JournalofMedicalPhysics/AssociationofMedicalPhysicistsofIndiaJMedPhys.35(3):154-163

Quantitationofnormalmetaboliteconcentrationsinsixbrainregionsbyin-vivo1H-MRspectroscopyLudovicoMinati12,DomenicoAquino2,MariaGraziaBruzzone2,AlessandraErbetta2

1.ScientificDepartmentFondazioneIRCCSIstitutoNeurologicoCarloBesta,Milano,Italy2.NeuroradiologyUnit,FondazioneIRCCSIstitutoNeurologicoCarloBesta,Milano,Italy

Addressforcorrespondence:LudovicoMinati,ScientificDepartmentandNeuroradiologyUnit,FondazioneIRCCSIstitutoNeurologicoCarloBesta,viaCeloria11,20133Milano,Italy.E-mail:lminati@istituto-besta.it

© JournalofMedicalPhysicsDOI:10.4103/0971-6203.62128Publishedinprint:Jul-Sep2010

Abstract

Thisstudyexaminedtheconcentrationsofbrainmetabolitesvisibletoin-vivo1H-MagneticResonanceSpectroscopy(1H-MRS)at1.5Tinasampleof28normalsubjects.Quantitationwasattemptedforinositolcompounds,cholineunits,totalcreatineandN-acetylmoieties,usingopen-sourcesoftware.Sixbrainregionswereconsidered:frontalandparietalwhitematter,medialtemporallobe,thalamus,ponsandcerebellum.Absoluteconcentrationswerederivedusingtissuewaterasaninternalreferenceandusinganexternalreference;metabolitesignalintensityratioswithrespecttocreatinewerealsocalculated.Theinter-individualvariabilitywassmallerforabsoluteconcentrations(internalreference)ascomparedtothatforsignalintensityratios.Significantregionalvariabilityinconcentrationwasfoundforallmetabolites,indicatingthatseparatenormativevaluesareneededfordifferentbrainregions.Thevaluesobtainedinthisstudycanbeusedasreferenceinfuturestudies,providedthesamemethodologyisfollowed;itisconfirmedthatdespiteunsuccessfulattemptsinthepast,smallercoefficientsofvariationcanindeedbeobtainedthroughabsolutequantification.

Introduction

Inprotonmagneticresonancespectroscopy(1H-MRS),thesignalintensityisnotonlyproportionalto

metaboliteconcentrationbutisalsoaffectedbyalargenumberofvariables,includingmetaboliterelaxationrates,pulsesequenceparametersandradio-frequencycoilsensitivity.Asaconsequenceoftheinherentdifficultiesinobtainingreliableabsoluteconcentrations,themajorityofin-vivoMRspectroscopystudiesofthebrainreportrelativemeasurements,calculatedassumingthattheconcentrationofcreatineisconstantandtakingitssignalasreference.However,theinterpretationofrelativedatacanbeambiguouswhentheconcentrationofcreatinechanges,suchasinearlydevelopmentandinbraintumors.[12]Inadditiontoremovinginterpretationambiguities,theprovisionofabsolutemeasurementsconsiderablysimplifiesthecomparisonofdatarecordedunderdifferentexperimentalconditionsandthecomparisonwithin-vitrobiochemicalmeasurements.[3]

Absolutemetaboliteconcentrationsmaybederivedusinganinternalreference,usuallyunsuppressedtissuewater,oranexternalreference,generallywaterorsodiumacetatecontainedinatubepositionednexttotheheadofthepatient.Bothmethodshaveimportantlimitations:theformercansufferfrombiasduetoalteredtissuewatercontent,forexamplecausedbyedema,andthelatterisassociatedwithlargeinter-subjectvariabilityduetocoilsensitivityinhomogeneities.Othermethodsbasedonreplacementphantomsandcoil-loadingmeasurementsalsoexistbutarelessfrequentlyused.[4–8]

Inordertoobtainabsoluteconcentrations,itisalsonecessarytodeterminetherelaxationratesofthemetabolitesofinterestinordertocorrectfortransverseandlongitudinalmagnetizationeffects.Directmeasurementwithrelaxometryisthemostaccurateoptionbutisoftenimpracticalduetoscantimelimitations.Asaconsequence,metaboliterelaxationratesarefrequentlyassumedtobeconstant,andstandardvaluesfromliteratureareused;thiscanbeasourceoferrorduetopathology-relatedchangesandregionaldifferences.[9–12]

Despitethesedifficulties,inrecentyearsquantitative1H-MRSofthebrainhasgainedincreasedacceptanceintheclinicaldomainandhasbeenappliedtothestudyofaging,seniledementia,epilepsy,multiplesclerosisandneuropsychiatricdisorders,amongothers;itprovidesatoolforquantitativemonitoringofdiseaseprogressionandtreatmentresponseandcansupportdifferentialdiagnosisbetweenconditionscharacterizedbysimilarimagingfindings.[13–17]

