AROMATIC AMIN0 ACID METABOLISM in the HUMAN: Estlmatlon of TYROSINE REQUIREMENT in the NEONATE and ADULT
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
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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 and Methods ...... 96
5.2.1. Patients and Nutrient lntake ...... 96
5.2.2. Çtudv Protocol ...... 98
5.2.3. Analvtical Procedures and Calculations ...... 102
5.2.3.1. Urinary Amino Acid Enrichment ...... 102
5.2.3.2. Urinary Phenylalanine and Tyrosine Concentrations ...... 102
5.2.3.3. Expired 13C0, Enrichment ...... 103
5.2.3.4. Mode1 of Amino Acid Metabolism and Calculations ...... 103
5.2.4. Statistical Analvses ...... 103
5.3. Results ...... 105
viii 5.3.1. Clinical Characteristics and Nutrient lntake ...... 105
5.3.2. Urinary Amino Acid and Expired CO? Enrichment ..... 105
5.3.3. Phenvlalanine and Tvrosine Kinetics ...... 105
5.3.4. Urinarv Arnino Acid Concentrations ...... 109
5.4. Discussion ...... 114
Conclusions ...... 121
6. WROSINE REQUIREMENT OF ADULT MALES AT A FlXED PHENYLALANINE INTAKE USlNG INDICATOR AMINO AClD OXlDATlON ...... 122
6.1. Introduction ...... 122
6.2. Materials and Methods ...... 125
6.2.1. Subiects ...... 125
6.2.2. Exoerimental Desian and Dietary lntake ...... 125
6.2.3.1. Tracer Protocol ...... 130
6.2.3.2. Sample Collection ...... 132
6.2.4. Analvtical Procedures and Calculations ...... 333
6.2.4.1. Plasma Amino Acid Enrichment ...... 133
6.2.4.2. Plasma Phenylalanine and Tyrosine Concentrations ...... 133
6.2.4.3. Expired CO, Enrichment ...... 136
6.2.4.4. Model of Amino Acid Metabolism and Calculations ...... 136
6.2.5. Statistical Analvses ...... 136 6.3. Results ...... 137
6.4. Discussion ...... 144
6.4.1. Methodolociical Considerations ...... 149
6.4.2. Plasma Amino Acids ...... 151
6.4.3. Tvrosine Requirernent ...... 153
6.4.4. Present Knowledae in Aromatic Amino Acid Metabolism and Reauirement ...... 153
6 Conclusions ...... 156
7. GENERAL DISCUSSION AND FUTURE DIRECTIONS ...... 158
7.1 . General Discussion ...... 158
7.2. Future Directions ...... 168
8 . REFERENCES ...... 171
9.1. Clinical Investigation Information Fom: Neonatal Study 1 ..... 193
9.2. Subject Enrolment Fom: Neonatal Studies ...... -196
9.3. Tracer Dose Calculation ...... 198
9.4. Correction for lsotoporner Overlap ...... 199
9.5. Clinical Investigation Information Form: Neonatai Study II .... 203
9.6. Regression Models Applied to Data in Chapters 5 and 6 ..... 206
9.7. Two-phase Linear Regression Crossover Model ...... 209
9.8. Information Sheet: Adult Study ...... 216
9.9. Diet Calculations and Recipes ...... 219 LIST OF TABLES
Table 2.1. Amino acid profile of cornmercially available amino acid solutions ...... 5
Table 2.2. Classification of amino acids according to requirernent ...... 17
Table 4.1 . Subject information of parenterally-fed neonates receiving Vamin orprimene ...... 57
Table 4.2. Amino acid profile of Vamin and Primene ...... 60
Table 4.3. Dietary intake of parenterally-fed neonates receiving Varnin or Primene ...... 61
Table 4.4. Phenylalanine and tyrosine kinetics in neonates receiving Vamin or Prirnene ...... 78
Table 4.5. Phenylalalnine oxidation in neonates receiving Vamin or Primene ...... 79
Table 5.1. Subject information of parenterally-fed neonates receiving varying intakes of glycyl-L-tyrosine as a source of tyrosine ...... 97
Table 5.2. Amino acid profile of Primene ...... 99
Table 5.3. Individuai subject tyrosine. phenylalanine. amino acid. lipid and carbohydrate intakes ...... 100
Table 5.4. lndividual subject L-[1.13C]phenylalanine. L-[l .13CJtyrosineyL[3. 3. 'HJtyrosine and I3CO, enrichment ...... 106
Table 5.5. lndividual subject phenylalanine and tyrosine kinetics ...... 107
Table 5.6. lndividual subject urinary creatitinine. phenylalanine. tyrosine. glycyl-L-tyrosine and glycine concentrations ...... 113
Table 6.1. Subject information of healthy adult male volunteers ...... 126
Table 6.2. Composition of crystalline L-amino acid mixture ...... 129
Table 6.3. Order of assignment of tyrosine intake ...... 131 Table 6.4. Study day CO2 production rate (FCOJ of individual subjects . . 138
Table 6.5. The effect of tyrosine intake on lysine F13C0, from the oxidation of L-[1-'3C]lysine ...... 140
Table 6.6. The effect of tyrosine intake and subject on L-[l -13C]lysine oxidation ...... 142
Table 6.7. The effect of tyrosine intake and subject on lysine fiux ...... 145
Table 9.1. Summary statistics for regression models from Chapter 5 Dependent variable: F13C02 ...... 206
Table 9.2. Summary statistics for regression models from Chapter 5 Dependent variable: phenylalanine oxidation ...... 206
Table 9.3. Summary statistics for regression models from Chapter 5 Dependent variable: phenylalanine hydroxylation ...... 207
Table 9.4. Summary statistics for regression models from Chapter 5 Dependent variable: tyrosine flux ...... 207
Table 9.5. Summary statistics for regression models from Chapter 6 Dependent variable: FI3CO, ...... 208
Table 9.6. Summary statistics for regression models from Chapter 6 Dependent variable: phenylalanine oxidation ...... 208
xii LIST OF FIGURES
Figure 2.1. lndicator amino acid oxidation ...... 28
Figure 2.2. Principal pathway of aromatic amino acid catabolism ...... 36
Figure 2.3. Alternative pathway of phenyialanine catabolism ...... 37
Figure 2.4. Alternative pathway of tyrosine catabolism ...... 40
Figure 4.1. Study protocol ...... 59
Figure 4.2. Urinary L-[1.13C]phenylalanine. L-[1.13C]tyrosine. and L43.3- 2HJtyrosine enrichment time course from a study subject ..... 64
Figure 4.3. Chrornatograms of mass peakç of phenylalanine and tyrosine from GCMS set to selected ion monitoring mode ...... 68
Figure 4.4. Urinary phenylalanine and tyrosine enrichment at plateau ..... 76
Figure 4.5. The relationship between energy intake on percent L-[1. 13C]phenylalaninedose oxidized in neonates receiving Vamin or Primene ...... 81
Figure 4.6. Urinary phenylalanine and tyrosine concentrations in neonates receiving Vamin or Prirnene ...... 82
Figure 4.7. Urinary concentration of alternate metabolites of phenylalanine catabolism ...... 84
Figure 4.8. Urinary concentration of alternate metabolites of phenylalanine catabolism ...... 85
Figure 5.1. Study protocol ...... 101
Figure 5.2. The effect of increasing tyrosine intake via glycyl-L-tyrosine on phenylalanine hydroxylation in neonates receiving TPN ...... 108
Figure 5.3. The effect of increasing tyrosine intake via glycyl-L-tyrosine on tyrosine flux in neonates receiving TPN ...... 110
xiii Figure 5.4. The effect of increasing tyrosine intake via glycyl-L-tyrosine on F3C0, production from phenylalanine oxidation in neonates receiving TPN . . . . - ...... - - - . . - - - ...... II1
Figure 5.5. The effect of tyrosine intake via glycyl-L-tyrosine on phenylalanine oxidation in neonates receiving TPN ...... 112
Figure 6.1. Study protocol
Figure 6.2. Chromatograms of mass peaks of phenylalanine, tyrosine and lysine analyzed by GCMS set to selected ion monitoring mode 134
Figure 6.3. Mean F13C0,frorn oxidation of L-[l-'3]lysine at graded tyrosine intakes in healthy adult male subjects ...... 141
Figure 6.4. Mean lysine oxidation rate at graded tyrosine intakes in healthy adult male subjects ...... - . . . 143
Figure 6.5. Response of plasma phenylalanine and tyrosine concentration throughout study ...... 146
Figure 6.6. Plasma phenylalanine concentration after 8 h of feeding the study diet ...... 147
Figure 6.7. Plasma tyrosine concentration after 8 h of feeding the study diet ...... 148
Figure 9.1. The mass spectral distribution of tracee and tracer standards . 200
xiv ABBREVIATIONS
Fe 1ron DA Dopamine NE Norepinephrine € Epinephrïne PKU Phenylketonuna KIC a-ketocaproic acid IAAO lndicator amino acid oxidation IAA Indispensable amino acid CO2 Cahon dioxide BBB Blood Brain Banier N Nitrogen vc02 Rate of CO, production SD Standard deviation SEM Standard error of the mean MP Molecule percent MPE Molecule percent excess NlCU Neonatal intensive care unit GT Glycyl-L-tyrosine Phe Phenylalanine Tyr Tyrosine LYS Lysine 1. INTRODUCTION
The neonatal period is a time of rapid growth requiring great nutrient intakes. The minimal nutrient stores that exist in the premature neonate require the provision of an adequate and consistent source of nutrient intake. Parenteral nutritional support therefore is a integral component of treatment of the prernature andlor surgical neonate who is unable to feed enterally or take in sufficient nutrition to meet needs (Hughes and Ducker 1981; Hack et al, 1991).
Since the first documented case of successful implementation of total parenteral nutrition in a paediatric patient (Helfrick and Abelson 1944), ongoing irnprovements to have been made. While sorne of these irnprovements involve technical aspects such as hypertonic nutrient infusion into the central vein
(Dudrick et al, 1968). and use of improved catheters for delivery (Superina et al, i988),other changes have involved irnproved means of meeting nutrient needs.
An example of the latter is the evolution from protein hydrolysates of casein or fibrin to crystalline amino acids (Heird et al, 1972; Johnson et al, 1972) to meet amino acid needs. Such irnprovements have allowed for manipulation of levels of individual amino acids and resulted in better control over amino acid intake.
The early parenteral arnino acid solutions were designed to sustain the adult unable to receive entera1 feeds for extended lengths of tirne. These solutions were considered to be general purpose in nature. Recognizing that these adult amino acid solutions resulted in some abnormalities to the plasma amino acid concentration in the neonate led to the development of paediatric
1 2 amino acid solutions. These solutions were designed to meet the needs of the neonate with consideration for their immature state. They largely reflect the pattern of neonatal reference proteins and have resulted in improved plasma amino acid profile (Heird et al. 1987; Rigo et al. 1987; Adamkin et al, 1991).
Nevertheless, there is little empirical evidence supporting their irnproved efficacy in meeting neonatal needs.
The piglet model of the neonate receiving TPN (Wykes et al. 1993) has been invaluable in providing a controlled paradigm to investigate issues related to parenteral nutriture. The piglet is considered to be an appropriate model to the study of neonatal amino acid requirement due to similarities in physiology
(MacDonald 1986; Groner et al, 1WO), amino acid requirement (FAOMIHOIUNU
1985; ?Ailler and Ullrey 1987; National Research Council 1988). and stage of developmental maturity (Book and Bustad 1974; Moughan and Rowan 1989).
Using an animal model allows for improved study control and can be more invasive and therefore provides a great deal of information with less variation
(increased sensitivity) than similar studies carried out in the human neonate. In addition, the rapid growth rate of the piglet can more readily identiw small differences that exist between dietary regimens. This model (Wykes et al, 1993) has identified total aromatic intake as limiting net protein gain in parenteral paediatric amino acid solutions designed for the neonatal population (Wykes et al. 1994a). Further to this. House et al, (1997a; 1997b) carried out studies in the model to determine more precisely the phenylalanine and tyrosine requirement 3 of the parenterally-fed neonate. The piglet safe (upper 95% confidence limit) levels of intake were found to represent 3.2% and 2.7% of total amino acids for p henylalanine and tyrosine, respectively.
In this thesis. we bring the investigation of aromatic amino acid metabolism and needs to the bedside. The objective of these studies was to investigate phenylalanine and tyrosine metabolism in neonates receiving TPN with differing arnino acid intakes, and to detemine the tyrosine requirernent at a phenylalanine intake typical to the paediatric arnino acid solutions. Furthermore, we extended our investigation of arornatic amino acid metabolism and requirement into the healthy adult to allow us to compare the immature systern of the neonate to the mature system of the adult with respect to total aromatic amino acid needs and balance.
2. LITERATURE REVIEW
This review of the literature covers the evolution of parenteral amino acid nutrition with a specific focus on the aromatic amino acid needs of the neonatal population. Both the classical and more recent methods used to assess amino acid requirements are described, including their strengths and limitations.
Factors affecting amino acid utilization and hence requirement are considered following background information on phenylalanine and tyrosine general metabolism. Finalty, it concludes with more specific kinetic studies of amino acid metabolism in the neonate and adult. 2.1. Parenteral Arnino Acid Nutrition in the Neonate
2.1 -1. Parenteral Amino Acid Solutions
The goal when providing parenteral amino acids in the neonate is to achieve an intake of indispensable arnino acids and non-specific nitrogen that supports optimal growth rates without giving amounts of any individual arnino acid that would result in an overloading of the catabolic enzyme systems and have potential deleterious effects. Early arnino acid solutions were composed of protein hydrolysates of casein or fibrin (Johnson et al, 1972). Several limitations existed with these hydrolysate solutions. Firstly, there was no standardization to the hydrolysis process, resulting in non-specific peptides of variable chah length
(Winters et al, 1983),therefore the composition of infused amino acids and peptides were unknown. Secondly, the high ammonia concentration within these solutions resulted in hyperarnmonernia (Johnson et al, 1972). Finally, the extent of rnetabolism of the spectrum of peptides that were formed was equally limited as evidenced by considerable urinary peptide excretion (Christensen et al,
1946), leading to uncertainty in both delivery and metabolisrn.
The introduction of crystalline L-arnino acids in the 1970s resulted in a major improvement to amino acid delivery (Table 2.1). The Rexibility associated with developing formulations from individual amino acids resulted in better defined solutions. Cornparison between protein hydrolysates and crystalline amino acid solutions demonstrated improved nitrogen rnetabolism as assessed by greater nitrogen retention and a reduction in the proportion of nitrogen flux Table 2.1 Arnino Acid Profile of Commercially Available Amino Acid Solutions (% amino acid by weight) Product VamIn Novamine Travasol Aminosyn FreAmine Primene Aminosyn-PF TrophAmine Vaminotact PharmaciaZ Baxter Baxter Abbott McGaw Baxter Abbott McGaw Pharmacia I Ile I Leu Val LYS Met i CYS Phe
a Tyr NAT3 Ii I Thr i T~P l His Arg l G~Y I Ala 1 A~P 1 Glu 1 Pro 1I Ser Tau i lI Orn - O O 2.2 O O O ! O 1, Human lilk, Calculated from Rassin, 1989; 2. Pharmacia & Upjohn; 3. NAT: N-acetyl-tyrosine as tyrosine 6 from breakdown (Duffy et al, 1981).
In general, plasma arnino acid profiles of the TPN-fed neonate follows the pattern of the infused amino acids (Stegink and Baker 1971; Anderson et al,
1977). Therefore, an appropriate balance of arnino acids rnust be provided.
Although early general purpose solutions provided all nine of the indispensable amino acids in amounts similar to high-quality reference proteins (egg and rnilk), the composition of their indispensable amino acid content varied greatly. In several of these solutions a large proportion of the indispensable cornponent was provided by glycine, primarily due to its low cost. Infants receiving such high glycine intakes were observed to experience hyperg lycinaemia (Chessex et al,
1985). Furthemore, those solutions were elevated in methionine to compensate for the absence of cysteine in solution (due to poor stability), resulted in hypemethioninemia (Chessex et al. 1985). Of these amino acid solutions. Vamin, patterned against the amino acid profile of egg protein, seemed to support the best balance of plasma amino acids (Chessex et al,
1985) with growth rates comparable to that observed in the third trimester fetus
(Widdowson 1981).
None of these general purpose formulations contain taurine. Although not a component of protein, taurine is an arnino acid that has been shown to be essential for the developing rat brain (Stuman et al, 1977), and retinal function in cats (Hayes et al. 1975). Furthemore, children receiving long-term taurine- free total parenteral nutrition (TPN) develop retinal abnormalities reversible upon 7 supplementation (Geggel et al, 1985). Auditory neurological impairment has also been found in low birth-weight infants receiving taurine-free formula (Tyson et al,
1989). Although the absence of taurine in TPN has not been associated with
reduced growth or nitrogen balance, low plasma taurine levels in neonates after long-term taurine-free TPN (Zelikovic et al, 1990) is suggestive of inadequate intake.
Tyrosine (Snydeman 1971; House et ai, 1997a) and, although not conclusive (Zlotkin and Anderson l982), cysteine (Snyderman IWI; Vina et al,
1995) are considered conditionally indispensable in the neonate. Due to poor stability and solubility issues, al1 parenteral arnino acid solutions possess low to absent quantities of these amino acids. Low intake is reflected in low plasma levels of these amino acids. The amino acid solution Vamin has cornpensateci for the low level of tyrosine by elevating the content of phenylalanine, the endogenous metabolic precursor of tyrosine via hepatic hydroxylation.
Observations have shown that the metabolic tolerance of this approach is limited. Some infants experience elevated levels of plasma phenylalanine
(Chessex et al, 1985; Puntis et al, 1986; Walker et al, 1986; Rigo et al, 1987;
Mctntosh and Mitchell 1990; Walker and Mills 1990) compared tc infants receiving rnother's milk. Tyrosine levels have also been found to be excessively above (Mclntosh and Mitchell 1990; Mitton et al, 1993) or below normal (Heird et al, 1987).
Recognizing the limitations of the general purpose solutions, newly 8 formulated "paediatric" amino acid solutions were designed. In general, the new paediatric solutions have been formulated to reflect the amino acid pattern of neonatal reference patterns such as hurnan milk (Vaminolact, Pharnacia and
Upjohn, Stockholm, Sweden), cord blood (Primene, Baxter Corporation,
Deerfield IL) (Rigo et al, 1987) or to result in plasma amino acid profiles that are similar to those found in healthy term infants receiving breast milk (TrophArnime,
McGaw Laboratories, Irvine, CA) (Heird et al, 1987) (Table 2.1). Other solutions have made minor modifications to original formulations to better suit the needs of the neonate (e.g., Aminosyn PF, Abbott Laboratories, Montreal, QC). Each of these approaches is based on a slightly different rationale for deciding what the optimal balance of dietary amino acids should bel yet none were developed based on direct measurements of parenteral amino acid metabolism and requirernent.
The amino acid profiles of plasma from infants receiving paediatric amino acid solutions seern to be more normal than those of infants receiving general purpose solutions when compared to the breast-fed neonate (Mitton et al, 1993).
Aside from differences in plasma amino acid profile, there has not been much evidence suggesting that paediatric amino acid solutions better meet the needs of the neonate. Studies by Mitton and colleagues (Mitton and Garlick 1992;
Mitton et al, 1993) examined to rates of nitrogen balance, plasma amino acid levels and protein turnover using L-[1-13C]leucine in neonates receiving Vamin or
Vaminolac, a paediatric amino acid solution patterned against human milk 9 protein. No significant differences in nitrogen balance, nitrogen flux, synthesis or catabolism were found. Heird (1 989b) pointed out that the presence of limiting levels of certain amino acids in both solutions may have masked any differences that exist between the quality of solutions.
Development of the paediatric formulation TrophArnine was based on the observation that plasma amino acid levels tend to reflect that of amino acid intake (Stegink and Baker 1971; Anderson et al, 1977). Also critical to the evolution of TrophAmine, was the establishment of mathematical models which, when given sufficient data, can theoretically deduce the composition of a formula that should result in plasma amino acid levels equivalent to healthy, normal growing, 30-day old breast-fed term infants (Heird et al, 1987). Although
TrophAmine-fed infants demonstrated good nitrogen retention rates and a plasma amino acid profile approaching that of the breast-fed infant, it did not result in greater nitrogen retention rates when compared to Aminosyn-PF
(Adamkin et al, 1991). Notable was the greater, although not completely normalized, plasma tyrosine levels in infants receiving TrophAmine, apparently due to the addition of N-acetyltyrosine, a soluble derivative of tyrosine.
Increases in both plasma and urinary N-acetyltyrosine levels were also apparent.
Similarly, the arnino acid composition of Primene was fashioned upon cord amino acid levels in an attempt to simulate in utero amino acid uptake (Rigo et al, 1987; Mclntosh and Mitchell 1990). The resultant solution differs from other paediatric solutions in its greater content of lysine and cysteine, while it IO differs from Vamin in its lower proline and phenylalanine content and higher tryptophan, glycine, alanine and serine levels (Table 2.1). Primene also has added taurine and ornithine (Uauy et al, 1993). While evidence for the inclusion of ornithine is unclear, and possibly lies in its role as a urea cycle intemediate, taurine is considered an arnino acid for brain developrnent and function (Hayes et al, 1975; Sturman et al, 1977; Geggel et al, 1985). Infants receiving Prirnene demonstrated nitrogen retention rates and plasma amino acid levels close to that found in the fetus during the third trimester of gestation (Rigo et al, 1987). In a double-blind, randomized, controlled trial of parenterally-fed infants receiving
Vamin vs Primene as source of amino acids during the first week of life,
Primene-fed infants were able to maintain plasma amino acid levels more closely to the normal reference range. In contrast, there was a concern for the high levels of phenylalanine, tyrosine, proline, serine and aspartic acid in the plasma of Vamin-fed infants (Mclntosh and Mitchell 1990). Although Primene demonstrated improved balance of the arnino acid intake, like the other paediatric amino acid solutions, tyrosine and cysteine levels were not comparable to cord blood or to the plasma of infants fed human rnilk.
The consistently low levels of plasma tyrosine in neonates receiving paediatric arnino acid solutions suggests that intake of the tyrosine is inadequate to meet needs. Wykes et al (1 993) using a piglet mode1 of the neonate receiving
TPN tested the hypothesis that total arornatic arnino acid intake is Iimiting in the paediatric arnino acid solutions (Wykes et al, 1994a). To determine if the 11
paediatric solution Vaminolact was deficient in total aromatic amino acids a second solution was designed for comparison. This solution was equal in amino acid composition to Vaminolact, but contained added phenylalanine to the level found in Vamin. Vamin was also compared in this study to these two solutions,
representative of a general purpose amino acid solution commonly used in the
neonatal population. Piglets receiving the phenylalanine supplernented solution gained significantly more weight (0.86 kg vs 1.1 8 kg, Vaminolact vs Vaminolact + phenylalanine; pc0.02). and retained significantly more nitrogen (1435 mg-kg-'-d-
' vs 1601 mg -kg-'-d-', Vaminolact vs Vaminolact + phenylalanine, pc0.0001) than did those receiving the unsupplemented paediatric amino acid pattern. This finding was supported by phenylalanine kinetic data which demonstrated that the percent of phenylalanine label oxidized by piglets receiving Vaminolact was only
10% compared to 23 and 24% for the Vamin and Vaminolact + phenylalanine groups, respectively (pc0.001). This low oxidation rate of the infused label likely
represents the obligatory phenylalanine oxidation loss in the parenterally fed piglet reflecting inadequate intake and arnino acid conservation. These results prove that the total aromatic arnino acid intake of the paediatric amino acid solution is below neonatal piglet needs. Further to these findings, this
investigation also exarnined the metabolic tolerance of the aromatic amino acid
load by monitoring the presence of urinary alternate metabolites of phenylalanine and tyrosine catabolism as well as plasma amino acid levels. It was observed that the neonates receiving Vamin, or the phenylalanine supplernented solution 12 had greater plasma phenylalanine levels (Vamin= 2234 pmol-L-' ; Varninolact =
156 pmol-L-' ; Vaminolact + phenylalanine = 399 prnol-L-'; p<0.0001).
Further support for the poor tolerance of large intakes of parenteral phenylalanine was demonstrated by the presence of alternate metabolites of phenylalanine catabolisrn that appeared in the urine of piglets receiving Vamin
(62 I30 prnol-kg-'-d-')compared to Vaminolact (1 I3 prnol-kg-'-d-')and
Vaminolact + phenylalanine (19 I 32 pmol-kg"-d-'). The excretion of alternate metabolites of phenylalanine (Walker and Mills 1990) and tyrosine (Chessex et al, 1985; Walker and Mills 1990) has also been observed in the neonate receiving Varnin. These alternate metabolites are not usually detected in the urine, but appear when catabolic pathways have been overloaded (Scriver et al,
1995). Furthermore, the high phenylalanine plasma concentration and presence of significant amounts of alternate metabolites of phenylalanine catabolism, suggests that increasing phenylalanine intake may not be the best rneans of meeting total aromatic amino acid needs in the parenterally-fed neonate.
2.1.2. Sources of Tvrosine
Elevated plasma phenylalanine concentrations without normalization of plasma tyrosine suggests that increasing parenteral phenylalanine intake is not the best means of meeting total aromatic amino acid needs. Therefore, an alternative soluble precursor source of tyrosine is necessary. Several sources have been suggested. 13
2.1.2.1 . N-acetyltyrosine
The addition of an acetyl group to tyrosine increases its solubility. lm and colleagues (1985) provided encouraging results when they demonstrated that N- acetyltyrosine increased plasma tyrosine levels, was rapidly utilized, and resulted in urinary losses of 16.8% when infused in rats. Heird et al, (1987) included this compound in TrophAmine in an attempt to better meet the total aromatic amino acid needs of the parenterally-fed neonate. Unfortunateiy, further research with
N-acetyltyrosine in the neonatal rnodel of the piglet receiving parenteral nutrition
(Wykes et al, 1994b), the human neonate (Heird et al, 1987; Helrns et al, 1988;
Adarnkin et al, 1991; Sulkers et al, 1991; Hanning 1993), and adults (Magnusson et al, 1989) have demonstrated that the compound is poorly utilized. This was evidenced by high plasma N-acetyltyrosine levels and its excretion in the urine suggesting limited deacylation capacity (Endo 1978; Endo 1980).
2.1 -2.2. Dipeptides
Evidence that hydrolysates of protein were poorly utilized led to the belief that peptides, in general, were not suitable for parenteral use. Work by Stegnik
(1975) and Adibi (1 987) have dispelled this fallacy by demonstrating that specific peptides are well utilized and provide the flexibility that will allow the needs of different population groups to be met. In contrast to N-acetyltyrosine, dipeptides of tyrosine are highly soluble and have been shown to be well utilized by rats
(Neuhauser et al, 1985; Adibi 1989; Jiang et al, 1993; Stehle et al, l996), dogs 14
(Abumrad et al, 1989), pigs (Wykes et al, i994b; House et al, 1997a; House et al, 1997b), primates (Steinhardt et al, l984), and humans (Albers et al, 1989;
Steininger et al, 1989; Druml et al, 1991; Lochs et al, 1992; Morlion et al, 1998).
Albers et al (1988; 1989) demonstrated in healthy adult hurnans that alanyl-L- glutamine and glycyl-L-tyrosine are hydrolysed with first order constants of 0.1 9
I0.02 and 0.20 * 0.02 min" respectively, and hence do not accumulate in the body. Albers and colleagues (1989) also demonstrated a high metabolic clearance rate of alanyl-L-g lutamine (35.9 k 9.5 mL-kg-'min-l) and g IycyCL- tyrosine (33.7 I 9.5 ml-kg-'-min-'), resulting in low plasma levels and undetectable urinary excretion. The rapid clearance rate from the circulation, with equimolar rises in plasma levels of individual amino acids suggest that the dipeptides are likely hydrolysed extracellularly than intracellularly (Albers et al,
1989).
There are several mechanisms for the metabolism of dipeptides. The major site of hydrolysis depends largely on the peptide's chemical structure
(Adibi et al, 1986; Kee et al, 1994). Organs are responsible for removal and hydrolysis of dipeptides, with the liver and kidneys being the major sites of hydrolysis (Adibi et al, 1986). The brain is the only organ that does not take part in dipeptide clearance (Zlokovic et al, 1983; Vazquez et al, 1992). Plasma membrane hydrolases are largely responsible for peptide cleavage, while hydrolysis by plasma enzymes is responsible for very little cleavage (Stegink
1975; Lochs et al, 1986). 15
Several long-term studies using dipeptides support the evidence that dipeptides are safe and efficacious (Steinhardt et al, 1984; Vazquez et al. 1986;
Petersson et al, 1994). The greater effectiveness of glycyltyrosine and alanyltyrosine over N-acetyltyrosine as tyrosine precursors in humans was finally dernonstrated in a cornparison study by Druml et al (1991). Their study revealed that dipeptides were not excreted in the urine, while more than half of the N- acetyltyrosine infused was excreted in the urine. More recently, Wykes et al
(1994b), cornpared the feasibility of N-acetyltyrosine versus glycyltyrosine as a precursor of tyrosine in the piglet, and revealed 28% greater protein accretion and 14% greater nitrogen retention in the piglets receiving the glycyl-L-tyrosine.
