Amino Acid and Protein Active, Trained the (IAAO) · The amino acid composition in the blood and extracellular and intracellular fluid is commonly known as the free amino acid pool,

Copyright2015JeffreyPacker

AminoAcidMetabolismandProteinRequirementsinActive,Trained

AdultMalesUsingtheIndicatorAminoAcidOxidation(IAAO)Technique

Packer,JeffreyE.MScThesis

DepartmentofExerciseSciencesUniversityofToronto

Supervisor:Dr.DanielMoore

CommitteeMember:Dr.ScottThomasCommitteeMember:Dr.MariusLocke

CommitteeMember:Dr.GlendaCourtney‐Martin

DistributedDate:September4,2015DefenseDate:September23,2015

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Aminoacidmetabolismandproteinrequirementsinactive,trainedadultmalesusingthe

IndicatorAminoAcidOxidationTechnique,MSc2015,JeffreyPacker,Departmentof

ExerciseScience,UniversityofToronto

Abstract:

ThepresentstudyutilizedtheminimallyinvasiveIndicatorAminoAcidOxidation(IAAO)

techniquetoevaluatetheimpactofvariableintensityexerciseonproteinrequirementsin

trained,youngadultmales.Sixtrainedmalespartookin1‐10metabolictrialseach

consistingofavariableintensityexerciseprotocolfollowedby8hourlymealsprovidinga

variableamountofprotein(0.2‐2.6g/kg/d),6g/kgofcarbohydrate,andsufficientenergy.

Proteinwasprovidedascrystallineaminoacidswiththeexceptionoftyrosine

(40mg/kg/d)andphenylalanine(30.5mg/kg/dwith5.46mg/kgover4hasL‐

[13C]phenylalanine).Theestimatedaveragerequirement(EAR)wasdeterminedfromthe

breakpointofthe13C02excretionafterapplicationofbi‐phaselinearregression.Analysis

forthecorrelationbetweenproteinintakeand13CO2excretionrevealedtheEARtobe1.35

g/kg/d(r2=0.64),andthesafeintakeencompassingtheupper95%CItobe1.64g/kg/d.

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TableofContents

TableofContents………………………………………………………………………………………………………......iiiListofAbbreviations…………………………………………………………………………………………….………...vListofFigures&Tables..........................................................................................................................................vChapter1.Introduction......................................................................................................................................1Chapter2.ReviewofLiterature…………………………………………………………….……………………..2 2.1Introduction…………………………………………………………………………………………………..2 2.2MetabolicFatesofProtein………………………………………………………………………………3 2.2.1OverviewofProteinMetabolism 2.2.2ProteinSynthesis 2.2.3ProteinDegradation 2.2.4ProteinOxidation 2.2.5EstimatedAverageRequirement&RecommendedDietaryAllowance 2.3MethodsDeterminingProteinRequirements…………………………………………..............9 2.3.1NitrogenBalance(NBAL) 2.3.2StrengthsofNBAL 2.3.3LimitationsofNBAL 2.3.4OverviewofInfusionTechniques 2.3.5Conclusions

2.4Minimally‐InvasiveIndicatorAminoAcidOxidation(IAAO)Technique………..…15 2.4.1HistoryoftheMinimally‐InvasiveIAAOTechnique 2.4.2MethodologicalApplicationoftheMinimallyInvasiveIAAOTechnique 2.4.3TheMinimally‐InvasiveIAAOTechnique&ProteinRequirements 2.4.4Conclusions

2.5EffectofExerciseonMuscleProteinMetabolism……………………………………...........20 2.5.1GeneralOverview 2.5.2EffectofResistanceExerciseonProteinMetabolism,RDA 2.5.3EffectofEnduranceExerciseonProteinMetabolism,RDA 2.5.4EffectofVariableIntensityExerciseonProteinMetabolism,RDA 2.6Conclusions,GapsintheLiterature………………………………………………………………..32 Chapter3.ResearchProposal......................................................................................................................34 3.1Introduction………………………………………………………………………………………………....34 3.2PhasesofResearch.………………………………………………………………………………………35 3.3ResearchSchematic(ParticipantTimeline)……………………………………………………36 3.4ProjectTimeline…………………………………………………………………………………………...37

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Chapter4.Methodology……………………………………………………………………………………………...38 4.1Introduction…………………………………………………………………………………………………38 4.2Participants&GeneralStudyDesign…………………………………………………………...…39 4.3PhaseI,IntroductorySession………………………………………………………………………...39 4.4PhaseII,BodyCompositionAnalysis&FitnessAssessment…………………………....40 4.5PhaseIII,MetabolicTrials……………………………………………………………………………..41 4.6DataOrganization…………………………………………………………………………………………46 4.7StatisticalAnalysis………………………………………………………………………………………..48Chapter5.Results……………………………………………………………………………………………………….49 5.1SubjectCharacteristics………………………………………………………………………………….49

5.2F13CO2Excretion……………………………………………………………………………………..........505.3PhenylalanineFlux……………………………………………………………………………………..…525.4PhenylalanineOxidation……………………………………………………………………………….53

Chapter6.Discussion………………………………………………………………………………………….……...55 6.1Introduction………………………………………………………………………………………………....55 6.2Results,ComparisontoNBAL&IAAOStudies………………………………………………...56 6.3ResultsExplained,MethodologicalConsiderations…………………………………………57 6.4PhysiologicalConsiderations,&ImplicationsforActiveIndividuals………………..59

6.5RateofAppearanceandPhenylalanineFlux……………………………………………..……636.6Strengths,Limitations,&FutureAvenuesofResearch…………………………………....65

6.7Conclusion…………………………………………………………………………………………………....68References………………………………………………………………………………………….……………………….69

Appendices……………………………………………………………………………….………………………………...831. RawData2. PilotStudy3. IAAOManual

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ListofAbbreviationsBCAA–Branch‐ChainAminoAcidsBCOAD–Branched‐ChainOxo‐AcidDehydrogenaseEnzymeBIA–BioelectricalImpedanceAnalysisDAAO–DirectAminoAcidOxidationDEXA–Dual‐EnergyX‐RayAbsorptiometryEAA–EssentialAminoAcidsEAR–EstimatedAverageRequirementFAO–FoodandAgricultureOrganizationoftheUnitedNationsFFM–Fat‐FreeMassFM–FatMassIAAO–IndicatorAminoAcidOxidation(Technique)IPAQ–InternationalPhysicalActivityQuestionnaireLIST–LoughboroughIntermittentShuttleTestNBAL–NitrogenBalance(Technique)NEAA–Non‐EssentialAminoAcidsPA–PhysicalActivityPAR‐Q+–PhysicalActivityReadinessQuestionnaireRDA–RecommendedDietaryAllowanceREE–RestingEnergyExpenditureWBPS–Whole‐BodyProteinSynthesisWHO–WorldHealthOrganizationListofFiguresFigure1.ProteinTurnover,andAminoAcidFlux.Figure2.TheEARandRDA.Figure3.TrendsinSynthesisandOxidationDuringtheIAAOTechnique.Figure4.ImpactofResistanceExerciseandAminoAcidConsumptionofProteinTurnoverFigure5.ImpactofResistanceExerciseandAminoAcidConsumptiononProteinBalanceFigure6.ThemodifiedLoughboroughIntermittentShuttleTest(LIST)Figure7.EffectofproteinintakeonF13CO2(kg)Figure8.EffectofproteinintakeonF13CO2(FFM)Figure9.PhenylalanineFluxFigure10.Effectofproteinintakeonphenylalanineoxidation(kg)Figure11.Effectofproteinintakeonphenylalanineoxidation(FFM)ListofTablesTable1.SubjectCharacteristics

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Chapter1.Introduction:

Theadequateingestionofdietaryproteinisacriticalfactorinensuringthehealthygrowth

anddevelopmentofleanbodymassinindividualsofallages.Proteinrequirementsarenot

universalacrossdemographicgroupsasdifferencesinage,physicalactivitylevels,andsex

mayresultinchangestooptimalproteinrequirements(FAO,WHO2007).Globalinitiatives

seektoincreasephysicalactivityinindividualsofallagesinordertoenhancetheirgeneral

healthandwell‐being,yetamajorknowledgegapexistsastheimpactofchronicandacute

physicalactivityonproteinrequirementsisnotwellunderstood.Researchsuggeststhat

methodssuchasNitrogenBalance(NBAL)oftenusedtoestimateproteinrequirements

maynotbeaccurate,andcanpotentiallyproduceunderestimatesoftruerequirements

(Huymayunetal.,2007).Itisthereforeessentialtoutilizenovel,alternativetechniquesto

evaluateproteinrequirementsinthepresenceofexerciseinordertoprovideoptimal

nutritionalguidancetoactivepopulations.Thus,thepresentstudyutilizedthegold

standardminimallyinvasiveIAAOtechniquetoevaluatetheeffectsofanacuteboutof

variableintensityexerciseonproteinrequirementsinactive,trainedyoungadultmales.It

washypothesizedthatproteinrequirementswouldbe:1)greaterthanthecurrent

requirementsestablishedonthebasisofNBAL,and;2)greaterthanthoserequirements

previouslydeterminedbytheIAAOtechniqueinnon‐activepopulations.Thisproject

representsacomprehensiveexaminationoftheeffectofvariableintensityexerciseon

proteinrequirementsinactive,trainedyoungadultmales.Itdemonstratesthefeasibilityof

applicationoftheIAAOtechniqueinactiveindividuals,whilealsoprovidingupdated

guidelinesforproteinconsumptioninactivepopulations.

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Chapter2‐ReviewofLiterature:

2.1Introduction

Thesubsequentliteraturereviewwillfirstprovideascientificbackgroundofthemetabolic

fatesofingestedprotein,inadditiontoestablishingthecurrentrecommendeddietary

allowance(RDA)(Section2.2).Anexplanationofthevarioustechniquesoftenusedto

developproteinrequirements,includingNBALandinfusionprotocolswillthenbe

summarized(Section2.3),followedbyadescriptionofthedevelopmentandpracticalityof

theminimally‐invasiveIndicatorAminoAcidOxidation(IAAO)technique(Section2.4).

Lastly,theeffectsofbothenduranceandresistanceexerciseonproteinmetabolismwillbe

provided(Section2.5).Afocusonyoung,adultmaleswillbeemphasizedthroughoutthe

review.Itwashypothesizedthatproteinrequirementswouldbe:1)greaterthanthe

currentrequirementsestablishedonthebasisofNBAL,and;2)greaterthanthose

requirementspreviouslydeterminedbytheIAAOtechniqueinnon‐activepopulations.

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2.2MetabolicFatesofProtein

2.2.1OverviewofProteinMetabolism

Proteinisamacronutrientthatservesmanyregulatoryandstructuralpurposeswithinthe

humanbody,includingbeinganintegralcomponentofenzymes,antibodies,cellreceptors,

hormones,aswellasmuscleandbone.Asingleproteiniscomprisedofacombinationof

aminoacids,allofwhichcontainanaminogroup(‐NH2),acarboxylicacidgroup(‐COOH),

andanR‐groupthatvariesdependingontheaminoacid.Proteinistheonlydietarysource

ofnitrogen,andthereforeitmustbeconsumedinadequatequantitiestoensureoptimal

health.Thereexist20aminoacids,ofwhich9areconsideredessentialaminoacids(EAA)

thatmustbeobtainedthroughone’sdiet(histidine,isoleucine,leucine,lysine,methionine,

phenylalanine,threonine,tryptophan,andvaline).Theremaining11non‐essentialamino

acids(NEAA)canbesynthesizedtovaryingdegreeswithinthehumanbody,andunder

mostconditionsdonotneedtobeconsumedinlargequantities.

Withinthebody,proteinisinacontinuousstateofmetabolicflux,commonlyreferredtoas

proteinturnover,whichischaracterizedbyconcurrentproteinsynthesisanddegradation

(breakdown)(M.Tarnopolsky,2004)(Figure1).Proteinturnovercanbeinfluencedby

variousexternalstimuliincludingexercise,dietarycomposition,andenergyintake.The

contentofwhole‐bodyproteinisbasedonthealgebraicdifferencebetweentherateof

proteinsynthesisfromfreeaminoacidsandtherateofproteindegradationtoaminoacids.

Thus,thefreeaminoacidpoolisconstantlybeingrecycled,asitservesnotonlytoprovide

aminoacidsforproteinsynthesis,butisalsocontinuouslybeingreplenishedbythe

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breakdownofendogenousprotein.Thefollowingsectionswilloutlinethemajorfatesof

protein(synthesis,degradation,andoxidation).

2.2.2ProteinSynthesis

Proteinsynthesisisahighlyregulatedtwo‐stepprocessconsistingoftranscriptionand

translation.Transcriptionoccursinthenucleusofacell,whereasignalinducesthe

expressionofDNAencodingforaspecificproteinorgroupofproteinsbygeneratinga

complementarymRNAtemplate.TranslationthenturnsthemRNAtemplateintoa

functionalproteinthroughthestringingtogetherofseveralaminoacidsviapeptidebond

formation.Forthemostpart,theacuteregulationofproteinsynthesisoccursatthelevelof

mRNAtranslation.

Figure1.Proteinturnoverandaminoacidflux.Intracellularandblood‐basedaminoacidscomprisetheaminoacidpool.Branch‐chainaminoacidscanbepreferentiallytransaminatedandoxidizedwithinthemuscle,whileotheraminoacidsmustbe

transportedtotheliverorkidneystobetransaminatedandoxidized.

AdaptedFrom:Phillips,S.M.(2004).Proteinrequirementsandsupplementationinstrengthsports.Nutrition,20(7‐8),689‐695.

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A‘fed‐state’maybeaccomplishedthroughthedietaryconsumptionofproteinorthrough

theingestionorinfusionofexogenousaminoacids(primarilyEAA)(Burd,Tang,Moore,&

Phillips,2009).Uponoralingestion,proteinisbrokendownintoitsindividualaminoacid

componentsandsmallpeptidefragmentsbythedigestivesystemandabsorbedviathe

smallintestinesandtransportedtotheliverbytheportalvein.Theseaminoacidsand

peptidefragmentsmaybesequesteredwithintheintestinesorliver(collectivelyreferred

toasthesplanchnicbed)foressentialtissuefunction(e.g.constitutiveand/orexport

proteinsynthesis)inaprocesscallsplanchnicextractionpriortobeingreleasedinto

circulationformetabolisminothercentralandperipheraltissues.Exogenousaminoacids

thatareadministeredviaingestionorinfusionarealsoabsorbedintotheblood.Theamino

acidcompositioninthebloodandextracellularandintracellularfluidiscommonlyknown

asthefreeaminoacidpool,whichservesastheprimaryfacilitatorofwholebodyprotein

synthesis(M.Tarnopolsky,2004).Aminoacidswithinthecellorthosecirculatinginthe

bloodstreamcanbeusedforthesynthesisofprotein.Thus,wholebodyproteinsynthesis

isaugmentedinafed‐statemainlyduetoanenhancedaminoacidpoolavailablefor

synthesis(Bohe,Low,Wolfe,&Rennie,2003;Svanberg,1998;Tipton&Wolfe,1998).

Freeaminoacidsareprovidedtotheaminoacidpoolviaproteindegradationinafasted

state,ofwhichsomeaminoacidswillberecycledtosynthesizeprotein(M.Tarnopolsky,

2004).However,freeaminoacidsmayalsobeusedtoprovideenergyviaoxidation(See

Section2.2.4),whichultimatelyremovesthemfromthefreeaminoacidpools,andrenders

themunavailabletosupportproteinsyntheticratesduringfasting.

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2.2.3ProteinDegradation

Withinthehumanbody,proteindegradationisaccomplishedbyproteases.Threemajor

systemsservetodegradeprotein,whichincludetheubiquitin‐proteosomepathway,the

lysosomalsystems,andthecalpainsystems(Belcastro,Shewchuk,&Raj,1998;Lecker,

Solomon,Mitch,&Goldberg,1999).Proteasesfunctionbyhydrolyzingthepeptidebonds

thatholdaminoacidstogether,ultimatelycleavingindividualaminoacidsfromtheprotein

molecule.Oncecleavedfromprotein,aminoacidsentertheaminoacidpoolinthebloodor

extracellularfluid.Uponenteringtheaminoacidpool,aminoacidscanbere‐synthesized

intostructuralorregulatoryproteinsand/oroxidizedforthepurposeofgenerating

energy,thelatterofwhichresultsinhepaticureasynthesisandultimatelynitrogen

excretion(Belcastroetal.,1998;Leckeretal.,1999).

2.2.4AminoAcidOxidation

Theoxidationofaminoacidsisaprocessthatcanuseproteinasafuelsourceforthe

productionofATP(Millward,1998).Additionally,incontrasttofatandcarbohydrate

metabolism,aminoacidscannotbestoredtoagreatextentwithinthehumanbodyand

thereforeanyexcessaminoacids(i.e.thosethatcannotbeincorporatedintoanyprotein

pools)areremovedviaoxidativepathways(Millward,1998).Duringaminoacidoxidation,

aminoacidsarerequiredtolosetheiraminogroupeitherthroughtransaminationor

deamination.Transaminationinvolvesthetransferoftheaminogroupfromanaminoacid

toanothermolecule(usuallyα‐ketoglutarate).Thetransaminationprocessdiffers

dependingonthetypeofaminoacid.Branch‐chainaminoacids(leucine,isoleucine,and

valine)arepredominantlyoxidizedwithinmuscletissue,whileotheraminoacids,suchas

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phenylalanine,mustfirstbeprocessedbytheliverbeforetransaminationoccurs(M.

Tarnopolsky,2004).Phenylalanineoxidationisalsounique,asitmustfirstbehydroxylated

intotyrosinepriortotransamination(Shiman&Gray,1998).Regardless,transamination

leavesbehindacarbon‐skeletonoftheaminoacidthatmaybereassembledintoCitricAcid

Cycleintermediates(e.g.acetylCoA)andbeconsumedwithinthemitochondriaasa

substratefortheproductionofATP.Deaminationisaprocessthattakesplaceprimarilyin

theliver,andservestoremovetheaminogroupfromanaminoacid,whichultimately

producesammonia.Ammoniacanthenbeexcreteddirectlybythekidneys,orconvertedto

ureawithintheliverviatheadditionofCO2priortoitssubsequenturinaryexcretion;

collectively,theseprocessesrepresentthemajorrouteofnitrogenlossinhumans.The

remainingcarbon‐skeletonoftheaminoacidcanthenbeusedtogenerateATP.

Severalcircumstancesmaycauseaminoacidstoundergooxidation.Instatesofexcessive

aminoacidconsumption,anysurplusaminoacidswillbedeaminatedandexcreted,asany

additionalaminoacidconsumptionabovetherequirementwillnotbeusedforprotein

synthesis(Millward,1998).Additionally,duringprolongedperiodsoffastingand/or

enduranceexercise,skeletalmuscleproteinmaybedegradedintoitsaminoacids

components,whichmaythenbetransaminatedwiththeircarbon‐skeletonsbeingutilized

asasourceofenergy(McKenzieetal.,2000;Smith&Rennie,1996).Carbohydrate

availabilitymayalsoinfluenceaminoacidoxidationduringexercise,asstudieshave

determinedthataminoacidoxidationisheightenedinresponsetolowdietary

carbohydrateintakes(Howarthetal.,2010).Insummary,anycondition(i.e.exercise,

fasting)thatincreasesaminoacidoxidationmayostensiblyincreaseproteinrequirements.

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2.2.5EstimatedAverageRequirement&RecommendedDietaryAllowance

Therecyclingofaminoacidsinthehumanbodyisnotfullyefficient(FAO,WHO2007).

Duringnormalprocesses,proteinsthathavebeendegradedintoaminoacidsmaynot

immediatelybere‐synthesized(FAO,WHO2007).Sincefreeaminoacidscannotbestored,

deaminationresultsindailylossesofnitrogenviatheexcretionofurea.Additionally,

miscellaneousroutesofnitrogenlossoccurviahair,nail,skin,digestivesystem,andfecal

losses.Asaresult,humansmustconsumeadailyestimatedaveragerequirement(EAR)of

0.66g/kg/day(FAO,WHO2007).TheEARrepresentstheminimumlevelofproteinto

offsetdailylossesofnitrogenin50%ofthepopulation(Figure2).

However,theEARmaynotsufficientlydefineadequateproteinintakeforallindividuals

withinapopulation(FAO,WHO2007).Therefore,arecommendeddietaryallowance(RDA)

hasbeenestablishedastheEARplustwostandarddeviationstocover97.5%ofthe

population,andiscurrentlysetat0.8g/kg/day(FAO,WHO2007).Itembodiestheamount

Figure2.TheEARandRDA.TheEARcorrespondstotheaveragerequirementof50%ofthepopulation,whiletheRDAencompassesthelevelofproteinthatensures97.5%ofthepopulationisnotdeficient.

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ofdietaryproteinsufficienttosupportthesynthesisandoxidationofprotein,whilealso

accountingforthefundamentalinefficiencyinherenttotherecyclingofproteinandamino

acids,andthedailyobligatorylossesofnitrogen.

2.3MethodsDeterminingProteinRequirements

2.3.1NitrogenBalance

NitrogenBalance(NBAL)hasbeenthemostcommonlyusedmethodtodetermineprotein

requirementssincethe20thcentury(Rand,Pellett,&Young,2003).NBALinvolvesthe

measurementofthetotalnitrogenintake(NIN)viadietaryconsumptionorinfusion,andthe

totalnitrogenexcretion(NOUT),thelatterofwhichprimarilyoccursthroughfeces,urine,

andsweatlosses(Randetal.,2003;Young,Bier,&Pellett,1989).Nitrogenequilibrium

(nitrogenbalance,netzerobalance)isobtainedwhenthesumofallavenuesfornitrogen

intakeisequaltothatofthesumofnitrogenlosses(NetZeroBalance:NIN=NOUT).NBALis

positivewhenNINexceedsNOUT,providingforanabolismoranetgainofprotein.

Conversely,NBALisnegativewhenNOUTisgreaterthanNIN,leadingtoacatabolicstate,or

lossofprotein(M.Tarnopolsky,2004).Theestimatedproteinrequirementisdetermined

fromtheNBALtechniqueusingstrictdietarycontrolsthatprovidesubjectswithvarying

levelsofproteinintakesfor~7dayswiththesubsequentdirectmeasurementand/or

estimatedexcretionofnitrogen.ThispermitsthecalculationofNBALateachlevelof

dietaryintake(M.Tarnopolsky,2004).Aplotofnitrogenintake(x‐axis)vs.nitrogen

balance(y‐axis)isgenerated,andlinearregressionanalysiscanthenbeusedtodetermine

thex‐intercept,whichcorrespondstotheEAR,orzeronet‐balance.Abufferoftwo

standarddeviationsisaddedasa‘safetyfactor’totheEARtoaccountforvariability

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betweenindividualsresultinginanRDAthatencompassesapproximately97.5%ofthe

population(Randetal.,2003;M.Tarnopolsky,2004;Youngetal.,1989).ThecurrentEAR

andRDAprovidedbytheFoodandAgricultureOrganizationoftheUnitedNations(FAO)

andtheWorldHealthOrganization(WHO)arebasedonacollectionofstudiesthathave

predominantlyemployedNBALtodetermineproteinrequirements(FAO,WHO2007).

Ameta‐analyticalreviewbyRandetal.soughttoexaminestudiesthatemployedNBALasa

meanstodetermineproteinrequirements(EARandRDA)inhealthyadults(Randetal.,

2003).Themeta‐analysisgatheredtheresultsfromatotalof19studies,accountingfor235

individualsubjects.Eachofthe19studiesincorporatedintothemeta‐analyticalreview

wererequiredtohaveprovidedsubjectswithaminimumofthreedifferentnitrogen

intakesrangingfromdeficient(resultinginnegativeNBAL)toexcessive(resultingin

positiveNBAL)intake.Additionally,thestudiesutilizedsimilarprotocols,whichrequired

subjectstoconsumeatestdietfor10‐14days.A10‐14daybufferperiodisrequiredforthe

participants’metabolismtoadjusttothealtereddietaryintakeandtoensureconsistent

nitrogenequilibriumisobtained(Randetal.,2003).Nitrogenintake(NIN)couldtherefore

bereadilydeterminedandregulated.Urinaryandfecalnitrogensamples,whichtypically

accountfor90‐95%oftotalexcretion(M.A.Tarnopolskyetal.,1988),areusedtoassess

themetabolicresponsetothevaryingdietaryintakestoallowforanestimationofthetotal

nitrogenexcretion.Only12ofthe19studiesaccountedformiscellaneouslossesofnitrogen

(suchashair,dermal,skin,andexhaledlossesofnitrogen).Therelationshipbetween

nitrogenintakeandnitrogenbalancewascomputedforeachofthe235subjects,providing

foranunderstandingoftheestimatedproteinrequirementsforeachofthe19studies.The

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meta‐analysisestablishedanEARandRDAtobe0.65and0.83g/kg/drespectively.These

aresimilartocurrentEARandRDAvaluesproposedbytheFAOandWHO,corresponding

to0.66g/kg/dayand0.80g/kg/dayrespectively.

2.3.2StrengthsofNBAL

ThereexistseveraladvantagesinherenttotheapplicationoftheNBALtechniqueforitsuse

indeterminingproteinrequirements.First,NBALisanon‐invasivemeansofdetermining

proteinrequirements,asintravenousinfusionsandmusclebiopsiesarenotrequiredas

partoftheapplicationoftheNBALtechnique.Thus,NBALpresentsveryminimalriskto

participants,andisthereforeintheorypracticalinalldemographicgroups.Additionally,

NBALrepresentsarelativelystraightforwardmeansofdeterminingproteinrequirements

astheindependentvariable(dietarynitrogenconsumption)canbeeasilycontrolledfor.

