THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 264, No. 33, Issue of November 25, pp. 19822-19827, 1989 Printed in U. S. A.

Endogenous Epoxyeicosatrienoic Acids CYTOCHROME P-450 CONTROLLED STEREOSELECTIVITY OF THE HEPATIC ARACHIDONIC ACID EPOXYGENASE*

(Received for publication, June 12, 1989)

Armando KararaS, Elizabeth Dishman$, Ian Blair#, J. R. FalckT, and Jorge H. Capdevila$)I** From the Departments of $Medicine (Division of Nephrology), §Pharmacology and I( Biochemistry, Vanderbilt University, Nashville, Tennessee 37232 and the 7Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75235

Chiral analysis of the endogenous rat liver epoxyei- cosatrienoic acids shows the biosynthesis of 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids in a 4:1, 2:1, and 3:l ratio of antipodes, respectively. Animal treatment with phenobarbital results in a 3.7-fold in- crease in microsomal cytochrome P-450 concentration and a concomitant, regioselective 6.8- and 3.4-fold increase in the liver concentration of 8,9- and 14,15- epoxyeicosatrienoic acids, respectively. Phenobarbital induces the in vivo synthesis of both regioisomers as nearly optically pure enantiomers. These results dem- onstrate the enzymatic origin of the epoxyeicosatri- enoic acids present in rat liver and document a novel metabolic function for cytochrome P-450 in the regio- and enatioselective epoxygenation of endogenous pools of arachidonic acid.

Lipid-derived mediators play a central role in the control of cell and tissue physiology (1). Hence, specific information is transduced to the arachidonic acid template via distinct regio- and stereoselective oxygenations. Mammalian cells uti- lize this chemical information to convey physiologically rele- vant intra- and/or intercellular messages (2). The cytochrome P-450 arachidonic acid epoxygenase catalyzes the NADPH- dependent, stereoselective, epoxidation of the fatty acid to generate 5,6-, 8,9-, 11,12-, and 14,15-cis-epoxyeicosatrienoic acids (EETs)' (3-5).* The potential significance of this novel

Grants GM37922 (to J. H. C.) and GM31278 (to J. R. F.) and North * This work was supported in part by National Institutes of Health

Atlantic Treaty Organization Grant RG 26/0085 (to J. H. C. and J. R. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

** To whom correspondence should be addressed Medical Center North s-3223, Vanderbilt University, Nashville, TN 37232.

' The abbreviations used are: EETs, cis-epoxyeicosatrienoic acids; EET-Me, methyl cis-epoxyeicosatrienoate; EET-PFB, pentafluoro- benzyl cis-epoxyeicosatrienoate; PB, phenobarbital; HPLC, high pressure liquid chromatography; NICI/MS, electron capture negative ion chemical ionization mass spectrometry; GC/gas-liquid chroma- tography; PC, phosphatidylcholine; PI, phosphatidylinositol.

* As initially defined (3), the term epoxygenase refers only to the catalysis of arachidonic acid epoxidation by Cytochrome P-450 to generate regioisomeric cis-epoxyeicosatrienoic acids. To avoid con- fusion (5), and in keeping with the uniqueness of the oxygenation step catalyzed by cytochrome P-450, i.e incorporation of one atom of molecular oxygen into the fatty acid, we would like to introduce the term arachidonic acid monooxygenase to identify all arachidonic acid oxygenation reactions catalyzed by cytochrome P-450. Thus, the arachidonic acid monooxygenase encompasses the following, isoen- zyme-specific, reactions: (a) olefin epoxidation or epoxygenase reac-

cytochrome P-450 activity has been highlighted by the mul- tiplicity of potent in vitro biological activities associated with the EETs (5), inter alia, effects on renal physiology, vasoactive properties, and secretagogue activity for several peptide hor- mones ( 5 ) .