Underscoring reduced utilization of N-acetyltyrosine was the excretion of 65% of intake (Wykes et al, 1994b).
2.2. AMIN0 AClD REQUIREMENTS
2.2.1. Dispensable. Indis~ensableand Conditionallv Indispensable Amino Acids
With the availability of crystalline L-arnino acids, studies into individual amino acid requirements became possible. The studies of Rose (1957) and
Leverton (1 959) in adult humans carried out in the 40s and 50s identified the body's requirement for eight indispensable amino acids (leucine, isoleucine, valine, methionine, tryptophan, lysine, phenylalanine, and threonine). The study approach was to provide a diet devoid of a single amino acid and examine nitrogen balance. The indispensable arnino acids were those that when 16 removed resulted in negative nitrogen balance. Requirernent was later predicted by adding back the amino acid in a graded fashion until nitrogen balance was re- established-
Recently a classification of arnino acids has been established identifying the conditionally indispensable amino acids (Table 2.2) (Pencharz et al, 1996).
This category was deemed necessary since it was found that during certain stages of developrnent, pathophysiological conditions, or modes of feeding, preformed sources of amirio acids considered dispensable may become necessary. Examples of these amino acids include arginine in the young pig
(Southern and Baker 1983) and neonate (Heird et al. 1972), proline in the piglet
(Bal1 et al, 1986), and tyrosine in the neonate (Snyderrnan 1971). Although the activity of cystathionase in extrahepatic tissues is similar to that of to the adult
(Zlotkin and Anderson 1982), low hepatic cystathionase activity in conjunction with low plasma cysteine concentration (Vina et al, 1995). results in cysteine being generally considered indispensable for the parenterally-fed premature neonate (Snyderman 1971 ; Vina et al, 1995). Studies in the piglet mode1 of the neonate have confirmed that tyrosine is the first limiting amino acid in paediatric amino acid solutions with low phenylalanine content (Wykes et al, 1994a). Early studies of Snyderrnan (1971) demonstrated that tyrosine was indispensable in the enterally-fed neonate as well. Table 2.2 Classification of Amino Acids According to Requirement' Indispensable Conditionally Indispensable Alanine Histidine Arginine Asparagine Isoleucine Cysteine As partate Leucine Glutamine Glutamate Lysine Glycine Serine Methionine Proline Phenylalanine Tyrosine Threonine Taurine2 Tryptophan VaIine 1. Adapted from Pencharz et al, (1996). 2. Taurine is not a component of protein. 18
In defining the needs for the various population groups that now have been identified, the methods used have to be sufficiently sensitive to quant@ requirernents. The following section reviews the rnethods used to detemine arnino acid requirement, including their strengths and weaknesses.
2.2.2. Methods Used in Estimatina Amino Acid Requirements
2.2.2.1. Nitrogen Balance and Growth
The classical and by far the most widely used means of assessing amino acid requirements is nitrogen balance. Nitrogen balance is estimated by the difference between total nitrogen intake and loss. Studies using this method are the foundation of the current estimates of amino acid requirernents in adults
(FAOWHOIUNU 1985). While these studies provide valuable information, they have been criticised for methodological difficulties (Young and Bier 1987b;
Millward et al, 1990). In summary, some of the major limitations of the nitrogen balance method include:
1. The estimate of nitrogen balance represents a srnaIl value as a result of the difference between two very large but similar measurements of nitrogen intake and output. Furtherrnore, the accurate measurement of nitrogen input and output are notoriously difficult to measure. Input measurement is ofien overestimated due to spillage and incornplete recovery of uneaten food, while output is often underestimated due to the difFiculty in achieving complete recovery (Hegsted 1976). That the two measurernents determining nitrogen 19 balance are associated with considerable error and the final difference is a very small value leading to large error of the estimated requirement.
2. Increasing the error of this srnall nitrogen balance difference, is the omission of integurnental and other nitrogen losses frorn the body in the output estirnate. When dealing with very small differences, these minor losses (0.3-0.5 g N-day4)(Calloway et al, 1971) are potentially significant.
3. The early studies of Rose (1957) and Leverton et al (1959) upon which curent amino acid estimates are based, were perfomed in very few individuals
(3-6 subjects per arnino acid).
4. The energy intakes of these individuals were excessive and likely to result in an underestimate of arnino acid needs due to the sparing effect that excess energy exerts on arnino acid utilization (FAOMIHOIUNU 1985).
5. The possibility exists that nitrogen equilibrium will occur at various states of protein dynamics, making it difficult to identify the significance of the nitrogen balance estimate. In view of this limitation to the model, Millward and Rivers
(1988) proposed a new model defining indispensable amino acid metabolism.
Their belief is that a further understanding of indispensable amino acid needs cannot be achieved without the development of a new biological model that better describes the nature of amino acid requirements.
6. Other limitations include the use of racemic mixtures of D and L-amino acids in the studies of Rose (1957). More recent knowledge of amino acid rnetabolisrn has dernonstrated poor utilisation of most arnino acids of the D form 20
(Darling 1997), placing into question the validity of these studies.
7. The amino acid mixtures used a disproportionate arnount of indispensable arnino acids in cornparison to a typical foodstuff (FAOMIHO 1990). This factor could have led to further underestirnation of amino acid requirernent by reducing apparent needs. The interaction of indispensable amino acid intake and amino acid oxidation was investigated by Young et al (1 987b). L-[l-'3C]leucine oxidation was reduced from 14 to 11 pmol-kg-'-h-' in individuals receiving greater intake of al1 indispensable amino acid from 0.6 to 1.1 g protein-kg-l-h-'.
Furthemore, the amount of leucine coming from tissue breakdown was lower
(72 vs 127 pmol-kg-'- h-l) in individuals receiving the hig her indispensable amino acid intake showing that non-specific nitrogen has an important impact on arnino acid losses.
8. The lack of an indispensable amino acid in the diet . Histidine was found to be indispensable (Giordano et al, 1973; Kopple and Swendseid 1975) almost
20 years after the studies of Rose (1957) and Leverton (1959). The fact that nitrogen balance methods could not detect the body's need for histidine, puts into question both the sensitivity of the method and the validity of the results for al1 studies.
Two approaches have been used to determine amino acid requirements of the neonate. Holt and Snydeman (1961) used growth rates in conjunction with nitrogen balance are used to assess amino acid in the neonate
(FAOMIHOIUNU 1985). Body weight gain, length and head circumference are 21 measures taken to quantify growth. Although they are crude estimates at best, they are easily performed and furnish a gross evaluation of the infant's nutritional status. Fornon (1991) used breast milk intake as a further assessrnent of adequacy of protein intake in the neonate. These results represent lower estimates for protein requirement than those detetmined by Hoft and Snyderman
(1961) using nitrogen balance and growth estimates which were those adopted by the international cornmittee of the FAOMRIOlUNU (1985).
Since nitrogen balance is associated with many recognized limitations, it was concluded by expert international cornmittees (FAOMIHO 1990;
FAOMIHOIUNU 1985) that there was a need to identify a more sensitive rnethod to determine amino acid requirements.
2.2.2.2. Plasma Amino Acids
Plasma arnino acids represent 0.2-0.696 of the body's total free arnino acid pool (Munro 1970). The liver is largely responsible for tight control of plasma amino acid levels (Denton and Elvehjem 1954; Peraino and Harper
1965). When individual arnino acid intake increases above requirement. the plasma concentration tends to increase (Young et al, 1971; Brookes et al, 1972).
Plasma arnino acid levels have therefore been used to assess amino acid requirernent in the rat (Morrison et al, 1961) and humans (Young et al, 1971;
Brookes et al, 1972), as well as to judge protein quality (Eggum 1973).
Unfortunately, the static nature of this rneasurement does not allow for a 22 complete understanding of the dynamic amino acid pool it represents. and hence altered plasma amino acid levels may be difficult to interpret and often misleading when presented on their own. Because of these issues, plasma amino acid levels have been considered to be too insensitive to be used as a predictor of amino acid requirement (Young et al, 1972; Young and Scrimshaw
1977). Nevertheless, plasma amino acid data in conjunction with other measures of nitrogen economy such as nitrogen balance and kinetics can be very helpful in understanding the body's response to varying amino acid and protein intakes.
2.2.2.3. Stable Isotope Tracer Methods
In response to the need for a more functional framework within which to define indispensable amino acid requirements, stable isotope tracer modeis have been developed.
2.2.2.3.1. Direct Amino Acid Oxidation (DAAO)
Young and colleagues have extensively investigated amino acid requirement by applying the stable isotope tracer infusion method of Waterlow et al (1978). The model, known as the 'Yracer balance rnethod", defines the physioiogic need for an indispensable amino acid as the intake needed to balance it's irreversible oxidative loss from the body (Le., daily intake - oxidation
= O) (Young and Bier 1987b). Loss of any amino acid is at minimal levels 23
(obligatory oxidative loss) at deficient intakes, and increases once requirement
intake has been reached. This model assumes that oxidation is the only major
route of amino acid disposal.
Direct amino acid oxidation methods have been used to determine the
requirements of leucine (Meguid et al, 1986), valine (Meguid et al, 1986), lysine
(Meredith et al, 1986; Kurpad et al, 1W8), threonine (Zhao et al, 1986) and more
recently phenylalanine (Zello et al, 1WOc; Basile-Filho et al, 1998). Results from these studies suggest that the current estimates of the FAONVHOIUNU (1985)
are underestimated by a factor of 2-3 (Young and Bier 1987a; Young and Bier
1987b; Young and Pellett 1987).
Methodologically, oxidation of the essential amino acid is measured using
an infusion of a L-[1J3C] label of the test amino acid. After correcting for CO,
retention in the body's bicarbonate pool, the label is released as expired 13C02
upon oxidation of the amino acid, collected and quantified. Oxidation is measured from either expired 13C02production or precursor pool enrichment estimates (Matthews et al, 1980).
Although direct amino acid oxidation studies have provided a wealth of
information regarding arnino acid kinetics and requirement, the model has been
criticised by several groups on a methodological and theoretical basis (Millward
and Rivers 1986; Millward et al, 1990; Fuller and Garlick 1994). The fact that the oxidation estimate relies on the precursor pool enrichment is one of the
methodological concerns. The problem of precursor pool enrichment is that the 24 sample site for precursor enrichment measurernent is the plasma free amino acid pool. However. this enrichment may not reflect the true amino acid enrichrnent at the point of oxidation. Leucine, isoleucine and valine are the only amino acids that are in reversible equilibrium with their transamination products, a-ketocaproic acid (KIC),a-keto-p-methylvaleric acid, and aketoisovaleric acid, respectively, which can be used as a more reliable eçtimate of precursor pool enrichment (Matthews et al, 1982; Schwenk et al, 1985; Pacy et al, 1991; Nair et al, 1992;Goulet et al, 1993; el-Khoury et al, 1995a). In fact, enrichment of KIC is consistently lower than that of leucine suggesting that when the enrichment of the tracer in plasma is used to predict precursor pool enrichment for oxidation, it results in an erroneously higher oxidation estimate. Other approaches have been used to better estimate intracellular enrichrnent such as hippurate for glycine (Gatley and Sherratt 1977; Stein et al, 1978; Arends et al, 1990) amino adipic acid for lysine (Arends and Bier 1991), and rapidly turning-over hepatic
(Ballmer et al, IWO; Reeds et al, 1992) and pancreatic (Bennet et al, 1993) proteins.
The often large amount of tracer amino acid infused to achieve sufficient enrichment in expired CO, is considered problematic. This additional amino acid source may underestirnate intake and therefore have a significant impact on the oxidation response to the test level of amino acid intake, overestimating requirement. Furthemore, as the test amino acid intake is increased, expansion of the plasma amino acid pool rnay result in dilution of the isotope and therefore 25 falsely increase the oxidation estimate. Further contributing to potential inaccuracies in the estimate is the omission of gastrointestinal and other non- oxidative losses that may add up to be a significant cornponent of needs.
There has been concern with whether fed state oxidation estimates truely reflect 24 h amino acid balances (Millward and Rivers 1988; Millward et al,
1989). Young and colleagues have attempted to deal with this issue by studying both fasted and fed periods in their cornprehensive 24 h studies
(el-Khoury et al, 1994a; el-Khoury et al, 1994b; el-Khoury et al, 1994c; Sanchez et al, 7 995; Sanchez et al, 1996; Basile-Filho et al, 1997; Basile-Fiiho et al, 1998;
Kurpad et al, 1998). Although their initial findings suggested that the four hour estimate was representative of the 24 h findings (el-Khoury et al, 1994a; el-Khoury et al, 1994b), more recent studies using the 24 h protocol have demonstrated that this may not always be the case (Sanchez et al, 1995;
Sanchez et al, 1996). The irnbalanced intake of phenylalanine to tyrosine of these studies rnay be responsible for the greater oxidation rates of phenylalanine resulting in negative balance estimates. Further studies are required to better understand these findings.
Zello et al, (1995) have further criticised this rnethod for its long adaptation periods that rnake these studies very difficult to apply to groups other than healthy adult volunteers. Furtherrnore, its very few levels of test amino acid intake used in several of their studies (Sanchez et al, 1995; Sanchez et al, 1996;
Basile-Filho et al, 1997; Basile-Filho et al, 1998) rnakes it dificult to corne to 26 conclusive estimates regarding amino acid requirements.
While there are several criticisms associated with the methodology, it must be recognised that the errors associated with many of them may be only srnall (Fuller and Garlick 1994). In fact, Zello et al (1 993) and Duncan et al,
(1996) using the independant isotope mode1 of indicator amino acid oxidation have supported the estimate of lysine obtained by Meredith et al (1986) using direct arnino acid oxidation methods. Overall, these studies suggest that the current estimates of indispensable amino acid requirement (FAONVHONN U
1985) are underestimated, and that actual requirements are approximately 2-3 fold h igher.
2.2.2.3.2. 1 ndicator Amino Acid Oxidation (IAAO)
lndicator amino acid oxidation methodology (Kim et al. 1983a; Kim et al,
1983b; Kim and Bayley 1983; Bal1 and Bayley 1984) is a separate rnethod to estimate amino acid requirements and is based on a distinct set of theoretical principles. The premise is that the partition of any indispensable amino acid between oxidation and protein synthesis is sensitive to the intake of the first limiting amino acid. lndicator amino acid studies require that adequate protein and energy is supplied and that the test amino acid is known to be the first limiting amino acid for protein synthesis. The mode1 involves using a suitable carboxyl carbon label that is released as CO, upon oxidation and without distributing significantly in other metabolic pools. This amino acid is identified as 27 the "indicator" and has been shown to respond to the level of adequacy of the first limiting amino acid or test amino acid (Kim et al, 1983a;Kim et ai, 1983b;
Kim and Bayley 1983; Bal1 and Bayley 1984). When the intake of the test amino acid is below requirement, oxidation of the indicator amino acid is dependent on its excess relative to the test amino acid level. Once requirement has been met, maximum utilization of the indicator has been achieved and it's oxidation no longer responds to increasing intake (Figure 2.1).
Selection of an appropriate "indicator" arnino acid is critical to the function of the model. The indicator must be an indispensable amino acid whose catabolic pathway leads primarily to CO,, with the labelled carboxyl carbon is irreversibly oxidized to CO,. Phenylalanine, lysine, and the branched-chain amino acids meet these criteria. It was found that methionine was unsuitable
(Brookes et al, 1972) since no relationship was found between [14C-methyl] methionine oxidation and lysine intake. Presumably this was due to label incorporation into several reactions besides oxidation.
A major strength of the model lies in the studying of 6 or 7 levels of the test amino acid intake within the same individual. Such a design allows for sufficient intake levels to bracket the expected breakpoint or the point at which oxidation of the indicator begins to plateau. Statistical rnodelling (Seber 1977;
Zello et al, 1990a) objectively identifies the intersection point of two regression lines defined by the data. The intersection point is considered to be the "mean requirernent". Using the estimate of variation about the intersection point, a 95% Breakpoint =
Oxidation
L-[1-I3C]Lysine
Dietary Tyrosine lntake (mg*kg-lad4)
Figure 2.1 Model of indicator amino acid oxidation adapted from Ball and Bayley, 1984. The mode1 is based on the concept that al1 amino acids, including the indicator, are oxidized relative the the limiting amino acid. Intakes of the limiting amino acid above requirement does no longer affects oxidation of the indicator. 29
upper confidence level is calculated and used as representative of a typical
recommended intake (Figure 2.1) (Zello et al, 199Oa; Zello et al, 1993; Duncan et
al, 1996; Lazaris-Brunner et al, 1998).
While sharing sorne of the limitations associated with direct arnino acid oxidation studies, the indicator amino acid oxidation method overcomes some of the pitFalls. lsotope dilution does not occur since the label is on the indicator amino acid, separate from the test amino acid. Therefore, the intake of the
indicator remains constant with respect to dietary and isotope intake for each study. In fact indicator studies have demonstrated that the flux of the indicator
pool remains constant regardless of the level of test amino acid intake (Zello et al, 1990a).
The IAAO method is a mode1 used to identify rapid changes in amino acid oxidation in response to changes in dietary intake and therefore is perfomed only in the fed state. It does not require prior adaptation to a test level of amino acid and thus is suitable for use in more vulnerable populations. Furthermore, the requirement for any arnino acid can be tested since the label is on the
indicator amino acid and not the test amino acid. Any level, including a diet devoid of the test arnino acid, can be studied since the Iower limit of intake is no
longer restricted to the isotope dose.
Phenylalanine has been used extensively as an effective indicator amino
acid in piglet studies investigating requirements for lysine (Kim et al, 198%;
House et al, 1998), histidine (Kim et al, 1983b), threonine (Kim et al, 1983a), 30 methionine (Kim and Bayley 1983), and arginine and proline (Bal1 et ai, 1986).
Similar estimates of tryptophan requirement were found in piglets when either L-
[1-'4C]phenylalanine or L-[l -'4C]lysine was used as indicator (Bail and Bayley
1984). The indicator amino acid oxidation rnethod has been extended to estimate lysine (Zello et al, 1993; Duncan et al, 1996), tyrosine (Roberts et al,
1WB), and threonine (Wilson et al, 1997) requirement in adult males, as well as tryptophan in adult females (Lazaris-Brunner et al, 1998). IAAO using lysine as an indicator, was used for the first time in children with PKU to determine tyrosine requirements (Bross et al, 1997). Estimates of requirement using indicator amino acid oxidation have supported the view of investigators using direct arnino acid oxidation methods, suggesting that the FAONVHOIUNU (1985) report underestimates requirements.
The indispensable amino acid oxidation method is a sensitive funetional method used to investigate indispensable arnino acid requirement. The advantages associated with the indicator rnethod allows it to be applied in vulnerable groupç who are receiving adequate nutrition.
2.2.2.4. Methodological lmprovements
The rnethodologies for stable isotope tracers have improved enormously over the past couple of decades. While sorne researchers have managed to reduce the tracer infusion time with primer infusions (Matthews et al, 1980;
Thompson et al, 1989), others have validated the use of urinary arnino acid 31 enrichment for representation of plasma amino acid enrichment waterlow and
Stephen 1967; De Benoist et al, 1984; Wykes et al, 1990; Darling 1997), still others have simplified the diet protocol (Thorpe et al, 1997; Bross et al, 1998).
These methodological irnprovements, by reducing the tirne and invasiveness of the studies, have allowed the ethical application of amino acid requirement studies to more vulnerable groups (e.g., PKU, neonates).
2.2.3. Factors Affectina Amino Acid Requirements
Studies investigating amino acid metabolism and req uirement must ensure adequate control for factors that can potentially confound results and lead to erroneous conclusions. Some important confounding factors faced in studies investigating amino acid metabolisrn include the level of energy intake, amount of non-specific nitrogen, and route of nutrient intake.
2.2.3.1. Energy lntake
When energy intake is inadequate, amino acids are oxidized as a source of energy substrate. Thus, the level of energy consumed is an important factor deterrnining the utilization of dietary amino acids. Zlotkin et al, (1981 a) exarnined the interactive effect of energy and protein intake on growth and nitrogen retention in intravenously-fed preterm infants. Their findings showed that nitrogen retention, at various protein levels is improved by increasing the level of non-protein energy from 50 to 80 kcalskg-'.d-'. In a sirnilar group of 32
neonates, DufQ et al (1981) also demonstrated increased nitrogen retention at greater non-protein energy intakes of 93 vs 68 kcal-kg-l-d-lusing a [15N]glycine stable isotope tracer. The improvement in nitrogen retention observed at higher energy intakes in low birthweight infants was likely due to the body's increased ability to use amino acids for protein synthesis because they were no longer required as energy substrate. Similar findings have been made in adult humans
(Motil et al, 1981a). These studies reveal, that for pretem neonates, non-protein energy intakes of 80-85 kcal-kg".d-' along with sources of good quality protein at
3.0 g-kg-l-d-'are required to attain in utero growth rates (320 mg N-kg-'sd")
(Widdowson 1981).
Milethese studies have unveiled the interaction that exists between the level of energy intake and nitrogen retention, more recently, a more subtle effect of energy intake on individual amino acid utilization (Clark et al, 1989; Lucas et al, 1993) has been reported. Clark and colleagues (1989) measured weekly plasma amino acid levels in 109 preterm infants receiving parenteral nutrition in an attempt to establish a database of plasma arnino acid levels of preterm neonates. They noted that the incidence of hyperphenylalaninaemia was low in the presence of adequate energy intake (up to 119 kcal-kg-'-d-'). Further supporting these findings, Lucas et al, (1993) using a more in-depth analysis of the relationship between energy intake and plasma phenylalanine utilization, recognized that hyperphenylalaninaernia was more prevalent in infants consuming Vamin than in a reference population of infants consuming breast 33 milk or formula. Further analysis of their data led them to conclude that the high concentrations of plasma phenylalanine were only observed when the total energy:protein energy ratio was below 8.5:l k~al-kg-~-d"and that energy intakes of 34 kcal-g-' protein could in most cases minimize the incidence of hyperphenylalaninaemia.
2.2.3.2. Non-Specific Nitrogen lntake
lnadequate intake of non-protein nitrogen in a diet is thought to increase requirements of indispensable arnino acids by reducing the efficiency upon which they are retained in the body (Kies 1974). Kies (1972a; 1974) demonstrated improved nitrogen retention with the addition of diammonium citrate and glycine to diets containing the same level of indispensable amino acids but low in total nitrogen. Similarly, Snyderman (1962) demonstrated improved nitrogen balance in neonates on low protein intake when receiving supplemental non-protein nitrogen in the form of urea or glycine. The recycling of urea in the neonate has been shown by Jackson (1989) to represent an important cornponent of nitrogen homeostasis in the neonate receiving human milk. These findings have led
Jackson to suggest (Jackson 1989) that glycine may be conditionally essential during the neonatal period.
While benefits of nonspecific nitrogen are apparent at low to deficient protein intake, Hiramatsu et al (1994) have shown that when intake of nonspecific protein is considered adequate, additional intake does not provide further benefit to the individual's amino acid economy.
2-2.3.3. Route of Nutrient Delivery
Studies investiyating indispensable amino acid metabolism have concluded that needs rnay differ under certain pathophysiological conditions or developmental stages of maturity (Penchan et al, 1996). Recent work comparing intragastric and intravenous metabolism of amino acids have highlighted the fact that an individual's needs for an arnino acid may differ due to route of nutrient administration (Bertolo et al, l998a; Bertolo et al, 1998b). This rnay be due to considerable splanchnic amino acid extraction and first-pass metabolisrn (Krempf et al, IWO;Reeds et al, 1996; Stoll et al, 1997: Stoll et al,
1998; Dudley et al, 1998) largely attributed to physiological changes in the gut
(Buchman et al, 1995; Dudley et al, 1998) resulting in reduced peripheral availability which suggests reduced needs in the parenterally-fed individual.
A short term study comparing enterally and parenterally-fed neonates did not demonstrate differences in ieucine kinetics (Denne et al, 1994). In contrast,
Duffy and Pencharz (1986) and Jeevanandam et al (1987) found increases in parameters of protein metabolism. These studies are particularly pertinent to efforts made toward the development of an ideal parenteral amino acid formulation for the neonate. It is now apparent that the traditional reference protein of human milk, does not apply to individuals sustained on parenteral nutrition. 2.2. AROMATlC AMiNO AClD METABOLISM
2.3.1. Phenvlalanine
The essentiality of phenylalanine in the human was first identified by Rose
(1957). Normal plasma phenylalanine concentration in the adult is 58 * 15 pM
(Scriver et al, 1995). for the child and adolescent 62 I18 FM (Scriver et al, 1995) and a similar range of levels is found in the neonate 20-70 FM (Wu et al, 1986).
Phenylalanine uptake into the cell is carrier rnediated in a Na+-dependentand independent manner depending on cell types (Kragh-Hansen et al, 1984;
Christensen 1986).
Hepatic phenylalanine hydroxylation is the first and limiting reaction in the catabolic pathway of phenylalanine ultimately leading to CO, and H,O (Moss and
Schoenheimer 1940; Milstien and Kaufrnan 1975) (Figure 2.2). When phenylalanine hydroxylase (EC 1.I4.16.l) is defective, as in the case of phenylketonuria, or overloaded due to intake outside the limits of tolerance, transamination products of phenylalanine (phenylpyruvate, phenyllactate. phenylacetate, phenylacetylglutarnine) are produced and excreted in the urine to rid the body of accumulated phenylalanine (Vavich and Howell 1971; Chessex et al. 1985; Walker and Mills 1990; Wykes et al, 1994a) (Figure 2.3). Normally, these compounds are found in low to trace quantities in the urine (Chalmers and
Lawson 1988; Mitchell et al, 1995).
Successful phenylalanine hydroxylation requires the presence of several components which include phenylalanine hydroxylase, dihydrobiopterin Tetrahydro biopterin Phenylalanine Dihydmpteridne Hydmxy/ase Dihydrobiopterin
no - O\ / - CH, -;Fi3; COO- a-Ketog lutarate Tpshe Aminotransferase
Glutamate
4-Hydroxyphenylpymate Dioxygenase
,CH, - COO- Homogentisate
-0OC . H C=C H* COD
Fumarate Acetoacetate
Figure 2.2 Principal pathway of aromatic amino acid catabolism Identifies fate of the carboxyl carbon a-Ketog lutarate Aminotransferase Glutamate
O Phenylpyruvate = - CH, - EH - coo-
NAD4 NADH + H'
Ho . ,CH, - COO- P henylacetate <=> Phenyllactate
2-Hydroxyphenylacetate
Figure 2.3 Alternative pathway of phenylalanine catabolism 38
reductase (to reduce oxidized BH,), and pterin tetrahydrobiopterin (BH,)
(Kaufman 1971). A dehydratase enzyme associated with the cornplex is believed to stimulate hydroxylation activity (Lazarus et al, 1983),although not considered essential. The hydroxylation step is considered to be the most . important determinant of phenylalanine homeostasis (Scriver et al, 1995) and therefore has several regulatory controls. It's activity is sensitive to its substrate through allosteric modulation (Shiman and Gray 1980). Down regulation occurs in the presence of the cofactor BH, (Scriver et al, 1995). These properties protect the individual from both high and low phenylalanine concentrations.
While early studies examining the enzyme's developmental maturity in the rat suggested low activity (Kenney and Kretchmer 1959), later studies have demonstrated that phenylalanine hydroxylase reaches levels of activity similar to that of the adult by the second trimester (Ryan and Orr 1966; Friedman and
Kaufman 1971; Jakubovic 1971). More recent studies investigating in vivo neonatal hydroxylation with isotopically labelled amino acids have found significant hydroxylation capacity (Castille et al, 1994; Shortland et al, 1994;
Kilani et al, 1995; van Toledo-Eppinga et al, 1996; Clark et al, 1997). However low plasma tyrosine concentration in infants receiving TPN with very low tyrosine levels and high phenylalanine content suggests that the neonate may not hydroxylate sufficient phenylalanine to meet needs. In fact, these neonates have been seen to occasionally experience increases in plasma phenylalanine (Evans et al, 1986; Puntis et al, 1986; Walker et al, 1986; Mitton et al, 1988; Mclntosh 39 and Mitchell 1990), indicating decreased capacity to metabolize high quantities of phenylalanine. Although hyperphenylalaninaemia associated with elevated phenylalanine intake has not been shown to be associated with deleterious effects (Lucas et al, 1993), concern remains in view of the fact that it has been demonstrated that even moderate elevation in plasma phenylalanine levels inhibit myelination in the developing rat brain (Agrawal et al, 1970). Finally, it would be preferable to avoid hyperphenylalaninaernia, as longer-term consequences are unknown and research into the issue is lirnited.
