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Aromatic amino acid requirements in human pregnancy and implications in maternal phenylketonutia management Ennis, Madeleine 2020

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AROMATIC AMINO ACID REQUIREMENTS IN HUMAN PREGNANCY AND IMPLICATIONS IN MATERNAL PHENYLKETONURIA MANAGEMENT by  Madeleine Ennis  B.Sc., Bishop’s University, 2015  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSPOHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Reproductive and Developmental Sciences)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  August 2020  © Madeleine Ennis, 2020  ii  The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: Aromatic Amino Acid Requirements in Human Pregnancy and Implications in Maternal Phenylketonuria Management  submitted by Madeleine Ennis in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Reproductive and Developmental Sciences  Examining Committee: Dr Rajavel Elango, PhD, Department of Pediatrics Supervisor  Dr Alexander Beristain, PhD, Department of Obstetrics and Gynaecology Supervisory Committee Member  Dr William Gibson, PhD, Department of Medical Genetics University Examiner Dr David Kitts, PhD, Faculty of Land and Food Systems University Examiner  Additional Supervisory Committee Members: Dr Dan Rurak, PhD, Department of Obstetrics and Gynaecology Supervisory Committee Member Dr Kenneth Lim, MD, Department of Obstetrics and Gynaecology Supervisory Committee Member      Dr Angela Devlin, PhD, Department of Pathology and Laboratory Medicine Supervisory Committee Member    iii  Abstract Background: During pregnancy there is an increased dietary requirement for most nutrients to allow for healthy growth and development of both the fetus and mother. Phenylalanine and tyrosine, two aromatic amino acids required for protein synthesis, are also key amino acids in the metabolic disorder phenylketonuria (PKU), which can be detrimental during pregnancy. The dietary requirements of phenylalanine and tyrosine for healthy human pregnancies have not been experimentally determined and publications about maternal PKU are limited.  Objectives: (1) Determine the minimum dietary phenylalanine requirements during early and late gestation in healthy women, (2) determine the dietary requirements for phenylalanine and tyrosine (total aromatic amino acids, TAA) during early and late gestation in healthy women, and (3) describe current practices for managing maternal PKU and assess their efficacy. Methods: (1) Healthy pregnant women (n=23) were studied at a range of phenylalanine intakes (in the presence of excess tyrosine) in early and late pregnancy using stable isotope-based techniques (totaling 76 study days). (2) Healthy pregnant women (n=19) were studied at a range of phenylalanine intakes (in the absence of tyrosine) in early and late pregnancy using stable isotope-based techniques (totaling 51 study days). (3) A retrospective analysis of the dietitian’s records for 16 maternal PKU subjects was conducted at the Adult Metabolic Diseases Clinic at Vancouver General Hospital. Management practices, blood analyses, and dietary records were collected and analyzed. Results: (1) The minimum phenylalanine requirements were 15 and 21 mg·kg−1·d−1 during early and late pregnancy, respectively. (2) The TAA requirements were 44 and 50 mg·kg−1·d−1 in early and late pregnancy, respectively. (3) Current practices at the Adult Metabolic Diseases Clinic are working well to achieve metabolic control in individuals with maternal PKU.  iv  Conclusion: The results of the requirement studies will contribute to improved dietary recommendations for phenylalanine and tyrosine during pregnancy, as current recommendations are underestimated. The natural history provides reference values and management practices of maternal PKU patients, contributing to the limited data available and potentially improving clinical care and allow for future dietary requirement studies in this population. This dissertation adds novel data on phenylalanine and tyrosine metabolism and requirements during human pregnancies.       v  Lay Summary During pregnancy, there is an increased need for nutrients in the diet to allow for healthy growth of the baby. This includes 2 building blocks of protein, phenylalanine and tyrosine, which are also called aromatic amino acids. Due to their important role in growth, development and brain health, we set out to determine phenylalanine and tyrosine requirements during early and late pregnancy, to help optimize birth outcomes. We found that in early pregnancy, women needed to consume more phenylalanine and tyrosine than is currently recommended. We also found that in late pregnancy the requirements are significantly higher than in early pregnancy. Phenylketonuria is a genetic disorder where phenylalanine cannot be removed from the body, and it builds up to toxic levels. Pregnant women with phenylketonuria need to monitor their phenylalanine intake. Therefore, we reviewed management practices at Vancouver General Hospital for these pregnancies and showed they are working well.  vi  Preface These studies were developed and conducted by myself, Madeleine Ennis, under the supervision of Dr Rajavel Elango. All three studies were approved by the UBC ethics board with certificate numbers: H15-02558, H17-02924 and H19-01225.  A version of Chapter 3 has been published in: Ennis MA, Rasmussen BF, Lim K, Ball RO, Pencharz PB, Courtney-Martin G, and Elango R. Dietary phenylalanine requirements during early and late gestation in healthy pregnant women. Am J Clin Nutr. 2020 Feb 1;111(2):351–9. The majority of this manuscript was written by me, with edits from the other authors. Occasionally, study prep and/or analytic support was provided by other members of the Elango lab: Betina Rasmussen, Peter Radonic, and Erin Gilbert. A version of chapter 4 is being prepared for journal submission: Ennis MA, Ong AJ, Lim K, Ball RO, Pencharz PB, Courtney-Martin G, and Elango R. Dietary aromatic amino acid requirements during early and late gestation in healthy pregnant women. The majority of this manuscript was written by me, with edits from the other authors. Occasionally, study prep and/or analytic support was provided by other members of the Elango lab: Betina Rasmussen, Katia Caballero, and Abrar Turki. A version of Chapter 5 is being prepared for journal submission. Ennis MA, Schmidt Z, Rosen-Heath A, Branov J, Bosdet T, Lehman A, Sirrs A, and Elango R. A Natural History of mPKU Patients at Vancouver General Hospital. The majority of this manuscript was written by me, with edits from the other authors. Data collection and input support was provided by Elango Lab member, Zoe Schmidt and Vancouver General Hospital Metabolic Dietitian, Annie Rosen-Heath. vii  Table of Contents  Abstract ......................................................................................................................................... iii Lay Summary .................................................................................................................................v Preface ........................................................................................................................................... vi Table of Contents ........................................................................................................................ vii List of Tables ............................................................................................................................... xii List of Figures ............................................................................................................................. xiii List of Abbreviations ...................................................................................................................xv Acknowledgements ................................................................................................................... xvii Dedication ................................................................................................................................. xviii Chapter 1: Introduction ................................................................................................................1 Chapter 2: Literature Review .......................................................................................................4 2.1 Overview of Protein and Amino Acids ........................................................................... 4 2.2 Phenylalanine and Tyrosine ............................................................................................ 5 2.2.1 Dietary Sources of Phenylalanine ............................................................................... 6 2.2.2 Metabolism of Phenylalanine and Tyrosine ............................................................... 6 2.2.3 Catecholamines ........................................................................................................... 8 2.3 Phenylketonuria .............................................................................................................. 8 2.3.1 Maternal Phenylketonuria ........................................................................................... 9 2.4 Maternal Nutrition ........................................................................................................ 11 2.4.1 Metabolic Adaptations during Pregnancy ................................................................. 11 2.4.2 Impacts of the Maternal Diet on the Fetus ................................................................ 11 viii  2.4.3 Impact of Dietary Protein and Amino Acids Intake during Pregnancy .................... 12 2.4.4 Maternal Amino Acid Metabolism ........................................................................... 13 2.4.5 Placental and Fetal Amino Acid Metabolism ........................................................... 14 2.4.5.1 Placental Transport of Phenylalanine and Tyrosine ......................................... 16 2.5 Methods to Determine Amino Acid Requirements ...................................................... 17 2.5.1 The Nitrogen Balance Technique ............................................................................. 17 2.5.2 Stable Isotopes .......................................................................................................... 18 2.5.3 Indicator Amino Acid Oxidation Technique ............................................................ 18 2.5.4 Direct Amino Acid Oxidation Technique ................................................................. 20 2.5.5 24-Hour Indicator Amino Acid Balance and Oxidation Technique ......................... 20 2.5.6 Importance of Excess Tyrosine ................................................................................. 21 2.5.7 The Importance of Bicarbonate ................................................................................ 22 2.6 Protein and Amino Acid Requirements during Pregnancy ........................................... 22 2.6.1 Current Phenylalanine and Tyrosine Recommendations .......................................... 25 2.7 Previous Phenylalanine and Tyrosine Requirement Studies ........................................ 26 2.8 Overall Objectives ........................................................................................................ 28 Chapter 3: Dietary Phenylalanine Requirements during Early and Late Gestation in Healthy Pregnant Women (90) ...................................................................................................30 3.1 Introduction ................................................................................................................... 30 3.1.1 Objectives and Hypothesis ........................................................................................ 31 3.2 Subjects and Methods ................................................................................................... 32 3.2.1 Participants ................................................................................................................ 32 3.2.2 Experimental Design ................................................................................................. 32 ix  3.2.3 Preliminary Assessment of Participants .................................................................... 35 3.2.4 Study Diets ................................................................................................................ 35 3.2.5 Isotope Protocol ........................................................................................................ 36 3.2.6 Sample Collection and Analysis ............................................................................... 37 3.2.7 Isotope Kinetics ........................................................................................................ 38 3.2.8 Statistical Analysis .................................................................................................... 38 3.3 Results ........................................................................................................................... 40 3.3.1 Participants ................................................................................................................ 40 3.3.2 Tracer Oxidation ....................................................................................................... 42 3.3.3 Plasma Amino Acids................................................................................................. 44 3.4 Discussion ..................................................................................................................... 46 Chapter 4: Dietary Aromatic Amino Acid Requirements during Early and Late Gestation in Healthy Pregnant Women.......................................................................................................51 4.1 Introduction ................................................................................................................... 51 4.1.1 Objectives and Hypothesis ........................................................................................ 52 4.2 Methods......................................................................................................................... 52 4.2.1 Participants ................................................................................................................ 52 4.2.2 Experimental Design ................................................................................................. 53 4.2.3 Preliminary Assessment of Participants .................................................................... 54 4.2.4 Study Diets ................................................................................................................ 55 4.2.5 Isotope Protocols ....................................................................................................... 56 4.2.6 Sample Collection and Analysis ............................................................................... 56 4.2.7 Isotope Kinetics ........................................................................................................ 57 x  4.2.8 Statistical Analysis .................................................................................................... 58 4.3 Results ........................................................................................................................... 59 4.3.1 Participants ................................................................................................................ 59 4.3.2 Tracer Oxidation ....................................................................................................... 61 4.3.3 Plasma Amino Acids................................................................................................. 62 4.4 Discussion ..................................................................................................................... 63 Chapter 5: A Natural History of Maternal Phenylketonuria Patients at Vancouver General Hospital .........................................................................................................................................69 5.1 Introduction ................................................................................................................... 69 5.1.1 Objectives and Hypothesis ........................................................................................ 70 5.2 Methods......................................................................................................................... 71 5.2.1 Population ................................................................................................................. 71 5.2.2 Dietary Management ................................................................................................. 72 5.2.3 Original Sample Analysis ......................................................................................... 73 5.2.4 Statistical Analysis .................................................................................................... 73 5.3 Results ........................................................................................................................... 74 5.3.1 Subject Characteristics .............................................................................................. 74 5.3.2 Dietary Recommendations and Intake ...................................................................... 75 5.3.3 Blood Spot and Plasma Analysis .............................................................................. 79 5.4 Discussion ..................................................................................................................... 82 Chapter 6: Discussion and Conclusion ......................................................................................89 6.1 Discussion ..................................................................................................................... 89 6.2 Limitations and Strengths ............................................................................................. 93 xi  6.3 Future Directions .......................................................................................................... 95 6.4 Conclusion .................................................................................................................... 95 Bibliography .................................................................................................................................97 Appendices ..................................................................................................................................114 Appendix A - Recruitment Materials for Chapter 3 ............................................................... 114 Appendix B - Consent Form for Chapter 3 ............................................................................. 117 Appendix C - Preliminary Assessment Form for Chapter 3 ................................................... 124 Appendix D - Study Day Form for Chapter 3......................................................................... 126 Appendix E - Master List for Chapter 3 ................................................................................. 127 Appendix F - Dietary Record for Chapter 3 ........................................................................... 128 Appendix G - Recruitment Materials for Chapter 4 ............................................................... 129 Appendix H - Consent Form for Chapter 4 ............................................................................ 131 Appendix I - Preliminary Assessment Form for Chapter 4 .................................................... 139 Appendix J - Study Day Form for Chapter 4 .......................................................................... 141 Appendix K - Master List Chapter 4 ....................................................................................... 142 Appendix L - Diet Record Chapter 4 ...................................................................................... 143  xii  List of Tables  Table 1 – Classification of the amino acids as adapted from (1) .................................................... 5 Table 2 - DRI recommendations for TAA intake in adults and pregnant women ........................ 26 Table 3 - Summary of aromatic amino acid requirement studies done in adults .......................... 27 Table 4 - List of study participants with individual test intakes ................................................... 34 Table 5 – Participant Characteristics1 ........................................................................................... 41 Table 6 - Study day assessments1 ................................................................................................. 41 Table 7 - Participant Characteristics1 ............................................................................................ 60 Table 8 - Study day assessments of healthy pregnant women during early and late gestation1 ... 60 Table 9 - Subject Characteristics .................................................................................................. 75 Table 10 - Summary of Gestational Weight Gain in mPKU Pregnancies .................................... 75 Table 11- Prescribed vs. Actual Dietary Intake in mPKU Pregnancies Adjusted for Body Weight....................................................................................................................................................... 76 Table 12 - Prescribed vs. Actual Dietary Intake in mPKU Pregnancies on a Per Day Basis ....... 77 Table 13 - Blood Spot Phenylalanine and Tyrosine Values in mPKU Pregnancies ..................... 79 Table 14 - Plasma Amino Acid Panel in mPKU Pregnancies ...................................................... 81 Table 15 - Biochemical Variables in mPKU Pregnancies ............................................................ 82  xiii  List of Figures  Figure 1 - Conversion of phenylalanine to tyrosine via PAH as adapted from (16) ...................... 7 Figure 2 - Phenylalanine and Tyrosine metabolism as adapted from (16) ..................................... 7 Figure 3 - Amino acid transportation. Extracted from (65). ......................................................... 16 Figure 4 - Schematic of 13CO2 production (orange star represents 13C) during phenylalanine oxidation. Adapted from (75). ...................................................................................................... 19 Figure 5- Response pattern of increasing intake of the test amino acid, extracted from (71). ..... 21 Figure 6 – Estimated average protein requirement in early (13-19wk) gestation. Extracted from (4). ................................................................................................................................................. 23 Figure 7 – Estimated average protein requirement during late (33-39wk) gestation. Extracted from (4). ........................................................................................................................................ 23 Figure 8 – Estimated average lysine requirement in early (13-19wk) gestation. Extracted from (6). ................................................................................................................................................. 24 Figure 9 – Estimated average lysine requirement in late (33-39wk) gestation. Extracted from (6)........................................................................................................................................................ 24 Figure 10 - CONSORT flow chart depicting enrollment and allocation of participants .............. 