Vol. 263, No. 27, Issue of September 25, pp. 13733-13738 1988
Printed in ~. S.A.
THEJOURNAL
OF BIOLOGICAL
CHEMISTRY
0 1988 by The American Society for Biochemistry and Molecular Biology, Inc.
Cytochrome P-450-catalyzed Desaturation of Valproic Acid in Vitro
SPECIES DIFFERENCES, INDUCTION EFFECTS, AND MECHANISTIC STUDIES*
(Received for publication, May 16,1988)
Allan E. Rettie, Michael Boberg$, Albert
W. Rettenmeiers, andThomas A.Baillie
From the Department of Medicinal Chemistry, School of Pharmacy, University of Washington, Seattle, Washington 98195
Thecytochrome P-460-mediated desaturationof
valproic acid (VPA)to its hepatotoxic metabolite,2-npropyl-4-pentenoic acid (4-ene-VPA), was examined
in liver microsomes from rats, mice, rabbits and humans. The highest substrate turnoverwas found with
microsomes from rabbits(44.2 2 2.7 pmol of product/
nmol P-460/15 min), while lower activities were observed in preparations from human, mouse, and rat
liver, in that order. Pretreatment of animals
with phenobarbital led to enhanced
rates of formation of
4-eneVPA in vitro and yielded inductionratios for desaturation ranging from 2.6 to 8.4, depending upon the
species. Comparative studies in the rat showed that
phenobarbital is a more potent inducer of olefin formation than either phenytoin or carbamazepine. The
mechanism of the desaturation reaction
was studied by
inter- and intramolecular deuterium isotope
effect experiments, which demonstrated that removal ofa hydrogen atom from the subterminal
C-4 position of VPA
is rate limiting in the formation of bothI-ene- and 4hydroxy-VPA. Hydroxylation at the neighboring C-6
position, on the other hand, was highly sensitive to
deuterium substitutionat that site, but not to deuteration at (3-4. Based on these
findings, it is proposed that
4-ene- and 4-hydroxy-VPA are products
of a common
P-450-dependent metabolic pathway, in which a carbon-centered free radicalatC-4 serves as the key
intermediate.6-Hydroxy-VPA,incontrast,
derives
from an independent hydroxylation reaction.
Valproic acid (VPA;’ Fig. I), first synthesized by Burton
(1882), was used initially as an organic solvent until the
serendipitous discovery of its anticonvulsantactivity by Meuni&e et al. (1963). Since its introduction into clinical use in
Europe in the late 19609, it has proven effective, both as a
component of polytherapy and as sole medication, in the
treatment of simple and complex absence seizures (Sat0 et
al., 1982; Covanis et al., 1982; Ramsay, 1984). During recent
years, however, it has become evident that VPA therapy may
* These studies were supported by National Institutes of Health
Research Grants GM 32165 and NS 17111. The costs of publication
of this article were defrayed in part by the payment of page charges.
This article must therefore be hereby marked “advertisement” in
accordance with 18 U.S.C. Section 1734 solelyto indicate this fact.
$Present address: Institut f i r Pharmakokinetik, Bayer AG, D5600 Wuppertal, Federal Republic of Germany.
$ Present address: Institut f i r Arbeits- und Sozialmedizin der
Universitat Tubingen, D-7400 Tubingen, Federal Republic of Germany.
‘ The abbreviations used are: VPA, 2-n-propylpentanoic acid, valproic acid 4-ene-VPA, 2-n-propyl-4-pentenoic acid; 4 (or 5)-hydroxyVPA, 2-n-propyl-4 (or 5)-hydroxypentanoic acid; PB, phenobarbital;
CBZ, carbamazepine; DPH, 5,5-diphenylhydantoin, phenytoin; TMS,
trimethylsilyl; GC/MS, gas chromatography/mass spectrometry.
be associated with a rare,but potentially fatal,hepatic toxicity
(Zafrani and Berthelot, 1982; Zimmerman and Ishak, 1982).
The risk of fatal hepatic dysfunction has been assessed at 1
in 37,000 in patients receiving VPA as monotherapy, and as
high as 1 in 500 in children younger than 2 years of age who
are receiving VPA in combination with other anticonvulsants
(Dreifuss et al., 1987).
The most common histopathological feature of VPA-induced liver injury is microvesicular steatosis, similar to that
produced by the toxic metabolite of hypoglycin A and by 4pentenoic acid. The close structural similarity between these
two compounds and 4-ene-VPA, an unsaturated metabolite
of VPA, aroused speculation that the lattercompound could
be responsible for the observed toxicity of the anticonvulsant
(Gerber et al., 1979; Zimmerman and Ishak, 1982). In support
of this contention, 4-ene-VPA was shown to be the most toxic
metabolite of VPA in rat hepatocytes in culture (Kingsley et
al., 1983) and to be considerably more potent an inducer of
steatosis in young rats than was the parent compound (Kesterson et al., 1984). Additional evidence of a pathological role
for 4-ene-VPA was furnished by the finding that plasma levels
of unsaturated VPA metabolites were elevated dramatically
in an epileptic patient who had died from hepatic failure
following a treatment regimen of phenobarbital and VPA
(Kochen et al., 1983).
