Discovery of serum biomarkers of alcoholic fatty liver in a rodent model: C-reactive protein

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Description

Excessive consumption of alcohol contributes to alcoholic liver disease. Fatty liver is the early stage of alcohol-related liver disease. The aim of this study was to search for specific serological biomarkers of alcoholic fatty liver (AFL) compared to healthy controls, non-alcoholic fatty liver (NAFL) and liver fibrosis in a rodent model. Methods Serum samples derived from animals with AFL, NAFL, or liver fibrosis were characterized and compared using two-dimensional differential gel electrophoresis. A matrix-assisted laser desorption ionization-time of flight tandem mass spectrometer in conjunction with mascot software was used for protein identification. Subsequently, Western blotting and flexible multi-analyte profiling were used to measure the expressions of the putative biomarkers present in the serum of animals and clinical patients. Results Eight differential putative biomarkers were identified, and the two most differentiated proteins, including upregulated C-reactive protein (CRP) and downregulated haptoglobin (Hp), were further investigated. Western blotting validated that CRP was dramatically higher in the serum of AFL compared to healthy controls and other animals with liver disease of NAFL or liver fibrosis ( p < 0.05). Moreover, we found that CRP and Hp were both lower in liver fibrosis of TAA-induced rats and clinical hepatitis C virus-infected patients. Conclusion The results suggest that increased levels of CRP are an early sign of AFL in rats. The abnormally elevated CRP induced by ethanol can be used as a biomarker to distinguish AFL from normal or otherwise diseased livers.

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Publié le 01 janvier 2011
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Langue English
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Liu
etal
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JournalofBiomedicalScience
2011,
18
:52
http://www.jbiomedsci.com/content/18/1/52

RESEARCH

OpenAccess

Discoveryofserumbiomarkersofalcoholicfatty
liverinarodentmodel:C-reactiveprotein
Shu-LinLiu
1
,Chun-ChiaCheng
2,3
,Chun-ChaoChang
4
,Fu-DerMai
2,5,6
,Chia-ChiWang
7
,Shui-ChengLee
3
,
Ai-ShengHo
8
,Ling-YunChen
1*
andJungshanChang
2,5,9,10*

Abstract
Background:
Excessiveconsumptionofalcoholcontributestoalcoholicliverdisease.Fattyliveristheearlystage
ofalcohol-relatedliverdisease.Theaimofthisstudywastosearchforspecificserologicalbiomarkersofalcoholic
fattyliver(AFL)comparedtohealthycontrols,non-alcoholicfattyliver(NAFL)andliverfibrosisinarodentmodel.
Methods:
SerumsamplesderivedfromanimalswithAFL,NAFL,orliverfibrosiswerecharacterizedandcompared
usingtwo-dimensionaldifferentialgelelectrophoresis.Amatrix-assistedlaserdesorptionionization-timeofflight
tandemmassspectrometerinconjunctionwithmascotsoftwarewasusedforproteinidentification.Subsequently,
Westernblottingandflexiblemulti-analyteprofilingwereusedtomeasuretheexpressionsoftheputative
biomarkerspresentintheserumofanimalsandclinicalpatients.
Results:
Eightdifferentialputativebiomarkerswereidentified,andthetwomostdifferentiatedproteins,including
upregulatedC-reactiveprotein(CRP)anddownregulatedhaptoglobin(Hp),werefurtherinvestigated.Western
blottingvalidatedthatCRPwasdramaticallyhigherintheserumofAFLcomparedtohealthycontrolsandother
animalswithliverdiseaseofNAFLorliverfibrosis(
p
<0.05).Moreover,wefoundthatCRPandHpwereboth
lowerinliverfibrosisofTAA-inducedratsandclinicalhepatitisCvirus-infectedpatients.
Conclusion:
TheresultssuggestthatincreasedlevelsofCRPareanearlysignofAFLinrats.Theabnormally
elevatedCRPinducedbyethanolcanbeusedasabiomarkertodistinguishAFLfromnormalorotherwise
diseasedlivers.
Keywords:
alcoholicfattyliver,biomarker,C-reactiveprotein,haptoglobin,two-dimensionaldifferentialgel
electrophoresis

