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PopulationandAssayThresholdsforthePredictiveValueofLipoprotein(a)
forCoronaryArteryDisease:TheEPIC-NorfolkProspectivePopulationStudy
RutgerVerbeek1,S.MatthijsBoekholdt2,RobertM.Stoekenbroek1,G.KeesHovingh1,JosephL.
Witztum,3NicholasJ.Wareham4,ManjinderS.Sandhu5,6,Kay-TeeKhaw5,SotiriosTsimikas7
1.DepartmentofVascularMedicine,AcademicMedicalCenter,theNetherlands
2.DepartmentofCardiology,AcademicMedicalCenter,theNetherlands
3.DivisionofEndocrinology,DepartmentofMedicine,UniversityofCaliforniaSanDiego,La
Jolla,California,USA
4.MedicalResearchCouncilEpidemiologyUnit,Cambridge,UnitedKingdom
5.DepartmentofPublicHealthandPrimaryCare,UniversityofCambridge,Cambridge,United
Kingdom
6.GeneticEpidemiologyGroup,WellcomeTrustSangerInstitute,Hinxton,UnitedKingdom
7.VascularMedicineProgram,SulpizioCardiovascularCenter,UniversityofCaliforniaSan
Diego,LaJolla,California,USA
Correspondingauthor:Sotirios Tsimikas, MD, Vascular Medicine Program, Sulpizio
CardiovascularCenter, University of California San Diego, 9500 Gilman Drive, BSB 1080,
La Jolla, CA 92093-0682. E-mail [email protected]
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Abstract
VariableagreementexistsbetweendifferentLp(a)measurementmethods,buttheirclinical
relevanceremainsunclear.ThepredictivevalueofLp(a)measuredbytwodifferentassays
(RandoxandUCSD)wasdeterminedin623CADcasesand948controlsinacase-controlstudy
withintheEPIC-Norfolkprospectivepopulationstudy.Participantsweredividedintosex-
specificquintiles,andbyLp(a)<50versus>50mg/dL,whichrepresentsthe80thpercentilein
northernEuropeansubjects.RandoxandUCSDLp(a)levelswerestronglycorrelated,with
Spearman’scorrelationcoefficientsformen,womenandsexescombinedwere0.905,0.915
and0.909,respectively(p<0.001foreach).The>80thpercentilecut-offvalues,however,were
36mg/dLand24mg/dLfortheRandoxandUCSDassays,respectively.Despitethis,Lp(a)levels
weresignificantlyassociatedwithCADrisk,withoddsratiosof2.18(1.58-3.01)and2.35(1.70-
3.26)forpeopleinthetopversusbottomLp(a)quintilefortheRandoxandUCSDassays,
respectively.ThisstudydemonstratesthatCADriskispresentatlowerLp(a)levelsthanthe
currentlysuggestedoptimalLp(a)levelof<50mg/dL.Appropriatethresholdsmayneedtobe
populationandassayspecificuntilLp(a)assaysarestandardizedandLp(a)thresholdsare
evaluatedbroadlyacrossallpopulationsatriskforCVDandaorticstenosis.
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Introduction
Elevatedlevelsoflipoprotein(a)[Lp(a)]areagenetic,independentriskfactorforthe
developmentofcardiovasculardisease(CVD)(1).TheEuropeanAtherosclerosisSociety(EAS)
ConsensusPanelhasrecommendedthatLp(a)levelsshouldbemeasuredinpatientswithan
intermediateorhighCVDriskandthatdesirableLp(a)levelsarebelowthe80thpercentileof
thepopulationdistribution,whichroughlycorrespondedto50mg/dLinaCopenhagen
populationof6000subjects(2).Preciseandreliablemeasurementmethodsaretherefore
essentialtoguideclinicaldecision-making.Yet,previousstudieshaveindicatedsubstantial
differencesinLp(a)valuesasmeasuredbyvariousassaysandamongdifferentracialgroups(3,
4).Animportantlimitationintheinconsistencyofmeasurementsisthefactthatmanyassays
areaffectedbythesizeofapolipoprotein(a)[apo(a)],themajorproteincomponentofLp(a),
andthatantibodiesandcalibratorsvaryacrossplatforms.Basedongeneticvariantsofthis
protein,largeinter-individualdifferencesinapo(a)isoformsizeexist.
Previousstudieshaveattemptedtoelucidatetheclinicalrelevanceofthepoorinter-
assayagreementintermsofcardiovascularriskclassification.Marcovinaetal.assessedthe
concordanceinLp(a)resultsamong22differentLp(a)assays(3).Considerableheterogeneity
wasobserved,inparticularbetweenapo(a)size-sensitiveassays.Theauthorsalsospeculated
thatthisinaccuracymighttranslateintoinaccurateriskassessment.Adirectcomparisonof
threeLp(a)assaysinasubsetoftheFraminghamOffspringcohortdemonstratedlargeinter-
assaydifferencesinLp(a)valuesandconsequentlyinthe80thpercentilecut-offvalues,which
waslargelydeterminedbydifferencesinapo(a)isoformsize(4).However,thenumberof
coronaryarterydisease(CAD)eventsinthatstudywaslimited.Arecentmeta-analysis
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evaluatedtheCVDpredictivevaluebetweenpooledisoform-sensitiveandisoform-insensitive
assays(5)andobservedasubstantialheterogeneitybetweenthedifferentstudies.However,no
formalassaycomparisonsweremadeandassayswereperformedindifferentstudy
populations,makingitimpossibletodirectlycomparethepredictivevalueofdifferentassays.
