Download effect of -fluorination of valproic acid on valproyl- s-acyl

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DRUG METABOLISM AND DISPOSITION
Copyright © 2001 by The American Society for Pharmacology and Experimental Therapeutics
DMD 29:1210–1215, 2001
Vol. 29, No. 9
344/923951
Printed in U.S.A.
EFFECT OF ␣-FLUORINATION OF VALPROIC ACID ON VALPROYL-S-ACYL-COA
FORMATION IN VIVO IN RATS
MARK P. GRILLO,1 GRAZIA CHIELLINI, MARCO TONELLI,
AND
LESLIE Z. BENET
Departments of Biopharmaceutical Sciences (M.P.G., L.Z.B.) and Pharmaceutical Chemistry (G.C., M.T.), University of California,
San Francisco, California
(Received January 24, 2001; accepted May 19, 2001)
This paper is available online at http://dmd.aspetjournals.org
ABSTRACT:
tandem mass spectrometry analysis of liver extracts from VPAdosed rats showed the presence of VPA-CoA that was maximal
after 0.5 h (185 nmol/g of liver) and was still measurable 5-h postadministration (90 nmol/g of liver). In agreement with our hypothesis, F-VPA did not form the corresponding acyl-CoA derivative as
determined by the absence of F-VPA-CoA upon HPLC analysis of
liver extracts from F-VPA-dosed rats. Further examination of liver
tissue for the presence of free acids revealed that the differences
in acyl-CoA formation cannot be explained by differences in VPA
and F-VPA free acid concentrations. From these observations and
related studies showing the lack of toxicity due to ␣-fluoro substitution, we propose that metabolism of VPA by acyl-CoA formation
may mediate the hepatotoxicity of the drug.
The use of valproic acid (2-n-propylpentanoic acid, VPA2; Fig. 1)
for the treatment of seizures has been associated with an idiosyncratic
hepatotoxicity that is usually characterized by hepatic microvesicular
steatosis (Zimmerman and Ishak, 1982). It has been proposed that a
metabolite (or metabolites) of VPA induce the associated hepatotoxicity (Lewis et al., 1982; Zimmerman and Ishak, 1982; Baillie, 1992).
⌬4-VPA, an unsaturated metabolite of VPA, elicits hepatic steatosis in
rats to a greater degree than VPA or other metabolites tested (Kester-
son et al., 1984). The mechanism by which ⌬4-VPA causes its
hepatotoxic effects is proposed to involve metabolic activation of
⌬4-VPA by the enzymes of fatty acid ␤-oxidation, resulting in the
formation of reactive species that bind covalently to important enzymes involved in fatty acid metabolism (Fig. 2). Evidence for the
formation of chemically reactive metabolites of VPA and ⌬4-VPA
comes from covalent binding studies in isolated rat hepatocytes.
Covalent binding was abolished in cells pretreated with 4-pentenoic
acid (a potent inhibitor of ␤-oxidation) and increased in incubations
with hepatocytes from rats pretreated with clofibrate (an inducer of
␤-oxidation) (Porubek et al., 1989). In addition, similar studies in
isolated rat liver mitochondria showed that ⌬4-VPA covalently binds
to proteins by a process that is dependent on the presence of the
cofactors of ␤-oxidation (CoA, ATP, L-carnitine, and Mg2⫹) (Kassahun et al., 1994). Other experiments revealed that treatment of rats
with ⌬4-VPA leads to a depletion of total hepatic and mitochondrial
glutathione (GSH), which supports a proposal that the depletion of
GSH by reactive metabolites of ⌬4-VPA may lead to the associated
hepatotoxicity (Kassahun et al., 1991). In agreement with this finding
are observations identifying the GSH conjugate of ⌬2,4-VPA in the
bile of ⌬4-VPA-dosed rats and the excretion of the corresponding
N-acetylcysteine conjugate in the urine of patients treated with VPA
(Kassahun et al., 1991). The authors proposed that a reactive diene
metabolite coming from the ␤-oxidation of ⌬4-VPA-CoA, namely
⌬2,4-VPA-CoA, represents the reactive intermediate undergoing conjugation with GSH (Fig. 2). Acyl-CoA dehydrogenase enzymes catalyze the first step in mitochondrial fatty acid ␤-oxidation by converting fatty acyl-CoA thioesters to their corresponding trans-2,3-
This work was supported in part by National Institutes of Health Grant
GM36633. Preliminary accounts of this work were presented at the Millennial
World Congress of Pharmaceutical Sciences, San Francisco, CA, April 2000. The
University of California at San Francisco Mass Spectometry Facility is supported
by the Biomedical Research Technology Program of the National Center for
Research Resources, National Institutes of Health Grant RR01614, and National
Science Foundation Grant DIR 8700766.
