Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Application of stable isotope tracers to examine phenylalanine metabolism and protein requirements in… Turki, Abrar Mohammed 2015

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
24-ubc_2015_september_turki_abrar.pdf [ 2.69MB ]
Metadata
JSON: 24-1.0167276.json
JSON-LD: 24-1.0167276-ld.json
RDF/XML (Pretty): 24-1.0167276-rdf.xml
RDF/JSON: 24-1.0167276-rdf.json
Turtle: 24-1.0167276-turtle.txt
N-Triples: 24-1.0167276-rdf-ntriples.txt
Original Record: 24-1.0167276-source.json
Full Text
24-1.0167276-fulltext.txt
Citation
24-1.0167276.ris

Full Text

APPLICATION OF STABLE ISOTOPE TRACERS TO EXAMINE PHENYLALANINE METABOLISM AND PROTEIN REQUIREMENTS IN CHILDREN WITH PHENYLKETONURIA (PKU)  by  Abrar Mohammed Turki  B.Sc., King Abdulaziz University, 2009  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (HUMAN NUTRITION)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  May 2015 © Abrar Mohammed Turki, 2015 ii  Abstract Phenylketonuria (PKU) is an inherited inborn error of phenylalanine (PHE) metabolism caused by deficiency of hepatic enzyme phenylalanine hydroxylase (PAH). Therefore, PHE accumulates in plasma leading to mental retardation and developmental delay. Kuvan® (Sapropterin dihydrochloride), a synthetic form of tetrahydrobiopterin (BH4), has been shown to reduce plasma PHE levels in PKU, but not all patients respond to sapropterin treatment. The major mode of treatment remains nutritional management with dietary restriction of PHE and provision of sufficient protein. The dietary protein requirement in children with PKU remains unknown. Therefore the objectives of the current thesis were: 1) to identify sapropterin responsiveness in PKU children using a minimally invasive L-[1-13C] phenylalanine breath test (13C-PBT), and 2) to determine protein requirements in PKU children using the indicator amino acid oxidation (IAAO) technique. Experiment 1- Nine children with PKU (5-18y) underwent 13C-PBT tracer protocols twice, once before and once after 1-2 weeks of sapropterin therapy. 13CO2 was measured using isotope ratio mass spectrometer (IRMS). The study protocol was tested in healthy children (n= 6) as proof of principle. Experiment 2- Four PKU children (5-18y) were recruited to participate in test protein intakes (ranging from deficiency to excess 0.2 – 3.2 g/kg/d) with the IAAO protocol using L-[1-13C] leucine, followed by collection of breath and urine samples over 8 hours.  Results 1- 13CO2 productions in all children with PKU pre-sapropterin treatment were low, except in one child (PKU04). Five children with PKU showed a significantly higher peak enrichment after sapropterin treatment at 20min. Three PKU children had no change in 13CO2 production post sapropterin therapy. Results 2- The mean protein requirement, identified using 2-phase linear regression analysis was determined to be 1.85 g/kg/d. This result is significantly iii  higher than the most recent PKU recommendations (2014) (1.14 – 1.33g/kg/d, based on 120-140% above current recommended dietary allowance RDA). These findings show that the 13C-PBT can be a minimally invasive method to examine in vivo PHE metabolism in PKU children responsive to sapropterin therapy. Also, current recommendations for optimal protein intake may be underestimated.      iv  Preface This thesis has been written in partial fulfillment of the requirement for the degree of Master of Science in Human Nutrition. I have written this thesis under the direction and supervision of Dr. Rajavel Elango from September 2012 to April 2015. Dr. Rajavel Elango, Dr. Sylvia Stockler-Ipsiroglu and Dr. Tim Green reviewed this thesis. The current thesis consists of two experiments which were approved by the Children’s and Women’s Health Centre and the University of British Columbia Clinical Research Ethics Board (CW12-0150 / H12-01421 and CW13-0165 / H13-00220). Research dietitian, Gayathri Murthy, was responsible for subject recruitment, consent, assent, brief study day questionnaire and sample collection for 1st experiment in collaboration with myself. All experiment procedures for 2nd experiment related to study coordinator, pre-study day, test diet preparation (amino acid preparation), study day, sample collection and sample analysis (isotope ratio mass spectrometer (IRMS, Isoprime Ltd, Cheadle, UK) were performed by me at Child and Family Research Institute (CFRI) and Clinical Research and Evaluation Unit (CREU). I performed data entry and statistical analysis with the help of my supervisor Dr. Rajavel Elango and research dietitian Gayathri Murthy. The results for 1st experiment were presented as an oral presentation at Clinical Nutrition Week 2015 (CNW 2015) organized by the American Society for Parenteral and Enteral Nutrition (ASPEN) held in Long Beach, CA (February 14-17, 2015). I received a certificate of honor for an International Abstract of Distinction Award. The abstract was published in Journal of Parenteral and Enteral Nutrition (JPEN) online. "CLINICAL NUTRITION WEEK 2015: Long Beach, CA February 14-17, 2015: AWARDED ABSTRACTS: Vars Candidates, Trainee Awards, International Awards, and Abstracts of v  Distinction." Journal of Parenteral and Enteral Nutrition 39.2 (2015): 231-56. In addition, the results for 1st experiment, "Minimally Invasive 13C-Breath Test to Examine Phenylalanine Metabolism in Children with Phenylketonuria", has been accepted for publication (April 25, 2015) in "Molecular Genetics and Metabolism". The Results for 2nd experiment were presented as a poster presentation at Experimental Biology 2015 (EB 2015) / American Society for Nutrition Annual Meeting (ASN) in March 27-April 1, 2015 in Boston, MA (FASEB J April 2015 29:742.9). The same poster was used for presentation in 16th Annual Biological Mass Spectrometry Symposium organized by Cambridge Isotope Laboratories held in Boston, MA (March 29, 2015) entitled "Protein Requirements in Children with Phenylketonuria (PKU)". The poster was chosen as the best poster at the 2015 Dinner Symposium poster session.    vi  Table of Contents  Abstract .......................................................................................................................................... ii Preface ........................................................................................................................................... iv Table of Contents ......................................................................................................................... vi List of Tables ................................................................................................................................ xi List of Figures .............................................................................................................................. xii List of Abbreviations ................................................................................................................. xiv Acknowledgements .................................................................................................................... xvi Dedication .................................................................................................................................. xvii Chapter 1: Introduction ................................................................................................................1 1.1 Introduction and Overview ................................................................................................ 1 Chapter 2: Background .................................................................................................................3 2.1 Classification of Inborn Errors of Metabolism (IEM) ....................................................... 3 2.2 Phenylketonuria (PKU) ...................................................................................................... 4 2.3 Classification of PKU ........................................................................................................ 7 2.4 Nutritional Approaches for Management of Patients with PKU ....................................... 9 2.4.1 Sapropterin (Tetrahydrobiopterin-BH4) Therapy ....................................................... 9 2.4.1.1 Clinical Trials Determining the Efficacy of Sapropterin Treatment in PKU .... 10 2.4.1.2 Challenges Determining the Efficacy of Sapropterin Treatment in PKU.......... 13 2.4.1.3 Stable Isotope Techniques Determining PAH Activity in PKU ........................ 14 2.4.1.4 Phenylalanine Breath Test ................................................................................. 15 2.4.2 Dietary Therapy ........................................................................................................ 17 vii  2.4.3 Protein Requirements in PKU ................................................................................... 19 2.5 The Indicator Amino Acid Oxidation Technique (IAAO) .............................................. 21 2.5.1 Amino Acid Requirements in Children .................................................................... 23 2.5.2 Amino Acid Requirements in Disease ...................................................................... 23 2.5.3 Protein Requirements in Humans ............................................................................. 24 2.6 Choice of Indicator Amino Acid in PKU ........................................................................ 27 Chapter 3: Rationale, Objectives and Hypotheses....................................................................30 3.1 Rationale .......................................................................................................................... 30 3.2 Objectives ........................................................................................................................ 31 3.3 Hypothesis........................................................................................................................ 31 Chapter 4: Experiment 1 – Minimally Invasive 13C-Breath Test to Examine Phenylalanine Metabolism in Children with Phenylketonuria .........................................................................32 4.1 Methods and Materials ..................................................................................................... 32 4.1.1 Study Principle .......................................................................................................... 32 4.1.2 Subjects ..................................................................................................................... 33 4.1.2.1 Inclusion Criteria ............................................................................................... 33 4.1.2.2 Exclusion Criteria .............................................................................................. 34 4.1.3 Experimental Design – Healthy Children ................................................................. 35 4.1.4 Experimental Design – Children with PKU.............................................................. 37 4.1.5 Sample Collection ..................................................................................................... 39 4.1.6 Analytical Procedures ............................................................................................... 40 4.1.7 Calculations............................................................................................................... 40 4.1.8 Statistical Analysis .................................................................................................... 41 viii  4.2 Results .............................................................................................................................. 41 4.2.1 Subject Characteristics .............................................................................................. 41 4.2.2 % Dose Oxidized of L-[1-13C] Phenylalanine .......................................................... 43 4.3 Discussion ........................................................................................................................ 48 4.3.1 Limitations ................................................................................................................ 51 Chapter 5: Experiment 2 – Protein Requirements in Children with Phenylketonuria Using L-[1-13C] Leucine as IAAO .........................................................................................................53 5.1 Methods and Materials ..................................................................................................... 53 5.1.1 Subjects ..................................................................................................................... 53 5.1.1.1 Inclusion Criteria ............................................................................................... 53 5.1.1.2 Exclusion Criteria .............................................................................................. 54 5.1.2 Experimental Design ................................................................................................. 54 5.1.3 Pre – Study Day and Study Protocol......................................................................... 58 5.1.3.1 Pre – Study Day Protocol ................................................................................... 58 5.1.3.2 Study Protocol and Isotope Infusion Studies ..................................................... 59 5.1.4 Sample Collection ..................................................................................................... 60 5.1.4.1 Breath Samples .................................................................................................. 60 5.1.4.2 Urine Samples .................................................................................................... 61 5.1.5 Analytical Procedures ............................................................................................... 62 5.1.6 Isotope Kinetics ........................................................................................................ 62 5.1.7 Statistical Analysis .................................................................................................... 62 5.2 Results .............................................................................................................................. 63 5.2.1 Subject Characteristics .............................................................................................. 63 ix  5.2.2 Protein Requirements in Children with PKU............................................................ 64 5.2.3 Phenylalanine and Tyrosine Levels .......................................................................... 65 5.3 Discussion ........................................................................................................................ 66 5.3.1 Protein Requirements in Children with PKU............................................................ 66 5.3.2 Phenylalanine and Tyrosine Levels .......................................................................... 66 5.3.3 Limitations ................................................................................................................ 69 5.3.4 Significance and Clinical Implications ..................................................................... 69 Chapter 6: Conclusions and Future Directions.........................................................................73 Bibliography .................................................................................................................................75 Appendices ....................................................................................................................................89 Appendix A : Subject Consent Form – Healthy Children ........................................................ 89 Appendix B : Subject Assent Form – Healthy Children ........................................................... 96 Appendix C : Advertisement – Healthy Children ..................................................................... 99 Appendix D : Study Day Form – Healthy Children ............................................................... 101 Appendix E : Subject Consent Form – Children with PKU ................................................... 104 Appendix F : Subject Assent Form – Children with PKU ...................................................... 113 Appendix G : Advertisement – Children with PKU ............................................................... 116 Appendix H : Patient Code Form – Children with PKU ........................................................ 118 Appendix I : Study Day Form – Children with PKU.............................................................. 120 Appendix J : Subject Consent Form – Protein Requirement in PKU ..................................... 123 Appendix K : Subject Adolescent Assent Form – Protein Requirement in PKU ................... 131 Appendix L : Subject Assent Form – Protein Requirement in PKU ...................................... 139 Appendix M : Pre – Study Day Questionnaire – Protein Requirement in PKU ..................... 142 x  Appendix N : Advertisement – Protein Requirement in PKU ................................................ 145 Appendix O : Subject Code Master List – Protein Requirement in PKU ............................... 147 Appendix P : Dietary Record Sheets – Protein Requirement in PKU .................................... 150 Appendix Q : Study Day Form – Protein Requirement in PKU ............................................. 152  xi  List of Tables  Table 1. Categories of Some IEM That Respond to Dietary Treatment ......................................... 3 Table 2. Prevalence of PKU by Population .................................................................................... 6 Table 3. PKU Classification Based on Dietary PHE Tolerance ..................................................... 8 Table 4. Plasma PHE Levels and Estimation of Initial Dietary PHE Intake ................................ 18 Table 5. Recommended Intakes of PHE, TYR, and Protein for Individuals with PAH Deficiency....................................................................................................................................................... 19 Table 6. Recommended Intakes of Protein in Children with PKU Calculated from (Table 5) .... 20 Table 7. Comparison of Healthy Children Amino Acid Requirements Identified by IAAO and DRI, 2005 ...................................................................................................................................... 23 Table 8. Comparison of Protein Requirements Identified by IAAO and DRI, 2005 in Healthy Adults, Children, and Pregnant Women ....................................................................................... 24 Table 9. Characteristics of Healthy Children (Nsubjects= 6)1 .......................................................... 42 Table 10. Characteristics of Children with Phenylketonuria (PKU) (Nsubjects= 9) ........................ 42 Table 11. L-[1-13C] Phenylalanine Oxidation (% of dose) Area Under the Curve (AUC) in Healthy Children (Nsubjects= 6) ...................................................................................................... 45 Table 12. L-[1-13C] Phenylalanine Oxidation (% of dose) Area Under the Curve (AUC) in Children with Phenylketonuria (PKU) (Nsubjects= 9) ..................................................................... 47 Table 13. Amino Acid Mixture Based on the Egg Protein Pattern Used on Study Day (Excluding L-Leucine, and L-Phenylalanine) ................................................................................................. 57 Table 14. Subject characteristics of children with PKU (Nsubjects = 4) .......................................... 63  xii  List of Figures  Figure 1. Phenylalanine Metabolism in Patient with Phenylketonuria (PKU) ............................... 5 Figure 2. PKU Classification Based on Pretreatment Blood PHE Levels ...................................... 8 Figure 3. Chemical Structure of Sapropterin Dihydrochloride ..................................................... 10 Figure 4. BH4 Treatment Efficacy in Subjects with PKU. Response Rate (%) Based on Pre - treatment Blood PHE Levels ........................................................................................................ 11 Figure 5. Mean Change in Blood PHE Levels in Sapropterin Group vs. Placebo Group for a Period of 6 Weeks ......................................................................................................................... 12 Figure 6. Illustration the Phenylalanine Hydroxylation System and L-[1-13C] Phenylalanine Oxidation to 13CO2  ....................................................................................................................... 14 Figure 7. Phenylalanine Breath Test Protocol Representing L-[1-13C] Phenylalanine and BH4 Consumption Time. PBT, Phenylalanine Breath Test .................................................................. 16 Figure 8. Phenylalanine Breath Test in Control, Heterozygotes and Subjects with PAH Deficiency. Δ 13C (‰) During Two hours Study Protocol. Solid Lines Without BH4 and Dashed Lines With BH4 ............................................................................................................................. 16 Figure 9. Concept of the Indicator Amino Acid Oxidation Technique (IAAO) ........................... 22 Figure 10. Protein Requirements in Healthy Children Using IAAO ............................................ 26 Figure 11. Leucine Catabolism. BCKAD, Branched-Chain α-Keto Acid Dehydrogenase. ........ 29 Figure 12. Concept of the 13C Phenylalanine Breath Test (13C-PBT) .......................................... 32 Figure 13. Experimental Design in Healthy Children .................................................................. 36 Figure 14. Study Day Protocol for the 13C-Phenylalanine Breath Test (13C-PBT) ...................... 37 xiii  Figure 15. Experimental Design in Children with PKU ............................................................... 39 Figure 16. 13C-Phenylalanine Breath Test (13C-PBT) in Healthy Children .................................. 44 Figure 17A. 13C-Phenylalanine Breath Test (13C-PBT) in Children with Phenylketonuria (PKU). Rate of L-[1-13C] Phenylalanine Oxidation (% of dose) in Children with PKU Prior to Treatment with Sapropterin (Kuvan®) ........................................................................................................... 46 Figure 18. Experimental Design ................................................................................................... 55 Figure 19. Study Day Protocol for Protein Requirements in Children with PKU Using L-[1-13C] Leucine as IAAO .......................................................................................................................... 60 Figure 20. Estimated Average Protein Requirement in Children with PKU (Nsubjects = 4, nstudies = 27) ................................................................................................................................................. 64 Figure 21. PHE and TYR Levels (μmol/L) in Children with PKU at the End of Each Study Day....................................................................................................................................................... 65  xiv  List of Abbreviations 13C-PBT – 13C-Phenylalanine Breath Test AAA – Aromatic Amino Acids ACMG – American College of Medical Genetics and Genomics APE – Atom Percent Excess  AUC – Area Under the Curve  BCAA – Branched Chain Amino Acids  BH4 – Tetrahydrobiopterin BIA – Bioelectrical Impedance Analysis BMI – Body Mass Index Cmax – The Maximum Peak Enrichment in13CO2 Oxidation DRI – Dietary Reference Intakes EAR – Estimated Average Requirement  ECO2 – 13CO2 Isotopic Enrichment (APE) F13CO2 – Rate of L-[1-13C] Phenylalanine or Leucine Tracer Oxidation FAO – Food and Agriculture Organization FCO2 – CO2 Production Rate Using Indirect Calorimetry  FFM – Fat Free Mass  FM – Fat Mass HPA – Hyperphenylalaninaemia IAAO – Indicator Amino Acid Oxidation Technique IEM – Inborn Errors of Metabolism IRMS – Isotope Ratio Mass Spectrometry   xv  MET+CYS – Methionine with Cysteine MHP – Mild Hyperphenylalaninaemia  MSUD – Maple Syrup Urine Disease PAH – Phenylalanine Hydroxylase PBT – Phenylalanine Breath Test PHE – Phenylalanine PKU – Phenylketonuria RDA – Recommended Dietary Allowance REE – Resting Energy Expenditure  SD – Standard Deviation SE – Standard Error  TEE – Total Energy Expenditure tmax – Time to Reach Maximum 13CO2 Oxidation TSAA – Total Sulfur Amino Acids TYR – Tyrosine VCO2 – Rate of Carbon Dioxide Production W – Weight          xvi  Acknowledgements First, I would like to express my special gratitude to my supervisor Dr. Rajavel Elango for his guidance and encouragement throughout my master journey. Dr. Rajavel Elango giving me this wonderful opportunity to pursue a master degree in Human Nutrition and to delve deeper in research that can help me earn a higher proficiency level as a researcher in clinical nutrition field. I would also like to thank my committee members, Dr. Sylvia Stockler-Ipsiroglu and Dr. Tim Green for their support and knowledge that extremely valuable and greatly enrich my work. I sincerely thank the Ministry of Higher Education in Saudi Arabia for providing a scholarship and tuition funding to continue my graduate studies in Canada. I would like to thank the multidisciplinary team at the Biochemical Diseases Department at BC Children’s Hospital. I also like to thank all children with PKU and their parents who participated in these two experiments without of them this research could not have been accomplished.  To the University of British Columbia, the Child and Family Research Institute and BC Children’s Hospital I would like to express my appreciation for allowing this research to take place. I offer thank to Rare Disease Foundation and Saudi Arabian Cultural Bureau for supporting funds. I would like to acknowledge my past and present lab mates: Trina Stephens, Maggie Payne, Leah Cooper, Cindy Wong, Gayathri Murthy, Betina Rasmussen and Erin Gilbert for help and support during my master. Finally, I owe a special thank to my parents and siblings for their love and support. No words can express how grateful I am having a wonderful and amazing family.   xvii  Dedication I would like to dedicate this thesis to all those who believing in me and supporting me to complete all stages of my master journey My mother, Amal My father, Mohammed My brothers, Moayed, Maan and Majd My little sister, Ghala To my supervisor Dr. Rajavel Elango, my lab mates and my friends. 1  Chapter 1: Introduction 1.1 Introduction and Overview Inherited metabolic disorders or inborn errors of metabolism (IEM) are a group of genetic conditions characterized by a block in metabolic pathways, which leads to accumulation of toxic substrates and deficiency of essential metabolites (Jurecki et al, 2009). A British physician Sir Archibald Garrod (1857 – 1936) was the first to use the IEM term to illustrate the inheritable nature of enzyme deficiencies in metabolic pathways (Garrod AE., 1902).The primary cause of these disorders is a gene mutation that results in the total or partial impairment of a certain enzyme or cofactor (Trahms, 2004). The accumulation of toxic substrate leads to several health problems including neurological problems, which might result in death or disability in some cases (Das S., 2013). Generally, IEM are rare conditions with low incidence rates (Demirdas et al, 2013). The incidence rate of IEM is more than 1:1000 worldwide (Alfadhel et al, 2013) and it affects 1 in 4000 births in USA and 1:9300 in Japan (Frazier et al, 2006; Ya maguchi, 2008). Consanguinity within the population may play a major role in increasing the incidence rate in some countries like the Middle East (Selim et al, 2014). For instance, the prevalence of IEM is 1:1380 in Saudi Arabia, 1:1130 in Lebanon and 1:2497 births in India (Rashed et al, 1999; Karam et al, 2013; Latheef SA., 2010). However, the incidence rate of disorders of amino acid metabolism varies from one disease to the other and within the same disorder depending on a certain population.  