Bile Acid Pro fi les Are Distinct among Patients with Di ff erent Etiologies of Chronic Liver Disease - Sang et al. - 2021 - Unknown

《Bile Acid Pro fi les Are Distinct among Patients with Di ff erent Etiologies of Chronic Liver Disease - Sang et al. - 2021 - Unknown》由会员上传分享，免费在线阅读，更多相关内容在学术论文-天天文库。

pubs.acs.org/jprArticleBileAcidProfilesAreDistinctamongPatientswithDifferentEtiologiesofChronicLiverDiseaseChaoSang,XiaoningWang,KejunZhou,TaoSun,HuaBian,XinGao,YixingWang,HuaZhang,WeiJia,PingLiu,*GuoxiangXie,*andTianluChen*CiteThis:J.ProteomeRes.2021,20,2340−2351ReadOnlineACCESSMetrics&MoreArticleRecommendations*sıSupportingInformationABSTRACT:Asignificantincreaseofbileacid(BA)levelshasbeenrecognizedasageneralmetabolicphenotypeofdiverseliverdiseases.MonitoringofBAprofileshasbeenproposedforetiologydifferentiationonliverinjury.Here,wequantitativelyprofiledserumBAsofhealthycontrolsand719patientswithchronicliverdiseaseoffiveetiologies,hepatitisBvirus(HBV),hepatitisCvirus(HCV),nonalcoholicsteatohepatitis(NASH),alcohol-inducedliverdisease(ALD),andprimarybiliarycirrhosis(PBC),andinvestigatedthegeneralityandspecificityofdifferentetiologies.TherawdatahavebeendepositedintoMetaboLights(ID:MTBLS2459).WefoundthatpatientswithHBV,HCV,andNASHappearedtobemoresimilar,andALDandPBCpatientsclusteredtogether.BAprofiles,consistingofatotalconcentrationofthe21quantifiedBAs[totalBAs(TBAs)],21BAproportions,and24BArelevantvariables,werehighlydifferentamongtheetiologies.Specifically,thetotalBAswashigherinALDandPBCpatientscomparedwiththeotherthreegroups.TheproportionofconjugateddeoxycholateswasthehighestinHBV-infectedpatients.Theratioof12α-hydroxylated(12α-OH)tonon-12α-OHBAswasthehighestinNASHpatients.Theproportionoftaurine-conjugatedBAswasthehighestinALDpatients.ForPBCpatients,theproportionofursodeoxycholatespecieswasthehighest,andtheratioofprimarytosecondaryBAswasthelowest.Comparatively,thedifferenceofBAprofilesamongcirrhosispatientswasconsistentbutweakerthanthatofallpatients.ThecorrelationsbetweenBAprofilesandclinicalindiceswerealsoquitedifferentindifferentpathologicalgroups,bothinallpatientsandinpatientswithcirrhosis.Overall,ourfindingssuggestedthatBAcompositionsaredistinctamongpatientswithdifferentetiologiesofchronicliverdisease,andsomeBA-relevantvariablesareofclinicalpotentialsforliverinjurytypedifferentiation,althoughfurthervalidationsonmoreetiologiesandpopulationsareneeded.KEYWORDS:Bileacids,alcohol-inducedliverdisease,NASH,HBV,HCV,primarybiliarycirrhosisDownloadedviaUNIVOFNEWMEXICOonMay16,2021at10:08:29(UTC).1.INTRODUCTIONcouldprovidecrucialinsightsfortheprediction,diagnosis,6−9Seehttps://pubs.acs.org/sharingguidelinesforoptionsonhowtolegitimatelysharepublishedarticles.treatment,andprognosisofchronicliverdisease.BileacidsChronicliverdiseaseiscausedbycontinuousandrepeated(BAs),afamilyofmetabolitesproducedintheliver,areinsultstotheliverthatcanleadtofibrosisandcirrhosis.Everymetabolizedbyenzymesderivedfromintestinalbacteriaandyear,chronicliverdiseaseanditscomplicationsleadto1,2arecriticallyimportantformaintaininghealthygutmicrobiotaapproximately2milliondeathsglobally.Hepatocellularandabalancedlipidandcarbohydratemetabolism,insulincarcinoma,theworstconsequenceofchronicliverdisease,issensitivity,andinnateimmunity.Mountingevidenceobtainedthefifthmostcommoncancerinmen(∼554,000cases)andfromstudiesinhumansandanimalmodelshasrevealedthetheninthmostcommoncancerinwomen(∼228,000cases)3closeassociationofBAsandchronicliverdisease,regardlessofgloballyandisthesecondleadingcauseofcancer-related10−124etiology.Monitoringoftotal,individual,andproportiondeathsinlessdevelopedcountries.AmongthevarietyofofBAshasbeenproposedfordifferentiationofvariousetiologies,hepatitisBandCvirusinfections(HBV,HCV),nonalcoholicsteatohepatitis(NASH),alcohol-inducedliverdisease(ALD),andimmunesystemabnormalities(suchasReceived:October28,2020primarybiliarycirrhosis,PBC),areleadingcausesofliverPublished:March23,2021injury.Itiswidelyacceptedthatthepathogenesisofchronicliverdiseaseinvolvesextensiveandcomplexgene−environment5interactions,andmetabolomicsandmetagenomicsstudies©2021AmericanChemicalSocietyhttps://doi.org/10.1021/acs.jproteome.0c008522340J.ProteomeRes.2021,20,2340−2351

