NADPH-dependent microsomal metabolism of 14,15-epoxyeicosatrienoic acid to diepoxides and epoxyalcohols

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 261, No. 1, February 15, pp. 122-133,1988

NADPH-Dependent Microsomal Metabolism of 14,i SEpoxyeicosatrienoic

Acid to Diepoxides and Epoxyalcohols’~*

JORGE H. CAPDEVILA,* PAUL MOSSET, PENDRI YADAGIRI, SUN LUMIN, AND J. R. FALCK3

*Division of Nephrology, Vanderbilt Medical School, Nashville, Tennessee 37232 and Departments of Molecular Genetics and Phamacology, University of Texas Health Science Center, Dallas, Texas 75235

Received June 29, 1987, and in revised form October 28, 1987

The arachidonic acid epoxygenase metabolite 14,15-epoxyeicosatrienoic acid is further metabolized by rat liver microsomal fractions to regioisomeric diepoxides and epoxyalcohols. Diepoxides result from epoxidation at the 5,6-, 8,9-, or 11,12-olefins. Hydroxylation leading to epoxyalcohols with a cis, trans-conjugated dienol occurs at carbons 5,8,9, or 12. Structural assignments were established by chromatographic and mass spectral comparisons with synthetic standards. The reaction requires NADPH and is inhibited by typical cytochrome P-450 inhibitors. Analysis of the time course of product formation during arachidonic acid oxidation by rat liver microsomal fractions indicated that all four regioisomeric epoxyeieosatrienoic acids can be further metabolized by the enzyme system. B 19s~ Academic press, I~C.

The initial oxidative metabolism of arachidonic acid is catalyzed by cyclooxygen- ase to generate a highly reactive endoper- oxide or by a group of lipoxygenases to form any of several isomeric hydroperoxides. These intermediates are further metabolized to a series of biologically impor- tant molecules such as prostaglandins, thromboxanes, prostacyclin, lipoxins, and leukotrienes (1). An alternative route of eicosanoid production catalyzed by cytochrome P-450 has been described (2-4). Designated the epoxygenase pathway, this route generates four regioisomeric epoxyeicosatrienoic acids (EETs)~ (5) with po-

’ This work was supported by USPHS Grants GM 31278 and 33541 and the Robert A. Welch Foundation (I-782).

ZThe chemical syntheses and spectral data of the oxygenated standards appear as a Miniprint Supple- ment.

3 To whom correspondence should be addressed. ’ Abbreviations used: EET, epoxyeicosatrienoic

acid; DHET, vie-dihydroxyeicosatrienoic acid; 5-OH-14,15-EET, 5-hydroxy-14,15-epoxy-6(E),8(2),

tential physiological relevance (6). The EETs possess potent in vitro biological activities (2, 7-10) and have been detected in mammalian tissue (11, 12) and in human urine (13).

The EETs are enzymatically hydrated to the corresponding vic-dihydroxyeicosatrienoic acids (DHETs) by cytosolic epoxide hydrolase (14) and arc actively conjugated to GSH in a reaction catalyzed by cytosolic GSH-S-transferases (15). As part of our studies of the arachidonic acid

11(Z)-eicosatrienoic acid; 8-OH-14,15-EET, 8-hydroxy-14,15-epoxy-5(Z),S(E),ll(Z)-eicosatrienoic acid; 9-OH-14,15-EET, 9-hydroxy-14,15-epoxy- 5(2),7(E),ll(Z)-eicosatrienoic acid; 12-OH-14,15- EET, 1!2-hydroxy-14,15-epoxy-5(Z),8(Z),lO(E)-eicosatrienoic acid; 5,6-epoxy-14,15-EET, 5,6:14,15-diepoxy-8(Z),ll(Z)-eicosadienoic acid; 8,9-epoxy-14, 15-EET, 8,9:14,15-diepoxy-5(Z),ll(Z)-eicosadienoic acid; 11,12-epoxy-14,15-EET, 11,12:14,15-diepoxy- 5(Z),8(Z)-eicosadienoic acid; TMS, trimethylsilyl; GC, gas chromatography; pC1, positive ion chemical ionization; MS, mass spectrometry; amu, atomic mass unit; Rt, retention time.

