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Dose-response relationships of inhaled salbutamol in competitive non-asthmatic athletes : effects on.. Sporer, Benjamin Carson 2006-02-03

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DOSE-RESPONSE RELATIONSHIPS OF INHALED SALBUTAMOL IN COMPETITIVE NON-ASTHMATIC ATHLETES: EFFECTS ON PERFORMANCE AND URINE CONCENTRATIONS. by Benjamin Carson Sporer M.Sc., University of Victoria, 2001 B.Sc.(Hons)., University of Victoria, 1998 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPY in FACULTY OF GRADUATE STUDIES (Human Kinetics) THE UNIVERSITY OF BRITISH COLUMBIA November 2006 © Benjamin Carson Sporer, 2006 ABSTRACT Currently, the World Anti-Doping Agency (WADA) permits asthmatic athletes to use inhaled salbutamol (SAL) to help attenuate compromised lung function during exercise. Although the majority of previous research shows no benefit in non-asthmatic athletes, there lacks an examination of the dose-response effect of SAL on performance using a sport-specific evaluation. Additionally, there lacks a description of how dose affects the concentration of SAL in the urine (cSAL). We hypothesized that salbutamol would have no effect on performance in non-asthmatic athletes and that cSAL would be affected by dose and be highly variable. Three projects were completed. Study 1 established the typical error and reliability of time-trial performance using the Velotron cycle ergometer. Highly trained, male cyclists performed three 20-km time-trials (TT) demonstrating the test to be highly reliable with low coefficients of variance for power and time (1.8-2.0% and 0.8-1.0% respectively). In Study 2, lung function was positively affected by SAL and urine analysis revealed a dose-response relationship with cSAL while at rest, up to a dose 800p.g. Peak values were observed at 60min post-inhalation and cSAL was highly variable at each time point. Although several samples approached the WADA limit of 1000 ng-ml"1, none exceeded this value. Using doses of 200Ltg, 400fig, and 800|ag, Study 3 revealed no effects of SAL on time-trial performance or physiological measures over placebo. Additionally, athlete perception of leg and breathing effort was unaffected across conditions. Similar to Study 2, cSAL was related to dose and highly variable, with no samples resulting in a doping violation. SG was found to be significantly related to cSAL and when corrected to a dehydrated state, several samples exceeded the WADA limit. In summary, these findings allow us to accept the hypothesis that acute inhalation of SAL lacks ergogenic properties in non-asthmatic athletes and does not affect ventilation or metabolic parameters during exercise. Additionally, inhaled SAL does not appear to alter athlete perceptions of effort. The findings further suggest that urine samples will generally fall below the WADA limit following therapeutic doses of SAL, although this may be affected by hydration. IV TABLE OF CONTENTS ABSTRACT ii TABLE OF CONTENTS iv LIST OF TABLES .' vi LIST OF FIGURES x ACKNOWLEDGEMENTS xDEDICATION xii CHAPTER ONE - GENERAL INTRODUCTION 1 B2-AGONISTS IN COMPETITION 1 SALBUTAMOL AND PERFORMANCE IN NON-ASTHMATICS 2 SALBUTAMOL AND DOPING CONTROL 4 STATEMENT OF THE PROBLEM 5 PURPOSE . 6 PROJECT 1PROJECT 2..... : 6 PROJECT 3 7 CHAPTER 2 - REVIEW OF THE LITERATURE 8 INTRODUCTION 8 THE RESPIRATORY SYSTEM AND EXERCISE 9 //2-AGONISTS AND MECHANISM OF ACTION 11 /?2-AGONISTS AND ATHLETIC COMPETITION •. 13 SALBUTAMOL AND PERFORMANCE IN NON-ASTHMATICS 4 SALBUTAMOL AND DOPING CONTROL 20 .SUMMARY AND FUTURE DIRECTIONS FOR RESEARCH 23 CHAPTER 3 - 20KM TIME TRIAL RELIABILITY 25 INTRODUCTION 25 MATERIALS AND METHODS 27 Subjects 27 Study DesignMaximal Aerobic Power Test.. 28 Simulated 20km Time Trial 29 Data Analysis 30 RESULTSMaximal aerobic power test .' 30 20km time-trial performance 31 Relationships between peak power and performance 33 DISCUSSION 35 CHAPTER 4 - DOSE RESPONSE OF SALBUTAMOL AT REST 40 INTRODUCTIONMATERIALS AND METHODS 42 Subjects : 42 Study Design 42 Days 1-3 - Drug Administration and Urine Collection 43 Urine Analysis 44 Data Analyses 45 RESULTS 45 Subjects ( 45 Dose Response Effects 46 DISCUSSION 50 CHAPTER 5 - DOSE RESPONSE OF SALBUTAMOL DURING EXERCISE .... 55 INTRODUCTIONMATERIALS AND METHODS 58 Subjects 58 Study DesignLung Function and Airway Hyper-responsiveness 59 Maximal Exercise Test 60 Dose Response Evaluation - Exercise Protocol 61 Urine Collection and Analysis 63 Data Analysis 65 RESULTS 65 Subject Characteristics and Airway Hyperresponsiveness 65 20km Time Trial Performance 67 Urine Concentrations of Salbutamol 70 DISCUSSION 74 CHAPTER 6 - SUMMARY AND CONCLUSIONS 83 REFERENCES 86 APPENDIX A - RAW DATA FOR PROJECT 1 94 APPENDIX B - RAW DATA FOR PROJECT 2 9 APPENDIX C - RAW DATA FOR PROJECT 3 105 APPENDIX D - SCALE FOR MEASURING PERCEIVED EXERTION 121 APPENDIX E - COPIES OF UBC RESEARCH ETHICS APPROVALS 122 vi LIST OF TABLES Table 2.1. Table 3.1. Table 3.2. Table 4.1. Table 4.2. Table 5.1. Table 5.2. Table 5.3. Table 5.4. Table 5.5. Summary of studies examining ergogenic effects of salbutamol. TTE = Time to Exhaustion; PP = Peak Power; MP = Mean Power; TW = Total Work; TTC = Time to Completion 15 Measured Variables During each 20km Time Trial Performance: Mean ± SD for Total Time (Ttot), First and Second Lap Times (Tu and TL2), Mean Velocity (VEL), Heart Rate (HR) and Absolute and Relative Power Output (Pmean and Prel) 31 Reproducibility Statistics Including Change in Means (A Means), Coefficient of Variance (CV) and Pearson Correlation Coefficients (r) along with 95% Confidence Intervals (C.I.) for TT1, TT2, and TT3 33 Specific Gravity for all Urine Samples at 30, 60, and 120 Minutes (T30, T60, T120 Respectively) Post-Inhalation of Salbutamol 46 Urine Concentrations of Salbutamol (non-sulfated) at 30 (T30), 60 (T60), and 120 Minutes (T120) Post-Inhalation of 200Lig (D2), 400u.g (D4), and 800u.g (D8) of Salbutamol. Mean, SD, Minimum (Min), and Maximum (Max) for Raw and Corrected for Specific Gravity (SG) Values are Reported in ng-ml'1 48 Subject Characteristics for Positive and Negative Responders to a Eucapnic Voluntary Hyperpnea (EVH) Test. Values presented are Means, Standard Deviations (SD), Maximums (Max), and Minimums (Min) 66 Lung Function Measures Including Percent Predicted Values for Forced Vital Capacity (FVC), Forced Expiratory Volume in One Second (FEVi), and Fraction of FVC Expired in One Seconds (FEVj/FVC), and Decrease in FEVi (Max AFEVi) for Positive and Negative Responders to a Eucapnic Voluntary Hyperpnea (EVH) Test. Values presented are Means, Standard Deviations (SD), Maximums (Max), and Minimums (Min) 66 Baseline Performance Characteristics of Negative EVH Subjects (n=30). 67 The Effects of Salbutamol Dose (D2=200ug, D4=400ug, D8=800u.g) on 20km Mean Power Output (Pmean), Total Time (Ttot), and Lap Times (TLi, TL2), Heart Rate (HR) and Rate of Perceived Exertion for Legs (RPEL) and Breathing (RPED). Values Reported are Means and (SD) 67 The Effects of Salbutamol Dose (D2=200ug, D4=400ug, D8=800pg) on Mean Metabolic and Ventilatory Parameters over 20km. Oxygen Consumption (VO2), Expired Carbon Dioxide (VCO2), Ventilation Rate (VE), Ventilatory Equivalents for Oxygen and Carbon Dioxide (VE/V02, Vll VE/VC02), Respiratory Rate (RR), and Tidal Volume (VT). Values are Reported as Means and (SD) 68 Table 5.6. Urine Concentrations of Salbutamol (non-sulfated) at 60 Minutes (T60) Post-Inhalation of Placebo (DP), 200p.g (D200), 400p.g (D400), and 800Lig (D800) of Salbutamol. Mean, SD, Minimum (Min), and Maximum (Max). Mean, Standard Deviation (SD), Maximum (Max), and Minimum (Min) Values are Reported Raw and Corrected for Specific Gravity (SG) Formats. Values are Reported in ng-mf1 70 Table A.2. Individual Subject Performance Characteristics Including Peak Oxygen Consumption (P^max) in Relative (Rel) and Absolute (Abs) terms, Maximal Ventilation (PEmax), Maximal Heart Rate (HRmax), and Peak (Ppeak) and Relative (Prei) Power Output 95 Table A.3. Individual Performance Times in Minutes for Each Time-trial (TT) Including Lap (Tu and TL2) and Total Times (Ttot) 96 Table A.4. Individual Mean Performance Power (Pmean) in Watts for Each Time-trial (TT) 97 Table A.5. Individual Mean Heart Rate for Each Time-trial (TT) 98 Table B.l. Individual Subject Characteristics and Baseline Lung Function Measures with Percent of Predicted Values (% Pred). Lung Function Measures Include Forced Vital Capacity (FVC), Forced Expiratory Volume in One Second (FEV,), and the Ratio of FEV, to FVC (FEVi/FVC) 99 Table B.2. Individual Urine Concentrations of Non-sulphated Salbutamol (ng-mf1) at 30 (T30), 60 (T60), and 120 Minutes (T120) Post-Inhalation of 200Lig (D2), 400(xg (D4), and 800ug (D8) of Salbutamol. Group Means and SD for Each Condition are Included 100 Table B.3. Individual Urine Concentrations of Non-sulphated Salbutamol (ng-mf1) Corrected for Specific Gravity (1.005) at 30 (T30), 60 (T60), and 120 Minutes (T120) Post-Inhalation of 200|ag (D2), 400Lig (D4), and 800|ag (D8) of Salbutamol. Mean and SD for Each Condition are Included 101 Table B.4. Individual Urine Concentrations of Non-sulphated Salbutamol (ng-mf1) Corrected for Specific Gravity (1.025) at 30 (T30), 60 (T60), and 120 Minutes (T120) Post-Inhalation of 200ug (D2), 400p.g (D4), and 800ug (D8) of Salbutamol. Mean and SD for Each Condition are Included 102 Table B.5. Specific Gravity of Individual Urine Samples at 30 (T30), 60 (T60), and 120 Minutes (T120) Post-Inhalation of 200p.g (D2), 400p.g (D4), and 800ug (D8) of Salbutamol. Mean and SD for Each Condition are Included 103 viii Table C.l. Individual Lung Function Measures Including % Predicted Values for Forced Vital Capacity (FVC), Forced Expiratory Volume in One Second (FEVi), Ratio of FEVi to FVC (FEV,/FVC), and Decrease in FEV, (Max AFEVi) for Negative Responders to a Eucapnic Voluntary Hyperpnea (EVH) Test. Included are Means and SD (n=30) 105 Table C.2. Individual Lung Function Measures Including % Predicted Values for Forced Vital Capacity (FVC), Forced Expiratory Volume in One Second (FEVi), Ratio of FEV, to FVC (FEVi/FVC), and Decrease in FEVi (Max AFEVi) for Negative Responders to a Eucapnic Voluntary Hyperpnea (EVH) Test. Included are Means and SD (n=7) 106 Table C.3. Individual Subject Characteristics and Training History of Negative EVH Subjects (n=30) 107 Table C.4. Baseline Performance Characteristics of Negative EVH Subjects Inducing Peak Oxygen Consumption ( Fcfemax), Maximum Heart Rate (HRmax), and Peak Absolute (Pmax) and Relative (Prei) Power Outputs. Group Means and SD are Included. (n=30) 108 Table C.5. Individual Mean Power Output (W) During a 20km Time-trial Post-Inhalation of Placebo (DP), 200u,g (D2), 400p.g (D4), and 800ug (D8) of Salbutamol. Group Means and SD for Each Condition are Included. (n=30) 109 Table C.6. Individual 20km Performance Times (min) Post-Inhalation of Placebo (DP), 200Lig (D2), 400u.g (D4), and 800Lig (D8) of Salbutamol ( Including Lap (TLI and TL2) and Total Times (Ttot). Group Means and SD for Each Condition are Included. (n=30) 110 • Table C.7. Individual Mean Oxygen Consumption (mL-kg"'-min"') During a 20km Time-trial Post-Inhalation of Placebo (DP), 200Lig (D2), 400u_ (D4), and 800Lig (D8) of Salbutamol. Group Means and SD for Each Condition are Included. (n=30) Ill Table C.8. Individual Mean Ventilation (L-min"1) During a 20km Time-trial Post-Inhalation of Placebo (DP), 200u_ (D2), 400u.g (D4), and 800u_'(D8) of Salbutamol. Group Means and SD for Each Condition are Included. (n=30) 112 Table C.9. Individual Mean Heart Rate (bpm) During a 20km Time-trial Post-Inhalation of Placebo (DP), 200u.g (D2), 400ug (D4), and 800Lig (D8) of Salbutamol. Group Means and SD for Each Condition are Included. (n=30) 113 ix Table C.10. Individual Mean Speed (knvhr"1) During a 20km Time-trial Post-Inhalation of Placebo (DP), 200Lig (D2), 400u.g (D4), and 800Lig (D8) of Salbutamol. Group Means and SD for Each Condition are Included. (n=30).. 114 Table C.ll. Individual Rate of Perceived Exertion for Breathing Effort (1-10) During a 20km Time-trial Post-Inhalation of Placebo (DP), 200ug (D2), 400u.g (D4), and 800ug (D8) of Salbutamol. Group Means and SD for Each Condition are Included. (n=30) 115 Table C.12. Individual Mean Rate of Perceived Exertion for Leg Effort (1-10) During a 20km Time-trial Post-Inhalation of Placebo (DP), 200p.g (D2), 400|ug (D4), and 800jLtg (D8) of Salbutamol. Group Means and SD for Each Condition are Included. (n=30) 116 Table C.13. Individual Urine Concentrations of Non-sulphated Salbutamol (ng-mf1) at 60 Minutes (T60) Post-Inhalation of Placebo (DP), 200u.g (D2), 400Lig (D4), and 800Lig (D8) of Salbutamol. Group Means and SD for Each Condition are Included 117 Table C.14. Individual Urine Concentrations of Non-sulphated Salbutamol (ng-mf1) Corrected for Specific Gravity (1.005) at 60 Minutes (T60) Post-Inhalation of Placebo (DP), 200|ag (D2), 400p,g (D4), and 800p.g (D8) of Salbutamol. Mean and SD for Each Condition are Included 118 Table C.15. Individual Urine Concentrations of Non-sulphated Salbutamol (ng-mf1) Corrected for Specific Gravity (1.025) at 60 Minutes (T60) Post-Inhalation of Placebo (DP), 200u.g (D2), 400u-g (D4), and-800u.g (D8) of Salbutamol. Mean and SD for Each Condition are Included 119 Table C.16. Specific Gravity of Individual Urine Samples for Placebo (DP), 200Lig (D2), 400u.g (D4), and 800u.g (D8) Conditions. Mean and SD for Each Condition are Included 120 X LIST OF FIGURES Fig. 3.1. A Bland-Altman style plot showing individual performance times for all three time trials (TT1, TT2, TT3) 32 Fig. 3.2. Relationships between peak power during an incremental exercise test (Ppeak) and (a) performance time (Ttot) and (b) mean power (Pmean) for TT1 (n=20). Lines represent 95% confidence intervals 34 Fig. 4.1. Individual Urine Concentrations of Salbutamol (cSAL) for Raw Samples (a), Samples Corrected to Specific Gravity of 1.005 (b), and Samples Corrected to a Specific Gravity of 1.025 (c). Individual Samples are Shown for 30 minutes post (T30), 60 minutes post (T60), and 120 minutes post (T120) for Doses of 200|ag (D2), 400>g (D4), and 800u_ (D8). Dashed Line Represents Doping Control Limit of 1000 ng-ml'1 49 Fig. 5.1. Experimental protocol timeline 5Fig. 5.2. Timeline for treatment and time trials 61 Fig. 5.3. Mean ratings of perceived exertion for breathing (RPED) and legs (RPEL) at 2km intervals. Rating of difficulty ranged from 1 (nothing at all) to 10 (maximal)..... .69 Fig. 5.4. Urine Concentrations of Salbutamol (cSAL) for Raw Samples (a), Samples Corrected to Specific Gravity of 1.005 (b), and Samples Corrected to a Specific Gravity of 1.025 (c). Individual Samples are Shown for Placebo, 200u_ (D200), 400p.g (D400), and 800Lig (D800). Dashed Line Represents Doping Control Limit of 1000 ng-ml-1 72 Fig. 5.5. Relationships between specific gravity and urine concentrations of salbutamol (cSAL) 1 hour post-inhalation of 400 pg (a) and 800 pg (b) doses 73 Fig. B.l. Force expiratory volume in 1 second (FEVi) (a) and the ratio of FEVi to forced vital capacity (FVC) as a percentage (b) prior to (pre) and at 30, 60, and 120 minutes following inhalation of salbutamol. Values are shown for doses of 200 ug (-T-), 400 \ig (-o-), and 800 ug (-•-) 104 xi ACKNOWLEDGEMENTS I would like to acknowledge and thank my supervisor, Dr. Don McKenzie, for his support, guidance, and mentorship over the past 4 years. He finds balance in both personal and professional aspects of his life and I leave UBC with more than simply an education in exercise physiology. His ability to mentor in quite, powerful ways has furthered my development as both a person and academic. I would also like to thank my committee members for their insights and ideas that shaped this dissertation. A special thanks to Dr. Sheel, with whom I collaborated on several projects outside of this dissertation and gained valuable insights on successfully balancing the many challenges faced early in an academic career. Thanks to the many subjects that willingly provided their time, effort and sweat to my education. Without them, none of this would be possible. Thanks also to Diana and my fellow lab mates, Alastair, Mike, and Lianne, who each played a role in shaping me as person and researcher. A special thanks to Kirstin Lane. It's been a great 10 years and I look forward to the years ahead of friendship, collaboration, and of course, coffee breaks! Finally, thank you to my best friend and wife, Trina. She has always provided unwavering support in my endeavours and aspirations while challenging my thoughts and ideas. This work is a reflection of her support and understanding. Xll DEDICATION This dissertation is dedicated to everyone that pursues their passion in life and supports others to do the same without judgement. 1 CHAPTER ONE - GENERAL INTRODUCTION Optimum performance in the elite athlete can be limited by pulmonary, cardiovascular, muscular, psychological, nutritional and/or environmental factors. In asthmatic athletes and individuals suffering from exercise induced-bronchospasm, lung function is reduced, thereby possibly limiting performance capabilities [6]. Currently four P2-agonists, salbutamol, formoterol, salmeterol, and turbutaline, have been approved by the International Olympic Committee Medical Commission (IOC-MC) and the World Anti-Doping Agency (WADA) for use by asthmatics in competition to minimize the negative effects of asthma on exercise. In order to use these medications (at the Olympic Games), the athlete must provide clinical evidence of variable airflow obstruction that is assessed by an independent medical panel [2]. Appropriate tests include bronchodilator response and bronchial provocation (eucapnic voluntary hyperpnea (EVH), lab/field exercise, or chemical challenge). At the 2002 Winter Olympics in Salt Lake City, 165 athletes (6.6% of all participants) applied to use an inhaled P2-agonist (increased from 5.6% in Nagano in 1998). Increased applications from athletes competing in the summer games have also been observed (Los Angeles (1984) - 1.7%, Atlanta (1996) - 3.6%, and Sidney (2000) -5.5%). At the most recent games in Athens, 4.6% of all athletes applied to use a p2-agonist [4]. BVAgonists in Competition For asthmatic athletes, p2-agonists permit them to compete at an elite level by minimizing the effects of asthma on performance. Of the four p2-agonists allowed in competition, salbutamol is the most commonly used and is the only one considered to have anabolic effects as well as act as a bronchodilator. The 2006 World Anti Doping Code (WADC) [82] states that salbutamol is allowable only when a therapeutic use exemption (TUE) has been obtained in advance and that it may only be administered through inhaled means. There is growing concern that non-asthmatic athletes are using inhaled salbutamol in an attempt to gain a competitive edge [2]. Furthermore, anecdotal evidence suggests that both asthmatic and non-asthmatic athletes believe in its ability to enhance performance and are using doses that substantially exceed therapeutic recommendations. This poses not only an ethical question but also raises concerns of athlete safety due to the possible negative side-effects associated with excessive doses (e.g. hyperkalemia, arrhythmia). Salbutamol and Performance in Non-Asthmatics Although a few studies exist demonstrate an ergogenic effect [7, 68, 78], the current research overwhelmingly suggests that inhaled salbutamol, in therapeutic doses, does not enhance athletic performance in non-asthmatics [10, 11, 22, 24, 48, 53, 61, 74]. It has been shown that the ventilatory response to salbutamol in both non-asthmatics and asthmatics is related to dose [35, 42] however, it is not clear whether a dose-response effect exists with respect to performance in elite athletes. Additionally, the majority of studies have evaluated performance using one, or a combination of, maximal oxygen consumption (Vcfrmax), Wingate, lactate threshold, or run to exhaustion tests (3-5 min). The validity of a test to be representative of performance is an important factor when evaluating the potential ergogenic effects of a treatment [34]. Two studies have investigated the effects of inhaled salbutamol using a simulated sport-specific performance test [53, 78]. Norris and colleagues [53], showed a non-significant 12-second improvement in 20-km time-trial performance time following a dose of 400 pg. In comparison, a dose of 800 (J.g has been shown to decrease time to complete a specific amount of work [78]. If salbutamol has an ergogenic effect, it may be related to dose. It has been shown that ventilatory response to salbutamol in both non-asthmatics and asthmatics is enhanced as dose increases [35, 42]. However, Goubault and colleagues showed no effect of dose (placebo, 200 \ig, and 800 |xg) on cycling time to exhaustion even though forced expiratory volume in one second (FEVi) was enhanced following salbutamol administration [24]. More research examining the dose-response effects of inhaled salbutamol using a sport-specific performance test is needed to determine if it has ergogenic properties. It is suggested that an improvement of 0.7-1.5 times the coefficient of variance (CV) in performance at the elite level could be a worthwhile enhancement in performance, potentially increasing the likelihood of winning for an athlete who averages 10th place [34]. Depending on the length of race, the typical CV for top performers in simulated cycling time-trials is approximately 1-1.7% [37, 58, 59, 69]. Although the majority of research to date has shown no significant improvement in performance with the use of inhaled salbutamol, the dose-response effect on performance has not been evaluated in a homogenous group of highly trained athletes with a sport specific performance test. 4 Salbutamol and Doping Control WADA currently requests that laboratories report all cases in which the urine concentration of salbutamol exceeds 200 ng/mL. Regardless of whether or not the athlete has a TUE, a urine concentration of greater than 1000 ng/mL (nonsulfated) is considered a doping violation [82]. A recently published case study has questioned whether or not this cut off point is appropriate as it may result in a positive doping test and subsequent 2-year ban from competition [65]. Schweizer and colleagues [65] reported an in-competition urinary salbutamol concentration of 8000 ng/mL in a male athlete with a TUE and were able to reproduce this positive test in a controlled, non-exercising trial. This is in agreement with other reports of positive test results using therapeutic doses, all with urine concentrations between 1000 and 3000 ng/mL following exercise [45].. High inter-subject variability (-38%) has been shown in urine recovery of salbutamol [77] and this may explain the recent occurrence of false positive tests. It is possible that differences in renal function, lung absorption, and/or dehydration from exercise [45] are responsible for the high variability. Furthermore, differences in time between inhalation and sample collection may affect urine concentrations. Up to 40% of the dose may be excreted in the first 4-6 hours post inhalation [21, 83] and depending on hydration, urine concentrations may vary. Despite the wealth of research on salbutamol, there lacks a clear characterization of the dose-response effect on urine concentrations as utilized by WADA at different time intervals post-inhalation for both rest and exercise. 