Protein, Energy and Their Interaction in Critically Ill Children

Sascha Cornelis Antonius Theodorus Verbruggen The studies as presented in this thesis were financially supported by the Sophia Children’s Hospital Fund (SSWO; grant 537; institutional grant, Erasmus MC – Sophia Children’s Hospital, Rotterdam, The Netherlands). The grant supplier had no involve- ment whatsoever in the study design, in the collection, analysis, and the interpretation of data, and in the decision to submit the reports for publication.

The printing of this thesis was financially supported by Nutricia Advanced Medical Nutrition, Nestlé Nutrition and Cambridge Isotope Laboratories, Inc.

ISBN: 978-90-8559-168-9

Layout: Optima Grafische Communicatie, Rotterdam, The Netherlands Cover: Sascha Verbruggen, Arnold van den Berg; www.fotoberg010.nl Printed by: Optima Grafische Communicatie, Rotterdam, The Netherlands

Copyright: S.C.A.T. Verbruggen , The Netherlands, 2010

All rights reserved. No part of this book may be reproduced, stored in a retrieval sys- tem, or transmitted, in any form or by any means, electronic, mechanical, photocopy- ing, recording, or otherwise, without the prior written permission of the holder of the copyright. Protein, Energy and Their Interaction in Critically Ill Children

Eiwit, Energie en Hun Interactie in Kritisch Zieke Kinderen

Proefschrift

ter verkrijging van de graad van doctor aan de Erasmus Universiteit Rotterdam op gezag van de rector magnificus

Prof. Dr. H.G. Schmidt

en volgens besluit van het College voor Promoties.

De openbare verdediging zal plaatsvinden op woensdag 22 december 2010 om 15.30 uur

door

Sascha Cornelis Antonius Theodorus Verbruggen

geboren te Nijmegen Promotiecommissie:

Promotor: Prof. Dr. J.B. van Goudoever

Co-promotor: Dr. K.F.M. Joosten

Overige leden: Prof. Dr. A.J. van der Heijden Prof. Dr. D. Tibboel Prof. Dr. P.J.J. Sauer “The less you know, the more you believe...” ‘last night on earth’ Bono, U2

Dit boek is voor mijn ouders

1. Table of contents 2. 3. 4. 5. 6. Chapter 1 General introduction and outline of the thesis 9 7. Part I Glucose metabolism 8. 9. Chapter 2 Insulin therapy in the Pediatric Intensive Care Unit 27 10. Chapter 3 Efficacy and safety of a tight glucose control protocol in critically 53 11. ill term infants 12. Chapter 4 Reducing glucose intake safely prevents hyperglycemia in 65 13. post-surgical infants 14. Part II Amino Acid metabolism 15. 16. Chapter 5 Ontogeny of methionine utilization and splanchnic uptake in 85 17. critically ill children 18. Chapter 6 Parenteral amino acid intakes in critically ill children; a matter of 109 19. convenience 20. 21. Part III Protein and Energy interactions 22. Chapter 7 Current recommended parenteral protein intakes do not 133 23. support protein synthesis in critically ill septic, insulin resistant 24. adolescents with tight glucose control 25. Chapter 8 Albumin synthesis rates in critically ill infants and adolescents; 157 26. influence of amino acids, energy, and insulin 27. 28. Part IV General discussion & summary

29. Chapter 9 General discussion and future perspectives 177 30. Chapter 10 Summary 205 31. Chapter 11 Samenvatting voor niet-medici 211 32. 33. Dankwoord 221 34. Curriculum vitae 227 35. 36. Affiliation of co-authors 229 37. List of publications 231 38. Portfolio 233 39.

7

Chapter 1

General introduction and Outline of the thesis

“In all maladies, those who are well nourished do best” Hippocrates, (460-377 BC.)

Introduction

1. Metabolism during critical illness 2. 3. Critically ill patients are in a catabolic state, characterized by three major metabolic 1 4. changes. First, there is an increased protein turnover with enhanced hepatic protein 5. synthesis and muscle protein breakdown1‑3. Second, during critical illness there is 6. increased lipolysis, or the breakdown of triglycerides to free fatty acids (FFA) and glyc- 7. erol2‑5. And third, insulin resistance causes hyperglycemia due to ongoing endogenous 8. glucose production (glycogenolysis and gluconeogenesis) and blunted peripheral 9. uptake6‑9. These metabolic derangements are caused by various endogenous and ex- 10. ogenous triggers, including increased inflammatory cytokines (Tumor Necrosis Factor 11. α, interleukin-1, interleukin-6, and interleukin-8), catecholamines and glucocorticoids, 12. all in which insulin resistance plays a central role9‑11. This response to injury is universal 13. and has been beneficial all through evolution at the acute onset of severe disease or 14. trauma. However, modern medicine has improved survival rate and critical illness has 15. become a process which lasts not just mere hours but can last for days or even weeks. 16. Early last century Sir David P. Cuthbertson described the short initial hypometabolic 17. “ebb” phase, followed by the prolonged hypermetabolic “flow” phase during adult 18. critical illness12. Persistent glucose overload and breakdown of skeletal muscle and 19. adipose tissue releasing large amounts of amino acids and FFA are the result. Un- 20. fortunately, although plasma substrate levels may be increased, their availability to 21. peripheral tissues may be blunted (because of factors such as insulin resistance and 22. inhibition of lipoprotein lipase), while plasma levels of other substrates (e.g. specific 23. amino acids, cholesterol) may be insufficient to meet metabolic demands13‑15. As a 24. result, these metabolic changes, beneficial in the initial phase from a teleological view- 25. point, become detrimental during prolonged critical illness. 26. 27. 28. Nutritional challenges on the PICU 29. 30. Pediatric patients are particularly vulnerable to prolonged metabolic stress, as their 31. muscle and fat mass is lower than in adults16, while they have higher resting energy 32. requirements17‑19. It has been shown extensively that children admitted to the pediatric 33. intensive care unit (PICU) accumulate substantial energy and protein deficits18, 20‑23, and 34. nutritional goals24 as well as glycemic control25 are still not met. The factors contributing 35. to inadequate nutritional and metabolic support are 1) poor knowledge and prescrip- 36. tion of adequate energy and protein amounts, 2) the lack of nutritional assessment, 37. 3) inability of adequate nutrient delivery, and 4) fear of causing iatrogenic complica- 38. tions25‑28. The lack of optimal nutritional and metabolic support does not acutely affect 39. the patient’s condition as obvious as inadequate mechanical ventilation, insufficient

11 Chapter 1

1. inotropic support or withholding the proper medication. However there is compelling 2. evidence that nutrient deficiencies and metabolic deteriorations have both short- as 3. well as long-term consequences. They lead to muscular and physical inability29, 30, 4. higher risk of infections30, prolonged mechanical ventilation31 and prolonged hospital 5. stay32, 33, and are associated with increased mortality33. Timely and adequate initiation 6. of nutritional and metabolic support in the PICU is therefore essential. Nonetheless, it 7. remains a poorly investigated area with little evidence for specific nutritional manage- 8. ment. Many recommendations and guidelines are based on small pediatric studies or 9. data extrapolated from adult studies34‑36. 10. Hence, a clear definition of optimal nutritional and metabolic support for critically ill 11. children is necessary. The overall goal of nutrition in critically ill conditions reaches 12. beyond the replacement of nutrient losses enabling individuals to maintain basal physi- 13. ological functions. Critically ill children should be fed the amount of nutrients to addi- 14. tionally counteract catabolism and supply sufficient substrate to help recover from the 15. disease process, while enabling normal growth and development. The macronutrient 16. requirements can be roughly divided between energy (glucose and fat) and protein (in 17. essence amino acid) requirements. To be able to create guidelines for these children, 18. nutritional goals should be based upon37; 1) nutritional therapy aiming to providing 19. adequate amounts of energy, especially when energy stores are depleted, 2) nutritional 20. therapy manipulating insulin metabolism, and 3) nutritional therapy aiming to conserve 21. or restore the body protein mass to enable growth and development. 22. The nutritional support is not simply providing more nutrients. Although inadequate 23. supply of nutrients can potentially lead to catabolism of protein and/or adipose tissue, 24. “overfeeding” also has several potential detrimental side effects. Providing excessive 25. amounts of energy can cause fat accumulation, dyslipidemia and exacerbation of 26. insulin resistance leading to hyperglycemia. Furthermore, excessive amounts of free 27. amino acids may also result in toxicity and long-term use of parenteral nutrition results 28. on cholestasis and liver failure38‑41. It has been demonstrated that L-methionine is 29. hepatotoxic42, whereas L-arginine, through L-ornithine production induces necrotizing 30. pancreatitis in rats43, and L-Lysine at large doses induces renal failure in dogs44. Provid- 31. ing the requirements in a tight balance to prevent underfeeding as well as overfeeding 32. is a major challenge. 33. 34. 35. Glucose metabolism and control 36. 37. Energy requirements of the human body, and especially the brain, depend on glu- 38. cose as the major fuel. Plasma glucose levels are the resultant of a balance between 39. exogenous glucose intake and endogenous glucose production (glycogenolysis and

12 Introduction

1. gluconeogenesis) on the one hand and glucose utilization (oxidation or storage as 2. glycogen and triglycerides) on the other. 3. Of the metabolic derangements occurring during critical illness, hyperglycemia has 1 4. gained the most attention in recent years. Hyperglycemia (> 110 mg.dL‑1 ~ > 6.1 5. mmol.L‑1) was considered a relatively benign response to stress and, although associ- 6. ated with increased morbidity and mortality in critically ill patients, no clear causal- 7. ity was shown45, 46. Critical illness hyperglycemia is predominantly caused by insulin 8. resistance, due to suppression of insulin receptor signalling induced by an increased 9. cytokine release11, which results in increased endogenous glucose production and 10. decreased peripheral glucose uptake. Additionally, the amount of glucose intake 11. provided on the intensive care unit is also independently related to hyperglycemia and 12. poor outcome47. Since a landmark paper showed that a tight glucose regimen with 13. insulin improved morbidity and mortality in an adult surgical Intensive Care Unit (ICU)48, 14. and subsequently in other populations49, the implementation of insulin administration 15. has been widely adopted on ICU’s50. Also in PICU’s, hyperglycemia is a frequent 16. complication which is associated with increased morbidity33, 51, 52. Large randomized 17. outcome studies of a tight glucose regimen with insulin therapy in the critically ill 18. pediatric population are limited to one recently published trial53 and one other trial 19. which started this year54, of which the results are eagerly anticipated for. The recently 20. published study showed an improved outcome53, despite an increase in hypoglycemia 21. (defined as blood glucose ≤ 2.2 mmol.L‑1) and severe hypoglycemia (defined as blood 22. glucose ≤ 1.7 mmol.L‑1). 23. Even though the evidence in favour of a tight glucose regimen in adult ICU’s is more 24. robust than the single study in children, especially in surgical ICU’s, there still remains 25. wide controversy regarding the replicability of earlier results and the increased inci- 26. dence of hypoglycemia49, 55, 56. Hypoglycemia as a consequence of a tight glucose regi- 27. men is of even greater importance in the critically ill pediatric population. The child’s 28. developing brain is more susceptible to hypoglycemia which can result in permanent 29. damage57‑59. Furthermore, young age is a risk factor for developing hypoglycemia, 30. especially when the child is ill60‑62. It is therefore essential to prevent hypoglycemia 31. in these children. Studies which present safe glucose control protocols or alternative 32. approaches to treat hyperglycemia in children are scarce63‑65. 33. In the mean time, fear of hypoglycemia discourages actual practice habits regard- 34. ing glycemic control in the PICU25. More insight in the risks of hyperglycemia, the 35. feasibility and safety of tight glucose protocols in the PICU, and strategies to prevent 36. hypoglycemia might help increase the awareness of the potential benefits of glycemic 37. control in critically ill children. 38. 39.

13 Chapter 1

1. Amino acid metabolism and requirements 2. 3. Proteins are made of amino acids as their building blocks. Almost the entire amino acid 4. pool (98%) resides in proteins, which are in a constant process of degradation and 5. synthesis, the so-called protein turnover. Protein turnover allows for a continuous flow 6. of amino acids available for necessary new proteins. In addition, specific amino acids 7. serve so-called “non-protein” actions. Amino acids act as precursors for the biosyn- 8. thesis of substrates such as nitric oxide and polyamines66, and as signalling molecules 9. in signal transduction pathways67, 68. They also regulate energy metabolism69, and help 10. protect against oxidative stress and protect endothelial function cells70, 71. 11. During critical illness, protein turnover is markedly increased to maintain the enhanced 12. hepatic protein synthesis, triggered by pro-inflammatory cytokines causing a cascade 13. stimulating the production of inflammatory/immune proteins72‑74. To provide substrate 14. for these proteins, amino acids are being mobilized from muscle, which is the largest 15. reservoir of peptide-bound and free amino acids15. Activation of the ubiquitin-protea- 16. some proteolytic pathway (UPP) triggers a profound catabolic response characterized 17. by increased muscle protein breakdown75, 76. Simultaneously, the synthesis of myofi- 18. brillar and sarcoplasmic muscle proteins is decreased due to changes in translation 19. initiation and reduction of translation efficiency77‑79. Despite the increased hepatic 20. protein synthesis, the disproportionate high loss of muscle protein results in a negative 21. whole body protein balance. 22. Currently, there is consensus on the total amount of protein that critically ill children 23. should receive35, 36. This is however based on limited evidence and is still subject to 24. debate28. To be able to develop guidelines for protein and amino acids requirements in 25. critically ill children various aspects need to be taken into consideration. 26. First, the ideal nutrition would provide every individual amino acid in amounts specifi- 27. cally required during a state of critically illness, not only to maintain protein balance but 28. also to maintain the abovementioned “non-protein” functions. 29. Second, amino acid utilization and metabolism is directed to different pathways de- 30. pending on the anatomical presentation80. Via the enteral route there is a first-pass 31. utilization of amino acids in the splanchnic area (liver, stomach, intestines and their 32. microbiota, pancreas and spleen), the so-called splanchnic uptake. Amino acids 33. provided through parenteral nutrition are not subjected to splanchnic uptake and 34. are presented to the liver through the arterial circulation instead of the portal venous 35. circulation. Therefore, requirements are different when provided via the enteral vs. 36. the parenteral route. Finally, protein and amino acid requirements differ with age. The 37. youngest individuals have the highest requirements even in health81‑83. Due to the age- 38. dependent difference in body composition, the mass of protein/amino acids available 39. through catabolism triggered during critical illness is lower in young children.

14 Introduction

1. To summarize, protein/amino acid requirements depend on the clinical condition, route 2. of administration and developmental stage of the patient. There is limited knowledge 3. of the specific amino acid requirements in critically ill children of different age groups 1 4. and on the amounts they are currently provided with. 5. 6. 7. Protein and energy interactions 8. 9. A close interrelationship exists between protein and energy metabolism. The World 10. Health Organisation has provided guidelines on protein-energy ratios to prevent 11. stunting and wasting in acute (9-11.5 energy% protein) and chronically malnourished 12. children (11-15% energy% protein)84. However, it is not known whether these interac- 13. tions change during critical illness. 14. A lack in energy supply (such as glucose) might enhance an already increased protein 15. catabolism during critical illness. However, an increase in the energy supply will not 16. promote nitrogen retention unless the protein supply is adequate, and conversely an 17. increased protein supply will be useless if energy is limiting. Moreover, free amino 18. acids provided in excessive amounts or released from proteolysis, and exceeding 19. the incorporation into proteins are oxidized and/or channelled into the gluconeogenic 20. pathway. So, although an increased amino acid intake might improve whole body 21. protein balance in critically ill children20, potentially there is a detrimental influence 22. on glucose metabolism. Finally, the increased lipolysis during critical illness results 23. in the excessive release of FFA, which exceeds their oxidation rates4, 5. Furthermore, 24. the ability of the utilization of substrates, such as glucose and FFA, in the peripheral 25. tissue is diminished due to insulin resistance and lipoprotein lipase inhibition4, 7, 13, 14. 26. The consequences of excessive FFA are decreased mitochondrial function, increased 27. insulin resistance, as well as an inhibition of glucose oxidation4, 85. The resulting effect 28. on the hampered energy metabolism potentially also influences protein metabolism. 29. These close interactions between the metabolism of the macronutrients, also in relation 30. to insulin resistance and metabolism during critical illness, are not fully understood. 31. 32. The role of insulin 33. Under physiological circumstances, the anabolic hormone insulin inhibits endogenous 34. glucose production and lipolysis, and improves whole body protein balance86, 87. More- 35. over, maintaining normoglycemia improves muscle protein anabolism independent of 36. insulin plasma levels88. It has been hypothesized that the anti-catabolic effects exerted 37. during a tight glucose regimen with intravenous insulin partially explain the beneficial 38. effects in critically ill patients. In critically ill adults insulin administration decreased 39. lipolysis and limited endogenous glucose production89, 90, but had no protein-sparing

15 Chapter 1

1. effects90. However, these studies were performed without providing amino acids to the 2. patients. Furthermore, there are age specific differences in insulin resistance following 3. an inflammatory or traumatic insult91. 4. Therefore, it remains unclear whether the anabolic response to insulin and nutrients 5. differs in the pediatric population, although it has been shown that there is a clear age- 6. related response to both nutrients and insulin on muscle protein synthesis in favour of 7. younger individuals92. 8. There is limited knowledge on the effects of different intakes of either glucose or amino 9. acids on one each other’s metabolism in critically ill children. Furthermore, whether 10. substrate metabolism differs in insulin resistant children treated with a tight glucose 11. regimen is currently unknown. These questions evidently contain essential information 12. to provide tools for nutritional and metabolic support in the PICU. 13. 14. 15. Outline of the thesis 16. 17. The ultimate goal of research related to pediatric intensive care is to improve morbidity 18. and mortality. As can be deducted from the introduction, nutrition for the critically ill 19. child is a major uncharted field in pediatric intensive care. Inherently, this means that 20. extra insight into the requirements and interactions of macronutrients potentially will 21. have widespread implications on the support given on the PICU. This notion has lead 22. to the following studies presented in this thesis. 23. The overall aim is to optimize nutritional and metabolic support in critically ill children. 24. With this in mind, the following hypotheses are tested: 25. - Hyperglycemia in critically ill children is caused by insulin resistance and can be 26. treated according to age-related glucose control protocols. 27. - Glucose and protein/amino acids are currently provided in inadequate amounts to 28. critically ill children of different age groups. 29. - Insulin exerts its anabolic properties also in critically ill children and can be used as 30. an additive tool to deflect catabolism. 31. 32. Part I Glucose metabolism 33. The first part of this thesis aimed to describe the consequences of hyperglycemia and 34. its treatment. Chapter 2 aimed to evaluate the causes and complications of hyper- 35. glycemia in critically ill children. Additionally we wanted to provide an overview on the 36. outcome and (non-)metabolic effects of insulin therapy in the PICU. 37. The following two chapters focus on the treatment and/or prevention of hyperglycemia 38. in infants. Chapter 3 sets out to evaluate the effectiveness and safety of insulin therapy 39. by means of a tight glucose protocol in infants less than one month old. Chapter 4

16 Introduction

1. describes a study investigating the effects of a reduced glucose intake in post-surgical 2. infants. The aim of this study was to determine whether this approach is a safe alterna- 3. tive for insulin therapy to prevent and/or treat hyperglycemia. Glucose homeostasis, 1 4. glucose kinetics and protein/amino acid catabolism were determined by means of 5. stable isotope tracer techniques. 6. 7. Part II Protein and amino acid metabolism 8. After the first part of the thesis the focus shifts from glucose to protein and amino 9. acid metabolism. In the two following chapters the effect of route of administration 10. are evaluated, and the age related differences are highlighted. Chapter 5 aimed to 11. provide insight into the ontogeny of the splanchnic uptake of the essential amino acid 12. methionine in three different age groups of critically ill children using stable isotope 13. tracer techniques. In chapter 6 we aimed to evaluate the actual parenteral intake of all 14. amino acids provided to children admitted to the PICU and to compare these with the 15. recommended amino acid intakes by the Institute of Medicine (IOM)93. 16. 17. Part III Protein and energy interactions 18. In the third part of this thesis part I and part II are taken a step further as it describes 19. the close interaction between the metabolism of glucose and protein/amino acids. 20. Two studies are presented which show how different amounts of glucose and amino 21. acid intake affect one another’s metabolism. Furthermore, these studies shed light on 22. the role of insulin resistance and insulin treatment on substrate metabolism. Chapter 23. 7 aimed to describe the effects of increased amino acids and hyperinsulinemia on 24. glucose, lipid and protein kinetics in insulin resistant septic adolescents. They were 25. provided total parenteral nutrition with two different amounts of amino acids. The study 26. used a combination of stable isotope tracer techniques and hyperinsulinemic eugly- 27. cemic clamps. In chapter 8 we set out to investigate the effect of different amounts of 28. glucose, amino acids and insulin infusion on albumin synthesis rates in post-surgical 29. infants and septic adolescents. 30. 31. Part IV General discussion and future perspectives 32. The final part of this thesis provides a general discussion and recommendations 33. for nutritional and metabolic support in addition to suggestions for future research 34. in chapter 9. A summary of our studies and major findings completes this thesis in 35. chapters 10 and 11. 36. 37. 38. 39.

17 Chapter 1

1. References

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18 Introduction

metabolism, nitrogen balance, and sub- 30. Villet S, Chiolero RL, Bollmann MD, et al. 1. strate utilization in critically ill children. Negative impact of hypocaloric feeding 2. Am J Clin Nutr 2001;​74(5):​664‑9. and energy balance on clinical outcome 3. 21. Hulst J, Joosten K, Zimmermann L, et al. in ICU patients. Clin Nutr 2005;​24(4):​ 1 4. Malnutrition in critically ill children: from 502‑9. 5. admission to 6 months after discharge. 31. Pichard C, Kyle UG, Morabia A, Perrier Clin Nutr 2004;​23(2):​223‑32. A, Vermeulen B, Unger P. Nutritional as- 6. 22. Hulst JM, van Goudoever JB, Zimmer- sessment: lean body mass depletion at 7. mann LJ, et al. The effect of cumulative hospital admission is associated with an 8. energy and protein deficiency on anthro- increased length of stay. Am J Clin Nutr 9. pometric parameters in a pediatric ICU 2004;​79(4):​613‑8. 10. population. Clin Nutr 2004;​23(6):​1381‑9. 32. Heyland DK, MacDonald S, Keefe L, 23. Oosterveld MJ, Van Der Kuip M, De Drover JW. Total parenteral nutrition in 11. Meer K, De Greef HJ, Gemke RJ. Energy the critically ill patient: a meta-analysis. 12. expenditure and balance following pe- Jama 1998;​280(23):​2013‑9. 13. diatric intensive care unit admission: a 33. Wintergerst KA, Buckingham B, Gandrud 14. longitudinal study of critically ill children. L, Wong BJ, Kache S, Wilson DM. Asso- 15. Pediatr Crit Care Med 2006;​7(2):​147‑53. ciation of hypoglycemia, hyperglycemia, 16. 24. de Neef M, Geukers VG, Dral A, Linde- and glucose variability with morbidity boom R, Sauerwein HP, Bos AP. Nutri- and death in the pediatric intensive care 17. tional goals, prescription and delivery in unit. Pediatrics 2006;​118(1):​173‑9. 18. a pediatric intensive care unit. Clin Nutr 34. Skillman HE, Wischmeyer PE. Nutrition 19. 2008;​27(1):​65‑71. therapy in critically ill infants and chil- 20. 25. Preissig CM, Rigby MR. A disparity dren. JPEN J Parenter Enteral Nutr 2008;​ 21. between physician attitudes and practice 32(5):​520‑34. regarding hyperglycemia in pediatric 35. Koletzko B, Goulet O, Hunt J, Krohn K, 22. intensive care units in the United States: Shamir R. 1. Guidelines on Paediatric 23. 23. a survey on actual practice habits. Crit Parenteral Nutrition of the European 24. Care 2010;​14(1):​R11. Society of Paediatric Gastroenterology, 25. 26. Hulst JM, Joosten KF, Tibboel D, van Hepatology and Nutrition (ESPGHAN) 26. Goudoever JB. Causes and conse- and the European Society for Clinical 27. quences of inadequate substrate supply Nutrition and Metabolism (ESPEN), to pediatric ICU patients. Curr Opin Clin Supported by the European Society of 28. Nutr Metab Care 2006;​9(3):​297-303. Paediatric Research (ESPR). J Pediatr 29. 27. Taylor B, Krenitsky J. Nutrition in the Gastroenterol Nutr 2005;​41 Suppl 2:​ 30. intensive care unit: year in review 2008- S1‑87. 31. 2009. JPEN J Parenter Enteral Nutr 2010;​ 36. Mehta NM, Compher C. A.S.P.E.N. Clini- 32. 34(1):​21‑31. cal Guidelines: nutrition support of the 28. Stroud M. Protein and the critically ill; do critically ill child. JPEN J Parenter Enteral 33. we know what to give? Proc Nutr Soc Nutr 2009;​33(3):​260‑76. 34. 2007;​66(3):​378‑83. 37. Sauerwein HP, Strack van Schijndel RJ. 35. 29. Pollack MM, Ruttimann UE, Wiley JS. Nu- Perspective: How to evaluate studies on 36. tritional depletions in critically ill children: peri-operative nutrition? Considerations 37. associations with physiologic instability about the definition of optimal nutri- 38. and increased quantity of care. JPEN J tion for patients and its key role in the Parenter Enteral Nutr 1985;​9(3):​309‑13. comparison of the results of studies on 39.

19 Chapter 1

nutritional intervention. Clin Nutr 2007;​ 48. van den Berghe G, Wouters P, Weekers 1. 26(1):​154‑8. F, et al. Intensive insulin therapy in the 2. 38. Kelly DA. Intestinal failure-associated critically ill patients. N Engl J Med 2001;​ 3. liver disease: what do we know today? 345(19):​1359‑67. 4. Gastroenterology 2006;​130(2 Suppl 1):​ 49. Griesdale DE, de Souza RJ, van Dam 5. S70‑7. RM, et al. Intensive insulin therapy and 39. Baker DH. Comparative species utiliza- mortality among critically ill patients: a 6. tion and toxicity of sulfur amino acids. J meta-analysis including NICE-SUGAR 7. Nutr 2006;​136(6 Suppl):​1670S-5S. study data. Cmaj 2009;​180(8):​821‑7. 8. 40. Garlick PJ. Assessment of the safety of 50. Wilson M, Weinreb J, Hoo GW. Intensive 9. glutamine and other amino acids. J Nutr insulin therapy in critical care: a review of 10. 2001;​131(9 Suppl):​2556S-61S. 12 protocols. Diabetes Care 2007;​30(4):​ 41. Munro IC, Renwick AG. The 5th workshop 1005‑11. 11. on the assessment of adequate intake of 51. Faustino EV, Apkon M. Persistent hyper- 12. dietary amino acids: general discussion glycemia in critically ill children. J Pediatr 13. 2. J Nutr 2006;​136(6 Suppl):​1755S-7S. 2005;​146(1):​30‑4. 14. 42. Moss RL, Haynes AL, Pastuszyn A, Glew 52. Srinivasan V, Spinella PC, Drott HR, Roth 15. RH. Methionine infusion reproduces liver CL, Helfaer MA, Nadkarni V. Associa- 16. injury of parenteral nutrition cholestasis. tion of timing, duration, and intensity of Pediatr Res 1999;​45(5 Pt 1):​664‑8. hyperglycemia with intensive care unit 17. 43. Rakonczay Z, Jr., Hegyi P, Dosa S, et al. mortality in critically ill children. Pediatr 18. A new severe acute necrotizing pancre- Crit Care Med 2004;​5(4):​329‑36. 19. atitis model induced by L-ornithine in 53. Vlasselaers D, Milants I, Desmet L, et al. 20. rats. Crit Care Med 2008;​36(7):​2117‑27. Intensive insulin therapy for patients in 21. 44. Asanuma K, Adachi K, Sugimoto T, Chiba paediatric intensive care: a prospective, S. Effects of lysine-induced acute renal randomised controlled study. Lancet 22. failure in dogs. J Toxicol Sci 2006;​31(2):​ 2009;​373(9663):​547‑56. 23. 23. 87‑98. 54. Macrae D, Pappachan J, Grieve R, et al. 24. 45. Capes SE, Hunt D, Malmberg K, Ger- Control of hyperglycaemia in paediatric 25. stein HC. Stress hyperglycaemia and intensive care (CHiP): study protocol. 26. increased risk of death after myocardial BMC Pediatr 2010;​10:​5. 27. infarction in patients with and without 55. Finfer S, Chittock DR, Su SY, et al. diabetes: a systematic overview. Lancet Intensive versus conventional glucose 28. 2000;​355(9206):​773‑8. control in critically ill patients. N Engl J 29. 46. Capes SE, Hunt D, Malmberg K, Pathak Med 2009;​360(13):​1283‑97. 30. P, Gerstein HC. Stress hyperglycemia 56. Wiener RS, Wiener DC, Larson RJ. Ben- 31. and prognosis of stroke in nondiabetic efits and risks of tight glucose control in 32. and diabetic patients: a systematic over- critically ill adults: a meta-analysis. Jama view. Stroke 2001;​32(10):​2426‑32. 2008;​300(8):​933‑44. 33. 47. der Voort PH, Feenstra RA, Bakker AJ, 57. Bober E, Buyukgebiz A. Hypoglycemia 34. Heide L, Boerma EC, der Horst IC. In- and its effects on the brain in children 35. travenous glucose intake independently with type 1 diabetes mellitus. Pediatr 36. related to intensive care unit and hospital Endocrinol Rev 2005;​2(3):​378‑82. 37. mortality: an argument for glucose toxic- 58. Vannucci RC, Vannucci SJ. Glucose me- 38. ity in critically ill patients. Clin Endocrinol tabolism in the developing brain. Semin (Oxf) 2006;​64(2):​141‑5. Perinatol 2000;​24(2):​107‑15. 39.

20 Introduction

59. Flykanaka-Gantenbein C. Hypoglycemia 70. Biolo G, Antonione R, De Cicco M. Glu- 1. in childhood: long-term effects. Pediatr tathione metabolism in sepsis. Crit Care 2. Endocrinol Rev 2004;​1 Suppl 3:​530‑6. Med 2007;​35(9 Suppl):​S591‑5. 3. 60. Solomon T, Felix JM, Samuel M, et al. 71. Wu G. Amino acids: metabolism, func- 1 4. Hypoglycaemia in paediatric admissions tions, and nutrition. Amino Acids 2009;​ 5. in Mozambique. Lancet 1994;​343(8890):​ 37(1):​1‑17. 149‑50. 72. Hasselgren PO, Pedersen P, Sax HC, 6. 61. Chaussain JL, Georges P, Calzada L, Job Warner BW, Fischer JE. Current con- 7. JC. Glycemic response to 24-hour fast cepts of protein turnover and amino acid 8. in normal children: III. Influence of age. J transport in liver and skeletal muscle dur- 9. Pediatr 1977;​91(5):​711‑4. ing sepsis. Arch Surg 1988;​123(8):​992‑9. 10. 62. Faustino EV, Bogue CW. Relationship 73. Vary TC. Regulation of skeletal muscle between hypoglycemia and mortality protein turnover during sepsis. Curr Opin 11. in critically ill children. Pediatr Crit Care Clin Nutr Metab Care 1998;​1(2):​217‑24. 12. Med 2010. 74. Vary TC, Kimball SR. Regulation of he- 13. 63. Preissig CM, Hansen I, Roerig PL, Rigby patic protein synthesis in chronic inflam- 14. MR. A protocolized approach to identify mation and sepsis. Am J Physiol 1992;​ 15. and manage hyperglycemia in a pediatric 262(2 Pt 1):​C445‑52. 16. critical care unit. Pediatr Crit Care Med 75. Lecker SH, Solomon V, Mitch WE, 2008;​9(6):​581‑8. Goldberg AL. Muscle protein breakdown 17. 64. Preissig CM, Rigby MR, Maher KO. Gly- and the critical role of the ubiquitin- 18. cemic control for postoperative pediatric proteasome pathway in normal and dis- 19. cardiac patients. Pediatr Cardiol 2009;​ ease states. J Nutr 1999;​129(1S Suppl):​ 20. 30(8):​1098-104. 227S-37S. 21. 65. Verhoeven JJ, Brand JB, van de Polder 76. Voisin L, Breuille D, Combaret L, et al. MM, Joosten KF. Management of hyper- Muscle wasting in a rat model of long- 22. glycemia in the pediatric intensive care lasting sepsis results from the activation 23. 23. unit; implementation of a glucose control of lysosomal, Ca2+ -activated, and 24. protocol. Pediatr Crit Care Med 2009;​ ubiquitin-proteasome proteolytic path- 25. 10(6):​648‑52. ways. J Clin Invest 1996;​97(7):​1610‑7. 26. 66. Morris SM, Jr. Arginine: beyond protein. 77. Lang CH, Frost RA, Vary TC. Regulation 27. Am J Clin Nutr 2006;​83(2):​508S-12S. of muscle protein synthesis during sepsis 67. Tokunaga C, Yoshino K, Yonezawa K. and inflammation. Am J Physiol Endocri- 28. mTOR integrates amino acid- and ener- nol Metab 2007;​293(2):​E453‑9. 29. gy-sensing pathways. Biochem Biophys 78. Cooney RN, Kimball SR, Vary TC. Regu- 30. Res Commun 2004;​313(2):​443‑6. lation of skeletal muscle protein turnover 31. 68. Tremblay F, Lavigne C, Jacques H, during sepsis: mechanisms and media- 32. Marette A. Role of dietary proteins and tors. Shock 1997;​7(1):​1‑16. amino acids in the pathogenesis of 79. Vary TC, Kimball SR. Sepsis-induced 33. insulin resistance. Annu Rev Nutr 2007;​ changes in protein synthesis: differential 34. 27:​293-310. effects on fast- and slow-twitch muscles. 35. 69. Jobgen WS, Fried SK, Fu WJ, Meininger Am J Physiol 1992;​262(6 Pt 1):​C1513‑9. 36. CJ, Wu G. Regulatory role for the argi- 80. Stoll B, Burrin DG. Measuring splanchnic 37. nine-nitric oxide pathway in metabolism amino acid metabolism in vivo using 38. of energy substrates. J Nutr Biochem stable isotopic tracers. J Anim Sci 2006;​ 2006;​17(9):​571‑88. 84 Suppl:​E60‑72. 39.

21 Chapter 1

81. Dewey KG, Beaton G, Fjeld C, Lonnerdal 88. Biolo G, De Cicco M, Lorenzon S, et al. 1. B, Reeds P. Protein requirements of in- Treating hyperglycemia improves skeletal 2. fants and children. Eur J Clin Nutr 1996;​ muscle protein metabolism in cancer 3. 50 Suppl 1:​S119-47; discussion S47‑50. patients after major surgery. Crit Care 4. 82. Pencharz PB, Ball RO. Amino acid needs Med 2008;​36(6):​1768‑75. 5. for early growth and development. J Nutr 89. Chambrier C, Laville M, Rhzioual Ber- 2004;​134(6 Suppl):​1566S-8S. rada K, Odeon M, Bouletreau P, Beylot 6. 83. te Braake FW, van den Akker CH, Riedijk M. Insulin sensitivity of glucose and fat 7. MA, van Goudoever JB. Parenteral amino metabolism in severe sepsis. Clin Sci 8. acid and energy administration to pre- (Lond) 2000;​99(4):​321‑8. 9. mature infants in early life. Semin Fetal 90. Whyte MB, Jackson NC, Shojaee- 10. Neonatal Med 2007;​12(1):​11‑8. Moradie F, et al. Metabolic effects of 84. WHO. Management of severe malnutri- intensive insulin therapy in critically ill 11. tion: a manual for physicians and other patients. Am J Physiol Endocrinol Metab 12. senior health workers. Geneva: World 2009;​298(3):​E697-705. 13. Health Organization 1999. 91. Zhai L, Messina JL. Age and tissue spe- 14. 85. Delarue J, Magnan C. Free fatty acids cific differences in the development of 15. and insulin resistance. Curr Opin Clin acute insulin resistance following injury. 16. Nutr Metab Care 2007;​10(2):​142‑8. J Endocrinol 2009;​203(3):​365‑74. 86. Bell JA, Fujita S, Volpi E, Cadenas JG, 92. Wilkes EA, Selby AL, Atherton PJ, et al. 17. Rasmussen BB. Short-term insulin and Blunting of insulin inhibition of proteolysis 18. nutritional energy provision do not stimu- in legs of older subjects may contribute 19. late muscle protein synthesis if blood to age-related sarcopenia. Am J Clin Nutr 20. amino acid availability decreases. Am J 2009;​90(5):​1343‑50. 21. Physiol Endocrinol Metab 2005;​289(6):​ 93. Food and Nutrition Board Institute of E999-1006. Medicine. Panel on Macronutrients. 22. 87. Fujita S, Rasmussen BB, Cadenas JG, Standing Committee on the Scientific 23. 23. Grady JJ, Volpi E. Effect of insulin on hu- Evaluation of Dietary Reference Intakes. 24. man skeletal muscle protein synthesis is Dietary Reference Intakes for Energy, 25. modulated by insulin-induced changes in Carbohydrate, Fiber, Fat, Fatty acids, 26. muscle blood flow and amino acid avail- Cholesterol, Proteins and Amino Acids. 27. ability. Am J Physiol Endocrinol Metab Washington DC: The National Press; 2006;​291(4):​E745‑54. 2005. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39.

22

Part I Glucose metabolism

Chapter 2

Insulin therapy in the Pediatric Intensive Care Unit

Sascha CAT Verbruggen Koen FM Joosten Leticia Castillo Johannes B van Goudoever

Clin Nutr. 2007;26(6):677-90.

Koen FM Joosten Sascha CAT Verbruggen Jennifer J Verhoeven

Lancet. 2009; 373(9673):1423-4 Chapter 2

1. Abstract 2. 3. Background & aims 4. Hyperglycemia is a major risk factor for increased morbidity and mortality in the inten- 5. sive care unit. Insulin therapy has emerged in adult intensive care units and several 6. pediatric studies are currently being conducted. This review discusses hyperglycemia 7. and the effects of insulin on metabolic and non-metabolic pathways, with a focus on 8. pediatric critical illness. 9. 10. Methods 11. A PubMed search was performed by a using the following keywords and limits 12. ((“hyperglycemia”[MeSH Terms] OR (“insulin resistance”[MeSH Major Topic]) AND 13. (“critical care”[MeSH Terms] OR “critical illness”[MeSH Terms])) in different combina- 14. tions with (“metabolism”[MeSH Terms] OR “metabolic networks and pathways”[MeSH 15. Terms]) and (“outcome”[All Fields]) and (“infant”[MeSH Terms] OR “child”[MeSH Terms] 16. OR “adolescent”[MeSH Terms]). Quality assessment of selected studies included clini- 17. cal pertinence, publication in peer-reviewed journals, objectivity of measurements and 18. techniques used to minimize bias. Reference lists of such studies were included. 19. 20. Results 21. The magnitude and duration of hyperglycemia are associated with increased morbidity 22. and mortality in the Pediatric Intensive Care Unit (PICU). Although a large randomized 23. trial showed clear benefit of insulin therapy, (severe) hypoglycemia occurred frequently 24. and warrants other multi-center studies and long-term follow-up. Evidence concerning 25. the mechanism and effect of insulin on glucose and lipid metabolism in pediatric criti- 26. cal illness is scarce. More is known about the positive effect on protein homeostasis, 27. especially in severely burned children. The effect in septic children is less clear and 28. seems age dependent. Some non-metabolic properties of insulin such as modula- 29. tion of inflammation, endothelial dysfunction and coagulopathy have not been fully 30. investigated in children. 31. 32. Conclusion 33. Further studies on the effect of insulin on morbidity and mortality as well as on the 34. mechanisms through which insulin exerts these effects are necessary in critically ill 35. children. We propose these studies to be conducted under standardized conditions 36. including precise definitions of hyperglycemia and rates of glucose intake. 37. 38. 39.

28 Insulin therapy in the PICU

1. Introduction 2. 3. Critical illness triggers an acute phase response, which is associated with severe 4. metabolic abnormalities. These changes are characterized by hyperglycemia, dyslipid- 5. emia and increased protein turnover1‑8. Recently, hyperglycemia has become the focus 6. of interest due to its relationship to outcome. The onset of “stress hyperglycemia” in 2 7. previously non-diabetic critically ill patients has been attributed to peripheral and he- 8. patic insulin resistance, certain drugs (steroids, catecholamines, thiazides), increased 9. stress hormone release and excessive dextrose administration via total parenteral 10. nutrition3, 5, 6. 11. Previously, hyperglycemia in these patients was considered to be part of the adaptive 12. stress response and even to be beneficial for the patient. It was thought to provide 13. glucose-dependent organs adequate energy supply and to compensate for volume 14. loss by increasing the osmotic pressure and so promote fluid movement into the 15. intravascular compartment5, 9, 10. As such, most clinicians accepted moderate hyper- 16. glycemia in these patients. In 2001 van den Berghe et al. described a regimen of tight 17. control of glucose levels in a surgical Intensive Care Unit. Strict insulin therapy reduced 18. overall in-hospital mortality (by 34 percent), and also reduced bacteremia, acute renal 19. failure, red-cell transfusions and critical-illness polyneuropathy11. 20. Since then, the use of insulin therapy has increased as standard therapy in adult inten- 21. sive care units and several studies are currently in progress in pediatric and neonatal 22. intensive care units. There is a definitive association between hyperglycemia and out- 23. come in the Pediatric Intensive Care Unit (PICU)12‑20. However, some questions need to 24. be answered before we can accept insulin administration as standard care in pediatric 25. critical illness. Are the beneficial effects and the safety of tight glucose control with 26. insulin in critically ill children the same as in adults? What specific mechanisms in the 27. developing pediatric population are responsible for the beneficial of glycemic control? 28. Finally, what metabolic and non-metabolic effects, other than glycemic control, does 29. insulin exert in critical illness? With this review we want to address these issues by 30. summarizing the pediatric literature concerning hyperglycemia in the PICU and the 31. relevant adult and animal studies concerning the different properties of insulin in criti- 32. cal illness. 33. 34. 35. Pediatric studies 36. 37. Hyperglycemia in critical illness 38. Several studies have investigated hyperglycemia in pediatric and neonatal ICU’s 39. in relation to morbidity and mortality. Most of these studies were done in the PICU

29 Chapter 2

1. with a diverse spectrum of diseases and injuries (Table 1)12‑17, 20. Except for the study 2. performed by Branco and colleagues all studies were retrospective. A total of 2599 3. children were studied with an average age of approximately 3 years, ranging from 4. 0 – 21 years. All studies report an association between hyperglycemia and morbidity 5. 6. Table 1. Summary of studies addressing hyperglycemia in Pediatric Intensive Care Unit. 7. Author Study Year Diagnosis Median age N= Glucose Threshold Outcome type yrs (range) intake mg/ mg/dl 8. kg/min (mmol/l) (range) 9. Branco 12 Prospec- 2005 Septic 2.8 57 2.8 178 (9.9) Peak glucose > 178 mg/dl 10. tive shock (0 – 7.1) (1.8 – 3.9) was associated with 2.59 fold 11. increase in risk of death Cochran 13 Retro- 2003 Head 4.0 170 n.m. 135, 267 Nonsurvivors had higher 12. spective trauma (0.1 – 17) (7.5, 14.8) admission glucose levels than 13. survivors (267 mg/dl vs. 135 mg/dl, p = 0.000) and an 14. admission blood glucose level 15. 200 mg/dl associated with worse neurological outcome 16. Faustino 14 Retro- 2005 Mixed 3.2 942 n.m. 120, 150 Maximum glucose levels 17. spective PICU (0.3 – 10.8) and 200 increased relative risk (RR) for (6.7, 8.3 dying (within 24 hrs > 150 mg/ 18. and 11.1) dl; RR 2.50; 95% confidence 19. interval (CI) 1.26 – 4.93 and within 10 days >120 mg/dl: RR 20. 5.68; 95% CI 1.38 – 23.47). 21. Srinivasan Retro- 2004 Mixed 6 152 n.m. 126 (7.0) Peak blood glucose in 15 spective PICU (1 – 12) nonsurvivors were higher (311 22. ± 115 vs. 205 ± 80 mg/dl, p < 23. 0.001) and lasted longer (71% ± 14% vs. 37% ± 5% of PICU 24. days, p < 0.001) 25. Wintergerst Retro- 2006 Mixed 2.8 1094 n.m. 65, 120, Both high peak glucose levels 16 spective PICU (0 – 21) 150, 200 (mortality rate 9.9% at > 200 26. 2 (3.6, 6.7, mg/dl, p < 0.0001 X test) 27. 8.3, 11.1) and low peak glucose levels (mortality rate 16.5% at < 65 2 28. mg/dl, p < 0.0001 X test) worsened outcome. LOS was 29. associated with hyper- and 30. hypoglycemia. Increased glucose variability had the 31. strongest association with 32. increased mortality and LOS. Yates 17 Retro- 2006 Post 0.3 184 n.m. 126 (7) Nonsurvivors had higher 33. spective cardiac (0.1 – 0.6) peak glucose levels (256 ± 34. surgery 196 vs. 179 ± 87 mg/dl, p < 0.001) and longer duration 35. of hyperglycemia > 126 mg/ dl (2.95 ± 2.28 vs. 1.19 ±1.3 36. days, p < 0.001). 37. Duration of hyperglycemia was associated with longer ventilator 38. use (p < 0.001, R2 = 0.10) and 2 39. LOS (p < 0.001, R = 0.21).

30 Insulin therapy in the PICU

Table 2. Summary of studies addressing hyperglycemia in Neonatal Intensive Care Unit 1. Author Study Year Diagnosis Gestational N= Intake Threshold Outcome 2. type age kcal/kg/day mg/dl (mmol/l) 3. Alaedeen 18 Retro- 2006 Septic Very Median 26 37 83 120 (6.7) Peak glucose levels over 120 4. spective Low Birth (23 – 34) (64 – 102) mg/dl associated with longer 5. Weight LOS (p = 0.006). Nonsurvivors infants had higher peak glucose levels 6. (241 ± 46 mg/dl vs. 141 ± 47 2 mg/dl, p < 0.0001). 7. Hall 19 Retro- 2004 Necrotizing Mean 29.1 88 n.m. 214 (11.9) Peak glucose levels over 214 8. spective enterocolitis SEM 0.5 mg/dl associated with a higher 9. late mortality > 10 admission (29% vs. 2%, p = 0.0009) and 10. LOS (p < 0.0001). 11. 12. (e.g. Length of stay (LOS), length of ventilator use, neurological outcome). Altogether 13. peak glucose levels and duration of hyperglycemia were higher in non-survivors and 14. two studies found hyperglycemia to be associated with a 2.5 fold increase in mortal- 15. ity risk. Wintergerst et al. also reported an increased mortality rate in children with 16. hypoglycemia16. 17. Two retrospective studies were performed in NICU patients, involving respectively 18. neonates with sepsis and necrotizing enterocolitis (Table 2)18, 19. A total of 125 neonates 19. were studied with an average gestational age of 28.2 weeks. Both studies show an 20. association between hyperglycemia, longer LOS and higher mortality rate. 21. Overall one can say that hyperglycemia is frequently present in critically ill children and 22. it is associated with an increased morbidity and mortality rate (Table 1, 2). However, 23. there are some controversial issues on hyperglycemia in critically ill children. First, 24. most studies are retrospective and could not demonstrate causality between glucose 25. levels and outcome measures, only associations were demonstrated. Second, only the 26. study performed by Branco and colleagues documented data on glucose intake (Table 27. 1)12. Moreover, excessive glucose intake might partially be responsible for hyperglyce- 28. mia. Finally, these retrospective studies mention different glucose thresholds, most of 29. them above 120 mg.dL‑1 (6.7 mmol.L‑1) (Table 1, 2). This makes it difficult to compare 30. the data and draw conclusions. Although various thresholds are reported, a glucose 31. level of 150 mg.dL‑1 (8.3 mmol.L‑1) seems to have the strongest association between 32. hyperglycemia and increased morbidity and mortality14‑16. A standardized approach 33. including definition of hyperglycemia, values that require intervention, length of hy- 34. perglycemia and rates of glucose intake, is necessary to make statements concerning 35. the risks of hyperglycemia and the necessity of insulin therapy. In anticipation for more 36. evidence we propose to use 150 mg.dL‑1 (8.3 mmol.L‑1) as a limit to study and treat 37. hyperglycemia children in the ICU with insulin. 38. 39.

31 Chapter 2

1. Insulin therapy 2. Initially studies on insulin therapy in the PICU focused primarily on burn patients. Pham 3. et al. compared in a retrospective study conventional treatment (n = 31) versus inten- 4. sive insulin treatment (n = 33) in severely burned children (Table 3). Their study reported 5. lower infection rates and even a positive association with survival in the intensive treat- 6. ment group. Patients who received intensive insulin therapy were more than five times 7. more likely to survive than those receiving conventional therapy (adjusted Odds Ratio 8. = 5.52; p = 0.06)21. 9. In the neonatal ICU there have been several smaller studies which looked at the effect 10. and safety of insulin therapy22‑27. These studies showed improved glycemic control 11. and all, except the study performed by Ng and colleagues, reported improved caloric 12. intake and even growth22‑27. Hypoglycemia (glucose < 40 mg.dL‑1 (< 2.2 mmol.L‑1)) 13. was the major complication, but this happened infrequently (0 – 5%) and no other 14. serious adverse effects were noted. To date, two large randomized studies have in- 15. vestigated the effect of tight glucose control with insulin on morbidity and mortality in 16. the pediatric population20, 28. The study by Beardsall and colleagues in very low birth 17. weight neonates where hyperglycemia was treated with insulin was discontinued early 18. because of an increased incidence of hypoglycemia and parenchymal abnormalities 19. detected on cranial ultrasound images in the infants treated with insulin28. Vlasselaers 20. and colleagues showed an improved short-term outcome, by decreasing PICU length 21. of stay (PICU LOS), attenuating the inflammatory response and even a decrease 22. 23. Table 3. Prospective studies of insulin therapy on critically ill children 24. Author Year Diag- Age yrs N= Intake Threshold Outcome nosis mg/dl (mmol/l) 25. Vlasselaers 20 2009 Various 1.3 700 20 – 80 kcal/ Conservative 11.9 Intensive insulin therapy decreased 26. (0.3 – 5.5) kg/day (214) duration of PICU stay, attenuated the 27. Intensive Infants inflammatory response as measured 0 - 1 y 2.8 - 4.4 with C-reactive protein on day 5, 28. (50 - 79) Children and decreased mortality from 6% in 1 - 16 y 3.9 - 5.5 the conservative group to 3% in the 29. (70 - 99) treatment group

30. Pham 21 2005 Burn 6.0 64 35 kcal/kg/ Conservative 200 Patients who received intensive 31. (1.0 – 11.2) day (11.1) insulin therapy were more than five Intensive 140 (7.8) times more likely to survive than those 32. receiving conventional therapy Intensive insulin therapy showed a 33. trend toward lower infection rates.

34. Jeschke 69 2004 Burn 5.4 28 1500 kcal/m2 180 mg/dl (10) in Insulin therapy improved lipid 35. (0.4 – 10.4) body surface + the treatment group metabolism and had a positive 1500 kcal/m2 and non-treatment effect on inflammatory response and 36. area burn group hepatic constitutive proteins. 37. Wu 114 2004 Burn 9.5 18 1500 kcal/m2 140 mg/dl (7.8) Prolonged hepatic acute phase (2 – 17) body surface + response decreased, no effect on 38. 1500 kcal/m2 constitutive proteins 39. area burn

32 Insulin therapy in the PICU

1. in mortality rate (3% vs. 6%)20. However, insulin therapy led to hypoglycemia (≤ 40 2. mg.dL‑1 ~ ≤ 2.2 mmol.L‑1) and severe hypoglycemia (≤ 31 mg.dL‑1 ~ ≤ 1.7 mmol.L‑1) 3. in 87 (25%) and 17 (5%) children, respectively. The high incidence of hypoglycemia 4. in the various studies with tight glucose control and insulin therapy remains a matter 5. of major concern. Neonates and young children are at particular risk for developing 6. hypoglycemia. Furthermore, as brain development is most profound in younger chil- 2 7. dren the complications caused by hypoglycemia are potentially highest in this patient 8. group. Hypoglycemia is a risk factor of thalamic infarction in neonates29. Hypoglycemia 9. has also been reported in the adult population during insulin therapy30‑33. In a large 10. Australian cohort, a U-shaped outcome curve showed that both high and low blood 11. glucose concentrations worsen outcome34. The reasons for hypoglycemia in the single 12. large randomized tight glucose study on the PICU are diverse. First, the target ranges 13. for plasma glucose levels concentrations were low (2.8 – 4.4 mmol.L‑1 for infants and 14. 3.9 – 5.6 mmol.L‑1 for children) in the study of Valsselaers and colleagues20. Second, 15. the infants’ glucose intake was lower than recommended (median 3.5 mg.kg‑1.min‑1 on 16. day 1 compared with 5.5 mg.kg‑1.min‑1 according to the guidelines from the European 17. Society for Pediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN))35. Third, 18. the treatment algorithm for insulin was adjusted by the nurses on the basis of their 19. experience. In our institution, hyperglycemia is treated (target glucose concentration 20. 4-8 mmol.L‑1) by use of a detailed stepwise algorithm to adjust insulin therapy without 21. occurrence of hypoglycemia (< 2.2 mmol.L‑1)36. Finally, the starting dose of insulin was 22. high (0.1 – 0.2 IU.kg‑1.h‑1), whereas a lower starting dose of 0.02 – 0.05 IU.kg‑1.h‑1 would 23. seem safer. An appropriate glucose intake, especially in infants, according to weight 24. and age, in combination with a step-wise (computer-assisted, or nurse driven) pro- 25. tocol and slightly higher glucose target concentrations might decrease the incidence 26. of hypoglycemia without losing the beneficial effects of insulin therapy in critically ill 27. children. However, in critically ill children, hypoglycemia is a frequent complication 28. even in the absence of insulin therapy15, 16, 37, 38. In a heterogeneous group of 1094 29. PICU patients a relatively higher prevalence of hypoglycemia of 18.6% was associated 30. with mortality16. In one study with 26 children with meningococcal septic shock (MSS) 31. they found lower glucose levels of 3.9 mmol.L‑1 (70 mg.dL‑1) with a range from 2.5 to 32. 6.5 mmol.L‑1 (45 to 117 mg.dL‑1) on admission in the non-surviving group, whereas in 33. both the surviving and non-surviving group the insulin-glucose ratios were normal37. 34. In another smaller study on 10 children diagnosed with meningococcal septic shock 35. (MSS) compared to a group diagnosed with meningococcal sepsis (MS), insulin and 36. insulin-glucose ratios remained lower in the meningococcal septic shock patients, 37. although both groups had hyperglycemia38. These studies indicate that in the acute 38. phase of septic shock in children hypoglycemia can occur and subsequently that the 39. mechanism of hyperglycemia is more complicated than mere insulin resistance.

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1. Hypoglycemia is a realistic complication as it occurs in a relatively high prevalence 2. in critically ill children without insulin therapy, especially in the acute phase of septic 3. shock. More large multi-center prospective studies are warranted to determine whether 4. the beneficial effects and safety of a tight glucose control shown in adults can be 5. extrapolated to the pediatric population. Adequate glucose intake depends on age and 6. clinical situation, such as prematurity and critical illness35. Therefore, especially in the 7. pediatric situation glucose intake is essential information to adequately interpret the lit- 8. erature. Younger children and neonates should be provided with higher glucose intake. 9. The ESPHGAN guidelines also state that critically ill children probably should receive 10. lower amounts of glucose (limited to 5 mg/kg/min) than their healthy peers35. However, 11. this recommendation was based on one study performed in burned children39. For 12. other disease processes (e.g. sepsis, trauma) in critically ill children no extensive stud- 13. ies regarding glucose requirements have been performed. These guidelines encourage 14. to correct caloric needs according to various disease processes35. We propose to use 15. standardized parenteral glucose intake in critically ill children on admission. In critically 16. ill children below 30 kg we propose to use a glucose intake of 4 – 6 mg.kg‑1.min‑1 and 17. in children above 30 kg we propose to use 2 – 4 mg.kg‑1.min‑1. Once tolerated in the 18. succeeding days enteral nutrition is preferred according to the child’s condition. 19. 20. Metabolic properties of insulin 21. 22. Lowering blood glucose levels 23. Critical illness is a condition where both hepatic and peripheral insulin resistance 24. contribute to the development of hyperglycemia. Although the effects of insulin are 25. peripheral (muscle and adipose tissue) and hepatic, the liver becomes more insulin 26. resistant than the peripheral tissues in critical illness (Figure 1)40, 41. The expression of 27. phosphoenolpyruvate carboxy kinase (PEPCK), which is the rate-limiting enzyme of 28. gluconeogenesis, is increased in critical illness due to elevated levels of cortisol and 29. catecholamines. Insulin is one of the most potent inhibitors of PEPCK42. However in 30. critically ill patients both the expression of PEPCK and hepatic glucokinase, which 31. controls glucose uptake and glycogen synthesis, remains unaltered by intensive insulin 32. therapy41, 43, 44. In contrast, insulin therapy increases expression in muscle of an insulin 33. dependent glucose transporter (GLUT-4), and of hexokinase–II, which is the rate limiting 34. enzyme of intracellular insulin-mediated glucose metabolism44. This suggests that in 35. critically ill patients exogenous insulin does not affect hepatic insulin resistance, but low- 36. ers blood glucose levels mainly through stimulation of skeletal muscle glucose uptake. 37. The explanation for the difference in insulin sensitivity between tissues remains unclear. 38. In order to understand how glycemic control with insulin proves to be beneficial in 39. critical illness one has to start with the effect of hyperglycemia in these patients.

