Thesis-Hayden White

EVALUATION OF A KETOGENIC FORMULATION

FOR TREATING PATIENTS WITH ACUTE BRAIN

INJURY

Hayden Thomas Wesley White

MBBCH MMED (Wits) FCICM FRACP

A thesis submitted for the degree of Doctor of Philosophy at

The University of Queensland in 2018

Faculty of Medicine Evaluation of a Ketogenic Formulation for Treating Patients with Acute Brain Injury

Abstract

Acute brain injury (ABI), including cerebral vascular accidents (CVA) and traumatic brain injury (TBI), are relatively common diseases which may lead to death, significant morbidity and place a heaving burden on health care systems globally, both in suffering and cost. Evidence has emerged from basic science research that ketones (beta-hydroxybutyrate and acetoacetate) which are water-soluble molecules produced by the liver during times of starvation, may have neuroprotective effects. It is therefore theoretically possible that increasing plasma ketone concentrations via intravenous or oral supplementation, may lead to improved outcomes following ABI.

This thesis documents a systematic investigation into the possibility that ketone supplementation may lead to benefits in patients with a variety of chronic and acute neurological conditions. While the main emphasis is on ABI, other conditions where ketones may be useful are also reviewed. Beginning with a review of ketones, their potential mechanisms of action and likely neuroprotective roles in disease, this thesis examines the effects of a novel hypertonic intravenous ketone solution on plasma, cerebrospinal fluid (CSF) and brain ketone concentrations in animals, then looks at the hurdles in producing such an intravenous solution as opposed to enteral formulations for humans, before investigating baseline ketone concentrations in patients with ABI and finally culminating in an interventional study where a modified enteral ketone supplement is administered to patients with ABI to determine whether adequate plasma ketone concentrations are attainable.

The animal study performed in rats involved the administration of hypertonic saline/ketone solutions of varying concentrations to anaesthetised rats. The results demonstrated that both CSF and beta-hydroxybutyrate (BHB) concentrations increased over time (p<0.0001) and that this was dependent on the BHB concentration in the solution (p<0.0001). Measurement of BHB concentrations in brain tissue noted a dose dependent increase compared to control with the highest concentration almost double 0.15 mmol/l versus 0.28 mmol/l. Significantly, the infusions did not lead to a metabolic acidosis which has been a concern amongst researchers.

These findings led to an attempt to produce an intravenous solution for administration to humans. However, the cost of producing this formulation proved prohibitive. Another source i Evaluation of a Ketogenic Formulation for Treating Patients with Acute Brain Injury of ketones was therefore sort. While this was underway, as baseline data was lacking, we undertook a study to measure ketone concentrations in patients with ABI.

The subsequent study recruited 38 patients with ABI and followed them for up to 7 days. Both plasma and CSF (where possible) concentrations of BHB and Acetoacetate were measured on a daily basis. 22 patients completed the full 7 days of observations, 7 CVA, 7 subarachnoid haemorrhage (SAH) and 8 TBI. During the study period, BHB concentrations increased initially but normalized by day 3 while acetoacetate (AcAc) concentrations remained within the normal range. There was a weak correlation between blood and CSF BHB (Spearman’s rho = 0.62, P=0.054). It was therefore clear that as blood ketone concentrations remained low, an external source would be required to increase blood ketone concentrations to clinically relevant concentrations.

Therefore, a study investigating enteral based supplementation with the view to increasing ketone concentrations in ABI patients was designed. The study was to look at the feasibility of administering a ketogenic enteral formulation to patients with ABI over a 6 day period. Outcomes included ketone concentrations in blood and CSF (where possible) and monitoring for adverse events resulting from the ketone administration. It was determined that the commercially available product Ketocal (Nutricia) would be the most suitable enteral supplement. However, as this product was lacking in sufficient protein, a protein supplement was added. The study was entitled “Induction of ketogenesis in patients with acute brain injury via oral administration of a ketogenic feed” or KABI and intended to recruit 20 patients.

While the KABI study was under development, 2 further reviews were undertaken in order to further examine the current literature on cerebral nutrition and the place of ketogenesis in this process. The first reviewed the place of metabolic substrates other than glucose on cerebral energetics. These included ketones, lactate, non-esterified fatty acids, branch- chain amino acids, and TCA cycle intermediates including triheptanoin. It was noted that while glucose is the main substrate for the brain in health, other metabolic substrates may prove beneficial in the injured brain. While ketones have received the most in-depth study and appear to be an excellent substitute for glucose, exploration into the field of cerebral energetics is in its infancy and further research is necessary.

The second was a systematic review of publications focusing on the use of ketones in both acute and chronic neurological disorders in adults (excluding chronic poorly controlled epilepsy). As expected, the data is very limited, consisting of mainly case series and small

ii Evaluation of a Ketogenic Formulation for Treating Patients with Acute Brain Injury randomised controlled trials. Study subjects included Alzheimer’s disease, severe refractory status epilepticus, intracranial neoplasms, TBI and Parkinson’s disease. Although outcomes were largely positive, the poor quality of the evidence prohibits firm recommendations other than that further research is necessary to explore the potential benefits of ketones in a number of neurological conditions.

The KABI study was subsequently undertaken and completed. We recruited 20 patients, 5 females and 15 males, 3 with stroke, 2 with subarachnoid haemorrhage and 15 with traumatic brain injury. We were able to demonstrate a significant increase in both plasma beta-hydroxybutyrate and acetoacetate to 0.61 ± 0.53 mmol/l (p =0.0005) and 0.52 ± 0.40 mmol/l (p<0.0001) over the 6 day period. The total daily Ketocal® caloric intake was positively correlated with plasma beta-hydroxybutyrate concentrations (p=0.0011). The feeds were well tolerated in 19 out of the 20 patients with no clinically significant changes in acid/base status over the 6 days with pH remaining within normal range. One patient developed a metabolic acidosis and feeds were ceased. We concluded that in patients with acute brain injury, although an enterally administered ketogenic formulation was well tolerated more work was required to determine the optimal concentration of ketones necessary to impact on cellular energetics and the best means of inducing ketosis.

One of the main issues limiting progress in investigating ketones as a therapeutic tool is the difficulty in attaining high plasma ketone concentrations rapidly and consistently using the current enteral approach. The ketone monoester (R)-3-hydroxybutyl (R)-3-hydroxybutyrate, may provide a solution to this problem. The thesis concludes by highlighting gaps in knowledge, challenges and avenues for future research.

iii Evaluation of a Ketogenic Formulation for Treating Patients with Acute Brain Injury

Declaration by author

This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis.

I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award.

I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School.

I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis and have sought permission from co- authors for any jointly authored works included in the thesis.

iv Evaluation of a Ketogenic Formulation for Treating Patients with Acute Brain Injury

Publication during candidature

Peer Reviewed Manuscripts:

1. White H, Venkatesh B. Clinical review: ketones and brain injury. Crit Care. 2011; 15:219

2. White H, Sosnowski K, Bird R, Jones M and Solano C. The utility of thromboelastography in monitoring low molecular weight heparin therapy in the coronary care unit. Blood Coagul Fibrinolysis. 2012; 23:304-10.

3. Reeves A, White H, Sosnowski K, Leveritt M, Desbrow B and Jones M. Multidisciplinary evaluation of a critical care enteral feeding algorithm. Nutrition and Dietetics. 2012; 69:242-9

4. Ming Y, Wysocki P, Hogan J and White H. An audit of characteristics and outcomes in adult intensive care patients following tracheostomy. IJCCM. 2012;16;100-105

5. White H, Venkatesh B, Jones M, Worrall S, Chuah T, Ordonez J. Effect of a hypertonic balanced ketone solution on plasma, CSF and brain beta-hydroxybutyrate levels and acid-base status. Intensive Care Med. 2013 Apr;39(4):727-33.

6. Reeves A, White H, Sosnowski K, Tran K, Jones M, Palmer. Energy and protein intakes of hospitalised patients with acute respiratory failure receiving non-invasive ventilation. M.Clin Nutr. 2014; 33:1068-73

7. Sosnowski K, Lin F, Mitchell M, White H. Early rehabilitation in the intensive care unit: an integrative literature review. Aust Crit Care 2015; 28:216-25

8. Aroney N, Ure S, White H, Sane S. Recurrent undifferentiated shock: Idiopathic systemic capillary leak syndrome. Clin Case Rep. 2015; 3:527-30

9. Sane S, Saulova A, McLaren R, White H. A fatal case of primary dengue infection with myocarditis and cerebral oedema. Australas Med J. 2015; 8:299-303

v Evaluation of a Ketogenic Formulation for Treating Patients with Acute Brain Injury

10. Bandeshe H, Boots R, Dulhunty J, Dunlop R, Holley A, Jarrett P, Gomersall CD, Lipman J, Lo T, O'Donoghue S, Paratz J, Paterson D, Roberts JA, Starr T, Stephens D, Stuart J, Thomas J, Udy A, White H. Is inhaled prophylactic heparin useful for prevention and management of pneumonia in ventilated ICU patients?: The IPHIVAP investigators of the ANZICS CTG. J Crit Care. 2016; 34:95-102

11. White H, Bird R, Sosnowski K, Jones M. An in vitro analysis of the effect of acidosis on coagulation in chronic disease states – a thromboelastograph study. Clin Med. 2016; 16: 230-4

12. White H, Venkatesh B, Jones M & Fuentes H. Serial changes in plasma ketone concentrations in patients with acute brain injury. Neurological Research 2017; 39:1- 6

13. White H, Venkatesh K, Venkatesh B. Systematic Review of the Use of Ketones in the Management of Acute and Chronic Neurological Disorders. J Neurol Neurosci. 2017, 8:2.

14. Hawkins A, Jackson R, White H, Vardesh D. SGLT-2 Inhibitor induced euglycemic ketoacidosis in acute surgical patients. J Case Rep Images Surg 2017; 3:41-46

15. Sosnowski K, Mitchell M, White H, Morrison L, Sharratt J, Lin F. A feasibility study of randomised controlled trial to examine the impact of the ABCDE bundle on quality of life in ICU survivors. Pilot and Feasibility studies. 2018; 4:32

Book Chapters

16. Reeves A, Kiss C, White H, Sosnowski K, Josephson C. Aid to Enteral Feeding in Critical Care: Algorithm. Diet and Nutrition in Critical Care. Springer Science 2014

17. White H, Venkatesh B. Traumatic Brain Injury. Oxford Textbook of Neurocritical Care Ed. Smith M, Oxford, Jan 2016

vi Evaluation of a Ketogenic Formulation for Treating Patients with Acute Brain Injury

18. White H, Kruger P, Venkatesh B. Novel metabolic substrates for feeding the injured brain In: Annual update in Intensive Care and Emergency Medicine 2017 Yearbook of Intensive Care and Emergency Medicine – Edited by J.L. Vincent, Springer-Verlag, 2017 pp 329 - 341

Publications included in this thesis

Each of these manuscripts and the studies they reflect were primarily conceived, driven and written up by me. I assumed primary responsibility for them all and the individual contributions of co-authors are specifically acknowledged in the citation section of each chapter of this thesis.

Peer Reviewed Manuscripts:

19. White H, Venkatesh B. Clinical review: ketones and brain injury. Crit Care. 2011; 15:219

20. White H, Venkatesh B, Jones M, Worrall S, Chuah T, Ordonez J. Effect of a hypertonic balanced ketone solution on plasma, CSF and brain beta-hydroxybutyrate levels and acid-base status. Intensive Care Med. 2013 Apr; 39(4):727-33 – incorporated into Chapter 2

21. White H, Venkatesh B, Jones M & Fuentes H. Serial changes in plasma ketone concentrations in patients with acute brain injury. Neurological Research 2017; 39:1- 6 – incorporated into Chapter 4

22. White H, Kruger P, Venkatesh B. Novel metabolic substrates for feeding the injured brain In: Annual update in Intensive Care and Emergency Medicine 2017– Edited by J.L. Vincent, Springer-Verlag, 2017 pp 329-341 – incorporated into Chapter 5

23. White H, Venkatesh K, Venkatesh B. Systematic Review of the Use of Ketones in the Management of Acute and Chronic Neurological Disorders. J Neurol Neurosci. 2017, 8:2. doi:10.21767/2171-6625.1000188 – incorporated into Chapter 6.

vii Evaluation of a Ketogenic Formulation for Treating Patients with Acute Brain Injury

Contributions by others to the thesis

This research could not have been completed without the support and assistance I have received from so many people over the past few years

I would like to acknowledge particularly:

The research co-ordinator teams at both the Princess Alexandra Hospital and Logan Hospital Intensive Care Units, Brisbane. They have assisted in screening, managing and data collection and include; Kellie Sosnowski, Lyn Morrison, Jo Sutton and Jason Myer.

Dr James Walsham who oversaw the recruitment and management of patients at the Princess Alexandra Hospital Intensive Care Unit.

Goce Dimeski from the Queensland Health Pathology Services who assisted with sample collation and storage.

Tom Robertson and Jenny Ordenez from the Therapeutics Research Center (University of Queensland) helped formulate the hypertonic ketone solution for the animal studies.

Dr Simon Worrall from University of Queensland Faculty of Science who process the brain specimens.

Dr Mark Jones for providing the statistical support and expertise throughout this project.

Dr Peter Kruger and Dr Jeremy Cohen for their participating in the milestone committees and ongoing support.

I would like to acknowledge the help and assistance from my Supervisor Professor Bala Venkatesh who provided the drive and focus for this work.

viii Evaluation of a Ketogenic Formulation for Treating Patients with Acute Brain Injury

Statement of parts of the thesis submitted to qualify for the award of another degree

None

ix Evaluation of a Ketogenic Formulation for Treating Patients with Acute Brain Injury

Research Involving Human or Animal Subjects

Project Title: Comparison of varying concentrations of sodium betahydroxybutyrate on metabolism in the rat UQ University Animal Ethics Committee Ref number: PAH/029/09/UQ

Project Title: Blood and CSF ketone levels following acute brain injury Metro South HREC Ref number: HREC/13/QPAH/150

Project Title: Inducing Ketogenesis in patients with acute brain injury via oral administration of a ketogenic feed Metro South HREC Ref number: HREC/16/QPAH/30

x Evaluation of a Ketogenic Formulation for Treating Patients with Acute Brain Injury

Acknowledgements

I would like to thank my wife Eleonore, and children Matthew, Julia, Timothy and Elisabeth for putting up with me during this long and arduous journey. This would not have been possible without their unceasing love and support.

I would also like to offer my gratitude to my colleagues, research and clinical staff at the Logan and Princess Alexandra Intensive Care Units for their ongoing support and dedication.

xi Evaluation of a Ketogenic Formulation for Treating Patients with Acute Brain Injury

Financial support

This work was supported in part by the following research grant:

Intensive care foundation grant 2010 - $20000- Development of a new hyperosmolar solution for use in neurotrauma (A/Prof Hayden White, Prof Bala Venkatesh, Dr Teong Chuah)

xii Evaluation of a Ketogenic Formulation for Treating Patients with Acute Brain Injury

Keywords

Acetoacetate, Beta-hydroxybutyrate, Brain injury, Critical Care, Ketone, Ketone Bodies, Neuroprotection.

Australian and New Zealand Standard Research Classifications (ANZSRC)

110904 Neurology and Neuromuscular Diseases (40%)

320503 Clinical Pharmacology and Therapeutics (40%)

111199 Nutrition and Dietetics not elsewhere classified (20%)

Fields of Research (FoR) Classification

1103 Clinical Sciences (30%)

1115 Pharmacology and Pharmaceutical Sciences (30%)

1109 Neurosciences (40%)

xiii Evaluation of a Ketogenic Formulation for Treating Patients with Acute Brain Injury

Table of Contents

Abstract ...... i

Declaration by author ...... iv

Publication during candidature ...... v

Peer Reviewed Manuscripts: ...... v

Publications included in this thesis ...... vii

Contributions by others to the thesis ...... viii

Statement of parts of the thesis submitted to qualify for the award of another degree ...... ix

Acknowledgements ...... xi

Keywords ...... xiii

Australian and New Zealand Standard Research Classifications (ANZSRC) ...... xiii

Fields of Research (FoR) Classification ...... xiii

Table of Contents...... xiv

Appendices ...... xviii

List of Figures ...... xix

List of Tables ...... xx

List of Abbreviations ...... xxi

Preamble – Scope of Thesis ...... xxiii

Chapter 1: The Role of Ketones in Brain Injury ...... 1

1.1. Synopsis and Citation ...... 1

1.2. Introduction ...... 2

1.3. Ketones, metabolism and the brain ...... 2

xiv Evaluation of a Ketogenic Formulation for Treating Patients with Acute Brain Injury

1.4. Current state of research - Changes in ketone metabolism during brain injury ...... 9

1.5. Potential clinical applications for ketone solutions – traumatic brain injury ...... 18

1.6. Conclusion ...... 19

Chapter 2: Prospective Evaluation of a Hypertonic Balanced Ketone Solution for use in Neuroprotective Strategies ...... 20

2.1 Synopsis and Citation ...... 20

2.2 Introduction ...... 23

2.3 Materials and Methods ...... 24

2.4 Statistical analysis ...... 27

2.5 Results ...... 27

2.6 Discussion ...... 30

2.7 Conclusion ...... 34

Chapter 3: Proposed Project: Hypertonic Ketone Infusion for the Management of Acute Brain Injury ...... 35

3.1 Synopsis and Citation ...... 35

3.2 Development of a hypertonic ketone solution for humans with brain injury ...... 36

3.3 Proposed Project Plan ...... 38

3.4 Challenges to PhD in its current design and proposed changes to PhD title and project focus ...... 41

Chapter 4: Serial changes in plasma ketone concentrations in patients with acute brain injury ...... 43

4.1Synopsis and Citation ...... 43

4.2 Introduction ...... 45

4.3 Materials and Methods ...... 46 xv Evaluation of a Ketogenic Formulation for Treating Patients with Acute Brain Injury

4.4 Results ...... 47

4.5 Discussion ...... 50

Chapter 5: Novel metabolic substrates for feeding the injured brain ...... 54

5.1 Synopsis and citation ...... 54

5.2 Introduction ...... 55

5.3 Cerebral Metabolism ...... 56

5.4 Potential substrates capable of maintaining cerebral energetics ...... 59

5.5 Challenges in developing new fuel substrates for the injured brain ...... 63

5.6 Conclusion ...... 64

Chapter 6: Systematic Review of the use of ketones in the management of acute and chronic neurological disorders ...... 66

6.1 Synopsis and citation ...... 66

6.2 Introduction ...... 68

6.3 Methods ...... 69

6.4 Results...... 70

6.5 Discussion ...... 78

6.6 Conclusion ...... 81

Chapter 7: Inducing ketogenesis via an enteral formulation in patients with acute brain injury: A phase II study ...... 82

7.1 Synopsis and citation ...... 82

7.2 Introduction ...... 84

7.3 Methods ...... 85

7.4 Results ...... 88

7.5 Discussion ...... 96 xvi Evaluation of a Ketogenic Formulation for Treating Patients with Acute Brain Injury

7.6 Conclusion ...... 98

Chapter 8: Conclusions and Future Directions ...... 99

Reference List ...... 106

Appendices ...... 132

xvii Evaluation of a Ketogenic Formulation for Treating Patients with Acute Brain Injury

Appendices

Appendix A: Contributions for publications in which I was not the sole author………………………131

Appendix B: Animal ethics approval certificate ...... 135

Appendix C: Human Research Ethics Committee Approval: Blood and CSF ketone

levels following acute brain injury ...... 136

Appendix D: Human Research Ethics Committee Approval: Inducing Ketogenesis in patients with Acute Brain Injury via oral administration of a ketogenic feed ...... 139

xviii Evaluation of a Ketogenic Formulation for Treating Patients with Acute Brain Injury

List of Figures

Figure 1.1 Chemical structure of ketone bodies ...... 2

Figure 1.2 Fate of circulating ketones during fasting ...... 3

Figure 1.3 Potential neuroprotective mechanisms of ketones ...... 8

Figure 2.1 Plasma beta-hydroxybutyrate profiles ...... 30

Figure 2.2 Plasma and CSF BHB concentrations with different fluids at various time points ...... 31

Figure 5.1 Ketones as an alternative cellular fuel source ...... 56

Figure 5.2 Sequence of cerebral metabolic changes following traumatic brain injury ...... 57

Figure 6.1 Evaluation of papers for inclusion into this review ...... 70

Figure 7.1 Distribution of acetoacetate by day ...... 89

Figure 7.2 Distribution of BHB by day ...... 90

Figure 7.3 Distribution of glucose by day ...... 91

Figure 7.4 Comparison of daily plasma BHB concentration with total daily intravenous catecholamine infusion (mg/day) ...... 91

Figure 7.5 Comparison of daily plasma BHB concentration with total daily intravenous insulin infusion (units/day) ...... 92

Figure 7.6 Daily plasma BHB concentration compared with total daily calories from Ketocal (kcal/day) ...... 92

Figure 7.7 Distribution of pH by day ...... 93

Figure 7.8 Daily ICH index compared with daily CPP index ...... 95

xix Evaluation of a Ketogenic Formulation for Treating Patients with Acute Brain Injury

List of Tables

Table 1.1 Animal and basic science studies on effects of ketone administration on brain injury .... 13

Table 1.2 Human studies investigating effects of exogenous ketone administration ...... 16

Table 2.1 Baseline data (mean +/- SD) ...... 24

Table 2.2 Formulations of intravenous solutions ...... 25

Table 2.3 BHB data for 3 time points (mean +/- SD) ...... 28

Table 2.4 Acid base and electrolyte data for 3 time points (mean +/- SD) ...... 29

Table 4.1.Demographic data of patients with acute brain injury ...... 48

Table 4.2.Clinical and biochemical observations measured daily over trial period ...... 48

Table 4.3 Trends over time based on mixed models analysis ...... 49

Table 4.4 Correlations between parameters based on mixed models ...... 50

Table 5.1 Potential substrates for enhancing cerebral energy supply ...... 55

Table 6.1 Summary of studies selected for inclusion into this review ...... 72

Table 7.1 Demographic data of patients with acute brain injury (mean +/- SD) ...... 88

Table 7.2 Plasma ketones and glucose profiles over trial period ...... 89

Table 7.3 Clinical and biochemical observations measured daily over the trial period ...... 94

Table 7.4 Adverse events ...... 95

xx Evaluation of a Ketogenic Formulation for Treating Patients with Acute Brain Injury

List of Abbreviations

AAA aromatic amino acids AA amino acids ABI acute brain injury ABG arterial blood gas AcAc acetoacetate acetyl CoA coenzyme A AD Alzheimer’s disease AKBR arterial ketone body ratio Ag anion gap ALT alanine transferase ANZICS Australia New Zealand Intensive Care Society ANZICS CTG Australia New Zealand Intensive Care Society Clinical Trials Group APO-4 apolipoprotein-E4 AST aspartate transferase ATP adenosine triphosphate BBB: blood-brain barrier BCCA branch-Chain amino acids BE base excess BHB β-hydroxybutyrate BHD β-hydroxybutyrate dehydrogenase CBF cerebral blood flow CSF cerebrospinal fluid CVA cerebrovascular accident DKA diabetic ketoacidosis FFA free fatty acids GABA gamma-aminobutyric acid GBM Glioblastoma Multiforme GCS Glasgow coma scale HL hypertonic lactate HREC Human Research Ethics Committee HTS hypertonic saline ICP intracranial pressure xxi Evaluation of a Ketogenic Formulation for Treating Patients with Acute Brain Injury

KB ketone bodies LGIT low glycemic index treatment MAD modified Atkins diet MCA middle cerebral artery MCI mild cognitive impairment MCT monocarboxylic acid transporter MRI Magnetic resonance imaging MRS magnetic resonance spectroscopy Na sodium NaAcAc sodium acetoacetate NaBHB sodium β-hydroxybutyrate NaCl sodium chloride NAD nicotinamide adenine dinucleotide NADH nicotinamide adenine dinucleotide hydrogenase NEFA non-esterified fatty acids; OKC oral ketogenic compounds PD Parkinson’s disease PET positron emission tomography PPAR Peroxisome Proliferator Activated Receptors QEMRF Queensland Emergency Medicine Research Fund ROS reactive oxygen species SADRs serious adverse drug reactions SAH subarachnoid haemorrhage SRSE severe refractory status epilepticus TBI traumatic brain injury TCA tricarboxylic acid cycle TPN total parenteral nutrition

xxii Evaluation of a Ketogenic Formulation for Treating Patients with Acute Brain Injury

Preamble – Scope of Thesis

This thesis documents my interest in the subject of cerebral energetics which initially began with discussions with Professor Venkatesh in 2008. Having always considered (as most doctors) ketones to be bad, I was surprised to discover the many potential therapeutic benefits provided by this small molecule. The published literature at that stage was sparse but suggested that energy provided by ketones could readily be used by the brain as a substitute for glucose, especially in the circumstances of brain injury. Given the poor outcomes from brain injury and the paucity of positive interventions, it seemed to me that ketones could provide a long sought-after therapeutic modality. The chapters that follow represent the state of the art knowledge of ketone therapeutics at this time.

This thesis covers the period from 2009 to 2018 and includes clinical and animal studies completed in an attempt to produce an intravenous and enteral ketone formulation suitable for patients with ABI. These studies include a hypertonic BHB saline solution administered to animals (chapter 2), an observational study examining ketone concentrations in adult ABI patients (chapter 4) and an interventional study investigating the effects of a ketogenic enteral feed in adults with ABI (chapter 7).

Ketones, which are naturally produced in times of starvation, represent a potential source of energy to the injured brain.

xxiii The Role of Ketones in Brain Injury

Chapter 1: The Role of Ketones in Brain Injury

1.1. Synopsis and Citation

Aim:

This chapter explores the physiology of ketone body utilization by the brain in health and a variety of neurological conditions and further discusses the potential for ketone supplementation as a therapeutic option in traumatic brain injury.

Citation:

This chapter is based on a publication reviewing the use of ketone supplementation in traumatic brain injury. Figures and tables have been inserted into the text at different positions and the numbering of pages, figures and tables has been adjusted to fit the thesis. The references have been incorporated with the other references of the Thesis.

The original manuscript was:

White H, Venkatesh B. Clinical review: ketones and brain injury. Crit Care. 2011; 15:219. Professor Venkatesh assisted with preparation of and co-authored the original manuscript

1 The Role of Ketones in Brain Injury

1.2. Introduction

Ketogenesis is the process by which ketone bodies, during times of starvation, are produced via fatty acid metabolism. Although much feared by physicians, mild ketosis can have therapeutic potential in a variety of disparate disease states. The principal ketones include acetoacetate (AcAc), β-hydroxybutyrate (BHB) and acetone. In times of starvation and low insulin concentrations, ketones supply up to 50% of basal energy requirements for most tissues, and up to 70% for the brain. Although glucose is the main metabolic substrate for neurons, ketones are capable of fulfilling the energy requirements of the brain. The purpose of this review is to explore the physiology of ketone body utilization by the brain in health and a variety of neurological conditions and to discuss the potential for ketone supplementation as a therapeutic option in traumatic brain injury.

