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Publicadoen:ChildsNervousSystem27(6):879891;2011
MeasurementofMelatonininBodyFluids:Standards,Protocols,andProcedures
EduardoAlvesdeAlmeida1,PaoloDiMascio2,TatsuoHarumi3,AdamMoscovitch4,Rdiger
Hardeland5,DanielP.Cardinali6,GregoryM.Brown7,S.R.PandiPerumal8,CA
DepartamentodeQumicaeCinciasAmbientais,IBILCE,UNESP,RuaCristvoColombo
2265,CEP15054000,SoJosdoRioPreto,SP.BRAZIL.
DepartamentodeBioqumica,InstitutodeQumica,USP.Av.Prof.LineuPrestes,748,CEP
05513970,SoPaulo,SP.BRAZIL.
DepartmentofAnatomy,AsahikawaMedicalCollege,JAPAN.
CanadianSleepInstitute,Toronto,ON,CANADA.
JohannFriedrichBlumenbachInstituteofZoologyandAnthropology,UniversityofGttingen,
Gttingen,GERMANY.
DepartamentodeDocenciaeInvestigacin,FacultaddeCienciasMdicas,Pontificia
UniversidadCatlicaArgentina,1107BuenosAires,ARGENTINA.
DepartmentofPsychiatry,UniversityofToronto,100BronteRd.Oakville,ON,L6L6L5,
CANADA.
SomnogenInc,8790112thStreet,NewYork,NY11418,USA.
CA
S.R.PandiPerumal
200LansdowneAvenue
Toronto,
ONM6K2V9
CANADA
sleepresearch@gmail.com
1
Abstract
Thecircadianrhythmofmelatonininsalivaorplasma,orofthemelatoninmetabolite6
sulphatoxymelatonininurine,isadefiningfeatureofsuprachiasmaticnucleusfunction,the
endogenousoscillatorypacemaker.Thesemeasurementsareusefultoevaluateproblems
relatedtotheonsetoroffsetofsleepandforassessingphasedelaysoradvancesofrhythmsin
entrainedindividuals.Additionally,theyhavebecomeanimportanttoolforpsychiatric
diagnosis,itsusebeingrecommendedforphasetypinginpatientssufferingfromsleepand
mooddisorders.Thus,thedevelopmentofsensitiveandselectivemethodsfortheprecise
detectionofmelatoninintissuesandfluidsofanimalsemergesasnecessary.Duetoitslow
concentrationandthecoexistenceofmanyotherendogenouscompoundsinblood,the
determinationofmelatoninhasbeenananalyticalchallenge.Thisreviewdiscussescurrent
methodologiesemployedfordetectionandquantificationofmelatonininbiologicalfluidsand
tissues.
Keywords:Melatonin;Circadianrhythms;Radioimmunoassay;EnzymeLinkedImmunoassay
Highperformanceliquidchromatography;Massspectrometry;Capillaryelectrophoresis
1.Introduction
Melatonin(Nacetyl5methoxytryptamine)isacompoundsecretedmainlybythepineal
gland,butsynthesizedalsoinmanyothertissuesandcellslikeretina(CardinaliandRosner,
1971;TosiniandMenaker,1998;Liuetal.,2004),humanandmurinebonemarrowcells(Conti
etal.,2000),platelets(Champieretal.,1997),gastrointestinaltract(Bubenik,2002),skin
(Slominskietal.,2005)orlymphocytes(CarrilloVicoetal.,2004).Withregardtothe
multiplicityofsitesofformationandthepresenceofmelatoninreceptorsinvariousdifferent
places,melatoninisaversatilephysiologicalsignalthathasbeenrelatedtothecontrolof
numerousphysiologicprocesses(DubocovichandMarkowska,2005;PandiPerumaletal.,
2008).Inmammals,photoperiodicinformationisrelayedthroughthesecretionofthis
hormonebythepinealgland,whichthenactsonthebrainandtheneuroendocrinesystemto
produceadaptivechangesinendocrinology,anatomyandphysiology,thusaffectingsleep,
reproduction,moulting,immuneresponses,energybalanceandbehavior,amongothers(Lewy
etal.,2006;CarrilloVicoetal.,2006;Arendt,2006;Reiteretal.,2009b).Moreover,melatonin
exhibitsdirectandindirectantioxidantproperties,andthereisstrongevidencethatthis
compoundcancounteractthedeleteriouseffectsofreactiveoxygenandnitrogenspeciesin
differentsystems(deAlmeidaetal.,2003;Onukietal.,2005;Reiteretal.,2007;Hardelandet
al.,2009;Reiteretal.,2009a).
Althoughnumerousphysiologicalfunctionshavebeenattributedtomelatonin,
mechanismsinvolvedinsuchfunctionsarefrequentlyunclear,especiallywhenparallel
signalingpathwaysareinitiatedviathemembranereceptorsMT1orMT2,orwhenother
melatoninbindingsitesareinvolved(Hardeland,2009).Therefore,furtherinvestigationat
cellularandmolecularlevelsisneededtoelucidatehowthiscompoundreallyactsasa
relevantphysiologicalregulatorysignal.
Althoughinvitrostudiescanfurnishimportantinformationontheeffectsofmelatonin
incellcultureorperfusedtissues,studiesonthefluctuationsofmelatoninconcentrationin
bodyfluidsandtissuescanbethemostrelevantissuetocomprehenditsfunctionin
organisms.Thus,thedevelopmentofsensitiveandselectivemethodsfortheprecisedetection
ofthesecompoundsintissuesandfluidsofanimalsemergesasnecessary.However,duetoits
lowconcentrationandthecoexistenceofmanyotherendogenouscompoundsinblood,the
determinationofmelatoninhasbeenananalyticalchallenge.Thisreviewdiscussescurrent
methodologiesemployedfordetectionandquantificationofmelatonininbiologicalfluidsand
tissues.