Miscellaneouslossesofnitrogen(i.e.sweat,hair,exhalationlosses)althoughdifficultto

measuredirectly,areoftenestimatedbasedonpre‐existingdatathatmaybegeneralizable

tothepopulation.Thus,NBALisarguablyapracticalmethodofdeterminingprotein

requirementsthatgenerallypresentsminimalrisktohealthyparticipants.

2.3.3LimitationsofNBAL

AlthoughNBALhasbeenthemethodmostfrequentlyusedtoestablishthecurrentEARand

RDA,ithasseverallimitationsthatmaybringintoquestionitsabilitytoobtainaccurate

estimatesofproteinrequirements.SeveralstudiessuggestthatNBALoftenunderestimates

thetruevalueofnitrogenexcretion(NOUT),asitisdifficulttomeasuremiscellaneouslosses

ofnitrogen(sweat,hair,exhalation,etc)(Forbes,1973;Humayun,Elango,Ball,&Pencharz,

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2007).Theimpactofthiserrorinmiscellaneouslossesresultsinanunderestimationof

NOUT,whichsubsequentlyresultsinanunderestimationofnitrogenexcretionandan

erroneouslyhighnitrogenbalance.Thismayultimatelyleadtoanunderestimationoftrue

proteinrequirements(Humayunetal.,2007).Thismayespeciallybethecaseinathletes,

whohaveexercise‐inducedincreasesinmetabolismandsweatloss(ConsolazioF.,Neslon

A.,MatoushL.,HardingR.,&CanhamJ,1963).NBALstudiesalsorequiresubjectsto

consumearegulatedstudydietforanextendedperiodoftime,usuallylastingonetothree

weeksinanattempttocontrolfornitrogenconsumption(NIN),andtoallowsubjectsto

adapttothenewproteinconsumption(Randetal.,2003;FAO,WHO2007).Subject

adherencetotheprescribedstudydietisthereforeessential,andanydeviationfromthe

studydietcouldresultininaccurateproteinrequirements.Thisisespeciallyproblematic

whenutilizingNBALtodetermineproteinrequirementsinchildren,astheirdieting

regimenismoresporadicthanadults.NBALalsoprovidesverylittleinformationregarding

thedynamicprocessofwhole‐bodyproteinturnoverandaminoacidflux(M.Tarnopolsky,

2004).NBALfailstoprovideinformationpertainingtotheadaptivechangesinwhole‐body

proteinsynthesisandoxidationinresponsetoprolongeddietaryorexercisestimuli.Lastly,

theworkofHumayanetal.hashighlightedapotentialstatisticallimitationwithrespectto

theutilizationofNBALtodetermineproteinrequirements(Humayunetal.,2007).The

currentEARandRDAforproteinconsumptionhasbeenestimatedthroughtheuseofa

meta‐analysisthatemployedsinglelinearregressiononseveralstudiesthatemployed

NBAL.TheEARwasdeterminedtobethepointwheretheregressionlineintersectedwith

zerobalance.However,theuseofsinglelinearregressionanalysistodeterminetheEARon

suchcomprehensiveNBALdatamayunderestimatetrueproteinrequirements.Theresults

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concludedthatthecurrentEAR(0.66g/kg/day)andRDA(0.80g/kg/day)determinedby

singlelinearregressionwereunderestimatedby29%and33%respectively.

2.3.4OverviewofInfusionTechniques

SomeofthelimitationsassociatedwithNBALhaveprovidedtheimpetusforresearchersto

developalternativemethodsthatcouldpotentiallybeusedtodetermineprotein

requirements.Infusiontechniquesinvolvetheprovisionofstableisotopesofaminoacids

thatoftenhavea13Cratherthanthenormal12C‐carbonononeoftheatomsofprimarilyan

EAA(FAO,WHO2007).Subjectsarerequiredtopartakeinadietaryadaptationperiod

priortoengagingintheinfusionprotocol.However,thisadaptationperiodisoftenshorter

inlengththanthatofNBAL(Elango,Humayun,Ball,&Pencharz,2009).Generally,atest(or

indicator)aminoacidisinfusedintravenouslyforaperiodusuallyrangingfrom4to24

hourswhereasubjectcanbeinafastedorfedstate.WhentheEAAisoxidized,the13Cis

cleavedfromthemoleculeinthefirststepofoxidationwhenitislabeledonthefirstcarbon

(e.g.L‐[13C]leucineorL‐[13C]phenylalanine).Breath(for13CO2enrichment)andurineor

bloodsamples(forestimatesofprecursorenrichment)canthenbecollectedandanalyzed

fortheirrespectiveenrichments.Thesedatacanthenbemodeled(typicallywithsingle‐

poolsteadystatekinetics)todeterminetheturnoverand,moreimportantly,rateof

oxidationoftheinfusedaminoacid,whichcollectivelyprovideanunderstandingofits

metabolismwithinthehumansubjectandresultantlytheirproteinrequirement

(Wagenmakers,1998).

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Aswithanymethod,thereexistbothstrengthsandlimitationsinherenttoinfusion

protocols.IncontrasttoNBAL,infusionprotocolsareabletoprovideimportant

informationpertainingtothedynamicprocessofwhole‐bodyproteinturnoverandamino

acidflux(M.Tarnopolsky,2004).Additionally,infusionprotocolsmaynotrequirethe

dietaryadaptationperiodtobeaslengthyasthoseanalogousadaptationperiods

associatedwithNBAL.Thismayenhancethepracticalityofinfusionprotocols(FAO,WHO

2007).However,infusionprotocolsaresomewhatinvasiveinnature,astheyrequire

participantstopartakeinintravenousinfusionsthatcanlastfrom4‐24hours.Additionally,

thecostsassociatedwithinfusionprotocolsoftenexceedthatofNBAL,representingwhat

couldbeperceivedasalimitation.

2.3.5Conclusions

AlthoughNBALhasbeenvaluedasthegoldstandardwhendeterminingprotein

requirements,thereareseveralflawsinherenttoitsmethodology.Also,despitebeinga

formidablealternativetotheutilizationofNBALfordeterminingproteinrequirements,

infusionstudiesareratherinvasive.Asaresult,theuseofalternativemethodologies,such

asthenon‐invasiveIndicatorAminoAcidOxidation(IAAO)technique,hasbeendeveloped

todetermineproteinrequirementsinhumans.

15

2.4TheMinimallyInvasiveIndicatorAminoAcidOxidation(IAAO)Technique

2.4.1HistoryoftheMinimallyInvasiveIAAOTechnique

Untilthelate1980’s,nearlyallofthestudiesthatsoughttodetermineprotein

requirementsinhumansusedtheNBALtechnique(Pencharz&Ball,2003).However,once

thelimitationswithNBALwererealized,alternativemethodsthatrelyonstableisotopesto

determineproteinrequirementsweredeveloped(Pencharz&Ball,2003);oneofthese

alternativemethodswastheindicatoraminoacidoxidationtechnique.Initialstudies

performedbyKimetal.(Kim,McMillan,&Bayley,1983),andBall&Bayley(Ball&Bayley,

1984)intheearly80’sfirstimplementedtheIAAOtechniqueinyoungpigsinorderto

determinetheiraminoacidrequirementsforfeeding.In1986,aseriesofdirectaminoacid

oxidation(DAAO)studiesreportedsignificantlyhigheraminoacidrequirementsfor

leucine,valine,andlysineforhumansthanthosethatwerebeingprescribedbytheFood

andAgricultureOrganizationoftheUnitedNations(FAO),andtheWorldHealth

Organization(WHO)(Pencharz&Ball,2003).TheDAAOstudiesrequiredparticipantsto

consumecrystallineaminoacidmixturessufficienttomaintainNBAL.The13C‐labelledtest

aminoacids(leucine,valine,andlysine)weretheninfusedatvaryingintakestodetermine

theirindividualrequirements(Meguidetal.,1986;Meredith,Wen,Bier,Matthews,&

Young,1986).Duetotheresultsofthesestudies,therewasashifttowardsdevelopinga

stable,non‐invasiveisotopeprotocolthatcouldbeutilizedfortheobtainmentofaccurate

proteinrequirementsinhumans.ThefirstpublishedreportoftheminimallyinvasiveIAAO

protocolforhumansubjectswasperformedbyPencharzandBallin1993(Pencharz&Ball,

2003).TheresultsfromthisstudysupportedthepreviousDAAOstudies,suggestingthat

16

proteinrequirementsmayexceedthosevaluestypicallyreportedinNBALstudies(Zello,

Pencharz,&Ball,1990;Zello,Pencharz,&Ball,1993).TheminimallyinvasiveIAAO

techniquehassubsequentlybeenrefinedandutilizedinseveralproteinrequirement

studiesinavarietyofpopulationsrangingfromyoungadultmales(Humayunetal.,2007)

toelderlyfemales(Rafiietal.,2015).

2.4.2MethodologicalApplicationoftheMinimally‐InvasiveIAAOTechnique

TheminimallyinvasiveIAAOtechniqueutilizesstableisotopes(mostcommonly13C)to

specificallylabelasingle‘indicator’aminoacid(Pencharz&Ball,2003).Theminimally

invasiveIAAOtechniquedictatesthatanindicatoraminoacidisalwayssupplementedin

excessoftheproteinrequirement,whiletheremainingintakeofaminoacidswillrange

frombeingdeficienttosurfeitoftheproteinrequirementthroughtheconsumptionof

crystallineaminoacidsbasedontheaminoacidcompositionofeggprotein(FAO,WHO

2007).Whenasingleaminoacid(oftenanEAA)fromthedietislimiting(belowthe

requirement)thentheotheraminoacids(includingtheindicator)cannotbeoptimally

utilizedforproteinsynthesisandaresubsequentlydirectedtowardsoxidation(FAO,WHO

2007)(Zello,Wykes,Ball,&Pencharz,1995).Accordingly,inasituationwherethe

indicatoraminoacidisconsumedinexcess,whiletheremainingtestproteinconsumption

isdeficient,anyamountoftheindicatoraminoacidinexcessofthetestproteinwillbe

directedtowardsoxidation.Inthisscenario,onewouldexpectthelevelsof13Cfromthe

oxidizedindicatoraminoacidtobehighandoverallproteinsynthesistobecompromised.

Astheconsumptionofthelimitingaminoacid(s)inthetestproteinisincreased,alesser

proportionoftheindicatoraminoacidwillbeoxidized;thiswouldresultina

17

correspondingdecreaseintheappearanceofthe13Clabelfromtheindicatoraminoacid

appearinginbreathasagreaterproportionoftheingestedaminoacidsareutilizedfor

proteinsynthesis(Figure3)(Zelloetal.,1995).However,oncetheproteinrequirementfor

thetestaminoacid(s)isreached,anyadditionalintakeofthetestproteinabovetheprotein

requirementshouldnotresultinafurtherdecreaseintheindicatoraminoacidoxidationas

thisaminoacidwillalwaysbeconsumedinexcessofitsrequirement(Zelloetal.,1995).

Additionally,thereshouldbenofurtherincreasesinproteinsynthesisoncethetestprotein

intakeexceedsthatoftheproteinrequirement.Thepointwherenofurtherdecreasesin

indicatoraminoacidoxidation(asmeasuredvia13CO2excretion)areseendespite

increasesinthetestproteinintakeistermedthe‘breakpoint’andismodeledbybi‐phase

linearregression(Figure5)(Zelloetal.,1995).ItisthispointthatdeterminestheEARof

thetestprotein,essentiallyallowingforproteinrequirementstobeestablishedfromthe

minimallyinvasiveIAAOtechnique.Theminimumrequirementcanthenbedeterminedby

addingtwostandarddeviationstotheEAR(breakpoint).

18

Theisotopicallylabeled[1‐13C]‐phenylalanineisthemostcommonlyusedindicatoramino

acidwhenadministeringtheminimallyinvasiveIAAOtechnique(Zelloetal.,1995).

Subjectsarerequiredtoconsumetheindicatoraminoacid,alongwithseveraldifferenttest

proteinintakesthroughouttheminimallyinvasiveIAAOprotocol.Subjectspartakein

roughly7isolatedtrialswherebythetestproteinconsumptionwillrangefromdeficientto

excessiveproteinintake(Zelloetal.,1995).Therelationshipbetweenthetestprotein

intakeandtheindicatoraminoacidoxidationisdeterminedduringeachtrial,allowingfor

abreakpoint(EAR)tobeestablished,andminimumproteinrequirementstobeelucidated.

Figure3.Trendsinaminoacid(AA)synthesisandoxidationduringIAAOtechnique.Thebreak‐point(EAR)hasbeendenotedwiththedashedline.

TheminimumrequirementisdeterminedbyaddingtwostandarddeviationstotheEAR.

19

2.4.3TheMinimallyInvasiveIAAOTechnique&ProteinRequirements

SeveralstudieshaveimplementedtheminimallyinvasiveIAAOtechniqueforthepurpose

ofestablishingproteinrequirementssinceitthefirstpublishedreportinhumanswas

introducedin1993.AstudybyHumayunetal.in2007usedthetechniquetoestimate

proteinrequirementsforthefirsttimeinhealthy,youngadultmen(Humayunetal.,2007).

Atotalofeighthealthyyoungadultmenofdifferentethnicitieswererecruitedforthe

study.InaccordancewithmoststudiesthathaveusedtheminimallyinvasiveIAAO

technique,L‐[1‐13C]‐phenylalaninewasusedastheindicatoraminoacid.Subjectsreceived

oneof7dietarytestproteinintakesrangingfromdeficient(0.10g/kg/d)toexcess(1.8

g/kg/d)overthe7trialdays.Uponanalysis,theestimatedEARandRDAcorrespondedto

0.93g/kg/dand1.2g/kg/drespectively.Bothofthesevaluesaregreaterthanthe

analogousvaluesoftenreportedfromNBALtechniques(EAR:0.66g/kg/d;RDA:

0.80g/kg/d),suggestingthatthecurrentEARandRDAmaybeunderestimatesoftrue

proteinrequirements.Thesevaluesalsocorrespondedwellwiththereanalysisofhistorical

NBALdatausingbi‐phaselinearandlinearanalysisthatincludedNBALstudiesconducted

athighproteinintakes(Humayunetal.,2007).

AlthoughtheminimallyinvasiveIAAOtechniqueisanovelnon‐invasivemethodto

determineproteinrequirements,thereexistsomelimitationstoitspracticability.Firstly,

thenatureoftheminimallyinvasiveIAAOprotocolrequiresparticipantstoengagein

several(~7)metabolictrialdayslastingatleast6‐8hourseach.Therearethereforeissues

relatedtoparticipantwithdrawalfromthestudybeforeitscompletion.Additionally,

participantsarerequiredtoconsumehourlyliquidmealsontrialdays.Althoughthese

20

mealsprovideasufficientenergy,theymaynotadequatelymimicatypicaldietary

regimen,whichprimarilyconsistofthreesubstantialmealsandunbalancedprotein

distribution(deCastro,Bellisle,Feunekes,Dalix,&DeGraaf,1997).

2.4.4Conclusions

TherecentutilizationoftheminimallyinvasiveIndicatorAminoAcidOxidationtechnique

hasconsistentlydeterminedthatproteinrequirementsexceedtheanalogousvalues

previouslydeterminedbystudiesthathaveimplementedNBALmethodologies.However,

anyeffectthatphysicalactivityelicitsonproteinrequirementshasyettobedetermined

usingtheminimallyinvasiveIAAOtechnique.Therefore,giventhepotential

underestimationofproteinrequirementsbyNBALandthepotentialforenhanced

requirementsinactivepopulations,itisimportanttoapplytheminimallyinvasiveIAAO

techniquetodetermineifexerciseiscapableofimpactingproteinrequirements.

2.5EffectofExerciseonProteinMetabolism

2.5.1GeneralOverview

Proteinsynthesis,degradation,andoxidationarehighlyregulatedprocessesthatmust

accommodatetherapidlychangingrequirementsofthemusclecellduringphysicalactivity

(Burd,Tang,Moore,&Phillips,2009).Exerciseiscapableofelicitinganeffectonboth

muscleproteinsynthesisandoxidation,whichwouldaffectmuscleandpotentiallywhole‐

bodyproteinbalance(i.e.synthesis&breakdown)whencomparedtorestingconditions

(Phillips,2004;M.Tarnopolsky,2004).Sinceexerciseisincorporatedintotrainingregimes

21

forathletesandisavitalcomponentofahealthylifestyle,theimpactthatexercisehason

currentminimumrequirementmustbeexaminedinordertoensureadequateprotein

consumptionforhealthy,activepopulations.Thefollowingsectionswilloutlinetheimpact

ofresistanceandenduranceexerciseonproteinmetabolismandtheRDAbyprovidinga

reviewofimportantliterature.

2.5.2EffectofResistanceExerciseonProteinMetabolism,RDA

Resistanceexerciseiswelldocumentedtoelicitchangesinproteinmetabolism,primarily

withinthemuscle(Burdetal.,2009).Variousstudieshaveprovidedevidencethatasingle

boutofresistanceexercisesignificantlyenhancesmuscleproteinsynthesiswhencompared

tobaselinevalues,shiftingmuscleproteinbalancetoamorepositivestateforupto48

hoursfollowingresistanceexercise(Figure4)(Phillips,Tipton,Aarsland,Wolf,&Wolfe,

1997;Biolo,Maggi,Williams,Tipton,&Wolfe,1995;Chesley,MacDougall,Tarnopolsky,

Atkinson,&Smith,1992;Yarasheski,Zachwieja,&Bier,1993).However,despitethe

augmentedstateofmuscleproteinsynthesis,netproteinbalanceremainsnegativeinthe

fastedstate(i.e.intheabsenceofdietaryproteinoraminoacids)asmuscleprotein

breakdownisalsoincreasedinresponsetoasingleboutofresistanceexercisetoprovidea

sourceofaminoacidstosupportproteinsynthesis(Bioloetal.,1995;Phillips,Tipton,

Aarsland,Wolf,&Wolfe,1997;Phillips,Tipton,Ferrando,&Wolfe,1999).

22

Muscleproteinbalanceremainsnegativefollowingresistanceexerciseunlessaminoacids

areeitheradministeredviainfusionorthroughoralconsumption(Figure5)(Bioloetal.,

1995;Borsheim,Tipton,Wolf,&Wolfe,2002;Rasmussen,Tipton,Miller,Wolf,&Wolfe,

2000).Thus,resistanceexerciseandaminoacidconsumptionelicitasynergisticeffecton

proteinsynthesis(Borsheimetal.,2002;Miller,Tipton,Chinkes,Wolf,&Wolfe,2003),

wherebynetproteinbalanceissufficientlypositivetosupportmusclehypertrophy.

Thechangesinmuscleproteinmetabolismwithresistanceexerciseandtheassociated

musclegrowthwithtraininghavepromptedquestionsregardingthesufficiencyofthe

currentRDA(0.8g/kg/d)forindividualspartakinginsuchformsofexercise(Gibalaetal.,

2000;M.A.Tarnopolskyetal.,1992).Someevidencesuggeststhatindividualsthat

regularlypartakeinresistanceexercisemayrequireproteinconsumptioninexcessofthe

currentRDA(asdiscussedbelow)(Gibalaetal.,2000;M.A.Tarnopolskyetal.,1992).Itis

hypothesizedthatthisadditionalproteinwouldberequiredtosupporttheenhancedlevels

Figure4.Theimpactofresistanceexercise,andaminoacidconsumptiononproteinsynthesisandbreakdown.AA=AminoAcids,RE=Resistance

Exercise.AdaptedFrom:Phillips,S.M.(2004).Proteinrequirementsandsupplementationinstrengthsports.Nutrition,20(7‐8),689‐695.

Figure5. Theimpactofresistanceexercise,andaminoacidconsumptiononproteinbalance.AA=

AminoAcids,RE=ResistanceExercise.AdaptedFrom:Phillips,S.M.(2004).

Proteinrequirementsandsupplementationinstrengthsports.Nutrition,20(7‐8),689‐695.

23

ofproteinsynthesisrequiredformusclehypertrophywhilealsoservingtoreplenishany

additionalcataboliclossesofproteininresponsetoresistancetraining(Gibala,

MacDougall,Tarnopolsky,Stauber,&Elorriaga,1995;M.A.Tarnopolskyetal.,1992).

Conversely,variousstudieshavesuggestedthatregularexercise(andpotentially

resistanceexercise)mayimprovetheefficiencyofproteinuse(Butterfield&Calloway,

1984;Torun,Scrimshaw,&Young,1977).TheseminalworkperformedbyButterfieldand

Callowayin1984revealedthatanimprovedefficiencyofnitrogenutilizationoccurredasa

resultofphysicalactivity(Butterfield&Calloway,1984).Sixhealthy,youngadultmales

wererequiredtoconsumetheFAO/WHOsafeproteinintakeof0.57g/kg/dinaninitial

dietaryadaptationperiod.Uponadaptationtothestudydiet,participantsthenengagedin

lighttreadmillbasedexerciseconsistingof320‐minutebriskwalks.Towardthelatter

portionofthestudyanadditionalexercisestimuluswasaddedtoincreasethesubjects’

energyoutput,whichconsistedoftwo30‐minutephasesofbicycling.Uponanalysisof

urinary,fecal,andsweatnitrogenvaluesthroughouttheprotocol,resultsconcludedthat

theimplementedphysicalactivityresultedinanimprovementintheover‐allnitrogen

economyoftheexercisingsubjects,effectivelysparingnitrogen(Butterfield&Calloway,

1984).Ifasimilarmetabolicadaptationoccursasaresultofresistanceexercise,itis

conceivablethatthecurrentRDAmaybesufficientforindividualsthatregularlypartakein

resistanceexercise.

Severalstudieshavedemonstratedthatproteinrequirementsareenhancedinresponseto

resistanceexercise.AstudybyLemonetal.foundevidenceofagreaterprotein

24

requirementassociatedwithstrengthtraininginnoviceathletes(Lemon,Tarnopolsky,

MacDougall,&Atkinson,1992).Twelveyoung,adultmaleswererequiredtoconsumea

proteinintakeof1.35g/kg/dor2.62g/kg/dduringtheinitialstagesofanintensive

bodybuildingregimen.NBALwasusedtodeterminebothzeroNBALandprotein

requirements.Despiteconsuming1.35g/kg/d,whichis~69%greaterthanthecurrent

RDA,participantswereinanetnegativenitrogenbalance.Theresultsestablishedanatural

NBALwasachievedonadietproviding1.4‐1.5gprotein/kg/d,whichresultedinan

estimatedproteinrequirementof1.6‐1.7g/kg/d.Thisfindingsuggeststhatprotein

requirementsduringtheinitialstagesofabodybuildingregimenaretwotimeshigherthan

thecurrentRDAof0.8g/kg/day(Lemonetal.,1992).AstudyperformedbyTarnopolskyet

al.(M.A.Tarnopolsky,MacDougall,&Atkinson,1988)soughttoevaluateprotein

requirementsinyoungadultmalesofdifferenttrainingstatuses.Threegroupsofsixmales

wererecruitedcorrespondingtobodybuilders,enduranceathletes,andsedentarycontrols.

Eachgroupparticipatedinaninitialexperimentdesignedtoevaluatetheprotein

requirementsusingNBALwhiletheparticipantsconsumedtheirhabitualdietaryintake.A

supplementalexperimentperformedbyTarnopolskyetal.employedastudydietthat

alteredtheproteinintakeforeachgroupofparticipants,whilealsousingNBALto

determineproteinrequirements(M.A.Tarnopolskyetal.,1988).Allparticipantswere

requiredtomaintaintheirhabitualtrainingroutinesthroughoutbothexperiments.The

resultsfromtheNBALdatarevealedthattheproteinrequirementsofbodybuilderswere

1.12timesgreaterthanthoserequiredbysedentarycontrols(M.A.Tarnopolskyetal.,

1988).Similarresultsofelevatedproteinrequirementshavealsobeendemonstratedin

eliteweightliftersandpowerlifters(Dohm,Williams,Kasperek,&vanRij,1982).

25

Additionally,inasubsequentstudy(M.A.Tarnopolskyetal.,1992),thirteenhealthy,young

adultmaleswererecruited,ofwhichsevenwerecategorizedasstrength‐trainedathletes

(SA),andsixassedentarycontrols(S).SAsubjectswererequiredtohaveperformed

regularexercisetoincreasetheirstrengthforatleasttwomonthspriortothestudy.

Subjectspartookinthreeexperimentswheretheywererandomlyassignedtoconsumeone

ofthreelevelsofproteinintake:alowproteindiet(LP)of0.86g/kg/d,amoderateprotein

diet(MP)of1.40g/kg/d,andahighproteindiet(HP)correspondingto2.40g/kg/d.

Strength‐trainedathleteswererequiredtomaintaintheirhabitualactivityduringallthe

experiments,whichbasedontheinclusionofteamsportathletesincorporatedboth

anaerobicandaerobiccomponents(i.e.circuitweights,rugbydrills,etc).Protein

requirementswereassessedusingNBAL.ResultsindicatedthatzeroNBALforthe

strength‐trainedathletegroupfarexceededthatofthesedentarygroup(SA:1.41g/kg/d

vs.S:0.69g/kg/d).Additionally,therecommendeddietaryintakeforthestrength‐trained

athletegroupexceededthecurrentRDAof0.8g/kg/d(SA:1.76g/kg/d).

