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ChemistryandPhysicsofLipids8919971311399Hydroxy1012octadecadienoica


1IntroductionThegenerationoflipidhydroperoxidesLOOHofunsaturatedfattyacidsisconsideredtobeinvolvedinthedevelopmentofnumerousCorrespondingauthorTel49921552680fax921552671e-mailgerhardspitelleruni-bayre

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Document on Subject : "ChemistryandPhysicsofLipids8919971311399Hydroxy1012octadecadienoica"— Transcript:

1 ChemistryandPhysicsofLipids89(1997)131±
ChemistryandPhysicsofLipids89(1997)131±1399-Hydroxy-10,12-octadecadienoicacid(9-HODE)and13-hydroxy-9,11-octadecadienoicacid(13-HODE):excellentmarkersforlipidperoxidationPeterSpiteller,GerhardSpiteller*LehrstuhlOrganischeChemieIÈtBayreuthÈtsstraûeReceived30April1997;receivedinrevisedform11August1997;accepted20August1997Variousconditionsforconversionof(9)-9-hydroperoxy-10,12-octadecadienoicacid(9-HPODE)and)-13-hydroperoxy-9,11-octadecadienoicacid(13-HPODE)intothecorrespondinghydroxyacids,)-9-hydroxy-10,12-octadecadienoicacid(9-HODE)and(13acid(13-HODE),wereinvestigatedinvitro.9-HODEand13-HODEweresubjectedtolipidperoxidationundervariousconditions:oxidationwascarriedoutinaironly,andinairascorbate,air,air,and.Incontrasttothecorrespondinghydroperoxides(9-HPODEand13-HPODE),9-HODEand13-HODEprovedtobestableinalltheseoxidationexperiments.Unexpectedly,hydroxycompoundsobtainedbyreductionofhydroperoxidesderivedfromarachidonicacidwerenotattackedbyairascorbateorair.Thus,forinstance,(15)-15-hydroxy-5,8,11,13-eicosatetraenoicacid(15-HETE)remainedunchangedinspiteofpossessingthestructuralprerequisitesforattackbyradicals,i.e.aCH-grouplocatedbetweentwodoublebonds.Consequently,metal-inducedairoxidationreactionsofthesesystemsseemtoberestrictedtohydroperoxidesofunsaturatedacids(LOOH)andnottocorrespondinghydroxycompounds(LOH).Thereportedexperimentsexplainwhyhydroxyderivativesofunsaturatedacids,especially9-HODEand13-HODE,areenrichedinnaturallyoccurringlipidperoxidation(LPO)processestoagreaterextentthananyotherLPOproductandwhytheyarenearlyidealmarkersforLPO.ƒ1997ElsevierScienceIrelandLtd.Lipidperoxidation;Linoleicacid;9-HODE;13-HODE;9-HPODE;13-HPODE 1.IntroductionThegenerationoflipidhydroperoxides(LOOH)ofunsaturatedfattyacidsisconsideredtobeinvolvedinthedevelopmentofnumerous*Correspondingauthor.Tel.:49921552680;fax:921552671;e-mail:gerhard.spiteller@uni-bayreuth.de$17.00ƒ1997ElsevierScienceIrelandLtd.Allrightsreserved. ChemistryandPhysicsofLipids89(1997)131±139chronicdiseases,suchasatherosclerosis(Moreletal.,1983,Steinbrecheretal.,1984),rheumatoidarthritis(Wingardetal.,1993),diabetes(Nishi-gakietal.,1981),multiplesclerosis(Mickel,1978)andothernervousdiseases(BraughlerandHall,LOOHisnotstable.Forexample,itiscleavedinthepresenceofFeinaFenton-likereactiontoalkoxy(LO)radicals(Gardner,1989):Otherwise,Femayproducelipidperoxylradicals(LOO)(Esterbaueretal.,1989):radicalsundergonumerousreactions(Mar-nettandWilcox,1995):theyabstracthydrogensfromothermolecules,especiallyfromCHactivatedbytwoadjacentdoublebondstoformthecorrespondinghydroxyacids(LOH)(Frankel,1982,Esterbauer,1982);theyaredecomposedbycleavageofadjacentC±Cbondsbygenerationofaldehydes(Frankel,1985,Esterbaueretal.,1991);theyreactwithadjacentdoublebondsbyformingepoxyradicalswhicharetransformedtoepoxyhydroxyacids(Hamberg,1973,GardnerandJursinic,1981);andtheyundergoalargenumberofsecondaryoxidationreactions.Onlyafewoftheresultingdegradationproductsareknown:e.g.malondialdehyde,4-hydroxynonenal(Esterbaueretal.,1991)and-hydroxyaldehydes(Loidl-StahlhofenandSpiteller,1994,Loidl-Stahlhofenetal.,1994).Researchontheoccurrenceandphysiologicalactivityoflipidperoxidation(LPO)productsinmammalianshaspreviouslybeenalmostexclu-sivelyrestrictedtothosederivedfromarachidonicacid,inspiteofthefa

2 ctthatlinoleicacidismuchmoreabundantthan
ctthatlinoleicacidismuchmoreabundantthanarachidonicacid(e.g.theratiooflinoleicacidarachidonicacidinlow-den-sitylipoprotein(LDL)wasfoundtobeabout7:1)(Esterbaueretal.,1992)andthatLOseemtobenearlyequallyabletoabstracthydro-genatomsfromactivatedCH-groupsoflinoleicacidasfromthoseofarachidonicacid.Thus,forinstance,productsobtainedbyreductionoflinoleicacidhydroperoxidesÐ(9-hydroxy-10,12-octadecadienoicacid(9-HODE)and13-hydroxy-9,11-octadecadienoicacid(13-HODE)Ðweredetectedinlargeamountsinatheroscleroticplaques(Brooksetal.,1970,Harlandetal.,1973,Belkneretal.,1991,KuÈhnetal.,1992)orthearteriesandbloodofratsafterfeedingadietrichincholesterol(WangandPowell,1991).[Note:theexpressions9-HODEand13-HODEareusedifthestereochemistryandcon®gurationareunknown.Thisisthecaseforproductsgeneratedinnon-enzymicLPOprocesses.]Recentlywedetected9-HODEand13-HODEinunusuallyhighamountsinhearttissueaftermyocardialinfarction(Duddaetal.,1996b),inLDLofelderlypeople(JiraandSpiteller,1996)andinLDLofpatientssufferingfromatheroscle-rosisorrheumaticarthritis(Jiraetal.,1997).Thus,oxidationoflinoleicacidseemstobeacommonprocessinmammaliansaftertissuein-jury,causingcelldeath.Thispointedtothepossibilitythathydroxyfattyacids()suchas9-HODEand13-HODEarestabletofurtheroxidation.Apriorithiswasnotexpectedsincetheypossessthesameconju-gatedsystemasthecorrespondinghydroperoxyfattyacids(),namely9-hydroperoxy-10,12-oc-tadecadienoicacid(9-HPODE)and13-hydroper-oxy-9,11-octadecadienoicacid(13-HPODE),respectively.Theseeasilyundergofurtheroxida-tivetransformationreactionsobviouslycausedbyhydrogenabstractionfromC±Hbondsactivatedbytheconjugatedsystem(Scheme1).Inthispaperwereportonoxidationexperi-mentswhichdemonstratethat9-HODEand13-HODEareinfactstableagainstoxidationunderusualLPOconditions.Inaddition,wedemon-stratethat(155,8,11,13-eicosatetraenoicacid(15-HETE)isScheme1.TheconjugatedsystemsofHODE(RH)andHPODE(ROH)arethesame.Hydrogensoncarbonsadja-centtotheconjugatedsystemareexpectedtobeequallypronetoabstractionbyradicals. ChemistryandPhysicsofLipids89(1997)131±139alsostableagainstoxidation,incontrasttoits2.Materialsandmethods(MSTFA)wasobtainedfromMachereyandNagel(DuÈren,Germany).Soybeanlipoxygenase(5.3U),FeO,linoleicacid,arachidonicacidandsodiumascorbatewerepurchasedfromFluka(NeuUlm,Germany).HClandOwereobtainedfromMerck(Darm-stadt).Solventsweredistilledbeforeuse.Gaschromatography(GC)wascarriedoutwithaCarloErbaHRGC5160MegaSeriesgaschromatographequippedwitha¯ameionizationdetectorusingaDB-1fusedsilicagelcapillarycolumn(30m0.32mmi.d.)