1IO

BBALIP

53783

Metabolism of 12( R) -hydroxyeicosatetraenoic by rat liver microsomes Hemant

K. Jajoo I, Jorge H. Capdevila

‘, J.R. Falck 3, R.K. Bhatt

acid

’ and Ian A. Blair ’

’ Dtpurfm~vzts of Chemistry and Fharmuco1o.g~ und Center in Mokculur Tu.ricolo~~, Vcrndcrhiit Unic~r.sity, Nu.sh~~ilk, TN (U.S. A. 1, ’ Deportments ofMedicine (Oil ,ision ofNephrology) und Biochemistry, Vundcrhilr Unicxxsity, Nushr,illc, TN IU..S.A. I und .’ Department

of Molecular

Genetics,

(Revised

Key words:

Cytochrome

Unirersity of T&IS Sourhwcvtrrn Medical C’enter, Ddlcrs, TX (U.S.A. 1 (Received manuscript

P-450: Eicosanoid;

I6 April 19YlJ received 21 June

Metabolism;

t99lf

Rat liver microsome;

Epoxypenase

The in vitro meta~lism of 12(~)-hydro~eicosatetraenoic acid was studied using freshly isolated rat liver microsomes. Ten metabolites were isolated and identified by a combination of ultraviolet spectroscopy and gas chromatography/mass spectrometry. The two major metabolites were dihdroxyeicosatetraenoic acids generated by w/w - 1 hydroxylation. Oxidation at C-5 resulted in the formation of four leukotriene-like compounds, two of which differed from leukotriene B, in double-bond geometry alone. The other two differed from leukotriene B, in olefin geometry and C-5 configuration. Epoxidation at the 14,SoleBn resulted in the formation of two diastereomeric epoxy alcohols, while C-16 hydro~iation gave two diastereomeric dihydro~eicosatetraenoic acids.

Introduction Cytochrome P-4SO-mediated oxidation of arachidonic acid leads to the formation of EETs [ll and HETEs [2] many of which have potent biological activities [3]. There is increasing evidence for the involvement of cytochrome P-450-derived eicosanoids in

Abbreviations: AA, arachidonic acid; BSTFA, bisttrimethylhexane: DHETE, dihydroxyeicosatesilylhrifluoroacetamide; C,H,,, traenoic acid; S(R),12(R)-(EEEZ)-DHETE, 5tRJ,12(R)-dihydroxy6,8.iO,l4-(E,E,E,Z)-DHETE; 5(R),12(S)-(EEEZ)-DHETE. 5(R~,12(S~-dihydroxy-h.X,lI~,[4-(E.E,E,Z~-HETE; %SJ,12(RJ(EEEZ)-DANTE, %SJ,lZ( R)-dihydroxy-6,8,10,14-tE,E,E,Z)-HETE; S(R). 12(R)-(EZEZ)-DHETE, S(R),12(R)-dihydroxy-6,8,10.14(E,Z,E,ZJ-HETE; 5(S),lZ(S)-(EZEZ)-DHETE, S(S),12(S)-dihydroxy-6,8,10,14-(E,Z.E,Z)-HETE; 5(s),l2(R)-(EZEZ)-DHETE, 5(S),12(R)-dihydroxy-6,8,10,14-(E,Z,E,Z)-HETE: 5(R), 12(S)(EZEZJ-DHETE, 5fR),12(S)-dihydroxy-6,8,10,14-(E,Z.E.ZJ-HETE: EET, cis-epoxyeicosatrienoic acid: EL, electron ionization: CiC/MS, gas liquid chromatography/mass spectrometry; HETE, hydroxyeicosatetraenoic acid; iPrOW, i-propanol; LT, leukotriene; ME. methyl ester: NICI, electron capture negative ion chemical ionization: PFB. pentafluorobenzyl; PFBB, pentafluorohenzyl bromide; TMS, trimethylsilyl: fn, retention time. Correspondence: bilt University,

LA. Blair, Department of Pharmacology, Nashville. TN 37232, U.S.A.

