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
ii
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.
iii
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
iv
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
v
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
1
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.
2
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.
3
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
4
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.
5
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.
6
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
7
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.
8
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.
9
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
10
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
11
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,
12
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
13
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).
14
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
References
AmericanDieteticAssociation,DietitiansofCanada,AmericanCollegeofSportsMedicine,
Rodriguez,N.R.,DiMarco,N.M.,&Langley,S.(2009).Americancollegeofsports
medicinepositionstand.nutritionandathleticperformance.MedicineandSciencein
SportsandExercise,41(3),709‐731.
Areta,J.L.,Burke,L.M.,Ross,M.L.,Camera,D.M.,West,D.W.,Broad,E.M.,etal.(2013).
Timinganddistributionofproteiningestionduringprolongedrecoveryfrom
resistanceexercisealtersmyofibrillarproteinsynthesis.TheJournalofPhysiology,
591(Pt9),2319‐2331.
Armstrong,N.,&Welsman,J.R.(2006).Thephysicalactivitypatternsofeuropeanyouth
withreferencetomethodsofassessment.SportsMedicine(Auckland,N.Z.),36(12),
1067‐1086.
Ascensao,A.,Rebelo,A.,Oliveira,E.,Marques,F.,Pereira,L.,&Magalhaes,J.(2008).
Biochemicalimpactofasoccermatch‐analysisofoxidativestressandmuscledamage
markersthroughoutrecovery.ClinicalBiochemistry,41(10‐11),841‐851.
Ball,R.O.,&Bayley,H.S.(1984).Tryptophanrequirementofthe2.5‐kgpigletdetermined
bytheoxidationofanindicatoraminoacid.TheJournalofNutrition,114(10),1741‐
1746.
Bangsbo,J.,Mohr,M.,&Krustrup,P.(2006).Physicalandmetabolicdemandsoftraining
andmatch‐playintheelitefootballplayer.JournalofSportsSciences,24(7),665‐674.
70
Baxter‐Jones,A.D.,Eisenmann,J.C.,Mirwald,R.L.,Faulkner,R.A.,&Bailey,D.A.(2008).
Theinfluenceofphysicalactivityonleanmassaccrualduringadolescence:A
longitudinalanalysis.JournalofAppliedPhysiology(Bethesda,Md.:1985),105(2),734‐
741.
Belcastro,A.N.,Shewchuk,L.D.,&Raj,D.A.(1998).Exercise‐inducedmuscleinjury:A
calpainhypothesis.MolecularandCellularBiochemistry,179(1‐2),135‐145.
Biolo,G.,Maggi,S.P.,Williams,B.D.,Tipton,K.D.,&Wolfe,R.R.(1995).Increasedratesof
muscleproteinturnoverandaminoacidtransportafterresistanceexerciseinhumans.
TheAmericanJournalofPhysiology,268(3Pt1),E514‐20.
Bohe,J.,Low,A.,Wolfe,R.R.,&Rennie,M.J.(2003).Humanmuscleproteinsynthesisis
modulatedbyextracellular,notintramuscularaminoacidavailability:Adose‐response
study.TheJournalofPhysiology,552(Pt1),315‐324.
Borsheim,E.,Tipton,K.D.,Wolf,S.E.,&Wolfe,R.R.(2002).Essentialaminoacidsand
muscleproteinrecoveryfromresistanceexercise.AmericanJournalof
Physiology.EndocrinologyandMetabolism,283(4),E648‐57.
Boyer,B.,&Odessey,R.(1991).Kineticcharacterizationofbranchedchainketoacid
dehydrogenase.ArchivesofBiochemistryandBiophysics,285(1),1‐7.
Brouns,F.,Saris,W.H.,Stroecken,J.,Beckers,E.,Thijssen,R.,Rehrer,N.J.,etal.(1989).
Eating,drinking,andcycling.Acontrolledtourdefrancesimulationstudy,partI.
InternationalJournalofSportsMedicine,10Suppl1,S32‐40.
