UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Glycogen utilization in the athlete under conditions of accute, moderate hypoxia Hazlett, David Lorne 1995

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-ubc_1995-0511.pdf [ 5.43MB ]
Metadata
JSON: 831-1.0087095.json
JSON-LD: 831-1.0087095-ld.json
RDF/XML (Pretty): 831-1.0087095-rdf.xml
RDF/JSON: 831-1.0087095-rdf.json
Turtle: 831-1.0087095-turtle.txt
N-Triples: 831-1.0087095-rdf-ntriples.txt
Original Record: 831-1.0087095-source.json
Full Text
831-1.0087095-fulltext.txt
Citation
831-1.0087095.ris

Full Text

G L Y C O G E N UTILIZATION IN THE A T H L E T E UNDER CONDITIONS OF A C U T E , M O D E R A T E H Y P O X I A by DAVID LORNE HAZLETT BSC, University of British Columbia, 1984 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES DIVISION OF HUMAN NUTRITION SCHOOL OF FAMILY AND NUTRITIONAL SCIENCES We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August 1995 © David Hazlett, 1995 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at The University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Division of Nutrition, School of Family and Nutritional Sciences The University of British Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date: 01/09/95 Abstract It is common knowledge that diet and endurance training both have a major impact on body glycogen stores and carbohydrate metabolism. Usual nutritional recommendations for athletes participating in endurance activities are to consume at least 8 grams of carbohydrate per kilogram body weight. This level of carbohydrate intake has been found to optimally maintain muscle glycogen stores in actively training endurance athletes. This recommendation has been r based on athletic activity undertaken under sea level conditions. A survey of the literature shows that this recommendation has never been investigated in athletes exercising under hypoxic conditions such as at altitude. Theoretical and experimental evidence would predict that under hypoxic conditions, one strategy which could maintain A T P production in the face of decreased oxygen availability is to increase flux through the glycolytic pathway. Increased glycolytic flux could ultimately affect carbohydrate use which could in turn lead to enhanced muscle glycogen use. If this did occur, it could have implications for dietary advice given to athletes exercising at altitude. It was our intent to see i f these alterations occur with exercise under acute exposure to moderate altitudes such as those an athlete could conceivably face during competition. To investigate this possibility, muscle glycogen utilization was assessed during exercise at sea level (SL) and with acute exposure to simulated altitude ( A L T ) equivalent to 7500 feet above sea level. Six endurance trained cyclists exercised for 75 minutes at the same relative exercise intensity (65% of VO2 m a x) u n d e r each condition. In addition to glycogen use, plasma lactate, serum glucose, and respiratory exchange ratios (RER) were also evaluated to assess whether any shift in substrate use occurred between conditions. There was a trend towards decreased muscle glycogen use under A L T conditions, however the difference was not found to be significant (P = 0.09). Plasma lactate, serum glucose, and R E R findings were also found to be not significantly different between conditions. This data suggests that exercise at the same relative intensity at A L T as S L does not result in enhanced carbohydrate use. From this it is inferred that under the conditions used, oxygen supply to exercising muscle is not diminished. i i These findings also suggest that in highly trained cyclists undertaking exercise under moderate altitude conditions, no increase in carbohydrate intake is necessary above that recommended for exercise of equivalent intensity at sea level. i i i Table of Contents A b s t r a c t i i Table of Contents i v List of Tables v i List of Figures v i i List of Abbreviations viii A c k n o w l e d g e m e n t s x I n t r o d u c t i o n 1 Review of Literature 6 N o r m a l patterns of substrate uti l ization 6 Substrate util ization in the trained athlete 8 Rev iew of mitochondrial energy production 9 Overview of oxidative phosphorylation 9 Regulation of oxidative phosphorylation 12 The effect of A D P and P i on oxidative phosphorylation 13 The effect of N A D H on oxidative phosphorylation 18 The effect of O2 on oxidative phosphorylation 22 S u m m a r y 28 Studies looking at hypoxia and substrate utilization 29 H y p o x i a and R E R 31 H y p o x i a and Fue l Substrates 32 H y p o x i a and Hormona l Responses 35 S u m m a r y 36 Experimental Design and Methods 37 Subjec ts 37 Research D e s i g n 37 (1) Pre-test phase - VO2 max assessment 38 (2) Dietary Intervention 39 (3) Test phase - 75 minute test rides 40 B i o c h e m i c a l A n a l y s i s 42 Sta t i s t ica l A n a l y s i s 43 iv Table of Contents continued.. Resu l t s 45 Subject and Pretest Phase (VO2 max) Results 45 Test Phase Resul ts 45 P o w e r 45 C u m u l a t i v e W o r k 50 Ratings of perceived Exert ion (RPE) 51 Minute Ventilation (VE ) 52 Ventilatory Equivalent (VE/VO 2) 53 Oxygen Utilization (Vo 2) 55 Carbon Dioxide Production (VcO2) 56 Arter ia l Oxygen Saturation (Sao 2 ) 56 Heart Rate 57 Die ta ry Data 59 Lac ta t e 60 G l u c o s e 61 Respiratory Exchange Rat io 63 G l y c o g e n 65 Summary of Results 69 D i s c u s s i o n 70 Experimental design - relative versus absolute exercise intensity 70 Variations observed in muscle glycogen concentrations 73 Potential improvements to the experimental design 80 Inferences about muscle oxygen availability from substrate and physiological data.... 82 C o n c l u s i o n 86 B i b l i o g r a p h y 88 Appendices Appendix 1. Certificate of Approval from the Clinical Committee for Research and Other Studies Involving Human Subjects 98 Appendix 2. Subject Recruitment Poster 100 Appendix 3. Subject Consent F o r m 102 Appendix 4. Subject Information F o r m 105 Appendix 5. Subject Exercise History Recording Form 107 Appendix 6. Subject Data Col lec t ion F o r m 109 v List of Tables 1. Studies which have been undertaken on humans to look at the effects of acute hypoxic exposure on substrate and/or hormonal responses 30 2. Subject data and VO2 max data under sea level and simulated altitude conditions 45 3. Physiological values for 75 minute bicycle ride under sea level conditions 47 4. Physiological values for 75 minute bicycle ride under simulated altitude conditions 48 5. Results of two-way A N O V A (condition x time) for repeated measures across both factors 49 6. Ratings of perceived exertion (RPE) at sea level and simulated altitude, using the Borg 10 point scale 51 7. Dai ly dietary intake of subjects for 48 hours prior to each exercise bout 59 8. Venous plasma lactate values under sea level and simulated altitude conditions 60 9. Venous serum glucose values for sea level and simulated altitude conditions 62 10. Respiratory exchange ratio (RER) values for sea level and simulated altitude c o n d i t i o n s 63 11. Pre- and post-exercise muscle glycogen concentrations, and glycogen utilizations under sea level and simulated altitude conditions 65 12. Glycogen utilization expressed as a function of oxygen consumption under sea level and simulated altitude condit ions 67 vi. List of Figures 1. Power output during exercise under sea level and simulated altitude conditions 46 2. Cumulative work under sea level and simulated altitude conditions 50 3. Ratings of perceived exertion during exercise under sea level and simulated altitude c o n d i t i o n s 52 4. Ventilation during exercise under sea level and simulated altitude conditions 53 5. Ventilatory equivalents during exercise under sea level and simulated altitude conditions...54 6. Oxygen utilization during exercise under sea level and simulated altitude conditions 55 7. Arterial oxygen saturation at rest and during exercise under sea level and simulated altitude c o n d i t i o n s 57 8. Heart rates during exercise under sea level and simulated altitude conditions 58 9. Plasma lactate values at rest and during exercise under sea level and simulated altitude c o n d i t i o n s 61 10. Serum glucose values at rest and during exercise under sea level and simulated altitude c o n d i t i o n s 62 11. Respiratory exchange ratios (RER) at rest and during exercise under sea level and simulated altitude condit ions 64 12. Pre- and post- exercise muscle glycogen concentrations under sea level and simulated alt i tude condi t ions 66 13. Correlation between glycogen use during exercise and pre-exercise muscle glycogen concent ra t ions 68 14. Correlation between pre-exercise muscle glycogen concentrations and the amount of dietary carbohydrate consumed in the diet 68 15. Comparison of relative versus absolute exercise intensities under sea level and simulated al t i tude condi t ions 71 vi i List of Symbols and Abbreviations A change (delta) [ ] concentration A D P Adenine diphosphate A L T simulated altitude A N O V A analysis of variance A T P Adenine triphosphate b p m beats per minute C O 2 carbon dioxide cyt c 2+ reduced cytochrome c oxidase cyt c 3+ oxidized cytochrome c oxidase E epinephrine E T C mitochondrial electron transport chain F F statistic F A D H 2 reduced flavin adenine dinucleotide F F A free fatty acid F l o 2 Fractional concentration of inspired oxygen g gram(s) H + hydrogen ion H t height J joule(s) k c a l kilocalorie(s) k g kilogram(s) K m Michaelis - Menten constant 1 liter(s) m meter(s) mg milligram(s) m i n minute(s) m l milliliter(s) m m H g millimeter(s) of mercury m m o l millimole(s) N 2 . . nitrogen N A D + oxidized nicotinamide adenine dinucleotide N A D H reduced nicotinamide adenine dinucleotide N E norepinephrine vii i O 2 oxygen P a o 2 arterial partial pressure of oxygen P B barometric pressure P C O 2 partial pressure of carbon dioxide p H negative logarithm of hydrogen ion concentration P i inorganic phosphate P l O 2 pressure of inspired oxygen P O 2 partial pressure of oxygen P 0 2 crit minimal PO2 required for maximal turnover of cytochrome c oxidase R E R respiratory exchange ratio R P E rating(s) of perceived exertion S a o 2 arterial oxygen saturation S E standard error S L sea level T C A cycle.... Tricarboxylic acid cycle T o r r 1 Torr equals 1 mm H g u. M micromole(s) per liter umo l micromole(s) V E minute ventilation V C O 2 carbon dioxide production VO 2 oxygen consumption VO2 max maximal oxygen consumption W watt(s) W t weight X group mean ix Acknowledgements To Susan Barr, my supervisor, I extend one enormous thank you for your support, your sage advice, and above all, your patience! To Linda McCargar, who saw me through thick and thin, thank you for your initiative, your wonderful listening skills, and for your friendship. To Don "Biopsy K i n g " McKenz ie , thank you for your invaluable time, expertise, and advice. Data collection went exceptionally smoothly thanks in no small part to you. To Diana Jespersen, thanks for all your help along the way including all the time you donated helping with data collection. There were some long days/evenings in there! To Angelo Belcastro, special thanks for your help and advice. Even though you were not directly involved, you always generously made time for me and my questions. I can't tell you how much I appreciate it — you're a gem! To Ken Coutts who was a rock solid source of support, thanks for keeping a l l the equipment going for me, and thanks also for your physiological expertise and advice. PS . I still occasionally have nightmares about Beckman CO2 analyzers! To Gord Matheson, thanks for giving freely of your time whenever I needed expertise. Your generous nature, and your help in clarifying issues and physiological troubleshooting are very much appreciated. To the "World 's Best Subjects": Cam, Chris, Darren, Dave, Raf, Ray, and James, you guys are truly amazing! After all we did to you (how many biopsies was that again?), and not so much as one complaint! The gods must have been smiling on me when you answered my recruitment poster! Thanks you guys, happy trails to you all and keep carbo loading! Last but certainly not least, a huge thank you to Wade Simmons - lab rat extraordinaire! If only I could give you one functioning islet cell for every mile you rode for me while setting up the equipment x Introduction 1 Skeletal muscle glycogen stores are of critical importance to the endurance athlete. Depending on recent diet and training patterns, skeletal muscle contains between 10 and 30 grams of glycogen per kilogram of skeletal muscle. In addition, the liver stores approximately 80 grams of glycogen. For endurance exercise bouts of from one to three hours duration at moderate intensities (60-80% of maximal oxygen consumption (VO2 max))> exercise endurance time is directly related to depletion of these glycogen stores9. Once muscle glycogen is depleted, the athlete experiences profound muscle fatigue and diminished exercise performance 5 2 - 5 3 . Given this pivotal role of muscle glycogen in sustaining prolonged endurance exercise, an understanding of the various factors impacting on glycogen and carbohydrate utilization would prove invaluable in enhancing performance. Diet and endurance training are both known to have a major impact on body glycogen stores and carbohydrate metabolism. Usual nutritional recommendations for athletes participating in endurance activities are to consume greater than 8 g-kg-^d" 1 of carbohydrate, corresponding to approximately 65 - 70% of the diet in the form of carbohydrate 3 3. This recommendation is based on athletic activity undertaken under "normal" conditions. A survey of the literature shows that this recommendation has never been investigated in athletes exercising under hypoxic conditions such as at altitude. Theoretical and experimental evidence would predict that glycogen metabolism may be altered with exercise at altitude due to differences in the availablity of oxygen at skeletal muscle. A t the level of the mitochondria under hypoxic ambient conditions, cellular respiration is capable of maintaining sufficient A T P production to fuel exercise. Evidence does indicate however that this A T P production is maintained under adapted physiological conditions; to maintain adequate A T P production myocytes likely increase glycolytic flux and as a result increase carbohydrate utilization. Can such adaptations occurring at the mitochondria under hypoxic conditions translate into measurable increases in carbohydrate use? Can these relatively small adaptations at the mitochondria in turn necessitate an increased need for dietary carbohydrate intake to maintain Introduction 2 performance under hypoxic conditions? These are the questions being addressed in the present study. M u c h work has been done in the field of carbohydrate metabolism, particularly since the early 1960's when use of the muscle biopsy needle became common 8 . While much understanding has been gained in the intervening time period, there are still several areas where knowledge is lacking. In particular, knowledge of how glycogen metabolism is regulated is fragmentary. Environmental hypoxia, which ultimately can be reflected as cellular hypoxia, may prove to play an important role in this regulation. To date, the effect of environmental hypoxia on muscle glycogen metabolism has received relatively minimal attention. This is largely due to the fact that: (a) there is lack of useful information regarding the effects of acute hypoxia on fuel metabolism, (b) most studies done to date have utilized conditions of advanced hypoxia (< 14% F1O2) which only a mountaineer would be likely to encounter, and (c) no studies have been done on highly trained subjects (who are known to have slightly different patterns of substrate metabolism compared to untrained subjects). Thus, the present study was undertaken to examine this relationship in trained cyclists under conditions of acute, moderate hypoxia. A number of studies have shown that for moderate altitudes such as those being simulated in this study there is no difference in physiological responses between "simulated" altitude (normobaric hypoxia) and actual altitude (hypobaric hypoxia) e x p o s u r e 4 7 ' 5 1 , 5 7 , 1 1 4 . Therefore, for the purposes of convenience and cost it was decided to use simulated altitude in the study rather than running the experiment at actual altitude. This was achieved by having the subjects breathe bottled air containing a lower fractional concentration of oxygen than air at sea level which is approximately 21% oxygen. The fraction of inspired oxygen (F1O2) used to simulate altitude was 16.0%, which is equivalent to an altitude of approximately 7500 feet. This level of hypoxia was chosen as it represents a situation that an athlete could conceivably be exposed to during an endurance athletic event. For example, cyclists in the Tour de France often traverse mountain Introduction 3 passes of this altitude. Also, this is only slightly higher than the altitude of Mexico City (7347 feet) where the Olympic games were held in 1968. Other studies investigating substrate use at altitude have used the same absolute exercise intensities under both conditions. As discussed in the review of literature, this equates to a higher relative exercise intensity under altitude conditions, which in turn is equivalent to a greater stress on the body. This results in an augmented hormonal stress response (catecholamines, Cortisol) which is capable of altering substrate use over and above the effect of altitude. To allow a more equitable comparison of substrate use between conditions, the same relative rather than absolute exercise intensity wi l l be used under each condition in the present study. The purpose of this study was to examine whether glycogen utilization is altered in an athlete when exercising under conditions of acute, moderate hypoxia. If any alterations in glycogen use were found, they could be used to infer whether oxygen plays a l imit ing role in cellular carbohydrate metabolism. Depending on the outcome of the experiment, the results may be useful in establishing dietary guidelines for athletes competing at altitude. If oxygen availabilty were limited at the muscle cell level there would be two possible options available to increase the efficiency of substrate metabolism: • (1) reduce anaerobic metabolism to make more efficient use of substrate (oxidative metabolism of glucose yields 38 moles of A T P , while anaerobic metabolism to lactate yields only 3 moles of ATP) . • (2) utilize the substrate that leads to the greatest energy yield per mole of O2 (complete oxidation of glycogen yields almost 15% more A T P per mole of O2 than complete oxidation of fat). This first option is more commonly found after chronic exposure to altitude, a phenomenon known as the lactate paradox in which lactate production declines at any given exercise intensity after acclimatization17,61,62,63,66,94 virtually every exercise study done to date on unacclimatized low-landers exposed acutely to hypoxia has found an increase in plasma l ac t a t e 6 0 ' 6 7 ' 8 2 ' 9 0 ' 9 2 . Introduction 4 A n increased lactate production would be expected i f the second option was employed by the muscle. Wi th an increase in glycogenolysis, there would be a concommitant increase in flux through glycolysis. Due to the near equilibrium position of lactate dehydrogenase, an increase in glycogenolysis would translate into an increase in lactate production through a mass action effect. Additionally an increased breakdown of glycogen would be reflected in an increased respiratory exchange ratio (RER) over that seen in exercise under normoxic conditions. A t the same relative exercise intensity under hypoxic conditions, VO2 levels are lower than those found for normoxic conditions. For example i f an athlete's VO2 max a t sea level is 5 1-min - 1, seventy percent of this value (ie 70% of VO2 m a x ) is 3.5 1-min"1. Wi th exercise under hypoxic conditions i f VO2 m a x measurements were to drop 20% from 5 1-min -1 to 4 1-min - 1, then 70% of this hypoxic VO2 max would be 2.8 1-min -1. This means that less oxygen w i l l be used by the body at 70% of VO2 max under hypoxic conditions compared to normoxic conditions (see Figure 15). This in turn means that on an overall absolute basis, less glycogen w i l l be used under hypoxic conditions. But, i f glycogen is preferentially used as a substrate under hypoxic conditions, on a relative basis it would be expected to contribute a proportionately larger share of the overall substrates oxidized. Based on these observations, the following null hypotheses were proposed: • There w i l l be no difference in absolute or relative muscle glycogen use between exercise performed at the same relative exercise intensity under sea level and simulated altitude conditions. • There wi l l be no difference in R E R values between exercise performed at the same relative exercise intensity under sea level and simulated altitude conditions. • There w i l l be no difference in serum glucose values between exercise performed at the same relative exercise intensity under sea level and simulated altitude conditions. Introduction 5 • There w i l l be nb difference in plasma lactate values between exercise performed at the same relative exercise intensity under sea level and simulated altitude conditions. To my knowledge, this study wi l l be the first to look at glycogen use in an athletic population under conditions of acute, moderate hypoxia. Review of Literature 6_ Normal Patterns of Substrate Utilization For the body to accomplish physical activity, it must be supplied with a sufficient source of energy to fuel muscular contraction. The energy source for this contraction is adenosine triphosphate (ATP) , with cleavage of this molecule releasing chemical energy which is captured and utilized by the actomyosin cross bridges. Wi th the onset of exercise, A T P utilization can increase more than one hundredfold leading to depletion of stores in a matter of seconds 1 0 9 . It is therefore necessary to oxidize the various energy producing nutrients present in the body; carbohydrates, fats, and proteins, to replenish A T P so that muscle contraction can continue. The importance of fats and carbohydrates as oxidative sources of energy during exercise has been appreciated since as early as 1920 by Krogh and Lindhard 8 4 . Determination of which nutrient source is being oxidized to drive A T P production can be obtained by measuring the respiratory exchange ratio (RER) of a person through indirect calorimetry. The R E R provides an indirect method of assessing the body's macronutrient utilization in the steady state. It is calculated by measuring the amount of CO2 expelled, and dividing it by the amount of O2 taken up (utilized) by the body over a given time period; providing a gross indication of which fuel(s) are being oxidized by the body. The general formula for a carbohydrate is C6H12O6, so that O2 and H are present in the same proportions as in water (H2O). Oxidation of carbohydrate yields: C6H12O6 + 6O2 -> 6CO2 + 6H2O Oxidation of a carbohydrate therefore produces the same amount of CO2 as it uses O2 (6:6), so that the R E R is unity (1). B y comparison, fat could be considered to be in a far less oxidized state than carbohydrate so that oxidation of fat requires relatively more oxygen. Oxidation of a typical source of fat (tripalmitin) yields: 2C51H98O6 + 14502 -» 102CO2 + 98H2O Review of Literature 7_ producing a R E R of 102:145 or approximately 0.7. Protein (amino acid) oxidation produces an intermediary R E R , between that of carbohydrates and fats, yielding a value of 0.82. Compared to carbohydrates and fats, protein sources are thought to play a far less important role in normal energy metabolism, and because the R E R of protein is half way between that of fat and carbohydrate, ignoring protein's contribution w i l l not significantly alter fuel metabolite determination. In the resting state, the R E R is approximately 0.70, signifying that the predominant source of energy is from fat oxidation in the form of free fatty acids ( F F A ) 1 0 9 ' 2 3 . During the transition from rest to exercise, the muscles shift from using mostly F F A ' s to using a blend of F F A , extramuscular glucose, and g lycogen 1 2 4 . A s exercise intensity increases, there is a continued progressive shift away from fat as a source of energy towards carbohydrate as is evidenced by the progressive rise in R E R . In general, with increasing intensity of exercise there is an increased reliance on muscle glycogen as an energy source 7 3. Saltin and Karlsson have reported a curvilinear (exponential) relationship between exercise intensity, and glycogen utilization between 65-150% of maximal oxygen consumption (VO2 max)111 • The importance of carbohydrates in fending off fatigue in endurance exercise has been appreciated since as early as 1939, when Levine and others 8 7 noticed that blood glucose levels in some runners fell precipitously by the end of a 25 mile race. Those with the greatest fatigue and disorientation at the finish were found to have the lowest blood glucose levels. Around the same time, Christensen and Hansen determined that a high carbohydrate diet could significantly enhance endurance during prolonged exercise 2 3. One of the first to associate muscle glycogen with fatigue was Grollman in 1955 5 6 . Working with rats, he found that aerobic endurance was significantly better in those rats with the highest muscle glycogen concentrations. Further understanding came with the development of the biopsy needle in the early 1960's. With this tool, it became possible to measure muscular glycogen levels before and after exercise and correlate fatigue to glycogen utilization. Much of the early work was Review of Literature 8_ pioneered by a Scandinavian group in the the m i d - 1 9 6 0 ' s 1 ' 2 ' 9 , 1 0 ' 5 9 ' 7 2 . They found that with increasing intensity of exercise, as measured by percentage of VO2 m ax> there was an increasing reliance on muscle glycogen. These authors determined that the capacity to maintain exercise intensity rates between 70 to 85% of VO2 m a x was related to the initial level of muscle glycogen stores. They also found that the enhanced endurance seen in response to training and increased dietary intake of carbohydrate was partially due to increased levels of muscle