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Determination of protein requirements of healthy pregnant women during early and late gestation using… Stephens, Trina 2013

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Determination?of?protein?requirements?of?healthy?pregnant?women?during?early?and?late?gestation?using?the?indicator?amino?acid?oxidation?technique?? by??Trina?Stephens??B.Sc.H.?(SSP),?Queen?s?University,?2010?B.A.?(Min),?Queen?s?University,?2010?? A?THESIS?SUBMITTED?IN?PARTIAL?FULFILLMENT?OF?THE?REQUIREMENTS?FOR?THE?DEGREE?OF??MASTER?OF?SCIENCE??in??The?Faculty?of?Graduate?and?Postdoctoral?Studies??(Experimental?Medicine)??THE?UNIVERSITY?OF?BRITISH?COLUMBIA?(Vancouver)??October?2013????Trina?Stephens,?2013?? ii?Abstract?? Adequate?maternal?dietary?protein?(PRO)?intake?is?necessary?to?support?rapid?tissue?accretion?during?a?healthy?pregnancy.?Both?insufficient?and?excessive?maternal?PRO?intake?during?pregnancy?is?associated?with?intrauterine?growth?restriction?(IUGR)?of?the?fetus.?IUGR?increases?the?risk?of?neonatal?morbidity?and?mortality,?and?is?associated?with?an?increased?risk?of?future?health?problems,?including?cardiovascular?disease,?kidney?disease,?obstructive?airway?disease,?and?obesity.?However,?current?PRO?intake?recommendations?for?healthy?pregnant?women?are?based?on?factorial?calculations?of?nitrogen?balance?data?derived?from?non?pregnant?adults.?Thus,?an?estimate?of?PRO?requirements?based?on?pregnancy?specific?data?is?needed.?PRO?requirements?of?healthy?pregnant?women?at?11?20?(early)?and?31?38?(late)?weeks?gestation?were?determined?using?the?indicator?amino?acid?oxidation?(IAAO)?method.?Twenty?nine?healthy?women?(age?24?37)?each?randomly?received?a?different?test?PRO?intake?(range?=?0.22?2.56?g/kg/day)?during?each?study?day?in?early?and?late?gestation?(nearly?=?37,?nlate?=?44).?The?diets?were?isocaloric?and?provided?energy?at?1.7?X?resting?energy?expenditure?(REE).?PRO?was?given?as?a?crystalline?amino?acid?mixture?based?on?egg?PRO?composition,?except?phenylalanine?and?tyrosine,?which?were?maintained?constant?across?intakes.?PRO?requirements?were?determined?by?measuring?the?oxidation?of?L?[1?13C]phenylalanine?to?13CO2?(F13CO2).?Breath?and?urine?samples?were?collected?at?baseline?and?isotopic?steady?state.?Linear?regression?crossover?analysis?identified?a?breakpoint?(requirement)?at?minimal?F13CO2?in?response?to?different?PRO?intakes.??? iii?The?estimated?average?requirement?(EAR)?for?PRO?in?early?and?late?gestation?was?determined?to?be?1.22?and?1.52?g/kg/d,?respectively.?Both?of?these?estimates?are?significantly?greater?than?the?EAR?of?0.88?g/kg/d?currently?recommended?by?the?Dietary?Reference?Intakes?(DRI?2005).?Our?results?indicate?increased?demand?for?PRO?before?20?weeks?gestation?(on?a?gram?per?kilogram?body?weight?basis),?a?consideration?that?has?not?been?addressed?by?current?DRI?recommendations.?This?study?is?the?first?to?directly?estimate?gestational?PRO?requirements?in?a?population?composed?solely?of?healthy?pregnant?women,?and?suggests?that?current?recommendations?based?on?the?nitrogen?balance?method?and?factorial?calculations?underestimate?PRO?requirements.??? iv?Preface???This?project?was?approved?by?the?University?of?British?Columbia?and?the?British?Columbia?Children?s?and?Women?s?Hospital?research?ethics?board?in?accordance?with?the?Declaration?of?Helsinki?principles.?Human?ethics?certificate?number:??H11?00070.?Clinical?Trials.gov?identifier:?NCT01784198.??Sections?of?this?thesis?are?already?published?in?abstract?form:?? 1. Stephens?TV,?Payne?M,?Ball?RO,?Pencharz?PB,?and?Elango?R.??Protein?requirement?in?healthy?pregnant?women?in?early?and?late?gestation?determined?by?indicator?amino?acid?oxidation?method.??The?FASEB?Journal,?26?(2012):?42.4.?2. Stephens?TV,?Payne?M,?Ball?RO,?Pencharz?PB,?and?Elango?R.??Protein?requirements?in?pregnancy?determined?using?the?indicator?amino?acid?oxidation?technique.??Applied?Physiology,?Nutrition,?and?Metabolism,?37.3(2012):?557?598.??3. Stephens?TV,?Payne?M,?Ball?RO,?Pencharz?PB,?and?Elango?R.??Determination?of?protein?requirements?in?healthy?pregnant?women?in?early?and?late?gestation.??Applied?Physiology,?Nutrition,?and?Metabolism,?38.4?(2013):?47? v??Table?of?contents?Abstract?......................................................................................................................................?ii?Preface?.......................................................................................................................................?iv?Table?of?contents?......................................................................................................................?v?List?of?tables?............................................................................................................................?vii?List?of?figures?.........................................................................................................................?viii?List?of?abbreviations?.............................................................................................................?ix?Acknowledgements?.................................................................................................................?x?Dedication?.................................................................................................................................?xi?Chapter?1.?Introduction?.........................................................................................................?1?Chapter?2.?Background?..........................................................................................................?3?2.1.?Metabolic?adaptations?to?pregnancy?..................................................................................?3?2.2.?Maternal?nutrition?....................................................................................................................?4?2.2.1.?Impact?of?maternal?nutrition?on?infant?health?.......................................................................?4?2.2.2.?Protein?and?amino?acid?nutrition?during?pregnancy?...........................................................?5?2.3.?Fetal?and?placental?amino?acid?metabolism?....................................................................?8?2.3.1.?Current?understanding?of?fetal?amino?acid?metabolism?....................................................?8?2.3.2.?Placental?transport?of?amino?acids?..............................................................................................?9?2.4.?Protein?metabolism?changes?during?different?stages?of?pregnancy?.....................?11?2.5.?Protein?requirements?during?pregnancy?......................................................................?12?2.5.1.?Current?protein?recommendations?during?pregnancy?....................................................?12?2.5.2.?Evaluation?of?DRI?during?pregnancy?.......................................................................................?14?2.5.3.?Fundamental?criticisms?of?nitrogen?balance?studies?........................................................?16?2.6.?Stable?isotopes?and?their?use?in?pregnancy?studies?...................................................?17?2.6.1.?Stable?isotopes?as?tracers?.............................................................................................................?17?2.6.2.?Protein?metabolism?during?pregnancy?studied?using?stable?isotopes?.....................?18?2.7.?The?IAAO?method:?a?robust?model?for?determining?protein?requirements?of?vulnerable?populations?................................................................................................................?20?2.7.1.?IAAO?method:?principles?and?protocol?...................................................................................?20?2.7.2.?Concerns?with?the?IAAO?method?...............................................................................................?21?2.7.3.?Advantages?of?the?IAAO?method?...............................................................................................?22?2.8.?Summary?of?background?.....................................................................................................?23?Chapter?3.?Objective?and?hypothesis?..............................................................................?24?Chapter?4.?Methods?...............................................................................................................?25?4.1?Subjects?......................................................................................................................................?25?4.1.1.?Age?demographic?.............................................................................................................................?25?4.1.2.?Pregnancy?considerations?............................................................................................................?27?4.1.3.?Gestational?stage?considerations?..............................................................................................?27?4.1.4.?Participant?health?............................................................................................................................?28?4.1.5.?Participant?recruitment?................................................................................................................?29?? vi?4.1.6.?Inclusion?and?exclusion?criteria?................................................................................................?29?4.2.?Experimental?design?.............................................................................................................?30?4.2.1.?Pre?study?day?protocol?..................................................................................................................?30?4.2.2.?Study?day?protocol?..........................................................................................................................?32?4.3.?Experimental?diet?..................................................................................................................?33?4.3.1.?Standardization?diet?.......................................................................................................................?33?4.3.2.?Test?protein?intakes?........................................................................................................................?34?4.3.3.?Study?day?diet?....................................................................................................................................?35?4.4.?Isotope?tracer?considerations?...........................................................................................?36?4.4.1.?Indicator?amino?acid?criteria?......................................................................................................?36?4.4.2.?Isotope?tracer?protocol?..................................................................................................................?36?4.4.3.?Sample?collection?and?analysis?..................................................................................................?38?4.4.5.?Statistical?analysis?...........................................................................................................................?40?Chapter?5.?Results?.................................................................................................................?42?5.1.?Subject?characteristics?.........................................................................................................?42?5.2.?Pre?study?assessment?...........................................................................................................?43?5.2.1.?Early?gestation?pre?study?results?..............................................................................................?43?5.2.2.?Late?gestation?pre?study?results?................................................................................................?45?5.3.?Phenylalanine?flux?and?protein?requirement?..............................................................?46?Chapter?6.?Discussion?...........................................................................................................?51?6.1.?Subject?characteristics?.........................................................................................................?51?6.2.?Indicators?of?metabolic?adaptations?to?pregnancy?.....................................................?51?6.3.?Estimation?of?protein?requirements?...............................................................................?53?6.3.1.?Protein?requirement?in?early?gestation?..................................................................................?53?6.3.2.?Sources?of?protein?demand?before?20?weeks?gestation?..................................................?54?6.3.3.?Protein?requirement?in?late?gestation?.....................................................................................?58?6.3.4.?Evidence?that?factorial?calculation?underestimates?protein?requirement?..............?59?6.4.?Comparison?of?protein?requirements?to?current?reported?dietary?intakes?......?59?6.5.?Comparison?of?protein?requirement?as?a?percentage?of?energy?to?DRI?energy?recommendations?..........................................................................................................................?61?6.6.?Strengths?and?implications?of?this?study?.......................................................................?62?Chapter?7.?Future?directions?.............................................................................................?64?Chapter?8.?Conclusion?..........................................................................................................?66?Bibliography?...........................................................................................................................?67?Appendices?..............................................................................................................................?76?Appendix?A???DRI?derivation?of?protein?intake?recommendations?for?healthy?pregnant?women?............................................................................................................................?77?Appendix?B???Pre?study?questionnaire?...................................................................................?79?Appendix?C???Recruitment?material?........................................................................................?83?Appendix?D???Subject?consent?form?.........................................................................................?86?Appendix?E???Food?record?template?........................................................................................?94?Appendix?F???Study?day?protocol?..............................................................................................?96?Appendix?G???Letters?of?contact?to?primary?care?provider?..............................................?98?Appendix?H???Protein?turnover?estimated?from?LC?MS?analysis?.................................?101??? vii??List?of?tables??Table?1.?Classification?of?amino?acids?as?dispensable,?conditionally?indispensable,?and?indispensable?in?the?diet?................................................................................................................?7?Table?2.?Comparison?of?dietary?protein?intake?recommendations?by?the?DRI?(2005),?WHO?(2007),?and?IAAO?method,?and?dietary?intake?patterns?of?pregnant?women?in?BC,?Canada.?.................................................................................................................................................?14?Table?3.?Subject?Characteristics?(Nearly?=?17,?Nlate?=?19)1?.........................................................?43?Table?4.?Pre?study?day?results?in?early?(N=17)?and?late?(N=19)?gestation?....................?44?Table?5.?Study?day?results?in?early?(n=37)?and?late?(n=44)?gestation?..............................?47????viii?List?of?figures??Figure?1.?Estimated?average?protein?requirement?analysis?using?IAAO?method?(from?Humayun?et?al.,?2007)?...........................................................................................................................?21?Figure?2.?Flow?diagram?of?our?experimental?design?................................................................?26?Figure?3.?Study?day?protocol?...............................................................................................................?33?Figure?4.?Biochemical?conversion?of?phenylalanine?to?tyrosine?.........................................?37?Figure?5.?Variation?in?phenylalanine?flux?across?protein?intakes?in?early?gestation?group?non?significant?(p=0.212)?as?determined?by?one?way?analysis?of?variance?in?SPSS?(Version?19,?SPSS?Inc,?Chicago,?IL)?........................................................................................?48?Figure?6.?Variation?in?phenylalanine?flux?across?protein?intakes?in?late?gestation?group?non?significant?(p=0.259)?as?determined?by?one?way?analysis?of?variance?in?SPSS?(Version?19,?SPSS?Inc,?Chicago,?IL)?........................................................................................?48?Figures?7.?Estimated?average?protein?requirement?in?early?gestation?derived?by?2?phase?linear?regression?cross?over?analysis?in?SAS?(Proc?Mixed,?Statistical?Analysis?Systems???SAS/STAT?version?9.0?SAS?Institute,?Cary,?NC)?.....................................................?49?Figure?8.?Estimated?average?protein?requirement?in?late?gestation?derived?by?2?phase?linear?regression?cross?over?analysis?in?SAS?(Proc?Mixed,?Statistical?Analysis?Systems???SAS/STAT?version?9.0?SAS?Institute,?Cary,?NC)?.....................................................?49?Figure?9.?Plasma?progesterone?and?estradiol?levels?throughout?human?pregnancy?(adapted?by?Nieman?L.K.?for?Endotext.org?from?Tulchinsky,?D.,?et?al.?"Plasma?estrone,?estradiol,?estriol,?progesterone,?and?17?hydroxyprogesterone?in?human?pregnancy.?I.?Normal?pregnancy."?American?Journal?of?Obstetrics?and?Gynecology?112.8?(1972):?1095)?.............................................................................................................................................................?56?Figure?10.?Mean?serum?hCG?levels?throughout?normal?pregnancy?(from?Braunstein,?G.?D.,?et?al.?"Serum?human?chorionic?gonadotropin?levels?throughout?normal?pregnancy."?American?Journal?of?Obstetrics?and?Gynecology?126.6?(1976):?678)?........?56??????? ix?List?of?abbreviations??BIA???bioelectric?impedance?analysis???DRI???Dietary?Reference?Intakes??EAR???estimated?average?requirement??EG???early?gestation??F13CO2???rate?of?appearance?of?heavy?carbon?