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Biochimica et Biophysica Acta 1771 (2007) 533 – 543
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Studies on the extra-mitochondrial CoA-ester formation of valproic
and Δ 4 -valproic acids
Cátia C.P. Aires a , Jos P.N. Ruiter c , Paula B.M. Luís a , Herman J. ten Brink b , Lodewijk IJlst c ,
Isabel Tavares de Almeida a , Marinus Duran c , Ronald J.A. Wanders c , Margarida F.B. Silva a,⁎
a
c
UBMBE, Centro de Patogénese Molecular, Faculdade de Farmácia da Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal
b
Free University Hospital, De Boelelaan, Amsterdam, The Netherlands
Laboratory of Genetic Metabolic Diseases, Department of Clinical Chemistry and Pediatrics, Academic Medical Centre, University of Amsterdam,
Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands
Received 30 October 2006; received in revised form 15 January 2007; accepted 17 January 2007
Available online 23 January 2007
Abstract
The hypothesis whether valproic acid (VPA) and its main microsomal metabolite, Δ4-valproic acid, can be activated to the respective CoA esters
in the cell cytosol was investigated. The valproyl-CoA formation was measured in different subcellular fractions obtained by differential
centrifugation of liver homogenates of rats treated with VPA (studies ex vivo) and digitonin fractionation of rat hepatocytes incubated with VPA and
cofactors (studies in vitro). The results show that VPA activation may occur in the cytosol and is not restricted to the mitochondrial matrix as
believed until now. Furthermore, the activation of Δ4-VPA is demonstrated in vitro. Valproyl-CoA and Δ4-valproyl-CoA were detected after in vitro
incubations and the former also in the mitochondrial and cytosolic fractions obtained from liver cells of treated rats. The activation to valproyl-CoA
was characterized in cytosolic fractions, optimized with respect to time and protein and the kinetic constants (Kapp
m ) were estimated for the reaction
substrates. Other medium-chain fatty acids decreased the formation of valproyl-CoA suggesting a competition for both mitochondrial and extramitochondrial VPA activating enzymes. The present findings suggest additional mechanisms of mitochondrial dysfunction associated with VPA,
and they may contribute to the further understanding of the toxic effects associated with this drug.
© 2007 Elsevier B.V. All rights reserved.
Keywords: Valproic acid; Δ4-Valproic acid; Fatty acid activation; Acyl-CoA synthetase; Drug induced steatosis; Steatosis mechanism
1. Introduction
Mitochondrial β-oxidation is the major process by which
fatty acids are oxidized, thus providing a major source of
energy for the heart and for skeletal muscle [1]. This pathAbbreviations: VPA, 2-n-propylpentanoic acid or valproic acid; Δ4-VPA,
2-n-propyl-4-pentenoic acid; CoA, coenzyme A; dephCoA, dephosphocoenzyme A; Pi, orthophosphate; AMP, adenosine-5′-monophosphate; ADP,
adenosine-5′-diphosphate; ATP, adenosine-5′-triphosphate; MES, 2-[N-morpholino]ethanesulfonic acid; MOPS, 3-[N-morpholino]propane-sulfonic acid;
EGTA, ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′,-tetraacetic acid;
SEM buffer, Sucrose/ EGTA/ MOPS buffer; BSA, bovine serum albumin;
BCA, bicinchoninic acid; DTNB, 5,5′-dithio-bis(2-nitrobenzoic acid) or
Ellman's reagent; ACS, acyl-CoA synthetase; RLM, rat liver mitochondria;
HPLC, high performance liquid chromatography; FAO, fatty acid β-oxidation;
MCFA, medium-chain fatty acids; LCFA, long-chain fatty acids
⁎ Corresponding author. Tel.: +351 21 794 64 91; fax: +351 21 794 64 91.
E-mail address: [email protected] (M.F.B. Silva).
1388-1981/$ - see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbalip.2007.01.010
way is also the main oxidative process of biotransformation
of the anticonvulsive drug, valproic acid (2-n-propylpentanoic acid, VPA), which is an eight-carbon branched-chain fatty
acid [2].
Conceptually, mitochondrial β-oxidation can be subdivided
into two parts: (a) the process of transferring acyl groups into
the mitochondria for oxidation via the carnitine shuttle and (b)
intra-mitochondrial chain shortening of the acyl-CoA by the
oxidative removal of two-carbon (acetyl) units [1]. Prior to
translocation into the mitochondria, all long-chain fatty acids
(LCFA) must be “activated” to their coenzyme A (CoA) esters.
However, the current opinion is that medium-chain fatty acids
(MCFA) enter the mitochondrial compartment as free acids,
being subsequently activated in the matrix, as it is reported to
happen with VPA [2].
