Health and Social Development, Faculty of (Okanagan)
Health and Exercise Sciences, School of (Okanagan)
DSpace
UBCO
Caldwell, Hannah Grace
Doctor of Philosophy - PhD
University of British Columbia
Metabolism describes the sum of biochemical reactions that take place within a living organism which provide energy for vital processes; this can be studied at the whole-body, organ specific, or cellular level. Energy availability, which is determined by the difference between energy intake and exercise energy expenditure, is affected by environmental and energetic stresses (e.g., high-altitude, exercise, diet/nutrition). Systemic energy deficits have implications for the regulation of organ-specific substrate utilization and function. The aim of this thesis was to integrate brain, skeletal muscle, and systemic substrate oxidation and relative fuel utilization in the context of selected environmental and energetic stress (high-altitude, exercise, nutritional caloric deficits). Study 1 addressed a fundamental question for integrative cerebrovascular physiology: How are the cerebral metabolic rates of oxygen and glucose utilization (CMRO₂ and CMRGlu, respectively) affected by alterations in arterial PCO₂ (PaCO₂)? The results of this study indicate that cerebral oxidative metabolism (i.e., CMRO₂) is altered by approximately 1 % per mmHg change in PaCO₂. These changes, in part, are explained by an increased compensatory contribution of anaerobic metabolic pathways. Study 2 investigated the research question: How does systemic fuel utilization at high-altitude affect the brain inflammatory response to exercise? The results of this study provide new evidence that 6-8 days of acclimatization at 3,800 m does not provoke immune suppression or exacerbate systemic pro-inflammatory responses at rest or with maximal exercise. Further, altitude-induced alterations in systemic inflammatory responses did not directly affect release of pro-inflammatory cytokines by the brain with maximal exercise. Study 3A showed that 14-days low energy availability (LEA) – involving effectively 50% daily caloric restriction – in endurance-trained females provokes immunological stress, systemic inflammation and impairs exercise performance. Study 3B further revealed that 14-days LEA in these same females provokes an increased reliance on fat oxidation to absolute energy expenditure during submaximal exercise that was independent of any changes in resting metabolic rate, insulin sensitivity, and skeletal muscle mitochondrial respiratory capacity. This dissertation advances our understanding of integrative brain, skeletal muscle, and systemic substrate oxidation and relative fuel utilization in the context of environmental and energetic stress.
SUBSTRATE UTILIZATION IN THE BRAIN AND SKELETAL MUSCLE DURING ENVIRONMENTAL AND ENERGETIC STRESS IN HUMANS by Hannah Grace Caldwell BHK., University of British Columbia, 2017 M.Sc., University of British Columbia, 2019 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE COLLEGE OF GRADUATE STUDIES (Kinesiology) THE UNIVERSITY OF BRITISH COLUMBIA (Okanagan) July 2023 © Hannah Grace Caldwell, 2023 ii The following individuals certify that they have read, and recommend to the College of Graduate Studies for acceptance, a thesis/dissertation entitled: SUBSTRATE UTILIZATION IN THE BRAIN AND SKELETAL MUSCLE DURING ENVIRONMENTAL AND ENERGETIC STRESS IN HUMANS submitted by Hannah Grace Caldwell in partial fulfillment of the requirements of the degree of Doctor of Philosophy in Kinesiology. Dr. Philip Ainslie, School of Health and Exercise Sciences Supervisor Dr. Jonathan Little, School of Health and Exercise Sciences Supervisory Committee Member Dr. Lasse Gliemann, University of Copenhagen Supervisory Committee Member Dr. Ylva Hellsten, University of Copenhagen Supervisory Committee Member Dr. Sarah Purcell, UBC Faculty of Medicine, Division of Endocrinology and Metabolism University Examiner Dr. José González-Alonso, Brunel University London External Examiner iii Abstract Metabolism describes the sum of biochemical reactions that take place within a living organism which provide energy for vital processes; this can be studied at the whole-body, organ specific, or cellular level. Energy availability, which is determined by the difference between energy intake and exercise energy expenditure, is affected by environmental and energetic stresses (e.g., high-altitude, exercise, diet/nutrition). Systemic energy deficits have implications for the regulation of organ-specific substrate utilization and function. The aim of this thesis was to integrate brain, skeletal muscle, and systemic substrate oxidation and relative fuel utilization in the context of selected environmental and energetic stress (high-altitude, exercise, nutritional caloric deficits). Study 1 addressed a fundamental question for integrative cerebrovascular physiology: How are the cerebral metabolic rates of oxygen and glucose utilization (CMRO2 and CMRGlu, respectively) affected by alterations in arterial PCO2 (PaCO2)? The results of this study indicate that cerebral oxidative metabolism (i.e., CMRO2) is altered by approximately 1 % per mmHg change in PaCO2. These changes, in part, are explained by an increased compensatory contribution of anaerobic metabolic pathways. Study 2 investigated the research question: How does systemic fuel utilization at high-altitude affect the brain inflammatory response to exercise? The results of this study provide new evidence that 6-8 days of acclimatization at 3,800 m does not provoke immune suppression or exacerbate systemic pro-inflammatory responses at rest or with maximal exercise. Further, altitude-induced alterations in systemic inflammatory responses did not directly affect release of pro-inflammatory cytokines by the brain with maximal exercise. Study 3A showed that 14-days low energy availability (LEA) – involving effectively 50% daily caloric restriction – in endurance-trained females provokes immunological stress, systemic inflammation and impairs exercise performance. Study 3B further revealed that 14-days LEA in these same females provokes an increased reliance on fat oxidation to absolute energy expenditure during submaximal exercise that was independent of any changes in resting metabolic rate, insulin sensitivity, and skeletal muscle mitochondrial respiratory capacity. This dissertation advances our understanding of integrative brain, skeletal muscle, and systemic substrate oxidation and relative fuel utilization in the context of environmental and energetic stress. iv Lay Summary Metabolism describes the sum of biochemical reactions that take place within a living organism which provide energy for vital processes; this can be studied at the whole-body, organ specific, or cellular level. Energy availability, which is determined by the difference between energy intake and exercise energy expenditure, is affected by selected environmental and energetic stresses (e.g., high-altitude, exercise, diet/nutrition). Systemic energy deficits have implications for the regulation of organ-specific metabolism. This thesis provides novel insights for integrative brain, skeletal muscle, and systemic substrate oxidation and relative fuel utilization in the context of environmental and energetic stress (e.g., high-altitude, exercise, nutritional caloric deficits). Experiments included: metabolism in the brain in response to CO2; the brain’s inflammatory response to maximal exercise following 6-8 days of acclimatization at 3,800 m; and a diet intervention in females involving 14-days of effectively 50% caloric restriction while maintaining 8 hours of endurance training per week. v Preface The University of British Columbia Clinical Research Ethics Board approved all experimental chapters in this thesis (CREB IDs: H16-01028, H18-01755, H15-00166, H21-00773, H22-01091). Chapter 1. Written by Hannah Caldwell with insightful and extensive feedback by Professor Philip Ainslie. Chapter 2. Published in The Journal of Physiology (Caldwell et al., 2021). Author contributions: HGC, JMJRC, and PNA drafted, edited, and revised the initial version of this review. JSM and ERS provided intellectual and expert feedback. All authors have read and approved the final version of this manuscript and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed. Published textbook chapter titled Exercise Metabolism by the American Physiological Society in partnership with Springer Nature (Caldwell et al., 2022). Author contributions: HGC, LG, and PNA drafted, edited, and revised the initial version of this review. All authors have read and approved the final version of this manuscript and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed. Chapter 3. Submitted to The Journal of Clinical Investigation at the time of dissertation submission. Author contributions: Study design: RLH, ARB, CAH, DMB, PNA. Data collection: HGC, RLH, ARB, CAH, JMJRC, TDG, MMT, BSS, DMB, MSS, DBM, PNA. Data analysis: HGC, CD, RLH, ARB, CAH. Data interpretation: HGC, CD, RLH, JMJRC, DMB, PNA. Drafted manuscript: HGC. Critically reviewed manuscript: HGC, RLH, ARB, CAH, JMJRC, TDG, CD, MMT, BSS, DMB, vi MSS, DBM, PNA. Approved final version: HGC, RLH, ARB, CAH, JMJRC, TDG, CD, MMT, BSS, DMB, MSS, DBM, PNA. Chapter 4. Author contributions: Study design: HGC, TDG, JPL, MS, PNA. Data collection: HGC, TDG, CAH, JST, EJ, LMB, ARS, JAM, KF, DBM, PNA. Data analysis: HGC, TDG. Data interpretation: HGC, TDG, JPL, PNA. Drafted manuscript: HGC. Critically approved manuscript: HGC, TDG, CAH, JST, EJ, LMB, ARS, JAM, JPL, MS, KF, DBM, PNA. Approved final version: HGC, TDG, CAH, JST, EJ, LMB, ARS, JAM, JPL, MS, KF, DBM, PNA. Chapter 5. Author contributions: Study design: HGC, JSJ, JPL, PNA, YH, LG. Data collection: HGC, JSJ, LOL, JPA. Data analysis: HGC, JSJ. Data interpretation: HGC, JSJ, JPL, PNA, YH, LG. Drafted manuscript: HGC, JSJ. Critically approved manuscript: HGC, JSJ, LOL, JPA, JPL, PNA, YH, LG. Approved final version: HGC, JSJ, LOL, JPA, JPL, PNA, YH, LG. Chapter 6. Author contributions: Study design: HGC, JSJ, JPL, PNA, YH, LG. Data collection: HGC, JSJ, LOL, JPA. Data analysis: HGC, JSJ. Data interpretation: HGC, JSJ, JPL, PNA, YH, LG. Drafted manuscript: HGC, JSJ. Critically approved manuscript: HGC, JSJ, LOL, JPA, JPL, PNA, YH, LG. Approved final version: HGC, JSJ, LOL, JPA, JPL, PNA, YH, LG. Chapter 7. Written by Hannah Caldwell, and edited by Professor Philip Ainslie. vii Table of Contents Abstract ...................................................................................................................................... iii Lay Summary ............................................................................................................................. iv Preface ......................................................................................................................................... v Table of Contents ...................................................................................................................... vii List of Tables ............................................................................................................................... x List of Figures ............................................................................................................................ xi Acknowledgements .................................................................................................................. xv Dedication ................................................................................................................................ xvii Chapter 1: Introduction ............................................................................................................. 1 1.1 Thesis objectives ......................................................................................................... 3 1.2 Individual study aims and hypotheses ........................................................................ 3 1.2.1 Study 1 – Evidence for direct CO2-mediated alterations in cerebral oxidative metabolism in humans ...................................................................................................... 3 1.2.2 Study 2 – How does systemic fuel utilization at high-altitude affect the brain inflammatory response to exercise? ................................................................................. 3 1.2.3 Study 3A – The importance of low energy availability for the immune system, systemic inflammation, and performance ability in endurance-trained female athletes .... 4 1.2.3.1 Study 3B – Substrate oxidation, skeletal muscle mitochondrial respiratory capacity, and insulin sensitivity following 14-days low energy availability in endurance-trained female athletes .................................................................................................. 4 Chapter 2: Literature Review .................................................................................................... 5 2.1 Acid-Base Balance and Cerebrovascular Regulation ................................................. 5 2.1.1 Abstract ............................................................................................................. 5 2.1.2 Introduction to acid-base physiology ................................................................. 7 2.1.3 Acid-base regulation in the brain ..................................................................... 11 2.1.3.1 Regulation of cerebral blood flow by arterial PCO2 versus pH .................... 13 2.1.3.2 Regulation of cerebral blood flow by HCO3- exchange ................................ 16 2.1.3.3 Acute alterations in cerebral blood flow stabilize CO2 gradients ................. 19 2.1.4 Human cerebrovascular regulation during acid-base disorders ...................... 21 2.1.4.1 Acute versus chronic cerebrovascular acid-base regulation ....................... 21 2.1.5 Clinical implications ......................................................................................... 26 2.1.6 Summary ......................................................................................................... 28 2.2 Metabolism in the brain during exercise in humans .................................................. 28 2.2.1 Abstract ........................................................................................................... 29 2.2.2 Background ..................................................................................................... 29 2.2.3 Cerebral metabolism and fuel utilization at rest .............................................. 31 2.2.3.1 Summary ..................................................................................................... 38 2.2.4 Regulation of cerebral metabolism during exercise ........................................ 39 2.2.4.1 Summary ..................................................................................................... 42 2.2.4.2 Techniques to assess cerebral metabolism during dynamic exercise in humans 42 2.2.5 Cerebral substrate oxidation during exercise .................................................. 45 2.2.5.1 Incremental versus steady-state exercise and cerebral metabolism ........... 47 2.2.5.2 Cerebral metabolic rate of oxygen during exercise ..................................... 48 2.2.5.3 Cerebral metabolic rate of glucose during exercise .................................... 49 2.2.5.4 Cerebral metabolic rate of lactate during exercise ...................................... 49 2.2.5.5 Ketone utilization during exercise? .............................................................. 50 2.2.5.6 Summary ..................................................................................................... 51 viii 2.2.6 Summary ......................................................................................................... 51 2.3 High-altitude .............................................................................................................. 51 2.3.1 Energy balance and metabolism at high-altitude ............................................ 53 2.3.1.1 Determinants of energy balance .................................................................. 54 2.3.1.2 Resting energy expenditure ......................................................................... 54 2.3.1.3 Diet-induced energy expenditure ................................................................. 54 2.3.1.4 Activity-induced energy expenditure ............................................................ 55 2.3.2 Inflammatory response to high-altitude ........................................................... 56 2.4 Low energy availability in endurance-trained athletes .............................................. 58 2.4.1 The immune system ........................................................................................ 59 2.4.2 Systemic inflammatory response .................................................................... 61 2.4.3 Exercise performance ..................................................................................... 65 2.4.4 Systemic substrate oxidation ........................................................................... 65 2.4.4.1 Skeletal muscle mitochondrial respiratory capacity ..................................... 66 2.4.4.2 Insulin sensitivity and glucose control ......................................................... 67 Chapter 3: Study 1 – Evidence for direct CO2-mediated alterations in cerebral oxidative metabolism in humans ............................................................................................................ 68 3.1 Introduction ............................................................................................................... 68 3.2 Methods .................................................................................................................... 70 3.3 Statistical analyses .................................................................................................... 75 3.4 Results ...................................................................................................................... 77 3.5 Discussion ................................................................................................................. 81 3.6 Conclusion ................................................................................................................ 85 Chapter 4: Study 2 – How does systemic fuel utilization at high-altitude affect the brain inflammatory response to exercise? ....................................................................................... 86 4.1 Introduction ............................................................................................................... 87 4.2 Methods .................................................................................................................... 88 4.3 Statistical analyses .................................................................................................... 95 4.4 Results ...................................................................................................................... 95 4.4.1 Part 1: Context for systemic and brain substrate utilization ............................ 96 4.4.2 Part 2: Trans-cerebral inflammatory stress and immune response ............... 100 4.5 Discussion ............................................................................................................... 108 4.6 Conclusion .............................................................................................................. 111 Chapter 5: Study 3A – The importance of low energy availability for the immune system, systemic inflammation, and performance ability in endurance-trained female athletes ........ 113 5.1 Introduction ............................................................................................................. 113 5.2 Methods .................................................................................................................. 114 5.3 Statistical analyses .................................................................................................. 123 5.4 Results .................................................................................................................... 124 5.5 Discussion ............................................................................................................... 136 5.6 Conclusion .............................................................................................................. 140 Chapter 6: Study 3B – Substrate oxidation, skeletal muscle mitochondrial respiratory capacity, and insulin sensitivity following 14-days low energy availability in endurance-trained female athletes ...................................................................................................................... 141 6.1 Introduction ............................................................................................................. 141 6.2 Methods .................................................................................................................. 143 6.3 Statistical analyses .................................................................................................. 146 ix 6.4 Results .................................................................................................................... 147 6.5 Discussion ............................................................................................................... 160 6.6 Conclusion .............................................................................................................. 163 Chapter 7: Conclusion .......................................................................................................... 164 7.1 Future perspectives ................................................................................................. 167 7.2 Integrated summary: ............................................................................................... 169 Bibliography ............................................................................................................................ 170 Appendices .............................................................................................................................. 213 Appendix A ........................................................................................................................ 213 x List of Tables Table 2-1. Chronological summary of key studies to investigate cerebral blood flow during acid-base disorders in humans. .......................................................................................................... 23 Table 2-2. Cerebral blood flow and global metabolic rate at rest and during exercise to exhaustion. .................................................................................................................................. 31 Table 2-3. Strengths, limitations, and utility of various techniques for the measurement of cerebral metabolism in humans. ................................................................................................. 44 Table 2-4. Summary of selected cytokines and their functions. .................................................. 62 Table 4-1. Resting arterial blood gas variables at SL and following 6-8 days at HA. ................. 95 Table 5-1. Diet composition during energy balance (EB) and low energy availability (LEA) interventions. ............................................................................................................................. 124 xi List of Figures Figure 2-1. Acid-base balance and cerebrovascular regulation in humans. ................................. 6 Figure 2-2. Time course of acid-base compensatory changes and cerebrovascular responses. .................................................................................................................................................... 10 Figure 2-3. Schematic diagram showing the changes in CSF pH with alterations in [HCO3-] and PCO2. .......................................................................................................................................... 11 Figure 2-4. Cerebral blood flow regulation is independent of arterial pH – rather, diffusion of CO2 across the blood-brain barrier to alter interstitial/cerebrospinal fluid pH. .................................... 14 Figure 2-5. The cerebral circulation is exceptionally sensitive to PaCO2 throughout the cerebrovascular tree. .................................................................................................................. 15 Figure 2-6. Overview of cerebrovascular acid-base exchange and the blood-brain barrier (BBB) epithelial transport. ...................................................................................................................... 18 Figure 2-7. Arterial-venous [HCO3-] and PCO2 differences with hypercapnic acidosis in humans. .................................................................................................................................................... 21 Figure 2-8. The relationship between [HCO3-], pH, and PCO2 in arterial blood and cerebrospinal fluid (CSF) with chronic metabolic acidosis/alkalosis in humans. ............................................... 22 Figure 2-9. Regulation of cerebral blood flow by metabolism. .................................................... 32 Figure 2-10. Summary of the bioenergetic requirements of the human brain, emphasizing its severely restricted energy stores of oxygen, glucose, and ATP, paired with high rates of metabolism. ................................................................................................................................. 34 Figure 2-11. Brain substrate metabolism of glucose, glycogen, and lactate. ............................. 35 Figure 2-12. Anatomy of the blood-brain barrier and cerebrovascular tree. ............................... 36 Figure 2-13. Relationship between the arterial D-b -hydroxybutyrate concentration and cerebral and leg, skeletal muscle, uptake rate per kg of tissue in healthy males. .................................... 38 Figure 2-14. Relationship between arterial-venous oxygen difference (AVDO2) and cerebral blood flow (CBF) at rest. ............................................................................................................. 39 Figure 2-15. Alterations in cerebral and systemic oxygen extraction during aerobic exercise to exhaustion. .................................................................................................................................. 40 Figure 2-16. Systemic and cerebrovascular variables affecting cerebral blood flow regulation during aerobic exercise to exhaustion. ....................................................................................... 42 Figure 2-17. Average cerebral metabolic rate and oxidative cerebral fuel utilization during incremental exercise. .................................................................................................................. 46 Figure 2-18. The influence of carbohydrate availability on cerebral substrate utilization during prolonged submaximal cycling exercise. .................................................................................... 48 xii Figure 2-19. Oxygen transport cascade. ..................................................................................... 52 Figure 2-20. Oxygen delivery cascade - emphasizing importance of O2 utilization in the skeletal muscle for exercise performance. ............................................................................................... 53 Figure 2-21. Daily energy expenditures (bars) and distance covered (stars) during a range of prolonged expeditions measured via doubly-labelled water. ...................................................... 56 Figure 2-22. The role of inflammation in high-altitude illness. ..................................................... 57 Figure 2-23. Unit-less illustration of an individual’s daily energy balance and energy availability (EA) when exercising and maintaining an energy balance of zero. ............................................ 58 Figure 2-24. Systemic effects of short-term (3-5 days) low energy availability in females. ........ 59 Figure 2-25. Cytokine network. ................................................................................................... 60 Figure 2-26. Relative contribution of carbohydrate and lipid to total energy expenditure with progressive increases in relative exercise intensity to max. ....................................................... 65 Figure 2-27. Interaction between fat oxidation and glycolysis; competition for substrate and inhibition of glucose utilization with high-intensity aerobic exercise. ........................................... 66 Figure 3-1. Schematic of gold-standard measurement of brain metabolism via the Fick approach by trans-cerebral internal jugular venous (IJV) and radial arterial blood sampling ..................... 75 Figure 3-2. Relationship between cerebral metabolic rate of oxygen (CMRO2) indexed against PCO2; insufficient O2 utilization to support oxidative metabolism. .............................................. 77 Figure 3-3. Increased reliance on anaerobic glycolysis during acute hypercapnia indicated via compensatory reduction in oxidative glucose index. ................................................................... 78 Figure 3-4. Exploratory analysis – influence of biological sex on the CMRO2 response to PaCO2. .................................................................................................................................................... 80 Figure 4-1. Testing locations at sea level (SL) and high-altitude (HA). ....................................... 92 Figure 4-2. Fat Max Study - Pre testing to establish FATmax and VO2max. ................................ 93 Figure 4-3. Fat Max Study – Exercise protocol schematic .......................................................... 94 Figure 4-4. Energy expenditure and substrate utilization at rest at sea level (SL) and following 3-5 days at high-altitude (HA). ....................................................................................................... 96 Figure 4-5. Absolute and relative exercise intensity achieved at FATmax at sea level (SL) and following 3-5 days at high-altitude (HA). ..................................................................................... 97 Figure 4-6. VO2 and HR achieved at FATmax relative to VO2max. .............................................. 98 Figure 4-7. Maximal fat and carbohydrate oxidation achieved during the FATmax test. ............ 99 Figure 4-8. Absolute and relative exercise energy expenditure normalized to workload during 60 minutes of steady-state cycling at altitude-specific FATmax exercise intensity. ......................... 99 xiii Figure 4-9. White blood cell count and cell-type distribution in response to FATmax and maximal cycling exercise at sea level and high-altitude. ......................................................................... 101 Figure 4-10. Inflammatory cytokines identified at rest at sea level (SL) and following 6-8 days at high-altitude (HA). ..................................................................................................................... 103 Figure 4-11. Inflammatory cytokines identified pre- versus post- the 60-minute FATmax exercise intensity cycling at sea level and high-altitude. ......................................................................... 104 Figure 4-12. Inflammatory cytokines identified pre- versus 5 minutes post- the max exercise protocol at sea level and high-altitude. ..................................................................................... 105 Figure 4-13. Total effect of 60-minutes FATmax in addition to an acute maximal exercise protocol on inflammatory cytokines. .......................................................................................... 106 Figure 4-14. Whole-body respiratory exchange ratio versus brain RQ response to maximal cycling exercise and recovery at sea level and high-altitude. ................................................... 108 Figure 5-1. Overview of study protocol and experimental methods .......................................... 118 Figure 5-2. LEA Study – Exercise protocol schematic .............................................................. 120 Figure 5-3. Endurance exercise training volume and intensity per week during the 14-day energy balance (EB) and low energy availability (LEA) diet interventions. ............................... 125 Figure 5-4. Dietary energy intake and related body mass changes pre- vs. post- 14 days of energy balance (EB) and low energy availability (LEA). ........................................................... 126 Figure 5-5. White blood cell count and cell-type distribution in response to exercise pre- vs. post- 14 days of energy balance (EB) and low energy availability (LEA). ................................ 127 Figure 5-6. Systemic stress and inflammatory biomarkers in response to exercise pre- vs. post- 14 days of energy balance (EB) and low energy availability (LEA). ......................................... 128 Figure 5-7. Inflammatory protein biomarkers identified at rest following 14-days of energy balance (EB) and low energy availability (LEA). ....................................................................... 130 Figure 5-8. Inflammatory protein biomarkers identified post- exercise following 14-days of low energy availability (LEA). .......................................................................................................... 131 Figure 5-9. Inflammatory protein biomarkers identified post- exercise following 14-days of energy balance (EB). ................................................................................................................ 133 Figure 5-10. Exercise performance during the 20-minute time trial and time to exhaustion tests pre- vs. post- 14 days of energy balance (EB) and low energy availability (LEA). ................... 136 Figure 6-1. Experimental methods - Skeletal muscle mitochondrial respiratory capacity. ........ 146 Figure 6-2. Energy expenditure and substrate utilization at rest pre- vs. post- 14 days of energy balance (EB) and low energy availability (LEA). ....................................................................... 147 Figure 6-3. Reductions in RER persist throughout submaximal cycling exercise post-LEA. .... 148 xiv Figure 6-4. Relative contribution of carbohydrate to energy expenditure is reduced at rest and during submaximal cycling exercise. ........................................................................................ 149 Figure 6-5. Relative contribution of fat to energy expenditure is increased at rest and during submaximal cycling exercise. ................................................................................................... 150 Figure 6-6. Energy Expenditure and absolute VO2 were not different at matched submaximal cycling exercise intensities pre- vs. post- EB and LEA diet interventions. ................................ 151 Figure 6-7. Skeletal muscle glycogen content at rest pre- vs. post- 14 days of energy balance (EB) and low energy availability (LEA) diet interventions. ........................................................ 152 Figure 6-8. Skeletal muscle enzyme activity at rest pre- vs. post- 14 days of energy balance (EB) and low energy availability (LEA) diet interventions. ........................................................ 153 Figure 6-9. Mitochondrial respiratory capacity pre- vs. post- 14 days of energy balance (EB) and low energy availability (LEA). .................................................................................................... 154 Figure 6-10. Regulation of normoglycemia and incidence of hypoglycemic events during days 7-14 of energy balance (EB) and low energy availability (LEA) diets. ......................................... 155 Figure 6-11. Daytime and nighttime regulation of glucose during days 7-14 of energy balance (EB) and low energy availability (LEA) diets. ............................................................................ 156 Figure 6-12. Metrics of glucose variability during days 7-14 of energy balance (EB) and low energy availability (LEA) diets. .................................................................................................. 157 Figure 6-13. Post-prandial glucose response to breakfast during days 7-14 of energy balance (EB) and low energy availability (LEA) diets. ............................................................................ 158 Figure 6-14. Insulin sensitivity ................................................................................................... 159 Figure 6-15. Exercise-induced glucose responses on the post- EB and LEA test days. .......... 160 Figure 7-1. Overview of key themes and overlapping techniques of this dissertation .............. 164 Figure 7-1. Venous-arterial [HCO3-] and PCO2 exchange with hypercapnic acidosis in humans. .................................................................................................................................................. 230 Figure 7-2. Venous-arterial [HCO3-] and PCO2 exchange with progressive submaximal to maximal cycling exercise. ......................................................................................................... 232 xv Acknowledgements First, I am forever thankful to my supervisor, Prof. Phil Ainslie. You are incredibly influential to me and have taught me to be ambitious, scientifically curious, and compassionate. Your prioritization of family, outdoor adventure pursuits, and scientific excellence is my inspiration. Thank you for every opportunity and for the exceptional environment of equally passionate and encouraging individuals with which I have had the privilege to work with the last 8 years: the Ainslie Army. Ryan and Michael, you two set the bar. You sold me my ticket for the ‘Phil Ainslie stoke train’ in 2015 and it has been such a thrilling ride. I’m inspired by you every day and consider it such a privilege to receive your mentorship and friendship. Jay – I am forever grateful to Phil for bringing the climber/philosopher Scotsman into his lab and into our Ainslie Fam. Your beautifully-written and awe-inspiring insights into physiology are remarkable and I am extremely privileged to learn from you. Travis – your curiosity, humility and scientific integrity is an inspiration. Everything you do is excellent. I just want to be like you when I grow up. Cody – words can’t express how much I have learned from you. As reluctant as I was, I seriously appreciate your persistent ‘scientific sparring’ and “but why?” Thank you for everything… Joshy – thank you for always supporting me, and teaching me that it’s ok to be a little neurotic… Alex – thank you for the adventures. “Cavtat Water Polo Championships 2019” Jan – I am extraordinarily grateful for your support and trust; it was a privilege to work with you on the LEA study. Endless 5 am test days and late afternoons at the Oxygraph, but at least we can always look forward to ‘kageklub’ … looking forward to our next adventure together in Copenhagen, and excited to see what you achieve. Lone & Jesper – we won the lottery with you two. Your support on the LEA study was invaluable; tak for i dag. Jonathan Little – thank you for your continuous support and encouragement over the years; I always look forward to working together. xvi Lasse, Ylva – I am exceptionally privileged that you welcomed me into your group; your warmth has created a ‘second home’ for me in Copenhagen and I am ecstatic for the possibilities and what we will achieve together in my upcoming Postdoctoral Fellowship, and further… I would like to give special thanks to my university examiner Dr. Sarah Purcell and my external examiner Dr. José González-Alonso. Thank you for the support and invigorating discussions. Thank you to my family, Lee Anne, Bruce – I appreciate your unconditional support. Ainslie Army Connor, you are my #1. I love you xvii Dedication To excellence. “Slow down, you crazy child You’re so ambitious for a juvenile…” INTRODUCTION 1 Chapter 1: Introduction Metabolism describes the sum of biochemical reactions that take place within a living organism which provide energy for vital processes; this can be studied at the whole-body, organ specific, or cellular level. Energy availability, which is determined by the difference between energy intake and exercise energy expenditure, is affected by environmental and energetic stresses (e.g., high-altitude, exercise, diet/nutrition). Systemic energy deficits have implications for the regulation of organ-specific substrate utilization. This thesis aims to integrate brain, skeletal muscle, and systemic substrate oxidation and relative fuel utilization in the context of environmental and energetic stress (high-altitude, exercise, nutritional caloric deficits). Although the human brain is reflective of <1/50th of total body mass, it accounts for approximately 15-20 % of the available cardiac output, body glucose, and oxygen consumption at rest. Cerebral oxygen consumption is approximately 3.5 ml/ 100 g brain/ min; therefore, an average sized brain, weighing approximately 1,400 g requires a rate of oxygen uptake (VO2) of 49 ml O2/ min (Sokoloff, 1960). As the majority of oxygen in the brain is utilized for carbohydrate oxidation – reflective of the brain’s respiratory quotient of 0.97 (Nybo et al., 2003; Dalsgaard et al., 2004a) – the energy equivalent of the total cerebral metabolic rate is approximately 20 watts or 0.29 kcal/ min. In contrast, the skeletal muscle is responsible for an equivalent 15 % resting cardiac output (750 ml/ min in absolute terms), however, when adjusted for organ size, this corresponds to approximately 2-3 ml/ 100 g/ min (versus 50 ml/ 100 g/ min brain blood flow). This can be contextualized in terms of organ-specific energy expenditure, where at rest; for example, the brain is exceptionally energetically costly (approximately 240 kcal/ kg/ day) compared to the skeletal muscle (13 kcal/ kg/ day), but less so than organs like the heart (440 kcal/ kg/ day) (Elia & Livesey, 1992). Additionally, skeletal muscle has extraordinary metabolic flexibility, with the ability to generate anaerobic and aerobic energy via a variety of substrate (e.g., phosphocreatine, glycogen, triglyceride, circulating glucose, lactate, free fatty acid, and ketone). Whereas, the brain relies nearly exclusively on blood glucose for metabolism at rest. The cerebral metabolic rate of oxygen (CMRO2) is reflective of oxidative metabolism and is calculated as: CBF × arterial-venous O2 content difference (Fick Equation). Importantly, the brain (unlike the muscle, for example) does not increase capillary recruitment and, therefore, the diffusive surface area for oxygen does not change (Kuschinsky & Paulson, 1992; Williams et al., 1993), requiring large increases in CBF and/or oxygen extraction to maintain or elevate CMRO2 (Buxton & Frank, 1997; Mintun et al., 2001). The brain has a remarkably restricted capacity for INTRODUCTION 2 substrate storage (e.g., glycogen <10 µmol/g), which when paired with an insufficient ability to increase oxygen extraction (via limited changes in capillary recruitment) and inability to utilize fat as substrate, necessitates continuous and tightly regulated CBF to support the high relative metabolic rate of brain tissue via oxidative respiration (Himwich & Homburger, 1947; Kety & Schmidt, 1948a; Siesjö, 1978; Gjedde et al., 2002; Brown & Ransom, 2007). Any alterations in CMRO2 – such as, for example, exercise or environmental stress – directly affect CBF regulation reflective of appropriate substrate delivery (Himwich & Homburger, 1947; Kety & Schmidt, 1948b; Siesjö, 1978; Madsen & Vorstrup, 1991; Buxton & Frank, 1997). It is well-established that CBF varies directly with PaCO2 (Lennox & Gibbs, 1932; Kety & Schmidt, 1948b; Willie et al., 2015) such that – provided the cerebral metabolic production of CO2 remains unaltered – hypercapnia will be related to reductions in the arterial-venous O2 content difference (as explained via the Fick Equation) (Posner et al., 1965; Siesjö, 1972). Additionally, exercise at high-altitude increases reliance on non-oxidative metabolism as indicated by larger carbohydrate uptake relative to oxygen, and in part due to elevated systemic arterial lactate availability (Smith et al., 2014). Taken together, the interplay between cerebral substrate delivery (via blood flow and arterial substrate content) and trans-cerebral exchange of nutrients is vital to support increases in fuel utilization in the brain during exercise and environmental stress. A further aim of this thesis was to explore the inflammatory consequences to exercise at high-altitude and during 14-days of low energy availability (50% less daily energy intake). The immune response to infection/inflammation is energetically taxing (Schmid et al., 2000); for example, the total energy requirements of the immune system is up to approximately 2000 kJ per day in states of ‘chronic inflammation’, which may account for up to 25-30% basal metabolism (Straub et al., 2010). Pro-inflammatory stress at high altitude is further implicated in the etiology of high-altitude illnesses (Pham et al., 2021; Turner et al., 2021); however, it is controversial whether/how exercise alters pro-inflammatory mediators and therefore the implications of exercise on high-altitude illnesses. Further, studies have shown that strenuous prolonged exercise training with low energy availability in normal weight individuals may suppress the immune system (Gleeson et al., 2004; Gleeson, 2007; 2016; Bermon et al., 2017; Peake et al., 2017). Thus, two experiments were performed to alter systemic metabolism and determine the impact on inflammation. INTRODUCTION 3 1.1 Thesis objectives The aim of this dissertation was to integrate brain, skeletal muscle, and systemic substrate utilization during environmental and energetic stress. The goal of this approach was to achieve a comprehensive understanding of these two regulatory systems in the context of systemic energy deficits. A secondary aim of this dissertation was to explore the inflammatory response – systemically and localized in the brain – with severe reductions in energy availability in endurance-trained females and to exercise at high-altitude. 1.2 Individual study aims and hypotheses 1.2.1 Study 1 – Evidence for direct CO2-mediated alterations in cerebral oxidative metabolism in humans The cerebral metabolic rates of oxygen and glucose utilization (CMRO2 and CMRGlu, respectively) are vital physiologic factors which may influence cerebrovascular CO2 reactivity (CVR) and related brain health in humans. How CMRO2 and CMRGlu are affected by alterations in arterial PCO2 (PaCO2) is equivocal and therefore was the primary question of this study. The aim of this study was to investigate CMRO2 indexed by the direct Fick approach via trans-cerebral internal jugular venous (IJV) and radial arterial blood sampling in response to steady-state alterations in PaCO2 in healthy humans. The objective of the current analysis was to test the hypothesis that CMRO2 would be directly affected by changes in PaCO2 such that CMRO2 would be reduced with progressive increases in PaCO2, and vice-versa. Further, to compensate for the expected reduction in CMRO2, we reasoned that the cerebral metabolic ratios for oxidative glucose and carbohydrate metabolism (i.e., OGI and OCI, respectively) would also be reduced. This latter observation would indicate that part of the glucose/lactate is not fully oxidized thereby indicating an increase in non-oxidative metabolism via anaerobic glycolysis and lactate oxidation. 1.2.2 Study 2 – How does systemic fuel utilization at high-altitude affect the brain inflammatory response to exercise? This study investigated systemic substrate oxidation and energy expenditure in addition to inflammatory stress in the brain during exercise at an intensity that elicits maximal fat oxidation (FATmax) and following acute exhaustive exercise at sea level and following 3-8 days at 3,800 m. Simultaneous radial arterial and internal jugular venous blood sampling was performed to assess trans-cerebral inflammatory cytokines (via 17-plex assay) and leukocytes (via INTRODUCTION 4 hematology analysis) prior to and following 60-minutes of low-intensity exercise, at maximal exercise, 5 minutes post-exercise, and following 60 minutes of passive recovery. It was hypothesized that: 1) 1-hour of cycling exercise at altitude-specific FATmax intensity will promote an anti-inflammatory response at both sea level and high-altitude (indicated by higher IL-4, IL-10, and IL-13), and 2) acclimatization to high-altitude will provoke an anti-inflammatory response at rest which will attenuate the pro-inflammatory and leukocyte mobilization responses to maximal exercise compared to sea level. 1.2.3 Study 3A – The importance of low energy availability for the immune system, systemic inflammation, and performance ability in endurance-trained female athletes This study examined the influence of 14-days low energy availability (LEA) in endurance-trained females on: 1) the immune system (white blood cell count via hematology analysis and targeted 96-panel inflammatory protein biomarker via high-multiplex immunoassay), 2) systemic inflammation (creatine kinase, cortisol, c-reactive protein), and 3) performance capacity (20-minute isokinetic cycling time trial and time to exhaustion tests). The immune and inflammatory responses were investigated at rest and post-exercise following two 14-day randomized, blinded, cross-over controlled dietary interventions: 1) energy balance (EB; 50 kcal/ kg fat free mass per day) and 2) low energy availability (LEA; 25 kcal/ kg fat free mass per day). It was hypothesized that LEA would suppress the immune system, provoke systemic inflammation, and impair exercise performance. 1.2.3.1 Study 3B – Substrate oxidation, skeletal muscle mitochondrial respiratory capacity, and insulin sensitivity following 14-days low energy availability in endurance-trained female athletes The objective of this study was to investigate the influence of 14-days LEA in endurance-trained female athletes on: 1) fuel utilization (e.g., respiratory exchange ratio, glucose, intramuscular glycogen, lactate, and b-OHB), 2) insulin sensitivity (HOMA-IR, mixed-meal tolerance test, continuous glucose monitoring), and 3) mitochondrial oxidative capacity in permeabilized human skeletal muscle fibers (via high-resolution respirometry, OROBOROS, Oxygraph-2k). It was hypothesized that 14-days LEA would suppress resting metabolic rate, impair insulin sensitivity, and increase relative energy expenditure during submaximal and exhaustive cycling exercise. Additionally, the influence of LEA on skeletal muscle mitochondrial oxidative capacity was measured as an exploratory outcome.LITERATURE REVIEW 5 Chapter 2: Literature Review In this chapter, topics relevant to the following studies within this dissertation are described. First, an overview of key regulators of cerebral blood flow is provided, with a focus on CO2 (2.1). Next, metabolism in the brain during exercise in humans is described (2.2), with attention to fuel utilization (2.2.5) in addition to energy balance and metabolism at high-altitude (2.3.1). Lastly, the interaction between energy availability (2.4), the immune/inflammatory response (2.4.1, 2.4.2), exercise performance (2.4.3) and systemic metabolism (2.4.4) in endurance-trained female athletes is summarized. 2.1 Acid-Base Balance and Cerebrovascular Regulation The following section has been published as an invited topical review in The Journal of Physiology (Caldwell et al., 2021a). 