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Determination of lysine requirements in healthy pregnant women using the indicator amino acid oxidation… Payne, Magdalene 2014

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!!   DETERMINATION OF LYSINE REQUIREMENTS IN HEALTHY PREGNANT WOMEN USING THE INDICATOR AMINO ACID OXIDATION TECHNIQUE by  Magdalene Payne B.Sc., Queen’s University 2011   A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF Master of Science in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (REPRODUCTIVE AND DEVELOPMENTAL SCIENCES) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)   June 2014 © Magdalene Payne, 2014      ii !Abstract Perinatal nutrient status influences the health of both mother and child. Lysine, an essential amino acid found mainly in animal derived products, is the first limiting amino acid in plant proteins. Inadequate lysine intakes during pregnancy may impact foetal health in both the short and long-term. Current Dietary Reference Intakes (DRI) recommendations extrapolate lysine requirements during pregnancy from non-pregnant adult data and may underestimate true requirements. Moreover, these recommendations remain constant throughout pregnancy and do not reflect the dynamic metabolic adaptations that occur during gestation. This study aims to define a quantitative lysine requirement in healthy pregnant women and to determine whether lysine requirements vary between phases of gestation.  Two phases of pregnancy (early gestation, 12-19 weeks; late gestation, 33-39 weeks) were studied using the indicator amino acid oxidation (IAAO) technique. Pregnant women (22-36y) consumed a diet containing a random lysine intake (range = 6 - 86 mg/kg/d) in a crystalline amino acid mixture based on egg protein profile. Diets were isonitrogenous with caloric and protein intakes maintained at 1.7 x resting energy expenditure and 1.5g/kg/d, respectively. Breath and urine samples were collected at baseline and isotopic steady state. Lysine requirements were determined by measuring oxidation of L-[1-13C]-phenylalanine to 13CO2 (F13CO2). Bi-phase linear regression crossover analysis was used to determine a breakpoint (estimated average requirement, EAR) in F13CO2 data. The breakpoint in early gestation (n=27) was determined to be 36.6 mg/kg/d (r2 = 0.484, Upper 95% CI = 46.2), similar to current non-pregnant recommendations of 41mg/kg/d. The breakpoint in late gestation (n=36) was determined to be 50.3 mg/kg/d (r2 = 0.664, Upper 95% CI = 60.4), and 25% higher than current DRI recommendations of 41 mg/kg/d. Urinary phenylalanine flux did not change in both early and iii !late gestation due to lysine intake. These data are the first to directly define a quantitative requirement for lysine during human pregnancy and to account for gestational age, describing an increase in requirement as pregnancy progresses. We expect these results will have significant implications for setting recommendations globally where plant based diets are the primary source of protein and amino acids.   iv !Preface  This thesis original, unpublished work by the author, M. Payne.      v !Table of Contents  Abstract ........................................................................................................................................... ii Preface ............................................................................................................................................ iv Table of Contents .............................................................................................................................v List of Tables ............................................................................................................................... viii List of Figures ................................................................................................................................ ix List of Abbreviations .......................................................................................................................x Acknowledgements ........................................................................................................................ xi Dedication ..................................................................................................................................... xii CHAPTER 1 Introduction  ...............................................................................................................1 1.1 Introduction ........................................................................................................1 1.2 Overview and Summary ....................................................................................2 CHAPTER 2 Background  ...............................................................................................................4 2.1 Maternal Adaptations During Pregnancy ...........................................................4 2.2 Lysine .................................................................................................................9 2.2.1 Overview of Protein ............................................................................9 2.2.2 Overview of Amino Acids ................................................................10 2.2.2 Structure and Properties of Lysine ....................................................12 2.2.3 Sources of Lysine  .............................................................................12 2.2.4 Transport of Lysine  ..........................................................................13 2.2.5 Synthesis of Lysine  ..........................................................................16 2.2.6 Metabolism of Lysine  ......................................................................17 2.2.7 Role in Carnitine Synthesis ...............................................................20 2.2.8 Roles of Lysine in Human Health2 .....................................................4 2.3 Current Recommendations for Lysine Intakes ................................................27 2.4 Protein and Amino Acid Requirement Research Techniques ..........................28 2.4.1 Nitrogen Balance Technique .............................................................29 2.4.2 Amino Acid Balance and Oxidation Techniques ..............................30 2.4.3 Indicator Amino Acid Oxidation Technique ....................................31 2.5 Current Research ..............................................................................................34 2.6 Rationale ..........................................................................................................36 2.7 Objectives ........................................................................................................36 2.8 Hypotheses .......................................................................................................36 vi ! CHAPTER 3 Methods ...................................................................................................................37 3.1 Subjects ............................................................................................................37 3.1.1Inclusion Characteristics ....................................................................37 3.1.2 Exclusion Characteristics ..................................................................38 3.2 Experimental Design ........................................................................................39 3.3 Study Protocol ..................................................................................................43 3.3.1 Pre-study Protocol .............................................................................43 3.3.2 Diet Standardization ..........................................................................44 3.3.3 Study Protocol and Isotope Infusion Studies ....................................45 3.4 Sample Collection ............................................................................................47 3.4.1 Breath Samples .................................................................................47 3.4.2 Urine Samples ...................................................................................47 3.5 Analytical Procedures ......................................................................................48 3.6 Isotope Kinetics ...............................................................................................48 3.7 Statistical Analysis ...........................................................................................49  CHAPTER 4 Results and Discussion ............................................................................................51 4.1 Results ..............................................................................................................51 4.1.1 Subject Characteristics ......................................................................51 4.1.2 Early Gestation ..................................................................................53 4.1.3 Late Gestation ...................................................................................55 4.2 Discussion ........................................................................................................57 4.2.1 Subject Characteristics and Pre-Study Data .....................................57 4.2.2 Lysine Requirements ........................................................................57 4.2.2 Supportive Evidence .........................................................................61 4.2.3 Limitations ........................................................................................62 4.2.4 Global Implications ...........................................................................63  CHAPTER 5 Trans-abdominal Ultrasound to Determine Post-Void Urine Retention .................66  5.1 Background ......................................................................................................66 5.1.1 Average Post-Void Urine Retention .................................................66 5.1.2 Measurement of Bladder Volumes ...................................................67 5.1.3 Objectives .........................................................................................68 5.1.4 Hypothesis.........................................................................................68 5.2 Methods............................................................................................................68 5.2.1 Subject Consent ................................................................................68 5.2.2 Methods.............................................................................................69 5.2.3 Analysis.............................................................................................69 5.3 Results and Discussion ....................................................................................69 vii !5.3.1 Results ...............................................................................................69 5.3.2 Discussion .........................................................................................76  CHAPTER 6 Conclusions and Recommendations for Future Work .............................................78    References ......................................................................................................................................80 APPENDIX A: Subject Consent Form ..........................................................................................97 APPENDIX B: Pre-study Day Protocol ......................................................................................106 APPENDIX C: Advertisement ....................................................................................................109 APPENDIX D: Brochure .............................................................................................................111 APPENDIX E: Subject Code Master List ...................................................................................114 APPENDIX F: Dietary Record Sheets ........................................................................................116 APPENDIX G: Letter of Contact to Primary Health Care Provider ...........................................118 APPENDIX H: Study Day Protocol ............................................................................................120 APPENDIX I: Pre-study Subject Demographics .........................................................................122   viii !List of Tables Table 1. Additional Energy Requirements during Gestation in women with an average weight gain of 12 kg  ..........................................................................................................6 Table 2. Categorization of Essential, Conditionally Essential, and Nonessential Amino Acids ..................................................................................................................................11 Table 3. Lysine content in a range of common foods................................................................13  Table 4. Cationic Amino Acid Transport Systems ...................................................................14 Table 5. Lysine Recommendations in non-pregnant adults and pregnancy ..........................28 Table 6. Amino Acid Composition of Egg Protein (not including L-Lysine, L-Alanine, and L-Phenylalanine) ..............................................................................................................42 Table 7. Results from Pre-Study Data in Early (N=13) and Late (N=19) Subjects ...............52 Table 8. Results from Study Day Data in Early (Nsubjects=13, nstudies=27) and Late (Nsubjects =19, nstudies=36) Gestation ...............................................................................................53 Table 9. Lysine Content in Egg and Rice Protein at Current Protein Recommendations (DRI, 2005) and Experimentally Derived Requirements .............................................64 Table 10a. Early Gestation: Post-Void Residual Urine Volume .............................................71 Table 10b. Early Gestation: Voided Urine Volume ..................................................................72 Table 10c.  Early Gestation: Percent Residual Urine Volume (Residual Urine/(Residual Urine + Void Volume)) ....................................................................................................73 Table 11a. Late Gestation: Post-Void Residual Urine Volume ...............................................74 Table 11b. Late Gestation: Voided Urine Volume ....................................................................75 Table 11c.  Late Gestation: Percent Residual Urine Volume (Residual Urine/(Residual Urine + Void Volume)) ....................................................................................................76        ix !List of Figures Figure 1. Graphic Representation of Protein Accretion in Various Tissues Throughout Pregnancy  ..........................................................................................................................7 Figure 2. Schematic of Amino Acid Structure ..........................................................................10 Figure 3. Schematic of Lysine Structure ....................................................................................12 Figure 4a. Illustration of Human Placental Cross-section .......................................................15 Figure 4b. Schematic of the Human Haemochorionic Placental Nutrient Transport ...........15 Figure 5. Schematic of Lysine Transport across the Human Placenta via the y+L and y+ Transport Systems ...........................................................................................................16 Figure 6. Lysine Catabolism in the Human Liver ....................................................................18 Figure 7.  Carnitine Biosynthesis in Mammals .........................................................................21 Figure 8. Representation of Response to Increasing Amino Acid/Protein Intake Using Direct Amino Acid Oxidation, Indicator Amino Acid Oxidation, and Nitrogen Balance Requirement Techniques ..................................................................................31  Figure 9. Example of Breakpoint Analysis Using the IAAO Technique ................................33 Figure 10. Schematic of Phenylalanine Metabolism and L-[1-13C]-Phenylalanine Tracer Oxidation to 13CO2 ...........................................................................................................34 Figure 11. Experimental Design .................................................................................................40 Figure 12. Outline of Study Day Protocol ..................................................................................46 Figure 13. Estimated average lysine requirement in early gestation (Nsubjects=13, nstudies=27) derived by bi-phase linear regression crossover analysis .............................................54 Figure 14. Variation in urinary Phenylalanine flux across all lysine intakes in early gestation determined using linear regression  ...............................................................55 Figure 15. Estimated average lysine requirement in late gestation (Nsubjects=19, nstudies=36) derived by bi-phase linear regression crossover analysis .............................................56 Figure 16. Variation in urinary Phenylalanine flux across all lysine intakes in late gestation determined using linear regression ................................................................................56 Figure 17. Plasma progesterone and estradiol profiles during pregnancy .............................60    x ! List of Abbreviations BIA – Bioelectric Impedance Analysis  DAAO – Direct Amino Acid Oxidation  DRI - Dietary Reference Intakes  EAR – Estimated Average Requirement F13CO2 – L-[1-13C] – Rate of Phenylalanine Oxidation Measured in Breath Samples FAO – Food and Agriculture Organization FFM – Fat-free Mass FM – Fat Mass hPL – Human Placental Lactogen IAAO – Indicator Amino Acid Oxidation Technique  IAAB – Indicator Amino Acid Balance Technique  IOM  - Institute of Medicine RDA – Recommended Dietary Allowance  REE – Resting Energy Expenditure UNU – United Nations University VCO2 - Volume of Carbon Dioxide Exhaled Per Minute VO2 – Volume of Oxygen Exhaled Per Minute WHO – World Health Organization       xi !Acknowledgements I sincerely thank the University of British Columbia, the Child and Family Research Institute, and BC Children’s Hospital and BC Women’s Hospital and Health Centre. These bodies, in addition to the Canadian Institutes of Health Research, allowed this research to take place. Externally, Ajinomoto and Mead Johnson generously provided essential study supplies, further permitting this research. I would also like to acknowledge my laboratory: my supervisor, Dr. Elango, and my colleagues Trina Stephens, Leah Cooper, Cindy Wong and Krystal Mehat. Finally, I sincerely thank all of my participants, without whom this research would not be possible.   