2.Melatoninfluctuationsinorganisms
3
Invertebrates,thechronobiologicallyrelevantfractionofmelatoninismainly
producedandreleasedintothecirculationbyeitherthepinealgland,especiallyinmammals,
orpinealglandplusretina,e.g.insomebirdsandamphibians(Bentley,2001;Doyleand
Menaker,2007).Nonmammalianpinealsaredirectlyphotosensitivewhilepinealglandsof
mammalsarecontrolledbyneuronalphototransductionpathwaysoriginatingintheretinaand
processedbythehypothalamiccircadianpacemaker,thesuprachiasmaticnucleus(SCN)(Doyle
andMenaker,2007)(Goldman,2001;Moore,2007).Invariousbirds,lightinfluencescircadian
oscillatorspresentinthepinealglandsandactadditionallyonneuronalpathwaysofretinalor
hypothalamicoriginmodulatingthegland(Zawilskaetal.,2004;Csernus,2006;Cassoneetal.,
2009).Thecontributionofextrapineal/extraretinalmelatonintobloodplasmaconcentrations
ofthehormoneareeitherverylowor,inthecaseofgastrointestinalorigin,onlyepisodicand
withoutprofoundchronobiologicalsignificance(Bubenik,2002).
Inhumans,nocturnallypeakinghighamplitudeoscillationsofmelatoninintheplasma
areparalleledbycorrespondingvariationsinsaliva(Benloucifetal.,2008).Althoughplasma
levelsaregenerallyabout10timeshigherthanthosefoundinsaliva,determinationsof
salivarymelatonincanbeofadvantage,especiallywheninvasiveprocedureshavetobe
avoided.Theprimarymelatoninmetaboliteintheurine,6sulphatoxymelatonin,alsooscillates
consistentlywithmelatoninconcentrationinplasmaandsaliva(Benloucifetal.,2008).
Interestingly,melatoninrhythmsinfishblood,whichlikewiseexhibitpeaksduringnighttime,
canbefollowedinthewaterofanaquarium,towhichthehormoneisreleased(Jamesetal.,
2004).
Melatoninlevelsinhumanplasmausuallybegintoincreasebetween18:00hand20:00
h,andpeakbetween00:00hand05:00h(Lewyetal.,1985;Laganaetal.,1995;Pandi
Perumaletal.,2007;Benloucifetal.,2008;Machidaetal.,2009),beingfollowedbyarapid
decrease.Thedurationofthenocturnalmelatoninpeakhasbeenshowntobethecrucial
signalforencodingseason(BartnessandGoldman,1989).Howeverchangesintheduration
alsooccurinnonseasonalanimals.Forexample,intheratthedurationofthemelatonin
increaseiscloselytiedtothephotoperiod(Hoetal.,1984).Seasonalchangesinthenocturnal
peakofmelatoninhavealsobeenwidelyreportedwiththeamountofchangevaryingbetween
species.ThenocturnalpeakofpinealmelatoninsecretionintheSiberianhamsterduring
winteris2timesgreaterthanduringsummer(Ribelaygaetal.,2000),whilethatofthe
Europeanhamstershowsa10foldincrease(VivienRoelsetal.,1992).Thusbothdurationand
magnitudeofmelatoninsecretionarestronglycorrelatedwiththechangingphotoperiodsof
theseason(Garidouetal.,2003;Srinivasanetal.,2008).Seasonalvariationsinmelatonin
durationandlevelsarealsofoundinhumans,butlessprominent.Again,thedurationand
4
heightofthemelatoninpeakisnegativelycorrelatedwiththelengthofthephotoperiod
(MoreraandAbreu,2006;UenoTowatarietal.,2007).
Anotherimportantaspectofmelatoninfluctuationinhumansconcernsvariable
environmentallightintensities.Ithasbeenproposedthatmelatoninsecretionduringdark
phasesisgreatlyinfluencedbydimlight,whereasverybrightlightcanmaskmelatonin
production(Lewyetal.,1980).Dimlightcanbeparticularlyimportantinthecircadian
entrainmentoftherhythm.Moreover,thesuppressionofmelatoninformationandreleaseby
nocturnallightrepresentsawellknownphenomenon,ofparticularimportanceinshiftwork
(Stevensetal.,2007).Thephoticshutoffmechanismsdependontherespectiveorganismand
maybebasedeitherondephosphorylationofakeyenzymeinmelatoninbiosynthesis,
arylalkylamineNacetyltransferase(AANAT),leadingtotheincapabilityofinteractingwitha
1433proteinfollowedbyrapidproteasomaldegradation,and/orondownregulationof
AANATexpression(Schomerusetal.,2000;Gangulyetal.,2001).Consequently,melatonin
actionsarenotonlyinfluencedbythephaseofthelightdarkcycle,butcanbestrongly
affectedbyvariationsinlightanddimlightintensities.Moreover,bothnormalmelatonin
patternsandtheinfluencesbylightcanvaryconsiderablybetweenindividuals,eitherinterms
ofapersonalcharacteristic(Grofetal.,1985;WaldhauserandDietzel,1985;Kolleretal.,1994;
LerchlandPartsch,1994)orasaconsequenceofagingoraprogressingdiseases(Brownetal.,
1979;Girottietal.,2000;KarasekandReiter,2002;Girottietal.,2003;Yapraketal.,2003;
Peschke,2008).Studiesinsiblingshaveindicatedthatsomeofthisvariationhasagenetic
origin(Griefahnetal.,2003).Diseasedependentdecreasesofmelatoninwerealsodetectedin
Alzheimerpatients,notonlyintheblood(SkeneandSwaab,2003;WuandSwaab,2005)but
alsointhecerebrospinalfluid(Zhouetal.,2003),anotherbodyfluidofdiagnosticrelevance.
