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Effect of medical foods used in dietary management of subjects with propionic acidemia (PROP) Saleemani, Haneen 2020

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 EFFECT OF MEDICAL FOODS USED IN DIETARY MANAGEMENT OF SUBJECTS WITH PROPIONIC ACIDEMIA (PROP)  by  Haneen Saleemani  B.Sc.,King Abdulaziz University, 2013  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)  January 2020 © Haneen Saleemani, 2020  ii  The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, a thesis entitled:  Effect of Medical Foods Used in Dietary Management of Subjects with Propionic Acidemia (PROP)  submitted by Haneen Saleemani in partial fulfillment of the requirements for the degree of Master of Science in Human Nutrition   Examining Committee: Dr. Rajavel Elango Supervisor  Dr. Sylvia Stockler Supervisory Committee Member  Dr. Gabriella Horvath Supervisory Committee Member Dr. Angela Devlin Additional Examiner   Additional Supervisory Committee Members: Dr. Crystal Karakochuk Supervisory Committee Member   iii  Abstract Introduction: Propionic Acidemia (PROP) is an inborn errors of metabolism disorder, caused by a defect in the enzyme propionyl-CoA carboxylase (PCC). PCC catalyzes two of the branched-chain amino acids (BCAA), valine, isoleucine. The management of PROP depends on dietary protein restriction and medical food consumption. Recently, concerns have been raised about medical foods due to imbalanced content of BCAA (high leucine – another BCAA, and no valine/isoleucine). It has been suggested that this imbalanced mixture of BCAA negatively impacts plasma concentrations of valine and isoleucine, and therefore growth in children with PROP. Studies on long-term growth outcomes in subjects with PROP are limited. Thus, a comprehensive assessment of dietary intake and its impact on growth in children with PROP is needed. Furthermore, since subjects with PROP depend on medical foods as an easily tolerable source of energy and protein, there is a need to determine the optimal BCAA ratio in medical foods to optimize protein synthesis and growth.  Methods & Results: A retrospective chart review was conducted on four subjects with PROP; longitudinal data on dietary intake and growth outcomes for 1999-2018 were collected. Results suggest that subjects had persistently low height Z scores, despite consuming protein intakes higher than guidelines. However, the high consumption of medical foods protein relative to intact protein impacted growth. A prospective study to test different BCAA (LEU: ILE: VAL) ratios was conducted using the indicator amino acid oxidation method. Eight healthy children participated at 7 different test intakes with the use of L-1-13C-Phenylalanine oxidation to 13CO2 as a marker of protein synthesis. ANOVA showed significant differences with different test  iv  intakes, with a ratio between 1: 0.26: 0.28 and 1:0.35:0.4 observed to be associated with optimal protein synthesis. Conclusion: Our results indicate that intact protein restriction together with overusing medical foods could have affected growth in children with PROP. Currently used medical foods are formulated to provide an imbalanced BCAA (1:0:0), which was associated with the highest oxidation rates (indicating low protein synthesis). Future studies should examine the effect of BCAA ratios between (1:0.26:0.28) and (1:0.35:0.4) in subjects with PROP to measure adequacy for protein synthesis.        v  Lay Summary Propionic Acidemia (PROP) is a genetic disorder in which the body is unable to process certain amino acids in protein rich foods. In order to provide a safe alternative to the regular diet for children with PROP, special medical foods have been developed. Recently, medical food consumption was reported to cause growth failure in children. In the first stage of this study, we reviewed dietary and growth charts of four children with PROP at BC Children’s Hospital. We found that children were consuming high amounts of medical foods and had poor growth outcomes. In the second stage, we showed that the currently used medical foods for PROP limit total body protein synthesis, and therefore restrict growth. To ensure normal growth for children with PROP we propose: 1) Reformulating the amino acid mixture in the medical foods; 2) Shifting the practice from using medical foods as the primary source of nutrition.   vi  Preface  I have written this thesis under the supervision of Dr. Rajavel Elango. My committee members, Drs. Sylvia Stockler, Gabriella Horvath and Crystal Karakochuk also provided substantial input regarding study design and methodology.  The current thesis consists of two studies which were approved by the University of British Columbia and BC Children's Hospital Research Ethics Board (H19-02912) and (H18-00439). In the first study, Dr. Gabriella Horvath and the dietitians in the Biochemical Disease Clinic at BC Children’s Hospital helped with the data collection. In the second study, all procedures including pre-study assessments, test diet preparation, sample collection and statistical analyses were done by me with the help of Dr. Rajavel Elango. Breath sample analysis (isotope ratio mass spectrometer (IRMS, Isoprime Ltd, Cheadle, UK) and urine sample analysis using the Amino Acid Analyzer (AAA) (Hitachi L8900, Tokyo, Japan) and HPLC (Chromaster 5430 Diode Array Detector, Hitachi, Tokyo, Japan) were done by Katia Caballero, Madeleine Ennis, Betina Rasmussen (researchers within the Elango Lab) and myself.       vii  Table of Contents  Abstract ......................................................................................................................................... iii Lay Summary .................................................................................................................................v Preface ........................................................................................................................................... vi Table of Contents ........................................................................................................................ vii List of Tables ................................................................................................................................ xi List of Figures .............................................................................................................................. xii List of Abbreviations ................................................................................................................. xiii Acknowledgements .................................................................................................................... xvi Dedication .................................................................................................................................. xvii Chapter 1: Introduction ................................................................................................................1 1.1 Introduction and Overview ............................................................................................. 1 Chapter 2: Background .................................................................................................................3 2.1 Inborn Errors of Metabolism .......................................................................................... 3 2.2 Propionic Acidemia ........................................................................................................ 5 2.3 Prevalence of PROP ........................................................................................................ 7 2.4 Overall Management of PROP ....................................................................................... 8 2.4.1 Nutritional Management of PROP ........................................................................ 10 2.4.1.1 Energy Requirements in PROP ......................................................................... 13 2.4.1.2 Protein and Amino Acid Requirements in PROP ............................................. 16 2.4.1.3 Medical Foods for PROP .................................................................................. 19  viii  2.5 Growth Outcomes in PROP .......................................................................................... 22 2.6 Branched-Chain Amino Acids ...................................................................................... 26 2.6.1 Branched-Chain Amino Acid Requirements in Humans ...................................... 28 2.6.2 Importance of Branched-Chain Amino Acid Ratio .............................................. 30 2.7 Indicator Amino Acid Oxidation (IAAO) Technique to Measure Total Body Protein Synthesis ................................................................................................................................... 32 2.7.1 Choice of Indicator Amino Acid in PROP ........................................................... 33 Chapter 3: Rationale, Objective and Hypothesis ......................................................................34 3.1 Rationale ....................................................................................................................... 34 3.2 Objectives ..................................................................................................................... 35 3.3 Hypothesis..................................................................................................................... 36 Chapter 4: Natural History Study on Subjects with Propionic Acidemia ..............................37 4.1 Subjects and Methods ................................................................................................... 37 4.1.1 Data Collection ..................................................................................................... 38 4.1.1.1 Growth Data ...................................................................................................... 38 4.1.1.2 Dietary Data ...................................................................................................... 38 4.1.2 Statistical Analysis ................................................................................................ 39 4.2 Results ........................................................................................................................... 40 4.2.1 Subject characteristics ........................................................................................... 40 4.2.2 Growth Data .......................................................................................................... 42 4.2.3 Dietary Data .......................................................................................................... 48 4.3 Discussion ..................................................................................................................... 58  ix  4.4 Limitations .................................................................................................................... 63 Chapter 5: Determining Ideal Balance among BCAA as a Proof of Concept Study in Healthy Children ..........................................................................................................................64 5.1 Methods and Materials .................................................................................................. 65 5.1.1 Study Principle ...................................................................................................... 65 5.1.2 Subjects ................................................................................................................. 65 5.1.2.1 Recruitment ....................................................................................................... 66 5.1.2.2 Inclusion and Exclusion Criteria ....................................................................... 66 5.1.3 Experimental Design ............................................................................................. 67 5.1.3.1 Pre-Study Day Protocol .................................................................................... 69 5.1.3.2 Study Day Protocol ........................................................................................... 70 5.1.3.3 Stable Isotope Protocol ..................................................................................... 72 5.1.3.4 Sample Collection ............................................................................................. 73 5.1.4 Sample Analysis .................................................................................................... 75 5.1.5 Data Calculations .................................................................................................. 75 5.1.6 Statistical Analysis ................................................................................................ 76 5.2 Results ........................................................................................................................... 76 5.2.1 Subject Characteristics .......................................................................................... 76 5.2.2 F13CO2 Oxidation .................................................................................................. 78 5.2.3 Urine BCAA Concentrations ................................................................................ 80 5.3 Discussion ..................................................................................................................... 82 5.3.1 Limitations ............................................................................................................ 87  x  Chapter 6: Conclusions and Future Directions ........................................................................88 Bibliography .................................................................................................................................90 Appendices ....................................................................................................................................98 Appendix A : Subject Consent Form ........................................................................................ 98 Appendix B : Subject Assent Form ........................................................................................ 107 Appendix C : Recruitment Poster ........................................................................................... 110 Appendix D : Pre-Study Day Assessment Form ..................................................................... 111 Appendix E : Study Day Form ................................................................................................ 113 Appendix F : Subject Code Form ........................................................................................... 114 Appendix G : Dietary Record Sheets ...................................................................................... 115 Appendix H : WHO Growth Charts for Canada (PROP-01 Weight for Age >10 years) ....... 116 Appendix I : WHO Growth Charts for Canada (PROP-02 Weight for Age >10 years) ......... 117 Appendix J : WHO Growth Charts for Canada (PROP-03 Weight for Age >10 years) ........ 118   xi  List of Tables   Table 2-1 Incidence Rate of PROP by Population .......................................................................... 8 Table 2-2 Dietary Reference Intakes for Energy (DRI 0-36 months) .......................................... 11 Table 2-3 Dietary Reference Intakes for Energy (DRI 3-18 years) .............................................. 12 Table 2-4 Dietary Reference Intakes for Protein (DRI) ................................................................ 13 Table 2-5 Recommended Energy Intakes for Well Individuals with PROP1 ............................... 15 Table 2-6 Recommended Protein Intakes for Subjects with PROP .............................................. 18 Table 2-9 BCAA Content in Regular milk, Eggs and Common Medical Foods for PROP ......... 21 Table 2-10: Review of Evidence for Long-term Dietary Management and Growth Outcomes in PROP ............................................................................................................................................ 23 Table 2-11 Dietary Reference Intakes for Branched-Chain Amino Acid Requirements (DRI) ... 29 Table 4-1 Subjects with PROP Characteristics ............................................................................. 41 Table 4-2 Subjects with PROP Growth Z Scores ......................................................................... 43 Table 4-3 Subjects with PROP Dietary Intakes ............................................................................ 50 Table 4-4 Protein to Energy Ratio for Subjects with PROP1 ........................................................ 57 Table 5-1 Subject Characteristics ................................................................................................. 77   xii  List of Figures Figure 2-1 Disorders of Intoxication ............................................................................................... 4 Figure 2-2 Metabolic Pathway of Propionic Acidemia .................................................................. 6 Figure 2-3 Catabolism of Branched-Chain Amino Acids ............................................................. 28 Figure 4-1 Growth Data Z scores for PROP-01 ............................................................................ 44 Figure 4-2 Growth Data Z Scores for PROP-02 ........................................................................... 45 Figure 4-3 Growth Data Z Scores for PROP-03 ........................................................................... 46 Figure 4-4 Growth Data Z Scores for PROP-04 ........................................................................... 47 Figure 4-5 Energy Intake for Subjects with PROP Compared with 80-120% of RDA ................ 52 Figure 4-6 A Comparison Among Different Guidelines for Total Protein Intakes for Subjects with PROP .................................................................................................................................... 52 Figure 4-7 PROP-01 Subject Protein Intakes ............................................................................... 53 Figure 4-8 PROP-02 Subject Protein Intakes ............................................................................... 54 Figure 4-9 PROP-03 Subject Protein Intakes ............................................................................... 55 Figure 4-10 PROP-04 Subject Protein Intakes ............................................................................. 56 Figure 5-1 Experimental Design ................................................................................................... 68 Figure 5-2 Study Day Protocol ..................................................................................................... 71 Figure 5-3 F13Co2 Oxidation Rate at Different Test Intakes ......................................................... 79 Figure 5-4 Urinary Amino Acids Concentrations ......................................................................... 