1.3. Ketones, metabolism and the brain

Ketone metabolism

Ketones are by-products of fat metabolism and are produced in hepatic mitochondria1. More specifically, the three ketone bodies (KB) are (see Figure 1.1);

• Acetoacetate (AcAc) - if not oxidized to form usable energy, is the source of the two other ketone bodies below.

• Acetone - is not an energy source, but is instead exhaled or excreted as waste.

• β-hydroxybutyrate (BHB) - is not technically a ketone but is reversibly produced from acetoacetate

Figure 1.1 Chemical structure of ketone bodies

2 The Role of Ketones in Brain Injury

The precursor for the synthesis of ketones is acetyl CoA, which is formed by ß-oxidation of free fatty acids in the liver (although the potential for extrahepatic ketone production is still under investigation)2. The two important determinants of ketogenesis are availability of acetyl CoA and mobilization of fatty acids liberated from white adipose tissue during fasting or catecholaminergic stress 3, 4.

Acetoacetate is the central ketone body. It is reduced to BHB by BHB dehydrogenase (BHD) in a NADH-requiring reversible reaction 4;

(R)-3-hydroxybutyrate + NAD+ ↔ acetoacetate + NADH + H+

The extent of this reaction depends on the state of the cellular pool of NAD+ 5. When highly reduced, most or all ketones will be in the form of BHB. The ratio of AcAc to BHB reflects the redox state within the mitochondrial matrix 6. BHB is non-volatile and is chemically stable. Its only metabolic fate is interconversion with AcAc. Once synthesized, ketones diffuse into the circulation.

Figure 1.2 Fate of circulating ketones during fasting

Prolonged fasting with subsequent inhibition of insulin release results in increased circulating free fatty acids (FFAs) which serve as substrate for ketogenesis in the hepatocytes. Ketone bodies are released into circulation and subsequently taken up by brain cells. As glucose levels are low, ketones bodies may provide up to 2/3 of cerebral energy requirements.

3 The Role of Ketones in Brain Injury

Fate of circulating ketones

Plasma concentrations of circulating ketone bodies range from < 0.1 mmol/l in the post prandial state to as much as 6 mmol/l during fasting with concentrations reaching 25 mmol/l in diabetic keto-acidosis 7-9. Under normal dietary conditions, ketogenesis from fatty acids is at very low concentrations. This is attributable to the relatively high blood concentrations of the ketogenesis-inhibiting hormone insulin, and low blood concentrations of ketogenesis- promoting hormones, glucagon and cortisol 3. Therefore, one of the prerequisites for ketogenesis is low concentrations of circulating insulin as seen during starvation and diabetic ketoacidosis.

During periods of low insulin, catabolic hormones such as glucagon, cortisol, catecholamines, thyroid hormone and growth hormone increase and induce ketogenesis via stimulation of lipolysis of FFA and subsequent stimulation of β-oxidation in the liver. The principle regulators for this cellular response are the Peroxisome Proliferator Activated Receptors (PPARs). These transcription factors are modulated directly by FFA’s 10. In the brain, astrocytes, which are capable of producing ketone bodies (see below), are also regulated by PPARs 11. Aside from the ketogenic properties, activation of PPARs have been shown to produce anti-inflammatory and anti-oxidant effects independently which may prove to have neuroprotective properties 12, 13

The majority of circulating ketone bodies will be taken up by extrahepatic tissues, although 10–20% may be lost in the urine 9, 14. KB’s provide an energy source for several tissues including brain, skeletal muscle, myocardium and liver although heart and kidney have the greatest capacity for utilization 15. The rate of utilization appears to be a function of the circulating plasma concentrations16-18.

Ketones and Critical Illness

The major illness associated by intensive care physicians with ketosis is diabetic ketoacidosis (DKA). Outside of DKA, ketone measurement is not routinely performed and has been largely used as a research tool to quantify cellular redox status. The arterial ketone body ratio (AKBR) is utilized to reflect the ‘mitochondrial redox status’ of hepatic cells and may reflect cellular energy status in a number of conditions including hemorrhage, hypoxia,

4 The Role of Ketones in Brain Injury sepsis and hepatic resection 19. The AKBR (ratio of acetoacetate to β-hydroxybutyrate) reflects the cellular NAD+/NADH ratio, which cannot be measured directly and is linked to the phosphorylation potential of the cell (see equation above). In critically ill patients the redox state of cellular mitochondria is further reduced inhibiting the entry of acetyl CoA into the tricarboxylic acid cycle (TCA). Consequently, acetyl CoA combines with another molecule of acetyl CoA to form acetoacetic acid, which in turn is converted to β- hydroxybutyrate leading to an altered NAD+/NADH ratio and decreased AKBR 19, 20. Therefore, a reduced AKBR is a sign of an inefficient TCA cycle. The AKBR has also been used as an indirect marker of the hepatocellular functional status as the predominant site of metabolism of the ketones is in the liver.

Not surprisingly, metabolic failure at the cellular level is associated with worse clinical outcomes. A low AKBR is associated with poor outcomes in both animal and human models of illness. Yassen et al measured AKBR in 20 critically ill patients 20. They demonstrated a progressive decrease in those patients with septic shock who died. In a study of critically ill heart failure patients, Limuro et al noted a survival of only 15% of patients with an AKBR of < 0.7 21. Dresing found AKBR’s to be significantly lower in non-survivors than survivors in 30 polytrauma patients (0.6 vs 1.3; p=0.001) 22. However, Levy et al failed to find a difference in AKBR levels between survivors and non-survivors in their cohort of septic patients 23. In conjunction with the lactate: pyruvate ratio, AKBR may provide further insight into the cellular redox potential of critically ill patients.

Ketones and the heart

Although the ability of heart muscle to utilize ketones as an energy substrate has been known for some time, only recently have the potential benefits of ketone supplementation in heart failure been explored. Animal research has demonstrated that the expression of the enzyme β-hydroxybutyrate dehydrogenase 1 is increased in heart failure suggesting a shift to ketone metabolism 24. Whether this is adaptive, or maladaptive is not known at this stage. Some early studies found that when ketones were utilized as the sole substrate, cardiac function actually decreased in an isolated rat heart model 25. This was thought to result from interference by ketones in several downstream intermediates of the Kreb cycle, a process which could be reversed by anaplerosis. Further research is required to better clarify the role of ketones in the failing heart.

5 The Role of Ketones in Brain Injury

Ketones and the brain

Glucose is the major fuel for the brain in humans on a balanced diet. More recently it was proposed that other substances such as lactate and pyruvate may be utilized by neurons to sustain their activity 26. Furthermore, during times of starvation, the brain has the capacity to adapt to the use of ketones as its major energy source (and in the metabolic disease diabetes mellitus). In starvation of long standing, ketones can provide 60 - 70% of the energy needs of the brain 27. The 1.5 kg human brain utilizes 100–150 g of glucose per day and 20% of the total body oxygen consumption at rest 8. Protein catabolism can supply somewhere between 17 and 32 g of glucose per day, well below the minimum daily cerebral glucose requirements 28. In fact, to supply sufficient glucose to support brain metabolic requirements from protein alone would lead to death in about 10 days instead of the 57–73 days 29. Moreover, in subjects undergoing total starvation for 30 days, the decrease in hunger coincides with the elevation of blood ketones to 7 mmol/l. During periods of ongoing starvation, an average size person produces about 150 g of ketone bodies per day 30. It should be noted however that ketones are incapable of maintaining or restoring normal cerebral function in the complete absence of glucose.

Astrocytes may play a significant role in the regulation of brain energy metabolism 31. Reports suggest that astrocytes can produce ketone bodies from fatty acids and leucine and are involved with other pathways of lipid metabolism, such as lipid synthesis and lipoprotein secretion 32-35. Furthermore, it appears that the production of lactate and ketone bodies by astrocytes may increase secondary to neurotransmitters released during enhance synaptic activity 36, 37. Neurons, astrocytes and oligodendrocytes are all capable of utilizing ketones as a substrate 38.

Although the implications of ketone body production by astrocytes are still unclear, the fact that astrocytes outnumber neurons approximately nine to one and occupy at least 50% of the cerebral volume suggests this constitutes a quantitatively important pathway in the brain 39.

Regulation of cerebral ketone uptake

As previously noted, humans can achieve very high circulating KB concentrations during prolonged fasting, up to 9 mmol/l (range 5.8-9.7) 27. The plasma concentration of KBs is a significant factor affecting the rate of cerebral uptake. The blood brain barrier (BBB) is

6 The Role of Ketones in Brain Injury relatively impermeable to most hydrophilic substances, such as KBs, unless they are transported by a carrier protein. A family of proteins involved in the transport of monocarboxylic acids such as lactic acid and pyruvate across cellular membranes called monocarboxylic acid transporters (MCT1 and 2) are thought to play an integral role.

It is not yet understood how MCT’s are regulated. KBs are transported at different rates with the uptake of acetoacetate being twice that of BHB at a given arterial concentration 17. Furthermore, prolonged fasting appears to increase the BBB uptake of ketones with some researchers demonstrating an 8-fold increase in the expression of MCT1 40. Similarly, Hasselbalch et al reported a 13-fold increase in cerebral uptake of BHB in humans following several days of fasting 41. In contrast, rapidly increasing plasma ketone concentrations does not lead to as significant an increase in cerebral ketone uptake. Pan et al noted a much smaller increase in cerebral ketone concentrations following a rapid intravenous infusion of BHB as compared to prolonged fasting implying that MCT upregulation is partly dependent on length of exposure to increased plasma ketone concentrations 42, 43. Animal studies have demonstrated an age related difference in MCT 1 expression with a switch from fatty acids and ketones preference in the young to carbohydrates metabolism with aging 44, 45.

Once across the BBB, KBs enter brain cells to support cellular energy requirements. Studies of the uptake of KBs by brain cells suggest entry occurs by two mechanisms: diffusion and a carrier-mediated process. MCTs have been demonstrated on neurons and glial cells although their role here appears less significant as compared to on the BBB. The impact of mitochondrial enzyme activity involved in ketone metabolism is less clear. Certainly, mitochondrial enzymes can be upregulated by increased concentrations of BHB as has been demonstrated following exposure to ketones through starvation or prolonged high fat diet 17, 46, 47. In summary therefore, a prolonged high blood ketone concentration leads to an increased rate of cerebral ketone uptake and metabolism.

Regional variation in KB metabolism

Data for this are based largely on animal studies. Hawkins et al noted that KB uptake varies from one brain region to another 48, 49. Following an intravenous administration of radioactive BHB to adult rats, uptake was greatest in the cerebral cortex (deep layers), the superior and inferior colliculi and regions where the BBB is absent 48, 49. Areas with a low metabolic rate such as the corpus callosum and other white-matter structures demonstrated much reduced uptake. Interestingly, the pattern of KB uptake resembled that of glucose except in the

7 The Role of Ketones in Brain Injury superficial layers of the cortex where glucose uptake was higher 50. Similarly, MCT1 expression in the brain may resemble the pattern of KB uptake with some exceptions as MCT1 is found on glia and neurons as well as on BBB epithelial cells and because the pituitary and pineal have no BBB.

Neuroprotective effects of ketones

Neuroprotection is the mechanisms and strategies that once implemented, may lead to salvage, recovery or regeneration of the nervous system. Ketones may have a neuroprotective effect (see Figure 1.3). Ketones represent an alternative fuel for both the normal and the injured brain 51. Firstly, β-hydroxybutyrate is more energy efficient than glucose and can stimulate mitochondrial biogenesis via the upregulation of genes encoding energy metabolism and mitochondrial enzymes 52.

Figure 1.3 Potential neuroprotective mechanisms of ketones

Ketones (1) require only three enzymatic steps to enter the TCA cycle; (2) reduce the NAD couple; (3) decrease free radical formation; (4) increase production of ATP; (5) increase mitochondrial uncoupling; (6) increase glutathione peroxidase activity; and (7) inhibit pyruvate entry into the TCA cycle.

8 The Role of Ketones in Brain Injury

There is a marked increase in free energy of ATP hydrolysis as demonstrated by a 25% increase in hydraulic work of isolated rat perfused heart preparations when exposed to KB 53. Also, ketones increase the intermediary metabolites delivered to the citric acid cycle with a 16 fold elevation in acetyl CoA content. Secondly, ketones protect against glutamate mediated apoptosis and necrosis through the attenuation of the formation of reactive oxidant species. Ketones can oxidize co-enzyme Q thus decreasing mitochondrial free radical formation. Additionally, the reduction of NAD favors reduction of glutathione, which 54, 55 ultimately favors destruction of H2O2 by glutathione peroxidase reaction . Thirdly, ketones enhance the conversion of glutamate to gamma-aminobutyric acid (GABA) with the subsequence enhancement of GABA-mediated inhibition 56, 57. Cerebral ketone uptake also increases cerebral blood flow (CBF) 58. Fourthly, cell death from apoptosis is attenuated by ketone ingestion 56. Mechanisms include reductions in activation of caspase-3, increases in calbindin and prevention of accumulation of the protein clusterin. Lastly, cerebral ketone metabolism alters cerebral blood flow (CBF) 58. Hasselbalch et al demonstrated a 39% increase in CBF following an infusion of NaBHB.

More recently, Greco et al demonstrated that the initial neuroprotective effects of BHB are secondary to the antioxidant properties while later benefits are related to substrate augmentation 59. There are also age related variations in ketone uptake and utilization which require further study. Furthermore, evidence suggests that not all effects of ketones are anti- inflammatory in nature and may depend on cell type, ketone concentration and other comodulators 2. Specifically, AcAc in certain circumstances appears to activate pro- inflammatory signalling 60. Further research to better delineate the impact of ketones on inflammatory pathways is necessary.

1.4. Current state of research - Changes in ketone metabolism during brain injury

Models of Injury - Animal studies

Adaptive changes to cerebral ketone metabolism during development and starvation are well documented. However, there is evidence that other conditions including traumatic brain injury (TBI), ischemia, hemorrhagic shock and hypoxia induce rapid changes in both vascular and cellular transporters that favour ketone uptake and metabolism. In addition, ketone-metabolizing enzymes demonstrate the ability to change in response to neuropathologic conditions. Collectively, these adaptations suggest that the brain may be more receptive to ketone metabolism under neuropathic conditions.

9 The Role of Ketones in Brain Injury

Researchers have demonstrated an increase in MCT1 and MCT2 expression following cerebral injury 61. Similarly, ketone metabolizing enzymes expression also appears to alter. Utilization of BHB in the brain is contingent on its conversion to acetoacetate by β- hydroxybutyrate dehydrogenase, which is scarce in the adult brain, especially in the basal ganglia. Studies confirm a significant increase in BHD activity following cerebral insult. 62

The brain’s ability to utilize exogenous ketone bodies is altered. At times of oxidative stress, the ability of the brain to use glucose as a substrate for metabolic activity may become compromised. Certainly, hyperglycemia has been repeatedly shown to be deleterious to the injured brain 63. During these times, an exogenous supply of ketones may force the brain to shift its reliance from glucose to ketones, thus taking advantage of improved transport and cellular metabolism. This has proven to be advantageous in both acute brain injury models (glutamate excitotoxicity, hypoxia/ischemia, TBI) and neurodegenerative conditions (Parkinson’s disease, Alzheimer’s disease).

Ketone supplementation has been investigated in a number of models of cerebral injury including glutamate toxicity, hypoxia/ischemia injury and traumatic brain injury. Glutamate neurotoxicity is implicated in a number of neurodegenerative disorders associated with brain ischemia, hypoglycemia and cerebral trauma. During injury glutamate and aspartate are released through the reverse action of their transport systems or via enhanced exocytotic release. This in turn leads to excitotoxicity and the production of reactive oxidant species. Administration of ketones appears to limit the extent of damage 64-68. For example, Massieu et al demonstrated that acetoacetate effectively protects against glutamate neurotoxicity both in vivo and in vitro in rats chronically treated with a glycolysis inhibitor 65.

Hypoxic/ischemic injury leads to a decrease in oxidative metabolism by reducing oxygen availability 69, 70. Ketone administration ameliorates many of the sequelae of this process including lactate generation, free radical damage and apoptotic cascade activation 71, 72. Suzuki et al demonstrated that BHB prolonged survival time in rat models of hypoxia, anoxia and global ischemia 73. They further showed that BHB reduced infarct size after MCA occlusion, irrespective of whether BHB administration was delayed.

More recently, traumatic brain injury models have been developed 74, 75. TBI is associated with impaired cerebral energy production with prolonged glucose metabolic depression and reduction of ATP. Cerebral injury is further exacerbated by free radical production, and activation of poly-ADP-ribose polymerase via DNA damage 76. As such, under these

10 The Role of Ketones in Brain Injury conditions of impaired glycolytic metabolism shifting the brain toward ketone metabolism may limit the extent of cerebral injury 73, 77-80 (see Table 1.1). Much of this work is attributable to Prins et al who demonstrated that administration of intravenous BHB after TBI in adult rats led to an increase in uptake of BHB and alleviated the decrease in ATP after injury. In a subsequent study they found that a ketogenic diet was effective in reducing the cortical contusion volume in a rat model of TBI 74, 75. Of concern is a recent finding that BHB administration to rats with TBI may have detrimental effects on BBB permeability 81. The study investigated the effect of BHB on BBB integrity following TBI noting that while nuclear factor-kappa B expression decreased, BBB permeability as evidenced by an increase in Evans blue extravasation in the ipsilateral cortex was noted. Although the mechanisms are unclear, the diverse effects of BHB may be due to its ability to variably change local cerebral oxidant/antioxidant capacity. More research is needed to clarify this issue.

Neurodegenerative conditions may also benefit from ketone administration as there is evidence of altered cerebral metabolism. Studies of Parkinson’s and Alzheimer disease confirm the encouraging effects of ketones. Mechanisms are complex but include decreasing free radical production and providing acetyl-CoA for energy production. 53, 62, 82- 85. Other conditions which may be responsive to ketones include amyotrophic lateral sclerosis and certain brain tumors (see Chapter 6).

Models of injury – human research

Mild ketosis is induced in humans by either fasting or the consumption of a high fat, low carbohydrate diet. Most studies in humans have delivered ketones via the enteral route 86. In adults, achieving blood concentrations of 5–7 mmol/l total ketone bodies requires the production or intake of about 150 g per day 30. This however is subject to the influences of glucose, insulin, cortisol and catecholamines. It has been suggested that to achieve a therapeutic response in refractory epilepsy, the concentration of blood ketone should be above 4 mmol/l 87.

Salts of ketone bodies can be produced. Several authors (see below) have administered Na AcAc or Na BHB successfully with few side effects. A new formulation called KTX 0101 consists of a combination of NaCl and BHB although relevant concentrations are not described 88. Main issues related to IV administration of ketone solutions include; volume loading, Na loading and the potential for significant metabolic alkalosis. This will depend on the relevant concentrations of anions and cations in the mixture and the osmolality.

11 The Role of Ketones in Brain Injury

Two studies have examined baseline ketone concentrations in humans with TBI. The first was an observational study of 38 patients with acute brain injury (ABI) on a normal diet which demonstrated that BHB concentrations were raised on admission but normalized rapidly 89.

12 The Role of Ketones in Brain Injury

Table 1.1 Animal and basic science studies on effects of ketone administration on brain injury

Mechanism of injury Reference Ketone Study Outcome source

Glutamate induced injury Massieu et al., IV AcAc infusion for 14 days in adult rodent model ↓ neuronal damage, ↓ lesion volume and ↑ prior to and during glutamate induce injury 2003 65 cellular ATP levels

Noh et al., PO 21 day old Mice fed ketogenic diet prior to kianic Preservation of neurons in hippocampus induced neuronal damage by inhibiting caspase-3-mediated 2006/2003 67, 68 apoptosis

Mejia-Toiber et al., * BHB applied before glutamate induced neuro- BHB reduces injury and lipoperoxidation toxicity. 2006 66

Maalouf et al., * Addition of BHB to invitro model of glutamate Reduction in free radical formation and neurotoxicity enhance mitochondrial respiration 2007 64

Cerebral hypoxia Puchowicz et al., PO Rats fed ketogenic diet 3 weeks prior to hypoxic Decreased cerebral lactate and increased 2005 70 injury tolerance of hypoxia

Masuda et al., * Rat neurons exposed to hypoxia following Decreased cell death and number of treatment with BHB apoptotic cells with maintenance of 2005 69 mitochondrial membrane

Cerebral ischemia Suzuki et al., IV Rats infused with BHB 3-6 hours after bilateral Decreased cerebral oedema and sodium carotid artery occlusion content and increased ATP levels 2001 80

Suzuki et al., IV BHB infusions following occlusion of Middle 50% reduction in cerebral infarct volume cerebral artery 2002 73

Marie et al., Fasted Adult rats fasted for 24 hours before four vessel Decreased mortality, post-traumatic occlusion seizures and brain lactate 1990 79

13 The Role of Ketones in Brain Injury

Mechanism of injury Reference Ketone Study Outcome source

Go et al., Fasted Starvation induced ketosis followed by carotid Fasting protected rats from brain infarction occlusion 1988 78

Traumatic brain injury Prins et al., IV BHB infusion following TBI in adult rats Increase in serum BHB and cerebral BHB and improved cortical ATP levels 2004 75

Prins et al., PO Rats with TBI fed ketogenic diet 50% reduction in cortical contusion volume 2005 74

Biros et al., PO Rats of differing ages fed ketogenic diet following Younger rats demonstrated larger TBI reduction in contusion volume 1996 77

Hu et al, PO Rats fed ketogenic diet prior to TBI Reduced brain oedema and cellular apoptosis in rats fed ketone diet 2009 71, 72

14 The Role of Ketones in Brain Injury

The second investigated the impact of targeting tight glycemic control on BHB concentrations post TBI. They noted an increase in BHB concentrations with improved glycemic control although this was in the setting of increased insulin usage and permissive underfeeding 90.

There is a paucity of research examining the effects of ketone administration in humans. Interpretation of results is further complicated by variable routes of administration, concentrations and age variability of research subjects. For instance, research on the effects of intravenous ketone preparations are confined almost exclusively to adults whereas, dietary studies include a large number of children. As far as we are aware, only two clinical studies examining the neuroprotective effects of KB in humans have been undertaken. The first by Ritter et al examined the effects of a ketogenic diet on biochemical markers of metabolism in head injury patients but did not include clinical outcomes 91. The second reported by Smith et al has never been published but reported on the use of a sodium BHB solution for neuroprotection during cardiac surgery 88. The bulk of the research has taken place using the rodent model.

Some general statements however, can be made. It has been demonstrated that; ketones can be given enterally or intravenously with minimal side effects, utilization of ketone bodies increase with rising blood concentrations, with a plateau at approx. 128g/24 hours, hyperketotic states are associated with improved glucose control, increased plasma concentrations lead to increase in cerebral uptake and ketogenic diets may lead to improved cognitive function in patients with chronic dementia (see Table 1.2). However, well designed outcome studies are lacking 42, 43, 88, 91-98.

Adverse effects of ketones

There is minimal data on the adverse effects of ketone administration, much of which is based on chronic intake via the enteral, rather than the parenteral route. In the acute setting, effects of parenteral formulations on pH, Na, lipids and glucose are likely to dominate e.g. IV formulations would theoretically be alkalinising, depending on the ketone concentration. This was confirmed by Hiraide et al who noted a significant increase in pH and Na concentrations following the IV administration of a 20% solution of NaBHB to severe trauma patients 94. Also of potential concern is the reduction in glucose cerebral metabolism and increase in CBF demonstrated by Hasselbalch et al during administration of intravenous BHB 58. The long term consequences of this are not known.

15 The Role of Ketones in Brain Injury

Table 1.2 Human studies investigating effects of exogenous ketone administration

Pathophysiological endpoints Reference Study Outcome

Metabolic effects Owen et al., Effect of ketones on insulin and free fatty AcAc levels 4.74 mmol/l after 30 min acid release. Sodium AcAc given as IV 1973 96 Utilization of ketones increased with rising infusion 1 mmol/kg over 2 hrs to 12 normal blood concentrations to a maximum of 13 g/24 adults after overnight fast. hr. Glucose remained normal

Miles et al., Evaluation of protein sparing effects of Ketone levels reached 2.33 mmol/l 1983 95 IV infusion of NaBHB in 6 healthy adults. No protein sparing, significant alkalosis

Hiraide et al., 20 pts following severe trauma. 11 pts Ketone levels reached 1.5 mmol/l. Alanine received IV 20% solution NaBHB at 25 release decreased in ketone group suggesting 1991 94] umol/kg/min for 3 hrs. 9 pts received Na a suppression of posttraumatic protein lactate. catabolism. There was a significant increase in Na and pH

Dashti et al., 64 health obese adults with and without BMI, glucose, total and LDL cholesterol diabetes fed ketogenic diet for 56 weeks. decreased significantly whereas HDL 2007 93 increased

CNS effects Pan et al., 6 healthy adults infused with NaBHB at a BHB blood levels reached 2.12 mmol/L bolus rate of 80 umol/kg/min, then 20 2001 43 BHB brain levels increased to 0.24mol/L umol/kg/min for 75 min. BHB brain levels measure by MRI Levels lower then noted in fasted individuals suggesting differences in brain transport of

ketones in fasted state

Pan et al., 5 healthy adults. Ketones measured in Plasma and brain BHB levels correlated well brain using MRI in non-fasted state and with brain ranging from 0.05 mmol/l in 2000 42 after 2 and 3 day fast. nonfasted state to 0.98 mmol/l after 3 days fasting

16 The Role of Ketones in Brain Injury

Pathophysiological endpoints Reference Study Outcome

Blomqvist et al., 6 healthy and 6 diabetic pts received Utilization rate of ketones increase infusion of BHB at 3-6 mg/kg/min for 60 proportionally with plasma concentrations. 2002 92 min followed by bolus of B-[11C] HB. PET Rate limiting step for ketone body utilization is scan was performed transport into the brain

Pathophysiological endpoints Hasselbalch et al., Permeability of blood brain barrier to BHB Increase cerebral ketone influx during was assessed following a 3.5 day fast. starvation determined by amount of ketone 1995 98 present in blood.

Ritter et al., 20 head injured patients assigned to Group receiving ketogenic diet demonstrated conventional or ketogenic diet for 2 weeks. improved glucose control with higher ketone 1996 91 levels. Cerebral lactate was similar

Smith et al., NaBHB IV solution known as KTX 0101 for Phase IA study not published neuroprotection post CABG. Details of 2005 88 study protocol not available

Hasselbalch et al., 8 healthy adults received IV infusion of Increased cerebral uptake of ketones was NaBHB at 4-5 mg/kg/min for 3 hrs. counterbalanced by reduction in glucose 1996 58 Cerebral blood flow measured using Kety- metabolism. Cerebral metabolic rate was Schmidt technique unchanged however cerebral blood flow increased.

Reger et al., 20 adults with Alzheimer’s disease were On cognitive testing, MCT treatment facilitated randomized to receive a MCT diet or 2004 97 performance for 4- but not 4+ subjects. Also placebo on two occasions. greater improvement with MCT treatment relative to placebo

17 The Role of Ketones in Brain Injury

Freeman has reported a significant rise in mean blood cholesterol to over 250 mg/dl following prolonged intake a of ketogenic diet. 99. This in turn may be atherogenic, leading to lipid deposition in blood vessels. In addition there are reports of dilated cardiomyopathy in patients on the ketogenic diet, possibly as a consequence of the toxic effects of elevated plasma free fatty acids. Finally, an increased incidence in nephrolithiasis in patients on the ketogenic diet as well as increases in serum uric acid secondary have been reported 76, 100.