Thesechangesinnocturnalmelatonincouldberesponsibleforgreatvariationsinthe
pacemakerprocessescontrollingcircadianandannualrhythms.Thiscouldbealso
extrapolatedtomanyhealthdisorders.Inetiologicalterms,changesinmelatoninhavebeen
repeatedlysuspectedtobeinvolvedinnumerousdiseases,inthesusceptibilityto
inflammatoryprocessesoringeneticpredispositions.Thehealthrelatedrolesofmelatonin
seemtoreflectamixtureofhormonal,immunomodulatory,neuromodulatoryandvarious
typesofantioxidantactions,anditsefficacyinsafeguardingtheseunderlyingprocessesis
observedatverylowconcentrations(Hardeland,2009).Therefore,itseemsthatfluctuations
inmelatonindurationandlevelswhichmayappear,atfirstglance,tobeofonlyminorextent,
maycause,onthelongrun,importantpathophysiologicalchanges.Becausemelatoninlevels
arerelativelylowevenatnighttimehighlyspecificandsensitivemethodsformelatonin
measurementsinbiologicalsamplesareamatterofnecessity.
5
3.Determinationofmelatonininbiologicalsamples
3.1.Samplepreparation
Consideringitslowlevelsanimportantissueofmelatoninmeasurementsisitsadequate
extractionfrombiologicalsamples.Inserumsamples,melatonincanbeextractedbysimple
liquid/liquidprocedures,suchastheadditionofdichloromethane(1:1,v/v).Samplesarethen
vigorouslymixedandcentrifugedtoobtainaqueousandorganicphases.Withthisprocedure,
melatoninisretainedinthedichloromethanephasesthatarecollectedanddriedunder
nitrogenatmospheretoconcentratemelatonin.Thisyieldsasatisfactoryrecoveryrate
(generallymorethan70%)andcanbealsoappliedtobufferhomogenizedtissues.However,
Rizzoetal.(2002)reportedlowprecisionandaccuracywithsingleliquidliquidextractionsof
melatoninforhighperformanceliquidchromatography(HPLC)coupledtofluorescence
detector(Rizzoetal.,2002).Formultipleanalysesofmelatoninanditsprecursorsor
metabolites,moreprofoundliquidliquidextractionshavebeenalsodescribedusinga
combinationofdifferentsolvents(HarumiandMatsushima,2000).
Inolderinvestigations,chloroform(trichloromethane)wasmostlyusedformelatonin
extractionandisstillinusetoday.Althoughthismethodcanbeused,dichloromethaneis
preferredforreasonsoflowertoxicity.Generally,chlorinatedmethaneshouldbeofhighest
purityandprotectionfromlightandredoxactivecompoundsisofutmostimportancetoavoid
formationofreactiveintermediateswhichcandestroymelatonin.
Laganetal.(1995)describedanextractionprocedureforserumsamplesthroughan
LC18cartridgeplusaCarbographcartridgewitharecoveryrangingfrom86.3to91.7%for10
to200pgofmelatoninmL1.Briefly,2mLofserumsampleispassedthroughanLC18
cartridge,whichisthenwashedwith2mLofwaterand2mLofwatermethanol(90:10,v/v)
(Laganaetal.,1995).Thereafter,melatonincanbeelutedfromthecolumnwithpure
methanol,driedandresuspendedinanappropriatesolutionforanalysis(Sieghartetal.,1987)
orbefurtherpurifiedbyelutingwith2mLofwatermethanol(40:60,v/v)andloadingontoa
Carbographcartridge,asdescribedby(Laganaetal.,1995).Thecartridgeisthenwashedwith
10mLofmethanoland3mLofmethanoldichloromethane(80:20,v/v),andmelatoninis
finallyelutedwith1.5mLofmethanoldichloromethane(10:90,v/v).Theeluateisevaporated
todrynessunderN2atmosphereandresuspendedin100 Lofwatermethanol(75:25,v/v)for
analyses.
Samplepreparationwillalsodependonthemethodusedforanalysis,sincethe
presenceofothercompoundsinthesamplecaninterferewiththemelatoninsignal.The
extentofmelatoninprepurificationfrombiologicalsamplescan,insomecases,be
6
fundamentalforthesensitivityofthemethodused.Theproceduredescribedaboveallows
melatonindetectionwithhighsensitivityandwithoutinterferenceofothercomponentsinthe
sample(Laganaetal.,1995).Ithasbeenshownthathomogenizationin10volumesoficecold
0.1Mperchloricacidcanalsorepresentagoodmeansformelatonindeterminationintissues
byHPLCcoupledtoelectrochemicalorfluorescencedetection(Harumietal.,1996).Inthis
case,thehomogenateiscentrifugedat10000gfor20minat4Candtheresulting
supernatantcanbedirectlyinjectedintotheHPLCsystem.Itwasalsosuggestedtomix90 L
ofthesupernatantfractionwith10 Lof1Msodiumphosphate,pH4.3,forbetterresolution
ofpeaks.