Phenylketonuria (PKU) is one of the most common rare disorders and is classified as one of the treatable inborn errors of metabolism presenting with intellectual disability. PKU affects 1:15,000 newborn infants in the general population (Bélanger-Quintana et al, 2011; van Karnebeek and Stockler, 2012; Blau et al, 2010). This disorder is caused by deficiency of the 2  hepatic enzyme phenylalanine hydroxylase (PAH). Therefore, phenylalanine (PHE) accumulates in plasma leading to neurocognitive and developmental delay. Kuvan® (Sapropterin dihydrochloride), a synthetic form of the cofactor for PAH, (tetrahydrobiopterin BH4), has been shown to reduce plasma PHE levels and improve dietary PHE tolerance in patients with PKU. But not all patients respond to sapropterin treatment (Ponzone et al, 2010). Therefore there is a need to identify sapropterin responsive PKU patients in a robust and sensitive manner, which is the first goal of this research. The primary treatment for children with PKU involves a PHE restricted diet and an adequate supply of protein (Blau et al, 2010). However, growth impairment occurs in children with PKU and protein insufficiency has been implicated as a cause in reduced linear growth (Arnold et al, 2002; Huemer et al, 2007). Recently the American College of Medical Genetics and Genomics (ACMG) Guidelines for PKU management recommended that protein intake for children (> 4 y to adult) should be 120-140% of the recommended dietary allowance (RDA) for age (Vockley et al, 2014; Singh et al, 2014). These are simply predicted values. Protein requirements in children with PKU have not been determined directly and need to be examined, which is the second goal of this research.    3  Chapter 2: Background 2.1 Classification of Inborn Errors of Metabolism (IEM) IEM can be classified based on the dietary treatment (Table 1). Table 1. Categories of Some IEM That Respond to Dietary Treatment Disorder Enzyme Defect Dietary Treatment Urea Cycle Disorders Carbamoyl-phosphate synthetase deficiency Carbamoyl-phosphate synthetase  Protein diet for all urea cycle disorders Ornithine transcarbamoylase deficiency Ornithine transcarbamoylase Citrullinemia Argininosuccinic acid synthetase Argininosuccinic aciduria Argininosuccinic acid lyase Argininemia Arginase Organic Acidaemias Methylmalonic acidemia Methylmalonyl-CoA mutase  Protein diet;  isoleucine, methionine, threonine, valine Propionic acidemia Propionyl-CoA carboxylase  Protein diet;  isoleucine, methionine, threonine, valine Amino Acid Disorders Phenylketonuria Phenylalanine hydroxylase  Phenylalanine,  tyrosine Tyrosinemia I Fumaryl-acetoacetate hydroxylase  Phenylalanine,  tyrosine 4  Disorder Enzyme Defect Dietary Treatment Maple syrup urine disease Ketoacid decarboxylase  Leucine, isoleucine, valine Homocystinuria Cystathionine synthase  Protein,  methionine Carbohydrate Disorders Galactosemia Galactose-1-phosphate uridyl transferase  Galactose, lactose-free diet Hereditary fructose intolerance Fructose-1-phosphate aldolase Fructose, sucrose, sorbitol-free diet Glycogen storage disease Ia Glucose-1-6-phosphatase Glucose from uncooked cornstarch, avoidance of fructose, lactose Data adapted from (Trahms, 2004) This thesis covers disorders of amino acid metabolism, specifically PKU in children, the dietary needs for protein in PKU and the metabolism of PHE examined using stable isotope tracers.  2.2 Phenylketonuria (PKU) Phenylketonuria (PKU; OMIM 261600) is an autosomal recessive disorder and is caused by a partial/complete deficiency of the hepatic enzyme phenylalanine hydroxylase (PAH) activity, which is important to convert PHE into tyrosine (TYR) (Figure 1). In untreated individuals PHE accumulates in blood and in the brain, resulting in hyperphenylalaninaemia (HPA) leading to serious problems such as mental retardation, seizures and developmental delay (Acosta & Yannicelli, 2001; van Rijn et al, 2007). TYR concentrations will be reduced in 5  patients with PKU, which could lead to TYR deficiency (MacDonald et al, 2011). HPA results from mutations in the PAH gene and pterin-requiring enzymes gene (Walter et al, 2012).  Asbjørn Følling in 1930 was the first who illustrated that the main cause of neuropsychological deficits in patients with PKU is due to an increase in blood PHE concentrations (Blau et al, 2010). In 1950, Horst Bickel was the first to identify PHE-restricted diet as a major treatment for PKU (Bickel et al, 1953).    Figure 1. Phenylalanine Metabolism in Patient with Phenylketonuria (PKU). Adapted From (Walter et al, 2012). DHPR, Dihydropteridine Reductase; PCD, Pterin-4a-Carbinolamine Dehydratase.  The incidence rate of PKU varies globally (Table 2). The overall incidence in Europe is around 1: 10,000 births, but still this incidence varies within the same continent (Loeber, 2007). 6  For instance, Turkey has the highest prevalence in Europe with 1: 4000 births due to increased consanguinity (Ozalp et al, 2001). However, in other regions in Europe the incidence rate is low, such as in Finland 1: 100,000 births (Guldberg et al, 1995). Similarly, the incidence rate varies in other continents such as Latin America from 1: 50,000 to 1: 25,000 live births (Borrajo, 2007). The incidence rate of PKU in the USA is about 1: 15,000 (National institutes of health consensus development panel, 2001). In Asia the incidence differs from one area to the other with around 1: 200,000 in Thailand, 1: 100,500 in China, and 1: 70,000 live births in Japan (Pangkanon et al, 2009; Jiang et al, 2003; Aoki et al, 2007). The Middle East seems to have a high incidence rate of PKU, which is about 1: 4,400 (Tayeb, 2009).    Table 2. Prevalence of PKU by Population Population Incidence rate of PKU References Thailand 1: 200,000 Pangkanon et al, 2009 China 1: 100,500 Jiang et al, 2003 Finland 1: 100,000 Guldberg et al, 1995 Japan 1: 70,000 Aoki et al, 2007 USA 1: 15,000 National institutes of health consensus development panel, 2001 Turkey 1: 4000 Ozalp et al, 2001  In 1961, the Guthrie bacterial inhibition assay was developed to detect PKU. This is a simple technique, which uses dried blood spots to measure blood phenylalanine concentrations (Guthrie et al, 1963). Since the 1990’s, new techniques based on tandem mass spectrometry (MS/MS), which is a rapid method and can screen several IEM at the same time, have been 7  developed (Millington et al, 1990). The cut-off blood levels of PHE to detect PKU vary between laboratories. However, based on the international database, which represents 133 laboratories, mean blood PHE levels of 130 μmol/L and PHE: TYR ratio > 3 is considered abnormal (Vockley et al, 2014).   2.3 Classification of PKU The classification for PKU was established in 1980 in order to distinguish between several phenotypes of this disorder (Güttler, 1980). The classification is based on pretreatment PHE concentrations in blood during newborn screening, and is currently used in the Biochemical Diseases Clinic at BC Children’s Hospital (Stockler-Ipsiroglu et al, 2015). Patients with blood PHE levels > 1200 μmol/L prior to initiation of dietary treatment are classified as classic PKU (Figure 2); those with pretreatment PHE levels between 900 – 1200 μmol/L is referred to as moderate PKU; and levels between 600 – 900 μmol/L is defined as mild PKU. Lastly, if the pretreatment PHE levels are less than 600 μmol/L this is referred to as mild hyperphenylalaninaemia (HPA) (Blau et al, 2011). Classification of phenotypes in this disorder is not simple, especially in newborn infants, due to insufficient opportunity for dietary PHE to build up in their bodies to reach diagnostic cut-off levels (Blau et al, 2010). Other classifications of PKU based on dietary PHE tolerance, can be identified later in life, usually after age 5. PHE tolerance is defined as the amount of dietary PHE that the patient can consume, while the blood PHE levels stay within the therapeutic range (Guldberg et al, 1994; Blau et al, 2011). There are four different phenotypes that might be classified on the basis of PHE tolerance as shown in (Table 3). 8   Figure 2. PKU Classification Based on Pretreatment Blood PHE Levels  Table 3. PKU Classification Based on Dietary PHE Tolerance Classification PHE Tolerance Classic PKU < 20 mg/kg/day (250 – 300 mg/day) Moderate PKU 20 – 25 mg/kg/day (350 – 400 mg/day) Mild PKU 25 – 50 mg/kg/day (400 – 600 mg/day) Mild HPA Patients not on diet therapy Data adapted from (Blau et al, 2011)  Classic PKU PHE > 1200 μmol/L ( > 20 mg/dL) Moderate PKU PHE 900-1200 μmol/L (15-20 mg/dL) Mild PKU PHE 600-900 μmol/L (15-20 mg/dL) Mild HPA PHE < 600 μmol/L ( < 10 mg/dL)   9  2.4 Nutritional Approaches for Management of Patients with PKU 2.4.1 Sapropterin (Tetrahydrobiopterin-BH4) Therapy The major mode of treatment still remains nutritional management with dietary restriction of PHE supplemented with PHE free amino acid formula to prevent neurological problems (Scriver et al, 2001; Shannon, 2007). PKU is considered as one of the treatable inborn errors of metabolism presenting with intellectual disability (Bélanger-Quintana et al, 2011; van Karnebeek and Stockler, 2012). Cognitive / behavioral deficiency is prevented by early administration of PHE-restricted diet supplemented with PHE free amino acid formula to control blood concentrations of phenylalaine  (Scriver et al, 2001, Shannon, 2007). But dietary treatment for PKU imposes considerable burden on the patients with PKU and their families due to the expensive cost of both modified foods (PHE free formula) and low protein products (Simon et al, 2008). Besides social considerations, compliance with diet therapy is not satisfactory in adolescent and adult patients with PKU, due to the unpleasant palatability and the total amount that must be consumed of the PHE free formula. This might lead to growth retardation, osteoporosis and several nutritional deficiencies such as calcium, iron, selenium, zinc, vitamin D and vitamin B12 (Acosta et al, 2003; Arnold et al, 2002; Barat et al, 2002; Levy et al, 2007; MacDonald et al, 2011). In clinical practice, PHE restriction is determined depending on the amount of PHE intake which allows for stable blood PHE levels within the therapeutic range 2 – 6 mg/dl (120 – 360 μmol/L). Determination of the individual patient’s PHE tolerance may be challenging because it is affected by several factors such as, growth rate, age, gender, diet compliance, PHE hydroxylation rate, amount of PHE – free amino acids formula and requires detailed food records 10  from the patients/caregivers (MacDonald et al, 2011). For all of these reasons, there is a need to develop new treatment approaches at the enzyme level (Bélanger-Quintana et al, 2011). Sapropterin (Sapropterin dihydrochloride, Kuvan®, Biomarin Pharmaceutical, Novato, CA) is a synthetic form of the BH4 cofactor of PAH and chaperone (Figure 3) (Blau, 2013; Vernon et al, 2010), with the ability to enhance residual enzyme activity and improve dietary PHE tolerance (Burton et al, 2010; Okano et al, 2004; Lindner et al, 2001; Zurflüh et al, 2008; Levy et al, 2007) and thus improve metabolic control and quality of life for many patients with PKU. However, not all patients with PKU respond to sapropterin (Ponzone et al, 2010).   Figure 3. Chemical Structure of Sapropterin Dihydrochloride. (Blau N., 2013)  2.4.1.1 Clinical Trials Determining the Efficacy of Sapropterin Treatment in PKU Many clinical trials have assessed the effectiveness of sapropterin treatment for reducing blood PHE concentrations and improving PHE tolerance in patients with PKU. One such study by Burton and colleagues (2007) involved administration of 10 mg/kg/d of sapropterin dihydrochloride to 489 subjects with PKU (age range 8 – 48 years) for a period of 8 days. The inclusion criteria included patients with PHE levels ≥ 450 μmol/L and were in poor compliance to a PHE – restricted diet. 485 patients completed the phase II, multicenter, open – label, 11  screening study and results revealed that ≥ 30% reduction in blood PHE levels in 20% of subjects with mean blood PHE levels 391.8 ± 185.3 μmol/L as shown in (Figure 4).    Figure 4. BH4 Treatment Efficacy in Subjects with PKU. Response Rate (%) Based on Pre - treatment Blood PHE Levels. Figure From (Blau et al, 2009); Data Based on (Burton et al, 2007) A phase III randomized, double – blind, placebo – controlled trial was conducted (Levy et al, 2007) of the effectiveness of sapropterin among 88 subjects with PKU (age range 8 – 49 years), who responded to sapropterin from a previous screening study. 41 subjects were given oral administration of 10 mg/kg/d of sapropterin and 47 subjects were given placebo for 6 weeks. At the end of the study duration, the sapropterin group had a reduction in blood PHE levels by – 235.9 ± 257 μmol/L and a significant blood PHE reduction after first week versus the placebo group, which had a reduction of 2.9 ± 239.5 μmol/L (Figure 5). The results revealed a ≥ 30% 12  reduction in blood PHE levels in 44% subjects who received treatment, compared with 9% of placebo group.    Figure 5. Mean Change in Blood PHE Levels in Sapropterin Group vs. Placebo Group for a Period of 6 Weeks. Adapted From (Levy et al, 2007)  Another phase III international, double – blind, randomized, placebo – controlled study by Trefz et al (2009) involved 2 parts. 90 children with PKU (age range 4 – 12 years) were eligible for part 1 and received 20 mg/kg/d of sapropterin for 8 days to identify the responders to the treatment. Subjects with ≥ 30% reduction in blood PHE levels between day 1 – day 8 and had blood PHE ≤ 300 μmol/L on day 8 were enrolled for part 2. 46 responsive children with PKU were enrolled in part 2, continued on a PHE – restricted diet, and were randomized to receive sapropterin (20 mg/kg/d) or placebo for a duration of 10 weeks. PHE supplements were introduced in the third week in part 2 and added every 2 weeks if the blood PHE levels were 13  within the therapeutic range. The results of this trial indicated that, the sapropterin group had a significant increase in PHE supplement tolerance (21.0 ± 2.3 mg/kg/d) while maintaining blood PHE levels < 360 μmol/L, compared to the placebo group who only tolerated 3.3 ± 3.9 mg/kg/d of PHE supplement. The authors concluded that, 20 mg/kg/d of sapropterin helped improve PHE tolerance and reduced blood PHE levels in children with PKU.  On the whole, the results of these clinical trials revealed that ≥ 30% decrease in blood PHE levels in 20 – 50% subjects with PKU on sapropterin treatment. It has been suggested that this can be considered a clinically significant response to the treatment (Burton et al, 2007; Levy et al, 2007; Trefz et al, 2009). But some medical centers have considered a lesser reduction in blood PHE levels (≥ 20% decrease) as a significant response. Thus, there is a lack of consensus on what is considered a clinically significant response to sapropterin therapy (Levy et al, 2007; Blau et al, 2009).   2.4.1.2 Challenges Determining the Efficacy of Sapropterin Treatment in PKU  In current clinical practice most patients with PKU are tested with a sapropterin challenge and monitoring of blood PHE concentrations to determine the responsiveness of such a treatment (Levy et al, 2007). Determination of responsiveness may be challenging, particularly in young patients and adolescents whose dietary PHE tolerance is subject to rapid change due to growth spurts as well as in subjects with poor adherence to dietary prescriptions and inability to provide exact diet protocols.   14  2.4.1.3 Stable Isotope Techniques Determining PAH Activity in PKU In vivo stable isotope techniques may provide a sensitive and robust tool for determination of changes in PAH activity in response to sapropterin treatment. Curtius et al (1972); Trefz et al (1979); Matalon et al (1982) were able to determine in vivo PAH activity in humans measuring 2H-phenylalanine conversion to 2H-tyrosine in plasma. However, this is an invasive procedure requiring repeated blood samples and administration of large dosages of phenylalanine (10-200 mg/kg). On the other hand, a rapid noninvasive breath test in humans based on conversion of L-[1-13C] phenylalanine to tyrosine by PAH and the subsequent release of the carboxyl labeled 13C to 13CO2 in breath (Figure 6), has been developed by Treacy et al (1997) to identify phenylalanine oxidative capacity, which is an indicator of PAH activity in subjects with PKU.   Figure 6. Illustration the Phenylalanine Hydroxylation System and L-[1-13C] Phenylalanine Oxidation to 13CO2 . Adapted From (Okano et al, 2007) 15  2.4.1.4 Phenylalanine Breath Test Muntau et al (2002) studied, 38 children with different classification of PKU aged 1 day – 17 years for a period of one year. The experimental design was based on phenylalanine and BH4 loading test 100 mg/kg and 20 mg/kg, respectively, followed by the measurement of L-[1-13C] phenylalanine (6 mg/kg) oxidation to 13CO2 for 24 hours. Breath samples were collected every three hours. The rate of phenylalanine oxidation was identified once without BH4 therapy and once with BH4 therapy (10 mg/kg). The author’s concluded that BH4 therapy improves PAH activity in patients with mild HPA, and the breath test reliably identified patients who were responsive. Okano et al (2004) studied 20 patients with PAH deficiency among a wide age range (1 – 23 years old) and used oral administration of 10 mg/kg of L-[1-13C] phenylalanine dose and a maximum amount of 200 mg with or without sapropterin dose for three days. For BH4 loading, 10 mg/kg was given orally for two days prior to the study day, as well on the study day (Figure 7). The researchers collected two blood samples to determine the effect of isotope dose on serum PHE levels, once before L-[1-13C] phenylalanine dose, and the other one, 1-hour after the isotope dose. The results indicated that the breath test in healthy controls had a higher peak of Δ 13C (13CO2/12CO2 ratio) compared to the heterozygotes, occurring at 20 min (Figure 8). Patients with mild PKU showed a significantly higher peak of Δ 13C (8.87 ± 8.99 ‰) occurring at 20 – 30 min after sapropterin. Sapropterin treatment increased the peak time from 45 min to 20 – 30 min for mild HPA. There was no significant difference on serum PHE levels pre and post L-[1-13C] phenylalanine dose in patients with classical PKU and mild PKU/HPA.  16   Figure 7. Phenylalanine Breath Test Protocol Representing L-[1-13C] Phenylalanine and BH4 Consumption Time. PBT, Phenylalanine Breath Test. Figure From (Okano et al, 2004)    Figure 8. Phenylalanine Breath Test in Control, Heterozygotes and Subjects with PAH Deficiency. Δ 13C (‰) During Two hours Study Protocol. Solid Lines Without BH4 and Dashed Lines With BH4. Adapted From (Okano et al, 2004)  17       The previous breath test studies have tested this new method to identify PAH activity in subjects with PKU in response to sapropterin treatment; but the studies differed in the isotope L-[1-13C] phenylalanine doses they provided, and sapropterin loading to identify responsiveness to treatment. Also, no measurements of actual CO2 production were made. The absolute measures identified earlier in the release of the carboxyl labeled 13C as 13CO2 in breath in patients with PKU cannot be measured with any confidence. Therefore, there is a need to systematically establish this stable isotope based method at BC Children’s Hospital as a minimally invasive technique to be used routinely to identify sapropterin responders, as well as monitor the progress of treatment in patients with PKU.   2.4.2 Dietary Therapy The medical nutrition therapy protocol for PKU patients has been around for 60 years (Van Spronsen & Enns, 2010). The principle of medical nutrition therapy in PKU is to decrease PHE levels in blood to the therapeutic range to avoid neurological problems (Walter et al, 2012). Therefore, nutritional therapy is administered within the first two weeks of life to prevent such consequences (Vockley et al, 2014).  Nutritional management is a major mode of treatment of PKU. There are two steps in the PKU diet to achieve the therapeutic goals. Firstly, it includes restricting the amounts of natural protein in order to reduce phenylalanine intake. Supplying restricted PHE with the minimum recommended dietary intake is important to enhance normal growth for PKU children and improve PHE tolerance. PHE tolerance is different among patients, based on the residual enzyme activity and plasma PHE concentration levels. Plasma PHE concentration is utilized for determining the initial amount of dietary PHE intake in newly diagnosed patients (Table 4).  18  The second step in the dietary treatment of PKU patients is that patients consume amino acid modified medical foods (PHE free formula or protein substitute) that can provide the recommended daily allowance of total protein intake within the safe limit and prevent energy, protein and vitamin/mineral deficiencies due to natural food restriction  (Jameson & Morris, 2011).   Table 4. Plasma PHE Levels and Estimation of Initial Dietary PHE Intake Plasma PHE (µmol/L) Plasma PHE (mg/dL) Dietary PHE (mg/kg) ≤ 605 ≤10 70 > 605 to ≤1210 > 10 to ≤ 20 55 > 1210 to ≤1815 > 20 to ≤ 30 45 > 1815 to ≤2420 > 30 to ≤ 40 35 >2420 > 40 25 Data from Acosta & Yannicelli (2001) Ross Metabolic Formula System:  Nutrition Support Protocols  Recently the American College of Medical Genetics and Genomics (ACMG) Guidelines with the Genetic Metabolic Dietitians International and Southeast Regional Newborn Screening and Genetics Collaborative (GMDI/SERC) for PKU management recommended, that nutrient intake should not differ from general population other than PHE, TYR and protein as demonstrated in (Table 5).   19  Table 5. Recommended Intakes of PHE, TYR, and Protein for Individuals with PAH Deficiency Age PHE (mg/day) TYR (mg/day) Protein (g/kg) Infant to < 4 years 0 to < 3 months 130 – 430 1,100 – 1,300 3 – 3.5 3 to < 6 months 135 – 400 1,400 – 2,100 3 – 3.5 6 to < 9 months 145 – 370 2500 – 3000 2.5 – 3 9 to < 12 months 135 – 330 2,500 – 3,000 2.5 – 3  (mg/day) (mg/day) (g/day) 1 to < 4 years 200 – 320 2,800 – 3,500 ≥ 30 After early childhood > 4 years to adult 200 – 1,100 4,000 – 6,000 120 – 140 % RDA for age Data from (Vockley et al, 2014; Singh et al, 2014)  2.4.3 Protein Requirements in PKU Recently the American College of Medical Genetics and Genomics (ACMG) Guidelines with the Genetic Metabolic Dietitians International and Southeast Regional Newborn Screening and Genetics Collaborative (GMDI/SERC) for PKU management recommended that protein intake for children (> 4 y to adult) should be 120-140% of the RDA for age (Table 5, Table 6) to enhance normal growth and development (Vockley et al, 2014; Singh et al, 2014).   20  Table 6. Recommended Intakes of Protein in Children with PKU Calculated from (Table 5)  Age RDA (DRI, 2005) Calculated Recommendation in Children with 120-140% of the RDA for age 4 – 13 years 0.95 1.14 – 1.33 14 – 18 years 0.85 1.02 – 1.19 Data calculated from (Vockley et al, 2014; Singh et al, 2014)  The adequate dose of protein substitute in children with PKU has not been identified (MacDonald et al, 2006). Some studies support higher intake of protein substitutes. MacDonald et al (2006) concluded that, the high dose of protein equivalents provided by the medical food mixture could lead to decreased plasma phenylalanine concentrations. Acosta & Yannicelli  (1994) state that, 25 infants with PKU and receiving 3.12g of protein/100 kcal had improved PHE tolerance and growth. Kindt et al (1984) gave two groups of infants (15 – 18 days) with PKU, two different amounts of protein. The first, RDA group followed the US recommended dietary allowances (median intake 2g/kg/day), and the second Food and Agriculture Organization (FAO) group received median intake 1.46g/kg/day. The RDA group tolerated more PHE compared with the FAO group, and the authors concluded that higher protein might be beneficial.  On the other hand, there are studies, which support the safety of lower amino acid intake from incomplete protein substitutes (vitamins/minerals free formula). Prince et al (1997) studied, 25 children with PKU aged between 4-10 years for a period of 5 years. The intake of amino acids from medical food mixture was reduced from 0.9 g/kg/day to 0.4 g/kg/day. The authors hypothesized that metabolic control; nutritional status and patient compliance would be 21  enhanced due to a reduction in the amount of sulphurous and dicarboxylic amino acids from the protein substitute, which have an unpleasant taste. Prince et al (1997) concluded that, growth status and biochemical results of serum proteins and minerals for children with PKU were not reduced because these children consumed more natural protein, and they received vitamin/mineral tablets in order to better maintain metabolic control. In contrast, plasma PHE concentrations was elevated in these children and the authors explained the increase of PHE concentrations in the plasma is related to the increase in age of patients, but it might be associated with reduced intake of medical food mixture.  Thus, there is no consensus on the actual amount of protein to be recommended in PKU and the protein needs for PKU patients are usually calculated according to the guidelines as illustrated previously in Table 5.  Protein requirements have not been directly determined in PKU children, because the traditional method to determine protein needs, Nitrogen Balance, is an invasive technique. It requires ~ 7 days of feeding each test diet prior to study day (DRI, 2005; Rand et al, 1976). This is not possible in children with disease. Newer stable isotope based methods have become available, and will be discussed in the following pages.  2.5 The Indicator Amino Acid Oxidation Technique (IAAO) The indicator amino acid oxidation (IAAO) technique uses a stable isotope labeled amino acid as an indicator amino acid (an essential amino acid, for e.g. L-[1-13C] lysine) to indicate protein synthesis. It is based on the principle that when an essential amino acid and protein intake is below the requirement, then all other essential amino acids including the indicator amino acid will be oxidized because amino acids cannot be stored in the body, and should be 22  partitioned between protein synthesis or oxidation. In other words, with increasing intake of the limiting amino acid/protein, oxidation will continue to decrease due to increasing protein synthesis. Once the requirement for amino acid is met then there will be no change in the indicator amino acid oxidation. As a result, an inflection point occurs when the change in oxidation of indicator amino acid takes place. This inflection point is known as the breakpoint, which represents the mean (estimated average requirement EAR) requirement of amino acid/protein intake (Figure 9) (Elango et al, 2008).   Figure 9. Concept of the Indicator Amino Acid Oxidation Technique (IAAO)  The IAAO technique requires 8 hours as the adaptation time for the test amino acid intakes to determine protein and amino acid requirements, compared with other methods like nitrogen balance technique, which requires 7 days as the adaptation period (DRI, 2005; Rand et al, 1976). Also, the IAAO method is non-invasive because the isotope tracer is given orally and 23  the protein requirement is measured in CO2 release in breath (Bross et al, 1998). Therefore, this method is appropriate for children with PKU.  2.5.1 Amino Acid Requirements in Children The IAAO method has been used to determine several amino acid requirement, such as branched chain amino acids BCAA (Riazi et al, 2004), total sulfur amino acids TSAA (Turner, 2006), methionine (with cysteine) MET+CYS (Humayun et al, 2006) and lysine (Elango et al, 2007) in healthy children as shown in (Table 7). The IAAO studies are the first studies to identify the optimal amino acid requirements in healthy children. Table 7. Comparison of Healthy Children Amino Acid Requirements Identified by IAAO and DRI, 2005 EAR based on BCAA (mg/kg/day) TSAA (mg/kg/day) MET+CYS mg/kg/day Lysine (mg/kg/day) IAAO 147 12.9 5.8 35 DRI, 2005 81 18 ‒ 37  2.5.2 Amino Acid Requirements in Disease The IAAO technique has been used to identify phenylalanine (Courtney-Martin et al, 2002) and tyrosine (Bross et al, 2000) requirements in children with PKU, which were determined to be 14 and 19 mg/kg/day, respectively. Therefore, the ratio of TYR: PHE is 60:40% compared with the current recommended ratio is 80:20%. By using the same IAAO method, branched-chain amino acid BCAA (leucine, isoleucine and valine) requirements was indicated to be 45 mg/kg/day for patients with maple syrup urine disease MSUD (Riazi et al, 2004), compared with BCAA requirements in healthy people of 144 mg/kg/day. 24  BCAA needs are hypothesized to increase in children who suffer from liver disease because their plasma BCAA concentrations are reduced. However, aromatic amino acids (AAA) needs are decreased in children with liver disease due to an increase in their plasma AAA concentrations. The IAAO technique was used to identify the requirement of BCAA in children with cholestatic liver disease, and was determined to be 209 mg/kg/day (Mager et al, 2006). In the same children after liver transplantation, the BCAA requirement using the IAAO technique was determined to be 172 mg/kg/day (Mager et al, 2006). Thus, requirement of BCAA in children with cholestatic liver disease is higher compared with children after liver transplantation. Furthermore, the BCAA requirements in children with liver disease and after liver transplantation are still higher than the requirement in healthy children of 147 mg/kg/day (Mager et al, 2003). Thus, the IAAO method has been applied in children with disease before, and is well suited for children with PKU. 2.5.3 Protein Requirements in Humans The IAAO method has not only been used to identify amino acid requirement, but also used to determine total protein requirements in healthy and vulnerable populations like pregnancy (Table 8) Table 8. Comparison of Protein Requirements Identified by IAAO and DRI, 2005 in Healthy Adults, Children, and Pregnant Women  EAR (DRI, 2005) (g/kg/day) IAAO (g/kg/day) References Healthy Adults 0.66 0.93 Humayun et al, 2007 Healthy Children 0.76 1.3 Elango et al, 2011 Healthy Pregnant Women 0.88 1.22 Stephens et al, 2015 25   The first study was to apply the IAAO technique to determine total protein requirements in healthy adult humans. Humayun et al (2007) studied, eight healthy men who consumed graded protein intakes from 0.1 to 1.8 g/kg/day, and L-[1-13C] phenylalanine was used as an indicator amino acid. Each subject participated in seven different study days. The oxidation of 13C to 13CO2 was measured on each study day. The diets involved 1.5 × resting energy expenditure as energy needs, 33% of energy from fat, (48-66%) of energy from carbohydrate and (1-19%) as protein. The intake of phenylalanine was constant with sufficient tyrosine intake. The mean and the recommended dietary allowance (RDA) were determined to be 0.93-1.2 g/kg/day compared with the current protein recommendation from DRIs 2005/FAO 2007 of 0.66-0.83 g/kg/day. The IAAO technique was then applied in healthy school-aged children (6-10 years old) to determine protein requirements. The current protein recommendations from DRIs/FAO are 0.76-0.85 g/kg/day. However, the mean and the RDA of protein requirements were determined to be 1.3-1.5 g/kg/day (Elango et al, 2011) as in (Figure 10).  