xii !Dedication  I would like to dedicate this work to all those who supported me during the completion of my research. To my family, my friends, my supervisor and my research associates.1 !Chapter 1 - Introduction 1.1 Introduction!There are few periods of dynamic development in our lifetimes that are comparable to gestation. This unique stage of life combines complex and multifactorial adaptations to mature a cluster of cells into a living, breathing child. Completion of gestation is dependent on successful fertilization, implantation and maintenance of an adequate in-utero environment; failure in any of these areas may significantly or completely impede the child’s success. Of noted importance in the maintenance of an adequate environment is maternal nutrient status. It is clear that poor nutrient delivery to the foetus predisposes a child to a multitude of negative effects; negative outcomes ranging from neural tube defects as a result of dietary folate deficiency to global growth impediment resulting from an array of conditions including poor placentation or maternal malnutrition. Birth weight is a significant predictor of neonatal morbidity and mortality (Fanaroff et al, 2007), and maternal nutritive status during gestation is a major influencing factor on infant birth weight (FAO, 2005). Although the most prevalent global cause of low infant birth weight is sub-optimal nutrition, over-nutrition in many “Western” diets is detrimental as well. Maternal overweight and obesity are associated with both neonatal macrosomia and low birth weight, though the aetiology linking one outcome over the other is unknown. Increasingly, importance is placed in nutritionally dense foods that provide an optimal amount of nutrients, rather than the idea that “more is always better”.  As gestation involves rapid physiological and metabolic changes, tissue anabolism, and increased nutrient and energy requirements, expansion of maternal and foetal tissues results in significant increases in maternal basal metabolic rate (BMR) and total energy expenditure (TEE), thereby contributing to an overall increase in energy requirements (King, 2000; IOM, 2009). Dietary protein and amino acids are crucial to develop both maternal and foetal structures 2 !including placental, uterine and breast tissues, expanding blood and extracellular fluid volume, and fuel for the entirety of the foetus. Lysine, the amino acid of focus in the current study, is of particular importance as neither the mother nor the foetus is capable of endogenous synthesis of this essential amino acid. Additionally, dietary lysine is known to be limiting in plant-based diets; assessments of global protein quality find that it is the first limiting amino acid in diets in all developing countries (Ghosh et al, 2012). Accurate estimates of energy and nutrient intakes are crucial during pregnancy to adequately support the deposition of new protein and maintenance of newly created tissues and to ensure optimal maternal and foetal development.   1.2 Overview and Summary Protein and amino acid requirement values and analytical techniques are currently a topic of intense debate (DRI, 2005; FAO, 2007). A traditional method used to determine requirements includes the Nitrogen Balance model, but this technique may underestimate the true requirement and is invasive in nature, thereby rendering it unethical to conduct in a vulnerable population such as pregnant women (Young et al, 1989). Recent research has focused instead on applying novel tracer techniques involving the use of stable isotopes in hopes of determining a consensus for these recommendations. Among amino acids of interest, lysine has been studied extensively mainly due to its role as the first limiting amino acid in cereal-based diets. This is particularly relevant in the developing world as the primary sources of protein are rice and wheat, and diets in a majority of these countries have been found to be limiting in lysine (Ghosh et al, 2012; Meredith et al, 1986; Zello et al, 1995; Pillai et al, 2010; Rand and Young, 1999). Lysine is an essential amino acid, thus it is critical for sufficient consumption in the diet to ensure adequate protein synthesis. Recently, experimental evidence from pig studies suggest that lysine 3 !requirements may be increased during later stages of pregnancy compared to the earlier stages (Samuel et al, 2012). The indicator amino acid oxidation (IAAO) technique has been used to determine amino acid requirements in adults, young children, neonates, and research in this thesis describes the first application of this technique in a pregnant population. There is very little information regarding amino acid requirements during pregnancy, and the research that has been conducted is insufficient or contradictory. The Institute of Medicine (IOM) issues Dietary Reference Intakes (DRI) for macronutrients only for populations living in North America, while the United Nations conglomerate of the World Health Organization/Food and Agriculture Organization/United Nations University (WHO/FAO/UNU) defines global recommendations. Current recommendations for lysine in pregnancy are only defined by the North American Dietary Reference Intakes and are based on factorial calculations extrapolated from adult values based on body weight, without experimental evidence (DRI, 2005). These recommendations are not only not defined by direct data, but the studies on which they are based study pregnancy using the Nitrogen balance technique, which may underestimate requirements (King, 2000). The Institute of Medicine has recommended that further research be conducted to determine protein and amino requirements during pregnancy to provide more accurate recommendations (DRI, 2002); thus, using the IAAO method, this project is the first to address this recommendation and to determine the lysine requirements in healthy pregnant women.      4 !Chapter 2 – Background 2.1 Maternal Adaptations During Pregnancy Pregnancy is a period of rapidly occurring metabolic, endocrine and physiological transformations resulting in new tissue accretion and formation (King, 2000). These adaptations result in altered maternal cardiovascular, renal, respiratory, immune and endocrine systems and are essential in promoting and maintaining successful implantation of the conceptus.  Hormonal influence is significant immediately following conception to allow for successful implantation of the conceptus, including thickening of the endometrium and immunological down-regulation, both mediated by progesterone (Sadler, 2006). Aside from maintaining pregnancy, adaptations to maternal endocrine systems modify metabolism resulting in a hyperinsulinemic state and diversion of nutrients to the foetus, placenta and supporting structures, in every effort to support a healthy pregnancy. Even so, adequate energy intake during gestation is critical to ensure full-term parturition of a neonate of adequate weight and body composition and to preserve maternal health during gestation (DRI, 2005). As maternal and foetal tissues develop, nutritional and metabolic requirements may change as the need for energy and nutrients increase.  During pregnancy, protein and fat accretion in maternal, foetal and placental tissues contribute to maternal weight gain (FAO, 2005). This additional weight gain is required to supply increased foetal needs and maternal energy expenditure associated with an increased BMR (FAO, 2005). The World Health Organization (WHO) describes a mean weight gain of 12 kg with a range of 10-14 kg; this amount correlates with neonates at optimal birth weights of 3.3 – 3.6 kg (WHO, 1995). It is estimated that 77 000 additional kilocalories are required during pregnancy (FAO, 2005; Table 1); however, this supplementary energy is not necessarily evenly distributed across the trimesters. Current recommendations based on a factorial model advocate 5 !360 and 475 additional kilocalories/day in trimesters two and three, respectively (FAO, 2005; DRI, 2005). This may reflect a trend of increased requirements for other nutrients, such as protein, amino acids, and lipids. It should be noted, however, that energy requirements may be population specific as lifestyle, maternal nutrition status, and body size will vary greatly between groups, and that weight gain during pregnancy varies depending on pre-pregnancy Body Mass Index (BMI) (FAO, 2005).  The average weight gain in healthy pregnancy is approximately 12 kg, with 0.6 kg (597 g) of which is estimated to be new protein accreted in maternal and foetal tissues (WHO, 1995). However, the current DRI estimates that an average pregnancy amasses 925 g of protein mass (DRI, 2005). Dietary and endogenous protein is required for tissue synthesis, creating new foetal, uterine, and placental tissues while adding to maternal extracellular fluid, blood and amniotic fluid (DRI, 2002; Figure 1). As energy requirements are not consistent throughout pregnancy, the FAO (2007) asserts the rate of protein storage is similarly variable. Protein synthesis increases by 1% (1.2 g/d), 15% (6.1 g/d) and 25% (10.7 g/d) in trimesters one, two, and three, respectively (FAO, 2007); as the rates of protein synthesis increase from early to late gestation, could be hypothesized that protein and amino acid requirements will also increase during later stages of pregnancy.    !!6 !Table 1. Additional Energy Requirements during Gestation in women with an average weight gain of 12 kg. Adapted from FAO, 2005.  Rates of tissue deposition    1st trimester g/d 2nd trimester g/d 3rd trimester g/d Total deposition g/280d Weight gain Protein deposition Efficiency of energy utilization 17 0 5.2 60 1.3 18.9 54 5.1 16.9 12 000 597 3741 Energy cost of pregnancy estimated form the increment in BMR and energy deposition  1st trimester kJ/d 2nd trimester kJ/d 3rd trimester kJ/d Total energy cost MJ Kcal Protein deposition Fat deposition Efficiency of energy utilization Basal metabolic rate Total energy cost of pregnancy (kJ/d) 0 202 20 199 421 30 732 76 397 1235 121 654 77 993 1845 14.1 144.8 15.9 147.8 322.6 3370 34600 3800 35130 77100 Energy cost of pregnancy estimated from the increment in TEE and energy deposition  1st trimester kJ/d 2nd trimester kJ/d 3rd trimester kJ/d Total energy cost MJ Kcal Protein deposition Fat deposition Total energy expenditure Total energy cost of pregnancy 0 202 85 287 30 732 350 1112 121 654 1300 2075 14.1 144.8 161.4 320.2 3370 34600 38560 76530 7 ! Figure 1. Graphic Representation of Protein Accretion in Various Tissues Throughout Pregnancy. Pitkin, 1976. Maternal nutrition throughout pregnancy has profound short and long-term impacts on the developing foetus; inadequate maternal nutrition has been linked to the complex, multifactorial condition known as intrauterine growth restriction (IUGR). Most famously, epidemiological studies involving long-term analysis of families following the “Hunger Winter”, a period of extreme food deprivation in the Netherlands during World War II, revealed substantial evidence that inadequate nutrition and energy during pregnancy is associated with long-term deleterious health effects in the offspring (Schultz, 2010; Roseboom et al, 2001). Infants born to famine-exposed mothers had reduced head circumference and birth length as well as increased rates of coronary heart disease later in life (Roseboom et al, 2001). Similarly, neonatal mortality was increased following the1974-75 Bangladesh famine (Razzaque et al, 1990), and offspring conceived during the Great Chinese Famine (1959-1961) were more likely to suffer from hyperglycemia and type II diabetes as adults (Li et al, 2010). Importantly, many of these negative consequences are inter-generational, and are described in grandchildren of famine survivors (Lumey et al, 2011).  8 !Excessive nutrition has recently also been associated with both intrauterine growth restriction (IUGR) and foetal macrosomia. Higher prevalence of IUGR, requirement of neonatal intensive care, spontaneous pregnancy loss, caesarean section and thromboembolic complications are noted in mothers with a BMI >30 (Radulescu et al, 2013; Wu et al, 2004; Leddy et al, 2008). However, obese or overweight mothers are also more likely to develop preeclampsia, gestational diabetes mellitus, and/or give birth to macrosomic offspring. Macrosomic infants, or neonates with a birth weight >4500g, also experience negative perinatal outcomes including increased rates of caesarean section and shoulder dystocia as well as greater incidence of metabolic syndrome later in life (Leddy et al, 2008). Placental insufficiency is believed to be involved in both extremes of the outcomes seen in overnutrition (Wu et al, 2004), although it is not clear why one outcome occurs over the other. Regardless, like maternal undernutrition, overnutrition is of significant concern in development of chronic disease.  While maternal nutrition certainly plays a crucial role in infant outcome, IUGR is a multifactorial complication that may result from a variety of etiologies. Regardless of cause, infants born growth restricted are at increased risk of perinatal morbidities including increased neonatal death, necrotizing enterocolitis and respiratory distress. As discussed in the aforementioned epidemiological studies, those offspring are also more likely to be diagnosed with adult-onset diseases such as metabolic syndrome, hypertension, ischemic heart disease, diabetes, kidney disease, and obstructive lung disease, indicating that in-utero nutrition has the capacity to “program” the offspring for life (Barker et al, 1993a; Barker et al, 1993b; Phillips, 1998; Belkacemi et al, 2010; Reis et al. 2002, Murphy et al., 2006). In relation to protein nutrition, IUGR is associated with both decreased placental transport of amino acids and reduced fetomaternal concentration ratios of essential amino acids (Jansson, 2001). In healthy 9 !pregnancies, maternal amino acid concentrations decrease in mid- to late-gestation while foetal plasma concentrations increase, indicating preferential and active transport of amino acids to the fetus, placenta and supporting tissues. However, this adaptation does not occur during IUGR pregnancies (Young and Prenton, 1969; Jansson, 2001). This decreased fetomaternal concentration ratio could be a result of decreased transporter activity or altered fetoplacental metabolism, or some combination (Cetin, 2001). While it is possible that amino acid and/or protein supplementation may help improve growth, the exact amount to be provided remains unknown.   2.2 Lysine 2.2.1 Overview of Protein   As one of the three basic macronutrients, protein acts in an essential role in human health. Protein and its component amino acids serve in a broad variety of functional roles, including structural, metabolic, immune and kinetic. Proteins, peptides, and amino acids are crucial aspects of many enzymes, membranes, cellular support structures, transport carriers, some hormones, connective and other organizational tissues. Dietary protein provides much of the amino acids and energy required for these various responsibilities to occur.   To utilize dietary amino acids, dietary protein must first be digested and absorbed by the gastrointestinal system and the peptide bonds be hydrolysed. The breakdown of protein begins in the mouth with mechanical digestion by chewing action of the jaw and teeth (Whitney and Rolfes, 2011). Once protein enters the stomach, it is digested by proteases, enzymes that break protein into smaller polypeptides. Pepsin is the primary protease present; however it must be converted from inactive pepsinogen to pepsin through the action of HCl in the stomach (Voet et 10 !al, 2008). Digestive enzymes trypsin and chymotrypsin further degrade peptide bonds once protein reaches the small intestine (Voet et al, 2008). This upper section of the small intestine, the duodenum, is the primary site of absorption where amino acids are absorbed directly into enterocytes and into the mesenteric capillaries (Whitney and Rolfes, 2011). Digested amino acids will be discussed further below.  2.2.2 Overview of Amino acids Amino acids are the most basic components of protein, forming important components of immune mediators, tissue structures and cell signalling. Each amino acid contains a central carbon to which an amino group (NH3+) and a carboxyl group (COO-) are bonded (Figure 2). A variable group determines the properties of the specific amino acid, deemed the R-group.    There are twenty standard amino acids, nine of which are considered essential; that is, they cannot be synthesized by animals and therefore must be consumed in the diet. This distinction is important in amino acid nutrition, as some amino acids are necessary in the diet for normal growth and metabolism. Based on the above, amino acids are categorized as essential, conditionally essential or nonessential (Table 2; Reeds, 2000). Protein synthesis cannot occur Figure 2. Schematic of Amino Acid Structure. Voet et al, 2008.  11 !unless all required amino acids are present in sufficient quantities; thus if one is limiting, anabolism will not occur. The first limiting amino acid is a significant factor as it is present in the diet in least amounts relative to the requirement. Consequently, as lysine is the first limiting amino acid in plant-based diets, it plays a critical role in protein synthesis and oxidation (Young and Pellett, 1994).  Table 2. Categorization of Essential, Conditionally Essential, and Nonessential Amino Acids. Adapted from Reeds, 2000. Essential Conditionally Essential Nonessential Lysine Threonine Tryptophan Phenylalanine Methionine Valine Histidine Leucine Isoleucine Tyrosine Glutamine Cysteine Glycine Proline Arginine   Glutamic Acid Aspartic Acid Serine Alanine Asparagine   All standard α-amino acids, with the exception of glycine, are classified by their chirality of optical activity as either a D- or L-amino acid. Having optical activity refers to asymmetrical compounds with the ability to rotate a plane of polarized light, i.e. are compounds that are chiral enantiomers (Solomons and Fryhle, 2008). There are three classification systems to describe these isomers: by configuration (R- and S- system), by optical activity ((+)- and (-)- system) and by configuration (D- and L- system) (Solomons and Fryhle, 2008).  The D- and L- system is used to describe the absolute configuration of the molecule compared to a standard molecule. In the 12 !case of amino acids, the standard amino acid used is serine (Voet et al, 2008). All amino acids derived from protein have the L-amino acid configuration, whereas D-amino acids comprise short polypeptide chains constituting bacterial cell walls (Voet et al, 2008). Also, D-amino acids are not synthesized by transcription and translation, but rather by specific bacterial enzymes (Voet et al, 2008), and are therefore not involved in human metabolism.  2.2.3 Structure and Properties of Lysine Lysine (abbreviations: Lys, K) is an essential α-amino acid with the formula HO2CCH(NH2)(CH2)4NH2 . Due to its basic ε-side chain, it acts as one of the three basic amino acids and is able to participate in hydrogen bonding as well as acid-base reactions. In addition, L-leucine and L-lysine are the only amino acids that are purely ketogenic, that is, their metabolites produce only ketone bodies through ketogenesis (Voet et al, 2008). Due to the positively charged polar butylammonium side chain, L-lysine is categorized as both a cationic and hydrophobic amino acid (Figure 3; Voet et al, 2008). !   2.2.3 Sources of Lysine Lysine is present in a variety of protein-rich foods, predominantly in animal derived foods such as eggs, chicken and beef. In addition, minimal amounts are also present in plant proteins such as wheat, rice and legumes (Table 3). However, in these cereal-based proteins, Figure 3. Schematic of Lysine Structure. 13 !lysine is the first limiting amino acid; consequently, these foods are a poor source of lysine (Young and Pellett, 1994). As the majority of the world is heavily reliant on cereal-based protein, lysine deficiency is a considerable threat among these populations.  