0003-9861/88 $3.00 Copyright 0 1988 by Academic Prew, Inc. All rights of reproduction in any form reserved.

122

__. ---- ----._--._- ,.yTI- .-yn.7 AT NAUl’H-UIWr;NUr;N’I‘ UXILJA’I’IUN VI’ 14,15-EPOXYEICOSATRIENOIC ACID 123

epoxygenase reaction and of the fate of its reaction products, we report here the NADPH-dependent conversion of 14,15- EET to epoxyalcohols and diepoxides. An increased awareness of the metabolic fate of the epoxygenase products should prove useful for the analysis of this pathway in viva as well as assist in the elucidation of novel metabolites.

230°C at a rate of 5”C/min. He and CH4 were utilized as carrier and reagent gases, respectively. The injec- tion port, transfer line, and source temperatures were 220,240, and 2OO”C, respectively.

RESULTS

MATERIALS AND METHODS

Racemic [l-i4C]14,15-EET was synthesized accord- ing to (16) from [1-‘Vlarachidonic acid (Amersham, Arlington Heights, IL) and it was diluted with arachidonic acid (Nu-Chek Prep, Elysian, MN). The chemical syntheses and spectral data of the oxygenated standards are included in the Miniprint Supple- ment. The radioactivity of the HPLC eluants was monitored using a Radiomatic Flow-One/Beta detec- tor (Radiomatic Instruments, Tampa, FL).

Microsomal fractions were isolated as described (17) from the livers of male Sprague-Dawley rats. Animals were injected daily with phenobarbital (80 mg/kg body wt) for 4 days prior to sacrifice (17). Incubation mixtures containing 50 mM Tris-Cl (pH 7.5), 150 mM KCl, 10 mM MgClz, 8 mM sodium isocitrate, 0.8 IU/ml isocitrate dehydrogenase, and 0.5 mg of microsomal protein/ml were maintained at 25°C for 1 min prior to the addition of 100 pM (final concentration) of HPLC purified [1-“‘Clarachidonic acid (0.2 pCi/mmol) or [l-i4C]14,15-EET (0.2 &i/mmol). Reactions were initiated by the addition of NADPH (1 mM final concentration) and continued under constant mixing for specified time periods. The reaction products were extracted twice with an equal volume of acidified ethyl acetate and, after solvent evaporation under argon, dissolved in ethanol and resolved by reverse-phase HPLC on a PBondapak Cis column (0.39 X 30 cm, Waters Associates, Milford, MA) utilizing a linear gradient of 49.95% HzO, 49.95% CH,CN, 0.1% CH,COOH to 99.9% CH,CN, 0.1% CHaCOOH at a rate of change of 1.25% per minute and a flow rate of 1 ml/min. The 14,15-EET derived metabolites were additionally fractionated by normal-phase HPLC utilizing a PPorasil column (0.39 X 30 cm, Waters Associates) and a linear gradient of 2.5% isopropanol, 97.4% hexane, 0.1% CHaCOOH to 5% isopropanol, 94.9% hexane, 0.1% CHaCOOH at a rate of change of 0.83% per minute and a flow rate of 2 ml/min.