Dependingonthemethodused,furthertreatmentofmelatoninextractsmaybe
needed.Gaschromatographycoupledtomassspectrometry(GCMS)detectionofmelatonin
requiressamplederivatizationformelatoninvolatilizationby,forexample
pentafluoropropionicanhydrideorheptafluorobutyrylimidazole(Degenetal.,1972;Covaciet
al.,1999).Inanothercase,humanplasmasamplesweredirectlyinjectedandevaluatedinan
HPLCsystemwithfluorescencedetectionwithoutpriorextractionorpurification,achievinga
detectionlimitof1ngpermlofhumanplasma(4pmol/ml)(Bechgaardetal.,1998).Also,it
hasbeenreportedthatderivatizationofmelatoninwithsodiumcarbonateandhydrogen
peroxideincreasessensitivityalmosttenfoldformeasurementinHPLCsystemscoupledto
fluorescencedetectors(Tomitaetal.,2003).
Roliketal.(2002)describedahighlyspecificmethodformelatoninisolationand
purificationfromcomplexbiologicalmatricesbyimmunoaffinitychromatography(Rolciketal.,
2002).PolyclonalantibodieshighlyspecificagainstmelatoninwereraisedbyMannich
synthesis(Grotaetal.,1981)andusedforpreparationofimmunoaffinitygel,witha95%
recoveryrateformelatoninextraction.Inthesesamples,melatoninconcentrationwas
determinedbyHPLCcoupledtomassspectrometrywithadetectionlimitof10fmol.
Regardingsamplepreparationforanalysisbymassspectrometry,theuseofadequate
isotopicallylabeledinternalstandardsrepresentsanimportantissue,improvingquantification
ofthehormoneandavoidingunderestimationofactuallevelsofmelatoninduetolosses
whichmighthaveoccurredinthesamplesduringextraction.
Finally,thecorrecthandlingandmaintenanceofsamplesisimportant,withsamples
keptconstantlyoniceandprotectedfromlightradiation,inordertoavoidmelatonin
degradation.Despiteitsrelativestability,melatoninoxidationcanoccurovertime,including
thereactionwithsingletoxygenthatcanbegenerateddependingonoxygenavailabilityand
lightincidence.Forsamplefreezing,itisrecommendedthatsamplesbedriedand
preferentiallykeptundervacuumornitrogenatmosphere.
7
3.2.Immunoassay
3.2a.Preparationofantisera
Forthemonitoringofmelatonininbiologicalfluids,useofimmunologicalmethodsisthe
mostwidespreadmethod.Severalcommercialkitsbasedonthesemethodshavebeen
availableformelatonindetermination.Someofthesemethodsarehighlysensitiveandsimple
touse(lowerlimitofdetection:0.5pgmL1)butmaysufferfromapotentialriskofcross
reactivitytostructurallysimilarcompoundsifmelatoninisnotextracted(Lemaitreand
Hartmann,1980;Dietal.,1998).
Themostcrucialaspectofimmunoassaysisthepreparationoftheantiserum.Because
melatoninistoosmalltobecapableofproducingantiseraonitsownitmustbecoupledtoan
antigenicprotein.Insuchaconjugatethesmallmolecularweightsubstanceiscalledahapten.
Theresultingantiserumbindsboththeproteinandthehaptenplusaportionoftheadjacent
protein.Thehaptenhasfewantigenicdeterminantsrelativetotheprotein.Specificitystudies
ofantiseraproducedbysteroidproteinconjugatesshowedthatantiseraarenotableto
discriminatestructuraldifferencesinthehaptenthatareimmediatelyatorclosetothesiteof
coupling(Grotaetal.,1983).
Thechoiceofthehaptenandconjugationreactionshouldthereforebedeterminedby
thetypeofdiscriminationthatisrequired.Indolealkylamineshaveincommonaringnitrogen
(position1)andanadjacentcarbon(Position2).Thusformelatonin,couplingviatheposition1
orposition2shouldallowresultingantiseratodiscriminatedifferentindolesthatare
commonlyfoundintissues.
StudiesofantiseraresultingfromMannichcouplingofmelatonintobovineserum
albumin(BSA)revealthatthisapproachleadstoahighlyspecificmelatoninantiserumas
shownbycrossreactivitystudiesinradioimmunoassay(RIA)(Bubeniketal.,1976;Pangetal.,
1977;LemaitreandHartmann,1980;Yangetal.,2006).Todeterminethelocusofattachment
ofmelatonintoprotein,modelreactionswereconductedandresultingproductsanalyzedby
nuclearmagneticresonanceandinfraredspectroscopy(Grotaetal.,1983).Resultsindicated
thatcouplingwaslikelyatposition2.Furtherstudiesweredoneofcrossreactivityof
intermediatereactionproductsrevealingthathighestcrossreactivityoccurredwithC2
substitutedmelatoninderivatives.Thusitwasconcludedthatthemethylenebridge
conjugatingmelatonintoBSAoccurredatthe2positionoftheindolemolecule.Thisapproach
hasbeenusedwidelyformelatoninimmunoassays.Recentlytwodifferentgroupshaveused
thisapproachingeneratingmonoclonalantiseraagainstmelatonin(Yangetal.,2006;
Soukhtanlooetal.,2008).