Understandingappropriateassayandpopulationthresholdsforriskareessentialas
noveltherapies,includingantisenseoligonucleotides,PCSK9inhibitorsandCETPinhibitorsand
mipomersen(6-10),lowerLp(a).Therefore,ourobjectivesweretwofold:firsttodetermineif
thepopulationthresholdspreviouslysuggestedforoptimallevelsapplybroadlytoothergroups
ofpatients;andsecondtodeterminetheconcordancebetweenaclinically-availableand
commonlyusedimmonoturbidimetricLp(a)assaycomparedtoaresearch-basedenzyme-linked
immunosorbentassay(ELISA)inpredictingCADrisk.Weaddressedthisobjectiveinacase-
controlstudynestedintheEPIC-Norfolkprospectivepopulationstudy.
Methods
Studydesign
TheEuropeanProspectiveInvestigationofCancer(EPIC)-Norfolkprospectivepopulation
studyhasbeendescribedindetailpreviously(11).Inbrief,theEPIC-Norfolkprospective
populationstudyincluded25,663menandwomenagedbetween45and79yearsold,and
livinginNorfolk,UnitedKingdom.Thestudywasdesignedtodeterminetheeffectsofdietand
otherlifestylefactorsontheriskofdevelopingcancer,andadditionaldatawereobtainedto
studydeterminantsofotherdiseases.Participantswererecruitedusingregistersfromgeneral
practicesandwereincludedafterwritteninvitation.Allsubjectshavebeenflaggedfordeath
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certificationattheUnitedKingdomOfficeofNationalStatistics,withvitalstatusascertainedfor
theentirecohort.Inaddition,allhospitalizationsforstudyparticipantswererecordedusing
theiruniqueNationalHealthServicenumberbydatalinkagewithENCORE,theEastNorfolk
HealthAuthoritydatabase,whichidentifiesallhospitalcontactsthroughoutEnglandandWales
forNorfolkresidents.Trainednosologistscodedtheunderlyingcauseofhospitaladmissionor
deathaccordingtothe10threvision(ICD-10)oftheInternationalClassificationofDiseases.
FatalCADduringfollow-upwasdefinedasdeathwiththeunderlyingcausecodedasICD-10
codesI20toI25.NonfatalCADwasdefinedashospitalizationwiththeunderlyingcausecoded
asI20toI25.
Participants
Forthisspecificstudy,weanalyzedthedataofacase-controlstudyoriginallydescribed
byBoekholdtetal(12).Briefly,participantsweremarkedasacaseiftheywereapparently
healthyatbaseline,andhaddiedorbeenhospitalizedwithCADastheunderlyingcauseduring
follow-up.Controlsweredefinedasstudyparticipantswhowereapparentlyhealthyatbaseline
anddidnotdevelopanyCVDduringfollow-up.Intheoriginalstudydesign,weattemptedto
matchtwocontrolstoeachcase,bysex,age(within5years),anddateofbaselinevisit(within
3months).Inthisstudyset,Lp(a)levelsweremeasuredin2160participantsattheUniversityof
CaliforniaSanDiego,aspreviouslydescribed(13).Severalyearslater,itwasdecidedto
measureLp(a)levelsintheentireEPIC-NorfolkcohortattheUniversityofCambridge,
dependingonsampleavailability,aspreviouslydescribed(14).Theseincludedsamplesfrom
thecase-controlstudy.Duetoinsufficientsamplematerialhowever,notallsampleswere
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measuredwithbothassays.Thereforethecurrentanalysisisbasedontheoverlapbetween
thesetworoundsofLp(a)measurements.
Biochemicalanalysis
Atthebaselinestudyvisit,bloodwasdrawnfromstudyparticipants,aspreviously
described(11).Totalcholesterol,high-densitylipoprotein(HDL)cholesterolandtriglycerides
weredeterminedwiththeRA1000(BayerDiagnostics,Basingstoke,UnitedKingdom).The
Friedewaldformulawasusedforthecalculationoflow-densitylipoprotein(LDL)cholesterol
levels.Laterintime,Lp(a)levelsweredeterminedinnon-fastingbaselinesampleswhichhad
beenstoredat-80°C.
IntheRandoxassay,Lp(a)levelsweredeterminedonaOlymposAU640analyserwithan
immunoturbidimetricmethod(RandoxlaboratoriesLtd.Crumlin,CountyAntrim,United
Kingdom)(14).Thisassayuseslatexparticlescontainingrabbitanti-humanLp(a)polyclonal
antibodyasareagent(licensedfromDenkaSeikenCo.,Ltd.,Niigata,Japan)andRandox'sown
calibratorsandcontrolsfromscreenedplasmadonatedbyvolunteers.Therabbitanti-human
Lp(a)polyclonalantibodyistechnicallyisoformsensitivebyvirtueoftheantiserabindingto
multiplesitesofKIV2repeats,buttheassayistheoreticallymadenearlyisoformindependent
bythecalibratorsystem.Thisassayformat,likemostcommercialassays,bindstobothfree
apo(a)andtrueLp(a)(i.e.apo(a)covalentlyboundtoapolipoproteinB-100),thereforeitisbest
describedasmeasuring"totalapo(a)"ratherthan"Lp(a)".