1
Present address: Pharmacia, Global Metabolism and Investigative Sciences,
301 Henrietta St., Kalamazoo, MI 49007.
2
Abbreviations used are: VPA, valproic acid; VPA-CoA, valproyl-S-acyl-CoA;
F-VPA, ␣-fluorovalproic acid; F-VPA-CoA, ␣-fluorovalproyl-S-acyl-CoA; ⌬4-VPA,
unsaturated metabolite of VPA; F-⌬4-VPA, ␣-fluorinated analog of ⌬4-VPA; ⌬2,4VPA, hepatotoxic metabolite of ⌬4-VPA; GSH, glutathione; BSTFA, bis(trimethylsilyl)trifluoroacetamide; CID, collisionally induced dissociation; THF, tetrahydrofuran; ESI/MS/MS, electrospray ionization/tandem mass spectrometry; GC/MS,
gas chromatography/mass spectrometry; HPLC, high-performance liquid chromatography.
Address correspondence to: Leslie Z. Benet, Department of Biopharmaceutical Sciences, School of Pharmacy, University of California, 513 Parnassus Ave.,
San Francisco, CA 94143-0446. E-mail: [email protected]
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Studies designed to compare valproic acid (VPA) with its ␣-fluorinated derivative (F-VPA) for their abilities to form acyl-CoA thioester derivatives in vivo are described. Recent studies have shown
that ␣-fluorination of a hepatotoxic metabolite of VPA (⌬4-VPA)
resulted in a nonhepatotoxic derivative. We hypothesize that the
decrease in hepatotoxicity may be related to a lack of formation of
the intermediary acyl-CoA thioester. To determine the effect of
␣-fluoro substitution on acyl-CoA formation, we synthesized FVPA and compared it with VPA for its ability to form the acyl-CoA
thioester derivative in vivo in rat liver. Thus, after dosing rats with
VPA or F-VPA, animals were sacrificed (0.05-, 0.5-, 1-, 2-, and 5-h
postadministration) for the analysis of liver tissue. High-performance liquid chromatography (HPLC) and electrospray ionization/
␣-FLUORO SUBSTITUTION AND VALPROYL-CoA FORMATION
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4
FIG. 1. Structures of compounds cited in the text.
FIG. 2. Proposed scheme for the metabolic activation of VPA.
Questionable effect of ␣-fluoro substitution on the metabolic activation of
⌬4-VPA. The F-⌬4-VPA-CoA intermediate shown in brackets has not been identified.
Experimental Procedures
Materials. VPA, EDTA, triethylamine, ethyl chloroformate, potassium
bicarbonate, diisopropylamine, butyllithium (1.6 M in hexanes), hydroxylamine hydrochloride, BSTFA, ibuprofen, and THF (anhydrous) were purchased from Aldrich Chemical (Milwaukee, WI). F-VPA, VPA-CoA, and
F-VPA-CoA were synthesized as described below. CoA was purchased from
Sigma (St. Louis, MO). N-Fluorobenzenesulfonimide was a gift from Allied
Signal, Inc. (Buffalo, NY). All solvents used for HPLC were of chromatography grade.