34 Insulin therapy in the PICU

a 1. 2. 3. 4. 5. 6. 2 7. 8. 9. 10. 11. 12. 13. 14. 15. b 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. Figure 1. Glucose and lipid metabolism during critical illness (Panel A) and the effect of insulin 32. therapy on metabolism during critical illness (Panel B). * Gluconeogenic amino acids such as 33. Alanine. HSL = hormone sensitive lipase; LPL = lipoprotein lipase; VLDL = very-low-density lipoprotein; GLUT = glucose transporters; FFA = free fatty acids; PEPCK = phosphoenolpyruvate 34. carboxylase 35. 36. Hyper­glycemia has been shown to be more acutely toxic in critically ill patients than in 37. healthy individuals or even patients with diabetes41. Under physiological circumstances 38. glucose uptake in the liver is directly proportional to blood glucose concentration, 39. while peripheral uptake is insulin dependent. Hyperglycemia down regulates insulin

35 Chapter 2

1. independent glucose transporters (GLUT-1, GLUT-2 and GLUT-3). In contrast, critical 2. illness causes an over expression of these transporters and leads to glucose overload 3. in organ systems that express these transporters. The upregulation of these insulin 4. independent glucose transporters is seen in central and peripheral nervous system, 5. as well as in endothelial, hepatic and immune cells, renal tubules and gastrointes- 6. tinal mucosa41. Glucose overload causes more excessive glycolysis and oxidative 7. phosphorylation resulting in larger generation of oxygen radicals such as peroxynitrite 8. and superoxide generation in these cells. These reactive species cause mitochondrial 9. dysfunction and disturbed energy metabolism which leads to increased apoptosis and 10. might explain cellular and organ system failure in critically ill patients41, 45, 46. 11. Insulin therapy has been shown to protect the function and structure of the mitochon- 12. drial compartment45, 47. The mitochondrial changes described were seen in hepatic 13. and not in muscular cells. These data suggest that maintaining normoglycemia, rather 14. than a direct effect of the administered insulin, is responsible for the major beneficial 15. effects of a tight glucose control45, 48‑50. However, many of the proposed benefits of 16. normoglycemic control in critical illness are difficult to separate from the direct effects 17. of insulin on several cell and organ systems. Insulin administration in adults show a 18. definitive direct effect on critical illness polyneuropathy and bacteremia51. The potential 19. improvement of lipid and protein metabolism as we will describe next are also direct 20. effects of insulin. 21. The abovementioned data are from adult studies. Pediatric ICU patients in general 22. have a lower morbidity and mortality rate than adults admitted to an ICU52‑56. Still, it is 23. likely that the mechanisms of hyperglycemia also apply for the PICU population. 24. 25. Insulin improves lipid metabolism 26. Critical illness is a condition accompanied by dyslipidemia (Figure 1). This is character- 27. ized by two main disturbances in the serum lipid profile. The first is an increase in plasma 28. triglycerides and very low density lipoproteins (VLDL)57‑59. Increased lipolysis, de novo 29. fatty acid synthesis and decreased oxidation of fatty acids increase hepatic triglyceride 30. production and VLDL secretion. Additionally, inflammatory responses inhibit the activity 31. of endothelial lipoprotein lipase, an enzyme responsible for triglyceride clearance57‑59. 32. In critical illness there is lipolysis, or the breakdown of triglycerides to free fatty acids 33. (FFA) and glycerol, and this exceeds fatty acid oxidation rates. The consequences of 34. excessive FFA are decreased mitochondrial function and increased oxidative stress60. 35. Moreover, elevated FFA are implicated in generating insulin resistance, as well as 36. diminishing insulin secretion by the pancreas and inhibiting glucose oxidation8, 42, 60, 61. 37. Furthermore, there is a decreased cholesterol content of high density lipoprotein (HDL) 38. and low density lipoprotein (LDL)62, 63. The etiology of hypocholesterolemia in critical 39. illness is not completely understood and is probably multifactorial. Caloric and protein

36 Insulin therapy in the PICU

1. deficiency, decreased hepatic synthesis, redistribution due to capillary leakage and an 2. increased utilization in cell recovery and endotoxin scavenging are potential explana- 3. tions7, 62, 64, 65. 4. In physiological conditions triglycerides have an important role in energy provision and 5. the lipoproteins play a key role in innate immunity, in endotoxin scavenging and trans- 6. portation of lipid products64, 66. Besides decreasing the HDL and LDL concentrations, 2 7. acute illness also changes the composition of these lipoproteins57, 59. Modified LDL 8. particles are directly cytotoxic to the endothelium, whereas modified HDL particles lose 9. their anti-atherogenic and anti-inflammatory conditions57, 59. The substantial change in 10. lipid metabolism in critical illness has a potential negative effect on outcome50, 62, 63, 67. 11. Specifically, hypocholesterolemia in critically ill patients is associated with increased 12. mortality62, 63. The exact role of circulating triglycerides in critical illness remains am- 13. biguous as they reflect severity of illness, but also play an important role in energy 14. provision and altering endotoxins61, 68. 15. Intensive insulin therapy corrects the disturbed serum lipid profile in critical illness 16. (Figure 1). Foremost the increased triglycerides and FFA are almost completely sup- 17. pressed44, 60. Insulin administration in critically ill adults increased, but not fully restored, 18. concentrations of LDL and HDL cholesterol44. The effect was ascribed to a direct effect 19. of insulin. Moreover, a multivariate logistic regression analysis showed that the positive 20. effect of insulin therapy on dyslipidemia in critically ill patients surpassed the effect of 21. glycemic control on morbidity and mortality44. Future investigations are necessary to 22. fully understand the mechanism(s) responsible for the effects of insulin on dyslipidemia. 23. The dyslipidemia seen in critically ill adults is also seen in septic children and correlates 24. with disease severity and outcome. In fifty-seven children with severe meningococcal 25. disease (MS or MSS) very low levels of total cholesterol, HDL and LDL were found65. 26. Another study in ICU patients reported increased rates of lipolysis comparable with 27. adult studies2. In severely burned children insulin therapy was reported to decrease 28. serum triglycerides and free fatty acids (FFA). Because glucose levels were between 29. 120 to 180 mg.dL‑1 (6.7 - 10 mmol.L‑1), both in the insulin and in the control groups, the 30. decreased triglycerides and FFA can be ascribed to insulin per se (Table 3)69. The effect 31. of insulin on pediatric lipid metabolism needs to be further investigated. 32. 33. Improve anabolism and protein balance 34. Critical illness is a highly catabolic state leading to loss of lean body mass mainly 35. due to muscle wasting and a negative protein balance. In pediatric critical illness the 36. increase in catabolism and subsequent loss of lean body mass has been extensively 37. reported70‑76. Often PICU patients have already a deprived nutritional status compared 38. to healthy children75, 77. Malnutrition and muscle wasting lead to prolonged mechanical 39. ventilation, weakness and organ dysfunction1, 75, 76, 78, 79. After ICU discharge malnu-

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1. trition and loss of lean body mass persists, especially in younger children and neo- 2. nates73, 75‑77, 80, 81. Development of enteral and parenteral nutrition has made it possible 3. to provide large amounts of energy along with proteins to critically ill patients. How- 4. ever, increasing caloric intake or protein supply alone seems insufficient to prevent or 5. reverse muscle breakdown76, 82, 83. The catabolic state and the negative protein balance 6. are not solely explained by insufficient nutritional support but is aggravated by both 7. hormonal and inflammatory factors. The catabolic state in critical illness is due in part 8. to dysregulation of growth hormone (GH), insulin like growth factor (IGF) and insulin like 9. growth factor binding protein (IGFBP). Pro-inflammatory cytokines play a significant 10. role, inducing growth hormone resistance, decreased insulin like growth factor-I (IGF-I) 11. and muscle sensitivity to IGF-I84, 85. In pediatric critical illness comparable derange- 12. ments are reported2, 69, 74, 86, 87. These changes are not related to nutritional support and 13. a lack of IGF-I recovery was associated with poor outcome74, 86‑88. Insulin has anabolic 14. properties mediated largely by its suppressive effect of insulin growth factor binding 15. protein-1 (IGFBP-I), whereby increasing IGF-I concentration41. Insulin normalized low 16. IGF-I levels in patients with diabetes mellitus89. In pediatric burn patients, serum IGF-I 17. concentration was found to decrease 3–4 fold. Insulin therapy increased IGF-I and 18. its major binding protein IGFBP-3 but had no significant effect on serum IGFBP-1 19. (Table 3)69. Critically ill adults treated with insulin had a reduced incidence of critical 20. illness myopathy and prolonged mechanical ventilation occurred less frequently90. 21. However, the somatotropic axis in these adult patients was further suppressed with 22. insulin therapy and no obvious anabolic effects were observed, despite the beneficial 23. effects on outcome91. Others have reported no effect of insulin therapy on IGFBP-I 24. serum concentrations, which might explain why anabolic effects of insulin were not 25. obvious in these patients43. The mechanisms through which insulin therapy affects the 26. somatotropic axis remain to be established. 27. In critical illness whole body protein synthesis is enhanced although there is a net 28. negative protein balance due to increased protein breakdown, which aggravates 29. muscle wasting. There is an increased visceral (especially hepatic) protein synthesis 30. of acute phase proteins. Pro-inflammatory cytokines stimulate the production of acute 31. phase proteins at the expense of constitutive proteins (e.g. albumin, transferrin)92‑95. 32. Simultaneously, critical illness reduces protein synthesis and increases proteolysis 33. in muscle85, 96. Reduced muscular protein synthesis is caused by a profound reduc- 34. tion of the translation initiation pathway85, 96‑98. Mobilization of amino acids through 35. protein breakdown serves to provide substrate for gluconeogenesis, oxidation and 36. acute phase protein synthesis93, 94. In contrast with glucose and fat, no storage pool 37. for protein exists. As a result, the amino acid pool is maintained by increased protein 38. breakdown94. Proteolysis is regulated in several pathways, among these the ubiquitin- 39. proteasome pathway has been recognized to be important in critical illness79. Insulin

38 Insulin therapy in the PICU

1. has been shown to be important in the regulation of protein homeostasis, both in 2. healthy individuals as well as in critically ill patients. Insulin promotes a positive protein 3. balance provided there is a sufficient amino acid availability99, 100. Administration of 4. amino acids and insulin independently stimulate protein anabolism and a combination 5. of both appears to be more beneficial than either stimulus alone101. The mechanism 6. of insulin-stimulated protein synthesis is multifactorial and reflects gene transcription, 2 7. translation initiation and activation of pre-activated enzymes102. Insulin has also been 8. shown to inhibit protein breakdown and does this through reducing the ubiquitin-pro- 9. teasome dependent pathway of proteolysis102. The effect of insulin on muscle protein 10. synthesis in healthy individuals decreases with age103‑105. There appears to be a differ- 11. ent response to insulin-related protein synthesis in muscle between different disease 12. conditions. Adult septic patients have a lesser response compared to adult burn pa- 13. tients93, 106, 107. Severe thermal injury induces a pro-inflammatory acute phase response 14. for a prolonged period extending the catabolic period74. Additionally, in severely burned 15. patients hyperglycemia exacerbates muscle protein catabolism108, 109. The ability of in- 16. sulin therapy to stimulate muscle protein synthesis and increase wound healing in adult 17. burn patients has been well established109‑112. The effect of insulin therapy on muscle 18. protein metabolism and lean body mass has not been investigated in burned children. 19. However, high-carbohydrate feeding in severely burned children resulted in decreased 20. muscle protein breakdown, probably due to endogenous insulin response113. Insulin 21. therapy in burned children decreases prolonged hepatic acute phase protein levels, 22. but did not have a positive effect on the hepatic constitutive proteins114. Jeschke et 23. al. however did report increased synthesis of hepatic constitutive proteins (Table 3)69. 24. Insulin also attenuates the inflammatory response in burned children decreasing the 25. pro-inflammatory and increasing the anti-inflammatory response69, 115. 26. In adult sepsis, muscle protein synthesis is profoundly decreased and exogenous 27. insulin has little effect in reversing muscle catabolism93, 106, 107. The effect of sepsis and 28. insulin on protein synthesis in neonates has been studied in greater detail in an elegant 29. animal model with piglets116‑118. These studies showed that, compared to adults, sepsis 30. in neonatal pigs elicits only modest reduction in muscle protein synthesis118‑120. This dif- 31. ference can in part be explained by a maintained sensitivity to insulin stimulation121, 122. 32. Similar to adult models decreased muscle protein synthesis was accompanied by a 33. reduced translation efficiency96, 118‑120, 123. Endotoxemia lead to an increase in hepatic 34. protein synthesis in these piglets despite repression of translation initiation. Further- 35. more, this response was not influenced by insulin levels121. In healthy piglets insulin 36. also did not enhance protein synthesis in the liver124. In contrast, in endotoxemic rats 37. insulin significantly altered hepatic protein synthesis, increasing albumin and decreas- 38. ing c-reactive protein125. The effect of insulin on protein synthesis in neonatal sepsis 39. may be explained by processes other than translation initiation.

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1. Insulin-related protein homeostasis has been studied in two different conditions in criti- 2. cally ill children. In extreme low birth weight infants (ELBW) receiving no protein intake, 3. insulin reduced protein breakdown but did not enhance protein synthesis126. In neonates 4. on extracorporeal membrane oxygenation insulin did improve the net protein balance, 5. mainly by reduction of protein breakdown127, 128. The effect of insulin therapy on protein 6. metabolism and lean body mass remain to be investigated in septic children of different 7. age groups. Whether the anabolic properties of insulin can, at least partially, explain the 8. beneficial outcome of insulin therapy in critically ill patients remains to be established. 9. 10. Non-metabolic properties of insulin 11. 12. Improving inflammatory reactions 13. Tight glucose control with insulin therapy prevented serious infections and sepsis re- 14. lated multi organ failure and death in adults11, 31. Hyperglycemia impairs leukocyte func- 15. tion, most specifically the capacity of monocytes and neutrophils to phagocytose and 16. to generate oxidative bursts and decreases complement function129, 130. Also certain 17. distinctive pro-inflammatory alterations, such as an increase in early pro-inflammatory 18. cytokine levels (eg. TNF-α, IL-1b, IL-6), activation of the three major pro-inflammatory 19. transcription factors and elevation of complement products are seen during hypergly- 20. cemia130‑133. 21. Improving glucose levels would therefore improve inflammation in critically ill patients. 22. However insulin also emerges as a molecule with strong direct immunomodulating 23. properties, improving the innate immunity. Insulin has strong anti-inflammatory 24. properties, suppressing a range of pro-inflammatory products and some major pro- 25. inflammatory transcription factors (eg. NF-κB), while increasing anti-inflammatory 26. cytokines (such as IL-10)69, 115, 134‑136. 27. This coincides with a suppression of the hepatic acute phase response, as shown 28. by a reduced circulating C-reactive protein (CRP) seen in patients treated with insu- 29. lin69. Whereas CRP is an acute phase protein used as an indicator for inflammation, 30. mannose-binding lectin (MBL) is an acute phase protein related to innate immunity and 31. host defense by recognizing and initiating opsonization. Low MBL levels may predict 32. poor outcome and were increased significantly by intensive insulin therapy137. Whether 33. this fully explains the reduction in bacteremia and sepsis related illness seen in these 34. patients, remains to be established. 35. The effect of insulin therapy on immunomodulation has not been investigated in 36. pediatric critical illness. One study reported a significant inversed relation between en- 37. dogenous insulin and an inflammatory response in children with severe meningococcal 38. disease38. Further investigations are needed to determine the effect of insulin therapy 39. on immunomodulatory processes of pediatric critical illness.

40 Insulin therapy in the PICU

1. Insulin improves vascular endothelial function and coagulopathy 2. Multi organ failure and death in critical illness is largely attributable to vascular endo- 3. thelial dysfunction138, 139. The endothelium controls (micro-) vascular tone and blood 4. flow and regulates permeability to nutrients and bioactive substrates140. Critical illness 5. causes endothelial dysfunction via reactive oxygen species (ROS) and pro-inflammatory 6. cytokines and growth factors, such as vascular endothelial growth factor (VEGF)138, 139. 2 7. Endothelial dysfunction then leads to coagulopathy, decreased organ perfusion and 8. cellular ischemia138‑141. Nitric oxide (NO) plays an essential role in endothelial function139. 9. Its function however is very delicate, as both low as well as high concentrations are 10. detrimental. Under normal circumstances the endothelium generates low concentra- 11. tions of NO through endothelial nitric oxide synthase (eNOS). Critical illness changes 12. NO concentrations and effects through several mechanisms. First, it induces very high 13. concentrations of NO through inducible nitric oxide synthase (iNOS). Uncontrolled 14. high concentrations increase cellular and vascular injury through inflammation, ROS 15. and VEGF142, 143. Secondly, asymmetric dimethylarginine (ADMA) concentrations are 16. increased in critical ill and insulin resistant patients144‑147. ADMA is produced by meth- 17. ylation of arginine parts of proteins and is released to the free amino acid pool during 18. proteolysis. ADMA inhibits NOS, directly and non-specifically144, 145, 147, 148. High ADMA 19. levels were recognized as an independent risk factor for ICU mortality144. The probable 20. mechanism by which increased ADMA attributes to adverse outcome is suppression of 21. endothelial NOS and interference with physiologic functions of NO145. 22. Insulin therapy maintaining normoglycemia has been shown to protect the endothelium 23. and thus contributed to prevention of multi organ failure and poor outcome in critically 24. ill patients143. This favorable effect of insulin can substantially be ascribed to improve- 25. ment of the delicate NO balance. Insulin induced a dose-dependent induction of eNOS 26. in human aortic cells (and possibly arterial/endothelial cells)149. This was in contrast 27. with another study where insulin reduced plasma NO concentrations by suppressing 28. iNOS expression, while eNOS expression was not altered143. The latter study however 29. could not exclude a beneficial effect on eNOS expression as tissue biopsies were only 30. taken from non-survivors. Another effect of insulin on the NO balance is the reduction of 31. ADMA levels in critically ill patients and this effect contributed to the beneficial effects 32. of insulin therapy150. Insulin causes this modulation probably by reduced protein break- 33. down, preservation of the degrading enzyme dimethylarginine dimethylaminohydrolase 34. (DDAH) and increased uptake. Finally, insulin also reduces ROS, such as superoxide 35. which binds with NO to form peroxynitrite151, 152. All these effects are beneficial to the 36. endothelial function and thus organ function and patient outcome. 37. Another consequence of endothelial damage in critical illness is an increased co- 38. agulopathy141. Hyperglycemia induces a pro-thrombotic state with increased vascular 39. constriction, oxidative stress and elevated platelet aggregation153. Insulin improves

41 Chapter 2

1. coagulopathy in critically ill patients. Insulin has anti-thrombotic and fibrinolytic effects 2. by suppressing plasma tissue factor and plasminogen activator inhibitor-1152, 154. Endo- 3. thelial dysfunction and vascular complications are seen in children with diabetes155. The 4. detrimental effects of poor glycemic control on oxidative stress have been reported in 5. pediatric studies. Markers for endothelial (intracellular cell adhesion molecule (ICAM), 6. vascular endothelial growth factor (VEGF) as well as oxidative stress (Malondialde- 7. hyde, MDA) were increased in poorly controlled diabetic children156‑158. Furthermore, 8. anti-oxidant capacity and glutathione-peroxidase were significantly lower in diabetic 9. children compared with healthy children158. However, the impact of insulin therapy on 10. endothelial function, nitric oxide balance and coagulopathy in critically ill children has 11. not been reported yet. 12. 13. 14. Conclusion 15. 16. The beneficial effect of insulin administration on the outcome in critically ill patients 17. has led to widespread implementation of this therapy in the ICU. Since then, our 18. knowledge of several changes occurring in metabolic pathways during critical illness 19. has increased significantly. However, these mechanistic and therapeutic approaches 20. cannot be simply extrapolated to the pediatric population without validation. Most 21. of the changes in metabolic pathways occurring in adult critical illness are likely to 22. occur in critically ill children as well. However, some important differences are ap- 23. parent for an attending pediatric intensivist in the treatment of a critically ill child and 24. many questions remain to be answered. Hyperglycemia has been shown to be associ- 25. ated with increased morbidity and mortality in a diverse clinical spectrum of pediatric 26. critical illness. Although a recent large randomized trial showed clear improvement of 27. short-term outcome, the high frequency of (severe) hypoglycemia remains reason of 28. concern. The results of other prospective studies in children on a tight glucose regimen 29. are in anticipation to indicate whether the positive effects shown in adult patients will 30. also count for the pediatric population. Moreover, the responsible pathways explaining 31. the beneficial outcome of insulin treatment in critically ill patients need to be elucidated 32. and compared with pediatric research data. Finally, the effects of various metabolic and 33. non-metabolic properties of insulin in critically ill children of different age groups need 34. to be studied. Although the effect of insulin on protein homeostasis and dyslipidemia 35. in critical illness seems promising, the possible consequences for nutritional support 36. need to be resolved. Furthermore, these metabolic properties are closely related to 37. endothelial function and inflammation, both of which are affected by insulin as well. 38. As pediatric critical illness involves changes in these pathways as well, these effects 39. should also be addressed in future studies.

42 Insulin therapy in the PICU

1. To achieve comparable results and answers, these studies should use and report 2. glucose and energy requirements adapted to age and clinical condition. In anticipation 3. of further evidence on thresholds of blood glucose levels, we propose to treat children 4. in the ICU with insulin using a blood glucose threshold of 150 mg.dL‑1 (8.3 mmol.L‑1) 5. and are currently conducting studies with these thresholds. 6. 2 7. 8. References 9. 10. 1. Biolo G, Toigo G, Ciocchi B, et al. 10. Preiser JC, Devos P, Van den Berghe G. 11. Metabolic response to injury and sepsis: Tight control of glycaemia in critically ill changes in protein metabolism. Nutrition patients. Curr Opin Clin Nutr Metab Care 12. 1997;​13(9 Suppl):​52S-7S. 2002;​5(5):​533‑7. 13. 2. Cogo PE, Carnielli VP, Rosso F, et al. Pro- 11. van den Berghe G, Wouters P, Weekers 14. tein turnover, lipolysis, and endogenous F, et al. Intensive insulin therapy in the 15. hormonal secretion in critically ill chil- critically ill patients. N Engl J Med 2001;​ 16. dren. Crit Care Med 2002;​30(1):​65‑70. 345(19):​1359‑67. 17. 3. Klein GW, Hojsak JM, Rapaport R. 12. Branco RG, Garcia PC, Piva JP, Casartelli Hyperglycemia in the pediatric intensive CH, Seibel V, Tasker RC. Glucose level 18. care unit. Curr Opin Clin Nutr Metab Care and risk of mortality in pediatric septic 19. 2007;​10(2):​187‑92. shock. Pediatr Crit Care Med 2005;​6(4):​ 20. 4. Mizock BA. Metabolic derangements in 470‑2. 21. sepsis and septic shock. Crit Care Clin 13. Cochran A, Scaife ER, Hansen KW, 22. 2000;​16(2):​319-36, vii. Downey EC. Hyperglycemia and out- 23. 5. Mizock BA. Alterations in fuel metabolism comes from pediatric traumatic brain in critical illness: hyperglycaemia. Best injury. J Trauma 2003;​55(6):​1035‑8. 24. Pract Res Clin Endocrinol Metab 2001;​ 14. Faustino EV, Apkon M. Persistent hyper- 25. 15(4):​533‑51. glycemia in critically ill children. J Pediatr 26. 6. Montori VM, Bistrian BR, McMahon MM. 2005;​146(1):​30‑4. 27. Hyperglycemia in acutely ill patients. 15. Srinivasan V, Spinella PC, Drott HR, Roth 28. Jama 2002;​288(17):​2167‑9. CL, Helfaer MA, Nadkarni V. Associa- 7. van Leeuwen HJ, Heezius EC, Dallinga tion of timing, duration, and intensity of 29. GM, van Strijp JA, Verhoef J, van Kessel hyperglycemia with intensive care unit 30. KP. Lipoprotein metabolism in patients mortality in critically ill children. Pediatr 31. with severe sepsis. Crit Care Med 2003;​ Crit Care Med 2004;​5(4):​329‑36. 32. 31(5):​1359‑66. 16. Wintergerst KA, Buckingham B, Gandrud 33. 8. Wolfe RR, Martini WZ. Changes in inter- L, Wong BJ, Kache S, Wilson DM. Asso- 34. mediary metabolism in severe surgical ciation of hypoglycemia, hyperglycemia, illness. World J Surg 2000;​24(6):​639‑47. and glucose variability with morbidity 35. 9. Gore DC, Chinkes D, Heggers J, Hern- and death in the pediatric intensive care 36. don DN, Wolf SE, Desai M. Association of unit. Pediatrics 2006;​118(1):​173‑9. 37. hyperglycemia with increased mortality 17. Yates AR, Dyke PC, 2nd, Taeed R, et 38. after severe burn injury. J Trauma 2001;​ al. Hyperglycemia is a marker for poor 39. 51(3):​540‑4. outcome in the postoperative pediatric

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metabolism in neonates on extracorpo- 137. Hansen TK, Thiel S, Wouters PJ, Chris- 1. real life support. Ann Surg 2006;​244(4):​ tiansen JS, Van den Berghe G. Intensive 2. 536‑44. insulin therapy exerts antiinflamma- 3. 128. Agus MS, Javid PJ, Ryan DP, Jaksic T. tory effects in critically ill patients and 4. Intravenous insulin decreases protein counteracts the adverse effect of low 5. breakdown in infants on extracorporeal mannose-binding lectin levels. J Clin membrane oxygenation. J Pediatr Surg Endocrinol Metab 2003;​88(3):​1082‑8. 6. 2004;​39(6):​839-44; discussion ‑44. 138. Aird WC. The role of the endothelium in 7. 129. Weekers F, Giulietti AP, Michalaki M, et severe sepsis and multiple organ dys- 8. al. Metabolic, endocrine, and immune function syndrome. Blood 2003;​101(10):​ 9. effects of stress hyperglycemia in a rab- 3765‑77. 10. bit model of prolonged critical illness. 139. Vallet B. Bench-to-bedside review: endo- Endocrinology 2003;​144(12):​5329‑38. thelial cell dysfunction in severe sepsis: 11. 130. Turina M, Fry DE, Polk HC, Jr. Acute a role in organ dysfunction? Crit Care 12. hyperglycemia and the innate immune 2003;​7(2):​130‑8. 13. system: clinical, cellular, and molecular 140. Vallet B. Endothelial cell dysfunction and 14. aspects. Crit Care Med 2005;​33(7):​ abnormal tissue perfusion. Crit Care Med 15. 1624‑33. 2002;​30(5 Suppl):​S229‑34. 16. 131. Dandona P. The link between insulin 141. Vallet B, Wiel E. Endothelial cell dysfunc- resistance syndrome and inflammatory tion and coagulation. Crit Care Med 17. markers. Endocr Pract 2003;​9 Suppl 2:​ 2001;​29(7 Suppl):​S36‑41. 18. 53‑7. 142. Cersosimo E, DeFronzo RA. Insulin 19. 132. Dandona P, Aljada A, Dhindsa S, Garg resistance and endothelial dysfunction: 20. R. Insulin as an anti-inflammatory and the road map to cardiovascular diseases. 21. antiatherosclerotic hormone. Clin Cor- Diabetes Metab Res Rev 2006;​22(6):​ nerstone 2003;​Suppl 4:​S13‑20. 423‑36. 22. 133. Turina M, Miller FN, Tucker C, Polk HC. 143. Langouche L, Vanhorebeek I, Vlasselaers 23. Effects of hyperglycemia, hyperinsu- D, et al. Intensive insulin therapy protects 24. linemia, and hyperosmolarity on neu- the endothelium of critically ill patients. J 25. trophil apoptosis. Surg Infect (Larchmt) Clin Invest 2005;​115(8):​2277‑86. 26. 2006;​7(2):​111‑21. 144. Nijveldt RJ, Teerlink T, Van Der Hoven 27. 134. Su F, Wang Z, Bruhn A, Verdant C, Cai Y, B, et al. Asymmetrical dimethylarginine Vincent JL. Euglycemic hyperinsulinemia (ADMA) in critically ill patients: high 28. in severe sepsis and septic shock. Eur plasma ADMA concentration is an inde- 29. Surg Res 2006;​38(6):​495-502. pendent risk factor of ICU mortality. Clin 30. 135. Jeschke MG, Klein D, Bolder U, Nutr 2003;​22(1):​23‑30. 31. Einspanier R. Insulin attenuates the 145. Nijveldt RJ, Teerlink T, van Leeuwen 32. systemic inflammatory response in endo- PA. The asymmetrical dimethylarginine toxemic rats. Endocrinology 2004;​145(9):​ (ADMA)-multiple organ failure hypoth- 33. 4084‑93. esis. Clin Nutr 2003;​22(1):​99-104. 34. 136. Soop M, Duxbury H, Agwunobi AO, 146. Stuhlinger MC, Abbasi F, Chu JW, et al. 35. et al. Euglycemic hyperinsulinemia Relationship between insulin resistance 36. augments the cytokine and endocrine and an endogenous nitric oxide synthase 37. responses to endotoxin in humans. Am inhibitor. Jama 2002;​287(11):​1420‑6. 38. J Physiol Endocrinol Metab 2002;​282(6):​ 147. Siroen MP, Teerlink T, Nijveldt RJ, Prins E1276‑85. HA, Richir MC, van Leeuwen PA. The 39.

50 Insulin therapy in the PICU

clinical significance of asymmetric di- 153. Dandona P, Aljada A. Advances in diabe- 1. methylarginine. Annu Rev Nutr 2006;​26:​ tes for the millennium: diabetes and the 2. 203‑28. endothelium. MedGenMed 2004;​6(3 3. 148. Nijveldt RJ, Teerlink T, Siroen MP, van Suppl):​6. 4. Lambalgen AA, Rauwerda JA, van 154. Aljada A, Ghanim H, Mohanty P, Kapur 5. Leeuwen PA. The liver is an important N, Dandona P. Insulin inhibits the pro- organ in the metabolism of asymmetrical inflammatory transcription factor early 6. dimethylarginine (ADMA). Clin Nutr 2003;​ growth response gene-1 (Egr)-1 expres- 2 7. 22(1):​17‑22. sion in mononuclear cells (MNC) and 8. 149. Aljada A, Dandona P. Effect of insulin on reduces plasma tissue factor (TF) and 9. human aortic endothelial nitric oxide syn- plasminogen activator inhibitor-1 (PAI-1) 10. thase. Metabolism 2000;​49(2):​147‑50. concentrations. J Clin Endocrinol Metab 150. Siroen MP, van Leeuwen PA, Nijveldt RJ, 2002;​87(3):​1419‑22. 11. Teerlink T, Wouters PJ, Van den Berghe 155. Pomilio M, Mohn A, Verrotti A, Chiarelli 12. G. Modulation of asymmetric dimethyl- F. Endothelial dysfunction in children 13. arginine in critically ill patients receiving with type 1 diabetes mellitus. J Pediatr 14. intensive insulin treatment: a possible Endocrinol Metab 2002;​15(4):​343‑61. 15. explanation of reduced morbidity and 156. Erciyas F, Taneli F, Arslan B, Uslu Y. 16. mortality? Crit Care Med 2005;​33(3):​ Glycemic control, oxidative stress, and 504‑10. lipid profile in children with type 1 diabe- 17. 151. Dandona P, Mohanty P, Chaudhuri A, tes mellitus. Arch Med Res 2004;​35(2):​ 18. Garg R, Aljada A. Insulin infusion in 134‑40. 19. acute illness. J Clin Invest 2005;​115(8):​ 157. Seckin D, Ilhan N, Ilhan N, Ertugrul S. 20. 2069‑72. Glycaemic control, markers of endothelial 21. 152. Dandona P, Aljada A, Mohanty P, et cell activation and oxidative stress in chil- al. Insulin inhibits intranuclear nuclear dren with type 1 diabetes mellitus. Diabe- 22. factor kappaB and stimulates IkappaB tes Res Clin Pract 2006;​73(2):​191‑7. 23. in mononuclear cells in obese subjects: 158. Varvarovska J, Racek J, Stetina R, et al. 24. evidence for an anti-inflammatory effect? Aspects of oxidative stress in children 25. J Clin Endocrinol Metab 2001;​86(7):​ with type 1 diabetes mellitus. Biomed 26. 3257‑65. Pharmacother 2004;​58(10):​539‑45. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39.

51

Chapter 3

The efficacy and safety of a tight glucose control protocol in critically ill term infants

Sascha CAT Verbruggen Lonneke J Landzaat Irwin KM Reiss Johannes B van Goudoever Koen FM Joosten

Submitted Chapter 3

1. Abstract 2. 3. Objectives 4. Treating hyperglycemia in critically ill children improved outcome, despite an increased 5. incidence of hypoglycemia, especially in infants. We evaluated the effectivity and 6. safety of a tight glucose protocol in critically ill term infants. 7. 8. Design 9. Term hyperglycemic infants (0 – 28 days) treated with a tight glucose protocol during a 10. 3.5-year period in a tertiary PICU were retrospectively analyzed. 11. 12. Participants 13. 73 term hyperglycemic infants (age 0 days (0-6 d), 3.2 ± 0.8 kg, PRISM III 16 (11 – 20)) 14. were included for analysis. 15. 16. Main outcome measures 17. Protocol efficacy and incidence of hypoglycemia. 18. 19. Results 20. The initial glucose level was 11.1 (9.6-15.2) mmol.L‑1, and normoglycemia (< 8 mmol.L‑1) 21. was reached within 5.3h (range 1 – 25h) with an overall treatment duration of 27h (10 22. – 57h). Seven hypoglycemic incidents (5 times ≤ 2.2 mmol.L‑1, 2 times < 1.7 mmol.L‑1) 23. occurred in five (6.7%) infants, with no effect on clinical outcome. Eighteen infants 24. died (25%); one infant who had developed hypoglycemia before and 17 infants without 25. hypoglycemia died. Three hypoglycemic incidents were directly explained due to a 26. protocol violation. One hypoglycemic incident occurred in the onset of sepsis, while no 27. apparent cause was identified for three hypoglycemic incidents. 28. 29. Conclusions 30. Our glucose protocol was effective, but hypoglycemia occurred more frequently than 31. in older children reported previously. Potential differences in glucose and insulin me- 32. tabolism in term infants appear to justify additional safety approaches, while awaiting 33. further studies assessing the benefits of tight glucose protocols in this population. 34. Meanwhile, we decreased the initial insulin starting doses in our protocol. 35. 36. 37. 38. 39.

54 Tight glucose control in infants

1. Introduction 2. 3. Hyperglycemia has been associated with increased morbidity and mortality in criti- 4. cally ill adults1, children2 and neonates3. A tight glucose regimen with insulin therapy 5. improved outcome in critically ill children4, despite the occurrence of hypoglycemia 6. (≤ 2.2 mmol.L‑1 (40 mg.dL‑1)) and severe hypoglycemia (≤ 1.7 mmol.L‑1 (31 mg.dL‑1)) in 7. 25% and 5% of the children, predominantly (>80%) in infants4. A study in hypergly- 8. cemic very low birth weight (VLBW) neonates was discontinued early because of an 9. increased incidence of hypoglycemia (29% vs. 17%) and parenchymal abnormalities 3 10. detected with cranial ultrasound in those treated with insulin5. 11. Young age has been recognized for long as a risk factor for developing hypoglycemia6, 7. 12. Furthermore, the young child’s developing brain is more susceptible to hypoglycemia 13. and may result in permanent damage8‑10. 14. Glucose control protocols designed to maintain normoglycemia while minimizing 15. glucose variability and hypoglycemic incidents are therefore of the essence in this 16. population. Simple model-based, computer-assisted or nurse driven protocols have 17. been reported in critically ill adults11‑16 and children17‑19. To date no studies have shown 18. feasibility and safety of these protocols specifically in critically ill newborn infants. 19. Therefore we evaluated the efficacy (time to achieve normoglycemia, duration of 20. therapy) and safety (hypoglycemic incidents, protocol violations) of a tight glucose 21. protocol in hyperglycemic term infants less than 28 days old. 22. 23. 24. Methods 25. 26. Patients 27. The Pediatric Intensive Care Unit (PICU) at Erasmus MC-Sophia Children’s hospital in 28. Rotterdam, The Netherlands is a 34-bed medical and surgical ICU. Term infants less 29. than 28 days old, admitted to our PICU from January 2006 to September 2009, and 30. treated with our tight glucose protocol were retrospectively evaluated. 31. 32. Insulin Protocol 33. The term infants received glucose according to our protocol (4-6 mg.kg‑1.min‑1) and 34. were treated with a step-wise nurse driven glucose control protocol which was previ- 35. ously published19. Briefly, infants with sepsis, multiple organ failure, and/or receiving 36. mechanical ventilation were treated after two consecutive blood glucose levels > 8 37. mmol.L ‑1 (>145 mg.dL‑1) within one hour. Depending on the blood glucose level at start 38. of treatment, insulin was started at a dose ranging from 20 to 50 mIU.kg‑1.h‑1. There- 39. after, the nurse was allowed to adjust the insulin rate according to the nurse-driven

55 Chapter 3

1. protocol up to a rate of 200 mIU.kg‑1.h‑1 after which the attending physician needed 2. to be consulted. Blood glucose levels were checked hourly until the target range (4–8 3. mmol/L‑1 (72–145 mg.dL‑1)) was achieved for three consecutive measurements, after 4. which measurements were performed every 3h. Insulin therapy was stopped at any 5. time according to clearly defined criteria in the protocol19. 6. 7. Blood glucose analysis and definitions 8. Blood glucose measurements were obtained as soon as possible after admission from 9. indwelling arterial catheters or from a capillary puncture and measured on a blood gas 10. analyzer (ABL 625; Radiometer, Copenhagen, Denmark) or a point-of-care bedside 11. system (HemoCue AB, Sweden). 12. Normoglycemia was defined as blood glucose levels between 4 and 8 mmol.L‑1 (72–145 13. mg.dL‑1). Time to reach normoglycemia was defined as the time from start of insulin 14. therapy until the first blood glucose level < 8 mmol.L‑1 (< 145 mg.dL‑1). Hypoglycemia 15. was defined as blood glucose ≤ 2.2 mmol.L‑1 (≤ 40 mg.dL‑1) and severe hypoglycemia 16. was defined as blood glucose < 1.7 mmol.L‑1 (< 31 mg.dL‑1)4. Symptoms of hypo- 17. glycemia were defined as mild (sweating, agitation, lethargy), severe (hemodynamic 18. deterioration, neurological deteriorations (convulsions, coma)) or death. Measurements 19. showing blood glucose levels < 2.6 mmol.L‑1 (47 mg.dL‑1) or > 15 mmol.L‑1 (272 mg.dL‑1) 20. were repeated immediately on the blood gas analyzer. Infants with blood glucose levels 21. < 2.6 (47 mg.dL‑1) were given a bolus of dextrose 10% 5 ml.kg‑1 over 10 minutes. 22. 23. Data collection 24. Patients were assessed by the Pediatric Risk of Mortality III (PRISM III) score20, which 25. is a validated measure of the severity of multiple organ dysfunction in PICU’s. Hypo- 26. glycemic incidents were recorded and analyzed for protocol violations. Based on our 27. previous evaluation of the glucose protocol in children19, we focused on two types 28. of protocol violations (incorrect insulin starting dose, inadequate insulin adjustment/ 29. discontinuation). 30. 31. Statistical analysis 32. Data are presented as medians with interquartile ranges, unless specified otherwise. 33. Statistical significance was considered at p < 0.05. Comparisons between infants 34. with and without hypoglycemia were made with the Mann Whitney U test. Data were 35. analyzed using a standard analysis software program (SPSS version 17.0 for Windows, 36. SPSS, Chicago, IL.). The study was approved by the Medical Ethics Committee of 37. Erasmus Medical Center, and was registered in the Dutch trial register (www.trialregis- 38. ter.nl) under registration number NTR2400. 39.

56 Tight glucose control in infants

1. Results 2. 3. Patients 4. In the 44 month period 4919 children were admitted to our PICU, of which 799 (16.2%) 5. were less than 28 days old. In total 383 children (7.8%; age 4.9 y; 0–18) were treated for 6. hyperglycemia. Of the 799 infants, 73 (9.1%; 0 days (0–6 d), weight 3.2 ± 0.8 kg) were 7. treated with the nurse-driven glucose control protocol. Their characteristics are shown 8. in Table 1. Overall mortality was high (25%) in the hyperglycemic infants, diagnosed 9. with various medical and surgical diagnoses (Table 2). 3 10. 11. Intake 12. Overall glucose intake was 4.7 ± 2.3 mg.kg‑1.min‑1 and not different in the infants who 13. developed hypoglycemia (Table 1). At start of the insulin therapy, 5 infants received 14. full continuous enteral feeding, 37 received full parenteral nutrition with amino acids 15. (Primene; Baxter inc. Utrecht, The Netherlands) and lipids (Intralipid; Fresenius Kabi 16. inc., Utrecht, The Netherlands), and 4 were receiving combined (par)enteral nutrition. 17. Thirty-three patients received no nutrients other than parenteral glucose. 18. 19. Table 1. Characteristics of infants (0 – 28 days) treated according to glucose control protocol* 20. Normoglycemic Hypoglycemic All infants p ( n = 68) (n = 5) (n = 73) value‡ 21. Male : Female 40 : 28 2 : 3 42 : 31 .41 22. Age on admission to PICU (days) 0 (0–5) 1 (0–21) 0 (0–6) .38 23. Weight (kg; mean ± SD) 3.2±0.8 3.0±0.5 3.2±0.8 .31 24. PRISM III † 15 (12–22) 10 (5–12) 14 (11–20) .02 25. Glucocorticoids n = (%) 40 (59%) 2 (40%) 42 (58%) .41 26. Glucose intake 4.7±2.4 5.0±1.1 4.7±2.3 .37 27. (mg.kg‑1.min‑1; mean ± SD)

28. Time to start insulin infusion after first 5.8 (2.5–11) 9.3 (2.3–25.4) 5.8 (2.5–11.0) .53 29. hyperglycemic incident (h) 30. Time to achieve normoglycemia (h) 5.3 (2.6–8.0) 6.1 (3.7–10.1) 5.3 (2.7–8.5) .40 31. Length of stay (days) 15.5 (6.3–29.3) 7.0 (4.5–30.0) 15.0 (6.5–29.5) .54 32. Glucose level at start of insulin therapy 11.2 (9.6–15.4) 10.8 (9.6–11.7) 11.1 (9.6–15.2) .49 33. Initial insulin dose (mIU.kg‑1.h‑1) 20 (20–30) 20 (15–20) 20 (20–30) .13 34. Maximum insulin dose (mIU.kg‑1.h‑1) 50 (30–88) 60 (45–75) 50 (30–80) .49 35. Duration of insulin therapy (h) 26 (10–56) 33 (13–33) 27 (10–57) .37 36. Mortality n = (%) 17 (27%) 1 (20%) 18 (25%) .75 37. *Values are median and interquartile range unless depicted otherwise. † PRISM III; Pediatric Risk of Mortality III score 20, ‡ p values for differences between normoglycemic and hypoglycemic infants. 38. 39.

57 Chapter 3

Table 2 Diagnoses of infants treated according to glucose control protocol 1. N 2. Surgical 3. Non-ECMO 4. Congenital diaphragmatic hernia 18

5. Congenital heart defect 7

6. Congenital abdominal malformation 10

7. Congenital pulmonary malformation 2

8. Necrotizing enterocolitis 1

9. ECMO

10. Congenital diaphragmatic hernia 9 11. Meconium aspiration 8 12. Persistent pulmonary hypertension 5 13. Congenital heart defect 1 14. Medical 15. Infection 16. Viral bronchiolitis 3 17. Meningitis 1 18. Sepsis 1 Meconium aspiration 1 19. Persistent pulmonary hypertension 3 20. Other 3 21. ECMO; Extracorporeal Membrane Oxygenation 22. 23. Glucose control protocol 24. Insulin treatment was initiated 5.8h (1–38h) after the first episode of hyperglycemia 25. (12.5 ± 4.1 mmol.L‑1 (225 ± 74 mg.dL‑1)). The initial insulin starting dose was 20 mlU. 26. kg‑1.h‑1 (10–100 mlU.kg‑1.h‑1), the maximum dose reached was 50 mlU.kg‑1.h‑1 (20–450 27. mlU.kg‑1.h‑1). Normoglycemia was reached within 12h of initiating insulin therapy in 65 28. infants (90.3%), and the median time to reach normoglycemia was 5.3h (1–25h). One 29. infant died before reaching the glucose target range. The overall duration of insulin 30. therapy was 27h (1–308h), while 50 (68.5%) of the infants were treated less than 48h 31. and only twelve (15.1%) infants received insulin for > 72h. 32. 33. Hypoglycemic incidents and protocol violations 34. Episodes of moderate hypoglycemia occurred in three (4.1%) infants and severe hy- 35. poglycemia occurred in two (2.7%) infants, without any severe clinical symptoms. Four 36. infants were recorded to be pale and lethargic. The intervention with a bolus of glucose 37. was sufficient to treat the hypoglycemic incidents. One (20%) infant who developed 38. hypoglycemia died, not directly related to the hypoglycemic incident. In the group 39. who did not develop hypoglycemia 17 (27%) infants died. Blood glucose levels, age,

58 Tight glucose control in infants

1. use of glucocorticoids, and clinical diagnoses at initiation of insulin therapy, dose and 2. duration of insulin therapy, and time to achieve normoglycemia did not differ between 3. hypoglycemic and non-hypoglycemic infants (Table 1). Hypoglycemia occurred twice 4. in the two infants with severe hypoglycemia, although severe hypoglycemia occurred 5. no more than once in these infants. Thus, a total of 5 hypoglycemic and 2 severe 6. hypoglycemic incidents were recorded. The hypoglycemic incidents occurred at day 7. 2 (1-5 d) of admission at the age of 4 days old (1-30 d), 8h (2-13 h) after initiation of 8. insulin therapy. 9. Of these 7 hypoglycemic incidents, no apparent cause could be identified in three 3 10. incidents. In retrospect, one hypoglycemic infant was diagnosed with sepsis within 11. several hours after the hypoglycemic incident. Three protocol violations were identified 12. which could have been responsible for the (severe) hypoglycemic incidents. In two 13. infants insulin was decreased too late or not at all after plasma glucose levels dropped, 14. whereas in one infant insulin was not discontinued according to the protocol. 15. In the 73 infants receiving insulin, 31 protocol violations were identified at the initiation 16. of insulin therapy, which were not associated with the hypoglycemic incidents. The 17. starting dose of insulin was below protocol recommendations in 26 (34.7%) infants 18. and above protocol recommendations in three (4%) infants. In two patients the start of 19. insulin should have been cancelled as the blood glucose was < 8 mmol.L‑1 immediately 20. prior to the start of the insulin treatment. 21. 22. 23. Discussion 24. 25. This is the first evaluation of the efficacy and safety of a tight glucose protocol specifi- 26. cally in critically ill term infants. Consistent with previous evaluations of tight glucose 27. protocols in older children17‑19, we showed that our nurse-driven glucose control 28. protocol achieved normoglycemia within 5.3h and in 90% of the infants within 12h. 29. Remarkably, the target ranges were achieved while a large proportion (34.7%) of the 30. insulin starting doses was below protocol recommendation. Furthermore, the overall 31. treatment duration was short, which makes it less likely that these infants could have 32. benefited from the tight glucose regimen. 33. It is obvious that the infants described, with high mortality rates and a large proportion 34. of infants treated on ECMO, are indeed one of the sickest populations on our PICU. 35. This is consistent with a recent study showing that hyperglycemia was highly prevalent 36. in children requiring mechanical ventilation, vasopressors, and support of continuous 37. renal replacement therapy or ECMO21. Critical illness hyperglycemia, strongly associ- 38. ated with disease severity22, is primarily caused by insulin resistance23, induced by 39. endogenous and exogenous triggers such as catacholamines, glucocorticoids and

59 Chapter 3

1. inflammatory cytokines. While hyperglycemia is associated with a poor outcome in 2. infants3, 24, 25, outcome studies of tight glucose protocols have not focused specifically 3. on this population. Additionally, no mechanistic studies have explored the effects of 4. hyperglycemia or insulin therapy in infants. The principal cause of cellular and organ 5. system failure in critical illness hyperglycemia is glucose overload resulting in excessive 6. generation of oxygen radicals, which leads to mitochondrial dysfunction, increased 7. generation of inflammatory cytokines and disturbed energy metabolism26‑28. Insulin 8. therapy has been shown to protect the function and structure of the mitochondrial 9. compartment in adults and children27, 29. It is not likely that there will be substantial 10. differences in the mechanistic effects of hyperglycemia or insulin therapy in infants. 11. Therefore, despite the lack of beneficial results in outcome studies, potential benefits 12. of insulin therapy in the infant population should not be ignored. 13. However, we recognize the 7 (severe) hypoglycemic incidents in 5 infants (6.7%). This 14. was markedly lower than previously described by Vlasselaers and colleagues, who 15. reported an incidence of hypoglycemia in 25% of children treated, predominantly 16. (>80%) in infants4. Using a treatment algorithm in critically ill older children by us and 17. others the reported incidence of hypoglycemia was 0-4%17‑19. As the timeframe of 18. the evaluation was similar for the older children without any hypoglycemic incidents19 19. as for the infants in the present study, neither the protocol implementation, nor the 20. experience working with the protocol by nursing staff and physicians explains this 21. difference. So, it appears that infants are more susceptible to hypoglycemia following 22. insulin administration. 23. Investigating the hypoglycemic incidents showed us that three of the hypoglycemic 24. incidents were explained by protocol violations, all due to inadequate insulin dose 25. adjustments, emphasizing the importance of adherence to the protocol. Notwithstand- 26. ing the iatrogenic causes of these hypoglycemic incidents, we did find that insulin 27. doses above recommendation occurred much less in our infants (4%), compared to 28. our previous report in older children (21%)19. Remarkably, we found a disproportionate 29. high incidence (34.7%) of insulin doses below the recommended dose. Although our 30. study was not designed to show the grounds for protocol violations, a certain fear of 31. iatrogenic hypoglycemia in these infants might be responsible for this latter violation. It 32. has been shown recently that a disparity exists between physicians beliefs and actual 33. practice habits regarding glycemic control in PICU’s30. Although a clear causal relation- 34. ship between hyperglycemia and poor outcome has become more evident, among pe- 35. diatricians hypoglycemia is still considered more detrimental than hyperglycemia30, 31. 36. Fear of hypoglycemia is a major barrier for the successful implementation of glycemic 37. control in critically ill children30. 38. Although three hypoglycemic incidents were explained by protocol violations, three hy- 39. poglycemic incidents occurred without apparent cause and one occurred in the onset

60 Tight glucose control in infants

1. of sepsis. Furthermore, in two infants hypoglycemia occurred twice. These observa- 2. tions allow us to speculate that infants indeed are a specific and vulnerable age group 3. on the PICU. Possibly the etiology in these infants is different from the “regular” insulin 4. resistance induced critical illness hyperglycemia. For instance, in the pediatric popula- 5. tion the initial phase of sepsis can cause insulin deficiency, rather than resistance32. 6. Additionally, high parenteral glucose intake can, independent of insulin resistance, 7. produce hyperglycemia. Under such circumstances, insulin infusion would decrease 8. plasma glucose levels faster than under insulin resistant conditions. Furthermore, in- 9. fants lack the precise control of glucose homeostasis as they undergo major changes 3 10. in glucose and insulin metabolism. Glycogen stores are low and glucose homeostasis 11. primarily depends on gluconeogenesis, partially explaining why young age itself is a 12. risk factor for developing hypoglycemia7. Furthermore, a transformation in β-cell popu- 13. lation, due to a transient wave of apoptosis and repopulation, causes a wide varia- 14. tion of insulin secretory capacities in newborn infants33. Moreover, hyperinsulinemic 15. euglycemic clamp studies have shown that infants have greater peripheral glucose 16. utilization34 than older children35 and adults36. Insulin receptors in infants are higher in 17. number as well as affinity, partially explaining this increased sensitivity37. Additionally, it 18. has recently been shown that there exists a developmental change in peripheral insulin 19. signalling38. Although reduced contents of glucose transporters (GLUT) 1 and 4 were 20. found, the proximal insulin signalling proteins (IR-β, IRS-1 and Akt) were increased in 21. muscle of neonatal baboons38. These wide variations in the ability of glucose produc- 22. tion, insulin secretory capacity, and insulin sensitivity might at least partially explain 23. the differences from older children and adults. This suggests that when insulin therapy 24. is considered in critically ill infants, increased awareness is needed. With the results 25. of the present study we have adjusted the protocol on our PICU for infants less than 26. 28 days old; we decreased the initial insulin starting doses by 10 mIU.kg‑1.h‑1 and the 27. minimum insulin dose as stop criteria (< 5 mIU.kg‑1.h‑1, or < 20 mIU.kg‑1.h‑1 > 24h) 28. (Figure 1). Our study has limitations. It is an observational study. It adds insight in 29. the safety and effectivity of tight glucose protocols for critically ill infants, but it was 30. not designed to show whether outcome improved with our tight glucose protocol. We 31. further acknowledge that our glucose target range was higher than that of Vlasselaers 32. and colleagues4, which could at least partially explain the difference in hypoglycemic 33. incidents. However, as the ideal target range of tight glucose regimens still needs to be 34. established less strict protocols might be sensible. 35. Based on this study, we conclude that term infants can be treated with a tight glu- 36. cose protocol, as target ranges were met, and overall treatment was short. However, 37. hypoglycemia occurred more frequently in infants than in older children, alerting us 38. that this is a vulnerable population where additional safety approaches are warranted. 39. Future studies assessing the outcome of a tight glucose regimen in the infant popula-

61 Chapter 3

1. Two subsequent (1 h pause) measurements of blood glucose 2. 8 mmol.L-1 (145 mg.dL-1) 3. with a glucose intake: 4-6 mg/kg/min 4. 5. 6. Insulin initial dose (mIU.kg-1.h-1) Glucose Insulin 7. >8-11 mmol.L-1(145-200 mg.dL-1)  10 >11-15 mmol.L-1(200-272 mg.dL-1)  20 8. >15-20 mmol.L-1(272-364 mg.dL-1)  30 -1 -1 9. >20 mmol.L (364 mg.dL )  40

10. After start or adjustment of insulin infusion: 11. Check glucose hourly until 3 times within target range 4-8 mmol.L-1 (72-145 mg.dL-1), after which glucose should be checked every 3 12. hours 13. Glucose < 4 mmol.L-1 Glucose >50% Glucose >25% Glucose increase or 14. (72 mg.dL-1) decrease decrease ≤ 25% decrease 15.

16. -Stop insulin Decrease Glucose Insulin Glucose Insulin -Glucose < 2.6 mmol.L-1 insulin dose 4-8 mmol.L-1  1/2 mmol.L-1 mIU.kg-1.h-1 17. (47 mg.dL-1): by 50% >8-11 mmol.L-1  2/3 4-6  -50% -1  18. bolus glucose 10% > 11 mmol.L  sqa >6-8 sqa 5 ml.kg-1 in 10 min >8-11  + 10 19. >11-15  + 20 >15-20  + 30 20. Check glucose every 30 min until >20  + 40 -1 -1 21. glucose >4 mmol.L (72 mg.dL )

22. Insulin >200 mIU.kg-1.h-1  consult -1 -1 23. Stop criteria: physician (max. dose = 600 mIU.kg .h ) -blood glucose < 4 mmol.L-1 (72 mg.dL-1) 24. -insulin rate < 5 mIU.kg-1.h-1 at any time -insulin rate < 20 mIU.kg-1.h-1 during 24 h 25. -start enteral bolus feeding -interruption of continuous feeding or glucose infusion 26. -discharge from pediatric intensive care unit 27. Figure 1. Adjusted step-wise nurse driven glucose control protocol for infants less than 28 days 28. old. Glucose; blood glucose level in mmol.L‑1 (mg.dL‑1), Insulin; Insulin dose in mIU.kg‑1.h‑1. 29. 30. tion, and addressing approaches which will help prevent hypoglycemia are of utmost 31. importance. Meanwhile, we will evaluate our adjusted protocol (Figure 1) and will have 32. a long-term follow up of the infants. 33. 34. 35. Acknowledgements 36. 37. We would like to thank Marjolein Augustus for her help with data collection. 38. 39.

62 Tight glucose control in infants

1. References

2. 1. Krinsley JS. Association between 12. Chase JG, Shaw GM, Lin J, et al. Adap- 3. hyperglycemia and increased hospital tive bolus-based targeted glucose regu- 4. mortality in a heterogeneous population lation of hyperglycaemia in critical care. 5. of critically ill patients. Mayo Clin Proc Med Eng Phys 2005;​27(1):​1‑11. 2003;​78(12):​1471‑8. 13. Chee F, Fernando T, van Heerden PV. 6. 2. Verbruggen SC, Joosten KF, Castillo L, Closed-loop control of blood glucose 7. van Goudoever JB. Insulin therapy in the levels in critically ill patients. Anaesth 8. pediatric intensive care unit. Clin Nutr Intensive Care 2002;​30(3):​295-307. 9. 2007;​26(6):​677‑90. 14. Lin J, Chase JG, Shaw GM, et al. Adap- 3 10. 3. Hays SP, Smith EO, Sunehag AL. Hyper- tive bolus-based set-point regulation of 11. glycemia is a risk factor for early death and hyperglycemia in critical care. Conf Proc morbidity in extremely low birth-weight IEEE Eng Med Biol Soc 2004;​5:​3463‑6. 12. infants. Pediatrics 2006;​118(5):​1811‑8. 15. Wong XW, Singh-Levett I, Hollingsworth 13. 4. Vlasselaers D, Milants I, Desmet L, et al. LJ, et al. A novel, model-based insulin 14. Intensive insulin therapy for patients in and nutrition delivery controller for gly- 15. paediatric intensive care: a prospective, cemic regulation in critically ill patients. 16. randomised controlled study. Lancet Diabetes Technol Ther 2006;​8(2):​174‑90. 2009;​373(9663):​547‑56. 16. Vogelzang M, Loef BG, Regtien JG, et 17. 5. Beardsall K, Vanhaesebrouck S, Ogilvy- al. Computer-assisted glucose control in 18. Stuart AL, et al. Early insulin therapy in critically ill patients. Intensive Care Med 19. very-low-birth-weight infants. N Engl J 2008;​34(8):​1421‑7. 20. Med 2008;​359(18):​1873‑84. 17. Preissig CM, Hansen I, Roerig PL, Rigby 21. 6. Solomon T, Felix JM, Samuel M, et al. MR. A protocolized approach to identify 22. Hypoglycaemia in paediatric admissions and manage hyperglycemia in a pediatric in Mozambique. Lancet 1994;​343(8890):​ critical care unit. Pediatr Crit Care Med 23. 149‑50. 2008;​9(6):​581‑8. 24. 7. Chaussain JL, Georges P, Calzada L, Job 18. Preissig CM, Rigby MR, Maher KO. Gly- 25. JC. Glycemic response to 24-hour fast cemic control for postoperative pediatric 26. in normal children: III. Influence of age. J cardiac patients. Pediatr Cardiol 2009;​ 27. Pediatr 1977;​91(5):​711‑4. 30(8):​1098-104. 8. Bober E, Buyukgebiz A. Hypoglycemia 19. Verhoeven JJ, Brand JB, van de Polder 28. and its effects on the brain in children MM, Joosten KF. Management of hyper- 29. with type 1 diabetes mellitus. Pediatr glycemia in the pediatric intensive care 30. Endocrinol Rev 2005;​2(3):​378‑82. unit; implementation of a glucose control 31. 9. Vannucci RC, Vannucci SJ. Glucose me- protocol. Pediatr Crit Care Med 2009;​ 32. tabolism in the developing brain. Semin 10(6):​648‑52. 33. Perinatol 2000;​24(2):​107‑15. 20. Pollack MM, Patel KM, Ruttimann UE. 10. Flykanaka-Gantenbein C. Hypoglycemia PRISM III: an updated Pediatric Risk 34. in childhood: long-term effects. Pediatr of Mortality score. Crit Care Med 1996;​ 35. Endocrinol Rev 2004;​1 Suppl 3:​530‑6. 24(5):​743‑52. 36. 11. Chase JG, Shaw GM, Lin J, et al. 21. Preissig CM, Rigby MR. Pediatric critical 37. Targeted glycemic reduction in critical illness hyperglycemia: risk factors as- 38. care using closed-loop control. Diabetes sociated with development and severity Technol Ther 2005;​7(2):​274‑82. of hyperglycemia in critically ill children. 39.

63 Chapter 3

J Pediatr 2009;​155(5):​734‑9. regarding hyperglycemia in pediatric 1. 22. Klein GW, Hojsak JM, Schmeidler J, Ra- intensive care units in the United States: 2. paport R. Hyperglycemia and outcome in a survey on actual practice habits. Crit 3. the pediatric intensive care unit. J Pediatr Care 2010;​14(1):​R11. 4. 2008;​153(3):​379‑84. 31. Hirshberg E, Lacroix J, Sward K, Willson 5. 23. Matsuda N, Yamamoto S, Yokoo H, D, Morris AH. Blood glucose control in Tobe K, Hattori Y. Nuclear factor-kappaB critically ill adults and children: a survey 6. decoy oligodeoxynucleotides ameliorate on stated practice. Chest 2008;​133(6):​ 7. impaired glucose tolerance and insulin 1328‑35. 8. resistance in mice with cecal ligation and 32. van Waardenburg DA, Jansen TC, Vos 9. puncture-induced sepsis. Crit Care Med GD, Buurman WA. Hyperglycemia in 10. 2009;​37(10):​2791‑9. children with meningococcal sepsis 24. Alaedeen DI, Walsh MC, Chwals WJ. and septic shock: the relation between 11. Total parenteral nutrition-associated plasma levels of insulin and inflamma- 12. hyperglycemia correlates with prolonged tory mediators. J Clin Endocrinol Metab 13. mechanical ventilation and hospital stay 2006;​91(10):​3916‑21. 14. in septic infants. J Pediatr Surg 2006;​ 33. Lucas A, Bloom SR, Aynsley-Green A. 15. 41(1):​239-44; discussion ‑44. Metabolic and endocrine events at the 16. 25. Hall NJ, Peters M, Eaton S, Pierro A. Hy- time of the first feed of human milk in perglycemia is associated with increased preterm and term infants. Arch Dis Child 17. morbidity and mortality rates in neonates 1978;​53(9):​731‑6. 18. with necrotizing enterocolitis. J Pediatr 34. Farrag HM, Nawrath LM, Healey JE, et 19. Surg 2004;​39(6):​898-901; discussion al. Persistent glucose production and 20. 898-901. greater peripheral sensitivity to insulin in 21. 26. Van den Berghe G. How does blood the neonate vs. the adult. Am J Physiol glucose control with insulin save lives in 1997;​272(1 Pt 1):​E86‑93. 22. intensive care? J Clin Invest 2004;​114(9):​ 35. Danadian K, Balasekaran G, Lewy V, 23. 1187‑95. Meza MP, Robertson R, Arslanian SA. 24. 27. Vanhorebeek I, De Vos R, Mesotten D, Insulin sensitivity in African-American 25. Wouters PJ, De Wolf-Peeters C, Van children with and without family history 26. den Berghe G. Protection of hepatocyte of type 2 diabetes. Diabetes Care 1999;​ 27. mitochondrial ultrastructure and function 22(8):​1325‑9. by strict blood glucose control with insu- 36. Wolfe RR, Allsop JR, Burke JF. Glucose 28. lin in critically ill patients. Lancet 2005;​ metabolism in man: responses to intrave- 29. 365(9453):​53‑9. nous glucose infusion. Metabolism 1979;​ 30. 28. Brealey D, Brand M, Hargreaves I, et al. As- 28(3):​210‑20. 31. sociation between mitochondrial dysfunc- 37. Thorsson AV, Hintz RL. Insulin receptors 32. tion and severity and outcome of septic in the newborn. Increase in receptor af- shock. Lancet 2002;​360(9328):​219‑23. finity and number. N Engl J Med 1977;​ 33. 29. Fram RY, Cree MG, Wolfe RR, et al. 297(17):​908‑12. 34. Intensive insulin therapy improves insulin 38. Blanco CL, Liang H, Joya-Galeana J, 35. sensitivity and mitochondrial function in DeFronzo RA, McCurnin D, Musi N. 36. severely burned children. Crit Care Med The ontogeny of insulin signaling in the 37. 2010;​38(6):​1475‑83. preterm baboon model. Endocrinology 38. 30. Preissig CM, Rigby MR. A disparity 2010;​151(5):​1990‑7. between physician attitudes and practice 39.

64 Chapter 4

Reducing Glucose Intake Safely Prevents Hyperglycemia In Post-Surgical Children

Sascha CAT Verbruggen Carlijn TI de Betue Henk Schierbeek Shaji Chacko Leon NA van Adrichem Jennifer J Verhoeven Johannes B van Goudoever Koen FM Joosten

Submitted Chapter 4

1. Abstract 2. 3. Objective 4. To investigate the effects of two different glucose intakes on glucose homeostasis and 5. amino acid metabolism in post-surgical children. 6. 7. Design 8. A single center, randomized crossover study. 9. 10. Setting 11. A pediatric intensive care unit (PICU) in a tertiary university hospital. 12. 13. Patients 14. Eight children (age 9.8 ± 1.9 months, weight 9.5 ± 1.1 kg) admitted after surgical cor- 15. rection for non-syndromal craniosynostosis. 16. 17. Interventions 18. Patients were randomized to receive low (LG; 2.5 mg.kg‑1.min‑1) and standard (SG; 5.0 19. mg.kg‑1.min‑1) glucose intake in a crossover setting. After a bolus (4 g.kg‑1) of deuteri- 2 20. umoxide, we conducted a primed, constant, 8 h tracer infusion with [6,6- H2]Glucose, 13 2 2 21. [1- C]Leucine, [ring- H5]Phenylalanine and [3,3- H2]Tyrosine. 22. 23. Measurements and Main Results 24. SG resulted in hyperglycemia (defined as > 110 mg.dL‑1), while during LG plasma 25. glucose levels were normoglycemic (105 ± 10 vs. 133 ± 30 mg.dL‑1; LG vs. SG respec- 26. tively, p = .02). Hypoglycemia did not occur during LG intake. Endogenous glucose 27. production (EGP) was not fully suppressed during the hyperglycemic state under SG 28. and increased with reduced glucose intake (2.6 ± 1.5 vs. 1.1 ± 1.4 mg.kg‑1.min‑1; LG 29. vs. SG; p = .05). Leucine kinetics did not differ between glucose infusion rates. Phenyl- 30. alanine hydroxylation was higher during LG (8.4 ± 1.7 vs. 7.4 ± 1.6 µmol.kg‑1.h‑1; LG vs. 31. SG; p = .04). Whole body protein balance derived from with leucine and phenylalanine 32. kinetics was slightly negative but not further affected with a decrease in glucose intake. 33. 34. Conclusions 35. The current recommended glucose intake induces hyperglycemia in post-surgical 36. children. A reduced glucose intake safely prevented hyperglycemia, while infants were 37. capable to sustain normoglycemia with increased endogenous glucose production due 38. to a combined increase in gluconeogenesis and glycogenolysis. The reduced glucose 39. intake did not exacerbate the mild catabolic state in which the patients were.

