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Correlations between Peripheral Polyunsaturated Fatty Acid Content and in Vivo Membrane Phospholipid Metabolites Jeffrey K. Yao, Jeffrey A. Stanley, Ravinder D. Reddy, Matcheri S. Keshavan, and Jay W. Pettegrew Background: There is evidence for membrane abnormal- ities in schizophrenia. It is unclear whether the observed membrane deficits in peripheral cells parallel central membrane phospholipid metabolism. To address this ques- tion we examined the relations between red blood cell polyunsaturated fatty acids and brain phospholipid me- tabolites from different regions of interest in schizophre- nia and healthy subjects. Methods: Red blood cell membrane fatty acids were measured by capillary gas chromatography and in vivo brain phospholipid metabolite levels were measured using a multi-voxel 31 P Magnetic Resonance Spectroscopy tech- nique on 11 first-episode, neuroleptic-naı ¨ve schizophrenic subjects and 11 normal control subjects. Results: Both the total polyunsaturated fatty acids and the individual 20:4(n-6) contents were significantly correlated with the freely-mobile phosphomonoester [PME(s- c )] levels (r .5643, p .0062 and r .6729, p .0006, respectively). The 18:2(n-6) polyunsaturated fatty acids content correlated positively with freely-mobile phos- phodiester [PDE(s- c )] levels (r .5573, p .0071). The above correlations were present in the combined right and left prefrontal region of the brain, while other regions including the basal ganglia, occipital, inferior parietal, superior temporal and centrum semiovale yielded no significant correlations. Conclusions: Our preliminary data support the associa- tion between the decreased red blood cell membrane phospholipid polyunsaturated fatty acids content and the decreased building blocks [PME(s- c )] and breakdown products [PDE(s- c )] of membrane phospholipids in the prefrontal region of first-episode, neuroleptic-naı ¨ve schizophrenic subjects. Biol Psychiatry 2002;52: 823– 830 © 2002 Society of Biological Psychiatry Key Words: Red blood cell, polyunsaturated fatty acids, membranes, in vivo 31 P magnetic resonance spectroscopy, phosphomonoesters, prefrontal, schizophrenia Introduction T here is substantial evidence for peripheral membrane abnormalities in chronic schizophrenic patients (Vaddadi et al 1989; Glen et al 1994; Yao et al 1994, 1996; Yao and van Kammen, 1994, 1996; Peet et al 1996), and a relative paucity of such evidence in early schizophrenia (Reddy et al 1999). On the other hand, there is convincing evidence for abnormal brain phospholipid metabolism in first-episode and chronic schizophrenic patients (Pet- tegrew et al 1991; Keshavan et al 2000). The relation between peripheral findings and brain membrane alter- ations is unknown. If confirmed, such a relationship provides a unique opportunity to simultaneously investi- gate central and peripheral biochemistry, and their rela- tions to clinical measures. Relations between peripheral and central phospholipid metabolism, if found, will also have several important implications for schizophrenia research. Firstly, the findings to date in peripheral tissue will assume greater relevance for understanding aspects of pathophysiology of schizophrenia. This issue has been vigorously debated because of examples in the literature, where peripheral measures either failed to adequately reflect central pathophysiology or did not serve as reliable biological markers. Secondly, the findings will support the notion that membrane abnormalities are present in both neural and extra-neural tissues. However, the functional consequences of the membrane deficits may differ across the tissue types. There are several conditions such as Down’s syndrome, phenylketonuria, and various lipidoses (Scriver et al 1989), where the metabolic abnormalities are expressed in both neural and peripheral tissues, but the functional consequences are most profound in the central nervous system. This paradigm may also apply to schizo- From the VA Pittsburgh Healthcare System, Pittsburgh, Pennsylvania and the Department of Psychiatry, Western Psychiatric Institute & Clinic, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania. Address reprint requests to Dr. J. K. Yao, VA Pittsburgh Healthcare System, Neurochemistry and Psychopharmacology Laboratory, 7180 Highland Drive, Building 13, Pittsburgh PA 15206. Received October 19, 2001; revised February 18, 2002; accepted February 22, 2002. © 2002 Society of Biological Psychiatry 0006-3223/02/$22.00 PII S0006-3223(02)01397-5

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Page 1: Correlations between peripheral polyunsaturated fatty acid content and in vivo membrane phospholipid metabolites