The enzymes responsible for the activation process are
members of the acyl-CoA synthetase (ACS) family [3]. The
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overall reaction catalysed by the acyl-CoA synthetases includes
firstly an adenylyl transfer step from ATP to the fatty acid,
forming an acyl-adenylate and pyrophosphate (PPi). The
activated intermediate (acyl-AMP) is then attacked by CoASH, yielding the acyl-CoA product and AMP [4,5]. ACS are
generally classified according to their specificities towards fatty
acids of varying chain length [3,4]. Short-chain (ACSS) and
medium-chain acyl-CoA synthetases (ACSM) may activate
fatty acids containing 2–4 or 4–10 carbons, respectively, and
long-chain acyl-CoA synthetase (ACSL) specifically activate
fatty acids with more than 10 carbon atoms [4,6].
These enzymes are located in various cell compartments and
exhibit wide tissue distribution, with highest activity associated
with liver and adipose tissue [4,6]. The mitochondrial matrix
contains several ACSS and ACSM [1,3,4,7], and ACSL have
been identified in the outer mitochondrial membrane [4,6]. Other
ACSL have been found in the cytosol, smooth endoplasmatic
reticulum, peroxisomes, mitochondria associated membranes
(MAM) [4,6,8] and also in the plasma membrane. Recent
experiments have shown that some fatty acid transport proteins
(FATPs) have acyl-CoA synthetase activity together with their
ability to translocate fatty acids across the cellular membrane.
Hall et al. reported in 2003 that fatty acid transport protein 1
(FATP1) exhibits very long-chain acyl-CoA synthetase activity
with broad substrate specificity, which supports the hypothesis
that fatty acid uptake into the cells is linked to their esterification
with coenzyme A [9]. Other proteins of this FATP family have
also been associated with activation of very long-chain fatty
acids [10–13]. Soluble proteins capable of synthesize acyl-CoA
esters have already been described [14,15], and cytosolic acylCoA synthetases were also identified in rat liver [16].
ACSM are involved in glycine conjugation of aromatic fatty
acids in mouse kidney mitochondria, one of the most important
routes for the detoxification, not only of many xenobiotic
carboxylic acids but also of endogenous acids [17–19]. Liver
and kidney contain ACSM that are capable of forming the CoA
thioester of not only short- and medium-chain fatty acids but
also of numerous carboxylic acid xenobiotics. These enzymes
have been referred to as xenobiotic/medium-chain fatty acidCoA synthetases (XM-synthetases) [20].
In patients treated with valproic acid, the oxidative metabolism of this xenobiotic involves its β-oxidation in liver
mitochondria. Numerous reports have described the interference
of VPA (or its metabolites, as valproyl-CoA) with biochemical
pathways, like fatty acid β-oxidation (FAO), the tricarboxylic
acid cycle and oxidative phosphorylation, affecting mitochondrial functions [21,22]. It has been assumed until now that this
drug can enter the mitochondria by simple diffusion, like most of
the MCFA, crossing the outer and inner mitochondrial
membranes independently of carnitine. Once inside the mitochondria VPA is activated to valproyl-CoA by a presumed
ACSM and then metabolised via β-oxidation to acetyl-CoA and
propionyl-CoA [2]. The formation of valproyl-CoA and
metabolites has been suggested to sequester the limited
concentration of mitochondrial free CoA, thereby interfering
with the CoA-dependent metabolic processes of other endogenous fatty acids [23–26] as well as pyruvate and 2-oxoglutarate. In
fact, the branched-chain acyl-CoA esters formed during VPA
biotransformation seem to have a greater resistance to hydrolysis
than straight-chain acyl-CoAs, probably due to steric hindrance
[27–29], a fact that would aggravate the CoA depletion.
In the present study we show that valproyl-CoA can be
synthesized not only in mitochondria but also outside this
organelle. One of the possible consequences to this fact is the
depletion of the extra-mitochondrial levels of CoA, which could
explain the observed FAO impairment in VPA treated patients.
This would be especially important since the extra-mitochondrial CoA concentration is much lower than the intramitochondrial CoA one. Also, the extra-mitochondrial VPA
activation can affect numerous cellular functions, bringing consequences for the metabolic fate of the drug.
Thus, the intracellular compartmentalization of VPA and Δ4VPA (its main microsomal metabolite) activation was studied by
monitoring the respective CoA conjugates either in vitro or ex
vivo, and an estimate of the kinetic parameters of the enzymatic
reactions was obtained. In addition, the studies were expanded
with the aim to confirm the formation in vivo and in a whole
cellular model of valproyl-dephospho-CoA, a new mitochondrial metabolite of VPA recently reported by our group [30].
2. Materials and methods
2.1. Materials
VPA, BSA, Coenzyme-A, ATP, Bicinchoninic acid (BCA) and other
standard biochemicals were obtained from Sigma-Aldrich. Lichrosolv-grade
solvents for high performance liquid chromatography (HPLC) were obtained
from Merck (Darmstad, Germany).