In recent animal studies on the metabolic origin of 4-eneVPA, we found that this hepatotoxic olefin was generated by
the microsomal cytochrome P-450 mixed-function oxidase
system of phenobarbital-treated rat liver and that the
responsible isoenzyme was P-45h (Rettie et al., 1987). While this
finding has profound implications with respect to the role of
polytherapy in inducing the formation of a hepatotoxic metabolite of VPA in epileptic patients, it also reveals that
cytochrome P-450 can actin the novel capacity of a desaturase
enzyme. Interestingly, this catalytic function of P-450 was
detected independently in studies on the metabolism of testosterone, where it was found that formation of the 6-ene
derivative of this steroid hormone was P-450-dependent and
appeared to be linked tothe GB-hydroxylation pathway
through a common carbon-centered free radical intermediate
(Nagata et al., 1986). Weproposed that an analogous mechanism may operate inVPA metabolism, namely that a carboncentered free radical, localized at either C-4 or C-5, partitions
between recombination (4- or5-hydroxy-VPA formation) and
elimination (4-ene-VPA formation) processes to yield the
observed reaction products (Rettie et al., 1987).
In viewof the general importance of cytochrome P-450mediated reactions in the metabolism of a wide spectrum of
both endogenous and foreign compounds, and in light of our
particular interest in 4-ene-VPA as a hepatotoxic metabolite
of VPA, the present study was carried out to provide fundamental information on the dual desaturase/hydroxylase functions of cytochrome P-450 in VPA biotransformation. Specif-
13733
P-450-catalyzed
Desaturation
13734
CO2H
I
VPA
'H,-VPA
COzH
I
'H3- VPA
ofAcid
Valproic
mass spectra (70 eV ionizing energy) were recorded using a HewlettPackard Model 5970 mass selective detector connected to a Model
59970C Chem StationTMdata system. Quantitative GC/MS analyses
and of 5-hydroxyof the diastereomeric 4-hydroxy-VPA-y-lactones
VPA were performed on the above instrument, which was operated
in the selected ion monitoring mode. In situations where enhanced
sensitivity was required, as was the case with 4-ene-VPA derived
metabolically from unlabeled and deuterated VPA, and with 4-hydroxy-VPA-y-lactone produced from [4,4-'H2]VPA,quantitation was
performed using a VG 7070H double-focusing mass spectrometer online to a VG Model 2035data system. Gas chromatographic conditions
used in the GC/MS analysis of TMS derivatives of VPA metabolites
were identical to those described previously (Rettie et al., 1987).
Quantitation of 4-ene-VPA was achieved by reference to calibration
curves prepared by adding known amounts of authentic 4-ene-VPA
and VPA to microsomes (lacking NADPH) and extractingthese
mixtures as described above. Calibration curves were linear over the
concentration range 0-400 ng ml-'. Retention times of compounds
were measured relative to a homologous series of n-alkanes, coinjected with each sample, and are expressed as methylene unit values.
Synthetic Procedures
CO,H
2
H,- VPA
CO2H
2
H,-VPA
FIG. 1. Structures of VPA and deuterated analogs thereof.
ically, we sought to establish the influence of the following
factors on the P-450-dependent hydroxylation and desaturation pathways: (i) source of microsomal enzymes, (ii) induction status of the enzyme source, and (iii) sensitivity to
deuterium substitution at theC-4 and C-5 positions.
EXPERIMENTAL PROCEDURES
Materials
Valproic acid was obtained from Abbott Laboratories (North Chicago, IL), while the derivatizing reagent bis(trimethylsily1)
trifluoroacetamide was purchased from Supelco Inc. (Bellefonte, PA).
[2,2-'Hz]Propylbromide (98 atom % excess) and [3,3,3-'H3]propylbromide (99 atom % excess) were obtained from Merck, Sharp and
Dohme Laboratories (St. Louis, MO). Resomfin and diethyl-2-propylmalonate were obtained from Aldrich. Resorufin was further purified by the method of Klotz et al. (1984) to give a final extinction
coefficient of73.2mM
cm-' a t 572 nm. Pentoxyphenoxazone was
prepared by alkylation of resorufin with pentyl iodide, as described
previously (Burke and Mayer, 1983). 4-Hydroxy-VPA, 5-hydroxyVPA, and 4-ene-VPA were synthesized as described by Rettenmeier
et al. (1985), while the labeled compounds [5,5,5,5',5',5'-'H~]VPA
and [4,4,4',4'-'H4]VPA were gifts from Dr. Bruce A. Mico (SmithKline and French Laboratories, Philadelphia, PA). The deuterium
contents of the lattercompounds were determined by GC/MS analysis of their respective TMS derivatives, and were found to be as
follows: ['H4]VPA 92.9% 'H4, 6.3% 'H3, 0.5% 'HZ, and 0.3% 'H,.