Based on the documentation of the EETs as endogenous constituents of rat liver, rabbit kidney, and human urine, we proposed a role for the microsomal cytochrome P-450 epoxy- genase as an additional member of the arachidonic acid cas- cade (6-8). While several control experiments indicated that the EETs present in samples extracted and purified from rat liver were of endogenous origin (6), a decisive criterion for their enzymatic origin would be the demonstration of their enantiselective biosynthesis. Utilizing recently developed methodology for the direct chiral analysis of all four EET regioisomers (22), we report here the absolute configurations of endogenous rat liver EETs as evidence for their enzymatic origin and additionally, confirm cytochrome P-450 as the in vivo arachidonic acid epoxygenase. As a participant in the arachidonate cascade, cytochrome P-450, a ubiquitous and important group of hemeprotein(s) may thus play an hitherto unrecognized physiological role in the control of cell and tissue homeostasis.

MATERIALS AND METHODS

Sample Extraction and Purification-Male Sprague-Dawley rats (250-300 g) were maintained at 22 "C with alternating cycles of light and darkness and fed ad libitum Purina Rat Chow and water. For PB induction studies, animals were injected once with PB (intraperito- neal, 40 mg/kg body weight) and then maintained 10 days as above but with their drinking water replaced by a 0.05% (w/v) solution of the sodium salt of PB. Rats, selected at random, were killed by decapitation, and their livers were perfused in situ with ice-cold 0.15 M KC1 and immediately homogenized in CH30H/Hz0 (2:3, 20 ml/g wet tissue) containing triphenylphosphine (0.1 mM, final concentra- tion). An aliquot of the homogenate, corresponding to 0.5 g of liver, was transferred to a test tube containing an equivalent volume of a 2.0 mM solution of triphenylphosphine in CHCh and the following mixture of [l-"C]EETs (58-60 mCi/mmol) internal standards: 14,15- (0.04-0.06 pCi, 200-300 ng), 11,12- (0.02-0.03 pCi, 100-150 ng) and 8,9-EET (0.04-0.06 pCi, 200-300 ng). After mixing, HCl was added to a final concentration of 0.75 N and the suspension centrifuged to effect phase separation. The aqueous phase was extracted once more with two volumes of CHC13/CH30H (2:l) and the combined organic phases evaporated under argon. To the resulting residue, 1 ml of 0.2 N KOH/CH30H (2%) was added. After 60 min at 50 "C, the pH of the mixture was adjusted to 4.0, the organic material extracted into ethyl acetate, concentrated under argon, dissolved in HOAc/CH&N (0.1:99.9) and loaded onto a Cis Sep-Pak cartridge (Waters Assoc.,

tion (cis-epoxyeicosatrienoic acids, EETs), (b) allylic oxidation or

traenoic acids, and (c) hydroxylations at C-19 or C-20 or W/W-l lipoxygenase-like reaction (cis-trans-conjugated hydroxyeicosate-

oxygenase reaction (19- and 20-hydroxyeicosatetraenoic acids).

Cytochrome P-450 Arachidonate Epoxygenase, Stereochemical Characterization 19823

Milford, MA) pre-equilibrated with the same solvent. The EETs were eluted with 10-12 ml of HOAc/CH&N (0.1:99.9). After solvent evap- oration, the residue was dissolved in HOAc/hexane (0.5:99.5) and applied to a Si02 column (200-400 mesh, 5 X 20 mm). The column was then washed with 10-15 ml of HOAc/hexane (0.5:99.5) and the radiolabeled EETs eluted with 10 ml of HOAc/EtnO/hexane (0.52079.5). The eluent, containing the l-14C-labeled internal stand- ards was resolved into 14,15-EET and a coeluting mixture of 11,12- and 8,9-EET by reverse-phase HPLC on a 5-pm Dynamax Microsorb Cle column (4.6 X 250 mm, Rainin Inst. Co., Woburn, MA) and a linear solvent gradient from HOAc/H20/CH3CN (0.1:49.95:49.95) to HOAc/CH&N (0.1:99.9) over 40 min at 1 ml/min. Fractions were collected every 30 s and the radioactivity of 5% aliquots from each fraction assessed by liquid scintillation. Fractions with retention times corresponding to 14,15-EET (28 min) and the mixture of 11,12- and 8,9-EET (29-31 min) were pooled and dried under an argon stream. The mixture of 11,12- and 8,9-EET was resolved by normal- phase HPLC as described (9). The methyl ester of 14,15-EET was prepared as in Ref. 6. The pentafluorobenzyl esters of 8,9-EET-, 11,12-, and 14,15-EET werepreparedin 280% yield, by a modification of Waddell et al. (10). Briefly, to the neat EETs were added sequen- tially, 30 pl of dry CH,CN, 5 p1 of dimethylformamide, 5 pl of N,N'- diisopropylethylamine and 30 pl of a 6% (v/v) solution of pentafluo- robenzyl bromide in CH,CN. After 40 min at 40 "C, 2 ml of hexane were added followed by 2 ml of H20. After mixing, the organic phase was collected and the aqueous phase extracted two additional times with hexane. The combined organic phases were evaporated under argon. Prior to chiral analysis, the EET esters were further purified by reverse-phase HPLC as described (11).

