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Relationship between exhaled and inhaled nitric oxide and exercise-induced hypoxemia Sheel, Andrew William 1999

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RELATIONSHIP BETWEEN EXHALED AND INHALED NITRIC OXIDE AND EXERCISE-INDUCED HYPOXEMIA by ANDREW WILLIAM SHEEL B.P.E., The University of New Brunswick, 1993 M.Sc, The University of British Columbia, 1995 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES SCHOOL OF HUMAN KINETICS We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August 1999 © Andrew William Sheel, 1999 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, 1 agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. of The University of British Columbia Vancouver, Canada Date AW DE-6 (2788) ABSTRACT The consensus in the literature is that exercise-induced hypoxemia (EIH) occurs secondary to ventilation-perfusion (VA/Q) inequalities and diffusion limitations resulting from elevated pulmonary pressures causing the development of interstitial pulmonary edema, or decreased pulmonary transit time in the pulmonary vasculature. Endogenously produced pulmonary nitric oxide (NO) has been hypothesized to have several physiological functions including VA/Q matching and maintenance of low pulmonary vascular resistance. Respiratory derived NO is detectable in exhaled gases. Inhaled NO, a selective pulmonary vasodilator is used in the treatment of diseases characterized by pulmonary hypertension and hypoxemia. Short-term inhalation of NO causes selective pulmonary vasodilation without any systemic effects. Given that athletes with EIH are thought to have altered pulmonary hemodynamics during exercise, the relationship between endogenously produced and exogenously delivered NO and EIH was examined in two separate studies. It was hypothesized that subjects with EIH would have a decreased production rate of NO (VNO) compared to subjects who maintained normal oxyhemoglobin saturation (Sa02) and that Sa02 would be correlated with VNO. A group of highly-trained male cyclists (n = 18), some of whom develop EIH performed a maximal cycle test. VNO was determined during the cycle test. No significant differences were observed between those with and those without EIH. There was also no observed linear relationship between delta Sa02 and delta VNO. It can be concluded that NO present in exhaled air is not related to the etiology of EIH. In a subsequent study, delivery of NO was accomplished using highly trained male cyclists (n = 7) with EIH who performed four 5-min cycle tests at V02max under conditions of normoxia (N), normoxia + 20 ppm nitric oxide (N/NO), hypoxia (H), and hypoxia + 20 ppm nitric oxide (H/NO). It was hypothesized that: (i) inhaled NO would reverse EIH during normoxia, and (ii) inhaled NO would improve arterial oxygenation during hypoxia. Inhalation of NO during normoxic or hypoxic conditions did not significantly affect gas ii exchange, cardiorespiratory variables, or power output. These findings imply that pulmonary capillary blood volume reaches a maximal morphological limit during exercise and further dilation is not possible. TABLE OF CONTENTS Abstract ii List of Tables v List of Figures viiDedication x CHAPTER 1 General Introduction 1 CHAPTER 2 Exhaled Nitric Oxide during Exercise 12 2.1 Introduction 12.2 Methodology 4 2.3 Results 7 2.4 Discussion 21 CHAPTER 3 Inhaled Nitric Oxide during Exercise 25 3.1 Introduction 23.2 Methodology3.3 Results 31 3.4 Discussion 4CHAPTER 4 General Summary and Conclusions 52 REFERENCES 54 APPENDIX A Review of Literature: Exercise-Induced Hypoxemia 75 APPENDIX B Review of Literature: Exhaled Nitric Oxide 96 APPENDIX C Review of Literature: Inhaled Nitric Oxide 108 During Exercise APPENDIX D Individual subject data: Exhaled Nitric Oxide 116 APPENDIX E Individual subject data: Inhaled Nitric Oxide 122 APPENDIX F Group data: Inhaled Nitric Oxide 138 iv LIST OF TABLES TABLE 1. Descriptive and peak exercise values in all subjects (ALL), subjects with normal (NOS) and low (LOS) oxyhemoglobin saturation. TABLE 2. Concentration of nitric oxide (CNO) and production rate of nitric oxide (VNO) at rest and during incremental exercise in all subjects (n = 1 TABLE 3. Descriptive and resting pulmonary function data. TABLE 4. Peak exercise values during maximal cycle ergometer tests. TABLE 5. Power output (watts) during rest and at each min of 5-min cycle ergometry tests under conditions of normoxia (N), normoxia + 20 ppm nitric oxide (N/NO), hypoxia (H), and hypoxia + 20 ppm nitric oxide (H/NO). TABLE 6. Published values for exhaled nitric oxide during rest and exercise. TABLE 7. Actual and potential uses of inhaled nitric oxide. TABLE 8. Individual subject data for age, height, mass and sport. TABLE 9. Individual subject data for respiratory variables during maximal cycle ergometer exercise. TABLE 10. Individual subject data for heart rate, saturation, and power during maximal cycle ergometer exercise. TABLE 11 Individual subject data for concentration of nitric oxide (CNO) at rest and during incremental exercise (watts). TABLE 12. Individual subject data for production rate of nitric oxide (VNO) at rest and during incremental exercise (watts). TABLE 13. Concentration of nitric oxide (CNO) (ppm) in subjects with normal (NOS) and low (LOS) oxyhemoglobin saturation at rest and during incremental exercise. TABLE 14. Production rate of nitric oxide (VNO) (nmol-min"1) in subjects with normal (NOS) and low (LOS) oxyhemoglobin saturation at rest and during incremental exercise. TABLE 15. Individual subject data for age, height, mass, sport, level of competition, and amount of training per week. TABLE 16. Individual subject data for pulmonary function. TABLE 17. Individual subject data for respiratory variables during normoxic (F1O2 = 21%) maximal cycle ergometer exercise. TABLE 18. Individual subject data for heart rate, saturation, 123 and power during normoxic (F1O2 = 21%) maximal cycle ergometer exercise. TABLE 19. Individual subject data for respiratory variables 123 during hypoxic (F1O2 = 14%) maximal cycle ergometer exercise. TABLE 20. Individual subject data for heart rate, saturation, 124 and power during hypoxic (F1O2 = 14%) maximal cycle ergometer exercise. TABLE 21 to TABLE 46. Individucal subject data for blood 124-137 gas and cardiorespiratory measurements during rest and at each min of 5-min cycle ergometry tests under conditons of normoxia (N), normoxia + 20 ppm nitric oxide (N/NO), hypoxia (H), and hypoxia + 20 ppm nitric oxide (H/NO). TABLE 47. Oxygen consumption (VO2, lmin"1) during rest and 138 at each min of 5-min cycle ergometry tests under conditions of normoxia (N), normoxia + 20 ppm nitric oxide (N/NO), hypoxia (H), and hypoxia + 20 ppm nitric oxide (H/NO). TABLE 48. Oxygen consumption (VO2, ml-kg"1 min"1) during rest 138 and at each min of 5-min cycle ergometry tests under conditions of normoxia (N), normoxia + 20 ppm nitric oxide (N/NO), hypoxia (H), and hypoxia + 20 ppm nitric oxide (H/NO). TABLE 49. Minute ventilation (VE, lmin"1) during rest and at 139 each min of 5-min cycle ergometry tests under conditions of normoxia (N), normoxia + 20 ppm nitric oxide (N/NO), hypoxia (H), and hypoxia + 20 ppm nitric oxide (H/NO). TABLE 50. Respiratory exchange ratio (RER, VCO2/VO2) 139 during rest and at each min of 5-min cycle ergometry tests under conditions of normoxia (N), normoxia + 20 ppm nitric oxide (N/NO), hypoxia (H), and hypoxia + 20 ppm nitric oxide (H/NO). TABLE 51. Heart rate (beats-min"1) during rest and at each min of 140 5-min cycle ergometry tests under conditions of normoxia (N), normoxia + 20 ppm nitric oxide (N/NO), hypoxia (H), and hypoxia + 20 ppm nitric oxide (H/NO). TABLE 52. Arterial pressure of 02 (Pa02, mmHg) during rest and 140 at each min of 5-min cycle ergometry tests under conditions of normoxia (N), normoxia + 20 ppm nitric oxide (N/NO), hypoxia (H), and hypoxia + 20 ppm nitric oxide (H/NO). vi TABLE 53. Alveolar-arterial difference for 02 ([A-a]D02, mmHg) 141 during rest and at each min of 5-min cycle ergometry tests under conditions of normoxia (N), normoxia + 20 ppm nitric oxide (N/NO), hypoxia (H), and hypoxia + 20 ppm nitric oxide (H/NO). TABLE 54. Oxyhemoglobin saturation (Sa02, %) during rest 141 and at each min of 5-min cycle ergometry tests under conditions of normoxia (N), normoxia + 20 ppm nitric oxide (N/NO), hypoxia (H), and hypoxia + 20 ppm nitric oxide (H/NO). TABLE 55. Arterial pressure of C02 (PaC02, mmHg) during rest 142 and at each min of 5-min cycle ergometry tests under conditions of normoxia (N), normoxia + 20 ppm nitric oxide (N/NO), hypoxia (H), and hypoxia + 20 ppm nitric oxide (H/NO). TABLE 56. Values for pH during rest and at each min of 5-min 142 cycle ergometry tests under conditions of normoxia (N), normoxia + 20 ppm nitric oxide (N/NO), hypoxia (H), and hypoxia + 20 ppm nitric oxide (H/NO). TABLE 57. Bicarbonate (HCO3", mmolT1) during rest and at each 143 min of 5-mincycle ergometry tests under conditions of normoxia (N), normoxia + 20 ppm nitric oxide (N/NO), hypoxia (H), and hypoxia + 20 ppm nitric oxide (H/NO). TABLE 58. Base excess (mmolT1) during rest and at each min of 143 5-min cycle ergometry tests under conditions of normoxia (N), 121 normoxia + 20 ppm nitric oxide (N/NO), hypoxia (H), and hypoxia + 20 ppm nitric oxide (H/NO). vii LIST OF FIGURES FIGURE 1. Concentration of nitric oxide (CNO) at rest and 19 during incremental exercise in subjects with normal (NOS, n =12) and low (LOS, n - 6) oxyhemoglobin saturation. FIGURE 2. Production rate of nitric oxide (VNO) at rest and 19 during incremental exercise in subjects with normal (NOS, n = 12) and low (LOS, n = 6) oxyhemoglobin saturation. FIGURE 3. Relationship between change in percent 20 oxyhemoglobin saturation (delta Sa02) and change in nitric oxide production (delta VNO) in all subjects (n = 18, r = - 0.12, P > 0.05). FIGURE 4. Overview of experimental protocol, repeated 30 for each condition of normoxia (N), normoxia + 20 ppm nitric oxide (N/NO), hypoxia (H), and hypoxia + 20 ppm nitric oxide (H/NO). S = sample for blood and cardiorespiratory variables. FIGURE 5. Arterial pressure of O2 (Pa02) during rest and at each 34 min of 5-min cycle ergometry tests under conditions of normoxia (N), normoxia + 20 ppm nitric oxide (N/NO), hypoxia (H), and hypoxia + 20 ppm nitric oxide (H/NO). FIGURE 6. Percent oxyhemoglobin saturation (Sa02) during rest 35 and at each min of 5-min cycle ergometry tests under conditions of normoxia (N), normoxia + 20 ppm nitric oxide (N/NO), hypoxia (H), and hypoxia + 20 ppm nitric oxide (H/NO). FIGURE 7. Alveolar-arterial difference for O2 ([A-a]D02) during 36 rest and at each min of 5-min cycle ergometry tests under conditions of normoxia (N), normoxia + 20 ppm nitric oxide (N/NO), hypoxia (H), and hypoxia + 20 ppm nitric oxide (H/NO). FIGURE 8. Arterial pressure of C02 (PaC02) during rest and 37 at each min of 5-min cycle ergometry tests under conditions of normoxia (N), normoxia + 20 ppm nitric oxide (N/NO), hypoxia (H), and hypoxia + 20 ppm nitric oxide (H/NO). FIGURE 9. Oxygen consumption (V02) during rest and at 38 each min of 5-min cycle ergometry tests under conditions of normoxia (N), normoxia + 20 ppm nitric oxide (N/NO), hypoxia (H), and hypoxia + 20 ppm nitric oxide (H/NO). FIGURE 10. Heart rate during rest and at each min of 5-min 39 cycle ergometry tests under conditions of normoxia (N), normoxia + 20 ppm nitric oxide (N/NO), hypoxia (H), and hypoxia + 20 ppm nitric oxide (H/NO). viii DEDICATION to Judy and Edith, who taught me the value of learning and Jen, the most gentle creature 1 CHAPTER 1. GENERAL INTRODUCTION Respiratory limitations to exercise and the biology of nitric oxide are contemporary topics of interest to