['Hs]VPk 97.1% 'Hs, 2.4% 'H4, 0.2% 'Hs, 0.2% 'H3,
and 0.1% 'Hz.
Carbamazepine (CBZ) was a gift from Ciba-GeigyLaboratories (Summit, NJ), and phenytoin (DPH) was purchased from Sigma.
Instrumentation
Proton nuclear magnetic resonance (NMR) spectra were recorded
using a Varian VXR-300 instrument at300 MHz. Deuterochloroform
was employed as solvent, and chemical shifts are expressed as parts/
million downfield from internal tetramethylsilane. Electron impact
[4,4-'HJ VPA-This asymmetrically labeled compound was prepared by a malonic ester synthesis, as follows. Sodium hydride (336
mg, 14 mmol) in dry tetrahydrofuran (40 ml), was added to diethyl
2-propylmalonate (2.02 g, 10.0 mmol) at room temperature under a
nitrogen atmosphere. After the evolution of hydrogen had ceased,
[2,2-'H2]propylbromide(969 pl, 10.5 mmol) was added, and the mixture was heated under reflux for 11 h. Further portions of sodium
hydride (20 mg) and [2,2-'H~]propylbromide(40 pl) were added, and
heating was continued for 6 h. The reaction mixture was cooled to
room temperature, 10% H2S04 (20 ml) was added, and the solution
was extracted three times with ethyl acetate. The combined organic
phases were washed with saturated NaC1 solution, dried over MgSO,,
filtered, and evaporated under reduced pressure. This afford diethyl
2-pr0pyl-2([2,2-~H~]propyl)malonate
in quantitative yield. This ester
(2.7 g) was then heated under reflux with ethanol (20 ml) and 40%
NaOH solution (10 ml) for 15 h. The reaction product was cooled to
ambient temperature, and the ethanol was removed under reduced
pressure. The residue was treated with water (20 ml), and theresulting
solution was extracted with CHzC1, (5 ml). The organic extract was
washed with water and discarded, after which the combined aqueous
phases were acidified (to pH1)with concentrated HZSO4 and cooled
to 4 "C. The product, which precipitated from solution, was collected
by filtration, washed with water and redissolved in ethyl acetate,
dried over MgSO4, and evaporated under reduced pressure. This
afforded the free diacid as a white solid in 70% yield, which wastaken
to thefinal step without further purification.
The above intermediate was dissolved in acetonitrile (20 ml), and
copper(1)oxide(60 mg, 0.4 mmol) was added under a nitrogen atmosphere. The resulting mixture was heated under reflux for 11 h, and,
after cooling to room temperature, the solvent was evaporated under
reduced pressure. The resulting slurry was acidified with 1 M HzSO,
and extracted three times with ethyl acetate. The combined organic
extracts were washed with saturated NaCl solution, dried (MgSO,),
and evaporated under reduced pressure to give the desired product.
Final purification by low pressure column chromatography on silica
gel (Merck, 60-200 mesh), using petroleum ether/ethyl acetate (9:1,
v/v) as eluent, yielded [4,4-'Hz]VPA (810 mg; 51%) which was homogeneous on gas-liquid chromatography analysis (single peak for
the TMS ester, methylene units = 11.52). MS (TMS derivative):
m/z 218 (M?, 0.1%), 203 ([M - CH$, 35%), 176 ([M - C3Hs]', 9%),
and 175 ([M - C3H2HJt, 8%).NMR 0.90 (s, 3H, CH3-C2Hz-), 0.92
(t, J = 7.2 Hz, 3H, CH3-CHz-), 1.38 (m, 4H, -CHz-CH2-), 1.60 (m,
2H, -C2Hz-CHz-), and 2.38 d (m, lH, -CH-). The isotopic composition of this compound was determined by GC/MS analysis to be 3.8%
'H,, 90.0% 'Hz, 1.0% 'HI, and 5.2% 'Ho.
&5,5-'HJVPA"This compound was prepared by a similar procedure to thatdescribed above for [4,4-'Hz]VPA, but used [3,3,3-'H3]
propylbromide and diethyl 2-propylmalonate as starting materials.