glycogen. A s exercise intensity increases, muscle glycogen utilization increases exponentially 1 1 1 . Typical rates of muscle glycogen utilization at 50%, 75%, and 100% VO2 m a x a r e 0.7, 1.4, and 3.4 mmol-kg^-min - 1 respectively. At prolonged moderate workloads (70% V 0 2 m a x , heart rate -160-170 bpm) muscle glycogen depletion occurs in a triphasic manner 1 1 0 . A t the onset of exercise glycogen depletion occurs at a high rate. This rate declines slightly after approximately 20 minutes as a steady state is reached in exercise 1 1 1 . Towards the end of an exhaustive bout of moderate exercise (2 hrs), glycogen depletion rate again increases slightly. This would be equivalent to the point at which the more oxidative fibers are glycogen depleted and starting to fatigue as discussed above. To meet energy needs, the more anaerobic, less energy efficient fast twitch fibers enhance their glycogen utilization rate which results in a more rapid decline in glycogen. Substrate Utilization in the Trained Athlete While the general pattern of fuel utilization outlined above applies to both trained and untrained humans, there are some differences which take place in response to training which are noteworthy. Perhaps the first and most obvious change in response to training is the increased metabolic potential of the body, as witnessed by the enhanced VO2 max m trained athletes. Training has several effects on the body, and in particular on skeletal muscle, which make such a rise in VO2 max possible. Skeletal muscle responds to aerobic exercise with an increase in the number of m i t o c h o n d r i a 7 1 , 9 8 , as well as concomitant increases in the levels of enzymes responsible for Review of Literature 9_ oxidizing fuel substrates. In particular there are increases in activities of enzymes of the tricarboxylic acid ( T C A ) cycle, the malate-aspartate shuttle, the respiratory chain, and p-oxidat ion 6 9 . Wi th these enhancements in enzymatic levels, comes an increased ability to oxidize pyruvate (the end product of glycolysis), and fatty acids. This latter effect is particularly important in the athlete, as an increase in fatty acid oxidation can forestall glycogen depletion. B y sparing glycogen breakdown, this increased oxidation of fatty acids has the effect of prolonging endurance time. Indeed this is one fundamental difference which accounts for the increase in endurance seen between trained and untrained subjects during aerobic exercise tasks 6 9 . Proof for this comes from studying the R E R of the same individuals both before training and after training as was done by Hol loszy 6 8 . He found a decrease in the R E R for the same absolute and relative exercise intensities in the same individuals after undergoing an endurance exercise training program. This signifies that in a trained individual, for any given workload compared to a nontrained person, more fatty acids are oxidized. The overall effect of such a metabolic alteration would be to preserve muscle glycogen and thereby prolong endurance. Another effect of training is to lower lactate levels for any given exercise intensity (i.e the same percent of VO2 m a x ) m comparison to the untrained state. Lactate accumulation has been shown to correlate with the development of fatigue, so this may be another factor responsible for the increase in endurance. The mechanism for this reduction in lactate has proven elusive, some authors have implicated an enhanced O2 delivery to the muscle in the trained state as being responsible 7 8, while others argue in favour of increased utilization of lactate by peripheral tissues as a substrate 1 1 5. Review of Mitochondrial Energy Production Overview of Oxidative Phosphorylation. To understand the impact of hypoxia at the cellular level on whole body substrate use it w i l l be necessary to examine the end point of nutrition - A T P production via oxidative phosphorylation. This is the point in cell metabolism where oxygen and substrate (now in the form of N A D H ) Review of Literature W actually converge. Oxygen has by far its most important interaction with cellular metabolism as the terminal electron acceptor of the electron transport chain (ETC). In the resting state it is here that about 85%-90% of whole body oxygen consumption occurs 3 6 . Without sufficient oxygen, the rate limiting cytochrome c oxidase reaction cannot proceed and A T P production through the E T C wi l l be stalled. For sustained exercise rates to be maintained, the rate of A T P utilization at the muscle cel l must be exactly balanced by A T P production. If this were not the case the very modest endogenous A T P reserves, present in the cell mainly as creatine phosphate, would be quickly exhausted and fatigue would ensue. In the working muscle cell two main systems are active: (i) an energy demanding system, largely a result of cytosolic ATPase activity which ultimately enables muscular contraction, and (ii) an energy producing system predominantly in the mitochondria. The ATPase demand occurs predominantly in the cell cytosol and is comprised mainly by three exergonic reactions; actomyosin ATPase , N a + / K + ATPase and C a + + ATPase. These latter two ATPases provide essential homeostatic functions restoring ion gradients and electric potential within the cell. The production of A T P to meet these demands is accomplished predominantly in the mitochondria via oxidative phosphorylation. Substrate level phosphorylations via glycolysis in the cytosol also contribute albeit to a small degree to the total supply of A T P . Before a consideration of how oxidative phosphorylation is regulated and how glycogenolysis is theoretically involved, a brief review of the general plan of oxidative phosphorylation w i l l be undertaken. A n overall net equation describing oxidative phosphorylation is: N A D H + \ 0 2 + 3 A D P 3 " + 3Pi 2 " +4H+ -> NAD+ + 3 A T P 4 " + H 2 0 (1) From this it can be seen that three "substrates" are required to produce A T P v ia oxidative phosphorylation: (i) reducing equivalents in the form of reduced nicotinamide adenine dinucleotides ( N A D H ) obtained through carbohydrate, l ipid and amino acid oxidation, (ii) A D P and inorganic phosphate (Pi) obtained for example from A T P hydrolysis during muscle Review of Literature 11 contraction, and (iii) oxygen which acts as the terminal acceptor of electrons to produce H2O in the rate limiting cytochrome c oxidase reaction of the E T C . Reducing equivalents obtained through substrate oxidation are produced both in the cytosol via glycolysis as well as in the mitochondria via the tricarboxylic acid ( T C A ) cycle and p-oxidation of fatty acids. The inner mitochondrial membrane is impermeable to N A D H produced in the cytosol. To gain entrance, N A D H must be translocated into the mitochondria where the enzymes of oxidative phosphorylation reside. In skeletal muscle this is thought to occur predominantly through the action of the electrogenic malate - aspartate shut t le 2 7 ' 9 9 . This shuttle acts to transfer the hydrogen atoms carried by N A D H across the inner mitochondrial membrane by reducing an oxidized substrate (oxaloacetate) to a reduced substrate (malate) which is capable of traversing the membrane to deposit the hydrogen atoms inside the mitochondria. The glycerol phosphate shuttle is also active in transporting reducing equivalents into the mitochondria however activity of this shuttle is higher in brain and liver. The glycerol phosphate shuttle is thought to have a very limited role in skeletal muscle N A D H transport9 9. Once inside the mitochondria, the electrons derived from N A D H w i l l be passed in a step-wise fashion down the electron transport chain. A s this occurs, according to Mitchel l ' s chemiosmotic theory, the difference in redox potentials between the various electron carriers is harnessed and used to pump protons outwards across the inner mitochondrial membrane 9 7 . This process creates an electrochemical proton gradient across the inner mitochondrial membrane. This gradient is used to phosphorylate intramitochondrial A D P at three putative sites along the chain. This is accomplished by coupling the energy released from the protons moving down their electrochemical gradients to the endergonic phosphorylation reaction thereby producing A T P . The ratio of oxidized nucleotide adenine dinucleotides to reduced, or [ N A D + ] / [ N A D H ] , is known as the redox potential of the cell . It can be expressed as either the redox state of the cytosolic compartment or as that inside the mitochondria. A typical value for this ratio in the cytosol is approximately 1000. The mitochondria however is reduced about two orders of Review of Literature 72 magnitude more than the cytosol, yielding a typical value of about 8. In the cytosol it is important to have N A D + present at sufficiently high levels to allow oxidation to proceed through glycolysis, while in the mitochondria it is advantageous to have higher levels of N A D H to act as a gradient to ensure flow of electrons from N A D H to flavin carriers at the start of the E T C . A s mentioned earlier, the A D P and P i necessary to synthesize A T P inside the mitochondria arise predominantly in the cytosol from ATPase activity. When the cell moves from respiratory state 4 towards state 3, increasing levels of A D P and P i are generated through ATPase activity. (Respiratory state 4 is the resting state in mitochondria in which A T P production is limited by the supply of A D P and P i , mitochondria in state 3 respiration have ample A D P and P i — in this case the respiratory chain itself is the rate limiting factor 2 2.) These metabolites are translocated into the mitochondria by an electfogenic adenine nucleotide translocase with A D P 3 " being transported into the mitochondria and A T P 4 " out, into the cytosol, and an electroneutral phosphate transporter". This latter transporter is a symport system in which an equal number of oppositely charged protons are transported into the mitochondria along with the negatively charged phosphate. Regulation of Oxidative Phosphorylation While most of the details of how oxidative phosphorylation produces A T P are relatively well known, what remains more difficult to unravel is how this process is regulated. In other words, how does an increase in A T P demand by cytosolic ATPases lead to an exactly equal increase in A T P supply by the mitochondria? In theory, the only way to regulate flux through a metabolic pathway is to alter the levels of substrate involved in the pathway, and/or to alter the activity (or number) of the enzymes involved in the pathway. Traditionally the enzymes of oxidative phosphorylation have been assumed to follow Michaelis-Menten kinetics. This being the case, activity of these enzymes and therefore regulation of the pathway would be dependent largely upon the levels of substrates involved: N A D H , A D P + P i , and O2. While the assumption of Michaelis-Menten kinetics has more recently come under quest ion 6 5 , i f for the moment we take this Review of Literature 13_ assumption to be correct, it provides a convenient point to begin our discussion on the regulation of oxidative phosphorylation by looking at the substrates involved. A n understanding of the regulation of oxidative phosphorylation is of utmost importance for our purposes in that it w i l l allow us to predict (if only theoretically) what effect alterations in levels of any of the three substrates of oxidative phosphorylation ( N A D H , A D P + P i , and O2) w i l l have on cell energy metabolism. For example, we are interested in the effects of hypoxia on glycogen util ization. If O2 was reduced to such a level as to be in short supply at the level of the mitochondria, could alterations in the other two metabolites ( N A D H , A D P + Pi) compensate for this deficiency? More specifically, could an increased delivery of N A D H to the mitochondria (i.e. an increase in substrate oxidation) compensate for a shortage of O2 by driving the reaction described above to the right, much as Le Chatelier's principle might suggest? If so, which of the three substrates (carbohydrate, l ipid, or amino acid) would be preferentially used to provide these reducing equivalents? Before we can answer these questions it wi l l be necessary to look at some in vitro and in vivo studies which have been undertaken to elucidate the effects of altering substrate levels on oxidative phosphorylation. The effect of ADP and Pi on oxidative phosphorylation Classically, oxidative phosphorylation has been considered to be largely controlled by A D P and P i ; the hydrolysis products of A T P . It has been clearly shown in vitro that adding A D P to a suspension of isolated mitochondria stimulates respiration. Based on work done by Lardy and co-w o r k e r s 8 6 and Chance and co-workers 2 2 forty years ago, it was hypothesized that these by-products of A T P hydrolysis could act as signals from the cytosolic compartment to provide feedback to the mitochondria and stimulate oxidative phosphorylation. The rationale behind this theory is elegantly simple; an increase in A T P consumption in the cell wi l l result in an exactly equal increase in A T P production because the flux through the mitochondrial pathway w i l l be directly proportional to the flux through the cytosolic pathway due to direct feedback of A D P and P i . Review of Literature 14 Exactly how A D P and P i exert their effects on respiration is not known with certainty. Some favour a kinetic mechanism where alterations in concentrations of A D P and P i directly affect one or more of the reactions involved in A T P synthesis 2 2 ' 8 6 . For example increases in the levels of A D P as a result of increased ATPase activity in the cytosol may act to directly stimulate the adenine nucleotide translocator thereby increasing both intramitochondrial levels of A D P and in turn oxidative phosphorylation. Others favour a thermodynamic control model where the ratio [ATP] / [ADP][P i ] , known as the cytosolic phosphorylation potential, is thought to be the controlling parameter 4 0 ' 8 5 ' 1 2 9 . Proponents of this hypothesis view the reactions up to and including the first two sites of A T P production to be in a near equilibrium state. Therefore, another pseudonym for this thermodynamic control model is the near equilibrium hypothesis. In this scheme, the reactions up to cytochrome c are thought to be in near equilibrium while the final reaction of the E T C , cytochrome c oxidase, is not near equilibrium and as such acts as the rate limiting step of the pathway 4 0. A reaction summarizing the near equilibrium events up to cytochrome c can be written as: N A D H + 2 cyt c 3 + + 2 A D P + 2 Pi <-» NAD+ + 2 cyt c 2 + + 2 A T P (2) Wi th this hypothesis, increases in ATPase activity in the cytosol lead to decreases in the cell phosphorylation state, thereby upsetting the thermodynamic equilibrium between A T P , A D P , and P i . This equilibrium in turn is believed to be linked to the near equilibrium reactions of the E T C so that an alteration in the phosphorylation state wi l l be transmitted via this series of near equilibrium reactions through the E T C . The overall effect would be to draw reducing equivalents down the E T C to reestablish the equilibrium, which in turn lowers the cell redox state and stimulates oxidation of substrate to restore the redox state. Which (if either) of these two hypotheses is correct has been a matter of much debate. Critics of the near equilibrium hypothesis point out that for this hypothesis to work, the adenine nucleotide translocator must be a near equilibrium process i f it is to effectively link near equilibrium reactions Review of Literature 15 in the cytosol to those inside the mitochondria. They argue that evidence from a number of laboratories has shown that this is not the case 1 2 0 . On the other hand, other interpretations of data obtained from studies on isolated mitochondria have concluded that this translocator is near equil ibrium 1 1 7 ' . A t present it is not known with certainty whether the adenine translocator is or is not rate limiting. A s was mentioned earlier, it has been widely assumed that the enzymes of oxidative phosphorylation obey hyperbolic Michaelis-Menten kinetics such that increased levels of substrate w i l l cause first order increases in reaction rate which gradually decline to zero order as substrate concentration is increased and enzyme saturation is achieved. If this were the case, a hypothetical 5 fold change in the levels of A D P and P i should result in a maximal 5 fold increase in A T P production (provided [ADP] and [Pi] concentrations are within the first order range). However this pattern is not seen experimentally. In heart muscle in vivo, Katz et al. using 3 1 P-Nuclear Magnetic Resonance Spectroscopy ( N M R ) have found virtually no change in A D P or P i levels despite several fold changes in A T P production 7 9 . N M R studies are particularly informative for this purpose in that they allow the calculation of free A D P and P i rather than total A D P and P i . In vitro biochemical measurements typically yield total cell [ADP] and [Pi], which includes both free and bound forms. A large portion of adenylates in the cell exist in the bound form, while it is only the free form which participates in cellular reactions. This makes biochemical measurements somewhat misleading due to a resultant overestimation of A D P and Pi In skeletal muscle A D P and Pi levels are more malleable but patterns that fit Michaelis-Menten kinetics still are not seen. Studies on skeletal muscle from a variety of mammalian species under a variety of work conditions have typically shown that for any given change in [ A D P ] , there is anywhere from a 2 to 20 fold change in A T P f l u x 3 7 - 3 9 ' 9 4 ' 1 0 0 . This belies the 1:1 change that would be expected i f Michaelis-Menten kinetics were operating. It is interesting to note that metabolically, as skeletal muscle is trained it approaches the behaviour of cardiac muscle — increased A T P production but with lower levels of A D P and P i 3 9 . This finding, in addition to Review of Literature 16_ placing Michaelis-Menten kinetics in doubt, suggests that something in addition to A D P and P i is contributing to control of respiration. Based on the current available data, the evidence is consistent that energy production is operating under a sigmoidal (allosteric) rather than hyperbolic (Michaelis-Menten) k ine t i c s 5 ' 6 4 ' 6 5 . This could potentially explain why such relatively small changes in A D P and P i can lead to much larger fluxes in A T P production. However the identity of the allosteric effector is at present u n k n o w n 1 3 2 . In addition to the ability of A D P and P i . to stimulate cell respiration there are two, often overlooked, aspects about the by-products of A T P hydrolysis that are very important in a discussion of cell energy metabolism. Firstly, these metabolites are capable of inhibiting muscle contractile activity particularly when cell p H drops. Diprotonated phosphate (H2PO4") is formed as cell p H drops during muscle contraction. Current evidence suggests that this compound is capable of inhibiting contractile act ivi ty 1 2 7 . Additionally, protons produced from A T P hydrolysis over and above the cell's buffering ability are also capable of interfering with cytosolic activity. Acidosis is thought to inhibit C a 2 + binding to troponin 4 4 as well as interfere with release and binding of C a 2 + at the sarcoplasmic reticulum 4 3 . Protons can also interfere with phosphocreatine (PCr) production by altering the creatine kinase e q u i l i b r i u m 1 0 0 , 1 2 7 . The combined effect of these actions is to reduce actomyosin ATPase activity and therefore A T P demand3. The second important aspect of A D P and P i is that they are capable of stimulating glycolysis allosterically at phosphofructokinase 1 1 6. This latter factor is particularly important for our purposes, as it can result in increased carbohydrate (glycogen) utilization. What role do A D P and P i play in hypoxia? Studies by Wilson and c o - w o r k e r s 1 2 8 ' 1 2 9 , 1 3 2 on isolated mitochondria have shown that a decrease in O2 concentration at cytochrome c oxidase is paralleled by a decrease in the ratio of [ATP]/[ADP]-[Pi] . This has been interpreted by these investigators as signifying that increases in the concentration of free A D P and P i are capable of Review of Literature 17_ counteracting the effects of hypoxia and allowing a constant rate of A T P synthesis (as w i l l be explained in an upcoming section, a very similar pattern is thought to exist for N A D H ) . Taken together what does this mean so far as the role of A D P and P i in regulating A T P production? In skeletal muscle there definitely seems to be a relationship between A D P and P i levels and the rate of A T P production, although it does not seem to obey Michaelis-Menten kinetics. A s w i l l be explained in the upcoming sections, it is feasible that in addition to A D P and P i , reducing equivalents and O2 availability also play significant roles in regulating cell energy production. The additional control of these latter two substrates may partially explain why such small A D P changes can result in larger changes in A T P flux. Apart from changes in substrate concentrations, it may be that some as yet unidentified metabolite is acting to allosterically modify the enzymatic machinery of oxidative phosphorylation. This could also explain why small changes in A D P and P i can result in much larger increases in A T P production. Another possible explanation is that a labile pool of enzymes are present which can be activated in response to increased cell demand. In a recent review by Harris et al., two intramitochondrial proteins are identified which are thought to play such a role in heart muscle 5 8 . These proteins, IF] and CaBI , are capable of inhibiting the activity of A T P synthase (FjFo ATPase) by binding to one of its subunits. Alterations in inner mitochondrial membrane potential can reverse the inhibition caused by IF] allowing increased production of A T P when cell demand rises. In a similar manner, the inhibition caused by CaBI can also be reversed by elevations in intramitochondrial calcium. Therefore the inhibition caused by these proteins at A T P synthase can be regulated through signals acting at the mitochondrial level allowing increased production of A T P to occur at times of increased demand. It is entirely possible that a similar method is utilized in skeletal muscle mitochondria which acts to regulate their A T P production. Superimposed on all of these potential regulators (which all affect A T P production by increasing supply) there is also the effect of decreased demand caused by A T P hydrolysis products as Review of Literature 18 discussed earlier. It is apparent that the control of energy production in the cell is an extremely intricate matter not easily ascribed to any one modulator. The effect of NADH on oxidative phosphorylation O f the substrates involved in oxidative phosphorylation, N A D H is the most important for our purposes as it is a direct reflection of fuel (carbohydrate, l ipid, amino acid) oxidation. If, for example, increased mitochondrial N A D H levels can enhance energy production then this w i l l manifest itself as an increase in use of either carbohydrate, triglyceride, or protein in the body depending on which fuel is preferred given the conditions under consideration. Similarly, i f increased N A D H levels in the mitochondria can make up for limiting O2 levels as the equations (1) and (2) above suggest, then it is possible that increased fuel use could be seen under these conditions. Again, the fuel that would be used would be that which was most efficiently used by the cell under hypoxic conditions. For reasons that w i l l be explained later in this section, this fuel would likely be glycogen. The energy to make A T P ultimately comes from the energy released from highly reduced substrates oxidized by intermediary metabolism. Reducing equivalents in the form of N A D H (and F A D H 2 ) are produced through the oxidation of carbohydrates, lipids, and amino acids. The majority of these reducing equivalents are produced inside the mitochondria via p-oxidation and the T C A cycle. Those reducing equivalents produced in the cytosol via glycolysis must be transported into the mitochondria, against a 100 fold concentration gradient to be ultimately utilized in the process of oxidative phosphorylation. This transport is accomplished in skeletal muscle primarily through the activity of the malate-aspartate shuttle 2 7 ' 9 9 . Once inside the mitochondria, it has been shown that reducing equivalents can affect the rate of oxidative phosphorylation. In vitro experiments on isolated mitochondria 8 3 , and in vivo in cardiac m u s c l e 7 9 , have found that increased levels of intramitochondrial N A D H can increase A T P production without a significant change in extramitochondrial A D P or P i levels. Exactly how Review of Literature 19_ increased N A D H levels are enhancing oxidative phosphorylation is not known 5 . It may occur via alterations in mitochondrial membrane potential 2 1 (which can affect proton gradients whose electrochemical free energy is used to power A T P synthase). Alternatively elevated N A D H may take its effect on the cytochrome chain through either thermodynamic or kinetic mechanisms 4 0. Studies by Wi l son and co-workers on isolated mitochondria have shown that the state of reduction of cytochrome c (the second to last cytochrome in the E T C which passes electrons to cytochrome c oxidase which reduces molecular oxygen to water) is highly dependent on both [ATP]/[ADP]-[Pi] and the oxygen concentration at cytochrome c o x i d a s e 1 3 0 , 1 3 1 . When levels of A D P and P i are low in the mitochondria the level of cytochrome c reduction increases almost as i f to counteract the low levels of A D P and P i . The state of reduction of cytochrome c can be related to the intramitochondrial [ N A D + ] / [ N A D H ] ratio through the near equilibrium reaction 4 0 . Reductions in this ratio (increased intramitochondrial N A D H ) results in an increased level of reduction of cytochrome c. Reduction of cytochrome c is important for maintaining mitochondrial A T P production as it