81      xiii  List of Abbreviations AAA- Amino Acid Analyzer  AI- Adequate Intake ANOVA- Analysis of Variance  APE- Atom Percent Excess BCAA- Branched-Chain Amino Acids  BCKA- Branched-Chain Keto Acids  BCKD- Branched-Chain Keto Dehydrogenase  BIA- Bioelectrical Impedance Analysis  BMI- Body Mass Index DRI- Dietary Reference Intakes ECO2-13CO2 Isotopic Enrichment (APE) F13CO2 -Rate of L-[1-13C] Phenylalanine or Leucine Tracer Oxidation  EER- Estimated Energy Requirements  FAO -Food and Agriculture Organization FCO2 -CO2 Production Rate Using Indirect Calorimetry FFM-Fat Free Mass FM- Fat Mass GMDI- Genetic Metabolic Dietitians International  HT- Height IAAO- Indicator Amino Acid Oxidations  xiv  IB-CoA- Isobutyryl-CoA IEM- Inborn Error of Metabolism  ILE- Isoleucine IRMS- Isotope Ratio Mass Spectrometry IV-CoA- Isovaleryl-CoA KIC- ketoisocaproic Acid KIV- keto- Isovalerate KMV- keto-Methylvalerate LEU- Leucine MB-CoA- a- methylbutyryl-CoA MET- Methionine NBS- Newborn Screening  OMIM- Online Mendelian Inheritance in Man PCC- Propionyl-CoA Carboxylase  PFD- Protein Free Powder PHE- Phenylalanine  PKU- Phenylketonuria PROP- Propionic Acidemia  RDA- Recommended Dietary Allowance  SD- Standard Deviation TCA- Tricarboxylic Acid Cycle  TEE- Total Energy Expenditure    xv  THR-Threonine  VAL- Valine VCO2 - Rate of Carbon Dioxide Production WHO- World Health Organization  WT- Weight     xvi  Acknowledgements I would like to express my very great appreciation and gratitude to my advisor Dr. Rajavel Elango, who has been a great mentor through my studies. I thank him for the ongoing guidance, encouragement and the kind advice he has provided throughout my time in his lab. I would also like to thank my committee members, Dr. Sylvia Stockler, Dr. Gabriella Horvath and Dr. Crystal Karakochuk, for their time and support during my research. I would like to acknowledge the physicians and dietitians in the Biochemical Diseases Clinic at both BC Children’s Hospital and Vancouver General Hospital for all their guidance and contributions to my research. Very special thanks to all my wonderful participants and their families, without whom this research could not have been completed. I would like to specially thank my colleagues in the Elango lab: Katia Caballero, Madeleine Ennis, Abrar Turki, Zoe Schmidt, Betina Rasmussen, Maggie Kinshella, and Kerri Scherbinsky for their help, constructive suggestions, and all the inspirational coffee breaks. I would also like to express my gratitude to King Abdulaziz University and the Saudi Arabian Cultural Bureau for their generous funding throughout my study.  Finally, special thanks are owed to my family, who have supported me throughout my years of education, both morally and financially. No words can express how grateful I am to my parents for all their love and emotional support.      xvii  Dedication  This thesis is dedicated to My wonderful parents, Rabab & Hassan who have raised me to be the person I am today My brothers, Rami & Hazem My sisters Rasha, Futoon & Yara My friends All who have supported me through this journey    1  Chapter 1: Introduction  1.1 Introduction and Overview Propionic Acidemia (PROP) is an inherited metabolic disorder, caused by a defect in the mitochondrial enzyme propionyl-CoA carboxylase (PCC). PCC converts propionyl-CoA to methylmalonyl-CoA, an intermediate in the catabolism of isoleucine (ILE) and valine (VAL), two of the three branched-chain amino acids (BCAA include leucine, isoleucine and valine), as well as threonine (THR), methionine (MET), and odd-chain fatty acids.  A block in the catabolic pathway of these compounds leads to an accumulation of excess propionic acid metabolites that can disrupt the tricarboxylic acid cycle (TCA), as well as the urea cycle, causing hyperammonemia (Baumgartner et al., 2014). PROP is considered an ultra-rare disorder, with similar rates across all regions estimated to be 1 in 100,000, except for regions in the Middle East, where most inherited metabolic disorders are more frequent (Almási et al., 2019). The goal of nutritional management of PROP is to reduce the accumulation of toxic metabolites by restricting dietary protein sources of the propiogenic amino acids (ILE, VAL, MET, and THR), while maintaining their normal plasma concentrations (Jurecki et al., 2019). In addition, subjects with PROP are usually supplemented with special medical foods that are formulated to contain no propiogenic precursors (ILE, VAL, MET, and THR) and normal to high amounts of other amino acids to ensure sufficient protein intake for optimal growth (Manoli et al., 2016). Specifically, medical foods for PROP are formulated to contain minimal or no valine and isoleucine, and high amounts of leucine. Due to the imbalanced content of the three BCAA in these formulations, there have recently been arguments against their use (Manoli et al., 2016;  2  Myles et al., 2018). High leucine intakes can negatively impact the other two BCAA (isoleucine and valine), by suppressing their plasma concentrations below normal ranges, limiting total protein synthesis and restricting growth (Harper et al., 1984). Studies on the long-term growth outcomes of individuals with PROP are limited. Thus, the objective of the first study in this thesis was to describe dietary therapeutic practices and growth outcomes in the management of subjects with PROP through a natural history. Recent dietary guidelines for PROP were released in 2019 and the recommendation is to use medical foods as supplementation only in subjects who tolerate less than 100% RDA from intact protein (Jurecki et al., 2019). However, most subjects with PROP are at risk for malnutrition and depend on medical foods as an easily tolerable source of energy and protein (Daly et al., 2017). Therefore, there is a need to determine the optimal ratio of BCAA in medical foods to optimize protein synthesis and growth, and to prevent the accumulation of toxic metabolites. The objective of the second study in this thesis was to determine a BCAA ratio at which total body protein synthesis is optimized using a stable isotope-based method: the indicator amino acids oxidation method (IAAO).     3  Chapter 2: Background 2.1 Inborn Errors of Metabolism Inborn errors of metabolism (IEMs) are a large, diverse group of disorders caused by a gene mutation that can result in total or partial impairment of a certain enzyme or cofactor. A greater number of IEMs are inherited in an autosomal recessive manner, due to a defect in single or multiple enzymes (Baker, 2015). As a result of this defect, a block in the metabolic pathway of some compounds may cause an accumulation of toxic metabolites. This accumulation can cause a wide range of symptoms in one or multiple organs, including neurological problems, disabilities, and even death in some cases. Most IEMs symptoms often start at or soon after birth, but also may appear at any time during adulthood (Nasser et al., 2012). The basic principle of management is to reduce the plasma and tissue accumulations of these toxic metabolites, either by limiting the intake of nutrients that produces them, or by increasing their excretion from the body (Boyer et al., 2015). Considering that IEMs are extremely diverse, it is challenging to classify them. A single universal classification system for IEMs does not exist. However, there are approaches used to classify IEMs according to different criteria (Lanpher et al., 2006). Pathophysiologically, IEMs are divided into three groups. The first group includes IEMs that occur due to defects in the intermediary metabolic pathways, which result in accumulation of toxic compounds; these are mostly referred to as disorders of intoxication (Figure 2-1). The second group includes IEMs that result in an energy deficiency (disorders of energy metabolism). The third group comprises IEMs caused by defects in the synthesis or catabolism of complex molecules in certain cellular organelles (El-Hattab, 2015; Jameson and Morris, 2011). IEMs can also be classified as treatable  4  verses untreatable inborn metabolic diseases. Whereas most of the intoxication disorders are treatable, they require immediate removal of the accumulated toxins by extracorporeal procedures, cleansing drugs or vitamins (Saudubray et al., 2006), and lifelong medical and dietary management.  Figure 2-1 Disorders of Intoxication  Adapted from (Jameson and Morris, 2011)             Disorders of IntoxicationAmino Acid DisordersMaple Syrup Urine Disease (MSUD)HomocystinuriaPhenylketonuria (PKU)Organic Acidemias Propionic & Methylmalonic Acidemias (PROP/MMA)Urea Cycle DisorderCarbohydrates Disorders 5  2.2 Propionic Acidemia  Propionic Acidemia (PROP) (Online Mendelian Inheritance in Man (OMIM) number #606054) is an autosomal recessive, inherited metabolic disorder that is serious and life-threatening. It is one of the most common forms of organic acidemias. PROP is caused by a defect in the mitochondrial enzyme propionyl-coenzyme A (CoA) carboxylase (PCC) (EC 6.4.1.3), which results in the accumulation of toxic metabolites such as propionic acid and methylcitrate. PCC catalyzes the reversible biotin-dependent conversion of propionyl-CoA to D- methylmalonyl-CoA. Propionyl CoA is an intermediate in the catabolism of isoleucine (ILE) and valine (VAL), two of the three branched-chain amino acids (BCAA include leucine, isoleucine and valine), as well as threonine (THR), methionine (MET), and odd-chain fatty acids. When the catabolic pathway of the propiogenic amino acids is blocked by the loss of the catalytic activity of PCC, the excess propionic acids will accumulate and disrupt the tricarboxylic acid cycle (TCA), as well as the urea cycle, causing hyperammonemia (Figure 2-2). The defect in the PCC enzyme can occur due to a deficiency in the enzyme, or its cofactor biotin, as a result of mutations in either the PCCA or PCCB gene (Almási et al., 2019; Baumgartner et al., 2014; Jurecki et al., 2019; Shchelochkov et al., 1993).  The onset of the disease varies depending on the disorder phenotypes. Early neonatal onset occurs frequently and can be severe with high mortality rates. The first days of life are characterized by hypotonia, lethargy, vomiting and poor feeding. If left untreated, it can progress to encephalopathy and death. While late onset PROP is usually milder, it can present with a variety of symptoms including developmental delay, intellectual disability, failure to thrive, movement disorders and protein intolerance (Shchelochkov et al., 1993). Prognosis of PROP is  6  poor, and severe cases may result in death in the newborn period or later, due to metabolic decompensations. Newborn screening (NBS) is used for early diagnosis, and can be an effective approach in reducing overall mortality rate. NBS can identify cases of PROP before it shows any symptoms, which allows for treatment to prevent metabolic decompensation (Heringer et al., 2016). In addition to NBS, another means of diagnosing PROP is to use acylcarnitine analysis by tandem mass spectrometry (MS/MS) on dried blood spots, which will report high levels of propionylcarnitine (C3) (Almási et al., 2019).  Figure 2-2 Metabolic Pathway of Propionic Acidemia  Adapted from (Jurecki et al., 2019) TCA- Tricarboxylic Acid Cycle    7  2.3 Prevalence of PROP Although individual IEM disorders are rare, collectively they represent a large and diverse group of genetic diseases, with new disorders and disease mechanisms being described regularly (Vernon, 2015). Their cumulative incidence is believed to be high, with very high mortality rates if not diagnosed early. The global birth prevalence is 50.9 per 100,1000 live births (Waters et al., 2018). However, in some populations, where consanguinity is very common, the incidence rate can be much higher. For instance, in Saudi Arabia the overall incidence is 1:1043 births (Alfadhel et al., 2016). Moreover, in a 13-year retrospective cohort study in Saudi Arabia 2001-2014, researchers reported an IEM incidence rate of (1:591). Among the cases, 64.7% were small molecule IEMs, where organic acidemia was the second most common category, with PROP as the most common in that group. This rate is the highest reported so far (Mak et al., 2013). Combining these results with another epidemiological study done in Saudi Arabia by Moammar et al., an IEM incidence rate of (1:667) was reported (Moammar et al., 2010). Another study from the Middle East done in the Emirates between January 1995 and May 2013, concluded a birth prevalence of IEM among the Emiratis to be 1 per 1329 live births (Al-Shamsi et al., 2014). Due to the rarity of PROP, prevalence-based studies are not available. However, reports from newborn screening programs across different regions provided valuable data on the incidence rate of the disorder (Table 2-1). According to the most recent systematic literature review and meta-analysis, PROP is considered an ultra-rare disorder. PROP had similar rates across all regions, estimated to be 1 in 100,000, except for regions in the Middle East and North  8  Africa, where most inherited metabolic disorders are more frequent (Almási et al., 2019; Chapman and Summar, 2012). Table 2-1 Incidence Rate of PROP by Population Population Incidence Rate Reference US 1:105,000 - 1:130,000 (Couce et al., 2011) Italy 1:166,000 (Dionisi-Vici et al., 2006) Germany 1:250,000 (Schulze et al., 2003) United Arab Emirates ~1:20,000 -1:45,000 (Al-Shamsi et al., 2014) Saudi Arabia 1: 12,500 1:2000 - 1:5000 (Alfadhel et al., 2016; Zayed, 2015) Japan 1:17,400 (Yorifuji et al., 2002)  2.4 Overall Management of PROP  The goal of managing PROP is to prevent/minimize the accumulation and production of propionyl-CoA and its metabolites. Propionyl-CoA is derived from three main sources: 1- the catabolism of the amino acids ILE, VAL, MET, and THR, 2- catabolism of odd-chain fatty acids and cholesterol side chains, 3- bacterial anaerobic fermentation of carbohydrates in the gut. Precisely, using stable isotope techniques to measure propionate production in individuals with PROP showed that the catabolism of the offending amino acids represents approximately 50% of propionate, while 25% represents propionyl-CoA derived from catabolism of odd-chain fatty acids, and the last 25% derives from bacterial production of propionate. Therefore, to improve  9  overall metabolic control in individuals with PROP, it is necessary to reduce and control propionate production from all sources (Daly et al., 2017; Jurecki et al., 2019; Leonard, 1997) Dietary therapy (which will be discussed in detail below) along with carnitine supplementation, antibiotics and ammonia scavengers, represent the main components of management. Carnitine plays a crucial role in the body. Not only does it transport long-chain fatty acids across the inner mitochondrial membrane to be oxidized via b-oxidation to produce energy, but it is also essential in the detoxification and removal of the accumulated acylcarnitine esters, wherein PROP will be found in the form of propionylcarnitine. Therefore, carnitine supplementation is used in PROP to enhance removal of accumulated propionic acid by converting it to propionylcarnitine, which is then excreted in urine (Sugiyama et al., 1990). This will increase carnitine requirements in the body, which, if not compensated for, can lead to secondary carnitine deficiency, which is not uncommon in PROP (Mardach et al., 2005). Carnitine supplementation is generally safe and the recommended dose ranges from 50-100 mg/kg/d. However, in critically ill individuals the dose can range from 100-300 mg/kg/d divided into 2-4 doses. The amount of carnitine provided should always be adjusted according to plasma total and free carnitine levels (Baumgartner et al., 2014; Jurecki et al., 2019). Metronidazole is commonly used to prevent propionate production from the bacterial fermentation in the gut. Although no data are available on clinical outcomes compared between patients on metronidazole and those not managed with antibiotics, the recommended dose of 20 mg/kg/d in 2-3 doses can be used for 1-2 weeks alternating with few weeks off (De Baulny et al., 2005; Jurecki et al., 2019). To treat and/or prevent hyperammonemia associated with acute decompensation in PROP,  ammonia scavengers such as sodium benzoate are cautiously used  10  (Baumgartner et al., 2014; Shchelochkov et al., 1993). Moreover, since the PCC enzyme is biotin-dependent, biotin supplementation is recommended to be used until definitive diagnosis is made that rules out biotin-responsive PROP (Jurecki et al., 2019). Finally, for severely affected individuals with PROP, liver transplantation may be a potential treatment option to provide increased enzyme capacity (Baumgartner et al., 2014; Shchelochkov et al., 1993).   2.4.1 Nutritional Management of PROP  Nutritional intervention is the cornerstone of management for most IEM disorders. The goal of nutritional management of PROP is to reduce the accumulation of toxic metabolites by restricting dietary protein sources of the propiogenic amino acids (ILE, VAL, MET, and THR), while maintaining their normal plasma concentrations (Jurecki et al., 2019). Moreover, it is essentially important to prevent endogenous catabolism, particularly protein catabolism, by providing sufficient energy to meet metabolic demands (Feillet et al., 2000). Sometimes, individuals with PROP need tube feeding to supplement their oral intake of nutrients and fluids, and to reduce fasting (Jurecki et al., 2019). Nutrient and energy intakes should be adjusted to ensure optimal growth and development in early years of infancy and childhood, and to maintain positive clinical outcomes during adulthood.  Given the lack of suitable nutrition-based studies on PROP, the dietary recommendations are provided based on an individualized clinical and laboratory assessment (Sutton et al., 2012). As with most IEMs, nutrient and energy requirements for individuals with PROP have not been studied fully, which is partly due to the variability of individuals’ tolerance and clinical status (Evans et al., 2017). The absence of such recommendations therefore leads to the application of  11  the current Dietary Reference Intakes (DRI) for energy and protein in healthy populations, adapted to address the needs of individuals with PROP (Table 2-2,Table 2-3,Table 2-4).  