Background
relatedliverdisease.However,themethodologyofhisto-
Excessivealcoholconsumptionaffectslipidmetabolismlogicalassessmentsneedstoovercomeseveraldraw-
intheliver[1,2],contributingtothedevelopmentofbackssuchasitsinvasivecharacterandsamplingerror
alcohol-relatedliverdiseases.Therearethreemaintypes[3].Moreover,ithasdifficultyindistinguishingAFL
ofalcohol-relatedliverdisease,theseare:alcoholicfattyfromnon-alcoholicfattyliversolelythroughahistologi-
liver(AFL),alcoholichepatitis,andalcoholiccirrhosis.calassessment.Ontheotherhand,predictingethanol-
AFListheearlystageofalcohol-relatedliverdiseases.inducedoxidativestressandtissueinjuryintheliver
Therefore,identifyingputativeserumbiomarkersofAFLrequireparticularlysensitivemarkers[4].Aprevious
forearlyandaccuratediagnosticmethodsisvital.studysuggestedthatglutamyl-transpeptidase(GGT)and
Histologicalassessmentofliverbiopsyspecimensalanineaminotransferase(ALT)arebiomarkersfordiag-
remainsthegoldstandardfordeterminingalcohol-nosingalcoholicliverdisease[5].Severalreportspro-
posedthatserumC-reactiveprotein(CRP),tissue
*Correspondence:chenly@csmu.edu.tw;js.chang@tmu.edu.tw
polypeptide-specificantigen(TPS),andinterleukin-6are
1
InstituteofBiochemistryandBiotechnology,ChungShanMedical
noninvasivebiomarkersofalcoholichepatitis[6-10].
University,Taichung,Taiwan
2
GraduateInstituteofMedicalSciences,CollegeofMedicine,TaipeiMedical
Nevertheless,areliablebiomarkertopredicttheearly
University,Taipei,Taiwan
Fulllistofauthorinformationisavailableattheendofthearticle
A©tt2ri0b1u1tiLoinuLeitcealn;sleic(ehntstep:e//BciroeaMtievdecCoemntrmalonLst.do.rgT/hliisceisnsaens/Obyp/e2n.0)A,cwcehsischarptiecrlemidtissturinbruetsterdictuenddeurset,hedistterribmustioofn,thaendCrreeaptirvoeduCcotimonmionns
anymedium,providedtheoriginalworkisproperlycited.

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stageofalcoholichepatitis,i.e.,AFL,andtodistinguish
AFLfromothertypesofliverdiseaseisneeded.
Aproteomicsstrategybasedontwo-dimensionaldiffer-
entialgelelectrophoresis(2D-DIGE)[11]allowsforthe
simultaneousresolutionofthousandsofproteinsfrom
sampleswithhighprecisionandreplication.2D-DIGE
wasusedtoscreenanddetermineputativebiomarkersof
manydiseases[12-15].Thoseproteinsamplesofinterest
displayedon2D-DIGEcanbeextractedandacquired
fromgelsforidentificationandfurtherinvestigation.In
total,wediscoveredeightdifferentialproteinsassociated
withAFLusing2D-DIGE,includingthemostdifferen-
tiatedproteins:CRPandhaptoglobin(Hp).
ThisstudyrevealedthatCRPisanovelearlybiomarker
ofalcohol-inducedfattyliver,andthatCRPandHpwere
bothparticularlydecreasedwithliverfibrosis.Inconclu-
sion,wepresentCRPasasurveillancemarkerofalcohol-
inducedfattyliverinarodentmodel,whichmayhelp
diagnoseearlyalcohol-inducedpathophysiologicalaltera-
tionsinclinicalpractice.
Materialsandmethods
Animalmodelandsamplepreparation
Animalexperimentationwasperformedaccordingto
approvedproceduresoftheInstituteofNuclearEnergy
Research,AtomicEnergyCouncil,Taoyuan,Taiwan
(approvalno.:98053).Wistarratswereusedtogenerate
animalswithAFL,non-AFL(NAFL),andliverfibrosis.
AFLrats(
n
=6)wereorallyfed5mlofa36%alcohol
solutionfor4weeks(6g/kg/day)[16].Forratswith
NAFL,animalsweregivenfoodcontaining60%fructose
(
n
=4)or45%fat(n=6)for12weeks.Theliver-fibrosis
rats(
n
=6)werefed0.04%thioacetamide(TAA)-contain-
ingdrinkingwaterfor12weeks.Controlanimalswerefed
normaldietswithnoadditivesintheirfood(
n
=7).Sera
andlivertissueswerecollectedforfurtherinvestigation.
Clinicalserumcollection
Theseraofhealthyvolunteers(
n
=16),patientswithnon-
alcoholicsteatohepatitis(
n
=19)andpatientswithhepatitis
Cvirus(HCV)-infectedliverfibrosis(
n
=17)were
obtainedfromChengHsinGeneralHospitalinTaiwan
(approvalno.97016).Aliverbiopsyandsubsequenthisto-
logicalexaminationwereusedtoassessthestageofliver
fibrosisaccordingtotheMetavirclassification,andalsoto
determinethefattychangeandmodifiedHAIgrade.A
liverbiopsywasnotperformedinhealthycontrolsdueto
ethicalissues.
2D-DIGE
Each50
μ
gofproteinfromanormalcontrolorAFLrat
waslabeledwith400pmolofCy3orCy5andtheinter-
nalpooledstandard(100
μ
g)waslabeledwith800pmol
ofCy2for30min.Thethreelabeledsampleswere