5 Statement of the Problem Although the majority of research suggests salbutamol has no performance enhancement in non-asthmatics, the dose-response effect on performance has not been evaluated in a homogenous group of highly trained athletes with a sport specific performance test. It is important that the study is conscious of the minimal enhancement that would be capable of increasing the likelihood of improving performance in competition (~0.7-1.5x the CV) [34]. A secondary problem is that there is limited data describing the effects of dose on urine concentrations of salbutamol as used in doping control at rest and after exercise. Recovery of salbutamol in the urine has shown to be highly variable between subjects [77, 80] which may help explain reports of positive doping violations for salbutamol when using therapeutic doses [45, 65]. There lacks a clear characterization of the dose-response effect on urine concentrations post-inhalation during both rest and exercise. 6 Purpose The overall purpose of this study was to determine the dose-response effects of inhaled salbutamol on exercise performance in elite non-asthmatic athletes using a sport specific test of performance. A sport-specific 20km cycling time-trial was used as the method of performance evaluation. Three projects were completed. The purpose and hypotheses of each were as follows: Project 1 The purpose of Project 1 was to develop a test for evaluating elite cyclists in a controlled environment. A cycle ergometer was used to simulate a sport specific 20km time-trial using a flat course with no wind effect. The reliability and reproducibility of this test was evaluated. A secondary goal was to determine appropriate performance criteria for Project 3 to ensure a homogenous subject group. It was hypothesized that a 20km time-trial would be reproducible in competitive cyclists and show a low coefficient of variation between tests. Project 2 The purpose of Project 2 was to describe the dose-response relationship of urine salbutamol concentrations at rest and at 30, 60, and 120 minutes post-inhalation from a metered dose inhaler (MDI). This provided data to compare the exercise response to in Project 3. The hypotheses stated there would be a positive effect of dose on salbutamol concentrations at all time periods and that urine salbutamol concentrations would 7 increase at each time interval post-inhalation. It was also hypothesized that there would be high inter-subject variability in urine concentrations across all three doses. Project 3 Once a reliable test of cycling performance had been obtained in competitive cyclists, it was used in the evaluation of salbutamol on exercise performance. The purpose of Project 3 was to examine the dose-response relationship of salbutamol on exercise performance in a sport-specific test and to examine the effects of exercise on urine concentrations of salbutamol. There were 3 hypotheses: 1. No change would occur in 20km time-trial performance following inhaled salbutamol and this would not be affected by dose. 2. Urine concentrations of salbutamol would be affected by dose following exercise and this relationship would be linear. 3. There would be high inter-subject variability in urine concentrations of salbutamol following exercise. 8 CHAPTER 2 - REVIEW OF THE LITERATURE Introduction Applications for the use of inhaled p2-agonists in international athletic competition have been increasing for the past 20 years and there is concern that this increase may be due to. attempts by non-asthmatic athletes to gain a competitive advantage [2]. P2-agonists have potent effects on bronchodilation, myocardial contractility, glycogenolysis, and membrane excitability which may enhance exercise performance. Of the four p2-agonists approved for competition, salbutamol is the most commonly prescribed. Recent reports suggest that the therapeutic use of salbutamol may result in doping violations [45, 65] and this is of concern to avoid false positive tests. There is a significant amount of research that has contributed to the understanding of the effects of p2-agonists on exercise performance in non-asthmatics [7, 10, 11, 15, 16, 22, 44, 48, 51, 53, 61, 68, 74, 76] with the majority of it suggesting no ergogenic benefit. Some of this research is limited in its applicability to elite athletes due to experimental design limitations. The literature is also lacking an evaluation of the dose-respOnse effects on a sports specific performance test and urinary concentrations of salbutamol following exercise, and the potential relationships between performance, urine concentration, and doping violations. This review summarizes the current research on the effects of P2-agonists and in particular, salbutamol, on exercise performance. It also examines the respiratory system with respect to exercise, and the relationships between salbutamol dose, urine concentration, and doping control. 9 The Respiratory System and Exercise The primary respiratory structures include the nasal cavity and nostrils, the mouth, pharynx, larynx, trachea, and the right and left lung with their respective bronchi. Beyond the larynx, the airways are often divided into two different zones: the conducting zone and the respiratory zone. The conducting zone includes the trachea, bronchi, bronchioles and terminal bronchioles, while the respiratory zone contains the respiratory bronchioles, alveolar ducts, and alveolar sacs. Gas-exchange occurs in the alveolar capillary unit which has a density of capillaries to alveolus of nearly 1000:1. Of the respiratory system's functions, two are particularly important to exercise: gas exchange (CO2 for O2) and regulation of blood pH [40]. For purposes of this review, only gas exchange will be discussed as it is this function that may potentially be affected by pVagonist use in non-asthmatics. The typical respiratory response to exercise is a linear increase in ventilation with increases in workload up to ventilatory threshold, after which increases in ventilation accelerate non-linearly with respect to oxygen consumption. As the demand for oxygen and cardiac output increases, greater demands are placed on the respiratory system to maintain the alveolar-arterial pressure gradient in order to maintain PA02. Furthermore, as oxygen metabolism increases, there is a greater need to eliminate CO2. Increased ventilation accommodates both these needs. 10 Resting ventilation (VE) is approximately 5-6 Lmin"1 and during strenuous exercise this can be increased to as much as 150 Lmin"1 or more for a short period of time. Early on in exercise, increases in ventilation are primarily accomplished through increases in tidal volume. As exercise progresses and becomes more difficult, higher ventilations are achieved through an increased breathing rate with very little further increase in tidal volume beyond the 50-60% increase over rest [40]. As ventilation increases, the airway resistance component of the work of breathing is augmented, primarily due to dynamic compression and increases in turbulence. Normally this is somewhat reduced by an exercise-induced bronchodilation [66]. It can generally be said that the respiratory system does not limit exercise capacity at sea level and it is built with plenty of reserve to provide adequate alveolar ventilation. Two such situations, where the respiratory system may limit exercise are respiratory disease [67] or exercise-induced arterial hypoxaemia (EIAH) [20]. In both these situations, exercise capacity may be limited due to inadequacies of the respiratory system to maintain arterial oxygen pressure (PaCh). In a large number of elite athletes (incidences of up to 50% depending on sport) either asthma and/or an exercise-induced bronchoconstriction results in reduced airway calibre and greater resistance to breathing [85]. This bronchoconstriction may have two detrimental effects on exercise. First, reductions in airway calibre may increase the work of breathing thereby shifting oxygen delivery from the working muscles. Harms and colleagues [25] have shown that with increased inspiratory muscle work during exercise total K02 doesn't change, however, the percentage of VQI directed to the legs is reduced from 81% to 71%. This was 11 accompanied by a significant decrease in leg blood flow from 17.8 Lmin"1 to 16.9 Lmin" 1. In a follow-up study, it was shown that at sustained high workloads (90% Fo^max) increased work of breathing decreased time trial performance by approximately 15% [27]. With a greater work of breathing, respiratory muscles require a greater amount of oxygen for energy production. This leads to a redistribution of cardiac output at the cost of the working muscles. Secondly, increased bronchoconstriction may lead to inadequate alveolar ventilation [3]. Inability to maintain a high alveolar oxygen pressure (PA02) will result in a reduced PA- ap2 gradient. At higher levels of exercise this may lead to a lowering of Pa02 as seen in EIAH [20]. EIAH has been shown to result in compromised performance both at sea level [26] and in hypoxia [13]. In individuals with asthma or exercise-induced bronchoconstriction, use of pVagonists is encouraged during exercise to ensure normal ventilation and help alleviate this effect. In noh-asthmatics, it is doubtful that this medication would have an ergogenic benefit as bronchodilation is not likely a factor that limits exercise performance. Both salbutamol and formoterol provided no performance benefit or attenuation of EIAH during a cycle to exhaustion test in non-asthmatic male athletes [75]. p2-Agonists and Mechanism of Action For the asthmatic athlete, bronchodilators provide rapid relief from bronchoconstriction. Bronchodilators relax the smooth muscle of the airway thereby increasing airway calibre. This has been shown to occur both in asthmatics and non-asthmatics and can easily be 12 confirmed using a bronchodilator response test [43]. The primary bronchodilator prescribed is often a pVagonist and these can be divided into both short and long acting. Short acting pVagonists include salbutamol and terbutaline with salbutamol being the most commonly prescribed P2-agonist worldwide [43]. They are characterized by a rapid onset of action with a relatively short duration of effectiveness, most effective in the first 2-3 hours and complete cessation within 5-6 hours[43]. Conversely, long acting P2-agonists (salmeterol and formoterol) have a mechanism of action lasting approximately 12 hours [8]. Both short and long acting P2-agonists are frequently used in conjunction with inhaled glucocorticoids in asthma management. The mechanism of action of P2-agonists is through the P2-receptor that is found in high concentrations in both the bronchial epithelium and bronchial smooth muscle [41, 43]. Normally these receptors are activated by the adrenergic fibres of the sympathetic nervous system [40]. The P2-receptor is a seven transmembrane molecule that allows for intracellular signalling via a G-protein. Binding of an agonist leads to a conformational change that triggers a cascade of effects involving cAMP. The end result is an inhibition of myosin-actin binding in the bronchial smooth muscle and subsequent relaxation [43]. It has been suggested that stimulation of large potassium channels (maxi-K) by p2-agonists may play a major role in smooth muscle relaxation [41]. It has been shown in both asthmatics and non-asthmatics that airway function is increased with salbutamol administration [11, 61, 83]. This effect has also been shown to occur 13 following exercise in both asthmatic and non-asthmatic individuals, [11,61] however, any additive effect of salbutamol to the normal bronchodilatory response during exercise is questionable [11, 24, 29, 48, 78]. pVAgonists and Athletic Competition For asthmatic athletes, pVagonist use allows them to compete at an elite level by minimizing the effects of asthma on performance. Of the four pVagonists that have been approved by the IOC and World Anti Doping Agency (WADA) for use in competition, salbutamol is the only one that has been shown to have an anabolic effect [44]. The 2006 WADA code [82] states that salbutamol is allowed only when a therapeutic use exemption (TUE) has been obtained in advance and that it may only be administered through inhaled means. Oral administration has been shown to have greater systemic side effects [43] and potential anabolic responses [44] and is therefore banned. Salbutamol's peak bronchodilatory effect is seen 15-30 minutes post inhalation [54]. Its effects on airway smooth muscle relaxation and resulting bronchodilation help minimize or eliminate the limitations of asthma and/or exercise induced bronchoconstriction on breathing and alveolar ventilation. There is growing concern that non-asthmatic athletes are using inhaled salbutamol in an attempt to gain a competitive edge [2]. It is speculated that by increasing airway caliber and reducing the work of breathing, a greater percentage of whole body VQI can be utilized by the working muscles and/or alter perception of dyspnea [25]. Also, inadequate ventilation has been suggested as a possible reason for the performance 14 limiting exercise-induced arterial hypoxaemia (EIAH) that is often seen in elite athletes [20], Although recent research suggests that salbutamol use does not reduce the impact of EIAH [75], anecdotal evidence suggests that non-asthmatic athletes believe in its ability to enhance performance and are using doses that substantially exceed therapeutic recommendations. This poses not only an ethical question but also raises concerns of , athlete safety. Salbutamol and Performance in Non-Asthmatics Interest in the performance enhancing qualities of pVagonists in non-asthmatics has increased in the past 10 years, likely due to the increased use of pVagonists in competition. An extensive review of literature examining (32-agonists as ergogenic aids has recently been published [47] and for this reason, this review will only focus on studies examining the effects of salbutamol. In this respect, some studies report an ergogenic benefit [7, 14, 17, 68, 78, 79], while the majority of the research suggests that acute salbutamol administration does not enhance athletic performance in non-asthmatics [11, 16, 22, 24, 29, 46, 48, 53, 61, 74, 75] (Table 2.1) Of the five studies that have shown a positive effect, three [14, 17, 79] utilized oral administration of salbutamol. Oral administration is banned due to its known anabolic effects and will therefore not be discussed. Bedi and colleagues [7] utilized a sport specific test to determine whether or not 180 \ig of salbutamol had any effects on performance. The test consisted of 45 minutes of cycling at 75% Fornax followed by a sprint to exhaustion. Salbutamol treatment resulted in an improvement in sprint time of approximately 23%. These results 15 have been questioned for the use of a non-homogenous group and in particular two outliers that affected mean data [47]. Table 2.1. Summary of studies examining ergogenic effects of salbutamol. TTE = Time to Exhaustion; PP = Peak Power; MP = Mean Power; TW = Total Work; TTC = Time to Completion Measure Administration Dose Performance Effect Reference TTE, V02max Inhaled 1200pg No Change Sandsun et al. [61] TTE Inhaled 360ng No Change Fleck et al. [22] AT & VC_max Inhaled 200pg No Change McKenzie et al. [46] VC_max, PP Inhaled 400(ig No Change Stewart et al. [74] VC_max, PP, TW Inhaled 200ug No Change Meeuwise et al. [48] 20km TT Inhaled 400(j.g No Change Norris et al. [53] VC^max Inhaled 400jig No Change Stewart et al. [75] TTE Inhaled 200 & 800 ug No Change Goubault et al. [24] TTE Inhaled 800ug Negative Carlsen et al. [11] TTE Inhaled 180ug Positive Bedi et al. [7] TW Inhaled 800pg Positive van Baak a/. [78] PP Inhaled 180^g Positive Signorile et al. [68] TTE Nebulized 0.05mg/kg No Change Heir et al. [29] 10 min MP Oral 6mg No Change Collomp et al. [16] TTE, Strength Oral 4mg Positive Van Baak et al. [79] TTE Oral 6mg Positive Collomp etai. [14] PP, MP Oral 4mg Positive Collomp etai. [17] Strength (9 weeks) Oral 16mg/day Positive Caruso et al. [12] TTE (3 weeks) Oral 12mg/day Positive Collomp etai. [15] PP (3 weeks) Oral 12mg/da Positive Le Panse et al. [38] 16 Signorile and colleagues [68] examined the effects of inhaled salbutamol (180 (ig) on sprint performance. Recreationally active male and female subjects performed two all-out sprints of 15 seconds on a bike separated by 10 minutes. The salbutamol trial showed significant improvements over placebo in peak power but no difference in total work. With respect to competitive performance enhancement, this data should be interpreted with caution for two reasons. Recreational athletes were used in this study and the inference to elite athletes would be unjustified. Secondly, several studies have since shown that salbutamol has no effect on peak power [48, 53, 75]. Both of the above mentioned studies that have reported a positive effect on performance have reported it in anaerobic type activities. Although Bedi and colleagues [7] performed 45 minutes of submaximal exercise, they only showed significant improvement in the sprint to exhaustion (-23%) with no effect on the submaximal exercise session. The majority of athletes that are using salbutamol participate in endurance sports with the top four at the Sydney 2000 Olympics being triathlon, swimming, modern pentathlon, and cycling [19]. Only one study has reported a performance enhancing effect of inhaled salbutamol in an endurance performance test. After a dose of 800 Lig, van Baak and colleagues [78] demonstrated that time complete a set amount of work cycling was 1.9% faster than following placebo. This result is questionable though as two subjects in this study were significant outliers while the majority of subjects follow the line of identity in comparing 17 trials. Other than this study, research examining the effects of salbutamol on endurance performance unequivocally demonstrates no enhancement. The validity of a test to be representative of performance is an important factor when evaluating the ergogenic effects of a treatment [34]. Majority of the research that has looked at endurance performance use one of, or a combination of, a Fc^max, Wingate, lactate threshold, or a run to exhaustion (3-5 min) test. No study to date has demonstrated an increased endurance performance using one of these measures. Although these are valuable in a laboratory setting to look at physiological changes, rarely are these measures indicative of performance in events [34]. Carlsen and colleagues [11] compared the effects of salbutamol (800 ug) to salmeterol (50 pg) in 18 male runners and cross-country skiers (Fo2max = 73.9 ml-kg"'-min"'). All subjects had normal lung function. Each person was required to perform a Vc^max test as well as run at anaerobic threshold. Results showed that although lung function (FEVi) was increased by both drugs prior to exercise when compared to placebo, there was no effect on either Vc^max or anaerobic threshold. Several other studies have shown similar effects on Fornax [22, 48, 53, 61, 75] following doses of 200 ug[48], 360 ug[22], 400 pg[53, 75], and 1200 ug[61]. It is clear that across a variety of doses, salbutamol does not have an effect on Vchmax . 