The product, which was obtained in 50% overall yield, was found to
be homogeneous by gas-liquid chromatography (single peak for the
TMS ester methylene units = 11.52). MS (TMS derivative): m/z 219
(M?, 0.1%), 204 ([M - CH$, 33%), 177 ([M - C3Hs]', 8%), and 174
([M - C3H3'H31t, 8%).NMR: 0.91 (t, J = 7.2 HZ, 3H, CH3-), 1.281.60 (m, 8H, -CHz- groups), and 2.38 8 (m, lH, -CH-). The isotopic
P-450-catalyzed
Desaturation
composition of this compound was determined by GC/MS analysis
to be: 3.3% 'H8, 0.4% 'Hs, 89.0% 'H3, 1.8%'Hz, 0.2% 'HI, and 5.3%
'H,.
Tissue Source and Animal Treatment
Male Sprague-Dawley rats (150-250 g), female BALB/c mice (2025 g), and female New Zealand White rabbits (2-3 kg) were used in
these experiments. Mice were injected intraperitoneally with phenobarbital (PB; 80 mg kg" day" in isotonic saline) for 4 days prior to
killing. Rabbits were given PB orally (0.1% w/v in their drinking
water, adjusted to pH 7) for 7 days prior to sacrifice. Rats were
injected intraperitoneally with CBZ, DPH, or PB (80 mg kg" day"
in propylene glycol) for 4 days prior to killing. Control rabbits and
mice were not pretreated,while control rats received propylene glycol
alone. Human liver was obtained from six transplant donors (three
males and three females, aged 13-45 years) within 30 min of respiratory cessation. The causes of death were either gunshot wound,
automobile accident, head injury, or cerebral hemorrhage. In each
case, the liver wasperfused with cold saline, divided into small cubes,
washed with cold isotonic KC1 solution, and frozen rapidly in liquid
nitrogen. These samples were then stored at -80 "C for up to 2
months before use. Hepatic microsomes from human and animal
tissue were prepared as described previously (Dean et al., 1986) and
were stored as a suspension at -80 "C in potassium phosphate buffer
(100 mM, pH 7.4) containing 20% glycerol.
Enzyme Purification
Cytochrome P - 4 5 0 ~ 2was isolated from hepatic microsomes from
PB-induced rabbits by the method of Haugen and Coon (1976) with
minor modifications. Briefly, the flow-through fraction of an 8-12%
polyethylene glycol 8000 precipitation of cholate-solubilized microsomes applied to a Whatman DE52 column as described by Haugen
and Coon (1976), was loaded directly onto a hydroxylapatite (BioGel HPT) column equilibrated with 10 mM potassium phosphate (pH
7.4), 20% glycerol, 0.2%Lubrol PX, and 0.1 mM EDTA. The column
was washed with equilibration buffer which had the phosphate concentration increased sequentially to 40, 90, and 150 mM. Essentially
homogeneous P-450LM2 was eluted with the 90 mM wash. Minor
contaminants were removed following adsorption of the hemoprotein
to CM-Sepharose CL-GB after dialysis against 10 mM potassium
phosphate buffer (pH 6.5), 20% glycerol, 0.2% Lubrol PX, and 0.1
mM EDTA. Bound P-450 was washed extensively (20 column volumes) with the equilibration buffer minus Lubrol PX andwas eluted
in wash buffer which had the phosphate concentration increased to
200 mM. At each stage of the purification, only those fractions which
appeared homogeneous on10% sodium dodecyl sulfate-polyacrylamide gels were pooledfor further purification. Cytochrome P-45oLMZ
was purified to a specific content of 14.7 nmol of P-450 mg" protein
by this method. The rate of benzphetamine N-demethylation obtained with a 3:l molar ratio of cytochrome P-450 reductase (see
below) to P-450LM2 (measured in the absence of cytochrome b6) was
found to be 46 nmol of product nmol-I P-450 rnin". Furthermore,
the purified P-450LMZ catalyzed the metabolic conversion of ( R ) warfarin regioselectively to the 4"hydroxy derivative, and of ( S ) warfarin to the corresponding 6-hydroxy product. Thus, metabolic
ratios for phenol formation at the4'-, 6-, and 7-positions of warfarin
were264.3:1.4 for the R isomer, and 7.9261.0 for the S isomer.
These ratios are very similar to the values reported for this isozyme
by Fasco et al. (1978). NADPH-dependent cytochrome P-450-reductase was isolated from liver microsomes from PB-induced rabbits and
was purified to a specific content of 10.5 nmol mg" protein by the
method of Shephard et al. (1983). Cytochrome bg, isolated from the
same source, was purified to a specific content of 61 nmol mg"
protein by the method reported by Waxman and Walsh (1982).
of Valproic Acid
ples were extracted with ethyl acetate (2 X 3 ml). The combined
organic extracts were concentrated (to -20 pl) under a stream of dry
nitrogen, and TMS derivatives were prepared by treatment with
bis(trimethylsily1)trifluoroacetamide (100 pl) a t 90 "C for 45 min.