26   Figure 10. Protein Requirements in Healthy Children Using IAAO. Adapted from (Elango et al, 2011)  Recently, the IAAO technique has been used to determine protein requirements in healthy pregnant women during early and late gestation. Stephens et al (2015) tested, 29 healthy pregnant women (age range 24 – 37 years). 17 women participated in early gestation (n= 35 observation) and 19 women in late gestation (n= 43 observations); they consumed protein intakes from 0.22 to 2.56 g/kg/day. The test protein was given based on egg protein composition, except PHE and TYR, which were maintained constant. Each study day, the test protein was provided as 8 hourly meals. With the fifth meal, L-[1-13C] phenylalanine as the indicator amino acid was provided orally with collection of breath and urine samples. The oxidation of L-[1-13C] phenylalanine to 13CO2 was measured on each study day and the protein requirement was determined by using a two-phase linear regression analysis on the change in breath 13C 27  enrichment, reflecting protein synthesis. The mean protein requirements were determined to be 1.22g/kg/d in early gestation and 1.52g/kg/d in late gestation, which are higher than the current protein requirement from DRIs 2005, which is 0.88 g/kg/day. These results are the first to directly define a quantitative requirement for protein in healthy pregnant women, and indicate that current recommendations are underestimated. Whether PKU children have a protein requirement of 1.3-1.5 g/kg/day is still not known. The IAAO method is well suited to be applied in children with PKU for the determination of protein requirement.   2.6 Choice of Indicator Amino Acid in PKU There is one significant issue to deal with, before applying the IAAO method in children with PKU.  There are three important criteria to choose the ideal indicator amino acid (Zello et al, 1995): 1) The indicator amino acid must be an essential amino acid 2) The indicator amino acid should have a carboxyl-labeled carbon that can be oxidized in an irreversible way during catabolism and produce CO2 to measure it in breath 3) The indicator amino acid should not have a large amino acid pool in the body and must not be involved in many other significant pathways in the body except protein synthesis or oxidation to CO2 L-[1-13C] phenylalanine (with excess tyrosine) is considered to be an ideal indicator amino acid to reflect protein synthesis because it covers the three criteria described above. Lysine covers the first and second criteria as an indicator amino acid; while Leucine covers only the first. Lysine has a large pool in the body and leucine has an unstable pool, involved in 28  reversible reaction with keto acids, and involved in stimulation of protein synthesis and insulin secretion. In the current study, L-[1-13C] phenylalanine cannot be used as an indicator amino acid because phenylalanine cannot be metabolized in children with PKU. Therefore, the other two options were considered L-[1-13C] lysine is an expensive option, as a stable isotope tracer. L-[1-13C] leucine is widely used as a tracer to examine protein metabolism (El-Khoury and Young et al, 1995) and in particular as an indicator amino acid (Kurpad et al, 1998; Kurpad et al, 2006). If leucine is used as an indicator, the dosage (natural leucine) is usually around the requirement (for children = 80 mg/kg/day) and would not stimulate protein synthesis or insulin secretion. Furthermore, L-[1-13C] leucine is less expensive; therefore in the current thesis it will be used as the indicator amino acid (Fig 11).       29   Figure 11. Leucine Catabolism. BCKAD, Branched-Chain α-Keto Acid Dehydrogenase.  30  Chapter 3: Rationale, Objectives and Hypotheses 3.1 Rationale    Phenylketonuria (PKU) is characterized by mutations in the phenylalanine hydroxylase (PAH) gene, leading to accumulation of phenylalanine in plasma.  This could be caused by deficiency of the hepatic enzyme phenylalanine hydroxylase (PAH). Kuvan® (Sapropterin dihydrochloride), a synthetic form of BH4 (a cofactor for PAH), has been shown to reduce plasma PHE levels in PKU, but not all patients respond to sapropterin treatment (Treacy et al, 1997; Muntau et al, 2002; Okano et al, 2004). There is a need to develop a more appropriate method to detect patients who respond to the co-factor therapy. With the use of stable isotopes, minimally-invasive measures (oral dosing of L-[1-13C] phenylalanine and its oxidation to 13CO2 ) can be used to examine in vivo phenylalanine metabolism. Oxidation of L-[1-13C] phenylalanine to 13CO2 would reflect whole body phenylalanine disposal, and represents a clinical phenotype which would help to identify children who are more responsive to sapropterin treatment, and help in individualizing treatment to this disorder.    Medical nutrition therapy for children with PKU involves a PHE restricted diet with provision of sufficient protein. However, the exact amount of protein to be provided is unknown. Currently, a factorial method is used to arrive at a protein intake recommendation. The recent American College of Medical Genetics and Genomics (ACMG) PKU guidelines recommend, for children aged 4y and above, to consume 120-140% above the DRI 2005 RDA for age. The current RDA for children (4-13y) is set at 0.95 g/kg/d. Recently using the stable-isotope based minimally invasive indicator amino acid oxidation (IAAO) technique, the current RDA for healthy children was shown to be underestimated by ~70%. It is likely that the protein recommendations in children with PKU are also underestimated. Thus, there is a need to do 31  direct studies in children with PKU to determine protein requirement and improve nutritional management.  3.2 Objectives 1: Our first objective is to establish the use of L-[1-13C] phenylalanine and its oxidation to 13CO2 as a minimally-invasive breath test (13C-PBT) to measure PHE metabolism in children with PKU being treated with sapropterin. 2: Our second objective is to determine dietary protein requirements in children with PKU using the indicator amino acid oxidation (IAAO) technique, with L-[1-13C] leucine as the indicator amino acid.  3.3 Hypothesis 1: We hypothesize that children who are responsive to sapropterin (co-factor treatment) will have increased 13CO2 in breath, reflecting increased PAH activity. Children who do not respond will have no change or minimal change in 13CO2 in breath before and after treatment. 2: We hypothesize that the dietary protein requirement in children with PKU will be higher than the current recommended intake.  32  Chapter 4: Experiment 1 – Minimally Invasive 13C-Breath Test to Examine Phenylalanine Metabolism in Children with Phenylketonuria 4.1 Methods and Materials 4.1.1 Study Principle The study design was based on the oxidation of L-[1-13C]phenylalanine, a stable isotope tracer, to 13CO2 to examine phenylalanine metabolism. The principle of the 13C-phenylalanine breath test (13C-PBT) is based on quantifying the enzyme dependent (phenylalanine hydroxylase, PAH) conversion of L-[1-13C]phenylalanine to tyrosine and the subsequent catabolism of tyrosine to release the carboxyl labeled 13C as 13CO2 in breath (Figure 12).   Figure 12. Concept of the 13C Phenylalanine Breath Test (13C-PBT) The current study consists of two experimental designs the first one in healthy children and the other in children with PKU. 33  4.1.2 Subjects The original proposal was to study 6 healthy children and 10 children with PKU at the Clinical Research and Evaluation Unit at the British Columbia (BC) Children’s Hospital in Vancouver. The healthy children participated in the study once, while children with PKU were studied once prior to start of BH4 (Sapropterin dihydrochloride, Kuvan®, Biomarin Pharmaceutical, Novato, CA) therapy, and once after a minimum of a week of BH4 therapy. All procedures were reviewed and approved by the Committee for Ethical Review of Research involving Human Subjects at the University of British Columbia and BC Children’s Hospital. The written informed consent was provided to parents and children aged 14-18 years old (Appendix A, E); the assent form was obtained from children aged 7-13 years old (Appendix B, F). The purpose and potential risks of the study were explained to the children and their parents before participating the study. Advertisements about study details and contact information were distributed in the community such as BC Children’s and Women’s Hospital, the University of British Columbia and coffee shops to recruit healthy children (Appendix C). Other advertisements were distributed to the Biochemical Diseases staff at BC children’s Hospital to recruit children with PKU (Appendix G). A master list of participants and their assigned alphanumeric code (Appendix H) was kept in a locked cabinet at the Child and Family Research Institute. In appreciation of the time that it takes to complete this study participants received a $10 gift card.   4.1.2.1 Inclusion Criteria Healthy Children:  Healthy Children 4 – 18 years of age 34   Children who have no medical conditions  Children who currently free from any concurrent illness such as fever or cold  Children with PKU:  Children 4 – 18 years of age who are diagnosed with PKU   Clinically stable children with PKU with no concurrent illness such as fever, cold, vomiting or diarrhea  Children with PKU who have had the clinical decision made to start the BH4 treatment (Kuvan®)  4.1.2.2 Exclusion Criteria Healthy Children:  Healthy children under age 4 years  Healthy children above age 18 years  Healthy children but are currently ill with a fever, cold, vomiting or diarrhea Children with PKU:  Children with PKU under age 4 years  Children with PKU above age 18 years  Children diagnosed with PKU, but are currently ill with a fever, cold, vomiting or diarrhea  Children diagnosed with PKU, but where the clinical decision has been made not to start on Kuvan® therapy In subjects under age 4, it might be difficult to take breath samples and perform indirect calorimetry. During the indirect calorimetry test subject is requested to lie down for 20 – 30 35  minutes with an open hood/canopy over the face and head. Young children may feel uncomfortable during this period. Subjects above age 18 moved to Vancouver General Hospital (VGH).  4.1.3 Experimental Design – Healthy Children As a proof of principle, the 13C-PBT study day protocol was tested in six apparently healthy children recruited from the community (Appendix C). Participants were instructed to arrive for the study day after an overnight fast (~12 h) to standardize measurements at the Clinical Research and Evaluation Unit at BC Children’s Hospital. Basic anthropometric measurements (body weight and height) were recorded and a brief study day questionnaire was administered to collect information on medical, diet and physical activity history (Appendix D). Baseline breath samples were collected to determine natural background 13C abundance (Figure 13). Participants received an oral dose of 6mg/kg of L-[1-13C]phenylalanine (99 atom% 13C enrichment, Cambridge Isotope Laboratories Inc., Andover, MA) dissolved in sterile water. Participants remained fasting and rested in the Clinical Research and Evaluation Unit for the entire period of the study to eliminate variability in CO2 production. Breath samples in quadruplicates were collected at 20,40,60,80,100 and 120 min after oral administration of the labeled isotope (Figure 14).  During the study visit, the rate of carbon dioxide production (VCO2) was measured for 20 minutes, one hour after the oral isotope dose using an indirect calorimeter (Vmax Encore, Viasys Healthcare Inc. Yorba Linda, CA). Assessment of body composition was performed using the Bioelectrical Impedance Analysis (BIA-Quantum IV, RJL Systems, MI). BIA calculated resistance, reactance and impedance by applying electrodes were placed at the right wrist and 36  ankle. Three readings for resistance, reactance and impedance were taken for each child then the mean of these readings was used to determined fat free mass (FFM) and fat mass (FM). FFM and FM were calculated using the manufacturer’s software system (RJL Systems, Body Composition Analysis V.2.1).    Figure 13. Experimental Design in Healthy Children   37   Figure 14. Study Day Protocol for the 13C-Phenylalanine Breath Test (13C-PBT)  4.1.4 Experimental Design – Children with PKU Twelve children with PKU were recruited from the Biochemical Diseases Clinic at BC Children’s Hospital (Appendix G). Three children excluded from the study PKU07 moved to Vancouver General Hospital VGH, PKU08 was not eligible because it was difficult to perform indirect calorimetry in that subject and PKU02 who did not come for the study day 2 after Kuvan® treatment. Nine children with PKU participated in the study twice, once before and once after a prescription of 6R-BH4 (Sapropterin dihydrochloride Kuvan®, Biomarin Pharmaceutical, Novato, CA) separated by a week or more. Thus, 9 children were studied twice for a total of 18 isotope tracer studies. All studies were conducted after an overnight fast  (~12hr) at the Clinical Research and Evaluation Unit at BC Children’s Hospital. Basic anthropometric measurements (body weight and height) were recorded and a brief study day questionnaire was administered to collect information on medical, diet and physical activity history (Appendix I). Baseline breath 38  samples were collected to determine natural background 13C abundance (Figure 15). Participants received an oral dose of 6mg/kg of L-[1-13C]phenylalanine (99 atom% 13C enrichment, Cambridge Isotope Laboratories Inc., Andover, MA) dissolved in sterile water. Participants remained fasting and rested in the unit for the entire period of the study to eliminate variability in CO2 production. Breath samples in quadruplicates were collected at 20,40,60,80,100 and 120 min after oral administration of the labeled isotope (Figure 14). During each study visit, the rate of carbon dioxide production (VCO2) was measured for 20 minutes, one hour after the oral isotope dose using an indirect calorimeter (Vmax Encore, Viasys Healthcare Inc. Yorba Linda, CA). Assessment of body composition was performed using the Bioelectrical Impedance Analysis (BIA-Quantum IV, RJL Systems, MI). FFM and FM were calculated using the manufacturer’s software system (RJL Systems, Body Composition Analysis V.2.1).  39   Figure 15. Experimental Design in Children with PKU  4.1.5 Sample Collection Breath samples were collected using breath bags (single use collection bags, EasySampler System, QuinTron Instrument Company, Inc. Milwaukee, WI) in disposable glass Exetainer® tubes (Labco Limited, Buckinghamshire, UK) using a collection mechanism that permits removal of dead air space (Elango et al, 2011). Baseline breath samples were collected -0, -5 and 40  -10 minutes prior to oral isotope dose (Figure 13, 15). After oral administration of L-[1-13C]phenylalanine, breath samples were collected at 20,40,60,80,100 and 120 minutes. All breath samples were stored at room temperature until analyzed by isotope ratio mass spectrometer (IRMS, Isoprime Ltd, Cheadle, UK).   4.1.6 Analytical Procedures Expired 13CO2 enrichment was measured using a continuous flow isotope ratio mass spectrometer (IRMS, Isoprime Ltd, Cheadle, UK) and expressed as atom percent excess (APE) when compared against a reference standard of compressed CO2.  4.1.7 Calculations Phenylalanine oxidation was measured as the rate of L-[1-13C]phenylalanine tracer oxidation, F13CO2 (µmol/kg/h): F13CO2 = (FCO2) (ECO2) (44.6)(60)/(W)(0.82)(100) where FCO2 is the CO2 production rate (ml/min) on study day as measured by indirect calorimetry, ECO2 is the 13CO2 isotopic enrichment above baseline (atom percent excess, APE) obtained from breath samples at each time point, W is the body weight (kg) of each subject, 44.6 (µmol/L) and 60 (min/h) are constants used to convert FCO2 to µmol/h, 0.82 is the correction factor for carbon dioxide retained by the body due to bicarbonate fixation, and 100 is used to convert APE to a fraction (Hoerr et al, 1989). In order to quantify the tracer oxidation, the % of dose oxidized was calculated as: (F13CO2/isotope dose) *100 41  4.1.8 Statistical Analysis Subject characteristics are expressed as the mean (SD), which was completed by using independent t-tests. L-[1-13C]phenylalanine oxidation as 13CO2 (% of dose) was the primary outcome measure, as this best describes the whole body oxidation capacity for phenylalanine. Area under the curve (AUC) for each subject’s 13CO2 oxidation from t0 to t120, the time to reach maximum 13CO2 oxidation (tmax) and the maximum peak enrichment in13CO2 oxidation (Cmax) was calculated. A Paired t-test was used to compare the 13CO2 oxidation before and after BH4 treatment. All values are presented for individual subjects, and significance was set at P<0.05. Statistical analysis was performed using GraphPad Prism 4.0 (GraphPad Software Inc, CA).  4.2 Results  4.2.1 Subject Characteristics A total of 6 healthy children were studied, completing 6 study days (Nsubjects = 6, nstudies = 6). The six healthy children, had a range in age from 7 to 17y, and all were in apparent good health and normal body mass index BMI for age (Table 9). A total of 9 children with PKU were studied, completing 18 study days (Nsubjects = 9, nstudies = 18). The demographic and anthropometric characteristics of the children with PKU ranging in age from 8-17y (Table 10) were within normal values for age, except for PKU06 who had low FFM and high FM when compared with reference values (Ellis et al, 2000).     42  Table 9. Characteristics of Healthy Children (Nsubjects= 6)1 Subject Age (y) Gender Weight (kg) Height (cm) BMI (kg/m2) Fat Free Mass (kg) Fat Mass (%) HC01 9 M 27.4 130.5 16.2 21.5 21.7 HC02 7 M 22 119.5 15.3 16.7 24.1 HC04 9 M 41.2 137.6 21.6 25.9 37.1 HC05 12 M 44 157.8 17.63 29.4 33.3 HC07 17 F 65 162 24.77 37.7 42 HC08 13 F 49.4 155.8 20.30 33.4 32.4 1Healthy children were selected to represent the wide range in age of children with PKU recruited for the study   Table 10. Characteristics of Children with Phenylketonuria (PKU) (Nsubjects= 9) Subject Age (y) Gender Weight (kg) Height (cm) BMI (kg/m2) Fat Free Mass1 (kg) Fat Mass (%) PKU01 13 M 66.7 166.3 24.1 44.8 32.9 PKU03 10 M 39.7 152 17.2 27.7 30.3 PKU04 8 M 23 126 14.5 18.2 21 PKU05 15 F 51 158 20.4 31.3 38.6 PKU06 17 M 63.7 159 25.2 40.3 36.8 43  Subject Age (y) Gender Weight (kg) Height (cm) BMI (kg/m2) Fat Free Mass1 (kg) Fat Mass (%) PKU09 9 M 26.3 132.4 15.0 22.2 15.5 PKU10 12 M 43.3 149.7 19.3 31 28.4 PKU11 14 F 54.3 157.6 21.9 34.2 36.9 PKU12 15 M 52 168.2 18.4 40.2 22.7 1 Fat free mass measured using Bioelectrical Impedance Analysis (BIA-Quantum IV, RJL Systems, MI)  4.2.2 % Dose Oxidized of L-[1-13C] Phenylalanine  Production of a peak enrichment (Cmax) of 13CO2 (% of dose) in all healthy children occurred at 20 minutes ranging from 17 – 29% of dose, with a subsequent return to ~5% by the end of 2h (Figure 16, Table 11). These findings indicate that the oral dose of stable isotope L-[1-13C] phenylalanine was sufficient and reached the liver at 20 minutes and was metabolized to tyrosine with a first-pass effect.  44   Figure 16. 13C-Phenylalanine Breath Test (13C-PBT) in Healthy Children Rate of L-[1-13C] Phenylalanine Oxidation (% of dose) During 120 Minutes Study Day Protocol        0 20 40 60 80 100 120 14005101520253035HC02 HC04HC05HC01HC07HC08Time (min)L-1-13C-Phenylalanine Oxidation(% of dose) 45  Table 11. L-[1-13C] Phenylalanine Oxidation (% of dose) Area Under the Curve (AUC) in Healthy Children (Nsubjects= 6) Subject 13CO2 Oxidation  AUC1201 (% of dose) tmax2 (min) Cmax3 (% of dose) HC01 1164 20 22.5 HC02 1217 20 27.5 HC04 1149 20 22.6 HC05 1201 20 22.1 HC07 1200 20 17.67 HC08 1457 20 29.75 1area under the curve for 13CO2 oxidation from t0 to t120 2time to reach maximum 13CO2 oxidation 3maximum peak enrichment in13CO2 oxidation  Production of 13CO2 from L-[1-13C] phenylalanine in all children with PKU before receiving sapropterin treatment remained low, except in one subject (PKU04) who had peak enrichment (Cmax) at t40 of 4.82%, representing residual PAH activity in the liver (Figure 17A, Table 12). After sapropterin treatment for a week, production of 13CO2 significantly increased in five children (PKU03, PKU05, PKU06, PKU11 and PKU12) suggesting improved PAH activity (Figure 17B, Table 12). Two children with PKU (PKU09 and PKU10) had decreased 13CO2 production (Cmax of 0.62 – 0.3%, and 0.23 – 0.07%, respectively) following sapropterin 46  supplementation, representing negligible PAH activity. PKU04, who had moderate 13CO2 production before sapropterin treatment showed a non-significant increase in peak enrichment (Cmax: 4.82% to 6.71%), although the time to reach maximum enrichment (tmax) improved from t40 to t20 (Table 12).    Figure 17A. 13C-Phenylalanine Breath Test (13C-PBT) in Children with Phenylketonuria (PKU). Rate of L-[1-13C] Phenylalanine Oxidation (% of dose) in Children with PKU Prior to Treatment with Sapropterin (Kuvan®)   0 20 40 60 80 100 120 14005101520PKU01PKU03PKU04PKU05PKU06PKU09PKU10PKU11PKU12ATime (min) L-1-13C-Phenylalanine Oxidation(% of dose) 47  Table 12. L-[1-13C] Phenylalanine Oxidation (% of dose) Area Under the Curve (AUC) in Children with Phenylketonuria (PKU) (Nsubjects= 9)  Subject  13CO2 Oxidation   P value*  AUC1201 (% of dose) tmax2 (min) Cmax3 (% of dose)  Pre BH4 Post BH4 Pre BH4 Post BH4 Pre BH4 Post BH4 PKU01 25.49 50.65 80 20 0.45 0.58 0.03 PKU03 173.4 842.6 60 20 1.95 15.14 0.03 PKU04 360.8 417.8 40 20 4.82 6.71 0.50 PKU05 152.9 637.9 40 40 1.52 9.09 0.01 PKU06 111.3 655.2 40 20 1.32 9.38 0.01 PKU09 31.97 12.02 40 20 0.62 0.3 0.23# PKU10 12.22 15.26 60 0 0.23 0.07 0.01# PKU11 32.34 147.2 20 20 0.53 1.43 0.01 PKU12 94.56 363.5 20 20 1.33 5.33 0.01 *significantly different as denoted by paired t test (P<0.05) at each time point before and after sapropterin supplementation 1area under the curve for 13CO2 oxidation from t0 to t120 2time to reach maximum 13CO2 oxidation 3maximum peak enrichment in13CO2 oxidation;  #significant decline in 13CO2 production 48   Figure 17B. 13C-Phenylalanine Breath Test (13C-PBT) in Children with Phenylketonuria (PKU). Rate of L-[1-13C] Phenylalanine Oxidation (% of dose) in Children with PKU After Treatment with Sapropterin (Kuvan®)  4.3 Discussion The objective of the current study was to establish in a pediatric population, a minimally invasive and sensitive test to measure phenylalanine metabolism in vivo. The results from the healthy children confirmed that the 13C-PBT study protocol was sufficient and robust to detect phenylalanine disposal within a 2h study period. Furthermore, in PKU children, who are 0 20 40 60 80 100 120 14005101520PKU01PKU03PKU04PKU05PKU06PKU09PKU10PKU11PKU12BTime (min)L-1-13C-Phenylalanine Oxidation(% of dose) 49  responsive to pharmacologic treatment (sapropterin therapy), the 13C-PBT was able to identify reliably the whole body in vivo oxidative capacity of phenylalanine metabolism.  Previous studies have used similar breath tests in PKU children to identify responders to sapropterin treatment. Muntau et al (2002) studied, 38 children with PKU aged 1 day-17years for period of one year. The experiment was based on phenylalanine (100mg/kg) and tetrahydrobiopterin (20mg/kg) loading followed by measurement of 1-13C-phenylalanine oxidation to 13CO2 within a 24h period. The authors’ concluded that sapropterin improves PAH activity in patients with mild HPA, and the breath test reliably identified patients who were responsive. Okano et al (2004) studied 20 PKU patients with a wider age range (1-23y) and used a 10 mg/kg of L-[1-13C] phenylalanine dose, but had a restrictive maximum amount (200 mg). Their study also involved sapropterin loading (10mg/kg) for two days, as well on the study day (Okano et al 2004), and the results indicated a wide range in phenylalanine oxidative capacity which was reflective of the type of mutation in the PAH gene. Both the studies did not measure actual CO2 production rates, and basal metabolic rate. Furthermore the varying amounts of doses provided make it difficult to compare different responses among PKU patients. Our study was designed to test a constant dose of L-[1-13C] phenylalanine, measure actual CO2 production rates, conduct the study under a standardized condition (fasting) in all subjects and the first step in making the 13C-PBT routine, as part of regular PKU management.   The 13C-PBT is reflective of PAH activity in the liver, but is more representative of whole body phenylalanine oxidative capacity, and as such represents the clinical phenotype. The current test protocol of 2h, with a peak enrichment (Cmax) occurring consistently at 20 min in healthy children suggests that the oral dose of L-[1-13C] phenylalanine is absorbed in the intestine, and primarily metabolized in the liver as a first pass effect. The 1-13C-label is 50  transferred to 1-13C-tyrosine by PAH, and subsequently converted to p-hydroxyphenylpyruvate, and the 13C is released as 13CO2 during the formation of homogentisate (Figure 12). Comparison of the peak enrichment and the time to achieve peak enrichment between healthy children (Figure 16, Table 11) and PKU children (Figure 17A, Table 12) suggests that the clearance rate of phenylalanine is clearly different prior to sapropterin treatment. After sapropterin therapy the Cmax was at 20 minutes in children who were responsive to the treatment (PKU03, PKU04, PKU05, PKU06, PKU11 and PKU12) (Figure 17B). While measurement of blood phenylalanine levels offer a more global picture of phenylalanine metabolism, the stable isotope based breath test offers more dynamic and patient specific details of in vivo disposal of phenylalanine. PKU is a complex and heterogeneous disorder, with almost 500 different mutations identified in the PAH gene. While genotypes, which are more responsive to sapropterin therapy, have been identified and classified (Zurflüh et al 2008), the genotype-phenotype correlations are not perfect. Thus, the 13C-PBT adds value to the clinical management of PKU children.  The use of stable isotope based breath tests in clinical conditions are well known, with the 13C-urea breath test for the detection of H Pylori infection in the stomach being the most well established as a routine test (Graham et al, 2001). 