a 1771 (2007) 533–543www.elsevier.com/locate/bbalip

Biochimica et Biophysica Act

Studies on the extra-mitochondrial CoA-ester formation of valproicand Δ4-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 UBMBE, Centro de Patogénese Molecular, Faculdade de Farmácia da Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugalb Free University Hospital, De Boelelaan, Amsterdam, The Netherlands

c 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 2007Available 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 estersin the cell cytosol was investigated. The valproyl-CoA formation was measured in different subcellular fractions obtained by differentialcentrifugation of liver homogenates of rats treated with VPA (studies ex vivo) and digitonin fractionation of rat hepatocytes incubated with VPA andcofactors (studies in vitro). The results show that VPA activation may occur in the cytosol and is not restricted to the mitochondrial matrix asbelieved until now. Furthermore, the activation ofΔ4-VPA is demonstrated in vitro.Valproyl-CoA andΔ4-valproyl-CoAwere detected after in vitroincubations and the former also in the mitochondrial and cytosolic fractions obtained from liver cells of treated rats. The activation to valproyl-CoAwas characterized in cytosolic fractions, optimized with respect to time and protein and the kinetic constants (Km

app) were estimated for the reactionsubstrates. Other medium-chain fatty acids decreased the formation of valproyl-CoA suggesting a competition for both mitochondrial and extra-mitochondrial 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 whichfatty acids are oxidized, thus providing a major source ofenergy for the heart and for skeletal muscle [1]. This path-

Abbreviations: VPA, 2-n-propylpentanoic acid or valproic acid; Δ4-VPA,2-n-propyl-4-pentenoic acid; CoA, coenzyme A; dephCoA, dephosphocoen-zyme A; Pi, orthophosphate; AMP, adenosine-5′-monophosphate; ADP,adenosine-5′-diphosphate; ATP, adenosine-5′-triphosphate; MES, 2-[N-mor-pholino]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) orEllman'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: mbsilva@ff.ul.pt (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 biotransformationof the anticonvulsive drug, valproic acid (2-n-propylpenta-noic acid, VPA), which is an eight-carbon branched-chain fattyacid [2].

Conceptually, mitochondrial β-oxidation can be subdividedinto two parts: (a) the process of transferring acyl groups intothe mitochondria for oxidation via the carnitine shuttle and (b)intra-mitochondrial chain shortening of the acyl-CoA by theoxidative removal of two-carbon (acetyl) units [1]. Prior totranslocation 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 tohappen with VPA [2].

The enzymes responsible for the activation process aremembers of the acyl-CoA synthetase (ACS) family [3]. The

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overall reaction catalysed by the acyl-CoA synthetases includesfirstly an adenylyl transfer step from ATP to the fatty acid,forming an acyl-adenylate and pyrophosphate (PPi). Theactivated intermediate (acyl-AMP) is then attacked by CoA-SH, yielding the acyl-CoA product and AMP [4,5]. ACS aregenerally classified according to their specificities towards fattyacids of varying chain length [3,4]. Short-chain (ACSS) andmedium-chain acyl-CoA synthetases (ACSM) may activatefatty acids containing 2–4 or 4–10 carbons, respectively, andlong-chain acyl-CoA synthetase (ACSL) specifically activatefatty acids with more than 10 carbon atoms [4,6].

These enzymes are located in various cell compartments andexhibit wide tissue distribution, with highest activity associatedwith liver and adipose tissue [4,6]. The mitochondrial matrixcontains several ACSS and ACSM [1,3,4,7], and ACSL havebeen identified in the outer mitochondrial membrane [4,6]. OtherACSL have been found in the cytosol, smooth endoplasmaticreticulum, peroxisomes, mitochondria associated membranes(MAM) [4,6,8] and also in the plasma membrane. Recentexperiments have shown that some fatty acid transport proteins(FATPs) have acyl-CoA synthetase activity together with theirability 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 activitywith broad substrate specificity, which supports the hypothesisthat fatty acid uptake into the cells is linked to their esterificationwith coenzyme A [9]. Other proteins of this FATP family havealso been associated with activation of very long-chain fattyacids [10–13]. Soluble proteins capable of synthesize acyl-CoAesters have already been described [14,15], and cytosolic acyl-CoA synthetases were also identified in rat liver [16].

