Chronic clozapine reduces rat brain arachidonic acid metabolism by reducing plasma arachidonic acid availability

Authors

  • Hiren R. Modi,

    Corresponding author
    • Brain Physiology and Metabolism Section, National Institute on Aging, Laboratory of Neurosciences, National Institutes of Health, Bethesda, Maryland, USA
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    • Both authors contributed equally to this article.
  • Ameer Y. Taha,

    1. Brain Physiology and Metabolism Section, National Institute on Aging, Laboratory of Neurosciences, National Institutes of Health, Bethesda, Maryland, USA
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  • Hyung-Wook Kim,

    1. Brain Physiology and Metabolism Section, National Institute on Aging, Laboratory of Neurosciences, National Institutes of Health, Bethesda, Maryland, USA
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  • Lisa Chang,

    1. Brain Physiology and Metabolism Section, National Institute on Aging, Laboratory of Neurosciences, National Institutes of Health, Bethesda, Maryland, USA
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  • Stanley I. Rapoport,

    1. Brain Physiology and Metabolism Section, National Institute on Aging, Laboratory of Neurosciences, National Institutes of Health, Bethesda, Maryland, USA
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  • Yewon Cheon

    1. Brain Physiology and Metabolism Section, National Institute on Aging, Laboratory of Neurosciences, National Institutes of Health, Bethesda, Maryland, USA
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    • Both authors contributed equally to this article.

Errata

This article is corrected by:

  1. Errata: Corrigendum Volume 125, Issue 3, 486, Article first published online: 21 April 2013

Address correspondence and reprint requests to Hiren R. Modi, Brain Physiology and Metabolism Section, National Institute on Aging, National Institutes of Health, 9000 Rockville Pike, Bldg. 9, 1S126, Bethesda, MD 20892, USA. E-mail: modihr@mail.nih.gov

Abstract

Chronic administration of mood stabilizers to rats down-regulates the brain arachidonic acid (AA) cascade. This down-regulation may explain their efficacy against bipolar disorder (BD), in which brain AA cascade markers are elevated. The atypical antipsychotics, olanzapine (OLZ) and clozapine (CLZ), also act against BD. When given to rats, both reduce brain cyclooxygenase activity and prostaglandin E2 concentration; OLZ also reduces rat plasma unesterified and esterified AA concentrations, and AA incorporation and turnover in brain phospholipid. To test whether CLZ produces similar changes, we used our in vivo fatty acid method in rats given 10 mg/kg/day i.p. CLZ, or vehicle, for 30 days; or 1 day after CLZ washout. [1-14C]AA was infused intravenously for 5 min, arterial plasma was collected and high-energy microwaved brain was analyzed. CLZ increased incorporation coefficients inline image and rates Jin,i of plasma unesterified AA into brain phospholipids i, while decreasing plasma unesterified but not esterified AA. These effects disappeared after washout. Thus, CLZ and OLZ similarly down-regulated kinetics and cyclooxygenase expression of the brain AA cascade, likely by reducing plasma unesterified AA availability. Atypical antipsychotics and mood stabilizers may be therapeutic in BD by down-regulating, indirectly or directly respectively, the elevated brain AA cascade of that disease.

Abbreviations used
AA

arachidonic acid

AA-CoA

arachidonoyl-CoA

ChoGpl

choline glycerophospholipid

CLZ

clozapine

CLZ-W

clozapine with washout

COX

cyclooxygenase

cPLA2

cytosolic phospholipase A2

DHA

docosahexaenoic acid

EPA

eicosapentaenoic

EtnGpl

ethanolamine glycerophospholipid

FAME

fatty acid methyl esters

GC

gas chromatography

NMDA

N-methyl-D-aspartate

OLZ

olanzapine

PG

prostaglandin

PtdIns

phosphatidylinositol

PtdSer

phosphatidylserine

PUFA

polyunsaturated fatty acid

sn

stereospecifically numbered

sPLA2

secretory phospholipase A2

TLC

thin layer chromatography

Bipolar disorder (BD) is a progressive neuropsychiatric illness characterized by recurrent episodes of depression and mania (BD I) or hypomania (BD II) [reviewed in (Rapoport et al. 2010)]. It is treated with the mood stabilizers lithium, valproate, carbamazepine or lamotrigine (Greil et al. 1997; Bowden et al. 2000; Calabrese et al. 2003; Cipriani et al. 2010; Geddes et al. 2010), or with the atypical antipsychotic olanzapine (OLZ), all of which are FDA-approved (Bowden et al. 2000; Scherk et al. 2007). Another atypical antipsychotic, clozapine (CLZ), a tricyclic dibenzodiazepine, is not FDA-approved but has been reported effective in acute BD mania (Calabrese et al. 1996; Scherk et al. 2007), in rapid cycling and in patients with refractory BD (Zarate et al. 1995).

Studies in unanesthetized rats indicate that the chronically administered mood stabilizers selectively down-regulate various aspects of the brain arachidonic acid (AA, 20:4n-6) cascade (Chang et al. 1996, 2001; Bosetti et al. 2002, 2003; Ghelardoni et al. 2004; Bazinet et al. 2006; Shimshoni et al. 2011). As the cascade is up-regulated in the BD brain, in association with neuroinflammation, excitotoxicity, apoptosis, and synaptic loss (Kim et al. 2010, 2011b; Rao et al. 2010, 2012), this down-regulation may contribute to the therapeutic efficacy of the mood stabilizers (Basselin et al. 2010). This interpretation is supported by evidence that topiramate, initially thought effective in BD but later shown ineffective in phase III clinical trials (Kushner et al. 2006), did not alter any measured parameter of the brain AA cascade in rats (Ghelardoni et al. 2005; Lee et al. 2005a), and that lithium pre-treatment dampened AA cascade up-regulation in animal models of neuroinflammation (Basselin et al. 2007, 2010).

As OLZ and CLZ also are effective in BD (see above) (Calabrese et al. 1996; Frye et al. 1998; Cipriani et al. 2010; Hegerl 2012), we thought it of interest to test the hypothesis that, like the FDA-approved mood stabilizers, these atypical antipsychotics can down-regulate the rat brain AA cascade. Supporting this hypothesis, we reported recently that chronically administration of OLZ to rats, to produce a plasma drug level therapeutically relevant to BD, reduced AA turnover and incorporation in brain phospholipid, total brain cyclooxygenase (COX) activity and prostaglandin E2 (PGE2) concentration, markers of the brain AA (Cheon et al. 2011). These effects of OLZ were ascribed to a concomitant reduction of the plasma concentration of unesterified AA [the form that enters the brain] (Washizaki et al. 1994; Purdon et al. 1997), thus of AA availability to brain (Cheon et al. 2011). We also reported that chronic CLZ, like OLZ, decreased COX activity and PGE2 concentration in rat brain (Kim et al. 2012).