Instrumentation and Analytical Methods. HPLC was carried out on a
Shimadzu LC-600 isocratic system coupled to a Shimadzu SPD-6AV UV-Vis
detector (Shimadzu, Kyoto, Japan). All HPLC analyses were performed on a
reverse-phase column (Beckman C8, 15 cm, 5 ␮, 1 ml/min; Beckman Coulter,
Inc., Fullerton, CA). Tandem liquid secondary-ion mass spectrometry was
performed on a Kratos Concept IIHH four-sector tandem mass spectrometer
(Kratos Analytical Instruments, Chestnut Ridge, NY) equipped with a cesium
ion source and a continuous flow liquid secondary-ion mass spectrometry
probe. The solvent used contained acetonitrile (5%), thioglycerol (2%), and
trifluoroacetic acid (0.1%) in water. Samples were delivered at a flow rate of
3 ␮l/min using a syringe pump (Applied Biosystems, Foster City, CA).
ESI/MS/MS analysis was performed on a Finnigan-MAT TSQ 7000 (San Jose,
CA). HPLC purified samples were introduced into the ESI source via a
Harvard Apparatus syringe pump (Holliston, MA) at a flow rate of 0.3 ml/min.
CID of the 12C component of the protonated molecules-of-interest was performed in the collision cell. GC/MS analysis was conducted on a HP 5890
chromatograph (Hewlett Packard, Palo Alto, CA) interfaced with a Varian
70/70 magnetic sector mass spectrometer (Varian, Palo Alto, CA). Analyses
were performed in the positive ion EI mode with an emission current of 350
␮A. The GC column used was a fused silica capillary column (60 m ⫻
0.32-mm i.d., 0.25-mm film thickness) coated with a DB-1 bonded stationary
phase (J&W Scientific, Rancho Cordova, CA). Samples were analyzed as their
trimethylsilyl esters after derivatization with BSTFA. GC conditions were
60°C for 0.5 min, 20°C/min to a final oven temperature of 290°C. Ibuprofen
was used as the internal standard for quantitation of VPA and F-VPA, and all
ions monitored corresponded to the [M ⫺ CH3]⫹ fragment. Quantitative
measurements were based on the ratios of peak areas (free acid/internal
standard) relative to a linear calibration curve.
1
H NMR spectra were recorded (in deuterochloroform) with a General
Electric QE300 spectrometer (Fairfield, CT) operating at 300 MHz. Proton
chemical shifts are reported in parts per million (ppm; ␦) relative to the
tetramethylsilane internal standard.
Synthesis of ␣-F-VPA. F-VPA was synthesized by electrophilic ␣-fluorination of ethyl valproate by an analogous method to that described by Tang et
al. (1995) for the synthesis of 2-fluoro-2-propyl-4-pentenoic acid. GC/MS
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enoyl-CoA derivatives (Schulz, 1990). These enzymes are believed to
function by a mechanism where removal of the ␣-proton by an
active-site base is followed by transfer of the ␤-hydride to a noncovalently bound flavin adenine nucleotide group (Thorpe, 1990).
The present work was prompted by an interesting study by Tang et
al. (1995) on ␣-fluorinated analogs as mechanistic probes in VPAinduced hepatotoxicity. The substitution of a fluorine atom at the
␣-position to the carboxylic acid group provides a derivative that
contains a chemically and enzymatically inert carbon center. The
authors proposed that fluorine substitution at the ␣-carbon would
block its metabolism to the reactive ⌬2,4-VPA-CoA diene metabolite,
via acyl-CoA dehydrogenases, and thus markedly reduce hepatotoxicity. Results from their studies showed that the ␣-fluorinated analog
of ⌬4-VPA (F-⌬4-VPA) does not cause hepatic steatosis, nor does it
metabolize to the glutathione conjugate of the ⌬2,4-VPA diene in vivo
in rats. These data support the acyl-CoA dehydrogenase-mediated
metabolic activation of ⌬4-VPA-CoA hypothesis. A critical part of the
hypothesis that may explain the lack of hepatotoxicity induced by the
fluoro derivative involves the formation of F-⌬4-VPA-CoA (Fig. 2),
although the ability of F-⌬ -VPA to form the corresponding acyl-CoA
thioester derivative has never been shown.
There have been reports indicating that ␣-fluorinated carboxylic
acid-containing compounds do not form acyl-CoA thioester conjugates. These reports include studies with ␣-fluoropalmitic acid (Soltysiak et al., 1984) and perfluorooctanoic and perfluorodecanoic acids
(Kuslikis et al., 1992) showing the lack of formation of their respective acyl-CoA thioester conjugates in vitro.