2.3.2. Tvrosine
Tyrosine is derived endogenously from hepatic hydroxylation of phenylalanine (Figure 2.2) (Moss and Schoenheimer 1940). Normal fasting plasma concentration of tyrosine is 35-90 pM in the adult, 30-90 pM in children and 25-103 pM in the neonate (Mitchell et al, 1995). Alternate products of tyrosine catabolism are excreted in the urine when the pathway is overloaded.
These products include 4-hydroxyphenylpyruvate, 4-hydroxyphenyllactate, 4- hydroxyphenylacetate, N-acetyltyrosine and tyramine (Figure 2.4). Normal concentrations for 4-hydroxyphenyllacetate, 4-hydroxyphenylpyruvate, and N- acetyltyrosine are c2 mmol~molcreatinine-' and 4-hydroxyp henylacetate is 6-28 rnrnol-mol creatinine-'. Tyrosine is the precursor of the catecholamines, dopamine (DA). norepinephrine (NE), and epinephrine (E), thyroid hormones and - CH, CH COO' - * - NH,' Tyrosine Tyrosine
,CH, - CH2 - NH; (=)
Tyrarnine NADH + H' 0, + H20 Dehydrogenase a Dehydrogenase NAD* NADH + H+ NH, + H202
,CH, - coo- (=)
Figure 2.4 Alternative pathway of tyrosine catabolism 41 the melanin pigments (Murray et al, 1996). Although important to the organism, these quantitatively represent a small portion of tyrosine requirement.
The site of tyrosine catabolism is pnrnarily within the cytosolic cornparbnent of the Iiver. Tyrosine arninotransferase (E.C. 2.6.1 -5.)is the first and rate limiting enzyme in the catabolisrn of tyrosine, possessing an activity that is 1/34 that of the next enzyme, 4-hydroxyphenylpyruvate dioxygenase, within the pathway (Taniguchi and Gjessing 1965). Tyrosine aminotransferase catalyses the conversion of tyrosine to 4-hydroxyphenylpyruvate. The enzyme requires pyridoxal5'-phosphate and is rnodulated hormonally (Mitchell et al,
1995). Low enzyme activity exists in the fetus, but seems to increase rapidly after birth (Ohisalo et al, 1982). Although tyrosine amino transferase is the rate- limiting step in the tyrosine catabolic pathway, Chydroxyphenylpyruvate dioxygenase can become limiting where there is tyrosine loading and maximum enzyme induction (Know et al, 1963).
A cornplex reaction catalysed by 4-hydroxyphenylpyruvate dioxygenase converts 4-hydroxyphenylpyruvate to homogentisate. The enzyme is present in the cytoplasm of hepatocytes and renal tubules. Molecular oxygen is used to contribute one oxygen to the hydroxyl and the other to the carbonyl group of homogentisate (Lindblad et al, 1970). Ascorbic acid is required as cofactor and ferric iron (Fe'') functions at the catalytic site (Lindstedt and Odelhog 1987). The enzyme is inhibited by the en01 form of its substrate and phenylalanine (Lindstedt and Rundgren 1982; Mitchell et al, 1995). Homogentisate's ring is then cleaved 42 forrriing maleylacetoacetate by homogentisate oxidase (EC 1.1 3.1 1.1 5). The enzyme is localized in the cytoplasrnic cornpartment of cells within the kidney and Iiver (Mitchell et al, 1995). Formation of fumarylacetoacetate, the more stable trans isomer is catalyzed by maleylacetoacetate isomerase (EC 5.2.1.2), which is subsequently hydrolysed by fumarylacetoacetate hydrolase (EC 3.7.1 -2) to fumarate and acetoacetate, gluconeogenic and ketogenic substrates, respectively.
Although several inborn errors of metabolism known to result in hypertyrosinaemia have been identified, transient tyrosinemia of the newborn is the most common cause of high plasma tyrosine levels (Mitchell et al, 1995). lmmaturity of 4-hydroxyphenylpyruvate dioxygenase is thought to be responsible for the neonatal condition. Predisposing factors include prematurity, elevated protein intake and deficient vitamin C intake (Mitchell et al, 1995). In fact, neonatal4-hydroxphenylpyruvate dioxygenase has been shown to possess 18-
30% the activity of the adult (Ohisalo et al, 1982) alternate products of 4- hydroxyphenylpyruvate are excreted in the urine in these neonates (Chalmers and Lawson 1988). While elevated plasma tyrosine levels are generally thought to be benign, there has been links to poor intellectual performance (Menkes et al, 1972; Mamunes et al, 1976).
2.3.3. Neonatal Phenvlalanine and Tvrosine Metabolic Studies
Estimates of phenylalanine and tyrosine flux in the enterally fed neonate 43 are few, but they indicate that the phenylalanine flux ranges from 90-145 pal-kg-'-h-' (Denne et al, 1992; Wykes et al, 1992; Darling 1997) and tyrosine flux has been estimated to be 93 prnol-kg-'-h" (Denne et al, 1993).
Phenylalanine hydroxylation rate has been estimated to lie between 11 -22 pmol-kg-'-h-'representing 8-25% of phenylalanine flux (Denne et al, 1992; van
Toledo-Eppinga et al, 1996).
Concerns with developmental imrnaturity of enzymes in the phenylalanine catabolic pathway combined with difficulty in maintaining nomal plasma concentrations in the parenterally-fed neonate has led to considerably more data on aromatic amino acid kinetics in the parenterally-fed condition. Phenylalanine and tyrosine flux with this route of feeding ranges from 86-195 pm~l-kg"-h-~and from 46-1 02 pm~l.kg-~.h-'for phenylalanine and tyrosine, respectively (Wykes et al, 1992; Denne et al, 1993; Shortland et al, 1994; Kilani et al, 1995; Denne et al,
1996; van Toledo-Eppinga et al, 1996; Clark et al, 1997).
The parenteral phenylalanine intake during these studies differs considerably due largely to the range of phenylalanine content within commercially available parenteral amino acid solutions. While differing phenylalanine intake does not obviously affect the estimates of phenylalanine and tyrosine Rux, differing intake does seem to impact on neonatal hydroxylation rates. The hydroxylation rate observed in the neonate receiving paediatric amino acid solutions with rnodest levels of phenylalanine range from 11-22 pmol-kg-l-h-'
(Denne et al. 1996; Clark et al, 1997). Sirnilarly, in studies where neonates 44 received parenteral amino acid solutions elevated in phenylalanine, hydroxylation was found to be greater, and in the range of 37-48 prnol-kg-'-h-'
(Castille et al, 1994; Shortland et al, 1994). These results suggest that the neonate is capable of significant hydroxylation and responds to increased substrate availability by increasing hydroxylation. However, recent investigations into the phenylalanine hydroxylation model suggest that it overestimates hydroxylation at high phenylalanine intakes (House et al, 1998). The difficulty in accurately sampling the precursor pool is considered the most likely reason for the uncertainties associated with the phenylalanine hydroxylation estimate.
While plasma is the sampling pool typically used in isotope studies, recent evidence suggests that it rnay not be a true representation of precursor pool enrichment (Ballmer et al, 1990; Reeds et al, 1992). There have not been any studies to date comparing the aromatic amino acid metabolism of infants fed parenteral amino acid solutions that differ in levels of aromatic amino acid concentration.
Even with the greater rates of phenylalanine hydroxylation observed in neonâtes receiving increased phenylalanine intake, plasma tyrosine is not normalized during parenteral nutrition often remaining low or becoming excessively higher than the breast-fed neonate (Chessex et al, 1985; Puntis et al, 1986; Mitton et al, 1988; Clark et al, 1989; Mclntosh and Mitchell 1990; Mitton et al, 1993; Murdock et al, 1995). This suggests that a preformed source of tyrosine is necessary in the parenterally-fed neonate, as was shown for the 45 enterally fed infant by Snydeman (1971). Since parenteral nutrition induces tyrosine aminotransferase. the first step in the catabolism of tyrosine (Hilton et al.
1998). greater induction of this enzyme rnay play a role in tyrosine's rapid disposal within the hepatocyte rather than it's immediate distribution in the plasma pool.
Concern with the abnormal plasma phenylalanine and tyrosine lies in the potential effects that aromatic amino acid imbalances may have on brain neurotransmitter homeostasis. The developrnental and neurological impairment manifested in phenylketonuria is well documented (Krause et al. 1985; Krause et al, 1986; Smith 1994). while the effects of low tyrosine levels are speculative.
Recently animal studies have begun to examine brain tyrosine and catecholamine levels with parenteral feeding (Radmacher et al. 1993; Lopez and Rassin 1995). Since tyrosine is the direct precursor of brain neurotransrnitter synthesis. whose effects are important to normal growth and function of the developing brain (Lauder 1983), consideration of the impact of parenteral amino acid intake on the balance of brain amino acids is important.
2.3.4. Adult Phenylalanine and Tvrosine Metabolic Studies
Similar to the neonate. there is sparse information on the phenylalanine and tyrosine kinetics of the adult. Serious doubt with regard to the current estirnates of indispensable amino acid requirements have resulted in several investigations into the area of arnino acid requirements using newer methods 46
(Zello et al, 199Oa; Sanchez et al, 1995; Sanchez et al, 1996; Basile-Filho et al,
1997; Basile-Filho et al, 1998). In vivo estirnates of phenylalanine hydroxylase activity has also provided information in the area. primariiy by developing a new kinetic model to rneasure phenylalanine hydroxylation rate (Clarke and Bier
1982; Thompson et al, 1989).
Zello et al, (1990a) estimated phenylalanine requirernent using direct amino acid oxidation methods, provided graded levels of p henylalanine to adult subjects receiving an adequate protein and excess tyrosine intake. The mean phenylalanine requirernent was found to be 9.1 mg-kg-'ad-' with a 95% upper confidence Iimit, representative of a safe population intake, of 14 mg - kg-' -d".
In several extensive studies frorn the same group (Sanchez et al, 1995;
Sanchez et al, 1996; Basile-Filho et al, 1997; Basile-Filho et al, 1998), phenylalanine and tyrosine kinetics and adequacy of total aromatic amino acid intake waç examined in healthy adult males. Assessrnent of adequate intake was based on 24 h phenylalanine hydroxylation, oxidation estimates and amino acid balance. Colle~tively~this body of work (Sanchez et al, 1995; Sanchez et al, 1996; Basile-Filho et al, 1997; Basile-Filho et al, 1998) concluded that a phenylalanine intake of 21.9 mg-kg-'-d-' in a diet devoid of tyrosine does not maintain whole body phenylalanine balance. whereas subjects receiving an intake of 39 mg-kg-l-d-'or 100 mg-kg-'-d'' experienced kinetics characteristic of adequate intake and were able to maintain arnino acid balance. These conclusions were confirmed using a second independent isotope protocol 47
(Basile-Filho et al, 1998). These authors suggested that 39 mg-kg-'-d-' be used as the tentative estimate of requirement until more information is available.
Although this work represents valuable information and a great research undertaking, their estimate lacks precision due to the very few phenylalanine intake levels studied. While 21-9 rng.kg-'-d" of phenylalanine seemed inadequate and 39 mg-kg-'-d-' seemed adequate, the large difference of 17.9 mg-kg-'-d-'that separates the two estimate leaves a big gap within which true requirement may actualty lie.
It is difficult to make cornparisons between the studies that exist on aromatic amino acid kinetics because of differences in isotope protocols which include issues of tracer selection and route of administration. The studies to date that examine aromatic amino acid requirement using kinetic approaches, have either involved scenarios where tyrosine intake is in excess (Zello et al,
1990a) or low (Sanchez et al, 1995; Sanchez et al, 1996; Basile-Filho et al,
1997; Basile-Filho et al, 1998). To date, there have not been any studies that examine the ideal balance of aromatic amino acids in meeting requirement. 3. RATIONALE, HYPOTHESES AND OBJECTIVES
3 1 - Rationale
AI1 parenteral nutrition solutions are low in tyrosine relative to the content
of standard neonatal reference proteins due to its poor solubility (0.453 mgeml-'
at 2S0C,Merck Index, 1983). The standard parenteral amino acid solution used
at The Hospital for Sick Children, Vamin, has compensated for the low tyrosine
levels by elevating the phenylalanine content. This approach assumes that there
will be adequate endogenous phenylalanine hydroxylation by the neonate to
synthesize tyrosine and dispose of excess intake. Occasionally, it has been
observed that the level of phenylalanine in Vamin is poorly tolerated as indicate
by elevated plasma phenylalanine concentration (Evans et al, 1986; Puntis et al,
1986; Walker et al, 1986; Mitton et al, 1988; Mclntosh and Mitchell 1990) and the
excretion of alternate metabolites of phenylalanine and tyrosine catabolism
(Chessex et al, 1985; Walker and Mills 1990; Wykes et al, 1994a). Paediatric
amino acid solutions, in contrast (Primene, is an example of this type of
solution), have been developed with special consideration for the irnmaturities of
the neonate and therefore have only modest phenylalanine content. Although
plasma amino acid levels in infants receiving these solutions are similar to
breast-fed infants, the low total aromatic amino acid intake of these solutions
may be inadequate for neonatal needs (Wykes et al, 1994a). Therefore an
evaluation of the phenylalanine and tyrosine rnetabolism of neonates receiving
Vamin or Primene as source of amino acids is required, and makes up the first 49 studies of this thesis.
The FAONVHOIUNU (1985) indicated a primary need was an assessment of amino acid requirements of the neonate. Generally, present parenteral amino acid solutions are patterned against accepted reference proteins or upon an attempt to duplicate plasma amino acid concentrations of the breast-fed neonate. Recent data have demonstrated that amino acids infused parenterally are metabolized differently from those given through the natural enteral route
(Krempf et al, 1990; Reeds et al, 1996; Stoll et al, 1997; Bertolo et al, 1998a;
Dudley et al, 1998; Stoll et al, 1998). Therefore, the requirernent of individuai amino acids administered parenterally must be determined to establish a basis upon which an ideal balance of amino acids for parenteral use can be developed. Of particular concern is the tyrosine content of parenteral arnino acid solutions since it has been demonstrated to be the first limiting amino acid of the commonly used paediatric amino acid solutions (Wykes et al, 1994a).
There are several reasons for expanding the investigations into the adult.
Firstly, because the experiment follows a similar design to the neonatal glycyl-L- tyrosine supplementation study, a cornparison can be made between the mature adult and the immature neonate with respect to the optimal balance of aromatic amino acid intake. Secondly, an ongoing controversy exists surrounding currently accepted aromatic amino acid requirements. The present population estimate of the total aromatic amino acids is 14 mg-kg-'-de'(FAONVHOIUNU
1985). This estirnate is founded on very few studies of small sarnple size using 50
nitrogen balance methods (Rose 1957). Applying direct oxidation methods, Zello
et al (1990a) estimated phenylalanine requirement in the presence of excess
tyrosine intake. The phenylalanine mean requirement was found to be 9.1
mg-kg-l-d-'with an upper 95% confidence limit of 14 mg-kgelad-'.These results
suggest that the FAOMRIOIUNU (1985) estimate of total aromatic amino acid
requirernent is underestimated. We investigated this issue by examining the total aromatic requirement as met by providing adequate phenylalanine intake
(9.1 mg-kg-' .de') and graded tyrosine intakes from deficient to excess. 3.2. Hypothesis and Objectives
Experiment I
Hvpotheses
1. Phenylalanine hydroxylation and oxidation will be greater in infants receiving parenteral nutrition with high phenylalanine (Vamin) versus rnoderate phenylalanine content (Primene).
2. The appearance of alternate metabolites of phenylalanine and tyrosine catabolism will increase in neonates receiving Vamin cornpared with that of those receiving Primene.
Obiectives a) To compare phenylalanine hydroxylation and oxidation in neonates receiving Vamin or Primene as source of arnino acids.
b) To quantify the presence of alternate metabolites of phenylalanine and tyrosine catabolism to assess the tolerance of phenylalanine intake. Experiment II
Hvpothesis
The tyrosine requirement of the TPN-fed neonate can be estimated by partitioning the oxidation of phenylalanine to a Wo-phase Iinear-regression analysis whereby the intersection of the two Iines is representative of the mean req uirement.
Objectives
1. To determine the tyrosine requirement of the TPN-fed neonate receiving a fixed p henylalanine intake.
2. To calculate the proportional balance of phenylalanine to tyrosine at the mean tyrosine requirement intake.
Experiment III
Hvpothesis
The tyrosine requirement of the adult male receiving a fixed phenylalanine intake, can be estirnated by patitioning the oxidation of the indicator amino acid, lysine to a two-phase linear-regression analysis whereby the intersection of the two lines is representative of the mean requirernent. 53
Objectives
1. To detemine the tyrosine requirement of the adult male receiving a fixed phenylalanine intake.
2. To calculate the proportional balance of phenylalanine to tyrosine at the mean tyrosine requirement intake. 4. PHENYLALANINE AND TYROSINE METABOLISM IN NEONATES
RECEIVING PARENTERAL NUTRITION THAT DIFFERS IN SOURCES OF
AMIN0 AClDS
4.1. INTRODUCTION
Tyrosine is considered indispensable during the neonatal period
(Snyderman 1971; Uauy et al, 1993; Castillo et al, 1994). Providing adequate tyrosine intake in the parenterally-fed infant is particularly difficult due to its poor
solubility, restricting maximum tyrosine intake to less than 1% of total amino
acids. This level of intake is significantly lower than the estirnated requirement of
Snydeman (1971) in neonates (2.0%). of House et al (1997a) in the piglet
(2.7%) or the content of hurnan milk (5.9%) (Rassin 1989). lncreasing parenteral phenylalanine intake is the most common means of providing a precursor source of tyrosine. This approach relies on the neonate's ability to hydroxylate
phenylalanine to tyrosine. Unfortunately, several investigations have observed that this approach may result in poor tolerance as indicated by elevated plasma
phenylalanine (Chessex et al, 1985; Evans et al, 1986; Puntis et al, 1986;
Walker et al, 1986; Mitton et al, 1988; Rigo et al, 1987; Puntis et al, 1989;
Mclntosh and Mitchell 1990; Walker and Mills 1990; Wykes et al, 1994a) and
urinary excretion of alternate catabolites of phenylalanine (phenylpyruvate, phenylacetate. phenyllactate, 2-hydroxyphenylacetate) and tyrosine (4-
hydroxyphenylpyruvate, 4-hydroxyp henylacetate, 4-h ydroxyp henyllactate)
54 (Chessex et al, 1985; Walker and Mills 1990; Wykes et al, 1994a).
Early studies examining phenylalanine hydroxylase enzyme (EC
1.14.16.1) activity from fetal liver tissue demonstrated that the phenylalanine hydroxylase activity of the fetus is only 57% that of the adult (Delvalle and
Greengard 1977). In contrast, recent studies investigating in vivo neonatal phenylalanine hydroxylation have found significant hydroxylation capacity in the neonate (Castillo et al, 1994; Shortland et al. 1994; Kilani et al, 1995; van
Toledo-Eppinga et al, 1996; Clark et al, 1997). These studies examined the rate of phenylalanine hydroxylation in response to parenteral nutrition (Shortland et al, 1994; Kilani et al, 1995; Denne et al, 1996; Clark et al, 1997), route of nutrient administration (van Toledo-Eppinga et al, 1996). maturity (Castillo et al, 1994;
Denne et al, 1996). and septic state (Castillo et al, 1994). While Shortland et al
(1994) have demonstrated that phenylalanine hydroxylation increases in response to a supply of amino acid substrate, other studies have not shown such an increase (Denne et al, 1996;Clark et al, 1997). These latter studies (Denne et al, 1996; Clark et al, 1997) used arnino acid solutions that had lower phenylalanine levels; the available phenylalanine may be an important factor in determining the phenylalanine hydroxylation response to parenteral nutrition.
Therefore, it is still unclear how the neonate responds to the differing phenylalanine intakes that are commonly provided by cornmercially available amino acids solutions. Of the solutions used in neonatal populations, "general purpose" amino acid solutions contain large amounts of phenylalanine to 56 compensate for their fow tyrosine levels. "Paediatric" amino acid solutions, in contrast, have been developed with particular concem for the immaturities of the
neonate, and therefore contain only modest levels of phenylalanine. Infants
receiving paediatric solutions generally demonstrate good tolerance of aromatic amino acid intake, however, evidence exists that these solutions may be inadeqvate in their content of total arornatic arnino acids (Wykes et al, 1994a).
To better understand the utilisation of phenylalanine as a parenteral precursor of tyrosine, this study examined the parenterally-fed neonates' aromatic amino acid metabolism, using infusions of stable isotope labelled phenylalanine and tyrosine, while receiving amino acid solutions that differed in their content of phenylalanine. It was hypothesised that the parenterally-fed neonate responds to increased phenylalanine intake by increasing their rate of phenylalanine hydroxylation and oxidation. Furthermore, urinary excretion of phenylalanine and tyrosine and alternate catabolites of phenylalanine and tyrosine were measured to assess tolerance of the arornatic amino acid intake.
4.2. MATERIALS AND METHODS
4.2.1. Patients and Nutrient lntake
Sixteen parenterally-fed infants (Table 4.1) who had been referred to the
Neonatal Intensive Care Unit of The Hospital for Sick Children were enrolled into Table 4.1 Subject information of parenterally-fed neonates receiving Vamin or P~irnenel*~
n Birth weight (kg) Gestational age (wk) Postnatal age (d) Postconceptional age (wk) Sex (M:F) Weight (kg) Length (cm) Head circumference (cm) Size for gestational age (A:S, A=appropriate, S=small) Ventilatory support (#) Diagnoses Necrotizing Enterocolitis Respiratory Distress Syndrome Intestinal Atresia Gastroschisis Tracheoesophageal fistula 'Mean ISD qhere were no significant differences between groups for continuous parameters (pO.05). 58
a randomized trial comparing two cornmercially available parenteral amino acid solutions currently used for neonates. All infants were clinically stable (afebrile, stable vital signs within normal range, absence of acute illness, and if surgical, 3 days post surgery) during the study period and were maintained in standard servo-controlled incubators or infant cribs. Gastroschisis, intestinal atresia, necrotizing enterocolitis and respiratory distress syndrome were the rnost common diagnoses in each group (Table 4.1). The study protocol was reviewed and approved by the Human Subject Review Cornmittee of The Hospital for Sick
Children. and written, informed consent was obtained from one or both parents
(a copy of the consent form appears in Appendix 9.1).
Infants were randomized to receive one of two parenteral feeding regimens differing only in arnino acid profile (Figure 4.1). The amino acid solutions were: Varnin (Vamin N, Pharmacia and Upjohn, Stockholm, Sweden), the standard amino acid solution used at The Hospital for Sick Children and patterned after the amino acid profile of egg protein, and Primene (Baxter,
Deerfield, IL), an amino acid solution patterned after human umbilical cord blood and designed for the paediatric population (Table 4.2). Previous studies from the present laboratory have used the amino acid pattern of Varninolact
(Pharmacia and Upjohn. Stockholm. Sweden). However, this solution was not available for use in Canada, and therefore Primene was selected as paediatric amino acid solution with a similar amino acid profile. The Primene was donated by Baxter.
60 Table 4.2 Amino acid profile of Varnin and Primene (9.1 009-' amino acid)' Vamin Primene lsoleucine 5.6 6.7 Leucine 7.5 9.9 Valine Lysine 5.5 10.9 Methionine 2.7 2.4 Cysteine 2.0 1.9
Threonine 4.3 3 -7 Tryptophan Histidine Arginine Glycine Alanine As partate 5.9 6.0 Glutamate Proline Serine Taurine O 0.6 Ornithine O 2.2 'Vamin 7% arnino acid concentration (Pharmacia and Upjohn, Stockholm), Primene 6.5% arnino acid concentration (Baxter, Deerfield, IL). Table 4.3 Dietary intake of parenterally-fed neonates receiving Vamin or Primene'** Vamin Primene
Energy (kJ-kg".d-') 360 i 56 350 * 79 Glucose (g -kg-'-d*') 13.7 I 2-9 13.0 I 3.1 Amino acids (g-kg-'-d-') 2.9 i 0.6 2.7 1: 0.6 Fat (g -kg-'- d-' ) 2.3 * 0.8 2.4 i 0.6 'Mean * SD. *Energy value of anhydrous glucose, 15.69 kJ-g-'; amino acids, 16.74 kJ-gel; lntralipid 20%. 41.84 kJ-g*'. There were no significant differences between groups (p>0.05). 62
Intakes of parenteral nutrition were as prescribed by the attending physician (Table 4.3). Parenteral intake included a solution of amino acids and glucose with a complete vitamin and mineral supplernent in combination with a
20% lipid emulsion (Intralipid, Phanacia and Upjohn) to meet nutrient needs.
Parenteral nutrition solutions were infused into a peripheral or central vein using an lmed pump (Imed Corporation, San Diego, CA). Infants were enrolled when receiving adequate amino acid and energy intake (335400 kJ-kg-'-d-'; 2.5-3.5 g protein -kg4-d-';Zlotkin et al, 1985). Minimal enteral feeds were consumed in six infants, and represented no greater than 10% of total amino acid requirement (A copy of the subject enrolment fom is found in Appendix 9.2).
4.2.2. Studv Protocol
Isotopically-labelled amino acids used for the tracer study were L-[1-
'3C]phenylalanine(99%, 13C, Tracer Technologies, Sornrnen/illel MA) and L-[3,3-
2H,]tyrosine (98%, 2H21Cambridge lsotope Laboratories, Andover, MA). Quality control tests were performed by the manufacturers. Chemical purity, specified isotopic enrichment and position was confirmed by nuclear magnetic resonance
(NMR), while isomeric purity and a second confirmation of isotopic enrichment was performed by gas-chromatography mass-spectrometry (GCMS). Solutions of each tracer amino acid in 3.3% dextrose and 0.3% NaCl were prepared by the
Manufacturing Phamacy at The Hospital for Sick Children. lsotope solutions were sterilized by passage through a 0.22 prn filter prior to dispensing into single 63 use vials for storage at 4°C until use. All solutions were dernonstrated to be sterile and free of bacterial growth over seven days in culture, and to be pyrogen-free by the Limulus Amebocyte Lysate test (Pearson 1979).
Two pilot studies were perforrned to detemine the dose of phenylalanine required to achieve measurable expired 13C0,. The L-[3,3-'HJtyrosine dose used was that of Castillo et al (1991). At Ieast 24 h after the start of the infusion of TPN; pheny!alanine flux, hydroxylation, oxidation, percent dose oxidized, and tyrosine flux were determined using a primed, 24 h constant infusion of L-[1-
'3C]phenylalanine (15.6 prnol.kg-' and 13 pmol-kg-'-h-', respectively), and L-[3,3-
2HJtyrosine (3.6 pmol-kg-' and 3.0 pmol-kg-'-h-', respectively). A period of 24 h of adaptation to the new parenteral amino acid solution was considered suffcient since the two arnino acid solutions are similar in overall nutrient intake. An isotope infusion time of 24 h was used for practical reasons. It took 9 h to achieve isotopic steady state in the urine (Figure 4.2). Sufficient samples collected after this point was necessary to define the plateau. As diapers are changed every three to four hours, running the infusion for 24 h allowed for 3 to
5 plateau urine specimens to be collected. The forms used to calculate the study tracer dose are shown in Appendix 9.3. The priming dose was infused over the first 15 min. Urine sarnples were collected for the rneasurement of background and steady state arnino acid enrichment, creatinine, and alternate catabolites of phenylalanine and tyrosine. Urine was used for measuring amino acid enrichment since it has been shown to be a valid representation of plasma A Time (h) 1 Start of isotope infusion
Figure 4.2 Urinary L-[l -13C]phenylalanine (W),L-[1-lC&rosine (O) and L-[3,3-W2ltyrosine (A} enrichment tirne course frorn a study subject. Isotope infusion started at time Oh. The start time was at least 24h after the neonate was receiving a stabte and adequate nutrient intake with either Vamin or Primene as source of amino acid intake. Plateau was achieved at 9h and maintained until the end of the infusion at 24h. 65 endchment (Waterlow and Stephen 1967; De Benoist et al, 1984; Wykes et al,
1990; Darling 1997). One to three baseline urine samples were collected prior to parenteral isotope infusion, followed by one sample every three to four hours throughout the isotope infusion. Amino acid enrichment measurement was performed on ail the samples collected from the first study subject to detemine the timing of plateau (Figure 4.2). Once the time to reach plateau was detemined, only plateau samples were analysed by GCMS. Urine was stored at
-20°C until analyses were performed. Expired CO, was collected and its production rate was measured before and after 18-20 h of constant isotope infusion. Breath measurements were only possible in twelve subjects since four infants were on ventilators. Ventilation prevented the reliable estimation of
F13C0, due to the dilution of CO, with high fiow rates of air and 0, through the ventilator systern and also because of the unrecoverable losses around the uncuffed endotracheal tube resulting in undetermined CO, production.
Nevertheless, as indicated in the statistical analyses section of this chapter, there was sufficient power to detect a significant difference of 50%.