33 Figure 11 - Estimated average requirement of phenylalanine in early pregnancy as determined by the DAAO method ........................................................................................................................ 43 Figure 12 - Estimated average requirement of phenylalanine in late pregnancy as determined by the DAAO method ........................................................................................................................ 43 Figure 13 - Estimated average requirement of phenylalanine in late pregnancy as determined by the IAAO method .......................................................................................................................... 44 xiv  Figure 14 – Plasma concentrations of phenylalanine and tyrosine in early pregnancy (DAAO) . 45 Figure 15 – Plasma concentrations of phenylalanine and tyrosine in late pregnancy (DAAO) ... 45 Figure 16 – Plasma concentrations of phenylalanine and tyrosine in late pregnancy (IAAO) ..... 46 Figure 17 - CONSORT flow chart depicting enrolment and allocation of participants ............... 53 Figure 18 - Estimated average requirement of TAA in early gestation using the Indicator Amino Acid Oxidation (IAAO) method in healthy pregnant women. ..................................................... 61 Figure 19 - Estimated average requirement of TAA in late gestation using the Indicator Amino Acid Oxidation (IAAO) method in healthy pregnant women. ..................................................... 62 Figure 20 -Plasma concentrations of phenylalanine and tyrosine in early pregnancy in response to graded phenylalanine intakes in healthy pregnant women. ...................................................... 63 Figure 21 - Plasma concentrations of phenylalanine and tyrosine in late pregnancy in response to graded phenylalanine intakes in healthy pregnant women. .......................................................... 63 Figure 22 - Phenylalanine Tolerance Throughout mPKU Pregnancies ........................................ 78 Figure 23 - Phenylalanine Concentrations in Blood Spots Throughout mPKU Pregnancies ....... 80  xv  List of Abbreviations AAA – Amino Acid Analyzer AIC – Akaike information criterion AICc – Corrected Akaike information criterion APE – Atoms percent excess BH4- Tetrahydrobiopterin BIA – Bioelectric impedance analysis BMI – Body mass index CF-IRMS – Continuous flow isotope ratio mass spectrometer DRI – Dietary Reference Intakes DAAO – Direct amino acid oxidation EAA – Essential amino acids EAR- Estimated average requirement FFM – Fat free mass FM – Fat mass GWG – Gestational weight gain IAAB – Indicator amino acid balance IAAO – Indicator amino acid oxidation IOM – Institute of Medicine IUGR – Intrauterine growth restriction mPKU – Maternal phenylketonuria NEAA – Nonessential amino acids PAH – Phenylalanine hydroxylase xvi  PKU – Phenylketonuria RDA – Recommended dietary allowance REE – Resting energy expenditure TAA – Total aromatic amino acids   xvii  Acknowledgements I offer my sincere gratitude to Dr Rajavel Elango for helping me deepen my understanding and appreciation of reproductive medicine and nutrition. The opportunities you provided to me, especially when I was in the role of an educator, have inspired me to pursue a career in science education. As well, thank you to all of the wonderful individuals I have had the opportunity to work with throughout the past five years: Betina, Katia, Abrar, Haneen, Kerri, Maggie, Anna, Alejandra, Annie, Taryn, Jennifer, Kevin and Roger. The support, coffee breaks, and friendship made my time at UBC a joy.  I realize now how unbelievably lucky I was with my Supervisory Committee and Chair. Dr Beristain, Dr Devlin, Dr Rurak, and Dr Lim: my education, research projects, and post-graduate prospects have been vastly improved by having you as part of my support system. Thank you for going above and beyond as often as you did.  My research projects would not have been possible without the support of BC Children’s Hospital Research Institute, The University of British Columbia, Canadian Institutes of Health Research and all of my wonderful participants. The gratitude I feel for my family cannot fit in this dissertation. Mom, Dad, Gen, Kev, Rob, Alisha, Mark, Kara, Marilyn, Bruce, Amelia, Liam, Garnet, Lea, Batman, and Pretzel. The family dream team. Thank you all so much.  Lastly, I need to thank my wonderful husband, Aubrey. You were the perfect combination of listener, advocate, personal chef, comedian, and dance partner over the past 5 years. Your active interest in my work, your encouragement to take every opportunity I was offered, and your indescribable amount of support got me here.    xviii  Dedication  For Aubrey, Mom, and Dad.1  Chapter 1: Introduction Protein is vital to the human body. It is integrated into cell membranes and enzymes while also a major building block for muscles, skin, hair, and bone. There is a continuous and well-regulated balance of protein metabolism in the body. Synthesis and degradation are continually occurring processes, balanced by the body’s health and regulatory mechanisms. There are 20 amino acids that make up protein in mammals, all of which are required for protein synthesis to occur. (1). Amino acids are classified into 3 groups based on a dietary perspective (1). Dispensable (nonessential), amino acids can be synthesized by the human body to meet the full physiological requirement (1). Conversely, indispensable (essential) amino acids must be obtained through the diet. The remaining amino acids are termed conditionally indispensable, because endogenous synthesis is limited under certain physiological conditions such as disease, pregnancy, or indispensable amino acid deficiency.  While dietary protein is required during adulthood for healthy body maintenance, it is especially significant during periods of critical growth such as pregnancy, childhood, and adolescent years (2). During a successful pregnancy, two individual cells come together, multiply and differentiate to create a healthy baby. A healthy maternal diet with adequate protein intake for normal growth and development is a critical factor in this process (2). Under-nourished women, over-nourished women, or women with health complications resulting in inadequate nutrient delivery to the fetus are at an increased risk of adverse birth outcomes such as intrauterine growth restriction (IUGR) and preeclampsia (3). These complications do not solely affect the fetus into infancy, but have life-long health implications. This includes, but is not limited to, an increased risk of cardiovascular disease, developmental delays, and diabetes. Therefore, it is extremely important to study, understand, 2  and educate pregnant women about proper nutrition and protein/amino acid requirements to optimize birth outcomes.  It was only recently that total dietary protein requirements during different stages of human pregnancy were determined (4). The results suggested that mean protein requirements during early pregnancy are 1.22 g·kg-1·d-1, and progressively increase with later stages of gestation to 1.52 g·kg-1·d-1. These findings are higher than the estimated average requirement (EAR) currently provided in the Dietary Reference Intakes (DRI) of 0.88 g·kg-1·d-1, and suggest the individual amino acid requirements increase as pregnancy progresses (5). Following the protein requirement study, lysine requirements (a key indispensable amino acid) were determined during early and late gestation (6). Similarly, the dietary lysine requirement was higher in late (50.3 mg·kg-1·d-1) when compared to early (36.6 mg·kg-1·d-1) pregnancy, and did not match current DRI recommendations of 41 mg·kg-1·d-1 (5). The results of these 2 studies highlighted that the DRI’s current method of factorially calculating protein and amino acid requirements during pregnancy is not appropriate. As well, it illustrates that static recommendations during pregnancy do not account for potential differences in requirement between early and late pregnancy, when there are dynamic changes in metabolism and rate of tissue accretion. Therefore, it is imperative that the requirements for the remaining 8 indispensable amino acid are determined experimentally during pregnancy.  Phenylalanine is another key indispensable amino acid. Though its principal role is as a building block for proteins, it is also hydroxylated by the enzyme phenylalanine hydroxylase (PAH) to synthesize tyrosine (a conditionally indispensable amino acid). Tyrosine, in turn, is the precursor for the catecholamines dopamine, epinephrine and norepinephrine, which play important roles in physiological processes (7). Plasma tyrosine concentrations are directly related 3  to phenylalanine intake and a dietary deficiency of phenylalanine has severe growth consequences (8). Currently, the EAR for phenylalanine and tyrosine requirements during pregnancy are paired in the DRI under Total Aromatic Amino Acids (TAA) and set at 36 mg·kg-1·d-1. There is no recommendation for minimum phenylalanine (the dietary phenylalanine requirement when the tyrosine requirement is met in the diet). The requirements for minimum phenylalanine and TAA have never been experimentally determined during human pregnancy, and whether these requirements differ between stages of gestation is unknown (9,10). Determining the minimum phenylalanine requirement could have significant impacts to health outcomes during pregnancy, particularly in those with specific inborn errors of metabolism.  A congenital deficiency of PAH elevates plasma phenylalanine and its related metabolites to toxic levels (11). This inherited disorder, known as phenylketonuria (PKU), results in intellectual impairment, seizures, and motor deficits when untreated (11). For pregnant women with PKU (maternal PKU, mPKU), there are further complications due to the teratogenic effects of elevated phenylalanine concentrations on fetal development. This condition leads to mPKU syndrome, and can result in fetal microcephaly, spontaneous abortions, heart defects, and postnatal morbidity (12,13). Therefore, it is imperative that women with PKU are properly monitored and managed with phenylalanine and tyrosine throughout pregnancy.  The objectives of this dissertation were to determine minimum phenylalanine and TAA requirements in healthy pregnant women using non-invasive novel stable isotope-based techniques. As well, I conducted a retrospective natural history analysis of current management practices and the efficacy of these practices for women with mPKU attending the Vancouver General Hospital’s Adult Metabolic Diseases Clinic, in Vancouver, BC Canada.  4  Chapter 2: Literature Review 2.1 Overview of Protein and Amino Acids Protein plays an essential role in the human body. They are key components in enzymes, hormones, transport carriers, cellular membranes, support structures, and connective tissues. Protein is made up of 20 amino acids, 9 of which must be consumed in the diet (1). These indispensable, or essential, amino acids enter the body as intact protein and must be broken down by the digestive system into amino acids before being absorbed through the gastrointestinal wall (14). Protein digestion begins in the mouth, where teeth allow for mechanical breakdown during chewing (15). After swallowing, protein moves into the stomach where proteases such as pepsin cleave it into shorter peptides. Pepsin must first be converted from its zymogen, pepsinogen, by hydrochloric acid before protein cleaving can occur. The bolus, with its polypeptides, will then pass into the duodenum for further breakdown of the peptide bonds by trypsin into amino acids. Following this, the amino acids are absorbed into enterocytes and mesenteric capillaries in the duodenum. Once these amino acids are circulating in the blood stream they can be used for protein synthesis, used for energy, oxidized, or excreted (14). Amino acids are the most basic components of protein. Each contains a central carbon to which a carboxyl (COO-) and amine (NH3+) group are bonded. The R-group, a side chain, is variable and depending on its configuration determines what amino acid is formed. Amino acids that make up mammalian protein fall into 3 aforementioned categories: indispensable, conditionally indispensable, or dispensable (Table 1). All 20 amino acids must be present for endogenous protein synthesis to occur. If even one amino acid is limiting, there will be suboptimal anabolism.  5  Indispensable Conditionally Indispensable Dispensable Histidine  Isoleucine Leucine Lysine Tryptophan Phenylalanine Valine Threonine Methionine Arginine Tyrosine Glutamine Glycine Proline Cysteine Serine Alanine Asparagine Aspartic Acid Glutamic Acid Table 1 – Classification of the amino acids as adapted from (1)  With the exception of glycine, all standard amino acids are classified by their chirality or optical activity. Depending on the direction of the isomer, amino acids are classified as either D- or L-, referring to an asymmetrical compound’s ability to rotate in a certain plane of light (15). All amino acids found in protein have an L-configuration. D-configuration amino acids are not involved in human metabolism because they are not synthesized by transcription and translation, but by specific bacterial enzymes.   2.2 Phenylalanine and Tyrosine Phenylalanine (C9H11NO2) and tyrosine (C9H11NO3) are indispensable and conditionally indispensable aromatic amino acids, respectively (16). These are relatively hydrophobic (non-polar) because of their aromatic side chains.   6  2.2.1 Dietary Sources of Phenylalanine Due to the metabolic disorder PKU (described in detail below), phenylalanine sources in food have been widely studied. Foods containing high quantities of phenylalanine (>50mg/serving) include: meat, fish, chicken, eggs, cheese, wheat flour, dried fruits, spinach, soya, and jelly (17). Other foods containing between 30mg and 50mg per serving include: milk, potatoes, corn, oats, rice, peas, and chocolate (17). Of note, aspartame is a synthetic dipeptide sweetener that is the product of L-aspartate reacting with L-phenylalanine (18). This creates an unexpected source of phenylalanine in foods with this additive.   2.2.2 Metabolism of Phenylalanine and Tyrosine As illustrated in Figure 1, the liver and kidney can metabolize phenylalanine to tyrosine via the enzyme PAH (19). PAH is a mixed-function oxidase, which simultaneously hydroxylates a substrate with one oxygen atom from O2, and reduces the remaining oxygen atom to H2O (16). PAH requires the cofactor tetrahydrobiopterin (BH4), which is oxidized to dihydrobiopterin, allowing electrons to be carried from NADPH to O2 (20). Following this, tyrosine can be converted to p-hydroxyphenylpyruvate via tyrosine aminotransferase, and eventually into fumarate and acetoacetyl-CoA (Figure 2). Alternatively, tyrosine can be converted to dihydroxyphenylalanine (DOPA) via tyrosine hydroxylase, allowing for the consecutive formation of important neurotransmitters dopamine, noradrenaline and adrenaline (Figure 2). Not detailed in Figure 2, is the formation of melanin from tyrosine. 7   Figure 1 - Conversion of phenylalanine to tyrosine via PAH as adapted from (16)    Figure 2 - Phenylalanine and Tyrosine metabolism as adapted from (16)  An alternative pathway for catabolism of phenylalanine, which is well studied in individuals with PKU, is the conversion to phenylpyruvate. This, in turn, can be converted into either phenylacetate or phenyllactate (Figure 2). 8   2.2.3 Catecholamines Phenylalanine and tyrosine are the precursors the catecholamines dopamine, norepinephrine, and epinephrine (21). The link between these amino acids and catecholamines is of importance, as rate of synthesis and release of these neurotransmitters are directly impacted by brain concentrations of phenylalanine and tyrosine, which is directly related to their availability in plasma (22). Both a deficiency and an excess of TAA can result in decreased catecholamine production.  The classic “fight or flight” response, mediated by the sympathetic nervous system, is the result of catecholamine release. Catecholamines are monoamine neurotransmitters that have both a side chain amine and a catechol (consists of a benzene and 2 hydroxyl side groups). These neurotransmitters have several physiological effects.  For example, they regulate vascular                smooth muscle contraction impacting blood pressure and regulate cardiac muscle contraction impacting heart rate (23). Catecholamines have further effects on the musculoskeletal system including piloerection, gastrointestinal and urinary smooth muscle relaxation, and expansion of the smooth muscle in the bronchioles. Catecholamines can influence blood glucose concentrations by increasing glucagon secretion from pancreatic alpha cells and decreasing insulin secretion from pancreatic beta cells which stimulates glycogenolysis and gluconeogenesis in the liver (23).   2.3 Phenylketonuria PKU is an inherited metabolic disorder characterized by variants in the gene that encodes PAH (11). Reduction or loss of PAH function results in increased concentrations of 9  phenylalanine in the blood that are toxic to the brain. PKU is an autosomal recessive disorder, arising when both alleles contain mutations. As of 2007, 548 different variants have been found to cause PKU (24). This disorder is treatable with dietary phenylalanine restriction and, for some individuals, BH4 supplementation. It is critical to determine individual phenylalanine tolerance, which is defined as the amount of phenylalanine (in mg/kg of body weight/day) that can be ingested while maintaining healthy blood phenylalanine concentrations (120-360 µmol/L). Given that tyrosine is an indispensable amino acid in this population, plasma levels and dietary intake should also be monitored. If left untreated, PKU symptoms can include, but are not limited to, intellectual impairment, developmental problems, seizures, and motor problems. Due to newborn screening, early diagnosis and intervention has greatly improved the outcomes for individuals with PKU over the last 60 years (11).   2.3.1 Maternal Phenylketonuria Elevated phenylalanine levels during pregnancy, caused by untreated PAH deficiency, are teratogenic to the fetus. Complications that arise include, but are not limited to, IUGR, developmental delays, facial dysmorphia, microcephaly, and congenital heart defects (25). A direct relationship between plasma phenylalanine concentrations and the frequency of fetal abnormalities has been reported in in women with untreated PKU during pregnancy (26). This is likely due to the active transport of phenylalanine across the placenta, which results in a 70% increase in fetal plasma concentrations in comparison to maternal concentrations (27). It is well established that blood phenylalanine concentrations between 120 and 360 µmol/L (or between 2 and 6 mg/dl) result in the best health outcomes for the developing fetus (28,29). While this is ideal to achieve preconception, the benefits are apparent if achieved by 8 wk gestation (28,29). 10  This is true, with the exception of congenital heart defects, as the heart is almost completely formed by 8 wk gestation (30). Therefore, plasma phenylalanine and tyrosine concentrations; dietary protein, phenylalanine and tyrosine intake; and fetal growth and development must be carefully monitored in pregnant women with PKU. This is especially critical as phenylalanine tolerance is known to increase throughout gestation (31). Due to a combination of the recent rise in mPKU cases due to better PKU management practices, ethical constraints when studying women with mPKU, and small sample sizes, little data are available on mPKU. The maternal PKU collaborative study, which aimed to ascertain the efficacy of dietary phenylalanine restriction on birth outcomes, reported that maternal protein, phenylalanine, and caloric intake were negatively correlated with plasma phenylalanine throughout pregnancy (13). Inadequate consumption of medical foods (a food specifically formulated and intended for consumption by a diseased population with specific nutritional needs), often due to untreated nausea and vomiting, led to low overall nutrient intake, increased incidence of congenital anomalies and poor birth outcomes (28). Conversely, appropriate phenylalanine intake (which increases throughout pregnancy), paired with adequate energy and fat intake are associated with improved birth outcomes for both mother and fetus (13). Interestingly, there is no current evidence that dietary tyrosine intake or tyrosine plasma concentrations are related to health outcomes in mPKU subjects (32). However, tyrosine supplementation is still recommended if blood spot tyrosine concentrations drop below the normal range, in case there is a role in fetal damage (33).  11  2.4 Maternal Nutrition 2.4.1 Metabolic Adaptations during Pregnancy During pregnancy, there are extensive changes to most physiological systems within the body to establish an ideal environment for the growing fetus and to prepare the mother for labor, delivery, and lactation (34,35). It has been well established that these adaptations require increased consumption of energy and most nutrients (36). Several metabolic adaptations have arisen to facilitate this increased demand. During the first half of pregnancy, anabolic adaptations facilitate an increase in fat stores to prepare for the impending energy demands of the fetus (37,38). The mother will begin to live in a hyperinsulinemic state, redirecting nutrients in the blood through the placenta to the fetus. There is a decrease in insulin sensitivity, with an increase in insulin secretion, glucose utilization, and lipogenesis (39). However, towards the end of pregnancy there is a shift in the mother to a catabolic state, as the nutritional demands of the fetus increase. Glucose and amino acids are directed towards the fetus and maternal lipid utilization increases (38,40).   2.4.2 Impacts of the Maternal Diet on the Fetus The maternal diet plays a key role in the development of the fetus (41). Insufficient maternal nutrition has been linked to IUGR, which is associated with a greater risk of fetal hypoglycemia and hypoxia (42,43). The complex issue of the effects of maternal undernutrition during pregnancy on the health of the adult child was most famously studied longitudinally in women who conceived during the “Hunger Winter” in the Netherlands during World War II (44). During this time, there was extreme food deprivation in areas of the Netherlands, due to the German army cutting off the food supply. Obstetrical records collected during this famine 12  allowed scientists to illustrate the short and long term health effects in the descendants of those undernourished from both nutrients and energy (44). The short-term effects included reduced head circumference and body length at birth due to IUGR (45). Long term effects included increased risk of developing coronary heart disease, altered blood coagulation, obesity, and obstructive airway diseases through adulthood (46,47). To note, these effects are likely due to the combined effect of maternal undernutrition and stress, and it is not possible to determine which played a greater role. Similarly, other studies have been done of offspring conceived during the Great Chinese Famine and the Bangladesh Famine, showing similar detrimental effects of malnourished and stressed mothers (48).   