ACCUMULATION OF FREE FATTY ACIDS FROM SEA WATER BY MARINE INVERTEBRATES

ACCUMULATION OF FREE FATTY ACIDS FROM SEA WATER BY MARINE INVERTEBRATES
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  Reference:Biol.Bull.,142:160—177.February,1972)ACCUMULATIONOFFREEFATTYACIDSFROMSEAWATERBYMARINEINVERTEBRATES JOHNK.TESTERMAN' DepartmentofDevelopmentalandCellBiology,UniversityofCalifornia, Irvine,Irvine,California92664 Numerousinvestigatorshavereporteduptakeofdissolvedfreeaminoacids,glucose,andothersmallorganicmoleculesfromseawaterbymarineinvertebrates (reviewedinStephens,1971).OtherthanthebriefobservationsbySouthwardandSouthward(1970)onuptakeofpalmiticandbutyricacidbythreespeciesofPogonophora,therearenopublishedinvestigationsontheabilityoffree-living marineinvertebratestotakeupfattyacidsfromseawater. Dissolvedfattyacidsareavailableinthemarineenvironment.Slowey,JeffreyandHood(1962)foundfattyacidsof12—18carbonnumberinethylacetateextractsofmilliporefilteredseawatertakenfromtheGulfofMexico.Freefattyacids,longchainhydrocarbons,fattyacidesters,sterols,andmanyothersubstances,includingphosphorusandnitrogencontainingcompoundswhich mayhavebeenphospholipids,wereidentifiedbyJeffrey,Pasby,Stevensonand Hood(1963).Totalconcentrationofethylacetate/petroleumether-extractablesubstancesintheseinshoreGulfsampleswas4—14mg/liter.Jeffrey(1966)hydrolyzedlipidsandidentifiedacidshavinglessthan10to22carbonatoms,withupto6unsaturatedlinkagesinthelongeracids.Totaldissolvedlipidconcentrationsrangedfrom0.5to6.0mg/i,thelowervaluesbeingintheoffshoreGulfsamples.Williams(1965)foundonly1—9@g/lofdissolvedfattyacidsinsamplesfromwithinandjustoutsideofPugetSound.Inviewoftheexistenceoffattyacidsinthemarineenvironment,itseemeddesirabletodetermineifsoft-bodiedmarineinvertebratesotherthanpogonophoranscouldtakeupandassimilatefreefattyacids.WhenpreliminaryexperimentsdemonstratedtheabilityofseveralpolychaetousannelidstoaccumulateC'4intheformofpalmiticandoleicacid,itbecameofparticularinteresttodeterminewhethera“¿ saturable―ptakesystemwasinvolved,inwhichtilevelocityislimitedbytheavailabilityofafinitenumberoftransportsites.Inhibitoryinteractionsamongfattyacidswereinvestigatedtocastlightonthespecificityoftheuptakemechanism.Labelledanimalswereextractedandfractionedintothemajor biochemicalclassesbysolventpartitionandthin-layerchromatographyinorder todetermineifthefreefattyacidsparticipateinenergymetabolismandsyntheticpathways.Accumulationofaradioactivelabelisnotadequateevidencethatnetaccumulationofthecompoundoccurs.Whenexchangediffusionorlabelexchangeareoperating,anorganismmayshowinitialaccumulationoflabeleventhoughthecompoundisleakingoutfasterthanitisbeingtakenup.Thedisappearanceof IPresentaddress:DepartmentofBiology,LomaLindaUniversity,Riverside,California 92505. 160  FATTYACIDUPTAKE161 unlabelledfreefattyacidfromthemediumwasmonitoredinordertocheckforthispossibility. Sinceuptakerateisafunctionofsubstrateconcentration,oncethisfunction hasbeendefined,dataontheconcentrationoffattyacidsinthenaturalenvironnlentoftheorganismcanbeusedtomakeanestimateofthesignificanceofthe uptakeprocessrelativetotilemetabolicrequirementsoftheorganism.Inviewofthevariabilityoffattyacidlevelsreportedintheliteratureandthefailureof thesereportstospecifyhowmuchoftilefattyacidwasinthefreeorcombined form,itwasdeemeddesirabletocollectseawatersamplesfromthehabitatoftheorganismbeingstudiedandassaytileillforfreefattyacids. MATERiALSANt)METHODS