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Neurological damage or changes to the brain as a result of bipolar disorder

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Psychopharmacology (2006) 184: 122-129
DOI 10.1007/s00213-005-0272-4
Richard P. Bazinet . Margaret T. Weis .
Stanley I. Rapoport . Thad A. Rosenberger
Valproic acid selectively inhibits conversion of arachidonic acid
to arachidonoyl-CoA by brain microsomal long-chain fatty
acyl-CoA synthetases: relevance to bipolar disorder
Received: 31 August 2005 / Accepted: 10 November 2005 / Published online: 13 December 2005
# Springer-Verlag 2005
Abstract Rationale: Several drugs used to treat bipolar
disorder (lithium and carbamazepine), when administered
chronically to rats, reduce the turnover of arachidonic acid,
but not docosahexaenoic acid, in brain phospholipids by
decreasing the activity of an arachidonic acid-selective phospholipase
A2. Although chronic valproic acid produces
similar effects on brain arachidonic acid and docosahexaenoic
acid turnover, it does not alter phospholipase A2
activity, suggesting that it targets a different enzyme in the
turnover pathway. Materials and methods/Results: By isolating
rat brain microsomal long-chain fatty acyl-CoA
synthetases (Acsl), we show in vitro that valproic acid is a
non-competitive inhibitor of Acsl, as it reduces the maximal
velocity of the reaction without changing the affinity
of the substrate for the enzyme. While valproic acid inhibited
the synthesis of arachidonoyl-CoA, palmitoyl-
CoA, and docosahexaenoyl-CoA, the Kifor inhibition of
arachidonoyl-CoA synthesis (14.1 mM) was approximately
one fifth the Ki for inhibiting palmitoyl-CoA
(85.4 mM) and docosahexaenoyl-CoA (78.2 mM) synthesis.
As chronic administration of valproic acid in bipolar
disorder achieves whole-brain levels of 1.0 to 1.5 mM,
inhibition of arachidonoyl-CoA formation can occur at
brain concentrations that are therapeutically relevant to
this disease. Furthermore, brain microsomal Acsl did not
produce valproyl-CoA. Conclusions: This study shows that
valproic acid acts as a non-competitive inhibitor of brain
microsomal Acsl, and that inhibition is substrate-selective.
The study supports the hypothesis that valproic acid acts in
bipolar disorder by reducing the brain arachidonic acid
cascade, by inhibiting arachidonoyl-CoA formation.
Keywords Valproate . Bipolar disorder . Arachidonic
acid . Acyl-CoA synthetase . Docosahexaenoic acid .
Valproic acid (2-propylpentanoic acid) has been used for
more than 40 years to treat generalized epilepsy and, more
recently, bipolar disorder (Calabrese et al. 1995; Loscher
1985; Meunier et al. 1963; Pope et al. 1991). However, its
mechanisms of action are not fully understood. Increases in
GABAergic neurotransmission (Johannessen 2000; Loscher
1993), inhibition of neuron growth cone collapse (Williams
et al. 2002), interference with brain energy and lipid
metabolism (Bolanos and Medina 1997; Friel 1990), and
alteration of membrane order through esterification into
brain lipids (Siafaka-Kapadai et al. 1998) have all been
proposed to explain its therapeutic effects.
Valproic acid can inhibit the β-oxidation of fatty acids
by up to 94% in rat liver, through mechanisms that involve
either coenzyme A (CoA) sequestration or direct inhibition
of β-oxidizing enzymes (Becker and Harris 1983; Silva et
al. 2001). Studies of cultured neurons exposed to radiolabeled
valproic acid suggested that it is esterified into
membrane phospholipids (Siafaka-Kapadai et al. 1998),
indirectly implying the formation of valproyl-CoA, because
a fatty acid can be esterified only after it is activated
by an acyl-CoA synthetase (Yamashita et al. 1997). However,
this study measured only radioactivity in lipid-extractable
material, without confirming that radioactive
valproic acid could be recovered from phospholipid. A sub-
R. P. Bazinet . S. I. Rapoport . T. A. Rosenberger
Brain Physiology and Metabolism Section,
National Institute on Aging,
National Institutes of Health,
Bethesda, MD 20892, USA
M. T. Weis
Department of Pharmaceutical Sciences,
School of Pharmacy,
Texas Tech University Health Science Center,
Amarillo, TX 79106, USA
T. A. Rosenberger (*)
Department of Pharmacology, Physiology, and Therapeutics,
School of Medicine and Health Sciences,
University of North Dakota,
501 North Columbia Road, Rm. 3742A,
Grand Forks, ND 58203, USA
Tel.: +1-701-7770591
Fax: +1-701-7774490
sequent study from our laboratory failed to find valproyl-
CoA or esterified valproic acid in the brain of rats following
chronic administration of the drug, at detection
limits of 25 and 37.5 pmol/g brain, respectively (Deutsch
et al. 2003).