2.1.1 Abstract The regulation and defense of intracellular pH is essential for homeostasis. Indeed, alterations in cerebrovascular acid-base balance directly affect cerebral blood flow (CBF) which has implications for human health and disease. For example, changes in CBF regulation during acid-base disturbances are evident in conditions such as chronic obstructive pulmonary disease and diabetic ketoacidosis. The classic experimental studies from the past 75+ years are utilized to describe the integrative relationships between CBF, carbon dioxide tension (PCO2), bicarbonate (HCO3-), and pH. These factors interact to influence 1) the time course of acid-base compensatory changes and the respective cerebrovascular responses (due to rapid exchange kinetics between arterial, extracellular fluid, and intracellular brain tissue). We propose that alterations in arterial [HCO3-] during acute respiratory acidosis/alkalosis contribute to cerebrovascular acid-base regulation; and 2) the regulation of CBF by direct changes in arterial versus extravascular/ interstitial PCO2 and pH – the latter recognized as the proximal compartment which alters vascular smooth muscle cell regulation of CBF. Taken together, these results substantiate two key ideas: first, that the regulation of CBF is affected by the severity of metabolic/respiratory disturbances, including the extent of partial/full acid-base compensation. Second, that the regulation of CBF is independent of arterial pH and that diffusion of CO2 across the blood-brain barrier is integral to altering perivascular extracellular pH. Overall, by realizing the integrative relationships between CBF, PCO2, HCO3-, and pH, experimental studies may provide insights to improve CBF regulation in clinical practice with treatment of systemic acid-base disorders. LITERATURE REVIEW 6 Figure 2-1. Acid-base balance and cerebrovascular regulation in humans. The cerebral circulation is exceptionally sensitive to arterial PCO2 (PaCO2) throughout the cerebrovascular tree; the integrative relationship between PaCO2 and pH on cerebral blood flow (CBF) regulation acts to stabilize CO2 gradients and thus regulate pH across the blood-brain barrier (BBB) (Fencl et al., 1969; Hoiland et al., 2019). Collectively, we aim to substantiate the following key theses: 1) regulation of CBF is independent of arterial pH – rather, diffusion of CO2 across the BBB is integral to alter perivascular extracellular pH; 2) alterations in arterial [HCO3-] during acute respiratory acidosis/alkalosis contribute to cerebrovascular acid-base regulation; and 3) regulation of CBF is affected by the severity of metabolic/respiratory disturbances and extent of respective compensation. (A) Elevations in PaCO2 increase CBF; note the left and right x-intercept shifts in resting PaCO2 induced by chronic (5 days) metabolic acidosis (via NH4Cl) and alkalosis (via NaHCO3), respectively. The CBF was calculated using the reciprocal of the arterial-venous O2 content (CaO2 – CvO2; ml/ 100 ml blood) – via arterial and internal jugular vein catheterization – by rearranging the Fick Equation with the assumption of constant cerebral metabolic rate of oxygen; e.g., CBF = CMRO2 / (CaO2 – CvO2). (B) The CBF response to elevated PaCO2 can be expressed as a function of CSF pH across all conditions. These data indicate that CBF varies directly with acute changes in PaCO2, the principal stimulus for which is changes in CSF/extravascular pH (Fencl et al., 1969). (C) The highly vascularized and vasoactive network of pial arterioles on the brain surface are sensitive to perivascular pH via changes in PCO2/[H+] (Wolff & Lennox, 1930; Wahl et al., 1970; Betz et al., 1973; Kontos et al., 1977b; 1977a). The PaCO2-mediated cerebrovascular responses are dependent on the rapid passive diffusion of CO2 across the vascular wall to alter perivascular extracellular pH rather than direct changes in arterial pH per se (Wolff & Lennox, 1930; Lambertsen et al., 1961; Severinghaus & Lassen, 1967; Betz & Heuser, 1967; Wahl et al., 1970; Kontos et al., 1977b; 1977a). Pial arterioles are surrounded by the subarachnoid space and are innervated by perivascular nerves acting on vascular smooth muscle cells (VSMCs). (D) Pial arterioles penetrate the pia mater via Virchow-Robin spaces where they are encapsulated by astrocytic processes and end-feet, as well as perivascular macrophages. Astrocytes release HCO3- in response to localized increases in CO2 produced by oxidative metabolism via electrogenic Na+/HCO3- cotransporter 1 to regulate extracellular pH (Theparambil et al., 2020). I Intraparenchymal arterioles and branching capillaries are covered by neural processes and pericytes within the basement membrane of the endothelium. Recent novel pre-clinical studies utilizing the cranial window technique suggest that in vivo contractile pericytes may facilitate the regulation of capillary blood flow (Peppiatt et al., 2006; Hall et al., FExtra-cranial arteriesCPial arteriolesECapillariesDPenetrating arterioles↑CO2↑[H+]o ↑CBFIntra-cranial arteriesAB105907560453030 40 50 60PaCO2 (mmHg)AlkalosisAcidosisNormalCBF (ml/ 100 g/ min)10590756045307.36 7.32 7.28 7.24Calculated CSF pHCBF (ml/ 100 g/ min)AlkalosisAcidosisNormalLITERATURE REVIEW 7 2014; Gonzales et al., 2020; Hartmann et al., 2021). (F) Increases in PaCO2 elicit shear stress mediated VSMC vasodilation (Fujii et al., 1991; 1992) of the intra/extra- cranial arteries via endothelial nitric oxide synthase (eNOS) activation (Sprague et al., 1996; Mashour & Boock, 1999; Paravicini et al., 2005) by increases in shear-dependent serine threonine specific protein kinase (Akt) activation of eNOS (Dimmeler et al., 1998; 1999; Fulton et al., 1999). Additionally, red blood cell release of ATP in response to reductions in pH (Bergfeld & Forrester, 1992; Ellsworth et al., 1995) causes endothelial purinergic (P2Y2) receptor binding to facilitate NO-mediated vasodilation. Created with BioRender.com 2.1.2 Introduction to acid-base physiology Regulation and defense of intracellular pH is critical for homeostasis in the human body. The tight control of extracellular pH – which directly affects cells via local changes in intravascular or extravascular/interstitial conditions – is achieved via four important processes: 1) chemical buffers (intra- and extracellular) which rapidly react to compensate for the addition or loss of H+ (changes which occur in seconds); 2) active intracellular H+ export into the extracellular space by membrane Na+/H+ exchangers and H+ translocating ATPases; 3) removal or retention of CO2 via alterations in alveolar ventilation (V̇A) (changes which occur in minutes) (Mitchell & Singer, 1965; Fencl et al., 1969); and 4) excretion or retention of HCO3- via renal compensation (changes which occur in hours to days) (Barker et al., 1957; Krapf et al., 1991).These topics have been expertly summarized previously (Berend et al., 2014; Hamm et al., 2015; Quade et al., 2021). The time course of acid-base compensatory changes and resultant cerebrovascular responses are outlined below and illustrated in (Figure 2-2). As described by the Henderson-Hasselbalch equation below, changes in [HCO3-] will directly affect pH and changes in PCO2 will inversely affect pH. pH = 6.1 + log [HCO3-]0.0314 × PCO2 Equation 2.1.1 Where 6.1 is the pKa (i.e., -log of the acid dissociation constant) at 37.0°C and 0.0314 mEq×l-1 per mmHg PCO2 is the solubility factor for dissolved CO2 plus carbonic acid (H2CO3) at 37.0°C. Increases in PaCO2 facilitate rapid passive diffusion of CO2 across the endothelial and vascular smooth muscle cells (VSMC) leading to increases in intra- and extracellular hydrogen ion concentration ([H+]I and [H+]o, respectively), thus causing both intra-/ extracellular acidosis (Jensen et al., 1988; Vorstrup et al., 1989) in accordance with Le Chatelier’s Principle (Le Chatelier, 1884) via shifting the reaction equilibrium through continuous conversion of CO2. CO2 + H2O CA!" H2CO3 ↔ HCO3- + H+ Equation 2.1.2 LITERATURE REVIEW 8 The rapid inter-conversion of CO2 and H2O to carbonic acid (H2CO3) is catalyzed by carbonic anhydrase (CA) which rapidly dissociates to produce bicarbonate and hydrogen ions (HCO3- and H+, respectively). While this reaction can occur without the catalysis by CA, it is much slower and may be rate-limiting and too slow for many physiological purposes. For example, the increase in the reaction rates of the reversible hydration of CO2 and dehydration of H2CO3 is 103-6 faster by the catalytic activity of CA (Swenson, 2014). The PaCO2 is directly proportional to whole-body CO2 production (V̇CO2) and inversely related to alveolar ventilation (V̇A) as described by (Equation 2.1.3) (West & Luks, 2020; Carr et al., 2021a). PaCO2 = V̇CO2V̇A Equation 2.1.3 A reduction in V̇A will cause an increase in PaCO2 and an increase in V̇A will cause a reduction in PaCO2. Additionally, a lesser increase in V̇A relative to V̇CO2 (e.g., alveolar hypoventilation relative to elevated V̇CO2) will cause an increase in PaCO2. Alterations in systemic metabolic changes to acid-base balance will directly affect V̇A through compensatory respiratory changes. Acute acidosis is reflective of an increase in [H+] thereby initiating the drive to breathe and alkalosis causes respiratory suppression. For example, chronic metabolic acidosis and alkalosis cause relative increases and decreases in V̇A, respectively, to alter PaCO2 and thus partially regulate pH. Whether the initial disturbance in pH is caused by an alteration in [HCO3-]/[H+] (e.g., metabolic) or PCO2 (e.g., respiratory), the compensatory variable will act to re-establish pH back towards a normal value of 7.40. Although the Henderson-Hasselbalch equation is the standard method for interpreting acid-base equilibrium in clinical practice, there are other experimental approaches, including the physico-chemical approach that was proposed by Peter Stewart (Stewart, 1978; 1983). According to Stewart’s analysis, three independent variables determine the dependent variables [H+] and [HCO3-]: 1) PaCO2, via the Henderson-Hasselbalch equation; 2) the total concentration of the weakly dissociated ions (e.g., from proteins, albumin, phosphate); and 3) the difference between the sum of the concentrations of the strongly dissociated positive and negative ions (e.g., Na+, K+, Cl-) called the strong ion difference. In any fluid compartment LITERATURE REVIEW 9 containing CO2, changing the concentration of any of the main strongly dissociated ions must change the bicarbonate ion concentration until electroneutrality is achieved (Hughes & Brain, 2013). This physico-chemical approach has major implications. For example, it changes the perspective of many cellular and molecular processes including the many membrane transporters (Cordat & Casey, 2008) that assume hydrogen and bicarbonate ions are physically transported across membranes. In the Stewart approach analysis, all regulation of [H+] and [HCO3-] is achieved by the transport of strongly dissociated ions across membranes. While Stewart’s physico-chemical approach is clinically relevant (Rastegar, 2009) – as well as provides an alternative explanation for regulation of [H+] (Duffin et al., 2021) – this review has adopted the traditional bicarbonate-centered approach to acid-base balance as this is likely to be familiar to physiology readers. Details on this important debate are provided elsewhere (Sirker et al., 2002; Story, 2004; Dubin et al., 2007; Kurtz et al., 2008; Guidet et al., 2010; Cove & Kellum, 2020; Swenson, 2020; Rubin, 2021). A practical illustration of the effects of physiological changes on blood acid-base chemistry is outlined below (Figure 2-3). These intuitive schematics provided by Siesjö (1972) illustrate how complete or full compensation of pH may never occur in some severe acid-base disturbances; as such, only partial restoration of pH can be achieved. This is explained by the relatively large compensatory changes in absolute [HCO3-] and PCO2 theoretically required to correct severe alterations in pH; e.g., Figure 1 (Robin, 1963). LITERATURE REVIEW 10 Figure 2-2. Time course of acid-base compensatory changes and cerebrovascular responses. (A) The swift cerebral blood flow (CBF) response to stepwise changes in PaCO2 (<15-30 seconds) with acute respiratory acidosis/alkalosis (Shapiro et al., 1966; Severinghaus & Lassen, 1967; Poulin et al., 1996; Hoiland et al., 2017) is attributable to rapid exchange kinetics between arterial, extracellular fluid (ECF), and intracellular brain tissue. Data from (Caldwell et al., 2021c). (B) Regulation of CBF is affected by the severity of chronic metabolic/respiratory disturbances and extent of subsequent respiratory/renal compensation, respectively (e.g., days to weeks). For example, patients with compensated chronic acid-base disorders showed normal resting CBF; e.g., respiratory acidosis (CSF PCO2 63.8 mmHg; [HCO3-]CSF 29.6 mEq/l; CSF pH 7.30; CBF 49 ml/ 100 g per min) and metabolic acidosis (CSF PCO2 36.1 mmHg; [HCO3-]CSF 16.2 mEq/l; CSF pH 7.30; CBF 48 ml/ 100 g per min). Alternatively, patients with uncompensated chronic acid-base disorders showed approx. 2.5 fold higher resting CBF than healthy controls; e.g., respiratory acidosis (CSF PCO2 76.8 mmHg; [HCO3-]CSF 32.3 mEq/l; CSF pH 7.26; CBF 113 ml/ 100 g per min) and metabolic acidosis (CSF PCO2 29.4 mmHg; [HCO3-]CSF 12.5 mEq/l; CSF pH 7.27; CBF 122 ml/ 100 g per min). Figure redrawn with data from (Skinhøj, 1968). Figure adapted/redrawn from (Dubose, 2007). 25 30 35 40 45 50 55250500750100012501500PaCO2 (mmHg)CBF (ml ⋅ min-1)0 2 4 6 8050100Completion of individual process (%)Distribution and buffering in ECFCellular buffering processesH+ or HCO3-loadHours8 10 12 14 24 48 72050100Completion of individual process (%)HoursRenal base excretionRespiratory compensationRenal acid excretionABAcuteChronicNormalResp. acidosisMetab. acidosisResp. acidosisMetab. acidosis255075100125150CBF (ml ⋅ 100 g-1 ⋅ min-1)UncompensatedCompensatedLITERATURE REVIEW 11 Figure 2-3. Schematic diagram showing the changes in CSF pH with alterations in [HCO3-] and PCO2. (A) The changes in CSF pH with variation of the [HCO3-]CSF at constant PCO2. The normal CSF pH was assumed to be 7.337 at PCO2 of 45 mmHg and [HCO3-] of 23 mEq/liter (closed circle). The arrows show the absolute PCO2 values which would give a normal pH at any given [HCO3-]. (B) The changes in CSF pH with variation of the CSF PCO2 at constant [HCO3-]. The arrows show the absolute [HCO3-] at which any given PCO2 would give a normal pH. Figure redrawn and caption adapted from (Siesjö, 1972). 2.1.3 Acid-base regulation in the brain Alterations in PaCO2 regulate cerebral blood flow (CBF) via localized changes in perivascular pH; this was first evidenced by pre-clinical in vivo experiments utilizing the cranial window technique to directly apply acidic and alkaline superfusates to the pial circulation (Wolff & Lennox, 1930; Wahl et al., 1970; Betz et al., 1973; Kontos et al., 1977b; 1977a). Additionally, classic experiments in the 1950s by Leusen in anesthetized dogs showed the importance of the CO2/HCO3- ratio in cerebrospinal fluid (CSF) acid-base balance and control of breathing. For example, they showed that intraventricular perfusion with an acidic or alkaline solution causes increases and decreases in ventilation, respectively (Leusen, 1954a; 1954b); these findings were later substantiated by others (Mitchell et al., 1963; Pappenheimer et al., 1965). Collectively, the close regulation of CSF pH is vital for ventilatory and cerebrovascular control; e.g., a change in CSF pH of just 0.2 units reportedly covers the full ventilatory response between life-threatening hypoventilation and maximal hyperventilation (Pappenheimer, 1967) and CBF increases up to 2.5-fold with a 0.1 unit reduction in CSF pH (Fencl et al., 1969). 7.57.47.37.27.17.0+10+8+6+4+20-2-4-6-8-10D [HCO3-]CSF (mEq/liter)CSF pH2530354050556065A 7.77.67.57.47.37.27.17.0CSF pH20 30 40 50 60 70 80CSF PCO2 (mmHg)10152031364126BLITERATURE REVIEW 12 The volume of freely diffusible and rapid flux of CO2 out of the brain tissues and across the blood-brain barrier (BBB) is the product of CBF, total arterial concentration of CO2 + H2CO3 + HCO3- ([total-CO2]a) in whole-blood, and the fractional extraction of [total-CO2]a across the BBB (Friis et al., 1980; Johnson et al., 1983). The extent of CO2 diffusion across the BBB is regulated by the gradient between cerebral tissue and arterial partial pressures of CO2; i.e., PbtCO2-PaCO2 gradient; 6.5 mmHg (Pontén & Siesjö, 1966) – as such, the prevailing PaCO2 and cellular CO2 production principally govern [H+] and pH in any of the fluid compartments. Experimental evidence utilizing CA inhibition (via acetazolamide) supports that CA mediates CO2 extraction (i.e., free diffusion) from the brain tissue across the BBB in vivo in humans (Friis et al., 1980). The brain has a restricted capacity for substrate storage (e.g., <10% nonoxidative metabolism), thus necessitating continuous and tightly regulated CBF to support the high relative metabolic rate of brain tissue (Himwich & Homburger, 1947; Kety & Schmidt, 1948a; Siesjö, 1978; Gjedde et al., 2002; Brown & Ransom, 2007). The cerebral vasculature is particularly sensitive to alterations in PaCO2 (Hoiland et al., 2019) such that increases and decreases in PaCO2 (i.e., hyper- and hypocapnia) rapidly increase and decrease CBF, respectively (Lennox & Gibbs, 1932; Kety & Schmidt, 1946; 1948b). The integrative relationship between PaCO2 and pH on CBF regulation acts to stabilize CO2 gradients and thus regulate pH across the BBB (Fencl et al., 1969; Hoiland et al., 2019). As CO2 travels freely and rapidly across the BBB – indicated by the swift changes in CBF (<15-30 seconds) following alterations in PaCO2 (Shapiro et al., 1966; Severinghaus & Lassen, 1967; Poulin et al., 1996; Hoiland et al., 2017) – perivascular/interstitial fluid (ISF) and intracellular brain tissue pH are tightly related to PaCO2. Although acute changes in respiratory acidosis/alkalosis elicit changes in interstitial/intracellular pH (Fencl et al., 1966; Betz & Heuser, 1967; Arieff et al., 1976), CSF pH is stable across chronic metabolic acidosis and alkalosis. The tight regulation of CSF pH is achieved via active changes in choroid plexus ion transport to support a narrow range of extravascular pH irrespective of marked changes in arterial pH (Mitchell et al., 1965; Fencl et al., 1969; Siesjö, 1972). As the CSF and extracellular fluid (ECF) contain HCO3- but are protein-poor, they are reasonably well-regulated with metabolic acid-base changes but poorly buffered against changes in CO2. This is a key consideration with respect to acute and spontaneous changes in PaCO2/pH in individuals with chronic respiratory/metabolic acid-base pathologies. LITERATURE REVIEW 13 2.1.3.1 Regulation of cerebral blood flow by arterial PCO2 versus pH It is noteworthy that PaCO2-mediated cerebrovascular responses are dependent on the rapid diffusion of CO2 across the vascular wall to alter perivascular extracellular pH rather than direct changes in arterial pH per se (Figure 2-4) (Wolff & Lennox, 1930; Lambertsen et al., 1961; Severinghaus & Lassen, 1967; Betz & Heuser, 1967; Lassen, 1968; Wahl et al., 1970; Kontos et al., 1977b; 1977a; Caldwell et al., 2021c; 2021d). Overall, CBF regulation is dependent on VSMC intracellular [H+] which is directly affected by extracellular/perivascular pH, and its relationship to PaCO2 such that PaCO2-dependent changes in pH predicate cerebrovascular reactivity to CO2 (Ainslie & Duffin, 2009; Hoiland et al., 2019). As such, appropriate changes in cerebrovascular CO2 reactivity (i.e., change in CBF for a given change in PaCO2) with respect to changes in buffering capacity are essential to tightly regulate cerebral interstitial pH to support critical enzymatic function (Fencl et al., 1966; Fencl & Rossing, 1989). The cerebral circulation is exceptionally sensitive to PaCO2 throughout the cerebrovascular tree as evidenced by vasomotion in: 1) large extra-cranial conduit vessels (Willie et al., 2012; Carter et al., 2016; Hoiland et al., 2017; Carr et al., 2020); 2) intra-cerebral arteries (Giller et al., 1993; Coverdale et al., 2014; Verbree et al., 2014; Coverdale et al., 2015; Al-Khazraji et al., 2019); and 3) pial vessels (Wolff & Lennox, 1930; Wahl et al., 1970; Shapiro et al., 1971; Betz et al., 1973; Kontos et al., 1977b; 1977a; Faraci & Heistad, 1990) mediated via endothelial cells (Mashour & Boock, 1999; Paravicini et al., 2005), astrocytes (Howarth et al., 2017; Marina et al., 2020), as well as pericyte regulation of capillary blood flow (Peppiatt et al., 2006; Hall et al., 2014; Gonzales et al., 2020; Hartmann et al., 2021) and pericapillary astrocyte control of interstitial flow via localized aquaporin-4 channels (Igarashi et al., 2013; Nakada, 2015) (Figure 2-5). Collectively, varied contraction/relaxation of arteriolar VSMCs across the cerebrovascular tree allows for tight regulation and localized prioritization of CBF – and thus acid-base balance – while sustaining relatively constant microvascular pressure (Heistad et al., 1978; Faraci et al., 1987; Faraci & Heistad, 1990). LITERATURE REVIEW 14 Figure 2-4. Cerebral blood flow regulation is independent of arterial pH – rather, diffusion of CO2 across the blood-brain barrier to alter interstitial/cerebrospinal fluid pH. (A) Blood flow through the cerebral cortex (via the Kety-Schmidt technique using Krypton-85) was unaltered across a wide range of arterial pH via infusion of lactic acid and sodium bicarbonate (metabolic acidosis and alkalosis, respectively) when PaCO2 was maintained by controlled passive ventilation in anaesthetized dogs. The calculated linear regression line is y = 0.002x + 0.773 and the dotted lines show one standard deviation from the mean. Figure redrawn with data from (Harper & Bell, 1963). (B) Estimated relative change in CBF, induced by CO2 inhalation, with various conditions of acid-base balance in humans are plotted against the calculated CSF pH. The CBF response to elevated PaCO2 can be expressed as a function of CSF pH across all conditions; i.e., irrespective of chronic (5 days) metabolic acidosis/alkalosis. The reciprocal of the arterial-venous O2 content (CaO2 – CvO2; ml/ 100 ml blood) – via arterial and internal jugular vein catheterization – was used to estimate CBF by rearranging the Fick Equation with the assumption of constant cerebral metabolic rate of oxygen; e.g., CBF = CMRO2 / (CaO2 – CvO2). Figure redrawn with data from (Fencl et al., 1969). (C) Overview of the regulation of smooth muscle [H+] – PaCO2 directly affects intracellular PCO2; oxygen is required to fuel Na+-K+-ATPase exchange; and intracellular [H+] dictates [Ca2+] and vascular smooth muscle cell (VSMC) tone, which controls CBF. Figure and caption adapted/redrawn from (Duffin et al., 2021). Created with BioRender.com CO2 + H2O ⇄ HCO3- + H+Cl-Cl-2K+2K+3Na+3Na+Na +-K +-ATPaseATPADP⇄GlucoseO2CO2CO2H+ → [Ca2+] → VSMC ToneInterstitial/ Extracellular FluidC10590756045307.36 7.32 7.28 7.24Calculated CSF pHCBF (ml/ 100 g/ min)AlkalosisAcidosisNormalA6.8 7.0 7.2 7.4 7.6 7.8020406080100120Arterial pHCortex CBF (ml/ 100 g/ min)BLITERATURE REVIEW 15 Figure 2-5. The cerebral circulation is exceptionally sensitive to PaCO2 throughout the cerebrovascular tree. Inlay A: relative diameter change of murine cerebral precapillary arterioles and capillaries at the end of hypercapnia (5% CO2 in air for 6 minutes), and following return to normocapnia (Hartmann et al., 2021). These data show the vasodilator action of both ensheathing pericytes which wrap precapillary arterioles (also synonymous with precapillary vascular smooth muscle cells), and capillary pericytes, which control flow to individual capillary branches. Hartmann and colleagues (2021) showed that both pericyte phenotypes relax with hypercapnia and constrict upon return to normocapnia, and precapillary arteriole pericytes constricted 3.5 fold faster than capillary pericytes. Inlay B: photographs of feline pial vessel. Left to right: at normoxic normocapnic rest; minor vasoconstriction with hypoxic hypocapnia; extreme dilation with hypoxic hypercapnia (Wolff & Lennox, 1930). Inlay C: high-resolution magnetic resonance images of human middle cerebral artery (MCA). Left to right: white box highlights the area of interest; zoomed images of MCA at -7.5, normocapnia, +7.5, and +15 mmHg end-tidal partial pressure of CO2. Note, likely due to poor resolution of MRI to measure the MCA cross-sectional area, only moderate hypercapnia appears to elicit marked dilation (Verbree et al., 2014). Inlay D: Mean (■) and individual (○) Duplex ultrasound measurements of internal carotid artery diameter across the vasomotor response to steady state changes in PaCO2 (Willie et al., 2012). Inlay E: Overview of the cellular sites of action and vasoactive mediators involved in respiratory hypercapnia/hypocapnia induced vascular smooth muscle cell (VSMC) relaxation/contraction. Overall, [H+] and/or direct PCO2 changes act on the VSMCs as well as the endothelium, astrocytes, and nerves. These latter three cell types release vasoactive factors, including prostacyclin (PGI2), hydrogen sulfide (H2S), nitric oxide (NO), endothelium-derived hyperpolarizing factor (EDHF), and endothelin-1 (ET-1). Anatomical diagram from (Willie et al., 2014). Figure and caption adapted/redrawn from (Yoon et al., 2012; Carr et al., 2021a). Created with BioRender.com Internal carotid arteryMiddle cerebral artery Hypocapnia NormocapniaMildHypercapniaModerateHypercapniaPial vesselsCPrecapillary arterioles and capillary pericytesADCO2/[H+] PGI2 H2S NOEDHF ET-1EndotheliumAstrocyteNerveVSMCENormoxicNormocapniaHypoxicHypocapniaHypoxicHypercapniaBLITERATURE REVIEW 16 2.1.3.2 Regulation of cerebral blood flow by HCO3- exchange To a certain extent, intravascular (arterial), interstitial (ISF), CSF, and intracellular fluid compartments equilibrate relatively separately and with varying time-sensitivity. For example, studies report rapid (within seconds to minutes) changes in CSF [H+]/PCO2 when PaCO2 is altered whereas changes in [HCO3-]CSF are slower (several hours) when arterial [HCO3-] is altered (Robin et al., 1958; Bradley & Semple, 1962; Bradley et al., 1965; Hornbein & Pavlin, 1975; Pavlin & Hornbein, 1975; Nattie & Romer, 1978; Abeysekara et al., 2012). To provide context, changes in CSF/extracellular [HCO3-] respond slowly and only partially to systemic changes in arterial [HCO3-], such that an intravenous infusion of sodium bicarbonate (NaHCO3) at maintained PaCO2 in anesthetized cats provokes an initial rapid increase in extracellular [HCO3-], which is quickly restored (within 1 hour) irrespective of the persistently elevated arterial [HCO3-] (Ahmad & Loeschcke, 1982a). Additionally, several studies investigating [HCO3-] exchange during metabolic acid-base alterations report better buffering of CSF versus arterial pH as indicated by the ratio of the change in [HCO3-]CSF to the change in arterial [HCO3-] (e.g., D [HCO3-]CSF/ D [HCO3-]a); e.g., the maximal change in [HCO3-]CSF was approximately 40% of the [HCO3-]a response (Fencl et al., 1966; Siesjö & Pontén, 1966; Pontén & Siesjö, 1966; Mitchell & Severinghaus, 1967; Kronenberg & Cain, 1968; Chazan et al., 1969; Fencl et al., 1969; Pavlin & Hornbein, 1975; Hasan & Kazemi, 1976; Nattie & Romer, 1978). Collectively, these studies indicate that with an acute systemic change in [HCO3-], CSF/extracellular [HCO3-] shows faster and more complete compensation than arterial [HCO3-]. The transport of HCO3- across the BBB is complex and unresolved; see (Siesjö, 1972; Hladky & Barrand, 2016) for excellent and detailed reviews on the topic. The regulation of CSF [HCO3-] and [H+] involves electrochemical differences between blood and CSF with a series of ionic exchanges or transports at the blood-CSF barrier (Figure 2-6) (Leusen, 1972), thus explaining the slower rate of exchange of HCO3- (versus CO2 diffusion). For example, the net amount of HCO3- transferred across the BBB from the systemic circulation is only approximately 10 mmol/day as estimated from an average value of 22 mmol/l [HCO3-] produced by specialized epithelial tissue and a total choroid plexus secretion rate of approximately 500 ml/day; this is markedly less than the exchange of CO2 transferred (Hladky & Barrand, 2016). As a result, this slow exchange of [HCO3-] reduces variation of pH within the extracellular fluids of the brain to allow tight regulation (Chesler, 2003). This, however, leaves control of CSF/ISF pH largely via PaCO2, CBF, and CO2 production via the metabolic rate (Pontén & Siesjö, 1966; Siesjö, 1972). LITERATURE REVIEW 17 Several pathways of cerebral HCO3- transport are advocated, including: 1) buffering of ISF by neurons and astrocytes, most of which involves inter-conversion of CO2 and HCO3- within the cells and exchange of HCO3- and Cl- across their membranes; 2) production of lactic acid within neurons and astrocytes and its transfer to the ECF where the H+ is buffered by the CO2/HCO3- system; 3) removal of lactate- together with H+ by monocarboxylate transporter 1 (equivalent to addition of a HCO3-); 4) transport of HCO3- (or equivalently of H+ in the opposite direction) across the choroid plexuses (blood-CSF barrier, BCSFB) or the BBB; and 5) the ISF and CSF can themselves be removed by fluid outflow from the brain. These pathways are elegantly reviewed in detail elsewhere (Hladky & Barrand, 2016). The first four of these routes are relevant to transient changes in [HCO3-]; whereas, the fourth and fifth pathways likely contribute to [HCO3-] exchange on time scales of several hours or longer (Severinghaus et al., 1963; Severinghaus, 1965; Mitchell et al., 1965; Pappenheimer, 1970; Hasan & Kazemi, 1976; Kazemi & Choma, 1977; Bledsoe et al., 1981). At least acutely, intravenous infusion of NaHCO3 causes increases in resting CBF irrespective of higher arterial pH and [HCO3-] (Schieve & Wilson, 1953; Wollman et al., 1968; Harp et al., 1975; Caldwell et al., 2021c) likely in part attributable to rapid reductions in periarteriolar ECF pH (Betz & Heuser, 1967). The NaHCO3 mediated vasodilatory response is practically explained by: 1) elevated PaCO2 via partial respiratory compensation (Gesell et al., 1930; Shock & Hastings, 1935; Bernthal, 1937; Hesser, 1949; Singer et al., 1956); 2) intracellular acidosis via exogenous HCO3- mediated rapid diffusion of CO2 and consequent increases in [H+]i (Arieff et al., 1976; 1982; Jensen et al., 1988); and/or 3) reductions in hematocrit due to sodium facilitated diffusion of H2O from the intra- to extracellular space (Young et al., 1984; Nakashima et al., 1996). Collectively, while the slow transport of HCO3- typically acts to stabilize ECF pH, exogenous introduction of systemic NaHCO3 may actually facilitate changes in ECF pH via continuous inter-conversion to CO2 which rapidly diffuses across the BBB. There are several clinical populations or scenarios where acute NaHCO3 infusion is indicated such as severe hyperkalemia, improvement of toxic excretion or neuronal membrane stabilization in certain drug overdoses. Additionally, chronic oral NaHCO3 administration is indicated in chronic end-stage renal failure, renal tubular acidosis, and severe hypochloremic metabolic acidosis. Previously, NaHCO3 was used in circulatory insufficiency due to shock, lactic acidosis, ketoacidosis, and in cardiac arrest states, but later studies reveal no benefit in these conditions (Swenson, 2001). Importantly, at least pre-clinically, alterations in extracellular LITERATURE REVIEW 18 [HCO3-] can directly affect cerebrovascular tone – via VSMC contractility and Ca2+ sensitivity (Boedtkjer et al., 2016a; Boedtkjer, 2018) – therefore the regulation of CBF at rest and with increases in exogenous arterial [HCO3-] requires investigation in pathology. There is recent clinical evidence to support the use of 8.4% NaHCO3 to acutely reduce intracranial pressure in traumatic brain injury (Zeiler et al., 2018). As such, it would be relevant to consider how spontaneous changes in PaCO2 following acute cerebrovascular injury further interact with NaHCO3 therapy in this population (Williamson et al., 2015; Salinet et al., 2019; Hextrum et al., 2020). Recent work has shown that hyperventilatory hypocapnia may be a promising intervention to improve CBF regulation in acute intracerebral hemorrhage (Minhas et al., 2018; 2020a); however, prospective studies are necessary to establish whether NaHCO3 may produce an equivalent effect in acute ischemic stroke patients (Minhas et al., 2020b). These approaches can only be considered as temporizing strategies until definitive neurosurgical options can be undertaken. Figure 2-6. Overview of cerebrovascular acid-base exchange and the blood-brain barrier (BBB) epithelial transport. (A) The cerebrovasculature can be divided into five compartments: 1) intravascular (arterial); 2) interstitial fluid (ISF); 3) extracellular fluid (ECF); 4) cerebrospinal fluid (CSF); and 5) intracellular (brain tissue). Exchange between the systemic and cerebral circulations occurs at the blood-brain barrier (BBB) and blood-CSF barrier (BCSFB). Pial arterioles – involving endothelial and vascular smooth muscle cells (VSMC) – penetrate the pia mater through the Virchow-Robin space where they are encapsulated by astrocytic glial-like processes termed end-feet within the perivascular ISF. The CSF interfaces with the intravascular and ISF/ECF circulations via the choroid plexus and ependymal cells, respectively. Alterations in arterial PCO2 (PaCO2) facilitate rapid passive diffusion of CO2 across the BBB (Johnson et CO2↑[H+]i ↑[H+]oBlood Interstitial/Extracellular FluidHCO3-[HCO3-]o ↓pHiBrain TissueCerebrospinal FluidBBBBCSFB[HCO3-]CSFH+H2CO3CO2HCO3-H2OH2CO3CO2HCO3-H+H2OH2CO3CO2 H2OCAHCO3- H+CAA BH2CO3H2O CO2H+ HCO3-H2CO3H2OCO2CAEndothelial CellNa+ Na+K+Cl- Cl-Na+K+Cl-Interstitial/ Extracellular FluidBlood-Brain BarrierBloodNa+-K+-ATPaseH+ HCO3-CA⇄ ⇄LITERATURE REVIEW 19 al., 1983) leading to changes in intra- and extracellular hydrogen ion concentration ([H+]i and [H+]o, respectively) in the VSMC and ECF (Jensen et al., 1988; Vorstrup et al., 1989) in accordance with Le Chatelier’s Principle (Le Chatelier, 1884) via shifting the reaction equilibrium through continuous conversion of CO2. The rapid inter-conversion of CO2 and H2O to carbonic acid (H2CO3) is catalyzed by carbonic anhydrase (CA) which rapidly dissociates to produce bicarbonate and hydrogen ions (HCO3- and H+, respectively). Additionally, alterations in extracellular [HCO3-]o can directly affect intracellular pH (pHi) (Betz & Heuser, 1967; Nakashima et al., 1996) as well as VSMC tone via changes in Ca2+ sensitivity (Boedtkjer et al., 2016a). (B) The Na+-K+-ATPase and the Na+/H+ exchangers are localized to the membranes of the endothelial cells at the BBB. The removal of H+ is an example of secondary active transport as the energy for Na+/H+ exchange is derived from passive entry of Na+. The electrochemical gradient driving Na+ entry is maintained by the Na+-K+-ATPase (e.g., primary active transport) at the membrane facing extracellular or cerebrospinal fluid (ECF and CSF, respectively). Transport of Na+ out of cells via Na+-K+-ATPase results in a low intracellular [Na+] which facilitates Na+ entry down its gradient in exchange for H+. Permeability of the ECF-facing membrane to H+ and HCO3- allows for differential net exit of HCO3- to ECF and H+ to blood. The production of H+ and HCO3- via hydration of CO2 is catalyzed by carbonic anhydrase (CA). Dark arrows indicate active transport; light arrows indicate passive transport. Figure and caption adapted from (Bledsoe et al., 1981; Hladky & Barrand, 2016; Carr et al., 2021a). For excellent reviews on the signalling pathways regulating intra-/extracellular HCO3- exchange, pH control, and cerebrovascular tone see (Boedtkjer et al., 2016b; Boedtkjer, 2018). Created with BioRender.com 2.1.3.3 Acute alterations in cerebral blood flow stabilize CO2 gradients It is well-established that CBF varies directly with PaCO2 (Lennox & Gibbs, 1932; Kety & Schmidt, 1948b; Willie et al., 2015) such that, provided the cerebral metabolic production of CO2 remains unaltered, hypercapnia or hypocapnia will be related to reductions or increases in both the arterial-venous and CSF-PaCO2 differences, respectively (via Fick Equation) (Posner et al., 1965; Siesjö, 1972). At rest, CSF PCO2 is 6 to 11 mmHg higher and pH is 0.05 to 0.08 units lower than that of the arterial blood (Bradley & Semple, 1962; van Heijst et al., 1966; Siesjö, 1972). Experimental data and results in clinical pathology indicates the arterial-venous PCO2 difference is altered during acute respiratory acidosis/alkalosis (Kety & Schmidt, 1948b; Lambertsen, 1960; Merwarth et al., 1961; Manfredi, 1962; Posner et al., 1965; Pontén & Siesjö, 1966; Fencl et al., 1969; Messeter & Siesjö, 1971). Whereas such regulatory changes in the arterial-venous PCO2 difference reported during chronic respiratory acidosis/alkalosis and metabolic acid-base disorders are inconsistent and likely physiologically trivial (Bradley & Semple, 1962; Agrest et al., 1962; Mitchell et al., 1965; Posner et al., 1965). Taken together, appropriate CBF responsiveness to acute alterations in PaCO2 are essential to tightly regulate a narrow range of cerebral interstitial pH (Fencl et al., 1966; Fencl & Rossing, 1989). Three key regulatory responses are apparent with acute respiratory acidosis: 1) arterial [HCO3-] increases (via inter-conversion of CO2) in response to elevated PaCO2 (Figure 2-7A); 2) rapid exchange of HCO3- and Cl- across the BBB and between brain tissue and ECF (Ahmad & Loeschcke, 1982a; 1982b); and 3) total CBF increases thus reducing the gradient between arterial and CSF PCO2 and limiting the rise in intracellular tissue PCO2. According to the LITERATURE REVIEW 20 Henderson-Hasselbalch relationship (Equation 2.1.1), acute elevations in arterial [HCO3-] would theoretically increase the buffering capacity; that is, with higher arterial [HCO3-] a given change in PaCO2 will result in a smaller change in arterial [H+]/pH (Siesjö, 1972). Additionally, elevated arterial [HCO3-] may conceivably contribute to localized reductions in CBF with severe respiratory acidosis – via increases in extravascular [HCO3-] – and thus help explain the maximal vasodilatory response to hypercapnia in vivo in humans; e.g., +15-20 mmHg PaCO2 equates to 150% increase in CBF (Willie et al., 2012). The internal jugular venous-arterial [HCO3-] difference is also reduced with acute hypercapnic acidosis (Figure 2-7B) – a change likely attributable to increased intracellular HCO3- ‘wash-out’ as the reduction in trans-cerebral [HCO3-] difference is related to the hypercapnic CBF response (Caldwell et al., in-review). Arvidsson and colleagues (1981) showed that intravenous infusion of NaHCO3 in hypercapnic dogs causes a 50-70% reduction in CBF (from hypercapnic elevation) paralleled by a reduction in arterial-venous O2 difference such that CMRO2 was unaltered. Additionally, [HCO3-]CSF was appreciably higher following NaHCO3; these data indicate that hypercapnia may facilitate the transport of exogenous HCO3- across the BBB (via higher CBF) leading to cerebral vasoconstriction due to increased extravascular pH (Arvidsson et al., 1981). The dual-contribution theory of [HCO3-]CSF regulation in response to elevated PaCO2 involves: 1) passive diffusion of HCO3- from the systemic circulation; and 2) production of HCO3- within the central nervous system (e.g., via choroid plexus and glial cells) (Hasan & Kazemi, 1976). Reports indicate that the regulatory rise in [HCO3-]CSF in response to elevated PaCO2 can be reduced or abolished by inhibition of CA via acetazolamide given intravenously or intraventricularly (Maren, 1972; Wichser & Kazemi, 1975; Kazemi et al., 1976b; Shibata et al., 1976; Hasan & Kazemi, 1976; Kazemi & Choma, 1977; Javaheri et al., 1984). Additionally, several investigators have hypothesized that the choroid plexus and glial cells are involved in [HCO3-]CSF production (Kazemi et al., 1967; Maren & Broder, 1970; Maren, 1972; Wichser & Kazemi, 1975; Hasan & Kazemi, 1976; Maren, 1979) in response to elevated CSF PCO2 to regulate CSF [H+]/pH – in addition to the previously substantiated contribution of arterial [HCO3-] to affect CSF (Wichser & Kazemi, 1975; Kazemi et al., 1976a; 1976b; Hasan & Kazemi, 1976; Nattie & Tenney, 1976; Nattie & Edwards, 1981). With respect to localized interstitial acid-base regulation, astrocytes release HCO3- via electrogenic Na+/HCO3- cotransporter 1 in response to localized increases in CO2 produced by oxidative metabolism to regulate extracellular pH (Theparambil et al., 2020). LITERATURE REVIEW 21 Figure 2-7. Arterial-venous [HCO3-] and PCO2 differences with hypercapnic acidosis in humans. Arterial [HCO3-] increases with progressive elevations in arterial PCO2 (PaCO2) (A); this response would theoretically contribute to localized reductions in CBF with severe respiratory acidosis – via increases in extravascular [HCO3-] – and thus contribute to the restricted maximal vasodilatory responses to hypercapnia in vivo in humans. There is an inverse relationship between reductions in arterial pH (e.g., respiratory acidosis) and narrowing of the venous-arterial HCO3- (B) and PCO2 (C) differences. This narrowing is likely attributable to increases in [HCO3-] and CO2 ‘wash-out’ due to higher CBF with hypercapnia; e.g., the venous-arterial HCO3- and CO2 differences were each related to increases in CBF (both P<0.001). Data are from (Caldwell et al., 2021b). 2.1.4 Human cerebrovascular regulation during acid-base disorders 2.1.4.1 Acute versus chronic cerebrovascular acid-base regulation A few relevant considerations for the application of acute and chronic experimental paradigms to clinical cerebrovascular acid-base pathologies are: 1) the extent of respiratory/metabolic compensation; 2) the turnover rate of CSF and ISF; and 3) the kinetics of [H+], [HCO3-], and CO2 across the BBB. The turnover rates of CSF and ISF are approximately 2-3 and 12 hours, respectively (Rosenberg et al., 1980; Cserr et al., 1986). An integrative and sophisticated study by Fencl and colleagues (1969) showed that the CBF response to elevated PaCO2 can be expressed as a function of CSF pH across experimental chronic (5 days) metabolic acidosis/alkalosis – via oral administration of NH4Cl and NaHCO3, respectively – in healthy male volunteers. Additionally, these data further substantiate the remarkably stable CSF pH across chronic metabolic acidosis and alkalosis to regulate a narrow range of extravascular pH levels irrespective of marked changes in arterial pH (Figure 2-8) (Mitchell et al., 1965; Fencl et al., 1969; Siesjö, 1972). Alternatively, acute changes in respiratory acidosis/alkalosis provoke rapid changes in interstitial/intracellular pH to affect CBF (Shapiro et al., 1966; Severinghaus & Lassen, 1967; Arieff et al., 1976). Experimental studies investigating CBF regulation in various acid-base disorders are mixed and likely influenced by the severity and extent of metabolic/respiratory disturbances and compensation, age and sex related differences, and 7.25 7.30 7.35 7.40 7.45 7.50-20246810pHv-a [HCO3-] (mEq ⋅ l-1)P<0.00120253035[HCO3 -] (mEq ⋅ l -1)ArterialVenous7.25 7.30 7.35 7.40 7.45 7.50-5051015202530pHv-a PCO2 (mmHg)P<0.00135455565PCO2 (mmHg)ArterialVenous35 40 45 50 55 60 6520222426283032PaCO2 (mmHg)[HCO3-] a (mEq ⋅ l-1)P<0.001A B CLITERATURE REVIEW 22 standardization of experimental methods. Table 2-1 summarizes the key studies to date that have examined the influence of alterations in acid-base balance on CBF regulation. Figure 2-8. The relationship between [HCO3-], pH, and PCO2 in arterial blood and cerebrospinal fluid (CSF) with chronic metabolic acidosis/alkalosis in humans. The severity of metabolic acidosis/alkalosis is indicated on the abscissa by the arterial [HCO3-] which is then influenced by the resultant (rapid) respiratory compensation (c.f., Figure 2-2). It is noteworthy that [HCO3-]CSF varies linearly with arterial [HCO3-] (C); however, there is considerable regulation of [HCO3-]CSF such that each 4-5 mM change in arterial blood is associated with only 1 mM change in CSF. The CSF is tightly regulated with metabolic acid-base changes due to the presence of HCO3- but is poorly buffered against changes in CO2 as CSF is practically protein-free (Mitchell et al., 1965; Fencl et al., 1969; Siesjö, 1972). The [HCO3-]CSF is approximately 5 mM higher than arterial [HCO3-] during severe acidosis and 10 mM less than arterial blood during severe alkalosis. The arterial PCO2 (B) can be expressed as a reciprocal of the steady-state alveolar ventilation, which increases twofold as arterial [HCO3-] changes from 40 mM in alkalosis to 10 mM in acidosis; this respiratory compensation explains the small (1 pH unit) absolute change in CSF pH across a wide range of chronic metabolic acid-base disturbances (A). Figure redrawn with data summarized by (Fencl et al., 1966); see original text for jugular PCO2 and CSF pH calculations. Data from: (Bradley & Semple, 1962; Schwab, 1962; Agrest et al., 1962; Pauli et al., 1962; Cowie et al., 1962; Fisher & Christianson, 1963; Severinghaus et al., 1963; Mitchell et al., 1965; Posner et al., 1965). 7.357.307.25504030201030201010 20 30 40Calculated CSF pHPCO2 (mmHg)Calculated jugular(= cisternal)Arterial CSF [HCO3-]mmol/kg H2OArterial [HCO3-] mmol/kg H2OBradley & Semple, 1962Fisher & Christianson, 1963Cowie et al., 1962Severinghaus et al., 1963Schwab, 1962Pauli et al., 1962Agrest et al., 1962Posner et al., 1965Mitchell et al., 1965BACLITERATURE REVIEW 23 Table 2-1. Chronological summary of key studies to investigate cerebral blood flow during acid-base disorders in humans. Study Experimental Design Main Findings Kety & Schmidt, 1946 - Acute respiratory alkalosis via active and passive hyperventilation (18 to 19 mmHg reduction in PaCO2) 1. - 34% reduction in CBF 2. +58% increase in arterial-venous O2 difference 3. +15% increase CMRO2 during active hyperventilation only Kety et al., 1948 - Resting CBF and CMRO2 in patients with severe diabetic acidosis and diabetic coma 1. Marked reduction in PaCO2 and pH (i.e., uncompensated metabolic acidosis) 2. -17% CBF with diabetic acidosis (PaCO2 18 mmHg; [HCO3-] 6.4 mEq/l; pH 7.13) 3. +20% CBF with diabetic coma (PaCO2 19 mmHg; [HCO3-] 4.7 mEq/l; pH 6.98) 4. Changes in CBF were related to arterial pH (r = -0.70) and explained by CVR 5. Reduced arterial pH was related to lower CMRO2 (r = 0.52) Kety & Schmidt, 1948b - Acute respiratory acidosis and alkalosis via 5-7% inspired CO2 and hyperventilation, respectively 1. +9 mmHg PaCO2 increased CBF by 75% 2. -19 mmHg PaCO2 reduced CBF by 35% 3. The PaCO2-induced changes in CBF occurred with equivalent alterations in the arterial-venous O2 difference (i.e., CMRO2 was unaltered) Patterson et al., 1952 - Resting CBF and CMRO2 before and during 85-100% O2 therapy in patients with severe COPD 1. Severe respiratory acidosis and hypoxemia in COPD (PaCO2 58 mmHg; PaO2 39; arterial pH 7.36) 2. 30-40% higher resting CBF and lower CMRO2 in COPD vs. healthy controls 3. The O2 therapy caused ventilatory suppression, higher PaCO2, lower pH, elevated CBF and unaltered CMRO2 in COPD Scheinberg et al., 1953 - Resting CBF and CMRO2 in moderate COPD vs. healthy controls - Effects of exercise on CBF and CMRO2 in COPD 1. Unaltered resting CBF, arterial-venous O2 difference, CMRO2, and CVR in less severe well-compensated COPD (PaCO2 46 mmHg; PaO2 69 mmHg; pH 7.37) vs. controls 2. Resting CBF was related to PaCO2 (rather than SaO2, PaO2, or pH) 3. 0.02 unit reductions in arterial pH during exercise did not alter CBF Schieve & Wilson, 1953 - Acute metabolic acidosis and alkalosis via NH4Cl and NaHCO3 1. 65% increase in resting CBF via 3% NaHCO3 2. -20 to 25% reduction CBF with 0.8% NH4Cl 3. Increases in CBF unexplained by changes in intravascular volume or hypertonic solution per se 4. The NaHCO3 mediated increase in CBF was further exacerbated with hypercapnic breathing although arterial pH was restored to 7.40 5. Evidence that acute PaCO2 rather than arterial pH regulates CBF Posner & Plum, 1960 - Acute respiratory acidosis (via 5% inspired CO2) and metabolic acidosis (via 0.5 to 2 g i.v. acetazolamide) in patients with hepatic encephalopathy 1. Arterial metabolic alkalosis with compensatory hyperventilation at rest (pH 7.57; PaCO2 25 mmHg) 2. Severity of hepatic encephalopathy/ alkalosis results in reduced CMRO2 (via lower CBF and/or a-v O2 difference) 3. -22% reduction in CMRO2 with acute respiratory acidosis in patients with hepatic encephalopathy LITERATURE REVIEW 24 Study Experimental Design Main Findings 4. Arterial metabolic acidosis evoked by 2 g i.v. acetazolamide increased CBF by 75% and reduced CMRO2 by -25% in patients with hepatic encephalopathy 5. Higher CO2 reactivity response in cirrhotic vs. noncirrhotic controls (+46% vs. +33% increase CBF) Lambertsen et al., 1961 - Acute respiratory acidosis (PaCO2 50 mmHg) with and without i.v. NaHCO3 to abolish the CO2-induced acidosis 1. Overall, CBF varied directly with alteration in PaCO2 and was unaffected by changes in arterial pH not produced by PaCO2 change Cohen et al., 1967 - Acute isocapnic hypoxia (6.9-7.5% inspired O2; i.e., 34.6 mmHg PaO2) 1. Total CBF increased by 71% (e.g., 45.0 to 77.1 ml/100 g per min) 2. Cerebral glucose and lactate utilization/uptake increased by 28% and 4.5-fold, respectively 3. Hypoxia caused -16% absolute reduction in oxygen/glucose index and +14% absolute increase in lactate/glucose index; CMRO2 was unchanged Skinhøj, 1968 - Resting CBF and arterial/CSF blood parameters in patients with chronic respiratory and metabolic acidosis 1. Unaltered CBF in patients with fully compensated chronic respiratory acidosis (i.e., via elevated arterial/CSF [HCO3-]) 2. Approx. 2 to 2.5-fold increase in resting CBF in patients with uncompensated chronic respiratory and metabolic acidosis (irrespective of markedly higher and lower arterial/CSF PaCO2, respectively) 3. Controlled hyperventilation in patients with chronic respiratory acidosis elevates CSF pH – in part explained by the slow [HCO3-] exchange kinetics across the BBB to acutely correct [HCO3-]CSF – thus CBF was markedly reduced Wollman et al., 1968 - A: 20 mins respiratory alkalosis (via mechanical hyperventilation) at PaCO2 = 19 and 10 mmHg - B: Additive metabolic/respiratory alkalosis, i.v. NaHCO3 with controlled PaCO2 at 19 mmHg 1. -43% and -52% CBF with PaCO2 19 and 10 mmHg, respectively 2. Metabolic alkalosis raised arterial pH from 7.63 to 7.79 and CBF increased +17% (vs. PaCO2 19 mmHg) 3. Severe alkalosis causes cerebral vasodilation (via reduced hematocrit and/or localized hypoxia) Ekström-Jodal & Häggendal, 1969 - Acute respiratory acidosis (5% inspired CO2) and alkalosis via hyperventilation in patients with chronic respiratory insufficiency 1. Normalization of CBF with chronic respiratory insufficiency attributed to restored CSF pH 2. Acute cerebrovascular CO2 reactivity was not different than control subjects Fencl et al., 1969 - Acute respiratory acidosis with chronic (5 days) metabolic acidosis and alkalosis via NH4Cl and NaHCO3 1. At a given PaCO2, both V̇E and CBF were higher in acidosis and lower in alkalosis 2. Both CBF and V̇E can be expressed as a single function of CSF pH King et al., 1974 - Serial arterial/CSF lactate, pH, PCO2, HCO3-, and PO2 sampling for 4 days immediately post- head injury 1. Arterial metabolic acidosis ([HCO3-] = 20.9 mEq/l) with compensatory respiratory alkalosis (PCO2 = 36.8 mmHg) on admission; spontaneous hyperventilation likely facilitated by persistent CSF lactic acidosis (e.g., range: 52.7 to 68.0 mg/100 ml up to day 3) LITERATURE REVIEW 25 Study Experimental Design Main Findings 2. Arterial [La] was initially markedly elevated, however, returned towards normal values by day 3; [La]CSF was elevated up to 3 days post- injury 3. Arterial [La] was inversely related to PCO2 and HCO3-, independent of pH 4. [La]CSF was inversely related to HCO3- but not PCO2 Harp et al., 1975 - Acute isocapnic metabolic alkalosis via i.v. NaHCO3 1. +27% increase in CBF with acute metabolic alkalosis at constant PaCO2 40 mmHg Wade et al., 1981 - Pre- and post- venesection in patients with polycythemia secondary to hypoxic lung disease - Resting CBF via i.v. 133Xe, blood viscosity, hematocrit, cerebral O2 delivery 1. Resting CBF and cerebral O2 delivery were 24% and 17% higher, respectively, than polycythemic controls without lung disease; likely attributable to chronic respiratory acidosis (PaCO2 = 56 mmHg; pH = 7.37) 2. Reductions in hematocrit via venesection (e.g., 49.5% vs. 61.3%) increased CBF by 21%; cerebral O2 delivery was unaltered Friberg et al., 1990 - An acute oral dose of 1,000 mg acetazolamide followed by 1,000 mg daily for 10 days 1. An oral dose of 1,000 mg acetazolamide acutely increased CBF by +38% – likely mediated by transient extracellular acidosis 2. Collectively, CBF is normalized within 2 days of chronic acetazolamide (500 mg twice daily) via respiratory compensation; i.e., progressive -30% reduction in alveolar PCO2 Hirakata et al., 1992 - Hemodialysis patients were treated with 3x per week i.v. recombinant human erythropoietin (EPO) to achieve 10% absolute value increase in hematocrit - Resting CBF, O2 extraction, and CMRO2 via PET 1. Pre- EPO, CMRO2 was -32% lower, whereas CBF and O2 extraction were +25% and +29% higher than controls, respectively 2. Post- EPO, CBF and O2 extraction were reduced to control values; however, CMRO2 remained -28% lower than controls Vorstrup et al., 1992 - Resting CBF via 133Xe inhalation with PET in patients on long-term hemodialytic treatment 1. Resting CBF was +21% higher than controls 2. Resting hematocrit was -13% absolute values lower (e.g., 30% vs. 43%), such that CBF and hematocrit were inversely correlated (r = -0.71) 3. Calculated CBF using a hematocrit of 43% revealed -20% below expected CBF, indicating higher CBF necessary to offset reduced O2 carrying capacity – consistent with reportedly lower CMRO2 in hemodialysis patients Nakashima et al., 1996 - Acute (15 mins) metabolic alkalosis via 7% i.v. NaHCO3 - Effects of CBF and intracellular pH via stable Xe-CT and 31P-MRS, respectively 1. Resting CBF and PaCO2 increased; hematocrit and intracellular pH decreased 2. Attributed increases in CBF to: 1) CO2-mediated vasodilation; 2) reduction in hematocrit; 3) intracellular acidosis Kuwabara et al., 2002 - Acute 5% inspired CO2 in anemic patients with chronic renal failure - Total CBF, O2 extraction, and CMRO2 via PET 1. The CO2 reactivity response was reduced by approx. 