Chem. Res. Toxicol. 1989,2, 35-40
35
Enantiotopic Differentiation during the Biotransformation of Valproic Acid to the Hepatotoxic Olefin 2-n -Propyl-4-pentenoic Acid David J. Porubek,* Hope Barnes, G. Patrick Meier, Louis J. Theodore, and Thomas A. Baillie Department of Medicinal Chemistry, BG-20, School of Pharmacy, University of Washington, Seattle, Washington 98195 Received September 15, 1988
The enantiomers of 2-( [3-13C]-n-propyl)pentanoic acid [ (R)- and (S)-[13C]VPA]were employed as metabolic probes to investigate stereochemical aspects of the biotransformation of valproic acid (VPA) to 2-n-propyl-4-pentenoic acid (A4-VPA),a hepatotoxic metabolite of VPA. When incubated with hepatocytes freshly isolated from untreated male rats, each labeled substrate (initial concentration 1.0 mM) underwent metabolism to [13C]-A4-VPA,the formation of which was time-dependent and occurred a t a rate of ca. 20 ng/(106 cells.4-h incubation). Analysis of this unsaturated metabolite by GC-MS techniques revealed that, following incubation of (R)-[W]VPA, desaturation had taken place preferentially (by a factor of -4) on the labeled propyl group (i.e., on the R side chain). Parallel incubations with (S)-[13C]VPAsupported this conclusion, in that metabolism of this isotopic variant of VPA led to a terminal olefin that also was predominantly (83 f 2%) of R configuration (in this case oxidized selectively on the unlabeled side chain). Hence, biotransformation of VPA to A4-VPA in rat hepatocytes occurs with marked enantiotopic differentiation, favoring production of the R enantiomer of this chiral metabolite. When rats were pretreated with phenobarbital (80 mg kg-' day-' ip for 3 days) prior to isolation of hepatocytes, the overall rate of metabolism of VPA to A4-VPAover the 4-h incubation period increased approximately 3-fold, while the degree of product enantioselectivity was unchanged. These findings provide the first evidence that cytochrome P-450 mediated 4,5-desaturation of VPA discriminates between the two prochiral propyl groups of this drug and also support the results of previous experiments with subcellular preparations which indicated that pretreatment of rats with phenobarbital induces biotransformation of VPA to the hepatotoxic terminal olefin
A4-VP A.
Introduction 2-n-Propyl-4-pentenoic acid (A4-VPA;' 1, Figure l),a hepatotoxic olefinic metabolite of the antiepileptic drug valproic acid (VPA; 2, Figure l),has been shown to act as an inhibitor of both cytochrome P-450 and @-oxidation enzymes in rat liver (1,2). The mechanisms underlying these inhibitory actions are believed to involve, in both cases, enzyme-catalyzed activation of A4-VPAto reactive, electrophilic intermediates which bind covalently to, and thereby destroy, their respective target enzymes. For cytochrome P-450, the alkylation metabolite of A4-VPA most likely is free radical in nature, generated by oxidative attack of the terminal olefin functionality (I,3), whereas in the case of &oxidation enzymes, the reactive species is believed to possess an a,p-unsaturated ketone structure which serves as a Michael acceptor for nucleophilic centers on protein ( 4 , 5 ) . Unlike the parent drug, A4-VPA is an asymmetric molecule and, as such, can exist in optically active forms. Recently, the R and S enantiomers of this metabolite were obtained by stereoselective synthesis, and preliminary metabolic studies with isolated rat hepatocytes demonstrated that these two isomers undergo further biotransformation by cytochrome P-450 and 0-oxidation-dependent pathways to different extents, as judged by the profile of end-product metabolites generated in this model system *To whom correspondence should be addressed.
(6). The implication of this finding is that the two enantiomers of A4-VPA should exhibit differences in their respective potencies as irreversible inhibitors of these liver enzymes and, consequently, may exert different hepatotoxic effects in vivo. The present study represented an extension of these stereochemical investigations and focused on the cytochrome P-450 mediated formation of A4-VPA from the parent drug (7). Specifically, information was sought on whether the desaturation process discriminates between the two prochiral propyl side chains of VPA to generate a preponderance of one enantiomer of A4-VPA over its antipode. Such information was considered to be essential in assessing the overall role of stereochemistry in A4VPA-mediated enzyme inhibition and hepatic damage (8).