2.4.3 Impact of Dietary Protein and Amino Acids Intake during Pregnancy Proper protein consumption during pregnancy is key for placental and fetal tissue development, mammary growth, and the increase in blood volume (10,49). IUGR has been linked to both excessive and insufficient protein consumption during pregnancy (50). Similarly, IUGR has been connected to a decrease in transport of amino acids across the placenta (poor availability of amino acids for the fetus) as well as a reduced ratio of fetal to maternal indispensable amino acid concentrations (51). Throughout a healthy pregnancy, fetal plasma amino acid concentrations increase and maternal concentrations decrease. This illustrates preferential and active transport of amino acids to the fetus and placenta (51,52). However, in the case of IUGR, this adaptation often does not occur to the same extent (51). This can be because of a deficiency of dietary protein of a specific amino acid, where fetal uptake reduces maternal supply of amino acids. If this issue is chronic, it can increase the risk of IUGR because there are suboptimal concentrations of amino acids for healthy tissue accretion. On the other hand, 13  excessive consumption of protein can lead to saturation of the placental amino acid transporters. Certain amino acids will out compete others for the use of the transporters. Without all the amino acids properly crossing the placenta, insufficient protein is formed, again increasing the risk of IUGR (53). While it is understood maternal protein intake plays a key role in healthy fetal development, it is also important to remember there are other factors in fetal amino acid supply. Firstly, the placental syncytiotrophoblast (the epithelial covering of the placental villi) requires facilitated, exchange, and accumulative transporters on both the apical and basal membranes to function harmoniously (52). As well, factors such as blood flow, metabolism, and placental structure can play key roles.   2.4.4 Maternal Amino Acid Metabolism Nitrogen (protein) that is retained during healthy human pregnancy has previously been calculated; 40% of protein gain during pregnancy is represented by the fetus, placenta, and amniotic fluid (54). Maternal tissues represent the other 60%. This includes breast tissue, uterine tissue, blood, and extracellular fluids (10,55). The amount of protein accretion that occurs during this life stage indicates dynamic shifts in protein and amino acid metabolism.  At 3 wk gestation, plasma amino acid concentrations are significantly lower in pregnant women compared to non-pregnant women, with the exception of threonine, lysine and histidine (56). This trend continues throughout pregnancy for most amino acids, with the greatest decline for glucogenic amino acids and amino acids in the urea cycle (57). Additionally, there is a decrease in maternal urea synthesis and urea excretion starting in early pregnancy (39,55). Therefore, increases in protein synthesis, paired with decreased amino acid catabolism (and urea synthesis), may be a compensation mechanism to increase protein retention during pregnancy. 14  2.4.5 Placental and Fetal Amino Acid Metabolism Studying amino acid metabolism in human placentas and fetuses poses too many ethical and technical constraints to be widely researched. The majority of current knowledge on fetal and placental amino acid metabolism comes from studies conducted using animal models. However, with the development of non-invasive stable isotope techniques there has been some insight into this poorly understood process. We know that fetal plasma amino acid concentrations are higher than maternal plasma concentrations. This is in part due to active uptake of amino acids from maternal circulation by the placenta, preferentially delivering amino acids to the fetus (56). As well, Chien et al. have showed that fetal whole-body protein synthesis was approximately 13 g·kg-1·d-1 through infusion of stable isotope labelled amino acids into the umbilical cord (58). They also showed that protein accretion in the fetus was occurring at a rate of 2-4 g·kg-1·d-1 (measured just prior to delivery). More recently, it has been shown that protein accretion may be closer to 1.7 g·kg-1·d-1 in the fetus near delivery (59). It is obvious that current data are limiting and conflicting, indicating fetal protein metabolism needs to be studied more in depth.  The placenta is a highly metabolically active organ. It forms a border between maternal and fetal circulation, enabling metabolite and gas exchanges as well as waste disposal for the fetus (60). While it acts as a barrier against the maternal immune system and is an endocrine powerhouse, the placenta also plays a huge role in protein metabolism including: synthesis, oxidation, transamination, and dispensable amino acid production (61–65). The placenta contains even higher concentrations of amino acids than the fetal compartment (66). Depending on the amino acid, transplacental transport occurs at different rates with a variety of transporters and mechanisms (65). As well, amino acids that are taken up from maternal circulation by the 15  microvillous membrane of the syncytiotrophoblast may either be directly transferred to the fetus (through the basal membrane) or utilized by the placenta. This means that fetal supply of amino acids isn’t solely dependent on placental uptake from maternal circulation, but also depends on utilization and rate of metabolism by the placenta. In fact, the placenta’s ability to metabolize amino acids is necessary for fetal supply in the case of amino acids like glycine (67).  Each of the transport systems used has high substrate specificity. Exchange transporters alter the amino acid concentrations on either side of the placenta without altering the osmolality across the membrane (67). This utilizes an antiport mechanism to allow for the efflux of accumulated amino acids from the trophoblast cytosol and allows for other amino acids to cross into the placenta. Accumulative transporters are activated by electrochemical differences across the membranes to draw amino acids into the trophoblast cytosol. Lastly, there are also facilitated transporters to move amino acids down their concentration gradients, allowing more amino acids to cross into the fetal compartment (65). All of these transporters contribute to an overall concentration gradient, and shows that placental amino acid transporter movement is highly interdependent (Figure 3). For example, increased transport of phenylalanine into the fetal compartment is accompanied by shifts in electrochemical and osmolality gradients, having a cascading effect on the rate of the other amino acids’ transfer between the maternal and fetal compartments (68). The placenta is such an integral organ in gestational protein metabolism.  16   Figure 3 - Amino acid transportation. Extracted from (65).  2.4.5.1 Placental Transport of Phenylalanine and Tyrosine Placental transport of amino acids has been fairly well studied, especially in animal models. It is clear that amino acid flux for the fetus depends on multiple factors such as: nutrient availability, transport proteins on the microvillous membrane and basal membrane, blood flow, and maternal and placental amino acid metabolism (69). Phenylalanine and tyrosine utilize the exchangers LAT1 and LAT2 and the facilitated transporters TAT1, LAT3, and LAT4 to cross the placenta. Both of these transport mechanisms are dependent on concentration gradients. While it is possible for phenylalanine to be utilized by the placenta to synthesize protein, there is negligible PAH present in a human placenta, meaning loss due to oxidation is little to none (19). This also means that tyrosine used in the placenta and fetus is mainly from maternal supply.  17  2.5 Methods to Determine Amino Acid Requirements Recently, there has been significant research done to determine protein and amino acid requirements in humans. There has been a keen focus of vulnerable populations such as children, the elderly, pregnant women, and diseased patient populations. There are a number of techniques available to determine these requirements, some of which are described in detail below.  2.5.1 The Nitrogen Balance Technique The nitrogen balance technique is the original method used to determine protein and amino acid requirements. This refers to the difference in dietary nitrogen consumption and nitrogen leaving the body via urine, feces, skin, etc. (70). Current DRI recommendations for protein and amino acids in non-pregnant adults are based on a meta-analyses of previously conducted nitrogen balance studies. Requirements are determined using a linear regression model analysis on the data, and measuring the point at zero nitrogen balance as the point of nutritional adequacy. This technique causes a shift in the urea pool, which is large and slow-changing, usually following a lengthy adaptation diet of the test amino acid (5-7 day period) (71). While used extensively in adult human populations, it is too invasive for vulnerable populations, such as pregnant women. The nitrogen balance method is also problematic because it underestimates protein and amino acid requirements. This mainly occurs because of an over-estimation of nitrogen intake and an underestimation of nitrogen excretion. The amount of food not consumed over a 7-day period is significant, and not properly accounted for in these studies. Additionally, not all nitrogen lost from the body can be accounted for (i.e. that incorporated into urine, breath, fecal matter, hair, skin, mucus, etc). While the nitrogen balance method is 18  functional in theory, its execution was more difficult than predicted. Now that stable isotope-based carbon oxidation methods have evolved, more studies need to be conducted.  2.5.2 Stable Isotopes Stable isotopes occur naturally in the environment. Humans are exposed to them daily, in the water we drink and the food we eat. There are stable isotope versions of the four most common atoms making up living organisms: carbon, hydrogen, oxygen, and nitrogen. These isotopes differ from their common atomic forms by containing an extra neutron, and therefore extra mass, which can be measured using mass spectrometers. Stable isotopes are an attractive alternative to radioactive tracers, which are carcinogenic, emitting radiation as they decay over time.  2.5.3 Indicator Amino Acid Oxidation Technique The IAAO technique was first developed in maturing pigs. By measuring the oxidation of one amino acid (the indicator amino acid; usually phenylalanine, leucine or lysine), we are able to determine the requirements of the test amino acid (72). As amino acids are unable to be stored within the body, the indicator amino acid will either be incorporated into protein or oxidized. Therefore, when providing subjects with graded doses of the test amino acid, we can determine the requirement by indirectly measuring oxidation of the indicator amino acid. As the test amino acid intake increases, oxidation of the indicator will decrease as more protein is able to be synthesized (Figure 5). Eventually, oxidation will plateau, creating a breakpoint, where amino acid requirements have been met (71). This method involves a 2-day adaptation diet to a set test 19  amino acid intake prior to an 8-hour study day that will allow for measurement of the oxidation of the indicator amino acid at a specific intake.  The amino acid used as the indicator should fulfill three specific requirements. Firstly, it must be an indispensable amino acid. Secondly, it must have a carboxyl labelled carbon that can be oxidized upon catabolism, which will be released quickly as CO2, so it can be measured in breath samples (Figure 4). Lastly, it must have a small pool within the body and not be involved in significant pathways other than protein synthesis and oxidation (71). This reduces the loss of our indicator amino acid to non-measurable pathways. In reality, there is only one amino acid that fits all three of these criteria, phenylalanine (in the presence of excess tyrosine). Lysine and leucine also closely fit the criteria with the following drawbacks: lysine has a large pool within the body and leucine has the potential to underestimate results because it has been shown to stimulate protein synthesis (73). However, leucine has been shown to provide clear breakpoints if provided at the EAR of the population being studied (74).  Figure 4 - Schematic of 13CO2 production (orange star represents 13C) during phenylalanine oxidation. Adapted from (75). 20  2.5.4 Direct Amino Acid Oxidation Technique The direct amino acid oxidation (DAAO) technique has a similar concept to that of the IAAO technique, with a few key differences. Here, the indicator and test amino acids are the same. As an example, L-[1-13C]phenylalanine is used as an indicator while determining phenylalanine requirements. Oxidation of the indicator amino acid will start at a slow constant state (Figure 5) at low intakes of the test amino acid, and once the requirement is reached, oxidation of the indicator (and test) amino acid will begin the rise progressively with increasing intakes (5). There are drawbacks to this approach. Unlike the other methods described above, indicator oxidation will never be measured in the absence of the test amino acid in the diet because a small portion will be required with the stable isotope label. Of course, this is observable in the graph depicting oxidation versus intake and is not a significant problem, except when the breakpoint (requirement) is at a low intake.  2.5.5 24-Hour Indicator Amino Acid Balance and Oxidation Technique Young et al. modified the IAAO technique, adding a six day adaptation diet for the test amino acid followed by a 24 hour study period to measure oxidation of the indicator amino acid (76). This accounts for oxidation and balance of the indicator amino acid over fasting and fed states; balance is determined by subtracting the indicator oxidation from intake (71). This method requires 6 days of an adaptation diet while the urea cycle metabolism of the nitrogen components adapt to new protein intakes (77). Conversely, when only measuring oxidation, a 2-day adaptation diet is required because changes to oxidation are more immediate. The protocol for this method is more demanding and invasive, making it not ideal for pregnant populations. 21  As well, differences in requirement between this method and the 8-hour IAAO method are not significant (73).   Figure 5- Response pattern of increasing intake of the test amino acid, extracted from (71).  2.5.6 Importance of Excess Tyrosine When using L-[1-13C]phenylalanine as the indicator amino acid, it must be in the presence of excess dietary tyrosine. Phenylalanine, as described above, is endogenously converted to tyrosine via PAH. Therefore, when providing doses of phenylalanine in the presence of excess tyrosine, it prevents the use of phenylalanine to meet the tyrosine requirement, allowing for the release of the carboxyl carbon and a more accurate measure of phenylalanine oxidation.  As well, when trying to determine minimum phenylalanine requirements (not phenylalanine and tyrosine requirements together), excess tyrosine needs to be provided. In a study measuring minimum phenylalanine requirements in adult humans, a low conversion of 22  phenylalanine to tyrosine was seen when providing excess tyrosine in the diet, measured using [13C]tyrosine enrichment in plasma (78).Another study has also shown that tyrosine provided in excess of the requirement spares ~75% of the phenylalanine requirements in adults (79). These findings highlight that accurately determining minimum phenylalanine requirements directly relies on tyrosine intake.  2.5.7 The Importance of Bicarbonate When using 13C labelled compounds, such as amino acids, to track metabolism, it has been well documented that a portion of 13C that is removed during oxidation is not expelled in the breath as CO2. The portion that is retained by the body is lost to bicarbonate fixation or slow exchanging pools such as bone (80). This is taken into account in our oxidation equation detailed in Chapters 3 and 4.  As well, providing a dose of labelled bicarbonate (NaH13CO3) allows us to prime (or enrich) the bicarbonate pool within the body to reach steady state of our product pool and to reduce loss from our labelled amino acid(s) (81).   2.6 Protein and Amino Acid Requirements during Pregnancy Only two studies (both conducted through the Elango Laboratory at BC Children’s Hospital’s Research Institute) have been published focusing on protein and amino acid requirements during human pregnancy. The first study, conducted by Trina Stephens (MSc 2013), determined overall protein requirements during pregnancy using the IAAO technique. Results indicated an EAR of 1.22 g·kg-1·d-1 and 1.52 g·kg-1·d-1 (Figures 6 and 7) of protein in early and late pregnancy, respectively (4). When comparing these values against the DRI’s current static recommendation of 0.88 g·kg-1·d-1, it is apparent that factorial calculations and 23  static recommendations are not appropriate for producing pregnancy specific protein guidelines (5).  Figure 6 – Estimated average protein requirement in early (13-19wk) gestation. Extracted from (4).  Figure 7 – Estimated average protein requirement during late (33-39wk) gestation. Extracted from (4).  The second study, conducted by Magdalene Payne (MSc 2014), achieved similar results while determining the requirement for the amino acid lysine using the IAAO method. The DRI’s 24  current EAR for lysine is 41 mg·kg-1·d-1 throughout all of pregnancy. This study determined a requirement of 36.6 and 50.3 mg·kg-1·d-1 (Figures 8 and 9) of lysine during early and late pregnancy respectively (6). The results from these 2 studies highlight the importance of experimentally determining protein and amino acid requirements during pregnancy, while accounting for potential differences in requirement at different stages of gestation.  Figure 8 – Estimated average lysine requirement in early (13-19wk) gestation. Extracted from (6).  Figure 9 – Estimated average lysine requirement in late (33-39wk) gestation. Extracted from (6). 25    Given the IAAO method has only recently been developed, there have been few studies performed in pregnant women. However, animal models have been studied more extensively. Reviewing studies conducted on pregnant sows provides some additional insight. Pigs are similar to humans in their dental characteristics as well as the anatomy and physiology of their skin, renal, cardiovascular, and digestive systems (82). As well, new-born pigs and humans have very similar metabolic features (such as trans and para cellular permeability in the intestines) and body composition. These parallels allow for the pig to be used as a model for human health and disease, and especially nutrition.   Similar to our human data, a series of studies on pregnant sows highlighted both the increased requirement for amino acids during pregnancy and the increase in requirement between early and late gestation. This was true for all indispensable amino acids that have been investigated, including isoleucine (by 63%), lysine (by 45%), threonine (by 55%), and tryptophan (by 35%) (83–86).  2.6.1 Current Phenylalanine and Tyrosine Recommendations Currently, nutrient intake recommendations in North America are published by the Institute of Medicine in the DRI (5). The most recent recommendations, published in 2005, provide both an EAR and a recommended dietary allowance (RDA) for TAA intake during pregnancy. The EAR is an estimated daily intake that would meet the dietary requirement for approximately half of the people of a particular life-stage and sex. This value, while useful for determining potential nutrient deficiencies, would not meet the requirement for the remaining 26  50% of a particular population. Therefore, the RDA will meet the requirement for a greater portion of individuals of a particular life stage and sex (5).  The current EAR for TAA intake during pregnancy is 36 mg·kg-1·d-1. Since this had not yet been experimentally determined, which was true for all amino acid requirements during pregnancy, this value was determined by multiplying amino acid requirements for non-pregnant women by 1.33, which is based upon nitrogen balance and potassium metabolism studies performed in a pregnant population (5). The RDA is then extrapolated by multiplying the EAR by 1.24 (the assumed CV), providing a recommendation for TAA of 44 mg·kg-1·d-1 (Table 2).   Adult (mg·kg-1·d-1) Pregnant Women (mg·kg-1·d-1) EAR 27 36 RDA 33 44 Table 2 - DRI recommendations for TAA intake in adults and pregnant women  2.7 Previous Phenylalanine and Tyrosine Requirement Studies Multiple stable isotope-based studies have been performed determining both minimum phenylalanine or TAA (phenylalanine and tyrosine) requirements in non-pregnant populations. To the best of our knowledge, only two studies have been done to determine minimum phenylalanine requirements. Firstly, Zello et al. provided graded intakes of phenylalanine in the presence of excess tyrosine to healthy adults (78). Oxidation of L-[1-13C]phenylalanine was low and constant below 10 mg·kg-1·d-1. After performing breakpoint analysis, the requirement was found to be 9.1 mg·kg-1·d-1. Though they only experimentally determined minimum phenylalanine requirements, they theorized that since tyrosine makes up 66% of the TAA 27  requirement, it is possible the TAA requirement is ~30 mg·kg-1·d-1. The second study determined minimum phenylalanine requirements in term and preterm neonates (9). Employing the IAAO technique with L-[1-13C]lysine 2HCl as the indicator, Hogewind-Schoonenboom et al observed breakpoints (the requirements) at 58 and 80 mg·kg-1·d-1 for term and preterm neonates respectively.   Reference Amino Acid(s) Method EAR (mg·kg-1·d-1) Sample Size (participants, individual observations) (78) Phenylalanine DAAO 9.1 10, 41 (87) Tyrosine IAAO 6 6, 42 (77) TAA 24h-DAAB 39 16, 16 (79) TAA IAAO 48 5, 40 (74) TAA IAAO 42 5, 35 (88) TAA 24h-IAAB 38 32. 64 Table 3 - Summary of aromatic amino acid requirement studies done in adults  The first study to investigate TAA requirements in adults, published in 1998, used a 24-hour tyrosine balance as the physiological endpoint with only three dietary phenylalanine intakes. Following 6 days of controlled phenylalanine intake, this study suggested a requirement of 39 mg·kg-1·d-1 (77). In 2001, Roberts et al used the IAAO technique (using L-[1-13C]lysine) determined the TAA requirement in healthy adults (87). Fixing the phenylalanine intake at 9.0 mg·kg-1·d-1 based on Zello et al. (78), they determined a breakpoint at a tyrosine intake of 6.0 mg·kg-1·d-1 and paired with their fixed phenylalanine intake, they determined a TAA requirement of 15 mg·kg-1·d-1 (87). In 2006, three studies were published determining TAA requirements in adults. Firstly, Hsu et al provided L-[1-13C]lysine while employing the IAAO technique and determined a requirement of 48 mg·kg-1·d-1 (79). No dietary tyrosine was provided 28  on the study days, and phenylalanine provided met the tyrosine requirement. Secondly, Hsu et al provided L-[1-13C]leucine while employing the IAAO technique and determined a requirement of 42 mg·kg-1·d-1, in the absence of dietary tyrosine (74). Lastly, Kurpad et al. determined a TAA requirement in healthy Indian men using the 24-hour IAAB technique (with L-[1-13C]leucine) finding a breakpoint of 38 mg·kg-1·d-1 (88). A review paper, published in 2007, considered all of these studies and suggested the requirement is 43 mg·kg-1·d-1 based on a combination of the most recent publications (89). There are no experimental data available for phenylalanine and/or tyrosine requirements during pregnancy.  2.8 Overall Objectives The rationale, hypotheses, and results of the following objectives will be described in Chapters 3, 4 and 5. 