passes its electrons on to the rate limiting cytochrome c oxidase step to allow reduction of oxygen to water. Therefore in order for the mitochondrial respiratory rate to change, it is necessary for the rate of oxygen reduction by cytochrome c oxidase to change by the same amount and in the same direction 1 3 2 . A similar pattern has been found by Wilson and co-workers in isolated mitochondria faced with low levels of o x y g e n 1 2 9 ' 1 3 0 , 1 3 1 . When O2 levels are decreased, the level of reduction at cytochrome c increases as i f to offset the low oxygen concentration and maintain A T P production. This has important ramifications for mitochondria in working skeletal muscle under hypoxic conditions as it suggests that increased mitochondrial N A D H (ie increased oxidation of substrates) can contribute to maintaining A T P production in the face of reduced oxygen tensions at cytochrome c oxidase. If increased fuel substrate oxidation can counteract the effects of hypoxia on mitochondrial A T P production by increasing reducing equivalent delivery as the above discussion suggests, would one Review of Literature 20 substrate be preferentially oxidized to supply N A D H over others? This question is to a large part the basis of this research. To answer this question it is necessary to look at the substrates of intermediary metabolism to see i f there is any reason to suggest why one would be preferred over the others. Triglycerides are metabolized to fatty acids and glycerol, and this is the form that they enter intermediary metabolism. Glycero l enters glycolysis as glyceraldehyde-3-phosphate, is metabolized to pyruvate which enters the mitochondria, and is converted to acetyl C o A which enters the T C A cycle. Glycerol oxidation produces sufficient reducing equivalents to produce 19 molecules of A T P 9 5 . The fatty acid component of a triglyceride is converted to a neutral carnitine ester for transportation into the mitochondria. Once inside the mitochondria, the fatty acid is catabolized via p-oxidation to acetyl C o A subunits which are ultimately metabolized via the T C A cycle to yie ld reducing equivalents. Each acetyl C o A molecule produces enough reducing equivalents to synthesize 12 A T P molecules. Therefore using the 16 carbon fatty acid palmitate as an example, each fatty acid molecule would yield sufficient reducing equivalents to produce 129 A T P 9 9 . In total, one entire l ipid molecule of tripalmitate completely oxidized w i l l yield 406 A T P molecules. Carbohydrates are used in the cell to produce energy either as exogenously supplied glucose, or as endogenously stored glycogen. After transport into the cell, glucose is converted to glucoses-phosphate through the action of hexokinase. This step requires one A T P molecule, and effectively traps the glucose molecule in the cell. Glucose-6-phosphate is then metabolized in the cytosol via glycolysis to pyruvate. On an overall net basis, glycolysis produces two molecules of A T P through substrate level phosphorylations, and two reducing equivalents as N A D H . If glycogen is the initial substrate, on a net basis there wi l l be one extra A T P produced. The initial catabolism of glycogen to glucose-1-phosphate does not require A T P , instead inorganic phosphate can be used. This effectively saves one A T P allowing a greater overall net yield of three A T P molecules produced per glucosyl unit metabolized to pyruvate. If glucose is completely oxidized through the T C A cycle Review of Literature 27_ enough reducing equivalents to produce 38 A T P molecules w i l l be liberated. If glycogen is the starting fuel 39 A T P wi l l result for the reasons discussed above. Protein is broken down to its constituent amino acids prior to being used in intermediary metabolism. In skeletal muscle, the primary amino acids utilized for fuel are the branched chain amino acids (leucine, isoleucine, and valine), as well as glutamate, alanine, and aspartate. These six amino acids are transaminated to remove their nitrogen groups and the remaining carbon skeletons are passed on to be further metabolized. O f these six amino acids, leucine being the only truly ketogenic amino acid is converted to acetyl C o A . The carbon skeletons of isoleucine, valine, glutamate, and aspartate are all ultimately metabolized to the T C A intermediate oxaloacetate. Much of the amino acid metabolism in skeletal muscle does not proceed to full oxidation. Rather, many of the amino acids are catabolized to generate acceptors such as pyruvate and 2-oxoglutarate which are used to combine with ammonia to form alanine and glutamine. These latter amino acids are subsequently transported via the bloodstream to liver and intestinal cells where they are involved in gluconeogenesis and gut mucosal barrier functions respectively. For these reasons the A T P yield from amino acid carbon skeleton metabolism is variable, depending on the original amino acid, where it enters the cells metabolism , and whether it remains in the cell. On a quantitative basis, the energy production from amino acid metabolism is not large in comparison to that of lipids and carbohydrates. Due to this relatively small contribution to overall muscle energy metabolism 4 , amino acids w i l l be excluded from the remainder of this discussion. A comparison of fatty acid versus glucose oxidation shows that palmitate yields over three times more A T P (129 versus 38) per molecule oxidized. A l l of the reducing equivalents formed through fatty acid oxidation are produced in the mitochondria while in the case of carbohydrate oxidation some of the A T P and reducing equivalents are produced in the muscle cell cytosol. It would appear based on net energy yield that it would be beneficial to the cell to utilize fatty acids as an energy source. In fact this is exactly the case when oxygen supply to the cell is not limited. However in Review of Literature 22 the case where oxygen supply is limited, as in hypoxia, it is not as advantageous to the cell to rely on lipid as an energy source due to the relatively larger O2 requirement to metabolize lipids. Relative to carbohydrates, lipids are in a much more reduced state. This is why they are a much more energy dense form of substrate. However as a result of this, relatively more oxygen is required to release this energy. This is evident when the two substrates are compared based on the amount of A T P produced per mole of O2. For oxidation of fatty acids, the number of moles of oxygen necessary to produce one mole of A T P is 0.177. For oxidation of glucose this value is 0.158, and for glycogen 0.154 4 . This shows that oxidation of fatty acids (palmitate) requires approximately 12% more oxygen when compared to aerobic oxidation of glucose. Metabolism of glycogen is even more oxygen efficient in comparison to glucose by approximately 2.5% due to the fact that one extra molecule of A T P is produced by glycogen catabolism. Clearly when the cell is faced with hypoxic conditions, it is beneficial to metabolize carbohydrates. In particular, glycogen oxidation would be the most efficient substrate source for muscle under hypoxic conditions. The effect of O2 on oxidative phosphorylation The previous section on the role of reducing equivalents in maintaining A T P production during hypoxia was predicated on the assumption that there is a shortage of O2 at the mitochondria. A s we shall see in this section, the whole subject of oxygen limitation at the mitochondria is controversial. (For two recent and exhaustive references taking opposing views see the reviews by Stainsby and B r o o k s 1 1 5 and by Katz and Sahlin 7 8 ) . Given the present uncertainty it may not be possible to say definitively that in an athlete exercising at 70% VO2 m a x while breathing 16% oxygen at sea level, O2 concentration at the mitochondria of the exercising muscle is limiting energy production. This confusion to a large part stems from the fact that the apparent K m for O2 at cytochrome c oxidase of isolated mitochondria is very low - in the order of 0.07 - 0.7 u M 5 . Technical considerations make it very difficult to measure such low oxygen concentrations with accuracy. Review of Literature 23 Before discussing the level of oxygen at cytochrome c oxidase, it w i l l prove instructive to briefly review the physics of oxygen in air as well as the pathway oxygen takes from inspired air down to the mitochondria. A surprisingly large gradient exists from the ambient air taken in at the mouth down to the site of energy production - the mitochondria. The partial pressure of oxygen (P02) at any of these steps can best be considered as a driving force or pressure head which facilitates transfer from one step to the next 1 2 5 . A reduction in this pressure head at any step leads to a decrease in oxygen tension at the next and remaining steps of the pathway. Such a reduction in oxygen tension can ultimately affect energy production by reducing available molecular O2 at cytochrome c oxidase. Partial pressure of oxygen (P0 2 ) in ambient air follows Dalton's law which states: (P02) = atmospheric pressure x percentage of O2 in air = 760 mm Hg x 20.93% = 159 mm Hg Therefore at sea level where there is a barometric pressure of 760 Torr (1 Torr = 1 mm Hg) and a fractional percentage of oxygen of 20.93%, oxygen exerts a partial pressure of 159 Torr. Wi th an increase in altitude, there is a exponential decrease in barometric pressure due to a progressively smaller amount of atmosphere pressing down from above. Added to this, there is a decrement in temperature as altitude is increased so the overall effect is a larger than exponential decrease in barometric pressure with a rise in alt i tude 1 2 3 . A t all altitudes, the percentage of oxygen in air is constant at 20.93% so that the reduction in the pressure of inspired oxygen (P1O2) is due to the reduction in barometric pressure rather than a change in fractional concentrations of the constituent gases. The type of hypoxia described in the previous paragraph is known as hypobaric hypoxia. This can be simulated physiologically by lowering the fractional concentration of inspired oxygen (F1O2) to below 21%, while maintaining barometric pressure. For example the barometric pressure Review of Literature 24 at Mexico City with an altitude of 7382 feet, is 580 Tor r 1 8 . This can be simulated by lowering the F l 0 2 f r o m 21% to 16% while maintaining barometric pressure at the sea level value of 760 Torr. This is known as normobaric hypoxia, and is frequently utilized in experimental studies to simulate acute exposure to altitude. A number of studies have found that the physiological responses to normobaric hypoxia are equivalent to those resulting from hypobaric hypoxic exposu re 4 7 ' 5 1 ' 5 7 ' 1 1 4 . Such findings validate the use of normobaric hypoxia as an experimental tool to simulate acute altitude exposure. Once inspired, oxygen travels down the trachea towards the alveoli becoming fully saturated with water vapour on the way. A t sea level, this lowers the PO2 from 159 Torr at the mouth by 47 Torr to 112 Torr. A further reduction in PO2 to about 100 Torr results from mixing of the moist inspired air with residual air in the a lveo l i 9 5 . It is this 100 Torr pressure of oxygen that acts as a pressure head to equilibrate with plasma and hemoglobin in pulmonary capillary blood. Once equilibration has taken place, there is a further slight reduction in arterial PO2 to approximately 95 Torr due to factors such as ventilation:perfusion inequalities in the lung, and intra- and extra-pulmonary arteriovenous s h u n t i n g 3 5 , 1 0 2 . In an individual with normal hemoglobin levels, approximately 99% of the oxygen carried in blood is bound to hemoglobin, with the remainder physically dissolved in plasma. U p to the point of arterial blood, nearly 40% of the initial pressure of oxygen has been dissipated. It is the remaining PO2 of 95 Torr which reaches the proximal side of the active muscle tissue vascular bed. A s blood traverses the arterioles toward the capillaries, oxygen begins to diffuse down its concentration gradient to the tissues. Once the venous side of the capillary bed is reached, the PO2 w i l l have been reduced to roughly 40 Torr. Evidence suggests that the oxygen in blood does not equilibrate with the tissue it suppl ies 5 0 . Studies using the technique of cryomicrospectroscopy which measure the saturation of myoglobin in quick frozen muscle have shown that the PO2 in resting muscle is typically in the range of 20 Torr yet venous PO2 is almost twice this suggesting that blood and tissue do not equilibrate. Review of Literature 25 It is currently thought that a very large gradient exists over the course of a few micrometers from the red blood cell to the muscle cell cytoplasm. In this so called carrier-free region, oxygen diffuses out across the plasma membrane of the red blood cell and across several barriers (plasma, capillary endothelial cell, extracellular space, and sarcolemma) to reach the cytoplasm of the cell it supplies. The vast majority of the loss in oxygen pressure at the cell level is thought to occur over this relatively small distance from red blood cell to sarcolemma (~3 um) that oxygen travels by simple diffusion in the carrier free region. Over this region there is a drop in PO2 from greater than 50 Torr in the capillary to less than 20 Torr in the cell cytosol 5 0 . This represents a very large gradient in the order of 10 Torr • unr 1 4 9 . Once inside the muscle cell, oxygen is rapidly bound to myoglobin and transported once again by a process of facilitated diffusion. Work by Connett et al. suggests that once oxygen reaches the cytoplasm and is bound to myoglobin, there is little further loss in PO2 from cytoplasm to mi tochondr ia 3 1 . However this conclusion is questioned by others 7 5 . The existence of oxygen gradients within the cell remains a controversial subject 1 3 2. Once oxygen reaches the mitochondria it "sees" a surface area over 200 times larger than that of the effective capillary surface area feeding the muscle c e l l 4 8 . Mitochondria therefore act as a very large physical sink for sequestration of O2 which themselves presents little i f any impediment to oxygen transport2 5. The discussion in the previous paragraphs has highlighted the decrement in the pressure head from ambient air down to the level of the mitochondria in resting muscle. This drop is magnified even further in active muscle; at maximal work rates cytosolic P 0 2 may drop as low as 0.5 to 2 T o r r 3 0 , 4 8 . This is due to the rapid consumption of oxygen by the cell to produce A T P via oxidative phosphorylation. This drop in intracellular PO2 with exercise is even further magnified by exercise at altitude due to the lower PO2 of inspired oxygen which effectively reduces the oxygen cascade gradient at the initial step. It should be pointed out that the analysis given above is somewhat oversimplistic as the region immediately surrounding the muscle cell is heterogeneous with respect to capillary supply. Review of Literature 26 Additionally, the oxygen tension in the capillaries surrounding the muscle cell varies as to the distance the muscle cell is away from the afferent end of the capillary bed. The further from the afferent end of the capillary bed the lower the oxygen tension is in the supplying capillary. This is a direct result of the drop in oxygen pressure seen from the arterial side of the capillary bed to the venous side as mentioned earlier. A further factor which complicates an analysis of intracellular is the heterogeneous distribution and clustering of mitochondria within muscle ce l l s 7 5 . The end result of the heterogeneous supply and distribution of oxygen at the cell is that PO2 within a cell is not constant. Some regions of the muscle cell can be relatively more hypoxic (or even anoxic) compared to others. Part of the problem in establishing whether oxygen is limiting to cell respiration relates to the uncertainty of the measurements of intracellular oxygen tension. A variety of methods, both invasive and noninvasive have been developed to assess intracellular P02. Oxygen tension has been measured invasively with oxygen microelectrodes, while noninvasive techniques include measurements of myoglobin saturation, cytochrome redox states, mitochondrial monoamine oxidase activity, 0 2 - quenched fluorescence, electron spin resonance 7 5 , 1 3 3 . Unfortunately it seems that each of these methodologies suffers from some drawback making the measurements obtained questionable. For example the oxygen microelectrode has been criticized for its invasiveness. It is thought that the cell disruption caused by the microelectrode leads to overestimations of the amount of oxygen present. Criticisms have also been levelled at the optical technique of measuring myoglobin saturation. With this technique working muscle is rapidly frozen so that oxygen bound to myoglobin is effectively trapped in this state. Spectrophotometry is then used to measure the percent oxygen saturation of myoglobin. These values can be used to calculate intracellular PO2 as the two are proportional. Unfortunately the spectra of myoglobin overlaps with that of hemoglobin and is also susceptible to light scattering effects making estimations of PO2 by this method questionable 5 , 1 3 3 . Despite this impressive inventory of measurement techniques, several questions still remain regarding oxygen gradients as well as absolute oxygen levels in actively working Review of Literature 27 muscle cells. These outstanding questions have resulted in a great deal of controversy over the role of oxygen in energy production via oxidative phosphorylation. We have seen that exercise, and in particular exercise at altitude, can reduce the pressure of oxygen reaching the mitochondria to very low levels. This raises the question as to whether this w i l l affect oxidative energy production by restricting the amount of molecular oxygen at the rate limiting cytochrome c oxidase step. As alluded to above, this contention is quite controversial. The apparent Km of cytochrome c oxidase for oxygen is thought to be exceedingly low due to the very high affinity this enzyme has for oxygen. The affinity of cytochrome c oxidase is about 100 times greater than that of myoglobin which supplies the E T C with oxygen, and approximately 500 times greater than that of hemoglobin 7 0 . Studies on isolated m i t o c h o n d r i a 1 1 8 ' 1 2 9 and intact musc le 4 9 have shown the apparent Km* to range from 0.05 to 0.5 Torr. The concept of a critical PO2 (PO2 crit) has also been employed to quantitate the oxygen requirements of the cytochrome c oxidase reaction. PO2 crit differs from Km in that it is the minimum PO2 required for maximal turnover of cytochrome c oxidase. A n y drop in oxygen tension below P0 2crit w i l l result in a detectable decrease in V 0 2 . Values for P02crit are slightly higher than those for Km which is defined as the PO2 which results in half maximal velocity of VO2 when oxygen is not limiting the cytochrome c oxidase reaction. Values for PO2 crit for working muscle are in the range of 0.5 Tor r 7 0 . A number of studies by Connett et al 2 6 , 2 8 , 2 9 and Gayeski et al 4 8 , 4 9 , 5 0 on maximally working canine red muscle (gracilis) have consistently shown that intracellular PO2 levels are above PO2 crit. These workers have found oxygen tensions (under normoxic conditions) to be in the range of 1 to 2 Torr, which would suggest that cytochrome turnover is not limited by oxygen tension under these conditions. But, again the question of how valid the measurements are given the potential artifacts arising from the optical technique used must * The Michaelis-Menten constant (Km) of cytochrome c oxidase is the PO2 at 50% of VC^max when O2 is not limiting. In this case it is referred to as an apparent Km because rather than being a constant value, it changes depending on the surrounding media. If high levels of ADP, Pi, and/or NADH are present, the Km of cytochrome c oxidase for oxygen will be at the low end of the 0.05 to 0.5 mm Hg range. If these mitochondrial substrates are present in low concentrations, more oxygen is required to allow the cytochrome c oxidase reaction to turnover at maximal rates. Review of Literature 28 be kept in mind. No studies have been done under conditions of moderate hypoxia which would be expected to decrease cell oxygen tensions below those seen with normoxia. Whether oxygen levels at the mitochondria are below PO2 crit during muscle work may not be the most important consideration however. Work by Wi l son and co-workers suggests that mitochondrial oxidative phosphorylation can still be affected at O2 tensions wel l above those required to reduce cytochrome t u r n o v e r 1 2 8 ' 1 2 9 , 1 3 1 . According to their data, cell metabolism (but not cell respiration) can be affected at oxygen tensions up to 20 Torr in isolated mitochondria. Therefore A T P flux can be maintained under these conditions but not without alterations at the level of the mitochondria. Such alterations involve the reduction state of cytochrome c. A s PO2 is lowered below 20 Torr which is well above PO2 crit , cytochrome c reduction is observed to increase. Such an adjustment is thought to assist in keeping the rate limiting cytochrome c oxidase reaction supplied with electrons to allow the E T C to maintain high flux rates (as was discussed in the section on reducing equivalents). This brings us back to the role of reducing equivalents and foodstuff oxidation in maintaining energy production. These findings suggest that an increase in substrate oxidation can, by keeping the E T C supplied with electrons in the form of N A D H , maintain energy production when oxygen supplies are low. As was discussed earlier, under conditions of hypoxia the substrate that would be most favoured to supply these reducing equivalents would be glycogen. Summary A n y consideration of the effects of hypoxia on substrate metabolism must ultimately end up at the point where oxygen and substrates (now in the form of N A D H ) interact - the mitochondria. It is here where 85-90% of the cell 's oxygen is consumed through the process of oxidative phosphorylation. This brief review on the regulation of oxidative phosphorylation has illustrated that there are many unanswered questions about this process. Having said this, it is l ikely that all three of the substrates involved in oxidative phosphorylation; A D P and P i (as a by-product of Review of Literature 29 muscle contraction); N A D H (as a by-product of substrate oxidation); and O2 (as a product of the surrounding environment), in combination play a role in regulating A T P production. Whether this is the major method of control in skeletal muscle mitochondria is at this time still very much open to question. It may be that the major site of control is by some sort of allosteric modification of components of the E T C . Or it may be, as Arthur et a l . 3 and H o c h a c h k a 6 4 , 6 5 have proposed, that cell energy levels are controlled more through alterations in A T P demand rather than through modulations in A T P supply as most schemes propose. Alternatively it is possible that a major site of regulation is at the level of A T P synthase, through inhibition/deinhibition, as discussed earlier. If these latter three mechanisms are active in controlling cell energy levels, this would relegate substrate control to more of a fine tuning position in the control of A T P production. This does not necessarily mean however that quantitatively, the role played by alterations in mitochondrial substrate w i l l be small and inconsequential. For example, in the case of hypoxia it is stil l theoretically possible that alterations in cell redox (likely brought about by glycogen breakdown) can aid in counteracting low O2 levels at cytochrome c oxidase. Given the large flux capable through glycolysis and through the E T C even small alterations in N A D H necessary to counteract hypoxia may lead to quantitatively measurable changes in glycogen oxidation over that seen under normoxic conditions. This forms the biochemical basis for the assertion that glycogen utilization wi l l change in an athlete exercising under acute hypoxic conditions. Studies looking at hypoxia and substrate utilization Most of the studies which have been done in this area have looked at the effects of chronic hypoxic exposure on fuel metabolism. The purpose of these studies was to look more at the physiological adaptations which occur over time as the body acclimatizes to hypoxia. In contrast, we are concerned here more with the effects of acute hypoxia. Table 1 below represents a review of studies undertaken to identify the effects of acute hypoxic exposure on substrate metabolism. Not all of the studies included in this table were specifically designed to look solely at this Review of Literature 30_ relationship. For example, a few of the studies were looking at some auxiliary aspect of substrate metabolism such as hormonal responses to acute hypoxic e x p o s u r e 1 9 ' 2 4 ' 4 1 , 1 0 8 . These studies were included for their predictive value in explaining the relationship between hypoxia and substrate utilization. Many of these parameters were measured in the present study, thus it w i l l be instructive to examine them at this time. Table 1. The effects of acute hypoxic exposure on substrate and/or hormonal responses in humans. STUDY n FI0 2 VQ2 R/A R GLYC- GLU- FFA GLYC- NE E HSBU GLUC-(%) max OGEN COSE EROL -LIN AGON Jones et al 7 6 4 11-13 A t NC T t Linnarsson et a l 9 0 6 14.3 52 A — NC — McManus et al 9 6 6 14.5 48 A T — — — — NC NC — — Clancy et al 2 4 4 14.0 a A T — — — — NC NC — — Sutton 1 1 9 8 11.0 57 A — — T T — — — — Young et al 1 3 6 8 12.3 45 R NC ? — NC NC — — — — Bubb et al 1 9 6 13-19 48 A — — — — — NC NC — — Escourrou et al 4 1 7 12.0 56 R — — — — — T T — — Rowell et al 1 0 7 1 1 11.0 — A — — t — — T t — — Bouissou et al 1 5 8 14.5 55 R — — NC NC — NC NC NC NC Cooper et al 3 2 10 15.0 43 A — — t — — T T NC NC Rowell et a l 1 0 8 6 11.0 — A — — — — — t t — — Bouissou et al 1 4 6 14.3 50 R NC — — — — NC NC — — Barstow et al 7 7 15.0 44 A t — Katz et al 7 7 8 11.4 44 A — — T — — — — — — n = number of subjects in study, FI02 = fractional concentration of 02 utilized in experiment, V02max = approximate mean V02max of subjects in the study, included to give an indication of the overall fitness level, R/A = relative or absolute work loads utilized, see text for details, R= respiratory exchange ratio, GLYCOGEN = muscle glycogen level after exercise compared to before, GLUCOSE = plasma glucose level, FFA = plasma free fatty acid level GLYCEROL = plasma glycerol level, NE = norepinephrine, E = epinephrine, Dashes (—) signify measurements that were not reported in the study, NC = no change observed, a = V02max was not reported, but author stated that subjects were "non-athletic males". A n overview of Table 1 reveals two common characteristics among most of the studies: (1) most were done on subjects that were not highly trained, as the Vo 2 max values indicate, and (2) the column beside VO2 max indicates that 11 of the 15 studies utilized the same absolute work rates under both normoxic and hypoxic conditions. This latter point deserves emphasis, as it can Review of Literature 31 obscure the relationship between hypoxia and substrate metabolism for the following reason. When a subject exercises at altitude, there is a decrease in VO2 m a x - This means that for a given work rate at sea level, that same work rate at altitude requires a greater percentage of VO2 m a x to accomplish. Stated another way, the same absolute work rate at altitude must be performed at a relatively higher exercise intensity. This places a greater physiological demand on energy production which in turn places a greater demand on macronutrient catabolism. A s described earlier, the dependence of muscle on glycogen as an energy source increases as exercise intensity (expressed as % VO2 m a x ) increases. If the intention is to measure the amount of glycogen used, it is not surprising then that under the same absolute conditions, more wi l l be used at altitude because exercise intensity is higher. For this reason, it would be important in a study looking at glycogen utilization to use the same relative work rates for a fair comparison to be made. Hypoxia and RER O f those studies that measured R E R , four found an increase in this value signifying a shift towards increased carbohydrate u t i l i z a t i o n 7 ' 2 4 , 7 6 ' 9 6 , while two found no change in R E R between exercise at sea level, and exercise under hypoxic condi t ions 1 5 , 1 3 6 . O f those finding an increase in R E R , all of them had used the same absolute work rates under both conditions so it is not surprising that carbohydrate utilization appeared to increase. The two studies that found no change in R E R , used the same relative work loads, a condition which would make a comparison of substrate utilization more favourable. Unfortunately these studies were flawed to some extent which makes the validity of these results questionable. The study by Young et a l . 