Thereremains,however,arelativepaucityofnormativestudiescoveringmultiplecorticalandsubcorticalbrainregions,aswellasalargeinter-studyvariabilityinreportedmetaboliteconcentrations(forthecerebralhemispheres,intherange4-8mMforinositolcompounds,1-5mMforcholineunits,6-14mMfortotalcreatine,and10-25mMforN-acetylmoieties)andinter-subjectvariationcoefficients(betweenabout10%andmorethan50%).[35–8]Ithasbeenshownthatdifferencesindataprocessingareadominantsourceofinter-studyvariability;forexample,LCModel

andAMARES,twowidelyusedcommerciallyavailablespectroscopytoolkits,mayperformdifferentlyintermsofsensitivitytonoise,linewidthandbaseline.[1819]

Recently,anopen-sourcetoolforquantitationofshortecho-timespectra,knownasAQSES,hasbecomeavailable.InAQSES,thequantitationproblemisformulatedasaseparablenonlinearleast-squaresfittingproblem,andfacilitiesforbaselinefitting,removalofresidualwaterandfilteringareprovided.[20]

Inordertocorroborateandextendtheexistingliterature,inthisstudywedeterminedthenormalabsoluteandrelativemetaboliteconcentrationsinanextendedsetofbrainregions:frontalandparietalwhitematter,medialtemporallobe,thalamus,ponsandcerebellum.Importantly,anotheraimwastocomparetheinter-subjectvariabilityofabsoluteconcentrationscalculatedusingtheinternalandexternalreferenceswiththatofrelativeconcentrations.Thiswasdonewiththepurposeofdeterminingwhetherperformingabsolutequantificationistrulyjustified,inspiteoftheneedtoapplymultiplecorrectionsinvolvingparameterswhichinjectmeasurementuncertainty.

MaterialsandMethods

Participants

Twenty-eightright-handedhealthyvolunteers,14femaleand14male,aged41.2±11.9years(mean±SD),wereenrolled.Allsubjects,unpaid,wereinformedaboutthepurposeandclinicalrelevanceofthestudy,andwritteninformedconsentwasobtainedfromthemaccordingtoinstitutionallyapprovedproceduresandregulations.Noneofthemhadapositiveanamnesisforneurologicalorpsychiatricdisorders,andallhadnormalmagneticresonanceimagingfindings(seebelow).

Dataacquisition

DataacquisitionwasperformedwithaSiemensMagnetomAvanto1.5Tscanner(SiemensAG,Erlangen,Germany)equippedwithatransceiverbirdcageheadcoil.Forthepurposeofpositioningthevoxelsforspectroscopyandtoexcludepathology,allsubjectswereimagedwithaxial,coronalandsagittalT2-weightedturbospin-echosequences,withTR=6270ms,TE=113ms,field-of-view

260×190mm,25slices,slicethickness4mmandnogap.Acoronalfluid-attenuatedinversion-recovery(FLAIR)sequencewasalsoused,withTR=8700ms,TE=121ms,TI=2400ms,field-of-view240×190mm,20slices,slicethickness4mmandnogap.High-resolutionT1-weighted

volumetricimageswereacquiredbymeansofaninversion-recoveryrapidgradient-echosequencewithTR=1160ms,TE=4.1ms,TI=600ms,field-of-view270×203mm,192slices,slicethickness1.1

mmandnogap.

Spectroscopyofthe1Hnucleuswasperformedusingasingle-voxel(SVS)point-resolvedspectroscopy(PRESS)sequence,withTR=1500ms,TE=30msandvoxelsize20×20×20mm[withtheexceptionofthemedialtemporalloberegion(seebelow),forwhichitwas40×10×20mm];foreachvoxel,1024datapointswereacquiredwithadwelltimeof1msand128averages.Metabolitespectrawereobtainedsuppressingthewatersignalbymeansofachemicalshift-selectivesaturation(CHESS)pulsewithbandwidth35Hz.Foreachspectroscopyvoxel,thesignalofunsuppressedwaterwasalsoacquired,with16averages.First-(X,YandZ),aswellassecond-order(XY,ZX,XY,Z2andX2-Y2),shimmingwasperformedautomaticallybymeansofafieldmap-basedalgorithmandthenrefinedmanuallybyanexperiencedoperator;thewidthathalfheightofthewaterpeakwasgenerallybelow10Hz(7±2.2Hz).A50-mLpolystyrenetubecontainingdistilledwater,positionedincontactwiththeheadnexttotheleftearlobe,servedasexternalreference;thecorrespondingwatersignalwasacquiredusinga10×10×10-mmvoxel.

Thestaticfieldhomogeneity,coilsensitivitymaps,tuningandsignal-to-noiseratio,radio-frequencychainlinearityandgradientperformancewereroutinelycheckedusingthestandardsoftwareandproceduresprovidedbythescannermanufacturer,withtheprescribedperiodicity.

Sixspectroscopyvoxelswerepositionedinthefollowingregions,showninFigure1,togetherwiththecorrespondingstereotacticcoordinatesgiveninMontrealNeurologicalInstitute(MNI)format:frontalwhitematter(FWM),parietalwhitematter(PWM),medialtemporallobe(MTL),thalamus(THAL),pons(PONS)andcerebellarhemisphere(CEREB).Notably,asaconsequenceofchoosingequalvolumeof8mLforallvoxels,itwasnotpossibletopositionthevoxelentirelywithintheponsandthalamus;asvisibleinFigure1,acontributionofsurroundingstructureswasalsopresent.TheNiewenhuysatlas[21]servedasanatomicalreference.Thespectroscopyvoxelswerenormallypositionedinthelefthemisphere;however,for9subjectsthecerebellarvoxelwasplacedintherighthemispherebecausemagneticsusceptibilityeffectsresultedinawaterpeakwidth>10Hzinthelefthemisphere.[22]For19subjects,spectrafromtherightmedialtemporallobewerealsoavailable;theywerenotacquiredforallsubjectsduetovariablescan-timelimitations.

