Heki Nagao

Izumi Hyonosuke

Kanoe Mikio

Kurakaane Kyuichi

Saito Yoshimasa

Kanazawa University Repository for Academic Resources

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、BIOCHEMICALSTUDYONMUqNOUSPLEURALEFFUSIONINACASEOFALVEOLARCELLCARCINOMAOFTHELUNG叩PartI．AnalyticaldataontheeffusioninarawstateHyonosukelZUMI*',MikioKANOE*1,YoshimasaSAITO*1,NagaoHEKI*1andKyuichiKURAKANE*1Receivedforpublication,June15,1969.ABSTRAm－InacaseofalveolarceUcarcinomaofthelungwithmucinouspleuraleffusion,abiochemicalstudywasconductedtoelucidatethechemicalnatureofthemucin.Carbohydrateanalysisoftheeffusionrevealedanincreasedcontentofprotein-boundhexose,especiallyinaformofso-calledmucOproteinhexose.Ouchterlony'smethoddisclosedthepresenceofserumproteincomponentsinthefluid・Electrophoreticstudycouldnotseparatemucinousprinciplebecauseofthehighviscosityofthefluid・Hyaluronidaseeliminatedhighviscosityofthefluid,andalsohighcontentofhyaluronicacidinthefluidwasobserved.Sometumorshaveactivityofproducingmucin.Butthissu"tancehasbeenrath-errarelyasubjectforchemicalanalysis・AfeWchemicalstudies'-5)carriedoutfromtheviewpointofglycoproteinormucopolysaccharidehavebeenpublishedonbronchialsecretaorpleuraleffusioninpatientswithlungcancer・Gernez-Rieuxandhisassoci-atesanalysedsputafrompatientswithalveolarcellcarcinomaofthelung,atumorwithremarkableactivityofproducingmucin,andtheyconcludedthatthesputacon-tainedsialicacidinhighratioandserumproteincomponents.TheauthmsrecentlyexperiencedanunusualcaseofalveolarceUcarcinomaofthelunginwhichpleuraleffusionwascharacterizedbyextremelyhighviscosityandbyformationofmucinclotindiluteaceticacid.Clinicalandpathologicalfindingswillbepublishedelsewhere､6)Attemptshavebeenmadetoseparatethesubstancecausinghighviscosityandelucidateitschemicalproperties・ThepresentpaperdealswiththeresultsofChemicalanalysisonraweffusionobtainedfromthepatient.De"rtmentofClinicalResearch,CancerResearchlnstitute,KanazawaUniversity,(Head:Prof・KyuichiKURAKANE)泉彪之助，鹿江幹雄，斉藤好正，日置長夫，倉金丘一金沢大学がん研究所臨床部（主任：倉金丘一教授）－97－98 IZUMIetal.Insomeofpulmonaryorpleuraltumors,suchasmesothelioma,afewauthors7-'')observedhighlyviscouspleuraleffusionwhichcontainedhyaluronicacidmhighcon-centration・DetailedstudyonthissubstanceinthepresentcasewillbethethemeoffurtherinvestigationoMATERTALSANDMETHODSMaterials:Eachsampleofpleuralfluidobtainedintravitamwasimmediatelyfrozenandstoredinafreezer,dividedintoaliquotsorasitswhole,untilitwasused.RepeatedfreezingandthawingwaSavoidedbecauseitwasfoundthatvaluesofpro-tein-boundhexcsedecreasedthrough.theSeprccedures.ApProximately51intotalweredrawnoutinquantityof300to500mlateachtapping,ofwhichabout31weresparedandstored・Thickviscousfluidwasfoundintheleftpleuralcavityonautopsyandwasalsofrozenandstored.Beforeanalysis,thawedmaterialwascentrifugedatp4,000rpmfor30minat4Ctoremovecenulardebrisandsubjectedtouse.Methods:I)Roughfatcontent:Thethawedmaterialwasdilutedwithanequalvolumeofphysiclcgicalsaline・Thedilutedfluidwasvigorcuslyshakenin20volumesofchloro-form-methanolmixture(2:1,v/v)andthenletstandingstillovernight･Thesolvent･layerwascollectedandfilteredthroughafilterpaper(ToyofilterpaperNo、51).AfterseveralwashingswithonequartervolumeofdistiUedwatertheextractwasdriedandthentheresiduewasweighed.II)Proteincontent:Estimatedbyarefractometer.III)Nitrogencontent:Estimatedbyamicro-Kjeldahl'smeth".IV)Carbohydratecontent:1)Protein-boundhexcse:PrecipitationprocedureswerecarriedoutbythemethodofWeimerandMoshin.'2'Winzler'sprecipitationprccedure'3)wasappliedforso-CaUedmuccprcteinhexose.Orcinol-sulfuricacidreactionwasusedforcolorime"icdeter皿一nationaccordingtoWinzler.'3)2)Methylpentose:DischeandShettles'cystein-sulfuricacidreaction'4)wasused,adoptinglOminforboilingtime.3)Hexosamine:EIscn-MorganreacticnwasappliedaccordingtoWmzler.'8)4)Sialicacid:Forroughestimation,DPAreaction'3)wasusedwithoutanycoITec-tion.Andfcrmcreprecisevalue,resorcinolreactionofSyennerholm'5)wasused.5)HexUrcnicacid:QuantitativestudywasnotcarriedOut・Forqualitativepurpose,Fishmanreacticnwasused.6)Hyalurcnicacid:EstimatedaccordingtothemetllodofJensen・'7)V)EnZymaticdegradationbyhyaluronidase:Hyaluronidasewasobtainedincom-merciallyavailablepharmaceuticalproductbyTakedaPharmaceuticalCompany<Hal｡d-ase).Incubationwascarriedoutat37Cfor30min.VI)Electrophoresis:1)Paperelectrophoresis:ItwasdoneonTWofilterpaperNo､51inveronalbuffer下可Bi"hemicalstudyonmucinouspleuraleffusion ”ofpH8．6("=0.05).2)Cenuloseacetateelectrophoresis:CenuloseacetatemembranewasuSedinbufferwiththeconstitutionabovedescribed.3)Freeelectrophoresis:UsingaTiseliusapparatus(HitachiHTD1),itwasper-formedatvariouspH'sofbuffersolution.VI1)Immunodiffusion:Ouchterlony'sstandardprocedurewascarriedoutemployingcommerciallyavailableantibodysolution(Behringwerke).REsULTS1)Physicalcharacteristics:Theeffusionwasbrownishyellow,semitranSparentorslightlyturbidfluidwithextremelyhighviScosity.IntheearlierdateoftheclinicalCourse,tappedflUiddidnotflowoutfromtesttubeevenwhenitwasupSidedown・ItdidnotcoagUlatethoughfibrinnetwasformed・Anticoagulantsuchaspotassiumoxalatehadnoinfluenceontheviscosity・Pleuralfluidobtainedatautopsywasmoremucinousandcontaminatedbydestroyedtissuebutbasicallysimilartothepunctate.Thepunctatewasnotcompletelyhomogenous,asitwasnotedthatwhitelesstrans-parentstringysubstancewasratherunevenlydispersed,especiallyinmaterialobtainedatautopsy・Butmostofthefluidwasalsoviscousandstringy.Specificgravityofthefluidcouldnotbedeterminedbyahydrometerbecauseofitshighviscosity.Bypycnometer,itwasl.019atl3C.I1)Chemicaldata:WhendrOppedintodiluteaceticacidsolution,thepunctateformedthread-likewhiteamorphousmass,i、e・mucinclot,whichwasdissolvedoutwhenalkalinesolutionwasfurtheradded.AnalyticaldataareshowninTablel.Electrophoresisfailedtoachievecompleteseparationofmucinoussubstance・Onelectrophoresis,viscoussubstanceremainedattheoriginalpointofrunandwasinter-feredwithmovementofothercomponentS.OnlyfreeelectrophoresiscoulddisclosetheTablel．AnalyticaldataOfraweffusionDryweight(%)Protein(%)Nitrogen(%)Fat(mg/dl)TOtalprotein-boundhexose(mg/dl,Winzler)MucoprOteinhexose(mg/dl,WinZler)Methlpentose(mg/dl)ILx"amine(mg/dl)DPAreactingsubstance(mgﾉdl)Hyaluronicacid(mg/dl)HexuronicacidSerumproteincomponentsValue5．52－34．92501401058~10．18438．8ca.400＋＋100 IZUMIetal.fluidwascomposedoftwoconstituents.mmunodiffusiondisclosedthepresenceofserumproteincomponentsintheeffusion.Whentheeffusionincubatedwithhyaluronidase,itsvis"itydecreasedremarkablyandfinallytothatofcommoneffusion・QuantitativesmdyWasnotcarriedout.Hyaluronicacidinthematerialrecoveredatautopsywasestimatedtobe400to500mg/dl.