1. I aimed to determine minimum phenylalanine (in the presence of excess tyrosine) requirements during early and late gestation in healthy pregnant women. Simultaneously, I wanted to compare the breakpoints, or requirements, determined by the IAAO (with L-[1-13C]leucine) technique and the DAAO (with L-[1-13C]phenylalanine) technique.  2. I aimed to determine the TAA (total aromatic amino acids; in the absence of dietary tyrosine) requirements during early and late gestation in healthy pregnant women. This was done using the IAAO (with L-[1-13C]leucine) technique. 3. I aimed to describe current management practices, from a dietitian’s perspective, and their efficacy for mPKU subjects at Vancouver General Hospital’s Adult Metabolic 29  Diseases Clinic, located in Vancouver British Columbia. This included dietary intake and recommendation data as well as blood and plasma analyses. These data allowed me to examine the relationship between dietary intake of protein, phenylalanine and tyrosine on blood concentrations of amino acids in mPKU pregnancies, as well as compare the intake data against protein and phenylalanine requirements determined in healthy pregnant women.       30  Chapter 3: Dietary Phenylalanine Requirements during Early and Late Gestation in Healthy Pregnant Women (90) 3.1 Introduction Pregnancy, a state of rapid tissue accretion, is accompanied by changes in energy and nutrient requirements (91–95). Increased tissue accretion from the fetus, placenta, uterine and breast tissues paired with expansion of blood and extracellular fluid volume require increased nutrient supply. Recently, we determined that when compared to early pregnancy requirements, there are significant higher requirements in late pregnancy for dietary protein (25% higher) and lysine (37% higher) in healthy human pregnancies (4,6).  Phenylalanine, an indispensable amino acid, cannot be endogenously synthesized in humans, requiring adequate consumption in the diet. Phenylalanine is required for protein synthesis, and is intracellularly converted to the conditionally indispensable amino acid, tyrosine. In turn, tyrosine is either used for protein synthesis, oxidized or is converted to the important neurotransmitters epinephrine, norepinephrine, and dopamine (21). The dietary requirement for phenylalanine in healthy human pregnancies has not been experimentally determined (10). Previously used techniques to determine amino acid requirements were considered too invasive for vulnerable populations such as pregnant women (5,96). Current phenylalanine recommendations by the DRI are factorially calculated, and based upon nitrogen balance studies in non-pregnant adults and total potassium accretion studies during pregnancy (96). Furthermore, the DRI do not provide a recommendation for minimum phenylalanine (phenylalanine in the presence of excess tyrosine), but rather a requirement for the total aromatic amino acids (TAA, phenylalanine + tyrosine) (5). The current recommendations for TAA during pregnancy are set at 31  36 mg·kg-1·d-1, compared to non-pregnant adult recommendations of 27 mg·kg-1·d-1 (5). In addition, these recommendations are static throughout pregnancy, and do not account for potential changes in requirement due to the dynamic rate of tissue accretion and protein metabolism. There is a need to determine phenylalanine requirements directly in pregnant women using minimally-invasive stable isotope based methods (73,97).  Earlier using the DAAO technique (with L-[1-13C]Phenylalanine) in healthy adult males the requirement for minimum phenylalanine (in the presence 40 mg·kg-1·d-1 of tyrosine) was determined to be 9.1 mg·kg-1·d-1 (78). To our knowledge, the DAAO technique has never been used in a human pregnant population. The DAAO has the drawback of not being able to test low intakes of the test amino acid, because of the mass of the tracer needed determines the lowest possible test intake (5,98). However, the IAAO technique has been used previously in our laboratory in human pregnant women, and provided robust requirement estimates when tested over a range of test intakes (4,6)   3.1.1 Objectives and Hypothesis The primary objective of the current study was to determine the minimum phenylalanine (in the presence of an excess of tyrosine at 61 mg·kg-1·d-1) requirements during early and late gestation using the DAAO technique (using L-[1-13C]Phenylalanine). The secondary objective was to compare the DAAO results with IAAO (using L-[1-13C]Leucine) in late pregnancy. We hypothesized that late stages of pregnancy would have higher needs for phenylalanine compared to early stages of pregnancy, and the late gestation stage DAAO and IAAO based phenylalanine requirement estimates will not be different. 32  3.2 Subjects and Methods 3.2.1 Participants The majority of study preparation, procedures, and analyses were carried out by myself, with occasional assistance from other Elango Lab members (see the preface).  Women experiencing healthy singleton pregnancies participated in this study at BC Children’s Hospital within the Clinical Research and Evaluation Unit (Appendix A). The participants were screened to ensure they had no significant nausea or vomiting, including hyperemesis gravidarum, gestational diabetes, pre-eclampsia, or other underlying health conditions. Women had to be between 20 and 40 years old, with self-reported prepregnancy body mass index (BMI) between 18 - 28 kg/m2. Information was collected about prescription medication and supplement use from each participant. A flow chart detailing the screening and enrolment of pregnant participants is provided in Figure 10. All women were confirmed to be consuming prenatal vitamins. Informed and written consent was obtained from all participants (Appendix B), and at the completion of each study day an honorarium was provided to participants. All participants were assigned an alpha numeric code to maintain confidentiality (Appendix E). The study protocol was approved by British Columbia Children’s and Women’s Hospital’s Research Ethics Board (H15-02558) and the study was registered through ClinicalTrials.gov (NCT02669381).  3.2.2 Experimental Design The study design was modelled after previous DAAO and IAAO studies (6,78). The DAAO technique measures the oxidation of the test amino acid (phenylalanine) in response to graded test intakes. The IAAO technique indirectly measures the oxidation of the indicator amino acid (in this study, leucine), to determine the requirement of a different indispensable 33  amino acid (in this study, phenylalanine). Both techniques are based on the concept that when one indispensable amino acid is deficient for protein synthesis, the remaining amino acids are oxidized since they cannot be stored in the body (73,98). In early (13-19 wk) and late (33-39 wk) pregnancy, the DAAO method was implemented by providing graded test phenylalanine (in the presence of excess tyrosine, provided at 61 mg·kg-1·d-1) intakes to participants ranging from 5.5 to 30.5 mg·kg-1·d-1. In late pregnancy, the IAAO method was implemented by providing graded test phenylalanine (in the presence of excess tyrosine) intakes from 2.5 to 30.5 mg·kg-1·d-1. Tyrosine levels were set at 61 mg·kg-1·d-1, based on egg protein tyrosine content (40.73 mg/g) x1.5 to ensure excess. Since phenylalanine is the precursor to tyrosine, and this study aimed to measure the phenylalanine requirement and not the TAA requirement, excess tyrosine was provided to ensure the tyrosine requirement was met (99). Participants completed up to 6 study days within a gestational stage, with at least 5 days between study days (Table 4). These studies are similar to our earlier pregnancy protein (4) and lysine (6) requirement studies.   Figure 10 - CONSORT flow chart depicting enrollment and allocation of participants 34  Study Group Study Code Intakes (mg·kg-1·d-1) DAAO Early Pregnancy PHE-T1-01 5.5, 10.5, 15.5, 25.5 PHE-T1-04 7.5, 13.5, 18.5 PHE-T1-05 9.5, 12.5, 16.5, 22.5 PHE-T1-06 11.5 PHE-T1-07 6.5, 8.5, 14.5, 20.5, 21.5, 24.5  PHE-T1-08 18.5 PHE-T1-09 23.5, 30.5 PHE-T1-11 5.5, 17.5, 28.5 PHE-T1-12 10.5, 15.5 DAAO Late Pregnancy PHE-T2-01 2.5, 12.5, 20.5 PHE-T2-02 8.5 PHE-T2-04 14.5, 30.5 PHE-T2-05 4.5 PHE-T2-06 16.5, 26.5 PHE-T2-07 10.5, 24.5 PHE-T2-08 6.5 PHE-T2-10 9.5, 22.5 PHE-T2-21 3.5, 28.5 PHE-T2 22 5.5, 18.5, 21.5, 25.5 PHE-T2-23 7.5 PHE-T2-24 11.5, 15.5 PHE-T2-26 17.5, 23.5 IAAO Late Pregnancy PHE-T2-09 5.5, 25.5 PHE-T2-11 10.5, 13.5, 15.5, 16.5 PHE-T2-16 7.5, 9.5, 18.5, 22.5 PHE-T2-17 6.5, 8.5, 12.5, 24.5 PHE-T2-18 14.5 PHE-T2-19 11.5, 15.5, 19.5, 20.5, 28.5 PHE-T2-20 17.5, 30.5 PHE-T2-25 23.5 PHE-T2-27 21.5, 26.5 Table 4 - List of study participants with individual test intakes 35   3.2.3 Preliminary Assessment of Participants During a preliminary assessment visit, eligibility was assessed (Appendix C). A fasted (10-12 h) blood glucose measurement was taken using a finger prick blood glucose monitor (One Touch® Ultra® 2 LifeScan, Canada Ltd), and a blood glucose cut-off of 5.3 mmol/L was used to screen for gestational diabetes (100). Participants were weighed using a digital scale to the nearest 0.1 kg, height was determined to the nearest 0.1 cm using a stadiometer, and body composition was determined using skinfold analysis. Three skinfold thickness sites (subscapular, triceps, and biceps) were measured using a Harpenden Skinfold Caliper (Baty International, UK) with gender, age, and gestational age specific calculations to assess fat mass (101). In the fasted state, resting energy expenditure (REE, kcal/d) was determined with an open-circuit indirect calorimeter with a ventilated hood system (Vmax Encore, VIASYS, CA). Glucose and protein in urine were assessed using Chemstrip®7 Urinalysis Strips (Roche Diagnostics, USA) to help rule out gestational diabetes and risk for preeclampsia, respectively. Two-day diet records (Appendix F) were obtained to create personalized diet recommendations to achieve a prescribed protein intake recommendation of 1.5 g·kg-1·d-1 (4). Participants were also instructed using food models, on how to maintain a two-day standardized diet for the two days prior to each study day, with prenatal vitamins to ensure adequacy of dietary intake.   3.2.4 Study Diets Participants arrived in the morning after a 10-12h overnight fast. Height, weight, fasted blood glucose, and urine test strip measurements were repeated at the beginning of each study day (Appendix D). A randomized phenylalanine intake, by pulling a predetermined test intake 36  from an envelope, was provided during each study day (DAAO test intake range: 5.5 - 30.5 mg·kg-1·d-1, IAAO test intake range: 2.5 - 30.5 mg·kg-1·d-1) as eight hourly meals, as a flavored liquid formula and protein-free cookies. Each meal provided 1/12th of the participant’s daily energy and nutrient requirement, allowing them to maintain isotopic steady state in the fed condition. Protein was provided at 1.5 g·kg-1·d-1 and energy was provided at measured REE of each participant x 1.7 (6). The liquid formula contained protein-free powder (PFD1: Mead Johnson Nutrition), orange flavored drink crystals (Tang and Kool-Aid: Kraft Canada), corn oil (Mazola: ACH Food Companies) and test protein as a mixture of crystalline L-amino acid mixture (Ajinomoto, Japan) based on egg-protein composition with the exception of phenylalanine, tyrosine, leucine (only when employing the IAAO method), and alanine. L-alanine content was altered for each study day to ensure all meals were isonitrogenous.  The macronutrient distribution of the diet was approximately 53% carbohydrates, 37% fat and 10% protein. Experimental diets were prepared at BC Children’s Hospital Research Institute. During each study day only the study diets and water were consumed.   3.2.5 Isotope Protocol Baseline measurements of breath were obtained before isotope consumption began at meal 5. Priming doses of NaH13CO3 (0.176mg/kg; 99 atom percent excess (APE) Cambridge Isotope Laboratories, MA), L-[1-13C]Phenylalanine (for the DAAO experiment; 0.664mg/kg; 99 APE Cambridge Isotope Laboratories, MA), and L-[1-13C]Leucine (for the IAAO experiment; 1.727mg/kg; 99 APE Cambridge Isotope Laboratories, MA). A continuous dose of L-[1-13C]Phenylalanine (for the DAAO experiment; 1.2 mg·kg-1·h-1) and L-[1-13C]Leucine (for the IAAO experiment; 1.727 mg·kg-1·h-1) were provided in meals 5-8. A mass equivalent to the 37  indicator amino acid labelled with 13C was removed from the diet to provide a constant intake across all 8 meals. The total leucine intake was 80 mg·kg-1·d-1. This quantity of leucine, similar to the amount present in egg protein, was chosen to ensure the dietary requirement was met, to increase its sensitivity as an indicator amino acid (74).   3.2.6 Sample Collection and Analysis Breath samples were obtained for baseline and isotopic plateau enrichment measurements. Breath bags (Single use collection bags, Easy Sampler System, QuinTron, Terumo Medical) that allow for removal of dead space air were used to collect the breath samples in exetainer tubes (Labco, UK). Baseline breath samples were collected 45, 30, and 15 minutes prior to isotope administration. Following isotope administration, breath samples were obtained at minutes 150, 180, 195, 210, 225 and 240 during isotopic steady state. Samples were stored at room temperature until analysis.   Venous blood samples were obtained on each study day by a certified phlebotomist, in tubes containing EDTA, at the 6th hour of the study day. The timing allowed for stabilization of plasma amino acid concentrations to be reached. Plasma was obtained after centrifugation (10 min, 3000 x g, 4°C; Sorvall® Biofuge Stratos, Mandel Scientific Co. LTD.) and stored immediately at -80°C until analyzed.   The breath samples were analyzed for expired 13C-enrichment using a continuous flow isotope ratio mass spectrometer (CF-IRMS IsoPrime100, Cheadle, UK). 13CO2 was quantified in atoms percent excess over a reference CO2 standard.   Plasma amino acid concentrations were determined with ion exchange chromatography with post-column ninhydrin derivatization using an Amino Acid Analyzer (Hitachi L8900, 38  Tokyo, Japan) as previously described (102). Briefly, 100 µL of 6% trichloroacetic acid was added to 50 µL of plasma, vortexed and centrifuged (Centrifuge 5424R, Eppendorf) at 10000RPM for 15 minutes at 4°C. The supernatant was collected, centrifuged through a filter (0.2µm) for 2 minutes at 10000 RPM at 4°C. Fifty µL of the resulting sample was injected into the Amino Acid Analyzer for analysis. The plasma free amino acids were compared against a standard amino acid mix (Sigma, St. Louis, MO). The areas under the individual peaks were integrated on EZChrom Elite Software (version 3.3.2 SP2, Agilent, ON, Canada), and concentrations measured.  3.2.7 Isotope Kinetics F13CO2, the rate of 13CO2 released during tracer oxidation (µmol 13CO2/kg/h) was calculated as:   F13CO2=(FCO2)(ECO2)(44.6)(60)/(W)(0.82)(100) where FCO2 is the rate of production (mL/minute) of CO2, ECO2 is 13CO2 enrichment (APE) in expired breath at isotopic steady state, W is the participant’s body weight (kg), the constants 44.6 µmol/mL and 60 min/hr convert F13CO2 to µmol/hr, and the factor of 100 converts the APE to a fraction. Lastly, the factor of 0.82 accounts for bicarbonate fixation of 13CO2 in the fed state (81).   3.2.8 Statistical Analysis Subject characteristics and study day assessments are presented as mean ± SD. Estimations for the mean requirement for phenylalanine in both early and late (DAAO and IAAO) gestation were derived from breakpoint analysis of the F13CO2 data by using a bi-phase linear regression crossover analysis using SAS (SAS/STAT Ver 9.4), with subject as a random 39  variable, because not all women participated in multiple study days (103–106). The method selects the model with minimum residual standard error in a stepwise partitioning of phenylalanine intake values (x) between two regression lines. The lines are estimated for each candidate breakpoint using mixed models in order to account for repeated measures within subject. Using I as an indicator variable equal to 0 for x values left of the breakpoint and 1 for x values to the right, the model is Y= β0+β1x+β2I+β3Ix , where Y= F13CO2 or phenylalanine oxidation, x = phenylalanine intake, β0 = left line intercept, β0+β2 = right line intercept, β1 = left line slope, β1+β3 = right line slope. Therefore, Y= β0+β1x for the left line and Y= (β0+β2) + (β1+β3)x for the right. Equating these, β0+β1x = (β0+β2) + (β1+β3)x and solving for x yields the breakpoint at x= -( β2/β3). Mixed-models regressions were used with estimates for multiple candidate breakpoints, and the model that minimizes the Akaike information criteria (AIC), with a minimum residual standard error and highest adjusted R2 was used to determine the final breakpoint model. The 95% CI was determined using Fiellers Theorem (4): 95% CI=breakpoint ± tdf,/2 X SE, where SE is the SE of the combined regression lines, df is the degrees of freedom associated with the residual mean square of the best fit model, and  is the 95%CI level (4,6). The statistical difference between the DAAO breakpoints for early and late pregnancy was determined using the comparison of a pooled two sample t-test as described previously (107,108). Briefly, t = (BP1 − BP2)/[√Sp2 (1/nBP1 + 1/nBP2)], with df = nBP1 + nBP2 − 2, and BP = breakpoint, Sp2 = pooled variance, and nBP = sample size of each breakpoint. The pooled variance is calculated with the average of each sample variance with weights equal to its df and is calculated using Sp2 = [(nBP1 − 1) SBP12 + (nBP2 − 1) SBP22]/(nBP1 + nBP2 − 2), where SBP is the SD of the combined regression lines from the bi-phase crossover analysis. Effect of test 40  phenylalanine intakes on plasma phenylalanine and tyrosine concentrations were analyzed using linear regression analysis (Graphpad Prism 6, Graphpad Software, La Jolla, CA). Significance was set at p ≤ 0.05.  3.3 Results 3.3.1 Participants Twenty-three women were studied in early and late gestation (nearly=9, nlate-DAAO=9, nlate-IAAO=13), completing a total of 76 individual study days (Table 5 and 6). Five participants were studied in both early and late gestation; 3 participants were studied using both the IAAO and DAAO method; otherwise all participants were unique between stages. Women were aged 26-37y and had self-reported pre-pregnancy BMIs between 18 and 28 kg/m2 (mean=22.86±3.3 kg/m2). No participants reported pregnancies in the 6 months prior to their current pregnancy. Gestation weight gain was appropriate (3.9 ± 1.2 kg in early pregnancy and 11.6 ± 3.6 kg in late pregnancy) when compared to current gestational weight gain recommendations (109). Protein standardization goals as indicated by dietary record analysis showed that participants ate ~1.2 – 1.3 g·kg-1·d-1 (Table 6), and less than our target of 1.5 g·kg-1·d-1. However, within-subject variation of protein intake prior to study days was low, and consistent across all three studies, including a consistent Phenylalanine intake of ~40-44 mg·kg-1·d-1. All women had normal fasted blood glucose ranges at recruitment (Table 5) and on study days (Table 6). One participant had elevated levels of glucose in urine at the end of study day, but had normal fasting blood glucose levels at study start. None of the women reported use of illicit drugs, alcohol, or cigarettes during the pregnancy. Two women reported use of Diclectin (doxylamine succinate-pyridoxine) for 41  morning sickness and four women reported use of Synthroid (levothyroxine) for mild hypothyroidism, although no prescription medications were taken on the study day.  Characteristic Early Gestation  (DAAO) Late Gestation  (DAAO) Late Gestation  (IAAO) Participants, n (n=9) (n=9) (n=13) Age, y 29.3 ± 2.6 29.5 ± 2.4 30.9 ± 3.8 Gestational age, wk 17.5 ± 1.9 36.1 ± 1.9 35.9 ± 2.0 Pre-pregnancy BMI2, kg/m2 23.5 ± 2.9 22.2 ± 2.5 22.0 ± 3.0 Fasting blood glucose, mmol/L 4.7 ± 0.5 4.9 ± 0.4 4.8 ± 0.4 Fat mass3, % 28.5 ± 6.6 25.7 ± 4.0 22.8 ± 4.9 Resting energy expenditure4, kcal/d 1265 ± 237 1516 ± 237 1493 ± 228 Table 5 – Participant Characteristics1 1Values are mean ± SD. DAAO, direct amino acid oxidation; IAAO, indicator amino acid oxidation. 3Based on participant-reported pre-pregnancy weight. 3Determined by skinfold measurements (Harpenden Skinfold Caliper, Baty International). 4Determined by open-circuit indirect calorimetry (Vmax Encore, Viasys).  Variable Early Gestation  (DAAO) Late Gestation  (DAAO) Late Gestation (IAAO) (n=26)  (n=25) (n=25) Weight, kg 64.8 ± 10.9 72.5 ± 10.5 73.3 ± 8.6 Fasting blood glucose, mmol/L 4.6 ± 0.5 4.9 ± 0.4 4.6 ± 0.5 Energy intake, kcal/d 2151 ± 403 2578 ± 403 2623 ± 361 Phenylalanine intake prior to study day2, mg∙kg-1∙d-1 40.8 ± 18.7 43.5 ± 19.9 42.8 ± 12.7 Protein intake prior to study day2, g∙kg-1∙d-1 1.21 ±0.40 1.28± 0.37 1.19± 0.31 Table 6 - Study day assessments1 1Values are mean ± SD. DAAO, direct amino acid oxidation; IAAO, indicator amino acid oxidation; n, number of individual observations. 42  2Amount of protein and phenylalanine consumed by participants in the 2-days before the study day as indicated by dietary records.  3.3.2 Tracer Oxidation L-[1-13C]-phenylalanine oxidation remained on a plateau at ~0.25 F13CO2 (µmol·kg-1·h-1) when employing the DAAO method with increasing test phenylalanine intakes. Oxidation began to rise at an intake of ~15 mg·kg-1·d-1 in early gestation and ~20 mg·kg-1·d-1 in late gestation (Figures 11 and 12), For the IAAO protocol during late gestation, L-[1-13C]-leucine oxidation decreased until ~2.5 F13CO2 (µmol·kg-1·h-1), and then plateaued at a phenylalanine intake of ~ 20 mg·kg-1·d-1 (Figure 13).   Bi-phase linear regression cross-over analysis of the DAAO tracer oxidation determined a mean phenylalanine requirement of 15.14 mg·kg-1·d-1 (rounded to 15 for dietary recommendation) in early gestation (R2=0.87, 95% CI:10.4, 19.9 mg·kg-1·d-1). The mean requirement in late pregnancy was determined to be 21.05 mg·kg-1·d-1 (DAAO, rounded to 21 for dietary recommendation; R2=0.79, 95% CI:17.4, 24.7 mg·kg-1·d-1) and 21.36 mg·kg-1·d-1 (IAAO, rounded to 21 for dietary recommendation; R2=0.37, 95% CI:10.5, 32.2 mg·kg-1·d). Comparison of the mean requirement estimates obtained by the DAAO method were significantly different (P<0.0001) between gestation stages.   43   Figure 11 - Estimated average requirement of phenylalanine in early pregnancy as determined by the DAAO method Biphase linear regression crossover analysis of l-[1-13C]phenylalanine tracer oxidation (F13CO2, μmol·kg−1·h−1) was used to determine the phenylalanine requirement using the mixed and regression procedure in SAS (SAS/STAT version 9.4). Phenylalanine requirements were determined to be 15 mg·kg−1·d−1 (R2=0.87; 95% CI: 10.4, 19.9 mg·kg−1·d−1; n=9, individual study days=26). Dashed line indicates the mean requirement.   Figure 12 - Estimated average requirement of phenylalanine in late pregnancy as determined by the DAAO method Biphase linear regression crossover analysis l-[1-13C]phenylalanine tracer oxidation (F13CO2, μmol·kg−1·h−1) was used to determine the phenylalanine requirement using the mixed and regression procedure in SAS (SAS/STAT version 9.4). Phenylalanine requirements were determined to be 21 mg·kg−1·d−1 (R2=0.79; 95% CI: 17.4, 24.7 mg·kg−1·d−1; n=9, individual study days=25). Dashed line indicates the mean requirement. 44     Figure 13 - Estimated average requirement of phenylalanine in late pregnancy as determined by the IAAO method Biphase linear regression crossover analysis of l-[1-13C]phenylalanine tracer oxidation (F13CO2, μmol·kg−1·h−1) was used to determine the mean phenylalanine requirement using the mixed and regression procedure in SAS(SAS/STAT version 9.4). Phenylalanine requirements were determined to be 21 mg·kg−1·d−1 (R2=0.37; 95% CI: 10.5, 32.2 mg·kg−1·d−1; n=13, individual study days=25). Dashed line indicates the mean requirement.   