Viewlargerversion

Figure1.Positioningofthevoxelsusedforacquisitionofthespectra,shownonthetransverse,coronalandsagittalplanesforarandomlychosensubject.ThestereotacticcoordinatesofthevoxelcentersaregiveninMNIformat.Thepolystyrenetubepositionednexttotheleftearlobeisvisible

(1),

Preprocessingandquantitationof1Hspectra

SpectrawereprocessedbymeansofthefreelyavailableAQSESsoftware(KatholiekeUniversiteitLeuven,Leuven,Belgium)runningunderMatLab7(TheMathWorksInc.,Natick,MA,USA)onaSunUltra80workstation(SunMicrosystemsInc.,PaloAlto,CA,USA).[20]Thesignalwaszero-paddedto4096points,apodizedwithaGaussianfunctionandFourier-transformed.Zero-orderrephasingandfrequencycorrectionwereperformedmaximizingthesymmetryofthemetabolitepeaksandcenteringtheN-acetylmoietiespeak(seebelow)on2.02ppm.Theresidualwatersignal(4.7ppm)wasremovedintherange4.3-5.1ppmbymeansoftheHankel-Lanczossingular-valuedecomposition(HLSVDpro).[20]Remainingbaselinefluctuationswereremovedbyfittingwithafifth-degreepolynomial.AsshowninFigure2,themetabolitespectrumwasfitintherange1.2-4.3ppmwiththelinearsuperpositionoffivecomponents,correspondingtoinositolcompounds(MI),cholineunits(CHO),creatineandphosphocreatine(CR),glutamateandglutamine(GLX),andN-acetyl(NA)moieties.Theintensityofthemetabolitesignalswasdeterminedintegratingthefollowingresonancepeaks:3.50ppmforMI,3.20ppmforCHO,3.00ppmforCR,2.70-1.70(range)ppmforGLX,and2.02ppmforNA.[35–6]Thebasisset,providedwiththesoftware,wasderivedfromphantommeasurements.[20]

Viewlargerversion

Figure2.Measuredandfittedspectrafromarandomlychosenacquisition.Thecontributionofeachcomponent(MI,CHO,CR,GLXandNA),thebaselineandtheresultingresidualsarevisible

VolumetricimageswerenormalizedtotheMNIspaceandsegmentedusingtheSPM5program(WellcomeNeuroimagingDepartment,London,UK),andtherelativecontentofwhitematter(WM),graymatter(GM)andcerebrospinalfluid(CSF)ineachspectroscopyvoxelwasdetermined.

Absoluteconcentrations,expressedinmillimolarities(mmol/L,mM),werecalculatedforMI,CHO,CRandNA(notforGLX,duetotheinabilitytoseparateglutamateandglutamineatafieldstrengthof1.5T),usingtheintensityofunsuppressedwatersignalinthespectroscopyvoxel(internalreference)orinthepolystyrenetube(externalreference)asreference.MetaboliteconcentrationsCmetweredeterminedusing

.

(2),

wherekmetandkH2OarecorrectionfactorsforT1recoveryandT2relaxationofmetaboliteandwater

(seebelow),SmetandSH2Oarethesignalintensities,nmetandnH2Oarethenumbersofprotons(4for

MI,9forCHO,3forCRandNAand2forwater),fCSFisthefractionofcerebrospinalfluidinthe

spectroscopyvoxel,Vmetisthevolumeofthespectroscopyvoxel(8mL),VH2Oisthevolumeofthe

voxelusedforwatersignalacquisition(8mLforinternalreference,1mLforexternalreference),andCH2Oisthereferencewaterconcentration.

Thecorrectionfactorformetabolite,kmet,wasdeterminedusing

.

wherefGMandfWMarethefractionsofgrayandwhitematterinthespectroscopyvoxel,and,T1,met,

GM,T2,met,GM,T1,met,WM,andT2,met,WMaretherelaxationtimesforthemetabolite,obtained

from[5]andprovidedinTable1.Notably,thefactor1/(1-fCSF)appearsinbotheq.1andeq.2;

whereasineq.1itaccountsforthefactthatmetabolitesignalisnotreceivedfromCSF,ineq.2itaccountsforthefactthattheweighingfactorsfortherelaxationterms,fGMandfWM,maynotsumto

1.

Seefulltable

Table1.Assumptionsaboutrelaxationtimesandwaterconcentrationsmadeforabsolutequantificationofmetaboliteconcentrations

Forquantitationwiththeinternalreference,thecorrectionfactorforwater,kH2O,wasdetermined

using

(3),

(4),

.

whereT1,H2O,GM,T2,H2O,GM,T1,H2O,WM,T2,H2O,WM,T1,H2O,CSF,andT2,H2O,CSFaretherelaxation

timesforwater,providedinTable1.Forquantitationwiththeexternalreference,inthecalculationofkH2Otherelaxationtimesofcerebrospinalfluidwereused,withfGM=fWM=0andfCSF=1,andkH2Owasmultipliedbyafactorof0.80,determinedfromcoilsensitivitymapsacquiredduringqualityassessmentchecks,toaccountforthedifferenceincoilsensitivitybetweenthetwopositions:centeroftheheadandcoilandaveragepositionofthepolystyrenetube.

