Alteration of Bile Acid

TheFASEBJournal•ResearchCommunicationAlterationofbileacidmetabolismintheratinducedbychronicethanolconsumptionGuoxiangXie,*,†,‡WeiZhong,*HoukaiLi,*QiongLi,*YunpingQiu,*XiaojiaoZheng,*HuiyuanChen,*XueqingZhao,*ShuchaZhang,§ZhanxiangZhou,*,1StevenH.Zeisel,§andWeiJia†,‡,1*CenterforTranslationalBiomedicalResearch,UniversityofNorthCarolinaatGreensboro,NorthCarolinaResearchCampus,Kannapolis,NorthCarolina,USA;†CenterforTranslationalMedicine,ShanghaiJiaoTongUniversityAffiliatedSixthPeople’sHospital,Shanghai,China;‡UniversityofHawaiiCancerCenter,Honolulu,Hawaii,USA;and§NutritionResearchInstitute,UniversityofNorthCarolinaatChapelHill,Kannapolis,NorthCarolina,USAABSTRACTOurunderstandingofthebileacidme-tabolismislimitedbythefactthatpreviousanalyseshaveprimarilyfocusedonaselectedfewcirculatingbileacids;thebileacidprofilesoftheliverandgastrointestinaltractpoolsarerarelyinvestigated.Here,wedeterminedhowchronicethanolconsump-tionalteredthebileacidsinmultiplebodycompart-ments(liver,gastrointestinaltract,andserum)ofrats.RatswerefedamodifiedLieber-DeCarliliquiddietwith38%ofcaloriesasethanol(theamountequivalentof4–5drinksinhumans).Whileconjugatedbileacidspredominatedintheliver(98.3%),duodenum(97.8%),andileum(89.7%),unconjugatedbileacidscomprisedthelargestproportionofmeasuredbileacidsinserum(81.2%),thececum(97.7%),andtherectum(97.5%).Inparticular,taurine-conjugatedbileacidsweresignif-icantlydecreasedintheliverandgastrointestinaltractofethanol-treatedrats,whileunconjugatedandglycine-conjugatedspeciesincreased.Ethanolconsumptioncausedincreasedexpressionofgenesinvolvedinbileacidbiosynthesis,effluxtransport,andreducedexpres-sionofgenesregulatingbileacidinfluxtransportintheliver.Theseresultsprovideanimprovedunderstandingofthesystemicmodulationsofbileacidmetabolisminmammalsthroughthegut-liveraxis.—Xie,G.,Zhong,W.,Li,H.,Li,Q.,Qiu,Y.,Zheng,X.,Chen,H.,Zhao,X.,Zhang,S.,Zhou,Z.,Zeisel,S.H.,Jia,W.Alterationofbileacidmetabolismintheratinducedbychronicethanolconsumption.FASEBJ.27,3583–3593(2013).www.fasebj.orgKeyWords:gastrointestinaltract
liver
blood
ultraperformanceliquidchromatographymassspectrometry
metabolomicsBileacidsaregainingincreasingrecognitionasimportantmetabolicsignalingmoleculesthatmodulatelipid,glucose,andenergymetabolism(1).Also,theyregulatetheirownhomeostasisthroughbindingwiththenuclearreceptorfarnesoidXreceptor(FXR),theplasmamembrane-boundbileacidreceptorTGR5/M-BAR(2).ActivationofintestinalFXRbybileacidsleadstoup-regulationoffibroblastgrowthfactor15(FGF15)(3,4),andsecretedFGF15suppresseslivertranscrip-tionofcholesterol7