In this study, we used our in vivo kinetic method to test whether CLZ, like OLZ, also would reduce rat brain AA kinetics (turnover and incorporation of AA in phospholipid) and plasma unesterified AA concentration. Showing this would argue further that the AA cascade is a common target of anti-BD atypical antipsychotics as well as of mood stabilizers (Rapoport and Bosetti 2002; Rapoport et al. 2009), and that our in vivo fatty acid model could be used to screen for new drug candidates by measuring AA cascade kinetics in rodents (Robinson et al. 1992).

CLZ was injected i.p. daily in rats for 30 days to produce a therapeutically relevant plasma concentration. Radiolabeled AA was infused intravenously for 5 min in unanesthetized rats after the last CLZ injection, and brain AA kinetics and brain and plasma concentrations were determined (Robinson et al. 1992; Chang et al. 2001). Studies were performed also in a vehicle-treated group, and in a washout group (CLZ-W) that received CLZ for 30 days and was injected with vehicle 24 h later, sufficient time for CLZ to have entirely disappeared from blood and brain, where its half-lives are 1.5 h and 1.6 h, respectively (Baldessarini et al. 1993; Kontkanen et al. 2002). An abstract of part of this work has been published (Modi et al. 2011).

Methods and materials

Animals

The study was conducted following the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals (Publication no. 80–23) and was approved by the Animal Care and Use Committee of the Eunice Kennedy Shriver National Institute of Child Health and Human Development. Chemicals and reagents were purchased from Sigma Chemicals (St. Louis, MO, USA) unless otherwise indicated. Male CDF-344 rats, weighing 180–200 g (Charles River, Wilmington, MA, USA), were acclimatized for 1 week in an animal facility with controlled temperature, humidity and light cycle, and had ad libitum access to water and NIH-31 diet, which contains 4% crude fat by weight. Dietary fatty acids (% of total fatty acid) consisted of 20.1% saturated, 22.5% monounsaturated, 47.9% linoleic, 5.1% α-linolenic, 0.02% AA, 2.0% eicosapentaenoic and 2.3% docosahexaenoic acid (DHA) (Demar et al. 2005; Igarashi et al. 2006). Rats were divided randomly into three groups, a vehicle control group, a CLZ treatment group and a CLZ washout group (CLZ-W) that was given vehicle 24 h after the last CLZ injection.

CLZ (NIMH Chemical Synthesis and Drug Supply Program, Bethesda, MD, USA) was dissolved in 1% glacial acetic acid to a concentration of 10.0 mg/mL and then neutralized with 0.1 N NaOH to pH 6.0. A drug solution was prepared once weekly and stored at 4°C. Chronic CLZ-treated rats (CLZ) received 10 mg/kg/day CLZ in 0.5 mL vehicle once daily for 30 days intraperitoneally (i.p.) as in our prior study (Kim et al. 2012) and were killed 1 h after the last injection. The dose was chosen on the basis of D2 receptor occupancy by CLZ (Farde and Nordstrom 1992), and is consistent with chronically administered clinically relevant doses used previously in rats (Halim et al. 2004; Levant et al. 2006; Tulipano et al. 2007; Cooper et al. 2008). A control group received the same volume of vehicle under parallel conditions. A third washout group (CLZ-W) received CLZ for 30 days followed by vehicle injection on the surgery day (24 h washout). On the last day of injection, the rat was injected with its appropriate treatment 1 h before its brain was removed for analysis.

Surgical procedure

A rat was anesthetized with 1–3% halothane, and polyethylene catheters (PE50; Clay Adams, Becton Dickinson, Sparks, MD, USA) filled with heparinized isotonic saline were inserted into the right femoral artery and vein (Chang et al. 1996; Cheon et al. 2011). The rat was allowed to recover from surgery with its hindquarters loosely wrapped and taped to a wooden block for 3 h in a temperature-controlled recovery chamber maintained at 25°C, while body temperature was maintained at 37°C with a rectal probe and a feedback heating element (TACT-2DF Temperature controller; Physitemp Instruments, Clifton, NJ, USA). CLZ or vehicle was injected 1 h before [1-14C]AA infusion. Heart rate and blood pressure were monitored after recovery from surgery using a CyQ BPM02 system (CyQ 103/302; CyberSense, Nicholasville, KY, USA).

[1-14C]AA infusion

[1-14C]AA (50 mCi/mmol, > 98% pure; Moravek Biochemicals, Brea, CA, USA) was dissolved in saline containing 50 mg/mL fatty acid-free bovine serum albumin by sonicating for 10 min (Sigma-Aldrich) (DeGeorge et al. 1989). One h after the last injection, an unanesthetized rat was infused intravenously for 5 min with 1.3 mL containing 170 μCi/kg of AA, at a rate of 0.223(1 + e−0.032t) ml/min (t = s) with a computer-controlled variable rate infusion pump (No. 22; Harvard Apparatus, South Natick, MA, USA), to achieve a steady-state plasma specific activity within 1 min (Washizaki et al. 1994). Arterial blood samples were collected at 0, 15, 30, 45, 90, 180, 240, and 300 s during infusion to determine radioactive and unlabeled concentrations of unesterified AA in plasma. Five min after starting infusion, the rat was anesthetized with sodium pentobarbital (50 mg/kg, i.v.) and subjected to head-focused microwave irradiation to stop brain metabolism (5.5 kW, 4.8 s; Cober Electronics, Norwalk, CT, USA) (Deutsch et al. 1997; Bazinet et al. 2005a). The brain was excised, dissected sagittally, and stored at −80°C for further analysis.