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pooledtogetherforanalysis.IPGstrips(18cm)atpH
4~7forthefirst-dimensionIEF(EttanIPGphorSystem,
GEHealthcare)and12.5%polyacrylamidegelsforthe
seconddimensionwereusedtoseparateserumproteins.
TheCy2,Cy3,andCy5-labeledimageswereacquiredon
aTyphoonTRIOVariableModeImager(GEHealth-
care)using488-,532-,and633-nmlaserswithrespec-
tiveemissionfiltersof520,532,and670nm.Images
wereanalyzedusingDeCyder6.5software(GEHealth-
care)toselectthedifferentialproteins.Proteinspotsof
interestwereselectedaccordingtoanindependentStu-
dent

st-testwithasignificantvalueof<0.05.
Proteinidentification
In-geldigestionandMALDI-TOFMSanalysiswereper-
formedaspreviouslydescribed[13].
Westernblotting
Eachserumsamplewasdiluted1:1withasodiumdode-
cylsulfate(SDS)buffercontaining50mMofTris-Cl,8M
urea,30%glycerol,2%SDS,20mMofdithiothreitol,and
0.1%bromophenolblue.A4%~12%SDS-polyacrylamide
gelelectrophoresis(PAGE)(Invitrogen)wasperformedto
separatetheproteins.Theiblot(Invitrogen)wasusedto
transferproteinstoapolyvinylidenedifluoride(PVDF)
membrane.Afterusing0.5%milktoblotthePVDFmem-
branefor30min,CRPandHpweredetectedbyamouse
anti-CRPimmunoglobulinG(IgG)(AffinityBioReagents)
andamouseanti-HpIgG(Sigma)foratleast1hrespec-
tively.Thesecondantibodyconjugatedwithhorseradish
peroxidase(HRP)wasincubatedfor1hatroomtempera-
ture.Themembraneswerewashedthreetimesinphos-
phate-bufferedsaline(PBS;10mMsodiumphosphate
(pH7.4)and0.9%NaCl)betweenaddingantibodies.The
ImagingSystem(GelDocXRSystem,Bio-Rad)wasused
toacquireimagesdependingonamoderateexploration
timeandtosemi-quantifyproteinexpressions.
MeasurementofCRPandHpconcentration
Flexiblemulti-analyteprofiling(xMAP)wasperformed
tomeasureserumconcentrationsofCRPandHpin
clinicalsamplesusingthecommercialBio-PlexPro
HumanAcutePhase4-PlexPanel(Bio-Rad).Themea-
surmentprocedurefollowedinstructionsinthemanual.
Statisticalanalysis
Thestatisticalsoftware,SPSS,wasusedtocalculatethe
significanceaccordingtoStudent

st-test.Significance(
p
value)wasacceptedas<0.05.
Results
Animalmodels
Eachexperimentalanimalbearingaspecificliverdisease,
namely:AFL,NAFLand,liverfibrosiswasanalyzedand

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compared.Toensurethecorrectestablishmentofthe
animalmodels,severalindicatorsintheserumincluding
aspartateaminotransferase(AST),alanineaminotransfer-
ase(ALT),totalbilirubin(TBIL),totalcholesterol
(TCHO)andtriglyceride(TG)weremeasuredandcom-
pared(Table1).LevelsofAST,ALT,andTBILincreased
inLFrats[199±37U/L(
p
<0.01),74.4±19U/L(
p
<
0.01),and0.84±0.10mg/dl(
p
<0.05),respectively]com-
paredtothoseinnormalcontrols(AST154±25U/L;
ALT56±15U/L;andTBIL0.70±0.06mg/dl),indicat-
ingthattheliverfunctionofLFratswasimpaired.How-
ever,nosignificantchangesinthesethreeserum
indicatorsinAFLandNAFLwereobserved.Otherindi-
cators,TCHOandTG,weremeasuredtoobservelipid
accumulationintheliver.AnincreasedTGlevelwas
observedonlyinthegroupofratsfedthehighconcen-
trationoffructose(91±14mg/dl,
p
<0.05)comparedto
otherratsfeddifferentdiets,buttheTCHOlevel
remainedunchanged.TheseresultsshowthatAST,ALT,
andTBILincreasedintheserumofLFrats,andTG
increasedinratsthatwerefedahighlevelfructose.
Therefore,theseexistingserumindicatorssofarcould
notbeusedtodistinguishAFLfromthenormalcontrols,
whichmeansthatfindingdifferentialbiomarkersofAFL
isvital.
Ontheotherhand,weobservedthattherewasnosig-
nificantdifferenceinthemorphologyofliversamong
normal,AFLand,NAFLratsexceptforratswithliver
fibrosisshowingexcessscars(Figure1).Tomore-deeply
assessourestablishedrodentmodelsusinghistological
examinations,theresultsshowedthatliversofratswith
AFLappearedtospecificallybefilledwithmacrovesicu-
larfatwithinhepatocytescomparedtonormalcontrols
accordingtohistologicalstainingwithhematoxylinand
eosin(H&E)(Figure1),demonstratingthatethanol
treatmentinducedlipidaccumulationintheliver.
Furthermore,signsoffocalnecroinflammationwere
absentfromthelivertissuesofratswithAFL(Figure1).