Chiral Phose HPLC Analysis-The optical isomers of 14,15-EET- Me, 8,9-, and 11,12-EET-PFB were resolved by chiral-phase HPLC on either a Chiracel OB or OD column (4.6 X 250 mm, J. T. Baker Chemical Co.) as described (22). The HPLC chromatography condi- tions and the retention times of the different EET enantiomers are summarized in Table I. For analysis, the biological samples containing the 1-"C-radiolabeled internal standards were dissolved in the HPLC mobile phase and individually injected onto the chiral column. Frac- tions of the column eluents were collected each min and their radio-

TABLE I Chiral-phase HPLC conditions for EET resolution

Enantiomer resolution was achieved utilizing either a Chiralcel OB (14,15-EET-Me) or Chiralcel OD (8,9- and 11,12-EET-PFB) column. Optimal resolution requires extensive column equilibration with the mobile Dhase. Drior to use (300-400 ml).

Regioisomer Mobile phase Flow rate Retention

S R R.S

% i-PrOH/hexane mllmin rnin

8,g-EET-PFB 0.080 2.0 49 42 11,12-EET-PFB 0.150 1.0 60 68 14,15-EET-Me 0.014 1.1 59 49

FIG. 1. Mass spectral fragmenta- tion properties of [1-'4C]11,12- EET-PFB. Synthetic [1-'4C]11,12- EET-PFB (59 mCi/mmol, 0.24 nCi, 2 ng) was analyzed by GC/NICI/MS exactly as described under "Materials and Methods." Shown is the fragmenta- tion pattern of the material eluting from the GC column with a retention time of 12 min. The n / 2 and the percent abun- dance relative to the base peak (m/z 321, 100) were: 322, 21.2; 319, 1.8; 305, 3.8; 303, 8.3.

activity estimated by liquid scintillation of 10% aliquots from each fraction. Radiolabeled fractions with retention times corresponding to those of each individual enantiomer were pooled and evaporated under argon. Absolute configurations were assigned as described (22). Control experiments utilizing racemic samples of [1-14C]14,15-EET- Me, [1-14C]11,12-EET-PFB, and [l-"C]8,9-EET-PFB (59 mCi/ mmol, each) as well as enantiomerically pure forms of the correspond- ing [12C20]EET derivatives demonstrated, for each regioisomer, coe- lution of the corresponding labeled and nonlabeled enantiomers. For all cases, better than 85% of the injected radioactivity was recovered as a 1:1 mixture of enantiomers. The purified 8,9- and 11,lZ-EET- PFB enantiomers were analyzed and quantified by GC/NICI/MS without further manipulations. The resolved 14,15-EET-Me enantio- mers were dissolved in 0.2 N KOH/CH,OH (2:8), incubated 30 rnin at 50 "C, and acidified to pH 4.0. Extractive isolation with ethyl acetate and derivatization as above gave the corresponding PFB esters.