IntheUCSDassay,designedandconductedattheUniversityofCaliforniaSanDiego,
Lp(a)levelswerealsodeterminedwithanLp(a)assaydesignedbyTsimikasetal(15),which
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incorporatesthemurinemonoclonalantibodyLPA4withachemiluminescentELISA(13).LPA4
isamurinemonoclonalIgGantibodytoapo(a)thatwasgeneratedbyimmunizingmicewiththe
apo(a)sequenceTRNYCRNPDAEIRP.ThissequenceispresentasonecopyonKIV5,KIV7,and
KIV8ofapolipoprotein(a),anddoesnotcross-reactwithplasminogen.LPA4doesnotbindto
KIV2,andthereforeitisisoformindependent.Thisassayisalsoatrue“Lp(a)”assayasitusesa
captureantibodytoapolipoproteinB-100anddetectsapo(a)withbiotinylatedLPA4.Free
apo(a)isnotdetectedwiththisassay,butitisusuallynotpresenttoanysignificantamountin
humansingeneralpopulations.BecauseLPA4bindsto3identicalsitesonapo(a),whichare
presentinallpatientsandallisoformstothesameextent,thisassayhashighsensitivityanda
broadlinearrangefrom0.5-180mg/dL.Thisassayuses9calibrators,rangingfrom6-168mg/dL
tocaptureawiderangeofvalues,identifiedfromindividualhumanplasmasamples.Thevalues
assignedtothecalibratorswerevalidatedbyboththeDiasorin(Stillwater,Mn)and
Technoclone(Vienna,Austria)calibrators.Thisassaycorrelateswellwithcommercially
availableLp(a)assays,hasacoefficientofvariabilityof3-5%,andhasbeenusedinover20
studiesand20,000patients(13,16-19).
Statisticalanalysis
ThecurrentanalysisisbasedonavailabilityofbothLp(a)measurementsinthecohort
database.TheselectionofcasesandcontrolsisthereforebasedontheoverlapbetweenLp(a)
measurementsperformedinanestedcase-controlsetasdescribedabove,andtheentireEPIC-
Norfolkcohort.Byvirtueofthisselectionprocedure,theoriginal1:2matchingbysex,ageand
enrollmenttimewaspartiallylost.
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Baselinecharacteristicswerecalculatedforcasesandcontrolsseparately,stratifiedby
sex.ThecorrelationbetweenLp(a)levelsasmeasuredbytheRandoxandUCSDassaywas
quantifiedusingSpearmancorrelationcoefficients.BecausetheEASConsensusPanel
recommendedusingthe80thpercentileasthecut-offvalueforanelevatedLp(a)level(2),we
performedanalysesusingbothsex-specificandpooledLp(a)quintiles.Inaddition,becausethe
80thpercentileinDanishpopulationsisthoughttocorrespondroughlytoanLp(a)levelof50
mg/dL,wealsoperformedanalyseswhereparticipantswerestratifiedusingtheLp(a)threshold
of50mg/dL.ToassesstheassociationbetweenLp(a)levelsandtheriskofdevelopingCAD,
logisticregressionanalyseswereperformedtodeterminetheoddsratiosandcorresponding
95%confidenceintervalsperLp(a)quintile,usingthelowestquintileasreferencecategory.
Furthermore,oddsratiosweredeterminedforparticipantswithLp(a)levels>50mg/dL,using
those<50mg/dLasreferencecategory.Analyseswereperformedseparatelyformenand
womenusingsex-specificcut-offs,andalsoformenandwomencombinedusingpooledcut-
offs.Inaddition,weassessedtheconcordancebetweentheassaysinclassifyingstudy
participantsas<50mg/dLversus>50mg/dL.AllstatisticalanalyseswereperformedwithSPSS
statisticalsoftware(Version21.0,IBMCorporation,Armonk,NY).
RESULTS
Atotalof1571studyparticipantshadLp(a)measurementsdonebyboththeRandox
andUCSDassays.Thissetcomprised623CADcasesand948matchedcontrols.Baseline
characteristicsareshowninTable1.Asexpected,caseshadhigherlevelsofLDL-cholesterol,
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higherbloodpressureandhigherbodymassindex,andweremorelikelytosmokeandhave
diabetesmellitus,comparedtocontrols.Median(interquartilerange)Lp(a)levelsmeasuredby
theRandoxassaywere14.3(7.2-35.9)mg/dLformalecases,11.2(6.1-23.2)mg/dLformale
controls,15.5(8.5-37.9)mg/dLforfemalecasesand11.4(6.8-21.9)mg/dLforfemalecontrols.
MedianLp(a)levelsmeasuredbytheUCSDassaywere9.3(6.8–23.9)mg/dLformalecases,
8.1(6.4-13.4)mg/dLformalecontrols,10.4(7.4-29.8)mg/dLforfemalecasesand8.5(6.5-12.9)
mg/dLforfemalecontrols.
Lp(a)levelsasquantifiedbytheRandoxandUCSDassayswerestronglycorrelated.