Forquantitationwiththeinternalreference,theconcentrationofwaterinthevoxel,CH2O,was

determinedusing

.cH2O = fGMcH2O,GM + fWMcH2O,WM + fCSFcH2O,CSF

whereCH2O,GM,CH2O,WM,andCH2O,CSFaretheconcentrationsofwateringraymatter,whitematter

andcerebrospinalfluid,providedinTable1.[8]Forquantitationwiththeexternalreference,inthecalculationofCH2Oweset

fGM = fWM = 0 and fCSF = 1

Inadditiontotheabsoluteconcentrations,thesignalintensityratiosMI/CR,CHO/CR,GLX/CRandNA/CRwerealsoobtained.Asthesewerenotnormallydistributed,theratioswerelogarithm-transformed.

Statisticalanalysis

Foreachparameterofinterest,meanandstandarddeviationwerecomputed.ToensurethattherewerenosignificantdeviationsfromGaussiandistribution,Kolmogorov-Smirnovnormalitytestswere

(5),

performed.

Inter-subjectcoefficientsofvariation(CVs)werecalculatedfortheabsoluteconcentrationsandforthesignalintensityratiosusingtheformulaCV=SD/mean,andaveragedforeachmetaboliteacrossthesixregions.Totestforsystematicdifferencesbetweenthemetaboliteconcentrationscalculatedwiththeinternalandexternalreferences,pairedt-testswereperformed.

ForthosesubjectsforwhomspectrawereavailableforboththeleftandtherightMTL,lateralizationindices(LIs)fortheconcentrationsofMI,CHO,CRandNA,determinedusingtheinternalreference,werecomputedwiththeformula

.LI = 2(cL - cR)/(cL + cR)

whereCLandCRrefertotheconcentrationsintheleftandrightvoxels;foreachmetabolite,the

statisticalsignificanceoflateralizationwasevaluatedbymeansofpairedt-tests.

Totestfordifferencesinmetaboliteconcentrations(internalreference)andsignalintensityratiosamongbrainregions,analysisofvariance(ANOVA)wasperformed,followedbyBonferroni-correctedpost-hoct-tests.

ResultsTheabsolutemetaboliteconcentrationsandintensityratiosaregiveninTable2,andthecorrespondingbarchartsareshowninFigure3.Allconcentrationsandlogarithm-transformedintensityratioswerenormallydistributed.Acrossthespectraoftheparticipants,thefollowingaveragemetabolitesignal-to-noiseratios(SNRs)wereobtained:32.1±5.1forCEREB,28.7±6.6forFWM,26.8±6.7forMTL,27.3±7.5forPONS,28.6±6.9forPWMand26.2±4.8forTHAL.

Seefulltable

Table2.Absoluteconcentrations(internalandexternalreferences)andlogarithm-transformedintensityratiosofthemetabolites,andrelativevoxelcontents

Viewlargerversion

Figure3.Barchartsoftheabsoluteconcentrations(internalreference)andlogarithm-transformedsignalamplituderatios.Theerrorbarscorrespondto1SD

Therewerenosignificantdifferencesbetweentheaveragemetaboliteconcentrationscalculatedwiththeinternalandexternalreferences.TheaverageCVsforconcentrationsdeterminedwiththeinternalreferencewere32%forMI,23%forCHO,20%forCRand15%forNA.Incomparison,theCVsobtainedwiththeexternalreferencewerelarger:50%forMI,44%forCHO,41%forCRand41%forNA.TheCVsforthecorrespondingsignalintensityratioswereintermediate:41%forMI/CR,27%forCHO/CR,28%forNA/CRand48%forGLX/CR.

ThelateralizationindicesfortheMTLwere0.01±0.18forMI,0.05±0.18forCHO,-0.01±0.18forCRand0.01±0.10forNA;therewasnosignificantlateralization.

Thedifferenceinmetaboliteconcentrations(internalreference)amongregionswaslargestforNA(F5,27=16.1,P<.001),intermediateforCR(F5,27=13.5,P<.001)andCHO(F5,27=11.0,P<.001),

andsmallestforMI(F5,27=5.0,P<.001).TheconcentrationofMIrangedbetween5.8mM(PWM)

and8.8mM(PONS),thatofCHOrangedbetween2.9mM(PWM)and4.7mM(PONS),thatofCRrangedbetween10.5mM(PONS)and15.7mM(CEREB),andthatofNArangedbetween14.0mM(PWM)and18.4mM(PONS).

ThedifferenceintransformedintensityratiosamongregionswaslargestforCHO/CR(F5,27=19.3,

P<.001),intermediateforNA/CR(F5,27=13.9,P<.001)andMI/CR(F5,27=7.4,P<.001),and

smallestforGLX/CR(F5,27=5.2,P<.001).Thetransformedintensityratioln(MI/CR)ranged

between–0.35(CEREB)and0.15(PONS),ln(CHO/CR)rangedbetween–0.29(CEREB)and0.33(PONS),ln(GLX/CR)rangedbetween1.07(THAL)and1.56(PONS),andln(NA/CR)rangedbetween0.10(CEREB)and0.66(PONS).