GC/MS Analysis and Quantification-Aliquots of the resolved EET-PFB enantiomers were dissolved in dodecane and analyzed by GC/NICI/MS on a Nermag RlOlOC quadrupole instrument inter- faced to a Varian Vista gas chromatograph utilizing He and CH4 as carrier and reagent gases, respectively. Splitless injections were made onto a 30-m SPB-30 fused silica capillary column (0.32 mm inner diameter, 0.25-pm coating thickness, Supelco Inc., Bellefonte, PA). After 1 min at 100 "C, the oven temperature was raised to 300 "C at 20 "C/min and then held at 300 "C for 15 min. Quantifications were done by GC/NICI/selected ion monitoring at m/z 319 (loss of PFB from the endogenous EET-PFB) and 321 (loss of PFB from the 11- "CIEET-PFB internal standard). The EET-PFB/[l-"C)EET-PFB sample ratios were calculated from the integrated values of the corresponding ion current intensities. The isotopic composition of each individual [1-14C]EET-PFB (59-60 mCi/mmol) standard was analyzed by GC/NICI/MS. All three standards showed better than 94 atom % enrichment in I4C, with the expected base peak at m/z 321 (M-PFB) and a small contribution from isotopic 12Czo at m/z 319 (M-PFB from contaminant, nonlabeled EET-PFB) (less than 6% of the base peak at m/z 321) (Fig. 1, shown for [l-"C]11,12-EET-PFB).

Synthetic Procedures-Enantiomerically pure 8,9-, 11,12-, and 14,15-EET were prepared by total asymmetric synthesis according to published procedures (12, 13). Racemic samples of the EETs were prepared as described (14, 15). Racemic [l-'IC]EET standards were synthesized from [l-l4CJarachidonic acid (59-60 mCi/mmol; 94-96 I4C atom% abundance; Amersham Corp.) by nonselective epoxidation (16) followed by regioisomer HPLC purification as in Ref. 9. Samples of l-~tearoyl-2-[l-~~C]arachidonyl-PC (58 mCi/mmol) and of l-stea- r0yl-2-[l-'~C]arachidonyl-PI (59 mCi/mmol) were epoxidized in 20% overall yield, by reaction with two equivalents of 3-chloroperoxyben- zoic acid in CH2C12/0.5 M NaHC03 aqueous (1:l) for 60 min at 25 "C. Excess reagent was quenched with 2 equivalents of triphenylphos- phine and the lipids extracted into CHC13/CH30H (2:l). The epoxi- dized PC and PI were separated from the precursor lipids by reverse- phase HPLC on a pBondapak Cla column (4.6 X 300 mm, Waters Assoc. Milford, MA) with the following solvent systems: (a) for PC,

m / z

19824 Cytochrome P-450 Arachidonate Epoxygenase, Stereochemical Characterization

O’” 1 8.9-EET-PFB

1

0.05 i n n 0 I r C _ _ h _ _ _ J V L

500 1 250 1

0 , UVk I

O’” 1 11,lP-EET-PFB W Y %

-x 0.05

a m

0 1 ” v \-

loo0 1 1

2 500 0

0

O’” ] 14.15-EET-Me

looo 1

0 20 40 60 80 100

RETENTION (Mink

FIG. 2. Comparisons of the chiral-phase HPLC chromato- graphic properties of l-’‘C-labeled and nonlabeled synthetic EET esters. Samples of 1-”C-labeled 8,9- and 11,12-EET-PFB and of 14,15-EET-Me (58 mCi/mmol, 0.2 pg each) were mixed with their matching nonlabeled EET derivatives (50 pg, each). Enantiomeric resolution was achieved under the chromatographic conditions de- scribed in Table I. Simultaneous on-line, monitoring of column eluents was performed utilizing a UV detector set at 210 nm and a Radiomatic Flo-One @-Detector (Radiomatic Instruments, Tampa, FL). The signal from the UV detector was delayed 0.2 min to correct for its physical position, upstream from the radioactivity detector.

a linear solvent gradient from 20 mM choline in CH30H/CH&N/ HzO (96.2:2.3:1.5) to 20 mM choline in CROH/CH&N/H,O (95:1.3:3.7) over 30 min at 1 ml/min (arachidonyl-PC, Rt: 32-33 min; epoxyeicosatrienoyl-PC, R,: 23-24 min); ( b ) for PI, isocratic with 20 mM choline in CH30H/CH3CN/H,0 (87.5:2.5:10) at 2 ml/min (arach- idonyl-PI, R,: 20-21 min; epoxyeicosatrienoyl-PI R,: 11-12 rnin).