1.5. Potential clinical applications for ketone solutions – traumatic brain injury

Traumatic brain injury is a major cause of mortality and morbidity in young adults 101. Cerebral edema and subsequent intracranial hypertension is an important factor influencing patient outcome, and despite considerable research, has proven difficult to manage. Over the past 30 years, osmotherapy has become an important tool in the management of intracranial hypertension after TBI. 101. Various substances, including urea, glycerol, sorbitol and mannitol have been utilized 102, 103. More recently hypertonic saline (HTS) has proved useful in controlling ICP.

HTS has been extensively studied in animal and human models of TBI 104-107. These experiments suggest that small volume resuscitation with HTS solution may be beneficial for increasing cerebral perfusion pressure and cerebral blood flow and decreasing ICP while maintaining hemodynamic stability following TBI. Animal studies comparing HTS and mannitol suggest that HTS is more effective in reducing ICP and has a longer duration of action 108, 109.

In humans, HTS appears effective for reducing ICP in cases of refractory intracranial hypertension following TBI. Several studies have confirmed a significant decrease in ICP correlated with an increasing serum osmolality 110-112.

In the past, researchers have combined hypertonic sodium with a variety of anions including acetate and lactate 113, 114. Acetate, a source of hydrogen ion acceptors, is an alternate source of bicarbonate by metabolic conversion in the liver. However, acetate is a myocardial depressant and can lead to hypotension in susceptible individuals. Hypertonic

18 The Role of Ketones in Brain Injury sodium/lactate solutions have also been investigated. Whilst HTS/lactate is generally considered safe and may provide cerebral nutrition, there is some limited animal data suggesting that fluids containing lactate may be associated with significant toxic effects 115.

Further research is required to clarify these issues. There is therefore a need for a solution able to address two fundamental problems related to TBI namely, refractory intracerebral hypertension and low cerebral energy levels. This could theoretically be accomplished by incorporating ketones into a hypertonic solution such as hypertonic saline.

Firstly, the impact of the constituents on individual metabolism needs to be evaluated. The hypertonicity induced by 3% NaCl is well documented and desirable. However, the target Na is not known and various authors have reported concentrations as high as 185 mmol/l. Secondly, although the long term metabolic effects of ketone administration are described, little exists on the acute changes induced by intravenous infusions. For example, the entry of ketones into CSF is partly concentration dependent and therefore a certain serum concentration (possibly above 4 mmol/l) may be required for maximum utilization. What then is the effect of high ketone concentration on blood pH? It would be expected that such a solution would be alkalinizing, but this will be altered by the rate of metabolism. Thirdly, which ketone should be incorporate, acetoacetate or BHB? Both have been administered to humans in the past without ill effects. Fourthly, should CSF or plasma concentrations be utilized to guide therapy? And lastly, ketones are expensive to produce, therefore, can a commercially viable solution be developed?

1.6. Conclusion

There is good theoretical evidence that administering ketones to patients with cerebral injury may provide a clinical benefit. While the use hypertonic saline has been demonstrated to improve intracranial hypertension in a number of clinical studies, the addition of ketones to the solution has the potential to improve cerebral metabolism. There are however, a number of outstanding questions requiring further consideration. These include dosing, timing, the route and duration of administration. Further research is necessary to clarify these issues and determine whether an HTS-ketone solution could be employed in the management of acute brain injury.

19 Prospective Evaluation of a Hypertonic Balanced Ketone Solution for use in Neuroprotective Strategies

Chapter 2: Prospective Evaluation of a Hypertonic Balanced Ketone

Solution for use in Neuroprotective Strategies

2.1 Synopsis and Citation

Background:

There is a need for a solution capable of addressing two fundamental problems related to TBI namely, intracranial hypertension and impaired cerebral energy production. While hypertonic saline has been utilized for a number of years to control intracranial hypertension, little attention has been paid to the provision of metabolic substrates capable of improving cerebral energetics. This could theoretically be accomplished by incorporating ketones into a hypertonic solution such as hypertonic saline. There are however, a number of challenges associated with the development of new parenteral solutions.

Firstly, the impact of the constituents on individual metabolism needs to be evaluated. The hypertonicity induced by 3% NaCl is well documented and desirable. However, the target Na is not known and various authors have reported plasma concentrations as high as 185 mmol/l (normal plasma Na range is 135 – 145 mmol/l). Secondly, although the long term metabolic effects of ketone administration are described, little published data exist on the acute changes induced by intravenous ketone infusions. For example, the entry of ketones into CSF is partly concentration dependent and therefore a certain serum concentration (possibly above 4 mmol/l) may be required for maximum utilization. Thirdly, the impact of an intravenous ketone solution on blood pH has not been fully evaluated. It would be expected that such a solution would be alkalinizing but this will be altered by the rate of metabolism. Fourthly, which ketone should be incorporate, acetoacetate or BHB? Both have been administered to humans without ill effects. Fifthly, should CSF or plasma concentrations be utilized to guide therapy? And finally, what are the effects on clinically measurable outcomes in humans?

Aim:

This was a proof of principle study to develop and investigate the effects of a hypertonic, ketone solution which can be used in the management of traumatic brain injury to treat/prevent intracranial hypertension and improve cerebral cellular energetics.

20 Prospective Evaluation of a Hypertonic Balanced Ketone Solution for use in Neuroprotective Strategies

Design:

Adult Sprague-Dawley rats (420-570g) were divided into 3 groups of 5, one control and 2 study arms. The control group received an intravenous infusion of 3% NaCl at 5 ml/kg/h. The animals in the two study arms were assigned to receive one of the two formulations of ketone solutions, containing hypertonic saline with 40 mmol/l and 120 mmol/l beta- hydroxybutyrate respectively. This was infused for 6 hours and then animal was euthanized and brains removed and frozen.

Key Findings:

Both blood and CSF concentrations of BHB demonstrated strong evidence of a change over time (p<0.0001). There was also strong evidence of a difference between groups (p<0.0001). Multiple comparisons showed all these means were statistically different (p<0.05). Measurement of BHB concentrations in brain tissue found strong evidence of a difference between groups (p<0.0001) with control: 0.15 mmol /l (0.01), BHB 40: 0.19 mmol /l (0.01), and BHB 120: 0.28 mmol /l (0.01). Multiple comparisons showed all these means were statistically different (p<0.05). There was no difference over time (p=0.31) or between groups (p=0.33) or an interaction between groups and time (p=0.47) for base excess.

Conclusion:

IV infusions of hypertonic saline/BHB are feasible and lead to increased plasma, CSF and brain concentration of BHB without significant acid/base effects.

Citation:

The information in this chapter has been adapted and combined from published and previously presented sources that include a previously published paper as an original manuscript. Figures and tables have been inserted into the text at slightly different positions and the numbering of pages, figures and tables has been adjusted to fit the overall style of the thesis. The references have been incorporated with the other references of the Thesis.

The manuscript “Effect of a hypertonic balanced ketone solution on plasma, CSF and brain beta-hydroxybutyrate levels and acid-base status.” by White H, Venkatesh B, Jones M, Worrall S, Chuah T, Ordonez J was published in Intensive Care Medicine. 2013 Apr;39(4):727-33 and extracts are reproduced with kind permission from Springer-Verlag copyright 2013 21 Prospective Evaluation of a Hypertonic Balanced Ketone Solution for use in Neuroprotective Strategies

An oral presentation titled “A novel solution for the management of brain injury. Preliminary animal study” by White H, Venkatesh B, Jones M, Worrall S, Chuah T, Ordonez J, was presented at the College of Intensive Care Medicine National Annual Scientific Meeting, Melbourne 2012.

Contributions:

This prospective study was performed at the Biological Research Facility of the University of Queensland. I commenced work in July 2009 and was involved in the initial concept, all aspects of study design, data collection, data analysis and manuscript preparation. This work was carried out with the assistance of Dr Teong Chuah (assistance with study design and management), Ms Jenny Ordonez (intravenous fluid production), Dr Simon Worrall (analysis brain specimens and data analysis, manuscript preparation), Dr Mark Jones (statistical analysis) and Professor Bala Venkatesh (study design, data analysis and manuscript preparation).

22 Prospective Evaluation of a Hypertonic Balanced Ketone Solution for use in Neuroprotective Strategies

2.2 Introduction

Glucose is the major fuel for the brain in normally fed humans. However, during periods of fasting, ketones provide a significant contribution to cerebral energetics, up to 70% in prolonged starvation 27. Furthermore, during times of severe stress or cerebral injury, ketones become a significant source of energy. There is evidence to suggest preferential uptake of ketones over glucose by the injured brain 116. Several studies in animals have confirmed the benefit of exogenous ketone supplementation in terms of improvement in contusion size, cerebral blood flow and concentrations of adenosine tri-phosphate in traumatic brain injury and stroke models 53, 58, 73, 117.

As the blood brain barrier (BBB) is relatively impermeable to ketones, cerebral uptake is via monocarboxylate transporter (MCT) proteins and increases with prolonged fasting 40, 41. Cerebral concentrations are therefore largely determined by plasma ketone concentration. Most studies of supplementation have examined a single bolus or a short term infusion of enteric or intravenous ketones on cerebrospinal fluid (CSF) and brain metabolism 42, 43, 75, 118.

Previous attempts to use ketones have been complicated by problems of hyperosmolarity and acid-base disturbances 42, 118. Acetoacetate and -hydroxybutyrate (BHB) are strong anions and sodium salts of these when administered as a hypertonic IV infusion will tend to lower the strong ion difference of the plasma and may results in an initial metabolic acidosis, particularly if the rate of reduction of strong ion difference overwhelms the metabolic capacity of the liver to metabolise these anions. With time, metabolic clearance of these anions will result in an increase in plasma strong ion difference resulting in a metabolic alkalosis. For ketone supplementation to be a potentially therapeutic agent in patients with traumatic brain injury (TBI), a few challenges remain: achieving a formulation which can be administered as a prolonged infusion, which rapidly increases ketone concentrations in the brain, achieves a target hyperosmolarity and maintains neutral acid-base status.

Hypertonic Saline (HTS) has been extensively studied in animal and human models of TBI and may be beneficial for increasing cerebral perfusion pressure and cerebral blood flow and decreasing intracranial pressure while maintaining hemodynamic stability 105, 107, 110. A solution containing both hypertonic saline and ketones would theoretically provide a number of advantages over current solutions including the ability to reduce intracranial pressures while providing a substrate capable of improving cerebral energetics. As no published data

23 Prospective Evaluation of a Hypertonic Balanced Ketone Solution for use in Neuroprotective Strategies exist on the effect of prolonged infusions of hypertonic NaBHB, the purpose of this study was to investigate hypertonic sodium ketone solutions with the aim of assessing the metabolic effects in general and specifically, on the brain. The aim of this study was to produce an intravenous hypertonic NaBHB (sodium D-3-hydroxybutyrate) formulation, to compare the impact of varying concentrations of BHB on plasma, CSF and cerebral BHB concentrations and examine their effect on plasma sodium and acid-base status.

2.3 Materials and Methods

This study was approved by the Animal Ethics Committee of the University of Queensland (PAH/029/09/UQ). Adult Sprague-Dawley rats weighing between 420 and 570g were utilized in the study. All animals were housed at the Biological Research Facility of the University of Queensland and were maintained according to the Standard Operating Procedures for the humane care of animals by the facility. Animals were provided with free access to food and water up to the time of the experiment.

BHB solution

The animals were divided into 3 groups of 5, one control and 2 study arms (see Table 2.1).

Table 2.1 Baseline data (mean +/- SD)

Control (n=5) BHB 40 (n=5) BHB 120 (n=5)

Weight (g) 498 ± 49 570 ± 64 420 ± 60

HR (bpm) 266 ± 42 272 ± 11 274 ± 41

SPO2 (%) 95 ± 2 98 ± 1 96 ± 1

BHB, beta-hydroxybutyrate; BHB 40, 3% NaCl with 40 mmol/l; BHB 120, 3% NaCl with 120 mmol/l

The control group received an intravenous infusion of 3% NaCl at 5 ml/kg/h. The animals in the two study arms were assigned to receive one of the two formulations of ketone solutions. The composition of the solutions is listed in Table 2.2.

The rats in the control arm received 3% NaCl only while the two intervention groups received a 3% NaCl solution containing 40 mmol/l and 120 mmol/l of BHB respectively at 5 ml/kg/h.

The D-3-hydroxybutyrate acid was manufactured by Sigma-Aldrich Pty. Ltd. (Castle Hill, NSW, Australia). The infusions were maintained for 6 hours. Bloods and CSF were sampled

24 Prospective Evaluation of a Hypertonic Balanced Ketone Solution for use in Neuroprotective Strategies at baseline, 1 hour and 6 hours. At the end of the 6 hour period, the animals were euthanized.

Table 2.2 Formulations of intravenous solutions

Na (mmol/l) Cl (mmol/l) BHB (mmol/l)

Control 514 514 0

BHB 40 514 474 40

BHB 120 514 394 120

BHB,  -hydroxybutyrate; BHB 40, 3% NaCl with 40 mmol/l; BHB 120, 3% NaCl with 120 mmol/l

Preparation of solutions

The solutions were prepared by the Therapeutic Research Center, University of Queensland. The solutions were prepared as follows. For the 40 mmol/l solution, 5.0436g/L of D-3-hydroxybutyrate Acid Sodium Salt was combined with 27.70 g/l NaCl to create a 3% solution (514 mmol/l) (see Table 2.1).

Similarly, for the 120 mmol/l solution, the relative concentrations were 15.13 g/l D-3- hydroxybutyrate Acid Sodium Salt and 23.03 g/l NaCl respectively. The appropriate amounts were weighted for the particular volume required. The solution was mixed thoroughly until dissolved and prepared the same day of the experiments.

Anaesthesia and vascular access

The animals were anaesthetized using a facemask and 4% isoflurane in 100% oxygen. The isoflurane was subsequently decreased to 0.5 – 1.5% for maintenance. Intraperitoneal xylazine 3 mg/kg was also administered. Body temperature was maintained with an animal heating blanket. A 24 gauge BD Insyte IV catheter (Franklin Lakes, NJ USA) was inserted into a tail vein for continuous drug administration. A midline incision was made in the neck and a single common carotid artery was exposed and separated from surrounding tissues. A 24 GA BD Insyte catheter was inserted into the artery to monitor blood pressure and to collect blood samples. In order to maintain adequate oxygenation and control ventilation, a tracheostomy was performed. The trachea was dissected out and a small incision made between the rings. A 14 gauge BD Insyte IV catheter was shortened and inserted into the trachea and secured. The animals were attached to a Harvard Small Animal Ventilator

25 Prospective Evaluation of a Hypertonic Balanced Ketone Solution for use in Neuroprotective Strategies

(Holliston, Massachusetts USA) using tidal volumes of between 1.5 and 2.0 ml per breath and rates from 80 – 120 bpm. Heart rate and oxygen saturations were monitored with the Nellcor N-65 pulse oximeter (Covidien, Boulder, CO USA)

Specimen collection

Arterial blood specimens were taken at baseline, 1 hour and prior to euthanasia. Tests included arterial blood gas with acid/base status (pH, base excess (BE) and HCO3), oxygenation, serum sodium and chloride and BHB concentrations. CSF specimens were obtained by performing a puncture into the cisterna magna using a 27 GA VanishPoint insulin syringe (Little Elm, Texas USA) and aspirating approximately 20 ul of CSF. BHB concentrations in the CSF specimens were measured. At completion of the study, the animals were euthanized using pentobarbital sodium at a dose of 15 mg/kg. Following euthanasia, the animals were decapitated and the brains were removed immediately, and stored at – 80 degrees Celsius for later analysis.

Assays

Plasma BHB

Plasma BHB was measured quantitatively using the Stanbio Laboratory β-Hydroxybutyrate LiquiColor Assay Kit (Boerne, TX, USA) on the Beckman DxC800 Unicell (Brea, CA, USA) analyser using a spectrophotometric endpoint assay. The linearity of the assay is 0.0-4.0 mmol/l with the coefficient of variation (CV) 3.0% at 0.38 mmol/l and CV 2.1% at 1.38 mmol/l.

Brain BHB

Brain tissue was homogenised into ice-cold saline and the clarified extracts were appropriately diluted for use in the BHB assay kit. Brain BHB concentrations were measured using the Cayman Chemical - β-Hydroxybutyrate (Ketone Body) Assay Kit (Ann Arbor, Michigan, USA). Briefly, the method for BHB determination is based upon the oxidation of D-3-Hydroxybutyrate to acetoacetate by the enzyme 3-hydroxybutyrate dehydrogenase. Concomitant with this oxidation, the cofactor NAD+ is reduced to NADH. In the presence of diaphorase, NADH reacts with the colorimetric detector WST-1 to produce a formazan dye with an absorbance maximum at 445-455 nm. The absorbance of the dye is directly proportional to the BHB concentration. The range for the assay is 0-0.5 mmol/l. All samples were diluted to fall within this range.

26 Prospective Evaluation of a Hypertonic Balanced Ketone Solution for use in Neuroprotective Strategies

Acid-Base status and sodium

Arterial blood gases and electrolytes were measured using the EC8+ cartridge with the Abbott Point of Care system, iSTAT (Abbott Park, Il, USA) (CV < 2.5%). The device was calibrated prior to use as per manufactures instructions.

2.4 Statistical analysis

Analysis of variance was used for analysis of BHB for blood, CSF and brain, as well as base excess (BE) and sodium (Na). Factors assessed were group (control, BHB 40, and BHB 120), time (baseline, 1 hour, and 6 hour) as well as their interaction for all variables except brain which was only measured at study completion, hence only the group factor was included. A statistically significant interaction implies that changes over time differed between groups. Tukey’s method was used for multiple comparisons of significant (p<0.05) factors. Spearman’s correlation was used to assess the correlation between BHB for blood and CSF at the three time-points as well as brain at the final time-point. SAS version 9.1 for Windows and Stata Version 10.1 for Windows were used for the analysis.

2.5 Results

Plasma BHB

Blood concentrations of BHB demonstrated strong evidence of a change over time (p<0.0001) with least squares means (standard error) at baseline: 0.19 mmol/l (0.03), 1 hour: 0.29 mmol /l (0.03) and 6 hours: 0.47 mmol /l (0.03) (Figure 2.1).

Multiple comparisons showed all these means were statistically different (p<0.05). There was also strong evidence of a difference between groups (p<0.0001) where the group least squares means (standard errors) were control: 0.21 mmol /l (0.03), BHB 40: 0.26 mmol /l (0.03), and BHB 120: 0.48 mmol /l (0.03). BHB 120 mean was statistically larger than that for BHB 40 (p<0.05) and control (p<0.05) but there was no evidence of a difference between control and BHB 40. There was no evidence of an interaction between group and time (p=0.071) (Table 2.3).

CSF BHB

BHB concentrations in CSF demonstrated strong evidence of a change over time (p<0.0001) with least squares means (standard error) at baseline: 0.15 mmol /l (0.02), 1 hour: 0.23 27 Prospective Evaluation of a Hypertonic Balanced Ketone Solution for use in Neuroprotective Strategies mmol /l (0.02) and 6 hours: 0.36 mmol /l (0.02). Multiple comparisons showed all these means were statistically different (p<0.05).

Table 2.3 BHB data for 3 time points (mean +/- SD)

BHB blood (mmol/l) BHB CSF (mmol/l) Brain BHB (mmol/l)

Control (n=5)

Baseline 0.16 ± 0.05 0.22 ± 0.04

1 hour 0.18 ± 0.08 0.18 ± 0.08

6 hours c 0.28 ± 0.08 0.28 ± 0.11 0.15 ± 0.02

BHB 40ab (n=5)

Baseline 0.14 ± 0.01 0.14 ± 0.01

1 hour 0.22 ± 0.04 0.26 ± 0.05

6 hours cd 0.43 ± 0.05 0.38 ± 0.05 0.19 ± 0.01

BHB 120ab (n=5)

Baseline 0.26 ± 0.05 0.10 ± 0

1 hour 0.48 ± 0.13 0.24 ± 0.09

6 hours c 0.70 ± 0.27 0.43 ± 0.11 0.28 ± 0.02

BHB, beta-hydroxybutyrate; BHB 40, 3% NaCl with 40 mmol/l; BHB 120, 3% NaCl with 120 mmol/l a = In both groups, change over time was significant: values at 6 hrs significantly > 1 hr significantly> baseline for both plasma and CSF b = There was a significant difference in plasma BHB concentrations between BHB 120 and BHB 40 at 1hr and 6 hrs. c = For brain BHB concentrations there was a strong evidence of a difference between groups (p<0.0001) d n = 4 for this group. A single specimen returned a blood BHB concentration of 3.8 mmol/l and CSF BHB concentration of 1.8 mmol/l at 6 hours. As the outlier had values that were 28 standard deviations greater than the mean for CSF BHB (based on the other 4 rats in the group) and 67 standard deviations greater than the mean for blood BHB (based on the other 4 rats in the group), the results were removed from the analysis

There was no difference between groups (p=0.46) however there was evidence of an interaction between group and time (p=0.009) where there was an increase in BHB CSF over time in the BHB 40 and 120 groups but not the control group. To investigate the change in BHB CSF over time separate analyses were conducted on the 3 groups. Results showed evidence of change over time for BHB 40 group (p=0.001) and for the BHB 120 group (p=0.0005) but not for the control group (p=0.21) (Figure 2.2).

28 Prospective Evaluation of a Hypertonic Balanced Ketone Solution for use in Neuroprotective Strategies

Brain BHB

For BHB concentrations there was strong evidence of a difference between groups (p<0.0001) where the group least squares means (standard errors) were control: 0.15 mmol /l (0.01), BHB 40: 0.19 mmol /l (0.01), and BHB 120: 0.28 mmol /l (0.01). Multiple comparisons showed all these means were statistically different (p<0.05).

There were no differences over time (p=0.31) or between groups (p=0.33) or an interaction between groups and time (p=0.47) for base excess (see Table 2.4). An analysis of just the final time-point also showed no difference between groups for base excess (p=0.24). For Na there was evidence of a difference over time (p<0.0001) but not between groups (p=0.24) or interaction between groups and time (p=0.15). Least squares means (standard error) were, at baseline: 137 mmol /l (1.5), 1 hour: 143 mmol/l (1.5) and 6 hours: 154 mmol/l (1.5). Multiple comparisons showed all these means were statistically different (p<0.05).

Table 2.4 Acid base and electrolyte data for 3 time points (mean +/- SD)

a a b a pH BE (mmol/l) Serum Na HCO3 (mmol/l) (mmol/l)

Control (n=5)

Baseline 7.49 ± 0.17 2.8 ± 3.7 137 ± 7 24.6 ± 2.3

1 hour 7.55 ± 0.07 0 ± 2.6 145 ± 2 20.6 ± 3.3

6 hours 7.50 ± 0.10 - 1.2 ± 3.7 153 ± 7 21.0 ± 4.1

BHB 40 (n=5)

Baseline 7.35 ± 0.09 3.0 ± 4.3 137 ± 4 28.8 ± 4.6

1 hour 7.32 ± 0.10 1.6 ± 3.4 142 ± 6 27.6 ± 3.2

6 hours 7.34 ± 0.08 1.0 ± 2.9 150 ± 4 26.4 ± 3.0

BHB 120 (n=5)

Baseline 7.37 ± 0.19 1.4 ± 3.5 138 ± 5 26.4 ± 2.6

1 hour 7.41 ± 0.42 3.2 ± 1.6 141 ± 5 27.8 ± 1.4

6 hours 7.38 ± 0.05 2.0 ± 1.9 160 ± 9 26.8 ± 1.6 BHB, beta-hydroxybutyrate; BHB 40, 3% NaCl with 40 mmol/l; BHB 120, 3% NaCl with 120 mmol/l a There was no statistically significant difference over time or between groups b Multiple comparisons showed all for all groups, the means were statistically different from baseline to 6 hours (p<0.05).

29 Prospective Evaluation of a Hypertonic Balanced Ketone Solution for use in Neuroprotective Strategies

In a final analysis there was evidence of correlation between BHB blood and CSF at 6 hours (correlation = 0.81, p<0.0001) but not at baseline (correlation = 0.0, p=0.99) or 1 hour (correlation = 0.31, p=0.26). There was evidence of a correlation between BHB blood and brain at 6 hours (correlation = 0.67, p=0.012) as well as between BHB CSF and brain at 6 hours (correlation = 0.62, p=0.023). In both groups, change over time was significant: values at 6 hrs significantly > 1 hr significantly > baseline for both plasma and CSF. There was a significant difference in plasma BHB concentrations between BHB 120 and BHB 40 at 1hr and 6 hrs.

Figure 2.1 Plasma beta-hydroxybutyrate profiles

BHB, beta-hydroxybutyrate; BHB 40, 3% NaCl with 40 mmol/l; BHB 120, 3% NaCl with 120 mmol/l

2.6 Discussion

We were able to demonstrate that a continuous infusion of BHB in non-fasted rats led to steadily increasing plasma and CSF concentrations of BHB. Furthermore, BHB concentrations in both plasma and CSF increased as the BHB concentration in the intravenous formulations increase. Despite this, there were no significant effects on acid- base metabolism at concentrations used. Furthermore, the solution achieved a desirable level of hypernatremia necessary for managing intracranial hypertension.

Prior studies have only examined the effects of short term BHB infusions on BHB concentrations. Suzuki et al used infusions of BHB to examine effects in acute brain injury but blood concentrations were not reported 73. Katayam et al administered an isotonic 30 Prospective Evaluation of a Hypertonic Balanced Ketone Solution for use in Neuroprotective Strategies solution of BHB at 30 umol/kg/min for 2 hours in a hemorrhagic model 119. This led to BHB concentrations of approximately 1.5 mmol/l. However, the treatment animals demonstrated a significant alkalosis with final pH 7.54. Prins et al while examining the cerebral uptake of BHB in rat model of TBI administered 30 mg/kg/h for 3 hours 75.

Figure 2.2 Plasma and CSF BHB concentrations with different fluids at various time points.

BHB, beta-hydroxybutyrate; BHB 40, 3% NaCl with 40 mmol/l; BHB 120, 3% NaCl with 120 mmol/l

Comparison with previously published data

There was an increase in BHB concentrations, to a peak of 0.41 mmol/l with no alteration in pH. As part of a study examining effects of BHB on cerebral blood flow and cerebral oxygen delivery, Linde et al examined the effect of administering 37.5mg/kg/min of BHB to rats over a period of 2 hours 118. They demonstrated a rapid increase in pH to 7.52 which loosely correlated with the increase in serum BHB concentrations to 6.08 mmol/l.

