AROMATIC AMIN0 ACID METABOLISM IN THE HUMAN: ESTlMATlON OF TYROSINE REQUIREMENT IN THE NEONATE AND ADULT Susan Ann Roberts A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Nutritional Sciences University of Toronto O Copyright by Susan Ann Roberts 1999 National Library 8ibliothèque nationale 1*1 of Canada du Canada Acquisitions and Acquisitions et Bibliographie Services services bibliographiques 395 Wellington Street 395, nie Wellington Ottawa ON K1A ON4 Ottawa ON KIA ON4 Canada Canada Yow fiie Votre Merenff Our file Notre reférence The author has granted a non- L'auteur a accordé une licence non exclusive licence allowing the exclusive permettant à la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or sel1 reproduire, prêter, distribuer ou copies of this thesis in microform, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/fih, de reproduction sur papier ou sur format électronique. The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fkom it Ni la thèse ni des extraits substantiels may be printed or othemise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. AROMATIC AMIN0 ACID METABOLISM IN THE HUMAN: ESTIMATION OF TYROSINE REQUIREMENT IN THE NEONATE AND ADULT Doctor of Philosophy, 1998 Susan Ann Roberts Graduate Departrnent of Nutritional Sciences University of Toronto ABSTRACT Tyrosine is indispensable in the neonate. The provision of an adequate source of tyrosine in the parenterally-fed neonate however, is complicated by its poor solubility. While some of the presently available parenteral amino acid solutions have cornpensated with increased phenylalanine levels, paediatric amino acid formulations have not. Using a 24h primed, continuous intravenous infusion of isotopically labelled phenylalanine and tyrosine, aromatic amino acid metabolism was examined in neonates (n=16) receiving total parenteral nutrition, containing high or modest levels of phenylalanine. Neonates responded to increased phenylalanine intake by elevating phenylalanine hydroxylation and oxidation rates. Neonates also increased urinary excretion of aromatic amino acids and alternate metabolites of phenylalanine and tyrosine catabolism. These data suggest that phenylalanine supplementation may not be the most appropriate approach to meeting total aromatic amino acid needs of the parenterally- fed neonate. To improve the aromatic arnino acid iotake of the moderate phenylalanine-containing amino acid solution, tyrosine requirement was estimated in neonates (n=13) using graded intakes of glycyl-L-tyrosine as a soluble source of tyrosine. It was hypothesized that a tyrosine requirement could be estimated by pariitioning the oxidation of phenylalanine to a two-phase linear regression analysis whereby the intersection of the lines would be representative of the mean requirement. Identical rnethods to the previous study were used. The mean tyrosine requirement was 74 mg-kg-'-d-' and the corresponding safe level of intake (upper 95% confidence limit) was 94 mg-kg-'-d" representing 3.1 and 3.9% of total amino acids, respectively. Using a similar study design, but with an oral isotope protocol, an estimate of the tyrosine requirement within healthy adult male volunteers (n=6) receiving a fked phenylalanine intake was estirnated. The mean tyrosine requirement was found to be 6 mg-kg-'-d" and the corresponding safe intake was 7 mg-kg".d". The ideal intake of the aromatic amino acids was found to be in a phenylalanine to tyrosine balance of 56:44 to 60:40. This is very different from that provided to parenterally-fed neonates. Data from these studies form the foundation upon which new parenteral amino acid formulations can be developed. iii ACKNOWLEDGEMENTS I wish to thank my thesis supervisors Dr. Paul Pencharz and Dr. Ron 8alI for the opportunity to train under their excellent guidance. I am certain that the experience will serve me well for years to corne. I would also Iike to thank the members of my advisory committee, Dr. Stephen Cunnane, Dr. Mitch Halperin and Dr. Stan Z lotkin for their constructive advice. I am grateful to the surgeons, neonatologists and nurses of the Neonatal Intensive Care Unit at The Hospital for Sick Children for their support and valuable advice throughout the study. The contribution and technical expertise of the staff in the Pharmacy and Genetic Metabolics Departments at The Hospital for Sick Children is gratefuliy acknowledged. I appreciate the support and confidence expressed by the parents in agreeing to have their precious newborns participate in a research investigation. Working with Rachelle Bross, Glenda Courtney-Martin, Pauline