Hypotheses requiring the formation of the VPA-CoA thioester have
been put forward to explain the mechanism of VPA-mediated hepatotoxicity and include VPA-induced CoA depletion (Thurston et al.,
1985), carnitine depletion (Coulter, 1984), as well as competitive
inhibition of one or several of the enzymes of fatty acid ␤-oxidation
by VPA-CoA or VPA-CoA metabolites (Becker and Harris, 1983;
Bjorge and Baillie, 1985). Therefore, we propose that an absence of
formation of the acyl-CoA derivative for the ␣-fluoro analogs of VPA,
or its hepatotoxic metabolites, could account for the associated lack of
toxic effects. To test this hypothesis, the ␣-fluoro analog of VPA was
used in in vivo metabolic studies in the rat. The studies presented here
were performed to determine whether F-VPA is able to be converted
to F-VPA-CoA in vivo in rat liver.
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GRILLO ET AL.
FIG. 3. Representative chromatograms from the reverse-phase isocratic HPLC
analysis of rat liver extract from a rat dosed with VPA (100 mg/kg, 0.5-h
postadministration) (A) and synthetic VPA-CoA standard (B).
mixture reacted with BSTFA (300 ␮l) overnight at room temperature. Aliquots
(10 ␮l) of these derivatized extracts were analyzed by GC/MS as described
above.
Results
Analysis of Acyl-CoA Metabolites in Liver Tissues of VPA- and
F-VPA-Treated Rats. Livers of rats treated with VPA (100 mg/kg) or
F-VPA (110 mg/kg) were analyzed for their respective acyl-CoA
thioester derivatives at 0.05-, 0.5-, 1-, 2-, and 5-h post i.p. administration. HPLC analysis of liver extract from a VPA-dosed rat at the
0.5-h time point revealed the presence of VPA-CoA eluting at an
HPLC retention time of ⬃22 min (Fig. 3). No substance was detected
at this retention time in control liver extracts of rats treated only with
water (pH 7.0, 1 ml i.p., data not shown). The identity of this
metabolite was confirmed by comparison of the HPLC and tandem
mass spectrometric characteristics of the biological and corresponding
synthetic standard (Figs. 3 and 4). CID of the parent MH⫹ ion (m/z
894) of the HPLC-purified biological extract provided a mass spectrum characteristic of the VPA-CoA synthetic standard, which included ions at m/z 285, m/z 387, and m/z 428 originating from the
proposed cleavages (Grillo, 1993) as shown in Fig. 4. The hepatic
concentration-time profile of VPA-CoA in rat liver (Fig. 6) showed
that the presence of VPA-CoA was maximal in liver tissue after 0.5 h
(185 nmol/g of liver) and was still measurable 5-h postadministration
(90 nmol/g of liver). The concentrations of VPA-CoA at the 1- and
5-h time points are approximately equal and are consistent with the
relative VPA free acid concentrations in livers 1- and 5-h post-VPA
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analysis of the synthetic product showed it to be 99% F-VPA with approximately a 1% impurity of VPA. GC/MS mass spectrum of F-VPA-trimethylsilyl
ester, m/z (%): 73 (100), 219 (12) (M⫹-15), 117 (8). No molecular ion was
detected. 1H NMR (CDCl3): ␦ 0.90 (t, 6H, J ⫽ 7.2 Hz, OCH3 groups), 1.2 to
1.6 (m, 4H, CH3OCH2Ogroups), 1.7 to 2.0 (m, 4H, ⫺CH2OCH2Ogroups).
The 1% impurity of VPA could not be detected by 1H NMR (complete absence
of the ␦ 2.39 chemical shift for the ␣-proton of VPA).
Synthesis of S-Acyl-CoA Thioester Derivatives of VPA and F-VPA.