Spearman’scorrelationcoefficientsformen,womenandsexescombinedwere0.905,0.915
and0.909,respectively(p<0.001foreach).ThedifferencesinLp(a)levelsmeasuredbythe
RandoxandUCSDassaysweremostprominentinthehighestquintiles,withtheRandoxassay
yieldinghigherLp(a)levelsthantheUCSDassay,asshowninTable2.The80thpercentile
thresholdswerealsodifferent.TheRandoxassayyielded80thpercentilethresholdvaluesof35
mg/dL,37mg/dLand36mg/dLformen,womenandsexescombined,respectively.The
corresponding80thpercentilethresholdsaccordingtotheUCSDassaywere23mg/dL,26mg/dL
and24mg/dLformen,womenandsexescombined,respectively.Importantly,withboth
assays,the80thpercentilethresholdswerewellbelow50mg/dL.UsingtheRandoxassay,the
distributionofcasesversuscontrolsamongthepooledsexesrangedfrom107/208(34.0%)in
thelowestquintileto166/148(52.9%)inthehighestquintile.UsingtheUCSDassaythe
correspondingnumberswere102/212(32.5%)inthelowestquintileand166/148(52.9%)in
thehighestquintile.Inthegroup≥50mg/dL,thenumbersofcasesversuscontrolswere95/82
(55.2%)fortheRandoxassay,and53/34(60.9%)fortheUCSDassay.
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OddsratiosfortheriskofCADbyLp(a)quintiles,aswellasforthecomparison≥versus
<50mg/mlareshowninTable3andFigure1.Oddsratiosforpeopleinthetopquintile(i.e.
>80thpercentile)comparedtothoseinthelowestquintilewere2.18(95%CI1.58–3.01)using
theRandoxassayand2.35(95%CI1.70–3.26)usingtheUCSDassay.Oddsratiosforpeople
withLp(a)≥50mg/dLversus<50mg/dLwere2.29(95%CI1.56–3.36)fortheRandoxassay
and2.85(95%CI1.66–4.90)fortheUCSDassay.
Theconcordance/discordancebetweenthetwoassaysinclassifyingstudyparticipants
accordingtoanLp(a)levelaboveorbelow50mg/dLareshowninTable4.Atotalof1465study
participantshadconcordantresultsforbothassays.Discordantresultswereobservedin106
studyparticipants(6.9%oftotalsubjects),with8havingLp(a)<50mg/dLontheRandoxassay
butLp(a)≥50mg/dLontheUCSDassay,whereas98havingLp(a)≥50mg/dLontheRandox
assaybutLp(a)<50mg/dLontheUCSDassay.
ABland-Altmananalysiswasperformedtodeterminethelevelofagreementbetween
theUCSDandtheRandoxassay(Figure2).Themeanaveragewas-5.9mg/dL,thelower95%
limitofagreementwas-16.3mg/dLandtheupper95%limitofagreementwas-28.2mg/dL.At
lowaverageconcentrations,thelevelofagreementwasgood,butwithincreasing
concentrations,thelevelofagreementdiminished.
DISCUSSION
ThisstudyevaluatedpopulationandassaythresholdsforthepredictivevalueofLp(a)
fortheriskofcoronaryarterydiseasewith2differentassaysmeasuredinthesamesubjects.
Interestingly,nearlyidenticalpredictivevalueforidentifyingsubjectswithorwithoutCADwas
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evidentbutthiswastruewhenexaminedinthecontextofthewholepopulationstudiedforthe
givenassay,e.g.intermsofquintiles,butnotwhencomparingabsoluteLp(a)levelsand80th
percentilecut-offvalues.Finally,forbothassaysthe80thpercentilecut-offsweresignificantly
lowerthan50mg/dL,theEASproposedthreshold,whichwasbasedontheCopenhagen
GeneraPopulationStudy(2,20,21).ThisthresholdwasbasedonprevalenceofLp(a)valuesin
thegeneralpopulationandnotnecessarilybasedonwhenriskofLp(a)begins.Epidemiological
andgeneticstudiessuggesttheriskthresholdsstartat25-30mg/dLinprimarycarepopulations
(1,14,20),but>50mg/dLinsecondarypreventionpopulationsthathavebeentreatedwith
severalsecondarypreventionmeasuressuchasaspirin,clopidogrel,statinsandanti-
hypertensivemedications(22,23).Ourfindingssuggestthatthethresholdsfordetermining
whatisahighlevelandwhoisatriskshouldbereportedasassayspecificthresholdsuntil
assaysfromallmanufacturersaresufficientlystandardizedeachassayprovidesthesame
absolutevaluesforagivenplasmasample.Preferably,asetofinternationallyaccepted
standardsandcalibratorsshouldbeagreeduponandimplementedacrossallassayplatforms.
Lp(a)consistsofanLDL-likeparticle,inwhichapoB-100iscovalentlyboundtoapo(a).
Apo(a)consistsofseveralkringledomains,inwhichthenumberofkringledomainIVtype2
(KIV2)repeatsisthedominantsize-determiningdomainofapo(a);eachapo(a)isoformcan
containfrom3upto>40KIV2repeats(24,25).ThenumberofKIV2repeatsisinverselyrelated
withtheplasmalevelofLp(a),andexplains25-50%ofplasmaLp(a)levels(20,26).Additional
geneticdeterminantsofvariabilityarepresentintheLPAgeneandincluderegulatoryelements
andsinglenucleotidepolymorphismsthatmediateeitherhighorlowLp(a)levels(27-31).These
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uniquepropertiesofLp(a)greatlycontributetothedifficultyofestablishingcommonstandards
foritsclinicalmeasurement.
