02.05.2017 LpoprotenSubfractonatonAnalyssAACC.

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LaboratoryNews
Lipoprotein
Subscrbe
Subfractionation Analysis
CLNDaly TheContnungQuestforImprovngCardovascularRskPredcton
CLNStat
Author:AnnaWolska,PhD,andAlanRemaley,MD,PhD// RELATED ITEMS
CLNArtcles Date:JAN.3.2017//Source:ClncalLaboratoryNews

WEBINAR
BoardofEdtors Topcs:CardovascularDsease,LpdsandLpoprotens
TheClncalChemst's
Cardovasculardsease(CVD)sthenumber1causeofdeath PerspectveonCardac
Permssonsand ntheworld.By2030,almost23mllonpeoplearepredctedto TroponnRuleOut/Rule
Reprnts defromCVD,manlybecauseofheartdseaseandstroke(1). InStrategesandUsen
Becauselpoprotenpartclesplayakeyrolenatherogeness, AcuteandChronc
ACCENTCE Dsease
theyareusefulbomarkersforassessngCVDrskaswellasfor
CredtforCLN APR.26.2017
montornglpdlowerngtherapy.
Artcles
Prortotheearly1950s,clncansusedtotalcholesterolnall ARTICLE
ContactUs thedfferentlpoprotenfractonsasthemanmarkerforCVD StrategesforUsng
rsk.However,nasemnalpublcatonn1950,JohnGofman HghSenstvty
Advertse frstshowedusngdenstygradentultracentrfugatonthat
TroponnAssays
APR.20.2017
cholesterolnlowdenstylpoprotens(LDL)waspostvely
assocatedwthCVDrsk,whereascholesterolnhghdensty
PRESSRELEASE
lpoprotens(HDL)wasnverselyrelatedtoCVDrsk(2).Durng
theensungdecades,researchershavenvestgatedthe DagnossofandCare
dfferentsubfractonswthnthemajorlpoprotenclasses, forHeartAttacksCould
namelychylomcrons,verylowdenstylpoprotens(VLDL),
BeAdvancedbyNew
StudyPublshedn
LDL,andHDL,todetermnewhetheranalyssoflpoproten
AACCsJournalof
subfractonatonsmprovesCVDrskpredcton. AppledLaboratory
Medcne
OnlyabouthalfofpatentswhodevelopCVDappeartobeat APR.3.2017
rskbasedontotalcholesterol,maknglpdsubfractonaton
attractvefortspotentaltodentfyatrskpatentswhowould
notbedetectedbyconventonallpdmarkers(3).Researchers
alsoareexplorngwhetherlpdsubfractonatonmghthelp
clncansdecdewhchpatentsatntermedaterskshouldbe
treatedwthlpdlowerngmedcatonsorothertherapes.

Thereareseveraldfferentmethodsforlpoproten
subfractonatonanalyssthatrelyuponthedfferentphyscal
andchemcalpropertesoflpoprotens.Thsartclefocuseson
thelpoprotensubfractonatonmethodsshownnTable1that
routneclncallaboratoresreadlyperformorwhchreference
labsoffer,ncludnggelelectrophoress,denstygradent

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ultracentrfugaton,nuclearmagnetcresonance(NMR)
spectroscopy,andonmobltyanalyss.

GelElectrophoress
Gelelectrophoressoneoftheoldestmethodstoseparate
lpoprotensdoessobasedonacombnatonofchargeand
sze(4).Althoughtsarelatvelylowresolutontechnque,
electrophoressonagarosegelsseparatesnotonlythemajor
lpoprotenclassesbutalsorevealssomelpoproten
subfractons(Fgure1).Typcallygelelectrophoresssa
qualtatvetest,asthelpoprotensarestanedwthadyesuch
asSudanblackthatprmarlystanstrglycerdesandcholesteryl
esters.Thecholesterolcontentoftheseparatedlpoprotens
can,however,bequanttatvelyandenzymatcallymeasuredby
atechnquethattreatsthegelwthcholesteroloxdase.Two
FoodandDrugAdmnstraton(FDA)approvedcommercalkts
areavalable.

TheorgnalFredrcksonphenotypcclassfcatonoflpd
dsorders,whchwasbasedonagarosegelelectrophoress(5),
haslargelybeenreplacedbymoreaccurateclassfcatons
basedonDNAsequenceanalyssand/orotherlpoproten
phenotypngtests.Lpoproten(a)[Lp(a)],apartcularlypro
atherogencLDLlkelpoproten,canbedetectedbyagarose
gels,butothermorequanttatvemethodsexstformeasurng
thssubfracton.