RESULTS AND DISCUSSION

Whereas in vitro studies are an indispensable tool for the enzymatic characterization of metabolic pathways, they pro- vide only limited information with regards to the in vivo significance of the products and enzymes involved. This view is underscored by the fact that microsomal cytochrome P-450 accounts for approximately 4-5% of the total rat liver micro-

somal protein, but at present, only limited information is available concerning a clearly defined endogenous function for these hemeproteins. As part of a comprehensive study of the relevance of microsomal cytochrome P-450 in the metab- olism of endogenous arachidonic acid, we reported epoxygen- ase products as endogenous constituents of rat liver. It was estimated that the concentration of EETs in rat liver was on the order of 1 pg/g wet tissue (6). A unique feature of these endogenous EETs was the observation that approximately 85% of their total mass was recovered esterified to the sn-2 position of cellular glycerophospholipids such as PI and phos- phatidylethanolamine (11). The studies reported here describe the influence of cytochrome P-450 isoenzyme composition on the chirality of endogenous rat liver 8,9-, 11,12-, and 14,15- EET. Since the labile 5,6-EET suffers extensive decomposi- tion during the extraction and purification procedures utilized (6-8), it was not further investigated. To minimize autoxida- tion, the peroxide reducing agent triphenylphosphine was included in all purification steps, prior to reverse-phase HPLC. We were able to demonstrate in control experiments using 0.5 mg of HPLC-purified arachidonic acid, 1 mg of arachidonyl-PC, or 0.5 mg of [2Hs]arachidonic acid added to the liver homogenate that under the conditions of extraction, purification, and analysis described under “Materials and Methods,” artifactual EET formation was negligible.

For chromatographic and GC/MS quantification, synthetic [1-14C]EETs (58-60 mCi/mmol) were added to the liver ho- mogenate as internal standards. This choice was based on the following: (a) they are readily synthesized from [l-14C]arachi- donic acid, commercially available in high isotopic purity; ( b ) the radiolabel is chemically stable; (e) they are easily quanti- fied at any step of the purification protocol by liquid scintil- lation (285% counting efficiency); (d ) in contrast with or [2H8]EET standards, the [1-14C]EETs coelute with the corresponding [12C20]EETs during all chromatographic steps, including chiral-phase HPLC; ( e ) the molecular mass differ- ence (2 atomic mass units) between the [1-14C]EET-PFB standards and the corresponding unlabeled EETs permits quantification by GC/NICI/MS. Relative endogenous EET concentrations were calculated as described under “Materials and Methods” from the sample 14C/12C ratio computed from the respective ion intensities at m/z 321 and 319. The validity of this experimental approach was documented by performing the following control experiments.

1)”Samples of l-l4C-1abeled 8,9- and 11,12-EET-PFB and of 14,15-EET-Me (59 mCi/mmol, 0.2 pg each) were mixed with their matching nonlabeled EET derivatives (50 pg, each). The antipodes were resolved by chiral-phase HPLC as shown in Table I. For each EET-regioisomer simultaneous, on-line monitoring of column eluent absorbance (210 nm) and radio- activity demonstrated the coelution of the UV absorbance and the radioactivity associated with the nonlabeled and labeled synthetic material, respectively (Fig. 2). Integration of the UV absorbance and of the radioactivity associated with the HPLC fractions containing the individual enantiomers demonstrated that both standards were 1:l mixture of ster- eoisomers. Moreover, in a different experiment, equimolar mixtures of l-’4C-labeled (59 mCi/mmol) and nonlabeled 8,9- and 11,12-EET-PFB and of 14,15-EET-Me (total mass 50 ng) were resolved as described in Table I, and fractions of the column eluents were collected every minute. Enantiomer elu- tion profiles were established by scintillation counting of 10% aliquots from each fraction. For each peak corresponding to an EET stereoisomer, fractions eluting 1 min before, at the peak maximum and 1 min after the maximum were collected. Selected ion monitoring at m/z 321 and 319 demonstrated