ACSM are involved in glycine conjugation of aromatic fattyacids in mouse kidney mitochondria, one of the most importantroutes for the detoxification, not only of many xenobioticcarboxylic acids but also of endogenous acids [17–19]. Liverand kidney contain ACSM that are capable of forming the CoAthioester of not only short- and medium-chain fatty acids butalso of numerous carboxylic acid xenobiotics. These enzymeshave been referred to as xenobiotic/medium-chain fatty acid-CoA synthetases (XM-synthetases) [20].

In patients treated with valproic acid, the oxidative metabo-lism of this xenobiotic involves its β-oxidation in livermitochondria. Numerous reports have described the interferenceof VPA (or its metabolites, as valproyl-CoA) with biochemicalpathways, like fatty acid β-oxidation (FAO), the tricarboxylicacid cycle and oxidative phosphorylation, affecting mitochon-drial functions [21,22]. It has been assumed until now that thisdrug can enter the mitochondria by simple diffusion, like most ofthe MCFA, crossing the outer and inner mitochondrialmembranes independently of carnitine. Once inside the mito-chondria VPA is activated to valproyl-CoA by a presumedACSM and then metabolised via β-oxidation to acetyl-CoA andpropionyl-CoA [2]. The formation of valproyl-CoA andmetabolites has been suggested to sequester the limitedconcentration of mitochondrial free CoA, thereby interferingwith the CoA-dependent metabolic processes of other endogen-ous fatty acids [23–26] as well as pyruvate and 2-oxoglutarate. In

fact, the branched-chain acyl-CoA esters formed during VPAbiotransformation seem to have a greater resistance to hydrolysisthan 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 besynthesized not only in mitochondria but also outside thisorganelle. One of the possible consequences to this fact is thedepletion of the extra-mitochondrial levels of CoA, which couldexplain the observed FAO impairment in VPA treated patients.This would be especially important since the extra-mitochon-drial CoA concentration is much lower than the intra-mitochondrial CoA one. Also, the extra-mitochondrial VPAactivation can affect numerous cellular functions, bringing con-sequences for the metabolic fate of the drug.

Thus, the intracellular compartmentalization of VPA andΔ4-VPA (its main microsomal metabolite) activation was studied bymonitoring the respective CoA conjugates either in vitro or exvivo, and an estimate of the kinetic parameters of the enzymaticreactions was obtained. In addition, the studies were expandedwith the aim to confirm the formation in vivo and in a wholecellular model of valproyl-dephospho-CoA, a new mitochon-drial 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 otherstandard biochemicals were obtained from Sigma-Aldrich. Lichrosolv-gradesolvents for high performance liquid chromatography (HPLC) were obtainedfrom Merck (Darmstad, Germany).

2.2. Synthesis of valproyl-CoA, valproyl-dephosphoCoA, Δ4-valproicacid and Δ4-valproyl-CoA

Valproyl-CoA, valproyl-dephosphoCoA (valproyl-dephCoA) and Δ4-val-proyl-CoA were synthesized according to published procedures [29,30] fromVPA and Δ4-VPA, respectively. Δ4-Valproic acid was obtained by chemicalsynthesis following a reported procedure [31].