Drugs effective against bipolar disorder, such as lithium
carbamazepine and valproic acid, have therapeutic and
biochemical overlap, which may indicate a common mechanism
of action. When administered chronically to produce
therapeutically relevant plasma and brain levels in rats,
they reduce incorporation rates and turnover of arachidonic
acid (20:4n−6) in brain phospholipids. Reductions in turnover
were 80% by lithium, 33% by valproic acid, and 30%
by carbamazepine (Bazinet et al. 2005b; Chang et al. 2001;
Chang et al. 1996). In contrast, none of these drugs modifies
the incorporation or turnover of docosahexaenoic acid
(22:6n−3) in brain phospholipids (Bazinet et al. 2005b,c;
Chang et al. 1999).
Arachidonic acid turnover in brain phospholipids can
be modified by three critical enzyme-catalyzed reactions
(Lands and Crawford 1975). These involve arachidonic
acid release from phospholipids catalyzed by a phospholipase
A2 (PLA2) (Rapoport et al. 2001), activation of
arachidonoyl-CoA, catalyzed by long-chain fatty acyl-
CoA synthetase (Acsl) (Harris and Stahl 1983; Robinson et
al. 1992), and transfer of the arachidonoyl-CoA to lysophospholipid,
catalyzed by acyl-CoA transferase
(Yamashita et al. 1997). Both lithium and carbamazepine
likely decrease brain arachidonic acid incorporation
and turnover in phospholipids by decreasing the gene expression
and activity of arachidonic-acid-specific calciumdependent
cytosolic PLA2 (cPLA2), sparing secretory
PLA2(sPLA2) and docosahexaenoic acid-selective calcium-
independent PLA2 (iPLA2) (Ghelardoni et al. 2004;
Rintala et al. 1999). In contrast, valproic acid does not alter
the expression of any PLA2 enzymes (Bosetti et al. 2003;
Chang et al. 2001). Therefore, its downregulation of arachidonic
acid incorporation and turnover might be explained
by reduced synthesis of arachidonoyl-CoA by Acsl (vide
supra) (Mashek et al. 2004), as reducing the rate of any step
in the cyclic series of reactions will downregulate the cycle as
a whole.
Chronic administration of valproic acid to rats decreases
brain levels of CoA, supporting the hypothesis that acyl-
CoA metabolism may be a therapeutic target of valproic
acid (Deutsch et al. 2003). In a learned-helplessness animal
model of depression, brain mRNA levels of Acsl2 are
increased (Setnik and Nobrega 2004), while arachidonic
acid levels are elevated in the Flinders Sensitive Line rat
model of depression (Green et al. 2005), suggesting that
this enzyme and the regulation of arachidonic acid incorporation
and turnover in phospholipids may be involved
in mood regulation. To see if acyl-CoA synthetase is a
therapeutic target of valproic acid, and to further rule out
direct esterification of valproic acid into membrane phospholipids,
we measured activities in two fractions of brain
microsomal Acsl in rats treated with valproic acid (200 mg/kg)
for 30 days. This dosing regimen (approximately tenfold
higher than for humans) produces a plasma concentration
of valproic acid (31 μg/ml) in the rat, which is close the
human therapeutic plasma concentration effective in bipolar
disorder (32.5 μg/ml), and decreases the turnover of
arachidonic acid but not of docosahexaenoic acid in brain
phospholipids of the unanesthetized rat (Bazinet et al.