3.3.3 Plasma Amino Acids In early pregnancy, plasma phenylalanine concentrations rose linearly in response to graded phenylalanine intakes (R2=0.78; Figure 14A). A similar trend was seen in late pregnancy when for both the DAAO (R2=0.82; Figure 15A) and IAAO (R2=0.79; Figure 16A) studies, a linear increase in plasma phenylalanine concentrations occurred. Plasma concentrations for tyrosine remained stable in early pregnancy (R2=0.03, x̅=48.4 ± 8.7 µmol/L; Figure 14B), late pregnancy when using the DAAO technique (R2=0.009, x̅=55.5 ± 11.2 µmol/L; Figure 15B), and late pregnancy when using the IAAO technique (R2=0.02, x̅=58.3 ± 11.6 µmol/L; Figure 16B).  45   Figure 14 – Plasma concentrations of phenylalanine and tyrosine in early pregnancy (DAAO) Linear regression analysis of (A) phenylalanine concentrations (R2=0.78) and (B) tyrosine concentrations (R2=0.03; mean ± SD: 48.8 ± 8.7); n=9, individual study days=26.    Figure 15 – Plasma concentrations of phenylalanine and tyrosine in late pregnancy (DAAO)  Linear regression analysis of (A) phenylalanine concentrations ((R2=0.82) and (B) tyrosine concentrations ((R2=0.009; mean ± SD: 55.5 ± 11.2); n=9, individual study days=25. 46   Figure 16 – Plasma concentrations of phenylalanine and tyrosine in late pregnancy (IAAO) Linear regression analysis of (A) phenylalanine concentrations (R2=0.79) and (B) tyrosine concentrations (R2=0.023; mean ± SD: 58.3 ± 11.6); n=13, individual study days=25.  3.4 Discussion To the best of our knowledge, this is the first study to experimentally determine minimum phenylalanine (in the presence of excess tyrosine) requirements in healthy pregnant women. In early pregnancy (13-19 wk gestation), the requirement was determined to be 15 mg·kg-1·d-1 (95% CI:10.4, 19.9). In late pregnancy (33-39 wk gestation), the requirement was determined to be 21 mg·kg-1·d-1 (95% CI:17.4, 24.7), using the DAAO technique. In comparison, when employing the IAAO technique in late pregnancy, a similar requirement of 21 mg·kg-1·d-1 (95% CI:10.5, 32.2) was determined.  Previously, using the DAAO technique, the adult male requirement for minimum phenylalanine was found to be 9.1 mg·kg-1·d-1 (95%CI: 4.6, 13.6) (78), which is considerably lower than what we determined in early pregnancy. Zello et al. (78) used a tyrosine intake that matched egg protein composition when determining minimum phenylalanine requirements in adult males. To account for a potential increase in requirement during pregnancy, we set the 47  tyrosine intake to 61 mg·kg-1·d-1 (1.5 times the egg protein content) to minimize the use of phenylalanine as a source of tyrosine. A low conversion of phenylalanine to tyrosine in the presence of excess tyrosine in their study (78) was illustrated by measuring [13C]tyrosine enrichment in plasma. This finding highlights that the minimum phenylalanine requirements found in adult males and pregnant women directly depends on the tyrosine intake. Previously in adults, tyrosine provided in excess of the requirement was shown to spare ~75% of the phenylalanine requirement in adults (79). However, it was also suggested that the ideal dietary ratio of phenylalanine to tyrosine is 60:40 (89). It is currently unknown whether phenylalanine can cover the complete dietary requirements for tyrosine during human gestation, but it is intriguing that in spite of providing a higher tyrosine intake than adult males, during pregnancy the requirements for phenylalanine is elevated even in early stages of pregnancy. A possible explanation could be that pregnancy specific tissues, such as the placenta, have a higher quantity of phenylalanine. Additionally, due to phenylalanine’s role in catecholamine synthesis, the requirements by the fetal brain may draw from the phenylalanine supply.  Recently we reported two requirement studies in pregnant women using the IAAO method. In the first study, we determined mean protein requirements during early (13-19 wk) and late (33 to 39 wk) pregnancy to be 1.22 and 1.52 g·kg-1·d-1 respectively (4). This suggested that the current DRI recommendation of 0.88 g·kg-1·d-1 during the course of pregnancy were underestimates for both early and late pregnancy, and that the static recommendation throughout pregnancy may not be appropriate (10). In the second study, we determined mean lysine requirements comparing the same two distinct phases of pregnancy, and found the requirement in early and late pregnancy to be 36.6 mg·kg-1·d-1 and 50.3 mg·kg-1·d-1, respectively. These values are different from the current DRI recommendation of 41 mg·kg-1·d-1. Earlier studies in pregnant 48  pigs have shown similar results, with late pregnancy resulting in higher requirements for threonine (110), lysine (85), isoleucine (84), and tryptophan (83), although proportional increases varied from 35-63% among all these amino acids.   Plasma amino acid concentrations responses to determine amino acid requirements can be highly variable and unreliable, as observed in the current study. We were unable to discern a breakpoint for phenylalanine intake from plasma phenylalanine concentrations, although tyrosine concentrations were fairly stable and constant across all test intakes in all 3 studies. When employing the DAAO method, the test amino acid is the same as the tracer amino acid. In comparison, the IAAO method uses one indispensable amino acid as the tracer (in this case, L-[1-13C]-leucine) to determine the requirement of a second amino acid (the test amino acid). The rationale behind testing both techniques in our study is three-fold. Firstly, due to the expected increase in demand for minimum phenylalanine in late pregnancy, we wanted to ensure we could provide a wide enough range of intakes using the DAAO method to create a robust breakpoint. Very low intakes of the test amino cannot be studied in the DAAO because a fixed amount of test amino acid must be provided as the tracer (in the case of the DAAO arm of this study, 5.5 mg·kg-1·d-1 of L-[1-13C]-phenylalanine), which has been suggested to impact the non-sloping plateau portion of the two-phase linear regression analysis (111). This problem does not occur with the IAAO technique. Secondly, to the best of our knowledge, these two techniques have never been compared by the same research laboratory within the same population of humans. Lastly, The DAAO technique has never been used in pregnant women before, whereas the IAAO technique has been used by our laboratory in recent studies (4,6).   The DRI provides recommendations for TAA (phenylalanine + tyrosine), and does not provide phenylalanine specific (minimum phenylalanine) recommendation. While for a healthy 49  adult population this does not pose a significant issue when conducting dietary evaluations, this is not useful for individuals with phenylketonuria (PKU). PKU a heritable disorder resulting in a reduced ability or inability to convert phenylalanine to tyrosine, requires dietary restriction of phenylalanine to minimize negative health outcomes (28). Providing phenylalanine specific recommendation during all life stages would decrease the risk of people with PKU developing neuropsychiatric disorders. Women with PKU have further complications during pregnancy due to the toxicity of phenylalanine on the developing fetus’s brain paired with a teratogenic effect (112) that result in a defined syndrome - maternal phenylketonuria (mPKU). Currently only two other studies have been reported on minimum phenylalanine requirements. One determined phenylalanine requirements in the presence of excess tyrosine in adult males to be 9.1 mg·kg-1·d-1 (78). The other, determined minimum phenylalanine requirement in enterally fed term and pre-term neonates to be 58 mg·kg-1·d-1 and 80 mg·kg-1·d-1, respectively (113). It will be beneficial to determine TAA requirements in healthy human pregnancy, which will help update the DRI’s recommendations, and set recommendations in women with mPKU, as tyrosine is an indispensable amino acid in this population.   In our current study, early pregnancy phenylalanine requirements were higher compared to non-pregnant adults (78). We showed earlier that mean protein requirements in early pregnancy are higher compared to non-pregnant adults (7), and whole-body protein turnover has been reported to increase early in pregnancy (114–117), with a 15% increase in protein synthesis by the onset of the second trimester (118, 57). However lysine requirements in early pregnancy are similar to non-pregnant adults (6,107). This could potentially be due to differences in the rate of development of fetal enzyme systems for phenylalanine and lysine catabolic pathways (119). Amino acid transporters involved in phenylalanine transport, both across the placenta and blood-50  brain barrier, have a high affinity for phenylalanine (119,120). However, these differences in indispensable amino acid demands at early stage pregnancy must be investigated further. The increase in phenylalanine requirement (40%) in late pregnancy when compared to early pregnancy corresponds well with the increase in lysine requirement (37%) previously determined by us (6).  Our study has a few limitations, including the relatively small number of subjects studied. Not all women participated in multiple test intakes, and the determined phenylalanine requirements have wide 95%CI which overlap. However, the relatively higher R2 values from the DAAO studies show that the data are robust and according to the best of our knowledge, provides the first identification of phenylalanine needs in different stages of human pregnancy. The low R2 of the IAAO study indicates that leucine intake may have been too high, and caused increased variability in the IAAO responses. In conclusion, phenylalanine requirements (in the presence of excess tyrosine) determined in healthy pregnant women during early and late gestation were 15 (95%CI:10.4, 19.9) and 21 (95%CI:17.4, 24.7) mg·kg-1·d-1, respectively. The phenylalanine requirements are higher than that previously found in adult males (78), and the mean late gestation requirement was higher compared to early gestation stage requirement. This has potential implications for future dietary recommendation guidelines during pregnancy and for women with mPKU.  51  Chapter 4: Dietary Aromatic Amino Acid Requirements during Early and Late Gestation in Healthy Pregnant Women 4.1 Introduction Pregnancy is associated with changes in dietary energy and nutrient requirements as a result of changes in maternal metabolism and increased tissue accretion (39,55). This dynamic period in life is accompanied by an increase in blood and extracellular volume, development of the placenta, changes in breast and uterine tissues, and fetal growth. Previously, using the IAAO and DAAO techniques, we have determined protein, lysine, and phenylalanine (in the presence of excess tyrosine) requirements during early and late pregnancy (4,6,90).  Phenylalanine and tyrosine are required for protein synthesis and are the precursors for neurotransmitters dopamine, norepinephrine, and epinephrine. Phenylalanine is an indispensable amino acid and is converted intracellularly to tyrosine, a conditionally indispensable amino acid, via the enzyme phenylalanine hydroxylase (20). Until now, the phenylalanine and tyrosine requirements (determined in the absence of tyrosine in the diet) have not been experimentally determined in human pregnancies.  Currently, the DRI provides an EAR for the total aromatic amino acids (TAA, phenylalanine + tyrosine) at 36 mg·kg-1·d-1 for pregnant women, compared to a recommendation of 27 mg·kg-1·d-1 for non-pregnant adults (5). Techniques used to determine protein and amino acid requirements in humans prior to stable isotope-based methods were considered too invasive to allow for experimental pregnancy studies. Therefore, the values for protein and amino acid recommendations that the DRIs provide for pregnant women are factorially calculated and based on total potassium accretion during pregnancy and nitrogen balance studies done in non-pregnant 52  adults (10). These DRI recommendations are static throughout pregnancy, not accounting for potential changes in requirements at different stages of gestation, which have dynamic differences in metabolism and development. Our laboratory has determined that there are significant differences in requirements between early and late gestation for protein (25% increase), lysine (37% increase), and phenylalanine (40% increase), indicating that current recommendations should be reevaluated, and the remaining indispensable amino acid requirements should be experimentally determined in early and late pregnancy (4,6,90).   4.1.1 Objectives and Hypothesis The objective of the current study was to determine the dietary requirement for TAA (phenylalanine in the absence of tyrosine) in early (13-19 wk) and late (33-39 wk) gestation. This was done using the minimally invasive IAAO technique (with L-[1-13C]Leucine). We hypothesized that the requirement for TAA in late pregnancy would differ from the requirement in early pregnancy, and that stage-specific requirements would differ from current recommendations.  4.2 Methods 4.2.1 Participants The majority of study preparation, procedures, and analyses were carried out by myself, with occasional assistance from other Elango Lab members (see the preface).  Healthy women who were pregnant with a single child participated in this study at BC Children’s Hospital Research Institute within our Clinical Research and Evaluation Unit (Appendix G). All women were ensured to be between 20 and 40 years old, with self-reported pre-pregnancy BMIs between 53  19 and 28 kg/m2. The participants had no significant nausea or vomiting, gestational diabetes, pre-eclampsia, or other chronic health conditions, and reported all prescription medication and supplement use. All women were ensured to be taking pre-natal vitamins. Written and informed consent was gathered from all participants (Appendix H). An honorarium was provided to participants at the end of each completed study day. This study was approved by British Columbia Children’s and Women’s Hospital’s Research Ethics Board (H17-02924) and was registered with clinicaltrials.gov (NCT03409939). A flow chart with the details of the screening and enrolment process is outlined in Figure 17.  Figure 17 - CONSORT flow chart depicting enrolment and allocation of participants  4.2.2 Experimental Design The study design was modelled after previous IAAO studies (90,121). The IAAO technique indirectly measures oxidation of an indicator amino acid (leucine in this study) to 54  determine the dietary requirement of the test amino acid (/s, TAA). The underlying principle of this method is that when an indispensable amino acid intake is deficient for protein synthesis to occur, the remaining amino acids (including the indicator amino acid), will be oxidized since amino acids are not stored in the body for later use (71). With increasing intakes of the test amino acid(s) IAAO will decrease, as amino acids will be incorporated for protein synthesis until the requirement is reached, after which the IAAO will achieve a plateau. The point at which this change occurs represents the mean requirement of the test amino acid(s).   In early (13 to 19 wk) and late (33 to 39 wk) gestation, 8 phenylalanine intakes (in the absence of dietary tyrosine; 5, 25, 40, 50, 60, 70, 85, 100 mg·kg-1·d-1) were repeated multiple times by different participants. Since phenylalanine is the precursor to tyrosine, and we aimed to measure the TAA requirement and not the minimum phenylalanine requirement, no tyrosine was provided in the diet.   Participants completed up to 6 study days within a gestational stage, with at least 5 days between study days. This study is similar to previous pregnancy studies performed by our lab determining protein, lysine, and minimum phenylalanine (in the presence of excess tyrosine) requirements (4,6,90).   4.2.3 Preliminary Assessment of Participants Eligibility of each participant was evaluated during a preliminary assessment (Appendix I). Participants were weighed using a digital scale to the nearest 0.1 kg, height was determined to the nearest 0.1 cm using a stadiometer, and body composition was determined using skinfold analysis. Gender, age, and gestational age were all accounted for in calculations to assess fat mass after three skinfold thickness sites were measured (triceps, biceps, and subscapular) using 55  Harpenden Skinfold Calipers (Baty International, UK) (101). Fasted (10-12 h) blood glucose was assessed by a finger prick blood glucose monitor (One Touch® Ultra® 2 LifeScan, Canada Ltd), and a blood glucose cutoff of 6.0 mmol/L was used to screen for gestational diabetes. REE, kcal/d, was assessed by an open circuit indirect calorimeter with a ventilated hood (Vmax Encore, VIASYS, CA). Glucose and protein in urine were determined by Chemstrip®7 Urinalysis Strips (Roche Diagnostics, USA) to rule out gestational diabetes and risk for preeclampsia. With the assistance of food models, two-day diet records were obtained to create a personalized diet recommendation that prescribed protein intake at 1.5 g·kg-1·d-1. Participants were instructed on how to maintain a two-day standardized diet prior to each study day (Appendix L), and ensured to take prenatal vitamins to ensure adequacy of dietary intake. Analysis of the diet records was carried out using the Food Processor Nutrition Analysis Software (ESHA).  4.2.4 Study Diets Participants arrived to the Clinical Research and Evaluation Unit after an over-night fast (Appendix J and K). Weight, height, urine test strip and fasted blood glucose were repeated at the beginning of each study day. A randomized phenylalanine intake, by pulling from an envelope, was provided on each study day (5, 25, 40, 50, 60, 70, 85, 100 mg·kg-1·d-1) as eight hourly meals consisting of flavoured liquid formula and protein-free cookies. Each of these meals provided 1/12th of the participant’s daily requirement for energy and nutrient requirements. Protein was provided at 1.5 g·kg-1·d-1 and energy was provided at 1.7X the participant’s measured REE from the preliminary assessment. The macronutrient distribution on study days was approximately 53% carbohydrates, 37% fat, and 10% protein. The formula contained 56  protein-free powder (PFD1: Mead Johnson Nutrition), orange flavoured drink powder (Tang and Kool-Aid: Kraft Canada), corn oil (Mazola: ACH Food Companies) and protein as a crystalline L-amino acid mixture (Ajinomoto, Japan) modelled after on egg-protein composition with the exception of phenylalanine, tyrosine, leucine, serine, and glutamine. Serine and glutamine content were altered depending on the phenylalanine intake to ensure all meals were isonitrogenous. The diets were prepared at BC Children’s Hospital Research Institute. On study days, only the experimental diets and water were consumed by participants.  4.2.5 Isotope Protocols Isotope consumption started at meal 5 with priming doses of NaH13CO3 (0.176mg/kg; 99 APE Cambridge Isotope Laboratories, MA) and L-[1-13C]Leucine (1.727mg/kg; 99 APE Cambridge Isotope Laboratories, MA). A continuous dose of L-[1-13C]Leucine (1.727 mg·kg-1·h-1) was provided in meals 5-8. The mass of non-labelled L-leucine equivalent to the L-[1-13C]Leucine was removed from the diet to provide a constant leucine intake across all 8 meals. Total leucine intake was 65 mg·kg-1·d-1. This quantity of the indicator amino acid was chosen to both ensure the dietary requirement was met and to increase its sensitivity to changes in phenylalanine consumption (74,90).  4.2.6 Sample Collection and Analysis Breath samples were collected and analyzed for baseline and isotopic plateau enrichment measurements. Breath bags (Single use collection bags, Easy Sampler System, QuinTron, Terumo Medical) that removed any dead space air were used to collect breath samples in exetainer tubes (Labco, UK). 3 baseline samples were collected 45, 30 and 15 minutes before 57  isotope administration (meal 5). Breath samples were then collected at 150, 180, 195, 210, 225, and 240 minutes after isotope administration began, during isotopic steady state. Samples were stored at room temperature. The samples were then analyzed for 13C-enrichment in expired breath 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, taken at baseline.   One venous blood sample was obtained on each study by a certified phlebotomist in EDTA tubes at the 6th hour of the study day. Samples were taken at this time point to allow for plasma amino acid concentration stabilization. Plasma was isolated via centrifugation (10 min, 2000G, 4°C; Sorvall® Biofuge Stratos, Mandel Scientific Co. LTD.) and stored at -80°C until analysis. Ion exchange chromatography with post-column ninhydrin derivatization was performed using an Amino Acid Analyzer (Hitachi L8900, Tokyo, Japan) to determine plasma amino acid concentrations, as previously described (102).  4.2.7 Isotope Kinetics The F13CO2 (rate of 13CO2 released from L-[1-13C]Leucine during oxidation) was expressed in µmol 13CO2/kg/h and calculated using the following equation:    F13CO2=(FCO2)(ECO2)(44.6)(60)/(W)(0.82)(100)  where FCO2 is the CO2 rate of production (mL/minute), ECO2 is 13CO2 enrichment (APE) in exhaled breath at isotopic steady state, W is the participant’s weight (kg), the constants 44.6 58  µmol/mL and 60 min/hr convert F13CO2 to µmol/hr, and the factor of 100 converts the APE to a fraction. Then 0.82 factors in bicarbonate fixation of 13CO2 in the fed state (81).   4.2.8 Statistical Analysis Subject characteristics and study day assessment results are presented as mean ± SD. The mean requirement for TAA in both early and late pregnancy were estimated from breakpoint analysis of F13CO2 data using a bi-phase linear regression crossover analysis in SAS (SAS/STAT Ver 9.4), with subject code as a random variable, because not all women participated in multiple study days (103–106). This analysis selects the model with the minimum residual standard error in a stepwise partitioning of phenylalanine intakes between two regression lines. These lines are assessed for a candidate breakpoint with mixed models in order to account for repeated measures within a participant. Using I as the indicator variable, it is equal to 0 for x values left of the breakpoint and 1 for x values to the right of the breakpoint. The model is Y= β0+β1x+β2I+β3Ix, where Y=phenylalanine oxidation or F13CO2, x=phenylalanine intake, β0 = left line intercept, β0+β2 = right line intercept, β1 = left line slope, β1+β3 = right line slope. Therefore, Y= β0+β1x for the left line and Y= (β0+β2) + (β1+β3)x for the right. Equating these, β0+β1x = (β0+β2) + (β1+β3)x and solving for x yields the breakpoint at x= -( β2/β3). We used regression with mixed models by selecting estimates for multiple breakpoint candidates. Then the model that minimizes the Akaike information criteria (AIC), with the highest adjusted R2 and lowest coefficient of variation was used to determine the final breakpoint model.   Using Fiellers Theorem, the 95% CI was determined: 95% CI= breakpoint ± tdf,/2 X SE, where SE is the SE of the combined regression lines, df is the degrees of freedom associated with the residual mean square of the best fit model, and  is the 95%CI level (4,6). The statistical 59  difference between the early and late stage breakpoints was assessed using a pooled 2-sample t test as described previously (90,122,123). Linear regression analysis (Graphpad Prism 6, Graphpad Software, LA Julla, CA) was used to analyze the effect of graded phenylalanine intakes on plasma concentrations of phenylalanine and tyrosine. Significance was set at p≤ 0.05.  