Table 3. Lysine Content in a Range of Common Foods. Data Collected From the USDA.  2.2.4 Transport of Lysine  Following digestion of protein in the gastrointestinal tract as previously described, transport systems are required to move macromolecules and lipid-insoluble particles, including amino acids, across the hydrophobic domain of the plasma membrane and into intestinal cells. Christensen and colleagues first described mammalian amino acid transport systems (Christensen, 1963; Christensen and Antonioli, 1969; Palacín et al, 1998), and since then, five transport systems involving lysine have been described in various tissues: y+, y+L, b°,+ B°,+ and b+ (Palacín et al, 1998; Table 4). These are classified by both Na+ dependency and substrate specificity (cationic only versus cationic and neutral amino acid accepting) (Devés and Boyd, 1998).   14 !Table 4. Cationic Amino Acid Transport Systems. Adapted from Palacín et al, 1998; Devés and Boyd, 1998.   Transport systems in pregnancy are critical to provide nutrients to ensure normal foetal growth; decreased amino acid transfer has been associated with intrauterine growth restriction (Paolini et al, 2001). The placenta plays a crucial role in transporting amino acids between maternal and foetal circulations, yet mechanisms for transplacental transport are not well understood, particularly for essential amino acids (Moe, 1995; Jansson, 1998). Humans and rodents are haemomonochorial; foetal chorion is in direct contact with maternal blood in the intervillous space (Figure 4b). In early pregnancy, multiple layers of tissue separate maternal and foetal circulation: continuous syncytiotrophoblast, discontinuous cytotrophoblast, stromal connective tissue and foetal capillary endothelium (Sengers et al, 2010). Foetal capillary endothelial cells are continuous, though amino acids are free to pass through this cell layer via 15 !pores in the interendothelial cleft (Jansson, 2001). Consequently, the principal mechanism and the rate-limiting step of amino acid transport are via specific proteins located on the basal and microvillus membranes of the syncytiotrophoblast (Moe, 1995). As pregnancy progresses, dilation of foetal villous capillaries occurs and cytotrophoblast is lost (Sengers et al, 2010), leaving only the polarized syncytium and the foetal capillary endothelium to separate direct mixing of maternal and foetal blood (Figure 4a, b; Sadler, 2006). This progression increases the efficiency of nutrient transfer through the placenta (Castellucci and Kaufmann, 2006).    Figure 4a. Illustration of Human Placental Cross-section.  Figure 4b. Schematic of the Human Haemochorionic Placental Nutrient Transport. As plasma amino acid concentrations are greater in the foetus than maternal tissues, it is assumed that active transport must play a role in transplacental amino acid transfer (Jansson, 2001). Multiple transport systems have been implicated in placental amino acid transport, and all appear to be Na+-independent (Jansson et al, 1998). Two major systems, y+ and y+L, seem to be involved in lysine transport in the syncytium; however there seems to be unequal distribution between the microvillus membrane and the basal membrane of syncytiotrophoblasts (Figure 5; 16 !Jansson et al, 1998). Nutrients must be transported from maternal circulation across the microvillous membrane and into the multinucleated syncytium, from which they are shuttled across the basal membrane and into foetal circulation (Sengers et al, 2010). Furesz et al (1995) showed evidence of both y+L and y+ involvement in cationic amino acid uptake in the microvillus membrane, though y+ is the primary system at this level. The y+L system is suggested to regulate and mediate the net transport of lysine across the basal membrane (Furesz and Smith, 1997; Eleno et al, 1994). Furesz et al (1991) show evidence for the b0,+ system as a third process engaged in syncytial transport, however the level of involvement of total lysine uptake was not determined. In conclusion, five transport systems have been identified in the transport of lysine in mammalian models, three of which have been identified in human placenta (Table 4; Figure 5).   Figure 5. Schematic of Lysine Transport across the Human Placenta via the y+L and y+ Transport Systems. Jansson, 2001.  !!17 !  2.2.5 Synthesis of Lysine  L-Lysine cannot be synthesized in mammalian tissues. There are two primary routes for lysine biosynthesis, which evolved separately. The first pathway, involved in the aspartate family, is the diaminopimelic acid route (DAP). The second pathway is a member of the glutamate family, the alpha-aminoadipic pathway (AAA) (Miyazuki et al, 2001). These pathways are utilized primarily by bacterium, and have been used commercially to produce lysine via a fermentation process using Corynebacterium glutamicum (Pfefferle et al, 2003). 2.2.6 Metabolism of Lysine L-Lysine catabolism occurs primarily in the mammalian liver (Voet et al, 2008; Tomé and Bos, 2007) in the mitochondrial matrix (Scislowski et al, 1994), yielding two moles of CO2 and acetyl-CoA and/or acetoacetate. First-pass metabolism accounts for approximately 30-42% of –lysine requirements (Tomé and Bos 2007). L-Lysine catabolism involves oxidation following deamination (Tomé and Bos, 2007; Figure 6): 18 ! Figure 6. Lysine Catabolism in the Human Liver. Qiagen, accessed 2014. (<!http://www.qiagen.com/products/genes%20and%20pathways/pathway%20details.aspx?pwid=274>) 19 !The primary route of L-lysine breakdown is via a saccharopine-dependent pathway (Figure 6; Fjellestedt and Robinson 1975a). L-Lysine and α-ketoglutarate are combined in an NADPH-dependent reaction catalyzed by L-lysine α-ketoglutarate reductase (LKR). This first step commits lysine to oxidation and to the formation of the stable intermediate, saccharopine (Gahl et al, 1997). Saccharopine is then reduced to α-aminoadipate-6-semialdehyde and glutamate via saccharopine dehydrogenase (SDH) action and NAD+ reduction. Aminoadipate semialdehyde dehydrogenase then reduces α-aminoadipate-6-semialdehyde to α-aminoadipate. This product then undergoes a PLP-dependent reaction producing α-ketoadipate, which is then oxidized by a multi-enzyme complex. The following four reactions are standard fatty-acyl oxidation, producing glutaconyl-CoA, crotonyl-CoA, β-hydroxybutyryl-CoA and acetoacetyl-CoA. Two molecules of CO2 are produced, one in reaction five and the second in reaction seven. The final two reactions involve ketone body formation, yielding acetoacetate and acetyl-CoA (Voet et al, 2008). Acetyl-CoA may then act as a substrate in the citric acid cycle, producing 2 molecules of CO2 for every molecule of acetyl-CoA.  LKR and SDH are critical in L-lysine catabolism and have been described in detail. LKR was first discovered in rat liver and is only found in the mitochondrial matrix of cells (Manangi et al, 2005). During L-lysine catabolism, LKR requires NADPH as a cofactor and has a pH optimum of 7.8 (Fjellestedt and Robinson, 1975b). SDH has a pH optimum between 8.5 and 8.9 and requires NAD reduction as a cofactor for L-lysine catabolism (Fjellestedt and Robinson 1975b). Together, these two enzymes initiate the breakdown of L-lysine.  There remains controversy regarding sites of lysine metabolism in the human body. There is some evidence in animal models that many tissues may have the capacity to catabolize lysine yet there have been few in human studies providing similar results. Lysine metabolism has 20 !been described in piglet enterocytes (Tomé and Bos 2007; van Goudoever et al, 2000) during excessive protein intake, but similar results were not found in human infant enterocytes (Tomé and Bos, 2007; van der Schoor et al, 2004). A study by Manangi et al (2005) in chickens studied a much broader range of tissues and found evidence of α-ketoglutarate reductase in the liver, kidney, lung, skin, intestine, heart, pancreas, brain, spleen, and breast muscle. LKR activity was discovered in chicken skeletal muscle, heart liver, lung, and intestine; however the lung and small intestine showed less complete oxidation of lysine than the other tissues. Manangi et al (2005) suggests that the first steps of lysine oxidation could occur in some tissues and the products be shuttled to the liver to complete the catabolic pathway. Sacksteder (2000) also saw expression of α-aminoadipic semialdehyde synthase (AASS) in various tissues; mRNA was highest in the liver with some in the heart and kidney, while detectable in all other tissues examined (ovary, small intestine, colon, peripheral blood leukocyte, pancreas, spleen, skeletal muscle, placenta, lung, brain, testis, prostate and thymus). Although the results are inconclusive, it is clear that there is some extraheptatic involvement in the oxidation of lysine (Manangi et al, 2005; Pink et al, 2011). 2.2.7 Role of Lysine in Carnitine Synthesis Carnitine, or γ-N-trimethyl-β-hydroxybutyrate, is a biocatalyst that exists in all tissues in the human body and is essential for the initiation of long-chain fatty acid oxidation via β-oxidation in the mitochondrion. The preferential energy source for heart and skeletal muscle is β-oxidation; therefore carnitine is crucial in providing adequate energy to these structures. (Hutzler and Dancis, 1975; Borum and Broquist, 1976). Carnitine also plays a role in the oxidation of pyruvate and the branched chain amino acids (leucine, isoleucine and valine). Carnitine is synthesized de novo in the human body from precursors L-lysine (peptide linked) and S-21 !Adenosyl-L-Methionine (Figure 7), thereby classifying it as a nonessential nutrient. However, the majority of carnitine is obtained through the diet and can be found in significant quantities in animal derived foods. As a majority of the world’s population is subsided on a cereal-based diet that is low in the precursor, lysine, and the peptide itself, deficiency of carnitine may be a serious concern.   Figure 7. Carnitine Biosynthesis in Mammals. Corresponding Enzymes: 1. S-adenosylmethionine:L-lysine methyltransferase; 2. Protein hydrolysis; 3. Ε –N-trimethyllysine hydroxylase; 4. Β-hydroxy- ε –N-trimethyllysine aldolase, 5. Γ-trimethylaminobutyraldehyde dehydrogenase; 6. Γ –butyrobetaine hydroxylase. From Carter et al, 1995. 22 ! Synthesis of carnitine requires lysine, as a pre-cursor; consumption and utilization of lysine therefore has an impact on the production of carnitine (Tanner et al, 2008). Rats fed a diet deficient in both carnitine and lysine were smaller and had lower concentrations of carnitine in heart and skeletal muscle while carnitine concentrations in the liver were elevated (Borum and Broquist, 1976). Another characterization of a lysine and carnitine deficient diet in rats is the development of hepatic steatosis, or fatty liver (Tanphaichitr et al, 1976). This condition is typically caused by poor nutritive status, drug abuse, or metabolic disorders (Spaniol et al, 2003). This disease is also seen in patients with kwashiorkor or protein-energy malnutrition, an extreme state of malnutrition that is associated with low plasma triglycerides (Tanphaichitr et al, 1976, Spaniol et al, 2003). This development is due to impaired lipoprotein complex formation, resulting in decreased triglyceride secretion from the liver into the plasma, increased plasma VLDL, increased plasma long-chain acyl-CoA concentrations and impairment of hepatic fatty acid oxidation (Tanphaichitr et al, 1976, Spaniol et al, 2003). Thus, if carnitine biosynthesis is impaired, lipids are retained in the liver causing it to become fatty and develop steatosis (Spaniol et al, 2003). It appears that low carnitine concentrations in kwashiorkor are related to deficient protein intakes, which includes the precursor amino acids protein-lysine and S-adenosyl-methionine (Spaniol et al, 2003). The outcome of hepatic steatosis is varied, ranging from hepatomegaly to hepatocarcinoma.   During pregnancy, plasma carnitine concentration profiles change significantly. At parturition, plasma carnitine concentrations have been shown to decline to approximately half of those in nonpregnant women (Cederblad et al, 1986; Cederblad et al, 1985; Bargen-Lockner et al, 1981; Genger et al, 1988; Marzo et al, 1994; Schoderbeck et al, 1995; Keller et al, 2009). The process behind this decline is not known, though some have suggested that low plasma carnitine 23 !concentrations noted during pregnancy are a result of diminished rates of carnitine synthesis due to reduced precursor activity or concentration (Keller et al, 2009; Ringseis et al, 2010). Precursors trimethyllysine and γ-butyrobetaine (Figure 7) are significantly correlated with concentrations of plasma carnitine, suggesting decreased carnitine biosynthesis due to a lack of availability of these substrates (Ringseis et al, 2010). Additional studies have shown decreased plasma concentrations of trimethyllysine and γ-butyrobetaine during pregnancy (Keller et al, 2009; Hirche et al, 2009), perhaps indicating altered plasma lysine concentrations during pregnancy influence maternal plasma carnitine concentrations. Alternatively, as β-oxidation increases during pregnancy, it is possible that carnitine may be diverted from the plasma to be used in initiation of fatty acid metabolism in the liver (Strauss and Barbieri, 2009).  Evidence suggests that carnitine is transported across the placenta to supply the foetus with carnitine (Novak et al, 1981; Bargen-Lockner et al, 1981), as the foetus expresses limited-at-best capability of endogenous carnitine synthesis. Studies in neonates indicate that carnitine is essential for foetal growth and maturation, as low plasma free carnitine concentration in the neonate is associated with intrauterine growth restriction (Xi et al, 2008). In neonates, cord blood concentrations of plasma total carnitine, free carnitine and total acylcarnitines are associated with low birth weight (Meyburg et al, 2001). This relationship is not clear, however, as studies vary in the direction of this relationship (Shenai et al, 1983; Meyburg et al, 2001). Finally, foetal development of metabolic pathways such as hepatic ketone synthesis allow glucose and lactate to be used for synthetic functions, an essential adaptation for adequate neonatal energy metabolism (Arenas et al, 1998).  The profile and role of carnitine during pregnancy and the perinatal period is continuing to be investigated. What is clear, though, is the importance it has in establishing adequate energy 24 !supplementation to both maternal and foetal tissues. Dietary deficiency of carnitine or its precursors may have deleterious effects on foetal outcomes, and it is therefore imperative that mothers consume adequate intakes of quality protein during gestation.  2.2.8 Roles of Lysine in Human Health As an essential amino acid, lysine must be obtained from an exogenous source to allow for normal body function. Lysine has multiple functions in human health, including the aforementioned role in the formation of carnitine. In addition, lysine is critical to ensure regular growth and formation of tissues, immune function, calcium absorption, and stress responses.  Populations living in developing countries are especially susceptible to deficient lysine intakes as dietary protein is primarily plant-based, a diet in which lysine is the first limiting amino acid for protein synthesis. Deficient lysine intakes are associated with numerous negative health outcomes, including decreased immune function, increased diarrheal morbidity, and increased anxiety (Ghosh et al, 2010; Smriga et al, 2004). Stress and anxiety are complex responses to a variety of stimuli, resulting in 24ehavioural, cardiovascular and gastrointestinal reactions. Serotonin (5-hydroxytryptamine, 5-HT) plays important roles in multiple emotional responses including the stress response. Receptors are located throughout the central and enteric nervous systems, and antagonism or agonism of these receptors drives the response. Studies indicate that lysine is a partial-antagonist at serotonin receptor 4 (5-HT4), a receptor located on enteric cholinergic neurons, enterochromaffin cells, enterocytes and smooth muscle cells. Given that 90% of 5-HT is stored in the gastrointestinal tract, in enterochromaffin cells, regulation of gut motility via 5-HT signalling is logical. Agonism of this receptor increases intestinal contraction and motility, 25 !causing increased transport of contents (Smriga, 2003). Indeed, for patients experiencing constipation, bloating or abdominal pain as a result of gastrointestinal morbidity, evidence has indicated that ingestion of 5-HT4 receptor agonists may be efficacious treatment (Spiller et al, 2007).  However, in healthy participants, excessive stimulation at the 5-HT4 receptor may lead to a detrimental increase in gut contraction and motility, thereby increasing diarrheal morbidity (Smriga and Torii, 2003). Animal studies have indicated lysine possesses partially antagonistic action at this receptor, thus blunting excessive peristalsis (Smriga and Torii, 2003). In rodents, deficient lysine consumption was associated with increased fecal excretion mediated by 5-HT4 (Sanger, 2000; Smriga and Torii, 2003). In guinea pigs, lysine was found to act as a partial antagonist at the level of the ileum (Smriga and Torii, 2003), and Goldhill and colleagues (1998) found involvement of the 5-HT4 receptor in stimulation of intestinal fluid transport during stress exposure.  Further studies have indicated lysine exhibits effects by acting at 5-HT4 receptor sites in the central nervous system, specifically in the limbic system, amygdala and corpus striatum. At this level, 5-HT4 receptors are involved in behavioural stress responses. In rats, plasma lysine concentrations below normal interfered with the circadian release of serotonin (5-HT) while increasing stress measures (Smriga et al, 2002). In both Syrian (Smriga et al, 2004) and Eastern European (Jezova et al, 2005) sample populations, increased lysine consumption lowered chronic anxiety and improved stress outcomes relative to controls. In the rural Syrian population, the t-STAI chronic anxiety questionnaire indicated a reduction in anxiety among males who indicated a high level of baseline anxiety upon lysine fortification (Smriga et al, 2004). Additionally, short-term measures of anxiety and sympathetic arousal – plasma cortisol and skin 26 !conductance in males and children, plasma cortisol concentration in females – were also noted upon lysine fortification in wheat-flour (Smriga et al, 2004).   In addition to the effects of lysine via 5-HT4 antagonism in the gastrointestinal system, lysine modulation of intestinal opioid peptide transport systems has been noted in intestinal pathologies (Ghosh et al, 2010; Miyauchi et al, 2007). Ghosh et al (2010) determined that lysine supplementation in peri-urban households in Accra, Ghana reduces the diarrheal morbidity in children and respiratory morbidity in men. The group proposed that these changes in the gastrointestinal tract of those that are lysine deficient are occurring independently from malnutrition or poor hygiene. Instead, this observed reduction in diarrheal prevalence and morbidity of those consuming lysine-fortified diets was suggested to be a result of enhanced intestinal repair and effects on opioid peptide transport systems, which are involved in fluid secretion in the intestines (Ghosh et al, 2010). These findings suggest that in both animal and human populations, lysine supplementation significantly decreases diarrheal morbidity via action on intestinal receptors 5-HT4 and opioid peptide transporters.  