1-13C-phenylalanine oxidation to 13CO2 is also used as a test for chronic liver disease (Moran et al, 2009) and gastric emptying (Bonfrate et al, 2014). While it is likely that measurement of blood phenylalanine levels will remain the key measure of clinical management, there is a need for a routine, simple test to help better manage PKU children.  Future work is necessary before a standardized protocol can be recommended for routine use in PKU children. For example, validation studies of different isotope doses within the same subject, prediction of CO2 production values versus actual measured values, length of study protocol etc need to be tested. New treatment modalities, such as polyethyleneglycol-51  phenylalanine ammonia lyase (PEG-PAL), are constantly being explored for the management of PKU children (Vockley et al, 2014). Thus newer stable isotope based dynamic tests would be required to help understand and manage patients with PKU. In summary, the current study is a first step in establishing the 13C-PBT as a routine test in PKU children. The results suggest that the 2h-stable isotope based breath test provides reliable data on whole body in vivo phenylalanine metabolism. In five of the nine PKU children tested, an increase in 13CO2 production from L-[1-13C] phenylalanine was measured due to sapropterin treatment. The breath data are corroborated by decline in blood phenylalanine levels in children who had increased responses in 13CO2 production, as reviewed post-hoc from clinical charts (data not shown). Future work should focus on further validation of the 13C-PBT and as a routine test for management of children with PKU.  4.3.1 Limitations There are some limitations in the current study. First, this is a short-term study, which is completed, once before and once after the administration of sapropterin; so future studies are needed to identify the effectiveness of the treatment in subjects with PKU over a longer period of time. Another reason for needing long-term studies is that PHE tolerance plays a major role in diet therapy in children with PKU and might be influenced by several factors including increasing age, adherence to the diet therapy along with treatment effectiveness. The current study was done in children aged 8 – 18 years, and therefore the study results cannot be generalized to all ages. Another limitation in this study is the small sample size. Due to the rarity of the disease, most patients are not local to Vancouver, and three of them were excluded from the study, since they did not meet study criteria that influenced the sample size. In general, 52  sapropterin therapy is expensive, which is not covered by the medical plan for some children with PKU. Finally, by nature PKU is a complex and heterogeneous disorder, with more than 853 different mutations in PAH gene (http://www.biopku.org/home/pah.asp), that possibly increased the data variance.          53  Chapter 5: Experiment 2 – Protein Requirements in Children with Phenylketonuria Using L-[1-13C] Leucine as IAAO 5.1 Methods and Materials 5.1.1 Subjects The original proposal was to study 5 children with PKU at the Clinical Research and Evaluation Unit at the British Columbia (BC) Children’s Hospital. Three children with PKU participated in 7 separate study days and one child participated in 6 study days; thus, four children were studied for a total of n = 27 isotope tracer IAAO studies. All procedures were reviewed and approved by the Committee for Ethical Review of Research Involving Human Subjects at the University of British Columbia and BC Children’s Hospital. Posters about study details and contact information were distributed to the Biochemical Diseases staff at BC Children Hospital to recruit study subjects (Appendix N). The purpose and potential risks of the study were explained to the children and their parents before participating in the study during a pre-study day. A master list of participants and their assigned alphanumeric code (Appendix O) was kept in a locked cabinet at the Child and Family Research Institute. In appreciation of the time that it takes to complete this study the participants received $ 100 per study day to a maximum of $ 700 for 7 study days.  5.1.1.1 Inclusion Criteria   Children 4 – 18 years of age who are diagnosed with PKU  Clinically stable with no current illness 54  5.1.1.2 Exclusion Criteria  Children under age 4 years and who are diagnosed with PKU  Children above age 18 years and who are diagnosed with PKU  Children diagnosed with PKU, but are currently ill, with a fever, cold, vomiting or diarrhea In subjects under age 4, it might be difficult to take breath samples and perform indirect calorimetry in very young children. Subjects above age 18 were transfer to Vancouver General Hospital (VGH). Illness such as fever, cold, vomiting or diarrhea might result in a reduced intake of protein substitute and calories, which leads to increased protein catabolism and amino acid release into plasma pool, primarily PHE; and therefore children were ensured to be clinically stable (Cleary et al, 2013).   5.1.2 Experimental Design Subjects participated in seven separate study days and each study was separated by ≥ 1 week. However, this was not possible in female participants who were studied once a month, because previous evidence has suggested that the phases of the menstrual cycle might impact the amino acid and protein requirements in females (Kriengsinyos et al, 2004). Kriengsinyos et al (2004) studied, 5 healthy females aged between 24 – 38 years and received seven levels of lysine intakes in both follicular and luteal phases. Thus, women participated twice for a total of 14 studies per participant. The lysine requirement was determined to be 37.7 mg/kg/d in luteal phase, which was higher than the follicular phase requirement (35.0 mg/kg/d). The authors hypothesized that amino acid catabolism would be enhanced during luteal phase (Kriengsinyos et al, 2004). Thus, in the current study all studies in female children were standardized to be during 55  the follicular phase (~d3-7 after the start of menstruation). The protein intakes ranged from deficient to excess, from 0.2 g/kg/d to 3.2 g/kg/d of protein (0.2, 0.6, 1.2, 1.8, 2.4, 3, 3.2 g/kg/d protein) (Figure 18).   Figure 18. Experimental Design  56  The test meals were consumed hourly to maintain a metabolic steady state during fed condition (Elango et al, 2008). The experimental diet involved a flavored liquid formula (protein – free powder PFD1, Mead Johnson, Evansville, IN; Tang, Don Mills, Canada; Kool – Aid, Don Mills, Canada; corn oil; and amino acid mixture) and protein – free wheat starch cookies represented the majority of the caloric content (Zello et al, 1990). Protein was provided as a crystalline L-amino acid mixture based on the egg protein pattern (Table 13). Phenylalanine was given separately to ensure it is provided at the current PHE tolerance for each participant - 55 mg/kg for PKU01, PKU02, PKU03 and 44 mg/kg for PKU04. Leucine was also provided at a constant amount - 82.64 mg/kg, since it is used as the indicator amino acid. Carbohydrate intake was adjusted with varying protein intakes to maintain an isocaloric diet. The macronutrient content of the diet provided as a percentage of dietary energy 33%, 44-66% and 1-19% as fat, carbohydrates and protein, respectively. The study day diets were weighed by using a digital scale (Mettler-Toledo) in the Child and Family Research Institute at BC Children’s Hospital. Participants did not consume any other foods during the study day; only water was consumed prior to arriving on the study day, and during the study day.     57  Table 13. Amino Acid Mixture Based on the Egg Protein Pattern Used on Study Day (Excluding L-Leucine, and L-Phenylalanine) Amino Acid Mixture mg/g Reference mg/0.2 g mg/0.6 g  mg/1.2 g mg/1.8 g mg/2.4 g mg/3 g mg/3.2 g L-Alanine 61 12.2 36.6 73.2 109.8 146.4 183 195.2 L-Arginine-HCL 74.54 14.9 44.7 89.4 134.2 178.8 223.6 238.5 L-Asparagine 33 6.6 19.8 39.6 59.4 79.2 99 105.6 L-Aspartic acid 33 6.6 19.8 39.6 59.4 79.2 99 105.6 L-Cysteine 21.94 4.4 13.2 26.3 39.5 52.7 65.8 70.2 L-Glutamine 56.2 11.2 33.7 67.4 101.2 134.8 168.6 179.8 L-Glutamic acid 56.2 11.2 33.7 67.4 101.2 134.8 168.6 179.8 L-Glycine 33 6.6 19.8 39.6 59.4 79.2 99 105.6 L-Histidine 22.53 4.5 13.5 27.0 40.6 54.1 67.6 72.1 L-Isoleucine 62.35 12.5 37.4 74.8 112.2 149.6 187.0 199.5 L-Lysine-HCL 75.12 15.0 45.0 90.1 135.2 180.3 225.4 240.4 L-Methionine 29.45 5.9 17.7 35.3 53.0 70.7 88.4 94.2 L-Proline 41.62 8.3 24.9 49.9 74.9 99.9 124.9 133.2 L-Serine 83.24 16.6 49.9 99.9 149.8 199.8 249.7 266.4 L-Threonine 46.73 9.3 28.0 56.1 84.1 112.2 140.2 149.5 L-Tryptophan 15.48 3.1 9.3 18.6 27.9 37.2 46.4 49.5 L-Tyrosine 40.42 8.1 24.3 48.5 72.8 97.0 121.3 129.3 L-Valine 69.72 13.9 41.8 83.7 125.5 167.3 209.2 223.1 58  5.1.3 Pre – Study Day and Study Protocol 5.1.3.1 Pre – Study Day Protocol Written informed consent was provided by parents and children (14 – 18 years of age); assent was obtained from children (7 – 13 years of age) before participating in the study (Appendix J, K, L). All children were requested to fast overnight (~ 12 hours) and were asked to come to the Clinical Research and Evaluation Unit (CREU) for a preliminary assessment (pre-study day) to measure basic anthropometry (weight and height), body composition and resting energy expenditure (REE). Weight and height were measured by using a digital scale and a stadiometer, respectively.  Energy requirements were calculated from resting energy expenditure (REE, kcal/d), which was measured by continuous, open-circuit indirect calorimetry (CareFusion Vmax Encore, VIASYS), which was calibrated prior to use. Total energy expenditure (TEE) and basal metabolic rate (BMR) are associated to fat free mass (FFM); therefore body composition was determined using bioelectrical impedance analysis (BIA) (Quantum IV RJL systems). BIA measures resistance, reactance and impedance by applying electrodes placed at the right wrist and right ankle. Three readings for resistance, reactance and impedance were taken for each child - then the mean of these readings was used to determine FFM and fat mass (FM). FFM and FM were calculated using the manufacturer’s software system (RJL Systems, Body Composition Analysis V.2.1).  A general questionnaire was used during the pre-study day (Appendix M) to give a clear idea about medical history, nutritional status, supplement intake and physical activity.  Finally, dietary record sheets were distributed to the participants to record food consumption for one day prior to the actual study day (Appendix P). In the current study, there was an advantage that all 59  subjects were following the metabolic dietitian’s instructions according to their clinical condition and age, and had a prescribed diet compared to healthy children.    5.1.3.2 Study Protocol and Isotope Infusion Studies All studies were conducted at the Clinical Research and Evaluation Unit at BC Children’s Hospital in Vancouver, BC. Basic anthropometric measurements (height, weight) were measured in the morning prior to start of the study. 8 hours were needed to complete the whole study day, which consisted of 6 hours of adaptation to the test protein intake and 2 hours of breath and urine sampling (Elango et al, 2007). The diet was consumed on the study day as 8 hourly isocaloric and isonitrogenous meals. Each meal represented one-twelfth of the subject’s daily needs (Appendix Q). During each study day subjects were randomly assigned to receive one of 7-test protein intakes. NaH13CO3 (99% atom excess, Cambridge Isotopes Laboratories, Woburn, MA) and L-[1-13C] leucine (99% atom excess, Cambridge Isotopes Laboratories, Woburn, MA) were given orally. The oral isotope infusion was started with the fifth meal on each study day. Four hourly meals were consumed before the oral isotope infusion. Priming oral doses of NaH13CO3 (0.176 mg/kg/d) and L-[1-13C] leucine (3.453 mg/kg/d) were consumed with the fifth meal (Figure 19; Appendix Q; Courtney-Martin et al, 2002). Continuous doses of L-[1-13C] leucine (1.726 mg/kg/h) were given with subsequent meals until the end of the study. The amount of leucine were deducted from the diet in the last 4 hour due to provide leucine as L-[1-13C] leucine in the last four meals.   60   Figure 19. Study Day Protocol for Protein Requirements in Children with PKU Using L-[1-13C] Leucine as IAAO  5.1.4 Sample Collection On each study day, breath and urine samples were collected as baseline and plateau samples. One blood dot was provided by each participant at the end of study day, which was forwarded to the Biochemical Diseases Clinic for analysis of blood phenylalanine and tyrosine concentration.  5.1.4.1 Breath Samples Breath samples were collected in disposable vacuum Exetainer tubes (Labco Ltd) by using breath bags (Single use collection bags, EasySampler System, QuinTron, Terumo 61  Medical). Each subject was taught to keep his/her mouth closed over the mouthpiece and take a normal breath then blow the air into air bag, which also had the capability to remove dead space air. After that, the Exetainer tube was pressed into the needle, which is located in the lower part of the mouthpiece till the rubber of the Exetainer was punctured at the same time as the subject breath. Breath samples were collected three times as a baseline 45, 30 and 15 min prior to oral isotope infusion protocol (Figure 18). After 2.5 h of starting the tracer protocol, 6 breath samples were collected at isotopic plateau (Figure 19; Bross et al, 1998). All breath samples were stored at room temperature until analyzed by isotope ratio mass spectrometry (IRMS, IsoPrime 100). On the study day, after the fifth meal was consumed, indirect calorimeter (Vmax Encore, Metabolic cart; VIASYS) was used to measure the rate of carbon dioxide production (VCO2) for 20 min.  5.1.4.2 Urine Samples Urine samples were collected two times as a baseline 45 and 15 min prior to start of oral isotope infusion protocol. After 2.5 h of starting isotope infusion protocol, four urine samples were collected as plateau (Figure 19). Samples were collected in urine hats (Specimen Container w/pour spout, 6.5 oz, Medegen) and transferred into sterile cups. 10 mL of urine was transferred into 15 mL conical tubes (BD Falcon, Mississauga ON), which consist of 200 μL 10% HCL to suppress bacterial growth. 1 mL of HCL urine mix was transferred to microcentrifuge tubes. All urine samples were stored at -80°C for later analysis.   62  5.1.5 Analytical Procedures Expired 13CO2 enrichment was measured using a continuous flow isotope ratio mass spectrometer (CF-IRMS IsoPrime100, Cheadle, UK). 13CO2 enrichment was represented as atom percent excess (APE) compared with a reference CO2 gas standard. Co-efficient of variant < 5% in breath 13CO2 enrichment was ensured at isotopic steady state on all study days.   5.1.6 Isotope Kinetics F13CO2 is the rate of 13CO2 released in breath after L-[1-13C] leucine oxidation (μmol/kg/h) and was calculated as: F13CO2 = (FCO2)(ECO2)(44.6)(60) / (W)(0.82)(100) where FCO2 is the CO2 production rate (mL/min), ECO2 is the 13CO2 enrichment in expired breath at isotopic steady state atom percent excess (APE); W is the subject body weight (kg). The constants 44.6 (μmol/mL) and 60 (min/h) were used to convert FCO2 to micromoles per hour. The factor 0.82 is the correction for CO2 retained in the body because of bicarbonate fixation (Hoerr et al, 1989), and the factor 100 converts APE to a fraction.  5.1.7 Statistical Analysis Subject characteristics were expressed as the mean (SD), which was tested using independent t-tests, and P < 0.05 was considered significant.  Estimates of the mean protein requirement for children with PKU were derived by breakpoint analysis of the F13CO2 by using a 2-phase linear regression crossover model (Seber, 1977) using SAS software (SAS/STAT; Version 9.2). The first regression line has a negative slope and the second one does not have a significant slope. Mixed models regressions are used to 63  estimate each candidate breakpoint. The model with a minimum residual standard error (SE) and highest R2 coefficient was used to determine the final breakpoint estimate (Elango et al, 2011).  5.2 Results 5.2.1 Subject Characteristics A total of 4 children with PKU were studied, completing 27 study days (Nsubjects = 4, nstudies = 27). The four subjects had a range in age from 9 to 18 y. The demographic and anthropometric characteristics (Table 14) were within normal values for age (Ellis et al, 2000).  Table 14. Subject characteristics of children with PKU (Nsubjects = 4) Subject Age (y) Gender Weight (kg)  Height (cm) Fat Free Mass (kg)1 REE (kcal/d)2 PPKU01 9 M 25.2 131.3 20.8 890 PPKU02 18 M 68.4 161 42.9 1147 PPKU03 16 F 54.5 159.3 33.5 1125 PPKU04 16 F 49.7 154.9 32.8 1195 1Determined by bioelectrical impedance analysis 2REE, resting energy expenditure determined by open-circuit indirect calorimetry64  5.2.2 Protein Requirements in Children with PKU F13CO2 (μmol/kg/h) represents the rate of 13CO2 from the oxidation of L-[1-13C] leucine. A 2-phase linear regression of the F13CO2 data resulted in a breakpoint (mean protein requirement) of 1.85 g/kg/d (r2 = 0.66) (Figure 20). L-[1-13C] leucine oxidation decreased with increasing test protein intakes until there was no further change and a plateau in oxidation occurred at 1.85 g/kg/d. Leucine enrichment in breath represents whole body protein synthesis including first pass metabolism by the gut and the liver. Thus, tracer kinetics from breath enrichment is more suitable compared to the rate of tracer oxidation from urine or plasma enrichments. (Elango et al, 2011).     Figure 20. Estimated Average Protein Requirement in Children with PKU (Nsubjects = 4, nstudies = 27) EAR = 1.85 g/kg/d R2=0.66  65  5.2.3 Phenylalanine and Tyrosine Levels By the end of each study day participants were asked to provide blood dots. PHE concentrations (range 81-353 μmol/L) were within the therapeutic range of 120 – 360 μmol/L. However, the PHE concentrations slightly fluctuated among study days in all participants as shown in (Figure 21). TYR concentrations (range 33 – 112 μmol/L) increased with increasing intake of the test protein until the test protein intake reached 1.8 g/kg/d, after which TYR concentrations stabilized with no further increases (Figure 21).    Figure 21. PHE and TYR Levels (μmol/L) in Children with PKU at the End of Each Study Day  66  5.3 Discussion 5.3.1 Protein Requirements in Children with PKU Protein requirements in children with PKU were determined to be 1.85 g/kg/d. The most recent recommendation for children with PKU (Singh et al, 2014) are 1.14 – 1.33 g/kg/d for 4 – 13 y and 1.02 – 1.19 g/kg/d for 14 – 18 y. This is based on 120 – 140% above current RDA for healthy children. Our results are ~ 60 – 70% higher than the above recommendation. The IAAO technique has been previously used to determine total protein requirements in healthy adult men (Humayun et al, 2007). The mean and the population-safe protein requirements in young men were determined to be 0.93-1.2 g/kg/day compared with the current protein recommendations from DRI (2005), of 0.66 – 0.83 g/kg/day. Elango and colleagues (2011) applied the IAAO method in healthy school-age children (6 – 11 years old) to determine protein requirements. The results revealed that the mean protein requirements in these healthy children are underestimated by 71 – 63% with the mean and the population-safe protein requirements of 1.3 – 1.55 g/kg/day, respectively, compared to the current recommendations from DRI (2005) of 0.76 – 0.95 g/kg/day (Elango et al, 2011). Thus, the current results in children with PKU are consistent with previous studies that current recommendations are lower than the actual requirements for protein intake.  5.3.2 Phenylalanine and Tyrosine Levels Blood PHE levels monitoring still remain the key measure to identify metabolic control, dietary PHE tolerance and clinical management in patients with PKU (Vockley et al, 2014; Singh et al, 2014). From our study results the PHE levels ranged from 1.4 – 5.9 mg/dL (81 – 353 μmol/L) and were within the therapeutic range 2 – 6 mg/dL (120 – 360 μmol/L), which is 67  important for optimal clinical outcomes. On the other hand, the findings show that PHE levels slightly fluctuated in all subjects between study days, but it is also important to remember that we had a range of protein intake (0.2 – 3.2 g/kg/d) (Figure 21). It has been reported blood PHE levels in subjects with PKU differ hourly, day-by-day and monthly (Cleary et al, 2013). There are many factors, which influence such fluctuations in PHE levels: age, dietary PHE intake, timing of protein substitutes, dietary compliance, changes in protein metabolism and concurrent illness. We believe that some of the previous factors might not be related to the current study and must be excluded. Firstly, dietary PHE intake was given at a constant amount in all study days (provided at current PHE tolerance for each participant 55 mg/kg/d for PKU01, PKU02, PKU03 and 44 mg/kg/d for PKU04). Secondly, the test L-amino acid mixtures were consumed hourly to maintain metabolic steady state during fed condition (Elango et al, 2008). Finally, one of the inclusion criteria is that these children should be free of any concurrent illness such as fever, cold, vomiting or diarrhea, which could lead to increased protein catabolism and amino acid release into the plasma pool (Cleary et al, 2013; Spronsen et al, 1993). Therefore, the fluctuation in blood PHE concentrations in our study must have been primarily due to the test protein intake, but were within the therapeutic range for all patients on all study days. Some fluctuations in PHE levels may be due, at least on some study days, to liberalization and relaxation of the diet therapy by some subjects prior to study days. Three out of the four subjects with PKU were on sapropterin therapy and had a reduction of blood PHE levels in response to sapropterin therapy (69, 51 and 69% reduction in blood PHE levels after the treatment for PKU01, PKU02 and PKU03, respectively). Sapropterin therapy helps to stabilize and decrease the fluctuations in blood PHE levels (Cleary et al, 2013). A retrospective study by Burton et al (2010) showed that, 37 subjects with PKU, administered with sapropterin treatment 68  had a reduction in the mean (SD) of blood PHE levels from 6.67 mg/dL (4.20) to 5.16 mg/dL (3.78) after the treatment. However, in the current study PKU01, PKU02 and PKU03 missed the sapropterin dose on some days prior to the study day and that might have contributed to the variations in blood PHE levels. TYR is a conditionally indispensable amino acid in patients with PKU due to the minimal supply from PHE through PHE hydroxylation (MacDonald et al, 2011). Elevated blood PHE levels have been suggested to reduce TYR uptake into brain leading to decreased TYR levels in blood and brain (Webster et al, 2010). Therefore, the recent ACMG Guidelines (2014) for PKU management recommend that TYR intake in children (4 – 18 y) should be 4,000 – 6,000 mg/day to maintain blood TYR levels within the normal range (Vockley et al, 2014; Singh et al, 2014). Quantitatively the mean TYR requirement in children with PKU aged 6 – 9 years has been identified about 19 mg/kg/d by using the minimally invasive IAAO technique (Bross et al, 2000). In the current study we observed that blood TYR levels range from 33 – 112 μmol/L (0.597 – 2.029 mg/dL) (Figure 21). The results showed that blood TYR levels were progressively elevated in response of graded increase in test protein intakes. However, once the requirement level is reached (1.85 g/kg/d) then TYR levels remained constant with no further increase, suggesting that needs for TYR was met at the IAAO determined protein requirements. Thus, blood TYR levels offer a strong support to the determined breakpoint of 1.85 g/kg/d, based on production of 13CO2 from the oxidation of orally administered of L-[1-13C] leucine (F13CO2). Once the requirement for protein synthesis is met there is no further increase in protein anabolism and thus TYR levels stabilized beyond the protein requirement.      69  5.3.3 Limitations There are some limitations that must be addressed in the current study. First, this is a short-term study, so long-term studies are needed to determine whether the protein requirement of 1.85g/kg/d are adequate and sufficient to enhance optimal growth and development in children with PKU. Furthermore, PHE tolerance plays a major role in dietary protein prescription in children with PKU; it is recognized that the results from the current study represents children with mild PKU. Children with the classical phenotype of PKU who do not have any residual enzyme activity might have a different protein requirement. Due to the rarity of the disease and most of these children were not local to Vancouver, this was a major factor, which affected the sample size of the study. Finally, by nature PKU is a complex and heterogeneous disorder, with more than 853 different mutations in PAH gene (http://www.biopku.org/home/pah.asp) that possibly increased the data variance.   5.3.4 Significance and Clinical Implications  The primary mode of treatment in PKU is nutritional management with dietary restriction of PHE and provision of sufficient protein (Walter et al, 2012; Vockley et al, 2014). Despite careful diet management, growth impairment still occurs in children with PKU because of protein insufficiency (Arnold et al, 2002; Verkerk et al, 1994). Identification of the optimal protein requirement directly in children with PKU might help with recommendations to help better growth outcomes. Acosta and Yannicelli (1994) assessed the correlation between protein intake, metabolic status and growth in 25 infants with classical PKU for a period of 6 months. The researchers gave two groups of infants’ two different amounts of protein. The first group identified as infants 70  fed PHE- free medical food A received 3.12 g protein/100 kcal; and the second group identified as infants feed PHE-free medical food B received 2.74 g protein/100 kcal. Three-day food records, growth measures and plasma amino acid concentrations were collected monthly. The results showed that the mean protein and PHE intakes of the first group were significantly higher compared with the second group. Infants fed food A had better growth measures (weight, head circumference and crown-heel length) compared to infants fed food B. On the other hand, plasma PHE levels were similar in both groups. Acosta and Yannicelli (1994) concluded that, greater protein intakes might be correlated with improved PHE tolerance and growth. Dhondt and colleagues (1995) in France analyzed the physical growth in 94 children with PKU. Patients were on PHE-restricted diet until 8 years of age, then a relaxed diet was introduced. Dhondt and colleagues reported that improved physical growth, with enhanced height z-scores was observed after liberalization of the diet in 8 years old children with PKU (Dhondt et al, 1995); thus a liberalized diet, which is more rich in protein would be beneficial to children with PKU for longer term health outcomes. Growth impairment and protein insufficiency, specifically at the early years of life, have been reported in patients with PKU. Arnold and colleagues (2002) prescribed protein from medical food formulas in children with classical PKU (age range 2 – 18 years) based on RDA for age, as 2 – 4 years: 30 g/d; 4 – 7 years: 35 g/d; 7 – 11 years: 40 g/d; ≥ 12 years: 50 and 55 g/d for girls and boys, respectively. Arnold et al (2002) reported that, there is a positive correlation between protein insufficiency, which was represented by low plasma prealbumin concentrations (< 20 mg/dL) and linear growth restriction. Rocha et al (2010) investigated that prealbumin concentration in patients with PKU (aged 1 – 27 years) and its correlation to protein nutritional status. Protein substitute intake, blood prealbumin and blood PHE levels were collected 71  retrospectively from 69 patients with PKU. The researchers used two different definitions to describe protein insufficiency. The first was prealbumin z-score under the 5th percentile and the second was prealbumin level under 20 mg/dL. The results showed that 9 of 69 ( ~ 13%) patients with PKU  had a prealbumin z-score under the 5th percentile. 