Experimental Procedures Materials. (RS)-A4-VPAwas obtained by alkylation of ethyl valerate with allyl bromide, as described previously (5). Chemicals obtained form the Aldrich Chemical Co. (Milwaukee,WI)included @)-(+)-valine,(lS,2R)-(+)-norephedrine, boron trifluoride etherate, borane-methyl sulfide complex (10.0-10.2 M), diethyl
'
Abbreviations: A'-VPA, 2-n-propyl-4-pentenoic acid; VPA, 2-npropylpentanoic acid; 3-oxo-A4-VPA, 2-n-propyl-3-oxc-4-pentenoic acid;
SGOT, serum glutamate-oxaloacetate transaminase; GC, gas chromatography; GC-MS, gas chromatography-mass spectrometry; SIM, selected ion monitoring; MU, methylene unit index; TMS, trimethylsilyl; BSTFA, N,O-bis(trimethylsily1)trifluoroacetamide;THF,tetrahydrofuran; Pd/C, palladium on carbon; Ar, aromatic.
0893-228x/89/2702-0035$01*50/0 0 1989 American Chemical Society
36 Chem. Res. Toxicol., Vol. 2, No. I , 1989 COlH
I
Porubek et al.
AH
C02H
I
0
1
I
Figure 1. Structures of A4-VPA (1) and VPA (2). carbonate, diphenyl carbonate, n-butyllithium (1.6 or 2.5 M, in hexane), diisopropylamine, allyl bromide, lithium aluminum hydride, [ Wlmethyl iodide (99 atom % excess), benzenesulfinic acid (sodium salt), thionyl chloride, tert-butyldimethylsilyl chloride, dichlorodimethylsilane, deuteriochloroform, sodium metal, silica gel (230-400 mesh), and the internal standard for analytical work, 1-methyl-1-cyclohexanecarboxylic acid. Mercury was purchased from the D. F. Goldsmith & Metal Corp. (Evanston, IL), and Pd/C catalyst (5%) was from Alfa Products (Danvers, MA). The silylating agent BSTFA was obtained from Supelco, Inc. (Bellefonte, PA) and 6-glucuronidase (type VII) from the Sigma Chemical Co. (St. Louis, MO). All other chemicals used were of reagent grade. Dry THF was obtained by distillation from sodium benzophenone ketyl prior to use. The chromic acid solution (Na2Cr207in HzS04)was prepared according to Brown et al. (9), and the sodium amalgam (5%) was prepared according to published procedures (10). Instrumentation. Proton (300 MHz) and carbon-13 (75 MHz) NMR spectra were recorded in CDC13 with a Varian VXR-300 spectrometer (Varian Associates Inc., Palo Alto, CA), and chemical shifts are reported relative to internal tetramethybilane (0.0 ppm) and to the central chloroform line (77.0 ppm), respectively. Gas chromatography was performed with a Hewlett-Packard 5710A instrument, equipped with a fused silica capillary column (60 m X 0.32 mm i.d., DB-5, J & W Scientific, Ventura, CA). Helium (20 psi head pressure) was used as both carrier and detector makeup gas. Samples were injected by using the splitless mode of operation (250 "C injection port temperature) and "coldtrapped on the column at 50 "C. The oven temperature was then raised linearly at 20 "C min-' to 280 "C. Mass spectrometric analyses were carried out under GC-MS conditions using a VG 70-70H double-focusing mass spectrometer (VG Analytical, Manchester, England), interfaced to a Hewlett-Packard 5710 gas chromatograph and on-line to a VG Model 2035 data system. A DB-1 fused silica column (source and dimensions being identical with those cited above) was used with helium (20 psi head pressure) as carrier gas. Samples were injected in the splitless mode, as described above, and cold-trapped on the column at 40 "C for 0.5 min. The temperature of the column oven was raised rapidly to 80 "C, then programmed at 2 "C min-' to 105 "C and at 40 OC min-' to 250 "C, and finally held at 250 "C for 5 min. Mass spectra were recorded in the electron impact mode (ionization energy 70 eV) at a scan rate of 1s decade-' and accelerating potential of 4 kV. The trap current was held at 200 MA, and the ion source and GC interface temperatures were maintained at 200 and 250 "C, respectively. Quantitative analyses of 13C-labeledA4-VPA formed in hepatocyte incubations were carried out by selected ion monitoring (SIM) GC-MS and were based upon the ratios of ion currents at m/z 200 and 199, which correspond to the [M - CH3]+fragment ions of the trimethylsilyl (TMS) derivatives of [13C]-A4-VPAand the internal standard 1-methyl-1-cyclohexanecarboxylic acid, respectively (6). Measurementa of '% enrichment in metabolically generated A4-VPA also were based on SIM GC-MS analysis of the TMS derivative, in this case using the following pair of diagnostic fragment ions: m / z 173 ([M - C3H6]'+)and 172 ([M 12C23CHJ+). The ion at m / z 171 ([M - '%23CH,]+) was included in the group of ions monitored in order that appropriate corrections could be made to the intensity of the signal at m / z 172, resulting from natural abundance contributions. In randomly selected examples, the [M - C2H5]+ions (at m / z 186 and 185) were monitored in addition to those at m / z 173 and 172. Essentially identical results (in terms of R:S ratios) were derived from both pairs of ions. However, the enhanced sensitivity associated with the use of m / z 173 and 172 ion pair favored its use for routine purposes. Synthesis. As described in detail elsewhere (11),(R)-['3C]VPA and (S)-[13C]VPAwere obtained by enantioselective synthesis
0
( R ) - 1 3 C- V P A
0
(S)-13C - V P A
Figure 2. Scheme for the synthesis of (R)- and (S)-[13C]VPA (adapted from reference 11). In the preparation of the R enantiomer, R1 = H, Rz = 2-propyl (p), and R3 = allyl (a),while in the synthesis of the S enantiomer, R1 = phenyl ( a ) ,Rz= methyl ( a ) ,and R3 = allyl (p). using the chiral auxiliaries (4S)-4-isopropyl-2-oxazolidone and (4R,5S)-4-methyl-5-phenyloxazolidone (Figure 2). The isotopic purity of each product was determined by GC-MS to be 99 atom % excess, and the stereochemical purity to be >96% enantiomeric excess for the R isomer and 95% enantiomeric excess for the S form [asjudged by GC analysis of the respective diastereoisomeric 3-(2-allylvaleryl)-2-oxazolidonederivatives (S)]. Both enantiomers of [13C]VPAgave identical 'H NMR, 13CNMR, and mass spectra, details of which are as follows: 'H NMR 6 0.91 (t, J H - H = 7.5 Hz, 3 H, -'%H3), 0.91 (dt, Jq-H = 123 Hz, JH-H = 7.5 Hz, 3 H, -'%H3), 1.39 and 1.62 (m, 8 H, -CH2CH2- groups), and 2.39 (m, 1 H, -CHC02H); I3C NMR 6 13.97 (-13CH3), 20.53 (-CH,CHJ, 20.53 (d, 'J = 34 Hz, -CHZl3CH3), 34.31 (-CH2CH213CH3and -CHzCHzCH3),44.78 (d, 3J = 3.7 Hz, -CHCH2CH213CH3),and 180.95 (-COzH); MS (TMS ester) m / z 202 ([M - CH3]+, 13%), 188 ([M - CZHb]+,0.6%), 187 ([M- 1zC'3CH5]+,0.6%), 175 ([M - C3Hs]'+, 5%), 174 ([M- 12C~13CH6]'f, 5 % ) , 145 ([M - C3Hs 1zC13CH5]+and [M - 1zC2'3CHs- CzH5]+,7%), 129 (6%), 117 (CO2TMS+,3%), and 73 (Me3Si+,100%). Verification that 13C had been incorporated enantioselectively at the terminal position in each of the above analogues of VPA was accomplished by 13C NMR analysis of the diastereoisomeric products formed by recondensation of the VPA enantiomers with a chiral auxiliary. The procedure, which is illustrated in Figure 3, was as follows: (R)-[I3C]VPAand (S)-[13C]VPA(5 pL) were placed in separate dry 1-mL Reacti-Vials and treated with thionyl chloride ( 5 pL). The mixtures were swirled gently and allowed to stand at room temperature for 90 min. Into a separate 5-mL (40 mg) Reacti-Vial was placed (4S)-4-isopropyl-2-oxazolidone and dry T H F (2 mL). The vial was sealed with a septum and cooled to 0 "C, and n-butyllithium (2.5 M, 126 pL) was added