Plasma and brain lipid extraction and separation

Total lipids were extracted from frozen plasma and from one cerebral hemisphere by the Folch method (Folch et al. 1957). Heptadecanoic acid (17 : 0) was added as an internal standard prior to extraction. Neutral lipids were separated from the total lipid extracts by thin layer chromatography (TLC) on silica gel plates (Silica Gel 60A TLC plates; Whatman, Clifton, NJ, USA) using a mixture of heptane : diethyl ether : glacial acetic acid (60 : 40 : 3 by volume) (Skipski et al. 1968). Authentic standards of cholesteryl ester, triacylglycerol, unesterified fatty acids, cholesterol, and phospholipids were run in separate lanes to identify lipid bands. Phospholipid classes (ChoGpl, choline glycerophospholipid; PtdSer, phosphatidylserine; PtdIns, phosphatidylinositol; EtnGpl, ethanolamine glycerophospholipid) were separated in chloroform : methanol : H2O : glacial acetic acid (60 : 50 : 4 : 1 by volume) (Skipski et al. 1967) and identified with unlabeled standards in separate lanes. The lipid bands were visualized under ultraviolet light after spraying the plates with 0.03% (w/v) 6-p-toluidine-2-naphthalene sulfonic acid (Acros, Fairlawn, NJ, USA) in 50 mM Tris buffer (pH 7.4). Each band was scraped, and the silica gel was used directly to quantify radioactivity by scintillation counting or to prepare fatty acid methyl esters (FAMEs) (see below). Prior to methylation, appropriate quantities of di-17 : 0-PC were added as an internal standard to quantify brain esterified lipids, and 17 : 0 (heptadecaenoic acid) was added to quantify brain unesterified fatty acids.

Quantification of radioactivity

Radioactivity in plasma total lipid extracts collected over the course of the 5 min infusion was determined using a liquid scintillation analyzer (2200CA, TRI-CARB®; Packard Instruments, Meriden, CT, USA) following reconstitution in 5 mL Cocktail mix.

FAME preparation and gas chromatography analysis

FAMEs were formed by heating the scrapes in 1% H2SO4 in methanol at 70°C for 3 h. FAMEs were separated on a SP-2330 fused silica capillary column (30 m × 0.25 mm inner diameter, 0.25 μm film thickness) (Supelco, Bellefonte, PA, USA), using gas chromatography (GC) with a flame ionization detector (Model 6890N; Agilent Technologies, Palo Alto, CA, USA). Runs were initiated at 80°C, with a temperature gradient to 150°C (10°C /min) and 200°C (6°C /min), and held at 200°C for 10 min, and then increased to 240°C for a total run time of 38 min. Fatty acid concentrations (nmol/g brain or nmol/mL plasma) were calculated by proportional comparison of the GC peak areas to that of the 17 : 0 internal standard.

Quantification of labeled and unlabeled acyl-CoA

Acyl-CoA species were extracted from the remaining microwaved half-brain using an affinity chromatography method (Deutsch et al. 1994). After adding 10 nmol heptadecanoyl-CoA (17 : 0-CoA) as an internal standard to the weighed half-brain (~0.8 g), the sample was homogenized in 2 mL of 25 mM potassium phosphate and sonicated for 20 s with a probe sonicator (Model W-225; Misonix, Farmingdale, NY, USA). Isopropanol (2 mL) was added to the homogenate, which was sonicated for another 20 s. Proteins were precipitated with saturated ammonium sulfate and shaking the sample lightly by hand. Then, acetonitrile (4 mL) was added and the sample vortexed for 10 min prior to centrifugation. The supernatant was collected and diluted with 10 mL of 25 mM potassium phosphate. Each sample was passed three times through an activated oligonucleotide purification cartridge (ABI Masterpiece, OPC®; Applied Biosystems, Foster City, CA), and the cartridge was washed with 10 mL of 25 mM potassium phosphate. Acyl-CoA species were eluted with 500 μL of elution buffer (75% isopropanol / 25% 1 mM glacial acetic acid by volume). Samples were dried under nitrogen and reconstituted in 100 μL of elution buffer for HPLC analysis. Extracted acyl-CoA species were separated on a reverse-phase HPLC column (Symmetry C-18, 5-μm particle size, 250 × 4.6 mm; Waters-Millipore, Milford, MA, USA), using HPLC (Beckman, Fullerton, CA, USA) and a pump coupled with a UV/VIS detector (System Gold, Model 168; Beckman). HPLC was performed using a linear gradient system composed of: (A) 75 mM potassium phosphate, pH 4.9 and (B) 100% acetonitrile. The composition of the initial solvent system (44% B, 1 min) was changed to 49% B over 25 min and then to 68% B over 10 min, maintained at 68% B for 4 min, returned to 44% B over 6 min, and held at 44% B for 6 min (52 min total run time). UV detection was set at 260 nm for integration of concentrations and at 280 nm for identification of acyl-CoAs (260/280 = 4 : 1). Peaks were identified from retention times of authentic acyl-CoA standards. Endogenous acyl-CoA concentrations (nmol/g brain) were calculated by direct proportional comparison of the peak areas with the peak area of the 17 : 0-CoA internal standard. The arachidonoyl-CoA (AA-CoA) peak was collected, its concentration quantified and its radioactivity determined by liquid scintillation counting in order to calculate the specific activity.

Calculations

We used our established in vivo kinetic model for quantifying brain fatty acid kinetic parameters (Robinson et al. 1992). Unidirectional incorporation coefficients, inline image (mL/s/g) of AA, representing incorporation from plasma into brain lipid i (phospholipid, triacylglycerol, or cholesteryl ester), were calculated as follows:

display math(1)

inline image (nCi/g) represents radioactivity of brain lipid i at time T = 5 min (time of termination of experiment), t is time after starting infusion, and inline image (nCi/mL) is the plasma concentration of labeled unesterified AA during infusion. Integrals of plasma radioactivity were determined by trapezoidal integration. Since AA synthesis within brain from its dietary precursor linoleic acid (18:2n-6) represents less than 0.5% of the plasma AA flux into brain (DeMar et al. 2006), the rate of incorporation Jin,i (nmol/s/g) of plasma unesterified AA into brain lipid i represents the rate of metabolic loss by the brain, and is calculated as follows:

display math(2)

Cpl (nmol/mL) is the concentration of unlabeled unesterified AA in plasma. The ‘dilution factor’ λ, defined as the steady-state ratio during [1-14C]AA infusion, of specific activity of the brain arachidonoyl-CoA pool to the specific activity of plasma unesterified AA, was determined as follows:

display math(3)

Net rates of incorporation of unlabeled unesterified AA from brain arachidonoyl-CoA into brain lipid i, JFA,i (nmol/s/g) equal:

display math(4)

The fractional turnover of AA within phospholipid i, because of deacylation and reacylation, FFA,i (%/h) is defined as:

display math(5)

Statistical analysis

Data are presented as mean ± SD. Data were analyzed with a one-way analysis of variance (anova) followed by Bonferroni's post-hoc test to compare differences between: (i) the CLZ and control group, and (ii) the CLZ-W and control group. Statistically significant differences between CLZ with or without washout relative to controls are indicated by asterisks; *p < 0.05, **p < 0.01, ***p < 0.001. Statistical analysis was performed on GraphPad Prism (version 4.03; GraphPad Software, San Diego, CA, USA).