66 Reduced glucose intake prevents hyperglycemia

1. Introduction 2. 3. Plasma glucose levels are the resultant of a balance between exogenous glucose sup- 4. ply and endogenous glucose production on the one hand and glucose oxidation or 5. storage as glycogen and triglycerides on the other. Critically ill children are at increased 6. risk to a disturbance in this balance leading to hyper- as well as hypoglycemia1. 7. Hyperglycemia is a frequent complication and associated with increased morbidity 8. and mortality in pediatric intensive care units (PICU’s)2. A tight glucose regimen with 9. insulin showed reduced morbidity and mortality in an adult Intensive Care Unit (ICU)3, 4, 10. although a number of subsequent studies were unable to replicate these results5, 6. 11. Notwithstanding the widespread implementation of the tight glucose regimen7, con- 12. cerns regarding hypoglycemia have been raised8. Recently, insulin therapy to achieve 4 13. normoglycemia has been shown to improve morbidity as well as mortality in critically 14. ill children, but also led to hypoglycemia (≤ 40 mg.dL‑1 ~ ≤ 2.2 mmol.L‑1) and severe 15. hypoglycemia (≤ 31 mg.dL‑1 ~ ≤ 1.7 mmol.L‑1) in 87 (25%) and 17 (5%) children, re- 16. spectively9. 17. The child’s developing brain is more susceptible to hypoglycemia which can result 18. in permanent damage10‑12. Furthermore, young age is a risk factor for developing 19. hypoglycemia, especially when the child is ill13, 14. It is therefore essential to prevent 20. hypoglycemia, which led to a debate questioning the risks of insulin therapy in the 21. pediatric population15, 16. 22. An alternative to insulin therapy is to reduce the amount of glucose intake in criti- 23. cally ill young children. This approach, however, also has two potential detrimental 24. side-effects; an increased risk for hypoglycemia and an amplification of an already 25. increased protein catabolism. Currently no data exist on the impact of different glucose 26. intakes on glucose kinetics and amino acid metabolism in critically ill children. 27. We hypothesized that in post-surgical children, reduced glucose intake will improve 28. plasma glucose levels without affecting glucose production rates or amino acid 29. metabolism. Therefore, our first objective was to determine the impact of standard 30. or low glucose intakes on glucose homeostasis and kinetics. Our second objective 31. was to determine whether a low glucose intake would affect protein and amino acid 32. catabolism. 33. 34. 35. Methods 36. 37. Patient characteristics 38. The study protocol was approved by the Medical Ethical Committee of the Erasmus 39. Medical Center, Rotterdam, the Netherlands. The studies were performed in children

67 Chapter 4

1. after surgical correction for non-syndromal craniosynostosis within 6 hours after ad- 2. mission to the Pediatric Intensive Care Unit (PICU) of the Erasmus Medical Center 3. - Sophia Children’s Hospital. Written informed consent was obtained from the parents. 4. Patients had infusing and drawing lines in place for clinical purposes. All were as- 5. sessed by the Pediatric Logistic Organ Dysfunction (PELOD) score17, Pediatric Index 6. of Mortality (PIM2)18, 19 and the Pediatric Risk of Mortality III (PRISM III) score20, which 7. are validated measures of the severity of multiple organ dysfunction in PICU’s. Patients 8. with metabolic diseases, diabetes mellitus, primary liver, or renal failure were excluded. 9. 10. Study design 11. The experimental design, shown in Figure 1, consisted of a cross-over design, with a 4 12. h period of intravenous low glucose intake (LG; 2.5 mg.kg‑1.min‑1) versus a 4 h period 13. of standard glucose intake (SG; 5.0 mg.kg‑1.min‑1)21. Patients were randomized for the 14. order of glucose intake through a computer generated envelop. Laboratory personnel, 15. nursing staff and investigators were blinded until analyses were finished. Six hours 2 16. after admission (t=0) to the PICU, an intravenous deuterium oxide infusion ( H2O; 4 17. gr.kg‑1) was administered in one hour to prime the body water pool. Two hours later 18. (t=120), after obtaining baseline blood samples, the intravenous glucose intake as per 19. standard care (4.0 – 6.0 mg.kg‑1.min‑1)21 was stopped and the study glucose intake 20. (SG or LG) started. Simultaneously, the patients received a primed, continuous, 8 hour 21. intravenous tracer infusion (see below). Four hours after start of the tracer infusion 22. (t=360) the glucose intake was switched. 23. 24. Tracer Infusion studies 25. All isotope tracers were purchased from Cambridge Isotope Laboratories (Andover, 26. MA, USA) and tested for sterility and pyrogenicity after they were compounded at the 27. investigational pharmacy at Erasmus Medical Center, Rotterdam, the Netherlands. 28. 29. 30. Blood samples

31. VCO2 measurement VCO2 VCO2 32. Intravenous glucose 4-6 mg.kg-1.min-1 LG or SG SG or LG 33. 2 Isotope tracers H2O 34. 35. Minutes 0 120 320 340 360 520 540 600 36. Figure 1. Schematic presentation of the tracer infusion study in eight post-surgical infants 37. receiving either low (LG, 2.5 mg.kg‑1.min‑1) or standard (SG, 5.0 mg.kg‑1.min‑1) glucose intake. 38. Black triangles indicate time points for plasma collection for laboratory parameters and isotopic enrichment measurements. Square boxes represent the time period in which carbon

39. dioxide production (VCO2) measurements took place.

68 Reduced glucose intake prevents hyperglycemia

1. At t = 120, the bicarbonate pool was primed with 2.1 µmol.kg‑1 13C sodium bicarbonate. 2 2. This was followed by a 8-hour primed, continuous tracer infusion of [6,6- H2]glucose ‑1 ‑1 ‑1 13 ‑1 ‑1 ‑1 2 3. (40 μmol.kg ; 48 μmol.kg .h ), L-[1- C]leucine (8 μmol.kg ; 8 μmol.kg .h ), [ring- H5] ‑1 ‑1 ‑1 2 ‑1 4. Phenylalanine (5.4 μmol.kg ; 4.1 μmol.kg .h ), [3,3- H2]Tyrosine (3.6 μmol.kg ; 3.0 ‑1 ‑1 2 5. μmol.kg .h ). The [ring- H5]Phenylalanine derived tyrosine pool was primed with [ring- 2 ‑1 6. H4]Tyrosine (2.5 μmol.kg ). 7. 8. Measurements and sample analysis 9. Blood samples were obtained at standard frequent intervals (Figure 1.), centrifuged (2 10. min 6000 rpg) and frozen at -80°C until samples were analyzed. 11. Isotopic enrichment of deuterium in plasma body water was determined by isotope 12. ratio mass spectrometry (IRMS) (Delta+XP IRMS Thermo Fisher, Bremen, Germany) as 4 13. an average of multiple measurements. Enrichments of glucose labeled with 2H were 14. measured by gas chromatography mass spectrometry (GC-MS) (GC 6890, MS 5973N; 15. Agilent Technologies, Wilmington, DE) using the penta-acetate derivative and analyzed 16. in the positive chemical ionization scan mode of GC-MS as previously described22, 23. 2 17. Plasma isotopic enrichment of [6,6- H2]glucose (M+2) was determined by monitoring 18. fragment ions at a mass-to-charge ratio (m/z) of 169 and 171. Selective ion monitoring 19. of m/z 170/169 was performed to determine the M+1 enrichment of deuterium in the 20. circulating glucose carbons (C-1,3,4,5,6,6)23. 21. Leucine kinetics were calculated from plasma alpha-ketoisocaproate (α-KIC) enrich- 22. ment, which is intracellularly produced from the leucine tracer infused. Plasma isotopic 23. enrichment of [1-13C]α-KIC were, after derivatization to butyldimethyl-silylquinoxalinol 24. derivatives, determined by gas chromatography mass spectrometry (GCMS)24. Plasma 2 2 2 25. isotopic enrichments of [ring- H5]Phenylalanine, [ring- H4]Tyrosine and [3,3- H2]Tyro- 26. sine were determined by GCMS. Samples and calibration curves were analyzed with 27. an Agilent 5975C GCMS (Agilent technologies, Amstelveen, The Netherlands) on a VF- 28. 17ms, 30m x 0.25mm ID capillary column (Varian Inc., Middelburg, The Netherlands) 29. after using the N-ethoxycarbonylethylester derivative according to a modified method 30. of Husek25.

31. Carbon dioxide production (VCO2), oxygen consumption (VO2) and respiratory quotient 32. (RQ), were obtained with a metabolic monitor in canopy mode (DeltatracTM I MBM-200, 33. Datex Division Instrumentarium Corp., Finland) during the last 40 minutes of study 13 34. periods. To determine the enrichment of CO2 in whole blood, 1.5 mL of perchloric

35. acid 10% was added to 1.5 mL of whole blood in a vacutainer to release the CO2. 13 36. The released gas was transferred to a vacuum impermeable glass tube and CO2 was 37. determined with isotope ratio mass spectrometry (IRMS)26, 27. 38. 39.

69 Chapter 4

1. Plasma samples for glucose, insulin, cortisol, triglycerides, and free fatty acids were 2. determined by standard in house protocols. Plasma glucose levels higher than > 110 3. mg.dL‑1 (> 6.1 mmol.L‑1) were considered hyperglycemic4. 4. 5. Calculations 6. Whole body kinetics of protein were calculated by conventional isotope dilution equa- 7. tions using a stochastic model during steady state enrichment28 and glucose kinetics 8. were estimated using the Steele equation29. At steady state plateau rate of appearance 9. (Ra) is equal to rate of disappearance (Rd). The rate of appearance (Ra) of unlabeled 10. substrate can be derived from the plasma isotope enrichment calculated by: 11.

12. Ra = Rd = i x (Einf/Epl – 1) (1) 13.

14. where i is the infusion rate of the labeled tracer, Einf is the tracer enrichment of the

15. infusate and Epl the tracer enrichment in plasma. 16. 17. Glucose kinetics 18. Glucose kinetics were calculated during the last 40 min of both study periods. Endog- 19. enous glucose production (EGP) rate is calculated by subtracting the glucose infusion 20. rate from the steady state glucose appearance (Ra) 21.

22. EGP = RaGlucose – GIR (2) 23. 24. , where GIR is the total glucose infusion rate in mg.kg‑1.min‑1. 25. Fractional gluconeogenesis is calculated using the average deuterium enrichment 26. method previously described23. Briefly, the average enrichment of 2H on each glucose 27. carbon is calculated with the following equation: 28.

29. Average (M+1)d = (M+1)d(m/z 169)/6 (3) 30.

31. where (M+1)d(m/z 169) is the M+1 enrichment of deuterium of glucose measured using m/z 32. 170/169 and “6” is the number of 2H labeling sites on the m/z 169 fragment of glucose. 33. Because body water is the precursor pool for deuterium or hydrogen, the extent of 2 34. deuterium labeling of glucose during the gluconeogenic process when H2O is infused 35. is a measure of fractional gluconeogenesis. Therefore, with the average deuterium 36. enrichment in m/z 170/169 for calculating fractional gluconeogenesis (Frac GNG), the 37. equation is 38.

39. Frac GNG = average(M+1)d/EH2O (4)

70 Reduced glucose intake prevents hyperglycemia

1. where EH2O is the deuterium enrichment in body water.

2. The absolute rate of appearance of gluconeogenic glucose in plasma (RaGNG) is then 3. calculated by multiplying the rate of appearance of glucose in plasma by the fraction 4. of gluconeogenesis: 5.

6. Gluconeogenesis = RaGluc x Frac GNG (5) 7. 8. Glycogenolysis is than calculated by subtracting the gluconeogenesis from the EGP. 9. 10. Glycogenolysis = EGP – Gluconeogenesis (6) 11. 12. Leucine kinetics 4 13. Whole body leucine flux was calculated during the last 40 min of both study periods30. 14. Leucine oxidation rates were calculated as follows; 15. 13 13 16. Leucine oxidation = VCO2 x (E CO2/69.18)/[ C]α – KIC (7) 17. 13 31 18. where 69.18 is the CO2 refraction correction factor for critically ill children . VCO2 19. (carbon dioxide production) is measured in milliliters per minute and converted to mil- 20. limoles per hour by multiplying by 60 min and dividing by 22.4, which is the number of 21. 1 in 1 mole of an ideal gas at standard temperature and pressure to convert to milliliters 22. per minute. Non-oxidative leucine disposal (NOLD; leucine converted into protein 23. synthesis) is the leucine oxidation subtracted from the leucine rate of disappearance. 24.

25. NOLD = Raleu – Leucine Oxidation (8) 26. 27. Phenylalanine and tyrosine kinetics 28. Whole body phenylalanine and tyrosine fluxes were calculated during the last 40 min 29. of both study periods32, 33. 30. Phenylalanine hydroxylation is the rate of phenylalanine conversion to tyrosine and is 31. calculated as follows; 32.

33. Hydroxylation = Ratyr x (E[2H4]tyr/E[2H5]Phe) x (Raphe/(iphe + Raphe) x 2.2 (9) 34.

35. where Raphe and Ratyr are the phenylalanine and tyrosine fluxes, calculated as described 2 2 36. with (1) using [ring- H5]phenylalanine and [3,3- H2]tyrosine respectively; E[2H4]tyr and E[2H5]

37. Phe are the plasma enrichments; and iphe is the rate of infusion of labeled phenylalanine

38. and the term (Raphe/(iphe + Raphe) corrects for the contribution of the tracer infusion to

39. Raphe. The factor 2.2 is to correct for the secondary deuterium-isotope kinetic effect for

71 Chapter 4

1. in vivo hydroxylation in fasted state as described and validated previously34, 35. Non- 2. hydroxylation phenylalanine disposal (NHPD; phenylalanine converted into protein 3. synthesis) is the phenylalanine hydroxylation subtracted from the phenylalanine rate 4. of disappearance. 5.

6. NHPD = Raphe – Hydroxylation (10) 7. 8. Whole body protein metabolism 9. Under the assumption that 1 gram of protein contains approximately 621 µmol of 10. leucine36 and 280 µmol of phenylalanine37, it is then possible to convert leucine and 11. phenylalanine kinetics (µmol.kg‑1.h‑1) into protein kinetics (g.kg‑1.d‑1). Whole body 12. protein turnover was calculated from the model described by Golden and Waterlow38. 13. Briefly, the model describes the following equation; 14. 15. Ra = Rd = S + O = B + I. (11) 16. 17. Whole body protein synthesis (S) is equal to NOLD and NHPD subtracted by leucine 18. oxidation and phenylalanine hydroxylation (O), respectively, converted into protein 19. kinetics as described above. Protein breakdown (B) can be calculated by subtracting 20. the leucine or phenylalanine intake (I) from the leucine or phenylalanine rate of appear- 21. ance (Ra), respectively, and converted again into protein synthesis. In our study the 22. intake is equal to the tracer infusion as our patients were fasted and did not receive any 23. amino acids. Protein balance can then be calculated by subtracting whole body protein 24. breakdown from whole body protein synthesis. 25. 26. Statistical analysis 27. A prospective power analysis revealed that 8 patients with complete data, would detect 28. a difference of 20% of plasma glucose levels (80% power, type I error of 5%). The Sha- 29. piro-Wilk normality test was used to determine whether data were normally distributed. 30. Comparisons between the two different glucose intakes at both infusion rates were 31. made using the paired student’s t-test. For non-parametric data the Wilcoxon matched 32. pairs test was used. Data are presented as the mean ± standard deviation unless 33. non-parametric in which case they are presented as median and interquartile range. 34. Statistical significance was considered at p < 05. Repeated measures ANOVA were 35. used to analyze the effect of glucose infusion on parameters of glucose and protein 36. metabolism over time and between LG and SG. Data were analyzed with Graphpad 37. Prism 5.0.3 (Graphpad Software, La Jolla, CA., USA). This trial was registered in the 38. Dutch trial register (www.trialregister.nl) under number NTR2079. 39.

72 Reduced glucose intake prevents hyperglycemia

1. Results 2. 3. Patient characteristics 4. The patients’ characteristics are shown in Table 1. A total of 8 children (9.8 ± 1.9 5. months) admitted to the PICU after surgical correction for non-syndromal craniosyn- 6. ostosis were included. All patients were hemodynamically stable without vasoactive 7. drugs and breathing spontaneously with an inspiratory oxygen fraction of less than 0.6. 8. They were receiving either opioids or acetaminophen as pain relief and were not se- 9. dated or receiving muscle relaxation at the time of study. Patients did not receive (par) 10. enteral nutrition other than intravenous glucose infusion in the range of 4-6 mg.kg‑1. 11. min‑1 as per standard care21. The resting energy expenditure and respiratory quotient 12. did not change between glucose intake protocols (Table 1). 4 13. 14. 15. Table 1. Demographic and nutritional data of 8 post-surgical children*. 16. Age (months) 9.8 ± 1.9 17. Gender (male:female) 6 : 2 Weight (kg) 9.5 ± 1.1 18. Height (cm) 74.3 ± 3.0 19. PELOD† 10.1 ± 7.6 20. PRISM III‡ 7.4 ± 3.7 21. PIM2^ 14.2 ± 2.8 22. LG£ SG£ P value 23. Glucose intake (mg.kg‑1.min‑1) 2.6 ± 0.1 5.2 ± 0.1 < .0001

24. ‑1 VO2 (mL.min ) 69 ± 24 67 ± 17 .79

25. ‑1 VCO2 (mL.min ) 59 ± 11 55 ± 14 .29 26. Resting energy expenditure (kcal.kg‑1.d‑1) 49.8 ± 17.6 49.7 ± 14.9 .98

27. Respiratory quotient 0.88 ± 0.14 0.83 ± 0.10 .37

28. Caloric intake (kcal.kg‑1.day‑1) 12.7 ± 0.2 25.3 ± 0.5 < .0001

29. Caloric intake (%) # 24 ± 1 47 ± 2 < .0001

30. Glucose (mg.dL‑1) 105 ± 10 133 ± 30 .02 31. Triglycerides (mmol.L‑1) 0.38 ± 0.25 0.43 ± 0.20 .54 32. Free fatty acids (mmol.L‑1) 0.72 ± 0.20 0.63 ± 0.12 .34 33. C-reactive protein (mg.dL‑1) 24 ± 13 26 ± 16 .61 34. Cortisol (nmol/L‑1) 649 ± 160 681 ± 205 .77 35. Insulin (µU.mL‑1) 64 ± 48 90 ± 51 .16 36. *All values are mean ± SD. £LG = Low glucose, £SG = Standard glucose, †PELOD = Pediatric Logistic Organ Dysfunction 17, ‡PRISM III = Pediatric Risk of Mortality III 20, ^PIM2 = Pediatric index of Mortality 18, 19, # Caloric intake as percentage of 37. requirements according to the Schofield equation 59. 38. 39.

73 Chapter 4

1. Laboratory values and hormone concentrations 2. Patients were hyperglycemic during standard glucose intake (SG), while during LG 3. plasma glucose levels were lower and normoglycemic (Figure 2, Table 1). LG did not 4. cause hypoglycemia; the lowest plasma glucose was 91.8 mg.dL‑1. Insulin plasma 5. concentrations did not differ significantly (Table 1). 6. 7. A B

] 200 5 -1

8. ]

-1 ‡ 4 150

9. .min

† -1 3 10. 100 2 11. 50 1

12. [mg.kg EGP

Plasma glucose [mg.dL glucose Plasma 0 0 13. LG SG LG SG 14. Figure 2. Glucose metabolism during low and standard glucose intake in eight post-surgical 15. infants. LG = Low glucose, SG = Standard glucose, EGP = Endogenous glucose production. 16. Panel A. Plasma glucose concentration, mg.dL‑1, mean ± SD, † p = .02, Panel B. Endogenous glucose production mg.kg‑1.min‑1, mean ± SD, ‡ p = .05. 17. 18. Glucose kinetics 19. The deuterium enrichment of body water was 0.59 ± 0.02 and 0.58 ± 0.03 MPE, at LG 20. and SG respectively. Glucose rate of appearance (Ra) did not differ between glucose 21. protocols (Table 2). Endogenous glucose production (EGP) increased during LG (Figure 22. 2, Table 2). Fractional gluconeogenesis was higher during LG. Absolute gluconeogen- 23. esis and glycogenolysis were not significantly different (p =.08) and glycogenolysis was 24. not significantly different from zero (Table 2). 25. 26. Table 2. Glucose kinetics in eight post-surgical infants during two different glucose intakes.* £ £ 27. LG SG p value Glucose Ra^ (mg.kg‑1.min‑1) 5.3 ± 1.5 6.4 ± 1.5 .14 28. Endogenous Glucose Production (mg.kg‑1.min‑1) 2.6 ± 1.5 1.1 ± 1.4 .05 29. Fractional gluconeogenesis (% of Ra^) 43 ± 2 29 ± 7 < .01 30. Absolute Gluconeogenesis (mg.kg‑1.min‑1) 2.3 ± 0.6 1.8 ± 0.4 .08 31. Glycogenolysis (mg.kg‑1.min‑1) 0.3 ± 0.9 -0.7 ± 1.1 .08 32. *All values are depicted as mean ± SD), ^Ra; rate of appearance, £LG; Low glucose, SG; Standard glucose 33. 34. 35. Leucine flux and oxidation rates 36. Leucine Ra and Leucine oxidation were not significantly affected by the change in 37. glucose intake. As a result, NOLD did not differ between glucose intake rates (Table 3). 38. 39.

74 Reduced glucose intake prevents hyperglycemia

Table 3. Leucine, phenylalanine and tyrosine kinetics in eight post-surgical infants during two 1. different glucose intakes.*

2. £LG £SG P value

3. Leucine Ra ^ 160 ± 15 158 ± 16 .68 4. Leucine oxidation 33 (22-82) 29 (22-62) .38 5. NOLD † 122 ± 14 124 ± 20 .76 6. Phenylalanine Ra 70 ± 6 68 ± 5 .25 7. Tyrosine Ra 39 ± 2 37 ± 2 .06 8. Phenylalanine hydroxylation 8.4 ± 1.7 7.4 ± 1.6 .04 9. NHPD ‡ 61 ± 6 61 ± 6 1.0 ‑1 ‑1 £ £ 10. *All values are measured in µmol.kg .h and depicted in mean ± SD, ^Ra; rate of appearance, LG; Low glucose, SG; Standard glucose, †NOLD; non-oxidative leucine disposal, ‡ NHPD; non-hydroxylation phenylalanine disposal 11. 12. Phenylalanine and tyrosine flux and hydroxylation rates 4 13. Phenylalanine Ra and tyrosine Ra did not differ. Phenylalanine hydroxylation was sig- 14. nificantly higher at the lower glucose intake rate (Table 3). However, non-hydroxylative 15. phenylalanine disposal (NHPD) did not differ between glucose intake rates (Table 3). 16. The phenylalanine hydroxylation fraction of the total phenylalanine Rd did not differ (12 17. ± 3% vs. 11 ± 3%; LG vs. SG, p = .07). 18. 19. Protein metabolism 20. Whole body protein metabolism derived from with leucine kinetics was as follows (Fig- 21. ure 3). Whole body protein synthesis (4.7 ± 0.5 vs. 4.8 ± 0.8 g.kg‑1.d‑1) and breakdown 22. (5.9 ± 0.6 vs. 5.8 ± 0.6 g.kg‑1.d‑1) did not differ between LG and SG respectively. The 23. whole body protein balance was negative and did not differ between LG and SG (-1.2 24. ± 0.8 vs. -1.0 ± 0.6 g.kg‑1.d‑1, p = .38). Whole body protein metabolism correspond- 25. ing with phenylalanine and tyrosine kinetics was as follows (Figure 3). Whole body 26. protein synthesis (5.3 ± 0.6 vs. 5.2 ± 0.5 g.kg‑1.d‑1) and breakdown (5.6 ± 0.5 vs. 5.4 ± 27. 28. A B C ] ] ] -1 -1 LeuPhe LeuPhe 29. -1 .d .d 0.0 8 .d -1 8 -1 30. -1 31. 6 6 -0.5 †

32. 4 4 -1.0 33. 2 2 -1.5 34. 0 0 -2.0

35. LeuPhe LeuPhe LeuPhe LeuPhe balanceProtein [g.kg Protein synthesis [g.kg synthesisProtein LG SG breakdownProtein [g.kg LG SG LG SG 36. 37. Figure 3. Whole body protein metabolism during low and standard glucose intake in 38. eight post-surgical infants. LG = Low glucose, SG = Standard glucose, Leu = leucine, Phe = Phenylalanine. Panel A. protein synthesis, Panel B. protein breakdown, Panel C. protein 39. balance; Values are g.kg‑1.d‑1, mean ± SD, † p = .04; LG vs. SG

75 Chapter 4

1. 0.4 g.kg‑1.d‑1) did not differ between LG and SG respectively. The whole body protein 2. balance was negative and showed a statistically significant but not relevant difference 3. (-0.3 ± 0.1 vs. -0.2 ± 0.1 g.kg‑1.d‑1; LG vs. SG, p =.04) (Figure 3). 4. 5. 6. Discussion 7. 8. Tight glucose control improves morbidity and mortality in critically ill children, although 9. hypoglycemia is a frequent and serious side effect of insulin therapy9. In this study 10. we showed that in post-surgical children, normoglycemia could safely be achieved 11. by a reduced glucose intake, without occurrence of hypoglycemia. The low glucose 12. intake (LG; 2.5 mg.kg‑1.min‑1), half of what is considered standard practice for age21, 13. can be considered an alternative to insulin therapy in the initial phase of glycemic 14. management. Additionally we observed that endogenous glucose production was not 15. fully suppressed, despite high plasma glucose levels. Moreover, reducing the glucose 16. intake induced an increase in endogenous glucose production due to an increase of 17. both gluconeogenesis as well as glycogenolysis. Furthermore, a reduced glucose did 18. not notably affect the negative whole body protein balance, measured with leucine and 19. phenylalanine kinetics. 20. Our study is the first to show the feasibility of reduced glucose intake in post surgical 21. children to prevent or treat hyperglycemia. We found that during LG plasma glucose 22. levels were lower and normoglycemic, not due to an increased rate of disappearance or 23. decreased glucose production, but solely through a reduced glucose infusion rate. Due 24. to the paucity of data in children it is difficult to define lower limits of glucose intake. 25. The current recommendations are based largely upon data for infants and children and 26. extracted from the relation between glucose intake and 1) glucose uptake by the brain, 27. 2) glucose oxidation, 3) endogenous glucose production (EGP), and 4) protein and 28. amino acid catabolism21. Although conditions are different in post-surgical children, 29. we did use these considerations to determine whether LG was within safe limits in our 30. population. We acknowledge that we did not quantify cerebral glucose uptake. Addi- 31. tionally, exact determination of glucose oxidation with [13C] tracer data was not possible 32. in our study, because of interference with our [13C]Leucine tracer. Therefore, we cannot 33. provide clear insight in the impact of LG on cerebral glucose uptake and utilization or 34. oxidation. However, glucose oxidation calculated from the oxygen consumption and 35. carbon dioxide production did not change. Of greater importance, hypoglycemia did 36. not occur during low glucose intake. This might suggest that a reduced glucose intake 37. did not negatively impact cerebral glucose uptake or utilization. 38. Endogenous glucose production rates in our infants, receiving 2.5 mg.kg‑1.min‑1, were 39. lower than those measured in healthy infants, fasted for 8 – 9 h (7.1 ± 0.3 mg.kg‑1.

76 Reduced glucose intake prevents hyperglycemia

1. min‑1)39. So, although an increased EGP was observed during LG, their production 2. was not at their full potential. During both glucose intakes glycogenolysis rates were 3. futile. Glycogenolysis was probably suppressed by elevated insulin levels and sup- 4. pressed glucagon levels during glucose infusion40, although the latter could not be 5. measured in our study due to blood draw limitations in our infants. Considering the 6. exogenous glucose our patients received it is not likely that glycogen stores were 7. already depleted. Therefore, it appears safe to conclude that our infants were capable 8. of sustaining normoglycemia, without a direct risk of developing hypoglycemia. 9. Of interest, even during SG, despite supraphysiological glucose and insulin levels, EGP 10. was not fully suppressed. During both glucose intakes, gluconeogenesis constituted 11. almost all of the EGP and it appears that gluconeogenesis in contrast to glycogenolysis 12. was unresponsive to insulin, high glucose concentrations and/or intake. These obser- 4 13. vations might indicate hepatic insulin resistance in our patients. A clear association 14. exists between increased contribution of gluconeogenesis and insulin resistance41. The 15. expression of phosphoenolpyruvate carboxy kinase (PEPCK), the rate-limiting enzyme 16. of gluconeogenesis, is increased and less sensitive to insulin during critical illness42. 17. Therefore, we hypothesize that the increase in EGP and moreover gluconeogenesis 18. during LG does not indicate that low glucose intake was below the threshold, but that 19. it rather indicates a certain state of insulin resistance in these post-surgical infants. 20. We did not find relevant differences in amino acid or protein metabolism during low 21. glucose intake. Although LG did not affect leucine kinetics and more specifically 22. leucine oxidation, phenylalanine hydroxylation was slightly but statistically significant 23. higher during LG. Increased availability of plasma amino acids, a known stimulant of 24. hydroxylation43‑45, is not a likely cause for this increase as our patients did not receive 25. amino acids and their proteolysis was not increased. One might hypothesize that the 26. increased hydroxylation of phenylalanine is a physiological response to cope with a 27. shortage of energy, providing energy by oxidation of the carbon skeleton of amino 28. acids. However, this does not explain the indifference in leucine oxidation which would 29. be expected to rise as well. Possibly, phenylalanine catabolism was induced to provide 30. gluconeogenic precursors. Products derived from phenylalanine hydroxylation can be 31. used for gluconeogenesis, while oxidized leucine can merely be used for the ketogenic 32. pathway46. This could explain the difference in catabolism between phenylalanine 33. and leucine. Although not statistically different, we did observe a trend towards an 34. increased gluconeogenesis during LG. 35. Whole body protein balance corresponding with leucine and phenylalanine was slightly 36. negative during both study periods and consistent with those obtained from septic 37. infants and children36, 37. In contrast with our data, normoglycemia, independent of 38. plasma insulin levels, improved protein turnover after abdominal surgery in adults47. 39. Our different results may be explained by differences in patient populations and the

77 Chapter 4

1. approach used. Furthermore, there are tissue and age specific differences in the meta- 2. bolic processes48. However, consistent with our data, it has been shown in neonates 3. that different glucose intakes do not affect protein turnover49. Although under physi- 4. ological conditions a clear relation exists between energy and protein metabolism, it 5. has been recognized that a deficiency in energy supply is not solely responsible for 6. protein catabolism during critical illness50. Proteolysis during critical illness is usually 7. caused by activation of the ubiquitin-proteasome proteolytic pathway (UPP) in muscle 8. initiated by activation of caspase 351, 52, and pathophysiological triggers include activa- 9. tion of lysosome-53 and calpain-dependent pathways54. In addition, there appears to be 10. a link between muscle wasting and insulin resistance55‑57. 11. There are some limitations to our study which need to be taken into account, some 12. of them inherent to studying critically ill children. Our sample size was small and con- 13. clusions from our study are restricted to post-surgical infants. Substrate metabolism 14. greatly differs between infants, children and adolescents both for glucose and amino 15. acid metabolism21, 48. Furthermore, various diagnoses, such as trauma, burns or sepsis, 16. as well as differences between single and multi organ failure yield a different response. 17. We must also note that the negative glycogenolysis rates are physiological not pos- 18. sible. This is a consequence of a consistent underestimation of the EGP by the glucose 19. tracer model, as part of the diluted tracer pool due to newly produced glucose that is 20. taken up by the liver again58. 21. 22. 23. Conclusion 24. 25. Reduced glucose intake, half of what is considered standard practice for age, in the initial 26. post-surgical phase safely prevented hyperglycemia in infants without occurrence of 27. hypoglycemia. Additionally we observed that the post-surgical infants appeared insulin 28. resistant, where endogenous glucose production was not fully suppressed and almost 29. entirely relied on gluconeogenesis, despite high plasma glucose levels and exogenous 30. glucose infusion. Furthermore, protein and amino acid catabolism was present but 31. not exacerbated during the reduced glucose intake. A reduced glucose intake can be 32. considered as an alternative to insulin therapy in the initial phase of glycemic manage- 33. ment. Further studies on (long term) clinical implications are warranted. 34. 35. 36. Acknowledgements 37. 38. Our gratitude goes out to the patients and their families at Erasmus MC Sophia Chil- 39. dren’s hospital for their selfless contribution to this study. We thank the nursing staff,

78 Reduced glucose intake prevents hyperglycemia

1. Marianne Maliepaard, Hansje Bredero-Boelhouwer, Gardi Minderman-Voortman and 2. Kristien Dorst for their contribution. 3. 4. 5. References 6. 7. 1. Zijlmans WC, van Kempen AA, Serlie randomised controlled study. Lancet 8. MJ, Sauerwein HP. Glucose metabolism 2009;​373(9663):​547‑56. 9. in children: influence of age, fasting, and 10. Bober E, Buyukgebiz A. Hypoglycemia infectious diseases. Metabolism 2009;​ and its effects on the brain in children 10. 58(9):​1356‑65. with type 1 diabetes mellitus. Pediatr 11. 2. Verbruggen SC, Joosten KF, Castillo L, Endocrinol Rev 2005;​2(3):​378‑82. 12. van Goudoever JB. Insulin therapy in the 11. Vannucci RC, Vannucci SJ. Glucose me- 4 13. pediatric intensive care unit. Clin Nutr tabolism in the developing brain. Semin 14. 2007;​26(6):​677‑90. Perinatol 2000;​24(2):​107‑15. 3. Van den Berghe G, Wilmer A, Hermans 12. Flykanaka-Gantenbein C. Hypoglycemia 15. G, et al. Intensive insulin therapy in the in childhood: long-term effects. Pediatr 16. medical ICU. N Engl J Med 2006;​354(5):​ Endocrinol Rev 2004;​1 Suppl 3:​530‑6. 17. 449‑61. 13. Solomon T, Felix JM, Samuel M, et al. 18. 4. van den Berghe G, Wouters P, Weekers Hypoglycaemia in paediatric admissions 19. F, et al. Intensive insulin therapy in the in Mozambique. Lancet 1994;​343(8890):​ 20. critically ill patients. N Engl J Med 2001;​ 149‑50. 345(19):​1359‑67. 14. Chaussain JL, Georges P, Calzada L, Job 21. 5. Finfer S, Chittock DR, Su SY, et al. JC. Glycemic response to 24-hour fast 22. Intensive versus conventional glucose in normal children: III. Influence of age. J 23. control in critically ill patients. N Engl J Pediatr 1977;​91(5):​711‑4. 24. Med 2009;​360(13):​1283‑97. 15. Joosten K, Verbruggen SC, Verhoeven 25. 6. Preiser JC, Devos P, Ruiz-Santana S, et JJ. Glycaemic control in paediatric criti- al. A prospective randomised multi-centre cal care. Lancet 2009;​373(9673):​1423-4; 26. controlled trial on tight glucose control by author reply 4. 27. intensive insulin therapy in adult intensive 16. Preissig C, Rigby M. Glycaemic control 28. care units: the Glucontrol study. Intensive in paediatric critical care. Lancet 2009;​ 29. Care Med 2009;​35(10):​1738‑48. 373(9673):​1423; author reply 4. 30. 7. Wilson M, Weinreb J, Hoo GW. Intensive 17. Leteurtre S, Martinot A, Duhamel A, et al. 31. insulin therapy in critical care: a review of Validation of the paediatric logistic organ 12 protocols. Diabetes Care 2007;​30(4):​ dysfunction (PELOD) score: prospective, 32. 1005‑11. observational, multicentre study. Lancet 33. 8. Wiener RS, Wiener DC, Larson RJ. Ben- 2003;​362(9379):​192‑7. 34. efits and risks of tight glucose control in 18. Shann F, Pearson G, Slater A, Wilkinson 35. critically ill adults: a meta-analysis. Jama K. Paediatric index of mortality (PIM): a 36. 2008;​300(8):​933‑44. mortality prediction model for children in 9. Vlasselaers D, Milants I, Desmet L, et al. intensive care. Intensive Care Med 1997;​ 37. Intensive insulin therapy for patients in 23(2):​201‑7. 38. paediatric intensive care: a prospective, 39.

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19. Slater A, Shann F, Pearson G. PIM2: a radioactive glucose. Am J Physiol 1956;​ 1. revised version of the Paediatric Index of 187(1):​25‑31. 2. Mortality. Intensive Care Med 2003;​29(2):​ 29. Steele R, Wall JS, De Bodo RC, Altszuler 3. 278‑85. N. Measurement of size and turnover rate 4. 20. Pollack MM, Patel KM, Ruttimann UE. of body glucose pool by the isotope dilu- 5. PRISM III: an updated Pediatric Risk tion method. Am J Physiol 1956;​187(1):​ of Mortality score. Crit Care Med 1996;​ 15‑24. 6. 24(5):​743‑52. 30. Matthews DE, Motil KJ, Rohrbaugh DK, 7. 21. Kalhan SC, Kilic I. Carbohydrate as Burke JF, Young VR, Bier DM. Measure- 8. nutrient in the infant and child: range of ment of leucine metabolism in man from 9. acceptable intake. Eur J Clin Nutr 1999;​ a primed, continuous infusion of L-[1-3C] 10. 53 Suppl 1:​S94-100. leucine. Am J Physiol 1980;​238(5):​ 22. Argoud GM, Schade DS, Eaton RP. Un- E473‑9. 11. derestimation of hepatic glucose produc- 31. Sy J, Gourishankar A, Gordon WE, et al. 12. tion by radioactive and stable tracers. Bicarbonate kinetics and predicted en- 13. Am J Physiol 1987;​252(5 Pt 1):​E606‑15. ergy expenditure in critically ill children. 14. 23. Chacko SK, Sunehag AL, Sharma S, Am J Clin Nutr 2008;​88(2):​340‑7. 15. Sauer PJ, Haymond MW. Measurement 32. Clarke JT, Bier DM. The conversion of 16. of gluconeogenesis using glucose frag- phenylalanine to tyrosine in man. Direct ments and mass spectrometry after measurement by continuous intravenous 17. ingestion of deuterium oxide. J Appl tracer infusions of L-[ring-2H5]phenyl- 18. Physiol 2008;​104(4):​944‑51. alanine and L-[1-13C] tyrosine in the 19. 24. Matthews DE, Schwarz HP, Yang RD, postabsorptive state. Metabolism 1982;​ 20. Motil KJ, Young VR, Bier DM. Relation- 31(10):​999-1005. 21. ship of plasma leucine and alpha-ke- 33. Thompson GN, Pacy PJ, Merritt H, et al. toisocaproate during a L-[1-13C]leucine Rapid measurement of whole body and 22. infusion in man: a method for measuring forearm protein turnover using a [2H5] 23. human intracellular leucine tracer enrich- phenylalanine model. Am J Physiol 1989;​ 24. ment. Metabolism 1982;​31(11):​1105‑12. 256(5 Pt 1):​E631‑9. 25. 25. Husek P. Amino acid derivatization and 34. Marchini JS, Castillo L, Chapman TE, Vogt 26. analysis in five minutes. FEBS Lett 1991;​ JA, Ajami A, Young VR. Phenylalanine 27. 280(2):​354‑6. conversion to tyrosine: comparative deter- 26. el-Khoury AE, Sanchez M, Fukagawa NK, mination with L-[ring-2H5]phenylalanine 28. Young VR. Whole body protein synthesis and L-[1-13C]phenylalanine as tracers in 29. in healthy adult humans: 13CO2 tech- man. Metabolism 1993;​42(10):​1316‑22. 30. nique vs. plasma precursor approach. 35. Marchini JS, Cortiella J, Hiramatsu T, 31. Am J Physiol 1995;​268(1 Pt 1):​E174‑84. Castillo L, Chapman TE, Young VR. 32. 27. Spear ML, Darmaun D, Sager BK, Par- Phenylalanine and tyrosine kinetics for sons WR, Haymond MW. Use of [13C] different patterns and indispensable 33. bicarbonate infusion for measurement amino acid intakes in adult humans. Am 34. of CO2 production. Am J Physiol 1995;​ J Clin Nutr 1994;​60(1):​79‑86. 35. 268(6 Pt 1):​E1123‑7. 36. Argaman Z, Young VR, Noviski N, et al. 36. 28. Altszuler N, De Bodo RC, Steele R, Wall Arginine and nitric oxide metabolism in 37. JS. Carbohydrate metabolism of hy- critically ill septic pediatric patients. Crit 38. pophysectomized dogs as studied with Care Med 2003;​31(2):​591‑7. 39.

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37. Castillo L, Yu YM, Marchini JS, et al. 46. Garrett RHCMG. Principles of Biochem- 1. Phenylalanine and tyrosine kinetics in istry with a Human Focus.: USA: Brooks/ 2. critically ill children with sepsis. Pediatr Cole; 2002. 3. Res 1994;​35(5):​580‑8. 47. Biolo G, De Cicco M, Lorenzon S, et al. 4. 38. Golden MH, Waterlow JC. Total protein Treating hyperglycemia improves skeletal 5. synthesis in elderly people: a comparison muscle protein metabolism in cancer of results with [15N]glycine and [14C] patients after major surgery. Crit Care 6. leucine. Clin Sci Mol Med 1977;​53(3):​ Med 2008;​36(6):​1768‑75. 7. 277‑88. 48. Verbruggen S, Sy J, Gordon WE, et 8. 39. Bier DM, Leake RD, Haymond MW, et al. al. Ontogeny of methionine utilization 9. Measurement of “true” glucose produc- and splanchnic uptake in critically ill 10. tion rates in infancy and childhood with children. Am J Physiol Endocrinol Metab 6,6-dideuteroglucose. Diabetes 1977;​ 2009(297):​E1046-E55. 11. 26(11):​1016‑23. 49. Hertz DE, Karn CA, Liu YM, Liechty 12. 40. Grasso S, Fallucca F, Mazzone D, EA, Denne SC. Intravenous glucose 4 13. Giangrande L, Romeo MG, Reitano G. suppresses glucose production but not 14. Inhibition of glucagon secretion in the proteolysis in extremely premature new- 15. human newborn by glucose infusion. borns. J Clin Invest 1993;​92(4):​1752‑8. 16. Diabetes 1983;​32(6):​489‑92. 50. Jahoor F, Shangraw RE, Miyoshi H, Wall- 41. Boden G, Chen X, Stein TP. Gluconeo- fish H, Herndon DN, Wolfe RR. Role of 17. genesis in moderately and severely insulin and glucose oxidation in mediat- 18. hyperglycemic patients with type 2 dia- ing the protein catabolism of burns and 19. betes mellitus. Am J Physiol Endocrinol sepsis. Am J Physiol 1989;​257(3 Pt 1):​ 20. Metab 2001;​280(1):​E23‑30. E323‑31. 21. 42. Van den Berghe G. How does blood 51. Lecker SH, Solomon V, Mitch WE, glucose control with insulin save lives in Goldberg AL. Muscle protein breakdown 22. intensive care? J Clin Invest 2004;​114(9):​ and the critical role of the ubiquitin- 23. 1187‑95. proteasome pathway in normal and dis- 24. 43. Cortiella J, Marchini JS, Branch S, Chap- ease states. J Nutr 1999;​129(1S Suppl):​ 25. man TE, Young VR. Phenylalanine and 227S-37S. 26. tyrosine kinetics in relation to altered 52. Voisin L, Breuille D, Combaret L, et al. 27. protein and phenylalanine and tyrosine Muscle wasting in a rat model of long- intakes in healthy young men. Am J Clin lasting sepsis results from the activation 28. Nutr 1992;​56(3):​517‑25. of lysosomal, Ca2+ -activated, and 29. 44. Young VR, Marchini JS. Mechanisms and ubiquitin-proteasome proteolytic path- 30. nutritional significance of metabolic re- ways. J Clin Invest 1996;​97(7):​1610‑7. 31. sponses to altered intakes of protein and 53. Deval C, Mordier S, Obled C, et al. 32. amino acids, with reference to nutritional Identification of cathepsin L as a differ- adaptation in humans. Am J Clin Nutr entially expressed message associated 33. 1990;​51(2):​270‑89. with skeletal muscle wasting. Biochem J 34. 45. Tessari P, Kiwanuka E, Zanetti M, Baraz- 2001;​360(Pt 1):​143‑50. 35. zoni R. Postprandial body protein synthe- 54. Smith IJ, Lecker SH, Hasselgren PO. 36. sis and amino acid catabolism measured Calpain activity and muscle wasting in 37. with leucine and phenylalanine-tyrosine sepsis. Am J Physiol Endocrinol Metab 38. tracers. Am J Physiol Endocrinol Metab 2008;​295(4):​E762‑71. 2003;​284(5):​E1037‑42. 39.

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55. Wang X, Hu Z, Hu J, Du J, Mitch WE. 57. Crossland H, Constantin-Teodosiu D, 1. Insulin resistance accelerates muscle Greenhaff PL, Gardiner SM. Low-dose 2. protein degradation: Activation of the dexamethasone prevents endotoxaemia- 3. ubiquitin-proteasome pathway by de- induced muscle protein loss and impair- 4. fects in muscle cell signaling. Endocri- ment of carbohydrate oxidation in rat 5. nology 2006;​147(9):​4160‑8. skeletal muscle. J Physiol 2010;​588(Pt 56. Crossland H, Constantin-Teodosiu D, 8):​1333‑47. 6. Gardiner SM, Constantin D, Greenhaff 58. Burns SP, Cohen RD. To: Krebs M, 7. PL. A potential role for Akt/FOXO signal- Brehm A, Krssak M et al. (2003) Direct 8. ling in both protein loss and the impair- and indirect effects of amino acids on 9. ment of muscle carbohydrate oxidation hepatic glucose metabolism in humans. 10. during sepsis in rodent skeletal muscle. Diabetologia 46:​917-925. Diabetologia J Physiol 2008;​586(Pt 22):​5589-600. 2004;​47(1):​141; author reply 2‑3. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39.

82 Part II Amino Acid metabolism

Chapter 5

Ontogeny of methionine utilization and splanchnic uptake in critically ill children

Sascha CAT Verbruggen Jama Sy William E Gordon Jean Hsu Manhong Wu Shaji Chacko David Zurakowski Douglas G Burrin Leticia Castillo

Am J Physiol Endocrinol Metab. 2009;297:E1046-E1055 Chapter 5

1. Abstract 2. 3. To determine the rates of methionine splanchnic uptake and utilization in critically ill 4. pediatric patients we used two kinetic models: the plasma methionine enrichment and 5. the “intracellular” homocysteine enrichment. Twenty four patients, eight infants, eight 6. children and eight adolescents were studied. They received simultaneous, primed, 2 13 7. constant, intravenous infusions of L-[ H3] methyl methionine and enteral L-[1- C] 8. methionine. The ratio of [13C]homocysteine to [13C]methionine enrichment (13C Hcy/13C 9. meth) was 1.0±0.15, 0.80±0.20 and 0.66±0.10, respectively for the infants, children 10. and adolescents, and different between the infants and adolescents (p < 0.01). Methio- 11. nine splanchnic uptake was 63, 45 and 36% respectively in the infants, children and 12. adolescents, and higher (p < 0.01) in the infants when compared to the adolescents. 13. The infants utilized 73% of methionine flux for non-oxidative disposal (NOD), while 14. 27% was used for transsulfuration (p < 0.001). Conversely, in the adolescents 40% 15. was utilized for NOD while 60% was used for transsulfuration. There is ontogeny on 16. the rates of methionine splanchnic uptake and on the fate of methionine utilization in 17. critically ill children, with greater methionine utilization for synthesis of proteins and 18. methionine-derived compounds (p < 0.01) and decreased transsulfuration rates in the 19. infants (p < 0.01), while the opposite was observed in the adolescents. The plasma 20. model underestimated methionine kinetics in children and adolescents, but not in 21. the infants, suggesting lesser dilution and greater compartmentation of methionine 22. metabolism in the infant population. All patients were in negative methionine balance, 23. indicating that the current enteral nutritional support is inadequate in these patients. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39.

86 Methionine splanchnic uptake in children

1. Introduction 2. 3. The portal-drained viscera involving the liver, stomach and intestines is profoundly 4. deranged during critical illness. Altered splanchnic function, whether elicited by hypo- 5. perfusion, hypoxia or inflammation has been associated with systemic inflammatory 6. response syndrome (SIRS) and multiple organ dysfunction syndrome (MODS)1, 2. Under 7. physiological conditions, the splanchnic tissues are important for whole body amino 8. acid metabolism and selectively modify the pattern of absorbed amino acids that 9. is subsequently presented to the peripheral circulation3. Thus, the pattern of amino 10. acids in the diet is different from that in portal venous blood and does not reflect 11. their availability to extraintestinal tissues. This concept has important implications for 12. defining protein and amino acid requirements for nutritional and functional purposes. 13. An extensive catabolism and/or utilization of dietary amino acids in the first pass by 14. the small intestine results in decreased nutritional efficiency and will influence their 15. requirements. The sulfur amino acids serve important protein and non-protein func- 16. tions4‑7. Methionine, an indispensable sulfur amino acid serves as the precursor in 5 17. the synthesis of S-adenosylmethionine (AdoMet), which through the transmethylation 18. pathway is the primary methyl group donor for methylation reactions involved in signal 19. transduction, protein repair, chromatin regulation and gene silencing4. Methyl groups 20. are required for biosynthesis of polyamines, choline, creatine, DNA and RNA intermedi- 21. ates4. Homocysteine, the demethylated product of methionine can be utilized through 22. the transsulfuration pathway and serve as precursor for cystathionine and cysteine, 23. which under physiological conditions is a limiting precursor in the synthesis of the 24. tripeptide Glutathione (γ-glutamyl-cysteinyl-glycine;GSH), a major antioxidant with 25. detoxifying and signaling properties that serves a key role in the control of apoptosis 26. and inflammation8. Alternatively homocysteine can be remethylated to methionine with 27. betaine or methyltetrahydrofolate as methyl donors4‑7, 9. Therefore, the sulfur amino 28. acids have major functional significance. 29. It is known that the splanchnic area is greatly affected during critical illness, but the 30. uptake of methionine by the splanchnic area under these conditions has not been de- 31. termined. Given the limited knowledge on the nutritional support provided to pediatric 32. critically ill patients, and the nutritional and functional significance of methionine, it is 33. important to examine the quantitative impact of the splanchnic area on methionine 34. availability to the peripheral tissues under conditions of critical illness, and whether 35. this is affected by age. Therefore, the objectives of these studies were: i) to determine 36. the rates of methionine first-pass disappearance and oxidation (transsulfuration) by 37. the splanchnic tissues of critically ill children by conducting simultaneous primed, con- 13 2 38. tinuous, enteral and intravenous tracer infusions of L-[ C] and L-[ H3] methyl labels of 39. methionine ii) To determine the difference between two kinetic models: The frequently

87 Chapter 5

1. used plasma model which uses plasma methionine enrichment and the intracellular 2. model using plasma homocysteine enrichment. Because plasma homocysteine and 3. cystathionine arise from intracellular metabolism of methionine, enrichment of these 4. substrates can be used to define intracellular methionine enrichment during an infu- 5. sion of labeled methionine. Therefore plasma homocysteine enrichment serves as an 6. indicator of “true” intracellular methionine enrichment10 and iii) we aimed to ascertain 7. whether the developmental stage from young infants to adolescents affects the rates of 8. methionine splanchnic uptake and its utilization through different metabolic pathways. 9. 10. 11. Material and Methods 12. 13. Subjects 14. These studies were conducted at the Pediatric Intensive Care Unit (PICU), Texas 15. Children’s Hospital, Baylor College of Medicine and at the Children’s Nutrition Re- 16. search Center, USDA. Twenty four consecutive critically ill children admitted to the 17. PICU receiving enteral feedings, supplying complete protein and energy needs as 18. per recommended standard of care, for at least 24 hours previous to the study were 19. included. All patients were studied when hemodynamically stable. Three age groups 20. were included: Infants 0-12 months, children >1-3 years and adolescents 13-18 years. 21. In the infant group all patients had diagnosis of respiratory failure, five of them second- 22. ary to respiratory syncitial virus (RSV) bronchiolitis, and two patients had influenza A 23. pneumonia. Another patient underwent a surgical procedure complicated by pneumo- 24. nia. All infants had been receiving enteral feedings for an average of 8±5 days, and 25. all had received complete feedings for at least 24 hours before the study. Six infants 26. required mechanical ventilation, but only 2 patients required an inspiratory fraction of

27. oxygen (FiO2) greater than 0.6. One patient required vasopressin to maintain blood 28. pressure. In the group of children >1 to 3 years the main diagnoses were respira- 29. tory failure secondary to Parainfluenza or influenza A pneumonia in 6 patients, septic 30. shock due to methicillin resistant staphylococci aureus (MRSA) in one patient and 31. acute lung injury secondary to aspiration pneumonia and viral sepsis in one patient. 32. Six children received mechanical ventilation and two patients required non-invasive 33. ventilatory support. One child required high frequency oscillatory ventilation and all

34. others received conventional ventilation. The FiO2 was 0.6 or below and none received 35. vasopressors. They had been receiving feedings for an average of 9 ± 3 days, provid- 36. ing complete nutritional support as per current clinical standard for at least 24 hours 37. previous to the study. In the adolescent group the main diagnoses were neurological 38. conditions such as seizures and aspiration pneumonia, post-surgical patients from 39. brain tumor resection and acute lung injury, post-tracheal reconstruction and pneu-

88 Methionine splanchnic uptake in children

1. monia. Two previously healthy adolescents had MRSA sepsis. Six were mechanically

2. ventilated on a conventional mode with less than 0.6 FiO2. Two patients were breathing 3. spontaneously. All adolescents were studied when hemodynamically stable and none 4. were receiving vasopressors at the time of the study. The study was approved by 5. the Baylor College of Medicine Institutional Review Board, and informed consent was 6. obtained from parents or guardians. All patients had drawing and infusing intravascular 7. lines and a post-pyloric feeding tube placed for clinical indication, and all had received 8. full enteral feedings for at least 24 hours. All were assessed for severity of disease by 9. the pediatric risk mortality score (PRISM III)11, which predicts mortality rates in relation 10. to acuity of disease. High scores indicate higher probability of mortality. Patients with 11. metabolic diseases, diabetes mellitus, primary liver or renal failure, as well as those 12. requiring renal replacement therapies were excluded. The demographic characteristics 13. of the patients are shown in Table 1. Because of ethical constraints, studies in healthy 14. infants and children were not conducted. 15. 16. Table 1. Patient characteristics 5 17. Infants Children Adolescents 18. Variable n = 8 n = 8 n = 8 Sex (male : female) 5 : 3 3 : 5 5 : 3 19. Age, yr 0.5 ± 0.3 2.8 ± 1.3 15.0 ± 2.0 20. Weight, kg 5.5 ± 1.5 13.6 ± 3.1 63.3 ± 23 21. BMI, kg/m2 27.9 ± 9.0 22. PRISM III 3.0 ± 3.0 8.0 ± 2.9 6.0 ± 5.0 23. All values are means ± SD. BMI, body mass index; PRISM III, pediatric risk mortality score 24. 25. Diets 26. Patients received nutritional support according to current standard clinical practice. All 27. patients were receiving complete enteral nutrition supplied through continuous 24 hour 28. feedings, via a nasojejunal tube placed for clinical indication. The position of the tube 29. was confirmed by x-ray. Protein and energy intake in all groups was directed by the 30. attending physician(s) as per standard clinical care, in collaboration with the Nutrition 31. Service. In the infant group, the formulas used were Enfamil (Meadjohnson, Atlanta, GA) 32. in 4 patients, Prosobee (Meadjohnson, Atlanta, GA) in one patient, Goodstart (Nestle, 33. Oakland CA) in one patient, Pregestamil (Meadjohnson, Atlanta, GA) in one patient and 34. Portagen (Meadjohnson, Atlanta, GA) in one patient. In the children group, all patients 35. received Pediasure (Abbott Laboratories, Grove city, OH). In the adolescent group 2 36. patients received Ensure (Abbott Laboratories, Grove city, OH), 4 patients received 37. Jevity (Abbott Laboratories, Grove City, OH) and Osmolyte (Abbott Laboratories, Grove 38. city, OH) was supplied to 2 patients. The protein and specific methionine and cysteine 39. content of the formulas supplied were recorded and they are shown in Table 2. Average

89 Chapter 5

Table 2. Enteral nutritional support 1. Kcal.kg‑1.day‑1 Glucose, Protein, Lipids, Methionine, Cysteine, Total Sulfur Amino 2. g.kg‑1. g.kg‑1. g.kg‑1. mg.kg‑1.day‑1 mg.kg‑1.day‑1 Acid Intake, day‑1 day‑1 day‑1 mg.kg‑1.day‑1 3. 0-1 yr (n = 8) 87.6 ± 28.0 9.3 ± 3.4 2.3 ± 1.0 4.4 ± 1.5 51.1 ± 34.8 19.9 ± 12.4 70.9 ± 40.0 4. 1-3 yr (n = 8) 66.1 ± 9.7 6.8 ± 2.0 2.0 ± 0.3 2.5 ± 0.4 55.2 ± 8.0 17.2 ± 2.6 72.3 ± 8.2 5. 13-18 yr (n = 8) 37.1 ± 15.5 4.0 ± 1.8 1.4 ± 0.6 1.4 ± 1.0 39.2 ± 16.2 11.0 ± 3.9 50.1 ± 19.9 6. All values are mean ± SD. 7. 8. protein intakes were 2.3 ± 1.0, 2.0 ± 0.3 and 1.4 ± 0.6 g.kg‑1.d‑1 while energy intakes 9. were 87.6 ± 28, 66.1 ± 9.7 and 37.1 ± 15.5 kcal.kg‑1.d‑1 respectively, for the infants, 10. children and adolescents. Sulfur amino acid intakes were variable in the infant group 11. due to the variable content of sulfur amino acids in the formulas used, while similar 12. formulas were used among children and adolescents, as shown in Table 2. The total 13. sulfur amino acid (methionine and cysteine) intakes were 70.9 ± 40, 72.3 ± 8 and 50.1 14. ± 19.9 mg.kg‑1.d‑1, respectively for infants, children and adolescents and above the 15. Institute of Medicine12 recommended dietary intakes of 43, 28 and 22 mg.kg‑1.d‑1 for 16. infants, children and adolescents. 17. 18. Materials 13 2 13 19. L-[ C] methionine (99 atom %), L-[ H3 methyl] methionine (99 atom %) and NaH CO3 20. were purchased from Cambridge isotopes (Andover, MA) 21. 22. Tracer Study Protocol 23. The experimental design is shown in Figure 1. Twenty four patients distributed in 24. three groups of 8 patients each, participated in this study. Each patient received one 25. tracer infusion study after they had received complete, continuous, enteral feedings 26. for at least 24 hours. On the day of the study priming doses of L-[13C] methionine at 27. 28. 24 hours continuous enteral feeding via nasojejunal feeding tube

29. Prime 30. -[1-13C]Methionine 13 -NaH CO2 2 31. -[ H3Methyl] Methionine 32. VCO2 measurement VCO2 33. Blood samples 34. 35. Enteral L-[1-13C] Methionine

2 36. Intravenous L-[ H3 Methyl] Methionine 37. 0 460 480 500 520 540 38. Minutes

39. Figure 1. Experimental design. VCO2, rate of CO2 production

90 Methionine splanchnic uptake in children

‑1 13 ‑1 1. 5 µmol.kg and NaH CO3 at 0.8 µmol.kg were administered by the nasojejunal tube 2 2. simultaneously with the enteral feedings. Concurrently L-[ H3 methyl] methionine was 3. intravenously primed at 2.5 µmol.kg‑1, through a pre-existing indwelling intravenous 4. catheter placed for clinical indication. These doses were immediately followed by a 5. 9-hour, continuous nasojejunal infusion of the L-[13C] methionine tracer at 5 µmol.kg‑1. ‑1 2 ‑1 ‑1 6. hr and intravenous infusion of L-[ H3 methyl] methionine at 2.5 µmol.kg .hr , infused 7. by means of calibrated infusion pumps (Gemini PC-2TX infusion pump; Alaris Medical 8. System, San Diego, California). 9. The tracers were prepared in sterile physiological saline solution by the Research 10. Pharmacy at Texas Children’s Hospital and filtered through a 0.22 µm-filter. Aliquotes 11. of the infused solution were collected at the end of the tracer study for determination 12. of infusate concentration. Exact tracer infusion doses were determined from analysis 13. of the amino acid concentrations in the infusates. At times –30 and 0 min, two baseline 14. blood samples were obtained, from a pre-existent intravascular catheter (arterial line) 15. placed for clinical indication, followed by several samples at 500, 520 and 540 minutes 16. for the infants, 480, 500, 520 and 540 minutes for the children and 460, 480, 500, 5 13 17. 520 and 540 for the adolescents. For determination of blood CO2 enrichment it was 18. ensured that air entered neither the blood drawing nor the collecting tube during the 19. blood transfer. Samples of 0.5 ml were transferred from the collection syringe into a 20. 3 ml sodium-heparin-coated, capped, evacuated tube and maintained at room tem- 21. perature. Samples were then processed as previously described13. For the plasma iso- 22. topic enrichment of the methionine tracers given and of derived homocysteine, blood 23. samples were kept on ice immediately centrifuged, and plasma was then withdrawn 24. and stored at -80°C until used for analysis. 25.