Vander-

pathophysiological processes [4,5] and in signal transduction pathways such as those associated with growth inhibitor cytokines 161. 12(R)-HETE, a major product of hepatic cytochrome P-450 metabolism [7], has also been identified in human psoriatic skin lesions [Xl. It has enhanced chemotactic and chemokinetic activity when compared with 12(S)-HETE [9]. Further potent biological activities of 12(K)-HETE include: stereoselective inhibition of Na’/K+-ATPase [IO],stereoselective stimulation of lymphocyte chemotaxis [l I], and stereoselective modulation of vascular tone [ 121. The possibility exists that after formation of 12(R)HETE, the monohydroxy fatty acid could undergo further metabolism to other potential eicosanoid autocoids. Biotransformations could occur either in the liver or in other organs rich in cytochrome P-450 such as the kidney or lung. Previous metabolism studies on 12-HETE have focused entirely on the plateletor macrophage-derived 12(S)-enantiomer. Thus, Mathur et al. 1131 have shown that 12(S)-HETE undergoes a series of peroxisomal-mediated P-oxidation reactions to give chain shortened metabolites. Marcus et al. [ 14,151 have demonstrated that 5- and 20-hydroxylation of platelet-derived 12(S)-HETE can occur in neutrophiIs through Iipoxygenase and cytochrome P-450mediated o~genation reactions, respectively. In spite

111 of the interest in 12-HETE metabolism [13-151, and the potential for forming additional biologically active metabolites, there have been no studies to date on the metabolism 12(R)-HETE. We report the characterization of major metabolites formed during incubations of 12(R)-HETE with freshly isolated rat liver microsomes. Materials

and Methods

General Authentic 12(R)-HETE,12(S),20-DHETE and 12(S),19(R)-DHETE were synthesized as previously described [ 16,171. [l- 14ClAA (50-59 mCi/mmol) was purchased from Amersham (Arlington Hts, IL). The 5(S),l2-(EEEZ>-DHETEs formed from LTA, and 5@>,12(R>-(EZEZ)-DHETE were kind gifts of Dr A.R. Brash (Vanderbilt University). [1-‘4C]12(R)-HETE was prepared from [l-14C]AA as described previously [181. 12(R)-HETE and its 14C analog used in the microsoma1 incubations were > 99% enantiomerically pure as judged by chiral chhromatography 1181. Microsomal incubations Male Sprague-Dawley rats (250-300 g> were used. They were fed, ad libitum, Purina rat chow and tap water. Microsomal fractions were isolated from either control or phenobarbital-induced rats as in [19]. For phenobarbital induction, animals were given a single i.p. injection of phenobarbital (40 mg/kg) and then maintained for 10 days with their drinking water replaced by a 0.05% (w/v) solution of sodium phenobarbital. Microsomal protein content was determined as previously described [19]. The incubation medium consisted of 0.05 M Tris-HCl (pH 7.41, 10 mM MgCl,, 0.15 M KCl, 2 mg/ml isocitric acid, and 0.8 IU of

TABLE

isocitric acid dehydrogenase. Incubations were carried out with 50 PM substrate in a shaking water bath at 37 “C. Reactions were initiated by the addition of NADPH (1 mM, final concentration). Metabolites were extracted into ethyl acetate (3 x 10 ml>, evaporated to dryness on a Speed-Vat concentrator, dissolved in the appropriate HPLC mobile phase and analyzed using the appropriate HPLC conditions (Table Il. Dericatization, GC/MS

and ultraviolet spectrophotome-

try ME derivatives were prepared in methanol with ethereal diazomethane. Hydrogenations were carried out in methanol over Rh/Al,O, by saturation with hydrogen for 1 min. After 5 min, the catalyst was removed by filtration over celite. TMS and PFB derivatives were prepared as described previously [20]. GC/MS was carried out on a Nermag RlOlOC quadrupole mass spectrometer (Delsi, Houston, TX) interfaced with a Varian Vista gas chromatograph equipped with a 15-m SPB-5 fused silica column (0.32 mm i.d.; 0.25 pm coating thickness) using helium as carrier gas at a flow of 1 ml/min. Injections (in BSTFA or hexane) were made in the splitless mode with the injector temperature held at 250°C. The oven was temperature programmed from 100 to 330°C at lS”C/min. EI spectra were obtained at 70 eV with the analyzer pressure at 2.0. lo-’ T, source temperature at 230°C and the conversion dynode at -5 kV. NICI spectra were obtained with methane as reagent gas, with the analyzer pressure at 5.0. 10ph T, source temperature at 230°C and the conversion dynode at +5 kV. Ultraviolet Spectra were obtained in methanol utilizing a Beckman (Waldwick, NJ) DU-7 single-beam spectrophotometer.

