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Arginine metabolism in human infants Christopher Tomlinson Supervisors Dr. Paul Pencharz, Dr Ron Ball Background • Arginine is a cationic amino acid Metabolic functions of arginine 1. Protein synthesis 2. Amidino group for creatine synthesis 3. Polyamine synthesis via ornithine and agmatine 4. Synthesis of nitric oxide 5. Urea Synthesis 6. Activates N-acetyl glutamate synthase 7. Post-transational modification of proteins through methylation and citrullination Background Protein ADMA Agmatine SDMA Argininosuccinate arginine decarboxylase ASL Arginine Citrulline NOS arginine : glycine amidinotransferase Excretion in urine ASS NO Glycine arginase Guanidinoacetate Carbamoyl Phosphate Urea Glutamine OTC Ornithine Creatine Glutamate P5C dehydrogenase OAT ODC Putrescine Spermidine Spermine, Cadaverine Pyrroline 5-carboxylate P5C reductase Proline proline oxidase non-enzymic reaction. Glutamylsemialdehyde P5C synthetase Arginine Requirements • Conditionally essential amino acid • Arginine deficient diet in humans – Healthy adults • no ill effects (Beamier et al 1996) • arg, pro, gln, asp free diet - no ill effects (Castillo 2008) • azospermia (Holt 1944) – Arginine deficient TPN leads to hyperammonemia – Essential during critical illness Carcillo 2003, Zello 2003, Morris 2004 Arginine Requirements • Arginine deficient diet – Rats • Poor growth, fatty liver, orotic aciduria, hyperammonemia Milner et al 1974 – Piglets • Hyperammonemia and death Brunton 1999, Prior 1995 – Obligate carnivores • Hyperammonemia and death Morris 1978 Arginine Requirements • Healthy human neonates – Essential / non-essential? – 3 infants fed arginine free diet • No apparent ill effects Snyderman 1959 • Neonates in ICU – Essential • TPN deficient in arginine leads to hyperammonemia Heird 1972, Batshaw 1984 • Arginine low in infants in ICU Arginine post cardiac surgery 120 100 80 60 40 20 0 Pre op post op 1 post op 2 Arginine Requirements • Neonates in ICU – Persistent pulmonary hypertension • Hypoargininemia, ? Reduced NO production • Treatment with arginine improved oxygenation – Necrotising enterocolitis • Development of NEC related to low arginine • Arginine supplementation reduced incidence? Arginine Synthesis Argininosuccinate ASL ASS Arginine Citrulline Carbamoyl Phosphate Glutamine OTC Ornithine Glutamate P5C dehydrogenase OAT Pyrroline 5-carboxylate P5C reductase Proline non-enzymic reaction. proline oxidase Glutamylsemialdehyde P5C synthetase Arginine Synthesis • Adult mammals – Intestinal – renal axis • Citrulline synthesised in enterocyte from glutamate • Citrulline not taken up by liver but by kidney • Converted to arginine Arginine Synthesis • Neonatal mammals – Kidney low levels of ASS and ASL – Enterocyte has high levels of ASS and ASL, low arginase – Hence, arginine synthesis takes place in enterocyte – Precursor proline or glutamate? Arginine Synthesis • Neonatal piglet model – In vitro studies • Glutamate is significant and main precursor of arginine synthesis Wu 1995, Wu 2000 Arginine Synthesis • Neonatal piglet model – In vivo studies of arginine metabolism • Arginine deficiency ameleriorated by enteral proline and dependant on enteral function Brunton1999, Bertolo 2003 • Arginine not synthesised from glutamate enteral or parenteral Brunton1999, Wilkinson 2004 Arginine Synthesis • Neonatal piglet model – Arginine synthesis upregulated but not able to completely synthesise sufficient arginine Wilkinson 2004 – Proline deficient diet • Arginine converted to proline • No conversion of glutamate to proline Murphy 1996 – Proline ~60% of arg synthesis Urschel 2007 Components of arginine requirements in human infants Neonatal Pigs (7 days) Arginine use Protein synthesis Arginine catabolism (arginase and NO synthase) Creatine synthesis Total Arginine use Arginine intake Arginine synthesis All units mg/kg/day 750 260 70 1080 400 680 Components of arginine requirements in human infants Neonatal Pigs (7 days) Arginine use Protein synthesis Arginine catabolism (arginase and NO synthase) Creatine synthesis Total Arginine use Arginine intake Arginine synthesis All units mg/kg/day Neonatal