50% in anemic patients versus controls 2. Reduced CO2 reactivity was correlated with the severity of anemia (e.g., CO2 reactivity vs. hematocrit, r = 0.79) 3. The reduced CO2 reactivity response was correlated with higher resting CBF and lower CMRO2; however, CO2 reactivity was unrelated to O2 extraction LITERATURE REVIEW 26 Study Experimental Design Main Findings Peebles et al., 2008 - Trans-cerebral vasoactive exchange with acute stepwise respiratory acidosis (4% and 8% inspired CO2) and alkalosis (-6 and -15 mmHg via voluntary hyperventilation) 1. Respiratory acidosis but not alkalosis caused a net cerebral release of nitrite indicated by an increase in venous-arterial nitrite difference (57 and 150 µmol/l at 4% and 8% CO2, respectively) 2. Higher cerebral release of C-natriuretic peptide during respiratory acidosis vs. alkalosis Yildiz et al., 2012 - Resting CBF via Duplex ultrasound in exacerbated vs. stable COPD patients 1. Resting CBF was higher in exacerbated vs. stable COPD patients 2. Resting CBF was related to PaCO2 in patients with exacerbated COPD (r = 0.53) Lewis et al., 2019 - Cerebrovascular regulation pre- and post- 8 weeks of aerobic exercise training in COPD patients 1. Resting CBF was unaltered between COPD patients and controls (791 vs. 658 ml/min, respectively) 2. Reduced CBV response to respiratory alkalosis in COPD patients versus controls (-1.1 vs. -1.6 cm/s per mmHg PETCO2) 3. Peak VO2 was improved post- 8 weeks aerobic exercise training (+1.7 ml/kg/min); however, CBF regulation was unaltered in COPD patients Abbreviations: Cerebral blood flow, CBF; cerebral blood velocity, CBV; cerebral metabolic rate of oxygen, CMRO2; cerebrovascular resistance, CVR; chronic obstructive pulmonary disease, COPD; arterial carbon dioxide tension, PaCO2; arterial oxygen tension, PaO2; end-tidal carbon dioxide tension, PETCO2; arterial oxygen saturation, SaO2; References: (Kety & Schmidt, 1946; Kety et al., 1948; Kety & Schmidt, 1948b; Patterson et al., 1952; Schieve & Wilson, 1953; Scheinberg et al., 1953; Posner & Plum, 1960; Lambertsen et al., 1961; Cohen et al., 1967; Skinhøj, 1968; Wollman et al., 1968; Fencl et al., 1969; King et al., 1974; Harp et al., 1975; Wade et al., 1981; Friberg et al., 1990; Hirakata et al., 1992; Vorstrup et al., 1992; Nakashima et al., 1996; Kuwabara et al., 2002; Peebles et al., 2008; Yildiz et al., 2012; Lewis et al., 2019). 2.1.5 Clinical implications As highlighted in Table 2-1, there are several clinical acid-base disturbances, but the implications of these on CBF are poorly understood and a full description of each is beyond the scope of the current review. There are, however, a few key experiments that have investigated alterations in CBF during the acid-base disturbances associated with chronic obstructive pulmonary disease (COPD) and diabetic ketoacidosis. These pathologies were chosen as exemplar respiratory and metabolic disorders, respectively, and are briefly summarized with respect to their influence on CBF regulation. Severe COPD is characterized by progressive arterial hypoxemia, often in the presence of respiratory acidosis; resting arterial blood parameters in COPD are typically within the following ranges: PaCO2 40 to 58 mmHg; [HCO3-] 25 to 32 mmol/l; pH 7.36 to 7.40 (Baldwin et al., 1949; Patterson et al., 1952; Scheinberg et al., 1953; Delclaux et al., 1994; McKeever et al., 2016). Relatively few studies to date have investigated cerebrovascular regulation utilizing direct LITERATURE REVIEW 27 volumetric CBF in patients with COPD thus resulting in mixed findings and the marked heterogeneity in pathophysiology/ disease progression of COPD restricts generalizability of results (Ainslie & Ogoh, 2010; Beaudin et al., 2017). An early study by Patterson and colleagues (1952) reported that patients with emphysema have 30-40% higher resting CBF and lower CMRO2 versus healthy controls (Patterson et al., 1952); these changes were likely attributable to the severe respiratory acidosis and hypoxemia (PaCO2 58 mmHg; PaO2 39 mmHg; arterial pH 7.36; calculated [HCO3-] 31.7 mmol/l) and these findings were later substantiated by others (Albayrak et al., 2006). Work by Scheinberg and colleagues (1953) reported that patients with less severe well-compensated COPD (PaCO2 46 mmHg; PaO2 69 mmHg; pH 7.37; calculated [HCO3-] 25.7 mmol/l) have unaltered resting CBF, trans-cerebral arterial-venous O2 difference, CMRO2, and cerebrovascular resistance compared to healthy controls. Additionally, resting CBF was best explained by PaCO2 (rather than arterial oxygen saturation, PaO2, or pH) in these patients (Scheinberg et al., 1953). Taken together, the severity of acidosis/hypoxemia and extent of metabolic compensation likely dictates CBF regulation in patients with COPD; e.g., (Yildiz et al., 2012). Acute oxygen therapy reportedly improves cerebral oxygen delivery and neurovascular coupling in hypoxemic COPD patients (Hoiland et al., 2018); these changes may be in part explained by alterations in intracellular pH in COPD patients following oxygen therapy (Hamilton et al., 2003). Additionally, patients with COPD have chronically raised [HCO3-] thus facilitating tolerance of elevated resting PaCO2 and hypoxemia; therefore, how this desensitization to PaCO2/PaO2 affects cerebral vasoactive reactivity and acute CBF regulation merits exploration (Skinhøj, 1968). Diabetic ketoacidosis is a life-threatening complication of diabetes – occurring at the onset in 15-67% of type-1 diabetes cases, and is the leading cause of death in newly-diagnosed children (Lévy-Marchal et al., 1992; Dahlquist & Källén, 2005; Rewers et al., 2008), although ketoacidosis may also occur with poor treatment compliance in type-2 diabetes. The condition is characterized by hyperglycemia, hyperketonemia, hyperventilatory-induced reductions in PaCO2 and very low arterial [HCO3-] and pH; i.e., uncompensated metabolic acidosis (Kety et al., 1948; Tasker et al., 2005). Although controversial, exogenous NaHCO3 is commonly prescribed in severe cases with pH <6.9 (Assal et al., 1974; Reddy & Orti, 1977; Hale et al., 1984; Duhon et al., 2013; Patel et al., 2018). The regulation of CBF is particularly relevant in pediatric type-1 diabetes as cerebral vasogenic edema – which is reportedly related to NaHCO3 therapy (Assal et al., 1974; Bureau et al., 1980) – is a devastating complication of diabetic ketoacidosis (Glaser et al., 2001; 2004; Levin, 2008). An influential study by Kety and colleagues (1948) utilized LITERATURE REVIEW 28 invasive trans-cerebral arterial-venous measures of CBF and CMRO2 in patients with severe diabetic acidosis and diabetic coma (Kety et al., 1948). These authors reported that changes in CBF and CMRO2 were related to arterial pH (i.e., severity of acidosis). For example, compared to healthy controls, resting CBF was 17% lower in patients with diabetic acidosis (PaCO2 18 mmHg; [HCO3-] 6.4 mEq/l; pH 7.13) and CBF was 20% higher in patients with diabetic coma (PaCO2 19 mmHg; [HCO3-] 4.7 mEq/l; pH 6.98). It is noteworthy that cerebral utilization of oxygen was reduced by 48% in patients with diabetic coma – across all patients, reductions in CMRO2 were related to low arterial pH and high ketone levels rather than CBF. Taken together, these data show that pH and/or ketone related changes in cerebrovascular tone affect CBF regulation in severe uncompensated diabetic acidosis regardless of the competitive remarkably low PaCO2. The human brain can readily uptake and oxidize ketone bodies with increases in the systemic circulation (Owen et al., 1967; Mikkelsen et al., 2015) such that acute ketone infusion increases CBF by 30-40% (Hasselbalch et al., 1996; Svart et al., 2018). As such, the results by Kety and colleagues (1948) are likely explained by the markedly elevated systemic ketone levels which were 50% higher in the diabetic coma patients versus the awake individuals with severe ketoacidosis; e.g., diabetic coma: 98.4 mg/100 ml vs. ketoacidosis: 66.3 mg/100 ml vs. healthy: 1.5 mg/100 ml (Kety et al., 1948). 2.1.6 Summary Overall, the integrative relationships between arterial and extravascular/interstitial pH, PCO2, and HCO3- affect cerebrovascular regulation in various acid-base pathology. We reviewed herein the relevant evidence in an attempt to substantiate the following key theses: 1) acute CBF regulation is likely independent of arterial pH – rather, diffusion of CO2 across the blood-brain barrier is integral to alter perivascular extracellular pH; 2) regulatory changes in arterial [HCO3-] during acute respiratory acidosis/alkalosis contribute to cerebrovascular acid-base regulation; and 3) regulation of CBF is affected by the severity of metabolic/respiratory disturbances and extent of respective compensation. Future research is needed to extend the basic understanding of the implications of pathological acid-base disturbances on cerebrovascular regulation in humans. 2.2 Metabolism in the brain during exercise in humans The following section has been published as an invited textbook chapter titled Exercise Metabolism by the American Physiological Society in partnership with Springer Nature (Caldwell et al., 2022). LITERATURE REVIEW 29 2.2.1 Abstract This chapter discusses the regulation of cerebral metabolism and fuel utilization at rest and during dynamic whole-body exercise in humans. The relative contributions of cerebral metabolic rates of key substrates (oxygen, glucose, lactate, ketone bodies) are outlined with respect to rest and exercise. A brief overview of the current gold-standard techniques to assess cerebral metabolism during dynamic exercise in humans is also provided and future research areas are highlighted throughout. 2.2.2 Background Regulation of the human circulation during exercise is astonishingly complex and is dictated by a balance between systemic sympathetic vasoconstriction and localized vasodilation. The tight regulation of blood flow and pressure prioritizes both: 1) systemic arterial blood pressure to provide adequate organ-specific perfusion pressure; and 2) sufficient delivery of oxygen and metabolic substrates to satisfy the increased tissue metabolism and the subsequent removal of metabolic end-products. Although the human brain is reflective of <1/50th of the body mass, it accounts for approximately 15-20% of the available cardiac output, body glucose, and oxygen consumption in the resting condition (LITERATURE REVIEW 30 Table 2-2). Cerebral oxygen consumption is approximately 3.5 ml/100 g brain/min; therefore, an average sized brain, weighing approximately 1,400 g requires a rate of oxygen uptake (V̇O2) of 49 ml O2/min (Sokoloff, 1960). As the majority of oxygen in the brain is utilized for carbohydrate oxidation, the energy equivalent of the total cerebral metabolic rate is approximately 20 watts or 0.29 kcal/min. This high cerebral metabolic rate, paired with a remarkably restricted capacity for substrate storage (e.g., glycogen <10 µmol/g) and inability to utilize fat as a fuel substrate, necessitates tight regulation of cerebral blood flow. Loss of consciousness occurs within 10-20 seconds of cerebral ischemia and brain death can ensue if adequate restoration of blood supply is not made. As an example, if the supply of glucose to the brain were theoretically abolished, the brain’s total substrate stores – from free glucose, lactate, and astrocytic glycogen – would only sustain cerebral metabolism for approximately 12 minutes (Barros & Deitmer, 2010). As such, the interplay between cerebral substrate delivery (via cerebral blood flow and arterial substrate content) and trans-cerebral exchange is essential to support increases in fuel utilization in the brain during exercise. LITERATURE REVIEW 31 Table 2-2. Cerebral blood flow and global metabolic rate at rest and during exercise to exhaustion. Per 100 g brain tissue Per whole brain (1,400 g) Relative to whole-body Max increase from rest with exercise Cerebral blood flow (ml/min) 57 798 15% cardiac output +15-25% Cerebral O2 consumption (ml/min) 3.5 49 20% total V̇O2 +30-35% Cerebral glucose utilization (mg/min) 5.5 77 15% total glucose +35-40% 1Reviewed in Clarke & Sokoloff (1999) with data derived from the literature published by Sokoloff (1960). Relative increases during exercise reviewed by Smith & Ainslie (2017). 2.2.3 Cerebral metabolism and fuel utilization at rest Cerebral metabolism is regulated by blood flow and the arterial to venous trans-cerebral exchange of primary substrates (i.e., oxygen, glucose, lactate, and ketones) across the cerebral circulation. The cerebral storage capacity of oxygen is exceptionally small relative to its rate of utilization; therefore, continuous cerebral circulation is essential to support oxidative metabolism. The regulation of cerebral substrate delivery is achieved via the neurovascular unit; involving neurons, glial cells, and cortical penetrating arterioles, which relays vascular signalling to tightly regulate cerebral blood flow in response to alterations in local cerebral metabolism (Figure 2-9). Effective regional cerebral blood flow control is requisite for coordinated substrate delivery and local paracrine regulation in response to alterations in cerebral metabolism. At least in pre-clinical models, novel in vivo evidence utilizing the cranial window technique suggests that contractile pericytes may facilitate the regulation of capillary blood flow (Gonzales et al., 2020); however, little is known about whether this occurs during exercise. At rest, the brain relies almost exclusively on oxygen and glucose. Although lactate in the arterial blood is relatively low at rest (e.g., <1 mmol/l), lactate utilization reportedly contributes to approximately 8% of cerebral metabolism (van Hall et al., 2009). It is noteworthy that the brain does not rely on fatty acid oxidation (Mitchell et al., 2011; Schönfeld & Reiser, 2013). The reasons for this are likely four-fold: 1) the rate-limiting passage of fatty acids across the blood-brain-barrier; 2) the rate of adenosine triphosphate (ATP) production derived from fatty acids is slower and less efficient than glucose utilization with respect to oxygen requirements; 3) there is relatively low enzymatic capacity of necessary fatty acid b-oxidation enzymes (e.g., carnitine palmitolytransferase 1, 3-ketoacyl-CoA thiolase, acyl-CoA dehydrogenase, enoyl-CoA LITERATURE REVIEW 32 hydratase); and 4) the b-oxidation of fatty acids generates superoxide and can contribute to severe oxidative stress. However, while fatty acid oxidation does not contribute substantially to ATP production, recent in vitro cultured cell work and in vivo PET imaging evidence support a role for localized insulin-sensitive astrocytes in cerebral fatty acid uptake and metabolism in humans (Rebelos et al., 2020; Heni et al., 2020). Figure 2-9. Regulation of cerebral blood flow by metabolism. PO2 brain tissue20-30 mmHgCaO2 - CvO26.5 mL/ 100 mLPO2 arterial<20 mmHgPO2 capillary (venous) 15 mmHgPO2 gradient10 mmHgPO2 mitochondria1 mmHgMetabolismCerebral blood flow(Coma) (Seizure)Nerve Cell ActivityLITERATURE REVIEW 33 The cerebral oxygen cascade highlights the necessity for tightly controlled cerebral blood flow regulation. Regulation of cerebral blood flow is affected by changes in localized neuronal activity and metabolism. As an example, the linear relationship between cerebral blood flow and CMRO2 is evidenced by very low to high levels of nerve cell activity; e.g., coma/deep sleep/anesthesia (Madsen & Vorstrup, 1991) and exercise/seizure (Siesjö, 1978). Figure adapted from (Bailey, 2019). Created in BioRender.com At rest, trans-cerebral uptake of oxygen and glucose each contribute approximately 50% to cerebral metabolic rate (Figure 2-10). The contribution of cerebral lactate utilization to cerebral metabolism is less than 10% at rest. At rest, oxygen is utilized in the brain almost exclusively for the oxidation of carbohydrates consistent with the respiratory quotient of 0.97 (Nybo et al., 2003; Dalsgaard et al., 2004a). The stable resting cerebral metabolic ratio for oxidative glucose metabolism (often termed OGI) is approximately 5.7 (Dalsgaard et al., 2004c; Smith et al., 2014). This value indicates near complete oxidation of glucose: 6 O2 + C6 H12O6 (glucose) ® 6 H2O + 6 CO2; approximately 5-10% of the glucose taken up undergoes glycolysis as evidenced by a slight lactate efflux at rest (Dalsgaard et al., 2004c; van Hall et al., 2009). Energy-yielding substrates within the arterial blood enter the brain principally via the blood-brain-barrier (Figure 2-11), albeit other less important pathways also include extracellular fluid, choroid plexuses, and interstitial/cerebrospinal fluid exchange. The blood-brain-barrier consists of endothelial cells of the capillary wall, astrocyte end-feet surrounding the capillary, and pericytes outside the capillary basement membrane (Edvinsson & Krause, 2002; Simard et al., 2003) (Figure 2-12). Gases like oxygen and carbon dioxide readily cross the blood-brain-barrier via passive diffusion. Larger molecules like glucose, however, crosses the blood-brain-barrier via specialized transporters within the endothelial cells (e.g., glucose transporter; GLUT- 1, 3, and 5). The glucose transporter GLUT1 is also found on astrocytes and the choroid plexus (Simpson et al., 2007), GLUT3 is found on neurons and to some extent the endothelium (Simpson et al., 2007), and GLUT5 is localized to the microglia. The majority of glucose entering the brain is distributed via the interstitial fluid where it is taken up via facilitated diffusion by neurons (GLUT3) and astrocytes (GLUT1). The rate of glucose transport is higher in the neuron versus astrocytes due to larger catalytic capacity of GLUT3 compared to GLUT1. The regulation of glucose transport at the blood-brain-barrier has been recently reviewed in detail elsewhere (Patching, 2017; Koepsell, 2020). LITERATURE REVIEW 34 Figure 2-10. Summary of the bioenergetic requirements of the human brain, emphasizing its severely restricted energy stores of oxygen, glucose, and ATP, paired with high rates of metabolism. Figure adapted from (Bailey, 2019). The majority of signaling-related ATP costs involve the Na+/K+ ATP pump to regulate synaptic ion fluxes and maintain the resting membrane potential as well as conversion of glutamate into glutamine via astrocytes. Non-signaling ‘housekeeping’ tasks include ATP-dependent actin treadmilling, microtuble dynamic instability, protein synthesis, lipid turnover, microglial motility, mitochondrial proton transfer, and cytoskeletal rearrangements (Attwell & Laughlin, 2001; Engl & Attwell, 2015). Created in BioRender.com AllocationRest ContentMetabolismGlucose1,000-4,000 nmol/mLATP1,000-3,000 nmol/mL2.8 mM/2 gOxygen1.5 mmol/min/g3 mol/dayGlucose0.4 µmol/min/g56 g/dayGlucose51.7%Oxygen48.3%ATP8.1 µmol/min/g8.3 kg/daySignaling60%Housekeeping40%Oxygen30 nmol/mL2% body mass25% basal metabolic rate15-25 Watts powerLITERATURE REVIEW 35 Figure 2-11. Brain substrate metabolism of glucose, glycogen, and lactate. (A) At rest, glucose is the primary cerebral substrate as the uptake ratio O2/glucose is close to 6. (B) Additionally, glucose fuels glycolysis in astrocytes producing lactate that is apparently oxidized by neurons. (C) Astrocytic breakdown of glycogen supports astrocyte metabolism, (D) particularly in astrocyte enveloping end-feet of synaptic clefts that are devoid of mitochondria. The spatial arrangement of the neurovascular unit may explain why lactate production from glycogen is separate from the breakdown of blood glucose. (E) Increases in cerebral lactate uptake during exercise may contribute to astrocytic glycogen-derived lactate oxidation during hypoglycemia experienced with exhaustive exercise. Figure and caption adapted from (Dalsgaard, 2006). Created in BioRender.com NeuronAstrocyteCapillaryLactateABCDEGlycogenGlucoseLITERATURE REVIEW 36 Figure 2-12. Anatomy of the blood-brain barrier and cerebrovascular tree. At the brain surface there is a highly vascularized capillary network of pial arterioles that are responsive to vasoactive stimuli (e.g., cerebral perfusion pressure, PO2/CaO2, PCO2/[H+]). (A) Pial arterioles are surrounded by the subarachnoid space and are innervated by perivascular nerves acting on vascular smooth muscle cells (VSMC). (B) Pial arterioles penetrate the pia mater through the Virchow-Robin space where they are encapsulated by astrocytic glial-like processes termed end-feet as well as perivascular macrophages. (C) Intraparenchymal arterioles and branching capillaries are covered by neural processes and pericytes within the basement membrane of the endothelium. Created in BioRender.com At rest, circulating ketone bodies, principally acetoacetate and its reduced form b-hydroxybutyrate (bHB), are <0.1 mmol/l; whereas, following prolonged aerobic exercise in the glycogen depleted state, post-exercise ketosis can arise depending on the intensity and BCALITERATURE REVIEW 37 duration of exercise as well as aerobic fitness and nutritional status in the range of 0.3 to 2.0 mmol/l (Evans et al., 2016). During conditions of hypoglycemia; e.g., fasting/ starvation (Owen et al., 1967), post-exhaustive exercise (Askew et al., 1975), and ketogenic diet (Yancy et al., 2004), increases in systemic ketone bodies act as an alternative fuel source for cerebral metabolism. The human brain can readily uptake and oxidize bHB linearly with increases in the systemic circulation (Figure 2-13) (Owen et al., 1967; Mikkelsen et al., 2015). Acute ketone infusion increases cerebral blood flow by approximately 30-40% and reduces cerebral metabolic rate of glucose by 30% with no change in oxygen utilization in humans (Hasselbalch et al., 1996; Svart et al., 2018). These ketone-induced increases in cerebral blood flow are perhaps in part due to changes in the brain cytosolic NAD+/NADH ratio (Vlassenko et al., 2006; Xin et al., 2018), reductions in oxidative stress (Shimazu et al., 2013), and/or direct effects of ketones on the vasculature (Kimura et al., 2011; Han et al., 2018; Wu et al., 2020). The majority of bHB oxidized by the human brain is supplied by the liver via the arterial blood (Robinson & Williamson, 1980; Laffel, 1999); however, bHB can be synthesized to a lesser extent endogenously in astrocytes via fatty acid oxidation (Edmond et al., 1987). With reductions in carbohydrate availability – and lower insulin levels – elevations in free fatty acid release and utilization results in excess acetyl-CoA in hepatocytes, where it will be converted to ketone bodies. Although, as discussed above, the brain does not typically rely on fatty acid oxidation at rest, the dependency and utilization of ketone bodies as a substrate during/following prolonged exercise is related to elevations in acetyl-CoA and upregulated free fatty acid availability. LITERATURE REVIEW 38 Figure 2-13. Relationship between the arterial D-b -hydroxybutyrate concentration and cerebral and leg, skeletal muscle, uptake rate per kg of tissue in healthy males. Brain ketone uptake and subsequent oxidation was linearly related to the arterial ketone concentration. Resting skeletal muscle ketone uptake was markedly less and showed saturation kinetics. These findings substantiate the selective utilization of ketone bodies as a fuel for cerebral metabolism with increased systemic availability. It is noteworthy that the brain readily uptakes D-b-hydroxybutyrate approximately 10-fold higher than skeletal muscle at rest. Figure redrawn from (Mikkelsen et al., 2015). 2.2.3.1 Summary At rest, the brain relies almost exclusively on aerobic oxidative metabolism as reflected by the large 50% contributions of oxygen and glucose trans-cerebral uptake to the total cerebral metabolic rate. The effective localized control of cerebral blood flow, in part achieved by the highly sensitive pial arteriolar capillary network and tightly regulated blood-brain-barrier, allows for coordinated cerebral substrate delivery. As discussed next, the regulation of cerebral blood flow and substrate delivery is critically important to support increases in total cerebral metabolism during exercise. 8060402000.0 0.4 0.8 1.2 1.6 2.0D-b-hydroxybutyrate uptake (µmol/(kg tissue mass.min))Arterial D-b-hydroxybutyrate (mmol/L)BrainSkeletal muscleLITERATURE REVIEW 39 2.2.4 Regulation of cerebral metabolism during exercise At rest and during exercise, adequate cerebral oxygen delivery (CDO2) is necessary to support an appropriate cerebral oxygen reserve (i.e., capillary oxygen volume sufficient to match cerebral metabolic rate of oxygen; Figure 2-14). CDO2 (ml/min) = CBF × CaO2 Equation 2.2.1 The cerebral metabolic rate of oxygen (CMRO2) reflects the oxidative metabolic rate of the brain and is derived from the product of oxygen delivery (i.e., cerebral blood flow) and the cerebral oxygen extraction (i.e., arterial-venous oxygen content difference; CaO2 – CvO2). CMRO2 (ml/min) = CBF 100⁄ × CaO2 – CvO2 Equation 2.2.2 Typically, the resting oxygen extraction fraction (OEF) of the brain is within 20-30% (Gjedde, 2005; Ainslie et al., 2014) and is dictated by the fixed oxygen conductivity of cerebral tissue (e.g., capillary surface area and distance for diffusion) and tissue metabolism for a given PO2 gradient between the tissues and capillaries (Gjedde, 2005). OEF (%) = CaO2 – CvO2 CaO2⁄ × 100% Equation 2.2.3 Figure 2-14. Relationship between arterial-venous oxygen difference (AVDO2) and cerebral blood flow (CBF) at rest. (A) If a coupled change in cerebral metabolic rate of oxygen (CMRO2) and CBF occurs, then AVDO2 remains unchanged and the relationship between CBF and AVDO2 shifts to a new CMRO2 curve (horizontal arrows). If CMRO2 remains constant, then changes in AVDO2 reflect uncoupled changes in CBF (curved arrows). (B) Model diagramming the relationship between CBF and cerebral metabolism in comatose patients. In the absence of cerebral ischemia, the AVDO2 and CBF have the relationship 0.02.04.06.00.0 0.4 0.8 1.2AVDO2(µmol/mL)CBF (mL/g/min)CMRO21.81.51.20.90.60.3Coupled ↓ in CBFCoupled ↑ in CBFUncoupled ↑ in CBFUncoupled ↓ in CBFA0.02.04.06.00.0 0.4 0.8 1.2AVDO2(µmol/mL)CBF (mL/g/min)CMRO21.81.51.20.90.60.3HypoperfusionNormalHyperemiaIschemiaInfarctionBLITERATURE REVIEW 40 illustrated by the solid curve, with CMRO2 averaging 0.9 µmol/g/min. In the presence of cerebral ischemia/infarction (open arrows), AVDO2 and CBF have an unpredictable relationship. Figure and caption adapted from (Robertson et al., 1989). It is noteworthy that the brain (unlike the muscle, for example) does not increase capillary recruitment and, therefore, the diffusive surface area for oxygen does not change (Kuschinsky & Paulson, 1992; Williams et al., 1993), necessitating large increases in cerebral blood flow and/or oxygen extraction to maintain or elevate CMRO2 (Buxton & Frank, 1997; Mintun et al., 2001). As such, increases in cerebral oxygen delivery and metabolism may be described by an exponential relationship with increases in cerebral blood flow in excess of relative increases in CMRO2 (Buxton & Frank, 1997). In support of this, the cerebral oxygen extraction is inversely proportional to cerebral blood flow when metabolism is unaltered and directly proportional to metabolism when cerebral blood flow is unaltered. However, although the brain has the capacity to somewhat alter cerebral extraction throughout submaximal cycling exercise to exhaustion; e.g., <5% change in fractional O2 extraction (Figure 2-15), it is noteworthy that this response is markedly less than other organs such as the heart or skeletal muscle where oxygen extractions up to 80-90% have been reported (Proctor et al., 1998; Calbet et al., 2004; Lundby et al., 2006). As such, the brain requires sufficient increases in cerebral oxygen delivery (cerebral blood flow × CaO2) to support elevated CMRO2 at maximal exercise. Figure 2-15. Alterations in cerebral and systemic oxygen extraction during aerobic exercise to exhaustion. (A) The relationship between cerebral oxygen extraction – via arterial-jugular venous O2 difference – throughout incremental cycling exercise to exhaustion (expressed as % maximal workload; %Wmax). Each symbol represents individual data from n=9 with data from (Smith et al., 2014) with permission. Note the relatively small change in O2 extraction across a large range of exercise intensities; e.g., <5% change in fractional O2 extraction (%). (B) The relationship between systemic oxygen extraction (calculated via the 1009590858075706560555002 3 4 5 6 7Systemic O2extraction (%)Maximal oxygen uptake (L × min-1)0BA0 20 40 60 80 10001020304050Exercise Intensity (%Wmax)Cerebral O2 Extraction (%)LITERATURE REVIEW 41 Fick equation) and the respective pulmonary maximal oxygen uptake; note the very high attainable systemic O2 extraction values at maximal exercise. Data are individual values from n=115 healthy young men at whole-body maximal exercise across 17 studies summarized by (Skattebo et al., 2020). Figure redrawn from (Skattebo et al., 2020). With progressive increases in exercise intensity up to approximately 60-70% maximal oxygen uptake (V̇O2max), cerebral blood flow increases steadily (+10-20%) (Sato & Sadamoto, 2010) to regulate cerebral substrate delivery (Ide & Secher, 2000; Fisher et al., 2013) (Figure 2-16). The increases in cerebral blood flow during submaximal exercise are mediated via relative alveolar hypoventilation (i.e., small elevations in arterial PCO2) and increases in cerebral oxidative metabolism (CMRO2) (Nybo et al., 2002a; Smith et al., 2014). With heavy exercise intensity (e.g., >70% V̇O2max), cerebral blood flow is attenuated via hyperventilatory-induced reductions in arterial PCO2 and resultant cerebral vasoconstriction (Larsen et al., 2008; Smith et al., 2014). Relative increases in arterial oxygen content (CaO2) during progressive exercise (Ekblom et al., 1975; Smith et al., 2014) likely offset countervailing reductions in cerebral blood flow to stabilize cerebral oxygen delivery at maximal exercise intensities. Throughout various exercise intensities, the CMRO2 response is coupled to cerebral oxygen delivery (cerebral blood flow × CaO2) and oxygen extraction (CaO2 – CvO2) rather than cerebral blood flow per se. This is further support by the nonlinear relationship between cerebral blood flow/oxygen delivery and CMRO2 (Fox & Raichle, 1986; Fox et al., 1988). LITERATURE REVIEW 42 Figure 2-16. Systemic and cerebrovascular variables affecting cerebral blood flow regulation during aerobic exercise to exhaustion. Generalized relative changes in arterial oxygen content (CaO2) and cerebral oxygen delivery (CDO2) (top), cerebral metabolic rate of oxygen (CMRO2) and oxygen extraction fraction (O2EF) (middle), global cerebral blood flow (gCBF) and partial pressure of arterial carbon dioxide (PaCO2) (bottom) responses to incremental exercise. The vertical dotted line indicates the point at which ventilatory threshold is typically achieved during exercise. Note the related temporal responses of CBF and PaCO2 during exercise. Figure adapted from (Smith & Ainslie, 2017). 2.2.4.1 Summary The brain’s inability to increase capillary recruitment and relatively small capacity to increase oxygen extraction necessitates large increases in blood flow to support elevations in CMRO2. The increases in cerebral blood flow during submaximal exercise are principally attributed to temporal changes in arterial PCO2 and CMRO2. With maximal exercise, the CMRO2 response is closely related to cerebral oxygen delivery (cerebral blood flow × CaO2) and oxygen extraction (CaO2 – CvO2) rather than cerebral blood flow per se. 2.2.4.2 Techniques to assess cerebral metabolism during dynamic exercise in humans The first experiments to accurately measure cerebral blood flow and metabolism during exercise in humans utilized the nitrous oxide dilution technique developed by Seymour Kety and Carl Schmidt in 1945 (Kety et al., 1945; Kety & Schmidt, 1948a). With this approach, Rest 20% 40% 60% 80% 100%Exercise intensity (%VO2max)030150301503015gCBFCMRO2CaO2PaCO2O2EFCDO2%ΔChangeLITERATURE REVIEW 43 inhalation/intravenous infusion of a freely diffusible tracer (e.g., nitrous oxide, xenon, hydrogen) and serial arterial and internal jugular venous blood sampling (i.e., Fick Principle) allows for calculation of absolute cerebral blood flow. The cerebral arterial-venous difference of inert tracer indicates the rate of appearance and clearance of the tracer from the cerebral circulation, respectively and is used to calculate cerebral blood flow. Cerebral substrate metabolism can thus be calculated as: cerebral metabolic rate (CMR) = cerebral blood flow × arterial-venous content difference of oxygen/glucose/lactate/ketones (Equation 2.2.2). Throughout the 1950s this reliable and robust technique was recognized as the gold-standard method of quantifying cerebral blood flow; however, notably, there are three key considerations: 1) the Kety-Schmidt technique has poor temporal resolution as each steady-state measurement requires serial blood sampling across 10 minutes; 2) there is no indication of regional contributions to total cerebral blood flow; and 3) this technique assumes that venous outflow is symmetrical between both jugular veins, although this assumption is likely inappropriate (Lichtenstein et al., 2001). Within the last decade, Duplex ultrasound has been utilized to quantify regional volumetric blood flow in the extra-cranial circulation (e.g., carotid and vertebral arteries). Duplex ultrasound has excellent temporal resolution and facilitates synchronized beat-by-beat arterial diameter and pulse-wave blood velocity measurements to calculate volumetric blood flow in the anterior and posterior cerebral circulations, responsible for approximately 70% and 30% of total cerebral blood flow, respectively (Thomas et al., 2015a). To address the previously described drawbacks of the Kety-Schmidt technique, cerebral metabolic rate of substrates can be calculated with this non-invasive ultrasound-derived cerebral blood flow – now inclusive of regional differences in flow – paired with direct arterial-venous blood sampling (via the Fick Principle). Table 2-3 outlines the strengths, limitations and utility of various techniques for the measurement of cerebral metabolism in humans. It is important to note that the Kety-Schmidt technique and the Fick Principle paired with Duplex ultrasound derived cerebral blood flow are the only techniques available to quantify cerebral substrate metabolism during whole-body exercise involving large muscle mass. LITERATURE REVIEW 44 Table 2-3. Strengths, limitations, and utility of various techniques for the measurement of cerebral metabolism in humans. Method Strengths Limitations Utility Exercise? Kety-Schmidt 1. Good reliability 2. Robust technique 3. Volumetric flow measurement 1. Poor temporal resolution (requires steady-state) 2. Invasive 3. Requires highly trained personnel Measure arterial-venous cerebral metabolism and global CBF at rest and during steady-state whole-body exercise Yes Fick principle with Duplex ultrasound 1. Excellent temporal resolution 2. High spatial resolution 3. Volumetric flow measurement 1. Reliability largely based on sonography experience 2. Difficulty getting ultrasound measures at higher exercise intensities 3. Extra-cranial assessment 4. Invasive catheterizations Measure arterial-venous cerebral metabolism and regional dynamic CBF responses at rest and during whole-body exercise Yes MRI 1. Excellent reliability 2. High spatial resolution 2. Non-invasive 1. Fair temporal resolution 2. Influenced by changes in CMRO2 3. No volumetric flow measurement High-quality dynamic (BOLD) and steady-state (ASL) volumetric, regional, and global CBF at rest No PET 1. Excellent reliability 2. High-quality images 1. Poor temporal resolution 2. Invasive 3. Unable to measure during whole-body dynamic exercise Optimum technique for regional changes in CBF and cerebral metabolism at rest No NMR spectroscopy 1. Very high reproducibility 2. Wide application for clinical diagnosis 3. Non-invasive 1. Low sensitivity Measure rates of substrate transport and metabolism in the brain No Microdialysis 1. Direct cerebral metabolism within the interstitial space 1. Highly invasive Only applicable in critical care patients (e.g., stroke, TBI) No LITERATURE REVIEW 45 2.2.5 Cerebral substrate oxidation during exercise The absolute change in cerebral metabolic rate and relative changes in oxidative cerebral fuel utilization during incremental exercise have been elegantly reviewed by Smith and Ainslie (2017) and are summarized in Figure 2-17. With incremental exercise to exhaustion, the cerebral metabolic ratios for oxidative glucose and oxidative carbohydrate metabolism (i.e., OGI and OCI, respectively) are progressively attenuated from their stable resting values of approximately 5.7 by up to 40-50% (Dalsgaard et al., 2004c; Smith et al., 2014). OGI = (CaO2 – CvO2) (Glua – Gluv)⁄ Equation 2.2.4 OCI = (CaO2 – CvO2) (Glua – Gluv) + ½ (Laca – Lacv)⁄ Equation 2.2.5 The reduction in OGI and OCI indicates part of the glucose/lactate was not fully oxidized suggesting an increase in anaerobic non-oxidative metabolism; i.e., larger increases in the respective glucose and carbohydrate (glucose + ½ lactate) uptakes relative to oxygen (Dalsgaard et al., 2004c). This reduction in OGI and OCI during exercise appears to be unaffected by changes in arterial lactate availability (Volianitis et al., 2011); however, both OGI and OCI are reportedly altered with severe high-altitude hypoxia (e.g., 5,050 m) (Smith et al., 2014). Collectively, these data indicate that the contribution of oxidative carbohydrate metabolism to cerebral substrate utilization is influenced by the balance between exercise intensity and arterial lactate availability (Smith & Ainslie, 2017). With very intensive rowing exercise, elevated arterial levels of lactate (e.g., >15 mmol/l) contribute upward of 30-40% of total cerebral metabolism (Ide & Secher, 2000; Volianitis et al., 2008). Pre-clinical studies indicate astrocytic glycogen-derived lactate may contribute to cerebral metabolism via lactate oxidation during hypoglycemia experienced with exhaustive exercise (Brown & Ransom, 2007; Matsui et al., 2017). To date, unlike at rest (Figure 2-13 above) no in vivo study in humans has investigated the role of ketone bodies as a substrate for cerebral metabolism during exercise. LITERATURE REVIEW 46 Figure 2-17. Average cerebral metabolic rate and oxidative cerebral fuel utilization during incremental exercise. (A) The cumulative oxidative (CMRglu + ½ CMRlac) and non-oxidative (1/6 CMRO2 – (CMRglu + ½ CMRlac) cerebral metabolic ratio (CMR; oxidative plus non-oxidative in glucose equivalent units) and cerebral oxidative carbohydrate ratio (OCI) during rest, light (20-40% Wmax), moderate (40-60% Wmax) and maximal (100% Wmax) cycling exercise. An increase in CMR indicates an increase in global cerebral metabolism, whereas a reduction in OCI indicates a reduction in oxidative metabolism. (B) the percentage contribution of oxygen, glucose, and lactate trans-cerebral uptake to the CMR. Sizes of the circles are proportional to the percentage difference in CMR from rest. All values are means and SDs calculated from the eight studies quantifying CMR during incremental exercise (Ide et al., 2000; Nybo et al., 2003; Larsen et al., 2008; Rasmussen et al., 2010b; Brassard et al., 2010; Rasmussen et al., 2010a; Fisher et al., 2013; Trangmar et al., 2014). Figure and caption from (Smith & Ainslie, 2017). LITERATURE REVIEW 47 2.2.5.1 Incremental versus steady-state exercise and cerebral metabolism The cerebral metabolic ratio (O2 uptake/glucose + ½ lactate) decreases during prolonged exhaustive exercise where arterial lactate remains low; however, progressive vigorous exercise above lactate threshold facilitates higher cerebral lactate uptake and oxidation due to increases in systemic lactate availability. Glucose is the preferential substrate at rest; yet, oxidation of monocarboxylic acids such as lactate, pyruvate, and ketone bodies can contribute to cerebral metabolism when glucose utilization is restricted and/or when these substrates become available during exhaustive exercise. For example, as shown by Nybo and colleagues (2003), exercise-induced hypoglycemia during prolonged submaximal exercise (via 3 hours of cycling at 60% VO2max with and without glucose supplementation) reduces cerebral glucose uptake and CMRO2 with increases in cerebral b-hydroxybutyrate uptake (Figure 2-18). It is noteworthy that systemic free fatty acids increased by up to 3-fold after 3 hours of cycling exercise without glucose supplementation; however, this did not result in an increase in cerebral arterial-venous free fatty acid exchange (Nybo et al., 2003). These results substantiate that the brain does not rely on fatty acid oxidation to contribute substantially to ATP production (Mitchell et al., 2011; Schönfeld & Reiser, 2013). Overall, the relative contribution of cerebral substrates to fuel utilization during progressive versus steady-state exercise is markedly affected by systemic carbohydrate availability, exercise intensity, and nutritional status. The majority of studies to date have investigated trans-cerebral metabolism during acute (15-30 minutes) incremental/steady-state exercise, therefore, it is difficult to extrapolate these findings to prolonged continuous exercise where marked changes in systemic fuel utilization occur. LITERATURE REVIEW 48 Figure 2-18. The influence of carbohydrate availability on cerebral substrate utilization during prolonged submaximal cycling exercise. (A) Cerebral glucose uptake is reduced with exercise-induced hypoglycemia during prolonged submaximal cycling exercise at 60% VO2max. (B) At the same time, cerebral ketone uptake is markedly increased during exercise with restricted carbohydrate availability. bHB, b-hydroxybutyrate. *P<0.05 versus resting value; +P<0.05 versus carbohydrate trial. Figure redrawn with data from (Nybo et al., 2003). 2.2.5.2 Cerebral metabolic rate of oxygen during exercise Collectively, progressive relative increases in CMRO2 throughout various exercise intensities occur in parallel with elevated cerebral oxygen delivery (i.e., blood flow × CaO2) and cerebral O2 extraction (Smith & Ainslie, 2017). At maximal exercise intensities, CMRO2 is linearly increased by approximately 30% – albeit with considerable reported variability – to prioritize substrate utilization in the face of reductions in cerebral blood flow. This relates to an increase in CMRO2 in excess of cerebral blood flow and emphasizes the importance of adequate cerebral oxygen delivery and extraction at maximal exercise intensities. It is noteworthy that the relative increases in CMRO2 are reflective of the total increase in cerebral metabolism during exercise. The contribution of trans-cerebral oxygen uptake to total cerebral metabolism is reduced by 30% at maximal exercise versus rest, thus emphasizing the increased contribution of anaerobic non-oxidative substrate utilization and a promising role for partial compensation via ketone body oxidation when carbohydrates are restricted. Aa-v glucose (mmol/L)0.30.40.50.60.7Rest 180Exercise duration (min)30*-204812a-v bHB(µmol/L)Rest 180Exercise duration (min)302610CarbohydratePlacebo*+BLITERATURE REVIEW 49 2.2.5.3 Cerebral metabolic rate of glucose during exercise Taken together, in concert with the elevated oxidative metabolism (CMRO2) described above, the cerebral metabolic rate of glucose (CMRglu) is markedly increased from rest to maximal exercise (Figure 2-17). Importantly, the relative increase in glucose utilization is a result of increases in total cerebral metabolism, whereas the relative contribution of glucose to trans-cerebral uptake and total fuel oxidation is reduced with progressive exercise to exhaustion. The data presented in Figure 2-17 summarize eight studies utilizing trans-cerebral arterial venous glucose values (Ide et al., 2000; Nybo et al., 2003; Larsen et al., 2008; Rasmussen et al., 2010b; Brassard et al., 2010; Rasmussen et al., 2010a; Fisher et al., 2013; Trangmar et al., 2014) as well as calculated cerebral blood flow (via Duplex vascular ultrasound) and/or the relative change in cerebral blood velocity derived estimates of blood flow during incremental exercise. It is noteworthy that the elevated CMRglu from rest to maximal exercise was principally attributable to an increased trans-cerebral exchange of glucose (i.e., higher glucose extraction) as these changes were unrelated to the cerebral glucose delivery (Dalsgaard et al., 2004a; Rasmussen et al., 2011b). Indeed, previous studies consistently report glucose delivery at rest and during various exercise intensities (submaximal to exhaustion) to be in excess of cerebral glucose demand (Rasmussen et al., 2011b; Fisher et al., 2013; Trangmar et al., 2014; Smith et al., 2014); therefore, quite unlike lactate availability, these data indicate that glucose uptake does not depend on arterial glucose levels. 2.2.5.4 Cerebral metabolic rate of lactate during exercise At rest, the <10% utilization of lactate does not appreciably contribute to cerebral metabolism; however, cerebral lactate uptake can increase almost 10-fold during progressive exercise to exhaustion (Dalsgaard et al., 2004c; Smith et al., 2014). This exponential rise in cerebral lactate uptake during incremental exercise is closely related to the elevated systemic arterial concentration (Larsen et al., 2008; Fisher et al., 2013). In support of this, van Hall and colleagues (2009) reported a highly significant relationship between arterial lactate availability and lactate extraction at rest and during 30 minutes of cycling at 75% maximal exercise intensity when systemic lactate was previously elevated via intravenous infusion (<1 mmol/l vs. 7 mmol/l). The arterial-venous lactate gradient changes from a small net ‘release’ at rest to a 10 and 17% ‘uptake’ during exercise and recovery, respectively. These increases in cerebral lactate uptake/oxidation occur with corresponding reductions in cerebral glucose uptake, thereby acting to maintain total carbohydrate oxidation (van Hall et al., 2009). LITERATURE REVIEW 50 Although lactate uptake is related to systemic lactate availability, previous studies show that total cerebral lactate uptake is not affected by the maximal arterial lactate value achieved during exhaustive exercise. Smith and colleagues (2014) showed that arterial lactate availability was approximately 50% higher at high altitude versus sea level for a given absolute exercise intensity; however, cerebral lactate metabolism was not different at matched absolute workloads throughout progressive submaximal cycling exercise to exhaustion. These findings are further substantiated by Volianitis and colleagues (2011) who reported that total lactate uptake is not altered when both arterial pH and lactate values (approx. +10 mmol/l) were elevated (via intravenous sodium bicarbonate infusion) during a 2000-meter maximal rowing time trial. Lastly, pre-clinical evidence shows that exercise induces cerebral-specific vascular endothelial growth factor (VEGF) via lactate signalling with direct relevance to exercise-mediated neovascularization in the brain (Morland et al., 2017). 2.2.5.5 Ketone utilization during exercise? Ketone body utilization as an alternative substrate for cerebral metabolism during exercise has received little attention with no in vivo human study to date investigating the relative contribution of cerebral metabolic rate of ketones during exercise. Influential work by Cahill and colleagues have established that at rest over 60% of the metabolic energy needs of the brain can be supplied by ketone bodies rather than glucose with situations of carbohydrate restriction (Owen et al., 1967; Cahill, 1976); these results are further substantiated by the observation that ketone infusion during acute controlled experimental hypoglycemia (<6 hours) lowers counter-regulatory hormone responses and neuroglycopenic symptoms (Amiel et al., 1991; Veneman et al., 1994). As such, ketones may also contribute as a key alternative fuel substrate to glucose during prolonged exhaustive exercise alongside progressive reductions in cerebral glucose oxidation. As previously described, circulating ketones can reach 1-3 mmol/l during 3-6 days fasting (Owen et al., 1969; Garber et al., 1974; Haymond et al., 1982), prolonged exercise (Johnson et al., 1969; Volek et al., 2016), and very high fat ketogenic diet (Langfort et al., 1996; Johnston et al., 2006). Within these conditions of ketosis, ketones can supply between 50-80% of brain energy requirements (Hasselbalch et al., 1994; 1995; Blomqvist et al., 1995; Hasselbalch et al., 1996; Pan et al., 2000). Nevertheless, the existing literature on fuel utilization during exercise has focused almost exclusively on skeletal muscle, which can resynthesize ATP from ketone bodies, and shows that ketone body disposal in skeletal muscle is elevated up to 5-fold during exercise (Hagenfeldt & Wahren, 1968; 1971; Balasse et al., 1978; Féry & Balasse, 1983; Wahren et al., 1984; Féry & Balasse, 1986; 1988). The ketogenic response to fasting LITERATURE REVIEW 51 prioritizes skeletal muscle protein, which would otherwise be catabolized via gluconeogenesis to produce glucose for cerebral substrate utilization (Veech, 2004); this glycogen-sparing effect of ketone metabolism is also advantageous following exhaustive exercise. Lastly, exercise-induced bHB oxidation reportedly improves brain-derived neurotropic factor expression (Sleiman et al., 2016; Marosi et al., 2016), therefore, ketone body utilization may contribute to the therapeutic/neuroprotective effects of exercise (Nay et al., 2021). 2.2.5.6 Summary The cerebral metabolic ratios for oxidative glucose and oxidative carbohydrate metabolism (i.e., OGI and OCI, respectively) are progressively attenuated from their stable resting values of approximately 5.7 by up to 40-50% with incremental exercise to exhaustion; such changes indicate an increase in anaerobic non-oxidative metabolism. As a compensatory action, increases in cerebral lactate uptake/oxidation occur with corresponding reductions in cerebral glucose uptake, thereby acting to maintain total carbohydrate oxidation. At rest, cerebral ketone body utilization increases with situations of carbohydrate restriction and/or increases in systemic ketone levels; therefore, it is likely that ketones may also contribute as a key alternative fuel substrate to glucose during prolonged exhaustive exercise alongside progressive reductions in cerebral glucose oxidation; however, this has yet to be experimentally tested in vivo in humans. 2.2.6 Summary The interaction between cerebral substrate delivery (via cerebral blood flow and arterial substrate content) and trans-cerebral exchange is essential to support fuel utilization in the brain during exercise. The high cerebral metabolic rate, paired with a remarkably restricted capacity for substrate storage (e.g., glycogen <10 µmol/g), insufficient ability to increase oxygen extraction and inability to utilize fat as substrate, necessitates tight regulation of cerebral blood flow to support oxidative metabolism. Future work is needed to ascertain the relative contribution of ketone utilization and/or astrocytic glycogen-derived lactate oxidation during hypoglycemia experienced with exhaustive exercise in vivo in humans. 2.3 High-altitude Exposure to high-altitude provokes an integrative systemic physiological stress in response to hypoxia (Figure 2-19). A previous study by Lundby & Steensberg (2004) has shown that systemic pro-inflammatory stress (in particular IL-6) during exercise in hypoxia is intensity-dependent (Lundby & Steensberg, 2004); however, the interaction between hypoxia and brain LITERATURE REVIEW 52 tissue oxygen extraction during maximal exercise and the implications for inflammatory stress in the brain has not been investigated. A recent meta-analysis substantiates that hypoxic exposure does not evoke a consistent change in carbohydrate/fat utilization during exercise when matched for relative exercise intensity (Griffiths et al., 2019b), thus emphasizing the importance of absolute exercise intensity in addition to nutritional and acclimatization status on preferential substrate utilization at high-altitude (Young et al., 2019; Griffiths et al., 2019a). Further, anecdotal reports suggests that viral or bacterial infections are slow to resolve at high-altitude (Basnyat et al., 2001; Basnyat & Starling, 2015), which is supported by pre-clinical work illustrating sensitization of inflammatory responses to infection as a result of prolonged exposure to hypoxia. How exercise – which alters both systemic fuel utilization and the immune system – affects the brain inflammatory response at high-altitude has yet to be investigated. Figure 2-19. Oxygen transport cascade. The oxygen transport cascade can be interrogated at the whole-body, organ specific, or cellular level. As oxygen travels from the atmosphere to mitochondria, various aspects of transport, exchange, and utilization of oxygen may contribute to localized or systemic reductions in oxygenation; i.e., hypoxia. LITERATURE REVIEW 53 The oxygen transport cascade describes the path of oxygen from the atmosphere to the tissues (Figure 2-20). The overall behaviour of the cascade can be described via the Fick equation which states that oxygen consumption (VO2) = cardiac output (Q) ´ arteriovenous oxygen difference (a-vO2 diff). The implications of oxygen utilization on exercise performance at high-altitude have been expertly summarized (Calbet et al., 2003; González-Alonso & Calbet, 2003; Travers et al., 2022). Figure 2-20. Oxygen delivery cascade - emphasizing importance of O2 utilization in the skeletal muscle for exercise performance. Key aspects of the oxygen delivery cascade to transport oxygen from the environment to cells involve minute ventilation, pulmonary O2 diffusion, cardiac output, circulatory O2 delivery, muscle O2 diffusion, muscle O2 utilization, and muscle ATP turnover. 