J.Physiol.(1969),204,pp.159-168159With3textfiguresPrintedinGreatBritai - PDF document

J.Physiol.(1969),204,pp.159-168159With3textfiguresPrintedinGreatBritainCOMPOSITIONOFALVEOLARLIQUIDINTHEFOETALLAMBBYT.M.ADAMSON,R.D.H.BOYD,H.S.PLATTANDL.B.STRANGFromtheDepartmentofPaediatrics,UniversityCollegeHospitalMedicalSchool,London,W.C.1(Received24February1969)SUMMARY1.Experimentswereperformedonfoetallambsatgestationsbetween125daysandterm.ThefoetuswasexteriorizedatCaesareansectionwiththeumbilicalcordandplacentalattachmentmaintainedintact.Samplesofliquidfromthealveolarpartsofthelungwerewithdrawnthroughatrachealcannulaandsamplesoflunglymph,plasmaandamnioticliquidwerealsoobtained.Measurementsweremadeoftotalosmolality,concen-trationsofelectrolytesandurea,pHandPco2.TitrationswerecarriedoutwithN/10HalandN/10NaOH.ThewatercontentoftheliquidswasestimatedandconcentrationsexpressedperkgH20.2.Inalveolarliquid[H+],[K+]and[CO-]werehigherand[Ca2+],[phosphates]and[HC03-]werelowerthaninplasmaorlymph.Inamnioticliquidosmolality[Na+],[Cl-]and[Ca2+]werelowerand[phos-phates]higherthaninplasmaorlymph.Alveolarliquid/plasmaratiosof[HCO3-],[Ca2+],[CI-]and[K+]differedfromultrafiltrate/plasmaratiosoftheseions.3.TitrationcurvesdemonstratedaverysmallamountofbufferinginalveolarliquidatitsinvivopHof6-27mostlyduetoHCO3-atanaverageconcentrationof2-8mM/kgH20.4.Itisconcludedthatfoetalalveolarliquidisnotanultrafiltrateofplasmanoramixtureofamnioticliquidandplasmaultrafiltrate,butaspecialmaterialelaboratedbythefoetallung.INTRODUCTIONThelungsoffoetalmammalsincludinghumansarefilledwithliquid(Addison&Howe,1913;Potter&Bohlender,1941;Adams,Moss&Fagan,1963).Inthelambattermthevolumewhichcanbeaspiratedthroughthetracheaisabout25ml./kgbodywt.(Humphreys,Normand,Reynolds&Strang,1967).Sinceligationofthetracheaintherabbit(Jost&Policard, T.M.ADAMSONANDOTHERS1948;Carmel,Friedman&Adams,1965)orcongenitalbronchialatresiainhumans(Potter&Bohlender,1941)leadstooverdistensionofthefoetallungs,itisverylikelythattheliquidisformedinsitu.Alveolarliquidhasalowconcentrationofprotein(0.03g/100ml.;Boston,Humphreys,Normand,Reynolds&Strang,1968)whichsuggeststhatitcouldbeformedbyultrafiltrationfromlungcapillaries.ButAdamsetal.(1963)reportedthattheliquiddiffersstrikinglyfromplasmainitslowtotalCO2contentandpH.Themeasurementsreportedinthispresentpaperweremadetoestablishhowmuchtheconcentrationsofsubstances,andthebufferinginalveolarliquiddifferfromplasmaandfromlunglymph,whichwehavetakentorepresentinterstitialfluid.Wehavealsocomparedalveolarandamnioticliquids.Thesemeasurementssuggestthatalveolarliquidisaspecialmaterialelaboratedbythefoetallung,andneitherasimpleultrafiltratenoramixtureofamnioticliquidandultra-filtrate.METHODSExperimentalprocedureExperimentswereperformedonfortyexteriorizedfoetallambswithgestationalagesbetween115daysandterm(meanweight3-9kg,range1-87-5-7kg),manyofwhichwereusedalsoforotherexperiments.Inthirty-oneexperimentsanaesthesiaintheeweswasinducedwiththiopentoneandmaintainedwithpentobarbitoneaspreviouslyreported(Humphreysetal.1967);butinnineexperimentshalothanewasgiventotheewebyinhalationinsteadofpentobarbitone.ACaesareansectionwasperformed,asampleofamnioticliquidtakenanaerobicallyintoasyringe,andthefoetusdeliveredontoaheatedtablewiththeumbilicalcordandplacentalattach-mentintact.ThefoetuseswereshowntobeingoodconditionbymonitoringP°02P0Co2andpH.incarotidarteryblood,withresultssimilartovaluespreviouslypublished(Humphreysetal.1967).ThethoracicductcomponentoflunglymphwascollectedasdescribedbyHumphreysetal.