Lp(a)measurementmethodsarebasedonavarietyoftechniques,including
immunonephelometry,immunoturbidometry,andELISA’s(32).Incontrasttootherprotein
quantificationassays,Lp(a)assaysarenotoriouslychallengingbecauseofthelargevariationin
sizeandstructureoftheapo(a)proteinsize,whichismediatedbytheallelicheterogeneityin
thenumberofKIV2repeats.Additionally,eachindividualgenerallyhastwoexpressedalleles
thatusuallycodefortwodifferentsizedapo(a)proteinsinplasma.Over40differentsized
isoformsofapo(a)havebeenreportedandapproximately80%ofindividualshavetwodifferent
sizedapo(a)isoformsinplasma(29).Furthermore,althoughthemolecularweightofapoB-100
isfixed,differencesbesidesvariabilityofKIV2repeatsmayexistinadditionalcomponentsof
Lp(a),suchasthecarbohydratecontentonapolipoprotein(a)andthelipidcompositionofthe
LDL,whichmayfurthercomplicatetheaccuratequantificationofLp(a)levelsbymass(33).
Otheraspectsthatimpactprecisionandreproducibilityaretheuseofdifferentdetection
antibodiesandimportantly,thelackofcommonstandardsandparticularlyuniformcalibrators.
Almostallcommerciallyavailablemethodsusepolyclonalantibodies,whichare,by
definition,isoformdependentsincepolyclonalantibodypreparationscontainheterogeneous
mixtureofantibodiesthatbindtodifferentsitesonapo(a)protein.SinceKIV2repeatsarethe
generallythemostprevalentkringles,itisexpectedthatamajorityofbindingsitesofsuch
polyclonalantibodieswillbeatKIV2repeats.Theonlyisoformindependentantibodiesusedin
recentlypublishedassays,asdefinedbyvirtueofnotbindingtoKIV2repeats,aremonoclonal
antibodya6fromtheNorthwestLipidMetabolismandDiabetesResearchLaboratoriesthat
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bindstoKIV9andmonoclonalantibodyLPA4fromUCSD.Itisalsoimportanttoemphasizethat
mostcommerciallyavailableassaysmeasure“totalapo(a)”ratherthanLp(a).Sinceall
polyclonalantibodieswilllikelybindtoKIV2,ideallymonoclonalantibodiesnotbindingKIV2
shouldbeusedtoavoidtheissueofisoformsensitivity.Althoughtheissueofisoformsensitivity
hasbeenaddressedtosomeextentbycarefulselectionofcalibrators,itisquitelikelythatuse
ofpolyclonalantibodieswillalwayshavesomeisoformsensitivityifcarefullyanalyzed.
Althoughmanufacturersdonotusuallypublicallyreporttheprocessofgenerating
appropriatecalibrators,theygenerallyusepooledplasmasamplesfrommanydonorstoisolate
Lp(a).Often,suchcalibratorsetsresultfromserialdilutionsofconcentratedLp(a)values,but
canalsobeseparatepoolsofplasmathathaveameanvaluesrangingfromlowtohigh.Itis
importantthatthecalibratorsusedreflectthepresenceofthewholerangeofdifferentsized
allelesbothbetweenandwithinindividuals,andthatsuchallelesarepresentinthesample
beingmeasured.Afurtherdisadvantageofusinghumanpooledplasmaforcalibratorsisthat
eachbatchchangesaccordingtotheavailabilityofdonorsovermonthsandyears,and
therefore,theremaybevariationeachtimeanewbatchismade.Thisissuewillbecome
increasinglyimportantasthemeasurementofLp(a)ispredictedtoincreasesubstantiallywith
availabletherapiestolowerlevelsandthemeasurementofLp(a)levelsincreasesglobally.
IdeallycalibratorsshouldbelinkedtothereferencematerialfromtheWorldHealth
Organizationtoensurerelativeisoformindependence.
Toaddressthelimitationofinter-assayvariation,manyinvestigatorssuggesttheuseof
apolipoprotein(a)isoform-independentassayscorrectedwithinternationalsecondaryLp(a)
references.AlsotheproposalhasbeenmadetodeterminetheconcentrationofLp(a)as
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particlenumberreportedinnmol/L,preferablyusingthereferencestandardofthe
InternationalFederationofClinicalChemistryasawaytominimizetheissuesrelatedto
variabilityinLp(a)mass(33,34).ThisWorldHealthOrganizationapprovedstandard,withan
Lp(a)concentrationof107nmol/Land21KIVrepeats,ensuresafixedandapo(a)isoform
independentmeasurementofthemolarconcentrationsofLp(a)(3).Althoughonecanroughly
convertmg/dLtonmol/Lwitharatioof~2.4,inreality,thisisnotappropriateandshouldnot
becarriedoutasitdependsonknowingtheunderlyingisoformnumbersaswellashavinga
fixedconcentrationofthelipidandcarbohydratecontentoftheLp(a),whichareusuallynot
known(35).Ultimately,theuseofmolar-basedmeasurescoupledwithadvancesinappropriate
gold-standardcalibratorswithouttheneedofpooledplasma,willpavethewaytofully
standardizetheseassays,improveclinicaldecision-making,aswellasoptimizeclinicaltrial
designwithnewtherapeuticapproaches.