Althoughsomeevidencehassuggestedaheightenedproteinrequirementinresponseto

resistancetraining(Lemonetal.,1992;M.A.Tarnopolskyetal.,1988;M.A.Tarnopolskyet

al.,1992),severalstudieshavedemonstratedthatthecurrentRDAmayinfactbeadequate

forthosewhopartakeinregularresistanceexercise(Campbell,Crim,Young,Joseph,&

Evans,1995;Phillipsetal.,1997;Phillips,2004;Torunetal.,1977).Thereexistsevidence

thatthecurrentRDAisadequateforresistance‐trainedindividuals,asregularresistance

exercisecanresultinproteinbeingusedmoreefficientlybythehumanbody(Campbellet

al.,1995;Phillipsetal.,1999;Phillipsetal.,2002).AstudybyPhillipsetal.(Phillipsetal.,

26

2002)supportsamoreefficientuseofdietaryproteininhealthy,resistance‐trainedmales.

Forexample,subjectsperformedanacuteboutofsingle‐legresistanceexercisebeforeand

followingan8‐weekresistance‐trainingprogram.Followingtheexercisestimulus,subjects

werefedandreceivedaninfusionof[d5]‐and[15N]‐phenylalaninetodeterminemuscle

proteinsynthesisandmuscleproteinbreakdown.Uponcomparisonoftheresultspre‐to‐

posttraining,itappearedthatresistance‐exerciseincreasedrestingmuscleprotein

synthesisandbreakdownwithoutimpactingproteinbalance,whilealsoattenuatingthe

acuteresponsetoanacuteboutofresistance‐exercise.Thisprovidesevidenceofamore

efficientuseofproteininresponsetoregularresistanceexercise.Studiespublishedby

Mooreetal.(Mooreetal.,2007),andHartmanetal.(Hartman,Moore,&Phillips,2006)

havereportedgreaterwholebodyproteinanabolismwhenconsumingamoderate(i.e.1.2‐

1.4g/kg/d)proteindietafterascomparedtobefore12‐wkofresistancetraining.The

resultsfrombothstudiesprovidedevidenceofamoreefficientwholebodyutilizationof

aminoacidsinresponsetotheresistancetraining.

Insummary,itappearsthereislittleconsensusastotheeffectofresistanceexerciseon

wholebodyproteinrequirementsgiventhedichotomybetweenstudiesthatsuggestthe

RDAissufficient(Campbell,Crim,Young,Joseph,&Evans,1995;Phillipsetal.,1997;

Phillips,2004;Torunetal.,1977),whereasothersdonot(Lemonetal.,1992;M.A.

Tarnopolskyetal.,1988;M.A.Tarnopolskyetal.,1992).Althoughthereisageneral

consensusthattheuseofproteinbecomesmoreefficientwithregularresistancetraining,it

isstillcontroversialastowhetherthisenhancedefficiencyofproteinuseisabletooffset

27

theincreasedproteinrequirementnecessaryforenhancedlevelsofproteinsynthesisand

potentialcataboliclossesofproteininresponsetoboutsofresistancetraining.

2.5.3EffectofEnduranceExerciseonProteinMetabolism,RDA

Enduranceexercisehasbeendocumentedtoelicitchangesinwholebodyandmuscle

proteinmetabolism.Forexample,studieshavedemonstratedthatthereareintensity‐

dependentincreasesinmuscleandmitochondrialproteinsynthesisandpotentially

correspondingincreasesmuscleproteinbreakdownaswellafterenduranceexercise

(Carraroetal.,1990;Carraro,Stuart,Hartl,Rosenblatt,&Wolfe,1990;DiDonatoetal.,

2014;Wilkinsonetal.,2008).Specifically,increasesinthesynthesisoftheenzymecitrate

synthase,andelectrontransportchainproteinsintegraltocomplexesI‐IIIresultfrom

enduranceexercise(McKenzieetal.,2000).Additionally,endurancetraininghasbeen

showntoelicitphysiologicalchangesthatcanimproveone’saerobiccapacity,including

increasedquantitiesofhemoglobin,myoglobin,andcapillaries.Somescientistsadvocate

thataheightenedRDAisrequiredtosupportthesephysiologicalchangesassociatedwith

enhancedproteinsynthesisfollowingenduranceexercise(Friedman&Lemon,1989;

Lamont,Patel,&Kalhan,1990;Meredith,Zackin,Frontera,&Evans,1989).

Enduranceexerciseisalsogenerallycharacterizedbyhigh(andsometimessustained)

levelsofenergyexpenditure.Althoughcarbohydratesandfatsarethepredominant

macronutrientsusedtoprovideenergyduringenduranceexercise,aminoacidsmay

contribute1‐6%ofthetotalenergytotheactiveskeletalmuscle(McKenzieetal.,2000;

Phillips,Atkinson,Tarnopolsky,&MacDougall,1993;L.J.Tarnopolsky,MacDougall,

28

Atkinson,Tarnopolsky,&Sutton,1990).Muscleiscapableofoxidizingcertainaminoacids

forthegenerationofATPtohelpfuelenduranceexercise(McKenzieetal.,2000).The

branch‐chainaminoacids(BCAA)isoleucine,leucine,andvalinearepreferablyoxidizedin

themusclebytherate‐limitingenzymebranch‐chainoxo‐aciddehydrogenase(BCOAD)

(Boyer&Odessey,1991;Lamont,McCullough,&Kalhan,1999;Smith&Rennie,1996).

StudieshavedemonstratedthatthereisanincreaseinthecapacityforBCOADactivity

followingendurancetraining(Lamontetal.,1999;McKenzieetal.,2000).Furthermore,

studieshaveshownincreasesintheoxidationoftheBCAAleucineandtheEAAlysine

duringenduranceexerciseandthattheseoxidativelossesmayberelatedtototaloxygen

consumption(Lamontetal.,1990;Lamontetal.,1999;McKenzieetal.,2000;Phillipsetal.,

1993).

Bothmuscleproteinsynthesisandaminoacidoxidationareenhancedinresponseto

enduranceexercise(Moore,Camera,Areta,&Hawley,2014).However,despitethese

changesinproteinmetabolism,thereexistssomedebateastowhetherdietaryprotein

requirementsarealteredinresponsetosuchanexercisestimulus(M.Tarnopolsky,2004).

Itappearsthatproteinrequirementsdifferdependingonthesex,trainingstatus,and

specificallythenature(frequency,intensity,&duration)oftheenduranceexercise(M.

Tarnopolsky,2004).Forthisreason,studieshavesoughttodeterminerequirements

associatedwithoneofthreedemographicgroups:sedentarytorecreationallyactive

individuals(low‐moderateintensityenduranceexercise)(el‐Khouryetal.,1997;Forslund

etal.,1999),moderatelytowell‐trainedenduranceathletes(training4‐5d/weekand≥60

29

min/d)(Lamontetal.,1990;Meredithetal.,1989;Phillipsetal.,1993),andeliteendurance

athletes(~12h/week)(Brounsetal.,1989;M.A.Tarnopolskyetal.,1988).

Forslundetal.performedwell‐controlledandcomprehensivestudyevaluatingtheeffects

ofbothproteinintakeandmoderateenduranceactivityon24‐hleucineturnoverand

macronutrientutilization(Forslundetal.,1999).Fourteenhealthyyoungadultmaleswere

splitintoeitheranormalproteinintakegroupconsuming1.0g/kg/d,orahighprotein

groupconsuming2.5g/kg/dfor6days.Subjectswererequiredtoperform90‐mincycling

exercisetwiceperdayat45‐50%VO2‐maxeachstudyday.Thesubjectsweretheninfused

withL‐[1‐13C]‐leucinefor24hoursonday7toevaluateproteinoxidation.Proteinbalance

wasdeterminedtobeslightlynegativeinthenormalproteingroup,yetwassignificantly

positiveinthehighproteingroup.Thiswasaccompaniedbyagreaterleucineoxidationin

thehighproteingroup,whichwouldsuggestanexcessconsumptionofthismacronutrient.

Additionally,thehighproteingroupsawafargreatercontributionofproteinforenergy

thanthatofthelowproteingroup.Theseresultssuggestthatadietaryproteinrequirement

of1.0g/kg/dshouldbesufficientforindividualspartakinginrecreationalmoderate

intensityenduranceexercise(el‐Khouryetal.,1997;Forslundetal.,1999).

Severalstudieshavealsosoughttodetermineifproteinrequirementsareelevatedin

moderatelytowell‐trainedenduranceathletesusingNBAL(Lamontetal.,1990;Meredith

etal.,1989;Phillipsetal.,1993).Phillipsetal.employedbothNBALandleucineinfusionto

determineiftheproteinrequirementof0.86g/kg/dwasadequateforendurancetrained

individuals(Phillipsetal.,1993).Itwasdemonstratedthatleucineoxidationwasgreaterin

30

maleascomparedtofemalesubjects,whichsuggeststhatmalesaremorereliantonthe

utilizationofproteinforenergyduringenduranceexercisethanfemales.However,both

sexeswerefoundtobeinnegativenitrogenbalance,suggestingthattheprotein

requirementof0.86g/kg/dwasinsufficientforenduranceathletes.Thiswassupportedby

astudypublishedbyMeredithetal.(Meredithetal.,1989),asyoungandmiddle‐agedmale

enduranceathleteswererecordedtohaveasafeproteinintakeof1.26g/kg/d.

Proteinrequirementsforeliteenduranceathletes(training~12h/week,VO2‐max

~70mlO2/kg/min)havealsobeendeterminedusingNBAL(Brounsetal.,1989;M.A.

Tarnopolskyetal.,1988).Tarnopolskyetal.conductedwell‐controlledandcomprehensive

studyevaluatingproteinrequirementsineliteenduranceathletes(M.A.Tarnopolskyetal.,

1988).SixtopsportrunnerandNordicskienduranceathletes(BodyFat%:7.1+/‐0.8;

TrainingLoad>125km/week)andsixsedentarycontrolswererecruited.Participants

wererequiredtoconsumetheirhabitualdietaryintakewhilemaintainingtheirhabitual

physicalactivitylevels.Anadditionalexperimentemployedastudydietthatalteredthe

proteinintakeforeachgroupofparticipantsinordertodetermineifvaryingthe

consumptionofproteinresultsinachangeinnitrogenbalance(M.A.Tarnopolskyetal.,

1988).Theresultssuggestedthattheeliteenduranceathletes’proteinrequirement(1.6

g/kg/g)farexceededtheanalogousrequirementforthesedentarycontrols(0.86g/kg/d)

(M.A.Tarnopolskyetal.,1988).StudiesbyMeredithetal.(Meredith,Zackin,Frontera,&

Evans,1989)andFriedmanandLemon(Friedman&Lemon,1989)havealsoreportedan

increasedproteinrequirementineliteenduranceathletes(TrainingVolume:12‐16km

runningperday).Meredithetal.recruited6youngand6middleagedmenwhoconsumed

31

0.6,0.9,and1.2g/kg/dofhigh‐qualityproteinoverthreeseparate10‐dayperiodswhile

maintainingtheirhabitualtrainingregimen(Meredithetal.,1989).Theresultssuggested

thattheparticipants’proteinrequirements(0.94g/kg/d)exceededthecurrentRDA

(0.80g/kg/d).InastudyconductedbyFriedman&Lemonin1989,fivewell‐trained

endurancerunners(VO2‐max~70)consumedeithertheRDA,or1.7timestheRDA(high‐

proteintrial)ontwoseparateoccasionsfor6dayswhilemaintainingtheirtypicaltraining

regimen(Friedman&Lemon,1989).ResultssuggestedthattheRDAof0.80g/kg/dwas

inadequatefortheenduranceathletesasnitrogenretentionwassignificantlyreduced,

whilenitrogenretentionremainedpositiveduringthehigh‐proteintrial.Insummary,there

appearstobeaheightenedproteinrequirementforhighlytrainedenduranceathletes.

Thereappearstobenosignificantincreaseinproteinrequirementsforindividualsthat

recreationallyengageinenduranceactivity,yetthereisconsiderableevidenceofa

heightenedproteinrequirementforendurance‐trainedindividualsandespeciallyelite

enduranceathletes.TheRDAforthesepopulationsmaybe~20‐100%greaterthanthe

currentRDAdependingonthenatureoftheenduranceexercisestimulus,thetraining

status,andsexoftheindividual.

2.5.4EffectofVariableIntensityExerciseonProteinMetabolism,RDA

Althoughawealthofstudieshaveexaminedtheindividualimpactsofeitherresistanceor

endurancetraining(exercise)onproteinmetabolismandrequirements,nostudyhas

soughttodeterminehowacombinationofbothtrainingtypescanimpactdietaryprotein

requirements.Sinceathletictrainingformostteamsportsoftenresemblesthatofvariable

32

intensityexercisethatincorporatesbothaerobic(e.g.increasedoxygenconsumption)and

resistive(e.g.highforcestop‐and‐go)components,itcouldbearguedthatnoprevious

studyhaselucidatedhowthistypeofexercisemodalityaltersproteinrequirements;this

wouldbeimportantfortheoptimizationnutritionintheseuniqueathletes.

AstudyperformedbyCoffeyetal.,in2010soughttodeterminetheimpactofcyclingsprint

intervaltrainingoncellsignalingandproteinsynthesis(Coffeyetal.,2011).Results

suggestedenhancedmyofibrillarandmitochondrialproteinsyntheticratesfollowingthe

sprintintervaltraining.Likevariableintensityexercise,sprintintervaltrainingiscapable

ofactivatingbothaerobicandanaerobicenergysystems.Assuch,itisplausiblethata

variableintensityexercisestimulusmimickingthatofsportcouldresultinenhancements

tomyofibrillarandmitochondrialproteinsynthesis,potentiallyincreasingprotein

requirementsinindividualswhoregularlypartakeinsuchanexerciseroutine(i.e.team‐

sportathletes)(M.A.Tarnopolskyetal.,1992).

2.6Conclusions,GapsinLiterature

Theimpactsthatbothresistanceandenduranceexercisehaveonproteinrequirements

havebeenextensivelystudiedbutwithlimitedconsensusreached.Theutilizationofthe

NBALtechniquesuggeststhatproteinrequirementsmaybeenhancedinindividualswho

regularlyperformsuchexercise.Nevertheless,nostudytodatehassoughttodetermineif

variableintensityweight‐bearingexercise,suchasthatperformedduringteamsportssuch

assoccer,rugby,andtosomeextenthockey,iscapableofchangingthedietaryprotein

requirement.Variableintensityexerciseresemblesthatoforganizedsportsandtrainingas

33

itiscapableofstimulatingboththeanaerobicenergysystemsassociatedwithresistance

training,andtheaerobicsystemsthatprovideenergyforendurancetraining(Bangsbo,

Mohr,&Krustrup,2006).Moreover,weightbearingsportssuchassoccerorfootballare

alsoassociatedwithhighforcemusclecontractionsandacceleration/decelerationevents,

whichmightbeastimulusformusclegrowthand/orinducemildlevelsofmuscledamage

(Ascensaoetal.,2008;Magalhaes,Rebelo,Oliveira,Silva,Marques,&Ascensao,2010a).

Therefore,thisexercisemodalitywithelementsofbothresistiveandaerobicexerciseis

uniquefromthosepreviouslystudied(i.e.resistancevs.endurance),whichprimarily

focusedonoppositeendsofthestrength‐endurancecontinuum.

TheutilizationoftheminimallyinvasiveIndicatorAminoAcidOxidationtechniquehas

consistentlydemonstratedthatproteinrequirementsmayexceedthosevaluesdetermined

usingtheNBALtechnique.However,theminimallyinvasiveIAAOtechniquehasnever

beenutilizedtodetermineifphysicalactivityelicitsaneffectonproteinrequirements.

Thus,itisessentialtoutilizetheminimallyinvasiveIAAOtechniquetodetermineprotein

requirementsinresponsetoavariableintensityexercisestimulus.Itishypothesizedthat

theresultsfromthepresentstudywillyieldproteinrequirementsthatarehigherthanthe

currentEARandRDAasestablishedthroughNBAL,inadditiontothoserequirements

establishedusingtheIAAOtechniqueinuntrainedpopulations.

34

Chapter3‐ResearchProposal:

3.1Introduction

Basedonthepreviouslydescribedknowledgebase,theresearchstudywasdividedintoa

totalof3sequentialresearchphases,encompassing3‐10laboratoryvisitsdependingon

theparticipant.Theresearchproposalsectionwillserveasageneraloutlineofthepurpose

andobjectivesassociatedwitheachresearchphase.Additionally,astudytimelineforthe

participantswillbepresentedintheformofaschematicoftheoverallstudy.Lastly,a

timelineofthecourseoftheprojectwillbepresented.Amorein‐depthreviewofthe

materialsandmethodologywillbeprovidedinChapter4.

35

3.2PhasesofResearch

Atotalof7active,trainedyoungadultmaleparticipantswererecruitedforthepresent

study.Participantspartookinarangeof3‐10trialsencompassingthethreephasesofthe

researchstudy.Thethreephasesoftheproposedstudyaredescribedbelow.

PhaseI–IntroductorySession(1x1.5h):

Objectives:PhaseIconsistedofasinglelaboratoryvisitlastingapproximately1.5hours,

andhadtwoprimaryobjectives.First,itservedtoprovidetheparticipantswitha

comprehensiveoralintroductiontothestudyprotocolinordertoensurethattheywere

properlyinformedbeforebeingaskedtoprovideconsent.Onceparticipantswereprovided

withadetailedoverviewofthestudyprotocol,theyweregiventheopportunitytoask

questions,andsubsequentlysignedtherequiredconsentdocumentindicatingtheir

participationinthestudy.Thefirstphaseofresearchwasalsousedtoobtainbackground

informationwithrespecttotheparticipants’generalhealthandhabitualactivitylevels.

PhaseII–BodyCompositionAnalysis,FitnessAssessments(1x2h):

Objectives:PhaseIIwascomprisedofasinglelaboratoryvisitofapproximately2hours,

andhadthreemainobjectives.Adetailedanalysisofbodycompositionwasfirst

performed,utilizingtheBodPod(AirDisplacementPlethysmography,COSMEDUSA,Inc.,

Concord,California),andbasicanthropometricmeasurementssuchasheight(cm)and

weight(kg).Uponcompletionofthevariousbodycompositionmeasurements,participants

wererequiredtoperformthebeeptest,whichwasusedtocharacterizetheparticipants’

fitness.Participantswerethengivenanintroductiontotheexercisestimulusandmetabolic

36

trialsthatwereperformedontheremainingmetabolictrialdays.Additionally,participants

wererequiredtoconsumea2‐daystudydietpriortoeachmetabolictrialday.

PhaseIII–MetabolicTrials(7x10h):

Objectives:PhaseIIIrequiredparticipantstovisitthelaboratoryforapproximately10

hourson1‐10differentoccasionsdependingontheparticipant.Eachmetabolictrial

followedthesameprotocol.Subjectswerefirstrequiredtocompleteanexercisestimulus

followedbyan8‐hourmetabolictrialwheretheyconsumedthestudydiet.Anin‐depth

reviewofthemetabolictrialmethodologywillbeprovidedinChapter4.

3.3ResearchSchematic

*Participantsengagedinarangeof1‐10PhaseIIImetabolictrials

37

3.4ProjectTimeline

Element StartDate EndDate

Proposal

Defense

September2014 Withcompleted

literaturereview

September2014

IAAO

Training

October2014 Incollaboration

withSickKids

Hospital

October2014

PilotData

Collection

October2014 Incollaboration

withSickKids

Hospital

January2015

Data

Collection

(PhasesI‐III)

February2015 Incollaboration

withSickKids

Hospital

June2015 Analysis

concurrentwith

datacollection

Writing July2015 Thesis,

journalarticle

submissions

August2015 Planforalldegree

requirementsmet

byAugust2015

*Participantscanbeindifferentphasesofthestudyatanygiventime‐point

38

Chapter4–Methodology:

4.1Introduction

ThissectionwillprovideadetaileddescriptionoftheprogressionfromPhaseItoPhaseIII

ofthepresentstudy.Thestudydesign,methodology,andmaterialsinherenttoeachphase

willbeclarified.Moreover,abriefexplanationofthestatisticalanalyseswillbeprovided.

39

4.2Participants&GeneralStudyDesign

7active,trainedmales(23±1years;177.5±6.2cm;83.3±6.3kg;13.5%±5.1%BF;52.3±

5.4mlO2/kg/min;mean±95%CI)wererecruitedfromvariousvarsitysportsteamsatthe

UniversityofToronto.ThePhysicalActivityReadinessQuestionnaire(PAR‐Q+)wasusedto

assessanyhealthriskspriortoparticipantenrollmentinthestudy.TheInternational

PhysicalActivityQuestionnaire(IPAQ)andVO2‐maxtest(BeepTest)confirmedthatthe

participants’fitnesslevelsmettherequirementsofthestudy(moderate‐vigorousactivity≥

5d/week,predictedVO2‐max~50mlO2/kg/minrespectively).Allparticipantspartookina

minimumofonemetabolictrial(PhaseIII)(range:1to10trials)inadditiontocompleting

theindependenttrialsassociatedwithPhaseIandPhaseII(detailsbelow).

4.3PhaseI,IntroductorySession

PhaseIconsistedofasinglelaboratoryvisitlastingapproximately1.5hours.Thisvisit

servedasanintroductorysessionthatallowedfortheparticipantstobecomefamiliarwith

thestudydesign.Participantswerefirstgivenacomprehensiveoralintroductiontothe

studydesigninadditiontoaclearexplanationoftheobjectivesofthestudy.All

participantswerethengivenanopportunitytoaskquestionsrelatedtothestudyasthey

reviewedtheconsentdocument.Thisensuredthattheyunderstoodwhattheir

commitmentwouldentailbeforeprovidinginformedconsent.Uponcompletionand

signatureoftheconsentdocument,backgroundinformationwasobtainedpertainingtothe

participants’generalhealthandhabitualfitnesslevelsusingthePhysicalActivityReadiness

Questionnaire(PAR‐Q+)andtheIPAQrespectively.Onceparticipantssatisfiedthecriteria

pertainingtoage,healthstatus,andfitnesslevelspresentedintheaforementioned

40

questionnaires,theyweretheninstructedtorecorda3‐daydietarylog,andtowearthe

SensewearBodyMediaArmbandAccelerometerforthreedayspriortothePhaseII

laboratoryvisit.Thesemethodsprovidedtheinvestigatorswithanunderstandingofthe

participants’free‐livingenergyexpenditure,whichhelpedtodesigntheparticipants’

individualfeedingprotocolstobeadministeredduringthemetabolictrialsofPhaseIII.

4.4PhaseII,BodyCompositionAnalysis&FitnessAssessment

PhaseIIconsistedofasinglelaboratoryvisitlastingapproximately2hours.Allparticipants

arrivedtothelaboratoryhavingworntheSensewearBodyMediaArmbandAccelerometer

for3days,andhavingcompleteda3‐daydietarylog.Participantswererequiredtohave

abstainedfromconsumingsolidfoodorliquid,andfromengaginginphysicalactivityfor2

hourspriortoPhaseII.AirDisplacementPlethysmography(BodPod)(COSMEDUSA,Inc.,

Model2007A,Concord,California)wasusedtodeterminebaselinewholebodymass(BW)

(kg),fatmass(FM),percentbodyfat(%BF)andfat‐freemass(FFM)ofeachparticipant

bothpriortoandafterthecompletionofthemetabolictrials.Anaveragedweight(kg)and

FFM(kg)atthetwotime‐pointswereusedforallsubsequentcalculationsforoutcome

variables(Table1)(SeeSection4.6).Therewasanaveragechangepre‐to‐post

correspondingto‐0.74%BF,‐0.077kgFFM,and‐0.83kgBW.Restingenergyexpenditure

(REE)wasestimatedusingtheBodPodforeachparticipant,andwascomparedtoREEdata

recordedbytheSensewearBodyMediaArmbandAccelerometerduringsleep(SeeSection

4.5).TheSensewearBodyMediaArmbandAccelerometerhasbeenvalidatedforitsusein

determiningREEandfree‐livingdailyenergyexpenditureinadults(Malavoltietal.,2007;

St‐Onge,Mignault,Allison,&Rabasa‐Lhoret,2007).Uponcompletionofallmeasurements,

41

participantswerefamiliarizedwiththeaerobicfitnessassessmentprotocoltoensurethat

theywerecomfortablewithallequipment(polar‐HRmonitor,SensewearBodyMedia

ArmbandAccelerometer).TheBeepTestwasusedtodetermineeachparticipant’s

predictedmaximaloxygenconsumption(VO2‐max).Thistestiswellestablishedinadults,

andconsistsofa10‐15minuterunningtestwithagradedincreaseinwork‐output,until

participantsreachvolitionalfatigue,oranage‐predictedmax‐HR(Leger&Lambert,1982).

Participantsweregiven15minutestorest,andwereprovidedwithanexplanationofthe

protocolforthesubsequenttrialdays.Participantsthenpartookinanintroductory

modifiedLoughboroughIntermittentShuttleTest(LIST)(Nicholas,Nuttall,&Williams,

2000).ThisensuredthatparticipantswereabletocompletethemodifiedLIST.

Additionally,itprovidedanestimateoftheirexerciseenergyexpenditurethatwas

determinedbyconvertingtheenergyexpenditureoffourpilotparticipantsduringthe

modifiedLISTstimulustoanaveragevalueinkcal/kg/minute.Thisinformationensured

thatthemetabolicdietineachofthemetabolictrialsofPhaseIIIprovidedsufficientenergy

tooffsetwhatwasexpendedduringthemodifiedLISTstimulus(SeeSection4.5).