(J&WScienti®c,Germany),coveredwitha0.1mlayerofliquidphase.Thetemperatureofthedetectorwaskeptat290 C,theinjectortemperaturewas280 C.Injectorvolumewas0.2±0.7lofa1%(msolution.Temperatureprogramme:3minisother-malat80 C,3 Cminfrom80 Cto280 C,then15minisothermalat280 C.Thecarriergaswashydrogenandthesplitratiowas1:30.Peakinte-grationanddatarecordingweredonewithaMerckHitachiChromatointegratorD-2500.Gaschromatographymassspectrometry(GCMS)wasperformedonaFinniganMAT95dou-blefocusingmassspectrometerwithinverseNier-Johnsongeometry,equippedwithanEIionsourceoperatedat70eV.AHewlettPackard5890SeriesIIgaschromatographwithafusedsilicaDB-05glasscapillarycolumn(30mmmi.d.,coveredwitha0.1mlayerofliquidphase;J&WScienti®c,Germany)wasusedforsampleseparati

3 on.Theinjectortemperaturewaskeptat280 C
on.Theinjectortemperaturewaskeptat280 C;injectionvolumeswere0.6±1.5ofa1±2%(mv)solution.Temperaturepro-gramme:3minisothermalat50 C,increasedwithin2minto100 C,then3 Cminuntil300 C,®nally10minisothermalat300 C.Kovatsindicesweredeterminedbyco-injectionofa0.2lsampleofastandardmixtureofsaturatedstraightchainalkanes(C)(Ko-vats,1958).layerchromatographyPreparativethin-layerchromatographywascar-riedoutonglassplates20cm20cmcoveredwitha0.75mmlayerofsilicagel60PF254.Theplateswereprechromatographedwithmethanol;the2cmzoneattheupperbordercontainingimpuritieswasthendiscarded.Beforeusetheplateswereactivatedfor2hatperformanceliquidchromatographyAnalyticalnormalphaseHPLCTheHPLCsystemcomprisedaPumpsystemBeckmanSystemGoldwithprogrammablesol-ventmodule126andaBeckmanSystemGolddiodearraydetector168,withaLKBBromma2210channelrecorder.ThecolumnwasaBischoffNucleosil3m(25cm4.6mmi.d.)column,withaBischoffSpherisorb5mSiNP(2cmmm)precolumn.eNPThiswascarriedoutwithaBeckmanSystemGoldwithprogrammablesolventmodule125,programmabledetectormodule166,aKipp&ZonenBD41recorder,aBischoffUltrasepFS100(6m)column(25cm20mmi.d.)andaBischoffprecolumnUltrasep(2cm20mmi.d.).SynthesisofoctadecadienoicacidSynthesisof13-HPODEwascarriedoutinaslightlymodi®edprocedureasdescribedbyHam-bergandSamuelsson(1967).Brie¯y,1goflinoleicacid(3.57mmol)wassuspendedin600mlof0.1Mboratebuffer(pH9.0)andemulsi®edbyultrasonicvibration.Afterremovaloftheultra-sonicbaththeemulsionwasvigorouslymagneti-callystirredat20 C,and100mgsoybeanlipoxygenase(5.3U)wereadded.Afewminuteslaterthesolutionclari®ed.After1hoxidation ChemistryandPhysicsofLipids89(1997)131±139wasstoppedbyadding2NHCluntilthesolutionbecameslightlycloudy.Thesolutionwasex-tractedthreetimeswithCHCl.TheCHClweredriedbyadditionofNaand®ltered.Thesolventwasthenremovedinvacuumat20 