Figure 1 shows the reverse-phase HPLC chromatograms of the organic soluble products extracted from comparable aliquots taken at 7.5 or 40 min from incu- bates containing 100 PM arachidonic acid and 0.5 mg of microsomal protein/ml. The profile in Fig. 1A was obtained after 7.5 min of incubation and is essentially identical to previously published data (1’7). Product generation remained linear within the first 10 min of incubation. With the exception of the DHETs, the profile shows primary oxygenation products. The cytochrome P-450 generated metabolites (Fig. 1A) were resolved by functionality into groups of (a) DHETs (R,, 16-18 min),

2- A

HETE EET

Methylations, silylations, and catalytic hydrogen- ations were performed as described (18). Packed column GC/MS analyses were obtained with a Finnigan 4021 spectrometer equipped with an INCOS 4000A data system. Samples were injected onto a 6 ft 5% SP 2100 DOH glass column (Supelco, Inc., Bellefonte, PA). The column temperature was raised from 210 to

FIG. 1. Reverse-phase HPLC of the organic soluble products of arachidonic acid metabolism generated after 7.5 and 40 min of incubation. Rat liver microsomal fractions (0.5 mg of protein/ml) were incubated with [1-‘“Clarachidonic acid in the presence of NADPH. Samples of the reaction mixture were withdrawn at 7.5 (A) and 40 min (B). After product extraction, comparable aliquots were analyzed by HPLC as indicated under Materials and Methods.

124 CAPDEVILA ET AL.

(b) w/w-l oxidation products (R,, 19 min), (c) HETEs (R,, 22-25 min), and (d) EETs (R,, 26-30 min). Comparison of the chro- matogram obtained at 7.5 min (Fig. 1A) with the profile at 40 min (Fig. 1B) demon- strates that with longer incubation times there was an increased production of more polar metabolites with a concomitant de- crease of low polarity metabolites. The data in Fig. 1 suggest that most of the primary metabolites are substrates for further metabolism by the microsomal fractions. Analysis of the time course of the reaction, under conditions identical to those in Fig. 1, showed that during the first 10 min of incubation the EETs repre- sent 30-35% of the total reaction products. This value decreased to 2-3% after 60 min. The initial rate of arachidonic acid metabolism remained constant at 4-6 nmol/ min/mg of microsomal protein during the first 10 min and then decreased contin- uously, reaching l-2 nmol/min/mg of microsomal protein after 60 min of incubation.

While all four EETs appeared to be further metabolized by the microsomal enzymes (Fig. l), 14,15-EET was selected as a model substrate for initial study of the secondary NADPH-dependent metabolism of the epoxygenase products. This isomer is the major product formed during arachidonic acid metabolism by rat liver microsomal fractions (18), is chemically stable and therefore amenable to a more straightforward analysis, and has been detected as an in vivo constituent of rabbit kidney (12) and human urine (13).

Figure 2 shows the result of an experi- ment in which rat liver microsomal fractions were incubated with racemic 14,15- EET in the presence (Fig. 2A) and in the absence of NADPH (Fig. 2B). After 5 min at 25”C, the reaction mixtures were extracted with ethyl acetate and the organic soluble material was analyzed by reverse- phase HPLC as described under Materials and Methods. The microsomal fractions catalyzed both NADPH-dependent and in- dependent reactions. Control experiments (not shown) performed under identical conditions, but utilizing heat denatured microsomes, demonstrated that both reac-

IO 1 +NADPH A

x LL IO 0

5

(

- NADPH B

\

\. n

IO 20 30

FIG. 2. Reverse-phase HPLC of the organic soluble products of 14,15-EET metabolism catalyzed by rat liver microsomal fractions. Microsomal fractions (0.5 mg of protein/ml) were incubated with [l-‘%]14,15- EET (50 pM) in the presence (A) or absence of NADPH (B). Samples of the reaction mixture were withdrawn after 5 min. Comparable aliquots of the organic soluble products were analyzed by HPLC as described under Materials and Methods.

tions are enzymatic. The radioactive material eluting at 16.5 min (Fig. 2B) has been previously identified as 14,15-DHET (14). To evaluate the role of cytochrome P-450 in the microsomal metabolism of 14,15- EET, incubations were performed in the presence of typical inhibitors of the heme- protein function. Ketoconazole and clotrimazole (19) produced a clear inhibition of the reaction (10% of controls, Table I). Metyrapone also significantly inhibits metabolism (35% of controls, Table I).

