colonic bacterial metabolism.docx

colonic bacterial metabolism.docx

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colonicbacterialmetabolism

AmJPhysiolGastrointestLiverPhysiol. 2012January; 302

（1）:

G1–G9.

Functionalanalysisofcolonicbacterialmetabolism:

relevanttohealth?

HenrikeM.Hamer, VickyDePreter, KarenWindey,and KristinVerbeke

TranslationalResearchCenterforGastrointestinalDisordersandLeuvenFoodScienceandNutritionResearchCenter,UniversityHospitalGasthuisberg,KatholiekeUniversiteitLeuven,Leuven,Belgium

Correspondingauthor.

Addressforreprintrequestsandothercorrespondence:

K.Verbeke,TARGID,Univ.HospitalLeuven,Herestraat49,3000Leuven,Belgium（e-mail:

 kristin.verbeke@med.kuleuven.be）.

ReceivedFebruary7,2011;AcceptedOctober16,2011.

Copyright ©2012theAmericanPhysiologicalSociety

Abstract

Withtheuseofmoleculartechniques,numerousstudieshaveevaluatedthecompositionoftheintestinalmicrobiotainhealthanddisease.However,itisofmajorinteresttosupplementthiswithafunctionalanalysisofthemicrobiota.Inthisreview,thedifferentapproachesthathavebeenusedtocharacterizemicrobialmetabolites,yieldinginformationonthefunctionalendproductsofmicrobialmetabolism,havebeensummarized.Toanalyzecolonicmicrobialmetabolites,themostconventionalwayisbyapplicationofahypothesis-driventargetedapproach,throughquantificationofselectedmetabolitesfromcarbohydrate（e.g.,short-chainfattyacids）andproteinfermentation（e.g., p-cresol,phenol,ammonia,orH2S）,secondarybileacids,orcolonicenzymes.Theapplicationofstableisotope-labeledsubstratescanprovideanelegantsolutiontostudythesemetabolicpathwaysinvivo.Ontheotherhand,atop-downapproachcanbefollowedbyapplyingmetabolitefingerprintingtechniquesbasedon 1H-NMRormassspectrometricanalysis.Quantificationofknownmetabolitesandcharacterizationofmetabolitepatternsinurine,breath,plasma,andfecalsamplescanrevealnewpathwaysandgiveinsightintophysiologicalregulatoryprocessesofthecolonicmicrobiota.Inaddition,specificmetabolicprofilescanfunctionasadiagnostictoolfortheidentificationofseveralgastrointestinaldiseases,suchasulcerativecolitisandCrohn'sdisease.Nevertheless,futureresearchwillhavetoevaluatetherelevanceofassociationsbetweenmetabolitesanddifferentdiseasestates.

Keywords:

 fermentation,metabolites,metabolomics,microbiota,short-chainfattyacids

thehumanintestinalmicrobiota complementsourphysiologywithfunctionsthatwehavenothadtodeveloponourown.Infact,theintestinalmicrobiotahaveametaboliccapacitythatiscomparabletothatoftheliver（83）.Thehumancoloncontainsanextremelycomplexanddynamicmicrobialecosystemwithhighdensitiesoflivingbacteriainconcentrationsof1011-1012 cells/gofluminalcontentsbelongingtomorethan1,000differentspecies.Inhealthyadults,80%oftheidentifiedfecalmicrobiotacanbeclassifiedintothreedominantphyla:

Firmicutes,Bacteroidetes,andActinobacteria,butthereissubstantialvariationinthespeciescompositionbetweenindividuals（30, 101）.

Theintestinalmicrobiotaplaysanimportantroleinhumanphysiology.Forexample,theintestinalmicrobiotaisresponsibleforthefurthermetabolismandenergyharvestfromnondigestednutrients,isinvolvedinthesynthesisofvitaminssuchasBandKandmetabolismofpolyphenols,providescolonizationresistancetowardpotentialpathogens,isinvolvedinthemetabolismofbileacids,andstimulatestheimmunefunctionofthehost（74, 83）.

Molecularapproaches,mainlybasedonthe16SribosomalRNAgene,haverevolutionizedthefieldofgutmicrobialecology.Nowadays,theunculturedandcomplexmicrobialcommunitiescanbecharacterizedwithgreatersensitivitybyusinghigh-throughputtechnologies,suchaspyrosequencing（5）andphylogeneticmicroarrays（79）,comparedwithformermolecularfingerprintingmethods,suchasPCR-denaturinggradientgelelectrophoresis（DGGE）.Complementaryquantitativetechnologies,suchasfluorescenceinsituhybridization（FISH）andreal-timequantitativePCR,canbeusedtoconfirmshiftsinparticulargroupsorspecies（113）.

Inrecentyearsanincreasingamountofliteraturehasdemonstratedthatseveraldiseasesarerelatedtoalterationsintheintestinalmicrobiota（knownasdysbiosis）,suchasirritablebowelsyndrome（54）,inflammatoryboweldisease（93）,diabetes（55）,atopicdiseases（76）,cancer（85）,andobesity（7）.Forexample,areductionintheabundanceanddiversityofFirmicutesisfrequentlyassociatedwithinflammatoryboweldiseaseandirritablebowelsyndrome（105, 113）.Thesestudieshavemainlyshownthatdifferencesinthecompositionoftheintestinalmicrobiotaareassociatedwithdisease.