26. Measurement of CO2 production rates (VCO2)

27. In the mechanically ventilated patients, the rates of VCO2 (ml/min) were measured 28. with a respiratory monitor (Cosmo, Respironics, Wallingworth, CT). The device was 29. calibrated and directly connected to the endotracheal tube. Continuous measurements 30. were obtained during the tracer study and the average value was recorded. This respi- 31. ratory monitor is routinely used as per standard clinical practice in ventilated patients 32. to adequately monitor pulmonary function. For the spontaneously breathing patients

33. VCO2 was determined by indirect calorimetry using a Vmax Encore (Vyassis Healthcare, 34. Yorbalinda CA), calorimeter, connected to a plastic canopy, which was placed over the 35. head and chest of the patients. None had air leaks through chest tubes. 36. 37. Analytical Methods 13 38. Analyses of blood samples for V CO2 enrichment were all conducted as previously 39. described13. In brief, the carbon dioxide was liberated from the blood bicarbonate

91 Chapter 5

1. by adding 2 mL of 85% (vol/vol) phosphoric acid into the evacuated tube and the 2. contents vortexed. The evacuated tube was then backfilled with nitrogen to bring it to 3. atmospheric pressure and let stand overnight. The liberated carbon dioxide was trans- 4. ferred to a plain, non-silicon coated 15 mL venoject tube, which was subsequently 13 5. backfilled with nitrogen to bring it to atmospheric pressure. The CO2 enrichment was 6. then measured by isotope ratio mass spectrometry (ThermoQuest Finnigan Deltaplus 7. XL Isotope Ratio Mass Spectrometer coupled with Gasbench-II, Bremen, Germany)13. 8. The plasma enrichment of the methionine tracers given was determined as previously 9. described10. In brief, the plasma methionine and homocysteine isotopic enrichments 10. were measured by tandem liquid chromatography-mass spectrometry (LC-MS/MS). 11. Plasma methionine and homocysteine were converted to their 5-(dimethylamino)-1- 12. napthalene sulfonamide (DANS) derivatives and analyzed on a triple quadrupole mass 13. spectrometer, TSQ Quantum Ultra (Thermo Fisher Scientific, San Jose, California), 14. equipped with an electrospray ionization (ESI) source, a Survey pump (Thermo Fisher 15. Scientific), and a HTC PAL autosampler (Leap Technologies Inc, Carrboro, NC, USA). 16. The ions were then analyzed by selected reaction monitoring (SRM) mode. The transi- 17. tions observed were precursor ions m/z 383, 384 and 386 to product ion m/z 170 for 18. methionine and precursor ion m/z 426 and 427 to product ion m/z 170 for homocyste- 19. ine. Instrumental control, data acquisition and analysis were done by XCalibur (version 20. 2.0) software package (Thermo Fisher Scientific). 21. 22. Calculations 23. 24. Plasma Fluxes 25. We used two models to estimate plasma fluxes, splanchnic uptake and oxidation 26. (transsulfuration) of methionine. In the first model, we used plasma methionine enrich- 27. ment as the precursor. In the second model the plasma enrichment of [13C] homocys- 28. teine intracellularly formed from transmethylation of the L-[13C] methionine infused was 2 29. used. This is the intracellular model. The methyl group of L-[ H3] methyl methionine is 30. lost during transmethylation, therefore intracellular homocysteine enrichment derived 2 13 31. from [ H3] methyl methionine tracer is estimated from the ratio of L-[ C] homocysteine 32. to L-[13C] methionine (13C hcy/13C meth), as previously described10. Hence, all calcu- 33. lations were obtained with the plasma methionine and homocysteine enrichments 34. (plasma and intracellular models). Plasma methionine fluxes obtained with the enteral 13 2 35. L-[ C] methionine or the intravenous [ H3] methyl methionine were calculated using the 36. steady-state isotope dilution equation and a simplified single-pool model14 as follows: 37. ‑1 ‑1 38. Ra (µmol.kg .h ) = I x [(Ei/Ep) – 1] (1) 39.

92 Methionine splanchnic uptake in children

13 2 1. Where I is the rate of appearance, I is the rate of enteral [ C] or intravenous [ H3] ‑1 ‑1 2. methylmethionine tracers infused (µmol.kg .hr ), Ei is the enrichment of the labeled 13 3. methionine tracer (99%) and Ep is the mean plasma isotopic enrichment of the [ C] 4. methionine for the plasma model, or [13C] homocysteine for the intracellular model. 2 13 13 5. For the intracellular model of the [ H3] methyl methionine tracer, the Chcy/ Cmeth 10 2 6. ratio was applied, as previously described , while the plasma [ H3]methyl methionine 7. enrichment was used for the plasma model. 8. 9. Splanchnic uptake 10. The splanchnic uptake of dietary methionine was estimated, as previously described3. 11. In brief, the plasma amino acid fluxes obtained with the enteral and intravenously 12. administered tracers, and the fraction of labeled tracer taken up in a first-pass through 13. the splanchnic area, before reaching the systemic circulation, can be determined as 14. follows3: 15. 5 16. Ra [2H3] methylmeth = IIV x [(EiIV/EPIV) – 1] (2) 17. 2 18. Where the rate of appearance or plasma flux from the intravenous [ H3] methyl methio- 19. nine tracer is obtained as in equation 1. 13 20. The rate of appearance or plasma flux from the enteral [ C] methionine tracer (Ra[13C]meth) 21. is determined from the enteral tracer’s plasma enrichment also as in equation 1: 22.

23. Ra [13C] meth = IE x [(EiE/EPE) – 1] (3) 24. 25. Because an unknown fraction (f) of the enterally infused tracer (IE) may be taken up 26. by the first pass through the splanchnic tissues before reaching the plasma pool, the 27. actual enteral tracer infusion rate into the plasma is IE x (1-f). Hence, IE x (1-f) is the 28. actual rate of isotope tracer infusion into the sampled plasma compartment, and the 29. true plasma methionine flux as estimated from the enteral infused tracer is: 30.

31. Ra [13C]meth = IE x (1 – f) x [(EiE/EPE) – 1] (4) 32. 33. For estimates of the first pass disappearance of methionine within the splanchnic 34. region, the following relationship stands: 35.

36. IIV x [(EiIV/EPIV) – 1] = IE x (1 – f) x [(EiE/EPE) – 1] (5) 37. 38. Rearranging and solving for (1– f) 39.

93 Chapter 5

1. (1 – f) = (IIV x [(EiIV/EPIV) – 1] ) / (IE x [(EiE/EPE) – 1]) (6) 2. 3. Therefore, the fraction of labeled tracer taken up during the first pass through the 4. splanchnic tissues, before reaching the systemic circulation, can be solved as the ratio 5. of the intravenous and enteral plasma methionine fluxes. 6.

7. (1 – f) = Ra IV [2H3] methyl meth / Ra E [13C] meth (7) 8. 9. And by rearrangement 10.

11. f = 1 – (Ra IV [2H3] methyl meth / Ra E [13C] meth) (8) 12. 13. Where, the unknown fraction is 1 minus the ratio of plasma flux of the intravenous 14. tracer over the plasma flux of the enteral administered tracer. The fraction of the tracer 15. taken up in a first pass should be the same as the fraction of unlabeled dietary amino 16. acid that is taken up in a first pass through splanchnic tissues. Therefore, the first 17. pass disappearance of dietary methionine may be estimated by multiplying dietary 18. methionine intake by the fraction of tracer taken up during the first pass. 19. 20. Splanchnic Methionine Oxidation (Transsulfuration of homocysteine) 13 ‑1 ‑1 21. The rate of CO2 appearance from methionine oxidation (Met ox µmol·kg .hr ) cor- 22. responds to irreversible methionine loss through transsulfuration (TS). Hence, TS 23. has been assumed to be equivalent to methionine oxidation10, 15. For determination 24. of methionine oxidation in critically ill children using the enteral L-[1-13C] methionine 13 25. tracer, it was necessary to establish a factor to be used for the retention of CO2 26. liberated from enterally supplied methionine, during its oxidative decarboxylation via 27. α-ketobutyrate7, 16. For this purpose, we used previous data by our group obtained in 13 28. enterally fed critically ill children receiving a primed, constant infusion of NaH CO3 29. which showed bicarbonate recovery of 69.80%13. Although the bicarbonate tracer in 30. the latter studies was provided by the parenteral route13, there is no difference on 31. bicarbonate recovery when the tracer is given through the enteral versus the parenteral 32. routes17. Therefore the mean value of 69.80 was used to estimate methionine oxidation 33. in the patients. Methionine oxidation (TS) was then calculated as follows: 34. 13 13 35. Meth ox = TS = (VCO2 x CO2) = [V CO2/69.80)]/Ep (9) 36. ‑1 ‑1 37. Where, methionine oxidation (TS) (µmol.kg .hr ) is determined by the rate of CO2

38. production (VCO2) measured in mL/min, obtained by using a respiratory monitor or 39. indirect calorimetry and converted to mmol/h by multiplying by 60 min and dividing by

94 Methionine splanchnic uptake in children

1. 22.4, which is the number of liters in one mole of an ideal gas at standard temperature 13 2. and pressure (STP) to convert mL to mmol/min; V CO2 is obtained by multiplying VCO2 13 13 3. by blood CO2 enrichment from the C methionine tracer infused and corrected by 4. 69.80, the bicarbonate retention factor obtained in critically ill children enterally fed13, 5. and Ep is the plasma enrichment of methionine for the plasma model or enrichment of 6. homocysteine for the intracellular model. 7. 8. Methionine balance 9. Methionine balance (mg.kg‑1.d‑1) was estimated as follows: 10. 11. Methionine Balance = Meth Intake – Meth ox (10) 12. 13. Where intake is estimated from dietary methionine and cysteine over the 24h period 14. and the intake of tracer infused. Cysteine is accounted for because it spares methio- 15. nine15, 16, 18, 19. The methionine tracer contribution, although minimal was also estimated 16. in the intake. 5 17. 18. Statistical Analyses 19. Continuous variables were tested for normality using the Kolmogorov-Smirnov good- 20. ness-of-fit statistic and no significant departures were found, indicating that means 21. and standard deviations are appropriate descriptive statistics. Two-way analysis of 22. variance (ANOVA) was used to assess differences between the plasma and intracel- 23. lular models for enrichment, fluxes, splanchnic uptake and oxidation within each age 24. group and the 13C homocysteine / 13C methionine ratio, plasma fluxes, splanchnic 25. uptake oxidative and non-oxidative disposal between groups (infants, children and 26. adolescents). A mixed-model ANOVA approach was applied to maximize statistical 27. power and to properly handle the within-subject correlation since each patient had 28. measurements obtained from both plasma and intracellular models. Two-tailed p < 29. 0.05 was considered statistically significant with Bonferroni correction to adjust for 30. multiple comparisons and protect against false positive results due to multiple testing. 31. Statistical analysis was performed using the SPSS software package (version 16.0, 32. SPSS Inc., Chicago, IL). 33. 34. 35. Results 36. 37. As shown in Table 3 the plasma isotopic enrichment in the critically ill infant group 38. was 0.057 ± 0.01 for [13C] methionine (plasma) and 0.056 ± 0.01 for [13C] homocys- 39. teine (intracellular), and there was no difference (p= 0.77) between the plasma and

95 Chapter 5

13 2 13 Table 3. Plasma isotopic enrichment for [ C] and [ H3]methylmethionine, enrichment of CO2 1. 13 13 in expired air, VCO2, V CO2, rates of oxidation, and methionine intakes after enteral L-[ C] methionine and intravenous [2H ]methyl tracer infusions in critically ill infants, children, and 2. 3 adolescents, obtained with the plasma and intracellular models 3. Infants Children Adolescents 4. Parameter Plasma Intracellular Plasma Intracellular Plasma Intracellular 5. ­Methionine Homocysteine ­Methionine Homocysteine ­Methionine Homocysteine 6. [13C] enrichment, 0.057 ± 0.01‡ 0.056 ± 0.01‡ 0.087 ± 0.02* 0.067 ± 0.01 0.11 ± 0.01* 0.073 ± 0.01 molar fraction 7. 2 [ H3] methyl enrichment, 0.081 ± 0.02 0.080 ± 0.02 0.084 ± 0.02* 0.068 ± 0.02 0.092 ± 0.02* 0.060 ± 0.01 8. molar fraction

9. 13 CO2 enrichment, atom 5.2 ± 1.2 5.2 ± 1.2 7.9 ± 0.1 7.9 ± 0.1 18.5 ± 0.7 18.5 ± 0.7 3 10. percent excess x 10 VCO , mmol.kg‑1.h‑1 15.5 ± 5.3 15.5 ± 5.3 14.8 ± 5.0 14.8 ± 5.0 8.1 ± 2.4 8.1 ± 2.4 11. 2 V13CO , µmol.kg‑1.h‑1 1.2 ± 0.3 1.2 ± 0.3 1.5 ± 0.0 1.5 ± 0.0 2.0 ± 0.9 2.0 ± 0.9 12. 2 Methionine oxidation 23.5 ± 10.6 23.0 ± 9.4 19.5 ± 6.6 * 28.4 ± 11.9 22.1 ± 7.8† 39.4 ± 16.6 13. (transsulfuration), 14. µmol.kg‑1.h‑1 15. Dietary methionine 14.3 ± 9.8 14.3 ± 9.8 15.4 ± 2.3 15.4 ± 2.3 11.0 ± 4.6 11.0 ± 4.6 intake, µmol.kg‑1.h‑1 16. Dietary splanchnic 9.4 ± 7.4 9.4 ± 7.4 6.7 ± 3.0 6.8 ± 2.9 3.5 ± 1.6 3.6 ± 1.5 17. methionine uptake, µmol.kg‑1.h‑1 18. 13 13 All values are means ± SD. VCO2, carbon dioxide production; V CO2, CO2 output. * P < .05 plasma vs. intracellular 19. model within a group; † P < .01 plasma vs. intracellular model adolescent group; ‡ P < .01 enrichments infants vs. 20. adolescents 21. 22. the intracellular models. In contrast, in the critically ill children there was a significant 23. difference (p < 0.05) between [13C] methionine and [13C] homocysteine enrichment, 24. with values of 0.087 ± 0.02 and 0.067 ± 0.01, respectively. Likewise, the plasma and 25. intracellular enrichment models were different (p < 0.01) in the critically ill adolescent 26. group with values of 0.11 ± 0.01 and 0.073 ± 0.01 respectively, for [13C] methionine and 27. [13C] homocysteine. 28. Hence, the ratio of [13C] homocysteine to [13C] methionine (13C Hcy/13C meth) was 29. 1.0 ± 0.15, 0.80 ± 0.20 and 0.66 ± 0.10, respectively for the infants, children and 30. adolescents, and different between the infants and adolescents (p < 0.01), but not 31. between the infants and children or the children and adolescents. The plasma isotopic 2 13 32. enrichments for the intravenously infused L-[ H3] methyl methionine and blood CO2 33. enrichments are also shown in Table 3. Plateau isotopic enrichment was achieved in 34. the three patient groups, but a difference was observed again between the plasma 35. and intracellular models in the children (p < 0.05) and adolescent (p < 0.05) groups. 36. From these enrichments the rates of appearance (Ra) of methionine obtained with both 13 2 37. tracers were estimated. The rates of appearance or plasma fluxes of the [ C] and [ H3] 38. methyl methionine obtained with the plasma and the intracellular models for the three 39. patient groups are shown in Figure 2, panels A and B.

96 Methionine splanchnic uptake in children

1. A 13 2. 120 # † # † C Met 13 3. C Hcy

4. -1]

.h 80 5. -1 * 6. * 7. 40 8. [µmol.kg

9. Flux Methionine Plasma 0 10. * p < .05 13C Methionine vs. 13C Homocysteine within the same age group; 11. # p < .05 13C Methionine or 13C Homocysteine infants vs. children; † p < .001 13C Methionine or 13C Homocysteine infants vs. adolescents 12. 13. B 14. 50 D3 Met 15. D3 Hcy 40

-1] 5 16. # .h

17. -1 30 * 18. 20 19. [µmol.kg 20. 10

21. Flux Methionine Plasma 0 22. 2 2 * p < .05 H3 Methyl methionine vs. estimated H3 Homocysteine children group; 2 2 23. # p < .001 H3 Methyl methionine vs. estimated H3 Homocysteine adolescent group;

24. Figure 2. A: Methionine fluxes estimated with the plasma 13[ C] methionine (Met) and the 25. intracellular [13C]homocysteine (Hcy) models during enteral administration of L- [13C]methionine 2 tracer. B: Plasma methionine fluxes obtained with the intravenous [ H3]methylmethionine tracer 26. (plasma model) and estimated homocysteine enrichment (intracellular model) in critically ill 27. infants, children and adolescents. 28. 29. In the infant group, there was no difference (p > 0.5) in the [13C] carbon methionine 30. fluxes between the plasma and the intracellular models, both yield rates of appearance 31. of 85.4 ± 18 and 86.0 ± 14 µmol.kg‑1.hr‑1 (mean ± SD), suggesting that there was no 32. significant intracellular dilution of the 13C methionine label. In contrast, in the children 33. group the plasma 13C methionine rate of appearance were 54.4 ± 12 and 69.7 ± 5 34. µmol.kg‑1.hr‑1 respectively, and significantly different (p< 0.05) for the plasma and the 35. intracellular models. Likewise in the adolescent group rates of appearance were 39.5 36. ± 3 vs. 63.7 ± 8 µmol.kg‑1.hr‑1 respectively for the plasma and intracellular models, 37. and significantly different (p< 0.001) among the models. The plasma fluxes obtained 38. with homocysteine as the precursor were 22 and 38% higher respectively for children 39. and adolescents, indicating a significant intracellular dilution of methionine enrichment

97 Chapter 5

1. 1.0 2. 0.8 # 3. * 4. 0.6 5. 0.4 6. 7.

splanchnic uptake splanchnic 0.2

8. methionine of Fraction 9. 0.0 Infants Children Adolescents 10. * p < .01 infants vs. children; # p < .001 infants vs. 11. adolescents.

12. Figure 3. Methionine splanchnic uptake in critically ill infants, children and adolescents. 13. 14. in these age groups. The plasma 13C methionine fluxes obtained with either model 15. were higher in the infants when compared to children (p < 0.05) and adolescents (p < 2 16. 0.001). The methionine rate of appearance obtained with the intravenous [ H3]methyl 17. methionine tracer were 29.7 ± 8.3 and 30.3 ± 8.2; 28.7 ± 8.5 and 38.7 ± 14; and 25.5 18. ± 6 and 40.5 ± 10 µmol.kg‑1.hr‑1, respectively for the plasma and intracellular models 19. in the infants, children and adolescents. While in the infant group there was no differ- 20. ence in the methyl fluxes obtained with either model, in the children and adolescent 21. groups the methyl methionine fluxes obtained with the intracellular model yield 25.6 22. (p < 0.05) and 37% (p < 0.01) higher values, respectively. We did not infuse intravenous 23. 13C methionine in these patients, thus for the intracellular model of the intravenously 2 13 13 24. infused [ H3]methyl methionine, we used the ratio of C hcy/ C meth obtained with 25. the enteral 13C methionine tracer, as previously described10. We cannot determine if the 26. 13C hcy/13C meth ratio is similar when the tracers are given by the intravenous10 versus 27. the enteral route in critically ill children. However, studies in the piglet model20 suggest 28. that the 13C hcy/13C meth ratio is 0.738 when the tracers are given intravenously and 29. 0.795 when given intragastrically, and therefore they appear to be comparable. The 30. methionine splanchnic uptake for the three groups using both models and estimated 31. as described in the methods section, is shown in Figure 3. Both models yield the same 32. fractional first-pass disappearance of methionine. Interestingly, the infant group had 33. the highest fractional methionine splanchnic uptake of 0.63 ± 0.14 when compared 34. to values of 0.45 ± 0.19 and 0.36 ± 0.13 for children (p < 0.01) and adolescents (p 35. < 0.001), respectively. The absolute first-pass disappearance or dietary methionine 36. splanchnic uptake in the infant group was 9.4 µmol.kg‑1.hr‑1 and highly variable due to 37. different methionine content in the infant formulas used, whereas in the children and 38. adolescents the dietary methionine splanchnic uptake was about 6.7 and 3.6 µmol. 39. kg‑1.hr‑1, respectively.

98 Methionine splanchnic uptake in children

1. Table 3 also summarizes the results for methionine oxidation (transsulfuration), the 13 3 13 13 2. blood CO2 enrichment (APE x 10 ) and the rates of CO2 production (V CO2; μmol. 3. kg‑1.hr‑1). In the infant group the rates of methionine oxidation (transsulfuration) were 4. about 23.5 µmol.kg‑1.hr‑1 with either the plasma or intracellular models, and not dif- 5. ferent from each other. In addition, the plasma model yielded comparable values of 6. methionine oxidation among infants, children and adolescents. However, the plasma 7. model in comparison to the intracellular model underestimated methionine oxidation 8. rates by 32% (p < 0.05) in the children and by 44% (p < 0.01) in the adolescents. 9. Hence, the model used had an impact on the children and adolescents, but not in the 10. infant group. Furthermore, although there was not statistical difference in the rates of 11. methionine oxidation obtained with the intracellular homocysteine model between the 12. infants, children and adolescents (p = 0.077), biologically there was a difference in 13. methionine oxidation rates of 16 µmol.kg‑1.hr‑1 between the infants and adolescents. 14. The fraction of plasma methionine flux utilization for oxidative (TS) and non-oxidative 15. disposal (NOD) and the proportion of plasma methionine flux originating from protein 16. breakdown are shown in Figure 4. The pattern of enteral methionine utilization was 5 17. different in the infants when compared to the adolescents. In the infants 26.7 ± 9.8% 18. of plasma methionine flux was utilized for oxidative disposal (transsulfuration) and 19. 73.3 ± 9.8% for NOD (p < 0.01). In contrast, in the adolescents 60.5 ± 21.2% was 20. utilized for oxidative disposal and 39.5 ± 16.6% for NOD, and there were intermediate 21. values of 40.7 ± 16.6% and 59.3 ± 16.6%, respectively, for the fraction of plasma 22. methionine flux utilization for oxidative and NOD in the children. There was no differ- 23. ence in the pattern of methionine flux utilization among the children and adolescents, 24. or children and infants. However, there was a significant difference in the pattern of 25. 26. Q for TS Q for NOD Q for PB 27. 100 28. 80 29. # 30. 60 # 31. 40

32. Percentage * 20 33. 34. 0 Infants Children Adolescents 35. * p < .001 Infants Q for TS vs. Q for NOD. 36. # p < .001 Q for TS adolescents vs. Infants. # p < .001 Q for NOD adolescents vs. infants. 37. 38. Figure 4. Percentage of splanchnic methionine flux (Q) utilized for nonoxidative disposal (NOD), oxidative disposal (transsulfuration (TS)) and methionine flux derived from protein breakdown 39. (PB) in critically ill infants, children and adolescents.

99 Chapter 5

1. Infants Children Adolescents 0 2. -25 ]

3. -1 #

.d -50 * 4. -1 5. -75 [mg.kg 6. -100 Methionine balance Methionine 7. -125 8. * p < .05 Infants vs. Adolescents; 9. # p < .05 Children vs. Adolescents 10. Figure 5. Methionine balance in critically ill infants, children and adolescents. 11. 12. methionine utilization between infants and adolescents, with a greater proportion of 13. methionine flux utilized for NOD in the infants when compared to adolescents (p < 14. 0.01) and conversely, greater utilization for oxidative disposal in adolescents when 15. compared to infants (p < 0.01). Most of the plasma methionine flux originated from 16. protein breakdown in all age groups despite that they were receiving standard enteral 17. nutritional intake. Methionine balance for the three age groups is shown in Figure 5. All 18. groups were in negative balance with significant differences observed between infants 19. and adolescents (p<0.01) and infants and children (p < 0.01). 20. Plasma amino acids and glutathione concentrations obtained in our patients and 21. compared to reference values21, 22 are shown in Table 4. Only the values that were 22. Table 4. Abnormal concentrations of plasma amino acids and plasma glutathione in critically ill 23. enterally fed children

24. Infants Children Adolescents

25. Essential 26. Histidine 13.4 ± 3.2 (61-91) 19.4 ± 10.8 (61-91) 18.6 ± 7.0 (77-107) 27. Methionine 40.5 ± 7.8 (14-38) 47.5 ± 13.2 (13-22) 42.9 ± 9.2 (20-34) 28. Tryptophan 21.8 ± 10.5 (34-73) 23.0 ± 11.9 (35-73) 29.5 ± 13.3 (54-93) 29. Nonessential 30. Cystine 18.0 ± 4.6 (21-53) 21.9 ± 9.3 (27-52) 26.3 ± 7.0 (36-61) 31. Cysteine 167 ± 52 (131 ± 40)* 159.7 ± 56.0 (209 ± 54)* 204.2 ± 34 (197 ± 56)* 32. Glutamine 622.5 ± 137.7 (474-747) 538.4 ± 159.3 (473-692) 513.1 ± 69.8 (551-797) 33. Glycine 172.1 ± 19.7 (138-276) 284.3 ± 148.2 (138-276) 162.7 ± 36.6 (183-322) Serine 127.2 ± 37.9 (98-160) 100.4 ± 24.0 (97-154) 83.9 ± 23.9 (101-177) 34. Taurine 29.3 ± 15.6 (39-111) 30.7 ± 11.4 (39-80) 32.0 ± 16.6 (41-66) 35. Other 36. Glutathione 2.21 ± 0.61 (12.5 ± 5.2)* 3.19 ± 0.94 (21.1 ± 8.9)* 2.94 ± 0.96 (12.2 ± 3.1)* 37. Homocysteine 3.37 ± 1.46 (5 ± 1.6)* 3.93 ± 1.26 (10)* 5.17 ± 2.25 (9)* 38. Values are means ± SD in micromoles. Nonreported amino acids were within normal range. Reference values are expressed 39. as 10th-90th quantile from Reference 22. *Values from Reference 28.

100 Methionine splanchnic uptake in children

1. abnormal or close to the abnormal range are reported. Among the essential amino 2. acids, histidine and tryptophan were lower in all age groups and methionine appeared 3. to be increased in the children and adolescents. For the nonessential amino acids, 4. cystine appeared to be lower in all groups. Glutamine, glycine and serine appeared 5. to be in the lower range only in the adolescent group. The children and adolescents 6. appear to have lower taurine values. All other non-reported amino acid values were 7. within normal range. 8. Plasma homocysteine appeared to be within normal range and plasma glutathione ap- 9. peared to be lower in all groups when compared to reference values. However, plasma 10. glutathione has limited value because the major pool of glutathione is intracellular. 11. 12. 13. Discussion 14. 15. We have investigated in vivo aspects of methionine metabolism in three different age 13 2 5 16. groups of critically ill children. We used the C and H3 methyl methionine tracers by 17. the enteral and parenteral routes, respectively. We acknowledge that both tracers have 18. different metabolic fates and given that they were not simultaneously administered by 19. the same route, the splanchnic rates of transmethylation and remethylation could not 20. be determined. It would have been difficult to obtain a second tracer study day using 13 2 21. the alternate route of tracer administration (intravenous C methionine and enteral H3 22. methyl methionine) in these critically ill children. This limits the scope of our results but 23. does not impact on our conclusions. Our data showed that the first pass disappear- 24. ance or splanchnic uptake of methionine was about 63% and significantly higher in the 25. critically ill infant population when compared to children and adolescents and almost 26. double the value of ~33% estimated from various studies in healthy adults15, 16, 18. There 27. are no comparable values on methionine splanchnic uptake in healthy children, infants 28. or adolescents, but our observations suggest that in critically ill children there is a 29. greater utilization of dietary methionine in the splanchnic tissues of sick infants when 30. compared to sick adolescents and that the rates of splanchnic methionine utilization 31. appear to decrease in the more mature individual. Greater utilization rates of methio- 32. nine in the splanchnic tissues of infants may be due to increased demand for synthesis 33. of methionine substrates in these critically ill patients. It has been demonstrated in 34. the piglet model that methionine main metabolic fate in the gut is transmethylation 35. to homocysteine and transsulfuration to cysteine20. Increased transmethylation rates 36. will render methyl groups available for the synthesis of polyamines, creatine and DNA 37. methylation and will increase production of homocysteine, which may be transsulfu- 38. rated to cysteine with production of glutathione (γ-glutamyl-cysteinyl-glycine;GSH), 39.

101 Chapter 5

6 1. taurine and H2S or remethylated to methionine for further availability of methyl groups, 2. with betaine or methyltetrahydrofolate as donors5, 6. 3. The intestine is a highly active metabolic tissue and due to its high proliferation rate, 4. the intestinal and colonic mucosa have a special demand for polyamines23. These low 5. molecular weight cations have major regulatory roles on tissue growth and differentia- 6. tion. Additionally, the use of broad spectrum antibiotics will result in changes in intesti- 7. nal flora, which is an important contributor to intestinal polyamines. The lack or limited 8. polyamines, creatine and choline content in the formulas supplied to critically ill infants 9. may also impose a greater demand in the splanchnic tissues for the synthesis of these 10. compounds5, 24‑26, and therefore the need for greater availability of methyl groups. 11. Interestingly, the elevated need for methionine precursor in the splanchnic tissues 12. appears to decrease at later developmental stages, as demonstrated by the lower 13. rates of methionine splanchnic uptake in adolescents. Hence, there appears to be 14. ontogeny of methionine splanchnic uptake. The expression of amino acid transporter 15. systems27, 28 and spatiotemporal patterns of enzymatic expression may explain the 16. ontogeny observed for amino acids and nutrients in the developing mammal29, 30. 17. In the infant population there was no difference among the enrichments of 13C methio- 18. nine and 13C homocysteine and therefore no difference in the plasma fluxes obtained 19. with the plasma or the intracellular models, suggesting that there was minimal or no 20. intracellular dilution of the 13C methionine tracer enterally infused. In addition, there was 21. no difference in the rates of transsulfuration (methionine oxidation) obtained with either 22. model, and about 27% of the plasma methionine flux was used for transsulfuration to 23. cysteine, while 73% was used for non-oxidative disposal, which involves the synthesis 24. of protein and methionine derived compounds. However, the absolute rates of methio- 25. nine transsulfuration of about 23 µmol.kg‑1.hr‑1 were significantly higher than the dietary 26. methionine splanchnic uptake of 9.4 µmol.kg‑1.hr‑1 indicating that dietary methionine is 27. insufficient to maintain transsulfuration rates, and therefore plasma methionine from 28. protein breakdown has to be utilized under these conditions. This is confirmed by 29. the fact that about 89% of the 13C methionine flux originates from protein breakdown 30. and that these critically ill infants are in a negative methionine balance of about 11.4 31. mg.kg‑1.d‑1. The fact that the enrichments obtained with the intracellular homocyste- 32. ine and the plasma methionine models do not differ in the infant population, despite 33. significant protein breakdown and expected intracellular dilution, suggests that there 34. is major compartmentation of methionine pools in the splanchnic tissues in this age 35. group. The mechanism(s) whereby this compartmentation occurs is beyond the scope 36. of this investigation. However, nuclear magnetic spectroscopy of subcellular fractions 37. in splanchnic tissues may help to better understand the underlying mechanisms31. 38. In contrast, in the children and adolescent patients there were clear differences in 39. methionine enrichments between the plasma and the intracellular models, and the

102 Methionine splanchnic uptake in children

1. ratio of 13C Hcy /13C meth of 0.80 in the children and 0.66 in the adolescents were 2. closer to values of 0.80 reported in healthy adult women32 and 0.58 reported in healthy 3. men10 suggesting that there was significant intracellular dilution of labeled methionine. 4. Therefore, there was a marked difference in plasma 13C methionine fluxes in children 5. and adolescents when obtained with the intracellular versus the plasma model. 6. Likewise, the rates of methionine oxidation (transsulfuration) obtained with the oral 13C 7. methionine tracer were 31% and 43% higher, respectively in children and adolescents 8. when the intracellular model was used. Therefore the plasma model underestimated 9. in vivo, whole body oxidation rates of methionine when the tracer was given by the 10. enteral route. 11. Irrespective of the model used, the rates of methionine transsulfuration (oxidation) in 12. the infants, children and adolescents were higher than the dietary sulfur (methionine 13. and cysteine) amino acid intake. In addition, the fraction of methionine flux utilized 14. for methionine transsulfuration, and therefore irreversible carbon losses, significantly 15. increased with age, from 27% in the infants to 61% in the adolescents. Conversely, the 16. infants utilized a higher proportion of methionine for non-oxidative disposal. 5 17. Taken together, these data suggest that the metabolic fate and utilization of methio- 18. nine differ in the critically ill pediatric population depending on age, and that there is 19. a greater contribution of plasma methionine for transsulfuration in the adolescents, 20. which may reflect a greater need in this age group for transsulfuration substrates,

21. such as cysteine, glutathione, taurine, and H2S, while there is a lesser utilization for 22. methylated substrates. Conversely, in the young infants the opposite holds true and 23. the group of children had intermediate values. Our infant population was 6 months of 24. age in average, fluctuating from 1 to 11 months, but provided that the same physiologi- 25. cal processes take place in the younger newborn and premature infant populations, 26. the decreased methionine transsulfuration and therefore limited glutathione availability 27. in young infants may contribute to make young infants more susceptible to oxidative 28. injury and necrotizing enterocolitis, which is seldom observed in older children. 29. It is known from animal data that nutrition and age influence enzymatic capacity and 30. therefore metabolic pathways. Finkelstein9 found in the rat model that the hepatic con- 31. tent of the methionine catabolizing enzymes methionine adenosyl transferase III (EC 32. 2.5.1.6), cystathionine β-synthase (EC4.2.1.22;CBA) and γ-cystathionase (EC 4.4.1.1) 33. increase with age and in response to higher dietary protein or methionine, or both. 34. It is also known that transsulfuration flux is increased by oxidative stress, whereas 35. antioxidants decrease it6. However, there was no difference in severity of disease or 36. in the acid base status among our patients, although this could have been limited by 37. our sample size. The redox regulation of the transsulfuration pathway may occur at 38. the level of cystathionine β-synthase, which contains a heme group that may serve 39. as a sensor of the oxidative environment33, 34. There is a tissue-specific metabolic

103 Chapter 5

1. distribution of methionine cycle enzymes5, 9 and the liver, kidney, small intestine, and 2. pancreas contain both cystathionine β-synthase and γ-cystathionase, key enzymes 3. in the transsulfuration pathway, which produces glutathione. Hence, the splanchnic 4. tissues in mature animals, in addition to the kidney, have the most rapid turnover of 5. glutathione35. Furthermore, It has been shown that hypoxia may affect remethylation 6. by altering methionine adenosyl transferase expression36, and that these changes are 7. comparable to those of a methyl-deficient diet37. However, none of our patients were 8. hypoxemic during the study or in the immediate preceding days. 9. Regardless of the rates and fate of methionine utilization, these critically ill patients 10. were in significantly negative balance despite receiving complete enteral feedings as 11. per current clinical standards, demonstrating that our understanding of enteral nutri- 12. tional support in critically ill patients is rather limited. 13. The oxidative irreversible loss of carbon moiety during transsulfuration is consistent 14. with a lesser efficiency in methionine utilization of critically ill children and adolescents 15. and probably a greater dietary need for sulfur amino acids. On the contrary, in young 16. infants there is a greater utilization of methionine for non-oxidative disposal, and there- 17. fore synthesis of protein and methylated compounds, which may impose a greater 18. need for neomethylgenesis, and therefore folate, betaine and vitamin B 12. These 19. observations are supported by the significantly negative methionine balance observed 20. in the adolescents when compared to the infants and children. Specific measurements 21. of transmethylation and remethylation in the splanchnic tissues of critically ill children 22. remain to be determined. 23. Jahoor and coworkers38 estimated methionine kinetics in malnourished, edematous 24. and non-edematous children, slightly older than our infant population, using intrave- 25. nous tracers. Although the malnourished population is physiologically different from 26. the critically ill population, these authors found that methionine transsulfuration was 27. maintained despite slower methionine turnover and as a consequence, less methionine 28. was available for protein synthesis and synthesis of methionine-derived compounds. 29. It is important to underscore that methionine metabolism occurs at the intracellular 30. level and therefore the homocysteine intracellular model is more accurate, while the 31. plasma model underestimates methionine kinetics in populations other than young 32. infants. 33. In summary, we investigated for the first time methionine splanchnic uptake in critically 34. ill infants, children and adolescents using the plasma and intracellular kinetic models, 35. and found that there is ontogeny on splanchnic uptake and methionine utilization in 36. critically ill children, characterized by a significant compartmentation of methionine 37. metabolism in young infants, with a greater utilization of methionine for non-oxidative 38. disposal, while in the adolescents methionine splanchnic uptake was lower and there 39. was greater methionine utilization for transsulfuration. Methionine balance was nega-

104 Methionine splanchnic uptake in children

1. tive in all age groups, indicating that current sulfur amino acid intakes provided to criti- 2. cally ill pediatric patients as per current standard care are insufficient to maintain sulfur 3. amino acid balance and probably functional needs. The plasma methionine enrichment 4. model underestimated methionine kinetics in children and adolescents. 5. 6. 7. References 8. 1. Chang JX, Chen S, Ma LP, et al. Func- 9. Finkelstein JD. Pathways and regulation 9. tional and morphological changes of the of homocysteine metabolism in mam- 10. gut barrier during the restitution process mals. Semin Thromb Hemost 2000;​26(3):​ 11. after hemorrhagic shock. World J Gastro- 219‑25. 12. enterol 2005;​11(35):​5485‑91. 10. MacCoss MJ, Fukagawa NK, Matthews 13. 2. Pastores SM, Katz DP, Kvetan V. DE. Measurement of intracellular sulfur Splanchnic ischemia and gut mucosal amino acid metabolism in humans. Am 14. injury in sepsis and the multiple organ J Physiol Endocrinol Metab 2001;​280(6):​ 15. dysfunction syndrome. Am J Gastroen- E947‑55. 5 16. terol 1996;​91(9):​1697-710. 11. Pollack MM, Patel KM, Ruttimann UE. 17. 3. Castillo L, Chapman TE, Yu YM, Ajami PRISM III: an updated Pediatric Risk 18. A, Burke JF, Young VR. Dietary arginine of Mortality score. Crit Care Med 1996;​ uptake by the splanchnic region in adult 24(5):​743‑52. 19. humans. Am J Physiol 1993;​265(4 Pt 1):​ 12. Food and Nutrition Board Institute of 20. E532‑9. Medicine. Panel on Macronutrients. 21. 4. Fenech M. Micronutrients and genomic Standing Committee on the Scientific 22. stability: a new paradigm for recom- Evaluation of Dietary Reference Intakes. 23. mended dietary allowances (RDAs). Food Dietary Reference Intakes for Energy, 24. Chem Toxicol 2002;​40(8):​1113‑7. Carbohydrate, Fiber, Fat, Fatty acids, 5. Finkelstein JD, Martin JJ. Methionine Cholesterol, Proteins and Amino Acids. 25. metabolism in mammals. Distribution Washington DC: The National Press; 26. of homocysteine between competing 2005. 27. pathways. J Biol Chem 1984;​259(15):​ 13. Sy J, Gourishankar A, Gordon WE, et al. 28. 9508‑13. Bicarbonate kinetics and predicted en- 29. 6. Stipanuk MH, Dominy JE, Jr., Lee JI, ergy expenditure in critically ill children. Coloso RM. Mammalian cysteine me- Am J Clin Nutr 2008;​88(2):​340‑7. 30. tabolism: new insights into regulation of 14. Matthews DE, Motil KJ, Rohrbaugh DK, 31. cysteine metabolism. J Nutr 2006;​136(6 Burke JF, Young VR, Bier DM. Measure- 32. Suppl):​1652S-9S. ment of leucine metabolism in man from 33. 7. Stipanuk MH, Ueki I, Dominy JE, Jr., a primed, continuous infusion of L-[1-3C] 34. Simmons CR, Hirschberger LL. Cysteine leucine. Am J Physiol 1980;​238(5):​ 35. dioxygenase: a robust system for regula- E473‑9. tion of cellular cysteine levels. Amino 15. Raguso CA, Ajami AM, Gleason R, Young 36. Acids 2009;​37(1):​55‑63. VR. Effect of cystine intake on methionine 37. 8. Deneke SM, Fanburg BL. Regulation of kinetics and oxidation determined with 38. cellular glutathione. Am J Physiol 1989;​ oral tracers of methionine and cysteine in 39. 257(4 Pt 1):​L163‑73.

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healthy adults. Am J Clin Nutr 1997;​66(2):​ files in human milk, infant artificial formu- 1. 283‑92. las, and semi-elemental diets. J Pediatr 2. 16. Hiramatsu T, Fukagawa NK, Marchini JS, Gastroenterol Nutr 1995;​21(1):​44‑9. 3. et al. Methionine and cysteine kinetics 26. Li Z, Vance DE. Phosphatidylcholine and 4. at different intakes of cystine in healthy choline homeostasis. J Lipid Res 2008;​ 5. adult men. Am J Clin Nutr 1994;​60(4):​ 49(6):​1187‑94. 525‑33. 27. Buddington RK, Diamond JM. Ontoge- 6. 17. Riedijk MA, Voortman G, van Goudoever netic development of intestinal nutrient 7. JB. Use of [13C]bicarbonate for meta- transporters. Annu Rev Physiol 1989;​51:​ 8. bolic studies in preterm infants: intragas- 601‑19. 9. tric versus intravenous administration. 28. Buddington RK, Diamond JM. Ontoge- 10. Pediatr Res 2005;​58(5):​861‑4. netic development of monosaccharide 18. Burrin DG, Davis TA. Proteins and amino and amino acid transporters in rabbit 11. acids in enteral nutrition. Curr Opin Clin intestine. Am J Physiol 1990;​259(4 Pt 1):​ 12. Nutr Metab Care 2004;​7(1):​79‑87. G544‑55. 13. 19. Di Buono M, Wykes LJ, Ball RO, 29. Reisenauer AM, Castillo RO. Regulation 14. Pencharz PB. Dietary cysteine reduces of the ontogenic and regional expression 15. the methionine requirement in men. Am of intestinal aminooligopeptidase. J Pe- 16. J Clin Nutr 2001;​74(6):​761‑6. diatr Gastroenterol Nutr 1994;​18(1):​1‑8. 20. Riedijk MA, Stoll B, Chacko S, et al. 30. Shenoy V, Roig JC, Chakrabarti R, Kubilis 17. Methionine transmethylation and trans- P, Neu J. Ontogeny of glutamine synthe- 18. sulfuration in the piglet gastrointestinal tase in rat small intestine. Pediatr Res 19. tract. Proc Natl Acad Sci U S A 2007;​ 1996;​39(4 Pt 1):​643‑8. 20. 104(9):​3408‑13. 31. Lapidot A, Gopher A. Cerebral metabolic 21. 21. Lepage N, McDonald N, Dallaire L, compartmentation. Estimation of glucose Lambert M. Age-specific distribution of flux via pyruvate carboxylase/pyruvate 22. plasma amino acid concentrations in a dehydrogenase by 13C NMR isotopomer 23. healthy pediatric population. Clin Chem analysis of D-[U-13C]glucose metabo- 24. 1997;​43(12):​2397-402. lites. J Biol Chem 1994;​269(44):​27198- 25. 22. Pastore A, Massoud R, Motti C, et al. Fully 208. 26. automated assay for total homocysteine, 32. Davis SR, Stacpoole PW, Williamson 27. cysteine, cysteinylglycine, glutathione, J, et al. Tracer-derived total and folate- cysteamine, and 2-mercaptopropionyl- dependent homocysteine remethylation 28. glycine in plasma and urine. Clin Chem and synthesis rates in humans indicate 29. 1998;​44(4):​825‑32. that serine is the main one-carbon donor. 30. 23. Loser C, Eisel A, Harms D, Folsch UR. Am J Physiol Endocrinol Metab 2004;​ 31. Dietary polyamines are essential luminal 286(2):​E272‑9. 32. growth factors for small intestinal and 33. Banerjee R, Zou CG. Redox regulation colonic mucosal growth and develop- and reaction mechanism of human 33. ment. Gut 1999;​44(1):​12‑6. cystathionine-beta-synthase: a PLP- 34. 24. Brosnan JT, Brosnan ME. Creatine: dependent hemesensor protein. Arch 35. endogenous metabolite, dietary, and Biochem Biophys 2005;​433(1):​144‑56. 36. therapeutic supplement. Annu Rev Nutr 34. Brosnan JT, Brosnan ME. The sulfur- 37. 2007;​27:​241‑61. containing amino acids: an overview. J 38. 25. Buts JP, De Keyser N, De Raedemaeker Nutr 2006;​136(6 Suppl):​1636S-40S. L, Collette E, Sokal EM. Polyamine pro- 39.

106 Methionine splanchnic uptake in children

35. Martensson J, Jain A, Meister A. Gluta- 37. Chawla RK, Jones DP. Abnormal me- 1. thione is required for intestinal function. tabolism of S-adenosyl-L-methionine 2. Proc Natl Acad Sci U S A 1990;​87(5):​ in hypoxic rat liver. Similarities to its 3. 1715‑9. abnormal metabolism in alcoholic cirrho- 4. 36. Blaise SA, Alberto JM, Audonnet-Blaise sis. Biochim Biophys Acta 1994;​1199(1):​ 5. S, Gueant JL, Daval JL. Influence of 45‑51. preconditioning-like hypoxia on the liver 38. Jahoor F, Badaloo A, Reid M, Forrester T. 6. of developing methyl-deficient rats. Am Sulfur amino acid metabolism in children 7. J Physiol Endocrinol Metab 2007;​293(6):​ with severe childhood undernutrition: 8. E1492-502. methionine kinetics. Am J Clin Nutr 2006;​ 9. 84(6):​1400‑5. 10. 11. 12. 13. 14. 15. 16. 5 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39.

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Chapter 6

Parenteral amino acid intakes in critically ill children: A matter of convenience

Sascha CAT Verbruggen Jama Sy Ana Arrivillaga Koen FM Joosten Johannes B van Goudoever Leticia Castillo

J Parenter Enteral Nutr. 2010;34(3):329-40. Chapter 6

1. Abstract 2. 3. Background 4. Parenteral and enteral amino acid requirements for nutritional balance and function 5. have not been defined in critically ill children or adults. In addition to their role in protein 6. synthesis, amino acids trigger signaling cascades that regulate various aspects of fuel 7. and energy metabolism and serve as precursors for important substrates. Amino acids 8. can also be toxic. We assessed parenteral intakes of essential (EAA) and non-essential 9. amino acids (NEAA) supplied to critically ill children as an initial step for further studies 10. aimed at establishing parenteral amino acid requirements. 11. 12. Methods 13. We conducted a retrospective review of parenteral amino acid intakes provided to 116 14. critically ill children and compared them to recommended EAA intakes by the Institute 15. of Medicine. There are no recommended intakes for NEAA. Hence, we compared NEAA 16. intakes to mixed muscle protein content in the older children, and breast milk amino 17. acid content in the infants. 18. 19. Results 20. Parenteral EAA are provided several fold in excess of recommended intakes for healthy 21. children except for phenylalanine and methionine, which although excessive, were 22. given in less generous amounts. NEEA were supplied in lower or higher amounts than 23. the content of mixed muscle proteins or breast milk. Parenteral amino acid formulas 24. are limited in taurine, glutamine and asparagine despite that inflammatory/immune 25. proteins are rich in these amino acids. 26. 27. Conclusions 28. Amino acid composition of parenteral formulas is variable and lacks scientific support. 29. Parenteral amino acid intakes should be based on measured requirements to maintain 30. nutritional and functional balance and on knowledge of toxicity. 31. 32. 33. 34. 35. 36. 37. 38. 39.

110 Parenteral amino acid intake

1. Introduction 2. 3. Amino acids are precursors for the biosynthesis of proteins and other substrates such 4. as nitric oxide, polyamines, collagen etc1. However, it is known that amino acids also 5. serve as signaling molecules in functionally diverse signal transduction pathways2. 6. Amino acids trigger signaling cascades that regulate various aspects of fuel and en- 7. ergy metabolism3, 4 and control the growth, proliferation, and survival of cells5. Given 8. their essential role, if they are not provided in sufficient amounts, they will be obtained 9. through protein breakdown, a process that in critically ill septic patients was described 10. as septic auto-cannibalism6. However, excessive administration of amino acids may 11. result in toxicity7‑9. 12. The dietary amino acid requirements of parenterally or enterally fed critically ill children 13. for nutritional balance or functional purpose have not been determined10. Knowledge 14. on amino acid toxicity is also limited11, 12. Current parenteral and enteral amino acid for- 15. mulas provided to critically ill children are based on sparse data despite that nutritional 16. support is an essential component in the management of these patients. Moreover, 17. critically ill patients present metabolic alterations of energy and protein homeostasis. 18. They undergo increased proteolysis unresponsive to the administration of protein 19. and energy intake, and there is increased hepatic synthesis of immune/inflammatory 6 20. proteins, at the expense of protein accretion, and decreased muscle protein synthesis 21. in fast-twitch fibers10, 13‑15. Hence, although whole body protein turnover is increased, 22. protein balance is often negative. In children, this situation has a major impact be- 23. cause in this population growth is an important function, which is suspended during 24. critical illness and results in the need to catch-up during convalescence. This may 25. have a developmental impact. In addition, critically ill patients present altered energy 26. metabolism manifested by glucose and lipid intolerance16 and changes in substrate 27. utilization16, 17. These alterations have also an impact on protein homeostasis because 28. protein and energy metabolism are closely interrelated. 29. Given the limited knowledge on the nutritional support of critically ill children, and the 30. significant role of amino acids, it is important to examine the specific parenteral amino 31. acid intakes that these patients receive. Therefore, the objective of this study was 32. to determine the parenteral amino acid intakes of essential (EAA) and non-essential 33. amino acids (NEAA) that critically ill children admitted to a Pediatric Intensive Care 34. Unit at a tertiary Hospital receive as per standard clinical care. We further contrasted 35. essential and non-essential amino acid intakes with the composition of representative 36. inflammatory/immune proteins, because these and not muscle proteins are the major 37. source of amino acid utilization for protein synthesis during critical illness. Therefore 38. protein synthesis composition may change the quality and quantity of specific amino 39. acid requirements during critical illness.

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1. Material and Methods 2. 3. Data Collection 4. This study was conducted at the Pediatric Intensive Care Unit (PICU), Texas Children’s 5. Hospital, Baylor College of Medicine. A retrospective chart and pharmacy records 6. review was performed in all patients that received parenteral nutritional support in 7. the PICU over a 12 month period from January 1st 2006 through December 31st 2006. 8. Only patients that received total parenteral nutritional support (TPN) for at least 24 9. hours providing 100% of their daily fluid requirements were included. The nutritional 10. support and the type of commercially available parenteral amino acid formulas used 11. were determined as clinically indicated by the attending(s) physicians and the nutri- 12. tion team. A total of 116 patients met these criteria. The study was exempt from full 13. Institutional Review Board review because it involved the use of non-identifiable exist- 14. ing records. Demographic information, including age, height and weight, as well as 15. the type, concentration and amount of parenteral amino acid formulas received, and 16. length of administration were recorded. The patient population was stratified within 17. the following five age groups: One month to 1 year, greater than one to three, four 18. to eight, nine to thirteen and fourteen to eighteen years. We used these age groups 19. because they are used by the IOM recommendations for dietary amino acid intakes, 20. which served as reference for established dietary intakes18. We compared parenteral 21. EAA intakes received by these patients with the Food and Nutrition Board, Institute 22. of Medicine (IOM), National Academy of Sciences recommendations for enteral EAA 23. intakes for age matched healthy children18. 24. There are no available recommended dietary intakes from the IOM, the World Health 25. Organization or any other official organization for NEAA intakes in the pediatric or adult 26. populations. Hence, the parenteral NEAA intakes supplied to critically ill children and 27. adolescents were compared with the composition of mixed muscle proteins, and in the 28. critically ill infant population the parenteral NEAA intakes supplied where compared 29. with the NEAA composition of breast milk. 30. 31. Calculations 32. Three different parenteral nutritional formulas were used in the PICU during the study 33. period: Aminosyn (Hospira Inc. Lake Forest, IL) and Trophamine (Braun Medical Inc. 34. Bethlehem, PA), which are commercially available as a 10% amino acid solution, and 35. Clinisol (Baxter Inc. Deerfield, IL), which is commercially available as a 15% amino acid 36. solution. Their amino acid content (mg/100mL) is shown in Table 1. From these solu- 37. tions, parenteral amino acid concentrations of 1.5, 2.5 or 3.5% are routinely prepared 38. and used by the Hospital Pharmacists, depending on the amount of protein and fluid 39. intake that patients receive.

112 Parenteral amino acid intake

Table 1. Amino Acid Composition of Formulas Administered to Critically Ill Children 1. TrophAmine Aminosyn Clinisol 2. Essential 3. Histidine 480 300 894

4. Isoleucine 820 660 749

5. Leucine 1400 1000 1040

6. Lysine 820 1050 1180

7. Methionine 340 172 749 8. Phenylalanine 480 298 1040 9. Threonine 420 400 749 10. Tryptophan 200 200 250 11. Valine 780 500 960 12. Non-essential 13. Arginine 1200 1018 1470 Alanine 540 993 2170 14. Asparagine 0 0 0 15. Aspartate 320 700 434 16. Cysteine 16 0 0 17. Glutamine 0 0 0 18. Glutamate 500 738 749 6 19. Glycine 360 500 1040

20. Proline 680 722 894

21. Serine 380 530 592 22. Tyrosine 240 270 39 23. Taurine 25 0 0 24. Total 10001 10051 14999 25. All values are mg/100mL. Composition provided by TrophAmine (Braun Medical, Bethlehem, PA), Aminosyn (Hospira, Lake Forest, IL), and Clinisol (Baxter, Deerfield, IL). 26. 27. The total parenteral amino acid intake of each patient over a 24 hour period (mL. 28. day‑1) was determined from the chart review. The specific parenteral EAA and NEAA 29. intakes (mg.kg‑1.d‑1) for each patient were derived from the known concentration of the 30. parenteral amino acid formulas provided to the patient (1.5, 2.5 or 3.5% ) and the total 31. volume of parenteral amino acid formula infused over the 24 hour period (mL.d‑1). The 32. EAA intakes were then compared with the recommended enteral dietary allowances 33. (RDA) established by the IOM for age matched healthy children. However, the IOM 34. does not provide recommendations on NEAA intakes. Therefore, in the children and 35. adolescents studied, the parenteral NEAA intakes were compared with data on the 36. free amino acid content in muscle (mg.kg‑1 wet tissue) of children obtained from the 37. literature19, 20, and as previously described in the adult population21, 22. In the infant 38. population we compared the parenteral NEAA intakes with the average NEAA compo- 39. sition of human breast milk23‑25, assuming an average intake of 100 ml.kg‑1.d‑1.

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1. Statistical analysis 2. Comparisons between parenteral EAA intakes and the IOM recommendations for EAA, 3. as well as NEAA intakes and NEAA content in mixed muscle proteins and breast milk 4. were analyzed using a one sample student t-test. Data were analyzed using standard 5. computer statistical software (Minitab 14, Minitab Inc. State College, PA). 6. 7. 8. Results 9. 10. Demographics and Nutritional Support 11. A total of 116 children admitted to the multidisciplinary PICU at the Texas Children’s 12. Hospital, and receiving parenteral nutrition providing complete nutritional support for 13. more than 24 hours were included in the study. The major diagnoses of the patient 14. population requiring parenteral nutrition were sepsis (n=58), malignancies (n=21), con- 15. genital anomalies (n=5), solid organ transplant (liver) (n=8) and chronic lung disease 16. (n=24). The demographic characteristics of the subjects, the parenteral amino acid for- 17. mulas received and the length of administration are shown in Table 2. The infants 0-12 18. months of age and children >1-3 years predominantly received Trophamine (100% and 19. 75%, respectively), while older children (4-8 and 9-13 yrs) and adolescents (> 14 yrs) 20.