In the presence of NADPH, the microsomal fractions catalyzed the time-dependent formation of several metabolites which were resolved by reverse-phase HPLC into four main radioactive fractions (Fractions A, B, C, and D, Fig. 2A). Prod- uct formation was linear within the first 10 min of incubation and proceeded at a

NADPH-DEPENDENT OXIDATION OF 14,15-EPOXYEICOSATRIENOIC ACID 125

TABLE I

EFFECT OF CYTOCHROME P-450 INHIBITORS ON THE

NADPH-DEPENDENT MICROSOMAL

METABOLISM OF 14,15-EET

TABLE II

REVERSE-PHASE HPLC R,‘s OF

SYNTHETIC STANDARDS

Rt structure (min)

Inhibitor

Rate (nmol/min/mg

protein) 5% of

control

None 3.1 100 Ketoconazole, 20 PM 0.3 10 Clotrimazole, 20 fiM 0.4 13 Metyrapone, 50 PM 1.1 35

Note. Rat liver microsomal fractions were incubated at 25°C with the inhibitors for 1 min prior to the addition of 14,15-EET (100 FM final concentration). Reactions were initiated by the addition of NADPH (1 mM, final concentration) and terminated after 5 min. Product extraction and resolution were done as described under Materials and Methods. Values shown are the averages of three different experiments. Standard deviations were ~5% of the averages.

5-OH-14,15-EET 15.9 Methyl 5-OH-14,15-EET 25.5

8- and 9-OH-14,15-EET 14.6 Methyl 8- and methyl 9-OH-14,15-EET 23.7

12-OH-14,15-EET 17.1 Methyl 12-OH-14,15-EET 25.0

5,6-Epoxy-14,15-EET 20.2 Methyl 5,6-epoxy-14,15-EET 29.3 5.6.Epoxide of 14,15-epoxyeicosanoic acid 25.0 5,6-Epoxide of methyl 14,15-epoxyeicosanoate 34.3

8,9-Epoxy-14,15-EET 22.1 Methyl 8,9-epoxy-14,15-EET 29.6 8,9-Epoxide of 14,15-epoxyeicosanoic acid 23.1 8,9-Epoxide of methyl 14,15-epoxyeicosanoate 33.2

11,12-Epoxy-14,15-EET 21.3 Methyl 11,12-epoxy-14,15-EET 30.6 11,12-Epoxide of 14,15-epoxyeicosanoic acid 26.0 11,12-Epoxide of methyl 14,15-epoxyeieosanoate 36.2

rate of 3.1 nmol/min/mg of microsomal protein. At 50-100 PM 14,15-EET, hydra- tion to 14,15-DHET accounted for less than 5% of the total products. However, at low substrate concentrations (~5 PM), hy- dration was a significant factor account- ing for 10-20s of the total products, thus severely limiting any meaningful kinetic analysis of the oxygenation reaction.

Fraction B (Fig. 2A; Rt, 13-15 min) (24% of the total reaction products) was collected batchwise and, after solvent evaporation, resolved by normal-phase HPLC as described under Materials and Methods. Fraction B was separated into two components: Bl (R,, 20.6 min) and B2 (R,, 22.0 min). Neither Bl nor B2 contained a uv chromophore. Analysis by pCI/GC/MS of the methyl ester TMS ether of Bl showed a major component with a GC Rt of 10.5 min and ions at m/x 451 (addition of CzH5), 423 (addition of H), 407 (loss of CH3), 391 (loss of OCHB), 333 (loss of OTMS), and 315 (base peak, loss of OTMS and HzO). The methyl ester TMS ether of hydrogenated Bl showed an increase of 6 amu for each of the ions described above, indicating the presence of three double bonds in the orig-

Note. Rts were obtained by reverse-phase HPLC utilizing the conditions described under Materials and Methods.

inal molecular structure. Analysis by pCI/GC/MS of the methyl ester TMS ether derivative of B2 and of its hydro- genation product gave results similar to

0.2

1

oh, I , , , , , , , , 14 18 22 26 30

RETENTION (Mini

FIG. 3. Normal-phase HPLC of the 14,15-EET metabolites contained in Fraction C. Fraction C (Fig. 2A) was purified by reverse-phase HPLC and then injected onto a pPorasi1 column. Product resolution was done as described under Materials and Methods. The uv absorbance of the eluant was monitored at 235 nm.