8
Couplingattheringnitrogenusing1(pcarboxybenzyl)melatonin(deSilvaand
Snieckus,1978)coupledtoBSAasantigen(Grotaetal.,1981)resultsinantiserathatbind
melatoninspecifically.Melatonin1propionicacidcoupledtoBSAalsostimulatesproduction
ofhighlyspecificantisera.Asimilarapproachcoupling1(4carboybutyl)melatonintoprotein
resultedinahighlyspecificRIA(Kawashimaetal.,1984).Themelatoninderivative,3(3(2
acetamidoethyl)5methoxyindollyl)propionicacidcoupledtobovinegammaglobulin
producesspecificantiserumthathasbeenusedwidelyinRIA(BlairandSeaborn,1979;
Kennawayetal.,1982).YetanotherderivativeN[3(2aminoethyl)5methoxyindole]
hemisuccinamidehasbeenusedtogenerateantiserumasthebasisforaspecificRIA(Manzet
al.,1989).ThusmelatonincoupledattheNpositiongivesrisetoantiserathatarehighly
specificformelatoninascomparedtootherindoles.
Couplingatthesidechainhasalsosuccessfullyproduceduseablemelatoninantiserum.
MethodsusedincludeNacetyl5methoxytryptophancoupledusingcarbodiimide(Wilkinson
etal.,1977;Vakkurietal.,1984),succinyl5methoxytryptaminecoupledtoprotein(Rollag
andNiswender,1976)andindomethacincoupledtoprotein(LevineandRiceberg,1975).
Melatonincoupledviaadiazolinkagehasalsobeenreportedtoproduceareasonablyspecific
antiserum,howeverthesensitivityoftheresultingassaywaslow(Lynchetal.,1975;
Wurzburgeretal.,1976).
CouplingofNacetylserotoninusingformaldehydegeneratesantiserumthatbinds
melatoninandNacetylserotoninequally;crossreactivitystudiesandmodelreactionsshowed
thatcouplingoccursatthe4positionofthemolecule(GrotaandBrown,1974;Kennawayet
al.,1977).ResultingantiserumhasbeenusedasthebasisofaRIAthatrequiredprior
extractionandcolumnchromatographytoeliminatethecrossreactingindole(Kennawayetal.,
1977).
Thechiefmetaboliteofmelatonininurine,6sulphatoxymelatonin(6hydroxymelatonin
sulphate)hasalsobeenmeasuredbyimmunologicmeans.Theantiserumtypicallyusedfor
thisassayisgeneratedbyuseoftheMannichreactionandishighlyspecific(GrotaandBrown,
1974;Arendtetal.,1985).Antiseraproducedusingtheseapproacheshavebeenused
extensivelynotonlyforRIA,butalsoforimmunohistochemistryandforenzymelinked
immunoassays(ELISA).
3.2b.Radioimmunoassay(RIA)
TheprincipleofRIAmethodformelatoninmeasurementisthataknownamountof
radioactivemelatonin(2I125iodomelatoninor3Hmelatonin,forexample)ismixedwithafixed
amountofantibodyraisedagainstmelatonin.Increasingconcentrationsofunlabeled
9
melatoninareaddedtothemixture,whichwillcompetewithlabeledmelatonincausingits
displacementfromtheantibody.Freelabeledmelatoninisthenseparatedfromremaining
antibodyboundradioactivemelatoninandradioactivityismeasured.Astheconcentrationof
unlabeledmelatoninincreasesinthemixture,competitionfortheantibodiesalsoincreases
andboundlabeledmelatonindecreases.Acalibrationcurveconstructedfromknownamounts
oflabeledandunlabeledmelatoninallowsthedeterminationofunknownmelatonin
concentrationsinbiologicalsamples.
Fraseretal.(1983)describedaprotocolformelatoninmeasurementbyRIAinplasma
thathasbeenadoptedbyseveralresearchers,somewithslightmodifications(Fraseretal.,
1983).Briefly,200 Lof1000folddilutedantibodyisaddedto500 Lofsolutionscontaining
differentamountofmelatoninstandard(2.5to250.0pg).Thesolutionisvortexedandkeptat
roomtemperaturefor30min.3Hmelatoninisaddedtothetubes(100 L,4000cpm),mixed
andkeptat4Cfor18h.Then,0.5mLofDextrancoatedcharcoalsolution(0.1gofdextran75
plus10gofcharcoalper500mLofbuffer)isaddedandthesolutioniscentrifugedfor15min
at1500gand4C,inordertoseparatetheantibodyboundmelatoninfromthefree
fraction.Thesupernatantfractionisfinallydecantedinto10mLofscintillationfluidand
radioactivityiscountedonabetascintillatorcounter(Fraseretal.,1983).
SeveralvariationsinRIAmethodshavebeendescribed,byusingdifferentantibodies(as
notedabove),bychanging3Hmelatoninto2I125iodomelatonin,orbyalteringtheseparation
procedure.Ingeneral,becauseofitshigherspecificactivity2I125iodomelatoninallowsalower
detectionlimitthusallowingtheuseofasmalleramountofsample.Theconcentrationof
melatoninduringdaylightcanbeaslowas0.2to0.3fM(Rolciketal.,2002).Thiscouldbe
especiallyimportantifmeasurementsarenotprecededbymelatoninpurification.However,
125
Iismorepronetononspecificbindingsothatsomedeterminationscanbefaulty.
Sieghartetal.(1987)reportedthatpriormelatoninpurificationfromplasmausing
reversedphasecolumnchromatographyreducesgreatlyproblemsofcrossreactivity(Sieghart
etal.,1987).MoreoverRoliketal.(2002)usedimmunoaffinitychromatographyemploying
specificantiseratoprocesssamplespriortoHPLCMSanalysis(Rolciketal.,2002).