4.2.3. Analytical Procedures and Catculations
4-2.3.1. Urinary Amino Acid Enrichment
Urine was analysed for phenylalanine and tyrosine enrichment by the method of Patterson et al (1991) using GCMS. Briefiy, urine samples (500 PL) were deproteinized with an equal volume of 20% (wlv) trichloroacetic acid 66 followed by centrifugation at 13 000 rprn x 5 min at 23°C in microcentrifuge tubes. The supernatant was transferred from the tubes to colurnns containing a cationic ion exchange resin (7.5 ml) (Dowex 50W-X8, 100-200 mesh, H'forrn,
BioRad Laboratories, Hercules, CA) for amino acid separation. The effluent solution was freeze-dn'ed (Freezone 12 L, Labconco Corp., Kansas City, MO) prior to derivatization to its N,O-heptafluorobutyryl n-propyl esters.
The first step of the derivatization process involved esterification of the arnino acids by the addition of 500 yL of acetylpropanol prepared fresh each day by reacting acetyl chloride with propanol over ice in a ratio of t5. Samples were vortexed, heated at llO°C for 1 h, and dried under a steady stream of N, gas at
~40°C.After drying, 50 pL of the derivative heptafluorobutyric anhydride was added to the samples, vortexed, and heated at 60°C for 20 min. Samples were then dried, topped with N, gas and stored at -20°C until analysis. When ready for analysis, samples were reconstituted in 100 pL of hexane.
Phenylalanine and tyrosine acid enrichment was measured on a gas chromatograph (GC, Hewlett Packard, Mode15890 Series 2, Mississauga, ON) attached to a quadrupole mass spectrometer (MS, VG Trio-2, Cheshire,
England). Derivatized amino acids were splitlessly introduced into the instrument by automatic injector (Hewlett Packard 7673 injector). Separation was perforrned on a 30 m X 0.32 mm (inner diameter) X 1.O prn (film thickness) fused silica capillary column (HP-5, Hewlett Packard) with helium serving as a carrier gas set at a head pressure of 7 psi. The injector port was maintained at 67
250°C. The GC temperature program contained two rates of temperature
increases. The first was set to start at 60°C and rise to a final temperature of
180°C within 6 min (20°C-min-'). The second increase conünued from 180°C to
260°C within 8 min (10°C-min-'). The column was maintained at 260°C for 2 min at the end of the run to clear the column of residual material.
The GC column was coupled directly to the ion source set at a temperature of 160°C and operated under conditions of negative chemical
ionization. Ammonia was used as reactant gas. Selected ion chromatograms were obtained by monitoring the m/z (rnass to charge) ratio of 383, 384 and 417,
41 8,419 for the M and M+1, and M, M+1, and M+2 species of phenylalanine and tyrosine, respectively (Figure 4.3). The M+1 peak of tyrosine represents L-[1-
13C]tyrosinederived from the hydroxylation of L-[l-13C]phenylalanine.
Phenylalanine and tyrosine had retention tirnes of 10.6 and 12.1min,
respectively. Areas under the peaks were integrated by a Digital DECp 450D,LP
cornputer (Digital Instruments, Santa, Barbara, CA), using Lab-Base software
(Vacuum Generator Biotech). lsotopomer overlap corrections were performed
based on the method of Rosenblatt et al (1 992) (Refer to Appendix 9.4 for details on isotopomer overlap corrections).
Standard curves of known concentration and enrichments of native and
L[1-13C]phenylalanineand L-[3,3-2HJtyrosine were perforrned to ensure the
proper functioning of the GCMS. While the limits of detection of the machine is
0.05 molecule percent (MP), sensitivity was measured at 0.5 rnolecule percent -.---1 - a-. -- * i b ---1.- \ ' . . . .----.! . ûrer 1- t; a;
Figure 4.3 Chromatograms of mass peaks of phenylalanine (mass 383, 384) and tyrosine (masses 417,418, 419) analyzed by GCMS set to selected ion monitoring mode. - 69
excess (MPE) based on duplicate injections of samples of vanous enrichment
(0.01 -1 % over native enrichement).
4.2.3.2. Urinary Phenylalanine and Tyrosine Concentrations and Alternate
Metabolites of Catabolisrn
Urinary phenylalanine and tyrosine concentrations were analysed by ion- exchange chromatography (Beckman 7300 Arnino Acid Analyzer) with post- column ninhydrin reaction (at 135°C) followed by spectrophotometrk detection of amino acids at A 570 nm and imino and sulfur amino acids at A 440 nm. The analysis was carried out in the Genetic Metabolics Laboratoty at The Hospital for
Sick Children.
Alternate metabolites of phenylalanine and tyrosine catabolism were expressed in pmol per mm01 of urinary creatinine. Creatinine was quantifieci by the Jaffe reaction (Butler 1975) using a Kodak Ektachem 700 Analyzer (Johnson and Johnson, Rochester, NY). The analysis involves reacting the sample containing creatining with picrate in alkaline medium (0.1 M NaOH). A red cornpound is formed that is measured spectrophotometrically at a wavelength of
470-550 nm. The detection limits of the analyzer is 88 pmol-L" with a linearity up to 291 80 pmol-L-'. Creatinine analysis was performed in the General
Biochemistry Laboratory at The Hospital for Sick Children.
Phenylalanine and tyrosine altemate metabolites of catabolism were measured by the method of Goodman and Markey (1981). The method has 70 been previously established in this laboratory using piglet urine samples (Wykes et al, 1994a). The organic acids in 1-3 mL of urine were first converted to oxirnes of ketoacids with hydroxylamine. a-Ketocaproate was used as intemal standard for oxime formation and pentadecanoic acid as the analytical intemal standard. Oxime formation was followed by acidification with 6 M HCI to pHc2.0, saturated with NaCI, and extracted in successive equal volumes of ethyl ether and diethyl ether. Extracted organiç acids were dried under N,, and subsequently derivatized to trimethylsilyl derivatives with bis(trimethylsilyl)- trifluoroacetamide containing 1% trimethylchlorosilane (TMS) as catalyst.
Tetracosane was used as external standard. The TMS derivatives were separated on a fused silica capillary column (HP-5, Hewlett Packard) and analysed by GCMS (Model 5890, Hewlett Packard) using electron impact ionization. Quantification of the phenylalanine and tyrosine alternate catabolites was performed using standard curves prepared for each compound.
4.2.3.3. Expired l3CO, Enrichment
Expired 13C0, enrichment was measured by isotope ratio mass spectrometry (IRMS) as described previously (Jones et al, 1985). CO, production, collected from a ventilated hood system was measured indirectly using a portable CO, analyser (1400 series, Servomex, Westech Industrial Ltd.,
Mississauga, ON) and rnass flowmeter (5860 series, Brooks, Trillium
Measurement and Control, Stouffville, ON). Complete CO, trapping was 71
perfoned by bubbling the exhaust from the CO, analyser at a rate of 500 ml-min", into a condenser coi1 containing 10 mL 1M NaOH for 10 min (Jones et al, 1985). 13C0, enrichment was rneasured on a dual inlet magnetic sector IRMS
(VG Micromass, Model602D, Cheshire, England). Trapped CO,, in the form of
Na,CO,, was released by reacting 0.5 mL of sample with an equal volume of
85% phosphoric acid within an evacuated Rittenburg tube. The CO, gas was isolated from the aqueous phase by immersing the tube into a bath of methanol and dry ice which functions to freeze the Iiquid, without restricting movernent of the gas. Enrichment measures were expressed as atoms percent excess (APE)
13C0, over a reference standard of compressed CO, gas. Regular repeat analysis of a standard from the National Bureau of Standards (PDB limestone,
NBS #20) demonstrated a coefficient of variation of 0.2%.
4.2.3.4. Model of Amino Acid Metabolisrn and Calculations
The model of arnino acid metabolism used in this study is a modification of the Waterlow et al (1978) model :
Q=B+I=S+PAH where, Q is the rate of phenylalanine flux (pmol-kg-'-h-l); S is the rate of phenylalanine non-oxidative disposal, a rneasure of the rate of phenylalanine incorporation into body protein; PAH is the rate of phenylalanine hydroxylation to tyrosine; B is the rate of phenylalanine released from body protein; and I is the rate of exogenous phenylalanine intake. 72
Whole body phenylalanine and tyrosine fluxes were calculated from the dilution of isotope in the body arnino acid pool at isotopic steady state (Matthews et al, 1980; De Benoist et al, 1984; Wkes et al, 1990):
Qphe = i [EJEp - 11
Whete, Q,, is the rate of phenylalanine or tyrosine (Q,) flux; i is the isotope infusion rate (pmol-kg-'- h-'), Ei is the enrichment of the infused isotope (APE) and E, is the enrichment of the amino acid in the urine (Wfaterlow and Stephen
1967; De Benoist et al, 1984; Wykes et al, 1990; Darling 1997) at isotopic steady state (plateau, in units of APE).
To measure the rate of phenylalanine hydroxylation, the model of Clarke and Bier (Clarke and Bier 1982) with the modification of Thompson et al (1989) was used:
Qphe-tyr = Qtyr [EtyCEphJ [Qpl~$~~he+ QphJ
Where, Ew,and E,,, are the urinary enrichments of L-[l-13C]tyrosineand L-[1-
'3C]phenylalanine, respectively; and i,, is the rate of infusion of the labelled phenylalanine.
The rate of phenylalanine oxidation was calculated as descrïbed by
Matthews et al (1980):
O,,, = Fq3C02(1 /Eph, - l/Ei)- 100 where, O,,, represents phenylalanine oxidation, and F13C0, represents the rate of 13C0, released by phenylalanine tracer oxidation (pmol-kg". h-') calculated from the following equation: FI3CO2= (FCO3 (ECU2) (44.6) (60) 1 (W) (RF) (100)
where, FCO, is the CO, production rate (cm3-min"); and ECO, is the 13C0,
enrichment in expired breath at isotopic steady state (APE). The constants 44.6
pmol-(cm3)-'and 60 min-h-l convert FCO, to micromoles per hour, and the factor
of 100 changes APE to a fraction. W is the weight (kg) of the infant. I3CO,
retained by the body as bicarbonate is corrected using a retention factor (RF) that was calculated from a regression equation using energy intake as the
dependent variable (y=0.64 + 0.1 667x; Van Aerde et al, 1985). The percent
phenylalanine isotope dose oxidized was calculated as:
% dose oxidized = (F'~COJ~,,)(100)
4.2.4. Statistical Analvses
Sample size was calculated using the following standard equation:
n = (Za,2+Zl-p)220 (Steel and Torrie 1980) where, n equals the estimated sarnple size for each group assuming a ho-tailed
comparison, a=0.05 and P=0.20 represents the probability allowed for
performing a type I and type II error, respectively. The o of phenylalanine
hydroxylation was used as the primary outcome measure in calculating sarnple
size, estimated from an approximate standard deviation observed in similar
studies in neonates (SD = 10 pmol-kg-'-h-l)(Castillo et al, 1994; Shortland et al,
1994; Denne et al, 1996; Clark et al, 1997). The desired difference to be
detected between treatment, 6, was chosen as 15 pmolokg-'-h" resulting in a 74 detection of a difference of 30%. The calculated required sarnple size for each
TPN treatrnent with the above criteria is 7.02 per group, rounded up to an estirnated sample size of 8 per group.
The dilution of the expired CO, with high oxygen flow rates of the neonatal ventilators in conjunction with unmeasurable leaks around the uncuffed endotracheal tube precluded the measurement of CO, production in vented neonates. Four infants were ventilated (2 per group) reducing the sample size to
6 per group. A post-hoc estimate of sample size for the type I and II errors permitted was calculated. The oxidation variation was determined from our own data since there does not presently exist phenylalanine F13C0, or oxidation data in the literature for these subjects. The SD was estimated to be 15 prnol-kge1-h-'.
A detectable difference between groups of 25 prnol~kg-'-h-',representing a difference of 50% resulted in an estimated sample size required within each TPN treatrnent group of 5.68 rounded up to an estirnated sample size of 6 per group.
The actual difference between groups for these rneasures was greater than
50%.
Data were tested for potential covariables (Le. gestational age, postnatal age, postconceptual age, weight. protein and energy intake) using analysis of covariance. None of these variables were fou nd to be significant covariables, although an interaction response was identified between energy intake and percent phenylalanine isotope dose oxidized. Since covariables were not identified, we presented data analyzed by an unpaired two-tailed Student's t-test 75
(Rosner 1990). Equality of variances was tested by the F-test. Alternate
metabolites of phenylalanine and tyrosine data were analyzed by the Wilcoxon
signed rank test (Rosner 1990) a non-parametric test that is analogous to the t-
test. A non-parametric test was conçidered to be the most appropriate analysis
for the data because it makes no assumptions regarding the distribution of the
data which in this case may not be normal as the neonates are not expected to
excrete large quantities of these alternate metabolites of phenylalanine and tyrosine catabolism. A p value less than or equal to 0.05 was considered
significant. Statistical analyses were performed using SAS (SAS Institute 1991).
4.3. RESULTS
4.3.1. Clinical Characteristics
Clinicai characteristics were not significantly different between neonates
receiving Vamin or Primene as source of arnino acids (Table 4.1). Body weights
reflected the predominance of premature infants enrolled in the study. Infants were enrolled into the study if their nutrient intakes were meeting amino acid and energy requirement levels (ZIotkin et al, 1985). Actual dietary intakes were not significantly different between groups (Table 4.3).
4.3.2. Urinary Amino Acid Enrichment
Urinary isotopic steady state was achieved for al1 neonates as defined by the absence of a significant slope (Figure 4.4). Sarnples at each time point Urine collection (h)
10 12 14 16 18 20 Urine collection (h)
Figure 4.4 Urinary phenylalanine and tyrosine plateau enrichment. Values represent molecuIes percent excess of the natural background enrichment (Le., plateau height). 80x A corresponds to babies receiving Vamin and Box B corresponds to those receiving Prirnene (L-[l -13Clphenylalanine (I), L-El-13C]tyrosine (a), and L-[3,3-2l+]tyrosine (A)). Urine coIlection is presented in the time in hours after the start of the isotope infusion. Lines represent the least-squares estimate of the mean. 77 represent urine collected closest to that hour. The average coeffkient of variation of individual plateau was 12% (range: 2.6-24%). The presence of isotope recycling was considered to be minimal as indicated by the lack of systematic increase (slope was not significantly different from zero) in enrichment at plateau for individuals or group rneans (Figure 4.4).
4.3.3. Phenvlalanine and Tvrosine Kinetics
There was no difference in phenylalanine flux (Table 4.4) between groups.
By design, phenylalanine intake was different between diet regimens (Table 4.4).
Protein breakdown was 33% greater, and approached significance (~~0.1)in the group receiving Primene (Table 4.4), however. percent flux from breakdown was found to be significantly greater in infants receiving Primene (pc0.001) (Table
4.4). Phenylalanine hydroxylation was significantly greater (pe0.004) in the neonates receiving Varnin (Table 4.4). Phenylalanine non-oxidative disposal (an estimate of phenylalanine incorporation into body protein) was not significantly different between groups (Table 4.4). No differences in tyrosine flux or intake were observed between the hnro groups (Table 4.4).
Isotopic steady state within the body HCO; pool was defined by the absence of significant dope in expired 13C02.Expired 13C0, enrichment was significantly greater (pe0.01) in infants receiving Vamin compared to those receiving Primene, while VCO, production rate was not significantly different between groups (p>0.5) (Table 4.5). Phenylalanine oxidation was significantly 78 Table 4.4 Phenylalanine (PHE) and tyrosine (TYR) kinetics in neonates receiving Varnin or Primene (prn~l=kg-~'h-')l~~
D< Vamin Primene - PHE Flux 158 * 39 158 I39 0.99 PHE appearance from: Intake 59 & 12 28 I7 3 Protein breakdown 99 I37 129 I49 0.1 Flux from breakdown (%) 61 i 10 81 *i? 0.001
PH€ disappearance to:
No n-oxidative disposal 116I46 135 * 37 0.3 TYR Flux 124k41 110 133 O. 5 TYR lntake 5I1 6 * 1 0.2 'Data were calculated from the enrichment values (Figure 4.4) inserted into the kinetic equations in the Model of Amino Acid Metabolism and Calculation Section. *Mean I SD; n=8/group 3Phenylalanine intake is different due to study design 4The estirnate of hydroxylation includes oxidation Table 4.5 Phenylalanine oxidation in neonates receiving Vamin or Primene'
Vamin Primene D<
13C0,Enrichment (APE) 0.008 I0.004 0,004 I 0.001 0.01 VCO, (mL-kg-' -min-') 7.0 I 1.1 6-6k 0.5 0.5
PHE oxidation (prnoi-kg4-h-') 27 I 17 1015 0.02 PHE dose oxidized (%)3 14*7 6i3 0.OA 'Mean ISD; n=6lgroup Amount of 13C0, expired Interaction effect with energy intake. See Figure 4.5. 80
greater in infants receiving Vamin compared with infants receiving Primene
(pc0.02) (Table 4.5). However, the actual precursor pool enrichment in the hepatocyte is unknown. Therefore, the percent phenylalanine isotope dose oxidized was measured and used as an index of phenylalanine oxidation that does not rely on precursor pool enrichment. Percent phenylalanine isotope dose oxidized was also found to be significantly greater @<0.01) in the infants receiving Vamin vs those receiving Primene.
The analysis of covariance identified a significant (p0.02)interaction response between energy intake and the percent L-[1 -I3C]phenylalaninedose oxidized in neonates receiving Vamin or Primene (Figure 4.5). Neonates receiving Varnin exhibited a reduction in the percent dose oxidized as energy intake increased (y = -0.12~+ 62; pc0.01), whereas those receiving Primene maintained a constant percent phenylalanine dose oxidized with increasing energy intake (y = -0.008~+ 3.2; pc0.7).
4.3.4. Urina- Phenvlalanine and Tvrosine Concentrations and Alternate
Metabolites of Catabolisrn
Urhary phenylalanine and tyrosine concentrations were significantly greater in infants receiving Vamin compared to those receiving Primene (Figure
4.6). There was no difference in creatinine excretion between groups, although
interindividual variation was great (Vamin: 742 i 31 5 prno1.L-' versus Primene:
791 I368 pmol-L-' ; p>0.7). 10-
1 O 1 I 1 1 300 350 400 450 500 Energy Intake (kJ-kg-7-d-1)
Figure 4.5 The interaction relationship (~~0.02)between energy intake and percent phenylalanine tracer dose oxidized in neonates receiving TPN with Vamin (i)or Primene (r). lncreasing energy intake reduced the tracer oxidation in neonates receiving Vamin but not those receiving Primene. Figure 4.6 Urinary phenylalanine and tyrosine concentrations in neonates receiving Vamin or Primene. Bars with different subscripts differ from one another (Phenylalanine p=0.05; Tyrosine p<0.05). Data is presented as Mean ISEM. 83
Alternate catabolite excretion of phenylalanine and tyrosine in urine were
standardized to urinary creatinine. The concentration of these compounds were greater in infants receiving Vamin versus infants receiving Primene (Figure 4.7 and Figure 4.8). Phenylpyruvate and 4-hydroxyphenylpyruvate are known to degrade upon storage (Vavich and Howell 1971). and were therefore not measured. Excretion of the phenylalanine catabolite 2-hydroxyphenylacetate was significantly greater in Varnin compared to Primene-fed infants (p<0.05), however the difference was primarily attributed to very large excretion of the cornpound within only two individuals. There was a tendency toward greater excretion of the phenyllactate (~~0.06)and phenylacetate (pc0.07). The tyrosine metabolite 4-hydroxyphenylacetate was significantly greater in the
Vamin compared to the Primene group (p<0.03), while 4-hydroxyphenyllactate levels were not significantly different between groups. It should be noted that one of the subjects in the Vamin group experienced the highest level of al1 the subjects for al1 the alternate catabolites. This subject did not demonstrate abnormal liver function tests.
One of the infants in the Vamin group and two in the Prirnene group experienced increased liver enzymes or unconjugated bilirubin levels during. or shortly before or afler the time of the study (closest available biochemistry result). Although these values are suggestive of impaired liver function, in general, these subjects did not dernonstrate evidence of increased urinary excretion of aromatic amino acids or alternate metabolites of their catabolism.
Figure 4.8 Urinary concentration of alternate metabolites of tyrosine catabolism. Statistical analysis by the Wilcoxon Sum Rank Test. 86
Only one of these subjects did have greater (although not the highest) excretion of tyrosine and altemate metabolites of tyrosine cataboiism.
4.4. DISCUSSION
The present study examined phenylalanine and tyrosine metabolism in stable neonates in a neonatal intensive care unit receiving parenteral nutrition differing in sources of amino acids. The difference in phenylalanine level in each amino acid solution, and therefore in total aromatic amino acid intake, is based on differing philosophies in the developrnent of these solutions. Vamin, an amino acid solution designed for the adult, has cornpensated for low tyrosine concentration by increasing the content of phenylalanine, the body's direct metabolic precursor of tyrosine. Although infants receiving Varnin exhibit good growth and nitrogen retention rates (Duffy et al, 1981), some infants experience high plasma phenylalanine concentrations (Puntis et al, 1986; Walker et al,
1986; Rigo et al, 1987; Mitton et al, 1988; Puntis et al, 1989; Walker and Mills
1990) and excrete alternate metabolites of phenylalanine and tyrosine catabolism (Chessex et al, 1985; Walker and Mills 1990; Wykes et al, 1994a). In neonates, these metabolites are normally found in low to trace amounts (Bremer et al, 1981; Chalmers and Lawson 1988; Mitchell et al, 1995). Such findings imply overloading of neonatal catabolic pathways with high parenteral phenylalanine intake. Since blood was not collected, urinary concentrations were measured as an index of plasma concentration. We found that the level of 87 urinary phenylalanine and tyrosine were elevated in infants receiving Vamin or
Primene cornpared with the expected output of enterally fed premature neonates
(Przyrernbel et al, 1973). However, while infants receiving Primene generally experienced excretion that was close to the upper range of normal, neonates receiving Vamin excreted phenylalanine and tyrosine at levels that were two and three times normal levels for phenylalanine and tyrosine, respectively. Similarly, piglets receiving Vamin experienced greater incidence of abnormal plasma phenylalanine and tyrosine levels with correspondingly higher levels of alternate metabolites of phenylalanine metabolism compared with piglets receiving the paediatric arnino acid solution Vaminolact (Wykes et al, 1994a). These metabolic responses are of concern because of the knowledge that infants with high plasma phenylalanine due to phenylketonuria experience neurological impairment. Lucas et al (1993) compared the neurological outcome (as assessed by the Bayley mental development index, psychomotor development index and social maturity quotient) of 93 infants who experienced high plasma phenylalanine levels when receiving parenteral nutrition containing Varnin to
(age-matched) controls. Lucas et al (1993) found no effect of prior hyperphenylalaninemia on these outcome measures at 18 rnonths corrected age. However, it would be preferable to avoid hyperphenylalanemia as longer- term consequences are unknown and research on the issue is limited.
In contrast to general purpose arnino acid solutions, solutions designed for paediatric use have been developed with concern for the immaturities of the 88 neonate. Due to the importance placed on the normalisation of plasma amino acid levels, these solutions have not compensated for low tyrosine concentrations with additional phenylalanine. It has therefore been suggested that these solutions rnay be limiting in total aromatic amino acids (Castillo et al,
1994; Wykes et al, 1994a). Although clinical studies cornparing general purpose with paediatric amino acid solutions have found no differences in growth or nitrogen balance, this rnay be due to diffïculties in nitrogen balance collections, small sample size, short study periods and large variation in the patient population due to illness. Conversely, the piglet, which grows at four tirnes the rate of the human neonate, has revealed significant differences in nitrogen balance and growth between groups receiving Vamin versus Vaminolact (a paediatric solution with an amino acid pattern similar to human milk protein), and provide evidence that the paediatric solution is Iimiting in total aromatic amino acids (Wykes et al, 1994a).
Infants receiving high phenylalanine solutions are assumed to be able to hydroxylate sufficient phenylalanine to meet tyrosine requirernents and dispose of excess phenylalanine that may have been infused. The neonates' ability to cope with a phenylalanine excess is dependent on the activity of the enzymes involved in the rnetabolism of phenylalanine and tyrosine. In the present study, infants receiving the greater intake of phenylalanine from Vamin had a significantly higher rate of hydroxylation compared to infants receiving Primene.
Recent studies examining phenylalanine hydroxylation rates using the 89
Thornpson modification (Thompson et al, 1989) of the Clarke and Bier model
(Clarke and Bier 1982). have found considerable hydroxylation in response to parenteral amino acid intake compared to a basal period when infants were receiving glucose alone (Shortland et al. 1994; Kilani et al, 1995). This response was not observed within a group of term infants (Denne et al, 1996) and recently in a group of 32 week-old neonates (Clark et al, 1997) receiving an arnino acid solution modest in phenylalanine supply. Although these results provide an indication that phenylalanine hydroxylase enzyme is not immature and is stirnulated by its substrate in the neonate as well as in the adult, the degree of response in relation to phenylalanine intake is unknown.
The hydroxylation rate observed in the neonates receiving Prirnene (22 pmol-kg-'- h-') supports other studies where infants receiving paediatric arnino acid solutions demonstrated phenylalanine hydroxylation rates ranging from 11 to 22 prnol-kg-'. h" (Denne et al, 1996; Clark et al, 1997). Similarly, in studies where neonates received parenteral nutrition with elevated phenylalanine. hydroxylation was found to be greater, and in the range of 37 to 48 pmol-kg-'. h-'
(present study: 42 prnol-kg"- kl) (Castillo et al. 1994; Shortland et al, 1994).
These results support the rationale that the neonate does respond to increased phenylalanine intake by increasing the rate of phenylalanine hydroxylation to tyrosine. Nevertheless, the concern exists for whether the increased phenylalanine hydroxylation that occurs in these infants is sufkient to meet tyrosine needs and to dispose of possible excess phenylalanine intake. 90
Of interest was the finding that the percent of dose oxidized decreased with increasing energy intake in infants receiving Vamin but not Primene. This suggests that increased energy intake supports improved utilization of the large supply of phenylalanine in the Varnin solution. Lucas et al (1993) demonstrated that poor tolerance of a high phenylalanine intake in neonates receiving Vamin did not occur when the total energy to protein energy intake ratio was 8.51 kJ-kg-'-d-' but occurred occasionally with a ratio of 6.5:l kJ-kg-'-d-'. In the present study the total energy to protein energy ratio was 8.1 I 2.0 kJkg-'-d-' for infants receiving Vamin and 7.8 I 0.8 kJ-kg-'-d-'for those receiving Primene.
The present data suggests that at an energy intake of 350 kJ-kg-'-d-', phenylalanine utilization was lirnited by energy intake. For infants receiving
Primene, the low percent dose oxidized (6%, Table 4.5) and lack of significant change due to increasing energy intake indicates that phenylalanine oxidation is at its basal level.
The greater urinary excretion of an alternate catabolite of tyrosine, and to a lesser extent the excretion of alternate catabolites of phenylalanine in neonates receiving Vamin supports the conclusion that the level of phenylalanine in Vamin is in excess of requirement. One individual in the Vamin group could be considered an outlier due to the greater excretion of al1 the alternate metabolites, however, this may reflect greater sensitivity to phenylalanine load or immaturity within this individual. Such a response may be expected in neonates found in typical neonatal intensive care units. Although there is concern for the 91 presence of these catabolites, there is no direct evidence that these compounds result in harm. Phenylalanine catabolites were also found in the urine of piglets receiving Vamin or a phenylalanine-supplemented paediatric amino acid solution, whereas piglets receiving the unsupplemented paediatric solution presented with only negligible arnounts of the metabolites (Wykes et al, 1994a).
Large concentrations of tyrosine catabolites were also found in infants (less than
33 weeks gestational age) receiving Vamin-based TPN (Walker and Mills 1990).
The presence of these metabolites in conjunction with the greater excretion of urinary phenylalanine and tyrosine, suggests an overload to the catabolic pathway of phenylalanine toward oxidation even in the presence of significant hydroxylation and oxidation. The parenteral route of administration of amino acids may also have a role in causing increased excretion of these catabolites.
Studies comparing amino acid rnetabolism in subjects receiving nutrient intake parenterally versus enterally demonstrate significantly different responses in metabolism (Beaufrere et al, 1992; Wykes et al, 1992; Adeola et ai, 1995;
Bert010 et al, 1998a).
Although this study specifically addresses phenylalanine and tyrosine metabolism in infants receiving Vamin or Primene, these solutions also differ in the level of other amino acids. The lysine content in Vamin (5.5% total amino acids), for example, is almost half that of Primene (10.9% total amino acids).