21. Table 2. Demographic Characteristics of the Patients, Formulas Used, and Days of Parenteral 22. Nutrition (PN) Administration 23. Age, y n Diagnoses Age, y Sex, BMI, Aminosyn, Trophamine, Clinisol, Days on PN F:M kg.m‑2 n (%) n (%) n (%) 24. 0 – 1 yr 9 Sepsis (n = 3); 0.8 ± 0.2 3:6 17.9 ± 5.0 0 9 0 42.8 ± 40.7 25. congenital anomalies (0) (100) (0) 26. (n = 3); solid organ transplant (n = 2); 27. CLD (n = 1) 28. 1 – 3 yrs 48 Sepsis (n = 21); 1.6 ± 0.8 20:28 21.8 ± 7.0 11 36 1 30.4 ± 37.9 congenital anomalies (23) (75) (2) 29. (n = 2); malignancies 30. (n = 8); CLD (n = 17) 4 – 8 yrs 12 Sepsis (n = 8); solid 6.3 ± 1.7 5:7 19.0 ± 4.8 12 0 0 24.1 ± 28.2 31. organ transplant (n = (100) (0) (0) 32. 1); CLD (n = 3) 33. 9 –13 yrs 27 Sepsis (n = 17); 11.0 ± 1.5 11:16 19.0 ± 5.2 26 1 0 36.8 ± 40.3 malignancies (n = 8); (96) (4) (0) 34. solid organ transplant 35. (n = 2) 14 –18 yrs 20 Sepsis (n = 9); 15.8 ± 1.2 12:8 24.8 ± 5.0 16 1 3 13.6 ± 11.8 36. malignancies (n = 5); (80) (5) (15) 37. solid organ transplant (n = 3); CLD (n = 3) 38. BMI, body mass index; CLD, chronic lung disease; SD, standard deviation. Data are given as mean ± SD, except where 39. noted otherwise

114 Parenteral amino acid intake

Table 3. Parenteral nutrition support received by critically ill children 1. n = Kcal.kg‑1.d‑1 Calorie distribution, % Glucose Protein Lipids 2. Carbohydrate:Protein:Fat mg.kg‑1.min‑1 g.kg‑1.d‑1 g.kg‑1.d‑1 3. 0 – 1 yr 9 77.5 ± 17.6 75.1%:10.6%:14.3% 11.3 ± 4.9 2.2 ± 0.4 1.4 ± 1.0 4. 1 – 3 yrs 48 68.1 ± 28.0 62.9%:15.4%:21.8% 8.5 ± 3.1 2.5 ± 1.0 1.6 ± 1.5 5. 4 – 8 yrs 12 51.8 ± 22.9 64.6%:16.0%:19.4% 7.0 ± 4.0 2.1 ± 0.9 0.9 ± 0.6 6. 9 –13 yrs 27 48.2 ± 21.5 62.2%:14.5%:23.2% 6.1 ± 3.0 1.8 ± 0.8 1.2 ± 0.8 7. 14 – 18yrs 20 35.2 ± 18.6 63.4%:16.2%:20.4% 4.6 ± 2.7 1.3 ± 0.6 0.7 ± 0.6 8. Data are given as mean ± SD, except where noted otherwise 9. 10. predominantly received Aminosyn (100%, 96% and 80%, respectively). Trophamine 11. was used occasionally in older children receiving long-term parenteral nutritional 12. support based on the fact that it contains small amounts of cysteine and taurine. All 13. available commercial amino acid formulas are devoid of glutamine and asparagine. 14. The specific protein and energy intakes received are shown in Table 3. and they were 15. within current recommendations for critically ill children26. Parenteral glucose adminis- 16. tration is also shown in Table 3. The infant group of 0-12 months received higher glu- 17. cose infusion rates of about 11 mg.kg‑1.min‑1 while the older children received between 18. 6 and 8.5 mg.kg‑1.min‑1 and the adolescents received about 4.6 mg.kg‑1.min‑1. Fat 19. intake was variable within the groups due to hypertriglyceridemia frequently observed 6 20. in these patients, but it was supplied between 0.93 and 1.62 g.kg‑1.d‑1 for infants, 21. children and adolescents. 22. 23. Parenteral Amino Acid Intakes 24. In the infants 0-12 months of age (Table 4), all parenteral essential amino acids were 25. provided in amounts one to four fold in excess (p < 0.0001) of the IOM recommended 26. enteral dietary intakes for healthy infants18, except for phenylalanine which was only 27. 120% (p = 0.045) of recommended enteral intakes. Parenteral intake of NEAA was vari- 28. able when compared to the content of NEAA in breast milk23, 24, but overall Trophamine 29. provided an excess of 6 fold the amount of arginine (p < 0.001), 2.6 fold of cysteine 30. (p < 0.005), 1.8 fold of proline (p < 0.001) and 1.5 fold of tyrosine (p < 0.005), while the 31. following amino acids were provided at deficient amounts when compared to those 32. supplied by breast milk: aspartate at 50% (p < 0.0001), serine at 71% (p < 0.0001), 33. taurine at 2% (p < 0.0001) and glutamate at 6% (p < 0.0001). Alanine and glycine were 34. provided in similar amounts to those of breast milk (p=NS). 35. Table 5 shows data on the 48 children >1 to 3 years of age. All the EAA were provided 36. in amounts of about 2 to 7 fold in excess (p < 0.0001) of the recommended DRI’s by 37. the IOM18, but phenylalanine and methionine were provided at a lower range than the 38. other EAA. The parenteral NEAA intakes were variable, but in general arginine was 39. provided at 5.7 (p < 0.0001), cysteine at 21.7 (p < 0.0001), glycine at 1.6 (p < 0.0001),

115 Chapter 6

Table 4. Parenteral essential (EAA) and non-essential amino acid (NEAA) intakes provided to 9 1. critically ill infants 0-12 months of age and enteral dietary recommended intakes (DRI) for EAAs 2. by the Institute of Medicine. 3. EAA Parenteral intake, Enteral DRI, † Parenteral Intake as a Excess or p value mg.kg‑1.d‑1 mg.kg‑1.d‑1 percentage of DRI deficiency 4. Histidine 100.6 32 314 ↑ < 0.0001 5. Isoleucine 171.8 43 400 ↑ < 0.0001

6. Leucine 293.4 93 315 ↑ < 0.0001

7. Lysine 171.8 89 193 ↑ < 0.0001

8. Methionine 71.3 43 166 ↑ < 0.0001

9. Phenylalanine 100.6 84 120 ↑ 0.045

10. Threonine 88.0 49 180 ↑ < 0.0001

11. Tryptophan 41.9 13 322 ↑ < 0.0001 12. Valine 163.5 58 282 ↑ < 0.0001 13. NEEA Parenteral intake, Breast milk NEAA Parenteral intake as a Excess or p value mg.kg‑1.d‑1 intake, ‡ mg.kg‑1.d‑1 percentage of breast deficiency 14. milk amino acid intake

15. Arginine 251.5 41.3 609 ↑ < 0.0001

16. Alanine 113.2 98.3 115 ↔ 0.095

17. Aspartate 67.1 133.1 50 ↓ < 0.0001 18. Cysteine 60.9 22.9 266 ↑ < 0.005 19. Glutamine 0.0 227.2 0 ↓ < 0.0001 20. Glutamate 104.8 1632.4 6 ↓ < 0.0001 21. Glycine 75.4 71.1 106 ↔ 0.43 22. Proline 142.5 77.6 184 ↑ < 0.0001 23. Serine 79.6 111.7 71 ↓ < 0.0001 ↑ 24. Tyrosine 50.3 32.1 157 < 0.005 ↓ 25. Taurine 5.2 334.7 2 < 0.0001 NEEAs are compared with breast milk amino acid content assuming intakes of mL‑1.kg‑1.d‑1 26. † Reference 18 27. ‡ References 23-25, assuming 100 mL.kg‑1.d‑1 intake 28. 29. proline at 3.4 (p< 0.0001), serine at 1.4 (p< 0.0001) and tyrosine at 84.8 (p< 0.0001) fold 30. in excess of the content of these amino acids in mixed muscle proteins19‑21. In contrast, 31. taurine (p< 0.0001), glutamate (p< 0.0001) and aspartic acid (p< 0.02) were given at 32. lower amounts than those found in mixed muscle proteins. Alanine (p=0.96) was given 33. in comparable amounts to the amino acid composition of mixed muscle proteins and 34. glutamine and asparagine were not supplied. 35. Over the study period twelve children age 4-8 years received TPN (Table 6). In this group 36. again, all the essential amino acids were provided between 3.4 and 6.8 fold higher (p< 37. 0.0001) than recommended DRI’s18, except for methionine and phenylalanine which 38. were provided at 1.6 (p<0.013) and 1.5 fold higher (p<0.026) than DRI’s recommenda- 39. tions18. While parenteral arginine (p< 0.0001), proline (p< 0.0001) and tyrosine were

116 Parenteral amino acid intake

Table 5. Parenteral essential (EAA) and non-essential amino acid (NEAA) intakes provided to 48 1. critically ill children 1 – 3 years of age and enteral dietary recommended intakes (DRI) for EAAs 2. by the Institute of Medicine. 3. EAA Parenteral intake, Enteral DRI, † Parenteral Intake as a Excess or p value mg.kg‑1.d‑1 mg.kg‑1.d‑1 percentage of DRI deficiency 4. Histidine 111.5 21 531 ↑ < 0.0001 5. Isoleucine 195.4 28 698 ↑ < 0.0001

6. Leucine 323.5 63 513 ↑ < 0.0001

7. Lysine 224.5 58 387 ↑ < 0.0001

8. Methionine 72.7 28 260 ↑ < 0.0001

9. Phenylalanine 112.2 54 208 ↑ < 0.0001

10. Threonine 106.6 32 333 ↑ < 0.0001

11. Tryptophan 50.7 8 634 ↑ < 0.0001 12. Valine 178.8 37 483 ↑ < 0.0001 13. NEAA Parenteral intake, Mixed muscle protein Parenteral intake as a Excess or p value mg.kg‑1.d‑1 amino acid content, percentage of mixed deficiency 14. ‡ mg.kg‑1 wet weight muscle amino acid intake

15. Arginine 291.9 51.2 570 ↑ < 0.0001

16. Alanine 177.1 177.9 100 ↔ 0.96

17. Asparagine 0.0 41.8 0 ↓ < 0.0001 18. Aspartate 107.0 132.6 81 ↓ 0.02 19. Cysteine 53.0 2.4 2177 ↑ < 0.0001 6 20. Glutamine 0.0 953.2 0 ↓ < 0.0001 21. Glutamate 143.7 278.8 52 ↓ < 0.0001 22. Glycine 104.5 64.9 161 ↑ < 0.0001 23. Proline 175.6 51.4 341 ↑ < 0.0001 ↑ 24. Serine 107.3 73.1 147 < 0.0001 ↑ 25. Tyrosine 61.2 7.2 848 < 0.0001 Taurine 4.5 1040.6 0 ↓ < 0.0001 26. NEEAs are compared with mixed muscle protein content 27. † Reference 18 28. ‡ References 19 and 20 29. 30. provided in amounts between 2.9 and 7 fold higher than the content of mixed muscle 31. proteins, serine was provided at about 1.5 fold higher (p< 0.03). Cysteine, taurine, 32. asparagine and glutamine are not supplied by Aminosyn. Glutamate was given 55% 33. lower (p<0.0001) and alanine and aspartic acid were given in comparable amounts 34. (p>0.1) to those of the amino acid composition of mixed muscle proteins. 35. Twenty seven patients received TPN in the age group of 9-13 years (Table 7). All EAA 36. were given between one and 6 fold higher than DRI’s, except again for methionine 37. and phenylalanine which were provided only at 1.48 (p< 0.005) and 1.39 fold higher 38. (p=0.005), respectively. In regard to the NEAA, arginine was given at 3.6 (p< 0.0001), 39. proline at 2.5 (p< 0.0001) tyrosine at 6.7 (p< 0.0001) and serine at 1.2 (p=0.01) fold

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Table 6. Parenteral essential (EAA) and non-essential amino acid (NEAA) intakes provided to 12 1. critically ill children 4 - 8 years of age and enteral dietary recommended intakes (DRI) for EAAs 2. by the Institute of Medicine. 3. EAA Parenteral intake, Enteral DRI, † Parenteral Intake as a Excess or p value mg.kg‑1.d‑1 mg.kg‑1.d‑1 percentage of DRI deficiency 4. Histidine 61.9 16 387 ↑ < 0.0001 5. Isoleucine 136.1 22 619 ↑ < 0.0001

6. Leucine 206.2 49 421 ↑ < 0.0001

7. Lysine 216.5 46 471 ↑ < 0.0001

8. Methionine 35.5 22 161 ↑ 0.013

9. Phenylalanine 61.5 41 150 ↑ 0.026

10. Threonine 82.5 24 344 ↑ < 0.0001

11. Tryptophan 41.2 6 687 ↑ < 0.0001 12. Valine 103.1 28 368 ↑ < 0.0001 13. NEEA Parenteral intake, Mixed muscle protein Parenteral intake as a Excess or p value mg.kg‑1.d‑1 amino acid content, percentage of mixed deficiency 14. ‡ mg.kg‑1 wet weight muscle amino acid intake

15. Arginine 209.9 51.2 410 ↑ < 0.0001

16. Alanine 204.8 178.0 115 ↔ 0.33

17. Asparagine 0.0 41.8 0 ↓ < 0.0001 18. Aspartate 144.3 132.6 109 ↔ 0.54 19. Cysteine 0.0 2.4 0 ↓ < 0.0001 20. Glutamine 0.0 953.2 0 ↓ < 0.0001 21. Glutamate 152.2 278.8 55 ↓ < 0.0001 22. Glycine 103.1 64.9 159 ↑ 0.02 23. Proline 148.9 51.4 289 ↑ < 0.0001 ↑ 24. Serine 109.3 73.1 150 0.03 ↑ 25. Tyrosine 55.7 7.2 771 < 0.0001 Taurine 0.0 1040.6 0 ↓ < 0.0001 26. NEEAs are compared with mixed muscle protein content 27. † Reference 18 28. ‡ References 19 and 20 29. 30. higher than the amounts found in mixed muscle proteins, while alanine and aspartate 31. were no different (p>0.1). Asparagine, cysteine, glutamine and taurine are not sup- 32. plied in Aminosyn. One patient received Trophamine which provides small amounts 33. of cysteine and taurine although in significantly (p< 0.0001) lower amounts than those 34. found in mixed muscle proteins. 35. Twenty children age 14-18 years received TPN during the study period (Table 8). Eighty 36. percent of these received Aminosyn and 5 and 15% received Trophamine and Clinisol, 37. respectively. Hence, negligible amounts of taurine and cysteine were given as a group, 38. but indeed those children receiving Aminosyn and Clinisol who were the majority, 39. received formulas devoid of cysteine and taurine, and all were devoid of glutamine and

118 Parenteral amino acid intake

Table 7. Parenteral essential (EAA) and non-essential amino acid (NEAA) intakes provided to 27 1. critically ill children 9-13 years of age and enteral dietary recommended intakes (DRI) for EAAs 2. by the Institute of Medicine. 3. EAA Parenteral intake, Enteral DRI, † Parenteral Intake as a Excess or p value mg.kg‑1.d‑1 mg.kg‑1.d‑1 percentage of DRI deficiency 4. Histidine 55.8 16 349 ↑ < 0.0001 5. Isoleucine 120.5 22 548 ↑ < 0.0001

6. Leucine 184.0 48 383 ↑ < 0.0001

7. Lysine 187.5 45 417 ↑ < 0.0001

8. Methionine 32.5 22 148 ↑ < 0.005

9. Phenylalanine 55.4 40 139 ↑ 0.005

10. Threonine 72.4 23 315 ↑ < 0.0001

11. Tryptophan 36.1 6 602 ↑ < 0.0001 12. Valine 92.7 28 331 ↑ < 0.0001 13. NEEA Parenteral intake, Mixed muscle protein Parenteral intake as a Excess or p value mg.kg‑1.d‑1 amino acid content, percentage of mixed deficiency 14. ‡ mg.kg‑1 wet weight muscle amino acid intake

15. Arginine 185.3 51.2 362 ↑ < 0.0001

16. Alanine 175.3 177.9 99 ↑ 0.859

17. Asparagine 0.0 41.8 0 ↓ < 0.0001 18. Aspartate 123.0 132.6 93 ↔ 0.374 19. Cysteine 0.1 2.4 6 ↓ < 0.0001 6 20. Glutamine 0.0 953.2 0 ↓ < 0.0001 21. Glutamate 131.1 278.8 47 ↓ < 0.0001 22. Glycine 89.0 64.9 137 ↑ < 0.005 23. Proline 129.9 51.4 253 ↑ < 0.0001 ↑ 24. Serine 94.3 73.1 129 0.01 ↑ 25. Tyrosine 48.5 7.2 672 < 0.0001 Taurine 0.2 1040.6 0 ↓ < 0.0001 26. NEEAs are compared with mixed muscle protein content 27. † References 18 28. ‡ References 19 and 20 29. 30. asparagine. As shown in Table 8, EAA were given between 2 and 4 fold in excess of 31. DRI’s18. However, methionine and phenylalanine intakes were provided at 1.9 (p>0.06) 32. and 1.4 (p>0.11) fold higher than DRI’s but not statistically significant. NEAA were 33. given in variable amounts, with an excess of arginine and proline of about 2.7 (p< 34. 0.0001) and tyrosine 4.1 fold (p< 0.0001), while alanine (p=0.12), glycine (p=0.96) and 35. serine (p=0.94) were given in comparable amounts to mixed muscle proteins. Aspartate 36. (p<0.0001) and cysteine (p<0.0001) were supplied in lower amounts when compared 37. to mixed muscle proteins. Glutamine and asparagine were not provided and taurine 38. was negligible. 39.

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Table 8. Parenteral essential (EAA) and non-essential amino acid (NEAA) intakes provided to 1. 20 critically ill children 14-18 years of age and enteral dietary recommended intakes (DRI) for 2. EAAs by the Institute of Medicine. 3. EAA Parenteral intake, Enteral DRI, † Parenteral Intake as a Excess or p value mg.kg‑1.d‑1 mg.kg‑1.d‑1 percentage of DRI deficiency 4. Histidine 49.3 15 329 ↑ < 0.0001 5. Isoleucine 85.5 20 428 ↑ < 0.0001

6. Leucine 130.0 46 283 ↑ < 0.0001

7. Lysine 129.6 42 309 ↑ < 0.0001

8. Methionine 38.6 20 193 ↔ 0.061

9. Phenylalanine 51.5 37 139 ↔ 0.115

10. Threonine 55.7 22 253 ↑ < 0.0001

11. Tryptophan 25.7 6 428 ↑ < 0.0001 12. Valine 73.2 26 282 ↑ < 0.0001 13. NEEA Parenteral intake, Mixed muscle protein Parenteral intake as a Excess or p value mg.kg‑1.d‑1 amino acid content, percentage of mixed deficiency 14. ‡ mg.kg‑1 wet weight muscle amino acid intake

15. Arginine 136.2 51.2 266 ↑ < 0.0001

16. Alanine 136.8 177.9 77 ↔ 0.12

17. Asparagine 0.0 41.8 0 ↓ < 0.0001 18. Aspartate 77.7 132.6 59 ↓ < 0.0001 19. Cysteine 0.2 2.4 9 ↓ < 0.0001 20. Glutamine 0.0 953.2 0 ↓ < 0.0001 21. Glutamate 88.8 278.8 32 ↓ < 0.0001 22. Glycine 69.1 64.9 106 ↔ 0.96 23. Proline 92.0 51.4 179 ↑ < 0.0001 ↔ 24. Serine 64.9 73.1 89 0.94 ↑ 25. Tyrosine 29.4 7.2 408 < 0.0001 Taurine 0.3 1040.6 0 ↓ < 0.0001 26. NEEAs are compared with mixed muscle protein content 27. † Reference 18 28. ‡ References 19 and 20 29. 30. Discussion 31. 32. Amino acids have active physiological roles not only in protein synthesis, but as signal- 33. ing molecules in various signal transduction pathways3 and regulate various aspects of 34. fuel and energy metabolism27. They also serve as precursors for important compounds 35. such as glutathione28, 29, creatine30, nitric oxide31, polyamines32, etc. Nutritional support, 36. whether it is provided through the enteral or parenteral route, remains an essential 37. component of the care of critically ill patients. There is consensus on the total amount 38. of protein that critically ill children should receive, although based on limited evidence26. 39. However, the parenteral or enteral specific amino acid requirements of critically ill

120 Parenteral amino acid intake

1. children have not been defined, and current parenteral amino acid administration is 2. rather variable depending on the amino acid formula used. 3. Except for Trophamine, which was designed to maintain the plasma amino acid pattern 4. of healthy, breast fed infants25 and therefore contains small amounts of cysteine and 5. taurine, the amino acid composition of commercial parenteral formulas provided to 6. critically ill children is based on the convenience of adequate solubility, stability, and 7. lack of side effects, such as hyperammonemia or metabolic acidosis, rather than on 8. detailed data on specific amino acid requirements, dose response and safe upper limits 9. of intake. Hence, cysteine, taurine and glutamine are not provided in most parenteral 10. amino acid formulas due to its lower solubility and/or poor stability. Trophamine is 11. mainly utilized in young infants and it contains small amounts of cysteine and taurine, 12. while aminosyn and clinisol were given to older children, and are devoid of these amino 13. acids. 14. Our data showed that most EAA are provided several fold in excess of recommended 15. intakes for enterally fed children18, except for phenylalanine and methionine, which 16. although excessive, were given in less generous amounts. NEAA are supplied in ex- 17. cess or in deficient amounts when compared to breast milk or the content or mixed 18. muscle proteins. Taurine and cysteine are provided in general in minimal amounts, 19. particularly in the older children while other important amino acids such as glutamine 6 20. and asparagine, which are important components of inflammatory/immune proteins, 21. are lacking in all age groups. 22. Several observations can be derived from these data and various issues need to be 23. taken into consideration. First, we acknowledge that it is difficult to compare enteral 24. amino acid intakes recommended for healthy children with parenteral amino acid in- 25. takes received by critically ill children, but in the absence of quantitative estimates of 26. parenteral amino acid requirements in critically ill children or adults, our comparisons 27. are a reasonable start point. Breast milk is the “gold” standard for adequate nutrition in 28. the infant population18, 24, 25 and muscle mass accretion and therefore growth, is an im- 29. portant source of amino acid utilization in healthy children. Therefore, comparing NEAA 30. from breast milk and muscle tissue composition with the NEAA intakes received by the 31. patients, although not ideal, it is reasonable as an initial comparison, particularly in light 32. that there are no official recommendations for the intakes of NEAA, which although 33. considered non-essential, many are indeed conditionally essential during stress. 34. Second, parenteral amino acid utilization and therefore requirements are different 35. when provided by the enteral versus the parenteral route. When the enteral route 36. is used, there is a first-pass disappearance of amino acids by the splanchnic area 37. or splanchnic uptake. The splanchnic area comprises the liver, stomach, intestines, 38. pancreas, and spleen. Except for the liver, all other organs constitute the portal- 39. drained viscera (PDV). Once extracted by the PDV, the first-pass disappearance or

121 Chapter 6

1. splanchnic uptake of amino acids is variable depending on its metabolic utilization 2. by the liver and PDV. For instance, in healthy humans 64% of dietary glutamate33, 3. and 96% of glutamine34, 35 undergo a first-pass disappearance indicating their active 4. metabolic role in the splanchnic area as a fuel for enterocytes36 and also as precursors 5. of citrulline, polyamines37, etc. In contrast, leucine, phenylalanine and arginine have a 6. splanchnic disappearance of 21, 29 and 38%38, 39, respectively, indicating that a large 7. proportion of these amino acids are utilized outside the splanchnic region, while the 8. splanchnic uptake of methionine and cysteine is about 33 and 44% respectively38, 40. 9. In recent studies by our group, we observed that in critically ill infants, children and 10. adolescents, methionine splanchnic uptake was 63, 43 and 36%, respectively, and it 11. was higher (p < 0.05) in the infant group when compared to critically ill adolescents41. 12. Furthermore, the children and adolescents had higher methionine splanchnic uptake 13. when compared to values of about 33% estimated in healthy adults40. These findings 14. suggest that developmental stage and critical illness will influence the metabolic needs 15. of the splanchnic area. Whether there is a higher requirement for substrates such as 16. polyamines, creatine, glutathione or methyl groups in the splanchnic area of critically 17. ill patients remains to be determined, but it is clear that specific amino acid needs are 18. different in critically ill patients and are influenced by age. 19. Leucine and phenylalanine splanchnic uptake is increased under conditions of endo- 20. toxemia in healthy subjects42; and in healthy adults subjected to a short, experimental 21. course of steroids the splanchnic uptake of glutamine was increased by 50%43. Hence, 22. conditions that are frequently seen in the ICU, such as sepsis and steroid therapy, will 23. influence amino acid requirements and utilization. However, it is not standard practice 24. to modify the nutritional support provided to critically ill children according to clinical 25. stage, medications received, etc. 26. The enteral or parenteral route of amino acid administration will significantly impact on 27. amino acid utilization and requirements, and the extraction and use of dietary amino 28. acids by the splanchnic area will have a major influence on their systemic availability 29. and requirements. Parenteral nutrition bypasses the splanchnic area and nutrients 30. are presented to the liver through the hepatic arterial circulation, instead of the portal 31. venous circulation44. Studies in piglets suggest that after enteral feeding, portal rather 32. than arterial phenylalanine is preferentially used for the synthesis of constitutive and 33. secretory hepatic proteins44. Spatial cellular localization will also influence amino acid 34. metabolism. It has been shown that in periportal hepatocytes excess portal ammonia 35. is scavenged by ureagenesis, whereas perivenous hepatocytes scavenge ammonia 36. for glutamine synthesis45. Hence, nitrogen metabolism will be directed to different 37. pathways depending on the anatomical presentation. 38. In the healthy piglet model, parenteral requirements of threonine, lysine, phenylalanine 39. and branch chain amino acids are lower than the enteral requirements46‑49, and clearly

122 Parenteral amino acid intake

1. the difference depends on the rates of the splanchnic uptake of these amino acids. 2. Thus, because the splanchnic metabolic needs are by-passed during parenteral nutri- 3. tion, the parenteral amino acid requirements will be greatly modified by its degree of 4. splanchnic uptake. Hence, the route of nutrient administration will have a profound 5. impact on amino acid requirements and their utilization. 6. As shown in Table 9, the composition of acute, inflammatory/immune proteins21 is 7. different from that of mixed muscle proteins21, 22 or from breast milk24, 50. Taurine is the 8. major component of polymorphonuclear cells, and except for Trophamine, the other 9. parenteral amino acid formulas used in children are devoid of taurine. NEAA and spe- 10. cifically glutamine are found in higher concentrations in inflammatory/immune proteins, 11. and this may explain the “conditional” essentiality of some amino acids traditionally 12. considered non-essential. Furthermore, synthesis of inflammatory/immune proteins 13. and not muscle accretion is a major source of amino acid utilization during critical 14. illness21, 51. Indeed, the majority of protein loss originates from muscle51. Therefore the 15. quality and quantity of protein synthesized during pediatric critical illness will have a 16. major impact on the adequacy of the parenteral or enteral amino acid formulas provided 17. to these patients. Reeds and coworkers21 compared the amino acid composition of 18. inflammatory proteins in relation to mixed muscle proteins, to estimate the amino acid 19. demand of a typical inflammatory response. They observed that various amino acids 6 20. among them phenylalanine, tryptophan, serine, cysteine and tyrosine were limiting. 21. Indeed, it would be difficult to determine the amino acid composition of the inflamma- 22. tory proteome in vivo, in critically ill children, given that many different inflammatory 23. proteins are expressed in different organs at the intracellular level or in organelles, and 24. they may not be detectable in plasma. However, the plasma inflammatory proteins 25. selected by Reeds21, 52, 53 are expressed in measurable concentrations and are grossly 26. representative of a clinical inflammatory response. 27. In our previous studies in critically ill infants54 we observed that greater parenteral 28. intakes of phenylalanine were associated with a lesser negative protein balance and 29. decreased rates of phenylalanine catabolism through hydroxylation to tyrosine. In the 30. present study, the EAA were provided several fold in excess, but phenylalanine and 31. methionine although excessive when compared to enteral recommendations, were 32. provided at lower rates. Furthermore, some NEAA were not provided at all, while others 33. were deficient and yet others were supplied in excess. 34. These observations raise the question of amino acid balance. For protein synthesis 35. to occur, all the necessary amino acids need to be available. If one amino acid is 36. limiting, protein catabolism will be maintained in order to support synthesis of pro- 37. teins essential for survival. Hence, if an EAA or a NEAA that becomes “conditionally” 38. essential during stress is limiting, protein catabolism will persist, despite adequate 39. protein intake, as defined by our current standards. Hence, a balanced composition

123 Chapter 6

1. † 2. 3.6 4.1 4.8 4.5 1.3 9.2 5.9 6.9 5.4 1.3 4.7 4.0 2.5 9.8 8.1 4.8 5.1 14.5

3. proteins, mg/100 mg 4. Skeletal muscle 5. ‑ 1 .day ‑ 1 32.1 77.6 71.1 22.9 98.3 41.3 66.1 20.5 71.5 39.4 15.1 68.9 92.1 49.9 6. 22.0 334.7 111.7 133.1 1859.6 mg.kg 7. Breast milk, ^

9

8. ‡ 0 0 0

9. cells 1.409 0.008 0.028 0.028 0.142 0.091 0.023 0.013 0.008 0.083 0.006 0.004 0.015 0.010 0.006 0.023 PMN, mg/10 10. 11. 12. 7.4 3.1 3.4 1.9 1.8 3.6 5.2 4.6 3.0 7.4 6.4 1.1 7.5 4.8 1.7 17.3 10.2 10.1 13. mg/100 mg † 14. α 1-glycoprotein, 15. 16. 3.6 6.2 5.0 4.0 9.6 1.8 6.5 4.6 7.5 9.1 1.8 6.8 5.3 2.9 9.6 5.8 2.4 11.2

17. mg/100 mg † Complement C3, 18. 19. 2.7 4.9 4.1 3.3 0.6 4.3 2.3 5.9 1.1 6.6 8.3 2.8 9.2 4.9 3.7 13.6 10.6 20. 12.4 mg/100 mg † 21. α 1-anti-trypsin,

22. †

23. 0 6.7 4.7 3.4 6.1 8.7 1.8 4.5 3.0 2.2 3.3 2.9 2.9 3.5 12.8 10.6 11.6 10.3

24. mg/100 mg Amyloid A, 25.

† 26. 7.0 4.0 4.4 4.4 2.4 5.4 2.8 8.4 3.2 5.4 3.0 1.6 9.2 8.2 4.7 3.8 11.5 27. 11.3 mg/100 mg

28. Haptoglobin,

29. † 30. 4.7 8.6 5.1 6.2 1.1 3.4 8.1 4.8 4.2 6.5 4.7 3.1 8.3 6.7 3.7 2.8 13.6 31. 12.8 per day intake mg/100 mg Fibrinogen, 32. ‑ 1

33. † 34. 5.5 8.6 4.6 4.6 1.1 8.2 3.1 3.9 7.8 3.2 6.0 9.6 1.4 7.3 8.2 5.2 1.4 11.7 Protein,

35. C-Reactive mg/100 mg 36. 37. 38. Reference 21 Reference 52 Taurine ASPX, aspartate plus asparagine; GLUX, glutamine glutamate; EAA, essential amino acid; NEAA, nonessential acid. Tyrosine Serine Proline Glycine GLUX Cysteine ASPX Alanine NEAA Arginine Valine Tryptophan Threonine Phenylalanine Methionine Lysine Leucine Isoleucine EAA Histidine ^Reference 24 , assuming 100 ml.kg 39. polymorphonuclear lymphocytes (PMNs), breast milk, and mixed muscle proteins 9. Amino acid composition of 7 major acute phase proteins, Table † ‡

124 Parenteral amino acid intake

1. of the amino acid formula tailored after the amino acid needs during the inflammatory 2. response will be critical to improving protein (nitrogen) homeostasis. It is not known if 3. a more appropriate supply of amino acids patterned after the patient’s specific needs 4. would have an impact on ameliorating the catabolic response, but it is evident that 5. the degree of stress response and interventions such as steroid therapy, will impose a 6. further demand on specific amino acid needs. 7. Courtney-Martin and coworkers55 estimated total sulfur amino acid requirements pro- 8. vided as methionine of about 47.4 mg.kg‑1.d‑1 and a safe level of intake of 56 mg.kg‑1.d‑1 9. in surgical neonates. We found in a previous study in critically ill infants 1-3 years of age, 10. that total parenteral sulfur amino acid requirements were 56 mg.kg‑1.d‑1 provided as 11. methionine and cysteine56. These values are about two fold higher than recommended 12. enteral intakes by the IOM, but lower than the total parenteral sulfur amino acid (TSAA: 13. methionine, cysteine, taurine) intakes of 137.4 mg.kg‑1.d‑1 provided to the infants in 14. the current study. Hence, although it is expected that critically ill children will require 15. higher parenteral amino acid intakes, given their increased demands, it is concerning 16. that intakes of several fold above requirements are provided to these patients. These 17. data raise questions about the adequacy of amino acids intakes provided to critically 18. ill children and the potential for toxicity. 19. Finally, whether the excessive amounts of amino acids provided to critically ill children 6 20. result in toxic effects cannot be established from this study, but it is well established 21. that amino acids can have toxic effects7 and that long-term use of parenteral nutrition 22. results on cholestasis and eventual liver failure57. Moss and coworkers58 demonstrated 23. that methionine is hepatotoxic in a rabbit model receiving large amounts of paren- 24. teral methionine with or without enteral feedings58, and it induced changes similar 25. to those observed in TPN cholestasis. Methionine toxicity appears to be related to 26. 3-methylthiopropionic acid (3-MTP), a known methionine transamination metabolite59. 27. L-arginine, through L-ornithine production is known to induce necrotizing pancreatitis 28. in a rat model60 and L-Lysine at large doses induces renal failure in dogs61. 29. The knowledge on safe levels of enteral amino acid intakes is sparse, and there is 30. further limitation on the knowledge on human parenteral amino acid toxicity, beyond 31. hyperammonemia, acidosis and seizures. Finally, in the original limited studies on the 32. development of parenteral amino acid formulas, it was decided that if the parenteral 33. amino acid formulas did not induce the latter complications, it was safe to use. This 34. limited evidence was accepted and has remained unchallenged for several decades. It 35. is clear that parenteral amino acid administration needs to be based on better knowl- 36. edge on the nutritional and functional requirements of individual amino acids under 37. specific pathophysiological conditions, as well as on a better understanding of toxicity 38. and safe upper limits of intake. 39.

125 Chapter 6

1. Conclusions 2. 3. We observed that the amino acid composition of commercially available parenteral 4. amino acid formulas provided to critically ill children are rather variable and unbal- 5. anced to support inflammatory/immune protein synthesis, resulting on excessive or 6. deficient supply of amino acids when compared to IOM recommendations, breast milk 7. and mixed muscle proteins. These formulas are based on the convenience of solubility 8. and stability and the lack of obvious, acute side effects such as acidosis and hyperam- 9. monemia. Some NEAA, the major component of inflammatory-immune proteins, were 10. not provided while other NEAA were provided in excess. Parenteral or enteral amino 11. acid intakes in critically ill children should be based on established requirements, not 12. only to maintain nutritional balance but also to maintain non-nutritional amino acid 13. functions. Dose-response, upper intake limits and surveillance of toxicity need also 14. to be defined in patients receiving parenteral amino acid solutions. These data have 15. also important implications for chronically ill children receiving long-term parenteral 16. nutrition, who may have increased amino acid needs for longer periods of time. 17. 18. 19. References 20. 21. 1. Morris SM, Jr. Arginine: beyond protein. balism. A failure of exogenous nutritional Am J Clin Nutr 2006;​83(2):​508S-12S. support. Ann Surg 1980;​192(4):​570‑80. 22. 2. Gulati P, Thomas G. Nutrient sensing 7. Baker DH. Comparative species utiliza- 23. in the mTOR/S6K1 signalling pathway. tion and toxicity of sulfur amino acids. J 24. Biochem Soc Trans 2007;​35(Pt 2):​236‑8. Nutr 2006;​136(6 Suppl):​1670S-5S. 25. 3. Tokunaga C, Yoshino K, Yonezawa K. 8. Garlick PJ. Assessment of the safety of 26. mTOR integrates amino acid- and ener- glutamine and other amino acids. J Nutr 27. gy-sensing pathways. Biochem Biophys 2001;​131(9 Suppl):​2556S-61S. Res Commun 2004;​313(2):​443‑6. 9. Munro IC, Renwick AG. The 5th workshop 28. 4. Tremblay F, Lavigne C, Jacques H, on the assessment of adequate intake of 29. Marette A. Role of dietary proteins and dietary amino acids: general discussion 30. amino acids in the pathogenesis of 2. J Nutr 2006;​136(6 Suppl):​1755S-7S. 31. insulin resistance. Annu Rev Nutr 2007;​ 10. Curley MA, Castillo L. Nutrition and 32. 27:​293-310. shock in pediatric patients. New Horiz 5. Marshall S. Role of insulin, adipocyte hor- 1998;​6(2):​212‑25. 33. mones, and nutrient-sensing pathways in 11. Bier DM. Amino acid pharmacokinetics 34. regulating fuel metabolism and energy and safety assessment. J Nutr 2003;​ 35. homeostasis: a nutritional perspective of 133(6 Suppl 1):​2034S-9S. 36. diabetes, obesity, and cancer. Sci STKE 12. Renwick AG, Walker R. An analysis of 37. 2006;​2006(346):​re7. the risk of exceeding the acceptable 38. 6. Cerra FB, Siegel JH, Coleman B, Border or tolerable daily intake. Regul Toxicol JR, McMenamy RR. Septic autocanni- Pharmacol 1993;​18(3):​463‑80. 39.

126 Parenteral amino acid intake

13. Vary TC. Inter-organ protein and carbo- in adult humans be approximated from 1. hydrate metabolic relationships during the amino acid composition of body 2. sepsis: necessary evils or uncanny mixed proteins? Proc Natl Acad Sci U S 3. coincidences? Curr Opin Clin Nutr Metab A 1995;​92(1):​300‑4. 4. Care 1999;​2(3):​235‑42. 23. Agostoni C, Carratu B, Boniglia C, Riva 5. 14. Vary TC, Hazen S. Sepsis alters pyruvate E, Sanzini E. Free amino acid content dehydrogenase kinase activity in skeletal in standard infant formulas: comparison 6. muscle. Mol Cell Biochem 1999;​198(1-2):​ with human milk. J Am Coll Nutr 2000;​ 7. 113‑8. 19(4):​434‑8. 8. 15. Vary TC, Jefferson LS, Kimball SR. Insulin 24. Chuang CK, Lin SP, Lee HC, et al. Free 9. fails to stimulate muscle protein synthe- amino acids in full-term and pre-term 10. sis in sepsis despite unimpaired signal- human milk and infant formula. J Pediatr ing to 4E-BP1 and S6K1. Am J Physiol Gastroenterol Nutr 2005;​40(4):​496-500. 11. Endocrinol Metab 2001;​281(5):​E1045‑53. 25. Heird WC, Dell RB, Helms RA, et al. 12. 16. Chiolero R, Revelly JP, Tappy L. Energy Amino acid mixture designed to maintain 13. metabolism in sepsis and injury. Nutrition normal plasma amino acid patterns in 14. 1997;​13(9 Suppl):​45S-51S. infants and children requiring parenteral 15. 17. Tappy L, Chiolero R. Substrate utilization nutrition. Pediatrics 1987;​80(3):​401‑8. 16. in sepsis and multiple organ failure. Crit 26. Koletzko B, Goulet O, Hunt J, Krohn K, Care Med 2007;​35(9 Suppl):​S531‑4. Shamir R. 1. Guidelines on Paediatric 17. 18. Food and Nutrition Board Institute of Parenteral Nutrition of the European 18. Medicine. Panel on Macronutrients. Society of Paediatric Gastroenterology, 19. Standing Committee on the Scientific Hepatology and Nutrition (ESPGHAN) 6 20. Evaluation of Dietary Reference Intakes. and the European Society for Clinical 21. Dietary Reference Intakes for Energy, Nutrition and Metabolism (ESPEN), Carbohydrate, Fiber, Fat, Fatty acids, Supported by the European Society of 22. Cholesterol, Proteins and Amino Acids. Paediatric Research (ESPR). J Pediatr 23. Washington DC: The National Press; Gastroenterol Nutr 2005;​41 Suppl 2:​ 24. 2005. S1‑87. 25. 19. Broyer M, Jean G, Dartois AM, 27. Jobgen WS, Fried SK, Fu WJ, Meininger 26. Kleinknecht C. Plasma and muscle CJ, Wu G. Regulatory role for the argi- 27. free amino acids in children at the early nine-nitric oxide pathway in metabolism stages of renal failure. Am J Clin Nutr of energy substrates. J Nutr Biochem 28. 1980;​33(7):​1396-401. 2006;​17(9):​571‑88. 29. 20. Delaporte C, Jean G, Broyer M. Free 28. Lyons J, Rauh-Pfeiffer A, Ming-Yu Y, et 30. plasma and muscle amino acids in ure- al. Cysteine metabolism and whole blood 31. mic children. Am J Clin Nutr 1978;​31(9):​ glutathione synthesis in septic pediatric 32. 1647‑51. patients. Crit Care Med 2001;​29(4):​ 21. Reeds PJ, Fjeld CR, Jahoor F. Do the 870‑7. 33. differences between the amino acid 29. Lyons J, Rauh-Pfeiffer A, Yu YM, et al. 34. compositions of acute-phase and muscle Blood glutathione synthesis rates in 35. proteins have a bearing on nitrogen loss healthy adults receiving a sulfur amino 36. in traumatic states? J Nutr 1994;​124(6):​ acid-free diet. Proc Natl Acad Sci U S A 37. 906‑10. 2000;​97(10):​5071‑6. 38. 22. Young VR, el-Khoury AE. Can amino acid 30. Brosnan JT, Brosnan ME. Creatine: requirements for nutritional maintenance endogenous metabolite, dietary, and 39.

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therapeutic supplement. Annu Rev Nutr and splanchnic uptake in critically ill 1. 2007;​27:​241‑61. children. Am J Physiol Endocrinol Metab 2. 31. Castillo L, Beaumier L, Ajami AM, Young 2009(297):​E1046-E55. 3. VR. Whole body nitric oxide synthesis 42. Fong Y, Matthews DE, He W, Marano MA, 4. in healthy men determined from [15N] Moldawer LL, Lowry SF. Whole body and 5. arginine-to-[15N]citrulline labeling. Proc splanchnic leucine, phenylalanine, and Natl Acad Sci U S A 1996;​93(21):​11460‑5. glucose kinetics during endotoxemia in 6. 32. Montanez R, Sanchez-Jimenez F, humans. Am J Physiol 1994;​266(2 Pt 2):​ 7. Aldana-Montes JF, Medina MA. Poly- R419‑25. 8. amines: metabolism to systems biology 43. Thibault R, Welch S, Mauras N, et al. 9. and beyond. Amino Acids 2007;​33(2):​ Corticosteroids increase glutamine uti- 10. 283‑9. lization in human splanchnic bed. Am J 33. Battezzati A, Brillon DJ, Matthews Physiol Gastrointest Liver Physiol 2008;​ 11. DE. Oxidation of glutamic acid by the 294(2):​G548‑53. 12. splanchnic bed in humans. Am J Physiol 44. Stoll B, Burrin DG. Measuring splanchnic 13. 1995;​269(2 Pt 1):​E269‑76. amino acid metabolism in vivo using 14. 34. Haisch M, Fukagawa NK, Matthews DE. stable isotopic tracers. J Anim Sci 2006;​ 15. Oxidation of glutamine by the splanchnic 84 Suppl:​E60‑72. 16. bed in humans. Am J Physiol Endocrinol 45. Haussinger D, Steeb R, Kaiser S, Wet- Metab 2000;​278(4):​E593-602. tstein M, Stoll B, Gerok W. Nitrogen 17. 35. Matthews DE, Marano MA, Campbell metabolism in normal and cirrhotic liver. 18. RG. Splanchnic bed utilization of gluta- Adv Exp Med Biol 1990;​272:​47‑64. 19. mine and glutamic acid in humans. Am J 46. Bertolo RF, Chen CZ, Law G, Pencharz 20. Physiol 1993;​264(6 Pt 1):​E848‑54. PB, Ball RO. Threonine requirement of 21. 36. Wang J, Chen L, Li P, et al. Gene expres- neonatal piglets receiving total parenteral sion is altered in piglet small intestine by nutrition is considerably lower than that 22. weaning and dietary glutamine supple- of piglets receiving an identical diet intra- 23. mentation. J Nutr 2008;​138(6):​1025‑32. gastrically. J Nutr 1998;​128(10):​1752‑9. 24. 37. Morris SM, Jr. Regulation of enzymes of 47. Brunton JA, Ball RO, Pencharz PB. Cur- 25. the urea cycle and arginine metabolism. rent total parenteral nutrition solutions for 26. Annu Rev Nutr 2002;​22:​87-105. the neonate are inadequate. Curr Opin 27. 38. Burrin DG, Davis TA. Proteins and amino Clin Nutr Metab Care 2000;​3(4):​299-304. acids in enteral nutrition. Curr Opin Clin 48. Elango R, Goonewardene LA, Pencharz 28. Nutr Metab Care 2004;​7(1):​79‑87. PB, Ball RO. Parenteral and enteral 29. 39. Matthews DE, Marano MA, Campbell RG. routes of feeding in neonatal piglets re- 30. Splanchnic bed utilization of leucine and quire different ratios of branched-chain 31. phenylalanine in humans. Am J Physiol amino acids. J Nutr 2004;​134(1):​72‑8. 32. 1993;​264(1 Pt 1):​E109‑18. 49. Elango R, Pencharz PB, Ball RO. The 40. Raguso CA, Ajami AM, Gleason R, Young branched-chain amino acid requirement 33. VR. Effect of cystine intake on methionine of parenterally fed neonatal piglets is 34. kinetics and oxidation determined with less than the enteral requirement. J Nutr 35. oral tracers of methionine and cysteine in 2002;​132(10):​3123‑9. 36. healthy adults. Am J Clin Nutr 1997;​66(2):​ 50. Agostoni C, Carratu B, Boniglia C, 37. 283‑92. Lammardo AM, Riva E, Sanzini E. Free 38. 41. Verbruggen S, Sy J, Gordon WE, et glutamine and glutamic acid increase in al. Ontogeny of methionine utilization human milk through a three-month lacta- 39.

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tion period. J Pediatr Gastroenterol Nutr human neonates. Am J Clin Nutr 2008;​ 1. 2000;​31(5):​508‑12. 88(1):​115‑24. 2. 51. Mansoor O, Breuille D, Bechereau F, et 56. Sy J, Gordon W, Griffin D, et al. Parenteral 3. al. Effect of an enteral diet supplemented sulfur amino acid requirements in septic 4. with a specific blend of amino acid on infants. FASEB J 2007;​21(6):​A1072-c-. 5. plasma and muscle protein synthesis in 57. Kelly DA. Intestinal failure-associated liver ICU patients. Clin Nutr 2007;​26(1):​30‑40. disease: what do we know today? Gas- 6. 52. Fukuda K, Nishi Y, Usui T. Free amino troenterology 2006;​130(2 Suppl 1):​S70‑7. 7. acid concentrations in plasma, erythro- 58. Moss RL, Haynes AL, Pastuszyn A, Glew 8. cytes, granulocytes, and lymphocytes RH. Methionine infusion reproduces liver 9. in umbilical cord blood, children, and injury of parenteral nutrition cholestasis. 10. adults. J Pediatr Gastroenterol Nutr Pediatr Res 1999;​45(5 Pt 1):​664‑8. 1984;​3(3):​432‑9. 59. Dever JT, Elfarra AA. L-methionine toxic- 11. 53. Gotschlich EC, Edelman GM. C-reactive ity in freshly isolated mouse hepatocytes 12. protein: a molecule composed of sub- is gender-dependent and mediated in 13. units. Proc Natl Acad Sci U S A 1965;​ part by transamination. J Pharmacol Exp 14. 54(2):​558‑66. Ther 2008;​326(3):​809‑17. 15. 54. Castillo L, Yu YM, Marchini JS, et al. 60. Rakonczay Z, Jr., Hegyi P, Dosa S, et al. 16. Phenylalanine and tyrosine kinetics in A new severe acute necrotizing pancre- critically ill children with sepsis. Pediatr atitis model induced by L-ornithine in 17. Res 1994;​35(5):​580‑8. rats. Crit Care Med 2008;​36(7):​2117‑27. 18. 55. Courtney-Martin G, Chapman KP, Moore 61. Asanuma K, Adachi K, Sugimoto T, Chiba 19. AM, Kim JH, Ball RO, Pencharz PB. Total S. Effects of lysine-induced acute renal 6 20. sulfur amino acid requirement and me- failure in dogs. J Toxicol Sci 2006;​31(2):​ 21. tabolism in parenterally fed postsurgical 87‑98. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39.

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Part III Protein and Energy interactions

Chapter 7

Current recommended parenteral protein intakes do not support protein synthesis in critically ill septic, insulin resistant adolescents with tight glucose control

Sascha CAT Verbruggen Jorge Coss-Bu Manhong Wu Henk Schierbeek Koen FM Joosten Johannes B van Goudoever Leticia Castillo

Provisionally accepted Crit Care Med Chapter 7

1. Abstract 2. 3. Objective 4. To investigate the effects of insulin infusion and increased parenteral amino acids 5. intakes on whole body protein balance, glucose kinetics, and lipolysis in critically ill 6. insulin resistant septic adolescents. 7. 8. Design 9. A single center, randomized, crossover study. 10. 11. Setting 12. A medico-surgical intensive care unit in a tertiary university hospital. 13. 14. Patients 15. Nine critically ill, septic adolescents (age 15.0 ± 1.2 years, BMI 20 ± 4 kg.m‑2) receiving 16. total parenteral nutrition. 17. 18. Interventions 19. Patients receive total parenteral nutrition with standard (SAA; 1.5 gr.kg‑1.day‑1) and high 20. (HAA; 3.0 gr.kg‑1.day‑1) amino acid intake in a two day crossover setting, randomized 21. to the order in which they received it. On both study days, we conducted a primed, 13 2 22. constant, 7 h. stable isotope tracer infusion with [1- C]Leucine, [6,6- H2]Glucose and 2 23. [1,1,2,3,3- H5]Glycerol, in combination with a Hyperinsulinemic Euglycemic Clamp 24. (HEC) during the last 3 hours. 25. 26. Measurements and Main Results 27. Insulin decreased protein synthesis at SAA and HAA intakes (p < .01), while protein 28. breakdown decreased with insulin at SAA (p < .05), but not with the HAA intake. HAA 29. intake improved protein balance (p < .05), but insulin did not have an additive effect. 30. There was significant insulin resistance with an M-value of ~3 mg.kg‑1.min‑1/µU.mL‑1, 31. which was 30% of reported normal values. At HAA intake endogenous glucose pro- 32. duction was not suppressed by insulin and lipolysis rates increased. 33. 34. Conclusion 35. The current recommended parenteral amino acid intakes are insufficient to maintain 36. protein balance in insulin resistant patients during tight glucose control. During sepsis 37. insulin decreases protein synthesis and breakdown, and while HAA intake improves 38. protein balance, its beneficial effects may be offset by enhanced endogenous glucose 39. production and lipolysis, raising concerns that insulin resistance may have been exac-

134 Insulin resistance and protein metabolism

1. erbated and that gluconeogenesis may have been favored by high amino acid intakes. 2. Dose response studies on the effect of the level of amino acid intakes on protein an 3. energy metabolism are needed. 4. 5. 6. Introduction 7. 8. During sepsis and inflammation, cytokine release through Nuclear Factor-Kappa 9. B activation induces suppression of insulin receptor signaling via reduced tyrosine 10. phosphorylation of IRS-1 and IRS-2, as well as decreased- activation of phosphati- 11. dylinositol 3-kinase (PI3-K) and protein kinase B (Akt), resulting in insulin resistance1. 12. Insulin has pleiotropic effects, and insulin resistance affects both, protein and energy 13. metabolism, in addition to multiple other processes2. 14. The inflammatory response elicited by sepsis induces altered whole body protein 15. turnover with increased hepatic synthesis of inflammatory/immune proteins and de- 16. creased synthesis of myofibrillar and sarcoplasmic muscle proteins3‑5, due to changes 17. in translation initiation and reduction of translation efficiency6‑8. Changes on protein 18. turnover during sepsis are organ specific and influenced by age9, 10. 19. Sepsis also triggers a profound catabolic response characterized by increased muscle 20. protein breakdown. Loss of lean body mass is caused by activation of the ubiquitin- 21. proteasome proteolytic pathway (UPP) in muscle initiated by activation of caspase 22. 311, 12. Furthermore, there appears to be a link between muscle wasting and insulin 7 23. resistance13‑15. 24. Most critically ill septic patients present with insulin resistance and receive insulin 25. therapy to maintain plasma glucose concentrations within acceptable range16. Never- 26. theless, protein and energy supply is rather variable and constrained by fluid restric- 27. tion, feeding intolerance and lack of evidence on appropriate nutrient requirements 28. under these conditions17. 29. There is a close interrelationship between protein (nitrogen) and energy metabolism. 30. Protein accretion will not occur without sufficient energy supply, and sufficient energy 31. supply will not support anabolism in the absence of adequate protein (nitrogen) intake. 32. We have shown that the parenteral protein requirements of critically ill children have 33. been based on limited data17 and that at currently recommended enteral protein in- 34. takes, critically ill children remain in significantly negative protein balance18. 35. Based on the knowledge that amino acid availability is a key component in the drive 36. for protein synthesis19, 20, we hypothesized that critically ill, insulin resistant, septic 37. adolescents would require higher parenteral amino acid intakes to maintain protein 38. balance in the presence of insulin administration. For this purpose we conducted a 39. two day, prospective, randomized, crossover study to compare the effects of a high

135 Chapter 7

1. parenteral amino acid intake of 3 g.kg‑1.d‑1 versus standard recommended amino acid 2. intake21 of 1.5 g.kg‑1.d‑1 on the rates of protein turnover, glucose and lipid kinetics 3. in critically ill insulin resistant septic adolescents, under basal conditions and while 4. receiving a hyperinsulinemic euglycemic clamp (HEC), aiming to maintain plasma 5. glucose concentrations at rates equivalent to a tight glucose control regimen (90-110 6. mg.dL‑1; 5.0 – 6.1 mM)22. 7. 8. 9. Material and Methods 10. 11. Patients 12. The study was approved by the Baylor College of Medicine Institutional Review Board, 13. and informed consent was obtained from parents. Studies were conducted at the Pe- 14. diatric Intensive Care Unit at Texas Children’s Hospital. Hyperglycemic (> 120 mg.dL‑1; 15. > 6.7 mM) adolescents (13-18 yrs of age) with a diagnosis of severe sepsis, septic 16. shock or Systemic Inflammatory Response Syndrome (SIRS)23, and receiving TPN. 17. Only adolescents age 13-18 years were included in the study as we have previously 18. observed that metabolic processes greatly varies with age18. All patients had drawing 19. and infusing intravascular lines and had received complete parenteral feedings for at 20. least 24 h before the study. All were assessed for severity of disease by the Pediatric 21. Logistic Organ Dysfunction (PELOD) score24 and the Pediatric Risk of Mortality III 22. (PRISM III) score25, and Tanner classification26, 27. Patients with metabolic diseases, 23. diabetes mellitus, primary liver, or renal failure were excluded. 24. A Figuretotal of 1 thirteen consecutive, hemodynamically stable, critically ill adolescents admit- 25. ted to the Pediatric Intensive Care Unit were included. From the thirteen adolescents, 26. nine patients (age 15.0 ± 1.2 years, BMI 20 ± 4 kg.m‑2, PRISM 11 ± 4) were enrolled in 27. the actual two-day study and randomized to receive first, either the standard (SAA) or 28. high (HAA) amino acid intake, and the alternate level of parenteral amino acid intake 29. was supplied over the next 24h (Figure 1), and four patients (age 16.5 ± 3.6 years, 30. Day 1 Day 2 31. 32. Amino acid intake SAA SAA

33. HAA HAA 34. Isotope tracer study tracer tracer 35. Time > 16 h 7 h > 16 h 7 h 36. 37. Figure 1. Schematic presentation of the study protocol in nine critically ill septic adolescents 38. randomized to either standard or high parenteral amino acid intake in a cross-over design. SAA = standard amino acid intake (1.5 g.kg‑1.d‑1), HAA = high amino acid intake (3.0 39. g.kg‑1.d‑1).

136 Insulin resistance and protein metabolism

1. BMI 25 ± 9 kg.m‑2, PRISM 6 ± 3) with similar conditions participated in a one-day pilot 2. study to determine the contribution of 13C carbon from the dextrose infusion to the 13 13 28 3. recovery of CO2 from C labeled leucine oxidation . The pilot patients received a 4. hyperinsulinemic euglycemic clamp without additional infusion of stable isotope trac- 5. ers (see below). 6. The characteristics of the patients are described in Tables 1 and 2. One patient ran- 7. domized to start first with the standard amino acid intake group (SAA) died the next 8. day after the first study (SAA) was completed, therefore the high amino acid intake 9. (HAA) arm of the study could not be conducted. In a second patient a technical error 10. occurred during the insulin infusion protocol on the study day with SAA intake. There- 11. fore, complete two-day study data were available on 7 patients. Data available from 12. the two incomplete studies were included according to intention-to-treat principle. In 13. three patients with severe insulin resistance, plasma glucose levels did not decrease 14. within the first hour of the HEC. Insulin infusion rates were increased until plasma 15. 16. Table 1. Demographic and nutritional data in nine critically ill septic adolescents* 17. Age (years) 15.0 ± 1.2 18. Gender (male:female) 3 : 6 ‑2 19. BMI (kg.m ) 20 ± 4 Tanner score 4.0 ± 0.9 20. REE^ according to Schofield (69)Preschool (kcal.kg‑1.day‑1) 29.3 ± 6.0 21. SAA£ HAA£ p-value 22. 7 PICU LOS† (days) 5.9 ± 3.6 7.0 ± 3.6 .53 23. PELOD‡ 9 ± 11 6 ± 7 .61 24. PRISM# 10 ± 4 8 ± 4 .65 25. C-Reactive Protein (mg.dL‑1) 16.5 ± 9.4 15.1 ± 12.0 .61 26. Highest Glucose prior study (mg.dL‑1) 182 ± 36 186 ± 81 .64

27. Catecholamines (n= ) 1 0

28. Glucocorticoids (n= ) 4 3

29. Protein intake (gr.kg‑1.day‑1) 1.5 ± 0.2 2.8 ± 0.4 < .001

30. Caloric intake (kcal.kg‑1.day‑1) 32.7 ± 10.0 37.8 ± 9.9 .36

31. Protein calories (kcal.kg‑1.day‑1) 5.8 ± 0.8 11.2 ± 1.4 < .001

32. Carbohydrate calories (kcal.kg‑1.day‑1) 22.5 ± 9.5 23.8 ± 10.3 .20 33. Lipid calories (kcal.kg‑1.day‑1) 5.7 ± 3.9 4.7 ± 3.5 .19 34. Glucose prior to HEC (mg.dL‑1) 168 ± 58 172 ± 62 .75 35. Glucose during HEC (mg.dL‑1) 98 ± 6 101 ± 15 .78 36. Insulin plasma levels baseline (µU.mL‑1) 32 (17 – 122) 51 (29 – 153) .16 37. Insulin plasma levels during HEC (µU.mL‑1) 144 (94 – 2385) 168 (93 – 2239) 1.0 38. *All values are mean ± SD. ^REE = Resting energy expenditure, £SAA = Standard amino acid intake, £HAA = High amino acid intake, †PICU LOS = Length of stay on the PICU at start of study, ‡PELOD = Pediatric Logistic Organ Dysfunction 24, 39. #PRISM III = Pediatric Risk of Mortality III 25

137 Chapter 7

Table 2. Characteristics of nine critically ill septic adolescents included in the two day study. 1. Patient Diagnosis Tanner BMI* Gender Mechanical Steroids Age Pressors 2. (kg.m‑2) Ventilation (years) 3. 1 MRSA† myositis 3 15.8 Male Yes Yes 17.0 Yes 4. 2 acute lymphatic leukemia, viral sepsis 5 24.2 Female Yes Yes 16.3 No 5. 3 peritonitis due to small bowel 4 20.7 Female Yes No 13.6 No perforation 6. 4 sepsis post lung and kidney 3 14.8 Male Yes Yes 15.9 No 7. transplantation

8. 5 sepsis post spinal surgery 5 29.3 Female Yes No 14.4 No 9. 6 MSSA‡ pneumonia 4 21.4 Female Yes Yes 15.8 No 10. 7 peritonitis due to small bowel 3 19.6 Male Yes No 13.9 No perforation 11. 8 MRSA† myositis 4 19.7 Female Yes No 14.4 No 12. 9 MSSA‡ pneumonia 5 18.5 Female Yes No 14.9 No 13. * BMI = Body mass index, † MRSA = methicillin resistant staphylococci aureus, ‡ MSSA = methicillin susceptible 14. staphylococci aureus 15. 16. glucose levels decreased to achieve normoglycemia. Due to this approach the tracer 17. infusion and clamp study in these three patients was extended to four hours instead 18. of three, to achieve steady state normoglycemia for at least one hour. Interestingly, the 19. same situation occurred in both study days for these three patients, who appeared to 20. be extremely insulin resistant. Two of these patients were receiving glucocorticoids. 21. 22. Experimental design 23. The experimental design followed, involved two 24h dietary study periods in a random- 24. ized cross-over fashion, where the subjects received for 24h a specific parenteral level 25. of amino acid intake (SAA or HAA) on study day 1, followed by the alternate parenteral 26. level of amino acid intake in the next day (Figure 1). Each patient received two dietary 27. study days and two tracer-clamp studies. 28. 29. Parenteral dietary intake 30. Patients were adapted to the level of study protein intake for at least 16hs before 31. the tracer infusion study, and continued to receive the randomized level of parenteral 32. amino acid intake during the tracer infusion study period at baseline and during the 33. HEC. The amino acid composition of the TPN provided to the patients is shown in 34. table 3. Energy intake provided as parenteral glucose and lipids were prescribed by 35. the clinical team according to standard care. The total energy intake supplied remained 36. unchanged during both study days (Table 1). 37. 38. 39.

138 Insulin resistance and protein metabolism

Table 3. Amino Acid Composition of Formulas Administered to the adolescents 1. Aminosyn (10%) Clinisol (15%) 2. (n=8) (n=1) 3. Essential 4. Histidine 300 894 5. Isoleucine 660 749 6. Leucine 1000 1040 7. Lysine 1050 1180 Methionine 172 749 8. Phenylalanine 298 1040 9. Threonine 400 749 10. Tryptophan 200 250 11. Valine 500 960

12. Non-essential

13. Arginine 1018 1470

14. Alanine 993 2170 15. Asparagine 0 0 16. Aspartate 700 434 17. Cysteine 0 0 18. Glutamine 0 0 19. Glutamate 738 749 Glycine 500 1040 20. Proline 722 894 21. Serine 530 592 22. 7 Tyrosine 270 39 23. Taurine 0 0

24. All values are mg/100mL. Composition provided by Aminosyn (Hospira, Lake Forest, IL), and Clinisol (Baxter, Deerfield, IL). 25. 26. Tracer infusion studies 27. On each study day the patients received a 7 hour, primed, continuous, stable isotope 28. tracer infusion study (Figure 2). The first 4 hours were the basal period and during the 29. last 3 hours of the tracer study the patients received the HEC. Isotope tracers were 30. purchased from Cambridge Isotope Laboratories (Andover, MA) and were tested for 31. sterility and pyrogenicity. On each tracer study the patients received an intravenous, 32. primed, continuous, 7 hour tracer infusion of L-[1-13C]leucine (6 µmol.kg‑1; 6 µmol. ‑1 ‑1 2 ‑1 ‑1 ‑1 2 33. kg .h ), D-[6,6- H2]glucose (25 µmol.kg ; 30 µmol.kg .h ), and [1,1,2,3,3- H5]glycerol 34. (30 µmol.kg‑1; 3 µmol.kg‑1.h‑1) for prime and constant infusion rates, respectively. The 35. bicarbonate pool was primed with 2.1 µmol.kg‑1 of 13C sodium bicarbonate as previ- 36. ously described 29. Four hours after initiation of the tracer infusions, a HEC was con- 37. ducted as described below (Figure 2). At the end of the first study day, the parenteral 38. protein intake was changed to the alternate level of protein intake and again, the tracer 39. infusion protocol was repeated (Figure 1).

139 Chapter 7

1. Hyperinsulinemic Euglycemic Clamp (HEC) 2. Four hours after the initiation of the tracer infusions a HEC was conducted as pre- 3. viously described28, 30. In brief, a 3h infusion of insulin (Actrapid, Novo Nordisk Inc., 4. Princeton, NJ), dissolved in sterile isotonic NaCl was started at 80 mU.m‑2.min‑1 in 5. order to achieve both normoglycemia between 90-110 mg.dL‑1 (5.0 – 6.1 mM) and 6. a plasma insulin concentration greater than 100 µU.mL‑1. During the insulin infusion, 7. whole blood glucose concentration was monitored at the bedside every 5-10 min. In 8. order to maintain the plasma glucose concentration between 90-110 mg.dL‑1 and the 2 9. enrichment of D-[6,6- H2]glucose at steady state for the duration of the study, a 30% 2 10. glucose solution (Baxter, Deerfield, IL) enriched at 3.5% with D-[6,6- H2]glucose was 11. infused as previously described (“hot Ginf”)30. 12. 13. Measurements and sample analysis 14. Arterial blood samples were obtained at frequent intervals (Figure 2), centrifuged 15. (12 min 3000 rpg at 4°C) and frozen at -80°C until analysis. We used plasma alpha- 16. ketoisocaproate (α-KIC) enrichment, which is intracellularly produced from the infused 13 2 17. leucine tracer. Plasma isotope enrichment of [1- C]α-KIC, D-[6,6- H2]glucose and 2 31‑34 18. [1,1,2,3,3- H5]glycerol were determined as previously described . Carbon dioxide

19. production (VCO2) was obtained with the respiratory profile monitor (CO2SMO Plus, 20. Novametrix Medical System, Wallingford, CT) during the last 40 minutes of the baseline 21. period and during the last 30 minutes of the HEC period. Enrichment of 13CO in whole Figure 2 2 22. blood was obtained as previously described29, 35. Plasma samples for insulin were 23. analyzed with standard human insulin specific RIA techniques. Amino acids in plasma 24. were determined as previously described36 by anion exchange chromatography with 25. ninhydrin detection on a Biochrom 30 Amino Acid Analyzer (Biochrom Ltd., Cambridge, 26. England). 27.