Evenwthtsshortcomngs,gelelectrophoressstllsthebest
methodfordetectngseveraldsordersandtypesoflpoproten
partcles.Chylomcrons,forexampledffculttomeasureby
mostothermethodsbecauseoftherlargeszecanbeeasly
observednagarosegelsbecausetheystaytrappednearthe
orgnofthegel.Incontrast,VLDL,theonlyotherpartclethat
carressgnfcantamountsoftrglycerdes,mgratesmuch
furtherntothegel.Patentswthdysbetalpoprotenemahavea
defectntheclearanceofchylomcronsandVLDLand
accumulatentermedatedenstylpoprotens(IDL),often
referredtoasremnantlpoprotens.Thesetendtomgrate
betweenLDLandVLDLonagarosegels.
Dysbetalpoprotenemasarelatvelycommondsorderthat
affectsabout1%ofthepopulaton.Identfyngndvdualswth
thscondtonsmportantbecausetheymghtbeneftfromboth
statnsanddrugsthatspecfcallylowertrglycerdes.

Agarosegelelectrophoresssalsooneofthefewmethods
avalablefordetectnglpoprotenX(LpX),anabnormal
lpoprotenpartclethataccumulatesnpatentswthcholestass
andfamlallecthncholesterolacyltransferasedefcency.
Unlkeotherlpoprotens,LpXmgratestowardthecathoden
agarosegel,makngteasytodstngushfromother
lpoprotens.

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Currently,oneFDAapprovedpolyacrylamdetubegel
electrophoresssystemsavalableforseparatngLDLnto
sevensubfractons:theLpoPrntsystemfromQuantmetrx(6).
Aswllbedscussedfurther,researchershavefoundthatsmall
denseLDLsubfractonsaremorecloselyrelatedtoCVDrsk
thanLDLC,mostlkelybecauseoftherncreasedabltyto
enteratherosclerotcplaques.Certanreferencelabsoffer2D
gelelectrophoressteststhatseparateHDLntoatleastfve
subfractons,andmayalsomeasuretheamountof
apolpoprotenAI(apoAI)neachofthefveHDLsubclasses
(7).

DenstyGradentUltracentrfugaton
Denstygradentultracentrfugatonnvolvesseparatng
lpoprotensaccordngtotherdenstyanddependsonthe
lpd/protenratooflpoprotens.Chylomcrons,forexample,are
almostcompletelycomprsedoflpdsandareverylght,wtha
denstylessthanwater.Incontrast,thesmallerlpoprotens,
suchasHDL,areonlyabout50%byweghtlpdswththeother
halfmadeupofdenserprotens,gvngHDLadenstyrange
between1.0631.25g/mL.Denstygradentultracentrfugaton
notonlyformsthebassforthemannomenclatureof
lpoprotensbutalsoservesasareferencemethodfor
measurnglpoprotens(8).Evenso,mostclncallaboratores
cannotreadlyperformthsmethod,anduntlrecentlytwas
avalablefromreferencelaboratoresastheVertcalAutoProfle
(VAP)testII,whchfrstseparateslpoprotensbydensty
gradentultracentrfugatonnavertcalrotor.

Thenextstepsenzymatcmeasurementofthecholesterol
dstrbutonthroughoutthedenstygradentspectrumby
contnuousflowanalyss.TheVAPtestncludesHDL,LDL,and
VLDLsubfractonatonbydrectlymeasurngtheamountof
cholesterolcontanedwthneachofthesesubfractons.Thetest
measuresIDLCandLp(a)Cusngsoftwarethatdeconvolutes
dataembeddedntheparenttracng.Italsoassessestotal
cholesterol,trglycerdes,themanprotencomponentsofHDL
andLDL,apoAI,andapolpoprotenB(apoB),respectvely,
whchcanbeusedtoestmatethepartclenumberofthese
lpoprotens.VAPclassfesLDLsubfractonsaspatternA,
patternB,orpatternA/B.PatternAmpleslarge,buoyant
LDLpartcles,whereaspatternBmplessmall,denseLDL
partcles(Table2),whchhavebeenstronglyassocatedwth
CVDrsk.ThetestalsoseparatescholesterolonHDLntoa
larger,lessdenseHDLsubfracton(HDL2)andsmall,denser
HDLsubfracton(HDL3)(9).AlthoughtheclncalutltyforHDL
subfractonssnotasclearasforLDLsubfractons,largersze
HDLnmoststudesappearstobemorestronglynversely
relatedtoCVDrsk(10).