the modern exercise physiologist. This is highlighted by the recent attention these subjects have received in the American College of Sports Medicine series "Exercise and Sport Sciences Reviews" [16, 79]. This dissertation examines the relationship between these two areas. Firstly, each topic is briefly introduced along with a description of the research questions that were addressed. Secondly, the details of the two experiments performed are described in separate chapters followed by a concluding chapter. Exercise-Induced Hypoxemia. The conventional paradigm held by exercise physiologists is that individuals engaged in strenuous activity at sea-level maintain blood gas homeostasis for both 02 and CO2 [8, 9]. Their respiratory system is able to precisely regulate ventilation and gas exchange. It is commonly held that the structural capacity of the normal lung is "over-built" and exceeds the demand for pulmonary O2 transport in the healthy, exercising human. That is, the upper limits to maximum exercise breathing frequency (/b), tidal volume (VT), minute ventilation (VE), and gas exchange are determined structurally by the lung's natural morphology and provide an adequate reserve for exercise demands. It is also believed that the respiratory system remains largely unchanged in response to chronic endurance training [45]. A notable exception to this adequate reserve concept is that in some highly-trained male aerobic athletes decreases in arterial pressure of O2 (Pa02), oxyhemoglobin saturation (Sa02), and a widened alveolar-arterial difference for O2 ([A-a]D02) are observed during heavy exercise [48, 91, 173]. In this population, it appears that demand exceeds the supply limits of the lung. This phenomenon, termed exercise-induced hypoxemia (EIH), occurs in approximately 50% of highly-trained male endurance athletes, while 2 un-trained and moderately-trained males do not experience EIH [173]. While EIH has been clearly established and well defined in the male athletic population, only recently has it been observed to occur in females (see APPENDIX A) [80]. EIH has been defined as a decrease in PaCh of 10-15 mmHg or an SaC^ less than 92% [174] with negative effects on maximal oxygen consumption (VChmax) [67, 96, 123, 132, 174, 216] and exercise performance [121]. Decreases in PaC>2 and SaC»2 during heavy exercise represent a physiological failure of the respiratory system. The pulmonary limitation to gas exchange is also manifested by an increase in the [A-a]DC>2, which can reach values as high as 50 mmHg [91]. The cause and significance of EIH have been the topics of a considerable research effort, yet the mechanism(s) responsible remain controversial. Four potential factors have been identified; (i) veno-arterial shunts, (ii) relative alveolar hypoventilation, (iii) ventilation/perfusion (VA/Q) inequalities, and (iv) diffusion limitations. Veno-arterial shunts [48, 176] and relative alveolar hypoventilation [176, 177] have been identified as minor contributors to the pathophysiology of EIH. In the ideal lung, PAO2 and PaC>2 are essentially identical where [A-a]D02 ~ 0. Any cause of inadequate gas exchange such as (i) incomplete diffusive gas mixing in the lung acini, (ii) diffusion limitation of O2 transfer across the blood-gas barrier, and (iii) intrapulmonary and (iv) extrapulmonary right-to-left shunt will cause an increase in [A-a]DC>2. Consequently, a widened [A-a]D02 is a general index of inadequate gas exchange and is not reflective of any one physiological condition [104]. The importance of a widened [A-a]DC>2 is useful in identifying a gas-exchange limitation but is limited by its lack of specificity. More specific identification of gas exchange limitations during exercise have been documented: worsening of VA/Q relationships [64, 89, 93, 204, 230], decreased red blood cell pulmonary capillary transit time [88, 234], and evidence of pulmonary edema [141, 204, 230]. 3 The presence of interstitial pulmonary edema during exercise is a favored hypothesis to explain the observed VA/Q inequality and gas exchange limitations [89, 106]. Anecdotal reports [137], depressed post-exercise diffusing capacity of the lung [185, 204], altered lung computerized tomography [39], and magnetic resonance images post-exercise [141] have been identified as possible indicators of pulmonary edema. However, these investigations can be collectively interpreted as indirect and circumstantial. Briefly, post-exercise measures may not correctly assess physiological events that are occurring during exercise. Identification of pulmonary edema during exercise, along with the possible causative factors has been difficult to achieve in exercising humans. The pulmonary blood-gas barrier is extremely thin to allow the least possible resistance to allow diffusion of 02 and CO2. At the same time, it must be strong to withstand the high capillary wall stresses that develop during exercise when capillary pressure rises [239]. In some athletic populations, the bioengineering of the pulmonary blood-gas barrier fails, most notably in racehorses where both pulmonary diffusion limitations and exercise-induced pulmonary hemorrhage occur [230, 238]. Exercise-induced pulmonary hemorrhage has also been reported in Shetland ponies [52], and greyhound dogs [114]. Identification of pulmonary stress failure in humans is a substantially more difficult task. Normal mean pulmonary artery pressure (PPA) in humans is ~ 15 mmHg, and any increase is regarded as representing pulmonary hypertension. Three principal mechanisms of pulmonary hypertension are (i) increase in left atrial pressure (PLA), (ii) increase in pulmonary blood flow, and (iii) increase in pulmonary vascular resistance (PVR) [235]. Alterations to systemic arterial pressure, via effects on left ventricular emptying, could alter pulmonary blood flow and PLA- It is well known that mean systemic arterial pressure increases with increasing exercise effort and left atrial and ventricle diastolic pressures also increase. Assuming capillary wedge pressures 4 (PWEDGE) reflect PLA, it is not surprising that an exercise-related increase in arterial pressure is accompanied by an increase in PWEDGE [190]. At a capillary transmural pressure of 40 mmHg ultrastructural changes to the blood gas barrier are seen using a rabbit model [225], including disruption of the capillary endothelial and alveolar epithelial layers, and in some cases all layers of the wall. Based on available human data, human PPA and PWEDGE can reach values ~ 40 and 27 mmHg respectively [189, 230]. Increased PPA with exercise in humans has been consistently reported [70, 101, 188, 222], while pulmonary capillary pressure must be deduced from the above mentioned pressures. The calculated capillary pressure at the base of the human lung can reach > 35 mmHg during exercise. These pressures observed in humans by Wagner and colleagues [230] are within the range where stress failure of pulmonary capillaries is seen in animal preparations [225, 239]. The net result of stress failure of the pulmonary capillaries at these elevated pressures is altered or disruption of the capillary endothelial and alveolar epithelial layers, collection of red blood cells in the alveolar wall interstitium, proteinaceous fluid and red blood cells in alveolar spaces, interstitial edema and fluid protrusions of the endothelium into the capillary lumen [237]. The cumulative effect of these events is presumably impaired gas-exchange. It has been demonstrated that high lung volumes increase stress failure in the pulmonary capillaries of an in situ vascular perfusion preparation of rabbit lung [61]. Possibly, the distention of lung capillaries during exercise, via pulmonary pressures, could increase permeability and promote fluid shifts into the interstitium [225]. Extrapolation of these data to exercising human athletes is not yet possible, but is suggestive of a putative mechanism for pulmonary edema. Accumulation of interstitial fluid is usually removed by lymph flow, but may be compromised during exercise [61]. Using sheep, it has been shown that 15-min after 5 prolonged heavy exercise (40-60 min) pulmonary lymph flow is significantly higher than at rest [153]. Possibly, the lymph system is unable to clear the interstitial space during exercise, resulting in mild pulmonary edema. This hypothesis has received support from recent studies [89, 95], but remains to be tested adequately in athletes with EIH. The pulmonary endothelium is exposed to the entire cardiac output (Q) and is possibly vulnerable to injury. Strenuous exercise can induce an acute inflammatory response marked by leukocytosis and neutrophil activation and release of inflammatory mediators. Inflammatory mediators such as eicosanoids, reactive oxygen species, cytokines all could negatively affect pulmonary vasomotor tone or membrane permeability. The potential for permeability edema and/or stress failure suggests the presence of an inflammatory mediator. One mediator in particular, histamine, has received attention in the EIH literature. Histamine can increase the permeability of the lung vascular membrane [181] and levels of histamine are increased in athletic populations during exercise [7, 83, 147, 149]. A significant correlation (r = 0.8) has been observed between the change in plasma histamine and the decrease in Pa02 in young and masters athletes compared to controls [7]. In addition, inhibition of histamine release via nedocromil sodium reduces the magnitude of decreased Pa02 and [A-a]D02 [180]. In this investigation EIH was not completely reversed, but oxygenation was significantly improved; indicating that histamine was involved but other factors also exist in the development of EIH. Other inflammatory mediators, such as leukotrienes may be involved and have been detected in the bronchoalveolar lavage of highly-trained athletes following maximal exercise [94]. Based on the available data, it is reasonable to suggest that pulmonary hypertension (i.e., increased PPA) during exercise in some highly-trained athletes may be responsible for the development of mild pulmonary edema and the ensuing EIH. 6 Exhaled Nitric Oxide. It is now well established that nitric oxide (NO) is produced in a variety of both human and animal cells with numerous biological functions. NO is believed to exert regulatory functions in the circulatory, pulmonary, nervous, and immune systems [244]. NO is produced from one of the nitrogen atoms of the N-guanidino terminals of the common amino acid L-arginine, and molecular oxygen. In addition to NO, citrulline is a product of this reaction. The formation of NO also requires an isoform from the family of enzymes, NO Synthase (NOS), and other co-factors (see APPENDIX B). NO is thought to be important in many regulatory processes because of its potent vasodilatory effect. The production of NO is believed to cause relaxation of smooth muscle cells via a signal transduction system. Here, NO stimulates the formation of 3',5'-cyclic guanosine monophosphate (cGMP), which in turn leads to a decrease in the concentration of free Ca^ in the smooth muscle cytosol resulting in relaxation. Endogenously produced pulmonary NO has been hypothesized to have several physiological functions. Possible roles include VA/Q matching, maintenance of low PVR, and airway relaxation. The idea that an optimal VA/Q ratio may be regulated by NO is supported by data showing arterial hypoxia in anesthetized animals upon administration of NOS inhibitors [163]. The importance of NO is evident when inspired NO induces pulmonary vasodilation and improves VA/Q relationships [60, 168, 182, 197]. Endogenous NO was recently found to be critical to the maintenance of a low PVR in endothelial NOS knockout mice