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DiscoveryofAFLbiomarkersusing2D-DIGE
Inordertoexplorethesignaturemolecularbiomarkers
ofAFL,aproteomicmethodology,2D-DIGE,described
above

Materialsandmethods

wasperformedtoana-
lyzeindividualserumfromtwonormalcontrolsortwo
AFLratsshownonFigure2Atosearchforputativebio-
markersofAFL.Inthe2D-DIGEanalysis,Cy3andCy5
wereusedtoindividuallylabelserumsamplesfromnor-
malcontrolsandAFLrats.Forsamplenormalization,
Cy2wasusedtolabeltheinternalstandardincluding
50%ofnormaland50%ofAFLrats.Theproteinimage
waspresentedasshowninFigure2B.Normalcontrol
serawerelabeledwithCy3andappearedcoloredgreen
inthegel.SamplesderivedfromratswithAFLwere
pre-labeledwithCy5andshowedasaredcolorinthe
gel.EightdifferentialproteinsincludingCRP,Hp,afa-
min,alpha-fetoprotein(AFP),inter-alpha-inhibitorH4
heavychain(ITIH4),serineproteaseinhibitorKazal-
type5(SPINK5),heakshockprotein75kDa(HSP75),
andvitaminDbindingproteinprepeptide(VDBP)were
acquiredaccordingtothestatisticalanalysiswithsignifi-
cant
p
values(
t
-test,
p
<0.05),andanintensitychange
ratioof>1.2-foldcalculatedwithDeCydersoftware.
ThelocationofeachproteinisshowninFigure2C.
Whenusingastereopictureanddetailedgelimagesto
presenttheproteinexpressionsofdifferentialbiomar-
kers(Figure3),CRP,AFPandafaminwereincreased
higherintheserumofAFLrats,andHp,ITIH4,
SPINK5,HSP75,andVDBPwereconverselylower.In
particular,Hp,ITIH4andSPINK5hadaseriesofspots
nearby,buttheproteinexpressionaltrendswerestillthe
same.Wespeculatedthatpost-translationalmodification
wouldnotaffectorinfluencetheproteinexpression.
Essentially,weidentifiedproteinspotsbycomparingthe
massspectrumobtainedfromMALDI-TOFMScoupled
withtheNCBIdatabase.Theproteinsidentifiedare
showninTable2.Accordingtothesetcalculationin
theDeCydersoftware,theupregulation(+)ispresented

Table1Levelsofsomeclinicalserumindicatorsintheanimals
IndicatorsNormalAFLHF-NAFL(
n
=4)HL-NAFL(
n
=6)LF
§
(
n
=7)(
n
=6)(
n
=5)
BW(g)270±5

/457±14
§
275±3

418±28
§
565±29
§,
*ND
AST(U/L)154±25148±34
c
164±31181±63199±37*
ALT(U/L)56±1541±13
c
48±1757±2274±19*
TBIL0.70±0.060.62±0.09
a,b,c
0.78±0.040.75±0.050.84±0.10*
(mg/dl)
TG51±1658±10
a
91±14**56±1148±14
(mg/dl)
TCHO61±1068±872±1071±1261±17
(mg/dl)
BW,bodyweight;AST,aspartateaminotransferase;ALT,alanineaminotransferase;TBIL,totalbilirulin;TG,triglyceride;TCHO,totalcholesterol;AFL,alcoholicfatty
liver;HF-NAFL,highfructose-inducednon-alcoholicfattyliver;HL-NAFL,highlipid-inducednon-alcoholicfattyliver;LF,liverfibrosis.Indicatorsforpredictingliver
functionincludeGOT,GPT

,andbil
§
irubinandforpredictingfatcellsincludetriglycerideandcholesterol.Ratsweretreatedbeforetheageof6weeksand
sacrificedbytheagesof10and18weeks.ND,Non-detection.The
p
valuewascalculatedaccordingtoStudent

s
t
-test.*
p
<0.05and**
p
<0.01ascompared
tonormalcontrols.Asignificantchange(
p
<0.05)wasalsodeterminedforAFLratscomparedto
a
HF-NAFL,
b
HL-NAFL,and
c
LFrats.