Cytochrome P-450 Arachidonate Epoxygenase, Stereochemical Characterization

100%=8068352 BT =11:57.8

BASE SPECTRUM=321 (8068352) TiC=17106496

100 3 2 1

8(R),S(S)-EET-PFB

19825

50-

303 305 FIG. 3. Mass spectral properties

of the 8,9-EET-PFB enantiomers 319 ~ isolated from rat liver. The 8,9-EET present in rat liver was extracted and purified as under “Materials and Meth- 0 ‘..‘u..-.,‘.. I “T*“l ...,l.!!,.,....I”..-,.. ., --r “I’. I ” ,.“‘I. 3 ads.” After esterification, the antipodes of 8,9-EET-PFB were resolved by HPLC on a Chiralcel OD column as described 100%=3102400 RT =,I’52 2

in Table I. Aliquots (2-3 ng) of each 10 0 BASE SPECTRUMr321 (3102400) TIC=71135960

stereoisomer were then analyzed by CC/ NW/MS. Shown are the fragmentation 3e1

patterns for biological 8(R),9(S)- and 8(S),S(A)-EET-PFB

B(S),S(R)-EET-PFB. Abscissa, abun- 319 dance as % of the base peak. Ordinate, m/z.

50 - 303

305 301

0 . . . . . . .,... ,... ,... .,. ..,I!! I,., .,!I.. .,.,, ,. . . .._.. .,_ ,, _._ . . . . . . ,4

240 260

that, regardless of the collection time, the YJjl*C ratio was constant. It was, therefore, concluded that (a) the synthetic procedures generated racemic mixtures of standards, (b) the labeled and nonlabeled standards coeluted as a single com- ponent during chiral-phase HPLC, (c) that the chiral milieu of the stationary HPLC phases did not alter the enantiomeric composition of the samples, and (d) that the GC/NICI/MS l”C/‘*C ratios were independent of sample collection pattern.

2)--Samples of 1-YJ-labeled 8,9-, 11,12-, and 14,15-EET (59 mCi/mmol) were mixed with the corresponding non- labeled EETs in a 1:l molar ratio (total mass 300 ng). Each regioisomer was individually processed by the extraction and purification protocol outlined under “Materials and Meth- ods.” After resolution by chiral-phase HPLC, the enatiomeric composition of the nonlabeled EETs was determined utilizing the GC/NICI/MS ‘YZ/12C ratios described under “Materials and Methods.” In all cases the analysis demonstrated that the synthetic material was composed of a 50% f 2 mixture of enantiomers. Consequently, it was concluded that during sam- ple extraction, purification, and analysis the enantiomeric composition of the samples was not altered.

3)-We have previously demonstrated that ~85% of the

280 300 320 340 360 380 400

m/z

EETs present in the rat liver are found esterified to the sn-2 position of cellular glycerolipids (11). The possibility that the chiral phospholipid environment could result in artifactual formation of optically active EETs was tested by epoxidizing samples of [l-‘4C]arachidonyl-PC and PI (59 mCi/mmol, 1 HCi, each) by reaction with 2 equivalents of 3-chloroperben- zoic acid as described under “Materials and Methods.” The resulting epoxyeicosatrienoyl-PC and PI were purified by HPLC and then hydrolyzed in 0.2 N KOH/CH30H (2:8) for 2 h at 50 “C. Acidification (pH 4.0) followed by extractive isolation, normal-phase HPLC resolution (9), derivatization, and chiral-phase HPLC resolution (Table I) demonstrated that the peracid reaction generated mixtures of 14,15-, 11, 12-, and 8,9-EET in a 5:3:2 ratio, respectively.