Synthesis of CoA thioesters was performed by conventional procedures employing ethyl chloroformate (Stadtman, 1957; Grillo, 1993). Briefly, triethylamine (1.6 mmol) followed by ethyl chloroformate (1.6 mmol) was added to
the free acid (1.6 mmol) dissolved in anhydrous THF (25 ml) at room
temperature and while stirring. After 30 min of continued stirring, the precipitate that formed (triethylamine hydrochloride) was removed by passing
through a glass funnel, fitted with a glass wool plug, and added directly into a
solution containing CoA (100 mg) and KHCO3 (1.6 mmol) in distilled water
(10 ml) and THF (15 ml). The solution was stirred continuously under nitrogen
gas at room temperature for 2 h, after which the reaction was terminated by the
addition of concentrated HCl (8 drops). The THF was then removed by
evaporation under reduced pressure, and the remaining aqueous phase was
extracted with diethyl ether (4 ⫻ 50 ml). Residual diethyl ether was removed
by evaporation under reduced pressure at room temperature. The solution was
adjusted to pH 7.0 by the addition of NaOH (1 N). Purification of the acyl-CoA
thioester standards was achieved by reverse-phase HPLC using isocratic elution with acetonitrile (20%) in 0.2 M ammonium acetate on a reverse-phase
column (C18, 25 cm ⫻ 4.6 mm, 5 ␮m, 1 ml/min) and detected by UV
absorbance (262 nm). CoA thioester-containing fractions were collected and
desalted by passing through a cation exchange solid phase extraction cartridge
(J. T. Baker, Phillipsburg, NJ). Desalted solutions containing CoA thioesters
were then frozen (⫺80°C) and lyophilized to dryness. MS/MS analysis VPACoA (CID of MH⫹ at m/z 894, 100%: m/z 136 ([adenine ⫹ H]⫹, 90%), m/z
387 ([M ⫹ H ⫺ adenosine triphosphate]⫹, 12%), m/z 428 ([adenosine diphosphate ⫹ 2H]⫹, 8%), and m/z 285 ([M ⫹ H ⫺ 609]⫹, 6%). F-VPA-CoA (CID
of MH⫹ at m/z 912, 100%: m/z 136 ([adenine ⫹ H]⫹, 100%), m/z 405 ([M ⫹
H ⫺ adenosine triphosphate]⫹, 100%), m/z 428 ([adenosine diphosphate ⫹
2H]⫹, 42%), m/z 508 ([adenosine triphosphate ⫹ 2H]⫹, 36%), m/z 303 ([M ⫹
H ⫺ 609]⫹, 14%), and m/z 565 ([adenosine monophosphate]⫹, 31%).
Analysis of Acyl-CoA Metabolites. After the administration of VPA or
F-VPA (0.7 mmol/kg i.p., in distilled water, pH 7.0) to male Sprague-Dawley
rats (200 –220 g), and at 0.05-, 0.5-, 1-, 2-, and 5-h time points, rodents were
anesthetized with diethyl ether, decapitated, and their livers immediately
excised. Livers were then quickly frozen at ⫺80°C and stored until analysis for
acyl-CoA derivatives by HPLC and mass spectrometry. The preparation of
liver samples for HPLC analysis was performed as follows: frozen livers were
broken into small pieces (approximately 0.5 g), and 1.0-g portions (duplicate)
were then added to ice-cold 7% perchloric acid (2.0 ml) and homogenized for
3 min on ice using a glass mortar and drill-driven Teflon pestle. The homogenized sample (1.5 ml) was then transferred to a microcentrifuge tube and
centrifuged (14,000 rpm, 3 min). The resulting supernatant was transferred to
a glass vial (20 ml) containing a solution of potassium phosphate buffer (0.05
M, pH 7.0, 5 ml). The pH of this solution was adjusted to 6.0 with 1 N NaOH.
An aliquot of this solution (200 ␮l) was then immediately injected onto the
HPLC (Beckman C8, 15 cm, 5 ␮, 1 ml/min). The acyl-CoA derivatives were
eluted by a linear gradient whereby the acetonitrile concentration of the 0.2 M
ammonium acetate mobile phase was increased from 0 to 50% over 50 min. All
acyl-CoA derivatives were detected by UV analysis at 262 nm. Quantitative
measurements of acyl-CoA derivatives detected in liver tissue were made
using a standard curve generated from absolute peak areas. Acyl-CoA metabolites formed in vivo were analyzed by tandem mass spectrometry after
purification by HPLC and further processing as described above (Grillo, 1993).