Nonethelessitshouldberecognizedthatevenweakcrossreactivitycanbeaproblemifthe
crossreactingmoleculeispresentinlargequantities.Thusindependentvalidationofthe
procedureisessentialwhenadifferentmatrixisassayed.
OneexampleofsuchadifferentmatrixissalivaforwhichseveralRIAshavebeen
described(Milesetal.,1985;Vakkuri,1985;Milesetal.,1989;Englishetal.,1993).Toobtain
salivadifferentmethodshavebeenused,fromchewinggum,chewingoncottonswabsor
usingcommercialapparatus.Againextractionisusuallyessentialespeciallysincelevelsin
10
salivaareabout40%ofthoseinplasma.Salivaisparticularlyusefulifrepeatedsamplingis
required:forexampletocharacterizethefull24hrhythmofmelatoninortodeterminethe
dimlightmelatoninonset(DLMO),ameasurethathasbeenshowntobeveryusefulinstudies
ofavarietyofsleepdisorders(LewyandSack,1989;Leibenluftetal.,1996;PandiPerumalet
al.,2007).
Severalvariantsofthetimeconsumingcharcoalseparationprocedurehavebeen
developedandsuccessfullyapplied.Inthesocalledscintillationproximityassay,themelatonin
antibodyisboundtoasecondaryantibody(e.g.,antisheep)attachedtoscintillatorcontaining
microbeads(fluomicrospheres)(PoeggelerandHuether,1992).Thisrelativelyconvenient
proceduredepends,however,usuallyonthecommercialavailabilityofsuitable
fluomicrospheres,sincepreparationandstandardizationofsuchbeadsistootieconsuming
foranaveragelaboratory.Intheproximityassay,boundradioactivityisdetecteddirectlyby
thescintillatorsystemofthemicrospheres.Forphysicalandgeometricalreasons,sucha
systemhastohavealowerscintillationefficiencythanahomogeneousscintillationcocktail.
However,thisprocedurehasotheradvantages.Apartfrombeingmorerapid,thesystemis
lessaffectedbynonspecificbinding(valuesclosetobackground)suchasoccurinthecharcoal
procedure,hasabetterreproducibilityandshowsamuchlowerassaydriftuponrepetitive
measurements(proximityassay:about10%changewithin84h;charcoalmethod:about25%
overthesameperiodoftime)(PoeggelerandHuether,1992).Othervariantsinclude
separationusingadoubleantibodyprocedure(Dietal.,1998)andammoniumsulfate
precipitation(Hoetal.,1984).
Althoughnotmelatoninitself,therehasbeenconsiderableinterestinthemajorurinary
metaboliteofmelatonin,6sulphatoxymelatonin(Arendtetal.,1985).The24hpatternof
excretionofthemetaboliteaccuratelyreflectsthepatternofmelatonininblood(Markeyet
al.,1985;Nowaketal.,1987).RIAsforthissubstanceareavailableandhavebeenusefulin
assessingpinealfunctioninvariousconditions(Martinetal.,1984;Johnetal.,1992;Cavallo
andDolan,1996;Girottietal.,2000;Girottietal.,2003;Fideleffetal.,2006).
3.3EnzymeLinkedImmunoassay(ELISA)
AvarietyofELISAsformelatoninhavealsobeenreportedthatemployantisera
identicaltothoseusedintheRIAdescribedabove.Onesuchimmunoassayemployed
melatoninhemisuccinatehumanserumalbuminabsorbedonpolystyrenespheres,withthe
melatonincompetingforafixedamountofperoxidaselabeledIgGantibodytomelatonin
(FerruaandMasseyeff,1985).Thismethodhadadetectionlimitof22fmolpertubeand
thereforerequiredextraction.AcompetitivesolidphaseELISAforhumanandratserumand
11
ratpinealglandhasbeendescribedandvalidatedusingmicrotitreplatesthathasamuch
lowerdetectionlimit(1.0fmolperwell)aswellasprecisioncomparablewithothermethods
andthatcanbeappliedwithoutextractiontoratserum(Yieetal.,1993).Aimprovedversion
ofthisassaywithashorterincubationtimewassubsequentlyreported(Shavalietal.,1999).A
comparativestudyofanRIAandacommercialELISAreportedthattheELISArequireda
purificationsteptobevalidwhenappliedtohumanserum,astepthatwasnotpartofthe
procedurerecommendedbythemanufacturer(Cheginietal.,1995).Withtheextractionstep,
theassayhaddistinctadvantages,Enzymeassayshavemajoradvantagesinthattheenzyme
conjugateisstable,ismoreconvenientthan3Hor125Iandpresentnoproblemwithdisposalof
radioactivewaste.Furthermoreifmicrotitreplatesareusedcentrifugationisnotnecessary.
Althoughnotanenzymeimmunoassay,itisofinterestthatatimeresolvedfluoroimmunassy
hasalsobeendescribed(Yamadaetal.,2002).Anenzymeimmunoassayfor6
sulphatoxymelatoninhasbeenreported(PenistonBirdetal.,1996)andcommercialkitsare
available.