4.5PhaseIII,MetabolicTrials

Pre‐Trial:PhaseIIIconsisted1‐10laboratoryvisits(dependingontheparticipant).Each

laboratoryvisitlastedapproximately10hoursandconsistedoftwocomponents:the

modifiedLISTexercisestimulusandasubsequentmetabolictrial.Twodayspriortoeach

ofthesevenmetabolictrials,participantswererequiredtoadheretoapredetermined

studydietproviding1.2g/kg/dofproteinandsufficientenergytocoverthehabitual

energyexpenditureaspreviouslymeasuredduringthe3‐daccelerometerrecord(see

42

below).Additionally,subjectswererequiredperformanovernightfastbeforeeach

metabolictrial.Uponreportingtothelaboratory,participantsingestedaliquid‐based,

protein‐freebreakfastproviding1g/kgofcarbohydrates(0.5g/kgPolycose,and0.5g/kg

OrangeGatoradePowder)toensurethattheywerenotexercisinginthefastedstate.

ModifiedLIST:ParticipantspartookinamodifiedLISTstimulus.TheLISTisavariable

intensityexercisetestconsistingofrepeated20‐mshuttlerunsresemblingplayin

organizedsportssuchasrugbyandsoccer(Armstrong&Welsman,2006;Castagna,

D'Ottavio,&Abt,2003;Stroyer,Hansen,&Klausen,2004).ThemodifiedLISTwas

comprisedof4segmentsof15minutesofvariableintensityexerciseincludingsprinting,

running(90%VO2‐maxspeed),jogging(60%VO2‐maxspeed)andwalkingpaces(Figure

6).ThemodifiedLISTintegratedcomponentsofbothresistanceandenduranceexercise

sufficienttostimulateenhancedmuscleandmitochondrialproteinturnover.

Figure6.ThemodifiedLoughbouroughIntermittentShuttleTest(LIST).

43

ThemodifiedLISTintensitieswerepreviouslydeterminedfromthespeedobtainedata

level12onthebeeptest(4.0m/s).ThetotaltimecommitmentfortheLISTexercise

stimuluswasapproximately75minutes(4x15minuteblocksofvariableintensityexercise

+3x5minutesofrestbetweenblocks).

StudyDiet:AftercompletingtheLISTtestinitsentirety,participantsreturnedtothe

laboratorywheretheywererequiredtoimmediatelyconsumethefirstof8hourlymeals

comprisingthestudydiet.Eachmetabolictrialstudydietprovidedavariableamountof

protein,6g/kgofcarbohydrate(accordingtocarbohydrateconsumptionguidelinesfor

generaltrainingneedsinmoderate‐highlyactiveindividualsestablishedbyBurkeetal.)

(Mountjoyetal.,2011)andsufficientenergytoensurethatparticipantswereinanet

positiveenergybalanceontrialday(seebelowfordetails).

StudyDietEnergyIntake

Thestudydaydietconsistedofeightliquidbeveragescontaininganassignedprotein

intakeandsixteenprotein‐freecookies.Thecookiesensuredthatparticipantsconsumed

somesolidfoodontrialday,thusreducingthelikelihoodofattritionorparticipant

withdrawal(SeeIAAOManualinAppendixforRecipeandNutritionalInformation).

Thetotalenergyintakewascalculatedusingtheequation:

TotalEnergyIntake=(REEx1.5)+[(0.1425(kcal/kg/min)xWeight(kg)x75(min))x1.1]

44

WhereREE=RestingEnergyExpenditureDuringSleep(kcal)(recordedusingthe

SensewearBodyMediaArmbandAccelerometer)(Malavoltietal.,2007);1.5=Activity

Factor(toaccountfortheactivenatureoftheparticipantsandtobeconsistentwith

previousIAAOstudiesinrestedadults)(Humayunetal.,2007);0.1425=AverageEnergy

ExpenditureDuringLISTExerciseStimulus(kcal/kg/min);75Minutes=Durationofthe

modifiedLISTExerciseStimulus;and1.1=10%BufferforEnergyExpendedDuringthe

LISTExerciseStimulus(toensureparticipantswereinasurplusofenergyandtoaccount

forindividualdifferencesinenergyexpenditure).

Proteinwasprovidedascrystallineaminoacidsmodeledonthebasisofeggproteinwith

theexceptionofexcesstyrosine(40mg/kg/d)andtheindicatoraminoacidphenylalanine

(30.5mg/kg/dwith5.46mg/kgasL‐[13C]phenylalanine)(Humayunetal.,2007).

Phenylalaninemetabolismoccursintheliver,suchthatitisfirsthydroxylatedtoform

tyrosine.Tyrosinecantheneitherbeusedasasubstrateforproteinsynthesis,orbe

transaminatedordeaminated,whichreleasesCO2containingthecarboxylcarbonfrom

phenylalanine.Whenextracellulartyrosineislowandplasmaconcentrationsof

phenylalaninearenormal,tyrosinewillbepreferentiallyusedinproteinsynthesisas

opposedtobeingdegradedorexported.Thus,tyrosinewasprovidedinexcesstoensure

thecarboxylcarbonofphenylalaninewouldappearinbreaththroughitsincorporation

intotyrosineandsubsequenttransaminationordeamination(Shiman&Gray,1998).All

participantswererandomlyassignedtoconsume1‐10differenttestproteinintakesina

randomorderoneachofthemetabolictrialdaysassociatedwithPhaseIII.Thetestprotein

intakesweredesignedtocoverdeficienttoexcessiveintake(0.2‐2.6g/kg/d),accordingto

45

previousstudiesimplementingIAAOandNBALprotocolsinadults(Humayunetal.,2007).

Atestproteinintakerangeof0.2‐2.6g/kg/dwasutilizedinconsiderationoftheCanadian

SocietyforExercisePhysiologyandAmericanCollegeofSportsMedicine’s

recommendationsforproteinconsumptionintrainedpopulationsof(1.2to1.7g/kg/d)

(AmericanDieteticAssociationetal.,2009),whicharebasedprimarilyonNBALstudies.

Therefore,anupperlevelof2.6g/kg/dwasselectedgiventhepotentialthatNBAL

underestimatestruerequirements(Humayunetal.,2007)andtoensureaplateauin

F13CO2wasachieved,thelatterofwhichisapre‐requisiteforrobustbi‐phaselinear

modelingandbreakpointanalysis.

Thefirst4hourlymealsconsumeddirectlyafterthemodifiedLISTexercisestimulus

containedthetestprotein,unlabeledphenylalanine,andtyrosineastraceringestionbegan

onthe5thhourlymeal.Wehadpreviouslyconfirmedthoughtpilotstudiesthatbackground

13CO2andcarbondioxideproductionwasstableoverthisperiodwhileconsumingthetest

diet(seeFigures4and5inAppendix),whichensuredastablebackgroundenrichmentand

metabolismwasachievedpriortotraceringestion.Priortothe5thmeal,4breathsamples

weretakenat15‐minuteintervals,and3urinesampleswerecollectedat30‐minute

intervalsinordertoestablishbaseline13CO2(viacontinuous‐flowisotoperatiomass

spectrometry)andL‐[13C]phenylalanineenrichment(vialiquidchromatographytandem

massspectrometry)respectively(Humayunetal.,2007).AprimingdoseofNaH13CO3

(0.176mg/kg)andL‐[13C]‐phenylalanine(1.86mg/kg)wereingestedonthe5thhourly

meal.TherateofCO2productionwasthenmeasuredovera20‐25minuteperiodfollowing

the5thhourlymeal,butbeforethe7thhourlymealusingindirectcalorimetry(MOXUS

46

MetabolicCart,AEITechnologies)todeterminesteadystatemetabolism.1.2mg/kgofL‐

[13C]‐phenylalaninereplacedanequivalentamountofunlabeledphenylalanineandwas

ingestedintheremainingthreehourlymealstoinduceisotopicsteadystate.Eightplateau

breathandfiveplateauurinesampleswerecollectedat15and30‐minuteintervals,

respectively,commencing2hoursaftertheonsetoftraceringestion(Humayunetal.,

2007).

4.6DataOrganization

Aninitialsetof42testproteinintakesrangingfrom0.20to2.25g/kg/dwereseparated

into7differentproteinintakeranges(i.e.0.20–0.45g/kg/d;0.50–0.75g/kg/d;0.80–

1.05g/kg/d;1.10–1.35g/kg/d,1.40–1.65g/kg/d,1.70–1.95g/kg/d,and2.00–2.25

g/kg/d).Eachproteinintakerangeencompassed6proteinintakelevelsseparatedby0.05

g/kg/dincrements.Forexample,proteinintakescorrespondingto0.20,0.25,0.30,0.35,

0.40,and0.45g/kg/drepresentedthe6proteinintakelevelswithintherangeof0.20–

0.45g/kg/d.Eachparticipantwasrandomlyassignedtoconsumeoneproteinintakelevel

withineachofthesevenproteinintakerangesoverthesevenmetabolictrials.

Atotalof40oftheinitial42testproteinintakeswerecompleted,asoneparticipantonly

completed5ofthe7trials.Asaresult,anadditional5trialswereperformedattestprotein

intakescorrespondingto0.225,2.45,2.50,2.55,and2.60g/kg/d.Atotalof45metabolic

trialscompletedby7participantswereconducted,exceedingthepreviousminimumtrials

(i.e.35)performedwiththeminimallyinvasiveIAAOprotocol(Elangoetal.,2011).

47

AtomsPercentExcess(A.P.E.)foreachmetabolictrialwasdeterminedbysubtractingthe

averagedbaselinebreath13CO2enrichmentfromtheaveragedplateaubreath13CO2

enrichment.13CO2excretion(F13CO2)wasthencalculatedusingtheformulabelow.

F13CO2=(FCO2)(ECO2)(44.6)(60)

(W)(0.82)(100)

FCO2=CO2ProductionRate(mL/min)

ECO2=13CO2Enrichment(A.P.E)

44.6(μmol/mL)&60(min/hour)=UsedtoconvertFCO2tomicromoles/hour

W=Massofparticipant(eitherinkgbodyweightorkgfat‐freemass,asappropriate)

0.82=CorrectionfactorforCO2retainedinthebodybecauseofbicarbonatefixation

(Elangoetal.,2011)

100=UsedtoconvertECO2intoafraction

F13CO2(perkgbodyweightorperkgfat‐freemass)servedastheprimaryoutcome

variable,andwasplottedasafunctionoftestproteinintake(relativetobodymass)in

ordertoestablishthebreakpoint(EAR)(SeeSection5.2).Phenylalanineoxidationwas

determinedbydividingF13CO2by[13C]‐phenylalanineenrichmentinurine,andwas

expressedasafunctionofmass(kg)orFFM(kg)versusproteinintakeinordertoestablish

thebreakpoint(EAR)inoxidationdata(SeeSection5.2).

BreakpointmeasurementsutilizingF13CO2havebeenshowntobesimilartobreakpoint

measurementsforphenylalaninehydroxylationbyusingenrichmentsinapolipoproteinB‐

100,whichisahepaticexportprotein(Rafiietal.,2008).Therefore,breathmeasurements

48

arepresumedtoberepresentativeoftheintracellularenrichmentofphenylalanineinthe

liverandarepreferabletotherateofphenylalanineoxidationcalculatedfromplasmaor

urineenrichments.Requirementsfortheproteinintakewerethereforedeterminedby

usingF13CO2data,astheyhavebeensuggestedtobemorerobustandappropriatefor

makingrecommendations(Rafiietal.,2008).

4.7StatisticalAnalyses

AllF13CO2andphenylalanineoxidationdatawasinputtedtoGraphPadPrismversion5.00

forWindows(GraphPadSoftware,SanDiegoCaliforniaUSA)toestimatewhetheralinear

orbi‐phaselinearregressionanalysisexplainedmorevariability.GraphPadversion5.00

forWindows(GraphPadSoftware,SanDiegoCaliforniaUSA)wassubsequentlyusedforall

statisticalanalyses.Biphaselinearregressionanalysiswasusedtodeterminetheeffectof

proteinintakeonF13CO2andphenylalanineoxidation.SignificancewasestablishedatP<

0.05.Todeterminethemeanproteinrequirement,abi‐phaselinearregressionanalysis

wasperformedonF13CO2(astheprimaryoutcomevariable)andphenylalanineoxidation

(asasecondaryoutcomevariable).

49

Chapter5–Results:

5.1SubjectCharacteristics

Sevenyoungadultmen(23±1y)participatedinthestudy.Allparticipantswereactiveand

inatrainedstate,mosthavingcompetedforavarsitysportsteam.Allparticipantsmetthe

inclusioncriteriapertainingtofitnesshavingobtainedaminimumlevelof10onthebeep

test(predictedVO2‐max~50)andhavingengagedinmoderate‐vigorousactivityforat

least5d/week.SubjectcharacteristicsarepresentedinTable1.Weight(kg),FFM(kg),and

bodyfat(%BF)measuresrecordedbeforeanduponcompletionofthestudywereaveraged

andusedforallprimaryoutcomecalculations.

Table1Subjectcharacteristicsandenergyintakesofyoungadultmen(n=7)whoparticipatedinthestudyCharacteristic: Value(mean±95%CI):

Age(y) 22.9±0.8

Height(cm) 177.5±6.2

Weight(kg) 82.3±5.7

FFM(kg) 71.1±5.1

PercentBodyFat(%) 13.5±4.3

PredictedVO2‐Max(ml/kg/O2/min) 52.3±5.4

HabitualEnergyExpenditure(kcal/d) 3596±446

RestingEnergyExpenditure(kcal/d) 2221±135

StudyDayEnergyIntake(kcal/d) 2867±177

50

5.2F13CO2Excretion:

Bi‐phaselinearregressionanalysisexplainedmoreofthevariabilityforproteinintake

versusF13CO2(perkgbodymass)thansimplelinearregression(r2Bi‐Phase=0.6434;r2

Linear=0.5921;p=0.0182).TheaverageparticipantVCO2expressedinbothabsoluteand

relativetermscorrespondedto276±16.9mL/min(mean±95%CI)and3.36±0.26

mL/kg/minrespectively.Therateof13CO2excretion(F13CO2)viatheoxidationofL‐[1‐13C]‐

phenylalaninedecreasedinparticipantswithincreasingproteinintakesupto1.35g/kg/d

(Figure7).Additionalincreasesinproteinintakedidnotresultinfurtherdecreasesin

F13CO2values.ThisindicatedthatnoadditionalexcretionofF13CO2occurredafterthe

proteinintakeof1.35g/kg/dwasreachedsuggestingnon‐oxidativedisposalof

phenylalanine(andbyextension,potentiallyproteinsynthesis)wasmaximized.Bi‐phase

linearregressionanalysisofF13CO2versusproteinintakecorrespondedtoabreakpoint

(estimatedaveragerequirement,EAR)of1.35g/kg/d(r2=0.64).Thesafepopulation

intakeestimatedbytheupper95%confidenceintervalofthebreakpointwascalculatedto

be1.64g/kg/dwithalowerCIof1.06g/kg/d.ExpressingthedataasafunctionofFFM

(kg)correlatedtoabreakpoint(EAR)of1.60g/kgFFM/d(r2=0.64),andasafepopulation

intakeof1.93g/kgFFM/dwithalowerCIof1.27g/kgFFM/d(Figure8).

51

Protein intake(g/kg FFM/day)

F1

3 CO

2

( m

ol/k

g F

FM

/hou

r)

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.00.0

0.5

1.0

1.5 Breakpoint = 1.60 g/kg BW/dayupper 95% CI = 1.93 g/kg BW/day

R2 = 0.6364p = 0.0075

Protein intake(g/kg BW/day)

F1

3 CO

2

( m

ol/k

g B

W/h

our)

0.0 0.5 1.0 1.5 2.0 2.5 3.00.0

0.5

1.0

1.5 Breakpoint = 1.35 g/kg BW/dayupper 95% CI = 1.64 g/kg BW/day

R2 = 0.6434p = 0.0182

Figure7.Influenceofdietaryproteinintakeontheproductionof13CO2 fromtheoxidationoforallyadministeredL‐[1‐13C]‐phenylalanine(F13CO2)inactive,youngadultmales.Thebreakpointestimatedthemeanproteinrequirement,andcorrespondedto1.35g/kg/d,withtheupper95%CI(orpopulation‐safeintake)estimatedtobe1.64g/kg/d.

Figure8.Influenceofdietaryproteinintakeontheproductionof13CO2 fromtheoxidationoforallyadministeredL‐[1‐13C]‐phenylalanine(F13CO2)inactive,youngadultmales.Thebreakpointestimatedthemeanproteinrequirement,andcorrespondedto1.60g/kgFFM/d,withtheupper95%CI(orpopulation‐safeintake)estimatedtobe1.93g/kgFFM/d.

Breakpoint=1.35g/kgBW/dUpper95%CI=1.64g/kgBW/dR2=0.6434

Breakpoint=1.60g/kgFFM/dUpper95%CI=1.93g/kgFFM/dR2=0.6364

52

5.3PhenylalanineFlux

Phenylalaninefluxmeasuredfromurinesamplesdidnotchangesignificantlywithdifferent

testproteinintakesrangingfrom0.5g/kg/dto2.6g/kg/d(Figure9).However,hightest

proteinintakes(2.0to2.3g/kg/d;2.45to2.60g/kg/d)weresignificantlydifferentfrom

thelowesttestproteinintakerangeof0.2g/kg/dto0.45g/kg/d(p<0.05)(Figure9).

Figure9.Phenylalaninefluxcorrespondingtotheeighttestproteinranges.Fluxdidnotchangesignificantlywithtestproteinintakesfrom0.5g/kg/dto2.60g/kg/d.Fluxfortestproteinintakesof2.0to2.30g/kg/dand2.45to2.60g/kg/dweresignificantlydifferentthantheintakerangeof0.20to0.45g/kg/d.

53

5.4PhenylalanineOxidation

Phenylalanineoxidationdeclinedduetoincreasingproteinintakesuntil1.4–1.5g/kg/d

and1.6–1.7g/kgFFM/d(Figures10&11),afterwhichadditionalincreasesinprotein

intakesdidnotfurtherimpactphenylalanineoxidation.Bi‐phaselinearregressionanalysis

identifiedabreakpointandupper95%CIfortherelationshipbetweenproteinintakeand

phenylalanineoxidationtobe1.44and1.76g/kg/d,respectively,forbodyweight(kg)(r2=

0.63),and1.62and1.96g/kgFFM/d,respectively,forfat‐freemass(r2=0.65).Areanalysis

ofphenylalanineoxidationdatawasconductedusinganaverageparticipanturinary

phenylalanineenrichmentvaluetoestimatethepotentialbiasthatthenegative

relationshipbetweenproteinintakeandphenylalaninefluxmayhavehadourbreakpoint

analysisofphenylalanineoxidation.Thisreanalysisresultedinabreakpointinthe

phenylalanineoxidationdataofthatwas~8%higherat1.56g/kg/d(SeeFigure1in

Appendix);thissuggeststhatthenon‐steadystatephenylalanineflux(derivedfromthe

urinaryphenylalanineenrichment)hadonlyaminoreffectonthebreakpoint(i.e.EAR)

estimatedfromphenylalanineoxidation(i.e.Figure10)

54

Protein intake(g/kg FFM/day)

Phe

Oxi

dati

on(

mol

/kg

FF

M/h

our)

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.00

10

20

30 Breakpoint = 1.62 g/kg BW/dayupper 95% CI = 1.96 g/kg BW/day

R2 = 0.6467p = 0.0109

Protein intake(g/kg BW/day)

Phe

Oxi

dati

on(

mol

/kg

BW

/hou

r)

0.0 0.5 1.0 1.5 2.0 2.5 3.00

5

10

15

20

25 Breakpoint = 1.44 g/kg BW/dayupper 95% CI = 1.76 g/kg BW/day

R2 = 0.6334p = 0.0172

Figure10.InfluenceofdietaryproteinintakeonPheoxidationinactiveyoungadultmales(n=45observations).Bi‐phaselinearregressionanalysisrevealedthebreakpointandupper95%tobe1.44and1.76g/kg/drespectively.

Figure11.InfluenceofdietaryproteinintakeonPheoxidationinactiveyoungadultmales(n=45observations).Bi‐phaselinearregressionanalysisrevealedthebreakpointandupper95%tobe1.60and1.96g/kgFFM/drespectively.

Breakpoint=1.44g/kgBW/dUpper95%CI=1.76g/kgBW/dR2=0.6334

Breakpoint=1.62g/kgFFM/dUpper95%CI=1.96g/kgFFM/dR2=0.6467

55

Chapter6–Discussion:

6.1Introduction

ThecurrentdietaryrequirementsforproteinconsumptionarebasedprimarilyonNBAL

data.Althoughnumerousstudiesexaminetheindividualimpactsofexclusivelyresistance

(Lemonetal.,1992;M.A.Tarnopolskyetal.,1988;M.A.Tarnopolskyetal.,1992)or

endurancetraining(exercise)(Brounsetal.,1989;M.A.Tarnopolskyetal.,1988)on

proteinmetabolismandrequirements,nostudyhassoughttodirectlydeterminehowan

exercisemodalitywithelementsofeach(i.e.variableintensityexercise)canimpactdietary

proteinrequirements.Thepurposeofthepresentstudywastoutilizetheminimally

invasiveIAAOtechniqueforthefirsttimeinthepresenceofavariableintensityweight

bearingexercisestimulusinactive,trainedyoungadultmales.Wereportabreakpointin

F13CO2dataof1.35g/kg/d(i.e.theEAR)andaminimumpopulationintakeestimatedby

theupper95%confidenceintervalofthebreakpoint(population‐safeprotein

requirement)tobe1.64g/kg/d,thelatterofwhichwouldrepresentadailyintakethat

wouldbesufficientfor>95%ofthisactivepopulation.Theresultsfromthisstudyyielded

proteinrequirementsthatare:i)inexcessofthecurrentEARandRDA;ii)greaterthan

thosepreviouslydeterminedininactivepopulationsusingtheminimally‐invasiveIAAO

method,and;iii)attheupperrange(ifnotgreater)thanthosepreviouslysuggestedfor

athletesaccordingtogeneralconsensusstatements(AmericanDieteticAssociationetal.,

2009).Thefollowingsectionswillcontextualizeourresultsinlightofpreviousstudies

determiningproteinrequirementsbyNBALandIAAO.Potentialavenuesforfuture

researchwillalsobeprovidedinlightofadiscussionofthestrengths,limitations,and

implicationsofthepresentstudy.

56

6.2Results,ComparisontoNBAL&IAAOStudies

Thisisthefirststudyto‐datethathasemployedtheminimallyinvasiveIAAOtechnique

bothinanactivepopulationandinthepresenceofanexercisestimulus.Weobservedthat

thebreakpointinF13CO2datawas1.35g/kg/d(Figure9),whichwouldrepresenttheEAR.

Theminimumpopulationintakeestimatedbytheupper95%confidenceintervalofthe

breakpoint(population‐safeproteinrequirement)was1.64g/kg/d,whichwould

subsequentlyrepresentadailyintakethatwouldbesufficientfor>95%ofthisactive

population.Theseresultsare105%higherthanthecurrentEARandRDA,whichare

0.66g/kg/dand0.80g/kg/drespectively(FAO,WHO2007).Thisdistinctionisalso

apparentwhentheproteinrequirementsdeterminedinthepresentstudyarecomparedto

thatofthemostcomprehensivemeta‐analysisofNBALdatato‐date(Randetal.,2003),

whichestablishedanEARof0.65g/kg/dandanRDAof0.83g/kg/d.Thus,ourestimateof

theproteinrequirementforouractivepopulationdeterminedbyIAAOissubstantially

greaterthanthatpreviouslydeterminedinnon‐activeindividualsutilizingNBAL,which

wouldbeconsistentwithpreviousNBALstudiesinactivepopulations(Lemonetal.,1992;

M.A.Tarnopolskyetal.,1988;M.A.Tarnopolskyetal.,1992)andcouldsuggestthatactive

individualsengagedinweight‐bearing,variableintensityexerciserequireagreaterprotein

intakethantheirnon‐activepeers.

Whileitispossiblethatourproteinrequirementsareelevatedbyourexercisestimulus,it

shouldbeconsideredthatthegreaterrequirementdeterminedhereinbytheminimally

invasiveIAAOtechniquemaybespecifictoourtracermodelgiventhepreviously

suggestedunderestimationofNBAL(Humayunetal.,2007).Thus,itmaybemorerelevant

57

tocompareourresultsinlightofpreviousstudiesinnon‐activeindividualsthatutilized

similarmethodologytoincreasetheinternalvalidityofourdata.Thedietaryprotein

intakesestablishedinthepresentstudywerealsogreaterthantherequirementsinall

publishedIAAOworkonadultsthusfar.AstudybyHumayunetal.in2007utilizedthe

IAAOtechniqueinthemostcomparabledemographicgrouptothepresentstudy:healthy,

youngadultmales(Humayunetal.,2007).Thestudyutilizedanidenticalcrystallineamino

acidmixturewhilefollowingthesame8‐hourfeedingandtraceringestionprotocolasthe

presentstudy.Themean(EAR)andpopulation‐safe(RDA)proteinrequirementswere

foundtobe0.93and1.2g/kg/drespectively.Theseresultsare31%and27%lowerthan

theEARandpopulation‐safeproteinrequirementdeterminedbythepresentstudy.Other

studiesthathaveemployedtheIAAOtechniquetodetermineproteinrequirementsinless

activepopulationsofolderadultshaveyieldedsimilarresultswherebytheprotein

requirementsestablishedarelowerthanthosefoundinthepresentstudy(Rafiietal.,

2015;Tangetal.,2014).Thus,thehypothesesthatthepresentstudywouldyieldprotein

requirementsinexcessofboththecurrentEARandRDAestablishedonthebasisofthe

NBALtechniqueandstudiesutilizingtheminimally‐invasiveIAAOtechniqueinlessactive

individualsweresupported.