Analysis of Free Acids. Liver homogenates (prepared as above, 1.5 ml,
duplicate) from VPA- and F-VPA-treated rats were added to 2 N NaOH (1.5
ml), to hydrolyze any acyl-CoA or acyl glucuronide esters, containing internal
standard (ibuprofen, 5 ␮g), and the samples were left to stand at room
temperature for 1 h. These solutions were then acidified (2 N HCl, 3 ml) and
extracted with ethyl acetate (2 ⫻ 5 ml). The combined ethyl acetate extracts
were dried (MgSO4) and evaporated to dryness under nitrogen gas at room
temperature. Diethyl ether (1 ml) was added to these dried samples, and the
␣-FLUORO SUBSTITUTION AND VALPROYL-CoA FORMATION
administration (Fig. 6). In addition, the nadir in hepatic VPA-CoA
concentration occurring at the 2-h time point is consistent with the
nadir in the hepatic VPA free acid concentration. The secondary rise
in VPA and VPA-CoA concentrations at the 5-h time point is due to
a reabsorption of VPA, which is released by hydrolysis of the VPA1-O-acyl glucuronide in the large bowel (Dickinson et al., 1979). In
agreement with our hypothesis, F-VPA-CoA was not detected in liver
extracts from F-VPA-treated rats (Fig. 5). The small amount of
VPA-CoA that is detected during the HPLC analysis of the F-VPA rat
liver extract (retention time 22 min) is presumably derived from the
metabolism of the 1% impurity of VPA in the F-VPA synthetic test
compound.
GC/MS Analysis and Quantitation of VPA and F-VPA in Rat
Liver. The hepatic concentration-time profile of VPA and F-VPA in
liver tissue at the 0.05-, 0.5-, 1-, 2-, and 5-h time points, as assessed
by GC/MS analysis of the liver extracts for the trimethylsilyl derivatives of the test compounds, indicated that the concentrations of the
VPA and F-VPA were similar throughout the 5-h experiment (Fig. 6).
The concentration of VPA and F-VPA free acids in liver tissue
extracts at the 1-h time point were determined to be 150 nmol/g of
liver and 141 nmol/g of liver, respectively, although only the acylCoA derivative of VPA was detected (Fig. 6) at the same time point.
Discussion
A mechanism that may explain the hepatotoxicity associated with
VPA therapy involves the formation of chemically reactive metabolites of the drug (Zimmerman and Ishak, 1982; Baillie, 1992). Most
attention has focused on the unsaturated metabolite of VPA, ⌬4-VPA,
FIG. 5. Representative chromatograms from the reverse-phase isocratic HPLC
analysis of rat liver extract from a rat dosed with F-VPA (110 mg/kg, 0.5-h
postadministration) (A) and synthetic F-VPA-CoA standard (B).