2.3. Animals

The study was conducted according to the National Guidelines for the careand use of laboratory animals (Faculty of Pharmacy animals laboratory). MaleWistar rats weighing about 100 g were kept under a standard diet with ad libitumaccess to food and water. Rats were allocated to either a single in vivo or achronic 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 sodiumvalproate (100 mg/kg) solution in 0.9% NaCl; (b) group 2 (n=5) was treateddaily 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 wereremoved and immediately rinsed into ice-cold homogenization medium (SEM:250 mM Sucrose, 0.5 mM EGTA, 5 mM MOPS, pH=7.4). After mincing andtwo to three washings, the chopped liver was homogenized in a precooled Teflonpestle glass homogenizer. The homogenate was centrifuged at 600×g (10 min,4 °C) and the obtained post-nuclear supernatant was further centrifuged at

Fig. 1. HPLC profiles of (A) purified standards; (B) mitochondria prepared fromrat liver after in vivo treatment with one single dose of VPA. (Peak identification:1-Valproyl-CoA; 2-Valproyl-dephCoA).

Fig. 2. Identification of Valproyl-CoA (peak 1) in (A) cytosolic fraction obtainedfrom control rat liver cells; (B) cytosolic fraction of rat liver cells after in vivotreatment with one single dose of VPA and (C) previous sample after spikingwith 2 μM of synthetic standard.

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3600×g (10 min, 4 °C). The pellet obtained was suspended and after one lastround of centrifugation at 2700×g (10 min, 4 °C), the mitochondria were finallyresuspended in the homogenization medium (25–50 mg/mL). The finalsupernatant was purified with a new centrifugation step at 105,000×g (1 h,4 °C) using a SW-41Ti rotor (Beckman Instruments), yielding the cytosolicfraction (supernatant) and a microsomal fraction (pellet).

Protein concentration of cellular fractions was determined using the BCAassay [32] with bovine serum albumin as a standard. Samples of mitochondrialand cytosolic fractions were diluted to a protein content of 20 mg/mL in SEMbuffer.

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 °Cwith Krebs–Ringer buffer according to established procedures. Hepatocyteswere isolated essentially as described by Berry and Friend [33]. The isolatedcells were washed and resuspended in SEM buffer (protein 20 mg/mL) and usedfor further in vitro incubations with VPA, which were performed at 37 °C in afinal volume of 250 μL using a shaking water bath. The reactions were started byaddition of VPA (0.5 and 1 mM, pH 7.4) and were stopped after 1 h by theaddition of 250 μL ice-cold digitonin (400 μg/mL, Calbiochem.). After 10 minat 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 PCAwhilein the supernatant (soluble fraction) the proteins were precipitated by theaddition of 20 μL of 11.8 M PCA. After 5 min on iced water, both fractions wereneutralized with 2 M KOH. To remove the precipitated protein, the fractionswere centrifuged at 17,000×g (5 min at 4 °C), and the supernatants weretransferred to HPLC vials.

2.6. Studies in vitro on the valproic acid activation

The characterization of the putative valproyl-CoA synthetase activity wasperformed in vitro by measuring the conversion of VPA into valproyl-CoA,using HPLC analysis as described below. The obtained mitochondria weresonicated (2×10 s; 8 W) prior to incubation.

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 of1 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 MHCl with vortexing. The samples were neutralized with 10 μL 2 M KOH/0.6 MMES and centrifuged at 17,000×g (5 min, 4 °C). The resulting supernatants weretransferred 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 thepresence 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 incubatedwith VPA (0–10 mM) for 60 min at 37 °C as described above, using 100 mMTris buffer at pH 9.0, 5 mM ATP, 5 mM CoA and 5 mM MgCl2. The obtainedsupernatants were analysed using HPLC.

Product formation was also determined as a function of CoA and ATPconcentration up to 10 mM and 20 mM respectively for both cofactors. A mixedsolution of ATP and MgCl2 in a 1:1 ratio was used in this case and incubationswere performed at 37 °C for 60 min, with 3.0 mg/mL protein, 5 mM VPA and100 mM Tris buffer (pH=9.0).

The formation of valproyl-CoA from VPA was also tested in microsomalfractions (20 mg/mL) using analogous conditions as described for mitochondria(2 mMVPA, 1 mMATP, 1 mM CoA, 1 mMMgCl2, potassium phosphate bufferat pH 7.4, for 5 and 10 min at 37 °C).