C.Theresiduewasdissolvedin5mlofhexanepropanolsolution(10:1,vv)andslowlypassedthroughasilicagelcartridgeapplyingaslightexcessofpressure.Thecartridgewasrinsedwithanother5mlofsolvent.Aftersolventevapora-tion,0.96mg(86%yield)ofraw13wasobtainedasacolourlessoil.SynthesisofoctadecanoicacidSynthesisof9-HPODEwascarriedoutasdescribedbyGalliard(Matthewetal.,1977).Brie¯y1.00goflinoleicacidwassuspendedin300mlof0.1Mphosphatebuffer(pH5.6)and1mlofTween20.Then1kgoffreshtomatoes,homogenizedin500mlofphosphatebuffer(pH5.6)usingaWaringblender,wereadded.Thesolutionwasvigorouslymagneticallystirredinairfor5h.ThereactionwasterminatedbyadjustingthepHto2±3byadditiono.Theaqueoussolutionwasextractedthreetimeseachwith100mlofdiethylether.Separationofthelayersrequiredcentrifugation(1500rpm,10min).TheetherealsolutionwasdriedwithNaAfter®ltrationthesolventwasremovedinvac-uumat20 C.Yieldwas0.91gofyellowoilwhichstillcontainedabouttwo-thirdsoflinoleicacid.SynthesisofeicosatetraenoicacidThisacidwaspreparedinasimilarwayasdescribedforsynthesisof13-HPODEfollowingthedescriptionsofVanOsetal.(1981)andFunketal.(1976).Insteadof1goflinoleicacid,100mgofarachidonicacidwereemulsi®edwith60mlofboratebufferpH9.0andonly4mglipoxyge-nasewereaddedbystirring.Completionofthereactionwascheckedafterstirringfor1h.Asamplewaswithdrawn,reducedwithP(OCH(seebelow)andtrimethylsilylatedandanalysedbyGC;ifarachidonicacidmethylatewasstillde-tected,another4mgoflipoxygenasewereaddedandthereactionmixturewasstirred

4 foranotherhour.Usuallythereactionwascomp
foranotherhour.Usuallythereactionwascompleteafter2h.Preparationof(9octadecadienoicacid(9-HODEand(13)-13-hydroxy,9,11-octadecadienoicacid(13HODE)wasdonebyreductionof13-HPODEand9-HPODE,respectively,withSnClOinamodi®edprocedureofHamberg(1971).First,0.50g(1.60mmol)of13-HPODE(obtainedbysoybeanlipoxygenaseoxidation)or9(obtainedbytomatolipoxygenase)wasdissolvedinamixtureof100mlofCHCl,200mlofmethanoland80mlofHO.ThepHofthesolutionwasadjustedto3byadditionof2NHCl.Immediatelyafterwards395mg(1.75mmol)ofSnClOwereadded.Thesolutionwasstirredfor1hatroomtemperature.Separationintoanaqueousandanorganiclayerwasachievedbyadditionof100mleachofHOand.Aftercentrifugationthechloroformlayerwasseparated.Theaqueouslayerwasextractedanothertimewith100mlCHCl.ThecombinedchloroformlayersweredriedwithNa®lteredandthenthesolventwasremovedat20 Cinvacuum.Theresidueobtainedafterreductionof9-HPODEcontainedlargeamountsoflinoleicacid.Thiswasremovedbythin-layerchromatography:0.47g(1.59mmol)ofraw9-HODEwasdis-solvedin2.5mlofamixtureofcyclohexane,ethylacetateandaceticacid596:400:4(vv)andappliedto®vethin-layerplates.Theplatesweredevel-opedwiththesolventmixturegivenabove.Imme-diatelyafterdrying,identi®cationof9wasachievedbyirradiationoftheplatewithUVlightof254nm.9-HODEshowed¯uorescenceinabandwithbetween0.33and0.45.Thisbandwasscrapedoff.Thesilicagelwaselutedthreetimeseachwith20mlofdriedethylacetate.After®ltrationthesolventwasremovedat30 Cusingarotorvap.Yield:0.11gof9Separationfromisomericbyproductsof9-HODEand13-HODEwasaccomplishedbypuri®cationwithHPLCinamodi®edprocedurereportedbyWuetal.