1 3 6 , used a work rate (relative) of 85% of V 0 2 m a x - For R E R measurements to be valid they must be made under "steady state" condit ions 7 , 2 0 . A t this work rate the subjects would have been past their anaerobic threshold, and as such would have been in non-steady state conditions i.e producing more lactate than they were oxidizing so that lactate was Review of Literature 52 accumulating. For this reason, the values obtained by Young et al. with respect to R E R must be interpreted cautiously. In the other study finding no change in R E R , by Bouissou et a l . 1 4 , the authors concluded that R E R values did not differ significantly between normoxia and hypoxia. These authors used a hypoxic level of 14.3% and an exercise bout of 60 minutes under each condition (normoxia and hypoxia). Their data appeared to show a rise in R E R towards the end of the exercise period, however the statistical techniques they used failed to show a significant difference overall. Perhaps i f the exercise period had been slightly longer they would have found a significant effect of hypoxia on R E R . In summary, in those studies that have measured R E R , the results are difficult to interpret. Either absolute work rates were used, which does not make for a fair comparison, or R E R was measured under non-steady state conditions which also tends to make the results questionable. Hypoxia and Fuel Substrates O f the studies in Table 1, six of them included measurements of plasma glucose l e v e l s 1 5 ' 3 2 ' 7 6 ' 7 7 ' 1 0 7 ' 1 1 9 . Unfortunately when those studies which utilized the same absolute work rates under both conditions are excluded, this leaves only one study by Bouissou et a l . 1 5 . Again, most of the other studies tended to show an increase in blood glucose levels perhaps indicating an enhanced release from the liver as well as enhanced utilization. But because these studies used a relatively higher work rate under hypoxic conditions, this result is not surprising. The study by Bouissou et al. did not find any difference in blood glucose levels with acute hypoxic exposure 1 5 . This result is not surprising however considering the short duration of exercise used in the experiment. The exercise bouts were only of 5 minute duration at 40, 60, 80, and 100 percent of VC-2 m a x - Due to this short duration, no changes were seen in any of the parameters measured (glucose, FFA ' s , catecholamines, insulin, and glucagon). Review of Literature 55 From the studies accomplished to date, no firm conclusions can be drawn about the effects of acute hypoxia on glucose metabolism. Two studies have incorporated measurements of plasma F F A ' s and have used the same relative work rates. These studies were done by Young and co-workers 1 3 6 , and Bouissou and co-workers 1 5 . Results from these two studies both indicate that there was no change in plasma F F A levels in response to acute hypoxia. In addition, the study by Young et al. also looked at plasma glycerol, and found no significant difference. Theoretically, i f oxygen was limited in these hypoxic exercise bouts you would expect the body to use the most efficient fuel for energy production. A s Hochachka et a l . 6 6 point out, under conditions where oxygen availability is potentially limited, the body has three potential strategies available to enhance the efficient use of oxygen: (1) use a fuel which wi l l yield the maximal amount of A T P produced per mole of substrate, (2) maximize the amount of A T P produced per mole of O2, and (3) maximize the amount of work achieved per mole of A T P . This latter strategy could be accomplished theoretically by increased efficiency in the mechanics of muscular contraction. This possibility is outside the scope of this paper, so only the first two possibilities w i l l be considered. The first option could be accomplished by reducing the energy contribution obtained through anaerobic metabolism. The second option could be accomplished by using carbohydrates as a fuel source rather than fat. Wi th respect to the first option, contribution of anaerobic pathways was not decreased in these experiments as there is more lactate produced under any given relative work load under acute hypoxic conditions compared to normoxic 1 5 . The body does not appear, based on this, to be increasing efficiency of energy production through the first option. The results would also tend to suggest that the second option is not utilized either. The oxidation of carbohydrates (glycogen, glucose) yields over 12% more A T P per mole of O2 compared to fat oxidation. So i f oxygen was limited it would be expected that more carbohydrate relative to fat Review of Literature 3£ would be oxidized. Based on the findings of Young et al. (no decrease in plasma fat levels), it would seem that O2 is not limited. However, these results should be interpreted cautiously. Plasma levels of any metabolite at any given time are not the best indicator of utilization. Rather, utilization is better defined as a balance between substrate production and cellular uptake. For example in the study by Young, production of free fatty acids may have been reduced (hypothetically) by the body under hypoxic conditions. If this were combined with a concomitant decrease in muscle cell uptake of an equal magnitude, plasma fatty acid levels would have remained stable yet overall utilization would have decreased. This drop in utilization would be impossible to determine from the static blood fatty acid measurement alone. This is precisely why static plasma or serum levels of a substrate are not an accurate reflection of cell utilization.To accurately determine cell utilization, kinetic tracer methods would need to be employed using labeled substrates. This method takes into consideration the more complex balance of substrate production and disappearance to give a more precise indication of utilization. The last substrate to be considered is glycogen. Muscle glycogen levels are easier to determine in one sense than plasma metabolite levels in that radioactive labels are not necessary. On the other hand, invasive muscle biopsies must be used so the technique is not without its drawbacks. A s mentioned above, i f oxygen was limited under hypoxic conditions the body would be expected to utilize the most efficient fuel to produce energy - glycogen. For this reason muscle glycogen measurements are an ideal tool to assess whether an increased dependency on carbohydrates is occurring. There have only been two studies done to date under acute hypoxic conditions looking at muscle glycogen l e v e l s 9 0 ' 1 3 6 . The study by Linnarsson et al. in 1974 found no change in muscle glycogen levels 9 0 . However, the exercise task was only a 4 minute submaximal test at a modest work rate (50% of VO2 max)-Under these conditions muscle glycogen utilization would only be minimal, and an effect of hypoxia would be difficult to discern 1 3 4 . Review of Literature 55 The other study, by Young et a l . 1 3 6 , reported that there was no significant difference in muscle glycogen utilization between acute hypoxic conditions and normoxic conditions. They did find however that there was a significant sparing of glycogen with chronic hypoxic exposure i.e after acclimatization had occurred. This finding is difficult to accept i f their results are closely scrutinized. Their data appear to show that there was significantly more glycogen sparing occurring under acute conditions than chronic. Little mention was made of this effect in their paper, as it seems their main objective was to study substrate utilization under chronic conditions. This discrepancy may have resulted because there was less muscle glycogen to begin with before exercise under chronic conditions. On the basis of this study, it seems more research is justified into the effects of acute hypoxia on muscle glycogen utilization. Hypoxia and Hormonal Responses Catecholamines (norepinephrine, epinephrine) are known to have a strong influence in the various metabolic pathways pertinent to substrate m e t a b o l i s m 1 3 4 . For example, both norepinephrine and epinephrine are known to accelerate muscle glycogenolysis, as wel l as lipolysis. For this reason, an understanding of any alterations in catecholamine levels in response to hypoxia would prove useful in explaining any changes seen in substrate metabolism. The argument used above, that substrate measurements using the same absolute work rates are not valid, also applies to catecholamine responses. If relatively more work is done at altitude, there would be a proportionately higher release of catecholamines for a given exercise intensity. This effect would apply only for work rates greater than 50% of VO2 m a x ; W e l c h 1 2 6 , and Galbo et al 4 5 point out that enhanced catecholamine release is usually only seen at work intensities above this point. Table 1 shows nine studies that have looked at catecholamine responses. O f these, six were done using absolute work rates and half of these found increases in plasma catecholamine levels. The remaining three found no change, however the exercise intensity used in these studies were all very l o w 1 5 ' 1 9 ' 9 6 . Review of Literature 36 More pertinent for our use, are the studies looking at catecholamines that used the same relative work rates. Only three studies in Table 1 did s o 1 4 ' 1 5 ' 4 1 , two of these found no change in catecholamine response 1 4 , 1 5 while the other found a significant increase in plasma catecholamine levels 4 1 . Clearly, more work needs to be done to define the exercise catecholamine response under acute hypoxic conditions. The hormones insulin and glucagon also obviously impact on substrate utilization, so it would prove helpful to understand i f there are any alterations in these hormones in response to hypoxia. A s Table 1 shows, there is a paucity of studies looking at this relationship. A s with the other parameters discussed above, it would seem that further study is warranted to try and define this relationship. If the levels of these hormones are affected by hypoxia, this may prove useful in predicting alterations in substrate metabolism. Summary There have not been a large number of studies undertaken to look at the effects of acute hypoxic exposure on substrate utilization. Virtually all of these are shown in Table 1. Most of these studies have used an F1O2 of 14% or less, which would be equivalent to an altitude of approximately 14,000 feet. Analysis of these studies to try and understand how acute hypoxia affects substrate utilization proves difficult for the following reasons: • the majority of these studies used the same absolute work loads under normoxic and hypoxic conditions. • of those few studies that measured R E R , either the work rates were very low and no change was seen, or the work rates were very high such that the measurements were made under non-steady state conditions. • the results based on actual substrate measurements have been conflicting between studies. On the basis of these findings, it would seem that further investigation in this area is justified. In particular, to the author's knowledge, no studies have been done looking at highly trained subjects under conditions which they may face in competition i.e moderate altitude. For this reason, as well as those mentioned above, the present study was undertaken. Experimental Design and Methods 37 Subjects Seven healthy, consenting, male cyclists comprised the study population. Due to a cervical vertebral fracture from a cycling accident, one of the subjects was unable to complete the study in its entirety. None of the data from this subject was included in the final results. Subjects were re-cruited on a volunteer basis from posters placed in local bicycle shops and sports venues. Criteria for participation included no previous physical or metabolic ailments such as cardiovascular, respiratory problems or diabetes. Additionally, subjects were required to achieve a maximal oxygen consumption (VO2 max) °f ^ 60 ml O2 • k g - 1 • m i n - 1 . A l l subjects were sea level natives, and had not been to altitude within the past six months. A l l subjects were informed of the nature and risks of the study both by a qualified physician as well as through a consent form which was signed by the subject. The experiment was approved by the University of Bri t ish Columbia C l in i ca l Screening Committee for Research and Other Studies Involving Human Subjects (Appendix 1). A l l subjects were discouraged from participating in heavy exercise for a period of 48 hours prior to each phase of the study and asked to undertake no exercise in the 24 hours prior to each exercise test. Additionally, subjects were asked to record their exercise activity during this 48 hour period. Research Design A two factor within-subjects design was employed with repeated measures across both factors. The independent variables were the F1O2 (21% or 16%) and exercise time. The dependent variables directly related to substrate use were muscle glycogen, plasma glucose, plasma lactate and respiratory Exchange Ratio (RER). In addition to these variables, physiological data were also collected such as minute ventilation (VE), oxygen consumption (VO2), carbon dioxide production ( V C 0 2 ) , respiratory exchange ratio (RER) , ventilatory equivalent ( V E / V 0 2 ) , heart rate, power output, cumulative work, and arterial oxygen saturation (Sao2)-The study proceeded in two phases: (1) a pre-test phase in which the VO2 max of e a c n subject was determined under both normoxic (sea level) and hypoxic (simulated altitude) conditions, (2) a Experimental Design and Methods 38_ test phase in which each subject performed 75 minute bicycle ergometer rides on a Mijnhardt K E M - 3 (Mijnhardt, Holland) electronically braked bicycle ergometer under normoxic and hypoxic conditions. A l l phases of the experiment took place in the Exercise Physiology Lab located in the Al lan McGav in Sports Medicine Centre at the University of British Columbia. (1) Pre-test phase - VO2 max assessment The pretest V o 2 max measurements were necessary to establish the exercise intensity to use in the test phase. V O 2 max w a s determined using a continuous incremental protocol on a Mijnhardt K E M -3 electronically braked bicycle ergometer (Mijnhardt, Holland). Subjects breathed through a sealed face mask connected to a precalibrated metabolic cart. Oxygen utilization was monitored with a Beckman L B - 2 Analyzer, (Beckman Instruments, Fullerton, C A ) . Carbon dioxide production was monitored with a SA-3 oxygen analyzer (Applied Electrochemistry, Sunnyvale, C A ) . Gas volumes were monitored with an air flow meter (Vacumetrics Mode l #17150, Ventura, C A ) The gas monitors were interfaced to a DOS-based personal computer equipped with data collection software (Rayfield Equipment Ltd. , Wilton, V A ) . Data were collected at 15 second intervals. Prior to the onset of exercise, subjects "acclimatized" for 10 minutes on the bicycle breathing either ambient room air (approximately 21% O 2 ) or hypoxic air (16% 0 2 balance N 2 ) through the face mask depending on which trial was being undertaken. During the simulated altitude test, hypoxic gas was supplied from a 7780 liter " T " cylinder (Medigas, Vancouver, B C ) . Hypoxic air was passed through a humidifying chamber prior to entering the subject's mask to prevent airway dessication during the exercise bout. The order of the test conditions (hypoxia or normoxia) was randomly assigned to prevent any sequencing effects from occurring. The VO^'max tests in phase 1 occurred no sooner than 48 hours apart. The 75 minute bike rides in phase 2 also did not occur within 48 hours of each other to prevent any possible sequencing effects. From the maximal exercise data, ventilatory thresholds for each subject were calculated. Based on the calculated ventilatory thresholds, a V O 2 level of 65% of the respective V O 2 max measured under each condition was chosen for each subject to ride at in the test phase described below. Experimental Design and Methods 39 Therefore subjects would be exercising at the same relative exercise intensity under sea level (SL) and simulated altitude (ALT) . This VO2 level was chosen as it represented a submaximal exercise level which was below the ventilatory threshold for al l subjects. Exercise intensity was intentionally kept below each subject's ventilatory threshold under each condition so that respiratory exchange ratio measurements could be made under steady state conditions. Beyond the ventilatory threshold relatively greater amounts of CO2 are produced relative to O2 consumed and R E R measurements are a less accurate indication of substrate utilization. (2) Dietary Intervention A number of studies have shown that the level of glycogen in pre-exercise muscle is an important factor in regulating subsequent glycogenolysis during e x e r c i s e 4 6 ' 5 4 ' 5 5 , 1 0 5 . These investigators, looking at both isolated rat muscle as well as human leg muscle have found that higher initial muscle glycogen levels result in an enhanced rate of glycogenolysis. In an effort to control for this effect in this study, subjects were instructed to maintain a target diet of between eight and nine grams of carbohydrate per kilogram body weight during the 48 hours prior to the 75 minute bicycle ride. Subjects were supplied with information describing typical diets containing 9.0 g carbohydrate • k g - 1 to help them in their menu planning over these two days. Additionally, 48 hour diet records were taken for the two days prior to the first 75 minute exercise test. To keep dietary intake constant for both 75 minute exercise tests, subjects were reissued their 48 hour diet histories after their first 75 minute test ride and were instructed to repeat the same diet during the 48 hours prior to the final test ride. Diet records were analyzed for nutrient intake using a nutritional analysis software package (Food Processor II, E S H A Research, Salem, OR.) . B y monitoring diet and activity in the days prior to the exercise tests it was hoped that large variations in pre-exercise muscle glycogen concentrations within and between subjects could be avoided between the sea level and simulated altitude tests. Experimental Design and Methods 40_ Three hours prior to the bicycle tests, each subject was supplied with a pre-exercise liquid meal (Boost Plus Calories 235 ml , Mead Johnson, Belleville, Ont.). They were instructed to consume no further food or caffeinated beverages after this meal until after the exercise test was completed. (3) Test phase - 75 minute test rides Immediately before the start of each of the 75 minute test rides, a 25 mg (approximately) pre-exercise muscle biopsy was taken from the vastus lateralis muscle using the technique outlined by K i r b y et a l . 8 0 A brief overview of the technique follows. After sterile skin preparation, approximately 10 ml of local anesthetic (0.5% marcaine without epinephrine) was infiltrated initially subcutaneously followed by intramuscular infiltration. Once pain sensation was lost, a 1.5 cm incision was made approximately 20 cm proximal to the patella, and 5 cm anterior to the iliotibial tract. A two piece Bergstrom needle was inserted perpendicularly into the incision site to a depth of approximately 4 cm. The window was opened by withdrawing the inner cutting needle 2 cm and then reinserted to "pinch o f f the muscle biopsy. Fol lowing withdrawal of the biopsy needle, the incision site was closed with butterfly bandages and wrapped with a tensor bandage prior to exercising. A l l muscle biopsies were immediately frozen in liquid N2 and later transferred to a -20° C freezer until analysis was undertaken. There were no complications associated with the muscle biopsies. Fol lowing the muscle biopsies, an indwelling venous catheter was placed in a forearm vein for withdrawal of blood samples. Subjects then mounted the cycle ergometer and put on a sealed adult sized Rudolph face mask (Hans Rudolph Inc., Kansas City, M O ) connected to a precalibrated metabolic cart. Oxygen utilization was monitored with a Beckman L B - 2 Analyzer, (Beckman Instruments, Fullerton, C A ) . Carbon dioxide production was monitored with a S A - 3 oxygen analyzer (Applied Electrochemistry, Sunnyvale, C A ) . Gas volumes were monitored with an air flow meter (Vacumetrics Mode l #17150, Ventura, C A ) The gas monitors were interfaced to a DOS-based personal computer equipped with data collection software (Rayfield, Wilton, V A ) . Experimental Design and Methods 41 With the face mask in place, subjects "acclimatized" for 10 minutes on the bicycle prior to the onset of exercise breathing either ambient room air (approximately 21% O2) or hypoxic air (16% O2 balance N2) depending on which trial was being undertaken. Hypoxic gas was supplied from a 7780 liter " T " cylinder (Medigas, Vancouver, B C ) . Hypoxic air was passed through a humidifying chamber prior to entering the subject's mask to prevent airway dessication during the exercise bout. A n ear oximeter was also applied to each subject's right ear (Hewlett-Packard 47201A, U S A ) to record Sa02 during the exercise bout. To facilitate Sa02 measurement, a vasodilatory cream (Finalgon, Boehringer, Ingelheim) was applied to the ear pinna to increase blood flow prior to fitting the apparatus. Venous blood samples (2.5 ml) were withdrawn at 0 (rest), 1, 3, 5, 10, 15, 30, 45, 60 and 75 minutes of exercise for the purpose of assessing plasma lactate and serum glucose levels. A l l blood samples were collected into heparinized tubes with sodium fluoride as preservative. Hematocrit was measured immediately using a capillary tube centrifuge. Physiological measurements ( V E , V O 2 , V C O 2 , R E R , VE/VO2, heart rate, power output, cumulative work, Sao 2) were recorded at rest, and continuously throughout the ride at 15 second intervals. A l l of these values with the exception of heart rate, Sao 2 . and cumulative work were recorded onto Rayfield software. Heart rate was recorded with a portable heart rate monitor (Polar Vantage X L , Finland). Cumulative work and Sao 2 data were recorded onto a second DOS-based personal computer equipped with Labtech Notebook software (Laboratory Technologies, Maryland, V A ) . In addition to the aforementioned physiological measurements, ratings of perceived exertion (RPE) were also monitored and recorded after 5, 15, 45, and 75 minutes of exercise utilizing a 10 point scale as outlined by B o r g 1 3 . A t these four time points during exercise, subjects were asked to indicate how difficult they perceived their exertion level to be by pointing to a large format Borg scale. The intention during the 75 minute test rides was to keep oxygen utilization and therefore exercise intensity constant throughout the exercise bout. A s prolonged endurance exercise Experimental Design and Methods 42 progresses, the muscular efficiency of an athlete typically declines and V 0 2 gradually drifts upward. To counteract this rise, VO2 levels were monitored as the exercise bout progressed, and as VO2 gradually increased, pedal resistance was gradually decreased to keep VO2 constant. This ensured that the relative exercise intensity remained constant throughout the 75 minutes of exercise. Immediately following the termination of the bicycle ride, a post-exercise muscle biopsy (-25 mg) was taken from the vastus lateralis muscle. Post-exercise muscle biopsies were obtained through the same initial incision site using the same procedure as described above for the pre-exercise biopsies. This biopsy was used in conjunction with the pre-exercise biopsy for the purpose of assessing net