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Figure1
Histologicalanalysisandcomparisonsamongfourgroupsofrats
.Imagesofliversfromnormalcontrol,ratswithalcoholicfatty
liver(AFL),non-alcoholicfattyliver(NAFL,fed60%fructose)andliverfibrosis(leftcolumn)wereanalyzedbyhematoxylinandeosin(H&E)
staining(rightcolumn).InAFLandNAFLrats,liverswerefilledwithprominentfattychange.Thescalebarrepresents25
μ
mfornormal,AFLand
NAFL;but100
μ
mforliverfibrosis.

asthelevelsofAFLdividedbythatofnormalcontrol,
CRPandHpvalidation
andthedownregulation(-)ispresentedasthelevelsofWewereinterestedincharacterizingtheroleofCRP
normalcontroldividedbythatofAFL.TheresultsandHpinAFLduetosignificantchangesintheirpro-
demonstratedthatCRPandHpweredramaticallyup-teinexpressions.IntheprocessofWesternblotting,we
anddownregulated,respectively(CRP:+4.71-fold;HP:preciselycontrolledtheloadingproteinto20
μ
g,and
-11.54-fold,Table2).thetotalproteinstainedbySYPRORubywasusedasa

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Figure2
Determinationofserumbiomarkersofalcoholicfattyliver(AFL)using2D-DIGE
.(A)Theexperimentaldesignusedtwoindividual
sampleseachfromhealthycontrolsandAFL.(B)Thecombinationoftwodifferentacquiredimages,inwhich,greencolorrepresentsCy3-labeled
normalcontrolandredcolorrepresentsCy5-labeledAFL.(C)Theidentifiedproteinsareindicatedbyarrowsonthe2Dgel.Ultimatelythere
wereeightproteinsselectedaccordingtostatisticalsignificancewitha
t
-testvalueof<0.05(
p
<0.05)asanalyzedbytheDeCydersoftware.
VitaminD-bindingprotein(VDBP),haptoglobin(HP),C-reactiveprotein(CRP),alpha-fetoprotein(AFP),inter-alpha-inhibitorH4heavychain(ITIH4),
serineproteaseinhibitorKazal-type5(SPINK5)andheakshockprotein75kDa(HSP75)andafamin.

loadingstandard(datanotshown).Figure4Aand4BtheexpressionofHpmayvarybetweenthenon-
showthatCRPparticularlyincreasedinAFLratscom-necroinflammatorystage(NAFL)andnecroinflamma-
paredtoallothergroupsincludingnormalratsandratstorystage(NASH).
withNAFLdiseaseorliverfibrosis(all
p
<0.05),
demonstratingthatCRPisaputativebiomarkerofAFL.
Discussion
Meanwhile,HpdidnotsignificantlydecreaseintheTheaimofthisstudywastodeterminetheputativesero-
serumofAFLratsaccordingtotheWesternblottinglogicalbiomarkersofAFLusing2D-DIGE,andthen
analysis(Figure4A).Interestingly,weobservedthatCRPthesecandidatemarkerswerevalidatedbyWesternblot-
andHpwerebothdownregulatedintheserumofliverting.TocharacterizeAFLdiseasemarkers,ourexperi-
fibrosisratscomparedtonormal,AFL,andNAFLratsmentalstrategywasfirsttoinducepatholophysiological
(Figure4B,C,all
p
<0.05).Ontheotherhand,wealsoabnormalitiesinanimalsbyadministeringalcohol,high
examinedthelevelofalpha1antitrypsin(AAT)tocaloriesofcompoundssuchas:fructoseorfats,and
excludetheelevationofCRPderivedfromethanol-drinkingwaterwithTAA,afibrosis-inducingchemical.
inducedgastrointestinalinflammation.TheresultsUnderthisexperimentalplatform,ratsshouldhave
showedthatAATlevelswerenotincreasedindevelopedAFL,NAFLandliverfibrosis,allowingusto
theserumofAFLratscomparedtohealthycontrolsdeterminethesignaturebiomarkersforAFL,whichisthe
(Figure4D).earlystageinthealcohol-inducedliverdisease.Fromour
Moreover,inordertoevaluatethedecreasedexpres-rodentmodels,wedeterminedthatCRPlevelsweresig-
sionsofCRPandHpintheserumofliverfibrosisrats,nificantlyelevatedintheserumofratswithAFL,pre-
wemeasuredserumCRPandHpconcentrationsinclin-sumablyasareliablebiomarkercomparedtothatin
icalpatientswithnon-alcoholicsteatohepatitis(NASH)liversofhealthyorsickratswithotherliverdiseases.
andHCV-inducedliverfibrosiscomparedtohealthyIndeed,CRPincreasesinotherconditions,suchas
controls.Table3showsthatCRPandHpwerelowerininflammation,obesity[17,18],andcardiovasculardisease
patientswithHCV-inducedliverfibrosis,whichwas[19,20];however,itisalsoaelevatedAFL-inducedpro-
consistentwiththeresultsdemonstratedbyWesternteininratsasdiscoveredinthisstudy.Here,ourdiscov-
blotting,suggestingthatCRPandHparereliablebio-eryprovidesahintthatCRPcanbeusedtodistinguish
markersofliverfibrosisasdownregulatedproteins.AFLfromnormalorotherwisediseasedlivers.Apre-
Interestingly,wefoundthatserumHpwaselevatedinviousreportindicatedthatCRPisanon-invasivemarker
NAFLrats,butlowerinNASHpatients,implyingthatofalcoholichepatitisinheavydrinkerscomparedto