(1995):100mgof9 ChemistryandPhysicsofLipids89(1997)131±139HODE,prepuri®edbythin-layerchromatogra-phy,or13-HODEobtainedafterreductionof-HPODE,weredissolvedinamixtureof500lofhexaneisopropanol95:5(vv).Thismixturewassubjectedtoseparationonasilicagelcolumn(25cm10mmi.d.)withsphericalbeads(6usingamixtureofhexaneaceticacid(99:1:0.1,vv).Detectionwasachievedbymeasur-ingtheUVabsorbanceat234nm(dienesystem):-HODE:elutionbetween10.8and12.0min.)-HODE:elutionbetween17.6and18.6min.-HODE:elutionbetween22.6and24.2min.)-HODE:elutionbetween25.8and27.4min.-HODEasitsTMSmethylate:RI2309;MS(70eV),(%):382(17,M),311(15),259(2),225(100),155(44),143(23),130(35),73-HODEasitsTMSmethylate:RI2297;MS(70eV),(%):382(25,M),311(100),225(40)155(17),143(23),130(50),73(80).-HETEwasobtainedbyreductionof15HPETE:20mgHPETEweredissolvedin0.5ml(topshapedvial),then50lofP(OCHwereadded.Thesolutionwasshakenfor15minatroomtemperature.Thesolventandexcessofreagentwereblownoffwithnitrogen.Theresiduewasdissolvedin200lofethylacetate;30lofthissolution(containing3mgofHETEpureenoughforuseinoxidationexperiments)weretransferredtoa250mlroundbottom¯askforfurtherreaction.AutoxidationexperimentswithFirst,15mg(51mol)9-HODEand13HODEwereemulsi®edin22mlofphosphatebuffer(pH7.4)and45mlof0.2MKClsolutionwereadded.Autoxidationwasstartedbyadditionof:(1)15mg(85mol)ofsodiumascorbateand0.6ml(1.0mM)ofFeSOsolution;(2)15mg(53mol)ofFeSOO;(3)15mg(53mol)ofOand100l(50mol)of3%Hsolution;(4)15mg(53mol)ofFeClAliquotsof1mlwereremovedafter0,1,12and24h,2and5days,and2weeks.Oxidationwasstoppedbytheadditionof10mlofP(OMe)Afteradditionof0.5mlofCHCl,thesolutionwasshakenatroomtemperaturefor15minandthenextractedthreetimeswith0.5mlofCHClTheCHCllayerwasseparatedan

5 ddriedwith.Thenthesolventwasremovedunder
ddriedwith.Thenthesolventwasremovedundera¯owofnitrogenatroomtemperature.Theresiduewassubjectedtomethylationandtrimethylsilyla-tionandinvestigatedbyGCandGCMSasde-scribedbelow.Autoxidationof15-HETEwasperformedinasimilarmanner,exceptthatinsteadof15mgonly3mgwereused;theamountsoftheotherreagentswereequallyreduced.Esteri®cationwithdiazomethaneThereducedaliquots(0.1±0.2mgsample),dis-solvedin10lofethylacetate,weretransformedtoatopshapedvialand200lofetherealdiazomethanesolutionwereadded;after10min,excessdiazomethaneandsolventwasblownoffundera¯owofnitrogen.MSTFA(10l)wasaddedtothemethylatedsampleandshakeninathermomixerfor60minat40 C.Compoundidenti®cationA0.2±1.5lvolumeoftheMSTFAsolutionobtainedaftermethylationandtrimethylsilylationwassubjectedtoGCandGCMS.MassspectrawererecordedfromallGCpeaks.3.ResultsSincehydroperoxidesinthepresenceoftracesofFereadilyundergofurtheroxidationtoproductswhichmightalsobegeneratedfromthecorrespondinghydroxyacids(seeScheme1),per-fectsamplepuri®cationwasobligatorytostudyoxidationofthelatter.