I

HPLC conditions used for unalysis of microsomal incubations and metabolite purification System

Column

A

C-18

B

Si

C

C-18

D

C-18

E

C-18

F

C-18

G

C-18

* Altex (Rainin,

*

Woburn,

Mobile phase

Flow (ml/min)

(nm)

Ultraviolet

solvent A

solvent B

gradient

H ,O/CH ,COOH (99.9: 0.1, v/v) C,H,,:CH,COOH (99.9: 0.1, v/v) CH,CN/H,O/CH,COOH (30.0: 69.9: 0.1,v/v) CH,CN/H,O/CH,COOH (40.0:59.9:0.1, v/v) CH,CN/H,O/CH,COOH (50.0:49.9:0.1, v/v) CH&N/H,O/CH,COOH (40.0: 59.9: 0.1, v/v) CH ,OH/H ,O/CH ,COOH (70.0: 29.9:0.1, v/v)

CH,CN/CH,COOH (99.9:0.1, v/v) C,H,, /iPrOH/CH,COOH (97.9: 2.0: 0.1, v/v) _

5O-100% B (40 min) 25-75% B (30 min) _

1.0

235/270

3.0

235

1.5

235

-

_

1 .o

235

-

_

1.o

270

_

_

1.5

235

1.0

270

MA) columns

(4.6 mmX250

-

mm; 5 pm).

112 Results

60

Incubation of rat liver microsomes with 12(R)-HETE in the presence of NADPH resulted in the formation of a number of metabolites that could be resolved into nine major radioactive peaks using HPLC system A (Figs. la and b). There was no metabolism of 12(R)HETE in the absence of NADPH or by boiled microsomes. The rate of product formation was linear for 40 min (Fig. 2). Longer incubation times resulted in the generation of un-identified more polar metabolites. NADPH was required to initiate the reactions and product formation was increased approx. 2-fold after animal treatment with phenobarbital. Metabolites 1 and 2 were further purified using HPLC system C (rn: 25.2 and 28.4 min, respectively). Metabolite 1 had a ultraviolet A,,;,, at 235.5 nm, indicating the presence of a conjugated diene system. The NICI mass spectrum of its TMS/PFB derivative showed an intense [M - PFBI- ion at m/z 479. After hydrogenation, the [M - PFBI- ion shifted to m/z 487 confirming the presence of four olefinic double bonds. The EI mass spectrum of metabolite 1 after hydrogenation, as its TMS/ME derivative, showed fragment ions at m/z 301 and 117 consistent with hydroxyl groups at C-12 and C-19, respectively. Metabolite 1 was con-

J

(b)

0 Fig. 1. HPLC incubations

chromatograms of 12(R)-HETE

tions were carried mg/ml

ried out on an Altex (99.9:0.1.

mobile v/v):

20

30

(mid

obtained

from the extract of microsmal

(al at 235 nm and (b) 270 nm. Incuba-

out for 30 min at a protein

and 12(R)-HETE

mm). The

10 time

concentration

ultrasphere

C,,

concentration

of SO PM.

HPLC

column (5 pm,

4.6 mmx250

phase consisted of solvent A: water/acetic

solvent B: acetonitrile/acetic

of 1

was car-

acid (99.9:O.I,

acid v/v).

A

gradient was run from StX? B to 100% B over a period of 40 min at a flow rate of 1 ml/min Peaks (a-e)

contained

with ultraviolet no radioactivity further.

detection

at 235 and 270 nm.

so they were not investigated

,

1

20

0

40 time

Fig.

2. Time

course

for

formation

60

lminl of

12(R)-HETE

mctsbolites.