because this metabolite is metabolically activated to reactive electrophilic species that may covalently bind to and injure important cellular
proteins (Fig. 2; Baillie, 1992). One hepatotoxic metabolite of ⌬4VPA is ⌬2,4-VPA, which has been shown to be reactive with nucleophiles such as glutathione (Kassahun et al., 1991). The conversion of
⌬4-VPA to ⌬2,4-VPA first involves the formation of ⌬4-VPA-CoA
thioester that is catalyzed by acyl-CoA synthetases and requires the
cofactors CoA, ATP, and Mg2⫹(Caldwell, 1984; Brass, 1994). ⌬4VPA-CoA then is metabolized by acyl-CoA dehydrogenases, the
enzymes involved in the first step of fatty acid ␤-oxidation (Thorpe,
1990), to ⌬2,4-VPA-CoA thioester. It has been proposed that ⌬2,4VPA-CoA reacts with and depletes mitochondrial GSH resulting in
hepatocellular injury (Kassahun and Abbott, 1993; Kassahun et al.,
1994). Although these data provide evidence for ⌬4-VPA-CoA metabolic activation to reactive and potentially toxic metabolites, there
are data that do not support this hypothesis. Evidence against reactive
unsaturated metabolites of VPA leading to VPA-induced hepatotoxicity comes from studies in rats (Loscher et al., 1993), where ⌬4-VPA
levels did not correlate with hepatic steatosis in VPA-treated animals,
and in humans (Siemes et al., 1993), where increased amounts of
⌬4-VPA could be detected only in one of the five patients with
fulminant liver failure. To clarify the role of ⌬4-VPA, and subsequently ⌬2,4-VPA, in VPA-induced hepatotoxicity, Tang et al. (1995)
synthesized F-⌬4-VPA and used it in histopathological, biochemical,
and metabolic studies in rats. Because of the chemical and metabolic
stability of the carbon-fluorine bond at the ␣-position, the authors
propose that fluorine substitution at the ␣-carbon would preclude its
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FIG. 4. Spectrum of product ions formed by CID of the MH⫹ ion (m/z 894) of
VPA-CoA synthetic standard (A) and VPA-CoA isolated by HPLC purification
from liver tissue of a rat dosed with VPA (B).
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Values are expressed as the average of duplicate experiments. The small amount
of CoA thioester measured in the liver extracts from F-VPA-treated rats is presumably due to VPA-CoA formation from the 1% impurity of VPA in the administered
synthetic F-VPA.
metabolism to the reactive diene metabolite, via acyl-CoA dehydrogenases, and thus markedly reduce hepatotoxicity. Their results, all of
which strongly support the ⌬4-VPA bioactivation hypothesis, showed
that F-⌬4-VPA when given to rats resulted in no hepatic steatosis,
depletion of liver mitochondrial glutathione, or formation of glutathione conjugates of the ⌬2,4-VPA metabolite (Fig. 2).
The ability of F-⌬4-VPA to be converted to F-⌬4-VPA-CoA thioester has not been directly determined but has been presumed to occur
because the amino acid conjugate of F-⌬4-VPA, N2-(␣-fluoro-2propyl-pentenoyl)glutamine, has been isolated as a urinary metabolite
from F-⌬4-VPA-treated rats (Tang et al., 1997). In similar studies with
mice, F-VPA was also shown to be converted extensively to the
F-VPA-glutamine amide conjugate (Tang et al., 1997). Amino acid
metabolites of acidic drugs are believed to occur through the intermediacy of the respective acyl-CoA thioester metabolites (Caldwell et
al., 1979; Caldwell, 1984), and therefore F-⌬4-VPA is proposed to
have formed the intermediate CoA thioester derivative. These results
are in contrast to literature reports showing examples that ␣-fluorinated derivatives of carboxylic acid compounds do not form acyl-CoA
thioester conjugates. These include studies with ␣-fluoropalmitic acid
(Soltysiak et al., 1984), shown not to be incorporated into triglycerides
in cultured mammalian cells, an acyl-CoA-mediated process, and in
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FIG. 6. Hepatic concentration-time profile of VPA, F-VPA, and VPA-CoA in rat
liver after i.p. administration of VPA (100 mg/kg) or F-VPA (110 mg/kg) to rats.
studies with perfluoro-medium chain fatty acids (Kuslikis et al.,
1992), shown not to form acyl-CoA thioesters in incubations with
liver microsomes and the cofactors of acyl-CoA formation (CoA,
ATP, and Mg2⫹). To our knowledge, it is not known whether amino
acid conjugation can occur independently of the formation of reactive
acyl-CoA intermediates, although results from studies with ␣-fluoropalmitate support involvement of CoA thiol ester-independent steps in
the modification of membrane lipids (Soltysiak et al., 1984), which
are processes requiring a reactive acylating intermediate. ␣-Fluoropalmitic acid was found to be incorporated without modification into
membrane lipids such as phosphatidylcholine, sphingomyelin, neutral
glycosphingolipids, and ceramides, independent of the formation of
␣-fluoro-palmitoyl-CoA. It may be that the formation of the glutamine-amide conjugate of F-VPA occurs by a CoA-independent
process.