Chem. Res. Toxicol., Vol. 2, No. 1, 1989 37
Enantioselective Formation of A4- VPA
300, 450, and 750 ng) to tubes containing 5% HC1 (50 pL), untreated hepatocytes (2 mL; 2 X lo6 cells mL-'), and the internal standard (1.86 pg). These mixtures were then centrifuged to sediment the protein pellet, and the supernatants were transferred to clean tubes, made alkaline by the addition of 0.1 M Na3P04 buffer (pH 12; 1mL), and warmed to 37 'C for 1h. The samples were acidified by addition of 5% HCl(600 pL) and then extracted with ethyl acetate (3 X 2 mL), dried (MgS04),and concentrated under a gentle stream of dry N2 to a small volume (ca. 0.5 mL). The extracts were then transferred to conical Reacti-Vials, where the volume was reduced further (to ca. 50 pL). BSTFA (100 pL) was added, the vials were capped, and the samples were heated at 90 O C for 30 min. Aliquots (typically 0.5 pL) of these derivatized samples were taken directly for analysis by SIM GC-MS. All analytical procedures were performed with glassware that had been silanized by treatment with dichlorodimethylsilane. Figure 3. Scheme for the recondensation of (R)- and (S)[13C]VPA(as their respective acid chlorides) with the lithium salt of the chiral auxiliary (4S)-4-isopropyl-2-oxazolidone for l3C NMR analysis. dropwise by syringe over 1 min. This mixture was swirled and allowed to stand at 0 OC for 30 min, following which 1-mL portions were added to each of the above vials containing (R)- and (S)[13C]VPA(as the acid chlorides). The resultant mixtures were swirled gently and allowed to stand at 0 "C for 30 min, after which 20% HCl(100 pL) was added to each. The reaction mixtures were worked up individually by transferring to small round-bottom flasks and removing the T H F in vacuo. The residues were dissolved in CH2C12(10 mL), and the resulting solutions were extracted with water (10 mL), 1 M NaOH (2 x 10 mL), water (10 mL), and finally brine (10 mL). The organic phases were then dried (MgS04)and filtered, and the solvent was removed in vacuo to leave viscous residues which were dissolved in CDC13 (0.7 mL) and analyzed directly by 13C NMR spectroscopy. Biological Experiments. Male Sprague-Dawley rats (180-200 g) were obtained from Charles River Laboratories, Inc. (Wilmington, MA) and were allowed free access to food (Rodent Blox, Wayne Pet Food Div., Continental Grain Co., Chicago, IL) and water prior to use. Pretreatment of animals with phenobarbital was carried out by ip injection of the sodium salt (80 mg kg-') in distilled water at 72,48, and 24 h prior to sacrifice. Hepatocytes were prepared according to Mold6us et al. (12), and greater than 90% cell viability was achieved routinely as assessed by trypan blue exclusion. Cells were incubated under an atmosphere of 95% 02/5% C 0 2 in 50-mL round-bottom flasks, rotated continuously in an incubator at 37 O C . Incubations were conducted at a concentration of 2 x lo6cells mL-' and with a substrate concentration [(I?)- or (S)-[13C]VPA]of 1 mM. At appropriate time points, aliquots (2 mL) were removed from the incubation flasks and added to concentration tubes at 0 OC containing 5% HCl(50 pL) to terminate metabolic activity. To each of these samples was then added internal standard (1-methyl-1-cyclohexanecarboxylic acid; 1.86 pg). Samples for the construction of calibration curves were prepared by adding known amounts of A4-VPA (150, 225, 14.245 114.220