(13C)?labeled?carbon?dioxide?in?breath??FAO???Food?and?Agriculture?Organization???FFM/LBM???fat?free?mass/lean?body?mass??hCG???human?chorionic?gonadotropin???IAAO???indicator?amino?acid?oxidation??IQR???interquartile?range??IUGR???intrauterine?growth?restriction??LBW???low?birth?weight??LG???late?gestation??RDA???recommended?dietary?allowance???REE???resting?energy?expenditure??UN???United?Nations???VCO2???volume?of?carbon?dioxide?production?per?minute??VO2???volume?of?oxygen?production?per?minute??WHO???World?Health?Organization???? x?Acknowledgements?? First?and?foremost,?I?would?like?to?thank?my?supervisor?Dr.?Rajavel?Elango?for?the?opportunity?to?work?on?this?novel?and?exciting?project.?His?guidance?over?the?past?three?years?will?not?be?forgotten.?I?would?also?like?to?thank?Dr.?Sheila?Innis?and?Dr.?Dan?Rurak?for?their?constant?support?and?insight?as?my?committee?members.?I?can?only?hope?to?aspire?to?a?career?in?research?as?productive?and?distinguished?as?theirs.??To?my?lab?mate?Maggie?Payne,?thank?goodness?he?found?a?Queen?s?girl!?I?could?not?have?done?this?without?you.?To?my?family,?thank?you?for?the?continued?love,?strength,?and?support?you?have?shown?me?throughout?my?academic?career.?Thank?you?to?all?of?the?amazing?individuals?at?the?Child?and?Family?Research?Institute?and?the?Experimental?Medicine?program?for?creating?such?a?wonderful?learning?environment.?Finally,?thank?you?to?all?of?the?women?who?participated?in?this?thesis?investigation.?Without?their?dedication,?patience,?and?open?minded?approach?to?our??creamsicle?flavored??protein?shakes,?this?research?project?would?not?have?been?possible.?? xi?Dedication?? ?????????For?Mom,?Dad,?and?Jeff???????? 1?Chapter?1.?Introduction?? Arguably?the?most?exquisite?event?in?the?human?lifecycle?is?the?series?of?highly?orchestrated?biological?pathways?that?transform?two?haploid?cells?into?an?autonomous?being?during?a?40?week?gestation?period.?Strong?genetic?selection?has?molded?human?reproduction?into?a?relatively?predictable?process,?but?many?environmental?influences?represent?modifiable?determinants?of?pregnancy?outcomes.?Increasingly?it?has?become?apparent?that?sub?optimal?maternal?nutrition?adversely?affects?maternal?and?fetal?health.?For?example,?insufficient?folic?acid?intake?before?and?during?pregnancy?is?associated?with?neural?tube,?cardiovascular,?and?urinary?tract?defects,?and?cleft?palate?(Hern?ndez?D?az?et?al.,?2000);?fetal?brain?development?and?early?childhood?visual?acuity?is?linked?to?docosahexaenoic?acid?(DHA)?intake?during?pregnancy?(Innis,?2007;?Innis?and?Freisen,?2008);?and?maternal?fat?and?carbohydrate?intake?is?associated?with?changes?in?hormone?balance?that?affect?fetal?growth?(King,?2006;?Jansson?et?al.,?2008).?Globally,?the?most?pervasive?affliction?caused?by?sub?optimal?maternal?nutrition?is?intrauterine?growth?restriction?(IUGR),?resulting?in?low?birth?weight?infants?(LBW;?WHO,?2002).?According?to?the?WHO,?24%?of?children?born?annually?are?classified?as?LBW?(WHO,?2002).?LBW?infants?are?at?an?increased?risk?of?neonatal?morbidity?and?mortality,?with?an?associated?increase?in?disability?adjusted?lifestyle?years?and?disease?susceptibility?in?later?life?(WHO,?2002).?Central?to?this?issue?is?the?availability?of?high?quality?or??complete??protein?in?the?diet,?because?it?has?been?suggested?that?dietary?protein?is?the?macronutrient?with?the?single?greatest?influence?on?birth?? 2?weight?(Cuco?et?al.,?2006).?Epidemiological?studies?have?linked?both?insufficient?and?excessive?dietary?protein?intake?to?an?increased?risk?of?IUGR,?although?the?mechanism?for?the?latter?remains?unclear?(Godfrey?et?al.,?1996;?Sloan?et?al.,?2001;?Kramer?and?Kakuma,?2003;?Moore?et?al.,?2004;?Imdad?and?Bhutta,?2011).?Dietary?protein?and?IUGR?are?related?in?developing?and?developed?countries?for?opposing?reasons.?In?developing?countries,?access?to?complete?dietary?protein?is?limited?by?socio?economic?status,?and?women?are?more?likely?to?suffer?from?insufficient?protein?intake?(Gebre?Medhin?and?Gobezie,?1975;?M?ller?and?Krawinkel,?2005).?In?developed?countries,?the?popularity?of?high?protein?diets?may?be?cause?for?concern?if?the?percentage?of?energy?from?protein?exceeds?a?healthy?intake?(Kramer?and?Kakuma,?2003).?However,?current?protein?intake?recommendations?for?healthy?pregnant?women?are?based?on?factorial?calculations?of?nitrogen?balance?data?derived?from?non?pregnant?adults.?Thus,?it?is?necessary?to?define?protein?requirement?directly?during?healthy?pregnancy,?and?thereby?encourage?maternal?nutritional?status?that?optimizes?pregnancy?outcomes.?The?objective?of?this?study?was?to?define?optimal?protein?requirement?during?healthy?pregnancy?using?a?modern,?state?of?the?art?stable?isotope?based?method???the?indicator?amino?acid?(IAAO)?method.??? ?? 3?Chapter?2.?Background??2.1.?Metabolic?adaptations?to?pregnancy?? Pregnancy?is?an?exceptional?stage?of?life?defined?by?rapid?growth?and?development,?and?enormous?maternal?physiological?changes?from?the?time?of?conception?to?parturition.?In?addition?to?fetal?development,?there?is?rapid?growth?of?maternal?tissues?like?the?heart,?blood,?breasts?and?uterus,?and?fetal?support?tissues?like?the?placenta?and?extra?embryonic?membranes?(Boron?and?Boulpaep,?2003).?To?accommodate?this?increased?demand,?maternal?metabolic?adaptations?to?pregnancy?arise.?The?overall?effect?of?these?adaptations?results?in?two?discrete?metabolic?phases:?anabolic?and?catabolic.??The?first?two?thirds?of?gestation?are?defined?by?anabolic?adaptations?that?prepare?the?pregnant?body?for?the?impending?energetic?demands?of?the?fetus?by?building?fat?stores?(Knopp?et?al.,?1981;?Herrera,?2000).?Fat?accumulation?is?facilitated?by?increased?insulin?release,?glucose?utilization,?and?lipogenesis,?normal?glucose?tolerance,?and?decreased?lipolysis?(Knopp?et?al.,?1981;?Butte,?2000).?During?this?stage,?the?demand?for?energy?is?high,?with?~36,000?kcal?of?energy?required?to?build?fat?stores?(King,?2006).?During?the?last?third?of?gestation,?nutritional?demands?of?the?fetus?increase?markedly.?In?response,?maternal?lipid?utilization?increases,?and?glucose?and?amino?acids?are?spared?for?the?fetus.?This?phase?is?defined?by?catabolic?adaptations?that?include?decreased?protein?catabolism,?increased?maternal?insulin?resistance,?hepatic?glucose?production,?lipolysis,?and?fat?mobilization,?and?decreased?glucose?tolerance?and?lipogenesis?(Knopp?et?al.,?1981;?Butte,?2000;?King,?2006).?Despite?this?elegant?metabolic?response,?mothers?must?? 4?consume?more?nutrients?in?order?to?provide?the?additional?biochemical?substrates?needed?to?support?a?healthy?pregnancy.??2.2.?Maternal?nutrition??2.2.1.?Impact?of?maternal?nutrition?on?infant?health?? To?support?gestational?demands,?healthy?pregnant?women?have?different?nutritional?requirements?than?non?pregnant?adults.?The?importance?of?maternal?nutrition?is?illustrated?in?a?number?of?epidemiological?studies,?perhaps?most?notably?by?the?exhaustive?follow?up?of?Dutch?famine?survivors.?During?the?winter?of?1944?1945,?German?troops?restricted?food?supply?to?many?areas?in?the?Netherlands,?resulting?in?adult?energy?intakes?of?less?than?600?kCal?per?day.?Prenatal?exposure?to?these?conditions?resulted?in?lower?birth?weight,?lower?placental?weight,?reduced?length?at?birth?and?reduced?head?circumference?in?the?offspring?(Stein?and?Susser,?1975).?Long?term?cohort?studies?have?demonstrated?an?increased?prevalence?of?glucose?intolerance,?coronary?heart?disease,?disturbed?blood?coagulation,?obesity,?and?obstructive?airway?disease?among?survivors?(Roseboom?et?al.,?2006).??As?evidenced?by?the?Dutch?famine?studies,?inadequate?maternal?nutrition?is?associated?with?a?variety?of?adverse?pregnancy?outcomes,?including?both?short??and?long?term?infant?morbidities?and?mortality.?In?the?short?term,?malnourished?mothers?are?more?likely?to?produce?IUGR?infants.?IUGR?infants?are?at?an?increased?risk?of?hypoxia/asphyxiation,?meconium?aspiration,?and?hypoglycemia,?which?may?result?in?death?without?adequate?medical?intervention?and?perinatal?care?(Kendig,?2007).?In?? 5?the?long?term,?inadequate?prenatal?nutrition?is?associated?with?an?increased?risk?of?hypertension,?coronary?heart?disease,?respiratory?disease,?kidney?disease?and?obesity?(Harding,?2001).?However,?the?hazards?of?poor?prenatal?nutrition?are?not?limited?to?insufficient?dietary?intake.??Unprecedented?food?security?in?recent?generations?has?revealed?that?excessive?maternal?nutrition?can?also?be?detrimental?(Godfrey?et?al.,?1996,?Kramer?and?Kakuma,?2003,?Moore?et?al.,?2004;?King,?2006).?Prospective?observational?studies?of?maternal?dietary?intake?patterns?have?revealed?that?superfluous?dietary?protein?or?carbohydrate?intake?is?also?associated?with?IUGR,?and?excessive?energy?intake?with?infant?macrosomia?(i.e.?birth?weight?>4.5?kg;?Godfrey?et?al.,?1996,?Sloan?et?al.,?2001;?Kramer?and?Kakuma,?2003,?Moore?et?al.,?2004;?King,?2006;?Imdad?and?Bhutta,?2011).?Macrosomia?is?associated?with?complicated?delivery?and?perinatal?hypoglycemia,?and?an?increased?risk?of?metabolic?syndrome?in?later?life?(Kendig,?2007).?Thus,?the?classic?conception?of??eating?for?two??during?pregnancy?has?been?debunked,?and?the?importance?of??optimal?and?adequate??nutrition?must?be?emphasized.??2.2.2.?Protein?and?amino?acid?nutrition?during?pregnancy??Dietary?protein?is?of?particular?importance?to?maternal?nutrition.?Dietary?protein?supports?fetal?and?placental?tissue?accretion,?permits?uterine?expansion?and?mammary?growth,?and?allows?for?an?increase?in?blood?volume?via?hematocrit?and?plasma?genesis?(Boron?and?Boulpaep,?2003).?Proteins?serve?a?variety?of?structural?and?functional?purposes?in?the?body,?acting?as?enzymes,?transporters,?signaling?molecules,?contractile?units,?connective?tissue,?and?many?other?roles.?The?vast?? 6?diversity?of?protein?types?and?functions?is?possible?because?of?their?composition.?20?amino?acids?act?as?the?building?blocks?from?which?mammalian?proteins?are?synthesized,?creating?the?potential?for?millions?of?unique?peptide?sequences?(DRI,?2005;?Otten?et?al.,?2006;?Table?1).?From?a?nutritional?perspective,?9?of?these?amino?acids?are?recognized?as?indispensable?in?the?diet,?owing?to?the?fact?that?they?cannot?be?endogenously?synthesized??out?of?materials?ordinarily?available?to?the?cell?at?a?speed?commensurate?with?normal?growth??(Reeds,?2000;?Borman?et?al.,?1946).?The?remaining?11?amino?acids?can?potentially?be?produced?in?nutritionally?sufficient?quantities?via?de?novo?synthesis?or?precursor?molecule?modification,?so?long?as?a?sufficient?nitrogen?source?is?provided?(Reeds,?2000).?Under?specific?conditions?the?human?body?may?require?arginine,?cysteine,?glutamine,?glycine,?proline?and?tyrosine???the?conditionally?indispensable?amino?acids???from?dietary?sources?(Reeds,?2000).?However,?the?dispensable/indispensable?paradigm?is?misleading?because?it?implies?that?a?healthy?diet?need?only?contain?sufficient?quantities?of?isoleucine,?leucine,?histidine,?lysine,?tryptophan,?phenylalanine,?valine,?methionine,?and?threonine,?in?order?to?meet?the?needs?of?protein?metabolism.?Protein?metabolism?in?humans?is?a?highly?integrated?process?dependent?upon?the?biological?availability?of?all?20?amino?acids?(WHO,?2007).?All??nonessential??amino?acids?require?an?amino?acid?source?of?nitrogen?for?biosynthesis?(Reeds,?2000).?In?this?regard,?all?20?amino?acids?required?for?protein?metabolism?in?humans?may?be?adversely?affected?by?limited?protein?intake.?Extremes?in?amino?acid?availability,?resulting?from?insufficient?or?excessive?protein?intake,?have?been?identified?as?underlying?mechanisms?for?IUGR.?Prolonged?exposure?to?insufficient?protein?intake?causes??? 7??Table?1.?Classification?of?amino?acids?as?dispensable,?conditionally?indispensable,?and?indispensable?in?the?diet?? Indispensable? Dispensable? Conditionally?Indispensable?Histidine? Alanine? Arginine?Isoleucine? Aspartic?Acid? Cysteine?Leucine? Asparagine? Glutamine?Lysine? Glutamic?Acid? Glycine?Methionine? Serine? Proline?Phenylalanine? ? Tyrosine?Threonine? ? ?Tryptophan? ? ?Valine? ? ??depletion?of?the?body?s?amino?acid?pool,?thereby?reducing?protein?synthesis?and?tissue?accretion.?Specifically,?the?biochemical?contributions?made?by?individual?amino?acids?can?hinder?intrauterine?growth.?For?example,?arginine?is?integral?to?the?synthesis?of?nitric?oxide?and?polyamines,?which?contribute?to?placental?angiogenesis?(Wu?et?al.,?2000).?While?healthy?pregnant?women?are?capable?of?synthesizing?arginine,?the?fetus?cannot?and?must?procure?arginine?via?the?placenta?(Wu?et?al.,?2000).?If?fetal?uptake?diminishes?maternal?arginine?supplies?more?quickly?than?they?can?be?replenished?via?endogenous?synthesis,?deficiency?will?ensue?unless?dietary?protein?intake?can?compensate?for?the?increased?demand.?Alternatively,?excessive?amino?acid?availability?may?cause?IUGR?due?to?competition?between?amino?acids?for?placental?transport?systems?(Ronzoni?et?al.,?2002;?Jozwik?et?al.,?2004).?Evidence?from?human?and?animal?studies?suggests?that?when?placental?transport?systems?are?saturated?with?amino?acids,?competition?arises?for?these?carriers?resulting?in?reduced?transport?of?certain?essential?amino?acids?(Ronzoni?et?al.,?2002;?Jozwik?et?? 8?al.,?2004).?This?can?then?lead?to?deficiencies?and?reduced?protein?synthesis.?Therefore,?finding?the??optimal??balance?among?all?amino?acids?during?pregnancy?is?critical?to?ensure?a?healthy?pregnancy.?2.3.?Fetal?and?placental?amino?acid?metabolism??2.3.1.?Current?understanding?of?fetal?amino?acid?metabolism?? Fetal?plasma?amino?acid?concentrations?are?significantly?higher?than?maternal?plasma?concentrations,?and?this?phenomenon?persists?throughout?pregnancy?(Cetin?et?al.,?1996).?Although?ethical?and?technical?constraints?have?hindered?in?vivo?studies?of?fetal?amino?acid?metabolism?in?humans,?the?development?of?stable?isotope?based?methods?has?revealed?new?information?(Chein?et?al.,?1993;?Marconi?et?al.,?1999;?Galan?et?al.,?2009;?van?den?Akker?et?al.,?2009?and?2011).?Using?13C?leucine?and?15N?phenylalanine?infusion?into?the?umbilical?cord?of?6?pregnant?women?at?term,?protein?synthesis?in?the?fetus?was?calculated?as?~13?g/kg/d,?with?net?protein?accretion?of?2?4?g/kg/d?(Chein?et?al.,?1993).?This?study?demonstrated?that?protein?turnover?is?increased?in?the?fetus,?and?similar?to?turnover?rates?observed?in?neonates.?A?more?recent?study?came?to?a?very?different?conclusion,?estimating?net?protein?accretion?as?1.7?g/kg/d?in?human?fetuses?at?term?(van?den?Akker?et?al.,?2009).?Amino?acid?kinetics?for?leucine,?phenylalanine,?tyrosine,?methionine,?and?valine?have?also?been?examined?in?human?fetuses?at?term?using?[1?13C]phenylalanine,?[ring?D4]tyrosine,?[1?13C,?15N]leucine,?[U?13C5]valine,?and?[1?13C]methionine??(van?den?Akker?et?al.,?2009?and?2011).??Net?accretion?of?40,?28,?9.2,?? 9?and?17.5??mol/kg/h?was?calculated?for?leucine,?valine,?methionine,?and?phenylalanine,?respectively.?The?metabolized?fraction?of?leucine,?valine,?methionine,?phenylalanine?and?tyrosine?was?considerably?less?than?what?was?available,?suggesting?a?large?amino?acid?reserve?capacity?in?the?fetus?(van?den?Akker?et?al.,?2009?and?2011).??Although?each?of?these?studies?provides?new?insight?into?how?the?fetus?uses?protein?and?amino?acids,?current?data?is?limited?to?the?fetus?at?term,?and?offers?conflicting?results.?It?is?clear?that?much?remains?to?be?determined?regarding?fetal?protein?and?amino?acid?metabolism.?2.3.2.?Placental?transport?of?amino?acids?The?placenta?is?a?highly?metabolically?active?organ?that?participates?in?protein?synthesis,?oxidation,?transamination,?and?nonessential?amino?acid?production?(Hay,?1991;?Battaglia?and?Regnault,?2001;?Cetin?2001;?Lewis?et?al.,?2012).?Amino?acids?are?more?concentrated?in?the?placenta?than?either?the?fetal?or?maternal?compartments,?and?transfer?of?amino?acids?is?asymmetric?but?preferentially?directed?towards?the?fetus?(Schneider?et?al.,?1987).?Evidence?suggests?that?while?some?amino?acids?move?rapidly?from?mother?to?fetus?(e.g.?lysine),?others?exhibit?no?net?transfer?across?the?placenta?(i.e.?glutamate,?aspartate)?and?others?still?appear?to?flow?from?the?mother?and?fetus?into?the?placenta?(i.e.?serine)?for?the?purpose?of?placental?amino?acid?production?(i.e.?glycine;?Cetin,?2001;?Jannson,?2001;?Avagliano?et?al.,?2012;?Lewis?et?al.,?2012).??Transplacental?amino?acid?transport?occurs?at?strikingly?different?rates?depending?on?the?amino?acid?(Galen?et?al.,?2009).?Although?not?fully?understood,?? 10?several?characteristics?of?the?placenta?contribute?to?this?variation.?The?placenta?uses?multiple?transport?systems?to?move?amino?acids?from?the?maternal?side?(across?the?microvillus?plasma?membrane)?to?the?fetal?side?(across?the?basal?plasma?membrane;?Jansson,?2001;?Avagliano?et?al.,?2012;?Lewis?et?al.,?2012).?For?example,?the?System?A?family?accumulative?transporters?move?small,?neutral?amino?acids,?while?the?System?X??AG?family?exchange?transporters?move?aspartic?acid,?glutamic?acid,?and?cysteine?(Lewis?et?al.,?2012).?Each?of?these?transport?systems?exhibits?high?substrate?specificity,?and?uses?a?different?mode?of?action.?Common?amongst?these?transporters?is?their?sensitivity?to?maternal?fetal?concentration?gradients.?Accumulative?transporters?are?driven?by?electrochemical?differences?across?the?microvillus?and?basal?plasma?membranes;?exchange?transporters?qualitatively?change?the?amino?acid?profile?across?the?membrane?without?altering?osmolality;?and?facilitated?transporters?move?amino?acids?down?their?concentration?gradient?(Lewis?et?al.,?2012).?The?unique?contribution?made?by?each?transport?system?to?the?overall?establishment?of?concentration?gradients?means?that?placental?amino?acid?transporter?activity?is?very?interdependent.?For?example,?increased?transport?of?lysine?into?the?fetal?compartment?will?alter?the?osmolality?and?electrochemical?gradients?across?the?basal?plasma?membrane,?causing?a?shift?in?the?rate?at?which?other?amino?acids?are?transferred.?Thus,?each?of?the?individual?placental?transport?systems?influences?amino?acid?transfer?rates?and?variability.???As?the?elaborate?relationships?governing?placental?transfer?of?amino?acids?are?unraveled,?it?becomes?clear?that?the?placenta?is?not?a?passive?conduit?but?an?integral?organ?in?the?highly?orchestrated?process?of?gestational?protein?metabolism.???? 11?2.4.?Protein?metabolism?changes?during?different?stages?of?pregnancy?? Maternal?glucose?and?fatty?acid?metabolism?becomes?increasingly?adapted?as?gestation?progresses?so?that?the?availability?of?these?energy?yielding?molecules?is?commensurate?with?the?increasing?demands?of?pregnancy?(Kalhan,?2000).?In?contrast,?adaptations?to?maternal?protein?metabolism?begin?early?in?gestation,?before?there?is?a?significant?increase?in?conceptus?demand?