Incubations

and analyses were carried out under the same conditions

as for Fig.

1. Total

metabolitea

metabolitc

2

(01, total of metabolites

3-8

(w

).

( A ). metabolitc I (v ).

firmed as 12(R),lY-DHETE by co-elution with a standard of 12(S),19-DHETE using HPLC system C and comparison of their EI mass spectra. The C-IY absolute configuration could not be determined since the epimers at this position are not resolved with system C. Metabolite 2 had a UV A”,;,, at 236.0 nm indicative of a conjugated diene system (Table II). The NICI mass spectrum of its TMS/PFB derivative showed an intense [M - PFB]- ion at m/z 479. After hydrogenation, the [M - PFB]- ion shifted to m/z 487 confirming the presence of four olefinic double bonds. The EI mass spectrum of metabolite 2 after hydrogenation, as its TMS/ME derivative, showed fragment ions at m/z 301 and 103, consistent with hydroxyl groups at C- 12 and C-20, respectively. Metabolite 2 was confirmed as 12(R),20-DHETE by coelution with a synthetic sample of its 12(S)-enantiomer using HPLC system B and comparison of their El mass spectra. Metabolites 3 and 4 were further purified on HPLC system D (t,: 18.3 and 20.4 min, respectively). Both metabolites had identical ultraviolet absorption characteristics to 12(R)-HETE with A,,;,, at 236.5 nm (Table II). This suggested that they retained the cis-tmns conjugated diene. The NICI mass spectra of their TMS/PFB derivatives showed intense [M - PFB]ions at m/z 479 which shifted to m/z 487 after hydrogenation, thus corroborating the presence of four olefinic double bonds. The EI mass spectra of the TMS/ME derivatives of 3 and 4 after hydrogenation showed fragment ions at m/z 301 and 159 (Fig. 3). These data were consistent with assignment of metabolites 3 and 4 as a pair of diastereomeric 12( HI, 1hDHETEs. Metabolite 5 was further purified using HPLC system G (t, 12.5 min). Its ultraviolet spectrum (Fig. 4a) was characteristic [21,22] of a conjugated triene system (Table II). The NICI mass spectrum of its TMS/PFB

113 TABLE

II

of 12(R)-HETE

Relutic,e umowzts

metabolites

from rat lirw microsomes

Metabolite

Relative amount (%‘I :’

Arni!X WV)

M-PFB

1

28.1 34.2 x.3 Il.7 3.1 5.2 5.1 4.2

235.5 236.0 236.5 236.5 268.5 268.5 235.5 235.5

419 479 479 479 479 479 407 407

2 3 4 5 6 I 8 ” h ’ ’

Calculated TMS/PFB TMS/PFB TMS/ME

from recovered radioactivity (mean derivative. derivative after hydrogenation. derivative after hydrogenation.

h

M-PFB’

El ’ diagnostic fragment

Metabolite structure

4x7 487 487 4x7 487 487 413 413

301 301 301 301 203 203 301 301

12( R),19-DHETE 12f R),20-DHETE 12t R),l&DHETE lZfR),l6DHETE 5.12tRkDHETE 5.12tRkDHETE 12(R)-OH-14,I5-EET 12t R)-OH-14.15.EET

1

159

was characteristic [21,22] of a conjugated triene system (Table II). Its mass spectral characteristics were identical with those of metabolite 5. When the ME derivative of 6 was chromatographed on HPLC system G, it resolved in two peaks, 6a and 6b in a ratio of 1: 1 (t,: 40.2 and 45.6 min, respectively) that coeluted with the ME derivatives of 5(s),l2(s)-(EZEZ)-DHETE (6a) (isolated from the incubation of S(S)-HETE with porcine leukocytes [24]) and authentic 5(S),12(R)(EZEZl-DHETE (6b), respectively. Based on the

I-

* _.._

I

.

1

I

*

&_=268.5nm

.