4.3 Results 4.3.1 Participants Nineteen women participated in early and/or late gestation (nearly=10 and nlate=10), with one participant studied at both stages. A total of 51 study days were completed (Table 7 and 8). No participants reported pregnancies in the 6 months prior to their current pregnancy, and self-reported pre-pregnancy BMIs were between 17 and 29 kg/m2 (mean=24.3±3.5 kg/m2). Women were aged between 22 and 38y and had appropriate gestational weight gain when compared to current recommendations (109). For the protein standardization diet, our dietary record analysis indicated that participants ate 1.22±0.34 and 1.22±0.49 g·kg-1·d-1 in early and late pregnancy respectively (Table 8). None of the participants reported use of alcohol, illicit drugs, or cigarettes during their current pregnancy. Two women reported use of pyridoxine/doxylamine, 1 reported use of levothyroxine and 1 reported use of fluoxetine, though no prescription medications were consumed on study days. All women had normal fasted blood glucose concentrations both during preliminary assessments (Table 7) and on study days (Table 8), and no abnormal glucose or protein concentrations in urine were observed.   60    Characteristic Early Gestation  Late Gestation  Participants, n (n=10) (n=10) Age, y 32.3 ± 3.0 30.0 ± 5.0 Gestational age, wk 17.2 ± 2.4 34.1 ± 2.5 Pre-pregnancy BMI2, kg/m2 25.0 ± 3.0 23.5 ± 3.8 Fasting blood glucose, mmol/L 4.8 ± 0.4 5.0 ± 0.4 Fat mass3, % 30.3 ± 4.6 30.2 ± 5.8 Resting energy expenditure4, kcal/d 1422 ± 203 1387 ± 252 Table 7 - Participant Characteristics1 1Values are mean ± SD  2Based on participant reported pre-pregnancy weight 3Determined by Skinfold Measurements (Harpenden Skinfold Caliper, Baty International, UK) 4Determined by Open-circuit Indirect-calorimetry (Vmax Encore, VIASYS, CA)   Variable Early Gestation  Late Gestation  (n2=24)  (n2=27) Weight, kg 72.4 ± 10.0 71.5 ± 12.7 Fasting blood glucose, mmol/L 4.9 ± 0.3 4.9 ± 0.3 Energy intake, kcal/d 2417 ± 345 2358 ± 430 Phenylalanine intake prior to study day3, mg∙kg-1∙d-1 52 ± 15 54 ± 23 Tyrosine intake prior to study day3, mg∙kg-1∙d-1 43 ± 13 43 ± 20 Protein intake prior to study day3, g∙kg-1∙d-1 1.22 ± 0.33 1.22 ± 0.49 Table 8 - Study day assessments of healthy pregnant women during early and late gestation1 1Values are mean ± SD 2n refers to number of individual observations  61  3Amount of protein and phenylalanine consumed by participants in the two days prior to study day as indicated by dietary records.  4.3.2 Tracer Oxidation In early pregnancy, L-[1-13C]-leucine oxidation decreased to a F13CO2 of 2.1 µmol·kg-1·h-1, and then plateaued at phenylalanine intakes after 40 mg·kg-1·d-1 (Figure 18). In late pregnancy, L-[1-13C]-leucine oxidation decreased to a F13CO2 of 2.0 µmol·kg-1·h-1, and then plateaued at phenylalanine intakes after 50 mg·kg-1·d-1 (Figure 19).   Bi-phase linear regression cross-over analysis of the early pregnancy data provided a breakpoint (mean requirement) or 43.57 mg·kg-1·d-1 (rounded to 44 for the dietary requirement; R2=0.56, 95%CI:28.3, 58.8 mg·kg-1·d-1). The mean requirement in late pregnancy was determined to be 49.56 mg·kg-1·d-1 (rounded to 50 for the dietary requirement; R2=0.67, 95%CI:36.1, 63.1 mg·kg-1·d-1). Comparison of the early and late stage mean requirements showed a significant difference (P < 0.01). 0 5 0 1 0 001234P h e n y la la n in e  In ta k e , m g ·k g-1·d-1F13CO2, umol·kg-1·d-1 T A A -T 1 -0 1T A A -T 1 -0 3T A A -T 1 -0 4T A A -T 1 -0 5T A A -T 1 -0 6T A A -T 1 -0 7T A A -T 1 -0 9T A A -T 1 -1 0T A A -T 1 -1 1T A A -T 1 -1 25 25 40 60 70 85B re a k p o in t= 4 4  m g ·k g-1·d-1R2= 0 .5 6 Figure 18 - Estimated average requirement of TAA in early gestation using the Indicator Amino Acid Oxidation (IAAO) method in healthy pregnant women.  Biphase linear regression crossover analysis of L-[1-13C]Leucine tracer oxidation (F13CO2, µmol·kg-1·h-1) was used to determine the TAA requirement using the mixed and regression procedure in SAS (SAS/STAT Ver 9.4). TAA 62  requirements were determined to be 44 mg·kg-1·d-1 (R2=0.56, 95%CI:28.3, 58.8 mg·kg-1·d-1; n=10, individual study days=24). Dashed line indicates the mean requirement.  0 5 0 1 0 001234P h e n y la la n in e  In ta k e , m g ·k g-1·d-1F13CO2, umol·kg-1·d-1 T A A -T 2 -0 1T A A -T 2 -0 2T A A -T 2 -0 4T A A -T 2 -0 5T A A -T 2 -0 7T A A -T 2 -0 8T A A -T 2 -0 9T A A -T 2 -1 0T A A -T 2 -1 1T A A -T 2 -1 25 25 40 60 70 85B re a k p o in t= 5 0  m g ·k g-1·d-1R2= 0 .6 7 Figure 19 - Estimated average requirement of TAA in late gestation using the Indicator Amino Acid Oxidation (IAAO) method in healthy pregnant women.  Biphase linear regression crossover analysis of L-[1-13C]Leucine tracer oxidation (F13CO2, µmol·kg-1·h-1) was used to determine the phenylalanine requirement using the mixed and regression procedure in SAS (SAS/STAT Ver 9.4). Phenylalanine requirements were determined to be 50 mg·kg-1·d-1 (R2=0.67, 95%CI:36.1, 63.1 mg·kg-1·d-1; n=10, individual study days=27) of. Dashed line indicates the mean requirement.   4.3.3 Plasma Amino Acids  In early pregnancy, plasma phenylalanine concentrations rose linearly in response to graded phenylalanine intakes (R2=0.81; Figure 20). A similar trend was seen in late pregnancy (R2=0.71; Figure 21). Plasma concentrations for tyrosine rose linearly in early pregnancy (R2=0.71; Figure 20) and late pregnancy rise was more variable (R2=0.31; Figure 21).   63  0 5 0 1 0 005 01 0 01 5 0P h e n y la la n in e  In ta k e , m g ·k g-1·d-1Plasma Phenylalanine, umol/L5 25 40 7060 85R2=  0 .8 10 5 0 1 0 001 02 03 04 05 0P h e n y la la n in e  In ta k e , m g ·k g-1·d-1Plasma Tyrosine, umol/LT A A -T 1 -0 1T A A -T 1 -0 3T A A -T 1 -0 4T A A -T 1 -0 5T A A -T 1 -0 6T A A -T 1 -0 7T A A -T 1 -0 9T A A -T 1 -1 0T A A -T 1 -1 15 25 40 60 70 85R2=  0 .7 1 Figure 20 -Plasma concentrations of phenylalanine and tyrosine in early pregnancy in response to graded phenylalanine intakes in healthy pregnant women.  Linear regression analysis of left graph, phenylalanine concentrations (R2=0.81) and right graph, tyrosine concentrations (R2=0.71) (n=10, individual study days=24).     0 5 0 1 0 005 01 0 01 5 0P h e n y la la n in e  In ta k e , m g ·k g-1·d-1Plasma Phenylalanine, umol/L5 25 40 60 70 85R2=  0 .7 10 5 0 1 0 001 02 03 04 05 0P h e n y la la n in e  In ta k e , m g ·k g-1·d-1Plasma Tyrosine, umol/LT A A -T 2 -0 1T A A -T 2 -0 2T A A -T 2 -0 4T A A -T 2 -0 5T A A -T 2 -0 7T A A -T 2 -0 8T A A -T 2 -0 9T A A -T 2 -1 0T A A -T 2 -1 1T A A -T 2 -1 25 25 40 60 70 85R2=  0 .3 1 Figure 21 - Plasma concentrations of phenylalanine and tyrosine in late pregnancy in response to graded phenylalanine intakes in healthy pregnant women.  Linear regression analysis of left graph, phenylalanine concentrations (R2=0.71) and right graph, tyrosine concentrations (R2=0.31) (n=10, individual study days=27).   4.4 Discussion This was the first study to experimentally determine TAA (phenylalanine in the absence of dietary tyrosine) requirements in healthy pregnant women, to the best of our knowledge. In early pregnancy (13-19 wk gestation), the mean requirement was determined to be 44 mg·kg-1·d-1. In late pregnancy (33-39 wk gestation), the mean requirement was determined to be 50 mg·kg-64  1·d-1. Both of these findings are different from the current DRI’s EAR for TAA intake during pregnancy of 36 mg·kg-1·d-1. However, further studies indirectly supporting our findings using longer-term approaches are needed to contribute to new recommendations.  Three studies have been previously conducted in our laboratory to determine protein and amino acids requirements in human pregnancies. Using the IAAO technique (with L-[13C]phenylalanine), we determined the mean protein requirements during early (13-19wk) and late (33-39wk) pregnancy as 1.22 and 1.52 g·kg-1·d-1 respectively (4). This was the first experimental study in pregnant humans to suggest that the current DRI recommendations (0.88 g·kg-1·d-1) were underestimated, and that the static recommendation during pregnancy was not appropriate (there was a 25% difference in requirement between stages). Therefore, determining the indispensable amino acid requirements during pregnancy became a priority. Next, lysine requirements during early and late pregnancy were determined to be 37 and 50 mg·kg-1·d-1, respectively (6). They were different from the current DRI recommendation of 41 mg·kg-1·d-1, and the late pregnancy requirement was 37% higher than early pregnancy requirement. Most recently, we determined the phenylalanine (in the presence of excess tyrosine at 65 mg/kg/d) requirements during early and late gestation in healthy pregnant women (90). Using L-[13C]phenylalanine, we employed the DAAO technique and determined breakpoints (requirements) of 15 and 21 mg·kg-1·d-1 in early and late pregnancy, respectively. The current study suggests that recommendations for TAA at 36 mg·kg-1·d-1 are underestimated (by 22% in early pregnancy and 39% in late pregnancy).  There are 5 studies that have been conducted earlier to determine TAA requirements in healthy non-pregnant adults using stable isotope-based techniques. The first, published in 1998, employed a 24h tyrosine balance method as a physiological endpoint for the TAA requirement 65  using 3 phenylalanine intakes (18.5, 35.6, and 96.6 mg·kg-1·d-1) (77). They determined a tentative requirement of 39 mg·kg-1·d-1, which was higher than the recommendation at the time of 35.6 mg·kg-1·d-1. Following this, in 2001, Roberts et al used the IAAO technique using L-[13C]lysine to determine the tyrosine requirement in healthy adults (87). Based on a study that had determined minimum phenylalanine requirements in healthy adults (78), they fixed the phenylalanine intake at 9.0 mg·kg-1·d-1. They determined a breakpoint at a tyrosine intake of 6.0 mg·kg-1·d-1 for phenylalanine, and when paired with the phenylalanine intake determined a TAA requirement of 15 mg·kg-1·d-1 (87). In 2006, three studies were published addressing TAA requirements in healthy adults. The first, using the IAAO technique with L-[1-13C]lysine, used a similar approach as the current study by providing no dietary tyrosine on study days (79). A requirement of 48 mg·kg-1·d-1 was found. Secondly, the IAAO technique was employed while providing L-[1-13C]leucine, to determine the TAA requirement (with an absence of dietary tyrosine). A requirement of 42 mg·kg-1·d-1 was determined. Lastly, Kurpad et al., using a cohort of healthy Indian men and the 24h – indicator amino acid balance method, found a TAA requirement of 38 mg·kg-1·d-1 (88). Following a thorough review of these data, an average requirement was deduced from the available data, of 43 mg·kg-1·d-1 for non-pregnant adults (89).  Thus, our early pregnancy TAA requirements (44 mg·kg-1·d-1) did not differ from requirements previously determined in non-pregnant adults, while late pregnancy requirements (50 mg·kg-1·d-1) increased by ~16%. Previously, both the protein and minimum phenylalanine requirements were higher for pregnant women compared to non-pregnant adults (4,90). This corresponds well to the fact that whole-body protein turnover has been reported to increase in early pregnancy with ~15% increase in protein synthesis by the end of the first trimester (114–117). Conversely, lysine and 66  TAA (the current study) requirements were similar between early pregnancy and non-pregnant adults. On the one hand, these findings suggest that while protein needs increase linearly from early stages of pregnancy, this is not true for all amino acids. It is not entirely clear why phenylalanine requirements increase early in pregnancy, but TAA requirements do not. It is potentially due to phenylalanine requirements increasing in early pregnancy as well as late pregnancy, while tyrosine requirements are held more constant throughout the pregnancy. Both minimum phenylalanine and TAA requirements increased by ~6 mg·kg-1·d-1 between early and late pregnancy, providing some evidence for this justification.  Since tyrosine is synthesized from phenylalanine in vivo, it has been suggested that dietary tyrosine can spare phenylalanine requirements. The minimum phenylalanine (in the presence of excess tyrosine) requirement in healthy adult males was reported as 9.1 mg·kg-1·d-1 (78). Paired with the data from the TAA requirement study estimates (43 mg·kg-1·d-1), it was reasoned earlier that the TAA requirement that can be met by tyrosine in nonpregnant adults is 78%, and phenylalanine must provide at least 22% of the requirement (89). When comparing our recent pregnancy studies on phenylalanine (15 and 21 mg·kg-1·d-1 in early and late pregnancy, respectively) with the current study, tyrosine spares 66% of the TAA requirement in early pregnancy, and 58% in late pregnancy. This adds to the idea that phenylalanine specific requirements are increasing more than tyrosine during pregnancy. Additionally, the study by Roberts et al provided another interesting component to the understanding of aromatic amino acids. Based on their findings (discussed above), protein synthesis was optimized when the dietary ratio of phenylalanine:tyrosine was 60:40, which is comparable to human tissue concentrations (87). Going forward, it would be interesting to investigate the effect of different dietary phenylalanine:tyrosine ratios during human pregnancies. 67  The plasma analysis of phenylalanine and tyrosine from both early and late pregnancy illustrated steady increases in concentrations with increasing phenylalanine intake. This was an expected result. With no tyrosine in the diet, dietary phenylalanine was being endogenously converted to tyrosine on the study days, supplying the majority of their tyrosine. Therefore, with low phenylalanine intake, limited tyrosine synthesis occurs. Conversely, as phenylalanine intake increases, there is more available to be converted to tyrosine, allowing for increased tyrosine synthesis. We had hoped to identify a change in plasma concentrations as an additional biomarker during pregnancy, however as apparent in Figure 3 and 4, we were unable to determine a breakpoint from our plasma data.  We are aware of a few limitations with this study, including a small number of subjects were studied, similar to previous pregnancy publications (4,6,90). Next, each participant could not participate in all test intakes due to the narrow gestational age limits in which they are able to be studied, paired with the huge time commitment and the 5-day wash-out period. As well, the determined requirements have a wide and overlapping 95% CI. However, the relatively high R2 values and low AICC and RMSE indicate robust data, providing the first experimentally determined TAA requirement at different stages of human pregnancy. Lastly, studies like these may use diets that are not like most natural foods. Natural foods do not contain phenylalanine without tyrosine, and do not contain protein that is as highly digestible as crystalline amino acids. However, our results provide a basis for future studies investigating metabolic availability and protein quality for formulas to treat individuals with phenylketonuria, who require life-long dietary management involving phenylalanine restriction and tyrosine supplementation, including during pregnancy – maternal Phenylketonuria (mPKU). 68    In conclusion, TAA requirements (phenylalanine in the absence of dietary tyrosine) determined in healthy pregnant women during early and late gestation were 44 and 50 mg·kg-1·d-1, respectively. The TAA requirements found in early pregnancy are similar to those found previously in adult males (79,87–89). As well, the mean late gestation requirement was higher compared to the early gestation requirement by 14%. This has the potential to contribute to improved future dietary recommendation guidelines during pregnancy.  69  Chapter 5: A Natural History of Maternal Phenylketonuria Patients at Vancouver General Hospital 5.1 Introduction  Phenylalanine, an indispensable amino acid, is essential for protein synthesis and fundamental for fetal development. Phenylalanine gets intracellularly converted to tyrosine, a conditionally indispensable amino acid, via the enzyme phenylalanine hydroxylase (PAH). In turn, tyrosine is the precursor for important neurotransmitters such as dopamine, norepinephrine, and epinephrine as well as thyroid hormones and melanin. A deficiency in PAH, more commonly known as phenylketonuria (PKU), is an inborn error of metabolism that elevates plasma phenylalanine and its metabolites to toxic levels.  PKU is the most common genetic cause of intellectual disability that is treatable, affecting approximately 1 in 15,000 live births in the USA, 1 in 10,000 in Europe and 1 in 12,000 in British Columbia (11,124). Due to low or no intracellular conversion of phenylalanine to tyrosine, people with PKU have a dietary requirement for tyrosine. Newborn screening has allowed for early diagnosis and implementation of effective treatments such as phenylalanine restriction in the diet and sapropterin dihydrochloride, a co-factor of PAH, supplementation (125). More people with PKU are now reaching adulthood in good health, allowing for a rise in pregnancies (maternal phenylketonuria, mPKU) in this population (126).  The syndrome resulting teratogenic effects of elevated maternal phenylalanine concentrations on fetal development is termed mPKU syndrome. Plasma phenylalanine concentrations have been set target ranges for management which are 120-360 µmol/L or 2-6 mg/dL, as determined by the maternal phenylketonuria collaborative study (13). In women who 70  have plasma phenylalanine above the target levels symptoms can include, but are not limited to, seizures, cognitive disabilities, microcephaly, spontaneous abortions, heart defects and postnatal morbidity (12,13,32). However, since phenylalanine and tyrosine are required for protein synthesis, and the pregnancy life stage is accompanied by rapid tissue accretion, phenylalanine is still required in the diet and must be closely monitored (127). In women with mPKU, there appears to be a direct correlation between maternal plasma phenylalanine concentrations and the frequency of abnormalities present in the fetus (26). This is likely due to that the fact that phenylalanine readily crosses the placenta via active transport, resulting in a 70% increase in fetal plasma concentrations in comparison to maternal concentrations (27).   5.1.1 Objectives and Hypothesis The objective of this retrospective analysis was to thoroughly review the dietary management of mPKU cases at Vancouver General Hospital from the last 20 years. We assessed dietary recommendations, blood analysis, treatment compliance and examined the relationship between dietary protein, phenylalanine and tyrosine intake on blood concentrations of amino acids in mPKU pregnancies. We also set out to increase the scope of this study by comparing the phenylalanine, tyrosine and protein intakes to the recently determined phenylalanine requirements (90) and protein (4) in healthy pregnant women. Detailed diet analysis of data from mPKU is scarce, and our results will provide insight and guidance prior to designing intervention studies determining dietary phenylalanine, tyrosine and protein requirements in mPKU subjects. We hypothesized that current management practices at Vancouver General Hospital are effectively allowing for metabolic control by 8 wk pregnancy in their mPKU patients.   71  5.2 Methods 5.2.1 Population The UBC Children’s and Women’s Research Ethics Board approved this study (H19-01225), as well as Vancouver Coastal Health Operations (V19-01225, Appendix M). The subjects included were those monitored for mPKU over the last 20 years (1999-2019) at the Adult Metabolic Diseases Clinic at Vancouver General Hospital. No genotype information was available to allow for classification. From a review of the dietitian’s health records, data was extracted on demographics, gestational weight gain (GWG), gestational age at delivery (method to determine gestational age likely differed over time, from dating ultrasound to last menstrual period), pregnancy outcomes, results of plasma analyses, reported health concerns during the pregnancy and dietary intake of phenylalanine, tyrosine and protein. Tyrosine intakes was solely based on medical foods and supplements, as tyrosine from natural foods had not been analyzed. Similarly, protein intake from natural foods was not available. In PKU management, dietitians estimate phenylalanine content in protein at 5% (128); therefore, protein intake from natural food was extrapolated based on phenylalanine intake. Total protein intake was calculated by adding protein from medical foods to calculated protein from natural foods.  For some subjects, the data collected included pre-pregnancy data. All data were grouped as (where available): preconception (in the 3 months prior), <8wk gestation (to mark desired timepoint of metabolic control), 13-19 wk gestation (early pregnancy stage as per our earlier studies) and 33-39 wk gestation (late pregnancy stage as per our earlier studies) (4,28,90). Body weight was not recorded regularly throughout the pregnancies; however total GWG was available for each mPKU pregnancy. Thus a modelled approach was used to estimate stage specific individual GWG based on previous pregnancy weight gain data throughout pregnancy 72  (90). Briefly, at time points less <8wk, prepregnancy weight was used, as no weight gain is expected at this stage; for 13-19 wk, 20% of their total GWG was added to their prepregnancy weight; and for 33-39 wk, 90% of their GWG was added to their pre-pregnancy weight. All collected information was saved to an encrypted and secured database at BC Children’s Hospital Research Institute.   5.2.2 Dietary Management The data utilized for this retrospective analysis was collected from the dietitians’ records at the Adult Metabolic Diseases Clinic at Vancouver General Hospital, Vancouver, British Columbia, BC, Canada. All subjects included were receiving some level of dietary management throughout their pregnancy. Pregnancies that received no dietary management are not included. As well, since this analysis is based on 20 years’ worth of data, some dietary management practices may have changed. Current practices are described below. All women of reproductive age are counselled to see the dietitians prior to becoming pregnant to achieve pre-conception metabolic control. Once a pregnancy was confirmed, initial recommendations were provided for calories based on Institute of Medicine Recommendations (5), protein (based on weight and set at 1.1 g·kg-1·d-1) and phenylalanine (based on amount of phenylalanine they can eat while maintaining healthy blood spot phenylalanine). Protein requirements were met with a combination of mostly medical phenylalanine free formula (a blend of indispensable and dispensable amino acids with additional tyrosine – since it is an indispensable amino acid in this population) and enough natural protein to support fetal growth (while keeping blood phenylalanine levels in the target range). As the pregnancy progresses, the subjects were expected to provide bi-weekly blood spots for their entire pregnancy to monitor 73  increases in phenylalanine tolerance. As well, daily diet records (including natural foods, medical foods, and drinks) were requested to be submitted to determine phenylalanine, protein, and calorie intake. The prescribed phenylalanine intake varies throughout pregnancy based on individual factors affecting phenylalanine tolerance such as: residual PAH activity, weight, illness, gestational age, and appetite. If two low phenylalanine concentrations are seen in a row in the blood spots, the last 3 food records submitted are analyzed, and the phenylalanine recommendations will be increased by 5 to 10%. Similarly, if tyrosine concentrations in blood spots are low 2 times in a row, tyrosine supplements will be provided in 2.5 g/d increments.  