Burd,N.A.,Tang,J.E.,Moore,D.R.,&Phillips,S.M.(2009).Exercisetrainingandprotein
metabolism:Influencesofcontraction,proteinintake,andsex‐baseddifferences.
JournalofAppliedPhysiology(Bethesda,Md.:1985),106(5),1692‐1701.
71
Burke,L.M.,Slater,G.,Broad,E.M.,Haukka,J.,Modulon,S.,&Hopkins,W.G.(2003).Eating
patternsandmealfrequencyofeliteaustralianathletes.InternationalJournalofSport
NutritionandExerciseMetabolism,13(4),521‐538.
Butterfield,G.E.,&Calloway,D.H.(1984).Physicalactivityimprovesproteinutilizationin
youngmen.TheBritishJournalofNutrition,51(2),171‐184.
Campbell,W.W.,Crim,M.C.,Young,V.R.,Joseph,L.J.,&Evans,W.J.(1995).Effectsof
resistancetraininganddietaryproteinintakeonproteinmetabolisminolderadults.
TheAmericanJournalofPhysiology,268(6Pt1),E1143‐53.
Carraro,F.,Hartl,W.H.,Stuart,C.A.,Layman,D.K.,Jahoor,F.,&Wolfe,R.R.(1990).Whole
bodyandplasmaproteinsynthesisinexerciseandrecoveryinhumansubjects.The
AmericanJournalofPhysiology,258(5Pt1),E821‐31.
Carraro,F.,Stuart,C.A.,Hartl,W.H.,Rosenblatt,J.,&Wolfe,R.R.(1990).Effectofexercise
andrecoveryonmuscleproteinsynthesisinhumansubjects.TheAmericanJournalof
Physiology,259(4Pt1),E470‐6.
Castagna,C.,D'Ottavio,S.,&Abt,G.(2003).Activityprofileofyoungsoccerplayersduring
actualmatchplay.JournalofStrengthandConditioningResearch/NationalStrength&
ConditioningAssociation,17(4),775‐780.
Chesley,A.,MacDougall,J.D.,Tarnopolsky,M.A.,Atkinson,S.A.,&Smith,K.(1992).
Changesinhumanmuscleproteinsynthesisafterresistanceexercise.JournalofApplied
Physiology(Bethesda,Md.:1985),73(4),1383‐1388.
72
Churchward‐Venne,T.A.,Murphy,C.H.,Longland,T.M.,&Phillips,S.M.(2013).Roleof
proteinandaminoacidsinpromotingleanmassaccretionwithresistanceexerciseand
attenuatingleanmasslossduringenergydeficitinhumans.AminoAcids,45(2),231‐
240.
Coffey,V.G.,Moore,D.R.,Burd,N.A.,Rerecich,T.,Stellingwerff,T.,Garnham,A.P.,etal.
(2011).Nutrientprovisionincreasessignallingandproteinsynthesisinhumanskeletal
muscleafterrepeatedsprints.EuropeanJournalofAppliedPhysiology,111(7),1473‐
1483.
ConsolazioF.,NeslonA.,MatoushL.,HardingR.,&CanhamJ.(1963).Nitrogenexcretionin
sweatanditsrelationtonitrogenbalancerequirements.TheJournalofNutrition,
79(63),399.
deCastro,J.M.,Bellisle,F.,Feunekes,G.I.J.,Dalix,A.,&DeGraaf,C.(1997).Cultureand
mealpatterns:Acomparisonofthefoodintakeoffree‐livingamerican,dutch,and
frenchstudents.NutritionResearch,17(5),807‐829.
DiDonato,D.M.,West,D.W.,Churchward‐Venne,T.A.,Breen,L.,Baker,S.K.,&Phillips,S.
M.(2014).Influenceofaerobicexerciseintensityonmyofibrillarandmitochondrial
proteinsynthesisinyoungmenduringearlyandlatepostexerciserecovery.American
JournalofPhysiology.EndocrinologyandMetabolism,306(9),E1025‐32.