Results

Body weight and physical parameters

Rats chronically administered CLZ with or without washout weighed 12% and 10% less, respectively, than controls (270.4 ± 10.49, 238.6 ± 21.37, 243.4 ± 13.6 g for control, CLZ and CLZ-W, respectively, p < 0.01) (Table 1). CLZ with or without washout reduced mean arterial blood pressure by 18% and 13%, respectively, compared to control (Table 1). Heart rate did not change significantly. Body temperature did not differ significantly among the three groups, since it was maintained at 37°C by a rectal probe and a heating element.

Table 1. Physiological parameters
 Control CLZCLZ-W
  1. Chronic clozapine-treated rats (CLZ, n = 8) received 10 mg/kg/day clozapine in 0.5 mL vehicle once daily for 30 days intraperitoneally (i.p.) and were killed 1 h after the last injection. 24 h washout group (CLZ-W, n = 8) received full period of clozapine, and followed by one vehicle injection on the surgery day. A control group (n = 10) received the same volume of vehicle under parallel conditions. Values are means ± SD. Data were analyzed by one-way anova followed by Bonferroni's post-hoc test, which compared differences between (i) control and CLZ, and (ii) control and CLZ-W.

  2. a

    p < 0.01.

  3. b

    p < 0.001 compared to controls.

Body weight (g)270.4 ± 10.49238.6 ± 21.4b243.4 ± 13.6a
Arterial blood pressure (mm Hg)138.1 ± 5.38113.3 ± 4.9b120.0 ± 8.67a
Heart rate (beats/min)424.4 ± 28.3389.1 ± 40.6448.2 ± 33.3
Body temperature (°C)37.0 ± 0.537.0 ± 0.436.8 ± 0.4

Plasma kinetics

Figure 1 shows steady-state plasma radioactivity during the 5-min [1-14C]AA infusion. Steady-state radioactivity was achieved within one minute in all groups, but was higher at all times for the CLZ and CLZ-W groups than control. Thus, integrated plasma radioactivity was significantly higher in the CLZ (148330 ± 19833 nC.s/mL) and CLZ-W (152507 ± 27312 nCi.s/mL) groups than in the control group (112057 ± 10684 nCi.s/mL) (p < 0.05), suggesting that CLZ prolonged the plasma half-life of unesterified plasma AA (Rapoport et al. 1982).

Figure 1.

Time course of arterial plasma radioactivity during 5 min [1-14C]AA infusion. Arterial plasma radioactivity (nCi/mL) was measured in total lipid extracts of plasma from control and clozapine-treated rats during intravenous infusion of 170 μCi/kg [1-14C]AA over 5 min at a rate of 0.223 (1 + e-0.032t) ml/min (t = seconds). CLZ rats received CLZ (10 mg/kg/day, i.p.), once daily for 30 days and were killed 2 h after the last drug injection. CLZ-W rats received full period of clozapine followed by a 24-h washout. A control group received the same volume of vehicle under parallel conditions. Values are mean ± SD (control, n = 10; CLZ, n = 8; CLZ-W, n = 8). Data were analyzed by one-way ANOVA followed by Bonferroni__s post-hoc test, which compared differences between (i) control and CLZ, and (ii) control and CLZ-W. **p < 0.01 compared to controls.

Plasma and brain fatty acids

As illustrated in Table 2, esterified 18 : 0 concentration was increased significantly by 2.5-fold by CLZ compared to control in plasma cholesteryl ester, without there being any other significant fatty acid change in either lipid. Cholesteryl ester 18 : 0 concentration did not differ significantly between the CLZ-W group and control. Esterified fatty acid concentrations in plasma triglycerides and phospholipids did not differ significantly (data not shown).

Table 2. Esterified fatty acid concentrations in plasma cholesteryl ester
Fatty acidControl (n = 10)CLZ (n = 8)CLZ-W (n = 8)
(nmol/mL plasma)(nmol/mL plasma)(nmol/mL plasma)
  1. ND, Not detected; SFA, Saturated fatty acid; MUFA, Monounsaturated fatty acid; PUFA, Polyunsaturated fatty acid. Values are means ± SD. Data were analyzed by one-way anova followed by Bonferroni's post-hoc test, which compared differences between (i) control and CLZ, and (ii) control and CLZ-W.

  2. a

    p < 0.05 compared to control.

16 : 045.6 ± 6.344.4 ± 2.840.7 ± 7.9
16 : 115.4 ± 4.613.7 ± 4.111.0 ± 7.6
18 : 06.9 ± 1.917.6 ± 12.9a7.2 ± 2.2
18 : 1n-9 21.1 ± 2.023.4 ± 2.822.1 ± 4.4
18 : 1n-75.8 ± 0.85.5 ± 0.75.5 ± 0.8
18 : 2n-6123.6 ± 15.1115.2 ± 5.1110.5 ± 20.6
18 : 3n-33.3 ± 1.92.9 ± 0.52.4 ± 0.9
20 : 4n-6267.4 ± 46.1251.2 ± 52.5254.8 ± 67.5
20 : 5n-322.6 ± 6.724.2 ± 5.121.3 ± 9.1
22 : 5n-38.6 ± 3.78.1 ± 2.69.2 ± 1.3
22 : 6n-313.9 ± 3.812.0 ± 1.613.1 ± 3.0
Total537.0 ± 73.6520.8 ± 56.1501.3 ± 108.6
SFA52.5 ± 7.060.0 ± 14.947.9 ± 9.5
MUFA42.2 ± 5.542.6 ± 5.338.5 ± 11.9
PUFA442.3 ± 68.5418.2 ± 52.8414.9 ± 93.3
n-6 PUFA402.7 ± 60.8379.0 ± 53.4378.1 ± 86.2
n-3 PUFA39.5 ± 11.539.2 ± 5.836.8 ± 11.9

As illustrated in Table 3, chronic CLZ compared with control caused a widespread decrease in the plasma concentrations of the majority of measured unesterified fatty acids, including net n-3 and n-6 PUFA concentrations. Many but not all of the reductions were significant even after the 1 day washout, indicating that they did not depend on the continued presence of CLZ in the body. With regard to unesterified AA, chronic CLZ significantly decreased the plasma concentration by 46% (p < 0.001) compared to control (13.6 ± 6.0 vs. 25.2 ± 8.7 nmol/mL) (Table 3), but this effect was not significant for the CLZ-W group (20.0 ± 5.4 nmol/mL). Unesterified 18 : 2n-6 and 18 : 3n-3 were decreased in both the CLZ and CLZ-W groups.

