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Algae Source of Docosahexaenoic Acid Increases (N-3) Fatty Acid Status

Algae Source of Docosahexaenoic Acid Increases (N-3) Fatty Acid Status

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dha from vegetarian source
dha from vegetarian source

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HumanandClinicalNutrition
SupplementationwithanAlgaeSourceofDocosahexaenoicAcidIncreases(n-3)FattyAcidStatusandAltersSelectedRiskFactorsforHeartDiseaseinVegetarianSubjects1'2
JULIEA.COHQÃœERANDBRUCEJ.HOLÃœB3
DepartmentofHumanBiologyandNutritionalSciences,UniversityofGuelph,Guelphi,Ontario,CanadaNIG2W1
ABSTRACTThepurposeofthisdouble-blindstudywastoinvestigatetheinfluenceofdietarysupplementationwithanalgaesourceofdocosahexaenoicacid[DMA;22:6(n-3)],devoidofanyeicosapentaenoicacid[EPA;20:5(n-3)],onserum/plateletDHAstatus,theestimatedretroconversionofDHAtoEPA,andriskfactorsforheartdiseaseinvegetariansubjects.Healthyvegetarians(12male,12female)consumedninecapsulesdailyofeitherDHA(1.62g/d)orcornoilfor6wk.ConsumptionofDHAcapsulesincreasedDHAlevelsinserumphospholipidby246%(from2.4to8.3g/100gfattyacids)andinplateletphospholipidby225%(from1.2to3.9g/100gfattyacids).EPAlevelsincreasedinserumphospholipidby117%(from0.57to1.3g/100gfattyacids)andinplateletphospholipidby176%(0.21to0.58g/100gfattyacids)viametabolicretroconversion;theestimatedextentofDHAretroconversiontoEPAwas11.3and12.0%,basedontheserumandplateletanalyses,respectively.Arachidonicacid[AA;20:4(n-6)Jlevelsinserumandplateletphospholipidsdecreasedmoderatelyduringthetrialperiod(DHAgroup)asdidbothdocosa-pentaenoicacids|22:5(n-6)and22:5(n-3)|.AlthoughnosignificantchangeswerefoundinthetotalandLDL-cholesterollevelswithDHAsupplementation,thetotalcholesterohHDL-cholesterolratioshowedamoderatedecreaseovertimeasdidtheLDL-cholesterol:HDL-cholesterolratioandserumtriglycérideconcentrations.DHAsupplementationdidnotalterthevariousthrombogenicfactorsmeasured.Inconclusion,DHAsupplementationmarkedlyenhancedtheDHAstatus(ofserumandplatelets),providedfortheformationofsubstantialEPA,andloweredthetotalandLDL-cholesterohHDL-cholesterolratios.J.Nutr.126:3032-3039,1996.
INDEXINGKEYWORDS:
â
¢humansâ
¢â
¢cholesterol
vegetarianâ
¢DHAsupplementationâ
¢hrombogenicfactors
Incontrasttotheomnivorousdiet,thevegandietisdevoidofthelong-chain(n-3)fattyacids,eicosapentaenoicacid[EPA,420:5(n-3)]anddocosahexaenoicacid[DHA,22:6(n-3)],commonlyfoundinfishandfishoils.AlthoughvegansconsumenoEPAorDHA(Panetal.1993,Sandersetal.1978),vegetariansconsumingeggs(SimopoulosandSalem1992)consumesmallamountsofboth.Thus,inthevegetariandiet,a-linolenicacid[aLNA,18:3(n-3)]isthemajordietary(n-3)fattyacid.TheefficiencyofconversionofthisfattyacidtoDHA,inhealthyadults,viadesaturationandelongation,appearstobe~5%(Emkenetal.1994).Variousstudieshaveconfirmedthatvegans/vegetarianshavelowerserumand/orplateletphospholipidlevelsofDHAthandoomnivores(Reddyetal.1994).DHAisfoundinhighlevelsinthebrainandretina,whereitfunctionsinmentalperformanceandvisualacuity,respectively(reviewedinNeuringeretal.1988).Severalinvestigatorshaveshownthatdietaryfish/fishoilscontainingEPAplusDHAmayhelpreducetheriskfordevelopingcardiovasculardiseases(CVD)(forreviewseeHeroldandKinsella1986).Fishoilscon-
1SupportedbytheHeartandStrokeFoundationofOntario.J.A.C,wasarecipientofaHeartandStrokeFoundationofOntarioresearchfellowship.2Thecostsofpublicationofthisarticleweredefrayedinpartbythepaymentofpagecharges.Thisarticlemustthereforebeherebymarked"advertisement"inaccordancewith18USCsection1734solelytoindicatethisfact.3Towhomcorrespondenceandreprintrequestsshouldbeaddressed.4Abbreviationsused:AA,arachidonicacid;ACD,acidcitratedextrose;CVD,cardiovasculardisease;DHA,docosahexaenoicacid;EPA,eicosapentaenoicacid;aLNA,a-linolenicacid;PPP,platelet-poorplasma;PRP,platelet-richplasma;PUFA,polyunsaturatedfattyacids;TxA2,thromboxaneA2;TxB2;thromboxaneB2.0022-3166/96$3.00©1996AmericanInstituteofNutrition.Manuscriptreceived5June1996.Initialreviewcompleted5July1996.Revisionaccepted22August1996.