2.2. Synthesis of valproyl-CoA, valproyl-dephosphoCoA, Δ4-valproic
acid and Δ4-valproyl-CoA
Valproyl-CoA, valproyl-dephosphoCoA (valproyl-dephCoA) and Δ4-valproyl-CoA were synthesized according to published procedures [29,30] from
VPA and Δ4-VPA, respectively. Δ4-Valproic acid was obtained by chemical
synthesis following a reported procedure [31].
2.3. Animals
The study was conducted according to the National Guidelines for the care
and use of laboratory animals (Faculty of Pharmacy animals laboratory). Male
Wistar rats weighing about 100 g were kept under a standard diet with ad libitum
access to food and water. Rats were allocated to either a single in vivo or a
chronic in vivo study group. Three groups of animals were used: (a) group 1
(n = 5) was treated with one single intraperitoneal (i.p.) injection of sodium
valproate (100 mg/kg) solution in 0.9% NaCl; (b) group 2 (n = 5) was treated
daily for 2 weeks with the same i.p. dose of sodium valproate; (c) group 3 (n = 7,
control rats) received equal volume of 0.9% saline solution by the same route.
2.4. Subcellular fractionation
Liver mitochondrial and cytosolic fractions were prepared from all animals.
One hour after a single injection of VPA (group 1) or the last injection of VPA
(group 2) the rats were killed after mild anesthesia with uretan. Livers were
removed and immediately rinsed into ice-cold homogenization medium (SEM:
250 mM Sucrose, 0.5 mM EGTA, 5 mM MOPS, pH = 7.4). After mincing and
two to three washings, the chopped liver was homogenized in a precooled Teflon
pestle glass homogenizer. The homogenate was centrifuged at 600×g (10 min,
4 °C) and the obtained post-nuclear supernatant was further centrifuged at
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535
The enzyme activity was determined as a function of the pH. To this end,
cytosolic fractions were incubated at 37 °C for 60 min with different buffers
(MES: pH 4.5, pH 5.0, pH 6.0; Tris: pH 7.0, pH 7.5, pH 8.0, pH 8.5, pH 9.0;
CHES: pH 9.0, pH 9.5, pH 10.0; final concentration 100 mM), in the presence of
1 mM VPA, 5 mM ATP, 5 mM CoA and 5 mM MgCl2, in a shaking water bath
(final volume of 100 μL). The reactions were stopped by adding 10 μL of 2 M
HCl with vortexing. The samples were neutralized with 10 μL 2 M KOH/0.6 M
MES and centrifuged at 17,000×g (5 min, 4 °C). The resulting supernatants were
transferred to microvials in order to be injected into the HPLC column.
Cytosolic rat liver samples were also used at different protein concentrations
(0.5 to 3.5 mg/mL) for different incubation periods at 37 °C (0 to 80 min), in the
presence of a fixed concentration of 5 mM VPA, CoA and ATP at pH = 9.0.
The enzyme activity was studied as function of the substrate (VPA)
concentration both in cytosol and mitochondria. The samples were incubated
with VPA (0–10 mM) for 60 min at 37 °C as described above, using 100 mM
Tris buffer at pH 9.0, 5 mM ATP, 5 mM CoA and 5 mM MgCl2. The obtained
supernatants were analysed using HPLC.
Product formation was also determined as a function of CoA and ATP
concentration up to 10 mM and 20 mM respectively for both cofactors. A mixed
solution of ATP and MgCl2 in a 1:1 ratio was used in this case and incubations
were performed at 37 °C for 60 min, with 3.0 mg/mL protein, 5 mM VPA and
100 mM Tris buffer (pH = 9.0).
The formation of valproyl-CoA from VPA was also tested in microsomal
fractions (20 mg/mL) using analogous conditions as described for mitochondria
(2 mM VPA, 1 mM ATP, 1 mM CoA, 1 mM MgCl2, potassium phosphate buffer
at pH 7.4, for 5 and 10 min at 37 °C).
Fig. 1. HPLC profiles of (A) purified standards; (B) mitochondria prepared from
rat liver after in vivo treatment with one single dose of VPA. (Peak identification:
1-Valproyl-CoA; 2-Valproyl-dephCoA).
3600×g (10 min, 4 °C). The pellet obtained was suspended and after one last
round of centrifugation at 2700×g (10 min, 4 °C), the mitochondria were finally
resuspended in the homogenization medium (25–50 mg/mL). The final
supernatant was purified with a new centrifugation step at 105,000×g (1 h,
4 °C) using a SW-41Ti rotor (Beckman Instruments), yielding the cytosolic
fraction (supernatant) and a microsomal fraction (pellet).
Protein concentration of cellular fractions was determined using the BCA
assay [32] with bovine serum albumin as a standard. Samples of mitochondrial
and cytosolic fractions were diluted to a protein content of 20 mg/mL in SEM
buffer.
2.5. Hepatocytes isolation and digitonin fractionation
Rat hepatocytes were isolated from male Wistar rats (approximately 350 g).