(1967);andsamplesofheparinizedcarotidarterybloodwerecollectedanaerobically;alveolarliquidwassampledbycannulatingthetrachea,clearingthedeadspacebywithdrawinganddiscardingbetween10and30ml.liquid,andgentlyaspiratingtheremainderanaerobicallyintoasyringe.AnalysisofliquidsAcid-basemeasurements.TotalCO2wasmeasuredinuncentrifugedanaerobicallycollectedsamplesofalveolarliquidbytheNatelsonmodification(1951)oftheVanSlykemethod(1917).ThishadanS.D.of+0-23mM/l.forfiverepeatedmeasure-mentsonthesamesample.ThemuchhigherconcentrationsoftotalCO2inlymph,plasmaandamnioticliquidweremeasured,afterequilibrationwith5%C02,inanAutoanalyser(Technicon)bythemethodgivenintheTechniconHandbook(fileN-21a).(S.D.ofrepeatedmeasurements1mm/1.).[HC03-]wascalculatedbysubtractingdissolvedCO2plusH2C03,estimatedas0-03xPCO2(VanSlyke,Sendroy,Hastings&Neill,1928).MeasurementsofPcolbythemethodofSeveringhaus&Bradley(1958)andofpHusingacapillaryglasselectrodeweremadeinsamplesofalveolarliquid,amnioticliquidandwholebloodcollectedanaerobicallybysyringe;thepHoflymphwasmeasuredinsamplescollectedunderoil.TitrationcurveswereobtainedbytitrationinroomairwithN/10HC1andN/10NaOH.160 ALVEOLARLIQUIDCOMPOSITIONMeasurementofelectrolyteandureaconcentrations.Themeasurementsweremadeonsamplesofalveolarandamnioticliquid,centrifugedat30,000gand00Cfor10min.Oncentrifugationamnioticliquid,paleyellowandfaintlyopalescentoncol-lection,retaineditscolourandsomeopalescence.Alveolarliquid,colourlessbutmoreopalescentoncollection,becamecrystalclearoncentrifugation.Measure-mentswerealsomadeonsixsamplesofuncentrifugedalveolarliquidandshowednoimportantdifferencefromresultsaftercentrifugation.ConcentrationsofNa+,K+Cl-,ureaandphosphates(asP)weremadeusinganAutoanalyser(Technicon)bymethodsgivenintheTechniconHandbook(filesN-21a,N-4bandN-ic)andCa2+bythemethodofHalse(1968).Thecoefficientsofvariationofrepeatedmeasurementswere1%orless.Osmolalitywasmeasuredbyfreezingpointdepression(AdvancedInstrumentsosmometer).ConcentrationsareexpressedperkgH20.Forthispurposeaveragewatercon-centrationsof956g/l.forplasmaand960g/l.forlymphwereobtainedfromtheequationofPeters(1953)relatingserumproteinandH20concentrations.Theseestimatesagreedtolessthan0.2%withmeasurementsmadebyevaporatingtodry-ness.Foralveolarliquidandamnioticliquid,waterconcentrationsof990and986g/l.respectivelywereobtainedbyevaporatingtodryness;thesecomparewithDavson's(1956)valueof990g/l.forc.s.f.whichhasasimilarproteinconcentrationtoalveolarliquid.RESULTSConcentrationof&ubstancesTable1givesameanvaluesforosmolality,electrolyteandureacon-centrationsandforpHandPC02inalveolarliquid,amnioticliquid,lunglymphandplasma.Nodifferencesrelatedtogestationalagewereobserved.Nodifferencesinconcentrationwereobserveddependingonhowmuchliquidwasaspiratedfromthetracheabeforetakingasample;andinoneparticularexperimentinapairofsamples,onetakenafterwithdrawing3ml.andtheotherafter30ml.,[Na+],[K+],[C1-]and[HC03-]differedbylessthan1mM/kgH20andpHby0-02.TheTableincludesvaluesforproteinconcentrationfromBostonetal.(1968)foralveolarliquid,andfromHumphreysetal.(1967)forlunglymphandplasma.Alveolarliquidandplasma.Inalveolarliquid,[protein],[HCO3j-,[Ca2+]and[phosphate]aresignificantlylowerthaninplasmawhile[Cl-],[K+]and[H+]aresignificantlyhigher(Poftheabovedifferences0-001exceptfor[K]whereP0.05).Inplasmathesumofmeasuredanions(excludingphosphates)is25m-equiv/kgH20lessthanthesumofcations;thedifferenceispresumablymadeupbyanionicprotein,organicacidsandphosphates(Gamble,1954).Inalveolarliquidthesumsofanionsandcationsaresimilar(anions2-6m-equiv/kgH20greaterthancations).ThusTable1probablyincludesallthequantitativelyimportantanionsinalveolarliquid.Figure1comparesultrafiltrateofplasma/plasmaratios(Rufp)givenbyDavspn(1956)withalveolarliquid/plasmaratios(Raiip)frommeasure-mentsonsimultaneouslycollectedpairsofsamplesfromsevenanimals.6Phy.204161 T.M.ADAMSONANDOTHERS*rqCo0o00-to+-P-0oqeq1o�C0-4coCM-aqco1r01-*-COtZ.CfC4E0r-t10-III5rto4*-d_-aqeN=aq0M-=�(=)cs0COCOo00o***...Cor=COrOtoot°q0COOC4--II0--IMMCoccl_so~-ccVLVtoICO-1INtC;oaq*-O-M_1I-(Mr-°:V_?_^-~OX.o-Xr-no0COc0)10COO1:HIOCOO+C)Mo1Co0+0C01:..6M:.CCO10Zo0-0O-0C-boM_=t-Mo_o0_~~~~~~~~~~~C)0o:3jolcerg.-�coE1621-:.516�,o--1-CD-0OICo009d0.C).~oCo.4000Cob.,0.0Hc;e�aOW.-oc)�*o3CO(MP-ZC)ao0It00oo506Co0A0*0S-32 ALVEOLARLIQUIDCOMPOSITIONTheRai/pvaluesfor[HC03-]and[Ca2+]aresubstantiallylower,andfor[C1-]and[K+]arehigherthanthecorrespondingRuvpvalues,andfromtheS.E.SgivenbyDavson(1956)thedifferencesappeartobesignificant.Lymph.Thereisalowerproteinconcentrationinlymphthaninplasma.Therewerenosignificantdifferencesbetweenlymphandplasmaintheconcentrationsoftheothersubstancesmeasuredinpairedsamples.1-6r-1-41-1-21-1-0I-0-81-061-0-41--0-21-.0F'NaOCastcI-....A...�e:.:�.:wen.�:::::of.i::.:.::.In,.'.-:'::''A:,.,.,',','He.|.-....-.-.-',...':,_,:,,:'m-:'::':'�er^Z.:,:,:.:.:-'1HC03-UreaOsm.Fig.1.Meanultrafiltrate/plasma(Rflp,filledcolumns)andalveolarliquid/plasma(Raiip,opencolumns)ratiosofconcentration(mM/kgH20)with+s.E.shownwhereavailableasverticalbar.RudpratiosfromDavson(1956);Raspratiosfromsimultaneouslycollectedpairsofsamplesinsevenfoetallambs.Amnioticliquid.Inamnioticliquid[Na+],[Cl-]andosmolalityarelower(P0-001)and[HC03-],[Ca2+],[phosphate]andpHhigher(P0-001)thaninalveolarliquid.TitrationcurvesFigure2showstypicaltitrationcurvesbetweenpH4andpH10foralveolarliquid,amnioticliquid,lymph,waterandasolutionofNaHCO3(4mm/l.).Theslopeofthetitrationcurveforalveolarliquidismuchlesssteepthanforlymphoramnioticliquidandindicatesasmallamountof6-2163T T.M.ADAMSONANDOTHERSbufferinginthisliquid.TheactualpositionofthetitrationcurveforlungliquidontheordinatedependedonthePco,atthestartofthetitration,whichwasnotcontrolled,butdifferencesinPco,donotaffecttheslopesofthecurve.Figure3givesbuffervalues(mm[H+]perlitreandunitpHchange)obtainedfromtheslopesofthecurvesinFig.2(VanSlyke,1922)..0-04_°0*03_e.0ozE:Amniotic0-02_0.010_0D01SIL£Water0*02_-Lymph003_45678910pHFig.2.Titrationcurvesof5ml.alveolarliquid(e),amnioticliquid(L),lunglymph(0),water(A),andNaHCO3solution,4mM/I.