Correlations between Peripheral Polyunsaturated FattyAcid Content and in Vivo Membrane PhospholipidMetabolites

Jeffrey K. Yao, Jeffrey A. Stanley, Ravinder D. Reddy, Matcheri S. Keshavan, andJay W. Pettegrew

Background: There is evidence for membrane abnormal-ities in schizophrenia. It is unclear whether the observedmembrane deficits in peripheral cells parallel centralmembrane phospholipid metabolism. To address this ques-tion we examined the relations between red blood cellpolyunsaturated fatty acids and brain phospholipid me-tabolites from different regions of interest in schizophre-nia and healthy subjects.

Methods: Red blood cell membrane fatty acids weremeasured by capillary gas chromatography and in vivobrain phospholipid metabolite levels were measured usinga multi-voxel 31P Magnetic Resonance Spectroscopy tech-nique on 11 first-episode, neuroleptic-naı̈ve schizophrenicsubjects and 11 normal control subjects.

Results: Both the total polyunsaturated fatty acids and theindividual 20:4(n-6) contents were significantly correlatedwith the freely-mobile phosphomonoester [PME(s-�c)]levels (r � .5643, p � .0062 and r � .6729, p � .0006,respectively). The 18:2(n-6) polyunsaturated fatty acidscontent correlated positively with freely-mobile phos-phodiester [PDE(s-�c)] levels (r � .5573, p � .0071). Theabove correlations were present in the combined right andleft prefrontal region of the brain, while other regionsincluding the basal ganglia, occipital, inferior parietal,superior temporal and centrum semiovale yielded nosignificant correlations.

Conclusions: Our preliminary data support the associa-tion between the decreased red blood cell membranephospholipid polyunsaturated fatty acids content and thedecreased building blocks [PME(s-�c)] and breakdownproducts [PDE(s-�c)] of membrane phospholipids in theprefrontal region of first-episode, neuroleptic-naı̈veschizophrenic subjects. Biol Psychiatry 2002;52:823–830 © 2002 Society of Biological Psychiatry

Key Words: Red blood cell, polyunsaturated fatty acids,

membranes, in vivo 31P magnetic resonance spectroscopy,phosphomonoesters, prefrontal, schizophrenia

Introduction

There is substantial evidence for peripheral membraneabnormalities in chronic schizophrenic patients

(Vaddadi et al 1989; Glen et al 1994; Yao et al 1994, 1996;Yao and van Kammen, 1994, 1996; Peet et al 1996), anda relative paucity of such evidence in early schizophrenia(Reddy et al 1999). On the other hand, there is convincingevidence for abnormal brain phospholipid metabolism infirst-episode and chronic schizophrenic patients (Pet-tegrew et al 1991; Keshavan et al 2000). The relationbetween peripheral findings and brain membrane alter-ations is unknown. If confirmed, such a relationshipprovides a unique opportunity to simultaneously investi-gate central and peripheral biochemistry, and their rela-tions to clinical measures. Relations between peripheraland central phospholipid metabolism, if found, will alsohave several important implications for schizophreniaresearch. Firstly, the findings to date in peripheral tissuewill assume greater relevance for understanding aspects ofpathophysiology of schizophrenia. This issue has beenvigorously debated because of examples in the literature,where peripheral measures either failed to adequatelyreflect central pathophysiology or did not serve as reliablebiological markers. Secondly, the findings will support thenotion that membrane abnormalities are present in bothneural and extra-neural tissues. However, the functionalconsequences of the membrane deficits may differ acrossthe tissue types. There are several conditions such asDown’s syndrome, phenylketonuria, and various lipidoses(Scriver et al 1989), where the metabolic abnormalities areexpressed in both neural and peripheral tissues, but thefunctional consequences are most profound in the centralnervous system. This paradigm may also apply to schizo-

From the VA Pittsburgh Healthcare System, Pittsburgh, Pennsylvania and theDepartment of Psychiatry, Western Psychiatric Institute & Clinic, University ofPittsburgh Medical Center, Pittsburgh, Pennsylvania.

Address reprint requests to Dr. J. K. Yao, VA Pittsburgh Healthcare System,Neurochemistry and Psychopharmacology Laboratory, 7180 Highland Drive,Building 13, Pittsburgh PA 15206.