who developed pulmonary hypertension [55]. It is also thought that NO is continuously released to maintain pulmonary arterial tone, systemic pressure, and Q [218] where patients with heart disease have reduced expiratory NO levels, perhaps explaining why they exhibit an increased vasoconstriction of their pulmonary blood vessels [220]. As alluded to above, endogenously produced NO has been detected in the exhaled air of humans [72] and numerous animal species [205]. Expiratory NO concentrations (CNO) and the production rate of NO (VNO) have been examined during physical exercise [130, 167]. It is believed that the formation of NO may play a role in the normal pulmonary response to exercise. Basal release of endogenous NO has been shown to have a role in low pulmonary vascular tone at rest and exercise in sheep [120]. In addition, it has been suggested that a defect in NO synthesis may contribute to high-altitude pulmonary edema (HAPE) [207]. It would seem that endogenously produced NO is necessary for maintenance of VA/Q matching and a low pulmonary vascular resistance, the extent to which expired concentrations of exhaled NO reflect this process requires clarification. During exercise, Q increases in proportion to exercise intensity, and the pulmonary circulation is able to accommodate the increase with little rise in pulmonary pressure. This is typically thought to be due to the distension and recruitment of the pulmonary capillaries. It has been traditionally thought that this process is passive in nature where the pulmonary circulation can be fully recruited. It is only possible to speculate at this stage, however it seems likely that NO may play a role in mediating the VA/Q response during exercise. Preliminary research into this area suggests the possibility that NO production is different between highly-trained athletes and un-trained individuals. It is widely accepted that physical conditioning affects the cardiovascular response to exercise and potentially training-induced alterations to Q, pulmonary blood flow, and VE may influence the production of NO. Basal CNO has been found to be identical between high aerobic capacity athletes and non-athletes [40, 130] and higher in one subject described as an athlete [19]. However, during exercise, it has been observed that athletes maintain a higher VNO than do moderately trained and sedentary individuals at a given exercise 8 intensity [130]. In disagreement with these data, no difference in VNO was found between trained men, sedentary women, and sedentary men at rest or during cycle exercise [40]. The dissimilar results may be related to the different levels of physical conditioning and maximal aerobic capacities of the respective athletic groups; 4.4 1-min"1 [40] and 5.6 1-min"1 [130]. These two groups would likely have different VE, Q, and pulmonary blood flow, thus they would have different rates of NO production. To date, these are the only data available addressing this issue, leaving the question of the effects of physical conditioning on basal and exercising NO unresolved. Athletes with EIH have impaired gas exchange, possibly related to alterations in pulmonary tone. The relationship between exhaled NO and EIH has yet to be examined. NO has also been detected in asthmatic patients and is elevated in comparison to matched non-asthmatics [4, 111, 112]. The increase in exhaled NO in asthmatics likely reflects increased activity of inducible NOS that may be mediated by inflammatory cytokines [73]. Exhaled NO may reflect airway inflammation in asthma, and may be used as means of monitoring inflammatory events [18]. The pulmonary endothelium is susceptible to injury given that it is exposed to the entire Q. Various inflammatory mediators may play a role, including NO, in altering pulmonary vascular tone and membrane permeability. Subsequent vasoconstriction or vasodilation may promote overperfusion, increased filtration pressure and edema formation. NO detected in exhaled air, as an indicator of lower airway inflammation, may be representative of these events but has not yet been clearly established. 9 Inhaled Nitric Oxide. Inhaled NO is a potent and selective pulmonary vasodilator [59, 60]. Inhaled NO therapy has received much clinical attention in recent years, and has been useful in the treatment of lung diseases characterized by pulmonary hypertension and arterial hypoxemia [2, 25, 194, 195, 198, 245]. Inhalation of NO in low concentrations (5-80 ppm) can cause selective pulmonary vasodilation because of the speed with which NO reacts with hemoglobin. Essentially, free NO never leaves the pulmonary circulation and only dilates vascular beds that are well-ventilated resulting in improved VA/Q matching [109]. The current consensus is that smooth muscle vasodilation is caused by the NO signal transduction system as described above. The clinical effects and applications of inhaled NO have been described extensively in recent reviews [24, 97, 143, 152]. To summarize, inhalation of NO is used effectively to treat a multitude of disorders including: primary pulmonary hypertension, pulmonary hypertension caused by embolism, pulmonary hypertension associated with cardiopulmonary bypass, adult respiratory distress syndrome, and HAPE [53, 107, 161, 198, 207] Only recently have the effects of inhaled NO been examined in exercising populations. Roger et al. [197] demonstrated that in chronic obstructive pulmonary disease (COPD) patients who inhaled NO (40 ppm), PPA was reduced both at rest and during submaximal exercise when compared to breathing only room air. During exercise, Pa02 was reduced while breathing room air (- 5 mmHg) and was slightly improved (+ 2 mmHg) with the addition of NO to the inspirate. Although small, both of these changes were considered statistically significant. It was also observed that when patients exercised while inhaling NO, the degree of VA/Q mismatch was lessened and likely was causative in improved Pa02 values. Inhaled NO (40 ppm) has also been shown to improve exercise capacity in patients with congestive heart failure [117]. Patients with 10 severe heart failure reduced the resistance of the pulmonary vessels leading to augmented maximum exercise workload and VO2 at the anaerobic threshold. In a separate investigation, Bocchi et al. [26] examined patients with left ventricular dysfunction and chronic heart failure during exercise conditions of control (room air) and NO inhalation (30 ppm). VT and ventilatory equivalent for oxygen (VE/VO2) were significantly reduced with NO compared to control. Unfortunately, other measures of gas exchange and pulmonary hemodynamics were not measured and the physiological significance of these findings is not clear. Nonetheless, based on these available data, it can be tentatively concluded that inhalation of NO during exercise in clinical populations can improve gas exchange and pulmonary hemodynamics. The effect is likely explained by a preferential distribution of inhaled NO to well-ventilated alveolar units, a reduction in the dispersion of ventilation distribution, and a lowering of pulmonary pressures. To date, only one study has sought to investigate the effects of NO inhalation on pulmonary gas exchange during exercise in highly trained athletes [49]. Male endurance athletes (n = 9: V02max ~ 65 ml-kg^-min"1) performed a maximal cycle test to exhaustion while inhaling either (i) room air, or (ii) room air combined with 15 ppm NO. No differences between conditions were observed for V02inax or maximal work. Inhalation of NO caused Pa02 to decrease at rest, 50%, 75%, and 100% V02max (I 9.5, 15.0, 14.6, and 5.1 mmHg). Interestingly, delta Pa02 continuously fell without NO (13.5%). With NO, after a preliminary drop, a stabilization of delta Pa02 was observed between 75 and 100% V02max. This trend was also reflected in [A-a]D02- Inhalation of NO also abolished histamine release between 75 and 100% V02max but EIH was not reversed. The physiological significance of these results remains to be determined. As concluded by Durand and co-workers [49], the negative effect of NO on gas 11 exchange at rest was unexpected and continued during exercise; and these preliminary results should be interpreted with caution. Statement of the Problem. Firstly, the relationship between endogenous pulmonary NO and EIH is not known. It is currently not clear if athletes with EIH have a different NO response to exercise compared to other highly-trained athletes without EIH. Based on the observation that those more susceptible to HAPE may have a defect in NO synthesis and that untrained, highly-trained athletes, and asthmatics have different levels of exhaled NO, it is reasonable to suggest that athletes with EIH may have an altered exhaled NO response to exercise. Secondly, while the mechanisms of EIH are debatable, the pathology may be related to elevations in pulmonary pressures. Thus inhaled NO, as a known pulmonary vasodilating agent, may reverse or attenuate the mechanisms underlying EIH if elevations in pulmonary pressures are involved. Hypotheses. The purpose of these two studies was to determine the relationship between EIH and NO. Specifically, the two fundamental hypotheses tested were: 1. Exhaled NO will be reduced in athletes with EIH compared to matched athletes without EIH. 2. Inhaled NO will improve arterial oxygenation in athletes with EIH during normoxic and hypoxic heavy exercise. 12 CHAPTER 2. EXHALED NITRIC OXIDE 2.1 INTRODUCTION During maximal exercise Pa02 and Sa02 are decreased significantly in some highly-trained male athletes [48, 91, 173] and an increase in [A-a]D02 is associated with the lowered Pa02. The mechanisms to explain the reduction in blood oxygenation remain controversial. It is generally agreed that VA/Q inequality and diffusion limitations resulting from elevated pressures in the pulmonary vasculature are a likely explanation [89]. It is now well established that NO is produced in a variety of both human and animal cells and has numerous biological functions. NO is a ubiquitous molecule thought to exert regulatory functions in the circulatory, pulmonary, nervous and immune systems [244]. Production of NO has vasodilatory effects causing relaxation of smooth muscle cells via a signal transduction system. In brief, NO is synthesized from the common amino acid L-arginine by several types of NO synthase (NOS). NO stimulates the conversion of GTP to cGMP leading to relaxation of smooth muscle by decreasing the concentration of free Ca in the cytosol. Endogenously produced NO was first detected in the exhaled air of humans by Gustafsson et al. [72] and both the concentration (CNO) and the production rate of NO (VNO) have since been examined during aerobic exercise [19, 40, 100, 130, 135, 167, 169]. Exercise has been shown to have no effect on CNO [19, 100], or cause a slight decrease [40, 130, 167, 169]. In all pulmonary NO-exercise investigations,VNO increases with physical work, however there is considerable variability among studies, possibly related to differences in training status and measurement techniques. Increases in VNO during exercise have been reported as different [19, 130] and the same [40] between athletes and non-athletes. A growing body of evidence suggests that exercise training increases basal NO production in the systemic vasculature [68, 108, 115, 172]. It is well known 13 that lowered systemic blood pressure and heart rate, increased coronary blood flow and capillary density of skeletal muscle, all characterize the effects of chronic exercise training. Decreased systemic blood pressure may be mediated by augmented basal production of NO and its subsequent vasodilatory effect [172]. An upregulation of NO production in the systemic vasculature caused