-DISCUSSIONThepleuraleffusioninthepresentcasewascharacterizedbyextremelyhighviscosity"andbyfOrmatiOnofmUbinblotin.dililteacetiCacid.TiiePlburalfluidwiththesecharacteriricshasrarelyappearedintheliterature.Ashighviscosityinbiologicalmaterialandmucinclotfonnationarecharacteris-ticsofhighmolecularcarbohydratecompoundssuchasglycOproteinorhyaluronicacid,'8)theeffusionwasanalysedtodeterminecarbOhydratecontents.Analyticaldatarevealedhighcontentofprotein-boUndhexose(Tablel)．AstOValueS℃f…carbOhydratecompoundsotherthanheXose,availabledataforcomparisonwereverylimitedintheliterature・Comparativestudyisbeingcontinuedinourlaboratory.ImmunodiffusionstUdydisclosedthepresenceofserumproteincomponentsintheeffusion.Someauthors'9)foundgoodcorrelationbetweenmucoproteinvalueandalpha-2-globulinineffusion・Butinthepresentcase,ratioofmucoproteinhexosetototalprotein-boundhexosevaluewasmuchhigherthanwouldbefoundinanyserumorexudate.Probably,thevaluesofso-calledmuc叩roteinhexoseinthepleuralfluidpre-sentlydescribedincludedthosederivedfromOthersubstancesthanorosomucoid.FurtherdetailedanalyticalstudiesalEnecessarytoClarifythispoint.Exudatewithhighviscosityandhighhyaluronicacidcontenthasbeenreportedonbyseveralauthors.Fromtheevidencethatincubationofthefluidwithhyaluroni-daseresultedinasignificantdecreaseinviscosity,andthathighcontentofhyaluronicacidwasfoundinthepresentcase,itwasassumedthatthemucinousprinciplemightbehyaluronicacid.Detailedstudyonthissubstancewillbepublishedsoon.SUMMARYAnalyticaldataonmucinouspleuraleffusioninarawstatewerepresented.CarbOhydrateanalysisrevealedincreasedcontentofprotein-boundhexose,especiallyinaformofso-calledmucoproteinhexose・Immunodiffusionrevealedthepresenceofserumproteincomponents・Electrophor℃ticalsmdycouldnotseparatemucinousprinciple.Additionofhyaluromdasetothefluideliminated.itshighviscosity,andhyaluronatecontentwashigh．・両’1マ1）2）3）4）5）6）7）8）9）10）1）12）13）14）15）16）17）18）19）Bi"hemicalstudyonmucinouspleuraleffusion 101REFFRENCESWerner,I.:ActaSoc.ed.Upsal.,58,1(1953).Have8,R.etal.:Clin.Chim.Acta,17,463,(1%7).GernezRieux,Ch.etal．:J.Franc・deMed.,29-30,799(1963).GernezRieux,Ch・etal.:J.Franc.Med.Chir・Thor・,1%3．citedbyExcerptaMedica,Sectionl5.17,477(1%4).GernezRieux,Ch.etaL:SelectedPapers(Ro"INetherl・Tuberc.Assoc.)1%9,citedbyExcerptaMedica,Sectionl5、15-16,317(1963).Kurakane,K.etal.:Ann.Rep・Cancerlnst.Kanazawa,3,(1969).Meyer,K・andChaffe,E､:J.Biol.Chem.,133,83(1940).Meyer,K・etal.:Bi"him.Biophys・Acta,21,506(1956).Blix,G.:ActaSoc.Med.Upsal.,56,47(1951).Balaz8,E.A､:FEderationProc.,17,1086(1958).Harrington,3．S.etal.:Brit.J.Exptl・Path.,44,81(1%3).WEimer,H6M.andMoghin,J.R・:Am.Rev.TUberc.,68,594(1953).Wii'zlr,R.J.:MethodsBi"hem・Anal.,2,279(1955).Dische,Z.andShettles,L.B・:J.Biol.Chem.,175,595(1948).SvennerhOlm,L､:Bicchim・Biophys・Acta,24,604(1957).Odin,L､:ActaSoc.Med・Upsal．,64,25(1959).Jengen,CdE.:ActaChim・Scand.,7,603(1953),CitedbyS・Sasaki,MethmsinEnzymeResearch,2,148p.2nded.,(1958),AsakuraShoten,Tokyo(inJapanese).Gottschalk,A・:GlyCoprotein,,1sted.,(1966),Elsevier,.Amsterdam.Zinneman,H､H.etal.:Am・Rev,Tnmberc.,76,247(1957).

Biochemical study on mucinous pleural effusion in a case of alveolar cell carcinoma of the lung: Part I. Analytical data on the effusion in a raw state

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Biochemical study on mucinous pleural effusion in a case of alveolar cell carcinoma of the lung: Part I. Analytical data on the effusion in a raw state

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