(M).Ordinate:mM-HClandmm-NaOHadded.Abscissa:pH.Inasolutionofasinglebuffer,pKvaluesaregivenbypointsofmaximumslope,andatapK,bufferconcentration(mM/l.)x0575=buffervalue(mm[H+]l.-lpH-')(VanSlyke,1922).ThisrelationshipisillustratedinFig.3wherethe4mM/l.solutionofNaHCO3hasabuffervalueof23atitspK.TheNaHCO3solutiongavea164 ALVEOLARLIQUIDCOMPOSITIONstandardbuffercurvewithanexpectedpKat6x37.ThecurveforalveolarliquidissimilartothatforNaHCO3solutionbutdiffersslightlyinshapefromthestandardcurve,indicatingthepresenceofsmallamountsofadditionalbufferinthelowpHrangeandrelativelylargeamountsintheupperpartoftherange.ThebufferingadditionaltobicarbonateinthelowpHrangeislikelytobeduetointermediarymetabolites(e.g.lacticacidandKrebscycleacids)withpKvaluesbelow6,andintheupperpHrange86I0.-1Ii4E0�3210Alveolar_WaterI_#45678910pHFig.3.BuffervaluesobtainedfromtheslopesofcurvesshowninFig.2ofalveolarliquid(0),water(A)andNaHCO3(4mm/l.)(M).Ordinate:buffervaluemmH+.1.pH-'.Abscissa:pH.toaminoacidswithpKvaluesmostlybetween8and12.TheclosenessofthepeakinbuffervalueofalveolarliquidtothepKofaqueousHC03-solutionsuggeststhatmostofthebufferinginalveolarliquidatthispHisduetoitsHC03-content.Themean[HC03-jinalveolarliquidswheretitrationcurveswereperformedwas2-7(±04s.E.)mM/kgH20.AtthepKofHC03-thiswouldgiveameanbuffervalueof1-5(±0.2)mmH+.l.-i.pH-1,whichcompareswithameasuredmeanbuffervalueof`-I--------h---H-1657�_A T.M.ADAMSONANDOTHERS2X2(±0-2)mM.H+.L.-'.pH-'atthepKofHCO-.ThusinalveolarliquidatitsinvivopHthereisasmallamountofbuffering(averagebuffervalue0.7)inadditiontoHCO3-.DISCUSSIONElectrolytecompositionofalveolarliquid.TheresultsgiveninTable1showthatalveolarliquidinthefoetallambhasanelectrolytecompositionwhichisquitedifferentfromeitheramnioticliquidorcarotidarteryplasma,whichinthefoetusislikelytodifferonlyslightlyfrompulmonaryarteryplasmaduetoacontributionwhichhaspassedthroughthelungs.Themixingtogetherofamnioticwithaplasmafiltrateformedinthelungscouldnotaccountforthecompositionofalveolarliquid,because[Ca2+],[phosphate]and[HCO3-]arelowerand[Cl-]and[H+]higherinalveolarliquidthanineitheroftheothertwoliquids.ByexpressingthemeasuredconcentrationsinalveolarliquidperkgH20andcomparingthemwithDavson's(1956)valuesforplasmaultra-filtrate,wehaveshownthatthedifferencesinionicconcentrationsofalveolarliquidfromplasmaarenotsimplyimposedbytheDonnanequili-briumasaresultofthedifferenceinproteinconcentration.Indeeditseemslikelythatalveolarliquidisaspecialmaterialactivelyelaboratedbythefoetallungincontrasttolunglymph,whichhasanelectrolytecompositionsimilartoplasma,andwhichhasbeenshownbyBoyd,Hill,Humphreys,Normand,Reynolds&Strang(1969)tohavethemacro-molecularcompositionexpectedfromthefiltrationofplasmathroughporouscapillarywalls.Thepresentexperimentsprovidenoevidenceastothepartoflunginwhichtheliquidisformed.Sincenovariationsincompositionwerefounddependingonhowmuchliquidwasaspiratedbeforetakingthesample,thewholecellularliningofthelungmustbecapableofmaintainingtheobservedelectrolyteconcentrationdifferencesfromplasma.Plainlythelargestareaacrosswhichtheconcentrationgradientsaremaintainedconsistsofalveolarwalls.Electronmicrographsoflung,suchasthosepublishedbyWeibel(1963),showdistinctcapillaryendothelialandalveolarepithelialcelllayers.Schneeberger-Keeley&Karnowsky(1968)haveshownthat,whilethereareporesorcleftsbetweenpulmonarycapillaryendothelialcells,thejunctionsbetweenalveolarcellsappeartobetight.Thelowproteinconcentrationandspecialelectrolytecompositionofalveolarliquidwouldappeartodependonthepropertiesofthealveolarcelllayer.Acidbasecompositionofalveolarliquid.Thetitrationcurveswereper-formedinairataneffectivelyzeroPCo2,butsincetheliquidwasnotthoroughlyequilibratedwithairbeforestartingthetitration,variable166 ALVEOLARLIQUIDCOMPOSITIONamountsofdissolvedC02wouldbepresentintheliquid,whichwouldaffectthepHatthetimeofstartingthetitrationandhencethepositionofthetitrationcurveontheordinate.ButtheslopeofthecurveatanypH,andhencetheestimateofbuffering,wouldbeunaffectedbythesediffer-encesin[C02],whichwerethereforeconsideredunimportant.ThetitrationcurvesshowthatmostoftheeffectivebufferinginalveolarliquidatitsinvivopHisprovidedbyitssmallconcentrationofHCO3-.ThepHof6-27canbeexplainedbyequilibrationofthispoorlybufferedliquidwithaPCO,of40mmHg.Thelow[HCO3-]couldbedueeithertotheactiveexclusionofthissubstanceortotheactiveadditionofastrongacid,whichwouldlower[HCO3-]byreleasingC02withitsdiffusionintoplasma.Iftheactiveadditionofacidtoalveolarliquidisthereasonforitslow[HCO3-],theacidisprobablyHClasanyotherwouldberepresentedasadeficitinmeasuredanionscomparedwithcations.Thefoetallungisderivedfromforegut(Hamilton,Boyd&Mossman,1962)andcontainscarbonicanhydrase(Berfemstam,1952).ItispossiblethatalveolarliquidissecretedbyaprocesssimilartoHClsecretioninlthestomach,whichhasbeenshowntodependontransferofH+fromH2CO3,andtorequirecarbonicanhydrase(Davies,1948).ThisworkwassupportedbyagrantfromtheMedicalResearchCouncilinlaboratoriesconstructedwithaidofagrantfromtheWellcomeTrust.REFERENCESADAMS,F.H.,Moss,A.J.&FAGAN,L.(1963).Thetrachealfluidinthefetallamb.BiologiaNeonat.5,151-158.ADDISON,W.H.F.&HowE,H.W.(1913).Ontheprenatalandneonatallung.Am.J.Anat.15,199-214.BERFEMSTAM,R.(1952).Carbonicanhydraseinfoetalorgans.Actapaediat.,Stockh.41,310-315.BOSTON,R.W.,HUMPHREYS,P.W.,NORMAND,I.C.S.,REYNOLDS,E.0.R.&STRANG,L.B.(1968).Formationofliquidinthelungsofthefoetallamb.Biologia.Neonat.12,306-35.BOYD,R.D.H.,HILL,J.R.,HUMPHREYS,P.W.,NORMAND,I.C.S.,REYNOLDS,E.0.R.&STRANG,L.B.(1969).Permeabilityoflungcapillariestomacromole-culesinfoetalandnew-bornlambsandsheep.J.Physiol.201,567-588.CARMEL,J.A.,FRIEDMAN,F.&ADAMS,F.H.(1965).Fetaltrachealligationandlungdevelopment.Am.J.Dis.Child.109,452-456.DAVIES,R.A.(1948).Hydrochloricacidproductionbyisolatedgastricmucosa.Biochem.J.42,609-618.DAVSON,H.(1956).PhysiologyoftheOcularandCerebralFluid,p.67.London:Churchill.GAMBLE,J.L.(1954).ChemicalAnatomy,PhysiologyandPathologyofExtracellularFluid,6thedn.,p.5.Cambridge,Mass:Harvard.HALSE,K.(1968).Automatedphotometricestimationofbloodcalcium.LiberationofzincbycalciumfromZnEGTAinthepresenceofazincindicator.InAuto-mationinAnalyticalChemistry.TechniconSymposium1967,vol.II,pp.143-149.NewYork:Medical.167 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