AswehavepreviouslyshownintheEPIC-Norfolkcohort,highLp(a)levelswerestrongly
associatedwithanincreasedriskofCAD(13,14).Theseobservationsareconsistentwiththe
EASconsensusstatement,andmultiplestudiesandmeta-analysesconsistentlydemonstratinga
curvilinearcorrelationbetweenLp(a)levelsandincreasedriskofcardiovasculardisease(2,5,
13).Inthecurrentstudy,independentofthetypeofassayorthesexofthestudyparticipants,
bothLp(a)levelsinexcessof50mg/dlorabovethe5thquintile(or80thpercentile)were
correlatedwithincreasedCADrisk.The80thpercentilecut-offvalues,inapopulationenriched
inCADcases,of36mg/dlfortheRandoxassayand24mg/dlfortheUCSDassaywere
considerablylowerthantheEASproposedthresholdof50mg/dl,butareconsistentwith
epidemiologicalandtrialdata(2,5,20,21).Thisdiscrepancyisrelevantbecauseclinical
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decision-makingissuggestedtobemadeonthe50mg/dlthresholdinEurope,althoughmost
USlaboratoriesuse>30mg/dLaselevatedrisk(36).Asshownbymendelianrandomization
studiesattheprimarycarelevel(20),thatCVDriskassociatedwithLp(a)beginsatmuchlower
levelsthan50mg/dLandimpliesthatthelevelof50mg/dLisarbitrarybasedonpopulation
cutoffsandnotevidencebasedonCVDrisk.Anotherwaytobypassspecificthresholdsisto
determineassayspecific80thpercentilethresholds.Inthisway,theeffectofinter-assay
variationisminimized,becauseeachassayhasitsowncut-offvalue.Forthistowork,itisvital
todetermineLp(a)levelsofabroadrangeofpersons.Althoughlessfavorablethanamethod
basedoninternationalstandardsandcalibratorsitcanbeagoodinterimsolution.Finally,it
wasrecentlyshownintheMESAstudythatrace-specificlevelsofLp(a)mayalsoneedtobe
institutedduetodifferencesinthethresholdsandpredictivevaluesforCHDacrossraces(37).
However,hewaslimitedbylownumberofcardiovasculareventsandwideconfidenceintervals
inthePointestimates,suggestinganeedforconfirmationofthesefindings.Futurestudies,as
wellasclinicaltrialswithnoveldrugs,(6-10)willhavetotakeintoaccountwhattheappropriate
riskthresholdistotestthehypothesisthatLp(a)loweringpreventsCVDevents.
Limitationsofthisstudyarethatitonlyincludedoneepidemiologicalcohort,whichwas
primarilyCaucasian,andalsoonlycomparedtwoassays.Furtherstudiescomparingavarietyof
assaysindiversepopulationswillbeneededtoconfirmtheseresults
Inconclusion,theseresultsdemonstratethatevenlowercut-offvaluesofLp(a)couldbe
clinicallysignificantandtheuseofdifferentassayscouldhavelargeimpactonclinicaldecision
making.WiththearrivalofpotentdrugstolowerLp(a)to“normal”levels,fullstandardization
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ofassaymethodologieswillbeneededtoultimatelyoptimizeclinicaldecision-makingforthe
estimated>1billionpeoplewithpotentiallyatherogenicLp(a)levels(38).
Funding
ThisworkwassupportedbytheEPIC-NorfolkStudyisfundedbyCancerResearchUK
grantnumber14136andtheMedicalResearchCouncilgrantnumberG1000143.Thefunders
hadnoroleinthestudydesign,datacollection,analysis,decisiontopublishorpreparationof
themanuscript.DrsTsimikasandWitztumaresupportedbyNIHR01grantsHL119828,P01-
HL088093,P01HL055798,R01-HL106579,R01-HL078610,andR01-HL124174.
Disclosures
Drs.TsimikasandWitztumareco-inventorsandreceiveroyaltiesfrompatentsowned
bytheUniversityofCaliforniaSanDiegoonoxidation-specificantibodiesandofbiomarkers
relatedtooxidizedlipoproteins.Dr.WitztumisaconsultanttoIonisPharmaceuticalsand
InterceptPharmaceuticals.Dr.TsimikascurrentlyholdsadualappointmentatUCSDandIonis
Pharmaceuticals.Theotherauthorshavenopotentialconflictstodeclare.
Acknowledgements
TheauthorswishtothanktheparticipantsandstaffoftheEPIC-Norfolkprospective
populationstudy.
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FIGURELEGENDS
Figure1.OddsratiosforCHDriskbylipoprotein(a)categoriesformen(A),women(B)andboth
sexescombined(C).Dataarepresentedassex-specific,age-adjustedoddsratioswith
corresponding95%confidenceintervallimits.Oddsratiosperquintilewiththelowestquintile
asreferencecategory.Inaddition,anoddsratioispresentedforpeoplewithLp(a)>50mg/dL,
usingthosewithLp(a)<50mg/dLasreferencecategory.Lp(a)=lipoprotein(a),UCSD=
UniversityofCalifornia,SanDiego.
Figure2.Bland-AltmananalysisoftheaverageoftheUCSDandRandoxassaysplottedagainst
themeandifference.Dataarepresentedasdifferenceinmeasurements(Randox-UCSD)in
mg/dL,mean,lower95%limitofagreement(mean-1.96SD)andupper95%limitofagreement
(mean+1.96SD).AverageofRandoxandUCSDassaywerecalculated.UCSD=UniversityofSan
Diego.