18 In an attempt to reproduce the findings of Bedi and colleagues [7] in a more sport specific test, Meeuwise et al [48] examined the effects of a 200 jug dose of inhaled salbutamol on sprint performance. Seven highly trained male cyclists (Fornax = 63.5 mlkg"'-min"') performed a sprint to exhaustion following 45 minutes of continuous cycling (-70% Fornax )• Wingate peak power and total work were also measured. No effect on sprint endurance time was seen nor were there any improvements in peak power or total work following salbutamol inhalation. Although this protocol is more likely to simulate endurance performance than a Fornax test, it still does not replicate the ability of the athlete to pace himself. A recent study by Goubault and colleagues [24] examined the effects of two different doses of salbutamol (200 ug and 800 Lig) on exercise performance in a time to exhaustion test. Twelve competitive triathletes (Fo^max = 57.9 mlkg'min"1) rode to exhaustion at 85% maximal aerobic power. No differences were noted in the time to exhaustion between placebo, 200 ug and 800ug conditions (23m31s, 21m45s, and 23m 18s respectively) indicating lack of a dose-response relationship. However, the variability in these results between trials questions the reliability of the measure used for performance in these subjects and it is difficult to determine the effects of dose with only two doses. The dose-response relationship should be re-examined in a reliable and reproducible measure using three or more doses. 19 Only one other study has investigated the effects of inhaled salbutamol on a simulated sport-specific performance test [53]. Norris et al., [53] demonstrated that a 400 pg dose had no effect on time-trial performance in competitive cyclists (Fornax = 63.4 mlkg" '•min"'). Although statistically not significant, salbutamol treatment resulted in a 12-second improvement during a 20km time-trial which equates to a 0.6% difference in average performance velocity. It is unlikely that this would be performance enhancing for the subjects used, however, at the elite level, athletes are a homogenous group physiologically within an individual sport. It is suggested that an improvement of 0.3 and 1.5 times the coefficient of variance (CV) in performance at the elite level could have a worthwhile effect on increasing the likelihood of winning for an athlete who averages 1st and 10th place respectively [34]. The typical CV for top performers in simulated cycling time-trials is approximately 1-1.7% [37, 58, 59, 69]. An analysis of the 2002 Tour de France prologue time-trial (7km) shows that a 0.6% difference in velocity (~ 4 seconds) is the difference between 1st and 4th place (unpublished analysis). Furthermore, the average difference in performance velocity in speed skating competitions at the 1988 Winter Olympics was 0.3% between 1st and 2nd place finishers, and 1.3% between 1st and 4th places [71]. If 30 seconds (1.5% improvement in speed) was used as a difference that would have a competitively significant effect for a 20km time-trial, retrospective analysis of the data from Norris et al, [53] would show that sample sizes utilized were inadequate to detect a difference that may have competitive significance. For statistical power of .80 and an alpha level of 0.05, 241 subjects would have been required to show an increase of 30 seconds as a significant 20 improvement in performance. For the sample size that was used (15), the standard deviation of the sample would need to be approximately 45 seconds, which is much more homogenous than the observed 186 seconds. Although it is extremely difficult to have sufficient statistical power with athletes to detect the smallest difference that may have a competitive effect (0.3-0.6 multiple of CV), this highlights the need to use as many subjects as possible while maintaining a low standard deviation. Future studies should be conscious of what is competitively significant and we suggest they be designed to detect at least and enhancement that would significantly increase the likelihood of winning for someone who averages 10th place (1.5 multiple of CV) [34]. In summary, the research clearly shows that inhaled salbutamol has no effect on endurance performance in highly trained athletes. However, the dose-response effect of salbutamol on performance has not been adequately evaluated and this needs to be assessed in a homogenous group of highly trained athletes with a sport specific performance test. Salbutamol and Doping Control The WADA code currently requests that laboratories report all cases in which the urine concentration of salbutamol exceeds 200 ng-mL"1. Regardless of whether or not the athlete has a TUE, a urine concentration of greater than 1000 ng-mL"1 is considered a doping violation due to salbutamol's anabolic effects [82]. At a competition, the athlete provides a urine sample after the event, with length of time since last inhalation not being standardized. Typically, this sample is then analyzed for the non-sulphated fraction of 21 SAL following glucuronidase enzymatic hydrolysis. This method allows for the determination of free and glucuronized forms of the drug only, and does not account for the sulphated portion. Currently the doping regulations do not specify that corrections are made for differences in urine specific gravity when analysing urine samples [65]. This would seem imperative when considering the potentially dehydrating effects of exercise. Furthermore, differences in time between inhalation and sample may affect urinary concentrations. Between 15% and 40% of the dose may be excreted in the first 4-6 hours post inhalation [21, 30, 83] and depending on both hydration and sample time, it is likely that urinary concentrations will vary. The pharmacokinetics of salbutamol are well researched and documented in both healthy and diseased populations [54]. The vast majority of urinary results are reported as a percentage of dosage, a percentage of dosage recovered, a ratio of free salbutamol and its metabolites, or as an absolute value [21, 30, 31, 77, 83]. However, little research actually reports values in concentrations as is used by WAD A. A recent examination of the dose-response effects on urinary salbutamol following 30 minutes of rest showed that inter-subject variability was quite high (36-38%) after both a single 100 pg dose and multiple doses (5 x 100 ug) [77]. Possible reasons for such high variability were thought to be due to variations in renal function and deposition of the drug in the lung between subjects. It was also shown that the absolute amount of salbutamol that was recovered in the urine was linearly related to dose inhaled. This is important to consider when analyzing urine for possible doping infractions as it will affect concentration. 22 Unfortunately, this study provided only absolute values and did not include volumes of urine samples so comparison to the WADC is impossible. A review of the literature revealed only two studies that have reported urine concentrations in their findings. In a recently published case study it was shown that inhaled salbutamol resulted in a positive doping test [65]. Schweizer and colleagues (2004) reported an in-competition measurement of 8000 ngmL"! in a male athlete with a TUE and were able to reproduce this positive test in a controlled non-exercising trial. Urine concentrations of non-sulphated salbutamol were found to be approximately 4000 ng-mL"1 urine up to 6 hours post inhalation. The majority of this was glucoronized salbutamol (up to 3400 ng-mL"1) with the remainder being free salbutamol. The subject in this case study was using three doses of three inhalations each (100 pg salbutamol/dose) over a period of 5 hours prior to the urine sample. This may be classified as a common treatment for asthma in sport [65] yet would appear to result in a positive doping infraction. Other similar cases have been reported in a variety of sports, all with urinary concentrations between 1000 and 3000 ng-mL"1 following exercise [45]. False positives may be a result of the previously mentioned interindividual differences in renal function and/or lung deposition [77] or in exercising cases, it may be due to dehydration from exercising in hot, humid environments [45] . Furthermore, exercise following inhalation increases lung absorption of pVagonists in healthy individuals [64]. In the second study, Ventura and colleagues [80] examined the effects of inhaled and oral administration of salbutamol on urine concentrations in swimmers post-training. Urine 23 concentrations of salbutamol (non-sulphated) following inhalation of a 200 jig dose were reported to be between 100 and 600 ng-mL"1 within one hour post-training (approximately 2-3 hours after drug administration). Similar values were found when the dose was increased to 1600 ug over the 4 hours prior to exercise. These values are much lower than those reported in the previously mentioned case study [65] and fall within the allowable limits, however, they still demonstrate high variability between subjects and would constitute a reportable doping result. Furthermore, they do not follow the linear dose-response relationship expected with increased dosage suggesting inconsistencies in urine analysis of salbutamol. Despite the wealth of research on salbutamol, there lacks a clear description of the dose-response effect on urine concentrations at different time intervals post-inhalation for both rest and exercise. Summary and Future Directions for Research The use of salbutamol in elite sport is on the rise and there are concerns of increased use by non-asthmatics in order to gain a competitive edge. Furthermore, there is anecdotal evidence of athletes using greater than the recommended therapeutic dose which raises both ethical and safety concerns. Although the majority of research suggests salbutamol has no performance enhancement in non-asthmatics, most studies have used non-specific laboratory measures rather than a test that effectively replicates sport performance. There is a need to re-examine the dose-response relationship using a sport-specific performance test with a homogenous group of highly trained athletes. 24 There is also limited data describing the effects of dose on urine concentrations of salbutamol at rest and after exercise. Recovery of salbutamol in the urine is highly variable between subjects [77, 80] which may help explain reports of positive doping violations for salbutamol when using therapeutic doses [45, 65]. There lacks a clear description of the dose-response effect on urine concentrations of salbutamol at specific time intervals post-inhalation following both rest and exercise. Future research should be directed at providing a description of these responses with respect to criteria used in doping control. 25 CHAPTER 3 - 20KM TIME TRIAL RELIABILITY Introduction Determining the effect of a treatment on exercise performance enhancement in athletes is best accomplished when two criteria have been met [32, 34, 58]. The first is to utilize a test that shows a strong relationship between competitive performance and performance in the test [34]. Laboratory based tests considered to have the highest performance validity in cyclists are simulated time trials that optimize the ability of a cyclist to best reproduce the shifting, inertia, and performance on the cycle ergometer [58]. Air braked ergometers that attach to the athletes own bike have provided the lowest typical error when comparing test results to performance [58]. The second criterion is that the test is highly reproducible to avoid large sample sizes and to detect small differences [34]. At the elite level small differences in performance can result in significant changes in placing. Both the Kingcycle and the Cyclosimulator, ergometers that attach to the cyclists own bikes, have been shown to be highly reproducible during simulated time trials with coefficients of variation (CV) of 1.0 or less [37, 58, 69]. Mean power in indoor time trials tends to demonstrate a higher CV (1.5% - 2.3%) when measured using either a Kingcycle or a Schoberer Rad Messtechnik (SRM) powermeter [70]. A new ergometer, the Velotron Pro, which uses a fully adjustable bike frame and is electronically braked rather than air braked, avoids some of the inherent problems with attaching a bike to a roller system: These include ensuring consistent air pressure in the rear tire, differences A version of this chapter has been accepted for publication. Sporer, B.C. & McKenzie, D.C (2006). Indoor Time Trial Reliability using the Velotron. International Journal of Sports Medicine. 26 in bearing friction between bikes, and controlling of movement of the cyclist during a test to avoid differences in rolling resistance from the calibration position. The reliability of the Velotron Pro has yet to be evaluated with respect to time trial performance. Several researchers have examined the predictive ability of peak power (Ppeak) achieved during an incremental exercise test in determining time trial performance [5, 28, 37]. In a laboratory setting, Ppeak has been shown to be highly related to 40km time trial performance [37]. In outdoor trials however there exists discrepancy. Hawley and Noakes [28] have reported Ppeak to be a strong predictor of 20km cycle time (r = -0.91) while others have shown it to be a poor predictor of performance time (r = -0.46) but an excellent predictor of mean power output (r = 0.99) [5]. Differences between indoor and outdoor predictability is not surprising as the majority of ergometers calculate speed from power output and demonstrate colinearity between the two (r = 0.999) [57] while other factors such as frontal area, rolling resistance, and topography are either held constant or not included. Smith and colleagues [70] showed mean power output, during lab based and outdoor 40km time trials, was not significantly different (303 W vs 312 W) even though performance time varied by more than 3 minutes. The relationship found between peak power in an incremental test and performance time in indoor time trials is likely due to a strong relationship between peak power and the mean power produced during the time trial. This has yet to be clarified in the literature. The purposes of this study were to determine the reproducibility of a laboratory based 20km cycle time-trial performance test in competitively trained cyclists using the .27 Velotron Pro cycle ergometer and to examine the relationships between Ppeak achieved in an incremental exercise test and time trial performance (time and power). Materials and Methods Subjects Twenty competitive, male cyclists participated in this study (mean ± SD: age = 31 ± 8 y). Subjects were determined to be competitive based on their ability to compete at the provincial level (Category 2 or higher for road cyclists and Pro/Elite for mountain bikers) with the average number of years competing being 9 ± 5 years. All subjects were required to have a maximal aerobic power (Vc^max) of at least 60 mlkg'-min"' or 5.0 L-min"1. This study was completed primarily during the off season and a period of training for local cyclists that averaged a volume of 274 ± 96 km-wk"1. Three subjects completed this study at the end of their competition phase. A medical history questionnaire and written informed consent were obtained from all subjects and procedures were approved by the University of British Columbia's Clinical Research Ethics Committee on Human Experimentation. Study Design This study utilized a repeated measures design. Each subject came to the lab on 4 different occasions at approximately the same time of day with a minimum of 72 hours between each visit. All trials were completed within a period of four weeks. Subjects were asked to refrain from intense exercise within 24 hours prior to each testing session and refrain from consuming food or caffeine for 3 hours prior. Cyclists were also 28 requested to maintain a consistent diet 24 hours prior to each testing session and instructed to prepare for each time trial as they would normally for a race. A self-selected warm-up of 30-45 minutes was used for each testing session and although this differed between subjects, the same warm-up was used prior to each test for any given subject. The first visit included medical screening and an assessment of Foimax. Height and weight was collected at the start of each visit. The remaining three visits involved a 20km simulated cycle time trial with each test being performed at the same time of day. Maximal Aerobic Power Test A Fo^maxtest was performed on the Velotron Pro cycle ergometer (Racermate Inc, Seattle). Prior to each test, factory calibration was verified using the Accuwatt "run down" verification program (Racermate Inc, Seattle) accompanying the ergometer software. Subjects were fitted to the ergometer based on the setup of their own bicycle. All settings were recorded and used in subsequent time trials. Bike settings included both seat and handle bar height and horizontal position, as well as crank length. Subjects were instructed to remain seated throughout the test. A 30 W-min"1 ramp protocol was utilized and controlled via the Velotron Coaching, Software (Version 1.5.186, RacerMate Inc, Seattle) with expired gases collected and analyzed every 15. seconds (TrueOne 2400 -Parvo Medics, Utah). Oxygen consumption (V02), minute ventilation ( VE ), production of carbon dioxide (VcOi), and respiratory exchange ratio (RER) were recorded. Air flow and gas calibrations were performed prior to each test using a 3 L calibration syringe and gases of known concentrations respectively. Standard indicators for achieving 29 Fornax were used including volitional fatigue, a plateau in Fc^with increasing work rate, HR > 90% of age predicted maximum, and a RER > 1.15. Vcnmax was recorded as the mean of the two highest consecutive 15-second samples. Heart rate (HR) was measured by telemetry (Polar Vantage XL, Kempele, Finland) and recorded. Peak power was recorded as the highest completed 15 second interval with power recorded in 7.5 watt intervals. Simulated 20km Time Trial All time trials were performed on the Velotron Pro cycle ergometer which was calibrated prior to each test as described previously. Subjects were required to perform 2 laps of a 10km course designed using the Velotron 3D software accompanying the ergometer (Version NB04.1.0.2101, RacerMate Inc, Seattle). The course was flat with no active wind effect. Subjects were able to change gears using the ergometer's electronic gearing system. A gearing system simulating a 53/39 front chain ring setup and 23/21/19/17/16/15/14/13/12/11 rear cog set was used. Throughout the time-trial, subjects were able to watch themselves racing the course on the computer monitor. Distance traveled and gear selected were displayed while all other feedback (speed, HR, power, and time) was blinded to the subject, although they were recorded by the ergometer software and downloaded afterwards for analysis. Subjects did not receive any information as to how well they performed until all three time trials were completed. Throughout the test, subjects were not required to remain seated and were permitted to drink water ad libitum. 