6.3ResultsExplained,MethodologicalConsiderations

Avarietyofmethodologicalconsiderationscanpotentiallyexplainthedifferencesin

proteinrequirementswhencomparingthepresentstudytotheexistingNBALandIAAO

data.SeveralstudieshavesuggestedthatNBALcanunderestimatethevalueofnitrogen

excretion,asmiscellaneouslossesofnitrogen(hair,sweat,exhalation,etc.)areoftenleft

58

unaccountedforsincetheyaredifficulttomeasureaccurately(Forbes,1973;Humayun,

Elango,Ball,&Pencharz,2007).Anunderestimationofnitrogenexcretioncanresultinan

erroneouslyhighnitrogenbalance,andconsequentlyanunderestimationoftrueprotein

requirements.ThesuggestionthatthecurrentEARandRDAmaybeunderestimatesof

proteinrequirementswassupportedbytheworkofHumayunetal.in2007who

reanalyzedhistoricalNBALdatausingbi‐phaselinearregressionwhileincludingstudiesat

highproteinintakesthatwerepreviouslydeemedoutliers(Humayunetal.,2007).Upon

theapplicationofbi‐phaselinearregressiontohistoricalNBALdatawhileincludingstudies

conductedathigherproteinintakes,theEARandRDAwereincreased(EAR–0.91g/kg/d;

RDA–1.00g/kg/d).Theserequirementswerecomparabletothoseestablishedusingthe

minimallyinvasiveIAAOtechniqueinyoung,adultmales(EAR–0.93g/kg/d;RDA–1.20

g/kg/d)(Humayunetal.,2007).Thissuggeststhatsimplelinearregressionforstatistical

analysisofNBALdatamaybeinappropriatefordeterminingtrueproteinrequirements

whenallstudiesareincluded,asthosepreviouslydeemedasoutliersmayhavea

pronouncedeffectonminimalestimateswhentheyareincludedwiththeotherNBALdata.

However,thebi‐phaselinearregressionmodelisnotbiasedbyover‐orunderestimates

andisthereforearguablyamorerobustmodeltodeterminetheEARandpotentiallytrue

proteinrequirements,providedthedataconformtothismodel.Insupportofthis,agreater

proportionofthevarianceinourF13CO2datawereexplainedbybi‐phasecomparedto

linearmodelingwhenexpressedtowholebody(r2=0.6434vs.0.5921,respectively;p=

0.0182)andrelativetoFFM(r2=0.6364vs.0.5679,respectively;p=0.0075).

59

ThestudyperformedbyHumayunetal.in2007recruitedeighthealthy,youngadultmales

andutilizedanidenticalcrystallineaminoacidmixturewhilefollowingthesame8‐hour

feedingandtraceringestionprotocolasthepresentstudy(Humayunetal.,2007).Each

participantconsumedsevenproteinintakescorrespondingto0.10,0.30,0.60,0.90,1.20,

1.50,and1.80g/kg/d.Incontrast,thepresentstudyassignedoneproteinintakewithin

eachofthesevenaforementionedproteinintakerangesfrom0.20g/kgto2.60g/kgtoeach

participant(SeeSection4.6).Thisallowedforamorecomprehensiveexaminationofthe

impactofproteinintakeonphenylalanineoxidationtobededucedasatotalof45protein

intakeswereconsumedcomparedtosevenintheHumayunetal.study.Theresultsfrom

thepresentstudy(EAR=1.35g/kg/d;RDA1.64g/kg/d)weresignificantlygreaterthan

thoseestablishedintheHumayunetal.study(EAR=0.93g/kg/d;RDA=1.20g/kg/d),and

maybeexplainedbythetraineddemographicgrouprecruitedinadditiontothe

incorporationofanacuteexercisestimulusintotheminimally‐invasiveIAAOprotocol.

6.4PhysiologicalConsiderations,&ImplicationsforActiveIndividuals

Humayunetal.(2007)andthepresentstudyemployedthesamecomponentsofthe

minimallyinvasiveIAAOtechnique.Assuch,thedifferencesinresultsmaybeattributedto

theincorporationoftheLISTexercisestimulusintotheIAAOprotocolofthepresentstudy.

NumerousNBALstudiessuggestthatproteinrequirementsareincreasedduetoresistance

(Lemonetal.,1992;M.A.Tarnopolskyetal.,1988;M.A.Tarnopolskyetal.,1992)and/or

enduranceexercise(Brounsetal.,1989;Friedman&Lemon,1989;M.A.Tarnopolskyetal.,

1988).ThemodifiedLISTexercisestimulusconsistedofrepeated20‐mshuttlesatvarious

intensitiesrangingfrombriskwalkingtomaximalsprints.Themostcomparablevariable

60

intensityexercisemodalitytothemodifiedLISTisthatofsprintintervaltraining.Coffeyet

al.soughtdeterminedtheimpactofcyclingsprintintervaltrainingoncellsignalingand

proteinsynthesis(Coffeyetal.,2011).Resultssuggestedenhancedmyofibrillarand

mitochondrialproteinsyntheticratesfollowingsprintintervaltraining.Similartothatof

sprintintervaltraining,thestop‐startnatureofthetestandrepeatedmaximalsprints

inherenttothemodifiedLISTexercisestimuluswereabletostimulaterapidmyofibrillar

contractionsinadditionactivatingtheanaerobicenergysystems(ATP‐PCr&Anaerobic

Glycolysis).Assuch,itisplausiblethatamodifiedLISTexercisestimulususedinthe

presentstudymayhaveresultedinenhancementstomyofibrillarandmitochondrial

proteinsynthesis,resultingintheheightenedproteinrequirement.Furthermore,astudy

performedbyMagalhaesetal.,alsoillustratedthattheLISTiscapableofinducingmuscle

damagesimilartothatcausedbyasoccermatch(Magalhaes,Rebelo,Oliveira,Silva,

Marques,&Ascensao,2010).Additionally,thestop‐startnatureoftheLISTexercise

stimuluscharacterizedbyaccelerationanddecelerationeventswerecapableofeliciting

rapidandrepeatedeccentriccontractionsthathavebeendemonstratedtoenhancemuscle

proteinbreakdown(Fieldingetal.,1991).Assuch,themodifiedLISTexercisestimulus

stimulatedmanyofthesamemetabolicandphysiologicalpathwaysinherenttoresistance

exercise,whichmayhavepartiallybeenresponsiblefortheheightenedprotein

requirementdeterminedinthepresentstudy.Alternatively,ifthepotentialexercise‐

inducedmuscledamageintheLISTresultedinagreaterproteinbreakdownthatwasnot

reciprocatedbyanequalstimulationofproteinsynthesis,thenagreaterproteinturnover

inducedbytheLISTintheabsenceofenhancedintracellularreutilizationofaminoacids

couldhavemanifestedinthegreaterproteinrequirementobservedhereinduring

61

recovery.Thesepossibilitieswouldrequirefuturestudiesthatincorporateparallel

measuresoftissue‐specificproteinturnoverandnetbalance(e.g.muscle)inorderto

enhanceourunderstandingofthephysiologicalrationaleforourheightenedprotein

requirement.

Theprolongednature(~75minutes)ofthemodifiedLISTexercisestimuluswouldbe

sufficienttostimulatethebody’saerobicenergysystemsthatareactivatedduring

prolongedboutsofenduranceexercise.DespitethemajorityofthemodifiedLISTexercise

stimulusbeingoflowerintensity(i.e.rest,walking,jogging),thisexercisetestwas

originallydevelopedtomimicthephysiologicaldemandsofasoccermatchinwhich

averageenergyexpenditureisgenerally~70%VO2‐max(Bangsbo,Mohr,&Krustrup,2006;

(Nicholas,Nuttall,&Williams,2000).Assuch,itshouldbenotedthattherelativelylong

durationandhighintensityofthemodifiedLISTexercisestimulus(75minutes)mayhave

increasedthebreakdownandoxidationofaminoacidsforenergy(vanLoon,Greenhaff,

Constantin‐Teodosiu,Saris,&Wagenmakers,2001).Althoughcarbohydratesandtoa

certainextentfatwouldbethepredominantmacronutrientsusedtoprovideenergyduring

enduranceexerciseof~70%VO2max,aminoacidsmaycontribute1‐6%ofthetotalenergyto

theactiveskeletalmuscleinmen(McKenzieetal.,2000;Phillips,Atkinson,Tarnopolsky,&

MacDougall,1993;L.J.Tarnopolsky,MacDougall,Atkinson,Tarnopolsky,&Sutton,1990;

vanLoonetal.,2001).Moreover,carbohydrateavailabilitycaninfluenceaminoacid

oxidationduringexercise,asstudieshavedeterminedthataminoacidoxidationis

heightenedinresponsetolowdietarycarbohydrateintakesandlimitedglycogen

availability(Howarthetal.,2010;Lemon&Mullin,1980).Althoughparticipantswere

62

providedwith1g/kgofacarbohydratebeveragepriortoengaginginthemodifiedLIST

stimulus,theywererequiredtohavefastedovernight,andassuchwerelikelyinanet

negativeenergybalancetowardsthelatterstagesoftheexercisestimulus.Giventhelikely

relativelyhighaverageVO2duringtheLIST(~70%VO2max)thatwouldhaverelied

heavilyonendogenouscarbohydratestorestofuelit(McKenzieetal.,2000;Phillips,

Atkinson,Tarnopolsky,&MacDougall,1993;L.;vanLoonetal.,2001),thepotentially

limitedmuscleglycogenavailabilitylateintheLISTcouldhaveresultedinarelatively

increasedutlilizationofaminoacidsasafuelsource.Therefore,itispossiblethat

participantsinthepresentstudyoxidizedahighproportionofaminoacids(i.e.aminimum

of5‐6%)forenergyduringthemodifiedLISTstimulus,especiallyduringthelaterstagesof

theexercisestimulusduetoaglycogen‐depletedstate.AssuminganapproximatetotalLIST

energyexpenditureof800kcal(basedoffanalysisoftheSensewearBodyMediaArmband

Accelerometer)whereby5%ofenergycontributionwasprovidedviaaminoacidoxidation,

atotalof40kcalofenergyduringthemodifiedLISTwouldhavebeenprovidedviaamino

acidoxidation.Thiswouldcorrespondto10gofoxidizedproteinforenergy,or

approximately0.13g/kg/dofproteinlostduringthemodifiedLISTforan80kgparticipant.

WhencomparingpresentstudythatoftheHumayunetal.,study,thereexistsa0.44g/kg/d

differenceinsafe‐levelproteinrequirements.Thedirectoxidationofaminoacidsfor

energyduringthemodifiedLISTmayaccountforapproximately30%ofthisdifference.

TheremainingdifferencesmaybeexplainedbytheuniquenessofthemodifiedLIST

exercisestimulus,asitincorporatedcomponentsofbothresistanceandendurance

exercisethatmayhavecausedsubstantialmetabolicstresssufficienttofurtherincrease

proteinrequirements.

63

AlthoughtheFAO/WHOrecognizeexercise‐inducedincreasesinaminoacidoxidationand

nitrogenlosses,theyhaveyettoreachaconsensusastowhetherexerciseiscapableof

increasingproteinrequirements(FAO,WHO2007).However,therequirements

determinedinthepresentstudy(EAR=1.35;upper95%CI=1.64)fallin‐linewiththe

CanadianSocietyforExercisePhysiologyandAmericanCollegeofSportsMedicinegeneral

recommendationsforendurance(1.2–1.4g/kg/d)andresistanceathletes(i.e.1.2‐

1.7g/kg/d)(AmericanDieteticAssociationetal.,2009),andasstatedpreviouslyarein

accordancewithawealthofNBALstudiesthathavesoughttoexamineprotein

requirementsinhabituallyactiveindividualsandathletes.Theselevelsofproteinintakes

aresignificantlygreaterthanthecurrentRDAof0.8g/kg/d.However,theRDA

encompassesaproteinintakeintendedsimplytopreventdeficiency(Phillips,Moore,&

Tang,2007),andmaythereforenotbeappropriateforsuchactiveindividuals.Elite

athletesoftenreportconsumingquantitiesofproteininexcessofthecurrentrequirements

(Burkeetal.,2003),andarethereforelikelytobemoreinterestedinoptimizingtheir

nutritionforenhancedsportperformancethansimplybeinginnetproteinbalance.Thus,it

remainstobeseenwhethertherequirementsestablishedinthepresentstudyarea

reflectionofa‘truerequirement’foractiveindividuals,orserveasaguidelineforoptimal

levelsofproteinintakerequiredforenhancingsportperformance.

6.5RateofAppearanceandPhenylalanineFlux

IncomparisontothestudyconductedbyHumayunetal.in2007,thepresentstudysaw

enhancedlevelsofphenylalaninefluxatallproteinintakelevels,asthemeanRaforthe

presentstudywas77.02µmol/kgBW/hourcomparedtoameanRaof56.93µmol/kg

64

BW/hourasdeterminedintheHumayunetal.study(Humayunetal.,2007).These

differencesarelikelyexplainedbytheincorporationofmodifiedLISTexercisestimulus

intotheIAAOprotocol,asresearchhassuggestedenhancedlevelsofwholebodyprotein

turnoverinresponsetoexercise(Rennieetal.,1981).Arequirementforstudiesthatutilize

theIAAOtechniqueisthatphenylalaninefluxisnotimpactedbyproteinintake.This

providesevidencethattheprecursorpoolforindicatoroxidationdoesnotchangeinsizein

responsetothetestproteinintake(Humayunetal.,2007).Resultsfromthepresentstudy

suggestthatthereweredecreasesinphenylalanineflux(rateofappearance,Ra)in

responsetoincreasingtestproteinintakes,asthemeanRainthetwohighesttestprotein

intakerangesweresignificantlydifferentfromthemeanRaatthelowestproteinintake

range(Figure9).AlthoughnosignificantdifferenceswereseeninRainresponseto

proteinintakeinthestudyconductedbyHumayunetal.,asimilartrendofdecreasing

phenylalaninefluxinresponsetoincreasingtestproteinintakewasapparent(SeeFigure

2inAppendix).Theinverserelationshipbetweenphenylalaninefluxandtestprotein

consumptionmayberelatedtoadietaryaminoacid‐inducedsuppressionofendogenous

(i.e.body)proteinbreakdown,whichhasbeenreportedpreviouslyatthewholebody

proteinlevels(Churchward‐Venne,Murphy,Longland,&Phillips,2013).Ifthiswerethe

caseinthepresentstudy,theprotein‐sparingeffectofexogenousaminoacidswouldhave

attenuatedtheappearanceofunlabeledaminoacidsintoplasma(andhenceurine)at

steadystate,whichwouldhavemanifestedinadecreaseinRafollowingexercise.

65

Toestimatethepotentialimpactoftheapparentreductioninphenylalaninefluxwith

increasingproteinintake,areanalysisofphenylalanineoxidationdatawasconductedusing

anaverageparticipanturinaryphenylalanineenrichmentvaluetodetermineifthe

breakpointwouldchangeunderthecircumstancewherephenylalaninefluxwasconstant

throughouttherangeoftestproteinintakes.Thereanalysiscorrespondedtoabreakpoint

inphenylalanineoxidationdataof1.56g/kg/d(SeeFigure1inAppendix).Thisvalueis

~8%greaterthanthebreakpointof1.44g/kg/dreportedinthepresentstudyfrom

phenylalanineoxidationdata(Figure10)andcouldsuggestthattheresultsfromthe

presentstudymayrepresentasmallunderestimateoftrueproteinrequirements.While

oneofthebenefitsoftheminimallyinvasiveIAAOisnothavingtosamplebloodforto

estimatetheprecursorenrichment,futurestudiesmaybenefitfromdeterminingplasma

andapolipoproteinB‐100enrichmentasarguablymorerobustsurrogatesofhepatic

intracellularenrichmenttodetermineifthedecreaseinurinaryphenylalanineenrichment

withincreasingproteinintakeobservedhereinistrulyreflectiveofalteredprecursor

enrichment.FuturestudiescouldalsoemploytheIAAOtechniqueinthepresenceofa

varietyofexercisestimuli,includingthemodifiedLIST,inordertodetermineifsimilar

trendsinphenylalaninefluxaredemonstratedinotherpopulationspost‐exercise.

6.6Strengths,Limitations,&FutureAvenuesofResearch

Thereexistseveralstrengthsinherenttothedesignofthepresentstudy.First,theIAAO

techniquewasaminimallyinvasivemeansfordeterminingproteinrequirements,deeming

itpracticalinbothhealthyindividualsandat‐riskpopulations.Additionally,theutilization

oftheminimallyinvasiveIAAOtechniquefordeterminingproteinrequirementsrequired

66

onlyatwo‐daydietaryadaptationperiodpriortoeachmetabolictrial(Elangoetal.,2009).

Thisallowedforasingleparticipanttobetestedoverarangeofdeficienttoexcessprotein

intakes.Incomparison,NBALrequiresa7‐14daydietaryadaptationperiod,whichoften

preventsparticipantsfromconsumingmorethanthreeproteinintakelevels(Randetal.,

2003;FAO,WHO2007).Asaresult,thepresentstudywasabletoprovideacomprehensive

determinationofproteinrequirements.Thepresentstudywasthefirsttoutilizethe

minimallyinvasiveIAAOtechniqueinthepresenceofanexercisestimulusthat

incorporatedcomponentsofbothresistanceandenduranceexerciseresemblingthatof

trainingandperformanceinorganizedteamsports(Armstrong&Welsman,2006;

Castagna,D'Ottavio,&Abt,2003;Stroyer,Hansen,&Klausen,2004).Thus,thepresent

study’sprotocolisapplicableinavarietyofathleticpopulations.Furthermore,the

simplicityofthefree‐living,running‐basedmodifiedLISTexercisestimulusdeemsthe

studydesignpracticableinyoungerpopulationsthatengageinteamsportvariable

intensityexercise;thismaybeasmuchas30%ofthegeneralpopulationwhoare

recreationallyactiveinteamsportsaccordingtothe2015PhysicalActivityCouncilReport.

TheIAAOtechniquewasanovelmethodfordeterminingproteinrequirementsinthe

presentstudy,yetthereexistlimitationsinherenttoitspracticabilityinfuturestudies.

Participantsarerequiredtoconsumehourlyliquidmealsontrialdays.Althoughthese

mealsprovideasufficientdailyamountoftotalenergy,theymaynotmimicatypical

dietaryregimen,whichoftenconsistsprimarilyofthreesubstantialmealsandunbalanced

proteinintakes(deCastroetal.,1997).However,thisfeedingpatternmaybeineffectiveat

maximizingnetproteinbalance,especiallyfollowingexercise,asrepeatedingestionof

67

moderateamountsofprotein(~20g)atregularintervalshavebeenshowntomaximize

proteinsynthesisandnetbalance(Aretaetal.,2013;Mooreetal.,2012).

SeeingasthepresentstudywasthefirsttoutilizetheIAAOtechniquetodetermineprotein

requirementsinatraineddemographicgroupandinthepresenceofanexercisestimulus,

thereareseveralpotentialavenuesforfutureresearch.Thepresentstudy’sprotocolcan

firstbeextendedtoincludeactive,youngadultfemalesinanattempttodetermineifthere

aresex‐baseddifferencesinproteinrequirementsforactiveindividuals,giventhat

differencesinaminoacidmetabolismduringexercisehavebeenreportedpreviously

(Phillips,Atkinson,Tarnopolsky,&MacDougall,1993).Additionally,theIAAOtechnique

hasneverbeenappliedtoactivepopulationsofchildrenoradolescents.Sincetheadequate

ingestionofdietaryproteinisessentialtosupporttheoptimalremodelinganddeposition

ofleanbodymassinactive,growingchildrenandadolescents,suchastudywouldhave

majorhealthandsportimplications(Rodriguez,2005).Beyondtheapplicationofthe

presentstudy’sprotocoltoalternativeagegroups,aninterestingfutureavenueofresearch

wouldbetoapplythetypicalIAAOtechniquetomorespecializedathleticpopulations.For

example,theIAAOtechniquecouldbeappliedtoeitherenduranceorresistance‐trained

athletes.SuchresearchwouldrequirethemodifiedLISTexercisestimulustobereplaced

withanalternativeexerciseroutinethatisrepresentativeofthestudypopulation’stypical

trainingregimen.Forresistance‐trainedathletes,aweight‐liftingregimencouldreplacethe

modifiedLISTexercisestimulus,whilea20‐kmruncouldbeusedasanexercisestimulus

inendurance‐trainedpopulations.Thepresentstudywillthereforeserveasabenchmark

forwhichallsubsequentIAAOstudiesinactivepopulationscouldbecomparedto.

68

6.7Conclusion

Theadequateingestionofdietaryaminoacidsisacriticalfactorinensuringthe

development,andmaintenanceofleanbodymassinindividualsofallages.Physical

activitycanalterproteinandaminoacidmetabolism,andcanincreaseprotein

requirementsinhighlyactiveindividuals(e.g.trainedathletes).Thepurposeofthepresent

studywastoutilize,forthefirsttime,theminimallyinvasiveIAAOtechniquetoevaluate

theimpactofavariableintensityexerciseonproteinrequirementsinactive,trainedyoung

adultmales.Thehypothesesthatthestudy’sresultswouldyieldproteinrequirementsin

excessofthecurrentrequirementsestablishedusingNBAL,inadditiontothoseestablished

inlessactivepopulationsusingtheIAAOtechniquewerevalidated.Futureavenuesof

researchwillseektoimproveupontheexistingIAAOprotocol,andtoemployitinactive

females,children,adolescents,andinmorespecializedathleticpopulations.