28. Plasma glucose 29. CO2 production 30. Blood samples 31. 32. HEC 33. Isotope tracer infusion

34. Minutes 0 200 220 240 390 405 420 35. Figure 2. Schematic presentation of the tracer infusion studies during both study days in nine 36. critically ill septic adolescents receiving either standard or high parenteral amino acid intake. Black triangles indicate time points of plasma glucose measurements, arrows indicate time 37. points for plasma collection for isotopic enrichment measurements and square boxes represent 38. the time period in which carbon dioxide production measurements take place. HEC = Hyperinsulinemic Euglycemic Clamp. 39.

140 Insulin resistance and protein metabolism

1. Calculations 2. Whole body kinetics of protein and lipids were calculated by conventional isotope dilu- 3. tion equations using a stochastic model during steady state enrichment37 and glucose 4. kinetics were estimated using the Steele equation38. The rate of appearance (Ra) of 5. unlabeled substrate can be derived from the plasma isotope enrichment calculated by: 6.

7. Ra = i x (Einf/Epl – 1) (1) 8.

9. where i is the infusion rate of the labeled tracer, Einf is the tracer enrichment of the

10. infusate and Epl the tracer enrichment in plasma (Mole fraction per cent excess), re- 11. spectively. 12. 13 13. CO2 recovery during 30% Dextrose infusion 14. To determine the contribution of 13C originating from the 30% dextrose infused dur- 13 15. ing the HEC to CO2 produced during leucine oxidation, we conducted an identical 16. clamp study without the tracer infusion28, in four patients with similar characteristics. 13 17. Using linear regression we established the CO2 background enrichment in relation to 18. the elevation of glucose infusion rate during HEC. For every additional mg.kg‑1.min‑1 13 19. of glucose infused during HEC in our pilot studies, CO2 enrichment increased by 20. 0.31 APE*103. Thus for every additional mg.kg‑1.min‑1 of glucose infused during HEC 21. in the patients enrolled in the actual study, we subtracted 0.31 APE*103 enrichment of 22. the 13CO blood enrichment (APE*103), because this amount was contributed by the 7 2 23. 30% dextrose infused, and the remaining enrichment originated from L-[1-13C]leucine 24. oxidation. 25. 26. Glucose metabolism 27. Estimates of whole-body glucose kinetics were made at isotopic steady-state, ef- 28. fectively attained during the last 40 min of the basal period and the last 30 min of the 2 29. HEC. Mean values of plasma D-[6,6- H2]glucose enrichment (Mole fraction percent 30. excess) and of exogenous glucose infusion rate were used for data calculation. Under 31. steady state conditions total glucose rate of appearance (Ra) is equal to the rate of 32. disappearance (Rd)38. The rates of glucose disappearance reflect glucose utilization. 33. Total endogenous glucose production (EGP) rate was calculated during the last 40 34. min of the basal period and the last 30 min of the HEC by subtracting the exogenous 35. glucose infusion rate from the glucose rates of appearance (Ra)39. 36. 37. EGP (mg.kg‑1.min‑1) = Glucose Ra – Glucose infusion rate (2) 38. 39.

141 Chapter 7

1. Insulin sensitivity (mg.kg‑1.min‑1/µU.mL‑1) as measured with the HEC and pointed out 2. as M-value, is the rate of glucose disappearance (Rd) divided by plasma insulin con- 3. centration during steady state40. 4. 5. Insulin sensitivity (mg.kg‑1.min‑1/µU.mL‑1) = Rd/Plasma insulin (3) 6. 7. Insulin stimulated glucose disposal (mg.kg‑1.min‑1) was considered equal to the sum of 8. EGP and GIR during steady state glucose rate of disappearance41. 9. 10. Insulin stimulated glucose disposal = EGP + GIR (4) 11. 12. Protein Metabolism 13. Whole body plasma leucine flux, an index of protein metabolism, was calculated using 14. plasma α-ketoisocaproate (α-KIC) enrichment expressed as mole fraction per cent 15. excess during the last 40 min of the baseline period and the last 30 min of the HEC 16. period, as described previously34. Leucine Oxidation was obtained as follows: 17. 13 13 18. VCO2 x (E CO2/69.18)/ C α-KIC (5) 19.

20. where VCO2 is the rate of carbon dioxide elimination measured in milliliters per minute 21. and converted to millimoles per hour by multiplying by 60 min and dividing by 22.4, 22. which is the number of 1 in 1 mole of an ideal gas at standard temperature and pres- 13 23. sure. We have previously shown that 69.18 is the correction factor for CO2 under 24. recovery obtained from parenterally fed critically ill children35. 25. Under the assumption that 1 gram of mixed muscle protein contains approximately 26. 621 µmol of leucine42, whole body protein turnover was calculated from the model 27. described by Golden and Waterlow43. Briefly, the model is described in the following 28. equation; 29. 30. Ra = S + O = B + I. (6) 31. 32. Where Ra is the rate of appearance into the plasma pool, S corresponds to protein 33. synthesis and O to oxidation, and therefore losses from the plasma amino acid pool, 34. while B represents protein breakdown and I dietary intake, and therefore entry of amino 35. acids into the plasma pool29. 36. Protein synthesis (S) was calculated by subtracting the rates of leucine oxidation (O) 37. from the plasma leucine flux (Ra). 38. 39. Prot Synthesis = Leucine Ra – Leu ox (7)

142 Insulin resistance and protein metabolism

1. Protein breakdown (B) was calculated by subtracting the leucine intake (I; tracer infu- 2. sion + leucine content of parenteral nutrition) from the leucine flux (Ra). 3. 4. Prot Breakdown = Leu flux – Leu intake (8) 5. 6. Protein balance was then calculated subtracting whole body protein breakdown from 7. whole body protein synthesis. 8. 9. Prot bal = Prot Synthesis – Prot Breakdown (9) 10. 11. Lipid Metabolism 12. Glycerol flux was calculated during the last 40 min of the baseline period, and during 13. the last 30 min of the hyperinsulinemic euglycemic clamp according to the steady 14. state tracer dilution equations reported previously32. As part of their parenteral nutrition 15. the patients were provided with lipids intravenously which contained 2.25 mg.mL‑1 16. glycerol. Lipolysis was calculated by subtracting the glycerol intake (tracer infusion + 17. glycerol intake through parenteral nutrition) from the glycerol flux (Ra). 18. 19. Lipolysis = Glycerol Ra – Total glycerol intake (10) 20. 21. Statistical analysis 22. From our previous data on protein turnover at random different levels of protein in- 7 23. take in critically ill children42, we estimated that 8 patients with complete data, would 24. detect a difference of 20% (80% power, type I error of 5%) on protein balance. The 25. Shapiro-Wilk normality test was used to determine data normality. Comparison be- 26. tween the two different amino acid intakes at baseline and during the hyperinsulinemic 27. euglycemic clamp were made using the repeated measurements ANOVA, after which a 28. student’s paired t test was used for normally distributed data. For non-parametric data 29. the Wilcoxon matched pairs test was used. Data are presented as the mean ± stan- 30. dard deviation. Statistical significance was considered at p < .05. Repeated measures 31. ANOVA were used to analyze the effect of insulin on parameters of glucose, lipid and 32. protein metabolism over time and between normal and high amino acid intake. Data 33. were analyzed with Graphpad Prism 5.0.3 (Graphpad Software, La Jolla, CA., USA). 34. 35. 36. 37. 38. 39.

143 Chapter 7

1. Results 2. 3. Patients 4. Between the two study days, there were no differences in PRISM, PELOD scores, CRP, 5. PICU length of stay at start of study (PICU LOS) or highest glucose values prior to the 6. onset of the tracer and clamp studies. There were no differences in the total energy 7. intake provided in both study days (Table 1). 8. 9. Insulin sensitivity and glucose kinetics 10. The maximum plasma glucose concentrations did not differ between both study days 11. prior to the onset of the tracer studies (Table 1). As expected, plasma glucose con- 12. centration differed between the baseline and HEC periods, respectively for SAA and 13.FigureHAA3 intakes (p < .001; Table 1). Plasma insulin concentrations for the baseline period 14. did not differ between the dietary groups (Table 1). However, there was considerable 15. variability in the plasma insulin concentrations during the HEC period in both, the SAA 16. and HAA groups because of three highly insulin resistant patients. 17. Endogenous glucose production (EGP) decreased significantly with insulin administra- 18. tion during the HEC, both at the SAA and the HAA intakes (p < .05) but it was not fully 19. suppressed during the HAA intake (Figure 3; Panel A). 20. Insulin stimulated glucose disposal, an index of peripheral insulin sensitivity40 was not 21. different between the dietary groups (p > .05). However, these values were about 30 - 22. 35% of reported values of 14.2 mg.kg‑1.min‑1 in healthy children44, confirming that our 23. patients presented significant peripheral insulin resistance (Figure 3; Panel B). 24. 25. A B C 26. ‡ 8 5 ]

4 ] -1 -1

] † †

27. -1 4

2 .min 6 28. -1 /uU.ml .min -1 -1 3 29. 0 4 .min 2 30. -1 -2 2 31. sensitivity Insulin 1 EGP [mg.kg EGP [mg.kg Insulin induced glucose induced Insulin 32. [mg.kg disposal -4 0 0 33. SAA HAA SAA HAA 34. SAA Base HAA Base SAA Insulin HAA Insulin 35. Figure 3. Glucose kinetics in nine critically ill septic adolescents with standard and high 36. parenteral amino acid intake at baseline and during HEC. Panel A; EGP (mg.kg‑1.min‑1), Panel B; 37. Insulin induced glucose disposal (mg.kg‑1.min‑1), Panel C; Insulin sensitivity depicted as M-value ‑1 ‑1 ‑1 38. (mg.kg .min /µU.mL ). SAA = Standard amino acid intake, HAA = High amino acid intake, HEC = hyperinsulinemic euglycemic clamp, EGP = Endogenous glucose production. Values are 39. mean ± SD, † p < .05; baseline vs. HEC, ‡ p < .05; SAA vs. HAA.

144 Insulin resistance and protein metabolism

1. There was no difference in the glucose infusion rate required during the HEC to main- 2. tain normoglycemia at steady state (6.2 ± 1.9 mg.kg‑1.min‑1 vs. 5.6 ± 2.2 mg.kg‑1.min‑1, 3. respectively for SAA and HAA; p > .05). Insulin sensitivity did not differ between study 4. days (M-value 3.1 ± 1.7 vs. 3.0 ± 1.7 mg.kg‑1.min‑1/µU.mL‑1, p > .05, respectively for 5. SAA and HAA) (Figure 3; Panel C). 6. 7. Protein turnover 8. 9. Plasma amino acid concentrations 10. As expected, the HAA intake resulted in higher (p < .05) plasma amino acid concentra- 11. tions for many but not all amino acids. Insulin administration only decreased tyrosine 12. levels at SAA (p < .05), while none of the measured plasma amino acids concentrations 13. decreased under hyperinsulinemia at HAA intake (Table 4). 14. Table 4. Plasma amino acid concentrations* 15. SAAa HAAb 16. Baseline HEC Baseline HEC 17. Leucine 134 ± 50 122 ± 42 197 ± 82 ‡ 185 ± 91 ‡ 18. Isoleucine 77 ± 28 73 ± 26 120 ± 48 ‡ 112 ± 51 ‡ 19. Valine 193 ± 74 174 ± 64 268 ± 102 ‡ 256 ± 114 ‡ 20. Alanine 296 ± 115 295 ± 116 363 ± 142 ‡ 364 ± 180 ‡

21. Arginine 110 ± 57 133 ± 85 149 ± 61 ‡ 172 ± 87

22. Ornithine 91 ± 38 87 ± 33 127 ± 53 120 ± 47 7

23. Citrulline 13 ± 10 12 ± 10 16 ± 17 16 ± 18

24. ASPX c 48 ± 14 45 ± 13 54 ± 19 52 ± 16

25. GLUX d 456 ± 131 455 ± 148 517 ± 103 504 ± 146

26. Glycine 203 ± 65 208 ± 70 236 ± 72 243 ± 96 27. Methionine 25 ± 28 22 ± 28 28 ± 26 24 ± 19 28. Cystine 6 ± 5 8 ± 6 8 ± 6 9 ± 8 29. Phenylalanine 104 ± 44 95 ± 38 109 ± 37 111 ± 41 30. Tyrosine 64 ± 33 55 ± 32 † 57 ± 27 ‡ 59 ± 36 31. Lysine 222 ± 98 231 ± 97 293 ± 82 ‡ 305 ± 116 ‡ 32. Histidine 63 ± 21 66 ± 20 78 ± 19 76 ± 23 33. Threonine 154 ± 80 155 ± 76 162 ± 74 172 ± 71 ‡ ‡ 34. Serine 99 ± 31 102 ± 29 128 ± 36 131 ± 49 Proline 236 ± 85 239 ± 62 376 ± 124 ‡ 361 ± 131 ‡ 35. Taurine 39 ± 36 30 ± 28 27 ± 20 30 ± 20 36. *Plasma amino acids depicted as µmol.L‑1; values are mean ± SD, † p < .05; Baseline vs HEC, ‡ p < .05; SAA vs HAA 37. aSAA = Standard amino acid intake, 38. bHAA = High amino acid intake, cASPX = Asparagine and Aspartate, 39. dGLUX = Glutamine and Glutamate

145 Figure 4

Chapter 7

‡ 1. A SAA Ra B ] ‡ 300 ] -1 300 † HAA Ra -1 2. † .hr

TPN SAA .hr -1 3. TPN HAA -1 200 200

4. mol.kg mol.kg   5. 100 100 6.

7. [ Ra Leucine 0 [ Ra Leucine 0

8. 200 220 240 390 405 420 Timepoints (min) 9. SAA Base HAA Base Base Insulin SAA Insulin HAA Insulin 10. C D ] ] -1 SAA -1 11. ‡ .hr 150 HAA .hr 150 -1 12. -1 ‡

13. mol.kg 100 mol.kg 100   14.

15. 50 50 16. 17. 0 0 Leucine oxidation [ oxidation Leucine 18. 200 220 240 390 405 420 [ oxidation Leucine Timepoints (min) Base Insulin SAA Base HAA Base 19. SAA Insulin HAA Insulin

20. Figure 4. Leucine kinetics in nine critically ill septic adolescents with standard and high 21. parenteral amino acid intake at baseline and during HEC. Panel A; Time curve of leucine rate of appearance through TPN and endogenous leucine appearance on both study days, Panel 22. B; Bars of total leucine rate of appearance during the study periods, Panel C; Time curve of 23. Leucine oxidation on both study days, Panel D; Total leucine rate of oxidation during the study periods, TPN = total parenteral nutrition, SAA = Standard amino acid intake, HAA = High 24. amino acid intake, HEC = hyperinsulinemic euglycemic clamp. Values are µmol.kg‑1.h‑1; mean ± 25. SD. † p < .05; baseline vs. HEC, ‡ p < .05; SAA vs. HAA. 26.

27. A ‡ B C ] ] ‡ -1 ] -1 ‡ 28. -1 10 10 2 ‡

.day † † .day † † -1 .day 29. -1 8 8 -1 1

30. 6 6 0

31. 4 4 -1 32. 2 2 -2 33.

0 0 [g.kg balance Protein -3 Protein Synthesis [g.kg Synthesis Protein 34. [g.kg breakdown Protein SAA Base HAA Base SAA Base HAA Base SAA Base HAA Base SAA Insulin HAA Insulin 35. SAA Insulin HAA Insulin SAA Insulin HAA Insulin 36. Figure 5. Protein metabolism in nine critically ill septic adolescents with standard and high 37. parenteral amino acid intake at baseline and during HEC. Panel A; Protein synthesis, Panel B; 38. Protein breakdown, Panel C; Protein balance, SAA = Standard amino acid intake, HAA = High amino acid intake, HEC = hyperinsulinemic euglycemic clamp. Values are g.kg‑1.d‑1, mean ± SD. 39. † p < .05; baseline vs. HEC, ‡ p < .05; SAA vs. HAA.

146 Insulin resistance and protein metabolism

1. Leucine Rate of appearance and oxidation 2. As shown in Figure 4 Panel A, plasma leucine Ra increased when amino acid intake 3. was higher, as it would be expected, both at baseline and during HEC (p < .01 SAA vs. 4. HAA), and during HEC the rate of appearance of leucine decreased both at SAA intake 5. and HAA intake (p < .05) (Figure 4; Panel A and B). 6. The oxidation rates of leucine were not affected by insulin infusion, but did increase 7. with the higher amino acid intake at baseline (p < .001) and during HEC (p < .001) 8. (Figure 4; Panel C and D). 9. 10. Protein metabolism 11. As shown in Figure 5 Panel A, whole body protein synthesis rates significantly decreased 12. when insulin was administered at SAA intake (p < .01), and this effect persisted even in 13. the presence of HAA intake (p < .01). The protein synthesis rates were improved by the 14. HAA intake during the baseline and the HEC period (p < .05). In contrast, as shown in 15. Figure 5 Panel B, whole body protein breakdown was not affected by the amino acid 16. intake, but significantly decreased (p < .05) with insulin administration during the SAA Figure 6 17. intake and the HAA intake. Protein balance improved (p < .05) when the HAA intake 18. was given, but insulin administered during the HEC at the HAA intake did not further 19. improve protein balance. Likewise, insulin administration during SAA intake failed to 20. significantly improve protein balance (Figure 5; Panel C). 21. 22. Lipolysis 7 23. The rates of lipolysis were not affected by insulin during SAA or HAA amino acid intake. 24. Interestingly, the high amino acid intake increased lipolysis (p < .05) (Figure 6). 25.

] 6 ‡

26. -1 ‡

27. .min -1 4 28. mol.kg 29.  2 30.

31. [ Lipolysis 0 32.

SAA base HAA Base 33. SAA Insulin HAA Insulin 34. Figure 6. Rate of lipolysis in nine critically ill septic adolescents with standard and high 35. parenteral amino acid intake at baseline and during HEC. SAA = Standard amino acid intake, 36. HAA = High amino acid intake, HEC = hyperinsulinemic euglycemic clamp. Values are µmol. kg‑1.min‑1; mean ± SD. ‡ p < .05; SAA vs. HAA. 37. 38. 39.

147 Chapter 7

1. Discussion 2. 3. Protein turnover 4. The negative whole body protein balance observed at standard amino acid intakes 5. in the present study is consistent with previous data by us and others45, 46. Our data 6. showed that parenteral amino acids intakes of 1.5 gr.kg‑1.day‑1 as per recommended 7. guidelines21 are insufficient to support protein turnover and balance in septic, insulin 8. resistant adolescents at baseline conditions and while receiving insulin. 9. In critically ill adults, increasing protein intake from about 1 to 1.5 gr.kg‑1.day‑1 improved 10. but did not normalize whole body protein balance, while a further increase to 2 gr.kg‑1. 11. day‑1 did not lead to a further improvement47. In contrast, our study demonstrated that 12. in these septic adolescents, increasing parenteral amino acid intakes from 1.5 to 3.0 13. gr.kg‑1.day‑1 in the presence of adequate caloric intake, showed a strong trend towards 14. stimulation of protein synthesis and significantly improved whole body protein balance, 15. even in the absence of insulin administration. 16. It is not surprising that whole body protein synthesis was increased with higher amino 17. acid intakes, since amino acids, especially branched-chain amino acids such as L- 18. leucine, and cationic amino acids such as L-arginine act as nutrient signals themselves, 19. modulating cellular processes that lead to protein synthesis via augmented mRNA 20. translation initiation20. Amino acids are known to alter the phosphorylation status 21. of mammalian target of rapamycin (mTOR) associated signaling proteins in muscle, 22. and inhibiting AMP-activated protein kinase (AMPK) signaling, resulting in increased 23. protein synthesis via enhanced translation initiation and translation elongation20. The 24. mTOR pathway is a key regulator of cell growth and proliferation48. 25. Under physiological conditions, the major regulator of muscle protein synthesis is in- 26. creased amino acid availability (particularly leucine), rather than insulin49. Hence, dietary 27. amino acid intake and intracellular transport play a key role in this process. However, 28. under conditions of sepsis and cytokine release, muscle protein synthesis is inhibited 29. by decreased mTOR kinase activity in muscle, as evidenced by reduced phosphoryla- 30. tion of both eukaryotic initiation factor (eIF)4E-binding protein (BP)-1 and ribosomal S6 31. kinase (S6K)1, which are mTOR downstream signaling proteins6. The ability of leucine to 32. increase 4E-BP1 and S6K1 phosphorylation is greatly attenuated by TNF and glucocor- 33. ticoids, which results in “leucine resistance”6. This effect, however, may be overcome at 34. some extent by administration of amino acids, as demonstrated in an endotoxemic piglet 35. model whereby supplying amino acids at concentrations comparable to those found in 36. the fed state, rescued the inhibitory response of sepsis on muscle protein synthesis50. 37. The same effect has been observed in an adult rat model of chronic sepsis by feeding 38. oral leucine51, and is observed in our current data where the septic adolescents receiving 39. a high amino acid intake, showed a strong trend towards increased protein synthesis.

148 Insulin resistance and protein metabolism

1. In regard to the effects of insulin on protein synthesis during sepsis, our data showed 2. that during sepsis and perhaps exacerbated by the use of glucocorticoids, insulin 3. decreased whole body protein synthesis, even when a high amino acid intake was 4. provided. These data are in agreement with that of septic animal models6, insulin 5. dependent diabetics52 and critically ill newborns53. Lang and coworkers6 reported that 6. insulin failed to stimulate protein synthesis in an animal model of sepsis via a defect in 7. insulin signaling to a step in translation initiation mediating the assembly of the active 8. eukaryotic initiation factor 4 F complex (eIF4F), which is a key protein complex in 9. translation initiation. Therefore, sepsis induces insulin resistance to protein synthesis. 10. We observed that insulin administration decreased protein breakdown, which is con- 11. sistent with previous reports in critically ill newborns53, 54 and in adults with diabetes 12. mellitus52. Using leucine kinetics in healthy humans, it has been shown that hyperin- 13. sulinemia decreased proteolysis but did not stimulate protein synthesis. By contrast, 14. elevation of plasma levels of amino acids, by infusion of an amino acid mixture, 15. stimulated the protein synthesis, but did not suppress endogenous proteolysis55. Thus, 16. amino acids and insulin appear to exert different and complementary effects in stimu- 17. lating protein anabolism. In healthy humans insulin is “permissive” for protein synthesis 18. and suppressive for protein breakdown56. 19. However, during sepsis and insulin resistance, the high amino acid intake did not over- 20. come insulin resistance. It appears then that in septic adolescents insulin suppresses 21. protein synthesis and breakdown, while high amino acid intake enhances protein 22. synthesis but diminishes insulin effect on protein breakdown. 7 23. Furthermore, there is evidence that insulin resistance is associated with muscle 24. breakdown. During inflammation, insulin resistance decreases Phosphoinositide 25. 3-kinase (PI3K) activity and this subsequently reduces the level of phosphorylated 26. protein kinase B (Akt)13. A low Akt relieves the inhibition of the expression of specific 27. E3 ubiquitin-conjugating enzymes atrogin-1/MAFbx and MuRF1 in muscle. Expression 28. of these E3 enzymes is found in conditions causing loss of lean body mass13. The 29. Ubiquitin Proteasome Pathway together with autophagy, are the main routes that cells 30. use for degrading intracellular proteins. 31. Endogenous glucocorticoids and impaired insulin signaling are required for stimulation 32. of muscle breakdown in inflammation57. These conditions were found in our patients, 33. and currently, a large proportion of critically ill patients are managed with glucocorti- 34. coids which exacerbates insulin resistance and protein breakdown. 35. Insulin did not have an additive effect to the HAA intake, in terms of net protein balance 36. in the septic adolescents. This was mainly due to a less pronounced suppression of 37. protein breakdown with insulin at HAA intake, while protein synthesis was simulta- 38. neously decreased at HAA intake. High protein intakes are known to induce insulin 39. resistance58.

149 Chapter 7

1. Glucose metabolism and insulin sensitivity 2. All septic patients showed considerable insulin resistance with about 65% decrease in 3. insulin sensitivity when compared to values of ~11 mg.kg‑1.min‑1/µU.mL‑1 reported in 4. fasted healthy adolescents59, 60, and the degree of insulin resistance is comparable to 5. that observed in critically ill adults (~ 4.6 mg.kg‑1.min‑1/µU.mL‑1)61. Despite significant 6. insulin resistance, when the standard amino acid intake was supplied in the presence 7. of insulin, endogenous glucose production was suppressed, which is in agreement 8. with previous studies in critically ill adults62‑65. 9. Interestingly, when a high amino acid intake was provided to our patients, insulin failed 10. to suppress the rates of endogenous glucose production and lipolysis rates were 11. increased. This observation, in addition to the lack of insulin suppression of protein 12. breakdown at high amino acid intakes raised concern that a high amino acid intake may 13. exacerbate insulin resistance, and that a greater supply of gluconeogenic amino acids 14. could preferentially trigger their use over glycogen for glucose production66. Amino 15. acid signaling is integrated by mTOR which operates a negative feedback loop toward 16. insulin receptor substrate 1 (IRS-1) signaling, promoting insulin resistance for glucose 17. metabolism67. Amino acid infusion in healthy subjects to achieve hyperaminoacidemia 18. decreases insulin sensitivity68. In contrast, we observed no effect of increased amino 19. acid intake on insulin plasma levels or insulin sensitivity, but our sample size was limited. 20. Hence, although a high amino acid intake increased protein synthesis, at the amounts 21. supplied may have negatively impacted glucose homeostasis. 22. 23. Lipolysis 24. Insulin did not decrease lipolysis and this is consistent with data in critically ill adults64, 69. 25. Furthermore, lipolysis increased at HAA intakes, again raising concern for increased 26. insulin resistance when the HAA intake was provided. 27. 28. 29. Conclusion 30. 31. We confirmed that critically ill septic adolescents are markedly insulin resistant and 32. conclude that recommended parenteral amino acid intakes in these patients are in- 33. sufficient to maintain protein balance. Increasing amino acid intakes to 3 g.kg‑1.d‑1 34. improved protein synthesis and balance, but it may have favored gluconeogenesis 35. and stimulated lipolysis, raising concern that this level of amino acid intake may have 36. enhanced insulin resistance. Insulin infusion and tight glucose control at standard 37. amino acid intakes decreased both protein synthesis and breakdown, and did not af- 38. fect protein balance. Dose-response studies on protein administration and their effects 39. on energy metabolism and insulin resistance are needed.

150 Insulin resistance and protein metabolism

1. Acknowledgements 2. 3. We are indebted to the patients and their families at Texas Children’s Hospital for their 4. selfless contribution to this study. We thank the nursing staff, Debra Griffin, David 5. Zurakowski, Wim Hop, Chaji Chacko, Gardi Minderman-Voortman and Kristien Dorst 6. for their contribution to these studies and Dr. Morey Haymond for his advice. 7. 8. 9. References 10. 1. Matsuda N, Yamamoto S, Yokoo H, Am J Physiol 1992;​262(6 Pt 1):​C1513‑9. 11. Tobe K, Hattori Y. Nuclear factor-kappaB 9. Kimball SR, Orellana RA, O’Connor PM, 12. decoy oligodeoxynucleotides ameliorate et al. Endotoxin induces differential 13. impaired glucose tolerance and insulin regulation of mTOR-dependent signaling 14. resistance in mice with cecal ligation and in skeletal muscle and liver of neonatal 15. puncture-induced sepsis. Crit Care Med pigs. Am J Physiol Endocrinol Metab 16. 2009;​37(10):​2791‑9. 2003;​285(3):​E637‑44. 2. Verbruggen SC, Joosten KF, Castillo L, 10. Orellana RA, Kimball SR, Nguyen HV, 17. van Goudoever JB. Insulin therapy in the et al. Regulation of muscle protein syn- 18. pediatric intensive care unit. Clin Nutr thesis in neonatal pigs during prolonged 19. 2007;​26(6):​677‑90. endotoxemia. Pediatr Res 2004;​55(3):​ 20. 3. Hasselgren PO, Pedersen P, Sax HC, 442‑9. 21. Warner BW, Fischer JE. Current con- 11. Lecker SH, Solomon V, Mitch WE, cepts of protein turnover and amino acid Goldberg AL. Muscle protein breakdown 22. 7 transport in liver and skeletal muscle dur- and the critical role of the ubiquitin- 23. ing sepsis. Arch Surg 1988;​123(8):​992‑9. proteasome pathway in normal and dis- 24. 4. Vary TC. Regulation of skeletal muscle ease states. J Nutr 1999;​129(1S Suppl):​ 25. protein turnover during sepsis. Curr Opin 227S-37S. 26. Clin Nutr Metab Care 1998;​1(2):​217‑24. 12. Voisin L, Breuille D, Combaret L, et al. 27. 5. Vary TC, Kimball SR. Regulation of he- Muscle wasting in a rat model of long- patic protein synthesis in chronic inflam- lasting sepsis results from the activation 28. mation and sepsis. Am J Physiol 1992;​ of lysosomal, Ca2+ -activated, and 29. 262(2 Pt 1):​C445‑52. ubiquitin-proteasome proteolytic path- 30. 6. Lang CH, Frost RA, Vary TC. Regulation ways. J Clin Invest 1996;​97(7):​1610‑7. 31. of muscle protein synthesis during sepsis 13. Wang X, Hu Z, Hu J, Du J, Mitch WE. 32. and inflammation. Am J Physiol Endocri- Insulin resistance accelerates muscle nol Metab 2007;​293(2):​E453‑9. protein degradation: Activation of the 33. 7. Cooney RN, Kimball SR, Vary TC. Regu- ubiquitin-proteasome pathway by de- 34. lation of skeletal muscle protein turnover fects in muscle cell signaling. Endocri- 35. during sepsis: mechanisms and media- nology 2006;​147(9):​4160‑8. 36. tors. Shock 1997;​7(1):​1‑16. 14. Crossland H, Constantin-Teodosiu D, 37. 8. Vary TC, Kimball SR. Sepsis-induced Gardiner SM, Constantin D, Greenhaff 38. changes in protein synthesis: differential PL. A potential role for Akt/FOXO signal- effects on fast- and slow-twitch muscles. ling in both protein loss and the impair- 39.

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ment of muscle carbohydrate oxidation consensus conference: definitions for 1. during sepsis in rodent skeletal muscle. J sepsis and organ dysfunction in pediat- 2. Physiol 2008;​586(Pt 22):​5589-600. rics. Pediatr Crit Care Med 2005;​6(1):​2‑8. 3. 15. Crossland H, Constantin-Teodosiu D, 24. Leteurtre S, Martinot A, Duhamel A, et al. 4. Greenhaff PL, Gardiner SM. Low-dose Validation of the paediatric logistic organ 5. dexamethasone prevents endotoxaemia- dysfunction (PELOD) score: prospective, induced muscle protein loss and impair- observational, multicentre study. Lancet 6. ment of carbohydrate oxidation in rat 2003;​362(9379):​192‑7. 7. skeletal muscle. J Physiol 2010;​588(Pt 25. Pollack MM, Patel KM, Ruttimann UE. 8. 8):​1333‑47. PRISM III: an updated Pediatric Risk 9. 16. Wilson M, Weinreb J, Hoo GW. Intensive of Mortality score. Crit Care Med 1996;​ 10. insulin therapy in critical care: a review of 24(5):​743‑52. 12 protocols. Diabetes Care 2007;​30(4):​ 26. Marshall WA, Tanner JM. Variations in 11. 1005‑11. pattern of pubertal changes in girls. Arch 12. 17. Verbruggen S, Sy J, Arrivillaga A, Joosten Dis Child 1969;​44(235):​291-303. 13. K, van Goudoever J, Castillo L. Paren- 27. Marshall WA, Tanner JM. Variations in the 14. teral Amino Acid Intakes in Critically pattern of pubertal changes in boys. Arch 15. Ill Children: A Matter of Convenience. Dis Child 1970;​45(239):​13‑23. 16. JPEN J Parenter Enteral Nutr 2010;​34(3):​ 28. DeFronzo RA, Tobin JD, Andres R. 329‑40. Glucose clamp technique: a method 17. 18. Verbruggen S, Sy J, Gordon WE, et for quantifying insulin secretion and 18. al. Ontogeny of methionine utilization resistance. Am J Physiol 1979;​237(3):​ 19. and splanchnic uptake in critically ill E214‑23. 20. children. Am J Physiol Endocrinol Metab 29. el-Khoury AE, Sanchez M, Fukagawa NK, 21. 2009(297):​E1046-E55. Young VR. Whole body protein synthesis 19. Davis TA, Fiorotto ML, Burrin DG, et al. in healthy adult humans: 13CO2 tech- 22. Stimulation of protein synthesis by both nique vs. plasma precursor approach. 23. insulin and amino acids is unique to Am J Physiol 1995;​268(1 Pt 1):​E174‑84. 24. skeletal muscle in neonatal pigs. Am J 30. Finegood DT, Bergman RN, Vranic M. 25. Physiol Endocrinol Metab 2002;​282(4):​ Estimation of endogenous glucose 26. E880‑90. production during hyperinsulinemic- 27. 20. Fujita S, Dreyer HC, Drummond MJ, et al. euglycemic glucose clamps. Comparison Nutrient signalling in the regulation of hu- of unlabeled and labeled exogenous 28. man muscle protein synthesis. J Physiol glucose infusates. Diabetes 1987;​36(8):​ 29. 2007;​582(Pt 2):​813‑23. 914‑24. 30. 21. Mehta NM, Compher C. A.S.P.E.N. Clini- 31. Argoud GM, Schade DS, Eaton RP. Un- 31. cal Guidelines: nutrition support of the derestimation of hepatic glucose produc- 32. critically ill child. JPEN J Parenter Enteral tion by radioactive and stable tracers. Nutr 2009;​33(3):​260‑76. Am J Physiol 1987;​252(5 Pt 1):​E606‑15. 33. 22. van den Berghe G, Wouters P, Weekers 32. Beylot M, Martin C, Beaufrere B, Riou JP, 34. F, et al. Intensive insulin therapy in the Mornex R. Determination of steady state 35. critically ill patients. N Engl J Med 2001;​ and nonsteady-state glycerol kinetics in 36. 345(19):​1359‑67. humans using deuterium-labeled tracer. 37. 23. Goldstein B, Giroir B, Randolph A, J Lipid Res 1987;​28(4):​414‑22. 38. International Consensus Conference on 33. Chacko SK, Sunehag AL, Sharma S, Pediatric S. International pediatric sepsis Sauer PJ, Haymond MW. Measurement 39.

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of gluconeogenesis using glucose frag- critically ill septic pediatric patients. Crit 1. ments and mass spectrometry after Care Med 2003;​31(2):​591‑7. 2. ingestion of deuterium oxide. J Appl 43. Golden MH, Waterlow JC. Total protein 3. Physiol 2008;​104(4):​944‑51. synthesis in elderly people: a comparison 4. 34. Matthews DE, Motil KJ, Rohrbaugh DK, of results with [15N]glycine and [14C] 5. Burke JF, Young VR, Bier DM. Measure- leucine. Clin Sci Mol Med 1977;​53(3):​ ment of leucine metabolism in man from 277‑88. 6. a primed, continuous infusion of L-[1-3C] 44. Danadian K, Balasekaran G, Lewy V, 7. leucine. Am J Physiol 1980;​238(5):​ Meza MP, Robertson R, Arslanian SA. 8. E473‑9. Insulin sensitivity in African-American 9. 35. Sy J, Gourishankar A, Gordon WE, et al. children with and without family history 10. Bicarbonate kinetics and predicted en- of type 2 diabetes. Diabetes Care 1999;​ ergy expenditure in critically ill children. 22(8):​1325‑9. 11. Am J Clin Nutr 2008;​88(2):​340‑7. 45. Castillo L, Yu YM, Marchini JS, et al. 12. 36. Moore S, Spackman DH, Stein WH. Phenylalanine and tyrosine kinetics in 13. Automatic recording apparatus for use in critically ill children with sepsis. Pediatr 14. the chromatography of amino acids. Fed Res 1994;​35(5):​580‑8. 15. Proc 1958;​17(4):​1107‑15. 46. van Waardenburg DA, Deutz NE, Hoos 16. 37. Altszuler N, De Bodo RC, Steele R, Wall MB, et al. Assessment of whole body JS. Carbohydrate metabolism of hy- protein metabolism in critically ill chil- 17. pophysectomized dogs as studied with dren: can we use the [15N]glycine single 18. radioactive glucose. Am J Physiol 1956;​ oral dose method? Clin Nutr 2004;​23(2):​ 19. 187(1):​25‑31. 153‑60. 20. 38. Steele R, Wall JS, De Bodo RC, Altszuler 47. Ishibashi N, Plank LD, Sando K, Hill GL. 21. N. Measurement of size and turnover rate Optimal protein requirements during the of body glucose pool by the isotope dilu- first 2 weeks after the onset of critical ill- 22. 7 tion method. Am J Physiol 1956;​187(1):​ ness. Crit Care Med 1998;​26(9):​1529‑35. 23. 15‑24. 48. Wullschleger S, Loewith R, Hall MN. TOR 24. 39. Bier DM, Leake RD, Haymond MW, et al. signaling in growth and metabolism. Cell 25. Measurement of “true” glucose produc- 2006;​124(3):​471‑84. 26. tion rates in infancy and childhood with 49. Fujita S, Rasmussen BB, Cadenas JG, 27. 6,6-dideuteroglucose. Diabetes 1977;​ Grady JJ, Volpi E. Effect of insulin on hu- 26(11):​1016‑23. man skeletal muscle protein synthesis is 28. 40. Matsuda M, DeFronzo RA. Insulin sensi- modulated by insulin-induced changes in 29. tivity indices obtained from oral glucose muscle blood flow and amino acid avail- 30. tolerance testing: comparison with the ability. Am J Physiol Endocrinol Metab 31. euglycemic insulin clamp. Diabetes Care 2006;​291(4):​E745‑54. 32. 1999;​22(9):​1462‑70. 50. Orellana RA, Kimball SR, Suryawan A, 41. Jani R, Molina M, Matsuda M, et al. De- et al. Insulin stimulates muscle protein 33. creased non-insulin-dependent glucose synthesis in neonates during endotox- 34. clearance contributes to the rise in fast- emia despite repression of translation 35. ing plasma glucose in the nondiabetic initiation. Am J Physiol Endocrinol Metab 36. range. Diabetes Care 2008;​31(2):​311‑5. 2007;​292(2):​E629‑36. 37. 42. Argaman Z, Young VR, Noviski N, et al. 51. Vary TC. Acute oral leucine administra- 38. Arginine and nitric oxide metabolism in tion stimulates protein synthesis during chronic sepsis through enhanced asso- 39.

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ciation of eukaryotic initiation factor 4G 60. Moran A, Jacobs DR, Jr., Steinberger J, 1. with eukaryotic initiation factor 4E in rats. et al. Insulin resistance during puberty: 2. J Nutr 2007;​137(9):​2074‑9. results from clamp studies in 357 chil- 3. 52. Charlton M, Nair KS. Protein metabolism dren. Diabetes 1999;​48(10):​2039‑44. 4. in insulin-dependent diabetes mellitus. J 61. Zauner A, Nimmerrichter P, Anderwald 5. Nutr 1998;​128(2 Suppl):​323S-7S. C, et al. Severity of insulin resistance in 53. Agus MS, Javid PJ, Piper HG, et al. The critically ill medical patients. Metabolism 6. effect of insulin infusion upon protein 2007;​56(1):​1‑5. 7. metabolism in neonates on extracorpo- 62. Shangraw RE, Jahoor F, Miyoshi H, et 8. real life support. Ann Surg 2006;​244(4):​ al. Differentiation between septic and 9. 536‑44. postburn insulin resistance. Metabolism 10. 54. Agus MS, Javid PJ, Ryan DP, Jaksic T. 1989;​38(10):​983‑9. Intravenous insulin decreases protein 63. Chambrier C, Laville M, Rhzioual Ber- 11. breakdown in infants on extracorporeal rada K, Odeon M, Bouletreau P, Beylot 12. membrane oxygenation. J Pediatr Surg M. Insulin sensitivity of glucose and fat 13. 2004;​39(6):​839-44; discussion ‑44. metabolism in severe sepsis. Clin Sci 14. 55. Tessari P, Inchiostro S, Biolo G, et al. Dif- (Lond) 2000;​99(4):​321‑8. 15. ferential effects of hyperinsulinemia and 64. Whyte MB, Jackson NC, Shojaee- 16. hyperaminoacidemia on leucine-carbon Moradie F, et al. Metabolic effects of metabolism in vivo. Evidence for distinct intensive insulin therapy in critically ill 17. mechanisms in regulation of net amino patients. Am J Physiol Endocrinol Metab 18. acid deposition. J Clin Invest 1987;​79(4):​ 2009;​298(3):​E697-705. 19. 1062‑9. 65. Thorell A, Rooyackers O, Myrenfors 20. 56. Greenhaff PL, Karagounis LG, Peirce N, P, Soop M, Nygren J, Ljungqvist OH. 21. et al. Disassociation between the effects Intensive insulin treatment in critically ill of amino acids and insulin on signaling, trauma patients normalizes glucose by 22. ubiquitin ligases, and protein turnover in reducing endogenous glucose produc- 23. human muscle. Am J Physiol Endocrinol tion. J Clin Endocrinol Metab 2004;​ 24. Metab 2008;​295(3):​E595-604. 89(11):​5382‑6. 25. 57. Hu Z, Wang H, Lee IH, Du J, Mitch 66. Chevalier S, Burgess SC, Malloy CR, 26. WE. Endogenous glucocorticoids and Gougeon R, Marliss EB, Morais JA. The 27. impaired insulin signaling are both re- greater contribution of gluconeogenesis quired to stimulate muscle wasting under to glucose production in obesity is re- 28. pathophysiological conditions in mice. J lated to increased whole-body protein 29. Clin Invest 2009;​119(10):​3059‑69. catabolism. Diabetes 2006;​55(3):​675‑81. 30. 58. Krebs M, Krssak M, Bernroider E, et al. 67. Tremblay F, Marette A. Amino acid and 31. Mechanism of amino acid-induced skel- insulin signaling via the mTOR/p70 S6 32. etal muscle insulin resistance in humans. kinase pathway. A negative feedback Diabetes 2002;​51(3):​599-605. mechanism leading to insulin resistance 33. 59. Arslanian SA, Saad R, Lewy V, Danadian in skeletal muscle cells. J Biol Chem 34. K, Janosky J. Hyperinsulinemia in afri- 2001;​276(41):​38052‑60. 35. can-american children: decreased insulin 68. Tessari P, Inchiostro S, Biolo G, et al. 36. clearance and increased insulin secretion Hyperaminoacidaemia reduces insulin- 37. and its relationship to insulin sensitivity. mediated glucose disposal in healthy 38. Diabetes 2002;​51(10):​3014‑9. man. Diabetologia 1985;​28(11):​870‑2. 39.

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69. Schricker T, Carli F, Lattermann R, Wa- 1. chter U, Georgieff M. Glucose infusion 2. does not suppress increased lipolysis 3. after abdominal surgery. Nutrition 2001;​ 4. 17(2):​85‑90. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 7 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39.

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Chapter 8

Albumin synthesis rates in post-surgical infants and septic adolescents; influence of amino acids, energy, and insulin

Sascha CAT Verbruggen Henk Schierbeek Jorge Coss-Bu Koen FM Joosten Leticia Castillo Johannes B van Goudoever

Submitted Chapter 8

1. Abstract 2. 3. Background and aims 4. To investigate the effects of glucose, parenteral amino acids, and intravenous insulin 5. on albumin synthesis rates in critically ill children. 6. 7. Methods 8. Two studies were performed in 8 post-surgical infants (age 9.8 ± 1.9 months; weight 9. 9.5 ± 1.1 kg) and 9 septic adolescents (age 15 ± 1 yr.; BMI 20 ± 4 kg.m‑2), respectively. 10. All received a primed, constant, tracer infusion with [1-13C]Leucine. The infants in study 11. 1 were randomized to receive low (2.5 mg.kg‑1.min‑1) and standard (5.0 mg.kg‑1.min‑1) 12. glucose intake in a crossover setting of two periods of 4 hours each. The adolescents 13. in study 2 were randomized to receive total parenteral nutrition with standard (1.5 14. g.kg‑1.day‑1) and high (3.0 g.kg‑1.day‑1) amino acid intake in a two day crossover setting. 15. On both study days, during the last 3 hours of the tracer study, they received insulin 16. infused at 80 mU·m‑2·min‑1. 17. 18. Results 19. The post-surgical infants and the septic adolescents were mildly hypoalbuminemic 20. (~2.5 g.dL‑1) with high synthesis rates, which were not affected by different intakes of 21. glucose, amino acids, or insulin infusion. 22. 23. Conclusions 24. Albumin synthesis rates in hypoalbuminemic critically ill children appear to be upregu- 25. lated through pathways other than nutrient signaling. In septic adolescents, albumin 26. synthesis rates appear insulin resistant. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39.

158 Albumin synthesis rates

1. Introduction 2. 3. Albumin is the most abundant protein in human plasma with a normal plasma con- 4. centration of around 4.0 g.dL‑1, while about 60% of the total albumin pool is located 5. in the interstitial space1. Albumin holds several important functions, both in health 6. as well as in critically ill patients. It is the main preserver of colloid oncotic pressure 7. (~75%), it functions as an anticoagulant and anti-oxidant and it is an important binding 8. transporter of metabolites and drugs1, 2. 9. Critically ill patients are often hypoalbuminemic, primarily due to dilution and redistri- 10. bution secondary to an altered vascular permeability1. In critically ill patients hypoal- 11. buminemia has been documented as a marker for disease severity, nutritional status, 12. prolonged ventilator support and prolonged length of stay3, 4. In critically ill adults and 13. children, low albumin plasma levels (< 3.3 g.dL‑1) are inversely related to morbidity and 14. mortality, where in adults each 1.0 g.dL‑1 drop in serum albumin raised the odds of 15. morbidity by 87% and mortality by 137%3‑5. However, plasma concentrations are static 16. measurements. Dynamic measurements by means of albumin synthesis rates actually 17. show a consistent increase in critically ill adults6, 7. 18. Despite the clear association between hypoalbuminemia and poor outcome, there 19. is still a debate on the benefits and safety of intravenous albumin administration, 20. partially due to the increased risk of escape in the extravascular space and inflamma- 21. tion2, 5. Therefore, stimulation of endogenous albumin synthesis seems an appealing 22. alternative. 23. Albumin synthesis can be stimulated by an increase in energy (glucose and fat) but 24. is particularly responsive to amino acid intake8‑10. Hyperinsulinemia has shown to 25. increase albumin synthesis as well, with an additive effect of increased amino acids 8 26. in healthy adults10. This latter intervention is of particular interest as insulin is more 27. frequently used to treat hyperglycemia in critically ill adults and children11. Moreover, 28. the increase in synthesis rates in response to these interventions is immediate and fast. 29. Albumin synthesis rates increased by 40% within 2 h following intravenous endotoxin 30. in healthy volunteers12. Furthermore, nutritional supplementation13, 14 and hyperinsu- 31. linemia15 increased albumin synthesis rates, within 4, 6, and 3 h respectively. 32. The effect of various nutritional interventions on albumin synthesis rates have not been 33. investigated in critically ill children, other than in premature infants16. Increasing amino 34. acid availability resulted in higher albumin synthesis rates, although not as high as 35. reached in utero16. Given the limited knowledge of albumin synthesis rates and the 36. impact of nutrition in the critically ill pediatric population, we set out to ascertain these. 37. We hypothesized that in critically ill children albumin synthesis rates are increased 38. and responsive to nutrients and hyperinsulinemia. Therefore, our first objective was 39. to quantify albumin synthesis rates in critically ill infants and adolescents. Our second

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1. objective was to determine the impact of nutrients on albumin synthesis rates in these 2. children, with special emphasis on parenteral glucose and amino acids. Our third ob- 3. jective was to determine whether additional hyperinsulinemia in combination with par- 4. enteral glucose and amino acid intake would increase albumin synthesis rates through 5. a synergistic fashion. The here described studies were part of two larger studies aiming 6. to investigate the effect of reduced glucose intake on glucose homeostasis and protein 7. catabolism (Study 1) and the effect and interactions of increased amino acid intake and 8. hyperinsulinemia on substrate metabolism and insulin resistance (Study 2). 9. 10. 11. Methods 12. 13. Patients 14. Study 1 was a one day study with infants (0.5 – 1.0 y) admitted after surgical repair of 15. non-syndromal craniosynostosis to the pediatric intensive care unit (PICU) at Erasmus 16. MC – Sophia Children’s Hospital in Rotterdam, The Netherlands. Study 2 was a two 17. day study in adolescents (13 – 18 y) who were admitted with a diagnosis of severe 18. sepsis or Systemic Inflammatory Response Syndrome (SIRS), as defined by the criteria 19. of the First International Pediatric Sepsis Forum17, to the PICU at Texas Children’s 20. Hospital, Houston, Texas. All patients had drawing and infusing catheters in place for 21. clinical purposes. Patients with metabolic diseases, diabetes mellitus, primary liver, or 22. renal failure were excluded. The study protocols were approved, respectively by the 23. Institutional Review Board of Erasmus Medical Center, Rotterdam, The Netherlands 24. and by the Institutional Review Board of Baylor College of Medicine, Houston, Texas. 25. Studies were carried out after written informed consent from the parents. 26. 27. Study design and data collection 28. The experimental design of study 1 is shown in figure 1 and consisted of an 8 hr glucose 29. infusion in a randomized, cross-over design, four hours with low (LG; 2.5 mg.kg‑1.min‑1) 30. and four hours with standard (SG; 5.0 mg.kg‑1.min‑1) glucose intake rates. Patients were 31. randomized for the order of glucose intake through a computer generated envelop. 32. Laboratory personnel, the clinical team and investigators were blinded for glucose 33. intake until after analyses were finished. Prior to the study, infants did not receive (par) 34. enteral nutrition other than intravenous glucose infusion in the range of 4-6 mg.kg‑1. 35. min‑1 as per standard care before start of the study. Eight hours after admission to the 36. PICU and after obtaining baseline blood samples, the intravenous glucose intake as 37. per standard care (4.0 – 6.0 mg.kg‑1.min‑1) was stopped and the study glucose intake 38. started (t=0), after which the patients received a primed, continuous, 8 h intravenous 39. tracer infusion with L-[1-13C]leucine at 8 μmol.kg‑1 and 8 μmol.kg‑1.h‑1 respectively. Four

160 Albumin synthesis rates

E t1 t2 t1 t2 1. leu-alb CO production VCO 2. 2 2 VCO2 3. Blood samples 4. LG or SG SG or LG 5. L-[1-13C]leucine 6. Minutes 0 120 200 220 240 360 440 460 480 7. Figure 1; Experimental design of study 1 in 8 post-surgical infants admitted to the pediatric 8. intensive care unit receiving only parenteral glucose as nutrition, LG = low glucose intake (2.5 mg.kg‑1.min‑1), SG = standard glucose (5.0 mg.kg‑1.min‑1), E = Enrichment of [1-13C]Leucine 9. leuc-alb incorporated into albumin, VCO = Carbon dioxide production 10. 2 11. hours after start of the study (t=240) the study glucose was switched as per cross-over 12. design (Figure 1). 13. Study 2 consisted of 2 study days in a randomized cross-over fashion, i.e. one with 14. standard (SAA; 1.5 g.kg‑1.day‑1) and one with high (HAA; 3.0 g.kg‑1.day‑1) amino acid 15. intake via total parenteral nutrition (TPN) (Figure 2a). Patients received full parenteral 16. nutrition (Aminosyn (Hospira Inc. Lake Forest, IL.) or Clinisol (Baxter, Deerfield, IL)) for 17. at least 24 hours before the start of the study. Both days consisted of two experimental 18. periods, a basal and a period with insulin infusion (see subsequent description at the 19. end of the paragraph) (Figure 2b). Amino acid intake was randomized by pharmacy 20. through a computer generated envelop. Insulin infusion always followed a baseline 21. period because of the expected wash-out effect of insulin on protein metabolism. 22. Laboratory personnel were blinded for both the amino acid intake and the insulin 23. allocation, while investigators were blinded for amino acid intake, but could not be 24. blinded for insulin allocation due to the risk of hypoglycemia. Energy intake provided 25. as parenteral glucose and lipids were prescribed by the clinical team according to 8 26. standard care. The total energy intake supplied remained unchanged during both study 27. days. After an adaptation period of at least twelve hours of the randomized TPN, the 28. patients received a primed, continuous, 7 h infusion with L-[1-13C]leucine at 6 µmol. 29. kg‑1 and 6 µmol.kg‑1.h‑1 respectively, of which the last three hours in combination with 30. a hyperinsulinemic euglycemic clamp as previously described18 (Figure 2b). Briefly, a 31. 3 h infusion of insulin (Actrapid, Novo Nordisk Inc., Princeton, NJ), dissolved in sterile 32. isotonic NaCl was started at 80 mU.m‑2.min‑1 in order to achieve both normoglycemia 33. between 90-110 mg.dL‑1 and a plasma insulin concentration greater than 100 µU.mL‑1. 34. During insulin infusion, small blood samples were obtained from the indwelling arterial 35. catheter every 5 - 10 minutes to monitor whole blood glucose concentration, at the 36. bedside with the aid of a Y.S.I. 2300 STAT Plus analyzer (YSI Life Sciences, Yellow 37. Springs, OH). To maintain the plasma glucose between 90-110 mg.dL‑1 (5.0 – 6.1 38. mM) for the duration of the study, a 30% glucose solution was infused with a Harvard 39. syringe pump (PHD 22/2000, Harvard apparatus, Holliston, MA)18.

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1. A 2. Day 1 Day 2 3. Amino acid intake SAA SAA 4. HAA HAA 5. Isotope tracer study 6. tracer tracer 7. Time > 16 h 7 h > 16 h 7 h 8. B 9.

10. Eleu-alb t1 t2 t1 t2

11. Plasma glucose

12. CO2 production VCO2 VCO2

13. Blood samples 14. HEC 15. L-[1-13C]leucine 16. 17. Minutes 0 140 200 220 240 330 390 405 420

18. Figure 2; Experimental design of study 2 in 9 adolescents admitted to the pediatric intensive 19. care unit receiving full parenteral nutrition with two different amino acid intakes at baseline and during hyperinsulinemia, Panel A Two day study protocol with two different amino acid 20. intakes in a randomized cross-over fashion, Panel B Isotope tracer infusion on both days 21. during baseline and during hyperinsulinemia, SAA = Standard amino acid intake (1.5 g.kg‑1. day‑1), HAA = High amino acid intake (3.0 g.kg‑1.day‑1), E = Enrichment of [1-13C]Leucine 22. leuc-alb incorporated into albumin, VCO2 = Carbon dioxide production 23. 24. Materials 25. L-[1-13C]leucine (99 atom%) was purchased from Cambridge Isotope Laboratories 26. (Andover, MA) and tested for sterility and pyrogenicity after they were compounded 27. at the investigational pharmacy at Texas Children’s Hospital or Erasmus MC – Sophia 28. Children’s Hospital. 29. 30. Measurements and sample analysis 31. Basal metabolic rate was predicted using the Schofield equation19. All were assessed 32. for severity of disease by the Pediatric Logistic Organ Dysfunction (PELOD) score20 and 33. the Pediatric Risk of Mortality III (PRISM III) score21. 34. Blood samples were obtained from the arterial line at standard frequent intervals (Fig- 35. ure 1) and immediately centrifuged and frozen at -80°C until samples were analyzed. 36. To isolate albumin from plasma, we used anti-human serum albumin affinity resin kits 37. (Vivascience; Sartorius Group, Hannover, Germany). Enclosed spin columns were filled 38. with 400 µL affinity resin and 25 µL of thawed plasma. The column was washed 3 times 39. with a tris-buffer, and albumin was thereafter eluted from the affinity resin with 0.1 mol

162 Albumin synthesis rates

1. glycine.L‑1 (acidified to pH 2.5 with HCl). Eluted albumin was precipitated with 750 µL of ‑1 ‑1 2. 2 mol HClO4.L . A washing step was performed with 0.2 mol HClO4.L by resuspending 3. and precipitating the pellet again. The protein pellet was then hydrolyzed in 140 µL of 4. 6 mol HCl.L‑1 for 22 h at 110°C. After hydrolyzation, the acid was evaporated by using

5. a speedvac, after which the dried amino acids were dissolved in H2O. Samples were 6. derivatized using propylchloroformate (commercial kits: Phenomenex for hydrolysates, 7. EZ:Faast, Bester BV, Amstelveen, the Netherlands) and measured in triplicate on a gas 8. chromatograph–combustion–isotope ratio mass spectrometer (GC-C-IRMS; Delta XP, 9. Thermo Electron, Bremen, Germany)22. As albumin precursor, we used plasma [1-13C] 10. α-ketoisocaproate (α-KIC; the keto-acid of leucine, a measure of intracellular leucine 11. enrichment) enrichment at a plateau. Liver amino acyl-tRNA enrichment forms the 12. true precursor, but its use requires tissue biopsies and technically demanding assays. 13. Nevertheless, α-KIC enrichment adequately represents leucyl-tRNA enrichment and 14. is valuable in this type of research23, 24. Plasma isotope enrichment of [1-13C]α-KIC 15. were, after derivatization to butyldimethyl-silylquinoxalinol derivatives, determined by 16. gas chromatography mass spectrometry (GC-MS) as previously described25. Plasma 17. albumin concentrations were routinely measured on a Hitachi 912 autoanalyzer (Roche 18. Diagnostics, Basel, Switzerland).

19. Carbon dioxide production (VCO2) was obtained with a respiratory profile monitor TM 20. (Deltatrac I MBM-200, Datex Division Instrumentarium Corp. Finland / CO2SMO Plus, 21. Novametrix Medical System, Wallingford, CT, USA) during the last 30 minutes of each 13 22. study period. To determine the enrichment of CO2 in whole blood, 1 mL of perchloric

23. acid 10% was added to 1.0 mL of whole blood in a vacutainer to release the CO2. The 13 24. CO2 in gas was subsequently determined on gas combustion isotope ratio mass 25. spectrometry (GC-IRMS)26, 27. 8 26. Plasma glucose level > 110 mg.dL‑1 (~ > 6.1 mmol.L‑1) were considered hyperglycemic 27. and plasma albumin levels < 3.5 g.dL‑1 were considered hypoalbuminemic. 28. 29. Calculations 30. Whole body kinetics of leucine were calculated by conventional isotope dilution 31. technique using a stochastic model during steady state plateau28. The rate of appear- 32. ance (Ra) of unlabeled leucine can be derived from the plasma isotope enrichment 33. calculated by: 34.

35. Ra = i x (Einf/Epl – 1) (1) 36. 13 37. where i is the infusion rate of [1- C]Leucine, Einf is the tracer enrichment of the infusate

38. and Epl the tracer enrichment in plasma, respectively. At steady state plateau rate of 39. appearance (Ra) is equal to rate of disappearance (Rd).

163 Chapter 8

1. The fractional albumin synthesis rate (FSR) reflects the fraction of the intravascular 2. albumin pool that is renewed per unit of time (%.d‑1) and can be calculated by using 3. the following equation16: 4.

5. FSR = (Eleu-alb,t2 – Eleu-alb,t1)/Eα-kic x (24 x 60)/(t2 – t1) x 100% (2) 6.

7. where Eleu-alb is the enrichment in mole percent excess (MPE.) of incorporated leucine

8. in albumin at t1 and t2 (Figure 1 and 2), and Eα-kic is the mean enrichment in MPE of the 9. precursor, ie, plasma α-KIC, at these time points in minutes. 10. The absolute albumin synthesis rate (ASR) represents the absolute amount of albumin 11. that is produced per day (mg.kg‑1.d‑1), and it can be calculated by using the following 12. equation16: 13. ‑1 14. ASR = FSR x Calb x volbl x (1 – Ht) x weight (3) 15. ‑1 16. where Calb is the plasma albumin concentration in gL , volbl is the child’s total blood 17. volume in mL (assumed to be 75 and 70 mL/kg body wt for the infants and adolescents 18. respectively), Ht stands for hematocrit and (1 – Ht) is the fraction of blood that is 19. plasma. 20. We also calculated the contribution (%) of albumin ASR in relation to whole-body protein 21. synthesis in percentage on the basis of measured leucine turnover data. To do so we 22. needed to determine leucine oxidation and the non-oxidative leucine disposal (NOLD) 23. representing leucine utilized into whole-body protein synthesis (in µmol.kg‑1.h‑1). 24. Leucine oxidation rates were calculated as follows; 25. 13 13 26. Leucine Oxidation = VCO2 x (E CO2/69.18)/[ C]α-KIC (4) 27. 28. NOLD is the leucine oxidation subtracted from the leucine rate of disappearance; 29.

30. NOLD = Raleu – Leucine Oxidation (5) 31. 13 29 32. where 69.18 is the CO2 refraction correction factor for critically ill children . VCO2 is 33. measured in milliliters per minute and converted to millimoles per hour by multiplying 34. by 60 min and dividing by 22.4, which is the number of 1 in 1 mole of an ideal gas at 35. standard temperature and pressure to convert to milliliters per minute. 36. This allows us to calculate the contribution of albumin synthesis in ratio to the whole 37. body protein synthesis according to the following equation16: 38. 39. Contribution = [(ASR x 0.104)/(NOLD x 131.2 x 24 x 0.001)] x 100% (6)

164 Albumin synthesis rates

1. where 0.104 represents the fraction of leucine residues in albumin on a weight basis, 131.2 2. is the mole mass of leucine, and 24 and 0.001 convert to day and milligram, respectively. 3. 4. Statistical analysis 5. A prospective power analysis on our previous data on albumin synthesis rates16, revealed 6. that 8 patients with complete data, would detect a difference of 20% (80% power, type 7. I error of 5%) in synthesis rates. Data are presented as the mean ± standard deviation 8. unless non-parametric, in which case they are presented as median and 95% CI. Due to 9. the variation between the two studies, not only in nutritional and metabolic support, but 10. moreover in age and diagnosis, statistical comparison would not be justified. Differences 11. within study groups were tested with the repeated measurements ANOVA, after which a 12. paired student’s t test was used. For non-parametric data the Wilcoxon matched pairs 13. or Mann Whitney was used. Statistical significance was considered at p < 0.05. Data 14. were analyzed with Graphpad Prism 5.0.3 (Graphpad Software, La Jolla, CA., USA). 15. 16. 17. Results 18. 19. Patients 20. The demographic characteristics of all patients are described in table 1. 21. Study 1. Study 1 included 8 post-surgical infants (age 0.8 ± 0.2 months; 9.5 ± 1.1 kg). 22. All patients were hemodynamically stable without vasoactive drugs and all patients 23. were breathing spontaneous with an inspiratory fraction of oxygen less than 0.6. 24. 25. Table 1. Patient demographics of 8 infants and 9 adolescents admitted to the PICU* 8 26. Study 1 Study 2 Age (years) 0.8 ± 0.2 15.0 ± 1.2 27. Gender (male:female) 6 : 2 3 : 6 28. Tanner score 4.0 ± 0.9 29. Body Mass Index (kg.m‑2) 20 ± 4 30. Weight (kg.) 9.5 ± 1.1 49.1 ± 13.1 31. Length (cm) 74.3 ± 3.0 154.3 ± 11.6 a b 32. SAA HAA PICU LOS c (days) 1 5.9 ± 3.3 6.3 ± 3.6 33. C-Reactive Protein (mg.dL‑1) 2.4 ± 1.3 16.5 ± 9.4 15.1 ± 12.0 34. PELOD d 10 ± 9 9 ± 11 6 ± 7 35. PRISM III e 7 ± 4 10 ± 4 8 ± 4 36. Catecholamines (n= ) 0 1 0 37. Glucocorticoids (n= ) 0 4 3 38. * All values are mean ± SD, a SAA = standard amino acid intake (1.5 g.kg‑1.d‑1), b HAA = high amino acid intake (3.0 g.kg‑1.d‑1), c PICU LOS = Length of stay in the PICU at start of the study, d PELOD = Pediatric Logistic Organ Dysfunction 20, 39. e PRISM III = Pediatric Risk of Mortality III 21

165 Chapter 8

1. Study 2. Study 2 included nine septic adolescents (age 15 ± 1 yr.; BMI 20 ± 4 kg.m‑2, 2. Tanner-score 4.0 ± 0.9). All patients were mechanically ventilated and hemodynami- 3. cally stable. One patient received epinephrine as vasopressor therapy and 4 patients 4. received glucocorticoids as additional therapy (Table 1). One patient randomized to 5. start first with the standard amino acid intake group (SAA) died the next day after the 6. first study (SAA) was completed, therefore the high amino acid intake (HAA) part of the 7. study could not be conducted. In a second patient a technical error occurred during 8. the insulin infusion protocol on the study day with SAA intake. Therefore, complete 9. two-day study data were available on 7 patients. Data available from the two incom- 10. plete studies were included according to intention-to-treat principle. 11. 12. Nutritional interventions 13. The nutritional characteristics of all patients are described in table 2. 14. Study 1. The infants received glucose intake in two different amounts and did not 15. receive any amino acids. As anticipated, the caloric intake was below the caloric 16. requirements according to the Schofield equations19, although significantly higher 17.

18. Table 2. Nutritional parameters of 8 infants and 9 adolescents admitted to PICU*

19. Study 1 Study 2

20. LGa SGb SAAc HAAd

21. Base Insulin Base Insulin

22. Schofielde 53.7 ± 5.3 29.3 ± 6.0 29.3 ± 6.0 ‑1 ‑1 23. (kcal.kg .day ) 24. Caloric intake 12.7 ± 0.2 25.3 ± 0.5 ^ 32.7 ± 10.0 41.8 ± 8.4 37.8 ± 9.9 43.0 ± 14.3 (kcal.kg‑1.day‑1)

25. Caloric intake 24 ± 1 47 ± 2 ^ 115 ± 39 147 ± 44 136 ± 31 156 ± 55 26. (% of Schofield) 27. Glucose intake 2.6 ± 0.1 5.2 ± 0.1 ^ 4.5 ± 1.7 6.2 ± 1.9 4.6 ± 1.5 5.6 ± 2.2 (mg.kg‑1.min‑1) 28. Protein intake 0 0 1.5 ± 0.2 1.5 ± 0.2 2.8 ± 0.4‡ 2.8 ± 0.4‡ 29. (gr.kg‑1.day‑1) 30. Glucose calories 12.7 ± 0.2 25.3 ± 0.5 ^ 21.8± 8.1 30.9 ± 8.9 22.4 ± 7.6 27.6 ± 10.8 (kcal.kg‑1.day‑1) 31. Amino acid calories 0 0 5.8 ± 0.8 5.8 ± 0.8 11.2 ± 1.4 ‡ 11.2 ± 1.4 ‡ 32. (kcal.kg‑1.day‑1)

33. Lipid calories 0 0 5.7 ± 3.9 5.7 ± 3.9 4.7 ± 3.5 4.7 ± 3.5 ‑1 ‑1 34. (kcal.kg .day ) Glucose plasma level 105 ± 10 133 ± 30 ^ 168 ± 58 98 ± 6 † 172 ± 62 101 ± 15† 35. (mg.dL‑1)

36. Insulin plasma levels 54 (19 -159) 73 (24 – 186) 32 (17 – 122)144 (94 – 2385)† 51 (29 – 153)168 (93 – 2239)† 37. (μU.mL‑1) 38. * All values are mean ± SD, a LG = low glucose (2.5 mg.kg‑1.min‑1), b SG = Standard glucose (5.0 mg.kg‑1.min‑1), c SAA = standard amino acid intake (1.5 g.kg‑1.d‑1), d HAA = high amino acid intake (3.0 g.kg‑1.d‑1), e Resting Energy Expenditure 39. according to Schofield equations19 , ^ LG vs. SG, p < .05, † Base vs. Insulin p < .05, ‡ SAA vs. HAA, p < .05

166 Albumin synthesis rates

1. during SG. During LG the infants were normoglycemic, and during SG glucose levels 2. were higher and hyperglycemic, while the insulin plasma levels did not significantly 3. differ. 4. Study 2. The adolescents received full parenteral nutrition in two different amounts 5. of amino acids (Table 2). The caloric intake was adequate or above requirements ac- 6. cording to the Schofield equations19 (Table 2), but did not significantly differ between 7. protocols. During baseline the adolescents were hyperglycemic, while during insulin 8. infusion they were normoglycemic and had significantly higher insulin concentrations 9. (Table 2). 10. 11. Albumin synthesis rates 12. All patients were hypoalbuminemic (Table 3). 13. Study 1. Albumin synthesis rates, non-oxidative leucine disposal (NOLD), and the 14. contribution of albumin synthesized in relation to the whole body protein synthesis 15. were not affected with different glucose intakes (Figure 3, Table 3). 16. Study 2. Neither an increased amino acid intake nor the supraphysiological insulin 17. concentrations affected albumin synthesis rates. NOLD was not significantly affected 18. by the different amino acid intakes, but was lower (p < .05) during insulin infusion in the 19. adolescents (Table 3). The contribution of albumin synthesized in relation to the whole 20. body protein synthesis was comparable between protocols and not affected by either 21. intervention (Figure 3, Table 3). 22. 23. 24. A B C 25. 8 40 500 26. ]

-1 12

] 400

27. -1 30 .day

28. -1 300 8 20 29. 200 4 30. 10 FSR [%.day FSR 100 31. ASR [mg.kg ASR 32. 0 0 0 33. LG SG LG SG LG SG Contribution of albumin synthesis albumin of Contribution to total body protein synthesis[%] bodyprotein total to

SAA BASE HAA BASE SAA BASE SAA BASE SAA Insulin HAA Insulin 34. SAA InsulinHAA BASEHAA Insulin SAA InsulinHAA BASEHAA Insulin 35. Figure 3; Albumin synthesis in 8 infants and 9 adolescents receiving various nutritional support and insulin, Panel A Albumin fractional synthesis rate (%.day‑1), Panel B Albumin absolute 36. synthesis rate (mg.kg‑1.day‑1), Panel C Contribution of albumin synthesis as ratio to total whole 37. body protein synthesis per day LG = low glucose intake (2.5 mg.kg‑1.min‑1), SG = standard ‑1 ‑1 ‑1 ‑1 38. glucose (5.0 mg.kg .min ), SAA = Standard amino acid intake (1.5 g.kg .day ), HAA = High amino acid intake (3.0 g.kg‑1.day‑1), Base = during baseline, Insulin = during insulin infusion 39. (80 mU.m‑2.min‑1).

167 Chapter 8

Table 3. Albumin synthesis of 8 infants and 9 adolescents admitted to the PICU* 1. Study 1 Study 2 2. LG a SG b SAA c HAA d 3. Base Insulin Base Insulin

4. Albumin concentration (g.dL‑1) 2.6 ± 0.4 2.6 ± 0.6 2.5 ± 0.3

5. FSR e (%.d‑1) 16.3 ± 3.2 16.0 ± 2.2 23.7 ± 13.8 27.5 ± 19.0 22.6 ± 8.3 23.8 ± 11.0

6. ASR f (mg.kg‑1.day‑1) 243 ± 45 243 ± 47 299 ± 172 330 ± 211 276 ± 99 296 ± 148

7. Non-oxidative leucine 122 ± 14 124 ± 20 153 ± 36 128 ± 38 † 173 ± 31 # 149 ± 38 †‡ ‑1 ‑1 8. disposal (µmol.kg .h ) 9. Contribution to total protein 6.7 ± 1.9 6.7 ± 1.9 6.9 ± 4.4 8.4 ± 4.4 5.3 ± 1.7 6.5 ± 2.5 synthesis (%)

10. * All values are mean ± SD, , a LG = low glucose (2.5 mg.kg‑1.min‑1), b SG = Standard glucose (5.0 mg.kg‑1.min‑1), c SAA = 11. standard amino acid intake (1.5 g.kg‑1.d‑1), d HAA = high amino acid intake (3.0 g.kg‑1.d‑1), e FSR = fractional synthesis rate, f ASR = absolute synthesis rate; † Base vs. Insulin; p < .05, ‡ SAA vs. HAA; p < .05, # SAA vs. HAA, p = .05 12. 13. 14. Discussion 15. 16. Our study provides insight in albumin synthesis rates in critically ill children of two 17. different age groups. The data obtained in our study are consistent with previous 18. observations in critically ill adults that, although in a hypoalbuminemic condition, al- 19. bumin synthesis rates were high during inflammation and metabolic stress. Nutritional 20. interventions with various levels of glucose and amino acid intake, and insulin infusion 21. did not further stimulate albumin synthesis rates, nor did they increase the contribution 22. of albumin to whole body protein synthesized. 23. It has been shown previously that fractional synthesis rates are higher in neonates 24. (12.5-23%)16, 22 and infants (15-20%)30, 31 than healthy adults (6-15%)7‑10, 14, 32, 33. This is 25. however not reflected in our study, where increased synthesis rates were more obvious 26. in the septic adolescents. The synthesis rates measured in our infants were consistent 27. with those measured in age-related peers30, 31. The synthesis rates in the adolescents 28. were even slightly higher than those in (critically) ill adults (11-18%)7, 34‑37. These relative 29. high synthesis rates might be explained by their condition, as an inflammatory and 30. catabolic insult, which usually follows after trauma, surgery or infection, is a strong 31. stimulant for albumin synthesis in patients12. These findings suggest that the ado- 32. lescents were in a higher inflammatory and catabolic state, compared to the infants, 33. explaining their relative high synthesis rates for their age. The latter is supported with 34. a higher leucine flux in the adolescents (data not shown), an indicator for amino acid 35. turnover as a derivative measure of catabolism. Furthermore, the C-reactive protein, a 36. hepatic acute phase protein, was substantially higher in the septic adolescents. 37. In contrast to our hypothesis and data in healthy adults and neonates9, 14, 16, increasing 38. both parenteral energy (glucose) as well as amino acid intake did not affect synthesis 39. rates. Although an unexpected finding at first, it is nevertheless consistent with pub-

168 Albumin synthesis rates

1. lished data in healthy38 as well as post-surgical37 adults receiving intravenous nutrients. 2. Albumin synthesis rates are less responsive to parenteral than enteral nutrients39, 40. 3. Parenteral nutrition bypasses the splanchnic uptake and amino acids are presented 4. to the liver through the portal venous circulation in lower concentrations41. Studies in 5. piglets suggest that after enteral feeding, portal rather than arterial amino acids are 6. preferentially used for hepatic protein synthesis41. Therefore, the parenteral route of 7. nutrient supply in our children might be responsible for the lack of effect of our nutri- 8. tional interventions, stressing the need for early enteral nutrition in critically ill children 9. even more. 10. The lack of nutritional effect could also have been caused by the medical or surgical 11. condition our patients were in. In health, the major regulator of (muscle and hepatic) 12. protein synthesis is increased amino acid availability, since amino acids themselves 13. modulate cellular processes leading to protein synthesis via enhanced translation 14. initiation as well as through promoting translation elongation42, 43. Under conditions 15. of inflammation and cytokine release, muscle protein synthesis is inhibited and less 16. responsive to amino acid supply, while hepatic synthesis of immune/inflammatory 17. proteins is greatly increased44‑46. An increased supply of amino acids moderately at- 18. tenuates the inhibitory response of inflammation on muscle protein synthesis47. In con- 19. trast, a pathway other than nutrient signaling is responsible for the increased hepatic 20. protein synthesis during an inflammatory insult48. Our study now shows that albumin 21. synthesis, already amplified in our patients, was also not further affected with changes 22. in nutrient supply. 23. Of further interest, administration of insulin, a strong anabolic hormone, previously 24. reported to stimulate albumin synthesis15, 49, did not have any effect. Insulin decreased 25. whole body protein synthesis, indicated by the NOLD, even when a high amino acid 8 26. intake was provided. Lang and coworkers50 reported that insulin failed to stimulate 27. protein synthesis in an animal model of sepsis via a defect in insulin signaling to a step 28. in translation initiation. Inflammation induces insulin resistance to protein synthesis, 29. which might explain why hyperinsulinemia in our septic adolescents did not increase 30. albumin synthesis rates. 31. There are some limitations to our study which need to be taken into account, some of 32. them inherent to studying critically ill children. As mentioned in the statistical descrip- 33. tion, statistical comparison between the two protocols was not justified due to the wide 34. variations in between the two groups. Furthermore, absolute albumin synthesis rates 35. are calculated with albumin plasma levels. However, as we mentioned in the introduc- 36. tion, hypoalbuminemia is primarily caused by dilution and redistribution secondary to 37. an altered vascular permeability. Therefore, we potentially underestimated the absolute 38. synthesis rates as our study was not able to correct for dilution and redistribution. 39. However, the data within the groups are consistent and justify our conclusions that

169 Chapter 8

1. synthesis rates were high, and unresponsive to change in parenteral nutrients as 2. well as hyperinsulinemia. We further acknowledge that we only performed short term 3. nutritional changes in our study. However, as has been shown in previous studies, 4. albumin synthesis rates can react acute and fast to these interventions12‑15. Finally, we 5. acknowledge that 4 septic adolescents received glucocorticoids as adjuvant therapy. 6. Glucocorticoids impair protein anabolism, also in the pediatric population51, and are 7. capable of obstructing the anabolic effects of insulin52. However, the catabolic prop- 8. erties are exerted primarily through increased proteolysis, more than suppression of 9. protein synthesis51. We did not find a difference in synthesis rates or effect of insulin 10. therapy in the 4 adolescents who received glucocorticoids, although our study was not 11. powered to discover such an effect. 12. 13. 14. Conclusion 15. 16. Although mildly hypoalbuminemic, albumin synthesis rates are increased in post- 17. surgical infants and even more in septic adolescents. Synthesis rates did not respond 18. to short term changes in parenteral intakes of glucose, amino acids, and insulin. The 19. data from our study, although the first in the critically ill pediatric population, confirm 20. previous observations that the low plasma albumin levels are not due to decreased 21. synthesis rates. Furthermore, critical illness appears to regulate albumin synthesis 22. rates through other pathways than nutritional signaling, and induces insulin resistance 23. to protein synthesis. 24. 25. 26. Acknowledgements 27. 28. Our gratitude goes out to the patients and their families at Texas children’s hospital 29. and Erasmus MC Sophia Children’s hospital for their selfless contribution to this study. 30. We thank the nursing staff, Gardi Minderman-Voortman and Kristien Dorst for their 31. contribution. 32. 33. 34. 35. 36. 37. 38. 39.

170 Albumin synthesis rates

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20. Leteurtre S, Martinot A, Duhamel A, et al. a primed, continuous infusion of L-[1-3C] 1. Validation of the paediatric logistic organ leucine. Am J Physiol 1980;​238(5):​E473‑9. 2. dysfunction (PELOD) score: prospective, 29. Sy J, Gourishankar A, Gordon WE, et al. 3. observational, multicentre study. Lancet Bicarbonate kinetics and predicted en- 4. 2003;​362(9379):​192‑7. ergy expenditure in critically ill children. 5. 21. Pollack MM, Patel KM, Ruttimann UE. Am J Clin Nutr 2008;​88(2):​340‑7. PRISM III: an updated Pediatric Risk 30. Jahoor F, Abramson S, Heird WC. The 6. of Mortality score. Crit Care Med 1996;​ protein metabolic response to HIV infec- 7. 24(5):​743‑52. tion in young children. Am J Clin Nutr 8. 22. Bunt JE, Rietveld T, Schierbeek H, Wat- 2003;​78(1):​182‑9. 9. timena JL, Zimmermann LJ, van Goud­ 31. Morlese JF, Forrester T, Badaloo A, Del 10. oever JB. Albumin synthesis in preterm Rosario M, Frazer M, Jahoor F. Albumin infants on the first day of life studied with kinetics in edematous and nonedema- 11. [1-13C]leucine. Am J Physiol Gastroin- tous protein-energy malnourished chil- 12. test Liver Physiol 2007;​292(4):​G1157‑61. dren. Am J Clin Nutr 1996;​64(6):​952‑9. 13. 23. Ahlman B, Charlton M, Fu A, Berg C, 32. Barle H, Nyberg B, Essen P, et al. The 14. O’Brien P, Nair KS. Insulin’s effect on synthesis rates of total liver protein and 15. synthesis rates of liver proteins. A swine plasma albumin determined simultane- 16. model comparing various precursors of ously in vivo in humans. Hepatology protein synthesis. Diabetes 2001;​50(5):​ 1997;​25(1):​154‑8. 17. 947‑54. 33. Ballmer PE, McNurlan MA, Milne E, et 18. 24. Barazzoni R, Meek SE, Ekberg K, Wahren al. Measurement of albumin synthesis 19. J, Nair KS. Arterial KIC as marker of liver in humans: a new approach employing 20. and muscle intracellular leucine pools in stable isotopes. Am J Physiol 1990;​259(6 21. healthy and type 1 diabetic humans. Am Pt 1):​E797-803. J Physiol 1999;​277(2 Pt 1):​E238‑44. 34. Dahn MS, Mitchell RA, Lange MP, 22. 25. Matthews DE, Schwarz HP, Yang RD, Smith S, Jacobs LA. Hepatic metabolic 23. Motil KJ, Young VR, Bier DM. Relation- response to injury and sepsis. Surgery 24. ship of plasma leucine and alpha-ke- 1995;​117(5):​520‑30. 25. toisocaproate during a L-[1-13C]leucine 35. Essen P, McNurlan MA, Gamrin L, et al. 26. infusion in man: a method for measuring Tissue protein synthesis rates in critically 27. human intracellular leucine tracer enrich- ill patients. Crit Care Med 1998;​26(1):​ ment. Metabolism 1982;​31(11):​1105‑12. 92-100. 28. 26. el-Khoury AE, Sanchez M, Fukagawa NK, 36. Barle H, Gamrin L, Essen P, McNurlan 29. Young VR. Whole body protein synthesis MA, Garlick PJ, Wernerman J. Growth 30. in healthy adult humans: 13CO2 tech- hormone does not affect albumin synthe- 31. nique vs. plasma precursor approach. sis in the critically ill. Intensive Care Med 32. Am J Physiol 1995;​268(1 Pt 1):​E174‑84. 2001;​27(5):​836‑43. 27. Spear ML, Darmaun D, Sager BK, Par- 37. Barle H, Nyberg B, Andersson K, et al. 33. sons WR, Haymond MW. Use of [13C] The effects of short-term parenteral nu- 34. bicarbonate infusion for measurement trition on human liver protein and amino 35. of CO2 production. Am J Physiol 1995;​ acid metabolism during laparoscopic 36. 268(6 Pt 1):​E1123‑7. surgery. JPEN J Parenter Enteral Nutr 37. 28. Matthews DE, Motil KJ, Rohrbaugh DK, 1997;​21(6):​330‑5. 38. Burke JF, Young VR, Bier DM. Measure- 38. Ballmer PE, McNurlan MA, Essen P, An- ment of leucine metabolism in man from derson SE, Garlick PJ. Albumin synthesis 39.

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rates measured with [2H5ring]phenyl- compositions of acute-phase and muscle 1. alanine are not responsive to short-term proteins have a bearing on nitrogen loss 2. intravenous nutrients in healthy humans. in traumatic states? J Nutr 1994;​124(6):​ 3. J Nutr 1995;​125(3):​512‑9. 906‑10. 4. 39. Bower RH, Talamini MA, Sax HC, Ham- 47. Orellana RA, Kimball SR, Suryawan A, 5. ilton F, Fischer JE. Postoperative enteral et al. Insulin stimulates muscle protein vs parenteral nutrition. A randomized synthesis in neonates during endotox- 6. controlled trial. Arch Surg 1986;​121(9):​ emia despite repression of translation 7. 1040‑5. initiation. Am J Physiol Endocrinol Metab 8. 40. Tsujinaka T, Morimoto T, Ogawa A, et al. 2007;​292(2):​E629‑36. 9. Effect of parenteral and enteral nutrition 48. Morlese JF, Forrester T, Jahoor F. Acute- 10. on hepatic albumin synthesis in rats. phase protein response to infection in Nutrition 1999;​15(1):​18‑22. severe malnutrition. Am J Physiol 1998;​ 11. 41. Stoll B, Burrin DG. Measuring splanchnic 275(1 Pt 1):​E112‑7. 12. amino acid metabolism in vivo using 49. De Feo P, Volpi E, Lucidi P, et al. Physi- 13. stable isotopic tracers. J Anim Sci 2006;​ ological increments in plasma insulin 14. 84 Suppl:​E60‑72. concentrations have selective and dif- 15. 42. Wilson FA, Suryawan A, Orellana RA, ferent effects on synthesis of hepatic 16. Gazzaneo MC, Nguyen HV, Davis TA. proteins in normal humans. Diabetes Differential effects of long-term leucine 1993;​42(7):​995-1002. 17. infusion on tissue protein synthesis in 50. Lang CH, Frost RA, Vary TC. Regulation 18. neonatal pigs. Amino Acids 2010. of muscle protein synthesis during sepsis 19. 43. Fujita S, Dreyer HC, Drummond MJ, et al. and inflammation. Am J Physiol Endocri- 20. Nutrient signalling in the regulation of hu- nol Metab 2007;​293(2):​E453‑9. 21. man muscle protein synthesis. J Physiol 51. Van Goudoever JB, Wattimena JD, Car- 2007;​582(Pt 2):​813‑23. nielli VP, Sulkers EJ, Degenhart HJ, Sauer 22. 44. Gabay C, Kushner I. Acute-phase pro- PJ. Effect of dexamethasone on protein 23. teins and other systemic responses to metabolism in infants with bronchopul- 24. inflammation. N Engl J Med 1999;​340(6):​ monary dysplasia. J Pediatr 1994;​124(1):​ 25. 448‑54. 112‑8. 8 26. 45. Lang CH, Frost RA. Sepsis-induced sup- 52. van Beek RH, Zimmermann LJ, Vergunst 27. pression of skeletal muscle translation van Keulen JG, et al. Leucine kinetics initiation mediated by tumor necrosis fac- during simultaneously administered 28. tor alpha. Metabolism 2007;​56(1):​49‑57. insulin and dexamethasone in preterm 29. 46. Reeds PJ, Fjeld CR, Jahoor F. Do the infants with severe lung disease. Pediatr 30. differences between the amino acid Res 2001;​49(3):​373‑8. 31. 32. 33. 34. 35. 36. 37. 38. 39.

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Part IV General discussion & summary

Chapter 9

General Discussion

General discussion

1. As summarized in chapter 1, critically ill children require specific nutritional and meta- 2. bolic support to counteract ongoing catabolism, regain control of glucose homeosta- 3. sis, and supply sufficient substrates to help recover from their disease process, while 4. maintaining growth and development1‑5. Nonetheless, it remains a poorly investigated 5. area with little evidence, and nutritional goals6 as well as glycemic control7 are still not 6. met in the Pediatric Intensive Care Unit (PICU). This leads to increased morbidity and 7. mortality in critically ill children5, 8‑13. 8. This thesis aimed to contribute to the development of future guidelines for the nutri- 9. tional and metabolic support in critically ill children of all age groups. 10. It was clear from the start that we needed to focus on the deficiencies of the support 11. currently provided and the impact this has on interactions between energy (glucose 12. and insulin metabolism) and protein (i.e. amino acids) in critically ill children. The main 13. hypotheses we set out to investigate were; 14. - Hyperglycemia in critically ill children is caused by insulin resistance and can be 15. treated according to age-related glucose control protocols. 16. - Glucose and protein/amino acids are currently provided in inadequate amounts to 17. critically ill children of different age groups. 18. - Insulin exerts its anabolic properties also in critically ill children and can be used as 19. an additive tool to deflect catabolism. 20. 21. 22. Role of insulin therapy on the PICU 23. 24. Sir Frederick G. Banting received the Nobel prize for the discovery of insulin and during 25. his acceptance speech in 1925 he stated; “Insulin is not a cure for diabetes, it is a 26. treatment. It enables the diabetic to burn sufficient carbohydrates, so that proteins and 27. fats may be added to the diet in sufficient quantities to provide energy for the economic 28. burdens of life”. 9 29. 30. The story on adult ICU’s so far… 31. In 2006, we developed the hypotheses on which the present thesis is based. By then 32. it had become clear that critical illness hyperglycemia, or “diabetes of the ill”, was not 33. merely an adaptive stress response, as was thought before. In 2001, a landmark paper 34. by van den Berghe and colleagues showed that a tight glucose regimen with insulin to 35. maintain blood glucose concentrations at 4.5 – 6.1 mmol.L‑1 reduced the morbidity and 36. mortality in a surgical intensive care unit (ICU)14. Since a subgroup analysis showed 37. the greatest gain was achieved in patients with sepsis and multisystem organ failure, 38. medical ICU patients were expected to benefit as well15. In 2004, the Surviving Sepsis 39. Campaign recommended glucose control for all patients with sepsis and explicitly

179 Chapter 9

1. stated; “There is no reason to think that these data are not generalizable to all severely 2. septic patients”16. Because few interventions in critically ill patients improved outcome 3. to this extent, the results were enthusiastically embraced, and many ICU’s, both surgi- 4. cal and medical, sought to institute intensive glucose control measures17‑19. 5. Also on our pediatric ICU (PICU), a step-wise nurse driven tight glucose control proto- 6. col to treat hyperglycemia was initiated in 2006. At that time, a subsequent large trial 7. by van den Berghe and colleagues was presented. It was a similar study on a medical 8. ICU and showed comparable beneficial organ protective results, although less striking 9. as in the surgical setting, revealing no mortality benefit from intensive glucose control, 10. except in a subgroup requiring critical care for 3 or more days20. This was an indication 11. that tight glucose control, which was thought to become one of the most successful 12. intensive care treatments, was more complex than anticipated. Since then, a number 13. of subsequent studies reported various results, some were terminated prematurely 14. due to the unacceptable high incidence of hypoglycemia21, others reported disparate 15. results22, 23. A large meta-analysis which includes a recent international multi-center 16. trial (NICE-SUGAR), the largest to date, showed that intensive insulin therapy had no 17. effect on overall risk of death, although it suggested that surgical patients do benefit 18. from tight glucose control (Figure 1)45. 19. Various arguments can be given for these differences in outcome; variation in popula- 20. tion, blood glucose target ranges, accuracy of insulin infusion and glucose monitoring 21. protocols, the form of (parenteral) nutrition, and methods used to measure blood 22. glucose levels46. Notwithstanding the ongoing debate, it has become undeniable that 23. both low as well as high blood glucose levels worsen outcome47, and several medi- 24. cal advisory boards recommend routine glycemic control as standard care48, 49. The 25. debate regarding the tight glucose control in adults is targeted on two major issues; 1) 26. uncertainty about the optimal glucose target ranges, and 2) the unacceptable high risk 27. of developing hypoglycemia. 28. 29. What to expect from “tight glucose control” on the PICU? 30. These two issues are of even greater concern in the pediatric population. So where do 31. we stand from a pediatric point of view? 32. In chapter 2 we have given a comprehensive overview of the causes and conse- 33. quences of hyperglycemia and the potential benefits of insulin therapy in critically ill 34. children. Similar to the pathofysiology in adults, hyperglycemia in critically ill children 35. is primarily caused by insulin resistance, as we have demonstrated in chapter 7 36. in septic adolescents, by means of a Hyperinsulinemic Euglycemic Clamp (HEC), 37. which is considered the “gold standard” to determine insulin resistance. Even the 38. post-surgical infants described in chapter 4 appeared in an insulin resistant condi- 39. tion, although their hyperglycemic state was shown to be aggravated predominantly

180 General discussion

1. No. deaths / total no. patients Favours IIT Favours control 2. Study IIT Control Risk ratio (95% CI) 3. Mixed ICU 4. Yu et al. 31 4/28 4/27 0.96 (0.27-3.47) 5. Henderson et al. 26 5/32 7/35 0.78 (0.28-2.22) 6. Mitchell et al. 29 9/35 3/35 3.00 (0.89-10.16) 30 7. Wang et al. 7/58 26/58 0.27 (0.13-0.57) Azevedo et al. 24 38/168 42/169 0.91 (0.62-1.34) 8. McMullin et al. 28 6/11 4/9 1.23 (0.49-3.04) 9. Preiser et al. 21 107/550 89/551 1.20 (0.93-1.55) 10. Brunkhorst et al. 22 98/247 102/288 1.12 (0.90-1.39) 11. Iapichino et al. 27 15/45 12/45 1.25 (0.66-2.36) 40 12. He et al. 16/58 29/64 0.61 (0.37-1.00) Zhang et al. 44 4/168 6/170 0.67 (0.19-2.35) 13. De La Rosa Gdel et al. 37 102/254 96/250 1.05 (0.84)-1.30) 14. Arabi et al. 32 72/266 83/257 0.84 (0.64-1.09) 15. Mackenzie et al. 31 39/121 47/119 0.82 (0.58-1.15) 16. NICE-SUGAR 23 829/3010 751/3012 1.10 (1.01-1.20) 17. All mixed ICU patients 1351/5051 1301/5089 0.99 (0.87-1.12) 18. 19. Medical ICU 35 20. Bland et al. 1/5 2/5 0.50 (0.06-3.91) Van den Berghe et al. 20 214/595 228/605 0.95 (0.82-1.11) 21. Walters et al. 43 1/13 0/12 2.79 (0.12-62.48) 22. Farah et al. 37 22/41 22/48 1.17 (0.77-1.78) 23. Oksanen et al. 42 13/39 18/51 0.94 (0.53-1.68) 24. Bruno et al. 36 2/31 0/15 2.50 (0.13-49.05) 25. All medical ICU patients 253/724 270/736 1.00 (0.78-1.28) 26. Surgical ICU 27. Van den Berghe et al. 14 55/765 85/783 0.66 (0.48-0.92) 28. Grey et al. 39 4/34 6/27 0.53 (0.17-1.69) 9 29. Bilotta et al. 34 6/40 7/38 0.81 (0.30-2.20) 25 30. He et al. 7/150 6/38 0.30 (0.11-0.83) Bilotta et al. 33 5/48 6/49 0.85 (0.28-2.60) 31. All surgical ICU patients 77/1037 110/935 0.63 (0.44-0.91) 32. All ICU patients 1681/6812 1681/6760 0.93 (0.83-1.04) 33. 34. 0.1 1 10 35. Risk ratio (95% CI) 36. 37. Figure 1. Meta-analysis from adult studies showing the risk-ratios of mortality in clinical trials comparing intensive insulin therapy (IIT) to conventional glycemic control stratified by type of 38. ICU. 39. CI; confidence interval. Adapted from reference 45.

181 Chapter 9

1. through their exogenous glucose intake, provided according to age-specific recom- 2. mendations50, 51. 3. In chapter 2 we review several studies that associated hyperglycemia in the pediatric 4. intensive care population with increased morbidity; increased rate of infections, pro- 5. longed mechanical ventilation and stay on the PICU and in the hospital, and worsened 6. neurological outcome9, 12, 52‑57. 7. Hyperglycemia, through intracellular glucose overload, causes more excessive gly- 8. colysis and oxidative phosphorylation resulting in an increased generation of oxygen 9. radicals such as peroxynitrite and superoxide in these cells. These reactive oxygen 10. species cause mitochondrial dysfunction and disturbed energy metabolism, which 11. leads to increased apoptosis and might explain cellular and organ system failure in 12. critically ill patients58‑60. To date, no studies have explored the (intra)cellular mecha- 13. nisms of hyperglycemia in children. However, it is not likely that there will be substantial 14. differences from adults, and recently two small mechanistic studies of insulin therapy 15. in the pediatric population have been reported. Intensive insulin therapy in burned chil- 16. dren has shown to improve insulin sensitivity, possibly through improved mitochondrial 17. function and oxidative capacity of fatty acids61. Additionally, it has been shown that in 18. infants undergoing congenital heart surgery prevention of hyperglycemia, independent 19. of insulin signaling pathways, protects myocardial cells and function, and reduces the 20. inflammatory response62. Yet, these mechanistic benefits in critically ill children do not 21. guarantee beneficial clinical implications. 22. A large proportion of chapter 2 consists of the review we published in 2007, in which we 23. could only report on smaller trials with insulin therapy, showing an improved glycemic 24. control, protein and lipid metabolism, caloric intake, and inflammatory response63‑68. 25. Since then, two large outcome studies of tight glucose protocols specifically designed 26. for the pediatric population have been published. A study in hyperglycemic very low 27. birth weight (VLBW) neonates was discontinued prematurely because of an increased 28. incidence of hypoglycemia (29% vs. 17%) and parenchymal abnormalities detected 29. with cranial ultrasound in those treated with insulin69. A large trial to study the outcome 30. of tight glucose control in critically ill children (< 16 years old) started recently, and 31. the results are eagerly anticipated for70. In the mean time, available data are limited to 32. one large prospective randomized trial, which enrolled 700 critically ill children, and 33. showed that tight glucose control with intensive insulin therapy shortened duration 34. of PICU stay (5.5 vs. 6.2 days) and decreased mortality (3% vs. 6%) as compared 35. with conventional treatment71. However, hypoglycemia (≤ 2.2 mmol.L‑1) occurred in 36. 25% of the children and severe hypoglycemia (≤ 1.7 mmol.L‑1) occurred in 5% of the 37. children. Although no severe clinical symptoms were reported, long-term follow up and 38. neurocognitive development of these children are eagerly awaited for. 39.

182 General discussion

1. Is insulin therapy in critically ill children dangerous? 2. There are several explanations for the high incidence of hypoglycemia as we sum- 3. marize in chapter 2. First, target ranges in the study by Vlasselaers and colleagues71 4. were low (2.8–4.4 mmol.L‑1 for infants and 3.9–5.6 mmol.L‑1 for children), and as the 5. ideal target range of tight glucose regimens still needs to be established less strict 6. protocols might be sensible. We currently treat hyperglycemia with help of a glucose 7. target range of 4.0-8.0 mmol.L‑1. Second, the starting dose of insulin was high 0.1-0.2 8. IU.kg‑1.h‑1, whereas a lower starting dose ranging from 0.02-0.05 IU.kg‑1.h‑1 appears to 9. be effective and safe72. Third, the insulin protocol was adjusted by nurses based on 10. their experience. Simple model-based, computer-assisted or nurse driven protocols 11. have been reported in critically ill adults to increase safety73‑78. There have been a few 12. studies which report tight glucose control protocols with a very low incidence of hy- 13. poglycemia (0-4%), one of which was the step-wise nurse driven protocol that started 14. on our PICU in 200672, 79, 80. These studies report effectivity and safety in older children, 15. while it drew our attention that the majority (> 80%) of the (severe) hypoglycemic in- 16. cidents in the study by Vlasselaers and colleagues71 occurred in infants. In chapter 3 17. we evaluated our glucose protocol specifically in this young population. Although our 18. protocol was equally effective in newborn infants as in older children72, hypoglycemia 19. occurred more frequently in the infants, potentially caused by less mature regulatory 20. capacities in glucose and/or insulin metabolism81‑83. This makes us aware that infants, 21. and possibly also young children, are a more vulnerable population where additional 22. safety approaches are necessary. For instance, tools that help identify individuals with 23. increased risk of developing hypoglycemia are warranted. In addition, we await the 24. development and implementation of (near-) continuous glucose monitoring devices 25. and “closed-loop” glycemic control systems for critically ill children. These would 26. provide additional safety in preventing hypoglycemic incidents. Moreover, combined 27. with validated protocols, these devices could help prevent glucose variability, which 28. would also be useful in older children. Fluctuations in blood glucose levels are highly 9 29. associated with increased mortality and may actually be worse than constant moder- 30. ate hyperglycemia84‑87. This novel aspect of tight glucose control therefore needs to be 31. taken into account in future tight glucose control studies. Furthermore, we observed 32. that overall insulin treatment in the infants in chapter 3 was short (median duration of 33. 27h, range 10 – 57h). Obviously it remains uncertain whether insulin therapy for such 34. a short period of time can be expected to exert any beneficial effect on outcome. An 35. alternative to insulin therapy in the initial phase of hyperglycemia might be to reduce 36. the glucose intake. We described this approach in chapter 4 and it concerns the 37. principal question what the specific requirements are for energy (glucose) in the initial 38. phase of injury (both trauma, surgery as well as infection). This subject is discussed 39. below in the paragraph “Glucose requirements; the initial phase”. Finally, it is important

183 Chapter 9

1. to understand that the optimal glucose target ranges are still uncertain and that ‘hypo- 2. glycemia’ in children and infants is still a controversial issue in regard to its definition88. 3. The complexity of the optimal target ranges becomes evident in patients with traumatic 4. brain injury, which is one of few specific diagnoses that have been investigated in 5. this setting. In this population, a reduction of systemic glucose below 6 mmol.L‑1 with 6. exogenous insulin has been found to exacerbate brain metabolic distress and increase 7. mortality, and a less restrictive target for systemic glucose control (6–10 mmol.L‑1) 8. seems more appropriate89, 90. Whether other diagnoses should be treated according to 9. specific target ranges is uncertain and also deserves further attention. 10. 11. Is insulin an anabolic tool during critical illness? 12. One of the main hypotheses of this thesis was that insulin exerts an anabolic function 13. in critically ill children, which at least partially could explain the beneficial effects of 14. insulin therapy. In healthy humans insulin is “permissive” for protein synthesis and sup- 15. pressive for protein breakdown91. During critical illness, insulin has multiple metabolic 16. effects, and insulin resistance affects both protein and energy metabolism, in addi- 17. tion to several other processes as we summarized in chapter 2. Some of the earlier 18. studies on the effects of insulin therapy in children receiving intensive care actually 19. used this pleiotropic property of insulin as outcome variable. These studies showed 20. that besides glycemic control insulin therapy improved inflammatory response, caloric 21. intake, dyslipidemia and growth, although no data on protein anabolism were avail- 22. able63, 64, 66, 68, 92, 93. 23. In contrast to our hypothesis, however, we found that whole body protein balance was 24. not affected during hyperinsulinemia, as described in chapter 7. Interestingly, although 25. hyperinsulinemia did reduce whole body proteolysis, it simultaneously blunted whole 26. body protein synthesis. Whole body protein synthesis can roughly be divided into 27. muscle and splanchnic protein synthesis, which largely consists of hepatic protein 28. synthesis; acute phase proteins and constitutive proteins. Although insulin has a differ- 29. ential effect on muscle and splanchnic protein synthesis94, our study was not designed 30. to make a differentiation for the effect of insulin on protein synthesis in these specific 31. organs. However, we did focus on one of the major hepatic constitutive proteins. In 32. chapter 8 we described fractional (FSR) and absolute (ASR) albumin synthesis rates in 33. post-surgical infants and septic adolescents. Both the FSR and the ASR were not af- 34. fected with plasma insulin changes. Interestingly, the contribution of albumin synthesis 35. to whole body protein synthesis increased slightly during insulin infusion, although 36. statistical significance was not achieved. Nonetheless, it appears that the decrease 37. in whole body protein synthesis during hyperinsulinemia was based on a decrease in 38. synthesis of hepatic acute phase proteins, a decrease in muscle protein synthesis or 39. a combination of both.

184 General discussion

1. The latter hypothesis is most likely, as both the influence on hepatic as well as muscle 2. protein synthesis are described in previous studies. A decrease in hepatic protein 3. synthesis would be in agreement with two clinical studies which showed that insulin 4. therapy reduced the hepatic pro-inflammatory cascade and reduced concentrations of 5. acute phase proteins such as c-reactive protein66, 71. The insulin resistance to muscle 6. protein synthesis is supported by data from septic animal models where, via a defect 7. in insulin signaling to mediate translation initiation, insulin failed to stimulate muscle 8. protein synthesis95. 9. Aside from the lack of protein anabolism, insulin insufficiently suppressed lipolysis in 10. septic insulin resistant adolescents as we reported in chapter 7, which is in agreement 11. with previous studies in critically ill adults96, 97. Therefore it appears safe to conclude 12. that during critical illness there are no arguments to use insulin therapy as an anabolic 13. tool to improve protein balance or the rate of lipolysis. 14. There are several conditions which were present in our study patients and in a large 15. proportion of patients on our ICU’s, which can be held responsible for the lack of 16. anabolic effect of insulin during critical illness. First, it is well known that exogenous 17. glucocorticoids induce insulin resistance both to glucose as well as protein anabo- 18. lism98, 99. Second, immobility per se causes a decreased muscle protein synthesis100‑102. 19. Finally, nutritive blood flow, depending on the opening of a vascular network to supply 20. nutrients and insulin to organs is essential to support protein anabolism103, while the 21. microcirculation is often compromised in our patients104, 105. These aspects should be 22. taken into account for future studies. 23. 24. Recommendations 25. In light of the large bulk of studies in adults, the available data in critically ill children, 26. and from our studies presented, we propose some recommendations on glycemic 27. control and insulin therapy on the PICU. 28. - In anticipation of subsequent trials identifying the optimal target range and the 9 29. specific population that benefits from tight glucose control, all critically ill children 30. requiring support for cardiac and/or respiratory failure should be treated with a 31. moderate glucose target range of 4-8 mmol.L‑1. Children with traumatic brain injury 32. should be treated with an even less strict glucose target range of 6-10 mmol.L‑1. 33. - A large proportion of children will exert hyperglycemia for a short period of time 34. (<24 h), and insulin therapy in this population will probably not exert the same 35. benefits, while it does hold the unnecessary risk of hypoglycemia. This population 36. might benefit from a reduced glucose intake in the initial phase. 37. - Young infants and children can effectively be treated with insulin, although adjusted 38. age-specific glucose control protocols to reduce the risk for hypoglycemia are war- 39. ranted, and we recommend to lower the insulin starting dose in infants.

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1. - Although organ specific anabolic effects cannot be ruled out, there are currently no 2. grounds to use insulin as an anabolic tool in insulin resistant critically ill children. 3. 4. 5. Macronutrient requirements 6. 7. Despite the clear association with in-hospital protein-energy malnutrition and increased 8. morbidity, the knowledge on specific requirements is poor and routine nutritional as- 9. sessment on the PICU is scarce5. 10. 11. Energy requirements and the importance of its assessment. 12. The energy requirement of a healthy child is the sum of basal metabolic rate, diet 13. induced thermogenesis, the energy needed for physical activity and the energy needed 14. for growth. Although the metabolic response is enhanced in critical illness, studies in 15. critically ill children have reported increased2, 106, but also equal107 or even decreased 16. energy expenditures108. These variations can be explained by the heterogeneous nature 17. of critical illness. The energy expenditure is impacted by diagnosis109, 110, therapy (seda- 18. tives, mechanical ventilation111, 112) and age113. Different methods for the assessment of 19. energy requirements in critically ill children have been developed. To estimate energy 20. requirements, two mathematical methods have been developed for children older than 21. 2 months of age. The predictive equation according to Schofield is based upon a large 22. number of healthy subjects114. An alternative equation based upon 100 mechanically 23. ventilated children was developed by White115. However, the predicted energy expen- 24. diture remains inaccurate in critically ill children due to the abovementioned wide varia- 25. tion in metabolic response. It is therefore preferred to measure energy requirements. 26. For instance with the indirect calorimetry, which is a non-invasive validated method 27. that can be used at the PICU bedside. This method has an additional advantage as it 28. also provides the respiratory quotient (RQ), which is the calculated quotient between

29. volume produced carbon dioxide (VCO2) and volume consumed oxygen (VO2). A RQ < 30. 0.8 indicates underfeeding with gluconeogenesis and protein catabolism and a RQ > 31. 1.0 suggests carbohydrate overfeeding with lipogenesis116. For most children, a single 32. measurement of the total daily energy expenditure is sufficient to provide a good in- 33. sight into energy requirements, although serial measurements are preferred due to the 34. effects of treatment, physical activity and type of nutrition117. The major limitation of 35. the indirect calorimetry is that it cannot be performed when patients are mechanically 118 36. ventilated with an endotracheal tube leak (>10%) and/or a FiO2 > 60% . Also, time 37. limitations and financial restraints prevent this method to be used on a wide scale in 38. every day practice. However, considering the valuable information on both the energy 39. requirements as well as the evaluation of the adequacy of the nutrients provided, it is

186 General discussion

1. a highly recommended tool to establish appropriate nutritional and metabolic support 2. on an individual basis. 3. 4. Glucose requirements; the initial phase. 5. Directly after children are admitted to a PICU with acute injury, regardless of the etiol- 6. ogy (e.g. trauma, surgery or infection), they are given intravenous glucose to provide 7. their fluid and glucose intake. Due to obvious reasons, such as insufficient intravenous 8. access, low priority in an acute situation, and lack of awareness, additional amino acids 9. and/or lipids are usually not provided in the initial phase. Hyperglycemia is a frequent 10. complication of excessive glucose intake in this initial phase. This led us to reassess 11. the glucose requirements in critically ill children. Due to the paucity of data in children 12. it is difficult to define lower limits of glucose intake. The current recommendations are 13. based largely upon “circumstantial evidence” such as the relation between glucose 14. intake and rate of glucose production, induced catabolism, and cerebral glucose 15. availability and utilization50. The latter is in essence the most crucial as hypoglycemia 16. primarily leads to neurological damage in children119‑121. In chapter 4 we showed that 17. hyperglycemia could safely be prevented by a reduced glucose intake, half of what 18. is considered standard practice for age50, without occurrence of hypoglycemia. The 19. children were well capable of sustaining normoglycemic blood glucose levels due to an 20. increased glucose production, primarily almost entirely through gluconeogenesis. We 21. acknowledge that our study only provides data for post-surgical infants, and that it is 22. premature to state that current glucose guidelines for children in the initial phase after 23. injury are too high. However, present recommendations on glucose requirements in 24. various age groups and diagnoses need to be reassessed, also in light of prevention of 25. hyperglycemia. We are currently performing confirmatory studies in children undergo- 26. ing congenital heart surgery and have planned additional studies in septic children. 27. Aside from the necessity to confirm the safety of reduced glucose in different age 28. groups and diagnoses, it is important to emphasize that the results from chapter 4 9 29. cannot be used for recommendations of glucose or energy requirements in critically 30. infants. Beyond the initial phase on injury it is still highly recommended to use the 31. abovementioned assessments to measure energy/glucose requirements on a regular 32. base. With use of the RQ, excessive glucose intake can be prevented and might help 33. avert complications such as hyperglycemia, fatty liver and increased oxygen consump- 34. tion or carbon dioxide production. 35. 36. Amino acid requirements; providing the right quantity. 37. It is well recognized that malnutrition and protein catabolism in critically ill patients lead 38. to muscle wasting which results in physical inability8, 10, higher risk of infections10, pro- 39. longed mechanical ventilation13 and hospital stay11, 12. The negative whole body protein

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1. balance observed in chapter 5 and 7 are consistent with previous data in critically ill 2. children122, 123. The enteral protein provided to the infants (~ 2.3 g.kg‑1.day‑1), children 3. (~ 2.0 g.kg‑1.day‑1) and adolescents (~ 1.4 g.kg‑1.day‑1) described in chapter 5 and 4. the standard parenteral amino acids provided to the adolescents (~ 1.5 g.kg‑1.day‑1) 5. described in chapter 7 were insufficient to prevent protein catabolism. These intakes 6. are current standard practice as we have shown in chapter 6 and are according to 7. current recommended guidelines by the American Society for Parenteral and Enteral 8. Nutrition (ASPEN), the European Society of Paediatric Gastroenterology, Hepatology 9. and Nutrition (ESPGHAN), and the European Society for Clinical Nutrition (ESPEN)51, 124. 10. This makes us conclude that current recommendations on (par)enteral protein / amino 11. acid requirements are inadequate to sustain an optimal protein balance. 12. In critically ill adults, increasing protein intake from 1.0 to about 1.5 g.kg‑1.day‑1 is 13. considered optimal, but did not normalize whole body protein balance, while a further 14. increase to about 2.0 g.kg‑1.day‑1 had no further benefit125‑128. In contrast to these stud- 15. ies in adults, our study in chapter 7 demonstrated that increasing parenteral amino 16. acid intakes to 3.0 g.kg‑1.day‑1 in the presence of adequate caloric intake stimulated 17. protein synthesis, and achieved a positive protein balance in septic adolescents. This 18. result is in agreement with a previous study in critically ill children who received a 19. higher protein intake (~ 2.8 g.kg‑1.day‑1) in combination with a higher ratio of energy 20. intake to energy expenditure (~ 1.7), which led to a positive protein balance3. 21. However, it is still premature to conclude that we should increase the amount of protein 22. and amino acids to critically ill children admitted to our PICU. First of all, we have 23. shown in chapter 5 that amino acid metabolism greatly differs between infants, chil- 24. dren and adolescents and confirmatory studies are therefore needed in children of all 25. age groups. Also, optimal protein and amino acid nutrition probably will differ between 26. different groups of diagnosis (e.g. burns, infection, trauma, post-surgery). Furthermore, 27. although it has been a long tradition to use protein or nitrogen balance as a primary 28. endpoint in nutritional studies evaluating protein and amino acid requirements, it needs 29. to be acknowledged that it merely is an intermediate endpoint. Therefore, clinical 30. outcome studies using maintenance of organ function, wound healing, length of stay 31. on the PICU or hospital among other parameters are awaited for. Finally, excessive 32. amounts of free amino acids may also result in toxicity, and long-term use of parenteral 33. nutrition results on cholestasis and liver failure129‑132. It has been demonstrated that 34. L-methionine is hepatotoxic133, whereas L-arginine, through L-ornithine production 35. induces necrotizing pancreatitis in rats134, and L-Lysine at large doses induces renal 36. failure in dogs135. Moreover, we observed in chapter 7 that the high parenteral amino 37. acid intake provided to the adolescents may have enhanced insulin resistance. We 38. will discuss this matter in further detail later in the paragraph “protein and energy 39. interactions”.

188 General discussion

1. Amino acid requirements: providing the right quality. 2. Protein and amino acid requirements in critically ill children reach beyond the traditional 3. areas of protein metabolism and balance. Individual amino acids exert “non-protein” 4. functions during health and critical illness. They are precursors for the biosynthesis of 5. substrates such as serotonin, nitric oxide and polyamines136, 137, they can act as signal- 6. ing molecules in signal transduction pathways that control the growth, proliferation, 7. and survival of cells138, 139, and they help protect against oxidative stress and protect 8. endothelial health140, 141. This emphasizes the importance to provide each individual 9. amino acid according to their specific requirements. Currently, however, the primary 10. goal of protein or amino acid administration is to try and meet current recommenda- 11. tions of nitrogen requirements. 12. The amino acid composition of the (parenteral) formulas is variable and lacks scientific 13. support as we demonstrated in chapter 6, and the specific amino acid requirements 14. of critically ill children have not been defined. This latter, however, is not an easy goal, 15. as these specific requirements of each individual amino acid differ with age, route of 16. administration and likely also diagnosis. In chapter 5 we demonstrated this at the 17. hand of the essential amino acid methionine. We showed that the splanchnic uptake 18. of enterally administered methionine decreased with age. This difference in first pass 19. disappearance in the splanchnic area illustrates the effect of route of administration. 20. The impact of age is clear from the differences in splanchnic uptake, but is also dem- 21. onstrated by the fate of methionine. While the infants utilized a high proportion of me- 22. thionine for non-oxidative disposal (synthesis of methylated compounds and protein), 23. the adolescents utilized a high fraction for transsulfuration. This might reflect a greater 24. need for other amino acids associated in the transsulfuration pathway and oxidative 25. stress (e.g. cysteine, taurine, glycine and glutathione) in the adolescents. Overall, we 26. observed that the rates of methionine transsulfuration (oxidation) in all infants, children 27. and adolescents exceeded the dietary intake of methionine and cysteine intake. In 28. some degree they were all in a negative balance, illustrating the age-specific inadequa- 9 29. cies of methionine currently provided. So, we can conclude that requirement studies 30. should focus on different age groups. 31. Furthermore, amino acid utilization and metabolism is directed to different pathways 32. depending on the anatomical presentation and requirements are different when 33. provided via the enteral vs. the parenteral route142. Therefore, differentiation between 34. parenteral and enteral administration is also required. Because this is just the example 35. for methionine, one can start to imagine the magnitude of the challenge that lies ahead 36. of us. 37. The preferred technique to determine amino acid requirements is the so-called indica- 38. tor amino acid oxidation (IAAO) method. This method has been used to determine 39. almost all essential amino acid requirements in adults143. Regarding the pediatric

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1. population, several essential amino acid requirements have been determined in healthy 2. school-aged children144‑147, parenterally fed post-surgical neonates148‑150, and enterally 3. fed premature neonates151. However, no data are available for critically ill children. 4. Aside from the lack of scientific basis on which the parenteral intakes of the essential 5. (EAA) amino acids are given, most non-essential (NEAA) amino acids are given in lim- 6. ited amounts as we showed in chapter 6. Moreover, several NEAA, such as cysteine, 7. taurine, glutamine, are sometimes not provided at all. It is widely assumed that the 8. NEAA must be less important, since they are the amino acids that are simply produced 9. by the body. However, this reasoning appears wrong from an evolutionary point of 10. view. It appears more plausible that human ancestors would have retained the capacity 11. to synthesize those amino acids that we now label NEAA, to maintain their supply even 12. when nutrition is scarce. These amino acids must really be important during periods of 13. illness, injury and catabolism. It is increasingly recognized that, both from a metabolic 14. as well as from a functional perspective, all amino acids are essential152‑156. Therefore, 15. the distinction between EAA and NEAA should be reassessed, as the NEAA deserve 16. special attention, particularly during critical illness. 17. 18. Protein and energy interactions 19. Aside from the abovementioned arguments there is another important aspect why 20. improving nutritional support is not simply a matter of providing more nutrients to our 21. patients. There is an interaction between protein and energy metabolism; they depend 22. on each other, but also influence one another. 23. 24. The effect of energy intake on protein metabolism. 25. Under physiological conditions, an increase in energy supply will not promote nitrogen 26. retention unless the protein supply is adequate, and conversely an increased protein 27. supply will be useless if energy is limiting. A lack in energy supply might even induce 28. protein catabolism to release substrates for the cost of energy. These conditions, how- 29. ever, might be different during conditions of critical illness. In chapter 4 we observed 30. that a 50% cut in energy (glucose) supply had no effect on the protein turnover in 31. post-surgical infants. This is in agreement with a previous study in neonates, which 32. showed that different glucose intakes did not affect protein turnover157. It is clear that 33. the reduced glucose intake described in chapter 4 was not meant for prolonged use, 34. considering that no amino acids were provided. Yet, although one should distinguish 35. between hypocaloric feeding (low energy, but sufficient protein delivery) and underfeed- 36. ing (energy and protein restriction), it is not likely that the provision of amino acids would 37. have affected the results. Chapter 7 shows that the negative protein balance could not 38. be deflected by sufficient or increased amounts of energy intake, even in combination 39. with insulin infusion and standard provision of amino acids. So, as we have discussed

190 General discussion

1. earlier, during conditions of critical illness insulin does not exert the expected anabolic 2. properties on protein balance, nor on albumin synthesis as shown in chapter 8, even 3. though the deficiencies in glucose homeostasis were largely overcome. This was in 4. contrast with our hypothesis, also because, independent of plasma insulin levels, nor- 5. moglycemia improved protein turnover after abdominal surgery in adults158. Our results 6. are supported by a study in insulin resistant adult ICU patients where different amounts 7. of glucose and insulin infusion did not affect protein metabolism97. Indeed, it has been 8. recognized that a deficiency in energy supply is not solely responsible for protein 9. catabolism159. Protein breakdown during critical illness is usually caused by activation 10. of the ubiquitin-proteasome proteolytic pathway (UPP) in muscle initiated by activation 11. of caspase 3160, 161, and pathophysiological triggers include activation of lysosome-162 12. and calpain-dependent pathways163. Recently, a mechanistic link has been proposed 13. between protein catabolism and insulin resistance164‑166. These findings suggest that 14. in (insulin resistant) patients, regulatory pathways controlling protein turnover change 15. and are less influenced by energy intake than in healthy individuals. 16. 17. Amino acid intake affects energy metabolism 18. It has been a long tradition of ignoring the contribution of protein metabolism to the 19. energy delivery of nutritional support. However a patient not only receives the energy 20. equivalent of 100% of the amino acids given in the diet, but also energy from the net 21. loss of nitrogen, which is even higher in a catabolic state. High amino acid feeding 22. might therefore add to any problems associated with high glucose or lipid provision, 23. such as increased insulin resistance. Moreover, free amino acids provided in excessive 24. amounts or released from proteolysis, and exceeding the incorporation into proteins 25. are oxidized and/or channeled into the gluconeogenic pathway167. So, increased amino 26. acid intake potentially might be a detrimental influence on insulin resistance and glu- 27. cose metabolism. 28. This was confirmed by the study we described in chapter 7. During the high amino 9 29. acid intake, endogenous glucose production and protein breakdown were not fully 30. suppressed during insulin infusion and lipolysis rates were increased. The high amino 31. acid intake may have favored gluconeogenesis, which raises concern that it may have 32. enhanced insulin resistance. An increased insulin resistance could also explain the 33. increased lipolysis rate we observed. Although this was not confirmed by increased 34. levels of plasma insulin or with our Hyperinsulinemic Euglycemic Clamp technique, 35. there is a strong indication from other studies that links the provision of amino acids 36. to insulin resistance. Amino acid infusion in healthy subjects to achieve hyperamino- 37. acidemia decreases insulin sensitivity for glucose metabolism168, possibly through a 38. negative feedback loop toward insulin-stimulated tyrosine phosphorylation of insulin 39. receptor substrate 1 and 2 (IRS-1, IRS-2)169, 170.

191 Chapter 9

1. Hence, although a high amino acid intake increased protein synthesis and improved 2. the protein balance, there is concern that it could negatively impact glucose and lipid 3. homeostasis. Dose-response studies on protein administration and their effects on 4. energy metabolism and insulin resistance are needed. Furthermore, these interactions 5. between protein and energy metabolism emphasize the importance of clinical outcome 6. studies, as this type of study does not focus on one aspect, such as protein balance, 7. but looks at the patient as a whole. 8. 9. Recommendations 10. Based on the studies presented in this thesis and in reflection with the arguments put 11. forward in the subsequent discussion we conclude and recommend the following: 12. - Current recommended glucose intakes can induce hyperglycemia in infants in the 13. initial post-surgical phase and can safely be reduced, while awaiting confirmatory 14. studies in different age groups and diagnoses. 15. - To evaluate glucose / energy requirements after the initial phase, routine assess- 16. ments by means of techniques such as indirect calorimetry, to measure Energy 17. Expenditure and Respiratory Quotient, are invaluable to prevent excessive glucose 18. intake. 19. - Both the quantity as well as the quality of currently provided amino acid intakes are 20. inadequate and lack scientific support. Future studies should focus on the require- 21. ments of specific amino acids based on age, diagnosis and route of administration. 22. - In light of protein balance as outcome variable, doubling the intake of amino acid 23. enhances protein anabolism in septic adolescents and should be considered in 24. future nutrition guidelines. However, before increased amino acid intakes are imple- 25. mented in the clinical setting, the potential detrimental effects on insulin sensitivity 26. and glucose and lipid metabolism demands further investigation. 27. 28. 29. Future perspectives 30. 31. There are two remaining issues not discussed in this thesis, we want to bring under 32. consideration. 33. First, to be worthwhile, recommendations and guidelines need to be followed by the 34. actual practice at the bedside. This is of even greater importance when we want to 35. demonstrate the impact on clinical outcome. However, there is an increased aware- 36. ness that the implementation of nutritional guidelines on ICU’s is complex and time- 37. consuming and therefore often unsuccessful7, 171‑173. Identifying effective methods to 38. improve guideline implementation should go hand in hand with the development of 39. these guidelines.