Studies in the piglet model of the neonate suggests that lysine may be the first lirniting amino acid in Vamin as lysine intake has been estimated to represent 92
5.6% total amino acids (House et al, 1998a). Proline in contrast, is rnuch greater
in Vamin (1 1.6% total amino acids) compared to Primene (3.0% total amino
acids). The low proline content of the paediatric amino acid solution may be a concem as growing animals have been shown to require a prefomed source of proline (Austic, 1976; Ball et al, 1986; Heger, IWO). However, the content of proline and its rnetabolic precursors (ornithine and arginine) are in a more similar concentration between amino acid solutions (Vamin, 16.3 and Primene, 13.6) possibly minirnizing the significance of a low proline level. It would be prudent however to maintain a greater level of proline in amino acid solutions designed for the rapidly growing individual.
In spite of these differences in amino acid levels, it is the total aromatic amino acids that have been shown to be limiting for protein synthesis in the piglet model of the parenterally-fed neonate (Wykes et al, 1994a). Therefore the kinetic measurements within this paper are largely responsive to the level of phenylalanine versus differences in overall pattern of amino acids. Mitton and
Garlick (1992) similarly examined leucine kinetics as an index of overall body protein turnover in premature infants receiving either Vamin or Vaminolact as source of parenteral amino acid intake. These solutions differ similarly to the present study in pattern of amino acids. These investigators did not find any difference in leucine kinetics or overall protein metabolism in infants receiving either solutions. Comparable to this situation, is the study of Wykes et al (1994a) in the piglet model of the parenterally-fed neonate receiving Vamin, Varninolact, 93 or Vaminolact supplemented with phenylalanine to the level of Vamin. Piglets receiving Vamin had similar differences in phenylalanine kinetics as those found in the present paper.
4.5. CONCLUSIONS
The first hypothesis of this study was suported as it was found that stable, parenterally-fed neonates receiving a high phenylalanine intake experienced greater phenylalanine hydroxylation and oxidation rates compared with neonates receiving a moderate phenylalanine intake. Supporting the second hypothesis, was the finding that greater phenylalanine intake also resulted in greater excretion of phenylalanine and tyrosine and alternate catabolites of their metabolism. This is the first tirne, to Our knowledge, that aromatic amino acid kinetics using stable isotope tracers were compared in human neonates receiving two cornmercially available amino acid solutions. Although the neonates responded to increase phenylalanine intake with greater hydroxylation and oxidation rates, the greater excretion of phenylalanine and tyrosine and the alternate metabolites of their catabolism suggests that increased parenteral phenylalanine intake may not be the rnost efficient or effective rneans of meeting totaI aromatic arnino acid requirement in the neonate. There is therefore a need to determine the tyrosine requirements of the parenterally fed human neonate. 5. THE EFFECT OF GRADED INTAKE OF GLYCYL-L-TYROSINE ON
PHENYULANINE AND TYROSINE METABOLISM IN PARENTERALLY-FED
NEONATES: ESTIMATION OF TYROSINE REQUlREMENT
5-1. INTRODUCTION
Almost three decades ago it was demonstrated by Snyderman that the neonate
requires a preformed source of tyrosine (Snyderman 1971). In the first study of the thesis, although neonates were found to increase their rate of hydroxylation and oxidation to increased phenylalanine intake, neonates receiving high phenylalanine
intakes excreted greater levels of alternate metabolites of phenylalanine and tyrosine catabolism, suggesting that the use of phenylalanine to cornpensate for low tyrosine
levels may not be the most appropriate approach to meeting neonatal aromatic arnino acid requirernents. The work of Wykes et al (1994b) in the piglet model of the neonate supports these findings.
The main impedirnent to meeting total aromatic amino acid requirement in TPN
is the poor solubility of tyrosine (0.453 gC' in H,O at 25°C) (Merck Index 1983), limiting
it's inclusion in parenteral arnino acid solutions to 4%total amino acids. When compared to neonatal reference proteins such as human milk or cord blood, which contain approxirnately equal quantities of phenylalanine and tyrosine (Brerner et al,
1981; Rassin 1989); the levels provided by parenteral amino acid solutions is grossly
inadequate. To circumvent this problem, soluble derivatives of tyrosine have been
94 95 investigated. N-acetyltyrosine has been included in one of the commercially available paediatric amino acid solutions, TrophAmine, as a soluble precursor of tyrosine (Heird et al, 1987). Unfortunately, it has been shown to be poorly utilized in the parenterally- fed piglet which excreted 65% of N-acetyltyrosine intake (Wykes et al, 1994b) and in the human neonate which also excreted large quantities of the derivative (Heird et al,
1987; Helms et al, 1988; Adarnkin et al, 1991; Sulkers et al, 4991 ; Hanning 1993; van
Goudoever et al, 1995). Dipeptides of tyrosine, in contrast, are a well utilized source of tyrosine with much evidence supporting their ability to serve as a soluble source of tyrosine in pigs (Wykes et al, 1994b; House et al, 1997a; House et al, 1997b), primates
(Steinhardt et al, 1984) and humans (Albers et al, 1989; Steininger et al, 1989; Druml et al, 1991; Lochs et al. 1992; Morlion et al, 1998). To improve the utilization of the presently available paediatric amino acid solutions, a soluble source of tyrosine must be included in these solutions. Dipeptides at present seem to be the most likely candidate to meet this objective.
Using graded intakes of tyrosine in the fom of glycyl-L-tyrosine, House et al,
(1997a) estimated that the safe tyrosine intake (95% upper confidence lirnit) in the piglet receiving a fixed phenylalanine intake of 4.1 % of total amino acids, to be 3.2% of total amino acids. This is significantly higher than the level of tyrosine in presently available paediatric amino acid solutions (0.9% of total amino acids). The low level of tyrosine is responsible for the reduced growth and nitrogen retention in piglets receiving the paediatric amino acid solution Vaminolact, versus the general purpose solution
Varnin (Wykes et al, 1994a). 96
Due to the immaturities in the tyrosine catabolic enzyme pathway of the neonate, tolerance of tyrosine intakes at levels greatly over requirement is limited. ln addition, due to the known neurological impairement of hypertyrosinaemia on the developing brain (Menkes et al, 1972; Mamunes et al, 1976) as assessed by lower IQ and psychological tests (Peabody Picture Vocabulary Test, McCarthy Scale of Children's
Abilitieç, and the Illinois Test of Psycholinguistic Ability), excess intakes must be avoided.
Tyrosine requirement in neonates receiving parenteral nutrition has not been determined. It was hypothesized that at a fixed phenylalanine intake, the tyrosine requirernent of the TPN-fed neonate can be estimated by partitioning the oxidation of phenylalanine to graded intake of tyrosine.
5.2. MATERIALS AND METHODS
5.2.1. Patients and Nutrient lntake
Thirteen parenterally-fed neonates (Table 5.1) who had been referred to the
Neonatal Intensive Gare Unit at The Hospital for Sick Children were enrolled into the study. Al1 infants were clinically stable (afebrile, stable vital signs within normal range, absence of acute illness, and if surgical, 3 days post surgery) during the study period and were rnaintained in standard sewo-controlled incubators or infant cribs. Intestinal atresia, necrotizing enterocoiitis, gastroschisis and malrotation were the diagnoses of infants enrolled in the study (Table 5.1). The study protocol was reviewed and approved by the Human Subject Review Cornmittee of The Hospital for Sick Children, Table 5.1 Subject information of parenterally-fed neonates receiving varying intake of glycyl-L-tyrosine as a source of tyrosine Mean SD n Birth weight (kg) Gestational age (wk) Postnatal age (d) Postconceptional age (wk) Sex (M:F) Weig ht (kg) Length (cm) Head circumference (cm) Size for gestational age (A:S, A=appropriate, S=smalI) Ventilatory support (#) O Diagnoses Necrotizing Enterocolitis 2 Intestinal Atresia 5 Malrotation 2 Gastroschisis 4 Amino Acid lntake (g-kg-'-d") 2.4 0.4 Energy 1 ntake (kJ-kg-'-d4) 340 38 98 and written, informed consent was obtained frorn one or both parents. A copy of the consent form appears in Appendix 9.5.
Study subjects were enrolled into the study when receiving adequate amino acid and energy intake (Zlotkin et al, 1985). Infants were randomized to receive total parenteral nutrition containing Primene with one of 5 levels of glycyl-L-tyrosine supplement as source of tyrosine (Figure 5.1) for two days. The base amino acid profile of Primene is shown in Table 5.2. The five levels of glycyl-L-tyrosine were 0.1 8,
0.35,0.52, 0.68, and 1.O1 mg-ml-' of parenteral amino acid solution. Nutrient intake was as prescribed by the attending physician. Actual individual intakes of amino acid, lipid, carbohydrate, phenylalanine and tyrosine are described in Table 5.3.
Phenylalanine intake was fixed at 4.2% of total arnino acids. There were no enteral feeds consumed by the subjects. The same enrolment form as used in the previous study was used in the present study. A copy of the form can be found in Appendix 9.2.
5.2.2. Studv Protocol
With the exception of the varying level of tyrosine as described in the previous section, an identical study protocol as that described in Section 4.2.2. was used in this study (Figure 5.1). The amino acid tracers and isotope protocol were also identical to that described in Section 4.2.2. Table 5.2 Amino acid profile of Primene (g-IOOg-' amino acid)' Primene lsoleucine Leucine Valine Lysine Methionine
Threonine Tryptophan Histidine Arginine Glycine Alanine As pa rtate Glutamate Proline Serine Taurine Ornithine 2.2 'Primene 6.5% amino acid concentration (Baxter, Deerfield, IL). Table 5.3 Individual subject tyrosine, phenylalanine, amino acid, lipid and 100 carbo hydrate intakes Subject Tyrosine1 Phenylalanine Amino Lipid3 CHO4 Energy Acid2 mg kg-' .d" g. kg-' .d-1 kJ kg-' .d-'
'Total tyrosine intake (includes the tyrosine component of glycyl-L-tyrosine and that from Primene) 2Arnino acid energy equivalent = 16.7 kJ& 31ntralipid (Phamacia and Upjohn, Stockholm, Sweden); energy equivalent = 41.8 kJg" 4CH0 = carbohydrate as dextrose; energy equivalent = 15.7 kJ-g" 'Mean and SD values for tyrosine intake was not provided because the tyrosine intake varied according to level of glycyl-L-tyrosine to which the neonate was assigned.
5.2.3. AnalSical Procedures and Calculations
5.2.3.1. Urinary Amino Acid Enrichment
Analysis of urinary amino acid 13C enrichment was perforrned by the method of
Patterson et al (1991) using GCMS as described in detail in Section 4.2.3.1.
5.2.3.2. Urinary Phenylalanine and Tyrosine Concentrations
Urinary phenylalanine and tyrosine concentrations were analysed by ion- exchange chromatography, expressed in pmol per rnmol of creatinine as described in
Section 4.2.3.2. The urinary excretion of the supplernental dipeptide, glycyl-L-tyrosine was also monitored within the arnino acid run. Using standards of known amounts of glycyl-L-tyrosine, the retention time of the peptide was determined to be 67.5 min. The concentration of the peptide standardized to creatinine excretion, was determined using an external standard and was calculated with the following equation:
((Area of sample peaWarea of peak of external standard of GT) x
(dilution factor) x 5 pgmyL-I ) 1 Creatinine mmol-L")
Where the external standard was analyzed in a separate run using a dilution factor of
80. The concentration of the external standard was 5 pg-pl-'. An interna1 standard, S,- amino ethyi cysteine was used for quality control of the run. Values different by 5% of the expected 400 pmol-L-' resulted in a repeat of the analysis. 1O3
5.2.3-3. Expired 13C02Enrichment
Expired l3CO, enrichment was measured by IRMS as described by Jones et al
(1985). CO, collection and enrichment was described in detail in section 4.2.3.3. The
CV of the I3CO, enrichment at plateau was ~5%.
5-2.3.4. Model of Amino Acid Metabolisrn and Calculations
The model of amino acid metabolisrn used in the study was a modification of that of Waterlow et al (1978) and has been described in detail in Section 4.2.3.4. along with calculations.
5.2.4. Statistical Analvses
The study followed a completely randomized design with tyrosine intake as the independent variable. Regression models (linear, quadratic, linear regression crossover model) were fitted to the data to determine the relationship that best describe the response of the dependent variables phenylalanine hydroxylation, phenylalanine
Fl3C0, and oxidation and tyrosine flux to increasing tyrosine intake. The regression analyses were performed by the method of least squares using the proc reg procedure of SAS (SAS lnstitute 1991). Selection of the best mode1 was determined by factors relating to fit (significance of the mode1 and ?) and estimates of variation about the model (coefficient of variation and percent standard error of the estimate).
Although there is no established estimate of sample size when performing regression analysis, insufficient data will likely result in a poor model as indicated by a number of rnodel-related parameters. The parameters for each of the rnodels are compared in table format within Appendix 9.6.1.
Where a significant two-phase regression crossover model (Seber 1977) was found to be the most appropriate analysis for the data (i.e.. phenylalanine F13C0, and oxidation), a breakpoint was objectively detemined. As described previously (Kim et al,
1983b; Zello et al, 1990a), the analysis involves partitioning the response of the independent variables to tyrosine intake into data points fitting either a Rat line (zero slope) representing obligatory phenylalanine oxidation when tyrosine intake is inadequate, and a sloped line representing phenylalanine oxidation when tyrosine intake was above requirement. The breakpoint of the two linear regression lines is considered representative of the group mean tyrosine requirement. The equation for the statistical model is:
Y=A1 + Blx+(A2 -Al)D+(B2-Bl)(Dx)+E where Y is the individual observation of the dependent variable phenylalanine F13C0, , or oxidation; Al and A2 are the intercepts of the first and second line, respectively; B1 and 82 are the slopes of the first and second lines, respectively; D equals O if the observation is from the first line and 1 if ii is frorn the second Iine; x is tyrosine intake and E is the residual error associated with the model. The safe population intake was estimated by detemining the upper 95% confidence limits using the Fieller theorem
(Seber 1977). The model and statistical analysis program are described in detail in
Appendix 9.7. 5.3. RESULTS
5.3.1. Clinical Characteristics and Nutrient lntake
Body weights reflect the predominance of prernature infants enrolled in the study
(Table 5.1). Parenteral nutrition was as prescribed by the attending physician and therefore the exact nutrient intakes were dependent on the total volume of parenteral nutrition infused. The average energy and protein intakes were 81 * 9 kcal-kg-'ad-' and
2.4 i 0.4 g-kg"-d*', respectively (Table 5.3). Mean phenylalanine intake was 97 i: 13 mg-kg-'-d-'. The tyrosine intake reflected the level of glycyl-L-tyrosine to which the neonate was assigned and ranged from 29 to 108 mg-kg-'-d-'(Table 5.3).
5.3.2. Urinarv Amino Acid and Expired CO, Enrichment
As in the previous study. plateau amino acid enrichment was achieved for al1 neonates by 9 h, as defined by the absence of a significant slope. The variation in phenylalanine and tyrosine arnino acid enrichrnent within the plateau was ~7%.
Individual amino acid enrichment of L-[l -13C]phenylalanine, L-[l-13C]tyrosine,L-[3,3-
2HJtyrosine and expired 13C0, are shown in Table 5.4.
5.3.3. Phenvlalanine and Tvrosine Kinetics
The rates of individual phenylalanine and tyrosine fluxes, phenylalanine hydroxylation, F13C0, and oxidation at graded tyrosine intake are shown in Table 5.5.
There was no significant effect of tyrosine intake on phenylalanine flux (Table 5.5).
There was however a significant quadratic response of phenylalanine hydroxylation to Table 5.4 Individual subject urinary L-[1-13C]phenylalanine, L-[1 -13C]tyrosine, L- i 06 [3,3-*HJtyrosine and expired I3CO2enrichment. ------.- Subject TYR1 13CPHE2 %TYR2 2H,TYR2 13C02 Intake mg-kg-'-d-' MPE APE 0.0828 0.0955 0.0837 0.1 142 0.0726 O.? 137 0.0949 0.1 785 0.0820 0.0833 0.0886 O. 1 O62 0.1 057 'TYR = tyrosine; Total tyrosine intake (includes the tyrosine component of glycyl-L- tyrosine and that from Primene) 213CPHE= L-[l -13C]phenylalaninel13CTYR = L-[l -13C]tyrosine, 'HJYR = L-[3,3- 2HJtyrosine 3Since consent was not given for breath collection in this subject, these data were not availab le. Table 5.5 Individual subject phenylalanine and tyrosine kinetics 4 A7 Subject TYR1 PHE PHEOH2 PHE PHE TYR Flux lntake Flux Fl3C0, Oxidation mg kg-' -d-' pmol. kg-lad-'
SD -4 21 -5 -5 -5 30 'TYR = tyrosine; Total tyrosine intake (includes the tyrosine cornponent of glycyl-L- tyrosine and that from Primene) *PHEOH = phenylalanine hydroxylation 3Sinceconsent was not given for breath collection in this subject, these data were not available. 4Mean and SD values for tyrosine intake were not provided because the tyrosine intake varied according to level of glycyl-L-tyrosine to which the neonate was assigned. 'Mean and SD values were not provided because the variables were related to the level of tyrosine intake as indicated in the results section. Tyrosine lntake (mg-kg-1-d-1)
Figure 5.2 The effect of increasing tyrosine intake via glycyl-L-tyrosine on phenylalanine hydroxylation in neonates receiving TPN. Tyrosine intake is in ternis of actual total tyrosine intake (tyrosine from TPN + tyrosine from glycyl-L-tyrosine). 1O9
increasing tyrosine intake (pc0.004 Table 5.5, Figure 5.2). The three regression models applied to the tyrosine flux data were significant (Appendix 9.6.1) . However. the best fit of the data was the linear relationship since it proved to have the best compromise between variation (coefficient of variation and percent error of the estimate) and correlation (pe0.006, Figure 5.3). Individual phenylalanine F13C0, and oxidation rates are shown in Table 5.5. The two-phase linear regression crossover model proved to be the best fit for the F13C0, data and the only significant mode1 for the phenylalanine oxidation data. Sumrnary statistics for al1 of the rnodels can be found in
Table 9.1-Table 9.4 in Appendix 9.6.1. A breakpoint in phenylalanine F13C0, at 66 mg-kg-l-d-'(Figure 5.4) was identified, and in phenylalanine oxidation at 82 mg-kg-'-d"
(Figure 5.5). lncreasing tyrosine intake above this level resulted in increased F13C0, and oxidation. These breakpoints, objectively identified by the two-phase linear regression crossover model, are representative of the mean tyrosine requirement for the neonate. The 95% upper confidence limits were found to be 90 and 97 mg-kg-'-d-' for the F13C0, and oxidation estimate, respectively (Figures 5.4 and 5.5). Analysis of the percent L-[l-13C]phenylalaninedose oxidized and energy intake did not demonstrate a significant linear (pc0.1) or quadratic (~~0.2)relationship.
5.3.4. Urinary Amino Acid Concentrations
Urinary phenylalanine, tyrosine and glycine excretion were generally within the normal range for neonates (phenylalanine = 0-1 51 pmol-mm01creatinine-', tyrosine = 0-
200 pmolmmol creatinine-l, glycine = 254-2341 pmol-mmol creatinine-') (Bremer et al, O 1 1 1 1 I O 25 50 75 1 O0 125 Tyrosine lntake (mg*kg-1-d-1)
Figure 5.3 The effect of increasing tyrosine intake via glycyl-L-tyrosine on tyrosine flux in neonates receiving TPN. Tyrosine intake is in terms of actual total tyrosine intake (tyrosine from TPN + tyrosine from glycyl-L-tyrosine). Tyrosine lntake (mg-kg-1-d-1)
Figure 5.4 The effect of increasing tyrosine intake via glycyl-L-tyrosine on F13C02 production f rom phenylalanine oxidation in neonates receiving TPN. The broken lines represent the 95% confidence limits of the breakpoint estimate. Tyrosine intake is in ternis of actual total tyrosine intake (tyrosine from TPN + tyrosine f rom glycyl-L-tyrosine). Tyrosine lntake (mg kgq-d-1)
Figure 5.5 The effect of increasing tyrosine intake via glycyl-L-tyrosine on phenylalanine oxidation in neonates receiving TPN. The broken lines represent the 95% confidence limits of the breakpoint estimate. Tyrosine intake is in ternis of actual total tyrosine intake (tyrosine from TPN + tyrosine f rom glycyl-L-tyrosine). Table 5.6 Individual subject urinary creatinine, phenylalanine, tyrosine, glycyl-L- 1 13 tyrosine and glycine concentrations - - - -- Subject Tyrosine Creatinine Phenylalanine Tyrosine Gv Glycine Intakel
'Total tyrosine intake (includes the tyrosine component of glycyl-L-tyrosine and that from Primene) 2GT = glycyl-L-tyrosine 3ND = Not detected 4Mean and SD values for tyrosine intake were not provided because the tyrosine intake varied according to level of glycyl-L-tyrosine to which the neonate was assigned. 'Mean and SD values for glycyl-L-tyrosine excretion were not provided due to the predominance of non-detectable quantities. Il4
1981). However, subject AL receiving a tyrosine intake of 52.5 mg-kg-'-d-',excreted an above average amount of phenylalanine and tyrosine in the urine suggesting that the plasma concentration of these amino acids may also have been elevated (Table 5.6).
This neonate did not demonstrate any evidence of abnormal liver function based on results of serum alanine aminotransferase or serum aspartate aminotransferase measured around the time of the study. The individual receiving the highest tyrosine intake (108.1 mg-kg-'-d-')demonstrated the greatest excretion of tyrosine in the urine, although rernaining within the expected range of the neonate (Table 5.6). The additional glycine intake from the dipeptide increased the glycine intake from 4.2% to
5.3% total amino acids. Urinary glycine excretion did not differ according to level of glycyl-L-tyrosine intake (Table 5.6). Four (SI,CC,JR,CM) of the thirteen subjects demonstrated a srnall amount of dipeptide in the urine in each case representing 40 ymol-mm01creatinine-' (Table 5.6).
5.4. DISCUSSION
Using nitrogen balance and growth measures, Snyderman (1971) identified the that enterally-fed neonate requires a preformed source of tyrosine at levels ranging from 50-120 mgmkg-l-d-l. The parenteral requirement of the human neonate has not been determined. The estimate of the tyrosine requirement for the enterally-fed neonate does not necessarily apply to neonates fed parenterally as recent evidence has demonstrated that the splanchnic organs play a major role in amino acid rnetabolism. and its bypass can significantly alter the needs of individual amino acids lis (Elwyn 1970; Krempf et al, 1990; Reeds et al, 1996; Stoll et al, 1997; Dudley et al,
1998; Stoll et al, 1998).
The present data are, to the authors knowledge, the first estimate of tyrosine
requirement in the parenterally-fed human neonate. Using breakpoint analysis, the estimated average tyrosine requirement at a fixed adequate, but not excessive
phenylalanine intake of 4.0% of total arnino acids was 66 mg-kg-'-d'' and 82 mg-kg-'ad-' for the estimates based on phenylalanine oxidation measures of phenylalanine F13C0,, and oxidation, respectively. These intakes represent 2.8% and 3.4% of total amino acid
intake, respectively. The upper 95% confidence limit, representative of the safe population intake, was estimated as 90 and 97 mg-kg-'-d-',for phenylalanine F13C0, and oxidation data, respectively. These intakes represent between 3.8 and 4.0% of total amino acids, respectively.
The present resultç are supported by similar estimates of tyrosine in the parenterally-fed piglet. The estimated rnean requirement and safe level of intake
represented 2.1 and 2.4% total amino acids at a fixed phenylalanine intake of 4.0% of total amino acids. The present results are also within the estimated range of tyrosine
requirement in the orally-fed infant, determined by Snyderman (1 971) in the presence of adequate phenylalanine intake which was from 50-120 mg -kg" -d". Because tyrosine is considered indispensable in the neonate, the provision of an ideal balance of aromatic amino acids is important to a nutritional regirnen that supports maximum efficiency of utilization of the aromatic amino acids and in turn promotes maximal growth without gross imbalances in amino acid levels. Based on the data of the Il6 present study, the proportional balance of the rnean aromatic amino acid requirernents results in a phenylalanine to tyrosine ratio of 5644. This ratio is similar to that found in the piglet mode1 of the neonate (59:41) (House et al, 1997a; House et al, 1997b) and to the proportional phenylalanine to tyrosine content of fetal tissue (59:41) (Widdowson
1981). Wth respect to the safe level of intake, the proportional phenylalanine to tyrosine balance are similar between the human and porcine species with a ratio of
51:49 in the neonate and 54:46 in the piglet (House et al, 1997a; House et al, 1997b).
The similarity in aromatic amino acid requirement and balance suggests that an approximately equal amount of phenylalanine to tyrosine is required for maximum eficiency of utilization in the neonate. Provision of arornatic amino acids in this recomrnended balance should lower the incidence of hyperphenylalaninaemia and hypertyrosinaemia obsewed in neonates receiving high intakes of phenylalanine
(Chessex et al, 1985; Puntis et al, 1986; Walker et al, 1986; Rigo et al, 1987; Mclntosh and Mitchell 1990; Walker and Mills 1990; Wykes et al. 1994a) or tyrosine (Wykes et al,
1994b). Since the long-terni neurological effects of low or elevated tyrosine intake is unknown, efforts should be made to provide intakes that meet needs without great excess. These estimates of tyrosine requirement represent an important step toward a larger research programme that is gathering a database on neonatal amino acid requirements which can be used to develop a parenteral amino acid solution that best supports neonatal needs without excess.
The present estimate of tyrosine requirement was determined at a phenylalanine intake of 97 i 13 mg-kg-'d", representing a phenylalanine intake of 4% of total amino 717 acids, the levels of phenylalanine in Primene. This intake was detemined by House et al (1997a), in the piglet model, to be above the safe level of phenylalanine requirernent which was estimated at 3.2% of total amino acids. The impact of a small excess of phenylalanine on the tyrosine requirement is unknown, but rnay be to decrease the tyrosine requirement because phenylalanine can be hydroxylated, through hepatic phenylalanine hydroxylase, to fom tyrosine.
There was a different effect of energy intake on the percent L-[1-
'3C]phenylalanine dose oxidized in infants receiving Vamin versus infants receiving
Prirnene in the neonatal study of Chapter 4. In the present study there was no significant relationship between energy intake and the percent L-[l-'3C]phenylalanine dose oxidized. This is consistent with the results found in neonates receiving Primene as source of amino acids.
Although a significant quadratic response of phenylalanine hydroxylation to increasing tyrosine intake was observed, the estirnate of phenylalanine hydroxylation did not provide meaningful results towards estimating tyrosine requirement. The response of phenylalanine hydroxylation to graded tyrosine intake was unexpected.
The level of phenylalanine hydroxylation was similar at both the lowest and highest intake of tyrosine. The predicted response was that hydroxylation would be higher at the lowest intake of tyrosine and decrease as tyrosine intake approached requirement.
These data reveal that the estimate of phenylalanine hydroxylation using the Clark and
Bier (1982) model as modified by Thornpson et al, (1989) cannot be used as an index of the adequacy of tyrosine intake. Phenylalanine hydroxylation has been previously shown not to reflect net tissue phenylalanine and tyrosine accumulation in the parenterally-fed piglet (House et al, 1998). Such discrepancies are attributed to limitations of the model, possibly due to the fact that plasma (or urine as a surrogate) enrichment does not accurately reflect the precursor pool enrichment. As new approaches to quantify precursor pool enrichment surface, in particular the use of enrichrnent of rapid-turnover hepatic proteins such as albumin (Ballmer et al, 1990) and apolipoprotein BI00 (Reeds et al, 1992), the hydroxylation estimate will need to be reassed as a measure of the adequacy of tyrosine intake.
Urinary excretion of phenylalanine, tyrosine, glycine and glycyl-L-tyrosine were rnonitored to provide some insight into the rnetabolic handling of the doses of aromatic amino acids and glycyl-L-tyrosine. In general, the excretion of the three arnino acids were within normal ranges and the excretion of glycyl-L-tyrosine was either non- detectable, or appeared in trace amounts. These data suggest that the intakes of these amino acids were not at ievels that cause overload to the neonates' rnetabolic pathways. The lack of significant excretion of the dipeptide, even at the highest intake of glycyl-L-tyrosine supplementation, indicates the absence of glycyl-L-tyrosine accumulation since it was presurnably rapidly hydrolysed into it's respective arnino acids and available for protein synthesis or for other metabolic purposes
(catecholamine biosynthesis, thyroid hormones, melanins).
One subject (AL), who received a tyrosine intake below requirement level (GT =
52.5 mg-kg-'-d-'), experienced urinary phenylalanine and tyrosine that were above expected noms for the neonate. Glycine excretion in contrast, was within the normal 119 limits and glycyl-L-tyrosine peptide was not detected in the urine. This suggests that the plasma concentration of phenylalanine and tyrosine were elevated (Brerner et al,
1981). The fact that this greater than normal excretion of phenylalanine and tyrosine occurred with a modest phenylalanine intake and a tyrosine intake that was below requirement, in conjunction with the lack of evidence of impaired liver function frorn liver enzyme tests. suggests that this neonate may have demonstrated an imrnaturity in the tyrosine catabolic pathway leading to an accumulation of products of metabolism prior to the blockage. Tyrosine aminotransferase (Delvalle and Greengard 1977, Ohisalo et al, 1982) and 4-hydroxyphenylpyruvate dioxygenase (Kretchmer et al, 1956,Ohisalo et al, 1982) are both considered to be immature during the neonatal period.