Table 2-2 Dietary Reference Intakes for Energy (DRI 0-36 months)  (Institute of Medicine, 2005)  Age EER = TEE + Energy Deposition1 0-3 months (89 x weight [kg] – 100) + 175 kcal 4-6 months (89 x weight [kg] – 100) + 56 kcal 7-12 months (89 x weight [kg] – 100) + 22 kcal 13-36 months (89 x weight [kg] – 100) + 20 kcal 1 Estimated Energy Requirement EER (kcal/day) = Total Energy Expenditure TEE + Energy Deposition            12  Table 2-3 Dietary Reference Intakes for Energy (DRI 3-18 years)  (Institute of Medicine, 2005)      Estimated Energy Requirement (EER) Physical Activity Coefficient (PA) Boys 3-8 years EER = 88.5 - (61.9 x age [y]) + PA x {(26.7 x weight [kg]) + (903 x height [m])} + 20 Sedentary  1.00 Low Active  1.13 Active  1.26 Very Active  1.42 Boys 9-18 years EER = 88.5 - (61.9 x age [y]) + PA x {(26.7 x weight [kg]) + (903 x height [m])} + 25  1.00  1.11  1.25  1.56 Girls 3-8 years EER = 135.3 - (30.8 x age [y]) + PA x {(10.0 x weight [kg]) + (934 x height [m])} + 20   1.00   1.11   1.25   1.48 Girls 9-18 years EER = 135.3 - (30.8 x age [y]) + PA x {(10.0 x weight [kg]) + (934 x height [m])} + 25  1.00  1.12  1.27  1.45  13  Table 2-4 Dietary Reference Intakes for Protein (DRI)  (Institute of Medicine, 2005)  Age Protein Requirements g/kg/day  EAR  Protein Requirements g/kg/day AI/RDA 0-6 months  N/A 1.52 7-12 months  1 1.2 1-3 y 0.87 1.05 4-8y 0.76 0.95 9-13 14-18 19-30 0.76 M: 0.73/ F: 0.71 M: 0.66/ F: 0.66 0.95 0.85 0.80 EAR- Estimated Average Requirement AI- Adequate Intake RDA- Recommended Dietary Allowance    2.4.1.1 Energy Requirements in PROP Energy intake should always be provided in an adequate amount to prevent catabolism, which can lead to the release of propiogenic amino acids from endogenous protein, as well as the release of odd-chain fatty acids from lipid stores. However, it is equally important to avoid overfeeding, especially with inactive individuals, to prevent overweight and obesity (Phyllis B.Acosta and Steven Yannicelli, 2001). According to Feillet et al., using open circuit indirect calorimetry showed a 20% reduction in resting energy expenditure (REE) when compared to the energy requirement calculated using the Schofield equation among well PROP children. The Schofield equation has been used previously to determine the energy requirements for healthy  14  and diseased children (Kaplan et al., 1995). One reason for this reduction is that PROP children tend to have low muscle mass because they are consuming a restricted protein intake compared to healthy children, where up to 30% of REE is related to muscle mass. Another reason is that since most individuals with PROP have a neurological deficit, their neurological status may have an impact on their REE. On the other hand, energy requirements during illness can rise up to 30% of REE, especially during acute decompensation (Feillet et al., 2000). In 2004, van Hagen and colleagues refuted the findings of reduced REE in children with PROP, as they measured REE using indirect calorimetry in 6 PROP children and compared it to the predicted REE using the Schofield equation. The researchers did not find any major differences in the percentage of predicted REE versus measured REE. In fact, there was slight elevation in measured REE. Van Hagen et al. linked the REE increase with the high consumptions of synthetic amino acids mixtures, which could have been associated with higher muscle mass, leading to a rise in REE (van Hagen et al., 2004).  In a multicenter study on 13 children with PROP, an increase in length centile was reported in children who received 98% of the EER for age, while children who received about 87% of the recommended energy needs either remained the same or had a decrease in length centile. All children had received ³ 100% RDA of total protein, which suggests that adequate energy intake is needed to spare protein in order to support overall growth and development (Yannicelli et al., 2003). Using the protein to energy ratio (P:E ratio) -the proportion of energy derived from protein- can provide clinicians with additional guidance when making dietary prescriptions. However, this can be challenging in PROP, since protein intake may be low. Furthermore, the value of medical foods as a protein alternative should also be considered, given  15  the differences in their absorption and bioavailability compared with natural sources. Failure to consider this can lead to height reduction and higher incidence of overweight. Evans et al concluded that using a protein: energy ratio ranging between >1.5 - <2.9 g protein/100 kcal/day correlated with optimal growth among most patients with inborn errors of intermediate protein metabolism including PROP (Evans et al., 2017) The evidence for energy needs in PROP is based on limited research. However, the recent 2019 PROP guidelines state that energy requirement should meet 80-120% of the DRI age (Table 2-5) in order to spare protein catabolism. The guidelines take into consideration each individual’s energy goals for physical activity and medical condition, as well as for normal growth and weight management (Jurecki et al., 2019).  Table 2-5 Recommended Energy Intakes for Well Individuals with PROP1  (Jurecki et al., 2019)  Age Energy (Kcal/kg/day)2 0-6 months M: 72-109 / F: 72-108 7-12 months M: 65-97 / F: 64-96 1-3 years M: 66-99 / F: 66-99 4-8 years M: 59-88 / F: 56-84 9-13 years M: 43-65 / F:39-58 14-18 years M: 36-53 / F:30-45 ³ 19 years 80-100% of EER 1 Adapted from the Institute of Medicine 2 Represents 80-100% of estimated energy requirements (EER)for energy    16  2.4.1.2 Protein and Amino Acid Requirements in PROP Although a protein-restricted diet is the primary foundation for nutritional management in PROP, there is little evidence for how much protein to prescribe, and what percentage of total protein should be provided through natural sources or medical foods. By restricting the consumption of intact protein intake, the propiogenic amino acid intake will be limited. However, these propiogenic amino acids are essential; they cannot be synthesized in the body, but must be obtained from food, in sufficient amounts to maintain growth and anabolism. The dietary prescription should be adjusted based on clinical and laboratory parameters. Severely affected individuals may require greater restriction of intact protein; therefore, they will depend more on medical foods to provide all other essential amino acids and nutrients. On the other hand, individuals with milder mutations may tolerate more intact protein, and such cases will not need to consume medical foods (Sutton et al., 2012). In a report of 49 PROP individuals from 18 metabolic centers across Europe, patients with mild phenotypes were treated with only natural protein, while severely affected patients were given only one third of their requirements from intact protein and the rest was supplemented using medical foods. The authors did not report any differences in outcomes between the two groups. In fact, they concluded that higher protein intakes, whether from natural sources or medical foods, may have been beneficial (Sass et al., 2004). Moreover, Sass et al. provided protein intake recommendations based on their experience during long-term management of PROP patients rather than on confirmation from a large study population (Table 2-6). In 2014, a cross-sectional survey was used to collect dietary data from 47 centers across Europe, where 186 individuals with PROP were followed. Most centers provided below the RDA for natural protein, especially those treating adult patients  17  supplemented with medical foods. Medical foods provided almost half of the requirement for age, and with this supplementation, about 83% of centers provided more than 120% of the RDA for total protein. No clinical or biochemical outcomes were reported (Daly et al., 2017). Total protein intake for disorders of amino acid metabolism should be higher than the DRI for a number of reasons. DRI was not intended for children with lifelong illnesses including infections and stress that can impact both energy and protein requirements. Protein sources used by PROP individuals are often of low biological value, which negatively affect protein status (Yannicelli, 2006). Moreover, it’s been suggested that the current Dietary Reference Intakes (DRI) recommendation for protein requirements in both adults and children is underestimated. Compared to the DRI (2005), and WHO (2007), protein requirements for school-aged children (6-10 y) has been determined by Elango et al. to be 1.3g/kg/day (EAR), and 1.55g/kg/day (RDA) using the indicator amino acid oxidation technique, which is significantly higher than DRI previously determined using nitrogen balance technique (Elango et al., 2011; Humayun et al., 2007). According to the most recent 2019 PROP guidelines, protein and amino acid requirements should be met by providing 60-100% of the age appropriate DRI from sources of intact protein. For individuals who only tolerate < 100% of the DRI from intact protein, there should be supplementation with medical foods to meet 100-120% of the total protein requirements (Jurecki et al., 2019) (Table 2-6). Requirements may be greater than AI/RDA when L-amino acids supply the majority of protein, because there will be a rapid amino acid absorption that can result in an early and high peak of plasma amino acid concentrations, leading to rapid catabolism of amino acids (Table 2-6) (Phyllis B.Acosta and Steven Yannicelli, 2001).    18  Table 2-6 Recommended Protein Intakes for Subjects with PROP  GMDI Guidelines  (Jurecki et al., 2019) 1-3 Age Total Protein g/kg/d Intact Protein g/kg/d Protein from Medical Food g/kg/d 0-6 months 1.52-1.82 0.91-1.52 N/A 7-12 months 1.20-1.44 0.72-1.2 1-3 y 1.05-1.26 0.63-1.05 4-8y 0.95-1.14 0.57-0.95 9-13 y 0.95-1.14 0.57-0.95 14-18 y 0.85-1.02 0.51-0.85 ³ 19 years 0.80-0.96 0.66-1.10 Sass Recommendation (Sass et al., 2004) Age Total Protein g/kg/d Intact Protein g/kg/d Protein from Medical Food g/kg/d 0-12 months 1.8-2.2 0.7-1.5 0.7-1.5 1-4 years 1.5-2.0 1-1.5 0.5-1.0 4-7 years 1.2-1.5 1-1.5 0.2-0.5 > 7 years 1.2-1.5 0.8-1.2 0.0-0.4 The Ross Nutrition Support Protocol Recommendation (Phyllis B.Acosta and Steven Yannicelli, 2001) Age Total Protein g/kg/d Intact Protein g/kg/d Protein from Medical Food g/kg/d 0 - < 3 months 2.5-3.5 N/A N/A 3 - < 6 months 2.5-3.5 6 - <9 months 2.5-3.0 9 - <12 months 2.5-3.0 1 - <4 years  ≥ 30.0 * 4 - <7 years  ≥ 35.0 * 7 - <11 years  ≥ 40.0 * 11- <15 years  M: ³ 50/ F:  ³ 55 * 15- <19 years  M: ³ 65/ F:³ 55 * ³ 19 years  M: ³ 65/ F:³ 50 * 1 Adapted from the Institute of Medicine 2 Represents 60-100% of the adequate intake/ recommended dietary allowance AI/RDA 3 if < 100% AI/DRI from intact protein, supplement with medical food to provide 100-120% of AI/RDA for total protein. *g/d   19  2.4.1.3 Medical Foods for PROP  The term medical food is defined by the Food and Drug Administration in section 5 (b) [3] of the Orphan Drug Act (21 U.S.C 360ee (b) [3]) as “food which is formulated to be consumed or administered enterally under the supervision of a physician and which is intended for the specific dietary management of a disease or condition for which distinctive nutritional requirements, based on recognized scientific principles, are established by medical evaluation” (Office of Regulatory Affairs) (Office of Regulatory Affairs). Amino acid supplementation/mixtures, medical formula/food, and precursor-free amino acids supplements, all refer to the same type of supplementation, that is intended to be used as safe alternative to a regular diet in individuals with inborn errors of metabolism. These supplementations are formulated to contain no offending amino acids, in order to prevent the accumulation of toxic precursors metabolized through a blocked disease pathway. Medical foods specially formulated for individuals with PROP are produced to contain no propiogenic precursors, including valine, isoleucine, methionine, threonine, and to include normal amounts of the other amino acids to ensure sufficient protein intake for optimal growth and protein synthesis by meeting overall protein needs (Manoli et al., 2016). The most commonly used medical foods in PROP are: Propimex1/2 (Abbott), Maximaid/Maxamum (Nutricia) and OA1/2 (Mead Johnson (Table 2-7)  Although there are limited efficacy studies that support the use of medical foods in PROP, reports from the European survey stated that about 81% of 47 centers across Europe are prescribing medical foods regularly, with more than half of centers prescribing medical foods to provide more than 50% of total protein (Daly et al., 2017; Touati et al., 2006). The role of medical food use in PROP is still questionable, due to their formulation with imbalanced BCAA  20  content: that is, with minimal or no valine and isoleucine, and with high amounts of leucine (Myles et al., 2018). An average of 161, 164, and 184 mg of leucine per g of protein is found in three of the most commonly used medical foods [OA2 (Mead Johnson), Propimex 2 (Abbott), and Maxamum (Nutricia), respectively], compared to only 85 mg of leucine in 1 g of egg protein. Leucine appears to be the critical amino acid in most supplements, and until recently there have been no largely reported side effects to leucine-enriched supplements, other than a short-term rise in blood ammonia levels, a complication commonly reported in PROP. However, due to the lack of long-term side effect studies, especially studies related to the safe upper-intake levels of leucine in different health conditions, high leucine intake is still controversial (Elango et al., 2016; Komar et al., 2015). Moreover, high leucine intakes can negatively impact the other two BCAA (isoleucine and valine), by suppressing their plasma concentrations below normal ranges. This is referred to as BCAA antagonism, a well-known phenomenon that was first observed in animals when the addition of L-leucine to a low protein diet was found to reduce plasma concentration of isoleucine and valine, and cause growth restriction. These side effects were partially controlled with isoleucine and valine supplementation. These observations indicated that the consumption of high leucine in a low protein diet can increase the requirements for the other two BCAA (Harper et al., 1984). The practice of supplementing ILE and VAL is questionable, as these amino acids are the main contributors to the toxic metabolite pool and is likely not ideal for PROP patients (Myles et al., 2018).    Therefore, the most recent guidelines for the management of PROP are to discourage the use of medical food as a sole source of energy, and to only recommend their use as an additional source of energy and protein in patients with feeding difficulties, when protein  21  tolerance is less than 100% of DRI from intact protein (Jurecki et al., 2019). In a study using Propimex1(Abbot) as an additional source of protein for sixteen infants and toddlers diagnosed with PROP, they found that using medical foods for 6 months improved growth, which was measured using anthropometric measurements (weight, height centile), and improved nutritional status by measuring mean plasma indices for protein status (Yannicelli et al., 2003). However, others have reported that adding medical foods to a patient’s diet has not shown any additional effect or did not play an important role in nutritional or developmental status (Nyhan et al., 1973; Touati et al., 2006). Clearly, further research is needed to determine what the optimal intake of medical food is, relative to intact protein.  Table 2-7 BCAA Content in Regular milk, Eggs and Common Medical Foods for PROP Product Name Leucine g/100g Isoleucine g/100g Valine g/100g LEU: ILE: VAL Milk1 0.333 0.173 0.207 1: 0.5: 0.6 Eggs2 1.086 0.671 0.858 1: 0.6: 0.7 XMTVI Maxamum3 6.4 0.14 nil 1: 0.002: 0 Propimex24 2.76 0.24 0 1: 0.08: 0 OA25 3.4 0 0 1: 0: 0 1USDA (01106) milk, whole (3.25 milkfat), with added vitamin D  2 USDA (01123) Egg, whole, raw, fresh 3 Nutricia, an unflavored powder free from methionine, threonine and valine, low in isoleucine but containing a balanced mixture of the other essential and non-essential amino acids, carbohydrate, vitamins, minerals and trace elements, for MMA/PROP children over 8 years and adults. 4 Abbott, Nutrition support for children and adults with propionic or methylmalonic acidemia. Methionine- and valine-free; low in isoleucine and threonine. Use under medical supervision. 5 Mead Johnson, Medical food powder for children and adults with propionic or methylmalonic academia.   22  2.5 Growth Outcomes in PROP Poor growth outcomes in children with inborn errors of protein metabolism is well described (Evans et al., 2017). Severely restricted dietary protein consumption is probably the main contributor to growth failure in children with PROP. While the body needs adequate protein intake for optimal protein synthesis and growth, optimal growth can also improve protein tolerance and therefore reduces metabolic decompensation (Molema et al., 2019a). A review of evidence regarding long-term dietary and growth outcomes in subjects with PROP is found in (Table 2-8 ). Data from subjects with methylmalonic acidemia (MMA) -another form of organic acidemia- is also used in the review, because of similarities to disease pathway and dietary treatments with PROP.    23  Table 2-8: Review of Evidence for Long-term Dietary Management and Growth Outcomes in PROP Subjects Intact Protein Medical Foods Growth outcomes Reference 12 subjects with PROP (early onset) • Severely restricted • Reported in g/d • Mean ranges between (5-10.7 g/d) for 0-6 years • Reported in g/d • Mean ranges between (4.2-11.9 g/d) for 0-6 years  • Almost all subjects showed delay in at least one growth parameter (weight, height) • No difference seen in growth outcomes between time of onset • Mean height -2SD • Mean weight -1SD (van der Meer et al., 1996) 5 subjects with PROP (Late onset) • Reported in g/d • Mean ranges between (7.3-27.6 g/d) for 0-6 years • Reported in g/d • Mean ranges between 2-1.3 g/d) for 0-4 years • Supplemented with medical foods only until age 4 years 41 subjects with MMA 29 subjects with PROP • Severely restricted • Reported in g/d • Mean ranges between (5-11 g/d) for 0-6 years • Not reported • Mean height below -2SD in girls by age 10 years • Results in boys are not clear (De Baulny et al., 2005) 3 subjects with MMA 13 subjects with PROP • Reported in g/d • Mean ranges between (15.3-25.1 g/d) for 0-4 years  • Medical foods supplemented almost 50% of protein intake • Mean weight centile increased from 26 to 49% • Mean length centile increased from 25 to 33% (Yannicelli et al., 2003)  24  7 subjects with MMA 9 subjects with PROP • Reported in g/kg/d • Mean ranges between (0.92-0.77 g/kg/d) for 3-11 years • No medical foods • Most subjects had close to normal growth outcomes, with the exception of a few. • At 3 years: height: -0.47SD/ weight: 0.14SD • At 6 years: height: 0.45SD /weight:0.13SD • At 11 years: height: -0.75SD /weight: -0.04SD (Touati et al., 2006) 15 subjects with MMA 8 subjects with PROP • Reported in g/kg/d • Mean ranges between (0.75-0.54 g/kg/d) for 3-11 years • Reported in g/kg/d • Mean ranges between (0.58-0.34 g/kg/d) for 3-11 years 29 subjects with MMA • Reported in g/kg/d • Subjects had highly variable intakes • Mean ranges between (0.29-2.12 g/kg/d) for 2-35 years • Reported in g/kg/d • Subjects had highly variable intakes • Mean ranges between: (0.09- 0.82 g/kg/d) for 2-35 years • Mean height: -1.04SD • Mean weight: 0.01SD • Mean BMI: 0.77SD (Hauser et al., 2011) 55 subjects with PROP • Reported in g/kg/d • Median ranges between (0.8 to1.1 g/kg/d) for 0-5 years • Reported in g/kg/d • Median ranges between (0.7 to 0.9g/kg/d) for 0-5 years • 91% of subjects had median height ranges: -2SD to 2 SD • 6% had median height below -2SD • Median weight ranges: -3.8SD (Grünert et al., 2013)  25  SD- Standard Deviation MMA- Methylmalonic Acidemia  to 3.7SD • Median BMI ranges:1SD to 1.5SD 61 subjects with MMA • Reported in g/kg/d • Mean ranges between (0.99g/kg/d) for 2-18 years • Reported in g/kg/d • Mean ranges between (0.78 g/kg/d) for 2-18 years • Mean height: -2SD • Mean weight: -0.81SD • Mean BMI: 0.72SD (Manoli et al., 2016) 14 subjects with MMA/PROP • Reported in g/kg/d • Mean ranges between (1.5-0.95 g/kg/d) for 3-11 years • No medical foods • Mean height: -1SD (Evans et al., 2017) 263 subjects with MMA/PROP • Reported as g protein/100kcal • Median: 1.23g/100kcal • Reported as g protein/100kcal • Median: 0.6g/100kcal • Mean height below -2SD in 33% of subjects (Molema et al., 2019a)  26   2.6 Branched-Chain Amino Acids  Catabolism of the offending amino acids in PROP represents approximately 50% of propionate production, primarily from BCAA (Leonard, 1997). Leucine, isoleucine, and valine are collectively known as the branched-chain amino acids (BCAA) and are quantitatively by far the most important single group among the dietary indispensable amino acids, as they comprise about 35% of the indispensable amino acids in muscle proteins. These amino acids are “essential” amino acids, they cannot be synthesized in the body, necessitating their adequate intake from dietary sources like meat and dairy products. They make up almost 50% of the indispensable amino acids in the food supply, therefore deficiencies of them do not occur naturally (Cole, 2015). The principal roles of BCAA include tissue protein synthesis; carbon precursors for synthesis of tricarboxylic acid (TCA) cycle intermediates, ketone bodies and fat; carbon and nitrogen precursors for synthesis of alanine, glutamate and glutamine; and energy source via oxidation to CO2. The catabolic pathways of the three BCAA have some features in common, where the initial step for each is transamination by the enzyme branched-chain amino transferase. This is a reversible step that converts each amino acid into its corresponding branched-chain keto acid (BCKA). Each BCKA then undergoes an irreversible oxidative decarboxylation, controlled by the enzyme branched-chain a-ketoacid dehydrogenase, which yields acyl-CoA metabolites. After that, the pathways will yield end products that can enter the TCA cycle. The end products of isoleucine catabolism are propionyl-CoA and acetyl-CoA; leucine catabolism yields acetoacetate and acetyl-CoA; and valine catabolism yields succinyl-CoA (Figure 2-3).  