Theresultsofpost-hoccomparisonsbetweenregionsaregiveninTable3.

Seefulltable

Table3.Resultsofpost-hoccomparisonsforabsoluteconcentrations(internalreference)andsignalintensityratios(↑(higher)and↓(lower)indicatestatisticalsignificanceatP<.05;↑↑and↓↓indicatestatisticalsignificanceatP<.01)

DiscussionWhiletherewerenosystematicdifferencesbetweenthemetaboliteconcentrationsestimatedwiththeinternalandexternalreferences,theexternalreferencewasassociatedwithconsiderablylargerinter-individualvariability,reflectedincoefficientsofvariationabouttwiceaslarge.Thisfindingisinlinewiththeresultsobtainedatthemajorityofcentersthatparticipatedinapreviousmulti-centricstudy.[7]Imperfectrepeatabilityinthepositioningofthereferencetubeisthemainsourceofrandomerror,anditseffectisamplifiedbychangesinthecoilsensitivityprofilebetweensubjects,causedbyvaryingshapeandsizeofthehead.Itthereforeappearsgenerallypreferabletousetheinternalreferenceunlessalterationsintissuewatercontentareexpected;moresuccessfulresultswithexernalstandardshave,however,beenobtainedbysomegroups.[6723]

Theinter-subjectcoefficientsofvariationwerelarger(byabout40%)forthesignalintensityratiosthanforthecorrespondingabsoluteconcentrationsestimatedwiththeinternalreference.Thislikelyresultsfromthecombinationoftwofactors:1)theactualinter-individualvariabilityinconcentrationislowerforwater(asconfirmedbyprotondensity-mappingstudies)thanforcreatine,and2)thewaterpeakismuchlargerthanthecreatinepeakandthereforeassociatedwithasmallerrelativemeasurementerror.[24]

Onecommoncriticismtoabsolutequantificationisthatitinvolvestheuseofmultiplecorrectionparameters,namely,CH2O,fGM,fWM,fCSF,T1,met,GMT2,met,GM,T1,met,WM,T2,met,WMwhichcarry

uncertaintiesthataredifficulttoestimateandwhichgetpropagatedinthefinalmeasurementresult.Althoughthisisanimportantshortcoming,ourfindingsontheinter-subjectcoefficientsofvariation(representingrandomerror)demonstratethatperformingabsolutequantificationisneverthelessmotivated,potentiallyleadingtoincreasedsensitivitytopathologicalchangewhencomparedtometaboliteratios.

Severalstudiesreportedmetaboliteconcentrationsinthecerebralhemispheres:theaverageconcentrationsobtainedinthepresentwork(7mMforMI,3mMforCHO,11mMforCRand14mMforNA)andthecorrespondinginter-individualCVsarewithinthespreadofvaluesfoundinliterature

(4-8mMforMI,1-5mMforCHO,6-14mMforCRand10-25mMforNA).[35–8]

TheNApeakisfrequentlyassumedtocorrespondtoN-acetylaspartate,acompoundofinterestasaputativeneuronalmarker.Ex-vivostudiesofthehumanandratbrainhavereportedconcentrationsintherange5-8mM,significantlybelowtheestimatesobtainedinthisstudyandinmostotherin-vivoMRSstudies.[2526]Itishypothesizedthatthisdiscrepancyarisesbecauseofunresolvedcompoundscontributingtothe2.02ppmNAresonanceobservedat1.5T,andpossiblyduetoN-acetylaspartatelossduringex-vivosamplepreparation.[35]TheCRpeakat3.00ppmcorrespondstothesuperpositionofcreatineandphosphocreatine,whichhavewell-knownrolesincellmetabolism,intheconcentrationratiodeterminedbyphosphokinaseequilibrium.[5]Ex-vivostudiesofthehumanandcaninebrainhavereportedconcentrationsintherange9-12mM,inlinewiththeresultsofthisstudyandotherin-vivoMRSstudies.[3527–28]TheCHOpeakat3.20ppmisthemostcomplex,receivingcontributionsfromarangeofcholine-containingcompounds,includingphosphocholine,glycerophosphocholine,freecholine,acetylcholine,phosphatidylcholineandcholine-plasmalogen;itsintensityisfrequentlytakenasanempiricalmarkerofthedensityandturnoverofcellmembranes.[35]Invitro,theconcentrationofphosphocholine,glycerophosphocholineplusfreecholinewasdeterminedtobeintherange1-2mM,inlineonlywithsomein-vivostudiesbutbelowtheestimatesofothers(includingthepresentone);thedeterminantsofthisdiscrepancyremainunclear.[35–6829]TheMIpeakat3.50ppmcorrespondstoarangeofcompounds,includingphosphatidylinositol,inositolpolyphosphatide,inositolmonophosphate,myo-inositoland,toasmallerextent,glycine;asinositoliselevatedwithinastrocytes,theintensityofthepeakisoftentakenasanempiricalmarkerofglialdensityandproliferation.Studiesonhumanbrainsampleshavereportedconcentrationsintherange5-7mM,inlinewiththeresultsofthisstudyandotherin-vivoMRSstudies.[682530]TheGLXcomplexintherange2.70-1.70ppmincludesmultipleoverlappingresonancesfromglutamateanditsprecursorglutamine;asthesecannotberesolvedat1.5T,quantitationwasnotattempted.[6]