2.3.1 Energy balance and metabolism at high-altitude It is well-established that body mass is reduced with prolonged exposure to high-altitude, and this effect is exacerbated with increasing severity of altitude and/or duration of stay, largely as a result of a progressive negative energy balance (reviewed in: Luks et al., 2021). The altitude-specific effects which contribute to this energy deficit are discussed next. LITERATURE REVIEW 54 2.3.1.1 Determinants of energy balance The widely accepted view is that energy balance in humans is determined via the interplay between energy intake and energy expenditure. However, energy intake and expenditure are not independent parameters as they are dynamically influenced by each other in addition to homeostatic signals related to weight loss (Hall et al., 2012; Hall & Chow, 2013). With respect to the complexities of energy balance, it is useful to define the determinants of energy expenditure and energy intake. Total daily energy expenditure (TDEE) is dictated by: 1) expenditure for physiological processes, typically referred to as resting energy expenditure or resting metabolic rate (RMR); 2) thermogenesis from the processing of ingested food, referred to as diet-induced thermogenesis (DIT); and 3) energy cost of physical activity or activity-induced energy expenditure; this is further divided into A) non-exercise activity thermogenesis (NEAT) and B) exercise energy expenditure (EEE). Energy intake is influenced by appetite, including the palatability of food, gastrointestinal regulation, and the availability of food. 2.3.1.2 Resting energy expenditure Studies show increases in RMR by approximately 6-27% within the first several days following initial ascent to high-altitude (Nair et al., 1971; Stock et al., 1978; Butterfield et al., 1992). This elevation in RMR is directly proportional to altitude and is reportedly exacerbated by cold exposure. With acclimatization there is a variable reduction in RMR to a lower steady state, but typically remains above sea level values. For example, Butterfield et al. (1992) reported increases in BMR by 27% on day 2 at Pikes Peak (4,300 m) which remained elevated by 17% after 10-21 days of acclimatization (Butterfield et al., 1992). This was consistent with the 28% increase in resting O2 consumption reported in females after three days at Pikes Peak (4,300 m) (Hannon & Sudman, 1973). Further, a study in 16 females at 4,300 m – while standardizing exercise energy expenditure and diet in addition to controlling for menstrual cycle – reported an increase in RMR by 7% after three days at Pikes Peak (Mawson et al., 2000). Although RMR returned to sea level values by day six, an apparent 6% “energy requirement excess” (160 kcal/day above sea level) for weight maintenance and body composition persisted; therefore, it is possible that this energy excess may influence changes in RMR. Lastly, as vigorous activity at high-altitude is typically of short duration, RMR is effectively the largest contributor to TDEE. 2.3.1.3 Diet-induced energy expenditure Diet-induced thermogenesis (DIT) is the increase in metabolic rate observed for several hours following the ingestion of food (de Jonge & Bray, 1997; Westerterp, 2004). This thermic effect of LITERATURE REVIEW 55 food is reflective of the energy cost of digestion, absorption, storage, and metabolic fate of dietary macronutrients (Westerterp, 2004). Due to the ATP requirements for the initial steps of metabolism and storage, there is an apparent macronutrient hierarchy, with proteins requiring a larger energy expenditure increment (20-30% DIT) than carbohydrates (5-10% DIT), which is greater than that of fat (0-3% DIT) (Acheson, 1993; Westerterp, 2004). For typical diet compositions, DIT is approximately 10% of energy intake, which is equivalent to 10% TDEE when a subject is in energy balance. As energy and macronutrient intake are not particularly high at high-altitude, this expenditure likely accounts for less than 10% of TDEE in terms of absolute kcal. 2.3.1.4 Activity-induced energy expenditure Although maximum work rate is reduced at altitude (Pugh et al., 1964; West et al., 1983; Wolfel et al., 1991) and all activity is associated with increased breathlessness and perceived level of exertion compared to sea level, the daily energy expenditure is almost twice that at sea level (Figure 2-21) due to increased physical activity (e.g., several days trekking), increased work of breathing, and RMR. In particular, overall energy intake required to maintain body weight is reportedly increased by approximately 335 kcal at 4,300 m (sea level: 3118 kcal/day vs. high-altitude: 3452 kcal/day) after adjustment for the 17% increase in RMR (Butterfield et al., 1992). Collectively, the high-altitude environment; that is hypoxia, cold exposure, trekking, and food availability/palatability, rather than hypoxia per se, is necessary to induce the increases in RMR and higher energy outputs reported in field studies (reviewed in: Luks et al., 2021). For context, while energy expenditure is nearly two-fold higher at high-altitude compared to sea level (Westerterp et al., 1992; Pulfrey & Jones, 1996; Reynolds et al., 1999), this remains markedly lower than other pursuits such as ski-crossing of the Antarctic (Halsey & Stroud, 2012), ultra-endurance running (Eden & Abernethy, 1994), cycling in the Tour de France (Westerterp et al., 1986), or cross-country ski training (Sjödin et al., 1994) (reviewed in: Luks et al., 2021; Figure 2-21). Importantly, time spent exercising at extreme altitude is typically short by necessity (to avoid altitude illness), therefore, these differences are likely attributable to a lesser duration of time per day spent participating in these activities at high-altitude. LITERATURE REVIEW 56 Figure 2-21. Daily energy expenditures (bars) and distance covered (stars) during a range of prolonged expeditions measured via doubly-labelled water. Examples, from left to right, include climbing Mount Everest, unsupported crossing of the Antarctic, and elite endurance athletes racing or training. Examples in red (Everest ascent and long distance running) are relevant for studies 2 and 3 in terms of high-altitude exposure and endurance training, respectively. See references in text for further details. Figure redrawn and caption from: (Luks et al., 2021). Panel B illustrates the measured resting metabolic rate following 6-8 days at 3,800 m (Study 2 – How does systemic fuel utilization at high-altitude affect the brain inflammatory response to exercise?) and following 14-days of LEA (50% caloric restriction) with 8 hrs/week training (Study 3B – Substrate oxidation, skeletal muscle mitochondrial respiratory capacity, and insulin sensitivity following 14-days low energy availability in endurance-trained female athletes). 2.3.2 Inflammatory response to high-altitude Several studies have reported that candidate pro-inflammatory markers (e.g., IL-1RA, IL-6, C-reactive protein) are upregulated in the systemic circulation during the first few days of acclimatization to high-altitude (Faquin et al., 1992; Hartmann et al., 2000; Eltzschig & Carmeliet, 2011; Julian et al., 2011; Liu et al., 2017). Further, studies have reported immune system sensitization at high-altitude; i.e., exaggerated immune response for a given inflammatory stress (Facco et al., 2005; Feuerecker et al., 2019). Although there are significant interactions between hypoxia and inflammation with the immune system, how the immune/inflammatory responses adapt to high-altitude exposure is less definitive. LITERATURE REVIEW 57 Figure 2-22. The role of inflammation in high-altitude illness. Predicted contributions of inflammation to high-altitude illnesses, erythrocytosis, and immune function. Hypoxic conditions lead to increases in pro-inflammatory mediators, which may play a role in the development of high-altitude illnesses such as acute mountain sickness (AMS), high-altitude pulmonary hypertension (HAPH), high-altitude pulmonary edema (HAPE), and high-altitude cerebral edema (HACE), and erythrocytosis, or modified immune function (A). Panels B and C illustrate the theorized transition between the pathophysiology of AMS and HACE. Importantly, it is controversial whether/how exercise alters pro-inflammatory mediators (A) and therefore the implications of exercise on high-altitude illnesses (B and C). It is suggested that the pathophysiology of AMS involves dilation of intracranial and/or extracranial arteries and veins causing trigeminal vascular system (TVS) activation and the onset of mild headache. Thereafter, an increase in headache intensity and symptom progression is due to sensitization of the TVS due to local or systemic release of inflammatory factors and/or further arterial/venous dilation during periods of sleep due to oxygen desaturation. Arterial and venous dilation causes an increase in intracranial blood volume and decreased cerebral spinal fluid volume, leading to dynamic changes in arterial blood and brain tissue pressure (i.e., intracranial pressure), which may affect glymphatic fluid flux and cause transient or sustained compression of small white matter capillaries and transient ischemia (B). Central adrenergic signaling may affect both glymphatic fluid flux and white matter arteriolar vasoconstriction. Cytotoxic edema establishes an osmotic gradient for the development of ionic edema. The few individuals who develop symptomatic HACE are likely physiologically (lower PaO2 for a given altitude, greater vasodilation, inflammation/pro-inflammatory cytokine release, edema development or impaired clearance) or anatomically (smaller capacity to buffer increases in intracranial blood and water content) limited. Figure and caption redrawn/adapted from: (Pham et al., 2021; Turner et al., 2021). LITERATURE REVIEW 58 2.4 Low energy availability in endurance-trained athletes Low energy availability (LEA) – which describes inadequate energy intake relative to exercise energy expenditure (<30 kcal/kg fat free mass/day) – predicates relative energy deficiency in sports (Logue et al., 2018; Wasserfurth et al., 2020; Areta et al., 2021). The reported prevalence of LEA is high within athletic populations, particularly in female athletes, with reports of 31-60% in endurance sports (Koehler et al., 2013; Melin et al., 2015; Heikura et al., 2018), 53-58% in intermittent sports such as soccer (Braun et al., 2018; Cherian et al., 2018), and 23-31% in power and strength-based sports (Hoch et al., 2009; Sygo et al., 2018; Melin et al., 2019). LEA has a multifactorial etiology, resulting from intentional or unintentional restrictions in energy intake and/or increased energy expenditure (typically related to an increased training volume) (Burke et al., 2018). A frequently used threshold for LEA in females is <30 kcal/ kg FFM/ day (Loucks & Thuma, 2003; Ihle & Loucks, 2004). However, recent studies have suggested that adverse consequences occur with reductions in energy availability but without a universal LEA threshold (Reed et al., 2015; Lieberman et al., 2018; De Souza et al., 2022), therefore, although practically difficult (Heikura et al., 2022), an energy availability range may be better reflective of physiological implications of LEA (Melin et al., 2023). Figure 2-23. Unit-less illustration of an individual’s daily energy balance and energy availability (EA) when exercising and maintaining an energy balance of zero. Daily energy balance (EB) represents energy balance from a biological system while maintaining physiological function. Daily energy availability (EA) represents the energy available to a biological system for maintaining physiological function. This schematic illustrates that energy intake and exercise energy expenditure (EEE) are accounted for equally in EB and EA, but EB accounts for all other factors of energy expenditure. The illustration represents parameters of a hypothetical case of an individual performing approximately 1 hour of purposeful exercise, with energy expenditure from dietary induced thermogenesis and non-exercise activity thermogenesis that are 10% that of energy intake and 40% of resting metabolic rate, respectively. Cold-induced thermogenesis is not incorporated in this illustration due to minimal energy expenditure in thermo-neutral conditions, though in cold conditions contribution to energy expenditure can be significant. Figure redrawn and caption adapted from (Areta et al., 2021). Abbreviations: EA, energy availability; EEE, exercise energy expenditure; NEAT, non-exercise activity thermogenesis; DIT, dietary induced thermogenesis; RMR, resting metabolic rate. LITERATURE REVIEW 59 Figure 2-24. Systemic effects of short-term (3-5 days) low energy availability in females. Schematic of the effects of short-term (3-5 days) low energy availability (LEA) on hormones, blood-borne substrates and skeletal muscle as evidenced by prospective studies. LEA is a powerful stressor that triggers marked hormonal and metabolic responses. Downregulation of key energy homeostasis-related adipokine leptin may lay upstream of and partially regulate the hypothalamic-pituitary-thyroid, hypothalamic-pituitary-gonadal and GH-IGF-1 axes. LEA also regulates markers of bone formation (decrease), resorption (increase), substrate availability and reduces skeletal muscle protein synthesis. Different tissues/systems are affected in different ways, and their response appears to vary between males and females, potentially due to divergent susceptibility to different levels of LEA in different tissues and sexes. Figure redrawn and caption adapted from (Areta et al., 2021). Abbreviations: b-HOB, beta hydroxybutyrate; FSH, follicle stimulating hormone; GH, growth hormone; IGF-1, insulin-like growth-factor 1; LH, luteinizing hormone, T3, triiodothyronine. Bone formation markers refers to osteocalcin, carboxy-terminal propeptide of type 1 procollagen (P1CP) and N-terminal propeptide of type 1 procollagen (P1NP). Bone resorption markers refers to C-terminal telopeptide of type 1 collagen (b-CTX) and aminoterminal telopeptide of type 1 collagen (NTx). 2.4.1 The immune system Studies have shown that strenuous prolonged exercise training with LEA in normal weight individuals may suppress the immune system (Gleeson et al., 2004; Gleeson, 2007; 2016; Bermon et al., 2017; Peake et al., 2017). These studies indicate an increased susceptibility to infection due to altered immunity within 72 hours after exercise – in addition to longer-term 1. Loucks & Heath (1994b), 2. Loucks, Verdun and Heath (1998), 3. Loucks & Thuma (2003), 4. Loucks & Verdun (1998), 5. Loucks (2006), 6. Areta et al (2014), 7. Ihle & Loucks et al (2004), 8. Papageorgiou et al (2018), 9. Papageorgiou et al (2017), 10. Loucks & Heath (1994b), 11. Loucks & Callister (1993), 12. Murphy & Koehler (2020), 13. Hilton & Loucks (2000)LITERATURE REVIEW 60 reductions in systemic immunity such as studies assessed prospectively across a 7-month intensive training season in elite swimmers (Gleeson et al., 1995). Overall, it appears that downregulated innate immunity responses by suppressed release of inflammatory markers and overall chemokine production is evident after prolonged energy restriction with intense exercise (Sarin et al., 2019). This is particularly relevant due to the high prevalence of upper respiratory tract infections in elite athletes, either immediately prior to or during competition (Peters & Bateman, 1983; Peters, 1997; Spence et al., 2007; Cox et al., 2008; Peters et al., 2010). Figure 2-25. Cytokine network. Immune cells communicate with other immune cells by cytokines, which control proliferation, differentiation, and function. Further, cytokines are involved in processes of inflammation, hematopoiesis, neurogenesis, embryogenesis, and oncogenesis. Unlike hormones, cytokines are not stored in glands as pre-formed molecules but are rapidly synthesized and secreted mostly after stimulation. Cytokines frequently affect the action of other cytokines in an additive, synergistic, or antagonistic manner. Figure and caption from Thermo Fisher Scientific. LITERATURE REVIEW 61 2.4.2 Systemic inflammatory response Exercise provokes the release of a cascade of cytokines (Pedersen & Hoffman-Goetz, 2000; Ostrowski et al., 2001) – in particular IL-6, which is a pleiotropic cytokine with important actions to regulate energy status and inflammation (Pedersen et al., 2001; Febbraio & Pedersen, 2002; Pedersen et al., 2003). Early studies investigating exercise and immunological parameters indicate that the profiles of pro- and anti- inflammatory cytokines are influenced by the intensity and duration of exercise (Pedersen & Toft, 2000; Pedersen, 2000). For example, marathon runners reportedly show high post-exercise levels of IL-1b, IL-6, and IL-8 after six hours endurance exercise (Drenth et al., 1995; Suzuki et al., 2000; Pedersen, 2000), while there were no changes in systemic cytokines after a 5 km run (Drenth et al., 1998). Table 2-4 summarizes selected cytokines and their functions. LITERATURE REVIEW 62 Table 2-4. Summary of selected cytokines and their functions. Cytokine Classification Main Sources Receptor Target Cell Major Function Erythropoietin Endothelium EpoR Stem cells Red blood cell production G-CSF Pro-inflammatory Fibroblasts, endothelium CD114 Stem cells in BM Granulocyte production GM-CSF Adaptive immunity T cells, macrophages, fibroblasts CD116, CDw131 Stem cells Growth and differentiation of monocytes and eosinophil, granulocytes production IL-1 Pro-inflammatory Macrophages, B cells, DCs CD121a B cells, NK cells, T-cells Pyrogenic, pro-inflammatory, proliferation and differentiation, BM cell proliferation IL-2 Adaptive immunity Th1 cells CD25 Activated T and B cells, NK cells Proliferation of B cells, activated T cells, NK cell function IL-3 Adaptive immunity T cells CD123, CDw131 Stem cells Hematopoietic precursor proliferation and differentiation IL-4 Adaptive immunity Th cells CD124 B cell, T cell, macrophages Proliferation of B and cytotoxic T cells, enhances MHC class II expression, stimulates IgG and IgE production IL-5 Adaptive immunity Th2 cells and mast cells CDw125, 131 Eosinophils, B-cells B-cell proliferation and maturation, stimulates IgA and IgM production IL-6 Pro-inflammatory Th cells, macrophages, fibroblasts CD126, 130 B-cells, plasma cells B-cell differentiation IL-7 Adaptive immunity BM stromal cells, epithelial cells CD127 Stem cells B and T cell growth factor IL-8 Pro-inflammatory Macrophages IL-8R Neutrophils Chemotaxis for neutrophils and T cells IL-9 Adaptive immunity T cells IL-9R, CD132 T cell Growth and proliferation IL-10 Anti-inflammatory T cells, B cells, macrophages CDw210 B cells, macrophages Inhibits cytokine production and mononuclear cell function IL-11 Pro-inflammatory BM stromal cells IL-11Ra, CD130 B cells Differentiation, induces acute phase proteins LITERATURE REVIEW 63 Cytokine Classification Main Sources Receptor Target Cell Major Function IL-12 Anti-inflammatory T cells, macrophages, monocytes CD212 NK cells, macrophages, tumor cells Activates NK cells, phagocyte cell activation, endotoxic shock, tumor cytotoxicity, cachexia IL-17 Pro-inflammatory Th17 cells IL-17R Monocytes, neutrophils Recruits monocytes and neutrophils to the site of infection. Activation of IL-17 in turn activates downstream of many cytokines and chemokine, such as IL-1, IL-6, IL-8, IL-21, TNF-b, and MCP-1 IL-18 Pro-inflammatory Macrophages, dendritic cells, and epithelial cells CD218a (IL-18Ra) Monocytes and T cells Recruits monocytes and T lymphocytes. Synergist with IL-12 in the induction of IFN-g production and inhibition of angiogenesis. IL-22 Anti-inflammatory Activated T-cells and NK cells IL-22R Stromal and epithelial cells Stimulation of cell survival, proliferation IL-37 (1L-1F7) Anti-inflammatory B-cells, NK cells, and monocytes CD218a (IL-18Ra) and potentially SIGGR Believed to act as a negative regulator inside the cell where it interacts with SMAD3 that is activated downstream of TGFb activity. IL-38 (IL-1F10) Anti-inflammatory B cells and macrophages IL-1R1 Unknown IFN-a Pro-inflammatory Macrophages, neutrophils, and some somatic cells CD118 (IFNAR1, IFNAR2) Various Anti-viral IFN-b Pro-inflammatory Fibroblasts CD118 (IFNAR1, IFNAR2) Various Anti-viral, anti-proliferative IFN-g Pro-inflammatory T cells and NK cells CDw119 (IFNG R1) Various Anti-viral, macrophage activation, increases neutrophil and monocyte function, MHC-I and -II expression on cells M-CSF Adaptive immunity Fibroblasts, endothelium CD115 Stem cells Monocyte production and activation TGF-b Anti-inflammatory T cells and B cells TGF-bR1, 2, 3 Activated T and B cells Inhibits T and B cell proliferation, inhibits hematopoiesis, promotes wound healing LITERATURE REVIEW 64 Cytokine Classification Main Sources Receptor Target Cell Major Function TNF-a Pro-inflammatory Macrophages CD120a,b Macrophages Phagocyte cell activation, endotoxic shock TNF-b Pro-inflammatory T cells CD120a,b Phagocytes, tumor cells Chemotactic, phagocytosis, oncostatic, induces other cytokines Abbreviations: IL; interleukin, TNF; tumor necrosis factor, IFN; interferon, G-CSF; granulocyte colony stimulating factor, GM-CSF; granulocyte macrophage colony stimulating factor, M-CSF; macrophage colony stimulating factor, TGF; transforming growth factor, CD; cluster of differentiation; CDw; cluster of differentiation designated by only one monoclonal antibody, BM; bone marrow, DC; dendritic cells. Table and caption from Thermo Fisher Scientific. LITERATURE REVIEW 65 2.4.3 Exercise performance Reductions in total body mass are often viewed as a favorable result of LEA. Research supports a relationship between lower body mass and faster competition times for novice and experienced marathon runners (Hagan et al., 1981; Holly et al., 1986; Hagan et al., 1987; Hoffman, 2008; Knechtle et al., 2009a). Further, low body fat specifically is related to faster race times (Bale et al., 1985; 1986; Knechtle et al., 2009b; 2011a). However, this is not universal with studies reporting no relationship between body composition and performance in recreational ultra-triathletes, recreational ultra-runners, and ultra-endurance cyclists (Knechtle et al., 2011c; 2011b). The implications of intentional LEA for performance improvements have severe systemic implications for multi-organ regulatory systems (Figure 2-24). 2.4.4 Systemic substrate oxidation The systemic substrate effects of LEA include lower circulating insulin and glucose with higher free fatty acids (FFAs), glycerol, cholesterol, and b-hydroxybutyrate (b-OHB) (Loucks & Thuma, 2003; Koehler et al., 2016). Taken together, increases in lipolytic activity and reductions in anaerobic glycolysis are an adaptive response with LEA to preserve carbohydrate, which is likely a consequence of limited glycogen reserves (Kojima et al., 2020). A theorized schematic of LEA-induced preferential fat oxidation during exercise is illustrated in Figure 2-26. Further, the implications of a larger reliance on fat oxidation for impairments in high-intensity aerobic exercise performance are shown in Figure 2-27. Figure 2-26. Relative contribution of carbohydrate and lipid to total energy expenditure with progressive increases in relative exercise intensity to max. LITERATURE REVIEW 66 Theorized schematic illustrating LEA-induced preferential reliance on fat oxidation during exercise due to altered substrate availability (i.e., higher free fatty acids and ketone bodies due to increases in lipolytic activity; reductions in glucose/glycogen). Figure 2-27. Interaction between fat oxidation and glycolysis; competition for substrate and inhibition of glucose utilization with high-intensity aerobic exercise. 2.4.4.1 Skeletal muscle mitochondrial respiratory capacity Whole-body maximal oxygen uptake (VO2max) is both positively related to mitochondrial respiratory capacity (van der Zwaard et al., 2016) and a proxy for metabolic health (Lee et al., 2011). Further, ‘overreaching’ with training in the absence of LEA can adversely affect skeletal LITERATURE REVIEW 67 muscle as mitochondrial oxidative capacity is impaired with 4-weeks progressively excessive endurance training (Flockhart et al., 2021). Therefore, it is important to investigate prolonged LEA with high endurance training volume and the related implications on skeletal muscle respiratory capacity. 2.4.4.2 Insulin sensitivity and glucose control Recent studies have shown that excessive exercise training reduces glucose tolerance and insulin sensitivity (via OGTT) in addition to the glucoregulatory response to intense exercise (Flockhart et al., 2021; 2023). Further, these elite endurance athletes exhibited an impaired glucose control during free-living conditions as evidenced by increased time spent outside of the normoglycemic range (4-8 mmol/l) compared to healthy national-level endurance athletes (Flockhart et al., 2021). Additionally, a study by Pestell et al., (1993) showed that impaired glucose tolerance in highly-trained athletes within 2 hours of completing a 6-day ultramarathon race was related to reduced insulin secretion rather than altered insulin sensitivity (Pestell et al., 1993). Further, prioritization of glycogen resynthesis is implicated in the regulation of exercise-induced alterations in insulin sensitivity (Jensen et al., 2011; Hingst et al., 2022). It is therefore necessary to investigate persistent LEA with high training volume on glucose control, glycogen status, and insulin sensitivity in endurance-trained individuals. FICK DERIVED CMRO2 WITH ALTERED PaCO2 68 Chapter 3: Study 1 – Evidence for direct CO2-mediated alterations in cerebral oxidative metabolism in humans Abstract Background: The cerebral metabolic rates of oxygen and glucose utilization (CMRO2 and CMRGlu, respectively) are vital physiologic factors which may influence cerebrovascular CO2 reactivity (CVR) and related brain health in humans. How CMRO2 and CMRGlu are affected by alterations in arterial PCO2 (PaCO2) is equivocal and therefore was the primary question of this study. Methods: This retrospective analysis involved pooled data from four separate studies, involving 41 healthy adults (35 males/ 6 females). Participants completed stepwise steady-state alterations in PaCO2 ranging between 30 to 60 mmHg. The CMRO2 and CMRGlu were assessed via the Fick approach (CBF ´ arterial-internal jugular venous difference of oxygen or glucose content, respectively) utilizing duplex ultrasound of the internal carotid artery and vertebral artery to calculate cerebral blood flow (CBF). Results: The CMRO2 was altered by 0.5 ml × min-1 (95% CI: -0.6 to -0.3) per mmHg change in PaCO2 (P < 0.001) which corresponded to a 9.8% (95% CI: -13.2 to -6.5) change in CMRO2 with a 9 mmHg change in PaCO2 (inclusive of hypo- and hypercapnia). The CMRGlu was reduced by 7.7% (95% CI: -15.4 to -0.08, P = 0.045; i.e., reduction in net glucose uptake) and the oxidative glucose index (OGI; ratio of oxygen to glucose uptake) was reduced by 5.6% (95% CI: -11.2 to 0.06, P = 0.049) with a +9 mmHg increase in PaCO2. Conclusions: The CMRO2 is altered by approximately 1% per mmHg change in PaCO2. Further, glucose is not fully oxidized during hypercapnia, indicating reductions in CMRO2 are either met by a compensatory contribution of anaerobic metabolic pathways or explained by a reduction in total brain metabolism. These results provide new insights into the influence of PaCO2 on CMRO2 in humans and have important physiological and clinical implications for the assessment of CVR in health and disease. 3.1 Introduction As the brain’s storage capacity for oxygen is exceptionally small relative to its rate of utilization – paired with a severely restricted glycolytic capacity (e.g., glycogen <10 µmol/g) and inability to utilize fat as a fuel substrate – the brain requires tight regulation of blood flow (Siesjö, 1978; Gjedde et al., 2002; Brown & Ransom, 2007). Increases in the partial pressure of arterial carbon dioxide (PaCO2; hypercapnia) lead to rapid diffusion of CO2 through the vessel wall to reduce extracellular pH within the perivascular space (Lambertsen et al., 1961; Kontos et al., 1977b; FICK DERIVED CMRO2 WITH ALTERED PaCO2 69 1977a; Caldwell et al., 2021a; 2021b), which increases cerebral blood flow (CBF) (Lennox & Gibbs, 1932; Shapiro et al., 1966; Carr et al., 2022). For example, CBF increases by 6-8% per mmHg increase in PaCO2 (Kety & Schmidt, 1948b; Willie et al., 2012; Hoiland et al., 2019); and such mild-to-moderate hypercapnia is practical, typically safe and well-tolerated, and is therefore a popular stressor to provoke increases in CBF to assess cerebrovascular reactivity (CVR). Measures of CVR are used extensively, both in healthy and clinical populations, to assess the vasodilatory capacity of the cerebrovasculature (Barnes et al., 2013; Fisher, 2016; Fisher & Mikulis, 2021) and cerebrovascular health (Portegies et al., 2014; Wolters et al., 2016; Fisher et al., 2018; Hoiland et al., 2019; Sobczyk et al., 2021). A vital determinant of CBF that is rarely considered during CVR testing is the cerebral metabolic rate of oxygen (i.e., oxidative glucose metabolism; CMRO2). Any alterations in cerebral metabolism directly affect CBF regulation reflective of appropriate substrate delivery (Himwich & Homburger, 1947; Kety & Schmidt, 1948b; Siesjö, 1978; Madsen & Vorstrup, 1991; Buxton & Frank, 1997). However, the sole attribution of CVR-mediated changes in CBF to PaCO2-dependent alterations in cerebrovascular tone is predicated on the assumption that CMRO2 is unaltered with changes in PaCO2. Pre-clinical work on this topic is inconclusive with studies reporting that CMRO2 is unaltered (Alberti et al., 1975), reduced (Kogure et al., 1975; Kliefoth et al., 1979; Berntman et al., 1979; Artru & Michenfelder, 1980), or increased (Hemmingsen et al., 1979; Horvath et al., 1994; Yang & Krasney, 1995) with hypercapnia. Interpretation of this pre-clinical work is further muddied by the use of anesthesia and supraphysiological hypercapnia (e.g., PaCO2 80-100 mmHg) that cannot be replicated in awake humans; i.e., exceeding upper limits of tolerability (Willie et al., 2012), reviewed in: (Siesjö, 1980; Yablonskiy, 2011). Recent experiments in healthy and unanesthetized humans report reductions in CMRO2 by 13% in response to +4 to 9 mmHg PaCO2 during inspiration of 5% CO2 (Xu et al., 2011; Deckers et al., 2022). Further, an 18 mmHg reduction in PaCO2 reportedly increases CMRO2 by 14% (Kety & Schmidt, 1946). However, these findings are not universal as studies have reported unaltered CMRO2 in response +9 mmHg with both the Fick technique (Kety & Schmidt, 1948b) and MRI-derived metrics (Chen & Pike, 2010a; Jain et al., 2011). Inconsistency in these results is likely explained by small sample sizes (n £ 10) and a mixture of fixed inspired CO2 (which obviates accurate PaCO2 targeting) and indirect surrogate calculations of jugular venous O2 saturation. As the relationship between PaCO2 and CMRO2 remains equivocal, we sought to investigate the influence of PaCO2 on CMRO2 by directly quantifying CMRO2 with the Fick technique using FICK DERIVED CMRO2 WITH ALTERED PaCO2 70 a larger sample size compared to prior studies. The objective of the current analysis was to test the hypothesis that CMRO2 would be directly affected by changes in PaCO2 such that CMRO2 would be reduced with progressive increases in PaCO2, and vice-versa. Further, to compensate for the expected reduction in CMRO2 during hypercapnia, we reasoned that the cerebral metabolic ratios for oxidative glucose and carbohydrate metabolism (i.e., OGI and OCI, respectively) would also be reduced. This latter observation would indicate that part of the glucose/lactate is not fully oxidized thereby indicating an increase in non-oxidative metabolism via anaerobic glycolysis and lactate oxidation. 3.2 Methods Ethical Approval All participants provided written informed consent before participating in these studies. The experiments presented herein were approved by the University of British Columbia Clinical Research Ethics Board (CREB: H16-01028, H18-01755, H15-00166, H21-00773) and conducted according to the principles established by the Declaration of Helsinki, except for prior registration in a database. Participants These data were collated from four separate experimental studies previously conducted within the Centre for Heart, Lung and Vascular Health at the University of British Columbia, Kelowna, British Columbia, Canada. In total, 41 healthy adults were included in this study (n=35 males; n=6 females). Participants were included if they were 18 years of age or older, had no reported history of cerebrovascular, cardiovascular, or respiratory disease and were not taking any prescription medication at their time of participation. Participants were instructed to refrain from alcohol and caffeine consumption as well as vigorous exercise or activity for at least 12 hours prior to testing. Timing of menstrual cycle and use of contraceptives for the female participants was recorded but not controlled. Females were either naturally menstruating (n=2), using a contraceptive implant (n=2), or taking oral contraception (n=2). Experimental Overview The hypotheses addressed in this study involved retrospective analysis of participants (n=27) from previously published experiments, while others have not been previously reported on (n=14) (Bain et al., 2020; Hoiland et al., 2022; Carr et al., 2022). The current experimental FICK DERIVED CMRO2 WITH ALTERED PaCO2 71 questions, however, involved new objectives and data analyses that have not been previously reported. To directly quantify the CMRO2 with the Fick technique, each participant underwent measurements of CBF alongside simultaneous collection of arterial (brain inflow) and internal jugular bulb (brain venous outflow) blood samples (see below; Figure 3-1). Participants completed stepwise alterations in PaCO2 while maintaining iso-oxia with the following protocols: study 1) +3, +6, +9 mmHg PaCO2 (n = 11 males); study 2) +4.5 and +9 mmHg PaCO2 (n = 12 males); study 3) +10 and +20 mmHg PaCO2 (n = 4 males); and study 4) +9 mmHg PaCO2 (n = 6 females; n = 8 males). A subset of n = 11 participants from study 4 completed -10 mmHg PaCO2 (n = 5 females; n = 6 males); as the sensitivity of these separately – but linearly – related CVR responses was not evidently different, all data was included in the group analysis to test the effect of PaCO2 on CMRO2. The alterations in PaCO2 were calculated individually from the resting room air breathing end-tidal values for each participant. All cardiorespiratory and cerebrovascular measures and blood sampling were performed following confirmation that PaCO2 had been effectively clamped to the target value for a steady-state period of approximately 4-5 minutes (Carr et al., 2021b). Catheterization While resting supine, catheterization of the radial artery and internal jugular vein was performed using sterile technique, under local anesthesia (Lidocaine, 1.0%), and with ultrasound guidance. A 20G arterial catheter (Arrow, Markham, ON, Canada) was placed in the radial artery and a 13G central venous catheter (Cook Medical, Bloomington, IN, USA) was inserted into the internal jugular vein and advanced 15 cm cephalad to the jugular bulb. The same technique, performed by our group has been previously shown to lead to catheter tip placement in the jugular bulb, superior to the facial vein, with correct placement confirmed radiographically (Ainslie et al., 2014). Further, fulfillment of these techniques leads to <3% contamination by extra-cerebral (facial) blood (Schell & Cole, 2000). These catheters were attached to in-line waste-less blood sampling systems for repeated trans-cerebral blood sampling (Edwards Lifesciences, TruWave VAMP, CA, USA). This technique has been successfully performed by our group and has been previously described in detail elsewhere (Schell & Cole, 2000; Ainslie et al., 2014). Blood Sampling FICK DERIVED CMRO2 WITH ALTERED PaCO2 72 Approximately 1.0 ml of radial arterial and internal jugular venous blood were drawn simultaneously into pre-heparinized syringes (SafePICO, Radiometer, Copenhagen, Denmark) and analyzed immediately using a commercial blood gas analyzer (ABL90 FLEX, Radiometer) at each experimental stage. This analysis included measurements of the partial pressures of arterial and venous carbon dioxide (Pa/vCO2) and oxygen (Pa/vO2), oxygen saturation (Sa/vO2), oxygen and glucose content (Ca/vO2 and Glua/v, respectively), calculated bicarbonate ion concentration ([HCO3-]), base excess [Base(B)], hydrogen ion concentration ([H+]), hemoglobin concentration ([Hb]), and pH. Cardiorespiratory Detailed cardiorespiratory experimental methods and results have been previously reported elsewhere; e.g., (Bain et al., 2020; Hoiland et al., 2022; 2023). Briefly, PaCO2 and PaO2 were targeted via alteration of the partial pressures of end-tidal CO2 and O2 (i.e., PETCO2 and PETO2, respectively) with a computerized gas control system that effectively regulates end-tidal gases across wide ranges of PETCO2 and PETO2 (Tymko et al., 2015; 2016), independent of ventilation (V̇E) (Howe et al., 2020). Intravascular blood pressure was measured beat-by-beat from the radial artery, with pressure corrected to the height of the right atrium (Edwards Lifesciences, TruWave VAMP, CA, USA). Cerebrovascular Detailed cerebrovascular experimental methods and results have been previously reported elsewhere; e.g., (Bain et al., 2020; Hoiland et al., 2022; 2023). Total CBF was measured via extra-cranial blood velocity and diameter of the internal carotid artery (ICA) and vertebral artery (VA) using a 10-MHz multifrequency linear array duplex ultrasound (Terason uSmart 3300; Teratech, Burlington, MA, USA). Pulse-wave mode was used to measure beat-to-beat peak blood velocity and arterial diameter was instantaneously measured via B-mode imaging; data were analyzed using custom edge-detection and wall tracking software (BloodFlow Analysis, version 5.1). The vessel location was decided on an individual participant basis to allow for high-quality image acquisition, with the same location and consistent insonation angle (60°) repeated within participants and between trials. Calculations Arterial and venous oxygen content (CaO2 and CvO2, respectively) were calculated as: FICK DERIVED CMRO2 WITH ALTERED PaCO2 73 CaO2 (ml × dl-1) = [Hb] × 1.34 × [SaO2 (%) 100]⁄ + 0.003 × PaO2 Equation 3.1 CvO2 (ml × dl-1) = [Hb] × 1.34 × [SvO2 (%) 100]⁄ + 0.003 × PvO2 Equation 3.2 Where 1.34 is the O2 binding capacity of hemoglobin and 0.003 is the solubility of O2 dissolved in blood (Lumb, 2016; West & Luks, 2020). The relationship between PaCO2 and brain tissue PCO2 (PbtCO2) is influenced by CBF, where increases in CBF lead to a reduction in the difference between PaCO2 and PbtCO2. Therefore, PjvCO2 (CO2 washout), which is in pseudo equilibrium with PbtCO2 is a better representative of the actual PCO2 stimulus (Bradley & Semple, 1962; Bradley et al., 1965; Pontén & Siesjö, 1966; Shapiro et al., 1966; Friis et al., 1980; Hoiland et al., 2019; Carr et al., 2021a; 2022). An index of PbtCO2 was calculated using the below equation as previously explained and validated by Pontén & Siesjö (1966) in cat experiments at PaCO2’s of 30 to 90 mmHg: PbtCO2 index (mmHg) = [(PaCO2 + PjvCO2) 2]⁄ + 1 Equation 3.3 Blood flow (Q) was calculated as: Q (ml × min-1) = peak envelope blood velocity 2⁄ × (p(0.5 × diameter)2) × 60 Equation 3.4 With the assumption of symmetrical blood flow of contralateral ICA and VA, total cerebral blood flow (CBF) was calculated as: CBF (ml × min-1) = 2 × (QICA + QVA) Equation 3.5 Cerebral oxygen delivery (CDO2) was calculated as: CDO2 (ml × min-1) = CBF (ml × min-1) 100⁄ × CaO2 (ml × dl-1) Equation 3.6 Cerebral oxygen extraction fraction was calculated as: O2 extraction (%) = CaO2 – CvO2 CaO2⁄ × 100 Equation 3.7 Cerebral metabolic rate of oxygen (CMRO2) was calculated as: CMRO2 (ml × min-1) = CBF (ml × min-1) 100⁄ × CaO2 – CvO2 (ml × dl-1) Equation 3.8 FICK DERIVED CMRO2 WITH ALTERED PaCO2 74 The cerebral metabolic rate of glucose and lactate (CMRGlu and CMRLac, respectively) were calculated by the trans-cerebral exchange of arterial (a) and venous (v) values: CMRGlu (mmol × min-1) = (Glua - Gluv) (mmol × l-1) × CBF (ml × min-1) 1000⁄ Equation 3.9 CMRLac (mmol × min-1) = (Laca - Lacv) (mmol × l-1) × CBF (ml × min-1) 1000⁄ Equation 3.10 Where a positive value indicates a net uptake and a negative value indicates a net release (Groeneveld, 1998). Cerebral glucose delivery (CDGlu) was calculated as: CDGlu (mmol × min-1) = CBF (ml × min-1) 1000⁄ × Glua (mmol × l-1) Equation 3.11 Cerebral lactate delivery (CDLac) was calculated as: CDLac (mmol × min-1) = CBF (ml × min-1) 1000⁄ × Laca (mmol × l-1) Equation 3.12 The stable resting cerebral metabolic ratio for oxidative glucose metabolism (often termed OGI) is approximately 5.5 (Dalsgaard et al., 2004c; Smith et al., 2014; Blazey et al., 2018). This value indicates near complete oxidation of glucose: 6 O2 + C6 H12O6 (glucose) ® 6 H2O + 6 CO2; approximately 5-10% of the glucose taken up contributes to glycolysis as evidenced by a slight lactate efflux at rest (Dalsgaard et al., 2004c; van Hall et al., 2009). Oxidative glucose index was calculated as: OGI (arbitrary units) = (CaO2 – CvO2) (Glua – Gluv)⁄ Equation 3.13 Theoretically, OGI values at a 1:1 ratio of oxygen to glucose uptake would equal 6, indicating that all glucose is consumed by oxidative pathways, as the stoichiometry for the oxidation of glucose by oxygen requires one oxygen molecule per each of the 6 carbon atoms in glucose. Oxidative carbohydrate index was calculated as: OCI (arbitrary units) = (CaO2 – CvO2) (Glua – Gluv) + ½ (Laca – Lacv)⁄ Equation 3.14 The OCI indicates the ratio of oxygen to carbohydrate (glucose and lactate) uptake. The carboxylation of lactate to pyruvate into the tricarboxylic acid cycle results in only one molecule of pyruvate versus the two derived from the breakdown of glucose to pyruvate during glycolysis. FICK DERIVED CMRO2 WITH ALTERED PaCO2 75 The OCI in the resting human brain is approximately 5.6 (Blazey et al., 2018), suggesting that lactate efflux does not account for all non-oxidative glucose consumption. A reduction in OGI and OCI indicates that part of the glucose/lactate was not fully oxidized indicating an increase in non-oxidative metabolism; i.e., larger increases in the respective glucose and carbohydrate (glucose + ½ lactate) uptakes relative to oxygen (Dalsgaard et al., 2004c). Figure 3-1. Schematic of gold-standard measurement of brain metabolism via the Fick approach by trans-cerebral internal jugular venous (IJV) and radial arterial blood sampling Quantification of global cerebral blood flow (CBF) via Duplex ultrasound of the internal carotid artery (ICA) (A) and vertebral artery (VA) (B) and rapid sampling of trans-cerebral radial arterial (D) and internal jugular venous (C) blood provides a direct index of total cerebral metabolism via the Fick equation: CMRO2 (ml/min) = CBF (ml/100g/min-1) × (CaO2 – CvO2) where CMRO2, cerebral metabolic rate for oxygen; CaO2, arterial oxygen content; CvO2, cerebral venous oxygen content. 3.3 Statistical analyses All data are presented in-text as mean with 95% confidence intervals. Statistical analyses were performed using R (Version 4.2.1, R Core Team, 2022). Statistical significance was set at P < FICK DERIVED CMRO2 WITH ALTERED PaCO2 76 0.05. Relationships between select variables were analyzed with a linear mixed-effects model with fixed effects for sex and either PaCO2, PbtCO2 index, arterial pH, or IJV pH with the following key dependent variables: CMRO2, OGI, OCI, O2 extraction, CMRGlu, CMRLac, CDO2, and V̇CO2 (lme4, Version 1.1-30, Bates et al., 2022). Subject ID was included as a random intercept to account for repeated measures within participants. Model assumptions were checked via visual inspection of fitted values vs. residuals plots and normal-probability plots. If warranted to satisfy model assumptions, outcome values were log-transformed for the analysis. As a result of zero values for CMRLac (due to no differences between arterial-venous lactate exchange), it was not possible to log transform the residuals; therefore, non-parametric bootstrap analysis using 2000 resamples with replacement was performed and bias-corrected and accelerated 95% confidence intervals were calculated (boot, Version 1.3-28.1, Davison & Hinkley, 1997; Canty & Ripley, 2021). As such, no P-value is reported for the CMRLac vs. PaCO2 comparison and sex was not included as a fixed effect due to the high probability of resamples including only one sex. For OGI and OCI, outliers were identified as > upper quartile + 2.2 ´ interquartile range and < lower quartile – 2.2 ´ interquartile range (Hoaglin & Iglewicz, 1987); the excluded data corresponded to values considered unphysiological. All comparisons have 142 observations, except OGI and OCI which include 136. An exploratory linear mixed-effects analysis was performed with fixed effects of PaCO2, sex (male vs. female) and arterial Hb. Statistical mediation analysis was performed to investigate the extent to which the main effect of sex on CMRO2 was mediated through arterial Hb. This analysis separated the total effect of sex on CMRO2 into the average direct effect (ADE) and average casually-mediated effect (ACME) that is mediated by arterial Hb; the proportion mediated effect of arterial Hb was calculated as ACME divided by the total effect (mediation, Version 4.5.0, Tingley et al., 2019). To provide context to the results, estimated marginal means at the level of the observed baseline PaCO2 (i.e., 42.6 mmHg) and 51.6 mmHg (i.e., +9 mmHg) for CMRO2, OGI, O2 extraction, CDO2, and carbon dioxide production (V̇CO2) and the corresponding mean differences were calculated (emmeans, Version 1.7.0, Lenth, 2021) and presented with 95% confidence intervals. FICK DERIVED CMRO2 WITH ALTERED PaCO2 77 3.4 Results For clarity, results are interpreted directionally with respect to increases in PaCO2. However, due to the linearity of these relationships, these results are equally and oppositely valid with respect to proportional reductions in PaCO2 up to -10 mmHg in this dataset. The CMRO2 was reduced by 0.5 ml × min-1 (95% CI: -0.6 to -0.3) per mmHg increase in PaCO2 across a range of PaCO2 from 30 to 60 mmHg (P < 0.001; Figure 3-2A). For example, CMRO2 was reduced by 9.8 % (95% CI: -13.2 to -6.5) with a +9 mmHg increase in PaCO2 from 43.1 ml × min-1 (95% CI: 39.9 to 46.3) to 38.9 ml × min-1 (95% CI: 35.5 to 42.2). These reductions in CMRO2 with hypercapnic acidosis were apparent when expressed against PaCO2, arterial pH, PbtCO2 index, or IJV pH (all P < 0.001; e.g., Figure 3-2B). A +9 mmHg increase in PaCO2 resulted in increases in CDO2 by 56.0 % (95% CI: 51.2 to 60.8) (P < 0.001) and disproportionally greater reductions in O2 extraction by 41.7 % points (95% CI: -43.6 to -39.8) (P < 0.001), such that O2 extraction was decreased by 0.6 % (95% CI: -0.6 to -0.5) per ml × min-1 increase in CDO2 (P < 0.001; Figure 3-2C). Overall, O2 extraction was reduced by 5.8 % (95% CI: -6.2 to -5.5) per mmHg elevation in PaCO2 (P < 0.001). Figure 3-2. Relationship between cerebral metabolic rate of oxygen (CMRO2) indexed against PCO2; insufficient O2 utilization to support oxidative metabolism. The CMRO2 was altered by 0.5 ml × min-1 (95% CI: -0.6 to -0.3) per mmHg change in PaCO2 across a range of PaCO2 from 30 to 60 mmHg (P < 0.001) (A). These results were consistent when expressed as CMRO2 versus the PbtCO2 index (B). Collectively, CMRO2 was reduced by 9.8 % (95% CI: -13.2 to -6.5) with a +9 mmHg increase in PaCO2 from 43.1 ml × min-1 (95% CI: 39.9 to 46.3) to 38.9 ml × min-1 (95% CI: 35.5 to 42.2). The elevated CDO2 achieved via stepwise hypercapnia was reflected in reductions in O2 extraction by 0.6 % (95% CI: -0.6 to -0.5) per ml × min-1 increase in CDO2 (P < 0.001) (C). For example, a +9 mmHg increase in PaCO2 increased CDO2 by 56.0 % (95% CI: 51.2 to 60.8) (P < 0.001) and resulted in a disproportionally greater reduction in O2 extraction by 41.7 % points (95% CI: -43.6 to -39.8) (P < 0.001). Males are indicated by blue circles and females are indicated by red triangles. Individual regression lines for each unique ID are displayed (blue or red for males or females, respectively). FICK DERIVED CMRO2 WITH ALTERED PaCO2 78 Abbreviations: Cerebral metabolic rate of oxygen, CMRO2; partial pressure of arterial CO2, PaCO2; brain tissue PCO2, PbtCO2; oxygen extraction, O2 extraction; cerebral oxygen delivery, CDO2. The OGI was reduced by 0.03 a.u. (95% CI: -0.06 to -0.0004) per mmHg elevation in PaCO2 (P = 0.049; Figure 3-3A) which corresponded to a 5.6 % (95% CI: -11.2 to 0.06) reduction in OGI with a +9 mmHg increase in PaCO2. The CMRGlu was reduced by 0.003 mmol × min-1 (95% CI: -0.007 to 0.00002) per mmHg elevation in PaCO2 (P = 0.045; Figure 3-3C) which corresponded to a 7.7 % (95% CI: -15.4 to -0.08) reduction in CMRGlu with a +9 mmHg increase in PaCO2; i.e., reduction in net glucose uptake. There was no significant change in OCI across the range of PaCO2; e.g., rest: 5.1 a.u. (95% 4.6 to 5.5) versus +9 mmHg PaCO2: 4.8 a.u. (95% CI: 4.3 to 5.3) (P = 0.144; Figure 3-3B). Further, there was no relationship between CMRLac and PaCO2 (0.004 mmol × min-1; 95% CI: -0.004 to 0.01) per mmHg elevation in PaCO2 (P = N/A). Figure 3-3. Increased reliance on anaerobic glycolysis during acute hypercapnia indicated via compensatory reduction in oxidative glucose index. The OGI was reduced by 0.03 a.u. (95% CI: -0.06 to -0.0004) per mmHg elevation in PaCO2 (P = 0.049) (A) which corresponded to a 5.6 % (95% CI: -11.2 to 0.06) reduction in OGI with a +9 mmHg increase in PaCO2. There was no significant change in OCI across the range of PaCO2; e.g., rest: 5.1 a.u. (95% 4.6 to 5.5) versus +9 mmHg PaCO2: 4.8 a.u. (95% CI: 4.3 to 5.3) (P = 0.144) (B). The CMRGlu was reduced by 0.003 mmol × min-1 (95% CI: -0.007 to 0.00002) per mmHg elevation in PaCO2 (P = 0.045) (C) which corresponded to a 7.7 % (95% CI: -15.4 to -0.08) reduction in CMRGlu with a +9 mmHg increase in PaCO2; i.e., reduction in net glucose uptake. Males are indicated by blue circles and females are indicated by red triangles. Individual regression lines for each unique ID are displayed (blue or red for males or females, respectively). Abbreviations: Oxidative glucose index, OGI; oxidative carbohydrate index, OCI. To highlight the error associated with assuming a constant CMRO2 with alterations in PaCO2, Fick-derived CBF calculations were compared against the duplex measured CBF (Supplemental Figure 1). The reciprocal of the arterial-IJV O2 content difference (CBF = 1/ CaO2 – CvO2) was used to estimate CBF by rearranging the Fick Equation (Equation 3.8) with the now invalidated assumption of constant CMRO2; e.g., CBF = CMRO2 / (CaO2 – CvO2). This calculation indicates FICK DERIVED CMRO2 WITH ALTERED PaCO2 79 that percent changes in CBF are inversely proportional to the percent changes in arterial-IJV O2 content difference. Bland-Altman analysis revealed a bias of 11.59 D% (95% CI: -37.40 to 60.59) indicating an overestimation of Fick-derived CBF with changes in PaCO2. Exploratory analysis – influence of biological sex on the CMRO2 response to PaCO2 An exploratory analysis was performed including fixed effects of PaCO2 and sex (male vs. female) to investigate the influence of biological sex on the CMRO2 response to PaCO2. When sex is statistically accounted for in our linear mixed-effects model, there was a 0.5 ml × min-1 (95% CI: -0.6 to -0.3) reduction in CMRO2 per 1 mmHg increase in PaCO2 (PaCO2 effect: P < 0.001; Figure 3-4A). Additionally, males had a higher CMRO2 by 10.5 ml × min-1 (95% CI: 4.3 to 16.6) at any given PaCO2 (sex effect: P < 0.001; Figure 3-4A). To confirm that the effect of sex was not driven by a systematic bias arising from the study, study ID was included as a main effect in the primary statistical model and there were no differences due to study ID (P = 0.527); further, separate visual inspection of the relationship of PaCO2 on CMRO2 supported this conclusion as males in the study that includes both males and females (study 4) were not obviously different from males in the other studies (studies 1-3) (Supplemental Figure 2). There was no PaCO2´sex interaction (i.e., no difference between the male vs. female regression slopes; P = 0.135). Next, as CMRO2 = CBF ´ CaO2-CvO2 difference, the contribution of these two variables to CMRO2 was investigated to explain the main effect of sex on CMRO2 (Figure 3-4 B-D). There was no effect of sex on the relationship between CBF vs. PaCO2 (sex effect: P = 0.719; Figure 3-4 B-C). There was a 0.27 ml × dl-1 (95% CI: -0.29 to -0.25) reduction in CaO2-CvO2 difference per 1 mmHg increase in PaCO2 (PaCO2 effect: P < 0.001; Figure 3-4D). Additionally, males