glycogen utilization during the exercise bout. Substrate utilization during the exercise bout was assessed on the basis of (1) the pre- and post-exercise muscle biopsies (glycogen), (2) R E R measurements, (3) serum glucose, and (4) plasma lactate. Biochemical Analysis Blood samples were centrifuged and the resulting plasma was divided in half. One half was reserved for plasma lactate levels. The other half was treated with 1 M perchloric acid and centrifuged to yield serum samples for glucose determination. Samples were then placed in microcentrifuge tubes and flash frozen in liquid nitrogen and stored at -20° C until analysis. For serum glucose determination, frozen samples were thawed to room temperature and measured in duplicate in a Beckman Glucose Analyzer 2, utilizing Beckman Glucose Standard, and Beckman Glucose Reagent K i t #671640 (Beckman Instruments, Fullerton, C A ) . Plasma lactate was analyzed utilizing a commercially available enzymatic assay with slight modifications (Sigma Diagnostics K i t #735, St. Louis, M O ) . Samples were thawed to room temperature. Sample p H was adjusted to approximately 7 with 150 ul of Tr i s -OH buffer. To 20 ul of this buffered sample, 1 ml of lactate reagent was added and colour development was allowed to occur for 15 minutes. The absorbance of these samples was then measured in a spectrophotometer at 540 nm. (Shimadzu Experimental Design and Methods 43_ Corp., Model UV-160 , Kyoto, Japan). The spectrophotometer was precalibrated using a blank test tube containing lactate reagent only. Measured samples were compared to a control sample containing 10 ul of 4.44 mmol • H lactic acid. Muscle glycogen concentrations were determined using the technique of L o and co-workers 9 1 . This analytical technique has been found to be accurate to within ± 2% 3 8 , 9 1 . The following is a brief overview of the technique. A n y visible fat and/or connective tissue was trimmed from each sample prior to being weighed (wet weight) to the nearest 0.01 milligram. Each sample was then hydrolyzed with concentrated K O H for 30 minutes in a screw cap test tube immersed in a boiling water bath. Tubes were then cooled on ice followed by addition of 95% ethanol to precipitate digested glycogen from the alkaline solution. Samples were then centrifuged, supernatant was aspirated, and the glycogen precipitates were dissolved in 3.0 ml distilled H2O. To an aliquot of this was added 1 milliliter of 5% phenol solution, followed by 5 milliliters of 98% H2SO4. This mixture was then agitated gently in a 30° C water bath for 20 minutes. Absorbance was read from this mixture on a spectrophotometer at 490 um, using blanks comprised of 1 milliliter of distilled H2O. Muscle biopsy samples were measured in duplicate. A l l glycogen values are expressed as umol glucosyl units • gram wet weight muscle - 1 . Statistical Analysis To determine i f there were any differences between conditions, substrate and physiological data from sea level and simulated altitude was analyzed using a two-way (F1O2 x time) A N O V A for repeated measures across both factors. In this case, data were analyzed from rest (time 0) through to the end of exercise (75 minutes). Any significant F ratios were tested for statistical significance using a Newman-Keuls post hoc test. Physiological data were also analyzed over the course of the exercise bout only (time 0 excluded); overall mean values were computed over the exercise period and means were compared between conditions using Student's paired T tests. When appropriate, a one-way A N O V A (F1O2) for repeated measures across time was used to identify any time points which differed significantly during exercise within a condition. Linear regression and correlation Experimental Design and Methods 44_ analysis were done on glycogen data to see i f there were any relationships between either dietary carbohydrate intake and pre-exercise muscle glycogen concentrations or pre-exercise muscle glycogen concentration and muscle glycogen consumption. For all statistical tests employed, values were considered to be significantly different at the p < 0.05 level. Statistical analysis was done on a D O S based personal computer equipped with statistical software (Statsoft Software Version 3.0, Tulsa, O K ) . Group data are reported as mean ± SE. Results 45 Subject and Pretest Phase (VO2 m a x ) Results Descriptive physical characteristics of the 6 subjects who completed the study are shown in Table 2 as are the results of the VO2 max testing at sea level and simulated altitude. The subjects consisted of mountain bikers who were competitive at the elite level. Table 2. Subject data and VO2 max data under sea level (SL) and simulated altitude ( A L T ) conditions. Subject Age (yr) Ht (cm) Wt (kg) SL V02max (ml-kg-l -min-') ALT V02max (ml-kg-l -min-1) A VC>2niax (%) 1 27 180.3 77.2 65.96 55.72 15.5 2 28 172.8 72.8 58.11 54.12 6.9 3 33 184.1 71.7 66.57 60.81 8.7 4 27 178.4 70.1 67.30 56.78 15.6 5 22 180.2 78.3 64.74 57.15 11.7 6 26 177.8 73.7 66.74 60.11 9.9 Mean 27 178.9 74.0 64.90 57.45 ¥ 11.4 ± SE + 1 ± 1.5 ± 1.3 + 1.40 + 1.05 ± 1.5 ¥ = significantly different from SL value (P < 0.001) Test Phase Results Power Power outputs during S L and A L T exercise are shown in Figure 1 and Tables 3 and 4. Results of two-way A N O V A testing for repeated measures across condition and time revealed significant effects for condition (P = 0.003), time (P < 0.001), and for their interaction (P < 0.001) (Table 5). Post hoc testing revealed that at all time points during the exercise bout significantly more power output (P < 0.001) was generated at sea level (SL) compared to simulated altitude ( A L T ) conditions. Results 46 0 I & 1 ^ 1 1 1 1 -15 0 15 30 45 60 75 90 Time (minutes) Figure 1. Power output (mean + SE) during exercise under sea level (SL) and simulated altitude (ALT) conditions. ¥ = significantly different from SL (P < 0.001) a = significantly different from 45, 60, and 75 minute values under same condition (P < 0.002) b = significantly different from 60, and 75 minute values under same condition (P < 0.04) c= significantly different from 75 minute value under same condition (P = 0.001) d = significantly different from 75 minute value under same condition (P = 0.02) e = significantly different from 75 minute value under same condition (P = 0.003) Mean differences were largest in magnitude at 15 minutes into the exercise bout (A = 37 watts), with subsequent time points showing differences of slightly lower magnitudes which varied between 22 to 28 watts. Overall mean power output averaged over the entire exercise bout was found to be 12.5% lower with A L T versus S L conditions (182 vs 209 W respectively). This difference was significant at the P < 0.001 level (Table 5). From the 15 minute time point onwards there was a trend under both conditions for a slow progressive decline in power output which continued until the end of the exercise bout. This reflected the intentional reduction in cycle Results 47 Table 3. Physiological values for 75 minute bicycle ride under sea level (SL) conditions. Sea Level (FIQ2 = 0.21) Time (min) 0 15 30 45 60 75 X exercise Power (W) — 225 ± 3 216 + 5 • 208 b,f ± 6 204 b,c,f + 6 193 b,c ± 7 209 ± 3 Cumulative work (J) — 13862g + 251 27084 g ± 406 39759 g ± 677 52258 g ± 9 9 6 64182 g ± 1374 39429 ± 3323 VE (1-rnhr1) 13.92 g + 1.34 79.80 ± 3.17 82.14 ± 2.68 82.15 ± 3.49 81.90 + 3.61 80.09 ± 4.28 81.22 ± 1.46 VE/VO2 30.70 g ± 1.06 25.28 + 0.99 25.82 ± 1.12 25.73 ± 1.14 25.70 ± 1.03 25.22 ± 1.13 25.55 ± 0.45 V o 2 (1-min-1) 0.40 g + 0.03 3.14 g ± 0.09 3.17 a.b ± 0.08 3.17 a,b ± 0.06 3.16 a,b ± 0.11 3.15 a,b ± 0.07 3.16 ± 0.03 V C 0 2 (1-min-1) 0.32 g ± 0.02 2.92 a,f + 0.09 2.88 a,f ± 0.08 2.85 a,f + 0.07 2.78 a ± 0.08 2.68 a-d ± 0.07 2.82 ± 0.04 S a 0 2 ( % ) 97.2 + 0.3 96.0 + 0.4 96.3 + 0.5 96.3 ± 0.5 96.0 ± 0.2 96.3 ± 0.2 96.2 + 0.2 Heart rate (bpm) 64 g ± 5 146 + 5 150 + 5 153 b + 5 155 b ± 6 155 b ± 6 152 ± 2 Values are X ± SE, n = 6 a = significantly different from resting value under same condition (P < 0.001) b - significantly different from 15 minute value under same condition (P < 0.05) c = significantly different from 30 minute value under same condition (P < 0.05) d = significantly different from 45 minute values under same condition (P < 0.05) e = significantly different from 60 minute values under same condition (P < 0.05) f = significantly different from 75 minute value under same condition (P < 0.05) g = significantly different from all other values under same condition (P < 0.001) Results 48 Table 4. Physiological values for 75 minute bicycle ride under simulated altitude ( A L T ) conditions. Simulated Altitude ( F l 0 2 = 0.16) Time (min) 0 15 30 45 60 75 X Power (W) Cumulative work (J) VE (1-min-1) VE/VO 2 V o 2 (1-min-1) V c o 2 (1-min-1) Sa0 2 (%) Heart rate (bpm) 188 ¥.f 188 ¥.f 186 ¥,f 179 ¥ 170 ¥ 182 ¥ ± 6 ± 5 ± 6 ± 5 ± 7 ± 3 12078 ¥,g 23521 ¥,g 34749 ¥,g 45707 ¥,g 56233 ¥,g 34458 ¥ ± 251 ± 603 + 895 + 1188 + 1520 ± 2 9 3 2 16.59 8 77.03 77.93 77.88 75.54 t 77.27 77.13 ¥ + 1.95 ± 1.42 + 2.48 + 2.20 ± 2.25 ± 1.40 ± 0.85 44.76 ¥,g 27.30 27.25 27.10 26.85 26.76 27.05 ¥ ± 3.97 ± 0.82 ± 0.95 + 0.98 + 1.06 + 0.99 + 0.40 0.36 g 2.81 t 2.85 t 2.85 t 2.80 t 2.87 t 2.83 ¥ ± 0.02 ± 0.08 ± 0.09 ± 0.05 + 0.07 ± 0.09 ± 0.03 0.36 g 2.53 ¥ 2.51 ¥ 2.48 ¥ 2.38 ¥ 2.44 ¥ 2.47 ¥ + 0.03 ± 0.10 ± 0.11 ± 0.08 ± 0.07 ± 0.11 ± 0.04 95.5 g 83.6 ¥ 83.1 ¥ 83.3 ¥ 83.7 ¥ 82.8 ¥ 83.3 ¥ + 0.7 ± 1.5 ± 1.5 ± 1.5 ± 1.6 ± 1.6 ± 0.6 67 g 148 151 f 154 b 153 b 157 b 152 ± 4 ± 5 ± 5 ± 6 ± 5 ± 4 ± 2 Values are X ± SE, n = 6 f = significantly different from equivalent SL value (P < 0.01) t = significantly different from equivalent SL value (P < 0.005) ¥ = significantly different from equivalent SL value (P < 0.001) a = significantly different from resting value under same condition (P < 0.001) b = significantly different from 15 minute value under same condition (P < 0.05) c = significantly different from 30 minute value under same condition (P < 0.05) d = significantly different from 45 minute values under same condition (P < 0.05) e = significantly different from 60 minute values under same condition (P < 0.05) f = significantly different from 75 minute value under same condition (P < 0.05) g = significantly different from all other values under same condition (P < 0.001) Results 49 resistance employed in the study design to compensate for the anticipated upward drifts in VO2 characteristic of prolonged bouts of steady state exercise. Statistical analysis within each condition (one-way A N O V A ) revealed that the S L 15 minute power output differed significantly from the 45 (P = 0.002), 60 (P < 0.001), and 75 minute values (P < 0.001). The S L 30 minute value was found to be significantly greater than the power output at 60 (P = 0.04) and 75 minute values (P < 0.001) at sea level. The S L 45 minute power output was significantly greater than the S L 75 minute value (P < 0.001) as was the S L 60 minute value (P = 0.02). For data collected with simulated altitude, the A L T 15, 30, and 45 minute power output values were all found to be significantly greater than the power output at 75 minutes (P < 0.003). Table 5. Results of two-way A N O V A (condition x time) for repeated measures across both factors. Variable Condition F Time F (p) Interaction F (P) (P) Power 25.36 (0.003) 1066.25 (< 0.001) 8.45 (< 0.001) Cumulative work 43.70 (0.001) 2185.25 (< 0.001) 30.63 (< 0.001) R P E 0.42 (0.54) 4.84 (0.01) 0.67 (0.57) V E 2.21 (0.20) 274.34 (< 0.001) 4.15 (0.007) VE/VO2 13.05 (0.015) 18.60 (< 0.001) 8.00 (< 0.001) V 0 2 25.28 (0.004) 1003.87 (< 0.001) 14.80 (< 0.001) V C 0 2 43.17 (0.001) 695.17 (< 0.001) 12.35 (< 0.001) S a 0 2 89.77 (<0.001) 30.04 (< 0.001) 26.58 (< 0.001) Heart rate 0.44 (0.53) 937.53 (< 0.001) 0.98 (0.45) Plasma Lactate 1.93 (0.22) 9.16 (< 0.001) 0.65 (0.74) Serum Glucose 0.24 (0.65) 5.82 (0.001) 0.81 (0.57) R E R 1.52 (0.27) 4.40 (0.005) 24.10 (< 0.001) Glycogen 0.11 (0.77) 22.16 (.005) 4.19 (0.09) Results 50 Cumulative Work Figure 2 and Tables 3 and 4 show that as the exercise bout proceeded there was a linearly progressive increase in cumulative work under both sea level and simulated altitude conditions. Two-way A N O V A testing showed significant effects for condition (P = 0.001), time (P < 0.001), as well as an interaction effect (P < 0.001) (Table 5). Post hoc analysis revealed that cumulative work was greater under S L conditions than A L T conditions for all time points during exercise (P < 0.003). Overall mean accumulated work, averaged over the entire exercise bout, was found to be significantly lower by 12.6% (34458 vs 39429 J; P < 0.001). One-way A N O V A testing showed that all time points were significantly different from each other during both S L and A L T exercise. This is due to the cumulative nature of this measure as exercise progressed such that each measure was continually accruing during the exercise bout. 70000 ,-—e— SL • ALT 0 15 30 45 60 Time (minutes) 75 90 Figure 2. Cumulative work (mean ± SE) under sea level (SL) and simulated altitude (ALT) conditions. ¥ = significantly different from SL (P < 0.001) Results 51 Ratings of Perceived Exertion (RPE) Ratings of perceived exertion were measured at 5, 15, 45, and 75 minutes into the exercise bout (Figure 3 and Table 6). A t sea level, there was an initial rapid rise in mean R P E values from the 5 to 15 minute time point measurements followed by a slower progressive rise in R P E . Under simulated altitude conditions, this fast initial rise in mean R P E values was not observed. Instead there was a slow progressive rise in mean values throughout the exercise bout at simulated altitude. Mean values for R P E appeared to be greater at sea level for all time points past 5 minutes until the end of exercise, although none of these values differed significantly between the two conditions as two-way A N O V A testing showed a significant effect only for time (P = 0.01). Both the main effect of condition as wel l as the interaction effect were non significant (P = 0.54 and P = 0.57 respectively) (Table 5). Statistical analysis within conditions (one-way A N O V A ) showed a trend towards differences in R P E between time points during exercise at sea level, but this did not reach significance (P = 0.06). Wi th simulated altitude, the R P E values at 75 minutes were found to be significantly greater than those at either 5 or 15 minutes (both P = 0.05). Table 6. Ratings of Perceived Exertion (RPE) at sea level (SL) and simulated altitude ( A L T ) , using the Borg 10-point scale. Time (min) 5 15 45 75 S L R P E 2.9 ± 0.2 3.6 ± 0.3 3.9 ± 0.5 4.1 + 0.6 A L T R P E 3.0 a ± 0.2 3.1 a ± 0.3 3.7 ± 0.4 3.9 ± 0.5 Values are X ± SE, n=6 No significant differences found between conditions a = significantly different than ALT 75 minute value (P = 0.05) Results 52 ft-ps 4.5 n c o 9 SL - - -o- - - ALT - - f a 0 15 30  45 60 Time (minutes) 75 90 Figure 3. Ratings of perceived exertion (Mean ± SE) during exercise under sea level (SL) and simulated altitude ( A L T ) conditions. No points differ significantly between conditions a = significantly different from 15 minute value under same condition (P = 0.05) Minute Ventilation (VE) V E values are shown in Figure 4 and Tables 3 and 4. A t rest, mean V E values A L T were greater than mean S L values (13.92 ± 1.34 vs 16.59 ± 1.95 1-min - 1). Wi th the onset of exercise, mean values for minute ventilation under both conditions showed a rapid initial rise which plateaued within the first 15 minutes of exercise. Mean values from 15 minutes onwards until the end of exercise at sea level ranged from 79.80 ± 3.17 to 82.15 ± 3.49 1-min - 1. Equivalent values under simulated altitude conditions were slightly lower and ranged from 75.54 ± 2.25 to 77.93 ± 2.48 1-min - 1. Two-way A N O V A testing revealed there was no significant effect for condition (P = 0.20) although there was a significant main effect of time (P < 0.001) and a significant interaction effect (P = 0.007) (Table 5). Post hoc testing revealed that V E at 60 minutes was significantly different between conditions (P = 0.005). There were no other significant differences in V E values between sea level and simulated altitude conditions. Results 53 0 I i i i I I I I -15 0 15 30 45 60 75 90 Time (minutes) Figure 4. Ventilation (mean ± SE) during rest and exercise under sea level (SL) and simulated altitude (ALT) conditions. $ = significantly different from SL (P = 0.005) a = SL rest value significantly different from all other SL values (P < 0.001) b = ALT rest value significantly different trom all other ALT values (P < 0.001) Mean V E averaged over the exercise bout only (time 0 not included) was found to be significantly lower at A L T vs S L by 5.0% (77.13 vs 81.22 1-min"1; P < 0.001). One way A N O V A testing revealed that under both S L and A L T conditions, resting values were significantly lower than all other time points under the same condition. There were no other significant differences found within conditions. Ventilatory Equivalent (VE/VO2) Resting VE/VO2 values were greater than exercise values under both conditions (Figure 5 and Tables 3 and 4). Wi th the onset of exercise, VE/VO2 values dropped substantially under sea level conditions and even more markedly under simulated altitude conditions. Values then levelled off and remained constant for the duration of the exercise bout, with simulated altitude values remaining slightly greater than sea level values at all time points. Statistical analysis (Table 5) Results 54 revealed significant main effects for both condition (P = 0.015) and time (P < 0.001). The interaction effect was also significant (P < 0.001). Post hoc analysis revealed that VE/Vo2 was significantly greater at rest with simulated altitude versus sea level. There were no other significant differences between conditions. Analysis of mean VE/VO2 calculated over the exercise period only revealed that mean VE/VO2 was significantly greater (P < 0.001) at A L T compared to S L (27.05 vs 25.55 respectively). This difference is equivalent to a 5.9% increase in VE/VO2 at A L T . One-way A N O V A testing revealed that the resting sea level value was significantly different from all other sea level values (P < 0.001). There was a similar finding for the resting simulated altitude value compared with all other simulated altitude values (P < 0.001). 50 r-45 h- i ¥, a 40 h -e- SL O > 35 - - -D- - - ALT > 20 -15 0 15 30 45 Time (minutes) 60 75 90 Figure 5. Ventilatory equivalents (mean + SE) during rest and exercise under sea level (SL) and simulated altitude (ALT) conditions. ¥ = significantly different from SL (P < 0.001) a = significantly different than all other values under the same condition (P < 0.001) Results 55 Oxygen Utilization (VO2) There were no significant differences between mean VO2 values at rest under each condition (Figure 6 and Tables 3 and 4). With the onset of exercise there was a rapid initial increase in mean VO2 values under both conditions. Values plateaued and remained stable for the duration of the exercise bout under each condition. e S 6 > 3.5 3.0 2.5 -2.0 -1.5 1.0 0.5 0.0 -15 • -a SL -ALT 15 30 45 60 Time (minutes) 75 90 Figure 6. Oxygen utilization (mean ± SE) during rest and exercise under sea level (SL) and simulated altitude ( A L T ) conditions. t = significantly different from SL (P < 0.005) a = significantly different from all other values under same condition (P < 0.001) Two-way A N O V A testing revealed significant main effects for condition (P = 0.004), and time (P < 0.001), as well for their interaction (P < 0.001). Post hoc testing showed that mean VO2 values under sea level conditions remained significantly greater than simulated altitude conditions for all time points beyond rest (P < 0.005) (Table 5). Calculation of Vo 2 over the exercise period only (excluding time 0) revealed that VO2 was significantly lower at A L T by 9.5% (2.83 vs 3.16 1-min - 1 ; P < 0.001). Oxygen utilization values at sea level at both rest and 15 minutes were found to be significantly different from all other sea level values (P < 0.001). Wi th simulated altitude, VO2 values at rest Results 56 were significantly lower than all other simulated altitude values (P < 0.001). There were no other significant differences between A L T values. Carbon Dioxide Production (VCO2) Mean values for carbon dioxide production (Tables 3 and 4) showed a similar overall pattern to that seen for oxygen utilization. There was an initial rapid increase in values with the onset of exercise, followed by a plateauing of values. Values then remained constant until the termination of exercise. Two-way A N O V A testing revealed a significant main effect for condition (P = 0.001), for time (P < 0.001), as well as a significant interaction effect (P < 0001) (Table 5). Post hoc testing showed that for all time points beyond rest, simulated altitude values were significantly lower than equivalent sea level values (P < 0.001). There was no significant difference between conditions at rest. Analysis of over the exercise period only (excluding time 0) revealed that VCO2 was significantly lower at A L T by 12.4% (2.47 vs 2.82 1-min"1; P < 0.001). Under sea level conditions, resting VCO2 values were significantly lower than all other VCO2 values at sea level (P < 0.001) (Table 3). Values at 15, 30, and 45 minutes were found to be significantly different from resting (P < 0.001), as well as 75 minute sea level values (P < 0.05). Additionally, VCO2 at the 75 minute time point was significantly different from rest (P < 0.001), 15 (P = 0.006), 30 (P = 0.01), and 45 minute V c o 2 values (P = 0.05). Wi th simulated altitude, resting VCO2 values were significantly lower than all other V C 0 2 values throughout the exercise bout (P < 0.001). A l l other simulated altitude time points were not significantly different from each other. Arterial Oxygen Saturation (Sac>2) Mean Sao 2 values are shown in Figure 7 and Tables 3 and 4. Wi th the onset of exercise, Sao 2 values dropped slightly at sea level while there was a much more marked drop with simulated altitude. Values then plateaued and remained stable for the duration of the exercise bout under both conditions. Statistical analysis revealed a significant effect for condition (P < 0.001), time (P < Results 57 0.001) as well as the interaction of these factors (P < 0.001) (Table 5). Post hoc analysis revealed that mean Sao 2 measurements during simulated altitude were significantly lower than equivalent sea level measures (P < 0.001). Resting values under each condition (97.2 ± 0.3 vs 95.5 ± 0.7 respectively) were not significantly different from each other. A comparison of overall mean exercise values also revealed that A L T values were significantly lower at A L T vs S L (83.3 vs 96.2%). This represents a 13.4% difference between conditions, and was found to be significant at the P < 0.001 level. Analysis of values within conditions revealed that there were no significant differences between any of the Sao 2 measurements at sea level. Wi th simulated altitude, mean Sao 2 measurements during exercise were found to be significantly lower the mean value at rest (P < 0.001). s JO CS u 3 a 100 96 92 84 80 -15 SL ALT _ L 15 30 45 60 Time (minutes) 75 90 Figure 7. Arterial oxygen saturation (mean ± SE) during rest and exercise under sea level (SL) and simulated altitude ( A L T ) conditions. ¥ = significantly different from SL (P < 0.001) a = significantly different from all other values under same condition (P < 0.001) Heart Rate Figure 8 and Tables 3 and 4 show the heart rate data under each condition. Wi th the onset of exercise, mean heart rates jumped markedly and tended to drift very slowly upwards throughout Results 58 the exercise bout under both conditions. Statistical analysis revealed that the only significant effect was for time (P < 0.001). Both the effect for condition and interaction were non significant (P = 0.53 and 0.45 respectively) (Table 5). Analysis of mean heart rates during the exercise period only (time 0 excluded) also revealed no differences between conditions. 170 150 I 130 ' 0> « 110 u S 90 70 50 -15 0 15 30 45 60 75 90 Time (minutes) Figure 8. Heart rates (mean ± SE) at rest and during exercise under sea level (SL) and simulated altitude (ALT) conditions. No points differ significantly between conditions a = significantly different from all other time points under same condition (P < 0.001) b = significantly different than SL 45, 60, and 75 minute values (P < 0.02) c = significantly different than ALT 45 and 75 minute values (P < 0.01) d = significantly different than ALT 75 minute value (P = 0.02) Statistical analysis within conditions revealed that under sea level conditions, resting H R values were significantly lower than all other sea level H R values during exercise (P < 0.001). Sea level heart rate values at 15 minutes were significantly lower than at 45 (P = 0.02), 60 (P = 0.0005), and 75 minutes (P = 0.003) of exercise. Resting heart rate values with simulated altitude were also found to be significantly lower than all other exercise values (P < 0.001). Heart rate values at A L T 15 were also significantly lower than at A L T 45 (P = 0.01), 60 (P = 0.03), and 75 minutes (P < Results 59 0.001). Heart rate values at A L T 30 were also significantly lower than H R values at A L T 75 (P = 0.02). Dietary Data The target area for carbohydrate intake was 8.0 - 9.0 g-kg body we igh t 1 . Table 7 shows that despite detailed individual instructions (verbal and written) regarding the target carbohydrate intake and how to achieve it, subjects 2 and 3 were unable to achieve this level, although they came close consuming 7.4 and 7.7 g carbohydrate-kg body weight - 1 respectively. Also shown in Table 7 are the mean daily protein and fat intakes (expressed as percentages of total daily caloric intake) for the two days prior to the exercise bouts. Table 7. Average daily dietary intake (based on diet history recorded 48 hours prior to exercise bout). Subject Calories Calories Carbohydrate Protein Fat (kcal) (kcal-kg - 1) (%) (gm-kg - 1) (%) (%) 1 4376 56.7 70.5 9.8 12.5 17.5 2 4490 61.7 47.5 7.4 19.0 33.5 3 3544 49.4 62.0 7.7 12.5 25.5 4 3074 43.9 69.0 8.1 10.5 20.5 5 3960 50.6 69.5 8.9 11.5 19.5 6 4510 61.2 65.0 10.0 11.0 24.0 Mean 3992 53.9 63.9 8.7 12.8 23.4 ± S E + 239 ± 2 . 9 ± 3 . 5 ± 0 . 4 ± 1 . 3 ± 2 . 