Dohm,G.L.,Williams,R.T.,Kasperek,G.J.,&vanRij,A.M.(1982).Increasedexcretionof
ureaandNtau‐methylhistidinebyratsandhumansafteraboutofexercise.Journalof
AppliedPhysiology:Respiratory,EnvironmentalandExercisePhysiology,52(1),27‐33.
73
Elango,R.,Humayun,M.A.,Ball,R.O.,&Pencharz,P.B.(2009).Indicatoraminoacid
oxidationisnotaffectedbyperiodofadaptationtoawiderangeoflysineintakein
healthyyoungmen.TheJournalofNutrition,139(6),1082‐1087.
Elango,R.,Humayun,M.A.,Ball,R.O.,&Pencharz,P,B.(2011).Proteinrequirementof
healthyschool‐agechildrendeterminedbytheindicatoraminoacidoxidationmethod.
TheAmericanJournalofClinicalNutrition.94(6),1545‐1552.
el‐Khoury,A.E.,Forslund,A.,Olsson,R.,Branth,S.,Sjodin,A.,Andersson,A.,etal.(1997).
Moderateexerciseatenergybalancedoesnotaffect24‐hleucineoxidationornitrogen
retentioninhealthymen.TheAmericanJournalofPhysiology,273(2Pt1),E394‐407.
Fielding,R.A.,Meredith,C.N.,O'Reilly,K.P.,Frontera,W.R.,Cannon,J.G.,&Evans,W.J.
(1991).Enhancedproteinbreakdownaftereccentricexerciseinyoungandoldermen.
JournalofAppliedPhysiology(Bethesda,Md.:1985),71(2),674‐679.
Forbes,G.B.(1973).Anothersourceoferrorinthemetabolicbalancemethod.Nutrition
Reviews,31(10),297‐300.
Forslund,A.H.,El‐Khoury,A.E.,Olsson,R.M.,Sjodin,A.M.,Hambraeus,L.,&Young,V.R.
(1999).Effectofproteinintakeandphysicalactivityon24‐hpatternandrateof
macronutrientutilization.TheAmericanJournalofPhysiology,276(5Pt1),E964‐76.
Friedman,J.E.,&Lemon,P.W.(1989).Effectofchronicenduranceexerciseonretentionof
dietaryprotein.InternationalJournalofSportsMedicine,10(2),118‐123.
Gibala,M.J.,Interisano,S.A.,Tarnopolsky,M.A.,Roy,B.D.,MacDonald,J.R.,Yarasheski,K.
E.,etal.(2000).Myofibrillardisruptionfollowingacuteconcentricandeccentric
resistanceexerciseinstrength‐trainedmen.CanadianJournalofPhysiologyand
Pharmacology,78(8),656‐661.
74
Gibala,M.J.,MacDougall,J.D.,Tarnopolsky,M.A.,Stauber,W.T.,&Elorriaga,A.(1995).
Changesinhumanskeletalmuscleultrastructureandforceproductionafteracute
resistanceexercise.JournalofAppliedPhysiology(Bethesda,Md.:1985),78(2),702‐708.
Hartman,J.W.,Moore,D.R.,&Phillips,S.M.(2006).Resistancetrainingreduceswhole‐
bodyproteinturnoverandimprovesnetproteinretentioninuntrainedyoungmales.
AppliedPhysiology,Nutrition,andMetabolism=PhysiologieAppliquee,NutritionEt
Metabolisme,31(5),557‐564.
Howarth,K.R.,Phillips,S.M.,MacDonald,M.J.,Richards,D.,Moreau,N.A.,&Gibala,M.J.
(2010).Effectofglycogenavailabilityonhumanskeletalmuscleproteinturnover
duringexerciseandrecovery.JournalofAppliedPhysiology(Bethesda,Md.:1985),
109(2),431‐438.
Humayun,M.A.,Elango,R.,Ball,R.O.,&Pencharz,P.B.(2007).Reevaluationoftheprotein
requirementinyoungmenwiththeindicatoraminoacidoxidationtechnique.The
AmericanJournalofClinicalNutrition,86(4),995‐1002.