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DMAANDHEARTDISEASERISKFACTORS
3033
tainingEPAplusDHAlowerserumtriglycéridelevelsinhumans(forreviewseeHarris1989)andreducebloodplateletreactivity(forreviewseeWeaverandHolub1988).TheselattereffectshaveoftenbeenattributedindirectlytoEPAduetoitspredominanceinmostfishoilsupplements/concentratesstudiedtodate.IsolatedstudieswithDHA-enrichedpreparationscontainingsignificantlevelsofEPAhaveshownareductioninplateletaggregation(vonSchackyandWeber1985)andaloweringofserumtriglycérideconcentrationsinhumanvolunteers(SandersandHinds1992).DHAhasbeenfoundtoinhibithumanplateletreactivityinvitro(GaudetteandHolub1991).AlthoughvegetarianstendtobeatoveralldecreasedriskforCVD(Dwyer1988),duepartlytotheirlowerserumcholesterollevels,theirthrombogenicriskfactorsmaybesignificant(Panetal.1993).ConsideringalsothelowerDHAstatusinvegetarians,itwasofinteresttodeterminetheeffectoforalsupplementationwithaDHA-rich/EPA-freesource(frommicro-algae,non-fishderived)oftriglycérideoil(DHASCOâ¢)inthisgroup.InadditiontoevaluatingthepotentialforDHASCOâ¢toenhancetheDHAandEPA(viaretro-conversion)statusinvegetarians,theinfluenceofdietaryDHAonconventional(serumtotalcholesterol,lipoproteinsandtriglycérides)andthrombogeniccardiovascularriskfactors(plateletaggregation,throm-boxaneproduction,serumviscosity,factorVileandfi-brinogenlevels)wasdetermined.Measuresoftotalcho-lesterol:HDL-cholesterolandLDL-cholesterol:HDL-cholesterolratioswereofparticularinterestbecauserecentreportsindicatethattheyaresuperiorpredictorsofcoronaryheartdiseaseriskcomparedwithtotalcholesterolandLDL-cholesterollevels(Kinosianetal.1994and1995).
SUBJECTSANDMETHODS
Subjectsandexperimentaldesign.Thesubjects
were24healthyvegetarians(12male,12female)selectedfromtheGuelphcommunitywhoreportedhavingnomeat,poultryorfishforaperiodofatleast6mo.Approvalforthisdouble-blindstudywasgrantedbytheHumanEthicsCommitteeoftheUniversityofGuelphandwritteninformedconsentwasobtainedfromeachsubject.The24subjects(aged29.6±1.7y,mean±SE)wererandomlyassigned(12/group)tothetwosupplementationgroups,DHA-supplementedandcontrol(6males,6femaleseach).TheDHA-enrichedencapsulatedtriglycérideoil,DHASCOâ¢(algaederived),andplacebo(cornoil)capsuleswerekindlyprovidedbyDavidKyleofMartekBiosciences,Columbia,MD.Asconfirmedbyquantitativegas-liquidchro-matographicanalyses,theDHASCOâ¢oilcontainedDHAasthemajorfattyacid(39g/100gfattyacids)
VariableHeight,mWeight,kgBMI,2g/m?Protein,%ofnergyFat,%ofnergyCarbohydrate,%ofnergyAlcohol,%ofenergyControl1.7±.067.7±.422.9±.011.8±0.625.4±.758.4±.14.2±1.9DHASCO™1.767.122.910.822.963.60.3
TABLE1
Subjectcharacteristicsandestimateddietaryintakeduringdocosahexaenoicacidorplacebosupplementation!