Another potential mechanism for VPA-mediated hepatotoxicity
comes from studies showing that acyl-CoA thioester derivatives of
carboxylic acid-containing compounds are chemically reactive species that may contribute to the acylation of protein (Tishler and
Goldman, 1970; Faed, 1984; Hertz and Bar-Tana, 1988; Bharadwaj
and Bizzozero, 1995; Sallustio et al., 2000). In addition to the effects
of VPA on CoA depletion (Thurston et al., 1985), carnitine depletion
(Coulter, 1984), and competitive inhibition of fatty acid metabolism
(Baillie, 1992), the chemical reactivity of VPA-CoA in transacylationtype reactions with protein nucleophiles may contribute to the covalent binding of VPA to protein in vitro and in vivo (Porubek et al.;
1989; Kassahun et al.; 1994; Bailey and Dickinson, 1996) and the
associated hepatotoxicity (Nelson and Pearson, 1990; Baillie, 1992).
The ability of VPA-CoA to transacylate protein nucleophiles remains
to be evaluated.
We propose that the lack of hepatotoxic effects of the ␣-fluorosubstituted derivative of ⌬4-VPA may be due to a lack of the formation of the F-⌬4-VPA-CoA thioester conjugate, rather than a block in
the formation of the reactive diene, ⌬2,4-VPA-CoA. To test this
hypothesis, we synthesized F-VPA and in the present studies tested
for its ability to form the F-VPA-acyl-CoA derivative in vivo in rat
liver. We assumed that the effect on acyl-CoA formation that occurs
for F-VPA would also occur for F-⌬4-VPA. Analysis of liver tissue
extracts by HPLC from rats treated with VPA showed the presence of
VPA-CoA (Fig. 3), but no F-VPA-acyl-CoA was detected in extracts
from the F-VPA-treated animals (Fig. 5). Analysis for the presence of
VPA and F-VPA free acids in liver tissue showed only small differences in their concentrations, indicating that F-VPA-CoA thioester is
not formed in vivo in liver even though the free acid is present (Fig.
6). Furthermore, standard curves of the acyl-CoA synthetic standards
isolated from acidified liver tissue were identical for VPA-CoA and
F-VPA-CoA, indicating similar extraction efficiencies for both CoA
thioesters (data not shown).
The inability of the ␣-fluoro-substituted analogs to form acyl-CoA
derivatives may result from the increased acidity of the carboxyl
group (Schwartz et al., 1995; Tang et al., 1997), due to the electronegativity effects of the fluorine atom (Welch and Eswarakrishnan,
1991), which may influence the binding of the analog to the acyl-CoA
synthetase enzymes (Soltysiak et al., 1984). The F-VPA analog was
shown to be more acidic (pKa ⫽ 3.55) than VPA (pKa ⫽ 4.80), which
may also help to explain the lack of the formation of F-VPA-acyl
glucuronide relative to the extensive glucuronidation of VPA in vivo
(Tang et al., 1997).
From these observations showing that the ␣-fluoro analog of VPA
does not form the respective acyl-CoA derivative in vivo and that
␣-fluorination precludes hepatic steatosis (Tang et al., 1995), we
propose that the metabolism of VPA, and unsaturated metabolites of
␣-FLUORO SUBSTITUTION AND VALPROYL-CoA FORMATION
VPA, by acyl-CoA formation may mediate the hepatotoxicity of the
drug. Ongoing studies in our laboratory are designed to evaluate the
abilities of VPA and unsaturated hepatotoxic metabolites of VPA to
form acyl-CoA thioesters, in vitro and in vivo, in that differences in
acyl-CoA formation may be related to differences in the ability to
induce hepatotoxicity.
Acknowledgments. We thank Milagros Han for assistance in performing HPLC analysis. We also acknowledge the assistance in mass
spectrometry provided by the University of California, San Francisco,
Mass Spectrometry Facility (A. L. Burlingame, Director). Further
acknowledgment goes to Dr. Thomas A. Baillie (Merck Research
Laboratories) for guidance and discussions on acyl-CoA thioesters
concerning chemical reactivity and characterization by electrospray
mass spectrometry.
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