Results Chemical. Although details of t h e enantioselective preparation of (R)-and (S)-[13C]VPA have been reported elsewhere (1l ) ,salient features of the respective synthetic pathways are reproduced here in Figure 2. I n brief, treatment of t h e chiral auxiliaries (4R,5S)-4-methyl-5with phenyloxazolidone and (4S)-4-isopropyl-2-oxazolidone n-butyllithium, followed by [5-13C]valeryl chloride, afforded t h e 13C-labeled N-acyloxazolidones which were alkylated with allyl bromide. Reductive cleavage of t h e products with lithium aluminum hydride afforded t h e chiral 2-n-propyl-4-pentenols. Chromic acid oxidation of these alcohols and catalytic hydrogenation of the olefinic functionalities generated t h e title chiral acids, (R)-a n d (S)-[13C]VPA. In assessing t h e chemical purity of the final products, particular care was taken t o establish that the final hydrogenation step in t h e above syntheses had proceeded to completion, since any contamination of t h e synthetic [13C]VPA isomers with residual [13C]-A4-VPA clearly would have compromised t h e results of t h e subsequent biological experiments. Typically, >99% chemical purity was obtained, as assessed b y GC a n d GC-MS analysis of t h e TMS derivatives of (R)-and (S)-[13C]VPA. High isotopic enrichment in each of t h e products was achieved by t h e use of 13CH31(99 atom % excess 13C) as the source of 13Clabel (11),while high enantiomeric purity (195% enantiomeric excess) resulted from t h e chiral oxazolidone-based methodology developed by Evans a n d co-workers (13). Finally, when portions of (R)-a n d (S)[13C]VPA were recondensed with t h e chiral auxiliary (4S)-4-isopropyl-2-oxazolidone, it was shown by 13C NMR spectroscopy that two diastereoisomeric products were produced. Thus, as illustrated in Figure 4, t h e acylated oxazolidone derived from (R)-[13C]VPA afforded a single resonance for t h e 13C-enriched terminal methyl group at 14.220 ppm, whereas t h e corresponding signal in t h e 14.220
14i245
1- -14.4
14.2
14.4
14.2
14.4
14.2
ppm
Figure 4. Partial 13CNMR spectra of the diastereoisomeric acylated oxazolidones depicted in Figure 3 showing the resonance attributed to the terminal 13CH3carbon. The product derived from (R)-[13C]VPAgave the partial spectrum shown in the center frame, while that derived from (S)-[13C]VPAis reproduced in the right frame. A t left is the corresponding natural abundance 13C NMR spectrum of the reference (nonenriched) compound.
38 Chem. Res. Toricol.,Vol. 2, No. 1, 1989
Porubek et al.
80
1
Table I. Enantiotopic Differentiation in the Metabolism of (R)-['$]VPA to ['*C]-A4-VPA in Rat Hepatocytes corrected ion
1
2 3 4 80 I
0
I
1
3
2
4
5
Time (h)
Figure 5. Formation of 13C-labeledA*-VPAfrom (R)-[13C]VPA (A) and (S)-[l%]VPA(B)in isolated rat hepatocytes aa a function of incubation time. Data represent mean values f SD ( N = 3) for metabolite formation in cells from a control rat (open symbols) and from a phenobarbital-pretreated animal (closed symbols).