Preliminary experiments indicated that the formation of 4-ene-VPA
from VPA in liver microsomes from phenobarbital-induced rabbits
was linear for 15 min with protein concentrations up to 1.7 mg ml"
(4 nmol of microsomal P-450 ml-'1. A K , value of0.95 mM was
obtained for this reaction when product formation was analyzed
(Eadie-Hofstee plot) as a function of substrate concentration (data
not shown). However, the GC/MS assay conditions described above
only just allowed base-line separation of product from substrate when
the concentration of VPA was 1mM (Fig. 2), and increasing the VPA
concentration further led to severe dynamic range problems. Therefore, unless indicated otherwise, incubations were performed at the
subsaturating substrate concentration of 1 mM for 15 min with 4
nmol of microsomal P-450. Data for incubations containing VPA are
expressed as picomoles of product formed per nmol P-450 15 min"
to facilitate comparisons between species and induction status, since
linearity of product formation was not verified for all of the various
enzyme sources described herein.
Calculation of Isotope Effects
Intermolecular isotope effects on the metabolism of VPA to 4-eneVPA were calculated from the ratio of the rate of product formed
from unlabeled substrate to that produced from the deuterated analogs. These measurements were carried out by selected ion monitoring
a
BImk
L
c
I
Ea
0
C
P-45OLY2
11'02
Time (rnin)
Metabolic Experiments
Microsomal preparations (1.0 ml, final volume) contained potassium phosphate (100 mM), 4 nmol of microsomal P-450, 1 pmol of
NADPH, 1 pmol of NADH, 3 pmol of MgCl,, and 1 pmol of VPA or
a deuterated analog thereof. Incubations were carried out for 15-60
min at 37 'C and were terminated by the addition of 10%HCl(1 ml).
Incubations using purified enzyme contained 2 nmol of P-450LM2,2
nmol of P-450-reductase, and 60 mg of phosphatidylcholine in place
of microsomes. Where indicated, the reconstituted system was supplemented with an equimolar concentration of cytochrome bg. 2-nPropylhexanoic acid (94 ng) was added to all samples a t the end of
the incubation as internal standard for the GC/MS assay, and sam-
13735
12'32"
Time (min)
FIG. 2. Formation of 4-ene-VPA catalyzed by liver microsomes from PB-induced rabbits and by P - 4 6 0 ~ ~Selected
2.
ion
current chromatograms (m/z 199) were generated, and enzyme preparations were obtained and incubations performed, as described under
"Experimental Procedures." The chromatograms depicted are representative of incubations performed in the absence of P-450 (a),
incubations performed with liver microsomes from PB-induced rabbits ( b ) ,incubations performed with P-450 but excluding cytochrome
bg (c); andincubations performed with P-450 plus cytochrome bg ( d ) .
Rates of formation of 4-ene-VPA were: a, notdetectable; b, 235 pmol
nmol" P-450 15 min"; c, 71.2 pmol nmol" P-450 15 rnin"; and d,
76.2 pmol" nmol" P-450 15 rnin".
P-450-catalyzed Desaturationof Valproic Acid
13736
GC/MS, as outlined above, and were based on the abundances of the
respective [M - CH3]+ions of the various molecular species of 4-eneVPA TMS ( m / z 199 for the unlabeled metabolite). Intramolecular
isotope effects on the formation of 4-ene-VPA were calculated from
the ratio of ion intensities in the metabolite at m/z 200 ('H loss) and
201 ('H loss) when [4,4-'Hz]VPA served as substrate and at m / z 201
('H loss) and 202 ('H loss) when [5,5,5-'H3]VPA was employed as
substrate. Intramolecular isotope effects on the formation of 4-hydroxy-VPA-y-lactone were calculated from the ratio of the [M CH,]+ ion intensities at m / z 128 ('H loss) and 129 ('H loss) when
[4,4-'Hz]VPA was used, and from the [M - C3H6If ion intensities at
m / z 100 (metabolism on the unlabeled propyl group) and 103 (metabolism on the deuterated propyl group) when [5,5,5-'Ha]VPA was the
substrate. Intramolecular isotope effects on the formation of 5-hydroxy-VPA from [4,4-'Hz]- and [5,5,5-'H3]VPA were calculated from
the ratio of the [M - CzHb-TMSOH]+ ion intensities at m / z 185,
186, and 187. Isotope effects were corrected for incomplete deuterium
incorporation in the substrates and for the natural abundance of
carbon, oxygen, and silicon isotopes in the ions monitored.