Kim,K.I.,McMillan,I.,&Bayley,H.S.(1983).Determinationofaminoacidrequirementsof
youngpigsusinganindicatoraminoacid.TheBritishJournalofNutrition,50(2),369‐
382.
Lamont,L.S.,McCullough,A.J.,&Kalhan,S.C.(1999).Comparisonofleucinekineticsin
endurance‐trainedandsedentaryhumans.JournalofAppliedPhysiology(Bethesda,Md.:
1985),86(1),320‐325.
Lamont,L.S.,Patel,D.G.,&Kalhan,S.C.(1990).Leucinekineticsinendurance‐trained
humans.JournalofAppliedPhysiology(Bethesda,Md.:1985),69(1),1‐6.
75
Leger,L.A.,&Lambert,J.(1982).Amaximalmultistage20‐mshuttleruntesttopredict
VO2max.EuropeanJournalofAppliedPhysiologyandOccupationalPhysiology,49(1),1‐
12.
Lecker,S.H.,Solomon,V.,Mitch,W.E.,&Goldberg,A.L.(1999).Muscleproteinbreakdown
andthecriticalroleoftheubiquitin‐proteasomepathwayinnormalanddiseasestates.
TheJournalofNutrition,129(1SSuppl),227S‐237S.
Lemon,P.W.,&Mullin,J.P.(1980).Effectofinitialmuscleglycogenlevelsonprotein
catabolismduringexercise.JournalofAppliedPhysiology:Respiratory,Environmental
andExercisePhysiology,48(4),624‐629.
Lemon,P.W.,Tarnopolsky,M.A.,MacDougall,J.D.,&Atkinson,S.A.(1992).Protein
requirementsandmusclemass/strengthchangesduringintensivetraininginnovice
bodybuilders.JournalofAppliedPhysiology(Bethesda,Md.:1985),73(2),767‐775.
Magalhaes,J.,Rebelo,A.,Oliveira,E.,Silva,J.R.,Marques,F.,&Ascensao,A.(2010a).Impact
ofloughboroughintermittentshuttletestversussoccermatchonphysiological,
biochemicalandneuromuscularparameters.EuropeanJournalofAppliedPhysiology,
108(1),39‐48.
Magalhaes,J.,Rebelo,A.,Oliveira,E.,Silva,J.R.,Marques,F.,&Ascensao,A.(2010b).Impact
ofloughboroughintermittentshuttletestversussoccermatchonphysiological,
biochemicalandneuromuscularparameters.EuropeanJournalofAppliedPhysiology,
108(1),39‐48.
Malavolti,M.,Pietrobelli,A.,Dugoni,M.,Poli,M.,Romagnoli,E.,DeCristofaro,P.,etal.
(2007).Anewdeviceformeasuringrestingenergyexpenditure(REE)inhealthy
subjects.Nutrition,Metabolism,andCardiovascularDiseases:NMCD,17(5),338‐343.
76
McKenzie,S.,Phillips,S.M.,Carter,S.L.,Lowther,S.,Gibala,M.J.,&Tarnopolsky,M.A.
(2000).EnduranceexercisetrainingattenuatesleucineoxidationandBCOAD
activationduringexerciseinhumans.AmericanJournalofPhysiology.Endocrinology
andMetabolism,278(4),E580‐7.
Meguid,M.M.,Matthews,D.E.,Bier,D.M.,Meredith,C.N.,Soeldner,J.S.,&Young,V.R.
(1986).Leucinekineticsatgradedleucineintakesinyoungmen.TheAmericanJournal
ofClinicalNutrition,43(5),770‐780.
Meredith,C.N.,Wen,Z.M.,Bier,D.M.,Matthews,D.E.,&Young,V.R.(1986).Lysine
kineticsatgradedlysineintakesinyoungmen.TheAmericanJournalofClinical
Nutrition,43(5),787‐794.
Meredith,C.N.,Zackin,M.J.,Frontera,W.R.,&Evans,W.J.(1989).Dietaryprotein
requirementsandbodyproteinmetabolisminendurance‐trainedmen.Journalof
AppliedPhysiology(Bethesda,Md.:1985),66(6),2850‐2856.