(Kalhan,?2000).?Changes?to?maternal?protein?metabolism,?as?compared?to?non?pregnant?women,?are?evidenced?by?differences?in?plasma?amino?acid?concentration?as?early?as?5?weeks?gestation?(Shoengold?et?al.,?1978).?With?the?exception?of?lysine,?histidine?and?threonine,?maternal?plasma?amino?acid?concentrations?are?significantly?lower?than?those?of?non?pregnant?women?(Shoengold?et?al.,?1978;?Cetin?et?al.,?1996).?A?comparison?of?plasma?amino?acid?profiles?in?early?versus?late?pregnancy?has?also?indicated?significant?differences?in?amino?acid?concentration???namely?a?significant?increase?in?threonine,?methionine,?serine,?and?glycine?in?late?gestation?(Cetin?et?al.,?1996).?Similarly,?gestational?differences?in?protein?metabolism?are?evident?in?the?amino?acid?profile?of?amniotic?fluid?(Reid?et?al.,?1971;?Rabier?et?al.,?1996).?With?the?exception?of?aspartic?acid,?serine,?glutamine?and?glycine,?all?other?amino?acids?significantly?decrease?in?concentration?within?the?amniotic?fluid?as?gestation?progresses?(Reid?et?al.,?1971;?Rabier?et?al.,?1996).??Nitrogen?metabolism?studies?conducted?using?15?N?leucine?also?show?gestational?differences:?significantly?higher?nitrogen?retention,?and?significantly?lower?urea?synthesis?and?branched?chain?amino?acid?oxidation?in?late?versus?early?? 12?pregnancy?(Kalhan,?2000;?King,?2000).??These?findings?suggest?that?adaptations?to?protein?metabolism?in?the?late?stages?of?pregnancy?favor?nitrogen?conservation.?Recently,?stable?isotope?techniques?have?been?used?to?determine?threonine?and?lysine?requirement?in?pregnant?sows?(Levesque?et?al.,?2009;?Levesque?et?al.,?2010;?Samuel?et?al.,?2012).?These?studies?identified?a?200%?and?85%?increase?in?threonine?and?lysine?requirement,?respectively,?during?late?as?compared?to?early?gestation?(Levesque?et?al.,?2009;?Levesque?et?al.,?2010;?Samuel?et?al.,?2012).?Similar?experiments?have?not?been?published?in?human?pregnancy,?but?it?is?clear?that?in?addition?to?changes?in?maternal?protein?metabolism?there?exists?changing?demand?for?dietary?protein?and?amino?acids.?Therefore,?there?is?a?need?to?determine?protein?and?amino?acid?requirements?during?early?versus?late?gestation.??2.5.?Protein?requirements?during?pregnancy??? 2.5.1.?Current?protein?recommendations?during?pregnancy??The?United?Nations??(UN)?World?Health?Organization?(WHO)?is?the?leading?international?public?health?authority,?responsible?for?determining?human?nutrition?requirements?and?providing?dietary?intake?recommendations.?In?conjunction?with?the?UN?Food?and?Agricultural?Organization?(FAO),?the?WHO/FAO/UNU?expert?council?has?set?international?protein?intake?recommendations?during?healthy?pregnancy?as?an?additional?0.7,?9.6,?and?31.2?grams?of?high?quality?protein?per?day?(g/d)?above?the?healthy?non?pregnant?adult?recommended?protein?intake?of?0.66?grams?per?kilogram?per?day?(g/kg/d)?in?the?1st,?2nd,?and?3rd?trimesters?of?pregnancy,?? 13?respectively?(WHO,?2007;?Table?2).?This?estimate?was?derived?by?a?factorial?approach?using?data?from?nitrogen?balance?studies?of?non?pregnant?adults,?and?mean?protein?deposition?during?pregnancy?obtained?from?studies?of?total?body?potassium?(WHO,?2007).??In?North?America,?nutrient?intake?recommendations?are?set?by?the?Dietary?Reference?Intakes?for?macronutrients?(DRI),?which?is?published?by?the?Institute?of?Medicines??Food?and?Nutrition?Board?(DRI,?2005).?The?current?DRI?recommends?that?healthy?pregnant?women?consume?at?minimum?0.88?g/kg/d???the?equivalent?of?an?additional?21?g/d???of?high?quality?protein?beyond?20?weeks?gestation?(DRI,?2005).?This?value?represents?the?estimated?average?requirement?(EAR),?or?the?protein?intake?that?will?ensure?50%?of?the?populations??dietary?protein?needs?are?met.?To?ensure?that?the?dietary?protein?needs?of?95%?of?the?population?are?met,?the?DRI?recommends?consuming?an?intake?that?is?2?standard?deviations?beyond?the?EAR,?a?value?referred?to?as?the?recommended?daily?allowance?(RDA).?The?current?DRI?RDA?for?protein?intake?during?healthy?pregnancy?is?1.1?g/kg/d?beyond?20?weeks?gestation?(DRI,?2005).??The?DRI?protein?requirement?estimate?for?healthy?pregnant?women?is?also?derived?by?a?factorial?approach?using?data?from?nitrogen?balance?studies?of?non?pregnant?adults,?and?mean?protein?deposition?during?pregnancy?obtained?from?studies?of?total?body?potassium?(DRI,?2005;?Appendix?A).?The?study?described?in?this?thesis?was?conducted?in?a?North?American?context,?so?the?DRI?protein?intake?recommendations?will?serve?as?the?primary?point?of?comparison?for?our?estimation?of?protein?requirement?of?healthy?pregnant?women.?? 14??Table?2.?Comparison?of?dietary?protein?intake?recommendations?by?the?DRI?(2005),?WHO?(2007),?and?IAAO?method,?and?dietary?intake?patterns?of?pregnant?women?in?BC,?Canada.??Dietary?Protein?g/kg/d???? ?Adult??(19?50?y)?DRI?2005? Pregnancy??(all?age?groups)?DRI?2005?Pregnancy??(all?age?groups?by?trimester)?WHO?2007?Adult?(19?50?y)?Elango?et?al.,?2009?Dietary?protein?intake?of?pregnant?women?in?BC,?Canada?Stephens?et?al.?(unpublished)?EAR?? RDA? EAR? RDA? 1st?? 2nd?? 3rd?? EAR? RDA? Intake??16wk? Intake??36wk?0.66? 0.80? 0.88? 1.1? 0.71? 9.61? 31.21? 0.93? 1.2? 1.5? 1.3??1Additional?grams?of?protein?per?day?(g/d)?above?adult?recommended?intake?of?0.66?g/kg/d??? 2.5.2.?Evaluation?of?DRI?during?pregnancy?? As?previously?stated,?the?current?DRI?recommends?that?healthy?pregnant?women?consume?1.1?grams?of?dietary?protein?per?kilogram?of?body?weight?per?day?(Table?2;?Appendix?A).?This?estimate?is?largely?based?on?research?conducted?by?J.C.?King,?D.?Calloway?and?S.?Margen?in?pregnant?adolescent?girls?(King?et?al.,?1973).?King?et?al.?(1973)?examined?the?balance?of?nitrogen?and?total?body?potassium?in?order?to?determine?nitrogen?retention?in?10?pregnant?girls?aged?15?19?years.?The?accuracy?of?nitrogen?balance?studies?has?been?critically?examined?in?a?number?of?scientific?publications?(Hegsted,?1976;?Young,?1986;?Waterlow,?1999).?In?general,?nitrogen?balance?estimates?are?cumbersome?due?to:?the?difficulty?of?accounting?for?miscellaneous?losses?(e.g.?hair,?nails,?sweat)?leading?to?underestimates?of?nitrogen?loss;?the?tendency?to?overestimate?nitrogen?intake;?the?lengthy?period?(~7?days)?required?to?adapt?to?an?experimental?diet?before?nitrogen?excretion?data?can?be?? 15?collected;?the?necessity?for?subjects?to?strictly?adhere?to?the?experimental?diet;?and?the?decrease?in?protein?utilization?efficiency?that?is?associated?with?dietary?test?protein?intakes?approaching?zero?nitrogen?balance?(Hegsted,?1976;?Young,?1986;?Waterlow,?1999).??Overall,?these?variables?tend?to?confound?nitrogen?retention?estimates,?resulting?in?an?underestimate?of?protein?requirements.?The?King?et?al.?(1973)?study?rigorously?diminished?environmental?variability?by?confining?subjects?to?an?experimental?site,?allowing?for?complete?measurement?of?all?nitrogen?intakes?and?losses.?However,?according?to?the?United?Nations?University?Food?and?Nutrition?department?s?criteria?for?upholding?the?validity?of?nitrogen?balance?assessments?of?protein?requirements,?the?King?et?al.?(1973)?study?violated?at?least?two?criteria?(Scrimshaw,?1996).?First,?the?researchers?allowed?subjects?to?freely?consume?snacks?devoid?of?protein?(e.g.?juice,?taffy,?soda).?This?violates?the?criterion?that?energy?intake?must?match?energy?requirements.?Second,?because?subjects?were?free?to?consume?beverages?ad?libitum,?urinary?nitrogen?retention?estimates?may?have?been?artificially?increased?due?to?uncontrolled?fluid?intake.?The?DRI?recommendations?also?take?into?account?six?studies?of?total?body?potassium?(TBK)?content?during?pregnancy,?as?an?indirect?estimate?of?protein?accretion.?In?each?of?these?studies,?TBK?was?measured?using?the?4??liquid?scintillation?counter?method,?which?is?well?validated?(Barnaby?and?Jasani,?1968;?Smith?et?al.,?1979).?However,?the?potassium/nitrogen?ratio?used?in?the?DRI?s?calculation?of?average?protein?deposition?(2.15?mmol?K/g?N)?is?based?on?infant?carcass?analysis?(Hamilton?and?Moriarty,?1929;?Iob?and?Swanson,?1934;?Fee?and?? 16?Weil,?1963;?Widdowson?and?Dickerson,?1964).??The?problem?with?using?this?value?when?investigating?pregnant?women?is?that?it?differs?from?the?value?obtained?using??adult?tissue?(2.7?mmol?K/?g?N),?and?thus?may?not?accurately?represent?adult?protein?deposition?(Reifenstien?et?al.,?1945).??Additionally,?the?DRI?derivation?for?protein?requirements?during?pregnancy?assumes?a?protein?utilization?efficiency?value?of?0.43,?which?is?less?than?the?efficiency?value?used?in?calculating?non?pregnant?adult?protein?requirement?(0.47;?DRI,?2005;?Otten?et?al.,?2006).?It?is?contradictory?that?during?a?period?of?increased?protein?requirement?(gestation),?the?pregnant?body?would?adapt?by?decreasing?protein?utilization?efficiency?(King,?2000).?Given?that?the?0.43?efficiency?value?is?based?on?the?slope?of?a?regression?line?for?protein?intake?versus?nitrogen?balance?from?the?King?et?al.?(1973)?data,?it?is?possible?that?this?value?was?distorted?by?the?aforementioned?violations?of?criteria?necessary?for?upholding?the?validity?of?nitrogen?balance?assessments?of?protein?requirements.?Alternatively,?the?biologically?implausible?efficiency?value?may?be?due?to?the?fact?that?King?et?al.?(1973)?used?a?linear?regression?analysis?to?determine?protein?utilization?efficiency.?Because?there?is?a?decline?in?protein?utilization?efficiency?from?test?protein?intakes?as?zero?balance?is?approached,?linear?regression?analysis?is?not?a?valid?statistical?tool?for?assessing?the?relationship?between?protein?intake?and?nitrogen?balance?(Rand?and?Young,?1999).?2.5.3.?Fundamental?criticisms?of?nitrogen?balance?studies?It?is?important?to?address?three?fundamental?criticisms?of?methodology?used?hitherto?when?determining?human?protein?requirements.?First,?nitrogen?balance?? 17?studies?are?highly?invasive.?Subjects?must?strictly?adhere?to?experimental?diets?over?relatively?long?periods?of?time,?must?limit?their?physical?activity,?and?are?often?confined?to?experimental?units?in?order?to?reduce?environmental?sources?of?variability?in?the?data.?Second,?in?order?to?determine?protein?requirements,?subjects?must?adapt?to?diets?designed?to?include?test?protein?intakes?both?above?and?below?the?expected?protein?requirement.?For?vulnerable?populations?such?as?pregnant?women,?a?prolonged,?induced?state?of?protein?deficiency?or?excess?could?pose?serious?risk,?and?as?such?is?unethical?(Bruton?et?al.,?1998).?Third,?nitrogen?balance?and?total?body?potassium?assessments?have?fundamental?experimental?flaws?that?cannot?be?easy?corrected.?Therefore,?it?is?essential?to?the?study?of?nutrition,?to?public?health?initiatives,?and?to?the?clinical?application?of?dietetics,?that?novel?techniques?be?employed?in?reassessing?protein?requirements.?In?this?pursuit,?the?indicator?amino?acid?oxidation?(IAAO)?method???with?the?use?of?stable?isotopes???has?emerged?as?a?compelling?technique?due?to?its?minimally?invasive?procedure.??2.6.?Stable?isotopes?and?their?use?in?pregnancy?studies?? 2.6.1.?Stable?isotopes?as?tracers?Stable?isotopes?are?naturally?occurring?compounds?present?in?the?foods?we?eat?and?the?environment?we?live?in.?For?the?4?most?common?atoms?that?make?up?living?organisms?(carbon,?hydrogen,?oxygen?and?nitrogen)?there?are?stable?isotopes.?For?example,?carbon?13?(13C)?is?the?stable?isotope?of?the?commonly?occurring?carbon?12?(12C),?and?15N?is?the?stable?isotope?to?the?more?common?14N.??Stable?isotopes?are?present?in?living?things?at?very?low?enrichments???expressed?as?atomic?percent?? 18?(atom%).?For?example?the?natural?abundance?of?13C?is?1.11?atom%,?and?15N?is?0.37?atom%.??These?isotopes?differ?from?their?common?atomic?counterparts?in?that?they?posses?an?extra?neutron,?and?therefore?extra?mass.?The?atomic?mass?difference?can?be?detected,?and?overall?isotope?enrichment?quantified,?from?biological?samples?using?a?mass?spectrometer.?Thus,?stable?isotopes?can?be?used?as?labels?to?follow?substrate?metabolism,?making?them?ideal?experimental?tracers.?Stable?isotope?tracers?have?become?an?alternative?to?radioactive?tracers,?which?decay?over?time?and?emit?radiation,?making?them?carcinogenic.???2.6.2.?Protein?metabolism?during?pregnancy?studied?using?stable?isotopes?In?addition?to?the?aforementioned?studies?of?fetal?amino?acid?metabolism?using?stable?isotopes?(Section?2.3.1.),?several?studies?have?examined?whole?body?protein?metabolism?during?pregnancy.?Using?stable?isotope?labeled?amino?acids,?oxidative?disposal?(e.g.?catabolism?of?amino?acids?to?urea?and?CO2)?and?non?oxidative?disposal?(e.g.?anabolic?uptake?of?amino?acids?for?protein?synthesis)?are?measured,?revealing?changes?to?amino?acid?usage?with?test?protein?intakes.?Maternal?whole?body?protein?metabolism?was?studied?at?13,?24?and?35?weeks?gestation?in?six?healthy?women?using?a?continuous?infusion?of?[1?13C]leucine?(Thompson?and?Halliday?1992;?Appendix?H).?Mean?protein?synthesis?increased?from?5.3?(?0.6?SD)?grams?per?kilogram?fat?free?mass?per?24?hours?(g/(kg?FFM*24h))?at?13?weeks?to?5.9?0.5?g/(kg?FFM*24h)?(p<0.1)?at?24?weeks?and?6.1?0.6?g/(kg?FFM*24h)?at?35?weeks.?Protein?synthesis?at?24?and?35?weeks?gestation?was?significantly?greater?when?compared?to?a?group?of?17?healthy,?non?pregnant?women?(4.9?0.6?g/(kg?FFM*24h);?p<0.001).?These?data?indicate?that?there?are?substantial?increases?in?? 19?protein?turnover?during?pregnancy.??However,?some?recent?studies?have?found?no?significant?increase?in?protein?turnover?in?the?3rd?trimester,?suggesting?that?protein?conservation?occurs?in?the?final?stage?of?gestation?(Whittaker?et?al.,?2000;?Appendix?H).?Whittaker?and?colleagues?infused?6?pregnant?women?with?[1?13C]leucine?between?34?38?weeks?gestation.?They?studied?the?same?women?post?partum?and?concluded?that?protein?turnover?was?not?significantly?different?between?the?3rd?trimester?and?non?pregnant?state.??This?study?also?suggested?that?the?sum?of?leucine?kinetic?differences?(oxidative?disposal?versus?non?oxidative?disposal)?resulted?in?a?net?increase?in?protein?conservation.?Similar?results?have?been?demonstrated?by?Jolly?et?al.?(2004)?using?[1?13C]leucine?and?Whittaker?et?al.?(1999)?using?[ring?2H5]phenylalanine.?To?investigate?the?apparent?decrease?in?protein?turnover?in?the?3rd?trimester?of?pregnancy,?Kalhan?and?Parimi?(2006)?studied?protein?and?amino?acid?metabolism?using?1?[13C]leucine,??1?[13C]phenylalanine?and?[15N]glycine.?Kalhan?and?Parimi?s?data?suggests?that?during?pregnancy?major?physiological?adaptations?result?in?changes?to?maternal?protein?metabolism?that?conserve?protein?for?protein?synthesis?in?the?maternal?and?fetal?compartments.??The?combined?results?show?that?stable?isotopes?can?be?used?to?examine?protein?and?amino?acid?metabolism?in?pregnant?populations?reliably.?However,?no?study?has?yet?applied?stable?isotope?techniques?to?determine?protein?or?amino?acid?requirements?during?pregnancy.?Two?possible?reasons?for?why?stable?isotope?techniques?have?not?been?applied?more?broadly?to?consider?requirements?during?pregnancy?are?that?1)?most?of?the?studies?already?conducted?involve?intravenous?infusion?of?isotopes?and?2)?studies?to?date?have?been?conducted?in?the?fasted?state?? 20?when?the?rate?of?protein?synthesis?decreases.?A?relatively?non?invasive,?fed?state?method?is?necessary?to?conduct?nutrient?requirement?studies?in?pregnant?women,?and?the?IAAO?technique?is?well?suited?for?this?purpose.?It?has?been?applied?previously?to?study?protein?requirements?of?healthy?adult?men?and?healthy?school?age?children?using?oral?stable?isotope?(1?[13C]phenylalanine)?infusion,?and?collection?of?breath?and?urine?to?measure?protein?synthesis?(Humayun?et?al.,?2007;?Elango?et?al.,?2011).?2.7.?The?IAAO?method:?a?robust?model?for?determining?protein?requirements?of?vulnerable?populations?? 2.7.1.?IAAO?method:?principles?and?protocol?The?IAAO?method?is?based?on?the?principle?that?if?a?single?indispensable?amino?acid?is?deficient?for?protein?synthesis,?then?all?other?amino?acids?will?be?oxidized?(Ball?and?Bayley,?1984;?Elango?et?al.,?2008).?This?is?because?excess?amino?acids?are?not?effectively?stored?in?the?body?and?are?therefore?disposed.?In?practice,?an?isotopically?labeled?amino?acid?(usually?L?[1?13C]phenylalanine)?is?taken?orally,?along?with?an?amino?acid?mixture?representative?of?a?complete?protein?(complete?protein?defined?here?as?the?amino?acid?combination?present?in?egg?protein;?Elango?et?al.,?2008).?The?amount?of?protein?administered?is?graded?to?give?intakes?both?above?and?below?the?predicted?requirement.?Subsequent?changes?in?protein?dynamics?are?measured?as?the?amount?of?heavy?carbon?(13C)?produced?from?the?indicator?amino?acid?in?breath?(13CO2)?and?urine?(13C?amino?acid)?samples?(Elango?et?al.,?2008).?Exhaled?13CO2?is?plotted?against?the?range?of?administered?test?protein?intakes?(Figure?1).?When?these?data?points?are?analyzed?using?a?two?phase?linear?regression?crossover?model,??? 21???Figure?1.?Estimated?average?protein?requirement?analysis?using?IAAO?method?(from?Humayun?et?al.,?2007)??a?breakpoint?is?produced?where?the?rate?of?IAAO?plateaus.?Because?IAAO?is?inversely?proportional?to?protein?synthesis,?an?increase?in?the?amount?of?protein?intake???when?deficient???is?associated?with?a?corresponding?decrease?in?the?rate?of?IAAO.?Where?the?rate?of?IAAO?plateaus,?it?can?be?inferred?that?the?test?intake?is?sufficient?for?protein?synthesis?to?occur,?and?thus?represents?the?estimated?average?protein?requirement?(Brunton?et?al.,?1998;?Elango?et?al.,?2008).?2.7.2.?Concerns?with?the?IAAO?method?Criticism?of?the?IAAO?method?has?focused?heavily?on?the?minimal?adaptation?period?to?the?test?protein/amino?acid?intake?(Elango?et?al.,?2010).?In?response?to?? 22?these?concerns,?Elango?et?al.?(2009)?compared?the?results?of?IAAO?in?healthy?young?men?following?8?hours?(current?minimal?invasive?IAAO?model),?3?days?and?7?days,?adaptation.?The?results?yielded?no?significant?difference?in?F13CO2?(L?[1?13C]phenylalanine?indicator?used),?indicating?that?breakpoint?determination?was?not?affected?by?adaptation?period.??Additionally,?the?validity?and?sensitivity?of?measuring?breath?13CO2?flux?as?an?indication?of?protein?dynamics?has?been?contested.?Recent?evidence?has?helped?to?dispel?these?concerns?(Elango?et?al.,?2009;?Rafii?et?al.,?2008).?When?the?breakpoint?for?IAAO?measured?using?F13CO2?flux?from?breath?samples?was?compared?with?the?breakpoint?for?phenylalanine?hydroxylation?measured?using?apolipoprotein?B?100???a?protein?synthesized?from?intrahepatocyte?amino?acids???the?two?values?were?found?to?be?very?similar?(Elango?et?al.,?2009).?This?indicates?that?F13CO2?measured?from?breath?samples?reflects?changes?in?protein?synthesis?similar?to?an?intracellular?index.??2.7.3.?Advantages?of?the?IAAO?method?? The?benefits?of?utilizing?the?minimally?invasive?IAAO?model?are?substantial?