8 out of these 9 had the milder forms of PKU. When the researchers followed the second definition of protein insufficiency, they found that 38 of 69 (~55%) patients with PKU had prealbumin levels under 20 mg/dL. Rocha et al (2010) concluded that, patients with milder phenotypes of PKU might be at risk of protein insufficiency. It is possible that patients with milder forms of PKU might have insufficient amounts of protein substitute (high protein biological value foods) and increased consumption of fruits and vegetables in order to control blood PHE levels. That means PHE-restricted diet may be beneficial for metabolic control, but it’s inadequate to maintain optimal nutritional status, especially protein (Rocha et al, 2010). Verkerk et al (1994) in Netherlands observed that, growth impairment and small head circumference in Dutch patients with PKU at the time of diagnosis and additional growth retardation happened at the age of 3 years. Verkerk et al (1994) suggested that, the growth impairment happened in this specific period in patients with PKU due to the strictness of the diet treatment, which leads to deficiencies in indispensable amino acids such as PHE and TYR. Results from Huemer and colleagues (2007) support the pervious study; 34 children with classical PKU (age range 2 months – 14 years) consumed protein around 20 – 40% above the RDA for age and had normal growth and body composition patterns compared with healthy populations. In addition FFM was significantly associated with natural protein intake. On the other hand, there are several studies, which support the correlation between enhancement of protein quality and normal growth in children with PKU rather than focusing on improving the 72  quantity of protein intake. Hoeksma et al (2005) studied, 174 infants with PKU retrospectively (aged < 21 days) for a period of 3 years. Head circumference, height and protein intake were analyzed. The mean total protein intake was 2.33 g/kg/day and the mean natural protein was 0.99 g/kg/day. Hoeksma and colleagues (2005) determined that, head circumference was significantly associated with total protein and natural protein intake, but there was no association between protein intakes and height. The authors concluded that increasing the quality of PHE-free formula is necessary to improve the head circumference growth in patients with PKU instead of increasing the quantity of protein intake (Hoeksma et al, 2005).  In summary, the current finding from this thesis has significant implications for prescribing protein intake in children with PKU, consuming a PHE-restricted diet. Optimal protein requirements determined directly in children with PKU add new and novel data to the literature. The higher protein recommendation has significant implications to prevent growth retardation due to protein insufficiency. But it is recognized that both quantity and quality of protein intake are important when giving a recommendation, and our study focussed only on quantity, and more research is needed to identify the ideal protein quality recommendations.  73  Chapter 6: Conclusions and Future Directions In summary this thesis covers two different aspects related to medical management in children with PKU. The first experiment is considered a first step in establishing the 13C-PBT as a minimally invasive, safe and reliable technique to be used routinely as part of regular PKU management at BC Children’s Hospital. The two-hour 13C-PBT helps to examine in vivo PHE metabolism in children with PKU responsive to sapropterin therapy. In five of the nine children with PKU the 13CO2 production from L-[1-13C] phenylalanine was significantly increased suggesting improve PAH enzyme activity after sapropterin therapy. The breath test results corroborated the long-term blood PHE monitoring in place to identify children being treated with sapropterin.  The second experiment’s findings are the first to directly define a quantitative requirement for protein intake in children with PKU, and indicate that current recommendations for optimal protein intake in children with PKU are insufficient and may be underestimated. Protein requirements in children with PKU were determined to be 1.85 g/kg/d. The mean protein requirements in young school-age children with PKU are 59 – 45% greater than the most recent recommendations in 2014 of 1.14 – 1.33 g/kg/d, respectively, based on 120 – 140% above current RDA (Singh et al, 2014). The mean protein requirement results in older children for both genders are 69 – 55% higher than the recent 2014 recommendations for PKU of 1.02 – 1.19 g/kg/d (Singh et al, 2014).  Blood PHE levels monitoring remain the key measure to identify metabolic control in patients with PKU. From our results the PHE levels (range 81 – 353 μmol/L) were within the therapeutic range (120 – 360 μmol/L) for all test protein intakes tested. Blood tyrosine levels 74  increased with increasing intake of test protein until it reached 1.85 g/kg/d, and plateaued after; suggesting that needs for tyrosine were met at the IAAO determined protein requirement. Future directions for research regarding the 1st experiment include studies with larger sample size and long-term follow up to understand the efficacy of sapropterin and it’s response to the 13C-PBT. More studies need to be done on validating the 13C-PBT as a routine test for management of children with PKU; different isotope doses within the same subject, repeatability of the tests within the same subject, prediction of CO2 production values versus actual measured values, length and duration of study day protocol need to be examined. It is also necessary to examine phenylalanine oxidation capacity using 13C-PBT and it’s relation to the genotype in order to identify the discordance between phenotype and genotype. Future recommendations for research related to the 2nd experiment include determining protein requirements in children with classical phenotype. PHE tolerance is different in patients with mild or moderate form of PKU versus patients with classical PKU and definitely impacts the dietary protein prescription. This is the first study to determine protein requirements in children with PKU leading the way to investigate protein requirements in adults with PKU, specifically maternal PKU, where adequate protein may be necessary to ensure proper growth and development of the fetus. Furthermore, our study used a crystalline amino acid base as the diet, and does not add any information to the protein quality to be recommended for children with PKU and needs to be studied further. Finally this study also paves the way for studies in other inborn errors of amino acid metabolism such as maple syrup urine disease (MSUD), propionic acidemia (PPA) and methylmalonic acidemia (MMA), all of which involve restriction of amino acids and provision of protein in order to improve nutritional management in patients with IEM. 75  Bibliography Acosta, P. B., and S. Yannicelli. 1994. Protein intake affects phenylalanine requirements and growth of infants with phenylketonuria. Acta paediatrica (Oslo, Norway : 1992).Supplement 407, : 66.  Acosta, PB., & Yannicelli, S. 2001. The Ross Metabolic Formula System: Nutrition Support Protocols, 4th Edition. Columbus, Ohio: Ross Products Division, Division of Abbott Laboratories.  Acosta, Phyllis B., Steven Yannicelli, Rani Singh, Shideh Mofidi, Robert Steiner, Ellen DeVincentis, Elaina Jurecki, et al. 2003. Nutrient intakes and physical growth of children with phenylketonuria undergoing nutrition therapy. Journal of the American Dietetic Association 103, (9): 1167-1173.  Alfadhel, Majid, Khalid Al-Thihli, Hiba Moubayed, Wafaa Eyaid, and Majed Al-Jeraisy. 2013. Drug treatment of inborn errors of metabolism: A systematic review. Archives of Disease in Childhood 98, (6): 454.  Aoki, K., M. Ohwada, and T. Kitagawa. 2007. Long-term follow-up study of patients with phenylketonuria detected by the newborn screening programme in japan. Journal of inherited metabolic disease 30, (4): 608-608.  Arnold, Georgianne L., Catherine J. Vladutiu, Russell S. Kirby, Eileen M. Blakely, and Jane M. DeLuca. 2002. Protein insufficiency and linear growth restriction in phenylketonuria. The Journal of pediatrics 141, (2): 243-246.  Barat, Pascal, Nicole Barthe, Isabelle Redonnet-Vernhet, and Françoise Parrot. 2002. The impact of the control of serum phenylalanine levels on osteopenia in patients with phenylketonuria. European journal of pediatrics 161, (12): 687-688.  76  Bélanger-Quintana, Amaya, Alberto Burlina, Cary O. Harding, and Ania C. Muntau. 2011. Up to date knowledge on different treatment strategies for phenylketonuria. Molecular genetics and metabolism 104 Suppl, : S19-S25.  Bickel, H., J. Gerrard, and E. M. Hickmans. 1953. Influence of phenylalanine intake on phenylketonuria. Lancet 265, (6790): 812.  Blau, Nenad, Amaya Bélanger-Quintana, Mübeccel Demirkol, François Feillet, Marcello Giovannini, Anita MacDonald, Friedrich K. Trefz, and Francjan J. van Spronsen. 2009. Optimizing the use of sapropterin (BH 4) in the management of phenylketonuria. Molecular genetics and metabolism 96, (4): 158-163.  Blau, Nenad, Francjan J. van Spronsen, and Harvey L. Levy. 2010. Phenylketonuria. The Lancet 376, (9750): 1417-1427.  Blau, Nenad, Julia B. Hennermann, Ulrich Langenbeck, and Uta Lichter-Konecki. 2011. Diagnosis, classification, and genetics of phenylketonuria and tetrahydrobiopterin (BH4) deficiencies. Molecular genetics and metabolism 104 Suppl, : S2-S9.  Blau, Nenad. 2013. Sapropterin dihydrochloride for the treatment of hyperphenylalaninemias. Expert Opinion on Drug Metabolism & Toxicology 9, (9): 1207-1218.  Bonfrate, Leonilde, Ignazio Grattagliano, Giuseppe Palasciano, and Piero Portincasa. 2015. Dynamic carbon 13 breath tests for the study of liver function and gastric emptying. Gastroenterology report 3, (1): 12-21.  Bross, R., RO Ball, and PB Pencharz. 1998. Development of a minimally invasive protocol for the determination of phenylalanine and lysine kinetics in humans during the fed state. Journal of nutrition128, (11): 1913-1919.  77  Bross, Rachelle, Ronald O. Ball, Joe T. R. Clarke, and Paul B. Pencharz. 2000. Tyrosine requirements in children with classical PKU determined by indicator amino acid oxidation. American Journal of Physiology - Endocrinology And Metabolism 278, (2): 195-201.  Burton, B. K., D. K. Grange, A. Milanowski, G. Vockley, F. Feillet, E. A. Crombez, V. Abadie, et al. 2007. The response of patients with phenylketonuria and elevated serum phenylalanine to treatment with oral sapropterin dihydrochloride (6R-tetrahydrobiopterin): A phase II, multicentre, open-label, screening study. Journal of inherited metabolic disease 30, (5): 700-707.  Burton, Barbara K., Heather Bausell, Rachel Katz, Holly LaDuca, and Christine Sullivan. 2010. Sapropterin therapy increases stability of blood phenylalanine levels in patients with BH4-responsive phenylketonuria (PKU). Molecular genetics and metabolism 101, (2): 110-114.  Borrajo, G. J. C. 2007. Newborn screening in latin america at the beginning of the 21st century. Journal of inherited metabolic disease 30, (4): 466-481.  Cleary, M., F. Trefz, AC Muntau, F. Feillet, FJ van Spronsen, A. Burlina, A. Belanger-Quintana, et al. 2013. Fluctuations in phenylalanine concentrations in phenylketonuria: A review of possible relationships with outcomes. Molecular genetics and metabolism 110, (4): 418-423.  Courtney-Martin, G., R. Bross, M. Raffi, JTR Clarke, RO Ball, and PB Pencharz. 2002. Phenylalanine requirement in children with classical PKU determined by indicator amino acid oxidation. American journal of physiology-endocrinology and metabolism 283, (6): E1249-E1256.  Curtius, H. -Ch, J. A. Völlmin, and K. Baerlocher. 1972. The use of deuterated phenylalanine for the elucidation of the phenylalanine-tyrosine metabolism. Clinica Chimica Acta 37, : 277-285.  Demirdas, Serwet, Imke N. van Kessel, Marjolein J. Korndewal, Carla E. M. Hollak, Hanka Meutgeert, Anja Klaren, Margreet van Rijn, Francjan J. van Spronsen, Annet M. Bosch, and 78  Dutch working Group. 2013. Clinical pathways for inborn errors of metabolism: Warranted and feasible. Orphanet journal of rare diseases 8, (1): 37-37.  Dhondt, JL, C. Largilliere, L. Moreno, and JP Farriaux. 1995. Physical Growth in Patients with Phenylketonuria. Journal of inherited metabolic disease 18, (2): 135-137.  DRI. Dietary reference intakes for energy, carbohydrate, fiber, fat, fatty acids, cholesterol, protein, and amino acids (macronutrients). In 2005. MyiLibrary Ltd  Elango, R., MA Humayun, RO Ball, and PB Pencharz. 2007. Lysine requirement of healthy school-age children determined by the indicator amino acid oxidation method. American journal of clinical nutrition 86, (2): 360-365.  Elango, R., RO Ball, and PB Pencharz. 2008. Indicator amino acid oxidation: Concept and application. Journal of nutrition 138, (2): 243-246.  Elango, R., MA Humayun, RO Ball, and PB Pencharz. 2011. Protein requirement of healthy school-age children determined by the indicator amino acid oxidation method. American journal of clinical nutrition 94, (6): 1545-1552.  Ellis, KJ., Roman J. Shypailo, Steven A. Abrams, and William W. Wong. 2000. The reference child and adolescent models of body composition: A contemporary comparison. Annals of the New York Academy of Sciences 904, (1): 374-382.  Das, Subir Kumar. 2013. Inborn errors of metabolism: Challenges and management. Indian Journal of Clinical Biochemistry 28, (4): 311-313.  Frazier, D. M., D. S. Millington, S. E. McCandless, D. D. Koeberl, S. D. Weavil, S. H. Chaing, and J. Muenzer. 2006. The tandem mass spectrometry newborn screening experience in north carolina: 1997–2005. Journal of inherited metabolic disease 29, (1): 76-85. 79   Garrod, ArchibaldE. 1902. The incidence of alkaptonuria: a study in chemical individuality. The Lancet 160, (4137): 1616-1620.  Graham, David Y., Hoda M. Malaty, Rhonda A. Cole, Robert F. Martin, and Peter D. Klein. 2001. Simplified 13C-urea breath test for detection of helicobacter pylori infection. The American Journal of Gastroenterology 96, (6): 1741-1745.  Guldberg, P., and F. Güttler. 1994. Mutations in the phenylalanine hydroxylase gene: Methods for their characterization. Acta Paediatrica 83, (s407): 27-33.  Guldberg, P., K. F. Henriksen, I. Sipilä, F. Güttler, and A. de la Chapelle. 1995. Phenylketonuria in a low incidence population: Molecular characterisation of mutations in finland. Journal of medical genetics 32, (12): 976-978.  Guthrie, R., and A. Susi. 1963. A simple phenylalanine method for detecting phenylketonuria in large populations of newborn infants. Pediatrics 32, : 338.  G ttler F. 1980. Hyperphenylalaninemia diagnosis and classification of the various types of phenylalanine hydroxylase deficiency in childhood. Acta Pediat Scand 280:1–80.  Hoeksma, M., M. Van Rijn, P. H. Verkerk, A. M. Bosch, M. F. Mulder, J. B. C. de Klerk, T. J. De Koning, et al. 2005. The intake of total protein, natural protein and protein substitute and growth of height and head circumference in dutch infants with phenylketonuria. Journal of inherited metabolic disease 28, (6): 845-854.  Hoerr, R. A., Y. M. Yu, D. A. Wagner, J. F. Burke, and V. R. Young. 1989. Recovery of 13C in breath from NaH13CO3 infused by gut and vein: Effect of feeding. American Journal of Physiology - Endocrinology And Metabolism 257, (3): 426-438.  80  Huemer, M., C. Huemer, D. Möslinger, D. Huter, and S. Stöckler-Ipsiroglu. 2007. Growth and body composition in children with classical phenylketonuria: Results in 34 patients and review of the literature. Journal of inherited metabolic disease 30, (5): 694-699.  Humayun, MA, JM Turner, R. Elango, M. Rafii, V. Langos, RO Ball, and PB Pencharz. 2006. Minimum methionine requirement and cysteine sparing of methionine in healthy school-age children. American journal of clinical nutrition 84, (5): 1080-1085.  Humayun, MA, R. Elango, R. Ball, and PB Pencharz. 2007. Reevaluation of the protein requirement in young men with the indicator amino acid oxidation technique. American journal of clinical nutrition 86, (4): 995-1002.  Jameson, Elisabeth, and Andrew A. M. Morris. 2011. Nutrition in metabolic disease. Paediatrics and Child Health 21, (9): 401-405.  Jiang, Jianhui, Xieqin Ma, Xiaochun Huang, Xinyan Pei, Haipin Liu, Zhiwei Tan, and Lanfang Zhu. 2003. A survey for the incidence of phenylketonuria in guangdong, china. The Southeast Asian journal of tropical medicine and public health 34 Suppl 3, : 185.  Jurecki, Elaina, and Wong, Joyce. 2011. Medical Disorders in “Nutrition Therapy and Pathophysiology”. Nahikian-Nelms, Marcia. Belmont, CA: Wadsworth, Cengage Learning.  Karam, Pascale E., Mohammad-Zuheir Habbal, Mohamad A. Mikati, Ghazi E. Zaatari, Najwa K. Cortas, and Rose T. Daher. 2013. Diagnostic challenges of aminoacidopathies and organic acidemias in a developing country: A twelve-year experience. Clinical biochemistry 46, (18): 1787-1792.  el-Khoury, A. E., M. Sanchez, N. K. Fukagawa, and V. R. Young. 1995. Whole body protein synthesis in healthy adult humans: 13CO2 technique vs. plasma precursor approach. American Journal of Physiology - Endocrinology And Metabolism 268, (1): 174-184. 81   Kindt, Elisabeth, Kristina Motzfeldt, Sverre Halvorsen, and Sverre O. Lie. 1984. Is phenylalanine requirement in infants and children related to protein intake? British Journal of Nutrition 51, (3): 435-442.  Kriengsinyos, Wantanee, Linda J. Wykes, Laksiri A. Goonewardene, Ronald O. Ball, and Paul B. Pencharz. 2004. Phase of menstrual cycle affects lysine requirement in healthy women. American Journal of Physiology - Endocrinology And Metabolism 287, (3): 489-496.  Kurpad, AV, AE El-Khoury, L. Beaumier, A. Srivatsa, R. Kuriyan, T. Raj, S. Borgonha, AM Ajami, and VR Young. 1998. An initial assessment, using 24-h [C-13]leucine kinetics, of the lysine requirement of healthy adult indian subjects. American journal of clinical nutrition 67, (1): 58-66.  Kurpad, Anura V., Meredith M. Regan, Tony Raj, and Justin V. Gnanou. 2006. Branched-chain amino acid requirements in healthy adult human subjects. The Journal of nutrition 136, (1 Suppl): 256S.  Levy, Harvey, Barbara Burton, Stephen Cederbaum, and Charles Scriver. 2007. Recommendations for evaluation of responsiveness to tetrahydrobiopterin (BH 4) in phenylketonuria and its use in treatment. Molecular genetics and metabolism 92, (4): 287-291.  Levy, Harvey L., Andrzej Milanowski, Anupam Chakrapani, Maureen Cleary, Philip Lee, Friedrich K. Trefz, Chester B. Whitley, et al. 2007. Efficacy of sapropterin dihydrochloride (tetrahydrobiopterin, 6R-BH4) for reduction of phenylalanine concentration in patients with phenylketonuria: A phase III randomised placebo-controlled study. The Lancet 370, (9586): 504-510.  82  Lindner, M., D. Haas, J. Zschocke, and P. Burgard. 2001. Tetrahydrobiopterin responsiveness in phenylketonuria differs between patients with the same genotype. Molecular genetics and metabolism 73, (1): 104-106.  Loeber, J. Gerard. 2007. Neonatal screening in europe; the situation in 2004. Journal of inherited metabolic disease 30, (4): 430-438.  MacDonald, A., A. Chakrapani, C. Hendriksz, A. Daly, P. Davies, D. Asplin, K. Hall, and IW Booth. 2006; 2005. Protein substitute dosage in PKU: How much do young patients need? Archives of Disease in Childhood 91, (7): 588-593.  MacDonald, A., J. C. Rocha, M. van Rijn, and F. Feillet. 2011. Nutrition in phenylketonuria. Molecular genetics and metabolism 104 Suppl, : S10-S18.  Mager, D.R., Wykes, L.J., Roberts, E.A., Ball, R.O., & Pencharz, P.B. (2006). Branchedchain amino acid needs in children with mild-to-moderate chronic cholestatic liver disease. The Journal of Nutrition, 136(1), 133-9.  Mager, DR, LJ Wykes, EA Roberts, RO Ball, and PB Pencharz. 2006. Effect of orthotopic liver transplantation (OLT) on branched-chain amino acid requirement. Pediatric research 59, (6): 829-834.  Mager, Diana R., Linda J. Wykes, Ronald O. Ball, and Paul B. Pencharz. 2003. Branched-chain amino acid requirements in school-aged children determined by indicator amino acid oxidation (IAAO)1,2. The Journal of nutrition 133, (11): 3540.  Matalon, R., D. E. Matthews, K. Michals, and D. Bier. 1982. The use of deuterated phenylalanine for the in vivo assay of phenylalanine hydroxylase activity in children. Journal of inherited metabolic disease 5, (1): 17.  83  Millington, D. S., N. Kodo, D. L. Norwood, and C. R. Roe. 1990. Tandem mass spectrometry: A new method for acylcarnitine profiling with potential for neonatal screening for inborn errors of metabolism. Journal of inherited metabolic disease 13, (3): 321-324.  Moran, S., I. Gallardo-Wong, G. Rodriguez-Leal, P. Mccollough, J. Mendez, B. Castaneda, P. Milke, J. Jacobo, and M. Dehesa. 2009. L-[1-C-13]phenylalanine breath test in patients with chronic liver disease of different etiologies. Isotopes in environmental and health studies 45, (3): 192-197.  Muntau, Ania C., Wulf Röschinger, Matthias Habich, Hans Demmelmair, Björn Hoffmann, Christian P. Sommerhoff, and Adelbert A. Roscher. 2002. Tetrahydrobiopterin as an alternative treatment for mild phenylketonuria. The New England journal of medicine 347, (26): 2122-2132.  National Institutes of Health Consensus Development Panel, and National Institutes of Health Consensus Development Panel. 2001. National institutes of health consensus development conference statement: Phenylketonuria: Screening and management, october 16-18, 2000. Pediatrics 108, (4): 972-982.  Okano, Yoshiyuki, Yutaka Hase, Mie Kawajiri, Yasuaki Nishi, Koji Inui, Norio Sakai, Yoko Tanaka, Kazuhiko Takatori, Masahiro Kajiwara, and Tsunekazu Yamano. 2004. In vivo studies of phenylalanine hydroxylase by phenylalanine breath test: Diagnosis of tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency. Pediatric research 56, (5): 714-719.  Okano, Yoshiyuki, Kazuhiko Takatori, Satoshi Kudo, Tomoko Sakaguchi, Minoru Asada, Masahiro Kajiwara, and Tsunekazu Yamano. 2007. Effects of tetrahydrobiopterin and phenylalanine on in vivo human phenylalanine hydroxylase by phenylalanine breath test. Molecular genetics and metabolism 92, (4): 308-314.  84  Ozalp, I., T. Coşkun, A. Tokatli, H. S. Kalkanoğlu, A. Dursun, S. Tokol, G. Köksal, M. Ozg c, and R. Köse. 2001. Newborn PKU screening in turkey: At present and organization for future. The Turkish journal of pediatrics 43, (2): 97.  Pangkanon, S., W. Charoensiriwatana, N. Janejai, W. Boonwanich, and S. Chaisomchit. 2009. Detection of phenylketonuria by the newborn screening program in thailand. The Southeast Asian journal of tropical medicine and public health 40, (3): 525.  Ponzone, Alberto, Francesco Porta, Alessandro Mussa, Alessandra Alluto, Silvio Ferraris, and Marco Spada. 2010. Unresponsiveness to tetrahydrobiopterin of phenylalanine hydroxylase deficiency. Metabolism 59, (5): 645-652.  Prince, A. P., M. P. McMurry, and N. R. M. Buist. 1997. Treatment products and approaches for phenylketonuria: Improved palatability and flexibility demonstrate safety, efficacy and acceptance in US clinical trials. Journal of inherited metabolic disease 20, (4): 486-498.  Rand, W. M., V. R. Young, and N. S. Scrimshaw. 1976. Change of urinary nitrogen excretion in response to low-protein diets in adults. The American Journal of Clinical Nutrition 29, (6): 639  Rashed, Mohamed S., Zuhair Rahbeeni, and Pinar T. Ozand. 1999. Application of electrospray tandem mass spectrometry to neonatal screening. Seminars in perinatology 23, (2): 183-193.  Riazi, Roya, Mahroukh Rafii, Joe T. R. Clarke, Linda J. Wykes, Ronald O. Ball, and Paul B. Pencharz. 2004. Total branched-chain amino acids requirement in patients with maple syrup urine disease by use of indicator amino acid oxidation with l-[1-13C]phenylalanine. American Journal of Physiology - Endocrinology And Metabolism 287, (1): 142-149.  Rocha, Júlio César, Manuela Ferreira Almeida, Carla Carmona, Maria Luís Cardoso, Nuno Borges, Isabel Soares, Graça Salcedo, Margarida Reis Lima, Isabel Azevedo, and Francjan J. van 85  Spronsen. 2010. The use of prealbumin concentration as a biomarker of nutritional status in treated phenylketonuric patients. Annals of Nutrition and Metabolism 56, (3): 207-211.  Scriver CR, Kaufman S. Hyperphenylalaninemia: phenylalanine hydroxylase deficiency. 8th ed. In: Scriver CR, Baudet AL, Valle D, Sly WS eds. 2001. The Metabolic and Molecular Bases of Inherited Disease. McGraw-Hill, New York, 1667–1724.  Seber, G. A. F. 1977. Linear regression analysis. New York: Wiley.  Selim, Laila A., Sawsan Abdel-Hady Hassan, Fadia Salem, Azza Orabi, Fayza A. Hassan, Fatma El-Mougy, Iman Gamal-Eldin Mahmoud, et al. 2014. Selective screening for inborn errors of metabolism by tandem mass spectrometry in egyptian children: A 5 year report. Clinical biochemistry 47, (9): 823.  Shannon, Joyce Brennfleck. 2007.Endocrine and Metabolic Disorders: Inborn Errors of Metabolism. 2nd ed. Detroit, MI: Omnigraphics.  Simon, Eva, Martin Schwarz, Judith Roos, Nico Dragano, Max Geraedts, Johannes Siegrist, Gudrun Kamp, and Udo Wendel. 2008. Evaluation of quality of life and description of the sociodemographic state in adolescent and young adult patients with phenylketonuria (PKU). Health and quality of life outcomes 6, (1): 25-25.  Singh, Rani H., Fran Rohr, Dianne Frazier, Amy Cunningham, Shideh Mofidi, Beth Ogata, Patricia L. Splett, et al. 2014. Recommendations for the nutrition management of phenylalanine hydroxylase deficiency. Genetics in medicine : official journal of the American College of Medical Genetics 16, (2): 121.  Stephens, TV, M. Payne, RO Ball, PB Pencharz, and R. Elango. 2015. Protein requirements of healthy pregnant women during early and late gestation are higher than current recommendations. Journal of nutrition 145, (1): 73-78. 86   Stockler-Ipsiroglu, Sylvia, Nataliya Yuskiv, Ramona Salvarinova, Delia Apatean, Gloria Ho, Barbara Cheng, Alette Giezen, Yolanda Lillquist, and Keiko Ueda. 2015. Individualized long-term outcomes in blood phenylalanine concentrations and dietary phenylalanine tolerance in 11 patients with primary phenylalanine hydroxylase (PAH) deficiency treated with sapropterin-dihydrochloride. Molecular genetics and metabolism 114, (3): 409.  Trahms, CM. 2004. Medical Nutrition Therapy for Metabolic Disorders in “Krause’s Food, Nutrition, & Diet Therapy”. Mahan, L.K., & Escott-Stump, S., USA: Elsevier. 1143-1168.  Treacy EP, Delente JJ, Elkas G, Carter K, Lambert M, Waters PJ, & Scriver CR. 1997. Analysis of phenylalanine hydroxylase genotypes and hyperphenylalaninemia phenotypes using L-[1–13C] phenylalanine oxidation rates in vivo: a pilot study. Pediatr Res 42:430–435.  Trefz, Friedrich K., Theo Erlenmaier, Donald H. Hunneman, Klaus Bartholomé, and Peter Lutz. 1979. Sensitive in vivo assay of the phenylalanine hydroxylating system with a small intravenous dose of heptadeutero l-phenylalanine using high pressure liquid chromatography and capillary gas chromatography/mass fragmentography. Clinica Chimica Acta 99, (3): 211-220.  Trefz, FK, and Sapropterin Study Group. 2009. Efficacy of sapropterin dihydrochloride in increasing phenylalanine tolerance in children with phenylketonuria: A phase III, randomized, double-blind, placebo-controlled study. J Pediatr 154, (5): 700-707.e1.  Turner, JM, MA Humayun, R. Elango, M. Rafii, V. Langos, RO Ball, and PB Pencharz. 2006. Total sulfur amino acid requirement of healthy school-age children as determined by indicator amino acid oxidation technique. American journal of clinical nutrition 83, (3): 619-623.  87  van Karnebeek, Clara D. M., and Sylvia Stockler. 2012; 2011. Treatable inborn errors of metabolism causing intellectual disability: A systematic literature review. Molecular genetics and metabolism 105, (3): 368-381.  van Rijn, Margreet, Marieke Hoeksma, Pieter Sauer, Beate Szczerbak, Martina Gross, Dirk-Jan Reijngoud, and Francjan van Spronsen. 2007. Protein metabolism in adult patients with phenylketonuria. Nutrition 23, (6): 445-453.  Vanspronsen, FJ, M. Vanrijn, T. Vandijk, GPA Smit, DJ Reijngoud, R. Berger, and HSA Heymans. 1993. Plasma Phenylalanine and Tyrosine Responses to Differentiate Nutritional Conditions (Fasting/Postprandial) In Patients with Phenylketonuria - Effect of Sample Timing. Pediatrics 92, (4): 570-573.  van Spronsen, Francjan J., and Gregory M. Enns. 2010. Future treatment strategies in phenylketonuria. Molecular genetics and metabolism 99, : S90-S95.  Verkerk, P. H., F. J. van Spronsen, G. P. Smit, and R. C. Sengers. 1994. Impaired prenatal and postnatal growth in dutch patients with phenylketonuria. the national PKU steering committee. Archives of Disease in Childhood 71, (2): 114-118.  Vernon, H. J., C. B. Koerner, M. R. Johnson, A. Bergner, and A. Hamosh. 2010. Introduction of sapropterin dihydrochloride as standard of care in patients with phenylketonuria. Molecular genetics and metabolism 100, (3): 229-233.  Vockley, Jerry, Hans C. Andersson, Kevin M. Antshel, Nancy E. Braverman, Barbara K. Burton, Dianne M. Frazier, John Mitchell, et al. 2014. Phenylalanine hydroxylase deficiency: Diagnosis and management guideline. Genetics in medicine : official journal of the American College of Medical Genetics 16, (2): 188.  88  Walter, John H., Robin H. Lachmann, and Peter Burgard. 