Stauronereisrudoiphi(DellaChiaje)(Annelida,Polychaeta,Dorvilleidae)is especiallyplentifulduringtilesummerandfallnlofltilSamongthematsofgreenalgaeandassociateddetritusthatcoatpilingsandlogsfloatinginLosAngeles Harbor.Theanimalsburrowintotilesubstrateandingestit,butdonotmain tampermanentburrows(Reish,1959).Specimenscollectedfromtheharborwerekeptat20°Cingallonjarsofaeratedandcontinuouslyfilteredseawater andfedpelletsofdriedalfalfaadlib.Wormswereusedwithinsixweeksofcollection. Nainereisdendritica(Annelida,Polychaeta,Orbiniidae)aboundsatandbelow thelowtidelineonexposedsandyl)eaches.Organismswerecollectedfromunder rootsofPhyllospadixonemilesouthofCoronaDelMarandkeptat15°Cinalargebucketofaeratedseawaterwithseveralinchesofsandinthebottom.Speci fll@t1Swereusedwithinthreeweeksofcollection. Oleic,palmitic,stearicandlinoleicacidslabelledinthe1-carbonpositionwith C14weresuppliedbyAmersham/Searletogetherwithdataattestingtoatleast 98%radiochemicalpurity.Theclaimed99%purityofuniformlylabelledpalmitic acidfromthesamecompanywasaffirmedbythin-layerchromatography(TLC) onsilicagelinpertoleumether,ethylether,aceticacid:70:30:2(v/v/v).Caproicacid-i-C14wasobtainedfromCaibiochemandtheaceticacid-1,2-C'4wassuppliedbyVolkRadiochemicai. AccumulationofC14-labelledfreefattyacids Glassexperimentalvesselswerecoatedwith“¿ Siliclad―Clay-Adams,Inc.)to minimizefattyacidsstickingtotheglass.Teflonbeakerswerealsousedinsomeexperiments. Artificialseawater(M.B.L.S.\V.)madeupindistilledwaterfromreagentgradesaltsaccordingtothestandardMarineBiologicalLaboratory,WoodsHole, forniula(Cavanaugll,1964)wasmilliporefilteredtwicejustbeforeuse.Animalswereallowedtorenîainatleast24hoursincleanM.B.L.S.W.toemptythegutbeforetransfertotheexperimentalbeakers.SmallwormssuchasStauronereiswereincubatedingroupsof1to2dozensothateachbeakercontainedabout300mgofanimalmaterial.Enoughradioactivefattyacidwasintroducedintoeach 50mlbeakerofM.B.L.S.W.togiveanactivityof20sc/I.Carrierforthe radioactivestockwas0.1mlof85%ethanol.Unlabelledfattyacid(suppliedbyCalbiochem)wasaddedtobriiigthesolutionuptothedesiredconcentration.  162JOHNK.TESTERMAN Afterincubation,rinsingandweighing,thewormsweredroppedintoscintillationvialscontaining10nilofscintillationfluid(6gramsPPO/ltoluene,2partsaddedto1partTriton-Xdetergent)andleft2daysbeforecounting.Ifquenching wastoogreat,theextractwasdiluted10Xbeforecounting.Thismethodex tracted97%oftheradioactivityintheworms.Largerinvertebratesweredigested withformicacid.ThevialswerecountedinaBeckmanCPM-100liquidscintillationspectrometer.EfficiencyofC'4countingofexperimentalsampleswas approximately80%. Assimilationofpalmiticacid-U-C14 SixspecimensofNainereisdendritica,weightinganaverageof110mgeach, werepreincubated24hoursinM.B.L.S.W.containing200mgstreptomycinsulfateperliter.M.B.L.S.W.containing40@&curies/lpalmiticacid-U-C'4(suppliedbyNewEnglandNuclear)wasusedastheincubationmedium. HomogenizationandextractionwereaccordingtothestandardprocedureofRoberts,Abelson,Cowie,BoltonandBritten(1963),whichseparatesthetissue intofractionssolubleincold5%trichloroaceticacid(TCA),85%ethanol,ether,hotTCA,andaresidualproteinprecipitate.Theether-solublefractionwaschromatographedalongwithsuitablestandardsonMerck250silicagel“¿ G―plates(BrinkmannInstruments,Inc.)developedinchloroform,methanol,water:65:25:4(v/v/v).Theradioactivespotswerelocatedbyautoradiography,thenscrapedintoscintillationvialsandcounted. Lossofunlabelledoleicacidfrontthemedium Unlessotherwisenoted,allorganicsolventsusedinthisandthefollowingexperimentswereMallinckrodt“¿ nanograde―glassdistilled,nonvolatilematter <5x10-@%).Allglasswarewascleanedinchromicacid,coatedwithSiliclad, rinsedinglassredistilledwaterandthenwithhexane.TheM.B.L.S.W.wasmadeupfromreagentgradesalts,glassredistilledwater,andwasmilliporefilteredthreetimesbeforeuse. 