Received October 19, 2001; revised February 18, 2002; accepted February 22, 2002.

© 2002 Society of Biological Psychiatry 0006-3223/02/$22.00PII S0006-3223(02)01397-5

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phrenia. Thirdly, if peripheral membrane indices parallelcentral phospholipid metabolism, and perhaps also neuro-morphometric findings, then there exists the possibilitythat alterations in peripheral membrane indices on longi-tudinal follow ups (repeated measures) can reflect centralnervous system membrane function over the course ofillness.

While a correlation between red blood count (RBC)polyunsaturated fatty acids (PUFAs) and brain phospho-lipid metabolites may not indicate a direct causal relation-ship, there is a growing body of evidence suggesting thatRBC membrane fatty acid changes parallel the brainmembrane fatty acid changes. In an earlier study byCarlson et al (1986), fatty acid-enriched diet in ratsresulted in a similar relative changes in phospholipid fattyacids of both neural and RBC membranes. Later, in fattyacid deficient juvenile rhesus monkeys fed with fishoil-rich diet, a parallel increase of 22:6(n-3) was alsoshown in brain and RBC membranes (Connor et al 1990).Following modification of maternal diet with enrichmentin fish oil, parallel increases of 22:6(n-3) levels werefound in brain and RBC membranes of the piglets (Ar-buckle and Innis 1993) and suckling rats (Araya et al1994). Similarly, breastfed infants had a greater proportionof 22:6(n-3) in their erythrocytes and brain cortex relativeto those fed formula (Makrides et al 1994). Recently, Yehet al (1998) have shown an increased accretion of 20:4(n-6) and 22:6(n-3) levels in brain and RBC membranesafter infant rats fed with rat milk formulas supplementedwith either 20:4(n-6) or 22:6(n-3). In two double-blindplacebo-controlled pilot studies of eicosapentaenoic acid(EPA) in the treatment of schizophrenia, Peet et al (2001)demonstrated that patients taking EPA had significantlylower scores on the first-episode, neuroleptic-naı̈veschizophrenic (FENNS) rating scale. Moreover, there wasa positive correlation between clinical improvement andrise in RBC arachidonic acid concentration (Peet et al2002). Taken together, these findings provide somewhatindirect support for the notion that peripheral measures ofRBC PUFAs could indeed be reflecting central PUFAmetabolism. However, few previous studies have directlyexamined the central-peripheral correlation in phospho-lipid metabolism in humans.

Using in vivo phosphorus magnetic resonance spectros-copy (31P MRS), it has been reported that schizophrenicpatients show alterations in membrane phospholipid me-tabolism in the prefrontal (Pettegrew et al 1991) and otherregions (for review, see Keshavan et al 2000). Thus, thepurpose of the present study is to test whether RBC levelsof phospholipid polyunsaturated fatty acids are associatedwith in vivo 31P spectroscopy measures of brain phospho-lipid metabolites, particularly in the prefrontal region.

Methods and Materials

Subject Selections and Diagnoses

Eleven FENNS subjects (males, n � 6; females, n � 5; meanage, 26; and age range, 17–44) were recruited through WesternPsychiatric Institute and Clinic (WPIC) inpatient and outpatientunits, after informed consent. A structured clinical interview(SCID) and available clinical information was utilized to derivea “best estimate” DSM-IV diagnosis of patients with schizophre-nia, schizoaffective, or schizophreniform disorder. AmmonsQuick Test was performed to estimate IQ (range was 82–107).The following criteria for inclusion and exclusion were appliedfor recruitment of schizophrenic patients:

INCLUSION CRITERIA. a) IQ of 75 or greater. For schizo-phrenic subjects, intellectual functioning was evaluated clinicallyat initial assessment; formal testing was deferred until patientshad been clinically stabilized; b) age between 12 and 45 years ofeither gender and any ethnicity; c) no prior history of neuroleptictreatment; d) DSM-IV criteria for schizophrenia, schizophreni-form disorder or schizoaffective disorder.

EXCLUSION CRITERIA. a) significant drug or alcohol usewithin 4 weeks of initial assessment; b) significant history of, orcurrent medical illness; c) hyperlipidemias at baseline, obesity,starvation in the previous 2 weeks; d) neurologic disorders,including head injury with loss of consciousness; e) history ofpsychosis longer than 2 years, defined by report of symptoms, inorder to control for possible confound of an extended psychoticprocess on outcome and study variables; and f) comorbidity forDSM-IV Axis I diagnosis.