Finally, lysine is involved in proper immune function and maintenance. A randomized, controlled trial in a low-income Syrian community compared an experimental diet containing lysine-fortified flour was tested against a control diet consisting of approximately 57% protein from cereal source and reported changes in immune factors including immunoglobulins, complement proteins and lymphocytes (Ghosh et al, 2008). The lysine-fortified population showed no change in C3 complement concentrations, whereas controls showed immunological compromise, specifically a decrease in C3 complement protein concentrations, indicative of illness (Ghosh et al, 2008). Additional studies have described immune modulations while consuming a lysine fortified diet: a Chinese population showed an increase in total CD3 27 !lymphocte levels in women and children; an increase in complement and IgG in men; an increase in IgA in women; and IgG, IgA, IgM and complement C3 increases in children (Zhao et al, 2004). Of a sample population in Pakistan, lysine-fortified diets saw improvement in immune indicators including T lymphocytes with CD4 and CD8 receptors and in complement C3 (Hussain et al, 2004). While it appears to be a complex relationship, it is clear that inadequacies in lysine intake have detrimental effects on the immune system.  2.3 Current Recommendations for Lysine Intakes Globally, the United Nations’ joint consultation of the World Health Organization/Food and Agriculture Organization/United Nations University (WHO/FAO/UNU) set nutritional recommendations. North American recommendations are defined by the Institute of Medicine’s Dietary Reference Intakes for macronutrients (DRI). The DRI has set an estimated average requirement (EAR) and a recommended daily allowance (RDA) for lysine intakes in pregnant women, summarized in Table 5. The EAR is defined as “median daily intake value that is estimated to meet the requirement of half the healthy individuals in a life-stage and gender group.” (DRI, 2005). This may be problematic, as 50% of the population will require greater intakes than the EAR recommends. Alternatively, the RDA “average daily dietary intake level that is sufficient to meet the nutrient requirement of nearly all (97 to 98 percent) healthy individuals in a particular life-stage and gender group.” (DRI, 2005). It is defined by the upper 95% CI of the EAR. In addition, these recommendations incorporate findings from a study completed in adolescent pregnancies (King, 1973). These requirements do not reflect a healthy adult population, as adolescent pregnancy is a metabolically disparate state, and the assumption that requirements are the same in mothers who are growing themselves is imprudent.   28 !As there is currently no known data defining a lysine requirement in human pregnancy, current lysine recommendations are a calculated estimate based on non-pregnant adult data. The DRI recommends 0.88 g/kg/d protein during pregnancy, which is 1.33 times higher than non-pregnant adult recommendations of 0.6 g/kg/d. This factorial estimates the requirement for growth using data from nitrogen balance studies in non-pregnant adults, pregnant adolescents, and from potassium metabolism studies (DRI, 2005). This same factor of 1.33 is applied to non-pregnant adult data to define the EAR for lysine during pregnancy (DRI, 2005).  Table 5. Lysine Requirements in non-pregnant adults and pregnancy. DRI, 2005.  EAR (mg/kg/d) RDA (mg/kg/d) Non-pregnant adult (age 19+) 31 38 Pregnant Adult 41 51  2.4 Protein and Amino Acid Requirement Research Techniques Current methods in determining protein and amino acid requirements range in adaptation periods, study duration, and invasiveness. There is continuing controversy regarding the optimal technique, as each method possesses advantages and disadvantages (Pencharz and Ball, 2003). The four primary techniques include the Nitrogen Balance model, on which current recommendations are based; the direct amino acid model (DAAO), indicator amino acid balance model (IAAB), and the indicator amino acid oxidation technique (IAAO). Although the Nitrogen Balance model is the current basis for DRI recommendations (DRI, 2005), it holds several limitations (Fuller and Garlick, 1994; Young, 1986; DRI, 2005) and the Institute of Medicine has recommended that new methods be investigated to determine amino acid requirements (DRI, 29 !2005). In the latest DRI, the IAAO and 24h-IAAO/IAAB techniques were accepted as appropriate techniques for the study of amino acid requirements (DRI, 2005). 2.4.1 Nitrogen Balance Technique The Nitrogen Balance technique was the first method designed to measure protein and amino acid requirements in human subjects (Rose, 1947) and currently defines protein and amino acid requirements (DRI, 2005). Protein is the major nitrogen-containing substance in the body, therefore allowing protein and amino acid turnover to be quantified by measuring nitrogen content and flux in the body (DRI, 2005). Subjects consume a diet containing test protein or amino acid intakes, and all forms of N excretion (urine, feces, sweat, and other miscellaneous losses) are collected and quantified (DRI, 2005). As described in section 2.3, current recommendations for lysine in pregnant women are based on lysine recommendations are based upon such studies (Rand and Young, 1999) as well as more modern methods (Meredith et al, 1986; Zello et al, 1993; Kurpad et al, 2001; Kriengsinyos et al, 2002; DRI, 2005).  There are several caveats to the N balance technique that suggest this method may not be appropriate for determining protein and amino acid requirements (Hegsted, 1976; Millward, 2001; Pencharz and Ball, 2003; DRI, 2005). Primarily, N intake tends to be overestimated while excretion underestimated; creating a falsely positive N balance that underestimates the requirement (Hegsted, 1976; DRI, 2005; FAO, 2007).  Nitrogen equilibrium may occur at various states of protein dynamics, thus it may not indicate adequacy of intake. N balance utilizes a linear regression analysis model, where the requirement is the point where the data intersects with the zero balance line (Figure 8). However, the physiologic response reaction between N intake and balance is not linear; the efficiency of protein utilization decreases as zero 30 !balance is approached (Rand et al, 2003; Young et al, 1973). The data therefore is better suited to a curvilinear model (DRI, 2005), which has been applied to many previous studies (Rand et al, 2003). In addition, as balance is a small value obtained by subtracting a large value of all N excreted from a large value of all N intakes, considerable potential for error may be a relevant issue (Forbes, 1973; Wallace, 1959; Humayun et al, 2007). Finally, this technique requires an adaptation period of at least 5-10 days to allow body urea pool to adapt to the test amino acid intake (FAO, 1985; Rand et al, 1976). Together, these factors indicate N balance models may cause underestimation of protein and amino acid requirements and are inappropriate in vulnerable populations such as pregnant women (Elango et al, 2008b).  2.4.2 Amino Acid Balance and Oxidation Techniques In the last thirty years, isotope based carbon oxidation techniques have been introduced and applied to a variety of populations (Pencharz and Ball, 2003). These techniques are based on the concept that amino acids cannot be stored effectively, and excess of what can be synthesized into new protein is preferentially oxidized. These methods include direct amino acid oxidation (DAAO) measurements, measurement of the oxidation of an indicator amino acid (IAAO), and measurements of daily amino acid balances (IAAB and DAAB).  These techniques are carried out in the sedentary fasted and fed state. Each of these methods has limitations; for example, in the DAAO technique, the tracer cannot participate in any reactions other than protein synthesis or oxidation, thus excluding the amino acids methionine and threonine (Brunton et al, 1998). In addition,the DAAO technique requires infusion of high concentrations of  the labelled amino acid to overcome natural concentrations of 13C, which is problematic when testing low dietary concentrations of the amino acid. To manage this issue, the tracer could only be infused for a few hours of the study (Brunton et al, 1998). As for the balance techniques, the IAAB and DAAB 31 !studies are completed over 24 hours in both the fasted and fed states and require adaptation to the test amino acid intake for 5-7 days. Therefore, these techniques are harder to conduct in vulnerable populations (Kurpad and Thomas, 2011).    Figure 8. Representation of Human Response to Increasing Amino Acid/Protein Intake Using Direct Amino Acid Oxidation, Indicator Amino Acid Oxidation, and Nitrogen Balance Requirement Techniques. Adapted from Pencharz and Ball, 2003.      32 !2.4.3 Indicator Amino Acid Oxidation Technique The indicator amino acid oxidation (IAAO) technique was first described 1983 to determine histidine requirements in pigs (Kim et al, 1983) and was later adapted for application in human subjects (Zello et al, 1993). Since then, the IAAO technique has been adapted to allow the study of vulnerable populations, namely children (Mager et al, 2003; Turner et al, 2006; Elango et al, 2007; Pillai et al, 2010) and disease states (Bross et al, 2000; Riazi et al, 2004), as it is non-invasive and requires a relatively short adaptation period (Brunton et al, 1998). Tracer amino acid is provided in oral doses (Kriengsinyos et al, 2002) and breath and blood/urine (Bross et al, 1998) samples are taken to determine 13CO2 and tracer enrichment, respectively.    The IAAO technique is based on essential amino acid (EAA) characteristics; specifically, the principle that when one essential amino acid is deficient for protein synthesis, all other EAAs will be in present in relative excess. As amino acids cannot be stored in substantial amounts (Holt et al, 1962), they must be partitioned between protein synthesis and oxidation for excretion (Brunton et al, 1998; Pencharz and Ball, 2003). Thus, if an amino acid is limiting for protein synthesis, all others must be oxidized as they cannot be stored or undergo protein synthesis (Zello et al, 1995). Simultaneously, plasma urea is high at low amino acid intakes, indicating amino acid catabolism (Pencharz and Ball, 2003; Figure 8)As limiting amino acid intake increases, oxidation of all other amino acids decreases reflecting their incorporation into protein (Gahl et al, 1997). Eventually an inflection point is reached; a change in the linearity of the data from a negative slope to a plateau (Figure 9). At this plateau, the once limiting amino acid is no longer in shortage and its concentration will not alter oxidation in the other amino acids (Duncan et al, 1996; Elango et al, 2008a,b). This inflection point, termed the breakpoint, represents the 33 !estimated average requirement (EAR) or mean requirement for the test amino acid (Pencharz and Ball, 2003; Elango et al, 2008a,b).   Figure 9. Example of Breakpoint Analysis Using the IAAO Technique. Elango et al, 2009. The indicator amino acid follows the same pattern: as more test amino acid is added to the diet, the rate of indicator amino acid oxidation levels will decrease. The indicator amino acid is chosen based on the presence of a carboxyl group that is irreversibly removed and excreted as CO2 in early catabolic stages.  Of the essential amino acids, only phenylalanine, lysine and the branched chain amino acids (leucine, isoleucine and valine) irreversibly lose a carboxyl carbon during oxidation (Figure 10) (Zello et al, 1990a). L-[1-13C]-phenylalanine is frequently used as the indicator amino acid as the tracer at carbon 1 is irreversibly lost and will not participate in further reactions. Thus, the tracer will be exhaled as measurable 13CO2. This characteristic allows for the quantification of the rate of amino acid oxidation and utilization in a non-invasive manner.  To ensure that phenylalanine will not participate in other reactions than oxidation to 13CO2, excess of its derivative tyrosine is provided. In the case of limiting tyrosine, phenylalanine may be modified to produce this nonessential amino acid (Figure 10). 34 ! Figure 10. Schematic of Phenylalanine Metabolism and L-[1-13C]-Phenylalanine Tracer Oxidation to 13CO2. 2.5 Current Research Few protein and amino acid requirement studies have been conducted in women using carbon oxidation techniques. Kriengsinyos et al (2004) studied lysine requirements in women during the luteal and follicular phases of the menstrual cycle and were the first to do so – the only similar study was conducted to determine tryptophan requirements during the follicular phase of the menstrual cycle. Of the five studies conducted prior to Kriengsinyos et al (2004) that looked at essential amino acid requirements in women, three had not controlled for the phases of the menstrual cycle. The two that did control for menstruation were conducted 7-10 days after the onset of menses. This, as Kriengsinyos et al (2004) discovered, was a critical flaw; there are changes that occur during the menstrual cycle that alter the requirements of lysine. Given the metabolic changes associated with the menstrual cycle, namely increased basal metabolic rate, increased 24 hour energy expenditure, higher nitrogen excretion, and lower plasma amino acid concentrations in the luteal vs. the follicular phase (Kriengsinyos et al, 2004), it is reasonable to question if amino acid requirements are altered between menstrual phases.  Furthermore, progesterone and estradiol have been shown to have an impact on amino acid oxidation and turnover. Both serum progesterone and estradiol increase during the menstrual cycle from the follicular to the luteal phase, by 17 fold and 3 fold, respectively (Kriengsinyos et 35 !al, 2004). These fluctuations in sex hormones seem to influence the rate of tracer oxidation, as a positive correlation was seen between the concentration of sex hormone and F13CO3 in the luteal phase. The concept of sex hormone influence on metabolism certainly is not unprecedented; Toth et al (2000) observed a positive correlation between serum estradiol concentrations and leucine oxidation and turnover. Kriengsinyos and colleagues (2004) found that progesterone has a great impact on amino acid catabolism in the luteal phase, similar to findings in healthy males upon injection of progesterone resulting in lower plasma amino acid concentrations (Landau and Lugibihl, 1967). This could potentially be a similar trend in pregnancy, as progesterone concentrations rise exponentially during healthy pregnancy.   The current calculated DRI recommendations might not be sufficient over the course of pregnancy. Lysine requirements have been shown to fluctuate even between phases of the menstrual cycle, with 35.0 and 37.7 mg/kg/day in the follicular and luteal phases, respectively (Kriengsinyos et al, 2004). Given this significant difference between phases of the menstrual cycle, it seems likely there is a difference between trimesters during gestation. A recent study in sows using the IAAO method described a threonine requirement that was 2 times higher in late gestation than in early gestation (12.3 g/d vs. 5.0 g/d) (Levesque et al, 2010).  Furthermore, an IAAO study investigating lysine requirements in sows found an estimated average requirement of 10.1 g/kg/d in early gestation vs. 16.5 g/kg/d in late gestation, suggesting that the trend of increasing requirements throughout gestation applies to lysine requirements as well (Samuel et al, 2012).    36 !2.6 Rationale There is currently no data to provide a dietary intake recommendation for lysine in pregnant women. Current recommendations are produced using a factorial method based on normal adult requirements and may not be adequate. Furthermore, the recommendations remain constant throughout pregnancy (DRI, 2005), and these static recommendations do not reflect the dynamic adaptations that take place during pregnancy and may not be adequate in later stages of gestation. Recent animal studies using the IAAO method have revealed that lysine requirements increase by 60% during later stages of gestation, compared to early stages (Samuel et al, 2012).  2.7 Hypothesis Current recommendations for lysine intake in pregnant women are underestimated. We also hypothesize that the lysine requirement will be greater during later stages of pregnancy, compared to the early stages of pregnancy.  2.8 Objectives The objective of the current study is to determine the lysine requirements using the indicator amino acid oxidation (IAAO) method in healthy pregnant women. The secondary objective is to compare lysine requirements during early (15-18 weeks since last menstrual period) and late (33-36 weeks since last menstrual period) stages of pregnancy.        37 !Chapter 3 – Methods 3.1 Subjects The initial proposal was to study 20 healthy subjects during both early and late gestation at the Clinical Research and Evaluation Unit at BC Children’s Hospital and BC Women’s Hospital and Health Centre in Vancouver. These 20 participants were expected to participate in up to four studies each: two in early and two in late gestation; thus, the total number of studies were, n = 80. The purpose and potential risks of the study were clearly stated to the subjects prior to commencement of the study during a pre-study day. Informational posters and brochures outlining study features and supplying contact information were distributed at BC Women’s Hospital and Health Centre and BC Children’s Hospital, local community centres, schools, stores (Coffee shops, book stores, supermarkets) and at the University of British Columbia (staff) to recruit study participants (Appendix C, D). No identifying information was kept on record; however a master list of participants and their assigned alphanumeric code (Appendix E) was kept in a locked cabinet in a secure area at the Child and Family Research Institute. Remuneration of $100 per study day was offered to participants. 3.1.1 Inclusion Characteristics " Healthy women 19 – 40 years of age pregnant with a single child It is recognized that maternal age impacts obstetric outcome, particularly after age 35; however, greater numbers of women are having children later in life. Over the past 10 years, fertility rates have been increasing in Canada – a trend primarily attributed to increased fertility among older Canadian women. In 2004, 35% of first births were to women over age 30 (Statistics Canada, 2007). In the last two decades, rates of first births in women over age 35 have 38 !nearly tripled from 4% in 1987 to 11% in 2005 (Statistics Canada, 2007). This allowance of women aged 35 – 40 expanded our potential subject pool, and precaution was taken to verify maternal and foetal health prior to study day. We monitored fasted blood glucose as a preliminary screening tool for gestational diabetes prior to participation, and subjects experiencing abnormal pregnancies as verified by medical history screening were not included in the study.  3.1.2 Exclusion Characteristics " Subjects under age 19  " Subjects above age 40  " Women who are pregnant with more than one child " Subjects who have a history of cardiovascular disease, metabolic disorder, endocrine disorder or recent weight loss " Less than 18 months between current pregnancy and last pregnancy  " Substance dependence (e.g. alcohol, cigarettes, illicit drugs) " Allergic to eggs or egg protein  " Severe nausea and/or vomiting throughout pregnancy  " History of spontaneous abortion, pre-term birth, preeclampsia/eclampsia, gestational diabetes, pregnancy-related anaemia, or pregnancy-related jaundice " Subjects who are underweight (<18.5 kg/m2), overweight (>25 kg/m2) or obese (>30 kg/m2) Populations with unique requirements were excluded from participation. In subjects under age 19, nutritional recommendations must be different as teenage mothers have higher 39 !requirements because they themselves are still growing (DRI, 2005). Multiple pregnancies were ineligible as these pregnancies may also require unique nutrient requirements (FAO, 2005). The partial exception was underweight, overweight or obese participants.  