2012. Hyperphenylalaninaemia. In , 251-264. Berlin, Heidelberg: Springer Berlin Heidelberg.  Webster, D., and J. Wildgoose. 2010. Tyrosine supplementation for phenylketonuria. Cochrane database of systematic reviews (8): CD001507.  Yamaguchi, Seiji. 2008. Newborn screening in japan: Restructuring for the new era. Annals of the Academy of Medicine, Singapore 37, (12 Suppl): 13.  Zurflüh, Marcel R., Johannes Zschocke, Martin Lindner, François Feillet, Céline Chery, Alberto Burlina, Raymond C. Stevens, Beat Thöny, and Nenad Blau. 2008. Molecular genetics of tetrahydrobiopterin‐ responsive phenylalanine hydroxylase deficiency. Human mutation 29, (1): 167-175.  Zello, Gordon A., Paul B. Pencharz, and Ronald O. Ball. 1990. The design and validation of a diet for studies of amino acid metabolism in adult humans. Nutrition Research 10, (12): 1353-1365.  Zello, GA, LJ Wykes, RO Ball, and PB Pencharz. 1995. Recent advances in methods of assessing dietary amino-acid-requirements for adult humans. Journal of nutrition 125, (12): 2907-2915. 89  Appendices  Appendix A  : Subject Consent Form – Healthy Children   90     SUBJECT INFORMATION AND CONSENT FORM  USE OF A SIMPLE BREATH TEST TO EXAMINE PHENYLALANINE DIGESTION IN HEALTHY CHILDREN  Principal Investigator:   Dr. Rajavel Elango, PhD       Department of Pediatrics      The University of British Columbia      Telephone: 604-875-2000 x4911 Co-Investigators:   Dr. Sylvia Stockler, MD      Professor and Head, Division of Biochemical Diseases      British Columbia Children's Hospital       Telephone: 604-875-2628            Barbara Cheng, RD      Biochemical Diseases Clinic      British Columbia Children's Hospital       Telephone: 604-875-2345 x7598  Primary Contact:   Gayathri Murthy, RD CDE      Research Dietitian      Child & Family Research Institute      Telephone: 604-875-2000 x4607      Email:gmurthy@cfri.ubc.ca  Emergency Phone Number:  Rajavel Elango 778-986-8655 available 24 hours per day and seven days per week  Sponsors:    Rare Disease Foundation  Site:     Oak Street Campus, UBC      Child & Family Research Institute   1. INVITATION  “You” refers to “you” or “your child” throughout the consent form. You are being invited to take part in this research study because you are a healthy individual between the ages of 4-18 years, free of any medical condition and are currently free from fever or having a cold. You will act as a control subject for a research study involving children with a medical condition called phenylketonuria (PKU).  2. YOUR PARTICIPATION IS VOLUNTARY   D e p a r t m e n t  o f  P e d i a t r i c s  950 West 28th Avenue, Room 170A Vancouver, BC, V5Z 4H4 Tel: (604) 875-2000 x4911  Fax: (604) 875-3597    91  Your participation is entirely voluntary, so it is up to you to decide whether or not to take part in the study. Before you decide, it is important for you to understand what the research involves. This consent form will describe the study, why the research is being done, what will happen to you during the study and the possible benefits, risks, and discomforts. If you wish to participate, you will be asked to sign this consent form within 7 days. If you decide to take part in this study, you are still free to withdraw at any time and without giving any reasons for your decision(s). If you do not wish to participate, you do not have to provide any reason for your decision(s) nor will you lose the benefit of any medical care to which you are entitled or are presently receiving. Please take time to read the following information carefully and to discuss it with your child, family and friends before deciding.   3.  WHO IS CONDUCTING THE STUDY?  The Principal Investigator [Rajavel Elango] has received financial compensation from the sponsor [Rare Disease Foundation] for the work required in doing this clinical research and/or for providing advice on the design of the study/travel expenses/etc. Financial compensation to researchers for conducting the research is associated with obligations defined in a signed contractual agreement between the researchers and the sponsor. Researchers must serve the interests of the subject and also abide by their contractual obligations. For some, the payment of financial compensation to the researchers can raise the possibility of a conflict of interest. You are entitled to request any details concerning this compensation from the Principal Investigator.  4. BACKGROUND  The purpose of the study is to see how the body digests phenylalanine (an amino acid in protein) in healthy children. The test uses a simple breath test, where you will be asked to drink a liquid which has 13C-phenylalanine, a stable isotope. The 13C stable isotopes are completely safe, they are present in the air we breathe, water we drink and food we eat. 13C is a type of carbon; amino acids are made of mostly 12C, so the 13C can be measured in your breath with special equipment.   The protein in our food is made up of 20 amino acids, including phenylalanine. Phenylalanine is digested in our body by an enzyme called phenylalanine hydroxylase. All healthy children have this enzyme. But some children do not have this enzyme, or the enzyme is not working properly, and they develop a condition called phenylketonuria (PKU). We are doing this breath test in children with PKU and comparing it to healthy children.  5.  WHO CAN PARTICIPATE IN THE STUDY?    Healthy children (4 -18years), who have no medical condition and currently free from any infection such as fever or cold.  6. WHAT DOES THE STUDY INVOLVE? 92  This study will be conducted at the Oak Street Campus of UBC at the Child and Family Research Institute (CFRI). If you agree to participate in this study, then you will be asked to complete the study procedures described below which will take about 2 hours.  Study Day Procedures:    If your child agrees to participate in this study then the following procedures will be followed:   The study will be conducted in the Clinical Research Evaluation Unit (CREU) at the Child & Family Research Institute located in BC Children’s Hospital.  You will be asked to come after an overnight fast of about 12 hours. The whole test time should take approximately 2 hours.   A Research Dietitian/Assistant will measure your child’s weight and height.    The Research Dietitian/Assistant will conduct a simple questionnaire to obtain your child’s basic medical history, current nutrient supplement and activity level.    You will drink the test liquid made up of an oral dose of the isotope dissolved in sterile water. The test liquid will have no taste to it and will be diluted with water. To measure how your body responds to the test liquid, the Research Assistant will collect your breath samples 7 times at 0,20,40,60,80,100 and 120 minutes after the isotope dose. To collect breath you will have to breathe into a bag - just like blowing through a straw into a bag. When we are not collecting samples, you can watch television, listen to music, read or use a computer.   The Research Assistant will also measure the rate at which you are breathing out carbon dioxide (VCO2) using an indirect calorimetry machine at the end of 1 hour (60 minutes). This will require you to relax on the examination table and lie still while a clear hood/canopy is placed on your face and head. This has room air freely flowing in and out. You can watch television/DVD player during the testing. The entire test will take about 20-30 minutes.   To measure your body muscle, a test (Bioelectrical Impedance Analysis) will be performed. The whole test will be for 30 seconds – 1 minute, during which a very small current is sent across from the right arm to the right leg, while you lie down on an examination table. You will not feel anything during the test procedure. This test will allow us to measure how much of your body is muscle, versus fat.     In total, you can expect to dedicate approximately 2 hours for the entire study.   7. WHAT ARE THE POSSIBLE HARMS AND SIDE EFFECTS OF PARTICIPATING IN THIS STUDY? 93   The breath test requires you to consume a small dose of isotope. This is a safe form of isotope and is widely used in medical research even in children and pregnant women. During the indirect calorimetry test you are requested to lie down for 20-30 minutes with an open hood/canopy over the face and head. Some children may feel uncomfortable during this period. The hood/canopy has outside air freely moving in and out. If you are uncomfortable, then the test will be stopped immediately. There are no other known risks involved with participating in this research. We recognize that the length of the study day may pose an inconvenience to you.    8.  WHAT ARE THE BENEFITS OF PARTICIPATING IN THIS STUDY?  There are no direct benefits by participation in the study. The test may help us understand the enzyme activity in healthy children and also help us examine phenylalanine digestion in humans. We hope that the information learned from this study can also be used in the future to improve management of children with PKU.  9.  WHAT HAPPENS IF I DECIDE TO WITHDRAW MY CONSENT TO PARTICIPATE IN THIS STUDY?  Your participation in this research is entirely voluntary. You may withdraw from this study at any time and without providing any reasons for your decision. If you decide to enter the study and then withdraw, there will be no penalty or loss of benefits, if any, to which you are otherwise entitled. If you choose to enter the study and then decide to withdraw at a later time, all data collected about you during the enrolment part of the study will be retained for analysis, after which the study information may be shredded.  10. WHAT WILL THE STUDY COST ME?  Participation in the study will not cost you anything. In appreciation of the time that it takes to complete this study, you will receive a $10 gift card.   11. WILL MY TAKING PART IN THIS STUDY BE KEPT CONFIDENTIAL?  Your confidentiality will be respected. No information or records that disclose your identity will be published without your consent, nor will any information or records that disclose your identity be removed or released without your consent unless required by law.   You will be assigned a unique study number as a subject in this study.  Only this number will be used on any research-related information collected about you during the course of this study, so that your identity [i.e. your name or any other information that could identify you] as a subject in this study will be kept confidential.   Information that contains your identity will remain only with the Principal Investigator and/or designate.    94  Your rights to privacy are legally protected by federal and provincial laws that require safeguards to insure that your privacy is respected and also give you the right of access to the information about you that has been provided to the sponsor and, if need be, an opportunity to correct any errors in this information.  Further details about these laws are available on request to your study doctor. Signing this consent form in no way, limits your legal rights against the sponsor, investigators, or anyone else.  12. WHO DO I CONTACT IF I HAVE QUESTIONS OR CONCERNS ABOUT MY/MY CHILD’S RIGHTS AS A SUBJECT DURING THE STUDY?  Your/your child’s rights to privacy are protected and guaranteed by the Child, Family and Community Services Act. This Act lays down the safeguards respecting your/your child’s privacy and also gives you the right of access to the information about you/your child that has been provided to the study, and if needed, you have the chance to correct any errors in the information. Further details about this legislation are available on request. If you have any concerns about your/your child’s rights as a research subject and/or your/your child’s experiences while participating in this study, contact the Research Subject Information Line, in the UBC Office of Research Services at 604-822-8598, if long distance, call the toll free number of the Research Subject Information Line (1-877-822-8598) or e-mail to RSIL@ors.ubc.ca.  If I have any further questions or desire further information about the study, you can contact Dr. Rajavel Elango at 778-986-8655.  95  13.  SUBJECT CONSENT TO PARTICIPATE    My signature on this consent form means that I:   have had this study explained to me and my child, read this form and understand the information concerning this study.  have had sufficient time to consider the information provided, get advice and ask questions if necessary and I/my child have received satisfactory responses to our questions.  understand that all of the information collected will be kept confidential and that the results will only be used for scientific objectives.  understand that my/my child’s participation in this study is voluntary and that I/my child am/is completely free to refuse to participate or to withdraw from this study at any time without giving any reason(s) and my/my child’s decision to withdraw will not change in any way the quality of care that I/my child receive.  understand that signing this consent form in no way limits my/my child’s legal rights against the sponsor, investigators or anyone else.  understand that there is no guarantee that this study will provide any benefits to myself/my child.  understand that if I have any further questions or desire further information I should contact Dr. Rajavel Elango at 778-986-8655.  Been told that I will receive a dated and signed copy of this form for my own record.  I, ___________________________________    voluntarily give consent for my and my child’s      (Subject-Parent-Guardian.  Please print your name)     ___________________________________ participation in the research study entitled:             (Please print child’s name)  USE OF A SIMPLE BREATH TEST TO EXAMINE PHENYLALANINE DIGESTION IN HEALTHY CHILDREN                     Signature of Subject-Parent-Guardian or person legally authorized to give consent       _________________________________________________         ____________________ Relationship to Child (Mother, Father ,legal guardian)                                        Date  _______________________    __          ___________________ Investigator Signature            Printed Name of principal investigator/ Designate                   Date       96   Appendix B  : Subject Assent Form – Healthy Children   97   SUBJECT ASSENT FORM (7-13 years of age)  USE OF A SIMPLE BREATH TEST TO EXAMINE PHENYLALANINE DIGESTION IN HEALTHY CHILDREN  1. INVITATION  I am being invited to be part of a research study. No one will make me be part of the study. Even if I agree now to be part of the study, I can change my mind later. No one will be mad at me if I choose not to be part of this study.  2.   WHY ARE WE DOING THIS STUDY?  The protein in our food is made up of 20 amino acids, including phenylalanine. Phenylalanine is digested in our body by an enzyme called phenylalanine hydroxylase. All healthy children have this enzyme. But some children do not have this enzyme, or the enzyme is not working properly, and they develop a condition called phenylketonuria (PKU). We are doing this breath test in children with PKU and comparing it to healthy children, such as myself.  3.  WHAT WILL HAPPEN IN THIS STUDY?   I will have to come without eating anything for 12 hours    The whole test time will take about 2 hours   A Research Assistant will measure my weight and height.    I will be requested to drink a test liquid (1 small cup) and then breathe into a bag several times like blowing through a straw into a bag. This liquid will have no taste to it and will be diluted with water. When samples are not being collected, I can watch television, listen to music, read or use a computer.   How much muscle I have will be measured. The whole test will only take 30 seconds – 1 minute. While I lie down on the bed two wires will be put on my right hand and foot, like band-aids. I will not feel anything during this test.     While I lie down on the bed a clear hood (like a space-man’s suit) will be placed on my head. This is a clear hood with air freely coming in and out. I can watch television during the testing, but am expected to lie still throughout the test. This test will take about 20 minutes. If I want to stop the test anytime, then I can just pull the hood away. This test will collect and measure the air I breathe out.  D e p a r t m e n t  o f  P e d i a t r i c s  950 West 28th Avenue, Room 170A Vancouver, BC, V5Z 4H4 Tel: (604) 875-2000 x4911  Fax: (604) 875-3597     98  4. WHO IS DOING THIS STUDY?  Dr. Rajavel Elango, Dr. Sylvia Stockler and other members of the research team from Children’s Hospital will be doing this study. They will answer any questions I have about the study. I can also call them at 778-986-8655, if I am having any problems or if there is an emergency and I cannot talk to my parents.   5.  CAN ANYTHING BAD HAPPEN TO ME?  There is nothing in this study which will make anything bad happen to me.  6.  WHAT SHOULD I DO IF I AM NOT FEELING WELL?    I can simply pull the hood away when the test is being done, or call the research assistant, Dr. Rajavel Elango / Gayathri Murthy, the Research Dietitian who will be standing next to me.  7.  WHO WILL KNOW I AM IN THE STUDY?  Only the doctors and people who are involved in the study will know I am in it.  When the study is finished, the doctors will write a report about what was learned.  This report will not say my name or that I was in the study. My parents and I do not have to tell anyone I am in the study if we don’t want to.  8. WHEN DO I HAVE TO DECIDE?  I have 7 days from today to decide if I want to be part of the study. If I am interested, I can contact the researchers and set up a time that is convenient for the both of us. I have also been asked to discuss my decision with my parents.   9.  SIGNATURES    If I put my name at the end of this form, it means that I agree to be in the study.   ___________________         __          ___________________   Printed Name    Signature     Date        99  Appendix C  : Advertisement – Healthy Children   100        101  Appendix D  : Study Day Form – Healthy Children   102  Study Day Form  USE OF A SIMPLE BREATH TEST TO EXAMINE KUVAN RESPONSIVENESS IN CHILDREN WITH PKU- CONTROL SUBJECTS Principal Investigator: Dr. Rajavel Elango       604-875-2000 ext. 4911 (office) Co-investigators:     Dr. Sylvia Stockler                         604-875-2628 (office)        Barbara Cheng       604-875-2345 ext. 7598 (office)  Research Assistant:       Gayathri Murthy       604-875-2000 ext. 4607 (office)  PRELIMINARY ASSESSMENT Subject ID: ________________________________          Date:___________________________  Gender: ________  Birthday (mo/year): ______/________           Age: ___________                      Height (cm):______________         Weight (kg):___________  BMI: _________________       Medical History  Details of health condition(s)__________________________________________________  Are you currently taking any prescription medications?  Yes_______ No_________  List of medications:____________________________________________________________  Are you currently having vomiting episodes?  Yes______ How many/day __       No______  Are you currently having fever/cold?  Yes______ No______  Nutritional Supplement Intake  Are you currently taking vitamins? Yes_______ No_______  Are you taking any other nutritional supplements? Yes_______ No________  If yes, please list all nutritional supplements: 1.__________________________________ 3.__________________________________  2.__________________________________ 4.__________________________________  Activity Level  Daily play/exercise (minutes)________________________  Sedentary_____________  Moderate_________________ High__________________ 103  Stable Isotope Protocol  Dose (@ the rate of 6mg/kg/day): __________________________      Time of dose: ______________________  Breath Sample Collection:  Breath Sample Time interval  (minutes) Time Comments 1st breath (3x)baseline Start    2nd breath (3x) 20    3rd breath (3x) 40   4th breath (3x) 60   VCO2 measurement at the end  of 60 minutes   Reached steady state @ ______ minutes 5th breath (3x) 80   6th breath (3x) 100   7th breath (3x) 120    Bioelectrical Impedance Analysis  BIA:R_____________________________   XC______________________________ (resistance)      (reactance)  BIA: Z_____________________________    (Impedance)    Body Composition Profile  % Body fat (BIA):______________________ Lean body mass (BIA):________________________  Indirect Calorimetry (at 60 minutes into the study)  Measured REE (kcal/day):_________________     Estimated REE (kcal/day):____________________  VCO2 (ml/min): _________________     VO2 (ml/min):____________________    RQ:  _________________  Comments:   104  Appendix E  : Subject Consent Form – Children with PKU   105   SUBJECT INFORMATION AND CONSENT FORM  USE OF A SIMPLE BREATH TEST TO EXAMINE KUVAN® RESPONSIVENESS IN CHILDREN WITH PKU  Principal Investigator:  Dr. Rajavel Elango, PhD       Department of Pediatrics      The University of British Columbia      Telephone: 604-875-2000 x4911 Co-Investigators:   Dr. Sylvia Stockler, MD      Professor and Head, Division of Biochemical Diseases      British Columbia Children's Hospital       Telephone: 604-875-2628            Barbara Cheng, RD      Biochemical Diseases Clinic      British Columbia Children's Hospital       Telephone: 604-875-2345 x7598  Primary Contact:   Gayathri Murthy, RD CDE      Research Dietitian      Child & Family Research Institute      Telephone: 604-875-2000 x4607      Email:gmurthy@cfri.ubc.ca  Emergency Phone Number:  Rajavel Elango 778-986-8655 available 24 hours per day and seven days per week  Sponsors:    Rare Disease Foundation  Site:     Oak Street Campus, UBC      Child & Family Research Institute   1. INVITATION  “You” refers to “you” or “your child” throughout the consent form. You are being invited to take part in this research study because your child has been diagnosed with Phenylketonuria or PKU. We are inviting children who have a known diagnosis of the disease, ranging in age from 4 -18 years, and are currently free from fever or having a cold.  2. YOUR PARTICIPATION IS VOLUNTARY   D e p a r t m e n t  o f  P e d i a t r i c s  950 West 28th Avenue, Room 170A Vancouver, BC, V5Z 4H4 Tel: (604) 875-2000 x4911  Fax: (604) 875-3597    106  Your participation is entirely voluntary, so it is up to you to decide whether or not to take part in the study. Before you decide, it is important for you to understand what the research involves. This consent form will describe the study, why the research is being done, what will happen to you during the study and the possible benefits, risks, and discomforts. If you wish to participate, you will be asked to sign this consent form within 7 days. If you decide to take part in this study, you are still free to withdraw at any time and without giving any reasons for your decision(s). If you do not wish to participate, you do not have to provide any reason for your decision(s) nor will you lose the benefit of any medical care to which you are entitled or are presently receiving. Please take time to read the following information carefully and to discuss it with your child, family and friends before deciding.    3.  WHO IS CONDUCTING THE STUDY?  The Principal Investigator [Rajavel Elango] has received financial compensation from the sponsor [Rare Disease Foundation] for the work required in doing this clinical research and/or for providing advice on the design of the study/travel expenses/etc. Financial compensation to researchers for conducting the research is associated with obligations defined in a signed contractual agreement between the researchers and the sponsor. Researchers must serve the interests of the subject and also abide by their contractual obligations. For some, the payment of financial compensation to the researchers can raise the possibility of a conflict of interest. You are entitled to request any details concerning this compensation from the Principal Investigator.  4. BACKGROUND  Phenylketonuria is a rare condition caused by the body’s inability to properly breakdown an amino acid called phenylalanine, due to a missing enzyme, Phenylalanine Hydroxylase (PAH). When the enzyme is missing and/or not functioning properly, it increases the level of phenylalanine in the blood. High levels of phenylalanine can cause severe brain or nerve damage unless the children are on a strict low phenylalanine diet. A low Phenylalanine diet restricts the intake of high protein foods and can pose a significant burden of both the patient and the family.  A new drug called Kuvan® (Sapropterin dihydrochloride) has shown to reduce the amount of phenylalanine in the body, although the response to the drug varies among children. Thus, there is a need to develop a test that will actually measure the drug responsiveness. This study will help develop a simple breath test to identify children who respond to treatment and thereby aid in the medical management of children with PKU.   5.  WHAT IS THE PURPOSE OF THE STUDY?   The purpose of the study is to determine Kuvan® responsiveness in patients with PKU. Although, a new drug called Kuvan® (Sapropterin dihydrochloride), a synthetic form of the tetrahydrobiopterin (BH4) which is used by the Phenylalanine Hydroxylase enzyme has shown 107  promise in decreasing the levels of plasma phenylalanine, the response to the drug is quite varied in patients with different forms of PKU. In some children there is a very small decrease in plasma phenylalanine concentrations. In some children, even when there is no change in plasma phenylalanine, the child seems to improve in cognitive functions. Thus, there is a need to develop a test that will measure whether the drug is actually responsive. The new stable isotope (a very safe form of isotope, widely used in medical research) test uses phenylalanine, with a 13C label. (13C is a type of carbon; phenylalanine is made of mostly 12C, so the 13C can be detected in breath and urine samples with special equipment). The 13C stable isotope is considered safe and is naturally present at lower concentration in the air we breathe, water we drink and food we eat. If the child is responding to Kuvan®, then there will be increased amounts of 13CO2 in the breath, as the 13C-Phenylalanine will be metabolized by the liver and the 13C released in breath as 13CO2. We are conducting this study to help us find out whether this simple breath test before and after the provision of Kuvan® will help in identifying children who respond to treatment.   6.  WHO CAN PARTICIPATE IN THE STUDY?     Children (4 -18y) who are diagnosed with PKU and who have had the clinical decision made to start the drug Kuvan®.  7. WHO SHOULD NOT PARTICIPATE IN THE STUDY?     Children < 4 years of age and who are diagnosed with PKU, as it may be difficult to take breath samples and perform indirect calorimetry in very young children.  Children diagnosed with PKU, but are currently ill, with a fever, cold, vomiting or diarrhea.  Children diagnosed with PKU, but where the clinical decision has been made not to start on Kuvan® therapy.  8. WHAT DOES THE STUDY INVOLVE?  This study will be conducted at the Oak Street Campus of UBC at the Child and Family Research Institute (CFRI). If you agree to participate in this study, then you will be asked to complete the study procedures described below which will take about 2 hours . Study Day Procedures:   If your child agrees to participate in this study then the following procedures will be followed:   The study will be conducted in the Clinical Research Evaluation Unit (CREU) at the Child & Family Research Institute located in BC Children’s Hospital.  You will be asked to come after an overnight fast of about 12 hours. The whole test time should take approximately 2 hours.   A Research Dietitian/Assistant will measure your child’s weight and height.  108    The Research Dietitian/Assistant will conduct a simple questionnaire to obtain your child’s basic medical history, current nutrient supplement and activity level.    You will drink the test liquid made up of an oral dose of the isotope dissolved in sterile water.  To measure how your body responds to the test liquid, the Research Assistant will collect your breath samples 7 times at 0,20,40,60,80,100 and 120 minutes  after the isotope dose. To collect breath you will have to breathe into a container - just like blowing through a straw into a bag. When we are not collecting samples, you can watch television, listen to music, read or use a computer.   