27  Although the three BCAA have similar metabolism, leucine is the key regulator among the BCAA (Harper et al., 1984). Excessive intakes of leucine, in a protein inadequate diet, causes not only a marked depression in growth, but also a drop in the plasma and tissue pools of isoleucine and valine, along with their corresponding ketoacids, keto-methylvalerate (KMV) and keto-isovalerate (KIV), respectively. The antagonistic effect of leucine is unique in that the effects are not observed with an adequate protein intake. Also, increased concentrations of isoleucine and valine do not decrease plasma leucine concentrations. Although the exact mechanism for this is yet to be elucidated, overall BCAA oxidation leading to depressed levels seems the most probable. Increased leucine supplies transaminase to form ketoisocaproic acid (KIC), and increased KIC levels stimulate transamination of valine and isoleucine. This increases the branched-chain keto dehydrogenase (BCKD) activity, thereby cell channeling of the keto acids of valine and isoleucine into their catabolic pathways takes place (Harper et al., 1984; Torres et al., 1998).         28  Figure 2-3 Catabolism of Branched-Chain Amino Acids Adapted from  (Shimomura et al., 2001) KIV- keto- Isovalerate / IB-CoA- Isobutyryl-CoA KMV- keto-Methylvalerate / MB-CoA- a- methylbutyryl-CoA KIC- ketoisocaproic Acid / IV-CoA- Isovaleryl-CoA   2.6.1 Branched-Chain Amino Acid Requirements in Humans The FAO/WHO/UNU 1985 recommendations for amino acid intakes were based on the original technique using nitrogen balance studies in adults with total BCAA requirements of 34 mg/kg/d. The nitrogen balance technique involves the determination of the difference between the intake of amino acid nitrogen and the amount excreted in urine, faeces, and sweat, together with minor losses by other routes. This technique has many limitations, including overestimation of nitrogen intake and an underestimation of nitrogen excretion, leading to an overly positive balance, and therefore, underestimation of requirements, especially of the BCAA requirements. This led to the application of stable isotope-labeled amino acid studies, which have proven to be more rapid and sensitive to changes in amino acid intakes, and more importantly, have resulted  29  in higher requirements than those determined by the nitrogen balance studies (Elango et al., 2012a). The current Dietary Reference Intakes (DRI 2005) report and FAO report (2007) on protein and amino acid intake recommendations are based on requirement estimates for adults using stable isotope studies(Institute of Medicine, 2005, 2007) (Table 2-9). However, to reduce the possibility of interaction between the BCAA in the mixture used to determine the estimate of their requirements, Riazi et al. determined the total BCAA requirements in healthy adult men using indicator amino acid oxidation (IAAO), where the participants received a balanced mixture of the BCAA based on egg protein pattern ( 38.5, 32.5 and 29% for leucine, valine and isoleucine, respectively). They concluded that 144 mg/kg/d is the mean requirement of the total BCAA (55.4, 46.8 and 41.8 mg/kg/d for leucine, valine, and isoleucine, respectively), which is higher than both (FAO/WHO/UNU 1985) and (DRI 2005)(Riazi et al., 2003a)  Table 2-9 Dietary Reference Intakes for Branched-Chain Amino Acid Requirements (DRI)  (Institute of Medicine, 2005)           EAR- Estimated Average Requirement          RDA- Recommended Dietary Allowance   Amino acids Adults 19 years and older (mg/kg/d) EAR RDA Leucine 34 42 Isoleucine 15 19 Valine 19 24  30   In children, the current WHO/FAO/UNU recommendation for BCAA requirements are also based on nitrogen balance studies with low estimates. On the other hand, the DRI recommendations for BCAA are determined using factorial approach based on the adult requirements plus the additional needs for growth in children with the assumption that maintenance rates are the same in adults and children. The current DRI recommendations for the mean intake of total BCAA for school-aged children is 81 mg/kg/d. The IAAO method was used to determine total BCAA requirements in healthy, school-aged children with the same model in adults, where the dietary BCAA were fed in the same proportion as those in egg protein, and concluded that mean requirements of total BCAA is 147 mg/kg/d (56.5, 42.6, and 47.7 mg/kg/d for leucine, isoleucine, and valine respectively) (Mager et al., 2003). Moreover, using the same IAAO method and BCAA dietary model, Mager et al. determined the total BCAA requirements in children with chronic liver disease to be 209 mg/kg/d, significantly higher when compared to healthy children, which suggests that children with liver disease may have an increased dietary needs for BCAA (Mager et al., 2006). Although, this model assumes that BCAA proportion in egg protein is optimal for total body protein synthesis in healthy children and adults, further research is needed to determine whether these proportions are truly optimizing protein synthesis (Mager et al., 2003).   2.6.2 Importance of Branched-Chain Amino Acid Ratio  Due to antagonism among BCAA, dietary supplementation with high levels of leucine might not only enhance metabolism of all BCAA and further increase the nutritional needs for isoleucine and valine, but may also aggravate consequences of their deficiencies. Therefore, an  31  appropriately balanced ratio of BCAA is very important to maintain overall health, especially when a low protein diet is used (Duan et al., 2018b). Wessels et al. examined the effect of excessive leucine intake in three different BCAA ratios on BCAA plasma and tissue concentrations in pigs. They fed the pigs either a control diet with BCAA ratio of (LEU: ILE: VAL = 1: 0.5: 0.7), or 2-fold and 4-fold higher in leucine associated with these ratios (1: 0.3: 0.4) and (1: 0.1: 0.2), respectively. They showed that pigs fed high leucine diets (both 2&4 fold) had lower food intake, which may be associated with their low growth rates. Moreover, leucine plasma concentrations in both groups fed high leucine were 2-4-fold higher compared to the control group. Conversably, plasma concentration of isoleucine and valine and their corresponding keto acids were low in response to high leucine diets, and they were deficient in almost all tissues (Wessels et al., 2016). On the other hand, Duan et al. showed that a BCAA ratio of (LEU: ILE: VAL= 1: 0.75: 0.75) in a protein reduced diet improved growth performance in growing piglets (Duan et al., 2016), and also improved their intestinal health and absorption area (Duan et al., 2018a), when compared to different ratios of (1:1:1) (1:0.51:0.63) and (1:0.25: 0.25). Another study using the IAAO technique to determine BCAA requirements in piglets fed enterally and parentally, where Elango et al. used a fixed BCAA ratio of (LEU: ILE: VAL= 1: 0.6: 0.7). The researchers showed different responses in plasma BCAA concentration during enteral and parental feedings. In parentally fed piglets, all BCAA plasma concentration were low until they reached the total BCAA requirement. At the requirement intake, all BCAA plasma concentrations increased and continued to increase with higher intakes. However, in enterally fed piglets, leucine concentration was low until requirements were met, then it started to increase, while isoleucine and valine were high even with low intakes. This might be explained by the  32  high demand for leucine in the gut, which made it unavailable for protein synthesis. In contrast, the other two BCAA were excreted into circulation, and were high in plasma (Elango et al., 2002). In another study, Elango et al. used IAAO to test the effect of providing deficient intake of all the BCAA in the fixed ratio of (LEU: ILE: VAL = 1: 0.6: 0.7), then supplementing each BCAA individually to test the effect of different BCAA ratios. They did not find any significant differences in the oxidation of the amino acid indicator before and after supplementing with all three BCAA, which suggests that during enteral feeding, the ratio of (LEU: ILE: VAL = 1: 0.6: 0.7) may be the most appropriate, compared to intravenous route (Elango et al., 2004). Despite all the work described above on BCAA ratios in adult humans and in animals, there is no evidence for the optimal BCAA ratio in children’s diets. This still needs to be experimentally determined.  2.7 Indicator Amino Acid Oxidation (IAAO) Technique to Measure Total Body Protein Synthesis  The Indicator amino acid oxidation (IAAO) technique has been used recently to determine protein and amino acid needs in healthy adults and children, as well as in disease, such as in PKU, and MSUD (Courtney-Martin et al., 2002; Elango et al., 2007, 2011; Humayun et al., 2007; Riazi et al., 2004). It can also be used with multiple test intakes to compare relative protein synthesis. The IAAO method is based on the concept that when one essential amino acid is limited in the body, all other amino acids, including the indicator amino acid, are oxidized. This is because amino acids cannot be stored in the body, and the amount of the limiting amino acid controls the partitioning of the other essential amino acids between protein synthesis and  33  oxidation. Therefore, when the limiting amino acid is provided, protein synthesis will increase, and the oxidation of the indicator amino acid will decrease (Elango et al., 2008). The IAAO technique is minimally invasive, as it uses hourly oral doses of stable isotope (1-13C-labeled essential amino acid) and breath sampling in order to measure isotopic enrichment. Therefore, it can be used to determine the effect of multiple test amino acid intakes on total body protein synthesis in vulnerable populations such as school-aged children and pregnant women (Elango et al., 2008; Stephens et al., 2015).  2.7.1 Choice of Indicator Amino Acid in PROP The choice of the indicator amino acid is critical, and its selection relies on the following criteria:  1- The indicator amino acid must be an essential amino acid. 2- The indicator amino acid must have a carboxyl-labeled carbon unit that is irreversibly oxidized upon catabolism and is released to CO2 that can be measured in breath. 3-The indicator amino acid should not have a large pool in the body, and is not involved in main pathways other than being used for protein synthesis or oxidized to CO2 (Zello et al., 1995). Lysine and leucine fulfill the first and second criteria, but lysine has a large pool in the body, and leucine has been shown to stimulate protein synthesis and insulin secretion (Casperson et al., 2012; Columbus et al., 2015). However, phenylalanine in the presence of excess tyrosine is the preferable indicator amino acid, as it fits the criteria required for choosing the indicator amino acid (Elango et al., 2012b; Zello et al., 1995).  34  Chapter 3: Rationale, Objective and Hypothesis   3.1 Rationale Propionic Acidemia (PROP) is an inborn error of metabolism disorder, caused by a defect in the enzyme propionyl-CoA carboxylase (PCC), resulting in the accumulation of propionic acid metabolites. PCC catalyzes propionyl-CoA, oxidative product of two of the branched-chain amino acids (valine, isoleucine), as well as methionine, threonine, odd-chain fatty acids and cholesterol (Baumgartner et al., 2014). The management of PROP primarily depends on dietary protein restriction to prevent the accumulation of propiogenic amino acids. However, to ensure optimal protein synthesis and proper growth, special medical foods were developed for PROP that are formulated to contain all essential amino acids and nutrients, but no propiogenic compounds. Recently, concerns have been raised about the use of medical foods, due to imbalanced content of amino acids; in particular, medical foods containing high leucine contents, and minimal or no valine and isoleucine. It has been suggested that this imbalanced mixture of BCAA negatively impacts plasma concentrations of valine and isoleucine, and therefore restricts growth in children with PROP (Manoli et al., 2016; Myles et al., 2018). Although PROP is one of the most frequent forms of organic acidemias, studies on the long-term growth outcomes of affected individuals are limited. Thus, a comprehensive assessment of dietary intake (including intact protein vs medical foods) and impact on anthropometric data through a natural history is needed. A cohort of subjects with PROP at BC Children’s Hospital treated from 1990-2018 will be included in this analysis.   35  Recently the PROP dietary guidelines (2019) were released and the recommendation is to reduce reliance on medical foods as a primary source of energy and protein. However most individuals with PROP are at risk of malnutrition and depend on medical foods as an easily tolerable source of energy and protein (Jurecki et al., 2019). Thus, there is a need to determine the optimal ratio of BCAA in medical foods to optimize protein synthesis and growth, and to prevent the accumulation of toxic metabolites. With the application of stable isotope-based minimally invasive methods in children, the effect of different BCAA ratios (LEU: ILE: VAL) on whole body protein synthesis can be tested. The indicator amino acid oxidation technique (IAAO), already established to determine amino acid requirements, uses oxidation of L-13C-Phenylalanine as an indicator for protein synthesis, in response to different test amino acid intakes. Different BCAA ratios ranging from the current high leucine: no/minimal valine and isoleucine to a more balanced ratio found in intact protein foods will be tested as part of a proof-of-concept study using healthy children. This approach allows for a safe method to examine multiple test BCAA ratios, determine outcomes based on protein synthesis and help design future studies on subjects with PROP.  3.2 Objectives The specific objectives of the thesis were: Objective 1: To describe dietary therapeutic practices and their impact on anthropometric (growth) outcomes in the management of four subjects with PROP at BC Children’s Hospital Biochemical Diseases Clinic from 1990 - 2018.   36  Objective 2: To examine the effect of different LEU: ILE: VAL ratios on whole body protein synthesis in healthy children using the IAAO method (oxidation of L-13C-Phenylalanine to 13CO2), by reducing leucine levels from the current high doses in medical foods, sequentially, while providing isoleucine and valine at PROP recommended intake levels.  Objective 3: To examine the effect of different LEU: ILE: VAL ratios on urinary concentrations of BCAA, by reducing leucine levels from the current high doses in medical foods, sequentially, while providing isoleucine and valine at PROP recommended intake levels.  3.3 Hypothesis 1- We hypothesize that subjects with PROP would have poor growth outcomes (low height for age and weight for age/height Z scores) associated with high intake of medical food compared to intact protein consumption, during childhood 2- We hypothesize that IAAO (oxidation of L-13C-Phenylalanine to 13CO2) will be high (suggesting low protein synthesis) when BCAA ratio is imbalanced and will be low (suggesting optimal protein synthesis) when leucine levels are reduced and at a better-balanced ratio  3- We hypothesize that participants will have low urinary concentrations of isoleucine and valine, with high urinary leucine when BCAA ratio is imbalanced and would normalize at a better-balanced ratio  37  Chapter 4: Natural History Study on Subjects with Propionic Acidemia  Subjects with PROP, treated with protein-restricted diets, are prone to growth failure (Grünert et al., 2013). Numerous factors, including physiological, genetic, and environmental aspects, can influence growth outcomes. However, consuming a diet that is severely restricted in protein is probably the main contributor to growth failure in children with PROP (Lui et al., 2015). Even though PROP is one of the most frequent forms of organic acidemias, information on the long-term growth outcomes of affected individuals is limited. Thus, a comprehensive assessment of dietary and anthropometric data of children with PROP at BC Children’s Hospital through a natural history study was needed. In this chapter, I discuss the methods and results of conducting a retrospective chart review for the years 1999-2018, on four subjects with PROP.   4.1 Subjects and Methods  This study was approved by the University of British Columbia and BC Children's Hospital Research Ethics Board (H19-02912). A retrospective chart review was conducted on a sample of four pediatric patients with propionic acidemia followed in the Biochemical Diseases Clinic at BC Children’s Hospital. These were two sibling pairs, with the older siblings diagnosed in the newborn period with an acute metabolic decompensation, and the younger siblings screened right after birth. We collected longitudinal data on dietary intake and growth outcomes for the years 1999-2018, following the cohort from age 0 to 18 years via a natural history study. Data were collected from medical and dietetic clinic records when subjects were metabolically stable.    