3 Results 60 Lactate Values for plasma lactate during exercise under each condition are shown below in Table 8 and Figure 9. Results of the two-way A N O V A are shown in Table 5. There was a significant effect found for time (P < 0.001) while the main effect for condition was found to be nonsignificant (P = 0.22), as was the interaction effect (P = 0.74). Table 8. Venous plasma lactate values (mmol-1"1) under sea level (SL) and simulated altitude (ALT) conditions. Time (min) S L A L T 0 0.49 + 0.07 0.49 ± 0.09 1 0.43 ± 0.05 0.42 ± 0.14 § 3 0.85 ± 0 . 1 0 a 0.83 ± 0.34 § 5 1.20 ± 0 . 1 4 b 1.14 ± 0.24 c 10 0.82 ± 0.13 a 1.01 ± 0.16 c 15 0.68 + 0.14 0.85 ± 0 . 1 8 30 0.65 ± 0.09 0.76 ±0 .11 45 0.62 ± 0.09 0.73 ± 0.07 60 0.61 ± 0.09 0.70 ± 0.07 75 0.72 ± 0.13 0.92 ± 0.07 d Values are X ± SE, n=6 except § (n=5) No significant differences found between conditions a = significantly different from SL 0 and 1 minute values (P < 0.05) b = significantly different from all other SL values (P < 0.05) c = significantly different from ALT 0 and 1 minute values (P < 0.05) d = significantly different from ALT 1 minute value (P < 0.05) One way A N O V A testing revealed that at S L , resting lactate values were significantly lower than the 3 minute (P = 0.02), 5 minute (P = 0.04), and 10 minute (P < 0.001) values under the same condition. Values at 1 minute were also found to be significantly lower than the 3 (P = 0.003), 5 (P < 0.001), and 10 minute (P = 0.009) values. Lactate values peaked at 5 minutes at sea level. This time point was significantly greater than all other sea level time points (P < 0.001). Results 61 With simulated altitude, values at rest were significantly lower than values measured at 5 (P = 0.005) and 10 minutes (P < 0.04). Similarly, 1 minute values were also significantly lower than the 5 (P = 0.001) and 10 minute (P = 0.01) values.The 1 minute time point was also found to be significantly lower than the 75 minute time point at altitude (P < 0.05). 0.20 I 1 1 1 1 1 1 1 -15 0 15 30 45 60 75 90 Time (minutes) Figure 9. Plasma lactate values (mean ± SE) during rest and exercise under sea level (SL) and simulated altitude (ALT) conditions. No points differ significantly between conditions Under SL conditions: the 3, 5, and 10 minute values are significantly greater than rest (P < 0.05) and 1 minute (P < 0.05) values Under ALT conditions: the 5 and 10 minute values are significantly greater than rest (P < 0.04) and 1 minute (P < 0.01) values. The 75 minute value is significantly greater than the 1 minute value (P < 0.05) Due to difficulties with blood withdrawal at the beginning of the exercise bout in subject 4, there were only 5 plasma lactate samples at 1 and 3 minutes under simulated altitude conditions rather than the 6 samples which comprise all other time points. 40 e SL - - -a- - - ALT Glucose Venous plasma glucose values are shown below in Table 9 and Figure 10. There was an initial Results 62 Table 9. Venous serum glucose values (mmol-1" 1) for sea level (SL) and simulated altitude (ALT) conditions. Time (min) S L A L T 0 4.22 ± 0.17 4.04 + 0.10 15 3.73 ± 0.29 4.03 ± 0 . 1 3 30 4.14 + 0.13 4.28 ± 0.29 45 4.29 ± 0.14 4.42 ± 0.37 60 4.44 ± 0.21 a 4.72 ± 0.34 b 75 4.18 + 0.27 4.07 ± 0.26 Values are X ± SE, n=6 No significant differences found between conditions a = significantly greater than SL 15 minute value (P = 0.02) b = significantly greater than ALT 0, 15, and 75 minute values (P < 0.05) 5.2 1 4.4 £ N—•> | 4.0 o s 5 3.6 3.2 -15 0 15 30 45 60 75 90 Time (minutes) Figure 10. Serum glucose values (mean ± SE) at rest and during exercise under sea level (SL) and simulated altitude ( A L T ) conditions. No points differ significantly between conditions a = significantly different from 15 minute value (P = 0.02) b - significantly different from rest, 15, and 75 minute values (P < 0.05) drop in serum glucose under sea level conditions which reached a nadir of 3.73 m m o l l " 1 at 15 minutes. Serum glucose values then gradually increased through the following 45 minutes to peak Results 63 at 60 minutes. In the last 15 minutes of exercise at sea level, mean plasma glucose values decreased to 4.18 ± 0.27 m m o l - H . Values under simulated altitude conditions did not show the initial drop seen in the first 15 minutes under sea level conditions. Otherwise, the general trend at simulated altitude was similar to that seen at sea level. Results of the two-way A N O V A are shown in Table 5. There were no significant differences found for the effect of condition or interaction effects between the two conditions (P = 0.65 and P = 0.55 respectively). There was a significant main effect for time (P = 0.001). One-way A N O V A testing revealed that plasma glucose levels at 60 minutes into exercise were significantly greater than the 15 minute value at sea level (P = 0.02). Under simulated altitude conditions, values at 60 minutes were significantly greater than values at rest (P = 0.03), 15 (P = 0.04), and 75 minute (P = 0.03) values. Respiratory Exchange Ratios Respiratory exchange ratio (RER) values are shown in Table 10. Figure 11 shows the general pattern of R E R values under sea level and simulated conditions. Table 10. Respiratory Exchange Ratio (RER) values for sea level (SL) and simulated altitude (ALT) conditions. Time (min) S L A L T 0 0.80 ± 0.02 a 1.03 ± 0.06 ¥,e 15 0.93 + 0.01 b 0.90 ± 0.01 30 0.91 ± 0.01 c 0.88 ± 0.01 45 0.90 ±0 .01 d 0.87 ± 0.01 60 0.88 ±0.01 d 0.85 ± 0.01 75 0.85 ± 0.01 a 0.85 ± 0.01 Values are X ± SE, n=6 ¥ = significantly greater than SL rest value (P < 0.001) a = significantly different from all other SL values (P < 0.001) b = significantly greater than SL 0, 45, 60, and 75 minute values (P < 0.02) c = significantly greater than SL 0 and 75 minute values (P < 0.001) d = significantly different than SL 0, 15, and 75 minute values (P < 0.02) e = significantly greater than all other ALT values (P < 0.001) Results 64 Two-way A N O V A evaluation showed significant effects for time (P = 0.005) as wel l as the interaction of condition and time (P < 0.001). The effect of condition alone was not significant (P = 0.27) (Table 5). Figure 11 shows the wide disparity between resting R E R measurements at sea level (0.80 ± 0.02) versus altitude (1.03 ± 0.06) suggesting that there was a greater degree of hyperventilation prior to exercise under simulated altitude conditions. Post hoc testing showed that this was the only significant difference among R E R measurements between S L and A L T (P < 0.001). Significant differences in R E R measurements within conditions (one-way A N O V A testing) are shown graphically in Figure 11. 1.10 1.05 1.00 « 0.90 0.85 0.80 0.75 -15 0 15 30 45 60 75 90 Time (minutes) Figure 11. Respiratory exchange ratios (RER) (mean ± SE) at rest and during exercise under sea level (SL) and simulated altitude ( A L T ) conditions. ¥ = significantly different from SL (P < 0.001) a = significantly different than all other values under same condition (PS 0.001) b = significantly different than all other SL values ( P < 0.01) c = significantly different than 0, 45, 60, and 75 minute values (P < 0.02) d = significantly different than 0 and 75 minute values (P < 0.001) e = significantly different than 0, 15, and 75 minute values (P < 0.02) Results 65 Glycogen Mean pre- and post-exercise muscle glycogen concentrations for each condition are shown in Figure 12. Glycogen utilization under sea level and simulated altitude conditions are shown in Table 11. Pre-exercise muscle glycogen concentrations for both conditions ranged from 57.95 to 141.88 umol glucosyl units • gram wet weight muscle tissue - 1. This corresponded to a mean value of 93.29 ± 6 . 8 2 u m o l - g - 1 . Table 11. Pre- and post-exercise muscle glycogen concentrations, and glycogen utilization (A Glycogen) under sea level (SL) and simulated altitude (ALT) conditions. Subject SL Pre-[ Glycogen] * SL Post-[ Glycogen] SL A Glycogen A L T Pre-[ Glycogen] A L T Post-[ Glycogen] A L T A Glycogen 1 112.74 88.92 23.82 97.31 87.38 9.93 2 57.95 17.70 40.25 66.33 56.92 9.41 3 76.26 43.12 33.14 101.41 50.20 51.21 4 141.88 64.48 77.40 90.41 35.13 55.28 5 111.12 83.40 27.72 90.82 76.68 14.14 6 68.85 12.19 56.66 104.45 71.44 33.01 Mean 94.80 51.64 a 43.17 91.79 62.96 b 28.83 ± SE ± 13.14 ± 13.35 ± 8.32 ± 5.58 ± 7.81 ± 8.50 * All glycogen values in umol glucosyl units • gram wet weight muscle tissue-1 No significant differences found between conditions for glycogen utilization (A glycogen) (P = 0.09) a = significantly different from SL pre-exercise glycogen value (P = 0.003) b = significantly different from ALT pre-exercise glycogen value (P = 0.02) A one-way A N O V A revealed that pre-exercise values differed significantly from post-exercise values at sea level (P = 0.003) and at simulated altitude (P = 0.02). Results from two-way analysis of variance (Table 5) indicate a statistically significant difference for the main effect of time (P = 0.005). There was no significant effect for condition (P = 0.77) and the interaction effect approached, but did not reach significance (P = 0.09). Results 66 Figure 12. Pre- and Post-exercise muscle glycogen concentrations (mean ± SE) under sea level and simulated altitude conditions. No significant differences were found between conditions a = significantly different from pre-exercise value (P = 0.003) b = significantly different from pre-exercise value (P = 0.02) The same relative exercise intensities were utililized under each condition during the 75 minute exercise bouts. Under simulated altitude conditions, this translates into a lower VO2 at any given exercise intensity. Table 12 shows glycogen utilization expressed as a function of oxygen consumption ( V o 2 ) . The purpose of this transformation was to see i f there were any differences in glycogen utilization between sea level and simulated altitude when oxygen consumptions was standardized across conditions. A one-way A N O V A comparing conditions revealed that there was also no significant difference between S L and A L T (P = 0.20) when the glycogen data was expressed adjusted for V 0 2 . Results 67 Table 12. Glycogen utilization expressed as a function of oxygen consumption (umol glucosyl units-gm wet wt"1 -1 Vc>2-1 ) under sea level (SL) and simulated altitude (ALT) conditions. Subject S L A L T 1 7.05 3.40 2 14.32 3.67 3 10.52 17.42 4 24.49 21.26 5 8.58 4.76 6 18.16 11.75 Mean 13.85 10.38 ± S E ± 2.69 ± 3.14 No significant differences found between conditions (P = 0.20) Linear regression and correlation analysis failed to show a significant relationship between pre-exercise glycogen levels and the amount of muscle glycogen that was utilized during exercise (r = 0.32, P = 0.18) (Figure 13). Similar analysis on the relationship of pre-exercise muscle glycogen concentration to the percentage of carbohydrate consumed in the diet (Figure 14) did find a significant correlation (r = 0.70, P = 0.01). However, this correlation was not found to be significant when dietary carbohydrate was expressed as grams • kilogram body weight" 1 (r = 0.27, P = 0.39). Although all glycogen measurements were done in duplicate, measurements were recorded only as means rather than individual values. For this reason, an inter-assay coefficient of variance could not be calculated for the data obtained. 50 75 100 125 150 Pre-exercise muscle [glycogen] (umol • g-1) Figure 13. Correlation between glycogen use during exercise and pre-exercise muscle glycogen concentrations. c M 0 u — M , .2 O 5 s 1 e u u 150 125 k •S 100 L. 75 50 y = 2.0x - 34.48, r = 0.70, P = 0.01 45 50 70 75 55 60 65 Dietary carbohydrate (% of total caloric intake) F i g u r e 14. Correlation between pre-exercise muscle glycogen concentrations and the amount of dietary carbohydrate consumed in the diet. Results 69 Summary of Results Mean maximal oxygen consumption with exercise at A L T was 11.4% lower than determined at S L . A s a result, to achieve the same relative exercise intensity at A L T as S L , resistance on the electronically braked bicycle was lowered by 12.5% from a mean value at S L of 209 W to 182 W at A L T . This resulted in a corresponding drop in several physiological variables with exercise at A L T : mean cumulative work dropped by 12.6% , mean exercise minute ventilation dropped by 5%, mean exercise V 0 2 dropped by 9.5%, mean exercise V C 0 2 dropped by 12.4%, and mean exercise S a o 2 dropped by 13.4%. A l l of these mean exercise differences were found to be significantly different at the P < 0.001 level. The only physiological variables which remained unchanged statistically between conditions were mean exercise heart rate and ratings of perceived exertion. Analysis of dietary data shows that the mean carbohydrate intake for subjects was 8.7 g-kg body weight - 1 , a level which should have been capable of maintaining optimal muscle glycogen concentrations in the subjects. A t this level of dietary carbohydrate intake, a mean pre-exercise muscle glycogen concentration of 93.29 ± 6.82 umol • g" 1 was determined for both conditions. Wi th respect to related measurements related to substrate utilization, no difference was found between S L and A L T for exercise plasma lactate or serum glucose levels. Similarly, exercise R E R measurements were also found to be not significantly different between conditions. There was a trend observed towards decreased exercise glycogen use under simulated altitude conditions however the difference did not reach statistical significance (P = 0.09). Discussion 70 Based on teleological reasoning as well as experimental evidence, it was hypothesized that exercise under simulated moderate altitude conditions would have resulted in enhanced muscle glycogen use compared to equivalent exercise undertaken at sea level. Results of the current study reveal that mean glycogen utilization was not significantly different between S L and A L T conditions. In fact, contrary to expectations there was actually a trend towards decreased glycogen utilization with A L T although the observed difference did not reach significance (P = 0.09). Experimental design - relative versus absolute exercise intensity In designing an experiment to compare muscle glycogen use with exercise at sea level versus altitude, one is faced with the problem of how to control for the lower VO2 m a x values which occur with exercise at altitude. There are two potential ways to proceed. The first possibility would be to use the same power output at A L T as S L . This would be equivalent to the same absolute exercise intensity under each condition. For example, i f 200 watts of power output is maintained at both S L and A L T , the result w i l l be roughly the same oxygen utilization (VO2) under each condition. This is shown in Figure 15 on the following page. In this diagram, line A is equivalent to the VO2 achieved at S L under a given power output, while line B represents the VO2 achieved at the same power output under A L T conditions. It can be seen that the same VO2 level at A L T is actually being achieved at a relatively greater percentage of Vo 2 max than the exercise undertaken at S L . In other words, this sets up a situation where the exercise with A L T is being done at a relatively greater intensity than that done at S L . A greater relative exercise intensity at A L T would result in roughly the same amount of total substrate consumed on a net basis. However due to the curvilinear relationship between exercise intensity and glycogen consumption 1 1 1 , a greater relative exercise intensity would result in proportionately greater glycogen consumption. This experimentally induced switch towards glycogen as fuel would make a comparison of S L and A L T glycogen consumptions difficult. Discussion 71 The second possibility for an experimental design would be to measure V o 2 m a x under each condition and have the athletes exercise at the same percentage of VO2 max found under each respective condition (lines A and A ' in Figure 15). This would be equivalent to using the same relative exercise intensity under each condition. £ 8 > SL V02 max ALT VO2 max -A B A ' --Sea Leve l A l t i t ude Figure 15. Relative versus absolute exercise intensities under altitude conditions. Line A represents a given exercise intensity (VO2 level) at sea level, line A' represents the same relative exercise intensity under altitude conditions. Line B represents the same absolute exercise intensity under altitude conditions. See text for explanation. To accomplish this, the power output at A L T would need to be reduced so that exercise VO2 is dropped proportionately to the reduction observed in A L T VO2 m a x - Such a reduction in VO2 at A L T introduces its own problem however. Although there is now no difference in relative exercise intensity between the two conditions (so that the proportion of energy coming from carbohydrate should be roughly equivalent under each condition), the lower exercise VO2 at A L T would result in an overall decrease in the amount of all substrates consumed, including glycogen. Therefore, i f this method was employed one would expect to see an overall net decrease in muscle glycogen Discussion 72 consumption with exercise at A L T . This also can make a comparison of glycogen use between conditions problematic. W h i c h method is chosen depends on what aspect of glycogen metabolism one is trying to elucidate. In the present study, the intent was to see i f reducing the amount of ambient oxygen available to the body results in alterations in substrate use by skeletal muscle. If the drop in ambient oxygen concentration at the mouth translates into a drop in oxygen availabilty at the skeletal muscle mitochondrion, it would be expected that the mitochondrion would preferentially metabolize a fuel which most efficiently allows it to maintain energy (ATP) production with decreased oxygen tensions. O f the fuels available to exercising muscle, this preferential fuel would be glycogen as it is capable of producing the largest amount of A T P per unit of oxygen consumed (for details, see review of literature). For the purposes of the present experiment, the latter method (same relative exercise intensity under each condition) was chosen for two reasons. Firstly, exercise at A L T using the same absolute intensity as at S L is equivalent to a greater stress on the body. This results in an augmented stress response with enhanced release of such hormones as catecholamines, Cortisol, and B-lipotropin. This altered hormonal milieu by itself could result in altered substrate metabolism in exercising muscle. If the experiment were carried out and a difference were found in substrate use, it would be impossible to conclude whether the difference was due to altered hormone levels or to changes in ambient oxygen levels. Secondly, previous work has shown that the principle determinant of muscle glycogen consumption during exercise is the relative ( % V 0 2 max) a ° d not absolute exercise intensity 5 9 ' i n . These authors studied subjects with different VO2 max values and found that when they exercised at the same percentage of their own VO2 max> they consumed similar amounts of glycogen. For these reasons, the present experiment was designed with the intent of exercising the athletes at the same relative rather than absolute intensities under each condition. Discussion 73 To achieve the same relative exercise intensity at A L T in this study, it was necessary to lower the exercise intensity (pedal resistance) at A L T by an average of 26 W (12.5%) to compensate for the 11.4% drop in VO2 max observed with simulated altitude conditions. A s described above, one drawback of using the same relative exercise intensity is the resulting lower exercise VO2 values at A L T . Mean VO2 values with A L T were found to be 9.5% lower than mean S L Vo 2 values. Lower exercise VO2 levels result in a concomitant reduction in substrate use, which may in part explain why mean glycogen utilization was lower under A L T conditions. In an effort to correct, for this drop in exercising Vo 2 levels at A L T , glycogen use was also expressed as a function of VO2. When expressed this way, mean glycogen use was also not significantly different between conditions. There was a trend towards decreased glycogen use with A L T with five of the six subjects displaying lower glycogen use with A L T . Perhaps i f more subjects had been used in the study, sufficient statistical power would have been achieved to show a significant difference between conditions. Variations observed in muscle glycogen concentrations Through the design of the experiment, every effort was made to control any foreseen factors which could have caused fluctuation in muscle glycogen levels. For example, diet is capable of altering muscle glycogen levels 2 ' 9 , therefore dietary carbohydrate levels were controlled in the study in an effort to maintain muscle glycogen levels at optimal levels. Studies have indicated that diets consisting of a minimum of 8 grams of carbohydrate • kilogram of body weight" 1 • d a y 1 are required to maintain optimal muscle glycogen levels in athletes training for endurance events 8 1 ' 1 0 1 . For this reason we set this as a target range for our subjects. Dietary analysis revealed a mean dietary carbohydrate intake of 8.7 gm • kg" 1 • d a y 1 in this study. The importance of maintaining similar pre-exercise muscle glycogen concentrations relates to the finding by several workers that higher pre-exercise concentrations of muscle glycogen result in increased exercise muscle glycogen consumpt ion 4 6 ' 5 4 ' 5 5 ' 1 0 4 . In order to control for this effect, an Discussion 74 effort was made to keep dietary carbohydrate intake the same for both conditions in the hope that pre-exercise muscle glycogen concentrations would remain constant. This was accomplished by returning each subject's 48 hour diet record prior to their second exercise test and asking them to repeat the same diet. With these dietary measures taken, mean pre-exercise muscle glycogen levels were very similar between S L and A L T . Although mean (group) values were similar, in two of the subjects there were marked within-subjects variations observed. In subject 4, pre-exercise glycogen levels for the S L test were 56.9% greater than pre-exercise values for the A L T test (141.88 vs 90.41 umol • g" 1 respectively). For subject 6, A L T pre-exercise glycogen values were 51.7% greater than equivalent S L values (104.45 vs 68.85 umol • g _ 1 respectively). W h y muscle glycogen levels fluctuated so markedly in these two subjects is not readily apparent. It is conceivable that dietary non-compliance was responsible for these observed differences. Forty-eight hour diet histories were recorded prior to the first exercise test. Dietary compliance prior to the second exercise test was not checked; subjects were simply reissued their diet books and asked to repeat the same diet. Therefore, i f dietary compliance was a problem, it more l ikely would have occurred prior to the the second rather than the first exercise test. However, in both subjects, the lower pre-exercise value was observed on the first test, presumably when dietary information was most accurate. This would tend to rule out dietary non-compliance as a likely source of the observed differences. Another possible explanation for the observed glycogen levels in these subjects could be the effect of training by the subjects on the days prior to their exercise test. During a typical two hour training ride at ventilatory threshold level, a trained cyclist w i l l use roughly half of their muscle glycogen stores; equivalent to approximately 75 umol glucosyl units • g" 1 muscle 3 4 . Fol lowing such exercise, glycogen is resynthesized at a rate of approximately 5-6 umol • g" 1 • h o u r 1 , provided adequate dietary carbohydrate is consumed 1 1 ' 7 4 . A t this rate, it would take 12 to 15 hours to replenish the muscle glycogen consumed during the training session. Exercise of a higher Discussion 75 intensity could deplete muscle glycogen stores to an even greater degree, and would take even longer to replenish. If the subjects undertook heavy training the day before their exercise test(s) it could have impacted on their muscle glycogen levels the day of the test. In an effort to circumvent this muscle glycogen depletion in the days and hours prior to the exercise tests, subjects were strongly encouraged to avoid strenuous exercise during this time. A l so , exercise history forms were administered in which the subjects recorded their exercise activity in detail over the 48 hours prior to each exercise test. According to these records none of the subjects undertook any heavy exercise over the 48 hours prior to their tests. Additionally, the training that was undertaken by each subject was found to be similar prior to both exercise tests. Therefore, muscle glycogen levels in each subject should have been similarly affected by training prior to their exercise tests. Providing these records were accurately filled out along with their dietary records, these interventions should have ruled out any perturbations in muscle glycogen due to diet or training. The precautions mentioned above should have controlled for potential within subject differences in muscle glycogen concentrations prior to each of their exercise tests. It seems that these measures were successful in the majority of subjects. Why subjects 4 and 6 had such wide discrepancies in pre-exercise muscle glycogen concentrations between conditions is not known. In addition to the within-subject variations mentioned above for subjects 4 and 6, there were also wide between-subject variations found in pre-exercise muscle glycogen concentrations. Mean pre-exercise muscle glycogen concentration (both conditions combined) was 93.29 + 6.82 umol • g - 1 , with values ranging from 57.95 to 141.88 umol • g" 1 . Other studies using highly trained cyclists as subjects have found higher mean glycogen concentrations in their studies. For example, a recent study by Sherman et a l . 1 1 3 in which cyclists were fed diets of 10 grams of carbohydrate (CHO) per kilogram body weight found mean pre-exercise glycogen concentrations of 186.7 ± 13.1 umol • g _ 1 . Another recent study by Rauch et a l . 1 0 3 , in which cyclists were fed diets of 10.52 g C H O • k g - 1 , found mean levels of 159 ± 9 umol • g" 1, while a study by Coyle et a l . 