Table 3. Unesterified fatty acid concentrations in plasma and esterified fatty acid concentrations in total brain phospholipid
Fatty acidUnesterified fatty acids in plasmaEsterified fatty acids in brain total phospholipids
Control (= 10)CLZ (n = 8)CLZ-W (n = 8)Control (n = 10)CLZ (n = 8)CLZ-W (n = 8)
(nmol/mL plasma)(nmol/mL plasma)(nmol/mL plasma)(nmol/g brain)(nmol/g brain)(nmol/g brain)
  1. ND, Not detected; SFA, Saturated fatty acid; MUFA, Monounsaturated fatty acid; PUFA, Polyunsaturated fatty acid. Values are means ± SD. Data were analyzed by one-way anova followed by Bonferroni's post-hoc test, which compared differences between (i) control and CLZ, and (ii) control and CLZ-W.

  2. a

    p < 0.05.

  3. b

    p < 0.01.

  4. c

    p < 0.001 compared to control.

16 : 0 451.3 ± 73.8195.3 ± 55.8c259.9 ± 54.2c24674 ± 98525013 ± 98525313 ± 1656
16 : 165.0 ± 20.221.1 ± 8.3c33.7 ± 12.6bNDNDND
18 : 0 89.8 ± 17.450.7 ± 6.6c71.3 ± 24.925258 ± 148925655 ± 182726841 ± 2236
18 : 1n-9 329.9 ± 56.9139.0 ± 45.3c209.0 ± 46.0c22900 ± 189924033 ± 216926946 ± 3351b
18 : 2n-6 356.4 ± 74.6141.1 ± 47.1c217.9 ± 37.0c724 ± 100750 ± 63927 ± 87c
18:3n-3 24.6 ± 5.08.8 ± 3.0c14.7 ± 3.5cNDNDND
20 : 1n-9NDNDND2491 ± 5412814 ± 4393496 ± 999a
20 : 4n-6 25.2 ± 8.713.6 ± 6.0b20.0 ± 5.410017 ± 82210106 ± 53910310 ± 761
20 : 5n-3 14.7 ± 4.95.1 ± 1.6c8.3 ± 1.8bNDNDND
22 : 4n-6NDNDND3149 ± 2123292 ± 2583383 ± 368
22 : 5n-6NDNDND230 ± 21235 ± 25208 ± 24
22 : 5n-3 18.2 ± 4.66.5 ± 2.8c8.4 ± 4.2b118 ± 28121 ± 11123 ± 9
22 : 6n-3 37.0 ± 10.914.6 ± 5.5c23.3 ± 5.4b13281 ± 97514078 ± 99214181 ± 1157
Total 1423.2 ± 296.2600.1 ± 171.4c872.2 ± 146.6c108810 ± 5791112551 ± 7417118868 ± 9579a
SFA 552.3 ± 89.4250.2 ± 62.9c336.9 ± 64.2c49932 ± 231550668 ± 279252153 ± 3727
MUFA 394.9 ± 74.3160.2 ± 52.1c242.7 ± 57.7c31358 ± 282833301 ± 325537584 ± 5150b
PUFA 476.1 ± 107.0189.7 ± 63.6c292.6 ± 45.9c27519 ± 196528582 ± 173529131 ± 2233
n-6 PUFA 381.5 ± 82.5154.7 ± 51.8c237.9 ± 35.6c14120 ± 104514384 ± 77214828 ± 1135
n-3 PUFA 94.5 ± 24.935.1 ± 12.3c54.7 ± 11.5c13399 ± 99414198 ± 99914303 ± 1155

Esterified fatty acid concentrations within brain phospholipids were not affected significantly by chronic CLZ treatment (Table 3). After CLZ-W, however, esterified concentrations of 18 : 1n-9, 18 : 2n-6 and 20 : 1n-9 and of total monounsaturated fatty acids were significantly higher than in the control group (Table 3).

Chronic CLZ had few significant effects on esterified concentrations of fatty acids in each of four brain phospholipids, but none on esterified AA or DHA, the major PUFAs (Tables 4 and 5). In ChoGpl, oleate (18 : 1n-9) and 20 : 1n-9 were increased by CLZ-W but not CLZ compared with control). In EtnGpl, palmitate (16 : 0), 18 : 1n-9, 18 : 1n-7, 18 : 2n-6, 20 : 1n-9 adrenate (22 : 4n-6) and 22 : 6n-3 were increased by CLZ-W but not CLZ (Table 4). In PtdIns, 16 : 0, 18 : 1n-9 and 18 : 2n-6 were increased by CLZ, whereas 16 : 0 and 18 : 1n-7 were increased by CLZ-W (Table 5). In PtdSer, 16 : 0 was decreased by CLZ, whereas 18 : 1n-9 was increased in CLZ-W (Table 5).

Table 4. Fatty acid concentrations in brain choline glycerophospholipids and ethanolamine glycerophospholipids
Fatty AcidCholine glycerophospholipidsEthanolamine glycerophospholipids
Control (n = 10)CLZ (n = 8)CLZ-W (n = 8)Control (n = 10)CLZ (n = 8)CLZ-W (n = 8)
(nmol/g brain)(nmol/g brain)(nmol/g brain)(nmol/g brain)(nmol/g brain)(nmol/g brain)
  1. Values are means ± SD. Data were analyzed by one-way anova followed by Bonferroni's post-hoc test, which compared differences between (i) control and CLZ, and (ii) control and CLZ-W.

  2. a

    p < 0.05.

  3. b

    p < 0.01.

  4. c

    p < 0.001 compared to controls.