Animals were anaesthetized with Nembutal and livers were perfused at 37 °C
with Krebs–Ringer buffer according to established procedures. Hepatocytes
were isolated essentially as described by Berry and Friend [33]. The isolated
cells were washed and resuspended in SEM buffer (protein 20 mg/mL) and used
for further in vitro incubations with VPA, which were performed at 37 °C in a
final volume of 250 μL using a shaking water bath. The reactions were started by
addition of VPA (0.5 and 1 mM, pH 7.4) and were stopped after 1 h by the
addition of 250 μL ice-cold digitonin (400 μg/mL, Calbiochem.). After 10 min
at 4 °C, the cells were centrifuged at 17,000×g (5 min, 4 °C). The obtained pellet
(organelle fraction) was further deproteinized with 100 μL of 0.5 M PCA while
in the supernatant (soluble fraction) the proteins were precipitated by the
addition of 20 μL of 11.8 M PCA. After 5 min on iced water, both fractions were
neutralized with 2 M KOH. To remove the precipitated protein, the fractions
were centrifuged at 17,000×g (5 min at 4 °C), and the supernatants were
transferred to HPLC vials.
2.6. Studies in vitro on the valproic acid activation
The characterization of the putative valproyl-CoA synthetase activity was
performed in vitro by measuring the conversion of VPA into valproyl-CoA,
using HPLC analysis as described below. The obtained mitochondria were
sonicated (2 × 10 s; 8 W) prior to incubation.
Fig. 2. Identification of Valproyl-CoA (peak 1) in (A) cytosolic fraction obtained
from control rat liver cells; (B) cytosolic fraction of rat liver cells after in vivo
treatment with one single dose of VPA and (C) previous sample after spiking
with 2 μM of synthetic standard.
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A competition study using hexanoic acid (C6:0) and phenylpropionic acid,
in both rat liver mitochondria and cytosol was also performed, by measuring the
formation of valproyl-CoA. The samples (3.0 mg/mL) were incubated for
60 min, 37 °C, with 5 mM VPA, CoA and ATP:MgCl2 (1:1) using 100 mM Tris
buffer pH 9.0, in the presence and absence of each MCFA (5 mM). The obtained
deproteinized supernatants were analysed by HPLC.
In all studies, the valproyl-CoA formed in the different incubations was
quantified by means of an external calibration curve previously performed with
the purified compound.
2.7. Studies in vitro on the Δ4-valproic acid activation
Rat liver subcellular fractions were obtained as described above, from the
control group of animals. Mitochondrial fractions suspended in SEM buffer
were sonicated (2 × 10 s; 8 W). Samples were incubated in a standard reaction
medium containing 5 mM ATP, 5 mM CoA, 5 mM MgCl2, 100 mM Tris buffer
(pH 9.0) and Δ4-valproic acid (0–10 mM) for 1 h at 37 °C (final reaction
volume of 200 μL). The reactions were stopped by adding 20 μL of 2 M HCl
with vortexing and the samples were neutralized with 20 μL of 2 M KOH/
0.6 M MES, centrifuged for 17,000×g (5 min, 4 °C) and the supernatants
transferred to microvials in order to analyse the formation of Δ4-valproyl-CoA
using HPLC.
In all studies, the Δ4-valproyl-CoA formed in the different incubations was
quantified by means of an external calibration curve previously performed with
the purified compound.
2.8. Analysis of VPA acyl-CoA esters using HPLC
Analysis of VPA acyl-CoA and valproyl-dephCoA esters was performed
according to published methodology [29,30]. Basically two different HPLC
systems were used in the present study.
(a) Analysis of mitochondrial and cytosolic fractions obtained from livers of
VPA treated rats and controls: samples of subcellular fractions were acidified
with 50 μl of 2 M HCl in a vortex mixer and protein was removed by centrifugation at 17,000×g (5 min, 4 °C) in an Eppendorf centrifuge. The supernatant was
neutralized with 50 μl of 2 M KOH/0.6 M MES during mixing on a vortex. After
removing the precipitated material by centrifugation at 17,000×g (5 min, 4 °C)
the supernatants were transferred to microvials in order to be analysed by HPLC.
Two HPLC pumps (Waters, models 510 and 501) were used as the solvent
delivery system. The samples (100 μL) were introduced by an autosampler
injector (Merck AS-2000A) on a C18-reverse phase column (Supelcosil LC-18-S:
5 μm, 250 mm, 4.6 mm) and the system was controlled by the Waters
Millennium32 chromatography manager software.