Humans 750 260 140 50 70 16 1080 400 680 206 45 160 Research Rationale • Few studies on arginine metabolism in human neonates • Lack of understanding of arginine physiology and role in pathophysiology • High rate of arginine synthesis is implied • What is the actual rate of arginine synthesis? • Where does it occur? • What is/are the precursors? Research objectives 1. To determine if human neonates can synthesise arginine and what the precursors are. 2. To determine arginine flux and rate of synthesise in healthy newborns 3. To determine if the premature infant has a greater dietary requirement for arginine. Stable isotope study design – Multi tracer experiment • (2-15N-amidino) Arginine • (1-15N) Proline • (2,4,4-2H3) Glutamate Stable isotope study design Argininosuccinate ASL ASS M+1 Arginine M+3 M+1OrnithineM+3 M+1 CitrullineM+3 Carbamoyl Phosphate Glutamine OTC Glutamate P5C dehydrogenase OAT Pyrroline 5-carboxylate P5C reductase M+1 Proline non-enzymic reaction. proline oxidase Glutamylsemialdehyde P5C synthetase M+3 Methods • Labelled amino acids fed via NG tube 2 or 3 hourly before feeds • Urine collected at baseline and with diaper changes throughout study Urine collection Time 0 12 hours Labelled amino acids pre feed Subjects • • • • • 12 preterm infants studied Fully enterally fed, no resp support Gestational age at birth 33+1 weeks Birthweight 1.82 Kg Age at study 13 days Arginine Flux • Average 90 mg/kg/hr • Range 55-150 mg/kg/hr • No correlation with gestational age at birth, age at study – Significantly higher than IV flux rate Castillo 1995 Proline Flux • Average • Range - 26 mg/kg/hr 19.5-47 mg/kg/hr Glutamate flux • No enrichment seen – In keeping with literature – All glutamate metabolised on first pass by splanchnic organs Arg synthesis from Pro/Glu Argininosuccinate ASL ASS M+1 Arginine M+3 M+1OrnithineM+3 M+1 CitrullineM+3 Carbamoyl Phosphate Glutamine OTC Glutamate P5C dehydrogenase OAT Pyrroline 5-carboxylate P5C reductase M+1 Proline non-enzymic reaction. proline oxidase Glutamylsemialdehyde P5C synthetase M+3 Pro/Glu conversion to Orn • No M+3 Orn seen • M+1 Orn seen in all infants – Mean 2.9% – Range 1.4 – 5.6 % Pro/Glu conversion to Cit • No M+3 Cit seen • M+1 Cit seen in all infants – Mean 2.9% – Range 0.1 – 5.6 % Pro/Glu conversion to Arg • No M+3 Arg seen • M+1 Arg seen in all infants – Mean 0.9% – Range 0.2 – 2.7 % Arginine Synthesis Argininosuccinate ASL ASS 0.9% Arginine Citrulline 2.9% Carbamoyl Phosphate 2.9% Ornithine Glutamine OTC Glutamate P5C dehydrogenase OAT Pyrroline 5-carboxylate P5C reductase 8% Proline non-enzymic reaction. proline oxidase Glutamylsemialdehyde P5C synthetase Arg rate of synthesis • Calculated from arginine and proline enrichments • ~7 mg/kg/hr = 168 mg/kg/day NO Production Argininosuccinate ASL ASS M+2 Arginine Citrulline M+1 Carbamoyl Phosphate Glutamine OTC Ornithine Glutamate P5C dehydrogenase OAT Pyrroline 5-carboxylate P5C reductase Proline non-enzymic reaction. proline oxidase Glutamylsemialdehyde P5C synthetase NO Production Argininosuccinate ASL ASS 3.98% Arginine Citrulline 2.5% Carbamoyl Phosphate Glutamine OTC Ornithine Glutamate P5C dehydrogenase OAT Pyrroline 5-carboxylate P5C reductase Proline non-enzymic reaction. proline oxidase Glutamylsemialdehyde P5C synthetase Conclusions • Arginine is a conditionally essential amino acid in human neonates. • Healthy growing preterm infants are able to synthesise arginine. • The dietary precursor is proline. • Glutamate entirely metabolised by splanchnic organs. Future studies • TPN results in gut mucosal atrophy • Repeat studies at HSC in infants as they transition to oral feeding – Comparison with data from study 1 Future studies • All neonatal evidence shows proline is only dietary precursor for arginine – Study in adults using same protocol to determine the dietary precursor for arginine • Currently available evidence is from studies in fasted, intraoperative patients Thank You • Supervisors – Paul Pencharz – Ron Ball • Committee – Keith Tanswell – Harvey Anderson • Pencharz lab – – – – Mahroukh Rafii Glenda Coutrney-Martin Raja Elango Karen Chapman • Staff at St Mikes NICU – Mike Sgro • CIHR