30 Total time to completion (Ttot), time for each 10km lap (TLI and T1.2), mean performance velocity (VEL), mean performance power in watts (Pmean), and mean relative performance power in watts/kg (Prei) were recorded for each time trial. Data Analysis Mean values for all performance variables were compared using a one way repeated measures analysis of variance. CVs between trials were calculated for the log-transformations of each variable measured as described by Hopkins and colleagues [34]. Relationships between trials were calculated using Pearson's product moment correlations. The relationships between peak power and both Ttot and Pabs were examined for TT1 only as both tests were completed during the same week. A multiple linear regression was performed to determine the predictive capability of peak power and Pabs for Ttot- Reliability and reproducibility statistics were performed using an Excel (Microsoft Corporation) spreadsheet [33] with confidence intervals being set at 95%. Analysis of variance and regressions were performed using Statistica software (Version 5.0, StatSoft Inc.). For all tests, a was set at 0.05 and results shown are mean ± SD unless otherwise noted. Results Maximal aerobic power test All subjects met the required minimum Fc^max criteria of 60 mlkg"'min"' with a mean value of 68.5 ± 3.6 ml-kg"1-min"1 (absolute 5.25 ± 0.61 L-min"'). Mean absolute and 31 relative Ppeak was 469 ± 33 W and 6.16 ± 0.49 W/kg respectively while peak HR was 186 - 9 bpm. 20km time-trial performance . Measured variables for each trial are shown in Table 3.1. There were no statistical differences in any measured variables across trials. Mean performance time was slightly faster during TT1 than both TT2 and TT3 with the mean difference equal to approximately six seconds (0.10 min) however this was not statistically significant (p=0.33). This difference was predominantly due to a lower TLI in TT1 compared to TT2 and TT3 which was also not statistically significant (p=0.47). Fig. 3.1 shows no apparent trend for one trial being faster than the others. Table 3.1. Measured Variables During each 20km Time Trial Performance: Mean ± SD for Total Time (Ttot), First and Second Lap Times (Tu and TL2), Mean Velocity (VEL), Heart Rate (HR) and Absolute and Relative Power Output (Pmean and Prei). Ttot TLI TL2 VEL HR Pmean Prel (min) (min) (min) (km/hr) (bpm) (W) (W/kg) TT1 30.03 ± 1.24 14.93 ±0.71 15.10 ± 0.56 40.0 ± 1.7 171 ±8 326 ±35 4.27 ± 0.35 TT2 30.12 ± 1.21 15.01 ±0.73 15.11 ±0.55 39.9 ± 1.6 170 ±9 323 ±35 4.24 ± 0.42 TT3 30.14 ± 1.21 15.03 ±0.71 15.11 ±0.55 39.9 ± 1.6 170 ±7 . 322 ± 34 4.23 ± 0.42 * - denotes statistical difference between trials, p < 0.05. 32 4 ^ 2H E © o c m E I 0) CL G CD. <3) s-.s EE o o c 0) w ffl 5 <s 3. -4 o « • < 5 0 -2 H © o 0 Q 9' rs • 8 9 S I • • 0 • • J » 1 1 1 1 1 1 1 1 1 1 +2SD Mean -2SD Subject* Fig. 3.1. A Bland-Airman style plot showing individual performance times for all three time trials (TT1, TT2, TT3). All reliability measures are reported in Table 3.2. Ttot was highly reproducible and strongly related between TT1 and TT2 (CV= 0.8%; r = 0.96) as well as TT2 and TT3 (CV - 0.7% r = 0.97). When separated into the first and second lap (TLi and TL2), the CV with respect to time to complete lap one was noticeably larger between TT1 and TT2 (2.1%) than TT2 and TT3 (1.3%). Power output (Pmean) demonstrated a higher CV than performance time between trials as did HR and both were strongly related between trials (Table 3.2). 33 Table 3.2. Reproducibility Statistics Including Change in Means (A Means), Coefficient of Variance (CV) and Pearson Correlation Coefficients (r) along with 95% Confidence Intervals (C.I.) for TT1, TT2, and TT3. A Means (units) CV (%) r (95% C.I.) (95% C.I.) (95% C.I.) Ttot (min) TT1 vs TT2 0.09 (-0.06; 0.25) 0.8 (0.6; 1.1) 0.96 (0.90; 0.98)* TT2 vs TT3 0.02 (-0.11; 0.15) 0.7(0.5; T.0) 0.97 (0.92; 0.99)* TLI (min) TT1 vs TT2 0.06 (-0.07; 0.20) . 2.1 (1.6; 3.1) 0.79 (0.52; 0.91)* TT2 vs TT3 0.03 (-0.04; 0.11) 1.3 (1.0; 1.9) 0.92 (0.80; 0.97)* TL2 (min) TT1 vs TT2 0.03 (-0.05; 0.11) 0.8 (0.6; 1.2) 0.94 (0.88; 0.98)* TT2 vs TT3 0.00 (-0.10; 0.11) 1.0 (0.8; 1.5) 0.93 (0.85; 0.98)* Pmean (watts) TT1 vs TT2 -3 (-7; 2) 2.1 (1.6; 3.1) 0.96 (0.91; 0.99)* TT2 vs TT3 -1 (-5; 3) 1.9(1.4; 2.8) 0.97 (0.91; 0.99)* HR (bpm) TT1 vs TT2 -2 (-4; 0) 2.0(1.6; 3.0) 0.90 (0.75; 0.96)* TT2 vs TT3 i (-i; 2) 1.4(1.1 2.1) 0.95 (0.86; 0.98)* * - Denotes statistically significant relationship, p<0.05 Relationships between peak power and performance Fig. 3.2 shows the relationships between Ppeak and both Ttot and Pmean for TT1. Peak power was significantly correlated to Ttot (r = -0.89, p<0.05) and Pmean (r = 0.91, p<0.05), while Pmean demonstrated colinearity with Ttot (r = 0.996, p<0.05). Multiple linear regression demonstrated that Pmean primarily accounted for predictability of Ttot (R = 0.993) by the equation T,ot (min) = 40.96 - 1.1 (Pmean) + 0.06(Ppeak). 34 Fig. 3.2. Relationships between peak power during an incremental exercise test (Ppeak) and (a) performance time (Ttot) and (b) mean power (Pmean) for TT1 (n=20). Lines represent 95% confidence intervals. 35 Discussion The main finding of this study was that in trained, competitive cyclists, completion time in three 20km time trials performed a minimum of 72 hours apart on the Velotron Pro cycle ergometer are not significantly different from each other. It was also demonstrated that performance was highly reproducible with respect to time, power, and heart rate. Total performance time demonstrated the lowest CV between trials (0.8% or less) with power and HR being slightly higher (<2.1% and <2.0% respectively). Often a familiarization trial is suggested when doing lab based performance tests. The Ttot data, and that of others [57, 70], do not suggest this is necessary in competitively trained cyclists. Although none of the subjects had used this ergometer before, all had previously used other ergometers, completed time trials, and trained with sustained of efforts of approximately 30 minutes. Furthermore, subjects reported the feeling of riding on the ergometer as being similar to riding on the road. Subjects were required to pace themselves based on perceived effort rather than heart rate, speed, or power. As an index of exercise intensity, HR did not vary between trials and demonstrated a CV similar to that previously reported when HR feedback was provided [70], suggesting trained cyclists are capable of pacing themselves without feedback. The higher CV seen for TLi between the first and second trials (2.3%) is likely due to differences between the resistance produced by electronic gearing system and the equivalent gear on a bicycle over flat ground. Some subjects reported relying on gear selection initially for pacing but soon afterwards switched to perceived exertion. In competitive cyclists, a full 36 familiarization trial may not be necessary when using the Velotron, but we recommend an opportunity to become familiar with the resistance produced by the gearing system. The high reproducibility of performance time across trials is comparable to that found in other reliability tests using air braked ergometers [37, 57, 70]. Over both 20 and 40km distances, performance time has been shown to have a CV of 1.1% and 1.0% respectively [57]. Following a familiarization trial, Laursen and colleagues [37] demonstrated a CV of 0.9%o in time to complete a simulated 40km time trial using the Cyclosimulator (Cateye) air braked ergometer. Using a Kingcycle ergometer and over three 40km trials, Smith et al. [70] reported a similar CV (0.7% and 0.9%). An important aspect for evaluation of a performance test is that it is more reliable than the event itself [34]. When compared to reported values for outdoor trials (1.1% - 2.2%) [70], indoor trials appear to demonstrate higher reproducibility. This is likely due to the control of several factors that can affect speed (wind, topography, temperature, rolling resistance, and aerodynamics). Rather than time or speed, mean power is apt to be a better variable for comparisons between indoor and outdoor efforts as it represents the performance capabilities of the cyclist. Indeed, when compared between the two, mean power (SRM) does not vary over 40km, with each demonstrating similar CV (indoor = 1.9% - 2.1%; outdoor = 2.1% - 2.4%) [70]. These values are similar to the CV shown in the present study (2.1 % and 1.9%) for TT1-TT2 and TT2-TT3 and suggest that the Velotron provides a reliable measure of power output over 20km. Further research is necessary to determine the validity of mean power using to the Velotron to mean power produced during an outdoor time trial. 37 Although power demonstrated a higher CV than performance time, it should not be assumed that power is a less reliable variable. The relationship between power and speed during cycling is non-linear and is described by the equation P = kV" where P = power, V= velocity while k and n are constants for a particular ergometer [34]. Hopkins et al. [34] simplify this equation to demonstrate that the percent change in power is approximately equal to the percent change in speed multiplied by a factor of n [100AP/P ~ n(100AV/V)]. Unfortunately we do not know the value of n for the Velotron but reported values for other ergometers range between 1.5 and 2.2 for speeds around 40km-hr"1 [34]. Assuming a similar value for the Velotron, the CV for Pmean would be expected in comparison to the CV for Ttot-The second purpose of this study was to examine the relationships between Ppeak, Pmean, and Ttot. It has previously been suggested that Ppeak is a good predictor of time trial performance [28, 37]. In a laboratory setting, Ppeak has been shown to be related to 40km time trial performance [37] (n=43) and is in agreement with our findings over 20km (r = -0.89). However, regression analysis suggests that the predictability of Ttot is primarily due to Pmean rather than Ppeak, as it accounted for ~ 99% of the variance. This is not unexpected as the majority of ergometers calculate speed from power output and demonstrate colinearity between the two (r = 0.999) [57]. With respect to outdoor trials, Hawley and Noakes [28] reported Ppeak to be highly related to 20km cycle time (r = -0.91) which is surprising considering all the factors that can influence speed during cycling. Their results are likely influenced by the heterogeneity of the subjects (Ppeak - 175 - 440 38 W; Ttot ~ 31 min - 45 min). With a more homogenous group of cyclists (Ppeak = 304 -480 W; Ttot ~ 21 min - 25 min), Balmer et al. [5] demonstrated a weak relationship between the two (r = -0.46) over 16.1 km, but Ppeak was an excellent predictor of mean power output (r = 0.99) [5]. This coincides with the relationships demonstrated between Ppeak and Pmean during an indoor trial in the present study. Rather than suggesting that Ppeak is a predictor of performance time, we echo the comments of Balmer et al. [5] that Ppeak is a good predictor of Pmean. Further, any relationships between Ppeak and performance time are dependent on factors that affect the conversion of power output into speed. An important aspect when evaluating reliability of a performance test is a clear distinction of the population the test is designed for [32]. The cyclists used in this study were defined as trained, competitive male cyclists based on their ability to compete at a provincial level or higher. The time taken to complete the 20km time trial averaged 30.1 minutes which equates to an average speed of 39.9 km/hr. This is slower than what would be expected for competitive cyclists during time trial events (>43km/hr) [52] and is likely due to the calculation of speed from power in the software. Input of mean power to a commonly used web-based speed calculator [18] resulted in an average speed of ~ 44 km/hr, similar to typical speeds seen in time trials reported by the cyclists in this study. On a physiological basis, they are comparable to competitive cyclists previously defined in the literature [73, 86]. Others have reported higher values in relative Fcfemax and relative peak power in professional male cyclists [36, 39, 52, 56], however, the number of these cyclists across the world is relatively small. We believe that the cyclists used in this 39 study represent the most plausible highly trained group from which sufficient sample size (>20) could be obtained for future studies examining performance enhancement. Furthermore, the CV demonstrated for Ttot and Pmean appears to be equal to or better than that demonstrated for actual performances [70] which is important when evaluating the applicability of interventions [34]. In conclusion, the present study has shown that a flat 20km time trial performed on the Velotron Pro cycle ergometer is highly reproducible over three trials in competitive cyclists and comparable to other frequently used ergometers. Although the results do not suggest a familiarization trial is necessary, we recommend cyclists become accustomed to the gearing system as it does not appear to reproduce speeds found on the road. Furthermore, there is strong relationship between Ppeak and sustainable power during time trials and this relationship is primarily responsible for the predictability of performance time for ergometer based time trials. Predictability of outdoor performance from Ppeak is questionable. 40 CHAPTER 4 - DOSE RESPONSE OF SALBUTAMOL AT REST Introduction For several years pVagonists have been approved by the World Anti Doping Agency (WADA) for use in competitive sport by athletes experiencing asthma and/or exercise induced bronchospasm (EIB). Salbutamol (SAL) is one of the approved pVagonists and was the most commonly used asthma medication in athletes selected for doping control at the Sydney 2000 Olympic Games [19]. The doping code specifies that administration of SAL must be through inhaled devices as oral administration may potentially have performance enhancing anabolic effects [12, 44, 79]. As applications for therapeutic use exemption (TUE) of SAL have been increasing [2], there are concerns that it may be used as an ergogenic aid by both asthmatics and non-asthmatics and is therefore monitored closely by WADA through urine sampling. Currently WADA requests that laboratories report all cases in which the urine concentration of SAL (cSAL) exceeds 200 ng-ml"1. Regardless of whether or not the athlete has a TUE, a urine concentration of greater than 1000 ng-ml"1 (nonsulfated) is considered a doping violation [82]. This is likely due to previously published research indicating values over 1000 ng-ml"1 are only observed following oral administration [80]. A recently published case study has questioned whether or not this cut off point is appropriate as it may result in a false-positive doping test and subsequent 2-year ban from competition [65]. Schweizer and colleagues [65] reported an in-competition measurement of 8000 ng-ml"1 in a male athlete with a TUE and were able to reproduce A version of this chapter has been submitted for publication. Sporer, B.C., Sheel, A.W., Taunton, J., Rupert, J.L., & McKenzie, D.C (2006). Variability in Urine Concentrations of Salbutamol: Implications for Doping Control. International Journal of Sports Medicine. Submitted September 2006. 41 this excessive test in a non-exercising trial. This is in agreement with other reports of positive test results using therapeutic doses, all with urine concentrations between 1000 and 3000 ng-ml"' following exercise [45]. High inter-subject variability (-38%) has been shown in urine recovery of SAL [77] and this may in part explain the recent occurrence of false-positive tests. It is possible that differences in renal function, lung absorption, and/or dehydration from exercise [45] are responsible for the high variability. Furthermore, differences in time between inhalation and sample collection may affect urine concentrations. Between 15% and 40% of the dose may be excreted in the first 4-6 hours post inhalation [21, 30, 83] and depending on hydration status, cSAL may vary. Currently WADA does not correct for hydration and only requires that urine samples have a minimum specific gravity (SG) of 1.005. Correcting urine samples for SG may provide insight as to the effects of hydration on cSAL. The pharmacokinetics of SAL are well defined and documented in both healthy and diseased populations [54]. The vast majority of urinary results are reported as a percentage of dosage, a percentage of dosage recovered, a ratio of free SAL to its metabolites, or as an absolute value [21, 30, 31, 77, 83]. However, little research actually reports values in concentrations as used by WADA. Despite the wealth of research on SAL, there lacks a description of the dose-response effect on urine concentrations at different time intervals post-inhalation. Therefore, the purpose of this study was to examine the dose-response relationship of urine SAL concentrations while resting at 30, 60, and 120 minutes post-inhalation. A secondary purpose of this study was to correct 42 urine samples for hydration status using a specific gravity measure and compare these values to the current WADA doping criteria. Materials and Methods Subjects Healthy, male subjects (n=8) aged between 19 and 35 years were recruited for this study. All subjects were not previously diagnosed with asthma, or any other lung disease and had normal lung function with FEVi > 80% of the predicted value (ATS criteria [72]). Each subject was required to perform a eucapnic voluntary hyperpnea (EVH) challenge test to confirm no susceptibility to bronchospasm. This test has previously been described in detail [2] and is one of the allowable methods by the International Olympic Committee to provide evidence of need for use of asthma medication during competition. Written informed consent was obtained from all subjects and procedures were approved by the University of British Columbia's Clinical Research Ethics Committee on Human Experimentation. Study Design A randomized, non-blinded, repeated measures design was used with 3 different treatment protocols; 200 pg (D2), 400 pg (D4), and 800 pg (D8) of inhaled SAL. Each subject came to the lab on 3 different occasions with a minimum of 72 hours between each visit. On each day, subjects received one of the three doses of SAL and provided urine samples at 30, 60, and 120 minutes post inhalation (T30, T60, and T120 respectively). An additional pre-treatment urine sample was provided on Day 1 to act as 43 a baseline measure for all conditions and confirm subjects were not currently taking SAL. Spirometry manoeuvres following ATS criteria [72] were also completed prior to inhalation and at T30, T60, and T120 to confirm drug delivery and action. Days 1-3 - Drug Administration and Urine Collection AH subjects were asked to refrain from intense exercise for 24 hours prior to each testing session. As the primary purpose of this study was to relate the findings to both in and out of competition testing, control for ingestion or food and water did not occur. Subjects were only asked to avoid alcohol or caffeine containing drinks for at least 12 hours prior to each testing session. SAL was administered using a metered dose inhaler (MDI) and spacer with each subject receiving training on proper use prior to starting the study. To avoid any potential side effects of the propellant, inhalations were done in sets of two with a period of 30 seconds used between each set. Each condition required 8 total inhalations of either 100 pg of SAL or placebo. The exact number of each was dependant on the condition with the required number of SAL inhalations administered first. Throughout the two hour period, subjects remained seated and were allowed to drink ad libitum. Subjects were asked to provide a urine sample of ~15 ml at T30, T60, and T120. It was requested that the sample be obtained mid-stream and that the bladder was voided of urine after each sample. Once the urine sample was obtained, specific gravity (SG) was measured using a refractometer (Pocket PAL-1 OS, Atago, USA). All samples were then frozen to -20° C until laboratory analysis. 44 Urine Analysis Samples were analyzed by a third party laboratory for SAL concentration using a hydrolysis method, accounting for non-sulfated forms (free and glucuronized forms only) The non-sulfated portion is the value measured by WADA at the time of this study [82]. Concentrations were determined by liquid chromatography-mass spectrometry. Urine was incubated with glucuronidase (from Helix pomatia, Sigma-Aldrich Co, St. Louis, MO, USA) at 37° C for 2 hours prior to addition of the internal standard. The internal standard (propionylprocanamide) was added to 1ml of the urine specimen. The mixture was acidified with 0.5 ml 10% trichloroacetic acid and 8 ml chloroform added. The mixture was vortexed, centrifuged and the aqueous phase recovered for SAL assay. The mass spectrometry instrument (Agilent model 1100 MSD) was coupled to a liquid chromatograph (Agilent model 1090), with both instruments controlled by Agilent ChemStation software. The mobile phase used for the chromatography was 10 mmol/L aqueous ammonium acetate adjusted to pH 3.2 and acetonitrile (95:5 ramping to 75:25) and the column employed was an Eclipse XDB-C8 (4.6 mm x 30 mm x 3.5 pm) (Agilent Technologies, Wilmington, DE, USA). Primary ions used for the quantitation were 240 m/z (SAL) & 292 m/z (propionylprocanamide). Flow rate for LC MS was 0.3 mlmin"1 with a retention time for SAL of 2.30 minutes. Concentrations were determined by comparison to a standard curve of the relative intensities of the SAL ion to that of the internal standard ion for standard solutions of the drugs prepared in drug free urine. 