192 General discussion

1. Finally, there is one other aspect which is getting increasingly more attention in nutri- 2. tion science. It is the concept of using specific nutrients to help modulate key pro- 3. cesses such as immunity, inflammation, endothelial health and oxidative stress. This 4. concept, known as “pharmaconutrition”, is becoming more accepted to play a future 5. role in nutritional and metabolic therapy. Several macro- as well as micronutrients have 6. drawn attention in this matter; glutamine, cysteine, arginine, selenium, zinc, vitamin 7. C and E, and omega-3 fatty acids. We will mention those which have been cited as 8. most promising. First, glutamine, the most abundant amino acid in plasma, which has 9. numerous metabolic functions174. It is an important fuel for rapidly dividing cells, is a 10. precursor for glutathione, protects intestinal mucosa and augments cellular immune 11. functions141, 174, 175. A meta-analysis of glutamine supplementation in critically ill patients 12. have revealed a reduction in mortality176. Second, arginine, which is also an amino 13. acid that exerts multiple functions141. It is a precursor for creatine, polyamines, it is 14. an essential product in the urea cycle and it is the sole precursor for nitric oxide141, 174. 15. This latter property is subject of concern, because arginine supplementation has lead 16. to inconsistent results in clinical studies, possibly because of the potential toxic effect 17. of arginine as a substrate for inducible nitric oxide synthase (iNOS)175. Increased NO 18. production might deteriorate microcirculation and organ dysfunction. In contrast, there 19. has been extensive evidence that certain patient groups might benefit from arginine 20. supplementation177. Finally, the use of the omega-3 fatty acids (primarily eicosapen- 21. taenoic acid (EPA) and docosahexaenoic acid (DHA)) is at the centre of attention. Their 22. biological effects are related to anti-inflammatory effects. Enteral supplementation of 23. fish oil, rich in omega-3 fatty acids, have improved outcome in patients with Acute 24. Respiratory Distress Syndrome (ARDS)175. 25. However, despite the promising results in adult patients, ‘pharmaconutrition’ studies 26. in critically ill children are limited to three small studies in burned children178‑180. These 27. studies are too small and limited to draw any conclusions and larger studies are neces- 28. sary. 9 29. 30. As a rule, future perspectives and recommendations always stress the importance of 31. forthcoming studies. Despite, and probably also due to, the studies and discussions 32. presented in this thesis, this isn’t any different in case of nutritional and metabolic 33. support in critically ill children. Therefore we want to summarize the most important 34. issues in future studies to follow. 35. - In respect to the mechanisms and benefits of tight glucose control in critically ill 36. children, a limited amount of data is available. Attention of future investigation 37. should be focused on; identification of children at risk for prolonged periods of hy- 38. perglycemia, optimal glucose target ranges, and the efficacy and safety of protocols 39. in specific age groups and diagnoses (e.g. post-cardiac surgery and septic shock).

193 Chapter 9

1. - Our studies described in chapter 4, 5, 7 and 8 have focused on specific populations; 2. varying in age, diagnosis and route of nutrition. Obviously, confirmatory studies are 3. needed to be able to make recommendations on glucose and amino acid intakes 4. and the impact they exert on each other for the entire pediatric population. 5. - Aside from well designed smaller mechanistic studies using intermediate endpoints 6. such as protein balance or plasma concentrations, we are in need of clinical out- 7. come studies. Since comparable pediatric ICU populations are small, multicenter 8. studies are therefore necessary. This type of study will show us whether we can 9. actually affect morbidity and may be even mortality, which in the end should be the 10. ultimate goal. 11. 12. These studies will provide further knowledge which is imperative to develop clear 13. guidelines on nutritional and metabolic support on the PICU. For now, the implementa- 14. tion of our results, regarding the reduced glucose intake and the increased amino acid 15. intake, would be premature for upcoming guidelines. Additionally, it is our responsibil- 16. ity as researchers to start providing scientific data with which the nutritional industry 17. can improve the composition of (par)enteral formulas. 18. However, we did set the scene for further examination of these approaches to improve 19. nutritional care in critically ill children. Furthermore, the recommendation to decrease 20. the insulin starting doses in infants is a small but valuable contribution. Once we 21. establish the effect of nutritional interventions not only on narrow endpoints but also 22. on the interaction between the different substrates and the patient as a whole, we are 23. confident about the future of nutritional therapy to improve the outcome of the critically 24. ill child. 25. 26. 27. References 28. 1. Cogo PE, Carnielli VP, Rosso F, et al. Pro- strate utilization in critically ill children. 29. tein turnover, lipolysis, and endogenous Am J Clin Nutr 2001;​74(5):​664‑9. 30. hormonal secretion in critically ill chil- 4. Hulst J, Joosten K, Zimmermann L, et al. 31. dren. Crit Care Med 2002;​30(1):​65‑70. Malnutrition in critically ill children: from 32. 2. Coss-Bu JA, Jefferson LS, Walding D, admission to 6 months after discharge. 33. David Y, Smith EO, Klish WJ. Resting Clin Nutr 2004;​23(2):​223‑32. energy expenditure and nitrogen bal- 5. Hulst JM, Joosten KF, Tibboel D, van 34. ance in critically ill pediatric patients on Goudoever JB. Causes and conse- 35. mechanical ventilation. Nutrition 1998;​ quences of inadequate substrate supply 36. 14(9):​649‑52. to pediatric ICU patients. Curr Opin Clin 37. 3. Coss-Bu JA, Klish WJ, Walding D, Nutr Metab Care 2006;​9(3):​297-303. 38. Stein F, Smith EO, Jefferson LS. Energy 6. de Neef M, Geukers VG, Dral A, Linde- metabolism, nitrogen balance, and sub- boom R, Sauerwein HP, Bos AP. Nutri- 39.

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lasting sepsis results from the activation kinase pathway. A negative feedback 1. of lysosomal, Ca2+ -activated, and mechanism leading to insulin resistance 2. ubiquitin-proteasome proteolytic path- in skeletal muscle cells. J Biol Chem 3. ways. J Clin Invest 1996;​97(7):​1610‑7. 2001;​276(41):​38052‑60. 4. 162. Deval C, Mordier S, Obled C, et al. 170. Patti ME, Brambilla E, Luzi L, Landaker 5. Identification of cathepsin L as a differ- EJ, Kahn CR. Bidirectional modulation entially expressed message associated of insulin action by amino acids. J Clin 6. with skeletal muscle wasting. Biochem J Invest 1998;​101(7):​1519‑29. 7. 2001;​360(Pt 1):​143‑50. 171. Doig GS, Simpson F, Finfer S, et al. Effect 8. 163. Smith IJ, Lecker SH, Hasselgren PO. of evidence-based feeding guidelines on 9. Calpain activity and muscle wasting in mortality of critically ill adults: a cluster 10. sepsis. Am J Physiol Endocrinol Metab randomized controlled trial. Jama 2008;​ 2008;​295(4):​E762‑71. 300(23):​2731‑41. 11. 164. Wang X, Hu Z, Hu J, Du J, Mitch WE. 172. Jain MK, Heyland D, Dhaliwal R, et al. 12. Insulin resistance accelerates muscle Dissemination of the Canadian clinical 13. protein degradation: Activation of the practice guidelines for nutrition support: 14. ubiquitin-proteasome pathway by de- results of a cluster randomized controlled 15. fects in muscle cell signaling. Endocri- trial. Crit Care Med 2006;​34(9):​2362‑9. 16. nology 2006;​147(9):​4160‑8. 173. Jones NE, Suurdt J, Ouelette-Kuntz 165. Crossland H, Constantin-Teodosiu D, H, Heyland DK. Implementation of the 17. Gardiner SM, Constantin D, Greenhaff Canadian Clinical Practice Guidelines for 18. PL. A potential role for Akt/FOXO signal- Nutrition Support: a multiple case study 19. ling in both protein loss and the impair- of barriers and enablers. Nutr Clin Pract 20. ment of muscle carbohydrate oxidation 2007;​22(4):​449‑57. 21. during sepsis in rodent skeletal muscle. 174. Obled C, Papet I, Breuille D. Metabolic J Physiol 2008;​586(Pt 22):​5589-600. bases of amino acid requirements in 22. 166. Crossland H, Constantin-Teodosiu D, acute diseases. Curr Opin Clin Nutr 23. Greenhaff PL, Gardiner SM. Low-dose Metab Care 2002;​5(2):​189‑97. 24. dexamethasone prevents endotoxaemia- 175. Jones NE, Heyland DK. Pharmaconutri- 25. induced muscle protein loss and impair- tion: a new emerging paradigm. Curr 26. ment of carbohydrate oxidation in rat Opin Gastroenterol 2008;​24(2):​215‑22. 27. skeletal muscle. J Physiol 2010;​588(Pt 176. Novak F, Heyland DK, Avenell A, Drover 8):​1333‑47. JW, Su X. Glutamine supplementation 28. 167. Chevalier S, Burgess SC, Malloy CR, in serious illness: a systematic review of 9 29. Gougeon R, Marliss EB, Morais JA. The the evidence. Crit Care Med 2002;​30(9):​ 30. greater contribution of gluconeogenesis 2022‑9. 31. to glucose production in obesity is re- 177. Heyland DK, Novak F, Drover JW, Jain M, 32. lated to increased whole-body protein Su X, Suchner U. Should immunonutrition catabolism. Diabetes 2006;​55(3):​ become routine in critically ill patients? A 33. 675‑81. systematic review of the evidence. Jama 34. 168. Tessari P, Inchiostro S, Biolo G, et al. 2001;​286(8):​944‑53. 35. Hyperaminoacidaemia reduces insulin- 178. Marin VB, Rodriguez-Osiac L, Schless- 36. mediated glucose disposal in healthy inger L, Villegas J, Lopez M, Castillo- 37. man. Diabetologia 1985;​28(11):​870‑2. Duran C. Controlled study of enteral argi- 38. 169. Tremblay F, Marette A. Amino acid and nine supplementation in burned children: insulin signaling via the mTOR/p70 S6 39.

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impact on immunologic and metabolic 180. Mayes T, Gottschlich MM, Kagan RJ. An 1. status. Nutrition 2006;​22(7-8):​705‑12. evaluation of the safety and efficacy of 2. 179. Sheridan RL, Prelack K, Yu YM, et al. an anti-inflammatory, pulmonary enteral 3. Short-term enteral glutamine does not formula in the treatment of pediatric burn 4. enhance protein accretion in burned patients with respiratory failure. J Burn 5. children: a stable isotope study. Surgery Care Res 2008;​29(1):​82‑8. 2004;​135(6):​671‑8. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39.

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Summary of the thesis

Summary

1. Chapter 1 2. Optimal nutritional and metabolic support to counteract ongoing catabolism and re- 3. gain control of glucose homeostasis is essential in critically ill children. Nevertheless, 4. nutritional goals as well as glycemic control are still not met in the Pediatric Intensive 5. Care Unit (PICU), resulting in increased morbidity and mortality. 6. In the introduction of this thesis an overview is given on the major metabolic changes 7. occurring during critical illness. Subsequently, the challenges and pitfalls in the devel- 8. opment of recommendations and guidelines for nutritional and metabolic support on 9. the PICU are described. This is followed by a summary on glucose metabolism and 10. the potential benefits and risks of tight glucose control regimens. Finally, an overview 11. is provided on the function of protein and amino acids, and the diverse variables that 12. should be taken into account when investigating their specific requirements. 13. The first chapter ends with the aims and outline of the thesis. 14. 15. Chapter 2 16. As is often the case in research as well as in clinical practice, the foundations are laid 17. in adult medicine. This also applies to tight glucose control by means of insulin therapy. 18. Based on the promising results of glycemic control on adult ICU’s, we provided an 19. extensive review that describes the causes and consequences of hyperglycemia in 20. critically ill children. Additionally we provided an overview of the diverse (non)metabolic 21. properties of insulin and speculated of their potential benefits to critically ill children. 22. From a mechanistic point of view, several benefits are to be expected from insulin 23. therapy and tight glucose control on the PICU. Finally, we provided a summary of the 24. studies that have investigated insulin therapy in the pediatric population, with special 25. emphasis on the single large outcome trial performed in critically ill children. This trial 26. showed that although tight glucose control with insulin improves outcome in a similar 27. fashion as reported in adult ICU’s, hypoglycemia was a serious and frequent complica- 28. tion. 29. 30. Chapter 3 31. Despite the early striking results of the tight glucose regimens in adult ICU’s, there is 32. still an ongoing debate focusing on the unacceptable high risk of hypoglycemia. The 10 33. same reaction followed when the results of the large trial on the PICU reported an 34. incidence of hypoglycemia of 25%, which occurred predominantly (>80%) in infants. 35. We evaluated a step-wise nurse driven glucose protocol on our PICU and focused 36. specifically on infants less than one month old. We concluded that the young infants 37. could be treated according to the protocol equally effective as older children. However, 38. in contrast to studies evaluating glucose control protocols in older children we found a 39. higher incidence of hypoglycemia (6.8%). Based on previous studies in infants as well

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1. as animal models we proposed that infants are more vulnerable to develop hypoglyce- 2. mia following insulin administration, due to a wide variation in the capacity to regulate 3. both glucose and insulin metabolism. Therefore, infants and potentially also young 4. children require a more cautious approach when treated for hyperglycemia with insulin. 5. We adjusted the insulin starting dose for infants in our protocol. 6. 7. Chapter 4 8. One of the observations in chapter 3 was that a large proportion of the infants treated 9. according to the tight glucose protocol required insulin therapy for a short period of 10. time. It is less likely that this subset of infants and children can benefit from this short 11. treatment period, while they still are put at increased risk to develop hypoglycemia. 12. In chapter 4 we described a study that showed that a reduced glucose intake, half of 13. the current recommendations, could prevent hyperglycemia effectively and safely. By 14. means of isotope tracer techniques we showed that the post-surgical infants (n=8) 15. were well capable to sustain normoglycemia, and increased their endogenous glucose 16. production. We also showed, with help of the relatively novel average deuterium en- 17. richment method, that these infants predominantly used gluconeogenesis to produce 18. glucose. Furthermore, we showed that reducing the glucose intake did not exacerbate 19. the mild catabolic state these infants were in. This approach could therefore be useful 20. to prevent or treat hyperglycemia in the initial phase of treatment when children are 21. admitted to the PICU. 22. 23. Chapter 5 24. This chapter showed the ontogeny of the splanchnic uptake and utilization of the es- 25. sential amino acid methionine in critically ill children (n=24). We used stable isotope 26. techniques in enterally fed infants, children and adolescents, and applied two kinetic 27. models: the plasma methionine enrichment and the “intracellular” homocysteine en- 28. richment. The plasma model underestimated methionine kinetics in children and 29. adolescents but not in the infants. 30. The rates of methionine splanchnic uptake were higher in infants than adolescents. 31. We also showed that the fate of methionine utilization in critically ill children differed 32. with age. In the infants methionine was used primarily for synthesis of proteins and 33. methionine-derived compounds, while the adolescents had high transsulfuration (oxi- 34. dation) rates. All patients were in a negative methionine balance, a clear indication that 35. the enteral supply of this specific amino acid is currently insufficient. 36. 37. Chapter 6 38. In this chapter, we provided an exploratory overview of the amino acid intake of 39. children of all age groups receiving full parenteral nutrition who were admitted to the

208 Summary

1. PICU during a 12-month period. Using the recommended intakes from the Institute 2. of Medicine (IOM) as reference we compared the intakes of the essential amino acids 3. (EAA) to those actually provided to critically ill children. The non-essential amino acids 4. (NEAA) were compared with the content of breast milk (infants) or mixed muscle pro- 5. tein (older children).. As the recommended intakes from the IOM are not developed for 6. critically ill children we further contrasted EAA and NEAA intakes with the composition 7. of representative inflammatory/immune proteins, because these proteins, not muscle 8. proteins, are the major source of amino acid utilization for protein synthesis during 9. critical illness. Our data showed that currently, EAA as well as NEAA are not provided 10. through evidence based guidelines but based on convenience. This results in both 11. insufficient, but also excessive amounts of intakes. The latter is also of importance as 12. amino acids potentially can be toxic. 13. 14. Chapter 7 15. In the study described in this chapter, the effect of an increased parenteral amino acid 16. intake, with and without insulin infusion, on glucose metabolism, protein kinetics and 17. lipolysis was investigated. On two subsequent days a stable isotope tracer infusion in 18. combination with a hyperinsulinemic euglycemic clamp (HEC) was performed in nine 19. septic adolescents. This two day study allowed us to show that providing parenteral 20. amino acids, twice the amount currently recommended, improved whole body protein 21. balance independent of hyperinsulinemia. 22. By means of the HEC technique we showed that hyperglycemic septic adolescents 23. were insulin resistant. Moreover, insulin infusion in these adolescents did not lead to 24. whole body protein anabolism and did not suppress lipolysis. 25. Despite the positive effect on whole body protein balance, the increased parenteral 26. amino acid intake also led to increased lipolysis and potentially enhanced gluconeo- 27. genesis. These aspects make us aware that increasing the amino acid intake can 28. potentially lead to increased insulin resistance. 29. 30. Chapter 8 31. The same eight post-surgical infants and nine septic adolescents described in chapter 32. 4 and 7 were used for this study. In these two separate studies the stable isotope tracer 10 33. of the amino acid leucine was infused to measure leucine and protein kinetics. In the 34. study described in chapter 8 albumin synthesis rates were calculated by measuring 35. the increase of incorporated tracer leucine into albumin over time. This allowed us to 36. describe the albumin synthesis rates in post-surgical infants as well as septic ado- 37. lescents. We described that both the infants as well as the adolescents were slightly 38. hypoalbuminemic, but exerted high synthesis rates. This latter was more obvious in 39. the septic adolescents, which is in agreement with their higher catabolic state. In the

209 Chapter 10

1. infants, the intake of glucose changed over time. In the adolescents, the amount of 2. parenteral amino acids as well as the plasma insulin levels changed over time. We 3. observed no differences in fractional as well as absolute albumin synthesis rates with 4. any of these interventions. We concluded that the synthesis rates are regulated through 5. other pathways than nutrient signaling. 6. 7. Chapter 9 8. This chapter provides a general discussion in which the major findings of the studies 9. presented in this thesis are critically analyzed and put into a wider perspective with 10. help of the current literature. With help of these considerations some recommenda- 11. tions and future perspectives for the clinicians as well as researchers are presented. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39.

210 Chapter 11

Samenvatting voor niet-medici

Samenvatting

1. Hoofdstuk 1 2. Kritisch zieke kinderen op de intensive care (IC) worden behandeld voor hun ziekte, 3. terwijl ze vaak met beademing en medicijnen ondersteund worden voor hun ernstig 4. zieke organen zoals longen, hart, nieren en lever. Kinderen die zo ziek zijn kunnen niet 5. op een normale manier eten en drinken, terwijl een goede voeding wel belangrijk is 6. voor hun herstel. Het voeden van kritisch zieke kinderen wordt nog verder gecompli- 7. ceerd omdat ook hun stofwisseling van suikers, eiwitten en vetten niet meer normaal 8. verloopt. In hoofdstuk 1 wordt kort een overzicht gegeven van de belangrijkste stofwis- 9. selingsproblemen bij kritisch zieke kinderen op de IC. Dit zijn een te hoge bloedsuiker, 10. en afbraak van vetten en lichaamseiwitten, voornamelijk de eiwitten in spieren. Deze 11. problemen kunnen worden veroorzaakt door medicijnen, door onvoldoende (goede) 12. voeding, maar ook door veranderingen van het lichaam als reactie op het kritisch ziek 13. zijn. Eén van deze veranderingen is een verminderde gevoeligheid voor het hormoon 14. insuline, wat belangrijk is voor een normale stofwisseling. Door deze veranderingen 15. in de stofwisseling worden de patiënten nog zieker, waardoor ze vaak langer op de 16. intensive care of in het ziekenhuis behandeld moeten worden, of waardoor ze soms 17. zelfs overlijden. Optimale voeding en ondersteuning van de stofwisseling zijn daardoor 18. van het grootste belang. Echter, de kennis van voeding voor kinderen op de IC is 19. nog onvoldoende, en veel voedingsproducten worden waarschijnlijk niet in de goede 20. hoeveelheid gegeven of zijn van onvoldoende kwaliteit. De samenstelling en de hoe- 21. veelheid van veel van de huidige voedingen voor kritisch zieke kinderen zijn gebaseerd 22. op het beleid van volwassenen of op kleine en soms zelfs verouderde studies. De 23. belangrijkste problemen en vraagtekens die er momenteel nog zijn rondom de voeding 24. en ondersteuning van de stofwisseling bij kritisch zieke kinderen worden samengevat 25. in hoofdstuk 1. Allereerst in een korte uitleg van de verstoorde suiker stofwisseling 26. en de mogelijke voordelen maar ook risico’s van de behandeling met het hormoon 27. insuline, zoals dat bij suikerpatiënten wordt gedaan. Van insuline is bekend dat het 28. bloedsuikerwaarden kan normaliseren, maar ook dat het de afbraak van eiwitten en 29. vetten kan tegen gaan. Er zijn ook risico’s verbonden aan een dergelijke behandeling, 30. zoals te lage bloedsuiker waarden. Een te lage bloedsuiker waarde kan gevaarlijk zijn, 31. terwijl het niet eens zeker is of insuline hetzelfde gunstige effect heeft bij kritisch zieke 32. kinderen als dat het heeft bij kritisch zieke volwassenen. Daarna wordt in hoofdstuk 33. 1 kort ingegaan op de functie van eiwitten en aminozuren (dit zijn de bouwstenen 34. van eiwitten), welke belangrijk zijn voor kritisch zieke kinderen. Het bestuderen van 35. de voedingsbehoeften van eiwitten en aminozuren is lastig en er wordt kort uitgelegd 11 36. waar men bij dit soort onderzoeken rekening mee moet houden. Het eerste hoofdstuk 37. eindigt met de doelstellingen en het overzicht van de studies verricht in dit proefschrift. 38. Het proefschrift bestaat grofweg uit vier delen; deel 1 (hoofdstuk 2 t/m 4) gaan over de 39. stofwisseling van suikers en de mogelijke effecten en risico’s van insuline behandeling.

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1. Deel 2 (hoofdstuk 5 en 6) gaat over de eiwitten en aminozuren in de voeding, en over 2. de gevolgen die de leeftijd en de manier van voeden van het kind hebben op de eiwit 3. stofwisseling en voeding. Deel 3 (hoofdstuk 7 en 8) beschrijft hoe het veranderen van 4. de hoeveelheid energie en eiwitten in de voeding ook effect heeft op elkaar. En deel 4 5. (hoofdstuk 9 en 10), tot slot, geeft een uitgebreide discussie en samenvatting van de 6. belangrijkste onderwerpen en conclusies van het gehele proefschrift. 7. 8. Hoofdstuk 2 9. Vaak worden zowel in onderzoek, als ook in de kliniek, behandelingen en ideeën voor 10. kritisch zieke kinderen gevolgd die succesvol zijn bij volwassen patiënten. Dit geldt 11. ook voor de behandeling van te hoge bloedsuikers met insuline op de intensive care, 12. ook bij patiënten die geen suikerziekte hebben. Bij kritisch zieke volwassenen is sinds 13. een aantal jaren bekend dat het verlagen van hoge bloedsuikers met insuline zorgt 14. voor een verbetering van het herstel en zelfs de overleving. Gebaseerd op de veelbe- 15. lovende resultaten van deze behandeling bij patiënten op de volwassenen IC, geven 16. wij in hoofdstuk 2 een uitgebreid overzicht van de oorzaken en de gevolgen van hoge 17. bloedsuikers in kritisch zieke kinderen. Bovendien geven wij een overzicht van de di- 18. verse effecten van insuline bij kritisch zieke zuigelingen en kinderen. Op basis van deze 19. gegevens speculeerden wij over de mogelijke voordelen van het geven van insuline aan 20. kritisch zieke kinderen. Daarnaast vatten we de studies samen die insuline als therapie 21. hebben onderzocht bij zuigelingen en kinderen op de IC. De nadruk van dit gedeelte 22. van hoofdstuk 2 ligt dan op de enige grote studie die in kritisch zieke kinderen werd 23. uitgevoerd. Deze studie toonde aan dat het behandelen van hoge bloedsuiker waarden 24. met insuline een verbetering gaf van de overleving van kritisch zieke kinderen, zoals dat 25. ook op de volwassen IC werd gezien. Echter, een gevolg van deze insuline behandeling 26. was wel dat veel kinderen lage bloedsuiker waarden kregen, iets wat bij kinderen een 27. ernstige complicatie kan zijn. De oorzaken van deze te lage bloedsuiker waarden lig- 28. gen naar onze mening in de lage bloedsuiker grenzen die worden nagestreefd in deze 29. studie, de hoge startdoseringen van insuline en het feit dat de verpleegkundigen in 30. deze studie zelf de insuline mochten aanpassen op basis van hun ervaring. 31. 32. Hoofdstuk 3 33. Ondanks de goede resultaten van de insuline behandelingen bij volwassen patiënten 34. op de IC, is er nog steeds een discussie gaande over de onaanvaardbare risico’s van 35. lage bloedsuikers. Dezelfde reactie volgde toen de resultaten van de enige grote studie 36. op de kinder IC een lage bloedsuiker bij 25% van de kinderen veroorzaakte, waarvan 37. het merendeel (meer dan 80%) bij zuigelingen. Zoals wij al beschreven in hoofdstuk 2 38. zijn de oorzaken van deze lage bloedsuikers naar onze mening; de lage bloedsuiker 39. grenzen die worden nagestreefd in deze studie, de hoge startdoseringen van insuline

214 Samenvatting

1. en het feit dat de verpleegkundigen in deze studie zelf de insuline mochten aanpas- 2. sen. Op onze kinder IC bestaat er een insuline protocol voor kritisch zieke kinderen 3. welke effectief en veilig is gebleken bij oudere kinderen. Omdat het merendeel van 4. de lage bloedsuikers in de beschreven studie ontstonden bij zuigelingen, evalueerden 5. wij het protocol dat op onze eigen kinder IC wordt gebruikt, en concentreerden ons 6. specifiek op zuigelingen jonger dan één maand oud. Wij concludeerden dat de jonge 7. zuigelingen volgens het protocol even goed behandeld kunnen worden als de oudere 8. kinderen. Echter, terwijl hetzelfde protocol bij de oudere kinderen geen te lage bloed 9. suikers deed ontstaan, vonden wij bij 6,8% van de zuigelingen een te lage bloedsuiker. 10. Door deze studie en door andere studies gedaan in zuigelingen, maar ook in dieren 11. stelden wij dat de zuigelingen kwetsbaarder zijn voor insuline behandeling, door een 12. onrijpe stofwisseling van suiker en het hormoon insuline. Daarom vinden wij dat de 13. zuigelingen, en mogelijk ook jonge kinderen, voorzichtiger behandeld moeten worden 14. met insuline. Wij verlaagden de insuline dosering in ons protocol voor zuigelingen om 15. in de toekomst te lage bloedsuiker waarden te voorkomen. 16. 17. Hoofdstuk 4 18. Eén van de observaties in hoofdstuk 3 was dat een groot deel van de zuigelingen 19. die met insuline werden behandeld, maar voor een hele korte periode (minder dan 20. een dag) te hoge bloedsuiker waarden hadden. Het is minder waarschijnlijk dat deze 21. zuigelingen en kinderen baat hebben van deze korte behandeling, terwijl het risico op 22. de lage bloedsuiker waarden door de insuline blijft bestaan. Veel kinderen die maar 23. voor een korte tijd te hoge bloedsuiker waarden hebben, zijn de kinderen in de begin 24. periode van de opname op de kinder IC. Het verlagen van de hoeveelheid suiker in het 25. infuus is voor deze kinderen mogelijk een manier om hoge bloedsuikers te voorkomen 26. zonder dat insuline nodig is. In hoofdstuk 4 beschrijven wij een studie waarin we minder 27. suiker gaven via het infuus aan zuigelingen na een operatie. We gaven deze kinderen 28. gedurende vier uur een normale hoeveelheid suiker volgens de huidige aanbevelingen 29. en gedurende vier uur ongeveer de helft van deze hoeveelheid. Wij toonden aan dat het 30. geven van minder suiker tot normale bloedsuiker waarden leidde, zonder dat er lage 31. bloedsuikers ontstonden. Deze studie maakte ook gebruik van infusen van stabiele 32. isotopen. Dit zijn natuurlijke producten die overal, ook in ieders lichaam, voorkomen 33. en die ons helpen om te kijken wat er met eiwitten en suikers gebeuren in zieke kin- 34. deren. Door middel van deze infusen met stabiele isotopen toonden wij aan dat de 35. zuigelingen hun eigen bloed suikers prima op peil konden houden, en zij verhoogden 11 36. hun eigen suiker productie tot het niveau waarop hun bloed suiker waarden normaal 37. waren. Wij toonden ook aan dat deze zuigelingen voornamelijk geheel nieuwe suiker 38. aanmaakten, en niet oude opgeslagen suiker gebruikten. Als laatste toonden wij aan 39. dat het minder geven van suiker de afbraak van eiwit (door het ziek zijn) in hun lichaam

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1. niet verergerde. Deze manier van voorkomen van hoge bloedsuikers kan daarom nuttig 2. zijn als alternatief voor insuline behandeling in de beginfase wanneer de kinderen op 3. de kinder IC worden opgenomen. 4. 5. Hoofdstuk 5 6. De studie in dit hoofdstuk gebruikt ook weer de techniek met stabiele isotopen. Dit- 7. maal om te kijken wat de invloed van leeftijd is op het verbruik van de darm van een 8. aminozuur (bouwsteen van eiwitten) in kritisch zieke kinderen van verschillende leef- 9. tijden. De interesse in deze studie ging vooral uit naar Methionine, een aminozuur wat 10. behalve voor de opbouw van eiwitten in het lichaam ook wordt gebruikt voor andere 11. functies. Zo kan Methionine ook gebruikt worden om andere bouwstoffen te maken die 12. je lichaamscellen en bloedvaten beschermen tegen schade van buitenaf. Dit geldt voor 13. gezonde maar ook zieke individuen. Wij hebben in onze studie onderzoek gedaan naar 14. zieke zuigelingen, kinderen en adolescenten die gevoed werden met sondevoeding. 15. Wij toonden aan dat het met de leeftijd verschilt hoeveel Methionine in de darm wordt 16. opgenomen en wat de darm ermee doet. De zuigelingen namen veel meer Methionine 17. op in hun darmen dan adolescenten. Ook zagen we dat de zuigelingen de Methionine 18. hoofdzakelijk gebruikten voor het maken van eiwitten, terwijl de adolescenten veel 19. methionine verbruikten om mogelijk omgezet te worden naar de bouwstoffen die be- 20. schermen tegen schade van buitenaf. Tot slot zagen wij in deze studie dat de patiënten 21. van alle leeftijden onvoldoende Methionine kregen via de voeding. Dit is een duidelijke 22. aanwijzing dat de samenstelling van de voeding voor dit specifieke aminozuur mo- 23. menteel onvoldoende is. Met deze studie toonden we aan dat het per leeftijd verschilt 24. hoeveel aminozuren een ziek kind nodig. 25. 26. Hoofdstuk 6 27. In dit hoofdstuk hebben we gedurende één jaar de voedingsstoffen, en dan voorna- 28. melijk de aminozuren, van alle kinderen op de kinder IC die voeding via het infuus 29. ontvingen, onderzocht. In totaal zijn er 20 standaard aminozuren. Deze kunnen op 30. verschillende manieren worden ingedeeld, en één manier om dat te doen is door ze te 31. verdelen in essentiële (EAA) en niet-essentiële aminozuren (NEAA). Essentieel zijn de 8 32. aminozuren die niet door het lichaam zelf gemaakt kunnen worden, en niet-essentieel 33. zijn de overige 12 die wel door het lichaam zelf gemaakt kunnen worden. We hebben 34. gekeken naar de hoeveelheid van elke individueel aminozuur die aan de kinderen werd 35. toegediend via het infuus. Deze waarden hebben we vervolgens vergeleken met de 36. voedingsadviezen van het Instituut van Geneeskunde (IOM) zoals die gelden in de Ver- 37. neigde Staten. Verder hebben we gekeken naar de samenstelling van borstvoeding als 38. vergelijkingsmateriaal voor de voeding van zuigelingen. Echter, deze voedingsadviezen 39. zijn niet bedoeld voor kritisch zieke kinderen en de samenstelling van voeding dient

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1. anders te zijn als je ziek bent. Daarom hebben we gekeken hoeveel aminozuren er 2. ongeveer nodig zijn om de belangrijkste ontstekingseiwitten te maken. Hoewel dit een 3. theoretische manier is van het vergelijken van wat de kinderen mogelijk nodig zouden 4. hebben en wat ze daadwerkelijk binnen krijgen, is dit momenteel het best mogelijke 5. bewijs. Onze gegevens toonden aan dat momenteel, EAA evenals NEAA, niet worden 6. verstrekt zoals de behoefte bij de kinderen van verschillende leeftijden vermoedelijk is, 7. maar dat de samenstelling van voedingsinfusen is gebaseerd op gemak. Gemak voor 8. de fabrikant omdat deze rekening moet houden met hoe oplosbaar de aminozuren zijn 9. en hoe lang houdbaar de voeding is. Hierdoor worden een aantal aminozuren te weinig 10. of helemaal niet gegeven. Ook worden andere aminozuren in grote hoeveelheden ge- 11. geven, dit terwijl zij mogelijk giftig kunnen zijn als ze teveel gegeven worden. Hiermee 12. onderbouwden we ons vermoeden dat de aminozuren die zieke kinderen via het infuus 13. krijgen niet in de juiste hoeveelheid wordt gegeven en dat er voor ieder aminozuur 14. moet worden gekeken wat een kind op een bepaalde leeftijd nodig heeft als hij of zij 15. ziek is. 16. 17. Hoofdstuk 7 18. In de studie van hoofdstuk 7 hebben we gekeken of het geven van meer aminozuren, 19. via het voedingsinfuus, met of zonder het toedienen van insuline de verstoorde eiwit 20. balans in kritisch zieke adolescenten zou verbeteren. Normaal gesproken heeft het 21. enkel geven van meer aminozuren of eiwitten geen effect als het lichaam ook niet meer 22. energie krijgt om wat met die extra bouwstoffen te doen. Zoals uitgelegd in hoofdstuk 23. 2 is van insuline bekend dat het de stofwisseling van suiker maar ook van eiwitten en 24. vetten kan verbeteren. Daarom hebben we gekeken of we insuline kunnen gebruiken 25. om ook de stofwisseling van eiwitten weer te normaliseren. Dit hebben we wederom 26. gedaan met gebruik van stabiele isotopen infusen. De zieke adolescenten kregen 27. twee dagen hun voedingsinfuus, waarvan één met een normale hoeveelheid eiwit 28. en de andere met een dubbele hoeveelheid eiwit. Op beide dagen werd een infuus 29. van stabiele isotopen gegeven, waarvan een periode in combinatie met een insuline 30. infuus. De resultaten van de studie toonden aan dat er momenteel nog te weinig eiwit 31. aan zieke adolescenten wordt gegeven en dat ze een negatieve eiwit balans hadden, 32. terwijl het voedingsinfuus met de hoge eiwit een duidelijke verbetering gaf. Er waren 33. hoge doseringen insuline nodig voor het verbeteren van de bloedsuiker waarden, maar 34. insuline had geen positief effect op de eiwitbalans en verminderde de vet afbraak ook 35. niet. Dit betekent dat de adolescenten ongevoelig waren voor insuline, niet alleen 11 36. voor de suiker maar nog meer voor de eiwit en vetstofwisseling. Verder zagen we nog 37. dat het geven van meer eiwit via het voedingsinfuus de eiwit balans verbeterde maar 38. dat het ook ervoor zorgde dat er meer suiker werd gemaakt en dat er meer vet werd 39. afgebroken. Hierdoor kunnen we ondanks de verbetering op de eiwitbalans nog niet

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1. zeggen dat er in de toekomst zomaar meer eiwit gegeven moet worden aan zieke 2. kinderen, want het heeft ook een mogelijk nadelig effect op de insuline gevoeligheid en 3. dus de suiker en vetstofwisseling. 4. 5. Hoofdstuk 8 6. In dit hoofdstuk wordt een studie beschreven die is verricht in dezelfde postchirurgi- 7. sche zuigelingen en zieke adolescenten die in hoofdstuk 4 en 7 worden beschreven. 8. In deze twee afzonderlijke studies werd gebruikt gemaakt van stabiele isotopen. Het 9. ging onder andere om het aminozuur Leucine om naar de eiwit balans te kijken in 10. deze patiënten. Het aminozuur Leucine is een aminozuur wat ook gebruikt wordt om 11. eiwitten op te bouwen. Van dit stabiele isotoop van Leucine konden we in hoofdstuk 12. 8 gebruik maken om de aanmaaksnelheid te berekenen van één van de belangrijk- 13. ste eiwitten in ons lichaam; albumine. Zoals eerder uitgelegd zijn aminozuren, zoals 14. Leucine, bouwstenen voor eiwitten, zoals albumine. Als albumine wordt gemaakt 15. in het lichaam, dit gebeurd in de lever, dan wordt dus ook het stabiele isotoop van 16. Leucine ingebouwd in het eiwit albumine. Door dit isotoop van het aminozuur Leucine 17. te meten konden we de aanmaaksnelheid van albumine berekenen in de zuigelingen 18. en de adolescenten. Wij zagen dat de zowel de zuigelingen als de adolescenten een 19. lage bloedwaarde van albumine hadden, iets wat vaak voorkomt als je ernstig ziek 20. bent. Echter de aanmaaksnelheid was in alle patiënten hoog, dus de lage waarden 21. worden niet verklaard door een lage aanmaak. Omdat de studies waren gedaan in 22. dezelfde patiënten als in hoofdstuk 4 en 7 konden we meteen zien wat het effect van 23. het geven van suiker, eiwit en insuline was op de aanmaak snelheid van albumine. 24. Wij zagen in de zuigelingen geen verschillen tussen de veranderde suikerinfusen, en 25. ook bij de adolescenten veranderde de hoeveelheid eiwit in het voedingsinfuus en de 26. insuline niets aan de albumine aanmaak snelheid. Wij denken dan ook dat de aanmaak 27. snelheid van albumine, en mogelijk andere eiwitten, in de lever van zieke patiënten, 28. door andere signalen in het lichaam worden aangestuurd dan door voedingsstoffen. 29. 30. Hoofdstuk 9 31. Dit hoofdstuk bespreekt de belangrijkste resultaten van alle studies die verricht zijn in 32. dit proefschrift. Er wordt met behulp van de studies maar ook van de huidige literatuur 33. ingegaan op de belangrijkste conclusies. Deze zijn onderverdeeld in een gedeelte over 34. de suiker en insuline stofwisseling en een gedeelte over de aminozuren en eiwitten. Met 35. een slag om de arm concludeerden wij dat kinderen, ook jonge zuigelingen effectief 36. behandeld kunnen worden met insuline om hoge bloedsuiker waarden te behandelen, 37. maar dat er meer studies nodig zijn om het gunstige effect hiervan aan te tonen. Ook 38. denken wij dat er extra maatregelen nodig zijn, in het bijzonder bij jonge zuigelingen, 39. om lage bloedsuikers te voorkomen. Een voorbeeld van een dergelijke maategel is het

218 Samenvatting

1. verlagen van de insuline dosering zoals wij dat hebben gedaan op onze kinder IC. Een 2. ander voorbeeld is het verminderen van het glucose infuus direct na een operatie als 3. alternatief voor insuline. Wij toonden aan dat deze maatregel effectief en veilig was. In 4. het gedeelte over aminozuren en eiwitten concludeerden we dat zowel de kwantiteit 5. als ook de kwaliteit van de aminozuren in de voeding onvoldoende zijn. Daarnaast 6. toonden wij aan dat naast de leeftijd van het zieke kind ook de manier van voeden (via 7. infuus of sonde) vraagt om een aangepaste voeding. In de broodnodige toekomstige 8. studies dient dan ook rekening gehouden te worden met de leeftijd van het kind en de 9. manier van voeden. In het laatste gedeelte concludeerden wij dat het geven van insu- 10. line om de stofwisseling van vetten en eiwitten te verbeteren bij ernstig zieke kinderen 11. geen zin heeft. Tot slot geven we aan dat we denken dat er meer eiwit gegeven kan en 12. moet worden aan zieke kinderen om hun eiwit balans te verbeteren. Echter omdat een 13. hogere eiwitsamenstelling in de voeding mogelijk nadelige effecten heeft op de insuline 14. gevoeligheid en de suiker- en vetstofwisseling, zijn er meer studies nodig voordat we 15. dit als aanbeveling kunnen opnemen in voedingsadviezen en richtlijnen. Met hulp van 16. deze overwegingen worden enkele aanbevelingen gedaan als opzet en richting voor 17. toekomstig onderzoek. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 11 36. 37. 38. 39.

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Words of Thanks - Dankwoord

“I not only use all the brains that I have, but all that I can borrow” Woodrow Wilson, (1856-1924, 28th President of the USA)

Dankwoord

1. Hoewel dit voor mij de allerlaatste woorden zijn die ik aan “mijn boekje” toevoeg, zullen 2. velen van jullie dit als eerste lezen. Dit zegt genoeg over het belang van deze laatste 3. woorden. Zoals zo velen al voor mij hebben geschreven; ‘een proefschrift schrijven doe 4. je niet alleen’, en ook dat van mij heb ik te danken aan velen. 5. 6. Allereerst wil ik alle kinderen en hun ouders bedanken die hebben meegewerkt aan 7. mijn studies. 8. 9. Daarnaast, al is het alleen maar omdat ik niemand wil vergeten, wil ik iedereen bedanken 10. voor de hulp, geruststelling, felicitaties, reddingen, ideeën, bezoeken in Houston, tips, 11. etentjes, gezelligheid of gewoon simpelweg het tonen van interesse. Niet alleen tijdens 12. de soms frustrerende vooruitgang van de onderzoeken, maar ook tijdens de soms hec- 13. tische en vermoeiende perioden van de afgelopen jaren waren deze meer dan welkom. 14. 15. Een aantal mensen wil ik hierbij in het bijzonder bedanken. 16. 17. Prof. Dr. van Goudoever, beste Hans, ‘die kliniek geloof ik nu wel, maar nu moet je 18. je maar eens wetenschappelijk bewijzen’. Zo prikkelde je mijn ambitie op een NVK- 19. congres in 2005. Ruim een jaar later, lag er een onderzoeksplan, was ik welkom op één 20. van ‘s werelds meest gerenommeerde voedingsinstituten (even een nachtelijke dienst 21. email) en hadden we zelfs de financiering rond. Je ongekende optimisme, energie en 22. drang naar nieuwe ideeën zijn ontzettend inspirerend en je vertrek uit Rotterdam zal 23. dan ook een gemis zijn. Ik hoop dat we nog lang kunnen samenwerken en ik wil je 24. bedanken voor de kansen en mogelijkheden die je hebt gegeven om in ieder geval 25. deze eerste stap te maken met dit proefschrift onder jouw bezielende leiding. 26. 27. Dr. Joosten, beste Koen, voeding en intensive care, ik prijs me gelukkig dat jij mijn 28. co-promotor wilde zijn. Je rust, geduld en kritiek hebben me meermaals gedwongen 29. om te stoppen met haasten en te kijken waar ik nu werkelijk stond met mijn studies, 30. resultaten en nieuwe ideeën. Dat bracht dan op het goede moment de rust en het juiste 31. overzicht, wat dit proefschrift zeker ten goede is gekomen. Ik hoop nog veel meer 32. studies met je te kunnen doen en te zorgen dat voeding op de kinder intensive care de 33. aandacht krijgt die het verdient. 34. 35. Prof. Castillo, dear Leticia, it all started with you welcoming me to come and work with 36. you at the CNRC and the PICU at Texas Children’s Hospital. I cannot thank you enough 37. for that invitation and opportunity. Everyone who reads this thesis can see the influence 38. the work performed in Houston has on my research. Thank you for your patience to D 39. make me a better researcher and for forgiving me my impatience to become one.

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1. Dr. Coss-Bu, dear Jorge, ‘amigo’, thank you for all your help during the studies in 2. Houston. I miss all our conversations during the early hours of rounds or during the 3. clamps, on the world’s economic situation, American culture, cars and of course Eu- 4. ropean soccer and ‘la Naranja Mechanica’. Hopefully, Patrycja and I will see you soon. 5. 6. Verder wil ik de kleine commissie, Prof. Dr. van der Heijden, Prof. Dr. Tibboel en Prof. 7. Dr. Sauer bedanken voor hun deelname in de promotiecommissie en voor hun snelle 8. beoordeling van het manuscript. 9. 10. When you spend such an important part of your life in a strange and huge city such 11. as Houston, Texas, you get to learn the dearest, strangest and most hard-working 12. people. First, I want to thank everyone at the CNRC and the Texas Children’s Hospital 13. who helped me survive as a ‘junior’ researcher. My own research group, Debra, Jama, 14. Eddie, Danny, Manhong and Jean, thanks for all the help at work and making sure I 15. would finish the studies in time! Doug Burrin, your calm and sharp focus on research 16. and nutrition are a true inspiration, and I am proud that you will be part of my thesis 17. defense committee. Barbara and mr. Bob, thanks for all the Adobe margarita nights, 18. the cave-man experiences and getting us to eat more health food and run a bit faster. 19. Oh, and mr. Bob, thanks for keeping my lovely wife out of prison. The ‘people down- 20. stairs’, Shaji, Mahmoud, Trina, Lisa, Jean, and Gert; thanks for all the answers to my 21. questions, helping out with the samples, the Wednesday Chipotle experiences, the 22. Friday Gingerman beers, and of course the Breckenridge snowboard event. Monique 23. and Robert, you guys showed us that in contrast to our prejudice, Houston is actually 24. one large culinary happening. Thanks for all the dinners, drinks, conversations and 25. taking us to those really strange and unique spots Houston has to offer. And finally, 26. Marta, Afonso and Gert, you have become the definition of ‘love conquers all’. Thanks 27. for all of the above and so much more! The hours at the pool, the long days at work, 28. the Texas camping weekend, crawfish festivals, the Rodeo, the world championship 29. BBQ, all the beers, the talks and the never-ending Portuguese Noddy-experience. 30. What would Houston have been without you guys? 31. 32. Maar ook in Rotterdam is er natuurlijk een groep mensen die ik zeer dankbaar ben 33. voor de afgelopen jaren. Ineke, die me wegwijs heeft gemaakt in de bureaucratische 34. machine van de METC en de CCMO. Daniëlla, die al die tijd mijn life-line is geweest in 35. de communicatie en agenda van Hans. De mensen achter de schermen, of in deze dan 36. ‘op het lab’, zonder wie al dit werk nooit iets tastbaars was geworden; Henk, Gardi en 37. Kristien, hartelijk dank. En natuurlijk de onderzoekers die nu nog zitten te dromen over 38. het schrijven van hun dankwoord; Denise, Hester, Willemijn en Carlijn, hou vol, maar 39. geniet ook van het maken van jullie eigen boekje. Margriet, leuk om betrokken te zijn

224 Dankwoord

1. bij jouw eigen project. En natuurlijk, de mensen die me voor gingen; ‘the boys’ Frans 2. en Chris en natuurlijk Maaike. Dank voor alle borrels, humor en congresbelevenissen. 3. En bedenk, “We will always have Hawaii…!”. 4. 5. Al mijn collega’s en verpleegkundigen op de intensive care kindergeneeskunde, harte- 6. lijk dank voor alle interesse in de voortgang van mijn onderzoek en jullie medeleven als 7. ik er weer eens iets slechter uitzag dan in mijn vorige dienst. Nu dit is afgerond kan ik 8. me weer volledig storten op onze prachtige afdeling. Ik heb er ontzettend veel zin in. 9. 10. Mijn Paranimfen, Chris en Maurits. Chris, ik ben ongelofelijk blij dat je op zo’n bijzon- 11. dere dag naast me staat. Voelt meteen een stuk gemakkelijker, alsof Johan Cruijf zelf 12. op het schoolpleintje verschijnt om te coachen. Geweldig dat je de komende jaren nog 13. in Rotterdam bent zodat we nog maar veel kunnen samenwerken op allerlei fronten. 14. Maurits, held, weer moet je dan voor de zoveelste keer voor me opdraven in je mooie 15. pak. Je hebt nu vrijwel alle mijlpalen wel meegemaakt, het is dat we elkaar ‘pas’ 15 16. jaar kennen, anders zou je nog gespeecht hebben bij mijn zwemdiploma’s. Ik beloof 17. je dat dit de laatste keer is geweest. Laten we nu maar weer eens een kroeg induiken. 18. 19. Pap en mam, dit boekje is niet voor niets aan jullie opgedragen. Zonder jullie steun en 20. hulp zou ik niet zijn wat ik nu ben en niet staan waar ik nu sta. Dit geldt niet alleen voor 21. het afgelopen hectische jaar, maar zover als ik me kan herinneren. 22. 23. Er wordt vaak gezegd; “Promoveren is net als trouwen in je eentje”. Nou dat lijkt me dus 24. geen donder aan! Lieve Patriets, wat geweldig om ook dit samen gedaan te hebben. 25. Je vroeg me om in het dankwoord niet teveel kleffe woorden voor je te schrijven…… 26. Lief, wees niet bang, want er zijn geen woorden klef genoeg om duidelijk te maken hoe 27. belangrijk jij voor me bent. 28. Allerliefste Isa, wat heerlijk dat het afgelopen half jaar lekker aan je voorbij is gegaan. 29. Als je toch eens wist hoe ongelofelijk veel leuker jij het leven van mama en mij hebt 30. gemaakt tussen al dat werken en schrijven door. “Nog meer jij is fantastig toch..” 31. Jet, dank dat je me zo veel achter de laptop hebt weggehaald en in beweging hebt 32. gehouden…..vang!!.... 33. Sascha 34. 35. 36. 37. 38. D 39.

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1. Curriculum Vitae 2. 3. 4. 5. 6. Sascha Verbruggen was born on the 31st of March 1976 in Nijmegen. He finished high 7. school at the Dominicus College in Nijmegen in 1994 and obtained his medical degree 8. at the Maastricht University in Maastricht in 2000 (clear pass). During his medical 9. training he finished a research project at the Pediatric Intensive Care Unit of the Red 10. Cross Children’s Hospital in Cape Town, South Africa in 1998, and a clinical internship 11. pediatrics at the Witbank Hospital in Pretoria, South Africa in 2000. He worked as a 12. pediatric resident (ANIOS) at the department of pediatrics at the Orbis Medical Center 13. in Sittard in 2001 and in the St. Radboud Academic Hospital in Nijmegen in 2002. In 14. January 2003 he started as a pediatric resident (AIOS) at the Erasmus MC – Sophia 15. Children’s Hospital in Rotterdam (Prof. Dr. A.J. van der Heijden, Dr. M. de Hoog) and 16. at the Amphia hospital in Breda (Dr. A.A.P.H. Vaessen-Verberne). After he finished his 17. training as a pediatrician in July 2007, he started to work on his dissertation (promo- 18. tor Prof. Dr. J.B. van Goudoever, co-promotor Dr. K.F.M. Joosten) on ‘Nutritional and 19. metabolic support in critically ill children’ at the Children’s Nutrition Research Center, 20. Baylor College of Medicine, and the Texas Children’s Hospital in Houston, Texas, USA, 21. under supervision of Prof. Dr. L. Castillo. In September 2008 he started his clinical 22. work as pediatrician on the Pediatric Intensive Care Unit at the Erasmusm MC – Sophia 23. children’s hospital and continued his work on his dissertation. Sascha lives with his 24. wife Patrycja Puiman and their daughter Isa in Rotterdam. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. Cv 39.

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1. Affiliation of co-authors 2. 3. 4. 5. 6. Author Affiliation 7. 8. 9. Arrivillaga, Ana Department of Critical Care Medicine, Texas Children’s Hospi- 10. tal, Houston, TX, USA 11. Burrin, Douglas Children Nutrition Research Center, Baylor College of Medi- 12. cine, Houston, TX, USA 13. Castillo, Leticia Division of Critical Care Medicine, Children’s Medical Center 14. Dallas, University of Texas Southwestern, Dallas, TX, USA 15. Chacko, Shaji Children Nutrition Research Center, Baylor College of Medi- 16. cine, Houston, TX, USA 17. Coss-Bu, Jorge Department of Critical Care Medicine, Texas Children’s Hospi- 18. tal, Houston, TX, USA 19. de Betue, Carlijn Department of Pediatric Surgery, Erasmus MC – Sophia Chil- 20. dren’s Hospital, Rotterdam, The Netherlands 21. Gordon, William E. Department of Critical Care Medicine, Texas Children’s Hospi- 22. tal, Houston, TX, USA 23. Hsu, Jean Children Nutrition Research Center, Baylor College of Medi- 24. cine, Houston, TX, USA 25. Joosten, Koen Department of Pediatric Critical Care Medicine, Erasmus MC 26. – Sophia Children’s Hospital, Rotterdam, The Netherlands 27. Landzaat, Lonneke Department of Pediatric Critical Care Medicine, Erasmus MC 28. – Sophia Children’s Hospital, Rotterdam, The Netherlands 29. Reiss, Irwin Department of Pediatric Surgery, Erasmus MC – Sophia Chil- 30. dren’s Hospital, Rotterdam, The Netherlands 31. Schierbeek, Henk Department of Pediatrics, Erasmus MC – Sophia Children’s 32. Hospital, Rotterdam, The Netherlands 33. Sy, Jama Department of Critical Care Medicine, Texas Children’s Hospi- 34. tal, Houston, TX, USA 35. van Adrichem, Leon Department of Plastic surgery, Erasmus MC – Sophia Chil- 36. dren’s Hospital, Rotterdam, The Netherlands 37. van Goudoever, Hans Department of Pediatrics, Erasmus MC – Sophia Children’s 38. Hospital, Rotterdam, The Netherlands A 39.

229 Affiliation of co-authors

1. Department of Pediatrics, Academic Medical Center – Emma 2. Children’s Hospital, Amsterdam, The Netherlands 3. Department of Pediatrics, VU University Medical Center, Am- 4. sterdam, The Netherlands 5. Verhoeven, Jennifer Department of Pediatric Critical Care Medicine, Erasmus MC 6. – Sophia Children’s Hospital, Rotterdam, The Netherlands 7. Wu, Manhong Stanford University, San Francisco Bay Area, CA, USA 8. Zurakowski, David Departments of Anesthesia and Surgery, Children’s Hospital 9. Boston, Harvard Medical School, Boston, MA, USA 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39.

230 1. List of Publications 2. 3. 4. 5. 6. - Verbruggen SC, Wijnen RM, van den Berg P. Megacystis-microcolon-intestinal 7. hypoperistalsis syndrome. J Matern Fetal Neonatal Med. 2004;16(2):140-1. 8. - Verbruggen SC, Corel LJ, Tiddens HA, Joosten KF, de Hoog M. Fataal astma op de 9. kinderleeftijd te voorkomen door herkenning van risicofactoren en presentatiewijze. 10. Ned Tijdschr Geneeskd. 2006;150(5):225-9 11. - Verbruggen SCAT, Tiddens HAWM, Hanff LM, de Hoog M. Te veel salbutamol, te 12. weinig inhalatiesteroïden. Pharm Weekbl 2006;141(42):1416-8. 13. - Verbruggen SC, Catsman CE, Naghib S, Lequin MH, van der Lely N, Buysse 14. CMP. Acute Disseminerende EncephaloMyelitis (ADEM). Ned Tijdschr Geneeskd. 15. 2006;150(20):1134-8 16. - Verbruggen SC, Joosten KF, Castillo L, van Goudoever JB. Insulin therapy in the 17. pediatric intensive care unit. Clin Nutr. 2007;26(6):677-90. 18. - Joosten K, Verbruggen SC, Verhoeven JJ. Glycaemic control in paediatric critical 19. care. Lancet. 2009;373(9673):1423-4 20. - Verbruggen S, Sy J, Gordon WE, Hsu JW, Wu M, Chacko SK, Zurakowski D, Burrin 21. DG, Castillo L. Ontogeny of methionine utilization and splanchnic uptake in criti- 22. cally ill children. Am J Physiol Endocrinol Metab. 2009;297; E1046-E1055 23. - Verbruggen S, Sy J, Arrivillaga A, Joosten K, van Goudoever J, Castillo L. Parenteral 24. Amino Acid Intakes in Critically Ill Children: A Matter of Convenience? J Parenter 25. Enteral Nutr. 2010;34(3):329-40. 26. - Verbruggen S, Coss-Bu J, Wu M, Schierbeek H, Joosten K, van Goudoever J, 27. Castillo L. Current recommended parenteral protein intakes do not support protein 28. synthesis in critically ill septic, insulin resistant children with tight glucose control. 29. Provisionally accepted Crit Care Med 30. - Verbruggen S, de Betue C, Schierbeek H, Chacko S, Verhoeven J, van Goudoever 31. J, Joosten K. Reducing Glucose Intake Safely Prevents Hyperglycemia In Post- 32. Surgical Children. Submitted 33. - Verbruggen S, Schierbeek H, Coss-Bu J, Joosten K, Castillo L, van Goudoever J. 34. Albumin synthesis rates in post-surgical infants and septic adolescents; influence 35. of amino acids, energy, and insulin. Submitted 36. - Verbruggen S, Landzaat L, Reiss I, van Goudoever J, Joosten K. Efficacy and safety 37. of a tight glucose control protocol in critically ill term infants. Submitted 38. P 39.

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1. PhD Portfolio 2. 3. 4. 5. 6. Summary of PhD training and teaching 7. Name PhD student: S.C.A.T. Verbruggen, MD. PhD period: 2007 – 2010 8. Erasmus MC Department: Pediatric Intensive Care Promotor: Prof. J.B. van Goudoever, MD. PhD. 9. Research School: Erasmus MC Supervisor: K.F.M. Joosten, MD. PhD. 10. 1. PhD training 11. Year Workload 12. ECTS 13. General courses 14. - Clinical Investigation Course; Baylor College of Medicine, Houston, USA 2007 2.0 15. - Classical Methods for Data Analysis; NIHES, Erasmus MC 2009 4.0 16. Specific courses 17. - Isotope Tracers in Metabolic Research; University of Arkansas for 2007 2.0 18. Medical Sciences, Little Rock, USA 19. Seminars 20. - Research seminars, Children’s Nutrition Research Center, Baylor College 2007- 2008 1.0 21. of Medicine, Houston, USA 22. - Research Fellow Symposium, Baylor College of Medicine, Houston, USA 2007- 2008 0.8 23. - Research bespreking kindergeneeskunde, Erasmus MC 2008 - 2010 0.6 24. - Research bespreking ‘Moeder en Kind Centrum’, Erasmus MC 2008 - 2010 0.6 25. International conferences 26. - American Society of Nephrology, San Francisco, USA 2007 1.0 27. - Society of Critical Care Medicine, Honolulu, USA 2008 1.0 28. - Federation of American Societies for Experimental Biology, San Diego, 2008 1.0 29. USA 30. - Society of Pediatric Research, Honolulu, USA 2008 1.0 31. - European Society of Pediatric Research, Nice, France 2008 1.0 32. - Federation of American Societies for Experimental Biology, New Orleans, 2009 1.0 33. USA 34. - European Society of Pediatric Research, Hamburg, Germany 2009 1.0 35. - Benelux Association of Stable Isotope Scientist, Arnhem, The 2010 0.4 36. ­Netherlands 37. - European Society of Pediatric Research, Copenhagen, Denmark 2010 1.0 38. P 39.

233 PhD Portfolio

1. Poster Presentations 2. - Methionine Cycle Kinetics and Arginine Supplementation in Endothelial 2007 1.0 3. Dysfunction of ESRD, American Society of Nephrology, San Francisco, 4. USA 5. - Effect of Arginine or Folic Acid Supplementation on Cysteine ­kinetics 2007 1.0 6. in End-Stage Renal Disease, American Society of Nephrology, 7. San Francisco, USA - Energy Expenditure in Patients with End-Stage Renal Disease, American 2007 1.0 8. Society of Nephrology, San Francisco, USA 9. - Methionine Splanchnic Uptake in Critically Ill Infants, Society of Critical 2008 1.0 10. Care Medicine, Honolulu, USA 11. - Insulin Resistance and Protein Metabolism in Pediatric Critical Illness, 2008 1.0 12. Society of Critical Care Medicine, Honolulu, USA 13. - Parenteral Amino Acid Intakes in Critically Ill Children, Federation of 2008 1.0 14. American Societies for Experimental Biology, San Diego, USA 15. - Methionine Splanchnic Uptake is increased in Critically Ill Children, 2008 1.0 16. Federation of American Societies for Experimental Biology, San Diego, 17. USA 18. - Parenteral Amino Acid Intakes in Critically Ill Children: A Matter of 2008 1.0 19. Convenience?, Federation of American Societies for Experimental 20. Biology, San Diego, USA 21. - Arginine supplementation improves insulin resistance in Obese 2009 1.0 22. Adolescents, Federation of American Societies for Experimental Biology, 23. New Orleans, USA 24. Oral Presentations 25. - Insulin Resistance and Protein Metabolism in Critically Ill Children, 2008 1.4 26. European Society of Pediatric Research, Nice, France 27. - Albumin Synthesis Rates in Critically Ill Adolescents; Effect of Insulin 2009 1.4 28. and Protein Intake, European Society of Pediatric Research, Hamburg, 29. Germany 30. - Albumin Synthesis Rates in Critically Ill Adolescents; Effect of Insulin and 2010 1.4 31. Protein Intake, Benelux Association of Stable Isotope Scientist, Arnhem, 32. The Netherlands 33. - Tight glucose regimen in critically ill infants, European Society of 2010 1.4 34. Pediatric Research, Copenhagen, Denmark 35. 36. 37. 38. 39.

234 PhD Portfolio

1. 2. Teaching activities 2. Year Workload 3. ECTS 4. Lecturing 5. - Education for PICU nursing staff; ‘Tight Glucose regimen on the PICU’, 2008 1.5 6. Erasmus MC 7. - Pediatric Intensive Care Fellow Course, Nijmegen; ‘tight glucose control’ 2009 0.8 8. on our PICU’s, what’s the evidence?’ 9. Supervising Master’s theses 10. - Marianne Koenraads, medical student, University of Maastricht 2009 1.0 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. P 39.

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