Two su bjects (CK. g lycyl-L-tyrosine level = 28.8 mg - kg-' -d-' ; SS, g lycyl-1-tyrosine level 60.3 mg-kg-'-cl-')that were receiving tyrosine intakes below requirement, had no detectable amounts of phenylalanine in the urine. Although the lack of detectable phenylalanine in neonatal urine is rare, it is nevertheless considered within the normal range (Bremer et al, 1981). Perhaps enhanced utilization of the body's phenylalanine with addition of the first Iimiting arnino acid, tyrosine, is responsible for reduced plasma phenylalanine, causing less of the amino acid to be filtered and lost during tubular reabsorption within the kidney.
The subject receiving the highest intake of tyrosine demonstrated urinary phenylalanine output within the normal range (Bremer et al, 1981), but excreted the highest quantity of tyrosine. The greater urinary tyrosine excretion may be suggestive of increased plasma tyrosine level. Although impossible to confirrn due to the lack of 120 plasma upon which to perform amino acid analysis, the elevated urinary tyrosine observed rnay be due to a primary deficiency in one of the enzymes of the tyrosine catabolic pathway as a result of immaturity, or secondy to an overload of the catabolic pathway, or a combination of both factors. This rationale is supported by the work of d Wykes et al (1994b) in the piglet mode1 where a dose of glycyl-1-tyrosine in the amount of 4.9% of total aromatic amino acids increased plasma tyrosine concentrations and the excretion of alternate metabolites of tyrosine catabolism in the urine. Although the intake of this neonate represents tyrosine a concentration of only 4.5%, it may nevertheless represent a level above the neonate's capacity to tolerate tyrosine.
Most of the subjects had non-detectable excretion of the dipeptide glycyl-L- tyrosine in the urine. Four individuals excreted trace amounts of glycyl-L-tyrosine and of the subjects with dipeptide excretion, the presence of the dipeptide was not related to the intake. In fact, the two individuals with the greatest intake of the dipeptide did not present with detectable amounts of glycyl-L-tyrosine in the urine. This provides evidence that the dipeptide glycyl-L-tyrosine is well utilized in the hurnan neonate.
Additional glycine, associated with the dipeptide was infused. This amounted to amounting to 4 to 29 mg-kg-'-d-' and reflected an increase of glycine intake from 4.2 to
5.3% of total amino acids. Although the glycine intake was increased, the excretion of glycine did not correlate with increasing glycyl-L-tyrosine infused, suggesting that the additional glycine load did not greatly impact the plasma glycine pool. 5.5. CONCLUSIONS
An estimate of tyrosine requirernent in the parenteraliy-fed neonate was estimated by partitiming the oxidation of phenylalanine to a two-phase linear regression analysis. This is the first estimate of tyrosine requirernent in the parenterally-fed human neonate. These data specifically demonstrate that the mean tyrosine requirernent of the parenterally-fed neonate lies between 66 and 82 mg-kg-'-& l, representing 2.8% to 3.4% of total amino acids. The corresponding safe estimate of intake was 90 to 97 mg-kg4-d-'corresponding to 3.8 to 4.0% of total arnino acids. The ideal balance of phenylalanine to tyrosine was found to be 56:44, based on the mean requirement estimate. This balance of aromatic amino acid intake at the mean requirement level is similar to that found in the piglet model of the neonate (59:41) as well as that of fetal tissue composition (59:41).
The lack of significant dipeptide excretion in the urine suggests that the dipeptide was a well utilized source of tyrosine in the human neonate. The total aromatic amino acid intake that met neonatal needs approximated 7.1% of total arnino acids. This total aromatic amino acid intake is below the intake of the phenylalanine-supplemented solutions such as Vamin, which contain 8.6% of the total amino acids as phenylalanine and tyrosine. In contrast, this level is well above that found in sorne paediatric amino acid solutions (5.1% of total arnino acids). These data provide valuable information toward the development of a parenteral amino acid solution that better meets the needs of the neonate. 6. TYROSINE REQUIREMENT OF ADULT MALES AT A FlXED
PHENYLALANINE INTAKE USlNG INDICATOR AMINO ACID OXlDATlON
6.1. INTRODUCTION
In the second study of this thesis (Chapter 5),the tyrosine requirement of the human neonate was estimated using stable isotope tracer methods. The mean estirnate of requirement was found to represent 2.7 to 3.4% of total amino acids in neonates receiving a modest phenylalanine intake (4.0% of total amino acids). The distribution of phenylalanine to tyrosine was 56:44, similar to that found in the piglet mode1 of the neonate (59:41) (House et al, 1997a; House et al, 1997b), and that of human fetal tissue protein (59:41) (Widdowson 1981).
Cornparison between the mature adult metabolic pathways and the immature neonatal metabolic pathways is ofien used to give insight into developmental status. This approach has been applied in the past to assess the maturity of phenylalanine hydroxylase in the neonate (Raiha 1973; Delvalle and
Greengard 1977). More recently in vivo studies using stable isotope tracers of phenylalanine and tyrosine metabolisrn have put forth evidence of significant hydroxylation in the neonate with rates equal to or higher than that found in the adult (Castillo et al, 1994a; Shortland et al, 1994; Kilani et al, 1995; van
Toledo-Eppinga et al, 1996; Clark et al, 1997).
An ongoing controversy exists surrounding currently accepted arornatic amino acid requirements (Millward and Rivers 1986; Millward et al, 1989; Young 123 et al, 1989; Young and Marchini 1990; Zello et al, 199Oa; Zello et al, 1993; Zello et al, 1995; Fuller and Garlick 1994). The international reference requirement estimate of the FAONVHOlUNU expert consultation (1985) was based on nitrogen balance studies (Leverton 1959; Reeds et al, 1992) which are known to have inherent rnethodological difficulties (Fuller and Garlick 1994). The limitations associated with the nitrogen balance method have necessitated the search for more sensitive methods to determine arnino acid requirernent (Zello et al, 1995).
Studies applying isotopically labelled amino acids have enabled the estimate of amino acid requirements in adult hurnans based on direct (Meguid et al, l986a; Meguid et al. 1986b; Meredith et al, l986a; Zhao et al, 1986; Zello et al, 1990a) or indirect (Zello et al, 1993; Duncan et al, 1996; Lazaris-Brunner et al, 1998) changes in the metabolism of specific amino acids in response to intake. Requirement estimates derived by these rnethods are considered more robust than those based on traditional nitrogen balance methods and are up to 3 times greater than that of the FAONVHOIUNU (1985) recommendation (Meguid et al, l986a; Meguid et al, 1986b; Meredith et al, 1986a; Zhao et al, 1986; Zello et al, 1990a; Duncan et al, t 996; Lazaris-Brunner et al, 1998).
Recently, there has been a resurgence of interest in the area of aromatic amino acid requirernents (Zello et al, 199Oa; Sanchez et al, 1995; Sanchez et al,
1996; Basile-Filho et al, 1997; House et al, 1997a; House et al, 1997b;
Basile-Filho et al, 1998). These studies have focused largely on the body's need for phenylalanine when given a diet completely lacking or with very low tyrosine 124 content (Sanchez et al, 1995; Sanchez et al, 1996; Basile-Filho et al, 1997;
Basile-Filho et al, 1998). The study of Zello et al (1990a) is the only study carried out in adult humans which examines the body's minimum phenylalanine requirement, under circumstances of excess tyrosine, using stable isotope tracer methods. When an excess of tyrosine is given, excess phenylalanine is believed to be preferentially oxidized without first equilibraüng with the whole-body tyrosine pool (Moldawer et al, 1983). Such a design isolates the component of minimum phenylalanine requirement from that of total aromatic amino acids.
The estimated mean and safe level of phenylalanine intake derived from this work was 9.1 mg-kg-'-d" and 14 mg-kg-'-d-', respectively. None of the above experiments were able to establish the ideal balance of phenylalanine and tyrosine as components of total aromatic amino acid requirements. This balance will help to define the intake of aromatic amino acids that would promote maximum efficiency of protein utilization. Such information would be beneficial for the production of commercial nutrition solutions whose goal is to meet nutritional needs in the most efficient and well tolerated manner or to optimize the quality of arnino acid intake when dietary intake is poor.
The goal of this study was to estimate the adult tyrosine requirement at a fixed and adequate but not excessive phenylalanine intake, using indicator amino acid oxidation methodology. This is the first time that tyrosine requirement has been examined in the adult receiving an adequate phenylalanine intake. The investigation should therefore also contribute further information toward resolving the ongoing controversy that exists over indispensable amino acid requirements.
6.2. MATERIALS AND METHODS
6.2.1. Su bjects
Six healthy adult male volunteers were enrolled into the study. Subjects
participated in the study on an outpatient basis with al1 studies carried out in the
Clinical Investigation Unit at The Hospital for Sick Children, Toronto. Subjects were excluded from the study if they had a previous history of weight loss or
chronic disease, or if they had endocrine disorders or were receiving drug
therapy. The purpose of the study and the potential risks associated with the
protocol were explained to the subjects. Participants were encouraged tu
maintain their usual physical activity pattern throughout the study period. A written, infonned consent was obtained from al1 subjects (Appendix 9.7). The
study protocol was approved by both the University of Toronto Human
Experimentation Cornmittee and the Human Review Cornmittee of The Hospital for Sick C hildren. Remuneration was provided to al1 volunteers. Subject
characteristics are summarized in Table 6.1
6.2.2. Experirnental Desian and Dietary lntake
Each subject was allocated to each of seven different tyrosine intakes in
random order. Studies were separated by at least one week with al1 studies
carried out within 3 months. For two days prior to each study day subjects 126 Table 6.1 Subject information of healthy adult male volunteers' Subject Age Weight Height BMR Energy lntake (Y) (kg) (m) (M J -d-') (MJ-d" )
Mean ISD2 2919 79112 1.73i0.1 7.7I0.7 13.1*1.2 'Calculated using WHOIFAORINU (1985) predictive equations with an activity factor of 1.7. 2SD = standard deviation 127
consumed a controlled formula intake that was adequate in energy and protein intake. The level of energy intake was estimated using predictive equations for basal rnetabolic rate (FAOMMOIUNU 1985) multiplied by a coefficient of 1.7 to account for usual activity level. Protein was provided at an intake of 'tg-kg".d".
The formula was composed of a low-protein milkshake (Scandishake,
Scandipharm, Inc., Birmingham, AL) prepared with skim milk with added carbohydrate (Caloreen, Baxter Corporation, Mississauga, ON) and protein
(Promod, Ross Laboratories, Columbus, OH) to tailor the formula to the exact energy and protein level required. The diet was given as four meals spread out throughout the day. With the exception of water, no additional food or beverages were consumed.
The experimental protocol of the study day is shown in Figure 6.1.
Starting at 6 am of the study day, subjects consumed their dietary intake in the form of the experimental formula diet and protein-free cookies (Zello et al,
199Ob). lntake was given in hourly rneals representing 1/12th of the daily requirement. The macronutrient distribution of the diet was 37% carbohydrate,
53% fat and 10% protein. The recipe for the formula diet is provided in Appendix
9.9. The main source of energy came from a non-protein Iiquid formula (Product
80056, Protein-Free Pl Mead Johnson, Evansville, IN). The nitrogen cornponent was provided as crystalline L-amino acids with an amino acid profile largely reflecting egg protein (Table 6.2). The only amino acids that diverged from this profile were phenylalanine, lysine, tyrosine and alanine. Phenylalanine intake was fixed at 9.0 mg-kg-'-d-'. The level of phenylalanine was chosen to represent
129 Table 6.2 Composition of crystalline L-amino acid mixture Amino Acidl Amino Acid Content (gokg-') Egg Protein Study Mix L-Alanine 61.O0 -3
L-Asparagine 33.00 33.00 L-As partic Acid 33.00 33.00
L-Glutarnic Acid 56.20 56.20
L-Valine 69.72 69.72 'All amino acids purchased through United States Biochemical Corporation. Cleveland, OH 2Actual concentration of amino acid in HCI forrn: L-Arg=61.66 g-kg", L-His=22.53 g-kg" and L-Lys = 60.12 gokg-' 3Amountvaries to balance nitrogen intake according to tyrosine level 41ntake dependent on study level (3.0.4.5,6.0,7.5,9.0,10.5,or 12.0 mg-kg-l-d-l) 130
the estimated mean requirement based on direct amino acid oxidation methods
(Zello et al, 1990a). The intake of the indicator amino acid for of the study, lysine, was fixed at 45 mg-kg-l-d-',the estimated safe level for adult males (Zello et al, 1993; Duncan et al, 1995). This lysine intake was chosen to ensure that al1 subjects received their lysine requirernent and because it has been shown by
Kim et al (1983a) that the oxidation of an indicator amino acid is rnost sensitive to changes in dietary amino acid intake when given near its requirement level.
Subjects received each of 7 levels of test tyrosine intake (3.0, 4.5, 6.0, 7.5, 9.0,
10.5, 12.0 mg-kg-'-d-') in random order. The order in which tyrosine intakes were assigned to each individual is indicated in Table 6.3. Alanine intake was modified to maintain a constant nitrogen intake. The intake of lysine was corrected for the L-[1-'3C]lysine given as part of the isotope infusion protocol.
Since this study forms part of a larger study that includes the examination of phenylalanine hydroxylation at graded intakes of tyrosine, and uses L-
['5N]phenylalanine and L-[3,3-*HJtyrosine, the intake of these isotopically labelled cornpounds were also subtracted from the dietary intake. Calculations of dietary arnino acid and isotope intake are shown in Appendix 9.9.
6.2.3.1. Tracer protocol
The isotopically labelled compounds used in the study were NaH13C0,
(99%, 13C, lsotec Inc., Miamisburg, OH) and L-[l-13C]lysine(99%, I3C, lsotec Inc.,
Miamisburg, OH). Quality control tests were perforrned by the manufacturers.
Chernical purity, specified isotopic en richment and position was confirmed by Table 6.3 Order of assignment of tyrosine intakel Tyrosine lntake (mg -kg" ad") Subject 3 4-5 6.0 7.5 9 10.5 12
'There was no significant effect of order on any of the outcome rneasures analysed. 132
nuclear rnagnetic resonance (NMR), while isorneric purity was performed by
GCMS. Solutions of each tracer were prepared in deionized water and stored at
-20°C until use. Before dispensing, isotope solutions were sterilized by passing through a 0.22 Hm filter (Millipore Corporation, Bedford, MA).
The oral tracer infusion protocol was commenced at 10 am on the study day. A bolus dose of 0.1 72 mg-kg-' NaH13C0, and 2.4 mg-kg" of L-[1 -'3C]lysine was given to prime the bicarbonate and lysine pools. In addition to the prime, a dose of L-[l-'3C]lysine (1-2 mg-kg-'.d-') was given and continued on an hourly basis throughout the remaining 6 h of the study. Isotopes were given with the hourly rneals.
6.2.3.2. Sample Collection
Three baseline blood and expired breath 13C0, samples were collected
10, 20, and 30 min before the isotope protocol was initiated. lsotopic steady state (plateau) samples were collected every 30 min during the period 210 to 330 min following commencement of the isotope protocol.
Blood specimens were sampled from a 21-gauge needle inserted into a superficial dorsal vein on the hand. The blood in the hand region was arterialized by placing the subjects hand in a warming device (Zello et al, 1990c) maintained at 60°C for 15 min prior to sarnple collection. Blood specimens (2 rnL) were drawn into heparinized syringes (Aspirator, Marquest Medical
Products, Inc., Englewood, CO) and immediately placed on ice until centrifugation. Plasma was separated from the sample with centrifugation at 133 1500 x g for 20 min at 4"C, transferred into microcentrifuge tubes and stored at -
20 "C until analysis for L-[1-13C]lysine enrichment and phenylalanine and tyrosine concentration.
Expired CO, production rate (FC0-J was measured during the baseline period of each study day using an indirect calorimeter (2900 Computerized
Energy Measurement System, Sensorrnedics, Yorba Linda, CA). CO, collection was performed by bubbling a sample (500 mlrnin-') of the expired CO, collected from a ventilated mask through a modified reflux container containing 10 mL
NaOH for 7 min (Jones et al, 1985) with subsequent transfer into collection tubes
(Vacutainer Brand 6441, 100 X 16 mm, Becton Dickison Inc., Mississauga, ON) for storage at -20°C until analysed for 13C enrichment.
6.2.4. Analvtical Procedures and Calculations
6.2.4.1. Plasma Amino Acid Enrichment
The enrichment of plasma was analysed by the rnethod of Patterson et al
(1991) using gas chromatography mass spectrometry (GCMS). Details of the method is described in Section 4.2.3.1. Distinguishing features of plasma analysis versus urinary analysis is the use of a 200 pl sample aliquot. The lysine retention time was 12.0 min (Figure 6.2).
6.2.4.2. Plasma Phenylalanine and Tyrosine Concentrations
Concentrations of phenylalanine and tyrosine were determined by GCMS using the interna1 standard technique described by Wolfe (1 992). A fixed Pre n r! 562 ~31
Figure 6.2 Chromatograms of mass peaks of phenylalanine (masses 383. 393), tyrosine (masses 417,427) and lysine (masses 560, 561) analyzed by GCMS set to selected ion monitoring mode (other phenylalanine masses were scanned for for the purpose of phenylalanine kinetic estimates perfomed as part of a collaborative study). 135 amount of internal standard (0.15 pg = T) in the form of an isotopically labelled phenylalanine and tyrosine that can be distinguished from the native species was added to the plasma sample (200 PL) to be derivatized for analysis by negative chemical ionization, selected ion monitoring GCMS. L-[U-13Cg,15N]phenylalanine and ~-[~-~~C,,~~bl]tyrosineisotopes (98% '3C,96-99% 15N, Cambridge Isotope
Laboratories, Andover, MA) were selected since they possess a rnolecular weight that is distinct from the native species of tyrosine (165 vs 175, and 181 vs
191 for phenylalanine and tyrosine, respectively) yet behave in an identical manner. The ratio (R) of the areas of the internal standard tracer to the unlabelled species is then calculated and used to estimate the concentration of amino acid in the original mixture.
NaWo = TlR*Vo
where, Nat is the amount of the tracee in the sample, Vo is the total volume of the original mixture, T is the arnount of internal standard added to the original mixture, and R is the ratio of tracer to tracee from GCMS measurements. GCMS analysis of the amino acids associated with the internal standard is similar to that described for lysine in Section 6.2.4.1. The masses scanned were 383 and 393, representing the M and M+10 rnass units of phenylalanine and 417 and 427 representing the M and M+10 mass units of tyrosine. The retention times of phenylalanine and tyrosine were 70.8 and 12.2 min, respectively (Figure 6.2). 6.2.4.3. Expired CO, Enrichment
Expired CO, enrichment was measured by IRMS as described in Section 4.2.3.3.
6.2.4.4. Model of Arnino Acid Metabolism and Calculations
Lysine flux and oxidation estimates are based on the same theory and equations as described in Section 6.2.4.1.
6.2.5. Statistical Analvses
The study followed a completely randomized design with tyrosine intake as the independent variable. A three-way analysis of variance was carried out to determine the effect of individual, order of study and tyrosine intake on FCO,,
F13C0,, lysine flux and oxidation outcome measures. Linear regression models
(linear, quadratic, linear regression crossover) were fitted to the data to detemine the relationship that best describe the response of the dependent variables F'3C0, production and lysine oxidation to increasing tyrosine intake.
The regression analyses were perfomed by the method of least squares using the regression procedure of SAS (SAS lnstitute 1991). As was done for the data from the previous chapter, the best model was determined based on factors relating to model fit and variation. These factors for all models are in Table 9.5 and Table 9.6 in Appendix 9.6.2.
The relationship that best described the F13C0, data was the linear 137 regression crossover analysis while none of the models resulted in a good fit for the phenylalanine oxidation. The estimate of the rnean and safe level of intake for the adult male population was derived by breakpoint analysis of the F13C0, data using a two-phase linear regression crossover rnodel, similar to that used in the previous studies (Bail and Bayley 1984; Duncan et al, 1996; Zello et al, 1993;
Wilson et al, 1997; Lazaris-Brunner et al, 7998). This model defines a breakpoint by minimizing the residual standard error in a stepwise partitioning of data points between two regression lines. The safe level of intake (95% confidence limits) for the mean tyrosine intake was calculated using Fieller's theorem (Seber 1977). Further details of the breakpoint analysis can be found in
Appendix 9.6. Al1 statistical analyses and modelling of the data were perfomed using SAS (SAS lnstitute 1991).
Plasma amino acid concentrations were analysed by two-way ANOVA with tyrosine intake and subject as main effects. Post-hoc analysis was perforrned with Duncan's Multiple Range test when warranted by a significant model.
6.3. RESULTS
CO, production rate (FCO,) for individuals at al1 tyrosine intakes are shown in Table 6.4. FCO, was similar between studies within subject for al1 individuals. lndividual variation of FCO, between study days ranged from 5% to
23%, while interindividual variation was 30%. There was no significant effect of tyrosine intake or order of study on FCO,, however there was a significant Table 6.4 Study day CO, production rate (FCOJ (mL-min-') of individual su bjectsl Tyrosine lntake (mg-kg-'-d-') Subject 3 4.5 6.0 7.5 9.0 10.5 12 Mean*SD2
------. -. 'There was no significant effect of tyrosine intake or order of study on FCO,, but there was a significant subject effect on FCO, production (pc0.0001). 2SD = standard deviation difference in FCO, between individuals (p<0.0001).
Table 6.5 shows the individual 13C0, production rate from L-[l -"Cl lysine oxidation at different tyrosine intakes. Subject DK is rnissing the value for level 3 mg-kg"-d" due to technical difkulty in sample collection. There was a main effect of tyrosine intake (pe0.003) on F'3C0,, but no effect of individual or order of study on the estimates of FI3CO,were observed. Figure 6.3 shows the mean
(* SEM) F'~CO, at each tyrosine intake level. In general, F13C0,decreased from tyrosine intakes of 3.0 mg-kg"-d" to 6.0 mg-kg"-d-', affer which point it remained steady with further increases in tyrosine intake. Regression analysis revealed the two-phase linear regression crossover model as the best fit to the data (Appendix 9.6.2). The breakpoint estimate, representative of the mean requirernent, was estimated to be 6.0 mg-kg-'sd". Calculated from variation about the estimate, the upper 95% confidence interval, representative of the safe population requirement was estimated to be 7.0 mg-kg-l-d-'. The proportion of tyrosine represented 40% of the total aromatic amino acid requirements.
Table 6.6 shows the individual lysine oxidation rate estimate, calculated from F13C0, and plasma [l-'3C]lysine at varying tyrosine intakes. Once again the value for level 3 rng-kg-l.d-l of subject DK is missing since oxidation is calculated from F'3C0, estimates which are not available for the reason described above. In general, the oxidation estimate was more variable than
F13C0,. Nevertheless, analysis of variance revealed a significant effect of tyrosine intake (p<0.001) and individual (~4.006)on oxidation. Figure 6.4 graphically shows the pattern of the data. Linear regression was obviously a Table 6.5 The effect of tyrosine intake on F13C02from the oxidation of L-[1- 13C]lysine(pmol-kg-' -d-')'. -. . .- -- Tyrosine lntake (mg - kg-' -d-') - -- - - Subject 3 4.5 6.0 7.5 9 10.5 12
- - 'There was no significant effect of subject or order of study on F'3C0,, there was a significant effect of tyrosine intake on F13C0, (p~0.003).Means with different subscripts differ from one another. 2N/A = data not available, refer to results section for details 3SD = standard deviation 1 I r2 = 0.4 1 I Breakpoint = 6.0 mg-kg-1-d-1 1
Tyrosine lntake (mg-kg-1-d-1)
Figure 6.3 Mean F13Co2 from the oxidation of L-[1-iJC]lysine at graded tyrosine intakes. Data are presented as mean ISEM. Dotted lines represent the 95% confidence limits of the breakpoint estirnate. Table 6.6 The effect of tyrosine intake and subject on lysine oxidation (pmol-kg" -d''). Tyrosine lntake (mg - kg-' -d-l) Subject 3 4.5 6.0 7.5 9 10.5 12 Mean ISD
'There was no significant effect of order of study on lysine oxidation, but there was a significant effect of tyrosine intake (p<0.001) and subject (pc0.006) on lysine oxidation. Means with different subscripts differ from one another. 'N/A = data not available, refer to results section for details 3S~= standard deviation Tyrosine lntake (mg-kg-1 - d-1)
Figure 6.4 Mean lysine oxidation rate ai graded tyrosine intakes. Data are presented as mean + SEM. 144 poor fit as is reflected in the ? of the analysis, nor did the quadratic analysis fit the data. The two-phase linear regression crossover analysis also did not result in a significant rnodel, therefore estimates of requirement were not made using the oxidation estimate.
Lysine flux estimates are shown in Table 6.7. Tyrosine intake and order of study did not effect lysine flux within individual. There was however, an subject effect on lysine flux estimates.
Figure 6.5 shows the effect of feeding our dietary protocol on plasma phenylalanine and tyrosine concentrations for a representative subject. Formula was consumed from 6 h until 16 h the day of the study. In general, both phenylalanine and tyrosine concentration decreased upon feeding, experiencing a plateau aiter 7-8 h of feeding (representing 13-14 h on the figure). Although there were significant differences in the phenylalanine concentration at differing tyrosine intakes, there was no trend to these differences (Figure 6.6).
Conversely, there were significant differences in plasma tyrosine concentration which dernonstrated higher concentration at tyrosine intakes above requirement
(6 mg-kg-'-d") except for the highest tyrosine intake (12 mg-kg-'-d") which was not significantly different from the concentration at an intake of 6 mg-kg-'.d"
(Figure 6.7).
6.4. DISCUSSION
The goal of the present study was twofold. Firstly. to determine the tyrosine requirement of the adult male receiving phenylalanine at the estirnated Table 6.7 The effect of tyrosine intake on lysine flux (ymol-kg-'-d"). Tyrosine lntake (mg-kg-'qd") Su bject 3 4.5 6.0 7.5 9 10.5 12 Mean*SD2
'There were no significant effect of tyrosine intake or order of study on lysine flux. There was however, a significant effect of subject on flux (p< 0.0001). Means with different subscripts differ from one another. *SD = standard deviation PLASMA PHENYLALANlNE SUBJECT: NM
1 O 1 I I I 1 I 1 8 9 10 11 12 13 14 15 16 Tirne (h).
PLASMA TYROSINE SUBJECT: NM
Tirne (h)
Figure 6.5 Response of plasma phenylalanine and tyrosine concentration throughout study. Data shown is that of a typical subject. Tirne is in hour of the day. abc abc 1
- 9.0 Tyrosine lntake (mg-kg-1-d-1)
Figure 6.6 Plasma phenylalanine concentration after 8 h of feeding the study diet. Bars with different subscripts differ significantly (p~0.05). Data are presented as meanSEM. Tyrosine lntake (mg-kg-1-d-3)
Figure 6.7 Plasma tyrosine concentration after 8 h of feeding the study diet. Bars with different letters differ significantly (pc0.05). Data are presented as mean+SEM. 149
mean population requirement. From these data. the ideal balance of aromatic
arnino acids for the mature metabolic system of the adult could be compared to
that of the immature metabolic system of the neonate. To allow for such a
comparison. a similar study design as in Chapter 5 was employed. Secondly, there has been a resurgence of interest into aromatic arnino acid rnetabolism
and requirements. This is the first tirne tyrosine requirements have been
determined in the adult using stable isotope tracer methods.
6.4.1. Methodotoaical Considerations
The mean phenylalanine requirement as estimated by Zello et al (1 990a)
in adult males receiving excess tyrosine is 9.0 mg-kg-'-&'. The conversion of
phenylalanine to tyrosine is an open metabolic pathway, the provision of excess tyrosine has been shown to cause excess phenylalanine to be preferentially oxidized over equilibrating with the tyrosine pool (Moldawer et al, 1983). This
characteristic of phenylalanine metabolism within the hepatocyte enabled the
isolation of phenylalanine needs from that of total aromatic arnino acid acids.
Fixing the phenylalanine intake at the mean requirement provided an adequate
phenylalanine intake without excess that could potentially be used as a source of tyrosine. This intake results in an inadequate total aromatic amino acid intake
until an adequate level of tyrosine is provided. This design in turn allowed for the
isolation of tyrosine requirement frorn that of phenylalanine.