Eleven healthy control subjects (males, n � 6; females, n � 5;mean age, 26; age range, 19–39) were matched for age, gender,and race distribution and screened for psychiatric illness with theuse of the SCID-NP. Prestudy evaluations included a completemedical history, physical examination, serum lipid analysis, andany other blood or laboratory tests as indicated by the history andphysical examination.

INCLUSION CRITERIA. a) age between 12 and 45 years ofeither gender and any ethnicity; and b) no history of currentpsychiatric or neurologic disorder.

EXCLUSION CRITERIA. a) a lifetime history of psychosis ormajor mood disorder; b) family history of psychosis or majormood disorder in a first-degree relative (family history-researchdiagnostic criteria); c) recent history (within past 6 months) ofalcohol or substance abuse (DSM-IV); and d) hyperlipidemias atbaseline, obesity, starvation/excessive dieting in the previous 2weeks.

Sample Preparation

Freshly drawn blood with anticoagulant citrate dextrose (ACD)was centrifuged at 750 � g for 7 minutes to remove platelet-richplasma and leukocytes. Immediately after fractionation, the redblood cell (RBC) samples were stored at �70°C ultracold freezer

824 J.K. Yao et alBIOL PSYCHIATRY2002;52:823–830

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until further analyses. All RBC samples used in this study havenever thawed previously. Hemoglobin-free RBC ghost mem-branes were prepared by the method of Dodge et al (1963). Lipidextraction of RBC ghost membranes was performed according tothe procedure of Rose and Oklander (1965). Fatty acid methylesters (FAME) were prepared using BF3-CH3OH reagent asdescribed by Morrison and Smith (1964). Diheptadecanoyllecithin (Matraya, Inc.) was used as an internal standard. Theresulting FAME were further purified by thin-layer chromatog-raphy, using the developing solvent systems described by Met-calfe et al (1966) to remove any excess catalysts and byproducts.This step can eliminate spurious peaks occurring during gaschromatographic analysis.

Quantitative Analyses of Fatty Acids by CapillaryGas ChromatographyAll the fatty acid methyl esters were analyzed on a Hewlett-Packard capillary gas chromatograph, Model 5890A, equippedwith a hydrogen flame ionization detector according to theprocedure published elsewhere (Yao et al 1994). Briefly, a30-meter, fused silica SP-2330 column, with an inner diameter(ID) of 0.32 mm and a 0.20 �m film thickness (Supelco,Bellefonte, PA) was used. Each sample was run under a splitlessinjection mode with hydrogen as the carrier gas (30 mL/min) andwith an inlet pressure of 6.5 psi. Oven temperature was pro-grammed under three stages: Stage 1, from 50 to 150°C at a rateof 25°C/min; Stage 2, from 150 to 175°C at a rate of 5°C/min;and Stage 3, from 175 to 235°C at a rate of 6°C/min., with a finaltime of 3 min at 235°C. Peaks on the chromatograms wereidentified by comparing the retention times with those ofstandard mixtures (Supelco, Inc.) and were calculated by aHewlett-Packard 3396 computing integrator using an internalstandard mode.

In Vivo 31P SpectroscopyACQUISITION OF DATA. A whole body 1.5 Tesla MR imager

equipped with a doubly tuned transmit/receive volume head coil(Isaac et al 1990) was used in this study. A self-refocused,slice-selective spin echo pulse sequence (Lim et al 1994) with aneffective flip angle of 60° and an echo time of 2.5 ms and withphase encoding gradients, was used to acquire the multivoxel 31Pspectroscopy data in a localized axial slice. Based on sagittalscout images, the axial slice was positioned parallel to the anteriocommissure-posterior commissure line to include the right andleft prefrontal, basal ganglia, superior temporal, inferior parietal,centrum semiovale regions (Figure 1). The experimental param-eters include: FOV 360mm, slice thickness � 30mm, 8 � 8phase encoding steps (nominal voxel dimension � 45 � 45 �30mm3), TR � 2 sec, 1024 data points, 4.0 KHz spectralbandwidth and NEX � 16.

