Heki Nagao

Izumi Hyonosuke

Kanoe Mikio

Kurakane Kyuichi

Saito Yoshimasa

Kanazawa University Repository for Academic Resources

Ann･Rep･Cancerlnst.Kanazawa,3,102-108(1％9)．BIOCHEMICALSTUDYONMUCINOUSPLEURALEFFUSIONINACASEOFALVEOLARCELLCARCINOMAOFTHELUNGPartll・TrialsforpurificationofglycoproteinHyonosukeIZUMI,MikioKANOEnYOshimasaSAITO,NagaoHEKI,andKyuichiKURAKANEReceivedforpublication,Junel5,1%9.Absmact-AttemptsweremadetoseparateandpUrifymucinousprin-ciplefromthemucmouspleuraleffusionObtainedinacaseofalveOlarcencarcinomaofthelung.Threedifferentchemicalmethodswereappliedforthispurpose・ChemicalanalysiscarriedoutfromtheviewpointofglycoproteindisclosedthatthecontentofhexOsaminewas.thehighestofcarbohydrateconstituentsofthepreparations,andthatsialicacidorfUcosecouldnotbeestimatedathighratio・Thepreparationsprovedtobeimpureandcontaminatedbyserumproteincomponents・Inconclusion,itseeemsleastprobablethatepithelialmucinisamucinousprinciplemtheeffUsion.INTRODUCTIONMucinouspleuraleffusionhasbeenveryrarelyexperiencedbyclinicians・Onlyafewcasesappearedintheliterature・Theauthorshaveencounteredthisunusualmani-festationinacaseofalveolarcellcarcinomaofthelung・Studieswereconductedtoelucidatethechemicalcharacteristicsofthemucinobtainedinthiscase.Inthepreviouspaper'),theauthorspresentedanalyticaldataontherawmaterial.Theresultswerecharacterizedbyincreasedcontentofprotein-boundhexose.Butthehighviscosityandmucinclotformationofthepleuralfluidcouldnotbeexplainedsolelyonthebasisofelevatedprotein-boundhexose.Cellsofalveolarcellcar℃inomaproducemucin,andtherefore,theauthorsassumedthatthemucinhereinvestigatedmightbelongtoepithelialmucinwhichofferedtheclosestanalogywiththeglycopro-teinfoundinbronchialsecretafrompatientswithcommonrespiratorydiseases・Inourendeavorstoseparateandpurifythesubstancecausinghighviscosityandformingmucinclot,asafirststepofinvestigationanattemptwasmadetoseparateglycopro-teinfractionpossessingthechemicalpropertiesOfepithelialmucinfromthepunctate.ThepresentpaperisconcernedwiththeresultsofafewseriesofexperimentsfromDepartmentofClinicalResearch,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）－102－侭可BiochemicalstudyOnmucinouspleuraleffuSion 103thisviewpomt.MATERIALSANDMETHODSMateriais:ThepleuralfluidstoredinafrOzenstatewasusedasamaterialafterCellUlarelementshavingbeenremovedbycentrifugationat4,000rpmfor30minat4C・Analyticalprocedureshavebeenpreviouslydescribed・Inaddition,protein-boundhexose2)wasdeterminedbyreactionphenolsulfuricacid3>,anduronicacidWcarbazolreactionaccordingtoDische4)andbyorcinolreactionaccordingtoBrown5>.Methods:Threedifferentmethodswereappliedtotheseparationofglycoprotein.I)Mucinclotfonnationindiluteaceticacid:Afewmillilitersofglacialaceticacidwereaddedtoseverallitersofwaterandthetwoweremixedthoroughly.Thepunctatewasdr"pedintotheaceticacidsolutionundercontinuousstirringMUcinclOtthusformedwaspickedupwithaglassrodandkeptinchilledwater.AceticacidsOlutionwasrenewedeverytimemucinclotwasformed・ThemucinclotwaswashedinChilleddistilledWateranddissolvedinslighUyalkalinesolutionandthen←Tablel．PurificationprocedureformucinousprincipleusingthemethodofmucinclotformationmdiluteaceticacidsolutionEffusion(234g)lDropintoalargeamountofdiluteaceticacidsolutionundermechanicalstirringMucinclotWashinchilleddistilledwaterlDissolveinaminimumamountof5%Na2CO3solutionlDropintoalargeamountofdiluteaceticacidsolutionMucinclotWashinchilleddistilledwaterDisOlvein5%INa2CO3solutionlDropintoalargeamOuntofchilled70%ethanollMucinclotlDissolvein5%Nam3solutionlDropintoalargeamountofchiUed70%ethanolIMucinclotDehydrateinether･ethMnoimixture(1:1,'/v)lDryinvacuo－wher(3.2gWhitepo104 IZUMI,etal.precipitatedbyaddingethanolinexcess.Thefinalstepoftheprocedurewasrepeatedtwice・Theprecipitatewasdriedwithether-ethanolmixture(1:1)．