38  4.1.1 Data Collection 4.1.1.1 Growth Data  Anthropometric data were collected from medical and dietetics records during clinic visits. Weight and length for children under 2 years of age were obtained by standard techniques using digital baby weighing scales and crown-heel length on a scale length board. Weight and height for children greater than 2 years of age were measured using digital scale and a stadiometer, respectively. Body mass index (BMI) was calculated using the equation kg/m2. Measurements were performed by the dietitian or clinic nurse. Anthropometric measurements were expressed as age-and sex-specific Z-scores, using the WHO Anthro and Anthroplus software for 0-5 years of age and 5-19 years of age, respectively. Indicators used between 0-5 years of age includes: weight for age, weight for height, and height for age. Between 5 and 10 years of age, indicators used include: weight for age, height for age, and BMI for age (WHO AnthroPlus for personal computers Manual: Software for assessing growth of the world's children and adolescents. Geneva: WHO, 2009) (http://www.who.int/growthref/tools/en/)..  4.1.1.2 Dietary Data  Dietary data was collected on the basis of formula recipes delivered via tube feeding and food records for the oral intake, which was analyzed by the metabolic dietitian using the MetabolicPro software from Genetic Metabolic Dietitian International (GMDI). MetabolicPro is the only web-based nutrient analysis software program designed specifically for the metabolic nutritionist. Dietary data was only collected when subjects were metabolically stable. We excluded data during sick days as subjects were consuming a special sick day formula, which  39  was supplying 120% EER for energy and no or very low intact protein. Number of sick days for each subject was calculated and presented with subject characteristics (Table 4-1). Formula composition information was obtained from the respective manufacturers. The data represent reported, rather than prescribed intake. Dietary intake of protein was expressed in grams per kilogram body weight per day (g/kg/d) as total protein, which was calculated by adding the intake of intact protein, protein from medical food, as well as single amino acid supplements (L-isoleucine and L-valine). Protein intake was also reported as g/kg/d of intact protein vs. protein from medical food separately. Energy intake was collected as kcal/kg/d. The protein to energy (P: E) ratio was calculated on the basis of the amount of total protein in g per 100 kcal per day. The calculated P: E ratio values were compared with P: E values associated with optimal growth in subjects with inborn errors of protein metabolism described by Evans et al ( > 1.5 - < 2.9g/100kcal/day) (Evans et al., 2017). Since subjects were nutritionally managed using different guidelines at different time periods, subjects’ actual intakes were compared with the Ross recommendation 2001(Phyllis B.Acosta and Steven Yannicelli, 2001), the Sass recommendation 2004 (Sass et al., 2004) and with the most recent guidelines for PROP from GMDI 2019 (Jurecki et al., 2019). For comparison purposes, protein and energy intakes were grouped for all four subjects according to age (0-6 months, 7-12 months, 1-3 years, 4-8 years, 9-13 years, and 14-18 years).  4.1.2 Statistical Analysis  Statistical analysis was performed using GraphPad Prism 4.0 (GraphPad Software Inc, CA). Descriptive statistics were used to compare actual energy and protein intakes with different  40  recommendation guidelines; all data were expressed as median and range (minimum -maximum). Different growth Z scores were reported according to age groups, for 0-5 years old (Weight for height Z scores, Weight for age Z scores, and Height for age Z scores). For 5 -10 years old (Height for age Z scores, Weight for age Z scores, and BMI Z scores). For 10-18 years old (Height for age Z scores, and BMI Z scores). Growth data were also expressed as median and range (minimum -maximum).  4.2 Results  4.2.1 Subject characteristics  Four subjects with PROP were followed in the Biochemical Disease Clinic at BC Children’s Hospital. PROP-01 and PROP-02 are a girl and boy sibling, respectively, and PROP-03 and PROP-04 are two sisters. All subjects had gastrostomy tubes, receiving part or all of their daily nutritional needs though bolus or continuous feeds; one subject (PROP-03) had received growth hormone treatment at 9 years of age for documented growth hormone deficiency. Although all subjects were well controlled with no major metabolic crises, one subject (PROP-04) died of cardiomyopathy at 10 years of age. Characteristics of the four subjects including age, time of diagnosis and mutations are presented in (Table 4-1)  41  Table 4-1 Subjects with PROP Characteristics  1 Sick day formula was used to supply 120%EER for age, no or low protein intake 1 Calculated as number of days from data points collected 2 Propionyl Co-A Carboxylase Alpha subunit  3 Propionyl Co-A Carboxylase Beta subunit  Subjects no. Age (Years) Age at Diagnosis Gender Number of Sick Days 1 Mutation PROP-01 19 4 weeks F • Birth-1 year (45 days) • 1-3 years (22 days) • 4-8 years (165 days) PCCA2 Homozygous c.134_135delTA p. Leu45TyrX PROP-02 18 2 weeks M • Birth-1 year (12 days) • 1-3 years (42 days) • 4-8 years (28 days) PCCA Homozygous c.134_135delTA p. Leu45TyrX PROP-03 19 6 months F • Birth-1 year (43 days) • 1-3 years (13 days) • 4-8 years (47 days) PCCB3 Homozygous c.337C>T p. Arg113X PROP-04 18 prenatal F • Birth-1 year (39 days) • 1-3 years (63 days) • 4-7 years (26 days) PCCB Homozygous c.337C>T p. Arg113X  42   4.2.2  Growth Data All four children had poor growth outcomes with persistently reduced height Z scores, and elevated weight and BMI Z scores. During the first 5 years of life, all subjects had a median Z score of 1.6, ranging from 1.04 to 2 for weight for height, and -0.717 (range: -1.36 to -0.2) for height. From 5 to 10 years of age, height Z scores declined to a median of -1.03 (range: -1.78 to - 0.23) for all subjects. However, their median BMI Z scores were at 1.35 with a range of 1.01 to 2.03 that translates to a BMI percentile of (> 84 and < 95) and classify them as overweight. Between the ages of 10 and 18 years, height Z scores decreased even more to a median of -1.4 ranging from -1.84 to -4.6. All growth Z scores are presented in more detail for each subject individually in (Table 4-2) and (Figure 4-1Figure 4-2Figure 4-3Figure 4-4).  After 10 year of age, the WHO recommends stopping using weight for age, as it does not distinguish between height and BMI during period of age where children are experiencing pubertal growth spurt. Although the Canadian Pediatric Endocrine Group extended the WHO growth charts to include weight for age beyond age 10y, WHO indicated that the new extended charts are not considered WHO charts (Appendix H, I &J). Thus our data are only presented as BMI for age from 10-18y. PROP-03 Subject, who had received growth hormone replacement therapy at age 9, had an improvement in height and BMI Z sores (Figure 4-3).      43  Table 4-2 Subjects with PROP Growth Z Scores * Age Variable Median (Range) PROP-01 0-5 years Weight for Age Height for Age Weight for Height 0.32 (-3.01– 1.28) -1.09 (-4.81– -0.8) 1.88 (0.94 – 2.35) 5-10 years Weight for Age Height for Age BMI for Age 0.775 (-0.21– 1.45) -0.79 (-1.08- -0.29) 1.015 (0.46 – 2.24) 10-18 years Height for Age BMI for Age -1.4 (-1.82 – -1.18) 0.85 (0.39 – 1.28) PROP-02 0-5 years Weight for Age Height for Age Weight for Height 0.48 (-3.23 – 1.61) -0.20 (-1.97 – 0.48) 1.04 (-2.61 – 2.4) 5-10 years Weight for Age Height for Age BMI for Age 0.94 (0.56 – 1.85) -0.23 ( -0.58 – 0.08) 1.38 (1.26 – 2.46) 10-18 years Height for Age BMI for Age -1.405 (-1.69 – -0.86) 1.655 (0.96 – 2.04) PROP-03 0-5 years Weight for Age Height for Age Weight for Height 0.42 (-0.25 – 1.54) -1.365 (-1.72 – 1.21) 1.365 (0.44 – 1.95) 5-10 years Weight for Age Height for Age BMI for Age -0.11 (-0.38 – 0.49) -1.78 (-2.16 – -1.42) 1.33 (0.82 – 1.89) 10-18 years Height for Age BMI for Age - 1.84 (-2.21 – -0.8) 0.435 (-0.72 – 0.62) PROP-04 0-5 years Weight for Age Height for Age Weight for Height 1.535 (0.24 – 2.79) -0.345 (-1.25 – 1.82) 2.015 (1.54 – 2.86) 5-10 years Weight for Age Height for Age BMI for Age 1.03 (0.61 – 1.28) -1.275 (-1.53 – -0.91) 2.035 (1.67 – 2.61) *Assessed using sex-specific growth charts  44   (A)      (B)        (C)      Figure 4-1 Growth Data Z scores for PROP-01 (sex-specific charts)                                       A) Weight for Height, Height for Age, and Weight for Age from 0-5 years old. B) BMI, Height and Weight for age from 5-10 years old. C) BMI and Height for age from 10-18 years old. 0 3 6 9 11 15 18 21 24 27 30 33 36 39 41 46 47 51 54 57 60-6-4-2024PROP-01 (0-5 years) Age (months)Z scoresWt for Age Ht for Age Wt for HT63 66 69 72 75 77 80 83 86 90 94 97 100105108111115118-2-10123PROP-01(5-10 years) Age (months)Z scoresWt for Age Ht for Age BMI for Age 123127129136139141147151154156160163165167174177180183187190193195198202205209210215218-2-1012PROP-01(10-18 years) Age (months)Z scoresHt for Age BMI for Age  45  (A)      (B)       (C)            Figure 4-2 Growth Data Z Scores for PROP-02 (sex-specific charts)                                        A) Weight for Height, Height for Age, and Weight for Age from 0-5 years old. B) BMI, Height and Weight for age from 5-10 years old. C) BMI and Height for age from 10-18 years old. 0 4 7 10 13 15 19 22 25 27 31 34 37 40 42 46 49 51 54 58 61-4-2024PROP-02 (0-5 years) Age (months)Z scoresWt for Age Ht for Age Wt for HT64 67 70 72 75 82 84 87 95 102106109115-10123PROP-02 (5-10 years) Age (months)Z scoresWt for Age Ht for Age BMI for Age 125128134137142147150160164167174177180187189192195205-2-10123PROP-02 (10-18 years) Age (months)Z scoresHt for Age BMI for Age  46   (A)      (B)      (C)        Figure 4-3 Growth Data Z Scores for PROP-03 (sex-specific charts)                                        A) Weight for Height, Height for Age, and Weight for Age from 0-5 years old. B) BMI, Height and Weight for age from 5-10 years old. C) BMI and Height for age from 10-18 years old.  63 66 68 72 75 78 81 84 87 89 92 95 99 101108111116120-3-2-1012PROP-03 (5-10 years) Age (months)Z scoresWt for Age Ht for Age BMI for Age 124128133139148153156164167170175180186193199204206211218-3-2-101PROP-03 ( 10-18 years) Age (months)Z scoresHt for Age BMI for Age 5 8 12 15 17 20 23 26 29 33 36 39 41 45 48 50 53 56 60-2-10123PROP-03 (0-5 years)Age (months)Z scoresWt for Age Ht for Age Wt for HT 47    (A)        (B)         Figure 4-4 Growth Data Z Scores for PROP-04 (sex-specific charts)                                        A) Weight for Height, Height for Age, and Weight for Age from 0-5 years old. B) BMI, Height and Weight for age from 5-10 years old.    63 66 69 72 77 81 90 94 98 102104107112-2-10123PROP-04 (5-10 years) Age (months)Z scoresWt for Age Ht for Age BMI for Age 0 0.2 3 6 9 12 15 18 21 24 27 30 31 36 39 42 45 48 51 54 57 60-2-101234PROP-04 (0-5 years) Age (months)Z scoresWt for Age Ht for Age Wt for HT 48  4.2.3 Dietary Data  Energy and protein intakes, including total, intact, and protein from medical foods in g/kg/d are all presented for each subject in (Table 4-3). Energy intake for all subjects was within 80-120% of the EER for age, as defined in the National Research Council Washington DC, 2005 (Institute of Medicine, 2005) (Figure 4-5). Protein intake was compared with three different guidelines; The Ross recommendation guidelines for total protein intake for 0-12 months of age (Phyllis B.Acosta and Steven Yannicelli, 2001), the Sass recommendation for both total and intact protein intake for all ages (Sass et al., 2004), and lastly with the Genetic Metabolic Dietitian International (GMDI) guidelines in PROP (Jurecki et al., 2019). A comparison among these guidelines for total protein intake is presented in (Figure 4-6). There are no major differences in total protein recommendations between the (GMDI.2019, and Sass et al.2004) guidelines. However, the Ross recommendations for total protein is 92-108% higher than GMDI recommended intakes, and 59-50% higher than Sass recommendations for 0-6 months and 7-12 months, respectively. This indicates that compared to the most recent guidelines, the Ross recommendations were significantly higher in total protein intakes.  All subjects had low intakes of intact protein compared with both guidelines (GMDI.2019, and Sass et al.2004), and were supplemented with medical foods and single L-amino acids (Valine and/or Isoleucine), which led to the excess consumption of total protein in comparison to both guidelines. It must be noted here that only the Ross recommendation guidelines were used to manage infants with PROP at BC Children’s Hospital between 1999 and 2003. Median percentages of intact protein vs. protein from medical foods varied for each patient and for each age group (Figure 4-7  49  Figure 4-8Figure 4-9Figure 4-10). However, as children grew older, their intakes from intact protein increased and, accordingly, medical food consumption decreased. Median intact protein consumption in g/kg/d for 1-3 years was 0.74 (0.62-0.85) 70% of RDA for intact protein. Whereas median total protein intake in g/kg/d for the same age was 2.26 (2.02-2.37) 179% of RDA for total protein (GMDI.2019). This indicates that for 1-3 years of age, 67% of total protein consumption was supplied from medical foods rather than intact protein sources. (Figure 4-7 Figure 4-8Figure 4-9Figure 4-10 Breakdown of Protein Intake). The protein to energy ratio (P: E) in g/100kcal/d was calculated for both intact vs. total protein, and presented for all subjects in (Table 4-4). A protein to energy ratio of (> 1.5 to <2.9 g/100kcal) was found by Evans at el. to be associated with optimal growth outcomes. In our results, median total protein: energy ratio for 1-3 years was 2.75/100kcal, within the optimal ratio. However, the median intact protein: energy ratio for the same age was 0.9g/100kcal; well below the reference for optimal growth.         50  Table 4-3 Subjects with PROP Dietary Intake  Total Protein g/kg/d  Age Subjects’ Actual Intakes 1 Recommended Intakes PROP-01 PROP-02 PROP-03 PROP-04 ROSS 2 SASS 3 GMDI 4 0-6 months 2.67 (0.44-3.23) 1.96(1.47-2.83) 1.8(0.51-2.25) 1.92(1.31-2.54) 2.50 - 3.50 1.8-2.2 1.52-1.82 7-12 months 2.25(1.93-2.61) 2.08(1.53-2.13) 1.52(1.46-1.71) 1.96(1.3-2.3) 2.50 - 3 1.20-1.44 1-3 years 2.02(1.18-2.18) 2.29(1.62-2.51) 2.37(1.52-2.61) 2.23(2.06-2.5) ³30* 1.5-2 1.05-1.26 4-8 years 2.07(1.26-2.24) 1.76(1.21-2.22) 2.33(1.22-2.51) 1.84(0.88-2.34) ³35* 1.2-1.5 0.95-1.14 9-13 years 1.25(0.96-1.46) 1.02(0.92-1.21) 1.23(1.1-1.44) 0.98(0.95-1.05) ³40* 0.95-1.14 14-18 years 1.04(0.77-1.13) 0.93(0.82-1.06) 1.13(1.05-1.3) N/A M: ³65 / F: ³55* 0.85-1.02 Intact Protein g/kg/d Age Subjects’ Actual Intakes 1 Recommended Intakes PROP-01 PROP-02 PROP-03 PROP-04 ROSS 2 SASS 3 GMDI 4 0-6 months 0.95(0.44-1.41) 1.44(0.5-1.72) 1.2(0. .32-1.36) 1.17(0.53-1.21) N/A 0.7-1.5 0.91-1.52 7-12 months 0.67(0.58-0.83) 0.87(0.21-0.97) 1.06(0.76-1.29) 0.87(0.43-1.03) 0.72-1.2 1-3 years 0.62(0.31-0.71) 0.85(0.42-0.93) 0.72(0.42-0.81) 0.77(0.67-0.88) 1-1.5 0.63-1.05 4-8 years 0.76(0.43-1.50) 0.86(0.75-1.01) 0.91(0.75-1.18) 0.76(0.58-0.86) 0.57-0.95 9-13 years 0.66(0.56-0.80) 0.70(0.55-0.96) 0.78(0.7-0.87) 0.63(0.61-0.69) 0.8- 1.2 0.57-0.95 14-18 years 0.75(0.54-0.89) 0.77(0.65-1.01) 0.75(0.73-0.84) N/A 0.51-0.85 Protein from Medical Foods g/kg/d  Age Subjects’ Actual Intakes 1 Recommended Intakes PROP-01 PROP-02 PROP-03 PROP-04 ROSS 2 SASS 3 GMDI 4 0-6 months 1.18(1.23-3.06) 0.63(0-1.8) 0.83(0.22-1.15) 0.74(0.44-1.32) N/A 0.7-1.5 N/A  51  7-12 months 1.58(1.36-1.77) 1.12(0-1.2) 0.48(0.22-0.81) 1.10(0.65-1.42) 1-3 years 1.37(1.22-1.63) 1.43(1.15-1.7) 1.63(0.76-1.89) 1.46(1.3-1.72) 0.5-1 4-8 years 1.30(0.62-1.54) 0.86(0.46-1.37) 1.24(0.47-1.64) 1.03(0.30-1.54) 0.2-0.5 9-13 years 0.61(0.40-0.68) 0.38(0-0.46) 0.44(0.38-0.57) 0.34(0.32-0.37) 0-0.4 14-18 years 0.22(0.19-0.36)  0.15(0-0.19) 0.34(0.33-0.44) N/A Energy Kcal/kg/day  Age Subjects’ Actual Intakes 1 Recommended Intakes PROP-01 PROP-02 PROP-03 PROP-04 ROSS 2 SASS 3 GMDI 4 0-6 months 97(97-97) 95(95-95) 95(95-95) 92.76(78.36-120.4) 95-145 N/A M: 72-109/F: 72-108 7-12 months 91.2(93.9-111.6) 94.1(90-95.2) 90.34(84.30-100) 70.59(65.96-88.24) 80-135 M: 65-97/F: 64-96 1-3 years 75.9(72.9-87.6) 89.5(72.7-97.9) 83.7(77.30-95.48) 74.31(65.36-85.54) 900-1800† M: 66-99/F: 66-99 4-8 years 66.8(40.4-80.7) 56.9(51-66.1) 57.08(49.03-82.16) 56.96(50.65-70.8) 1300-2300† M: 59-88/F: 56-84 9-13 years 46(34.4-56.9) 40(34.96-65.8) 47.07(42-55.05) 48.82(45.71-52.52) 1650-3300† M: 43-65/F:39-58 14-18 years 34.6(32.1-40.7) 34.9(30.2-37.2) 37.97(33.28-44.70) N/A M:2100-3900 / F:1200-3000† M: 36-53/F:30-45 1 Subjects’ actual intakes reported in median and range (Minimum- Maximum) 2 Recommended intakes adapted from the Ross Nutrition Support Protocol (Phyllis B.Acosta and Steven Yannicelli, 2001) 3 Recommended intakes based on the Sass Recommendation (Sass et al., 2004) 4 Recommendedn intakes based on the GMDI guidelines (Jurecki et al., 2019)  * g/d † kcal/d   52                 1 (Jurecki et al., 2019) 2 (Sass et al., 2004) 3 (Phyllis B.Acosta and Steven Yannicelli, 2001). Recommendations were reported in g.kg/d only for 0-12 months of age.  Figure 4-5 Energy Intake for Subjects with PROP Compared with 80-120% of RDA       Figure 4-6 A Comparison Among Different Guidelines for Total Protein Intakes for Subjects with PROP  0-6 months 7-12 months1-3 years4-8 years9-13 years14-18 years0.51.01.52.02.53.03.5g/kg/dTotal ProteinGMDI. 