The identification of a breakpoint resulting from the partitioning of the data points lying below and above the tyrosine requirement (Appendix 9.6) was made. 150
Two end product estirnates of catabolisrn of the indicator were rneas~red:'~CO, production from [l-'3C]lysine oxidation, and lysine oxidation. These two estimates differ in that the lysine oxidation measure takes into account the variation in the enrichment of the plasma pool. while 13C0, production makes no assumptions regarding the precursor pool's enrichment or location. The 13C0, production data yielded a linear regression crossover model while the oxidation data did not. Therefore, an estimate of the mean and safe tyrosine requirement could only be detemined from the FI3CO, data. The lack of fit of the oxidation data to a significant rnodel is believed to be due to the greater variability associated with the plasma [l-13C]lysine;this has been observed in previous studies examining the lysine (Zello et al. 1993; Duncan et al, 1996) requirement of the adult male leading the primary use of 13C0, production when defining the tryptophan requirement in the adult male (Wilson et al, 1997), and female
(Lazaris-Brunner et al, 1998).
The greater variation about the oxidation estimate may be related to a weak relationship between the plasma enrichrnent and the precursor oxidation pool enrichment. Because the 13C0, can only arise from the enriched amino acid at the site of oxidation, it is considered to be a less variable responder to changes in the test amino acid intake. In fact. a study identifying issues related to precursor pool enrichment in piglets has shown that estimates of phenylalanine hydroxylation were incompatible with actual direct body accretion measurements (House et al, 1998b). Further exploration into the matter with the examination of the phenylalanine hydroxylation response to graded tyrosine 151 intakes seem to further suggest uncertainty with measures based on precursor pool enrichment (Thorpe et al, 1998). Ongoing investigations using rapidly- turnover hepatic proteins (Ballrner et al, 1990; Reeds et al, 1992; Cayol et al,
1996) are being investigated with respect to their feasibility to serve as an improved reflection of precursor pool enrichment for protein synthesis. There is no information regarding the exact precursor pool enrichment for oxidation presently available. lndicator amino acid oxidation using I3CO, production as the outcome measure is useful because it does not rely on indirect estimates of precursor pool enrichment, and therefore serves as a direct indicator of amino acid oxidation.
6.4.2. Plasma Amino Acids
Plasma phenylalanine and tyrosine consistently decreased and plateaued after 7-8 h of feeding. While there was no pattern to the plasma phenylalanine response to tyrosine intake, there was an effect on plasma tyrosine concentration. Generally, the plasma tyrosine concentration was affected by the adequacy of the tyrosine intake. Tyrosine intakes that were at or below the mean requirement for tyrosine resulted in lower plasma tyrosine levels than when subjects received tyrosine intakes that were above requirement (Le., 6 mg-kg-'-d-
) The exception to this response was with subjects receiving the greatest tyrosine intake of 12 mg-kg-'-d". Plasma levels within these subjects were numerically greater, but not significantly different from the tyrosine intakes of 6 mg-kg"-d" and below. This suggests that at a higher tyrosine intake of 12 mg-kg* 152
'-d", tyrosine catabolic enzymes were induced such that plasma levels were maintained at a lower level. Such a response to elevated intakes are characteristic of enzyme behaviour and has been observed previously (House et al, 1997a).
The recent studies of the MIT group have undertaken the investigation of phenylalanine and tyrosine kinetics in healthy adult males receiving three levels of phenylalanine in diets with minimal tyrosine intake. They noted that while phenylalanine intake in the fed state responded with an expansion of the plasma phenylalanine pool, plasma tyrosine was less responsive and consistently decreased even at phenylalanine intakes that were largely in excess of total aromatic amino acid requirement (100 mg-kg-'-d-'). These observations led these authors to speculate that a preformed source of tyrosine may be required to maintain tyrosine balance and aromatic amino acid homeostasis (Sanchez et al, 1996). Results of the present study reveal that the decrease in tyrosine during the fed state occurs even when an adequate exogenous preformed source of tyrosine is provided in the diet. This suggests that a decrease in plasma tyrosine is a normal physiological response in the fed state. This is unlike many other of the indispensable arnino acids whose plasma concentrations are known to increase with intake that is above requirement level
(Morrison et al, 1961; Young et al, 1971; Brookes et al, 1972). These results suggest that the plasma response of tyrosine can be differentiated from that of phenylalanine and other indispensable amino acids in the fed state. 153
6.4.3. Tvrosine Reauirement and Arornatic Amino Acid Balance
When receiving a fixed phenylalanine intake of 9.0 mg-kg-'-d-', the mean and safe tyrosine requirement in the adult male was found to be 6.0 mg-kg-l-d-' and 7.0 mg-kg-'.d-', respectively. The resultant total mean requirement and safe level of aromatic amino acid intake is 15 mg-kg-l-d-'and 21 mg-kg-'d", respectively. The balance of aromatic arnino acids detemined from the mean estimates of phenylalanine and tyrosine requirernent was 60:40. This balance of aromatic amino acids is not restricted to the human adult, but is sirnilar to that of the piglet mode1 of the neonate (56:44) (House et al, 1997a; House et al, 1997b). piglet tissue (57:43) (Aumaitre and Duee 1974), and human fetal tissue (59:41)
(Widdowson 1981). Prelirninary evidence in children with PKU suggests a similar phenylalanine to tyrosine ratio of 55:45 (Bross et al, 1997).
6.4.4. Present Knowledoe in Arornatic Amino Acid Metabolisrn and Reauirement
The recent resurgence of interest into aromatic arnino acid requirements of the adult (Zello et al, 1990a; Sanchez et ai, 1995; Sanchez et al, 1996;
Basile-Filho et al, 1997; Basile-Filho et al, 1998) make up part of a large body of research re-evaluating currently accepted requirement estirnates
(FAONVHOIUNU 1985). Zello et al, (1990a) were the first to establish the adult phenylalanine requirement when receiving excess tyrosine, thereby isolating the phenylalanine requirement from total aromatic amino acid needs. The mean and safe estirnate of requirement was detemined to be 9.1 rngokg-'-d-' and 14 mg-kg-
'-d-', respectively. This data forrned the first body of evidence that the aromatic 154 amino acid estimate of the FAOMRIOIUNU (1 985) may underestirnate true requirements.
Tyrosine is generally considered dispensable, itls needs being met readily through phenylalanine hydroxylation (Moss and Schoenheimer 1940). With this in mind, the early studies examining aromatic amino acid requirement were carried out wÎth diets free of tyrosine. Tyrosine was added to the diets to determine it's sparing impact on phenylalanine needs. Tyrosine was suggested to spare up to 75% of phenylalanine needs based on nitrogen balance rnethods
(Rose and Wixom 1955; Tolbert and Watts 1963). Others have found tyrosine to have a much lower replacement value of 50% for men and 35-50% for women, depending on the level of tyrosine fed (Burrill and Schuck 1964). Due to the errors associated with the nitrogen balance method in non-growing individuals
(Fuller and Garlick 1994), these data rernain inconclusive. In the growing rat
Stockland et al, (1971) found that tyrosine was able to provide 45% of the total aromatic amino acid requirernent. Likewise, Milner et al, (1984) in immature beagle dogs and Williams et al, (1987) in kittens found that tyrosine could spare
46% and 43% of phenylalanine, respectively. The balance of phenylalanine to tyrosine that approximates 55:45 to 60:40 appears to be similar across species and stage of development and rnay therefore reflect the actual sparing capacity of tyrosine on phenylalanine.
The estirnated safe total aromatic amino acid requirement from this study is 21 mg-kg-'-d". This estimate suggests that the currently accepted safe aromatic amino acid requirernent (FAOMR1OIUNU 1985) of 14 mg-kg-' -dm'is 155 underestimated by 50%. This is supported by other studies in the adult that show that the requirement estirnate of the FAOMRlOlUNU (1985) underestimates the requirement of leucine (Meguid et al, 1986a). valine (Meguid et al, 1986b). lysine (Kurpad et al, 1998; Meredith et al, 1986) and threonine
(Zhao et al, 1986; Wilson et al, 1997) and tryptophan (Lazaris-Brunner et al,
1998) in adult humans.
Sanchez et al (1995, 1996) and Basile-Filho et al (1997, 1998) have carried out extensive 24 h balance studies using stable isotope tracers of phenylalanine and tyrosine to examine aromatic amino acid metabolism and the adequacy of phenylalanine intake. The amino acid balance method was used to assess the adequacy of intake. This approach defines the requirement as the minimum intake needed to balance obligatory amino acid catabolism. In their studies they used two catabolic indices, phenylalanine hydroxylation and oxidation. These investigations studied subjects receiving one of three levels of phenylalanine intake (21.9, 39, 100 mg-kg-'-d") within diets that contained little tyrosine. The collective evidence from these studies suggests that an intake of
21 -9 mg-kg*'-d" was inadequate as indicated by the negative phenylalanine balance estimated from the difference of phenylalanine input and oxidation or hydroxylation. lndividuals receiving the 100 mg-kg-'.d" and 39 mg-kg-'-d-'diets in contrast, were in general positive phenylalanine balance. Therefore, the data from these studies suggest that the total aromatic amino acid requirernent as estimated with phenylalanine is greater than 21 -9rngwkg-'-d-'.
There are a few reasons why our results differ. Firstly and most 156 importantly, there is a fundamental difference in the study design. The design was established to estimate the tyrosine requirement given an adequate intake of phenylalanine as detemined previously (Zello et al, 1WOa). Providing the aromatic arnino acids in an ideal balance likely rnaximizes the efficiency of utilization of the aromatic amino acids so that the ultimate estimate of total aromatic amino acid requirement is lower than would occur if al1 aromatics were given as phenylalanine. In fact, within their paper, Sanchez et al (1996), discuss the likelihood that a preformed source of tyrosine may improve arornatic amino acid homeostasis. Secondly, their estimates of phenylalanine catabolism are either phenylalanine oxidation or hydroxylation. Both estimates are dependent on precursor pool enrichment estimates, which as discussed above are responsible for contributing considerable variation to the estimates and in the case of phenylalanine hydroxylation, will lead to results that are clearly incorrect
(House et al, 1998b).
6.5. CONCLUSIONS
The tyrosine requirement of the adult human was estimated by partitioning the oxidation of the indicator amino acid, lysine, to a two-phase linear regression crossover analysis. This is the first time that tyrosine requirements have been estimated in the adult male using stable isotope tracer methodology. The data from this experiment suggests that tyrosine accounts for 40% of total aromatic amino acid requirernent. The mean and safe tyrosine requirement was found to be 6.0 mg-kg"-d-' and 7.0 mg-kg-'-d", respectively, given a fixed adequate 157
p henylalanine intake of 9.0 mg-kg-' -d". The proportional balance of
phenyfalanine to tyrosine of 60:40 was similar to that identified in the neonate
receiving TPN, and to the piglet mode1 of the neonate (House et al, 1997a;
House et al, 1997b) and other species (Stockland et al, 1971; MiIner et al, 1984;
Wiiliams et al, 1987).
Furthemore, it was determined that the safe intake for total aromatic amino acids in the adult male is 21 mg-kg-'-d-'. This estimate is 50% greater than the current international aromatic amino acid requirements of the expert cornmittee of the FAOMRIOIUNU, (1985) suggesting that the internationally accepted value for aromatic arnino acids may be significantly underestirnated. 7. GENERAL DCSCUSSION AND FUTURE DlRECTlONS
7.1. General Discussion
The work within this thesis makes up part of a larger research programme aimed at determining aromatic amino acid metabolisrn and requirernent of the human. The purpose of this thesis was to investigate the effects of variations in the intake of aromatic amino acids on phenylalanine and tyrosine rnetabolisrn with a specific focus on tyrosine requirement. This was achieved by: (i) investigating phenylalanine and tyrosine metabolisrn in the parenterally-fed neonate receiving TPN differing in their content of phenylalanine, (ii) estimating the tyrosine requirernent of the TPN-fed neonate and (iii) estimating tyrosine requirement of the adult at a fixed phenylalanine intake.
The main findings from the first neonatal study support the hypotheses that the neonatal phenylalanine hydroxylation and oxidation rates were greater in infants receiving the parenteral nutrition with high phenylalanine versus moderate phenylalanine intake. Also supporting the hypotheses, was the greater excretion of urinary phenylalanine and tyrosine as well as alternate metabolites of phenylalanine and tyrosine catabolism. While the neonate responded to increased phenylalanine intake with increased phenylalanine hydroxylation and oxidation, the excretion of phenylalanine and tyrosine and the alternate metabolites suggested that phenylalanine is not the ideal precursor source of tyrosine when provided through parenteral routes.
Limitations of this study include differences in overall arnino acid profile 159 between treatment groups (i.e., Vamin and Primene). Because we cornpared two commercially available amino acid solutions, we did not have control over the overall amino acid composition. Notable differences were in the level of lysine, arginine and proline. However, Wykes et al (1994a) demonstrated that total aromatic amino acids were first limiting in the paediatric amino acid solution
Vaminolact. In addition, Mitton and Garlick (1992) performed a similar comparison between a general purpose (Vamin) and a paediatric (Vaminolact) amino acid solution and found no difference in nitrogen balance or leucine kinetics in infants.
The study of Wykes et al, (1994a) who cornpared arornatic amino acid rnetabolism in the piglet model of the parenterally-fed neonate receiving Vamin or Vaminolact supplemented with phenylalanine to the level in Varnin. Piglets receiving Vamin experienced similar increases in phenylalanine kinetics to the neonatal study within this thesis. However, due to improved overall quality of the balance of amino acids in the paediatric amino acid formulation, nitrogen balance and growth estimates were improved in piglets receiving the paediatric amino acid supplemented with phenylalanine. From this evidence, the impact of the difference in arnino acid pattern that exists between solutions, would likely result in Prirnene supporting improved amino acid metabolism and minimize any differences observed between groups.
Several general purpose arnino acid solutions such as Vamin have compensated for low tyrosine levels by increasing phenylalanine. With this approach, it is hoped that the neonate will convert sufkient phenylalanine to 160 tyrosine to meet total aromatic amino acid requirernents. lmmaturity of phenylalanine hydroxylase would prevent this method from being effective. This study is the first cornparison of aromatic amino acid metabolisrn within the parenterally-fed human neonate receiving different intakes of phenylalanine.
Results demonstrate that significant substrate induction of phenylalanine hydroxylation occurs in the neonate reducing concems of enzyme immaturity.
The concomitant increase in phenylalanine oxidation may suggest that excess phenylalanine was infused. However, the intravenous route of delivery and the imbalanced nature of the intake (very large load of phenylalanine with very little tyrosine) rnay have had an important impact on the utilization of phenylalanine.
The reason for the neonate's excretion of urinary phenylalanine and tyrosine and alternate catabolites of phenylalanine and tyrosine may be a primary immaturity in the enzymes of phenylalanine and tyrosine catabolism. or secondary to substrate inhibition. Although imrnaturity of phenylalanine hydroxylase is unlikely for the reasons indicated above, immaturities of enzymes of the tyrosine catabolic pathway are possible. In vitro studies comparing the enzyme activity of the neonate to the adult have dernonstrated that while human fetal Iiver possesses 57% the phenylalanine hydroxylation activity of the adult
(Delvalle and Greengard 1977). the tyrosine aminotransferase and hydroxyphenylpyruvate dioxygenase activity have been shown to be only 7%
(Delvalle and Greengard 1977; Ohisalo et al 1982) and 8% (Kretchmer et al,
1956; Ohisalo et al, 1982) that of the adult. respectively. However there is a post-natal stimulation of tyrosine aminotransferase that is not observed with 161 phenylalanine hydroxylase (Delvalle and Greengard 1977; Ohisalo et al, 1982).
Substrate inhibition may also be partially responsible for the increased excretion of tyrosine and the alternate metabolites of tyrosine. Phenylalanine and 4- hydroxyphenylpyruvate are known to inhibit 4-hydroxyphenylpyruvate dioxygenase. blocking the catabolism of 4-hydroxyphenylpyruvate to homogentisate. Partial inhibition of this enzyme could lead to a back-up in the catabolic pathway resulting in increased urinary excretion of phenylalanine and tyrosine as well as the alternate metabolites. Further studies would have to be performed to fully understand the reasons for the results obseived.
The observation (Figure 4.5) within this study that increased energy intake minimizes the percent phenylalanine isotope dose oxidized suggests that other factors may play important roles in the utilization of excess phenylalanine intake.
Increasing energy intake in parenterally-fed neonates resulted in improved amino acid economy by increasing protein synthesis (Duffy et al, 1981). This mechanism possibly contributes to the findings of Lucas et al (1993) that poor tolerance of phenylalanine in parenterally-fed neonates receiving Vamin is eliminated when at least 34 kcal are provided per g of protein consumed. The small sample size of the present study prevents more definitive conclusions to be made with regard to this issue. Further studies are required to pursue greater understanding of the relationship between energy intake and individual amino acid utilization. However, these data stress the importance of maintaining energy intake as a fixed variable within future study designs.
In general, results from this first study suggest that though the neonate 162 increased phenylalanine hydroxylation and oxidation rates in response to increasing phenylalanine intake, the imbalanced infusion of aromatic arnino acids was met with limited tolerance. Increased excretion of phenylalanine and tyrosine and alternate metabolites of phenylalanine and tyrosine catabolism suggests that using high levels of phenylalanine to meet total aromatic amino acids should be avoided. Providing an intake that better reflects the phenylalanine and tyrosine individual needs may improve efficiency of utilization, minimizing any possible deleterious effects of elevated plasma phenylalanine or tyrosine.
To improve the balance of phenylalanine and tyrosine for the parenterally- fed neonate, a second neonatal study was performed. The second study of this thesis estimated the tyrosine requirement of the parenterally-fed neonate. These data provide the first estimate of tyrosine requirement in the parenterally-fed human neonate. These data suggest that tyrosine accounts for approximately
44% of the total aromatic amino acid requirement. The objective was to try to improve the arornatic arnino acid balance of Primene through the provision of supplemental glycyl-L-tyrosine, a soluble precursor source of tyrosine. Various outcome estimates of the adequacy of tyrosine intake were used to increase the sensitivity and validity of the estimate. The outcome measures were: phenylalanine F13C0,, oxidation and hydroxylation.
An attempt was made to investigate several physiological indicators of amino acid adequacy. The traditional estimates of Fq3C0, and oxidation have been previously validated (Kim and Bayley 1983; Kim et al, 1983a; Kim et al, 163
1983b; Bal1 and Bayley 1984; Meguid et al, 1986a; Meguid et al, 1986b; Zhao et al, 7 986; Zello et al, 1WOc; Hoerr et al, 1991;Zello et al, 1993; Duncan et al,
1996; Bross et al, 1997; Wilson et al, 1997; Kurpad et al, 1998; Lazaris-Brunner et ai, 1998; Roberts et al, 1998) and were effective in estirnating a tyrosine requirement. Phenylalanine hydroxylase on the other hand, has never been used as an indicator of tyrosine adequacy, but when examined, was found to be an ineffective indicator of amino acid requirement (Figure 5.2). Recently there have been issues raised regarding the ability of the Clarke and Bier (1982) model to estirnate true phenylalanine hydroxylation rates when applied in the fed state (House et al, 1998b). Furthermore, phenylalanine hydroxylation has been found to be incompatible with net tissue phenylalanine and tyrosine accumulation in the parenterally-fed piglet (House et al, 19985). The discrepancy is attributed to limitations of the model, possibly relating to urinary enrichment measures being a poor refiection of the true intracellular pool enrichrnent. Furtherrnore, the study design involved changing intake levels of tyrosine which may have affected enrichrnent of the precursor tyrosine pool through dilution. There has been ongoing research attempting to obtain better estimates of intracellular enrichment of protein synthesis (Ballmer et al, 1990;
Reeds et al, 1992). Results from such studies may serve as an improved indicator of the precursor pool of hydroxylation. With better estimates of precursor pool enrichment, further studies can be carried out to detennine if phenylalanine hydroxylation proves to be an additional indicator of tyrosine adequacy. 164
The results from this second neonatal study provide information reg arding the tyrosine requirement and ideal balance of the aromatic amino acids in the parenterally-fed neonate. These findings are of value since providing aromatic amino acids in a balanced manner will support improved efficiency of amino acid utilization and in turn promote maximum protein deposition with minimal excess intake. Furthemore, these data provide empirical evidence for the optimal level of aromatic amino acid intake in the parenterally-fed neonate within a neonatal intensive care unit which can used towards the development of an improved parenteral amino acid solution.
Our interest in the study of the aromatic arnino acid metabolism has led to the investigation of aromatic arnino acid metabolism and needs of the adult human. This is the first time that tyrosine requirement has been estimated in the adult receiving an adequate, but not excessive phenylalanine intake. The mean and safe (upper 95% confidence limit) tyrosine requirement was found to be 6.0 and 7.0 mg-kg-'-d-',respectively. The proportional balance of aromatic amino acids was found to lie in a phenylalanine to tyrosine ratio of 60:40. The similar study design between the adult study (Chapter 6) and the second neonatal study
(Chapter 5) has allowed for a comparison to be made of the balance of aromatic amino acids between the hurnan adult and neonate. The adult ideal balance of arornatic arnino acids of 60:40 (Chapter 6) was found to be proportionally similar to that of the neonate (56:44) (Chapter 5) suggesting that while total aromatic arnino acid intake may differ, the balance of the aromatic amino acids supporting maximum effciency of amino acid utilization is not affected by maturity. This 165 balance of aromatic arnino acids is not restricted to the human adult and neonate, but is sirnilar to that of the pigiet mode1 of the neonate (59:41) (House et al, 1997a; House et al, 1997b), piglet tissue (57:43) (Aumaitre and Duee
1974), or human fetal tissue (59:41) (Widdowson 1981). Preliminary evidence in children with PKU demonstrates a similar phenylalanine to tyrosine ratio of 55:45
(Bross et al, 1997).
There is a thirteen-fold greater requirement for tyrosine in the neonate than for the aduit human. Similar difierences in needs have been recommended by the FAOMRlOlUNU (1985) for the aromatic amino acids (phenylalanine + tyrosine), leucine and threonine.
Considered as a whole, these results suggest that maxirnizing the efficiency of amino acid utilization occurs when the phenylalanine to tyrosine intake is approximately 60:40 to 55:45. Such information is particularly important in the parenterally fed neonate whose ability to handle excess aromatic amino acid intake is limited (Chessex et al, 1985; Puntis et al, 1986; Walker et al, 1986;
Rigo et al, 1987; Mclntosh and Mitchell IWO; Walker and Mills 4990; Mitton et al, 1993). These findings can also be beneficial to individuals with conditions associated with altered aromatic arnino acid metabolism such as sepsis
(Herndon et al, 1978; Mori et al, 1WZ), hepatic (O'Keefe et al, 1981;
Shanbhogue et al, 1987) or renal disease (Jones et al, 1978; Tizianello et al,
1990). Provision of phenylalanine and tyrosine intake that more closely meets requirement would possibly minimize the metabolic stress associated with irnbalanced aromatic amino acid intake. 166
It should be noted that the neonatal study was canied out in the parenterally-fed state while the adult study was performed in the enterally-fed state. However, there is no evidence of, or a priori reason for, a discriminatory splanchnic bed uptake or metabolisrn of phenylalanine over tyrosine. lleal digestibility data suggest that gut absorption of phenylalanine and tyrosine are sirnilar (Darragh and Moughan 1998). Studies exarnining phenylalanine splanchnic bed metabolisrn suggest 29-45% first pass extraction (Matthews et al,
1993; Stoll et al, 1997; Stoll et al, 1998), with no evidence of preferential metabolisrn of phenylalanine over tyrosine.
There has been a resurgence of interest in the aromatic amino acid requirements of the adult. Providing the aromatic amino acids in a balanced form, the safe level of intake of aromatic amino acids was found to be 21 mg-kg-
'.d-'. This was in contrast to the requirement found in adults when the aromatic amino acid intake was provided solely as phenylalanine. Under these dietary conditions, the total aromatic amino acid requirement was found to be greater than 21 -9mg-kg-'-de' but less than 40 mg-kg-'-d-'(Sanchez et al, 1995; Sanchez et al, 1996; Basile-Filho et al, 1997; Basile-Filho et al, 1998). Since oniy three levels of phenylalanine were used in their studies due to the very intensive 24 h balance study design, a more specific estimate of arornatic amino acid requirement was not possible. The indicator arnino acid study estimates have the advantage of being derived from 6-7 levels of tyrosine intake carried out in the same subject which allows for a precise mathematical estirnate of requirement. Although the studies used different experimental designs, similar 167 differences in study design of previous investigations resulted in comparable estimates of lysine requirement in adult males (Meredith et al, 1986; Zello et al,
1993; Kurpad et al, 1998). Therefore, the difference in total aromatic amino acid requirement seems to be largely attributed to the provision of the aromatic amino acids as either phenylalanine alone, or in a physiological balance. These results further support the importance of supplying the aromatic amino acids in a balanced intake of phenylalanine and tyrosine. This seems to apply to the neonate where tyrosine is considered to be an indispensable amino acid, or in the adult to maximize efkiency of amino acid utilization. The improved utilization of the aromatic arnino acids when supplied in a more balanced intake is not surprising as natural protein foodstuff in general provides a balance of phenylalanine and tyrosine. Finally, this work provides further evidence that the currently accepted safe requirement of 14 mg - kg-' .d" necessary for nitrogen balance maintenance as indicated by the expert consultation of the
FAONVHOIUNU (1985) is an underestimate of actual needs.
This body of work contributes to present knowledge in aromatic amino acid metabolisrn in the human. Further, estimating tyrosine requirement in the parenterally-fed neonate provides empirical evidence that can be used toward the development of an amino acid solution that best meet the needs of the neonate. Finally, data from the adult study provides further evidence that the currently accepted estimates of total aromatic amino acid needs in the adult is underestirnated. 7.2. Future Directions
The present study investigated the adequacy of the level of the first
limiting amino acid in presently available parenteral amino acid solutions
(tyrosine) and determined an estimate for its requirement. These experirnents
make up part of an ongoing research programme that addresses neonatal amino
acid requirements. The approach involves initial investigations carried out in the
piglet rnodel leading to refined protocols for studies in the hurnan neonate.
Recent data acquired from the piglet model has demonstrated reduced
amino acid requirement with parenteral versus enteral feeding (Stoll et al, 1997;
Bertolo et al, 1998a; House et al, l998a). The degree of difference between feeding models has been shown to be specific to the individual amino acid.
While the piglet parenteral lysine requirement is 72% that of the enteral
requirement (House et al, 1998a), the parenteral threonine requirernent has
been shown to be only 45% that of the enteral requirement (Bertolo et al,
1998a). The differences that are now known to exist between the enteral and parenteral route of feeding are important to consider when providing nutritional support to the neonate, who possess immature synthetic and catabolic enzymes and whose rapidly growing brain limits tolerance of amino acid intake to a narrow
range. Future studies should investigate the metabolisrn and requirement of these and other amino acids such as lysine, threonine and the sulfur amino acids. Data from such studies can provide the foundation upon which a new parenteral arnino acid formulation can be developed. The piglet model can also 169
be adapted to the gastrointestinal surgical neonate or other physiological
conditions frequently observed in neonatal intensive care units. Prelirninary studies carried out in these various states would better reflect the needs of the population of neonates that truly exist in the neonatal intensive care unit.
The variable maturity and clinical conditions (Le., surgical, cardiac, respiratory distress, etc) of neonates within these neonatal intensive care units depict a heterogeneous population that may have specific requirements. Further to using these methods to expand our knowledge into the isquirernent of other amino acids, the requirements of these subpopulation of neonates within the
NlCU need to be investigated.
Observations of interaction between energy intake and phenylalanine utilization suggest that energy has an impact on individual arnino acid utilization.
The findings with respect to this physiologic response were based on small numbers and therefore further study into the area would be of interest to determine the extent to which energy can improve the use of phenylalanine and perhaps other arnino acids.
Results from the second neonatal and adult investigation provided insight into the ideal balance of the aromatic amino acid when requirement is met. It would be of value to apply this infornation to further studies in the neonate and adult. Specifically, investigation into the metabolism of a balanced intake of aromatic arnino acids in individuals with liver (O'Keefe et al, 1981; Shanbhogue et al, 1987) or renal disease (Jones et al, 1978; Tizianello et al, 1990) would be of benefit, since these conditions are characterized by altered metabolism of the 170 arornatic arnino acids resulting in limited tolerance of intakes much greater than requirements. 8. REFERENCES
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Title of Research Proiect:
Amino Acid Metabolism in Infants Receiving Intravenous Nutrition
Investiaators: Name Contact Number Dr. Paul Pencharz Director, Nutritional Prograrn, 813-6169 Division of GI and Nutrition Susan Roberfs Dietitian, PhD Graduate Student 813-6175 Dr. Aideen Moore Neonatologist 813-7331 Dr. Robert M. Fîller Surgeon 8 13-6400 Joan Brennan Clinical Dietitian, NlCU 813-661 3 Constance Williams Research Nurse 813-6121
Purpose of the Research:
This study is being performed to improve the nutrition of infants needing intravenous nutrition. Infants fed with intravenous nutrition receive their protein needs from a solution made up of a careful balance of arnino acids (building blocks of protein). There are currently several solutions available that differ in both the quality and balance of their amino acids. The goal of this study is to determine which of two available amino acid solutions is best used by your child.