had a higher CaO2-CvO2 difference by 0.92 ml × dl-1 (95% CI: 0.33 to 1.51) at any given PaCO2 (sex effect: P = 0.002; Figure 3-4D). Role of arterial Hb to mediate the effect of sex on CMRO2 As per Equations 3.1 & 3.2, and due to lower arterial Hb in females (e.g., (Hoiland et al., 2023) Figure 3F), arterial Hb was included as a fixed effect to provide insight into whether the main effect of sex on CMRO2 (regulated by CaO2-CvO2 difference; Figure 3-4D) was explained by different arterial Hb. The main effects of PaCO2 (P < 0.001) and sex (P = 0.022) persisted; however, there was a main effect of arterial Hb (P = 0.030), therefore a further exploratory statistical mediation analysis was performed. Overall, the proportion mediated effect of sex on CMRO2 by arterial Hb was 20.2 % (95% CI: 0.1 to 68.2) (P = 0.042), such that approximately 80 FICK DERIVED CMRO2 WITH ALTERED PaCO2 80 % (P = 0.034) of this response was a direct effect of sex on CMRO2 which was unexplained by arterial Hb. Figure 3-4. Exploratory analysis – influence of biological sex on the CMRO2 response to PaCO2. An exploratory analysis was performed including fixed effects of PaCO2 and sex (male vs. female) to investigate the influence of biological sex on the CMRO2 response to PaCO2. There was a 0.5 ml × min-1 (95% CI: -0.6 to -0.3) reduction in CMRO2 per 1 mmHg increase in PaCO2 (PaCO2 effect: P < 0.001) (A). Additionally, males had a higher CMRO2 by 10.5 ml × min-1 (95% CI: 4.3 to 16.6) at any given PaCO2 (sex effect: P < 0.001) (A). There was no PaCO2´sex interaction (i.e., no difference between the male vs. female regression slopes; P = 0.135). Next, as CMRO2 = CBF ´ CaO2-CvO2 difference, the contribution of these two variables to CMRO2 was investigated to explain the main effect of sex on CMRO2 (B-D). There was no effect of sex on the relationship between CBF vs. PaCO2 (sex effect: P = 0.719); (B) illustrates the un-transformed observed values with regression lines for each sex, whereas, CBF was log-transformed to satisfy model assumptions (C). There was a 0.27 ml × dl-1 (95% CI: -0.29 to -0.25) reduction in CaO2-CvO2 difference per 1 mmHg increase in PaCO2 (PaCO2 effect: P < 0.001) (D). Additionally, males had a higher CaO2-CvO2 difference by 0.92 ml × dl-1 (95% CI: 0.33 to 1.51) at any given PaCO2 (sex effect: P = 0.002) (D). Regression lines are shown for males (black) and females (dashed black) where a significant effect of sex was observed. The black regression line for panels B-C shows the total effect of CBF versus PaCO2 averaging over sex. Males are indicated by blue circles and females are indicated by red triangles. FICK DERIVED CMRO2 WITH ALTERED PaCO2 81 Individual regression lines for each unique ID are displayed (blue or red for males or females, respectively). Abbreviations: Cerebral metabolic rate of oxygen, CMRO2; partial pressure of arterial CO2, PaCO2; cerebral blood flow, CBF; arterial-venous oxygen content difference, CaO2-CvO2 3.5 Discussion The results of this study indicate a direct effect of PaCO2 on cerebral oxidative metabolism (i.e., CMRO2). In particular, CMRO2 and CMRGlu are reduced indicating a reduction in cerebral metabolism that may be in part compensated by an increased contribution of anaerobic metabolic pathways during acute steady-state hypercapnic acidosis in healthy humans. This observation is supported by: 1) the CMRO2 was reduced by 9.8 % in response to +9 mmHg PaCO2 (Figure 3-2); 2) glucose uptake (via CMRGlu) was reduced to a lesser extent by 7.7 %; therefore 3) the corresponding OGI was reduced by 5.6 % (Figure 3-3), thus indicating a lesser reduction in glucose uptake relative to oxygen suggesting that part of the glucose was not fully oxidized. It is well-established that hypercapnic acidosis increases CBF (Lennox & Gibbs, 1932; Kety & Schmidt, 1948b; Shapiro et al., 1966; Willie et al., 2012; Hoiland et al., 2019). In vitro studies indicate that acidosis reduces Ca2+ sensitivity and thus intracellular [Ca2+] (Klöckner & Isenberg, 1994a; 1994b; Boedtkjer et al., 2016a) which contributes to vascular smooth muscle cell hyperpolarization and relaxation (Kerrick & Hoar, 1981; Boedtkjer et al., 2011). Although hypercapnic acidosis increases CBF, larger relative reductions in the arterial-venous oxygen content difference will theoretically contribute to lower CMRO2 (explained via the Fick principle: CMRO2 = CBF × arterial-venous O2 content). The conceptualization of these changes in the framework of the Fick principle mathematically explains that increases in PaCO2 will be related to reductions in the trans-cerebral arterial-venous PCO2 difference as: 1) CBF varies directly with PaCO2 (Lennox & Gibbs, 1932; Kety & Schmidt, 1948b; Willie et al., 2015) see Figure 3-4B; and 2) the cerebral metabolic production of CO2 (V̇CO2) is reportedly unaltered in the physiological range; e.g., CBF = V̇CO2 / PvCO2 – PaCO2 (Posner et al., 1965; Siesjö, 1972). These novel findings, however, show that CMRO2 is reduced with severe hypercapnic acidosis, thus affecting V̇CO2 as well. Indeed, calculated V̇CO2 (CBF ´ CvCO2-CaCO2 difference) was reduced by approximately 21 % with +9 mmHg PaCO2 (10.4 ml × min-1 reduction in V̇CO2; 95% CI: -14.2 to -6.6, P < 0.001). Acute hypercapnic acidosis reduces cerebral oxidative metabolism FICK DERIVED CMRO2 WITH ALTERED PaCO2 82 The reported reductions in CMRO2 with hypercapnic acidosis are likely explained by: 1) extracellular pH-mediated reductions in glycolytic flux (Folbergrová et al., 1972; Miller et al., 1975; Borgström et al., 1976); 2) pH-sensitive alteration of glutamatergic signalling via increases in adenosine (Dulla et al., 2005); and 3) higher circulating catecholamines via sympathetic nervous activity with CO2-mediated acidosis (Sechzer et al., 1960; Seifert et al., 2009). The decreases in cerebral glucose utilization are likely in part mediated by reductions in phosphofructokinase activity and thus glycolytic flux (Folbergrová et al., 1972; Miller et al., 1975; Borgström et al., 1976). Additionally, CO2-mediated extracellular acidosis increases adenosine concentrations (Dulla et al., 2005); these in vitro data indicate that glutamatergic signalling is regulated in part by extracellular pH-mediated activation of adenosine A1 receptors (Dulla et al., 2005) whereby hypercapnic acidosis reduces cortical excitability (Thesen et al., 2012) (thus explaining lower CMRO2). Elevations in sympathetic nervous activity (e.g., high dose intravenous adrenaline; 0.08 µg per kg × min-1 for 15 mins) reportedly increase the contribution of non-oxidative carbohydrate uptake by 8-fold (Seifert et al., 2009) and the larger reliance on non-oxidative metabolism during exhaustive exercise is at least in part attributable to adrenaline (Larsen et al., 2008). As such, acidosis-induced increases in sympathetic activity via chemoreflex activation (Steinback et al., 2009; 2010a; 2010b) – in addition to hypercapnia-mediated elevations in circulating catecholamines (Sechzer et al., 1960; Metz, 1969; Davidson et al., 1986; Brofman et al., 1990) – may explain a shift towards anaerobic cerebral metabolism with CO2-mediated acidosis. Indeed, CMRO2 is reportedly reduced by 29-44% with hypercapnic acidosis achieved during a maximal breath-hold (PaCO2 approximately 58 mmHg) (Bain et al., 2016; 2017). In the current study, CMRGlu was reduced (i.e., lower net uptake) yet, to a lesser extent than CMRO2, which further supports the reductions in OGI during hypercapnic acidosis; however, unaltered CMRLac and OCI indicates that lactate oxidation was not substantial (Figure 3-3). Whether total energy production is affected – i.e., if anaerobic pathways are insufficient to sustain total rate of energy flux – with increasing severity/duration of hypercapnia requires further exploration. Clinical and research implications The effect of hypercapnia on cerebral metabolism is of critical interest to the validity of functional imaging techniques. In particular, hypercapnia is routinely used in calibrated blood oxygen-level dependent (BOLD) functional magnetic resonance imaging (fMRI) as an index of the vasodilatory health of the brain vasculature; e.g., (Mandell et al., 2008; Fierstra et al., 2018; Sobczyk et al., 2021). Importantly, this technique relies on the assumption that tissue metabolic FICK DERIVED CMRO2 WITH ALTERED PaCO2 83 activity and, therefore CMRO2, remains unchanged; such that any change in the BOLD signal accurately reflects changes in CBF (Davis et al., 1998; Hoge et al., 1999; Chen & Pike, 2010b; Duffin et al., 2017). This assumption was previously accepted due to work by Chen et al. (2010a) who non-invasively estimated CMRO2 changes from CBF and oxygenation data with a MR-equivalent of the Kety-Schmidt technique via arterial-spin labelling, phase-contrast flow imaging, as well as in vivo MR oximetry (Chen & Pike, 2010a). These authors reported unaltered estimated CMRO2 throughout the range of -6 to +9 mmHg PETCO2 (Chen & Pike, 2010a). This assumption has been tested (Kliefoth et al., 1979; Zappe et al., 2008; Xu et al., 2011; Bulte et al., 2012; Thesen et al., 2012; Xu et al., 2012; Driver et al., 2016; 2017; Peng et al., 2017; Deckers et al., 2022) with previous work showing significant error in estimating oxygen extraction and CMRO2 using calibrated BOLD techniques (Griffeth & Buxton, 2011; Blockley et al., 2015; Merola et al., 2016). Further, in addition to BOLD fMRI, T2-relaxation-under-spin-tagging (TRUST) and susceptibility-based oximetry MR techniques rely on a stable SvO2 to calculate CMRO2, which has recently shown systematic bias with direct jugular SvO2 during 5% inspired CO2 in n=2 subjects (Miao et al., 2019). Therefore, this study is the first to our knowledge to assess this comprehensively via direct arterial-IJV sampling with tightly-controlled PaCO2 and PaO2 in humans. Taken together, any PaCO2-induced alteration in CMRO2 is highly relevant in the interpretation of the vasodilatory response to PaCO2, as changes in CBF can be affected independently by the metabolic requirements (Himwich & Homburger, 1947; Kety & Schmidt, 1948b; Siesjö, 1978; Madsen & Vorstrup, 1991; Buxton & Frank, 1997). For example, alterations in any of the determinants of oxidative metabolism that affect CMRO2 could influence CVR independently of any vasodilatory responsiveness and therefore invalidate the clinical interpretation of such tests. The next important implication of these findings is the widespread use of CVR tests – often assessed by either MRI approaches (as outlined above) or with ultrasound (Koep et al., 2022; Carr et al., 2022; Hoiland et al., 2023) (Willie et al., 2012; Carr et al., 2021b; Koep et al., 2022) – as an index of the vasodilatory health of the brain vasculature (Lavi et al., 2006; Ainslie et al., 2007). It is worth recognizing that the utility and relevance of these steady-state CVR tests in identifying pathology is inexact as this test is unrelated to nitric oxide dependent shear-mediated dilation and therefore does not provide an accurate indication of endothelial-dependent vasodilatory responsiveness (Carr et al., 2020; Hoiland et al., 2022) and does not provide predictive value for “cerebrovascular risk” in otherwise healthy individuals (Markus & Cullinane, 2001; Portegies et al., 2014). With respect to the present findings, changes in CMRO2 are yet FICK DERIVED CMRO2 WITH ALTERED PaCO2 84 another important (and typically rarely measured, with few exceptions; e.g., (Peebles et al., 2007; Carr et al., 2022) variable that influences CVR. Future studies are needed to establish if hypercapnic-induced reductions in CMRO2 occur with aging and/or pathology. Lastly, it is noteworthy that a subset of n = 11 participants completed -10 mmHg PaCO2 (study 4); therefore, the linear relationship between variables can be equally interpreted as the reciprocal response with hypocapnia. For example, these results can be expressed as a 0.5 ml × min-1 increase in CMRO2 per mmHg reduction in PaCO2, which corresponds to 9.8 % increase in CMRO2 with a -9 mmHg reduction in PaCO2. These data emphasize a striking mismatch between lower O2 delivery (5.1 ml × min-1 reduction in CDO2 per mmHg decrease in PaCO2, P < 0.001) and higher tissue metabolic requirements with hypocapnia (e.g., 30 to 40 mmHg range PaCO2; Figure 3-2A). Brain tissue O2 extraction – the process that reflects a tightly-controlled balance between vascular (e.g., blood supply) and neural (e.g., oxygen consumption) regulation – was markedly higher with hypocapnia (Figure 3-2C). However, this process is energetically taxing to sustain and in patients with large-vessel diseases, such as carotid artery occlusion, a higher O2 extraction predicts a greater risk for stroke (Grubb et al., 1998). These results are relevant to the myriad of idiopathic hyperventilatory syndromes as well as incidental hypocapnia during mechanical ventilation and the controversial application of therapeutic hyperventilation to reduce intracranial hypertension (Rowed et al., 1975; Godoy et al., 2017). Experimental Considerations A key strength of this study was the gold-standard invasive Fick principle technique to quantify arterial-IJV exchange of fuel substrates in vivo in healthy humans (Kety & Schmidt, 1948a). The principal assumption of this technique is that IJV drainage is solely from the brain – in support of this, it is estimated that 97.3% of IJV blood is representative of cerebral sources (Shenkin et al., 1948) such that there is reportedly small bias (<3%) of contamination with extra-cerebral blood. Further, the extra-cranial duplex ultrasound derived metrics of cerebral blood flow utilized in the present experiment are assumed to indicate cerebral tissue nutritive flow. Likewise, with unilateral ICA, VA, and IJV assessments, we assume symmetrical arterial inflow and jugular venous drainage, and neglect possible extra-cranial anastomoses which would affect CBF and CMRO2 calculations (Gibbs et al., 1945; Ferris & Engel, 1946; Himwich & Homburger, 1947; Lichtenstein et al., 2001; Tartière et al., 2009; Thomas et al., 2015b; Friend et al., 2021). However, the former is approximately <5-10% (Lassen & Lane, 1961; Friend et al., 2021) and FICK DERIVED CMRO2 WITH ALTERED PaCO2 85 the latter will likely have a trivial influence on our CBF and CMRO2 values in healthy adults (Heistad & Kontos, 1983; Hartmann, 1985). An exploratory analysis was performed to investigate the influence of biological sex on the CMRO2 response to PaCO2. These data indicate CMRO2 is lower in females by approximately 10.5 ml × min-1 at any given PaCO2 (Figure 3-4A). Additionally, this effect was justified by 16% lower CaO2-CvO2 difference at rest in females (sex effect; Figure 3-4D) and was overall approximately 20% in part explained by arterial Hb (via mediation analysis). It is worth noting that these CMRO2 values reflect the brain’s absolute VO2; therefore, relative CMRO2 can be calculated assuming normative values of average male (1,400 g; (Sokoloff, 1960) and female (approximately 11% less, 1246 g; (Ruigrok et al., 2014; Eliot et al., 2021) adult brain mass. For example, absolute estimated CMRO2 at rest was approximately 22% lower in females (37.9 vs. 48.4 ml × min-1, respectively); that is, when expressed as relative CMRO2 (females: 3.0 vs. males: 3.5 ml × min-1 × 100 g-1 brain tissue), this difference is reduced substantially to 14% between sexes. Thus, it appears that any sex-related differences in the CMRO2 response to PaCO2 are in part explained by anatomical differences in brain tissue mass and, to a lesser extent, arterial Hb. 3.6 Conclusion These results provide evidence that cerebral oxidative metabolism (i.e., CMRO2) is altered by approximately 1 % per mmHg change in PaCO2. Further, CMRO2 and CMRGlu are reduced indicating a reduction in cerebral metabolism that may be in part compensated by an increased contribution of anaerobic metabolic pathways during acute steady-state hypercapnic acidosis in healthy humans. Utilizing the gold-standard direct Fick approach, with volumetric duplex ultrasound derived blood flow paired with IJV-arterial blood sampling, these results show a direct effect of PaCO2 on CMRO2. These results have implications for the physiological and clinical validity of CVR testing, in particular techniques which rely on the (incorrect) assumption of unaltered CMRO2. INFLAMMATORY STRESS IN THE BRAIN WITH EXERCISE AT HIGH-ALTITUDE 86 Chapter 4: Study 2 – How does systemic fuel utilization at high-altitude affect the brain inflammatory response to exercise? Abstract Background: Systemic substrate availability – which can be altered by hypoxia and/or exercise – affects the immune system, which has important relevance in situations of inflammation, such as with strenuous exercise and/or high-altitude exposure. How acclimatization to high-altitude affects immune sensitization/suppression and hypersensitivity of inflammatory responses in the brain has not been investigated, particularly following maximal exercise. Methods: This study investigated substrate oxidation and energy expenditure during exercise at an intensity that elicits maximal fat oxidation and following cycling exercise to exhaustion at sea level (SL) and following 6-8 days at 3,800 m (HA). Healthy adults (n=12, 6/6 females/males) received radial arterial and internal jugular venous (IJV) catheterizations to assess trans-cerebral exchange of inflammatory cytokines. Data were obtained prior to and following 60 minutes of steady-state semi-recumbent cycling at an altitude-specific intensity to elicit maximal fat oxidation (FATmax), and at 5 minutes post- an incremental maximal exercise test. Results: There was no altitude-effects on leukocyte count or cell-type distribution at rest or in response to exercise indicating that basic ‘immune status’ was unaltered at HA (P > 0.05). High-altitude per se provoked a reduction in pro-inflammatory cytokines at rest (indicated by reductions of approximately 40 % in arterial MCP-1 and IL-8; P = 0.057 and P = 0.001, respectively). Although, maximal fat oxidation achieved during the FATmax test was reduced by 30.1 % at HA (SL: 0.41 g/min, 95% CI: 0.36 to 0.48 vs. HA: 0.29 g/min, 95% CI: 0.25 to 0.34, P < 0.001), there was no evidence of an anti-inflammatory effect of 1-hour cycling exercise at FATmax intensity at either altitude. Although acute maximal exercise per se did not provoke inflammatory stress in the brain, it appears that the additive duration/intensity effect of prolonged low-intensity exercise followed by acute maximal exercise – irrespective of altitude – provoked a shift toward trans-cerebral net release by approximately 0.5 to 2.3 pg/ml of IL-6, IL-8, MCP-1, and MIP-1b (exercise effect: all P < 0.05). Conclusions: These results indicate that 6-8 days of acclimatization at 3,800 m does not provoke immune suppression or mediate elevations in systemic pro-inflammation with maximal exercise. Acclimatization to high-altitude attenuates systemic pro-inflammatory responses at rest; however, although independent of altitude, an additive effect of exercise duration/intensity provokes trans-cerebral net release of pro-inflammatory cytokines. INFLAMMATORY STRESS IN THE BRAIN WITH EXERCISE AT HIGH-ALTITUDE 87 4.1 Introduction Previous research on the relative contribution of substrate utilization to total energy expenditure during exercise at high-altitude is contradictory. For example, studies have reported increases in relative carbohydrate utilization at high-altitude (Brooks et al., 1991; Roberts et al., 1996; Lundby & van Hall, 2002; Péronnet et al., 2006) and that acclimatization alleviates the hypoxia-induced suppression of exogenous glucose oxidation during steady-state aerobic exercise (Young et al., 2018; O'Hara et al., 2019). In contrast, studies have reported an increased reliance on fat oxidation during 60-120 minutes of aerobic exercise at 50-55% relative VO2max with acute exposure to high-altitude (O'Hara et al., 2017; Matu et al., 2017). A recent meta-analysis substantiates that hypoxic exposure does not evoke a consistent change in carbohydrate/fat utilization during exercise when matched for relative exercise intensity (Griffiths et al., 2019b), thus emphasizing the importance of absolute exercise intensity as well as nutritional and acclimatization status on preferential substrate utilization at high-altitude (Young et al., 2019; Griffiths et al., 2019a). Hypoxia reduces VO2max (Wagner, 2000; Calbet et al., 2002; 2003); therefore, exercise performed at the same absolute exercise intensity would be reflective of a higher relative exercise intensity and metabolic rate at high-altitude, thus a higher physiological “exercise strain” per se. The immune response to infection/inflammation is energetically taxing (Schmid et al., 2000); for example, the total energy requirements of the immune system may account for up to 25-30% basal metabolism (e.g., approximately 2000 kJ per day) (Straub et al., 2010). The interplay between hypoxia and substrate utilization on the immune response is complex (Vats et al., 2006; Weinberg et al., 2015). While acute hypoxia-induced inflammation can be adaptive to initiate an immune response and promote tissue healing (Harris et al., 2014), acclimatization to high-altitude (> 3 days exposure) reportedly provokes a compensatory anti-inflammatory response to counteract the exacerbated pro-inflammatory response experienced with acute exposure upon initial ascent to high-altitude (Pham et al., 2021; 2022). Further, the anti-inflammatory and immunosuppressive effects of strenuous exercise (Shaw et al., 2018) may play a role in immune system sensitization and prevalence of infection at high-altitude (Facco et al., 2005; Feuerecker et al., 2019). Pro-inflammatory stress at high altitude is further implicated in the etiology of high-altitude illnesses (Pham et al., 2021; Turner et al., 2021); however, it is controversial whether/how exercise alters pro-inflammatory mediators and therefore the implications of exercise on high-altitude illnesses. How acclimatization to high-altitude, with and INFLAMMATORY STRESS IN THE BRAIN WITH EXERCISE AT HIGH-ALTITUDE 88 without exhaustive exercise, affects immune sensitization/suppression and hypersensitivity of inflammatory responses in the brain has not been investigated. Mitochondrial fatty acid oxidation is pivotal for immune cells anti-inflammatory status (Vats et al., 2006; Weinberg et al., 2015; Liepinsh et al., 2020). The current study utilized 1-hour semi-recumbent cycling exercise performed at maximal fat oxidation intensity (FATmax - approx. 30-35% VO2max corresponding to 20-25% Wmax) to increase whole-body fat oxidation rate. This experimental design allows us to investigate the influence of fat oxidation during exercise on the immune response, while abating the post-exercise inflammation provoked by high-intensity exercise (Nakajima et al., 2010; Bessa et al., 2016). Additionally, by utilizing the same relative exercise intensity at both sea level and high-altitude, this approach attempts to standardize the potential hypoxia-related effects on the post-exercise inflammatory response, as well as any alterations in relative substrate utilization. The latter which would be exacerbated when exercise is performed at matched absolute exercise intensity to sea level values (Young et al., 2019; Griffiths et al., 2019a). Next, participants completed a stepwise incremental exercise test at previously established altitude-specific Wmax. Radial arterial and internal jugular venous blood sampling was performed to assess trans-cerebral inflammatory cytokines at maximal exercise, 5 minutes post-exercise, and following 60 minutes of passive recovery. It was hypothesized that: 1) 1-hour of cycling exercise at altitude-specific FATmax intensity will promote an anti-inflammatory response at both sea level and high-altitude (indicated by higher IL-4, IL-10, and IL-13), and 2) acclimatization to high-altitude will provoke an anti-inflammatory response at rest which will attenuate the pro-inflammatory and leukocyte mobilization responses to maximal exercise compared to sea level. 4.2 Methods Ethical Approval Participants provided informed written consent prior to participating in this study. This study was approved by the University of British Columbia Clinical Research Ethics Board (CREB: H22-01091) and conducted according to the principles established by the Declaration of Helsinki, except for registration in a database. Participants Data were obtained at sea level (SL) within the Centre for Heart, Lung and Vascular Health at the University of British Columbia, Kelowna, British Columbia, Canada and following 3-8 days at INFLAMMATORY STRESS IN THE BRAIN WITH EXERCISE AT HIGH-ALTITUDE 89 3,800 m (HA) at the Barcroft Research Laboratory on White Mountain, California, USA (Figure 4-1). In total, 15 healthy adults (7/8 females/males) completed testing at SL and HA. A subset of 12 participants (6/6 females/males) completed the invasive arterial-internal jugular venous catheterization protocol at SL with n=11 repeating the protocol at HA (n=6/5 females/males). At HA, two participants became ill prior to testing and either the arterial/internal jugular venous catheter was unsuccessful in two participants, thus explaining the lower sample size. Participants had no reported history of cardiovascular, cerebrovascular, or respiratory disease and were not taking any prescription medication at their time of participation. Participants refrained from alcohol and vigorous exercise or activity for at least 12 hours prior to testing. Habitual caffeine users were provided a single black coffee with their standardized breakfast 1 hour prior to the experimental protocol, and this was repeated at both SL and HA. Experimental Overview Following at least 12-hour overnight fast and after having refrained from alcohol, caffeine, and vigorous exercise, participants completed a 25-minute resting metabolic rate (RMR) via indirect calorimetry with a facemask (Vyaire Vyntus CPX). Breath-by-breath VO2 data were analyzed as 1-minute averages – excluding the first 5 minutes – and the RMR was identified as the lowest energy expenditure value. Next, participants completed an incremental semi-recumbent cycling test to assess maximal fat oxidation during exercise (MFO) and the exercise intensity evoking MFO (FATmax) (Figure 4-2) (Amaro-Gahete et al., 2019). The MFO test involved an incremental cycling test whereby exercise intensity was increased by 20/25 W/min (females/males, respectively) until the respiratory exchange ratio (RER) reached 1.0. The MFO and FATmax was calculated as the maximal fat oxidation achieved and the associated exercise intensity expressed as percentage of the maximal oxygen uptake (VO2peak). Next, participants completed a VO2peak test whereby the workload will increase by 25 W/min until at least three of the following five criteria was met: individual perception of volitional exhaustion, respiratory exchange ratio >1.15, VO2 curve plateau, heart rate approaching age-predicted maximum, and inability to maintain pedaling frequency above 70 rpm (physical exhaustion). Lastly, participants completed a VO2peak verification (time to exhaustion) test at 110% VO2max. The RMR, FATmax, and VO2peak tests were performed on days 3-5 at HA. Participants completed the main experimental exercise test battery on a separate visit at least 24 hours following the VO2peak test. All participants completed this invasive protocol within 72 hours apart on days 6-8 at HA. Participants arrived at the laboratory overnight fasted and INFLAMMATORY STRESS IN THE BRAIN WITH EXERCISE AT HIGH-ALTITUDE 90 received radial arterial and jugular venous catheterizations (see Blood Sampling). Following a standardized breakfast, participants rested 1 hour before starting the exercise protocol. First, participants completed 60 minutes of steady-state cycling at individualized altitude-specific FATmax exercise intensity (Figure 4-3). After approximately 15 minutes of rest, participants completed three 4-minute steady-state exercise stages at individualized workloads corresponding to 30%, 60%, and 90% Wmax. Lastly, participants rested for 60 minutes of passive recovery. Volumetric CBF and paired arterial-IJV blood samples were collected at the following timepoints to assess CMRO2 and trans-cerebral exchange of inflammatory cytokines: pre/post 60 minutes of FATmax exercise; pre- max test, at 90% Wmax, and at 5- and 60-minutes recovery. Total CBF and paired arterial-IJV ABL samples were collected at 20 and 40 minutes of FATmax exercise and at the 30% and 60% Wmax stages to assess CMRO2. Blood Sampling While resting supine, catheterization of the radial artery and internal jugular vein was performed using sterile technique, under local anesthesia (Lidocaine, 1.0%), and with ultrasound guidance. A 20G arterial catheter (Arrow, Markham, ON, Canada) was placed in the radial artery and a 13G central venous catheter (Cook Medical, Bloomington, IN, USA) was advanced 15 cm into the internal jugular vein to the jugular bulb. These catheters were attached to in-line waste-less blood sampling systems for repeated trans-cerebral blood sampling (Edwards Lifesciences, TruWave VAMP, CA, USA). This technique has been successfully performed by our group and has been previously described in detail elsewhere (Schell & Cole, 2000; Ainslie et al., 2014). Arterial and jugular bulb blood samples were drawn into K2EDTA Vacutainers Ò (Becton Dickinson, USA). Whole blood was then immediately centrifuged at 600 g for 10 minutes at a temperature of 4°C. Plasma was aliquoted into cryovials (approximately 500 µl), flash frozen in liquid N2, and stored at -80°C. A Bio-Plex Pro Human Cytokine 17-plex Assay (Bio-Rad, Mississauga, Canada) was performed at Alberta Children’s Hospital BioCORE facility. Approximately 1.0 ml of radial arterial and internal jugular venous blood were drawn at the same time into pre-heparinized syringes (SafePICO, Radiometer, Copenhagen, Denmark) and analyzed immediately using a commercial blood gas analyzer (ABL90 FLEX, Radiometer) at each experimental stage. This analysis included measurements of the partial pressures of arterial carbon dioxide (PaCO2) and oxygen (PaO2), arterial oxygen saturation (SaO2), arterial INFLAMMATORY STRESS IN THE BRAIN WITH EXERCISE AT HIGH-ALTITUDE 91 oxygen content (CaO2), calculated bicarbonate ion concentration ([HCO3-]), base excess [Base(B)], hydrogen ion concentration ([H+]), hemoglobin concentration ([Hb]), and pH. All blood gas variables were temperature-corrected to rectal temperature. Rectal temperature was measured using a pediatric thermistor (Mon-a-therm general purpose temperature probe, Mallinckrodt Medical, St. Louis, MO, USA) inserted approximately 10 cm past the anal sphincter. Rectal temperature was sampled continuously at 1000 Hz using an analogue-to-digital converter (Powerlab, 16/30; ADInstruments, Colorado Springs, CO, USA) and data were interfaced with LabChart (Version 7.1) and analyzed offline. Cardiorespiratory Breath-by-breath VO2 data was collected via indirect calorimetry (Vyaire Vyntus CPX) during all testing procedures. Heart rate was measured via heart rate strap (Polar) with the metabolic cart and lead-II electrocardiogram (ECG; ML132 BioAmp, ADInstruments, CO, USA). Beat-by-beat arterial blood pressure was continuously acquired via the radial artery pressure transducer positioned at the height of the right atrium (Edwards Lifesciences, TruWave VAMP, CA, USA) and the arterial blood pressure waveform was averaged to calculate mean arterial blood pressure (MAP). Cerebrovascular Total CBF was measured via extra-cranial blood velocity and diameter of the internal carotid artery (ICA) and vertebral artery (VA) using a 10-MHz multifrequency linear array Duplex ultrasound (Terason t3000; Teratech, Burlington, MA, USA), as per technical recommendations (Thomas et al., 2015a). Pulse-wave mode was used to measure beat-to-beat peak blood velocity and arterial diameter was instantaneously measured via B-mode imaging; data were analyzed using custom edge-detection and wall tracking software (Cardiovascular Suite v3.5.3, Quipu, Pisa, Italy / BloodFlow Analysis, version 5.1). The ICA blood velocity and vessel diameter were measured ≥1.5 cm from the carotid bifurcation to avoid any turbulent or retrograde flow patterns, while VA blood velocity and diameter were measured between C4-C5 or C5-C6. The vessel location was decided on an individual basis to allow for reliable image acquisition, with the same location and consistent insonation angle (60°) repeated within participants and between trials. Calculations INFLAMMATORY STRESS IN THE BRAIN WITH EXERCISE AT HIGH-ALTITUDE 92 Brain respiratory quotient (RQ) was calculated as: RQ = (CvCO2 – CaCO2) (CaO2 – CvO22)⁄ Equation 4.1 Systemic respiratory exchange ratio (RER) was calculated as: RER = VCO2 VO2⁄ Equation 4.2 Figure 4-1. Testing locations at sea level (SL) and high-altitude (HA). INFLAMMATORY STRESS IN THE BRAIN WITH EXERCISE AT HIGH-ALTITUDE 93 Figure 4-2. Fat Max Study - Pre testing to establish FATmax and VO2max.INFLAMMATORY STRESS IN THE BRAIN WITH EXERCISE AT HIGH-ALTITUDE 94 Figure 4-3. Fat Max Study – Exercise protocol schematic Participants first completed a steady-state incremental submaximal exercise test to determine maximal fat oxidation and the corresponding exercise intensity (FATmax). Thereafter, an incremental maximal exercise test followed by a supramaximal time to exhaustion verification test was performed to determine maximal oxygen uptake (VO2max) (A). The main experimental exercise test battery was as follows: 1) 60 minutes of steady-state cycling at individualized FATmax exercise intensity; 2) three 4-minute steady-state exercise stages at individualized workloads corresponding to 30%, 60%, and 90% Wmax; and 3) 60 minutes of recovery at rest (B). Brain blood flow and paired arterial-IJV blood samples were collected at the following timepoints to assess CMRO2 and trans-cerebral exchange of inflammatory cytokines: pre/post 60 minutes of FATmax exercise; pre- max test, at 90% Wmax, and at 5 and 60 minutes recovery. Brain blood flow and paired arterial-IJV blood gas samples were collected at 20 and 40 minutes of FATmax exercise and at the 30% and 60% Wmax stages to assess CMRO2. All experimental measures were performed on a semi-recumbent cycle ergometer (C). INFLAMMATORY STRESS IN THE BRAIN WITH EXERCISE AT HIGH-ALTITUDE 4.3 Statistical analyses All data are presented in-text as mean with 95% confidence intervals and as individual values in figures. Data analyses were performed using RStudio (2021.09.1) and statistical analyses were performed using SPSS software (IBM statistics, Version 23.0). Statistical significance was set at P < 0.05. A linear mixed-effects model analysis with compound symmetry covariance structure with fixed effects of exercise (pre- vs. post- exercise) and altitude (SL vs. HA) was performed for repeated measures comparisons. Further, fixed effects of sample (arterial vs. IJV), altitude (SL vs. HA) and time (pre-fat, post-fat, max, post-5) were used to compare exercise responses between samples and altitude for hematology results. A Bonferroni correction was applied for multiple comparisons when significant interactions were detected. For selected variables, altitude (SL vs. HA) was the only fixed effect. Subjects were included as a random effect. Model assumptions were checked via visual inspection of fitted values vs. residuals plots. If warranted to satisfy model assumptions, outcome values were log-transformed for the analysis. 4.4 Results The results are first presented as Part 1 which describes the altitude-related effects on resting substrate utilization as well as the effectiveness of the FATmax intervention to alter the relative contribution of fat/carbohydrate oxidation to exercise energy expenditure. These results aid in quantifying the relative exercise stimulus in terms of metabolic strain to provide context for systemic and brain substrate utilization during the invasive arterial-IJV protocol. Next, after establishing that exercise intensity was matched for altitude-specific FATmax, Part 2 describes the physiologic and metabolic consequences of 60 minutes steady-state exercise at this low-intensity followed by a staged maximal exercise test to provoke respective immune and anti/pro- inflammatory responses. Table 4-1. Resting arterial blood gas variables at SL and following 6-8 days at HA. SL HA P-Value pH 7.431 (7.420-7.441) 7.456 (7.446-7.467) P < 0.001 PaCO2 (mmHg) 37.6 (35.8-39.4) 30.6 (28.7-32.4) P < 0.001 PaO2 (mmHg) 95.1 (92.1-98.0) 55.3 (52.2-58.3) P < 0.001 [Hb] (g/dl) 14.0 (13.3-14.7) 15.5 (14.9-16.2) P < 0.001 Hct (%) 43.0 (40.8-45.1) 47.7 (45.5-49.8) P < 0.001 SaO2 (%) 98.2 (97.5-99.0) 89.3 (88.6-90.1) P < 0.001 CaO2 (ml/dl) 18.7 (17.8-19.6) 18.8 (17.9-19.7) P = 0.810 [HCO3-] (mmol/l) 25.2 (24.3-26.0) 23.2 (22.3-24.1) P = 0.001 INFLAMMATORY STRESS IN THE BRAIN WITH EXERCISE AT HIGH-ALTITUDE 96 4.4.1 Part 1: Context for systemic and brain substrate utilization There was no difference in RMR between altitudes (SL: 1517 kcal/day, 95% CI: 1336 to 1699 vs. HA: 1599 kcal/day, 95% CI: 1420 to 1777, P = 0.293; Figure 4-4A). Additionally, there was no difference in resting RER between altitudes (SL: 0.83, 95% CI: 0.80 to 0.85 vs. HA: 0.85, 95% CI: 0.83 to 0.88, P = 0.177; Figure 4-4B). Further, there was no difference in relative contribution of fat (SL: 43 %, 95% CI: 34 to 51 vs. HA: 34 %, 95% CI: 26 to 42, P = 0.183; Figure 4-4C) or carbohydrate (SL: 33 %, 95% CI: 26 to 41 vs. HA: 39 %, 95% CI: 31 to 49, P = 0.334; Figure 4-4D) to resting energy expenditure between altitudes. Figure 4-4. Energy expenditure and substrate utilization at rest at sea level (SL) and following 3-5 days at high-altitude (HA). There were no differences in RMR (A), RER (B), relative fat (C) or carbohydrate (D) contribution per day at rest between SL and HA (all P > 0.05). Abbreviations: RMR, resting metabolic rate; RER, respiratory exchange ratio The absolute exercise intensity at which FATmax was achieved was reduced by 12.7 % at HA (SL: 55 W, 95% CI: 47 to 64 vs. HA: 48 W, 95% CI: 41 to 56, P = 0.020; Figure 4-5A). As expected, VO2max peak wattage was reduced by 10.9 % at HA (SL: 249 W, 95% CI: 219 to 282 vs. HA: 222 W, 95% CI: 196 to 251, P < 0.001; Figure 4-5B). Therefore, the FATmax exercise intensity relative to peak power output was nearly identical between altitudes (SL: 22 %, 95% CI: 20 to 25 vs. HA: 22 %, 95% CI: 20 to 24, P = 0.756; Figure 4-5C). SL HA5001000150020002500RMR (kcal/day)P = 0.293SL HA0.700.750.800.850.900.951.00RERP = 0.177SL HA20406080100Relative fat contributionper day (%)P = 0.183SL HA20406080100Relative CHO contributionper day (%)P = 0.334A DB CINFLAMMATORY STRESS IN THE BRAIN WITH EXERCISE AT HIGH-ALTITUDE 97 Figure 4-5. Absolute and relative exercise intensity achieved at FATmax at sea level (SL) and following 3-5 days at high-altitude (HA). There was a 12.7 % reduction in FATmax exercise intensity (P = 0.020) (A) and a 10.9 % reduction in VO2max peak wattage (P < 0.001) (B) at HA, such that the wattage achieved at FATmax relative to peak power output was not different between altitudes (P = 0.756). There was no difference between altitudes when VO2 at FATmax was normalized relative to VO2max (SL: 31 %, 95% CI: 29 to 34 vs. HA: 33 %, 95% CI: 31 to 36, P = 0.089; Figure 4-6A). Average HR at FATmax was not different between altitudes (SL: 97 bpm, 95% CI: 90 to 103 vs. HA: 100 bpm, 95% CI: 94 to 107; P = 0.181); however, as expected, HR at VO2max was lower at HA (SL: 181 bpm, 95% CI: 177 to 186 vs. HA: 176 bpm, 95% CI: 171 to 180; P = 0.033). As such, HR at FATmax relative to VO2max HR was 7.6 % higher at HA (SL: 53 %, 95% CI: 50 to 56 vs. HA: 57 %, 95% CI: 54 to 61, P = 0.018; Figure 4-6B). SL HA20406080FATmax exercise intensity (W)-12.7 %P = 0.020SL HA100200300400VO2max peak wattage (W)-10.9 %P < 0.001SL HA10203040FATmax relative toVO2max watts (%)P = 0.756A B CINFLAMMATORY STRESS IN THE BRAIN WITH EXERCISE AT HIGH-ALTITUDE 98 Figure 4-6. VO2 and HR achieved at FATmax relative to VO2max. There was no difference between altitudes when FATmax exercise intensity was normalized for VO2 relative to max (P = 0.089) (A); however, there was a 7.6 % higher FATmax exercise intensity at HA when normalized for HR relative to max (P = 0.018) (B). The maximal fat oxidation achieved during the FATmax test was reduced by 30.1 % at HA (SL: 0.41 g/min, 95% CI: 0.36 to 0.48 vs. HA: 0.29 g/min, 95% CI: 0.25 to 0.34, P < 0.001; Figure 4-7A). Additionally, maximal carbohydrate oxidation achieved during the FATmax test was reduced by 11.9 % at HA (SL: 2.14 g/min, 95% CI: 1.88 to 2.44 vs. HA: 1.89 g/min, 95% CI: 1.66 to 2.15, P = 0.005; Figure 4-7B). The reduction in maximal carbohydrate oxidation was not explained by external workload as there was no difference between altitudes when normalized for exercise intensity (W) relative to max (SL: 51 %, 95% CI: 46 to 56 vs. HA: 51 %, 95% CI: 46 to 56; P = 0.932). SL HA20304050FATmax relative toVO2max VO2 (%)P = 0.089SL HA3040506070FATmax relative toVO2max HR (%)+7.6 %P = 0.018A BINFLAMMATORY STRESS IN THE BRAIN WITH EXERCISE AT HIGH-ALTITUDE 99 Figure 4-7. Maximal fat and carbohydrate oxidation achieved during the FATmax test. Maximal fat oxidation was reduced by 30.1 % at HA (P < 0.001) (A) and maximal carbohydrate oxidation was reduced by 11.9 % at HA (P = 0.005) (B) during the FATmax test. Exercise energy expenditure during 60 minutes of steady-state cycling at altitude-specific FATmax exercise intensity was reduced by 12.8 % at HA (SL: 297 kcal, 95% CI: 260 to 340 vs. HA: 259 kcal, 95% CI: 226 to 297, P = 0.001; Figure 4-8A); however, this was proportional to the 12.7 % reduction in FATmax exercise intensity (Figure 4-5), such that there was no difference between altitudes when energy expenditure was normalized to external wattage (SL: 5.4 kcal/W, 95% CI: 5.0 to 5.8 vs. HA: 5.3 kcal/W, 95% CI: 4.9 to 5.8, P = 0.557; Figure 4-8B). Figure 4-8. Absolute and relative exercise energy expenditure normalized to workload during 60 minutes of steady-state cycling at altitude-specific FATmax exercise intensity. SL HA0.20.40.60.8Maximal fat oxidation (g/min)-30.1 %P < 0.001SL HA1234Maximal CHO oxidation (g/min)-11.9 %P = 0.005A BSL HA100200300400500Total energy expenditure (kcal)-12.8 %P = 0.001SL HA2468Energy expenditure / FATmax (kcal/W)P = 0.557A BINFLAMMATORY STRESS IN THE BRAIN WITH EXERCISE AT HIGH-ALTITUDE 100 Exercise energy expenditure during 60 minutes of steady-state cycling at altitude-specific FATmax exercise intensity was reduced by 12.8 % at HA (P = 0.001) (A); however, this was proportional to the 12.7 % reduction in FATmax exercise intensity (Figure 4-5), such that there was no difference between altitudes when energy expenditure was normalized to external wattage (P = 0.557) (B). These data indicate that exercise efficiency (kcal/W) was not different between altitudes during the 60 minutes of cycling at FATmax. 4.4.2 Part 2: Trans-cerebral inflammatory stress and immune response Immune response Figure 4-9 illustrates the WBC count and cell-type distribution in response to FATmax and maximal cycling exercise at SL and HA. There was no effect of sample (arterial vs. IJV, P = 0.607) or altitude (SL vs. HA, P = 0.173) between timepoints for WBC count. There was, however, an effect of time such that WBCs were higher post-fat versus pre-fat by 10.8 % (D 0.7 ´103 cells/µl, 95% CI: 0.2 to 1.2; P = 0.003) and higher at max compared to post-fat by 53.3 % (D 3.8 ´103 cells/µl, 95% CI: 3.3 to 4.3; P < 0.001). There were no differences between max and post-5 (P = 0.248), all inclusive of sample type and altitude (time ´ sample ´ altitude interaction: P = 0.745). Relative distribution of WBC types There was no difference between arterial versus IJV samples between timepoints for % distribution of lymphocytes, monocytes, neutrophils, eosinophils, or basophils (P = 0.660, P = 0.613, P = 0.801, P = 0.810, P = 0.575, respectively). At HA, there was an increased relative % distribution of monocytes by 23.8 % (D 1.8 % points, 95% CI: 1.4 to 2.1; altitude effect: P < 0.001) and eosinophils by 21.4 % (D 0.3 % points, 95% CI: 0.1 to 0.5; altitude effect: P < 0.001) with a reduction in % distribution of lymphocytes by 7.2 % (-D 2.2 % points, 95% CI: -3.2 to -1.2; altitude effect: P < 0.001) compared to SL, inclusive of sample type and timepoint. Pre-fat versus post-fat (time effect) There was a reduction in the % distribution of eosinophils at post-fat versus pre-fat by 20.1 % (-D 0.4 % points, 95% CI: -0.7 to -0.1; P = 0.010). Post-fat versus max (time effect) At maximal exercise, when compared to post-fat, there was a higher % distribution of lymphocytes by 27.7 % (D 7.2 % points, 95% CI: 5.3 to 9.0; P < 0.001), monocytes by 15.4 % (D 1.2 % points, 95% CI: 0.6 to 1.8; P < 0.001), and basophils by 21.7 % (D 0.1 % points, 95% CI: INFLAMMATORY STRESS IN THE BRAIN WITH EXERCISE AT HIGH-ALTITUDE 101 0.004 to 0.1; P = 0.031). There was a reduction in the % distribution of neutrophils by 12.9 % (-D 8.3 % points, 95% CI: -10.5 to -6.2; P < 0.001) at max compared to post-fat. Max versus post-5 (time effect) There was a reduction in the % distribution of monocytes at post-5 min versus max by 8.2 % (-D -0.7 % points, 95% CI: -1.4 to -0.1; P = 0.015). Figure 4-9. White blood cell count and cell-type distribution in response to FATmax and maximal cycling exercise at sea level and high-altitude. There was no difference between sample type (arterial vs. IJV) at any timepoint for WBC count or % distribution of cell types at either altitude (all P > 0.05). There was an effect of time such that WBCs were higher post-fat versus pre-fat by 10.8 % (P = 0.003) and higher at max compared to post-fat by 53.3 % (P < 0.001), with no difference between max and post-5 (P = 0.248), all inclusive of sample type and altitude (time ´ sample ´ altitude interaction: P = 0.745) (A & C). At HA, there were small but significant changes in the % distribution of monocytes, eosinophils, and lymphocytes (D range: -2.2 to 1.8 % points; altitude effect: all P < 0.001) compared to SL, inclusive of sample type and timepoint. At max versus post-fat, there was a higher % distribution of lymphocytes by D 7.2 % points (P < 0.001) and a reduction in the % distribution of neutrophils by -D 8.3 % points (P < 0.001). PRE-FATPOST-FATMAXPOST-551015WBC (×103 cells/µl)ArterialIJVSea LevelPRE-FATPOST-FATMAXPOST-551015WBC (×103 cells/µl)High-AltitudeArterialIJVPRE-FAT (A)PRE-FAT (V)POST-FAT (A)POST-FAT (V)MAX (A)MAX (V)POST-5 (A)POST-5 (V)255075100WBC (% distribution) LymphocytesMonocytesNeutrophilsEosinophilsBasophilsSea LevelPRE-FAT (A)PRE-FAT (V)POST-FAT (A)POST-FAT (V)MAX (A)MAX (V)POST-5 (A)POST-5 (V)255075100WBC (% distribution)LymphocytesMonocytesNeutrophilsEosinophilsBasophilsHigh-AltitudeA BC DINFLAMMATORY STRESS IN THE BRAIN WITH EXERCISE AT HIGH-ALTITUDE 102 Inflammatory response Overall, there were altitude effects indicating reductions in systemic (arterial) inflammatory cytokines at HA versus SL. There were few selected effects of exercise per se (e.g., IL-6, MCP-1, MIP-1b) and changes in a-v uptake/release of inflammatory cytokines (e.g., IL-6, IL-8, MCP-1, MIP-1b) which were independent of altitude. The following cytokines were >50% below the observable concentration ranges within these arterial/IJV samples: IL-4, IL-5, IL-10, IL-17A, G-CSF, GM-CSF. Rest (altitude effect) When compared with SL at rest, arterial IL-8 was reduced by 41 % (P = 0.001; Figure 4-10B) and there was a tendency for a reduction in arterial MCP-1 by 39 % (P = 0.057; Figure 4-10A) at HA. There were no changes in a-v uptake/release of inflammatory cytokines at rest between altitudes (all P > 0.05). Pre-fat versus post-fat (altitude effect) When compared to SL, there were reductions in arterial IL-7 (67 %, P = 0.032; Figure 4-11A), IL-8 (67 %, P = 0.001; Figure 4-11B), TNF-a (63 %, P = 0.003; Figure 4-11C), IFN-g (57 %, P = 0.001; Figure 4-11D), MCP-1 (43 %, P = 0.003; Figure 4-11E), and MIP-1b (35 %, P = 0.053; Figure 4-11F) at HA, irrespective of pre/post 60-minutes FATmax exercise. Additionally, there was a reduction in net uptake of IL-8 at HA when compared to SL (altitude effect: P = 0.034; Figure 4-11G). Post-fat versus post-max-5 (altitude effect) When compared to SL, there were reductions in arterial IL-7 (73 %, P = 0.030; Figure 4-12A), IL-8 (62 %, P = 0.002; Figure 4-12B), TNF-a (56 %, P = 0.006; Figure 4-12C), IFN-g (56 %, P < 0.001; Figure 4-12D), IL-6 (34 %, P = 0.057; Figure 4-12E), and MCP-1 (29 %, P = 0.052; Figure 4-12F) at HA, irrespective of pre/post max exercise. Post-fat versus post-max-5 (exercise effect) There was a tendency toward an exercise effect such that arterial IL-6 was increased by 51 % (P = 0.056; Figure 4-12E) at 5 mins post- max exercise; however, this was not different between altitudes. INFLAMMATORY STRESS IN THE BRAIN WITH EXERCISE AT HIGH-ALTITUDE 103 Figure 4-10. Inflammatory cytokines identified at rest at sea level (SL) and following 6-8 days at high-altitude (HA). In total, 8-10 out of 17 analyzed inflammatory cytokines were present in arterial and IJV samples at rest at each altitude. At rest, arterial IL-8 was reduced by 41 % (P = 0.001) (B) and there was a tendency for a reduction in arterial MCP-1 by 39 % (P = 0.057) (A) at HA compared to SL. There were no changes in a-v uptake/release of inflammatory cytokines at rest between altitudes (all P > 0.05). SL HA51015202530Arterial MCP-1 (pg/ml)-39 %P = 0.057SL HA5101525Arterial IL-8 (pg/ml)-41 %P = 0.001A BINFLAMMATORY STRESS IN THE BRAIN WITH EXERCISE AT HIGH-ALTITUDE 104 Figure 4-11. Inflammatory cytokines identified pre- versus post- the 60-minute FATmax exercise intensity cycling at sea level and high-altitude. In total, 9-11 out of 17 analyzed inflammatory cytokines were present in arterial and IJV samples during the 60-minute cycling protocol at each altitude. There were no significant exercise effects or exercise ´ altitude interactions. Overall, there were reductions in arterial IL-7 (67 %, P = 0.032) (A), IL-8 (67 %, P = 0.001) (B), TNF-a (63 %, P = 0.003) (C), IFN-g (57 %, P = 0.001) (D), MCP-1 (43 %, P = 0.003) (E), and MIP-1b (35 %, P = 0.053) (F) at HA compared to SL, inclusive of pre/post 60-minutes FATmax exercise. Additionally, there was a reduction in net uptake of IL-8 at HA (altitude effect: P = 0.034) (G). PRE-FATPOST-FATPRE-FATPOST-FAT2.55.07.510.012.5Arterial IFN-g (pg/ml)-57 %, Altitude:P = 0.001PRE-FATPOST-FATPRE-FATPOST-FAT51015202530Arterial MCP-1 (pg/ml)-43 %, Altitude:P = 0.003PRE-FATPOST-FATPRE-FATPOST-FAT10203040Arterial MIP-1b (pg/ml)-35 %, Altitude:P = 0.053PRE-FATPOST-FATPRE-FATPOST-FAT-5051015a-v [IL-8] (pg/ml)Altitude: P = 0.034UptakeReleasePRE-FATPOST-FATPRE-FATPOST-FAT1020304050150155Arterial IL-7 (pg/ml)-67 %, Altitude:P = 0.032PRE-FATPOST-FATPRE-FATPOST-FAT510151530Arterial IL-8 (pg/ml)-67 %, Altitude:P = 0.001PRE-FATPOST-FATPRE-FATPOST-FAT5101520256580Arterial TNF-a (pg/ml)-63 %, Altitude:P = 0.003E FA B CD GINFLAMMATORY STRESS IN THE BRAIN WITH EXERCISE AT HIGH-ALTITUDE 105 Figure 4-12. Inflammatory cytokines identified pre- versus 5 minutes post- the max exercise protocol at sea level and high-altitude. In total, 9-10 out of 17 analyzed inflammatory cytokines were present in arterial and IJV samples during the max exercise protocol at each altitude. There were no significant exercise ´ altitude interactions. Overall, there were reductions in arterial IL-7 (73 %, P = 0.030) (A), IL-8 (62 %, P = 0.002) (B), TNF-a (56 %, P = 0.006) (C), IFN-g (56 %, P < 0.001) (D), IL-6 (34 %, P = 0.057) (E), and MCP-1 (29 %, P = 0.052) (F) at HA compared to SL, inclusive of pre/post max exercise. There was a tendency toward an exercise effect such that arterial IL-6 was increased by 51 % (P = 0.056) (E) at 5 mins post- max exercise; however, this was not different between altitudes. Pre-fat versus post-max-5 – additive effect of duration/intensity of exercise The total effect of 60 minutes of low-intensity exercise with an acute maximal exercise test was compared (pre-fat versus post-max-5; Figure 4-13). Overall, the arterial IL-7, IFN-g, IL-8, TFN-a, and MCP-1 responses were consistent with the results described previously comparing post-fat versus post-max-5 (Figure 4-12). It is noteworthy, however, that the total exercise protocol POST-FATPOST-MAX-5POST-FATPOST-MAX-5102030405090155Arterial IL-7 (pg/ml)-73 %, Altitude:P = 0.030POST-FATPOST-MAX-5POST-FATPOST-MAX-52468Arterial IFN-g (pg/ml)-56 %, Altitude:P < 0.001POST-FATPOST-MAX-5POST-FATPOST-MAX-55101520Arterial IL-8 (pg/ml)-62 %, Altitude:P = 0.002POST-FATPOST-MAX-5POST-FATPOST-MAX-5123Arterial IL-6 (pg/ml)Post-Max-5 vs Post-Fat:+51%, P = 0.056HA vs SL: -34%, P = 0.057POST-FATPOST-MAX-5POST-FATPOST-MAX-551015203570Arterial TNF-a (pg/ml)-56 %, Altitude:P = 0.006POST-FATPOST-MAX-5POST-FATPOST-MAX-551015202530Arterial MCP-1 (pg/ml)-29 %, Altitude:P = 0.052A B CD E FINFLAMMATORY STRESS IN THE BRAIN WITH EXERCISE AT HIGH-ALTITUDE 106 resulted in a shift toward trans-cerebral net release of IL-6, IL-8, MCP-1, and MIP-1b (Figure 4-13 G-J; exercise effect: all P < 0.05). These exercise-induced changes in a-v release of cytokines were independent of altitude (all P > 0.05). Figure 4-13. Total effect of 60-minutes FATmax in addition to an acute maximal exercise protocol on inflammatory cytokines. In total, 9-11 out of 17 analyzed inflammatory cytokines were present in arterial and IJV samples during the full exercise protocol at each altitude. There were no significant exercise ´ altitude interactions. Overall, there were reductions in arterial IL-7 (73 %, P = 0.027) (A), IFN-g (59 %, P = 0.002) (B), IL-8 (54 %, P = 0.014) (C), and TNF-a (54 %, P = 0.006) (D) at HA compared to SL, inclusive of pre/post max PRE-FATPOST-MAX-5PRE-FATPOST-MAX-55101520253580Arterial TNF-a (pg/ml)-54 %, Altitude:P = 0.006PRE-FATPOST-MAX-5PRE-FATPOST-MAX-5-10-8-3-2-1012a-v [IL-6] (pg/ml)Post-Max-5 vs Pre-Fat:P = 0.048 UptakeReleasePRE-FATPOST-MAX-5PRE-FATPOST-MAX-5-2.50.02.55.07.510.0a-v [IL-8] (pg/ml)Post-Max-5 vs Pre-Fat:P = 0.007UptakeReleasePRE-FATPOST-MAX-5PRE-FATPOST-MAX-5-10-50510152025a-v [MCP-1] (pg/ml)Post-Max-5 vs Pre-Fat:P = 0.012UptakeReleasePRE-FATPOST-MAX-5PRE-FATPOST-MAX-5-20-10010203040a-v [MIP-1b] (pg/ml)Post-Max-5 vs Pre-Fat:P = 0.001UptakeReleasePRE-FATPOST-MAX-5PRE-FATPOST-MAX-51020304090155Arterial IL-7 (pg/ml)-73 %, Altitude:P = 0.027PRE-FATPOST-MAX-5PRE-FATPOST-MAX-5123451012Arterial IFN-g (pg/ml)-59 %, Altitude:P = 0.002PRE-FATPOST-MAX-5PRE-FATPOST-MAX-5510152530Arterial MCP-1 (pg/ml)Post-Max-5 vs Pre-Fat:-33%, P = 0.025HA vs SL: -27%, P = 0.078PRE-FATPOST-MAX-5PRE-FATPOST-MAX-5510152530Arterial IL-8 (pg/ml)Post-Max-5 vs Pre-Fat:-41%, P = 0.078HA vs SL: -54%, P = 0.014PRE-FATPOST-MAX-5PRE-FATPOST-MAX-510203040Arterial MIP-1b (pg/ml)Post-Max-5 vs Pre-Fat:-49%, P = 0.005A B CD E FG H I JINFLAMMATORY STRESS IN THE BRAIN WITH EXERCISE AT HIGH-ALTITUDE 107 exercise. Arterial MCP-1 and MIP-1b were reduced post- max exercise by 33 % (P = 0.025; E) and 49 % (P = 0.005; F), respectively, independent of altitude. The full exercise protocol resulted in a shift toward trans-cerebral net release of IL-6, IL-8, MCP-1, and MIP-1b (exercise effect: all P < 0.05), which was independent of altitude. Whole-body respiratory exchange ratio versus brain RQ This experiment provides a unique opportunity to compare whole-body respiratory exchange ratio (RER) versus brain RQ responses to maximal cycling exercise and recovery at SL and HA (Figure 4-14). Brain RQ was calculated as: v-aCO2 / a-vO2. Overall, brain RQ was consistently higher than whole-body RER at rest (pre-max) and max exercise, independent of altitude (both P < 0.001). Linear regression and Bland-Altman analysis reveal a weak but significant relationship between whole-body RER and brain RQ with a bias of 0.14 indicating a systematically higher brain RQ for any given whole-body RER. Whole-body RER was reduced by 0.15 at 60 minutes post-max compared to pre-max (Pre-Max: 0.83, 95% CI: 0.80 to 0.86 vs. Post-Max-60: 0.68, 95% CI: 0.65 to 0.71, exercise effect: P < 0.001) with no effect of altitude (P = 0.976). Brain RQ was fully recovered at 60-minutes post-max compared to pre-max at SL and HA (exercise ´ altitude interaction: P = 0.192). These data may suggest systemic prioritization of glycogen replenishment following maximal exercise; i.e., excess post-exercise oxygen consumption (indicated via RER 0.68); whereas, this is not particularly relevant for the brain given its preferential oxidative glucose metabolism (RQ > approx. 