(Zello?et?al.,?1995;?Brunton?et?al.,?1998;?Elango?et?al.,?2008).?Without?the?need?for?a?prolonged?adaptation?period,?subjects?are?only?required?to?fast?overnight?then?adhere?to?an?experimental?diet?for?8?hours?the?following?day.?By?reducing?the?amount?of?time?researchers?are?required?to?disrupt?the?subject?s?diet,?protein?requirements?can?be?directly?examined?in?vulnerable?populations.?During?the?study?day,?the?indicator?amino?acid?is?administered?orally,?and?does?not?involve?the?more?invasive?approach???intravenous?infusion.?Samples?are?collected?in?the?form?of?? 23?breath?and?urine,?and?all?samples?are?collected?during?the?8?hour?study?day,?reducing?subject?discomfort?and?lifestyle?disruption.?From?a?methodological?perspective,?the?IAAO?model?is?the?first?direct?measure?of?protein?metabolism?that?has?been?accepted?as?an?appropriate?application?for?determining?amino?acid?requirements?(Institute?of?Medicine?Food?and?Nutrition?Board,?2002;?Note:?the?IAAO?approach?to?determining?protein?requirements?is?still?the?subject?of?debate).?Furthermore,?by?measuring?an?end?product?of?protein?catabolism?(CO2)?we?arrive?at?a?single?estimate?of?protein?requirements?that?represents?the?pregnant?body?as?a?whole,?thereby?eliminating?complications?that?may?arise?due?to?differential?protein?metabolism?in?the?maternal,?placental?or?fetal?compartments.??Cumulatively,?the?minimally?invasive?qualities?that?characterize?the?IAAO?method?have?made?it?an?attractive?new?approach?to?determining?protein?requirements,?particularly?in?vulnerable?populations?such?as?pregnant?women.??2.8.?Summary?of?background??? The?critical?importance?of?maternal?nutrition?in?assuring?healthy?pregnancy?outcomes?is?undeniable.?Adequate?and?optimal?protein?intake?is?paramount?in?this?pursuit.?Currently,?there?is?a?paucity?of?evidence?supporting?protein?requirements?during?pregnancy,?and?no?studies?identifying?protein?requirements?during?different?stages?of?pregnancy.?This?is?a?direct?consequence?of?a?dependency?on?the?nitrogen?balance?technique,?which?has?many?assumptions?and?flaws,?and?is?unsuitable?for?vulnerable?populations.?The?minimally?invasive?IAAO?model?is?a?sensitive?and?robust?technique,?which?presents?an?opportunity?to?better?identify?protein?requirement?during?pregnancy.? ?? 24?Chapter?3.?Objective?and?hypothesis?? The?objective?of?this?study?was?to?determine?protein?requirements?of?healthy??women?aged?19?35y?during?early?(11?20?weeks)?and?late?(31?38?weeks)?gestation?by?use?of?the?minimally?invasive?IAAO?model,?where?oxidation?of?L?[13C]phenylalanine?to?13CO2?(collected?from?expired?breath)?in?response?to?a?graded?intake?of?crystalline?L?amino?acid?based?on?the?composition?of?egg?protein?is?measured.??IAAO?research?from?Humayun?et?al.?(2007)?indicates?that?the?current?DRI?underestimates?protein?requirements,?based?on?the?finding?of?a?41%?discrepancy?between?the?DRI?EAR?and?IAAO?derived?protein?requirement?estimated?from?healthy?adults.?Elango?et?al.?(2011)?achieved?similar?results?that?suggest?a?78%?discrepancy?between?the?DRI?estimated?average?protein?requirement?and?IAAO?derived?protein?requirement?of?healthy?children.?Based?on?these?findings,?we?hypothesize?that?the?current?recommendations?for?protein?intake?during?pregnancy?are?underestimated?(Table?2).??Nitrogen?retention?and?protein?deposition?studies?indicate?significantly?greater?protein?accretion?during?the?late?stages?of?pregnancy?(Pitkin,?1977;?Marino,?1983;?Denne?et?al.,?1991).?Based?on?these?findings,?we?hypothesize?that?protein?requirements?during?late?gestation?will?be?greater?than?early?gestation.???? 25?Chapter?4.?Methods?4.1?Subjects?4.1.1.?Age?demographic??This?study?examined?healthy,?pregnant?women?aged?19?to?35?years?(Figure?2).?The?upper?and?lower?limit?of?this?demographic?was?selected?to?reduce?the?risk?of?pregnancy?complications?that?might?skew?our?results.?Pregnancy?during?adolescence?is?associated?with?an?increased?risk?of?pre?term?and?very?pre?term?birth,?low?and?very?LBW,?small?for?gestational?age?neonates,?low?and?very?low?Apgar?score,?and?fetal?and?neonatal?mortality?(Otterbald?Olausson?et?al.,?1999;?Chen?et?al.,?2007).?Adolescents?are?more?likely?to?partake?in?social?behaviors?such?as?cigarette?use,?illicit?drug?use,?frequent?alcohol?consumption,?and?sexual?promiscuity,?all?of?which?adversely?affect?pregnancy?(McAnarney,?1987).?By?19?years?of?age,?the?adverse?affects?associated?with?adolescent?pregnancy?are?diminished?(Chen?et?al.,?2007).?For?this?reason,?a?lower?limit?of?19?years?was?selected?for?our?study?demographic.?In?recent?decades?there?has?been?a?significant?increase?in?the?proportion?of?women?in?Western?nations?who?delay?childbearing?(Johnson?and?Tough,?2012).?As?a?result,?there?has?been?increased?interest?in?the?implications?of?reproduction?in?the?latter?part?of?childbearing?years?(Seoud?et?al.,?2002,?Johnson?and?Tough,?2012).?Evidence?suggests?that?from?35?years?of?age?onwards,?maternal?age?is?associated?with?a?statistically?significant?increase?in?pregnancy?complications?(Hansen,?1986;??? ?? 26????????????Figure?2.?Flow?diagram?of?our?experimental?design????????????????????????? ????????????? ??????? ??????????????????????????????????????????????? ???????? ??????????????????? ??? ? ????? ????????????????????? ??????????????????????????? ??????????????? ????? ?????? ???????????? ????? ?????????? ?????? ???????????????????????????? ????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????? ? ??????????????????????? ? ??????????????????????????? ? ????????????????????????????? ? ??????????????????????????????????????? ???????????????????????????????????????????????????????????????????????????????????????????????????????????????? ???????????????????????????????????????????????????????????????????? 27??Seoud?et?al.,?2002;?Luke?and?Brown,?2007,?Johnson?and?Tough,?2012).?Complications?include?chronic?hypertension,?gestational?diabetes,?pregnancy?associated?hypertension,?low?and?very?LBW,?preterm?birth,?preeclampsia,?and?spontaneous?abortion?(Nybo?Andersen?et?al.,?2000;?Seoud?et?al.,?2002;?Luke?and?Brown,?2007).?Therefore,?the?upper?limit?of?our?study?demographic?was?set?at?35?years?of?age.??4.1.2.?Pregnancy?considerations?? This?study?included?women?pregnant?with?a?single?fetus?who?had?not?recently?given?birth?(<18?months).?Minimal?inter?pregnancy?interval?is?associated?with?maternal?nutrient?depletion?resulting?in?preterm?birth?and/or?fetal?growth?restriction?(King,?2003).?Multiple?pregnancy?augments?maternal?nutritional?needs,?and?is?associated?with?an?increased?risk?of?complications?such?as?preeclampsia,?spontaneous?abortion?and?IUGR?(Kahn?et?al.,?2003;?DRI,?2005;?Duckitt?and?Harrington,?2005).?Similarly,?use?of?assisted?reproductive?technologies?(ARTs)?for?conception?increases?the?risk?of?pregnancy?complications,?so?preference?was?given?to?naturally?conceived?pregnancies?(Reddy?et?al.,?2007).??4.1.3.?Gestational?stage?considerations??Pregnancy?can?be?divided?into?several?distinct?stages.?The?most?common?is?by?trimester,?defined?as?1?12?weeks?(1st),?13?28?weeks?(2nd),?and?29?40?weeks?(3rd).?Others?describe?the?quarters?of?pregnancy?(1?10th,?11?20th,?21?30th,?31?40th?weeks?gestation),?or?simply?the?halves?(1?20th?and?21?40th?weeks?gestation;?King,?2000).?Here,?we?refer?to?the?2nd?and?4th?quarters?of?pregnancy?as?early?and?late?gestation.??? 28?We?studied?women?from?11?20?weeks?and?31?38?weeks?gestation,?respectively?(i.e.?the?2nd?and?4th?quarters?of?pregnancy).?The?purpose?of?this?design?was?to?account?for?protein?requirements?during?different?stages?of?pregnancy,?allowing?for?a?temporal?gap?between?the?stages?studied?so?as?to?detect?significant?changes?in?protein?metabolism,?while?avoiding?most?of?the?first?trimester???when?many?women?experience?nausea?and?vomiting?(Gadsby?et?al.,?1993).?The?minimally?invasive?IAAO?model?requires?that?subjects?adhere?to?specific?dietary?consumption?patterns?before?and?during?the?study?day.?To?reduce?subject?discomfort,?and?to?increase?the?accuracy?of?our?results,?we?did?not?study?women?experiencing?severe?nausea,?food?avoidance?and/or?vomiting,?or?women?<11?weeks?gestation.?In?addition,?physiological?changes?associated?with?the?first?10?weeks?of?pregnancy?are?predominantly?characterized?by?up?regulation?of?hormone?production?and?secretion,?and?are?not?believed?to?substantially?affect?nutritional?needs?(Blackburn?and?Loper,?1992;?King,?2000;?Picciano,?2003).??4.1.4.?Participant?health?? Health?status,?medication?use,?and?social?behaviors?were?considered?during?subject?selection?(Appendix?B).?Women?studied?were?in?apparent?good?health,?with?preexisting?health?conditions?assessed?on?an?individual?basis.?Subjects?with?preexisting?health?conditions?directly?linked?to?metabolic?dysfunction?were?excluded?(e.g.?diabetes).?Medication?use?was?also?considered?on?a?case?by?case?basis.?The?most?commonly?used?medication?that?delayed?participation?was?Diclectin??(doxylamine?succinate/pyridoxine?hydrochloride),?which?is?prescribed?for?controlling?nausea.?Potential?participants?taking?Diclectin??were?asked?to?? 29?discontinue?use?(if?possible)?for?1?week?prior?to?the?study.?Potential?participants?were?asked?if?allergic?to?eggs?or?egg?protein?due?to?the?fact?that?the?amino?acid?composition?of?the?experimental?diets?followed?egg?protein?composition.?Participant?body?mass?index?(BMI;?kg/m2)?was?also?taken?into?consideration.?Low?maternal?BMI?is?associated?with?poor?pregnancy?outcomes,?including?preterm?labor,?IUGR?and?LBW?(Erhenberg?et?al.,?2003).?Maternal?obesity?disrupts?metabolic?adaptations?to?pregnancy,?disrupts?fetal?growth,?and?increases?the?risk?of?preeclampsia?(King,?2006;?Nelson?et?al.,?2010).?Therefore,?the?lower?and?upper?pregravid?BMI?limits?were?set?at?18.5?kg/m2?and?25?kg/m2,?respectively.???4.1.5.?Participant?recruitment?? Recruitment?of?participants?was?conducted?by?posting?advertisements?and?brochures?within?the?British?Columbia?Children?s?Hospital,?and?community?centers?such?as?prenatal?fitness?classes?and?coffee?shops?(Appendix?C).?Subjects?were?invited?to?participate?in?2?study?days?per?gestational?period,?for?a?total?of?4?studies?per?individual.?Subjects?were?compensated?for?transportation?costs?(public?transit?passes?or?parking?passes),?and?offered?an?honorarium?($100/?study?day)?for?their?participation.??4.1.6.?Inclusion?and?exclusion?criteria?We?included?women?who?were?19?to?35?years?of?age,?pregnant?with?a?single?fetus,?11?20?or?31?38?weeks?gestation,?in?apparent?good?health,?belonging?to?any?ethnicity.?Potential?participants?were?excluded?based?on?the?following?criteria:?women?who?were?not?pregnant?or?pregnant?with?more?than?one?fetus;?women?who?? 30?were?not?19?35?years?of?age;?women?with?a?metabolic,?neurological,?genetic,?or?immune?disorder?likely?to?affect?nutritional?requirements;?women?dependent?on?medication?that?affects?normal?metabolism;?women?experiencing?severe?nausea,?food?avoidance,?or?vomiting?throughout?their?pregnancy;?women?allergic?to?eggs?or?egg?protein;?women?classified?as?underweight?(<18.5?kg/m2),?overweight?(25?30?kg/m2)?or?obese?(>30?kg/m2);?and?women?smoking?cigarettes,?taking?illicit?drugs,?or?consuming?alcohol?regularly?during?their?pregnancy.?4.2.?Experimental?design?4.2.1.?Pre?study?day?protocol?Participants?were?asked?to?meet?for?a?brief?(1?hour)?pre?study?assessment?in?preparation?for?the?study?day?(Appendix?B).?Subjects?arrived?at?the?Child?and?Family?Research?Institute?s?Clinical?Research?and?Evaluation?Unit?having?fasted?overnight,?and?in?a?calm?physiological?state?(at?least?8?hours?since?last?caloric?intake;?no?morning?exercise,?and?assisted?transportation?to?minimize?stress).?During?this?appointment,?the?following?measurements?were?taken:?height?(Harpenden?Stadiometer;?Holtain?Limited,?UK)?and?weight?(electric?scale;?HealthOMeter??Professional,?Sunbeam?Products,?Inc.),?bioelectrical?impedance?analysis?(BIA;?Q4?Bioimpedance?Analyzer,?RJL?Systems,?MI),?3?site?skinfold?(Harpenden?Calipers;?Baty?International,?UK),?blood?glucose?(One?Touch??Ultra?,?LifeScan,?Inc),?and?resting?energy?expenditure?(REE;?Vmax?Encore,?Viasys,?CA).?REE?describes?the?caloric?requirement?for?vital?function?in?the?absence?of?physical?exertion,?and?was?used?to?calculate?the?total?energy?content?of?the?experimental?diets?in?order?to?ensure?that?the?specific?caloric?needs?of?each?participant?were?met?(Bross?et?al.,?1998).?Blood?? 31?glucose?was?assessed?to?screen?for?gestational?diabetes?mellitus?(GDM;?fasting?plasma?glucose?level?of??6.7?mmol/L?indicative?of?GDM;?Metzger?et?al.,?2007).?Skinfold?and?BIA?measurements?were?collected?to?determine?maternal?fat?and?fat?free?mass.?Maternal?body?composition?is?difficult?to?determine?as?a?result?of?pregnancy?related?physiological?changes?(McCarthy?et?al.,?2004).?Skinfold?assessment?is?affected?by?changes?to?maternal?hydration?and?skin?contours,?and?tends?to?overestimate?the?subcutaneous?fat?content?(McCarthy?et?al.,?2004).?Therefore,?we?selected?3?sites?minimally?affected?by?hydration:?the?biceps,?triceps?and?subscapular?region.?BIA?is?affected?by?changes?in?hydration?and?hematocrit?concentration,?but?correlates?well?with?other?body?composition?approaches?(i.e.?deuterium?water?and?hydrodensitometry),?and?is?safe?and?easy?to?conduct?(McCarthy?et?al.,?2004).?Therefore,?BIA?was?also?used?to?determine?body?composition.??A?questionnaire?was?completed?that?asked?for?information?about?recent?medical?history,?pregnancy?history,?medication?use,?supplement?use?and?exercise?patterns?(Appendix?B).?Prenatal?vitamin?use?was?requisite?for?participation?in?this?study?in?order?to?ensure?adequate?vitamin?and?mineral?availability.?If?necessary,?prenatal?vitamins?(Materna?,?Centrum)?were?available?for?the?duration?of?the?study?at?the?request?of?the?participant.??Informed?consent?was?obtained?from?each?participant?in?accordance?with?the?BC?Women?s?and?Children?s?Research?Ethics?Board?guidelines?(Appendix?D).?Lastly,?subjects?were?asked?to?provide?a?2?day?food?intake?diary?to?reveal?food?preferences.?These?were?used?to?inform?food?choices?when?designing?the?standardization?diet?(Appendix?E).?? 32?4.2.2.?Study?day?protocol?Each?study?day?was?carried?out?in?the?temperature?controlled?Clinical?Research?and?Evaluation?Unit?(Translation?Building,?CFRI;?Figure?3).?Subjects??arrived?fasted?and?in?a?calm?physiological?state.?Subjects?were?assessed?for?abnormal?fasting?blood?glucose?levels?using?a?glucometer?(Brody?et?al.,?2003).?If?blood?glucose?was?detected?at?values??6.7mmol/L?it?was?recommended?that?the?participant?contact?their?health?care?provider?for?further?testing?(Appendix?G).?Each?study?day?proceeded?in?accordance?with?the?minimally?invasive?IAAO?model?(Figure?3;?Appendix?F;?Bross?et?al.,?1998).?Meal?consumption?began?at??240?minutes,?followed?by?subsequent?meals?at??180,??120,??60,?0,?60,?120,?and?180??minutes.?Indirect?calorimetry?was?performed?at?0?minutes?to?determine?the?rate?of?CO2?production?(VCO2),?which?was?used?to?calculate?13C?enrichment.?The?first?4?meals?did?not?contain?our?indicator?amino?acid,?L?[1?13C]phenylalanine.?Urine?and?breath?samples?collected?prior?to?the?5th?meal?were?used?to?determine?background?13C?enrichment.?Continuous?oral?L?[1?13C]phenylalanine?infusion?was?introduced?in?the?5th?meal?along?with?a?priming?dose?of?L?[1?13C]phenylalanine?and?NaH13CO3?.?Each?subsequent?meal?(6th?8th)?contained?a?constant?dose?of?L?[1?13C]phenylalanine.?Urine?and?breath?samples?collected?from?150?240?minutes?post?infusion?were?used?to?determine?L?[1?13C]phenylalanine?derived?13C?enrichment.?Following?breath?and?urine?sampling?at?240?minutes?post?infusion,?the?study?was?complete?and?participants?were?free?to?leave.??? 33?????Figure?3.?Study?day?protocol???4.3.?Experimental?diet?4.3.1.?Standardization?diet?Participants?were?asked?to?standardize?their?dietary?protein?intake?to?1.5?g/kg/d?during?the?2?days?before?the?study?in?order?to?reduce?protein?intake?variability?amongst?subjects?prior?to?IAAO?analysis?(Elango?et?al.,?2009).?From?food?diaries?collected?during?the?pre?study?assessment,?each?participant?s??normal??dietary?protein?intake?was?quantified?using?a?nutrient?analysis?database?(Food?Processor?SQL?11,?ESHA?Research).?Participants?were?advised?to?increase,?decrease?or?maintain?their?dietary?protein?intake?in?order?to?achieve?a?total?intake?of?1.5?g/kg/d.?Food?choice?recommendations?were?provided?based?on?individual?food?preferences?in?order?to?help?participants?consume?the?standardized?protein?intake.??? 34?4.3.2.?Test?protein?intakes?To?identify?a?breakpoint?using?the?IAAO?method,?the?test?protein?intakes?must?be?varied?enough?to?have?sufficient?data?points?above?and?below?the?requirement?so?that?two?distinct?slopes?are?achieved?upon?statistical?analysis.?Conventionally,?IAAO?studies?have?selected?~7?protein?or?amino?acid?intake?levels?(3?above,?3?below?and?1?at?the?predicted?requirement),?and?performed?IAAO?analysis?on?each?subject?at?all?intake?levels?(Mager?et?al.,?2003;?Turner?et?al.,?2006;?Elango?et?al.,?2007;?Humayun?et?al.,?2007;?Pillai?et?al.,?2010).?However,?this?approach?was?not?possible?in?this?study?because?of?limitations?posed?by?pregnancy.?Within?a?matter?of?weeks,?protein?metabolism?in?the?pregnant?body?can?change?through?adaptive?physiological?strategies,?negating?the?efficacy?of?the?conventional?IAAO?experimental?approach?(Hadden?and?McLaughlin,?2009).?To?overcome?this?challenge,?we?provided?a?unique?protein?intake?for?each?study?day,?as?done?recently?in?school?aged?children?(Elango?et?al.,?2011)?and?neonate?(Chapman?et?al.,?2009,?2010)?IAAO?studies.??IAAO?studies?of?protein?requirements?of?healthy?children?and?healthy?adults?have?demonstrated?that?the?current?EAR?for?protein?intake?recommended?by?the?DRI?are?78%?and?41%?lower?than?IAAO?derived?protein?requirements,?respectively?(Elango?et?al.,?2011;?Humayun?et?al.,?2007).?A?recent?analysis?of?dietary?protein?intake?patterns?from?pregnant?women?in?British?Columbia,?Canada?indicates?that?the?DRI?RDA?for?protein?(1.1?g/kg/d)?may?be?underestimated,?given?that?women?are?consuming?1.5?g/kg/d?in?early?gestation,?and?1.3?g/kg/d?in?late?gestation?(Stephens,?Innis,?Elango?et?al.,?unpublished?data).?Based?on?these?findings,?we?predicted?a?protein?requirement?of?approximately?1.3?g/kg/d?for?healthy,?pregnant?women.?? 35?With?this?estimated?breakpoint?in?mind,?subjects?were?randomly?assigned?to?one?of?the?following?40?test?protein?intakes?