69

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Appendices

Raw Data All data in this appendix can be accessed via the excel document ‘Master Data (August 20th)’ 

Subject Characteristics: 

Participant #:  Age:  Height (cm):  Weight (kg):  % BF:  BodPod (FFM, kg):  BodPod RMR (kcal/d): 

1  23  178  88.18  13.15  76.58  2000 

2  22  176.5  79.78  7.1  74.115  1948 

3  23  183  87.09  11.1  77.4085  2036 

4  22  174  87.72  22  68.4075  1835 

6  24  185  84.82  15.2  71.914  1908 

7  22  165  75.36  10.9  67.149  1771 

5  24  181  73.41  15.1  62.344  1653  

Participant #:  Accelerometer RMR (J/min):  Accelerometer RMR (kcal/d):  Study Day E‐Expenditure (kcal): SD E‐Intake (kcal):

1  6.8834  2370  4592 3061

2  6.0865  2095  4080 2720

3  6.6316  2280  4444 2963

4  7.0298  2420  4661 3107

6  6.5149  2240  4357 2905

7  6.1158  2105  4043 2696

5  5.9278  2040  3923 2615

Participant #: Accelerometer E‐Expenditure (kcal): Beep Test Score: VO2‐Max: Max‐HR (bpm):

1 3700 10.7 48.9 186

2 3500 12.7 55.7 198

3 4200  15  64.1  210 

4 4200 10.6 48.6 180

6 3500 11.2 50.8 186

7 3100 10.2 47.4 162

5 2970 11 50.3 N/A

Subject #  Weight (kg)  FFM (kg)  PRO‐Intake (g/kg)  A.P.E.  VCO2 (ml/min)  F13CO2  F13CO2 (FFM) 6  84.82  71.91  0.2  0.01085376  281.7111111  1.176408538  1.387609126 2  79.78  74.115  0.225  0.009403998  280.6956522  1.079758034  1.16228963 1  88.18  76.58  0.25  0.011219405  241.2650602  1.001766307  1.15350944 4  87.72  68.407  0.3  0.009076886  272.5128205  0.920232248  1.180036733 3  87.09  77.408  0.35  0.006278581  243.0444444  0.571809227  0.643329702 7  75.36  67.15  0.4  0.006264227  241.9662921  0.656377536  0.73662861 2  79.78  74.115  0.45  0.010333844  266.7471264  1.127560605  1.213746003 3  87.09  77.408  0.5  0.004580104  354.2459016  0.607972847  0.684016577 2  79.78  74.115  0.55  0.00584702  267.9078521  0.640764176  0.689741159 4  87.72  68.407  0.6  0.006556779  308.0041119  0.751312662  0.96342694 6  84.82  71.91  0.65  0.006258226  310.0375  0.746516634  0.880538741 7  75.36  67.15  0.7  0.004601528  280.6818182  0.559303698  0.627686176 1  88.18  76.58  0.75  0.00647773  289.8823529  0.694939197  0.800205515 6  84.82  71.91  0.8  0.0061841  282.7808219  0.672822356  0.793614132 4  87.72  68.407  0.85  0.004972644  300.0138889  0.555011996  0.711705707 7  75.36  67.15  0.9  0.003493907  256.8838428  0.388668585  0.436188601 2  79.78  74.115  0.95  0.007702033  242.04  0.762554216  0.820840253 3  87.09  77.408  1  0.003982914  277.2615385  0.413803797  0.465561346 1  88.18  76.58  1.05  0.006152891  264.3580081  0.601968652  0.693152204 4  87.72  68.407  1.1  0.00425865  308.0041119  0.487980059  0.625748984 

1  88.18  76.58  1.15  0.007446627  240.4782609  0.662731418  0.763119045 3  87.09  77.408  1.2  0.002433046  239.0333333  0.217927819  0.245185688 6  84.82  71.91  1.25  0.004223053  277.3648649  0.450663058  0.531570582 2  79.78  74.115  1.3  0.006514465  279.9342105  0.745955559  0.802972873 7  75.36  67.15  1.35  0.002667755  252.7333333  0.291971112  0.327668548 2  79.78  74.115  1.4  0.004888827  277.6779661  0.55529574  0.597739919 6  84.82  71.91  1.45  0.003923703  292.4564367  0.441500635  0.52076323 4  87.72  68.407  1.5  0.003872376  311.3090909  0.448479892  0.575096936 7  75.36  67.15  1.55  0.003097448  248.6438356  0.333513299  0.374289832 1  88.18  76.58  1.6  0.005575369  262.5890411  0.541816707  0.623888707 3  87.09  77.408  1.65    0  0 1  88.18  76.58  1.7  0.004527471  264.3580081  0.442945548  0.510040982 

7  75.36  67.15  1.75  0.002290812  262.9027778  0.260805094  0.29269206 6  84.82  71.91  1.8  0.00499188  298.8333333  0.573940834  0.676980413 3  87.09  77.408  1.85    0  0 4  87.72  68.407  1.9  0.003949421  296.7529412  0.436015672  0.559113757 2  79.78  74.115  1.95  0.004440971  247.5633803  0.449720337  0.484094832 1  88.18  76.58  2  0.0044141  250.9333333  0.409923412  0.472016799 3  87.09  77.408  2.05  0.003917727  206.3382353  0.302912952  0.34080055 6  84.82  71.91  2.1  0.002819339  304.010989  0.329769577  0.388973099 4  87.72  68.407  2.15  0.003072847  359.4318182  0.410895247  0.52690121 7  75.36  67.15  2.2  0.001897828  254.375  0.209056055  0.234615998 2  79.78  74.115  2.25  0.002211918  280.6966292  0.253971233  0.273383593 5  73.41  62.34  2.45  0.001700551  278.3404255  0.210418053  0.247782953 4  87.72  68.33  2.5  0.0024627  308.0041119  0.282190062  0.362267119 2  79.78  74.115  2.55  0.003440106  267.9078521  0.376994956  0.405810667 2  79.78  74.115  2.6  0.003742096  267.9078521  0.410089465  0.441434764 

Highlighted trial indicates trial where participant became ill.  Any trials where F13CO2 values = 0 were not conducted 

FIGURE 1. Phenylalanine Oxidation Re‐Analysis 

Breakpoint = 1.56g/kg/d 

FIGURE 2. Comparison between Phenylalanine Flux Data and Protein Intake Including Humayun et al., 2007 

ValidationoftheIndicatorAminoAcidOxidationTechnique’sUtilizationInthePresenceofa1VariableIntensityExerciseStimulus2

3JeffreyPacker4

5TheUniversityofToronto6

789101112

Acknowledgements:DanMoore,DeniseWooding,HiroKato,MichaelMazzulla,&Sidney13AbouSawan1415KeyWords:IAAO,LIST1617AbstractWordCount:19818WordCount:128619

ABSTRACT20

ThepurposeofthepresentstudywastovalidatetheIndicatorAminoAcidOxidation21

(IAAO)Techniqueforitsusefollowingavariableintensityrunningtestthroughtheanalysis22

ofbackground13CO2enrichmentandrestingVCO2production.Itwashypothesizedthat23

13CO2enrichmentandVCO2productionwouldreachaplateauwithin2‐4hoursfollowingthe24

variableintensityexercisestimulusthatwouldbesustainedthroughouttheremainderofthe25

protocol,thusvalidatingtheIAAOTechnique’susefollowingsuchanexercisetest.Three26

activeadultmalesaged(22.7+/‐1.5years)partookinasinglemetabolictrialfollowinga27

two‐dayadaptationperiodproviding1.2g/kg/dofdietaryprotein.Afterfastingovernight,28

participantsengagedinamodifiedversionoftheLoughboroughIntermittentShuttleTest29

(LIST)followedbyan8‐hourmetabolictrialconsistingof8hourlyisocaloricbeverageseach30

containingtheequivalentof1.2g/kg/dofcompleteprotein.Breathsamplesweretaken31

every20‐minutesthroughoutthe8‐hourprotocolusingQuinTronbreathcollectionbags.32

VCO2productionwasmeasuredduringthefinal15‐20minutesofeveryhourofthe8‐hour33

protocolusingaMOXUSMetabolicCart(AEITechnologies).Analysisofmassspectrometry34

andVCO2datavalidatedtheuseoftheIAAOTechniquefollowingtheLISTstimulus.35

INTRODUCTION36

Theadequateingestionofdietaryaminoacids(AA)isthemostcriticalfactorin37

ensuringthehealthygrowthanddevelopmentofleanbodymassinindividualsofallages.38

Currentdietaryrecommendationsforproteinconsumptionarebasedpredominantlyonthe39

nitrogenbalancetechnique(NBAL).However,recentadvancesintheuseofstableisotopes40

haveprovidedforalternativemethodsthatcanbeusedtoevaluateproteinrequirements41

suchastheIAAOtechnique.42

AstudybyBrossetal.soughttodevelopandvalidatetheIAAOtechniqueforitsusein43

sedentaryadults(1).Thirteenhealthyadultfemales,andonehealthyadultmalereceiveda44

4‐horal,primed,equaldoseinfusionofeitherL‐[1‐13C]phenylalanineorL‐[1‐13C]lysineafter45

4hoursofsteady‐statefeedingwithoutthetracer.Isotopicplateauin13CO2wasachieved46

within120minutesofphenylalanineorlysineinfusion,suggestingthatprimed,equaldose,47

oralinfusionproducedsteady‐stateconditionsrequiredfortheimplementationoftheIAAO48

techniquewhendeterminingproteinrequirements.TheIAAOtechniquehassincebeen49

implementedinseveralstudiesforthepurposeofdeterminingproteinrequirements(2,5).50

OnestudybyHumayunetal.utilizedtheIAAOtechniqueinhealthy,youngadultmen(2).51

Theresultsfromthisstudysuggestthatcurrentproteinrequirementsestablishedusing52

NBALmightunderestimatetrueproteinrequirements.53

Physicalactivityhasbeenshowntopotentiallyincreaseproteinrequirementsin54

activeindividuals(3,4),yettheIAAOtechniquehasonlybeenvalidated,andassuch55

implementedinnon‐activepopulations(2).Thus,theobjectiveofthepresentstudyisto56

validatetheIAAOtechniqueforitsuseinactivepopulationsbyintegratingthevariable57

intensityLISTexercisestimulusintoatypicalIAAOprotocol.58

METHODS59

Threehealthy,youngadultmalesubjects(age22.7+/‐1.5years;height181.3+/‐3.560

cm;weight85.7+/‐9.3kg)wererequiredtowearaSensewearBodyMediaArmband61

Accelerometerforthreedayswhileengagingintheirhabitualexerciseanddietaryroutine.62

Subjectswerealsorequiredtocompleteathree‐daydietarylog.Habitualenergy63

expenditureswereaveragedoverthethree‐dayaccelerometerperiod,providingforan64

estimationofeachsubject’stypicaldailyenergyexpenditure.65

Subjectsengagedinanovernightfastpriortothemetabolictrial,andreportedto66

laboratoryintherestedstatehavingfastedandabstainedfromalcoholandcaffeine67

consumptionfor24‐hours.Uponarrival,subjectscompleteda15‐20minutebaselineVCO268

collection.Subjectsthenconsumedaprotein‐freebreakfastproviding1g/kgof69

carbohydratesfromPolycose(0.5g/kg)andGatoradepowder(0.5g/kg).Subjectsthen70

completedtheLIST,whichconsistedoffour15‐minuteperiodsofvariableintensityexercise71

(walking,sprinting,running,jogging)separatedby5minutesofrest(Figure1).72

UponcompletionoftheLISTexercisestimulus,subjectsconsumed8isocaloricand73

isoproteinichourlymealscontainingcaloriesandproteinequivalentto⅔oftheirdaily74

energyexpenditureasmeasuredontheSensewearBodyMediaArmbandAccelerometer,75

and⅔of1.2g/kg/dofproteinrespectively.Breathsampleswerecollectedevery20minutes76

throughoutthe8‐hourmetabolictrialusingQuinTronbreathcollectionbagsandvacuumed77

Serconexertainers.VCO2wascollectedduringthefinal15‐20minutesofeachhour78

throughoutthe8‐hourprotocolusingtheMOXUSMetabolicCart(AEITechnologies).79

Breathsampleswereanalyzedfor13CO2enrichmentusingmassspectrometry.VCO280

dataforeachtime‐pointwasexportedtoMicrosoftExcelandaveragedforanalyticpurposes.81

RESULTS82

13CO2enrichmentreachedaplateauinallsubjectswithin3hoursofthefirsthourly83

meal’sconsumption.Thisplateauwassustainedthroughouttheremainderoftheprotocolin84

allsubjects(Figure2).Baselinefastedandrestingstate13CO2enrichmentwaslowerthan85

thepost‐exercise,fed‐state13CO2enrichmentinallsubjects.VCO2datawasconsistentboth86

withinandbetweensubjectsthroughoutthe8‐hourprotocol(Figure2).87

DISCUSSION88

13CO2enrichmentdatafollowedasimilartrendforallsubjects,inthatafter89

approximately3hoursoffeedingfollowingtheLISTexercisestimulus,aplateauin13CO290

enrichmentoccurred.Thisplateauin13CO2enrichmentwasgreaterthanbaselineinall91

subjects,andwassustainedthroughouttheremainderoftheprotocol(Figure2).However,92

thereweredifferencesbetweensubjectswithrespecttohow13CO2enrichmentatbaseline93

comparedto13CO2enrichmentinthefirstone‐to‐twohoursfollowingtheLISTexercise94

stimulus.Onesubject’sbaseline13CO2enrichmentwasgreaterthanthoseanalogousvalues95

correspondingtothefirstone‐to‐twohoursoffeedingfollowingtheLISTexercisestimulus96

(Figure3).However,afterthreehoursoffeeding,thissubject’s13CO2enrichmenthad97

reachedaplateauinexcessoftheirbaseline13CO2enrichment,similartothatoftheother98

twosubjects(Figure3).Theseresultscouldpotentiallybeexplainedbysubject‐based99

differencesintrainingstatusorhabitualdietaryconsumption.100

VCO2datawasconsistentinallsubjectsthroughoutthe8‐hourprotocol(Figure2).101

However,twooutlierswereremovedfromthedata.SinceVCO2isdependentontheenergy102

expenditureoftheparticipant,subjectmovementduringVCO2collectionmustbestrictly103

controlledinordertoensureconsistencyinthedata.Itisconceivablethatthediscrepancies104

intheoutlierVCO2datamaybeattributedtoalackofconsistencyinmonitoringofthe105

subjectsthroughoutthe20‐minuteVCO2collection.Thisrepresentsalimitationofthe106

presentstudydespitetheVCO2databeingconsistentoncetheoutlierswereremoved.107

Thesustainedplateauin13CO2enrichmentexhibitedinallsubjectsincombination108

withthestableandconsistentVCO2datajustifythattheIAAOTechniqueisasuitablemethod109

fordeterminingproteinrequirementsinthepresenceofthevariableintensityLISTexercise110

stimulus.Thishasmajorresearchimplications,astheIAAOTechniquehasyettobeusedto111

determineproteinrequirementsinactivepopulations,andhasneverbeenusedfollowing112

anytypeofexercisestimulus.SincestudiesthathaveemployedtheIAAOTechniqueinnon‐113

activepopulations,andstudiesutilizingNBALinactivepopulationshavebothestablished114

thatproteinrequirementsmaybegreaterthanthecurrentrecommendeddietaryallowance115

(RDA)(2),itisplausiblethatfuturestudiesutilizingtheIAAOTechniquemayyieldprotein116

requirementsthatareinexcessofthepresentRDA.117

CONCLUSIONS118

TheoutcomesofthepresentstudyvalidatedthehypothesisthattheIAAOTechnique119

issuitableforitsuseinthepresenceofthevariableintensityLISTexercisestimulus,asboth120

13CO2enrichmentandVCO2productionreachedaplateauwithin3hoursfollowingthe121

variableintensityexercisestimulusthatwassustainedthroughouttheremainderofthe122

protocol.FutureavenuesofresearchshouldseektoemploytheIAAOTechniqueinathletic123

populationsinordertoestablishoptimalproteinrequirementsforsuchindividuals.Such124

studieswillhavemajorimplicationsonbothhealthandsportperformance.125

REFERENCES126

Bross,R.,Ball,R.,&Pencharz,P.B.(1998).Developmentofaminimallyinvasiveprotocolfor127

thedeterminationofphenylalanineandlysinekineticsinhumansduringthefedstate.128

JournalofNutrition,128,1913‐1919.129

Humayun,M.A.,Elango,R.,Ball,R.O.,&Pencharz,P.B.(2007).Reevaluationoftheprotein130

requirementinyoungmenwiththeindicatoraminoacidoxidationtechnique.The131

AmericanJournalofClinicalNutrition,86(4),995‐1002.132

Tarnopolsky,M.A.,Atkinson,S.A.,MacDougall,J.D.,Chesley,A.,Phillips,S.,&Schwarcz,H.P.133

(1992).Evaluationofproteinrequirementsfortrainedstrengthathletes.Journalof134

AppliedPhysiology(Bethesda,Md.:1985),73(5),1986‐1995.135

Tarnopolsky,M.A.,MacDougall,J.D.,&Atkinson,S.A.(1988).Influenceofproteinintakeand136

trainingstatusonnitrogenbalanceandleanbodymass.JournalofAppliedPhysiology137

(Bethesda,Md.:1985),64(1),187‐193.138

Zello,G.A.,Pencharz,P.B.,&Ball,R.O.(1993).Dietarylysinerequirementofyoungadult139

malesdeterminedbyoxidationofL‐[1‐13C]phenylalanine.TheAmericanJournalof140

Physiology,264(4Pt1),E677‐85.141

FIGURE3.ThemodifiedLoughboroughIntermittentShuttleTest(LIST).TheLISTconsistsoffour15‐minuteperiodsofvariableintensityexercise(walking,sprinting,running,jogging)separatedby5minutesofrest.Thetimetocompleteeach‘pattern’oftheLISTis90seconds,whichisrepeatedtentimestocompriseone15‐minuteblockofvariableintensityexercise.

FIGURE4.Theeffectofexperimentaldieton13CO2enrichmentexpressedasatomspercentexcess(A.P.E.;)andontherateofCO2production(VCO2;☐).Aplateauinaveraged13CO2enrichmentandaveragedVCO2wasachievedforallsubjectsbeginningat~180minand~60minrespectively.Timeof0equatestobaseline13CO2enrichmentandbaselineVCO2.

FIGURE5.Theeffectofexperimentaldieton13CO2enrichmentexpressedasatomspercentexcess(A.P.E.)foreachsubjectinthestudy(Subject1☐;Subject2;Subject3Δ).Aplateauin13CO2productionwasachievedby~180minutesinallsubjects.Subject2obtaineda13CO2enrichmentplateauatapproximately180minutesdespiteinitialpost‐exercisefed‐state13CO2enrichment(0‐180minutes)beinglowerthanbaseline13CO2enrichment.Timeof0equatestobaseline13CO2enrichment.

C: 647‐825‐7032 

IAAOStudyLabManual             

ParticipantName:ParticipantNumber:

 CreatedbyJeffPacker

E:[email protected]

Table of Contents Section1.IAAOStudyOverview(3)

‐1.1AnIntroductiontotheIndicatorAminoAcidOxidation(IAAO)Technique‐1.2PurposeofthePresentStudy

 Section2.PhaseIProtocol,IntroductorySession(6‐7)

‐2.1Trial‐2.2PhaseIDataTables

 Section3.PhaseIIProtocol,BodyComposition&FitnessAssessment(8‐9)

‐3.1Pre‐Trial‐3.2Trial‐3.3Post‐Trial‐3.4PhaseIIDataTables

 Section4.PhaseIIIProtocol,MetabolicTrials(9‐10)

‐4.1Pre‐Trial‐4.2Trial‐4.3Post‐Trial‐4.4PhaseIIIDataTables

 Section5.Appendices(11‐25)

‐ A)Consent,AssentForms 

‐ B)PAR‐Q+Questionnaire,IPAQQuestionnaires(Children+Adults) 

‐ C)DietaryLogSheet 

‐ D)Description&UseofSensewearBodyMediaArmbandAccelerometer 

‐ E)TheLISTExerciseStimulus 

‐ F)Descriptionsof2‐DayStudyDiet&Protein‐FreeBreakfast 

‐ G)HowtoMake1kgTestProteinMix&TestProteinAminoAcidComposition 

‐ H)HowtoMakeStudyDayMeals 

‐ I)CookieRecipe

Section 1. IAAO Study Overview: 1.1AnIntroductiontotheIAAOTechniqueTheIAAOtechniqueutilizesstableisotopes(13C)tolabelasingle‘indicator’aminoacid.TheIAAOtechniquedictatesthatanindicatoraminoacidisalwayssupplementedinexcessoftheproteinrequirement,whiletheremainingintakeofaminoacidswillrangefrombeingdeficienttoinexcessoftheproteinrequirementthroughtheconsumptionofcrystallineaminoacidsbasedontheaminoacidpatternofeggprotein.Whenasingleessentialaminoacidfromthedietislimiting(belowtherequirement),theotheraminoacids(includingtheindicator)cannotbeoptimallyutilizedforproteinsynthesis,andaredirectedtowardsoxidation.Inasituationwheretheindicatoraminoacidisconsumedinexcess,whiletheremainingtestproteinconsumptionisdeficient,anyamountoftheindicatoraminoacidinexcessofthetestproteinwillbedirectedtowardsoxidation.Inthisscenario,onewouldexpectthelevelsof13Cfromtheoxidizedindicatoraminoacidtobehigh,andoverallproteinsynthesistobecompromised.Astheconsumptionofthelimitingaminoacidsinthetestproteinisincreased,alesserproportionoftheindicatoraminoacidwillbeoxidized;thiswouldresultinadecreaseinbreathandurinary13Cfromtheindicatoraminoacid,asagreaterproportionoftheingestedaminoacidsareutilizedforproteinsynthesis(SeeFigureBelow).Oncetheproteinrequirementforthetestaminoacidisreached,anyadditionalintakeofthetestproteinabovetheproteinrequirementshouldnotresultinafurtherdecreaseintheindicatoraminoacidoxidationasthisaminoacidwillalwaysbeconsumedinexcessofitsrequirement.Additionally,thereshouldbenofurtherincreasesinproteinsynthesisoncethetestproteinintakeexceedsthatoftheproteinrequirement.Thepointwherenofurtherdecreasesinindicatoraminoacidoxidation(asmeasuredvia13CO2production)areseendespiteincreasesinthetestproteinintakeistermedthe‘breakpoint’(SeeFigureBelow).ItisthispointthatdeterminestheEstimatedAverageRequirement(EAR)ofthetestprotein,essentiallyallowingforproteinrequirementstobeestablishedfromtheIAAOtechnique.TheRecommendedDietaryAllowance(RDA)canbedeterminedbyaddingtwostandarddeviationstotheEARasdeterminedbythebreakpoint.

                        Isotopicallylabeled[1‐13C]‐phenylalanineisthemostcommonlyusedindicatoraminoacidwhenadministeringtheIAAOtechnique.Ingeneral,subjectsarerequiredtoconsumetheindicatoraminoacid,alongwithseveraldifferenttestproteinintakesthroughouttheIAAOprotocol.Subjectspartakein7isolatedtrialswherebythetestproteinconsumptionwillrangefromdeficienttoexcess.Therelationshipbetweenthetestproteinintakeandtheindicatoraminoacidoxidationisdeterminedduringeachtrial,allowingforthebreakpoint(EAR),andRDAtobeestablished.

 1.2PurposeofthePresentStudyThepurposeofthepresentstudyisto,forthefirsttime,utilizetheminimallyinvasiveIAAOtechniquetoevaluateoftheimpactofavariableintensityexerciseonproteinrequirementsinactivepopulations.Itishypothesizedthatthestudy’sresultswillyieldhigherproteinrequirementsthanthoseanalogousvaluesestablishedusingNBAL.

Section 2. Phase I Protocol, Introductory Session: Atotalof7participantswillberecruitedfromeachdemographicgroup(i.e.foryoung,adultmales,7participantsaged18‐35willberecruited).Therearethreephasesofthepresentstudy.Eachparticipantwilltakeintwopreliminarytrials,eachlastingone‐to‐twohours(PhaseIandPhaseII),followedby7metabolictrials(PhaseIII).

 2.1Trial‐ThepurposeofPhaseIwillbetoprovideanintroductiontothestudytotheparticipants.1)SubjectsarriveattheGoldringMetabolismandSportScienceLab2)Provideintroductiontothestudy&provideanopportunityforparticipantstoaskquestions3)Participants(andtheirparentsifapplicable)areprovidedwithconsent+assentformstosign(SeeAppendix)4)ParticipantscompletethePAR‐Q+surveytoassesstheirhealth(SeeAppendix)5)ParticipantscompletetheIPAQsurveytoassesstheirhabitualactivitylevels(SeeAppendix)6)Screenthesurveystoensurethattheparticipantsmeettherequirementsforthestudy

‐Criteria:Nohealthrisks(PAR‐Q+),&performmoderatetovigorousexercise≥5timesperweek(IPAQ)7)Onceparticipantshavebeenscreened,andmeettheinclusioncriteria,theyareofficiallyenrolledinthestudy8)Set‐UptheSensewearAccelerometerwithparticipant’sinformationtobewornpriortoPhaseII

‐Participantsmustweartheaccelerometerfor3dayspriortoPhaseII9)ExplaintheDietaryLogformtobefilledoutpriortoPhaseII(SeeAppendix)

‐Participantsmustcompletea3‐daydietarylogpriortoPhaseI

2.2PhaseIDataTable 

ParticipantNumber: Date: 

Height(cm): Weight(kg): 

Event Date(Time) Checklist1)SubjectsarriveatGoldring    

2)Introductiontostudy&opportunitytoaskquestions    

3)Participantssignconsent&assentforms(if applicable)    

4)ParticipantscompletethePAR‐Q+andIPAQ surveys    

5)Screensurveystoensureparticipantsmeetinclusion criteria‐InclusionCriteria:Nohealthrisks&moderate‐vigorous≥5/week

6)SetUpSensewearaccelerometerwithparticipant information‐Instructparticipantstowearaccelerometerfor3dpriortoPhaseII

7)Provideparticipantswithdietarylog‐Instructparticipantstocompletea3‐daydietarylogpriortoPhaseII

Section 3. Phase II Protocol, Body Composition & Fitness Assessment: 3.1Pre‐Trial*Subjectswillhavecompletedthe3‐daydietarylogandhavingworntheSensewearBodyMediaArmbandAccelerometerfor3dayspriortoreportingtotheGoldringLabforPhaseII.