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Figure3
Stereopicturesanddetailedimagesofeightputativebiomarkers
.VDBP,vitaminDbindingprotein;HP,haptoglobin;CRP,C-
reactiveprotein;AFP,alpha-fetoprotein;ITIH4,Inter-alpha-inhibitorH4heavychain;SPINK5,SerineproteaseinhibitorKazal-type5;HSP75,Heak
shockprotein75kDa.Theproteinspotsareindicatedbyarrows.Threeproteins,CRP,AFPandafamin,wereupregulatedwhereastheother
proteins,includingHp,ITIH4,APINK5,HSP75andVDBP,weredownregulated.

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Table2ProteinspotsidentifiedbyMALDI-TOF/TOFMS
GenenameProteinnameMr(Da)/pICoverageratioRegulation

p
value
CrpC-reactiveprotein25452/4.897%
§
+4.710.02
AfmAfamin49311/6.1459%+1.640.04
AfpAlpha-fetoprotein47195/5.473%
§
+1.460.03
HpHaptoglobin39052/6.1048%-11.540.04
GcVitaminD-bindingprotein53493/5.654%
§
-1.740.04
Itih4Inter-alpha-inhibitorH4heavychain103885/6.0848%-1.660.03
Spink5SerineproteaseinhibitorKazal-type5114816/8.6874%-1.670.04
Trap1Heakshockprotein75kDa,mitochondrial80639/6.5657%-1.960.02
§
ThoseproteinswereidentifiedbyMS/MS.

Presentedasup-(+)ordownregulation(-)comparedtonormalcontrols.
hepatitisunrelatedtoalcohol[7].Inthisstudy,wedis-examinedproteinlevelsofalpha1antitrypsin,anacute-
coveredthatCRPlevelswereelevatedinAFLratscom-phasemarker[24,25],determinedbyWesternblotting,
paredtohealthyanimals,andratswithotherformsofwhichprovidedapositivereferenceofaninflammatory
liverdiseases,suchasNAFLandTAA-inducedliverresponse.Theresultsshowedthatlevelsofalpha1anti-
fibrosis.Althoughtheuseofmoderatealcoholcon-trypsininratsamongthefourgroupswerethesame
sumptioncanlowerthelevelofCRPintheserumand(Figure4A,D),indicatingthattheelevationofCRPwas
decreasecardiovascularmortality[21],inourstudythenotduetogastrointestinalinflammation.Moreover,in
intakeofhighamountsofalcoholinthisrodentmodelvitrostudyrevealedthatethanolcandirectlytriggerthe
increasedthelevelofserumCRP,suggestingthatthesecretionofCRPinHepG2cells(datanotshown).
intakeofethanolispositivelyassociatedwiththelevelHerein,theresultssuggestthatethanol-inducedforma-
ofserumCRP.Toourknowledge,CRPisconsideredantionoffattyliverwasstronglyrelatedtotheinduction
inflammatoryproteinproducedfrommacrophagesinofserumCRPinratssuppliedwithexcessethanol.
theliverandadipocytes[22,23].Inordertoexcludeele-AnincreaseinCRPwasalsoreportedtobeassociated
vatedCRPderivedfromaresponsetogastrointestinalwithobesity[17,18].Toaddressourconcernforthe
inflammationbecauseofethanolconsumption,weobesityissue,wealsomeasuredanimalweightinall