1.00). INFLAMMATORY STRESS IN THE BRAIN WITH EXERCISE AT HIGH-ALTITUDE 108 Figure 4-14. Whole-body respiratory exchange ratio versus brain RQ response to maximal cycling exercise and recovery at sea level and high-altitude. Brain RQ (calculated as: v-aCO2 / a-vO2) is consistently higher than whole-body RER at rest (pre-max) and max exercise, independent of altitude (both P < 0.001). 4.5 Discussion This is the first study to date to directly interrogate inflammation in the human brain at rest and during exercise at both sea level and high-altitude. The results of this study indicate that 6-8 days of acclimatization at 3,800 m does not provoke immune suppression or mediate elevations in systemic pro-inflammation with maximal exercise. There was no altitude-effects on leukocyte count or cell-type distribution at rest or in response to exercise supporting that basic ‘immune status’ was unaltered at HA (Figure 4-9). Further, although high-altitude per se reduced arterial MCP-1 and IL-8 by approximately 40 % at rest (Figure 4-10), there was no evidence of an anti-inflammatory effect of 1-hour cycling exercise at FATmax intensity at either altitude (Figure 4-11). There were no changes in a-v uptake/release of inflammatory cytokines at rest between altitudes. Although acute maximal exercise per se did not provoke inflammatory stress in the brain (Figure 4-12), it appears that the additive duration/intensity effect of prolonged low-intensity exercise followed by acute maximal exercise provokes a shift toward trans-cerebral net release SL HA SL HAPre-Max Max0.60.81.01.21.41.6Whole-body RER / brain RQBrain RQWhole-body RER0.6 0.8 1.0 1.2 1.40.40.60.81.01.21.41.6Whole-body RERBrain RQLine of identityR2 = 0.19P < 0.001RER RQ RER RQSL HA0.40.60.81.01.21.41.6Whole-body RER / brain RQPre-MaxSLRERHA SLRQHA0.250.500.751.001.251.50Whole-body RER / brain RQPre-MaxPost-Max-60RER RQ RER RQSL HA0.40.60.81.01.21.41.6Whole-body RER / brain RQMax0.50 0.75 1.00 1.25 1.50-0.50-0.250.000.250.500.751.00AverageBrain RQ - Whole-body RER Bias: 0.14ADB CE FINFLAMMATORY STRESS IN THE BRAIN WITH EXERCISE AT HIGH-ALTITUDE 109 of IL-6, IL-8, MCP-1, and MIP-1b (Figure 4-13). These exercise-induced changes in a-v release of pro-inflammatory cytokines were, however, independent of altitude. Maximal fat oxidation during submaximal exercise is reduced at high-altitude, yet inflammatory responses to exercise are unaffected Several studies have reported that candidate pro-inflammatory markers (e.g., IL-1RA, IL-6, C-reactive protein) are upregulated in the systemic circulation during the first few days of acclimatization to high-altitude (Faquin et al., 1992; Hartmann et al., 2000; Eltzschig & Carmeliet, 2011; Julian et al., 2011; Liu et al., 2017). In particular, a recent study by Pham et al., (2022) characterized the systemic inflammatory profile (via transcriptomics) during 3 days acclimatization at the same high-altitude research station as the present study (3,800 m). The authors reported that acute high-altitude exposure upregulates novel inflammation-related genes (Pham et al., 2022), which may play a role in immune system sensitization at high-altitude (Facco et al., 2005; Feuerecker et al., 2019). As such, the objective of this study was to utilize exercise and related changes in systemic fuel utilization to alter the immune/inflammatory response to high-altitude. Fatty acid oxidative metabolism reportedly reduces inflammation via macrophage differentiation toward the anti-inflammatory alternative M2 state (Vats et al., 2006; Weinberg et al., 2015). Further, expression of enzymes important in mitochondrial oxidation of fatty acids are increased by IL-4 (anti-inflammatory cytokine) (Vats et al., 2006). It was therefore anticipated that 1-hour cycling at a personalized intensity to maximize fat oxidation would promote a favorable anti-inflammatory response which would attenuate the pro-inflammatory response to maximal exercise. Contrary to this hypothesis, however, there was no difference in WBCs at rest between altitudes indicating that basic ‘immune status’ was unaltered at HA (Figure 4-9). Further, there was no evidence of an altitude-induced brain inflammatory response at rest (Figure 4-10) – if anything, all arterial cytokines were approximately 35-75 % lower at rest at HA, indicating a systemic anti-inflammatory response. Interestingly, there was no specific effect of exercise on altering these responses. Calculated plasma volume contraction in these participants was approximately 600 ml at HA (indexed by blood volume via carbon monoxide-rebreathe technique; unpublished data) which would, if anything, further exaggerate the reductions in cytokines at altitude. Further, given the comparable relative exercise intensity and duration, any exercise-induced changes in plasma volume would not be expected to be different between altitudes. As IL-7 is implicated in adaptive immunity, the approximately 70 % reduction in IL-7 with HA may have INFLAMMATORY STRESS IN THE BRAIN WITH EXERCISE AT HIGH-ALTITUDE 110 important relevance for exposure to infection in altitude environments. There is a relative paucity of evidence with respect to the effects of high-altitude exposure on innate immunity against microbial infection in addition to the risk of communicable disease between low and high-altitude regions (Basnyat et al., 2001; Basnyat & Starling, 2015). Exercise per se – independent of altitude – provokes trans-cerebral net release of pro-inflammatory cytokines Previous experiments have shown that IL-6 is released from the brain following prolonged cycling exercise (Nybo et al., 2002b; 2003; Rasmussen et al., 2011a) – which is in part related to glycogen status – however, this is the first study to investigate inflammatory stress in the brain with exercise at high-altitude. The previously established systemic increase in arterial IL-6 was apparent with maximal exercise, irrespective of altitude (Figure 4-12). Work by Lundby & Steensberg (2004) has shown that systemic increases in IL-6 following 60-minutes cycling exercise in acute hypoxia and after 3.5-4.5 weeks exposure to 4,100 m is exercise intensity-dependent (Lundby & Steensberg, 2004); the novel results of this study extend this finding to show that trans-cerebral IL-6 release is provoked by an additive effect of exercise duration/intensity. Importantly, the exercise-induced changes in a-v release of pro-inflammatory cytokines were independent of altitude. Although acute maximal exercise per se did not provoke inflammatory stress in the brain (Figure 4-12) – which is consistent with previous data involving an incremental test to exhaustion (Dalsgaard et al., 2004b) – it appears that the additive influence of prolonged low-intensity exercise followed by acute maximal exercise provokes a shift toward trans-cerebral net release of IL-6, IL-8, MCP-1, and MIP-1b (Figure 4-13). It is important; however, that this is perhaps regulated by hyperventilatory-induced reductions in CBF at 5 minutes post- maximal exercise (PaCO2 reduced by approx. 9.5 mmHg independent of altitude), such that, contemporary calculations of exchange which incorporate CBF and plasma shifts (net exchange (pg/ml) = CBF (ml/min)/1000 ´ (1 – Hct) ´ a-v difference), would help in the interpretation of the a-v release of these cytokines; this will be explored with further analysis in this dataset. Regardless, these results are consistent with work by Rasmussen et al., (2011a) who reported an a-v difference of IL-6 of -2.2 pg/ml following 4-hours of ergometry rowing which persisted 1-hour into recovery (compared to 1.0 pg/ml release of IL-6 in the present study). Further, the reduction in cerebral uptake of [IL-8] at HA may be explained by the approximately 67 % lower systemic (arterial) availability of IL-8 (Figure 4-11) which would then necessitate less INFLAMMATORY STRESS IN THE BRAIN WITH EXERCISE AT HIGH-ALTITUDE 111 IL-8 uptake if inflammatory stress is reduced at altitude. Hypoxia-mediated VEGF expression reportedly regulates leukocyte transendothelial migration via upregulation of IL-8 in human brain microvascular endothelial cells in response to inflammation (Lee et al., 2002). Importantly, IL-8 within the CNS is relatively low, and the values reported in this study are within the physiological range of 0-170 pg/ml as measured in cerebrospinal fluid (CSF) (Kossmann et al., 1997). For reference, elevated IL-8 release in CSF in severe traumatic brain injury is reportedly as high as 8,000 pg/ml (Kossmann et al., 1997). Taken together, at least in young and healthy volunteers, it appears that exposure to high-altitude does not exacerbate pro-inflammatory stress in response to maximal exercise in the brain. Although not directly tested, the current exercise intervention did not initiate any symptoms of altitude illness or headache. As pro-inflammatory stress is implicated in the pathophysiology of altitude illness (Pham et al., 2021; Turner et al., 2021), these data support that, in the context of the current experimental study following acclimatization to 3,800 m, this duration and intensity of exercise is not a trigger for altitude illness via pro-inflammatory stress. Experimental considerations Maximal fat oxidation during submaximal cycling exercise was reduced at high-altitude, yet there were no anti-inflammatory implications of this alteration in systemic substrate utilization. In particular, although IL-4 is implicated in fatty acid oxidation (Vats et al., 2006), there were no changes in arterial or cerebral uptake/release of IL-4 during the 1-hour FATmax exercise (>50 % samples below the observable concentration range). Although exercise intensity was matched for altitude-specific FATmax and relative exercise intensities, it is noteworthy that these immune/inflammatory responses are influenced by the reduction in absolute exercise intensity (peak power output reduced by 27 W at HA; Figure 4-5). Further, FATmax was appreciably lower than previously reported values during upright cycling (Figure 4-5 and Figure 4-6 vs. approx. 45-50% VO2max corresponding to 35-40% Wmax (Croci et al., 2014)). This is perhaps attributable to postural-related differences in exercise efficiency, economy, and/or venous return with semi-recumbent cycling. However, this reduction in FATmax intensity compared to upright cycling was consistent between altitudes and did not alter systemic inflammatory responses. 4.6 Conclusion The results of this study provide new evidence that 6-8 days of acclimatization at 3,800 m does not provoke immune suppression or mediate elevations in systemic pro-inflammation with INFLAMMATORY STRESS IN THE BRAIN WITH EXERCISE AT HIGH-ALTITUDE 112 maximal exercise. Acclimatization to high-altitude attenuates systemic pro-inflammatory responses at rest; however, although independent of altitude, an additive effect of exercise duration/intensity provokes trans-cerebral net release of pro-inflammatory cytokines. The implications of these findings remain to be elucidated. IMMUNE/INFLAMMATORY RESPONSE AND EXERCISE PERFORMANCE WITH LEA 113 Chapter 5: Study 3A – The importance of low energy availability for the immune system, systemic inflammation, and performance ability in endurance-trained female athletes Abstract Background: Physical activity and energy intake are two key factors that can influence both the body’s immune system and systemic inflammation; thus, it is expected that a high level of exercise training together with low energy availability (LEA) would result in immune suppression and exacerbate the inflammatory response to exercise. Methods: Thirteen endurance-trained females (V̇O2max 53.6 ± 6.0 ml´min-1´kg-1) completed two 14-day randomized, blinded, cross-over, controlled dietary interventions: 1) energy balance (EB; 50 kcal/ kg FFM per day) and 2) low energy availability (LEA; 25 kcal/ kg FFM per day). Participants maintained their exercise training volume and intensity (approx. 8 hours running/cycling per week at approx. 78 % heart rate max) during both dietary interventions. Results: Dietary energy intake was reduced by 45.4 % during the LEA intervention (EB: 3251 kcal/day, 95% CI: 3054 to 3449 vs. LEA: 1774 kcal/day, 95% CI: 1579 to 1969, P < 0.001). The exercise-induced leukocyte mobilization was reduced by approximately 18 % following LEA compared to pre- LEA (P = 0.003) and post- EB (P = 0.005). At rest, LEA provoked a systemic inflammatory response (19/96 identified inflammatory biomarkers via targeted proteomics). Exercise performance during a 20-minute time trial and supramaximal time to exhaustion test was impaired by 7.8 % and 18.9 %, irrespective of the LEA-induced 2.5 kg reduction in total body mass. Further, performance impairments persisted following 3-days recovery in sufficient EB post- LEA (Post-LEA vs. Recovery, P = 0.409). Conclusions: Taken together, 14-days LEA in endurance-trained females provokes immunological stress, systemic inflammation and impairs exercise performance. 5.1 Introduction Regular physical activity leads to an improvement in the immune system. However, an excessive amount of exercise – particularly prolonged high-intensity exercise, such as long-distance running – can negatively affect the immune system by initiating a pro-inflammatory response and increasing the risk of infections (Barros et al., 2017). In addition to physical activity, the immune system is affected by dietary intake, whereby a reduced energy intake and thus limited energy availability can also lead to an impaired immune response. If continued over time, a chronic state of elevated systemic inflammation may result (Bermon et al., 2017). The immune response to infection/inflammation is responsible for up to approximately 25-30% total energy requirements (Schmid et al., 2000; Straub et al., 2010). Physical activity and energy IMMUNE/INFLAMMATORY RESPONSE AND EXERCISE PERFORMANCE WITH LEA 114 intake are therefore two key factors that can influence both the body’s immune system and systemic inflammation; thus, it is expected that a high level of exercise training together with low energy availability (LEA) would result in immune suppression and exacerbate the inflammatory response to exercise (Straub et al., 2010; Nieman & Wentz, 2019). It is widely accepted that exposure to LEA is related to a variety of health and performance impairments as supported by prospective interventional studies conducted in a tightly-controlled laboratory setting (Loucks et al., 1998; Ihle & Loucks, 2004) or with free-living conditions (Koehler et al., 2016; Papageorgiou et al., 2018; McKay et al., 2022), longitudinal observational studies (Vanheest et al., 2014), and cross-sectional data (reviewed in: (Logue et al., 2018; 2020; Areta et al., 2021; De Souza et al., 2022)). The relationship between body mass, training quality and performance is particularly relevant in weight-dependent endurance sports, such as long-distance running. For example, a recent study reported that short severe energy restriction (15 kcal/ kg fat free mass/ day for 9 days) with pre-race refueling reduces body mass without altering training-associated performance improvement in highly-trained race walkers (Burke et al., 2023). Importantly, however, chronic exposure to LEA may impair physical performance via a reduction in training/recovery capacity and adaptation (Vanheest et al., 2014), in addition to an acute reduction in muscle glycogen reserves (Kojima et al., 2020). To date, unlike the majority of reports in short-term (3-5 days, (Loucks & Heath, 1994; Loucks & Thuma, 2003; Areta et al., 2021) very few studies have investigated the effect of persistently LEA (>10 days) in female athletes and its implications for the immune system and systemic inflammation (Mountjoy et al., 2018b; 2018a). The aim of this study was to investigate the effect of LEA in endurance-trained female athletes on: 1) the immune system, 2) systemic inflammation, and 3) performance capacity. Additionally, an aim was to investigate the acute immunological response to a high-intensity training session to evaluate whether the inflammatory response is increased when maximal exercise is performed with low energy availability. It was hypothesized that low energy availability with high training volume will: 1) increase immunological stress; 2) elicit systemic inflammation; and 3) impair performance. Additionally, it was hypothesized that LEA-induced immune suppression would exacerbate the inflammatory response following high-intensity exercise. 5.2 Methods Ethical Approval IMMUNE/INFLAMMATORY RESPONSE AND EXERCISE PERFORMANCE WITH LEA 115 Participants provided informed written consent prior to participating in this study. The study was approved by the Ethics Committee of the Capital Region of Copenhagen (H-21032399) and conducted according to the principles established by the Declaration of Helsinki. Participants Thirteen young endurance-trained eumenorrheic females participated in this study (VO2max 53.6 ± 6.0 ml × min-1 × kg-1). Participants maintained their exercise training volume (approximately 8 hrs/week) during both dietary interventions. Inclusion Criteria 1. Females aged 18-35 years 2. BMI 18-23 3. Regular endurance training > 6 hours/week Exclusion Criteria 1. Irregular menstruation within the last 6 months (shorter than 24 days or longer than the 35 day cycle) 2. Ratio between estimated resting metabolic rate and measured resting metabolic rate of < 0.9 (Staal et al., 2018) 3. Low Energy Availability in Females Questionnaire (LEAF-Q) > 8 (Melin et al., 2014) 4. Injuries/pain that specifically prevents endurance training 5. Medication that can affect inflammatory parameters 6. Chronic disease and chronic medication related to metabolism or nutrient uptake 7. Smoking 8. Unstable weight / major weight loss (weight fluctuations of > 5 kg in the last six months) 9. Vegan 10. Eating disorder examination questionnaire (EDE-Q) > 2.3 (Fairburn & Beglin, 1994) 11. Pregnancy 12. Donated blood within the past 1 month 13. Cannot read or understand Danish Experimental Overview This study was performed within the Department of Nutrition, Exercise and Sports at the August Krogh Institute (University of Copenhagen, Denmark). This study was a randomized, single-IMMUNE/INFLAMMATORY RESPONSE AND EXERCISE PERFORMANCE WITH LEA 116 blinded, placebo-controlled, crossover design whereby n=13 endurance-trained females completed two 14-day diet interventions: 1) Energy balance (EB), 50 kcal/kg FFM per day; and 2) Low energy availability (LEA), 25 kcal/kg FFM per day (A). Overall, n=7 participants started with EB and n=6 started with the LEA diet intervention. Following each 14-day diet intervention, participants completed 3 days of recovery in energy balance. The diet interventions were separated by approximately ³14 days wash-out. Due to the 14-day diet intervention followed by 14 days wash-out, participants began each intervention at approximately the same time in their respective menstrual cycle, which was verified through blood analysis pre/post both diets (estrogen, progesterone, testosterone, transferrin, follicle-stimulating hormone, and luteinizing hormone). Participants were tested immediately prior to each diet intervention (T2/T6, day #1); after 7 days (T3/T7, day #8) and 14 days of diet (T4/T8, day #15); and following 3 days of recovery in energy balance (T5/T9, day #18) (B). Participants were instrumented with a continuous glucose monitor (CGM) on T3/T7 which recorded data during days 7-14 of the diet intervention and throughout the 3 days of recovery. Insulin sensitivity was assessed following each diet intervention on T4/T8 via fasting plasma glucose/insulin and mixed meal tolerance test. Muscle biopsies were performed pre- (T2/T6) and post- (T4/T8) each diet intervention. Test Day Procedures A brief overview of each test day is summarized below and illustrated in Figure 5-1 and Figure 5-2. Test day 1 (screening) Study explanation, questionnaire, sign consent; DXA scan; blood sample; RMR; 10-lead ECG; resting blood pressure; submaximal warmup, VO2max test, TTE, 20-min time trial familiarization. Test day 2 & 6 (pre- diet) DXA scan; blood sample; RMR; 5-lead ECG; resting blood pressure; breakfast; muscle biopsy; submaximal warmup, 20-min time trial, TTE; post- exercise blood sample, ECG, blood pressure. Test day 3 & 7 (diet day 7) DXA scan; blood sample; CGM insertion Test day 4 & 8 (post- diet) IMMUNE/INFLAMMATORY RESPONSE AND EXERCISE PERFORMANCE WITH LEA 117 DXA scan; blood sample; RMR; 5-lead ECG; resting blood pressure; breakfast; muscle biopsy; blood sample MMTT (60-mins post- breakfast); submaximal warmup, 20-min time trial, TTE; post- exercise blood sample, resting VO2, ECG, blood pressure. Test day 5 & 9 (recovery) DXA scan; blood sample; breakfast; 5-lead ECG; resting blood pressure; submaximal warmup, 20-min time trial, TTE; post- exercise blood sample, ECG, blood pressure. IMMUNE/INFLAMMATORY RESPONSE AND EXERCISE PERFORMANCE WITH LEA 118 Figure 5-1. Overview of study protocol and experimental methods IMMUNE/INFLAMMATORY RESPONSE AND EXERCISE PERFORMANCE WITH LEA 119 This study was a randomized, single-blinded, placebo-controlled, crossover design whereby n=13 endurance-trained females completed two 14-day diet interventions: 1) Energy balance, 50 kcal/kg FFM per day; and 2) Low energy availability, 25 kcal/kg FFM per day (A). Following each 14-day diet intervention, participants completed 3 days of recovery in energy balance. The diet interventions were separated by approximately ³14 days wash-out. Participants were tested immediately prior to each diet intervention (T2/T6, day #1); after 7 days (T3/T7, day #8) and 14 days of diet (T4/T8, day #15); and following 3 days of recovery in energy balance (T5/T9, day #18) (B). Participants were instrumented with a continuous glucose monitor (CGM) on T3/T7 which recorded data during days 7-14 of the diet intervention and throughout the 3 days of recovery. Insulin sensitivity was assessed following each diet intervention on T4/T8 via fasting plasma glucose/insulin and mixed meal tolerance test. Muscle biopsies were performed pre- (T2/T6) and post- (T4/T8) each diet intervention and mitochondrial oxidative capacity was assessed via high-resolution respirometry (OROBOROS, Oxygraph-2k). This study was performed within the Department of Nutrition, Exercise and Sports at the August Krogh Institute (University of Copenhagen, Denmark) (C). IMMUNE/INFLAMMATORY RESPONSE AND EXERCISE PERFORMANCE WITH LEA 120 Figure 5-2. LEA Study – Exercise protocol schematic IMMUNE/INFLAMMATORY RESPONSE AND EXERCISE PERFORMANCE WITH LEA 121 Participants completed an incremental step protocol maximal exercise test followed by a supramaximal time to exhaustion verification test to determine maximal oxygen uptake (VO2max) on the initial screening visit (T1) (A). The exercise performance test battery was as follows: 1) three 4-minute submaximal warmup exercise stages at individualized workloads corresponding to 50%, 60%, and 70% VO2max; 2) 20-minute isokinetic time trial; and 3) time to exhaustion at 110% VO2max (B). Venous ABL samples were taken at rest, within the last minute of each warmup stage, and immediately post- exercise (C). This exercise performance test was completed pre/post each diet intervention (T2/T4/T6/T8) and after three days of recovery in energy balance following each diet (T5/T9). IMMUNE/INFLAMMATORY RESPONSE AND EXERCISE PERFORMANCE WITH LEA 122 Dietary Intervention Participants were provided all foods throughout the experimental period based on individualized meal plans to achieve EB and LEA in the diet interventions. Diets were designed to provide a balanced macronutrient distribution (Table 5-1). Meals included breakfast (oats, raisins, milk), prepackaged meals for lunch and dinner (Getfitfood.dk, Skovlunde, Denmark), and snacks (unsalted almonds, dark chocolate, vanilla skyr). In an attempt to blind participants to the dietary intervention, highly-concentrated juice syrup containing either zero calories (LEA) or approx. 331 kcal/ 100 ml (82 g carbohydrate/ 100 ml) (EB) was provided. Additionally, maltodextrin and protein powder were provided (bodylab.dk, Hadsund, Denmark). All food/drink were individually weighed and provided in clear plastic bags/cups labeled for each day. The level of EB was selected based on an earlier study reporting an average energy availability of 50 kcal/ kg FFM per day in eumenorrheic female athetes (Melin et al., 2015). The level of LEA was based on previous literature showing disruption of the hypothalamic-pituitary-thyroid and ovarian axis at energy availability <30 kcal/ kg FFM per day (Loucks & Heath, 1994; Loucks & Thuma, 2003). Exercise Training Exercise energy expenditure was calculated from the predicted training volume each participant provided of their habitual training, subtracted by measured resting energy expenditure (via indirect calorimetry). A linear regression was calculated using energy expenditure (kcal/min) and heart rate measured during the last 60-s of the three 4-minute steady-state submaximal warmup intensities performed on the initial screening visit (100 W, 125 W, 150 W). This calculation was used to determine exercise energy expenditure at a given intensity and duration for each workout (as verified by heart rate and training session duration records). All endurance exercise was accounted for (running, cycling, swimming; via self-report with Strava/Garmin/Apple Watch), whereas daily physical activity (non-exercise activity thermogenesis; NEAT) was not measured and assumed to be relatively consistent between diet interventions (e.g., instructions were to maintain habitual cycle commuting and approximate steps/day). Experimental Procedures Dual-Energy X-ray Absorptiometry IMMUNE/INFLAMMATORY RESPONSE AND EXERCISE PERFORMANCE WITH LEA 123 DXA scans were performed using a Lunar iDXA (GE Healthcare, Brøndby, Denmark) which was calibrated daily according to manufacturer’s instructions. Participants were scanned following an overnight fast at the same time each morning within approximately 1 hour between visits. Resting Metabolic Rate Resting energy expenditure was determined by indirect calorimetry (Vyaire Vyntus CPX). A 25-minute measurement was performed and the first 5 minutes were excluded from data analysis. Breath-by-breath VO2 data were analyzed as 1-minute averages and the RMR was identified as the lowest energy expenditure value. RMR ratio was calculated as the ratio between measured RMR and predicted RMR, where predicted RMR was calculated by the Cunningham equation (Cunningham, 1980). An eligibility criteria of < 0.9 for the RMR ratio was used, which is currently accepted as a useful marker for LEA in physically active females (De Souza et al., 2007; 2008). Blood Samples An indwelling I.V. catheter (Venflon, 20 G, Becton Dickinson Infusion Therapy Systems, UT, USA) was placed in the antecubital vein to obtain venous blood samples at rest and during exercise. Approximately 1.0 ml of venous blood was drawn into pre-heparinized syringes (SafePICO, Radiometer, Copenhagen, Denmark) and analyzed immediately using a commercial blood gas analyzer (ABL800 FLEX, Radiometer) at rest, within the last minute of each submaximal warmup stage, and post- exercise. Blood b-OHB was measured with a handheld monitor and test strip (FreeStyle Precison Neo, Abbott Diabetes Care Ltd., Witney, UK). Blood samples were centrifuged at 20°C and 2000 g for 10 minutes before plasma was collected and stored at -80°C for later analysis. Samples were analyzed for HbA1c, creatine kinase, cortisol, C-reactive protein, high-sensitivity CRP, estrogen, FSH, LH, progesterone, testosterone, transferrin, insulin, and c-peptide within 2 hours (Rigshospitalet, Copenhagen, Denmark). 5.3 Statistical analyses All data are presented in-text as mean with 95% confidence intervals and as individual values in figures. Data analyses were performed using RStudio (2021.09.1) and statistical analyses were performed using SPSS software (IBM statistics, Version 23.0). Statistical significance was set at P < 0.05. A linear mixed-effects model analysis with compound symmetry covariance structure with fixed effects of time (pre vs. post) and treatment (EB vs. LEA) was performed for repeated measures comparisons. Further, fixed effects of exercise (pre- vs. post- exercise) and treatment (pre- EB, post- EB, pre- LEA, post- LEA) were used to compare exercise responses between IMMUNE/INFLAMMATORY RESPONSE AND EXERCISE PERFORMANCE WITH LEA 124 diet interventions. A Bonferroni correction was applied for multiple comparisons when significant interactions were detected. For selected variables, treatment (EB vs. LEA) was the only fixed effect. Subjects were included as a random effect. A two-tailed dependent sample Student’s t-test was used to compare the following inflammatory proteomic data: 1) pre- versus post- LEA at rest; 2) pre- versus post- EB at rest; 3) rest versus post- exercise on the post- LEA test day; and 4) rest versus post- exercise on the post- EB test day. Adjusted P-values were calculated for false discovery rate as: (original P-value / rank value) ´ 75. Data are expressed as log 2 values with 95% confidence intervals. 5.4 Results Diet intervention Table 5-1 shows the diet composition during the EB and LEA interventions. Overall, macronutrient quantity (g/kg) and total energy (kcal) were higher during EB (all P < 0.001). The relative contribution of protein, carbohydrate, and fat to dietary intake was significantly different between trials (all P < 0.05), although within approximately 5% for all macronutrients. Table 5-1. Diet composition during energy balance (EB) and low energy availability (LEA) interventions. EB LEA D LEA vs. EB P-Value PRO intake (g/kg/day) 2.5 (2.3-2.8) 1.8 (1.5-2.0) -0.7 (-0.9 to -0.6) P < 0.001 CHO intake (g/kg/day) 6.2 (5.6-6.7) 3.2 (2.7-3.7) -3.0 (-3.4 to -2.6) P < 0.001 FAT intake (g/kg/day) 1.7 (1.6-1.9) 0.9 (0.7-1.0) -0.9 (-1.0 to -0.7) P < 0.001 Energy PRO (kcal/day) 638 (577-698) 444 (384-503) -194 (-237 to -151) P < 0.001 Energy CHO (kcal/day) 1553 (1448-1659) 799 (697-901) -754 (-867 to -642) P < 0.001 Energy FAT (kcal/day) 986 (919-1053) 496 (431-562) -490 (-555 to -425) P < 0.001 PRO (Energy %) 20.0 (18.4-21.6) 25.5 (23.9-27.1) +5.5 (3.8 to 7.2) P < 0.001 CHO (Energy %) 48.9 (47.2-50.6) 45.9 (44.3-47.6) -3.0 (-5.7 to -0.2) P = 0.038 FAT (Energy %) 31.2 (29.4-32.9) 28.5 (26.9-30.2) -2.6 (-4.8 to -0.4) P = 0.025 Exercise training Participants maintained their exercise training volume comparable during both diet interventions (EB: 8.5 hrs/week, 95% CI: 7.5 to 9.5 vs. LEA: 8.6 hrs/week, 95% CI: 7.6 to 9.6, P = 0.380; Figure 5-3A). Average HR was not different during training between the diet interventions (EB: 146 bpm, 95% CI: 140 to 152 vs. LEA: 145 bpm, 95% CI: 139 to 151, P = 0.421; Figure 5-3B). Therefore, HR relative to max was not different (EB: 79 %, 95% CI: 76 to 83 vs. LEA: 78 %, 95% CI: 75 to 82, P = 0.431; Figure 5-3C). IMMUNE/INFLAMMATORY RESPONSE AND EXERCISE PERFORMANCE WITH LEA 125 Figure 5-3. Endurance exercise training volume and intensity per week during the 14-day energy balance (EB) and low energy availability (LEA) diet interventions. Participants maintained their exercise training volume during both diet interventions (P = 0.380) (A). Average HR was not different during training between the diet interventions (P = 0.421) (B). Therefore, HR relative to max was not different (P = 0.431) (C). Sex hormones There were no time ´ treatment interactions for estrogen (P = 0.426), progesterone (P = 0.962), testosterone (P = 0.054), transferrin (P = 0.132), follicle-stimulating hormone (P = 0.553), or luteinizing hormone (P = 0.309). There was a time effect of progesterone (Pre- diet: 1.2 nmol/l, 95% CI: -3.2 to 5.7 vs. Post- diet: 6.0 nmol/l, 95% CI: 1.6 to 10.3; P = 0.035), inclusive of both diet interventions (treatment effect: P = 0.878). Diet-induced weight loss Dietary energy intake was reduced by 45.4 % during the LEA intervention (EB: 3251 kcal/day, 95% CI: 3054 to 3449 vs. LEA: 1774 kcal/day, 95% CI: 1579 to 1969, P < 0.001; Figure 5-4A). Total body mass was reduced by 2.5 kg post- LEA (Pre-LEA: 62.3 kg, 95% CI: 57.1 to 67.4 vs. Post-LEA: 59.8 kg, 95% CI: 54.6 to 64.9, P < 0.001; Figure 5-4B) and to a lesser extent by 0.7 kg post- EB (Pre-EB: 61.9 kg, 95% CI: 56.8 to 67.1 vs. Post-EB: 61.2 kg, 95% CI: 56.1 to 66.4, P = 0.026; Figure 5-4B). There was no difference in total body mass between the pre- diet test EB LEA567891011Training volume (hr/week)P = 0.380EB LEA120130140150160170180Average training HR (bpm)P = 0.421EB LEA7075808590Average training HR (% max)P = 0.431CBAIMMUNE/INFLAMMATORY RESPONSE AND EXERCISE PERFORMANCE WITH LEA 126 days (P = 0.239). Fat free mass was reduced by 0.8 kg post- LEA (Pre-LEA: 48.0 kg, 95% CI: 44.7 to 51.2 vs. Post-LEA: 47.2 kg, 95% CI: 43.9 to 50.4, P = 0.001; Figure 5-4C). There was no difference in fat free mass during the EB intervention (Pre- vs. Post- EB: P = 0.394) or between the pre- diet test days (P = 0.088; Figure 5-4C). Prior to the diet interventions, body fat mass was higher by 0.5 kg pre- LEA (95% CI: 0.2 to 0.9, P = 0.004). Body fat mass was reduced by 1.1 kg post- LEA (Pre-LEA: 6.0 kg, 95% CI: 4.8 to 7.2 vs. Post-LEA: 4.9 kg, 95% CI: 3.7 to 6.1, P < 0.001; Figure 5-4D) while there was no change in body fat mass during the EB intervention (Pre- vs. Post- EB: P = 0.180). Figure 5-4. Dietary energy intake and related body mass changes pre- vs. post- 14 days of energy balance (EB) and low energy availability (LEA). The LEA intervention was effective in that dietary energy intake was reduced by 45.4 % (P < 0.001) (A). As a result, total body mass was reduced by 2.5 kg post- LEA (P < 0.001) and to a lesser extent by 0.7 kg post- EB (P = 0.026) with no difference between pre- EB and LEA test days (P = 0.239) (B). Fat free mass was reduced by 0.8 kg post- LEA (P = 0.001) (C). There was no difference in fat free mass during the EB intervention (P = 0.394) or between the pre- diet test days (P = 0.088) (C). Prior to the diet interventions, body fat mass was higher by 0.5 kg pre- LEA (P = 0.004) (D). Body fat mass was reduced by 1.1 kg post- LEA (P < 0.001) (D) while there was no change in body fat mass during the EB intervention (P = 0.180). Energy balance is shown in blue and LEA is shown in red. Immune response There was no diet-induced change in calculated plasma volume between the EB and LEA interventions (P = 0.628). Additionally, there was no difference in the exercise-induced reduction in calculated plasma volume pre- versus post- both EB and LEA interventions (P = 0.665). As such, the reported changes in WBC count are not explained by diet or exercise- induced changes in plasma volume. There was no difference in WBC count at rest (Pre-Ex) within or between diet interventions (all P > 0.05). The WBC count was higher post- exercise within each test day (all P < 0.001). The EB LEA10002000300040005000Dietary energy intake (kcal/day)-45.4 %P < 0.001PrePost PrePost4050607080Total body mass (kg)-Δ 2.5 kgP < 0.001-Δ 0.7 kgP = 0.026PrePost PrePost35404550556065Fat free mass (kg)-Δ 0.8 kgP = 0.001PrePost PrePost246810Body fat mass (kg)-Δ 1.1 kgP < 0.001A B C DIMMUNE/INFLAMMATORY RESPONSE AND EXERCISE PERFORMANCE WITH LEA 127 post- exercise WBC count on the post- LEA test day was lower than all other post-exercise WBC values on the respective test days (Post-Ex, Post-LEA: 7.2 vs. Pre-LEA: 8.9, Pre-EB: 10.0, Post-EB: 8.9, all P < 0.005; Figure 5-5A). Relative distribution of WBC types There were no exercise ´ treatment interaction effects for % distribution of lymphocytes, monocytes, neutrophils, eosinophils, or basophils (P = 0.752, P = 0.283, P = 0.784, P = 0.859, P = 0.548, respectively; Figure 5-5B). Pre- versus post- exercise (exercise effect) Post- exercise, there was a reduction in % distribution of monocytes by 11.3 % (-D 1.0 % points, 95% CI: -1.5 to -0.5; P < 0.001), eosinophils by 59.8 % (-D 1.5 % points, 95% CI: -2.0 to -1.1; P < 0.001), and basophils by 20.3 % (-D 0.15 % points, 95% CI: -0.2 to -0.1; P < 0.001). There was a higher % distribution of neutrophils by 8.4 % (D 4.0 % points, 95% CI: 1.4 to 6.5; P = 0.002) post- versus pre- exercise. Pre- versus post- LEA (treatment effect) Pre- versus post- LEA, there was a higher % distribution of lymphocytes by 11.9 % (D 4.6 % points, 95% CI: 0.02 to 9.1; P = 0.049) and basophils by 15.5 % (D 0.1 % points, 95% CI: 0.0 to 0.2; P = 0.048). Figure 5-5. White blood cell count and cell-type distribution in response to exercise pre- vs. post- 14 days of energy balance (EB) and low energy availability (LEA). Inflammatory/stress response EBPre- DietLEA EBPost- DietLEA5101520WBC (×103 cells/µl)Ex × treatment post-hoc:Pre- Ex: all P > 0.05Pre- vs. Post- Ex: all P < 0.001Post- Ex, Post- LEA vs. all: P < 0.05Pre-ExPost-ExPRE-EX (PRE-EB)POST-EX (PRE-EB)PRE-EX (PRE-LEA)POST-EX (PRE-LEA)PRE-EX (POST-EB)POST-EX (POST-EB)PRE-EX (POST-LEA)POST-EX (POST-LEA)255075100WBC (% distribution) LymphocytesMonocytesNeutrophilsEosinophilsBasophilsEx (pre/post): Mono, Neut, Eo, Baso, all P < 0.05Baso/Lymph, treatment post-hoc: Pre- vs. Post- LEA: both P <0.05A BIMMUNE/INFLAMMATORY RESPONSE AND EXERCISE PERFORMANCE WITH LEA 128 Creatine kinase was increased by 23.3 % post- exercise, pre/post both diet interventions (Pre-Ex: 135 U/l, 95% CI: 91 to 179 vs. Post-Ex: 166 U/l, 95% CI: 123 to 210, exercise effect: P = 0.004; Figure 5-6A). Additionally, creatine kinase was higher post- LEA compared to post- EB by 45.7 % (Post-EB: 127 U/l, 95% CI: 81 to 173 vs. Post-LEA: 185 U/l, 95% CI: 139 to 231, treatment effect: P = 0.002; Figure 5-6A), inclusive of pre/post exercise. There was no difference in creatine kinase between pre- diet test days (Pre-EB: 145 U/l, 95% CI: 99 to 190 vs. Pre-LEA: 146 U/l, 95% CI: 100 to 192, treatment effect: P = 1.000; Figure 5-6A). Cortisol was reduced by 15.6 % post- exercise on the post-LEA test day (Pre-Ex: 808 nmol/l, 95% CI: 654 to 962 vs. Post-Ex: 682 nmol/l, 95% CI: 528 to 836, P = 0.006; Figure 5-6B). There was no difference in cortisol pre- versus post- exercise during the EB intervention or on the pre- LEA test day (all P >0.05). There was no effect of exercise on high-sensitivity C-reactive protein (hs-CRP) within or between diet interventions (exercise effect: P = 0.929). There was an effect of treatment such that hs-CRP was lower by 42.4 % post- LEA compared to pre- EB (Pre-EB: 1.4 mg/l, 95% CI: 1.0 to 1.8 vs. Post-LEA: 0.8 mg/l, 95% CI: 0.4 to 1.2, treatment effect: P = 0.026; Figure 5-6C). Figure 5-6. Systemic stress and inflammatory biomarkers in response to exercise pre- vs. post- 14 days of energy balance (EB) and low energy availability (LEA). Inflammatory protein biomarkers Figure 5-7A shows the inflammatory proteins which were significantly changed pre- versus post- the LEA intervention at rest. After P-Value adjustment for false discovery rate, 6 out of 19 identified proteins remained significantly affected by LEA (shown in black versus red, respectively). There were no significantly changed inflammatory proteins pre- versus post- the EB intervention at rest after P-Value adjustment for false discovery rate (Figure 5-7B). EBPre- DietLEA EBPost- DietLEA100200300400500600Creatine kinase (U/l)Ex (pre/post): P = 0.004Treatment post-hoc:Post- LEA vs. Post- EB, P = 0.002Ex × treatment: P = 0.997EBPre- DietLEA EBPost- DietLEA500100015002000Cortisol (nmol/l)Ex (pre/post): P = 0.714Treatment: P = 0.170Ex × treatment post-hoc:Post-LEA, Pre- vs. Post- Ex: P = 0.006EBPre- DietLEA EBPost- DietLEA12345hs-CRP (mg/l) Pre-ExPost-ExEx (pre/post): P = 0.929Treatment post-hoc:Pre- EB vs. Post- LEA, P = 0.026Ex × treatment: P = 0.975A B CIMMUNE/INFLAMMATORY RESPONSE AND EXERCISE PERFORMANCE WITH LEA 129 Figure 5-8 and Figure 5-9 show the inflammatory proteins which were significantly changed pre- versus post- exercise during the post- LEA and post- EB test days, respectively. After P-Value adjustment for false discovery rate, 48 out of 52 identified proteins remained significantly affected post- exercise following the LEA diet (shown in black versus red, respectively; Figure 5-8) and 47 out of 53 identified proteins remained significantly affected post- exercise following the EB diet (Figure 5-9). There was considerable overlap between the post- exercise inflammatory protein responses following both diet interventions (Figure 5-8 and Figure 5-9). However, of the total identified proteins (P-Value adjusted), there were 9 which were selectively altered with exercise post- LEA only (CD40, FGF-5, FGF-19, IL-12B, MCP-4, MMP-10, SLAMF1, TNF, TNFB) and 8 which were altered with exercise post- EB only (CCL20, CCL28, CXCL11, GDNF, IFN-gamma, IL8, IL-10RA, MMP-1). IMMUNE/INFLAMMATORY RESPONSE AND EXERCISE PERFORMANCE WITH LEA 130 Figure 5-7. Inflammatory protein biomarkers identified at rest following 14-days of energy balance (EB) and low energy availability (LEA). Targeted 96 Inflammatory Protein Biomarker Panel via High-Multiplex Immunoassay. After P-Value adjustment for false discovery rate, 6 out of 19 identified proteins remained significantly affected by LEA (shown in black versus red, respectively) (A). There were no significantly changed inflammatory proteins pre- versus post- the EB intervention at rest after P-Value adjustment for false discovery rate (B). -0.10 -0.05 0.00 0.05 0.10IL-18R1IL-10RBVEGFAFlt3LMCP-1IL18CSF-1TWEAKSCFMCP-4TNFBLIF-RMMP-10Significantly changed proteins post- LEAAdjusted P-Value < 0.05-0.030 -0.025 -0.020 -0.015 -0.010 -0.005 0.000VEGFASignificantly changed proteins post- EB-1.0 -0.5 0.0 0.5 1.0IL6FGF-21OSMCDCP1Beta-NGFCCL28Log 2 changesAdjusted P-Value < 0.05-3 -2 -1 0 1 2 3IL-20RAIL2Log 2 changesA BNB: All P > 0.05when P-Value adjustedIMMUNE/INFLAMMATORY RESPONSE AND EXERCISE PERFORMANCE WITH LEA 131 Figure 5-8. Inflammatory protein biomarkers identified post- exercise following 14-days of low energy availability (LEA). 0.00 0.05 0.10 0.15 0.20MMP-10TRAILTWEAKDNERCD8AMCP-1CXCL9MCP-4SCFCSF-1VEGFAIL-10RBIL18OPGCD40IL-12BCST5ADAPD-L1CCL11MCP-2TNFBCD5CCL19LAP TGF-beta-1CCL23HGFCXCL11uPAIL-18R1CD244Flt3LFGF-19CXCL6Significantly changed proteins post- Ex following LEAAdjusted P-Value < 0.05-1.0 -0.5 0.0 0.5 1.0 1.5TNFCCL25CX3CL1CD6TGF-alphaLIF-RFGF-23FGF-5GDNFTNFSF14CDCP1IL10TSLPOSMEN-RAGESLAMF1CASP-8IL6Log 2 changesAdjusted P-Value < 0.05IMMUNE/INFLAMMATORY RESPONSE AND EXERCISE PERFORMANCE WITH LEA 132 Targeted 96 Inflammatory Protein Biomarker Panel via High-Multiplex Immunoassay. After P-Value adjustment for false discovery rate, 48 out of 52 identified proteins remained significantly affected post- exercise following the LEA diet (shown in black versus red, respectively). IMMUNE/INFLAMMATORY RESPONSE AND EXERCISE PERFORMANCE WITH LEA 133 Figure 5-9. Inflammatory protein biomarkers identified post- exercise following 14-days of energy balance (EB). -0.10 -0.05 0.00 0.05 0.10 0.15 0.20IFN-gammaMMP-10DNERCD8AMCP-1TWEAKSCFVEGFACSF-1TRAILIL-12BCXCL9IL18IL-10RBCD40CST5OPGCCL11MMP-1CCL20MCP-2ADACCL23CCL19TNFHGFFlt3LCD5uPACD244CXCL11CXCL6IL-18R1LAP TGF-beta-1IL8Significantly changed proteins post- Ex following EBAdjusted P-Value < 0.05-3 -2 -1 0 1 2IL-2RBIL5IL-10RACCL25CX3CL1LIF-RCD6FGF-23TGF-alphaTNFSF14CDCP1CCL28IL10GDNFOSMEN-RAGECASP-8IL6Log 2 changesAdjusted P-Value < 0.05IMMUNE/INFLAMMATORY RESPONSE AND EXERCISE PERFORMANCE WITH LEA 134 Targeted 96 Inflammatory Protein Biomarker Panel via High-Multiplex Immunoassay. After P-Value adjustment for false discovery rate, 47 out of 53 identified proteins remained significantly affected post- exercise following the EB diet shown in black versus red, respectively). Abbreviations: ADA, adenosine deaminase; AXIN1, axin-1; b-NGF, beta-nerve growth factor; CASP-8, caspase-8; CCL3, C-C motif chemokine 3; CCL4, C-C motif chemokine 4; CCL11, eotaxin; CCL19, C-C motif chemokine 19; CCL20, C-C motif chemokine 20; CCL23, C-C motif chemokine 23; CCL25, C-C motif chemokine 25; CCL28, C-C motif chemokine 28; CD5, T-cell surface glycoprotein CD5; CD6, T-cell surface glycoprotein CD6 isoform; CD8A, T-cell surface glycoprotein CD8 alpha chain; CD40, CD40L receptor; CD244, natural killer cell receptor 2B4; CDCP1, CUB domain-containing protein 1; CSF-1, macrophage colony-stimulating factor 1; CST5, cystatin D; CX3CL1, fractalkine; CXCL1, C-X-C motif chemokine 1; CXCL5, C-X-C motif chemokine 5; CXCL6, C-X-C motif chemokine 6; CXCL9, C-X-C motif chemokine 9; CXCL10, C-X-C motif chemokine 10; CXCL11, C-X-C motif chemokine 11; DNER, delta and Notch-like epidermal growth factor-related receptor; 4E-BP1, eukaryotic translation initiation factor 4E-binding protein 1; EN-RAGE, protein S100-A12; FGF-5, fibroblast growth factor 5; FGF-19, fibroblast growth factor 19; FGF-21, fibroblast growth factor 21; FGF-23, fibroblast growth factor 23; Flt3L, fms-related tyrosine kinase 3 ligand; GDNF, glial cell line-derived neurotropic factor; HGF, hepatocyte growth factor; IFN-g, interferon gamma; IL-6, interleukin-6; IL-7, interleukin-7; IL-8, interleukin-8; IL-10, interleukin-10; IL-10Ra, interleukin-10 receptor subunit alpha; IL-10Rb, interleukin-10 receptor subunit beta; IL-12b, interleukin-12 subunit beta; IL-15Ra, interleukin-15 receptor subunit alpha; IL-17A, interleukin-17A; IL-17C, interleukin-17C; IL-18, interleukin-18; IL-18R1, interleukin-18 receptor 1; IL-20Ra, interleukin-20 receptor subunit alpha; LIF-R, leukemia inhibitory factor receptor; MCP-1, monocyte chemotactic protein 1; MCP-2, monocyte chemotactic protein 2; MCP-4, monocyte chemotactic protein 4; MMP-1, matrix metalloproteinase-1; MMP-10, matrix metalloproteinase-10; NT-3, neurotrophin-3; OPG, osteoprotegerin; OSM, oncostatin-M; PD-L1, programmed cell death 1 ligand 1; SCF, stem cell factor; SIRT2, SIR2-like protein 2; SLAMF1, signaling lymphocytic activation molecule; ST1A1, sulfotransferase; STAMBP, STAM-binding protein; TGF-a, transforming growth factor alpha; TGF-b, transforming growth factor beta; TNF, tumor necrosis factor; TNF-b, tumor necrosis factor beta; TNFRSF9, tumor necrosis factor receptor superfamily member 9; TNFSF14, tumor necrosis factor ligand superfamily member 14; TRAIL, TNF-related apoptosis-inducing ligand; TRANCE, TNF-related activation-induced cytokine; TWEAK, tumor necrosis factor (Ligand) superfamily, member 12; uPA, urokinase-type plasminogen activator; VEGF, vascular endothelial growth factor. IMMUNE/INFLAMMATORY RESPONSE AND EXERCISE PERFORMANCE WITH LEA 135 Exercise performance Power output during the 20-minute time trial was reduced by 7.8 % post- LEA (Pre-LEA: 216 W, 95% CI: 198 to 234 vs. Post-LEA: 199 W, 95% CI: 181 to 218, P < 0.001; Figure 5-10A). There was no change in power output during the EB intervention (Pre-EB: 211 W, 95% CI: 193 to 229 vs. Post-EB: 210 W, 95% CI: 191 to 228, P = 0.766; Figure 5-10A) or between pre- EB and LEA test days (P = 0.124). These results persisted when expressed as power output relative to total body mass (Post- vs. Pre- LEA: -3.7 %, P = 0.014; Figure 5-10B) or fat free mass (Post- vs. Pre- LEA: -6.2 %, P < 0.001; Figure 5-10C) with no change pre- versus post- EB (P = 0.666 and P = 0.954, respectively). Power output relative to total body mass was not different between pre-diet test days (P = 0.237), although power output relative to fat free mass was higher pre- LEA compared to pre- EB by 3.7 % (P = 0.037). There was an effect of time for HR during the 20-minute time trial such that post- diet was reduced by 5 bpm (95% CI: -9 to 0, P = 0.030); however, there was no time ´ treatment interaction (P = 0.223; Figure 5-10D). This corresponded to a 2.6 % point (95% CI: -4.8 to 0) reduction in HR relative to max during the 20-minute time trial post- diet (time effect: P = 0.020; Figure 5-10E). Lastly, time to exhaustion was reduced by 18.9 % post- LEA (Pre-LEA: 121 s, 95% CI: 86 to 156 vs. Post-LEA: 98 s, 95% CI: 64 to 133, P = 0.009; Figure 5-10F). There was no change in time to exhaustion during the EB intervention (Pre-EB: 122 s, 95% CI: 87 to 157 vs. Post-EB: 123 s, 95% CI: 88 to 158, P = 0.925; Figure 5-10F) or between pre- EB and LEA test days (P = 0.911). IMMUNE/INFLAMMATORY RESPONSE AND EXERCISE PERFORMANCE WITH LEA 136 Figure 5-10. Exercise performance during the 20-minute time trial and time to exhaustion tests pre- vs. post- 14 days of energy balance (EB) and low energy availability (LEA). There was a reduction in power output during the 20-minute time trial by 7.8 % (P < 0.001) post- LEA with no change pre- versus post- EB (P = 0.766) or between pre- EB and LEA test days (P = 0.124) (A). This impairment in performance persisted when expressed as power output relative to total body mass (-3.7 %, P = 0.014) (B) or fat free mass (-6.2 %, P < 0.001) (C). There was no time ´ treatment interaction for average HR during the 20-minute time (P = 0.223); however there was a time effect such that HR was reduced by 5 bpm post- diet (P = 0.030) (D) which corresponded to a 2.6 % point reduction in HR relative to max post- diet (time effect: P = 0.020) (E). Lastly, time to exhaustion was reduced by 18.9 % (P = 0.009) post- LEA with no change pre- versus post- EB (P = 0.925) or between pre- EB and LEA test days (P = 0.911) (F). Energy balance is shown in blue and LEA is shown in red. 5.5 Discussion The results of this study indicate that 14-days LEA in endurance-trained females provokes immunological stress, systemic inflammation and impairs exercise performance. These results are supported by: 1) reduced WBC mobilization post- exercise following LEA (Figure 5-5); 2) higher creatine kinase with exercise and elevated systemic inflammatory biomarkers at rest PrePost PrePost50100150200250300Time to exhaustion (s)-18.9 %P = 0.009PrePost PrePost15017520022525027530020-TT power output (W)-7.8%P < 0.001PrePost PrePost2.53.03.54.04.5Relative power output (W/kg)-3.7 %P = 0.014PrePost PrePost14015016017018019020020-TT heart rate (bpm)Time × treatment:P = 0.223PrePost PrePost3.54.04.55.05.5Relative power output (W/kg FFM)-6.2 %P < 0.001PrePost PrePost70809010011020-TT heart rate (% max)Time × treatment:P = 0.256BDA CE FIMMUNE/INFLAMMATORY RESPONSE AND EXERCISE PERFORMANCE WITH LEA 137 post- LEA (via targeted proteomics) (Figure 5-6 and Figure 5-7); and 3) reductions in power output during a 20-minute time trial and overall time to exhaustion by 7.8 % and 18.9 %, irrespective of the LEA-induced 2.5 kg reduction in total body mass (Figure 5-10). Weight-loss implications of LEA Reductions in total body mass are often viewed as a favorable result of LEA. Research substantiates a relationship between lower body mass and faster competition times for novice and experienced marathon runners (Hagan et al., 1981; Holly et al., 1986; Hagan et al., 1987; Hoffman, 2008; Knechtle et al., 2009a). Further, low body fat specifically is related to faster race times (Bale et al., 1985; 1986; Knechtle et al., 2009b; 2011a). However, this is not universal with studies reporting no relationship between body composition and performance in recreational ultra-triathletes, recreational ultra-runners, and ultra-endurance cyclists (Knechtle et al., 2011c; 2011b). Total body mass was reduced by 2.5 kg following 14-days LEA in the current study – this was reflected by approximately equal reductions in fat free mass and body fat mass (-D 0.8 and 1.1 kg, respectively; Figure 5-4). It is noteworthy that the impairments in exercise performance during the 20-minute time trial persisted when expressed as power output relative to total body mass or fat free mass (Figure 5-10). As such, the LEA-induced reductions in body mass did not support a performance improvement in endurance-trained females. Further, body mass did not recover following three days of sufficient energy balance post- LEA (Post-LEA: 59.8 kg, 95% CI: 54.7 to 64.9 vs. Recovery: 60.4 kg, 95% CI: 55.3 to 65.5, P = 0.081; data not shown) and exercise performance remained equally impaired by 6.7 % (Pre-LEA: 216 W, 95% CI: 198 to 235 vs. Recovery: 202 W, 95% CI: 183 to 220, P < 0.001; data not shown) (Post-LEA vs. Recovery, P = 0.409). This is contradictory to a recent report by Burke et al., (2023) who showed that 9 days of severe energy restriction (15 kcal/ kg FFM/ day) with refueling prior to competition reduces body mass by 2 kg without altering training-associated performance improvement in male/female elite racewalkers (Burke et al., 2023). Acute restoration of carbohydrate availability for 24 hours prior to competition is standard sports nutrition practice (Burke et al., 2018); however, these novel data show that the adverse implications of 14-days LEA on exercise capacity persists even with three days of refueling in EB (50 kcal/ kg FFM/ day). Exercise-induced leukocytosis is reduced post- LEA IMMUNE/INFLAMMATORY RESPONSE AND EXERCISE PERFORMANCE WITH LEA 138 Studies have shown that strenuous prolonged exercise training with LEA in normal weight individuals may suppress the immune system (Gleeson et al., 2004; Gleeson, 2007; 2016; Bermon et al., 2017; Peake et al., 2017) in particular with evidence by integrated multi-omics approaches (Gleeson et al., 1995; Nielsen et al., 2016; Sarin et al., 2019). These data indicate an increased susceptibility to infection due to altered immunity within 72 hours after exercise – in addition to longer-term reductions in systemic immunity such as studies assessed prospectively across a 7-month intensive training season in elite swimmers (Gleeson et al., 1995). Overall, it appears that downregulated innate immunity responses by suppressed release of inflammatory markers and overall chemokine production is evident after prolonged energy restriction with intense exercise (Sarin et al., 2019). In the present study, resting WBC count was not different within or between diet interventions indicating that immune status at rest was unaffected by 14-days LEA (Figure 5-5). It is noteworthy, however, that the absolute neutrophil count at rest post- LEA (2.1 ´ 103 cells/µl) was approaching below normal reference values (< 2.0 ´ 103 cells/µl) (Lim et al., 2010; Coates et al., 2020) compared to pre- LEA and the EB intervention (all approx. 2.6 ´ 103 cells/µl). Further, immediately post- diet compared to following three days recovery in sufficient EB, neutrophils were not different between diets at recovery (P = 0.587) yet were reduced with LEA by approximately 10 % (EB: 2.5 vs. LEA: 2.2 ´ 103 cells/µl, treatment effect: P = 0.042; data not shown). Reductions in neutrophils would theoretically increase risk of infection, which is particularly relevant with respect to the high prevalence of upper respiratory tract infections in elite athletes, either immediately prior to or during competition (Peters & Bateman, 1983; Peters, 1997; Spence et al., 2007; Cox et al., 2008; Peters et al., 2010). There was a treatment effect of LEA such that % distribution of lymphocytes was higher than EB (inclusive of exercise), which may provide initial evidence for infection post- LEA. The post- exercise WBC response following LEA was reduced by 18.5 % compared to pre- LEA with no changes during the EB intervention (Figure 5-5A). These data may indicate that exercise-induced leukocytosis (mobilization of WBCs) is reduced with LEA (Peake et al., 2017). Whether this ‘insufficient’ post- exercise WBC response is responsible for the reported 45.7 % increases in creatine kinase post- LEA versus post- EB (indicative of muscle damage) requires further investigation (Tsai et al., 2001; Baird et al., 2012). As discussed next, it is worth considering that the exercise-induced alterations in the immune and inflammatory responses post- LEA are likely influenced by the reductions in performance. For example, reductions in IMMUNE/INFLAMMATORY RESPONSE AND EXERCISE PERFORMANCE WITH LEA 139 WBC mobilization may either indicate immune suppression or are explained by a lower absolute exercise intensity (e.g., approx. 17 W reduction in average power output during 20-minute time trial; Figure 5-10). 14-days LEA provokes systemic inflammation at rest Utilizing a targeted 96 inflammatory protein biomarker panel via high-multiplex immunoassay, these novel data provide evidence for elevated systemic inflammation at rest post- LEA (Figure 5-7). Further, by characterizing the post- exercise inflammatory response following both diet interventions, these results provide insight into the ‘typical’ inflammatory cytokines present following exercise and whether these proteins are apparent post- exercise with LEA (Figure 5-8 and Figure 5-9). Exercise provokes the release of a cascade of cytokines (Figure 5-9) (Pedersen & Hoffman-Goetz, 2000; Ostrowski et al., 2001) – in particular IL-6, which is a pleiotropic cytokine with important actions to regulate energy status (via exercise-induced lipolysis and hepatic glucose production) (Pedersen et al., 2001; Febbraio & Pedersen, 2002; Pedersen et al., 2003). Exercise-induced IL-6 is related to exercise intensity and is particularly evident with endurance activity (Ostrowski et al., 2000) with reports showing 100-fold increases in muscle-derived IL-6 following a marathon race (Ostrowski et al., 1998; 1999). Low carbohydrate availability and muscle glycogen depletion upregulate intramuscular gene expression (Keller et al., 2001; Steensberg et al., 2001) and systemic IL-6 levels (Steensberg et al., 2000). For example, Pasiakos et al., (2016) reported that increases in IL-6 after endurance exercise are negatively related to energy balance and glycogen stores (Pasiakos et al., 2016). Recent studies substantiate that exercise-induced IL-6 expression regulating inflammation is reduced by sufficient energy intake prior to exercise in order to spare muscle glycogen (Hennigar et al., 2017). Although the present results indicate systemic inflammation at rest post- LEA, the post- exercise IL-6 response with LEA was comparable to that achieved with exercise post- EB (Figure 5-8 and Figure 5-9). Further, as discussed in Study 3B – Substrate oxidation, skeletal muscle mitochondrial respiratory capacity, and insulin sensitivity following 14-days low energy availability in endurance-trained female athletes, intramuscular glycogen content was not different at rest post- diet interventions. Taken together, as IL-6 was not exacerbated following exercise post- LEA, this would be consistent with the reported ‘glycogen-sparing’ effects of IL-6. Exercise performance is impaired by LEA IMMUNE/INFLAMMATORY RESPONSE AND EXERCISE PERFORMANCE WITH LEA 140 An important practical implication of this study is the approximately 20 % reduction in time to exhaustion performance post- LEA (Figure 5-10F). A recent study reported that 3 days of LEA (19 kcal/ kg FFM/ day) with 75 minutes of daily endurance training at 70 % VO2max had no effect on exercise capacity in well-trained male runners (average VO2max 67.5 ml/ kg/ min) (Kojima et al., 2020). The performance test utilized was relatively comparable to the current study and involved a time-to-fatigue test at 90 % VO2max lasting approximately 20 minutes in duration (versus Figure 5-10E). Additionally, the study by Kojima et al., (2020) reported reductions in muscle glycogen content by approximately 30 % which was likely attributable to the additive effects of high training load and low-carbohydrate availability, rather than energy availability per se (Bergström et al., 1967; Olsson & Saltin, 1970; Areta & Hopkins, 2018). Although endurance training was high during both dietary interventions in the current study (approx. 8 hours/ week at 80 % HR max), daily carbohydrate intake exceeded the Nordic Nutrition Recommendations 2004 stating 100 g/day carbohydrate (Becker et al., 2004) (approx. 200 g/day during LEA, Table 5-1); therefore, carbohydrate availability was not limited in this intervention. The systemic and skeletal muscle metabolic implications of LEA are discussed in Study 3B – Substrate oxidation, skeletal muscle mitochondrial respiratory capacity, and insulin sensitivity following 14-days low energy availability in endurance-trained female athletes. For context, the impairment in exercise capacity was not explained by changes in intramuscular glycogen content, or skeletal muscle enzyme activity of citrate synthase (CS; mitochondrial content) b-Hydroxy acyl-CoA dehydrogenase (HAD; fatty acid oxidation), or phosphofructokinase (PFK; glycolysis). As such, unlike the reports by Kojima et al., (2020), the LEA-induced impairments in exercise performance observed in this study were independent of reductions in muscle glycogen availability. 5.6 Conclusion The results of this study provide evidence that 14-days LEA in endurance-trained females provokes immunological stress, systemic inflammation and impairs exercise performance. Further the implications of LEA on exercise performance impairments persisted following three days recovery in sufficient energy balance post- LEA. These results are supported by: 1) reduced post- exercise leukocyte mobilization following LEA; 2) higher creatine kinase with exercise and elevated systemic inflammatory biomarkers at rest post- LEA (via targeted proteomics); and 3) reductions in power output during a 20-minute time trial and overall time to exhaustion by 7.8 % and 18.9 %. METABOLISM, INSULIN SENSITIVITY AND MITOCHONDRIAL RESPIRATION WITH LEA 141 Chapter 6: Study 3B – Substrate oxidation, skeletal muscle mitochondrial respiratory capacity, and insulin sensitivity following 14-days low energy availability in endurance-trained female athletes Abstract Background: Exercise training improves metabolic health via both increases in mitochondrial oxidative capacity and improved glucose regulation. However, excessive exercise training impairs mitochondrial respiration and reduces glucose tolerance in healthy volunteers; therefore, the objective of this study was to investigate the influence of 14-days LEA in endurance-trained female athletes on fuel utilization, insulin sensitivity, and mitochondrial oxidative capacity in permeabilized human skeletal muscle fibers (via high-resolution respirometry, OROBOROS, Oxygraph-2k). Methods: Thirteen endurance-trained females (V̇O2max 53.6 ± 6.0 ml´min-1´kg-1) completed two 14-day randomized, blinded, cross-over, controlled dietary interventions: 1) energy balance (EB; 50 kcal/ kg FFM per day) and 2) low energy availability (LEA; 25 kcal/ kg FFM per day). Participants maintained their exercise training volume and intensity (approx. 8 hours running/cycling per week at approx. 78 % heart rate max) during both dietary interventions. Results: There was no effect of diet per se on resting metabolic rate (time ´ treatment: P = 0.910). Systemic ketone bodies were elevated post- LEA at rest by 4-fold following an overnight fast (P = 0.002) and increases in fat oxidation persisted during submaximal exercise selectively post- LEA (P = 0.002). Regulation of daily glucose control (via CGM), HOMA-IR/ HOMA-b, and insulin/glucose responses to a mixed-meal tolerance test were largely unaffected by LEA. Skeletal muscle mitochondrial respiratory capacity in addition to intramuscular glycogen or enzyme activity of CS, HAD, or PFK were unaltered with LEA (all P > 0.05). Conclusions: Taken together, 14-days LEA in endurance-trained females provokes an increased reliance on fat oxidation to absolute energy expenditure during submaximal exercise in addition to no differential impact on resting metabolic rate, insulin sensitivity, and skeletal muscle mitochondrial respiratory capacity. 6.1 Introduction Low energy availability (LEA) – which describes inadequate energy intake relative to exercise energy expenditure (30 kcal/kg fat free mass/day) – predicates relative energy deficiency in sports (Logue et al., 2018; Wasserfurth et al., 2020; Areta et al., 2021). The systemic substrate effects of LEA include lower circulating insulin and glucose with higher free fatty acids (FFAs), glycerol, cholesterol, and b-hydroxybutyrate (b-OHB) (Loucks & Thuma, 2003; Koehler et al., METABOLISM, INSULIN SENSITIVITY AND MITOCHONDRIAL RESPIRATION WITH LEA 142 2016). Taken together, increases in lipolytic activity and reductions in anaerobic glycolysis are an adaptive response with LEA to preserve carbohydrate, which is likely a consequence of limited glycogen reserves (Kojima et al., 2020). During conditions of low carbohydrate availability (e.g., fasting/ starvation (Hagenfeldt & Wahren, 1971; Cahill, 1976; Balasse et al., 1978) and during/post- exhaustive exercise (Hagenfeldt & Wahren, 1968; Koeslag et al., 1982; Féry & Balasse, 1983; 1986; 1988)) increases in systemic ketone bodies act as an alternative fuel substrate for skeletal muscle metabolism (Robinson & Williamson, 1980; Evans et al., 2016). Acute exogenous increases in circulating ketone bodies provide an alternative oxidative substrate via increases in skeletal muscle ketone utilization and reductions in lipolytic rate and glycolysis that are insulin-independent (Mikkelsen et al., 2015). Additionally, at least in pre-clinical experiments, the capacity to uptake and ability to utilize ketone bodies is higher in exercise-trained skeletal muscle (Winder et al., 1975; Askew et al., 1975). This observation is likely, in part, attributable to higher oxidative capacity (related to PGC1a expression) (Svensson et al., 2016) and is readily explained by the higher absolute exercise intensity/duration and thus extent of glycogen-depletion achieved with exhaustive aerobic exercise in trained individuals (Adams & Koeslag, 1988; Cox et al., 2016). With respect to exercise performance, elevated systemic ketone bodies reportedly lower the exercise-induced rise in [La-] with high-intensity exercise (Féry & Balasse, 1988; Lestan et al., 1994; Cox et al., 2016). Further, recent studies have shown that exogenous ketosis stimulates muscular angiogenesis during endurance training overload in recreationally active males (+40% increase in muscle capillarization explained via elevations in circulating erythropoietin in addition to protein and mRNA expression of pro-angiogenic factors) (Poffé et al., 2023). In addition, exogenous ketosis was protective against the training-induced energy deficit by increasing ad libitum energy intake proportionally by 20% to meet the increases in exercise energy expenditure (Poffé et al., 2019). Overall, it is expected that nutritional ketosis will occur following 14-days of LEA in endurance-trained female athletes, which will be reflective in systemic alterations in substrate utilization at rest and during exercise indicated by an increased reliance on fat oxidation. Exercise training improves metabolic health via both increases in mitochondrial oxidative capacity and improved glucose regulation. For example, whole-body maximal oxygen uptake (VO2max) is both positively related to mitochondrial respiratory capacity (van der Zwaard et al., 2016) and a proxy for metabolic health (Lee et al., 2011). A recent study has shown that excessive exercise training impairs mitochondrial respiration and reduces glucose tolerance in healthy volunteers (Flockhart et al., 2021). Reduced mitochondrial capacity is typically related to METABOLISM, INSULIN SENSITIVITY AND MITOCHONDRIAL RESPIRATION WITH LEA 143 insulin-resistance (Simoneau et al., 1999; Kelley et al., 2002; Bruce et al., 2003; Lowell & Shulman, 2005; Bhatti et al., 2017); therefore, LEA-induced impairments in insulin sensitivity may result in lower skeletal muscle respiratory capacity. Additionally, insulin-resistant skeletal muscle is related to lipid status (Furler et al., 2001; Kriketos et al., 2003; Poynten et al., 2005), and is implicated in a mismatch between FFA availability and mitochondrial fat oxidation (Pan et al., 1997). Thus, LEA-induced increases in FFA availability (Koehler et al., 2016) may explain impaired skeletal muscle oxidative capacity. Importantly, it is highly debated whether reduced mitochondrial capacity/efficiency is a cause or consequence of impaired blood glucose control and insulin sensitivity. An alternative explanation is that LEA would improve mitochondrial bioenergetic efficiency to sustain ATP production. The objective of this study was to investigate the influence of 14-days LEA in endurance-trained female athletes on: 1) fuel utilization (e.g., respiratory exchange ratio, glucose, intramuscular glycogen, lactate, and b-OHB), 2) insulin sensitivity (HOMA-IR, mixed-meal tolerance test, continuous glucose monitoring), and 3) mitochondrial oxidative capacity in permeabilized human skeletal muscle fibers (via high-resolution respirometry, OROBOROS, Oxygraph-2k). It was hypothesized that 14-days LEA would suppress resting metabolic rate, impair insulin sensitivity, and increase relative energy expenditure during submaximal and exhaustive cycling exercise. Additionally, the influence of LEA on skeletal muscle mitochondrial oxidative capacity was measured as an exploratory outcome. 6.2 Methods A detailed overview of the experimental protocol is outlined in Study 3A – The importance of low energy availability for the immune system, systemic inflammation, and performance ability in endurance-trained female athletes. Experimental procedures related to muscle biopsies and glucose control are described below. Experimental Procedures Muscle Biopsies Muscle biopsies were collected under local anaesthesia (20 mg/ml Xylocain without epinephrine; AstraZeneca, Cambridge, UK) through a small incision in the skin over the vastus lateralis. Biopsies were collected with a modified Bergström needle with suction (Bergström, 1975). Upon collection, the muscle biopsy was rinsed in saline (9 mg/ml, Fresenius Kabi, METABOLISM, INSULIN SENSITIVITY AND MITOCHONDRIAL RESPIRATION WITH LEA 144 Sweden) and divided in two. Approximately 5-6 mg was placed in ice cold BIOPS (Pesta & Gnaiger, 2012) (50 mM K+-MES, 20 mM taurine, 0.5 mM dithiothreitol, 6.56 mM MgCl2, 5.77 mM ATP, 15 mM phosphocreatine, 20 mM imidazole, pH 7.1, adjusted with 5 N KOH at 0°C, 10 mM Ca-EGTA buffer) for analysis of mitochondrial respiration, and the remainder was quickly frozen in liquid N2 and stored at -80°C for later Western blot analysis, glycogen content, and enzymatic activity assays. Glycogen content 1-2 mg d.w. muscle tissue was extracted in 0.5 ml 1M HCl and hydrolyzed at 100°C for 3 h, and the glucose content was determined by the hexokinase method using a glucose kit (TrioLab) on a PentraC 400 analyzer, TrioLab. The formatted NADPH is directly proportionally to the glucose content and is measured spectophotmetricaly at 340 nm. Citrate synthase activity Maximal enzyme activity of citrate synthase was determined from approximately 0.5 mg d.w. muscle tissue dissected free of visible blood and connective tissue, before homogenization (1:400) in a 0.3 mol/l phosphate buffer (pH 7.7) by two rounds of 30-seconds using a TissueLyser II (QIagen, Valencia, CA, USA). Maximal enzyme activity was determined fluorometrically by NAD-NADH coupled reactions (Fluoroscan Ascent, Thermo Fisher Scientific, Waltham, USA) at 25°C as previously described (Lowry & Passonneau, 1972). High-resolution mitochondrial respirometry Mitochondrial respiratory capacity was assessed in permeabilized muscle fibers by high-resolution respirometry (Oxygraph-2k; Oroboros Instruments, Innsbruck, Austria) prior to and following each diet intervention (Figure 6-1). Within two hours of muscle biopsy sampling, the muscle tissue in BIOPS was prepared for high-resolution respirometry as previously described (Pesta & Gnaiger, 2012). Muscle tissue was dissected free from connective tissue and fat and muscle fibers were carefully separated mechanically (in ice-cold BIOPS) and permeabilized by incubation with saponin (5 mg/ml) on ice for 25 min and then washed twice in 2 ml MiR06 (0.5 mM EGTA, 3 mM MgCl2.6H2O, 60 mM K+-lactobionate, 20 mM taurine, 10 mM KH2PO4, 20 Mm HEPES, 110 mM D-sucrose and 1 g/l of fatty acid free BSA, 280 U/mL catalase, pH 7.4) 10 min each. High-resolution respirometry were determined in duplicate with an internal chamber temperature of 37°C. Instrumental and chemical oxygen background fluxes were calibrated as a function of oxygen concentration and subtracted from the total volume-specific oxygen flux METABOLISM, INSULIN SENSITIVITY AND MITOCHONDRIAL RESPIRATION WITH LEA 145 (Datlab v.6.1 software; Oroboros Instruments). 2-3 mg of fibers were added to each chamber with oxygen concentrations maintained between 250-500 μM O2 to prevent potential oxygen diffusion limitations. A substrate-uncoupler-inhibitor titration protocol was used to measure specific mitochondrial respiration. Leak respiration (LN) was measured by adding malate (2 mM), pyruvate (5 mM), and glutamate (10 mM). Complex I (CI) respiration was determined after the addition of ADP (5 mM) with magnesium (5 mM). Maximal OXPHOS capacity through complex I and II combined (CI+CII) was determined after addition of succinate (10 mM) followed by addition of cytochrome C (10 mM) to test for mitochondrial outer membrane integrity. To determine the maximal electron transport system respiratory capacity (ETS), a series of stepwise carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) titrations (1.5–3.0 mM) until a plateau in respiration was reached. Complex II (CII) respiration was determined by inhibition of complex I-driven respiration by the addition of rotenone (0.5 µM). Finally, antimycin A (2.5 µM) was added to terminate respiration and for determination and correction of residual oxygen consumption, indicative of nonmitochondrial respiration. Continuous Glucose Monitoring Participants were instrumented with a flash glucose monitor (FreeStyle Libre 2, Abbott Diabetes Care Ltd., Witney, UK) on T3/T7 which recorded data during days 7-14 of the diet intervention and throughout the 3 days of recovery. Participants were instructed to mark when they ate breakfast, lunch, and dinner each day to identify post-prandial glucose responses. METABOLISM, INSULIN SENSITIVITY AND MITOCHONDRIAL RESPIRATION WITH LEA 146 Figure 6-1. Experimental methods - Skeletal muscle mitochondrial respiratory capacity. Mitochondrial respiratory capacity was assessed via high-resolution respirometry (OROBOROS, Oxygraph-2k) in permeabilized human skeletal muscle fibers (m. vastus lateralis). 6.3 Statistical analyses All data are presented in-text as mean with 95% confidence intervals and as individual values in figures. Data analyses were performed using RStudio (2021.09.1) and statistical analyses were performed using SPSS software (IBM statistics, Version 23.0). Statistical significance was set at P < 0.05. A linear mixed-effects model analysis with compound symmetry covariance structure with fixed effects of time (pre vs. post) and treatment (EB vs. LEA) was performed for repeated measures comparisons. Further, fixed effects of exercise (submaximal warmup stages: 50%, 60%, 70%) and treatment (pre- EB, post- EB, pre- LEA, post- LEA) were used to compare exercise responses between diet interventions. A Bonferroni correction was applied for multiple comparisons when significant interactions were detected. For selected variables, treatment (EB vs. LEA) was the only fixed effect. Subjects were included as a random effect. METABOLISM, INSULIN SENSITIVITY AND MITOCHONDRIAL RESPIRATION WITH LEA 147 6.4 Results The average glycated hemoglobin (HbA1c, %) of participants was 5.23 % (95% CI: 5.10 to 5.36) prior to starting the experiment. Energy expenditure and substrate utilization at rest and during submaximal cycling exercise following EB and LEA diet interventions The RMR was reduced by 11.3 % post- diet with both interventions (Pre: 1655 kcal/day, 95% CI: 1547 to 1764 vs. Post: 1468 kcal/day, 95% CI: 1360 to 1577, time effect: P < 0.001; Figure 6-2A). Resting RER was reduced by 5.8 % post- diet with both interventions (Pre: 0.83, 95% CI: 0.81 to 0.85 vs. Post: 0.78, 95% CI: 0.76 to 0.80, time effect: P < 0.001; Figure 6-2B). Additionally, RER was lower with the LEA intervention by 2.7 % (EB: 0.82, 95% CI: 0.80 to 0.84 vs. LEA: 0.80, 95% CI: 0.78 to 0.82, treatment effect: P = 0.021; Figure 6-2B). Ketone levels were increased by 0.3 mmol/l following an overnight fast post- LEA versus EB (Post-EB: 0.1 mmol/l, 95% CI: 0.02 to 0.3 vs. Post-LEA: 0.4 mmol/l, 95% CI: 0.3 to 0.6, P = 0.002; Figure 6-2C). Figure 6-2. Energy expenditure and substrate utilization at rest pre- vs. post- 14 days of energy balance (EB) and low energy availability (LEA). There were no significant time ´ treatment interactions for resting metabolic rate (RMR) (A) or respiratory exchange ratio (RER) (B); however, there were main effects of time, such that RMR and RER were PrePost PrePost5001000150020002500BMR (kcal/day)Time: P < 0.001Treatment: P = 0.157Time × treatment: P = 0.910PrePost PrePost0.700.750.800.850.900.95RERTime: P < 0.001Treatment: P = 0.021Time × treatment: P = 0.318EB LEA0.00.20.40.60.81.0Ketone (mmol/l)+Δ 0.3P = 0.002A B CMETABOLISM, INSULIN SENSITIVITY AND MITOCHONDRIAL RESPIRATION WITH LEA 148 reduced following both diet interventions. Ketone levels were increased by 4-fold (+300%) following an overnight fast post- LEA versus EB (C). Energy balance is shown in blue and LEA is shown in red. The reduction in RER observed at rest post-LEA (Figure 6-3A) persisted throughout all submaximal cycling exercise intensities (50%, 60%, 70% VO2max) post-LEA (treatment effect: P = 0.003). In contrast, there were no significant differences pre- vs. post- EB (treatment effect: P = 0.919) or between pre-LEA vs. pre-EB (treatment effect: P = 1.000) during submaximal exercise (Figure 6-3B). Figure 6-3. Reductions in RER persist throughout submaximal cycling exercise post-LEA. The reduction in respiratory exchange ratio (RER) observed at rest post-LEA (A) persisted throughout all submaximal cycling exercise intensities (50%, 60%, 70% VO2max) post-LEA (P = 0.003), whereas, there were no significant differences pre- vs. post- EB (P = 0.919) or between pre-LEA vs. pre-EB (P = 1.000) during submaximal exercise (B). Energy balance is shown in blue and LEA is shown in red (Panel A). Panel B shows pre- vs. post- diet changes within EB (blue) and LEA (red) conditions at each exercise intensity. The relative contribution of carbohydrate to resting energy expenditure was reduced by 42.4 % post- diet with both interventions (Pre: 36 %, 95% CI: 30 to 43 vs. Post: 21 %, 95% CI: 15 to 27, time effect: P < 0.001; Figure 6-4A). Additionally, there was a treatment effect such that the relative contribution of carbohydrate with the LEA intervention was lower by 19.9 % (EB: 32 %, 95% CI: 26 to 38 vs. LEA: 26 %, 95% CI: 19 to 32, treatment effect: P = 0.040; Figure 6-4A). Following LEA, the reduction in carbohydrate contribution to energy expenditure (Figure 6-4B) and total carbohydrate oxidation (Figure 6-4C) persisted throughout all submaximal cycling exercise intensities (50%, 60%, 70% VO2max) (treatment effects: P < 0.001 and P = 0.002, PrePost PrePost0.700.750.800.850.900.95RERTime: P < 0.001Treatment: P = 0.021Time × treatment: P = 0.318EB50%LEA EB60%LEA EB70%LEA0.800.850.900.951.001.05RERTreatment post-hoc:Pre- vs. Post- LEA: P = 0.003Pre- vs. Post- EB: P = 0.919Pre- LEA vs. Pre- EB: P = 1.000A BMETABOLISM, INSULIN SENSITIVITY AND MITOCHONDRIAL RESPIRATION WITH LEA 149 respectively). There were no significant differences pre- vs. post- EB or between pre- LEA vs. pre- EB during submaximal exercise (all P > 0.05). Figure 6-4. Relative contribution of carbohydrate to energy expenditure is reduced at rest and during submaximal cycling exercise. The relative contribution of carbohydrate (CHO) to resting energy expenditure was reduced following both dietary interventions (time effect: P < 0.001) (A); however, following LEA only, this reduction in CHO contribution to energy expenditure (B) and total CHO oxidation (C) persisted throughout all submaximal cycling exercise intensities (50%, 60%, 70% VO2max) (P < 0.001 and P = 0.002, respectively). There were no significant differences pre- vs. post- EB or between pre-LEA vs. pre-EB during submaximal exercise (all P > 0.05). Energy balance is shown in blue and LEA is shown in red (Panel A). Panel B and C show pre- vs. post- diet changes within EB (blue) and LEA (red) conditions at each exercise intensity. The relative contribution of fat to resting energy expenditure was increased by 34.5 % post- diet with both interventions (Pre: 43 %, 95% CI: 36 to 49 vs. Post: 57 %, 95% CI: 51 to 64, time effect: P < 0.001; Figure 6-5A). Additionally, there was a treatment effect such that the relative PrePost PrePost20406080Relative CHO contribution per day (%)Time: P < 0.001Treatment: P = 0.040Time × treatment: P = 0.561EB50%LEA EB60%LEA EB70%LEA20406080100Relative CHO contribution (%)Treatment post-hoc:Pre- vs. Post- LEA: P < 0.001Pre- vs. Post- EB: P = 1.000Pre- LEA vs. Pre- EB: P = 0.785ABCEB50%LEA EB60%LEA EB70%LEA0.00.51.01.52.02.53.0CHO oxidation (g/min)Treatment post-hoc:Pre- vs. Post- LEA: P = 0.002Pre- vs. Post- EB: P = 1.000Pre- LEA vs. Pre- EB: P = 1.000METABOLISM, INSULIN SENSITIVITY AND MITOCHONDRIAL RESPIRATION WITH LEA 150 contribution of fat with the LEA intervention was increased by 12.5 % (EB: 47 %, 95% CI: 40 to 54 vs. LEA: 53 %, 95% CI: 46 to 59, treatment effect: P = 0.052; Figure 6-5A). Following LEA, the increase in fat contribution to energy expenditure (Figure 6-5B) and total fat oxidation (Figure 6-5C) persisted throughout all submaximal cycling exercise intensities (50%, 60%, 70% VO2max) (treatment effects: P < 0.001 and P = 0.002, respectively). There were no significant differences pre- vs. post- EB or between pre- LEA vs. pre- EB during submaximal exercise (all P > 0.05). Figure 6-5. Relative contribution of fat to energy expenditure is increased at rest and during submaximal cycling exercise. The relative contribution of fat (FAT) to resting energy expenditure was increased following both dietary interventions (time effect: P < 0.001) (A); however, following LEA only, this increase in FAT contribution to energy expenditure (B) and total FAT oxidation (C) persisted throughout all submaximal cycling exercise intensities (50%, 60%, 70% VO2max) (P < 0.001 and P = 0.002, respectively). There were no significant differences pre- vs. post- EB or between pre-LEA vs. pre-EB during submaximal exercise (all P > 0.05). EB50%LEA EB60%LEA EB70%LEA0.00.20.40.60.81.0FAT oxidation (g/min)Treatment post-hoc:Pre- vs. Post- LEA: P = 0.002Pre- vs. Post- EB: P = 0.460Pre- LEA vs. Pre- EB: P = 1.000EB50%LEA EB60%LEA EB70%LEA20406080100Relative FAT contribution (%)Treatment post-hoc:Pre- vs. Post- LEA: P < 0.001Pre- vs. Post- EB: P = 0.659Pre- LEA vs. Pre- EB: P = 0.717PrePost PrePost20406080Relative FAT contribution per day (%)Time: P < 0.001Treatment: P = 0.052Time × treatment: P = 0.348ABCMETABOLISM, INSULIN SENSITIVITY AND MITOCHONDRIAL RESPIRATION WITH LEA 151 Energy balance is shown in blue and LEA is shown in red (Panel A). Panel B and C show pre- vs. post- diet changes within EB (blue) and LEA (red) conditions at each exercise intensity. Energy expenditure and absolute VO2 increased as expected with progressive increases in exercise intensity (exercise intensity effect: both P < 0.001; Figure 6-6) and these responses were not different within (pre- vs. post-) or between diet interventions (P = 0.913 and P = 0.904, respectively). Figure 6-6. Energy Expenditure and absolute VO2 were not different at matched submaximal cycling exercise intensities pre- vs. post- EB and LEA diet interventions. There were no differences in energy expenditure (A) or absolute VO2 (B) within (pre- vs. post-) or between energy balance (EB) and low energy availability (LEA) diet interventions. Data presented shows pre- vs. post- diet changes within EB (blue) and LEA (red) conditions at each exercise intensity. Skeletal muscle substrate content and enzyme activity There was no difference in skeletal muscle glycogen content within or between EB and LEA conditions (EB: -D 8.0 mmol/kg DW, 95% CI: -111.6 to 95.6 vs. LEA: -D 12.7 mmol/kg DW, 95% CI: -113.3 to 87.9, P = 0.947; Figure 6-7). EB50%LEA EB60%LEA EB70%LEA5101520Energy expenditure (kcal/min)Ex intensity: P < 0.001Treatment: P = 0.507Ex intensity × treatment: P = 0.913EB50%LEA EB60%LEA EB70%LEA1000200030004000VO2 (ml ⋅ min-1)Ex intensity: P < 0.001Treatment: P = 0.322Ex intensity × treatment: P = 0.904A BMETABOLISM, INSULIN SENSITIVITY AND MITOCHONDRIAL RESPIRATION WITH LEA 152 Figure 6-7. Skeletal muscle glycogen content at rest pre- vs. post- 14 days of energy balance (EB) and low energy availability (LEA) diet interventions. There was no difference in skeletal muscle glycogen content within or between EB and LEA conditions (P = 0.947). Energy balance is shown in blue and LEA is shown in red. There was no difference in skeletal muscle citrate synthase activity within or between EB and LEA conditions (EB: D 3.9 µmol/g protein/min, 95% CI: -3.9 to 11.6 vs. LEA: D 0.9 µmol/g protein/min, 95% CI: -6.6 to 8.4, P = 0.574; Figure 6-8A). Likewise, there was no difference in skeletal muscle b-Hydroxy acyl-CoA dehydrogenase (HAD) activity within or between EB and LEA conditions (EB: D 1.1 µmol/g protein/min, 95% CI: -2.9 to 5.1 vs. LEA: -D 1.1 µmol/g protein/min, 95% CI: -5.0 to 2.8, P = 0.422; Figure 6-8B). There was no difference in skeletal muscle phosphofructokinase (PFK) activity within or between EB and LEA conditions (EB: D 8.8 µmol/g protein/min, 95% CI: -64.3 to 82.0 vs. LEA: D 3.9 µmol/g protein/min, 95% CI: -66.9 to 74.7, P = 0.920; Figure 6-8C). PrePost PrePost2004006008001000Glycogen (mmol/kg DW)Time × treatment:P = 0.947METABOLISM, INSULIN SENSITIVITY AND MITOCHONDRIAL RESPIRATION WITH LEA 153 Figure 6-8. Skeletal muscle enzyme activity at rest pre- vs. post- 14 days of energy balance (EB) and low energy availability (LEA) diet interventions. There were no differences in skeletal muscle enzyme activity of citrate synthase (CS) (A), b-Hydroxy acyl-CoA dehydrogenase (HAD) (B), or phosphofructokinase (PFK) (C) within or between EB and LEA conditions (all P > 0.05). Energy balance is shown in blue and LEA is shown in red. Skeletal muscle mitochondrial respiratory capacity There were no differences pre/post within or between EB versus LEA conditions for complex I (P = 0.824), complex II (P = 0.893), complex I+II (P = 0.984), or the electron transport system (ETS) (P = 0.608) skeletal muscle mitochondrial respiratory capacity (Figure 6-9). PrePost PrePost255075100125150CS activity (µmol/g protein/min)Time × treatment:P = 0.574PrePost PrePost2030405060HAD activity (µmol/g protein/min)Time × treatment:P = 0.422PrePost PrePost200400600800PFK activity (µmol/g protein/min)Time × treatment:P = 0.920A B CMETABOLISM, INSULIN SENSITIVITY AND MITOCHONDRIAL RESPIRATION WITH LEA 154 Figure 6-9. Mitochondrial respiratory capacity pre- vs. post- 14 days of energy balance (EB) and low energy availability (LEA). There were no differences pre/post within or between EB versus LEA conditions (all P>0.05) for complex I, complex II, complex I+II, or the electron transport system (ETS) skeletal muscle mitochondrial respiratory capacity. Panel A shows pre- vs. post- delta changes within EB (blue) and LEA (red) conditions. Panels B and C show pre- vs. post- EB (B) and LEA (C) within diet comparisons, respectively. Regulation of glucose during days 7-14 of EB and LEA diet interventions There was no difference between diet interventions for time spent in range (3.9-10.1 mmol/l) (EB: 10 %, 95% CI: 9 to 11 vs. LEA: 11 %, 95% CI: 10 to 12, P = 0.506; Figure 6-10A), number of hypoglycemic events (EB: 4 events, 95% CI: 0 to 8 vs. LEA: 6 events, 95% CI: 2 to 10, P = 0.457; Figure 6-10B), or time spent hypoglycemic (EB: 3 %, 95% CI: 0 to 7 vs. LEA: 6 %, 95% CI: 2 to 10, P = 0.478; Figure 6-10C). CI CII CI+CII ETS255075100125150175O2 flux per mass (pmol/(s×mg)LEACI CII CI+CII ETS-40-20020406080Δ O2 flux per mass (pmol/(s×mg) Time × treatment:All P > 0.05EBLEACI CII CI+CII ETS255075100125150175O2 flux per mass (pmol/(s×mg)EBABCMETABOLISM, INSULIN SENSITIVITY AND MITOCHONDRIAL RESPIRATION WITH LEA 155 Figure 6-10. Regulation of normoglycemia and incidence of hypoglycemic events during days 7-14 of energy balance (EB) and low energy availability (LEA) diets. There were no differences between EB versus LEA for time spent in range (3.9-10.1 mmol/l) (P = 0.506) (A); number of hypoglycemic events (P = 0.457) (B); or time spent hypoglycemic (<3.9 mmol/l) (P = 0.478) (C). Time spent in ranges were expressed as percentage of available data for the specified dates (day 7-14) of each diet. There were no differences between diet interventions for daytime or nighttime AUC (Figure 6-11 A, D), average (Figure 6-11 B, E), or minimum (Figure 6-11 C, F) glucose values (all P > 0.05). EB LEA80859095100Time in range 3.9-10.1 mmol/l (%)P = 0.506EB LEA5101520Hypoglycemic events (#)P = 0.457EB LEA5101520Time spent <3.9 mmol/l (%)P = 0.478A B CMETABOLISM, INSULIN SENSITIVITY AND MITOCHONDRIAL RESPIRATION WITH LEA 156 Figure 6-11. Daytime and nighttime regulation of glucose during days 7-14 of energy balance (EB) and low energy availability (LEA) diets. There were no differences between EB versus LEA for daytime or nighttime AUC (A, D), average (B, E), or minimum (C, F) glucose values (all P > 0.05). There were no differences between diet interventions for glucose coefficient of variation (Figure 6-12A), total AUC (Figure 6-12B), mean amplitude of glycemic excursions (MAGE) (Figure 6-12C), or continuous overall net glycemic action (CONGA) (Figure 6-12D) (all P > 0.05). EB LEA1000020000300004000050000Daytime AUCP = 0.544EB LEA5000100001500020000Nighttime AUCP = 0.957EB LEA4.55.05.56.06.5Daytime average (mmol/l)P = 0.830EB LEA4.04.55.05.56.0Nighttime average (mmol/l)P = 0.376EB LEA2.53.03.54.04.55.0Daytime minimum (mmol/l)P = 0.813EB LEA2.53.54.55.5Nighttime minimum (mmol/l)P = 0.470A BD E FCMETABOLISM, INSULIN SENSITIVITY AND MITOCHONDRIAL RESPIRATION WITH LEA 157 Figure 6-12. Metrics of glucose variability during days 7-14 of energy balance (EB) and low energy availability (LEA) diets. There were no differences between EB versus LEA for glucose coefficient of variation (A), total AUC (B), mean amplitude of glycemic excursions (MAGE) (C), or continuous overall net glycemic action (CONGA) (D) (all P > 0.05). Glucose AUC was analyzed via CGM during breakfast throughout days 7-14 of the EB and LEA diet interventions. There were no differences between diet interventions for glucose AUC (P = 0.272; Figure 6-13A) or post- breakfast peak glucose (P = 0.241; Figure 6-13C); however, incremental glucose AUC was approximately 20.4 % higher (P = 0.022; Figure 6-13B) during the LEA diet. EB LEA0.00.10.20.3Coefficient of variationP = 0.413EB LEA20000400006000080000Total AUCP = 0.622EB LEA01234MAGEP = 0.243EB LEA0.00.51.01.52.0CONGAP = 0.151A B C DMETABOLISM, INSULIN SENSITIVITY AND MITOCHONDRIAL RESPIRATION WITH LEA 158 Figure 6-13. Post-prandial glucose response to breakfast during days 7-14 of energy balance (EB) and low energy availability (LEA) diets. Glucose AUC was sampled continuously via CGM every 15 minutes up to 60 minutes post- breakfast. There were no differences between EB versus LEA for glucose AUC (P = 0.272) (A) or post- breakfast peak glucose (P = 0.241) (C); however, incremental glucose AUC was approximately 20.4 % higher (P = 0.022) (B) during the LEA diet. Plasma insulin, c-peptide, and glucose were measured in the fasted state and 60 minutes post-prandially following a standardized breakfast (MMTT) on the post- diet test days (T4/T8). The HOMA-IR was calculated as: glucose (mmol/l) ´ insulin (mU/l) / 22.5. The HOMA-b was calculated as: (20 ´ insulin (mU/l)) / (glucose (mmol/l) – 3.5). There was no difference in HOMA-IR (P = 0.312) or HOMA-b (P = 0.817) between post- EB and LEA test days (Figure 6-14 A & B). There was an effect of time (P = 0.016) and treatment (P = 0.006) for fasting plasma glucose; however, there was no time ´ treatment interaction (P = 0.699) (Pre- EB: 4.9 mmol/l, 95% CI: 4.7 to 5.1 vs. Post- EB: 4.7 mmol/l, 95% CI: 4.5 to 4.9 and Pre- LEA: 4.7 mmol/l, 95% CI: 4.5 to 4.9 vs. Post- LEA: 4.5 mmol/l, 95% CI: 4.3 to 4.7) (Figure 6-14C). The delta change between fasted versus MMTT glucose, insulin, and c-peptide responses were not different between post- diet test days (P = 0.564, P = 0.603, P = 0.695, respectively; Figure 6-14 D-F). EB LEA678910Post- breakfast peak glucose (mmol/l) P = 0.241EB LEA255075100125150iGlucose AUC+20.4 %P = 0.022EB LEA300350400450500Glucose AUCP = 0.272A CBMETABOLISM, INSULIN SENSITIVITY AND MITOCHONDRIAL RESPIRATION WITH LEA 159 Figure 6-14. Insulin sensitivity There were no differences between post- diet test days for HOMA-IR (P = 0.312) (A), HOMA-b (P = 0.817) (B), fasting plasma glucose (time ´ treatment: P = 0.699; Post- EB vs. Post- LEA pairwise comparison: P = 0.081) (C), or the delta change in glucose (P = 0.564) (D), insulin (P = 0.603) (E), or c-peptide (P = 0.695) (F) in response to a MMTT. There were no differences between the exercise-induced reflexive hypoglycemia (i.e., delta change between rest vs. minimum glucose during submaximal exercise, P = 0.417; Figure 6-15C) or the post- exercise glucose responses (P = 0.514; Figure 6-15D) between diet interventions. EB LEA3.54.04.55.05.5Fasting plasma glucose (mmol/l)EB LEA-3-2-10123Δ Glucose (mmol/l) EB LEA20406080Δ Insulin (mU/l)EB LEA5001000150020002500Δ C-peptide (pmol/l)EB LEA0.00.51.01.52.02.5HOMA-IREB LEA50100150200HOMA-βA BEC DFMETABOLISM, INSULIN SENSITIVITY AND MITOCHONDRIAL RESPIRATION WITH LEA 160 Figure 6-15. Exercise-induced glucose responses on the post- EB and LEA test days. There were no differences between the glucose responses to submaximal exercise (A vs. B), exercise-induced reflexive hypoglycemia (i.e., delta change between rest vs. minimum glucose during submaximal exercise; P = 0.417) (C) or the post- exercise glucose responses (P = 0.514) (D) between diet interventions. 6.5 Discussion The results of this study indicate that 14-days LEA in endurance-trained females provokes an increased reliance on fat oxidation to absolute energy expenditure during submaximal exercise in addition to no differential impact on resting metabolic rate, insulin sensitivity, and skeletal muscle mitochondrial respiratory capacity. These results are supported by: 1) systemic b-OHB was elevated at rest, and increases in fat oxidation persisted during submaximal exercise post- LEA (Figure 6-2 and Figure 6-5); 2) regulation of daily glucose control (via CGM), HOMA-IR/ HOMA-b, and insulin/glucose responses to a mixed-meal tolerance test were largely unaffected by LEA; and 3) skeletal muscle mitochondrial respiratory capacity in addition to intramuscular glycogen or enzyme activity of CS, HAD, or PFK were unaltered with LEA (Figure 6-7, Figure 6-8 and Figure 6-9). Systemic substrate utilization – Why is exercise performance impaired with 14-days LEA? Aerobic exercise performance was impaired (Study 3A – The importance of low energy availability for the immune system, systemic inflammation, and performance ability in endurance-trained female athletes, Figure 5-10); further, the absolute VO2max achieved during time to exhaustion was reduced by 5.8 % with LEA (Pre: 3086 ml´min-1, 95% CI: 2843 to 3329 vs. Post: 2908 ml´min-1, 95% CI: 2665 to 3150; P = 0.009). As power output during the 20-minute time trial was reduced by 7.8 % (P < 0.001), it is likely that peak power output during a VO2max test would be reduced as well, however, this was not re-evaluated post- LEA. Therefore, Rest50%60%70%Post-Ex12345678Glucose (mmol/l)Post- EBRest50%60%70%Post-Ex12345678Glucose (mmol/l)Post- LEAEB LEA-3-2-101Δ Glucose (mmol/l)Rest vs.Ex MinEB LEA-2-101234Δ Glucose (mmol/l)Rest vs.Post- ExA B C DMETABOLISM, INSULIN SENSITIVITY AND MITOCHONDRIAL RESPIRATION WITH LEA 161 submaximal exercise performed at the same absolute workloads (corresponding to 50%, 60%, and 70% pre-intervention VO2max) would conceivably relate to higher exercise intensity relative to VO2 during the post- LEA submaximal cycling test (by approx. 7%). As such, it would be expected that RER would be higher during the submaximal exercise test post-LEA reflective of an increased relative exercise intensity; rather, however, higher fat oxidation and relative contribution of fats to energy expenditure persisted throughout submaximal exercise post- LEA versus the EB intervention (Figure 6-5). Additionally, energy expenditure and absolute VO2 were not different at matched submaximal cycling exercise intensities within or between diet interventions, indicating unaltered exercise efficiency/economy with LEA (Figure 6-6). An explanation for the LEA-induced impairments in exercise performance involves increased reliance on fatty acid oxidation (Burke et al., 2017; 2020); this was indicated by higher fat oxidation and relative contribution of fat to energy expenditure post- LEA during absolute intensity-matched submaximal cycling exercise (Figure 6-5). Further, the ‘fat adaption’ reported post- LEA during submaximal exercise was reversed following three days recovery in sufficient EB (e.g., fat contribution to energy expenditure, Pre-LEA: 30.8 %, 95% CI: 25.3 to 36.4 vs. Recovery: 30.8 %, 95% CI: 25.2 to 36.3, treatment effect: P = 1.000; data not shown) (Post-LEA vs. Recovery, treatment effect: P = 0.001). This is consistent with work from Burke et al., (2021) who showed that acute restoration of carbohydrate availability within 24 hours pre-race was ineffective at reversing the performance impairment provoked by 5-6 days of low carbohydrate high fat ‘keto adaptation’ (Burke et al., 2021). Burke et al., (2021) implicated a reduction in capacity for carbohydrate oxidation as a candidate for the persistent impairments in performance. The LEA-induced reduction in carbohydrate oxidation during exercise was restored to 99 % of the pre- LEA value following three days recovery in sufficient EB (treatment effect: P = 1.000; data not shown) compared to approx. 75 % restoration reported by Burke et al., 2021. These data indicate that carbohydrate availability was not a limiting factor for performance in the present study. It is worth noting that the relative contribution of macronutrients to dietary intake were statistically different between diet interventions, although within approximately 5 % for all macronutrients (Study 3A – The importance of low energy availability for the immune system, systemic inflammation, and performance ability in endurance-trained female athletes, Table METABOLISM, INSULIN SENSITIVITY AND MITOCHONDRIAL RESPIRATION WITH LEA 162 5-1). As such, the reported ‘fat adaptation’ persisted irrespective of the 2.6 % lower contribution of fat to total energy intake prescribed during LEA. Insulin sensitivity and glucose regulation are unaltered by 14-days LEA Regulation of glucose control and insulin sensitivity was largely unaffected by LEA. Further, 14-days LEA with high endurance training volume did not affect intramuscular glycogen content; as prioritization of glycogen resynthesis is implicated in the regulation of exercise-induced improvements in insulin sensitivity (Jensen et al., 2011; Hingst et al., 2022), the unaltered insulin sensitivity supports unaltered glycogen status post- LEA. Recent studies have shown that excessive exercise training reduces glucose tolerance and insulin sensitivity (via OGTT) in addition to the glucoregulatory response to intense exercise (Flockhart et al., 2021; 2023). Further, these elite endurance athletes exhibited an impaired glucose control during free-living conditions as evidenced by increased time spent outside of the normoglycemic range (4-8 mmol/l) compared to healthy national-level endurance athletes (Flockhart et al., 2021). Additionally, a study by Pestell et al., (1993) showed that impaired glucose tolerance in highly-trained athletes within 2 hours of completing a 6-day ultramarathon race was related to reduced insulin secretion rather than altered insulin sensitivity (Pestell et al., 1993). There were no differences in HOMA-IR, HOMA-b, or the absolute change in glucose, insulin, or C-peptide in response to a mixed-meal tolerance test between diet interventions in the present study (Figure 6-14). There was an approx. 20.4 % increase in post-prandial incremental glucose AUC in response to breakfast during days 7-14 of LEA versus EB (Figure 6-13). Although the composition of breakfast food was the same each day, it is important to note that this increase in incremental glucose AUC was apparent even though the total energy equivalent of the breakfast provided during LEA was effectively half that of EB; and further, is perhaps explained by the lower (although not statistically significant) fasting plasma glucose with LEA. A future perspective is to quantify circulating FFAs to calculate adipose tissue insulin resistance index (via Adipo-IR: [FFA] ´ fasting [insulin]) (Søndergaard & Jensen, 2016; Søndergaard et al., 2017) which would provide further insight into the interaction between excess FFA availability and insulin sensitivity with LEA. Skeletal muscle mitochondrial respiratory capacity does not explain impairments in exercise performance or systemic substrate utilization METABOLISM, INSULIN SENSITIVITY AND MITOCHONDRIAL RESPIRATION WITH LEA 163 Investigations by Areta et al., show that short-term LEA (1-5 days) downregulates myofibrillar protein synthesis by approximately 27 % (Areta et al., 2014) while upstream intracellular signalling of AMPK and mRNA expression of PGC-1a (mitochondrial biogenesis) are largely unaffected (Areta et al., 2014; 2020). Taken together, acute and short-term exposure to LEA apparently does not impair oxidative adaptive responses in skeletal muscle or affect aerobic capacity. This is further supported by field reports of endurance athletes with signs of acute and chronic LEA showing high aerobic capacity and performance (Fudge et al., 2006; Stellingwerff, 2018; Areta et al., 2020) in addition to comparable maximal aerobic capacity between ‘healthy’ athletes and those showing signs of chronic LEA (Loucks et al., 1989; Melin et al., 2015). The present study showed unaltered skeletal muscle mitochondrial respiratory capacity in addition to intramuscular glycogen or enzyme activity of CS, HAD, or PFK following 14-days LEA (Figure 6-7, Figure 6-8, and Figure 6-9). A recent study has shown that circulating and skeletal muscle microRNA profiles are sensitive to high levels of aerobic exercise-induced energy expenditure rather than energy status with 72 hours LEA (Margolis et al., 2022). Further, mitochondrial oxidative capacity is impaired with 4-weeks progressively excessive endurance training (Flockhart et al., 2021). Therefore, it appears that exercise rather than energetic strain per se affects skeletal muscle oxidative capacity. 