-HPODEwaspreparedusingsoybeanlipoxygenasewhichdevelopsitshighestactivityat ChemistryandPhysicsofLipids89(1997)131±139pH9.0(HambergandSamuelsson,1967).Linoleicacidisoflowsolubilityinboratebuffer.Thereforeanemulsionoflinoleicacidwaspre-paredapplyingvigorousstirringunderultrasoni®-cation.Afteradditionofsoybeanlipoxygenasethesolutionclari®ed;thusobviouslylinoleicacidwasboundtolipoxygenase.Itturnedouttobeunnecessarytoblowpureoxygenthroughthesolution,asrecommendedearlier(Hamberg,1983,Dolevetal.,1967),asthisdidnotincreasetheyieldof13-HPODE.Theoxidationwasterminatedbyadditionof2NHCl.HCladditionmustbedonecarefullyuntilslightcloudsbecomevisible(pH4.5).Ifmoreacidisadded,13-HPODEmaydecompose.Inaddition,thevoluminousprecipitatedenzymeisthentrans-ferredbyfollowingextractionwithCHCltheorganiclayer.Excessenzymewasremovedbycartrige®ltration.Thesynthesisof15-HETEwasperformedanalogoustothatof13-HPODEstartingwitharachidonicacidandsoybeanlipoxygenase.Theproductwasonlyobtainedinsatisfactoryyieldiftheamountofsoybeanlipoxygenasewaskeptlow.Theprogressofthereactionwascontrolledbysamplewithdrawalandproductanalysis.Ifafter2hofreactiontimestartingmaterialwasstillpresent,asecondportionofsoybeanlipoxy-genasewasaddedandafteranothecontrolwasperformed.Whenthereactionwasdonewiththesameorhigheramountsofenzymecomparedtoarachidonicacid,14,15-epoxy-13-hy-droxy-5,8,11-eicosatrienoicacidwasobtainedasthemainproduct,indicatedbyMS.-HPODEwaspreparedbyatomatolipoxy-genaseinboratebufferatpH5.6obtainedbyhomogenationoftomatoes.Tomatohomogenateisacidic,thereforethepHmustbeadjusteddur-ingthereactionbycarefuladditionof1NNaOHifnecessary.Unfortunately,onlyalimitedamountoflinoleicacidwasconvertedto9Hydroperoxidesreadilyundergoisomerization(HambergandSamuelsson,1967,Chanetal.,1979,Frankel,1982),evenwhenstoredinsolu-tionat0 C.DecompositionisprovokedinthepresenceoftracesofacidwhichisnecessarytoexcludepeakbroadeningduringHPLCseparation(Wuetal.,1995).Wethereforedecidedtoreducetherawhydroperoxideswithoutfurtherpuri®ca-tion,sincethehydroxyderivativesproducedarestabletoisomerization.EitherNaBH(HambergandSamuelsson,1965,Graveland,1970),SnCl(Hamberg,1971),(Porteretal.,1980,Neffetal.,1981),orP(CH(Shulman,1977)isrecom-mendedforreductionofhydroperoxidestothec

6 orrespondingalcohols:NaBHreducesnotonlyh
orrespondingalcohols:NaBHreducesnotonlyhydroperoxides,butalsoattacksoxocompounds.SincehydroxyoxoacidsaregeneratedinthecourseoffurtheroxidationofLOOH(Loidl-Stahlhofenetal.,1994),NaBHwasnotconsid-eredasareducingagent.ReductionwithSnClshouldbecarriedoutinaqueousacidicmediaatpH3±4toensuresolu-tionofSnCl.Incontrasttopreviousreportedreductions(Hamberg,1971),whereSnClinanethanolicemulsionwasapplied,weusedahomo-geneoussolutionforthispurpose.Hydroperoxyfattyacidsarealmostinsolubleinwater,thereforethereductionwasdoneinamixtureofchloro-form,methanolandwater(BlighandDyer,1959).InthissinglephasesolutionbothSnClandfattyacidarewellsoluble.Thereactionwasterminatedafterafewminutes.Separationofreactionprod-uctswasachievedbyaddingchloroformandwa-ter,formingtwolayers.Thisreductionproceedednearlycompletelyandwithoutanyization.Thereforethismethodwasusedforre-ductionofsampleamountsinmilligramscale.turnedoutnottobesuitableforreduc-tionoffattyacidhydroperoxides,sinceP(Ph)andP(Ph)O(producedasoxidationproductafterreductionofLOOH)aresolubleinorganicsol-ventsandthereforeP(Ph)Oisextractedwithorganicsolvents.TheGCretentiontimesof)Oarenearlyidenticalwiththoseofthemainoxidationproductsof9-HPODEand13-HPODE,causingseverepeakoverlapping.Incontrast,P(OCHwasvolatileenoughforremovalinthecourseofsolventevaporation.ThereductionwasachievedatpH7.4,avoidingthedangerofacid-induceddecompositionofLOOH,thereactionwascompletewithinminutes,butstillisomerizationwasobserved.Neverthe-less,thismethodwasalwaysappliedforreduction ChemistryandPhysicsofLipids89(1997)131±139inminiscale.ReductionwithP(CHwasalsochecked,butprovedlesssuf®cientthanthatwithduetothehighvolatilityofthereagent.Samplesof9-HODEhadtobepuri®edbythin-layerchromatographytoremovethelargeamountsoflinoleicacidbyuseofasolventmixtureconsistingofcyclohexane,ethylacetateandtracesofaceticacid.Puri®cationofallhy-droxyderivativeswasachievedwiththesamesolventmixture(Wuetal.,1995).Theairoxidationexperimentswith9and13-HODEwerecarriedoutbyadditionofamountsofironionsgreatlysurpassingtheFeconcentrationinbiologicalsamples.Conse-quently,wededucethatthemuchsmallerironionamountsinbiologicalmaterialarenotabletoinduceoxidationof9-HODEand13In®rstexperiments,9-HODEand13aswellas15-HETEweretreatedinairatpH7.4androomtemperaturewithFeascorbate,thususingtypicaloxidationconditionsappliedalsoformodeloxidationofunsaturatedacids(ThieleandHuff,1964,Yamagataetal.,1983,Esterbaueretal.,1986).Evenifthereactiontemperaturewasincreasedto45±50 C,andthereactiontimewasextendedto2weeks,oxidationproductswereonlyobservedintracesbyGCMS,and95%of-HODEand13-HODEwererecoveredun-changed.Undersimilaroxidationconditions15HETEalsoremainedunattacked.Sincepreliminaryexperimentswith9and13-HPODErevealedthatadditionofequimolaramountsofFeinducesoxidationreactionsmuchfasterthanFeascorbate,9HODE,13-HODEand15-HETEwerealsosubjectedtooxidationinairwithFeAlsointhesecasesoxidationwasnotobserved.andFeOHradicalsintheFentonreaction.ThereforeaswellasFealsoaddedHtosamplesof9-HODEand-HODE.Evenafter2weeks'reactiontime,GCrevealedonlythepresenceofunchangedstartingmaterial.Also,autoxidationexperimentsof13-HODEwithairusingFeascatalystshowedthatnoreactionoccurred:thestartingmaterialwasrecoveredunchanged.Additionofcausedalsonooxidation.Similarly,theoxidationexperiments

7 with9-HODEandwith-HETEwerenegative,onlyt