3.2 Experimental Design   Subjects participated in up to six separate study days. Subjects were studied in early gestation (12-19 weeks last menstrual period) or  late gestation, (33-39 weeks last menstrual period), and were permitted to participate in both (although this occurred only with one participant).. Test lysine intakes ranged from deficient to excess; from 6 mg/kg/d to 86 mg/kg/d lysine (6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86 mg/kg/d lysine) (Figure 11). To ensure participants consumed meals that spanned from deficient lysine intake to excess, a broad range of intakes were chosen with 51 mg/kg/d set as the median, as this is the current DRI (2005) recommended daily allowance for pregnant women. We assumed that should maternal weight and lysine requirement occur during trimester one, our breakpoint during early gestation would be similar to a recent IAAO-Lysine requirement study in women. Kriengsinyos et al (2004) estimated lysine requirements of 35.0 mg/kg/d and 37.7 mg/kg/d in luteal and follicular phases, respectively. Additionally, Samuel et al (2012) recently described an increase in estimated average requirement of lysine in sows of 60% between early and late gestation. We incorporated this finding into the range of test intakes, allowing a range that encompassed 37.7 mg/kg/d + 60% increase in EAR, which will lead to an EAR of ~58 mg/kg/d. The range of lysine intakes included values significantly above and below these initial, hypothesized requirements.  40 ! Figure 11. Experimental Design.   Meals were consumed hourly to maintain metabolic steady state in the fed condition (Elango et al, 2008b). The diet consisted of a flavoured liquid formula (protein-free powder, Product 80056, Mead Johnson, Evansville, IN; Tang, Don Mills, Canada; Kool-Aid, Don Mills, Canada; corn oil; and amino acid mixture) providing protein at 1.5 g/kg/d and protein-free wheat starch cookies, which constituted the majority of the caloric content (Zello et al, 1990b). Nitrogen was derived from the crystalline amino acid mixture, which was based on the amino acid composition of egg protein (Table 6). Each subject was assigned a specific lysine intake as part of this standard L-amino acid mixture. This standard amino acid mixture represented 77% of the amino acid requirements, while the remaining 23% was provided as phenylalanine, tyrosine, 41 !lysine (test amino acid) and alanine (Pillai et al, 2010). To maintain nitrogen intake across lysine intakes, L-alanine concentrations were modified (Pillai et al, 2010); L-alanine is an indispensable amino acid and is not involved in lysine metabolism to any significant extent. The indicator amino acid phenylalanine was provided at 25 mg/kg, and its derivative, tyrosine at 61 mg/kg to ensure phenylalanine oxidation was channelled towards oxidation or protein synthesis.      The macronutrient composition of the diet was approximately 53% carbohydrate, 37% fat, and 10 % protein, expressed as a percentage of dietary energy (Pillai et al, 2010). Energy intake was based on REE measured during the pre-study day multiplied by a factor of 1.7. This method for determining energy requirements was appropriate during short-term amino acid oxidation studies as it maintained subjects’ weight (Bell et al, 1985; Duncan et al 1996; Zello et al, 1993). Experimental diets were prepared and weighed (Mettler Scale) in the Child and Family Research Institute at BC Children’s Hospital and BC Women’s Hospital and Health Centre. Subjects did not consume any other foods during the study day with the exception of water.         42 !Table 6. Amino Acid Composition of Egg Protein used on Study Day (not including L-Lysine, L-Alanine, and L-Phenylalanine).      Amino Acid Mixture  mg/g L-Arginine L-Asparagine L-Aspartic acid L-Cysteine L-Glutamine L-Glutamic acid Glycine L-Histidine L-Isoleucine L-Leucine L-Methionine L-Proline L-Serine L-Threonine L-Tryptophan L-Valine Total 97.87 43.33 43.33 28.80 73.79 73.79 43.33 29.58 81.87 108.51 38.62 54.64 109.30 61.36 20.33 91.54 1000 43 ! 3.3 Pre-study and Study Day Protocol 3.3.1 Pre-Study Day  Prior to pre-study test protocol, subjects had the opportunity to ask questions and to give written informed consent (Appendix A). Subjects were then weighed to the nearest 0.1 kg using a digital scale and height was measured to the nearest 0.1 cm using a stadiometer. Following a 12-hour overnight fast, blood glucose was measured using a finger prick blood glucose meter (OneTouch Ultra 2, LifeScan Canada Ltd. Burnaby BC) and single-use lancets (Single-Let Disposable Lancet 23G, Bayer Inc. Toronto ON). Fasted blood glucose concentrations should not be ≥5.3 mmol/L (Canadian Diabetes Association Clinical Practice Guidelines Expert Committee, 2013) as this is indicative of gestational diabetes. If a subject exhibited fasted blood glucose test be ≥5.3 mmol/L, a second test was completed to confirm these results. Participants with elevated fasted blood glucose were not eligible to participate in the study. We recommended that subjects follow up with their primary health care provider (Appendix G).   Energy requirements were calculated from resting energy expenditure (REE, kcal/d), quantified in the fasted state using continuous, open-circuit indirect calorimetry (CareFusion Vmax Encore, VIASYS) with a ventilated hood system calibrated prior to use. Fat free mass (FFM) is the strongest predictor of total energy expenditure (TEE) and basal metabolic rate (BMR) (Butte, 1999), thus body composition was determined using bioelectrical impedance analysis (Quantum IV RJL systems) and skinfold thickness measurements (Harpenden Skinfold calliper. Baty international England). BIA calculated resistance, reactance and impedance using electrodes placed at the wrist and ankle. FFM was calculated from these values using the 44 !following equation described earlier in pregnant women (Lukaski and Bolonchuk 1987):  FFM = 0.734 Ht2/R + 0.096 Xc + 0.116 Wt – 4.033  where FFM is fat-free mass (kg), Ht is height (cm), R is resistance (Ω), Xc is reactance (Ω), and Wt is weight (kg). Fat mass was calculated as the difference between body weight and fat-free mass.   Skinfold measurement was taken at tricep, bicep, and subscapular sites using a Harpenden calliper to the nearest 0.1 mm (Durnin and Womersley, 1974). Density was derived from the sum of the three skinfold thickness measurements (Durnin and Womersley, 1974): Density = c – m x log skinfold    where c and m are constants dependent on maternal age. Maternal fat mass was determined using the following equations (Soltani and Fraser, 2000):   FM = W/100 × (497/D – 452) (12 weeks) FM = W/100 × (504/D – 460) (24 weeks) FM = W/100 × (516/D – 473) (36 weeks)      Further questioning was completed at this time (Appendix B) to provide background information regarding nutritional status, supplement intake, physical activity and dietary restrictions. Finally, subjects were provided dietary record sheets (Appendix F) to record daily food intakes over the two days immediately preceding the study day.  3.3.2 Two-Day Dietary Protein Standardization During the two days prior to study, subjects consumed a diet containing 1.5-g/kg/d dietary protein. This diet consisted of everyday foods, however an outline was given to subjects to ensure sufficiency based on foods commonly eaten as described in the subject food records (Pillai et al, 2010). This two-day standardization period to the study day protein level is required 45 !to ensure amino acid kinetics are not altered due to inappropriate protein intake (Thorpe et al, 1999; Moehn et al, 2004). However, standardization to the test amino acid level may not be required (Zello et al, 1990a) as lack of an amino acid storage compartment and short half-life of free amino acid pools allow for an immediate adaptation of metabolism to accommodate to a new intake level of nutrients (Moehn et al, 2004; Holt et al, 1962). Recently, Elango et al, (2009) experimentally determined an adaptation period of 8 hours is adequate to estimate amino acid requirements using the IAAO method and breath and urine sampling in adult humans. F13CO2 and phenylalanine flux did not change compared to adaptation periods of 8 hours, 3 days or 7 days (Elango et al, 2009). Furthermore, it may not be feasible or ethical to adapt pregnant women for longer periods on a test amino acid diet, especially on the deficient intake levels.  3.3.3 Study Day Protocol and Isotope Infusion Studies  Studies were conducted in the Clinical Research and Evaluation Unit at BC Hospital and BC Women’s Hospital in Vancouver, BC in a temperature-controlled environment. Anthropometric measurements (height, weight) were taken at the beginning of each study day to the nearest 0.1 cm and 0.1 kg, respectively. The study day required 8 hours to complete: 6 hours to adapt to the test lysine intake and 2 hours of breath and urine sampling (Elango et al, 2007). Subjects orally consumed eight isocaloric and isonitrogenous hourly meals, each providing 1/12 of the subject’s daily energy and nutrient requirements (Appendix H). Subjects were randomly allocated to one of the 40 lysine intakes on each study day.   NaH13CO3 (99 % atom excess, Cambridge Isotopes Laboratories, Woburn, MA) and L-[1-13C]-phenylalanine (99 % atom excess, Cambridge Isotopes Laboratories, Woburn, MA) were consumed orally. The manufacturer performed optical rotation tests to determine presence of D-lysine isomer as well as bacteriologic and limulus amebocyte lysate testing to ensure food safety. 46 !At CFRI, the isotopes were stored in a dry, sterile condition.  Three baseline measurements of breath and urine were taken before isotope consumption commenced at meal 5. At the time of baseline sampling, there was no isotope present in the meals. Priming doses of both NaH13CO3 (2.07 µmol/kg) and L-[1-13C]-phenylalanine (0.655 µmol/kg) were given at hour 4 (Figure 12; Appendix H; Hoerr et al, 1989; Pillai et al, 2010). After this priming dose, simulated continuous dose L-[1-13C]-phenylalanine was consumed at each subsequent meal at 18.0 µmol/kg/hr (Elango et al, 2007).  Figure 12. Outline of Study Day Protocol.     47 !3.4 Sample Collection       Samples of breath and urine were collected for baseline and isotopic enrichment measurements.  3.4.1 Breath Samples Breath samples were collected using breath bags (Single use collection bags, EasySampler System, QuinTron, Terumo Medical) in disposable Labco Exetainer tubes using a collection mechanism that removes dead space air. Baseline breath samples were collected 45, 30 and 15 minutes prior to isotope infusion protocol (Figure 12). Following tracer administration, breath samples were collected at minute 150, 180, 195, 210, 225 and 240 to ensure satisfactory isotopic steady state is achieved at approximately 2 hours after isotope infusion (Figure 12; Bross et al, 1998). Breath samples were stored at room temperature following collection until analysis by isotope ratio mass spectrometry (IRMS, IsoPrime 100). 3.4.2 Urine Samples Two urine samples were collected 45 and 15 minutes prior to isotope infusion and at minute 150, 180, 210 and 240 following isotope consumption (Figure 13; Kriengsinyos et al, 2002). Urine was collected in a specimen collection container (Specimen Container w/pour spout, 6.5 oz, Medegen) and transferred to sterile cups. 10 mL samples were transferred to 15 mL conical tubes (BD Falcon, Mississauga ON) containing 200 µL 10% HCl to quell bacterial growth. 1 mL of this HCl urine mixture was transferred to microcentrifuge tubes and stored at -80oC until analysis by liquid chromatography mass spectrometry (LC-MS).          48 !3.5 Analytical Procedures       Expired CO2 enrichment was analyzed using a continuous flow isotope ratio mass spectrometer (CF-IRMS IsoPrime100, Cheadle, UK). 13CO2 was quantified in atoms percent excess (APE) over a reference CO2 standard.   Urine samples continue to be analyzed at the Hospital for Sick Children in Toronto, ON. L-[1-13C]-Phenylalanine enrichment in urine samples is analyzed with a triple quadrupole mass analyzer API 4000 (Applied Biosystems/MDS SCIEX, Concord, Canada) coupled to an 1100 HPLC system (Agilent, Mississauga, Canada) (Humayun et al, 2007; Pillai et al, 2010). Urine samples are deproteinized with 200 µL methanol and centrifuged at 7000 x g for 5 min. The supernatant fluid is freeze-dried and then reconstituted in 1 mLwater containing 0.1% formic acid. L-[1-13C]-Phenylalanine enrichment is then analyzed by triple quadrupole mass analyzer API 4000 (Applied Biosystems/MDS SCIEX) operated in positive ionization mode (Turner et al, 2006). Isotopic enrichment is calculated from peak area ratios (M and M+1) at isotopic steady state and baseline plateaus, and is expressed in molecules percent excess (MPE) (Humayun et al, 2007; Pillai et al, 2010; Turner et al, 2006). 3.6 Isotope Kinetics       L-[1-13C]-Phenylalanine flux (Q, µmol.kg-1.h-1) were calculated from the dilution of orally administered L-[1-13C]-phenylalanine into the metabolic pool at steady state using enrichments if L-[1-13C]-phenylalanine in urine (Bross et al, 1998) using the equation: Q = S + O = B + I; 49 !where S is the rate of nonoxidative phenylalanine disposal, O is the rate of phenylalanine oxidation, B is the rate of phenylalanine released from body protein and I is the rate of exogenous phenylalanine intake (phenylalanine infusion). A secondary equation was used: Q = I x (Ei / Eu – 1);  where Q is the flux (in mmol×kg-1×h-1), i is the rate of L- [1-13C]-phenylalanine infused (mmol/kg/h), and Ei and Eu are the isotopic enrichments as mole fractions (molecules percent excess) of the infusate and urinary phenylalanine, respectively, at isotopic plateau (Kriengsinyos et al, 2002; Matthews et al, 1980). F13CO2 is the rate of 13CO2 released by phenylalanine tracer oxidation (µmol 13CO2/kg/h) calculated by the equation: F13CO2= (FCO2)(ECO2)(44.6)(60) / (W)(0.82)(100); where FCO2 is the CO2 production rate (mL/minute), ECO2is 13CO2 enrichment in expired breath at isotopic steady state in APE, W is the subject’s body weight (kg), and the constants 44.6 µM/mL and 60 min/hour convert F13CO2 to µM/h. the factor 100 changes APE to a fraction. Finally, the factor 0.82 accounts for bicarbonate fixation of 13C (Hoerr et al, 1989).   3.7 Statistical Analysis Subject characteristics are expressed as the mean (SD). Analysis of subject characteristics was completed using independent t-tests in SPSS ver. 19; significance was set at p ≤ 0.05.Estimations for the mean requirement for lysine in both early and late gestation were derived from breakpoint analysis of the F13CO2 data by using a bi-phase linear regression crossover model in SAS program software (SAS/STAT Ver 8.2) (Figure 10; Zello et al., 1993). The first regression line has a negative slope, while the second line does not have a significant slope 50 !(Elango et al, 2007). The point where the two regression lines meet is referred to as the breakpoint (requirement). Mixed models regressions are used with estimates for multiple candidate breakpoints, and the model with a minimum residual standard error and greatest coefficient of determination is used to determine the final breakpoint (requirement) (Elango et al, 2011). Standard error (SE) is determined using Fieller’s theorem, which is in turn used to calculate the upper 95% Confidence Interval (CI), which represent the RDA, as breakpoint + t x SE (Elango et al, 2011).              51 !Chapter 4 – Results and Discussion 4.1 Results 4.1.1 Subject Characteristics A total of 13 women were studied in early gestation, completing 27 individual study days (Nsubjects=13, nstudies=27). A total of 19 women were studied in late gestation, completing 36 individual studies (Nsubjects=19, nstudies=36) (Table 7). One participant was studied in both early and late gestation; all participants otherwise were unique between stages. Average gestational age was 16.3 ± 2.1 and 34.9 ± 1.6 weeks in early and late gestation, respectively. Prior to becoming pregnant, all but two women in the early and late gestation designations (Nwomen=32) had a mean BMI that fell into the “normal” range at 23.69 ± 3.84 and 21.47 ± 2.43 kg/m2, respectively. Mean weight gain increased significantly (p<0.001) from early to late gestation; participants gained a mean of 3.43 ± 2.83 kg in early gestation, compared to 12.44 ± 4.16 kg in late gestation. Fat mass determined using skinfolds were determined to be 28.8 ± 4.90% in early gestation and 25.08± 4.82% in late gestation. The higher fat mass % in early gestation was primarily due to two subjects who only participated in the early gestation study and had a BMI above 30%, as described below. Protein standardization goals as indicated by dietary record analysis indicated a mean of 1.44 ± 0.35 and 1.51 ± 0.34 g/kg/day protein consumption in early and late gestation, respectively; difference between gestational age groups was found to be insignificant (p=0.35) (Table 8).  Due to difficulty recruiting participants in early gestation, eligibility criteria was altered to allow two participants who were obese prior to becoming pregnant (BMI 30.1, 30.5). As previously noted, persons outside of the normal BMI range may have slightly altered 52 !requirements, thus care was taken to monitor these participants’ results for any aberrant or unusual findings, and none were observed. Additionally, one participant in early gestation used the anti-nausea drug, Diclectin®. In both gestational age groups, a majority of women self-reported a moderately active lifestyle. In early gestation, 4 were multiparous, 6 were multigravida (two miscarried previously). In late gestation, 8 were multiparous, 5 were multigravida (three miscarried previously).  Table 7. Results from Pre-Study Data in Early (Nsubjects =13) and Late (Nsubjects =19) Subjects. (SPSS Ver. 19, SPSS Inc, Chicago, IL).  Variable Early Gestation (Mean ± SD; 12-19 weeks) Late Gestation (Mean ± SD; 33-39 weeks) t value (df)1 p Age (years) 29.46 (3.23) 30.52 (3.83) -1.323 (79) 0.190 Gestational Age (weeks) 16.3 (2.1) 34.9 (1.6) -32.926 (78) <0.001 Pre-pregnancy BMI (kg/m2) 23.69 (3.84) 21.47 (2.43) 3.998 (76) <0.001 Gestation Weight Gain (kg) 3.43 (2.83) 12.44 (4.16) -10.626 (75) <0.001 Blood Glucose (mmol/L) 4.66 (0.32) 4.50 (0.39) 1.176 (30) 0.249 Fat-Free Mass (Skinfold Calliper) (kg) 47.08 (6.39) 50.44 (7.81) -0.172 (31) 0.865 Fat-Free Mass (BIA) (kg) 37.17 (3.28) 38.25 (3.27) -1.003 (31) 0.324 Fat Mass (Skinfold Calliper) (%) 28.8 (4.90) 25.08 (4.82) 2.09 (31) 0.045 Fat Mass (BIA) (%) 36.79 (6.06) 35.16 (7.02) -0.310 (32) 0.759 Resting Energy Expenditure 1353.43 (235.16) 1483.9 (209.55) -1.648 (32) 0.109 Respiratory Quotient 0.90 (0.05) 0.86 (0.17) 0.674 (31) 0.505 VO2 0.195 (0.036) 0.207 (0.03) -1.011 (31) 0.320 VCO2 0.176 (0.030) 0.225 (0.151) -1.120 (31) 0.271 1Results assume equal variances.  53 !Table 8. Results from Study Day Data in Early (Nsubjects=13, nstudies=27) and Late (Nsubjects =19, nstudies=36) Gestation. (SPSS Ver. 19, SPSS Inc, Chicago, IL). Variable Early Gestation (Mean ± SD; 12-19 weeks) Late Gestation (Mean ± SD; 33-39 weeks) t value (df) p Weight (kg) 67.97 (10.14) 69.12 (8.26) 1.171 (78) 0.245 Blood Glucose (mmol/L) 4.58 (0.27) 4.55 (0.40) 0.459 (78) 0.648 Standardized Dietary Protein Intake (g/kg/d)2 1.44 (0.36) 1.52 (0.33) -0.798 (78) 0.427 Energy Provided (kcal) 2372 (364) 2459 (347) -1.058 (77) 0.293 Respiratory Quotient 0.95 (0.04) 0.93 (0.03) 1.440 (75) 0.154 VO2 (L/min) 0.244 (0.041) 0.255 (0.03) -1.202 (75) 0.233 VCO2 (L/min) 0.232 (0.04) 0.238 (0.03) -0.783 (76) 0.436 1Results assume equal variances.  2Amount of protein consumed by participants in the two days prior to study day as indicated by dietary records.   4.1.2 Lysine Requirement in Early Gestation The results from the early gestation population show a mean lysine requirement in women 12-19 weeks gestation.  