Some authors have suggested that high plasma concentrations of BHB are necessary to produce a beneficial effect on cerebral energetics. As such many studies have administered large concentrations of BHB over short periods of time to rapidly increase blood BHB 42. This is not necessarily true as relatively small increases in BHB may be sufficient to improve cerebral metabolism. Although a correlation exists between blood BHB concentration and cerebral uptake there is period of adaptation as the MCT transfer molecules are upregulated over time 120, 121 Therefore, cerebral uptake of BHB is not only dependent on plasma concentration but also increases following prolonged hyperketonemia 42, 43. This was

31 Prospective Evaluation of a Hypertonic Balanced Ketone Solution for use in Neuroprotective Strategies demonstrated by Linde et al following an acute increase in peripheral BHB concentrations to greater than 6 mmol/l 118. They observed no decrease in glucose metabolism and no substitution of glucose by ketone bodies for oxidate metabolism, suggesting adaptation had not yet occurred. Pan et al demonstrated a small, but statistically significant increase in cerebral ketones (0.24 ± 0.04 mmol/l) following a 75 min infusion of BHB but this was considerably lower in comparison with previous data acquired from fasted adults 43. These findings suggest the way the brain handles BHB is different following acute as compared to chronic hyperketonemia and likely represents transport upregulation.

We demonstrated that ketones could be readily measured in CSF and that there was a progressive increase in CSF concentration. We also found an increase in CSF concentrations related to concentration of solution administered. This is despite the fact that the rats were not starved before the study. Furthermore, the effect was likely related to BHB infusion as CSF concentrations did not change significantly in the control group. Similarly, there was a significant increase in brain tissue BHB concentrations which depended on the amount of BHB administered. Pan et al using H+ magnetic resonance spectroscopy were able to show an increase in occipital lobe concentrations of BHB in healthy human subjects following a 75 min infusion of BHB 43. Although Pan reached blood concentrations of 2.12 mmol/l the cerebral BHB concentration was only 0.24 mmol/l whereas we achieved a significant increase by administering a lower concentration of BHB over a prolonged period. Furthermore, there was no comment on the effects of these concentrations on pH. Pan previously demonstrated the effects of prolonged ketone exposure on cerebral concentrations with significant increases in cerebral ketones after 48 and 72 hours starvation 42.

Several prior studies have demonstrated the development of a significant metabolic alkalosis following BHB infusion 95. Blood pH reached a maximum of 7.6 following BHB infusion during the study by Linde et al. Concentration, rate of administration and length of administration are likely to be important factors in determining acid/base response. Metabolic alkalosis has several potential deleterious effects on cerebral metabolism 122-124. These include a decrease in CBF, lethargy and confusion and if severe, coma and seizures. More specifically, Gillard et al found an alkaline pH exacerbated glutamate induce excitotoxic neuronal damage and appeared to sensitize neurones to ischemic injury and potentiate reperfusion injury 125. Alkalosis has been shown to decrease CBF in a number of animal studies by both Arvidsson et al and Pannier et al 122, 124. Furthermore, Schrock et al

32 Prospective Evaluation of a Hypertonic Balanced Ketone Solution for use in Neuroprotective Strategies demonstrated a decrease in CBF and glucose utilization during severe hypochloremic alkalosis 123. This is in contrast to the ketogenic diet which generates protons and pH- lowering metabolic products. The decreased pH was initially considered to be a key seizure preventing aspect of the KD. However, there is little evidence that ketone administration decreases cerebral pH other than briefly and less that this is associated with anticonvulsant activity.

Bourdeaux et al on the other hand, found infusions of 8.4% sodium bicarbonate effective at reducing intracranial pressure although whether the hypertonic nature of the solution or alkalinizing effects were responsible is not clear 126. We found that concentrations of BHB up to 120 mmol/l were well tolerated with no significant change in base excess over time. Others have observed BHB containing solutions to be alkalinizing at high doses when administered over short periods 118. This is likely due to the insufficient time for acid/base homeostasis to occur. The clinical effects of this have not been investigated.

As one of the potential uses of this solution includes the management of intracranial hypertension in patients with traumatic brain injury, a key outcome was to demonstrate the solution to be capable of producing hyperosmolar conditions. All solutions were able to achieve this and the concentration of BHB did not alter the outcome. Hyperosmotic therapy has been the corner stone of the management of intracranial hypertension for a number of years. In many units, hypertonic saline has replaced mannitol as the principle solution 127. HTS has a number of potential benefits including a high coefficient of reflection, maintenance of BBB integrity, modulation of inflammatory response and augmentation of volume resuscitation. Furthermore, a number of animal and human studies have confirmed it as an effective treatment for intracranial hypertension 128, 129. A combined solution of hypertonic saline and BHB could provide the benefits of hyperosmolar therapy with improved cerebral energetics 130.

Limitations

There are several limitations of our study. Although the infusion lasted 6 hours, longer than most prior studies, this is still relatively short. The effects of longer duration infusions are unknown although a number of studies have examined prolonged hyperketonemia induced by either diet or starvation. Secondly, we did not measure clinical endpoints such as intracranial pressure, cerebral perfusion pressure or CBF. Nor did we look at cerebral metabolic neuroprotective endpoints such as reductions in reactive oxygen species and

33 Prospective Evaluation of a Hypertonic Balanced Ketone Solution for use in Neuroprotective Strategies mitochondrial permeability transition complex although prior studies have demonstrated improved brain energetics from ketones as compared to glucose. And thirdly, we used healthy rats. The pharmacokinetic and pharmacodynamic effects cannot necessarily be extrapolated to head injury models as the blood brain barrier permeability and cerebral metabolism cannot be predicted. Experiments in acute brain injury models demonstrate an increase in uptake and utilization of ketones leading to decrease contusion size.

2.7 Conclusion

We have demonstrated that IV infusions of hypertonic saline/BHB are possible and lead to increased plasma and CSF BHB concentrations in healthy rats and that increases in brain concentrations of BHB are dependent on plasma concentrations. Solutions containing 40 and 120 mmol/l of hypertonic NaBHB do not lead to metabolic acidosis and are capable of producing adequate hypernatremia.

Following on from the animal work, the next stage of the project is the development of an intravenous hypertonic solution containing BHB that can be administered to humans. A number of challenges exist including determining the optimal concentration of ketones necessary to impact on cerebral energetics and the overall impact on outcome. The pitfalls of such a project are examined in the next chapter.

34 Proposed Project: Hypertonic Ketone Infusion for the Management of Acute Brain Injury

Chapter 3: Proposed Project: Hypertonic Ketone Infusion for the

Management of Acute Brain Injury

3.1 Synopsis and Citation

Aims:

The aim of this chapter was to examine the challenges involved in producing an intravenous hypertonic ketone solution and potential alternatives to the parenteral route for increasing the plasma ketone concentration.

Citation:

This chapter has been adapted from:

An oral presentation titled “Ketone infusion for the management of acute brain injury” by H White and B Venkatesh. This was presented at the ANZICS CTG Winter meeting Melbourne 2012. (Oral presentation by H White).

35 Proposed Project: Hypertonic Ketone Infusion for the Management of Acute Brain Injury

3.2 Development of a hypertonic ketone solution for humans with brain injury

Following the results from the animal study (see chapter 2), the next stage of the project was to develop and assess the effects of a hypertonic saline/BHB (HTS-BHB) solution in human subjects with brain injury. Intravenous ketone solutions have been evaluated in humans before 95. Outcomes differ but mainly relate to effects on cerebral concentrations of BHB and varying metabolic endpoints. Of note is that there are no reports of serious adverse events from administration of intravenous ketone containing solutions in humans. Below is a summary of human studies of intravenous ketones;

Studies of intravenous ketones in humans

Owen et al (1973) investigated the influence of ketone bodies on insulin and FFA release in response to rapid IV infusion of sodium acetoacetate (NaAcAc) 96. The ketones solution was made from ethyl acetoacetate. It was administered as a rapid solution of NaAcAc at 1.0 mmol/kg after overnight fast in 12 adults weighing between 88 – 215% of ideal body wt. Ketone concentrations peaked 30 min after infusion at 4.74 mmol/l and then rapidly declined (similar to long term starvation). Glucose concentrations remained stable. The main finding from this study was that the utilization of ketone bodies increased with rising blood concentrations, with a plateau at approximately 128g/24 hours. Also, when total ketone body concentrations were below 2.0 mmol/l, there was a linear relationship between ketone body utilization rates and concentration.

Miles et al (1983) investigated the potential protein sparing effects of ketone bodies using an intravenous infusion of sodium beta-hydroxybutyrate (NaBHB), prepared as a 3M solution 131. Six healthy volunteers received the infusion at a rate of 15-30 umol/kg/min for 3 hours. Blood ketone concentrations increased to 2.33 mmol/l while lactate peaked at 0.95 mmol/l. pH increased from 7.43 to 7.52. Contrary to the belief of many, the infusion of a ketone salt is likely to have an alkalinizing effect. Glucose remained stable at 4.7 mmol/l. No adverse effects were reported and the infusion did not appear to decrease proteolysis.

Hiraide et al (1991) looked again to further assess the potential protein sparing effects of a ketone infusion in 20 trauma patients 94. Eleven patients received a 3 hour infusion of BHB at 25 umol/kg/min while 9 control patients received a sodium lactate infusion at a similar rate. Total ketone concentrations in the intervention group reached a peak of 1.5 ± 0.42 mmol/l. Glucose concentrations remained unchanged but a decrease alanine release from

36 Proposed Project: Hypertonic Ketone Infusion for the Management of Acute Brain Injury muscle was demonstrated suggesting that ketones have a suppressive effect on posttraumatic protein catabolism.

CNS effects of intravenous ketone solution

A number of investigators have investigated the ability of exogenous ketone solutions to enter the cerebral tissue. Pan et al (2001) measured BHB in the human brain of 6 normal adult volunteers following an infusion of Na-BHB 200 mmol/l solution 42, 43. The 6 patients fasted overnight and then received an infusion of BHB at a bolus rate of 80 um/kg/min followed by 20 um/kg/min for 75 min. BHB blood concentrations reached a steady state of 2.12 ± 0.30 mmol/l. BHB concentrations in the occipital lobe of the brain were measured using MRI spectroscopy. BHB brain concentrations were measured at 0.24 ± 0.04 mmol. Although elevated, this proved to be lower than brain tissue concentrations measured after a 48 hour fast, suggesting that upregulation of MCT transporter is required before cerebral BHB concentrations can increase significantly.

Similarly, Blomqvist et al (2002) investigated the factors influencing BHB utilization and uptake by the brain in 6 patients with diabetes and 6 non-diabetic volunteers 132. Using positron emission tomography, they studied the effect of acute hyperketonemia (range 0.7- 1.7 mmol/l) on cerebral ketone body utilization. The volunteers received an infusion of BHB for one hour followed by a bolus injection of radiolabelled BHB. Utilisation rates were found to correlate with plasma ketone concentrations in both groups, confirming that BHB utilization by cerebral tissue is dependent on transportation into the brain.

Smith et al (2005) reported on a novel compound called KTX 0101 (which was undergoing development) 88. They reported that the compound was administered to 20 healthy volunteers, was well tolerated and with no serious adverse events. The company, KetoCytonyx was proposing to develop the solution for neuroprotection in cardiopulmonary bypass. However, no final publication was ever submitted and the product is not available commercially

As can be seen from the above studies, a number of patients have received ketone administrations intravenously without major side effects. The key points are:

1. Intravenous BHB appears well tolerated 2. Problems with alkalemia are likely due to the high relative concentrations of BHB administered over a short period. While the concentration of BHB in the brain is

37 Proposed Project: Hypertonic Ketone Infusion for the Management of Acute Brain Injury

related to plasma concentrations, there is also a relationship with time as protein transporters are required to carry BHB across the blood brain barrier and this process takes time to up regulate. 3. No outcome studies have been undertaken in patients with traumatic brain injury. 4. There are no studies evaluating hypertonic saline BHB solutions exist.

We therefore intended to undertake a study using a prolonged infusion of hypertonic saline/BHB which will examine a number of clinical endpoints.

3.3 Proposed Project Plan

Objectives: To safely deliver a 72 hour continuous infusion of 3% HTS-BHB (80 mmol/l) and demonstrate hyperosmolality could be achieved.

Secondary objectives:

• Demonstrate a significant increase in serum ketones as compared to control. • Demonstrate equivalent control of ICP and CPP compared to controls • Demonstrate stable acid-base status during infusion

Study design

The study was to take place at a tertiary ICU where neurological services manage patients with severe traumatic brain injury. Our aim was to recruit 40 patients with brain injury and intracranial pressure monitoring.

Following admission to ICU, patients would have been randomized into 2 groups. The control would have received standard care including 3% hypertonic saline for management of intracranial hypertension. The intervention group would have received 3% hypertonic saline/BHB (80 mmol/l). The solution infusions would have begun at 40 mls per hour. The subsequent rate of infusion would have been titrated to maintain Na in the range of 145 – 155 mmol/l. The infusions of HTS/BHB would have continued for 72 hours and then replaced with HTS at the discretion of the treating consultant. Observations were to continue for a further 24 hours.

Formulation of solution:

The NaBHB solution to be used for the infusion was to be formulated as follows;

38 Proposed Project: Hypertonic Ketone Infusion for the Management of Acute Brain Injury

10.08 g of sodium D-3-hydroxybutyrate will be combined with 24.78 g of NaCl in 1 L of water. Sterility and stability studies will be undertaken as part of the product development.

Data Collection

For all patients, data was to be collected at baseline and then 4 hourly for 96 hours. This included:

• Vital signs including GCS and temperature • Intracranial pressure • Medication used for sedation and paralysis • Inotropes and pressor use • Calorie intake

Laboratory data would have been collected a minimum of 12 hourly

• ABG • Electrolytes • Beta-hydroxybutyrate and acetoacetate • Lactate and glucose • Patients with intraventricular drains – CSF BHB, glucose, lactate

At the end of the study, a lipogram would have been performed to assess effects on lipid metabolism

Study population

Inclusion criteria;

• 18 years of older • Severe traumatic brain injury requiring intracranial pressure monitoring • Life expectancy of at least 72 hours

Exclusion criteria

• Failure to obtain written consent from patient or substitute decision maker • Pregnancy • Non-survivable injury or brain death • Patients considered for organ donation

39 Proposed Project: Hypertonic Ketone Infusion for the Management of Acute Brain Injury

• Type I diabetes

Data analysis

Categorical data would have been compared using Fishers Exact test. Continuous data would have been compared using Wilcoxon 2-sample test. Repeated measures continuous data was to be analysed using linear mixed models.

Outcomes and significance:

Study outcomes

Primary outcome

• Achievement of a target sodium of 145 – 155 mmol/l • Beta-hydroxybutyrate and acetoacetate concentrations in blood

Secondary outcome

• Acid-base status including pH and Base Excess • ICP and CPP • Glucose and lactate concentrations

As this study would be used to understand the power of a larger trial, we would be recording ICU/hospital length of stay and mortality although we do not expect to see significant differences in these outcomes.

Adverse events

There is minimal data on the adverse effects of ketone administration, much of which is based on chronic intake via the enteral, rather than the parenteral route. In the acute setting, effects of parenteral formulations on pH, Na, lipids and glucose are likely to dominate e.g. IV formulations would theoretically be alkalinising, depending on the ketone concentration. This was confirmed by Hiraide et al who noted a significant increase in pH and Na concentrations following the IV administration of a 20% solution of NaBHB to severe trauma patients94. Also of potential concern is the reduction in glucose cerebral metabolism and increase in CBF demonstrated by Hasselbalch et al during administration of intravenous BHB 58. The long term consequences of this are not known.

40 Proposed Project: Hypertonic Ketone Infusion for the Management of Acute Brain Injury

Some side effects are predictable following enteral administration such as dehydration and hypoglycemia. Others are less common and present following prolonged use, including growth retardation, obesity, nutrient deficiency, decreased bone density, hepatic failure, and immune dysfunction. Freeman has reported a significant rise in mean blood cholesterol to over 250 mg/dl following prolonged intake a of ketogenic diet 99. This in turn may be atherogenic, leading to lipid deposition in blood vessels. In addition, there are reports of dilated cardiomyopathy in patients on the ketogenic diet, possibly as a consequence of the toxic effects of elevated plasma free fatty acids. Finally, an increased incidence in nephrolithiasis in patients on the ketogenic diet as well as increases in serum uric acid secondary have been reported 76, 100.

The fact that patients are critically ill and monitored continuously in an ICU should mitigate against any unrecognized side effects. Patients will also receive continuous nutrition as per standard ICU protocol. Glucose, pH and Na will be regularly measured.

In this study, reporting of adverse events would have been restricted to events that are considered to be related to study treatment. Serious adverse drug reactions (SADRs) that were thought to be study treatment related would be reported immediately to the chief, or co-investigators. These would have then been reported to the HREC. The definitions of SADRs were as follows;

• Results in death • Is life-threatening • Requires inpatient hospitalisation or prolongation of existing hospitalisation • Results in persistent of significant disability • Is a congenital anomaly

3.4 Challenges to PhD in its current design and proposed changes to PhD title and project focus

Following discussions with several compounding chemists including Stenlake (Sydney, NSW), it became apparent that while the production of an intravenous hypertonic saline solution containing BHB was feasible, it was also extremely costly. The main reason for this was not related to the production of the solution as such but rather high cost of sourcing the BHB compound. The estimates at the time were approximately 1000 AUD per one litre bag (personal correspondence). Given that patients would require several bags over the course

41 Proposed Project: Hypertonic Ketone Infusion for the Management of Acute Brain Injury of their treatment, the costs for the proposed study were in the region of 100 000 AUD. Applications were made to both Queensland Emergency Medical Research Fund (QEMRF) and ANZICS Research Foundation for funding but was declined. A presentation was also made to the Australia and New Zealand Intensive Care Society Clinical Trials Group (ANZICS CTG) but did not receive favorable review. The reasons for rejecting the funding application included; concern about the lack of animal and human data required for a phase II study, the high cost of the product making the funding of further studies unlikely, the uncertainty related to the efficacy of the chosen concentration of BHB and concern regarding an appropriate outcome measure for the study.

The mechanism by which ketosis is induced does not appear to impact on the clinical outcome. Therefore, the oral route was investigated as an alternative option. A major challenge is that inducing ketosis in adult humans with conventional ketogenic feeds is difficult. Furthermore, the concentrations of BHB attainable purely through feeding may not be adequate for ABI patients. A group in Oxford University led by Professor Kieran Clarke was investigating the production of a ketone ester which had the potential for rapidly increasing plasma ketone concentrations. It was clear however that the product was still in development and it would be a number of years before it became available.

As such we were forced to look at the use of a commercially available ketogenic feed, KetoCal. This would require certain modifications as the feed was not designed for critically ill adults. This process is further discussed in Chapter 7.

It was therefore necessary to alter the focus of this PHD to evaluating ketosis induced by enteral formulation. As such, the title of the PHD was altered from

“Development of a novel solution for treating patients with acute brain injury” to

“Evaluation of a ketogenic formulation for treating patients with acute brain injury”

The following chapters will investigate the potential issues involved in providing ketogenic feeds enterally to critically ill patients with ABI, evidence supporting ketogenic feeds in this group of patients, alternatives to ketones for improving cerebral cellular energetics and the outcomes from providing a modified ketogenic feed to a cohort of patients with ABI.

42 Serial changes in plasma ketone concentrations in patients with acute brain injury

Chapter 4: Serial changes in plasma ketone concentrations in patients

with acute brain injury

4.1 Synopsis and Citation

Background:

Acute brain injury (ABI) is a catastrophic event, leading to disruption of the normal cerebral metabolic pathways and a subsequent cerebral energy deficit. Ketones (beta- hydroxybutyrate and acetoacetate) may represent an alternative metabolic substrate with the potential to improve cerebral energy supply and decrease injury.

Aim:

The purpose of this study was to evaluate baseline ketone concentrations in the ABI population.

Design:

This was a prospective cohort observational study of 38 patients with ABI followed for up to 7 days. We collected arterial blood samples immediately after admission and daily to measure the concentrations of beta-hydroxybutyrate and acetoacetate. Where possible, matching cerebrospinal fluid (CSF) specimens were also collected.

Key Findings:

During the study period, plasma BHB concentrations were increased initially but normalized by day 3 while acetoacetate concentrations remained within the normal range. The change in BHB was significant. There were 30 observations in 10 patients where BHB could be measured in both blood and CSF. When the data was averaged over patients there was a weak correlation between blood and CSF BHB (Spearman’s rho = 0.62, P=0.054).

Conclusion:

Blood ketone concentrations remain low within the ABI population. An external source of ketones will be required to increase blood concentrations to clinically relevant levels.

43 Serial changes in plasma ketone concentrations in patients with acute brain injury

Citation:

The material presented in this chapter has been previously published as an original manuscript. Figures and tables have been inserted into the text at slightly different positions and the numbering of pages, figures and tables has been adjusted to fit the overall style of the thesis. The references have been incorporated with the other references of the Thesis.

The manuscript “Serial changes in plasma ketone concentrations in patients with acute brain injury.” by Hayden White, Balasubramanian Venkatesh, Mark Jones & Hesly Fuentes was published in Neurological Research 2017;39:1-6 and extracts are reproduced with kind permission from Taylor and Francis, copyright 2017.

Contributions:

This prospective observational study was performed at Logan Hospital (Meadowbrook) and Princess Alexandra Hospital (Brisbane), Australia. I commenced this work in May 2013 and was involved in the initial concept, all aspects of study design, data collection, data analysis and manuscript preparation. This work was carried out with assistance from Ms Lynette Morrison (data collection), Dr Mark Jones (statistical analysis), Ms Kellie Sosnowski (data collection), Professor Bala Venkatesh (study design and manuscript preparation), Dr Hesley Fuentes (data collection) and Mr Jason Meyer (recruitment and data collection).

44 Serial changes in plasma ketone concentrations in patients with acute brain injury

4.2 Introduction

Acute brain injury (ABI) including, traumatic brain injury (TBI), cerebrovascular accident (CVA) and subarachnoid haemorrhage (SAH) are common and catastrophic events resulting in significant morbidity and mortality. Much of the pathophysiology leading to the ongoing injury relates to disruption of the normal metabolic pathways of the brain and the subsequent inability to adequately meet cerebral energy requirements. While glucose under normal circumstances provides the major substrate for cerebral metabolism, hyperglycemia clearly leads to worse outcomes following ABI133. Ketones may represent an alternative source of fuel in these instances 1.

Ketogenesis is the process by which ketone bodies (beta-hydroxybutyrate and acetoacetate), during times of starvation or catecholaminergic stress, are produced via fatty acid metabolism. In health, glucose is the brain’s main metabolic substrate for fulfilling energy requirements 4. During times of oxidative stress however, the brain may alter its ability to use glucose as a substrate for metabolic activity134. During these periods, ketones are capable of supplying up to 70% of basal cerebral energy requirements 51. There is evidence that after acute brain injury, ketone utilization may be preferred to glucose, that it improves cerebral energy production and decreases cerebral injury 135. Animal models simulating both rapidly developing pathologies (glutamate excitotoxicity, hypoxia/ischemia) and neurodegenerative conditions (Parkinson’s disease, Alzheimer’s disease) and more recently TBI have found ketosis produces a number of beneficial effects including; improved cerebral energetics, decrease cerebral injury and improved cerebral blood flow 73, 136.

There is a paucity of research examining ketones in critically ill patients with ABI and the role of ketone supplementation. Existing research has demonstrated a number of potential benefits to exogenous ketone administration. These include; that ketones can be given enterally or intravenously with minimal side effects, utilization of ketone bodies increase with rising blood concentrations, hyperketosis is associated with improved glucose control, increased plasma concentrations lead to increase in cerebral uptake and improvement in cognitive function in patients with chronic neurodegenerative disorders. However, well designed outcome studies are lacking 43, 83, 91, 94.

Many unanswered questions remain including optimum route of administration and target plasma concentration. Generally, plasma ketone concentrations can range from <0.1 mmol/l in the postprandial state to over 6 mmol/l during prolonged fasting/starvation states and may

45 Serial changes in plasma ketone concentrations in patients with acute brain injury reach 25 mmol/l in uncontrolled diabetes. However, little is known about ketone physiology during critical illness and ABI.

Tamaki et al investigated the physiologic changes to ketone bodies in patients with SAH 137. They noted a strong correlation between ketone body concentrations and catecholamine levels. Although the pathogenesis differs, many regulatory hormones are abnormally raised post ischemic stroke, TBI or SAH. As such, catecholamine, cortisol and glucocorticoid levels are universally increased acutely while there is evidence that the production of counter regulatory hormones such as insulin may be impaired 138-140.

The principle drivers for ketogenesis include fasting (glucopenia) and catecholaminergic stress1. We hypothesised that ketone concentrations would remain low in patients with ABI although, the impact of critical illness with its associated catecholaminergic stress is unpredictable. Furthermore, the unpredictability of enteral absorption during the critically ill period may lead to altered glucose and insulin metabolism. Prior to undertaking studies on ketone supplementation in these patients, it is important to quantify baseline concentrations. The purpose of this study is to examine the physiological changes in ketone concentrations following ABI.

4.3 Materials and Methods

This dual-site, prospective, observational study was conducted over a 12 month period to define baseline ketone concentrations in plasma and cerebrospinal fluid (CSF) following acute brain injury. The study setting was two metropolitan Intensive Care Units (ICU). Ethics approval was granted by the Princess Alexandra Hospital Human Research Ethics Committee (HREC/13/QPAH/150). The requirement for written informed consent was waived by the institutional review board.

Patients were enrolled as soon as possible following admission to ICU or following diagnosis of CVA. All patients > 18 years old with an ICP monitor admitted to ICU following TBI, SAH or acute CVA were included. (CVA patients did not require ICP monitor to be included). Exclusion criteria included type I diabetes and pregnancy.

Patients’ fulfilling the inclusion criteria had blood samples collected immediately following enrolment and then daily from the morning specimens. All clinical observations were taken on admission and then from 8 am daily. Patients remained on study for 7 days or until death or discharge from ICU. Samples were obtained simultaneously for ketones (both Beta-

46 Serial changes in plasma ketone concentrations in patients with acute brain injury hydroxybutyrate and acetoacetate), glucose and arterial blood gases: (pH, CO2, HCO3, base excess, anion gap, lactate). If an extraventricular device was present (SAH), CSF was tested for BHB and Acetoacetate.

Acetoacetate concentrations were determined using an automated modification of the original endpoint procedure described by Williamson et al on a Cobas Bio centrifugal analyser17 (Roche, Basel, Switzerland). Specimens were collected in lithium heparin tubes (#456083, Greiner Bio-One, Kremsmunster, Austria). The linearity of the assay is 0.008-1.0 mmol/L (CV 3.6% at 0.003 mmol/L and 2.5% at 0.437 mmol/L).

Beta Hydroxybutyrate (D-3-Hydroxybutyrate) was measured quantitatively using the Stanbio Laboratory β-Hydroxybutyrate LiquiColor Assay Kit (Boerne, TX, USA) on the Beckman DxC800 Unicell (Brea, CA, USA) analyser using a spectrophotometric endpoint assay. Specimens were collected in lithium heparin tubes (#456083, Greiner Bio-One, Kremsmunster, Austria). The linearity of the assay is 0.0-4.0 mmol/L with the coefficient of variation (CV) 3.0% at 0.38 mmol/L and CV 2.1% at 1.38 mmol/L

Patients were fed a weight based diet with a caloric goal of achieving 25 – 30 kcal/kg/d and a protein intake of 1 – 1.2 g/kg/d.