234 j- “.Yk

, ,,,I,, I,,,,, 210 250 290 210 es0 290 210 250 es0

WAVELENGTH (nm)

FIG. 4. Ultraviolet absorption spectra of the metabolites contained in Fraction C. Fractions Cl, C2, and C3 were purified by normal-phase HPLC (Fig. 3). After solvent evaporation they were dissolved in CHaOH and their absorption spectra were determined between 210 and 310 nm utilizing an Aminco DWU-2 spectrophotometer.

Bl. These data are consistent with a cy- The chromatographic properties of syn- tochrome P-450 catalyzed hydroxylation thetic standards suggested that fraction C at the C-20 or C-19 positions of 14,15-EET (R,, 15-19 min; Fig. 2A) was a mixture of (o and o-l oxidation products, respec- diene conjugated hydroxy-EETs (Table tively). The unequivocal identification of II). Fraction C was collected batchwise these metabolites will require compari- and, after solvent evaporation under sons with authentic standards. The corre- argon, was resolved into Cl, C2, and C3 sponding trihydroxy derivatives have been (Fig. 3) by normal-phase HPLC as de- previously reported (3). scribed under Materials and Methods.

100 150 200 250 300 350 400 450

FIG. 5. Mass spectrum of the 14,15-EET metabolite contained in Fraction Cl. An aliquot of the methyl ester TMS ether derivative of Cl (Fig. 3) was submitted to pCI/GC/MS analysis as described under Materials and Methods. Shown is the mass spectrum of the material with a GC Rt corresponding to that of the methyl ester TMS ether derivative of synthetic 12-OH-14,15-EET. Abscissa: mass scale, m/z; ordinate: abundance as percentage of the base peak.


FIG. 6. Mass spectrum of the methyl esters of Fraction D. An aliquot of the methyl esters of Fraction D (Fig. 2A) was submitted to pCI/GC/MS analysis as described under Materials and Methods. Shown is the mass spectrum of the material eluting with a GC R, corresponding to that of an equimolar mixture of the three diepoxides of synthetic 14,15-EET. Abscissa: mass scale, m/z; ordinate: abundance as percentage of the base peak.

Fractions Cl, C2, and C3 displayed in methanol a uv chromophore typical of a conjugated diene (Fig. 4). Cl was identified as 12-OH-14,15-EET based on (a) comparable reverse-phase HPLC Rt’s of its free acid and methyl ester with that of the corresponding synthetic standards (Table II); (b) a normal-phase HPLC retention time (Fig. 3) comparable with synthetic 12-OH-14,15-EET (18.7 min); and (c) a GC retention time (14.5 min) and a pCI/ MS fragmentation pattern of its methyl ester TMS ether derivative similar to that of a synthetic standard analyzed under identical conditions (Fig. 5). Upon methylation, Fraction C2 was resolved by reverse-phase HPLC into two components with retention times of 24.8 and 26.0 min. The earlier component was identified as 5-OH-14,15-EET based on (a) comparable reverse-phase HPLC Rt’s of its free acid and methyl ester with that of the corresponding synthetic standards (Table II); (b) a normal phase HPLC retention time (Fig. 3) comparable with synthetic 5-OH-14,15-EET (22.6 min); and (c) a GC Rt (16.7 min) and a pCI/MS fragmentation pattern of its methyl ester TMS ether derivative similar to that of a synthetic

standard analyzed under identical conditions (data not shown). The second component of methyl C2 (26.0 min) was shown to be 14,15-DHET methyl ester based on previously published data (14). Fraction C3, after methylation, eluted as a single

- FREE ACIDS -~--METHYL ESTERS

0’ ’ 7 20 25 30 35 40

TiME ,Mln”lesl

FIG. ‘7. Reverse-phase HPLC of hydrogenated Fraction D before and after methylation. Fraction D (Fig. 2A) was hydrogenated catalytically as described under Materials and Methods. An aliquot of hydrogenated Fraction D and its methyl ester were analyzed by reverse-phase HPLC as described under Materials and Methods.