by training could be mirrored in the pulmonary vasculature. Endogenously produced pulmonary NO may have several physiological functions including VA/Q matching and maintenance of low pulmonary vascular resistance [71]. The hypothesis that an optimal VA/Q ratio may be regulated by NO is supported by data showing the development of arterial hypoxia in anesthetized animals upon administration of NOS inhibitors [163]. The importance of NO is also evident when inspired NO induces pulmonary vasodilation and improves VA/Q relationships [60, 168, 182, 197]. The consequence of inhaled NO is underscored during submaximal exercise in patients with chronic obstructive pulmonary disease who inhaled 40 ppm NO. Improvements were observed in both VA/Q relationships and Pa02 compared to breathing room air [197]. Endogenous NO was recently found to be critical to the maintenance of a low pulmonary vascular resistance in NOS knockout mice who developed pulmonary hypertension [55], and may be continuously released to maintain pulmonary arterial tone, systemic pressure, and Q [218]. In addition, patients with heart disease have reduced expiratory NO levels, perhaps explaining why they exhibit an increased vasoconstriction of their pulmonary blood vessels [220]. The mechanism for increased VNO during exercise has been attributed to elevated shear stress on the pulmonary endothelium via increased pulmonary blood flow [19, 100, 130]. In the peripheral vasculature, an increase in shear stress leads to enhanced endothelial NO release [163] arising from the frictional force of blood flow, although other physical stimuli such as pulsatility 14 and changes in transmural pressure may be important [170, 201]. Deformation of endothelial cells is the resultant effect of shear stress and initiates the NO-cGMP signal transduction system [142, 242]. Data from an isolated pig lung preparation indicates that the pulmonary vascular endothelium likely contributes to the NO found in exhaled air [44]. Increased pulmonary endothelial NO production may enter the airway lumen via the alveoli. Endurance athletes with EIH are known to have altered VA/Q relationships during strenuous work [89]. Abnormalities in pulmonary vascular tone, resulting in EIH, may be mediated by a lowered NO response to exercise. Thus, the purpose of this study was to determine the relationship between exhaled NO and Sa02 in two groups of highly trained male cyclists. We hypothesized that subjects with EIH would have a decreased VNO compared to subjects who maintained normal Sa02 and EIH would be correlated with VNO. 2.2 METHODOLOGY Subjects. Highly-trained male competitive road and mountain cyclists and triathletes were recruited to participate in this study (n = 18). All subjects were required to have participated in elite level cycle competition (provincial, national, or international) and have no history of cardio-respiratory disease. Prior to any testing, subjects received a verbal description of the experiment and completed a written informed consent form. This study was approved by the Clinical Screening Committee for Research and other Studies Involving Human Subjects of the University of British Columbia. Maximal Cycle Ergometer Test. Prior to all testing, subjects abstained from exhaustive exercise for 24 h, ingestion of food or fluid other than water for 4 h, and alcohol and caffeine consumption for 12 h. V02max was 15 determined using an incremental test on an electronically braked cycle ergometer (Quinton Excalibur). Subjects pedaled at a self-chosen cadence at a progressing workload, which started at 0 watts and increased 30 watts-min"1. Subjects inspired through an air flowmeter (Vacumetrics model 17150, Ventura, CA) using a two-way non-rebreathing valve (Hans-Rudolph, model 2700B, Kansas City, KS) and expired air passed into a 5 litre mixing chamber from which gas samples were analyzed at a rate of 300 ml-min"1 for oxygen and carbon dioxide concentrations (S-3A oxygen analyzer and CD-3A carbon dioxide analyzer, Applied Electrochemistry, Pittsburgh, PA). Expired gases and minute ventilation (VE) were recorded using a computerized system (Rayfield, Waitsfield, VT). Gas analyzers were calibrated with gases of known concentration prior to each experiment, and the air flowmeter was calibrated by passing 100 litres of air through the system. Heart rate was recorded every 15 s using a portable heart rate monitor (Polar Vantage XL, Kempele, Finland). SaC<2 was measured by a pulse oximeter (Ohmeda Biox 3740, Louisville, CO) with values averaged and recorded every 5 s. This oximeter has previously been shown to be a valid and reliable predictor of Sa02 during cycling [133] and performs a self-calibration prior to usage. Prior to placement of the oximeter sensor to the pinna of the ear, a topical vasodilator cream (Finalgon, Boehringer/Ingeheim) was applied to increase local perfusion. Attainment of V02inax was considered when at least three of the four following criteria were met: (i) a plateau in VO2 with increasing workload, (ii) RER > 1.15, (iii) attainment of 90% of age predicted maximal heart rate, and/or (iv) volitional fatigue. Nitric Oxide. It is known that ambient air contains a variable concentration of NO. During the cycle ergometer test subjects inspired compressed air where NO concentration remained < 2 ppb, indicating that NO in expired air was of endogenous origin. The compressed medical air was 16 delivered from a large cylinder through water for humidification, then into a large meteorological balloon, which acted as a reservoir for inspired air. Expired air was mixed within a five litre mixing chamber placed distally to the expiratory port of the mouthpiece. Air samples were withdrawn from the chamber, through a column of Drierite to remove water vapor and into a three litre syringe (Hans Rudolph) equipped with a 3-way stop to prevent expired air becoming contaminated with room air. Approximately one litre of gas was then immediately infused into a Mylar collection bag shown to be impermeable to and non-reactive with NO [167]. A resting measurement was performed after an initial period of 10 min, during which the subject was seated and resting quietly on the cycle ergometer breathing compressed air. A five min self-selected cycling warm-up (30-100 watts) was performed prior to commencing the cycle test. Expired NO samples were obtained when subjects reached 100, 200, 250, 300, 350, 400, and 450 watts respectively. Collected expired air samples were analyzed for CNO using a chemiluminescent analyzer (NOA 280, Sievers Instruments, Boulder, CO). Briefly, chemiluminescence is based on a gas-phase chemiluminescent reaction between NO and ozone resulting in nitrogen dioxide (NO2). Emission from electronically excited NO2 is in the red and near-infrared region of the spectrum and is detected by a thermoelectrically cooled red-sensitive photomultiplier tube. The sensitivity of this analyzer for NO is < 1 ppb. The NO analyzer was calibrated prior to each use as per the manufacturer's specifications and is linear over a range of 1 ppb - 500 ppm. The output of the analyzer was connected to a personal computer for recording purposes. VNO was calculated as the product of CNO (ppb) and VE (1-min"1) and was expressed as nmol-min"1. The technique employed to examine NO in the present study was similar to those used in other exercise and NO investigations [130, 167]. 17 Statistical Analyses. Subjects were divided into two groups based on SaC>2 at maximal exercise. Those with SaC>2 > 92.0% were classified as normal saturation (NOS), those with Sa02 < 92.0% were assigned to the low saturation group (LOS). Comparisons between NOS and LOS for anthropometric, descriptive, and V02max data were made using paired Mests. Expired NO data were examined using a 2 (group) by 8 (time) analysis of variance, with repeated measures across time. When significant F values were obtained, Bonferroni's test was applied post-hoc to determine where the differences occurred. Pearson's product moment correlation coefficient was utilized to ascertain the relationship between change in percent oxyhemoglobin saturation (delta Sa02) and change in NO production (delta VNO). The level of significance was set at P < 0.05 for all statistical procedures. 2.3 RESULTS Descriptive and peak exercise values are shown in Table 1. Sa02 dropped significantly from a resting mean resting value of 97.8% to a peak exercise value of 92.7% (P < 0.05). When subjects were divided into NOS and LOS only Sa02 data were statistically different between groups (P < 0.05). Table 2 depicts values for CNO and VNO at rest and during incremental exercise for all subjects. CNO remained unchanged from rest throughout exercise. VNO was significantly greater than rest at 300, 350, 400, and 450 watts. NOS and LOS groups showed no significant differences for CNO (Figure 1) or VNO (Figure 2). The correlation between delta Sa02 and delta VNO (r = - 0.12) was non-significant (P > 0.05; Figure 3). All individual subject data is presented in Appendix D. 18 TABLE 1. Descriptive and peak exercise values in all subjects (ALL), subjects with normal (NOS) and low (LOS) oxyhemoglobin saturation. Definition of abbreviations: V02max = maximal oxygen consumption; VE = minute ventilation; RER = respiratory exchange ratio; HRmax = maximal heart rate; Sa02 = percent oxyhemoglobin saturation. Values are means ± SD. f significantly different than Resting Sa02, P < 0.05. * significantly different than NOS, P <0.05. ALL NOS LOS («=18) (n = 12) (n = 6) Age (yr) 26.7 ±4.6 27.8 ±4.9 24.7 ±2.6 Height (cm) 179.2 ±5.2 178.2 ±5.0 181.3 ±5.1 Mass (kg) 72.8 ± 7.9 71.0 ±8.8 76.5 ±4.0 V02max (1-min"1) 4.9 ±0.6 4.8 ±0.7 5.2 ±0.3 V02max (ml-kg"'-min"1) 67.7 ±5.2 67.7 ±4.8 68.7 ±5.7 VE (1-min"1) 174.6 ± 14.0 174.1 ± 14.3 175.6 ± 13.3 RER (VC02/V02) 1.16 ±0.04 1.16 ±0.05 1.16 ±0.03 HR (beats-min"1) 190.6 ±7.4 190.4 ±8.2 191.0 ±5.8 Resting Sa02 (%) 97.8 ± 0.6 97.9 ± 0.4 97.5 ±0.8 Lowest Sa02 (%) 92.7 ± 2.0 f 93.9 ±0.8 f 90.3 ± 1.0 t* Power (watts) 459.1 ±27.6 455.5 ±30.3 466.2 ± 19.9 TABLE 2. Concentration of nitric oxide (CNO) and production rate of nitric oxide (VNO) at rest and during incremental exercise in all subjects (n = 18). Values are means ± SE. * Significantly different than Rest, P < 0.05. WORK CNO VNO (watts) (ppm) (nmol-min"1) Rest 8.0 ± 1.4 3.3 ±0.5 100 6.5 ± 1.1 8.9 ± 1.6 200 6.1 ± 1.1 12.0 ±2.2 250 6.3 ±1.3 15.2 ±2.9 300 6.7 ± 1.2 20.6 ±3.7* 350 6.3 ± 1.2 24.1 ±4.5 * 400 7.0 ± 1.4 32.6 ± 7.2 * 450 7.6 ± 1.5 41.4 ±8.7 * 19 FIGURE 1. Concentration of nitric oxide (CNO) at rest and during incremental exercise in subjects with normal (NOS, n =12) and low (LOS, n = 6) oxyhemoglobin saturation. Open circles = NOS, closed squares = LOS. Values are means ± SE. 0 100 200 300 400 500 Work (watts) FIGURE 2. Production rate of nitric oxide (VNO) at rest and during incremental exercise in subjects with normal (NOS, n = 12) and low (LOS, n = 6) oxyhemoglobin saturation. Values are means ± SE. 20 FIGURE 3. Relationship between change in percent oxyhemoglobin saturation (delta SaOi) and change in nitric oxide production (delta VNO) in all subjects (n = 18, r = - 0.12, P > 0.05). 