1JournalofProteomeResearchpubs.acs.org/jprArticle±±±±0.25=57)±±±±±±±n±<0.05,7.902.985.87388.07398.88106.0182.3537.94218.652.4123.57ep61.3022.5531.83103.88119.28156.5998.7539.81136.0815.1180.28is;BMI,body±0.621.21=37)S4(±±±±±±±±±±n±8.003.775.2415.9337.9764.5753.2799.1698.755.5022.4056.7824.3229.0226.9752.27133.1184.8480.6792.5319.241.6457.38=8)S4(±±0.460.78±±±±±±n±±13.674.239.59122.9042.7224.4261.6777.70vestagesofHBVpatients.57.2529.9029.75132.0074.5096.38157.38108.38fi=25)S4(±±0.793.990.851.94±±±±±±n±±12.023.844.1065.1928.8727.2055.3428.61kalinephosphatase;PLT,platelet;TBIL,total51.7227.5927.8098.2059.1675.20221.4459.28=67)S3(±±1.254.482.511.62±±±±±±n±±<0.05,comparisonbetweenthecontrolgroupanddisease14.754.6612.8349.9432.2427.0147.10163.70p44.3429.4726.9183.4949.0774.72223.9397.90b=92)S2(±±1.324.983.692.60±±±±±±±±±±n±±15.174.884.702.4758.6933.2522.3250.255.2668.820.6919.2343.1126.594.802.7826.2142.8972.6742.9373.13225.2316.7865.7212.12124.78=22)S1(±e±±±±±e±±e±±e±±<0.05,multiplegroupscomparisonamongne±e±edp12.934.271.152.763.782.3947.6518.0426.1558.382.8084.150.5815.4882.09=1,3,10.1340.913.6227.604.9526.273.2045.2037.9141.5925.4973.0542.05215.954.8218.2868.4993.860.7312.1815.38122.80n1.100.335.182.79±±±±±±±±±±±±4(3,3,1)S0(±±−132.09S040.6423.4133.6440.73113.0953.7374.64171.2717.4356.6412.82111.27veetiologicalgroups.fi0.814.351.271.05±±=32)±±±±±±±±±±n±±9.103.426.024.64118.4267.1234.4850.7911.6987.591.1916.61a41.6923.7235.1640.26116.0167.9391.81127.8622.3186.9113.5494.550.944.660.641.40±±±=51)S4(±±±±±±±±±n±±11.482.894.253.60175.61118.1725.0445.8446.0067.971.3120.5738.8023.0534.7039.46180.44103.9287.34140.8231.9789.9413.5698.65=n0.874.900.461.27±±±±±±±±±±±±±±125)S3(S2(9.583.174.163.34230.68125.0934.5546.3615.4864.771.0415.9033.0222.2333.4241.44224.00118.3381.18179.1223.3666.2013.11103.95=<0.05,multiplegroupscomparisonamongn±±0.924.630.841.06±±±±±±±±±±cp±±154)S1(8.802.944.253.33214.6185.8125.3348.1611.1747.330.9516.8031.9721.5033.4442.51186.5987.6372.79183.1021.1647.3312.86107.62SD.HBV,hepatitisBvirus;HCV,hepatitisCvirus;NASH,nonalcoholicsteatohepatitis;ALD,alcoholicliverdisease;PBC,primarybiliarycirrhos±vestagesofNASHpatients.fi=38)0.964.680.921.17±ddd±dd±dn±d±d±±±d±d±±±d±±d9.312.243.593.18181.4376.2821.2439.258.5140.020.6513.4233.3221.764.581.2232.3943.13176.3975.6668.37183.3419.1242.1312.87109.00=n±±±±±±b±±bb±±bb±b±bbbbb153)S0(11.423.040.931.022.802.6015.326.3518.6272.494.9812.13healthy(108:4538.9233:522.805.22116:381.7090:3529.0648.7043:831.5122.2722:1088.058:3258.6315.1814:818.6549:4333:348:175:337:07:50cccccccc/L)cc)9stage2cccmol/μccpathologycontrolsex(M/F)Age(yrs)BMI(kg/mTC(mmol/L)TG(mmol/L)GLB(g/L)ALB(g/L)HBVALT(IU/L)AST(IU/L)ALP(U/L)PLT(10TBIL(L)GGT(IU/L)HCVPT(s)INRPTA(%)Note:ValuesareexpressedasmeanNASHALDTable1.DemographicandClinicalCharacteristicsofAllParticipantsamassindex;TC,totalcholesterol;TG,triglyceride;GLB,globulin;ALB,albumin;ALT,alaninetransaminase;AST,aspartatetransaminase;ALP,albilirubin;GGT,gamma-glutamyltransferase;PT,prothrombintime;INR,internationalnormalizedratio;PTA,prothrombintimeactivity.group(HBV+HCV+NASH+ALD+PBC).multiplegroupscomparisonamong2341https://doi.org/10.1021/acs.jproteome.0c00852J.ProteomeRes.2021,20,2340−2351

2JournalofProteomeResearchpubs.acs.org/jprArticleetiologiesofliverinjury,withasmallsamplesizeandasmallimprovecurrentunderstandingsofBAmetabolisminchronicnumberofBAs.LuoandcolleaguesquantifiednineserumBAsliverdiseaseswithdifferentetiologies.ofpatientswithvarioustypesofliverinjuryanddemonstratedthedistinctBAprofilesamongliverinjurypatientsofliver2.METHODOLOGYtransplants,hepaticcarcinoma,coronaryarterydisease,2.1.StudyPopulationscoronaryheartdisease,cirrhosis,liverinjury(hepatitisBorC,ethanolcirrhosis,drugabuse,ortransaminitis/hepaticAtotalof153healthycontrolsand719patientswithdifferentcongestion),pulmonarydiseases,andacetaminophentox-etiologiesfrom5independentcohortswereenrolledinthisicity.13Sugitaetal.reportedthatBAcompositionandseveralstudy(Table1).HealthycontrolswererecruitedfromAprilindividualBAshadpotentialsfordifferentialdiagnosisofliver2013toJune2015atShuguangHospitalaffiliatedtothediseasesbyanalyzing16serumBAsin150chronicliverdiseaseShanghaiUniversityofTraditionalChineseMedicine(Shang-patientswithHBVandHCVinfection,nonalcoholicfattyliverhai,China).Cohort1enrolledatotalof400patientswithdisease(NAFLD),alcoholconsumption,orbiliarytractHBVinfectionfromShuguangHospitalaffiliatedtothedisease.14Bathenaetal.exploredthealterationsof10BAs,5ShanghaiUniversityofTraditionalChineseMedicine(Shang-sulfatedBAs,andsomerelevantvariablesintheurinesampleshai,China)fromApril2013toJune2015.Cohort2wasof121chronicliverdiseasepatientswithvaryingseverityandrecruitedfromApril2013toJune2015atShuguangHospitaletiologies.Theyreportedthatthecomposition(i.e.,affiliatedtotheShanghaiUniversityofTraditionalChineseproportion),sulfation,andamidationofindividualBAswereMedicine(Shanghai,China),consistingof11patientswithHCVinfection.Cohort3withatotalof214NASHpatientsbetterassociatedwithliverdisease,comparedwiththeirabsoluteconcentrations.15wererecruitedfromFudanUniversity-affiliatedZhongshanhospital.Cohort4enrolledatotalof37patientswithALDInthepastdecade,ourgroupconductedaseriesofstudiesrecruitedfromShuguangHospitalaffiliatedtotheShanghaionBAsandliverdiseasesinbothhumansandanimals.WeUniversityofTraditionalChineseMedicine(Shanghai,China).examinedtheBAprofilesofthegastrointestinaltractcontent,Cohort5,including57patientswithPBC,wasrecruitedfromliver,andseruminnormalratsanddeterminedhowtheseShuguangHospitalaffiliatedtotheShanghaiUniversityofprofileswereimpactedbyethanolconsumption,whichTraditionalChineseMedicine(Shanghai,China).providedanimprovedunderstandingofthesystemicAlltheparticipantswereincludedaftersignedinformedmodulationsofBAmetabolisminmammalsthroughthe16consent.Thestudywasapprovedbytheinstitutionalreviewgut−liveraxis.ThedysregulatedBAsarecloselyassociated17boardsofcorrespondinghospitals(approvalno.ofhealthywithliverdiseasesandattributedtoalteredgutmicrobiota.controls,cohorts1,2,4,and5fromShuguanghospital:2012-WethenstudiedthedistinctlyalteredgutmicrobiotaandBAs206-22-01;approvalno.ofcohort3fromZhongshanhospital:atvariousliverpathologicalstagesusingastreptozotocinhigh-B2013-132).fatdiet-inducedNASH-hepatocellularcarcinomaC57BL/6Jmousemodel,whichishighlyrelevanttohumanliverdisease2.2.LiverBiopsyforDiagnosisandStagingprogressionfromsteatosistoNASH,fibrosis,andfinally,Allthepatientsreceivedaliverbiopsydirectedbyultra-18sonographywithin1weekafterenrollment.Thebiopsyhepatocellularcarcinoma.WeobservedthatBAprofilesaredifferentinhealthyChinesesubjectswithdifferentage,gender,specimenswerefixedwith10%formalin,embeddedinparaffin,19andstainedwithhematoxylinandeosin,picrosiriusred,andandbodymassindex(BMI).WeconstructedamachinelearningmodeltopredictadvancedhepaticfibrosisusingPerlsstaining.Examinationofaminimumlengthof1.5cmofmetabolitemarkersincludingBAswhichoutperformedseveraltheliverbiopsyandatleastsixportaltractswasrequiredforexistingscoresintheChinesepopulation.20,21Wealsodiagnosis.Histologicalstagingofliverfibrosis(S0−S4)was23investigatedtheserumandurineBAsfrom15healthycarriedoutaccordingtoScheuer’sclassificationasfollows:volunteersand7patientswithbiopsy-confirmedNASHandS0=none;S1=1aor1bperisinusoidalzone3or1cportalfoundthatpatientswithNASHhavehigherfastingandfibrosis;S2=perisinusoidalandperiportalfibrosiswithoutpostprandialexposuretoBAs,includingthemorehydrophobicbridging;S3=bridgingfibrosisandS4=cirrhosis.Allsamplesandcytotoxicsecondaryspecies.22wereindependentlyassessedbythreepathologistsfromtheOverall,existingreportshavedemonstratedthelinkageofcorrespondinghospitalsforsamplecollection,whowereBAprofilesandtheetiologiesandstagesofchronicliverblindedtothesampleID.Specimenswithdiscrepantdisease,althoughthespecificmechanismsandtheirdifferencesassessmentswerere-examineduntilaconsensuswasreached.Thefinalassessmentsofthethreepathologistswerefurtherunderlyingeachoftheseclinicalscenariosremainunclear.InprocessedusingtheKappaconcordancetest.thisstudy,wequantifiedserumBAsin153healthycontrolsand719patientswithdifferenttypesofchronicliverdisease2.3.SerumSampleCollectionandClinicalIndicesandinvestigatedtherelationshipbetweentheetiologiesofOvernightfastingbloodsampleswerecollectedfromallchronicliverdiseaseandBAprofiles.Differenceanalysiswassubjects.ThebloodandtheseparatedserumweredeliveredonusedtocompareBAprofilesinallpatientsandcirrhosisicetolaboratorieswithin2hofcollection.Allsampleswerepatients(S4)withdifferentetiologiesandtofindsomealiquotedandstoredat−80°Cuntilanalysis.Someoftheetiology-specificBA-relevantvariables.Randomforest(RF)clinicalindices,suchastotalcholesterol(TC),triglyceridemodelswereestablishedtodifferentiatedifferentetiologies.(TG),globulin(GLB),albumin(ALB),alaninetransaminasePartialSpearmancorrelationanalysiswascarriedouton(ALT),aspartatetransaminase(AST),alkalinephosphataseclinicalindicesandBA-relevantvariables.Toourknowledge,(ALP),platelet(PLT),totalbilirubin(TBIL),andgamma-thisisbyfarastudywiththelargestsamplesizeandmostBA-glutamyltransferase(GGT),wereobtainedbyanLH750relevantvariables.OurfindingsmayofferpotentialbiomarkershematologyanalyzerandaSynchronDXC800clinicalsystemforchronicliverdiseasediagnosisandtreatmentandmay(BeckmanCoulter,USA)accordingtothemanufacturers’2342https://doi.org/10.1021/acs.jproteome.0c00852J.ProteomeRes.2021,20,2340−2351