-hydroxylase1(CYP7A1),therate-limitingenzymeforliverbileacidbiosynthesis(5).Errorsinbileacidmetabolismareimplicatedinseveralhumandiseases,includingbothalcoholic(6)andnon-alcoholicfattyliverdiseases(7)andcoloncancer(8).Thegutmicrobiomemayplayaroleinthedevelop-mentofavarietyofdiseasessuchasobesity,diabetes1Correspondence:W.J.,UniversityofHawaiiCancerCen-ter,Honolulu,HI96813,USA.E-mail:wjia@cc.hawaii.edu;Z.Z.,UniversityofNorthCarolinaatGreensboro,NorthCarolinaResearchCampus,Kannapolis,NC28081,USA.E-mail:z_zhou@uncg.edudoi:10.1096/fj.13-231860Thisarticleincludessupplementaldata.Pleasevisithttp://www.fasebj.orgtoobtainthisinformation.Abbreviations:6,7-DKDCA,6,7-diketodeoxycholate;7-DCA,7-dehydrocholate;
-MCA,
-muricholate;-MCA,-murich-olate;-MCA,-muricholate;-MCA,-muricholate;ALT,alanineaminotransferase;ASBT,apicalsodiumdependentbileacidtransporter;BAAT,bileacidCoA:aminoacidN-acyltransferase;BACS,bileacidCoAsynthetase;BSEP,bilesaltexportpump;CA,cholate;CDCA,chenodeoxycholate;CYP7A1,cholesterol7
-hydroxylase1;CYP8B1,sterol12
-hydroxylase;CYP27A1,sterol27-hydroxylase;DCA,deoxy-cholate;DHCA,dehydrocholate;FGF15,fibroblastgrowthfactor15;FGFR4,fibroblastgrowthfactorreceptor4;FXR,farnesoidXreceptor;GCA,glycocholate;GCDCA,glycoche-nodeoxycholate;GDCA,glycodeoxycholate;GHDCA,glyco-hyodeoxycholate;GI,gastrointestinal;GLCA,glycolithocholate;GUDCA,glycoursodeoxycholate;HDCA,hyodeoxycholate;IS,in-ternalstandard;LCA,lithocholate;MDCA,murideoxy-cholate;methylDCA,methyldeoxycholate;MRP2,multidrugresistance-associatedprotein2;MRP4,multidrugresistance-associatedprotein4;MS,masssprectrometry;NDCA,norde-oxycholate;NTCP,sodium-taurocholatecotransportingpoly-peptide;OST
/,organicsolutetransporter
/;PCA,principalcomponentanalysis;T
-MCA,tauro
-muricholate;T-MCA,tauro-muricholate;TCA,taurocholate;TCDCA,taurochenodeoxycholate;TDCA,taurodeoxycholate;THDCA,tau-rohyodeoxycholate;TLCA,taurolithocholate;TUDCA,taur-oursodeoxycholate;UPLC,ultraperformanceliquidchroma-tography35830892-6638/13/0027-3583©FASEBDownloadedfromwww.fasebj.orgbyShanghaiInformationCenterforLifeSciences,CAS(202.127.20.56)onFebruary05,2018.TheFASEBJournalVol.27,No.9,pp.3583-3593.mellitus,fattyliverdisease,andcoloncancer(9).Primarilyharboredinthelargeintestine,thegutmicrobiotanotonlycontributetothesalvageofbileacidsthatescapereabsorptionbyactivetransportindistalileumbutalsomodifythechemicalstructureofabsorbedbileacidsthroughprocessesincludingdecon-jugation,dehydrogenation,dehydroxylation,anddes-ulfation(10–12).Perturbingthegutmicrobiomecanresultinadisturbanceofbileacidmetabolismandreabsorption,leadingtoalteredbileacidprofilesintheblood,liver,kidneys,andheart(2).Moreover,inhibit-ingintestinalmicrobiotawithampicillinincreasesmRNAandproteinexpressionoftheapicalsodium-dependentbileacidtransporter(ASBT/Slc10a2)inthebrush-bordermembraneoftheileum,whichinturnincreasesbileacidtransportintoportalblood(13).Chronicethanolconsumptionisassociatedwithal-coholicliverdiseasesandhasadverseeffectsonlipidmetabolism,bothinhepaticandextrahepatictissues.Thisleadstothedevelopmentoffattyliver,hepatitis,andcirrhosis(14,15),andethanol-inducedfattyliverisaccompaniedbyalterationsinthebileacidprofileandinthecompositionofthegutmicrobiome(15–17).Thesechangesintheenterohepaticcirculationofbileacidsareimportantnotonlyforfeedbackinhibitionofbileacidsynthesisbutalsoforwhole-bodylipidhomeo-stasis.However,thereareveryfewreportsthatsystem-icallydelineatethecompositionofbileacidprofilesinthewholegastrointestinal(GI)tract,liver,andbloodpool,noristhereanyinformationavailableonthedynamicchangesofthesebileacidprofilesduringchronicethanolconsumption.Thegoalofthisstudywastouseatargetedmetabo-lomicsapproachtocharacterizethebileacidprofilesoftheserum,GItract(duodenum,jejunum,ileum,ce-cum,colon,andrectum)andliverinmaleSpragueDawleyratsconsumingethanolchronicallyfor8wk.Wequantifiedapanelof20–30bileacidsusingultraper-formanceliquidchromatography–triple-quadrupolemassspectrometry(UPLC-TQMS)andfoundthateth-anolconsumptionsubstantiallyaffectedthebileacidprofilesofdifferenttissues.Inaddition,wemeasuredtheexpressionofdifferentgenesregulatingbileacidmetabolismintheliverandileum.MATERIALSANDMETHODSChemicalsandreagentsLithocholate(LCA),nordeoxycholate(NDCA),murideoxy-cholate(MDCA),hyodeoxycholate(HDCA),chenodeoxycholate(CDCA),deoxycholate(DCA),dehydrocholate(DHCA),glyco-cholate(GCA),taurolithocholate(TLCA),glycolithocholate(GLCA),
-muricholate(
-MCA),-muricholate(-MCA),-muricholate(-MCA),-muricholate(-MCA),cholate(CA),7-dehydrocholate(7-DCA),methyldeoxycholate(methylDCA),6,7-diketodeoxycholate(6,7-DKDCA),tauro
-muricholate(T
-MCA),tauro-muricholate(T-MCA),taurocholate(TCA),tauroursodeoxycholate(TUDCA),taurohyodeoxycholate(THDCA),taurochenodeoxycholate(TCDCA),taurodeoxy-cholate(TDCA),glycoursodeoxycholate(GUDCA),glycohyode-oxycholate(GHDCA),glycochenodeoxycholate(GCDCA),gly-codeoxycholate(GDCA),cholate-d4(CA-d4;5-cholanicacid-3
,7
,12
-triol-2,2,4,4-d4),glycocholate-d4(GCA-d4;5-cholanicacid-3
,7
,12
-triolN-(carboxymethyl)-amide-2,2,4,4-d4),lithocholate-d4(LCA-d4;5-cholanicacid-3
-ol-2,2,4,4-d4),anddeoxycholate-d4(DCA-d4;5-cholanicacid-3
,12
-diol-2,2,4,4-d4)werepurchasedfromSteraloids,Inc.(Newport,RI,USA).HPLC-grademethanol,acetonitrile,water,ammoniumacetate,andaceticacidwereobtainedfromSigma-Aldrich(St.Louis,MO,USA).AnimalsandethanolfeedingexperimentsMaleSprague-DawleyratswereobtainedfromCharlesRiver(Wilmington,MA,USA).AllratsweretreatedaccordingtoexperimentalproceduresapprovedbytheInstitutionalAni-malCareandUseCommittee.Three-month-oldratswerepair-fedamodifiedLieber-DeCarliliquiddietcontainingeitherethanolorisocaloricmaltosedextrinfor8wk.Thecaloriesoftheethanolliquiddietwerederived38%fromethanol,34%fromfat,16%fromprotein,and12%fromcarbohydrate.Theethanolcalorieswerereplacedbymaltosedextraninthecontrolliquiddiet.AllingredientsfortheliquiddietswereobtainedfromDyets(Bethlehem,PA,USA)withtheexceptionofethanol,whichwaspurchasedfromSigma-Aldrich.Theethanolcontent(%,w/v)wasinitially5%forthefirst2wkandwasincreasedby0.2%every2wkuptoaconcentrationof5.6%duringthefinal2wk.Thecontrolgroupwaspair-fedtheamountthatethanol-fedratshadinthepreviousday.Theratsweredeniedaccesstofoodfor4hbeforebloodwasdrawn.Serumsampleswerecollectedatwk2,4,and6aswellasattheendoftheexperiment.Rats(n5–9/group)wereanesthetizedwithisofluoraneandse-rum,andliverandintestinalcontentswereharvestedforanalysis.PreparationofsamplesandstandardsSerumsamplepreparationAnaliquotof100lofserumwasmixedwith400lofamixtureofmethanolandacetonitrile[5:3,contains5g/mlofaqueous4-chlorophenylalanine,usedastheinternalstan-dard(IS)].Themixturewasthenvortexedfor2min,allowedtostandfor10min,andcentrifugedat13,000rpmfor20min.ThesupernatantwasusedforUPLC-MS/MSanalysis.LiversamplepreparationLivertissuesamples(100mg)werehomogenizedonicein500lofamixtureofchloroform,methanolandwater(1:2.5:1,v/v/v).Thesampleswerethencentrifugedat13,000rpmfor10minat4°C,anda150-laliquotofthesupernatantwastransferredtoanLCsamplingvialcontaininganIS(10lL-4-chloro-phenylalanineinwater,5g/ml).Thedepositwasrehomogenizedwith500lofmethanol,anda150-laliquotofsupernatantwasaddedtothesamevialfordryingpriortoreconstitutionwithacetonitrile/H2O(6:4,v/v)toafinalvolumeof500l.IntestinalcontentsamplepreparationIntestinalcontents(100mg)weremixedwith500lofice-coldwater.Themixturewasvortexedfor4minandthencentrifugedat13,200rpmfor10minat4°C.A300-laliquotofsupernatantwastransferredtoa2-mltube,andthepellets3584Vol.27September2013XIEETAL.TheFASEBJournal
www.fasebj.orgDownloadedfromwww.fasebj.orgbyShanghaiInformationCenterforLifeSciences,CAS(202.127.20.56)onFebruary05,2018.TheFASEBJournalVol.27,No.9,pp.3583-3593.werefurtherextractedwithice-coldmethanolusingthesameprotocol.Another300-laliquotofsupernatantwasaddedtothesametubeastheinitialaliquot,and10lofIS(p-chlorophenylalanineinwater,5g/ml)wasadded.Theextractionwasvortexedfor30sandcentrifugedat13,000rpmfor20min.TheresultingsupernatantwasusedforUPLC-MSanalysis.StandardsolutionEachofthe32standardswasindividuallydissolvedinmeth-anolorwaterandpreparedasastocksolutionataconcen-trationof5mg/ml.MethodvalidationEachaliquotofstandardstocksolutionwasmixedtoobtainamixedstocksolution.Theresultingmixedsolutionwasdilutedtogenerateaseriesofconcentrationsof0.01,0.1,0.5,1,5,10,50,100,500,1000,and10,000ng/ml.LCA-d4wasusedasanIS,andthecalibrationcurveandthecorrespond-ingregressioncoefficientswereobtainedbyISadjustment(SupplementalTableS1).Allbileacidswerefoundtobelinearoverthemeasuredrange.InstrumentationAWatersAcquityUPLCsystemequippedwithabinarysolventdeliverymanagerandasamplemanager(Waters,Milford,MA,USA)wasusedthroughoutthestudy.Themassspec-trometerwasaWatersTQinstrumentwithanelectrosprayionization(ESI)source(Waters).TheentireLC-MSsystemwascontrolledbyMassLynx4.1software