Inagreementwiththeexistingliterature,significantregionaldifferenceswerefoundforallmetabolitesofinterest.[6822]ThesewerelargestforNA,intermediateforCRandCHO,andsmallestforMI.GiventhatN-acetylaspartateispresentinthesomaofneurons,indendritesandinaxons,itsregionalvariabilityislikelyrelatedtodifferencesinneuralarchitecture,populationanddensity;asimplelinearrelationshipwiththedensityofneuronsis,however,unlikelygiventhatitalsoreflectsreversiblemetabolicchanges.[31]Totalcreatineandcholinearelessspecificastheyincludecontributionsfrombothneuronsandglia,andtheirregionalvariabilityisprobablyrelatedtodifferencesinthedensityofthecellularmatrix;andinthecaseofcholine,alsointhelevelofmyelination.[32]Thevariabilityintheconcentrationofinositolcompoundscouldbemore

specificallyrelatedtotheglialpopulation,butitislikelyalsoinfluencedbyregionalmetabolicdifferences.[6]

TheconcentrationofN-acetylaspartatewashigherinthethalamus,ponsandcerebellumthaninthecerebralhemisphericregions;thiseffectcouldberelatedtohigherdensityofneuralsoma,axonsanddendritictreesintheseregions,andisfoundforthethalamusalsoin[8]butnotin.[6]Theconcentrationofcholineunitswasmarkedlyhigherintheponsthaninotherregions;thiseffectcouldberelatedtothehighmyelinationofthedenserostro-caudalpathwaysinthisregion[21]andisalsofoundin[6]and[8].Inagreementwiththesamestudies,theconcentrationofcholineunitswaslowestintheparietallobe;thesametrendwasobservedforinositol,andtheinterpretationisunclear.Theconcentrationofcreatinewasmarkedlyhigherinthecerebellumthaninotherregions;thiseffectcouldberelatedtotheveryhighdensityofneuralcellsinthecerebellum[21]andisalsofoundin[6]and[22].Thepresenceofsignificantregionaldifferencessignalstheneedtohaveseparatereferencevaluesforeachregionofinterest,especiallyforsubcorticalstructures.

Theconcentrationofmetabolitesinthehippocampuswasfoundtobesymmetrical,inlinewith[33]significantasymmetriesemergeinepilepsy,eveninabsenceoffrequentseizures.[15]

Thepresentstudyhasthreemainstrengths.First,incontrastwithseveralpreviousoneswhichconsideredspecificregionsonly,metaboliteconcentrationsweremeasured,forallsubjects,inanextendedsetofcorticalandsubcorticalbrainregions.Second,high-resolutionsegmentationwasperformedtocorrectforcerebrospinalfluidpartialvolumingandtoreportthewhite/graymattercontentsofeachvoxel.Third,afreelyavailablefittingprogramwasused,enablingcentersthatdonothaveaccesstocommercialspectroscopysoftwaretoreproduceourfindings.

Thereare,however,alsoseveralimportantlimitationsthatneedtobetakenintoconsideration.First,themetaboliterelaxationrateswerenotmeasured,duetoscan-timelimitationsrelatedtothelargenumberofvoxelsunderstudy,andthevaluesfromapreviousstudywereused.Whilethisisapotentialsourceoferror,itshouldbenotedthattheinter-studyvariabilityinrelaxationtimesisconsiderablysmaller(CV<20%)incomparisonwiththatinconcentrations.[9]Thereareknowndifferencesinrelaxivitiesamongbrainregions,whicharelessmarkedat1.5Tthanathigherfieldstrengths;thesewereindirectlytakenintoaccount,determining“effective”T1andT2foreach

metaboliteonthebasisofthecontentofeachvoxelandoftheaverageT1andT2valuesforwhiteand

graymatter.[59–11]Althoughthisapproachcannotrevealpathology-inducedchangesinthemetaboliteT2sasfound,forexample,instroke,thepotentialconfoundingeffectwasminimized

becausetheT2-weightingwasveryweak,giventhattheMRSechotime(30ms)wasconsiderably

shorterthanthemetaboliteT2s(200-500ms).[912]Second,theuncertaintyassociatedwitheach

correctionparameter(CH2O,fGM,fWM,fCSF,T1,met,GM,T2,met,GM,T1,met,WM,T2,met,WM)remains