5.2.3 Original Sample Analysis Phenylalanine and tyrosine concentrations in blood spots as well as amino acid concentrations in plasma were analyzed by the core pathology laboratory located at BC Children’s Hospital using a liquid chromatography quadrupole tandem mass spectrometer. Diet records submitted to the dietitians were analyzed using either: howmuchphe.org or metabolicpro.org.  5.2.4 Statistical Analysis Descriptive statistics were run for subject characteristics and each subject’s data for: phenylalanine recommendation, phenylalanine intake, phenylalanine and tyrosine in blood spots, protein and tyrosine intake from medical foods and caloric intake. Paired samples t-test was run to determine if there were differences between prescribed phenylalanine and phenylalanine intake at each stage of gestation. Percent difference was calculated between dietary intake of phenylalanine and protein (using a modelled weight) compared to previously determined results 74  (4,90). A generalized additive mixed models approach was used to look at changes in phenylalanine concentration in blood spots as well as phenylalanine tolerance over the course of the pregnancy. Participant ID was used as a random effect, with autoregressive autocorrelation structure of order 1 to model the temporal autocorrelation of observations within participants. With the resulting models, the average relationship between phenylalanine concentration or phenylalanine tolerance and the weeks of gestation was estimated. Analysis was run on R Core Team (Version 3.6.3). Significance was set at P>0.05.  5.3 Results 5.3.1 Subject Characteristics Eleven women, with a total of 16 pregnancies, were included in this natural history. Table 9 summarizes subject characteristics. One subject had 3 pregnancies, 4 subjects had 2 pregnancies, with the remainder only having had 1 pregnancy included in this analysis. Women were aged 20-40y with pre-pregnancy BMIs between 21-41 kg/m2. Total GWG was appropriate based on current Institute of Medicine guidelines (109). Based on our modelled approach, weight gain by 13-19 wk pregnancy was 3.0 ± 0.9 kg and by 33-39 wk pregnancy was 13.4 ± 3.9 kg, as shown in Table 10. One subject reported use of Diclectin. The following supplements were reported being taken during the pregnancies: prenatal vitamins (all pregnancies), docosahexaenoic acid (8 pregnancies), iron (8 pregnancies), folic acid (7 pregnancies), vitamin D (5 pregnancies), vitamin B6/12 (3 pregnancies), choline (2 pregnancies), fish oil (2 pregnancies), and calcium (1 pregnancy).    75  Characteristic Mean ± SD (range) Pregnancies, n 16 Age at conception, y 30.7 ± 5.3 (20-40) Height, cm 161 ± 5 (155-171) Pre-pregnancy weight1, kg 69 ± 12 (58-109) Pre-pregnancy BMI, kg/m2 26 ± 5 (21-41) Gestational weight gain1, kg 14.7 ± 4.7 (9-28) Table 9 - Subject Characteristics 1Based on medical records from the Adult Metabolic Diseases Clinic  Stage Characteristic Mean ± SD (range) <8wk1 GWG, kg - 13-19wk2 GWG, kg 3.0 ± 0.9 (1.7-5.6) 33-39wk3 GWG, kg 13.4 ± 3.9 (7.7-25.1) Table 10 - Summary of Gestational Weight Gain in mPKU Pregnancies 1Based on the prepregnancy weight from hospital records 2 Calculated by adding 20% of the total GWG to the prepregnancy weight 3Calculated by adding 90% of the total GWG to the prepregnancy weight   5.3.2 Dietary Recommendations and Intake Dietary intake and recommendation data are summarized in Tables 11 and 12. Prescribed phenylalanine increased from 6.5 ± 3.2 to 16.7 ± 4.8 mg·kg-1·d-1 between <8wk and 33-39wk gestation (Figure 22). Based on this graph, the rate of increase in phenylalanine tolerance rose after ~20 wk gestation. Similarly, phenylalanine intakes increased from 6.2 ±3.1 to 17.7 ±4.9 mg·kg-1·d-1 between these same 2 stages. No statistical difference was found between prescribed phenylalanine and phenylalanine intake for any of the 3 stages. When compared to our previously determined mean phenylalanine requirements in healthy women during early 76  pregnancy (15 mg·kg-1·d-1) and late pregnancy (21 mg·kg-1·d-1), phenylalanine intake was 50% lower at 13-19 wk pregnancy and 16 percent lower at 33-39 wk pregnant.   Stage Characteristic Participants, n Mean ±SD (range) <8wk Phenylalanine prescribed1 mg/kg/d 11 6.5 ± 3.2 (1.8-14.4) Phenylalanine intake2, mg/kg/d 11 6.2 ± 3.1 (2.5-15.3) Medical food protein intake3, g/kg/d 13 1.2 ± 0.2 (0.8-1.4) Natural protein intake4, g/kg/d 11 0.12 ± 0.06 (0.05-0.30) Total protein intake, g/kg/d 11 1.2 ± 0.2 (0.8-1.6) Tyrosine intake5, mg/kg/d 13 159 ± 77 (82-380) 13-19wk Phenylalanine prescribed1 mg/kg/d 11 7.2 ± 4.3 (2.4-18.3) Phenylalanine intake2, mg/kg/d 7 7.5 ± 4.3 (2.2-18.5) Medical food protein intake3, g/kg/d 12 1.2 ± 0.2 (0.9-1.7) Natural protein intake4, g/kg/d 7 0.15 ± 0.09 (0.04-0.37) Total protein intake, g/kg/d 7 1.4 ± 0.4 (0.9-2.0) Tyrosine intake5, mg/kg/d 12 204 ± 103 (32-521) 33-39wk Phenylalanine prescribed1 mg/kg/d 9 16.7 ± 4.8 (8.6-24.9) Phenylalanine intake2, mg/kg/d 7 17.7 ± 4.9 (7.0-25.1) Medical food protein intake3, g/kg/d 11 1.3 ± 0.2 (0.9-1.5) Natural protein intake4, g/kg/d 7 0.35 ± 0.1 (0.14-0.50) Total protein intake, g/kg/d 5 1.6 ± 0.3 (1.0-2.0) Tyrosine intake5, mg/kg/d 11 210 ± 124 (104-620) Table 11- Prescribed vs. Actual Dietary Intake in mPKU Pregnancies Adjusted for Body Weight 1 Based on all recommendations provided during that gestational stage 2 Based on all food records provided by subjects during that gestational stage, as analyzed by howmuchphe.org or metabolicpro.org 3 Protein consumed by medical foods, based on recommended formula intake during that gestational stage 4 Estimated from phenylalanine consumption (set at 5% of natural protein) during that gestational stage 5 Based on tyrosine from formula and supplements, does not account for tyrosine from natural foods. 77  Stage Characteristic Participants, n Mean ±SD (range) <8wk Phenylalanine prescribed1, mg/d 11 413 ± 169 (200-850) Phenylalanine intake2, mg/d 11 413 ± 185 (200-991) Medical food protein intake3, g/d 13 77 ± 9 (64-90) Natural protein intake4, g/d 11 8.3 ± 3.7 (4.0-7.6) Total protein intake, g/d 11 83 ± 8 (71-99) Tyrosine intake5, mg/d 13 10482 ± 4645 (6600-23160) Energy intake2, kcal/d 12 1916 ± 342 (1196-2655) 13-19wk Phenylalanine prescribed1, mg/d 11 475 ± 243 (175-1100) Phenylalanine intake2, mg/d 7 513 ± 234 (182-1112) Medical food protein intake3, g/d 12 88 ± 11 (70-109) Natural protein intake4, g/d 7 10.3 ± 4.7 (3.6-22.2) Total protein intake, g/d 7 98.5 ± 10.5 (80.7-124.8) Tyrosine intake5, mg/d 12 13880 ± 6251 (2081-33160) Energy intake2, kcal/d 11 2119 ±260 (1548-2856) 33-39wk Phenylalanine prescribed1, mg/d 9 1378 ± 458 (800-2150) Phenylalanine intake2, mg/d 7 1483 ± 443 (747-2095) Medical food protein intake3, g/d 11 101 ± 11 (75-115) Natural protein intake4, g/d 7 29.7 ± 8.9 (14.9-41.9) Total protein intake, g/d 5 133 ± 19 (92-157) Tyrosine intake5, mg/d 11 16480 ± 8788 (7823-45040) Energy intake2, kcal/d 7 2639 ± 313 (1888-3184) Table 12 - Prescribed vs. Actual Dietary Intake in mPKU Pregnancies on a Per Day Basis 1 Based on all recommendations provided during that gestational stage 2 Based on all food records provided by subjects during that gestational stage, as analyzed by howmuchphe.org or metabolicpro.org 3 Protein consumed by medical foods, based on recommended formula intake during that gestational stage 4 Estimated from phenylalanine consumption (set at 5% of natural protein) during that gestational stage 5 Based on tyrosine from formula and supplements, does not account for tyrosine from natural foods 78  Protein intake from medical foods rose from 1.2 ± 0.2 to 1.3 ± 0.2 g·kg-1·d-1 between <8wk and 33-39wk gestation, respectively. With the addition of the estimated natural protein intake, total protein intake rose from 1.2 ± 0.2 to 1.6 ± 0.3 g·kg-1·d-1 between <8wk and 33-39wk gestation, respectively.  There was negligible (<5%) difference between the 13-19 wk and 33-39 wk total protein intake when compared to previously determined mean protein requirements in healthy women during early (1.22 g·kg-1·d-1) and late (1.52 g·kg-1·d-1) pregnancy (4). Tyrosine and energy intake are summarized in Tables 11 and 12, with the mean intakes of both increasing from each stage of pregnancy to the next.    Figure 22 - Phenylalanine Tolerance Throughout mPKU Pregnancies The average relationship between phenylalanine tolerance (indicated by prescribed phenylalanine) and gestational age in weeks. 121 individual observations, n=16 pregnant women. The open circles indicate the raw data, the line indicates the best-fit from the generalized additive mixed models, and the shading indicates the 95%CI around the estimated line. Phenylalanine tolerance rose throughout pregnancy, with a steeper slope (the rate of increase in phenylalanine tolerance rose) beginning around 20 weeks gestation. 79  5.3.3 Blood Spot and Plasma Analysis Blood spot results are summarized in Table 13. Mean phenylalanine concentration in blood spots decreased as pregnancy progressed (Figure 23), with the mean concentration being highest preconception (403 ± 265 µmol/L), and a slight increase between 8 and 12 wk (the mean stayed below the upper cutoff level of 360 µmol/L). Tyrosine concentrations in blood spots went up slightly at each gestational stage, ranging from 49 ± 20 to 60 ± 25 µmol/L preconception to 33-39 wk pregnant, respectively (Table 5). The mean ratio between phenylalanine:tyrosine in blood spots decreased between each stage of pregnancy, ranging from 9 ± 7 preconception to 3 ± 2 at 33-39 wk pregnant.   Gestational Stage Characteristic Mean ± SD (range) Preconception2 (n=6) Phenylalanine1, µmol/L 403 ± 265 (36-1029) Tyrosine1, µmol/L 49 ± 20 (28-93) Phenylalanine:Tyrosine 9 ± 7 (1-29) <8wk (n=15) Phenylalanine1, µmol/L 220 ± 173 (48-999) Tyrosine1, µmol/L 52 ± 25 (22-152) Phenylalanine:Tyrosine 5 ± 4 (0.5-10) 13-19wk (n=16) Phenylalanine1, µmol/L 186 ± 98 (54-544) Tyrosine1, µmol/L 58 ± 35 (19-272) Phenylalanine:Tyrosine 4 ±3 (0.3-20) 33-39wk (n=14) Phenylalanine1, µmol/L 182 ± 77 (38-405) Tyrosine1, µmol/L 60 ± 25 (32-247) Phenylalanine:Tyrosine 3 ± 2 (0.6-10) Table 13 - Blood Spot Phenylalanine and Tyrosine Values in mPKU Pregnancies 1 Determined by liquid chromatography quadrupole tandem mass spectrometry 2 Within the 3 months prior to the reported conception date 80   Figure 23 - Phenylalanine Concentrations in Blood Spots Throughout mPKU Pregnancies The average relationship between phenylalanine concentration and gestational age in weeks. 886 individual observations, n=16 mPKU pregnancies. The open circles indicate the raw data, the line indicates the best-fit from the generalized additive mixed models, and the shading indicates the 95%CI around the estimated line.  Mean phenylalanine concentrations in blood spots were highest in early pregnancy, and decreased by 8 weeks gestation. There was a slight rise in concentration between 8 and 12 wk gestation (the mean still below the cutoff of 360 µmol/L), after which the mean plateaus.  Further results from plasma analysis performed during each trimester can be found in Table 14 displaying plasma amino acid concentrations and Table 15 displaying Biochemical Variables. While mean ferritin values did not signify iron deficient anemia (<15 ug/L) during any stage, they did drop below a concentration that indicates early iron depletion (<30 ug/L) in the 3rd trimester (129). Mean hemoglobin concentrations remained above anemia cutoff values at each stage of pregnancy (<110 g/L for 1st trimester and <105 g/L for 2nd and 3rd trimesters) (129). 81  Since these had variable records among different mPKU subjects, no statistical analysis was performed.   Preconception1 (n=3) 1st Trimester (n=5) 2nd Trimester (n=13) 3rd Trimester (n=13)   Mean ± SD (range) Phenylalanine, µmol/L 719 ± 488 (166-1090) 341 ± 273 (79-998) 185 ± 93 (79-343) 151 ± 72 (26-284) Tyrosine, µmol/L 37 ± 10 (27-49) 33 ± 5 (24-42) 37 ± 8 (19-51) 46 ± (21 (20-96) Alanine, µmol/L 305 ± 82 (227-390) 232 ± 50 (143-289) 239 ± 72 (65-345) 260 ± 52 (179-347) Arginine, µmol/L 57 ± 14 (49-73) 38 ± 8 (23-28) 39 ± 9 (21-59) 40 ± 19 (27-100) Asparagine, µmol/L - 43 ± 18 (31-70) 41 ± 11 (21-57) 49 ± 18 (18-80) Aspartic Acid, µmol/L - 3 ± 2 (2-6) 4 ± 2 (1-6) 4 ± 1.9 (1-6) Cysteine, µmol/L 40 ± 9 (32-50) 26 ± 12 (9-39) 27 ± 9 (13-38) 28 ±16 (4-66) Glycine, µmol/L 231 ± 34 (195-262) 197 ± 88 (88-365) 155 ± 51 (88-235) 175 ± 108 (98-500) Glutamic Acid, µmol/L 18 ± 15 (5-34) 22 ± 12 (9-51) 27 ± 13 (13-59) 34 ± 11 (21-58) Glutamine, µmol/L 408 ± 83 (346-503) 380 ± 99 (217-520) 416 ± 98 (274-612) 421 ± 119 (285-744) Histidine, µmol/L 63 ± 13 (51-77) 56 ± 10 (41-70) 71 ± 12 (54-90) 70 ± 19 (41-110) Isoleucine, µmol/L 46 ± 23 (30-72) 34 ± 8 (21-48) 42 ± 15 (18-85) 35 ± 6 (27-49) Leucine, µmol/L 83 ± 27 (65-114) 69 ± 14 (45-88) 81 ± 27 (46-166) 68 ± 10 (56-85) Lysine, µmol/L 125 ± 36 (100-166) 121 ± 23 (73-156) 149 ± 30 (102-212) 136 ± 24 (91-172) Methionine, µmol/L 18 ± 2 (17-20) 15 ± 4 (8-21) 17 ± 4 (11-24) 17 ± 3 (11-24) Proline, µmol/L 101 ± 23 (75-118) 78 ± 14 (54-99) 82 ± 22 (59-150) 80 ± 14 (58-101) Serine, µmol/L 82 ± 14 (70-97) 79 ± 19 (47-103) 67 ± 11 (45-81) 73 ± 17 (55-117) Threonine, µmol/L 75 ± 15 (65-92) 93 ± 46 (61-209) 159 ±75 (7-377) 191 ± 106 (58-420) Valine, µmol/L 200 ± 72 (157-283) 147 ± 33 (106-218) 159 ± 48 (94-307) 133 ± 21 (93-167) NEAA, µmol/L 1282 ± 193 (1102-1486) 1084 ± 196 (876-1322) 1116 ± 199 (790-1444) 1164 ± 337 (716-2095) EAA, µmol/L 1329 ± 630 (662-1914) 853 ± 121 (752-1020) 879 ± 223 (595-1276) 844 ± 158 (507-2095) Table 14 - Plasma Amino Acid Panel in mPKU Pregnancies 1 Within the 3 months prior to the reported conception date (-) Indicates missing data NEAA represents total nonessential amino acids, EAA represents total essential amino acids  82   Preconception1 (n=3) 1st Trimester (n=4) 2nd Trimester (n=13) 3rd Trimester (n=13)   Mean ± SD (range) Platelet Count 238 ± 103 (165-310) 214 ± 60 (128-285) 217 ± 78 (113-359) 233 ± 81 (122-399) Hematocrit, % 40 ± 4 (37-42) 39 ± 2 (35-44) 36 ± 3 (32-42) 35 ± 9 (32-40) RBC 4.3 ± 0.2 (4.1-4.4) 4.3 ± 0.3 (4.0-4.7) 3.9 ± 0.3 (3.3-4.5) 3.8 ± 0.2 (3.5-4.2) WBC 7.1 ± 1.2 (5.8-7.9) 7.5 ± 1.8 (2.5-9.8) 8.5 ± 2.4 (3.9-13.4) 10.6 ± 2.3 (5.1-14.3) Albumin, g/L - 42 ± 3 (36-47) 36 ± 4 (30-44) 33 ± 4 (25-40) Pre-albumin, mg/L - 238 ± 41 (209-267) - - Ferritin, ug/L - 44 ± 33 (14-124) 40 ± 36 (14-122) 22 ± 17 (4-62) Vitamin D, ng/mL - 87 ± 25 (60-121) 93 ± 11 (85-100) - Hemoglobin, g/dL 135 ± 13 (125-144) 132 ± 9 (117-151) 121 ± 9.6 (109-142) 119 ± 8 (105-131) Table 15 - Biochemical Variables in mPKU Pregnancies 1 Within the 3 months prior to the reported conception date (-) Indicates missing data  5.4 Discussion To the best of our knowledge, this is the first retrospective analysis of mPKU patients that includes detailed dietitians’ records (prescribed phenylalanine intakes, etc). This study described current dietary management practices of pregnant women with mPKU at Vancouver General Hospital, while demonstrating that adherence to phenylalanine intake recommendations is working well to achieve metabolic control before 8 wk pregnant, as per recommendations from the mPKU Collaborative Study (28,29). Ideally, in the future, management practices will be better able to target women pre-conception, as this study illustrated phenylalanine concentrations in blood spots were above the recommended range during preconception. Our analysis also shows that there is a more rapid increase in phenylalanine values around 20 wk gestation, highlighting the importance of continued monitoring of these subjects as the pregnancies progress.  83   The rise in phenylalanine tolerance throughout mPKU pregnancies has been documented in previous studies. Phenylalanine tolerance refers to the quantity of phenylalanine individuals with PKU can consume while maintaining healthy plasma phenylalanine levels (2-6 mg/dL or 120-360 µmol/L). However, it has also been recorded that the rise in tolerance was reduced in pregnancies where the fetus also has PKU, paired with a greater likelihood of having elevated phenylalanine concentrations in blood (130). This suggests that PAH activity in the fetus plays a role in maternal metabolic control, and that a smaller increase in phenylalanine tolerance towards the end of pregnancy may indicate fetal PKU. To the best of our knowledge, no offspring were diagnosed with PKU from the 16 pregnancies reported in this study. As well, we did not have access to the PAH genotype responsible for the PAH mutation, or the subjects’ original diagnosis. Therefore, differences in the subjects’ phenylalanine tolerance slope throughout pregnancy could be affected by differences among their ability, as well as their fetuses, to metabolize phenylalanine. Previously, our laboratory has determined minimum dietary phenylalanine (in the presence of excess tyrosine at 65 mg·kg-1·d-1) requirement for healthy pregnant women using stable isotope-based techniques. During early pregnancy (13-19 wk), we found the mean requirement to be 15 mg·kg-1·d-1, which is 65% higher than what has previously been determined in adult males (78,90). The mean requirement for phenylalanine during late pregnancy (33-39wk) is 21 mg·kg-1·d-1, which is 40% higher than the requirement for early pregnancy (90). These results provide insight into aromatic amino acid requirements and metabolism during pregnancy, having implications for future studies and current clinical practices for women with mPKU. When comparing against the data collected from this study, at 13-19 wk pregnant, subjects were consuming approximately 50% less phenylalanine than the previously determined 84  requirement (15 mg·kg-1·d-1) in healthy human pregnancies (90). At 33-39 wk pregnant, the difference was narrower, with subjects consuming 16% less phenylalanine than the previously determined requirement of 21 mg·kg-1·d-1 (90). A rationale for why these subjects consumed less phenylalanine than the requirements in pregnant women without PKU while maintaining good metabolic control was because there will be obligatory oxidation of phenylalanine to tyrosine when PAH is functioning properly. This remains true even when healthy women are provided with excess tyrosine in the diet, which allows for saturation of the body’s tyrosine pool, meaning there will be a decrease in loss of phenylalanine from conversion to tyrosine (131). There are data to support that some obligatory oxidation of phenylalanine through conversion to tyrosine occurs in healthy human populations, varying depending on method of determination and life stage, with previous studies showing it ranges anywhere from 2-26% (78,79,131,132). While this has never been measured during pregnancy, there is no way of knowing if this obligatory oxidation is affected during different stages of gestation, this accounts for the lower intake in comparison to previously determined requirements. Another potential rationale is that with the increase in phenylalanine seen in blood spots between 8 and 12 wk, the dietitians are recommending very low phenylalanine intakes to ensure the subjects achieve metabolic control during this critical stage of development. Lastly, though unknown, the decrease in percent difference from 50% to 16% may be due to the maturing fetus’s PAH capabilities, allowing for more conversion of phenylalanine to tyrosine.   Mean protein intake, as a function of kg of body weight, rose throughout the pregnancies.  This corresponds well to what we know about dietary protein requirements during pregnancy, as there are increases in nitrogen retention and tissue accretion throughout pregnancy (4,39). It is important to remember that the total protein values we report are estimated from phenylalanine 85  intake and added on to what is consumed as medical foods. There was negligible percent difference (less than 5%) between protein intake at 13-19wk and 33-39wk when compared to the previously determined protein requirement. Having a high protein intake in all people with PKU is recommended, since low phenylalanine intake can limit protein synthesis (133). Our laboratory has shown earlier that children with PKU have a mean protein requirement of 1.85 g·kg-1·d-1, which is substantially higher than the recommendations for PKU children of 1.33 g·kg-1·d-1 (134). While mPKU protein requirements are yet to be determined, ideally, protein and other amino acids should not be limiting, potentially further decreasing the potential for adequate protein synthesis (135). Therefore, ideally, protein intake should be higher in these mPKU pregnancies than the previously determined requirement in healthy pregnant women, to account for this. The current DRI recommendation is 0.88 – 1.1 g·kg-1·d-1 throughout all of pregnancy (5), and generally in PKU management the target is to achieve 120 – 140% above this recommendation. Clearly there is a need to determine protein requirements in mPKU during different gestation stages to ensure optimal protein intake recommendations can be provided.  Recently, a research group in Baltimore performed a retrospective analysis of mPKU subjects (n= 35) and determined that women who achieved metabolic control of phenylalanine prior to conception had improved phenylalanine plasma concentrations for the remainder of their pregnancy, similar to previously published studies (112,136,137). This indicated that early metabolic control was a good predictor for overall control throughout pregnancy. In the current study, we found that those who had good metabolic control (consistent phenylalanine blood levels between 120 and 360 µmol/L) and those who didn’t (consistent phenylalanine levels >360µmol/L) in early pregnancy both managed to achieve good metabolic control by mid-pregnancy. Another publication, from the Charles Dent Metabolic Unit in London, described 86  their clinic’s current mPKU management. Similarly, they highlight the importance of prepregnancy control, emphasizing that natural sources of dietary protein should be greatly restricted (138). As well, a research group in Turkey retrospectively analyzed data from mPKU subjects (n=71) (139). They determined that metabolic control worsens during the late first trimester, and that more frequent monitoring during this period may be one of the keys to improving metabolic control and birth outcomes. A similar trend is seen in our current analysis, where phenylalanine concentrations in blood rose around 10-12 wk pregnancy, and then reduced after 20 wk gestation and stabilized. However, our findings also highlight the importance of continued monitoring during late stages of pregnancy, where phenylalanine tolerance rises at a higher rate (Figure 2). Continuing to diligently monitor these subjects after 20 wk will prevent phenylalanine concentrations from dropping too low, which can increase the risk of complications such as intrauterine growth restriction (140).  