Figure4
Evaluationoftheexpressionsofalcoholicfattyliver(AFL)biomarkersusingwesternblotting
.(A)ConfirmationofC-reactive
protein(CRP)andhaptoglobin(Hp)expressionbyWesternblotting.AATwasusedasanindicatorrepresentedasapositiveinflammatory
proteinforgastrointestinalinflammation(B)Thesemi-quantificationoftheresultsofWesternblotting.CRPincreasedintheserumofAFLrats
comparedtonormal,NAFLandliverfibrosisones,butdecreasedinTAA-inducedliverfibrosis.(C)HpincreasedinratssufferingfromNAFL,but
decreasedinliverfibrosiscomparedtothecontrols.(D)AATwasnotaffectedamongtherats.Threeindividualsamplesofhealthycontrols,
NAFL,andliverfibrosis,andfivesamplesofAFLwereexaminedbyusingWesternblotting.N,normalcontrols;AFL,alcoholicfattyliver;NAFL,
#non-alcoholicfattyliver;CRP,C-reactiveprotein;Hp,haptoglobin;AAT,alph1antitrypsin.*
p
<0.05,comparedtohealthycontrols;p<0.05as
comparedtotheothergroups.

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:52
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Table3SerumconcentrationofCreactiveprotein(CRP)andhaptoglobin(Hp)inclinicalpatients
Patients(
n
)FibrosisP-steatosisHAICRPHp
(
n
,%)(
n
,%)(
n
,%)(mg/L)(g/L)
Healthy(16)NDNDND1.58±0.481.13±0.63
NASH(19)(19F,11~0F02%)S(t7a,g3e70%~)1S(t1a8,ge950%~)4ND0.74±0.39*
Stage2~3Stage5~13
(12,63%)(1,5%)
,#
liverfiHbrCoVs-is(17)(7F,14~1F2%)S(t1a5g,e880%~)1S(t2a,ge120%~)41.31±0.44*0.45±0.58*
(1F03,~5F94%)St(a2,ge122%~)3S(ta1g5,e858~%1)3
ThefibroticstagewasdeterminedaccordingtotheMetavirclassification.Thestageofp-steatosiswasdeterminedaccordingtothefattychange.HAI,
sniegcnriofiicnaflnacmemwaatsorcyalscculoartees.dNaAccSoHr,dinnogn-taolcSothuodliecntst

sea
t
t-toehset.pTathiteis;daNtaD,arneotprdeesteerntmeidneads.*th
p
e<me0.a0n5,±cSoEmMp.aredtohealthycontrols.
#
p
<0.05,comparedtoNASH.The

groups.Weobservedthatmacrovesicularfatwasappar-
entinthelivertissuesofratsfeddietscontainingahigh
fructosecontent(HF-NAFL)whichdidnotincrease
serumCRP.InAFLrats,macrovesicularfatwasalso
observed,butserumCRPwaselevated.Therefore,this
suggeststhatalcoholabusemaycausefattyliverand
inducehighserumCRPlevels,indicatingthatCRPmay
bequalifiedasauniquebiomarkerofAFL.
Administrationofethanolcanelicitoxidativestress
andinjurytotheliver[4,26],andthepresenceofpoly-
unsaturatedfatscaninducetheproductionofcyto-
chromeP4502E1(CYP2E1)[27,28].Underconditionsof
persistentethanolstimulation,CYP2E1seemstoplaya
criticalroleinmetabolizingandactivatingmanytoxico-
logicalsubstancessuchasreactiveoxygenspecies(ROS)
[26,29].Arecentstudyindicatedthatcytokinessuchas
tumornecrosisfactor-alphaandinterleukin-10inadi-
posetissuesofacutealcoholichepatitispatientswere
elevated,andwerecorrelatedwiththeserumCRPcon-
centration[30],implyingthatinflammationcausedthe
productionofCRP.Althoughthemechanismofhow
ethanolinducesCRPinserumisunclear,inthisstudy,
wediscoveredthatethanolconsumptionisanimportant
factorpositivelyassociatedwiththeproductionof
serumCRP.
InadditiontotheincreasedCRPlevelsinAFLrats,
serumHpwasalsodiscoveredtobeabiomarkerofAFL
withreducedexpressionlevelsintheserumofAFLrats
using2D-DIGE.However,Hpwaselevatedintheserum
ofNAFLratsandevendecreasedinthatofratswith
TAA-inducedliverfibrosis(Figure4C).Previousstudies
reportedbyChiellini

sgroupindicatedthatelevated
serumHpisrecognizedasamarkerofadiposity[31].
OurresultsdemonstratethatHpisnotonlyincreased
inNAFL,butalsodecreasedinTAA-inducedandHCV-
inducedliverfibrosis.Furthermore,Shuetal.indicated
thatHpwasoverexpressedinhepatocellularcarcinoma
comparedtothosewithhepatitisBvirus(HBV)-related
cirrhosis[32].Howeveranotherresearchgroupledby

