTB, was not converted to any products by a cytosolic fraction from PMNL, but was converted to both 10,l l-dihydro- LTB, and 10,ll-dihydro-12-oxo-LTB, by the 1500 X 8 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 l0,ll-double bond. Consistent with this hypothesis, 12-oxo-LTB, was rapidly metabolized to 10,l 1-dihydro-12-oxo-LTB, by the cytosolic fraction in the presence of NADH. Only small amounts of this product, along with some LTB,, were formed by the microsomal fraction. These results indicate that the initial step in the formation of l0,ll-dihydro products from 12-hydroxyeicosanoids is oxidation of the 12-hydroxyl group by a microsomal 12-hydroxyeicos-

* This work was supported by a grant from the Medical Research Council of Canada (WSP). SLW was supported by a studentship award from the Medical Research Council of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduer- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$To whom all correspondence should he addressed Meakins- Christie Laboratories, 3626 St. Urbain St., Montreal, Quebec, Canada H2X 2P2.

anoid dehydrogenase in the presence of NAD+, which is followed by reduction of the olefinic double bond by a cytosolic Alo-reductase in the presence of NADH.

Leukotriene B, (LTB,)’ is an arachidonic acid metabolite produced by the 5-lipoxygenase pathway in polymorphonuclear leukocytes (PMNL) (1). LTB4 is a potent proinflammatory agent, primarily due to its actions on PMNL, which include chemotaxis (2, 3), aggregation (2), degranulation (4, 5), and stimulation of adherence to vascular endothelium (6). 12-Hydroxy-5,8,10,14-eicosatetraenoic acid (12(S)-HETE) is another metabolite of arachidonic acid which is formed by the 12-lipoxygenase pathway in platelets and porcine PMNL. 12(S)-HETE has chemotactic and chemokinetic effects on PMNL, although it is about 3000 times less potent than LTB, (3). 12(R)-HETE, formed by a cytochrome P-450 (7), is a more potent chemotactic agent than 12(S)-HETE (8), and may be involved in psoriasis (9).

LTB, and 12-HETE are metabolized by several pathways. In human PMNL, both substrates are metabolized primarily by w-oxidation to the corresponding 20-hydroxy (10, 11) and w-carboxy compounds (12). LTB, is metabolized to 19-hydroxy-LTB, by rat PMNL (13), rat mononuclear cells (14), and rat liver microsomes (15), and to 18-hydroxy-LTB, by rat PMNL (16). In general, w-oxidation products derived from LTB, have considerably less biological activity than LTB,, with the exception of the contraction of guinea pig parenchymal strips in which case 20-hydroxy-LTB, and LTB, are equipotent (12).

There are at least two different reductase pathways for the metabolism of mono- and dihydroxy eicosanoids. The 6,11- reductase pathway, which was first described in human PMNL (17, 18), results in the metabolism of 12-epi-6-tram- LTB, to 5,12-dihydroxy-7,9,14-eicosatrienoic acid. However, LTB, and 12-HETE are not substrates for this pathway.’ Another reductase pathway is present in rat (13) and porcine (19) PMNL, which results in reduction of the 10,ll-double bond of eicosanoids containing a hydroxyl group in the 12

The abbreviations used are: LT, leukotriene; PG, prostaglandin; 12-HETE, 12-hydroxy-5,8,10,14-eicosatetraenoic acid 13-HODE, 13- hydroxy-9,11-octadecadienoic acid; 12-oxo-ETE, 12-oxo-eicosatetraenoic acid; 13-oxo-ODE, 13-oxo-octadecadienoic acid diHETE, dihydroxy-eicosatetraenoic acid PMNL, polymorphonuclear leukocytes; ETYA, 5,8,11,14-eicosatetraynoic acid; RP-HPLC, reversed- phase high pressure liquid chromatography; NP-HPLC, normal- phase high pressure liquid chromatography; tR, retention time.

W. S. Powell, unpublished work.

20899

20900 12-Oxo-leukotriene B4

position preceded by at least two conjugated double bonds (22). Both LTB, (19) and 12-HETE (20), as well as 6-tran.s isomers of LTB, (21), are metabolized by the A''-reductase, which, in porcine and rat PMNL, appears to be associated with 12-hydroxyeicosanoid dehydrogenase, resulting in the formation of 10,ll-dihydro- and 10,11-dihydro-12-oxo compounds. Porcine PMNL also catalyze inversion of the stereochemistry at Clz of the dihydro metabolites of LTB, (22) and 12-HETE (20), giving rise to the corresponding 10,11- dihydro-12-epi metabolites.

LTB, has also been reported to be metabolized by the A'"- reductase pathway in human alveolar macrophages (23), monocytes (24), and mesangial cells (25), rat mesangial cells and fibroblast tumour cells, and mouse T-lymphocytes and macrophages (26), as well as by human lung (27). The dihydro metabolite of LTB, produced by rat mesangial cells (28) and the two dihydro-LTB, isomers produced by human mesangial cells (25) appear to have little proinflammatory activity. 10,ll-Dihydro-LTB, and lO,ll-dihydro-12-epi-LTB, were found to be approximately 100 times less potent than LTB, in stimulating contraction of guinea pig lung parenchymal strips, as well as in increasing microvascular permeability in the hamster cheek pouch (27). On the other hand, the 10,ll- dihydro metabolite of 12(S)-HETE, which is formed by a different mechanism in bovine corneal microsomes (29), has been reported to be a potent proinflammatory agent (29, 30).

The mechanism for the formation of 10,ll-dihydro metabolites of LTB, is not clear. Experiments in which LTB,, containing a deuterium atom in the 12 position, was incubated with porcine PMNL indicated that about 65% of the deuterium was retained in the resulting 10,ll-dihydro metabolite (19). Similar results were obtained for the conversion of 12(S)-HETE to lO,ll-dihydro-12-HETE (31). This suggested that the l0,ll-double bond of these substrates could be directly reduced without the requirement for activation by the formation of a 12-oxo intermediate. On the other hand, the time course for the metabolism of LTB, by both porcine (19) and rat (16) PMNL indicated that 10,1l-dihydro-12-0~0- LTB, was initially formed more rapidly than l0,ll-dihydro- LTB,, whereas the latter product predominated later. This would be consistent with the initial oxidation of the 12- hydroxyl group of LTB,, followed by reduction of the 10,ll- double bond and finally reduction of the 12-oxo group back to a hydroxyl group to give 10,ll-dihydro-LTB,.

The major goal of the present investigation was to determine the mechanism for the formation of the 10,ll-dihydro metabolites of LTB, and 12-HETE. Our results indicate that the initial step in this reaction is the formation of a 12-oxo intermediate by a microsomal 12-hydroxyeicosanoid dehydrogenase. This is followed by reduction of the 10,ll-double bond by a cytosolic A''-reductase. In the case of LTB,, the 12-oxo- intermediate has been isolated and identified on the basis of its UV and mass spectra.