2019 1Sass et al.2004 2Ross. 2001 30-6 months7-12 months 1-3 years4-8 years 9-13 years14-18 years 306090120150Energy Intake Kcal/kg/dTotal Energy 80% of EERTotal Energy 120% of EERPROP-01PROP-02PROP-03PROP-04 53   Figure 4-7 PROP-01 Subject Protein Intakes A) Total Protein Intake in Comparison with GMDI 2019. B) Total Protein Intake in Comparison with Sass et.2004. C) Total Protein in Comparison with Ross.2001. D)Intact Protein in Comparison with GMDI.2019. E). Intact Protein in Comparison with Sass et al.2004. F) Breakdown of Protein Intake  0-6 months7-12 months 1-3 years4-8 years 9-13 years14-18 years 1.01.52.02.53.0g/kg/dTotal Protein Intake Recommended Intake(GMDI.2019)PROP-0147%56%60% 81%2%9%(A)0-6 months7-12 months 1-3 years4-8 years 9-13 years14-18 years 0.51.01.5g/kg/dIntact Protein Intake Recommended Intake (GMDI.2019)PROP-01-37%-44%-20%-30%-12%-41%(D)0.0 0.5 1.0 1.5 2.0 2.50-6 months7-12 months 1-3 years4-8 years 9-13 years14-18 years g/kg/dBreakdown of Protein Intake (PROP-01)Intact Protein Protein from Medical FoodTotal protein (F)0-6 months7-12 months 1-3 years4-8 years 9-13 years14-18 years 1.01.52.02.5Total Protein Intake g/kg/dPROP-01 Recommended Intake (Sass et al.2004)212.51 37-17-30(B)0-6 months7-12 months 1-3 years4-8 years 9-13 years14-18 years 0.51.01.5g/kg/dIntact Protein Intake PROP-01 Recommended Intake (Sass et al.2004)-37-55 -59-49-45-38(E)0-6 months7-12 months 1.01.52.02.5Total Protein Intake g/kg/dPROP-01 Recommended Intake (Ross. 2001)(C) 54  Figure 4-8 PROP-02 Subject Protein Intakes A) Total Protein Intake in Comparison with GMDI 2019. B) Total Protein Intake in Comparison with Sass et.2004. C) Total Protein In comparison with Ross.2001. D)Intact Protein in Comparison with GMDI.2019. E). Intact Protein in Comparison with Sass et al.2004. F) Breakdown of Protein Intake 0-6 months7-12 months 1-3 years4-8 years 9-13 years14-18 years 1.01.52.02.5g/kg/dTotal Protein Intake Recommended Intake (GMDI.2019)PROP-028%44%82%54%-10% -9%(A)0-6 months7-12 months 1-3 years4-8 years 9-13 years14-18 years 0.51.01.5g/kg/dIntact Protein Intake Recommended Intake  ( GMDI.2019)PROP-02-5%-27% -9%-26% -9%-19%(D)0.0 0.5 1.0 1.5 2.0 2.50-6 months7-12 months 1-3 years4-8 years 9-13 years14-18 years Breakdown Protein Intake (PROP-02)g/kg/dIntact Protein Protein from Medical FoodTotal protein (F)0-6 months7-12 months 1-3 years4-8 years 9-13 years14-18 years 1.01.52.02.5Total Protein Intake g/kg/dPROP-02 Recommended Intake (Sass et al.2004)-10 -51517-32 -38(B)0-6 months7-12 months 1-3 years4-8 years 9-13 years14-18 years 0.51.01.5g/kg/dIntact Protein Intake PROP-02 Recommended Intake (Sass et al.2004)-4-42 -43 -42-43-36(E)0-6 months7-12 months 1.01.52.02.5Total Protein Intake g/kg/dPROP-02 Recommended Intake (Ross .2001)(C) 55   Figure 4-9 PROP-03 Subject Protein Intakes A) Total Protein Intake in Comparison with GMDI 2019. B) Total Protein Intake in Comparison with Sass et.2004. C) Total Protein in Comparison with Ross.2001. D)Intact Protein in Comparison with GMDI.2019. E). Intact Protein in Comparison with Sass et al.2004. F) Breakdown of Protein Intake 0-6 months7-12 months 1-3 years4-8 years 9-13 years14-18 years 0.51.01.5g/kg/dIntact Protein Intake Recommended Intake (GMDI.2019)PROP-03-23%-11%-4%-18% -11%-31%(D)0-6 months7-12 months 1-3 years4-8 years 9-13 years14-18 years 1.01.52.02.5g/kg/dTotal Protein Intake Recommended Intake (GMDI.2019)PROP-03-1%5%88% 83%8% 11%(A)0.0 0.5 1.0 1.5 2.0 2.50-6 months7-12 months 1-3 years4-8 years 9-13 years14-18 years Breakdown Protein Intake (PROP-03)g/kg/dIntact Protein Protein from Medical FoodTotal protein (F)0-6 months7-12 months 1-3 years4-8 years 9-13 years14-18 years 0.51.01.5g/kg/dIntact Protein Intake PROP-03 Recommended Intake (Sass et al.2004)-22-29-52-39-35 -37(E)0-6 months7-12 months 1-3 years4-8 years 9-13 years14-18 years 1.01.52.02.5Total Protein Intake g/kg/dPROP-03 Recommended Intake (Sass et al.2004)-18-3019 55-18-25(B)0-6 months7-12 months 1.01.52.02.5Total Protein Intake g/kg/dPROP-03 Recommended Intake (Ross. 2001)(C) 56  Figure 4-10 PROP-04 Subject Protein Intakes A) Total Protein Intake in Comparison with GMDI 2019. B) Total Protein Intake in Comparison with Sass et.2004. C) Total Protein in Comparison with Ross.2001. D)Intact Protein in Comparison with GMDI.2019. E). Intact Protein in Comparison with Sass et al.2004. F) Breakdown of Protein Intake 0-6 months7-12 months 1-3 years4-8 years 9-13 years0.51.01.5g/kg/dIntact Protein Intake Recommended Intake  ( GMDI.2019)PROP-04-23%-27%-20%-34%-27%(D)0-6 months7-12 months 1-3 years4-8 years 9-13 years1.01.52.02.5g/kg/dTotal Protein Intake Recommended Intake (GMDI.2019)PROP-045% 36%77%61%-14%(A)0.0 0.5 1.0 1.5 2.0 2.50-6 months7-12 months 1-3 years4-8 years 9-13 yearsBreakdown Protein Intake (PROP-04)g/kg/dIntact Protein Protein from Medical FoodTotal protein (F)0-6 months7-12 months 1-3 years4-8 years 9-13 years1.01.52.02.5Total Protein Intake g/kg/dPROP-04 Recommended Intake (Sass et al.2004)-13 -111223-35(B)0-6 months7-12 months 1-3 years4-8 years 9-13 years0.51.01.5g/kg/dIntact Protein Intake PROP-04 Recommended Intake (Sass et al.2004)-22-42-49 -49-48(E)0-6 months7-12 months 1.01.52.02.5Total Protein Intake g/kg/dPROP-04 Recommended Intake (Ross. 2001)(C) 57  Table 4-4 Protein to Energy Ratio for Subjects with PROP1 Age Subjects’ Actual Intakes2 Recommended Protein to Energy Ratio3 (Evans et al., 2017) Total Protein: Energy Intact Protein: Energy PROP-01 0-6 months 2.75 0.98 >1.5 to < 2.9 7-12 months 2.47 0.73 1-3 years 2.66 0.81 4-8 years 3.09 1.13 9-13 years 2.7 1.4 14-18 years 2.99 2.1 PROP-02 0-6 months 2.06 1.5 >1.5 to < 2.9 7-12 months 2.21 0.92 1-3 years 2.55 0.94 4-8 years 3.09 1.5 9-13 years 2.55 1.7 14-18 years 2.66 2.2 PROP-03 0-6 months 1.89 1.23 >1.5 to < 2.9 7-12 months 1.68 1.17 1-3 years 2.85 0.86 4-8 years 4.2 1.66 9-13 years 1.61 1.65 14-18 years 2.97 1.98 PROP-04 0-6 months 2.06 1.26 >1.5 to < 2.9 7-12 months 2.77 1.23 1-3 years 3 1.03 4-8 years 2.23 1.33 9-13 years 2.06 1.29 1 g of protein/100 kcal/day 2 Subjects’ Actual Intakes are reported as medians 3 Protein to Energy ratio associated with optimal growth in subjects with inborn error of metabolism    58   4.3 Discussion Poor growth outcomes with respect to height rather than weight in subjects with PROP has been well described (De Baulny et al., 2005; Evans et al., 2017; Grünert et al., 2013; van der Meer et al., 1996; Molema et al., 2019a; Touati et al., 2006). Although physiological, genetic, and environmental factors can influence growth patterns, severely restricted intact protein consumption, especially during the first years of life, is probably the main contributor to growth failure in PROP (Lui et al., 2015). The purpose of this retrospective chart review was to describe dietary practices in the management of subjects with PROP and to establish their effects on growth outcomes. We collected longitudinal dietary and growth data for four pediatric patients for 1999-2018, following the cohort for 18 years through a natural history study. Results from the current study confirmed that all children had poor growth outcomes, with persistently reduced height-for-age Z scores, and elevated weight and BMI Z scores. Energy intakes for all children were within 80-120% of the EER for age. All children had low intakes of intact protein compared with most recent guidelines and were supplemented with medical foods and single L-amino acids (Valine and/or Isoleucine), which lead to the excess consumption of total protein in comparison to guidelines. Our results showed that with the current dietary intakes, all children had persistently low height Z scores. However, height data could be skewed by subject PROP-03 who received growth hormone treatments at age 9 in response to documented growth hormone deficiency. Low height for age Z score reflects a process of failure to reach linear growth potential due to suboptimal health and/or nutritional conditions, which is also an indication of stunting (Lui et al.,  59  2015). In agreement with our results, many reported a tendency toward a decrease in height Z scores and an overall growth failure in subjects with PROP (Table 2-8) (De Baulny et al., 2005; Evans et al., 2017; Grünert et al., 2013; van der Meer et al., 1996; Molema et al., 2019a; Sass et al., 2004). However, the one exception is the study, where Yannicelli et al. reported an improved weight and moderate improvement in height centile in infants and toddlers with PROP when medical food provided 50% of total protein intake (Yannicelli et al., 2003). It is unknown why these results differ, and may be related to the clinical severity of the disease. The strong restriction of intact protein seen in our subjects, which persisted beyond the first two years of life could have limited their linear growth. The association between severely restricted intact protein consumption and low height Z scores was also previously reported (De Baulny et al., 2005; Evans et al., 2017; Grünert et al., 2013; van der Meer et al., 1996; Molema et al., 2019a; Sass et al., 2004). In addition to low height Z scores, we also observed an elevated weight for height/age Z scores during the first five years of life, as well as elevated BMI Z scores after 5 years of age: a surprizing observation, since all children had an energy intake within 80-120% of EER at different ages. This discrepancy can be explained by two reasons; subjects could have been physically inactive due to their neurological deficits and accordingly would have needed less energy, but with the lack of data on physical activity from the charts, we cannot conclude whether or not their energy consumption was appropriate. In one study on 6 children with PROP, measured resting energy expenditure by indirect calorimeter was noted to be 20% less than calculated requirements (Feillet et al., 2000). Thus, subjects with PROP could only require 80% of their EER, except during periods of illness. Another explanation is the exclusion of dietary data from sick days, where subjects were supplied with energy at 120% of EER for age, which  60  could have affected their overall weight and BMI. The number of sick days varied among children: on average all children had 32 sick days in the first 6 months of life, and 35 days from 1-3 years (Table 4-1). During those days, children were consuming high energy (120%EER), none to low protein, which was increased gradually after the first two days. The impact of sick days and subsequent impact on growth cannot be quantified, however it is likely that the eventual outcomes were influenced by the episodes of sick days. Furthermore, protein requirements assume an adequate energy intake to ensure efficient protein utilization, thus protein and energy are interdependent (Uauy, 2013). This can be difficult to maintain in patient population with highly modified diets, which lead to the application of protein to energy ratio (P: E). This concept describes the proportion of energy derived from protein, if an individual is consuming a diet that meets energy needs, will the amount of protein be high enough to meet protein needs? Evans et al. showed that a ratio of > 1.5-<2.9 g of protein/100 kcal is associated with optimal growth in subjects with inborn errors of metabolism (Evans et al., 2017). In a natural history study on organic acidemia patients, the median intact protein to energy ratio was 1.23g/100kcal/d and it was positively associated with height Z score (Molema et al., 2019a). In the current study, calculated P: E ratios from total protein showed a median ratio that was within the optimal ratio. However, when intact protein intake was used to calculate the ratio, the results for all subjects were lower than the optimal ratio (Table 4-4). This further indicates that one of the reasons for the low height for age observed in all children were due to inadequate protein in relation to the energy supply. As stated above, protein and energy needs both need to be met for normal linear growth (Evans et al., 2017)   61  This suggests that the highly modified diets with the excess consumption of the nutritionally imbalanced medical food could have resulted in low P: E ratio, which can cause an increase in body weight and BMI, predisposing subjects to overweight. Although high weight for height is an indicator of obesity, in this case, with the lack of an adiposity measure, obesity should not be used to describe high weight for height seen in these children (de Onis and Blössner, 1997). Future studies could include body composition assessment in PROP subjects and observe changes longitudinally. In comparison to total protein consumption, intact protein intake was significantly lower than current guidelines, which indicates that the argument of increasing intact protein intake was translated into increasing protein from medical foods. In fact, the percentage of protein derived from medical foods was higher than intact protein (Figures 4-7 to 4-10 (E) Breakdown of total protein intake).However, it is also possible that these subjects did not tolerate higher than prescribed intact protein specially during infancy, and therefore medical foods were used as an alternative to increase total protein intake. It was taken into consideration that the Ross recommendation guidelines were used to manage children with PROP at BC Children’s Hospital between 1999 and 2003. The guidelines suggested 3.5 and 3 g/kg/d as the recommended intake values for total protein during infancy and childhood, respectively, especially if L-amino acids were used as the major source of protein intake. The Ross guidelines also advised clinicians to either supply all or half of the protein intake from medical foods (Phyllis B.Acosta and Steven Yannicelli, 2001). Therefore, total protein intake among all subjects were high in comparison to both the Sass and the GMDI guidelines. This high protein consumption can cause high nitrogen load especially for a patient cohort with a risk of chronic kidney disease and hyperammonemia.  62  It is also worth mentioning that with new guidelines, all subjects’ intakes of intact protein increased with age, and accordingly, medical food consumption decreased.  The use of medical food specially formulated for subjects with PROP can result in chronic imbalanced supplementation of leucine in comparison to the other BCAA, which can induce unintended effects on amino acid metabolism and transport (Myles et al., 2018). The effect of this imbalanced content of the BCAA was reported by many. In a cross-sectional study, Molema et al. reported that subjects receiving medical food had significantly lower plasma values of valine and isoleucine compared to subjects not receiving medical food. Moreover, plasma valine was positively associated with the amount of intact protein consumption and negatively associated with the amount of leucine in medical food used (Molema et al., 2019a, 2019b). Another study presenting the long-term outcomes and dietary data on PROP, reported low to very low plasma valine and isoleucine in all subjects (Touati et al., 2006). In this study, we did not report plasma amino acid concentrations, but the fact that subjects needed to be supplemented with single amino acids (valine and isoleucine) indicates that their plasma values were deficient. This can be explained by the antagonistic interaction among the BCAA (Harper, 1984). BCAA are essential for maintaining anabolism and supporting normal growth and development. A decrease in their plasma values can lead to an acute metabolic crisis, decompensation and growth impairment. In conclusion, despite adequate amount of total protein and energy intake, growth outcomes in all subjects were below population standards. Although various dietary protocols with different ranges of intact protein and medical foods were used, most studies reported growth outcomes similar to our results. This suggests that the restricted intact protein intake together  63  with the overuse of medical food could have affected subjects’ overall growth. Therefore, optimizing dietary management is the primary means to improving outcome in subjects with PROP. The imbalanced BCAA ratio in medical foods needs to be corrected by determining the ideal balance among LEU: ILE: Val to ensure children who do need to use medical foods are not compromised.  4.4 Limitations   There are some limitations associated with this study, including the small sample size of four patients. Due to the rarity of the disease, there were only six subjects with PROP being treated at BC Children’s Hospital. Two of them transitioned to adulthood in the early 2000’s; thus, collecting their data after 1999 would not have provided enough data for making any comparisons.  Although all four subjects were treated at the same clinic, there was high variability in their dietary treatments. Therefore, we could not perform any statistical correlations between diet and growth outcomes. While the dietary data represented actual intakes rather than prescribed, it did not describe intakes of the branched-chain amino acids. In this study, we assumed that subjects had low plasma isoleucine and/or valine concentrations that prompted the clinic to supplement them with single amino acids. Although the longitudinal growth data over 18 years in four subjects with PROP enabled us to confidently document growth pattern, there was no information on body composition. Moreover, one of the subjects (PROP-03) was found to be growth hormone deficient, which prompted the clinic to treat with growth hormones. This could have been the reason for their skewed height data.  64  Chapter 5: Determining Ideal Balance among BCAA as a Proof of Concept Study in Healthy Children  PROP is an inherited metabolic disorder, caused by a defect in the mitochondrial enzyme propionyl-CoA carboxylase (PCC), resulting in the accumulation of propionic acid metabolites. PCC catalyzes two of the branched-chain amino acids (valine, isoleucine), as well as methionine, threonine, odd-chain fatty acids and cholesterol (Baumgartner et al., 2014). The goal of nutritional management for PROP is to prevent the accumulation of toxic metabolites derived from propiogenic amino acids by restricting natural protein intake, while also maintaining normal plasma concentrations. Moreover, subjects with PROP usually need to supplement their diet with special medical foods, which are formulated to contain all essential amino acids and nutrients, but no propiogenic compounds. Due to the imbalanced content of BCAA (high leucine; minimal or no valine and isoleucine) in medical foods specially formulated for PROP, concerns have been raised about their use. An imbalanced mixture of BCAA reduces plasma concentrations of valine and isoleucine to below normal ranges, restricting total body protein synthesis and limiting growth in children with PROP (Manoli et al., 2016; Myles et al., 2018). Although recent dietary guidelines are recommending that medical foods only be used for individuals who cannot tolerate their RDA from intact protein, most of individuals with PROP are at risk for malnutrition and are depending on these medical foods as an easily tolerable source of energy and protein (Jurecki et al., 2019). Thus, there was a need to determine the ideal ratio of BCAA in medical foods to optimize protein synthesis and growth, and to prevent the accumulation of toxic metabolites. In this chapter, I discuss the methods and results of using the  65  stable isotope based minimally invasive technique in children, to determine the effect of different BCAA ratios (LEU: ILE: VAL) on body protein synthesis.  5.1 Methods and Materials  5.1.1 Study Principle The experimental design was based on the oxidation of the stable isotope L-[1-13C] phenylalanine to 13CO2 to compare protein synthesis in healthy children, under different test intakes (leucine) while other amino acids, including isoleucine and valine, are kept constant. This study was done as a proof of concept in healthy children, to allow for characterization of the metabolic response to different leucine test intakes, which will help us design different BCAA ratios to test in individuals with PROP. All procedures were reviewed and approved by the Committee for Ethical Review of Research involving Human Subjects at the University of British Columbia (H18-00439).  5.1.2 Subjects This study examined 8 healthy, school-aged children between 6 and 10 years. The upper limit for age range (10 y) was selected to avoid influence of hormones (puberty) on our results. Use of the IAAO to determine lysine requirement in women provided evidence that F13CO2 oxidation rate and therefore lysine requirement was affected by different menstrual cycle phases (luteal vs. follicular phase). Lysine requirement was found to be higher in the luteal phase, which may be attributed to the higher oxidation of amino acids during luteal phase compared to follicular phase (Kriengsinyos et al., 2004). Previous IAAO studies in children and adults have demonstrated that reliable results can be obtained by studying 5-6 subjects in a repeated measure  66  design (Elango et al., 2007; Mager et al., 2003; Rasmussen et al., 2016). Moreover, The ratio of BCAA was studied previously in adult men using the IAAO, where five healthy men were studied 7 times for a total of 35 studies (Riazi et al., 2003b).   