Miller,S.L.,Tipton,K.D.,Chinkes,D.L.,Wolf,S.E.,&Wolfe,R.R.(2003).Independentand
combinedeffectsofaminoacidsandglucoseafterresistanceexercise.Medicineand
ScienceinSportsandExercise,35(3),449‐455.
Millward,D.J.(1998).Metabolicdemandsforaminoacidsandthehumandietary
requirement:MillwardandrRvers(1988)revisited.TheJournalofNutrition,128(12
Suppl),2563S‐2576S.
Moore,D.R.,Areta,J.,Coffey,V.G.,Stellingwerff,T.,Phillips,S.M.,Burke,L.M.,etal.(2012).
Daytimepatternofpost‐exerciseproteinintakeaffectswhole‐bodyproteinturnoverin
resistance‐trainedmales.Nutrition&Metabolism,9(1),91‐7075‐9‐91.
77
Moore,D.R.,Camera,D.M.,Areta,J.L.,&Hawley,J.A.(2014).Beyondmusclehypertrophy:
Whydietaryproteinisimportantforenduranceathletes.AppliedPhysiology,Nutrition,
andMetabolism=PhysiologieAppliquee,NutritionEtMetabolisme,39(9),987‐997.
Moore,D.R.,DelBel,N.C.,Nizi,K.I.,Hartman,J.W.,Tang,J.E.,Armstrong,D.,etal.(2007).
Resistancetrainingreducesfasted‐andfed‐stateleucineturnoverandincreases
dietarynitrogenretentioninpreviouslyuntrainedyoungmen.TheJournalofNutrition,
137(4),985‐991.
Mountjoy,M.,Andersen,L.B.,Armstrong,N.,Biddle,S.,Boreham,C.,Bedenbeck,H.B.,etal.
(2011).Internationalolympiccommitteeconsensusstatementonthehealthand
fitnessofyoungpeoplethroughphysicalactivityandsport.BritishJournalofSports
Medicine,45(11),839‐848.
Nicholas,C.W.,Nuttall,F.E.,&Williams,C.(2000).Theloughboroughintermittentshuttle
test:Afieldtestthatsimulatestheactivitypatternofsoccer.JournalofSportsSciences,
18(2),97‐104.
Pencharz,P.B.,&Ball,R.O.(2003).Differentapproachestodefineindividualaminoacid
requirements.AnnualReviewofNutrition,23,101‐116.
Phillips,S.M.(2004).Proteinrequirementsandsupplementationinstrengthsports.
Nutrition(Burbank,LosAngelesCounty,Calif.),20(7‐8),689‐695.
Phillips,S.M.,Atkinson,S.A.,Tarnopolsky,M.A.,&MacDougall,J.D.(1993).Gender
differencesinleucinekineticsandnitrogenbalanceinenduranceathletes.Journalof
AppliedPhysiology(Bethesda,Md.:1985),75(5),2134‐2141.
78
Phillips,S.M.,Moore,D.R.,&Tang,J.E.(2007).Acriticalexaminationofdietaryprotein
requirements,benefits,andexcessesinathletes.InternationalJournalofSportNutrition
andExerciseMetabolism,17Suppl,S58‐76.
Phillips,S.M.,Parise,G.,Roy,B.D.,Tipton,K.D.,Wolfe,R.R.,&Tamopolsky,M.A.(2002).
Resistance‐training‐inducedadaptationsinskeletalmuscleproteinturnoverinthefed
state.CanadianJournalofPhysiologyandPharmacology,80(11),1045‐1053.
Phillips,S.M.,Tipton,K.D.,Aarsland,A.,Wolf,S.E.,&Wolfe,R.R.(1997).Mixedmuscle
proteinsynthesisandbreakdownafterresistanceexerciseinhumans.TheAmerican
JournalofPhysiology,273(1Pt1),E99‐107.