Dr.LeereportedthatHplevelsweredecreasedindepen-
dentlyinhepaticfibrosisinchronicliverdisease[33].
Therefore,thelevelofserumHpmayvaryundervar-
iouspathophysiologicalsituationsorstagesinclinical
liverdiseases.HpisalsousedinthepanelofAshTest
[34]asadown-regulatedprotein.Inthisstudy,we
foundthatHpwasadownregulatedproteininNASH
andHCV-infectedliverfibrosisalthoughwefoundthat
HpmaybehigherintheserumofNAFLrats.The
resultsdemonstratedthatHpisareliabledownregulated
biomarkerofNASHandliverfibrosisinclinicalcases.
Conclusions
Foradiagnosisofalcoholicliverdisease,abiopsyisthe
goldstandard.Currentstudiesfocusingonthediscovery
ofanoninvasivebiomarkerpanelfordiagnosisorprog-
nosisimplythatdevelopmentofanoninvasivemethod
isurgent.CRPisconsideredtobeamarkerofathero-
scleroticcardiovasculardiseaseinclinicalanalysis
[19,20],modulatingendothelialfunctionintheprocess
ofatherogenesis[35];therefore,wesuggestthatusing
CRPtodistinguishAFLfromtheotherliverdiseases
mayconsiderthecomplicationofcardiovasculardisease.
Inconclusion,thisisthefirstreporttorevealnewcan-
didatebiomarkersofAFLusingaproteomicsanalysis.
Inthisstudy,eightAFL-associatedserologicalproteins
weredisclosed,whichmaybeassociatedwithAFLin
rats.WesuggestthatCRPissuitabletoserveasacan-
didatebiomarkerofAFL,andHpisareliablebiomarker
thatdecreaseinNASHandliverfibrosis.Inparticular,
serumCRPmaybequalifiedtobeanelevatedsurveil-
lancetargetforearlydiagnosisofAFLinclinical
screening.
Acknowledgements
WewouldliketothankDr.Jyh-ChinYangandDr.Chiang-TingChienwho
kindlysupportedandprovidedtheanimalsfromNationalTaiwanUniversity,
Taipei,Taiwan.WealsothankDavidCookefromOxfordshire,UKforhelping
ustorevisethemanuscript.

Liu
etal
.
JournalofBiomedicalScience
2011,
18
:52
http://www.jbiomedsci.com/content/18/1/52

Authordetails
1
InstituteofBiochemistryandBiotechnology,ChungShanMedical
University,Taichung,Taiwan.
2
GraduateInstituteofMedicalSciences,College
ofMedicine,TaipeiMedicalUniversity,Taipei,Taiwan.
3
InstituteofNuclear
EnergyResearch,AtomicEnergyCouncil,Taoyuan,Taiwan.
4
Departmentof
InternalMedicine,SchoolofMedicine,CollegeofMedicine,TaipeiMedical
UniversityHospital,Taipei,Taiwan.
5
DepartmentofBiochemistry,Schoolof
Medicine,TaipeiMedicalUniversity,Taipei,Taiwan.
6
BiomedicalMass
ImagingResearchCenter,TaipeiMedicalUniversity,Taipei,Taiwan.
7
Division
ofGastroenterology,BuddhistTzuChiGeneralHospital,Taipeibranch,
Taiwan.
8
DivisionofGastroenterology,ChengHsinGeneralHospital,Taipei,
Taiwan.
9
ResearchCenterForBiomedicalImplantsandMicrosurgeryDevices,
TaipeiMedicalUniversity,Taipei,Taiwan.
10
NeuroscienceResearchCenter,
TaipeiMedicalUniversityHospital,Taipei,Taiwan.
Authors

contributions
SLL:revisedthearticleanddesignedtheexperiments.CCC:participatedin
articlewritingandperformedmostexperiments,including2D-DIGE,MALDI-
TOF/TOFMS,andWesternblotting.FDMandJS:Projectleadersand
correspondingauthors,participatedinthisprojectinrevisingthearticleand
providingopinionsandinterpretationofthedata.SCL:providedsuggestions
andanalysisregardingexperimentaloutcomesaswellasrevisedthearticle.
CCWandCCC:performedtissuesamplinganddiagnosis.ASH:participated
inthecollectionanddiagnosisofclinicalsamples.LYC:Lableaderand
articlefinalrevision,contributedtointerpretationofthedata.Allauthors
readandapprovedthefinalmanuscript.
Competinginterests
Theauthorsdeclarethattheyhavenocompetinginterests.
Received:25March2011Accepted:1August2011
Published:1August2011
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doi:10.1186/1423-0127-18-52
Citethisarticleas:
Liu
etal
.:
Discoveryofserumbiomarkersofalcoholic
fattyliverinarodentmodel:C-reactiveprotein.
JournalofBiomedical
Science
2011
18
:52.

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