 1)Inputtheaccelerometerdatatothelabcomputer,andsave2)Keepfileofdietarylogforfuturereference3)EnsurethatBodPodisavailableforuse4)EnsurethatFieldHouseattheAthleticCentre,orthegymatGoldringisavailableforuse

 3.2Trial–PhaseIIwillservetocollectbodycompositionmeasurements,andtoprovideafitnesstesttoparticipants1)SubjectsarriveattheGoldringMetabolismandSportScienceLab2)Recordsubject’sheight(cm)andweight(kg)3)ProvidesubjectswithaverbalintroductiontotheBodPod,beeptest,andLISTexercisestimulus4)PerformBODPODanalysis5)PlaceSensewearBodyMediaArmbandaccelerometerontheparticipantsleftarm6)ParticipantsperformtheBeepTest

‐Recordmax‐HR(usingcarotidpalpation)andbeeptestlevelobtained‐TimestampbothbeforeandaftertheBeepTest

7)Provide15‐20minutesofrestbeforepartakingintheLISTexercisestimulusintroduction(SeeAppendix)‐ParticipantscompletetheLISTexercisestimulus

8)ParticipantsreturntheGoldringMetabolismandSportScienceLab9)ProvidebriefexplanationofthemetabolictrialdaysinPhaseIII10)Provide2‐daystudydiettobeconsumedthetwo‐dayspriortothefirstmetabolictrial

 3.3Post‐Trial1)EnsurethatparticipantsreporttolabforPhaseIIIfollowinganover‐nightfast2)Ensurethatparticipantsrefrainfromconsumingalcohol,orcaffeinepriortoeachmetabolictrial3)Atime‐periodofatleast72hmustoccurbetweenPhaseIIandPhaseIII4)Saveallaccelerometerdata5)Ensurethatparticipantsmeetbeeptestinclusioncriteria

3.4PhaseIIDataTables ParticipantNumber: Date:

 Height(cm): Weight(kg):

BeepTestLevelObtained: Max‐HR(bpm):

Event Date(Time) Checklist1)SubjectsarriveattheGoldringMetabolism and Sport Science Lab‐Saveaccelerometerdata&collect3‐daydietarylog

2)Recordsubject’sheight(cm)andweight(kg) above    

3)ProvideanintroductiontotheBODPOD,beep test, and LIST exercise    

4)PerformBODPODanalysis    

5)OutfitSensewearAccelerometeronparticipants    

6)Participantsperformthebeeptest(timestamp before +after beep test)‐RecordBeepTestLevelObtained&Max‐HRonBeepTest

7)Participantsrestfor15‐20minutespriorto LIST introduction test    

8)ParticipantsperformoneblockoftheLIST for familiarization    

9)ParticipantsreturntotheGoldringMetabolism and Sport Science Lab    

10)Provideabriefexplanationtothetrialdays in Phase III    

11)Provideparticipantswithadaptationdiet to be consumed 2‐d prior toeachtrialinPhaseIII

POST‐TRIALSaveallaccelerometerdatafrom Phase II‐Ensurethatparticipantsmeetthebeeptestinclusioncriteria(≥level10)

Section 4. Phase III Protocol, Metabolic Trials: 4.1Pre‐Trial1)Subjectsmustreporttolabhavingconsumedthe2‐daystudydiet,andhavingfastedovernight2)Ensurethatallstudydaymealshavebeenportioned(SeeAppendix)3)EnsurethatMOXUSisset‐up,calibrated,andreadyforuse4)EnsurethattheFieldHouseor(preferably)theGoldringGymcanbeusedfortheLISTexercisestimulus5)Haveprotein‐freebreakfastreadyforconsumption(SeeAppendix)6)Prepareallexertainersforthetrial(~40),andurineeppendorfs(~25)

 4.2Trial0:00 ‐ParticipantsarrivetotheGoldringLabintheearlymorning

‐Subjectmusthaveconsumedthe2‐dadaptationdiet,andfastedovernight0:00‐Participantsareoutfittedwiththeaccelerometer,consumethePRO‐freebreakfast,andthetrialisexplained1:00to2:30‐ParticipantscompletetheLISTExerciseStimulus(SeeAppendix)

‐RemembertotimestamptheaccelerometerbothbeforeandaftertheLIST2:30‐Participantsconsumethefirstof8hourlymeals3:30‐Participantsconsumethesecondof8hourlymeals4:30‐Participantsconsumethethirdof8hourlymeals5:30‐Participantsconsumethefourthof8hourlymeals(collectbaselinebreathandurinesamples)

‐Breath:3Exertainers@5:30,5:45,6:00,6:15(3x4=12BaselineBreathSamplesfrom4time‐points)‐Urine:3Eppendorfs@5:30,6:00,6:30(3x3=9BaselineUrineSamplesfrom2time‐points)

6:30‐Participantsconsumethefifthof8hourlymeals‐ParticipantsconsumetheIsotopePrimer,theBicarbonatePrimer,andthefirstIsotopeCI

7:30‐Participantsconsumethesixthof8hourlymeals‐ParticipantsconsumethesecondIsotopeCI

8:00‐Breath(CO2)analysisfor20‐30minutesonMOXUSmetaboliccart‐Ensuretosavealldata

8:30–Participantsconsumetheseventhof8hourlymeals(collectplateaubreathandurinesamples)‐ParticipantsconsumethethirdIsotopeCI‐Breath:3Exertainers@8:30,8:45,9:00,9:15,9:30,9:45,10:00,10:15(3x8=24PlateauBreathSamples)‐Urine:3Eppendorfs@8:30,9:00,9:30,10:00,10:30(3x5=15PlateauUrineSamples)

9:30–Participantsconsumetheeighthof8hourlymeals(continuetocollectbreathandurineasabove)10:30–MetabolicTrialiscomplete

‐Provide2‐daystudydiettoparticipantsforthenextmetabolictrialifneeded 4.3Post‐Trial1)Inputallaccelerometerdataintothelabcomputer&savemetaboliccartdata2)Placenecessarysamplesintheappropriatefridgeorfreezer3)ReturnbreathandurinesamplestoSickKidsforanalysis

 Aminimumofa3‐daywashoutperiodmustoccurbetweenPhaseIIItrials

4.4PhaseIIIDataTables ParticipantNumber: TrialNumber: Date:

 ActualTime                    

Time(h) 0:00 1:00 2:30 3:30 4:30 5:30 5:45 6:00 6:15 6:30Breakfast                    

Drink                    

Breath                    

Urine                    

CO2                    

LIST   LIST1:00to2:30

  ActualTime                      

Time(h) 7:30 8:00 8:30 8:45 9:00 9:15 9:30 9:45 10:00 10:15 10:30Breakfast                      

Drink                      

Breath                      

Urine                      

CO2   CO28:00to8:30

LIST                      

ParticipantNumber: TrialNumber: Date: ActualTime                    

Time(h) 0:00 1:00 2:30 3:30 4:30 5:30 5:45 6:00 6:15 6:30Breakfast                    

Drink                    

Breath                    

Urine                    

CO2                    

LIST   LIST1:00to2:30

  ActualTime                      

Time(h) 7:30 8:00 8:30 8:45 9:00 9:15 9:30 9:45 10:00 10:15 10:30Breakfast                      

Drink                      

Breath                      

Urine                      

CO2   CO28:00to8:30

LIST                      

ParticipantNumber: TrialNumber: Date: ActualTime                    

Time(h) 0:00 1:00 2:30 3:30 4:30 5:30 5:45 6:00 6:15 6:30Breakfast                    

Drink                    

Breath                    

Urine                    

CO2                    

LIST   LIST1:00to2:30

  ActualTime                      

Time(h) 7:30 8:00 8:30 8:45 9:00 9:15 9:30 9:45 10:00 10:15 10:30Breakfast                      

Drink                      

Breath                      

Urine                      

CO2   CO28:00to8:30

LIST                      

ParticipantNumber: TrialNumber: Date: ActualTime                    

Time(h) 0:00 1:00 2:30 3:30 4:30 5:30 5:45 6:00 6:15 6:30Breakfast                    

Drink                    

Breath                    

Urine                    

CO2                    

LIST   LIST1:00to2:30

  ActualTime                      

Time(h) 7:30 8:00 8:30 8:45 9:00 9:15 9:30 9:45 10:00 10:15 10:30Breakfast                      

Drink                      

Breath                      

Urine                      

CO2   CO28:00to8:30

LIST                      

ParticipantNumber: TrialNumber: Date: ActualTime                    

Time(h) 0:00 1:00 2:30 3:30 4:30 5:30 5:45 6:00 6:15 6:30Breakfast                    

Drink                    

Breath                    

Urine                    

CO2                    

LIST   LIST1:00to2:30

  ActualTime                      

Time(h) 7:30 8:00 8:30 8:45 9:00 9:15 9:30 9:45 10:00 10:15 10:30Breakfast                      

Drink                      

Breath                      

Urine                      

CO2   CO28:00to8:30

LIST                      

ParticipantNumber: TrialNumber: Date: ActualTime                    

Time(h) 0:00 1:00 2:30 3:30 4:30 5:30 5:45 6:00 6:15 6:30Breakfast                    

Drink                    

Breath                    

Urine                    

CO2                    

LIST   LIST1:00to2:30

  ActualTime                      

Time(h) 7:30 8:00 8:30 8:45 9:00 9:15 9:30 9:45 10:00 10:15 10:30Breakfast                      

Drink                      

Breath                      

Urine                      

CO2   CO28:00to8:30

LIST                      

ParticipantNumber: TrialNumber: Date: ActualTime                    

Time(h) 0:00 1:00 2:30 3:30 4:30 5:30 5:45 6:00 6:15 6:30Breakfast                    

Drink                    

Breath                    

Urine                    

CO2                    

LIST   LIST1:00to2:30

  ActualTime                      

Time(h) 7:30 8:00 8:30 8:45 9:00 9:15 9:30 9:45 10:00 10:15 10:30Breakfast                      

Drink                      

Breath                      

Urine                      

CO2   CO28:00to8:30

LIST                      

ParticipantNumber: TrialNumber: Date: ActualTime                    

Time(h) 0:00 1:00 2:30 3:30 4:30 5:30 5:45 6:00 6:15 6:30Breakfast                    

Drink                    

Breath                    

Urine                    

CO2                    

LIST   LIST1:00to2:30

  ActualTime                      

Time(h) 7:30 8:00 8:30 8:45 9:00 9:15 9:30 9:45 10:00 10:15 10:30Breakfast                      

Drink                      

Breath                      

Urine                      

CO2   CO28:00to8:30

LIST                      

Section5.Appendices 5.1Consent,AssentForms

5.2 PAR‐Q+Questionnaire 

5.3IPAQQuestionnaires(Children+Adults) 

5.4Description&UseoftheSensewearBodyMediaAccelerometer 

5.5DietaryLogSheet(x3)&Instructions 

5.6TheLISTExerciseStimulus 

5.7Descriptionofthe2‐DayStudyDiet 

5.8DescriptionoftheProtein‐FreeBreakfast 

5.9HowtoMakethe1kgTest‐ProteinMix,1kgAminoAcidMix(Composition) 

5.10 HowtoMakeStudyDayMeals 

5.11 CookieRecipes

AppendixL–MetabolismStudyConsentFormforAdults

TitleofResearchProject:•Aminoacidmetabolismandbasicrequirementsinactive,youngadultsInvestigatorsContactnumber

DanMooreProfessor,FacultyofKinesiology,UniversityofToronto416‐946‐4088GlendaCourtney‐MartinPhD,SickKidsHospital,416‐813‐5744JeffPacker MScStudent,FacultyofKinesiology,UniversityofToronto647‐825‐7032PurposeoftheResearch:Proteinisanessentialnutrientweneedtoeatenoughofinourdiettomaintainimportant bodily functions and to support normal growth in children andadolescents and the recovery from exercise. Currently how much protein isneeded in healthy, active school‐age children and adolescents is not knownexactly. Additionally, active children tend to have greater amounts of muscleand strongerbones than childrenwho are inactive. Since proteinprovides thebuildingblocks for the growthofmuscleandbone, the effect that exercisehason how much protein children and adolescents need to eat is not wellunderstood with current recommendations being based mostly on estimatesfrom adults. Better understandingof the specific needs for protein during theimportant growth periods of childhood and adolescence up to and includingyoungadulthoodis neededto ensureyoung individualsaremeetingthebody’sprotein needs, including that for optimal growth and for the recovery fromexercise.DescriptionoftheResearch:Ifyoudecidetoenterthisstudywewillmeasurethetotalamountofproteinyouneedanduse that informationtodeterminewhat the requirementis for youngadults. The results from this study will be compared to that of children andadolescents to determine if protein requirements change with growth andmaturation. This study will involve a total of 9 separate trial days includingtoday’s informationsession. Thestudydayswillbe separatebutwillbecompletedoveraperiodspanningacoupleofmonths.

In the event that you agree to participate in the study, the remainder of thissession will serve to assess your physical activity levels and general healththroughthecompletionoftwodistinctsurveys.Followingthecompletionofthesesurveys,wewillprovideyouwithanaccelerometer(adeviceusedtomeasurenormal activity patterns and energy expenditure), and 3 days of food recorddocumentationalongwiththeappropriate instructions.Forthe3daysprior toreturning the Cardiovascular Regulation Labhere at theAthletic Centre at theUniversity of Toronto for Session 2, you will be required to wear theaccelerometer,aswellas fillouta3‐day foodrecordofwhatyounormallyeataccordingtotheinstructionswehaveprovidedyouwith.

 Onthesecondsession,youwillreturntotheCardiovascularRegulationLab.Wewillthenassessyourbodycomposition(amountoffatandfat‐freemass)usingone non‐invasive measure called the BODPOD The BODPOD is a non‐invasivemethodusedtoestimateyourbodycomposition,anddoesnotcauseanypain.TheBODPODrequiresyoutositinachamberforlessthanaminutetoestablishtheamountofairyoudisplacewithinthechamber.Thisallowsustoestimateyourbody composition. Upon completion of the body composition measures,youwillengageinanaerobicfitnesstestcalledtheBeepTest.Thebeeptestrequiresyoutorunbackandforthbetweenconesspaced20‐metresapartuntilvolitionalfatigue,andhasbeenvalidatedinallagegroups.Oncethebeeptestassessmenthasbeencompleted,youwillbegivenanopportunitytorestpriortoperformingadditional fitness assessments including a handgrip strength exercise and averticaljumptoassesslowerlimbmusclefunction.Youwillthenbeintroducedtotheexercisetestthatwillbeconductedonallsubsequenttrialdays,whichwillinvolve running at different speeds that will be similar to what you wouldencounterduringasoccergame.

 Priortosessionsthreetonine,youwillweartheaccelerometerfor2daysandwillconsume a controlled liquid diet designed to provide adequate energy andprotein.YouwillthenreturntotheCardiovascularRegulationLabinthemorninghavingrefrainedfromconsuminganybreakfast.Youwillthenbeequippedwiththe accelerometer andwill consume a small liquid breakfast. Youwill thenbepermittedtorestfor30minutespriortoengagingintheexerciseprotocol.TheexerciseprotocolwillbeconductedinopenenvironmentintheFieldHouseattheAthleticCentreattheUniversityofToronto.Itwillconsistofrunningatdifferentspeedsspanningfromasprint,toarun,toajog,toawalk,whichwillbesimilartothe typeofactivitypatterns thatwouldoccurduringagame of soccer. Theexercise protocolwill take approximately 1 hour tocompletewithatotalof15minofrestthroughout.Followingthecompletionoftheexerciseprotocol,youwillconsumeamealeveryhourfor8consecutivehours,whichwillprovideyouwithyourenergyneedsandavariableamountofprotein.Youwillberequiredtooccasionallybreatheintoagascollectionchamberandtoprovideurinesamplesforanalysis.Onceall8mealshavebeenconsumedandalldatahasbeencollected,thetrialdaywillbecomplete.

Potential Harms:•Weknowofnoharmthattakingpartinthisstudycouldcauseyou.

 Potential Discomforts or Inconvenience:•Whilethisstudydoesnotcauseharmwerecognizethatthelengthofthestudyday, the number of days required to complete the study and travel to theUniversityofTorontomightposeaninconveniencetoyou.

 Potential Benefits:•Youwillnotbenefitdirectlyfromparticipatinginthisstudy.•Howevertheresultsofthisstudywillbeusedtodeterminetherequirementforproteininhealthyactiveyoungadults.Aseparategroupofchildrenandadolescentswillalsobetestedinaparallelstudytodeterminetheirproteinrequirements. Thiswillhelpusunderstandhowmuchproteindifferentagegroups(i.e.children,adolescents,andyoungadults)shouldconsumeintheirdietstosupportgrowthduringanactivelifestyle.

 Confidentiality:•Wewillrespectyourprivacy.Noinformationaboutwhoyouwillbegiventoanyoneorbepublishedwithoutyourpermission,unlessrequiredbylaw.Forexample,thelawcouldmakeusgiveinformationaboutyouhavebeenabused,ifyouhaveanillnessthatcouldspreadtoothers,ifyouorsomeoneelsetalksaboutsuicide(killingthemselves),orifcourtordersustogivethemthestudypapers.

 Thedataproducedfromthisstudywillbestoredinasecure,lockedlocation.Onlymembersoftheresearchteam(andmaybethoseindividualsdescribedabove)willhaveaccesstothedata.Thiscouldincludeexternalresearchteammembers.FollowingcompletionoftheresearchstudythedatawillbekeptaslongasrequiredthendestroyedasrequiredbyUniversityofTorontopolicy.Publishedstudyresultswillnotrevealyouridentity.

 Reimbursement:

 •Theproposedcompensationismeanttoadequatelyreimburseyouforanycostsincurred(e.g.parkingandasmallpost‐studymeal)andtoprovideatokengiftofappreciationforyoureffort.Compensationwillbe$100permetabolictrial

 Participation:•Itisyourchoicetotakepartinthisstudy.Youcanstopatanytime. Youwillbecompensatedforalltrialsthatyoupartakesin.

 •Newinformationthatwegetwhilewearedoingthisstudymayaffectyourdecisiontotakepartinthisstudy.Ifthishappens,wewilltellyouaboutthisnewinformation.Andwewillaskyouagainifyoustillwanttobeinthestudy.

Conflict of Interest:•I,andtheotherresearchteammembershavenoconflictofinteresttodeclare.

 Consent:

 Bysigningthisform,Iagreethat:1)Youhaveexplainedthisstudytome.Youhaveansweredallmyquestions.2)Youhaveexplainedthepossibleharmsandbenefits(ifany)ofthisstudy.3) IknowwhatIcoulddoinsteadofhavingmyselftakepartinthisstudy.Iunderstand

that I have the right to refuse to letmyself take part in the study. I also have therighttotakemyselfoutofthestudyatanytime

4) Iamfreenow,andinthefuture,toaskquestionsaboutthestudy.5) Iunderstandthatnoinformationaboutmyselfwillbegiventoanyoneorbepublishedwithoutfirstaskingmypermission.6).Ihavealsobeenprovidedthestudytimelineandbeengivendemonstrationsofallthemeasurestobeused.7)Iagree,orconsent,thatI maytakepartinthisstudy.

PrintedNameofAdult Adultsignature&date    

PrintedNameofpersonwhoexplainedconsent SignatureofPersonwhoexplainedconsent&date 

PrintedWitness’name(iftheparent/legalguardian Witness’signature&datedoesnotreadEnglish)

Ifyouhaveanyquestionsaboutthisstudy,pleasecall at     Ifyouhavequestionsaboutyourrightsasasubject inastudyor injuriesduringastudy, please contact either of the investigators or the ethics review board [email protected] or 416 946 3273.

COPYRIGHT © 2012 1 / 4 

!

CSEP approved Sept 12 2011 version  

PAR-Q+  

The Physical Activity Readiness Questionnaire for Everyone Regular physical activity is fun and healthy, and more people should become more physically active every day of the week. Being more physically active is very safe for MOST people. This questionnaire will tell you whether it is necessary for you to seek further advice from your doctor OR a qualified exercise professional before becoming more physically active.

SECTION 1 - GENERAL HEALTH  

Please read the 7 questions below carefully and answer each one honestly: check YES or NO. YES NO

1. Has your doctor ever said that you have a heart condition OR high blood pressure?  

2. Do you feel pain in your chest at rest, during your daily activities of living, OR when you do physical activity?

 3.

Do you lose balance because of dizziness OR have you lost consciousness in the last 12 months? Please answer NO if your dizziness was associated with over-breathing (including during vigorous exercise).

 4.

Have you ever been diagnosed with another chronic medical condition (other than heart disease or high blood pressure)?

5. Are you currently taking prescribed medications for a chronic medical condition?

 6.

Do you have a bone or joint problem that could be made worse by becoming more physically active? Please answer NO if you had a joint problem in the past, but it does not limit your current ability to be physically active. For example, knee, ankle, shoulder or other.

7. Has your doctor ever said that you should only do medically supervised physical activity?

 If you answered NO to all of the questions above, you are cleared for physical activity.

 Go to Section 3 to sign the form. You do not need to complete Section 2.

 › Start becoming much more physically active – start slowly and build up gradually. › Follow the Canadian Physical Activity Guidelines for your age (www.csep.ca/guidelines). › You may take part in a health and fitness appraisal. › If you have any further questions, contact a qualified exercise professional such as a

CSEP Certified Exercise Physiologist® (CSEP-CEP) or CSEP Certified Personal Trainer®

(CSEP-CPT). › If you are over the age of 45 yrs. and NOT accustomed to regular vigorous physical activity,

please consult a qualified exercise professional (CSEP-CEP) before engaging in maximal effort exercise.

If you answered YES to one or more of the questions above, please GO TO SECTION 2.

Delay becoming more active if: › You are not feeling well because of a temporary illness such as a cold or fever – wait until you

feel better › You are pregnant – talk to your health care practitioner, your physician, a qualified exercise

professional, and/or complete the PARmed-X for Pregnancy before becoming more physically active OR

› Your health changes – please answer the questions on Section 2 of this document and/or talk to your doctor or qualified exercise professional (CSEP-CEP or CSEP-CPT) before continuing with any physical activity programme.

COPYRIGHT © 2012 2 / 4 CSEP approved Sept 12 2011 version

SECTION 2 - CHRONIC MEDICAL CONDITIONS  

Please read the questions below carefully and answer each one honestly: check YES or NO. YES NO   

1.

  Do you have Arthritis, Osteoporosis, or Back Problems?

If yes, answer

questions 1a-1c

If no, go to question 2

 1a.

Do you have difficulty controlling your condition with medications or other physician-prescribed therapies? (Answer NO if you are not currently taking medications or other treatments)

1b.

Do you have joint problems causing pain, a recent fracture or fracture caused by osteoporosis or cancer, displaced vertebra (e.g., spondylolisthesis), and/ or spondylolysis/pars defect (a crack in the bony ring on the back of the spinal column)?

 1c.

Have you had steroid injections or taken steroid tablets regularly for more than 3 months?

2.

  Do you have Cancer of any kind?

If yes, answer

questions 2a-2b

If no, go to question 3

 2a.

Does your cancer diagnosis include any of the following types: lung/bronchogenic, multiple myeloma (cancer of plasma cells), head, and neck?

2b. Are you currently receiving cancer therapy (such as chemotherapy or radiotherapy)?   

3.

 Do you have Heart Disease or Cardiovascular Disease? This includes Coronary Artery Disease, High Blood Pressure, Heart Failure, Diagnosed Abnormality of Heart Rhythm

If yes, answer

questions 3a-3e

If no, go to question 4

 3a.

Do you have difficulty controlling your condition with medications or other physician-prescribed therapies? (Answer NO if you are not currently taking medications or other treatments)

 3b.

Do you have an irregular heart beat that requires medical management? (e.g. atrial brillation, premature ventricular contraction)

3c. Do you have chronic heart failure?  

3d. Do you have a resting blood pressure equal to or greater than 160/90 mmHg with or without medication? (Answer YES if you do not know your resting blood pressure)

 3e.

Do you have diagnosed coronary artery (cardiovascular) disease and have not participated in regular physical activity in the last 2 months?

 4.  Do you have any Metabolic Conditions? This includes Type 1 Diabetes, Type 2 Diabetes, Pre-Diabetes

If yes, answer

questions 4a-4c

If no, go to question 5

4a. Is your blood sugar often above 13.0 mmol/L? (Answer YES if you are not sure)

 4b.

Do you have any signs or symptoms of diabetes complications such as heart or vascular disease and/or complications affecting your eyes, kidneys, and the sensation in your toes and feet?

 4c.

Do you have other metabolic conditions (such as thyroid disorders, pregnancy- related diabetes, chronic kidney disease, liver problems)?

5.

 Do you have any Mental Health Problems or Learning Difficulties? This includes Alzheimer’s, Dementia, Depression, Anxiety Disorder, Eating Disorder, Psychotic Disorder, Intellectual Disability, Down Syndrome)

If yes, answer

questions 5a-5b

If no, go to question 6

 5a.

Do you have difficulty controlling your condition with medications or other physician-prescribed therapies? (Answer NO if you are not currently taking medications or other treatments)

5b. Do you also have back problems affecting nerves or muscles?

COPYRIGHT © 2012 3 / 4 CSEP approved Sept 12 2011 version

Please read the questions below carefully and answer each one honestly: check YES or NO. YES NO   

6.

 Do you have a Respiratory Disease? This includes Chronic Obstructive Pulmonary Disease, Asthma, Pulmonary High Blood Pressure

If yes, answer

questions 6a-6d

If no, go to question 7

 6a.

Do you have difficulty controlling your condition with medications or other physician-prescribed therapies? (Answer NO if you are not currently taking medications or other treatments)

 6b.

Has your doctor ever said your blood oxygen level is low at rest or during exercise and/or that you require supplemental oxygen therapy?

 6c.

If asthmatic, do you currently have symptoms of chest tightness, wheezing, laboured breathing, consistent cough (more than 2 days/week), or have you used your rescue medication more than twice in the last week?

 6d.

Has your doctor ever said you have high blood pressure in the blood vessels of your lungs?

7.

  Do you have a Spinal Cord Injury? This includes Tetraplegia and Paraplegia

If yes, answer

questions 7a-7c

If no, go to question 8

 7a.

Do you have difficulty controlling your condition with medications or other physician-prescribed therapies? (Answer NO if you are not currently taking medications or other treatments)

 7b.

Do you commonly exhibit low resting blood pressure significant enough to cause dizziness, light-headedness, and/or fainting?

 7c.

Has your physician indicated that you exhibit sudden bouts of high blood pressure (known as Autonomic Dysreflexia)?

 8.  Have you had a Stroke? This includes Transient Ischemic Attack (TIA) or Cerebrovascular Event

If yes, answer

questions 8a-c

If no, go to question 9

 8a.

Do you have difficulty controlling your condition with medications or other physician-prescribed therapies? (Answer NO if you are not currently taking medications or other treatments)

8b. Do you have any impairment in walking or mobility?  

8c. Have you experienced a stroke or impairment in nerves or muscles in the past 6 months?

 9.  Do you have any other medical condition not listed above or do you live with two chronic conditions?

If yes, answer

questions 9a-c

If no, read the advice on page 4

 9a.

Have you experienced a blackout, fainted, or lost consciousness as a result of a head injury within the last 12 months OR have you had a diagnosed concussion within the last 12 months?

 9b.

Do you have a medical condition that is not listed (such as epilepsy, neurological conditions, kidney problems)?

9c. Do you currently live with two chronic conditions?   

Please proceed to Page 4 for recommendations for your current medical condition and sign this document.

COPYRIGHT © 2012 4 / 4 CSEP approved Sept 12 2011 version

!

PAR-Q+  

If you answered NO to all of the follow-up questions about your medical condition, you are ready to become more physically active: › It is advised that you consult a qualified exercise professional (e.g., a CSEP-CEP or CSEP-CPT) to help

you develop a safe and effective physical activity plan to meet your health needs. › You are encouraged to start slowly and build up gradually – 20-60 min. of low- to moderate-intensity

exercise, 3-5 days per week including aerobic and muscle strengthening exercises. › As you progress, you should aim to accumulate 150 minutes or more of moderate-intensity physical

activity per week. › If you are over the age of 45 yrs. and NOT accustomed to regular vigorous physical activity, please

consult a qualified exercise professional (CSEP-CEP) before engaging in maximal effort exercise.  