1Valuesarereportedasmeans±SEM,n=12(height,weight,BMI)orn=10(protein,fat,carbohydrate,andalcoholasapercentageofenergyintake).Nosignificantdifferencesbetweenthegroupswerefoundfortheabovevariables.2BMI,bodymassindex.
followedbymyristicacid(21%),palmiticacid(16%),oleicacid(11%)andlauricacid(8%);noEPAwasdetected.Linoleicacid(53%),oleicacid(21%)andpalmiticacid(10%)predominatedinthevegetableoil(cornoil)placebo.EachDHASCOâ¢capsulecontained0.5goilandwasestimatedtoprovide180mgDHA.TheexperimentalgroupconsumednineDHA-containingcapsulesperday(total1.62gDHA/d)withtheirmeals,andthecontrolgroupconsumedninevegetableoilplacebocapsulesperday.Eachgroupconsumedthecapsulesforaperiodof42dbeginningond0.After42dofcapsuleingestion,bothgroupscompletedawashoutperiodfor21dduringwhichtherewasnosupplementation.Subjectswereweighedoneachvisit(d0,21,42and63)andheightwasmeasuredatentry;therewerenosignificantdifferencesintheseorothercharacteristicsbetweenthegroupsatentry¡Table1).Theamountoffatconsumedinencapsulatedform(4.5g/d)represented<10%ofthedietaryfatintake.Therewerenodifferencesbetweendailyenergyintakes(8372kj/d)orintakesofsaturatedfat,cholesterol,monounsaturatedfatorpolyunsaturatedfatbetweenthetwogroups(datanotshown).Theweightofthesubjectswasnotaffectedthroughoutthesupplementationperiodineithergroup.AlldietaryrecordswereanalyzedbytheCanWestDietAnalysis-PlusprogramwhichincludescomparisonwiththeCanadianRecommendedNutrientIntakes(WestPublishing,St.Paul,MN).Only20/24subjectscompletedthedietaryquestionnaire.Allsubjectscompletedthestudy.Compliancewasmonitoredbydeterminingthefattyacidcompositionofserumandplateletphospholipidat0,6and9wk(3weekspostsup-plementation)andfromacapsulecountattheendofthestudy.
Bloodcollection.Atd0(presupplementation),21
and42(supplementation),and63(washout),bloodwascollectedbyantecubitalvenipuncturefromfastingsubjectsintosiliconizedtubescontainingnoanticoagulant(forserumisolation),ortheanticoagulantsacidcitrate
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3034
CONQUERANDHOLUB
dextrose(ACD)[25g/LNacitrate,20g/Ldextrose,14g/Lcitricacid(allfromFisherChemicals,Nepean,Canada)],orNacitrate(3.2%).Wholebloodwastreatedasfollows:Tubewithoutanticoagulant.Wholebloodwascentrifugedat1250xgfor15mintoobtainserum.Serumwasusedformeasurementofplasmatotal-,HDL-andLDL-cholesterollevels,triglycéridelevels,serumtotalphospholipidfattyacidcontentandserumviscosity.Serumwasstoredat-20°Cuntilallsampleswerecollectedandthawedjustbeforeanalysesoflip-ids.Serumwasstoredatroomtemperatureuntilviscositywasanalyzed(seebelow).
TubescontainingNacitrate.Wholebloodwascen
trifugedat200Xgfor17mintoobtainplatelet-richplasma(PRP)whichwasremoved,andtheremainingbloodwascentrifugedat1250xgfor15mintoobtainplatelet-poorplasma(PPP).ThePRPwasusedinaggregationstudiesandthePPPwasusedforanalysesoffactorVIIandfibrinogenlevels.ThePPPwasstoredbelow-20°CuntilanalysisbyMedChemLaboratories(Scarborough,Ontario).
TubecontainingACD.Washedplateletsuspensions
werepreparedaccordingtothemethodofTurinietal.1994.
Plateletaggregation.Plateletaggregationwasper
formedwithin2hofbloodcollection.PlateletswerecountedinPRPusingaCoulterCounter,modelZM(CoulterElectronics,Burlington,Canada)andadjustedtoafinalconcentrationof2.0-2.5xIO11platelets/LusingautologousPPP.Aliquots(0.5mL)ofadjustedPRPwerepreincubatedfor1mininsiliconizedcuvetteswithstirringat900rpmat37°Cnadual-channel800Baggre-gometer(PaytonInstruments,IonTrace,Scarborough,Canada)beforeadditionoftheaggregatingagent.Collagen(Hormone-Chemie,
¼
nchen,Germany)wasaddedattwolevelswiththefinalconcentrationsof2and10mg/L.Theplateletswereallowedtoaggregatefor2minfollowingtheadditionofagonistandthenthelevelofaggregationwasmeasured(Born1962).