3JournalofProteomeResearchpubs.acs.org/jprArticleprotocols.Theprothrombintime(PT),internationalnormal-2.6.DefinitionsofBA-RelevantVariablesizedratio(INR),andprothrombintimeactivity(PTA)wereAtotalof21BAswerequantifiedintheserumsamples.Takingmeasuredwithanautomaticcoagulationanalyzer(STAGOintoaccountthepossibledetectionbiasandindividualCompact,DiagnosticaStago,France).BMIwascalculatedbydifferencesandpreviousreports,proportionandratiovariablesdividingaperson’sweightinkilogramsbythesquareofthebutnotabsoluteconcentrationswereinvolvedfortheheightinmeters.subsequentanalysis.Atotalof46BA-relevantvariableswere2.4.Reagentsgenerated,includingtotalBAs(TBAs),21BAproportions(proportionofindividualBAsinTBAs),and24combinedAlloftheBAstandardswereobtainedfromSteraloidsInc.variables(e.g.,ratios,proportionsofBAspecies,and(Newport,RI)andTRCChemicals(Toronto,ON,Canada),proportionsofBAsubtypes).Detaileddefinitionsandandninestableisotope-labeledstandardswereobtainedfromtaxonomicrelationsarelistedinTableS1.C/D/NIsotopesInc.(Quebec,Canada)andSteraloidsInc.(Newport,RI).Methanol[Optimaliquidchromatography−2.7.StatisticalAnalysismassspectrometry(LC−MS)],acetonitrile(OptimaLC−Student’st(fornormalvariables),Mann−Whitney(forotherMS),andformicacid(OptimaLC−MS)werepurchasedfromcontinuousvariables),andChi-square(forcountvariables)Thermo-FisherScientific(FairLawn,NJ).UltrapurewaterwastestswereusedforcomparisonsbetweenhealthycontrolsandproducedbyaMilli-QReferencesystemequippedwithaLC−patientswithdiversechronicliverdiseases(twogroups),MSPakfilter(Millipore,Billerica,MA).respectively.One-wayanalysisofvariance(ANOVA)wasusedformultiplegroupcomparisonorvariousstagecomparisonfor2.5.QuantitationofBAsvariablesconformingtonormalityandhomogeneityofThequantitationofBAswasperformedaccordingtoourvariance.TheKruskalWallistestwasemployedforother24previouslypublishedprotocols.Atotalof41BAstandardsvariablesamongmultiplegroups.TheDunntestwasusedforwereincludedtoidentifyandquantifythelevelsofBAs,andposthocanalysisbetweenanytwogroups.Correlationsofamongofthem,21BAscanbeidentifiedandquantifiedinrealclinicalindicesandBA-relevantvariableswereassessedusingsamples.Inbrief,analiquotof50μLofserumwasmixedwithpartialSpearmancorrelationanalysis.Allthepvalueswere150μLofmethanol(containing0.10μMoflithocholate-adjustedbythefalsediscoveryrate(FDR)usingthe2,2,4,4-D4(LCA-D4),ursodeoxycholate-2,2,4,4-D4(UDCA-Benjamini−Hochbergmethod.Thecorrectedp-valueof0.05D4),andcholate-2,2,4,4-D4(CA-D4)fromC/D/NIsotopeswastakenasasignificancelevel.R(version3.6.2)andRStudioInc.asinternalstandards).Themixturewasvortexedfor2(version1.2.5033)wereusedforstatisticalanalysis.Dataaremin,allowedtostandfor10min,andthencentrifugedatexpressedasmean±standarddeviation(SD)orasratios.20,000gat4°Cfor10min.Analiquotof160μLofthe2.8.ModelConstructionandAssessmentsupernatantwastransferredtoacleantubeandvacuum-dried.RFmodelswereconstructedusingallorrepresentativeBATheresiduewasredissolvedwithanequalamountofvariablestodifferentiatedifferentetiologiesinallpatientsoracetonitrile(0.1%formicacid)andwater(0.1%formicacid)patientswithcirrhosis.Theleave-one-outmethodwasusedtotoafinalvolumeof40μL.Aftercentrifugation,thevalidatethemodelperformanceowingtothelackofsupernatantwasquantifiedbyultraperformanceliquidindependentvalidationdatasets.TheperformancesofthechromatographytriplequadrupolemassspectrometryRFmodelswereevaluatedbytheareaunderreceiveroperating(UPLC−TQMS).Eachaliquotof41standardstocksolutionscharacteristic(auROC)andPR(auPR)curvesofthewasmixedtoobtainamixedstocksolution.Calibrationpredictivescores.TheRFmodelwasbuiltbytherandom-solutionscontainingall41BAstandardswerepreparedataForest(v4.6-14)packagebasedonR(v4.0.2).Theseriesofconcentrationsof0.610,1.221,2.441,4.883,9.766,calculationofauROCandauPRwasperformedbythe19.531,39.063,78.125,156.250,312.5,625.0,1250.000,andpROC(v1.16.2)andPRROC(v1.3.1)packages.2500.00ng/mLinnaivepooledserumdepletedofBAsusing̈activatedcharcoal.Thecalibrationcurveandthecorrespond-3.RESULTSingregressioncoefficientswereobtainedbyinternalstandardadjustment.Pooledbiologicalsamples(QCsamples)and3.1.CharacteristicsoftheStudyPopulationinternalstandardswereusedforqualitycontrolofBAanalysis.Atotalof153healthycontrolsand719patientsfrom5TheQCsampleswerepreparedalongwiththestudysamplesindependentcohortswereinvolvedinthisstudy.Cohort1andwerekeptat10°Cduringtheentireanalysis.TheQCincludedatotalof400fibrosispatientswithHBVinfection,sampleswereevenlyinsertedineachsetoftheanalysisrunningconsistingof38,154,125,51,and32inthestageofS0−S4,sequencetomonitorthestabilityofthelarge-scaleanalysis.respectively.Cohort2includedatotalof11fibrosispatientsThenoisebaselinewasestablishedfromreagentblankwithHCVinfection(1,3,3,3,and1stagedasS0−S4,measurement,andanymetabolitewithsignal-to-noiseratiorespectively).Cohort3includedatotalof214NASHfibrosis≤3.0wasrejectedfromstatisticalanalysis.Relativestandardpatientswith22,92,67,25,and8diagnosedasS0−S4,deviations(RSDs)ofeachmetaboliteintheQCsamplesrespectively.Alltheparticipantsincohort4(37ALDpatients)measuredwerecalculated.TheRSDfortheQCswaslessthanandcohort5(57PBCpatients)werestagedasS4,cirrhosis15%foreachbatchofsampleanalysis.TheentireUPLC−patients.ThedemographicandclinicalindicesaresummarizedTQMSsystemwascontrolledbyMassLynx4.1.Allchromato-inTable1.ThereweresignificantdifferencesinmanyclinicalgraphicseparationswereperformedwithanACQUITYBEHindicesbetweenthehealthycontrolgroupandfiveetiologicalC18column(1.7μm,100mm×2.1mminternaldimensions)groupsexceptforageandsex.Especially,thelevelsofALB,(Waters,Milford,MA).AlltherawdatafileswereprocessedPLT,andGGTweremuchhigherandthelevelsofALT,AST,andthenquantifiedbythewebserverTargetedMetabolomeandTBILweremuchlowerinhealthycontrolsthaninBatchQuantification(http://119.136.25.134:9011).patients.Infiveetiologicalgroups,moremenwereinvolvedin2343https://doi.org/10.1021/acs.jproteome.0c00852J.ProteomeRes.2021,20,2340−2351