Demographic data was collected including participant age, sex and primary diagnosis. Further clinical data including intracranial pressure (ICP), Glasgow Coma Scale (GCS) and nutrition (number of days of enteral nutrition) were also collected.

With 33 included patients we had 80% power to detect a half standard deviation change in Ketone concentration over the 7 days of follow up assuming a 5% level of significance. A linear mixed model was used to estimate the change in Ketone concentration over the 7 days of follow up. Within patient correlations were accounted for by the inclusion of random effects for each patient.

4.4 Results

38 patients with ABI were recruited into the study (22 F and 16 M) and followed for up to 7 days (Table 4.1). The admission diagnoses included 19 CVA, 8 SAH and 11 with TBI. 22 patients completed the full 7 days observations, 7 CVA, 7 SAH and 8 TBI. The overall mortality rate was 23%. During this period the mean highest ICP varied between 18 mmHg and 31 mmHg and CPP from 66 mmHg to 82 mmHg (Table 4.2). There was no evidence of

47 Serial changes in plasma ketone concentrations in patients with acute brain injury an association between BHB and ICP based on Spearman correlation (p=0.46 for ICP lowest and p=0.96 for ICP highest).

Table 4.1. Demographic data of patients with acute brain injury

N Age Sex (m / f) LOS (hours) ICU Mortality (%)

Total 38 60 ± 19 16 / 22 266 ± 250 23.7

CVA 19 61 ± 19 179 ± 147 15.8

SAH 8 64 ± 13 456 ± 418 12.5

TBI 11 55 ± 21 277 ± 161 45.5

All data were expressed as mean± standard deviation or number (percentage). CVA: Cerebrovascular accident; SAH: subarachnoid haemorrhage; TBI: traumatic brain injury

Table 4.2. Clinical and biochemical observations measured daily over trial period

Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7

ICP (mmHg) 20±10 18±6 22±14 24±13 25±11 31±17 25±13

CPP (mmHg) 66±11 73±10 80±24 82±22

BHB(plasma) 0.29 ±0.33 0.32±0.58 0.19±0.24 0.2 ±0.23 0.22±0.41 0.1 ±0.06 0.08±0.03 (mmol/l)

Acetoacetate 0.19 ±0.16 0.16±0.29 0.09 0.05 0.12±0.10 0.13±0.15 0.09±0.08 0.05±0.04 (plasma) (mmol/l)

Glucose(mmol/l) 8.26 ±5.52 8.25±2.31 8.3 ±2.04 7.87±1.61 8.18±2.04 8.10±15.9 8.17±2.36

Lactate (mmol/l) 1.69 ±1.09 1.46±0.63 1.49±0.65 1.32±0.55 1.27±0.58 1.62 ±1.8 1.25±0.49

pH 7.41 ±0.05 7.43±0.06 7.44±0.07 7.45±0.07 7.45±0.05 7.44±0.07 7.44±0.05

HCO3(mmol/l) 22.67±3.24 23.7±3.45 24.9±6.14 25.3±3.76 25.8 ±3.8 25.8±4.08 25.8±3.58

Na(mmol/l) 140±4.8 141±4.9 141±6.1 143±5.9 142±6.9 142±8.04 142±7.28

Ag 6.5 ±3.0 5.4±2.45 5.6±3.15 10.6±2.05 6.5 ±2.7 5.9 ±3.30 6.5 ±2.7

CPP: cerebral perfusion pressure; ICP: intracranial pressure; BHB: beta-hydroxybutyrate; Ag: anion gap

During the study period, plasma BHB concentrations were increased initially but normalized by day 3 and acetoacetate concentrations remained within the normal range (normal BHB < 0.20 mmol/l, acetoacetate < 0.30 mmol/l). The change in BHB was significant (Table 4.3).

48 Serial changes in plasma ketone concentrations in patients with acute brain injury

There was no statistically significant difference in BHB concentrations between genders a gender difference in BHB (P>0.05). More specifically there was insufficient evidence of a difference at baseline (P=0.24) or over time (P=0.085). Plasma lactate concentrations peaked at 1.69 mmol/l and glucose varied between 7.87 mmol/l and 11.21 mmol/l. There were 30 observations in 10 patients where BHB could be measured in both blood and CSF (in patients with SAH). There is no evidence of differences in blood BHB by diagnostic group (P=0.39) or evidence of interaction between trend over time and diagnostic group (P=0.19). When the data are averaged over patients there is weak evidence of correlation (Spearman’s rho = 0.62, P=0.054).

Table 4.3 Trends over time based on mixed models analysis

Estimate SE P-value

BHB -0.033 0.011 0.004*

Acetoacetate -0.010 0.007 0.14

pH 0.0076 0.0017 <0.0001*

Lactate -0.044 0.032 0.16

Glucose 0.21 0.21 0.32

PaCO2 0.51 0.32 0.11

* PaO2 -3.7 0.91 <0.0001

Bicarbonate 0.62 0.081 <0.0001*

Sodium 0.15 0.13 0.25

Anion gap 0.11 0.34 0.76

BHB: beta-hydroxybutyrate

*P<0.05

These results as shown in Table 4.3 suggest BHB and PaO2 decreased over the 7 days; pH and bicarbonate increased; but there was insufficient evidence to show changes over time for any other variable.

The results in Table 4.4 were based on decomposing the time-dependent covariates (pH, bicarbonate, glucose) into components based on the mean values for each patient; and the differences within patients 141. There was no evidence of association between the time- dependent covariates and acetoacetate and also between glucose and BHB but there was

49 Serial changes in plasma ketone concentrations in patients with acute brain injury evidence of associations between the differences in pH and bicarbonate within patients and BHB, however there was no evidence of association between the mean values of each patient for pH and bicarbonate, and BHB. There is therefore evidence that both pH and bicarbonate are associated with BHB but that this association is due to differences within patients and not differences between patients.

Table 4.4 Correlations between parameters based on mixed models

Outcome Predictor Estimate SE P-value

BHB pH (mean) -0.99 0.86 0.25 pH (difference) -1.27 0.53 0.018*

BHB Bicarbonate (mean) -0.0071 0.013 0.58 Bicarbonate -0.033 0.0091 0.0004* (difference)

BHB Glucose (mean) -0.014 0.015 0.34 Glucose (difference) 0.00066 0.0038 0.87

Acetoacetate pH (mean) -0.21 0.58 0.71 pH (difference) -0.13 0.32 0.68

Acetoacetate Bicarbonate (mean) -0.0041 0.0083 0.62 Bicarbonate -0.0054 0.0052 0.31 (difference)

Acetoacetate Glucose (mean) 0.0074 0.011 0.50 Glucose (difference) 0.011 0.0066 0.10

BHB: beta-hydroxybutyrate

*P<0.05

Note that there were only 6 (3%) occasions when patients did not received nutrition. This occurred for 5 patients at day 1 and for 1 patient on day 2. Due to the small number of patients not fed over the follow up period this variable was not included in the analysis.

4.5 Discussion

The purpose of this study was to examine baseline ketone concentrations in blood and CSF following ABI. We noted a reduction in plasma BHB over the course of the study period with concentrations peaking at 0.32 ± 0.58 mmol/l on day 2 and subsequently decreased to 0.08 ± 0.03 mmol/l by day 7. This reduction was seen across all diagnostic groups. This finding may reflect a number of physiological changes during the acute phase of illness. Certainly,

50 Serial changes in plasma ketone concentrations in patients with acute brain injury catecholamine levels are raised following ABI. Termed “autonomic dysfunction syndrome” elevated catecholamine levels were initially described in SAH and stroke but also occur in patients with TBI. Raised catecholamine levels are associated with poor outcome and the use of Beta-blockers to limit secondary injury is associated with improved outcomes post TBI 142. This was confirmed by Tamaki et al who demonstrated significantly raised epinephrine and norepinephrine concentrations post SAH and the corresponding increase in ketones137. Despite only collecting data on admission and day 30, they noted significant decrease in ketone body concentrations which correlated with a decrease in plasma catecholamine concentrations.

Catecholaminergic stress is a major trigger for ketogenesis and leads to partial degradation of long-chain fatty acids liberated from white adipose tissue and subsequent generation of ketone bodies. The other potential trigger for ketone production is glucopenia brought about by fasting1. It is not uncommon for delays to occur in the establishment of adequate nutrition in the critically ill population. The reasons are numerous but include delayed initiation of feeds and a tendency to initial absorption delays in the ABI population20. We postulated that the combination of catecholaminergic stress and inadvertent fasting was responsible for the initial rise in BHB.

Ketone bodies can provide energy to the brain at times of glucose shortage and may displace glucose as preferred energy source in cerebral tissue following ABI. In fact, Owen et al demonstrated that ketone body uptake was sufficient to supply 70% of cerebral energy requirements 27. The optimal concentration of BHB is unknown although laboratory experiments suggest plasma concentrations of approximately 4.0 mmol/l or greater are required to protect against cerebral oxidative stress 143. Although controversial, a similar, plasma concentration is thought to be required to inhibit seizures in children with poorly controlled epilepsy 87. Our results suggest that post ABI, intrinsic concentrations of BHB are insufficient to provide significant cerebral energy augmentation. Exogenous BHB supplementation would be required to raise BHB concentrations to clinically significant concentrations.

The blood brain barrier is relatively impermeable to ketones and BHB is transported into the CSF by a carrier protein which regulates the rate of uptake. Cerebral BHB concentrations are therefore affected by overall plasma concentrations and time. Previous animal research from our group noted that a continuous IV infusion of saline/BHB leads to a dose dependent increase in plasma and CSF BHB concentrations in healthy rats and that increases in brain 51 Serial changes in plasma ketone concentrations in patients with acute brain injury concentrations of BHB are dependent on plasma concentrations 144. Furthermore, we found a correlation between the final plasma, CSF and brain concentrations of BHB, suggesting that cerebral BHB concentrations are dependent on plasma and CSF.

Of the current SAH patients we investigated, we were able to collect 30 matching plasma/CSF specimens noting a correlation coefficient of 0.62. The mean CSF concentration was 0.09 mmol/l. Lamers et al found a similar relationship between blood and CSF BHB in 58 children following a period of fasting (r=0.71). Similarly, Owens et al noted that the CSF ketone concentration was directly proportional to the blood ketone-body concentration in 8 obese patients undergoing a 21 day fast 30, 145. Owen noted relatively low blood (0.070 mmol/l) and CSF (0.042 mmol/l) ketone concentrations initially. However, after a 21 day fast, there was a significant increase in blood BHB (4.95 mmol/l) which was similarly reflected in the CSF (2.09 mmol/l). These and other studies confirm the relationship between blood and CSF BHB. Although starvation is an effective means of increasing blood ketones, the evidence suggests that early feeding improves outcome in critically ill patients 146. Therefore, in order to produce a sufficiently raised plasma concentration an external source of ketones will be required.

Plasma (and subsequently CSF) ketones may potentially be increased by several means. Traditionally ketogenic diets, which consist of high fat, low carbohydrates in varying concentrations, have been utilized for this purpose. Most experience has been gained from using ketogenic feeds to control seizures in children with poorly controlled epilepsy28. Although generally unpalatable, ketogenic diets are fairly effective at increasing plasma ketone concentrations. There is far less evidence in the adult population, where these diets have proven less ketogenic. Ritter et al examined several ketogenic diets in an animal model of ischemia, one of which was subsequently evaluated in adult patients with TBI91. Although the difference between the ketogenic diet and control groups was significant, plasma BHB concentrations remained low, at less than 0.6 mmol/l, well below what may be considered a therapeutic level. Another option is to provide ketones intravenously as a salt. Pan and Blomqvist have demonstrated an increase in plasma and brain concentrations of BHB following an intravenous infusion 43, 132. Hiraide et al were able to increase ketone concentrations to 1.5 mmol/l with a 3 hour infusion of BHB at 25 umol/kg/l94. However, the high cost of producing intravenous ketone formulations makes the IV route uneconomical. More recently Kieran et al have demonstrated significant increase in plasma ketones following the oral administration of a ketone ester, with BHB reaching plasma concentrations

52 Serial changes in plasma ketone concentrations in patients with acute brain injury of up to 3.30 mmol/l 147. In the future, this may provide an effective and affordable means for inducing ketosis.

This study has several limitations. Firstly, the high SD for BHB is due to 4 patients with particularly high values (>1 mmol/l). Three of those patients had cerebrovascular disease and the fourth patient had subarachnoid haemorrhage. It likely also reflects the relatively small sample size. Secondly, catecholamine levels were not measured. The only study of which we are aware comparing ketones and catecholamine levels in patients with SAH performed measurements on day 1 and 30 only. We therefore have no direct comparison over the shorter period. Certainly, it has been demonstrated catecholamine levels are initially high following TBI and then decrease steadily over the subsequent 7 days 138-140. Our results are similar to Ritter et al where ketone concentrations steadily decrease in their control group until about day 4 and then remained low until day 14 91. Thirdly, we did not record the total calorie intake per patient per day. We did however; observe that there were only 6 (3%) occasions when a patient wasn’t fed. The feeding regimens are fairly standard providing 25- 30 kcal/kg/d of energy and 1.0-1.2 g/kg/d of protein. As noted, the main determinant of ketogenesis is low insulin concentrations and high catecholamine levels. Although the average glucose concentrations remained above 7.5 mmol/l, insulin was not measured and its effects are unpredictable in the acute setting. Lastly, exogenous catecholamine administration was not recorded. Studies in health adults have demonstrated a correlation between exogenous catecholamine administration and ketone concentrations 148. It is therefore conceivable that this could play a role in ketogenesis in unstable ABI patients.

4.6 Conclusion

In this study, we have demonstrated that patients with ABI who are fed a standard diet fail to produce significant concentrations of ketosis intrinsically. Thus, to achieve elevated plasma concentrations of ketones, an external source of ketones is required. The results provide a basis to proceed to interventional studies of ketone supplementation in patients with brain injury. The challenge then is to design studies to demonstrate that; ketosis can be achieved using one of these formulations, and that the side effect profile is acceptable and finally, that outcomes can be improved.

Prior to undertaking a study of ketone supplementation, it was necessary to understand the role of other potential cerebral energy substrates with the potential to replace glucose as the preferred energy source. We therefore undertook a review of current literature.

53 Novel metabolic substrates for feeding the injured brain

Chapter 5: Novel metabolic substrates for feeding the injured brain

5.1 Synopsis and citation

Aim:

This chapter reviews the basics of cerebral energetics and examines alternative substrates capable of meeting cerebral energy requirements in both health and disease.

Citation:

This chapter is based on a publication reviewing the use of alternate substrates capable of meeting cerebral metabolic requirements. Figures and tables have been inserted into the text at different positions and the numbering of pages, figures and tables has been adjusted to fit the thesis. The references have been incorporated with the other references of the Thesis.

The original manuscript was:

White H, Kruger P, Venkatesh B. Novel metabolic substrates for feeding the injured brain. In Vincent JL, ed. Yearbook of Intensive Care and Emergency Medicine, 2017. Pa: Springer-Verlag; 2017: 477-487. Associate Professor Kruger and Professor Venkatesh assisted with preparation of the original manuscript.

54 Novel metabolic substrates for feeding the injured brain

5.2 Introduction

Brain injury is common with a high mortality and morbidity with implications for both society and individuals, in terms of cost, loss of production, and long term impairment 149. The cellular and molecular events initiated by cerebral injury are complex, restricting the precision of characterization into primary, secondary and long term as the duration of pathogenic events are variable and can overlap. Following injury, cellular energetics play a vital role in maintaining cerebral homeostasis 150. A better understanding of the impact of substrate supply on the injured brain may help improve management following brain injury and provide novel therapeutic options.

The adult brain consumes approx. 20% of basal metabolism, most of which is provided by the oxidation of 100-120 g of glucose/24 hrs 143. However, in times of starvation or injury, the primary cerebral metabolic substrates may alter. Following TBI, brain injury a number of biochemical changes take place in the brain which diverts the processing of glucose via the normal pathways.

The purpose of this review is to examine cerebral energetics and alternative substrates capable of supplying cerebral energy requirements (Table 5.1).

Table 5.1 Potential substrates for enhancing cerebral energy supply.

Substrate Proposed Mechanism of action

Ketones BHB is more energy efficient then glucose

Increase in free energy of ATP hydrolysis

Increase in intermediary metabolites delivered to citric acid cycle

Protect against glutamate mediated apoptosis by attenuation of reactive oxidant species

Enhancement of GABA mediated inhibition

Increase in cerebral blood flow

Lactate Sparing of cerebral glucose metabolism

Regulation of cerebral blood flow

Protects neural tissue against excitotoxicity as it attenuates neuronal death

55 Novel metabolic substrates for feeding the injured brain

BCAAs Limited protein loss from skeletal muscle

Contribute to synthesis of both inhibitory and stimulatory neurotransmitters

Lower cerebral levels of serotonin and catecholamines

Supplement intermediates in TCA cycle

Triheptanoin Replenish TCA cycle intermediates

Improvement in cerebral energetics

BHB (beta-hydroxybutyrate), GABA (gamma-aminobutyric acid), BCAA (branch chain amino acids)

5.3 Cerebral Metabolism

Despite comprising only 2% of total body weight, the brain receives 15% of cardiac output and can use up to 20% of total body oxygen 151. The main cerebral energy source is derived from ATP produced almost completely from the oxidative metabolism of glucose. As such, 25% of total glucose is metabolised by the brain. During starvation however, hepatic glycogen stores rapidly become exhausted. After several days, brain glucose is produced as a result of gluconeogenesis from amino acids derived from muscle metabolism 152. From a week onward however, hepatic gluconeogenesis decreases and ketone bodies become the dominant fuel source for the brain (Figure 5.1).

Figure 5.1 Ketones as an alternative cellular fuel source

56 Novel metabolic substrates for feeding the injured brain

Ketone bodies added to glucose fundamentally alter mitochondrial metabolism. When added to glucose, a physiological level of ketone bodies reduces the mitochondrial NAD couple, oxidizes the co-enzyme Q couple, and increases metabolic efficiency. These changes are shown on the right side of the figure. The arrows illustrate the effects of ketone bodies compared to glucose alone. Ketone bodies provide an alternative metabolic fuel which can act during blockade of glycolysis.

The injured brain

Following injury, a number of biochemical changes take place in the brain which diverts the processing of glucose via the normal pathways. Disruption of ionic equilibrium shortly following traumatic brain injury (TBI) requires energy to correct, reflected by an initial increase in cerebral glucose uptake 153. This is followed by a prolonged period of glucose metabolic depression. Further disruptions include; shunting of glucose through the pentose phosphate pathway, increase in production of reactive oxidant species and DNA damage and inhibition of glycolytic processing of glucose (See Figure 5.2) 150. As such, other substrates including ketones may provide the injured brain with much needed energy.

Figure 5.2 Sequence of cerebral metabolic changes following traumatic brain injury

57 Novel metabolic substrates for feeding the injured brain

Studying cerebral metabolic dysfunction

The metabolic dysfunction resulting from TBI has been extensively studied. In general, injury is still classified as primary, occurring at time of injury and secondary, a result of complex pathological processes occurring hours to days post injury. Experimental studies have revealed a number of changes including disruption to cellular membranes and homeostasis, release of excitatory neurotransmitters including glutamate, impaired mitochondrial function and generation of free radicals151, 154-156. Mitochondrial failure further exacerbates the cerebral energy deficit leading ultimately to apoptosis and cellular death. Much of the current research is aimed at restoring failing cerebral metabolic pathways and providing supplements to augment cerebral energy supply.

A number of techniques are employed to investigate both the underlying metabolic perturbations following TBI and impact of substrate supplementation in these patients 153. Arterio-venous gradient studies have investigated the cerebral metabolic rate of glucose, the uptake of a number of precursors including lactate, fatty acids, essential amino acids and ketones 157-159. Although simple to perform, they lack the precision and the ability to differentiate between injured from normal cerebral tissues. Microdialysis improves upon these limitations providing continuous sampling from localised areas of interest with the opportunity to analyse a variety of molecules 158. Features associated with adverse outcomes include high concentrations of lactate, high lactate/pyruvate ratio and both high and low glucose concentrations 160, 161. Despite these findings, microdialysis is still an experimental technique. Parameters may be difficult to interpret and owing to the focal nature of the findings, their impact on treatment options is uncertain.

Positron emission tomography (PET) scanning has the advantage of being non-invasive, quantitative and dynamic. PET imaging studies using labelled substrates including 2-deoxy- D-glucose have provided valuable information regarding glucose metabolism, confirming the relative depression in glucose utilisation post TBI 162. Newer techniques however allow PET studies of labelled β-hydroxybutyrate and acetoacetate to determine the metabolic fate of ketone bodies in the brain and other tissues 163. To date studies have confirmed that in the brain, ketosis reduces glucose utilization and increases ketone oxidation. With the steady increase in the availability of tracers, more research into cerebral metabolism will improve our understanding of these pathways.

58 Novel metabolic substrates for feeding the injured brain

Magnetic resonance imaging (MRI) techniques (such as magnetic resonance spectroscopy (MRS)) can be useful in detecting the uptake and metabolism of a number of substrates including N-acetylaspartate, choline-containing phospholipids, creatine and phosphocreatine, lactate, myo-inositol, glutamate, Ketones, gamma-aminobutyric acid (GABA), and lipids 164. An important advantage is their ability to provide biochemical information without the need for tracers and non-invasively. A number of studies have utilized MRS to provide data on cerebral metabolism of both lactate and BHB during starvation and following TBI 42. The main limitation of the technique is the need to transfer critically ill patients to the MRI scanner and the associated risks.

5.4 Potential substrates capable of maintaining cerebral energetics

Neuroprotective effects of ketones

Ketogenesis is the process by which ketone bodies are produced via fatty acid metabolism in the liver 1. Ketones or ketone bodies are by-products of fat metabolism. They consist of four carbon units where part of the molecule contains a carbon-oxygen bond (C= O). They provide an alternative pathway for the metabolism of acetyl CoA through the ß-oxidation of free fatty acids. Acetoacetate is the central ketone body. Subsequently acetoacetate can be reduced to BHB by β-hydroxybutyrate dehydrogenase in a NADH-requiring reaction.

In health, glucose is the major fuel for human brains. During times of starvation however, the brain has the capacity to adapt to the use of ketones as its major energy source. Once glucose stores are depleted, supplying sufficient glucose to support brain metabolic requirements from protein alone would lead to death in about 10 days. However there are reports of survival for 50–70 days in people undergoing prolonged fasting and ketones play a key role 152. During periods of ongoing starvation, an average size person produces about 150 g of ketone bodies per day, capable of supply 70% of cerebral metabolic requirements 27. Ketones therefore play a critical role in human evolution.

Aside from their evolutionary benefits, ketones may have neuroprotective effects as they represent an alternative fuel for both the normal and the injured brain 4, 51, 117. There are unique properties of ketone metabolism that may make it a more suitable cerebral fuel under various neuropathologic conditions:

1. Ketones are more energy efficient than glucose

59 Novel metabolic substrates for feeding the injured brain

2. Ketones protect against glutamate-mediated apoptosis

3. Ketones enhance GABA-mediated inhibition

4. Cerebral ketone metabolism alters cerebral blood flow

Exogenous ketone supplementation has been examined in a wide range of animal and human models of neurological disorders including autism, Alzheimer’s disease, migraine, strokes, hypoxic-ischemic encephalopathy, Parkinson disease, amyotrophic lateral sclerosis, epilepsy and traumatic brain 165, 166. More recent studies have examined the use of these diets in the management of brain tumours based on the fact that altered glucose metabolism may have anti-tumour effects 167. Most human research has looked at ketone supplementation in children for the management of refractory epilepsy. Although initially contentious, there is now good evidence supported by two randomized controlled trials that ketogenic diets in children improve seizure control 99, 168. The evidence in adults is less compelling but no good studies exist.

Non-esterified fatty acids (NEFA) as a cerebral energy supplement

The ability of ketones to improve cerebral energy metabolism is not shared by other fatty acid containing compounds. Despite being hydrogen rich, fatty acids are utilized poorly by the brain as fuel and may have a number of deleterious properties including the induction of apoptotic pathways 169. Although highly protein bound, the slow passage of NEFA across the blood brain barrier (BBB) does not appear to be the cause of poor fatty acid oxidation. Certainly, there is evidence that NEFA may actually damage the BBB by enhancing oxidative stress in endothelial cells. Furthermore, despite the rapid oxidation in peripheral tissues, NEFA is metabolised slowly by the brain and appears to require more oxygen than glucose, further exacerbating the hypoxic environment of the injured brain 170. The poor anti- oxidative defences in neurons may be considerably overwhelmed by the rapid accumulation of superoxides following β-oxidation of NEFA. Thus, the many benefits associated with ketone supplementation are not shared by free fatty acids which may prove detrimental in the TBI population.

Lactate as a glucose sparing fuel

Lactate is a dead-end metabolite of its redox partner, pyruvate 171. In mammals, physiological concentrations may vary between 0.05 – 5 mmol/l. Lactate is metabolised via

60 Novel metabolic substrates for feeding the injured brain lactate dehydrogenase with the extent and direction of the reaction determined by the NAD/NADH(H) ratio. Along with pyruvate, it forms the initial substrate for the tricarboxylic acid cycle and subsequently is critical for cellular energetics 172. Lactate has two optically active forms, L(-) and D(+). The predominant physiological form is L(-) and the metabolic effects resulting from metabolism of the 2 forms differs significantly. Lactate is actively transported across plasma membranes including red cells, kidney and liver and somewhat slower in the heart skeletal muscle and the brain.

Evidence suggests that lactate can act as an energy source for the brain, especially during periods of substrate deficiency. Data confirms a significant increase in brain lactate following 2-3 days of fasting 42. This may arise from a shift in lactate metabolism following the shift toward ketone oxidation. Others suggest a continuous production of lactate through glycolysis possibly via a complex interaction between astrocytes and oligodendrocytes (the so call astrocyte-neuron lactate shuttle) 173.

Studies in TBI patients seem to support a role for hypertonic lactate solution in reducing intracranial pressure (ICP) and demonstrate positive effects on cerebral energetics. However, this may depend on the baseline cerebral metabolic state 174. Studies in animals confirm the supposition that lactate may improve outcomes in TBI. Animal models of both trauma and hypoxic injury have demonstrated a reduction in functional deficits, decrease in lesion volumes and CBF augmentation 175, 176. Only a small number of studies have involved humans. Glenn et al demonstrated that peripheral lactate production accounted for approx. 70% of carbohydrate consumed for energy production by the traumatized brain and suggested lactate should be supplemented to compensate for decrease in glucose cerebral metabolism 177. To assess the impact of exogenous lactate on cerebral energy metabolism, Bouzat et al administered hypertonic lactate (HL) to 15 patients with TBI 178. Using cerebral microdialysis, they demonstrated an increase in lactate, pyruvate and glucose and noted a concomitant decrease in glutamate and ICP. They concluded that HL solution had a positive effect on cerebral energetics and may be a useful therapeutic intervention. Similarly, Ichai et al demonstrated HL to be more effective at lowering and maintaining ICP when compared with other hypertonic solutions 179. By sparing cerebral glucose and decreasing ICP, HL may provide an alternative means of controlling ICP while improving cerebral energy metabolism.