Resolution of the VPA acyl-CoA thioesters (valproyl-CoA, valproyldephCoA, Δ2-valproyl-CoA, Δ4-valproyl-CoA, 3-OH-valproyl-CoA and
3-keto-valproyl-CoA) was achieved by linearly increasing the acetonitrile
content of the 16.9 mM sodium phosphate (pH 6.9) elution buffer from 10% to
45% in 20 min at a flow rate of 1 mL/min. The column was washed for 5 min at
70% of acetonitrile and then reequilibrated for 15 min under the starting
conditions in a total runtime of 42 min. Acyl-CoA esters were detected with a UV
detector (Waters model 440; λ = 254 nm) and a Photodiode array UV-Vis detector
(Shimadzu, SPD-M6A).
(b) Analysis of hepatocytes fractions and enzyme characterization: an HPLC
pump system from Perkin Elmer (series 200 PUMP) was used. The samples
were injected with a Gilson 234 automatic injector on a Supelcosil LC-18-DB
column (5 μm, 250 mm, 4.6 mm) and the runs controlled with the Chromeleon
chromatografic software, from Dionex.
Resolution of the VPA acyl-CoA thioesters was achieved by linearly
increasing the acetonitrile content of the 16.9 mM sodium phosphate (pH 6.9)
elution buffer from 10% to 32% in 16 min at a flow rate of 1 mL/min. The
column was washed for 5 min at 70% of acetonitrile and then reequilibrated for
7 min under the starting conditions in a total runtime of 30 min. Acyl-CoA esters
were detected with a UV detector (Shimadzu, SPD-10A; λ = 260 nm).
Fig. 3. HPLC profile of VPA acyl-CoA esters: (A) in the organelle fraction obtained from rat hepatocytes incubated in vitro for 1 h at 37 °C with 1 mM VPA; (B) in the
same organelle sample after spiking with the purified standards; (C) in the respective soluble fraction; (D) in the same soluble sample after spiking with the purified
standards. (Peak identification: 1-Valproyl-CoA; 2-Valproyl-dephCoA).
C.C.P. Aires et al. / Biochimica et Biophysica Acta 1771 (2007) 533–543
2.9. Enzyme assay
Citrate synthase activity (CS; EC 4.1.3.7) was measured on a Cobas-Fara
centrifugal analyzer (Hoffmann-La Roche) monitoring the CoA formation using
a coupled reaction with DTNB, essentially as described by Haggie et al. [34]. The
reaction was started by adding 0.2 mM oxaloacetate in an incubation mixture
containing 100 mM Tris/HCl pH 7.6, 100 mM KCl, 0.1 mM DTNB, 0.2 mM
acetyl-CoA, 1 g/L Triton X-100 in a final volume of 250 μL. Incubations were
carried out at 37 °C and an assay without the substrate was used as blank. The
citrate synthase activity was calculated using the molar absorption coefficient
(λ = 412 nm) of the DTNB-CoA complex (ε = 13600 M−1 cm−1).
CS was used as marker enzyme for mitochondria and its activity was
determined in the subcellular fractions (rat liver mitochondria and cytosol).
2.10. Data analysis
The characterization of the multisubstrate reaction catalysed by the putative
“valproyl-CoA synthetase” activity in different subcellular fractions was performed by plotting the measured reaction rates as function of substrate concentration (at fixed concentration of the remaining components of the reaction).
The steady state kinetic data were analysed by nonlinear regression analysis
using the SigmaPlot® v10.0 Technical Graphing Software and the Enzyme
Kinetics module v1.3.
The Michaelis–Menten equation [35] was used to calculate kinetic parameters (Km and Vmax) of the enzyme reaction involving VPA, Δ4-VPA, CoA and
ATP. The results shown in Fig. 7 represent the estimated mean (± standard
deviation) of an apparent Km (Kapp
m ) obtained of at least three independent
substrate dependence curves. Values were compared by Student's t test (unpaired, two-tailed, unequal variance) and a statistical significance was defined as
p < 0.05.
537
and valproyl-dephCoA in the two subcellular compartments. As
expected, Fig. 3A clearly shows the formation of valproyl-CoA
in the organelle fraction, containing mitochondria, peroxisomes,
etc., obtained after digitonin fractionation of rat hepatocytes
incubated in vitro with VPA. The presence in the same chromatogram of valproyl-dephCoA indicates that the dephosphorylation of valproyl-CoA also occurs in hepatocytes [30]. The
analysis of the cytosolic fraction revealed traces of valproylCoA (Fig. 3C).
3.3. Kinetics of the VPA activation to valproyl-CoA
The activation of VPA to valproyl-CoA was determined in
vitro using mitochondria and cytosolic samples prepared from
control rats, using differential centrifugation methods. Firstly,
the pH optimum of the enzymatic reaction was studied. Fig. 4A
represents the obtained pH curve of the cytosolic VPA activating enzyme, which shows that the enzyme has a maximum
3. Results
3.1. Formation of valproyl-CoA and valproyl-dephCoA
(studies ex vivo)
As depicted in Fig. 1B, both valproyl-CoA and valproyldephCoA were detected in liver mitochondria of rats (RLM)
treated with one single dose of VPA (group 1). A virtually
identical profile was observed for all animals of the same group.