10 c> 8 CM O 6 re CO 4 re 2 Q 0 0 20 40 60 80 100 120 Delta VNO (nmol/min) 140 21 2.4 DISCUSSION The principal findings of this study were that CNO remained unchanged during incremental exercise to exhaustion, and that VNO increased in proportion to exercise intensity in highly-trained male cyclists. Unique to the findings of this study was the observation that changes to Sa02 during incremental exercise were unrelated to alterations in exhaled NO. Decreased arterial oxygenation has been widely reported in highly-trained male athletes [48, 91, 173]. Mechanisms to explain EIH remain debatable, however elevated pulmonary pressure resulting in VA/Q mismatch and diffusion limitations are a likely explanation [48, 89]. Endogenously produced pulmonary NO has been suggested to possibly have several physiological functions including VA/Q matching and maintenance of low pulmonary vascular resistance [71]. We hypothesized that exhaled NO may be reflective of the processes responsible for EIH. VNO was increased significantly in all subjects during exercise (Table 2), but was not different between NOS and LOS (Figure 2). Delta VNO was not correlated with delta Sa02 (r = -0.12, P > 0.05) (Figure 3). Collectively, these results show that EIH is not related the production rate of NO. We must therefore reject our original hypothesis that subjects with EIH have decreased VNO compared to non-EIH subjects during heavy physical work. The presence of NO in exhaled air has been widely reported in humans [19, 40, 72, 130, 167] and different animal species [206]. Human values of resting and exercising CNO and VNO are variable. The effect of exercise on CNO has been shown to cause a slight decrease [40, 130, 166, 169], or have no effect [19, 100]. The latter is in agreement with the results of the present study. The physiological importance of maintaining, or slightly reducing, CNO during exercise remains unclear but possibly reflects a shift in the site of NO production. CNO in the nasal passages is predominant in resting humans with the lower airways contributing a smaller fraction 22 [113, 193, 205]. The contribution of the nasal passages to exhaled NO has recently been observed to decrease during exercise [127, 168, 192], while the proportion from the lower airways increases [168]. Nasal cavity NO levels dropped rapidly (~ 50% resting values) after only one min of exercise compared with rest, and were lowered (~ 76% resting values) during heavy work [127]. This shift may be reflective of a change from nasal to oral breathing at the onset of exercise explaining the immediate decrease in nasal CNO [127], although total exhaled CNO remains unchanged or slightly reduced. This suggests that during exercise a shift occurs where a greater percentage of total CNO comes from a lower airway source rather than the nasal cavity [168]. An increase in pulmonary endothelial production could explain the stability of CNO, in spite of increased ventilation and a dilution effect, since NO derived from the endothelium may enter the airway lumen via the alveoli. Increased NO production may be the result of increased Q and flow through the pulmonary circulation. During exercise, Q increases in proportion to exercise intensity and the pulmonary circulation is able to accommodate the increase with little rise in pulmonary pressure due to the distension and recruitment of the pulmonary capillaries. It has been traditionally thought that this process is passive in nature where the pulmonary circulation is fully recruited. Although not yet fully understood possibly NO aids in regulating this process and is reflected in exhaled NO [71]. The production rate of NO has previously been shown to be elevated during exercise [19, 40,130,135,160, 167,168, 169, 224]. VNO values obtained in the present investigation are comparable to other studies [40, 130]. Comparison is limited however, as there is currently no consensus on measurement techniques for exhaled NO during exercise. As such, only studies using similar methods can be adequately compared, as it appears that various techniques can alter NO values. It is also unclear what effect training status has on VNO further hindering the 23 comparison of studies. It is widely accepted that physical conditioning affects the cardiovascular response to exercise. Training-induced alterations to Q, pulmonary blood flow, and VE all may influence the production rate of NO. Basal CNO has been found to be identical between high aerobic capacity athletes and non-athletes [40, 130] and higher in one subject described as an athlete [19]. During exercise, it has been observed that athletes maintain a higher CNO than do moderately trained and sedentary individuals at a given exercise intensity [130]. In disagreement with these data, no difference in CNO was found between trained men, sedentary women, and sedentary men at rest or during cycle exercise [40]. The dissimilar results may be related to different levels of physical conditioning and maximal aerobic capacities of the respective athletic groups; 4.4 1-min"1 [40] and 5.6 1-min"1 [130]. These two groups would likely have different VE, Q, and pulmonary blood flow; thus they would possibly have different rates of NO production. Additionally, disunity in results is possibly related to the method of reporting data. When VNO is expressed per unit of body mass (pmol-kg'^min"1), differences are seen between athletes and non-athletes [130]. However, when VNO is expressed in absolute terms (nmol-min"1), no differences between groups are observed [40]. In a preliminary study of unpublished observations Phillips et al [168], describe a statistically significant correlation between exhaled NO output and body surface area (r = 0.59). To date, there are few data available addressing this issue, leaving the question of the effects of physical conditioning on basal and exercising CNO and VNO unresolved. Power analysis was performed to determine the level of power (1 - (3) achieved in the present study. Utilizing a statistical computer program (SPSS), the effect size for VNO was 0.3 and that power for the analysis of variance was ~ 22%. This can be considered low given that a power of 80% represents reasonable protection against type II error. Given the small effect 24 size (large variability in VNO) in this study that to achieve 80% power, approximately 60 additional subjects would have to participate in the study. In summary, this study confirmed that VNO increases and that CNO remains unchanged with progressive exercise in highly trained male endurance athletes. This is the first study to show that no differences in VNO and CNO were observed between LOS and NOS. Changes to VNO during exercise were unrelated to exercise-induced alterations to Sa02. Increased NO production during exercise, determined via exhaled air, was not related to exercise-induced hypoxemia. 25 CHAPTER 3. INHALED NITRIC OXIDE 3.1 INTRODUCTION During maximal exercise Pa02 and Sa02 are decreased significantly in some highly-trained male athletes [48, 91, 173]. An increase in [A-a]D02 is associated with the lowered Pa02. The mechanisms to explain EIH remain controversial but it is believed that that VA/Q inequality and diffusion limitations resulting from elevated pressures and pulmonary edema and/or decreased pulmonary transit time with the pulmonary vasculature are likely [74, 89]. Inhaled NO, a selective pulmonary vasodilator, is used in the treatment of diseases characterized by pulmonary hypertension and hypoxemia [116]. If EIH and pulmonary edema is caused by elevations of pulmonary pressures, inhaled NO should diminish the observed hypoxemia during exercise. The observed hypoxemia during hypoxic exercise is more pronounced in athletes with EIH than in normal individuals [123, 132]. Therefore, the aim of this study was to test the effects of inhaled NO (20 ppm) on gas exchange in athletes with EIH during strenuous exercise under conditions of normoxia and hypoxia. We hypothesized that during short duration, high-intensity exercise: (1) inhaled NO would reverse EIH during normoxia, and (2) inhaled NO would improve arterial oxygenation during hypoxia. 3.2 METHODOLOGY Subjects. Highly-trained male cyclists were recruited to participate in this study (n = 8). One subject was forced to withdraw due to difficulties with placement of the arterial catheter, thus all data will be reported as such (n = 7). This investigation was divided into two parts. Subjects who met the inclusion criteria in Part 1 participated in Part 2. Inclusion criteria were (i) normal 26 spirometry, no history of asthma or cardiorespiratory disease, (ii) VC^max > 60 ml-kg" '•min'1 and/or 5 1-min"1, (iii) maximal exercise SaC»2 < 91.0%, (iv) between the ages of 18-40. Prior to any testing, subjects received a verbal description of the experiment, and completed a written informed consent form. This study was approved by the Clinical Screening Committee for Research and Other Studies Involving Human Subjects of the University of British Columbia. Preliminary Screening - Part 1. Subjects reported to the Applied Physiology Laboratory in the Allan McGavin Sports Medicine Center, UBC, having refrained from exhaustive exercise for 24 h, abstained from ingestion of food or fluid for 4 h except for water, and alcohol and caffeine for 12 h. Subjects were weighed and their height was recorded. Both spirometry, and pulmonary diffusion measurements for carbon monoxide (DLCO) were collected using the same commercial apparatus (Collins DS/PLUS II, Braintree, MA). DLCO was determined using the single-breath method [1991], and conformed to the American Thoracic Society standardization of DLCO measurement [215]. Prior to performing DLCO and spirometry measures subjects sat and rested for 30 min to stabilize their pulmonary system. Maximal oxygen consumption was determined using an incremental test on an electronically braked cycle ergometer (Quinton Excalibur, Lode, Groningen, Netherlands). Subjects pedaled at a self-chosen cadence at a progressing workload, which started at 0 watts and increased 30 wattsmin"1. Subjects inspired through an air flowmeter (Vacumetrics model 17150, Ventura, CA) using a two-way non-rebreathing valve (Hans-Rudolph, model 2700B, Kansas City, KS) and expired air passed into a 5 litre mixing chamber from which gas samples were analyzed at a rate of 300 ml-min"1 for oxygen and carbon dioxide concentrations (S-3 A oxygen analyzer and CD-3A carbon dioxide analyzer, Applied Electrochemistry, Pittsburgh, PA). Expired gases 27 and VE were recorded using a computerized system (Rayfield, Waitsfield, VT). Gas analyzers were calibrated with gases of known concentration, and the air flowmeter was calibrated by passing 100 litres of air through the system. Heart rate was recorded every 15 s using a portable heart rate monitor (Polar Vantage XL, Kempele, Finland). SaCh was measured by a pulse oximeter (Ohmeda Biox 3740, Louisville, CO) with values averaged and recorded every 5 s using a personal computer. This oximeter has previously been shown to be a valid and reliable predictor of Sa02 during cycling [133]. Prior to placement of the oximeter sensor to the pinna of the ear, a topical vasodilator cream (Finalgon, Boehringer/Ingeheim, Burlington, ON) was applied to increase local perfusion. Attainment of V02max was considered when at least three of the four following were met observed: (i) a plateau in VO2 with increasing workload, (ii) RER > 1.15, (iii) attainment of 90% of age predicted maximal heart rate, and/or (iv) volitional fatigue. During Part 1 cycle ergometry subjects inspired compressed air (F1O2 = 20.93%). The air was delivered from a large cylinder through a closed container of water for humidification, then into a large meteorological balloon, which acted as a reservoir for inspired air. Those who met the inclusion criteria returned on a separate day at least 72 h later to perform another maximal cycle ergometry test under hypoxic conditions (F1O2 = 14%). This F1O2 has previously been used to accentuate decreases in Sa02 in exercising trained males [123, 132]. Expired gases, heart rate, and Sa02 were determined during the hypoxic exercise session as detailed above. Inhaled Nitric Oxide - Part 2. Following completion of both V02tnax tests (normoxic and hypoxic) those subjects who met the inclusion criteria returned on a separate day at least 72 h later. Subjects were randomly assigned and blinded to each of the four following conditions: (i) Normoxia (N), (ii) Normoxia/Nitric Oxide (N/NO), (iii) Hypoxia (H), and (iv) Hypoxia/Nitric Oxide (H/NO). 