6.6 Conclusion The results of this study provide evidence that 14-days LEA in endurance-trained females provokes an increased reliance on fat oxidation to absolute energy expenditure during submaximal exercise in addition to no differential impact on resting metabolic rate, insulin sensitivity, and skeletal muscle mitochondrial respiratory capacity. These results are supported by: 1) systemic b-OHB was elevated at rest, and increases in fat oxidation persisted during submaximal exercise post- LEA; 2) regulation of daily glucose control (via CGM), HOMA-IR/ HOMA-b, and insulin/glucose responses to a mixed-meal tolerance test were largely unaffected by LEA; and 3) skeletal muscle mitochondrial respiratory capacity in addition to intramuscular glycogen or enzyme activity of CS, HAD, or PFK were unaltered with LEA. Taken together, while skeletal muscle O2 utilization or carbohydrate availability were not limiting factors, the apparent ‘fat adaptation’ to 14-days LEA with a high training volume in endurance-trained females may contribute to the reported reductions in aerobic exercise capacity and performance post- LEA. CONCLUSION 164 Chapter 7: Conclusion The aim of this dissertation was to integrate brain, skeletal muscle, and systemic substrate oxidation and relative fuel utilization in the context of selected environmental and energetic stress (e.g., high-altitude, exercise, nutritional caloric deficits). The goal of this approach was to achieve a novel and comprehensive understanding of these two regulatory systems in the context of systemic energy deficits. A secondary aim of this dissertation was to explore the inflammatory response – systemically and localized in the brain – with severe reductions in energy availability in endurance-trained females and to exercise at high-altitude (Figure 7-1). Thus, two experiments were performed to alter systemic metabolism and determine the impact on inflammation (Studies 2 & 3A). To address these questions, gold-standard invasive techniques for the direct assessment of in vivo trans-cerebral oxidative metabolism (via arterial-IJV exchange) and skeletal muscle mitochondrial respiratory capacity (via high-resolution respirometry, OROBOROS, Oxygraph-2k) were utilized. Further, state-of-the-art molecular techniques including trans-cerebral exchange of inflammatory cytokine biomarkers (via 17-plex assay) in addition to a targeted 96 inflammatory biomarker proteomics approach (via high-multiplex immunoassay) were performed to assess brain and systemic inflammatory responses to exercise, respectively. These novel experiments were performed in young healthy individuals, with balanced representation of males and females. The individual study objectives, experimental approaches, and overall results are summarized next. Figure 7-1. Overview of key themes and overlapping techniques of this dissertation CONCLUSION 165 Key themes of this dissertation include metabolism (brain, systemic, muscle), in addition to the immune system, inflammation, environmental stress, energetic stress, and exercise. Brain metabolism was measured via arterial-IJV sampling (A), systemic metabolism was assessed via indirect calorimetry (B), and muscle metabolism was quantified via high-resolution respirometry (C). Study 1 – Evidence for direct CO2-mediated alterations in cerebral oxidative metabolism in humans Study 1 addressed a long-standing fundamental question for integrative cerebrovascular physiology: How are the cerebral metabolic rates of oxygen and glucose utilization (CMRO2 and CMRGlu, respectively) affected by alterations in arterial PCO2 (PaCO2)? The objective of the current analysis was to test the hypothesis that CMRO2 would be directly affected by changes in PaCO2 such that CMRO2 would be reduced with progressive increases in PaCO2, and vice-versa. The results of this study indicate that CMRO2 is altered by approximately 1 % per mmHg change in PaCO2. Further, during hypercapnia, CMRGlu is reduced to a lesser extent than CMRO2 – a finding interpreted that reductions in cerebral metabolism that may be in part compensated by an increased contribution of anaerobic metabolic pathways during acute steady-state hypercapnic acidosis in healthy humans. These results have important implications for the physiological and clinical validity of CVR testing in health and disease. Study 2 – How does systemic fuel utilization at high-altitude affect the brain inflammatory response to exercise? This study investigated systemic substrate oxidation and energy expenditure in addition to inflammatory stress in the brain during exercise at an intensity that elicits maximal fat oxidation (FATmax) and following acute exhaustive exercise at sea level and following 3-8 days at 3,800 m. Radial arterial and internal jugular venous blood sampling was simultaneously performed to assess trans-cerebral inflammatory cytokines (via 17-plex assay) and leukocytes (via hematology analysis) prior to and following 60-minutes of low-intensity exercise, at maximal exercise, 5 minutes post-exercise, and following 60 minutes of passive recovery. It was hypothesized that: 1) 1-hour of cycling exercise at altitude-specific FATmax intensity will promote an anti-inflammatory response at both sea level and high-altitude (indicated by higher IL-4, IL-10, and IL-13), and 2) acclimatization to high-altitude will provoke an anti-inflammatory response at rest which will attenuate the pro-inflammatory and leukocyte mobilization responses to maximal exercise compared to sea level. The results of this study provide new evidence that 6-8 days of acclimatization at 3,800 m does not provoke immune suppression or mediate CONCLUSION 166 elevations in systemic pro-inflammation with maximal exercise. Acclimatization to high-altitude attenuates systemic pro-inflammatory responses at rest; however, although independent of altitude, an additive effect of exercise duration/intensity provokes trans-cerebral net release of pro-inflammatory cytokines. Study 3A – The importance of low energy availability for the immune system, systemic inflammation, and performance ability in endurance-trained female athletes This study examined the influence of 14-days low energy availability (LEA) in endurance-trained females on: 1) the immune system (white blood cell count via hematology analysis and targeted 96-panel inflammatory protein biomarker via high-multiplex immunoassay), 2) systemic inflammation (creatine kinase, cortisol, c-reactive protein), and 3) performance capacity (20-minute isokinetic cycling time trial and time to exhaustion tests). The immune and inflammatory responses were investigated at rest and post-exercise following two 14-day randomized, blinded, cross-over controlled dietary interventions: 1) energy balance (EB; 50 kcal/ kg fat free mass per day) and 2) low energy availability (LEA; 25 kcal/ kg fat free mass per day). It was hypothesized that LEA would suppress the immune system, provoke systemic inflammation, and impair exercise performance. The results of this study provide evidence that 14-days LEA in endurance-trained females provokes immunological stress, systemic inflammation and impairs exercise performance. Further the implications of LEA on exercise performance impairments persisted following three days recovery in sufficient energy balance post- LEA. These results are supported by: 1) reduced post- exercise leukocyte mobilization following LEA; 2) higher creatine kinase with exercise and elevated systemic inflammatory biomarkers at rest post- LEA (via targeted proteomics); and 3) reductions in power output during a 20-minute time trial and overall time to exhaustion by 7.8 % and 18.9 %. Study 3B – Substrate oxidation, skeletal muscle mitochondrial respiratory capacity, and insulin sensitivity following 14-days low energy availability in endurance-trained female athletes The objective of this study was to investigate the influence of 14-days LEA in endurance-trained female athletes on: 1) fuel utilization (e.g., respiratory exchange ratio, glucose, intramuscular glycogen, lactate, and b-OHB), 2) insulin sensitivity (HOMA-IR, mixed-meal tolerance test, continuous glucose monitoring), and 3) mitochondrial oxidative capacity in permeabilized human skeletal muscle fibers (via high-resolution respirometry, OROBOROS, Oxygraph-2k). It was CONCLUSION 167 hypothesized that 14-days LEA would suppress resting metabolic rate, impair insulin sensitivity, and increase relative energy expenditure during submaximal and exhaustive cycling exercise. Additionally, the influence of LEA on skeletal muscle mitochondrial oxidative capacity was explored. The results of this study provide evidence that 14-days LEA in endurance-trained females provokes an increased reliance on fat oxidation to absolute energy expenditure during submaximal exercise in addition to no differential impact on resting metabolic rate, insulin sensitivity, and skeletal muscle mitochondrial respiratory capacity. These results are supported by: 1) systemic b-OHB was elevated at rest, and increases in fat oxidation persisted during submaximal exercise post- LEA; 2) regulation of daily glucose control (via CGM), HOMA-IR/ HOMA-b, and insulin/glucose responses to a mixed-meal tolerance test were largely unaffected by LEA; and 3) skeletal muscle mitochondrial respiratory capacity in addition to intramuscular glycogen or enzyme activity of CS, HAD, or PFK were unaltered with LEA. Taken together, while skeletal muscle O2 utilization or carbohydrate availability were not limiting factors, the apparent ‘fat adaptation’ to 14-days LEA with a high training volume in endurance-trained females may contribute to the reported reductions in aerobic exercise capacity and performance post- LEA. 7.1 Future perspectives There are several techniques which can be utilized to further explore aspects of fat metabolism on energy expenditure and substrate utilization during LEA as well as fat oxidation at rest and during exercise at high-altitude. For example, it is possible to assess fatty acid oxidation in skeletal muscle tissue with high-resolution respirometry via a series of substrate-uncoupler-inhibitor-titration protocols which specifically interrogate mitochondrial respiration during b-oxidation. Acylcarnitines such as palmitolycarnitine or octanoylcarnitine are commonly used substrates (Ojuka et al., 2016). Further, it is possible to measure ETS capacity directly in adipose tissue (Kraunsøe et al., 2010; Watt et al., 2023); each of these techniques would provide further insight into the apparent preferential utilization of fat following 14-days LEA. Additionally, while skeletal muscle mitochondrial respiratory capacity was measured at rest post- diet, alterations in skeletal muscle bioenergetics post- exercise would be useful to better understand the performance impairments of LEA. While indirect calorimetry was utilized to measure substrate oxidation at rest and during exercise, the labeled isotope technique (via coingestion of 13C glucose and 13C fructose) provides a better indication of substrate utilization as this can differentiate between exogenous carbohydrate oxidation in addition to endogenous CONCLUSION 168 glucose oxidation derived from liver and muscle glycogen. Additionally, infusion of [5-2H]glycerol and [1-13C]palmitate provides an accurate measurement of total lipid oxidation, whole-body lipolysis, and rate of appearance of nonesterified fatty acids (Kurpad et al., 1994; Friedlander et al., 1999). Lastly, plasma and serum samples for free radical-mediated lipid peroxidation were collected during Study 2 which will be analyzed. These data may provide insight into the interaction between exercise intensity and oxidative stress in the brain (New et al., 2013). At extreme altitude > 5,000 m, sustained exposure to environmental hypoxia is associated with marked involuntary weight loss (i.e., high-altitude cachexia). This is apparent at lower altitudes although to a lesser extent. As such, future studies are necessary to explore energy availability by either caloric restriction and/or increases in energy expenditure on the inflammatory response to exercise following prolonged stay at high-altitude. As 14-days LEA provokes a systemic inflammatory response, it would be worth investigating whether the apparent anti-inflammatory response with acclimatization mediates LEA-induced inflammation at high-altitude. Additionally, had Study 2 investigated the acute high-altitude response (within 1-2 days following initial ascent) we may have expected immune suppression in addition to an exacerbated pro-inflammatory response both at rest and post- maximal exercise compared to sea level. Further, 1-hour cycling at a personalized intensity to maximize fat oxidation would be expected to promote a favorable anti-inflammatory response which would attenuate the expected systemic inflammation with acute exposure to high-altitude (Pham et al., 2021; 2022). The time-sensitivity of exposure to high-altitude on pro/anti- inflammatory responses is an important consideration; however, the multi-factorial hematological, respiratory, and acid-base compensatory changes which occur during acclimatization to high-altitude – in addition to the impracticality of performing maximal exercise within < 2 days initial ascent – was motivation to perform Study 2 following 6-8 days acclimatization instead of prioritizing a maximal inflammatory stress with acute exposure. This timeframe is consistent with guidelines on avoiding altitude illness. In addition, as pro-inflammatory stress at high altitude is implicated in the etiology of high-altitude illnesses (Pham et al., 2021; Turner et al., 2021), it would be relevant to further explore the brain inflammatory response in an acute context. It is noteworthy, however, that exposure to high-altitude does not exacerbate pro-inflammatory stress in response to maximal exercise in the brain, and in the context of acclimatization to 3,800 m, this duration and intensity of exercise is not a trigger for altitude illness via pro-inflammatory stress. CONCLUSION 169 7.2 Integrated summary Collectively, this thesis provides novel insights for integrative brain, skeletal muscle, and systemic substrate oxidation and relative fuel utilization in the context of environmental and energetic stress. For example, it appears that energy deficiency (i.e., caloric restriction) but not high-altitude affects the systemic inflammatory response to exercise. While maximal fat oxidation during exercise is reduced at high-altitude, 14-days of LEA results in ‘fat compensation’ to preferentially utilize fat oxidation during exercise. 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APPENDICES 213 Appendices Appendix A This study provides context for trans-cerebral acid-base regulation which involves participants from Chapter 3: Study 1 compared to participants completing incremental cycling exercise to exhaustion. Published in the Journal of Cerebral Blood Flow and Metabolism. Trans-cerebral HCO3- and PCO2 exchange during acute respiratory acidosis and exercise-induced metabolic acidosis in humans Hannah G. Caldwell1, Ryan L. Hoiland2,3, Kurt J. Smith4, Patrice Brassard5,6, Anthony R. Bain7, Michael M. Tymko8, Connor A. Howe1, Jay M.J.R. Carr1, Benjamin S. Stacey9, Damian M. Bailey9, Audrey Drapeau5,6, Mypinder S. Sekhon10, David B. MacLeod11, Philip N. Ainslie1 1Centre for Heart, Lung and Vascular Health, School of Health and Exercise Sciences, University of British Columbia Okanagan, Kelowna, BC, Canada, V1V 1V7 2Department of Anesthesiology, Pharmacology and Therapeutics, Vancouver General Hospital, West 12th Avenue, University of British Columbia, Vancouver, BC, Canada, V5Z 1M9 3Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, BC, Canada, V6T 1Z4 4Integrative Physiology Laboratory, Department of Kinesiology and Nutrition, University of Illinois Chicago, Chicago, IL, USA 5Department of Kinesiology, Faculty of Medicine, Université Laval, Québec, Canada 6Research center of the Institut universitaire de cardiologie et de pneumologie de Québec, Québec, Canada 7University of Windsor, Faculty of Human Kinetics, Department of Kinesiology, Windsor, Ontario, Canada 8Neurovascular Health Laboratory, Faculty of Kinesiology, Sport and Recreation, University of Alberta, Edmonton, Alberta, Canada 9Neurovascular Research Laboratory, Faculty of Life Sciences and Education, University of South Wales, Pontypridd, CF37 4BB, United Kingdom 10Division of Critical Care Medicine, Department of Medicine, Vancouver General Hospital, West 12th Avenue, University of British Columbia, Vancouver, BC, Canada, V5Z 1M9 11Human Pharmacology and Physiology Lab, Department of Anesthesiology, Duke University Medical Center, Durham, NC, USA, 27708 Corresponding author: Hannah G. Caldwell, Centre for Heart, Lung and Vascular Health, School of Health and Exercise Sciences, University of British Columbia – Okanagan, 3333 University Way, Kelowna, British Columbia, Canada, V1V 1V7 Email address: hannah.caldwell@ubc.ca APPENDICES 214 ABSTRACT This study investigated trans-cerebral internal jugular venous-arterial bicarbonate ([HCO3-]) and carbon dioxide tension (PCO2) exchange utilizing two separate interventions to induce acidosis: 1) acute respiratory acidosis via elevations in arterial PCO2 (PaCO2) (n=39); and 2) metabolic acidosis via incremental cycling exercise to exhaustion (n=24). During respiratory acidosis, arterial [HCO3-] increased by 0.15±0.05 mmol×l-1 per mmHg elevation in PaCO2 across a wide physiological range (35 to 60 mmHg PaCO2; P<0.001). The narrowing of the venous-arterial [HCO3-] and PCO2 differences with respiratory acidosis were both related to the hypercapnia-induced elevations in cerebral blood flow (CBF) (both P<0.001; subset n=27); thus, trans-cerebral [HCO3-] exchange (CBF × venous-arterial [HCO3-] difference) was reduced indicating a shift from net release toward net uptake of [HCO3-] (P=0.004). Arterial [HCO3-] was reduced by -0.48±0.15 mmol×l-1 per nmol×l-1 increase in arterial [H+] with exercise-induced acidosis (P<0.001). There was no relationship between the venous-arterial [HCO3-] difference and arterial [H+] with exercise-induced acidosis or CBF; therefore, trans-cerebral [HCO3-] exchange was unaltered throughout exercise when indexed against arterial [H+] or pH (P=0.933 and P=0.896, respectively). These results indicate that increases and decreases in systemic [HCO3-] – during acute respiratory/ exercise-induced metabolic acidosis, respectively – differentially affect cerebrovascular acid-base balance (via trans-cerebral [HCO3-] exchange). Word Count: 199/200 Key Words: Acidosis, bicarbonate, carbon dioxide, exercise, trans-cerebral exchange APPENDICES 215 INTRODUCTION The regulation of extracellular pH – which directly affects cells via local changes in intravascular or extravascular/interstitial conditions – is affected by rapid chemical buffering reactions, involving phosphate, glycolysis, and carbon dioxide tension (PCO2). The cerebral vasculature is exceptionally sensitive to changes in arterial PCO2 (PaCO2) 1-3, such that cerebral blood flow (CBF) rapidly increases by 6-8% per mmHg increase in PaCO2 (reviewed in: 4). This CBF responsiveness to PaCO2/[H+] acts to stabilize CO2 gradients and thus regulate pH across the blood-brain barrier (BBB) 4-6. As CO2 travels rapidly across the BBB – indicated by the swift changes in CBF (<15-30 seconds) following stepwise changes in PaCO2 7-10 – perivascular/interstitial fluid (ISF) and intracellular brain tissue pH are tightly related to PaCO2. The Fick principle explains that elevated PaCO2 will be related to reductions in the trans-cerebral venous-arterial PCO2 difference as: 1) CBF varies directly with PaCO2 1,3,11; and 2) the cerebral metabolic production of CO2 (V̇CO2) is unaltered in the physiological range; e.g., CBF = V̇CO2 / PvCO2 – PaCO2 12,13. Experimental results in several acid-base pathologies indicate the trans-cerebral venous-arterial PCO2 difference is reduced and increased with acute respiratory acidosis/alkalosis, respectively 3,5,12,14-18. These regulatory changes in the venous-arterial PCO2 difference contribute to tight regulation of a narrow range of cerebral interstitial pH 19,20 and are influenced by the responsiveness of CBF to acute alterations in PaCO2; i.e., cerebrovascular CO2 reactivity 6. Exercise-induced metabolic acidosis results when the rate of ATP hydrolysis (H+ release) eventually exceeds the maximal buffering capacity; the excess CO2 is then removed via hyperventilation 21,22 and these PaCO2 changes in part explain the CBF kinetics response with exercise 23-25. With exercise-induced acidosis, arterial [HCO3-] is markedly reduced due to excessive buffering of [H+] 21,22 – however, whether this change in arterial [HCO3-] is related to alterations in trans-cerebral [HCO3-] and PCO2 exchange as well as in vivo buffering capacity has not been experimentally addressed in humans. The cerebral metabolic rate of oxygen (e.g., CMRO2 = CBF × arterial-venous oxygen content difference) increases linearly by up to 30% at maximal exercise intensities to support substrate utilization in the face of reductions in CBF 26-33. At maximal exercise, there is a higher relative contribution of cerebral anaerobic glycolysis and lactate oxidation 34-36. There is evidence to support that maximal exercise-induced acidosis facilitates trans-cerebral lactate exchange and related increases in lactate oxidation mediated via pH-sensitive transcellular [H+] gradients 37-39 and increases in systemic lactate availability 28,32,40-42. Additionally, metabolic acidosis achieved with exhaustive exercise reportedly increases BBB permeability with direct relevance for transcellular CO2 transport 38,43. Overall, cerebrovascular acid-base regulation is likely interrelated with cerebral substrate prioritization during exercise via pH-sensitive utilization of lactate 44,45. The Henderson-Hasselbalch relationship (Equation 2) explains that acute elevations in arterial [HCO3-] would increase arterial buffering capacity; for example, with increases in arterial [HCO3-] any given change in PaCO2 will result in a lesser change in arterial [H+]/pH 13,46. This relationship would theoretically apply with reductions in arterial [HCO3-] and therefore be reflective of related decreases in arterial buffering capacity. Pre-clinical experiments in anesthetized and artificially ventilated cats show that rapid/transient [HCO3-]/Cl- exchange occurs within 15 seconds between intravascular and extracellular fluid in response to elevated systemic arterial [HCO3-] at maintained PaCO2 47 – these results indicate that extracellular [HCO3-] is partially and transiently altered with changes in arterial [HCO3-], however, such changes in extracellular [HCO3-] are restored (within 1 hour) and these [HCO3-]/Cl- exchange kinetics are appreciably less influential than persistent and freely diffusible CO2 transport 48,49. At rest, cerebrospinal fluid (CSF) PCO2 – typically indexed via internal jugular venous sampling – is 6 to 11 mmHg higher and pH is 0.05 to 0.08 units lower than that of the arterial blood 13,50,51. As such, resting trans-cerebral net exchange of [HCO3-] and PCO2 (e.g., CBF × venous-arterial difference) is a positive value reflective of net release. A narrowing of the venous-arterial APPENDICES 216 difference would indicate a shift toward net uptake and can be achieved, for example by: 1) larger increase in arterial relative to venous value; and 2) reduction in venous with a corresponding increase in arterial values. No study to date has investigated whether alterations in the trans-cerebral venous-arterial [HCO3-] and PCO2 differences during acute respiratory/ exercise-induced metabolic acidosis are related to CBF and regulatory systemic changes in arterial [HCO3-] in vivo in humans. The objective of this study was to investigate acid-base balance via alterations in trans-cerebral internal jugular venous-arterial [HCO3-] and PCO2 exchange utilizing two separate experimental interventions to induce acidosis: Study 1) acute respiratory acidosis via elevations in PaCO2 (range: 35 to 60 mmHg); and Study 2) metabolic acidosis via incremental cycling exercise to exhaustion (range: 7.45 to 7.20). We hypothesized that: 1) acute hypercapnic acidosis would reduce the venous-arterial [HCO3-] and PCO2 differences and this reduction would be related to higher CBF, less trans-cerebral [HCO3-] exchange, and elevated arterial [HCO3-]; and 2) arterial [HCO3-] would progressively decrease with maximal exercise-induced acidosis and – as explained by the non-linear CBF response with incremental exercise – this reduction in systemic [HCO3-] would be unrelated to any change in CBF or trans-cerebral venous-arterial [HCO3-] and PCO2 exchange. METHODS Ethical Approval All participants provided informed written consent before participating in these studies. The original research studies were approved by the University of British Columbia Clinical Review Ethical Board (CREB: H16-01028, H18-01755, H15-00166, H11-03287) and the Comité d’éthique de la recherche de l’Institut universitaire de cardiologie et de pneumologie de Québec (CER: 21557) and according to the principles established by the Declaration of Helsinki (except for registration in a database). As the current study included a secondary analysis from previous institutional review board approved studies and the use of de-identified data sharing, this study did not require additional institutional review board review. Participants These data were obtained during five separate experimental studies previously conducted at the University of British Columbia, Kelowna, British Columbia, Canada and the Université Laval, Québec City, Québec, Canada. Thirty-nine healthy adults completed the CO2 trials (n=34 males and n=5 females) and twenty-four healthy adults completed the exercise trials (n=19 males and n=5 females). Participants had no history of cerebrovascular, cardiovascular, or respiratory disease and were not taking any prescription medication at their time of participation. Participants refrained from alcohol and caffeine consumption as well as vigorous exercise or activity for at least 12 hours prior to testing. Experimental Overview The experimental questions addressed in this study involved post-hoc data analysis; therefore, few of the arterial and venous blood gas data have been reported separately in various context for previously published works from these studies; e.g., 36,52,53. Importantly, the current experimental questions involved new data analyses that have not been previously reported. Study 1: Respiratory Acidosis Protocols Participants completed one of the following protocols to elicit progressive stepwise steady-state elevations in PaCO2 via dynamic end-tidal forcing: 1) +3, +6, +9 mmHg PaCO2 (n=11 males); 2) +4.5 and +9 mmHg PaCO2 (n=12 males); 3) +8 mmHg PaCO2 (n=5 females; n=7 males); or 4) +10 and +20 mmHg PaCO2 (n=4 males). The alterations in PaCO2 were calculated from the resting eupneic breathing end-tidal values. All measurements were taken after at least 2-3 minutes of steady-state. Previous investigations from our group have shown that cerebrovascular CO2 reactivity (utilizing the same dynamic end-tidal forcing system) is highly linear in the hypercapnic range up to +20 mmHg 4,54. Additionally, recent retrospective analysis by APPENDICES 217 our research group (Carr et al., 2021; accepted) has shown that following a rapid stepwise change in PETCO2 (via the exact same experimental methods/techniques in the current studies), relevant cerebrovascular and cardiorespiratory variables achieve steady-state within 2-minutes of exposure duration to elevated inspired PCO2. As such, by applying a linear mixed model analysis, there is no statistical or physiological rationale to expect these results to be different whether participants completed the exact same stepwise steady-state elevations in PaCO2 compared to the varied stages utilized in this post-hoc analysis. Study 2: Exercise Protocols Participants completed supine incremental cycling exercise to exhaustion at various relative (0, 20, 40, 60, 80, 100% maximal workload; n=12 males) and fixed exercise intensities (0, 50, 75, 100 watts; n=5 females and 0, 75, 100, 125 watts; n=7 males). Each workload was 3-5 minutes in duration and steady-state blood samples were drawn within the last 20 s of each exercise stage. Blood Sampling Approximately 1.0 ml of radial arterial and internal jugular venous blood were drawn at the same time into pre-heparinized syringes (SafePICO, Radiometer, Copenhagen, Denmark) and analyzed immediately using a commercial blood gas analyzer (ABL90 FLEX, Radiometer) at each experimental stage (n=39). This analysis included measurements of PaCO2 and oxygen tension (PaO2), arterial oxygen saturation (SaO2), arterial oxygen content (CaO2), base excess, [H+], hemoglobin concentration ([Hb]), and pH. Data collected at the Université Laval were analyzed within 10-15 minutes for n=12 (ABL800 FLEX, ABL825, Radiometer). Arterial oxygen content (CaO2) was calculated as: CaO2 (mL×dL-1) = [Hb] × 1.34 × [SaO2 (%)/ 100] + 0.003 × PaO2 Equation 1 Where 1.34 is the O2 binding capacity of hemoglobin and 0.003 is the solubility of O2 dissolved in blood 55,56. Acid-Base Balance Data Analysis Blood gas analyzers do not typically have the capacity to directly measure [HCO3-]; instead, it is calculated from measured PaCO2 and pH, by rearranging the Henderson-Hasselbalch equation (Equations 2 & 3). pH = 6.1 + log [HCO3-]0.031 × PCO2 Equation 2 [HCO3-] = 0.031 × PCO2 × 10(pH-6.1) Equation 3 Where 6.1 is the pKa (i.e., -log of the acid dissociation constant) at 37.0°C 57 and 0.031 mEq×l-1 per mmHg PCO2 is the solubility factor for dissolved CO2 plus carbonic acid (H2CO3) at 37.0°C in plasma. The following calculations were used to quantify an index of in vivo buffering capacity termed “extracellular pH defense” 58,59. All in vivo buffering capacity calculations were analyzed with the average venous-arterial values of [HCO3-], [La], and pH as an estimate of cerebral tissue values. These results were consistent when expressed as arterial or internal jugular venous buffering capacity. Exercise: D [HCO3-] × D pH-1 Equation 4 The reduction in [HCO3-] per unit pH is reportedly attributable to hyperventilation and HCO3- buffering 58. - D [La] × D pH-1 Equation 5 This index includes lactate ([La]) and is considered total pH defense by hyperventilation, as well as HCO3- and non-bicarbonate buffers 58. APPENDICES 218 Respiratory acidosis: - D [HCO3-] × D pH-1 Equation 6 Equation 4 above has been adapted as [HCO3-] increases with respiratory acidosis. Cardiorespiratory Detailed cardiorespiratory experimental methods and results have been previously reported elsewhere; e.g., 36,52,53. Briefly, the partial pressures of end-tidal CO2 and O2 (i.e., PETCO2 and PETO2, respectively) were controlled during acute respiratory acidosis using a custom-designed dynamic end-tidal forcing system to effectively regulate end-tidal gases across wide ranges of PETCO2 and PETO2 60,61, independent of ventilation (V̇E) 62. Beat-by-beat arterial blood pressure was continuously acquired via the radial artery pressure transducer positioned at the height of the right atrium (Edwards Lifesciences, TruWave VAMP, CA, USA) and the arterial blood pressure waveform was averaged to calculate MAP. Cerebrovascular Detailed cerebrovascular experimental methods and results have been previously reported elsewhere; e.g., 36,52,53. Briefly, extra-cranial blood velocity and diameter of the internal carotid artery (ICA) and vertebral artery (VA) were measured using a 10-MHz multifrequency linear array Duplex ultrasound (Terason t3000; Teratech, Burlington, MA, USA). Pulse-wave mode was used to measure beat-to-beat peak blood velocity and arterial diameter was instantaneously measured via B-mode imaging; data were analyzed using custom edge-detection and wall tracking software (BloodFlow Analysis, version 5.1). The vessel location was decided on an individual basis to allow for reliable image acquisition, with the same location and consistent insonation angle (60°) repeated within participants and between trials. Blood flow was calculated as: Q (mL×min-1) = peak envelope blood velocity2 × (p(0.5 × diameter)2) × 60 Equation 7 Total cerebral blood flow (CBF) was calculated as: CBF (mL×min-1) = 2 × (QICA + QVA) Equation 8 Net [HCO3-] exchange was calculated as: Net [HCO3-] exchange (mmol×min-1) = ([HCO3-]v - [HCO3-]a) × CBF Equation 9 Net PCO2 exchange was calculated as: Net PCO2 exchange (mmHg×min-1) = (PvCO2 - PaCO2) × CBF Equation 10 Where a negative value indicates a net uptake and a positive value indicates a net release 63. Statistical Analyses All data are presented in-text as mean ± SD and as individual values in figures. Statistical analyses were performed using SPSS software (IBM statistics, Version 23.0) and Prism (GraphPad Software, Version 9.1.0). Normality was assessed using Shapiro-Wilk tests and by visual inspection of Q-Q plots. Statistical significance was set at P < 0.05. Relationships between select variables were analyzed using linear regression. A linear mixed-model analysis with fixed effects of either arterial [H+], pH, or PaCO2 was used to compare PCO2, [HCO3-], [La], in vivo buffering capacity, trans-cerebral exchange, and CBF separately for the respiratory acidosis and exercise interventions. Subjects were included as a random effect. RESULTS Study 1: Respiratory acidosis Total CBF increased by 4 ± 1 ml × 100 g-1 × min-1 per mmHg elevation in PaCO2 across a wide range from 35 to 60 mmHg PaCO2 (P < 0.001; Figure 1A); this hypercapnia-mediated APPENDICES 219 increase in CBF was related to narrowing of the venous-arterial PCO2 difference (P < 0.001; Figure 1B) such that trans-cerebral PCO2 exchange was unaltered with stepwise increases in PaCO2 (P = 0.057; Figure 1C). Arterial [HCO3-] increased by 0.15 ± 0.05 mmol × l-1 per mmHg elevation in PaCO2 (P < 0.001; Figure 1D). There was a relationship between respiratory acidosis and narrowing of the venous-arterial [HCO3-] and PCO2 differences; e.g., -0.16 ± 0.11 mmol × l-1 and -0.52 ± 0.22 mmHg reduction in venous-arterial [HCO3-] and PCO2 differences per nmol × l-1 increase in arterial [H+], respectively (both P < 0.001; Figure 1 E & F). The venous-arterial [HCO3-] and PCO2 differences were each related to hypercapnia-induced increases in CBF (both P < 0.001; Figure 1G). As such, [HCO3-] exchange was reduced by -0.05 ± 0.08 mmol × min-1 per nmol × l-1 increase in arterial [H+]; i.e., less venous [HCO3-] efflux/release with respiratory acidosis (P = 0.004; Figure 1H). Study 2: Exercise-induced metabolic acidosis Total CBF was related to PaCO2 throughout progressive cycling exercise to exhaustion corresponding to 1 ± 1 ml × 100 g-1 × min-1 per mmHg change in PaCO2 (P = 0.004; Figure 2A). There was no relationship between CBF and the venous-arterial PCO2 difference (P = 0.177; Figure 2B); therefore, trans-cerebral PCO2 exchange was unrelated to PaCO2 during exercise (P = 0.155; Figure 2C). Arterial [HCO3-] was reduced by -0.48 ± 0.15 mmol × l-1 per nmol × l-1 increase in arterial [H+] with exercise-induced acidosis at maximal cycling exercise (P < 0.001; Figure 2D). There was no relationship between the venous-arterial [HCO3-] difference and arterial [H+] with exercise-induced acidosis (P = 0.801; Figure 2E). There was, however, a relationship between widening of the venous-arterial PCO2 difference by 0.12 ± 0.20 mmHg per nmol × l-1 increase in arterial [H+] during incremental cycling exercise to exhaustion (P = 0.006; Figure 2F); i.e., increased PCO2 uptake reflective of acidosis. The exercise-induced CBF response was unrelated to the venous-arterial [HCO3-] difference (P = 0.682; Figure 2G); as such, there were no changes in trans-cerebral [HCO3-] exchange during exercise when indexed against arterial [H+] or pH (P = 0.933 and P = 0.896, respectively; Figure 2H). In vivo buffering capacity during acute respiratory acidosis and exercise Linear mixed-model analysis revealed no significant relationship between in vivo buffering capacity (D [HCO3-] × D pH-1) and the average venous-arterial pH during stepwise respiratory acidosis (P = 0.078; Figure 1I). Additionally, there were no relationships between in vivo buffering capacity (D [HCO3-] × D pH-1 or -D [La] × D pH-1) and the average venous-arterial pH during incremental cycling exercise to exhaustion (P = 0.187 and P = 0.392, respectively; Figure 2I). DISCUSSION The results of this study indicate that trans-cerebral [HCO3-] and PCO2 exchange are differentially regulated in response to acute respiratory and exercise-induced acidosis. This finding is supported by: 1) acute hypercapnic acidosis elevated arterial [HCO3-]; however, trans-cerebral [HCO3-] exchange was reduced (i.e., indicating a shift from net release toward net uptake of [HCO3-]) and this was reflective in narrower venous-arterial [HCO3-] and PCO2 differences with higher CBF (Figure 1); and 2) arterial [HCO3-] was progressively reduced with maximal exercise-induced acidosis and this was unrelated to venous-arterial [HCO3-] and PCO2 exchange (Figure 2). Additionally, these results show there are no appreciable changes in the in vivo buffering capacity across a wide range of respiratory acidosis (up to +20 mmHg PaCO2) or incremental cycling exercise to exhaustion in humans (Figures 1I & 2I). Respiratory versus metabolic acidosis – influence of [HCO3-] exchange: The two experimental interventions of acute respiratory acidosis and exercise-induced metabolic acidosis each provoke equivalent reductions in pH with a markedly disparate arterial [HCO3-] response. For example, the reductions in arterial [HCO3-] during exercise are reflective of a 3-fold larger rate of change in [HCO3-] APPENDICES 220 versus the small increases in [HCO3-] observed with respiratory acidosis (e.g., approx. -0.48 vs. +0.15 mmol × l-1 [HCO3-] per nmol × l-1 increase in [H+]). Additionally, it is noteworthy to consider how alterations in CMRO2 with exercise and respiratory acidosis will affect V̇CO2 and, therefore, PvCO2 25,35,64-66. Increases in PaCO2 facilitate rapid passive diffusion of CO2 across the BBB via reduced CSF driving pressure leading to increases in intra- and extracellular [H+], thus provoking acidosis through acid-base re-equilibration 67,68 in accordance with Le Chatelier’s Principle (rightward shift in Equation 7) 69. Overall, CO2 transport is the sum of the diffusion of 1) dissolved CO2; and 2) CO2 bound as HCO3- (i.e., “facilitated CO2 diffusion”) – this process involves a flux of H+ equivalent to that of HCO3- as per Equation 7 70-72. CO2 + H2O CA!" H2CO3 ↔ HCO3- + H+ Equation 7 Acute respiratory acidosis increases arterial [HCO3-] via continuous conversion of CO2 – rapidly catalyzed by carbonic anhydrase (CA) – shifting the equilibrium relationship toward the [HCO3-] buffer reaction. This is reflective of rapid increases in extracellular [HCO3-] due to intracellular [HCO3-] exchange with Cl- to buffer the [H+] produced via CO2 hydration 73. Importantly, however, the ratio of arterial [HCO3-] to PaCO2 is lower such that pH is reduced (as per Equation 2). At rest, the relative contributions of dissolved CO2, chemically bound to hemoglobin and protein carbamate, and HCO3- to total CO2 exchange are approximately 5%, 10% and 85%, respectively. During severe exercise, HCO3- facilitated CO2 diffusion is reduced to approximately 2/3 of total CO2 exchange (as the contribution of dissolved CO2 increases sevenfold with exercise-induced acidosis) 71,74. With maximal exercise-induced acidosis, arterial [HCO3-] is markedly reduced due to compensatory buffering of [H+] leading to production of CO2 and H2O (leftward shift in Equation 7); the excess CO2 is then removed via hyperventilation 21,22. Acute elevations in cerebral blood flow stabilize [HCO3-] and PCO2 gradients: Acute respiratory acidosis provokes three key regulatory responses: 1) arterial [HCO3-] increases (via inter-conversion of CO2) in response to elevated PaCO2 (Figure 1D); 2) there is rapid/transient exchange of HCO3- and Cl- across the BBB and between brain tissue and extracellular fluid 47,73; and 3) total CBF increases thus reducing the difference between arterial and extravascular PCO2, thereby limiting the rise in intracellular tissue PCO2 (Figure 1B). The increase in CBF in response to PaCO2 is restricted by the maximal vasodilatory response to hypercapnia in vivo in humans; e.g., +15-20 mmHg PaCO2 equates to 150% increase in CBF 54. Elevations in arterial [HCO3-] may theoretically contribute to localized changes in CBF with severe respiratory acidosis – via increases in extravascular [HCO3-] – and thus, regulatory changes in [HCO3-] may help explain the maximal cerebrovascular vasodilatory reserve to PaCO2. The internal jugular venous-arterial [HCO3-] difference is reduced with acute severe hypercapnic acidosis (Figure 1E) – a response likely explained by increased extravascular HCO3- ‘wash-out’ as the reduction in trans-cerebral [HCO3-] difference is related to the hypercapnic CBF response (P < 0.001; Figure 1G). Arvidsson and colleagues (1981) showed that intravenous infusion of NaHCO3 in hypercapnic dogs causes a 50-70% reduction from the PaCO2-induced higher CBF. Additionally, cerebrospinal fluid [HCO3-] ([HCO3-]CSF) was appreciably higher following NaHCO3; these data indicate that acutely elevated PaCO2 may facilitate the transport of exogenous HCO3- across the BBB (via higher CBF) resulting in cerebral vasoconstriction due to increased extravascular pH 75. The present results support that the CBF response to acute respiratory acidosis contributes to tight regulation of cerebrovascular pH (via narrowing of the trans-cerebral [HCO3-] and PCO2 differences) in vivo in humans. Regulation of cerebrovascular acid-base balance during exercise: APPENDICES 221 Trans-cerebral venous-arterial [HCO3-] exchange was unaffected by maximal exercise-induced acidosis (e.g., pH 7.20; Figure 2E) and there was a widening of the venous-arterial PCO2 difference during incremental cycling exercise to exhaustion (Figure 2F). Total CBF increases steadily by 10-20% up to intensities of approximately 60-70% of maximal oxygen uptake (V̇O2max) to regulate cerebral substrate delivery 32,76, and is mediated via relative alveolar hypoventilation (i.e., elevations in PaCO2) and increases in CMRO2 25,36,77. With progressive increases in cycling exercise intensity, the relatively linear CMRO2 response is coupled to cerebral oxygen delivery (CBF × CaO2) and oxygen extraction (CaO2 – CvO2 / CaO2) × 100%)) rather than CBF per se. At maximal exercise, hyperventilatory-induced reductions in CBF – together with marked acidosis – would conceivably adversely affect intracellular/extravascular [HCO3-] and CO2 ‘wash-out’. Previous work by Bisgard and colleagues (1978) reported unaltered [HCO3-]CSF and PaCO2-mediated increases in CSF pH (via hyperventilatory response) with severe near-maximal exercise in ponies 78 – these data emphasize the importance of the ventilatory response on cerebrovascular PCO2/[HCO3-] regulation during exercise 6. Additionally, albeit during resting conditions, several studies report that sustained changes in CSF/extracellular [HCO3-] respond slowly (e.g., several hours) and only partially to systemic changes in arterial [HCO3-] 50,79-84, thus explaining the steady [HCO3-]CSF during acute exercise (<10 minutes). Taken together with the remarkably consistent trans-cerebral [HCO3-] exchange (Figure 2H) and in vivo [HCO3-] and [La] buffering capacity throughout exercise (Figure 2I), these data indicate that the CBF response to exercise per se likely plays a lesser role in cerebrovascular acid-base regulation (versus respiratory acidosis, for example). Experimental considerations: A key strength of this study was the invasive direct Kety-Schmidt technique to quantify trans-cerebral venous-arterial exchange of [HCO3-], PCO2, and [H+] in vivo in healthy humans; we recognize that this relies on the assumption that these sampling sites are reflective of trans-cerebral exchange due to practical and ethical experimental constraints 85. Additionally, the Duplex ultrasound derived indexes of extra-cranial blood flow utilized in the current experiment are assumed to indicate cerebral tissue nutritive flow. Although we verified PaCO2 values with blood gas sampling, without temperature correcting to adjust for higher blood temperature during exercise, we may have underestimated PaCO2 by 1-2 mmHg (due to reduced CO2 solubility at higher temperatures) 86,87. Additionally, the influences of exercise 88-90 and temperature 91-95 may conceivably have a small effect on PCO2, H+, and HCO3- via alterations in carbonic anhydrase activity in the exercise trial 96,97 – however, this is unlikely to differentially affect the trans-cerebral venous-arterial exchange values within participants during this trial. A subset of female participants completed the +8 mmHg PaCO2 respiratory acidosis protocol as well as the fixed intensity submaximal exercise protocol (n=5 females and n=7 males). Previous literature is equivocal, with studies reporting that cerebrovascular CO2 reactivity is higher in females 98-100, higher in males 101-103, or not different between sexes 104,105. Whether sex-related differences in cerebrovascular CO2 reactivity exist between females and males in response to a fixed targeted elevation in PaCO2, this is unlikely to affect our results as we saw no change in the in vivo buffering capacity (D [HCO3-] × D pH-1) when indexed against the average venous-arterial pH during respiratory acidosis. These results indicate that any within-subject change in the sensitivity of CBF to PaCO2 would be reflected in an equivalent pH response (with which we indexed against our dependent variables). Additionally, recent studies have shown no difference in the CBF response to moderate-intensity exercise between females and males 106,107 with sex-related differences in CBF regulation only apparent at severe intensity exercise (80-100% maximal workload) 108. As such, we do not expect that the inclusion of 5 female participants in the fixed intensity submaximal exercise protocol (0, 50, 75, 100 watts) would affect the variability of our results. Future investigations are merited to address any sex-related differences in cerebrovascular acid-base regulation in response to respiratory and exercise-induced metabolic acidosis. APPENDICES 222 CONCLUSION Taken together, these results indicate that increases and decreases in systemic arterial [HCO3-] – during acute respiratory/ exercise-induced metabolic acidosis, respectively – differentially contribute to cerebrovascular acid-base balance. The previously recognized tight regulation of cerebral interstitial pH during acute hypercapnic acidosis 19,20 is likely attributable to: 1) narrower internal jugular venous-arterial [HCO3-] and PCO2 differences; 2) higher CBF; and 3) reductions in trans-cerebral [HCO3-] exchange indicating less venous [HCO3-] efflux/release. These results are supportive of unaltered trans-cerebral [HCO3-] exchange with exercise-induced acidosis – consistent with the unchanged in vivo buffering capacity – which was unrelated to the CBF response throughout progressive cycling exercise to exhaustion. Acknowledgements We would like to extend our thanks to the volunteers who participated in these studies. Additionally, we would like to thank past and present members of the Centre for Heart, Lung and Vascular Health at UBCO for their contribution to this series of studies by our group. Author Contributions Study design: RLH, KJS, PB, ARB, DMB, PNA. Data collection: HGC, RLH, KJS, PB, ARB, MMT, CAH, JMJRC, BSS, DMB, AD, MSS, DBM, PNA. Data analysis: HGC, RLH, KJS, PB, ARB. Data interpretation: HGC, RLH, JMJRC, DMB, PNA. Drafted manuscript: HGC. Critically reviewed manuscript: HGC, RLH, KJS, PB, ARB, MMT, CAH, JMJRC, BSS, DMB, AD, MSS, DBM, PNA. Approved final version: HGC, RLH, KJS, PB, ARB, MMT, CAH, JMJRC, BSS, DMB, AD, MSS, DBM, PNA. Funding Philip N. Ainslie was funded by the Natural Sciences and Engineering Research Council of Canada (NSERC) and a Canada Research Chair. Hannah G. Caldwell was funded by a NSERC PGS-Doctoral Scholarship. Patrice Brassard was funded by the Foundation of the Institut universitaire de cardiologie et de pneumologie de Québec and a NSERC Discovery Grant. Damian M. Bailey was supported by a Royal Society Wolfson Research Fellowship (#WM170007) and Higher Education Funding Council for Wales (Postdoctoral Fellowship for Benjamin S. Stacey). Conflict of Interests N/A. Data Availability Statement The data that support the findings of this study are available from the corresponding author upon reasonable request.APPENDICES 223 References 1. Lennox WG, Gibbs EL. THE BLOOD FLOW IN THE BRAIN AND THE LEG OF MAN, AND THE CHANGES INDUCED BY ALTERATION OF BLOOD GASES. 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Venous-arterial [HCO3-] and PCO2 exchange with hypercapnic acidosis in humans. Total cerebral blood flow (CBF) increases with stepwise elevations in arterial PCO2 (PaCO2) (A); this hypercapnia-mediated increase in CBF is related to narrowing of the venous-arterial PCO2 difference (B) such that – explained via the Fick principle – trans-cerebral PCO2 exchange is unaltered with respiratory acidosis (C). Arterial [HCO3-] increases with progressive elevations in PaCO2 (D); this response may theoretically contribute to localized changes in CBF with severe respiratory acidosis – via increases in extravascular [HCO3-] – and thus, regulatory changes in [HCO3-] may help explain the maximal cerebrovascular vasodilatory reserve to PaCO2. There is a relationship between reductions in arterial pH 35 40 45 50 55 60 65255075100125150PaCO2 (mmHg)CBF (ml ⋅ 100 g-1 ⋅ min-1)P<0.00135 40 45 50 55 60 6520222426283032PaCO2 (mmHg)[HCO3-] a (mmol ⋅ l-1)P<0.0017.25 7.30 7.35 7.40 7.45 7.50-1012345pH[HCO3-] exchange (mmol ⋅ min-1)P=0.003-5 0 5 10 15255075100125150v-a PCO2 (mmHg)CBF (ml ⋅ 100 g-1 ⋅ min-1)P<0.0017.25 7.30 7.35 7.40 7.45 7.50-20246810pHv-a [HCO3-] (mmol ⋅ l-1)P<0.00120253035[HCO3 -] (mmol ⋅ l -1)ArterialVenous-1 0 1 2 3 4 5255075100125150v-a [HCO3-] (mmol ⋅ l-1)CBF (ml ⋅ 100 g-1 ⋅ min-1)P<0.00140 45 50 55 60 65-5051015PaCO2 (mmHg)PCO2 exchange (mmHg ⋅ min-1)P=0.0577.25 7.30 7.35 7.40 7.45 7.50-5051015202530pHv-a PCO2 (mmHg)P<0.00135455565PCO2 (mmHg)ArterialVenous7.30 7.35 7.40 7.45-20020406080Avg v-a pH-Δ [HCO3-] × Δ pH-1P=0.078A B CD E FG H IAPPENDICES 231 (e.g., respiratory acidosis) and narrowing of the venous-arterial [HCO3-] (E) and PCO2 (F) differences. The reduction in venous-arterial [HCO3-] difference is achieved by a decrease in venous [HCO3-] and increase in arterial [HCO3-]; whereas, PaCO2 increases to a larger extent relative to PvCO2 with respiratory acidosis (illustrated by the coloured inlay figures). This narrowing is reflective of less [HCO3-] exchange (H) in part attributable to increases in [HCO3-] and CO2 ‘wash-out’ due to higher CBF with hypercapnia (G); i.e., the venous-arterial [HCO3-] and PCO2 differences were each related to increases in CBF (both P < 0.001; subset n=27). There was no relationship between the average venous-arterial in vivo buffering capacity (-D [HCO3-] × D pH-1) with stepwise respiratory acidosis when indexed against average venous-arterial pH (I). Data are individual values across stepwise progressive increases in PaCO2 for n=27 (A, B, C, G, H, I) and n=39 (D, E, F) participants via dynamic end-tidal forcing. APPENDICES 232 Figure 7-2. Venous-arterial [HCO3-] and PCO2 exchange with progressive submaximal to maximal cycling exercise. Total cerebral blood flow (CBF) is positively related to arterial PCO2 (PaCO2) throughout progressive cycling exercise to exhaustion (A); however, there is no relationship between CBF and the venous-arterial PCO2 difference (B), therefore, trans-cerebral PCO2 exchange is unrelated to PaCO2 during exercise (C). Arterial [HCO3-] is reduced with exercise-induced acidosis at maximal exercise (D). There is no relationship between the venous-arterial [HCO3-] difference and arterial pH (E) – as indicated by the equivalent reduction in arterial and venous [HCO3-] – however, there is widening of the venous-arterial PCO2 difference with exercise-induced acidosis (F) as indicated by a larger relative reduction in PaCO2 versus 30 35 40 4520304050607080PaCO2 (mmHg)CBF (ml ⋅ 100 g-1 ⋅ min-1) P=0.0047.20 7.25 7.30 7.35 7.40 7.451015202530pH[HCO3-] a (mmol ⋅ l-1) P<0.0017.25 7.30 7.35 7.40 7.45-3-2-1012pH[HCO3-] exchange (mmol ⋅ min-1)P=0.896-5 0 5 10 15 2020304050607080v-a PCO2 (mmHg)CBF (ml ⋅ 100 g-1 ⋅ min-1)P=0.1777.20 7.25 7.30 7.35 7.40 7.45-404812pHv-a [HCO3-] (mmol ⋅ l-1)P=0.820101520253035[HCO3 -] (mmol ⋅ l -1)ArterialVenous-4 -2 0 2 420304050607080v-a [HCO3-] (mmol ⋅ l-1)CBF (ml ⋅ 100 g-1 ⋅ min-1)P=0.68230 35 40 45-5051015PaCO2 (mmHg)PCO2 exchange (mmHg ⋅ min-1)P=0.1557.20 7.25 7.30 7.35 7.40 7.45010203040pHv-a PCO2 (mmHg)P=0.0072030405060PCO2 (mmHg)ArterialVenous7.15 7.20 7.25 7.30 7.35 7.4020406080100Avg v-a pHΔ [HCO3-] × Δ pH-1P=0.187A B CD E FG H IAPPENDICES 233 PvCO2 (illustrated by the coloured inlay figures). The exercise-induced CBF response was unrelated to the venous-arterial [HCO3-] difference (G); as such, there were no changes in trans-cerebral [HCO3-] exchange during exercise when indexed against arterial pH (H). There are no relationships between the average venous-arterial in vivo buffering capacity (D [HCO3-] × D pH-1 or -D [La] × D pH-1) during incremental cycling exercise to exhaustion (I). These data are reflective of the linear relationship between pH, reductions in [HCO3-], and increases in [La] with exercise-induced acidosis and indicate an unaltered in vivo buffering capacity at maximal cycling exercise. Data are individual values during supine incremental cycling exercise to exhaustion for n=12 (A, B, C, G, H, I) and n=24 (D, E, F). Exercise stages included various relative (0, 20, 40, 60, 80, 100% maximal workload; n=12 males) and fixed exercise intensities (0, 50, 75, 100 watts; n=5 females and 0, 75, 100, 125 watts; n=7 males). The in vivo buffering capacity was calculated at 60, 80, 100% maximal workload (I).
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Kinesiology
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Ainslie, Philip N., 1974-
Substrate utilization in the brain and skeletal muscle during environmental and energetic stress in humans
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