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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 theformation of valproyl-CoA. The samples (3.0 mg/mL) were incubated for60 min, 37 °C, with 5 mM VPA, CoA and ATP:MgCl2 (1:1) using 100 mM Trisbuffer pH 9.0, in the presence and absence of each MCFA (5 mM). The obtaineddeproteinized supernatants were analysed by HPLC.

In all studies, the valproyl-CoA formed in the different incubations wasquantified by means of an external calibration curve previously performed withthe purified compound.

2.7. Studies in vitro on the Δ4-valproic acid activation

Rat liver subcellular fractions were obtained as described above, from thecontrol group of animals. Mitochondrial fractions suspended in SEM bufferwere sonicated (2×10 s; 8 W). Samples were incubated in a standard reactionmedium 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 reactionvolume of 200 μL). The reactions were stopped by adding 20 μL of 2 M HClwith 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 supernatantstransferred to microvials in order to analyse the formation of Δ4-valproyl-CoAusing HPLC.

In all studies, the Δ4-valproyl-CoA formed in the different incubations wasquantified by means of an external calibration curve previously performed withthe purified compound.

2.8. Analysis of VPA acyl-CoA esters using HPLC

Analysis of VPA acyl-CoA and valproyl-dephCoA esters was performedaccording to published methodology [29,30]. Basically two different HPLCsystems were used in the present study.

Fig. 3. HPLC profile of VPA acyl-CoA esters: (A) in the organelle fraction obtained frsame organelle sample after spiking with the purified standards; (C) in the respectivestandards. (Peak identification: 1-Valproyl-CoA; 2-Valproyl-dephCoA).

(a) Analysis of mitochondrial and cytosolic fractions obtained from livers ofVPA treated rats and controls: samples of subcellular fractions were acidifiedwith 50 μl of 2 M HCl in a vortex mixer and protein was removed by centrifuga-tion at 17,000×g (5 min, 4 °C) in an Eppendorf centrifuge. The supernatant wasneutralized with 50 μl of 2 MKOH/0.6 MMES during mixing on a vortex. Afterremoving 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 solventdelivery system. The samples (100 μL) were introduced by an autosamplerinjector (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 WatersMillennium32 chromatography manager software.

Resolution of the VPA acyl-CoA thioesters (valproyl-CoA, valproyl-dephCoA, Δ2-valproyl-CoA, Δ4-valproyl-CoA, 3-OH-valproyl-CoA and3-keto-valproyl-CoA) was achieved by linearly increasing the acetonitrilecontent of the 16.9 mM sodium phosphate (pH 6.9) elution buffer from 10% to45% in 20 min at a flow rate of 1 mL/min. The column was washed for 5 min at70% of acetonitrile and then reequilibrated for 15 min under the startingconditions in a total runtime of 42min. Acyl-CoA esters were detected with a UVdetector (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 HPLCpump system from Perkin Elmer (series 200 PUMP) was used. The sampleswere injected with a Gilson 234 automatic injector on a Supelcosil LC-18-DBcolumn (5 μm, 250 mm, 4.6 mm) and the runs controlled with the Chromeleonchromatografic software, from Dionex.

Resolution of the VPA acyl-CoA thioesters was achieved by linearlyincreasing 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. Thecolumn was washed for 5 min at 70% of acetonitrile and then reequilibrated for7 min under the starting conditions in a total runtime of 30 min. Acyl-CoA esterswere detected with a UV detector (Shimadzu, SPD-10A; λ=260 nm).

om rat hepatocytes incubated in vitro for 1 h at 37 °C with 1 mMVPA; (B) in thesoluble fraction; (D) in the same soluble sample after spiking with the purified

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2.9. Enzyme assay

Citrate synthase activity (CS; EC 4.1.3.7) was measured on a Cobas-Faracentrifugal analyzer (Hoffmann-La Roche) monitoring the CoA formation usinga coupled reaction with DTNB, essentially as described byHaggie et al. [34]. Thereaction was started by adding 0.2 mM oxaloacetate in an incubation mixturecontaining 100 mM Tris/HCl pH 7.6, 100 mM KCl, 0.1 mM DTNB, 0.2 mMacetyl-CoA, 1 g/L Triton X-100 in a final volume of 250 μL. Incubations werecarried out at 37 °C and an assay without the substrate was used as blank. Thecitrate 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 wasdetermined 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 per-formed by plotting the measured reaction rates as function of substrate concen-tration (at fixed concentration of the remaining components of the reaction).