45 To account for differences in SG between samples and to compare values to those that might be observed in a doping control situation, all samples were adjusted for SG using the following equation [50]: SG-corrected cSAL = raw cSAL • ((SGtarget- 1.0)/(SGsampie - 1.0)) where SGtarget refers to the SG to which values are to be adjusted, while SGsampie refers to the actual SG of the sample. Corrections for SG targets of 1.005 and 1.025 were calculated. The lower value represents the minimum acceptable value for a doping control sample [81] while the higher value would be considered to be representative of moderate dehydration in athletes [55] and has commonly been seen following exercise in our laboratory. Data Analyses Means and standard deviations were computed for all descriptive variables. Urine concentrations of SAL and spirometry measures were compared across dose and time using repeated measures ANOVA. Post-hoc analyses were performed using Tukey's test for honest significance when a significant main effect was found. All statistical procedures were performed using Statistica software (Version 5.0, StatSoft Inc.) with a set a 0.05. Data are presented as means ± standard deviation. Results Subjects Eight subjects with a mean age, weight, and height of 27.9 ± 5.9 years, 77.4 ±5.4 kg, and 179.4 ± 5.1 cm respectively completed this study. FVC (5.58 ± 0.60) and FEVi (4.46 ± 46 0.41) were 101.3% and 97.2% of predicted values. All subjects had a negative EVH test with a mean maximal drop in FEVi of 5.65 ± 3.84%. Dose Response Effects Each dose demonstrated a significant enhancement of FEVi over baseline values at each time point post-inhalation confirming delivery and action of the drug. All baseline urine samples returned a cSAL value of zero and were therefore excluded from the remainder of the analysis. There was no difference in SG across doses (D2=1.011 ± 0.011, D4=1.012 ± 0.008, D8 = 1.012 ± 0.010) however, SG did decrease across time becoming significantly lower at T120 (Table 4.1). Table 4.1. Specific Gravity for all Urine Samples at 30, 60, and 120 Minutes (T30, T60, T120 Respectively) Post-Inhalation of Salbutamol. T30 T60 T120 Mean 1.015 1.011 1.009a SD (0.009) (0.010) (0.009) Min 1.002 1.001 1.002 Max 1.029 1.032 1.028 a - denotes significant difference from T30; p<0.05 As shown in Table 4.2, cSAL of urine samples (uncorrected for specific gravity) increased as dose increased with dose D8 being significantly greater than D2 at each time interval. No effect of time was demonstrated although the trend was for cSAL to peak at T60 for each dose. Large variability existed in cSAL across all doses with a minimum of 0 ng-ml"1 and a maximum of 904 ng-mf1 (Table 4.2). The variability of individual samples is shown in Fig. 4. la and of note is that no samples exceeded 1000 ng-ml 48 Table 4.2. Urine Concentrations of Salbutamol (non-sulfated) at 30 (T30), 60 (T60), and 120 Minutes (T120) Post-Inhalation of 200ug (D2), 400ug (D4), and 800>g (D8) of Salbutamol. Mean, SD, Minimum (Min), and Maximum (Max) for Raw and Corrected for Specific Gravity (SG) Values are Reported in ng-ml"1. D2 T30 D4 D8 D2 T60 D4 D8 D2 T120 D4 D8 Mean 58 173 196 + 66 181 272 + 21 74 194 + SD (77) (192) (142) (62) (159) (288) (31) (60) (176) Raw Min 0 29 83 0 21 47 0 0 31 Max 189 636 519 157 529 904 . 74 167 562 Mean 19 62 81 + 41 85 151 a'+'* 15 52 125+'* Corrected to SD (27) (47) (37) (53) (65) (58) (34) (46) (99) 1.005 SG Min 0 5 36 0 31 59 0 0 35 Max 79 141 138 157 228 230 98 143 347 Mean 98 309 406 + 206 423 754 a'+'* 74 259 624 +'* Corrected to SD (135) (237) (183) (266) (324) (291) (171) (232) (495) 1.025 SG Min 0 24 178 0 157 294 0 0 173 Max 394 706 692 785 1142 1150 492 713 1733 a - denotes significant difference from T30 at same dose, p<0.05 + - denotes significant difference from D2 at same time, p<0.05 * - denotes significant difference from D4 at same time, p<0.05 49 ^ 1500 H o S 8 D2T30 D2T60 O2T120 O4T30 04TBO D4T120 08T30 O8T60 D8T120 b) 8 2 S 8 8 $ 8 § 9' —i 1 1—i—i—i—i—i 1— D2T30 D2T60 D2T120 D4T30 D4T60 D4T120 O8T30 D8T60D8T12O Condition t> 1000 • 8 8 D2T30 D2T60 D2T120D4T30 D4T60 &4T120 D8T30 DBT60DBT1M Condition Fig. 4.1. Individual Urine Concentrations of Salbutamol (cSAL) for Raw Samples (a), Samples Corrected to Specific Gravity of 1.005 (b), and Samples Corrected to a Specific Gravity of 1.025 (c). Individual Samples are Shown for 30 minutes post (T30), 60 minutes post (T60), and 120 minutes post (T120) for Doses of 200pg (D2), 400pg (D4) and 800pg (D8). Dashed Line Represents Doping Control Limit of 1000 ng-ml"1. 50 Also noted in Table 4.2, a time effect was seen when samples were corrected for specific gravity to both the low and high targets (1.005 and 1.025 respectively) with T60 being significantly greater than T30 for dose D8 (754 ± 291 ng-ml"1 and 406 ± 183 ng-ml"1). Individual subject plots for corrected samples are shown in Fig. 4.1b-c. Of note is the change in order of the subjects from highest to lowest when compared to the raw urine samples. Corrections to 1.005, reduced the mean values across all doses with the maximum individual sample being 347 ng-ml"1 at dose D8. When corrected to 1.025, one subject exceeded the doping limit of 1000 ng-ml"1 at doses D4 and D8 with a total of five of the eight subjects producing at least one sample that was over 750 ng-ml"1. Discussion The purpose of this study was to examine the dose-response effect of inhaled SAL on cSAL while resting at 30, 60, and 120 minutes post-inhalation. A secondary purpose of this study was to correct urine samples for hydration status using a specific gravity measure and compare these values to the current WADA doping criteria. The main findings were that urine cSAL values were higher with higher doses; urinary cSAL was highly variable between subjects, and it appeared to peak at 60 minutes post-inhalation. Although none of the uncorrected samples exceeded the WADA doping control limit of 1000 ng-ml"1, when corrected to a dehydrated state using specific gravity (1.025), the maximum value observed was 1733 ng-ml"1. 51 Some of the concerns regarding SAL are that supra-therapeutic doses and/or oral doses are being used in attempts to gain a competitive advantage. Currently WADA stipulates that any urine samples containing greater than 1000 ng-ml"1 of SAL is considered an adverse analytical finding unless the athlete is able to prove the result was due to an inhaled therapeutic dose [82]. The rationale for the doping control threshold of 1000 ng-ml"1 is not clear but it may in part be based on evidence from prior research [9, 80]. Previously reported values for cSAL rarely exceed 500 ng-ml"1 following low (200 pg) and high (1600 pg) inhaled doses [80]. To our knowledge, this is the first study reporting the dose-response effect of inhaled SAL on urine concentrations as utilized in doping control. As dose increased to 800 pg, an increase in cSAL was observed at each time point (Table 4.2). This is in agreement with a recent examination of the dose-response effects of inhalation on absolute SAL excretion that reported a linear relationship with inhaled doses up to 500 pg following 30 minutes of rest [77]. While the values of Tomlinson et al. [77] are reported in absolute values, they are comparable to the present findings as urine SG was not different between doses suggesting the increases in cSAL observed were due to increases in absolute excretion. Our findings also suggest that at rest, cSAL concentrations peak at 60 minutes post-inhalation and begin to decrease afterwards. This was significant at higher doses when corrected for SG (Table 4.2). Hindle and Chrystyn [30] have shown that the rate of excretion of non-sulfated SAL following an inhaled dose of 400 pg is greatest in the first 52 hour and quickly tapers off after. They further show that the amount of drug excreted in the first 30 minutes is representative of the portion of the dose delivered to the lung. At 60 minutes, this is augmented by the portion of the dose swallowed as it becomes absorbed and available for first pass metabolism [30]. While the present data combined with excretion kinetics [30] support the idea that cSAL would continue to decrease beyond 120 minutes post-inhalation, this should not be assumed and requires further clarification. Peak values have been reported 2-6 hours post-inhalation in a recent-case study when 900 jig was administered over 5 hours [65]. Concentration can also be affected by hydration status, renal function, and individual variations in absorption and metabolism. Furthermore, the impact of the prior urine samples (T30 and T60) on subsequent cSAL cannot be discounted. While it is plausible that an athlete might pass urine post-inhalation prior to a doping control request, further work examining concentrations over longer time periods with and without repeated doses is necessary to fully characterize SAL kinetics. Although concentrations of SAL in the urine increased with dose at each time interval, no uncorrected samples exceeded the WADA threshold of 1000 ng-ml"1. Most samples presented here fall within the range demonstrated by Ventura and colleagues [80], yet cSAL was highly variable and several samples from one subject exceed 500 ng-ml"1 (Fig. 4.1a). Furthermore, one of the samples from this subject approached the WADA threshold (904 ng-ml"1). Inter-subject variability of urine recovery of SAL is high (-38%) [77] and can be affected by a variety of factors, one of which is hydration. Currently, with respect to SAL, WADA does not take into consideration hydration status 53 other than ensuring samples are not diluted by requiring SG > 1.005. Normal values for SG range between 1.005 and 1.030. To consider the impact of hydration on cSAL we measured SG and corrected each sample to a moderately dehydrated (1.025) and well hydrated condition (1.005) (Fig. 4.1b-c). When corrected to a moderately dehydrated state, three values from one subject exceeded 1000 ng-ml"1 with a maximum of 1733 ng-ml"1. Theoretically this subject could have produced a positive doping sample after a dose of only 400 ug, providing support for prior claims that dehydration may play a role in false-positive doping tests [45, 65]. Conversely, when corrected lower, mean values were consistently under 200 ng-ml"1 with a maximum value of 347 ng-ml"1. At the WADA minimum SG of 1.005 the potential for a false-negative test exists. It is interesting that after correcting for specific gravity, the peak value from the present study was still less than half of that reported by Schweizer and colleagues [65]. This may in part be due to the differences in the timing of the dose as well as the timing of the urine sample [45]. The subject identified in the case-study inhaled 300 pg at three different time points over 5 hours and the peak cSAL were from urine samples provided 2 and 6 hours after the last inhalation [65]. While SG is generally indicative of hydration and comparable to creatinine for correcting urine concentrations [50], we stress caution in applying these findings to doping control and in explaining false-positive doping violations previously reported in the literature. The relationships between hydration status and the absorption, metabolism, and excretion of SAL are complex and not well defined. It is possible that any of these rates may be altered with a change in hydration. Obtaining the volume of urine at each time point 54 (sample plus amount discarded) would assist evaluation of cSAL in doping control. Hindle and Chrystyn [30] have determined the percentage of dose recovered of both sulphated and non-sulphated forms at various time points post-inhalation. This information could be used in conjunction with cSAL and volume to determine the absolute values of salbutamol recovered and the likelihood that a doping sample was from inhaled administration. Future work exploring these relationships is suggested. Additionally, this study was performed at rest and although athletes can be tested out of competition, the most likely scenario is to provide a urine sample following an event. Exercise has been shown to increase lung absorption of the pVagonist terbutaline in healthy individuals [64]. Whether this holds true for SAL or if there are additional effects of exercise on metabolism and excretion is unclear and requires further examination. In conclusion, urine cSAL increased with inhaled dose and peaked at 60 minutes post-inhalation. There is marked variability between individuals with respect to cSAL and this is amplified at higher doses. While no samples exceeded the 1000 ng-mf1 limit, it was approached with a dose of 800 pg and seems plausible that it could be exceeded in some individuals. The data further suggest that hydration status should be considered when evaluating doping control samples for cSAL and that future work examining the relationships between timing and amount of dose inhaled, urine volume, salbutamol excretion, and individual variations in absorption, metabolism, and excretion be conducted. 55 CHAPTER 5 - DOSE RESPONSE OF SALBUTAMOL DURING EXERCISE Introduction Optimum performance in the elite athlete can be limited by pulmonary, cardiovascular, muscular, psychological, nutritional and/or environmental factors. In asthmatic athletes and individuals suffering from exercise induced-bronchospasm, lung function is reduced, thereby possibly limiting performance capabilities [6]. Currently four P2-agonists, salbutamol (SAL), formoterol, salmeterol, and turbutalihe, have been approved by the World Anti-Doping Agency (WADA) for use by asthmatics, providing the athlete obtain a therapeutic use exemption (TUE) prior. This is normally achieved by physician confirmation; however, in order to use these medications at the Olympic Games, athletes must provide objective evidence of variable airflow obstruction. This is assessed by an independent medical committee and appropriate tests include bronchodilator response and bronchial provocation (eucapnic voluntary hyperpnea (EVH), lab/field exercise, or chemical challenge) [2]. Athlete applications for use of P2-agonists have been increasing over the past 20 years with 6.6% and 4.6% of all participants at the 2002 (Salt Lake City) [2] and 2004 (Athens) [4] Olympic games requesting their use. Of the four p2-agonists allowed, SAL is most commonly prescribed and is only allowed to be administered through inhaled means for use in competition [82]. There is growing concern that non-asthmatic athletes are using inhaled SAL in an attempt to gain a competitive edge [2]. Anecdotal evidence suggests that both asthmatic and non-asthmatic athletes believe in its ability to enhance 56 performance and are using doses that substantially exceed therapeutic recommendations. This poses not only an ethical question but also raises concerns of athlete safety. The current research overwhelmingly suggests that acute inhaled salbutamol, in therapeutic doses, does not enhance performance in non-asthmatics [10, 11, 22, 24, 48, 53, 61, 74]. The majority of studies have evaluated performance using one, or a combination of, a Fc^max, Wingate, lactate threshold, or work to exhaustion test. The validity of a test to be representative of athletic performance is an important factor when evaluating the ergogenic effects of a treatment [34]. Two studies have investigated the effects of inhaled salbutamol using a simulated sport-specific performance test [53, 78]. Norris and colleagues [53], showed a non-significant 12-second improvement in 20-km cycling time-trial performance time following a does of 400 pg. In comparison, a dose of 800 pg has been shown to decrease time to complete a set amount of work on a cycle ergometer (-1.9%) [78]. If salbutamol has an ergogenic effect, it may be related to dose. It has been shown that ventilatory response to salbutamol in both non-asthmatics and asthmatics is enhanced as dose increases [35, 42]. However, Goubault and colleagues showed no effect of dose (placebo, 200 pg, and 800 pg) on cycling time to exhaustion even though FEVi was enhanced (-5%) following salbutamol [24]. More research examining the dose-response effects of inhaled salbutamol using a sport-specific performance test is needed. Unauthorized use of SAL is closely monitored through doping control. Even for athletes possessing a TUE, a urine concentration of non-sulphated SAL (cSAL) greater than 1000 57 ng-ml'1 is considered an adverse analytical finding resulting from oral administration and can result in a two year suspension. This cut-off point has been questioned of late with recent reports of positive test results using inhaled therapeutic doses, all with urine concentrations well over 1000 ng-ml"1 following exercise [45, 65]. Although the majority of urine samples reported in the literature rarely exceed 500 ng-ml"1 [80], it has been suggested that with variations in dose, individual differences in the ability to absorb, metabolize, and excrete salbutamol, and changes in hydration status following competition, the possibility for elevated concentrations exists [45]. Previous findings from our laboratory (Chapter 4) have shown that at rest, cSAL is related to dose, highly variable between subjects, and peaks at approximately 60 minutes post-inhalation. Furthermore, individual values can approach the WADA cut-off point following therapeutic doses. It is unclear whether or not similar responses would be observed following exercise. An examination of the dose-response effect of inhaled SAL on urine concentrations following exercise as used in doping control is lacking. Although research to date has shown no significant improvement in exercise performance with the use of inhaled salbutamol, the dose-response effect on performance has not been evaluated in a homogenous group of highly trained athletes with a sport specific performance test. Therefore the purpose of this study was to examine the effects of increasing doses of SAL on 20km time-trial performance and evaluate cSAL following exercise in competitive athletes. 