50specimensofStauronerei@rudoiphiwerecleanedbypreincubatingthemfor 24hoursinM.B.L.S.W.towhichstreptomycinsulfatewasaddedtomakea concentrationof200mg/l.Thewormswerethenrinsedandplacedintooneoftwoteflonbeakers,eachofwhichcontained50mlofM.B.L.S.W.inwhichwasdissolved2.5pniolesoleicacidperliter.Duplicate10mlsamplesweretakenfromthecontrolbeakeratthebeginningoftheexperimentandfrombothbeakersattheendofonehour.Thewatersampleswereacidifiedwith2NHCIandextracted3timeswith2mlportionsofhexane.Theextractswerepooledintoconicalcentrifugetubes,evaporatedtodrynessunderN2,redissolvedin0.1mlbenzeneandquantitativelyspottedontoAnaltech250silicagel“¿ G―thin-layerplateswhichhadbeendividedinto7mmlanes(methodofDowning,1968).Theplatesweredevelopedinhexane,ether,aceticacid:60:40:2(v/v/v),sprayedwith50%H2S04(v/v)andheated20minutesina180°Coventocharthelipidspots.ThefreefattyacidspotswerescannedwithaPhotovoltModel520Adensitometerusinga396m@filter.Theproductofthepeakwidthatheighttimestheheightwasalinearfunctionoftheweightofoleicacidstandard (TLCneutrallipidstandard,SigmaChemicalCo.)forvaluesabove2@grams.  FATTYACIDUPTAKE163 Analysisofdissolvedlipids Seawatersaniphe“¿ A―ascollectedinaglassbottleonSeptember28,1970,1footbelowthesurfacenearBerth 158ofLosAngelesHarbor.The1500mlsaniplewasputoniceandimmediatelybroughtbacktothelaboratoryandfilteredwithsuctionthroughmultiplesheetsofWhatman 1filterpaperandthenthroughWhatmanGF/Aglassfiberpaper.ThefiltratewasbroughttoaboutpH2with2NHC1andextracted3timesbyshakingwith150mlportionsofchloroform.Theextractswerefilteredthroughchloroform-extractedWhatman1-PSphaseseparatingpapertoremovedropletsofwater.Twogramsofchloroform-extractedSephadexG-25/40wereaddedtoremovewater,saltsamidnon-lipidcomitaminantsaccordingtothemethodofWilliamsandMerrihees(1970).Afterevaporationofthesolvemitundervacuuni,thepowderwasquantitativelywashedontoasinteredglassfilterwithalittlechloroformandthelipidmaterial wasehutedwithchloroformii.Thefiltratewasevaporatedtodrynesstinderastream ofN2,dissolvedin0.5nilbenzemieamidstoredumiderN2at—¿ 20°.Asolventblankwaspreparedbytreatimig500mlofchloroforminthemanmierdescribedabove fortheextract. Sample“¿ B―ascollectedonOctober1,1970,atthesamelocationbydrawing @@aterromumiderthealgalandniusselniatonfloatingpilingsusingalargesyrimige.The2litersaniphewastreatedasthefirstsample,exceptthatparticulates wereremovedbycemitrifugatiomiat0°CinSilicladcoatedNalgemiebucketsfor 25minutesat11,600G(cf.RudohfsamidBalmiiat,1952). Thechloroformextractionremovedniorethan99%ofpalmiticacid-C'4dissolvedinfilteredM.B.L.S.W.Glassfiberfiltrationremovesabouthalfoforganicacidsdissolvedinseawater(Quinnand@vieyers,1971).Recoveryofoleicacid-C'4 addedtosample“¿ B―eawaterbeforecemitrifugationwasalsoabout50%.Vir tuallynoactivitywasretainedbytheSephadexpowderafterelutionwith