Other Assays
Cytochrome P-450 concentrations were determined by the method
of Estabrook et al. (1972) using an extinction coefficient of 100 mM
cm". Protein concentrations were analyzed by the method of Lowry
et al. (1951) and by the modified method of Dulley and Grieve (1975)
when samples contained detergent. Warfarin metabolites were analyzed by the GC/MS method of Bush et al. (1983), and formaldehyde
production from benzphetamine was measured using the Nash reagent (Nash, 1953).
RESULTS
Induction Effects in the Formation of 4-ene-VPA-Assay
sensitivity for 4-ene-VPA was increased approximately 3-fold
over that reported previously (Rettie et al., 1987) by using a
magnetic sector GC/MS instrument for selected ion monitoring. This increase in sensitivity allowed us to quantify, for
the first time, rates of 4-ene-VPA formation in hepatic microsomes from untreated rats (Table I). The value of 12.8 1.0
pmol nmol" P-450 15 min" for control rat microsomes was
found to be the lowest rate of 4-ene formation catalyzed by
any of the enzyme sources used in this study, including human
liver. Induction of rats with PB, however, increased the rate
of hepatic microsomal 4-ene-VPA formation to 107 k 1.5
pmol nmol" P-450 15 min", which represents an 8-fold
induction. This maybe compared with the 6- and 3-fold
inductions reported previously (Rettie etal., 1987) for 4- and
5-hydroxylation, respectively, of VPA. Rats induced with CBZ
and DPH also exhibited elevated rates of 4-ene formation
compared to controls. CBZ did not induce total P-450 content
but increased 4-ene-VPA formation by90%over controls.
DPH induced total P-450 content by40% and 4-ene-VPA
formation by 230%. Rates of pentoxyphenoxazone O-dealkylase activity were also measured in rat liver microsomes from
each of the four treatment groups and gave the following
induction values: CBZ, 42-fold; DPH, 95-fold; and PB, 161fold. A good correlation (? = 0.92) was found between the
*
rates of formation of 4-ene-VPA and the rateof dealkylation
of pentoxyphenoxazone in the four treatment groups. Since
the pentyl ether of phenoxazone may be used as an indicator
of the levels of P-45% (Lubet et al., 1985; Wolf et al., 1986),
these data provide strong circumstantial evidence that the
induction of this specific form of P-450 occurs in each of the
anticonvulsant drug treatment groups and that it is responsible for the enhanced formation of 4-ene-VPA in the rat.
Species Differences in the Formation of 4-ene- VPA-Table
I1 illustrates the differential aspects of PB induction on the
microsomal formation of 4-ene-VPA in rat,mouse, and rabbit
and facilitates comparison of basal levels of metabolite formation in uitro between animals and man. Basal rates of 4ene-VPA formation increased in the order rat, mouse, human,
and rabbit. A 3-fold variation was observed in the rates of
product formation from six human liver samples, although
reaction rates in these human microsomal preparations did
not correlatesignificantly (? = 0.27) with total P-450 content.
Phenobarbital induction in mice, rats, and rabbits each resulted in approximately 2.5-fold increases in liver microsomal
P-450 content and in 2.5-, 8.4-, and 6.3-fold increases in the
rates of formation of 4-ene-VPA, respectively. Clearly, no
correlation existed across animal species between total P-450
content and induction of 4-ene-VPA formation.
The hypothesis that the induction of the P-45% ortholog
in therabbit, P - 4 5 0 ~was
~ ~ responsible
,
for the increased rate
of formation of 4-ene-VPA in PB-pretreatedrabbits was
tested by comparing turnover numbers in the purified preparation and in the microsomes from which P-450L~2
was isolated (Fig. 2). Rates of 4-ene-VPA formation by P-450L~z
were30% of those found inthe component microsomes.
Addition of an equimolar amount of cytochrome bg to the
reconstituted enzyme system did not result in a significant
increase in product formation (32% of microsomal values).
Deuterium Isotope Effect Studies-We suggested previously
that formation of 4-ene-VPA from VPA could result from
elimination of a hydrogen atom from a free radical metabolite
of VPA, in which the radical was centered on either carbon
C-4 or C-5. In order to distinguish between these two positions
in VPA as the site for initial radical formation (hydrogen
atom abstraction),we examined the deuterium isotope effects
on 4-ene-VPA formation using a number of selectively deuterated analogs of VPA (Fig. 1).It is clear from an appraisal
of the preceding data that the formation of 4-ene-VPA is
catalyzed most efficiently by microsomes prepared from PBinduced rabbits. Therefore, this enzyme source was chosen
for the deuterium isotope effect study.
Intramolecular isotope effects were calculated for 4-eneVPA, 4-hydroxy-VPA, and 5-hydroxy-VPA using [4,4-'Hz]
VPA and [5,5,5-2H3]VPAas substrates (Table 111). As expected, large primary kinetic isotope effects were found for
the formation of 4-hydroxy-VPA from [4,4-2H2]VPAand for
TABLEI
Effects of anticonvulsant drug pretreatment onmicrosomal P-450 content, 4-ene-VPA formation, and
pentoxyphenoxazone-0-dealkylationin rat liver
Assays were carried out as described under "Experimental Procedures." Values are the mean S.D. of three or
four determinations.