(in?g/kg/d):?0.22,?0.28,?0.34,?0.4,?0.46,?0.52,?0.58,?0.64,?0.7,?0.76,?0.82,?0.88,?0.94,?1,?1.06,?1.12,?1.18,?1.24,?1.3,?1.36,?1.42,?1.48,?1.54,?1.6,?1.66,?1.72,?1.78,?1.84,?1.9,?1.96,?2.02,?2.08,?2.14,?2.20,?2.26,?2.32,?2.38,?2.44,?2.50,?2.56.?Participants?were?studied?at?the?same?40?test?protein?intakes?during?both?early?and?late?gestation.?However,?in?order?to?confirm?the?breakpoint?analysis?in?late?gestation,?3?additional?test?intakes?were?included:?0.84,?0.98,?and?1.08?g/kg/d.?A?total?of?37?and?44?studies?were?completed?during?early?and?late?gestation,?respectively.??4.3.3.?Study?day?diet??Subjects?were?randomly?assigned?1?of?40?test?protein?intakes?comprised?of?a?crystalline?L?amino?acid?mixture?consistent?with?egg?protein?amino?acid?composition,?with?the?exception?of?phenylalanine?and?tyrosine.?Phenylalanine?intake?was?held?constant?at?30.5?mg/kg/d?and?tyrosine?was?held?constant?at?61?mg/kg/d.?The?test?protein?intake?constituted?3?21%?of?the?total?energy?provided?by?the?experimental?diet?(total?energy?for?the?day?calculated?as?1.7?X?REE).?The?remaining?energy?came?from?37%?fat?and?42?60%?carbohydrates,?provided?by?corn?oil,?protein?free?powder?and?protein?free?cookies.?The?diet?was?consumed?in?8?isocaloric?and?isonitrogenic?meals,?each?representing?1/12th?the?daily?requirement.?In?addition?to?the?experimental?diet,?subjects?were?allowed?to?consume?only?water?prior?to,?and?throughout,?the?study?day.???? 36?4.4.?Isotope?tracer?considerations?4.4.1.?Indicator?amino?acid?criteria?Selection?of?an?indicator?amino?acid?is?based?on?specific?criteria?(Zello?et?al.,?1995).?First,?in?order?to?measure?changes?in?protein?utilization?in?relation?to?amino?acid?intake,?an?indispensable?amino?acid?must?be?use?as?the?indicator.?The?body?cannot?synthesize?indispensable?amino?acids,?thus?allowing?for?experimental?control?of?indicator?availability.?Second,?for?IAAO?to?be?measured?from?breath?samples,?the?labeled?carboxyl?carbon?of?the?indicator?amino?acid?must?be?irreversibly?oxidized?to?CO2.?Third,?the?indicator?amino?acid?should?not?contribute?significantly?to?any?reactions?within?the?body?apart?from?protein?synthesis?or?oxidation?to?CO2.?If?an?alternate?biochemical?pathway?consumes?the?labeled?carbon,?tracer?collected?in?breath?samples?will?not?reflect?changes?in?whole?body?protein?utilization.?These?criteria?leave?phenylalanine?(in?the?presence?of?excess?of?tyrosine),?lysine,?and?the?branched?chain?amino?acids?as?possible?indicator?amino?acid?candidates?(Zello?et?al.,?1995;?Figure?4).?L?[1?13C]phenylalanine?was?successfully?applied?for?the?determination?of?protein?requirements?of?adults?and?children,?and?therefore?was?used?in?this?study?(Humayun?et?al.,?2007;?Elango?et?al.,?2011).??4.4.2.?Isotope?tracer?protocol??During?each?study?day,?a?priming?dose?(0.66?mg/kg)?and?continuous?dose?(1.2?mg/kg/d)?of?L?[1?13C]phenylalanine?(99?atom?percent?excess,?Cambridge?Isotope?Laboratories?Inc.,?Andover,?MA)?was?given?orally?with?the?5th?meal,?followed?thereafter?by?an?hourly?intake?of?1.2?mg/kg/d?(Humayun?et?al.,?2007).?Isotopic?steady?state?was?achieved?at?this?dose?during?each?of?the?81?study?days.?Isotopic??? 37????????????Figure?4.?Biochemical?conversion?of?phenylalanine?to?tyrosine??steady?state?was?calculated?as?<10%?coefficient?of?variation?in?13C?enrichment?during?the?4hr?tracer?protocol.??Tracer?phenylalanine?intake?was?subtracted?from?the?dietary?phenylalanine?content?supplied?by?the?experimental?diet?in?order?to?keep?the?total?phenylalanine?intake?constant.?Because?phenylalanine?is?normally?metabolized?by?conversion?to?tyrosine,?an?excess?of?tyrosine?(61?mg/kg/d)?was?provided?in?the?experimental?diet?(Figure?4).?This?ensured?that?phenylalanine?oxidation?was?sensitive?to?changes?in?protein?intake?(Pencharz?and?Ball,?2003).???A?priming?dose?of?NaH13CO3?(0.176?mg/kg;?99?atom?percent?excess,?Cambridge?Isotope?Laboratories?Inc.,?Andover,?MA)?was?given?orally?with?the?5th?meal?in?order?to?increase?the?recovery?of?13C?in?breath?(Hoerr?et?al.,?1989).?NaH13CO3?increases?the?recovery?of?13C?in?breath?based?on?the?following?relationship:?? 38?H+?+?HCO3?????H2O?+?CO2??CO2?is?transported?in?blood?as?HCO3??so?by?providing?NaH13CO3?we?primed?the?body?pool?to?more?rapidly?achieve?steady?state?in?13C?enrichment.?4.4.3.?Sample?collection?and?analysis?To?measure?the?oxidation?of?L?[1?13C]phenylalanine?to?13CO2??(F13CO2),?VCO2?was?measured?once?and?breath?samples?were?collected?at?9?times?points?during?each?study?day.?Open?circuit?indirect?calorimetry?was?performed?until??5?consecutive?steady?state?data?points?were?obtained?to?measure?VCO2?(VMAX?Encore?system?by?Viasys).?3?baseline?and?6?isotopic?steady?state?breath?samples?were?collected?at?time??45,??30,??15,?150,?180,?195,?210,?225?and?240?minutes?using?disposable?Labco?exetainer?tubes?(Elango?et?al.,?2007).?Breath?samples?were?stored?at?room?temperature?in?airtight?bags.??In?accordance?with?the?minimally?invasive?IAAO?model,?urine?samples?were?collected?in?place?of?plasma?to?assess?whole?body?amino?acid?pool?L?[1?13C]phenylalanine?enrichment?at?isotopic?steady?state?(Bross?et?al.,?1998).?2?baseline?and?4?isotopic?steady?state?urine?samples?were?collected?at?time??45,??15,?150,?180,?210,?and?240?minutes?using?sterile?collection?hats.?Urine?samples?were?preserved?using?200??L?of?3.4?mol/L?HCl?per?10?mL?sample,?then?stored?at??80?C.?Urine?samples?collected?at?time??45?minutes?and?240?minutes?were?analyzed?for?the?presence?of?protein?using?the?Chemstrip?7??dipstick?test?by?Roche?.?If?protein?was?detected?in?the?urine?at?values??300?mg/24?hour?(+1?or?higher?as?indicated?by?? 39?dipstick?analysis)?it?was?recommended?that?the?participant?contact?their?health?care?provider?for?further?testing?(Appendix?G).??Breath?13CO2?enrichment?was?determined?using?continuous?flow?isotope?ratio?mass?spectrometry?(Isoprime?Ltd,?UK)?and?expressed?as?atom?percent?excess?(APE)?when?compared?against?a?reference?standard?of?compressed?CO2?(Turner?et?al.,?2006;?Elango?et?al.,?2007;?Humayun?et?al.,?2007).?Urine?sample?[13C]phenylalanine?enrichments?were?analyzed?by?the?SickKids?(Toronto)?mass?spectrometry?facility.?Briefly,?urine?[13C?]phenylalanine?enrichment?was?determined?using?a?triple?quadruple?mass?spectrometer?(AB?Sciex,?ON)?coupled?to?a?high?precision?liquid?chromatography?system?(Bross?et?al.,?1998;?Turner?et?al.,?2006;?Elango?et?al.,?2007;?Humayun?et?al.,?2007).?Following?mass?spectrometry,?select?ion?chromatograms?were?obtained?by?monitoring?mass?to?charge?(m/z)?ratios?of?the?product?ions?165?and?166?for?[1?13C]phenylalanine,?corresponding?to?unenriched?(M)?and?enriched?(M+1)?peaks,?respectively.?The?area?under?these?peaks?at?baseline?and?plateau?isotopic?steady?state?was?used?to?calculate?[13C]phenylalanine?enrichment?in?urine?(expressed?as?molecules?percent?excess;?Turner?et?al.,?2006;?Elango?et?al.,?2007;?Humayun?et?al.,?2007).???4.4.4.?Calculation?of?isotope?kinetics??? To?calculate?L?[1?13C]phenylalanine?kinetics,?the?stochastic?models?described?by?Waterlow?et?al.?(1978)?and?Matthew?et?al.?(1980)?were?used.?These?models?have?been?used?to?assess?L?[1?13C]phenylalanine?kinetics?in?previous?IAAO?studies?(Bross?? 40?et?al.,?1998;?Kriengsinyos?et?al.,?2002;?Mager?et?al.,?2003;?Elango?et?al.,?2007;?Pillai?et?al.,?2009).?Whole?body?flux?will?be?calculated?as:??? ? ? ? Q=i[Ei/Eu???1]?Where?Q?represents?phenylalanine?flux?(?mol/kg*h),?i?is?the?rate?of?L?[1?13C]?phenylalanine?infusion?(?mol/kg*h),?and?Ei?and?Eu?are?the?istopic?enrichments?as?mole?fractions?(molecules?percent?excess)?of?the?infusate?and?urinary?phenylalanine,?respectively,?at?plateau?isotopic?steady?state.?The?rate?of?phenylalanine?oxidation?was?calculated?using?the?rate?of?13CO2?released?by?L?[1?13C]phenylalanine?oxidation?(F13CO2;?in??mol/kg*h).?F13CO2?is?calculated?as:??? ? F13CO2?=?(FCO2)(ECO2)(44.6)(60)/W(0.82)(100)?Where?FCO2?represents?carbon?dioxide?production?(mL/min),?ECO2?is?the?13CO2?enrichment?in?breath?at?plateau?isotopic?steady?state?(APE),?W?is?the?subjects?weight?(kg),?44.6?(?mol/L)?and?60?(min/h)?are?constants?used?to?convert?FCO2?to??mol/h,?0.82?is?the?correction?factor?for?carbon?dioxide?conserved?by?the?body?due?to?bicarbonate?fixation,?and?100?is?used?to?convert?APE?to?a?fraction?(Hoerr?et?al.,?1989).??4.4.5.?Statistical?analysis??Subject?characteristics?are?reported?as?the?mean?and?standard?deviation.?A?two?phase?linear?regression?cross?over?model?was?used?to?analyze?the?effect?of?protein?intake?on?F13CO2?(Proc?Mixed,?Statistical?Analysis?Systems???SAS/STAT?version?9.0,?SAS?Institute,?Cary,?NC).?A?breakpoint?was?determined?at?the?minimum?residual?standard?error?in?a?stepwise?partitioning?of?data?points?between?two?regression?? 41?lines?(Zello?et?al.,?1995;?Humayun?et?al.,?2007;?Elango?et?al.,?2009).?The?model?assumes?that?one?regression?line?is?sloped?and?the?other?is?horizontal?with?minimal?or?no?slope.?Protein?intake?at?the?breakpoint?is?the?estimated?average?protein?requirement.?The?pre?study,?study?day,?and?protein?intake?data?were?compared?using?Student?s?t?test,?and?phenylalanine?flux?data?were?compared?using?one?way?analysis?of?variance?(Version?19,?SPSS?Inc,?Chicago,?IL).?Significance?was?set?at?P?0.05.??? ?? 42?Chapter?5.?Results??5.1.?Subject?characteristics?? The?majority?of?subjects?enrolled?in?this?study?were?in?their?late?twenties?or?early?thirties?(Early?Gestation,?EG:?30.6?3.9?y;?Late?Gestation,?LG:?30.3?2.8y),?had?a?pre?pregnancy?BMI?of?18.5?25?kg/m2?(EG:?22.1?2.9;?LG:?21.8?2.9),?and?were?pregnant?with?their?first?child?(EG:?77%;?LG:?74%;?Table?3).??Due?to?recruitment?difficulties?the?final?sample?included?women??37?years?of?age,?and?women?were?not?studied?twice?during?each?gestational?period?as?expected.?Participants?were?given?the?option?of?participating?in?a?single?study?day?or?up?to?4?study?days?per?gestational?period.?This?flexibility?allowed?us?to?enroll?more?women?because?it?reduced?the?required?time?commitment?to?the?study.?Therefore,?in?the?early?gestation?group?4?women?were?studied?4?times,?2?women?were?studied?3?times,?4?women?were?studied?2?times,?and?7?women?were?studied?once?(n=37,?N=17).?In?the?late?gestation?group?3?women?were?studied?4?times,?3?women?were?studied?3?times,?10?women?were?studied?2?times,?and?3?women?were?studied?once?(n=44,?N=19).??None?of?the?women?studied?were?taking?Diclectin??or?any?Anatomical?Therapeutic?Chemical?(ATC)?code?A?medications?at?the?time?of?study.?None?of?the?women?studied?reported?any?previous?or?existing?pregnancy?complications,?ongoing?nausea?or?vomiting,?or?an?allergy?to?eggs?or?egg?protein.?One?participant?reported?the?use?of?assisted?reproductive?therapy?for?conception?(in?vitro?fertilization).?None?of?the?women?studied?reported?consuming?alcohol,?cigarettes?or?illicit?substances?at?any?time?during?their?pregnancy.?2?women?reported?using?an?antidepressant??? 43??Table?3.?Subject?Characteristics?(Nearly?=?17,?Nlate?=?19)1????Characteristic? ?????Early?Group??????????????????????????????????????????????????????????????????????Late Group ?? ? ? ?Age?(years)?Pre?pregnancy?weight?(kg)? 30.6?(3.9)?60.2?(10.4)? 30.3?(2.8)?59.0?(10.2)? ?Height?(cm)? 165.1?(7.2)? 165.8?(7.3)? ?Pre?pregnancy?BMI?(kg/m2)? 22.1?(2.9)? 21.8?(2.9)? ?Previous?pregnancies? ??????????????0???77?%???????????????????????????????????????????1???18%??2???5%? 0?? 74%1???21%??2???5%? ?? ??????????????????????????????????????????????? ?1All?values?are?mean?(?SD)?unless?otherwise?indicated???during?their?pregnancy?(Venlafaxine,?commonly?referred?to?as?Effexor?),?one?woman?reported?using?a?steroid?inhaler,?and?one?woman?reported?using?an?over?the?counter?stool?softener.?No?medications?were?taken?on?the?study?day.?Although?we?did?not?inquire?about?the?participants??ethnic?background,?participants?were?predominately?Caucasian.?5.2.?Pre?study?assessment??? ? 5.2.1.?Early?gestation?pre?study?results?In?the?early?gestation?group,?pre?study?BMI?was?predominantly?in?the?range?of?18.5?25?kg/m2?(23.1?2.8),?with?an?average?weight?gain?of?2.9?2.9?kg?above?pre?pregnancy?weight?(Table?4).?Body?composition?analysis?determined?average?percentage?of?FFM?(kg)?as?74.3%,?65.9%,?and?70.2%?via?skinfold,?BIA,?and?factorial??? ?? 44??Table?4.?Pre?study?day?results?in?early?(N=17)?and?late?(N=19)?gestation????Variable?? Early?Group?Mean?(SD)?? Late?Group?Mean?(SD)? ??t?value?(df)1? p?? ? ? ?? ??2.158?(34)???? ????2.554?(34)?????????1.722?(34)??? ?2.438?(33)?????? ?0.815?(34)????0.081?(34)???? ?????1.744?(34)???1.003?(34)?????????? ?1.091?(34)???0.827?(34)???? ?????0.215?(33)????????? ??????Weight?(kg)? ??????????????????????????63.2?(9.8)? ?70.5?(10.2)? ?0.038??????????????0.015???0.094??? ?? ?0.020?????????? 0.421??????????????0.936??????? ?????? 0.090???0.323????? ???0.283?????? 0.414????0.831???? ?????BMI?(kg/m2)???Blood?glucose?(mmol/L)??Skinfold?FFM?(kg)??BIA?FFM??(kg)??Pipe?et?al.?(1979)?FFM?(kg)??Resting?energy?expenditure???Respiratory?quotient??? 23.1?(2.8)?? ?4.85?(0.50)??? 46.9?(7.2)??? 41.7?(5.3)???44.8?(6.9)??? ?1370?(180)???0.90?(0.08)???25.6?(3.0)???4.58?(0.44)??? 53.0?(7.3)??? 43.1?(4.8)???44.6?(6.5)??? ?1480?(197)???0.88?(0.03)??VO2??(L/min)??VCO2?(L/min)??Protein?intake?before?pre?study?(g/kg/d)???????????????????????0.193?(0.027)??0.173?(0.023)??1.38?(0.38)??0.205?(0.038)??0.181?(0.035)??1.35?(0.51)?? ?????????????????????????????? ? ? ? ??1Reported?values?assume?equal?variances??? 45?calculation?based?on?Pipe?et?al.?(1979),?respectively.?Average?fasted?blood?glucose?levels?were?4.85?0.50?mmol/L,?and?fasted?indirect?calorimetry?revealed?an?average?rate?of?oxygen?consumption?of?0.193?0.027?L/min,?and?rate?of?carbon?dioxide?exhalation?of?0.173?0.022?L/min,?to?yield?an?average?respiratory?quotient?of?0.90?0.08.??Resting?energy?expenditure?was?1370?180?kcal/d.?Food?record?analysis?indicated?an?average?protein?intake?of?1.38?0.38?g/kg/d?amongst?participants?prior?to?the?pre?study.??5.2.2.?Late?gestation?pre?study?results?In?the?late?gestation?group,?most?participants?had?a?pre?study?BMI?in?the?range?of?18.5?25?kg/m2?(25.6?3.0),?with?an?average?weight?gain?of?10.8?2.6?kg?above?pre?pregnancy?weight?(Table?4).?Body?composition?analysis?determined?average?percentage?of?FFM?(kg)?as?75.1%,?61.1%,?and?63.3%?via?skinfold,?BIA,?and?factorial?calculation?based?on?Pipe?et?al.?(1979),?respectively.?Average?fasted?blood?glucose?levels?were?4.58?0.44?mmol/L,?and?fasted?indirect?calorimetry?revealed?an?average?rate?of?oxygen?consumption?of?0.205?0.038?L/min,?and?rate?of?carbon?dioxide?exhalation?of?0.181?0.035?L/min,?to?yield?an?average?respiratory?quotient?of?0.88?0.03.??Resting?energy?expenditure?was?1480?197?kcal/d.?Food?record?analysis?indicated?an?average?protein?intake?of?1.35?0.51?g/kg/d?amongst?participants?prior?to?the?pre?study.? ?????? 46?5.3.?Phenylalanine?flux?and?protein?requirement??Participants?had?study?day?blood?glucose?levels?of?4.77?0.39?and?4.56?0.42?mmol/L?in?early?and?late?gestation,?respectively?(Table?5).??Participant?protein?intake?standardization?compliance?in?the?2?days?preceding?each?study?day?was?high,??with?early?gestation?group?subjects?consuming?1.44?0.30?g/kg/d?and?late?gestation?group?subjects?consuming?1.47?0.53?g/kg/d,?on?average.???Phenylalanine?flux?(EG:?45.72?7.04mol/kg/h;?LG:?47.57?6.82?mol/kg/h)?did?not?vary?significantly?(EG:?p=0.212;?LG:?p=0.259)?across?protein?intakes,?as?required?by?the?IAAO?technique?(Figure?5?and?Figure?6).?Without?a?change?in??phenylalanine?flux,?changes?in?oxidation?with?protein?intake?reflect?a?shift?in?amino?acid?utilization,?from?amino?acid?oxidation?to?amino?acid?incorporation?for?protein?synthesis.??L?[1?13C]phenylalanine?oxidation?to?13CO2?varied?significantly?with?protein?intake?in?both?the?early?(p<0.001)?and?late?(p<0.001)?gestation?group,?declining?with?increasing?protein?intake?to?a?threshold?intake,?then?plateauing?(Figure?7?and?8).?Two?phase?linear?regression?cross?over?analysis?determined?a?breakpoint?in?the?L?[1?13C]phenylalanine?oxidation?to?13CO2?versus?protein?intake?data?at?1.22?and?1.52?g/kg/d?protein?intake?in?early?and?late?gestation,?respectively.?These?estimates?represent?minimum?protein?requirement?required?to?meet?the?needs?of?50%?of?the?population?(EAR).??Changes?in?L?[1?13C]phenylalanine?oxidation?to?13CO2??with?protein?intakes?are?modeled?in?SAS?(SAS/STAT?version?9)?according?to?a?protein?intake?cutoff.?The???? 47?Table?5.?Study?day?results?in?early?(n=37)?and?late?(n=44)?gestation????Variable?? Early?group?Mean?(SD)?? Late?group?Mean?(SD)? t?value?(df)1? p?? ? ? ?? ?3.260?(79)?????????????2.307?(79)??? ?0.317?(69)?????2.596?(79)????1.727?(79)?????? ??????Weight?(kg)? ??????????????????????????64.4?(10.5)? ?71.4?(9.1)? ?0.002????????? 0.024??? ??????????0.752????0.011????0.088?????? ??? ?????Blood?glucose?(mmol/L)??Protein?intake?before?study?day?(g/kg/d)??Energy?intake?on?study?day?(kcal/d)??Energy?intake?on?study?day?(kcal/kg/d)?? ?4.77?(0.39)??? 1.44?(0.30)????2304.9?(291.9)???36.1?(3.1)???? ?? ?4.56?(0.42)??? 1.47?(0.53)????2482.5?(318.6)????34.9?(3.2)??????? ? ?1Reported?values?assume?equal?variances??????????????? 48??Figure?5.?Variation?in?phenylalanine?flux?across?protein?intakes?in?early?gestation?group?non?significant?(p=0.212)?as?determined?by?one?way?analysis?of?variance?in?SPSS?(Version?19,?SPSS?Inc,?Chicago,?IL)????Figure?6.?Variation?in?phenylalanine?flux?across?protein?intakes?in?late?gestation?group?non?significant?(p=0.259)?as?determined?by?one?way?analysis?of?variance?in?SPSS?(Version?19,?SPSS?Inc,?Chicago,?IL)??0?10?20?30?40?50?60?70?80?90?100?0? 0.5? 1? 1.5? 2? 2.5? 3?Phenylalanine?Flux??(?mol/kg/h)?Protein?Intake?(g/kg/d)?0?10?20?30?40?50?60?70?80?90?100?0? 0.5? 1? 1.5? 2? 2.5? 3?Phenylalanine?Flux??(?mol/kg/h)?Protein?Intake?(g/kg/d)?? 49??Figures?7.?Estimated?average?protein?requirement?in?early?gestation?derived?by?2?phase?linear?regression?cross?over?analysis?in?SAS?(Proc?Mixed,?Statistical?Analysis?Systems???SAS/STAT?version?9.0?SAS?Institute,?Cary,?NC)???Figure?8.?Estimated?average?protein?requirement?in?late?gestation?derived?by?2?phase?linear?regression?cross?over?analysis?in?SAS?(Proc?Mixed,?Statistical?Analysis?Systems???SAS/STAT?version?9.0?SAS?Institute,?Cary,?NC)??0.0 0.5 1.0 1.5 2.0 2.5 3.00.000.250.500.751.001.251.501.752.00EAR = 1.22 g/kg/dr2=0.60Early GestationProtein Intake (g/kg/d)F13CO2 (umol/kg/h)0.0 0.5 1.0 1.5 2.0 2.5 3.00.000.250.500.751.001.251.501.752.00EAR = 1.52 g/kg/dr2=0.63Late GestationProtein Intake (g/kg/d)F13CO2 (umol/kg/h)? 50?cutoff?separates?the?data?into?two?groups:?oxidation?values?associated?with?protein?intakes?below?the?cutoff,?and?oxidation?values?associated?with?intakes?above?it.?With?the?oxidation?data?separated?into?2?groups?a?regression?is?applied,?producing?2?lines,?which?are?analyzed?using?a?two?phase?linear?regression?crossover?model.?4?different?parameters?are?assumed?for?each?cutoff:??un?weighted?where?one?line?has?slope,?weighted?where?one?line?has?slope,?un?weighted?where?both?lines?have?slope,?weighted?where?both?lines?have?slope.?A?breakpoint?is?determined?from?the?model?with?the?highest?coefficient?of?determination?