unknown,andthefindingofreducedinter-subjectcoefficientsofvariationdoesnotexcludethepresenceofsignificantsystematicerror,eventhoughtherewerenosystematicdifferencesbetweeninternalandexternalreferences.Thisisagenerallimitationofthetechniqueatthepresentstage,whichneedsaddressingthroughdedicatedrelaxometryandsegmentationstudies.Ourfindingsofreducedinter-subjectvariabilitymotivatesuchstudies.Third,withtheexceptionofthemedialtemporallobe,voxelswerepositionedinonehemisphereonly,preventingthedeterminationofpotentialmetabolicasymmetriesrelated,forexample,totheknowndifferenceinaxonaldensityinsomeregionsbetweenthedominantandthenon-dominanthemispheres;thislimitationisincommonwithmanyothersimilarstudies.[357–8]Fourth,thenumberofsubjectsandtheagerangeweretoosmalltoinvestigateage-relatedchanges.Theagerangeunderconsiderationis,however,relevanttoawiderangeofpathologies,andpreviousstudiesindicatethattheage-relatedchangesinmetaboliteconcentrationsarenegligibleuntiltheseventhdecade.[32]Anotherlimitationisthatthestudywasconductedat1.5T,whiletheuseofa3-TscannerwouldhaveprovidedbetterSNRandpeakseparation,reducingquantitationuncertainty;however,inthemajorityofcenters,clinicalspectroscopyisstillroutinelypracticedat1.5T.[34]Duetotheinabilitytoresolveglutamineandglutamateresonancesat1.5T,thedataontheGLX/CRratioshouldbeinterpretedwithcaution.Finally,thevoxelsize(8mL)wascomparativelylarge,especiallyconsideringtheponsandthalamusregions.Althoughwehavecorrectedforpartialvolumingwithfluid-containingspaces,inclusionofgrayandwhitematterfromsurroundingareasaffectedtheanatomicalspecificityofthemeasurements.Reducingvoxelvolume,e.g.,to4mL,wouldincreaseanatomicalspecificity,aswellaslimitsusceptibilityeffects.Inthisstudy,thechoiceof8mLwasmadeinordertomaximizeSNR,andallvoxelshadequalvolumetoavoidintroducingSNRdifferencesacrosstheregionalspectra.IthasbeenpreviouslyshownthatreducingtheSNRincreasesrandomerrorandmayalsoresultinsystematicconcentrationbiases.[1934]WhilecomprehensiveevaluationsoftheeffectsofSNRareavailableforLCModelandAMARES,atpresenttheseremainlackingforAQSES.[19]AdditionalworkisthereforenecessarybothtodeterminehowSNRchangesaffecttheaccuracyofconcentrationscalculatedusingtheproposedmethod,andtoderivefurtherreferencevaluesusingsmallervoxels.

Moregenerally,alimitationofthepresentstudy,whichisincommonwithallothersimilarnormativestudies,[35–8]isthatthereferencevaluesarevalidonlysolongasthesamemethodologyandconditionsareapplied.ReplicationofthequantificationprocedureemployedinthepresentstudyappearsunproblematicasafulldescriptionhasbeengivenandtheAQSESsoftwareisfreelyavailable.Theexactpositionofthevoxelsinnormalizedspacewasprovided[Figure1],andthe

acquisitionparametersarestandardforclinicalMRSat1.5T.Nevertheless,thereareanumberofpotentialsourcesoferror,suchasdifferencesinsignal-to-noiseratio,shimmingqualityandpulsesequenceimplementation,asaconsequenceofwhichourfindingscannotimmediatelybeusedasnormalreferenceeveninabsenceofexplicitmethodologicaldifferences;rather,theyshouldinitiallybecomparedwithlocallyacquiredcontrolvaluesandincludedasreferenceonlyafterconfirmingtheabsenceofstatisticallysignificantdifferences.Forsituationsinwhichthesamequantificationmethodologycannotbefollowed,forexamplewhenadifferentvalueoffieldstrengthisused,thepresentvaluescannotbeconsideredandtherelevanceofthepresentworkispurelymethodological.

Inconclusion,wehavereportedthenormalin-vivoconcentrationsofinositolcompounds,cholineunits,totalcreatineandN-acetylmoietiesinfrontalandparietalregions,medialtemporallobe,thalamus,ponsandcerebellum.Thesevaluescanbeusedasreferenceforfuturestudiesprovidedthesamemethodology,basedonopen-sourcesoftware,isfollowed.Theinter-individualcoefficientsofvariationwerelargestforquantitationbasedonanexternalreference,discouragingitsgeneraluse;therewas,however,nosystematicbiaswithrespecttoquantitationbasedontissuewater.Theinter-individualcoefficientsofvariationwerelargerformetaboliteratiosthanforabsoluteconcentrations,confirmingthepotentialusefulnessofquantitativeMRSinspiteofthecomplexityofthecorrectionprocess.Significantdifferencesinmetaboliteconcentrationswerefoundamongbrainstructures.Whiletheirexactdeterminantscannotbedeterminedbyin-vivostudiessuchasthepresentone,theyarehypothesizedtoberelatedtoregionalvariabilityinneuralandglialpopulation,andinmyelination.Separatereferencevaluesareneededfordifferentbrainregions.

AcknowledgmentsWearegratefultoEmmaLisaStanleyforheroperationalcontributioninthepreprocessingandfittingofthespectra.WewouldalsoliketoexpressourgratitudetoDr.FaustoCevolani,whosupportedthepreparationofsomephantomsthatwereusedforpreliminarytests.Thisworkisdedicatedtohismemory.Wethanktwoanonymousreviewersforprovidinginsightfulfeedbackonanearlierversionofthemanuscript.

FootnotesSourceofSupport:Nil

ConflictofInterest:Nonedeclared.