16 : 0 21095 ± 85621326 ± 75321311 ± 12882691 ± 1642759 ± 1722955 ± 284a
18 : 0 7714 ± 6087819 ± 6838333 ± 9938422 ± 7468821 ± 5328880 ± 552
18 : 1n-9 11029 ± 64811389 ± 78512054 ± 1031a7714 ± 9378193 ± 8539816 ± 1434b
18 : 1n-7 4066 ± 2544184 ± 3974480 ± 5081708 ± 2952004 ± 2602386 ± 435c
18 : 2n-6 401 ± 41421 ± 34448 ± 47248 ± 60229 ± 52372 ± 33c
20 : 1n-9593 ± 120646 ± 91771 ± 191a1468 ± 3361714 ± 2852139 ± 588b
20 : 4n-6 2582 ± 1932501 ± 1812479 ± 2085398 ± 5095418 ± 3235736 ± 319
22 : 4n-6223 ± 37234 ± 26210 ± 402383 ± 1862523 ± 1762635 ± 242a
22 : 6n-3 1741 ± 2021894 ± 2361895 ± 2528211 ± 6408650 ± 7299051 ± 556a
Total 49444 ± 234450413 ± 294951980 ± 398838502 ± 280940581 ± 287344222 ± 3412b
Table 5. Fatty acid concentrations in brain phosphatidylinositol and phosphatidylserine
Fatty AcidPhosphatidylinositolPhosphatidylserine
Control (n = 10)CLZ (n = 8)CLZ-W (n = 8)Control (n = 10)CLZ (n = 8)CLZ-W (n = 8)
(nmol/g brain)(nmol/g brain)(nmol/g brain)(nmol/g brain)(nmol/g brain)(nmol/g brain)
  1. Values are means ± SD. Data were analyzed by one-way anova followed by Bonferroni's post-hoc test, which compared differences between (i) control and CLZ, and (ii) control and CLZ-W.

  2. a

    p < 0.05 compared to controls.

16 : 0 470 ± 88616 ± 106 a618 ± 111a418 ± 117311 ± 54a428 ± 147
18 : 0 1746 ± 1781852 ± 2671916 ± 2947376 ± 5937163 ± 5167712 ± 741
18 : 1n-9 539 ± 198830 ± 225a744 ± 1613618 ± 4213621 ± 4584332 ± 853a
18 : 1n-7195 ± 66267 ± 42275 ± 67aNDNDND
18 : 2n-6 35 ± 1362 ± 31a50 ± 940 ± 1839 ± 1137 ± 9
20 : 1n-970 ± 3599 ± 25102 ± 42361 ± 90 354 ± 60 484 ± 244
20 : 4n-6 1471 ± 1921607 ± 2331507 ± 233565 ± 72579 ± 124588 ± 122
22 : 6n-3 134 ± 42167 ± 51153 ± 513195 ± 2263367 ± 1713082 ± 419
Total 4658 ± 7165501 ± 911a5366 ± 79116205 ± 114416055 ± 131417300 ± 1916

Brain acyl-CoA concentrations

Chronic CLZ, with or without washout, did not change significantly the brain concentration of unlabeled arachidonoyl-CoA compared with control (Table 6). CLZ alone, however, increased the concentration of labeled AA-CoA compared with control (p < 0.05), which was not significantly different between CLZ-W and control. There was no significant difference in concentrations of the other measured brain acyl-CoA species, except for palmitoyl-CoA, which was significantly reduced in the CLZ-W group compared with control (Table 6).

Table 6. Brain acyl-CoA concentrations and λ
 Control (n = 5)CLZ (n = 5)CLZ-W (n = 4)
(nmol/g brain)(nmol/g brain)(nmol/g brain)
  1. a

    λ (Eqn 3) is the steady-state ratio during [1-14C]AA infusion of specific activity of brain arachidonoyl-CoA pool to specific activity of plasma unesterified AA.

  2. Values are means ± SD. Data were analyzed by one-way anova followed by Bonferroni's post-hoc test, which compared differences between (i) control and CLZ, and (ii) control and CLZ-W.

  3. b

    p < 0.05 compared to control.

Mystearoyl-CoA 0.26 ± 0.070.28 ± 0.090.16 ± 0.11
Palmitoyl-CoA 9.19 ± 0.3510.08 ± 1.188.13 ± 0.87b
Stearoyl-CoA 6.29 ± 1.186.26 ± 0.636.62 ± 0.77
Oleayl-CoA 12.10 ± 0.5713.68 ± 1.7712.90 ± 1.45
Linoleoyl-CoA 0.49 ± 0.120.45 ± 0.110.51 ± 0.14
Docosahexaenoyl-CoA 0.83 ± 0.190.89 ± 0.130.70 ± 0.21
Arachidonoyl-CoA (nmol/g brain)0.63 ± 0.080.71 ± 0.110.61 ± 0.19
[14C]AA-CoA (nCi/g brain)0.38 ± 0.09 0.78 ± 0.16b0.68 ± 0.44
λa0.029 ± 0.0130.020 ± 0.0130.033 ± 0.013

Brain kinetics

CLZ significantly increased the AA incorporation coefficient, inline image (Eqn 1) into ChoGpl compared with control (p < 0.05; Table 7). CLZ-W significantly increased inline image into total phospholipids (p < 0.01), ChoGpl (p < 0.01), and PtdIns (p < 0.05) compared to control (Table 7). Reflecting the reduction in unesterified plasma AA concentration, CLZ significantly decreased Jin,i (Eqn 2), the incorporation rate of unesterified AA from plasma into brain total phospholipids, by 36%, compared with control (Table 7). CLZ-W did not significantly affect Jin,i for total or individual brain phospholipids (Table 7).

Table 7. Incorporation coefficients (ki*) and incorporation rates (Jin,i) of AA from plasma into brain phospholipids
 ki*(mL/g/s × 10−5)Jin,I (nmol/g/s × 10−4)
Control (n = 10)CLZ (n = 8)CLZ-W (n = 8)Control (n = 10)CLZ (n = 8)CLZ-W (n = 8)
  1. ChoGpl, choline glycerophospholipids; PtdSer, phosphatidylserine; PtdIns, phosphatidylinositol; EtnGpl, ethanolamine glycerophospholipids. Values are means ± SD. Data were analyzed by one-way anova followed by Bonferroni's post-hoc test, which compared differences between (i) control and CLZ, and (ii) control and CLZ-W.

  2. a

    p < 0.05.

  3. b

    p < 0.01 compared to control.