(R2),?and?the?lowest?coefficient?of?variation?(CV)?and?root?mean?squared?error?(RMSE).?Breakpoints?determined?for?our?early?and?late?gestation?groups?came?from?models?that?assumed?un?weighted?data?where?one?regression?line?was?sloped?and?the?other?was?not?(EG:?R2?=?0.60;?LG:?R2?=?0.63).?? ?? 51?Chapter?6.?Discussion??6.1.?Subject?characteristics??? Women?recruited?for?this?study?were?healthy,?30?years?of?age?on?average,?had?a?healthy?pre?pregnancy?BMI,?and?were?predominately?primigravida?(Table?3).?All?of?the?women?studied?were?taking?prenatal?vitamins?prior?to?their?first?study?day?so?protein?metabolism?is?unlikely?to?have?been?affected?by?cofactor?availability.?2?women?in?the?early?gestation?group?were?included?despite?using?Effexor??during?their?pregnancy.?Effexor?is?a?serotonin?norepinephrine?reuptake?inhibitor?that?alters?endocrine,?gastrointestinal?and?metabolic?function?(Series,?Micromedex?Healthcare).?Those?taking?Effexor??may?experience?weight?loss?(3?47%?of?users),?constipation?(8?15%),?loss?of?appetite?(8?22%),?or?sweating?(6.7?25%),?all?of?which?may?alter?protein?metabolism?(Series,?Micromedex?Healthcare).?Due?to?recruitment?challenges?in?our?early?gestation?group?we?decided?to?study?these?women.?These?women?did?not?take?their?medications?on?the?study?day.?Inspection?of?F13CO2?values?from?these?studies?revealed?coefficients?of?variation?less?than?5%,?and?valid?F13CO2?values.?Therefore,?these?studies?were?included?in?our?final?breakpoint?analysis.?None?of?the?other?medications?reportedly?used?by?our?participants?significantly?alter?protein?metabolism.??6.2.?Indicators?of?metabolic?adaptations?to?pregnancy?? During?the?preliminary?assessment?we?evaluated?several?metabolic?indicators?in?each?of?the?potential?participants.?Potential?participants?were?instructed?to?arrive?for?this?appointment?having?fasted?overnight,?and?in?a?? 52?physiologically?calm?state?(e.g.?no?morning?exercise;?assisted?transportation?to?minimize?stress).??As?expected,?women?in?the?late?gestation?group?weighed?more?and?had?a?higher?BMI?than?women?in?the?early?gestation?group?(Table?4).?Fat?deposition?occurs?predominantly?during?the?2nd?and?3rd?quarters?of?pregnancy?(Section?2.1.).?Fat?reserves?are?then?used?as?a?primary?source?of?maternal?energy?during?the?4th?quarter?of?pregnancy,?sparing?glucose?and?amino?acids?for?the?fetal?exponential?growth?phase?(Pipe?et?al.,?1979;?King,?2000).?FFM?is?expected?to?increase?during?the?last?10?weeks?of?pregnancy?as?the?fetus?more?than?doubles?in?size.?However,?only?skinfold?caliper?analysis?of?body?composition?indicated?a?significant?increase?in?FFM?in?the?late?versus?early?gestation?groups?(Table?4).?BIA?and?a?factorial?calculation?showed?a?trend?toward?higher?FFM?in?late?gestation,?but?this?was?not?significant.?Thus,?our?inability?to?detect?a?significant?increase?in?FFM?from?early?to?late?gestation?using?BIA?and?factorial?estimate?is?likely?due?to?the?relatively?small?sample?size.??Blood?glucose?measurements?were?collected?from?participants?while?fasted?during?the?pre?study?and?on?each?study?day?(Table?4?and?5).?None?of?the?participants?registered?a?blood?glucose?value??6.7mmol/L,?and?therefore?are?unlikely?to?have?been?experiencing?GDM?at?the?time?of?study.?Cumulatively,?these?measurements?revealed?a?significant?decrease?in?fasting?blood?glucose?levels?from?early?to?late?gestation.?Reduced?circulating?glucose?in?late?gestation?likely?reflects?increased?placental?uptake?in?order?to?accommodate?the?fetus?s?additional?energy?demands?during?this?gestational?period?(King,?2000).??? 53?The?resting?energy?expenditure,?rate?of?O2?inhaled?and?rate?of?CO2?exhaled?increased,?and?the?respiratory?quotient?decreased,?on?average?from?early?to?late?gestation?(Table?4).?However,?these?changes?were?not?significant.?Each?of?these?measurements?was?collected?once?in?the?fasted?state?during?the?pre?study?assessment,?and?thus?detection?of?significant?changes?was?likely?obscured?by?small?sample?size.?It?is?interesting?to?note?that?the?average?respiratory?quotient?in?the?early?and?late?gestation?groups?were?higher?than?that?expected?from?a?non?pregnant?adult.?During?the?fasted?state,?we?expect?the?body?to?derive?energy?primarily?from?fat?reserves,?with?an?expected?respiratory?quotient?closer?to?0.7.?The?relatively?high?respiratory?quotients?observed?in?this?study?reflect?maternal?adaptations?to?pregnancy?that?alter?energy?metabolism?to?favor?glucose?oxidation,?resulting?in?respiratory?quotients?closer?to?0.9?(Butte,?2000).??6.3.?Estimation?of?protein?requirements??? 6.3.1.?Protein?requirement?in?early?gestation?The?estimated?average?protein?requirement?during?early?gestation?was?determined?to?be?1.22?g/kg/d?(Figure?7).?These?results?suggest?that?dietary?protein?needs?are?increased?compared?to?non?pregnant?adults?from?the?first?trimester?onwards.?As?compared?to?the?DRI?recommended?EAR?(0.88?g/kg/d)?for?protein?intake?during?pregnancy,?our?estimate?is?39%?higher?(DRI,?2005).?Compared?to?the?IAAO?derived?EAR?for?protein?requirement?in?healthy,?non?pregnant?adults,?our?estimate?is?32%?higher?(Humayun?et?al.,?2007).?The?increased?need?for?dietary?protein?detected?in?this?data?set?indicates?that?maternal?adaptations?to?protein?? 54?metabolism?are?established?early?in?pregnancy,?and?agree?with?previous?findings?that?suggest?changes?to?protein?and?nitrogen?metabolism?occur?early?in?pregnancy,?in?anticipation?of?fetal?needs?(Section?2.4.;?Kalhan,?2000;?King,?2000).?Data?used?to?derive?this?estimate?was?collected?from?women?who?adhered?exceptionally?well?to?our?standardization?diet?recommendation?of?1.5?g/kg/d?in?the?2?days?preceding?the?study?(Table?5).?Liquid?chromatography?tandem?mass?spectrometry?urine?analysis?revealed?non?significant?tracer?phenylalanine?flux?(Figure?5).?This?is?important?because?if?phenylalanine?flux?varies?proportionally?with?increasing?intakes?of?protein?then?the?measured?13CO2?values?are?potentially?erroneous.?Therefore,?we?are?confident?that?the?appearance?of?tracer?phenylalanine?derived?13CO2?in?breath?reflects?the?partitioning?of?phenylalanine?between?protein?synthesis?and?oxidation?in?response?to?protein?intake.??The?physiologic?implications?of?these?results?are?compelling.?Despite?minimal?tissue?accretion?in?the?fetus?before?20?weeks?gestation,?it?appears?that?dietary?protein?requirements?(on?a?body?weight?basis)?are?significantly?increased.?The?additional?need?for?dietary?protein?may?be?explained?by?a?number?of?interrelated?adaptations?to?pregnancy,?as?described?below.??6.3.2.?Sources?of?protein?demand?before?20?weeks?gestation?The?placenta?is?an?essential?extra?embryonic?tissue?that?facilitates?nutrient?and?waste?exchange?between?the?mother?and?fetus.??In?order?to?meet?the?demands?of?the?fetus,?the?placenta?must?be?fully?formed?before?the?fetus?commences?its?exponential?growth?phase.?Therefore,?rapid?growth?of?the?placenta?occurs?in?early?gestation,?with?a?fully?functional?placenta?observed?by?18?20?weeks?gestation?? 55?(Porter?RS?and?Kaplan?JL,?2011).?On?average,?human?placenta?has?a?mass?of?approximately?470g,?suggesting?that?considerable?protein?deposition?occurs?in?utero?before?20?weeks?gestation.?As?the?placenta?develops,?its?trophoblast?cells?invade?the?maternal?decidua,?eventually?gaining?access?to?the?maternal?blood?supply?via?the?spiral?arteries?(Espinoza?et?al.,?2006).?By?10?12?weeks?gestation?maternal?blood?is?in?contact?with?the?terminal?villi?of?the?placenta,?allowing?for?gas?and?nutrient?exchange?between?the?mother?and?fetus,?and?increased?metabolic?activity?in?the?feto?placental?unit?(Gude?et?al.,?2004).?As?previously?stated?(Section?2.3.2.),?the?placenta?is?not?a?passive?conduit?but?rather?a?highly?metabolically?active?organ?that?is?also?nourished?by?the?maternal?blood?supply.?In?addition,?the?placenta?has?a?particularly?high?affinity?for?amino?acids,?which?concentrate?here.?Therefore,?dietary?protein?requirements?may?increase?at?this?time,?as?suggested?by?the?current?results.??Many?maternal?tissues,?including?uterine,?breast,?cardiovascular?and?kidney,?undergo?expansion?in?order?to?accommodate?the?physiological?demands?imposed?by?pregnancy.?These?changes?occur?progressively?throughout?gestation,?in?response?to?a?progressive?increase?in?progesterone?and?estrogen?levels?(Figure?9).?However,?human?chorionic?gonadotrophin?(hCG)?does?not?increase?progressively,?and?may?be?implicated?in?contributing?to?muscle?expansion?during?early?gestation.?HCG?is?produced?by?the?syncytiotrophoblast?layer?of?the?placenta?from?approximately?2?weeks?gestation?until?term?(Figure?10).?Unlike?progesterone?and?estrogen,?hCG?production?peaks?during?the?first?trimester,?between?8?13?weeks?gestation,?then?plateaus?beyond?20?weeks?gestation.?While?the?common?function?of?hCG?is?to?maintain?the?corpus?luteum,?it?may?play?a?role?in?regulating?protein?turnover?in??? 56???Figure?9.?Plasma?progesterone?and?estradiol?levels?throughout?human?pregnancy?(adapted?by?Nieman?L.K.?for?Endotext.org?from?Tulchinsky,?D.,?et?al.?"Plasma?estrone,?estradiol,?estriol,?progesterone,?and?17?hydroxyprogesterone?in?human?pregnancy.?I.?Normal?pregnancy."?American?Journal?of?Obstetrics?and?Gynecology?112.8?(1972):?1095)? ???Figure?10.?Mean?serum?hCG?levels?throughout?normal?pregnancy?(from?Braunstein,?G.?D.,?et?al.?"Serum?human?chorionic?gonadotropin?levels?throughout?normal?pregnancy."?American?Journal?of?Obstetrics?and?Gynecology?126.6?(1976):?678)?? 57?muscle?tissue.?HCG?has?been?shown?to?increase?leptin?release?(Dos?Santos?et?al.,?2007).?A?recent?study?conducted?in?mice?found?that?leptin?helped?regulate?muscle?protein?turnover?by?increasing?protein?synthesis?and?decreasing?degradation?(Mao?et?al.,?2012).?HCG?receptors?are?found?in?several?tissues?including?the?uterus?(Rao,?2010).?The?uterus?contains?a?substantial?muscular?layer,?the?myometrium,?which?undergoes?expansion?during?pregnancy.?If?leptin?released?in?response?to?hCG?production?stimulates?protein?synthesis?in?the?myometrium,?this?activity?would?be?most?pronounced?during?peak?hCG?release,?and?taper?off?until?20?weeks?gestation.?A?flurry?of?myometrium?expansion?during?the?11?20?weeks?period?studied?in?our?early?gestation?cohort?would?also?help?account?for?the?increased?protein?requirement?observed?in?this?data?set.??Pregnancy?is?characterized?by?hypoaminoacidemia?(Kalhan,?2000).?The?decline?in?plasma?amino?acid?concentrations?is?partially?due?to?elevated?progesterone?levels?(Figure?9).??Progesterone?increases?amino?acid?catabolism?at?the?liver?for?the?production?of?new?molecules.?This?is?evidenced?by?a?reduction?in?circulating?amino?acid?concentrations?without?the?concomitant?increase?in?urinary?amino?acid?nitrogen?excretion,?which?is?observed?when?protein?catabolism?is?stimulated?by?adrenocortical?or?thyroid?hormones?(Landau?and?Lugibihl,?1961).?Together?with?estrogen?and?insulin,?progesterone?also?decreases?lipolysis?and?increases?lipogenesis?in?early?gestation,?facilitating?maternal?fat?reserve?formation?(Butte,?2000).?As?discussed?in?Section?2.1.,?the?first?two?thirds?of?pregnancy?are?anabolic,?and?maternal?energy?demands?are?met?primarily?from?carbohydrate?and?protein?energy?sources?in?order?to?conserve?lipids?for?adipose?tissue?expansion.?It?is?? 58?possible?the?increased?amino?acid?catabolism?associated?with?progesterone?secretion?in?early?gestation?facilitates?fat?reserve?formation?by?favoring?protein?derived?energy.?This?too?would?help?explain?the?increased?need?for?dietary?protein?observed?in?our?early?gestation?cohort.??6.3.3.?Protein?requirement?in?late?gestation?The?estimated?average?protein?requirement?during?late?gestation?was?determined?to?be?1.52?g/kg/d?(Figure?8).?These?results?suggest?that?dietary?protein?needs?are?increased?above?those?of?non?pregnant?adults?during?the?last?quarter?of?pregnancy.?As?compared?to?the?DRI?recommended?EAR?(0.88?g/kg/d)?for?protein?intake?during?pregnancy,?our?estimate?is?73%?higher?(DRI,?2005).?Compared?to?the?IAAO?derived?EAR?for?protein?requirements?of?healthy,?non?pregnant?adults,?our?estimate?is?63%?higher?(Humayun?et?al.,?2007).??Data?used?to?derive?this?estimate?was?collected?from?women?who?adhered?exceptionally?well?to?our?standardization?diet?recommendation?of?1.5?g/kg/d?in?the?2?days?preceding?the?study?(Table?5).?Liquid?chromatography?tandem?mass?spectrometry?urine?analysis?revealed?non?significant?tracer?phenylalanine?flux?(Figure?6).?Therefore,?we?are?confident?that?the?appearance?of?tracer?phenylalanine?derived?13CO2?in?breath?reflects?the?partitioning?of?phenylalanine?between?protein?synthesis?and?oxidation?in?response?to?protein?intake.?Increased?tissue?accretion?during?the?final?quarter?of?pregnancy?is?essential?for?a?healthy?infant?birth?weight.?Given?the?considerable?difference?between?our?estimate?and?that?provided?by?the?DRI,?our?results?suggest?that?women?who?strictly?adhere?to?current?recommendations?may?not?be?consuming?adequate?protein.??? 59?6.3.4.?Evidence?that?factorial?calculation?underestimates?protein?requirement?The?discrepancy?between?our?IAAO?derived?EAR?for?late?gestation?and?the?DRI?EAR?for?pregnant?women?(73%)?is?similar?to?that?observed?in?a?study?by?Elango?et?al.?(2011)?that?examined?protein?requirements?of?school?aged?children.?Elango?et?al.?(2011)?determined?that?the?IAAO?derived?EAR?for?protein?requirements?of?children?was?78%?higher?than?current?DRI?recommendations.?The?current?method?used?by?the?DRI?for?estimating?protein?requirements?of?pregnant?women?and?children?is?a?factorial?calculation?that?incorporates?nitrogen?balance?data???derived?primarily?from?non?pregnant?adults???with?mathematical?constants?that?assume?protein?needs?associated?with?growth.?The?protein?needs?of?children?and?late?gestation?pregnant?women?are?significantly?influenced?by?rapid?tissue?accretion?(e.g.?juvenile?growth?phase?and?the?fetal?rapid?growth?phase).?Thus,?direct?measurement?of?protein?requirements?is?essential?to?identify?the??optimal??requirements?in?these?unique?populations.???6.4.?Comparison?of?protein?requirements?to?current?reported?dietary?intakes??? The?2003?2004?National?Health?and?Nutrition?Survey?(NHANES),?and?the?Canadian?Community?Health?Survey?(CCHS)?Cycle?2.2?reported?protein?intakes?in?the?United?States?of?America?and?Canada,?respectively,?but?excluded?pregnancy?specific?data?from?the?analysis.?We?recently?completed?a?prospective?analysis?of?270?pregnant?women?from?British?Columbia?who?were?recruited?for?a?separate?study?(Innis?SM?and?Friesen?RW,?2008;?Stephens,?Innis,?Elango?et?al.,?unpublished).?Dietary?food?frequency?questionnaires?were?administered?during?early?and?late?gestation?in?the?same?cohort?of?pregnant?women.?The?results?suggested?that?maternal?protein?? 60?consumption?adjusted?for?body?weight?(g/kg/day)?was?significantly?higher?at?16?weeks?gestation?than?36?weeks?gestation,?with?median?intakes?of?1.5?(Interquartile?Range,?IQR:?0.61)?and?1.3?(IQR:?0.56)?g/kg/d?respectively.??The?intakes?on?a?g/d?basis?equate?to?99.3?(IQR:34)?and?100.5?(IQR:37)?g/d,?respectively.??Since?infant?birth?weight?data?was?available,?we?compared?protein?intake?to?birth?weight?and?found?a?significant?negative?correlation?between?protein?intake?and?birth?weight.?The?results?from?our?Canadian?cohort?of?pregnant?women?can?be?compared?to?Godfrey?et?al.?(1996;?n=538?British?women)?and?Moore?et?al.?(2004;?n=429?Australian?women).?In?contrast?to?our?study,?both?of?these?studies?found?a?positive?correlation?between?protein?intake?and?infant?birth?weight.?The?differing?results?may?be?explained?by?the?substantial?difference?in?protein?consumption?between?the?sample?populations.?In?our?study,?the?median?total?protein?intakes?at?16?and?36?weeks?gestation?were?99.3?and?100.5?g/d,?respectively,?which?are?considerably?higher?than?the?intakes?of?84?87?g/d?reported?by?Godfrey?et?al?(1996)?and?Moore?et?al?(2004).?A?quadratic?relationship?between?maternal?protein?intake?and?infant?birth?weight?has?been?proposed,?which?suggests?that?both?insufficient?and?excessive?protein?intake?is?associated?with?LBW?(Imdad?and?Bhutta,?2011;?Sloan?et?al.,?2001).?This?suggests?that??optimal??or??balanced??protein?and?amino?acid?intakes?during?pregnancy?are?beneficial,?but?excessive?intake?could?have?negative?effects.?Thus,?maternal?protein?intake?patterns?of?women?from?British?Columbia?determined?during?early?and?late?gestation?compare?favorably?with?our?estimates?of?protein?requirements?of?healthy?pregnant?women.??? 61?6.5.?Comparison?of?protein?requirement?as?a?percentage?of?energy?to?DRI?energy?recommendations?? Protein?metabolism?does?not?operate?in?isolation?of?other?nutrients?but?rather?is?directly?affected?by?carbohydrate,?fat?and?total?energy?intake,?particularly?during?pregnancy?(Sec?2.1.).?Thus,?it?is?useful?to?consider?protein?requirements?as?a?percentage?of?total?energy.?Energy?was?provided?at?1.7?X?REE?for?each?participant.?This?meant?that?energy?provisions?were?specific?to?the?individual,?and?not?biased?by??test?protein?intake.?On?average,?we?provided?2305?kcal/d?and?2483?kcal/d?of?energy?in?early?and?late?gestation,?respectively?(Table?5).?Using?the?DRI?formula?to?calculate?estimated?energy?requirement?(EER)?for?pregnant?women?weighing?as?much?as?our?average?participant?in?early?and?late?gestation?(EG:?64.4?kg;?LG:?71.4?kg),?we?find?that?2488?kcal/d?and?2695?kcal/d?is?recommended?by?the?DRI,?respectively.?Thus,?the?energy?provided?in?this?study?is?comparable?to?DRI?energy?recommendations.?It?should?be?noted?that?DRI?recommendations?for?energy?intake?during?pregnancy?are?unlike?protein?recommendations?in?that?they?are?based?on?a?substantial?body?of?evidence?derived?from?studies?of?pregnant?women.??When?DRI?recommendations?for?protein?intake?during?pregnancy?are?converted?to?a?percentage?of?energy,?we?find?that?the?DRI?recommends?9%?energy?from?protein?in?both?early?and?late?gestation.?Our?estimated?protein?requirements?during?early?and?late?gestation?represent?14%?and?17.5%?energy?from?protein,?respectively.?The?current?DRI?recommends?an?acceptable?macronutrient?distribution?range?(AMDR)?of?10?30%?of?energy?from?protein.?Thus,?despite?our?estimates?of?protein?requirements?being?significantly?higher?than?current?DRI?? 62?protein?intake?recommendation?for?pregnancy?on?a?g/kg/d?basis,?our?estimates?are?more?consistent?with?the?AMDR?than?current?DRI?protein?intake?recommendations.??6.6.?Strengths?and?implications?of?this?study?? Use?of?the?minimally?invasive?IAAO?method?allowed?us?to?study?protein?requirements?of?a?healthy,?adult,?pregnant?population?for?the?first?time.?The?rapid?nature?of?IAAO?protocol?