After processing the biological samples as described under “Materials and Methods,” the overall recoveries of the re- solved l-14C-internal standard enantiomers, prior to GC/MS analysis, were 5-8, 10-13, and 13-15% for 14,15-, 11,12-, and 8,9-EET-PFB, respectively. Each rat liver EET enantiomer was characterized by full scan mass spectra under NICI con- ditions (Fig. 3, shown for 8,9-EET-PFB enantiomers). The material in the biological sample with the retention time of

Cy~ochrome P-450 Aruchi~o~u~e Epoxygenuse, Stereochemicat Churucteriz~~io~

I i __.-__--. i jz= - ,-__~~--~.-,~--,~~~,~.-. ,

fmo 2:oo 400 mo e:OO lo:oo 1200 14:oo

RETENTION TIMEhin)

FIG. 4. Selected ion current profiles under NICI conditions for the 8,9-EET-PFB enantiomers isolated from rat liver. The purified rat liver 8(R),9(S)- and S(~),S~~)-EET-PFB enantiomers were individually analyzed by NICI/GC/seIec~d ion monitoring at m/z 321 and 319 as described under “Materials and Methods.” The samples “C/1”C ratios were calculated from the integration of the area under the observed peaks. Abscissa, GC retention time in min- utes. Ordinate, abundance relative to the most intense peak.

TABLE II

~tere~~rn~a~ properties of the endage~~~ rat her EETs The enantiomers of rat liver 8,9-, 11,12-, and 14,15-EET were

extracted, purified, and quantified as described under “Materials and Methods.” EET regioisomer antipode percent distributions are given as the averages f SE. of four different experiments.

EET reeioisomer R.S S,R 8,9-EET 21 + 3 79 f 3

11,12-EET 37 f 3 63 2 3 14.15-EET 77 * 2 23 f 2

authentic 8(S),S(R)-EET-PFB showed [1-‘4C]8(S),9(R)- EET-PFB derived fragment ions at m/t 321 (base peak, loss of PFB) and 305 (22.3% abundance, loss of PBF and 0). Diagnostic ions for endogenous 8(S),9(~)-JET-PFB were at m/z 319 (77.9% abundance, loss of PFB) and 301 (15.8% abundance, loss of PFB and H,O). The fragment ion at m/z 303 (36.6% abundance) was common to both the internal standard (loss of PFB and I&O) and the endogenous EET (loss of PFB and 0). The fragment ions at m/z 322 and 320 (20.5 and 16.9% abundance, respectively) originated from the contribution of isotopic 13C (1.108 atom % natural abundance) present in the synthetic and endogenous EET, after loss of the PFB group. The results obtained with the other enan-

4.0 -

3.0 -

2.0 -

1.0 -

O-

11,12-EET

I v//1

FIG. 5. Effect of PB induction on the regioisomeric distri- bution of rat liver endogenous EETs. The 8,9-, l&12-, and 14,15- EET present in the livers of control and PB-induced rats were extracted and purified as described under “Materials and Methods.” After derivatization to the corresponding PFB esters, the individual EET-regioisomers were quantified by NICI/GC/MS as illustrated in Fig. 4. Values are the average 4 S.E. of at least three different experiments. Open bars, control animals. Hatched bars, PB-induced animats.

TABLE III

Stereochemical properties of P&induced endogenow rat liver EETs The enantiomers of 8,9-, 11,12-, and 14,15-EET were extracted

from the livers of PB-induced rats, purified, and quantified as de- scribed under “Materials and Methods.” For each EET regioisomer, the percent distribution of antipodes are given as the calculated mean + SE. from three different experiments.

EET m&isomer RS S.R

8,9-EET 1+0 99 5z 0 ll,lP-EET 17* 1 83 +- 1 14.15EET 94 -t 1 6+1

8‘9 -ET ll,lZ-EEI

FIG. 6. Effect of PB induction on the liver 8,9-, 11,12-, and l&15-EET enantiomer concentration. The antipodes of 8,9-, 11,12-, and 14,15-EET present in the livers of PB-induced and noninduced animals were extracted, purified, and quantified as de- scribed under “Materials and Methods.” Values shown are the ave- rages + S.E. obtained from at least three different experiments. Open bars, control animals. Hatched bars, PB-induced animals.

tiomer of 8,9-EET-PFB are shown in Fig. 3, top. The mass spectrum of this sample was dominated by ions derived from the internal standard m/z (% abundance) 321 (loo), 322 (21.3), 305 (19.6), and 303 (20.5). Additionally, fragment ions for the corresponding endogenous enantiomer were observed at m/z (% abundance) 319 (10.8), 320 (2.6), and 301 (2.2%).