The steady state kinetic data were analysed by nonlinear regression analysisusing the SigmaPlot® v10.0 Technical Graphing Software and the EnzymeKinetics module v1.3.

The Michaelis–Menten equation [35] was used to calculate kinetic para-meters (Km and Vmax) of the enzyme reaction involving VPA,Δ4-VPA, CoA andATP. The results shown in Fig. 7 represent the estimated mean (± standarddeviation) of an apparent Km (Km

app) obtained of at least three independentsubstrate dependence curves. Values were compared by Student's t test (un-paired, two-tailed, unequal variance) and a statistical significance was defined asp<0.05.

Fig. 4. Effect of (A) pH, (B) protein and the incubation time on the formationrate of valproyl-CoA in vitro in cytosol. (The represented values of valproyl-CoA were obtained with HPLC analysis and UV detection of (A) cytosolicsamples (0.3 mg) of a control rat liver, incubated with 5 mM VPA, 5 mM CoA,5 mM ATP and 5 mMMgCl2 (37 °C / 60 min) using different pH buffers at finalconcentration of 100 mM; (B) cytosolic samples of a control rat liver, incubatedwith 5 mMVPA, 5 mMCoA, 5 mMATP and 5 mMMgCl2, 100 mM Tris bufferpH=9 at 37 °C. The represented lines were obtained by linear regressionconsidering the values obtained at 20, 40 and 60 min: r2=0.993 and r2=0.992for 0.5 and 3.5 mg/mL of protein, respectively).

3. Results

3.1. Formation of valproyl-CoA and valproyl-dephCoA(studies ex vivo)

As depicted in Fig. 1B, both valproyl-CoA and valproyl-dephCoA were detected in liver mitochondria of rats (RLM)treated with one single dose of VPA (group 1). A virtuallyidentical profile was observed for all animals of the same group.Valproyl-CoA and the respective dephosphoCoA ester wereonly detected in RLM samples of two animals of group 2 thatwere subjected to chronic valproic acid treatment (2/5). Inuntreated rats (group 3) no corresponding peaks were detected.The analysis of the cytosolic fractions (Fig. 2B) revealed thepresence of valproyl-CoA in both groups of VPA-treated rats(groups 1 and 2), although in quite low concentrations. It must beemphasized that the cytosolic fraction was diluted in SEMduring isolation, which stresses the significance of this tracepresence of conjugates. The identification of valproyl-CoA andvalproyl-dephCoA was performed either by comparing theretention times with those of the synthetic CoA esters (Fig. 1A)or by the spiking of samples with the respective purifiedstandards (Fig. 2C). No VPA activation was observed inmicrosomes.

3.2. Formation of valproyl-CoA and valproyl-dephCoA(studies in vitro in rat hepatocytes)

A second experimental approach was used in order to obtainadditional evidence for the in vivo formation of valproyl-CoA

and valproyl-dephCoA in the two subcellular compartments. Asexpected, Fig. 3A clearly shows the formation of valproyl-CoAin the organelle fraction, containing mitochondria, peroxisomes,etc., obtained after digitonin fractionation of rat hepatocytesincubated in vitro with VPA. The presence in the same chroma-togram of valproyl-dephCoA indicates that the dephosphoryla-tion of valproyl-CoA also occurs in hepatocytes [30]. Theanalysis of the cytosolic fraction revealed traces of valproyl-CoA (Fig. 3C).