128 CAPDEVILA ET AL

component by reverse-phase HPLC with a Rt of 24.1 min. pCI/GC/MS analysis of the TMS ether methyl ester of C3 revealed two components with GC R/s of 14.8 and 15.3 min, each with similar fragmentation patterns. A mixture of authentic 8-OH- and 9-OH-14,15-EET TMS ether methyl esters also resolved into two components

with GC Rls and pCI/MS fragmentation patterns comparable with those of C3 methyl ester TMS ether. C3 is therefore a mixture of &OH- and 9-OH-14,15-EETs. The formation of 8-OH-14,15-EET by rabbit liver microsomal fractions has been previously suggested (3).

The chromatographic properties of syn-

A: BIOLa SAMPLE

loo 113 B: STANDARD

337

FIG. 8. Mass spectrum of the methyl ester of Fraction D3 and of synthetic methyl 11,12-epoxy- 14,15-epoxyeicosanoate. An aliquot of esterified Fraction D3 (Fig. 7B) was submitted to pCI/GC/MS analysis as described under Materials and Methods. Shown is the mass spectrum of the material eluting with a GC R, corresponding to that of a standard of the 11,12-epoxide of methyl 14,15-epoxyeicosanoate. Top: biological sample. Bottom: synthetic standard. Abscissa: mass scale, m/z; ordinate: abundance as percentage of the base peak.


TABLE III maior comnonents designated Dl, D2, and

RELATIVE DISTRIBUTION OF THE REACTION PRODUCTS D3”with Ri’s of 33.2, 34.3, and 36.2 ‘min, FORMED BY NADPH-DEPENDENT MICROSOMAL respectively (Figure 7, dashed line), corre-

METABOLISM OF 14,SEET sponding to those of the 8,9-, 5,6-, and 11,12-epoxides of synthetic methyl 14,15-

Fraction Reaction

type % of total

product structure

A ND” 4 B w/w-l oxidation 25 C Allylic oxidation” 10

10 14

D Epoxidation 6 17 14

ND 20-/19-OHb 12-OH 5-OH g-/g-OH 5,6-Epoxide 8,9-Epoxide 11,WEpoxide

Note. 14,15-EET (100 pM) was incubated with 0.5 mg microsomal protein/ml for 10 min at 25°C. Products were analyzed as described under Materials and Methods.

“Not determined. b Tentatively identified. ‘Corrected for the contribution of 14.15-DHET.

thetic standards (Table II) suggested that Fraction D (Fig. 2A) contained a mixture of epoxy-14,15-EETs. As expected, Frac- tion D did not contain a uv chromophore. After methylation, D eluted with a GC R, of 13.5 min and had a pCI/MS fragmentation pattern with major ions at m/x 379 (addition of C2H5), 351 (addition of H), 333 (loss of OH), 319 (loss of OCHB), and 301 (loss of OCH3 and HzO). A standard mixture of the three possible regioisomeric epoxy-14,lkEET methyl esters, analyzed under identical conditions, coeluted with a GC R, and displayed a pCI/MS fragmentation pattern similar to that of the biological sample shown in Fig. 6.