. ~ . ~
~~~
~~~~~~~~~~
Induction status
P-450 content
pmollnmol
P-450115
protein
nmollmg
0.80
Rate of formation
of 4-ene-VPA
min
12.8 f 1.0
Controls
0.82
24.8 k 0.9 (1.9)
CBZ
42.6 & 1.2 (3.3)
DPH
1.15 (1.4)
107 f 1.5 (8.4)
PB
1.83 (2.2)
a Values in parentheses represent -fold induction relative to control preparations.
Rate of dealkylation
of uentoxmhenoxazone
"
pmollnmol
P-4501min
4.4 f 0.9
184 f 29 (42)
416 k 40 (95)
707 & 38 (161)
P-450-catalyzed
Desaturation
TABLEI1
Species effects on the formation of 4-ene-VPA inhepatic microsomes
Assays were carried out as described under “Experimental Procedures.” Values are the mean f S.D. of three to seven determinations.
Species
Induction
status
Rat
Rat
Mouse
Mouse
Control
Rabbit
Rabbit
Human
Control
PB
Control
PB
PB
P-450
content
Rate of formation
of 4-ene-VPA
nmollmgproteinpmollnmol
P-450115 min
0.82
12.8 f 1.0
1.83 (2.2)”
107 f 1.5 (8.4)
0.72
18.3 f 3.1
1.70 (2.4)
45.2 f 3.6 (2.5)
44.2 f 2.7
0.94
280 f 16 (6.3)
2.36 (2.5)
24.6 f 7.7
0.38 f 0.06
[0.30-0.46]*
[11.9-35.81
Numbers in parentheses refer to -fold induction relative to control
preparations.
‘Numbers in square brackets represent the rangeof values obtained from six different human liver preparations.
TABLEI11
Intramolecular isotope effects on the formationof 4-hydroxy-VPA,
5-hydroxy- VPA,and 4-ene- VPAby liver microsomes from
PB-induced rabbits
Assays and isotope effect calculations were carriedout as described
under “Experimental Procedures.” Values are the mean f S.D. of
three to nine determinations as shown in parentheses.
Substrate
Metabolite
kdkD
[4,4-’H2]VPA
4-ene-VPA
4-OH-VPA
5-OH-VPA
4-ene-VPA
4-OH-VPA
5-OH-VPA
5.58 f 0.19 (8)
5.05 f 0.17 (3)
0.97 f 0.01 (5)
1.62 f 0.04 (6)
1.09 f 0.06 (9)
4.27 f 0.12 (5)
[5,5,5-’H3]VPA
ofAcid
Valproic
13737
second hydrogen/deuterium exerts on the overall kinetics of
the reaction is small.
Finally, additional data were sought to support the above
mechanistic interpretation through an analysis of intermolecular isotope effects on the formation of 4-ene-VPA from VPA.
In these experiments, the substrates consisted of analogs of
VPA in which one or both alkyl chains were fully substituted
with deuterium at the terminal or sub-terminal positions
(Table IV). In addition to [4,4-’H2]- and [5,5,5-2H3]VPA,
intermolecular isotope effects were determined with [4,4,4’,4’2H4]- and [5,5,5,5’,5’,5’-’Hs]VPA (Fig. 1).A small intermolecular isotope effect ( k H / k D = 2.20) was obtained for [4,4‘H2]VPA, which compares with a value of 5.58 derived from
the corresponding intramolecular experiment. This phenomenon, known as “masking,” is encountered frequently in isotope effect experiments which employ an intermolecular design. Substitution of both the C-4 and C-4’ protons of VPA
with deuterium resulted in a substantialincrease in themeasured intermolecular isotope effect for 4-ene-VPA formation
( k H / k=~ 6.6), a result which would bepredicted if the initial,
rate-limiting step inthe reaction sequence were the formation
of a radical centered on the sub-terminal carbon. If the above
model is consistent, one would not expect to observe appreciable intermolecular isotope effects for the desaturation of
[‘H3]- or [‘Hs]VPA. In fact, inverse isotope effects were found
for these substrates, the magnitude of which increased with
increasing deuterium substitution (Table IV).
DISCUSSION
Using an improved GC/MS assay procedure, we were able
to determine constitutive rates of formation of 4-ene-VPA in
liver microsomes from a variety of sources. Formation of this
toxic metabolite in vitrowas shown to occur in all of the
TABLEIV
hepatic microsomal sources studies, including human tissue.