NMRSpectroscopy

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LaboratoresalsouseNMRspectroscopytoperformlpoproten
subfractonanalyss.NMRspectroscopyquanttatvely
measuresthespectralsgnalsgeneratedbythetermnalmethyl
groupsonlpdswthnlpoprotenpartcles.Unlkemostother
methods,NMRspectroscopydoesnotrequrephyscal
separatonoflpoprotens,andasdefromseparatngplasma
frombloodcells,nopreanalytcsampleprocessngs
necessary.Atleastonereferencelaboratoryoffersthemethod,
andothersaredevelopngt.TheNMRLpoProfletests
currentlytheonlyNMRassayformeasurngLDLpartcle
number(LDLP),trglycerdes,andHDLCthathasbeen
approvedbyFDA.Theotherlpdandlpoprotenparameters
thatthsmethodmeasuresareshownnTable3.Thstestalso
reportsalpoprotennsulnresstancescorebasedonthe
lpoprotenproflethatsassocatedwthnsulnresstanceand
dabetesrsk.ThepostonoftheresonancentheNMRspectra
ofthetermnalmethylgroupsonlpdssaffectedbytheszeof
thelpoprotenpartcle,whchafteradeconvolutonalgorthm
enablesthelaboratorytocalculatethenumberofpartcles
wthneachlpoprotenszesubfracton.LDLPssmply
calculatedasthesumofallthendvdualnumbersofLDLsze
subfractons(11).

AsllustratednFgure2,twondvdualswthsmlarLDLC
levelscanhavequteasubstantalvarancenLDLP,because
largerszeLDLpartclescancarrymorecholesterol(12).
ResearchershavefoundthatCVDrsktracksmorecloselywth
LDLPwhentheresdscordancebetweenLDLCandLDLP
asoftenoccursnpatentswthmetabolcsyndromeand
dabetes(13).Inthefuture,laboratoresmghtobtanevenmore
dscrmnatonbyspecfcallymeasurngthedfferentsze
subfractonswthnVLDL,LDL,andHDL,butthssstllan
actveareaofnvestgaton.

IonMobltyAnalyss
Boththeszeandconcentratonsoflpoprotenpartcle
subfractonscanalsobemeasuredbymassspectroscopy,
usnggasphasedfferentalelectrcalmoblty(alsoknownas
onmoblty).Thsmethoddependsontheprncplethat
partclesofagvenszeandchargebehavedfferentlywhenput
nalamnarflowofarandsubjectedtoanelectrcfeld.Quest
Dagnostcshasadaptedthsmethodfordrectlymeasurngthe
szedstrbutonoflpoprotenpartclesnarangefrom7nmto
about120nm.Theanalysspartofthemethodsautomated
andgeneratesproflesofpartclenumberandpartclemass
versuspartcledameter.However,theplasma/serumsample
requresextensvepreanalytcalpreparatontosolatethe
lpoprotens.

Inthsstep,thelaboratoryfrstaddsanalbumnremovalreagent
tothesample,thenultracentrfugesthesampleforabout2

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hourstosolatethelpoprotenfracton.Thelaboratorythen
dlutesthesamplenavolatlebufferandelectrospraystna
dfferentalmobltyanalyser.Thsprocessyeldspartcle
numberdstrbutonswthn2mnutesforHDL,LDL,IDL,and
VLDLconvertedntopartclemassdstrbutons.Other
conventonallpdandapolpoprotentestsarealsoncludedn
thepanel(Table4)(14).

Concluson

Numerousmethodsnowexstformeasurnglpoproten
subfractonsandtheresgrowngevdenceforhowsuchtests
mproveCVDrskpredctonandcosteffectveness(15).Atths
tme,however,theyarenotwdelyusedandarenot
recommendedbynatonalandnternatonalgudelnesforuse
asscreenngtests.LkethestuatonwthCreactveprotenasa
CVDrskbomarker,theycanperhapsbebestusednpatents
wthntermedaterskand/orwhenthereappearstobe
dscordancebetweenapatentsclncalpresentatonand
conventonallpdbomarkermeasurements.InthecaseofNMR
andmassspectrometrybasedtestng,theabltytoaddnew
markersforCVDandotherdseaseswthoutmuchaddtonal
costmaymaketheseapproachesmoreattractventhefuture.

*AnnaWolska,PhD,andAlanT.Remaley,MD,PhD,have
recevedresearchsupportfromLpoScenceInc.,whchwas
acquredbyLabCorpInc.n2014.

AnnaWolska,PhD,sapostdoctoralfellowntheLpoproten
MetabolsmSectonofthePulmonaryandVascularMedcne
BranchattheNatonalInsttutesofHealth.
+Emal:anna.wolska@nh.gov

AlanT.Remaley,MD,PhD,sthesenornvestgatornthe
LpoprotenMetabolsmSectonofthePulmonaryandVascular
MedcneBranchattheNatonalInsttutesofHealth.+Emal:
aremaley1@nhlb.nh.gov

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