5.1.2.1 Recruitment  Eight healthy children were recruited to participate in the study. Recruitment posters (Appendix C) were posted in local coffee shops, community centers, and doctors’ offices to recruit children. The poster included contact information such as cellphone, email address, and office number where interested parents could contact us for additional information. Participants were invited to participate in 7 studies per child for a total of 42 studies. A master list of participants and their assigned alphanumeric code was kept in a locked cabinet at BC Children’s Hospital Research Institute (Appendix F). Participants were compensated for transportation costs, including public transit passes or parking passes, and offered an honorarium ($100/ study day) for their participation. 5.1.2.2 Inclusion and Exclusion Criteria  Inclusion Criteria  Ø Healthy children 6-10 years old. Ø Normal weight (3rd -85th percentiles for weight according to the World Health Organization (WHO) Ø Normal eating habits (no food allergies)  Exclusion Criteria   67  Ø Children under 6 years old, or over 10 years old. Ø Children who are currently ill with a fever, cold, vomiting or diarrhea. Ø Children outside of normal weight parameters (3rd -85th percentiles for weight according to the World Health Organization (WHO)). Ø Children with claustrophobia. Ø Children currently or recently taking prescription medication or antibiotics. Ø Children with food allergies.   5.1.3 Experimental Design  Each subject was studied at seven different BCAA ratios (LEU: ILE: VAL). A negative control BCAA ratio of (1: 0: 0) was chosen to resemble the BCAA ratio in medical foods for subjects with PROP. Following this ratio, subjects received five different BCAA ratios: (1: 0.14: 0.15), (1:0.19: 0.20), (1: 0.21: 0.24), (1: 0.26: 0.28), (1:0.35:0.4), during which isoleucine and valine intake were kept constant based on recommended intakes for subjects with PROP (Blau, 2006; Phyllis B.Acosta and Steven Yannicelli, 2001). Leucine was provided in high amounts based on observed intakes of subjects with PROP (Manoli et al., 2016; Myles et al., 2018) and then gradually decreased by 25%. Lastly, the BCAA ratio found in egg protein was tested as a positive control, where we reduced leucine more than 25% to reach a ratio of (1:0.6:0.7) while also keeping isoleucine and valine constant (Figure 5-1). All study days were separated by a minimum of 1 week to ensure sufficient washout period between intakes.     68              a Isoleucine and Valine intakes were based on the recommended intakes for subjects with PROP (Isoleucine:1000 mg/day, Valine:1100 mg/day). Leucine intake was based on the observed intake of subjects with PROP (Leucine:7104 mg/day)  b based on egg protein pattern.8 Healthy children 6-10 years old                  (3boys: 3 girls)Total Caloric Intake = 1.7x REETotal Protein Intake= 1.5g/kg/dCarbohydrates= 55% Fat: 38%Negative Control ExperimentLEU: ILE: VAL1: 0: 0 Leucine7104 mg/dExperiment 1aLEU: ILE: VAL1: 0.14 : 0.15Leucine7104 mg/dExperiment 225% reduction in LeucineLEU: ILE: VAL1: 0.19: 0.20Leucine5328 mg/dExperiment 335% reduction in LeucineLEU: ILE: VAL1: 0.21: 0.24Leucine4618 mg/dExperiment 445% reduction in LeucineLEU: ILE: VAL1: 0.26: 0.28Leucine3907 mg/dExperiment 560% reduction in LeucineLEU: ILE: VAL1: 0.35: 0.4Leucine2842 mg/dPostive Control Experiment bLEU: ILE: VAL   1: 0.6: 0.7Leucine1600 mg/dFigure 5-1 Experimental Design   69   5.1.3.1 Pre-Study Day Protocol  Before the studies were started, all participants had an initial assessment to determine eligibility. All pre-study day assessment was conducted after an overnight fast (~12 hours), and took place at the Clinical Research Evaluation Unit (CREU), BC Children’s Hospital Research Institute. During this visit, the following measurements were taken: anthropometric measurements (weight and height), body composition analysis, and resting energy expenditure (REE). Weight and height were measured using a digital scale and a stadiometer, respectively. Body composition was measured by bioelectrical impedance analysis (BIA model Quantum IV; RJL Systems), and, using the manufacturer’s software system (RJL Systems, Body Composition Analysis V.2.1), we calculated fat mass (FM) and fat free mass (FFM). REE, which describes the caloric requirement for body functions in the absences of any physical activity, was measured by continuous, open-circuit indirect calorimetry (Vmax Encore, Viasys, CA). REE was used to calculate total energy content of the study diet in order to ensure that caloric needs for each child were met. A general questionnaire was used during the pre-study day assessment to collect information about medical history, nutritional status, and physical activity (Appendix D). Participants were screened to have no history of recent weight loss or illness. The purpose of the study and the potential risks involved were explained in detail to each participant’s parent or guardian and written informed consent (Appendix A) and assent (Appendix B) forms were obtained for all children from their parents or guardians. A multivitamin was provided for each child to ensure adequate intake of vitamins and minerals (Centrum Junior Complete). A three-day food record was collected from each subject to allow the prescription of a standardized diet  70  to be consumed prior to each study day. (Appendix G). We used food models to advise parents or guardians on how to record food intakes.    5.1.3.2 Study Day Protocol  Two days before each study day, subjects consumed a maintenance diet supplying 1.7 x REE energy and 1.5 g protein/kg/day. Maintenance diet was used to standardize participants’ protein intake, which was determined by analysing the food record collected during the pre-study day using a nutrient analysis database (Food Processor SQL 11, ESHA Research). Participants were also asked to keep a two-day food record during their maintenance diet to ensure consistency of dietary protein intake among subjects. Parents were advised to increase, decrease or maintain their children’s protein intake in order to achieve a total of 1.5g/kg/d. Food choices were based on each child’s food preferences.  Each study was carried out at the Clinical Research Evaluation Unit (CREU), BC Children’s Hospital Research Institute, after an overnight fast (~12 hours). Weight and height measurements were taken, then each study proceeded in accordance with the minimally invasive IAAO model (Figure 5-2) (Appendix E) (Bross et al., 1998). The study day diet was consumed as hourly isocaloric meals Each meal was formulated to represent one-twelfth of each subject’s daily energy requirements, depicting a 12-hour feed condition. The experimental diet consisted of protein-free liquid formula made with protein-free powder (PFD1, Mead Johnson, Evansvile, IN), flavored with added drink (Tang and Kool-Aid, Kraft Foods, Toronto, Canada), corn oil and protein-free cookies. Energy was be provided at 1.7 x REE, based on each subject’s measured REE after 12-hour fast, as described earlier. The diet provided 55% of the energy as  71  carbohydrates, 38% as fat and 7-10% as protein. Protein was added as a crystalline L-amino acid mixture based on egg protein pattern, except for BCAA, which followed a special pattern determined according to the recommended BCAA intakes in individuals with PROP. The test leucine doses were provided as crystalline L-leucine graded into 7 different doses in 7 different BCAA ratios.                 Figure 5-2 Study Day Protocol   72  5.1.3.3 Stable Isotope Protocol  On each study day, participants consumed 4 hourly meals, before the consumption of the tracer isotope. The oral isotope consumption protocol started at the fifth meal on each study day. Subjects consumed a priming oral dose of 2.5 mg/kg of L-[1-13C] phenylalanine, a priming dose of NaH13CO3 of (0.176mg/kg/day) and a continuous dose of (1.4 mg/kg/h) of L-[1-13C] phenylalanine (99 atom percent excess, Cambridge Isotope Laboratories Inc., Andover, MA) until the end of the study. The quantity of phenylalanine provided in the isotope was subtracted from the diet to provide total amount of 30.5 mg/kg/day phenylalanine. Tyrosine was given at a total of 61 mg/kg/day to ensure that an excess amount of tyrosine was available to separate the carboxyl carbon of phenylalanine between incorporating into protein synthesis or oxidations (Elango et al., 2011). The priming dose of NaH13CO3 of (0.176mg/kg/day) was given with the fifth meal to prime the body pool to more rapidly achieve steady state 13C enrichment (Hoerr et al., 1989).        73  5.1.3.4 Sample Collection  On each study day, breath and urine samples were collected as baseline and plateau samples. On the study day, after the fifth meal was consumed, the indirect calorimeter (Vmax Encore, Viasys, CA) was used for 20 minutes to measure the rate of carbon dioxide production (VCO2).  Breath samples were collected three times as a baseline 45, 30, and 15 minutes before the consumption of the isotope, and 2.5 hours after the start of the tracer protocol, six breath samples were collected before study end (Figure 5-2) (Elango et al., 2007). Breath samples were collected in disposable vacuum Exetainer tubes (Labco Ltd) by using breath bags (Single use collection bags, EasySampler System, QuinTron, Terumo Medical). Participants were taught to keep their mouth closed over the mouthpiece and take a normal breath then blow the air into air bag, at the same time 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. The Exetainer tubes were prepared by using a vacuum needle pump to bring the interior pressure down to a very low internal pressure. This ensured that the exetainers draw in a ~10 ml breath sample during the sampling process. These samples were then labeled with individual subject ID, breath sample number, and set ID, and stored at room temperature following collection, until analyzed with isotope ratio mass spectrometry (IRMS, IsoPrime 100)  In accordance with the minimally invasive method, we collected urine samples in place of plasma samples to measure amino acid concentration (Rasmussen et al., 2016). Urine samples were collected at the beginning of the study day before the first meal, 15 minutes prior to the oral isotope consumption, and at the end of the study day (Figure 5-2). Samples were collected in  74  urine hats (Specimen Container w/pour spout, 6.5 oz, Medegen), and 10 mL aliquot of urine was transferred into 15 mL conical tubes (BD Falcon, Mississauga ON) which contained 200 μL 10% HCL to suppress bacterial growth. One mL of HCL urine mix was transferred to microcentrifuge tubes. In the event that participants were unable to produce the small amount of urine per sample time, participants were offered a cup of water, and asked to try again after a few minutes. Water was made freely available throughout the course of the study day to ensure proper hydration, and starting 20 minutes before an upcoming urine sample collection, participants were reminded to drink plenty of water. All urine samples were stored at -80°C for later analysis. Urinary concentrations of BCAA were determined by ion exchange chromatography with post-column ninhydrin derivatization using an Amino Acid Analyzer (AAA) (Hitachi L8900, Tokyo, Japan), and using a modified procedure as previously described (Le Boucher et al., 1997). 100 μL of 1% TCA was added to 1000 μL of urine, followed by centrifugation for 15 min at 10000 rpm at 4 °C. The resulting supernatant was filtered and then 440 μL dried down using a speed vac (Savant, Thermo Electron Corporation, SPD 131DDA). After the sample had dried, it was diluted with 55 μL water, vortexed, and 100 μL was aliquoted into glass vile insert, and 25 μL was injected into the AAA. The amino acids were separated using an ion exchange column (Hitachi Packed Column #2622 6.0 Å~ 40 mm Li Type, Tokyo, Japan,) and analyzed against an amino acid standard mix (AA-S- 18, Sigma, St Louis, MO). The areas under the peaks were integrated using the EZChrom Elite software (version 3.3.2 SP2; Agilent, ON, Canada). From the same aliquot of urine, urinary creatinine concentrations were measured using HPLC (Chromaster 5430 Diode Array Detector, Hitachi, Tokyo, Japan) using a modified procedure as previously described (Tsikas et al., 2004), on a C18 Agilent 2.1 x mm column (EC 125/2  75  Nucleosil 100-3 C18, Phenomenex, CA, USA), to allow standardization of urinary amino acid concentrations on a g creatinine basis. 375 μL of buffer (10 mmol/L of sodium salt of 1- octanesulfonic acid, pH 3.2) was added to 125 μL of filtered urine and vortexed, and 50 μL was injected into the HPLC.    5.1.4 Sample Analysis  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. (Elango et al., 2007; Humayun et al., 2007).  5.1.5 Data Calculations F13CO2 is the rate of 13CO2, that was released in the breath after L-[l-13C] phenylalanine oxidation (μmol/kg/h), and it 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 the expired breath at isotope steady state atom percent excess (APE), W is the subject body weight (kg). The constants 44.6 (μmol/mL) and 60(min/h) will be used to convert FCO2  to micromoles per hour. The factor 0.82 is the correction for CO2, and the factor 100 converts the APE to a fraction (Hoerr et al., 1989).  76   5.1.6 Statistical Analysis  Subject characteristics are expressed as means ± SDs. The Shapiro Wilk test was used to check for normality. Repeated Measures ANOVA was used to measure the difference among means of F13CO2  oxidations in response to 7 test intakes (BCAA ratios), as well as means of BCAA urinary excretions using GraphPad Prism 4.0 (GraphPad Software Inc, CA). In all cases, the difference was considered significant at P < 0.05. When warranted, post hoc analysis was performed using Tukey’s multiple comparisons test to confirm where the differences occur between different conditions (BCAA ratios).   5.2 Results  5.2.1 Subject Characteristics A total of 8 healthy children were studied, completing 7 study days (NSubjects = 8, NStudies = 42). Four subjects participated in all seven test intakes, two subjects participated in 4 test intakes and two subjects participated in three test intakes, completing a total of 42 oxidation study days. During the study period, two children moved out of the city and therefore we had to recruit additional children to complete all study test intakes. Children’s ages ranged from 7-9 years old with a mean of (8.3 ± 0.81). They were all in good health and had normal body mass index (BMI) for their age, mean BMI 16.02 ± 1.59. One participant was taking 30 mg of VYVANS (Lisdexamfetamine), a treatment for attention deficit hyperactivity disorder in children aged 6-10 years. This compound is bonded to L-lysine, an essential amino acid, that, after oral ingestion, realizes lysine into the circulation (K.A et al., 2007). The amount of lysine in the drug was  77  considered very low and did not significantly increase total protein intake during study day. The medication was not consumed on study days. Subjects had a mean of 1.78± 0.3 for protein intake during the two-day adaptation prior to study day. This variation in protein intake was not expected to affect our results. Thorpe et al., showed that consuming 0.8, 1.4 and 2 g/kg/d protein for two days prior to study, did not affect F13CO2 oxidation rates (Thorpe et al., 1999) (Table 5-1).  Table 5-1 Subject Characteristics  Characteristic Value1 Age (years) 8.4 ± 0.74 Male: Female  4: 4 Weight (kg) 29.4 ± 5.3 Height (cm) 132.8 ± 5.6 BMI (kg/m2) 16.7 ± 1.6 Fat Free Mass (%)2 76.5 ± 6.8 Fat Mass (%)2 23.4 ± 6.8 REE (Kcal/d)3 1073 ± 138 Protein Intake g/kg4 1.78± 0.3 1 Values are mean ± SD (N=8) 2 Determined by electrical impedance analysis (BIA) 3 Resting Energy Expenditure determined by open-circuit indirect calorimeter 4 Values derived from two-day food records prior to each study day    78  5.2.2 F13CO2 Oxidation  F13CO2 data were normally distributed among all test intakes (Shapiro Wilk test, p value >0.05). Repeated measures ANOVA showed significant differences in F13Co2 with different test intakes (BCAA ratios); P value < 0.001, R2 = 0.9281. F13Co2, which represents the rate of the release of 13CO2 from phenylalanine oxidation that reflects total body protein synthesis, was significantly higher when a ratio of 1:0:0 (LEU: ILE: VAL) was tested compared to all other ratios (p value <0.001), using Tukey’s multiple comparison. This indicates that total body protein synthesis at this ratio was low. Oxidation rates associated with these ratios (1:0.19: 0.20), (1: 0.21: 0.24), (1: 0.26: 0.28) were low with no significant differences between them. However, when the ratio of (1:0.35:0.4) was tested, oxidation rate as indicated by F13CO2 increased significantly compared with (1:0.19: 0.20), (1: 0.26: 0.28) p value <0.05. The data taken together suggests that a BCAA ratio between 1: 0.26: 0.28 and 1:0.35:0.4 may enhance total body protein synthesis (Figure 5-3).           79               a leucine (7100 mg/d), Isoleucine and Valine (0 mg/d) b Leucine (7100mg/d), Isoleucine (1000mg/d), Valine (1100 mg/d) c Leucine (5380mg/d), Isoleucine (1000mg/d), Valine (1100 mg/d) d Leucine (4618mg/d), Isoleucine (1000mg/d), Valine (1100 mg/d) e Leucine (3907mg/d), Isoleucine (1000mg/d), Valine (1100 mg/d) f Leucine (2842mg/d), Isoleucine (1000mg/d), Valine (1100 mg/d) g Leucine (1600mg/d), Isoleucine (1000mg/d), Valine (1100 mg/d)   Figure 5-3 F13Co2 Oxidation Rate at Different Test Intakes  Negative Control (1:0:0) a1: 0.14 : 0.15 b1: 0.19: 0.20 c1: 0.21: 0.24 d1: 0.26: 0.28 e1: 0.35: 0.4 fPositive Control (1: 0.6: 0.7) g0.00.51.01.52.02.53.0F13 Co 2 OxidationNSubjects= 8  NStudies= 42****** < 0.001   *  < 0.05*BCAA Ratio (Leucine: Isoleucine: Valine) 80  5.2.3 Urine BCAA Concentrations   Urinary BCAA concentrations were measured from the last time point sample at the end of each study day. Urinary BCAA data were all normally distributed according to the Shapiro Wilk test, p value > 0.05. ANOVA showed significant differences between urinary leucine concentrations (p value < 0.001). With decreasing intakes of leucine, urinary leucine concentrations (standardized to per g creatinine) continued to decrease (P value <0.05) (Figure 5-4A). However, urinary leucine concentrations were higher than reference values for 4-13-year-old children (BC Children’s Hospital Laboratories), except when a ratio of (1:0.6:0.7) was tested, where leucine intake was 1600 mg/d, equivalent to 57 mg/kg/d for a child weighing 28kg: in this case, urinary leucine concentration reduced significantly compared to all test intakes. Isoleucine urinary concentrations were always within normal reference values, with significant lower concentrations seen when a ratio of (1:0:0) was tested compared to (1:0.19: 0.20), and (1: 0.35:0.4) (Figure 5-4B). Valine urinary concentration was below normal reference values when a ratio of (1:0:0) was tested, and that was significantly lower than concentrations associated with a ratio of (1: 0.35:0.4), where valine urinary concentration increased to be within normal reference values (Figure 5-4C). 