3 4 which had Discussion 76 subjects ingesting a "high carbohydrate diet" found mean concentrations of 135 ± 12 umol • g" 1 . The calculated coefficients of variation for these studies were 21.0, 19.6, and 25.1% respectively, while the value obtained for this measure in the present study was 17.9%. From these data it can be seen that these studies had higher mean pre-exercise muscle glycogen concentrations with a slightly greater range in values than those obtained in the present study. These studies used slightly different glycogen assays than those used in the present study but the general technique was similar. It is possible that the different assay procedures are responsible for the observed differences in mean glycogen concentrations. Other than this possibility, no other explanation can be offered for why pre-exercise muscle glycogen concentrations were lower in this study. Given the relationship described earlier between pre-exercise muscle glycogen levels and glycogen use it is unfortunate that the pre-exercise muscle glycogen levels were relatively low (despite the efforts to maximize them via control of diet and training). Higher initial values may have resulted in greater glycogen consumptions in the study, which would have in turn made it more likely that a significant relationship would be found between glycogen use and exercise with simulated altitude. Correlation analysis showed no significant relationship between pre-exercise muscle glycogen concentrations and glycogen utilization in this study. One possible reason for this could be due to the low pre-exercise glycogen levels observed in the subjects. Additionally, the small and very homogeneous subject population in this study also l ikely contributed to the failure to find a significant relationship. It is interesting to note however that despite the fact that the subjects were a homogenous group, there was still a fairly wide range observed in pre-exercise muscle glycogen levels. Another finding that merits discussion is the pattern of glycogen use in subject 3. O f the six subjects participating in the experiment, all of them with the exception of subject 3 showed decreased glycogen use with exercise under A L T conditions. This pattern was reversed in subject 3 Discussion 77 however. A t S L , this subject showed the second lowest glycogen use, while at A L T , he showed the second highest. A s mentioned, every effort was made to have all subjects reproduce all controllable factors which could impact on glycogen use as closely as possible between trials. For example, al l subjects were encouraged to drive and not cycle to the sports medicine laboratory to conserve muscle glycogen prior to the exercise tests. Subject 3 was unable to drive to his first exercise test (SL) and therefore rode his bicycle from his place of employment which was approximately 15 kilometers from the laboratory. He notified me of this the day before, and at this time he was instructed to ride to the laboratory with the lowest possible intensity in an effort to conserve muscle glycogen. For his next exercise test (ALT) this subject drove a car to the laboratory. It was thought that perhaps cyling to the lab for his S L test may have depleted muscle glycogen and driving to his A L T test may have conserved it such that the observed discrepancy in glycogen use was obtained. This explanation is rendered somewhat less likely however after an examination of the glycogen data from subject 4. This subject insisted (despite much pleading) on cycling to both of his exercise tests, explaining that he preferred to ride because it allowed him to warm up. He rode a similar distance to the lab as subject 3, and as with subject 3, he was instructed to ride at as low an intensity as possible. His data shows that he had the greatest muscle glycogen use of all the subjects under both conditions. Given this finding it seems that for this subject, this amount of exercise had minimal impact on muscle glycogen stores. It is possible that subject 3 rode to the lab at a higher intensity however and this resulted in more pronounced muscle glycogen depletion. This may have been responsible for the unusual pattern of muscle glycogen use observed in this subject. Other than this difference in modes of transport to the laboratory, no other explanation can be offered for the difference in muscle glycogen use patterns observed in subject 3. One other finding deserves comment with respect to subject 3 related to the size of the muscle biopsies obtained from him. Of the 24 muscle biopsies taken in the study, the range in biopsy sizes was 12.7 - 38.9 milligrams which corresponded to a mean value of 24.0 ± 1.7 mg. It is interesting Discussion 78 to note that of these biopsies, those obtained from subject 3 at S L were the two smallest at 12.7 (pre-exercise) and 14.7 mg (post-exercise). Originally it was thought that perhaps there may have been a correlation between the small biopsy sizes and the resulting glycogen measurements. However, an examination of the muscle biopsy data revealed no pattern between biopsy size and glycogen values obtained. It is l ikely that the small biopsies from subject 3 and the unusual glycogen findings associated with them are nothing more than a coincidence. In addition to subject-induced sources of variation in muscle glycogen concentrations described above (diet and exercise), experiment-induced variation can also occur via the techniques employed to obtain and measure muscle glycogen. For example, the needle biopsy technique itself is indirectly capable of influencing muscle glycogen determination. During a needle biopsy, approximately 300 - 600 muscle fibers are removed. Within such a biopsy, muscle fiber types seem to be homogeneously distributed, however whether fiber types are the same between two separate needle biopsies in the same individual taken from the same site is equivocal 1 2 . Some studies have failed to show any variation in muscle fiber type distribution between duplicate biopsies from the same s i te 9 3 , while other studies have shown up to a 10% var i a t ion 1 2 1 . If different samples of muscle fibers were obtained from two sequential biopsies it could in theory affect the determination of glycogen consumption. Work by Lexel l et al. found that due to the large variation in the proportion of the fiber types within the muscle, a single needle biopsy is a poor estimator of the proportion of the fiber types for the whole musc le 8 8 ' 8 9 . These workers arrived at their conclusions by analyzing cross sections of whole human muscle. Given this finding, it is not surprising to find large discrepancies in the results of various studies when using needle muscle biopsies to look at muscle fiber type distributions in human skeletal muscle. These studies usually ascribe the wide variation in values obtained to natural variation between subjects. However given the findings of the study by Lexel l et al. mentioned above, it is entirely likely that the reason for the variation is due at least in part to the muscle biopsy technique itself rather than intersubject variation. Discussion 79 The fibers comprising skeletal muscles display a spectrum of metabolic activities. Based on these activities, muscle fibers have been arbitrarily subdivided into three types; type 1 (oxidative) fibers, type 2a (oxidative-glycolytic) fibers, and type 2b (glycolytic) fibers. Mitochondrial enzyme levels are approximately twice as high in the type 1 versus type 2 fibers conferring these fibers with an enhanced ability to oxidatively metabolize fuel 6 9 . During exercise intensities such as those used in this study, it would be expected that type 1 muscle fibers would be supplying the majority of energy via oxidative metabolism 6 . It would be from these fibers therefore that the greatest glycogen depletion would be expected. In theory, a difference in muscle fiber type between two biopsies from the same site could have a significant impact on muscle glycogen determination. Type 2 fibers have been reported to store up to 17% more glycogen than type 1 f i b e r s 1 2 2 (again it should be pointed out that this is equivocal, another often quoted study by Essen et al. found no difference in glycogen storage between fiber types although this conclusion was reached by studying only one subject 4 2 )! Assuming type 2 fibers do store more glycogen, the combination of a pre-exercise biopsy containing relatively more type 2 fibers with a post exercise biopsy from the same site containing relatively more type 1 fibers (more glycogen consumed) would result in the appearance that a greater amount of glycogen was consumed relative to another subject in which the order of these pre- and post-exercise muscle biopsy fiber type findings was reversed. Such variation could have been responsible, at least in part for the range of glycogen consumptions found within subjects in this study. Between subject variations in muscle glycogen levels observed in this study may also be in part due to the needle biopsy technique. For example, a study by Mahon and workers using the needle biopsy technique found a coefficient of variation of approximately 35% for the frequency of occurrence of muscle fiber types from biopsies coming from different individuals 9 3 . There is really no way of knowing how much of this variation is due to normal intersubject variation and how much is due to the needle biopsy technique. Applying a similar argument as that used above Discussion 80 correlating fiber type to glycogen use, it is possible that variations in muscle fiber samples from one needle biopsy to another could lead to variations in muscle glycogen determinations and therefore ultimately to variations in the level of glycogen consumed. This could be responsible for some of the observed variation in glycogen consumption found between subjects in this study. Another source of variation in the findings of this study is the glycogen assay itself. Published values for this technique indicate that the method is accurate to within + 2% 3 8 , 9 1 . Therefore the biochemical assay itself was likely a minor source of the variation observed in muscle glycogen determinations. A s mentioned in the results section, glycogen measurements were done in duplicate but unfortunately measurements were recorded only as means rather than individual values. For this reason, an inter-assay coefficient of variation could not be calculated or reported for the data obtained. With all of these potential sources of variation in glycogen determination (diet, exercise, biopsy technique, and assay), it is likely that six subjects was not a sufficient population to discriminate a statistically significant difference. The study revealed that there was a trend (P = 0.09) towards a decrease in muscle glycogen use with exercise under simulated altitude conditions. Had more subjects been used, it would have increased statistical power and increased the l ikelihood of obtaining a significant difference. Potential improvements to the experimental design Another way to maximize the l ikelihood of finding a significant difference in glycogen utilizations between conditions would have been to increase the intensity of exercise. There is a semilogarithmic relationship between exercise intensity and glycogen u s e 1 1 1 . In this study, subjects exercised at 65% of their condition specific VO2 m a x - This level was chosen intentionally to maintain each subject below their ventilatory threshold so that during exercise, their gas exchange values would be in a steady state. It was decided that this would maximize the accuracy of R E R measurements. However, at exercise intensities beyond 65% of VO2 m ax> progressively Discussion 81 greater amounts of glycogen are consumed. If a higher intensity had been used, for example 75% of VO2 max> it would have resulted in greater glycogen consumptions which may have improved the likelihood of achieving a significant difference between conditions. This potential gain would have been offset by potentially less accurate R E R measurements however. Ratings of perceived exertion values obtained in the study also support the contention that higher exercise intensities could have been employed without significant hardship to the subjects. On the ten point Borg scale, the highest mean rating of effort was only 4.1 ± 0.6 which suggests that the subjects were not working excessively during the exercise bouts. A t the design stage of this study, it was difficult to know how long the subjects would be able to maintain an exercise intensity of 65% of VO2 max under simulated altitude conditions. Due to the prohibitive cost of the bottled hypoxic air, it was not feasible to do a practice run to determine this. This served as another reason for choosing a lower exercise intensity (65% of VO2 m a x ) ; this intensity was chosen as it represented an intensity which all subjects would be likely to maintain for 75 minutes of exercise under simulated altitude conditions. In hindsight once it was known they were easily capable of it, it may have been more advantageous to have had the subjects exercising at a higher intensity to maximize their glycogen use. Studies which have compared ratings of perceived exertion for exercise at sea level and exercise with acute altitude exposure have found that R P E values at altitude tend to be lower when the same relative exercise intensity was employed for both c o n d i t i o n s 1 0 6 ' 1 1 2 , 1 3 5 . Although values obtained at A L T in these studies were lower, they were not found to be statistically different than S L values however. The same findings were obtained in the present study. Mean values for R P E with exercise at A L T tended to be slightly less than those achieved at S L , but the differences were not found to be statistically significant. These R P E findings provide further support that the same relative exercise intensities were achieved under both S L and A L T conditions. Another potential modification which may have maximized glycogen consumption and in so doing made it easier to achieve a significant difference would have been exercising the subjects for Discussion 82 a longer duration. Unfortunately, technical considerations precluded this from occurring. The capacity of the cylinders which supplied the hypoxic gas was only large enough to support 75 minutes of exercise at 65% of A L T VO2 m ax- It may have been possible to have mixed a 16% O2 mixture from tanks of 100% oxygen and nitrogen. This would have allowed longer durations of exercise, but would have introduced another potential source of error / variability into the experiment. It was decided when designing the experiment that due to the small differences expected in glycogen use between S L and A L T as many sources of variability should be eliminated to ensure the likelihood of achieving significance. For this reason, it was decided to use the more accurately measured 16% gas cylinders. Inferences about muscle oxygen availability from substrate and physiological data The original hypothesis in this experiment was that exercise at simulated altitude, i f it resulted in decreased available oxygen, would cause a shift in substrate utilization towards glycogen. The results obtained suggest that this did not happen. Based on the original hypothesis, this would imply that oxygen availability was not decreased at the muscle mitochondria when the same relative exercise intensity was undertaken under conditions of acute simulated moderate altitude. A n examination of the physiological data (in conjunction with other experimental evidence) also supports this conclusion. W i t h exercise under simulated altitude conditions there was a marked drop in mean Sao 2 compared to sea level (83.3 vs 96.2%). There was therefore less oxygen being carried in the blood during exercise under simulated altitude conditions. Faced with this situation, the body has two options available to maintain oxygen delivery to exercising muscles (1) increase heart rate so that blood and therefore O2 delivery are increased, or (2) increase oxygen extraction at the exercising muscle. Rowel l et al. have shown that under hypoxic conditions (10 - 11% O2), the first option is employed by exercising musc le 1 0 8 . These workers found oxygen extraction by muscle remains Discussion 83 largely unchanged under hypoxic conditions. Instead, oxygen delivery was maintained by increasing blood flow to active muscle. Muscle blood flow can be increased by increasing cardiac output, the product of stroke volume and heart rate. Data collected in this study shows that there was no difference in mean exercise heart rates between conditions. This suggests that oxygen delivery was not reduced to the vastus lateralis muscle during exercise under the simulated altitude conditions used in this study. It is possible however that blood flow and therefore oxygen delivery to muscle could have been maintained by other mechanisms. For example an increase in stroke volume with no change in heart rate, or redirecting more of the cardiac output towards active muscle by modulating vascular resistances could also have been responsible for maintaining oxygen delivery. These parameters were not measured in this study, so it cannot be concluded with certainty that oxygen delivery was not decreased. To completely rule out a decrease in oxygen delivery would require measurements of muscle blood flow similar to those done by Rowell et al. The fact that there was not a relative increase in glycogen use with simulated altitude conditions also tends to suggest that oxygen supply at the working muscle was not decreased. On a stoichiometric basis, glycogen is the most efficient fuel per mole of oxygen consumed for producing A T P . In fact complete oxidation of glycogen yields almost 15% more A T P per mole of O2 than complete oxidation of fat. If oxygen was limited at working muscle, an increase in the proportion of energy derived from glycogenolysis should have been observed. A n examination of venous plasma lactate values also suggests there was no increase in anaerobic metabolism under simulated altitude conditions. Lactate values tended to be slightly greater under simulated altitude conditions although this difference was not significant. This further supports the contention that oxygen availablity was not decreased with exercise at A L T . These lactate values must be interpreted with caution however. Plasma lactate values are a reflection of both the rate of appearance (Ra) as well as the rate of disappearance (Rd) of lactate (for a good discussion of this topic see the review paper by Brooks 1 6 ) . A static measurement of lactate without consideration of Discussion 84 these kinetic changes in Ra and R d can be misleading. It is theoretically possible that the Ra of lactate in our experiment increased but that the R d for lactate also increased proportionately such that there was no change in static blood lactate measurements. Without doing kinetic tracer studies with labelled lactate there is no real way of knowing i f lactate metabolism is actually changing. This same argument applies to glucose metabolism. In this study, blood samples were withdrawn and measurements were made on serum to determine static blood glucose levels. From these measurements it was found that although glucose values were slightly higher during exercise under simulated altitude conditions, this difference was not found to be significant. This suggests that there was no difference in glucose metabolism between the two conditions. However, the only accurate way to discern i f there has been a change in glucose metabolism would be to undertake kinetic tracer studies. For this reason it is difficult to draw any firm conclusions from the lactate and glucose data obtained. The other parameter followed to give an indication of possible substrate shifts was R E R . Resting mean R E R values with A L T were significantly greater than mean S L values. This difference is due to hyperventilation in the subjects at rest while breathing hypoxic air. From data obtained during exercise, analysis showed that there were no significant changes in R E R values between conditions. Had oxygen levels at the muscle been decreased, R E R values would have likely risen, signifying increased reliance on carbohydrate as a fuel source. The lack of change in R E R values further supports the conclusion that there were no measureable changes in substrate use patterns between conditions. Based on the data obtained, it appears as though oxygen availability to exercising muscle was not decreased under the simulated altitude conditions used in this study. Had oxygen availabilty been decreased, an increase in glycogen use as well as increased R E R values would have been expected with A L T . In addition, as discussed earlier, increases in heart rate would l ikely have occurred in an effort to deliver more O2 to the working muscle. Due largely to the lack of any of Discussion 85 these occurrences it is concluded by inference that under the conditions used in this study, oxygen levels to working muscle were maintained with exercise at A L T . The purpose of this study was to examine whether glycogen utilization is altered in exercising athletes exposed acutely to a moderate altitude. It was proposed that the findings could prove useful in establishing dietary guidelines for athletes exercising at altitude. The muscle glycogen data obtained in the study showed that there was no significant difference between the patterns of exercise muscle glycogen depletion between conditions. Based on these findings, it can be concluded that in highly trained cyclists performing steady state exercise under acute moderate altitude conditions at the same relative intensity as exercise at S L , there is no need to alter current dietary guidelines with respect to carbohydrate intake. A minimum of 8 grams of carbohydrate per kilogram of body weight should be sufficient to maintain optimal muscle glycogen stores for both of the conditions described above. Conclusion 86 This study looked at the effect of acute, simulated, moderate altitude exposure on glycogen and substrate utilization in six highly trained cyclists during prolonged exercise. The original hypothesis was that exercise under simulated altitude conditions may have resulted in decreased oxygen availabilty to exercising muscle which would have in turn resulted in a shift towards enhanced glycogen use. The results of this study showed that there was no significant difference in glycogen use found between conditions. There was a trend found towards decreased glycogen use under simulated altitude conditions; however, this did not reach significance. Results of this study (as well as other studies that have looked at glycogen utilization) have shown that muscle glycogen measurements are notoriously variable both within- as wel l as between-subjects. A number of factors responsible for this variablity have been discussed: most notably these include diet, training patterns, and the needle muscle biopsy technique. It is concluded that to overcome this variabil i ty and reach sufficient statistical power would l ikely require more subjects than were used in this study. If more subjects had been used, it is possible that the trend towards a lower glycogen use at A L T may have proven to be statistically significant. Other strategies that may have proven helpful would have been to either increase the exercise time and/or increase the exercise intensity in an effort to maximize glycogen utilization. Also , the mean pre-exercise muscle glycogen concentrations in the study were inexplicably low compared to results obtained in other studies. Due to the positive relationship between this value and glycogen use, it could have proven beneficial to have had more optimal pre-exercise muscle glycogen concentrations in this study. Achieving higher pre-exercise glycogen levels may have helped increased glycogen use. This, in combination with the other suggestions made above could have enhanced the study and strengthened the results. Wi th regards to the issue of whether oxygen delivery to exercising muscle was reduced with A L T , it is difficult to make firm conclusions from the data obtained. Kinetic studies on lactate metabolism as well as muscle blood flow studies would likely be required to determine this with certainty. However, based on the observed trend in glycogen use as well as the R E R data obtained Conclusion 87 it seems likely that oxygen delivery was not decreased at A L T under the conditions used in this study. The muscle glycogen data obtained in the study showed that there was no significant difference between the patterns of exercise muscle glycogen depletion between conditions. Based on these findings, it can be concluded that in highly trained cyclists performing steady state exercise under acute moderate altitude conditions at the same relative intensity as exercise at S L , there is no need to alter current dietary guidelines with respect to carbohydrate intake. The currently recommended 8 grams of carbohydrate per kilogram of body weight should be sufficient to maintain optimal muscle glycogen stores with exercise under both of the conditions described above. Bibliography 88 1. Ahlborg, B . , Bergstrom, J. , Ekelund, L . - G . and Hultman, E . Muscle glycogen and muscle electrolytes during prolonged physical exercise. Acta Physiologica Scandinavica 70: 129-142, 1967. 2. Ahlborg, B . G . Human muscle glycogen content and capacity for prolonged exercise after different diets. Foersvarsmedicin 3: 85-99, 1967. 3. Arthur, P. G . , Hogan, M . C , Bebout, D . E . , Wagner, P. D . and Hochachka, P. W . Model ing the effects of hypoxia on A T P turnover in exercising muscle. Journal of Applied Physiology 73: 737-742, 1992. 4. Astrand, P. O. and Rodahl, K . Textbook of Work Physiology, 3rd ed. New York : M c G r a w -H i l l , 1986. 5. Balaban, R. S. Regulation of oxidative phosphorylation in the mammalian cel l . American Journal of Physiology 258: C377-C389, 1990. 6. Ball-Burnett, M . , Green, H . J. and Houston, M . E . Energy metabolism in human slow and fast twitch fibres during prolonged cycle exercise. Journal of Physiology 437: 257-267, 1991. 7. Barstow, T. J . , Cooper, D . M . , Epstein, S. and Wasserman, K . Changes in breath 1 3 C 0 2 / 1 2 C 0 2 consequent to exercise and hypoxia. Journal of Applied Physiology 66: 936-942, 1989. 8. Bergstrom, J. Muscle electrolytes in man. Determined by neutron activation analysis in needle biopsy specimens. A study on normal subjects, kidney patients, and patients with chronic diarrhoae. Scandinavian Journal of Clinical Laboratory Investigation Supplement 68: 1-110, 1962. 