Branch-Chain amino acids and recovery post TBI The branch-chain amino acids consist of leucine, isoleucine and valine. They account for approximately 35% of essential amino acids and 14% of amino acids in skeletal muscle. 61 Novel metabolic substrates for feeding the injured brain

BCAA’s, while nutritionally essential cannot be synthesized by humans and must be supplied in the diet. They have been demonstrated to impact positively on protein metabolism by inhibiting muscle breakdown and promoting muscle and hepatic protein synthesis 180. They are essential in the nutritional support of the critically ill. BCAA’s impact on the production of cerebral neurotransmitters in several ways and contribute to the synthesis of both inhibitory and stimulatory amino acids 181. They are essential for synthesis of glutamate, which has been implicated in excess cellular damage following TBI, but also GABA, considered to be an inhibitory neurotransmitter and therefore potentially neuroprotective 182. Furthermore, by competing for transport across the blood brain barrier, BCAA’s lower cerebral concentrations of tryptophan, tyrosine and phenylalanine and therefore indirectly, concentrations of serotonin and catecholamines. They can also supplement intermediates in the citric acid cycle, potentially improving cerebral energetics. BCAA have been shown to improve cognitive function in a number of conditions. In animal studies, BCAA supplementation provided a sustained improvement from in cognitive function in a mouse model of TBI 183. In healthy, exercising people, BCAA’s lower exertional and mental fatigue. Patients with hepatic encephalopathy benefit from BCAA supplementation with improved mental status, asterixis orientation, speech and writing. In an observational study of the TBI population, Vuille-Dit-Bille et al demonstrated that raised levels of the aromatic amino acids (AAA) were associated with decreased ICP and increased jugular venous oxygen saturation as compared to BCAA’s 182. One small study (n = 20 ) from Ott et al demonstrated that TBI patients administered BCAA maintained a positive nitrogen balance but data on morbidity and mortality were not provided 184. Aquilani et al conducted 2 small studies (total of 42 patients in intervention group and 39 in control) examining the benefits of BCAA supplementation on cognitive function in the recovery period post TBI 185, 186. They were able to demonstrate a significant improvement in the Disability Rating Scale in patients receiving the supplements. However, the BCAA’s were administered anywhere from 19 – 140 days following injury, and therefore the studies do not address their use in the acute setting. There is therefore currently no strong evidence supporting BCAA use following acute brain injury.

Triheptanoin and anaplerosis

Anaplerosis, is the process by which intermediates in the TCA cycle are replenished involving the carboxylation of pyruvate and propionyl-CoA. Anaplerotic molecules may

62 Novel metabolic substrates for feeding the injured brain include amino acids and odd chain fatty acids. Pyruvate carboxylase appears to be the main anaplerotic enzyme in the brain. Cerebral ATP production is largely dependent on a functioning TCA cycle. Evidence suggests that during neurological injury, including seizures, energy production may be compromised. Therefore, improving cerebral energetics may be an effective therapeutic target.

Triheptanoin was discovered by Roe et al as an oral anaplerotic treatment for metabolic disorders 187. It is a triglyceride of heptanoate (C7 fatty acid) capable of being metabolized by β-oxidation to provide propionyl – CoA which can subsequently be carboxylated to produce succinyl-Coa, and entering the TCA cycle. Triheptanoin is a tasteless oil, which may enter the brain directly or following the following metabolism in the liver to C5 ketone bodies β-hydroxypentanoate and β-ketopentanoate. These may subsequently be taken up in the brain through MCT transporters 188. The result is an increase in acetyl-CoA and ATP production.

Triheptanoin has been proven effective in patients with different metabolic problems including cardiomyopathy and rhabdomyolysis in acyl-CoA dehydrogenase deficiency, pyruvate carboxylase deficiency and carnitine-palmitoyltransferease II deficiency. Several studies involving animal models of epilepsy found triheptanoin to be effective as an anticonvulsant 189, 190. Adenyeguh et al found that triheptanoin administered to patients with early stage Huntington’s disease was able to partially correct the abnormal brain energy profile noted in these patients 191. Efforts are underway to further evaluate the anticonvulsant effects of triheptanoin and to understand its clinical potential for management of epilepsy and other brain disorders 189, 192.

5.5 Challenges in developing new fuel substrates for the injured brain

While several examples exist that might make metabolic sense in the injured brain, the impact of their therapeutic use and other aspects of systemic metabolism remain to be seen. Probably the most researched substrates are ketones. Adverse effects related to chronic oral intake are well described from the paediatric literature. Less is known about the consequences of administering high doses in the acute brain injury population. Furthermore, there are a number of potential routes of administration which may present their own challenges. Several papers have looked at the administration of BHB as a sodium salt via the intravenous route 80, 88, 144. This allows for a rapid increase in peripheral BHB concentrations although long term infusions lead to an increase in pH. The effects of this on

63 Novel metabolic substrates for feeding the injured brain

ICP have not been investigated to date. Other potential routes of administration include various ketogenic enteral formulations, the commercial product KetoCal® and more recently, as a ketone monoester 193.

Lactate can be administered as an iso or hypertonic formulation. The benefits of hypertonic solutions in the head injury population are well described 194. In general, the likelihood of potential side effects including hypernatremia, increased osmolality and metabolic alkalosis will depend on the formulation provide and the length of time. Bouzat et al administered a hypertonic solution of lactate to head injury patients and did not find any change in 178 physiological variables including PaCO2 however, the infusion only ran for 3 hours .

The impact on systemic glucose metabolism is variable. Ketone administration is associated with decreased glucose concentration 91. Lactate is thought to produce a glucose sparing effect in the brain while NEFA may actually lead to hyperglycemia 195. BCAA seem to stimulate insulin production in the short term but may lead to insulin resistance following long term administration 185.

Consideration should also be given to the impact of organ dysfunction such as liver or renal failure when considering substrate supplementation. BCAA have been extensively studied in critically ill patients and appear to be well tolerated and certainly are demonstrated to be protein sparing 88. Similarly, NEFA may have a positive impact in critical illness although as previously noted, may be deleterious in the brain injury population 195. Ketones and lactate may accumulate and potentially lead to metabolic derangements such as systemic academia if liver/renal function markedly impaired, leading to an inability to metabolise these substrates.

It is therefore necessary to consider the implications of supplementing substrates in terms of other potential metabolic effects and in the setting of major metabolic derangements.

5.6 Conclusion

In healthy individuals, glucose is the main substrate for cerebral energy production. However, it is clear that in the damaged brain, a significant alteration takes place in metabolic pathways and other substrates may become more important. Of the various potential substances investigated, ketones have received the most in-depth study and would appear to be an excellent alternative to glucose. Research into the field of cerebral energetics however, is still in its infancy. More effort is required to determine which metabolic

64 Novel metabolic substrates for feeding the injured brain substrate or combinations of these might provide the damaged brain with the optimum energy requirements.

65 Systematic Review of the use of ketones in the management of acute and chronic neurological disorders

Chapter 6: Systematic Review of the use of ketones in the management

of acute and chronic neurological disorders

6.1 Synopsis and citation

Background:

Ketosis is currently being utilized as treatment option in paediatric patients with resistant epilepsy. However, the evidence for efficacy in adult epilepsy and other neurological conditions is lacking.

Aims:

The purpose of this systematic review is to examine the evidence for inducing ketogenesis in adult humans with neurological and neurosurgical disorders, both acute and chronic

Design:

We conducted a literature review through electronic databases, including Medline, the Cochrane Library and PubMed.

Key Findings:

14 publications met the selection criteria for inclusion into this study. Study subjects included Alzheimer’s disease, severe refractory status epilepticus, intracranial neoplasms, traumatic brain injury and idiopathic Parkinson’s disease. Although studies suggested ketosis may be beneficial, interpretation is limited by poor quality of evidence.

Conclusions:

Ketone administration may prove beneficial in the management of a number of neurological conditions. Better quality studies are required before firm conclusions can be drawn

Citation:

This chapter is based on a publication reviewing the evidence for inducing ketogenesis in adult humans with neurological disorders. Figures and tables have been inserted into the text at different positions and the numbering of pages, figures and tables has been adjusted

66 Systematic Review of the use of ketones in the management of acute and chronic neurological disorders to fit the thesis. The references have been incorporated with the other references of the Thesis.

The original manuscript was “Systematic review of the use of ketones in the management of acute and chronic neurological disorders” by H White, K Venkatesh and B Venkatesh was published in Journal of Neurology, and Neuroscience, April 27, 2017.

Contribution:

H White and K Venkatesh performed all data collection, collation and review of data and completion of manuscript. B Venkatesh reviewed manuscript and assisted with completion.

67 Systematic Review of the use of ketones in the management of acute and chronic neurological disorders

6.2 Introduction

Neurological disorders are common and sometimes catastrophic, leading to significant morbidity and mortality. At a pathophysiological level, many of these disorders are characterised by a disruption of normal metabolic pathways 130. Following injury, cellular energetics play a vital role in maintaining cerebral homeostasis. The adult brain consumes approximately 20% of basal metabolism, most of which is provided by the oxidation of 100- 120g of glucose over 24 hours 8. However, during times of starvation or injury, the primary cerebral metabolic substrates may alter. Ketones may represent an alternative fuel in these instances.

There are unique properties of ketone metabolism that may make it a more suitable cerebral fuel under various acute and chronic neuropathologic conditions 51, 58 (see chapter 1). Induction of ketosis has therefore been attempted in various neurological states. Ketosis may be induced via the intravenous or enteral route. However, largely owing to the high cost of producing intravenous IV solutions, most researchers have employed enteral feeding formulations. Ketogenic diets were initially employed in the management of refractory epilepsy in children 196, 197. This came about from the observation that starvation states were associated with improved seizure control 198. Until recently, the benefits of the ketogenic diet were contentious and restricted to specialist units such as John’s Hopkins and Harvard medical school. However, the benefits of the ketogenic diet for seizure control in children have now been demonstrated in two randomized controlled trials 99, 168. The evidence in adults however is far less clear with case reports and case series dominating the literature 199-201.

Ketogenic diets have been examined in a wide range of animal and human models of neurological disorders including autism, Alzheimer’s disease, migraine, strokes, hypoxic- ischemic encephalopathy, Parkinson disease, amyotrophic lateral sclerosis, and traumatic brain injury 56, 165, 202, 203. More recent studies have examined the use of these diets in the management of brain tumours based on the fact that altered glucose metabolism may have anti-tumour effects 167.

Most published research has investigated the role of ketogenesis in children for the management of refractory epilepsy 196, 204. Recent reviews have examined the use of ketogenic diets in the management of seizure disorders in adults 205-207. The purpose of this systematic review is to examine the evidence for inducing ketogenesis in adult humans with

68 Systematic Review of the use of ketones in the management of acute and chronic neurological disorders neurological and neurosurgical disorders, both acute and chronic. We have excluded seizure disorders (other than status epilepticus) as this was recently reviewed by others 204.

6.3 Methods

Data sources

We conducted a literature review through electronic databases, including Medline, the Cochrane Library and PubMed. Additionally, we performed a search through www.clinicaltrials.gov to check for new or ongoing studies. Our search was focussed using the following MeSH terms: ketones, ketonaemia, B-hydroxybutyrate, ketogenic diet, adults, brain trauma, traumatic brain injury, status epilepticus, carbohydrate-free diet, neurodegenerative disease, Parkinson’s disease, dementia, Alzheimer’s disease, mild cognitive impairment, intracranial bleed and subarachnoid haemorrhage.

We limited our search to papers published between January 1st, 1980 and June 30, 2016. We further limited our search to include only studies involving adult humans (over the age of 18).

Study selection

Two authors (HW and KV) performed the literature search as per the parameters listed above and compiled the relevant studies. Discrepancies were resolved by mutual discussions. Studies were selected if they were trials assessing ketosis in adult humans with current neurosurgical or neurological disorders. Both HW and KV were in consensus that for the purpose of this study, studies would be included if they assessed ketogenic diet, ketone supplementation, carbohydrate-free diet, induction of ketosis, or markers of ketone biochemistry in the study population.

Studies were only included if they were original trials or case reports; they were excluded if they were reviews, meta-analyses or letters to the editor. Furthermore, studies were excluded if they were not written in English.

Definitions

Neurosurgical disorders were defined as traumatic brain injury, space-occupying lesions and any type of intracranial haemorrhage. Neurological disorders included cerebrovascular

69 Systematic Review of the use of ketones in the management of acute and chronic neurological disorders accidents, status epilepticus, and any neurodegenerative conditions where the primary pathology was occurring within the brain.

6.4 Results

Study selection

From the literature search, we identified 14 publications that met the selection criteria for inclusion into this study (Figure 6.1). These are listed and summarised in Table 6.1 below.

548 potential papers identified and screened after literature search using defined search terms

Database Medline 138 PubMed 378 Cochrane Library 0 www.clinicaltrials.gov 0

Papers after duplicates removed (n = 472)

Papers after filters applied (n = 261) Papers excluded (n = 211)

Full-text papers assessed for eligibility (n = 261) Full-text papers excluded (n = 247)

Total number of papers included for review in this study (n = 14)

Figure 6.1 Evaluation of papers for inclusion into this review

Study characteristics

Of the 14 publications, 3 were randomised controlled trials, 10 were case series (6 prospective and 4 retrospective) and one was a non-randomised placebo control trial. The study populations of the 143 studies were as follows: 4 assessed patients with either mild cognitive impairment or Alzheimer’s disease, 5 investigated patients with severe refractory

70 Systematic Review of the use of ketones in the management of acute and chronic neurological disorders status epilepticus, 32 evaluated patients with intracranial neoplasms and there was one study each in patients with traumatic brain injury and idiopathic Parkinson’s disease.

In each study, the investigators aimed to induce hyperketonaemia either via carbohydrate restriction, or implementation of a ketogenic diet, or via the administration of oral ketogenic compounds (OKC). The OKCs were all high in lipids with low proportion of carbohydrates.

Mild cognitive impairment and Alzheimer’s disease

Four studies investigated the role of ketone supplementation in patients with either mild cognitive impairment (MCI) or Alzheimer’s disease (AD). Henderson et al. and Reger et al. both compared the effects of an OKC on memory and cognition compared to placebo, in double-blind trials 97, 208. In both trials, higher plasma ketone concentrations were associated with improvements in memory and cognitive function. Subanalysis in the cohorts was performed to assess for the Apolipoprotein-E4 (APO-4) status of the patients (APO-4 positivity is a mutation associated with increased risk of developing Alzheimer’s disease).

In both studies, APOE-positive status was associated with a reduced response to ketone supplementation. Krikorian et al. compared a low carbohydrate diet with a carbohydrate-rich diet in adult patients with MCI, and assessed each diet’s effects on cognitive function 209. The group in the low-carbohydrate diet arm had improvements in their memory compared to the other group, however there was no significant difference in executive cognitive function between the groups. Newport et al. report a case study of an adult patient with AD who was commenced on an oral ketone ester to stimulate ketosis and to assess the effects of this on cognitive function 210. Improvements were noted in the patient’s cognitive performance (however not assessed objectively, with additional amelioration in social and self-care functions).

Traumatic brain injury

Only 1 study investigated the role of ketosis in adult patients with traumatic brain injury. It investigated whether sufficient caloric intake could be met with a low carbohydrate diet in patients with TBI (compared to standard nutrition), and whether there were significant adverse effects between the two groups 91. Patients in the low carbohydrate arm received adequate dietary calories without hypoglycaemia.

71 Systematic Review of the use of ketones in the management of acute and chronic neurological disorders

Table 6.1 Summary of studies selected for inclusion into this review

Study Author, year Type of study Population Number Intervention Primary endpoint Main findings

1 Cervenka et al., Case report Adult patient 1 Administration of To assess whether Administration of enteral KD to 2011 221 with SRSE enteral KD after 58 administration of adult male patient after 58 days of

days of SRSE enteral KD may be SRSE was associated with effective in SRSE improvement in clinical and electroencephalographic seizure activity. Patient able to be weaned off parenteral anticonvulsants. No significant adverse effects reported related to KD.

2 Champ et al., Retrospective Adult patients 53 Ketogenic diet vs. To assess 6 out of the 53 patients reviewed 2014 217 case series with GBM normal diet in biochemical profile trialled the KD. There were no

patients with GBM and toxicity of KD in episodes of hypoglycaemia patients with GBM associated with the KD and there did not appear to be any issues related to toxicity.

3 Henderson et Randomised, Adult patients 152 OKC vs. placebo To assess whether Administration of oral ketogenic al., 2009 208 double blind, with mild- over 90 days oral ketogenic compound caused increased placebo- moderate AD compound could serum levels of B-hydroybutyrate controlled improve cognitive and significantly improved multicentre trial performance cognitive scores compared to compared to placebo. Reduced response to placebo oral ketogenic compound in APOE4+ patients.

4 Krikorian et al., Randomised- Adults with 23 High CHO diet vs. To assess Improved neurocognitive function 2012 209 controlled trial MCI low CHO diet over 6 differences in was observed in the low CHO diet weeks cognitive function arm. Low CHO diet was between patients on reasonably well-tolerated however

high CHO diet and long-term feasibility was low CHO diet questionable

72 Systematic Review of the use of ketones in the management of acute and chronic neurological disorders

Study Author, year Type of study Population Number Intervention Primary endpoint Main findings

5 Nam et al., Retrospective Patients with 5 (1 adult Administration of KD To assess the In the adult patient, there was 2011 212 case series SRSE and 4 in patients with RSE efficacy and safety complete resolution of SRSE children) over course of profile of the KD in within one month and return to admission patients with RSE normal function, with nil adverse effects reported in this patient

6 Newport et al., Singe-patient Patient with 1 Administration of Compare cognitive Administration of OKC was 2015 210 case study younger-onset OKC over 20 function pre and deemed safe, convenient and AD months post implementation tolerable. Improved cognitive of OKC as per function and activities of daily living were observed in the patient. However, nil discussion of pre and post formal cognitive function testing scores.

7 Reger et al., Double-blind Adult patients 20 Administration of To compare the Administration of OKC was 2004 97 placebo-control with AD or MCI either OKC or effects of increased associated with improved memory trial placebo ß-OHB on memory and cognitive function, compared and cognitive to placebo. However, it was noted function, compared that patients positive for APOE to placebo. Also to genotype were less responsive to assess effect of ß- OKC than those who were OHB on patients negative for the gene. with APOE genotype vs. those without.

8 Reiger et al., Prospective Adult patients 20 Administration of To assess feasibility Over the study time period, 17 out 2014 216 case-control with recurrent low carbohydrate of the KD as judged of 20 (85%) of patients were able pilot study GBM diet over 16 weeks by percentage of to adhere to the KD. No significant patients who adverse outcomes were reported discontinued diet in patients.

73 Systematic Review of the use of ketones in the management of acute and chronic neurological disorders

Study Author, year Type of study Population Number Intervention Primary endpoint Main findings

9 Ritter et al., Randomised Adult patients 20 Administration of Evaluate the Patients fed on the normal diet 1996 91 open-label with traumatic either OKC or biochemical had higher arterial glucose control trial brain injury normal diet parameters concentrations compared to the associated with group on the OKC diet. Arterial administration of the ketone concentrations were OKC compared to significantly higher in the OKC those in patients on group than the normal diet group. a normal diet, There was insufficient power to particularly draw conclusions on neurological assessing the outcomes with either group. glycaemic profile.

10 Schwartz et al., Case studies Adult patients 2 Administration of an Assess the efficacy The two patients studied in the 2015 215 with literature with GBM energy-restricted of KD in treating trial showed progression of their review KD over 12 weeks malignant gliomas, tumours while on the ketogenic and to assess the diet. The ketogenic diet was safety and feasibility deemed to be safe without major of a ketogenic diet. side effects and ketosis was easily inducible with ketogenic diet.

11 Strzelczyk et al., Retrospective Adult patient 1 Intravenous To assess the Administration of intravenous KD 2013 213 case study with SRSE administration of KD efficacy and safety was associated with resolution of profile of intravenous SRSE and was a safe and KD in treating SRSE feasible option for management of SRSE.

12 Thakur et al., Retrospective Adult patients 10 Administration of a Assess the effects of 9 out of 10 patients achieved 2014 201 case series with SRSE KD a KD on timing of ketosis, with all 9/10 achieving resolution of SRSE resolution of SRSE within median of 3 days of starting KD.

74 Systematic Review of the use of ketones in the management of acute and chronic neurological disorders

Study Author, year Type of study Population Number Intervention Primary endpoint Main findings

13 Vanitallie et al., Prospective Adult patients 7 Home-prepared KD Assess the feasibility 5 out of the 7 patients were able to 2005 211 case series with idiopathic over 28 days of patients preparing prepare the KD themselves and PD a KD themselves adhere to it over the study course.

and adhering to it There appeared to be some degree of improvement in symptoms of PD but insufficient sample size to draw conclusions on this.

14 Wusthoff et al., Prospective Adult patients 2 Patient 1: Assess the efficacy In patient 1, administration of KD 2010 214 observational with SRSE administration of of the KD in treating resulted in reduced seizure case series enteral KD, Patient patients with SRSE frequency with complete cessation 2: Initial induction of (both in hospital and of seizures by Day 11 post KD ketosis via after discharge) initiation. In patient 2, initiation of starvation and then ketosis resulted in a reduction of enteral seizure frequency and cessation of administration of KD seizures after 8 days of KD.

AD – Alzheimer’s disease, APOE – Apolipoprotein E, ß-OHB – ß-hydroxybutyrate, CHO – Carbohydrate, GBM – Glioblastoma multiforme, KD – Ketogenic diet, MCI – Mild cognitive impairment, OKC – oral ketogenic compound, PD – Parkinson’s disease, SRSE – Severe refractory status epilepticus

75 Systematic Review of the use of ketones in the management of acute and chronic neurological disorders

There were no differences in CSF and cerebral lactate concentrations between the two groups. The study had insufficient sample size to draw any conclusions regarding the nutritional effects on neurological outcome following TBI.

Parkinson’s disease

VaniItallie et al. prospectively investigated the feasibility of a ketogenic diet (KD) in adult patients with idiopathic Parkinson’s disease (PD) 211. Seven patients were assessed over one month and were required to prepare KDs at home by themselves. Five of the seven patients adhered to the diet over the course of the study. The diet was associated with improved symptoms of PD (as per the Unified Parkinson’s Disease Rating Scale) however there was insufficient power to draw significant conclusions from this finding.

Status epilepticus

Nam et al. retrospectively reviewed the effects of an oral KD in patients with severe refractory status epilepticus (SRSE) 212. Five patients were reviewed, of which one was an adult patient. In this patient there was complete resolution of seizures within one month with no significant adverse effects reported, and the patient was discharged home with normal neurological function. Strzelczyk et al. investigated the effects of an intravenous and enteral ketogenic diet in an adult patient with SRSE 213. Commencement of the KD was associated with a resolution of status epilepticus, however there was ongoing myoclonus and the patient still had generalised tonic-clonic seizures every 3-4 days. There were no episodes of status epilepticus in the 4 months of follow up. Wusthoff et al. reported a case series of two adults with SRSE treated with a ketogenic diet 214. The first patient was an adult female with SRSE despite 3 months of maximal medical therapy. An enteral ketogenic diet was commenced on hospital day 101 and within four days a reduction in seizure frequency was noted; there was complete resolution of seizures after 11 days of KD. At the time of discharge, the patient’s neurological function improved however did not return to her pre-hospital baseline. The second patient was an adult male with SRSE with no clinical response to maximal antiepileptic therapy after 18 days. Ketosis was induced on day 18 initially with fasting and then an enteral KD. Midazolam was weaned on day 26 and the patient was subsequently discharged on antiepileptics and a modified KD. At one year of follow up, only one self- limiting seizure was reported and he had returned to work.

76 Systematic Review of the use of ketones in the management of acute and chronic neurological disorders

Intracranial neoplasms

Schwartz et al. investigated whether the oral KD could alter glioma progression in two adult male patients with Glioblastoma Multiforme (GBM) 215. Both patients had advanced posterior GBM and had already received surgery, radiotherapy and chemotherapy. The first was commenced on an oral KD 20 months after his initial diagnosis. He continued the diet for 4 weeks but subsequently withdrew from the study after serial imaging revealed further progression of the tumour. The second patient was enrolled into the study 14 months after his initial diagnosis. He was commenced on the oral diet and maintained it for 12 weeks, however clinical and radiological assessment at this time point revealed tumour progression and he subsequently withdrew from the study. Rieger et al. prospectively investigated 20 adult patients with recurrent glioblastoma to assess the feasibility of a very-low carbohydrate diet as a potential treatment modality in this group, over a 16-week period 216. After 2-3 weeks, three patients (15%) discontinued the diet due to the impact on their quality of life. The remaining 17 patients adhered to the diet over the entire course of the study. Champ et al. 217 retrospectively reviewed a case series of 53 patients with GBM, 6 of whom trialled a KD during their treatment course. The purpose of the study was to assess the biochemical and toxicity profile of the ketogenic diet in this population. The authors reported no significant adverse effects related to the diet with no documented episodes of hypoglycaemia or toxicity related to the diet.

Adverse effects and issues related to ketosis

Poor compliance was associated with the KD due to poor palatability 211, 215. Henderson et al. reported a higher rate of diarrhoea in the ketogenic diet group compared to the placebo group however this was not discussed in the other studies 208; notably this was specifically not mentioned as an adverse outcome in the study by Rieger et al 216 and was retrospectively reported as well-tolerated in the study by Champ et al 217.

Krikorian et al. questioned the long-term feasibility and safety of the diet, particularly with respect to adequate nutrient intake. The other concern was regarding the potential for weight loss, particularly the effects this may have on elderly patients susceptible to sarcopenia 209.

There were no reports of hypoglycaemia in patients trialling the KD in any of the studies. Thakur et al. reported acidosis as a complication in one of their patients and in the study by Wusthoff et al., bicarbonate was supplemented on a daily basis to maintain acid-base

77 Systematic Review of the use of ketones in the management of acute and chronic neurological disorders balance 201, 214. There were no other reports of metabolic acidosis secondary to ketogenesis. Thakur et al. reported two patients developing complications of hypertriglyceridaemia 201.

6.5 Discussion

The purpose of this systematic review was to review the evidence pertaining to the use of ketogenic therapy in the management of neurological and neurosurgical disorders in adults. While ketogenic therapies have been extensively studied in epileptic children, evidence in adults is lacking. Of the papers included, there is significant heterogeneity between the studies, including differences in study populations, trial designs, methods of ketosis induction and chosen primary end points. The studies included in our review have focussed on the role of ketosis in status epilepticus, or in patients with mild cognitive impairment and Alzheimer’s disease, brain tumours or traumatic brain injury.