2005c; Chang et al. 2001; Jacobsen 1993). Furthermore,
the ability of valproic acid to inhibit two brain microsomal
Acsl as described previously (Laposata et al. 1985) was
determined in vitro. Identifying the enzymes by which
valproic acid reduces brain arachidonic acid turnover may
aid in understanding the pathogenesis of bipolar disorder
and in developing more effective treatments for it. An
abstract of part of this work has been published (Bazinet
et al. 2005a).
Materials and methods
Reagents [1-14C]Arachidonic acid (50 mCi/mmol), [1-14C]
docosahexaenoic acid (56 mCi/mmol), [1-14C]palmitic
acid (16:0) (56 mCi/mmol), and [4,5-3H]valproic acid
(55 Ci/mmol) were purchased from Moravek Biochemicals
(Brea, CA). Non-esterified arachidonic and docosahexae-
Fig. 1 Solubilized microsomal long-chain fatty acyl-CoA synthetase
(Acsl) activity elution profiles from valproic acid-treated (solid
line) and control (dashed line) rats. Assay substrates are a palmitic
acid, b arachidonic acid, and c docosahexaenoic acid. Data represent
the means of three animals per group
noic acids were purchased from Nu-Chek-Prep (Elysian,
MN). Reagent-grade palmitic and valproic acids, potassium
phosphate, ATP, and other chemical reagents were
from Sigma (St. Louis, MO). HPLC-grade 2 propanol and
n-heptane were from EMD chemicals (Gibbstown, NJ)
and sulfuric acid was purchased from Aldrich Chemicals
(Milwaukee, WI).
Animals The study was conducted according to the National
Institutes of Health Guidelines for the Care and Use of
Laboratory Animals (Publication no. 80-23), and was approved
by the National Institute of Child Health and
Development Animal Care and Use Committee. Male CDF-
344 rats, weighing 180-200 g (Charles River; Wilmington,
MA), were acclimatized for 1 week to controlled temperature
(22°C), humidity (25%), and light cycle (12-h
light/12-h dark) with ad libitum access to food (NIH-31
diet) and water. Rats were allocated to either a chronic in
vivo or an in vitro study group. Animals in the chronic in
vivo study were further assigned to either valproic-acidtreated
or control groups (saline-treated). Valproic-acidtreated
rats received 200 mg/kg valproic acid in 0.9%
saline i.p. for 30 days, as described (Chang et al. 2001).
Control rats received an equal volume of 0.9% saline. This
valproic acid dosing regimen has been shown to reduce
brain cyclooxygenase activity levels, PGE2 concentrations,
and turnover of arachidonic acid, but not of
docosahexaenoic acid in phospholipids of the awake rat
(Bosetti et al. 2003; Bazinet et al. 2005c; Chang et al.
2001). Each animal was anesthetized with sodium pentobarbital
(50 mg/kg, i.p.) and decapitated, and its brain was
removed and frozen in −40°C 2-methyl butane. Samples
were stored at −80°C until use.
Enzyme preparation Microsomal Acsl activity was measured
as reported (Wilson et al. 1982). Frozen whole brain
was homogenized in sucrose HEPES TRIS buffer, pH 7.4,
composed of 320 mM sucrose, 10 mM HEPES (pH adjusted
to 7.4 with 1 M TRIS base), and 20 μg/ml phenylmethylsulfonyl
fluoride, 1 mM benzamidine, 10 mM 2-
mercaptoethanol, and 0.01% soybean trypsin inhibitor.
The homogenate was centrifuged at 1,000×g for 10 min at
4°C to remove cell debris. The microsomal membranes
were isolated from the supernatant by centrifugation at
35,000×g at 4°C for 1 h. The pellets were solubilized by
re-suspension in 20 mM potassium phosphate buffer
(pH 7.4) containing 1% IGEPAL CA-630 detergent, 10 mM
EDTA, and 10 mM 2-mercaptoethanol, with stirring at
4°C for 2 h. Insoluble materials were removed by a second
centrifugation at 35,000×g at 4°C for 1 h. The Acsl activities
were separated on a 10-ml bed volume of Spectra/
Gel HA hydroxyapatite (Spectrum, Los Angeles, CA)
equilibrated with 20 mM potassium phosphate buffer
using a step gradient of 20 mM potassium phosphate,
80 mM potassium phosphate, and 300 mM potassium
phosphate, as described previously (Laposata et al. 1985).
All chromatography buffers had a pH of 7.4 and contained
1% IGEPAL CA-630 detergent and 10 mM 2-mercaptoethanol.