3.3. Kinetics of the VPA activation to valproyl-CoA

The activation of VPA to valproyl-CoA was determined invitro using mitochondria and cytosolic samples prepared fromcontrol rats, using differential centrifugation methods. Firstly,the pH optimum of the enzymatic reaction was studied. Fig. 4Arepresents the obtained pH curve of the cytosolic VPA acti-vating enzyme, which shows that the enzyme has a maximum

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activity at pH=9.0. Subsequent experiments concerning theenzyme characterization were thus always performed at pH9.0.

Formation of valproyl-CoA was found to be linear asfunction of the protein concentration of the cytosolic rat liversample (up to 3.5 mg/mL) and as function of time at 37 °C (upto 60 min), (Fig. 4B). Further experiments were performedusing a protein content of 3.0 mg/mL and a reaction time of60 min.

In order to study the affinity of the enzyme towards specificsubstrates, both mitochondrial and cytosolic samples wereincubated with increasing concentrations of VPA (or Δ4-VPA)and the cofactors CoA and ATP (Figs. 5 and 6 respectively). Foreach studied substrate at least three independent curves wereobtained.

Application of adequate computer software to the experi-mental results provided an estimate of kinetic constants and themean of the apparent Km (Km

app) and respective standarddeviation are represented in Fig. 7. These values provide anapproximation of the catalytic activity of the enzyme orenzymes within the two cellular compartments. The significantdifferences (p<0.05), observed in the values of the apparent Km

in mitochondria and cytosol for CoA and ATP, substantiate thehypothesis that the cytosolic enzyme which converts VPA tovalproyl-CoA is different from the mitochondrial one. In

Fig. 5. The initial reaction rate of valproyl-CoA synthetase activity was determined inof increasing concentrations of the substrates (0–10 mM): VPA (1A and 2A); Δ4-VP

addition, higher concentrations of ATP were found to inhibitvalproyl-CoA formation both in the cytosol as well as inmitochondria (see Fig. 6: 1B and 2B).

Subsequently we studied whether VPA competes with otherMCFA, like hexanoic acid and phenylpropionic acid, for bothmitochondrial and cytosolic CoA-synthesizing enzymes. Theamount of valproyl-CoA formed in vitro when VPA wasincubated simultaneously with hexanoic acid (Fig. 8: A-II andB-II) was clearly lower as compared with the amount of productresulting from the incubation with VPA alone, in both fractions(Fig. 8: A-I and B-I). Similar results were obtained withphenylpropionic acid (plus or minus VPA) and for this reasonthe chromatograms of in vitro incubations with this acid are notshown.

3.4. Citrate Synthase (CS) activity in RLM and cytosol

In order to establish that the valproyl-CoA synthetaseactivity measured in cytosolic fractions prepared from ratlivers by differential centrifugation is not due to leakage ofvalproyl-CoA present in mitochondria, we measured theactivity of the mitochondrial marker enzyme citrate synthase(CS) in mitochondrial and cytosolic fractions, respectively.The activity of CS as measured in mitochondrial and cyto-solic samples amounted to 1902±77 U/μL and 33±1.2 U/μL,

vitro either in rat liver mitochondria (1A, 1B) or in cytosol (2A, 2B) as functionA (1B and 2B). (Experimental details are described in Materials and methods.)

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 functionof 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.)

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respectively, whereas the activities of valproyl-CoA synthe-tase in the two fractions were 2.82 U/μL and 0.36 U/μL,respectively.

Fig. 7. Estimate of the apparent Km (Kmapp) 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 leastthree independent experiments and respective standard deviation. The reactionrate was measured as the amount of valproyl-CoA formed (nmol/min.mg) asfunction of the substrate concentration. The concentration of each substrate wasvaried 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 mMVPA (CoA variable). Significant differences between represented values:*p<0.05 and **p<0.001).

The results clearly show that the valproyl-CoA synthetaseactivity as measured in the cytosolic fraction is only partly(11%) due to leakage of valproyl-CoA synthetase from brokenmitochondria.