EXPERIMENTAL PROCEDURES

Materials

Diazald and A23187 were obtained from Aldrich and Calbiochem, respectively. Unlabeled arachidonic acid was purchased from Nuchek Prep Inc. (Elysian, MN), whereas [1-"C]arachidonic acid was obtained from Du Pont-New England Nuclear. Unlabeled PGF,, was obtained from The Upjohn Co. (Kalamazoo, MI). 5,8,11,14-Eicosa- tetraynoic acid (ETYA) was kindly provided by Dr. J. R. Paulsrud of Hoffman-La Roche. Dithiothreitol, phenylmethylsulfonyl fluoride, o-anisidine dihydrochloride, phenolphthalein glucuronic acid, PGB2, NADH, and NADP+ were purchased from Sigma. Ouabain, adeno- sine-5'-triphosphate, NAD+, and NADPH were purchased from Boehringer Mannheim (St. Laurent, Quebec).

Biosynthesis of Substrates Unlabeled LTB, was prepared by incubation of arachidonic acid

and A23187 (10 p ~ ) with porcine PMNL which were preincubated with ETYA (32, 33). Unlabeled 12(S)-HETE was prepared under identical conditions except that ETYA was not present. Unlabeled 12(R)-HETE was prepared by incubation of arachidonic acid with a homogenate of sea urchin eggs (Strongylocentrotus purpurutus) as described by Brash et al. (34). [1-"C]LTB4 was prepared by incubation of [l-'4C]arachidonic acid with purified human PMNL and A23187 (10 pM) for 5 min at 37 "C (35). 12(S)-[1-'4C]HETE was prepared by incubation of [l-'4C]arachidonic acid with human platelets (36). [9@-3H]PGF2, was prepared by reducing PGE, with tritiated NaBH4, followed by purification by RP-HPLC on a Spherisorb ODS- 2 column (Jones Chromatography Ltd.) with a gradient between acetonitrile/water/acetic acid (30:700.02) and acetonitrile/water/ acetic acid (4060:0.02) over 30 min at a flow rate of 1.5 ml/min.

Preparation of Porcine PMNL Porcine peripheral blood was collected in the presence of EDTA

(final concentration, 10 mM). Leukocytes were prepared as previously described (11) by treatment of the blood with Dextran T-500 (Phar- macia Fine Chemicals, Dorval, Quebec), to sediment the red blood cells. PMNL were purified by centrifugation over Ficoll-Paque (Phar- macia LKB Biotechnology Inc.). Any remaining red blood cells were lysed with 0.135 M NH4C1. After washing with 0.15 mM NaC1, the cells were resuspended in Tris-acetate-sucrose buffer (0.01 M Tris- acetate, 0.25 M sucrose, pH 7.4) containing 2 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, and 1 mM EDTA.

Cell Disruption and Fractionation Purified porcine PMNL (187.5 X 106/ml) were disrupted by soni-

cation (Model 4710 Ultrasonic Homogenizer; Sonics & Materials, Danbury, CT) for 2 X 10 s at a setting of 40 cycles/s. The disruptate was centrifuged at 1500 X g at 4 "C for 10 min to remove unbroken cells and nuclei. The postnuclear supernatant was centrifuged at 20,000 X g at 4 "C, for 20 min producing a granule-rich pellet. The 20,000 X g supernatant was centrifuged at 150,000 X g at 4 "C, for 120 min, to give microsomal and cytosolic fractions. The 150,000 X X g pellet was resuspended in a volume of Tris-acetate-sucrose buffer identical to the initial volume of the sonicate.

Preparation of IZ-oZO-LTB4

12-oxo-LTB4 was prepared by incubation of LTB4 (4 pM) with the microsomal fraction from porcine PMNL (approximately 1.5 mg of protein/ml) for 40 min at 37 "C. The incubation was terminated by the addition of MeOH (final concentration, 15%), followed by immediate extraction on a cartridge of ODS-silica at neutral pH (37). 12-Oxo-LTB4 was partially purified by RP-HPLC on a Novapak Cle column (3.9 X 150 mm; Waters-Millipore) with a mobile phase of acetonitrile/water/acetic acid (3862:0.02) at a flow rate of 1 ml/min (tR, 25 min). Further purification was achieved by NP-HPLC on a column of silicic acid (5-pm RoSil, Alltech Associates, Inc., Deerfield, IL) with a mobile phase of hexane/2-propanol/acetic acid (95.5:4.5:0.1) at a flow rate of 2 ml/min (tR, 19 min).

Gas Chromatography-Mass Spectrometry Purified 12-oxo-LTB4 was methylated with diazomethane and hy-

drogenated with rhodium on alumina (1 mg; Aldrich) in 0.6 ml of MeOH for 3 min at 23 "C. The reagent was subsequently removed by filtering the sample through a small column of silicic acid. Hydrogen- ated 12-oxo-LTB4 methyl ester was then converted to its trimethylsilyl ether derivative by treatment with N-methyl-N-trimethylsilyl- trifluoroacetamide (30 min, 23 "C). Electron impact GC-MS was carried out on a VG ZAB instrument located in the Biomedical Mass Spectrometry Unit of McGill University. The stationary phase was a column (20 m X 0.32 mm) of DB-1 (J and W Scientific, Inc.).

Marker Enzyme Assays Myeloperoridase (38)"Aliquots (100 pl) of subcellular fractions

from porcine PMNL were mixed with 1 mM H~02, 0.33 mM O- dianisidine, and 0.01 M phosphate buffer and the rate of increase of absorbance at 460 nm monitored. One unit of activity is equivalent to an increase in absorbance of O.OOl/min.

@-Glucuronidase (39)"Aliquots (200 pl) of subcellular fractions were incubated with 1 mM phenolphthalein glucuronic acid in 0.1 M

12-Oxo-leukotriene Bq 20901

acetate buffer (pH 4.6) in a total volume of 1 ml for 6 h at 37 "C. The reaction was stopped by the addition of 2 ml of ice-cold glycine buffer (0.2 M glycine and 0.2 M NaC1, pH 10.4). Phenolphthalein released during the incubation was quantitated by measuring absorbance at 550 nm.

Ouabain-sensitive Na+-R-ATPase (4O)"Aliquots (200 pl) of subcellular fractions were incubated with 0.2 mM ATP in 10 mM Tris- HCI, pH 8.6, containing 5 X lo-' M EGTA under the following conditions: (a) Mg+', Na+, K+; (b) Mg+', Na+, K+, ouabain; (c) Mg+*. The final ionic concentrations were: Mg+' 0.26 mM, Na+ 100 mM, K+ 10 mM, and ouabain 0.1 mM. All incubations were for 10 min at 37 "c in a final volume of 1 ml and were terminated by the addition of 0.15 ml of 30% trichloroacetic acid and immediately placed on ice. Re- leased phosphate was measured as described in the literature (41), except that the samples were left at 4 "C for 15 h to allow for color development with minimal ATP hydrolysis. Results were expressed as ouabain-inhibitable ATPase activity.