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Figure1.Positioningofthevoxelsusedforacquisitionofthespectra,shownonthetransverse,coronalandsagittalplanesforarandomlychosensubject.ThestereotacticcoordinatesofthevoxelcentersaregiveninMNIformat.Thepolystyrenetubepositionednexttotheleftearlobeisvisible

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Figure2.Measuredandfittedspectrafromarandomlychosenacquisition.Thecontributionofeachcomponent(MI,CHO,CR,GLXandNA),thebaselineandtheresultingresidualsarevisible

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Table1.Assumptionsaboutrelaxationtimesandwaterconcentrationsmadeforabsolutequantificationofmetaboliteconcentrations

T1,GM T2,GM cGM T1,WM T2,WM cWM T1,CSF T2,CSF cCSF

MI1130ms

279ms

-1200ms

197ms

- - - -

CHO1390ms

401ms

-1440ms

325ms

- - - -

CR1320ms

204ms

-1300ms

209ms

- - - -

NA1330ms

399ms

-1380ms

483ms

- - - -

H2O 670ms 76ms52.3mM

510ms 67ms45.8mM

2400ms

160ms

55.5mM

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Table2.Absoluteconcentrations(internalandexternalreferences)andlogarithm-transformedintensityratiosofthemetabolites,andrelativevoxelcontents

FWM PWM MTL THAL PONS CEREB

Internalreference(absoluteconcentrationsinmM):

 MI 7.6±2.0 5.8±2.0 7.9±3.0 6.6±1.8 8.8±2.8 8.0±2.5

 CHO 3.6±0.8 2.9±0.4 3.6±1.1 3.4±0.8 4.7±1.1 3.9±0.9

 CR 11.5±2.4 10.7±1.5 12.0±4.0 12.0±1.1 10.5±3.2 15.7±2.1

 NA 14.2±2.0 14.0±1.8 14.1±2.5 16.3±2.0 18.4±3.0 16.4±2.8

Externalreference(absoluteconcentrationsinmM):

 MI 7.5±3.3 5.9±2.0 8.1±4.1 7.1±4.9 8.2±3.2 8.4±5.5

 CHO 3.8±1.8 3.0±0.8 3.8±2.0 3.5±1.6 4.6±1.9 4.1±2.1

 CR 11.9±4.9 11.0±3.2 12.4±5.9 12.6±5.2 10.3±4.3 16.6±7.1

 NA 14.8±6.4 14.3±3.7 14.6±6.5 17.4±8.6 18.2±6.8 17.4±7.5

Logarithm-transformedintensityratios:

 ln(MI/CR) -0.14±0.35 -0.32±0.43 -0.04±0.38 -0.30±0.28 0.15±0.43 -0.35±0.31

 ln(CHO/CR) -0.07±0.27 -0.21±0.17 -0.07±0.31 -0.19±0.26 0.33±0.26 -0.29±0.27

 ln(GLX/CR) 1.32±0.47 1.38±0.43 1.50±0.49 1.07±0.34 1.56±0.51 1.17±0.40

 ln(NA/CR) 0.27±0.20 0.32±0.15 0.26±0.36 0.36±0.15 0.66±0.36 0.10±0.20

Voxelcontent:

 %WM 80.4±8.1 81.9±7.1 34.4±10.5 14.0±4.7 48.8±19.1 39.0±15.7

 %GM 18.9±7.3 16.2±7.3 61.7±10.7 85.5±5.5 46.4±18.7 60.1±15.1

 %CSF 0.7±1.3 1.8±1.9 3.9±3.1 0.5±1.3 4.7±3.4 0.8±1.8

(valuesareexpressedasmean±SD)

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Figure3.Barchartsoftheabsoluteconcentrations(internalreference)andlogarithm-transformedsignalamplituderatios.Theerrorbarscorrespondto1SD

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Table3.Resultsofpost-hoccomparisonsforabsoluteconcentrations(internalreference)andsignalintensityratios(↑(higher)and↓(lower)indicatestatisticalsignificanceatP<.05;↑↑and↓↓indicatestatisticalsignificanceatP<.01)

FWM PWM MTL THAL PONS

vs.PWM

- - - -

vs.MTL

MI↓↓,CHO↓, - - -

MI/CR↓

vs.THAL

NA↓, NA↓↓NA↓↓,

GLX/CR↑↑- -

vs.PONS

CHO↓↓,NA↓↓,

CHO/CR↓↓,NA/CR↓↓

MI↓↓,CHO↓↓,NA↓↓,

MI/CR↓↓,CHO/CR↓↓,NA/CR↓↓

CHO↓↓,NA↓↓,

CHO/CR↓↓,NA/CR↓↓

MI↓,CHO↓↓,NA↓,MI/CR↓↓,CHO/CR↓↓,NA/CR↓↓,GLX/CR↓↓

vs.CEREB

CR↓↓,NA↓↓,

CHO/CR↑

MI↓,CHO↓↓,CR↓↓,NA↓↓,NA/CR↑

CR↓↓,NA↓↓,MI/CR↑↑,CHO/CR↑↑,GLX/CR↑

CR↓↓,NA/CR↑↑

CHO↑↑,CR↓↓,NA↑,MI/CR↑↑,CHO/CR↑↑,NA/CR↑↑,GLX/CR↑

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