Cholesterolandtriglycéridemeasurement.Total
cholesterolwasmeasuredenzymaticallywithadiagnostictest(SigmaDiagnosticsProcedureNo.352,St.Louis,MO).HDL-cholesterolwasisolatedbyusingadextransulfateandMgionsolutiontoprecipitatetheVLDLandLDLfromtheserumsample.TheHDLfractionwasthenassayedbyanenzymaticassay(SigmaDiagnosticsProcedureNo.352-3).Triglycéridewasmeasuredenzymaticallywithadiagnostictest(SigmaDiagnosticsProcedureNo.339).LDL-cholesterolwascalculatedusingtheformulavalidatedbyFriedewaldetal.(1972).Analysesofallsamplesforeachvolunteerwereperformedinasingleassay.
Totalphospholipidanalysis.Thefattyacidcompo
sitionsoftotalphospholipidfromserumandwashedplateletsuspensionsweredeterminedfollowinglipidextraction,TLC,transmethylationoffattyacidresiduesandgasliquidchromatographybyproceduressimilartothosepreviouslydescribed(DonadÃ-oetal.1994).
Fibrinogen,factorVileandserumviscosity.Fibrin
ogen,factorVileandserumrelativeviscositywereanalyzedbyMed-ChemLaboratoriesLTD.Serumrelativeviscositywasdeterminedonserum(maintainedatroomtemperature)within16hofbloodwithdrawal.Therelativeviscosityistheflowtimeofspecimeninseconds/flowtimeofwaterinsecondsinanOswaldViscosimetertube(Gradwohl1970).Theconcentrationoffibrinogeninplasmasampleswasdeterminedbyadiagnostictestcomparingthrombintimevaluesofdilutedplasmasamples(dilutedwithOrthoQ.F.A.Buffer,OrthoDiagnosticSystems,JohnsonandJohnson,Raritan,NJ),towhichOrthoQ.F.A.Thrombinwasadded,toareferencecurve(Clauss1957).Thelogofthethrombintimevalueisinverselyproportionaltothelogofthefibrinogenconcentration.TodeterminefactorVilelevels,prothrombintimewasdeterminedusingThromborelS(BehringDiagnostics,Westwood,MA)inconjunctionwithplasmadeficientinfactorVile(Quick1935).
Thromboxaneproduction.AliquotsofPRP(0.5
mL)werestimulatedwithcollagen(2mg/L)for2minandreactionsstoppedbytheadditionof250¿¿mol/L(finalconcentration)indomethacin(Sigma)and4.5g/LNaCl(finalconcentration)andwereimmediatelychilledonice.Sampleswerecentrifugedat2000xgfor15minat0°C.Thesupernatantwasremovedandstoredat-20°CuntilanalysisusingtheThromboxaneB2Biotrackenzymeimmunoassaysystem(AmershamCanada,Oakville,Ontario,Canada).
Statisticalanalysis.Alldataarereportedasmeans
±SEM.Datathatwerenotnormallydistributedweretransformed(logorsquare)beforeanalysistoreachnormality.Whenobservationsweremissing,least-squaredmeanswerecalculatedsothatmeanscouldbecompared.Split-plotdesign,includingtimeandtreatmentasfactors,wasusedinallanalysesbeforeexaminationofspecificdifferencesbyFisher'sprotectedLSD.Specificdifferencesforpreplannedcomparisonsbetweentreatmentmeansaswellascomparisonbetweendietaryintakesandsubjectcharacteristicswereexaminedbyftest(Kirk1968).StatisticalanalysesweredoneusingtheSASsystem(SASInstitute,Cary,NC).Changeswithingroupsaswellascomparisonsbetweengroupsweremade.
RESULTS
Thefattyacidcompositionsoftheserumtotalphospholipid(controlsandDHA-supplemented)atentry(wk0)andafter3and6wkaregiveninTable2.Thelevelsofthevarious(n-3)polyunsaturatedfattyacids(PUFA)werenotdifferentbetweenthetwogroupsat
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