Analysis of Eicosanoids Formed by Subcellular Fractions from Porcine PMNL

Subcellular fractions from porcine PMNL were incubated at 37 "C for various times with various substrates in Tris-acetate-sucrose buffer, containing 1 mM MgClZ and 1.8 mM CaClZ. The reactions were terminated by the addition of 0.6 ml of ice-cold methanol and immediate cooling to -20 "C. PGBz (300 ng) was added to each sample as an internal standard. The samples were then analyzed by precolumn extraction/RP-HPLC (42) using an ULTRA WISP automatic injector and a WAVS automated switching valve (Waters-Millipore). UV absorbance was monitored with a Waters-Millipore Model 991 photodiode array detector. The material retained on the precolumn (CIS p-Bondapak Guard-Pak cartridge; Waters-Millipore) was analyzed on a Novapak Cle column (3.9 X 150 mm; Waters-Millipore). The mobile phase for each substrate was as follows: LTB, (acetonitrile/water/acetic acid (39:61:0.02); isocratic); 12(S)-HETE and 12(R)-HETE (linear gradient between acetonitrile/water/acetic acid (50:500.02) and acetonitrile/water/acetic acid (55:45:0.02) over 30 min); PGF,, (linear gradient between acetonitrile/water/acetic acid (26:74:0.02) and acetonitrile/water/acetic acid (4654:0.02) over 30 min). The flow rate was 1 ml/min in all cases. The products were quantitated either by measuring the radioactivity by liquid scintilla- tion counting in fractions collected every minute or by measuring UV absorbance. The extinction coefficients used were as follows; LTB,, 39,500,280 nm; 12-oxo-LTB4, 41,000,318 nm; 12-HETE, 30,500,235 nm; la-oxo-ETE, 36,500,280 nm; PGBz, 28,600,280 nm.

Incubations of 12-oxo-LTB4 with subcellular fractions were per- formed under identical conditions, except that they were terminated by the addition of 17% MeOH (10 ml), followed immediately by extraction on cartridges of ODS silica at neutral pH (37). Products were resolved by RP-HPLC on a Novapak c18 column (3.9 X 150 mm: Waters-Millipore), with a mobile phase of acetonitrile/water/ acetic acid (39:61:0.02) at a flow rate of 1.0 ml/min.

RESULTS

Tritium Isotope Effect in the Formation of 10,ll-Dihydro- 12-HETE-Previous experiments in which LTB, (19) and 12-HETE (31) labeled with deuterium in the 12 position were incubated with intact porcine PMNL indicated that the 10,ll- dihydro products retained between half and two-thirds of the deuterium in this position. Since oxidation of the 12-hydroxyl group would result in complete loss of the deuterium from the 12 position, this suggested that PMNL contained a reductase which could reduce the l0,ll-double bond of the substrate directly, without the requirement for a 12-oxo intermediate. The partial loss of deuterium from the 12 position could be explained by the subsequent conversion of the initially formed dihydro products to dihydro-oxo metabolites, which is a re- versible reaction (22). If this is true, we would predict that the presence of a deuterium or tritium atom in the 12 position would not affect the formation of the l0,ll-dihydro metabolite, but would retard the formation of the subsequently formed 10,11-dihydro-12-oxo metabolite. To test this hypothesis, we incubated a mixture of 12(S)-[1-14C]HETE and 12(S)-[5,6,8,9,11,12,14,15-3H]HETE with porcine PMNL and

analyzed the products by RP-HPLC (Fig. 1). Contrary to what we had predicted, the ratio of 3H to 14C in l0,ll-dihydro- 12-HETE and 10,ll-dihydro-12-oxo-ETE remained about the same. However, the proportion of 12(S)-[3H]HETE converted to tritiated dihydro products was much lower than the proportion of 12(S)-[14C]HETE converted to the corresponding 14C-labeled products (Fig. 1). This marked isotope effect suggests that the initial step in the formation of dihydro metabolites from 12(S)-HETE is oxidation of the 12-hydroxyl group, which would require cleavage of a carbon-tritium bond.

Effects of Different Cofactors on the Formation of Dihydro Metabolites of LTB, by the Postnuclear Supernatant Fraction from PMNL-If LTB4 and 12-HETE could be reduced directly by a lO,ll-reductase, we would expect the reaction to require cofactors such as NADH or NADPH, whereas oxidized cofactors such as NAD' or NADP+ would be required if oxidation of the 12-hydroxyl group were the initial rate- limiting step. To determine which cofactors are required for the formation of 10,ll-dihydro metabolites of 12-hydroxyeicosanoids, porcine PMNL were disrupted by sonication and the sonicate was centrifuged at 1500 x g to remove undis- rupted cells and nuclei. LTB, was incubated with the 1500 X x g supernatant fraction for 10 min at 37 "C either in the absence of cofactors or in the presence of NAD+, NADP', NADH, or NADPH (Table I). In the presence of NAD+, large amounts of 10,ll-dihydro-LTB, and l0,ll-dihydro-12-oxo- LTB, were formed from LTB4, whereas only a relatively small amount of 10,ll-dihydro-LTB, was formed in the presence of NADH and no 10,ll-dihydro products were detected in the absence of cofactors or in the presence of either NADP+ or NADPH. These results therefore also suggest that the initial step in the formation of 10,ll-dihydro metabolites is oxidation of the 12-hydroxyl group.

Metabolism of LTB, by Subcellular Fractions from Porcine PMNL-To attempt to clarify the individual steps involved in the formation of dihydro metabolites of 12-hydroxyeicosanoids, various subcellular fractions were prepared from porcine PMNL. The activities of marker enzymes for azurophilic granules (myeloperoxidase and @glucuronidase), and plasma membranes (ouabain-sensitive Na+-K+ ATPase) were measured in each fraction (Table 11). Myeloperoxidase and /I- glucuronidase activities were localized primarily in the 20,000 r- , \ f E T E ~

~

dh-fZ-HETE

IZISI-HETE

~ ~ ~ ~ ~ ~ , ~ ~ ~ ~ ~ ~ ~ , ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ i

g l~ p&~wr~';y?!?&z&6* IJ

TlME (mid

FIG. 1. RP-HPLC profile of the metabolites of 12(S)-[1-'4C] HETE and 12(S)-[5,6,8,9,11,12,14,15-3H]HETE produced by intact porcine PMNL. A mixture of 12(S)-[l-"C]HETE and 12(S)-[5,6,8,9,11,12,14,15-3H]HETE (2 p ~ ) was incubatedwith intact porcine PMNL (50 X lo6 cells/ml) for 30 min at 37 "C. Products were resolved by precolumn extraction/RP-HPLC. Products retained on the precolumn were separated on a Spherisorb ODS-2 column (Jones Chromatography Ltd.) with a linear gradient between acetonitrile/ water/acetic acid (20:80:0.02) and acetonitrile/water/acetic acid (52:480.02) over 60 min at a flow rate of 2.0 ml/min. Radioactivity in the column eluate was analyzed by a Ramona-5-LS liquid cell

HETE, 10,ll-dihydro-12-HETE; dho-l2-ETE, 10,11-dihydro-12- radioactivity detector (Raytest). The abbreviations used are: dh-12-

oxo-ETE.