3.5. Studies in vitro on the Δ4-VPA activation toΔ4-valproyl-CoA

Since VPAwas clearly activated to valproyl-CoA in the cellcytosol, it remained to be established whether metabolites ofVPA would undergo a similar conjugation with CoA. For thispurpose we investigated the formation of Δ4-valproyl-CoAfrom Δ4-VPA, the main microsomal metabolite formed bydesaturation of VPA.

Fig. 9 demonstrates that activation of Δ4-VPA to Δ4-valproyl-CoA clearly occurs in the cytosolic fraction. Interest-ingly, our results show that Δ4-valproyl-CoA is also formed inmitochondrial fractions (Fig. 5: 1B and 2B) suggesting that β-oxidation may be the strategy of further metabolism of certainmicrosomal acidic metabolites. Estimated Km

app values for Δ4-VPA are 0.28 mM in mitochondria and 0.07 mM in cytosol(using 5 mM ATP and 5 mM CoA). These data suggest that inthe cytosolic fraction there is a higher affinity of the enzyme forΔ4-VPA (low Km

app) than in mitochondria, although similar effi-ciency of catalysis (similar Vmax

app ) is attained in both subcellular

Fig. 8. HPLC profiles of (A) mitochondrial in vitro incubations with 5 mMVPA (A-I), 5 mMVPA 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-Valproyl-CoA; 3-Hexanoyl-CoA).

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compartments (0.02 and 0.03 nmol/min. mg for mitochondriaand cytosol, respectively).

4. Discussion

Mitochondrial dysfunction has been recognised as animportant mechanism of drug-induced liver injury [36,37].The VPA-induced inhibition of mitochondrial fatty acid β-oxidation [38] has sparked our renewed interest in the acyl-CoAmetabolism. In fact, the formation of xenobiotic-CoA esters mayoccur, either as a detoxifying pathway, or as a pathway gene-rating reactive metabolites and a cascade of secondary effects.

We and others have previously shown that VPA is activatedin vitro to valproyl-CoA in mitochondria. The primary results

described in this report show the presence of valproyl-CoA inmitochondria of liver cells isolated from rats subjected to VPAtreatment, as expected. Surprisingly, trace amounts of thismetabolite were also found in the cytosol of these cells. Thisresult was not a consequence of leakage from mitochondriaduring the homogenization procedure, since this finding wasconfirmed by another cellular model, in digitonin-fractionatedrat hepatocytes treated in vitro with VPA. In addition, valproyl-dephosphoCoA was also detected in these cells, a result thatconfirms our previous data obtained with isolated mitochondria[30].

The presence of two distinct valproyl-CoA pools clearlysuggests that the VPA activation reaction occurs in two differentsubcellular compartments, which lead to the investigation whe-

Fig. 9. Identification of Δ4-valproyl-CoA (Peak 4) in (A) the cytosolic fractionof rat liver cells incubated in vitro with 2 mMΔ4VPA (plus cofactors); (B) samesample after spiking with 2 μM of synthetic standard. (C) Blank (Δt=0).

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ther acyl-CoA derivates of VPA can be formed before enteringmitochondria. Indeed, our results also show the formation ofvalproyl-CoA in vitro, after incubation of both mitochondriaand cytosolic fractions with VPA and cofactors. Importantly,only 11% of the extra-mitochondrial valproyl-CoA synthetaseactivity can be attributed to mitochondrial contamination, ascalculated by the study of the mitochondrial marker enzymecitrate synthase. Furthermore, valproyl-CoAwas not detected inmicrosomal fractions after in vitro incubation with VPA, which

points to absent interference of microsomal proteins with thepresent results. Taken together, the results clearly suggest twodifferent subcellular locations for the activation of VPA: onein the mitochondria and another in the extra-mitochondrialspace.