1

_._._

I

1

301

I 75

100

117 103 159 159 215 215 na na

of two experiments)

derivative showed an intense [M - PFB]- ion at m/z 479, which shifted to m/z 487 after hydrogenation, confirming the presence of four olefinic double bonds. The EI mass spectrum of the TMS/ME derivative of hydrogenated 5, had diagnostic fragment ions at m/z 203 and 215 (Fig. 5), consistent with the presence of hydroxyl groups at C-5 and C-12, respectively. When the ME derivative of metabolite 5 was chromatographed on HPLC system G, it resolved into two equal peaks (t,: 31.6 and 38.1 min, respectively) that coeluted with the ME derivatives of S(S),12(R)-(EEEZ)DHETE and 5(S),12(S)-(EEEZ)-DHETE obtained by hydrolysis of LTA, [23]. Since both compounds retained the original 12(R) stereochemistry, metabolite 5 must be composed of a 1:l mixture of 5(S),12(R)(EEEZI-DHETE and 5(R>,12(R)-(EEEZI-DHETE (enantiomer of 5(s),l2@)-(EEEZ>-DHETE). Metabolite 6 was further purified using HPLC system E (tn 16.5 min). Its ultraviolet spectrum (Fig. 4b)

129

und muss spectral und ultrat?olet characteristics

200

I 500

400

600

ml2 Fig. 3. EI mass spectrum of metabolite 3 after hydrogenation as its TMS/ME derivative. The corresponding spectrum of metabolite 4 was identical.

Fig. 4. Ultraviolet

spectra

of (a) metabolite methanol.

5 and (b) metabolite

6 in

114

400

500

ml2 Fig. 5. EI mass spectrum

600

ml2

of the TMS/ME derivative after hydrogenation.

of metabolite

5

Fig. h. EI mass spectrum

12(R)-configuration of the starting material, 6a was identified as 5(R),12(R)-(EZEZ)-DHETE (enantiomer of 5(s>,l2(s)-(EZEZ)-DHETE) and metabolite 6b was identified as 5(s),l2(R)-(EZEZ>-DHETE. During the course of these investigations, it was found that metabolite 6 underwent conversion to metabolite 5 when stored at 0°C in methanol for four weeks. This is consistent with olefin isomerization to the thermodynamically more stable all-trans configuration and suggests that 6 contributes to the production of 5. Metabolites 7 and 8 were further purified on HPLC system F (t,: 24.9 and 26.7 min, respectively). Both metabolites absorbed maximally at 235.5 nm suggesting that the original conjugated diene system was preserved. The intense [A4 - PFB]- ions at m/z 407 in the NICI mass spectra of their TMS/PFB derivatives suggested that epoxidation had occurred at either of the non-conjugated olefinic double bonds. The hydrogenated TMS/PFB derivatives of metabolites 7 and 8 each showed an intense [M - PFBI- ion at m/z 413,

of the TMS/ME derivative after hydrogenation.

Discussion Platelets and macrophages metabolize arachidonic acid to 12(S)-HETE [25]. In contrast, arachidonic acid is metabolized to 12(R)-HETE by rat liver microsomes and by psoriatic skin [7,8]. Further metabolism of 12(S)-HETE by neutrophils and macrophages results in the formation of 5,12-DHETE [14] and 12,20-

(20.0 ‘70) lPR.lS-DHETE

\

3.4

COOH

(28.1 %I

1

CH20H

5,12R(EEEZ)-DHETE

OH

(3 1 70)

COO”

5

5,12R(EiiZ)-DHETE

lZR,PO-DHETE

(34.2 RI

(5.2 70)

6

Fig. 7. Scheme

for metabolism

7

confirming that both metabolites had three double bonds. The EI mass spectra of hydrogenated TMS/ME derivatives of metabolites 7 and 8 each showed prominent fragment ions at m/z 301 (Fig. 6), consistent with a hydroxyl group at C-12 and an oxido function at C-14,15. Consequently, metabolites 7 and 8 were identified as the diastereomeric 14,15-epoxides of 12(R)HETE. The last eluting compound was unchanged 12(R)-HETE. Peaks a - e (Fig. 1) were not radioactive and were not, therefore, investigated further.

OH

lZR,lG-DHETE

of metabolite

of 12(R)-HETE

by rat liver microsomes.