9. Bergstrom, J. , Hermansen, L . , Hultman, E . and Saltin, B . Diet, muscle glycogen and physical performance. Acta Physiologica Scandinavica 71: 140-150, 1967: 10. Bergstrom, J. and Hultman, E . A study of the glycogen metabolism during exercise in man. Scandinavian Journal of Clinical Laboratory Investigation 19: 218-228, 1967. 11. B l o m , P. C . S., Hostmark, A . T., Vaage, O., Kardel, K . R. and Maehlum, S. Effect of different post-exercise sugar diets on the rate of muscle glycogen synthesis. Medicine and Science in Sports and Exercise 19: 491-496, 1987. 12. Blomstrand, E . and Ekblom, B . The needle biopsy technique for fiber type determination in human skeletal muscle — a methodological study. Acta Physiologica Scandinavica 116: 437-442, 1982. 13. Borg, G . A . V . Psychophysical bases of perceived exertion. Medicine and Science in Sports andExercise 14: 377-381, 1982. 14. Bouissou, P., Guezennec, C. Y . , Defer, G . and Pesquies, P. Oxygen consumption, lactate accumulation, and sympathetic response during prolonged exercise under hypoxia. International Journal of Sports Medicine 8: 266-269, 1987. Bibliography 89_ 15. Bouissou, P., Peronnet, F. , Brisson, G . , Helie, R. and Ledoux, M . Metabolic and endocrine responses to graded exercise under acute hypoxia. European Journal of Applied Physiology 55: 290-294, 1986. 16. Brooks, G . A . Current concepts in lactate exchange. Medicine and Science in Sports and Exercise 23: 895-906, 1991. 17. Brooks, G . A . , Butterfield, G . E . , Wolfe, R. R., Groves, B . M . , Mazzeo, R. S., Sutton, J. R., Wolfe l , E . E . and Reeves, J. T. Decreased reliance on lactate during exercise after acclimatization to 4,300 m. Journal of Applied Physiology 11: 333-341, 1991. 18. Brotherhood, J. R. Human acclimatization to altitude. British Journal of Sports Medicine 8: 5-8, 1974. 19. Bubb, W . J., Howley, E . T. and Cox, R. H . Effects of various levels of hypoxia on plasma catecholamines at rest and during exercise. Aviation, Space and Environmental Medicine 54: 637-40, 1983. 20. Bursztein, S., E lwyn , D . H . , Askanazi, J. and Kinney, J. M . Energy metabolism, indirect calorimetry, and nutrition. Baltimore: Williams and Wilkins, 1989, pp. 27-84. 21. Chance, B . Pyridine nucleotide as an indicator of the oxygen requirements for energy-linked functions of mitochondria. Circulation Research 38 Suppl. 1:131-138, 1976. 22. Chance, B . and Wil l iams, C . M . The respiratory chain and oxidative phosphorylation. Advances in Enzymology 17: 65-134, 1956. 23. Christensen, E . H . and Hansen, O. Arbeitsfahfahigket and ermundung. Scandinavian Archives of Physiology 81: 160-171, 1939. 24. Clancy, L . J., Critchley, A . J. H . , Leitch, A . G . , Kirby, B . J., Ungar, A . and Flenly, D . C. Arterial catecholamines in hypoxic exercise in man. Clinical Science and Molecular Medicine 49: 503-506, 1975. 25. Clark, A . , Jr. and Clark, P. A . A . Loca l oxygen gradients near isolated mitochondria. Biophysical Journal 48: 931-938, 1985. 26. Connett, R. J. Glycolytic regulation during an aerobic rest-to-work transition in dog gracilis muscle. Journal of Applied Physiology 63: 2366-2374, 1987. 27. Connett, R. J. The cytosolic redox is coupled to V 0 2 . A working hypothesis. In: Advances in Experimental Medicine and Biology Vol.22: Oxygen transport to tissue X, edited by M . Mochizuki , C . R. Honig, T. Koyama, T. K . Goldstick, D . F . Bruley. New York: Plenum Press, 1988, pp. 133-142. 28. Connett, R. J., Gayeski, T. E . J. and Honig, C. R. Lactate accumulation in fully aerobic, working, dog gracilis muscle. American Journal of Physiology 246: H120-H128, 1984. 29. Connett, R. J., Gayeski, T. E . J. and Honig, C. R. Energy sources in fully aerobic rest-work transitions: a new role for glycolysis. American Journal of Physiology 248: H922-H929, 1985. Bibliography 90 30. Connett, R. J. and Honig, C. R. Regulation of V 0 2 in red muscle: do current biochemical hypotheses fit in vivo data? American Journal of Physiology 256: R898-R906, 1989. 31. Connett, R. J., Honig, C. R., Gayeski, E . J. and Brooks, G . A . Defining hypoxia: a systems view of V 0 2 , glycolysis, energetics, and intracellular P 0 2 > Journal of Applied Physiology 68: 833-842, 1990. 32. Cooper, D . W . , Wasserman, D . H . , Vranic, M . and Wasserman, K . Glucose turnover in response to exercise during high- and low- F T 0 2 breathing in man. American Journal of Physiology 286: E209- E214, 1986. 33. Coyle, E . F. Substrate utilization during exercise in active people. American Journal of Clinical Nutrition 61 (suppl): 968S-79S, 1995. 34. Coyle , E . F . , Hamilton, M . T., Alonso, J. G . , Montain, S. J. and Ivy, J. L . Carbohydrate metabolism during intense exercise when hyperglycemic. Journal of Applied Physiology 70: 834-840, 1991. 35. Dempsey, J. A . Is the lung built for exercise? Medicine and Science in Sports and Exercise 18: 143-155, 1986. 36. Denison, D . M . Oxygen supply and uses in tissues. In: Clinical aspects of 02 transport and tissue oxygenation, edited by K . Reinhart, K . Eyrich. New York: Springer-Verlag, 1989, pp. 37-43. 37. Dobson, G . P., Parkhouse, W . S., Weber, J. M . , Stuttard, E . , Harman, J. , Snow, D . H . and Hochachka, P. W . Metabolic changes in skeletal muscle and blood in greyhounds during 800m track sprint. American Journal of Physiology 255: R513-R519, 1988. 38. Dubois, M . , Gi l les , K . A . , Hamilton, S. K . , Rebers, P. A . and Smith, F . Colorimetric method for determination of sugars and related substances. Analytic Chemistry 28: 350-356, 1956. 39. Dudley, G . A . , Tullson, P. C. and Terjung, R. L . Influence of mitochondrial content on the sensitivity of respiratory control. Journal of Biological Chemistry 262: 9109-9114, 1987. 40. Erecinska, M . and Wilson, D . Regulation of cellular energy metabolism. Journal of Membrane Biology 70: 1-14, 1982. 41. Escourrou, P., Johnson, D . G . and Rowell , L . B . Hypoxemia increases plasma catecholamine concentrations in exercising humans. Journal of Applied Physiology 57: 1507-1511, 1984. 42. Essen, B . and Henriksson, J. Glycogen content of individual muscle fibers in man. Acta Physiologica Scandinavica 90: 645-647, 1974. 43. Fabiato, A . and Fabiato, F. Effects of p H on the myofilaments and the sarcoplasmic reticulum of skinned cells from cardiac and skeletal muscle. Journal of Physiology (London) 276: 233-255, 1978. 44. Fuchs, F. , Reddy, V . and Briggs, F . N . The interaction of cations with the calcium-binding site of troponin. Biochimica et Biophysica Acta 221: 407-409, 1970. Bibliography 91 45. Galbo, H . , Hoist, J. J. and Christensen, N . J. Glucagon and plasma catecholamine responses to graded and prolonged exercise in man. Journal of Applied Physiology 38: 70-76, 1975. 46. Galbo, H , Hoist, J. J. and Christensen, N . J. The effect of different diets and of insulin on the hormonal response to prolonged exercise. Acta Physiologica Scandinavica 107: 19-32, 1979. 47. Gale, G . E . , Torre-Bueno, J. R., Moon, R. E . , Saltzman, H . A . and Wagner, P. Ventilation-perfusion inequality in normal humans during exercise at sea level and simulated altitude. Journal of Applied Physiology 58: 978-988, 1985. 48. Gayeski, T. E . , Connett, R. J. and Honig, C. R. Oxygen transport in rest-work transition illustrates new functions for myoglobin. American Journal of Physiology 248: H914-H921, 1985. 49. Gayeski, T. E . J., Connett, R. J. and Honig, C. R. Min imum intracellular P 0 2 for maximum cytochrome turnover in red muscle in situ. American Journal of Physiology 252: H906-H915, 1987. 50. Gayeski, T. E . J. and Honig, C . R. 0 2 gradients from sarcolemma to cell interior in red muscle at maximal V 0 2 . American Journal of Physiology 251: H789-H799, 1986. 51. Giesbrecht, G . G . , Puddy, A . , Ahmed, M . , Younes, M . and Anthonisen, N . R. Exercise endurance and arterial desaturation in normobaric hypoxia with increased chemosensitivity. Journal of Applied Physiology 70: 1770-1774, 1991. 52. Gol lnick, P. D . , Armstrong, R. B . , Saubert, C. W. , I V , Sembrowich, W . L . , Shepherd, R. E . and Saltin, B . Glycogen depletion patterns in human skeletal muscle fibers during prolonged work. Pflugers Archives 344: 1-12, 1973. 53. Gol lnick, P. D . , Armstrong, R. B . , Sembrowich, W . L . , E . , S. R. and Saltin, B . Glycogen depletion pattern in human skeletal muscle fibers after heavy exercise. Journal of Applied Physiology 34: 615-618, 1973. 54. Gollnick, P. D . , Pernow, B . , Essen, B . , Jansson, E . and Saltin, B . Availabil i ty of glycogen and plasma F F A for substrate utilization in leg muscle of man during exercise. Clinical Physiology 1:27-42, 1981. 55. Gol lnick, P. D . , Piehl, K . , Saubert, C. W. , Armstrong, R. B . and Saltin, B . Diet, exercise, and glycogen in human muscle fibers. Journal of Applied Physiology 33: 421-425, 1972. 56. Grollman, S. A . A study of oxygen debt in the albino rat. Journal of Experimental Zoology 128: 511-523, 1955. 57. Hammond, M . D . , Gale, G . E . , Kapitan, K . S., Ries, A . and Wagner, P. D . Pulmonary gas exchange in humans during normobaric hypoxic exercise. Journal of Applied Physiology 61: 1749-1757, 1986. 58. Harris, D . A . and Das, A . M . Control of mitochondrial A T P synthesis in the heart. Biochemical Journal 280: 561-573, 1991. Bibliography 92 59. Hermansen, L., Hultman, E. and Saltin, B. Muscle glycogen during prolonged severe exercise. Acta Physiologica Scandinavica 71: 129-139, 1967. 60. Hermansen, L. and Saltin, B. Blood lactate concentration during exercise at acute exposure to altitude. In: Exercise at Altitude, edited by R. Margaria. Amsterdam: Excerpta Medica, 1967, pp. 63-70. 61. Hochachka, P. W. The lactate paradox: analysis of underlying mechanisms. Annals of Sports Medicine A: 184-188, 1988. 62. Hochachka, P. W. Patterns of Cydependence of metabolism. In: Advances in Experimental Medicine and Biology Vol.222: Oxygen transport to tissue X, edited by M. Mochizuki, C. R. Honig, T. Koyama, T. K. Goldstick, D. F. Bruley. New York: Plenum Press, 1988. 63. Hochachka, P. W. Principles of physiological and biochemical adaptation: high-altitude man as a case study. In: Physiological adaptations in vertebrates, edited by S. C. Wood, R. E. Weber, A. R. Hargens, R. W. Millard. New York: Marcel Dekker, Inc., 1992, pp. 21-35. 64. Hochachka, P. W., Bianconcini, M. S. C , Parkhouse, W. S. and Dobson, G. P. On the role of actomyosin ATPases in regulation of ATP turnover rates during intense exercise. Proceedings of the National Academy of Science USA 88: 5764-5768, 1991. 65. Hochachka, P. W. and Matheson, G. O. ATPases and energy metabolism as proactive and reactive components in control of ATP turnover rates. In: Hypoxia and Mountain Medicine, edited by J. R. Sutton, G. Coates, C. S. Houston. New York: Pergamon Press, 1992, pp. 171-185. 66. Hochachka, P. W., Stanley, C , Matheson, G. O., McKenzie, D. C., Allen, P. S. and Parkhouse, W. S. Metabolic and work efficiencies during exercise in Andean natives. Journal of Applied Physiology 70: 1720-1730, 1991. 67. Hogan, M. C , Cox, R. H. and Welch, H. G. Lactate accumulation during incremental exercise with varied inspired oxygen fractions. Journal of Applied Physiology 55: 1134-1140, 1983. 68. Holloszy, J. O. and Booth, F. W. Biochemical adaptations to endurance exercise in muscle. Annual Review of Physiology 38: 273-291, 1976. 69. Holloszy, J. O. and Coyle, E. F. Adaptations of skeletal muscle to endurance exercise and their metabolic consequences. Journal of Applied Physiology 56: 831-838, 1984. 70. Honig, C. R., Connett, R. J. and Gayeski, T. E. J. 0 2 transport and its interaction with metabolism; a systems view of aerobic capacity. Medicine and Science in Sports and Exercise 24: 47-53, 1992. 71. Hoppeler, H. P., Luthi, H., Claassen, H., Weibel, E. R. and Howald, H. The ultrastructure of normal human skeletal muscle. A morphometric analysis on untrained men, women, and well trained orienteers. Pflugers Archiv 344: 217-232, 1973. 72. Hultman, E. Studies on muscle metabolism of glycogen and active phosphate in man with special reference to exercise and diet. Scandinavian Journal of Clinical and Laboratory Investigation Supplements 19 Suppl. 94: 1-63, 1967. Bibliography 93 73. Ivy, J. L . Muscle glycogen synthesis before and after exercise. Sports Medicine 11: 6-19, 1991. 74. Ivy, J. L . , Lee, M . C , Brozinick, J. T. and Reed, M . J. Muscle glycogen storage after different amounts of carbohydrate ingestion. Journal of Applied Physiology 65: 2018-2023, 1988. 75. Jones, D . P. Intracellular diffusion gradients of 0 2 and A T P . American Journal of Physiology 250: C663-C675, 1986. 76. Jones, N . L . , Robertson, D . G. , Kane, J. W . and Hart, R. A . Effect of hypoxia on free fatty acid metabolism during exercise. Journal of Applied Physiology 33: 733-738, 1972. 77. Katz, A . and Sahlin, K . Effect of hypoxia on glucose metabolism in human skeletal muscle during exercise. Acta Physiologica Scandinavica. 136: 377-382, 1989. 78. Katz, A . and Sahlin, K . Role of oxygen in regulation of glycolysis and lactate production in human skeletal muscle. In: Exercise and Sport Science Reviews. Vol. 18, edited by K . B . Pandolf, J. O. Holloszy. New York: Academic Press, 1990, pp. 1-29. 79. Katz, L . A . , Swain, J. A . , Portman, M . A . and Balaban, R. S. Relation between phosphate metabolites and oxygen consumption in heart in vivo. American Journal of Physiology 256: H265-H274, 1989. 80. K i rby , R. L . , Bonen, A . , Belcastro, A . N . and Campbell , C . J. Needle muscle biopsy: technique to increase sample sizes, and complications. Archives of Physical Medicine and Rehabilitation 63: 264-268, 1982. 81. Ki rwan , J . P., Cost i l l , D . L . , Mi tchel l , J. B . , Houmard, J. A . , F lynn, M . G . , Fink, W . J. and Beltz, J. D . Carbohydrate balance in competetive runners during successive days of intense running. Journal of Applied Physiology 65: 2601-2606, 1988. 82. Knuttgen, H . G . and Saltin, B . Oxygen uptake, muscle high energy phosphates and lactate in exercise under acute hypoxic conditions in man. Acta Physiologica Scandinavica 87: 368-376, 1973. 83. Koretsky, A . P. and Balaban, R. S. Changes in pyridine nucleotide levels alter oxygen consumption and extramitochondrial phosphates in isolated mitochondria: a 3 1 P N M R and fluorescence study. Biochimica et Biophysica Acta 893: 398-408, 1987. 84. Krogh, A . and Lindhard, J. Relative value of fat and carbohydrate as a source of muscular energy. Biochemical Journal 14: 290-298, 1920. 85. Kushmerick, M . J. Energetics of muscle contraction. In: Handbook of Physiology Section 10: Skeletal Muscle, edited by L . D . Peachey, R. H . Adrian , S. R. Geiger. Bethesda, Maryland: American Physiological Society, 1983, pp. 189-236. 86. Lardy, H . A . and Wellman, H . Role of oxidative phosphorylations: role of inorganic phosphate as an acceptor systems in control of metabolic rate. Journal of Biological Chemistry 195: 215-224, 1952. Bibliography 94 87. Levine, S. A . , Gordon, B . and Derrick, C. L . Some changes in the chemical constituents of the blood following a marathon race: with special reference to the development of hypoglycemia. Journal of the American Medical Association 82: 1778-1779, 1924. 88. Lexe l l , J. , Hendriksson-Larsen, K . and Sjostrom, M . Distribution of different fibre types in human skeletal muscles. 2. A study of cross-sections of whole m. vastus lateralis. Acta Physiologica Scandinavica 117: 115-122, 1983. 89. Lexe l l , J. , Taylor, C. and Sjostrom, M . Analysis of sampling errors in biopsy techniques using data from whole muscle cross sections. Journal of Applied Physiology 59: 1228-1235, 1985. 90. Linnarsson, D . , Karlsson, J., Fagraeus, L . and Saltin, B . Muscle metabolites and oxygen deficit with exercise in hypoxia and hyperoxia. Journal of Applied Physiology 36: 399-402, 1974. 91. L o , S., Russell, J. C . and Taylor, A . W . Determination of glycogen in small tissue samples. Journal of Applied Physiology 28: 234-236, 1970. 92. Lundin, G . and Strom, G . The concentration of blood lactic acid in man during muscular work in relation to the partial pressure of oxygen in the inspired air. Acta Physiologica Scandinavica 13: 253-266, 1947. 93. Mahon, M . , Toman, A . , Wil lan , P. L . T. and Bagnall, K . M . Variability of histochemical and morphometric data from needle biopsy specimens of human quadriceps femoris muscle. Journal of the Neurological Sciences 63: 85-100, 1984. 94. Matheson, G . O., Al l en , P. S., Ellinger, D . C , Hanstock, C . C , Gheorghiu, D . , McKenz ie , D . C , Stanley, C , Parkhouse, W . S. and Hochachka, P. W . Skeletal muscle metabolism and work capacity: a 3 1 P N M R study of Andean natives and lowlanders. Journal of Applied Physiology 70: 1963-1976, 1991. 95. M c A r d l e , W . D . , Katch, F. I. and Katch, V . L . Exercise physiology: energy, nutrition, and human performance, 3rd ed. Philadelphia: Lea & Febiger, 1991. 96. M c M a n u s , B . M . , Horvath, S. M . , Bolduan, N . and M i l l e r , J. C . Metabol ic and cardiorespiratory responses to long-term work under hypoxic conditions. Journal of Applied Physiology 36: 177-182, 1974. 97. Mi tche l l , P. Coupling of phosphorylation to electron and hydrogen transfer by a chemical osmotic type of mechanism. Nature (London) 191: 144-148, 1961. 98. Morgan, T. E . , Cobb, L . A . , Short, A . , Ross, R. and Gunn, D . R. Effect of long-term exercise on human muscle mitochondria. In: Muscle Metabolism During Exercise, edited by B . Pernow, B . Saltin. New York: Plenum Press, 1971, pp. 87-95. 99. Newsholme, E . A . and Leech, A . R. Biochemistry for the medical sciences. New York: John Wiley & Sons, 1983. 100. Nioka , S., Argov, Z . , Dobson, G . P., Forster, R. E . , Subramanian, H . V . , Veech, R. L . and Chance, B . Substrate regulation of mitochondrial oxidative phosphorylation in hypercapnic rabbit muscle. Journal of Applied Physiology 72: 521-528, 1991. Bibliography 95 101. Pascoe, D . D. , Costi l l , D . L . , Robergs, R. A . , Davis, J. A . , Fink, W . J. and Pearson, D . R. Effects of exercise mode on muscle glycogen restorage during repeated days of exercise. Medicine and Science in Sports and Exercise 22: 593-598, 1990. 102. Powers, S. K . and Wil l iams, J. Exercise-induced hypoxaemia in highly trained athletes. Sports Medicine 4: 46-53, 1987. 103. Rauch, L . H . G . , Rodger, I., Wilson, G . R., Belonje, J. D . , Dennis, S. C , Noakes, T. D . and Hawley, J. A . The effects of carbohydrate loading on muscle glycogen content and cycling performance. International Journal of Sport Nutrition 5: 25-36, 1995. 104. Richter, E . A . and Galbo, H . High glycogen levels enhance glycogen breakdown in isolated contracting skeletal muscle. Journal of Applied Physiology 61: 827-831, 1986. 105. Richter, E . A . , Garetto, L . P., Goodman, M . N . and Ruderman, N . B . Enhanced muscle glucose metabolism after exercise: modulation by local factors. American Journal of Physiology 246: E476-E482, 1984. 106. Robertson, R. J. Central signals of perceived exertion during dynamic exercise. Medicine and Science in Sports and Exercise 14: 390-396, 1982. 107. Rowel l , L . B . , Blackman, J. R., Kenny, M . A . and Escourrou, P. Splanchnic vasomotor and metabolic adjustments to hypoxia and exercise in humans. American Journal of Physiology 247: H251-H258, 1984. 108. Rowel l , L . B . , Saltin, B . , Kiens, B . and Christensen, N . J. Is peak quadriceps blood flow in humans even higher during exercise with hypoxemia? American Journal of Physiology 251: H1038-H1044, 1986. 109. Sahlin, K . Muscle glucose metabolism during exercise. Annals of Medicine 22: 185-189, 1990. 110. Saltin, B . and Gollnick, P. D . Fuel for muscular exercise: Role of carbohydrate. In: Exercise, Nutrition, and Energy Metabolism, edited by E . S. Horton, R. L . Terjung. Toronto: Collier Macmillan, 1988, pp. 45-71. 111. Saltin, B . and Karlsson, J. Muscle glycogen utilization during work of different intensities. In: Muscle metabolism during exercise, edited by B . Pernow, B . Saltin. New York : Plenum Press, 1971, pp. 289-299. 112. Shephard, R. J. , Vandewalle, H . , G i l , V . , Bouhle l , E . and M o n o d , H . Respiratory, muscular, and overall perceptions of effort: the influence of hypoxia and muscle mass. Medicine and Science in Sports and Exercise 24: 556-567, 1992. 113. Sherman, W . M . , Doyle, J. A . , Lamb, D . R. and Strauss, R. H . Dietary carbohydrate, muscle glycogen, and exercise performance during 7 d of training. American Journal of Clinical Nutrition 57: 27-31, 1993. 114. Shigeoka, J. W. , Colice, G . L . and Ramirez, G . Effect of normoxemia and hypoxic exercise on renin and aldosterone. Journal of Applied Physiology 59: 142-148, 1985. Bibliography 9 5 115. Stainsby, W . N . and Brooks, G . A . Control of lactic acid metabolism in contracting muscles and during exercise. In: Exercise and Sport Science Reviews. Vol. 18, edited by K . B . Pandolf, J. O. Holloszy. New York: Academic Press, 1990, pp. 29-63. 116. Stanley, W . C . and Connett, R. J. Regulation of muscle carbohydrate metabolism during exercise. FASEB Journal 5: 2155-2159, 1991. 117. Stubbs, M . Inhibitors of the adenine nucleotide translocase. Pharmacology and Therapeutics 7: 329-349, 1979. 118. Sugano, T., Oshino, N . and Chance, B . Mitochondrial functions under hypoxic conditions. The steady states of cytochrome c reduction and of energy metabolism. Biochimica et Biophysica Acta 347: 340-358, 1974. 119. Sutton, J. R. Effect of acute hypoxia on the hormonal response to exercise. Journal of Applied Physiology 42: 587-592, 1977. 120. Tager, J. M . , Wanders, R. J. A . , Groen, A . K . , Kunz, W . , Bohnensack, R. , Kuster, U . , Letko, G . , Bohme, G . , Duszynski. and Wojtczak, L . Control of mitochondrial respiration. FEBS Letter 151: 1-9, 1983. 121. Thorstensson, A . , Hulten, A . B . , von Dobeln, W . and Karlsson, J. Effect of strength training on enzyme activities and fiber characteristics in human skeletal muscle. Acta Physiologica Scandinavica 96: 392-398, 1976. 122. Vollestad, N . K . , Vaage, O. and Hermansen, L . Muscle glycogen depletion patterns in type I and subgroups of type II fibres during prolonged severe exercise in man. Acta Physiologica Scandinavica 122: 433-441, 1984. 123. Ward, M . P., Mil ledge, J. S. and West, J. B . High Altitude Medicine and Physiology. Philadelphia: University of Pennsylvania Press, 1989. 124. Wasserman, D . H . and Abumrad, N . Physiological bases for the treatment of the physically active individual with diabetes. Sports Medicine 7: 376-392, 1989. 125. Weibel , E . R. The pathway for oxygen: structure and function in the mammalian respiratory system. Cambridge, Massachusetts: Harvard University Press, 1984. 126. Welch , H . G . Effects of hypoxia and hyperoxia on human performance. In: Exercise and Sport Science Reviews. Vol. 15, edited by K . B . Pandolf. Toronto: Col l ier Macmil lan Publishers, 1987, pp. 191-221. 127. Wilk ie , D . R. Muscular fatigue: effects of hydrogen ions and inorganic phosphate. Federation Proceedings 45: 2921-2923, 1986. 128. Wi l son , D . F . , Erecinska, M . , Drown, C. and Silver, I. A . Effect of oxygen tension on cellular energetics. American Journal of Physiology 233: C135-C140, 1977. 129. Wi l son , D . F. , Erecinska, M . , Drown, C. and Silver, I. A . The oxygen dependence of cellular energy metabolism. Archives of Biochemistry and Biophysics 195: 485-493, 1979. Bibliography 97 130. Wilson , D . F. , Owen, C. S. and Erecinska, M . Quantitative dependence of mitochondrial oxidative phosphorylation on oxygen concentration: a mathematical model. Archives of Biochemistry and Biophysics 195: 494-504, 1979. 131. Wi l son , D . F . , Owen, C . S. and Hol ian , A . Control of mitochondrial respiration: a quantitative evaluation of the roles of cytochrome c and oxygen. Archives of Biochemistry and Biophysics 182: 749-762, 1977. 132. W i l s o n , D . F . and Rumsey, W . L . Factors modulating the oxygen dependence of mitochondrial oxidative phosphorylation. In: Advances in Experimental Medicine and Biology Vol.22: Oxygen transport to tissue X, edited by M . Mochizuk i , C . R. Honig, T. Koyama, T. K . Goldstick, D . F. Bruley. New York: Plenum Press, 1988, pp. 121-131. 133. Wittenberg, B . A . and Wittenberg, J. B . Transport of oxygen in muscle. Annual Review of Physiology 51: 857-878, 1989. 134. Young, A . J. Energy substrate utilization during exercise in extreme environments. In: Exercise and Sport Science Reviews. Vol. 18, edited by K . B . Pandolf, J. O. Holloszy. New York: Academic Press, 1990, pp. 65-117. 135. Young, A . J. , Cymerman, A . and Pandolf, K . B . Differential ratings of perceived exertion are influenced by high altitude exposure. Journal of Applied Physiology 14: 223-228, 1982. 136. Young, A . J., Evans, W . J., Cymerman, A . , Pandolf, K . B . , Knapik, J. J. and Maher, J. T. Sparing effect of chronic high-altitude exposure on muscle glycogen utilization. Journal of Applied Physiology 52: 857-62, 1982. Appendix 2 Subject Recruitment Poster. Appendix 3 Subject Consent Form. Appendix 4 Subject Information Form. 106 M U S C L E G L Y C O G E N USE W H E N E X E R C I S I N G A T A L T I T U D E S U B J E C T I N F O R M A T I O N S H E E T Name Age D.O.B. Address Home phone hours at this number to_ Business phone hours at this number to Are you able to attend 3 test days? yes_ no Do you have any chronic illnesses? yes no If yes, please explain: Do you have thyroid disease? yes no Do you have diabetes? yes no Are you taking any medication which may affect study results? (ie. anti-depressants, l i thium, beta blockers) yes_ no If yes, please list medications: Appendix 5 Subject Exercise History Recording Form. 108 Muscle Glycogen When Exercising At Altitude 48 Hour Exercise Record Name: Please record in detail any exercise undertaken (training rides, races, etc.) in the 48 hours prior to your exercise test. Please include duration, intensity, and time of activity. Appendix 6 Subject Data Collection Form. 110 THESIS Subject Name: Experimental Condition: Normoxia / Hypoxia Date: Time: REST Glycogen. 1 3 5 L a . L a . L a 10 L a 15 L a . 30 L a . 45 L a . 60 L a 75 L a Glucose. Glucose. Glucose. Glucose. Glucose Borg . Borg . Borg Borg Hct Hct END Glycogen. Comments: 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0087095/manifest

Comment

Related Items