Since it was not possible to satisfacto- rily resolve Fraction D into its components by normal- or reverse-phase HPLC, an aliquot was catalytically hydrogenated. Reverse-phase HPLC resolved hydrogenated D into four fractions with Rt's of 20.9, 23.0, 25.0, and 25.9 min (Fig. 7, solid line) corresponding to those of authentic 5,6-dihydroxy-14,15-epoxyeicosanoic acid d-lactone and the 8,9-, 5,6-, and 11,12- epoxides of 14,15-epoxyeicosanoic acid, respectively (Table II). Upon esterification with diazomethane, the free acid fraction of hydrogenated D resolved into three

epoxyeicosanoate, respectively (Table II). Comparative pCI/GC/MS analyses of

esterified aliquots of D3 and a standard of the 11,12-epoxide of 14,15epoxyeicosan- oate are shown in Fig. 8. Both the synthetic compound and fraction D3 eluted at 11.2 min and had similar pCI/MS fragmentation patterns with major ions at m/x 355 (addition of H), 337 (loss of H,O), 323 (loss of OCH3), 305 (loss of OCH3 and HeO), 287, 253, 227, and 213. By a similar analysis, Dl and D2 were identified as the 8,9- and 5,6-epoxides of methyl 14,15- epoxyeicosanoate, respectively (data not shown). Based on the above chromatographic and MS data, it was concluded that Fraction D (Fig. 2A) contained a mixture of 5,6-, 8,9-, and 11,12-epoxy- 14,15-EETs.

Table III shows the relative distribution of the identified products generated during the NADPH-dependent oxidation of 14,15-EET. Products were generated under conditions favoring primary metabolism of the epoxide, i.e., low protein concentration and short incubation times. No structural information is as yet available for approximately 4% of the remaining reaction products (Fraction A, Fig. 2A).

DISCUSSION

The wide variety of structurally related compounds produced by the arachidonate cascade is one of the best examples of the importance of secondary metabolism in the biotransformation of physiologically relevant molecules. Secondary metabolism also plays a fundamental role in the subsequent inactivation and disposition of these metabolites (20, 21). Elucidation of the enzymology and cellular fate of eicosanoids is critical to our understanding of their mechanism of action and role in ho- meostasis.

The initial oxygenation step catalyzed by the microsomal, cytochrome P-450-dependent arachidonic acid epoxygenase re-


02 Dl 03

FIG. 9. Summary of the oxidative metabolism of 14,15-EET.

sults in the formation of four regioiso- merit EETs (5) with potent in vitro biological activities (2, ‘7-10). As part of a study to delineate their mechanism of action and possible physiological function, we initiated an investigation into their in vitro metabolic fate. The available evidence shows that the EETs are substrates for further metabolism by (a) microsomal and cytosolic forms of epoxide hydrolase (3, 14), (b) cytosolic GSH-S-transferases (15), and (c) prostaglandin synthetase (22). The data presented here further expand the contribution of cytochrome P-450 to the rich structural diversity that character- izes the arachidonic acid cascade.

The microsomal NADPH-dependent oxidation of 14,15-EET proceeds via three types of reactions: (a) w/o-l oxidation, (b) allylic oxidation to cis, trans-conjugated hydroxy-EETs, and (c) epoxidation (Fig. 9). All three reactions appeared to be catalyzed by the microsomal cytochrome P-450 enzyme system. The hydroxy-EETs and epoxy-EETs reported here are novel metabolites of arachidonic acid. While several epoxy-hydroxy eicosanoids have been reported as products of the enzymatic and nonenzymatic rearrangement of arachidonic acid hydroperoxides (23,24), the cytochrome P-450 derived epoxydienols are

structurally distinct. No attempt was made to characterize possible diastereo- isomers generated by the enzyme system from the racemic 14,15-EET substrate.

Figure 1 shows that the other isomeric EETs are also metabolized by the microsomal enzymes. Moreover, they are able to compete efficiently for the enzyme active site even in the presence of an excess of arachidonic acid. Preliminary evidence suggests that during 14,15-EET stimu- lated release of luteinizing hormone from a preparation of isolated rat anterior pitu- itary cells, the EET is oxidized to an epoxy-alcohol chromatographically similar to the type reported here (Gary D. Snyder, personal communication).

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