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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]

by guest, on March 23, 2019

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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

s ra

tio


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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------------------------

Odd

s ra

tio


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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--------------------------

Odd

s ra

tio


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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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