ThreelotswereObtainedbythismethcdwithminormcdificationoftheprccedure(Tablel).2)Ammoniumsulfatefractionation:SaturatedsolutionorpowderedcrystalsofammoniumsulfatewereaddedslowlywithmechanicalstirringtothedilutedeffUsion.Thefinalccncentraticnsofammoniumsulfatewere40%andlOO%ofsaturation,respectively・TherespectiveprecipitatewascoUectedbycentrifugationanddissolvedinaminimalamountofdistinedwater・ThesolutionofmucinoussubstancewasdialyzedagainstrUnningwaterfor48hours,andthento:thisw3Saddedethanolinexcess.OnelotwasobtainedfOreachoftheseconcentrations. ①3)Precipitaticn.withcetyltrimethylammoniumbromide(CTAB):Dilutedpunctatewasdeproteinizedtwicewithcmoroform-amylalCoholmixtureaccordingtoSevag.ThedeproteiniZedfluidwasstirredandtothiswasaddeddropwiseonetwentiethvolumeoflO%CTABsolution.DevelopedmucinclotwasWaShedwellindistilledwaterandthentransferredintoaminimalamountof4NNaClsolution.ThesolutionwasstirredatrccmtemFeraturefcr48hcurstogetahomogenoussolution・Thesolutionwascentrifugedandthesupernatantwasdialyzedovernightagainstrepeatedchangesofdistilledwaterat2-4C・Tothedialyzedsolutionanexcessamountofethanolwasaddedandallowedtostandovernightintherefrigerator・Theprecipitatewascollectedbycentrifugationandlyophilized・Fivepreparationsweremadebythismethod,twoofwhichweresufficientinquantityforsystemicanalysisofcarbohydrate.Analiquotofonepreparation(No.5G)wassubjectedtofurtherfractionationwithethanolinconcentrationsof50to70%ofsaturation，Eachprecipitatewasdissolvedinslightlyalkalinesolutionanddialyzedagainstdistilledwater・Thedialyzedsolutionwaslyo-philized.RESULTSPreparationsobtainedbythemethodl.Thepreparationswerewhitepowder,andalmostinsolubleindistilledwater,butsolubleinslightlyalkalinesolution.Thetotalcarbohydratecontentwaslowerthanexpected,buttheproteinwashigher・TheOuch-terlony'simmunodiffusionrevealedcontaminationbyserumproteincomponentSineve-rypreparation・Ofindividualcarbohydrateconstimentsthecontentofhexosaminewasthehighest,andthatofhexcseanduronicacidfollowedthis・DPAreactingsubstancewasfoundasmuchashexosebutresorcinolreactiongaveremarkablylowervallses.Fuccseuasdetectedinminuteamountscnly・FishmanreacticnwasnegativebutunnegligibleamountofuronicacidwasdetectedbyDische'scarbazolreaction(Table2).Preparationsmadebythemethod2・Theywerereddishbrownfineflake,andcontainedmorecarbohydratethanthosepreparedbythemethodl.Hexosaminewascontainedinthehighestvalue,andhexoseinthesecond・Sialicacidandmethylpent"eweredetectedinhigherproportionthaninthemethodl.Thepreparationsgaveposi-秤BiochemicalstudyonmucinouSpleuraleffusion 105;Table2．Chemicaldataonpre"rationsmadebythemethmofmucinclotformationLotm'mberり,ConstituentNo．3No．1 No.2?????????、???、??? ?? ?Nitrogen(%)Protein(%)Hexose(%),(Winzler)Methylpentose(%)ILxoSamine(%)Sialicacid(%),(DPA)Sialicacid(%),(Svennerholm)Hexuronicacid(FishmanreaCtion)Uronicacid(%),("rbazol)Uronicacid(%),(Orcin)Serumproteincomponents3.941.61．20．073．32．60．13(一）1．60．6（＋）2.442.31．20．032．31．10．09（－）0．90．4（＋）tiveFishmanreaction・Contaminationbyserumproteincomponentswasconfirmedbyimmunmiffusion(Table3).Preparationsmadebythemethod3.Carbohydrateswerecontainedinhigherratiothaninabovetwomethods・Neitherfucosenorsialicacidwasfoundinhighratio.Fishmanreactionwaspositive.Contaminationbyserumproteincomponentswasno-ticedbyimmunmiffusionbutmoredetailedstudycouldnotbeperformedlxcauseofTable3．