POST-PROCESSING AND QUANTIFICATION. To optimizethe right and left voxel position for each region, the 8 � 8 gridwas shifted accordingly with respect to the anatomical magneticresonance imaging (MRI) prior to the inverse Fourier transfor-mation. The remaining post-processing and quantification stepswere 100% automated. A mild spatial (Fermi window) and

spectral (5 Hz exponential line broadening) apodization wasapplied. The 31P resonances, phosphomonoester (PME), phos-phodiester (PDE), phosphocreatine (PCr), adenosine triphos-phate (�-, �-, and �-ATP as two doublets and one triplet), andinorganic orthophophate (Pi), were modeled in the time domainwith exponentially damped sinusoids using the Marquardt-Lev-enberg algorithm.

A typical in vivo 31P brain spectrum collected in this study(Figure 2), not only included signals from freely-mobilephosphomonoesters [termed PME(s-�c), which are precursors ofmembrane phospholipids], freely mobile phosphodiesters[termed PDE(s-�c), which are breakdown products of membranephospholipids], phosphocreatine (PCr), inorganic orthophos-phate (Pi), and adenosine triphosphate (ATP), but also broadersignals underlying the PDE and PME spectral region, which arisefrom less mobile molecules with PDE and PME moieties such assynaptic/transport vesicles and phosphorylated proteins [termedPDE(i-�c) and PME(i-�c), respectively] (Pettegrew et al 1994).Quantifying the 31P data in the time domain allows us to omitdata points at the beginning of the free induction decay (FID) inthe fitting routine, without requiring additional corrections to thebaseline as it would if quantifying in the frequency domainTherefore, by omitting several points (i.e., having a relativelylong delay time (DT), the broad underlying spectral components,PDE(i-�c) and PME(i-�c), are removed from the fit as illustratedin the residual of Figure 2 and as described by Stanley andPettegrew (2000). As a result of implementing this method, thePME(s-�c) and PDE(s-�c) levels were quantified by using a longDT of 2.75 ms. To obtain the combined PME(i-�c)PDE(i-�c)levels, the FIDs were modeled a second time but with omittingthe first 1.0 ms of the FID and then taking the difference between

Figure 1. a) Sagittal magnetic resonance imaging (MRI) imageshowing the cross-sectional location of the 3 cm thick

31P

spectroscopy axial slice. An axial MRI image located in themiddle of the 3cm thick axial slice is used to show the differentshifting of the 8 � 8 voxel matrix used in order to sample thefollowing right and left regions of interest (white box): b)prefrontal, c) basal ganglia, d) superior temporal and inferiorparietal, e) centrum semiovale, and f) occipital regions.

RBC Fatty Acids and Brain MRS Measures 825BIOL PSYCHIATRY2002;52:823–830

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the total fitted amplitudes of the two modeled results (Stanleyand Pettegrew 2000). The right and left side effect was elimi-nated by averaging the signal from the two voxels prior to fitting,

which included correcting for phase and frequency shift. Addi-tionally, metabolite levels are expressed as a mole % relative tothe total fitted amplitude of the 1.0 ms DT fit.

Statistical Analysis

Group comparisons were done using Students t tests. Separateregression analyses were conducted for each of the biochemicalmeasures to determine whether RBC membrane phospholipidPUFAs of both normal control subjects and FENNS subjectswere correlated to their brain 31P spectroscopy measures ofphospholipid metabolites. To reduce the potential for Type Ierrors, the standard significance levels of p � 0.05 were adjustedfor multiple comparisons using Bonferonni correction. This wasaccomplished for families of primary variables (e.g., RBC fattyacid measures or 31P MRS measures), and the degree ofadjustment depended on the number of variables in each family.

Results

Group Differences in RBC Membrane Fatty Acids

In agreement with our previous findings in chronic schizo-phrenic patients (Yao et al 1994), the RBC levels ofarachidonic acid, 20:4(n-6), were significantly lower inFENNS subjects than in normal controls (Table 1). Inaddition, levels of total polyunsaturated fatty acids werealso lower in FENNS patients than in normal controls,although it is not statistically significant (p � 0.08). Nosignificant differences were observed in either saturated ormonounsaturated fatty acids between FENNS and controlsubjects.