3.3.HPLCcoupledtoelectrochemicalandfluorescencedetection
Accordingto(Vitaleetal.,1996)RIAmethodologyhasbeenreplacedbyHPLCwith
electrochemicalandfluorescencedetectionformelatoninevaluationinbiologicalsamples,
duetoitsgreatsensitivityandspecificity.Howevertheyalsosuggestthatthisprocedureis
moreadequateformelatoninalone,andnotformixturesofseveralindoleslikeserotoninand
tryptamineamongothers,thatcancauseinterferencesintheassay.Indeed,theseauthors
reportedthatserotonin/melatoninratioishigherthan100inratpineal,whichcauses
disturbancesinchromatographicseparationsthatcandifficultmelatonindetection,so
requiringagoodprocedureformelatoninextraction.However,theavoidanceofpartial
coelutionwithotherindolesismostlyamatteroftheartofchromatography.Inourworkwe
havebeenabletodetectmelatoninwithgreataccuracyinbloodplasmaaftersimple
dichlorometaneextractionasdescribedabove,andusinganHPLCsystemconnectedto
electrochemicaldetection.Figure1showsachromatogramofmelatonindetectioninhuman
plasmabythisprocedure.GoodpeakseparationwasachievedbyusingaLC18columnand50
mMsodiumacetate100mMaceticacid(pH4.3),0.1mMNa2EDTA,andacetonitrile(75:25,
v/v)asmobilephasepumpedisocraticallyat1mL/min.
Harumietal.(1996)alsosuccessfullydeterminedmelatoninbyHPLCwith
electrochemicaldetection,withveryclearpeakseparationfordifferentindoleaminesamong
melatonin(Harumietal.,1996).However,thesensitivityofthisproceduredependsonthe
modelofelectrochemicalcell.Amperometricbasedelectrochemicalcellsaregenerallyless
12
sensitivethancoulometriccells,sothattheadequatepotentialshouldbepreviouslyoptimized
bytheconstructionofhydrodynamicvoltammograms.Withourcoulometricelectrochemical
system,thebestmelatoninsignalisobtainedat600mV.Sensitivitycanbealsogreaterwith
coulometricelectrochemicaldetectorslikeESAcoulochemIIImodel(ESA,Bedford,USA),
whichusesporouselectrochemicalcellsthatallowgreateraccuracyinmelatoninpeak
resolution.Harumietal.(1996)reportedtheuseofahigherpotential,900mV,forgood
melatoninsignalwiththeirgraphitecarbonworkingelectrode,andevensotheydetected
melatoninatverylowlevels(Harumietal.,1996).Rizzoetal.(2002)alsoused900mVfor
melatonindetectionwithanamperometricelectrochemicaldetector(Rizzoetal.,2002).
Withrespectoffluorescencedetection,somehighlysensitivemethodologieshavebeen
reportedformelatonindetectionatfmollevel(Simoninetal.,1999;Iinumaetal.,1999;Yang
etal.,2002;Luetal.,2002).MelatonincanbeseparatedonaC18columnbyusing75mM
sodiumacetatepH5.0andacetonitrile(72:28,v/v)asthemobilephasepumpedisocratically
at1.0mL/min,anddirectlydetectedbysettingupthefluorescencedetectoratanexcitation
wavelengthof275nmandanemissionwavelengthof345nm(Rizzoetal.,
2002).Nevertheless,insomecasesinwhichmelatoninconcentrationisverylow,derivatization
isrecommendabletoenhancethemelatoninsignal(Iinumaetal.,1999).Tomitaetal.(2003),
forexample,describesanoxidationprocedurethatcanenhancemelatoninfluorescenceby6.8
times,allowingitsdeterminationatamollevelsinbiologicalsamples(Tomitaetal.,2003).
Melatoninwasoxidizedtoanewfluorescentcompoundwithsodiumcarbonateandhydrogen
peroxide.However,precautionsshouldbetakenbyusingthiskindofapproach,because
otherscomponentsfromthebiologicalsamplemayleadforthegenerationoffluorophores
thatmayinterfereonthecorrectlevelevaluationcausinglackofmethodspecificity(Tomitaet
al.,2003).
Inanycase,carewithsamplepreparationcanimprovemelatoninsignal.Prepurification
ofmelatoninasdescribedbeforewilldecreasechromatogramnoisesandavoidthecoelution
ofmelatoninwithothercompoundsthatcaninterferewithmelatoninpeaks.Generally,the
useoffluorescencetechniquescanbeaffectednotonlybycoelutionwithotherfluorescent
compoundsinthesample,butalsobythepresenceofquenchers.Thisshouldnotbe
underratedsincethemajorityofaromatesabsorbaroundtheexcitationmaximumof
melatonin.Therefore,samplesshouldbetestedinadvanceforquenchingbyaddingknown
amountsofmelatonin.
3.4.Massspectrometry
13
TheGCMStechniqueisverysensitiveandoffersmorespecificitythanHPLCwith
electrochemicalorfluorescencedetectors;however,aproblematicdifficultyistheneedof
derivatizationandthusthistechniquehavebeengraduallysubstitutedbyliquid
chromatographymassspectrometryprocedures.Thus,alternativeHPLCMSmethods
appropriateforuseinbiologicalissueshavebeendeveloped(Yangetal.,2002;Erikssonetal.,
2003;Motoyamaetal.,2004;Almeidaetal.,2004).However,thisapproachislimitedbythe
needofadequateinternalstandards.Yangetal.(2002)reportedamethodologybyusing
acetyltryptamineasinternalstandard(Yangetal.,2002);however,thisisnottheideal
situation.Themostappropriateistheemploymentofalabeledinternalstandardwhose
structureisthesameoftheanalyteunlessthemassdifference.Theadditionofanisotopically
labeledinternalstandardpriortoanalysisimprovesthemethodsconfidencelevel.