chloroform. MeasuredahiquotsoftheseawaterextractsamidsolventblankwerechromatographedonAmialtechthin-layerplates,charredamidscannedasabove.PeakswereidentifiedandquamitifiedbycomparisonwithSigmaquantitativeneutral lipidstandards. Therestofthesamiiplesweresaponifiedbyheatimigat60—80°for30minutes with1ml10%KOHimi50%methanolandthemion-saponifiablesubstances removedbyhexaneextractiomi.Themixturewasthenacidified,2mlofwaterwereadded,amidthefattyacidsextractedwithhexane.EsterificationwascarriedoutbytheprocedureofMorrisonamidSmith(1964).After0.5mlofbenzeneand2mlof14%BF,inmethanol(AppliedSciencesLaboratories)wereaddedtothefattyacids,themriixturewasheatedat80°Cfor15minutes.2mlof waterwereaddedtothecooledreactionmiiixtureamidthemethylestersextracted 3timeswithhexane.Gas-liquidchroniatography(GLC)ofthefattyacidmethylesterswasdone usingaBarber-ColmamiModel5000imistrumentemployingasingleargonionization detector.Thecolumns,glassU-ttmbes6feetlongand3.5mniininsidediameter,werepackedwitheither10%Apiezon-Lon60/80meshGasChromQor10%ethylenesuccinatemethylsihiconepolymer(EGSS-X)on100/120meshGas ChromP(AppliedSciemicesLaboratories,Inc.).TheApiezoncolumnwas  SpeciesFattyacidconc.(pmoles/1)cpm/g tissue cpm/gmediumPer centof activitylost mediumPercentre organism coverynNainereis dendriticapalmitic oleic0. 100.6024.656.43445666Stauronereis rudoiphioleic0.3527.43475Glyceradibranchiatapalmitic0.141.81835Podarkepugettensispalmitic0.0816.22835Lumbrinerisspp.palmitic0.124.6942Cirriformiaspirabranchapalmitic0.124.02349Nereislimnicola*oleic0.686.6486Tubifextubifex**oleic caproic0.690.5010.612.76121015Urechis caupooleic 0.343.15292Amphipholis pugetanapalmitic0.094.41855Strongylocentrotuspur@uratuspalmitic0. 183.61845 164JOHNK.TESTERMAN operatedonatemperatureprogramstartingat175°Candrisingto270°Cattherateof2°C/minute.Argongasflowwas60mi/minuteandtheinjector portanddetectorwereheatedto280°Cand300°C,respectively.TheEGSS-X columnwasoperatedisothermallyat180°Cwithcarriergasflowingat50ml/minuteandinjectoranddetectorat220and235°C,respectively.Undertheseconditionsthelogarithmoftheretentiondistanceisalinearfunctionofthecarbonnumber(James,1960).Chromatographingthesamesampleonbothcolumnsfacilitatesidentificationofthepeaks,astheunsaturatedestersrunahead oftheirsaturatedanaloguesonthenon-polarApiezoncolumn,andbehindthe TABLEI Speciessurveyedforabilitytotakeu@dissolvedfreefattyacids.Unlessotherwisenoted,theorganismswereincubatedfor1hourinM.B.L.S.W.containinga04-labelledfattyattheconcentrationspecified 4Incubatedin0.4 NaCl.4*Incubatedindistilledwaterfor4hour. saturatedestersonthepolarEGSS-Xcolumn.Therelativeweightofeachmethylesterwascalculatedbymultiplyingthepeakheighttimestheretentiondistance(BrandtandLands,1968)ontheEGSS-XcolumnandbycomparisonwithK-101andK-108FAMEstandardmixturessuppliedbyAppliedSciences Laboratories. RESULTS Surveyoffattyaciduptake Anumberofspeciesofmarineworms,thefreshwaterohigochaeteTubifex tubifex,theechiuroidwormUrechi.scaupo,and2echinodermsweretestedfor abilitytotakeuponeormoreC'4-labelledfattyacids.Mostwereabletocon centratethelabelatleastseveralfoldoverthemediumactivity(TableI).Radio activityaccumulatedintheorganismasitwaslostfromthemedium.About10% oftheradioactivityinartificialseawatermediumadheredtothesurfaceoftheglasswareincontrolbeakerscontainingnoorganisms.Therestoftheradio activitycouldbeaccountedforintherinsewaterandmucusthrownoffbysome oftheorganisms.
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