Figure 2. A typical in vivo 31P spectrum with 5 Hz linebroadening from left and right prefrontal region of a controlsubject superimposed on the 1H MRI using the proposed proto-col. The modeled results using both the short (1.0 ms, top) andlong (2.75 ms, bottom) delay times are included by showing thefourier transform of the modeled free induction decays superim-posed on the acquired 31P spectra and the residuals below in eachvoxel.

Table 1. Comparison of RBC Membrane Fatty Acids (nmol/mL packed RBC) between NormalControls and First-Episode Neuroleptic-Naı̈ve Schizophrenic Patients

Fatty acidsNormal control subjects

(n � 11)FENNS subjects

(n � 11)p value

(two-tailed t tests)

Saturated:14:0 46 6 47 10 .784316:0 848 149 841 172 .918718:0 471 128 487 116 .757724:0 44 26 46 28 .8765Sub-totala 1420 271 1418 284 .9857

Monoenes:18:1 461 100 437 110 .591824:1 58 5 60 19 .8009Sub-totala 486 125 467 142 .7336

Polyenes:18:2(n-6) 327 80a 300 41 .337120:3(n-6) 48 16 45 17 .626020:4(n-6) 371 57 299 51 .0057b

22:4(n-6) 72 32 84 33 .381422:5(n-3) 59 17 47 18 .105022:6(n-3) 69 28 56 18 .2256Sub-total* 975 166 850 149 .0800

FENNS � first-episode neuroleptic-naı̈ve schizophrenic.aOther minor fatty acids, 15:0, 20:0, 22:0, 16:1 and 20:5(n-3), were also included in the final tabulation.bBold denotes statistically significant result.

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Correlations between RBC PhospholipidPolyunsaturated Fatty Acid Content and In Vivo31P Metabolite Levels

In the Prefrontal region, a significant positive correlationusing Bonferonni correction (p � 0.0125) was demon-strated between total PUFA contents and PME(s-�c) levels(r � 0.5643, p � 0.0062) and PCr/Pi ratios (r � 0.5604,p � 0.0067) (Table 2). There were no significant correla-tions in other regions including basal ganglia, occipital,inferior parietal, superior temporal, and centrum semi-ovale areas.

When the individual PUFA contents were analyzed inthe prefrontal cortex, arachidonic acid [20:4(n-6)] wassignificantly correlated with levels of PME(s-�c) (r �0.6729, p � 0.0006) and linoleic acid [18:2(n-6)] wassignificantly correlated with PDE(s-�c) levels (r � 0.5573,p � 0.0071) using Bonferonni correction (p � 0.0083)(Table 3). Moreover, there were marginal significantcorrelations between arachidonic acid [20:4(n-6)] andPCr/Pi (r � 0.5405, p � 0.0094), and between dihomo-gamma-linolenic acid [20:3(n-6)] and PCr/Pi (r � 0.5286,p � 0.0114). In contrast, arachidonic acid [20:4(n-6)]contents were inversely correlated with the PME(i-�c)PDE(i-�c) levels (r � �0.5448, p � 0.0088).

Regardless of normal control subjects or schizophrenicpatients, it is hypothesized that a generalized relationshipexists between peripheral and central membrane phospho-lipid turnover. Thus, we correlated RBC membrane phos-pholipid PUFAs of both normal control subjects andFENNS patients to their brain 31P MRS measures ofphospholipid metabolites (Tables 2 and 3). When subjectswere divided into the normal control and FENNS groups,stronger correlations appeared to be observed in theFENNS than in the normal control subjects in the presentsample size. In the FENNS group (Figure 3), significantcorrelations between arachidonic acid [20:4(n-6)] andPME(s-�c) level (r � 0.6230, p � 0.0406), and between

linoleic acid [18:2(n-6)] and PDE(s-�c) levels (r � 0.5928,p � 0.0546) were demonstrated. In the normal controlgroup, however, the correlations between arachidonic acid[20:4(n-6)] and PME(s-�c) level (r � 0.5618, p � 0.0721)and between linoleic acid [18:2(n-6)] and PDE(s-�c) levels(r � 0.5166, p � 0.1037) were only marginal.