28 Participants performed a 10-15 min cycling warm-up at a self-selected workload and then sat quietly on the cycle ergometer for 5 min when resting data were obtained (Rest). Cycling intensity was then manually increased to 100% of their respective maximum normoxic or hypoxic work load as determined in Part 1. The duration from rest to maximal workload was 1 min. Subjects cycled at this intensity for 5 min, and cardiorespiratory variables were recorded at each min in the same fashion as during Part 1. Arterial blood samples were drawn at rest and at each min of the 5 min test (see ARTERIAL BLOOD SAMPLING). Following each test condition subjects cycled easily (30-50 watts-min"1) for 10 min and then rested for 50 min prior to commencing the next test condition. An overview of the experimental protocol is depicted in Figure 4. During N subjects rested for 5 min and cycled while breathing normoxic gas. Condition N/NO consisted of normoxic gas with 20 ppm NO delivered to the inspiratory tubing. During condition H subjects rested for 5 min and cycled while inhaling hypoxic gas (F1O2 = 14%) and condition H/NO consisted of the same hypoxic gas with 20 ppm NO delivered to the inspiratory tubing. During all conditions the inspired air was delivered from a large cylinder through water for humidification, then into a large meteorological balloon, which acted as a reservoir prior to being inspired by the subject. NO was delivered at the distal end of the tubing, while inspired concentrations of O2, NO and nitrogen dioxide (NO2) were monitored continuously during each test condition ~ 5 cm from the subject's mouth using a commercial apparatus (PulmoNOx II, Pulmonox, Tofield, AB). The system was calibrated prior to each experiment as per the manufacturer's specifications. The flow rate of NO was calculated as follows: NO flow (1-min"1) = (VE X desired [NO]) / (source tank [NO]) For example, if VE = 145 1-min"1, desired [NO] = 20 ppm, and source tank [NO] = 2000 ppm 29 Then ... NO flow (1-min1) - (145 x 20) / 2000 = 1.45 1-min'1 The concentration of NO used in the present study (20 ppm) has previously been shown to improve VA/Q distributions, Pa02, and PVR in pigs [182], reverse hypoxic pulmonary vasoconstriction, and redistribute blood flow to better ventilated areas of the lung in sheep [168], and is thought to be an appropriate concentration for exercising humans (W. M. Zapol, personal communication). Furthermore, Durand et al. [49] recently utilized a comparable concentration of NO (15 ppm) in exercising athletes to evaluate the effect on gas exchange. Arterial Blood Sampling. A 20-gauge arterial catheter was inserted in the radial artery of the non-dominant hand by percutaneous cannulation using 1% local anesthesia (Lidocaine) and sterile technique, and was then secured to the skin. Adequate collateral circulation via the ulnar artery (Allen's test) was estimated before the cannula was inserted. A minimum volume extension tube, connected in series with two, three-way stopcocks arranged at right angles, was flushed with a saline-heparin solution. A rapid response (<0.01 s) thermister (18T, Physitemp Instruments, Clifton, NJ) used to measure peak arterial blood temperature was inserted through a Touhy-Borsch heparin lock (Abbott Hospitals, North Chicago, IL). Catheter patency was maintained with a continuous heparin infusion (1 ml 1:1000 units in 500 ml NS at 3 ml/hr). At the onset of sampling, 12 ml of blood was withdrawn, and the final 3 ml was collected in preheparinized plastic syringes. The remaining 9 ml was then slowly reinfused. Samples were withdrawn at rest and at 1 min intervals for the duration of each test (4 conditions x 6 samples per condition = 24 samples/subject). Blood samples were placed on ice until analyzed for H+ ion concentration, P02, PC02, base excess, and HC03" (CIBA-Corning 278 Blood Gas System, CIBA-Corning 30 Diagnostics Corporation, Medfield, MA). Pa02 was corrected for temperature and H+ ion concentration. Temperature increased 0.9 + 0.1 °C (mean ± SD) from rest to 5 min of exercise across all trials. Sa02 levels were calculated based on corrected Pa02. The alveolar gas equation was used to calculated alveolar partial pressure (PAO2) and [A-a]DC>2 [84]. FIGURE 4. Overview of experimental protocol, repeated for each condition of normoxia (N), normoxia + 20 ppm nitric oxide (N/NO), hypoxia (H), and hypoxia + 20 ppm nitric oxide (H/NO). S = sample for blood and cardiorespiratory variables. cycle 0 1 2 3 4 5 Warm up 10-15 min Rest 5 min V i V i V i V i Cycle easy 10 min rest 50 min 1 \ s s s s s s 31 Statistical Analyses. Mean values and measures of variability were determined for descriptive, anthropometric, and lung function variables obtained during preliminary screening. Maximal cycle ergometry data from Part 1 were compared using r-tests for dependent samples (normoxia vs. hypoxia). Experimental data were analyzed using 4 (condition) by 6 (time) two-way factorial analysis of variance with repeated measures on both factors. When sphericity was not assumed, Greenhouse-Geisser P-values were utilized. When significant F-ratios were observed, Scheffe's test was applied post-hoc to determine where the differences occurred. The level of significance was set at P < 0.05 for all r-tests and P < 0.01 for ANOVA procedures. Statistical power calculations were performed a priori to estimate an appropriate minimum sample size . A sample size of five was calculated. 3.3 RESULTS Physical and Maximal Exercise Data. Descriptive data and resting pulmonary function data are presented in Table 3. Data from normoxic and hypoxic maximal cycle ergometry tests are presented in Table 4. Significant differences were observed between normoxic and hypoxic conditions for VC^max, RER, HRmax, and power output while no significant differences were detected for VE. From rest to maximal exercise SaC»2 dropped significantly under both normoxia (97.7 to 90.2) and hypoxia (97.0 to 75.5). All individual subject data is presented in Appendix E and mean data in Appendix F. Blood Gases during 5-min Cycling. PaC"2 results are reported in Figure 5. Across all time points there were no significant differences between N and N/NO or between H and H/NO. Both hypoxic conditions were significantly lower than both normoxic conditions. PaCh values 32 were significantly lower at 1, 2, 3, 4, and 5 min of exercise compared to rest for all inspired gas conditions. Similar results were observed for SaC>2, except that values at 1 and 2 min were not significantly different from rest under conditions of N and N/NO (Figure 6). [A-a]DC>2 was significantly different at all time periods when compared to rest for all inspired gas mixtures (Figure 7) and significant differences were detected between N/NO and H at rest and during all exercise measurements. PaC02 was not significantly different between gas conditions, but was lower compared to rest throughout all exercise for H and H/NO, and at minutes 3, 4, and 5 for both N and N/NO (Figure 8). Metabolic and Power Output during 5-min Cycling. Oxygen consumption increased significantly from rest to 1-min during all conditions (Figure 9). Across all conditions, VO2 was also significantly higher at 2, 3, 4, and 5 min compared to rest and 1 min. Significant differences were detected between N/NO and H at 3, 4, and 5 min. Heart rate was significantly higher during exercise compared to rest (Figure 10) while no differences where observed between gas conditions. At minutes 4 and 5 heart rate was higher than minute 1 for all conditions. Power output was significantly lower during both hypoxic conditions compared to normoxic conditions (Table 5). 33 TABLE 3. Descriptive and resting pulmonary function data. Actual pulmonary function value with % predicted in parentheses. Values are means ± SD. Definition of abbreviations: FVC = forced vital capacity, FEVi = forced expired volume in 1 second, FEF25-75% = forced expiratory flow at 25-75% of FVC, FEFmax = maximal forced expiratory flow rate, DLCO = pulmonary diffusion capacity for carbon monoxide, VA = alveolar volume, DLCO/VA = pulmonary diffusion capacity for carbon monoxide/alveolar volume. Variable Mean ± SD Age (yr) 28.9 ±3.9 ~ Height (cm) 181.4 ±7.5 ~ Mass (kg) 74.7 ± 6.6 ~ FVC (L) 5.59 ±0.81 (102 ± 14) FEVi (L) 4.58 ±0.73 (102 ± 15) FEF25-75% (1-sec"1) 4.52± 0.91 (102 ± 18) FEVi/FVC (%) 80.54 ±5.82 (98 ± 7) FEFmax (1-sec"1) 9.95 ± 1.08 (102 ± 17) DLCO (ml-min'-mmHg'1) 36.99 ±5.04 (117 ± 19) VA(L) 5.26 ± 1.42 — DLCO/VA 7.33 ± 1.65 — TABLE 4. Peak exercise values during maximal cycle ergometer tests. Values are means ± SD. * Significantly different than NORMOXIA, | significantly different than Resting Sa02 (P < 0.05). Definition of abbreviations: V02max = maximal oxygen consumption; VEmax = maximal minute ventilation; RER = respiratory exchange ratio; HRmax = maximal heart rate; Sa02 = percent oxyhemoglobin saturation. NORMOXIA HYPOXIA Fi02=21% Fi02=14% V02max (lmin"1) 4.88 ± 0.43 4.24 ± 0.49 * V02max (ml-kg^-min"1) 65.3 ± 1.6 56.6 ±5.6* VEmax (1-min"1) 175.9 ± 10.2 168.2 ± 11.5 RER (VC02/V02) 1.20 ±0.02 1.12 ±0.07 * HRmax (beats-min"1) 188 ±4 179 ±3 * Resting Sa02 (%) 97.7 ±0.6 97.0 ± 0.8 Lowest Sa02 (%) 90.2 ± 0.9 f 75.5 ±4.5 * f Power (watts) 449 ± 39 371 ±38 * 34 FIGURE 5. Arterial pressure of 02 (Pa02) during rest and at each min of 5-min cycle ergometry tests under conditions of normoxia (N), normoxia + 20 ppm nitric oxide (N/NO), hypoxia (H), and hypoxia + 20 ppm nitric oxide (H/NO). Values are means ± SD. * indicates N and N/NO significantly different from REST, f indicates H and H/NO significantly different from REST. 35 FIGURE 6. Percent oxyhemoglobin saturation (Sa02) during rest and at each min of 5-min cycle ergometry tests under conditions of normoxia (N), normoxia + 20 ppm nitric oxide (N/NO), hypoxia (H), and hypoxia + 20 ppm nitric oxide (H/NO). Values are means ± SD. * indicates N and N/NO significantly different from REST, t indicates H and H/NO significantly different from REST. 5.0 H 1 1 1 1 1 REST 12 3 4 5 Time FIGURE 7. Alveolar-arterial difference for 02 ([A-a]D02) during rest and at each min of 5-min cycle ergometry tests under conditions of normoxia (N), normoxia + 20 ppm nitric oxide (N/NO), hypoxia (H), and hypoxia + 20 ppm nitric oxide (H/NO). Values are means ± * indicates N and N/NO significantly different from REST, f indicates H and H/NO significantly different from REST. FIGURE 8. Arterial pressure of C02 (PaC02) during rest and at each min of 5-min cycle ergometry tests under conditions of normoxia (N), normoxia + 20 ppm nitric oxide (N/NO), hypoxia (H), and hypoxia + 20 ppm nitric oxide (H/NO). Values are means ± SD. * indicates N and N/NO significantly different from REST, f indicates H and H/NO significantly different from REST. 38 FIGURE 9. Oxygen consumption (V02) during rest and at each min of 5-min cycle ergometry tests under conditions of normoxia (N), normoxia + 20 ppm nitric oxide (N/NO), hypoxia (H), and hypoxia + 20 ppm nitric oxide (H/NO). Values are means ± SD. * indicates N and N/NO significantly different from REST, f indicates H and H/NO significantly different from REST. 39 FIGURE 10. Heart rate during rest and at each min of 5-min cycle ergometry tests under conditions of normoxia (N), normoxia + 20 ppm nitric oxide (N/NO), hypoxia (H), and hypoxia + 20 ppm nitric oxide (H/NO). Values are means ± SD. * indicates N and N/NO significantly different from REST, t indicates H and H/NO significantly different from REST. 40 TABLE 5. Power output (watts) during rest and at each min of 5-min cycle ergometry tests under conditions of normoxia (N), normoxia + 20 ppm nitric oxide (N/NO), hypoxia (H), and hypoxia + 20 ppm nitric oxide (H/NO). Values are means (± SD). * indicates significantly different than H and H/NO. REST 1 MIN 2 MIN 3 MIN 4 MIN 5 MIN N 0 390.8 * 368.3 * 353.3 * 336.3 * 333.8 * 0 (36.1) (32.5) (44.6) (61.7) (60.9) N/NO 0 378.3 * 355.7 * 341.7 * 330.0 * 330.0* 0 (44.8) (59.0) (66.2) (49.0) (67.2) H 0 326.3 303.9 296.4 288.6 284.3 0 (33.4) (40.7) (55.4) (55.0) (52.3) H/NO 0 329.3 307.0 278.6 263.3 262.6 0 (30.3) (42.7) (29.3) (30.8) (35.6) 41 3.4 DISCUSSION The principal finding of this study was that inhalation of 20 ppm NO during normoxic or hypoxic high-intensity, short-duration exercise did not significantly affect gas exchange in highly-trained athletes with exercise-induced hypoxemia. 