A number of cellular mechanisms are thought to underlie the neuroprotective activity of ketones. Ketones enhance cerebral mitochondrial energy usage by providing enhanced energy stores and providing a more efficient source of energy per unit oxygen consumption as compared to glucose. Ketones may interfere with glutamate mediated toxicity, possibly via attenuation of glutamate-induced reactive oxygen species (ROS) formation. GABA levels are enhanced with subsequent increase in GABA mediated inhibition 218, a possible explanation for the anti-epileptic effects. ROS species may decrease via a number of mechanisms including a reduction in coenzyme Q semiquinone and increased activity of glutathione peroxidase activity 59, 219. Apoptosis or programmed cell death plays an important role in cellular damage following injury. The ketogenic diet may mitigate apoptosis by inhibition of kainic acid-induced accumulation of the protein clusterin, thought to influence apoptotic signalling 220. And lastly, carbohydrate restriction itself is neuroprotective via various mechanisms including suppression of ROS production, stabilization of intracellular calcium and improvements in mitochondrial function.

Status epilepticus is a serious and an often fatal condition. The use of KD to control status epilepticus in adults has been reported in several case reports and one case series, with a total of 13 patients (12 treated with an enteral diet, one treated with a parenteral KD) 201, 213, 214, 221. The underlying diagnoses in these cases included encephalitis, anoxia, neurocysticercosis and cortical dysplasia. Patients were generally in status for a number of days prior to the initiation of the ketogenic diet as a rescue therapy. Strzelczyk et al published the first and only description of emergency induction of an intravenous ketogenic diet

78 Systematic Review of the use of ketones in the management of acute and chronic neurological disorders utilizing a combination of commercially available TPN solutions 213. They describe the case of a 21-year-old female with a history of poorly controlled myoclonic and generalized seizures who presented in status epilepticus. The patient was treated for 16 days with multiple antiepileptic meds and general anaesthesia with little improvement. The IV KD was initiated with commercially available fat emulsion with medium-chain triglycerides; this was then converted to enteral feeds several days later. Her seizures gradually improved and discharged after 47 days with no further episodes of status after 4 months. Given the lack of large studies, it is difficult to draw any conclusion regarding the efficacy of ketosis on seizure control during status epilepticus. It is promising however that no significant issues were present with regards to safety of ketosis.

The ketogenic diet may have a role in modifying disease progression in a number of neurodegenerative disorders. Much of the pathophysiology of Parkinson’s and Alzheimer’s disease relates to mitochondrial dysfunction and ketones may play a neuroprotective role. In Parkinson’s disease for example, it is hypothesized that the death of the dopaminergic neurons may partly reflect impairment of mitochondrial complex I activity. Ketones may play a role by either bypassing complex I and providing an alternative fuel source or enhancing mitochondrial function with subsequent improvements in ATP production and neuroprotection 62, 83. The VanItallie study confirmed that patients able to tolerate a ketogenic diet improved their Unified Parkinson Disease Rating Scale scores with no harmful effects 211. Although the study had insufficient power to allow any conclusions on the clinical effects of the KD, the data appears promising and certainly invites further study into the potential therapeutic effects of ketones in this group of patients.

In Alzheimer’s disease, the ketogenic diet may be neuroprotective via a number of mechanisms. Castellano et al. have shown through nuclear imaging that patients with Alzheimer’s disease can have regional brain hypometabolism secondary to impaired glucose uptake 222. Subsequently, ketosis may offer an alternative metabolic substrate for the brain in this population. Secondly, by improving mitochondrial efficiency, ketones may provide protection against the deposition of Amyloid-B 1-42 (AB), which is thought to be crucial to the development of this disease 83. This has been observed in transgenic mice models of AD where animals fed the ketogenic diet had 25% less soluble AB in their brains after only 40 days 85. In humans, there is some evidence that this process may be determined by the presence or absence of the APO-4 allele; the presence of which is a risk factor for developing AD. Henderson et al found a beneficial effect of the ketone inducing

79 Systematic Review of the use of ketones in the management of acute and chronic neurological disorders diet only in individuals where the allele was absent 208. Confounding these findings is that dietary supplementation of fatty acids itself may improve cognitive function in patients with AD. More research is necessary to clarify the importance of various supplements in mitigating the ongoing neurodegeneration in patients with AD disease.

Despite a number of animal studies 72, 74 investigating the benefits of ketone administration in a variety of models of acute brain injury, only a single study has been published in humans. In Ritter et al.’s study, the intervention group, blood glucose concentration remained well controlled, arterial lactate concentrations were lower and plasma ketones higher compared to the control standard feeding group 91. Twenty patients were included and randomized to either trial feed or standard feeding. In the intervention group, blood glucose concentration remained well controlled, arterial lactate concentrations were lower and plasma ketones higher compared to the control standard feeding group. Otherwise, there was no difference between the groups and they did not report any major side effects. Animal studies have suggested that acute brain injury induces rapid changes in cellular transporters that favour ketone uptake and metabolism. In a rodent model, Prins et al. found that administration of intravenous -hydroxybutyrate after TBI mitigated the reduction in ATP availability after injury and an associated reduction in cortical contusion volume 74. Similar findings have been observed in a stroke model, where Suzuki et al found a decrease in stroke size in a hypoxic animal model treated with -hydroxybutyrate 73.

Adverse effect profile of ketogenic diets

In our review, induction of ketosis did not appear to have significant safety or adverse effects in the studied populations. Data on the adverse effects of ketone administration are limited in the adult population. In the acute setting, effects of parenteral formulations on pH, Na, lipids and glucose are likely to predominate. This was confirmed by Hiraide et al who noted a significant increase in pH and Na concentrations following the IV administration of a 20% solution of NaBHB to severe trauma patients 94. Also of potential concern is the reduction in cerebral glucose metabolism and increased cerebral blood flow demonstrated by Hasselbalch et al during administration of intravenous BHB 58. The long term consequences of this are not known, but may have implications in the short term to patients with traumatic brain injury, cerebral oedema or raised intracranial pressure. Some side effects are predictable following enteral administration such as dehydration and hypoglycaemia. Others are less common and present following prolonged use, including growth retardation, obesity,

80 Systematic Review of the use of ketones in the management of acute and chronic neurological disorders nutrient deficiency, decreased bone density, hepatic failure, and immune dysfunction. Freeman has reported a significant rise in mean blood cholesterol to over 250 mg/dl following prolonged intake a of ketogenic diet 99. This in turn may be atherogenic, leading to lipid deposition in blood vessels. Finally, an increased incidence in nephrolithiasis in patients on the ketogenic diet as well as increases in serum uric acid secondary have been reported 76, 100.

Limitations of this systematic review

This systematic review was limited by the small number of studies and a preponderance of case reports and case series. While results from the studies suggest a clinical benefit from ketosis, the paucity of well-designed clinical trials creates problems when interpreting the data. In many instances it is difficult to definitively prove a causal relationship between initiation of ketosis and neurological improvement including cessation of seizure activity. There is often a prolonged lag time between initiation of the ketogenic diet and seizure cessation (>1 week in 6 out of the 13 patients). Moreover, the studies did not have a sufficient sample size and power to show a treatment effect. Furthermore, there is a bias to publish positive results and it is not known how many patients failed to respond to treatment. Additionally, there are minimal prospective data available on this topic. Of the case reports on status epilepticus, 3 deaths were reported during hospitalization, however, the long term outcomes of the other patients were not reported.

6.6 Conclusion

The evidence for the use of ketogenic diets or induction of ketosis for the management of neurological conditions in adults is limited. Although there might be a suggestion of benefit in limiting the frequency of seizure episodes in patients with refractory status epilepticus, a number of case reports and case series are available, but high quality research is yet to be produced. Theoretically, the induction of ketosis is a promising therapeutic tool in the management of both acute and chronic neurological and neurosurgical disorders. It is time for a well-designed study to be undertaken in adults to determine if the potential benefits translate into clinical success.

81 Inducing ketogenesis via an enteral formulation in patients with acute brain injury: A phase II study

Chapter 7: Inducing ketogenesis via an enteral formulation in patients

with acute brain injury: A phase II study

7.1 Synopsis and citation

Background:

Although extensively studied in children, the safety and tolerability of ketone supplementation in adults is unclear, particularly in the acute brain injury population.

Aim:

To examine the feasibility of inducing ketosis using an enteric ketogenic formulation as measured by plasma beta-hydroxybutyrate and acetoacetate concentrations and determine its impact on cerebral hemodynamics and metabolic parameters

Design:

Prospective interventional Phase II trial of ventilated critically ill patients with acute brain injury administered a ketogenic feed over a 6 day period

Key Findings:

20 patients were recruited, 5 females and 15 males, 3 with stroke, 2 with subarachnoid haemorrhage and 15 with traumatic brain injury. Feeds were well tolerated with 19 patients completing study. There was a significant increase in both plasma beta-hydroxybutyrate and acetoacetate to 0.61 ± 0.53 mmol/l (p =0.0005) and 0.52 ± 0.40 mmol/l (p<0.0001) over the 6 day period. Total daily Ketocal® caloric intake was positively correlated with plasma beta- hydroxybutyrate concentrations (p=0.0011). There was no correlation between cerebral hemodynamic indices and plasma ketone concentrations. In 19 patients, there were no clinically significant changes in acid/base status over the 6 days with pH remaining within normal range. One patient developed a metabolic acidosis and feeds were ceased. No severe adverse events were reported.

82 Inducing ketogenesis via an enteral formulation in patients with acute brain injury: A phase II study

Conclusion:

In patients with acute brain injury, an enterally administered ketogenic formulation was well tolerated and can be safely administered.

Citation:

An oral presentation titled “Inducing ketogenesis via an enteral formulation in patients with acute brain injury: A phase II study” by H White, M Jones, P Kruger and B Venkatesh. This will be presented at the CICM ASM meeting Hobart 2018. (Oral presentation by H White)

Contributions:

Hayden White, Peter Kruger, Mark Jones and Balasubramanian Venkatesh made substantial contributions to the design, acquisition and analysis of data and writing of publication. James Walsham made significant contributions to data analysis and writing of publication. All authors approved final version.

83 Inducing ketogenesis via an enteral formulation in patients with acute brain injury: A phase II study

7.2 Introduction

Ketogenesis is the process by which ketone bodies (beta-hydroxybutyrate and acetoacetate), are produced via fatty acid metabolism. In health, glucose is the brain’s primary metabolic substrate. Ketones however, are capable of acting as an alternative fuel source, supplying up to 70% of basal cerebral energy requirements 27, 223. Acute brain injury (ABI) is a common and catastrophic event leading to significant morbidity and mortality. Much of the pathology relates to disruption of the normal cerebral metabolic energy supply leading to an inability to maintain cerebral energy requirements 224, 225. Ketones may represent an alternative fuel in these instances.

Ketogenic diets have demonstrated benefit in a wide range of neurological disorders including childhood epilepsy, autism, Alzheimer’s disease, migraine, strokes, hypoxic- ischemic encephalopathy, Parkinson disease, amyotrophic lateral sclerosis, and traumatic brain injury (TBI) 91, 168, 208, 211, 226-228. More recent studies have examined the use of ketogenic diets in the management of brain tumours based on the finding that as tumour cells are more able to utilize glucose, altered glucose metabolism may have anti-tumour effects 215.

The determinants of cerebral ketone concentrations include plasma concentration and duration of ketosis 1, 229. We have previously demonstrated in an animal model that infusion of a bespoke hypertonic sodium beta-hydroxybutyrate (BHB) intravenous (IV) solution produced a rapid increase in both plasma and cerebrospinal fluid (CSF) BHB concentrations with no perturbations in metabolic acid-base status 144. However, no commercial intravenous formulation of ketones is currently available. The induction of ketosis via the enteral route is well established, particularly for the management of refractory epilepsy in children 168, 230. The evidence for the administration of enteral ketones to adults with neurological disorders however is far less clear with case reports and case series dominating the literature 231, 232.

Despite evidence suggesting possible benefits to inducing ketosis in neurological disorders, there are no published data investigating the safety, feasibility, metabolic and detailed cerebral haemodynamic effects of inducing ketosis in adults with acute brain injury (ABI) defined as patients diagnosed with acute subarachnoid haemorrhage (SAH), Traumatic brain injury (TBI) and strokes or cerebrovascular accidents (CVA). We hypothesized that a commercially available enteral ketogenic feeding formulation could produce ketosis, without adversely affecting acid-base status or cerebral hemodynamics in patients with ABI.

84 Inducing ketogenesis via an enteral formulation in patients with acute brain injury: A phase II study

The KABI (Inducing ketogenesis in patients with acute brain injury) study was a Phase II trial with the primary aim of examining the feasibility of inducing ketosis using an enteric ketogenic formulation as measured by beta-hydroxybutyrate (BHB) and acetoacetate (AcAc) concentrations in plasma and CSF. Our secondary aims were to determine the impact of a ketogenic diet on the temporal profile in intracranial pressure (ICP) and cerebral perfusion pressure (CPP) during administration and to assess tolerability of this formulation by studying its impact on metabolic and acid-base parameters.

7.3 Methods

Patients

This single site, prospective, interventional study was conducted over a 12 month period to investigate the changes in ketone concentrations in plasma and CSF following acute brain injury. The study setting was a single metropolitan Intensive Care Unit (ICU). Ethics approval was granted by the Princess Alexandra Hospital Human Research Ethics Committee (HREC/16/QPAH/30). Written informed consent was obtained from the participants ‘person responsible’.

Protocol

Within 48 hrs of admission to ICU, 20 adult mechanically ventilated critically ill patients with ABI (defined as any patient admitted with a diagnosis of an acute stroke, SAH or TBI) and expected to survive for a minimum of 72 hours were enrolled. Patients were excluded if < 18 years of age, pregnant, or suffering from Acute or Chronic liver disease or type I diabetes. Following entry into study, the patients were administered the ketogenic formulation (see below) via a naso-gastric tube as a continuous infusion. The individual caloric targets were determined as per the ICU feeding protocol. Episodes of feed intolerance were managed according to the institutional feeding protocol guidelines. The infusion continued for a maximum of 6 days or until extubation.

Feeding formulation

Patients enrolled into the study received a combination of 2 commercial enteral formulations - Ketocal® (Nutricia, Macquarie Park, NSW, Australia) and Protifar® (Nutricia, Macquarie Park, NSW, Australia). Ketocal® is a commercially produced ketogenic formulation. The 4:1 ratio product consists of 8.3% protein, 3.1% CHO and 88.7% fat. It contains a blend of oils

85 Inducing ketogenesis via an enteral formulation in patients with acute brain injury: A phase II study

(high oleic sunflower, soy, palm oil), milk protein (casein and whey), supplemental amino acids, carbohydrate, vitamins, minerals and trace elements. The energy density is 1.5 kcal/ml consisting of 4 g of fat for every 1 g of protein and carbohydrate. Based on a caloric goal of 25-30 kcal/kg/d and protein gal 1.2 g/kg/d the formulation does not deliver adequate protein supplementation at target caloric intake; it was therefore combined with an additional feed, Protifar®, rich in proteins. Protifar® is a protein supplement containing 2.2 g protein (casein and whey) per scoop. Final mixture consisted of Ketocal® which provided 1500 kcal/l of energy and 31 g protein per litre, supplemented with 28 g/l of Protifar® providing a total protein of 59 g/l.

Patients were allowed to receive standard enteral feeds (Nutrison® Protein Plus Multifiber) (Nutricia, Macquarie Park, NSW, Australia) prior to enrolment or following the end of the study period. The use of sedation, analgesia, and other intravenous fluids were administered as determined by treating physician. The management of ICP and CPP were as per ICU guidelines based on Brain Trauma Foundation 2007 guidelines 233, 234. Glucose containing solutions were avoided where possible.

Physiological Analysis

Blood and CSF (if external ventricular device (EVD) present) samples were collected at 8 AM on study days and tested immediately. All clinical observations were recorded hourly for the duration of the study. The cerebral hypoperfusion (CHI) and intracranial hypertension indices (IHI) were based on hourly ICP and CPP measurements 235.

The intracranial hypertension index was calculated as follows;

= 푁푢푚푏푒푟 표푓 표푏푠푒푟푣푎푡푖표푛푠 표푓 퐼퐶푃 > 20 푚푚퐻푔 x 100 푇표푡푎푙 푛푢푚푏푒푟 표푓 표푏푠푒푟푣푎푡푖표푛푠

The cerebral hypoperfusion index was calculated as follows;

푁푢푚푏푒푟 표푓 표푏푠푒푟푣푎푡푖표푛푠 표푓 퐶푃푃<50 푚푚퐻푔 = x 100 푇표푡푎푙 푛푢푚푏푒푟 표푓 표푏푠푒푟푣푎푡푖표푛푠

For safety purposes, blood glucose and BHB were monitored with the Optium FreeStyle glucose/ketone hand held device (Abbott Diabetes Care Roma, Italy).

The total daily catecholamine dose (adrenalin and noradrenalin) was recorded in mg per day. Insulin infusions were similarly recorded in units per day.

86 Inducing ketogenesis via an enteral formulation in patients with acute brain injury: A phase II study

Biochemical analyses

Blood samples were obtained simultaneously for the measurement of plasma ketones (Beta- hydroxybutyrate (BHB) and acetoacetate (AcAc), glucose and arterial blood gases: (pH,

PaCO2, bicarbonate (HCO3,), anion gap and lactate), kidney and liver functions. If an EVD was present which facilitated CSF drainage and collection, CSF was tested for BHB and Acetoacetate concentrations.

Plasma BHB was measured quantitatively using the Stanbio Laboratory β-Hydroxybutyrate LiquiColor Assay Kit (Boerne, TX, USA) on the Beckman DxC800 Unicell (Brea, CA, USA) analyser using a spectrophotometric endpoint assay. Specimens were collected in lithium heparin tubes (#456083, Greiner Bio-One, Kremsmunster, Austria). The linearity of the assay is 0.0-4.0 mmol/l with the coefficient of variation (CV) 3.0% at 0.38 mmol/l and CV 2.1% at 1.38 mmol/l Plasma AcAc concentrations were determined using an automated modification of the original endpoint procedure described by Williamson et al on a Cobas Bio centrifugal analyser5 (Roche, Basel, Switzerland). Specimens were collected in lithium heparin tubes (#456083, Greiner Bio-One, Kremsmunster, Austria). The linearity of the assay is 0.008-1.0 mmol/l (CV 3.6% at 0.003 mmol/l and 2.5% at 0.437 mmol/l).

Arterial blood gases were measured quantitatively using Siemens RAPIDpoint 500 (Siemens Healthcare Diagnostics Inc., Frimley, Camberley, UK).

Statistical analysis

With 20 included patients there was an 80% power to detect a large (by convention a large effect size is given by Cohen as f 2 = 0.35) increase in ketone concentrations over the 6 days of follow up assuming a 1-sided 5% level of significance. Changes in ketone concentrations over time were described using boxplots. We used linear mixed models to estimate the linear change in ketone concentrations. Mixed models were also used to estimate associations between ketone concentrations and other variables of interest.

Safety evaluation

Regular safety analysis was undertaken by an independent monitor (after every 5 patients). The safety assessment of the enteral formulation included continuous monitoring for adverse events. Subjects were monitored for nausea or vomiting, constipation and diarrhoea, hypoglycaemia and metabolic acidosis. Laboratory tests were performed as

87 Inducing ketogenesis via an enteral formulation in patients with acute brain injury: A phase II study described above. Three safety analyses were performed and no safety concerns were identified.

7.4 Results

Baseline characteristics

Twenty patients with ABI were recruited into the study (5 F and 15 M). The admission diagnoses included 3 CVA, 2 SAH and 15 with TBI. The median APACHE II score was 21 (range 10-28). The mean (median, range) GCS at time of enrolment was 3 (median 3, range 3-6 and 13 (median 13, range 10-15) at ICU discharge. The overall ICU mortality rate was 20%. 15 patients had ICP monitoring. 4 of which were through an EVD. The mean length of time from admission to commencement of ketogenic feed was 33.2 ± 21.4 hours. The clinical characteristics and outcome data are summarised in Table 7.1

Table 7.1 Demographic data of patients with acute brain injury (mean +/- SD)

Variable CVA (N = 3) SAH (N = 2) TBI (N = 15) Total N = 20)

Sex Male 15 Female 5

Age 61 ± 19 64 ± 13 55 ± 21 51 ± 19

LOS (Hours) 142 ± 45 327 ± 192 237 ± 157 232 ± 150

ICU Mortality (%) 20

CVA: cerebrovascular accident; SAH: subarachnoid haemorrhage; TBI: traumatic brain injury; LOS: length of stay

Feeding tolerance

Total daily nasogastric aspirates (mls per day) were high on day one (404 ± 331 ml/d), by day 6 this had decreased to 193 ± 266 ml/day. Total caloric intake varied from 1362 ± 559 kcal on day 1 to peak at 1705 ± 857 kcal by day 5. 11 patients completed 5-6 days of ketogenic feed. 15 patients received other enteral feeding (Nutrison®) the day prior to inclusion in the above study.

88 Inducing ketogenesis via an enteral formulation in patients with acute brain injury: A phase II study

Plasma Ketone profiles The mean baseline plasma BHB and AcAc concentrations were 0.24 ± 0.31 mmol/l and 0.19 ± 0.16 mmol/l respectively (Table 7.2) (Figure 7.1 and 7.2).

Table 7.2 Plasma ketones and glucose profiles over trial period

Day 1 Day 2 Day 3 Day 4 Day 5 Day 6

Glucose 8.73 ± 1.95 6.99 ± 0.99 7.07 ± 1.13 6.93 ± 1.01 7.01 ± 1.53 7.50 ± 2.16

BHB 0.24 ± 0.31 0.45 ± 0.63 0.50 ± 0.51 0.50 ± 0.33 0.65 ± 1.09 0.61 ± 0.53

Acetoacetate 0.19 ± 0.16 0.31 ± 0.33 0.45 ± 0.30 0.48 ± 0.22 0.49 ± 0.64 0.52 ± 0.40

All data were expressed as mean ± standard deviation

BHB: beta-hydroxybutyrate

Figure 7.1 Distribution of acetoacetate by day

Boxplots show a decrease in glucose from day 1 to day 2 (p=0.0002) then a stabilised glucose for days 3 to 6. Mixed models analysis shows a consistent result. Horizontal lines within the boxes show the median, and the boxes show interquartile range. Mean represented by ◊

There was a significant increase in both plasma BHB and AcAc to 0.61 ± 0.53 mmol/l (p =0.0005) and 0.52 ± 0.40 mmol/l (p<0.0001) over the 6 day period. 90% of patients increased plasma BHB concentrations from day 1, while 95% increased plasma AcAc concentrations.

89 Inducing ketogenesis via an enteral formulation in patients with acute brain injury: A phase II study

Figure 7.2 Distribution of BHB by day

Boxplots and mixed model results suggest betahydroxybutyrate (BHB) increased approximately linearly over 6 days of follow up by 0.08 units per day (P=0.0005). Horizontal lines within the boxes show the median, and the boxes show interquartile range. Mean represented by ◊

60% patients demonstrated a peak plasma BHB concentration above 0.50 mmol/l and 25% above 1.00 mmol/l. There was a significant decrease in mean serum glucose (8.7 ± 1.9 mmol/l to 7.0 ± 1.0 mmol/l, p=0.0002) from day 1 to day 2 and then stabilised for days 3 to 6 (mean glucose on day 6 was 7.5 ± 2.6 mmol/l) (Figure 7.3). There was strong evidence of a negative association between glucose and plasma acetoacetate (p=0.0034) and BHB (p<0.0001). A positive association was noted between daily intravenous catecholamine infusions (mg per day) (Figure 7.4) and plasma BHB (p<0.0001) but no association with plasma acetoacetate. There was no association between plasma BHB/acetoacetate and daily dosage of insulin (p=0.9171) (Figure 7.5). Of patients who received Nutrison® feeds (either prior or at the completion of the study) when compared with Ketocal® feeds, there was a strong negative association between total daily caloric intake of Nutrison® and plasma BHB concentration (p<0.0001) while total daily Ketocal® caloric intake was associated with a positive correlation with plasma BHB concentrations (p=0.0011) (Figure 7.6).

CSF was obtained from 4 patients with EVD’s in place. CSF BHB and AcAc concentrations ranged from 0.05 – 0.68 mmol/l and 0.02 – 0.16 mmol/l although 50% of specimens were contaminated with blood.

90 Inducing ketogenesis via an enteral formulation in patients with acute brain injury: A phase II study

Figure 7.3 Distribution of glucose by day

Figure 7.4 Comparison of daily plasma BHB concentration with total daily intravenous catecholamine infusion (mg/day).

BHB betahydroxybutyrate Catecholamine infusion includes noradrenalin and adrenalin and represented as mg of catecholamine delivered per day

91 Inducing ketogenesis via an enteral formulation in patients with acute brain injury: A phase II study

Figure 7.5 Comparison of daily plasma BHB concentration with total daily intravenous insulin infusion (units/day) BHB – betahydroxybutyrate Insulin infusion represented as units of insulin delivered per day

Figure 7.6 Daily plasma BHB concentration compared with total daily calories from Ketocal (kcal/day)

BHB – betahydroxybutyrate

92 Inducing ketogenesis via an enteral formulation in patients with acute brain injury: A phase II study

Acid-base status and other biochemical analysis

In 19 patients, there were no clinically significant changes in acid/base status over the 6 days. The mean blood pH increased from 7.39 ±0.04 to 7.45 ±0.04 over the first 3 days and then remained within the normal range until day 6 (pH 7.45 ±0.06) (Figure 7.7).

Figure 7.7 Distribution of pH by day

Boxplots show an increasing pH from day 1 to day 3 (p = 0.0004) then a stabilised pH for days 4 to 6. Mixed models analysis shows a consistent result. Horizontal lines within the boxes show the median, and the boxes show interquartile range. Mean represented by ◊

Serum bicarbonate concentrations remained stable during the study period ranging from 23 ± 3 mmol/l on day one to 24 ±3 mmol/l by day 6 (p=0.26). Lactate was 1.4 ± 0.8 mmol/l on day 1 but decreased by day 2 and remained below 1.0 mmol/l for the rest of study period. One patient however, developed a metabolic acidosis with a peak anion gap of 20 mmol/l and BHB of 4.0 mmol/l (see below). Renal function remained within the normal range with a steady decrease in serum creatinine concentrations. Serum alanine transferase (ALT) and aspartate transferase (AST) concentrations increased over the period as did gamma- glutamyl transferase and alkaline phosphatase concentrations. The biochemical characteristics over the course of the study are summarised in Table 7.3.