Discussion

Combining the FENNS and control subjects in the corre-lation analysis, resulted in a significant association be-tween the total PUFA content and PME(s-�c) levels; afinding that only was present in the prefrontal and not inthe basal ganglia, superior temporal, inferior parietal,occipital or centrum semiovale regions. As for the associ-ation between the individual PUFA and 31P metabolites inthe prefrontal, the arachidonic acid [20:4(n-6)] contentcorrelated with the PME(s-�c) levels and PCr/Pi ratios andinversely correlated with the PME(i-�c) PDE(i-�c)levels. The PDE(s-�c) levels correlated with the linoleicacid [18:2(n-6)] content. In all, the above data demonstratesignificant correlations between RBC-PUFA contents andin vivo 31P spectroscopy measurements, which support thenotion that peripheral measurements of PUFA content inRBC parallel in vivo 31P spectroscopy measurements ofmembrane phospholipid metabolites in the brain not onlyin control subjects but also in FENNS subjects (Figure 3).Thus, longitudinal peripheral measurements of PUFAcontents in RBC could reflect longitudinal alterations inmembrane phospholipid metabolites and high-energy me-tabolism that may occur over the course of schizophrenia.

There is indeed support of an association betweenperipheral and central measurements. Richardson andcolleagues (2001) compared in vivo 31P spectroscopymeasurements averaged over 10 large voxels within thebrain and RBC PUFA composition in normal adult sub-jects and demonstrated an inverse correlation between

Table 2. Correlations of RBC Total Polyunsaturated Fatty Acids to the 31P Spectroscopy Measures of Brain PhospholipidMetabolite Levels from Different Regions of Interest

Regions of Interesta

Correlation coefficientsb (p value)

PME(s-�c) PDE(s-�c) PME(i-�c)PDE(i-�c) PCr/Pi

Basal ganglia �.0738 .0657 .0189 .4169Prefrontal .5643c (0.0062) .3796 �.4095 .5604c (.0067)Occipital �.1890 �.0248 .1831 �.2417Inferior parietal �.2121 .0853 .0898 �.0458Superior temporal �.0430 .0781 .0591 .2954Centrum semiovale �.3669 .1178 .2312 .2960

PUFAs, polyunsaturated fatty acids; PME(s-�c), freely-mobile phosphomonoester; PDE(s-�c), freely-mobile phosphodiester; PME(i-�c)PDE(i-�c), the relatively broadpeak underlying the PME and PDE resonances; PCr, phosphocreatine; Pi, inorganic orthophosphate.

aCombined right and left voxelsbFrom both normal control subjects and first-episode neuroleptic-naı̈ve schizophrenic subjects.cSignificant correlation with p � 0.0125 using Bonferonni correction.

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docosahexaenoic acid [22:6(n-3)] and eicosapentaenoicacid [20:5(n-3)] content and PDE levels. The quantifica-tion did not measure the PDE(s-�c) and the (i-�c) compo-nent of the PDE resonance, separately as did in this study.Therefore, it is possible that the (i-�c) component of thePDE resonance and not the PDE(s-�c) component isimplicated to the correlation, which would then be consis-tent with the inverse docosapentaenoic acid [22:5(n-3)]correlation with PME(i-�c) PDE(i-�c) levels (r �-0.4444, p � 0.0382) observed in the present findings(Table 3). This evidence suggests that peripheral PUFAmeasures are associated with larger, less mobile moleculeswith PDE and PME moieties and not the breakdown

products of membrane phospholipids, as suggested byRichardson et al (2001).

Of the six different PUFAs that were analyzed in RBC,the only significant group difference was a reduction inarachidonic acid [20:4(n-6)] in FENNS compared to con-trols. This specific reduction in arachidonic acid [20:4(n-6)] in RBC also has been reported in chronic medicatedschizophrenia subjects compared to controls (Yao et al1994). Using the identical spectroscopy protocol as in thisstudy, decreased PME(s-�c) and increased PME(i-�c) PDE(i-�c) in the prefrontal of FENNS subjects comparedto controls were reported in a preliminary study (Stanley etal 2001). In the prefrontal FENNS subjects showed sig-nificant increased PME(i-�c) PDE(i-�c) levels andtended to have lower PME(s-�c) and PDE(s-�c) levelscompared with controls (results not shown). The reductionof membrane phospholipid precursors and increasedPME(i-�c) PDE(i-�c) have also been observed by othersand in chronic medicated schizophrenia subjects (forreview, see Keshavan; et al 2000). These correlations andgroup differences suggest that decreasing membrane phos-pholipid precursor levels and increasing synaptic vesiclesand/or phosphorylated proteins are associated with de-creasing RBC arachidonic acid [20:4(n-6)] content.Though no group differences for PDE(s-�c) were seen inthis study, increasing breakdown products are associatedwith increasing linoleic acid [18:2(n-6)]. The significanceof these correlations will require further study.