58 Materials and Methods Subjects Healthy, competitive male cyclists and triathletes (n=37) were recruited for this study. An a priori power calculation was performed using 1.5 times the coefficient of variance for mean power over 20km (-2% as described in Chapter 3) as the minimum improvement that will make a competitive difference. It was calculated that approximately 30 subjects were required with an estimated standard deviation of 20 W, to identify significance at 0.05 with a power of 0.80. All athletes were competing at a provincial level or higher in the elite categories for their respective sport and disciplines. Exclusion criteria included a Fc^max of less than 60 mlkg"'-min"' and 5 L-min"1, previous history or diagnosis of asthma, abnormal resting spirometry, or a positive eucapnic voluntary hyperpnea (EVH) test, indicative of exercise induced bronchospasm (EIB). Written informed consent was obtained from all subjects and the methods and protocol were approved by the University of British Columbia Clinical Research Ethics Board. Study Design A randomized, double blind, repeated measures design was utilized with 4 different treatment protocols (placebo (DP), 200 pg (D2), 400 pg (D4), and 800 pg (D8) of inhaled salbutamol). Each subject came to the lab on 5 separate occasions with a minimum of 72 hours between visits. The first visit included medical screening, measurement of height and weight, pulmonary function, and an EVH test. Qualifying subjects then performed a ramped exercise test to determine maximal oxygen consumption on the same day. The remaining four sessions involved a simulated 20km cycling time trial following one of 59 the four treatments. At the end of each time trial, athletes were required to provide a urine sample that was analyzed for concentration of non-sulfated salbutamol. See Figure 5.1 for a timeline of the study. Med. clearance EVH V02max Familiarization (if needed) 20km TT 20km TT 20km TT T 1 T2 T3 T4 20km TT Dayl Day 2 Day 3 Day 4 Day 5 Fig. 5.1. Experimental protocol timeline. Lung Function and Airway Hyperresponsiveness Prior to completing the EVH test, subjects performed baseline pulmonary function measures. This was achieved via a flow-volume loop using a Medical Graphics CPX-D Metabolic cart (St. Paul, MN) with 1070 Pulmonary Function Software. Calibration was performed prior to each testing session and subjects were familiarized with the procedure prior to actual testing. Each subject performed three trials with the highest valid FEVi recorded. A trial was considered valid if it was greater than 80% of the predicted value and was reproducible using ATS criteria [72]. Subjects were then screened for susceptibility to bronchospasm using the EVH challenge test. This test has previously been described in detail [2] and is one of the methods approved by WADA and the IOC 60 Medical Committee to provide evidence for use of asthma medication during competition. Briefly, each subject was required to breathe a hypercapnic gas mixture (5% CO2, 21% O2, balance nitrogen) for a period of six minutes at a target ventilation which was calculated as 30 times the individuals pre-test FEVi (~ 85% maximal voluntary ventilation). Spirometry was measured immediately following and at 5, 10, 15, and 20 minutes post. A decrease in FEVi of greater than 10% from baseline measure was considered to be a positive test for bronchospasm and is usually observed in the first 10 minutes. For purposes of this study, the maximum decrease in FEVi at any time point of 5 minutes post or greater was identified and recorded as a percentage drop from pre test FEV,. Maximal Exercise Test A maximal exercise test was performed on the Velotron Pro cycle ergometer (Racermate Inc, Seattle, WA, USA). Prior to each test, factory calibration was verified using the Accuwatt "run down" verification program (Racermate Inc, Seattle) accompanying the ergometer software. Subjects were fitted to the ergometer based on the setup of their own bicycle. All settings were recorded and used in subsequent time trials. Bike settings included both seat and handle bar height and horizontal position, as well as crank length. Subjects were instructed to remain seated throughout the test. A 30 W-min"1 ramp protocol was utilized and controlled via the Velotron Coaching Software (Version 1.5.186, RacerMate Inc, Seattle, WA, USA) with expired gases collected and analyzed every 15 seconds (TrueOne 2400 - Parvo Medics, Utah, USA). Oxygen consumption (V02), minute ventilation ( VE ), production of carbon dioxide (FCO2), and respiratory 61 exchange ratio (RER) were recorded. Flow and gas calibrations were performed prior to each test using a 3 L calibration syringe and gases of known concentrations respectively. Standard indicators for achieving Fo^max were used including volitional fatigue, a plateau in Vcn with increasing work rate, HR > 90% of age predicted maximum, and a second samples. Heart rate (HR) was measured by telemetry (Polar Vantage XL, Kempele, Finland) and recorded. Peak power was recorded as the highest completed 15 second interval with power recorded in 7.5 watt intervals. Dose Response Evaluation - Exercise Protocol A timeline of events for Days 2-5 is depicted in Figure 5.2. Subjects were encouraged to prepare for each time trial as they would a competitive event with no strenuous exercise in the previous 24 hours. RER > 1.15. Fc^max was recorded as the mean of the two highest consecutive 15-Treatment • placebo • 200 ng • 400 |ig • 800 ug Begin 20km TT End 20km TT End Urine Cool Down Sample Warm-up Bike Setup (30 min) Height, Weight lOmin cycle (light) J Time Zero 15 min 45 min 55 min 60 min Fig. 5.2. Timeline for treatment and time trials. 62 Warm-up was self-selected and although this varied between individuals, it was the same for each subject for all trials. Immediately following the warm-up, subjects were weighed and began receiving a treatment. A total of 8 inhalations were administered each day from 3 different coded MDI for a dose equal to one of DP, D2, D4, or D8. Spacers were used to optimize delivery of the medication and subjects were trained in its proper use prior to participation. Following administration, bike fit was confirmed and subjects were allowed to keep loose by spinning freely. At 10 minutes post-inhalation a mask (Hans Rudolph 8930 Series, Kansas City, MO, USA) and two-way breathing valve (Hans Rudolph 2700 Series, Kansas City, MO, USA) were fitted to the subject and connected to a metabolic cart (TrueOne 2400 - Parvo Medics, Sandy, UT, USA). A complete seal of the mask was confirmed prior to testing. At 15 minutes post-inhalation, subjects began the simulated 20km time trial and were instructed to complete the distance as quickly as possible. All time trials were performed on the Velotron Pro cycle ergometer which was calibrated prior to each test. This performance test has been described previously and is highly reproducible in trained cyclists with a CV of <1% for time and <2% for mean power (Chapter 3). Approximately half of the subjects were familiar with this protocol in our laboratory and those that weren't performed a familiarization trial following a rest period at the end of Day 1. Subjects were required to perform 2 continuous laps of a 10km course designed using the Velotron 3D software accompanying the ergometer (Version NB04.1.0.2101, RacerMate Inc, Seattle, WA, USA). The course was flat with no active wind effect. Resistance was adjustable using the ergometer's electronic gearing system. A gearing system simulating a 53-39 front chain ring setup and 23-21-19-17-16-15-14-13-12-11 rear cog set was used. Throughout the time-trial, subjects were able to 63 watch themselves racing the course on the computer monitor. Distance traveled and gears selected were displayed while all other feedback was blinded to the subject. Power, speed, and time were recorded by the ergometer software and downloaded afterwards for analysis. The sampling rate for all ergometer variables was 1 sample-see"1. Heart rate was also recorded by the ergometer and confirmed by telemetry (Polar Vantage XL, Kempele, Finland) throughout the time trial. Subjects did not receive any information as to how well they performed until all trials were completed. Throughout the time-trial, expired gases were collected with metabolic parameters averaged every 20 seconds. Every 2km, subjects were asked to rate the perceived exertion (RPE) for leg (RPEL) and breathing (RPED) effort using a 10-point Borg RPE scale. Upon completion of the time-trial, subjects were requested to cool down and rest until the 55 minute mark post-inhalation. Subjects were allowed to rehydrate ad libitum during this time. Urine Collection and Analysis At the one hour mark post-inhalation (T60), subjects were requested to provide a urine sample of-15 ml. It was requested that the sample be obtained mid-stream and that the bladder was voided of urine following. Once the urine sample was obtained, specific gravity (SG) was measured using a refractometer (Pocket PAL-10S, Atago, USA). All samples were then frozen to -20° C until laboratory analysis. Samples were analyzed by a third party laboratory for total cSAL using a hydrolysis method, accounting for free and glucuronized forms only. This is the value that is reported by the World Anti-Doping Agency at the time of the study [82]. Concentrations were determined by liquid chromatography-mass spectrometry. Urine was incubated with glucuronidase (from 64 Helix pomatia, Sigma-Aldrich Co, St. Louis, MO, USA) at 37° C for 2 hours prior to addition of the internal standard. The internal standard (propionylprocanamide) was added to 1ml of the urine specimen. The mixture was acidified with 0.5 ml 10% trichloroacetic acid and 8 ml chloroform added. The mixture was vortexed, centrifuged and the aqueous phase recovered for SAL assay. The instrument used was an Agilent model 1100 MSD coupled to an Agilent model 1090 liquid chromatograph, both instruments are controlled by Agilent ChemStation software. The mobile phase used for the chromatography was 10 mmol/L aqueous ammonium acetate adjusted to pH 3.2 and acetonitrile (95:5 ramping to 75:25) and the column employed was an Eclipse XDB-C8 (4.6 mm x 30 mm x 3.5 um) (Agilent Technologies, Wilmington, DE, USA). Primary ions used for the quantitation were 240 m/z (SAL) & 292 m/z (propionylprocanamide). Flow rate for LC MS was 0.3 mlmin"1 with a retention time for SAL of 2.30 minutes. Concentrations were determined by comparison to a standard curve of the relative intensities of the SAL ion to that of the internal standard ion for standard solutions of the drugs prepared in drug free urine. To account for differences in SG between samples and to compare values to those that might be observed in a doping control situation, all samples were adjusted for SG using the following equation [50]: SG-corrected cSAL = raw cSAL • ((SGtarget - 1.0)/(SGsampie - 1.0)) where SGtarget refers to the SG to which values are to be adjusted, while SGsampie refers to the actual SG of the sample. Corrections for SG targets of 1.005 and 1.025 were calculated. The lower value represents the minimum acceptable value for a doping 65 control sample [81] while the higher value is considered to be representative of moderate dehydration and has commonly been seen following exercise in our laboratory. Data Analysis Mean and standard deviations (SD) were calculated for descriptive variables. A repeated measures analysis of variance was used to determine statistical significance across treatments for all performance variables and urine concentrations measured. Post-hoc analyses were performed using Tukey's test for significance when a main effect was found. Pearson product moment correlations were used to examine relationships between urine concentrations and specific gravity. Statistical procedures were completed using Statistica Software (Version 5.0, Statsoft Inc, USA). For all tests, a was set a 0.05. Values reported are means ± SD unless otherwise noted. Results Subject Characteristics and Airway Hyperresponsiveness Characteristics of subjects with negative (n=30) and positive (n=7) responses to the EVH test are shown in Tables 5.1 and 5.2. A total of seven subjects produced a positive EVH test resulting in a prevalence rate of -19% for airway hyperresponsiveness. Maximum drop in FEVi following the EVH test was 27.7%. All positive responders were excluded from the remainder of the study. Baseline performance characteristics of remaining subjects (n=30) are shown in Table 5.3. 66 Table 5.1. Subject Characteristics for Positive and Negative Responders to a Eucapnic Voluntary Hyperpnea (EVH) Test. Values presented are Means, Standard Deviations (SD), Maximums (Max), and Minimums (Min). Group Age (yrs) Height (cm) Weight (kg) Cycling Experience (yrs) Mean 29 182.2 76.0 8 Negative EVH (n=30) (SD) (6) (6.7) (7.6) (5) Max Min 51 18 195.3 166 95 62.7 25 2 Mean 25 183.7 76.2 8 Positive EVH (n=7) (SD) (5) (6.8) (8.62) (5) Max Min 35 20 195.6 176 92.6 68.7 16 3 Table 5.2. Lung Function Measures Including Percent Predicted Values for Forced Vital Capacity (FVC), Forced Expiratory Volume in One Second (FEVi), and Fraction of FVC Expired in One Seconds (FEVi/FVC), and Decrease in FEV, (Max AFEVi) for Positive and Negative Responders to a Eucapnic Voluntary Hyperpnea (EVH) Test. Values presented are Means, Standard Deviations (SD), Maximums (Max), and Minimums (Min). Group FVC % FEV, % FEVi/FVC % Max AFEV, (L) Predicted (L) Predicted (%) Predicted (%) Mean 5.86 103.8 4.86 103.0 82.8 99.4 3.9 Negative EVH (n=30) (SD) (0.77) (11.5) (0.72) (11.5) (5.0) (6.0) (2.7) Max Min 7.01 4.56 135.8 85.0 5.98 3.55 122.1 78.9 91.5 70.4 110.0 85.0 9.8 -1.4 Mean 6.09 101.8 4.67 94.7 76.8 92.9 15.5 Positive EVH (n=7) (SD) (0.80) (6.8) (0.63) (11.1) (6.2) (7.3) (5.9) Max Min 7.25 4.89 108.0 . 88.9 5.63 3.92 106.2 76.0 82.1 67.0 98.7 81.2 27.7 11.0 67 Table 5.3. Baseline Performance Characteristics of Negative EVH Subjects (n=30). Subject FQ2max FQ2max Max HR Max Power Max Power (mL-kg"'-min"1) (L-min1) (b-min1) (W) (W-kg1) Mean 67.1 5.08 186 457 6.06 (SD) (4.3) (0.54) (10) (31) (0.48) 20km Time Trial Performance Three subjects were unable to complete all conditions and therefore results of only 27 subjects are presented. Mean power (Pmean) over the 20km for each of the conditions ranged between 306 and 310 watts with no effect of salbutamol observed between conditions (Table 5.4). This was approximately 67% of max power (Pmax) and equal to roughly 4.05 W-kg"1. Table 5.4. The Effects of Salbutamol Dose (D2=200ug, D4=400ug, D8=800ug) on 20km Mean Power Output (Pmean), Total Time (Ttot), and Lap Times (TL;, TL2), Heart Rate (HR) and Rate of Perceived Exertion for Legs (RPEL) and Breathing (RPED). Values Reported are Means and (SD). Placebo D2 D4 D8 Pmean 306 310 307 307 (W) (29) (30) (29) (30) Ttot 30.72 30.55 30.67 30.70 (min) (1.06) (1.03) (1.06) (1.04) TLI 15.31 15.25 15.29 15.35 (min) (0.55) (0.54) (0.55) (0.58) TL2 15.40 15.31 15.38 15.35 (min) (0.53) (0.54) (0.55) (0.50) HR 172 173 171 171 (bpm) (9) (10) (9) (10) RPEL 5.9 6.1 6.0 6.1 (1.4) (1.5) (1.5) (1.4) RPED 6.1 6.2 6.2 6.2 (1.4) (1.5) (1.6) (1.4) 68 Similarly there was no effect of salbutamol on any of the metabolic or ventilatory parameters (Table 5.5). Mean Voi and HR throughout the time trials was approximately 55 mL-kg"'-min"1 and 172 beats-min"1. This equated to approximately 82% and 92% of the respective peak values (Fo2max= 67.1 ± 4.3 mL-kg"'-min"1; HRmax= 186 ± 10 beats-min" ') achieved on Day 1. As shown in Table 5.5, breathing frequency was similar across conditions (~45 bpm) as was tidal volume (~2.9 L) resulting in no differences in exercise ventilation with salbutamol. Table 5.5. The Effects of Salbutamol Dose (D2=200ug, D4=400pg, D8=800pg) on Mean Metabolic and Ventilatory Parameters over 20km. Oxygen Consumption (VO2), Expired Carbon Dioxide (VCO2), Ventilation Rate (VE), Ventilatory Equivalents for Oxygen and Carbon Dioxide (VE/V02, VE/VC02), Respiratory Rate (RR), and Tidal Volume (V-r). Values are Reported as Means and (SD). Placebo D2 D4 D8 vo2 54.5 55.4 54.6 55.0 (mL-kg"'-min"1) (4.3) (4.0) . (4.2) (3-6) VC02 4.03 4.10 4.02 4.05 (L-min"1) (0.45) (0-47) (0.42) (0.42) VE 102.1 104.7 101.9 102.1 (Lmin"1) (15.9) (12.8) (16) (13.1) vE/vo2 30.5 30.7 30.2 30.0 (3.7) (3.1) (3.5) (3.3) vE/vco2 30.9 31.3 30.8 30.7 (3.5) (3.2) (3.4) (3.2) RR 44 45 45 44 (breaths-min"1) (9) (8) (9) (8) VT 2.87 2.89 2.85 2.90 (L) (0.45) (0.49) (0.45) (0.50) 69 During each time trial subjects were requested to rate their rate of perceived exertion for both leg and breathing effort. Mean values over 20km were unaffected by salbutamol (Table 5.5) and there was no difference at any distance between conditions for both PvPEL and RPED (Fig. 5.3). 3 -2 • I • 0 J 1 1 1 1 1 1 1 1 1 1-0 2 4 6 8 10 12 14 16 18 20 Distance (Km) Placebo » D200 -* D400 -*- •800 3 • 2 • I • 0 J . 1 . 1 1 , . 1 r-0 2 4 6 8 10 12 14 16 18 20 Distance (Km) Fig. 5.3. Mean ratings of perceived exertion for breathing (RPED) and legs (RPEL) at 2km intervals. Rating of difficulty ranged from 1 (nothing at all) to 10 (maximal). 70 Urine Concentrations of Salbutamol Urine concentrations are shown in Table 5.6. There was no difference in SG across conditions (DP = 1.012 ± 0.008, D2=1.013 ± 0.008, D4=1.013 ± 0.008, D8 = 1.012 ± 0.007) with the minimum and maximum values obtained across all trials being 1.002 and 1.032 respectively. As shown in Table 5.6, cSAL of uncorrected urine samples increased as dose increased with D4 being greater than DP, and D8 being significantly greater than all other conditions. Large variability existed in cSAL across all doses with a minimum of 0 ng-ml"1 and a maximum of 831 ng-ml"1 (Table 5.6). Table 5.6. Urine Concentrations of Salbutamol (non-sulfated) at 60 Minutes (T60) Post-Inhalation of Placebo (DP), 200pg (D200), 400pg (D400), and 800pg (D800) of Salbutamol. Mean, SD, Minimum (Min), and Maximum (Max). Mean, Standard Deviation (SD), Maximum (Max), and Minimum (Min) Values are Reported Raw and Corrected for Specific Gravity (SG) Formats. Values are Reported in ng-ml"1. DP D2 D4 D8 Mean 7 46 115 + 210 +A* Raw SD (15) (73) (126) (177) Min 0 0 0 26 Max 54 347 627 831 Mean 2 19 52 + 104 +,a** Corrected to SD (6) (29) (49) (90) 1.005 SG Min 0 0 0 7 Max 25 145 210 425 Mean 12 97 261 + 520 +'a'* Corrected to SD (30) (147) (245) (451) 1.025 SG Min 0 0 0 33 Max 123 723 1050 2125 + - denotes significantly greater than DP, p<0.05 a - denotes significantly greater than D2, p<0.05 * - denotes significantly greater than D4, p<0.05 71 Fig. 5.4a shows the variability in individual samples and of note is that no samples exceeded 1000 ng-mf1 when uncorrected for specific gravity. A significant relationship between SG and cSAL was observed in conditions D4 (n=28) and D8 (n=30) (r = 0.42 and r = 0.37 respectively, p<0.05) (Fig. 5.5a-b). Alternatively, SG was not related to cSAL in either DP (n=30) or D2 (n=29) conditions (r = 0.18 and r = 0.11 respectively). Fig. 5.4b and 5.4c show the individual subject plots for corrected samples. Corrections to 1.005, reduced the mean values across all doses with the maximum individual sample being 425 ng-mf1 at dose D8. When corrected to 1.025, three subjects exceeded the doping limit of 1000 ng-ml"1 at doses D4 and D8 (max = 2125 ng-ml"1) while four other subjects produced samples of 900 ng-ml"1 or more at dose D8. a) 2500 i 72 "I) 1500 j Placebo D200 D400 Condition b) Place Do D200 O400 Condition D200 D400 Condition Fig. 5.4. Urine Concentrations of Salbutamol (cSAL) for Raw Samples (a), Samples Corrected to Specific Gravity of 1.005 (b), and Samples Corrected to a Specific Gravity of 1.025 (c). Individual Samples are Shown for Placebo, 200ug (D200), 400ug (D400), and 800ug (D800). Dashed Line Represents Doping Control Limit of 1000 ng-ml-1. a) 700 -I 600 • J 500 • 400 v_» r -w v ) 1 "'- "'"i i — —f . . •] 1.000 1.005 1.010 1.015 1.020 1 025 1.030 1.035 Specific Gravity b) 1000 -I 800 • # _i E |> 600 • 1.000 1.005 1.010 1.015 1.020 1.025 1.030 1.035 Specific Gravity Fig. 5.5. Relationships between specific gravity and urine concentrations of salbutamol (cSAL) 1 hour post-inhalation of 400 ug (a) and 800 ug (b) doses. Discussion The main purposes of this study were to examine the dose-response effect of inhaled SAL on exercise performance and urine concentration in competitive non-asthmatic athletes. The primary findings were that SAL had no effect on 20km time trial performance as measured by mean sustainable power nor did it have effects on metabolic and ventilatory parameters during exercise. Urine concentrations of SAL following exercise at 1 hour post-inhalation increased with dose and were highly variable. No subject exceeded the WADA cut-off of 1000 ng-ml"1, however, cSAL was related to hydration as measured by specific gravity and the possibility exists that dehydration could lead to increased values. Additionally it was found that the prevalence of hyper-reactive airways in cyclists and triathletes not previously diagnosed with asthma is approximately 19%. Although applications to use SAL during the Olympic Games have been increasing over the past 20 years [2], the percentage of all athletes requesting use of a pYagonist at the last two Olympic Games is within the range of the prevalence of asthma in the general population (-5-10%). Nonetheless, there are certain sports where this rate is much higher; specifically cycling and triathlon where the percentage of athletes requesting a TUE for SAL at the Sydney Olympic Games (2000) was approximately 17% and 20% respectively [2]. This has prompted concerns of misuse by athletes that may be trying to gain a competitive edge [19]. The present data suggest that these numbers are not out of the ordinary for this population, however evaluations with larger samples sizes would be needed to confirm this. Of the 37 cyclists and triathletes who participated in this study, seven had a >10% reduction in FEVi following an EVH test equating to -19% testing 75 positive for airway hyperresponsiveness. Furthermore, subjects were selected from a group of athletes that had not previously been diagnosed with asthma, suggesting the percentage of athletes that could benefit from SAL use may in fact be higher. Previous data from the 1996 US Olympic team report that prevalence of asthma in elite cyclists may be as high as 50% (10 of 20 athletes) [84]. Our findings suggest that there may be need for further education of competitive cyclists and triathletes as to the symptoms and complications of asthma and exercise-induced bronchospasm in sport. Although several factors are known to contribute to airway hyperresponsiveness, the reasons why cyclists have an increased prevalence are not clear and require further investigation. This is the first study to utilize a sport-specific evaluation method while examining the dose-response of inhaled SAL on performance in non-asthmatic athletes. Previous research has used standard laboratory evaluations such as maximal aerobic power, anaerobic threshold, or time to exhaustion [22, 24, 46, 48, 53, 61, 75]: Overwhelmingly these studies have found inhaled SAL to have no performance enhancing effects in athletes from a variety of different sporting backgrounds [22, 24, 46, 48, 53, 61, 75]. Furthermore, Goubalt et al. [24] showed a lack of a dose-response with doses up to 800 (ng in a time to exhaustion test at 85% of maximal oxygen consumption. Although in agreement with our current findings the applicability of non-specific test results to sport performance enhancement is questionable. The validity of a test to be representative of performance is an important