The in vivo Oxyhaemoglobin Dissociation Curve at Sea Level and High Altitude by Dahlia Balaban A thesis submitted in conformity with the requirements for the degree of Master’s of Science Institute of Medical Science University of Toronto © Copyright by Dahlia Balaban 2009 The in vivo Oxyhaemoglobin Dissociation Curve at Sea Level and High Altitude Dahlia Balaban Master‘s of Science Institute of Medical Science University of Toronto 2009 Abstract Some animals have adapted to hypoxia by increasing their haemoglobin affinity for oxygen, but in vitro studies have not shown any change of haemoglobin affinity for oxygen in human high altitude natives or lowlanders acutely acclimatized to high altitude. We conducted the first in vivo study of the oxyhaemoglobin dissociation curve by progressively reducing arterial PO2 while maintaining normocapnia in lowlanders at sea level, lowlanders sojourning at 3600m for two weeks and native Andeans at the same altitude. We found that the in vivo PO2 at which haemoglobin is half-saturated (P50) is higher in lowlanders at sea level (32 mmHg) than that measured in vitro (27 mmHg) and that lowlanders and highlanders do significantly increase the in vivo affinity of their haemoglobin for oxygen with exposure to high altitude. These results indicate the value of an in vivo approach for studying the oxyhaemoglobin dissociation curve. ii Acknowledgments I would like to thank a number of people who have played instrumental roles in the conceptualization, performance and preparation of my thesis studies. First and foremost I would like to thank my supervisor, Dr. Joe Fisher. He provided me with life-changing opportunities and his mentorship and guidance were essential to the completion of my thesis. Dr. James Duffin was a valuable mentor and committee member. He was always available to help, even on weekends and holidays, and his feedback was always useful. I would also like to thank Professor Scott Thomas, my other committed committee member. He always managed to make time to look over my drafts and attend my meetings, even on short notice or when on vacation. I would like to acknowledge all the people who played instrumental roles in data collection for my studies. Dr. Keita Ikeda, Sonia Capps and Dr. Dave McLeod hosted me and collected data for Experiments 1 and 2 at Duke University. Then there were the members of my expedition to Bolivia. We worked long, gruelling hours together under hypoxic conditions and overcame many obstacles that stood in the way of completion of my study. They also provided me with continuing support as I analyzed and wrote up my data. These members are Dr. David Preiss, Dr. Alex Vesely, Professor Richard Greene, Alexandra Mardimae, Marat Slessarev and, of course, my supervisor. I would especially like to acknowledge the contributions of Dr. David Preiss, who was always willing to help me with data analysis and conceptualization, long after the expedition. On the topic of Bolivia, I must also thank the Zubietas, who provided us with a laboratory, equipment and subjects for our study. Their hospitality was greatly appreciated. I would also like to acknowledge the contribution of Cliff Ansel, the president of Thornhill Research, Inc., for supporting the expedition Although they did not take part in the expedition, Cathie Kessler, Stephanie Dorner and David Le provided technical and logistical support for the expedition equipment. I also appreciate the contributions of my other lab mates, Jay Han, Matt Machina and Anne Battisti. I would like to extend a special thank you to Rosemary Regan, who started her MSc when I did and provided me with endless support throughout. iii I would also like to mention Dianne Fukanaga and Dr. Howard Mount of the Institute of Medical Science, who supported and assisted me in completing my thesis in a timely manner, despite the associated difficulties. Lastly, I want to thank the people who influenced my success less directly. My former supervisors, Professors Laurie Wilcox and Laurence Harris, instilled in me a love for research and inspired me to pursue graduate studies. However, this would not have been done without the positive experiences I had in their labs, where I met students who continue to inspire and help me today. I would like single out Hassan Masoom, who was always willing to help me out despite having no affiliation with my lab or project. His editing and advice were invaluable. I would also like to mention my friends and especially my family, who have supported me unconditionally through the ups and downs of my graduate studies. They gave me advice and went through every experience with me, no matter how trivial or difficult. My parents and sisters (and their families) have always been at my side no matter how stressed or busy I became. These and countless others have helped me get to where I am today. I could not have done it without them. iv Table of Contents Abstract ...................................................................................................................................... ii Acknowledgments ..................................................................................................................... iii Table of Contents ....................................................................................................................... v List of Tables ............................................................................................................................ xi List of Figures ......................................................................................................................... xiv List of Abbreviations ............................................................................................................... xvi 1 Literature Review .................................................................................................................. 1 1.1 Oxygen (O2) Transport.................................................................................................... 1 1.2 The Oxyhaemoglobin Dissociation Curve ....................................................................... 2 1.2.1 Temperature ........................................................................................................ 3 1.2.2 The Bohr Effect (pH) .......................................................................................... 4 1.2.3 Carbon Dioxide ................................................................................................... 5 1.2.4 2,3-Diphosphoglycerate (2,3-DPG) ..................................................................... 5 1.2.5 Inorganic Salts..................................................................................................... 6 1.2.6 Carbon Monoxide ................................................................................................ 6 1.2.7 Red Blood Cells (RBCs) and Haemoglobin ......................................................... 6 1.3 Measuring the ODC ........................................................................................................ 6 1.3.1 In vitro methods for measuring the shape and position of the ODC ...................... 8 1.3.2 Comparing P50s ................................................................................................. 10 1.3.3 Predicting Saturation ......................................................................................... 10 1.4 Physiological Effects of High Altitude .......................................................................... 11 1.4.1 Hypoxic Ventilatory Response .......................................................................... 11 1.4.2 Haemoglobin ..................................................................................................... 12 1.4.3 2,3-DPG ............................................................................................................ 13 v 1.5 Population Studies at High Altitude .............................................................................. 13 1.6 The ODC at Altitude ..................................................................................................... 16 1.6.1 The ODC in Lowlanders at High Altitude .......................................................... 16 1.6.2 The ODC in Highlanders ................................................................................... 19 1.6.3 What type of shift is beneficial at altitude? ........................................................ 22 2 Statement of the Problem ..................................................................................................... 25 2.1 Drawbacks of Measuring the ODC in vitro ................................................................... 25 2.2 Difficulty of Measuring the ODC in vivo ...................................................................... 26 2.3 Using Prospective End-Tidal Targeting to Measure the ODC in vivo ............................ 27 3 Research Aims and Hypotheses ........................................................................................... 30 3.1 Aim One: Method Demonstration ................................................................................ 30 3.2 Aim Two: Characterization of in vivo Curves and Comparison to in vitro Predictions .. 30 3.3 Aim Three: Comparison of the in vivo ODC Across Experimental Groups ................... 31 4 Methods ............................................................................................................................... 32 4.1 Subjects .......................................................................................................................