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

Liu Shu-Lin, Cheng Chun-Chia, Chang Chun-Chao, Mai Fu-Der, Wang Chia-Chi, Lee Shui-Cheng, Ho Ai-Sheng, Chen Ling-Yun, Chang, Chang Jungshan

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.

Sujets

• Fatty liver
• Biomarker
• Haptoglobin

Informations

• Publié par: biomed
• Publié le: 01 janvier 2011
• Nombre de lectures: 8
• Langue: English
• Poids de l'ouvrage: 7 Mo

Extrait

Liu
etal
.
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:[e-mail];[e-mail]
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.

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

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

Page2of10

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

Liu
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JournalofBiomedicalScience
2011,
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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±0.06