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Table1.Baselinecharacteristics
Men Women
Controls Cases Controls Cases
n=592 n=380 n=356 n=243
Age,years 63.8±8.0 65.0±8.0 66.7±6.9 67.7±6.6Bodymassindex,kg/m2 26.2±2.8 27.1±3.2 26.1±4.2 26.8±4.3Currentsmoker 47(8.0%) 55(14.5%) 23(6.5%) 43(18.0%)Diabetesmellitus 18(3.0%) 40(10.5%) 8(2.2%) 16(6.6%)Systolicbloodpressure,mmHg 137±17 144±18 137±18 142±20Diastolicbloodpressure,mmHg 83±10 87±12 81±11 84±12Totalcholesterol,mmol/L 6.0±1.1 6.3±1.1 6.7±1.2 6.9±1.3LDLcholesterol,mmol/L 3.9±0.9 4.2±1.0 4.3±1.1 4.5±1.1HDLcholesterol,mmol/L 1.3±0.3 1.2±0.3 1.6±0.4 1.5±0.4Triglycerides,mmol/L 1.9±0.9 2.1±1.0 1.8±0.9 2.1±1.0Lipoprotein(a)Randoxassay,mg/dL 11.2(6.1–23.2) 14.3(7.2–35.9) 11.4(6.8–21.9) 15.5(8.5–37.9)Lipoprotein(a)UCSDassay,mg/dL 8.1(6.4-13.4) 9.3(6.8–23.9) 8.5(6.5–12.9) 10.4(7.4–29.8) Dataarepresentedasmean±standarddeviationforcontinuousvariableswithanormaldistribution,median(interquartilerange)forcontinuousvariableswithanon-normaldistribution,andnumber(percentage)forcategoricalvariables.LDL=low-densitylipoprotein,HDL=high-densitylipoprotein.UCSD=UniversityofCalifornia,SanDiego.
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Table2A.DistributionofCHDcasesandcontrolsbylipoprotein(a)categories,UCSDassay
UCSDassay
Lp(a)quintiles
1 2 3 4 5 ≥30.0mg/dL ≥50.0mg/dL
Men Lp(a)range,mg/dL 3.4–6.2 6.2–7.6 7.6–9.8 9.8–23.1 23.1-89.5 30,4-89.5 50.0–89.5
Lp(a)mean,mg/dL 5.5±0.5 6.8±0.4 8.5±0.6 13.8±3.9 41.8±15.0 47.1±13.7 63.6±10.0Cases/controls,n/n 66/128 63/132 73/121 82/113 96/98 71/74 29/19Cases,%oftotal 34 32.3 37.6 42.1 49.5 49 60.4Total,n 194 195 194 195 194 145 48
Women Lp(a)range,mg/dL 4.6–6.4 6.4–8.0 8.0–10.6 10.6–25.7 25.8-99.7 30.1-99.7 50.4–99.7
Lp(a)mean,mg/dL 5.7±0.4 7.2±0.4 9.2±0.7 14.6±3.9 45.5±15.8 48.1±15.4 64.9±11.7Cases/controls,n/n 84/35 44/76 45/75 50/70 69/51 60/44 24/15Cases,%oftotal 29.4 36.7 37.5 41.7 57.5 57.7 61.5Total,n 119 120 120 120 120 104 39
Combined Lp(a)range,mg/dL 3.4–6.3 6.3–7.7 7.7–10.2 10.1–23.8 24.0–99.7 30.1-99.7 50.0–99.7
Lp(a)mean,mg/dL 5.6±0.5 7.0±0.4 8.8±0.6 14.1±3.9 43.2±15.0 47.5±14.4 64.2±10.7Cases/controls,n/n 102/212 109/205 116/199 130/184 166/148 131/118 53/34Cases,%oftotal 32.5 34.7 36.8 41.4 52.9 52.6 60.9Total,n 314 314 315 314 314 249 87Dataarepresentedasmean±standarddeviation,numberorpercentage.Forthecategorymenandwomen,sexspecificquintileswerecalculated.Forthecategorycombinedpooledquintileswerecalculated.Lp(a)=lipoprotein(a),UCSD=UniversityofCalifornia,SanDiego.Lp(a)rangesforeachquintilerepresentsthe~cutoffvalues.