Bi-phase linear regression of L-[1-13C]-phenylalanine tracer oxidation (F13CO2, umol/kg/h) determined a mean lysine requirement in this sample (nstudies =27) of 36.6 mg/kg/d (r2 = 0.484, Upper 95% CI 46.2) (Figure 13). L-[1-13C]-phenylalanine oxidation decreased with increasing lysine intakes until a plateau in obligatory oxidation of 0.5 F13CO2, umol/kg/h was reached at an intake of 36.6 mg/kg/d. Urinary sample analysis revealed no significant change in phenylalanine flux across lysine intakes (r2 = 0.091) (Figure 14). The breakpoint analysis involves categorizing L-[1-13C]-phenylalanine tracer oxidation (F13CO2, umol/kg/h) into different cut-off group. The selected model in this study had a cut-off between 44-46 mg in early gestation (intakes below cut-off of 6-44 mg/kg/d, intakes above the 54 !cut-off of 46-86 mg/kg/d) and a cut-off between 58-62 mg/kg/d in late gestation (intakes below cut-off of 6-58 mg/kg/d, intakes above the cut-off of 62-88mg/kg/d). Regression analysis is applied to both groups by considering four different models: un-weighted, one line has a slope; weighted, one line has a slope; un-weighted, both lines have a slope; and weighted, both lines have a slope. The model that defines the breakpoint is selected based on having the highest coefficient of determination (r2), lowest coefficient of variation (CV) and lowest root-mean-squared error (RMSE). From these results, the upper 95%CI, is calculated as the breakpoint + t x SE (Elango et al, 2011). This value represents the recommended dietary allowance for lysine, or the amount that satisfies the requirement of 97-98% of the population.  Figure 13. Estimated average lysine requirement in early gestation (Nsubjects=13, nstudies=27) derived by bi-phase linear regression crossover analysis in SAS (Proc Mixed, Statistical Analysis Systems SAS/STAT version 9.0 SAS Institute, Cary, NC.)     10 20 30 40 50 60 70 80 90 1000.00.10.20.30.40.50.60.70.80.91.01.1Early GestationBreakpoint = 36.6Upper 95%  CI = 46.2r2 = 0.484Lysine Intake (mg/kg/d)F13 CO2 (umol/kg/h)55 !  Figure 14. Variation in urinary Phenylalanine flux across all lysine intakes in early gestation determined using linear regression (r2 = 0.091).   4.1.3 Lysine Requirements in Late Gestation The results from the late gestation population show a mean lysine requirement in women 33-39 weeks gestation.  Bi-phase linear regression of L-[1-13C]-phenylalanine tracer oxidation (F13CO2, umol/kg/h) determined a mean lysine requirement in this sample (Nsubjects =19, nstudies =36) of 50.3 mg/kg/d (r2= 0.664, Upper 95% CI = 60.4) (Figure 15), and ~ 37% higher than requirements in early gestation. L-[1-13C]-phenylalanine oxidation decreased with increasing lysine intakes until a plateau in obligatory oxidation of 0.35 (F13CO2, umol/kg/h) was reached at an intake of 50.3 mg/kg/d. Urinary sample analysis revealed no significant change in phenylalanine flux across lysine intakes (r2 = 0.00028) (Figure 16).!0"10"20"30"40"50"60"70"80"90"0" 20" 40" 60" 80" 100"Phenylalanine Flux (umol/kg/h) Lysine Intake (mg/kg/d) 56 ! Figure 15. Estimated average lysine requirement in early gestation (Nsubjects=19, nstudies=36) derived by bi-phase linear regression crossover analysis in SAS (Proc Mixed, Statistical Analysis Systems SAS/STAT version 9.0 SAS Institute, Cary, NC.)   Figure 16. Variation in urinary Phenylalanine flux across all lysine intakes in late gestation determined using linear regression (r2 = 0.00028).    10 20 30 40 50 60 70 80 90 1000.00.10.20.30.40.50.60.70.80.91.01.1Late GestationLysine Intake (mg/kg/d)F13 CO2 (umol/kg/h) Breakpoint = 50.3Upper 95% CI = 60.4r2 = 0.66457 ! 4.2 Discussion 4.2.1 Subject Characteristics and Pre-Study Data Participants were healthy women in their late twenties-early thirties (Early Gestation: 29.46 ± 3.23y; Late Gestation: 30.52 ± 3.83y), and most were primigravid. Although mean pre-pregnancy BMI was significantly different between early and late gestation (Early Gestation: 23.69 ± 3.84 kg/m2; Late Gestation: 21.47 ± 2.43 kg/m2; p<0.001), both populations fell within the “normal” BMI range prior to becoming pregnant. Gestational weight gain was significantly different between the phases (Early Gestation: 3.43 ± 2.83 kg; Late Gestation: 12.44 ± 4.16 kg; p < 0.001), and the values were in accordance with the Institute of Medicine and Health Canada recommendations (IOM, 2009; Health Canada, 2013). Body composition differed significantly between methods of measurement; this discrepancy may be due to excess water retention during pregnancy, thereby decreasing the accuracy of the BIA technique. Additionally, protein standardization goals did not differ significantly between gestational ages (1.44 ± 0.36 g/kg/d in early gestation vs. 1.52 ± 0.33 g/kg/d in late gestation). While the standard deviation in these data is considerable, the mean intakes fall within current findings of estimated average requirements during pregnancy (Stephens, Elango et al, unpublished data).   4.2.2 Lysine Requirements Lysine requirements during pregnancy were determined to be 36.6 mg/kg/d (upper 95% CI = 46.2 mg/kg/d) and 50.3 mg/kg/d (upper 95% CI = 60.4 mg/kg/d) in early and late gestation, respectively. While the mean lysine requirement in early gestation is 11% lower than current DRI (2005) recommendations of 41 mg/kg/d, the mean lysine requirement in late gestation is 25% higher.  This increase in lysine requirement from early to late gestation is considerably large 58 !(~37% increase with progressive stages of gestation), though not as great as initially anticipated based on studies conducted in sows. Experimental evidence of a 60% increase was observed (Samuel et al, 2012), although these could be due to species differences as all our subjects were pregnant with singletons; sows generally have a litter of 6-10 piglets.  Baseline obligatory L-[1-13C]-phenylalanine oxidation decreased from early (0.5 umol/kg/h) to late gestation (0.3 umol/kg/h), similar to previous findings noted by Samuel et al (2012) in the animal study. The physiological basis for this shift may be due to the movement towards net protein deposition in late stages of gestation. As early as 5 weeks gestation, protein metabolism is altered to accumulate nitrogen to meet the future needs of the mother and foetus (Kalhan, 2000), requiring increased amino acid oxidation. As pregnancy progresses, protein is deposited into foetal and placental tissues as well as maternal structures including the uterus, breast, heart, kidney, liver, blood and extracellular fluid. Due to the fact that free amino acids cannot be effectively stored, metabolism shifts from heightened oxidation to preferential partitioning to synthesis of new protein, resulting in a decreased baseline oxidation rate (Kalhan, 2000). Indeed, progressive decline in amino acid oxidation from mid-to late-gestation is indicative of effective nitrogen and amino acid retention and preferential deposition of tissue protein (Duggleby and Jackson, 2002).  Maternal metabolic modification is necessary to ensure adequate energy and nutrient transfer to the foetus and maintenance of maternal health. Endocrine influence is a major driver of these adaptations; human chorionic gonadotropin (hCG), progestins, sex hormones, and the somatolactogenic hormones human placental lactogen (hPL) and placental growth hormone (PGH) are all believed to have some level of involvement. The first two trimesters of pregnancy are primarily anabolic as pregnancy is an energetically costly process. In early gestation, 59 !progesterone and human placental lactogen (hPL), which also increases maternal insulin production, facilitate the formation of maternal fat stores (Blackburn, 2007; Freemark, 2006; Butte, 2000). In early to mid-gestation, establishment of a diabetogenic state accompanied by a decrease in insulin sensitivity to approximately 45-70% of a non-pregnant state promoties maternal shuttling of glucose, ketones, and amino acids to the fetus for preferential energy (Freemark, 2006). Additionally, placental growth hormone (PGH) has a role in enhancing cell growth and proliferation in the mother by increasing nutrient uptake and preventing protein breakdown while also stimulating gluconeogenesis and lipolysis (Blackburn, 2007).  As pregnancy progresses, maternal insulin resistance accompanied by facilitated lipolysis, driven by increasing hPL concentrations (Figure 17). This allows for free fatty acids to be used as energy for the mother thereby sparing glucose for the fetus (Blackburn, 2007), as 80% of fetal energy is derived from glucose oxidation and the foetal rate of glucose utilization is higher than those of an adult (Blackburn, 2007). The final trimester of pregnancy involves major tissue deposition in the fetus accompanied by a significant rise in progesterone (Figure 17). Progesterone is involved in the characteristic hypoaminoacidemia of pregnancy, as evidenced by increased rates of amino acid catabolism following a rise in progesterone (Kriengsinyos et al, 2004; Landau and Lugibihl, 1961).  As much of early gestation involves expansion of maternal fat stores and establishment of a diabetogenic state, it seems as though lysine demand does not significantly increase until later in gestation. By late gestation, however, lysine demands increase paralleling a rise in absolute tissue deposition in maternal and foetal tissues. The findings from the current thesis aligns with studies in sows, which noted lower lysine requirements in early gestation followed by significant increase in requirements in late gestation (Samuel et al, 2012).  60 !  Figure 17. Plasma progesterone and estradiol profiles during pregnancy. From Guyton, A.C. 1982.   Urine samples were analysed for estimation of tracer kinetics, and analysis of carnitine concentrations and other biochemical/metabolite markers are ongoing. These results added robustness to the current results; however, the findings did not alter the estimated requirement in early or late gestation. This is because the breakpoint (requirement) measures are only based on breath data. Urinary data is primarily used to measure the flux of the L-[1-13C]-phenylalanine into the amino acid pool as lysine intake increases, as changes in flux need to be steady with increasing test intakes. Current non-significant results align with previous work by our group, which showed that flux remains unchanged using the IAAO technique in adults (Humayun et al 2007), children (Elango et al 2007), and during pregnancy (Stephens, Elango et al, unpublished data).  61 !4.2.2 Supportive Evidence These data are the first to directly define a quantitative requirement for lysine during human pregnancy. Lysine holds an important place in the field of protein and amino acid nutrition, due to its first limiting status. Thus lysine requirement studies using the indicator amino acid oxidation technique have been conducted previously in adult men (Zello et al, 1993), women (Kriengsinyos et al, 2004), undernourished men (Kurpad et al, 2001; Kurpad et al, 2003a), healthy children (Elango et al, 2007; Pillai et al, 2010) and post-surgical neonates (Huang et al, 2011; Chapman et al, 2010). Lysine requirements have been defined during two distinct phases of the menstrual cycle (Kriengsinyos et al, 2004) as well as in two gestational stages in pregnant sows (Samuel et al, 2012; Levesque et al, 2011). Additionally, nitrogen balance studies have been completed in gestating sows (Dourmad and Etienne, 2002); however, no such studies have been completed in a human pregnancy, until now. Our requirement estimates are comparable to the earlier studies, and are marginally higher than most studies due to the dynamic nature of pregnancy. The basis for the range of test lysine intakes in the current study was set upon two recent studies investigating lysine requirements in both humans and sows. A study by Kriengsinyos et al (2004) in healthy women found that lysine requirements are not constant over the course of the menstrual cycle as a result of metabolic adaptation during the later luteal phase. We estimated that requirements in early gestation would be similar to those in the luteal phase of the menstrual cycle as many of the significant metabolic adaptations that occur during gestation begin at the onset of the second trimester (Kalhan, 2000). Additionally, a recent study by Samuel and colleagues (2012) used the IAAO technique to investigate lysine requirements in two phases of gestation in sows and indicated a 60% increase in lysine requirement in late as compared to 62 !early gestation. While the current study did not find an increase of a similar magnitude as in the sow study, requirements for lysine were increased in late gestation by ~37 % in healthy pregnant women.   Recently, protein requirements in two phases of gestation were determined using the IAAO method in our lab. These data found that current protein recommendations are significantly underestimated in both early and late gestation (Stephens, Elango et al, unpublished data). In late gestation, the estimated average protein requirements were determined to be 1.52 g//kg/d, reflecting an underestimation of 74% compared to the current dietary recommendations. These results are in agreement with the current findings of an increased requirement for lysine in late gestation.  4.2.3 Limitations A few limitations can be identified.  Primarily, this is a short-term study completed over the course of a day in a healthy population in a developed country; it is recognized that future long-term studies in both developed and developing countries are necessary. All previous IAAO studies are also acute daylong studies, and therefore the current study is comparable on that aspect. Additionally, long-term studies will address the second limitation faced by this study, namely small sample size. This was more of an issue in early gestation, as inter-subject variability was exacerbated an already small pool of participants. Many more complications were noted in early gestation; nausea, vomiting, and availability (many mothers in late gestation were on maternity leave, thereby allowing them to participate in a greater number of studies) are believed to be the central factors influencing this small sample population.  Parity has been noted to alter gestational physiology in humans; for example, maternal 63 !iron stores are highly influenced by parity (Hindmarsh et al, 2000). Whether lysine requirements are altered by parity remains unclear; our study is not designed to analyze such effects. From previous experimental findings in sows, requirements for lysine and threonine decreased in both early and late gestation from 1st to 4th parity (Samuel et al, 2012; Levesque et al, 2011). However, much of the noted decrease was attributed to the age of the sow, rather than parity. It is likely to be the same in human subjects as well, although it remains to be determined. 4.2.4 Global Implications As the majority of the global population subsides on a plant-based diet (Young and Pellett, 1994), the findings from the current study have more significant implications in these areas. Primarily, this means that developing countries are most affected, however, vegan and vegetarian populations in developed countries may also be impacted. A protein assessment survey conducted in 2012 found that lysine was the first limiting amino acid in all (n=116) countries included in the study (Ghosh et al, 2012). When determining protein and amino acid requirements, both the amount of essential amino acid as well as food digestibility must be assessed. Egg protein is considered a high quality food source with high amounts of essential amino acids and highly digestible protein. Alternatively, plant proteins such as white rice are much lower quality, as greater amounts must be consumed in order to reach the same levels of essential amino acids, and the products are less digestible. Though pregnant Indian women are consuming an approximate average of 50 g protein/day (Swaminathan et al, 2012), the risk of lysine deficiency is still apparent if the primary source of protein is plant-based. Table 9 summarizes the lysine content in egg protein vs. rice protein at both the current recommended protein intake (DRI, 2005) and at requirements determined by this group (Stephens, Elango et al, unpublished data).  If we instead use an amino acid profile of white rice, the amount of lysine 64 !consumed in a diet of 50 g protein/day is approximately 18.1 g lysine/day. Table 9. Lysine Content in Egg and Rice Protein at Current Protein Recommendations (DRI, 2005) and Experimentally Derived Requirements (Stephens, Elango et al, unpublished data).   Lysine Content in Egg (mg/kg/d) Lysine Content in Rice (mg/kg/d) Current Protein EAR1 (DRI, 2005) 63 41 Studied Protein EAR (Stephens, Elango et al, unpublished)2    Early Gestation    Late Gestation    50.5 62.9    44.0 54.9 1 50 g/day 2 69.5 g/day (Early Gestation); 86.6 g/day (Late Gestation)  Populations in developing countries are susceptible to lysine deficiency not only due to low lysine concentration in foods, but also as a result of increased risk of parasitic infection, which itself increases lysine requirements (Kurpad et al, 2003a,b). Specifically, lysine requirements in chronically undernourished Indian males were found to be ~50% higher than their North American counterparts, largely due to the presence of intestinal parasites (Kurpad et al, 2003a,b). Following treatment, requirements were found to be comparable.  However, parasitic infection is a common morbidity in developing countries; studies in South Asian and rural Indian communities have found parasitic infection in up to 97.4% of the population (Kang et al, 1998). These infestations may increase lysine demands in an already lysine deficient population.  Significant negative health outcomes are associated with lysine deficiency. As described in section 2.2.8, Deficient lysine intakes are associated with decreased immune function, increased chronic and acute anxiety, and increased diarrhoea (Smriga et al, 2004; Ghosh et al, 2010; Ghosh et al, 2008). As parasitic infection is already rampant in many developing 65 !countries, decreased immune function has the potential to further morbidities faced by these populations (Zhao et al, 2004; Hussain et al, 2004; Ghosh et al, 2008). Lysine is also the precursor to the conditionally essential carnitine, essential for the initiation of fatty acid oxidation via β-oxidation and therefore normal lipid metabolism (Tanner et al, 2008). As pregnancy is a period with greater nutritional demands, dietary inadequacies may be exacerbated and influence both short- and long-term maternal and infant health.  In summary, the current findings from this thesis have the greatest significance in those consuming a plant-based diet comprised of low-quality protein, a diet common in developing countries. There is potential for implications among those in developed nations who have specific dietary restrictions, namely vegetarians or lacto-ovo-vegetarians (vegans). Besides the low amounts of indispensible amino acids in plant-based proteins, these foods tend to have lower digestibility. Moreover, the quantity of food required to meet nutritional requirements could be impractical from a low lysine food, again emphasizing the importance of protein quality when providing recommendations.   