20902 12-Oxo-leulzotriene B4 TABLE I

Metabolism of LTB, by the postnuclear supernatant fraction from porcine PMNL

LTB, (2 PM) was incubated with the 1500 X g supernatant (6.5 mg of protein/ml) for 10 min at 37 "C in a volume of 1 ml in the presence of various cofactors (1 mM). Incubations were terminated by the addition of MeOH (0.6 ml), and the products were analyzed by precolumn extraction/RP-HPLC as described under "Experimental Procedures."

Cofactor Products

10,ll-Dihydm-LTB, 10,11-Dih~dro-12-oxo-LTB. pnwl/rnin/mg ofprotein

None ND" ND NAD+ 1.40 NADP+ ND

1.56 ND

NADH 0.53 ND NADPH ND ND ND, no detectable activity.

TABLE I1 Activities of marker enzymes in subcellular fractions from porcine

PMNL Enzyme activity was measured as described under "Experimental

Procedures." Relative specific activity was calculated by dividing the specific activity in a given fraction by the specific activity in the Dostnuclear amernatant.

Relative specific activity ~~

Subcellular ~~

a Ouabain- fraction Myeloper. ci'u- sensitive

Oxidase ronidase ATPase Na'-K+-

20,000 X g pellet 3.10 2.14 5.50

150,000 x g pellet 1.76 0.70 4.35

150,000 X g supernatant ND" 0.62 ND

(granule-rich)

(microsomes)

(cytosol) a ND, no detectable activity.

X g pellet, indicating that this fraction was enriched with granules. Ouabain-sensitive Na'-K+ ATPase activity was present in both the granule-rich fraction and the 150,000 X g pellet (microsomal fraction). The 150,000 X g supernatant (cytosol) contained very little of the above enzyme activities.

LTB, was incubated with each subcellular fraction in the presence of NAD+ and the products were separated by NP- HPLC (Fig. 2), which enables the resolution of l0,ll-dihydro- LTB, and lO,ll-dihydro-lP-epi-LTB4. The 20,000 x g pellet did not convert LTB, to appreciable amounts of products (data not shown). On the other hand, the metabolism of [l- 14C]LTB4 by the 20,000 X g supernatant fraction was very similar to that of intact PMNL and the 1500 X g supernatant (Table I), in that lO,ll-dihydro-LTB, and 10,11-dihydro-12- oxo-LTB, were the major products (Fig. 2.4) . However, unlike intact PMNL (22), the 20,000 X g supernatant did not convert this substrate to any detectable 10,ll-dihydro-12-epi-LTB4. In contrast to the 20,000 X g supernatant, the 150,000 X g supernatant (cytosol) exhibited little metabolic activity to- wards LTB,, with only a very small amount of l0,ll-dihydro- 12-oxo-LTB, being detected (Fig. 2B). Neither were appreciable amounts of dihydro metabolites formed by the microsomal fraction in the presence of NAD+, but instead, this fraction converted LTB, to a substance (product X; t R 18 min) which absorbed in the UV at 318 nm (Fig. 2C). Finally, recombina- tion of the microsomal and cytosolic fractions restored the formation of the two dihydro products, but considerably reduced the amount of product X detected (data not shown). These results suggest that the formation of 10,ll-dihydro

dho-LTB4 dh-LTB4

dh-12e-LTE4 LTE 0 f

0 10 20 30 TIME (mid

0 10 20 30 VME (mid

0 w d A

n

0 f

0 10 20 30 TIME (mid

FIG. 2. Normal-phase HPLC profiles of LTBa metabolites produced by subcellular fractions from porcine PMNL. A,

incubated with [l-"CC]LTB, (2 PM) for 30 min at 37 "C with 1 mM 20,000 X g supernatant; B, cytosol; C, microsomes. Fractions were

NAD+. Products were extracted on a cartridge of octadecylsilyl silica and separated on a column of silicic acid (RoSil; Alltech Associates) with a mobile phase of hexane/2-propanol/acetic acid (95.5:4.5:0.1) at a flow rate of 2 ml/min. The abbreviations used are: dh-LTB4, 10,ll-dihydro-LTB,; dh-LTB4, l0,11-dihydro-12-oxo-LTB,; dh-12e- LTB,, l0,ll-dihydro-12-epi-LTB,.

metabolites of LTB, requires at least one enzyme present in the microsomal fraction and another enzyme present in the cytosolic fraction.

The above results describe the metabolism of LTB4 in the presence of NAD+. We also determined the total amounts of LTB, metabolites formed by various fractions in the presence of other cofactors (Table 111). There was no detectable metabolism of LTB, by the granule-rich fraction in the absence or presence of any of the cofactors investigated. The 20,000 x g supernatant converted LTB, to 10,ll-dihydro-LTB, and 10,11-dihydro-12-oxo-LTB~ in the presence of NAD+, but in its absence or in the presence of NADPH, no metabolites were detected. Only small amounts of metabolites were detected in the presence of NADP+ or NADH. The microsomal fraction converted LTB, to product X in the presence of NAD+ and to a lesser extent in the presence of NADP', but no metabolites were detected in the presence of reduced cofactors. No detectable l0,ll-dihydro products were formed by the microsomal fraction in the presence or absence of any of the cofactors tested. LTB, was not metabolized to an appreciable extent by the cytosolic fraction in the presence or absence of the above cofactors.

Identification of Product X-Product X had a retention time shorter than that of LTB4 when chromatographed by NP-HPLC, but longer than that of LTBl with RP-HPLC, indicating that it is less polar. The UV absorption spectrum

12-Oxo-leukotriene B4 20903

TABLE I11 Effects of different cofactors on the metabolism of LTB, by the

subcellular fractions from porcine PMNL LTB, (2 PM) was incubated with the granule-rich and 20,000 X g

supernatant fractions for 10 min and the microsomal and cytosolic fractions for 30 min in the presence or absence of cofactors (1 mM). Incubations were terminated by the addition of 0.6 ml of MeOH, and the products were analyzed by precolumn extraction RP-HPLC as described under “Experimental Procedures.” The values for products represent the sums of 10,ll-dihydro-LTB4, 10,11-dihydro-12-oxo- LTB, and product X (12-oxo-LTB4) formed.

Subcellular fraction

Products

NAD’ NADP+ NADH NADPH None ~ ~~ ~~

pmolfminfrngprotein ~~

Granule ND” ND ND ND ND 20,000 X g supernatant 93.3 5.0 14.1 ND ND Microsomes 52.9 29.0 ND ND ND Cvtosol ND ND ND ND ND a ND, no detectable activity.