The mitochondrial ACS and the extra-mitochondrial VPAactivating enzyme were characterized by monitoring the val-proyl-CoA formation. The study of the enzyme affinity to thesubstrate and cofactors (CoA and ATP) enabled the estimate ofthe respective apparent Km in both mitochondrial and cytosolicfractions. These values provide an approximation of thesubstrate concentration range yielding control of the enzymicactivity. Moreover, the significant differences obtained in theapparent Km values for CoA and ATP in both cellular com-partments suggest the existence of two different enzymes ineach fraction capable of activating VPA (and possibly Δ4-VPA)into the respective CoA esters.

Mitochondrial β-oxidation of VPA is the second mostimportant route of its biotransformation in man after glucur-onidation [21]. The CoA thioester intermediates formed in thisprocess constitute the biologically active metabolites andvalproyl-CoA is the first metabolite of the overall oxidativepathway, being mainly formed in the mitochondrial matrix [28].Thus, the passive diffusion of the free acid across mitochondriais most probably the predominant pathway. However, the datareported here are suggestive of a second VPA activation stepduring cellular trafficking (possibly in cytosol) before reachingmitochondria. The formation of the acyl-CoA esters seems to beextended to other microsomal acidic metabolites of VPA, asdemonstrated for the in vitro activation of Δ4-VPA to Δ4-val-proyl-CoA in the cytosol, a reaction that has not been reportedbefore in literature.

The recognition of the extra-mitochondrial activation of VPAnext to the intra-mitochondrial activation of VPA has conse-quences for the biotransformation of the drug. In mitochondria,valproyl-CoA can be further metabolized byβ-oxidation [2], butthe metabolic fate in the extra-mitochondrial fraction is still un-known. The valproyl-CoA as formed in the cytosol may enter themitochondria via the carnitine shuttle (carnitine palmitoyl-trans-ferase I (CPT I), carnitine-acylcarnitine translocase (CACT) andcarnitine palmitoyl-transferase II (CPT II)). This pathway iscurrently under investigation.

Secondary effects, like the trapping of CoA in mitochondriaand/or extra-mitochondrial compartments have different con-sequences in the cell. Eukaryotic cells contain sequestered poolsof CoA that are crucial for energy metabolism, namely for thepriming or activation of fatty acids, forming CoA derivatives inan ATP-dependent manner. Cytosolic concentrations of CoAvary between 0.02 and 0.14 mM in animal tissues, values thatare significantly lower than mitochondrial concentrations whichrange from 2.2 to over 5.0 mM [39]. Tissue levels of CoA arealso quite variable, the liver being one of the richest. However,hepatic CoA levels are among the most responsive to changes inseveral metabolic states [39] and therefore the extensive hepaticbiotransformation of VPA will certainly influence CoA regu-lation and intermediary metabolism. For instance, this drug mayinterfere with the activation of endogenous LCFA, mainly by

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CoA trapping, an effect that would account to the decreasedoxidation rate of these fatty acids induced by the drug [38].

The formation of these VPA acyl-CoA esters both inmitochondria and extra-mitochondrial compartments can alsohave different potential inhibitory targets. For instance, theextra-mitochondrial activation to valproyl-CoA could poten-tially have consequences by interference with fatty-acid elon-gation, peroxisomal metabolism, glyconeogenesis, ureagenesisand many other pathways involving cytosolic reactions.

The increasing recognition that formation of a CoA conju-gate increases chemical reactivity of a xenobiotic carboxylicacid [6,8] reinforces the absolute need to further investigatethe differences in toxicity of Δ4-valproyl-CoA in relation toΔ4-VPA.

From the present findings we expect that additional mecha-nisms of mitochondrial dysfunction associated with valproicacid will be clarified. This knowledge may contribute to thefurther understanding of the toxic effects associated with thisdrug, mainly having a broader perspective of the extra-mito-chondrial mechanisms.

Acknowledgments

This work was financially supported by Fundação para aCiência e a Tecnologia (FCT), Lisboa, Portugal (POCTI/FCB/48800/2002 with partial funding of FEDER and SFRH/BD/22420/2005).

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