66 !Chapter 5 - The use of trans-abdominal ultrasound to determine post-void urine retention during acute dietary studies in two stages of pregnancy 5.1 Background Maternal urine sampling is relatively non-invasive and urine samples have the potential to be tested for unique biomarkers in research studies. In the current thesis, urine samples were collected to measure urinary metabolites and enrichment of 13C-phenylalanine to measure flux. However, bladder function is altered significantly over the course of gestation. Beginning in early gestation, maternal glomerular filtration rate and urine output increases, increasing frequency and volume of urination (FitzGerald and Graziano, 2007). As pregnancy progresses, there may be a risk of incomplete voiding due to partial obstruction by the foetus or morphological changes to the bladder (Dietz and Benness, 2005). Currently, estimated residual urine volume in late gestation is approximately 15mL (Nel et al, 2001). If this is the case, results from urine testing are potentially inaccurate and may lead to inaccurate clinical and research findings. In this on-going study of lysine requirements during pregnancy, urine sampling is essential to our analysis of amino acid flux and measurement of the amino acid pool. If incomplete voiding is indeed common in pregnancy, urine samples taken at baseline and isotopic steady state have the potential to be confounded by urine retained post-void.  5.1.1 Average Post-Void Urine Retention: Current literature describes a decrease in average voided volumes in late gestation. A prospective observational study using translabial ultrasound found residual urine volumes did not change over the period of observation, describing a mean retention of 20 mL in early (6-18 weeks) gestation and 19.4 mL in late (32-39 weeks) gestation (Dietz and Benness, 2005). Weiniger and colleagues (2006) described a mean urine retention volume of 45 mL in 67 !intrapartum women, with a range of 13-250 mL urine retained (Weiniger et al, 2006). While a post-void residual volume of 20-45 mL would not have significant effects on the results of our study, volumes up to 250 mL would have substantial ramifications. 5.1.2 Measurement of Bladder Volumes Potential problems in assessing bladder volumes in pregnancy include the morphological changes that occur during later stages of gestation in urogenital system. Fluoroscopy has revealed that the pregnant bladder may become “dumbbell-shaped” as opposed to the non-pregnant elliptical shape (FitzGerald and Graziano, 2007). However, ultrasound has been used to accurately measure bladder volume even with this change in shape during parturition (Weiniger et al, 2006; Gyampoh et al, 2004). Catheterization may be the “gold standard” of bladder volume estimation, but it is invasive, uncomfortable, potentially introduces infection, and perhaps embarrasses the participant (Goode et al, 2000). Ultrasonography has been used to determine bladder volume in women during labour accurately and provides a minimally invasive alternative to catheterization (Gyampoh et al, 2004). Three types of ultrasound are available for use: transabdominal ultrasound, translabial ultrasound, and transvaginal ultrasound. In non-pregnant women, the estimation of urine retention is comparable between the transabdominal and transvaginal methods (Yip et al, 1998). In addition, transabdominal ultrasounds have been successful in previously measuring urine retention volumes in intrapartum patients. Finally, the use of transabdominal ultrasound over the more invasive bladder catheterization has been validated in postpartum women, with an indicated 97.7% specificity (Maymon et al, 1991). For our purposes, the least invasive procedure is ideal; given that these techniques do not produce significantly different results allows us to conclude that transabdominal ultrasound is the most appropriate technique.  68 !It is important to investigate urine retention in both early and late gestation for differing reasons. In early gestation, women with a retroverted uterus may experience urine retention if the fundus becomes trapped behind the sacrum, pinning the lower bladder closed; this usually resolves by 16 weeks gestation (Chaliha and Stanton, 2002). Additionally, there is minor distortion of the bladder dome in early gestation as the uterus remains in the pelvic region in late gestation, the fetal head modifies the shape of the maternal bladder (FitzGerald and Benness, 2005) and may cause urine to become trapped in the bladder resulting in post-void retention. Urine collection is imperative for our study as it provides the only method of determining amino acid flux. If complete urine samples are not provided at each time point, i.e. if >100mL of urine is retained post-void, the results of this study would be significantly affected. 5.1.3 Hypothesis We hypothesize that urine retention will be increased in late gestation due to increased disturbance to typical bladder morphology.  5.1.4 Objectives We hope to provide quantitative evidence of post-void urine retention in two distinct phases of pregnancy.  5.2 Methods 5.2.1 Subject Consent Subjects were part of the primary lysine requirement study. An amendment was approved to the subject consent form to include the use of transabdominal ultrasound post-void in 69 !participants. Subjects were consented either at pre-study or, following verbal consent, provided with the updated consent form on study day.  5.2.2 Methods We have used trans-abdominal ultrasonography to determine post-void urine retention in participants in both early and late gestation. In the Lysine Requirements study, urine samples are taken 4 times in the last 1.5 hours of the study day. Bladder volume was measured immediately post-void in a supine position using transabdominal ultrasonography to determine any residual urine present. Three measurements were obtained: sagittal, transverse, and perpendicular (orthogonal). A Sonosite Micromaxx portable ultrasound machine was used with a 5-1MHz curvilinear probe. Measurements were taken by Dr. Kenneth Lim, MD, FRCSC (or a trained sonographer under Dr. Lim’s supervision) during the study days.  5.2.3 Analysis Results are presented as means SD. Measurements were recorded and analysed using Microsoft Excel for Mac 2011 (14.3.8) and mean retention rates were compared using independent t-tests. Significance was defined at p<0.05.    5.3 Results and Discussion 5.3.1 Results We have completed a total of 20 studies in both early and late gestation. These results were separated by gestational age, with N=7, n=10 in early gestation and N=4, n=10 in late gestation. Current results indicate a mean bladder retention in early gestation of 27.55 (± 29.37) 70 !mL across all time points. This is significantly greater (p<0.001) than the results found in late gestation, with a mean of 13.57 (±8.24) mL. The range in early gestation was also much greater than late gestation, at 0 - 269.86 mL and 0.96- 28.19 mL, respectively. Finally, the mean ratio of volume of urine retained to total bladder volume at time of void is significantly greater in early gestation (p < 0.01).            71 ! Table 10a. Early Gestation: Post-Void Residual Urine Volume. Subject Time Point 1 (mL) Time Point 2* (mL) Time Point 3 (mL) Time Point 4 (mL) Day Mean ± SD (mL) Lys-T1-08       (17 weeks) 48.34 89.80 3.02 18.53 39.92 (35.33) Lys-T1-08 (18 weeks) 25.87 48.55 19.28 18.09 27.95 (13.22) US-01 (19 weeks) ND ND 52.66 72.85 65.26 (29.20) US-01 (20 weeks) 6.47 41.16 10.44 21.38 19.86 (15.40) Lys-T1-10   (15 weeks) 0** 0** 0** 0** 0 (0) Lys-T1-11 (19 weeks) 1.77 44.74 1.58 0 12.02 (20.68) Lys-T1-12 (17 weeks) 34.49 259.98 45.57 13.45 88.37 (104.48) Lys-T1-13 (18 weeks) ND ND 0** 0** 0 (0) Lys-T1-15 (17 weeks) ND 9.15 0** 0** 3.05 (4.31) Lys-T1-15 (18 weeks) ND 46.53 3.42 7.13 19.03 (19.50) Total     27.55 (29.37) *Measurement taken following a liquid meal (~10 minutes later post-void than other time points).  **No measureable post-void urine retention.     72 !  Table 10b. Early Gestation: Voided Urine Volume. Subject Time Point 1 (mL) Time Point 2* (mL) Time Point 3 (mL) Time Point 4 (mL) Day Mean ± SD (mL) Lys-T1-08 (17 weeks) 150 300 325 300 269 (69) Lys-T1-08 (18 weeks) 200 ND 200 225 208 (12) US-01 (19 weeks) ND ND 300 300 300 (0) US-01 (20 weeks) 45 300 400 200 236 (131) Lys-T1-10** (15 weeks) 100 150 50 100 100 (35) Lys-T1-11 (19 weeks)                                                                                                                                                                                    100 50 100 75 81 (21) Lys-T1-12 (17 weeks) 550 150 200 200 275 (160) Lys-T1-13 (18 weeks) ND ND 110 100 105 (5) Lys-T1-15 (17 weeks) ND 90 100 125 105 (15) Lys-T1-15 (18 weeks) ND 150 225 100 167 (42) Total     185 (79) *Measurement taken following a liquid meal (~10 minutes later post-void than other time points).  **No measureable post-void urine retention.     73 ! Table 10c.  Early Gestation: Percent Residual Urine Volume (Residual Urine/(Residual Urine + Void Volume)). Subject Time Point 1  Time Point 2* Time Point 3 Time Point 4 Day Mean ± SD  Lys-T1-08 (17 weeks) 14.71 13.93 5.60 5.69 9.98 (4.35) Lys-T1-08 (18 weeks) 19.46 ND 1.49 7.61 9.52 (7.46) US-01 (19 weeks) ND ND 14.93 20.60 17.77 (2.84) US-01 (20 weeks) 12.57 12.07 2.54 9.66 9.21 (4.00) Lys-T1-10** (15 weeks) 0 0 0 0 0 (0) Lys-T1-11 (19 weeks)                                                                                                                                                                                    1.74 47.22 1.56 0 12.63 (19.98)Lys-T1-12 (17 weeks) 5.90 63.41 18.56 6.30 23.54 (23.57) Lys-T1-13 (18 weeks) ND ND 0** 0** 0 (0) Lys-T1-15 (17 weeks) ND 9.22 0** 0** 9.22 (0) Lys-T1-15 (18 weeks) ND 23.67 1.50 6.65 10.61 (19.50) Total     9.41 (6.57) *Measurement taken following a liquid meal (~10 minutes later post-void than other time points). **No measureable post-void urine retention.      74 ! Table 11a. Late Gestation: Post-Void Residual Urine Volume. Subject Time Point 1 (mL) Time Point 2* (mL) Time Point 3 (mL) Time Point 4 (mL) Day Mean ± SD (mL) Lys-T2-17 (35 weeks) 0.96 3.83 7.05 6.13 4.49 (2.58) Lys-T2-17 (36 weeks) 10.46 7.26 2.58 4.79 6.27 (3.97) Lys-T2-17 (38 weeks) 5.21 8.07 15.91 7.16 9.09 (5.29) Lys-T2-18 (33 weeks) ND 16.12 4.49 5.19 8.60 (4.72) Lys-T2-22 (34 weeks) 15.55 17.86 18.20 16.64 17.06 (1.05) Lys-T2-22 (35 weeks) 20.14 25.96 2.98 2.10 12.80 (10.46) Lys-T2-23 ND ND ND 14.87 14.87 (0) Lys-T2-23 ND ND 7.75 ND 7.75 (0) Lys-T2-23 ND ND ND 26.55 26.55 (0) Lys-T2-23 ND ND ND 28.19 28.19 (0) Total     13.57 (8.24) *Measurement taken following a liquid meal (~10 minutes later post-void than other time points).       75 ! Table 11b. Late Gestation: Voided Urine Volume. Subject Time Point 1 (mL) Time Point 2* (mL) Time Point 3 (mL) Time Point 4 (mL) Day Mean ± SD (mL) Lys-T2-17 (35 weeks) 450 100 150 175 219 (136) Lys-T2-17 (36 weeks) 340 150 215 150 214 (78) Lys-T2-17 (38 weeks) 225 100 175 125 156 (48) Lys-T2-18 (33 weeks) ND 400 375 425 400 (20) Lys-T2-22 (34 weeks) 225 175 100 100 17.06 (1.05) Lys-T2-22 (35 weeks) 350 80 100 125 164 (109) Lys-T2-23 ND ND ND 150 150 (0) Lys-T2-23 ND ND 225 ND 225 (0) Lys-T2-23 ND ND ND 125 125 (0) Lys-T2-23 ND ND ND 125 125 (0) Total     176 (106) *Measurement taken following a liquid meal (~10 minutes later post-void than other time points).       76 !Table 11c. Late Gestation: Percent Residual Urine Volume (Residual Urine/(Residual Urine + Void Volume)). Subject Time Point 1  Time Point 2*  Time Point 3  Time Point 4  Day Mean ± SD Lys-T2-17 (35 weeks) 2.98 4.61 1.19 3.19 2.99 (1.22) Lys-T2-17 (36 weeks) 0.21 3.69 4.49 3.38 2.94 (1.63) Lys-T2-17 (38 weeks) 225 100 175 125 5.87 (2.34) Lys-T2-18 (33 weeks) ND 3.87 1.18 1.21 2.08 (1.26) Lys-T2-22 (34 weeks) 15.55 17.86 18.20 16.64 11.35 (0.04) Lys-T2-22 (35 weeks) 20.14 25.96 2.98 2.10 10.61 (11.34) Lys-T2-23 ND ND ND 14.87 3.33 (0) Lys-T2-23 ND ND 7.75 ND 9.02 (0) Lys-T2-23 ND ND ND 26.55 12.52 (0) Lys-T2-23 ND ND ND 28.19 18.40 (0) Total     7.91 (5.10) *Measurement taken following a liquid meal (~10 minutes later post-void than other time points).  5.3.2 Discussion The results from this study differ from our initial hypotheses; however, results are novel and relevant to our study. The observed decrease in mean urine retention in late gestation may be due increased pressure from the uterus, causing more complete emptying of the bladder. Using multichannel urodynamic testing in continent women, it has been seen that bladder pressure at 77 !maximum capacity increases from 9 cm H2O in the second trimester to 20 cm H2O at the end of pregnancy (Iosif et al, 1980). Indeed, this increase was related to the greater pressure from the enlarged uterus.   There may also have been slight differences in the time between voiding and ultrasound, which may have contributed to the wide range of retention data seen. Additionally, the amount of liquid consumed on the study day may have played a role, as it was noted that some women had noticeably increasing bladder volumes while the ultrasound was being performed. Indeed, the participant with the greatest retention in early gestation (269.86 mL) noted the urge to urinate during the ultrasound testing.   These findings will have significant implications for dietary studies using urine sampling as a non-invasive measure of metabolite, including the lysine requirement study described in the current thesis. In healthy non-pregnant adults, post-void residual volume is approximately 25 mL, and urologists suggest that volumes of 50-100 mL indicate some level of abnormal retention (Kelly, 2004). Our findings indicate a mean retention rate in both early and late gestation within normal parameters; however, some participants fall outside of this normal range. Notably, two participants in early gestation had post-void residual volumes >100 mL, indicating possible errors on study day measures based on urinary measures.            78 !Chapter 6 - Conclusions and Recommendations for Further Work  It is now apparent that current recommendations for lysine during pregnancy are inadequate, both quantitatively and theoretically. Lysine requirement during healthy pregnancy were determined to be 36.6 mg/kg/d (upper 95% CI = 46.2 mg/kg/d) and 50.3 mg/kg/d (upper 95% CI = 60.4 mg/kg/d) in early and late gestation, respectively. While the mean lysine requirement in early gestation is 11% lower than current DRI (2005) recommendations of 41 mg/kg/d, the mean lysine requirement in late gestation is 25% higher. Current recommendations do not account for gestational age, thereby not acknowledging the dynamic state of pregnancy. While these recommendations are adequate in the first half of pregnancy, they are estimated to be ~ 37% below requirement in late gestation. We hope that these findings will influence current recommendations to reflect true lysine requirement at distinct gestational ages.  Future recommendations for research include expansion upon perinatal health improvement by investigating lysine requirements during lactation. Lactation, like pregnancy, requires heightened macronutrient and energy intake to maintain maternal and neonatal health. A similar factorial approach based on nitrogen balance studies is applied to non-pregnant adult requirements to define current protein recommendations during lactation, and there is no data regarding amino acid requirements during lactation (DRI, 2005). Many of the same caveats with current recommendations during pregnancy may apply to lactation as well, and definitive requirements are strongly recommended.  Lysine is the first individual amino acid to be studied during healthy human pregnancy, leading the way to explore further essential amino acid requirements, including threonine and methionine. Experimental evidence from a study in sows indicates that threonine requirements 79 !are 2 times higher in late gestation than in early gestation (12.3 g/d vs. 5.0 g/d) (Levesque et al, 2010). Threonine is a precursor to glycine and serine, nonessential amino acids. Methionine is another amino acid that should be investigated due to its roles in human health and production of carnitine. Additionally, methionine has a significant role in gene regulation; it is apparent that methylation of genomic DNA resulting in epigenetic modifications of metabolism may occur due to donation of one-carbon units from methionine. The role of methionine in epigenetics may be influenced by nutrient intake and altered metabolic states such as obesity and type 2 diabetes. Due to the significant role that methionine intake may have on offspring epigenetic profiles and future onset of disease, investigation of optimal requirements of this essential amino acid is recommended.  Finally, the current study limited its investigation to healthy human pregnancy; future investigations hope to determine lysine requirements in complicated pregnancies, and in pregnant women living in developing countries. 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Carnitine deficiency and L-carnitine supplementation in lysinuric protein intolerance. Metabolism clinical and experimental. 2008. 57:549-554. Tanner LM, Näntö-Salonen K, Niinikoski H, Huoponen M, Simell, O. Long-term oral lysine supplementation in lysinuric protein intolerance. Metabolism Clinical and Experimental. 2007. 56:185–189. Tanner LM, Näntö-Salonen K, Niinikoski H, Jahnukainen T, Keskinen P, Saha H, Kananen K, Helanterä A, Metso M, Linnanvuo M, Huoponen K, Simell O. Nephropathy advancing to end-stage renal disease: A novel complication of lysinuric protein intolerance. Pediatrics 2007. 150:631 – 634.  Tanphaichitr V, Zaklama MS, Broquist HP. Dietary lysine and carnitine: relation to growth and fatty livers in rats. J Nutr. 1976. 106(1):111-7. Thorpe JM, Roberts SA, Ball RO, Pencharz PB. Effect of prior protein intake on phenylalanine kinetics. J. Nutr. 1999. 129:343–48. Tomé D, Bos C. Lysine Requirement through the human life cycle. J. Nutr. 2007. 137:1642S-1645S. Toth MJ, Tchernof A, Rosen CH, Matthews DE, Poehlman ET. Regulation of protein metabolism in middle-aged, premenopausal women: roles of adiposity and estradiol. J Clin Endocrinol Metab. 2000. 85:1382-1387. 95 !Turner JM, Humayun MA, Elango R, Rafii M, Langos V, Ball RO, Pencharz PB. Total sulfur amino acid requirement of healthy school-age children as determined by indicator amino acid oxidation technique. Am J Clin Nutr. 2006. 83(3):619-23. U.S. Department of Agriculture, Agricultural Research Service. 2010. USDA National Nutrient Database for Standard Reference, Release 23. Nutrient Data Laboratory Home Page, http://www.ars.usda.gov/ba/bhnrc/ndl van der Schoor SR, Reeds PJ, Stellaard F, Wattimena JD, Sauer PJ, Buller HA, van Goudoever JB. Lysine kinetics in preterm infants: the importance of enteral feeding. Gut. 2004. 53:38–43. van Goudoever JB, Stoll B, Henry JF, Burrin DG, Reeds PJ. Adaptive regulation of intestinal lysine metabolism. PNAS. 2000. 97(21):11620-11625. Voet, Voet, Pratt. Fundamentals of Biochemistry, 3rd edition. Life at the molecular level. Wiley. 2008.  Wallace WM. Nitrogen content of the body and its relation to retention and loss of nitrogen. Fed Proc. 1959. 18:1125–30. Weiniger C.F., Wand S., Nadjari M., Elchalal U., Mankuta D., Ginosar Y., Matot I. Post-void residual volume in labor: a prospective study comparing parturients in and without epidural analgesia. Acta Anaesthesiol Scand. 2006. 50: 1297-1303. Whitney EN, Rolfes SR. Understanding Nutrition, 12th Ed. Wadsworth Publishing: 2011. Print. WHO Collaborative Study. Maternal anthropometry and pregnancy outcomes. Bull World Health Organ. 1995; 73 (Suppl): 1–98.  Wolf G, Berger CRA. Studies on biosynthesis and turnover of carnitine. Archives of Biochemistry and Biophysics. 1961. 92(2):360-365. Wu G, Bazer FW, Cudd TA, Meininger CJ, Spencer, TE. Maternal Nutrition and Foetal Development. J. Nutr. 2004. 134(9):2169-2172. Xi L, Brown K, Woodworth J, Kwanseob S, Johnson B, Odle J. Maternal Dietary L-Carnitine Supplementation Influences Foetal Carnitine Status and Stimulates Carnitine 96 !Palmitoyltransferase and Pyruvate Dehydrogenase Complex Activities in Swine. J. Nutr.  2008. 138(12):2356-2362. Yip SK, Fung TY, Chung TK. Ultrasonographic estimation of postpartum postvoid residual bladder volume: a comparison between transabdominal and transvaginal ultrasonography. Int Urogynecol J Pelvic Floor Dysfunct. 1998. 9(1):9-12. Young VR. Nutrient Balance Studies: Indicators of human requirements of Adaptive Mechanisms? J Nutr. 1986. 