115 DHETE [ 151. These biotransformations are mediated by 5Jipoxygenase [14] and cytochrome P-450, respectively [ 151. 12,20-DHETE undergoes further metabolism to 12(S)-HETE-1,20-dioic acid through an NAD-dependent w-hydroxy fatty acid dehydrogenase located in neutrophils [26]. As shown in the present study, cytochrome P-450-dependent metabolism of 12(R)-HETE results mainly in w/w - 1 hydroxylation (62% of total products, Table II>. The identification of an w - 1 hydroxylated metabolite contrasts with the observation of Marcus et al. [15] for 12(S)-HETE where it was reported that neutrophil cytochrome P-450 gave exclusively the w-hydroxylated product. Further cytochrome P-450-dependent o-hydroxylation products of 12(R)-HETE were observed in the minor metabolites 3 and 4 that were characterized as 12(R),16DHETE diastereomers. There is a precedence for hydroxylation at C-16 as we [2] and others [27] have recently reported that AA undergoes cytochrome P450-mediated hydroxylation at this position. Hydroxylation of 12(R)-HETE at C-5 led to the formation of metabolites 5 (5,12(R)-(EEEZI-DHETE) and 6 (5,12(R)-(EZEZ)-DHETE). These metabolites were similar to 5,12-DHETE, the 5-lipoxygense product formed from 12(S)-HETE. However, the 5,12DHETEs formed from 12(R)-HETE were both found to be 1 : 1 mixtures of diastereomers. Thus, it appears that compared with 5-lipoxygenase-mediated hydroxylation of 12(S)-HETE 1141, cytochrome P-450-mediated hydroxylation of 12(R)-HETE is not stereoselective. During the course of the present study, it was observed that metabolite 6 underwent conversion to the thermodynamically more stable all-trans-triene olefin geometry to give metabolite 5. Thus, it seems likely that metabolite 6 is the enzymic product and that it undergoes non-enzymic conversion to 5 during the isolation and purification procedure. Two epoxide metabolites of 12(R)-HETE (7 and 8) were identified. Metabolites of this type derived from 12(S)-HETE have not been previously characterized. Interestingly, the epoxides were formed as a pair of 14,15-epoxide diastereomers rather than a mixture of regioisomers, suggesting that the cytochrome P-450 epoxygenase is not stereoselective for 12(R)-HETE. This differs from similar studies with AA where it was found that a mixture of regioisomers was formed and that 14(R),lS(S)-EET was the predominant 14,lSEET [ll. From data obtained in the present study it appears that the conjugated diene and 12(R)-hydroxyl group in 12(R)-HETE prevent cytochrome P-450-mediated epoxidation of olefinic double bonds other than the one at C-14. However, it is known that 5,6-epoxides are extremely unstable. The possibility that a 5,6-EET metabolite was formed in the microsomal incubation and that it decomposed during the isolation procedure cannot be completely ruled out.

In summary, we have demonstrated that 12(R)HETE undergoes cytochrome P-450-dependent oxidation to give a mixture of DHETEs, LT isomers and epoxy alcohols (Fig. 7). The structural similarity to other known eicosanoid autocoids suggests that the metabolites may possess biological activity. Metabolite 6 is particularly intriguing as one of its diastereomers only differs from the potent chemotactic agent, LTB, [28] in having a trans-cis-truns-conjugated triene rather than a cis-trams-tram triene. There is a precedence for the formation tram isomers of LTB, by Kupffer cells from rat liver [29]. Further studies will clearly be required to assess the biological significance of the metabolites and to determine whether they are formed in viva Acknowledgements This work was supported by National Institutes of Health Grant DK 38226. We acknowledge the invaluable advice given by Dr Alan R. Brash. References 1 Karara, 2