4JournalofProteomeResearchpubs.acs.org/jprArticleFigure1.(a,b)HeatmapanalysisofBA-relevantvariablesshowsvariousBApatternsacrossdifferentgroups.(c−h)RepresentativeBA-relevantvariablesinfiveetiologicalgroups.The*labeledonvariablenamerepresentsFDRadjustedp<0.05comparingthefivegroupsandthe*labeledforanytwogroupsrepresentsp<0.05fromtheposthocDunntest.2344https://doi.org/10.1021/acs.jproteome.0c00852J.ProteomeRes.2021,20,2340−2351

5JournalofProteomeResearchpubs.acs.org/jprArticleFigure2.(a,b)HeatmapanalysisofBA-relevantvariablesshowsvariousBApatternsincirrhosis(S4)patientsacrossdifferentetiologicalgroups.(c−h)RepresentativeBA-relevantvariablesincirrhosis(S4)patientsoffouretiologicalgroups.The*labeledonvariablenamerepresentsFDRadjustedp<0.05comparingthefivegroupsandthe*labeledforanytwogroupsrepresentsp<0.05fromtheposthocDunntest.2345https://doi.org/10.1021/acs.jproteome.0c00852J.ProteomeRes.2021,20,2340−2351

6JournalofProteomeResearchpubs.acs.org/jprArticleFigure3.(a−d)ROCandPRcurvesofRFmodelsinS0−S4orS4patients.(e)StackedbarofauROCsandauPRsofRFmodelsinS0−S4orS4patients.HBV,HCV,andALDgroups(M/F=304:96,8:3,and37:0,comparabletothoseofNASHpatients(M/F=109:105).Therespectively),andmorewomenwereinvolvedinthePBCpatientsofALDandPBCgroupswereolderthanthoseofgroup(M/F=7:50).Thenumbersofmenandwomenwereothergroups.Whenallpatientsinstage4werecompared,2346https://doi.org/10.1021/acs.jproteome.0c00852J.ProteomeRes.2021,20,2340−2351

7JournalofProteomeResearchpubs.acs.org/jprArticleFigure4.(a)ThenumberofBA-relevantvariablescorrelatedwitheachclinicalindex.(b)Thenumberofcorrelatedpairsinallpatientsoffourpathologicalgroups.(c)HeatmapofthenumberofcorrelatedpairsinS0−4andS4patientsoffourpathologicalgroups.thosewithHBVandHCVinfectionwereyoungerthanthosehigherinonegroupthanintheothers.ForALDpatients,theirofotheretiologies.Inaddition,thelevelsofage,BMI,andTGserumlevelsofprimary/secondaryandT-conjugatedBAs%ofNASHpatientswereallhigherthanthoseofHBVandotherwerehigherthanthoseoftheothergroups(Figure1d,e).Forgroups.NASHpatientshadlowerlevelsofGLB,AST,andPBCpatients,theirlevelsoftotalUDCA%(i.e.,UDCA%ALTinallstagescomparedtothatofHBVpatients.ForALD+GUDCA%+TUDCA%)werehigherandlevelsofprimary/andPBCpatients,thelevelsofALP,TBIL,andPTwerehighersecondarywerelowerthanthoseoftheothergroups(Figurethanthoseoftheothergroups,andthelevelsofALB,PLT,1d,f).ForHBVpatients,theirlevelsofconjugatedDCAs%andPTAwerelowerthanthoseoftheothers.Theclinicalwerehigherthanthoseoftheothergroups(Figure1g).ForindiceswereextremelydifferentamongdifferentetiologiesandNASHpatients,theirlevelsofratio12α-OH/non-12α-OHstageswhichpromptedustoexamineintensivelytheirwerehigherthanthoseoftheothergroups(Figure1h).ThesimilaritiesanddifferencesinBAprofiles.BAprofilesweredifferentamongthefiveetiologicalgroups,andsomevariableswerelikelytobemetabolicmarkersspecific3.2.BAProfilesoftheStudyPopulationtoeachetiology.Thereweresignificantdifferencesbetweenthehealthycontrol3.3.BAProfilesinCirrhosisPatientsgroupandthefiveetiologicalgroups,bothinabsoluteconcentrationandincompositionvariables(Figure1a,TableFurthermore,comparisonsofBAprofilesincirrhosis(S4)S2).Theabsoluteconcentrationsofalmostall(37of42)thepatientswereconductedaspatientswithPBCandALDwereBAvariablesincreasedgreatlyinthefiveetiologicalgroupsallcirrhosispatients.PatientswithHCVinfectionwerenotcomparingwiththehealthycontrolgroup,butthechangesofinvolvedinthecomparisonsconsideringtheverylowsampleBAcompositionswerediverse(TableS2).Thelevelsofnumber(n=1).Generallyspeaking,boththesimilarityandCDCA%,DCA%,unconjugatedBAs%,andtotalDCA%specificityofeachgroupwereroughlyconsistentwiththepreviousresultsderivedfromallpatients.HBVandNASHweremuchhigher,andthelevelsofconjugated/unconjugated,groupswereclusteredtogetherandALDandPBCgroupswereprimary/secondary,andconjugatedBAs%,especiallyGCA%alsoso(Figure2a,b).ThelevelsofTBAs,TCA%,LCA%,weremuchlowerinhealthycontrolsthaninpatientswithTCDCA%,TUDCA%,GUDCA%,conjugatedUDCAs%,diversechronicliverdiseases(FigureS1,TableS2).conjugatedBAs%,T-conjugatedBAs%,conjugated/ThereweresomesimilaritiesinBAprofilesamongthefiveunconjugated,andCA/CDCAremainedhigher,andthelevelsetiologicalgroups.HBV,HCV,andNASHgroupsseemedtoof12-ketoLCA%,CDCA%,NorDCA%,unconjugatedBAs%,becloser,andALDandPBCgroupsseemedtobemoretotalDCA%,andconjugatedDCAs%remainedlowerinPBCsimilartoeachother,asshowninFigure1a,b.IntermsofandALDgroups,comparingwithHBVandNASH(Figureabsoluteconcentration,thelevelsofTBAsinPBCandALD2b,c,TableS3).Thespecificityofthefiverepresentativegroups(bothmean>25,000ng/mL)weresignificantlyhighervariablesmentionedabovewasnotchangedeither(FigurethanthoseofHBV,HCV,andNASHgroups(allmean<2d−h).Certainly,thereweresomedifferencesbetweenthe10,000ng/mL)(Figure1c).IntermsofBAcomposition,theresultsfromallandS4patients.Comparatively,thenumberoflevelsoftaurocholate(TCA)%,LCA%,taurochenodeox-differentialvariablesderivedfromS4patientswaslowerthanycholate(TCDCA)%,tauroursodeoxycholate(TUDCA)%,thatofallpatients(36vs46),indicatingthatetiologicalglycoursodeoxycholate(GUDCA)%,conjugatedUDCAs%,differencewasweakerinS4patients.Somevariables,suchasconjugatedBAs%,T-conjugatedBAs%,conjugated/CDCA%,CA/CDCA,7-ketoLCA%,andtotalHCA%,wereunconjugated,andCA/CDCAwerehigherinPBCandALDdifferentamongetiologiesinallpatientsbutwerecomparablegroups,andthelevelsof12-ketoLCA%,7-ketoLCA%,CDCAinS4patients(TablesS2andS3).%,GDCA%,HCA%,NorDCA%,TLCA%,unconjugatedBAs%,totalDCA%,andconjugatedDCAs%werelowerin3.4.EtiologyDifferentiationwiththeRFModelPBCandALDgroups,comparingwiththeotherthreegroupsAseriesofRFmodelswereconstructedtodifferentiate(Figure1b).Detailedlevels(meanwithS.D.)ofalltheetiologiesofallandcirrhosispatients.PatientswithHCVvariablesinthefivegroupsarelistedinTableS2.Figure1d−hinfectionwerenotinvolvedconsideringthesmallsamplesize.arethebarplotsofsomerepresentativevariablesthatwereFirst,allthe46BA-relevantvariableswereusedand2RF2347https://doi.org/10.1021/acs.jproteome.0c00852J.ProteomeRes.2021,20,2340−2351

8JournalofProteomeResearchpubs.acs.org/jprArticlemodelswereconstructed,oneforallpatients,andoneforHCV,andNASHgroupsweremoresimilar,andALDandcirrhosis(S4)patients.Bothmodelsshowedgoodperform-PBCgroupswerecloserinserumBAprofiles.Therewerehugeanceswithhigh(>0.8)auROCandauPRvalues(exceptfordifferencesinBAprofilesofpatientswithfiveetiologies,andtheauPRofALDvstheothers)(Figure3a,b).Second,thethelevelsofsomevariableswereetiology-specific.Theaforementionedsixrepresentativevariables(TBAs,primary/correlationsbetweenBA-relevantvariablesandclinicalindicessecondary,T-conjugatedBAs%,totalUDCA%,conjugatedwerealsoquitedifferentinfourpathologicalgroups(HCVDCAs%,and12α-OH/non-12α-OH)wereused,andother2groupnotinvolved).OurfindingsprovidedbasicinformationRFmodelswereestablishedforetiologydiagnosisinallandforanimprovedunderstandingofthelinkageofBAprofilescirrhosis(S4)patients,respectively(Figure3c,d).Compara-andchronicliverdiseases.tively,theperformancesofthemodelsderivedfromallpatientsSomeofourmethodsandfindingswereconsistentwithweresuperiortothosefromcirrhosispatients,andthemodelsexistingstudies.First,proportionsandratiosbutnotthewithallvariablesweresuperiortothosewithsixvariablesabsoluteconcentrationsofindividualBAsweregeneratedand(Figure3e).Thisagreedwellwithpreviousresultsthattheusedinourstudy,takingintoaccountthepossibledifferencesetiologicaldifferenceinS4patientswasweakerthanthatofall-indetectionandstudypopulation.Bathenaetal.alsosuggestedstagepatients.SomemodelsperformednotwellforthatthecompositionofBAswasbetterassociatedwiththediscriminatingALDpatients(withlowauPRvalues).ThisseverityoftheliverdiseasecomparedwiththeabsolutemightbecausedbytherelativelylargevarianceandsmallconcentrationsofindividualandTBAs.BesidesabsolutesamplesizeofALDpatients.concentrations,therelevantvariablesofBAcompositions,3.5.CorrelationsbetweenBA-RelevantVariablesandwhichaccountforindividualdifferencesoftheBApool,wereClinicalIndicesmoreworthyofattentionforchronicliverdiseasestudy.15PartialSpearmancorrelationanalysis,whichadjustedforage,Second,thelevelofconjugatedDCAs%washigherinthesex,andBMI,wascarriedoutonclinicalindicesandBA-HBVgroupthanintheothers(Figures1gand2g),andthismaybeduetothereducedactivityofNa+-taurocholaterelevantvariables,usingpatientsfromHBV,NASH,PBC,andALDgroups(FiguresS2aandS2d).Figure4ashowsthecotransportingpolypeptide(NTCP),whichisresponsiblefornumberofBA-relevantvariablescorrelatedwitheachclinicalthemajorityofhepatocellularuptakeofconjugatedbilesalts.index,andFigure4bshowsthetotalnumberofcorrelatedpairsOehler’sstudyhasreportedthatHBVcouldbindtoNTCP,ineachetiology.Comparatively,whenallpatientswerelimitthefunctionofNTCP,andhindertheactivityandrecruited,theHBVgroup,followedbyPBC,NASH,andnucleartranslocationoftheBAsensor,farnesoidXreceptor26ALDgroups,hadadescendingnumberofvariablesassociated(FXR).Third,theproportionofconjugatedprimaryBAs,withclinicalindices.TheTBIL,PTA,PT,ALB,andASTwereespeciallyconjugatedCAs,washigherintheNASHgroupcorrelatedwithmostBA-relevantvariables.Interestingly,comparedtotheothers(Figures1band2b).TheyarealsodespitethelargestnumberofBAvariablescorrelatedwithhighlightedaspotentialmarkersforstagingNASHpatientsin27clinicalindicesintheHBVgroup(FiguresS2a),thereare26Caussy’sstudy.Fourth,thelevelsofTBAs,primaryBAs%,and27(thenumbersaremuchmorethantheotherthreeconjugatedBAs%,andT-conjugatedBAs%wereobviouslygroups)BA-relevantvariablescorrelatedwithPLTandALB,higherintheALDgroupthantheothers(Figures1b,eandrespectively,inthePBCgroup(FigureS2a−d),indicatingthat2b,e).FXRisgenerallythoughttobesuppressedbyethanolPLTandALBmaybepotentialriskfactorsforPBConsetandandthusresultsinincreasedBAsynthesisandBAlevelsduringprogression.Besidesallpatients,correlationanalysiswasalsoALD.28Interestingly,someresearchshowedthatCYP7B1conductedonS4patients.Figure4cshowsthespecificmightbethekeycontributortoincreasedBAsynthesisfornumbersofBA-relevantvariablescorrelatedwitheachclinicalCYP7B1,whichshowsamarkedinductionintheliver,whileindexindifferentscenarios.Comparatively,forbothHBVandotherBAsynthesisenzymes,CYP7A1,CYP8B1,andNASHgroups,thecorrelationstrengthbetweenBA-relevantCYP27A1,aresignificantlydownregulatedinALDpatients.29variablesandclinicalindiceswasstrongerinallsamplesthanFifth,inourstudy,CDCA-relevantvariables,includingtotalthatofinS4samples,implyingthatBA-relevantmarkersmayCDCA%,conjugatedCDCAs%,andespeciallyTCDCA%,bemoreefficaciousindiagnosisorpredictionintheearlywerehighestintheALDgroup(Figures1band2b).Huangetstagesofthedisease.al.foundthatthecontinuousincreaseofCDCAwillactivatetheFXR-SHPsignalingpathwayintheliver,andtheactivation4.DISCUSSIONofthissignalingpathwaywillfurtheractivatetheexpressionof30FibrosisisthecommonterminalstageformostchronicliverCYP7B1.Moreover,Yangetal.reportedthatdysregulationinjuriesincludingALD,NAFLD,viralhepatitis,andcholestaticofmethionine/homocysteinemetabolismmightleadtoliverdiseases.25Thedegreeofliverfibrosiscanbestagedfromincreasedproductionoftaurine,resultinginsubstantial29S0toS4accordingtoScheuer’sclassification,andpatientsinelevationoftaurine-conjugatedBAs.TheabundanceofS4arealsocalledcirrhosispatients.23Inthisstudy,wecriticalmicrobiotathatdecreasedisthepossiblemechanismtoinvestigatedtheserumBAprofilesinhealthycontrolsandexplainthehigherproportionofprimaryBAsandtheratioofbiopsy-confirmedpatientswiththeetiologyofHBV,HCV,primarytosecondaryBAsinALD.Sixth,differentfromALD,NASH,ALD,andPBC.WecomparedthedifferencebetweenthecompositionofsecondaryBAswashigherinthePBChealthycontrolsandpatientswithchronicliverdiseaseandthegroup(Figures1band2b).IthasbeenwellrecognizedthatthegeneralityandspecificityofBAprofilesamongthefivegutmicrobiomeplaysthepredominantroleinsecondaryBAetiologicalgroups,establishedseveralRFmodelstodiffer-synthesis.AsreportedbyTangetal.,gutmicrobiomedysbiosisentiatedifferentetiologies,andalsoexaminedthecorrelationsisobservedinPBCpatientsandeightgenera(Haemophilus,betweenBA-relevantvariablesandclinicalindicesinpatientsofVeillonella,Clostridium,Lactobacillus,Streptococcus,Pseudomo-9allstages(stages0−4)andthecirrhosis(S4)patients.HBV,nas,andKlebsiella)significantlyincreasedinPBC.2348https://doi.org/10.1021/acs.jproteome.0c00852J.ProteomeRes.2021,20,2340−2351

9JournalofProteomeResearchpubs.acs.org/jprArticleSomeofourfindingshavenotbeenreportedyet.First,inDCAs%,andT-conjugatedBAs%)showeddesirableBAprofiles,ALDandPBCgroupsseemedtobemoresimilarconsistencyamongallandage-and-BMI-matchedsamples,inallandinS4patients(Figures1a,band2a,b).Thismayandmalesandfemales(FigureS4).ConsideringthesmallresultfromtheseverecholestasiscausedbycirrhosisinALDorsamplesize,theresultsoncirrhosispatientsandonmalesandtheclosureofthebilesalt-exportpump.Second,thefemalesneedfurthervalidationandexamination.compositionsof12α-OHBAsandtheratioof12α-OHtoTherearelimitationsinourstudy.First,allthesampleswerenon-12α-OHBAswereapparentlyhigherinNASHthaninfromChinaandthesamplesizewasunbalanced.Second,someothergroups(Figures1b,hand2b,h).InHaeusler’sresearch,patientsinthePBCgrouphadahistoryofUDCAmedication.insulinresistance(IR)isassociatedwithincreased12α-OHWerepeatedalltherelevantanalysiswithpatientswhowere31notonmedication,andtheresults(FigureS3)wereBAsandtheratioof12α-OHtonon-12α-OHBAs.Ourrecentstudiesalsodemonstratedthatincreased12α-OHBAcomparabletothatofallpatients(Figures1and2).Even32though,theUDCA-relatedresultsneedfurthervalidation.levelsareassociatedwithanunhealthyhighBMIstatus.SeveralstudieshaveshownthatIRisamajorstepintheInconclusion,ourstudydemonstratesthattheBAprofiles33−35differsignificantlyamongliverdiseasepatientswithdifferentpathogenesisofNAFLD.Therefore,12α-OHBAsandtheratioof12α-OHtonon-12α-OHBAscanbepotentialmarkersetiologies,whichsuggeststhattheanalysisofBAshasthetodistinguishNAFLD/NASHpatientsfromotherpatientspotentialfordifferentiatingvariousformsofliverinjury.Ourwithchronicliverdiseases.Third,thereweremoredifferentialcomparisonsandfindingsprovidebasicinformationforfurthervariablesamongthefiveetiologieswhenallthepatientsweremechanisticstudyonBAhomeostasisandchronicliverdisease.involvedthaninthosewhereonlycirrhosispatientswereinvolved(TablesS2andS3).Theetiologydifferentiation■ASSOCIATEDCONTENTmodelsderivedfromthesameBA-relevantvariablesperformed*sıSupportingInformationbetterwithallpatientsthanwithcirrhosispatients(Figure3).TheSupportingInformationisavailablefreeofchargeatThecorrelationbetweenBAprofilesandclinicalindiceswashttps://pubs.acs.org/doi/10.1021/acs.jproteome.0c00852.alsostrongerinallpatientsthanincirrhosispatients(FigureAbbreviationsofBA-relevantvariablesandclinical4c).AllthesedemonstratethatBAprofilesandsomeoftheindices,levelsofBA-relevantvariablesinthecontrolrepresentativevariablesareofhighersignificanceforstudyongroupandfivepathologicalgroups,levelsofBA-relevantearly-stageliverdiseases.ThisisinlinewithSole’sresearch,variablesincirrhosis(stage4)patientsoffourwhichshowedcirrhosishasaremarkablereductioningeneandpathologicalgroups,PvaluesofBA-relevantvariablesMGSrichness,andthelossofrichnesscorrelatedwithdisease36comparingfivedifferentstages,.heatmapanalysisofBA-stages.Fourth,besidesetiologicaldifference,pathologicalrelevantvariablesinahealthycontrolgroupandfivestagedifferencewasalsoexamined.Withthesamemultigroupdifferentetiologicalgroups,heatmapsofpartialcomparisonandposthocanalysismethods,37differentialSpearmancorrelationcoefficientsbetweenallBAvariableswereidentifiedamongstagesS0−4(TableS4).Inrelevantvariablesandclinicalindiceswithfourtermsofthenumberofdifferentialvariables,theetiologicalpathologicalpatients,representativeBA-relevantvaria-differencewaslargerthanthestagingdifference(46vs37)inblesindifferentetiologicalgroupswithpatientswhohadBAprofilesinvolvedinthisstudy.However,itshouldbenotednomedicationhistory,andlevelsofrepresentativeBA-thatthereisnoS0−3intheALDandPBCgroups,andsowerelevantvariablesindifferentetiologicalgroupsbasedoncannotconcludewhichisthemaincauseofthedifferencesinage-and-BMI-matchedpatients(PDF)BAswiththecurrentresult.Therearedifferencesinage,BMI,gender,andsomeotherclinicalindicesamongdifferentetiologies.Hence,partial■AUTHORINFORMATIONSpearmancorrelationanalysisadjustingage,sex,andBMICorrespondingAuthorswascarriedoutanddifferenceanalysiswasperformedinallPingLiu−E-instituteofShanghaiMunicipalEducationsamples,age-and-BMI-matched(p>0.05)samples,andmalesCommittee,InstituteofInterdisciplinaryIntegrativeMedicineandfemales,respectively.Thereare231,105,9,and154BA-Research,ShanghaiUniversityofTraditionalChineserelevantvariablescorrelatedwithanyoftheclinicalindicesinMedicine,Shanghai201203,China;Email:liuliver@HBV,NASH,ALD,andPBCgroup,respectively.Thelevelsofvip.sina.comBA-relevantvariablesandtheirtrendsinfouretiologies(HBV,GuoxiangXie−HumanMetabolomicsInstitute,Inc.,NASH,ALD,andPBC)arecomparableinmatchedandallShenzhen518109,Guangdong,China;orcid.org/0000-samples.Fortheetiologicalcomparisonsbasedonpatientsin0002-0951-4150;Email:xieguoxiang@hmibiotech.comallstages,thenumberofdifferentialBA-relevantvariableswasTianluChen−CenterforTranslationalMedicine,Shanghaicomparableinmatchedandallsamples(42and46,JiaoTongUniversityAffiliatedSixthPeople’sHospital,respectively).ForthecomparisonsbasedoncirrhosispatientsShanghai200233,China;orcid.org/0000-0003-1798-(S4),therearemuchfewerdifferentialBA-relevantvariables5435;Email:chentianlu@sjtu.edu.cnidentifiedfrommatchedsamplescomparedwithallsamples(6and42,respectively).Furtheron,malesandfemales(ofAuthorsmatchedsamples)wereanalyzedseparatelyastherearenoChaoSang−CenterforTranslationalMedicine,ShanghaifemalesintheALDgroupandonlythreefemalecirrhosisJiaoTongUniversityAffiliatedSixthPeople’sHospital,patientsintheNASHgroup.ThelevelsandtrendsofsomeShanghai200233,ChinaBA-relevantvariableswereconsistentandsomeweredifferentXiaoningWang−E-instituteofShanghaiMunicipalinmalesandfemales.WearepleasedtoobservethattheresultsEducationCommittee,InstituteofInterdisciplinaryofthesixrepresentativeBA-relevantvariables(TBAs,primary/IntegrativeMedicineResearch,ShanghaiUniversityofsecondary,12α-OH/non-12α-OH,totalUDCA%,conjugatedTraditionalChineseMedicine,Shanghai201203,China2349https://doi.org/10.1021/acs.jproteome.0c00852J.ProteomeRes.2021,20,2340−2351

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