Darling, Larry Fisher, Sandra Parker, Mahroukh Rafii, Jane Thorpe and Connie Williams was a genuine pleasure as these individuals became true friends. This work entails rewards over and above the scientific findings held within. Exposure to a critical care unit such as the NlCU at The Hospital for Sick Children revealed to me the great hope that cornes with high quality medical care. This thesis is dedicated to the Roberts family for their encouragement in everything i do. The research was conducted with financial support from the Medical Research Council of Canada. The protein-free powder was generously donated by Mead Johnson, Canada and the Primene was generously donated by Baxter . Personal financial support from the University of Toronto (Simcoe Scholarship. Open Fellowship), The Fonds Pour la Formation de Chercheurs et l'Aide a la Recherche, and the Ontario Graduate Scholarship prograrn are gratefully acknowledged. The data presented in Chapter 4 have been accepted for publication: Roberts, S.A., Ball, R.O., Moore, A., Filler, R.M. and Pencharz, PB., 1998. Aromatic amino acid kinetics in parenterally-fed neonates. Ped. Res. 441-8. A portion of the research reported in Chapter 6 has been reported in abstract form: Roberts, S.A., Thorpe, J.M., Bal, R.O. and Pencharz, P.B., 1998. Tyrosine requirernents of adult males at a fixed phenylalanine intake using indicator amino acid oxidation. Faseb J. 1Z:A86O. TABLE OF CONTENTS 1. INTRODUCTION ........................................... 1 2 . LITERATURE REVIEW ...................................... 3 2.1. PARENTERALAMlNOAClDNUTRlTlONlNTHENEONATE ... 4 2.1.1. Parenteral Amino Acid Solutions .................... 4 2.1.2. Sources of Tyrosine ............................. 12 2.1.2.2. Dipeptides .......................... 13 2.2. AMINO ACID REQUIREMENTS ......................... 15 2.2.1. Dispensable. Indisriensable and Conditionallv Indis~ensable AminoAcids ................................... 15 2.2.2. Methods Used in Estimatina Amino Acid Requirements . 18 2.2.2.1. Nitrogen Balance and Growth ........... 18 2.2.2.2. Plasma Amino Acids .................. 21 2.2.2.3. Stable Isotope Tracer Methods .......... 22 2.2.2.3.1. Direct Amino Acid Oxidation ....... 22 2.2.2.3.2. lndicator Amino Acid Oxidation .... 26 2.2.2.3.3. Methodological Improvements ..... 30 2.2.3. Factors Affectina Amino Acid Reauirements .......... 31 2.2.3.1. Energy lntake ......................... 31 2.2.3.2. Non-specific Nitrogen lntake .............. 33 2.2.3.3. Route of Nutrient Delivery ................ 34 vi 2.3. AROMATlC AMIN0 AClD METABOLISM .................. 35 2.3.1. Phenylalanine ..................................35 2.3.2. Tvrosine ...................................... 39 2.3.3. Neonatal Phenvlalanine and Tvrosine Metabolic Studies 42 2.3.4. Adult Phenvlalanine and Tvrosine Metabolic Studies .... 45 3 . RATIONALE. HYPOTHESES AND OBJECTIVES ................ 48 3.1. Rationale ........................................... 48 3.2. Hypotheses and Objectives ............................ 51 4 . PHENYLALANINE AND TYROSINE METABOLISM IN NEONATES RECElVlNG PARENTERAL NUTRITION SOLUTIONS THAT DIFFERS IN SOURCES OF AMIN0 AClDS ................................ 54 4.1. Introduction ......................................... 54 4.2. Materials and Methods ................................ 56 4.2.1. Patients and Nutrient lntake ....................... 56 4.2.2. Studv Protocol ................................. 62 4.2.3. Analytical Procedures and Calculations .............. 65 4.2.3.1. Urinary Arnino Acid Enrichement .......... 65 4.2.3.2. Urinary Phenylalanine. Tyrosine and Alternate Metabolites of Catabolism Concentrations ... 69 4.2.3.3. Expired 13C0, Enrichment ................ 70 4.2.3.4. Model of Amino Acid Metabolism and Calculations ........................... 71 4.2.4. Statistical Analvses .............................. 73 vii 4.3. Results ............................................ 75 4.3.1. Clinical Characteristics ...........................75 4.3.2. Urinary Amino Acid Enrichment .................... 75 4.3.3. Phenvlalanine and Tvrosine Kinetics ................ 77 4.3.4. Urinary Phenvlalanine and Tvrosine Concentration and Alternate Metabolites of Catabolism ................. 80 4.4. Discussion .......................................... 86 Conclusions ......................................... 93 5 . THE EFFECT OF GRADED INTAKE OF GLYCYL-L-TYROSINE ON PHENYLALANINE AND TYROSINE METABOLISM IN PARENTERALLY- FED NEONATES: ESTIMATION OF TYROSINE REQUIREMENT ... 94 5.1. Introduction ......................................... 94 5.2. Materials