factor when evaluating the ergogenic effects of a treatment [34]. Only two studies have utilized sport specific protocols and they have provided conflicting results [53, 78]. Following a dose of 400 ug, Norris and colleagues [53] 76 showed no effect on 20-km time trial performance in competitive cyclists. At higher doses (800 pg) however, van Baak et al. [78] demonstrated an improvement in time to complete a set amount of cycling work suggesting the ergogenic effects of SAL may be related to dose. Our findings do not agree with this concept and are in agreement with the majority of other investigations that have failed to show an ergogenic effect. The difference noted by van Baak and colleagues [78] may be due to the length of the protocol utilized (> lhr) which would require different contributions from aerobic and anaerobic energy systems than -30 minutes of intense effort. However this seems unlikely as they showed no differences in lactate measures or substrate availability during exercise. Furthermore, we observed no differences in oxygen consumption or carbon dioxide production across doses which is in agreement with previous findings [11, 22, 24, 29, 61]. We feel their significant difference is likely due to the influence of two outliers who appear to have experienced an 8-10% improvement following SAL inhalation. This plus other data begs the question - are there specific athletes who may get an ergogenic effect. Genetics variations exist in pYreceptors which may be partially responsible for individual variability in response. One potential mechanism for SAL to have ergogenic properties may be related to its ability to act as a potent bronchodilator. Even in non-asthmatic individuals, SAL has the ability to increase airway calibre at rest resulting in a measurable increase in airway function (Chapter 4). Theoretically, this may lead to enhanced alveolar ventilation and/or a reduced work of breathing thereby increasing available oxygen for working muscles. However, previous reports have shown that during physical activity SAL does not have 77 an accumulative effect to the normal bronchodilatory response to exercise [11, 24, 29, 48, 78] nor does it reduce respiratory resistance during exercise [60]. Hence, our finding that exercise ventilation was unchanged with SAL and unaffected by dose was not surprising and is similar to previous findings at both maximal and sub-maximal intensities [11, 24, 53, 61, 75]. The finding that RPED was similar and that the pattern of ventilation (tidal volume and breathing frequency) did not change between conditions further supports the notion that SAL inhalation in non-asthmatics has minimal impact on ventilation during exercise. Two other studies in which subjects subjectively rated dyspnea during exercise found similar results [22, 24]. It has also been postulated that SAL may alter substrate utilization by mobilizing fatty acids and sparing glucose. Indeed, SAL has a stimulatory effect on lipolysis at rest and leads to increased fatty acid mobilization [63]. However, evidence to support that acute SAL treatment augments any normal response to exercise is lacking [24, 78, 79]. Blood measures were not obtained in this study so additional discussion is unwarranted. Lastly, the present study only examined the effect of acute administrations of inhaled SAL on exercise performance. Our findings cannot preclude the possibility that short term (~ 3 weeks) use by this means will not have an ergogenic effect. Continued oral administration of SAL for 3 weeks has resulted in enhanced endurance performance [15] and increases in peak and mean power during high-intensity cycling [38]. Unlike acute administrations of SAL, short term oral use has been shown to alter substrate availability and utilization during exercise [15], along with increasing strength capabilities [44]. Although oral administration is currently banned by WADA, it should not be assumed that continued inhaled administration is non-ergogenic. The likelihood that approved athletes would be using the drug regularly in training as part of an overall management program is high. A constant presence of SAL in the plasma following inhalation may lead to some of the adaptations that have been associated with oral administration. Further examinations of short term use of inhaled SAL on performance are necessary to eliminate this possibility. To our knowledge, this is the first study reporting the dose-response effect of inhaled SAL on cSAL following exercise. Previously reported post-exercise values for cSAL following low (200 pg) and high (1600 pg) inhaled doses show large variability between subjects with the majority of samples being less than 500 ng-mf1 [80]. Our findings are similar in both regards and not surprising as inter-subject variability of urine recovery of SAL is high (-38%) [77] . The finding that cSAL is related to dose is in agreement with our previous findings at rest (Chapter 4) and previous reports of absolute SAL recovery 30 minutes post-inhalation [77]. As dose increased so did the variability between subjects, particularly at higher doses (Fig. 5.4a) which may in part explain the recent reports of urine samples resulting in positive tests for athletes with a TUE [45, 65]. Currently WADA stipulates that any urine samples containing greater than 1000 ng-mf1 of SAL is considered an adverse analytical finding unless the athlete is able to prove the result was due to an inhaled therapeutic dose [82]. Although none of the subjects in this study exceeded the limit, there were 3 individuals who had one test over 500 ng-ml"1 with one subject approaching the limit at 831 ng-mf1. Considering this high value, it is plausible that an individual could exceed the WADA limit with a therapeutic dose. 79 However, cSAL values in this study are significantly less than those reported in recent positive tests (upwards of 3000 ng-ml"1) [45, 65]. The high variability observed in cSAL between subjects may be due to several factors as the pathway SAL must pass from inhalation to excretion is complex and involves several processes which may affect the time-course of passage. Lung absorption, metabolism, renal clearance, and hydration can all affect the amount of SAL that is excreted in the urine in the non-sulphated form. Interpretation is further complicated by the fact that urine cSAL following inhalation is a combination of local and systemic administrations due to a significant portion of the dose being swallowed. Approximately 20% of the dose is available to the lung following inhalation from a metered-dose inhaler and this can be enhanced when using a spacer device [49]. Although representing a potential explanation for the high variability observed we feel this is unlikely as spacer devices were implemented and each subject was instructed on proper use of the device prior. A more reasonable explanation is the individual differences in absorption, metabolism and renal clearance. Using charcoal ingestion to block gastrointestinal absorption, time to peak plasma concentrations post-inhalation have been shown to vary between 8 and 18 minutes [1]. Furthermore, exercise following inhalation of terbutaline (another B-agonist) has been shown to increase rate of lung absorption, likely due to increased blood flow to the microcirculation [64]. Damaged epithelium may further increase absorption in the lung [62] and considering the amount of time endurance athletes spend at high ventilation rates, it is plausible that variations in epithelium integrity may exist between individuals. Exercise can also adversely effect renal function as glomerular filtration rate, osmotic 80 clearance, and urine flow are compromised following 30 minutes of exercise at 85% Fornax [23]. Considering the multiple organs and processes that are involved prior to excretion of SAL, it is difficult to isolate a single reason to explain the variability in urine concentrations observed between subjects. However, a significant positive relationship was observed between SG and cSAL at both D4 and D8 (Fig. 5.5a-b) suggesting it may in part be due to hydration status. Currently, with respect to SAL, WADA does not take into consideration hydration status other than ensuring samples are not diluted by requiring SG > 1.005. Normal values for SG range between 1.005 and 1.030 and can have a significant impact on the concentration of urine specimens. To examine the impact of hydration all samples were corrected for SG to hydrated (1.005) and dehydrated states (1.025) (Fig. 5.4b-c). When corrected to a moderately dehydrated state, values from three different subjects exceeded 1000 ng-mf1 with a maximum of 2125 ng-ml"1. Theoretically these subjects could have produced a positive doping sample with a dose as low as 400 pg, providing support for the role of dehydration in false-positive doping tests [45, 65]. While SG is generally indicative of hydration and comparable to creatinine for correcting urine concentrations [50], we stress caution in applying these findings to doping control and in explaining false-positive doping violations previously reported in the literature. The low correlations between SG and cSAL would suggest that hydration status plays only a partial role and that values exceeding the WADA limit are likely due to the interplay of this and the several factors mentioned previously. The roles of length of time between inhalation and providing the urine sample, and the effects of multiple doses over time 81 need to be considered. Schweizer et al. [65], noted peak cSAL between 3 and 6 hours post-inhalation following multiple inhalations over 5 hours. Additionally, urine samples were taken 60 min post-inhalation following only 30 minutes of exercise. The values reported here may not be representative of events lasting several hours that may result in significant fluid shifts and dehydration. From a methodological standpoint, there is one other limitation to this study that is worth noting. Although the data demonstrate no impact of SAL on time-trial performance, it could be argued that the influence of the one-way valve for collection of ventilatory and metabolic parameters may have masked any benefits of bronchodilation during exercise. We expect that this impact would be minimal considering a low-resistance valve was utilized and the ventilation rates maintained during the time-trials was significantly lower than those achieved during maximal exercise and EVH tests. However, we cannot exclude this possibility and it is worth examining the effects of bronchodilators on expiratory flow resistance and work of breathing during exercise in non-asthmatics. The effect of small reductions in the work of breathing may not manifest into performance enhancement over 30 minutes of exercise, but may reduce overall fatigue in longer duration events (> 2 hours). In conclusion, this study failed to demonstrate any effects of SAL bn time trial performance and ventilatory/metabolic parameters. Furthermore the use of multiple doses up to 800 jj,g did not reveal trends related to dose, strengthening the consensus that acute administration of inhaled SAL to non-asthmatic athletes is not performance 82 enhancing in endurance sports. From a doping control standpoint, although urine cSAL will generally fall under 500 ng-ml"1 following inhaled therapeutic doses, the potential for exceeding the WADA limit does exist as individual responses are highly variable. This is partially related to hydration status but likely dependant more so on individual differences in absorption, metabolism and renal function. Lastly, the prevalence of asthma and airway hyperresponsiveness in cyclists and triathletes is significantly higher than that normally reported for the general population. As all athletes were previously undiagnosed with asthma, further education is suggested for athletes, coaches, and medical professionals to increase the awareness and/or education with respect to the symptoms, proper diagnosis, and consequences of airway sensitivity with respect to sport. 83 CHAPTER 6 - SUMMARY AND CONCLUSIONS The intention of this dissertation was to address two questions: what are the relationships between SAL dose and exercise performance in a simulated cycling time-trial, and what are the effects of dose on cSAL as used in doping control? A series of three projects was used to demonstrate that inhaled SAL does not enhance endurance performance in non-asthmatic athletes when using a highly reproducible and sport-specific test. This the first examination of the dose-response effect of inhaled salbutamol using a sport-specific performance evaluation and used a substantially larger sample size (n = 27) compared to most previous work (n = 8-16). The lack of a dose-response relationship further supports previous findings that acute SAL inhalation does not enhance exercise performance in non-asthmatics [11, 22, 24, 48, 53, 61, 74, 75]. It was also shown that cSAL following inhalation is highly variable both at rest and following exercise, and related to dose. At rest, cSAL seems to peak at approximately 60 minutes post-inhalation. These findings are unique in reporting the dose-response relationships of inhaled SAL on urine concentrations, as reported and utilized by WADA. Previous pharmacological reports are typically reported in absolute values recovered or as a percentage of total dose administered. Although observed values for cSAL were similar between Projects 2 and 3, suggesting minimal effects of exercise, this conclusion is limited. Each study was performed independently and fluid intake was not controlled between the two. Future studies are needed to delineate the impacts of exercise on SAL 84 excretion using a randomized cross-over design. Furthermore, the short duration of the time-trial may not have provided sufficient stimulus for changes in hydration status that can accompany longer duration exercise. Even though most urine samples generally fell well below the WADA limit of 1000 ng-mL"1, the possibility exists for individuals to exceed this value following inhaled administration. A significant relationship between cSAL and urine SG was observed at higher doses, signifying the potential impacts on hydration on values observed in doping control. As with exercise, the role of hydration and individual differences in absorption, metabolism, and excretion on cSAL require further investigation. It is also noted that the finding of SAL to be non-ergogenic cannot preclude the possibility that continued, short-term (>2-3 weeks) use of inhaled SAL would not be performance enhancing. Regular use of SAL during both training and competition would be expected and it is possible that continued elevated plasma levels following inhalation may increase ergogenic properties of SAL. Future research needs to be conducted to eliminate this possibility. Lastly, it was observed that a large portion (-19%) of the cyclists/triathletes tested were susceptible to airway hyperresponsiveness. Although a small number of cyclists and triathletes were recruited for these studies, the possibility exists that there is a significant portion of this athlete population competing with impaired airway function unbeknownst to them. 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Subject Age Height Weight Exp (yrs) (cm) (kg) (yrs) 1 36 191.5 98. 9 2 33 186.2 85 12 3 29 180.0 71 5 4 30 194.6 92 5 7 33 183.9 71 7 8 34 181.9 80 15 9 22 190.0 85 4 10 28 192.2 86 8 11 32 178.0 84 9 12 29 171.6 62 15 13 25 180.6 78 9 14 19 193.0 81 6 15 21 172.0 63 7 16 23 180.5 66 3 17 34 176.0 71 16 18 35 172.0 70 7 19 36 181.2 68 5 20 43 188.2 80 4 21 20 185.9 74 6 22 51 179.9 73 20 Mean 31 183.0 77 9 SD 8 7.1 9.7 5 95 Table A.2. Individual Subject Performance Characteristics Including Peak Oxygen Consumption (f^rnax) in Relative (Rel) and Absolute (Abs) terms, Maximal Ventilation (FE max), Maximal Heart Rate (HRmax), and Peak (Ppeak) and Relative (Prei) Power Output. Subject Rel Fc^max Abs Fckniax max HRmax Pmax Prel (mLkg"1min"1) (Lmin1) (L-min1) (b-min1) (W) (W/kg) 1 63.1 6.19 160.3 180 495 5.05 2 66.9 5.66 160.8 183 495 5.85 3 68.1 4.84 136.3 190 435 6.12 4 70.3 6.49 196.4 182 503 5.45 7 72.0 5.10 156.0 183 473 6.68 8 71.9 5.74 130.3 175 495 6.20 9 65.7 5.59 162.5 183 495 5.82 10 70.4 6.04 138.1 178 503 5.86 11 66.3 5.58 220.5 187 465 . 5.53 12 77.9 4.80 165.1 193 443 7.19 13 70.4 5.46 .157.7 188 495 6.38 14 68.2 5.50 164.9 197 503 6.24 15 71.9 4.50 131.9 207 405 6.47 16 67.1 4.43 129.0 189 420 6.36 17 65.1 4.60 147.8 190 425 ' 6.02 18 70.1 4.89 126.6 184 450 6.45 19 67.2 4.58 126.1 190 435 6.38 20 64.0 5.14 169.0 164 473 5.89 21 68.6 5.06 130.8 207 515 6.98 22 64.9 4.71 * 152.6 163 450 6.20 Mean 68.5 5.25 153.1 186 469 6T6 SD 3.6 0.61 25.1 9 33 0.49 96 Table A.3. Individual Performance Times in Minutes for Each Time-trial (TT) Including Lap (TLi and TL2) and Total Times (Ttot). TTl TT2 TT3 Subject TLI TL2 Ttot TLI TL2 Ttot TL2 Ttot 1 14.32 15.08 29.40 15.08 14.86 29.94 15.50 14.95 30.45 2 14.93 14.97 29.90 15.21 15.03 30.23 15.31 14.91 30.22 3 15.83 15.33 31.15 15.90 15.60 31.50 15.76 15.40 31.16 4 13.99 14,30 28.29 14.14 14.37 28.51 13.98 14.40 28.37 7 14.67 14.93 29.60 14.53 14.92 29.45 14.70 15.12 29.82 8 14.19 14.53 28.72 14.22 14.28 28.50 14.27 14.60 28.87 9 14.76 14.87 29.63 14.86 14.59 29.45 14.87 14.59 29.46 10 14.23 14.27 28.51 14.31 14.62 28.93 14,21 14.19 28.39 11 15.51 15.33 30.84 15.33 15.56 30.88 15.14 15.45 30.59 12 15.11 15.61 30.7L 15.32 15.75 31.06 15.19 15.63 30.82 13 14.40 15.12 29.51 14.42 15.16 29.58 14.42 15.08 29.50 14 14.70 14.70 29.38 . 14.80 14.85 29.65 14.93 14.80 29.73 15 15.53 16.07 31.60 15.65 16.07 31.72 15.82 15.68 31.48 16 16.27 16.13 32.40 14.73 15.95 31.70 15.80 15.90 31.70 17 16.37 15.95 32.33 16.98 15.77 32.77 16.82 16.22 33.03 18 15.08 15.33 30.42 14.98 15.20 30.20 15.00 15.10 30.10 19 15.22 15.45 30.67 15.25 15.40 30.67 15.23 15.55 30.78 20 14.82 14.77 29.58 14.37 14.77 29.13 14.43 14.70 29.13 21 14.17 14.30 28.47 14.18 14.37 28.55 14.48 14.65 29.13 22 14.60 14.91 29.52 14.88 15.17 30.05 14.90 15.23 30.13 Mean 14.93 15.10 30.03 14.96 15.11 30.12 15.04 15.11 30.14 SD 0.71 0.56 1.24 0.70 0.55 1.21 0.71 0.55 1.21 Table A.4. Individual Mean Performance Power (Pmean) in Watts for Each Time-trial (TT). Subject TT1 TT2 TT3 1 346 328 313 2 329 317 320 3 292 284 292 4 379 377 376 7 335 340 329 8 365 372 360 9 336 340 340 10 374 362 379 11 306 303 313 12 " 303 294 301 13 340 337 339 14 344 335 333 15 280 278 284 16 263 278 277 17 264 257 250 18 312 318 321 19 304 304 301 20 336 351 350 21 372 369 350 22 337 321 320 Mean 326 323 322 SD 35 35 34 Table A.5. Individual Mean Heart Rate for Each Time-trial (TT). Subject TTl TT2 TT3 1 175 161 166 2 169 172 169 3 167 173 168 4 167 166 166 7 171 172 169 8 170 165 170 9 168 165 165 10 166 162 163 11 167 165 171 12 174 180 176 13 182 178 178 14 182 172 171 15 195 194 192 16 175 171 172 17 162 159 164 18 169 168 169 19 179 176 179 20 149 153 155 21 191 189 189 22 153 153 152 Mean 172 170 170 SD 8 8 7 99 APPENDIX B - RAW DATA FOR PROJECT 2 Table B.l. Individual Subject Characteristics and Baseline Lung Function Measures with Percent of Predicted Values (% Pred). Lung Function Measures Include Forced Vital Capacity (FVC), Forced Expiratory Volume in One Second (FEVi), and the Ratio of FEV, to FVC (FEVi/FVC). Subject Age (y) Height (cm) Weight (kg) FVC (L) %Pred FEV, (L) % Pred FEVi/FVC (%) % Pred 1 22 182.2 74.8 6.66 112.1 5.28 106.5 79.3 95.0 2 33 186.0 87.5 5.63 9.4.9 4.15 84.9 73.71 89.4 3 24 181.5 73.5 6.15 105.7 4.86 100.2 79.12 94.9 4 23 179.6 67.9 4.92 86.5 4.01 84.2 81.46 97.5 5 34 176.6 77.0 5.06 99.0 4.40 103.8 86.97 104.9 6 27 183.4 78.1 5.05 85.7 4.31 88.1 85.25 102.7 7 35 176.1 80.2 5.45 107.5 4.27 101.7 78.36 94.5 8 25 169.9 79.8 5.73 119.1 4.38 108.1 76.57 90.9 Mean 28 179.4 77.4 5.58 101.32 4.46 97.2 80.09 96.2 SD 5.30 5.08 5.71 0.60 11.95 0.41 9.85 4.37 5.34 100 Table B.2. Individual Urine Concentrations of Non-sulphated Salbutamol (ng-mf1) at 30 (T30), 60TT60), and 120 Minutes (T120) Post-Inhalation of 200pg (D2), 400pg (D4), and 800pg (D8) of Salbutamol. Group Means and SD for Each Condition are Included. T30 T60 T120 Subject D2 D4 D8 D2 D4 D8 D2 D4 D8 1 189 621 185 157 529 904 59 98 562 2 38 27 95 39 107 97 0 28 64 3 0 213 178 0 137 138 0 57 208 4 28 182 232 85 255 403 33 167 194 5 0 90 519 0 164 139 0 20 58 6 180 132 189 154 182 369 74 147 310 7 0 64 86 61 45 47 0 73 31 8 27 58 83 32 29 78 0 0 121 Mean 58 173 196+ 66 181 272+ • 21 74 194+ SD 80 192 142 62 159 288 31 60 176 a - denotes significant difference from T30 at same dose, p<0.05 + - denotes significant difference from D2 at same time, p<0.05 * - denotes significant difference from D4 at same time, p<0.05 101 Table B.3. Individual Urine Concentrations of Non-sulphated Salbutamol (ng-ml"1) Corrected for Specific Gravity (1.005) at 30 (T30), 60 (T60), and 120 Minutes (T120) Post-Inhalation of 200ug (D2), 400ug (D4), and 800ug (D8) of Salbutamol. Mean and SD for Each Condition are Included. T30 T60 T120 Subject D2 D4 D8 D2 D4 D8 D2 D4 D8 1 79 141 36 157 126 161 98 98 140 2 8 5 79 32 31 162 0 23 80 3 0 118 99 0 228 230 0 142 347 4 7 41 45 18 61 72 6 38 35 5 0 18. 