If you answered YES to one or more of the follow-up questions about your medical condition: › You should seek further information from a licensed health care professional before becoming more

physically active or engaging in a fitness appraisal and/or visit a or qualified exercise professional (CSEP-CEP) for further information.

 Delay becoming more active if: › You are not feeling well because of a temporary illness such as a cold or fever – wait until you feel better › You are pregnant - talk to your health care practitioner, your physician, a qualified exercise profesional,

and/or complete the PARmed-X for Pregnancy before becoming more physically active OR › Your health changes - please talk to your doctor or qualified exercise professional (CSEP-CEP) before

continuing with any physical activity programme.

SECTION 3 - DECLARATION

› You are encouraged to photocopy the PAR-Q+. You must use the entire questionnaire and NO changes are permitted.

› The Canadian Society for Exercise Physiology, the PAR-Q+ Collaboration, and their agents assume no liability for persons who undertake physical activity. If in doubt after completing the questionnaire, consult your doctor prior to physical activity.

› If you are less than the legal age required for consent or require the assent of a care provider, your parent, guardian or care provider must also sign this form.

› Please read and sign the declaration below:

I, the undersigned, have read, understood to my full satisfaction and completed this questionnaire. I acknowledge that this physical activity clearance is valid for a maximum of 12 months from the date it is completed and becomes invalid if my condition changes. I also acknowledge that a Trustee (such as my employer, community/fitness centre, health care provider, or other designate) may retain a copy of this form for their records. In these instances, the Trustee will be required to adhere to local, national, and international guidelines regarding the storage of personal health information ensuring that they maintain the privacy of the information and do not misuse or wrongfully disclose such information.

NAME DATE   

SIGNATURE WITNESS   

SIGNATURE OF PARENT/GUARDIAN/CARE PROVIDER       

KEY REFERENCES

For more information, please contact: Canadian Society for Exercise Physiology

www.csep.ca

The PAR-Q+ was created using the evidence- based AGREE process (1) by the PAR- Q+Collaboration chaired by Dr. Darren E. R. Warburton with Dr. Norman Gledhill, Dr. Veronica Jamnik, and Dr. Donald C. McKenzie

1. Jamnik VJ, Warburton DER, Makarski J, McKenzie DC, Shephard RJ, Stone J, and Gledhill N. Enhancing the eectiveness of clearance for physical activity participation; background and overall process. APNM 36(S1):S3- S13, 2011. 2. Warburton DER, Gledhill N, Jamnik VK, Bredin SSD, McKenzie DC, Stone J, Charlesworth S, and Shephard RJ. Evidence-based risk assessment and recommendations for physical activity clearance; Consensus Document. APNM 36(S1):S266-s298, 2011.

(2). Production of this document has been made possible through financial contributions from the Public Health Agency of Canada and the BC Ministry of Health Services. The views expressed herein do not necessarily represent the views of the Public Health Agency of Canada or BC Ministry of Health Services.

1391IPAQ INTERNATIONAL RELIABILITY AND VALIDITY Medicine & Science in Sports & Exercise® 

APPENDIXB  

INTERNATIONAL PHYSICAL ACTIVITY QUESTIONNAIRE

(August 2002 version)

SHORT LAST 7 DAYS TELEPHONE FORMAT   

For use with Young and Middle-aged Adults (15-69 years)   

The International Physical Activity Questionnaires (IPAQ) comprises a set of 4 questionnaires. Long (5 activity domains asked independently) and short (4 generic items) versions for use by either telephone or self-administered methods are available. The purpose of the questionnaires is to provide common instruments that can be used to obtain internationally comparable data on health-related physical activity.

 Using IPAQ

 Use of the IPAQ instruments for monitoring and research purposes is encouraged. It is recommended that no changes be made to the order or wording of the questions as this will affect the psychometric properties of the instruments.

 Translation from English and Cultural Adaptation

 Translation from English is supported to facilitate worldwide use of IPAQ. Information on the availability of IPAQ in different languages can be obtained at www.ipag.ki.se. If a new translation is undertaken we highly recommend using the prescribed back translation methods available on the IPAQ website. If possible please consider making your translated version of IPAQ available to others by contributing it to the IPAQ website. Further details on translation and cultural adaptation can be downloaded from the website.

 Data Entry and Coding

 Attached to the response categories for each question are suggested variable names and valid ranges to assist in data management and interviewer training. We recommend that the actual response provided by each respondent is recorded. For example, "120 minutes" is recorded in the minutes response space. "Two hours" should be recorded as "2" in the hours column. A response of "one and a half hours" should be recorded as either "1" in hour column and "30" in minutes column.

 Further Developments of IPAQ

 International collaboration on IPAQ is on-going and an International Physical Activity Prevalence Study is in progress. For further information see the IPAQ website.

1392 Official Journal of the American College of Sports Medicine http://www.acsm-msse.org 

More Information  

More detailed information on the IPAQ process and the research methods used in the development of IPAQ instruments is available at www.ipaq.ki.se and Booth, M.L. (2000). Assessment of Physical Activity: An International Perspective. Research Quarterly for Exercise and Sport, 71 (2): s114-20. Other scientific publications and presentations on the use of IPAQ are summarized on the website.

Short Last 7 Days Telephone IPAQ   

READ: I am going to ask you about the time you spent being physically active in the last 7 days. Please answer each question even if you do not consider yourself to be an active person. Think about the activities you do at work, as part of your house and yard work, to get from place to place, and in your spare time for recreation, exercise or sport.

READ: Now, think about all the vigorous activities which take hard physical effort that you did in the last 7 days. Vigorous activities make you breathe much harder than normal and may include heavy lifting, digging, aerobics, or fast bicycling. Think only about those physical activities that you did for at least 10 minutes at a time.

 1. During the last 7 days, on how many days did you do vigorous physical

activities? Days per week [VDAY; Range 0-7, 8,9]

8. Don't Know/Not Sure

9. Refused   

[Interviewer clarification: Think only about those physical activities that you do for at least 10 minutes at a time.]

 Ilnterviewer note: If respondent answers zero, refuses or does not know, skip to Question 3]

 2. How much time did you usually spend doing vigorous physical activities

on one of those days? _ _ Hours per day [VDHRS; Range: 0-16]

  Minutes per day [VDMIN; Range: o-960, 998, 999]

 998. Don't Know/Not Sure 999. Refused

 [Interviewer clarification: Think only about those physical activities you do for at least 10 minutes at a time.]

1393IPAQ INTERNATIONAL RELIABILITY AND VALIDITY Medicine & Science in Sports & Exercise® 

[Interviewer probe: An average time for one of the days on which you do vigorous activity is being sought. If the respondent can't answer because the pattern of time spent varies widely from day to day, ask: "How much time in total would you spend over the last 7 days doing vigorous physical activities?"

Hours per week [VWHRS; Range: o-1121

Minutes per week [VWMIN; Range: o-6720, 9998, 9999]

9998. Don't Know/Not Sure 9999. Refused

READ: Now think about activities which take moderate physical effort that you did in the last 7 days. Moderate physical activities make you breathe somewhat harder than normal and may include carrying light loads, bicycling at a regular pace, or doubles tennis. Do not include walking. Again, think about only those physical activities that you did for at least 10 minutes at a time.

3. During the last 7 days, on how many days did you do moderate physical activities?

Days per week [MDAY; Range: o-7, 8, 9]

8. Don't Know/Not Sure 9. Refused

 [Interviewer clarification: Think only about those physical activities that you do for at least 10 minutes at a time]

[Interviewer Note: If respondent answers zero, refuses or does not know, skip to Question 5]

 4. How much time did you usually spend doing moderate physical activities

on one of those days? Hours per day [MDHRS; Range: 0-16]

Minutes per day [MDMIN; Range: o-960, 998, 999]

998. Don't Know/Not Sure 999. Refused

 [Interviewer clarification: Think only about those physical activities that you do for at least 10 minutes at a time.]

 [Interviewer probe: An average time for one of the days on which you do moderate activity is being sought. If the respondent can't answer because the pattern of time spent varies widely from day to day, or includes time

1394 Official Journal of the American College of Sports Medicine http://www.acsm-msse.org 

spent in multiple jobs, ask: "What is the total amount of time you spent over the last 7 days doing moderate physical activities?"

Hours per week [MWHRS; Range: 0-1121

Minutes per week [MWMIN; Range: 0-6720, 9998, 99991

9998. Don't Know/Not Sure 9999. Refused

 READ: Now think about the time you spent walking in the last 7 days. This includes at work and at home, walking to travel from place to place, and any other walking that you might do solely for recreation, sport, exercise, or leisure.

 5. During the last 7 days, on how many days did you walk for at least 10

minutes at a time? Days per week [WDAY; Range: o-7, 8, 91

8. Don't Know/Not Sure 9. Refused

 [Interviewer clarification: Think only about the walking that you do for at least 10 minutes at a time.]

 [Interviewer Note: If respondent answers zero. refuses or does not know, skip to Question 7]

6. How much time did you usually spend walking on one of those days? Hours per day [WDHRS; Range: o-161

Minutes per day [WDMIN; Range: o-960, 998, 999]

998. Don't Know/Not Sure 999. Refused

 [Interviewer probe: An average time for one of the days on which you walk is being sought. If the respondent can't answer because the pattern of time spent varies widely from day to day, ask: "What is the total amount of time you spent walking over the last 7 days?"

 _ _ _ Hours per week [WWHRS; Range: o-1121

Minutes per week [WWMIN; Range: o-6720, 9998, 9999]

9998. Don't Know/Not Sure 9999. Refused

 READ: Now think about the time you spent sitting on week days during the last 7 days. Include time spent at work, at home, while doing course work, and during leisure time. This may include time spent sitting at a desk, visiting friends, reading or sitting or lying down to watch television.

1395IPAQ INTERNATIONAL RELIABILITY AND VALIDITY Medicine & Science in Sports & Exercise® 

7. During the last 7 days, how much time did you usually spend sitting on a week day?

_ _ Hours per weekday [SDHRS; o-1s1  

Minutes per weekday [SDMIN; Range: o-960, 998, 9991

998. Don't Know/Not Sure 999. Refused

 [Interviewer clarification: Include time spent lying down (awake) as well

as sitting]    

[Interviewer probe: An average time per day spent sitting is being sought. If the respondent can't answer because the pattern of time spent varies widely from day to day, ask: "What is the total amount of time you spent sitting last Wednesday?"

Hours on Wednesday [SWHRS; Range o-161

_ _ _ Minutes on Wednesday [SWMIN; Range: o-960, 998, 9991

998. Don't Know/Not Sure 999. Refused

Supplements and/or Medications: 

Food Records  

Name: Age: Phone #:

Height: Weight: Date:

Notes:

First Meal Second Meal Third Meal Description of Food or

Drink Amount

Eaten Description of Food or

Drink Amount

Eaten Description of Food or

Drink Amount

Eaten            

Morning Snack Afternoon Snack Evening Snack            

INSTRUCTIONS FOR KEEPING A FOOD INTAKE RECORD  

1. Please record all food and drink consumed, except water, for 3 consecutive days. It is most accurate and easiest to record directly after a meal or snack. Record any vomit or “spit up”.

 2. Include the time of day when the food is eaten and the place where the food is eaten.

For example: Home, School, Restaurant, Watching TV, etc.  

3. Describe the foods accurately and give brand names if possible. For example: Margarine (Becel) – 1 teaspoon.

 4. State whether fruits and vegetables are fresh, canned (water packed, heavy or light syrup), cooked or frozen.

 5. Record the amount of food consumed by using household measures such as: cups, teaspoons, tablespoons, or simply

weigh the food. For example: Homo milk – ½ cup or 4 ounces (oz) or 125 grams (gm)

2 % Cottage cheese – 4 level tablespoons or 50 grams (gm)  

6. If weighing meat on a food scale, give detailed description. If there is a bone in the meat or if there is leftover food, weigh after eating and subtract from original weight.

For example: Broiled pork chop with bone – 75 gm, bone weighs – 21 gm (after eating), total weight of pork chop eaten is 54 gm. If weighing is unnecessary, approximate the number of ounces or record the measured size of meat: 2” x 2” x 1”

 7. Describe sandwiches in detail.

For example: Bologna sandwich Whole wheat bread 2 slices Bologna 1 slice (50 gm) Light mayonnaise (Kraft) 1 teaspoon Lettuce (Iceberg) 1 leaf Processed cheese (Kraft) 1 slice (30 gm)

 8. Be sure to record amounts of additional foods served with cereals, desserts, etc.

For example: Cereal Rice Krispies (Kellogg’s) ½ cup (15 gm) Milk 2% ¼ cup Brown Sugar 2 level teaspoons

 9. Include how the food is prepared especially for meats, fish, poultry, eggs, and vegetables. Methods of preparation include

Boiling, Roasting, Baking, Broiling, Frying or Steaming. When frying, be sure to mention the type of fat or oil used and quantity (be sure to measure any fat or oil left over).

For example: Scrambled eggs Eggs 1 large Milk 2 % 1 tablespoon Margarine – fried in (Becel) 1 teaspoon Left over margarine ¾ teaspoon

10. For mixed dishes such as casseroles, stews and baked goods (homemade cookies, cakes, pies, and other desserts), provide recipes on a separate sheet. State the amount of ingredients in the recipe, the number of servings made and the portion eaten by the child.

 11. If using commercial (store bought) baked goods, sate brand name and amount eaten.

 12. If eating out, name the restaurant/chain and record foods eaten with portion sizes.

 13. Give the name and amount of vitamin/mineral supplements and of medications/formulas if taken. Please inform us if

you are using a special supplement, food replacement or medication. Further information and breakdown of these products may be requested.

  Overview:

SensewearArmbandInstructions

TheSensewearArmband(SWA)analyzesphysiologicalparametersandusesalgorithmstoreportdailymovement,stepstaken,degreeofphysicalactivityandenergyexpenditure.Thearmbanddetectstriaxialacceleration,aswellasgalvanicskinresponse,skintemperatureandheatflux.ThisinformationisanalyzedusingalgorithmsperformedbytheSenseWearProfessional7.0software.AnexcelfilecanbeexportedfromtheSensewearsoftware,containingalloftheimportantresultsincludingenergyexpenditure.InstructionsforUse: Thearmbandmustbeinitializedandchargedpriortouseforeachparticipantoneachday.Removethewhitesquarefacefromthearmbandstrapbypushingdownonthefrontfacewithtwothumbs.InserttheSensewearsoftwareprogramUSBchiptothecomputerandconnectthearmbandtothecomputerusingtheUSBwireconnection.OpentheSenseWearSoftwareandclickthe“ConfigureArmband&Display”tabatthetop.Typeinallsubjectinfoandensure“DataChannels”aresetto“Research”(ensuresthearmbandwillcollectatthedefaultrateof1sample/min).Click“OK”andallowthearmbandtoinitializebeforeunplugging. Onceinitialized,removethecomputerconnectionandreinsertthearmbandfaceintothestrap.Wearontheupperlefttricepswiththelogofacinginthereadabledirectionandthesensorsonthebacksideindirectcontactwiththeskin.Adjustthestrapsoitfitscomfortably.Within10minutesofwearing(usuallyless),thearmbandaudiotoneswillsoundindicatingthebeginningofdatacollection.Thereisnopowerbutton.Afterremovalfromthebody,thearmbandtonewillsoundagain,indicatingtheendofdatacollection. Thearmbandmustberemovedatleastonehourperday(23hoursmaximumofcontinuouswear).Thereissufficientmemoryforapproximately28daysofsteadyuse,andenoughbatterypowerfor5‐7daysofuseiffullycharged.Memoryandbatterylightsonthefrontfaceindicatethefollowing:Battery:Green(solid)=morethan24hoursofbatteryliferemainAmber(flashing)=lessthan24hoursofbatteryliferemainRed(flashing)=batteryneedstobechargedimmediatelyandthearmbandcannotcollectdataMemory:Green(solid)=morethan24hoursofmemoryliferemainAmber(flashing)=lessthan24hoursofmemoryliferemainRef(flashing)=memoryisfullandthearmbandcannotcollectadditionaldata The‘timestamp’function(mainbuttononthefrontface)allowstheusertoflagtimepointsinthedataduringthecollectionprocess.Forexample,theresearchermighthavetheuserputonthearmbanduponarrivalatthelab,then‘timestamp’atthebeginningofanexerciseprotocol,and‘timestamp’theendoftheprotocol.Thenthedataofinterestcanbeeasilyandaccuratelylocatedwithintheminute‐by‐minuteexcelsummary.DataReview: Plugthearmbandintothecomputerandopenthesoftware.Click“Retrieve”tosaveallexistingdatafromthearmbandtothecomputer,andkeepthe“cleararmbandfornextuse,afterdatahasbeensaved”optionselected,sothearmbandmemoryisclearedfornextuse.ThiswillsavedataasaBodyMediafile.Tosaveasanexcelfile,proceedtothe“View&AnnotateArmbandData”tab,andclick“Export”.Thiswillsavethedatainexcelformat,whichcanthenbeviewedandanalyzedonothercomputerswithouttheSenseWearsoftware. ColumnAintheexcelfileshowsthetimeofeachminute‐by‐minutesample.ColumnRshowsEEinkcal/minandColumnWshowsMETs.ColumnXwilldisplaytimestampsasa“1”.

   OverviewofLIST:

TheLISTExerciseStimulus:

‐ TheLISTisavariableintensityexerciseteststhatsimulatestheactivitypatternofasoccergame‐ WewillbeusingamodifiedprotocoloftheLISTperformedbyNicholasetal.(referencebelow)‐ IntensitiesoftheLISTwillbebasedoffthemaximalspeedobtainedatVO2‐maxontheBeepTest

‐AVO2‐maxspeedof4.0m/s(obtainedatlevel12oftheBeepTest)willbeusedforadultsubjects‐ Thelistconsistsof‘shuttle‐running’betweentwoconesspaced20‐mapart

Intensities:Intensitiescorrespondtowalking,sprinting,90%VO2‐maxspeed,and60%VO2‐maxspeed

‐ Walking:2.0m/s(~10secondstotravel20‐m)‐ Sprinting:Maximaleffort(~2‐3secondstotravel20‐m,followedbya~5‐7restperiod)‐ 90%VO2‐MaxSpeed(Run):3.6m/s(~5.5secondstotravel20‐m)‐ 60%VO2‐MaxSpeed(Jog):2.4m/s(~8.5secondstotravel20‐m)

LISTProtocol:

‐ TheLISTwillcompriseofa5‐minutewarm‐up,then4blocksof15‐minutevariableintensityexerciseseparatedby5‐minuterestperiodsbetweeneachexerciseblock,thena5‐mincool‐down

‐ ThetimetocompletetheLISTis85minutes[(4x15minutes)+(5x5minutes)]=85minutes  

NicholasC,NuttallE,&WilliamsC.(2000).TheLoughboroughIntermittentShuttleTest:Afieldtestthatstimulatestheactivitypatternofsoccer.JournalofSportsSciences,19,97‐104.

The2‐DayAdaptationStudyDiet: 

Restingenergyexpenditure,andhabitualenergyexpenditurewillbemeasuredthroughtheuseoftheSensewear

BodyMediaArmbandAccelerometer.Thiswillprovideusthenecessaryinformationthatwillbeusedtodesignthe

2‐dayadaptationstudydiet.Theadaptationstudydietwillbeconsumedfortwodayspriortoeachmetabolictrial

day.Participantswillalsocompletea3‐dayfoodrecord,whichwillprovideimportantinformationregarding

dietaryintake,andthetypicalfoodsthateachparticipantconsumes.

For2‐daysbeforeeachmetabolictrial,participantswillconsumeadietproviding1.2g/kg/dofcompleteprotein,

withenoughenergytocovertheREEandhabitualenergyexpenditureasmeasuredbytheaccelerometer.The

adaptationdietwillbecomprisedofnormalsolidfoods.Thebackground13Ccontentwillbekeptrelativelystable

throughthefeedingoffoodswithsimilar13Ccompositions.

ThePRO‐FreeBreakfast:  Theprotein‐freebreakfastwillbecomprisedhalfofpolycose,andhalfofGatoradepowderdissolvedinwater.

Polycose(aglucosepolymer)providesacarbohydratesourcethatisrapidlyabsorbedwithinthebody.Thedosageof

bothpolycoseandtheGatoradepowderinthePRO‐freebreakfastwillbe0.5gofpolycoseperkgofbodymassofthe

participant(i.e.an80kgparticipantwillconsume40gofpolycosedissolvedinwater,40gGatoradepowder).This

willprovideenoughenergyfortheparticipanttocompletetheexercisestimulusafterfastingovernight(i.e.

80gCHO=~320kcal).Non‐sugarsweetenersmaybeusedtoenhancethetasteofthetasteofthesolution. 

HowtoMake1kgAminoAcidMix: 

‐ Thestudy‐dayaminoacidmixwillbecomprisedofallaminoacidsEXCEPTL‐PHEandL‐TYR 

‐Itismodeledfromtheaminoacidcompositionofeggprotein 

‐ Thequantitiesofeachaminoacidrequiredforthemixinmgarelistedbelowunder‘AAMIX#1’ 

‐ Ensuretouseaprecisescale(atleast3decimalplaces)whenaddingeachaminoacidtothemix 

‐ IMPORTANT:Washallimplementsafteraddingeachaminoacidtoavoidcontamination 

‐ IMPORTANT:Ensuretocleanthescale(withabrush)fromaminoacidresiduefrequently 

‐ IMPORTANT:Beforepartitioningtheaminoacidmixintomealsforstudyday,shakethemixthoroughly 

‐ FORPILOT:ThecorrespondingamountsofL‐PHEANDL‐TYRwillbeaddedintothemixseparately 

MakingtheStudyDayMeals: 

  TheStudyDayMealsareComprisedof9Ingredients:

1)Water 

2)Tang&Polycose 

3)ProteinFreePowder 

4)GrapeseedOil 

5)AminoAcidMix 

6)Tyrosine 

7)UnlabeledPhenylalanine 

8)13C‐PHE(LabeledPhenylalanine) 

9)Bicarbonate 

  DirectionsforMakingStudyDayMeals:

*Thebreakdownofallmealcomponentsisprovidedintheexceldocument,andwillbeprintedpriortoeachtrial 

*Thecomponentsineachmealwillchangedependingontheparticipant’sweight(kg),energyexpenditure

(kcal),andtestproteinintake(g/kg)thattheywillbeconsumingonthatparticulartrialday

*Ensuretoweighcookiesforeachtrialdaytobeconsumedinadditiontotheformula 

1)Weighandpartitiontheaminoacidmixpowderintoeachofthe8studydaycontainers 

2)Weighthetyrosine,phenylalanine,tracerPHE,andbicarbonaterequiredforeachofthe8studydaycontainers ‐TracerPHEonlyinmeals5‐8,Bicarbonateonlyinmeal5(BEVERYPRECISE)

3)Weighandaddtherequiredamountofwatertotheblenderfirst(topreventsticking) 

4)WeighandaddTang&Polycosetotheblender,blenduntilhomogeneous 

5)Weightheproteinfreepowder,butdonotaddittotheblender 

6)Beforeaddingtheprotein‐freepowdertotheblender,dissolvetheoilintheprotein‐freepowder 

7)Scrapetheprotein‐freepowderandoilintotheblender 

8)Blenduntilallingredientsareuniformlydissolvedintoahomogenoussolution 

9)Pourtheformulaequallyintoeachofthe8studydaycontainersusingtheweightonthefoodbalance 

‐Blendthemixperiodicallybetweenpouringitintoeachofthe8studydaycontainers 

10)Shakewell,andstoreinfreezer,orfridge(ifusingdrinkswithin24hours)

   CORNFLAKE CHERRY COOKIES               

WEIGHT:  INGREDIENTS:                

306 GM  WHEAT STARCH                

178 GM  MILK FREE MARGARINE                

100 GM  WHITE SUGAR                

10.5 GM  CORN SYRUP                

10 GM  WHIPPED TOPPING                

0.5 ML  SALT                

5 ML  ALMOND EXTRACT                

34 GM  CORNFLAKES‐crumbled                

100 GM   MARASCHINO CHERRIES‐finely chopped                

PREP METHOD:            

Cream together sugar and margarine, add corn syrup, almond extract & whipped topping.   

Stir in wheat starch, salt and crumbled cornflakes in creamed mixture.   

Add chopped cherries.            

Roll into 1 tbsp balls and place onto cookie sheet. Using a fork, lightly press down each cookie   

to flatten dough.            

Bake at 350 F  for approximately 10‐12 minutes or until fluffy and firm to the touch.   

***when weighing cookies, weigh frozen. These cookies crumble easily***      

   BUTTERSCOTCH COOKIES               

WEIGHT:  INGREDIENTS:                

240 GM  WHEAT STARCH                

113 GM  BUTTERSCOTCH PUDDING MIX                

95 ML  COLD WATER                

89 GM  VEGETABLE SHORTENING                

59 GM  MILK FREE MARGARINE                

41 GM  BROWN SUGAR                

25 GM  WHITE SUGAR                

7.5 ML  PURE VANILLA EXTRACT                

7.5 ML  BAKING POWDER                

6 GM  EGG REPLACER                

2 ML  SALT                

PREP METHOD:            

Cream together shortening and margarine. Add white, brown sugar, pudding mix and egg  

replacer.              

Add water and vanilla extract. Mix well.            

Mix in wheat starch, salt and baking powder to creamed mixture. Mix until fluffy. 

Roll into 1 tbsp balls and place onto cookie sheet. Using a fork, lightly press down each cookie   

to flatten dough.            

Bake at 375 F  for approximately 10‐12 minutes or until fluffy and golden brown.