(8?hour?studies)?made?it?possible?to?study?a?wide?range?of?test?protein?intakes?and?arrive?at?a?strong?intake?versus?response?curve.?The??quick?and?easy??approach?to?our?study?days?encouraged?participant?retention,?accommodated?participants?who?only?wanted?to?enroll?for?a?single?study?day,?and?allowed?us?to?conduct?more?than?80?studies?in?less?than?2?years.??Perhaps?the?most?unique?aspect?of?this?experimental?design?was?the?inclusion?of?2?gestational?phases?for?analysis?(i.e.?early?and?late).?The?paucity?of?protein?requirement?data?available?for?pregnancy?was?cited?by?the?2005?DRI.?The?early?gestation?data?is?particularly?important?given?the?relative?absence?of?similar?data?available?in?the?literature,?and?the?critical?importance?of?this?stage?to?fetal?development?and?placental?sufficiency.?With?our?data?we?are?the?first?to?provide?pregnancy?specific?dietary?protein?requirements?for?healthy?women?during?early?and?late?gestation.??Our?results?suggest?that?women?require?additional?protein?above?non?pregnant?adult?requirements?before?20?weeks?gestation,?and?that?protein?requirements?are?significantly?different?during?early?versus?late?gestation.?Thus,?our?? 63?results?support?a?stage?specific?approach?to?protein?intake?recommendations?for?pregnant?women.??These?data?suggest?that?current?DRI?and?WHO/FAO/UNU?recommendations?for?protein?intake?during?pregnancy?are?significantly?underestimated.?Mothers?in?many?regions?around?the?world?rely?on?government?and?non?government?organizations?to?supplement?their?diet?during?pregnancy?to?ensure?adequate?nutrition.?However,?exactly?what?constitutes?an??adequate??amount?of?a?particular?nutrient?is?predominantly?defined?by?DRI?and?WHO/FAO/UNU?guidelines.?These?guidelines?are?used?to?allocate?nutritional?resources?to?individuals,?and?therefore?directly?impact?the?amount?and?type?of?food?vulnerable?populations?have?access?to.?Thus,?these?data?indicate?that?current?recommendations?are?inappropriate?and?may?increase?the?risk?of?protein?deficiency.??In?North?America?the?relative?risk?of?protein?deficiency?is?low?(Fulgoni,?2008;?Stephens,?Innis,?Elango?et?al.,?unpublished).?However,?the?popularization?of?high?protein?diets?has?increased?the?risk?of?excessive?protein?intake.?As?previously?stated?(Section?2.2.;?Section?6.4.),?both?high?and?low?protein?diets?during?pregnancy?have?been?associated?with?LBW?infants.?Therefore,?by?providing?pregnancy?specific?protein?requirement?estimates?we?can?help?North?American?mothers?optimize?dietary?choices?and?improve?pregnancy?outcomes.????? ?? 64?Chapter?7.?Future?directions??? Using?the?IAAO?technique?to?determine?individual?amino?acid?requirements?throughout?pregnancy?is?a?natural?step?forward.?Similar?to?protein?requirements,?very?little?pregnancy?specific?data?exists?for?individual?amino?acid?requirements?during?this?dynamic?life?stage.?Currently,?our?first?study?of?amino?acid?requirements?is?ongoing?and?focused?on?lysine?requirements.?Lysine?is?an?indispensable?amino?acid,?and?the?first?limiting?amino?acid?in?plant?based?diets.?Therefore,?women?consuming?diets?devoid?of?animal?protein?are?susceptible?to?deficiency.?Women?who?fit?this?profile?include?those?following?vegetarian?and?vegan?diets,?as?well?as?women?with?low?food?security?that?rely?heavily?on?rice?or?grains?as?a?primary?food?source.?Lysine?deficiency?during?pregnancy?is?associated?with?decreased?immune?function,?increased?diarrhea,?and?increased?stress?(Smriga?et?al.,?2004;?Zhao?et?al.,?2004;?Ghosh?et?al.,?2010).?Thus,?we?hope?to?provide?pregnancy?specific?lysine?requirements?in?the?near?future.??Protein?intakes?in?developing?countries?are?very?low?during?pregnancy?(Swaminathan?et?al.,?2012).?Trends?in?protein?intakes?amongst?pregnant?women?in?India?indicate?that?on?average?~50?g/d?is?consumed,?as?compared?to?the?84?100?g/d?reported?by?pregnant?women?in?developed?countries?(Godfrey?et?al.,?1996;?Moore?et?al.,?2004;?Stephens,?Innis,?Elango?et?al.,?unpublished).???Furthermore,?60%?of?the?protein?consumed?in?developing?countries?arises?from?sources?such?as?rice?and?grains,?which?are?deficient?in?indispensable?amino?acids?and?thus?are?lower?quality?proteins.?Findings?of?the?current?study?will?be?significant?in?these?settings,?particularly?in?relation?to?the?incidence/prevalence?of?LBW,?which?has?been?? 65?implicated?in?adult?onset?chronic?disease.?Future?studies?will?need?to?be?conducted?in?pregnant?women?in?settings?where?access?to?protein?sources?is?limited?in?order?to?define?the??optimal??protein?and?amino?acid?requirements?of?these?unique?populations.??? ?? 66?Chapter?8.?Conclusion?In?conclusion,?we?have?determined?that?healthy,?pregnant?women?during?early?and?late?gestation?require?1.22?and?1.52?g/kg/d?of?protein,?respectively.?Each?of?these?estimates?is?significantly?higher?than?current?DRI?EAR?and?RDA?recommendation?for?protein?intake?during?pregnancy?of?0.88?g/kg/d?and?1.1?g/kg/d,?respectively.?The?large?discrepancy?between?our?estimate?of?protein?requirement?and?current?DRI?recommendations?suggests?that:?1)?the?factorial?approach?to?determining?protein?requirements?underestimates?protein?needs,?and?2)?emphasizes?the?importance?of?utilizing?evidence?derived?directly?from?the?population?an?estimate?is?meant?to?represent.??Our?successful?application?of?the?IAAO?method?in?pregnancy?demonstrates?the?value?of?this?novel?technique?for?determining?protein?requirements?of?vulnerable?populations.?The?IAAO?method?represents?a?new?avenue?for?collecting?data?for?these?nutritionally?unique?populations,?and?should?be?utilized?to?identify?protein?and?amino?acid?needs.??The?results?from?our?early?gestation?group?suggest?that?maternal?adaptations?to?pregnancy?increase?dietary?protein?needs?before?20?weeks?gestation,?a?gestational?stage?not?addressed?by?the?current?DRI.?In?addition,?the?substantial?difference?between?our?early?and?late?gestation?estimates?suggest?that?maternal?protein?needs?increase?as?pregnancy?advances.?Taken?together,?these?results?indicate?that?protein?intake?recommendations?should?be?provided?in?a?gestational?stage?specific?manner,?and?current?recommendations?may?be?significantly?underestimated.??? 67?Bibliography?? 1. 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Turner?JM,?Humayun?MA,?Elango?R,?Rafii?M,?Langos?V,?Ball?RO,?and?Pencharz?PB.??Total?sulfur?amino?acid?requirement?of?healthy?school?age?children?as?determined?by?indicator?amino?acid?oxidation?technique.??American?Journal?of?Clinical?Nutrition,?83?(2006):?619?623.?116. Thompson?GN?and?Halliday?D.??Protein?turnover?in?pregnancy.??European?Journal?of?Clinical?Nutrition,?46.6?(1992):?411?7.?117. van?den?Akker?CHP,?Schierbeek?H,?Dorst?KY,?Schoonderwaldt?EM,?Vermes?A,?Duvekot?JJ,?Steegers?EAP,?and?van?Goudoever?JB.?"Human?fetal?amino?acid?metabolism?at?term?gestation."?The?American?journal?of?clinical?nutrition,?89.1?(2009):?153?160.?118. van?den?Akker?CHP,?Schierbeek?H,?Minderman?G,?Vermes?A,?Schoonderwaldt?EM,?Duvekot?JJ,?Steegers?EAP,?and?van?Goudoever?JB.?"Amino?acid?metabolism?in?the?human?fetus?at?term:?leucine,?valine,?and?methionine?kinetics."?Pediatric?research,?70.6?(2011):?566?571.?119. Waterlow,?JC.??The?mysteries?of?nitrogen?balance.??Nutrition?Research?Review,?12?(1999):?25?54.?120. Waterlow?JC,?Garlick?PJ?and?Millward?DJ.??Protein?turnover?in?mammalian?tissues?and?in?the?whole?body.??North?Holland?Publishing?Co.,?Amsterdam?(1978).?121. Whittaker?PG,?Lee?CH,?Cooper?BG,?Taylor?R.??Evaluation?of?phenylalanine?and?tyrosine?metabolism?in?late?human?pregnancy.??Metabolism,?48.7(1999):?849?? 75?52.?122. Whittaker?PG,?Lee?CH,?Taylor?R.??Whole?body?protein?kinetics?in?women:?effect?of?pregnancy?and?IDDM?during?anabolic?stimulation.??American?Journal?Physiology,?Endocrinology,?and?Metabolism,?279.5(2000):?E978?88?123. Widdowson,?EM?and?Dickerson,?JWT.??Chemical?composition?of?the?body.??In:?Mineral?Metabolism:?An?Advanced?Treatise,?vol.?2,?part?A,?eds.,?Comar?CL?and?Bronner?F.?New?York:?Academic?Press?Inc.?(1964).?124. World?Health?Organization.??Protein?and?amino?acid?requirements?in?human?nutrition.??WHO?Technical?Report?Series,?935?(2007).?125. World?Health?Organization,?Geneva.??Report?of?the?advisory?group?meeting?on?maternal?nutrition?and?low?birth?weight.??(2002).?126. Wu?G,?Meininger?CJ,?Knabe?DA,?Baze?FW,?and?Marc?RJ.??Arginine?nutrition?in?development,?health?and?disease.??Current?Opinion?in?Clinical?Nutrition?and?Metabolic?Care,?3.1?(2000):?59?66.?127. Young,?VR.??Nutritional?balance?studies:?indicators?of?human?requirements?or?of?adaptive?mechanisms???Journal?of?Nutrition,?116?(1986):?700?703.?128. Zhao?W,?Zhai?F,?Zhang?D,?An?Y,?Liu?Y,?He?Y,?Ge?K,?and?Scrimshaw?NS.?"Lysine?fortified?wheat?flour?improves?the?nutritional?and?immunological?status?of?wheat?eating?families?in?northern?China."?Food?&?Nutrition?Bulletin?25.2?(2004):?123?129.?129. Zello?GA,?Wykes?LJ,?Ball?RO,?and?Pencharz?PB.??Recent?advances?in?methods?of?assessing?dietary?amino?acid?requirements?for?adult?humans.??Journal?of?Nutrition,?125.12?(1995):?2907?2915.?? ??? ?? 76?Appendices????????? ?? 77???????????Appendix?A???DRI?derivation?of?protein?intake?recommendations?for?healthy?pregnant?women?????????????????????????????? 78?????????a?Carmichael?et?al?(1997);?average?body?weight?gain?by?end?of?trimester;?divided?by?2?to?get?approximate?increase?mid?trimester?b?End?of?trimester?increase?in?body?weight?X?0.66?g/kg/d,?the?Estimated?Average?Requirement?(EAR)?for?maintenance?of?protein?in?adults.?c?From?Table?10?15?where?protein?deposition?=?total?potassium?accumulated?(mmol/day)?/?2.15?(mmol?potassium/g?nitrogen)?X?6.25;?and?assumption?that?nitrogen?accretion?during?second?trimester?is?~50%?that?of?third?trimester?d?Protein?deposition?/?0.43,?slope?of?regression?line?of?protein?intake?versus?nitrogen?balance?(recalculated?from?King?et?al.,?1973).?e?Average?required?additional?amount?needed?during?pregnancy?f?RDA?is?based?on?EAR?+?assumed?variation?in?requirements;?amount?needed?above?nonpregnant?needs???????????????? 79????????? ??? ??Appendix?B???Pre?study?questionnaire??????????? ?? 80??Pre?Study?Day?Assessment?Protein?Requirement:?Healthy?Pregnant?Women??Principal?Investigator:?Dr.?Rajavel?Elango? ? 604?875?2000?ext.?4911?(office)?Student?Investigator:?Trina?Stephens? ? 604?875?2000?ext.?4607(office)?? ? ? ? ? ? ? 778?998?2365?(cell)?? ?PRELIMINARY?ASSESSMENT??Subject?ID:________________________________? ? Date:___________________________??Birthday:?_______/______/________???????????Age:___________????????Last?Menstrual?Period:________??Ultrasound?Dating:?___________? Gestational?Age:?___________?? ? ? ? ? ? ???????Height?(cm):______________?????????Weight?(kg):___________?? BMI:_________________???????Fasting?Blood?Glucose?(mmol/L):_____________? Pre?Pregnancy?Weight?(kg):?_______??[Optional]?Primary?Health?Care?Provider?Details?(name,?phone,?address):_________________________?________________________________________________________________________________________________??Skinfold?measurement??MAC:_______________________________? ? ? TSF:____________________________?(mid?arm?circumference)? ? ? ? (tricep?skin?fold)??BSF:_______________________________? ? ? SSSF:____________________________?(bicep?skin?fold)? ? ? ? ? (sub?scapula?skin?fold)???Bioelectrical?Impedance?Analysis??BIA:R_____________________________? ? ? XC______________________________?(resistance)? ? ? ? ? ? (reactance)???Body?Composition?Profile??%?Body?fat?(skinfolds):_________________?? Lean?body?mass?(skinfolds):________________??%?Body?fat?(BIA):______________________? Lean?body?mass?(BIA):______________________?? 81?Indirect?Calorimetry??Measured?REE?(kcal/day):_________________? ????Estimated?REE?(kcal/day):_______________??Daily?energy?requirement?(kcal/day):_________________________??Medical?History??Are?you?currently?having?vomiting?episodes???Yes______?How?many/day?__???????No______??Are?you?currently?taking?Diclectin????Yes_____?How?many/day?__?????????No______??Are?you?currently?taking?any?other?medications???Yes_______??No_________??List?of?medications:________________________________________________________________________??Any?preexisting?health?condition(s)??Yes_________?No_____________??Details?of?health?condition(s)?______________________________________________________________??Do?you?smoke?cigarettes???Yes__________?No__________??Are?you?consuming?alcohol?on?a?regular?basis?during?your?pregnancy??Yes___?No___???Have?you?ever?been?diagnosed?with?any?of?the?following?health?conditions???Gestational?diabetes??? Pregnancy?associated?or?chronic?hypertension????Preclampsia/Eclampsia??? ? Pregnancy?related?anemia??????????Pregnancy?related?jaundice?????Pregnancy?Information??Is?this?your?first?pregnancy??Yes_______?No_______??If?no,?when?did?you?last?give?birth?(months)?______________________??Were?any?assisted?reproductive?therapies?(ARTs)?used?for?conception??Yes____?No_____??Details?of?ARTs?________________________________________________________________??? ?? 82?Nutritional?Supplement?Intake??Are?you?currently?taking?prenatal?vitamins??Yes_______?No_______??If?yes,?how?long?have?you?been?taking?them?__________________________??Which?brand?of?prenatal?vitamins?_____________________________??Are?you?taking?any?other?vitamins/?nutritional?supplements??Yes_______?No________??If?yes,?please?list?all?vitamin/nutritional?supplements:??1.__________________________________?2.__________________________________?3.__________________________________?4.__________________________________???Activity?Level??Daily?exercise?(minutes)________________________??Sedentary_____________? ? Moderate_________________? High__________________??Availability?for?4?studies??Yes___________? No______________??Comments:??? ?? 83???????????Appendix?C???Recruitment?material??????????? ?????????? 84??????????????? 85??????? 86???????????Appendix?D???Subject?consent?form??????? ???????? ?????? 87??????????????????????????INFORMED?CONSENT?AND?SUBJECT?INFORMATION??Determination?of?protein?requirements?in?healthy?pregnant?women?using?the?indicator?amino?acid?oxidation?technique??Principal?Investigator:? ?????????????Dr.?Rajavel?Elango,?PhD??? ? ? ? ? Department?of?Pediatrics?? ? ? ? ? Faculty?of?Medicine?? ? ? ? ? The?University?of?British?Columbia?? ? ? ? ? Telephone:?604?875?2000?x4911?Primary?Contact:? ? ? Trina?Stephens,?BScH?(SSP),?BA?(Min),?M.Sc?candidate??? ? ? ? ? Department?of?Pediatrics?? ? ? ? ? Faculty?of?Medicine?? ? ? ? ? The?University?of?British?Columbia?? ? ? ? ? Telephone:?604?875?2000?x4607???Emergency?Phone?Number:? ? Rajavel?Elango?778?986?8655?Trina?Stephens?778?998?2365?available?24?hours?per?day?and?seven?days?per?week??Sponsors:? ? ? ? Canadian?Institutes?of?Health?Research?(CIHR)??Site:? ? ? ? ? Oak?Street?Campus,?UBC?? ? ? ? ? Child?&?Family?Research?Institute???1.?INTRODUCTION??Protein?is?an?essential?part?of?our?diet.?It?is?used?to?build?muscle?and?body?tissue,?to?send?signals?throughout?the?body,?and?to?support?the?immune?system.?During?pregnancy,?dietary?protein?becomes?even?more?crucial,?as?it?is?needed?for?healthy?development?of?the?baby.?Inadequate?protein?intake?during?pregnancy?has?been?linked?to?a?future?risk?of?high?blood?pressure,?heart?disease?and?other?metabolic?problems?in?the?baby.?For?this?reason,?it?is?very?important?to?understand?how?much?protein?we?need?to?eat?during?pregnancy.?Even?though?it?is?well?known?that?pregnant?women?need?more?protein?in?their?diet,?it?is?not?exactly?known?how?much?additional?protein?is?required.?The?older?techniques?to?measure?how?much?protein?we?need?require?participants?to?eat?a?low?protein?diet?for?several?days?at?a?time.?Because?it?is?unethical?to?do?this?in?a?pregnant?woman,?there?is?very?little?information?about?protein?requirements?in?pregnant?women.?To?gain?a?better?understanding?of?protein?requirements?throughout?pregnancy,?we?plan?to?study?pregnant?women?from?all?ethnic?backgrounds,?aged?19?35?years,?in?their?2nd?and?3rd?trimesters?of?pregnancy?using?a?more?quick?and?modern?technique?called?the?? Depa r tmen t ? o f ? P e d i a t r i c s ?950?West?28th?Avenue,?Room?170A?Vancouver,?BC,?V5Z?4H4?Tel:?(604)?875?2000?x4607???Fax:?(604)?875?3597??? ? 88?indicator?amino?acid?oxidation?(IAAO)?technique.?This?technique?uses?a?test?liquid?meal?with?a?specific?amount?of?protein?mixed?with?a?stable?isotope?tracer.?A?stable?isotope?is?a?labeled?amino?acid?(building?block?of?protein).?These?labeled?amino?acids?are?colourless,?odourless?and?tasteless,?but?can?be?measured?in?breath?and?urine,?because?it?looks?different?than?the?rest?of?the?amino?acids?in?the?body.?This?allows?us?to?measure?if?you?are?eating?enough?protein?for?protein?synthesis?to?take?place,?and?we?can?study?protein?requirements?in?just?one?day.?Stable?isotopes?are?completely?safe,?because?they?are?present?in?the?air?we?breathe,?water?we?drink?and?food?we?eat.??This?technique?has?been?used?to?measure?protein?requirements?in?healthy?babies,?children?and?adult?human?beings.?More?details?about?the?stable?isotopes?are?available?in?point?4?below.??2.???YOUR?PARTICIPATION?IS?VOLUNTARY??You?are?invited?to?participate?in?our?study.?Your?participation?is?entirely?voluntary,?so?it?is?up?to?you?to?decide?whether?or?not?to?take?part?in?the?study.?Before?you?decide,?it?is?important?for?you?to?understand?what?the?research?involves.?This?consent?form?will?describe?the?study,?why?the?research?is?being?done,?what?will?happen?to?you?during?the?study?and?the?possible?benefits,?risks,?and?discomforts.?If?you?wish?to?participate,?you?will?be?asked?to?sign?this?consent?form?within?7?days.?If?you?decide?to?take?part?in?this?study,?you?are?still?free?to?withdraw?at?any?time?and?without?giving?any?reasons?for?your?decision(s).?If?you?do?not?wish?to?participate,?you?do?not?have?to?provide?any?reason?for?your?decision(s)?nor?will?you?lose?the?benefit?of?any?medical?care?to?which?you?are?entitled?or?are?presently?receiving.?Please?take?time?to?read?the?following?information?carefully?and?to?discuss?it?with?your?family?and?friends?before?deciding.???3.??WHO?IS?CONDUCTING?THE?STUDY???The?study?is?being?conducted?by?the?Nutrition?and?Metabolism?Research?Program?of?the?Child?and?Family?Research?Institute,?University?of?British?Columbia.?Funding?has?been?received?from?the?Canadian?Institutes?of?Health?Research?(CIHR)?to?complete?this?study?and?you?are?entitled?to?request?details?about?the?research?funding?from?the?Principal?Investigator.??4.??WHAT?IS?THE?PURPOSE?OF?THE?STUDY???This?study?is?about?how?much?protein?pregnant?women?need?in?early?(2nd?trimester)?and?late?pregnancy?