Descri~tionof Research:
The study will be performed over a period of 2 days. If you choose for your child to participate in the study (s)he will receive one of two different nutrition solutions. The 1st nutrition solution is the usual amino acid solution used at The Hospital for Sick Children, while the 2nd solution is a pediatric amino acid solution considered by many to have a better balance of building blocks of protein important for optimal growth. In order to see how your baby uses two of these building blocks (specifically known as phenylalanine and tyrosine), we have specially marked a small amount of each and will give them to your baby with the standard intravenous nutrition solution. This marker is a naturally occurring substance and is completely harmless. We will then look for the appearance of the rnarker in the babies breath and urine. Urine will be collected placing cotton swabs into the diaper. Periodically, breath will be collected by placing a clear ventilated hood around your babies head. Fast experience has shown that rnost babies sleep through this procedure. Throughout much of the study an additional nurse will be helping with the extra attention your child will be receiving.
Potential Harms:
There are no known hams associated with participation in this study.
Potential Benefits:
This study has no direct benefit to your infant, however it will provide information that should result in the design of better solutions for future infants in need of intravenous nutrition at The Hospital for Sick Children and in other hospital centres.
Confidentiality:
Confidentiality will be respected and no information that discloses the identity of the subject will be released or published without consent. For your information, the research consent form will be inserted in the patient health record.
Participation:
Participation in research rnust be voluntary. If you choose not to participate, you and your farnily will continue to have access to quality care at The Hospital for Sick Children. If you choose on behalf of your child to participate in this study you can withdraw your child from the study at any time. Again, you and your farnily will continue to have access to quality care at The Hospital for Sick Children. Consent:
I acknowledge that the research procedures described above have been explained to me and that any questions that I have asked have been answered to rny satisfaction. I have been informed of the alternatives to participation in this study, including the right not to participate and the right to withdraw without compromising the quality of medical care at The Hospital for Sick Children for rny child and for other members of rny family. As well, the potential harms and discomforts have been explained to me and I also understand the benefits (if any) of participating in the research study. I know that I may ask now, or in the future, any questions I have about the study or the research procedures. I have been assured that records relating to my child and my child's care will be kept confidential and that no information will be released or printed that would disclose personal identity without my permission.
I hereby consent for my child to participate.
The Person whom may be contacted Name of Parent about the research is:
Susan Roberts
Who may be reached at Signature Tel.: 813-6175 (W) 703-1960 (H) Pager: 375-5350
Name of person who obtained consent
Signature
Date 9.2. SUBJECT ENROLMENT FORM
Su bject Initiais: Study Number: Hospital Chart Nurnber:
ELIGIBILIN REQUIREMENTS Inclusion Criteria
[ ] Term or preterm AGA infant [ 1 Teml [ ] Preterm
[ ] Expected to be nounshed predorninantly by TPN for 2-5 days
[ ] Nutrient intake at requirement levels (85-95 kcaVkgId; 2.5-3.0 g amino acidslkgld) [ ] lnforrned consent
Exclusion Criteria
[ ] Infants receiving oral feeds with protein intake in excess of 10% of total aa intake
[ ] Infants with acute or chronic disease or major congenital abnormalities
[ ] Septic or otherwise rnetabolically unstable infants
[ ] Infants receiving medications that are known to affect amino acid metabolism
Entrv into the Studv
Start of adaptation to study TPN solution: DATE: Il TI ME:
Amino Acid Solution to which Infant is assiqned
Study: [ ] 1 LI2
Study 1
Study 2
[ ] Primene + GTI [ ] Primene + GT2 [ ] Primene + GT3 [ ] Primene + GT4 [ ] Primene + GT5
Notes
-- - -- Meds: DEMOGRAPHIC DATA
Sex: [ ] Male [ ] Female
Parent's name and address:
Prior Feeding: Amino acid solution: Infusion rate: mUkg1h Energy : AA: Lipid:
Staff Physician:
BASELINE PHYSICAL EXAMINATION (Date 1 1 )
Date of Birth: II
Birth Weight: kg Length: cm Head Circ.: cm Gestational Age: wks Age at Study: days Tirne since surgery: days
Briefly describe abnorrnal findings.
[ ] COMPLETED, NO ABNORMAL FINDINGS 9.3. TRACER DOSE CALCULATION
Subject Initiais: Study Nurnber. Hospital Chart Number:
lnfusion Date: 1 / Time: h
Study weight Length: cm Head circ: cm
Prescribed Intake: Parenteral ln take
Solution: Rate: rnUkgld Actual lntake: mUd
Protein: g/kg/d Energy: kcal/kg/d CHO: q/kg/d
Lipid: g/kg/d clld
Enteral lnfake
Source: Rate: mL Q h Actual Intake: mUd
Protein : glkgld Energy: kcallkgld Lipid: glkgld
Isotope Tracer Calculations: [ J L-(7-'%)~henylalanine [ ] L-(3, 3-2H3Tyrosine
Batch #: Batch #:
Concentration: mglm1 Concentration: mg/mL
Constant Infusion: mgtkglh Constant Infusion: mglkglh
X kg = mg/h X kg = mglh
C rnglrnL = mUh - rng/rnl= mUh
X h = mL tracer X h = mL tracer
Prime + overfill : 1.50 rnL Prime + overfill: 20.50 mL
Total Overfiil: 22 mL (12 rnL prime + ?O mL overfill)
Actual Start Time: hrs Actual Stop Tirne: hrs
Actual L-(13C)Phenylalanine Infused: Actual L-(3,3-2H2)Tyrosine Infused:
-vol inf (mL)X conc of tracer (mg!ml) vol inf (ml) X-conc of tracer (mg/mL)
- mg -... mg 9.4. CORRECTION FOR ISOTOPOMER OVERLAP
9.4.1. General
lsotopomer correction was performed by the method of Rosenblatt et al,
(1992). Isotopomers are different masses of the same molecular fragment which can be separated and quantified by GCMS set in selected ion monitoring mode.
This correction is necessary since some tracee isotopomers (natural compounds) have the same molecular mass as some tracer isotopomers
(exogenous isotopically-labelled compounds). The isotopomer weig ht distribution overlap complicates the accurate estimation of the tracerltracee ratio.
This can be overcome as long as the minimum possible weight of each pair of distinct tracer and tracee are different. The isotopomer problem faced in the two neonatal studies of this thesis (Chapter 4 and 5) involve correcting for the overlap of the M+l and M+2 peaks of the tracer and tracee of tyrosine.
To solve this problem the spectral data of 13C tyrosine and 2H, tyrosine is needed from the GCMS chromatograms. These spectra are corrected using expected abundances derived from standards run multiple times by GCMS.
Alternatively, theoretical calculations can be made by determining the theoretical abundances of the tracer, tracee, and their isotopomers and correcting with their theoretical overlap (see Figure 9.1). Figure 9.1 Mass spectral distribution of tracee and tracer standards. 9.4.2. Example Correction
Mathematical expression of correction
= Minimum mass, tyrosine = 417 amu, 13Ctyr = 418 arnu
= Tracer to tracee ratio
= Observed tracee abundance (TR) at (M+x) mass units (Le. baseline) in our case, x=1 ,2
= Observed tracer abundance (TR) at (M+x) mass units
(i.e. plateau)
= Probability of enrichment at (M+x) given "spillup" and "spilldown" effects
Spilldown = Contribution at M-x from tracer of mass M
Spillup = Contribution at M+x from tracer of mass M
Tabulated Observed Spectral Data with lsotopomer Corrections M O bserved 13CTyrosine *H,Tyrosine Observed
Tracee (B) Sarnple (S) 417 1O0 O O 1O0 41 8 20.82 3.2 O .O492 24.077 Then, estimated ITR,,+,, of sample is =
S,M+l,- B,,+,, = 24.08 - 20.82 = 3.26 Then correction for M+2 peak of tyrosine = [S,,+, - B,,,,] - (lTR,,+,, x P,,,(,+,,)]
(6.5 -3.1) - (3.26x 0.166) = 2.87 Uncorr = 3.4
With the corrected M+2 peak we can estirnate the arnount of spilldown that would have contarninated the (M+1) peak =
3.26 - (2.87x 0.0172)= 3.2 Uncorr = 3.26
In this case, correction of the 2H, tyrosine peak represents a 19% difference, while correction of the 13C tyrosine peak represents a 2% difference. 9.5. CLlNlCAL INVESTIGATION INFORMATION FORM
Title of Research Pmject:
Amino Acid Metabolisrn in Infants Receiving Intravenous Nutrition
Investiaators: Name Contact Number Dr. Paul Penchan Director, Nutritional Program, 813-6169 Division GI and Nutrition Susan Roberts Dietitian, Phi3 Graduate Student Dr. Aideen Moore Neonatologist Dr. Robert Filler Head, General Surgery Joan Brennan Clinical Dietitian, NlCU Sandra Parker Research Nurse
Purpose of the Research:
This study is being perfomed to improve the nutrition of infants needing intravenous nutrition. Infants fed with intravenous nutrition receive their needs from a solution made up of a careful balance of amino acids (building blocks of protein). There are currently several solutions available that differ in the balance of their amino acids. The goal of this study is to improve the use of your infants intravenous amino acid intake by modifying the balance and of the amino acids.
Descri~tionof Research:
The study will be perfomed over a period of 2 days. If you choose for your child to participate in the study (s)he will receive a special pediatric amino acid solution considered by many to have a better balance of amino acids. We will further improve this balance by adding tyrosine, an important amino acid whose concentration is presently included at levels below optimum. In order to see how your baby uses two of the building blocks (specifically known as phenylalanine and tyrosine), we have specially marked a small amount of each and will give them to your baby with the standard intravenous nutrition solution. This marker is a naturally occurring substance and is completely harmless. We will then look for the appearance of the marker in your babies breath and urine. Urine will be collected by placing cotton swabs in the diaper. On each day of the twoday study (1 h each day), breath will be collected by placing a clear ventilated hood over your babies head. Past experience with this study has shown that most babies sleep through this collection.
Potential Harms:
There are no known hams associated with participation in this study.
Potential Benefits:
This study has no direct benefit to your infant, however it will provide information that should result in the design of better nutrition solutions for future infants in need of intravenous nutrition at The Hospital for Sick Children and in other hospital centres.
Confidentialitv:
Confidentiality will be respected and no information that discloses the identity of the subject will be released without consent. For your information, the research consent form will be inserted in the patient health record.
Participation:
Participation in research rnust be voluntary. If you choose not to participate, you and your family will continue to have access to quality care at The Hospital for Sick Children. If you choose on behalf of your child to participate in this study you can withdraw your child from the study at any time. Again, you and your family will continue to have access to quality care at The Hospital for Sick Children. Consent:
I acknowledge that the research procedures described above have been explained to me and that any questions that I have asked have been answered to my satisfaction. I have been inforrned of the alternatives to participation in this study, including the right not to participate and the right to withdraw without compromising the quality of medical care at the Hospital for Sick Children for my child and for other members of my family. As well, the potential harms and discomforts have been explained to me and I also understand the benefits (if any) of participating in the research study. I know that I rnay ask now, or in the future, any questions I have about the study or research procedures. I have been assured that records relating to my child and rny child's care will be kept confidential and that no information will be released or printed that would disclose personal identity without my permission.
I hereby consent for rny child to participate.
The person whom may be contacted about Narne of Parent the research is:
Susan Roberts
Who rnay be reached at: Signature Te I: 813-6175 ON) 703-1960 (H) Pager: 375-5350
Name of person who obtained consent
Signature
Date 9.6. REGRESSION MODELS APPLIED TO DATA IN CHAPTERS 5 AND 6.
9.6.1. Summary Statistics of Rearession Models Applied to Data in Chapter 5
Table 9.1. Summary Statistics of Regression Models Dependent Variable: FI3CO, Model Linear Quadratic Linear Regression Crossover*
Pc 0.006 ? 0.6 CV 31 Standard error 0.003 Parameter estirnate 0.01 0 % Error of estimate 28 Breakpoint 66 Upper 95% CL 90 *Two-phase linear regression crossover rnodel
Table 9.2. Summary Statistics of Regression Models Dependent Variable: Phenylalanine Oxidation Model Linear Quad ratic Linear Regression Crossover*
P < 0.08 ? 0.3 cv 34 Standard error 0.028 Parameter estirnate 0.055 0.0016 -12 % Error of estimate 51 66 29 Breakpoint 82 Upper 95% CL 97 *Two-phase linear regression crossover model Table 9.3. Summary Statistics of the Regression Models Dependent Variable: Phenylalanine Hydroxylation Model Linear Quadratic Linear Regression Crossover Pc 0.63 does not appty ? 0.6 CV 22 Standard error 0.04 Estirnate 0.02 % Estimate 20
Table 9.4. Summary Statistics of the Regression Models Dependent Variab1e:Tyrosine Flux Model Linear Quadratic Linear Regression Crossover*
Pc 0.006 ? 0.5 CV 31 Standard error 0.25
Oh Error of estimate 29 176 60 Breakpoint 55 Upper 95% CL 94 "Two-phase linear regression crossover model 208 9.6.2. Surnmary Statistics of Remession Models Applied to Data in Chaoter 6
Table 9.5. Surnmary Statistics of Regression Models Dependent Variable: Fq3CO, Model Linear Quadratic Linear Regression Crossover* P< 0.001 0.05 0.001 ? 0.3 0 -2 0.4 CV 21 21 19 Standard error 0.0092 0.0006 0.1 6 Parameter estirnate 0,033 0.002 0.67 % Error of estirnate 28 30 22 Breakpoint 5.94 Upper 95% CL 6.94 'Two-phase linear regression crossover model
Table 9.6. Summary Statistics of Regression Models Dependent Variable: Phenylalanine Oxidation Model Linear Quadratic Linear Regression Crossover*
P < 0.004 i=L 0.2 CV 26 Standard error 0.168 Parameter estirnate 0.49 % Error of estimate 33 Breakpoint Upper 95% CL *Two-phase linear regression crossover model 209
9.7. TWO-PHASE LINEAR REGRESSION CROSSOVER MODEL
The mean and safe level of tyrosine requirement was determined for the neonate (Chapter 4) and the adult (Chapter 6) using a two-phase linear regression crossover model (Seber 1977), similar to that described previously
(Bal1 and Bayley 1984; Duncan et al, 1996; Zello et al, 1993; Lazaris-Brunner et al, 1998). The model defines a breakpoint by minirnizing the residual standard error in a stepwise partitioning of data points between two regression lines.
9.1 -1. Break~ointestimate of mean reauirement
The rnodel equation predicting the regression Iines are defined by:
Y=A1 +BIx+(A2-Al)D+(B2-BI)(Dx)+E where Y is the individual obsewation for the independent variable (F13C0, or
Oxidation), Al and A2 are the y-intercepts of the first and second line, respectiveiy, BI and B2 are the dopes of the first and second lines, respectively;
D=O if the data is frorn the line with zero slope and D=l if the data is from the sloping line; E represents the error associated with the rnodel. For the neonatal data of Chapter 4 the fist line has no dope and for the adult data of Chapter 6, the first line has a slope. Reducing this equation for each line gives:
Y=AI+BIx for the line that has no siope @=O), and
Y=(AI+A2Al) + (BI+B2-B1)x for the sloping line (D=l). Solving for the x parameter yields the location where the lines cross over identifying the "breakpoint" which represents the study population's mean requirernent, and is defined by:
x = -(A2-AI)I(BZ-BI).
Using the data from the neonatal study of Chapter 5 as an example, the breakpoint can be directly calculated from the SAS output parameters where,
(A2A1) = choose = -1.1276
(B2-BI) = choose*tyrin = 0.0171
The breakpoint is therefore = choose/choose*tyrin = -(-1.1276/0.0171) =
65.79 mglkgld. The SAS (SAS Institute 1991) program and output statement is shown below. SAS program for the Iinear crossover breakpoint analysis
Options pagesize=ôO; Iibname perm '\sr\bb\datat; data gtbbox; set perm.bbox; proc sort data=perm.bbox; by tyrin; proc print data=perm.bbox; var id fyrin f13co2 ox; titlel 'fI3co2 and oxidation'; data gtlv64; set pem-bbox; choose=O; if tyrin=108.1 or tyrin=104.3 or tyrin=101.5 or tyrin=81.4 or tyrin=72.4 or tyrin= 71-8 or tyrin = 66.6 or tyrin=64.2 then choose=l ; tyrchoo=tyrin*choose; proc glm; model f13co2 ox=choose tyrin*choose; titlei 'tyrin 108.1 +104.3+101.5+81.4+72.4+71.8+66.6+64.2 vs tyrin=restt; title2 "; Me3 'one phase has slope'; proc reg outest=outlin covout outsscp=outsums; model f13co2 ox=choose tyrchoo; plot r.*p.; proc print data=outlin; proc print data=outsums; run; SAS output statement of the linear regression crossover analysis
General Linear Models Procedure Dependent Variable: FI3CO2
Source DF Sum of Squares Mean Square F Value Pr> F
Model 2 8.64 0.0080 Error 9 Corrected Total 1 1
C.V. Root MSE 28.53877 0.23129135
Source DF Type 1 SS Mean Square F Value Pr> F
CHOOSE 4.74 0.0573 CHOOSE*TYRIN 12.54 0.0063
Source DF Type III SS Mean Square F Value Pr>F
CHOOSE 6.89 0.0276 CHOOSE*TYRIN 1254 0.0063
T for HO: Pr > lTI Std Error of Parameter Estimate Parameter=O Estimate
INTERCEPT 0.0005 0. 11564567 CHOOSE O,O276 0.4295451 1 CHOOSE*TYRIN 0.0063 0.00483989
9.1 -2. 95% confidence intewal An estimate representative of a safe intake for a similar population of individuals is taken as the upper 95% confidence Iirnit of the mean estimate (FAONVHOIUNU 1985). The 95% confidence limits were estimated using Fieller's theorem (Seber 1977) programmed with SAS (SAS Institute 1991). The program uses estimates of variation frorn the output statement of the breakpoint analysis:
covbeta =choose*tyrchoo The program and output are shown below.
SAS program for 95% confidence limits LIBNAME TO '\SR\bb\datal; DATA TO .b bcico2; input beta2 vbeta2 betal2 vbetal2 covbeta t; CARDS; -1.1276 0.21 O9 0.01 714 0.00003 -0.0022 1-796
1 proc print; run; options pagesize=60; libnarne perm '\sr\bb\data'; data perm.bbcico2; set perm. bbcico2; t=1.796; ratio=-(beta2lbetal2); aa=vbeta2/(beta2"2); b b=vbeta 12/(beta 12**2); ab=covbeta/(beta2*betal2); varratio=(ratio**Z)*(aa+bb-2*ab); seratio=sqrt(varratio); clower=ratio-t*seratio; cupper=ratio+t*seratio; proc print; titlel 'Upper and Lower Confidence Limits'; run; Output statement of the 95% confidence limits analysis OBS BETAî VBETA2 BETA12 VBETA12 COVBETA T RATIO AA 1 -1.1276 0.2109 0.01714 .O0003 -.O022 1.796 65.7876 0.16587
BB AB VARRATIO SERATIO CLOWER CUPPER 0.102120 -11383 174.535 13.21 12 42.0604 89.5 149
RAT10 = Breakpoint CLOVER = Lower 95% confidence limit CUPPER = Upper 95% confidence lirnit = 89.5 rngkgfd 9.8. INFORMATION SHEET
Phenvlalanine and Tvrosine Kinetics in response to araded intakes of Tvrosine
1 nvestiaators Contact number Paqer Paul Pencharz, MD PhD 813-6176 Susan Roberts, RD MSc 813-6175 Jane Thorpe, SRD MSc 813-6175
Purpose of research :
This study is being carried out to investigate various aspects of the metabolism of the aromatic amino acids (phenylalanine and tyrosine) at differing intakes of dietary tyrosine. The three prirnary aims of the study are :
1. to establish a requirement for the dietary intake of aromatic amino acids
Evidence has indicated that the published requirements for sorne essential amino acids may be too low. Our laboratory has developed a more sensitive method of measuring amino acid requirements, using labelled amino acids. We intend to use this methodology to establish a requirement for the aromatic amino acids in healthy adults.
2. to detemine how the rate of conversion of phenylalanine to tyrosine in the body responds to varying intakes of tyrosine
In certain patient groups the rate of conversion of phenylalanine to tyrosine rnay be impaired. It is therefore of interest to establish the normal range for this conversion so that any abnormalities may be identified. The dietary supply of both phenylalanine and tyrosine will influence this reaction, and therefore initially we need to determine the effect of diet on this rneasurement.
3. to investigate different ways of measuring amino acid rnetabolism in the body.
Our methods for investigating amino acid rnetabolisrn involve the use of labelled amino acids. How we measure these amino acids in the body influences the results of the study and therefore we need to determine the best rneasure to use Le. blood amino acids or proteins.
Description of the research :
Subjects will be asked to participate in seven studies, differing in the prescribed intake of tyrosine on each study day. For the two days prior to each study day, subjects will be required to take a formula diet consisting of rnilkshake drinks, which will be provided. Each study takes a day to complete, with at least one week between each of the study days. Subjects are required to consume an experimental formula diet hourly over a ten hour period, starting at 6.00 am on the day of the study. No other food will be consurned throughout the study. The first three hourly rneals can be consumed at home. The subject should arrive at the Clinical Investigation Room (4D045) at the Hospital for Sick Children at 8.30 am. Blood samples (to a total volume of -1 50 mUday) will be collected throug hout the day frorn an intravenous hand vein line established by the research nurse at the beginning of the study day. Breath sarnples will also be collected through a loose fitting mask at regular intervals throughout the day. Starting at 10.00 am stable isotope labelled amino acids will be given with each hourly meal. Potentiai hams :
There are no known hans associated with participation in this study.
Confidentiality :
Confidentiality will be respected and no information that discloses the identity of the subject will be released or published without consent.
Participation :
Participation in research must be voluntary. If you choose to participate, you may withdraw from the study at any time. Participants will receive $700 remuneration upon completion of al1 seven studies.
Consent :
I acknowledge that the research procedures described in the attached information sheet (and which I have a copy) have been explained to me and that al1 my questions have been answered satisfactorily. I understand the possible risks and discornforts associated with the study. I understand that I am free to withdraw from the study at any time without consequence. Withdrawal from the study prior to completion will result in forfeit of compensation.
Subject :
Date :
Signature : lnvestigators Signature : 9.9 DlET CALCULATIONS AND REClPES
Study Date : Subject : Height : m Weight : kg Age: yrs
Enerav Reauirements : kcallday
Wornen Men 18 - 30 yrs : 13.3W + 334H + 35 18 - 30 yrs : 15.4W - 27H + 717 30 - 60 yrs : 8.7W - 25H + 865 30 -60yrs : 11.3W- 16H + 901
kcalfday x 1.7 = kcallday
Diet Constituents
Amino Acids = 10% total energy intake Formula = 65% total energy intake Butterscotch Cookies = 12.5% total energy intake Cornflake Cookies = 12.5% total energy intake
Amino Acids = 1.O g Protlkglday x kg = g Protlday [BI
Formula = 0.427mUkcal x 65% x [A] kcallday = mUday [Cl
BS Cookies = 0.234 glkcal x 12.5% x [A]kcallday = glda~[Dl
CF Cookies = 0.229 glkcal x 12.5% x [A] kcallday = g/da~[El Amount Fat (g) CHO (g) Prot(g) Amino [BI - - Acid s Formula [CJ x 0.1 x 0.36 -
BS Cookies [Dl x0.223 x 0.565 -- - CF Cookies [El x0.244 x 0.537 - Total (g) Total (kcal)
Actual Energy lntake - kcal/d ay
Energy Distribution = % Fat, % CHO, %Pro Amino Acid Mix # 1 (studv dav)
Study day includes 83.3%of the total intake , a total of 70 (/72) meals.
g Protlday [BI x 0.833 = g Protlstudy day
Mix # 1 represents 77% of the total AA reqt = g Mix # 1 1 study day
g Mix # 1 1 study day + 1O meals = g Amino acid mix # 1 / meal
The total of the other amino acids (Lys, Phe, Tyr, and Ala) = 23% of reqt = s PB1
Lvsine lntake
Lys reqtlday = 45 mglkg x kg x 0.833 = mg [FI Tracer Lys intake = 2.4 + (6 x 1.2) mg/kglday x kg = mg [G] Dietary Lys = [FI - [G]= mg [Hl
The dietary Lys should be distributed between the meals to standardise the intake of each meal. Initially balance the intakes to the highest intake at meal #5.
Meal # 5 Lys isotope intake= (2.4 + 1.2 mglkg) x kg = mg [al
Meal # 6 - #10 isotope intake = 1.2 mglkg x kg = mg [bl
Meal #6 - #IO balance Lys = [a]- [b]= mg / meal #6 - #IO [cl
Meai #1 - #4 balance Lys = [a]= mg 1 meal #i- #4 [dl
XS Lys = [Hl - 4 x [dl - 5 x [cl = mg [el Dietary Lys is in the fom of Lys.HC1, therefore al1 amounts must be multiplied by a correction factor of 1.24. The lys isotope on the other hand is in the form of Lys.2HC1, in which case we must use a correction factor of 7.47.
Dietary intake meal#I - #4 = (M + [dl) = mgx 1.24= mg 1 Dietary lntake meal # 5 = [fl= mg x 1.24= mg Pl Dietary intake meal#6 - #IO = ([q + [cl) = mg x 1.24 = mg Pl
Isotope intake meal #5 = [a] x 1.47 = mg [a Isotope intake meal#6-10 = [b] x 1.47 = mg [bu
Phenvlalanine lntake The level of phenylalanine intake is fixed for ail studies at an intake of 9 rng/kg resulting in an hourly phe intake based on 12 rneals = 0.75 mglkglh Phe requirementlday = 9 rng/kg x kg x 0.833 = mg [gl
- Phelmeal = 0.75 mgJkg x -kg - mg [hl (meal #14 dietary, # 6-10 isotope)
Tracer intake meal #5 = 0.75 c0.75 mgkg x kg = mg [Il
Tvrosine lntake Level 3, 4.5, 6, 7.5, 9, 10.5, 12 rng/kg/d
Tyr intake/day - -mglkgfd x kg x 0.833 = mg [il
Tyr intakefmeal = Dl11 O = mg
Tracer Tyr intake = (Garlick et al, 1980; el-Khoury et al, 1995a)+ (6 x 0.25)mgkg/day x kg = mg [kl Dietary Tyr = D] - [6 x 0.25 x kg] = W [ml
Meal #5 Tyr isotope intake = 0.18 + 0.25 + 0.25 mglkg x kg - mg [n]
Meal #6 - #10 Tyr isotope intake = 0.25 mg/kg x kg = mg Io]
Meal #l- #4 dietary Tyr balance = [O] = mg [cl]
XS Tyr = [il - (Antoshechkin et al, 1991d) = mg Pl
XS Tyrlmeal = [SI + 10 = mglrneal [SI
Tyr diet intake rneal #1 -#4 = [Cl1 + Pl = mglmeal [7]
Tyr diet intake rneal #5-1 0 = [SI = pl
Alanine lntake
Ala reqt Formula Requisition [Studv dav) Need 83.3% tequirement i-e. 1Of1 2 meals
Formula required per study day = [Cl ml= mUday [Cl
Formula required per mealid = [Cl mL + 12 = mumeal [O]
" Each rneal is served in a 4 oz bottle.
Formula requisiiion = 11 x 4 oz bottles x mumeal [O]= mUday [Pl
Formula Reci~e
lngredient 1 Conc'n 1 Study day 1 Study day I (g/m~) 1 voi(rn~)[PI amt (g)* I I Product 1 0.281 1 I #80056 Orange 0.0832 Tang 1 Koolaid 0.0832 1 CrystaIs 1 Corn Oil 0.033 Sterile 0.64 water
* Study day amt (g) = Conc'n (giml) x study day vol [Pl Butterscotch Cookies (studv davl f3S Cookies required per study day = [Dl = g/da~[Dl g BS Cookies required per meal =[DI+12 = glmeal
Cornfiake Cookies [studv dav)
CF Cookies required per study day = [El = g/da~[El g CF Cookies required per meal =[E] + 12 = g/rneal
** Each rneal is setved in a separate plastic bag Diet Reauisition Form:
Subject's Narne: Delivery Date:
Formula Recipei Studv Dav
g Product #80056
g Orange Tang
9 Koolaid Crystals Flavour:
g Corn Oil
g Sterile Water
Total volume of formula: ml
-1 1- 40z Bottles ml per bottle -- -- - Subject's Name: Delivery Date:
Butterscotch Cookie: Studv Dav
g BS cookie per day
-10- meals per day g BS Cookie per meal
Cornflake Cookie: Studv Dav
g CF cookie per day
10- meals per day g CF Cookie per meal
*** All meals should be divided into separate plastic bags, each labelled as follows:
-Subject name
-Meal Number
-Type of Cookie
-Weight (grams) of cookie per bag Studv Dav Amino Acid Recipe First column = mg of amino acids Second column = mL of isotope solution
) Totals 1) 1 1 Isotope solution concentrations