Maintaining proper tyrosine levels is also important in mPKU patients. Tyrosine is the precursor for catecholamines, thyroid hormones, and melanin, with both low and high levels of tyrosine having potential health impacts on the mother and fetus. Tyrosine monitoring and supplementation occurs in both PKU and mPKU to reduce/mitigate any potential negative effects, though they are poorly understood. During mPKU pregnancies, a tyrosine deficiency may be particularly harmful to the fetus; not only is the mother unable to synthesize tyrosine, but the fetus also has reduced tyrosine synthesizing capacity due to its increased risk of having PKU and its premature hepatic enzymatic abilities (33,119). Therefore, it is currently recommended that if multiple low tyrosine concentrations in blood (<45 µmol/L) spots are observed, supplementation should be initiated (33,135,141,142). Though currently there is no evidence for tyrosine toxicity in mPKU pregnancies from overconsumption, there are arguments for 87  preventing blood concentrations from rising to high (142) . Active transport of placental tyrosine to the fetal compartment will allow fetal blood tyrosine concentrations to be 1.8 to 3.3 times greater than maternal concentrations (143). These high concentrations have unknown consequences on fetal development. As well, due to competitive inhibition of placental transporters that carry both phenylalanine and tyrosine, fetal phenylalanine concentrations may be lowered (144). In comparison with previous publications, the blood spot tyrosine values observed in our analysis indicate good monitoring and control of tyrosine, and will contribute to the body of reference values currently available for mPKU pregnancies (33,141,145).  Retrospective analyses are accompanied with a few limitations. Firstly, we did not have access to the health records of the babies born to allow us to analyze detailed birth outcomes and assess any long-term effects. We recognize this would have greatly improved the scope of our study, as the long-term complications of mPKU for offspring are well documented. It is difficult to determine if the current management mitigates these risks without including this piece of information. As well, our sample size was small, though this is a common problem with inherited metabolic disease studies. Lastly, we had to estimate both natural dietary protein intake and weight at each stage of pregnancy.  In conclusion, the current dietary management practices described in this natural history of mPKU subjects from Vancouver General Hospital are working well to achieve targeted metabolic control.  Mean phenylalanine and tyrosine concentrations in blood are within the target range by 8 wk gestation. The findings highlight the importance of early control and continued diligent management throughout pregnancy to ensure phenylalanine and tyrosine tolerance are being accounted for. This study provides a wealth of reference values to allow for comparison between clinics, an understanding of how well our own practices are working at the Adult 88  Metabolic Diseases Unit, and provides a basis for future experimental studies in our mPKU population.   89  Chapter 6: Discussion and Conclusion 6.1 Discussion This dissertation contributes a significant amount of new data to our current understanding of aromatic amino acid requirements and metabolism during pregnancy. The in-utero environment for fetuses plays an important role in their life long health. It is well documented that dietary protein intake during pregnancy is key for healthy development of fetal, placental, and maternal tissues particularly regarding indispensable amino acids. However, due to the technical and ethical constraints of previous experimental methods, they have never been determined during pregnancy. As well, data are limited on inborn errors of metabolism, such as PKU, during pregnancy. Due to newborn screening and an improvement in the clinical treatment of PKU (phenylalanine restriction in the diet and the advancement of medical foods), more women with PKU are reaching adulthood in good health, leading to a recent rise in pregnancies (mPKU) in this population. It is well documented that poor metabolic control during mPKU affected pregnancies has serious negative health impacts, but there are limited descriptions of current management.  The research described in this dissertation provides the following critical information: the dietary requirement for minimum phenylalanine (in the presence of excess tyrosine) during early and late pregnancy, the dietary requirement for TAA (in the absence of dietary tyrosine) during early and late pregnancy, and a description of current dietary management practices and their efficacy for mPKU patients at Vancouver General Hospital.   The dietary requirement for minimum phenylalanine was found to be ~15 and 21 mg·kg-1·d-1 in early and late pregnancy respectively. This translates to a 40% increase in requirement during gestation (90). This corroborates previous publications in humans and pigs, which 90  describe an increase in requirements of indispensable amino acids between early and late pregnancy (10). Interestingly, there was a 65% increase between the previously determined non-pregnant adult requirement of 9.1 mg·kg-1·d-1 and early pregnancy (78).  The dietary requirement for TAA was found to be ~ 44 and 50 mg·kg-1·d-1 in early and late pregnancy (a 14% increase), respectively. Though data from previous studies using stable isotopes on TAA requirements in non-pregnant adults are conflicting, it appears our early pregnancy requirement is similar to the mean recommendation provided based on a summary of the older studies of 43 mg·kg-1·d-1 (89). Both the early and late pregnancy EAR that we determined are higher than current DRI recommendations of 36 mg·kg-1·d-1.  Lastly, we described current dietary management practices for mPKU patients at Vancouver General Hospital and illustrated their efficacy. Though the findings indicated the diet practices are working very well to achieve metabolic control, they also highlighted the importance of targeting control during pre-pregnancy and continuing to diligently monitor patients throughout the pregnancy, as phenylalanine tolerance increases more rapidly after ~20wk gestation. The results of the first two studies (Chapters 3 and 4) have different implications for improving our understanding of nutrition during pregnancy. When determining minimum phenylalanine requirements, an excess of tyrosine is provided in the diet to prevent loss of phenylalanine to meet the body’s tyrosine requirement. Currently, recommendations are not available for minimum phenylalanine in the DRIs. In people with normally functioning PAH, the tyrosine requirement can be met from dietary phenylalanine or tyrosine, and both are present in the majority of natural protein sources. However, in individuals with PKU, the decreased ability or inability to convert phenylalanine to tyrosine means that the requirement for these two amino 91  acids must be met separately. Therefore, by determining minimum phenylalanine requirements we are able to provide insight into the increase in phenylalanine requirement that occurs in all pregnancies without unethically providing graded doses of phenylalanine to women with mPKU. It is important to note there will always be obligatory oxidation of phenylalanine to tyrosine in people unaffected by PKU, meaning the requirements we determined will likely be slightly higher than those for individuals with mPKU, although this needs to be established in future. Additionally, determining minimum phenylalanine requirements, paired with TAA requirements, allows us to determine how much of the requirement for phenylalanine can be spared by dietary tyrosine, and how much must be provided in the diet as phenylalanine. Determining TAA requirements will contribute to updated recommendations in the DRI, which are currently stated as ‘phenylalanine + tyrosine’ at 36 and 44 mg/kg/d, as EAR and RDA, respectively (5). The results from the current thesis adds support to our laboratory’s previous findings that suggest factorially calculating protein and amino acid requirements for pregnant women does not provide accurate values. As well, it highlights that static recommendations (one recommendation throughout all stages of pregnancy) for nutrients during pregnancy are not appropriate. Static recommendations do not account for the significant changes in metabolism and rate of tissue accretion that occurs throughout pregnancy (146). However, when discussing the results of these data, it is important to note that we do not know if phenylalanine can meet the full requirement for TAA during pregnancy. In 2007, Hsu et al published data suggesting that in healthy school-aged children, phenylalanine may not be able to provide the full needs for the aromatic amino acids (147). To verify these findings, in 2011 Hsu et al measured phenylalanine flux, hydroxylation, and oxidation by administering 13C-phenylalanine and 2H2 -tyrosine to children. They found that healthy children are capable of converting phenylalanine to tyrosine, 92  but the full tyrosine requirement cannot be met by phenylalanine. Seeing as both pregnancy and childhood are periods of rapid growth, it would be interesting to investigate whether tyrosine is a conditionally essential amino acid during healthy pregnancies. When pairing the results of Chapter 3 and 4 together, further insight into phenylalanine and tyrosine requirements and metabolism during pregnancy are uncovered. Both minimum phenylalanine and TAA requirements increased by ~ 6 mg·kg-1·d-1 between early and late pregnancy. It was a surprising result that TAA requirements did not increase more than minimum phenylalanine requirements. This is potentially due to an increase in phenylalanine requirement throughout pregnancy, but not tyrosine. Further justification for this rationale is provided after calculating the percent of the TAA requirement that can be met by supplying tyrosine in the diet. Based on previous studies in non-pregnant adults, it was calculated that 78% of the TAA requirement can be spared by tyrosine, with the remaining 22% being required in the diet as phenylalanine (89). Our data in pregnant women shows that tyrosine spares 66% and 58% of the TAA requirement in early and late pregnancy, respectively. This illustrates that as pregnancy progresses, more of the TAA requirement must be supplied as phenylalanine, possibly because the phenylalanine requirement is increasing disproportionately to the tyrosine requirement. It was an easy decision to design a mPKU project after beginning to implement the studies from Chapters 3 and 4. The majority of data available on aromatic amino acids has been conducted to provide further insight into this inborn error of metabolism. However, a literature review revealed gaps in descriptions of current practices for mPKU and limited data on phenylalanine and tyrosine intake and blood analyses. A natural history study (retrospective analysis of historical cases) would allow us to directly compare the results from the minimum 93  phenylalanine requirement study to phenylalanine intakes in mPKU affected pregnancies that achieved metabolic control. We found, as hypothesized, that at 13-19 and 33-39 wk gestation, mPKU subjects were consuming less phenylalanine than the determined EARs. Their mean phenylalanine consumption was ~ 50% and 16% less than the determined requirements during early and late gestation, respectively. A potential rationale for this large discrepancy is that during early pregnancy, the dietitians are recommending very low phenylalanine intakes because preconception metabolic control was poor, and lowering blood phenylalanine concentrations is a priority. As well, there are no data available on obligatory oxidation of phenylalanine to tyrosine during pregnancy, and potentially it is higher at early stages of gestation compared to late stages, and this needs to be determined in future.  6.2 Limitations and Strengths A limitation present in all three studies is a small sample size. Stable isotope-based requirement studies are a huge time commitment for both the participant and researcher. Additionally, when working with a pregnant population the targeted time window is narrow due to the dynamic nature of the physiological stage, meaning a very limited number of studies can be conducted per participant and a larger sample size is difficult to achieve. However, the breakpoint analyses in Chapters 3 and 4 indicate robust data with high R2 and low AICs. As well, to the best of our knowledge, these studies provide the first experimentally determined requirements for minimum phenylalanine and TAA during healthy human pregnancies. A small sample size is also expected for the natural history analysis, due to the rarity of the disease.  Another limitation for the requirement studies is that protein is provided as pure crystalline amino acids on the study days, which does not mimic normal intact dietary protein. 94  Our study day diets are more readily digested and absorbed, allowing for a higher bioavailability of the nutrients we study than what is found in normal food sources. However, the results from these studies can be used when designing future projects that investigate protein quality and bioavailability of different food sources for phenylalanine and tyrosine during pregnancy. Two key limitations are associated with the natural history project. As we did not have access to medical charts, but dietitian’s charts, we were unable to determine the genotype causing PKU in each subject. In a disease caused by over 500 different mutations in the gene encoding PAH, this could have provided additional information on the severity of each subject’s disease. Secondly, we were unable to assess long term offspring outcomes, beyond the Dietitian’s notes which indicated a healthy birth. Though we highlighted the success of the Adult Metabolic Disease Unit had at achieving metabolic control in their patients, long term follow-up of the children would have allowed for more significant conclusions to be made based on the ideal metabolic control observed in most mPKU patients. The strengths of Chapter 3 and 4 include: they are the first studies to experimentally determine minimum phenylalanine and TAA requirements during pregnancy, it was the first time the DAAO and IAAO technique have been compared in the same population, and they provided robust breakpoints with high R2 that were statistically different between early and late pregnancy. The strengths of Chapter 5 include: it was the first study to analyze dietitian’s charts for individuals with mPKU in a detailed manner, it provides a substantial number of reference values (dietary recommendations, dietary intake, and blood analyses), and includes 16 pregnancies.  95  6.3 Future Directions After presenting the above data, many questions about aromatic amino acids and pregnancy remain unanswered. Before conducting more studies that determine TAA requirements during mPKU pregnancy, it would be important to determine if phenylalanine can meet the full tyrosine requirement. We would also be able to draw more direct conclusions about how the minimum phenylalanine requirements translate to individuals with mPKU if we could investigate obligatory oxidation of phenylalanine to tyrosine that occurs at different stages of gestation. Additionally, it would be beneficial to compare the effect on protein synthesis of different ratios of phenylalanine to tyrosine in both healthy and mPKU affected pregnancies. Lastly, and more broadly, determining the dietary requirements for the remaining indispensable amino acids during pregnancy and their metabolic availability from different food sources will be necessary to translate findings to food based dietary guidance during such active stages of growth.  6.4 Conclusion The results of the series of studies presented in this thesis will contribute to updated recommendations for phenylalanine and tyrosine intake during pregnancy, as current recommendations are underestimated. These findings also illustrated that as pregnancy progresses, more of the TAA requirement must come from dietary phenylalanine. The natural history analysis provides feedback to the Adult Metabolic Disease Unit at Vancouver General Hospital on the efficacy of their current management practices for PKU women during pregnancies. Though optimal metabolic control is being achieved in their patients in early pregnancy, the results from this study highlight the importance of targeting pre-conception 96  control and continued monitoring throughout the entire pregnancy as phenylalanine tolerance rises quickly after ~20 wk gestation. As well, it provides other primary care facilities with substantial reference values for phenylalanine and tyrosine blood spot values, dietary phenylalanine and protein prescriptions, and allow for comparison of management techniques. 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Pediatr Res. 2007 Mar;61(3):361–5.   114  Appendices Appendix A   - Recruitment Materials for Chapter 3  115   116                       117  Appendix B   - Consent Form for Chapter 3  118   119   120   121   122   123   124  Appendix C   - Preliminary Assessment Form for Chapter 3 PRE-STUDY ASSESSMENT FORM DETERMINING PHENYLALANINE REQUIREMENTS AT DIFFERENT STAGES OF GESTATION IN HEALTHY PREGNANT WOMEN  Subject ID:________________________  Date:_______________________________ Birth date (Mo/yr):__________________  Age yr:_____________________________ Last menstrual period:________________  Ultra sound dating:___________________ Gestational age (week+days):___________   Pre-pregnancy weight:_________________  Pre-pregnancy BMI:__________________ Fasting glucose:______________________   Urine test strip: pH:___________ Glucose:_________ Ketones:__________ Leukocytes:___________ Nitrile:________ Protein:__________ Blood:____________  Height:___________________________  Weight:___________________________  Optional: Primary health care provider Name:___________________________  Phone:____________________________ Address:_____________________________________________________________________  Skinfold measurements Mid-arm circumference (cm):___________  Tricep(mm):_________________________ Bicep (mm):_________________________  Sub-scapula (mm):____________________  Bioelectric Impedance Analysis Resistance:_________________________  Reactance:_________________________ Impedance:_________________________  125   Indirect calorimetry Measured REE (kcal/day):______________  Medical history Are you currently taking any medications?  □Yes   □No If yes, list of medications:_________________________________________________________ Are you claustrophobic?  □Yes   □No Do you smoke?  □Yes   □No Are you currently taking any supplements?   □Yes   □No If yes, list of supplements and amount taken:__________________________________________ How long have you been taking them?_______________________________________________  Do you have any pre-existing health conditions?____________________________________________________________________ Have you ever been diagnosed with any of the following: □Hypertension □ Diabetes  □ Pregnancy related anemia □ Pregnancy related jaundice  Have you ever experienced any of the following? □ Spontaneous abortion □ pre-term birth  □ Severe pregnancy related nausea or vomiting  Do you have any food allergies:____________________________________________________ Do you have any diet preferences (vegetarian, gluten free etc.)?___________________________    126  Appendix D  - Study Day Form for Chapter 3 Study Day Form Determining Dietary Phenylalanine Requirements during Different Stages of Gestation in Healthy Pregnant Women Subject ID:______________ Date:__________________  Blood Glucose (mmol/L):_____________  Height (cm):_____________ Weight (kg):_________________ Phe. intake (mg/kg/d): ________  Energy intake (kcal/day): _________ Prescription medicine taken: ________________________  Time Sample Collection/ Anthropometry Meals and isotope  tracer Comments 8:00 Fasting blood glucose 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 + 1st oral dose  13:00 1st blood sample Meal #6 – 2nd oral dose  14:00  Meal #7 – 3rd oral dose  14:30 4th breath (3x) 3rd urine   15:00 5th breath (3x) Before meal 4th urine Meal #8 – 4th oral  dose  15.15 6th breath (3x)   15:30 7th breath (3x) 5th urine    15:45 8th breath (3x)   16:00 9th breath (3x) 6th urine    Morning urine Afternoon Urine Comments pH    Glucose    Ketones    Leukocytes    Nitrile    Protein    Blood    127  Appendix E  - Master List for Chapter 3             128  Appendix F  - Dietary Record for Chapter 3           129  Appendix G  - Recruitment Materials for Chapter 4           130             131  Appendix H  - Consent Form for Chapter 4  132   133   134   135   136   137   138    139  Appendix I  - Preliminary Assessment Form for Chapter 4 PRE-STUDY ASSESSMENT FORM Determining Dietary Total Aromatic Amino Acid (Phenylalanine and Tyrosine) Requirements during Different Stages of Gestation in Healthy Pregnant Women  Subject ID:____  ____________     Date:______    _______ Birth date (Mo/yr):__    ______  Age yr:____________________ Last menstrual period:__   _____  Previous Pregnancies:     . Gestational age (week+days):__         __   Pre-pregnancy weight:_    _______  Pre-pregnancy BMI:___________ Fasting glucose:______   __   Urine test strip: pH:___________ Glucose:_________ Ketones:__________ Leukocytes:___________ Nitrile:________ Protein:__________ Blood:____________  Height:_______________________  Weight:__________________  ptional: Primary health care provider Name:___________________________  Phone:____________________________ Address:_____________________________________________________________________  Skinfold measurements Mid-arm circumference (cm):___________  Tricep(mm):_________________________ Bicep (mm):_________________________  Sub-scapula (mm):____________________  Bioelectric Impedance Analysis Resistance:______              Reactance:_________________________ Impedance:________________________ 140    Indirect calorimetry Measured REE (kcal/day):______________  Medical history Are you currently taking any medications?  □Yes   □No If yes, list of medications:_________________________________________________________ Are you claustrophobic?  □Yes   □No Are you substance dependent (ie cigarettes, alcohol, illicit drugs)?  □Yes   □No Are you currently taking any supplements?   □Yes   □No If yes, list of supplements and amount taken:               _________ How long have you been taking them?                     _  Do you have any pre-existing health conditions?_______________________________________________________________ Have you ever been diagnosed with any of the following: □Hypertension □ Diabetes  □ Pregnancy related anemia □ Pregnancy related jaundice  Have you ever experienced any of the following? □ Spontaneous abortion □ pre-term birth  □ Severe pregnancy related nausea or vomiting  Do you have any food allergies: ____________________________ Do you have any diet preferences (vegetarian, gluten free etc.)?_____          __   141  Appendix J   - Study Day Form for Chapter 4 Study Day Form Determining Dietary Total Aromatic Amino Acid (Phenylalanine and Tyrosine) Requirements during Different Stages of Gestation in Healthy Pregnant Women Subject ID:______________ Date:________________  Blood Glucose (mmol/L):_____________   Height (cm):_____________ Weight (kg):_________________ Phe. intake (mg/kg/d): ____ ___    Energy intake (kcal/day): _________ Prescription medicine taken: ______________________ __ Time Sample Collection/ Anthropometry Meals and isotope  tracer Comments 8:00 Fasting blood glucose 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)   12:00 VCO2 measurement  Meal #5 – primer dose + 1st oral dose  13:00 1st blood sample Meal #6 – 2nd oral dose  14:00  Meal #7 – 3rd oral dose  14:30 4th breath (3x)   15:00 5th breath (3x) Before meal 2nd urine Meal #8 – 4th oral  dose  15.15 6th breath (3x)   15:30 7th breath (3x)    15:45 8th breath (3x)   16:00 9th breath (3x) 3rd urine    Morning urine Afternoon Urine Comments pH    Glucose    Ketones    Leukocytes    Nitrile    Protein    Blood    142  Appendix K  - Master List Chapter 4            143  Appendix L  - Diet Record Chapter 4                    

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