(3rd?trimester).?We?will?measure?your?protein?requirements?using?the?IAAO?technique,?which?uses?a?13C?stable?isotope?(labeled)?amino?acid?to?trace?whole?body?amino?acid?oxidation?(13C?is?a?type?of?carbon;?amino?acids?are?made?of?mostly?12C,?so?the?13C?can?be?detected?in?breath?and?urine?samples?with?special?equipment).?The?13C?stable?isotopes?are?completely?safe,?because?they?are?present?in?the?air?we?breathe,?water?we?drink?and?food?we?eat.??Oxidation?of?the?labeled?amino?acid?will?be?measured?from?breath?and?urine?samples.?This?will?allow?us?to?determine?the?correct?amount?of?dietary?protein?a?pregnant?women?needs?to?? 89?eat?to?maintain?health?in?early?and?late?pregnancy.???5.??WHO?CAN?PARTICIPATE?IN?THE?STUDY????? ? Women?who?are?19?to?35?years?of?age?and?are?pregnant?with?a?single?child??6.?WHO?SHOULD?NOT?PARTICIPATE?IN?THE?STUDY????? ? Women?who?are?not?pregnant?or?? Women?who?are?pregnant?with?more?than?one?child?(this?changes?protein?demands)?? Women?who?are?not?19?35y?or??? Women?not?in?good?health?or?have?a?metabolic,?neurological,?genetic,?or?immune?disorder,?including?gestational?diabetes?or?anemia??? Women?who?smoke?or?consume?alcohol?during?their?pregnancy?? Women?who?are?classified?as?underweight?(<18.5?kg/m2),?overweight?(25?30?kg/m2)?or?obese?(>30?kg/m2)?? Women?who?are?allergic?to?eggs?and?egg?protein?? Women?who?have?severe?nausea/vomiting?throughout?their?pregnancy??7. WHAT?DOES?THE?STUDY?INVOLVE???This?study?will?be?conducted?at?the?Oak?Street?Campus?of?UBC?at?the?Child?and?Family?Research?Institute?(CFRI).?If?you?agree?to?participate?in?this?study,?then?you?will?be?asked?to?complete?the?procedures?described?below.?You?may?participate?in?four?separate?study?days,?two?in?the?2nd?trimester?and?two?in?the?3rd?trimester.???Preliminary?Study?Day?Procedures?[Once?during?2nd?trimester?and?once?during?3rd?trimester]:???? The?preliminary?assessment?is?done?to?collect?basic?information?about?you,?make?sure?you?are?informed?about?the?study?details,?and?to?collect?information?about?you?to?design?the?study?diet?specifically?to?meet?your?body?needs.?? ? The?preliminary?assessment?will?be?conducted?at?the?Clinical?Research?Evaluation?Unit?(CREU)?at?the?Child?&?Family?Research?Institute?located?in?BC?Children?s?Hospital.?You?will?be?asked?to?come?at?8AM?after?having?fasted?overnight?(10?12h).?The?whole?procedure?will?take?1?hour?to?complete.???? During?the?preliminary?assessment,?a?Research?Assistant?will?measure?your?weight,?height,?blood?glucose,?body?fat?and?muscle?mass,?and?resting?metabolic?rate?which?tells?us?how?much?energy?your?body?needs.?Blood?? 90?glucose?will?be?measured?using?a?glucose?meter?that?reads?the?amount?of?sugar?in?your?blood?by?gently?pricking?the?finger.?Body?fat?will?be?measured?using?skin?fold?thickness?measured?from?the?arm?and?shoulder?using?a?caliper?(a?handheld?instrument?that?gently?pinches?your?skin?between?two?moving?arms).?Body?muscle?will?be?measured?using?bioelectrical?impedance?which?measures?the?passage?of?a?small,?safe?amount?of?current?(that?cannot?be?felt)?between?four?electrodes?on?the?arms?and?legs?while?you?lay?still?for?a?few?minutes.?The?body?fat?and?muscle?measurements?are?completely?safe?and?do?not?cause?any?discomfort?or?harm.?Metabolic?rate?is?measured?using?an?indirect?calorimetry?machine,?which?consists?of?a?clear?hood?that?is?placed?over?your?head?while?you?lay?on?a?bed,?breathing?normally.?You?can?see?everything?through?the?hood?and?breathe?normally?without?any?discomfort.?This?measurement?takes?about?20?minutes?to?complete.?? ? You?will?also?be?asked?health?related?questions?to?assess?your?medical?history.?If?you?are?not?taking?prenatal?vitamins,?we?will?provide?you?with?some?at?this?time.?? ? During?the?preliminary?assessment?you?will?meet?with?a?Dietitian?who?will?evaluate?your?normal?dietary?protein?intake.?The?dietitian?will?give?you?recommendations?on?how?you?can?meet?the?standard?protein?intake???required?in?the?two?days?prior?to?each?study?day???from?the?foods?you?normally?eat?in?your?diet.??Study?Day?Procedures:???? ? The?study?day?will?be?conducted?in?the?Clinical?Research?Evaluation?Unit?(CREU)?at?the?Child?&?Family?Research?Institute?located?in?BC?Children?s?Hospital.??You?will?be?asked?to?come?at?8AM?after?having?fasted?overnight?(10?12h).??? ? Only?water?may?be?consumed?prior?to?arriving?on?the?study?day,?and?during?the?study?day.?The?study?day?test?diet?as?described?below?will?provide?your?daily?energy?and?nutritional?needs.?At?the?end?of?the?study?day,?you?are?free?to?resume?your?normal?food?intake.??? On?the?study?day?a?Research?Assistant?will?again?measure?your?weight,?height?and?blood?glucose.?The?Research?Assistant?will?also?measure?the?rate?at?which?you?are?breathing?out?carbon?dioxide?(VCO2)?using?the?same?indirect?calorimetry?machine?that?was?used?to?determine?metabolic?rate?in?the?preliminary?assessment.???? You?will?eat?the?test?liquid?diet?as?eight?small?hourly?meals?on?the?study?day.?Each?meal?is?made?up?of?1)?a?mixture?of?amino?acids,?2)?an?amino?acid?free?flavored?liquid?and?amino?acid?free?cookies?that?provide?energy?and?? 91?other?nutrients,?and?3)?the?labeled?amino?acid?is?added?to?the?last?four?meals.?The?test?meals?will?meet?all?your?daily?energy,?vitamin?and?mineral?needs,?as?they?were?determined?during?the?preliminary?assessment.??? ? To?measure?how?your?body?responds?to?the?test?diet?we?will?collect?your?breath?sample?9?times?and?urine?sample?6?times?during?the?study?day.?To?collect?breath?you?will?have?to?breathe?into?a?container???just?like?blowing?through?a?straw?into?a?bag.?To?collect?urine?you?will?have?to?pass?urine?into?a?urine?sample?hat?in?the?privacy?of?the?washroom.?The?first?and?last?urine?samples?of?the?day?will?be?tested?for?protein?in?the?urine?using?a?dipstick?test.?When?we?are?not?collecting?samples,?you?can?watch?television,?listen?to?music,?read?or?bring?computer?related?work?to?complete.?? ? In?total,?you?can?expect?to?dedicate?approximately?9?hours?per?study?day?you?participate?in.?You?are?invited?to?participate?in?up?to?4?studies?over?the?course?of?your?pregnancy.?If?you?choose?to?participate?in?all?4?studies,?you?will?be?asked?to?dedicate?approximately?36?hours?to?this?project,?plus?an?hour?each?during?the?2?preliminary?study?visits.???8.?WHAT?ARE?THE?POSSIBLE?HARMS?AND?SIDE?EFFECTS?OF?PARTICIPATING?IN?THIS?STUDY???There?are?no?known?risks?involved?with?participating?in?this?research.?Some?women?may?find?the?finger?prick?used?for?blood?glucose?measurement?uncomfortable.?We?recognize?that?the?length?of?the?study?day,?and?travel?to?BC?Children?s?Hospital?might?pose?an?inconvenience?for?you.????9.??WHAT?ARE?THE?BENEFITS?OF?PARTICIPATING?IN?THIS?STUDY???There?are?no?direct?benefits?to?you?from?taking?part?in?this?study.?However,?we?hope?that?the?information?learned?from?this?study?can?be?used?in?the?future?to?improve?dietary?protein?recommendations?during?pregnancy.???10.??WHAT?HAPPENS?IF?I?DECIDE?TO?WITHDRAW?MY?CONSENT?TO?PARTICIPATE?IN?THIS?STUDY???Your?participation?in?this?research?is?entirely?voluntary.?You?may?withdraw?from?this?study?at?any?time?and?without?providing?any?reasons?for?your?decision.?If?you?decide?to?enter?the?study?and?then?withdraw,?there?will?be?no?penalty?or?loss?of?benefits,?if?any,?to?which?you?are?otherwise?entitled.?If?you?choose?to?enter?the?study?and?then?decide?to?withdraw?at?a?later?time,?all?data?collected?about?you?during?the?enrolment?part?of?the?study?will?be?retained?for?analysis,?after?which?the?study?information?may?be?shredded.????? 92?11.?WHAT?WILL?THE?STUDY?COST?ME???Participation?in?the?study?will?not?cost?you?anything.?In?appreciation?of?the?time?that?it?takes?to?complete?this?study?you?will?receive?$100?upon?each?study?day?completion?to?a?maximum?of?$400?for?4?study?days.?Vehicle?parking?coupons?for?the?pre?study?duration?at?BC?Children?s?Hospital?will?be?provided.??12.?WILL?MY?TAKING?PART?IN?THIS?STUDY?BE?KEPT?CONFIDENTIAL???Your?confidentiality?will?be?respected.?No?information?that?discloses?your?identity?will?be?released?or?published?without?your?specific?consent?to?the?disclosure.?However,?research?records?and?medical?records?identifying?you?may?be?inspected?in?the?presence?of?the?Investigator?or?his?or?her?designate?by?representatives?of?Health?Canada?and?the?UBC?Research?Ethics?Boards?for?the?purpose?of?monitoring?the?research.?No?records?that?identify?you?by?name?or?initials?will?be?allowed?to?leave?the?Investigators'?offices.?In?this?study?your?samples?and?questionnaires?will?be?identified?by?a?study?code?and?any?identifying?information?will?be?kept?in?a?locked?cabinet?in?a?secure?area.?Analyzed?sample?data?from?the?study?will?be?shared?with?specific?project?collaborators?at?the?University?of?Toronto?and?the?University?of?Alberta.?No?information?that?identifies?you?will?be?allowed?to?leave?the?study?center?or?be?used?in?any?reports?or?publications?about?the?study.?Signing?this?consent?form?in?no?way?limits?your?legal?rights?against?the?sponsor,?investigators,?or?anyone?else.???13.?WHO?DO?I?CONTACT?IF?I?HAVE?QUESTIONS?ABOUT?THE?STUDY?DURING?MY?PARTICIPATION????This?study?will?be?fully?explained?to?you,?and?you?will?be?given?the?opportunity?to?ask?questions.?If?you?have?questions?or?want?more?information?about?the?study?procedures?before?or?during?participation,?you?may?contact?Dr.?Rajavel?Elango?at?any?time?at?778?986?8655,?Trina?Stephens?at?any?time?at?778?998?2365,?and/or?the?toll?free?Research?Subject?Information?Line?at?any?time?at?1?877?822?8598.???14.?WHO?DO?I?CONTACT?IF?I?HAVE?QUESTIONS?OR?CONCERNS?ABOUT?MY?RIGHTS?AS?A?SUBJECT?DURING?THE?STUDY???Your?rights?to?privacy?are?protected?and?guaranteed?by?the?Child,?Family?and?Community?Services?Act.?This?Act?lays?down?the?safeguards?respecting?your?privacy?and?also?gives?you?the?right?of?access?to?the?information?about?you?that?has?been?provided?to?the?study,?and?if?needed,?you?have?the?chance?to?correct?any?errors?in?the?information.?Further?details?about?this?legislation?are?available?on?request.?If?you?have?any?concerns?about?your?rights?as?a?research?subject?and/or?your?experiences?while?participating?in?this?study,?contact?the?toll?free?Research?Subject?Information?Line?at?any?time?at?1?877?822?8598?or?via?e?mail?to?RSIL@ors.ubc.ca.???? 93??15.??SUBJECT?CONSENT?TO?PARTICIPATE????My?signature?on?this?consent?form?means?that?I:?? ? have?had?this?study?explained?to?me,?read?this?form?and?understand?the?information?concerning?this?study.?? have?had?sufficient?time?to?consider?the?information?provided,?get?advice?and?ask?questions?if?necessary?and?I?have?received?satisfactory?responses?to?my?questions.?? understand?that?all?of?the?information?collected?will?be?kept?confidential?and?that?the?results?will?only?be?used?for?scientific?objectives.?? understand?that?my?participation?in?this?study?is?voluntary?and?that?I?am?completely?free?to?refuse?to?participate?or?to?withdraw?from?this?study?at?any?time?without?giving?any?reason(s)?and?my?decision?to?withdraw?will?not?change?in?any?way?the?quality?of?care?that?I?receive.?? understand?that?signing?this?consent?form?in?no?way?limits?my?legal?rights?against?the?sponsor,?investigators?or?anyone?else.?? understand?that?there?is?no?guarantee?that?this?study?will?provide?any?benefits?to?myself.?? understand?that?if?I?have?any?further?questions?or?desire?further?information?I?should?contact?Dr.?Rajavel?Elango?at?778?986?8655.?? understand?that?if?I?have?any?concerns?about?my?rights?as?a?research?subject?or?my?experiences?while?participating?in?this?study,?I?may?contact?the?toll?free?Research?Subject?Information?Line?at?any?time?at?1?877?822?8598?or?via?e?mail?to?RSIL@ors.ubc.ca.?? ?have?been?told?that?I?will?receive?a?dated?and?signed?copy?of?this?form?for?my?own?record.???I,?___________________________________????voluntarily?give?consent?for?my?participation?in?the???????(Subject.??Please?print?your?name)?????????????????????????????research?study?entitled:?Determination?of?protein?requirements?in?healthy?pregnant?women?using?the?indicator?amino?acid?oxidation?technique????________________________________________________???????____________________????????????????????Signature?of?Subject?? ? ? ????????????????????Date????________________? ? ? ? ?????????? ????????????????????????___________________?Investigator?Signature? ?Printed?Name?of?Principal?Investigator/?????? ???????????????????????????Date????? ? ? ????Designated?Representative?? 94???? ?????????Appendix?E???Food?record?template??? ?? 95?????? ? ????????? 96??????? ????Appendix?F???Study?day?protocol????? ???????????????????????? 97?Study?Day?Schedule?Protein?Requirement:?Healthy?Pregnant?Women???Subject?ID:_______________________? ? ? Date:___________________________??Height?(cm):_______________?????Weight?(kg):__________?Blood?Glucose?(mmol/L):__________??Protein?intake?(g/kg/d):?_________________??Energy?intake?(kcal/day):?______________?? Time? Sample?Collection/?Anthropometry? Meals?and?isotope?tracer? Comments?8:00? ? Meal?#1? ?9:00? ? Meal?#2? ?10:00? ? Meal?#3? ?11:00? ? Meal?#4? ?11:15? 1st?breath?(3x)?1st?urine? ? ?11:30? 2nd?breath?(3x)? ? ?11:45? 3rd?breath?(3x)?2nd?urine? ? ?12:00? VCO2?measurement?? Meal?#5???primer?dose?and??1st?oral?dose?? ?13:00? ? Meal?#6???2nd?oral?dose? ?14:00? ? Meal?#7???3rd?oral?dose? ?14:30? 4th?breath?(3x)?3rd?urine? ? ?15:00? 5th?breath?(3x)?4th?urine? Meal?#8???4th?oral?dose? ?15.15? 6th?breath?(3x)? ? ?15:30? 7th?breath?(3x)?5th?urine? ? ?15:45? 8th?breath?(3x)?? ? ?16:00? 9th?breath?(3x)?6th?urine? ? ?Chemstrip?7??Dipstick?Test?(Roche?)?Morning?Urine? Afternoon?Urine?pH:? Nitrite:? pH:? Nitrite:?Glucose:? Protein:? Glucose:? Protein:?Ketones:? Blood:? Ketones:? Blood:?Leukocytes:? ? Leukocytes:? ??? 98????????????Appendix?G???Letters?of?contact?to?primary?care?provider?? ?? 99?   ??Date:_____________??To?the?Health?Care?Provider?of?Ms.____________________________??Ms.?______________________?volunteered?to?participate?in?a?research?study?titled??Determination?of?protein?requirements?in?healthy?pregnant?women?using?the?indicator?amino?acid?oxidation?technique??being?conducted?at?the?Child?&?Family?Research?Institute,?BC?Children?s?Hospital?and?Department?of?Pediatrics,?University?of?British?Columbia.??As?part?of?this?study?we?measure?fasting?blood?glucose?using?a?finger?prick?glucometer?to?screen?for?normal?glucose?values?(<6.7mmol/L).??Her?fasting?blood?glucose?on?the?date?specified?above?was:_______________mmol/L.?Her?second?measurement?5?minutes?later?was:_________________________mmol/L.??We?have?stopped?her?participation?in?our?study?and?requested?her?to?follow?up?this?fasting?glucose?measurement?values?with?you,?as?her?primary?health?care?provider.??If?you?require?further?information?or?any?clarification?regarding?this?please?do?not?hesitate?to?contact?me.??Sincerely,???Rajavel?Elango?Ph.D.?Assistant?Professor,?Department?of?Pediatrics?University?of?British?Columbia?Scientist,?Level?1,?Diabetes,?Nutrition?&?Metabolism?Child?&?Family?Research?Institute?BC?Children's?Hospital?Room?170A,?950?West?28th?Avenue?Vancouver,?BC,?V5Z?4H4?Ph:?604?875?2000?ext.4911;?Fax:?604?875?3597?Cell:?778?986?8655?Email:?relango@cfri.ubc.ca????? D e p a r t m e n t  o f  P e d i a t r i c s  BC Children's Hospital 4480 Oak St., Room 2D19 Vancouver, BC Canada Tel: (604) 875-3177  Fax: (604) 875-2890 ???100?   ?Date:_____________??To?the?Health?Care?Provider?of?Ms.____________________________??Ms.?______________________?volunteered?to?participate?in?a?research?study?titled??Determination?of?protein?requirements?in?healthy?pregnant?women?using?the?indicator?amino?acid?oxidation?technique??being?conducted?at?the?Child?&?Family?Research?Institute,?BC?Children?s?Hospital?and?Department?of?Pediatrics,?University?of?British?Columbia.??As?part?of?this?study?we?conduct?two?dipstick?tests?to?check?for?protein?in?the?urine:???Chemstrip?7?Dipstick?Test?(Roche)?? Morning?Urine? Afternoon?Urine?pH:? Nitrite:? pH:? Nitrite:?Glucose:? Protein:? Glucose:? Protein:?Ketones:? Blood:? Ketones:? Blood:?Leukocytes:? ? Leukocytes:? ??We?have?requested?that?she?to?follow?up?this?urine?analysis?with?you,?as?her?primary?health?care?provider.??If?you?require?further?information?or?any?clarification?regarding?this?please?do?not?hesitate?to?contact?me.??Sincerely,???Rajavel?Elango?Ph.D.?Assistant?Professor,?Department?of?Pediatrics?University?of?British?Columbia?Scientist,?Level?1,?Diabetes,?Nutrition?&?Metabolism?Child?&?Family?Research?Institute?BC?Children's?Hospital?Room?170A,?950?West?28th?Avenue?Vancouver,?BC,?V5Z?4H4?Ph:?604?875?2000?ext.4911;?Fax:?604?875?3597?Cell:?778?986?8655?Email:?relango@cfri.ubc.ca?? D e p a r t m e n t  o f  P e d i a t r i c s  BC Children's Hospital 4480 Oak St., Room 2D19 Vancouver, BC Canada Tel: (604) 875-3177  Fax: (604) 875-2890 ???101???????? ??Appendix?H???Protein?turnover?estimated?from?LC?MS?analysis??? ?????????????102???Figure?1.?Protein?synthesis?versus?protein?intake?in?early?gestation?group??????Figure?2.?Protein?breakdown?versus?protein?intake?in?early?gestation?group????? ?0?10?20?30?40?50?60?70?80?90?100?0? 0.5? 1? 1.5? 2? 2.5? 3?Non?oxidative?Phe?Removal?(?mol/kg/h)?Protein?Intake?(g/kg/d)?0?10?20?30?40?50?60?70?80?90?100?0? 0.5? 1? 1.5? 2? 2.5? 3?Phe?release?from?protein?(?mol/(kg/h))?Protein?Intake?(g/kg/d)??103???Figure?3.?Protein?synthesis?versus?protein?intake?in?late?gestation?group??????Figure?4.?Protein?breakdown?versus?protein?intake?in?late?gestation?group????????? ?0?10?20?30?40?50?60?70?80?90?100?0? 0.5? 1? 1.5? 2? 2.5? 3?Non?oxidative?Phe?Removal?(?mol/kg/h)?Protein?Intake?(g/kg/d)?0?10?20?30?40?50?60?70?80?90?100?0? 0.5? 1? 1.5? 2? 2.5? 3?Phe?release?from?protein?(?mol/(kg/h))??Protein?Intake?(g/kg/d)??104?Table?1.?Comparison?of?protein?turnover?estimates?during?pregnancy?? Author? Gestational?Age?(weeks)? Sample?size?? State? Synthesis? Breakdown? Turnover?Stephens?et?al.,?2013? ?11?20?? ?37? ?Fed? 4.37?g?PRO/kg/da? 3.98?g?PRO/kg/da? 0.39?Stephens?et?al.,?2013? ?31?38? ?44? ?Fed? 4.71?g?PRO/kg/da? 4.17?g?PRO/kg/da? 0.54??Thompson?and?Halliday?1992??35? ?6? ?Fasted? 3.87?g?PRO/kg/d? n/a? n/a?Jackson,?Duggleby?and?Grove?2000??31? ?5? ?Fed? 1.99??g?N/hour? 1.73??g?N/hour? 0.26??Willommet?et?al.,?1992??33? ?9? ?Fed? 5.4??g?PRO/kg/d? 4.1??g?PRO/kg/d? 1.3??Wittaker,?Lee?and?Taylor?2000??34?38? ?6? ?Fasted? 4.81?g?PRO/kg/d? 5.39?g?PRO/kg/d? ?0.58??a?Assuming?232?mol?Phe/g?muscle?protein?(Kaufman?S.??A?model?of?human?phenylalanine?metabolism?in?normal?subjects?and?in?phenylketonuric?patients.??Proceedings?of?the?National?Academy?of?Sciences?in?the?United?States?of?America,?96.6?(1999):?3160?3164)?

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