3.4.1 Rationale for the Study Exercise-Induced Hypoxemia. The consensus in the EIH literature is that hypoxemia during exercise likely occurs via VA/Q inequality and diffusion limitations resulting from elevated pulmonary pressures (stress failure) causing the development of interstitial pulmonary edema, or decreased pulmonary red blood cell transit time in the pulmonary vasculature [89]. Identification of pulmonary edema and pulmonary stress failure has been difficult to achieve in exercising humans but has been successfully documented in racehorses [230, 238], Shetland ponies [52], and greyhound dogs [114]. At a capillary transmural pressure of 40 mmHg ultrastructural changes to the blood gas barrier are seen using a rabbit model [225], including disruption of the capillary endothelial and alveolar epithelial layers, and in some cases all layers of the wall. Based on available human data, human pulmonary artery pressure (PPA) and capillary wedge pressures (PWEDGE) can reach values ~ 40 and 27 mmHg respectively [189, 230]. Increased PPA with exercise in humans has been consistently reported [70, 101, 188, 222], while pulmonary capillary pressure must be deduced from the above mentioned pressures. The calculated capillary pressure at the base of the human lung can reach > 35 mmHg during exercise. These pressures observed in humans by Wagner and colleagues [230] are within the range where stress failure of pulmonary capillaries is seen in animal models [225, 239]. The net result of stress failure of the pulmonary capillaries at these elevated pressures is altered or 42 disruption of the capillary endothelial and alveolar epithelial layers, collection of red blood cells in the alveolar wall interstitium, proteinaceous fluid and red blood cells in alveolar spaces, interstitial edema and fluid protrusions of the endothelium into the capillary lumen [237]. The cumulative effect of these events is presumably impaired gas-exchange. Following this line of reasoning, it is reasonable to hypothesize that a reduction of pulmonary pressures or increased transit time, via pulmonary vasodilation, may reduce or reverse the hypoxemia seen in some highly trained athletes. We attempted to achieve this by using inhaled NO. Effects of Inhaled NO. Inhaled NO, a selective pulmonary vasodilator, is used in the treatment of diseases characterized by pulmonary hypertension and hypoxemia [116]. Short-term inhalation of gaseous NO causes selective vasodilation without any systemic effects. In patients with chronic pulmonary hypertension the administration of intravenous vasodilators can have beneficial effects (4 PPA) but these are typically at the expense of systemic hypotension and worsening hypoxemia [183]. Inhalation of NO in adult respiratory distress syndrome (ARDS) patients (2-20 ppm for 2-27 d) has been shown to decrease mean PPA and improve arterial oxygenation [24]. A decrease in pulmonary vascular resistance (PVR), via NO, may also serve to improve right ventricular function [58]. Using normal volunteers, Frostell et al. [59] examined the effects of inhaled NO during normoxia and acute hypoxia (F1O2 = 12%). Normoxic inhalation of NO (40 ppm) had no effect on pulmonary or systemic hemodynamics. Hypoxia induced mild pulmonary hypertension by increasing PPA (+5 mmHg) and PVR (+50 dynes-s~'-cm ~5) and inhalation of NO decreased PPA and PVR to control values without causing systemic vasodilation. While it seems clear that inhaled NO can improve oxygenation in some patient populations, this effect should not be considered to be a general response but is dependent upon the pathophysiology of the disease/condition. Hopkins et al. [90], utilizing 43 anesthetized dogs, examined how inhalation of NO (80 ppm) could alter pulmonary gas exchange under conditions of: (i) normal lungs, (ii) shunt, and (iii) VA/Q inequality. In dogs with VA/Q inequality, NO variably affected matching which was improved in some dogs and worsened in others. These data are in agreement with other reports of NO inhalation in patients with chronic obstructive pulmonary disease (COPD) where Pa02 values have been shown to increase slightly [3], have no effect [145], and decrease with VA/Q inequality [17]. Critical analysis of COPD-NO inhalation studies reveals that grouped data may not be indicative of the NO-inhalation response. Based upon the above mentioned dog data [90], and the variable responses in gas exchange [3, 17, 145], it can be concluded that areas of low VA/Q matching contribute to the variable clinical effects of inhaled NO. Hopkins et al. [90], surmised that inhaled NO improved gas exchange when the normal areas of the lung increased blood flow, but worsened it when both the normal areas and the areas of low VA/Q ratio had a fall in PVR. This would likely explain why the response of NO inhalation is variable in COPD patients, and emphasizes the need to understand the pathophysiology of the lung disease/condition. Studies that have sought to examine the effects of inhaled NO during exercise have been few and have focussed on patient populations. It is well established that COPD patients may develop pulmonary hypertension when exercising [243]. Roger and colleagues [197], demonstrated that in COPD patients who inhaled NO (40 ppm), pulmonary hypertension was reduced and VA/Q ratios were improved during exercise. However, the effect of NO was different at rest than during exercise. During rest, Pa02 and VA/Q ratios were worsened while during exercise they both improved slightly. The authors concluded that during exercise the effect is likely explained by a preferential distribution of inhaled NO to well-ventilated alveolar units with faster time constants and normal VA/Q ratios. The results are in agreement with the 44 previous discussion of the variability of NO inhalation at rest in COPD patients. In a different patient population, subjects with left ventricular dysfunction and chronic heart failure were examined during exercise conditions of control (normoxia) and NO inhalation (30 ppm) [26]. VT and ventilatory equivalent for oxygen (VE/VO2) were significantly reduced during exercise with NO inhalation compared to control. There was also a strong, but non-significant, trend for VE to be reduced (P = 0.051). The results of this study are difficult to interpret physiologically. It is also unclear what mechanisms were responsible for alterations in ventilatory parameters. Hemodynamic measurements at rest were determined from a Swan-Ganz catheter placed in the pulmonary artery and Q was determined by the thermodilution technique. No statistical differences were observed for hemodynamic and gas exchange variables. It is also worth noting that the authors of this study utilized in excess of 20 paired t-tests (i.e., large probability for Type I error). In a separate investigation, Koelling et al. [118] examined patients undergoing cardiac transplantation evaluation in = 14). Right heart catheterization was performed and Q was determined by thermodilution. Exercise was performed under normal conditions and with NO (40 ppm). Patients with greater PPA, larger left ventricular end-diastolic volumes, and lower right ventricular ejection fractions were those who benefited the most from NO. Reduction of resistance, via NO, within the pulmonary vessels was the likely cause of augmented maximum workload and VO2 at the anaerobic threshold. This effect was not observed during treadmill exercise in sheep [120]. With inhalation of NO (30 ppm), pulmonary vascular tone was unchanged at rest, while during exercise the usual reduction in PVR was not observed. The implication of this observation is that the pulmonary vasculature was already maximally dilated during exercise by flow-related pressure effects [120, 187]. These results are consistent with those of Brett et al. [31] who recently characterized the response of the 45 pulmonary circulation in normal individuals to different doses of inhaled NO (n = 8; NO = 0, 20, and 40 ppm). No significant changes were observed for Q or DLCO implying that the normal pulmonary vascular bed is not amenable to vasodilation by inhaled NO. This is in agreement with the observation that in dogs with normal lungs NO does not alter Pa02 or VA/Q matching [90]. The rationale for this study was therefore based upon the following: (i) some athletes experience hypoxemia during exercise as result of abnormal lung function, and (ii) nitric oxide inhalation can reverse hypoxemia in some patient populations. 3.4.2 Effect of Inhaled NO during 5-min Cycling No differences in Pa02 were detected between N and N/NO in the present study. These findings do not support our original hypothesis that, if EIH is caused by elevations of pulmonary pressures, inhaled NO (20 ppm) should diminish the observed hypoxemia during exercise. No differences were observed between N and N/NO for other blood gas variables ([A-a]D02, Sa02, PaC02). Based upon these collective findings we can conclude that inhalation of 20 ppm NO during 5-min heavy normoxic exercise had no effect on gas exchange in subjects who exhibit EIH. Although pulmonary pressures were not measured in this study, we can speculate on two possible scenarios: (1) pulmonary pressures were not altered, or (2) pulmonary pressures were reduced with no effect on gas exchange (or development of pulmonary edema). It is more probable that the first scenario occurred. The pulmonary capillary bed in these athletes may have been already maximally dilated and inhalation of a vasodilator would have no effect. Therefore, no effect on pulmonary pressure would be expected nor would any alteration in gas exchange occur. Our results therefore agree with the hypothesis of Dempsey [45] that pulmonary capillary 46 blood volume reaches its maximal morphological limit and further dilation is not anatomically possible. This observation also agrees with the observation that when using sheep [120], inhalation of NO (30 ppm) did not change pulmonary vascular tone at rest and during exercise. The implication of this observation, in conjunction with the present study, is that the pulmonary vasculature is already maximally dilated during exercise by flow-related pressure effects [120, 187]. To date, Durand et al. have been the only group to investigate the effects of NO inhalation on pulmonary gas exchange during exercise in highly trained athletes [49]. Male endurance athletes (n = 9: V02max ~ 65 ml-kg^-min"1) performed a maximal cycle test to exhaustion while inhaling either (i) room air, or (ii) room air combined with 15 ppm NO. In agreement with our results, no differences between conditions were observed for VO2 (Figure 9) or power output (Table 5). Durand et al., found that inhalation of NO caused Pa02 to decrease at rest, 50%, 75%, and 100% V02max. Interestingly, delta Pa02 continuously fell without NO (13.5%). With NO, after a preliminary drop, a stabilization of delta Pa02 was