Valproyl-CoA and the respective dephosphoCoA ester were
only detected in RLM samples of two animals of group 2 that
were subjected to chronic valproic acid treatment (2/5). In
untreated rats (group 3) no corresponding peaks were detected.
The analysis of the cytosolic fractions (Fig. 2B) revealed the
presence of valproyl-CoA in both groups of VPA-treated rats
(groups 1 and 2), although in quite low concentrations. It must be
emphasized that the cytosolic fraction was diluted in SEM
during isolation, which stresses the significance of this trace
presence of conjugates. The identification of valproyl-CoA and
valproyl-dephCoA was performed either by comparing the
retention times with those of the synthetic CoA esters (Fig. 1A)
or by the spiking of samples with the respective purified
standards (Fig. 2C). No VPA activation was observed in
microsomes.
3.2. Formation of valproyl-CoA and valproyl-dephCoA
(studies in vitro in rat hepatocytes)
A second experimental approach was used in order to obtain
additional evidence for the in vivo formation of valproyl-CoA
Fig. 4. Effect of (A) pH, (B) protein and the incubation time on the formation
rate of valproyl-CoA in vitro in cytosol. (The represented values of valproylCoA were obtained with HPLC analysis and UV detection of (A) cytosolic
samples (0.3 mg) of a control rat liver, incubated with 5 mM VPA, 5 mM CoA,
5 mM ATP and 5 mM MgCl2 (37 °C / 60 min) using different pH buffers at final
concentration of 100 mM; (B) cytosolic samples of a control rat liver, incubated
with 5 mM VPA, 5 mM CoA, 5 mM ATP and 5 mM MgCl2, 100 mM Tris buffer
pH = 9 at 37 °C. The represented lines were obtained by linear regression
considering the values obtained at 20, 40 and 60 min: r2 = 0.993 and r2 = 0.992
for 0.5 and 3.5 mg/mL of protein, respectively).
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activity at pH = 9.0. Subsequent experiments concerning the
enzyme characterization were thus always performed at pH
9.0.
Formation of valproyl-CoA was found to be linear as
function of the protein concentration of the cytosolic rat liver
sample (up to 3.5 mg/mL) and as function of time at 37 °C (up
to 60 min), (Fig. 4B). Further experiments were performed
using a protein content of 3.0 mg/mL and a reaction time of
60 min.
In order to study the affinity of the enzyme towards specific
substrates, both mitochondrial and cytosolic samples were
incubated with increasing concentrations of VPA (or Δ4-VPA)
and the cofactors CoA and ATP (Figs. 5 and 6 respectively). For
each studied substrate at least three independent curves were
obtained.
Application of adequate computer software to the experimental results provided an estimate of kinetic constants and the
app
mean of the apparent Km (Km
) and respective standard
deviation are represented in Fig. 7. These values provide an
approximation of the catalytic activity of the enzyme or
enzymes within the two cellular compartments. The significant
differences (p < 0.05), observed in the values of the apparent Km
in mitochondria and cytosol for CoA and ATP, substantiate the
hypothesis that the cytosolic enzyme which converts VPA to
valproyl-CoA is different from the mitochondrial one. In
addition, higher concentrations of ATP were found to inhibit
valproyl-CoA formation both in the cytosol as well as in
mitochondria (see Fig. 6: 1B and 2B).
Subsequently we studied whether VPA competes with other
MCFA, like hexanoic acid and phenylpropionic acid, for both
mitochondrial and cytosolic CoA-synthesizing enzymes. The
amount of valproyl-CoA formed in vitro when VPA was
incubated simultaneously with hexanoic acid (Fig. 8: A-II and
B-II) was clearly lower as compared with the amount of product
resulting from the incubation with VPA alone, in both fractions
(Fig. 8: A-I and B-I). Similar results were obtained with
phenylpropionic acid (plus or minus VPA) and for this reason
the chromatograms of in vitro incubations with this acid are not
shown.
3.4. Citrate Synthase (CS) activity in RLM and cytosol
In order to establish that the valproyl-CoA synthetase
activity measured in cytosolic fractions prepared from rat
livers by differential centrifugation is not due to leakage of
valproyl-CoA present in mitochondria, we measured the
activity of the mitochondrial marker enzyme citrate synthase
(CS) in mitochondrial and cytosolic fractions, respectively.
The activity of CS as measured in mitochondrial and cytosolic samples amounted to 1902 ± 77 U/μL and 33 ± 1.2 U/μL,
Fig. 5. The initial reaction rate of valproyl-CoA synthetase activity was determined in vitro either in rat liver mitochondria (1A, 1B) or in cytosol (2A, 2B) as function
of increasing concentrations of the substrates (0–10 mM): VPA (1A and 2A); Δ4-VPA (1B and 2B). (Experimental details are described in Materials and methods.)