Total phospholipid28.5 ± 2.633.5 ± 2.335.2 ± 6.8b71.5 ± 23.745.7 ± 20.6a72.3 ± 30.8
ChoGpl12.3 ± 1.1515.4 ± 1.5a15.9 ± 3.1b30.7 ± 10.220.9 ± 9.432.6 ± 14.1
PtdSer2.7 ± 0.42.9 ± 0.33.2 ± 0.86.9 ± 2.74.0 ± 1.86.6 ± 3.3
PtdIns10.1 ± 0.911.3 ± 0.712.0 ± 2.3a25.2 ± 8.415.5 ± 7.124.7 ± 10.1
EtnGpl3.5 ± 0.53.9 ± 0.54.1 ± 0.88.7 ± 2.85.4 ± 2.68.4 ± 3.5

The dilution factor λ (Eqn 3) was not significantly changed by CLZ (0.020 ± 0.013) or CLZ-W (0.033 ± 0.013) compared with control (0.029 ± 0.013) (Table 6). Inserting λ into Eqn 5 provided rates of incorporation of non-esterified AA from the brain precursor AA-CoA pool into phospholipids, JFA,i (Eqn 4). JFA,i and AA turnover FFA,i due to deacylation-reacylation were not changed significantly in the CLZ or CLZ-W group compared with the control group (Table 8).

Table 8. Net incorporation rate of brain AA-CoA into brain phospholipids (JFA) and AA turnover (FFA)
  JFA (nmol/g/s × 10−2)FFA (% per hour)
Control (n = 5)CLZ (n = 5)CLZ-W (n = 4)Control (n = 5)CLZ (n = 5)CLZ-W (n = 4)
  1. Values are means ± SD. ChoGpl, choline glycerophospholipids; PtdSer, phosphatidylserine; PtdIns, phosphatidylinositol; EtnGpl, ethanolamine glycerophospholipids.

Total phospholipids21.6 ± 8.418.4 ± 3.918.5 ± 3.37.7 ± 2.86.5 ± 1.56.3 ± 1.3
ChoGpl9.4 ± 3.78.5 ± 2.18.2 ± 1.612.9 ± 4.611.9 ± 3.211.5 ± 2.6
PtdSer2.0 ± 0.81.6 ± 0.31.6 ± 0.413.2 ± 6.010.6 ± 2.99.6 ± 2.2
PtdIns7.5 ± 2.86.2 ± 1.16.5 ± 1.017.4 ± 6.215.0 ± 5.315.1 ± 2.7
EtnGpl2.7 ± 1.12.1 ± 0.42.2 ± 0.31.8 ± 0.71.3 ± 0.31.3 ± 0.2

Discussion

Baseline concentrations of unesterified and esterified plasma fatty acids, of esterified brain fatty acids in individual phospholipids, and of brain acyl-CoA species, as well as AA incorporation coefficients, rates and turnovers in brain phospholipids of control (vehicle-treated) rats, were comparable to published values in rats fed the NIH-31 diet in this study (Chang et al. 1996, 2001; Bazinet et al. 2005a, 2006; Basselin et al. 2007; Lee et al. 2008, 2010; Cheon et al. 2011). This diet has a high content of the n-3 PUFAs, EPA (2.0%) and DHA (2.3%) (see Methods), unlike some other rodent diets (e.g., Teklad 2018 diet, Harlan Laboratories, USA) that lack EPA and DHA. The unesterified plasma AA concentration in control rats on this diet also is within the published range of 16–42 μM, as are concentrations of other unesterified and esterified fatty acids, including palmitate (16 : 0), stearate (18 : 0), oleate (18 : 1n-9), linoleate (18 : 2n-6), and α-linolenate (18 : 3n-3) (Bazinet et al. 2005a, 2006; Demar et al. 2005; Basselin et al. 2007; Lee et al. 2008). This confirms the validity and reproducibility of our analytical methods, our kinetic model and the unanesthetized rat preparation (Robinson et al. 1992).

The major finding of this study is that chronic CLZ (10 mg/kg/day i.p. 30 days), administered at a dose that produced a therapeutically relevant plasma concentration, compared with vehicle, significantly decreased rates of AA incorporation (Jin,i) from plasma into brain total phospholipid, largely by decreasing the plasma concentration and thus availability to brain of unesterified AA (Washizaki et al. 1994). This effect was statistically insignificant after the 24-h washout period (CLZ-W), reflecting normalization of the plasma unesterified AA concentration. As AA is a substrate for COX and other oxidative enzymes within the brain AA cascade (Shimizu and Wolfe 1990), its reduced plasma AA availability likely contributes to the reported decreases in brain COX activity and PGE2 concentration following chronic CLZ treatment (Kim et al. 2012). These changes and their 24-h reversibility are similar to those caused by chronic OLZ (Cheon et al. 2011), suggesting that each of the two atypical antipsychotic agents used in BD reduce rat brain AA metabolism, and that they do so by decreasing plasma unesterified AA concentration. This mechanism deserves to be tested with other antipsychotics.

Whereas OLZ significantly reduced AA turnover within total phospholipid and PtdIns in rat brain (Cheon et al. 2011), CLZ's effect on turnover in total and individual phospholipids was statistically insignificant. The difference may have reflected drug dose effects, or the lower variance of the OLZ data because of more rats having been studied with it. Further, calculated turnover depends on the dilution coefficient λ, which was reduced insignificantly by 31% by CLZ compared to vehicle, which would tend to counterbalance the influence of reduced the Jin,i for AA (Eqn 5).

The rate of AA incorporation into phospholipid i, Jin,i, is the product of the incorporation coefficient inline image and plasma unesterified AA concentration (Eqn 2). It equals the rate of AA metabolic loss from brain, as AA cannot be synthesized de novo or converted significantly from its linoleic acid precursor in brain (Holman 1986; DeMar et al. 2006). Although inline image was increased by chronic CLZ, suggesting greater brain avidity for unesterified AA, Jin,i was reduced because of the decreased plasma unesterified AA concentration. That Jin,i returned to baseline following the 24 h washout suggests that CLZ's effect on plasma unesterified AA required significant drug in the body, since CLZ half-lives are 1.5 h and 1.6 h, respectively, in rat plasma and brain (Baldessarini et al. 1993; Kontkanen et al. 2002).