81  Figure 5-4 Urinary Amino Acids Concentrations A) Leucine Urinary Concentration. B) Isoleucine Urinary Concentration. C) Valine Urinary Concentration a leucine (7100 mg/d), Isoleucine and Valine (0 mg/d) b Leucine (7100mg/d), Isoleucine (1000mg/d), Valine (1100 mg/d) c Leucine (5380mg/d), Isoleucine (1000mg/d), Valine (1100 mg/d) d Leucine (4618mg/d), Isoleucine (1000mg/d), Valine (1100 mg/d) e Leucine (3907mg/d), Isoleucine (1000mg/d), Valine (1100 mg/d) f Leucine (2842mg/d), Isoleucine (1000mg/d), Valine (1100 mg/d) g Leucine (1600mg/d), Isoleucine (1000mg/d), Valine (1100 mg/d) 82   5.3 Discussion The purpose of this proof of concept study was to determine a BCAA ratio at which total body protein synthesis is optimized. We used the minimally invasive indicator amino acid oxidation technique (IAAO), and L-1-13C-Phenylalanine as the indicator amino acid for protein synthesis to test the effect of different BCAA ratios in healthy children. Results from the current study confirmed that a BCAA ratio of (LEU: ILE: VAL =1:0:0), similar to the BCAA ratio in medical foods specially formulated for PROP, limits total body protein synthesis as indicated by F13CO2. Moreover, a balanced BCAA ratio at which total protein synthesis is optimized may be found between (1: 0.26: 0.28) and (1: 0.35:0.4). By using the IAAO, this study provides the first direct test of the hypothesis that the current BCAA mixture found in medical foods for PROP is inadequate.   The IAAO is a functional method based on the concept that the amount of the limiting amino acid controls the partitioning of the other essential amino acids between protein synthesis and oxidation. Therefore, when the limiting amino acid is provided, protein synthesis will increase, and the oxidation of the indicator amino acid will decrease (Elango et al., 2008). In this study, we did not limit the intake of any amino acids directly (except in the negative control experiment) (Figure 5-1). Instead, we provided leucine in excess amounts that may increase the oxidation of isoleucine and valine and therefore limit their availability for protein synthesis (Harper et al., 1984). Thus, we hypothesized that the oxidation of the indicator amino acid indicated by F13CO2 would be high when leucine is given in high amounts and it would decrease when leucine intake decreased, reaching a balanced ratio with the other BCAA. The study design  83  included seven different test intakes (BCAA ratios LEU: ILE: VAL). We gradually decreased leucine intakes while keeping isoleucine and valine constant at the recommended levels for children with PROP.  When a ratio of (LEU: ILE: VAL=1:0:0) was tested, the oxidation rate of the indicator amino acid increased as indicated by F13CO2 compared to the rates associated with all other ratios tested (P value <0.001); this suggests that protein synthesis was extremely limited. To our knowledge, this is the first study to test the effect of BCAA ratio of (LEU: ILE: VAL =1:0:0) on total body protein synthesis using the IAAO. Moreover, isoleucine and valine urinary concentrations at this imbalanced ratio were significantly lower than concentrations associated with other ratios (P value <0.05). Although, isoleucine urinary concentration was still within normal reference values, valine was reduced to less than normal reference values at (LEU: ILE: VAL =1:0:0). In this study, urinary amino acids concentrations were compared with values obtained from the BC Children’s Hospital Laboratories for children 4-13 years of age. Although children consumed adequate protein diets (1.5 g/kg/d) including high leucine (~250mg/kg/d for a child weighing 28 kg) and all other essential and non-essential amino acids, their total body protein synthesis was still limited due to the absence of isoleucine and valine from the diet. Despite the fact that isoleucine and valine are considered among the offending compounds for PROP, that are metabolized to produce propionate, they are essential amino acids and the body needs them in an adequate amount to maintain homeostasis, protein synthesis, and growth. Therefore, if subjects with PROP were to depend on medical foods as a sole source of nutrition, they may have low plasma and urinary isoleucine and valine, limited body protein synthesis and restricted growth. In fact, many studies have reported that excess consumption of medical foods  84  was associated with low plasma isoleucine and valine concentrations in subjects with PROP, which prompted their clinics to supplement with single amino acids (L-isoleucine and/or valine) (Manoli et al., 2016; Molema et al., 2019a). Supplementing with isoleucine and valine to ameliorate side effects caused by excess leucine consumption was reported in animals fed low protein diet (Harper, 1984). However, in the case of PROP, supplementing with single L-isoleucine and valine can be harmful (Jurecki et al., 2019).  The BCAA have similar structures and share common enzymes for metabolism and transportations. Interactions between disproportionate BCAA intakes have been reported in human and animals. The addition of excess leucine amounts to a low protein diet depleted isoleucine and valine pools, and depressed food intake and growth in animals (Harper, 1984). High intakes of leucine enhanced the activity of the branched-chain keto acid dehydrogenase enzyme in various tissues, that increased the oxidation of isoleucine and valine, which limited their availability for protein synthesis (Harper, 1984). Similarly, excess leucine supplementations resulted in reducing plasma and urinary isoleucine and valine in healthy elderly men (Elango et al., 2016b). In subjects with PROP, the effect of high leucine intake associated with excess consumption of medical food on plasma valine and isoleucine and overall growth outcomes was reported by many (Molema et al., 2019a, 2019b; Touati et al., 2006). Just because leucine is not metabolized into propionic acids, we cannot assume that any amount of leucine is safe for subjects with PROP; as clearly shown by our 1:0:0 of LEU: ILE: VAL diet and negative impact on whole body protein synthesis.   In this study, by gradually decreasing leucine intakes by 25%, starting with high amounts, like those found in medical foods, while keeping isoleucine and valine constants, we tested the effect of the following BCAA ratios on total body protein  85  synthesis; LEU: ILE: VAL = (1:0.14: 1.15) , (1:0.19: 0.20), (1: 0.21: 0.24), (1: 0.26: 0.28), (1: 0.35:0.4) and (1:0.6: 0.7 reflecting egg protein pattern). Oxidation rates associated with these ratios (1:0.19: 0.20), (1: 0.21: 0.24), (1: 0.26: 0.28) were low with no significant differences between them. However, when the ratio of (1: 0.35:0.4) was tested, oxidation rate as indicated by F13CO2 increased significantly compared with (1:0.19: 0.20), and (1: 0.26: 0.28) p value <0.05. This may indicate that a BCAA ratio between (1: 0.26: 0.28) and (1: 0.35:0.4) enhanced total body protein synthesis. Moreover, by gradually decreasing leucine intakes, leucine urinary concentration decreased from higher than refence values to within normal values for healthy children 4-13 years old. As well, valine urinary concentrations improved when leucine intakes were reduced.  We can speculate that when we tested a ratio of (1:0.14: 1.15), there was a relative excess of leucine that stimulated the oxidation of isoleucine and valine through the branched-chain keto acid dehydrogenase enzyme, limiting their availability for protein synthesis. This ratio was also associated with leucine urinary concentration higher than normal reference values. The ratio of (1: 0.6 :0.7) derived from egg protein pattern was associated, though not significantly, with a numeric increase in oxidation rate. This may be explained by the fact that total BCAA intake at this ratio was low in some subjects (106-150 mg/kg/d), compared to the total BCAA requirement in healthy school-aged children, determined by the IAAO 147 mg/kg/d (Mager et al., 2003). Furthermore, at this ratio while leucine urinary concentration significantly decreased, isoleucine and valine urinary concentrations increased compared to other test intakes. Similarly, Elango et al. showed that plasma leucine concentration was low, while isoleucine and valine concentrations were high in piglets fed low BCAA test intakes. This indicated that while leucine  86  was extracted and used by the gut, thus limiting protein synthesis, isoleucine and valine were passing to the circulation. While leucine limited protein synthesis, isoleucine and valine were excreted in the urine (Elango et al., 2002). Another explanation would be that the BCAA ratio in egg protein (LEU: ILE: VAL = 1:0.6:0.7) is not optimal for protein synthesis. Using the IAAO, Riazi et al. tested different BCAA ratios to determine whether the BCAA ratio in egg protein was optimal for protein synthesis. They concluded that valine may be limited in a ratio of (LEU: ILE: VAL = 1:0.6:0.7), and that this could limit total body protein synthesis (Riazi et al., 2003b).   In the current study, the effect of the imbalanced BCAA ratios on F13CO2 oxidation rate was less than expected, and that may be explained by the small reduction in leucine between test intakes (25%). In addition, our subjects consumed adequate protein diets during study days (1.5g/kg/d), so the effect of the imbalanced BCAA ratio was smaller in comparison to that in other studies, where they supplied a deficient protein diet that could have exacerbated the BCAA antagonism interactions (Harper, 1984). In conclusion, the effect of different BCAA ratios on total body protein synthesis was tested for the first time on healthy children in a non-invasive repeated measure and it was concluded that the current BCAA ratio found in medical foods for PROP is inadequate. A ratio between (1:0.26: 0.28) and (1: 0.35:0.4), where leucine intake can be provided between (3907-2842 mg/d equivalent to 102-140 mg/kg/d for a child weighing 28 kg) could improve plasma and urinary BCAA concentration, total body protein synthesis, and therefore growth. Thus, with the current isoleucine and valine recommendations for PROP, metabolic dietitians are encouraged to gradually reduce leucine intake to 102-140 mg/kg/d for a child weighing 28 kg, by limiting the use of medical foods for subjects who cannot tolerate their RDA from intact protein. Moreover, reformulating the medical foods by reducing leucine content  87  by 50% would be a step forward for the manufacturers. Further research is needed to determine the optimal BCAA ratio in subjects with PROP to optimize their total protein synthesis.   5.3.1 Limitations This was a proof of concept study in healthy children to establish an adequate BCAA ratio that optimizes protein synthesis. Therefore, results from this study cannot be generalized to patient populations. This was a short-term with an acute dose response study, where each subject was studied 7 times with different BCAA ratios. Thus, future studies are needed to assess the long-term effect of these BCAA ratios on growth pattern. Another limitation is the small sample size (4 boys and 4 girls). We studied healthy children to characterize the metabolic response of different BCAA ratios in order to design different BCAA ratios to test in subjects with PROP.  88  Chapter 6: Conclusions and Future Directions Growth failure is one of the most common complications in subjects with propionic acidemia (Baumgartner et al., 2014). Although numerous factors can affect growth outcomes, including physiological, genetic and environmental factors, dietary protein restriction is probably the main contributor to growth failure in subjects with PROP (Lui et al., 2015). In the first study, we confirmed that despite adequate intake of total protein and energy, all four subjects with PROP had poor growth outcomes. All subjects had persistently low height for age Z scores combined with high weight and BMI Z scores, which are indications of stunting, overweight and an abnormal body composition. Analyzing dietary treatments in the management of four subjects with PROP demonstrated that subjects were consuming a diet that is restricted in intact protein. However, they were consuming total protein that exceeded the recommendations, due to their high intake of medical foods.  High consumption of medical foods could have resulted in a chronic and imbalanced supplementation of leucine relative to the other two BCAA (isoleucine and valine), therefore restricting overall growth. In the second study, we confirmed that the BCAA ratio of (LEU: ILE: VAL = 1:0:0), which is similar to the ratio found in the medical foods, limited total body protein synthesis. Using the IAAO method in healthy children, we tested for the first time the effect of BCAA ratio of (LEU: ILE: VAL = 1:0:0) on total body protein synthesis. Furthermore, a balanced BCAA ratio that optimized protein synthesis using the IAAO method was found to be between (1: 0.26: 0.28) and (1: 0.35:0.4). By decreasing leucine intake from the current high doses found in medical foods, results demonstrated a significant reduction in leucine urinary  89  concentrations to normal ranges, with a subsequent increase in both valine and isoleucine urinary concentrations within normal ranges.  To optimize dietary management in subjects with PROP, further research is needed to determine the optimal intake of medical foods relative to intact protein. Furthermore, in a cohort of subjects with PROP, the effect of different distributions between intact protein and medical foods should be studied with a focus on long-term clinical outcomes including growth and metabolic control. In addition, since the current isoleucine and valine requirements in subjects with PROP are based on an individualized clinical and laboratory assessment, there is a need to determine isoleucine and valine requirements using a direct measure like the IAAO. Future research is also needed to determine the optimal BCAA ratio to optimize total body protein synthesis in subjects with PROP. In summary, with the findings from this thesis, when current isoleucine and valine recommendations for PROP management are being followed, metabolic dietitians are encouraged to decrease leucine intake by limiting the use of medical foods. In subjects who tolerate less than 100%of the RDA from intact protein, medical foods should be added only to complement protein needs as a secondary source to achieve 100-120% of RDA. Finally, we propose reformulating the BCAA mixture in medical foods, by reducing leucine content by 50%.  90  Bibliography  Alfadhel, M., Benmeakel, M., Hossain, M.A., Al Mutairi, F., Al Othaim, A., Alfares, A.A., Al Balwi, M., Alzaben, A., and Eyaid, W. (2016). Thirteen year retrospective review of the spectrum of inborn errors of metabolism presenting in a tertiary center in Saudi Arabia. Orphanet Journal of Rare Diseases 11. Almási, T., Guey, L.T., Lukacs, C., Csetneki, K., Vokó, Z., and Zelei, T. (2019). 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Protein and amino acid requirements in human nutrition: report of a joint WHO/FAO/UNU Expert Consultation ; [Geneva, 9 - 16 April 2002] (Geneva: WHO).   98  Appendices Appendix A   : Subject Consent Form                                        99    100    101    102    103    104    105    106    107   Appendix B  : Subject Assent Form               108    109    110    Appendix C  : Recruitment Poster                 111  Appendix D  : Pre-Study Day Assessment Form                 112    113  Appendix E  : Study Day Form                                         Study Day Protocol                            BCAA Ratio in Medical Foods                             Page 1 of 1 Ver.1 April 11, 2018 		 	 Study	Day	Protocol	Determining	the	Optimal	Ratio	of	Branched-Chain	Amino	Acids	(BCAA)	in	Medical	Foods			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)		 	 	15:00	 5th	breath	(3x)		 Meal	#8	–	4th	oral	dose	Collect	breath	1st	 	15:15	 6th	breath	(3x)	 	 	15:30	 7th	breath	(3x)		 	 	15:45	 8th	breath	(3x)		 	 	16:00	 9th	breath	(3x)	3rd	urine	 	 		 114  Appendix F  : Subject Code Form                                        Subject Code Master List                   BCAA Ratio in Medical Foods                        Page 1 of 1 Ver.1     April 11, 2018                                                                                      Subject Code Master List  Determining the Optimal Ratio of Branched-Chain Amino Acids (BCAA) in Medical Foods   Subject Name  Code (Alpha-Numeric) Comments                                                                    115  Appendix G  : Dietary Record Sheets                 Dietary Record sheet                                                                   BCAA Ratio in Medical Foods                             Page 1 of 1 Ver.1                                                                                          April 11, 2018  Dietary Record           Subject ID:__________________________  Date: ____/_____/_____        Mon   Tues    Wed    Thu   Fri    Sat    Sun           Item            Amount                 Item              Amount  Breakfast   Snack           Snack   Dinner          Lunch               116  Appendix H  : WHO Growth Charts for Canada (PROP-01 Weight for Age >10 years)                                          GIRLS32 654 8 9 10 11 12 13 14 15 16 17 18 19732 654 8 9 10 11 12 13 14 15 16 17 18 19720304050195 1951901851801751701651601551501451401901851801701751651601551501451401351301251201151101051009590858075SOURCE: The main chart is based on World Health Organization (WHO) Child Growth Standards (2006) and WHO Reference (2007) adapted for Canada by Canadian Paediatric Society, Canadian Pediatric Endocrine Group (CPEG), College of Family Physicians of Canada, Community Health Nurses of Canada and Dietitians of Canada. The weight-for-age 10 to 19 years section was developed by CPEG based on data from the US National Center for Health Statistics using the same procedures as the WHO growth charts.© Dietitians of Canada, 2014. Chart may be reproduced in its entirety (i.e., no changes) for non-commercial purposes only. www.whogrowthcharts.ca2 TO 19 YEARS: GIRLSHeight-for-age and Weight-for-age percentilesWHO GROWTH CHARTS FOR CANADA GIRLSNAME:DOB: RECORD #1015202510152025304045505560657075808590AGE (YEARS)AGE (YEARS)WEIGHTHEIGHTWEIGHTHEIGHT3940414243444546474849505152535455565758596061626364656667686971707273747576777838373635343332313029in68697071727362636465666756555457585960617475767778in20304050608090100lb110120130140150160170180190200kglb kglbcm cm35MOTHER’S HEIGHTFATHER’S HEIGHTDATE AGE HEIGHT WEIGHT COMMENTS315508597315508597WHO recommends BMI as the best measure after age 10 due to variable age of puberty. Tracking weight alone is not advised.PROP-01 117  Appendix I  : WHO Growth Charts for Canada (PROP-02 Weight for Age >10 years)                                           118  Appendix J  : WHO Growth Charts for Canada (PROP-03 Weight for Age >10 years)  

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