The column eluate was collected in 1.75-ml fractions
and each fraction was assayed for enzyme activity as
described below. The protein content was measured by the
method of Bradford (1976) and the enzyme preparations
were stored at −80°C until assay.
Assay of long-chain fatty acyl-CoA synthetase Acsl activity
was measured as described previously (Saunders et
al. 1996; Wilson et al. 1982). Briefly, 50 μl of enzyme
preparation was added to 100 μl of an assay cocktail
having a final concentration of 100 mM Tris/HCl (pH 8.0),
2 mM Triton X-100, 0.55 mM CoA, 6.6 mM ATP, 2.5 mM
MgCl2, 10 μM radiolabeled (14C or 3H) fatty acid and
increasing concentrations of unlabeled fatty acids (0 to
120 μM). The reactions were allowed to proceed for 10 or
15 min at 37°C in a shaking water bath, and were terminated
by adding 2.25 ml of 2-propanol with n-heptane
and 2 M H2SO4 (40:10:1, by volume). Non-esterified
Table 1 Acyl-CoA synthesis
rates in rat solubilized microsomal
brain peaks 1 and 2
Data are means±SD of triplicate
analysis as described in the
Materials and methods
Brain peak 1 Brain peak 2
Substrate (pmol min−1 μg−1 protein)
Palmitic acid (16:0) 1.57±0.34 3.30±0.814
Arachidonic acid (20:4n−6) 0.727±0.224 2.27±0.819
Docosahexaenoic acid (22:6n−3) 0.581±0.121 1.06±0.381
Valproic acid 0 0
Table 2 Km and Vmax measured rat solublized microsomal brain peaks 1 and 2 in control and rats chronically treated with valproic acid,
using [14C] arachidonic acid as substrate
Brain peak 1 Brain peak 2
Control Valproic acid Control Valproic acid
Km (μM) 44.3±10.1 49.4±3.9 77.9±17.5 59.3±6.9
Vmax (nmol/min) 68.6±25.3 59.9±13.9 241±125 154±18.0
Km and Vmax were determined by fitting the data to the equation (v=Vmax[s]/(Km+[s]),where Vmax is the maximal velocity, [s]=a fixed
substrate concentration, and Km=the concentration of substrate at which the reaction is 1/2Vmax using arachidonic acid as a substrate. Data
are means±SD of triplicate analysis of n=3 rats per group
substrate was extracted from the reaction mixture with
1.5 ml n-heptane and 1 ml water. The aqueous layer was
re-extracted twice with 2 ml n-heptane containing 4 mg/ml
palmitic acid. Radioactivity in a 1-ml portion of the aqueous
layer was determined by liquid scintillation spectroscopy.
Initial reaction rates were calculated based on the
specific radioactivity of the substrate and are reported as
picomole of product formed per minute per microgram of
Analysis of results Data were plotted as velocity vs. substrate
for each fixed concentration of valproic acid, then
fitted to the hyperbolic model represented as (v=Vmax[s]/
(Km+[s])), where Vmax is the maximal velocity, [s]=a fixed
substrate concentration and Km=the concentration of substrate
at which the reaction rate is Vmax/2 (Segel 1975),
using GraphPad Prism version 4.00 (GraphPad Software,
San Diego, CA). Values for apparent Km and apparent
Vmax were replotted as a function of the valproic acid
concentration, and the enzyme inhibition constant (Ki) was
derived from the replots.
Statistics Data are presented as means±SD or 95% confidence
intervals. Comparisons between groups were made
using unpaired, two-tailed Student t-tests. Linear regression
analysis and all other calculations were made using
GraphPad Prism version 4.00 (GraphPad Software).
Elution profile of chronically treated rats Hydroxyapatite
elution profiles of brain microsomal Acsl activity (pmol/μg/
min), using 10 μM palmitic (a), 10 μM arachidonic (b), or
10 μM docosahexaenoic (c) acids as substrate, are presented
in Fig. 1. Two peaks of brain microsomal Acsl activity
were consistently detected in both the valproic-acid-treated
and control brains. The total enzymatic activities of the
brain Acsl using the different radiolabeled fatty acids are
outlined in Table 1. For a given substrate and peak of
activity, there was no difference in ...

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