The Research Assistant will also measure the rate at which you are breathing out carbon dioxide (VCO2) using an indirect calorimetry machine at the end of 1 hour (60 minutes). This will require you to relax on the examination table while a clear hood/canopy is placed on your face and head. This has room air freely flowing in and out. You can watch television/DVD player during the testing. The entire test will take about 20-30 minutes.   To measure your body muscle, a test (Bioelectrical Impedance Analysis) will be performed. The whole test will be for 30 seconds – 1 minute, during which a very small current is sent across from the right arm to the right leg, while you lie down on an examination table. You will not feel anything during the test procedure. This test will allow us to measure how much of your body is muscle, versus fat.     You are invited to participate in 2 study days separated by at least 1 week (7 days) to determine your drug responsiveness.   In total, you can expect to dedicate approximately 4 hours (2 hours per study day) for the entire study.   9. WHAT ARE THE POSSIBLE HARMS AND SIDE EFFECTS OF PARTICIPATING IN THIS STUDY?  The breath test requires you to consume a small dose of isotope. This is a safe form of isotope and is widely used in medical research even in children and pregnant women. During the indirect calorimetry test you are requested to lie down for 20-30 minutes with an open hood/canopy over the face and head. Some children may feel uncomfortable during this period. The hood/canopy has outside air freely moving in and out. If you are uncomfortable, then the test will be stopped immediately. There are no other known risks involved with participating in this research. We recognize that the length of each study day may pose an inconvenience to you. The Physician will also discuss the possible side effects, if any, due to the drug, Kuvan®.   10.  WHAT ARE THE BENEFITS OF PARTICIPATING IN THIS STUDY?  109  There are no direct benefits by participation in the study, because we do not know what the results mean for the overall effectiveness of Kuvan®. We hope that the information learned from this study can also be used in the future to improve management of children with PKU.   11.  WHAT HAPPENS IF I DECIDE TO WITHDRAW MY CONSENT TO PARTICIPATE IN THIS STUDY?  Your participation in this research is entirely voluntary. You may withdraw from this study at any time and without providing any reasons for your decision. If you decide to enter the study and then withdraw, there will be no penalty or loss of benefits, if any, to which you are otherwise entitled. If you choose to enter the study and then decide to withdraw at a later time, all data collected about you during the enrolment part of the study will be retained for analysis, after which the study information may be shredded.  12. WHAT WILL THE STUDY COST ME?  Participation in the study will not cost you anything. In appreciation of the time that it takes to complete this study, you will receive a $10 gift card for each study day.  13. WILL MY TAKING PART IN THIS STUDY BE KEPT CONFIDENTIAL?  Your confidentiality will be respected.  However, research records and medical records identifying you may be inspected in the presence of the Investigator or his or her designate by representatives of Rare Diseases Foundation, Health Canada, and UBC / Children’s and women’s Health Centre of BC Research Ethics Board for the purpose of monitoring the research. No information or records that disclose your identity will be published without your consent, nor will any information or records that disclose your identity be removed or released without your consent unless required by law.   You will be assigned a unique study number as a subject in this study.  Only this number will be used on any research-related information collected about you during the course of this study, so that your identity [i.e. your name or any other information that could identify you] as a subject in this study will be kept confidential.   Information that contains your identity will remain only with the Principal Investigator and/or designate.  The list that matches your name to the unique identifier that is used on your research-related information will not be removed or released without your consent unless required by law.  Your rights to privacy are legally protected by federal and provincial laws that require safeguards to insure that your privacy is respected and also give you the right of access to the information about you that has been provided to the sponsor and, if need be, an opportunity to correct any errors in this information.  Further details about these laws are available on request to your study doctor. Signing this consent form in no way, limits your legal rights against the sponsor, investigators, or anyone else.  110  14. WHO DO I CONTACT IF I HAVE QUESTIONS OR CONCERNS ABOUT MY/MY CHILD’S RIGHTS AS A SUBJECT DURING THE STUDY?  Your/your child’s rights to privacy are protected and guaranteed by the Child, Family and Community Services Act. This Act lays down the safeguards respecting your/your child’s privacy and also gives you the right of access to the information about you/your child that has been provided to the study, and if needed, you have the chance to correct any errors in the information. Further details about this legislation are available on request. If you have any concerns about your/your child’s rights as a research subject and/or your/your child’s experiences while participating in this study, contact the Research Subject Information Line, in the UBC Office of Research Services at 604-822-8598, if long distance, call the toll free number of the Research Subject Information Line (1-877-822-8598) or e-mail to RSIL@ors.ubc.ca.  If I have any further questions or desire further information about the study, you can contact Dr. Rajavel Elango at 778-986-8655.  111  15.  SUBJECT CONSENT TO PARTICIPATE    My signature on this consent form means that I:   have had this study explained to me and my child, read this form and understand the information concerning this study.  have had sufficient time to consider the information provided, get advice and ask questions if necessary and I/my child have received satisfactory responses to our questions.  understand that all of the information collected will be kept confidential and that the results will only be used for scientific objectives.  understand that my/my child’s participation in this study is voluntary and that I/my child am/is completely free to refuse to participate or to withdraw from this study at any time without giving any reason(s) and my/my child’s decision to withdraw will not change in any way the quality of care that I/my child receive.  understand that signing this consent form in no way limits my/my child’s legal rights against the sponsor, investigators or anyone else.  understand that there is no guarantee that this study will provide any benefits to myself/my child.  understand that if I have any further questions or desire further information I should contact Dr. Rajavel Elango at 778-986-8655.  understand that I authorize access to my health record and samples as described in this consent form.  understand that the results from the tests will be disclosed to you and treating physicians   have been told that I will receive a dated and signed copy of this form for my own record.  I, ___________________________________    voluntarily give consent for my and my child’s      (Subject-Parent-Guardian.  Please print your name)     ___________________________________ participation in the research study entitled:             (Please print child’s name)  USE OF A SIMPLE BREATH TEST USING STABLE ISOTOPES TO EXAMINE KUVAN® RESPONSIVENESS IN PATIENTS WITH PHENYLKETONURIA                     Signature of Subject-Parent-Guardian or person legally authorized to give consent       _________________________________________________         ____________________ Relationship to Child (Mother, Father ,legal guardian)                                        Date  _______________________    __          ___________________ Investigator Signature            Printed Name of principal investigator/ Designate                   Date    112  SUBJECT'S ASSENT TO PARTICIPATE IN RESEARCH  I have had the opportunity to read this consent form, to ask questions about my participation in this research, and to discuss my participation with my parents/guardians. All my questions have been answered. I understand that I may withdraw from this research at any time, and that this will not interfere with the availability to me of other health care. I have received a copy of this consent form. I assent to participate in this study.      _________________________________________________         ____________________ Signature of individual (aged 14y and above) to give assent    Date       113  Appendix F  : Subject Assent Form – Children with PKU   114   SUBJECT ASSENT FORM (7-13 years of age)  USE OF A SIMPLE BREATH TEST TO EXAMINE KUVAN® RESPONSIVENESS IN CHILDREN WITH PKU                1. INVITATION  I am being invited to be part of a research study. A research study tries to find better treatments to help children like me. It is up to me if I want to be in this study. No one will make me be part of the study. Even if I agree now to be part of the study, I can change my mind later. No one will be mad at me if I choose not to be part of this study.  2.   WHY ARE WE DOING THIS STUDY  I have a disease called phenylketonuria. This disease affects many other children.  This study is trying to find out how my body will respond to a new drug called Kuvan® so that I can better manage my condition.  3.  WHAT WILL HAPPEN IN THIS STUDY?   I will have to come without eating anything for 12 hours    The whole test time will take about 2 hours   A Research Assistant will measure my weight and height.    I will be requested to drink a test liquid (1 small cup) and then breathe into a container several times like blowing through a straw into a bag. When samples are not being collected, I can watch television, listen to music, read or use a computer.   How much muscle I have will be measured. The whole test will only take 30 seconds – 1 minute. While I lie down on the bed two wires will be put on my right hand and foot, like band-aids. I will not feel anything during this test.     While I lie down on the bed a clear hood (like a space-man’s suit) will be placed on my head. This is a clear hood with air freely coming in and out. I can watch television during the testing. This test will take about 20 minutes. If I want to stop the test anytime, then I can just pull the hood away. This test will measure how much carbon dioxide I produce.    D e p a r t m e n t  o f  P e d i a t r i c s  950 West 28th Avenue, Room 170A Vancouver, BC, V5Z 4H4 Tel: (604) 875-2000 x4911  Fax: (604) 875-3597     115   4. WHO IS DOING THIS STUDY?  Dr. Rajavel Elango, Dr. Sylvia Stockler and other members of the research team from Children’s Hospital will be doing this study. They will answer any questions I have about the study. I can also call them at 778-986-8655, if I am having any problems or if there is an emergency and I cannot talk to my parents.   5.  CAN ANYTHING BAD HAPPEN TO ME?  There is nothing in this study which will make anything bad happen to me.   6.  WHAT SHOULD I DO IF I AM NOT FEELING WELL?    I can simply pull the hood away when the test is being done, or call the research assistant, Dr. Rajavel Elango / Barbara Cheng, the Dietitian who will be standing next to me.  8.  WHO WILL KNOW I AM IN THE STUDY?  Only my doctors and people who are involved in the study will know I am in it.  When the study is finished, the doctors will write a report about what was learned.  This report will not say my name or that I was in the study. My parents and I do not have to tell anyone I am in the study if we don’t want to.  8. WHEN DO I HAVE TO DECIDE?  I have 7 days from today to decide if I want to be part of the study. When I come in for my next visit to the clinic at BC Children’s Hospital I can be part of the study, if I want to. I have also been asked to discuss my decision with my parents.   9.  SIGNATURES    If I put my name at the end of this form, it means that I agree to be in the study.   ___________________         __          ___________________   Printed Name    Signature     Date      116  Appendix G  : Advertisement – Children with PKU117            118  Appendix H  : Patient Code Form – Children with PKU   119  PATIENT CODE FORM   USE OF A SIMPLE BREATH TEST TO EXAMINE KUVAN® RESPONSIVENESS IN CHILDREN WITH PKU                Patient Code   Name of Patient Comments  First Name Last Name   BTPKU01      BTPKU02      BTPKU03      BTPKU04      BTPKU05      BTPKU06      BTPKU07      BTPKU08      BTPKU09      BTPKU10        120  Appendix I  : Study Day Form – Children with PKU   121  Study Day Form  USE OF A SIMPLE BREATH TEST TO EXAMINE KUVAN RESPONSIVENESS IN CHILDREN WITH PKU Principal Investigator: Dr. Rajavel Elango       604-875-2000 ext. 4911 (office) Co-investigators:     Dr. Sylvia Stockler                         604-875-2628 (office)        Barbara Cheng       604-875-2345 ext. 7598 (office)  Research Assistant:       Gayathri Murthy       604-875-2000 ext. 4607 (office)   PRELIMINARY ASSESSMENT  Subject ID: ________________________________          Date:___________________________  Birthday (mo/year): ______/________           Age: ___________                      Height (cm):______________         Weight (kg):___________  BMI: _________________       Medical History  Details of health condition(s)__________________________________________________  Are you currently taking any prescription medications?  Yes_______ No_________  List of medications:__________________________________________________________   Are you currently having vomiting episodes?  Yes______ How many/day __       No______  Are you currently having fever/cold?  Yes______ No______  Nutritional Supplement Intake  Are you currently taking vitamins? Yes_______ No_______  Are you taking any other nutritional supplements? Yes_______ No________  If yes, please list all nutritional supplements: 1.__________________________________ 3.__________________________________  2.__________________________________ 4.__________________________________  Activity Level  Daily play/exercise (minutes)________________________  Sedentary_____________  Moderate_________________ High__________________ 122  Stable Isotope Protocol  Dose (@ the rate of 6mg/kg/day): __________________________      Time of dose: ______________________  Breath Sample Collection:  Breath Sample Time interval  (minutes) Time Comments 1st breath (3x)baseline Start    2nd breath (3x) 20    3rd breath (3x) 40   4th breath (3x) 60   VCO2 measurement at the end  of 60 minutes   Reached steady state @ ______ minutes 5th breath (3x) 80   6th breath (3x) 100   7th breath (3x) 120    Bioelectrical Impedance Analysis  BIA:R_____________________________   XC______________________________ (resistance)      (reactance)  BIA: Z_____________________________    (Impedance)    Body Composition Profile  % Body fat (BIA):______________________ Lean body mass (BIA):________________________  Indirect Calorimetry (at 60 minutes into the study)  Measured REE (kcal/day):_________________     Estimated REE (kcal/day):____________________  VCO2 (ml/min): _________________     VO2 (ml/min):____________________    RQ:  _________________  Comments:   123  Appendix J  : Subject Consent Form – Protein Requirement in PKU   124   SUBJECT INFORMATION AND CONSENT FORM  Application of Stable Isotopes to Determine Protein Requirements in Children with Phenylketonuria (PKU)   Principal Investigator:   Dr. Rajavel Elango, PhD       Department of Pediatrics      The University of British Columbia      Telephone: 604-875-2000 x4911                                                                   Email: relango@cfri.ubc.ca  Co-Investigator:   Dr. Sylvia Stockler, MD      Professor and Head, Division of Biochemical Diseases      British Columbia Children's Hospital       Telephone: 604-875-2628  Primary Contact:   Abrar Turki, M.Sc candidate      Department of Human Nutrition, UBC      Child & Family Research Institute      Telephone: 604-875-2000 x4607      Email: aturki@cfri.ca   Emergency Phone Number:  Rajavel Elango 778-986-8655                                                                   Abrar Turki      778-885-8274 available 24 hours per day and seven days per week  Sponsors: Rare Disease Foundation and Royal Embassy of Saudi Arabia – Cultural Bureau, Ottawa   Site:     Oak Street Campus, UBC      Child & Family Research Institute   1. INVITATION  “You” refers to “you” or “your child” throughout the consent form. You are being invited to take part in this research study because your child has been diagnosed with Phenylketonuria or PKU. We are inviting children who have a known diagnosis of the disease, ranging in age from 5 -18 years, and are currently free from fever or having a cold.   D e p a r t m e n t  o f  P e d i a t r i c s  950 West 28th Avenue, Room 170A Vancouver, BC, V5Z 4H4 Tel: (604) 875-2000 x4911  Fax: (604) 875-3597    125  2. YOUR PARTICIPATION IS VOLUNTARY  Your participation is entirely voluntary, so it is up to you to decide whether or not to take part in the study. Before you decide, it is important for you to understand what the research involves. This consent form will describe the study, why the research is being done, what will happen to you during the study and the possible benefits, risks, and discomforts. If you wish to participate, you will be asked to sign this consent form within 7 days. If you decide to take part in this study, you are still free to withdraw at any time and without giving any reasons for your decision(s). If you do not wish to participate, you do not have to provide any reason for your decision(s) nor will you lose the benefit of any medical care to which you are entitled or are presently receiving. Please take time to read the following information carefully and to discuss it with your child, family and friends before deciding.    3.  WHO IS CONDUCTING THE STUDY?  The Principal Investigator [Rajavel Elango] has received financial compensation from the sponsor [Rare Disease Foundation and Royal Embassy of Saudi Arabia – Cultural Bureau, Ottawa] for the work required in doing this clinical research and/or for providing advice on the design of the study/travel expenses/etc. Financial compensation to researchers for conducting the research is associated with obligations defined in a signed contractual agreement between the researchers and the sponsor. Researchers must serve the interests of the subject and also abide by their contractual obligations. For some, the payment of financial compensation to the researchers can raise the possibility of a conflict of interest. You are entitled to request any details concerning this compensation from the Principal Investigator.   4. BACKGROUND  Phenylketonuria (PKU) is an inherited inborn error of phenylalanine metabolism caused by a decreased activity or impaired synthesis of the hepatic enzyme called phenylalanine hydroxylase (PAH) or its cofactor tetrahydrobiopterin (BH4), which is important to convert phenylalanine into tyrosine. This leads to accumulation of phenylalanine in the blood. High levels of phenylalanine can cause severe brain or nerve damage unless the child is on a low phenylalanine diet.  Medical nutrition therapy for children with PKU involves a phenylalanine restricted diet with sufficient protein. However, the exact amount of protein is still unknown. There is a need to do a test to determine how much protein a child with PKU requires to ensure adequate growth and development.   5.  WHAT IS THE PURPOSE OF THE STUDY? The purpose of the study is to determine the exact protein requirements in children with phenylketonuria. We will measure your child’s protein requirements using the IAAO technique, which uses stable isotope (lysine) with a 13C label. (13C is a type of carbon; amino acids are made 126  of mostly 12C, so the 13C can be detected in breath and urine samples with special equipment). The 13C stable isotopes are completely safe. They are present naturally in the air we breathe, water we drink and food we eat. The labeled amino acid oxidation will be measured from breath and urine samples. This will help us to determine how much protein a child with PKU needs to eat in a day.   6.  WHO CAN PARTICIPATE IN THE STUDY?     6 Children (5 -18y) who are diagnosed with PKU, and clinically stable with no current illness.  7. WHO SHOULD NOT PARTICIPATE IN THE STUDY?     Children < 5 years of age and who are diagnosed with PKU, as it may be difficult to take breath samples and perform indirect calorimetry in very young children.  Children diagnosed with PKU, but are currently ill, with a fever, cold, vomiting or diarrhea.  9. WHAT DOES THE STUDY INVOLVE?  This study will be conducted at the Oak Street Campus of UBC at the Child and Family Research Institute (CFRI). If you agree to participate in this study, then you will be asked to complete the study procedures described below. You may participate in 6 separate study days and 1 pre-study visit. Preliminary Study Day Procedures:    The preliminary assessment is done to collect basic information about you, make sure you are informed about the study details, and to collect information about you to design the study diet specifically to meet your body needs.   The preliminary assessment will be conducted at the Clinical Research Evaluation Unit (CREU) at the Child & Family Research Institute located in BC Children’s Hospital. You will be asked to come at 8AM after having fasted overnight (10-12h). The whole procedure will take 1 hour to complete.    During the preliminary assessment, a Research Assistant will measure your weight, height, body fat and muscle mass, and resting metabolic rate, which tells us how much energy your body needs. Body muscle will be measured using bioelectrical impedance which measures the passage of a small, safe amount of current (that cannot be felt) between four electrodes on the arms and legs while you lay still for a few minutes. The muscle measurements are completely safe and do not cause any discomfort or harm. Metabolic rate is measured using an indirect calorimetry machine, which consists of a clear hood that is placed over your head while you lay on a bed, breathing normally. You can see everything through the hood and breathe normally without any discomfort. This measurement takes about 20 minutes to complete. 127   You will also be asked health related questions to assess your medical history. Study Day Procedures:    If your child agrees to participate in this study then the following procedures will be followed:   The study will be conducted in the Clinical Research Evaluation Unit (CREU) at the Child & Family Research Institute located in BC Children’s Hospital.  You will be asked to come at 8AM after an overnight fast of about 12 hours.   Only water may be consumed prior to arriving on the study day, and during the study day. The study day test diet as described below will provide your child’s daily energy and nutritional needs. At the end of the study day, your child’s is free to consume his/her normal food intake.    A Research Assistant will measure your child’s weight and height.    You/ your child’s will eat the test liquid diet as eight small hourly meals on the study day. Each meal is made up of 1) a mixture of amino acids, 2) an amino acid-free flavored liquid and amino acid-free cookies that provide energy and other nutrients, and 3) the labeled amino acid is added to the last four meals. You/ your child’s will consume an oral dose of 2.5 mg/kg of L-[1-13C] lysine.HCL with the fifth meal (stable isotope – which is very safe to eat). Eight hourly doses of L-[1-13C] lysine (1.4 mg/kg/h) will be given orally mixed with meals until the end of the study. The test meals will meet all your/ your child’s daily energy, vitamin and mineral needs.   To measure how your body responds to the test diet we will collect your breath sample 9 times and urine 6 times during the study day. To collect breath you will have to breathe into a container – just like blowing through a straw into a bag. To collect urine you will have to pass urine into a urine sample hat in the privacy of the washroom.    The Research Assistant will also measure the rate at which you are breathing out carbon dioxide (VCO2) using an indirect calorimetry machine. This will require you to relax on the examination table while a clear hood/canopy is placed on your face and head. This has room air freely flowing in and out. You can watch television/DVD player during the testing. The entire test will take about 20-30 minutes.   You are invited to participate in 6 study days separated by at least 1 week (7 days) so the total study time will be at least 6 weeks.   In total, you can expect to dedicate approximately 8 hours per study day. You/ your child’s invited to participate in up to 6 studies. If you choose to participate in all 6 studies, you will be asked to dedicate approximately 48 hours to this project.  9. WHAT ARE THE POSSIBLE HARMS AND SIDE EFFECTS OF PARTICIPATING IN THIS STUDY? 128   The test requires you/ your child’s to consume a small dose of isotope. This is a safe form of isotope and is widely used in medical research even in children and pregnant women. During the indirect calorimetry test you are/ your child’s is requested to lie down for 20-30 minutes with an open hood/canopy over the face and head. Some children may feel uncomfortable during this period. The hood/canopy has outside air freely moving in and out. If you are/ your child’s is uncomfortable, then the test will be stopped immediately. There are no other known risks involved with participating in this research. We recognize that the length of each study day may pose an inconvenience to you.    10.  WHAT ARE THE BENEFITS OF PARTICIPATING IN THIS STUDY?  We will share the results of the study with you, and the Dietitian and Physician responsible for your treatment may use the protein requirement value determined in the study. Apart from that there are no direct benefits by participation in the study. However, we also hope that the information learned from this study can be used in the future to improve dietary protein recommendations of children with PKU.   11.  WHAT HAPPENS IF I DECIDE TO WITHDRAW MY CONSENT TO PARTICIPATE IN THIS STUDY?  Your participation in this research is entirely voluntary. You may withdraw from this study at any time and without providing any reasons for your decision. If you decide to enter the study and then withdraw, there will be no penalty or loss of benefits, if any, to which you are otherwise entitled. If you choose to enter the study and then decide to withdraw at a later time, all data collected about you during the enrolment part of the study will be retained for analysis, after which the study information may be shredded.  12. WHAT WILL THE STUDY COST ME?  Participation in the study will not cost you anything. In appreciation of the time that it takes to complete this study you will receive $100 upon each study day completion to a maximum of $600 for 6 study days. Vehicle parking coupons for the pre-study duration at BC Children’s Hospital will be provided.  13. WILL MY TAKING PART IN THIS STUDY BE KEPT CONFIDENTIAL?  Your confidentiality will be respected.  However, research records and medical records identifying you may be inspected in the presence of the Investigator or his or her designate by representatives of Rare Diseases Foundation and Royal Embassy of Saudi Arabia – Cultural Bureau, Ottawa, Health Canada, and UBC / Children’s and women’s Health Centre of BC Research Ethics Board for the purpose of monitoring the research. No information or records that disclose your identity will be published without your consent, nor will any information or 129  records that disclose your identity be removed or released without your consent unless required by law.   You will be assigned a unique study number as a subject in this study.  Only this number will be used on any research-related information collected about you during the course of this study, so that your identity [i.e. your name or any other information that could identify you] as a subject in this study will be kept confidential.   Information that contains your identity will remain only with the Principal Investigator and/or designate.  The list that matches your name to the unique identifier that is used on your research-related information will not be removed or released without your consent unless required by law.  