In patients with generalized peroxisomal disorders,there is drastic reduction of membrane 22:6(n-3) levels(Moser and Moser 1996). Following treatment with 22:6(n-3) ethyl ester, normalization of both blood levels of22:6(n-3) and brain myelin as observed by MRI weredemonstrated (Martinez and Vazquez 1998). Recently,dietary supplementation with essential fatty acids (EFA)has shown promise in ameliorating some of the clinicalsymptoms of schizophrenia (Peet et al 1996; Peet 1999;Puri et al 2000) as well as cognitive impairments associ-ated with dyslexia and attention deficit hyperactivitydisorder (Richardson et al 1999; Stordy 1999). Thus,investigating EFA metabolism in peripheral membranessuch as RBC has the potential to be useful in monitoringthe therapeutic progress during EFA treatment for patientswith schizophrenia.

Previously, we (Reddy et al 1999) demonstrated thatFENNS patients (n � 24) had significant reductions intotal RBC PUFAs (�13%; p � 0.02), but not in mono-unsaturated or saturated fatty acids, relative to normalsubjects (n � 31). Specifically, reductions were found inarachidonic acid (�18%; p�0.002), docosapentaenoicacid (�36%, p�0.002), and docosahexaenoic acid(�26%; p � 0.003) concentrations. These reductions werenot related to age, gender, smoking status, or cotinine

Figure 3. Correlations between RBC membrane n-6 polyunsat-urated fatty acid levels and the 31P spectroscopy measures in theprefrontal region of patients with schizophrenia. 18:2(n-6), lino-leic acid; 20:4(n-6), arachidonic acid; PME(s-�c), phosphomono-ester; PDE(s-�c), phosphodiesters; RBC, red blood cell.

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levels. In the present study, a significant reduction ofarachidonic acid also was demonstrated in the FENNSpatients (Table 1) even though the sample size was smaller(n � 11). In addition, both 22:5(n-3) and 22:6(n-3) werelower in patients than in normal control subjects, althoughit is not statistically significant due to the smaller samplesize.

This study was supported in part by grants from the National Institute ofMental Health MH58141 (JKY), MH45203 (MSK), and MH46614(JWP), NARSAD Young Investigator Award (RDR), Office of Researchand Development (Merit Review, JKY), Department of Veterans Affairs,and the Highland Drive VA Pittsburgh Healthcare System. The authorsare grateful to C. Korbanic and L. McElhinny for their technicalassistance. The authors also are grateful for the time domain fittingsoftware package, which was provided by Dr. Drost’s laboratory,University of Western Ontario, London, Ontario, Canada.

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Table 3. Correlations of RBC Individual Polyunsaturated Fatty Acid Levels to the 31P Spectroscopy Measures of PhospholipidMetabolites in the Combined Right and Left Frontal Lobe of the Brain

RBC-PUFAs

Correlation coefficientsa (p value)

PME(s-�c) PDE(s-�c) PME(i-�c)PDE(i-�c) PCr/Pi

18:2(n-6) .3980 .5573b (.0071) �.3947 .391620:3(n-6) .3233 .2512 �.2675 .5286c (.0114)20:4(n-6) .6729b (.0006) .3538 �.5448c (.0088) .5405b (.0094)22:4(n-6) .0673 �.1320 .0399 .225922:5(n-3) .3724 .2102 �.4444 .190622:6(n-3) .1805 .0896 .0098 .4531

PUFAs, polyunsaturated fatty acids; PME(s-�c), freely-mobile phosphomonoester; PDE(s-�c), freely-mobile phosphodiester; PME(i-�c)PDE(i-�c), the relatively broadpeak underlying the PME and PDE resonances; PCr, phosphocreatine; Pi, inorganic orthophosphate.

aFrom both normal control subjects and first-episode neuroleptic-naı̈ve schizophrenic subjects.bSignificant correlation with p�0.0083 using Bonferonni correction.

cMarginal significance using Bonferonni correction.

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