product derived from (S)-[13C]VPAwas centered at 14.245 ppm. These data served to verify the enantiomeric relationship between the two 13C-labeled analogues of VPA used in this study as metabolic probes. Biological. In incubations of (R)-or (S)-[13C]VPAwith control hepatocytes, [ 13C]-A4-VPAlevels were found to increase with time, although in a nonlinear fashion, and reached a plateau after approximately 2 h (Figure 5). Similar rates of formation [ca. 20 ng/(106 cells.4-h incubation)] were obtained for metabolite formation from both substrates. When hepatocytes from phenobarbital-pretreated rats were used, A4-VPA was formed a t an appreciably higher rate [ca.65 ng/(l@ cells.4-h incubation)] than was the case with control cells (Figure 5 ) . Moreover, metabolite formation in the induced cells continued to increase throughout the experiment and did not reach a plateau during the 4-h incubation. When the A4-VPA formed in incubations of (R)- and (S)-[13C]VPAwith control hepatocytes was extracted and analyzed (as its TMS ester) by GC-MS, the partial mass spectra depicted in Figure 6A,B were obtained. (For comparison, the corresponding partial mass spectrum of the reference, unlabeled metabolite is reproduced in Figure 6C.) A key feature of the spectra of the metabolically generated A4-VPA species is that whereas the relative abundance of the [M - C3H6]'+ion at mlz 173 is considerably greater than that of the [M - 12C2'3CH6]'+ion at m l z 172 in the compound derived from (R)-[13C]VPA (Figure 6A), the reverse is true in the spectrum of the metabolite formed from (S)-[13C]VPA (Figure 6B). A similar reciprocal relationship is evident in the abundances of the ions a t mlz 186 ([M - C,H,]+) and 185 ([M 12C13CH5]+).The elimination from the M'+ ion of an ethyl radical (to give mlz 18516) and the elimination of the elements of propene (to yield mlz 17213) are both processes that can result only from fragmentations involving the propyl (as opposed to the allyl) side chain of the A4-VPA derivative (via a-bond ionization and McLafferty rearrangement, respectively). Therefore, the relative abundances of these pairs of daughter ions reflect the relative populations of molecules that bear the 13Clabel
4.36 f 0.41b 3.66 f 0.67 phenobarbital 3.21 f 0.31 pretreated 3.44 f 0.45 control
81.3 f 1.3 78.5 f 2.71 76.3 f 1.6 77.5 f 2.11
79.9 f 2.2 x = 76.9 f - 2.3e x =
Corrected for natural abundance contributions from adjacent ions at m / z 172 and 171. Values represent means f S D ( N = 3). b N = 4. 'Not statistically different from control; p = 0.01. Table 11. Enantiotopic Differentiation in the Metabolism of (S)-[ '3C]VPA to [ '%]-A4-VPA in Rat Hepatocytes corrected ion abundance source of ratio ( m / z stereochemistry of A4-VPA( % R ) expt no. hepatocytes 173/172)" 1 control 0.18 f O.Olb 84.7 f 0.7 x 83.2 f 2 0.23 f 0.04 81.3 f 2.61 - ;.O 3 phenobarbital 0.27 f 0.02 78.7 f 1.2 z 79.7 f 4 pretreated 0.24 f 0.02 80.7 f 1.3 - ;.7c
1
-
Corrected for natural abundance contributions from adjacent ions at m / z 172 and 171. Values represent means f S D ( N = 3). * N = 4. CNotstatistically different from control; p = 0.01.
on propyl versus allyl groups of the metabolite. Consequently, since the 13C atom in each of the substrates is known to occupy a stereochemically defined position on one or the other of the two prochiral side chains of VPA and thus serves as a marker for absolute configuration, the above ion abundance ratios reflect the enantiomeric composition of the metabolite. Hence, the spectra shown in Figure 6A,B indicate that metabolism of VPA to A4-VPA discriminates between the two prochiral side chains of VPA and favors strongly desaturation of the pro-R propyl group. Accurate measurements of the ion abundance ratios at mlz 172 and 173 for metabolically generated A4-VPAwere obtained by SIM GC-MS analysis and were employed to calculate the degree of enantiotopic differentiation accompanying metabolism of VPA to A4-VPA. As shown in Table I, metabolism of (R)-[13C]VPAin control hepatocytes led to [13C]-A4-VPAwhich was predominantly (79.9 f 2.2%) of R configuration, while with cells from phenobarbital-pretreated rats, the degree of enantiotopic differentiation was essentially the same (76.9 f 2.3% R). These findings were reinforced by the results of parallel experiments in which (S)-[13C]VPAserved as substrate for metabolism, when (R);A4-VPA was again formed enantioselectively in both control and phenobarbital-induced hepatocytes (Table 11). The absolute values for the percentage of (R)-A4-VPAformed were slightly higher when the (S)-[13C]VPAsubstrate was used (cf. Tables I and 11), which is the result of small differences in the enantiomeric purities of (R)-and (S)-[13C]VPA.