Phillips,S.M.,Tipton,K.D.,Fero,A.A.,&Wolfe,R.R.(1999).Resistancetrainingreduces
theacuteexercise‐inducedincreaseinmuscleproteinturnover.TheAmericanJournal
ofPhysiology,276(1Pt1),E118‐24.
Porszasz,J.,Casaburi,R.,Somfay,A.,Woodhouse,L.J.,&Whipp,B.J.(2003).Atreadmill
rampprotocolusingsimultaneouschangesinspeedandgrade.MedicineandSciencein
SportsandExercise,35(9),1596‐1603.
Proteinandaminoacidrequirementsinhumannutrition.WorldHealthOrganTechRepSer
1‐265,back,2007.
Rand,W.M.,Pellett,P.L.,&Young,V.R.(2003).Meta‐analysisofnitrogenbalancestudies
forestimatingproteinrequirementsinhealthyadults.TheAmericanJournalofClinical
Nutrition,77(1),109‐127.
79
Rafii,M.,Chapman,K.,Owens,J.,Elango,R.,Campbell,W.W.,Ball,R.O.,etal.(2015).Dietary
proteinrequirementoffemaleadults>65yearsdeterminedbytheindicatoramino
acidoxidationtechniqueishigherthancurrentrecommendations.TheJournalof
Nutrition,145(1),18‐24.
Rafii,M.,McKenzie,J.M.,Roberts,S.A.,Steiner,G.,Ball,R.O.,&Pencharz,P.B.(2008).In
vivoregulationofphenylalaninehydroxylationtotyrosine,studiedusingenrichment
inapoB‐100.AmericanJournalofPhysiology‐EndocrinologyandMetabolism,294(2),
E475‐E479.doi:10.1152/ajpendo.00604.2007
Rasmussen,B.B.,Tipton,K.D.,Miller,S.L.,Wolf,S.E.,&Wolfe,R.R.(2000).Anoral
essentialaminoacid‐carbohydratesupplementenhancesmuscleproteinanabolism
afterresistanceexercise.JournalofAppliedPhysiology(Bethesda,Md.:1985),88(2),
386‐392.
Rennie,M.J.,Edwards,R.H.,Krywawych,S.,Davies,C.T.,Halliday,D.,Waterlow,J.C.,etal.
(1981).Effectofexerciseonproteinturnoverinman.ClinicalScience(London,England
:1979),61(5),627‐639.
Rodriguez,N.R.(2005).Optimalquantityandcompositionofproteinforgrowingchildren.
JournaloftheAmericanCollegeofNutrition,24(2),150S‐154S.
Shiman,R.,&Gray,D.W.(1998).Formationandfateoftyrosine:INTRACELLULAR
PARTITIONINGOFNEWLYSYNTHESIZEDTYROSINEINMAMMALIANLIVER.Journal
ofBiologicalChemistry,273(52),34760‐34769.
Smith,K.,&Rennie,M.J.(1996).Themeasurementoftissueproteinturnover.Bailliere's
ClinicalEndocrinologyandMetabolism,10(4),469‐495.
80
St‐Onge,M.,Mignault,D.,Allison,D.B.,&Rabasa‐Lhoret,R.(2007).Evaluationofaportable
devicetomeasuredailyenergyexpenditureinfree‐livingadults.TheAmericanJournal
ofClinicalNutrition,85(3),742‐749.
Stroyer,J.,Hansen,L.,&Klausen,K.(2004).Physiologicalprofileandactivitypatternof
youngsoccerplayersduringmatchplay.MedicineandScienceinSportsandExercise,
36(1),168‐174.
Svanberg,E.(1998).Aminoacidsmaybeintrinsicregulatorsofproteinsynthesisin
responsetofeeding.ClinicalNutrition(Edinburgh,Scotland),17(2),77‐79.
Tang,M.,McCabe,G.P.,Elango,R.,Pencharz,P.B.,Ball,R.O.,&Campbell,W.W.(2014).
Assessmentofproteinrequirementinoctogenarianwomenwithuseoftheindicator
aminoacidoxidationtechnique.TheAmericanJournalofClinicalNutrition,99(4),891‐
898.