(Waters).Allchro-matographicseparationswereperformedwithanAcquityUPLCC18column(1.7m,50mm2.1mminternaldimen-sions;Waters).LCandMSconditionsThemobilephaseconsistedof10mMammoniumacetateadjustedtopH4usingaceticacid(mobilephaseA)andmethanol(MeOH;mobilephaseB)runataflowrateof0.3ml/min.TheLCelutionconditionswereoptimizedasfol-lows:isocraticat40%B(0–0.5min),lineargradientfrom40to80%B(0.5–9.0min),80to100%B(9.0–12.0min),isocraticat100%B(12.0–12.5min);andisocraticat40%B(12.5–15.0min).Thecolumnwasmaintainedat40°C,andtheinjectionvolumeofallsampleswas10l.Themassspectrometerwasoperatedwithsourceanddesolvationtemperaturessetat120and350°C,respectively.Bileacidsweredetectedinthenegativemode.Thecapillary,extractor,andRFvoltageswere3000,4,and0V,respectively.Thedesolvationgas(nitrogen)wassetataflowrateof650L/h.Theconevoltages,collisionenergies,andmultiplereactionmonitoring(MRM)transitionsarelistedinSupple-mentalTableS2.qRT-PCRanalysisTotalRNAwasisolatedfromliverorileummucosausingTRIzolreagent(Invitrogen,LifeTechnologies,GrandIsland,NY,USA)andreversetranscribedwithTaqManReverseTranscriptionReagents(AppliedBiosystems,FosterCity,CA,USA).ExpressionoftargetmRNAwasmeasuredintriplicatebythecomparativecyclethresholdmethodontheAppliedBiosystems7500RealTimePCRSystem.TheforwardandreverseprimerswerepurchasedfromIntegratedDNATech-nologies(Coralville,IA,USA),andsequencesareshowninTable1.Targetgeneexpressionwasnormalizedto18srRNAlevelsandpresentedasfoldchangesrelativetocontrolvalues(whichweresetat1).DataanalysisUPLC-MSrawdataobtainedwithnegativemodewereanalyzedusingQuanLynx4.1applicationsmanager(Wa-ters).AStudent’sttestwasusedtoinvestigatedifferencesbetweenthegroupsinbileacidsmeasurements.Principalcomponentanalysis(PCA)wasperformedusingSIMCA-Psoftware(Umetrics,SanJose,CA,USA).RESULTSGeneralinformationabouttheanimalexperimentAnumberofserumparameters,includingserumala-nineaminotransferase(ALT),aspartateaminotransfer-ase(AST),alkalinephosphate(ALP),albumin,ammo-nia,and-glutamyltranspeptidase(GGT),weremeasured,andthevalueswerelistedinTable2.ALTactivitywasmarkedlyincreasedduetoethanolconsumption(P 0.05;Table2).GItractbileacidprofilesofcontrolratsTosystemicallycharacterizethebileacidprofileintheGItractofcontrolrats,wequantitativelymeasuredtheabundanceofbileacidsinintestinalcontentsfromtheduo-denum,ileum,cecum,andrectum(Table3).TherepresentativeUPLC-MS/MSchromatogramsoftheauthenticstandardsofbileacidsunderthefinalchromatographyanddetectionconditionswereshowninFig.1.TheduodenumisthefirstsitewherebileacidsaresecretedintotheGItract,andmostofthebileacidswillbereabsorbedthroughenterohepaticcir-culationbeforereachingthececum.Theconcentrationsofunconjugatedbileacids,suchasCA,CDCA,DCA,andHDCA,changedsignificantlyfromtheduodenumtotheileum,thececum,andtherectum.Taurine-conjugatedbileacidsaccountedforthegreatestportionofallmea-suredbileacidsfromtheduodenumandileum,whereastheirlevelsdecreaseddramaticallyinthececumandrectum.Forexample,theconcentrationofTCAwasthehighestintheduodenum(1.15nmol/mg)andileum(1.87nmol/mg),butonlyaverylowlevelofTCAwasdetectedinthececum(0.0007nmol/mg)andrectum(0.000004nmol/mg).Ingeneral,theabundanceofgly-cine-conjugatedbileacidswasverylow.GCAwasthemostabundantglycine-conjugatedbileacidintheduodenum(0.02nmol/mg)andileum(0.03nmol/mg),anditslevelsdecreasedto0.00005and0.000009nmol/mginthececumandrectum,respectively.TherelativecompositionofbileacidsdifferedbyregionoftheGItract(Fig.2).Taurine-conjugatedbileacidsaccountedfor97.1and89.1%ofalldetectedbileacidsintheduodenumandileum,butweremuchlessprevalentinthececumandrectum( 0.5%ineach3585BILEACIDPROFILESINMULTIPLEBODYCOMPARTMENTSDownloadedfromwww.fasebj.orgbyShanghaiInformationCenterforLifeSciences,CAS(202.127.20.56)onFebruary05,2018.TheFASEBJournalVol.27,No.9,pp.3583-3593.location).Incontrast,unconjugatedbileacidsconsti-tutedthelargestportionofallmeasuredbileacidsinthececumandrectum.TCAlevelsdecreased(46.6to29.5%),whereasTUDCA(1.2to10.0%),THDCA(14.6to17.2%),andCA(1.2to6.5%)levelsallincreasedbetweentheduodenumandileum.ThepredominantunconjugatedbileacidsinthececumandrectumincludedCDCA,DCA,andHDCA;theseallhadcom-parableabundancebetweenthececumandrectum.However,theproportionof-MCAand
/-MCAdecreasedsignificantlyfromthececumtotherectum(-MCA:9.4to0.7%,P 0.05;
/-MCA:3.7to0.5%,P 0.05).Thelevelsofthesebileacidsinthejejunumandcolon(SupplementalFig.S1)werealsomeasured.Chronicethanolconsumptionaltersthebileacidmetabolomeofliver,gastrointestinaltract,andserumWecomparedthebileacidprofileofthecontentsofGItract,liver,andserumfromcontrolandethanol-treatedTABLE1.PrimersetsforquantitativeRT-PCRanalysisGeneFullnameGeneBankIDSequences,forward/reverse5=–3=Ampliconsize(bp)CYP7A1CytochromeP4507A1NM_012942TGAAAGCGGGAAAGCAAAGACCAC/TCTTGGACGGCAAAGAGTCTTCCA117CYP8B1CytochromeP4508B1NM_031241TCCATATGTCCCGGCAGGTTCTTT/TGTCAGGGTCCACCAGTTCAAAGT97CYP27A1CytochromeP45027A1NM_178847ACCTTTCCTGAGCTGATCTTGGCT/GCATGTGGGCAAAGTCCTTGTTCT169StARSteroidogenicacuteregulatoryproteinNM_031558AAGCCAGCAGGAGAATGGAGATGA/TGACATTTGGGTTCCACTCTCCCA158Fatp2Fattyacidtransportprotein2NM_031736AGTACATCGGTGAACTGCTTCGGT/CAATGTTGCCTTCAGTGGAAGCGT180BACS/Fatp5BileacidCoAsynthaseNM_024143TTCAGGGACCACTGGACTTCCAAA/ACCACATCATCAGCTGTTCTCCCA105BAATBileacidCoA:aminoacidN-acyltransferaseNM_017300GGTTGGCATCCTTTCTGTGTGCAT/ATTCTTCACTGCAGGGTGTAGGCT167CYP3A11CytochromeP4503A11NM_153312TCCAGCTGATGCTGAACGCTCATA/AGGGTGAGTGGCCAGGAAATACAA164SULT2A1Sulfotransferasefamily2A1NM_131903TCAGTTCCAAGGCCAAGGTGATCT/AGTTCCCAGTGAGTCTGGCTTCTT116NTCP/Slc10a1Na-taurocholatecotransportingpolypeptideNM_017047AGGATGGAGGTGCACAACGTATCA/AGCCCAGTGAGAGCATGATAAGCA133BSEP/Abcb11BilesaltexportpumpNM_031760ATCTGTTAATCCTGGGCAGACGCT/TGGGAGACAATCCCGATGTTGGAA180MRP2/Abcc2Multidrugresistance-associatedprotein2NM_012833AACCGGGAAGGTCAAGTTCTCCAT/TTGTCAGAGTCACTGGTCCAAGCA153MRP4/Abcc4Multidrugresistance-associatedprotein4NM_133411ATTGTGGGAAGAACTGGAGCTGGA/TGGTTCCAGTGAACAGGACAGGTT175OST