93 Inducing ketogenesis via an enteral formulation in patients with acute brain injury: A phase II study

Table 7.3 Clinical and biochemical observations measured daily over the trial period

Day 1 Day 2 Day 3 Day 4 Day 5 Day 6

pH 7.39 ± 0.04 7.42 ± 0.03 7.45 ± 0.04 7.44 ± 0.05 7.44 ± 0.07 7.45 ± 0.06

pCO2 39.00 ± 3.88 37.55 ± 4.30 36.42 ± 5.31 38.00 ± 4.50 37.14 ± 5.71 36.25 ± 6.11

pO2 92.60 ± 22.14 89.80 ± 21.88 83.32 ± 21.79 79.93 ± 23.34 79.43 ± 29.01 84.00 ± 25.19

Bicarbonate 23.45 ± 2.74 23.95 ± 2.86 24.84 ± 2.75 25.63 ± 3.70 24.43 ± 3.11 24.33 ± 3.45

Anion Gap 7.35 ± 2.81 7.85 ± 3.38 8.05 ± 3.12 8.25 ± 3.32 9.50 ± 3.16 10.00 ± 3.07

Lactate 1.45 ± 0.82 0.91 ± 0.24 0.98 ± 0.23 0.90 ± 0.29 0.89 ± 0.33 0.87 ± 0.25

AST 48.05 ± 28.7 45.30 ± 28.71 49.35 ± 27.12 58.44 ± 32.66 80.71 ± 94.46 75.00 ± 50.28

ALT 49.20 ± 74.78 39.20 ± 48.16 41.50 ± 41.21 52.94 ± 42.58 68.57± 69.13 73.46 ± 58.59

GGT 37.80 ± 25.34 42.15 ± 24.08 57.85 ± 40.60 96.50 ± 58.33 120.36±95.23 131.85±100.37

ALP 52.50 ± 15.39 57.45 ± 15.74 65.20 ± 28.73 78.63 ± 39.18 91.14 ± 52.31 94.00 ± 50.17

LDH 284.75±93.71 274.65±79.37 294.65±28.73 336.88±104.33 395.43±136.29 421.00±139.61

Bilirubin 13.90 ± 4.85 12.70 ± 5.40 12.95 ± 4.67 13.38 ± 5.45 14.00 ± 5.79 15.46 ± 5.53

Sodium 141.50 ± 5.39 143.25 ± 5.07 142.15 ± 4.74 142.69 ± 5.53 143.14 ± 6.18 142.38 ± 6.19

Potassium 3.88 ± 0.32 3.77 ± 0.32 3.69 ± 0.39 3.79 ± 0.30 3.70 ± 0.34 3.62 ± 0.23

Chloride 111.60 ± 6.33 111.65 ± 5.34 109.55 ± 5.94 109.63 ± 6.87 109.71 ± 6.49 108.69 ± 7.70

Urea 6.25 ± 5.12 6.03 ± 4.87 6.27 ± 4.49 8.37 ± 10.13 9.27 ± 10.38 9.08 ± 9.97

Creatinine 80.00 ± 35.06 70.40 ± 29.25 67.35 ± 20.06 63.88 ± 11.52 61.29 ± 12.39 59.77 ± 11.84

All data were expressed as mean ± standard deviation AST: Aspartate aminotransferase; ALT: Alanine Aminotransferase; GGT: Gamma glutamyl transferase; ALP: Alkaline phosphatase; LDH: Lactate dehydrogenase

Cerebral hemodynamic indices

The mean peak ICP measurement decreased from 20 ± 6 mmHg on day 1 to 18 ± 5 mmHg (p=0.26) on day 6. The mean lowest CPP measurement increased from 55 ± 9 mmHg on day 1 to 57 ± 7 mmHg (p=0.073) on day 6. Neither change was statistically significant. The ICH and CPP indices were 12% ± 18% and 3% ± 7% respectively on admission and 2% ± 2% and 2% ± 4% on discharge (Figure 7.8). There was no correlation between ICH/CPP index and plasma ketone concentrations. 15 patients required an intravenous catecholamine infusion at some time during the study period to maintain CPP. 10 patients required hyperosmolar therapy to manage intracranial hypertension. The mean period for

94 Inducing ketogenesis via an enteral formulation in patients with acute brain injury: A phase II study which hyperosmolar therapy was necessary was 43.5 ± 74.3 hours although the range was wide (0 – 280 hours).

Figure 7.8 Daily ICH index compared with daily CPP index

ICH index – intracranial hypertension index. CPP index – cerebral hypoperfusion index (see text for calculation) Adverse effects

The ketocal feeds were well tolerated (Table 7.4). Only 8 episodes of vomiting and diarrhoea were recorded during the study period. No episodes of hypoglycaemia or seizures were reported during study period.

Table 7.4 Adverse events

Number of patients experiencing 1 or more event

Vomiting and diarrhoea 8

Hypoglycaemia 0

Seizures 0

Metabolic academia 1

Increase in hepatocellular or 12 cholestatic liver enzymes from baseline

95 Inducing ketogenesis via an enteral formulation in patients with acute brain injury: A phase II study

Feeds were prematurely ceased on one patient who developed a metabolic acidemia while on ketogenic diet in the setting of severe sepsis, malnutrition and vasopressor requirements. The ketogenic feed was stopped and the anion gap and BHB returned to normal within 36 hours. All other patients completed the study as per protocol. As noted above, there was an increase in the mean concentration of both hepatocellular and cholestatic liver enzymes during the study period. No severe adverse events were reported.

7.5 Discussion

This phase II study of ketogenic feeds in 20 ventilated ICU patients with acute brain injury, demonstrates that an enterally administered ketogenic formulation consistently increased plasma BHB and acetoacetate concentrations, above both baseline and the normal, non- fasting ranges. This formulation was associated with an improvement in glucose control, was well tolerated with few gastro-intestinal side effects and with no adverse impact on intracranial hemodynamics or acid-base parameters in 19 out of the 20 patients. An increase in several hepatocellular and cholestatic enzymes was noted although the clinical significance and causation of this is not clear. To our knowledge, this represents the most comprehensive study of ketone diets in patient with ABI to date.

Despite the lack of clinical studies, a body of experimental work supporting the benefits of ketone supplementation as an alternative source of fuel in acute and chronic brain disease exists 51, 130. BHB is more energy efficient then glucose and increases ATP production via an increase in citric acid cycle intermediates with a 16-fold elevation in acetyl CoA content 52, 53. Ketones reduce formation of reactive oxidant species and protect against glutamate mediated apoptosis and necrosis 54-56. They also improve cerebral blood flow by up to 39% 56. During times of oxidative stress, the ability of the brain to utilize glucose may become compromised and ketones may be preferentially taken up, an effect which may be augmented by the external supply of ketones 75.

Despite demonstrating a significant increase in plasma BHB concentrations to 0.61 ± 0.53 mmol/l, it is unclear whether this concentration is sufficient to provide significant cerebral metabolic substrate. Gilbert et al demonstrated a significant correlation (r=0.471 p = 0.007) between plasma BHB concentrations and seizure control in children on a ketogenic diet with a cut off of 4 mmol/l the most effective 87. This was supported by Cahill et al who suggested a therapeutic plasma BHB range of 3-5 mmol/l necessary for clinical benefit 143. However, other authors suggest that the clinical benefit of targeting a specific BHB plasma

96 Inducing ketogenesis via an enteral formulation in patients with acute brain injury: A phase II study concentration is controversial 196. In children with acyl CoA dehydrogenase deficiency, blood BHB concentrations of 0.19-0.36 mmol/l produced a therapeutic response 86. Furthermore, a study on the benefits of ketone supplementation in cognitively impaired adults demonstrated an improvement in cognitive function with a peak plasma BHB concentration of only 0.68 mmol/l 97. Ketone neuroprotection in various CNS injury animal models seems to demonstrate benefit across a broad spectrum of plasma BHB concentrations 73, 80, 236. Further study is required to clarify the optimum plasma ketone concentrations in humans.

A major concern of administering exogenous ketones is the induction of a metabolic acidosis. In our study, there was no evidence of perturbations in pH, plasma bicarbonate, lactate or the anion gap in 95% of the study population. As noted, one patient developed a metabolic acidemia during the trial, although it was in the context of severe sepsis and high vasopressor requirements. These findings are consistent with published data from a recent study of 20 obese patients placed on a low calorie ketogenic diet 237. These results are also in accord with those reported in a study by our group where rats were administered an intravenous ketone formulation over a 6 hour period 144.

A consistent finding in studies investigating the exogenous administration of ketones is the improvement in glucose control 91, 237. Our study noted a significant initial decrease in glucose from day one and this improvement was sustained throughout the study period with no documented episodes of hypoglycaemia. Evidence suggests that during times of injury, cerebral metabolism switches to favouring ketones, as glucose may no longer be sufficient during high energy requirements 61, 224, 225. High glucose concentrations have persistently been associated with poor outcome in critically ill patients with TBI as ischemic injury leads to a limited substrate supply and metabolism which is exacerbated by glucose leading to lactate accumulation and brain damage 133, 238.

Our study had a number of strengths. It is the most comprehensive study of a ketogenic feed in patients with ABI which included the systematic collection of data with detailed plasma biochemical and acid-base profiling and the reporting of metabolic outcomes and cerebral hemodynamic indices. A thorough safety assessment was undertaken with continuous monitoring for adverse events. The majority of patients completed their feeding protocol so that the primary aim of assessing the impact of a ketogenic feed on plasma ketone concentrations was successful. The only other published work investigating ketogenic diets in the TBI population was from a pilot study by Ritter et al in 1996 91. It was a proof of principle study, which examined the effects of a ketogenic diet consisting of 75% 97 Inducing ketogenesis via an enteral formulation in patients with acute brain injury: A phase II study lipid and 25% protein on biochemical markers of metabolism in patients with TBI. Only 8 patients in the experimental arm completed at least 5 days of the study. The authors were able to demonstrate better glucose control and significantly higher plasma BHB concentrations in the experimental group although there was no difference in neurological outcomes at 3 months.

Our study has several limitations. The limited number of EVD’s prevented an assessment of the impact of the ketogenic feed on CSF ketone concentrations. Secondly, as the study was not powered to look at clinical outcomes, our measure of outcome was limited to GCS on discharge from ICU. Longer outcomes involving more detailed clinical assessment would be desirable in future studies. And lastly, we did not address the question of the optimal plasma concentration of ketones for clinical benefit.

A number of questions still remain. Foremost is that although ketone concentrations were significantly increased as compared to patients fed a normal diet, it is unclear whether the concentrations reached are sufficient to improve cerebral energetics. Further studies are necessary to determine; the optimal concentration of ketones necessary to impact on cerebral energetics, the best means of inducing ketosis and the overall impact on outcome.

7.6 Conclusion

In conclusion, in patients with acute brain injury, an enterally administered ketogenic formulation was well tolerated and can be safely administered.

98 Conclusions and Future Directions

Chapter 8: Conclusions and Future Directions

The work presented in this thesis provides further evidence to the growing body of knowledge that exists regarding the role of ketones in cerebral cellular energetics in patients with neurological dysfunction. The synthesis of basic science research and results from both animal and human research provide compelling evidence to support ongoing investigation into potential treatments in a group of patients which have traditionally suffered from a lack of management options. Traditionally viewed as ‘metabolisms ugly duckling’, because of the association with DKA, this work has attempted to demonstrate the physiological and clinical importance of ketones.

Although it has been known for over 100 years that starvation states are associated with improved seizure control and that this might be due to an increase in blood ketone concentrations, detailed research into the mechanisms and possible benefits of administering exogenous ketones has only occurred in the past few decades. This was despite the 1928 paper which appeared in NEJM entitled “Ketogenic diet in the treatment of epilepsy’ 239. However, researchers at John’s Hopkins continued to advocate for the administration of these diets to children with refractory epilepsy and eventually the benefits of the ketogenic diet were demonstrated in two randomized controlled trials. A study of 145 children with poorly controlled epilepsy published in the Lancet Neurology, demonstrated that a ketogenic diet was capable of; significantly reducing seizure frequency compared to controls, that it was equally effective in both symptomatic generalised or symptomatic focal syndromes and was tolerated for extended periods with most frequent side-effects reported at 3-month review being constipation, vomiting, lack of energy, and hunger 168.

However, despite the evidence that ketones supplementation was beneficial in the paediatric epilepsy population, adult research remained elusive. Certainly there was encouraging data from animal and basic science research that suggested a potential role for exogenous administered ketones in the management of acute and chronic neurological diseases which may result from energy deficits. The translation of this basic science to treatments for adult humans however has been a slow process.

Our initial goal was to produce an intravenous ketone containing solution. The reasons for this was that by administering ketones intravenously, we could be confident that the drug would enter the plasma at a controlled concentration and be available to enter the CSF. We

99 Conclusions and Future Directions could also investigate the effects of dose in a predictable way. Hypertonic saline was chosen as it has largely replaced mannitol as the treatment of choice for intracranial hypertension and could theoretically improve ICP/CPP and correct cerebral energy deficits simultaneously. The data from our animal model was encouraging and confirmed that as hypothesized, at least in the concentration range we investigated, plasma, CSF and brain ketone concentrations were dependent on IV concentration 144. However, producing an IV ketone formulation for humans proved more complicated and expensive than anticipated and once it became clear that the costs would be prohibitory, we abandoned this route of administration and began searching for a less expensive and more feasible alternative.

We therefore turned our attention to enteral preparations. Ketogenic diets have been examined in a wide range of neurological disorders including autism, Alzheimer’s disease, migraine, epilepsy, hypoxic-ischemic encephalopathy, Parkinson disease, amyotrophic lateral sclerosis, and traumatic brain injury 91, 165, 208, 210. More recent studies have examined the use of these diets in the management of brain tumours based on the fact that altered glucose metabolism may have anti-tumour effects. A number of “ketogenic diets” have been described including: the traditional ‘classic’ ketogenic diet, the medium-chain triglyceride (MCT) diet, the modified Atkins diet (MAD) and the low glycemic index treatment (LGIT). However, it was unclear as to which diet would be the most ketogenic or appropriate in acutely ill ABI population. Furthermore, it was unclear what target concentration of BHB to aim for. This has not been well studied although there are some reports that concentrations closer to 4 mmol/l were necessary to improve cerebral energetics 52, 143.

There was very little evidence from the adult literature to guide our formulation. The only study to date looking at the effects of a ketogenic diet on TBI was conducted by Ritter et al in 1996 91. They were able to demonstrate better glucose control in the experimental group. Although the BHB concentrations were significantly higher in the intervention group, the maximum concentration was still under 0.6 mmol/l.

We therefore embarked on a number of investigations and reviews to further inform our study into the cerebral energy metabolism in ABI patients. As there was no data examining ketone concentrations in ABI patients, we undertook an observational study examining baseline ketone concentrations in this population 89. We measured ketone in plasma and CSF of 38 patients with CVA's, SAH or traumatic brain injury and found that aside from a small increase in ketones on admission, the concentrations remained low during their hospital stay. We also performed a systematic review of all studies in adults reporting the 100 Conclusions and Future Directions use of ketones to treat neurological disorders (acute or chronic) and found that although there appears to be evidence to support the role of ketones in these disorders, the evidence is of poor quality and relies largely on case reports/series 232. Furthermore, there was no evidence supporting a specific plasma ketone concentration in adults.

As ketones are not the only potential energy substrate that may augment cerebral metabolism, we undertook a review of other substances including glucose, NEFA, lactate, BCAA and anaplerotic molecules including triheptanoin and found again, that the evidence was of poor quality although research into lactate supplementation was encouraging 240. We also discussed the challenges in developing new fuel substrates for the injured brain. Our conclusions were however that ketones supplementation still represents the best option for improving cerebral energetics and should be studied further. We therefore set out to design a phase II interventional study to look at a commercially available ketogenic feed, Ketocal in the adult ABI population. This formulation has been available for many years but largely utilized in the paediatric population.

Our study consisted of 20 adult patients with severe ABI managed in a tertiary ICU. Although Ketocal was utilized as our source of calories, the formulation does not provide the necessary protein content required for an adult critically ill patient. The formulation was therefore modified by adding extra protein. Patients included in the study were fed the ketone formulation for a maximum of 6 days. From this study we were able to determine that; 1. the formulation was successful in increasing ketone concentrations, 2. the formula was well tolerated and there were no issues with delivery, 3. that no major metabolic abnormalities were detected especially changes in acid/base status and 4. there was no adverse impact on intracranial hemodynamics. Disappointingly, BHB concentrations peaked at approximately 0.6 mmol/l which is lower than we predicted but the clinical significance of this is not known.

In summary, the body of work from this thesis consists of;

1. A literature review of ketone physiology and potential clinical applications 2. An animal study demonstrating the proof of principle of an intravenous ketone formulation 3. An observational study (n=38 patients) documenting the plasma concentrations of ketones in patients with ABI 4. A literature review of the novel substrates for the injured brain

101 Conclusions and Future Directions

5. A clinical trial (n=20 patients) examining serial concentrations of plasma ketones in patients with ABI who are administered a ketone diet and their safety and feasibility 6. A systematic review of the trials of ketone supplementation in acute and chronic neurological states.

Our findings include;

1. Ketones are a potential energy substrate for the brain 2. We have been able to demonstrate that an intravenous ketone formulation is feasible and safe in animal models. However, the bench to bedside translation has been limited by costs and logistics 3. We have demonstrated that in an observational study of patients with acute brain injury on conventional feeds, plasma concentrations of ketones remain low 4. Although other potential cerebral energy substrates have been investigated, ketones appear to have the strongest evidence supporting their use 5. Although there is strong evidence for ketone supplementation in childhood epilepsy, evidence for the use in adults with acute and chronic neurological disease is limited to case series and small RCTs 6. We have demonstrated in a prospective interventional study that an enterally administered ketogenic formulation was well tolerated and can be safely administered

Gaps in our knowledge

There remain a number of gaps in our knowledge regarding the optimal use of ketones in acute and chronic brain injury.

• Foremost is mode of administration and dosing of ketone supplementation. • No studies exist demonstrating the optimum plasma concentration. During starvation, ketone levels may rise to 7 mmol/l. As noted in chapter 7, significant disagreement exists between authors with some supporting a therapeutic range of 3-5 mmol/l while others have noted improvements with plasma concentrations of less the 1 mmol/l. Secondly, although ketosis may be induced by a number of enteral formulations, their ability to consistently raise plasma ketone concentrations in adults is limited. Although an intravenous formulation would be the most likely to provide a rapid and sustained increase in plasma ketone concentrations, no such formulation is available and as previously noted, is unlikely in the near future. We therefore require an enteral

102 Conclusions and Future Directions

formulation with more predictable pharmacokinetics. It appears that ketone esters have the potential to provide the consistency needed and is explored further below in “Future directions”. • Thirdly, the optimum duration of ketone supplementation is not known. Although in the acute setting, periods of up to 2 weeks are described, no clear evidence exists to guide duration. In patients with chronic neurological dysfunction including Alzheimer’s, ketogenic diets have been continued for up to 90 days. • Although the adverse complications of inducing ketosis has been extensively studied in the epileptic population on long term supplementation, little is known of the acute effects in the ABI population. Specifically, on intracranial hemodynamics. In our study in chapter 7, we failed to demonstrate any significant adverse impact on ICP and CPP but our study was limited by the small numbers and short follow ups. A future study with the ability to perform cerebral microdialysis may be better able to examine the impact of ketosis on cerebral metabolites. Furthermore, studies with larger numbers and longer clinical outcomes are required to adequately address the long term impact of ketone supplementation. • There are no studies examining the term effects of ongoing ketone supplementation in the rehabilitation setting. However, as small studies in patients with chronic cognitive dysfunction have demonstrated improved functional outcomes following ketogenic diets this is a potential area for further research.

Future directions

Through this series of studies and reviews, we have been able to determine that exogenous ketones are a potential alternative source for cerebral energy supplementation in the injured brain. However, the means of administering and increasing plasma levels to theoretically beneficial concentrations is still elusive given the fact that an IV formulation is currently not feasible. There is however ongoing research in this field looking at other enteral formulations which may prove more ketogenic. These include ketone esters.

Ketone esters

As noted previously, established methods for inducing ketosis via the oral route consist of either adherence to a ketogenic diet or regular ingestion of medium chain triglycerides. These diets are generally poorly tolerated, associated with atherogenic lipids and relatively ineffective at raising the BHB concentrations in adults much higher than 0.5 mmol/l 241, 242.

103 Conclusions and Future Directions

Furthermore, the efficacy of these diets is dependent on the depletion of CHO from hepatic and muscle stores with a subsequent increase in FFA and thus ketone concentrations 243. This process takes time and therefore ketone concentrations may not rise for a number of hours. Given that evidence suggests higher blood concentrations of BHB are more effective, researchers have attempted to examine more efficient methods of increasing plasma ketone concentrations.

One such approach is the use of the ketone monoester (R)-3-hydroxybutyl (R)-3- hydroxybutyrate to elevate blood ketone concentrations. Following ingestion, the ketone monoester undergoes complete hydrolysis into D-b-hydroxybutyrate and R-1,3-butanediol) by carboxylesterases and esterases found within the gastrointestinal tract, blood, liver and other tissues 244. R-1,3-butanediol is subsequently metabolized to D-b-hydroxybutyrate and acetoacetate in the liver 245.

Significant differences exist between ketosis induced by starvation or high-fat diet and that resulting from the ingestion of ketone esters. Ketone bodies are endogenously produced in the liver from circulating free fatty acids (FFA) from adipolysis. Ketone esters in contrast are cleaved in the gut and are absorbed through the gut epithelium. Raised concentrations of ketone bodies inhibit further production of FFA. The subsequent metabolic fate of entering the TCA however, is the same.

Several recent publications have examined the safety and efficacy of a ketone monoester produced by the researcher Kieran Clarke from Oxford 147, 193. An initial toxicology study looking at the administration of KM for 28 days to rats noted a significant decrease in oral intake and weight, but no other significant changes, supporting the safety of KM. A subsequent study examined the kinetics, safety and tolerability of KM in 54 healthy adult subjects. Both single and multiple doses at varying concentrations (140, 357, or 714 mg/kg body weight) were examined. Maximum plasma concentrations of ketones were noted within 1–2 h, reaching 3.30 mmol/l and 1.19 mmol/l for beta-hydroxybutyrate and acetoacetate, respectively. The ketone monoester was subsequently administered at 140, 357, and 714 mg/kg body weight, three times daily, over 5 days. At the highest dose, BHB concentrations did not exceed 5.5 mmol/l. These patients did however experience significant gastrointestinal side effects, although this was believed to reflect the high quantities of milk ingested as the ketone monoester is administered in a milkshake.

104 Conclusions and Future Directions

A number of future areas of study therefore present themselves. A larger phase II study should be undertaken to investigate the limitations of the current research. The choice of potential ketogenic supplement needs to be determined but ketone esters, by providing a more stable plasma ketone concentration would provide a good starting point. Future studies would need to include a number of endpoints not previously investigated including cerebral biomarkers. Cerebral biomarkers including S100 and Neuron-specific enolase are markers of cerebral injury although clinical applications are still in early phase. Other significant endpoints would include detailed evaluation of cerebral hemodynamics and long term clinical outcomes, both cognitive and morbidity/mortality. The results of this detailed study could then be used to inform the design of a randomized controlled study with the main aim of determining whether ketone supplementation leads to an improvement in clinical outcomes of patients suffering from TBI.

In conclusion, investigations into the role of ketones in patients with brain injury remains in its infancy. Certainly, the scientific plausibility, animal studies and small human intervention studies are encouraging and justify pursuing a more rigorous research agenda into this fascinating molecule. Many unanswered questions remain including the most beneficial dose, plasma concentration, formulation and route of administration. On a more fundamental level, the interaction between ketones, glucose and other potential cerebral substrates including lactate require more vigorous investigation. Hopefully our research will lead to a greater awareness of this group of molecules, once thought of as a waste product of fat metabolism.

105 Reference List

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131 Appendices

Appendices

Appendix A Contributions for publications in which I was not the sole author

White H, Venkatesh B. Clinical review: ketones and brain injury. Crit Care. 2011; 15:219

Contributor Statement of Contribution

White (Candidate) Conception and design(80%) Analysis and interpretation (80%) Drafting and production (70%)

Venkatesh Conception and design (20%) Analysis and interpretation (20%) Drafting and production (30%)

White H, Venkatesh B, Jones M, Worrall S, Chuah T, Ordonez J. Effect of a hypertonic balanced ketone solution on plasma, CSF and brain beta-hydroxybutyrate levels and acid- base status. Intensive Care Med. 2013 Apr; 39(4):727-33 – incorporated into Chapter 2

Contributor Statement of Contribution

White (Candidate) Conception and design (65%) Analysis and interpretation (70%) Drafting and production (70%)

Worrall Conception and design (0%) Analysis and interpretation (5%) Drafting and production (0%)

Chuah Conception and design (5%) Analysis and interpretation (0%) Drafting and production (5%)

Ordenez Conception and design (5%) Analysis and interpretation (0%) Drafting and production (0%)

Jones Conception and design (5%)

132 Appendices

Analysis and interpretation (10%) Drafting and production (5%)

Venkatesh Conception and design (20%) Analysis and interpretation (15%) Drafting and production (20%)

White H, Venkatesh B, Jones M, Fuentes H. Serial changes in plasma ketone concentrations in patients with acute brain injury. Neurological Research 2017; 39:1-6 – incorporated into Chapter 4

Contributor Statement of Contribution

White (Candidate) Conception and design (75%) Analysis and interpretation (70%) Drafting and production (70%)

Fuentes Conception and design (0%) Analysis and interpretation (5%) Drafting and production (0%)

Jones Conception and design (5%) Analysis and interpretation (10%) Drafting and production (5%)

Venkatesh Conception and design (20%) Analysis and interpretation (15%) Drafting and production (25%)

White H, Kruger P, Venkatesh B. Novel metabolic substrates for feeding the injured brain In: Annual update in Intensive Care and Emergency Medicine 2017– Edited by J.L. Vincent, Springer-Verlag, 2017 pp 329-341 – incorporated into Chapter 5

Contributor Statement of Contribution

White (Candidate) Conception and design (80%) Analysis and interpretation (80%) Drafting and production (70%)

133 Appendices

Kruger Conception and design (10%) Analysis and interpretation (10%) Drafting and production (15%)

Venkatesh Conception and design (10%) Analysis and interpretation (10%) Drafting and production (15%)

White H, Venkatesh K, Venkatesh B. Systematic Review of the Use of Ketones in the Management of Acute and Chronic Neurological Disorders. J Neurol Neurosci. 2017, 8:2. doi:10.21767/2171-6625.1000188 – incoporated into Chapter 6.

Contributor Statement of Contribution

White (Candidate) Conception and design (65%) Analysis and interpretation (65%) Drafting and production (70%)

Venkatesh K Conception and design (25%) Analysis and interpretation (25%) Manuscript Preparation (20%)

Venkatesh B Conception and design (10%) Analysis and interpretation (10%) Drafting and production (10%)

134 Appendices

Appendix B Animal Ethics Approval Certificate

135 Appendices

Appendix C Human Research Ethics Committee Approval: Blood and CSF ketone levels following acute brain injury.

136 Appendices

Appendix C Human Research Ethics Committee Approval: Blood and CSF ketone levels following acute brain injury (continued)

137 Appendices

Appendix C Human Research Ethics Committee Approval: Blood and CSF ketone levels following acute brain injury (continued)

138 Appendices

Appendix D Human Research Ethics Committee Approval: Inducing Ketogenesis in patients with Acute Brain Injury via oral administration of a ketogenic feed.

139 Appendices

Appendix D Human Research Ethics Committee Approval: Inducing Ketogenesis in patients with Acute Brain Injury via oral administration of a ketogenic feed (continued)

140 Appendices

Appendix D Human Research Ethics Committee Approval: Inducing Ketogenesis in patients with Acute Brain Injury via oral administration of a ketogenic feed (continued)

141