ChemicaldataonpreparationsmadebyammoniumsulfatefractionationLotConstituent100％sa血ratio、40％Satu重tionProtein(%)Hexose(%)Hexosamine(%)Metmpentose(%)Sialicacid(Z),(DRA)Sialicacid(%),(Svennerholm)Hexuronicacid(Fishmanreaction)Uronicacid(%),("rbazol)Uronicacid(%),(Orcin)SerumproteincOmponents65．54．08．70．52．72．0(＋）3．42．3(＋）87．03．58．70．621．91．7(＋）4．14．5(＋）106 IZUMI,･_etal.Table4．ChemicaldataonpreparationsmadebyCTABprecipitaticnConstituentLotm'mberNo.3GNo.5GNo.記2Protein(%)Hexose(%)Hexosamine(%)Methylpentose(%)Sialicacid(%),(DPA)Sialicacid(%),(Svennerholm)Hexuronicacid(FYshmanreaction)Uronicacid(%),(Carbazol)Serumalbumin14．21127．10．081．0＜1.053.15.619.70．151．11．4（＋）4．1（十）68.02．52．30．180．8(－）paucityofthelot・Alcoholfractionedmaterialshowedlowercarbohydratecontentthantheoriginallot(Table4).DISCUSSIONThepleuralfluidinthecasedescribedwasviscousandsix･ingy,andformedmucinclOtindiluteaceticacid・Substancepossessingthesecharacteristicshavebeengenerallyacceptedasmucin,andarehighmolecularcarbohydratecompounds,suchasglycopro-teinwithhighcontentofsialicacidorfucose(epithelialorglandularmucin)6=9)orhyaluronateproteincomplex(synovialmucin)'0).Becausetheauthorssupposedthatcellsofaiveolarcellcarcinomamighthavebiologicalpropertiessecretingepithelialmucinjustastheepithelialcellsofthebronchiandbronchiolesdo,anattemptasafirststepofinvestigatiOnwasmadetoseparateglycoproteinfractioninwhichsialicacidorfucosewascontainedinhighratio・Buttheviscosity"thematerialwassohighthatitwasimpossibletoseparateandpurifyglycoproteinfractionbyusingelectrophoreticorchromatographicmethods・Therefore,severalchemicalmethodswereusedinthisreport.SincethestudyofVirchow,theseparationofepithelialmucinbymeansofpreci-pitationfromaqueoussolutionwithdiluteaceticacidhasbeenreportedbyseveralauthors'0-'4).Asamethodofseparation,thisclassicalonewasadoptedinthepresentstudy.Asshownintheresults,experimentsrevealedthatmuchproteinWasinvolvedintothemucinousprinciplewhenmucinclotwasformed.Contaminationbyserumproteinwasremarkableandcarbohydratecontentwaslowerthanexpected.Theana-lyticaldataweresimilartothoseofthemucinclotmadefrombovinesubmaxinarygland'3)exceptforlowsialicacidcontent.WithrespecttothecontentofcarbOhydrateconstituents,itwashighintheorder,hexosamine,hexoseanduronicacid・ButFish-man'sreactionwasnegative.『ノBi"hemicalstudyonmucinouspleuraleffusion 107Ammoniumsulfatefractionationhasbeenused'fortheseparationofglycoproteinormucopolysa℃charidesbyseveral!authors'5,'6).Inthe,presentinvestigation,thismeth-,"was･usedtoconfirm､roughlywhethertheglycoproteinrichinsialicacidorfucoseWaSprもsentornot・Thepreparationspreparedbythismethmgavehighercontentofcarbohydratethanthosemadebythemethodl.Highproportionofhexosaminetoprotein-boundhexoseaswellaslowvaluesinsialicacidandmethylpentoseintheselotswerefoundasinthefirstmethod.ItisnoteworthythatthepresenceofuronicacidwasdisclosedbybothFishman'sreactionandcarbazolmethod.CTABisareagenttoprecipitatespecificallyinorganicpolyanion'7,'8).Tsuikiandhisassociates7,'2)separatedbovinesalivarymucinbyusingthisreagent.Theauthorsalsoutilizedthisreagentforthepurificationofglycoproteininthepleuralfluid.Car-bohydrateinthepreparationsobtainedbythismethodshowedthehighestvalueofthethreemethods.Butthepreparationswereapparentlyimpureandimmunodiffusiondis-cl"edthepresenceofserumproteincomponents.Ofindividualcarbohydratecompo-nentscontentofhexosaminewasthehighestandthatofhexosethesecond.Smallamountofsialicacidandmethylpentosewasdetected・Hexuronicacidwaspresentbutremovedbyfurtherpurificationbyethanolfractionation.Allthepreparationspreparedbythethreemethodsabovedescribedprovedtoberichinhexosamineandscantyinsialicacidorfucoseandtobecontaminatedbyserumproteincomponents.Forthoughinotherexperimentswhichwillbepresentedelsewhereremovalof'251-labeledhumanserumalbuminaddedtotheraweffusionwastriedaccordingtothemethodofSevag,itresultedinafailure・Theaffinityofproteinformucinousprinciplewasconsideredtoostrongtoremovetheformerfromthelatterbyusingcommondeproteinizationprocedures・NishizawaandPigman'8)reportedthatbovinesubmaxillarymucinissupposedlyacoacervateofproteincationsandglycoproteinanions,andMeyerandhisassociates'0)foundhyaluronateproteincomplexinsynovialmucin.Inthepresentstageofourexperiments,thesignificanceofserumproteininvolvedinmucinclotwasnotexplained,andonthisproblemfurtherstudieswillbeneeded.Inconclusion,itseemsleastprobablethatepithelialmucinisamucinousprincipleinthiseffusion,becauseneithersialicacidnorfucosecouldnotbeestimatedinhighratio.Severalauthors'5,19-2')foundglycoproteinspecifictoneoplasminascitesorpleu-ralfluidinpatientswithtumors･Apartfromaroleplayedinproducinghighvisco-sity,presenceofabsenceofsuchaglycOproteinwillbesmdiedinfollowingexperi-mentS.SUMMARYSeparationofmucinousprinciplefrommucinouspleuraleffusionwascarriedoutfromtheviewpointofglycoprotein・Threedifferentchemicalmeth"s,thatis,mucinclotformationindiluteaceticacid,ammoniumsulfatefractionation,andprecipitationwithCTAB,wereusedforthispurpose・Obtainedpreparationsprovedtobeimpure~～三108 IZUMI,etal.－andcontaminatedbyserumproteincomponents.Ofcarbohydrateconstituentsofthepreparationsthecontentofhexosaminewasthehighestandthatofhexoseoruronicacidthesecond・Neithersialicacidnorfucosewasfoundinhighratio.Itisleastprobablethatepithelialmucinistheprincipleresponsiblefortheextremelyhighvis-cosityandmucinclotformationofthepleuraleffusion.1）2）3）4）5）6）7）8）9）10）1）12）13）14）15）16）17）18）19）20）21）REFERENCESIzumi,H.etal.:Ann.Rep・Cancerlnst.Kanazawa,3,96(1969).Hodge,J.E・etal.:MethodsinCarbohydrateChemistry,1,888(1962).Anno,K．andSemo,N．:Protein-Nucleicacid-Enzyme,Suppl・MethodsinBiologicalChemis-try,11,13(1968),(inJapanese).Dische,Z.:J.Biol.Chem.,167,189(1947).Brown,A.H.:Arch.Biochem.,11,269(1946).Gottschalk,A.:Glycoproteins,1ed.,(1966),Elsevier,Amsterdam.Tsuiki,S.:KagakunoRyoiki,16,947(1962),(inJapanese).Werner,I.:ActaSoc.Med.Upsal.,58,1(1953).odin,L.:ibid.,64,1(1959).Meyer,K・etal.:J.Biol.Chem.,128,319(1939).TBuiki,S.etal.:Nature,4762,399(1961).Tsuiki,S・etal.:J.Biol・Chem.,236,2172(1961).Nishizawa,K・etal.:Arch.OralBiol.,1,161(1959).Nishizawa,K.etal.:Biochem.J.,75,293(1960).Tsurumi,K、:RJkushimaJ.Med.Sci.,3,31(1956).JeanloZ,R.W・etal.:J.Biol.Chem.,186,495(1950).Scott,J.E.:MethodsBiochem・Anal.,8,145(1960).Scott,J.E.:MethodsinCarbohydrateChem.,5,38(1965).Masamune,H.etal.:TohokuJ.exp・Med.,59,87(1953).Takahashi,S.:Fukushimaj.Med.Sci.,3,43(1956).Nagai,Y・etal.:ibid.,6,257(1959).ilI

Biochemical study on mucinous pleural effusion in a case of alveolar cell carcinoma of the lung: Part II. Traials for purification of glycoprotein

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Biochemical study on mucinous pleural effusion in a case of alveolar cell carcinoma of the lung: Part II. Traials for purification of glycoprotein

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