Tarnopolsky,L.J.,MacDougall,J.D.,Atkinson,S.A.,Tarnopolsky,M.A.,&Sutton,J.R.
(1990).Genderdifferencesinsubstrateforenduranceexercise.JournalofApplied
Physiology(Bethesda,Md.:1985),68(1),302‐308.
Tarnopolsky,M.(2004).Proteinrequirementsforenduranceathletes.Nutrition(Burbank,
LosAngelesCounty,Calif.),20(7‐8),662‐668.
Tarnopolsky,M.A.,Atkinson,S.A.,MacDougall,J.D.,Chesley,A.,Phillips,S.,&Schwarcz,H.
P.(1992).Evaluationofproteinrequirementsfortrainedstrengthathletes.Journalof
AppliedPhysiology(Bethesda,Md.:1985),73(5),1986‐1995.
Tarnopolsky,M.A.,MacDougall,J.D.,&Atkinson,S.A.(1988).Influenceofproteinintake
andtrainingstatusonnitrogenbalanceandleanbodymass.JournalofApplied
Physiology(Bethesda,Md.:1985),64(1),187‐193.
81
Tipton,K.D.,&Wolfe,R.R.(1998).Exercise‐inducedchangesinproteinmetabolism.Acta
PhysiologicaScandinavica,162(3),377‐387.
Torun,B.,Scrimshaw,N.S.,&Young,V.R.(1977).Effectofisometricexercisesonbody
potassiumanddietaryproteinrequirementsofyoungmen.TheAmericanJournalof
ClinicalNutrition,30(12),1983‐1993.
vanLoon,L.J.,Greenhaff,P.L.,Constantin‐Teodosiu,D.,Saris,W.H.,&Wagenmakers,A.J.
(2001).Theeffectsofincreasingexerciseintensityonmusclefuelutilisationin
humans.TheJournalofPhysiology,536(Pt1),295‐304.
Wagenmakers,A.J.(1998).Muscleaminoacidmetabolismatrestandduringexercise:Role
inhumanphysiologyandmetabolism.ExerciseandSportSciencesReviews,26,287‐314.
Wilkinson,S.B.,Phillips,S.M.,Atherton,P.J.,Patel,R.,Yarasheski,K.E.,Tarnopolsky,M.A.,
etal.(2008).Differentialeffectsofresistanceandenduranceexerciseinthefedstate
onsignallingmoleculephosphorylationandproteinsynthesisinhumanmuscle.The
JournalofPhysiology,586(Pt15),3701‐3717.
Yarasheski,K.E.,Zachwieja,J.J.,&Bier,D.M.(1993).Acuteeffectsofresistanceexerciseon
muscleproteinsynthesisrateinyoungandelderlymenandwomen.TheAmerican
JournalofPhysiology,265(2Pt1),E210‐4.
Young,V.R.,Bier,D.M.,&Pellett,P.L.(1989).Atheoreticalbasisforincreasingcurrent
estimatesoftheaminoacidrequirementsinadultman,withexperimentalsupport.The
AmericanJournalofClinicalNutrition,50(1),80‐92.
Zello,G.A.,Pencharz,P.B.,&Ball,R.O.(1990).Phenylalanineflux,oxidation,and
conversiontotyrosineinhumansstudiedwithL‐[1‐13C]phenylalanine.TheAmerican
JournalofPhysiology,259(6Pt1),E835‐43.
82
Zello,G.A.,Pencharz,P.B.,&Ball,R.O.(1993).Dietarylysinerequirementofyoungadult
malesdeterminedbyoxidationofL‐[1‐13C]phenylalanine.TheAmericanJournalof
Physiology,264(4Pt1),E677‐85.
Zello,G.A.,Wykes,L.J.,Ball,R.O.,&Pencharz,P.B.(1995).Recentadvancesinmethodsof
assessingdietaryaminoacidrequirementsforadulthumans.TheJournalofNutrition,
125(12),2907‐2915.
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 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
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.