240 280 320

MVELENGTH (nm) 380

FIG. 3. UV spectrum of product X (12-oxo-LTB4).

1 00

80

t e 3 00

L e 40

5 @ 20

0 1

*os

209 14 1 OTMS

0 527

FIG. 4. Mass spectrum of the trimethylsilylether, methyl ester derivative of hydrogenated product X. TMS, trimethylsilyl.

of product X is shown in Fig. 3. Unlike LTB4, which has a typical triene chromophore with absorption maxima at 261, 270, and 282 nm, product X has a single absorbance maximum at 318 nm, indicating the presence of four conjugated double bonds. This suggests that one of the two hydroxyl groups of LTB4 had been converted to an oxo group. The mass spectrum of the trimethylsilyl ether, methyl ester derivative of hydrogenated product X (Fig. 4) exhibited intense ions at m/z 427

C,,), 203 (base peak, Cl-C6), 171, 141 (CI2-Cz0), 129, and 113. The ions at m/z 203 and 327 confirm the integrity of the Cs hydroxyl group, whereas the ion at m/z 141 is consistent with the presence of an oxo group in the 12 position. This mass spectrum is identical to the mass spectrum which we previously obtained for the corresponding derivative of 10,11- dihydro-12-oxo-LTB4 (19). The UV and mass spectra of product X, along with its chromatographic properties, indicate that it is identical to 12-oxo-LTB4.

Metabolism of 12-Oxo-LTB,-The results described above

(M - l ) , 413 (M - 15), 397 (M - 31), 381 (M - 47), 327 (Cb-

suggest that the first step in the formation of l0,ll-dihydro metabolites of LTB4 is the formation of 12-oxo-LTB4 by a microsomal 12-hydroxyeicosanoid dehydrogenase in the presence of NAD+. To investigate the next step in this pathway, 12-oxo-LTB4 was incubated with either the microsomal (30 min) or the cytosolic fraction (5 min) from porcine PMNL in the presence and absence of NADH and NADPH (Table IV). 12-Oxo-LTB4 was slowly metabolized by the microsomal fraction in the presence of either NADH or NADPH to two products with retention times and UV spectra identical to those of LTB, (tR = 16.0 min) and 10,11-dihydro-12-oxo- LTBl (tR = 26.5 min) (Fig. 5A). Neither of these products was formed in the absence of cofactors. Reduction of 12-oxo-

TABLE IV Metabolism of 12-oxo-LTB4 by the cytosolic and microsomal fractions

from porcine PMNL 12-Oxo-LTB4 (2 PM) was incubated with the microsomal fraction

(30 min) and the cytosolic fraction (5 min) in the presence or absence of cofactors (1 mM). Incubations were terminated by the addition of 17% MeOH (10 ml), followed by immediate extraction on a cartridge of ODs-silica at neutral pH. Products were resolved by RP-HPLC and quantitated by UV absorbance using PGB, (300 ng) as an internal standard. l0,ll-Dihydro-12-oxo-LTB4 was the only product formed by the cytosol, whereas the microsomal fraction produced a mixture of dihydroxyeicosatetraenoic acids (diHETEs) and 10,114hydro-12- OXO-LTB~ (dho-B,).

Product Subcellular

fraction NADH NADPH None

dho-B, diHETE dho-B, diHETE dh0-B. diHETE pmolll0 minfrng ofprotein

Microsomes 2.3 1.4 2.9 0.9 ND” ND Cytosol 38.9 ND 23.5 ND 22.3 ND

ND, no detectable activity.

4

0 10 20 30 TIME (mi”)

FIG. 5. Reversed-phase HPLC profiles of the metabolites formed after incubation of 12-oxo-LTB4 with the microsomal fraction ( A ) and cytosolic fraction ( B ) obtained from porcine PMNL. Porcine PMNL microsomes and cytosol were incubated with 12-oxo-LTB4 (2 pM) for 30 and 5 min, respectively, at 37 “C with 1 mM NADH. The samples were analyzed by precolumn extraction/ RP-HPLC. Products retained on the precolumn were separated on a Novapak CIS column with a mobile phase of acetonitrile/water/acetic acid (39:61:0.02). The flow rate was 1.0 ml/min. The abbreviations are as described in the legend to Fig. 2, except 12e-LTB4 represents 12-epi-LTB,.

20904 12-Oxo-leukotriene B4 LTB, to a diHETE could give either LTB4 or 12-epi-LTB4 which would probably not be resolved using RP-HPLC with acetonitrile/water/acetic acid as the mobile phase. To exam- ine the stereochemistry of the diHETE derived from 12-oxo- LTB,, this product was rechromatographed by NP-HPLC (Fig. 5A, inset). The diHETE peak consisted of one major component (90%) with a retention time identical to that of LTB, along with a minor component (10%) with a retention time identical to that of 12-epi-LTB,, which had been syn- thesized by reduction of 12-oxo-LTB, with sodium borohy- dride. The formation of LTB4/12-epi-LTB4 from 12-oxo- LTB, appeared to be favored by NADH, whereas the formation of 10,ll-dihydro-12-oxo-LTB, was greater with NADPH (Table IV).

The cytosolic fraction from porcine PMNL was much more active than the microsomal fraction in converting 12-oxo- LTB, to 10,ll-dihydro-12-oxo-LTB4 in the presence of NADH (Fig, 5 B ) , and to a lesser extent NADPH (Table IV). 12-Oxo-LTB, was also metabolized to some extent by the cytosolic fraction in the absence of added cofactors, probably due to the presence of endogenous cofactors. Neither LTB, nor 10,ll-dihydro-LTB, were detected after incubation of 12- oxo-LTB, with the cytosolic fraction.

Metabolism of 12(S)-HETE and 12(R)-HETE by Microso- mal Fractions from Porcine PMNL-12(S)-HETE was incubated with a microsomal fraction from porcine PMNL in the presence of NAD’ and the products were resolved by RP- HPLC (Fig. 6). A less polar metabolite (tR = 27 min) was produced with a single UV maximum at 279 nm (Fig. 6, inset). Based on its chromatographic properties and UV spectrum, and by analogy with the metabolism of LTB, to 12-oxo-LTB4, this metabolite is presumably identical to 12-oxo-5,8,10,14- eicosatetraenoic acid (12-oxo-ETE).