Your rights to privacy are legally protected by federal and provincial laws that require safeguards to insure that your privacy is respected and also give you the right of access to the information about you that has been provided to the sponsor and, if need be, an opportunity to correct any errors in this information.  Further details about these laws are available on request to your study doctor. Signing this consent form in no way, limits your legal rights against the sponsor, investigators, or anyone else.  14. WHO DO I CONTACT IF I HAVE QUESTIONS OR CONCERNS ABOUT MY/MY CHILD’S RIGHTS AS A SUBJECT DURING THE STUDY?  Your/your child’s rights to privacy are protected and guaranteed by the Child, Family and Community Services Act. This Act lays down the safeguards respecting your/your child’s privacy and also gives you the right of access to the information about you/your child that has been provided to the study, and if needed, you have the chance to correct any errors in the information. Further details about this legislation are available on request. If you have any concerns about your/your child’s rights as a research subject and/or your/your child’s experiences while participating in this study, contact the Research Subject Information Line, in the UBC Office of Research Services at 604-822-8598, if long distance, call the toll free number of the Research Subject Information Line (1-877-822-8598) or e-mail to RSIL@ors.ubc.ca.  130  15.  SUBJECT CONSENT TO PARTICIPATE    My signature on this consent form means that I:   have had this study explained to me and my child, read this form and understand the information concerning this study.  have had sufficient time to consider the information provided, get advice and ask questions if necessary and I/my child have received satisfactory responses to our questions.  understand that all of the information collected will be kept confidential and that the results will only be used for scientific objectives.  understand that my/my child’s participation in this study is voluntary and that I/my child am/is completely free to refuse to participate or to withdraw from this study at any time without giving any reason(s) and my/my child’s decision to withdraw will not change in any way the quality of care that I/my child receive.  understand that signing this consent form in no way limits my/my child’s legal rights against the sponsor, investigators or anyone else.  understand that there is no guarantee that this study will provide any benefits to myself/my child.  understand that if I have any further questions or desire further information I should contact Dr. Rajavel Elango at 778-986-8655 or email: relango@cfri.ubc.ca   understand that the results from the tests will be disclosed to you and treating physicians.   have been told that I will receive a dated and signed copy of this form for my own record.  I, ___________________________________    voluntarily give consent for my and my child’s      (Subject-Parent-Guardian.  Please print your name)     ___________________________________ participation in the research study entitled:             (Please print child’s name)  Application of Stable Isotopes to Determine Protein Requirements in Children with Phenylketonuria (PKU)  Signature of Subject-Parent-Guardian or person legally authorized to give consent       _________________________________________________         ____________________ Relationship to Child (Mother, Father ,legal guardian)                                        Date  _______________________    __          ___________________ Investigator Signature            Printed Name of principal investigator/ Designate                   Date        131  Appendix K  : Subject Adolescent Assent Form – Protein Requirement in PKU   132   SUBJECT INFORMATION AND ADOLESCENT ASSENT FORM (14-18y)  Application of Stable Isotopes to Determine Protein Requirements in Children with Phenylketonuria (PKU)   Principal Investigator:   Dr. Rajavel Elango, PhD       Department of Pediatrics      The University of British Columbia      Telephone: 604-875-2000 x4911                                                                   Email: relango@cfri.ubc.ca  Co-Investigator:   Dr. Sylvia Stockler, MD      Professor and Head, Division of Biochemical Diseases      British Columbia Children's Hospital       Telephone: 604-875-2628  Primary Contact:   Abrar Turki, M.Sc candidate      Department of Human Nutrition, UBC      Child & Family Research Institute      Telephone: 604-875-2000 x4607      Email: aturki@cfri.ca   Emergency Phone Number:  Rajavel Elango 778-986-8655                                                                   Abrar Turki      778-885-8274 available 24 hours per day and seven days per week  Sponsors: Rare Disease Foundation and Royal Embassy of Saudi Arabia – Cultural Bureau, Ottawa   Site:     Oak Street Campus, UBC      Child & Family Research Institute   1. INVITATION  You are being invited to take part in this research study because you have been diagnosed with Phenylketonuria or PKU. We are inviting children who have a known diagnosis of the disease, ranging in age from 5 -18 years, and are currently free from fever or having a cold.    D e p a r t m e n t  o f  P e d i a t r i c s  950 West 28th Avenue, Room 170A Vancouver, BC, V5Z 4H4 Tel: (604) 875-2000 x4911  Fax: (604) 875-3597    133  2. YOUR PARTICIPATION IS VOLUNTARY  Your participation is entirely voluntary, so it is up to you to decide whether or not to take part in the study. Before you decide, it is important for you to understand what the research involves. This consent form will describe the study, why the research is being done, what will happen to you during the study and the possible benefits, risks, and discomforts. If you wish to participate, you will be asked to sign this consent form within 7 days. If you decide to take part in this study, you are still free to withdraw at any time and without giving any reasons for your decision(s). If you do not wish to participate, you do not have to provide any reason for your decision(s) nor will you lose the benefit of any medical care to which you are entitled or are presently receiving. Please take time to read the following information carefully and to discuss it with your family and friends before deciding.    3.  WHO IS CONDUCTING THE STUDY?  The Principal Investigator [Rajavel Elango] has received financial compensation from the sponsor [Rare Disease Foundation and Royal Embassy of Saudi Arabia – Cultural Bureau, Ottawa] for the work required in doing this clinical research and/or for providing advice on the design of the study/travel expenses/etc. Financial compensation to researchers for conducting the research is associated with obligations defined in a signed contractual agreement between the researchers and the sponsor. Researchers must serve the interests of the subject and also abide by their contractual obligations. For some, the payment of financial compensation to the researchers can raise the possibility of a conflict of interest. You are entitled to request any details concerning this compensation from the Principal Investigator.   4. BACKGROUND  Phenylketonuria (PKU) is an inherited inborn error of phenylalanine metabolism caused by a decreased activity or impaired synthesis of the hepatic enzyme called phenylalanine hydroxylase (PAH) or its cofactor tetrahydrobiopterin (BH4), which is important to convert phenylalanine into tyrosine. This leads to accumulation of phenylalanine in the blood. High levels of phenylalanine can cause severe brain or nerve damage unless the child is on a low phenylalanine diet.  Medical nutrition therapy for children with PKU involve a phenylalanine restricted diet with sufficient protein. However, the exact amount of protein is still unknown. There is a need to do a test to determine how much protein a child with PKU requires to ensure adequate growth and development.   5.  WHAT IS THE PURPOSE OF THE STUDY?  The purpose of the study is to determine the exact protein requirements in children with phenylketonuria. We will measure your protein requirements using the IAAO technique, which uses stable isotope (lysine) with a 13C label. (13C is a type of carbon; amino acids are made of 134  mostly 12C, so the 13C can be detected in breath and urine samples with special equipment). The 13C stable isotopes are completely safe. They are present naturally in the air we breathe, water we drink and food we eat. The labeled amino acid oxidation will be measured from breath and urine samples. This will help us to determine how much protein a child with PKU needs to eat in a day.   6.  WHO CAN PARTICIPATE IN THE STUDY?     6 Children (5 -18y) who are diagnosed with PKU, and clinically stable with no current illness.  7. WHO SHOULD NOT PARTICIPATE IN THE STUDY?     Children < 5 years of age and who are diagnosed with PKU, as it may be difficult to take breath samples and perform indirect calorimetry in very young children.  Children diagnosed with PKU, but are currently ill, with a fever, cold, vomiting or diarrhea.  10. WHAT DOES THE STUDY INVOLVE?  This study will be conducted at the Oak Street Campus of UBC at the Child and Family Research Institute (CFRI). If you agree to participate in this study, then you will be asked to complete the study procedures described below. You may participate in 6 separate study days and 1 pre-study visit. Preliminary Study Day Procedures:    The preliminary assessment is done to collect basic information about you, make sure you are informed about the study details, and to collect information about you to design the study diet specifically to meet your body needs.   The preliminary assessment will be conducted at the Clinical Research Evaluation Unit (CREU) at the Child & Family Research Institute located in BC Children’s Hospital. You will be asked to come at 8AM after having fasted overnight (10-12h). The whole procedure will take 1 hour to complete.    During the preliminary assessment, a Research Assistant will measure your weight, height, body fat and muscle mass, and resting metabolic rate, which tell us how much energy your body needs. Body muscle will be measured using bioelectrical impedance which measures the passage of a small, safe amount of current (that cannot be felt) between four electrodes on the arms and legs while you lay still for a few minutes. The muscle measurements are completely safe and do not cause any discomfort or harm. Metabolic rate is measured using an indirect calorimetry machine, which consists of a clear hood that is placed over your head while you lay on a bed, breathing normally. You can see everything through the hood and breathe normally without any discomfort. This measurement takes about 20 minutes to complete. 135    You will also be asked health related questions to assess your medical history.   Study Day Procedures:    If you agree to participate in this study then the following procedures will be followed:   The study will be conducted in the Clinical Research Evaluation Unit (CREU) at the Child & Family Research Institute located in BC Children’s Hospital.  You will be asked to come at 8AM after an overnight fast of about 12 hours.   Only water may be consumed prior to arriving on the study day, and during the study day. The study day test diet as described below will provide your daily energy and nutritional needs. At the end of the study day, you are free to consume his/her normal food intake.    A Research Assistant will measure your weight and height.    You will eat the test liquid diet as eight small hourly meals on the study day. Each meal is made up of 1) a mixture of amino acids, 2) an amino acid-free flavored liquid and amino acid-free cookies that provide energy and other nutrients, and 3) the labeled amino acid is added to the last four meals. You will consume an oral dose of 2.5 mg/kg of L-[1-13C] lysine.HCL with the fifth meal (stable isotope – which is very safe to eat). Eight hourly doses of L-[1-13C] lysine (1.4 mg/kg/h) will be given orally mixed with meals until the end of the study. The test meals will meet all your daily energy, vitamin and mineral needs.   To measure how your body responds to the test diet we will collect your breath sample 9 times and urine 6 times during the study day. To collect breath you will have to breathe into a container – just like blowing through a straw into a bag. To collect urine you will have to pass urine into a urine sample hat in the privacy of the washroom.    The Research Assistant will also measure the rate at which you are breathing out carbon dioxide (VCO2) using an indirect calorimetry machine. This will require you to relax on the examination table while a clear hood/canopy is placed on your face and head. This has room air freely flowing in and out. You can watch television/DVD player during the testing. The entire test will take about 20-30 minutes.   You are invited to participate in 6 study days separated by at least 1 week (7 days) so the total study time will be at least 6 weeks.   In total, you can expect to dedicate approximately 8 hours per study day you participate in. You are invited to participate in up to 6 studies. If you choose to participate in all 6 studies, you will be asked to dedicate approximately 48 hours to this project. 136   9. WHAT ARE THE POSSIBLE HARMS AND SIDE EFFECTS OF PARTICIPATING IN THIS STUDY?  The test requires you to consume a small dose of isotope. This is a safe form of isotope and is widely used in medical research even in children and pregnant women. During the indirect calorimetry test you are requested to lie down for 20-30 minutes with an open hood/canopy over the face and head. Some children may feel uncomfortable during this period. The hood/canopy has outside air freely moving in and out. If you are uncomfortable, then the test will be stopped immediately. There are no other known risks involved with participating in this research. We recognize that the length of each study day may pose an inconvenience to you.    10.  WHAT ARE THE BENEFITS OF PARTICIPATING IN THIS STUDY?  We will share the results of the study with you, and the Dietitian and Physician responsible for your treatment may use the protein requirement value determined in the study. Apart from that there are no direct benefits by participation in the study. However, We hope that the information learned from this study can be used in the future to improve dietary protein recommendations of children with PKU.   11.  WHAT HAPPENS IF I DECIDE TO WITHDRAW MY CONSENT TO PARTICIPATE IN THIS STUDY?  Your participation in this research is entirely voluntary. You may withdraw from this study at any time and without providing any reasons for your decision. If you decide to enter the study and then withdraw, there will be no penalty or loss of benefits, if any, to which you are otherwise entitled. If you choose to enter the study and then decide to withdraw at a later time, all data collected about you during the enrolment part of the study will be retained for analysis, after which the study information may be shredded.  12. WHAT WILL THE STUDY COST ME?  Participation in the study will not cost you anything. In appreciation of the time that it takes to complete this study you will receive $100 upon each study day completion to a maximum of $600 for 6 study days. Vehicle parking coupons for the pre-study duration at BC Children’s Hospital will be provided.  13. WILL MY TAKING PART IN THIS STUDY BE KEPT CONFIDENTIAL?  Your confidentiality will be respected.  However, research records and medical records identifying you may be inspected in the presence of the Investigator or his or her designate by representatives of Rare Diseases Foundation and Royal Embassy of Saudi Arabia – Cultural Bureau, Ottawa, Health Canada, and UBC / Children’s and women’s Health Centre of BC Research Ethics Board for the purpose of monitoring the research. No information or records that 137  disclose your identity will be published without your consent, nor will any information or records that disclose your identity be removed or released without your consent unless required by law.   You will be assigned a unique study number as a subject in this study.  Only this number will be used on any research-related information collected about you during the course of this study, so that your identity [i.e. your name or any other information that could identify you] as a subject in this study will be kept confidential.   Information that contains your identity will remain only with the Principal Investigator and/or designate.  The list that matches your name to the unique identifier that is used on your research-related information will not be removed or released without your consent unless required by law.  Your rights to privacy are legally protected by federal and provincial laws that require safeguards to insure that your privacy is respected and also give you the right of access to the information about you that has been provided to the sponsor and, if need be, an opportunity to correct any errors in this information.  Further details about these laws are available on request to your study doctor. Signing this consent form in no way, limits your legal rights against the sponsor, investigators, or anyone else.  14. WHO DO I CONTACT IF I HAVE QUESTIONS OR CONCERNS ABOUT MY RIGHTS AS A SUBJECT DURING THE STUDY?  Your rights to privacy are protected and guaranteed by the Child, Family and Community Services Act. This Act lays down the safeguards respecting your privacy and also gives you the right of access to the information about you that has been provided to the study, and if needed, you have the chance to correct any errors in the information. Further details about this legislation are available on request. If you have any concerns about your rights as a research subject and/or your experiences while participating in this study, contact the Research Subject Information Line, in the UBC Office of Research Services at 604-822-8598, if long distance, call the toll free number of the Research Subject Information Line (1-877-822-8598) or e-mail to RSIL@ors.ubc.ca.  138  15.  SUBJECT CONSENT TO PARTICIPATE    My signature on this consent form means that I:   have had this study explained to me, read this form and understand the information concerning this study.  have had sufficient time to consider the information provided, get advice and ask questions if necessary and I have received satisfactory responses to our questions.  understand that all of the information collected will be kept confidential and that the results will only be used for scientific objectives.  understand that my participation in this study is voluntary and that I am completely free to refuse to participate or to withdraw from this study at any time without giving any reason(s) and my decision to withdraw will not change in any way the quality of care that I receive.  understand that signing this consent form in no way limits my legal rights against the sponsor, investigators or anyone else.  understand that there is no guarantee that this study will provide any benefits to myself.  understand that if I have any further questions or desire further information I should contact Dr. Rajavel Elango at 778-986-8655 or email: relango@cfri.ubc.ca   understand that the results from the tests will be disclosed to you and treating physicians.   have been told that I will receive a dated and signed copy of this form for my own record.  I, ___________________________________    voluntarily give consent for my participation in                      (Please print your name) the research study entitled:  Application of Stable Isotopes to Determine Protein Requirements in Children with Phenylketonuria (PKU)  Signature of Subject-Parent-Guardian or person legally authorized to give consent       _________________________________________________         ____________________ Signature of individual                                                                              Date  _______________________    __          ___________________ Investigator Signature            Printed Name of principal investigator/ Designate                   Date          139  Appendix L  : Subject Assent Form – Protein Requirement in PKU   140   SUBJECT ASSENT FORM (7-13 years of age)  Application of Stable Isotopes to Determine Protein Requirements in Children with Phenylketonuria (PKU)  1. INVITATION I am being invited to be part of a research study. The research study tries to find the exact amount of protein to be eaten to help children like me grow well. It is up to me if I want to be in this study. No one will make me be part of the study. Even if I agree now to be part of the study, I can change my mind later. No one will be mad at me if I choose not to be part of this study.  2.   WHY ARE WE DOING THIS STUDY I have a disease called phenylketonuria. This disease affects many other children. This study is trying to find out how much protein I need to eat in a day so that I can better manage my condition, and grow well.  3.  WHAT WILL HAPPEN IN THIS STUDY?  I will have to come to the hospital without eating anything for 12 hours    I will have to come 6 times, including 1 time before I start the study.    The whole test time will take about 8 hours.   A Research Assistant will measure my weight and height.    I will be requested to drink a test liquid (1 small cup eight times during a day). The liquid drink will taste a bit sweet and a bit sour because it has pure protein powders and orange tang added in. Small cookies will also be given with each liquid drink, which are regular cookies. Then I will breathe into a container several times like blowing through a straw into a bag. When samples are not being collected, I can watch television, listen to music, read or use a computer.   Urine samples will be collected in urine hate.   How much muscle I have will be measured. The whole test will only take 30 seconds – 1 minute. While I lie down on the bed two wires will be put on my right hand and foot, like band-aids. I will not feel anything during this test.     While I lie down on the bed a clear hood (like a space-man’s suit) will be placed on my head. This is a clear hood with air freely coming in and out. I can watch television during the testing. This test will take about 20 minutes. If I want to stop the test  D e p a r t m e n t  o f  P e d i a t r i c s  950 West 28th Avenue, Room 170A Vancouver, BC, V5Z 4H4 Tel: (604) 875-2000 x4911  Fax: (604) 875-3597     141  anytime, then I can just pull the hood away. This test will measure how much carbon dioxide I produce.   My Parents can stay with me in the Clinical Research Evaluation Unit (CREU) during the study.  4. WHO IS DOING THIS STUDY? Dr. Rajavel Elango and his research assistant Abrar Turki will be doing this study. Dr. Stockler (Head, Division of Biochemical Diseases) is also involved. They will answer any questions I have about the study. I can also call them at 778-986-8655 (Dr. Elango) or at 778-885-8274 (Abrar Turki) or at 604-875-2628 (Dr. Stockler), if I am having any problems or if there is an emergency and I cannot talk to my parents.  5.  CAN ANYTHING BAD HAPPEN TO ME? There is nothing in this study, which will make anything bad happen to me.  6. CAN I GET BETTER BY BEING ON THIS STUDY? This study is trying to find out how much protein I need to eat in a day so that I can better manage my condition, and grow well.  7.  WHAT SHOULD I DO IF I AM NOT FEELING WELL?   I can simply pull the hood away when the test is being done, or call the research assistant Abrar Turki who will be standing next to me.  8.  WHO WILL KNOW I AM IN THE STUDY? Only my doctors and people who are involved in the study will know I am in it.  When the study is finished, the doctors will write a report about what was learned.  This report will not say my name or that I was in the study. My parents and I do not have to tell anyone I am in the study if we don’t want to.  9. WHEN DO I HAVE TO DECIDE? I have 7 days from today to decide if I want to be part of the study. When I come in for my next visit to the clinic at BC Children’s Hospital I can be part of the study, if I want to. I have also been asked to discuss my decision with my parents.   10.  SIGNATURES   If I put my name at the end of this form, it means that I agree to be in the study.   ___________________         __          ___________________   Printed Name    Signature     Date      142  Appendix M  : Pre – Study Day Questionnaire – Protein Requirement in PKU   143  Pre-Study Day Assessment Protein Requirements in Children with Phenylketonuria  Principal Investigator: Dr. Rajavel Elango  604-875-2000 ext. 4911 (office) Student Investigator: Abrar Turki               604-875-2000 ext. 4607(office)        778-885-8274 (cell)   PRELIMINARY ASSESSMENT  Subject ID:________________________________        Date:___________________________  Birthday:               ______/________                                    Age:___________      Gender:____                     (month)   (year)           Height (cm):______________         Weight (kg):___________  BMI:_________________       Bioelectrical Impedance Analysis  BIA:R_____________________________   XC______________________________ (resistance)      (reactance)  BIA: Z_____________________________    (Impedance)    Body Composition Profile  % Body fat (BIA):______________________ Lean body mass (BIA):_______________________   Indirect Calorimetry  Measured REE (kcal/day):_________________     Estimated REE (kcal/day):__________________  Daily energy requirement (kcal/day):_________________________  VCO2 (ml/min): _________________     VO2 (ml/min):____________________    RQ:  _________________     144  Medical History  Details of health condition(s) _____________________________________________________________  Are you currently taking any prescription medications?  Yes_______ No_________  List of medications:_____________________________________________________________________  Are you currently having vomiting episodes?  Yes______ How many/day __       No______  Are you currently having fever/cold?  Yes______ No______   Nutritional Supplement Intake  Are you currently taking vitamins? Yes_______ No_______  Are you taking any other nutritional supplements? Yes_______ No________  If yes, please list all nutritional supplements:  1.__________________________________ 3.__________________________________  2.__________________________________ 4.__________________________________   Activity Level  Daily exercise (minutes)________________________  Sedentary_____________  Moderate_________________ High__________________   Availability for 6 studies  Yes___________ No______________   Comments:       145  Appendix N  : Advertisement – Protein Requirement in PKU   146       147  Appendix O  : Subject Code Master List – Protein Requirement in PKU   148   Subject Code Master List  Application of Stable Isotopes to Determine Protein Requirements in Children with Phenylketonuria (PKU)  Subject Name Code (Alpha- Numeric)  Comments                                                                                                                               149   Subject Code Master List  Application of Stable Isotopes to Determine Protein Requirements in Children with Phenylketonuria (PKU)  Subject Name Code (Alpha- Numeric)  Comments                                                                      150  Appendix P  : Dietary Record Sheets – Protein Requirement in PKU151  Dietary Record           Subject ID:__________________________  Date: ____/_____/_____        Mon   Tues    Wed    Thu   Fri    Sat    Sun           Item            Amount                 Item              Amount  Breakfast   Snack             Dinner   Snack             Lunch              152  Appendix Q  : Study Day Form – Protein Requirement in PKU   153  Study Day Protocol Protein Requirements in Children with Phenylketonuria   Subject ID:_______________________   Date:___________________________  Height (cm):_______________                                                Weight (kg):_______________      Protein intake (g/kg/d): ____________________  Energy intake (kcal/day): _______________   Time Sample Collection/ Anthropometry Meals and isotope tracer Comments 8:00  Meal #1  9:00  Meal #2  10:00  Meal #3  11:00  Meal #4  11:15 1st breath (3x) 1st urine   11:30 2nd breath (3x)   11:45 3rd breath (3x) 2nd urine   12:00 VCO2 measurement  Meal #5 – primer dose and  1st oral dose   13:00  Meal #6 – 2nd oral dose  14:00  Meal #7 – 3rd oral dose  14:30 4th breath (3x) 3rd urine   15:00 5th breath (3x) 4th urine Meal #8 – 4th oral dose  15.15 6th breath (3x)   15:30 7th breath (3x) 5th urine   15:45 8th breath (3x)    16:00 9th breath (3x) 6th urine      

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.24.1-0167276/manifest

Comment

Related Items