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Table2B.DistributionofCHDcasesandcontrolsbylipoprotein(a)categories,Randoxassay
Randoxassay
Lp(a)quintiles
1 2 3 4 5 ≥30.0mg/dL ≥50.0mg/dL
Men Lp(a)range,mg/dL 2.2–5.5 5.5–9.5 9.5–15.9 16.0–34.8 34.9–162.3 30.2-162.3 50.2–162.3
Lp(a)mean,mg/dL 3.8±0.8 7.4±1.1 12.2±1.8 23.1±5.3 58.3±23.5 55.2±23.6 72.9±23.6Cases/controls,n/n 65/128 70/125 65/130 83/112 97/97 108/113 52/52Cases,%oftotal 33.7 35.9 33.3 42.6 50 48.9 50Total,n 193 195 195 195 194 221 104
Women
Lp(a)range,mg/dL 2.2–6.4 6.4–10.3 10.3–16.6 16.7–36.8 37.0–143.5 30.2-143.5 50.1–143.5Lp(a)mean,mg/dL 4.4±1.1 8.3±1.1 12.9±1.8 24.8±6.1 63.2±24.5 57.8±24.8 75.6±24.0Cases/controls,n/n 38/81 43/77 46/74 51/69 65/55 82/65 43/30Cases,%oftotal 31.9 35.8 38.3 42.5 54.2 55.8 61.4Total,n 119 120 120 120 120 147 73
Combined
Lp(a)range,mg/dL 2.2–5.8 5.9–9.8 9.8–16.2 16.2–35.4 35.6–162.3 30.2-162.3 50.1–162.3Lp(a)mean,mg/dL 4.0±0.8 7.8±1.1 12.5±1.8 23.8±5.3 60.2±23.5 56.2±24.1 74.0±23.7Cases/controls,n/n 107/208 109/205 111/203 130/184 166/148 190/178 95/82Cases,%oftotal 34 34.7 35.4 41.4 52.9 51.6 55.2Total,n 315 314 314 314 314 368 177Dataarepresentedasmean±standarddeviation,numberorpercentage.Forthecategorymenandwomen,sexspecificquintileswerecalculated.Forthecategorycombinedpooledquintileswerecalculated.Lp(a)=lipoprotein(a).Lp(a)rangesforeachquintilerepresentthecutoffvalues.Lp(a)rangesforeachquintilerepresentsthe~cutoffvalues.
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Table3.OddsratiosforCHDriskbylipoprotein(a)categories Lp(a)quintiles
1 2 3 4 5 <50.0mg/dL ≥50.0mg/dL
Men Randoxassay 1.00 1.10 0.97 1.45 1.99
1.00 1.74
(0.72-1.67) (0.64-1.49) (0.96-2.19) (1.32-3.01)
(1.15-2.62)
UCSDassay 1.00 0.93 1.17 1.41 1.95
1.00 2.55
(0.61-1.42) (0.77-1.78) (0.93-2.13) (1.29-2.94)
(1.41-4.63)
Women Randoxassay 1.00 1.18 1.30 1.52 2.48
1.00 2.36
(0.69-2.01) (0.76-2.21) (0.89-2.58) (1.46-4.21)
(1.43-3.89) UCSDassay 1.00 1.36 1.39 1.63 3.21
1.00 2.50
(0.79-2.34) (0.81-2.39) (0.95-2.79) (1.88-5.49)
(1.28-4.87)
Combined Randoxassay 1.00 1.03 1.05 1.35 2.18
1.00 1.97
(0.74-1.43) (0.75-1.46) (0.98-1.87) (1.58-3.01)
(1.43-2.70) UCSDassay 1.00 1.10 1.20 1.45 2.35
1.00 2.53
(0.79-1.53) (0.86-1.67) (1.04-2.01) (1.70-3.26) (1.62-3.95) Dataarepresentedasoddsratioswithcorresponding95%confidenceintervallimits.Forthecategorymenandwomen,sex-specific,
age-adjustedoddsratioswerecalculated,withtheLp(a)cut-offvaluesaspresentedintable2.Forthecategorycombinedsex-andage-adjustedoddsratioswerecalculated,withtheLp(a)cut-offvaluesaspresentedintable2.Oddsratiosperquintilewiththelowestquintileasreferencecategory.Inaddition,anoddsratioispresentedforpeoplewithLp(a)>50mg/dL,usingthosewithLp(a)<50mg/dLasreferencecategory.Lp(a)=lipoprotein(a),UCSD=UniversityofCalifornia,SanDiego.
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Table4.NumberofstudyparticipantswithanLp(a)level<and≥50mg/dLperassay
UCSDassay
Randoxassay
Lp(a) <50.0mg/dL ≥50.0mg/dL
Men <50.0mg/dL 861 63 ≥50.0mg/dL 7 41
Women <50.0mg/dL 525 35 ≥50.0mg/dL 1 38
Combined <50.0mg/dL 1386 98 ≥50.0mg/dL 8 79
Datearepresentedasnumbers.Lp(a)=lipoprotein(a),UCSD=UniversityofCalifornia,SanDiego.
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Figure1A.OddsratiosforCHDriskbylipoprotein(a)categories,men
1 2 3 4 50
1
2
3
4
5
6
Randox assay
UCSD assay
< 50 m
g/dL
≥ 50 m
g/dL
Lp(a) quintiles--------------------------
Odd
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Figure1B.OddsratiosforCHDriskbylipoprotein(a)categories,women
1 2 3 4 50
1
2
3
4
5
6
Randox assay
UCSD assay
< 50 m
g/dL
≥ 50 m
g/dL
Lp(a) quintiles------------------------
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Figure1C.OddsratiosforCHDriskbylipoprotein(a)categories,sexescombined
1 2 3 4 50
1
2
3
4
5
6
Randox assay
UCSD assay
< 50 m
g/dL
≥ 50 m
g/dL
Lp(a) quintiles--------------------------
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Figure2.Bland-AltmanplotRandoxvs.UCSDRandoxassay
0 20 40 60 80 100
120
-40
-20
0
20
40
60
80
100
120
Mean - 1.96SD
Mean + 1.96SD
Mean
Average of Randox and UCSD assay (mg/dL)
Dif
fere
nce
in m
easu
rem
ents
(Ran
dox
- UC
SD) (
mg/
dL)
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