118 0 43 174 0 33 145 6 31 73 50 24 70 154 13 52 91 7 0 40 86 18 45 59 0 26 39 8 27 58 138 80 72' 195 0 0 121 Mean 19 62 81+ 41 85 151a'+'* 15 52 125+!* SD 27 47 37 53 65 58 34 46 99 a - denotes significant difference from T30 at same dose, p<0.05 + - denotes significant difference from D2 at same time, p<0.05 * - denotes significant difference from D4 at same time, p<0.05 102 Table B.4. Individual Urine Concentrations of Non-sulphated Salbutamol (ng-mf1) Corrected for Specific Gravity (1.025) at 30 (T30), 60 (T60), and 120 Minutes (T120) Post-Inhalation of 200pg (D2), 400pg (D4), and 800pg (D8) of Salbutamol. Mean and SD for Each Condition are Included. T30 T60 T.120 Subject D2 D4 D8 D2 D4 D8 D2 D4 D8 1 394 706 178 785 630 807 492 490 702 2 41 24 396 162 157 808 0 . 117 400 3 0 592 494 0 1142 1150 0 712 1733 4 35 207 223 89 304 360 32 190 173 5 0 90 590 0 216 869 0 167 725 6 155 367 249 120 350 769 66 262 456 7 0 200 430 90 225 294 0 130 194 8 135 290 692 400 362 975 0 0 605 Mean 95 309 406+ 206 423 754a'+>* 74 259 624+'* SD 135 237 183 266 324 291 171 232 495 a - denotes significant difference from T30 at same dose, p<0.05 + - denotes significant difference from D2 at same time, p<0.05 * - denotes significant difference from D4 at same time, p<0.05 103 Table B.5. Specific Gravity of Individual Urine Samples at 30 (T30), 60 (T60), and 120 Minutes (T120) Post-Inhalation of 200pg (D2), 400pg (D4), and 800ug (D8) of Salbutamol. Mean and SD for Each Condition are Included. T30 T60 T120 Subject D2 D4 D8 D2 D4 D8 D2 D4 D8 1 1.012 1.022 1.026 1.005 1.021 1.028 1.003 1.005 1.020 2 1.023 1.028 1.006 1.006 1.017 1.003 1.004 1.006 1.004 3 1.004 1.009 1.009 1.002 1.003 1.003 1.002 1.002 1.003 4 1.020 1.022 1.026 1.024 1.021 1.028 1.026 1.022 1.028 5 1.002 1.025 1.022 1.001 1.019 1.004 1.003 1.003 1.002 6 1.029 1.009 1.019 1.032 1.013 1.012 1.028 1.014 1.017 7 1.012 1.008 1.005 1.017 1.005 1.004 1.006 1.014 1.004 8 1.005 1.005 1.003 1.002 1.002 1.002 1.002 1.002 1.005 Mean 1.013 1.016 1.015 1.011 1.013 1.011 1.009 1.009 1.010 SD 0.010 0.009 0.010 0.012 0.008 0.011 0.011 0.007 0.010 104 Fig. B.l. Force expiratory volume in 1 second (FEVi) (a) and the ratio of FEVi to forced vital capacity (FVC) as a percentage (b) prior to (pre) and at 30, 60, and 120 minutes following inhalation of salbutamol. Values are shown for doses of 200 ug (-•-), 400 ug (-o-), and 800 ug (-•-). * - denotes all pre values are statistically significant from all post values for the same dose, p<0.05; + -denotes 800 ug at 60 min is greater than 200 ug at all time points, p<0.05. 105 APPENDIX C - RAW DATA FOR PROJECT 3 Table C.l. Individual Lung Function Measures Including % Predicted Values for Forced Vital Capacity (FVC), Forced Expiratory Volume in One Second (FEVi), Ratio of FEVi to FVC (FEVi/FVC), and Decrease in FEVi (Max AFEVi) for Negative Responders to a Eucapnic Voluntary Hyperpnea (EVH) Test. Included are Means and SD (n=30). Subject FVC (L) % Predicted FEVi (L) % Predicted FEVi/FVC (%) % Predicted Max AFEV, (%) 1 5.64 98.6 4.74 99.6 84.1 100.9 2.3 2 6.75 102.6 5.28 103.1 78.3 95.0 6.6 3 5.63 117.1 4.90 121.9 87.1 104.1 8.0 4 6.67 97.9 5.74 102.5 86.2 104.8 4.7 5 4.92 86.5 4.01 84.2 81.5 97.5 5.7 6 5.03 92.5 4.29 95.8 85.3 103.4 1.6 9 5.43 93.3 4.08 85.9 75.2 92.3 5.6 10 4.59 99.6 3.55 92.9 77.3 93.1 -1.4 13 6.84 101.6 5.96 104.6 87.1 105.5 4.5 15 6.77 112.1 5.81 115.7 85.8 103.2 1.2 16 5.96 109.6 4.97 109.5 83.4 100.0 3.4 18 5.06 85.0 3.92 78.9 77.5 92.7 6.4 19 4.97 135.8 3.76 122.1 75.7 90.0 9.8 20 4.85 94.4 4.44 103.7 91.5 110.0 -1.4 21 5.97 102.8 5.40 111.1 90.4 108.2 5.6 23 6.96 108.9 5.98 111.6 85.9 102.9 3.2 24 5.29 98.5 4.63 104.3 87.5 105.8 0.6 25 6.33 98.3 4.91 91.4 77.6 93.1 9.0 26 6.45 99.7 5.50 102.2 85.3 99.5 5.1 27 6.56 107.9 5.58 110.1 85.0 101.8 3.4 28 6.05 100.5 4.26 85.4 70.4 85.0 3.8 29 6.41 104.9 4.79 94.3 74.7 89.8 3.5 30 5.96 116.2 4.84 114.4 81.2 98.9 2.1 31 5.78 96.2 4.88 98.2 84.4 101.1 3.1 32 6.15 98.8 5.41 104.2 88.0 105.6 3.0 34 5.22 111.1 4.36 111.8 83.0 100.5 3.2 35 6.92 116.5 5.81 117.9 84.0 101.2 2.9 36 5.12 94.1 4.24 93.4 82.8 99.3 0.0 37 ' 7.01 129.6 5.67 115.0 80.9 97.3 7.1 38 4.56 93.8 3.95 100.3 86.7 106.8 4.1 Mean 5.86 103.5 4.86 102.9 82.8 99.6 3.9 SD 0.77 11.4 0.72 11.3 5.0 6.0 2.7 106 Table C.2. Individual Lung Function Measures Including % Predicted Values for Forced Vital Capacity (FVC), Forced Expiratory Volume in One Second (FEVi), Ratio of FEVi to FVC (FEVi/FVC), and Decrease in FEV, (Max AFEVi) for Negative Responders to a Eucapnic Voluntary Hyperpnea (EVH) Test. Included are Means and SD (n=7). Subject FVC (L) . % Predicted FEV, (L) % Predicted FEVi/FVC (%) % • Predicted Max AFEVi (%) 7 4.89 96.6 4.01 100.0 81.9 98.7 27.7 8 7.25 103.9 4.86 84.3 67.0 81.2 17.7 11 5.52 88.9 3.92 76.0 71.0 85.5 12.2 12 6.86 108.0 5.63 106.2 82.1 98.5 12.1 14 6.38 102.6 5.16 99.6 80.9 97.2 15.3 17 5.95 107.2 4.84 104.3 81.3 97.2 11.0 33 5.81 105.4 4.26 92.4 73.3 87.7 12.4 Mean 6.09 101.8 4.67 94.7 76.8 92.3 15.5 SD 0.80 6.8 0.63 11.1 6.2 7.3 5.9 107 Table C.3. Individual Subject Characteristics and Training History of Negative EVH Subjects (n=30). Subject Age (yrs) Height (cm) Weight (kg) Competitive Experience (yrs) Training Volume (km-week"1) 1 25 180.6 79.5 9 400 2 29 192.2 90.8 9 325 3 30 171.6 62.7 15 400 4 30 195.3 95.0 4 300 5 23 181.0 66.0 3 400 . 6 36 181.2 67.2 6 425 9 43 188.2 81.7 4 200 10 37 171.7 67.3 12 175 13 25 194.6 79.2 5 400 15 25 184.4 77.3 12 275 16 27 178.1 73.6 6 150 18 21 182.0 70.5 6 200 19 31 166.0 67.9 10 250 20 31 175.8 66.4 3 250 21 22 180.7 73.1 8 100 23 18 186.4 73.6 5 400 24 34 179.7 73.3 6 300 25 25 187.1 87.7 5 250 26 21 188.1 77.6 7 600 27 21 183.5 74.0 6 350 28 29 185.3 75.1 14 150 29 23 184.5 78.4 3 200 30 41 179.6 77.7 2 275 ; 31 30 185.9 73.3 10 250 32 21 185.2 83.6 5 100 34 34 172.4 69.4 12 • 225 35 27 184.0 82.9 8 120 36 27 178.7 81.9 10 150 37 30 178.7 76.0 10 325 38 51 179.6 73.5 25 270 Mean 29 182.1 75.9 8 274 SD 7.4 6.6 7.5 5 115 108 Table C.4. Baseline Performance Characteristics of Negative EVH Subjects Inducing Peak Oxygen Consumption (Fc^max), Maximum Heart Rate (HRmax), and Peak Absolute (Pmax) and Relative (Prei) Power Outputs. Group Means and SD are Included. (n=30) Fornax Fcfemax Max HR Max Power Max Power Mimect (mLkg"1min"1) (Lmin1) (b-min1) (W) (W-kg1) 1 71.0 5.64 191 488 6.14 2 66.7 6.06 180 517 5.69 3 72.6 4.62 190 435 6.94 4 72.0 6.84 184 533 5.61 5 72.8 4.88 189 465 7.05 6 67.0 4.42 190 442 6.58 9 61.4 5.02 163 472 5.78 10 66.1 5.11 183 420 6.24 13 70.0 5.54 179 495 6.25 15 71.2 . 4.54 183 495 6.40 16 64.2 4.73 180 443 6.02 18 67.2 4.74 201 450 6.38 19 62.7 4.26 190 405 5.96 20 65.4 4.34 198 435 6.55 21 72.6 5.3 191 450 6.16 23 69.7 5.13 178 473 6.43 24 69.0 5.06 184 473 6.45 25 57.4 5.04 201 465 5.30 26 65.1 5.05 177 435 5.61 27 70.9 5.25 204 465 6.28 28 67.7 5.08 190 480 6.39 29 68.5 5.37. 189 450 5.74 30 65.3 5.08 182 428 5.51 31 66.6 4.88 188 428 5.84 32 58.4 4.88 189 435 5.20 34 69.3 4.81 181 450 6.48 35 72.2 5.99 194 503 6.07 36 59.6 4.89 194 420 5.13 37 67.8 5.15 184 435 5.73 38 63.4 4.66 162 428 5.82 Mean 67.1 5.08 186 457 6.06 SD 4.3 0.54 10 31 0.48 109 Table C.5. Individual Mean Power Output (W) During a 20km Time-trial Post-Inhalation of Placebo (DP), 200pg (D2), 400pg (D4), and 800pg (D8) of Salbutamol. Group Means and SD for Each Condition are Included. (n=30) Subject DP D2 D4 D8 1 324 n/c 315 339 2 351 365 350 360 3 288 290 295 290 4 352 373 363 369 5 293 308 289 293 6 293 287 307 287 9 340 339 345 345 10 278 269 272 277 13 345 343 343 352 15 310 314 316 311 16 298 297 300 291 18 307 305 301 310 19 262 269 270 274 20 275 275 274 270 21 332 332 330 326 23 332 333 332 313 24 312 322 299 288 25 285 n/c n/c 281 26 297 304 290 285 27 269 293 284 267 28 321 331 n/c 343 29 314 306 316 324 30 270 287 279 279 31 293 296 302 296 32 260 280 252 287 34 306 307 299 306 35 364 379 365 374 36 307 313 309 312 37 298 307 299 305 38 310 289 307 300 Mean 306 311 307 308 SD 28 29 28 30 n/c- condition not completed 110 Table C.6. Individual 20km Performance Times (min) Post-Inhalation of Placebo (DP), 200ug (D2), 400ug (D4), and 800ug (D8) of Salbutamol (Including Lap (TLi and TL2) and Total Times (Ttot). Group Means and SD for Each Condition are Included. (n=30) DP D2 D4 D8 Subject TLI TL2 Ttot TL. TL2 Ttot TLI TL2 Ttot TLI TL2 Ttot 1 14.92 15.05 29.97 n/c n/c n/c 14.93 14.93 30.28 14.47 15.03 29.50 2 14.87 14.47 29.33 14.52 14.37 28.88 1.4.68 14.68 29.25 14.63 14.35. 28.98 3 15.43 15.85 31.28 15.25 16.00 31.23 15.20 15.20 31.03 15.37 15.87 31.23 4 14.57 14.52 29.07 14.12 14.35 28.47 14.43 14.43 28.73 14.13 14.45 28.58 5. 15.58 15.50 31.08 15.17 15.38 30.53 15.47 15.47 31.32 15.60 15.50 31.10 6 15.57 15.48 31.05 15.60 15.72 31.32 15.18 15.18 30.50 15.70 15.62 31.32 9 14.58 14.87 29.45 14.63 14.87 29.50 14.47 14.47 29.30 14.52 14.82 29.33 10 15.60 16.10 31.70 15.92 16.18 32.08 15.92 15.92 31.95 15.77 15.98 31.75 13 14.45 14.68 29.13 14.55 14.83 29.38 14.48 14.48 29.28 14.48 15.02 29.50 15 15.07 15.45 30.52 15.07 15.28 30.35 14.95 14.95 30.28 15.12 15.33 30.45 16 15.60 15.33 30.95 15.58 15.37 30.97 15.52 15.52 30.92 15.60 15.63 31.23 .18 . 15.17 15.45 30.62 15.33 15.38 30.72 15.37 15.37 30.90 15.25 15.30 30.55 19 16.17 16.27 32.45 16.08 16.03 32.12 15.98 15.98 32.08 16.02 15.88 31.92 20 15.75 16.07 31.83 15.83 16.00 31.85 15.75 15.75 31.90 15.98 16.12 32.10 21 14.83 14.85 29.70 14.85 14.83 . 29.68 14.85 14.85 29.75 14.97 14.95 29.90 23 14.68 15.02 29.70 14.80 14.85 29.67 14.78 14.78 29.68 15.20 15.17 30.37 24 15.27 15.12 30.40 14.82 15.23 30.05 15.68 15.68 30.92 15.90 15.42 31.30 25 15.73 15.72 31.45 n/c n/c n/c n/c n/c n/c 15.58 16.02 31.60 26 15.33 15.67 31.02 15.57 15.15 30.72 15.90 15.90 31.32 16.07 15.45 31.52 27 16.25 15.95 32.20 15.70 15.55 31.25 15.83 15.83 31.55 16.33 16.03 32.35 28 15.22 14.85 30.07 14.83 14.93 29.75 n/c n/c n/c 14.77 14.58 29.35 29 15.12 15.22 30.35 15.18 15.42 30.60 15.00 15.00 30.23 14.90 15.07 29.97 30 16.12 16.10 32.23 15.73 15.60 31.33 15.97 15.97 31.70 16.03 15.67 31.70 31 15.62 15.50 31.10 15.37 15.62 30.98 15.18 15.18 30.75 15.52 15.47 30.98 32 16.33 16.22 32.53 16.08 15.62 31.75 16.45 16.45 32.92 15.57 15.77 31.32 34 15.10 15.52 30.62 15.07 15.50 30.55 15.15 15.15 30.90 15.10 15.50 30.60 35 14.28 14.47 28.75 14.3.2 13.97 28.30 14.47 14.47 28.68 14.30 14.13 28.43 36 15.37 15.60 30.97 15.25 15.55 30.80 15.28 15.28 30.80 15.27 15.65 30.92 37 15.70 15.33 31.03 15.57 15.07 30.63 15.73 15.73 30,88 15.60 15.10 30.70 38 15.10 15.35 30.47 15.72 15.57 31.28 15.27 15.27 30.58 15.50 15.35 30.85 Mean 15.31 15.39 30.70 15.23 15.29 30.53 15.28 15.28 30.66 15.30 15.34 30.64 SD 0.54 o;52 1.03 0.53 0.53 1.02 0.54 0.54 1.04 0.58 0.52 1.05 n/c - condition not completed Ill Table C.7. Individual Mean Oxygen Consumption (mL-kg"1-min"1) During a 20km Time-trial Post-Inhalation of Placebo (DP), 200pg (D2), 400pg (D4), and 800pg (D8) of Salbutamol. Group Means and SD for Each Condition are Included. (n=30) Subject DP D2 D4 D8 1 56.5 n/c 54.6 44.4 2 49.4 53.9 47.3 53.5 3 58.8 55.0 57.7 57.6 4 53.9 57.7 54.7 57.2 5 55.0 50.5 52.2 58.4 6 56.3 59.2 58.9 56.6 9 53.0 55.7 57.4 54.9 10 58.4 55.4 56.7 55.5 13 53.0 50.8 52.0 55.7 15 57.9 56.5 58.2 56.7 16 53.5 54.5 53.7 50.9 18 57.9 . 57.4 56.2 57.2 19 51.0 51.1 51.5 52.4 20 53.5 62.0 54.6 55.3 21 62.3 62.5 61.5 62.0 23 60.7 61.1 61.6 59.3 24 56.6 57.1 53.9 53.3 25 42.1 n/c n/c 43.4 26 51.2 52.4 49.4 48.7 27 50.0 55.1 52.3 51.3 28 52.6 55.7 n/c 55.9 29 56.8 54.4 58.0 58.3 30 48.0 52.9 53.2 51.5 31 55.2 56.5 57.6 52.9 32 42.7 46.5 43.1 50.1 34 55.7 58.6 55.3 58.7 35 60.3 62.1 59.5 62.3 36 50.8 50.2 52.3 51.6 37 54.1 55.6 51.6 53.1 38 55.9 52.5 54.8 50.9 Mean 54.1 55.5 54.6 54.3 SD 4.7 3.9 4.1 4.4 n/c- condition not completed Table C.8. Individual Mean Ventilation (Lmin"1) During a 20km Time-trial Post-Inhalation of Placebo (DP), 200pg (D2), 400pg (D4), and 800pg (D8) of Salbutamol. Group Means and SD for Each Condition are Included. (n=30) Subject DP D2 D4 D8 1 115.5 n/c 106.7 90.1 2 79.2 86.9 77.3 86.2 3 105.5 93.4 101.7 99.0 4 107.0 115.5 107.6 121.4 5 89.9 86.7 83.2 95.6 6 83.6 93.7 93.6 83.7 9 113.5 114.0 113.7 114.2 10 110.1 105.3 112.9 109.9 13 104.0 105.5 99.3 106.1 15 125.7 128.6 130.4 124.6 16 84.3 89.9 88.3 84.0 18 99.8 102.8 98.4 98.9 19 78.5 83.5 80.7 90.9 20 87.6 107.6 89.6 92.5 21 108.7 106.1 104.9 107.6 23 142.6 138.6 145.4 129.3 24 106.3 107.3 96.8 94.3 25 82.5 n/c n/c 82.9 26 101.8 102.3 92.4 84.5 27 . 81.6 108.7 88.6 84.4 28 89.1 95.5 n/c 95.5 29 104.9 103.3 100.8 109.7 30 91.1 98.6 101.8 102.3 31 112.7 116.3 119.9 98.6 32 80.4 92.4 83.0 103.0 .34 97.7 99.4 92.2 96.3 35 115.7 119.5 110.4 109.9 36 119.6 115.5 123.0 124.6 37 106.2 100.2 98.7 102.3 38 119.4 104.5 118.1 103.7 Mean 101.5 104.3 102.1 100.9 SD 15.8 12.6 15.8 13.1 n/c- condition not completed 113 Table C.9. Individual Mean Heart Rate (bpm) During a 20km Time-trial Post-Inhalation of Placebo (DP), 200pg (D2), 400pg (D4), and 800pg (D8) of Salbutamol. Group Means and SD for Each Condition are Included. (n=30) Subject DP D2 D4 D8 1 174 n/c 178 178 2 157 166 164 162 3 173 169 169 163 4 167 171 165 174 5 172 176 172 168 6 172 177 179 174 9 155 155 158 157 10 175 170 170 169 13 166 166 168 171 15 171 167 174 170 16 165 170 173 181 18 184 187 189 188 19 171 175 177 175 20 185 186 186 186 21 180 182 181 175 23 175 173 173 166 24 177 179 166 166 25 173 n/c n/c 178 • 26 172 168 . 161 160 27 183 194 186 188 28 173 180 n/c 178 29 176 178 173 175 30 163 163 163 163 31 171 173 174 175 32 166 170 167 175 34 171 169 174 168 35 190 184 183 184 36 185 183 176 181 37 162 156 155 ' 155 38 154 150 155 152 Mean 172 173 172 172 SD 9 10 9 9 n/c- condition not completed 114 Table CIO. Individual Mean Speed (km-hr1) During a 20km Time-trial Post-Inhalation of Placebo (DP), 200pg (D2), 400pg (D4), and 800pg (D8) of Salbutamol. Group Means and SD for Each Condition are Included. (n=30) Subject DP D2 D4 D8 1 40.1 n/c 39.6 40.7 2 40.9 41.6 41.0 41.4 3 38.4 38.4 38.7 38.4 4 41.3 42.2 41.8 42.0 5 38.6 39.3 38.3 38.6 6 38.7 38.3 39.3 38.3 9 40.7 40.7 41.0 40.9 10 37.9 37.4 37.6 37.8 13 41.0 40.7 40.8 41.2 15 39.3 39.6 39.6 39.4 16 38.8 38.8 38.8 38.4 18 39.2 39.1 38.8 39.3 19 37.0 37.4 37.4 37.6 20 37.7 37.7 37.6 37.4 21 40.4 . 40.4 40.3 40.1 23 40.4 40.5 40.4 39.5 24 39.5 39.9 38.8 38.3 25 38.2 n/c n/c 38.0 26 38.7 39.1 38.3 38.1 27 37.3 38.4 38.0 37.1 28 39.9 40.3 n/c • 40.9 29 39.6 39.2 39.7 40.0 30 37.2 38.3 37.9 37.9 31 38.6 38.7 39.0 38.7 32 36.9 37.9 36.5 38.3 34 39.2 39.3 38.9 39.2 35 41.7 42.4 41.8 42.2 36 38.8 39.0 39.0 38.8 37 38.7 39.2 38.9 39.1 38 39.4 38.4 39.2 38.9 Mean 39.1 39.3 39.2 39.2 SD 1.3 1.3 1.3 1.4 n/c- condition not completed 115 Table C.ll. Individual Rate of Perceived Exertion for Breathing Effort (1-10) During a 20km Time-trial Post-Inhalation of Placebo (DP), 200pg (D2), 400pg (D4), and 800pg (D8) of Salbutamol. Group Means and SD for Each Condition are Included. (n=30) Subject DP D2 D4 D8 1 7.2 n/c 7.5 6.2 2 6.3 6.4 5.8 6.2 3 6.4 5.0 6.6 5.7 4 3.3 4.2 3.1 3.5 5 8.4 8.6 8.5 8.3 6 7.4 7.5 8.3 8.1 9 9.0 9.6 9.3 9.1 10 6.4 5.7 5.1 5.4 13 5.8 5.2 6.0 6.2 15 7.9 7.7 8.1 7.9 16 3.8 3.7 3.7 . 4.4 18 6.4 7.0 6.6 6.4 19 6.7 5.5 6.2 6.5 20 6.5 6.9 7.2 6.6 21 4.8 .4.0 4.9 7.3 23 5.5 7.3 5.9 5.9 24 4.4 5.6 4.7 4.5 25 4.7 n/c . n/c 4.7 26 5.5 5.7 6.2 5.3 27 5.5 5.5 5.5 5.9 28 * 7.1 n/c 6.4 29 6.3 7.1 7.1 7.3 30 6.0 5.5 5.1 6.0 31 4.6 4.8 5.2 4.9 32 6.0 5.1 6.5 5.9 34 8.5 8.4 8.6 8.0 35 4.7 4.7 4.3 3.8 36 6.3 8.6 8.3 7.1 37 5.7 6.1 5.8 6.2 38 5.9 5.7 5.9 5.5 Mean 6.0 6.2 6.3 6.1 SD 1.4 1.5 1.5 1.3 n/c- condition not completed * - data collection error 116 Table C.12. Individual Mean Rate of Perceived Exertion for Leg Effort (1-10) During a 20km Time-trial Post-Inhalation of Placebo (DP), 200ug (D2), 400ug (D4), and 800ug (D8) of Salbutamol. Group Means and SD for Each Condition are Included. (n=30) Subject DP D2 D4 D8 1 7.1 n/c 7.6 5.8 2 6.8 7.2 6.5 7.4 3 5.8 7.4 6.0 6.0 4 4.4 4.4 4.5 4.4 5 8.4 8.5 8.6 8.2 6 7.5 7.5 8.0 7.8 9 8.9 9.3 9.3 9.0 10 6.2 5.4 4.9 5.3 . 13 4.7 4.0 4.6 4.8 15 7.8 8.0 8.1 7.7 16 4.8 5.9 4.9 4.6 18 5.6 4.9 4.6 5.0 19 5.7 5.2 5.6 6.1 20 5.9 6.5 5.4 6.5 21 5.7 5.0 5.5 6.6 23 5.0 7.0 5.9 6.7 24 2.6 4.0 3.4 3.2 25 6.4 n/c n/c 5.2 26 5.3 5.3 5.7 5.0 27 . 6.1 5.1 5.6 7.0 28 * 7.0 n/c 6.4 29 6.6 6.6 7.0 6.6 30 6.5 5.7 5.5 6.1 31 5.7 6.2 6.6 6.0 32 5.8 5.0 5.4 5.8 34 8.6 8.3 8.4 8.3 35 4.6 4.6 4.2 3.8 36 5.7 8.8 8.6 7.0 37 4.4 5.8 4.9 5.1 38 5.4 4.9 5.6 4.6 Mean 6.0 6.2 6.1 6.1 SD 1.4 1.5 1.5 1.4 n/c- condition not completed * - data collection error 117 Table C.13. Individual Urine Concentrations of Non-sulphated Salbutamol (ng-mf1) at 60 Minutes (T60) Post-Inhalation of Placebo (DP), 200pg (D2), 400pg (D4), and 800pg (D8) of Salbutamol. Group Means and SD for Each Condition are Included. Subject DP D2 D4 D8 1 0 n/c 118 347 2 0 84 94 372 3 0 108 627 339 4 31 0 66 67 5 0 99 286 237 6 0 25 87 217 9 0 153 210 142 10 0 0 33 33 13 54 347 123 151 15 20 46 114 539 16 48 0 83 225 18 0 41 152 66 19 26 26 177 98 20 0 34 0 340 21 0 45 . 198 361 23 0 0 42 72 24 0 25 60 831 25 0 n/c n/c 250 26 0 0 55 121 27 0 90 100 165 28 0 56 n/c 52 29 0 0 22 '. 43 30 0 0 38 247 31 0 0 58 48 32 0 29 242 186 34 0 51 68 301 35 • 0 0 67 189 36 0 0 0 147 37 0 38 45 96 38 0 0 51 26 Mean 7 46 115 + 210 +'a'* SD 15 73 126 177 n/c- no urine sample collected + - denotes significantly greater than DP, p<0.05 a - denotes significantly greater than D2, p<0.05 * - denotes significantly greater than D4, p<0.05 11 Table C.14. Individual Urine Concentrations of Non-sulphated Salbutamol (ng-ml"1) Corrected for Specific Gravity (1.005) at 60 Minutes (T60) Post-Inhalation of Placebo (DP), 200ug (D2), 400ug (D4), and 800ug (D8) of Salbutamol. Mean and SD for Each Condition are Included. Subject DP D2 D4 D8 1 0 n/c 18 124 2 0 38 78 186 3 0 32 157 89 4 7 0 16 19 5 0 25 71 62 6 0 42 109 271 9 0 45 44 28 10 0 0 33 82 13 25 145 27 63 15 14 15 57 180 16 11 0 20 62 18 0 15 36 37 19 9 32 126 82 20 0 42 0 425 21 0 16 66 113 23 0 0 210 . 180 24 0 18 33 198 25 0 n/c n/c 54 26 0 0 12 36 27 0 21 25 92 28 0 70 n/c 65 29 0 0 12 43 30 0 0 47 103 31 0 0 24 30 32 0 6 55 116 34 0 8 15 68 35 0 0 67 105 36 0 0 0 37 37 0 24 45 96 38 0 0 21 6 Mean 2 19 52 + 104 +A* SD 6 29 49 90 n/c - no urine sample collected + - denotes significantly greater than DP, p<0.05 a - denotes significantly greater than D2, p<0.05 * - denotes significantly greater than D4, p<0.05 119 Table C.15. Individual Urine Concentrations of Non-sulphated Salbutamol (ng-mf1) . Corrected for Specific Gravity (1.025) at 60 Minutes (T60) Post-Inhalation of Placebo (DP), 200pg (D2), 400pg (D4), and 800pg (D8) of Salbutamol. Mean and SD for Each Condition are Included. Subject DP D2 D4 D8 1 0 n/c 92 620 2 0 191 392 930 3 0 159 784 446 4 35 0 82 93 5 0 124 357 312 6 0 208 544 1356 9 0 225 219 142 10 . 0 0 165 412 13 123 723 134 315 15 71 77 285 898 16 57 0 99 312 18 0 73 181 183 19 46 162 - 632 408 20 0 212 0 2125 21 0 80 330 564 23 0 0 1050 900 24 0 89 167 989 25 0 n/c n/c 272 26 0 0 60 178 27 0 107 125 458 28 0 350 n/c 325 29 0 0 61 215 30 0 0 237 515 31 0 0 121 150 32 0 29 275 581 34 0 42 77 342 35 0 0 335 525 36 0 0 0 184 37 0 119 225 480 38 0 0 106 32 Mean 12 97 261 + 520 +'a'* SD 30 147 245 451 n/c - no urine sample collected + - denotes significantly greater than DP, p<0.05 a - denotes significantly greater than D2, p<0.05 * - denotes significantly greater than D4, p<0.05 120 Table C.16. Specific Gravity of Individual Urine Samples for Placebo (DP), 200ug (D2), 400ug (D4), and 800ug (D8) Conditions. Mean and SD for Each Condition are Included. Subject DP D2 D4 D8 1 1.008 n/c 1.032 1.014 2 1.006 1.011 1.006 1.010 3 1.020 1.017 1.020 1.019 4 1.022 1.010 1.020 1.018 5 1.018 1.020 1.020 1.019 6 1.004 1.003 1.004 1.004 9 1.008 1.017 1.024 1.025 10 1.007 1.007, 1.005 1.002 13 1.011 1.012 1.023 1.012 15 1.007 1.015 1.010 1.015 16 1.021 1.023 1.021 1.018 18 1.011 1.014 1.021 1.009 19 1.014 1.004 1.007 1.006 20 1.003 1.004 1.005 1.004 21 1.012 1.014 1.015 1.016 23 1.002 1.002 1.001 1.002 24 1.020 1.007 1.009 1.021 25 1.021 n/c n/c 1.023 26 1.032 1.018 1.023 1.017 27 . 1.029 1.021 1.020 1.009 28 1.005 1.004 . n/c 1.004 29 . 1.003 1.004 1.009 1.005 30 1.009 1.011 1.004 1.012 31 1.013 1.008 1.012 1.008 32 1.024 1.025 1.022 1.008 34 1.008 1.030 1.022 1.022 35 1.004 1.005 1.005 1.009 36 1.003 1.023 1.004 1.020 37 1.005 1.008 1.005 1.005 38 1.021 1.028 1.012 1.020 Mean 1.012 1.013 1.014 1.013 SD 0.008 0.008 0.008 0.007 n/c - no urine sample collected APPENDIX D - SCALE FOR MEASURING PERCEIVED EXERTION 10 f Maximal 9 -j™ Very, very severe 8 7 —|— Very severe 6 5 •+• Severe T 4 Somewhat severe 3 Moderate 2 - - - Slight . 1 Very Slight 0.5 ™ ™ Very, very slight 0 * Nothing at all 

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