Anotheranalyticalmethodenabledthedeterminationofendogenousmelatoninin
humansaliva,byusingcolumnswitchingsemimicrocolumnliquidchromatography/mass
spectrometryandselectedionmonitoring(SIM).Melatoninwasmonitoredbasedonits
fragmentionatm/z174byinsourcedissociationandusingdeuteratedmelatoninasinternal
standard,andadetectionlimitof10fmolwasobtained(Rolciketal.,2002).Themain
limitationofthismethodologyistheuseofSIMmodetodetecttheionsgeneratedinthe
probe,whichdoesnotimplyanabsolutespecificity.Yet,Erikssonetal.(2003)reporteda
methodforthedeterminationofmelatonininhumansalivabyHPLCMS/MS,using7D
melatoninasinternalstandard(Erikssonetal.,2003).Thelimitofdetectionwas1.05pgmL1
andthelimitofquantificationwas3.0pgmL1.Oneofushasreportedthedevelopmentofa
newHPLCMS/MSassaywithelectrosprayionization(ESI)toquantitativelydetermine
melatoninandalsoitsdegradationproductN1acetylN2formyl5methoxykynuraminewith
highsensitivityandspecificity(Almeidaetal.,2004).Astableisotopicinternalstandard
melatoninD3(deuteratedmelatonin)waseasilysynthesizedbythereactionof5
methoxytryptaminewithdeuteratedacetylchloride(CD3COCl)(Figure2).
Thepredominantion[M+H]+inthefullscanmassspectraofmelatonin,andmelatonin
D3werelocated(Figure3AB).Thefragmentsgeneratedincollisioninduceddissociation
chamberrevealedapredominantfragmentatm/z=174formelatoninandmelatoninD3(loss
oftheNacetylgroup)(figure3CD).Them/ztransitionsfrom233to174(melatonin)andfrom
236to174(melatoninD3)werethereforechosenfortheMultipleReactionMonitoring(MRM)
detectionexperiments,whichensuredahigherspecificityandanaccuratequantificationof
melatonininhumanplasma(Figure3E).
3.4.Othertechniques
14
Somelaboratorieshavetakenanddevelopedcapillaryelectrophoresis(CE)fortheseparation
anddeterminationofmelstonininbloodplasma(Kimetal.,1999;Pobozyetal.,2005;
Musijowskietal.,2006)andpinealgland(Chenetal.,2001;Wuetal.,2006).Detectionof
analytewasperformedwithUVandfluoscencedetector(Kimetal.,1999;Pobozyetal.,2005;
Musijowskietal.,2006;Heviaetal.,2010)orelectrochemicaldetector(Chenetal.,2001;Wu
etal.,2006).DetectionlimitofmelatoninwithCEiscomparablewiththedataobtainedby
HPLCmethodsreportedpreviously.Recently,fortheseparationofmelatoninfromrelated
compounds,CEwithmicellarelectrokineticchromatographywasapplied(Pobozyetal.,2005;
Musijowskietal.,2006;Wuetal.,2006;Heviaetal.,2010).Thistechniqueallowedtoseparate
melatoninanditsprecursorsormetaboliteseffectively.Sodiumdodecylsulfateisusedto
produceapseudostationaryphase.
4.Conclusions
ThemostcommonmethodsfordeterminationofmelatonininbloodorsalivaareRIAs
andELISAs.Moreoverseveralcommercialkitsarenowavailablefortheseassays.Theyare
convenienttouse,especiallytheenzymebasedassays,butmustbeusedwithcarebecauseof
thepossibilityofcrossreactions,andnonspecificeffects.Thisisparticularlythecasebecause
oftheverylowlevelsofmelatoninthataretobemeasured.Thesepotentialproblemscanbe
reducedbydeterminingwhetherextractionisnecessaryandbycomparisonwithother
establishedmethods.Itistobeexpectedthatthesemethodswillcontinuetoimproveand
thatenzymeassayswillcontinuetogaingroundforroutinemeasurementsofmelatoninin
bloodandsaliva.
15
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Figure1Chromatogramofmelatoninstandard(2pmol;A)andmelatoninextractedfrom2
mLofaplasmasample(B),byHPLCwithcoulometricelectrochemicaldetector.
25
Figure2SynthesisoflabeledD3melatoninforitsuseasinternalstandardduringmelatonin
measurementinplasmasamples.
26
Figure3FullmassscanspectraofmelatoninandD3melatoninintheESI+mode
174
100
Daughters of 233ES+
[(M N-acetil) +
H]+
1.16e6
m/z = 174
H3C
O
N
H
CH3
m/z = 233
125
145
165
185
205
225
174
100
245
265
Daughters of 236ES+
1.16e6
m/z = 174
H3C
O
N
N
H
CD3
m/z = 236
125
145
165
185
m/z
205
225
245
265
27
Figure4DaughtersofmelatoninandD3melatoninionsafterfragmentation inthecollision
cellofthemassspectrometer.
28
Figure5Standardcalibrationcurveofmelatonin.OntheYaxis,datawasplotedbydividing
theareaoflabeledstandard(D3melatonin)bynotlabeledmelatoninstandard.
29
100
MRM ES+
236.00 > 174.00
MELD3 (1 pmol)
0
MRM ES+
233.00 > 174.00
100
MEL
2.00
4.00
6.00
8.00
10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00
Time (min)
Figure6ChromatogramofmelatoninanddeuteratedmelatoninbyHPLCMS/MS.
30