The esterified AA concentration within total brain phospholipids did not change despite the reduction in Jin,i, possibly because downstream AA metabolism was reduced proportionately to the reduction in Jin,i, as evidenced by the reduced brain COX activity and PGE2 concentration (Kim et al. 2012). This finding confirms a prior report that measured brain fatty acid concentrations at a daily CLZ dose of 20 mg/kg (Levant et al. 2006) and highlights the importance of measuring fluxes as well as concentrations when testing drug effects on brain fatty acid metabolism. In this regard, despite significant increases in expression of cytosolic phospholipase A2 (cPLA2)-IVA, secretory sPLA2-IIA, and COX-2 in post-mortem BD frontal cortex, suggesting disturbed AA metabolism, phospholipid and fatty acid concentrations were minimally different from control values (Igarashi et al. 2010; Kim et al. 2011b). In vivo PET imaging of inline image and Jin,i might be used to further examine antipsychotic drug effects on brain AA kinetics in bipolar patients (Thambisetty et al. 2012).

The widespread reductions in unesterified concentrations of plasma fatty acids following CLZ were not related to reductions in plasma esterified concentrations, which were found following chronic OLZ in rats (Cheon et al., unpublished observations). They thus may be related to CLZ's effects on hydrolysis of esterified circulating fatty acids by liver, adipose, or other tissue. In humans, CLZ decreased expression of hepatic lipase, which is involved in lipoprotein secretion, and of adipose lipases (Ferno et al. 2009) that regulate lipolysis and secretion of unesterified fatty acids (Gavino and Gavino 1992; Raclot 2003; Duncan et al. 2008). Similar peripheral actions have been proposed for OLZ, which also decreases plasma unesterified fatty acid concentrations in rats (Albaugh et al. 2011, 2012; Cheon et al. 2011) and in humans (Kaddurah-Daouk et al. 2007; Vidarsdottir et al. 2010; Albaugh et al. 2011).

Chronic CLZ with or without washout increased the AA incorporation coefficient inline image into rat brain ChoGpl, as did OLZ and valproate (Chang et al. 1996, 2001; Bazinet et al. 2006; Cheon et al. 2011). An increased inline image represents increased ‘affinity’ of serial reactions involving diffusion, transport, and enzymatic activation leading to entry of plasma AA into the sn-2 position of brain phospholipid (Sun and MacQuarrie 1989; Robinson et al. 1992; Rapoport 2008; Kirkilionis 2010). This increase may involve up-regulated expression of brain dopaminergic D2 and D4 receptors or of the NR2B subunit of N-methyl-d aspartate (NMDA) receptors caused by chronic drug administration (Janowsky et al. 1992; Meshul et al. 1996; Lidow and Goldman-Rakic 1997; Tarazi et al. 1997; Silvestri et al. 2000; Ossowska et al. 2002; Kabbani and Levenson 2006), given that D2-like and NMDA receptors can be coupled to cPLA2 and AA release from membrane phospholipid (Piomelli and Di Marzo 1993; Bhattacharjee et al. 2005; Basselin et al. 2006). It also could represent a compensatory response to reduced plasma unesterified AA, as also found following chronic valproate and OLZ (Bazinet et al. 2005b; Cheon et al. 2011; Ramadan et al. 2011).

Like chronic CLZ, chronic administration of OLZ to rats, to produce a plasma drug level therapeutically relevant to BD, reduced AA incorporation (Jin,i) into phospholipid, total brain COX activity and PGE2 concentration (Cheon et al. 2011; Kim et al. 2012). These effects also were ascribed to a reduced plasma concentration of unesterified AA, thus of AA availability to brain (Cheon et al. 2011). These similarities suggest that the AA cascade is a common target of anti-BD atypical antipsychotics as well as mood stabilizers (Rapoport and Bosetti 2002; Rapoport et al. 2009), and that our in vivo kinetic fatty acid method could be used to screen for new drug candidates in rodents (Robinson et al. 1992).

Fatty acid concentrations and inline image for AA remained elevated in some brain phospholipids 24 h following CLZ washout, suggesting a withdrawal effect of CLZ on membrane fatty acid concentrations as reported following OLZ (Cheon et al. 2011). CLZ is an amphiphilic molecule that is positively charged at physiologic pH, and can interact with acidic and neutral phospholipid polar head groups via electrostatic and repulsion forces (Soderlund et al. 1999; Jutila et al. 2001; Parry et al. 2008). This interaction may have caused long-lasting disruption in membrane phospholipid, despite CLZ's absence from brain after the 24-h washout period.

The reductions by CLZ and OLZ of plasma unesterified AA concentration and brain COX activity and PGE2 concentration are similar to the effects of chronic dietary n-6 PUFA deprivation in rats (Kim et al. 2011a). This suggests a possible therapeutic advantage of reducing dietary n-6 PUFA content as a stand-alone therapy or in combination with CLZ or OLZ, as each treatment reduces plasma unesterified AA availability.

A paradoxical aspect of atypical antipsychotics is that they cause weight loss in male rats but weight gain in humans, despite inducing similar metabolic disturbances related to insulin resistance and hyperlipidemia (Albaugh et al. 2006, 2011; Minet-Ringuet et al. 2006; Kaddurah-Daouk et al. 2007; Vidarsdottir et al. 2010). This discrepancy has not been resolved, although female rats appear more likely to develop obesity than male rats following i.p. OLZ in particular, but not following antipsychotic administration through the diet (Albaugh et al. 2006; Fell et al. 2007; Minet-Ringuet et al. 2006). Several effects of atypical antipsychotics relevant to this study have been reported in rats and humans, including reduced unesterified plasma fatty acid concentrations by OLZ in rats (Albaugh et al. 2011, 2012) and humans (Kaddurah-Daouk et al. 2007; Vidarsdottir et al. 2010). In this study, CLZ also reduced plasma unesterified fatty acid concentrations. Whether this occurs in humans remains to be tested.

In conclusion, application of our in vivo fatty acid kinetic model in unanesthetized rats showed that chronic CLZ reduced AA incorporation rates into brain phospholipids by decreasing the unesterified plasma AA concentration in plasma, despite increasing AA incorporation coefficients. This effect required the presence of CLZ in the body, since it was absent after the 24-h washout. The decreases in AA incorporation rates, brain COX activity and PGE2 concentration overlap with effects of OLZ (Cheon et al. 2011). When related to studies of the direct action of mood stabilizers on the rat brain AA cascade, this article further supports the overall hypothesis that targeting the brain AA cascade is a reasonable approach for treating BD and can be used to screen for novel drug candidates in rats.

Acknowledgements

This study was supported by the Intramural Research Program of the National Institute on Aging, NIH. Clozapine was supplied by National Institute of Mental Health's Chemical Synthesis and Drug Supply Program. The authors thank Dr. Mireille Basselin for her valuable comments. The authors have no conflict of interest.

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