with9-HODEandwith-HETEwerenegative,onlythestartingmate-rialwasrecovered.Incontrast,9-HODEand13-HODEarenotstableinanorganicsolvent.Ifasolutionof-HODEinethylacetatewasstoredfor4monthsatroomtemperatureindaylightandair,nearlyallthe13-HODEdisappeared.4.DiscussionHydroperoxidesoflinoleicacid(LOOH)andthecorrespondinghydroxyacids(LOH)possessanidenticalconjugateddienesystem(seeScheme1).ItwasthereforeassumedthatLOOHandLOHareequallypronetooxidation(Spiteller,1996,Duddaetal.,1996a).Thisassumptionwascheckedbysubjecting9-HODEand13-HODEtoairoxidationinthepresenceofcatalyticamountsofFeandascorbate.ThelatterisacommonreagenttoreduceFegeneratedinthecourseofthereaction(ThieleandHuff,1964,Wills,1965).9-HODEand13-HODEprovedtobestableinthissystem.Theyprovedstablealsoevenafterprolongedreactiontime(14days):onlytracesofoxidationproductsweredetected.SimilarresultswereobtainedbyoxidationwithequimolaramountsofFe,whileHPODEsareoxidizednearlyinstantlywiththisreagent.EvenadditionofHtoasolutionof13-HODEandFeOHradicals)wasnotsuccessful:95%ofstartingmaterialwasrecoveredunchanged.AlsoafteradditionofFeonlythestartingmaterialwasre-isolated.Thishighstabilityof9-HODEand13HODEseemstoindicatethateitherthehydro-gensinpositions9and13,respectively(seeScheme1),arenotequallyactivatedasinthecorrespondinghydroperoxides(sinceLOOHread-ilyundergodecomposition)orthattheoxidizingagentisperoxylradicals.Hydroxyacidsderivedbyreductionofcorre-spondinghydroperoxidesofarachidonicacid,e.g.)-15-hydroxy-5,8,11,13-eicosatetraenoicacid(15-HETE),possessacti-vatedCH-groupsinpositions7and10.WeassumedthereforethattheseCH-groups(Scheme2)wouldsufferhydrogenabstractionaseasilyas ChemistryandPhysicsofLipids89(1997)131±139Scheme2.CH-groupsinpositions7and10areactivatedbytwoadjacentdoublebonds.Inspiteofthis,15-HETEisnotattackedbyLO,incontrastto15theactivatedCH-groupsinarachidonicacidorlinoleicacid.Unexpectedlythisassumptionprovednottobetrue.Whenwesubjected15HETEtooxidationwithairascorbateorevenafter2daysnearlynolossofstartingmaterialwasrecognized.Thusweareforcedtoconlcudethatfurtherdegradationoflipidperoxi-dationproductsrequiresthepresenceofahy-droperoxygrouporaperoxylradical.Thehighstabilityof9-HODEand13HODEtofurtheroxidationexplainswhyin-creasedamountsofthesecompoundscomparedtootherLPOproductsaredetectedinbiologicalsamplesafteroxidativestress.Becauselinoleicacidusuallyoccursinbiologicalsamplesinmuchhigheramountsthanarachidonicacidandduetothehighstabilityof9-and13-HODE,thesehydroxyacidsarewellsuitedtoestimateoxidativestressexertedinhumanLDLsamplesandtissue.Belkner,J.,Wiesner,R.,KuÈhn,H.,Lankin,V.Z.,1991.Theoxygenationofcholesterolestersbythereticulocytelipoxy-genase.FEBSLett.279,110±114.Bligh,E.G.,Dyer,W.J.,1959.Arapidmethodoftotallipidextractionandpuri®cation.Can.J.Biochem.Physiol.37,911±917.Braughler,J.M.,Hall,E.D.,1989.Centralnervoussystemtraumaandstroke.I.Biochemicalconsiderationsforoxy-genradicalformationandlipidperoxidation.FreeRadic.Biol.Med.6,289±301.Brooks,C.J.W.,Harland,W.A.,Steel,G.,Gilbert,J.D.,1970.Lipidsofhumanatheroma:isolationofhydroxyoctadeca-dienoicacidsfromadvancedaortallesions.Biochim.Bio-phys.Acta202,563±566.Chan,H.W.-S.,Levett,G.,Matthew,A.,1979.Themecha-nismoftherearrangementoflinoleatehydroperoxides.Chem.Phys.Lipids24,245±256.Dolev,A.,Rohwedder,W.K.

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