The configurations of the hydroxyl groups at Clz in 12(S)- HETE and LTB4 are opposite to one another, yet intact cells appear to metabolize both substrates to a similar extent (20). To determine whether the stereochemistry of the Clz-hydroxyl group affects the rate of metabolism by 12-hydroxyeicosanoid dehydrogenase, LTB4, 12(S)-HETE, and 12(R)-HETE were incubated with porcine PMNL microsomes in the presence of NAD’ for various times (Fig. 7). The time courses for the formation of 12-oxo metabolites from all three substrates are

I n n 91 / \

0 10 20 T/M€ (mid

30

FIG. 6. RP-HPLC profile of the products formed after incubation of 12(S)-HETE (1 p ~ ) with a microsomal fraction from PMNL for 40 min at 37 OC in the presence of NAD+ (1 mM). The sample was analyzed by precolumn extraction/RP-HPLC after addition of PGB2 (300 ng) as an internal standard. Products retained on the precolumn were resolved on a column of octadecylsilyl silica (Novapak; Waters-Millipore) with a gradient between acetonitrile/water/acetic acid (5050:0.02) and acetonitrile/water/acetic acid (55:45:0.02) over 30 min at a flow rate of 1.0 ml/min. UV absorbance at 280 nm is 4-fold more sensitive than at 235 nm. The inset shows the UV spectrum of the peak eluting with a retention time of 27 min. is., internal standard.

i 0 10 20 30 40 50 60

TIME (mid

FIG. 7. Time course for the metabolism of LTB, (1 p ~ ) , 12(S)-HETE (1 p ~ ) , and 12(R)-HETE (1 p ~ ) by the microsomal fraction from porcine PMNL in the presence of 1 mM NAD+. Products were analyzed by precolumn extraction/RP-HPLC as described under “Experimental Procedures,” and quantitated on the basis of UV absorbance, using PGB, (300 ng) as an internal standard.

Microsomal

OH OH 12-Hydroxyelcosanold H OH

NADHINADPH

LTBd M-OXO-LTB~

H OH H OH

10,ll-dihydro-LTB4 10,11-dihydro-12-oxo-LTB4

Qf P f COOH

10, rl-dihydro-12-epi-LTB4

FIG. 8. Scheme for the metabolism of LTB4 by the 12-hydroxyeicosanoid dehydrogenase/lO,ll-reductase pathway.

quite similar, indicating that the orientation of the 12-hydroxyl group does not appear to affect the metabolism of the substrate by this enzyme. PGF2,, which is metabolized to a 15-oxo metabolite by the cytosolic enzyme, 15-hydroxyprostaglandin dehydrogenase (43), was not converted to any detectable products by PMNL microsomes (data not shown).

DISCUSSION

We have previously reported that the major pathway for the metabolism of LTB, (22) and 12(S)-HETE (20) by porcine PMNL is the formation of lO,ll-dihydro, 10,ll-dihydro- 12-epi, and 10,11-dihydro-12-oxo products. The present study has clearly shown that the initial step in the formation of dihydro products by this pathway is oxidation of the 12- hydroxyl group of the substrate by a microsomal 12-hydroxyeicosanoid dehydrogenase to give a 12-oxo intermediate (Fig. 8). We are unable to conclude from our results whether this enzyme is present in the plasma membrane or the endo- plasmic reticulum of PMNL, since experiments in which we measured the activities of enzymes (glucose-6-phosphatase and alkaline phosphatase) which are markers for the endo-

12-Oxo-leukotriene B4 20905

plasmic reticulum in other tissues gave unsatisfactory results with subcellular fractions from porcine PMNL.

The fact that a pronounced isotope effect is observed for the saturation of the l0,11-double bond when the substrate possesses a tritium atom in the 12-position indicates that oxidation of the 12-hydroxyl group is the rate-limiting step in the formation of dihydro metabolites. This result is in contrast to our previous findings that intact porcine PMNL convert LTB, (19) and 12(S)-HETE (31), both labeled with deuterium in the 12 position, to dihydro products in which the deuterium was retained to extents of 65 and 50%, respectively. In the present study, negligible amounts of dihydro metabolites were formed from LTB, by the microsomal and cytosolic fractions from PMNL, and substantial amounts of these products could be detected only when these two fractions were combined. The retention of deuterium which we previously observed was presumably due to transfer of a deuterium atom from carbon-12 of the substrate to NAD' to give NAD2H. In the subsequent reduction of the 12-oxo intermediate to a 10,11-dihydro-12-oxo product, the deuterium of NAD2H could be returned to the 12 position, resulting in partial apparent retention of the CI2 deuterium.

12-Oxo-LTB, is rapidly metabolized by a l0,ll-reductase in the cytosolic fraction, preferentially in the presence of NADH, to ~0,11-dihydro-12-oxo-LTB4. No l0,ll-dihydro- LTB, could be detected under these conditions, suggesting that the principal site of conversion of 10,11-dihydro-12-oxo- LTB, to 10,11-dihydro-LTB4 may be the microsomal fraction. In agreement with this, it was observed that 12-oxo-LTB, is converted to LTB,, along with a small amount of 12-epi- LTB,, by the microsomal fraction in the presence of NADH or NADPH. However, reduction of the 12-oxo group by the microsomal fraction was much slower than reduction of the l0,ll-double bond by the cytosolic reductase. It is not clear whether the reduction of the 12-oxo group by the microsomal fraction is catalyzed by the 12-hydroxyeicosanoid dehydrogenase responsible for oxidation of LTB4 and 12-HETE, acting in the reverse direction, or whether there is a separate ketoreductase in this fraction. Although we previously detected substantial amounts of l0,ll-dihydro-12-epi-LTB, after incubation of LTB, with intact porcine PMNL (22), we did not detect this compound after incubation of LTB, or 12- oxo-LTB, with any of the subcellular fractions investigated. The reason for this is not clear, but it may be related to the fact that l0,ll-dihydro-12-epi-LTB, is formed much more slowly than 10,ll-dihydro-LTB, (22), and the incubation times used in the present study may not have been long enough. Alternatively, an additional enzyme may be required for epimerization of the 12-hydroxyl group, and this enzyme could be localized in a different subcellular fraction.

Several eicosanoid dehydrogenases, all of which appear to be distinct from the 12-hydroxyeicosanoid dehydrogenase described here, have been described in the literature. 15-Hy- droxy prostaglandin dehydrogenase, which is present in large amounts in the cytosol from lung, liver (44), and placenta (45), oxidizes the 15-hydroxyl group of a variety of prosta- noids, as well as w6-monohydroxy fatty acids such as 12- hydroxy-5,8,10-heptadecatrienoic acid (46) and 15-HETE (47). 15-oxo prostaglandins can be further metabolized by a A13-reductase to 13,14-dihydro-15-0~0 products, which can then be reduced by a ketoreductase to 13J4-dihydroprosta- glandins. As with 10,ll-dihydro-LTB,, the latter substances cannot be formed directly from prostaglandins, but require prior oxidation of the 15-hydroxyl group (48,49). 15-Hydroxy prostaglandin dehydrogenase does not appear to be responsible for the 12-hydroxyeicosanoid dehydrogenase activity ob-

served in the present study, since PGF2, was not converted to any detectable products either by intact PMNL (20) or by PMNL microsomes.

Interestingly, 13-hydroxy-9,11-octadecadienoic acid (13- HODE), which, like prostaglandins, has an w6-hydroxyl group, is not a substrate for 15-hydroxyprostaglandin dehydrogenase (47). However, Earles et al. (50) have recently described a cytosolic dehydrogenase present in rat colon mucosa which oxidizes 13-HODE to the corresponding 13-oxo compound, 13-oxo-ODE (50). The reaction catalyzed by this dehydrogenase is highly specific for NAD' and was found to be essentially irreversible. 13-HODE is also metabolized by the 12-hydroxyeicosanoid dehydrogenase/lO,ll-reductase pathway in porcine PMNL to 11,12-dihydro and 11,12-dihy- dr0-13-0XO metabolites (20), presumably through a 13-oxo- ODE intermediate. The cytosolic localization of the 13-HODE dehydrogenase in colonic mucosa suggests that it is distinct from the 12-hydroxyeicosanoid dehydrogenase of PMNL. However, the relationship between the two enzyme activities remains to be investigated.

Oxo metabolites of polyunsaturated fatty acids can also be formed by the nonenzymatic dehydration of the corresponding hydroperoxy compounds. Microsomal fractions from aorta convert linoleic acid to g-oxo-ODE, presumably by this mechanism (51). Platelets have been shown to convert 12(S)- hydroperoxy-5,8,10,14-eicosatetraenoic acid to 12-oxo-ETE, along with two geometric isomers of 12-oxo-5,8,10-dodecatri- enoic acid (52).

Our results would suggest that the 12-hydroxyeicosanoid dehydrogenase responsible for the metabolism of LTB,, 12(R)-HETE, and 12(S)-HETE exhibits equal preference for 12(R)- and 12(S)-hydroxyl groups. However, we cannot rule out the possibility that porcine PMNL contain two 12-hydroxyl dehydrogenases, one of which is specific for a 12(R)- hydroxyl group, and the other for a 12(S)-hydroxyl group. The subsequent reduction of 12-oxoeicosanoids to 12-hydroxyeicosanoids appears to be quite stereospecific. 12-Oxo-LTB, was converted primarily to LTB4, with only a small amount of 12-epi-LTB, being formed. The latter substance is only about 5% as potent as LTB, as a chemotactic agent, and has a much higher Kd for the high affinity LTB, receptor on PMNL (53). Reduction of the 12-oxo group of 10,ll-dihydro- 12-oxo-LTB4 also appears to be stereospecific, since only 10,11-dihydro-LTB4 and not the corresponding 12-epi compound was detected when LTB, was incubated with various subcellular fractions. Previous studies with intact porcine PMNL also indicated that the stereochemistry of the 12- hydroxyl group of LTB, (22) and 12-HETE (20) was con- served in the initially formed 10,ll-dihydro products, although the 12-epi compounds were formed with longer incubation times. Similar results were obtained with intact rat PMNL (16). The stereospecific reduction of 12-oxo-ETE to 12(R)-hydroxy-5,8,14-eicosatrienoic acid3 (ie. l0,ll-dihydro- 12(S)-HETE) may be quite important, since the latter compound is a potent proinflammatory agent (29, 30).

Rat liver microsomes have been reported to possess 12- ketoreductase activity, resulting in the reduction of 12-0x0- ETE to a mixture of 12(S)-HETE and 12(R)-HETE in the presence of either NADH or NADPH (54). Rat peritoneal PMNL microsomes specifically reduce 12-oxo-ETE to 12(S)- HETE in the presence of NADH (55). Considering the cellular localization of the rat peritoneal ketoreductase, and the spec-

Due to the priority rules for assigning R and S configurations, the configuration of the 12-hydroxyl group of lP(S)-HETE is identical with that of 12(R)-hydroxy-5,8,14-eicosatrienoic acid [10,11- dihydro-lS(S)-HETE].

20906 12-Oxo-Zeukotriene B4 ificity of the reduction, this enzyme may be similar to that which we have described in porcine PMNL.

The 12-hydroxyeicosanoid dehydrogenase/lO,ll-reductase pathway may be viewed either as a pathway for the inactiva- tion of biologically active compounds such as LTB4, or as a pathway for the generation of biologically active compounds. Both 10,11-dihydro-LTB4 and l0,ll-dihydro-12-epi-LTB4 have less proinflammatory activity than LTB, (25, 27). On the other hand, the 10,ll-dihydro metabolite of 12(S)-HETE (i.e. 12(R)-hydroxy-5,8,14-eicosatrienoic acid),3 has been reported to have potent vasodilatory and angiogenic properties (29), and to be a more potent chemotactic agent for human neutrophils than LTBl (30). In contrast, 12(S)-hydroxy- 5,8,14-eicosatrienoic acid has little proinflammatory activity (29). 12(R)-hydroxy-5,8,14-eicosatrienoic acid is formed from arachidonic acid by corneal microsomes by a cytochrome P- 450-dependent mechanism involving 12(R)-HETE as an intermediate (29). This requires inversion of the stereochemistry of the 12-hydroxyl group, since the 12(R)-hydroxyl group of the dihydro product has the opposite configuration to the 12(R)-hydroxyl group of 12(R)-HETE? Our present results indicate that both 12(R)-HETE and 12(S)-HETE are equally good substrates for the 12-hydroxyeicosanoid dehydrogenase in PMNL and provide a mechanism for the conversion of 12(R)-HETE to the biologically active 12(R)-hydroxy-5,8,14- eicosatrienoic acid. 13-Oxo-ODE, produced by a cytosolic dehydrogenase in rat colon mucosa, has been shown to have mitogenic activity when administered intrarectally to rats (56). Work is currently in progress in our laboratory to investigate the biological activities of the products of the 12- hydroxyeicosanoid dehydrogenase/lO,ll-reductase pathway.

Acknowledgments-We are grateful to Dr. Orval Mamer, McGill University, for assistance with the mass spectrometry and to Dr. Alan Brash for helpful discussions concerning the synthesis of 12(R)- HETE.

1. 2.

3.

4.

5.

6.

7.

8.

10. 9.

11. 12.

REFERENCES Bor eat, P., and Samuelsson, B. (1979) J. Biol. Chem. 254,2643-2646 Fori-Hutchinson, A. W., Bray, M. A., Doig, M. V., Shipley, M. E., and

Smith, M. J. H. (1980) Nature 286,264-265 Palmer, R. M. J., Stepney, R. J., Higgs, G. A., and Eakins, K. E. (1980)

Prostaglandins 20,411-418 Feinmark, S. J., Lindgren, J. A., Claesson, H.-E., Malmsten, C., and

Samuelsson, B. (1981) FEBS Lett. 136 , 141-144 O'Flaherty, J. T., Wykle, R. L., Lees, C. J., Shewmake, T., McCall, C. E.,

and Thomas, M. J. (1981) Am. J. Pathol. 105,264-269 Dahlh, %-E., Bjork, J., Hedqvist, P., Arfors, K.-E., Hammarstrom, S.,

Lindgren, J.-A., and Samuelsson, B. (1981) Roc. Natl. Acud. Sci. U. S. A. 78.3887-3891

Capdeviii J., Marnett, L. J., Chacos, N., Prough, R. A,, and Estabrook, R.