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539
Fig. 6. The initial reaction rate of valproyl-CoA synthetase activity was determined in vitro either in rat liver mitochondria (1A, 1B) or in cytosol (2A, 2B) as function
of increasing concentrations of the CoA (0–10 mM) (1A and 2A) and ATP (0–20 mM) (1B and 2B). (Experimental details are described in Materials and methods.)
respectively, whereas the activities of valproyl-CoA synthetase in the two fractions were 2.82 U/μL and 0.36 U/μL,
respectively.
The results clearly show that the valproyl-CoA synthetase
activity as measured in the cytosolic fraction is only partly
(11%) due to leakage of valproyl-CoA synthetase from broken
mitochondria.
3.5. Studies in vitro on the Δ4-VPA activation to
Δ4-valproyl-CoA
Fig. 7. Estimate of the apparent Km (Kapp
m ) for “valproyl-CoA synthetase” in
subcellular fractions (mitochondria and cytosol of rat liver) as function of VPA,
ATP and CoA concentrations. (The represented values are the mean of at least
three independent experiments and respective standard deviation. The reaction
rate was measured as the amount of valproyl-CoA formed (nmol/min.mg) as
function of the substrate concentration. The concentration of each substrate was
varied independently fixing the remaining reactants: 5 mM ATP, 5 mM CoA
(VPA variable), 5 mM VPA, 5 mM CoA (ATP variable) and 5 mM ATP, 5 mM
VPA (CoA variable). Significant differences between represented values:
*p < 0.05 and **p < 0.001).
Since VPA was clearly activated to valproyl-CoA in the cell
cytosol, it remained to be established whether metabolites of
VPA would undergo a similar conjugation with CoA. For this
purpose we investigated the formation of Δ4-valproyl-CoA
from Δ4-VPA, the main microsomal metabolite formed by
desaturation of VPA.
Fig. 9 demonstrates that activation of Δ4-VPA to Δ4valproyl-CoA clearly occurs in the cytosolic fraction. Interestingly, our results show that Δ4-valproyl-CoA is also formed in
mitochondrial fractions (Fig. 5: 1B and 2B) suggesting that βoxidation may be the strategy of further metabolism of certain
app
microsomal acidic metabolites. Estimated Km
values for Δ4VPA are 0.28 mM in mitochondria and 0.07 mM in cytosol
(using 5 mM ATP and 5 mM CoA). These data suggest that in
the cytosolic fraction there is a higher affinity of the enzyme for
app
Δ4-VPA (low Km
) than in mitochondria, although similar effiapp
ciency of catalysis (similar Vmax
) is attained in both subcellular
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C.C.P. Aires et al. / Biochimica et Biophysica Acta 1771 (2007) 533–543
Fig. 8. HPLC profiles of (A) mitochondrial in vitro incubations with 5 mM VPA (A-I), 5 mM VPA and 5 mM hexanoic acid (A-II) and 5 mM hexanoic acid (A-III); (B)
cytosolic in vitro incubations with 5 mM VPA (B-I), 5 mM VPA and 5 mM hexanoic acid (B-II) and 5 mM hexanoic acid (B-III). (Peak identification: 1-ValproylCoA; 3-Hexanoyl-CoA).
compartments (0.02 and 0.03 nmol/min. mg for mitochondria
and cytosol, respectively).
4. Discussion
Mitochondrial dysfunction has been recognised as an
important mechanism of drug-induced liver injury [36,37].
The VPA-induced inhibition of mitochondrial fatty acid βoxidation [38] has sparked our renewed interest in the acyl-CoA
metabolism. In fact, the formation of xenobiotic-CoA esters may
occur, either as a detoxifying pathway, or as a pathway generating reactive metabolites and a cascade of secondary effects.
We and others have previously shown that VPA is activated
in vitro to valproyl-CoA in mitochondria. The primary results
described in this report show the presence of valproyl-CoA in
mitochondria of liver cells isolated from rats subjected to VPA
treatment, as expected. Surprisingly, trace amounts of this
metabolite were also found in the cytosol of these cells. This
result was not a consequence of leakage from mitochondria
during the homogenization procedure, since this finding was
confirmed by another cellular model, in digitonin-fractionated
rat hepatocytes treated in vitro with VPA. In addition, valproyldephosphoCoA was also detected in these cells, a result that
confirms our previous data obtained with isolated mitochondria
[30].