116(4):700-3. Young VL, Bier DM, Pellett PL. A theoretical basis for increasing current estimates of the amino acid requirements in adult man, with experimental support. Am J Clin Nutr. l989. 50:80-92.  Young VR, Pellett PL. Plant proteins in relation to human protein and amino acid nutrition. Am J Clin Nutr. 1994. 59:1203S-12S. Young M, Prenton MA. Maternal and fetal plasma amino acid concentrations during gestation and in retarded fetal growth. J Obstet Gynaecol Br Cwlth 1969. 76:333–44. Young VR, Taylor YS, Rand WM, Scrimshaw NS. Protein requirements of man: efficiency of egg protein utilization at maintenance and submaintenance levels in young men. J Nutr. 1973.103:1164–74. Zello GA, Pencharz PB, Ball RO. Phenylalanine flux, oxidation and conversion to tyrosine in humans studied with L-(1-13C)phenylalanine. Am. J. Physiol. 1990. 259:E835–43. Zello GA, Pencharz PB, Ball RO. The design and validation of a diet for studies of amino acid metabolism in adult humans. Nutrition Research. 1990. 10(12):1353-1365. Zello GA, Pencharz PB, Ball RO. Dietary lysine requirement of young adult males determined by oxidation of L-[1-13C] phenylalanine. Am J Physiol. 1993. 264(4 Pt 1):E677-85. Zello GA, Wykes LJ, Ball RO, and Pencharz PB. Recent advances in methods of assessing dietary amino acid requirements for adult humans. J Nutr. 1995. 125(12): 2907-2915. Zhao W, Zhai F, Zhang D, An Y, Ying L, He Y, Ge K, Scrimshaw NS. Lysine fortified wheat flour improves the nutritional and immunological status of wheat-eating families in Northern China. Food Nutr Bull. 2004. 252:123-9. 97 !        Appendix A: Subject Consent Form                98 ! SUBJECT!INFORMATION!AND!CONSENT!FORM!!Determination of lysine 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:! ! ! Maggie!Payne,!BScH,!M.Sc!candidate!!! ! ! ! ! Department!of!Pediatrics!! ! ! ! ! Faculty!of!Medicine!! ! ! ! ! The!University!of!British!Columbia!! ! ! ! ! Telephone:!604"875"2000!x4607!!Sponsors:! ! ! ! Canadian!Institutes!of!Health!Research!(CIHR)!!Emergency!Phone!Number:! ! Rajavel!Elango!778M986M8655!Maggie!Payne! 604M363M5143!available'24'hours'per'day'and'seven'days'per'week!!!Site:' ' ' ' ' Oak'Street'Campus,'UBC'' ' ' ' ' Child'&'Family'Research'Institute''!1.!INVITATION!!You!are!being! invited! to! take!part! in! this! research! study!because! there! is! currently! little!information!regarding!protein!nutrition!during!healthy!pregnancy.!Pregnancy! is!a!critical!time! that! necessitates! sufficient! nutrition! to! ensure! healthy! development! of! both! the!mother!and!baby.!There! is!virtually!no! information!regarding!amino!acids!needed! in!our!diet!during!pregnancy.!Amino!acids!are! the!building!blocks!of!protein,!which!are!used! to!build!muscle!and!body! tissue!and! to!support! the! immune!system.!Lysine! is!an! important!amino! acid! that! we! cannot!make! in! our! bodies,! and! that! we! need! to! get! from! our! diet.!Lysine! is! present! in!many! foods,! such! as!meat,! eggs! and! fish;! however,! there! is! not! very!much!lysine!in!plants!and!cereals!such!as!wheat!and!rice.!During!pregnancy,!dietary!lysine!becomes!even!more!crucial.!Not!consuming!enough!lysine!during!pregnancy!could!lead!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! know! how! much! lysine! we! need! to! eat! during!pregnancy.!!!! D e p a r t m e n 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 !! 99 !2.!!!YOUR!PARTICIPATION!IS!VOLUNTARY! Your!participation!is!voluntary.!You!have!the!right!to!refuse!to!participate!in!this!study.!If!you!decide!to!participate,!you!may!still!choose!to!withdraw!from!the!study!at!any!time!without!any!negative!consequences!to!the!medical!care,!education,!or!other!services!to!which!you!are!entitled!or!are!presently!receiving.!Before!you!decide,!it!is!important!for!you!to!understand!what!the!research!involves.!!This!consent!form!will!tell!you!about!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!in!this!study,!you!will!be!asked!to!sign!this!form!within!7!days.!Please!take!time!to!read!the!following!information!carefully!and!to!discuss!it!with!your!family,!friends,!and!doctor!before!you!decide.!!!!3.!!WHO!IS!CONDUCTING!THE!STUDY?!!The Principal Investigator, Dr. Rajavel Elango, and the Nutrition and Metabolism Research Program of the Child and Family Research Institute, University of British Columbia has received financial compensation from the sponsor Canadian Institutes of Health Research (CIHR) for the work required in doing this clinical research and/or for providing advice on the design of the study/travel expenses/etc. Financial compensation to researchers for conducting the research is associated with obligations defined in a signed contractual agreement between the researchers and the sponsor. Researchers must serve the interests of the subject and also abide by their contractual obligations. For some, the payment of financial compensation to the researchers can raise the possibility of a conflict of interest. You are entitled to request any details concerning this compensation from the Principal Investigator. !4. BACKGROUND   Although!it!is!well!known!that!pregnant!women!need!more!lysine!in!their!diet,!it!is!unclear!how! much! additional! lysine! is! required.! Older! techniques! used! to! measure! how! much!lysine!we!need!require!participants!to!eat!a!low!lysine!diet!for!several!days!at!a!time!and/or!remain! in! an! experimental! environment! for! an! extended! period! of! time.! Because! it! is!unethical! to! do! this! in! a! pregnant! woman,! there! is! very! little! information! about! lysine!requirements! in! this! population.! To! gain! a! better! understanding! of! lysine! requirements!throughout! pregnancy,! we! plan! to! study! pregnant! women! from! all! ethnic! backgrounds,!aged! 19"40! years,! in! early! and! late! pregnancy! using! a! modern! technique! called! the!indicator! amino! acid! oxidation! (IAAO)! technique.! This! technique! uses! a! test! liquid!meal!with! a! specific! amount!of! lysine!mixed!with! a! stable! isotope! tracer.!A! stable! isotope! is! a!labeled!amino!acid.!These!labeled!amino!acids!are!colourless,!odourless!and!tasteless,!and!are!completely!safe;!they!are!present!in!the!air!we!breathe,!water!we!drink!and!food!we!eat.!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!because!it!looks!different!than!the!rest!of!the!amino!acids!in!the!body.!This!allows!us!to!measure!if!you!are!eating!enough!lysine!for!protein!synthesis!to!take!place,!and!we!can!study!lysine!requirements!in!just!one!day.!This!technique! has! been! used! previously! to! measure! lysine! requirements! in! healthy! babies,!children! and! adult! human! beings.! Also,! as! pregnancy! progresses,! there! is! a! risk! of! urine!retention!after!voiding.!It!is!unclear!how!much!urine!may!remain!in!the!bladder!post"void!100 !as!most! studies! focus! on! urine! retention! postpartum! or! during! labor! rather! that! during!stages!of!pregnancy.!Transabdominal!ultrasound!reliably!and!quickly!determines!bladder!volumes!in!pregnancy!in!a!non"invasive!manner.!!!!!!!5.  WHAT IS THE PURPOSE OF THE STUDY? !The! purpose! of! the! study! is! to! determine! the! lysine! needed! in! our! diets! in! early! (15"18!weeks! from! last! menstrual! period)! and! late! (33"36! weeks! from! last! menstrual! period)!pregnancy.! The! results! from! this! study! may! allow! us! to! provide! accurate! lysine!recommendations! during! pregnancy.! There! is! currently! no! conclusive! data! for! lysine!intakes!in!pregnant!women.!Current!recommendations!are!produced!using!a!mathematical!calculation! method! and! remain! constant! throughout! pregnancy.! However,! during!pregnancy! our! body! undergoes! a! lot! of! changes! as! the! baby! grows,! and! therefore! one!recommendation!may! not! be! sufficient! in! all! stages! of! pregnancy.! Recent! animal! studies!have!shown!that!lysine!requirements!are!increased!in!late!versus!early!pregnancy.!The!final!aim!of!this!study!is!to!determine!whether!post"void!residual!urine!volume!is!a!significantly!impacts!urine!sampling!in!early!and!late!gestation.!!!6.!!WHO!CAN!PARTICIPATE!IN!THE!STUDY?!!!!You!may!be!able!to!participate!in!this!study!if:!!! You!are!19!to!40!years"of"age!!! You!are!pregnant!with!a!single!child!7.!WHO!SHOULD!NOT!PARTICIPATE!IN!THE!STUDY?!!!! ! Women!who!are!not!pregnant!or!! Women!who!are!pregnant!with!more!than!one!child!(this!changes!lysine!demands)!! Women!who!are!not!19"40!years"of"age!or!!! Women!not!in!good!health!or!have!a!metabolic,!neurological,!genetic,!or!immune!disorder,!including!gestational!diabetes!or!anemia!!! 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!!8. WHAT!DOES!THE!STUDY!INVOLVE?!!Overview!of!the!Study!!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. Following a Preliminary study day to ensure your eligibility, you may participate in four separate study days, two in early and two in late pregnancy. Each of these study days will be 8 hours in length and will involve hourly meals (Protein shake and cookies) and non-invasive breath and urine sampling.  !101 !If!You!Decide!to!Join!This!Study:!Specific!Procedures!! a. Preliminary!Study!Day!Procedures:!!! 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! CFRI! located! in! BC! Children’s!Hospital.! You!will! be! asked! to!come! at! approximately! 8AM! after! having! fasted! overnight! (10"12h).! The! whole!procedure!will!take!1!hour!to!complete.!!!! During!the!preliminary!assessment,!a!Research!Assistant!(M.!Payne)!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!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!we!will!evaluate!your!normal!dietary!protein!intake.! 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!will!be!provided.!! b. 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!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.! 102 !! 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!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.! When! we! are! not! collecting! samples,! you! can! watch!television,!listen!to!music,!read!or!bring!computer!related!work!to!complete.!! ! To!measure! post"void! residual! urine! volumes,! four! bladder! ultrasound! readings!will!be!obtained!immediately!following!afternoon!urine!samples.!A!physician!or!a!trained!sonographer!under!physician!supervision!will!obtain!these!readings.!!! ! In! total,! you! can! expect! to! dedicate! approximately! 8! 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! the! study! day! projects.! There! will! be! one!preliminary! study! in!each!stage! (early!and! late)!of!pregnancy,! each! requiring!an!hour!of!your!time.!!!9.!WHAT!ARE!THE!POSSIBLE!HARMS!AND!DISCOMFORTS?!!There are no known risks involved with participating in this research. Some women may find the finger prick used for blood glucose measurement or the transabdominal ultrasound uncomfortable. We recognize that the length of the study day, and travel to BC Children’s Hospital might pose an inconvenience for you.  !!10.!!WHAT!ARE!THE!POTENTIAL!BENEFITS!OF!PARTICIPATING?!!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!lysine!recommendations! during! pregnancy.!We! also! hope! to! draw! conclusions! regarding! post"void!urine!retention!in!early!and!late!pregnancy.!!!11.!WHAT!HAPPENS!IF!I!DECIDE!TO!WITHDRAW!MY!CONSENT!TO!PARTICIPATE?!!103 !You may withdraw from this study at any time without giving reasons. A decision to withdraw will not have any negative ramifications to your health care at any hospital, research centre or physician’s office. 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.  12.!CAN!I!BE!ASKED!TO!LEAVE!THE!STUDY?!!!If!you!are!not!able!to!follow!the!requirements!of!the!study!or!for!any!other!reason,!the!principal!investigator!may!withdraw!you!from!the!study.!If!the!principal!investigator!considers!withdrawal!to!be!in!your!best!interest!to!ensure!your!health!(e.g.!in!the!case!of!gestational!diabetes),!you!will!be!withdrawn!from!the!study!without!your!consent.!!!13.!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!information!or!records!that!disclose!your!identity!will!be!published!without!your!consent,!nor!will!any!information!or!records!that!disclose!your!identity!be!removed!or!released!without!your!consent!unless!required!by!law.!!!You!will!be!assigned!a!unique!study!number!as!a!subject!in!this!study.!!Only!this!number!will!be!used!on!any!research"related!information!collected!about!you!during!the!course!of!this!study,!so!that!your!identity![i.e.!your!name!or!any!other!information!that!could!identify!you]!as!a!subject!in!this!study!will!be!kept!confidential.!!!Information!that!contains!your!identity!will!remain!only!with!the!Principal!Investigator!and/or!designate.!!The!list!that!matches!your!name!to!the!unique!identifier!that!is!used!on!your!research"related!information!will!not!be!removed!or!released!without!your!consent!unless!required!by!law.!Urine!samples!will!be!analyzed!at!the!Hospital!for!Sick!Children!in!Toronto!and!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.!Your!rights!to!privacy!are!legally!protected!by!federal!and!provincial!laws!that!require!safeguards!to!insure!that!your!privacy!is!respected!and!also!give!you!the!right!of!access!to!the!information!about!you!that!has!been!provided!to!the!sponsor!and,!if!need!be,!an!opportunity!to!correct!any!errors!in!this!information.!!Further!details!about!these!laws!are!available!on!request!to!your!study!investigator.!  14.!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!104 !maximum!of!$400!for!4!study!days.!Vehicle!parking!coupons!or!Translink!passes!for!each!pre"study!and!study!day!duration!at!BC!Children’s!Hospital!will!be!provided.!!15.!WHO!DO!I!CONTACT!IF!I!HAVE!QUESTIONS!OR!CONCERNS!ABOUT!MY!RIGHTS!AS!A!SUBJECT?!!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.!!If!you!have!any!concerns!or!complaints!about!your!rights!as!a!research!subject!and/or!your!experiences!while!participating!in!this!study,!contact!the!Research!Subject!Information!Line!in!the!University!of!British!Columbia!Office!of!Research!Services!by!e"mail!at!RSIL@ors.ubc.ca!or!by!phone!at!604"822"8598!(Toll!Free:!1"877"822"8598).!!!!105 !19.!!SUBJECT!CONSENT!!!!!My!signature!on!this!consent!form!means:!!! ! I!have!read!and!understood!the!subject!information!and!consent!form!! I! have! had! this! study! explained! to! me,! read! this! form! and! understand! the!information!concerning!this!study.!! I!have!had!sufficient!time!to!consider!the!information!provided!and!to!ask!for!advice!if!necessary.!!! I!have!had!the!opportunity!to!ask!questions!and!have!had!satisfactory!responses!to!my!questions.!!! I!understand!that!all!of!the!information!collected!will!be!kept!confidential!and!that!the!results!will!only!be!used!for!scientific!objectives.!! I! 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.!! I!understand!that!signing!this!consent!form!in!no!way!limits!my!legal!rights!against!the!sponsor,!investigators!or!anyone!else.!! I!understand!that!there!is!no!guarantee!that!this!study!will!provide!any!benefits!to!myself.!! I! understand! that! if! I! have! any! further! questions! or! desire! further! information! I!should!contact!Dr.!Rajavel!Elango!at!778"986"8655.!! I!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.!!I!will!receive!a!signed!copy!of!this!consent!form!for!my!own!records.!!!I,!___________________________________!!!!voluntarily!give!consent!for!my!participation!in!the!!'''''(Subject.''Please'print'your'name)'!!!!!!!!!!!!!!!!!!!!!!!!!!!!research!study!entitled:'!!!!!!!!!!!!'Determination of lysine requirements in healthy pregnant women using the indicator amino acid oxidation technique   !________________________!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!________________________!!!!!!!! !_________________!Signature'of'Subject''''''''''''''''''''''''''''''''''''''''''''''''''''''Printed'Name''''''''''''''''''''''''''''''''''''''''''Date' ' ' '!__________________________! !!!!!!!!!!!!!!!!!!!!!!!!!_________________________________________________!Signature'of'person'obtaining'consent'' '''''''''''Printed'Name'' ' ''''Study'Role'' ''''''''''''Date!!!!' 106 !     Appendix B: Pre-Study Day Questionnaire                  !107 !PreMStudy!Day!Assessment!Lysine!Requirement:!Healthy!Pregnant!Women!!Principal!Investigator:!Dr.!Rajavel!Elango! ! 604"875"2000!ext.!4911!(office)!Student!Investigator:!Maggie!Payne! ! ! 604"875"2000!ext.!4607(office)!! ! ! ! ! ! ! !! !CONTACT!INORMATION!!!Email:!_____________________________________!!!!Phone!#:!____________________________________!!PRELIMINARY!ASSESSMENT!!Subject!ID:________________________________! ! Date:___________________________!!Birthday!(mo/yr):!_______/______!!!!!!!!!!Age:___________!!!!!!!!Last!Menstrual!Period:________!! !Ultrasound!Dating:!___________! Gestational!Age:!___________!! ! ! ! ! ! !!!!!!!Height!(cm):______________!!!!!!!!!Weight!(kg):___________!! BMI:_________________!!!!!!!Fasting!Blood!Glucose!(mmol/L):_____________!![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):________________________!!108 !!Indirect!Calorimetry!!Measured!REE!(kcal/day):_________________! !!!!Estimated!REE!(kcal/day):____________________!!Daily!energy!requirement!(kcal/day):_________________________!!                     109 !     Appendix C: Advertisement                   110 !  111 !      Appendix D: Brochure               !  112 ! 113 ! 114 !    Appendix E: Subject Code Master List                   !!115 !! Subject(Code(Master(List((Determination(of(lysine(requirements(in(healthy(pregnant(women(using(the(indicator(amino(acid(oxidation(technique(! Subject!Name! Code!(Alpha4Numeric)!! Comments!!! ! !!! ! !!! ! !!! ! !!! ! !!! ! !!! ! !!! ! !!! ! !!! ! !!! ! !!! ! !!! ! !!! ! !!! ! !!! ! !!! ! !!!!!   116 !       Appendix F: Dietary Record Sheets         !117 !  118 !     Appendix G. Letter of Contact to Primary Health Care Provider                 119 !   !!Date:_____________!!!!!To!the!Health!Care!Provider!of!Ms.____________________________!!!Ms.!______________________!volunteered!to!participate!in!a!research!study!titled!“Determination!of!lysine!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:!604d875d2000!ext.4911;!Fax:!604d875d3597!Cell:!778d986d8655!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 !!120 !     Appendix H. Study Day Protocol                    121 !Study&Day&Schedule&Lysine&Requirement:&Healthy&Pregnant&Women&&Subject!ID:_______________________! ! ! Date:___________________________!!Height!(cm):_______________!!!!!Weight!(kg):_______________!!!!Blood!Glucose!(mmol/L):____________!!Lysine!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!1st!Ultrasound! ! !15:00! 5th!breath!(3x)!4th!urine!2nd!Ultrasound! Meal!#8!–!4th!oral!dose! !15:15! 6th!breath!(3x)! ! !15:30! 7th!breath!(3x)!5th!urine!3rd!Ultrasound! ! !!15:45! 8th!breath!(3x)! ! !16:00! 9th!breath!(3x)!6th!urine!4th!Ultrasound! ! !! Morning!Urine! Afternoon!Urine! Comments!pH! ! ! !Glucose! ! ! !Ketones! ! ! !Leukocytes! ! ! !Nitrile! ! ! !Protein! ! ! !Blood! ! ! !!122 !      Appendix I. Pre-Study Subject Demographics                  123 ! Pre;Study&Day&Assessment&Lysine&Requirement:&Healthy&Pregnant&Women&&Principal!Investigator:!Dr.!Rajavel!Elango! ! 604d875d2000!ext.!4911!(office)!Student!Investigator:!Maggie!Payne! ! ! 604d875d2000!ext.!4607(office)!! ! ! ! ! ! ! !!CONTACT&INORMATION&&&Email:!_____________________________________!!!!Phone!#:!____________________________________!&PRELIMINARY&ASSESSMENT&1&!Subject!ID:________________________________! ! Date:___________________________!!Birthday!(mo/yr):!_______/______!!!!!!!!!!Age:___________!!!!!!!!Last!Menstrual!Period:________!! !Ultrasound!Dating:!___________! Gestational!Age:!___________!! ! ! ! ! ! !!!!!!!!![Optional]!Primary!Health!Care!Provider!Details!(name,!phone,!address):______________________________!!_____________________________________________________________________________________________________!&&&&&&&&&  

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