FunctionalCapacityoftheMicrobiota

Thehumanmicrobiotaischaracterizedbyasignificantdegreeoffunctionalredundancy,meaningthatdifferentbacteriacanperformsimilarfunctionsandmetabolizethesamesubstrate（60, 64）.Therefore,notonlythecompositionbutalsothefunctionalcapacityoftheintestinalmicrobiotaishighlyimportantregardingtheclinicalendpoints.

Next-generationpyrosequencingcanbeappliedtofurtherevaluatethefunctionalcapacityofthecolonicmicrobiotabycreatingacatalogofthegeneticpotentialofthemicrobiota.However,ithastoberealizedthatthedetectionofgenesinametagenomiclibrarydoesnotnecessarilymeanthatthesearefunctionallyimportant（113）.Togainbetterinsightintotheactivityandfunctionalityoftheintestinalmicrobiota,othermeta-“omics”approachescanbeappliedthatuseRNA,proteins,andmetabolitesastargets.Inthisreview,wesummarizethedifferentapproachesthathavebeenusedtoquantifyandcharacterizethemetabolitesproducedbythemicrobiota,whichyieldinformationontheactualendproductsofmetabolism.Colonicmicrobialfermentationresultsintheproductionoflargeamountsofdifferentendproducts.Thetypeandamountofthesefermentation-derivedmetaboliteslargelydependonthecompositionofthemicrobiota,transittime,andthesubstratesavailableforfermentation（95）.Someoftheseendproductshavebeenshowntobeprotectivetothecolonicepithelium,andothershaveprovedtobeproinflammatoryorprocarcinogenicmetabolites（3, 48）.Byusingknowledgeofthesespecificmetabolites,ahypothesis-driventargetedapproachcanbeappliedtoevaluatechangesincolonicmetabolismfollowingdietaryinterventionsorduringdifferentdiseasestates,forexample,throughquantificationofselectedmetabolitesfromcarbohydrateandproteinfermentation,secondarybileacids,orcolonicenzymes.Ontheotherhand,atop-downapproachcanbefollowedbyapplyingmetabolitefingerprintingtechniquesbasedon 1H-nuclearmagneticresonance（NMR）ormassspectrometricanalysis.Byfollowingthisapproach,novelmetabolitesandmechanismscanbeidentifiedthatareinvolvedinhealthanddisease.Thisis,however,notaneasytask,sincethesignalsfirsthavetobeidentifiedandtheirmetabolicroleselucidated.

Foranalysesofmetabolitesasendproductsofintestinalmetabolisminhumans,wemainlyrelyonfecalsamplesoronbreath（forexample,hydrogen,methane,andcarbondioxide）,urine,andplasmasamplesduetotherelativeinaccessibilityofthecolontosampleatdifferentlocations（Fig.1）（50）.Inthesehumanstudies,anelegantsolutiontostudymetabolicpathwaysinvivoistheapplicationofstableisotopetracers.

TypesofFermentation

Thecolonicmicrobiotafermentendogenoushost-derivedsubstratessuchasmucus,pancreaticenzymes,andexfoliatedepithelialcells,aswellasdietarycomponentsthatescapedigestionintheuppergastrointestinaltract.Twomaintypesofcolonicmicrobialfermentationcanbedistinguished,includingsaccharolyticfermentationofcarbohydrateandproteolyticfermentationofprotein（Fig.2）.Intheproximalpartofthecolon,mainlysaccharolyticfermentationtakesplace,sincemostmicroorganismspreferentiallyfermentcarbohydratesandswitchtoproteinfermentationwhencarbohydratesourcesaredepleted（75）.Themainproductsofcarbohydratemetabolismareshort-chainfattyacids（SCFA）,mainlyacetate,propionate,andbutyrate,whichhavebeenshowntocontributetocolonichealth.SCFAprovideenergytothecolonicepithelialcells,decreaseluminalpH,andimprovemineralabsorption.Furthermore,butyratehasbeenshowntopossessananti-inflammatoryandanticarcinogenicpotential（46, 63）.Besidestheircontributiontoguthealthandmaintenance,SCFAmayprovidefurtherbenefitsforthesystemicmetabolism.Forexample,ithasbeenshownthatacetateandpropionateaffecthepaticlipidmetabolism.Acetateistheprimarysubstrateforcholesterolsynthesis,whereaspropionatecaninhibitcholesterolsynthesis（27, 110）.SincethedifferentSCFAmayshowdistincteffects,notonlytheamountofSCFAproducedbutalsotheirratioisofimportancewithregardtothesehealtheffects.Furthermore,SCFAstimulateincreasedplasmalevelsofsatietyhormonessuchaspeptideYY（PYY）,leptin,andglucagon-likepeptide-1（GLP-1）andmayattenuateinsulinresistance（1, 34）.TheseeffectsmayoccurduetothefactthatSCFAareligandsforGprotein-coupledreceptors（GPR）41and43,expressedonadipocytes,enteroendocrineL-cells,andimmunecells（88）.Inaddition,recentstudieshavelinkedSCFAactivationofGPR43tothesuppressionofcoloncancer（94, 96）.ProteolyticfermentationalsoleadsnotonlytotheproductionofSCFA（loweramountsthanproducedfromcarbohydrates）andbranched-chainfattyacidsbutalsotopotentiallytoxicmetabolitessuchasphenoliccompounds,sulfur-containingcompounds,amines,andammonia（Fig.2）（48）.Thetoxicityoftheseproteinfermentationmetaboliteshasmainlybeenes