The presence of two distinct valproyl-CoA pools clearly
suggests that the VPA activation reaction occurs in two different
subcellular compartments, which lead to the investigation whe-
C.C.P. Aires et al. / Biochimica et Biophysica Acta 1771 (2007) 533–543
Fig. 9. Identification of Δ4-valproyl-CoA (Peak 4) in (A) the cytosolic fraction
of rat liver cells incubated in vitro with 2 mM Δ4VPA (plus cofactors); (B) same
sample after spiking with 2 μM of synthetic standard. (C) Blank (Δt = 0).
ther acyl-CoA derivates of VPA can be formed before entering
mitochondria. Indeed, our results also show the formation of
valproyl-CoA in vitro, after incubation of both mitochondria
and cytosolic fractions with VPA and cofactors. Importantly,
only 11% of the extra-mitochondrial valproyl-CoA synthetase
activity can be attributed to mitochondrial contamination, as
calculated by the study of the mitochondrial marker enzyme
citrate synthase. Furthermore, valproyl-CoA was not detected in
microsomal fractions after in vitro incubation with VPA, which
541
points to absent interference of microsomal proteins with the
present results. Taken together, the results clearly suggest two
different subcellular locations for the activation of VPA: one
in the mitochondria and another in the extra-mitochondrial
space.
The mitochondrial ACS and the extra-mitochondrial VPA
activating enzyme were characterized by monitoring the valproyl-CoA formation. The study of the enzyme affinity to the
substrate and cofactors (CoA and ATP) enabled the estimate of
the respective apparent Km in both mitochondrial and cytosolic
fractions. These values provide an approximation of the
substrate concentration range yielding control of the enzymic
activity. Moreover, the significant differences obtained in the
apparent Km values for CoA and ATP in both cellular compartments suggest the existence of two different enzymes in
each fraction capable of activating VPA (and possibly Δ4-VPA)
into the respective CoA esters.
Mitochondrial β-oxidation of VPA is the second most
important route of its biotransformation in man after glucuronidation [21]. The CoA thioester intermediates formed in this
process constitute the biologically active metabolites and
valproyl-CoA is the first metabolite of the overall oxidative
pathway, being mainly formed in the mitochondrial matrix [28].
Thus, the passive diffusion of the free acid across mitochondria
is most probably the predominant pathway. However, the data
reported here are suggestive of a second VPA activation step
during cellular trafficking (possibly in cytosol) before reaching
mitochondria. The formation of the acyl-CoA esters seems to be
extended to other microsomal acidic metabolites of VPA, as
demonstrated for the in vitro activation of Δ4-VPA to Δ4-valproyl-CoA in the cytosol, a reaction that has not been reported
before in literature.
The recognition of the extra-mitochondrial activation of VPA
next to the intra-mitochondrial activation of VPA has consequences for the biotransformation of the drug. In mitochondria,
valproyl-CoA can be further metabolized by β-oxidation [2], but
the metabolic fate in the extra-mitochondrial fraction is still unknown. The valproyl-CoA as formed in the cytosol may enter the
mitochondria via the carnitine shuttle (carnitine palmitoyl-transferase I (CPT I), carnitine-acylcarnitine translocase (CACT) and
carnitine palmitoyl-transferase II (CPT II)). This pathway is
currently under investigation.
Secondary effects, like the trapping of CoA in mitochondria
and/or extra-mitochondrial compartments have different consequences in the cell. Eukaryotic cells contain sequestered pools
of CoA that are crucial for energy metabolism, namely for the
priming or activation of fatty acids, forming CoA derivatives in
an ATP-dependent manner. Cytosolic concentrations of CoA
vary between 0.02 and 0.14 mM in animal tissues, values that
are significantly lower than mitochondrial concentrations which
range from 2.2 to over 5.0 mM [39]. Tissue levels of CoA are
also quite variable, the liver being one of the richest. However,
hepatic CoA levels are among the most responsive to changes in
several metabolic states [39] and therefore the extensive hepatic
biotransformation of VPA will certainly influence CoA regulation and intermediary metabolism. For instance, this drug may
interfere with the activation of endogenous LCFA, mainly by
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C.C.P. Aires et al. / Biochimica et Biophysica Acta 1771 (2007) 533–543
CoA trapping, an effect that would account to the decreased
oxidation rate of these fatty acids induced by the drug [38].
The formation of these VPA acyl-CoA esters both in
mitochondria and extra-mitochondrial compartments can also
have different potential inhibitory targets. For instance, the
extra-mitochondrial activation to valproyl-CoA could potentially have consequences by interference with fatty-acid elongation, peroxisomal metabolism, glyconeogenesis, ureagenesis
and many other pathways involving cytosolic reactions.
The increasing recognition that formation of a CoA conjugate increases chemical reactivity of a xenobiotic carboxylic
acid [6,8] reinforces the absolute need to further investigate
the differences in toxicity of Δ4-valproyl-CoA in relation to
Δ4-VPA.
From the present findings we expect that additional mechanisms of mitochondrial dysfunction associated with valproic
acid will be clarified. This knowledge may contribute to the
further understanding of the toxic effects associated with this
drug, mainly having a broader perspective of the extra-mitochondrial mechanisms.
Acknowledgments
This work was financially supported by Fundação para a
Ciência e a Tecnologia (FCT), Lisboa, Portugal (POCTI/FCB/
48800/2002 with partial funding of FEDER and SFRH/BD/
22420/2005).
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