For quanti~cation, aliquots of the biological EET-PFB enantiomers were analyzed by NICI/selected ion monitoring at m/z 321 and 319. The [l-‘*C]EET-PFB standardlendoge- nous EET-PFB ratio was calculated by integration of the

Cytochrome P-450 Arachidonate Epoxygenase, Stereochemical Characterization 19827

areas under the corresponding ion current profiles (Fig. 4, shown for 8,9-EET-PFB). The concentration of each EET enantiomer in rat liver followed directly from the calculated ratio and the amount of internal standard added to the liver homogenate.

Table I1 summarizes the absolute configurations of the rat liver endogenous 8,9-, 11,12-, and 14,15-EET. The antipodes of the two major EET regioisomers, i.e. 14,15- and 8,9-EET, are present in 3:l and 4:l ratios, respectively. On the other hand, the formation of 11,12-EET is less stereoselective. Comparison of these data with the results from an in vitro reconstituted system containing the major PB-inducible form of rat liver cytochrome P-450 indicates that although the absolute configurations are comparable, the products of the purified isoenzyme are generated with higher enantiofacial selectivity (17).

The multiple isoenzyme nature of rat liver cytochrome P- 450 has been established at the enzymatic, structural, and genetic level (18). Importantly, the liver cytochrome P-450 isoenzyme composition can be experimentally manipulated by animal treatment with inducers such as PB (19, 20). Stereochemical analysis of several cytochrome P-450 cata- lyzed reactions, utilizing isolated microsomal fractions or reconstituted systems containing purified cytochrome P-450 isoenzymes, have demonstrated the coexistence in the micro- somal membrane of isoenzymes with opposite enantiofacial selectivities (20). To further elucidate the influence of cyto- chrome P-450 isoenzyme composition on the biogenesis of EETs, livers from PB-induced animals were analyzed as above. Induction resulted in a 3.7-fold increase in the micro- somal concentration of cytochrome P-450 (from 0.6 to 2.2 nmol of cytochrome P-450/mg of microsomal protein) accom- panied by a 3.8-fold increase in the level of total endogenous EETs (Fig. 5). The PB inductive effect was restricted to 14,15- and 8,9-EET, with no significant change in 11,12-EET. Im- portantly, PB induction produced a general increase in en- antioselectivity, most dramatically seen by the biosynthesis of 8,9- and 14,15-EET as nearly optically pure enantiomers (Table 111). The influence of cytochrome P-450 isoenzyme composition in the control of EET stereochemistry is clearly illustrated when the liver EET enantiomer concentrations are compared before and after induction (Fig. 6). The PB-induced increase in the biosynthesis of EETs is the result of the stimulated formation of a single enantiomer of 14,15- and 8,9- EET.

From the foregoing evidence, as well as published data (6, l l ) , we conclude that the EETs present in rat liver are of enzymatic origin and that cytochrome P-450 is the endoge- nous epoxygenase. The experimental data, in conjunction with the high enantiofacial selectivity of a purified form of the liver cytochrome P-450 epoxygenase (l?), show that the regio- and stereoselectivity of the liver epoxygenase is cyto-

chrome P-450 isoenzyme dependent. The unprecedented high stereoselectivity of oxidation of such an unbiased, acyclic molecule points to arachidonic acid as one of the natural substrates for cytochrome P-450.

These observations, as well as the documented presence in rat liver of unique phospholipid pools containing an esterified EET moiety (1 l), may have broad implications for the role of these hemeproteins in ( a ) the biogenesis of novel lipid-derived mediators and their role in transmembrane signaling, and ( b ) the generation of enzyme-controlled changes in the physico- chemical microenvironment of membrane phospholipids (21, 22). The latter may have potentially important implications for the cellular control of localized changes in membrane permeability and/or membrane turnover.

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