OrganicsolutetransporteralphaNM_001107087AGCAATTTCCTTGCTGTGTCCACC/AGGATGACAAGCACCTGGAACAGA131OSTOrganicsolutetransporterbetaXM_238546TCCGTTCAGAGGATGCAACTCCTT/CATTCCGTTGTCTTGTGGCTGCTT140ASBT/Slc10a2ApicalsodiumdependentbileacidtransporterNM_017222TGGGCTTCCTCTGTCAGTTTGGAA/AGTGTGGAGCAAGTGGTCATGCTA196TGR5/Gpbar1Gprotein-coupledbileacidreceptor1NM_177936TTGCTCCTGTCAGTCTTGGCCTAT/TTGGGTCTTCCTCGAAGCACTTGT191FGF15Fibroblastgrowthfactor15NM_130753ACAATTGCCATCAAGGACGTCAGC/TGAAGATGATGTGGAGGTGGTGCT172FGFR4Fibroblastgrowthfactorreceptor4NM_001109904TCACAGTTGAGGCCTTCTGTTCCA/TGCTTCGCTCCTTTGAGGATGAGT10818srRNA18sribosomalRNANR_046237ACGGACCAGAGCGAAAGCAT/TGTCAATCCTGTCCGTGTCC120TABLE2.Serumvaluesofalanineaminotransferase(ALT),aspartateaminotransferase(AST),alkalinephosphate(ALP),albumin,ammonia,and-glutamyltranspeptidase(GGT)incontrolratsandratsunderethanolconsumptionfor8wkGroupALT(U/L)AST(U/L)ALP(U/ml)Albumin(mg/ml)Ammonia(M)GGT(mU/ml)Control25.023.4138.126.3839.985.9630.000.94815.7360.7843.876.48Ethanol41.907.03*44.837.0739.214.1129.900.97825.2941.8039.496.96*P 0.05vs.controlgroup.3586Vol.27September2013XIEETAL.TheFASEBJournal
www.fasebj.orgDownloadedfromwww.fasebj.orgbyShanghaiInformationCenterforLifeSciences,CAS(202.127.20.56)onFebruary05,2018.TheFASEBJournalVol.27,No.9,pp.3583-3593.rats.Asdescribedabove,theconcentrationofunconju-gatedbileacidsincontrolratsincreasedsteadilyfromtheduodenumtocecum(0.04to1.11nmol/mg),whereasadecreasingtrendwasobservedintheethanolgroup(1.30to0.54nmol/mg)(Fig.3).However,theconcentrationofunconjugatedbileacidsintherectumwascomparablebetweenthecontrolandethanolgroups.Ethanolcon-sumptionledtolowerlevelsoftaurine-conjugatedbileacidsintheduodenumandileum(0.15and0.02nmol/mg,respectively)relativetocontrolrats(2.39and5.66nmol/mg).Finally,averylowleveloftaurine-conjugatedbileacidswasdetectedinthececumsandrectumsofbothcontrolandethanol-treatedrats.Theliveristhecriticalsitewherebileacidsaresynthe-sizedandsecreted.Intheliversofcontrolrats,taurine-conjugatedbileacidspredominated;however,ethanolconsumptionresultedinasignificantincreaseinbothglycine-conjugatedaswellasunconjugatedbileacids(Fig.3).Inaddition,thelevelsofseveralbileacidsweredifferentbetweentheliversofcontrolandethanol-treatedrats,includingTCA(36.6vs.15.5%incontrolandetha-nol-treated,respectively),THDCA(27.6vs.15.3%),T
/-MCA(14.7vs.2.6%),GCA(1.1vs.9.6%),GUDCA(0.6vs.8.6%),andGHDCA(0.8vs.10.9%).Inserumfromcontrolrats,unconjugatedbileacidsweremostabundant,particularlyCAandHDCA.Incontrastwiththeliverandintestinalcontents,theserumlevelsofglycine-conjugatedbileacidsweregreaterthanthoseoft