Cunnineham. F. M.. and Woollard. P. M. (1986) Prostaelandins 34.71-78 W. (1982) Proc. Natl. Acud. Sci. U. S. A. 79 , 767-770

WoollaFd, P. 'M. (1986) Biochem. Biophys. Res. Commu;. 136 , 169-176 Lindgren, J. A., Hansson, G., and Samuelsson, B. (1981) FEBS Lett. 128 ,

Powell, W. S. (1984) J. Biol. Chem. 259,3082-3089 Hansson, G., Lindgren, J. A., Dahlh, %-E., Hedqvist, P., and Samuelsson,

329-335

B. (1981) FEBS Lett: 130,107-112 13. Powell, W. S. (1987) Blochem. Biophys. Res. Commun. 145,991-998 14. Maas, R. L., Brash, A. R., and Oates, J. A. (1982) Adu. Prostaglandin

Thromboxane Leukotriene Res. 9,29-44 15. Romano, M. C., Eckardt, R. D., Bender, P. E., Leonard, T. B., Straub, K.

M., and Newton, J. F. (1987) J . Biol. Chem. 2 6 2 , 1590-1595 16. Powell, W. S., and Gravelle, F. (1990) J. Biol. Chem. 265,9131-9139 17. Powell, W. S. (1986) Biochem. Biophys. Res. Commun. 136,707-712 18. Powell, W. S., and Gravelle, F. (1988) J. Biol. Chem. 263,2170-2177

20. Wainwright, S. L., Falck, J. R., Yadagiri, P., and Powell, W. S. (1990) 19. Powell, W. S., and Gravelle, F. (1989) J. Biol. Chem. 264,5364-5369

21. Powell, W. S., and Gravelle, F. (1990) Biochim. Biophys. Acta 1044 , 147- Biochemistry 29,10126-10135

157 22. Wainwright, S. L., Falck, J. R., Yadagiri, P., and Powell, W. S. (1990)

23. Schonfeld, W., Schluter, B., Hilger, R., and Konig, W. (1988) Immunology Biochemistry 29,1180-1185

LR F.'?Q-!P.G

24. Fader, J., Marx, K.-H., Kaever, V., and Frolich, J. C. (1989) Prostaglandins

25. Kaever, V., Bruuns, J., Wunder, J., Damerau, B., Zimmer, G., Fader, J., Leukotrienes Essent. Fatty Acids 37,193-196

Wessel, K., Floege, J., Topley, N., Radeke, H., and Resch, K. (1990) Life SCI. 4 6 , 1465-1470

26. Kaever, V., Martin, M., Fader, J., Marx, K.-H., and Resch, K. (1987) Biochem. Biophys. Acta 922,337-344

27. Kumlin, M., Falck, J. R., Raud, J., Harada, Y., DahlBn, S.-E., and Gran- strom, E. (1990) Biochem. Biophys. Res. Commun. 170,23-29

28. Kaever, V., Damerau, B., Wessel, K., and Resch, K. (1988) FEBS Lett.

", "" """

921 RQR-RQQ

29. Murphy, R. C., Falck, J. R., Lumin, S., Yada iri, P., Zirrolli, J. A., Balazy, M., Masferrer, J. L., Abraham, N. G., and &hwartzman, M. L. (1988) J. Biol. Chem. 263,17197-17202

30. Rimarachin, J. A,, Masferrer, J. L., Gerritsen, M. E., and Schwartzman, M. L. (1989) FASEB J. 3. A913

"-, """ ""

31. Wainwright, S. L., and'Powel1, W. S. (1989) in New Trends in Lipid Mediators Research (Zor, U., Naor, Z., and Danon, A,, eds), Vol. 3, pp. 200-203, Karger, Base1

32. Powell, W. S. (1983) Anal. Biochem. 128,93-103 33. Borgeat, P., Picard, S., Vallerand, P., and Sirois, P. (1981) Prostaglandins

34. Hawkins, D. J., and Brash, A. R. (1987) J. Biol. Chem. 262,7629-7634 35. Borgeat, P., and Samuelsson, B. (1979) Proc. Natl. Acud. Sci. U. S. A. 76 ,

36. Hamberg, M., and Samuelsson, B. (1974) Proc. Natl. Acud. Sci. U. S. A.

Med. 6,557-570

2148-2152

71.3400-3404 37. Powell, W. S. (1982) Methods Enzymol. 86,467-477

39. Brittinger, G., Hirschhorn, R., Douglas, S. D., and Weissmann, G. (1968) 38. Klebanoff, S. J. (1965) Endocrinology 76,301-311

40. Sha'afi, R. I., Naccache, P., Raible, D., Krepcio, A., Showell, H., and Becker, J. Cell Biol. 37 , 394-411

E. L. (1976) Biochim. BioDhvs Acta 448.638-641 41. Ames, B. N. (1966) Method; I h y m o l . 8 , 115-118 42. Powell, W. S. (1987) Anal. Biochem. 164,117-131 43. Hamberg, M., and Samuelsson, B. (1971) J. Biol. Chem. 246,1073-1077 44. Anggird, E., Larsson, C., and Samuelsson, B. (1971) Acta Physiol. Scand.

45. Braithwaite, S. S., and Jarabak, J. (1975) J. Biol. Chem. 260,2315-2318 46. Tai, H. H., Liu, Y., Tai, C. L., Yoden, K., and Shen, R.-F. (1985) Adu.

47. Bergholte, J. M., Soberman, R. J., Hayes, R., Murphy, R. C., and Okita, R.

48. Hamberg, M., and Samuelsson, B. (1971) J. Biol. Chem. 246,1073-1077 49. Westbrook, C., and Jarabak, J. (1978) Arch. Biochem. Biophys. 185,429-

8 1,396-404

Prostaglandin Thromboxane Leukotriene Res. 15 , 159-161

(1987) Arch. Biochem. Biophys. 257,444-450

442

Biochim. Biophys. Acta 1081,174-180

. .

50. Earles, S. M., Bronstein, J. C., Winner, D. L., and Bull, A. W. (1991)

51. Funk, C. D., and Powell, W. S. (1983) Biochim. Biophys. Acta 754,57-71 52. Fructeau de Laclos, B., Maclouf, J., Poubelle, P., and Borgeat, P. (1987)

53. Goldman, D. W., and Goetzl, E. J. (1984) J. Enp. Med. 159 , 1027-1041 54. Falgueyret, J.-P., Lehlanc, Y., Rokach, J., and Riendeau, D. (1988) Biochem.

55. Falgueyret, J. P., Leblanc, Y., and Riendeau, D. (1990) FEBS Lett. 2 6 2 ,

56. Bull, A. W., Nigro, N. D., and Marnett, L. J. (1988) Cancer Res. 48,1771-

Prostaglandins 33 , 315-337

Biophys. Res. Commun. 156,1083-1089

197-200

1776

the of chemistry vol. no. 5, pp. by for in u. s. a ... · mation of dihydro products by this...

Documents