Discussion Although a number of investigations (reviewed in reference 8) have shown that A4-VPA is hepatotoxic when administered to rats and serves as an inhibitor of cytochrome P-450 and @-oxidationenzymes in vitro, the role of this minor unsaturated metabolite in VPA-mediated liver injury remains to be established. In order to gain information on the toxicological significance of this olefin, we have conducted a series of investigations on the biological fate of racemic A4-VPA, the results of which have
Chem. Res. Toxicol., Vol. 2, No. 1, 1989 39
Enantioselective Formation of A*- VPA
1
100
A 200
%
173
50
1
.-- .
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xs M W 214
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.
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180
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.
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170
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. ,
180
,I.
,
190
,' 200
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, 210
214 I
,
,
assess the role of absolute stereochemistry in the mechanism by which VPA undergoes bioactivation. The results of the present study, which employed freshly isolated rat hepatocytes as the biological model system and the two enantiomers of a chiral 13C-labeled analogue of VPA as metabolic probes, demonstrated clearly that desaturation of VPA in hepatocytes exhibits a high degree of enantiotopic differentiation favoring metabolism on the pro-R side chain. Thus, it may be concluded that VPA interacts with the cytochrome P-450 enzyme(s) responsible for A4-VPA formation in a preferred orientation in order to generate a preponderance of the R enantiomer of the product. This report is the first to document enantiotopic differentiation in a cytochrome P-450 mediated desaturation reaction, although precedents exist for discrimination between prochiral phenyl (16), methyl (27), and methoxy groups (18) in hydroxylation and O-demethylation processes, respectively, catalyzed by this enzyme system. Of further interest were the results of the present experiments using hepatocytes from phenobarbital-pretreated rats. Incubation of VPA with these induced cells led to formation of A4-VPA at a rate that was almost %fold greater than that seen in control experiments, consistent with the results of previous in vitro studies which demonstrated that pretreatment of rats with phenobarbital induced the formation of this unsaturated metabolite of VPA in hepatic microsomal preparations (7,19). However, the enantiomeric ratios of the desaturation process were found to be unresponsiveto phenobarbital administration. This result may suggest that isozyme(s) of cytochrome P-450 induced by phenobarbital also select for the pro-R side chain during desaturation of VPA. Conversely, although less likely, phenobarbital may in fact induce cytochrome P-450 isozyme(s)which desaturate VPA with a change in stereoselectivity. If so, phenobarbital must also induce further biotransformation of the enantiomers of A4-VPA unequally such that the apparent level of enantiotopic differentiation is unchanged. Experiments with purified isozymes of cytochrome P-450 will be of great value in distinguishing these possibilities, and these experiments are currently in progress. The known potentiating effects of phenobarbital (and other enzyme-inducing antiepileptic drugs) on VPA-mediated liver injury in rats (20) and human subjects (21)may be due, at least in part, to induction of VPA metabolism. The formation of the hepatoxic metabolite, A4-VPA, is known to be induced by phenobarbital pretreatment of rats when measured in microsomes (7, 19) and hepatocytes (reported herein). While a selective increase of either enantiomer does not appear to accompany the enhanced rate of formation, this possibility requires further testing, as does the theoretical hypothesis that (S)-A4-VPAis more toxic than its antipode. Future investigations with the two enantiomers of A4-VPA (now accessible by synthesis) will allow a direct evaluation of the relative hepatotoxic properties of (R)- and (S)-A4-VPAin vivo.
Acknowledgment. We thank Mr. Robert Gollehon and Ms. Shannon West for assistance in the preparation of the manuscript. These studies were supported by National Institutes of Health Research Grants GM 32165 and DK 30699. Registry NO.2,9946-1; (R)-A4-VPA,117039-61-9;(S)-A4-VPA, 117039-65-3; cytochrome P-450, 9035-51-2.
References (1) Prickett, K. S., and Baillie, T. A. (1986) Metabolism of unsaturated derivatives of valproic acid in rat liver microsomes and
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