Intermolecular isotope effects on the formation of 4-em- VPAby liver In fact, the mean basal rate of product formation catalyzed
microsomes from PB-induced rabbits
by six samples of human liver microsomes was exceededonly
Assays and isotope effects calculations were performedas described by that found in rabbits. This finding suggests that it will be
under “Experimental Procedures.”Values represent the mean f S.D.
possible to use human liver microsomes as a model for future
of five or seven determinations, as shown in parentheses.
studies on the “4-ene pathway” of VPA metabolism in man.
Rate of formation
Substrate
kdko
Although pretreatment of rats, rabbits, and mice with PB
of 4-ene-VPA
led to anenhancement of the rateof 4-ene-VPA formation in
pmollnmol P-450115 min
liver microsomes, the extentof induction of the 4-ene pathway
VPA
280 f 16 (7)
varied
markedly among these species. In earlier studies, we
f
11
(5)
2.2
127
[4,4-*H2]VPA
established that the major form of P-450 induced in rat liver
332 f 14 (5)
0.8
[5,5,5-’H3]VPA
6.6
42.2 f 1.9 (5)
by PB treatment is the enzyme which participates in the
[4,4,4’,4’-’H4]VPA
0.7
386 f 20 (5)
[5,5,5,5’,5’,5’-’H6]VPA
formation of 4-ene-VPA (Rettie etal., 1987). However, in the
rabbit the major liver isozyme induced by PB, P“k!jOLMz,was
the formation of 5-hydroxy-VPA from [5,5,5-’H3]VPA. Con- responsible for only 30%of the microsomal turnover number,
versely, although again as expected, small P-secondary kinetic both in the presence and absence of cytochrome b5. That this
isotope effects were observed for the formation of 4-hydroxy- finding is not a consequence of general catalytic insufficiency
VPA from [5,5,5-2H3]VPA,and for the formation of 5-hy- was indicated by the favorable comparison of turnover numdroxy-VPA from [4,4-2Hz]VPA.The primary isotope effect bers for benzphetamine demethylation obtained with the
~ ~ ~ in this laboratory (46 nmol
associated with the formation of 4-ene-VPA from [4,4-2H2] preparation of P - 4 5 0 purified
VPA was 5.58, while the corresponding value for the termi- of product nmol-’ P-450 min”) with that of the enzyme
nally labeled substrate was 1.62. These figures are consistent prepared in Dr. Coon’s laboratory (22-41 nmol of product
with a mechanism in which a carbon-centered radical at C-4, nmol” P-450 min-’) (Koop and Coon, 1979; Gorsky and
rather than atC-5, serves as anintermediate in the formation Coon, 1986). Phenobarbital also induces the formation of P~ 5rabbit liver. It is not known at this time if P-450LM5
of 4-ene-VPA. Moreover, the larger isotope effect ( k ~ / =
k ~ 4 5 0 ~ in
1.62) associated with the formation of 4-ene-VPA from [5,5,5- is functional in thedesaturation of VPA catalyzed by hepatic
*H3]VPArelative to that observed for the formation of 4- microsomes from PB-induced rabbits. We are in the process
hydroxy-VPA from this substrate ( k ~ / =
k 1.09)
~
shows that of purifying other isozymes of P-450 from liver microsomes
the elimination of the second hydrogen atom isan isotopically from PB-induced rabbits in an attempt toidentify the forms
sensitive step. However, the fact that the isotope effect asso- of the cytochrome, other than P-~~OLM’,
which contribute to
ciated with 4-ene-VPA formation from [5,5,5-’H3]VPA is the elevated formation of 4-ene-VPA in this species. Unforsmall compared to that observed for the formation of this tunately, although isozymes of P-450 induced by PB pretreatolefin from [4,4-’Hz]VPA shows that theinfluence which the ment have been purified and characterized extensively from
13738
x
P-450-catalyzed Desaturationof Valproic Acid
Acknowledgments-We would like tothank
Dr. Bruce Mico
(SmithKline and French Laboratories, Philadelphia, PA) for generous gifts of labeled analogs of VPA, and Mr. William Howald of this
department for assistance with the mass spectrometry. We would also
like to thank Dr. A. Craig Eddy (Department of Surgery, University
of Washington, Seattle, WA) for supplying samples of human liver
from renal transplant donors. The efforts of Charlotte Widener in
manuscript preparation are gratefully acknowledged. One of us (M.
B.) would like to thank Bayer AG for support during a leave of
absence at theUniversity of Washington.
VPA
Fe'"- OH
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7939
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In conclusion, we have shown that the formation of 4-eneVPA from valproate in vitro is induced by a number of Waxman, D. J., and Walsh, C. (1982) J. Biol. Chem. 2 5 7 , 1044610457
anticonvulsant drugs which are commonly co-administered
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649
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2,591-597
human subjects receiving VPA are currently in progress.
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