3 4 5 6 7 8 9

10 I1 12 13 14

15 16 17

A., Dishman, E., Blair, I.A., Falck, J.R. and Falck, J.H. (1990) J. Biol. Chem. 264, 19822-19827. Falck, J.R., Lumin, S., Blair, IA., Waxman, D.. Dishman, E., Guengerich, F.P. and Capdevila, J.H. (1990) J. Biol. Chem. 26, 10244-10249. Fitzpatrick, F.A. and Murphy, R.C. (1989) Pharmacol. Rev. 40, 229-241. Escalante, B., Erlij, D. Falck, J.R. and McGiff, J.C. (1991) Science 251, 799-801. Catella, F., Lawson, J.A. and FitzGerald, G.A. (1990) Proc. Natl. Acad. Sci. USA 87, 2223-2229. Hannigan, G.E. and Williams, B.R.G. (1991) Science 251, 204207. Capdevila, J.C., Yadagiri, P.. Manna, S. and Falck, J.R. (1986) Biochem. Biophys. Res. Commun. 141, 1007-1011. Woollard, P.M. (1986) Biochem. Biophys. Res. Commun. 136, 169-176. Woollard, P.M., Cunningham, F.M., Murphy, G.M., Camp, R.D.R., Derm, F.F. and Greaves, M.W. (1989) Prostaglandins 38, 465-471. Masferrer, J.L., Rios, A.P. and Schwartzman, M.L. (1990) Biochem. Pharmacol. 39, 1971-1974. Bacon, K.B., Camp, R.D., Cunningham, F.M. and Woollard, P.M. (1988) Br. J. Pharmacol. 95, 966-974. Masferrer, J.L. and Mullane K.M. (1988) Eur. J. Pharmacol. 151, 487-490. Mathur, S.A., Albright, E. and Field, F.J. (1990) J. Biol. Chem. 265, 21048-21055. Marcus, A.J., Broekman, M.J., Safier, L.B., Ullman, H.L., Islam, N., Serhan, C.N., Rutherford, L.E., Korchak, H.M. and Weissmann, G. (19821 Biochem. Biophys. Res. Commun. 109, 130-137. Marcus, A.J., Safier, L.B., Ullman, H.L., Islam, N., Broekman, M.J. and von Schacky (1987) J. Clin. Invest. 79. 179-187. Yadagiri, P., Lumin, S., Mosset, P., Capdevila, J. and Falck, J.R. (1986) Tetrahedron Letts. 27, 6039. Manna, S., Viala, J., Yadagiri, P. and Falck, J.R. (1986) Tetrahedron Letts. 27, 2679.

116 IX Hawkins D.J. and Brash, A.R. (19X7) J. Biol. Chem. 262, 762Y7634. 19 Remmer. H.. Griem, H., Schenkman. J.B. and Estahrook. R.W. (1967) Methods Enzymol. 10, 703-708. 20 Blair. I.A. (1900) Methods Enzymol. 1X7, 13-23. 21 Borgeat, P. and Samuelsson, B. (1979) J. Biol. Chem. 254, 7X657869. 22 Borgeat, P., Picard, S., Vallerand, P. and Sinus, P. (1981) Prostaglandins Med. 6. 557-570. 2.1 Radmark, 0.. Malmsten C. and Samuelsson, B. (1980) Biochem. Biophys. Res. Commun. 92, 954-961. 24 Yoshimoto, T., Mitamoto. Y.. Ochi, K. and Yamomota, S. (lYX2) Biochim. Biophys. Acta 713, 63X-646.

25 Spector, A.A., Gordon, J.A. and Moore, S.A. (1088) Prog. Lipid Res. 27. 27 I-323. 26 Marcus, A.J., Sat’ier, L.B., Ullma, H.L., Ilm, N., Broekman, M.J.. Falck, J.R.. Fischer, S. and Schacky, J.R. (198X) J. Biol. Chem. 263, 2223-2229. 27 Glare, R.A.. Huang, S., Doiy, M.V. and Gibson, G.G. (IYYI) J. Chromatogr. 562, 237-247. 2X Ford Hutchinson. A.W., Bray, M.A., Doig, M.V., Shipley, M.E. and Smith, M.J.H. (1980) Nature 286, 264-265. 29 Gut. J.. Goldman. D.W. and Trudell. J.R. (19Xx) Mol. Pharmacol. 34. 256-264.

Metabolism of 12(R)-hydroxyeicosatetraenoic acid by rat liver microsomes.

The in vitro metabolism of 12(R)-hydroxyeicosatetraenoic acid was studied using freshly isolated rat liver microsomes. Ten metabolites were isolated a...
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