observed between 75 and 100% V02max. This trend was also reflected in [A-a]D02. These results are seemingly different than ours, but several points are worth noting prior to arriving at such a conclusion. The results of Durand et al. show that at 100% V02max, Pa02 values were not statistically different between exercise without NO (Pa02 = 84.7 mmHg) and with NO (Pa02 = 79.6 mmHg). These values are nearly identical to those in the present study during the 5th min of exercise in N (Pa02 = 84.2) and N/NO (Pa02 = 78.0) where we also observed no significant difference. What is different between the two studies is that Durand et al., observed a decrease in Pa02 and [A-a]D02 at rest with NO while we did not observe this drop. It is necessary to point out that it is not clear from the Durand manuscript how long subjects inspired NO prior to commencing exercise, whereas in 47 the present study subjects sat quietly on the cycle ergometer and breathed the gas mixture for 5 min prior to exercise. Possibly, differences in the duration of NO inhalation contributed to the reduced gas exchange at rest. In contrast to Durand et al., when delta Pa02 was calculated between time periods in the present study no stabilization was observed. Their conclusion of a "stabilization" must be made with caution as this is based only upon two time points (between 50 and 75% vs. 75 and 100% V02max) rather than a longer time course. It is also necessary to note that arterial blood gas measurements were corrected for temperature in the present study, while those of Durand et al. were not. This would likely overestimate the drop in Pa02 and Sa02 observed during exercise. Given that both studies utilized relatively small sample sizes, an overestimation of small magnitude can affect the interpretation of results. The apparent difference in delta gas exchange values between the two studies are difficult to reconcile, however another possible explanation is that each study utilized a different exercise protocol. The effect of different exercise protocol has been examined by Lama et al. [122] and no difference was found between three different incremental tests to exhaustion (20, 30, or 40 watts-min"1). However, in the present investigation, we employed a 5-min high intensity test while Durand et al. utilized an incremental test to exhaustion. In both studies Pa02 declined with exercise. Perhaps the cause of hypoxemia was different with a different mode/intensity of exercise and thus the effect of inhaled NO would not be the same. The result of this would possibly be reflected in a different delta response for Pa02 and [A-a]D02. This highly speculative conclusion requires further study but it remains possible that EIH can occur during short-duration high-intensity exercise via a different mechanism than during an incremental test to exhaustion. Presently, no data exist comparing these two modes of exercise. 48 Unique to this investigation was the delivery of NO to individuals with EIH during hypoxic exercise. As expected, during H and H/NO, Pa02 was significantly lower than N and N/NO at all time points (Figure 5). The drop in oxygenation with hypoxia (F1O2 = 14%) was consistent with that observed in previous EIH studies [123, 132]. The present study showed that inhaled NO did not alter Pa02, Sa02, AaD02, or PaC02 during hypoxia. Pison et al. [168], showed that addition of 20 ppm NO to a hypoxic (Fl02 = 12%) gas mixture returned PPA and the pulmonary gas exchange to baseline measures in mechanically ventilated sheep. These are in contrast to our results where no statistical difference was observed for gas exchange variables between H and H/NO. This was an unexpected result as we can assume that hypoxic pulmonary vasoconstriction (HPV) was induced by using a hypoxic inspiratory gas mixture at rest and during exercise. We hypothesized that inhaled NO would have reversed the transient HPV and enhanced pulmonary gas exchange by redistributing blood flow to better ventilated alveoli. Reversal of HPV has been demonstrated in sheep [168], humans with high-altitude pulmonary edema [207], rabbits [164], and in healthy humans breathing hypoxic gas [60]. Potentially, the vasoconstriction at rest in the present study was of a small enough magnitude to have had minimal effect on PPA and no measurable effect on gas exchange. Further speculation is not possible as we did not measure pulmonary hemodynamics in the present study. Ideally, measures of pulmonary artery pressure and cardiac output, along with gas exchange measures would reveal the effect of inhaled NO during hypoxic exercise. Cardiorespiratory data (V02, VE, heart rate) and power output were not affected by the inhalation of NO. As expected, power output was significantly lower during H and H/NO compared to N and N/NO. A lack of NO effect is consistent with the findings of Durand et al. [49]. These findings confirm that no significant alteration to gas exchange occurred. If gas 49 exchange was positively affected by NO inhalation (i.e., t Pa02, t Sa02, i [A-a]D02), the effect would presumably be reflected in whole-body exercise measures such as VO2, heart rate, VE, and power output. 3.4.3 Methodology A debatable point is: how much of the inhaled NO actually reached the lower airways? While it is not possible to measure the amount of inhaled NO that reaches the alveoli, it is assumed that subjects in the present study did inhale 20 ppm NO. We are confident that NO was delivered to the mouth as it was measured only ~ 5 cm from the point of inspiration. The experimental set-up and NO concentration employed in our study were similar to that of Durand et al. [49] (15 ppm). It is possible that the concentration of NO used in this study was not sufficient to induce vasodilation. However, this seems unlikely given that similar concentrations have been used previously to show significant alterations in gas exchange and pulmonary pressures [145, 168, 198]. Our choice of 20 ppm was based upon the above mentioned studies, and in light of the toxicity of higher oxides of nitrogen we chose to use a lower dose than some of the clinical studies, and in addition 20 ppm is thought to be an appropriate and safe concentration for exercising humans (W. M. Zapol, personal communication). 3.4.4 Inter-Subject Mechanisms for EIH. EIH has been defined as a reduction in Pa02 of at least 10 mmHg below resting values [48, 80, 175], or 5 mmHg below resting values [6], and when using pulse oximetry an Sa02 less than 92% [174], 91% [175], or 90% [81, 234]. While the criteria for EIH is debatable, the present study confirms that Pa02 is decreased and [A-a]D02 is widened during strenuous 50 normoxic exercise in highly-trained male athletes and is significantly worsened with hypoxic gas inhalation. Mean Pa02 values decreased approximately 20 mmHg from rest during exercise during normoxia and 22 mmHg during hypoxia and are consistent with the EIH literature [7, 48, 91]. Mean values for PaC02 were lower than rest at each exercise condition. While not statistically feasible to examine this relationship, it is interesting to note that in one subject (NS-subject 5) there appeared to be a definite hypoventilation response to exercise during N and N/NO (PaC02 ~ 40 mmHg) while during H and H/NO PaC02 dropped to more "appropriate" levels (~ 30 mmHg). It is also worth noting that this subject had the greatest drop in Pa02 during hypoxia and normoxia exercise compared to other subjects. In addition, one subject (LB-subject 6) developed EIH during the normoxic cycle test to exhaustion (Sa02 = 90.92%) but the Pa02 values decreased only modestly (98 to 92 mmHg) during normoxic 5 min cycling. It is possible that exercise protocol (intensity, duration) can variably affect the drop in arterial oxygenation among individuals. Examination of individual subject data or small groups (i.e., 2-3 subjects, see Dempsey et al. [48]) can provide insight into possible mechanisms of EIH and emphasizes the possibility that EIH may occur via different mechanisms in different individuals. It is conceivable that some individuals are "hypoventilators" while others experience diffusion limitations. The complex interaction between 02 kinetics, EIH, mode/intensity/duration of exercise, and pulmonary hemodynamics has not been examined. Future research involving single-subject, repeated-measures designs my provide new information regarding the mechanisms of EIH. 51 3.5.5 Summary In summary, inhalation of 20 ppm NO during normoxic or hypoxic high-intensity, short-duration cycle exercise did not significantly affect gas exchange in athletes with exercise-induced hypoxemia. Cardiorespiratory variables and power output were unaffected by NO inhalation. The implication of these findings is that pulmonary capillary blood volume reaches its maximal morphological limit during exercise and further dilation is not possible. 52 CHAPTER 4. GENERAL SUMMARY AND CONCLUSIONS General Summary Exercise-induced hypoxemia is a perplexing physiological paradox. Physical training is known to exert many health benefits, improve aerobic capacity and exercise performance. High aerobically powered athletes can be thought to represent the upper state of the health continuum. In contrast to this premise, some highly-trained endurance athletes engaged in strenuous work have an arterial blood gas profile which resembles that of the chronically ill respiratory patient. These two studies sought to determine potential mechanisms for this phenomenon. Specifically investigated was the relationship between endogenously produced exhaled NO and EIH and the effect of inhalation of exogenous NO on EIH. Exhaled Endogenous Nitric Oxide. A group of highly-trained male cyclists (n= 18, V02max = 67.7 ± 5.2 mlkg"1-min"1) some of whom develop EIH, performed a maximal cycle test. CNO and VNO were determined during the cycle test. No significant differences were observed between those with and those without EIH. There was also no observed linear relationship between delta Sa02 and delta VNO. Inhaled Nitric Oxide. Highly trained male cyclists (n = 7, V02max = 65.3 ± 1.6 ml-kg^min"1) performed four 5-min cycle tests at V02max under conditions of normoxia (N), normoxia + 20 ppm nitric oxide (N/NO), hypoxia (H), and hypoxia + 20 ppm nitric oxide (H/NO). Inhalation of 20 ppm NO during normoxic or hypoxic high-intensity, short-duration exercise did not significantly affect gas exchange in athletes with exercise-induced hypoxemia. 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In contrast, in well-trained athletes, the adequacy of the respiratory system has been challenged when decreases in arterial pressure of O2 (Pa02) and oxyhemoglobin saturation (Sa02) are observed [48, 91, 173]. These changes were first observed in the 50's [87] and 60's [200], however this physiological fact lay dormant until the work of Dempsey et al. [48] stimulated interest. This phenomenon, termed exercise-induced arterial hypoxemia (EIH), occurs in 50% of highly-trained male endurance athletes, while untrained and moderately-trained males do not experience EIH [173]. EIH is defined by a Pa02 less than 75 mmHg or an Sa02 less than 92% [174]. Individuals with EIH are also characterized by a widening of the alveolar-arterial difference for oxygen ([A-a]D02) which can reach as high as 50 mmHg [91]. A decline of Pa02 and Sa02 during heavy exercise represents a physiological failure of the respiratory system. The cause and significance of EIH have thus become topics of considerable interest to physiologists and athletes alike. Despite a considerable research effort, the mechanism(s) responsible for EIH remain controversial. Four primary factors have been reported in the literature; (i) veno-arterial shunts, (ii) relative hypoventilation, (iii) ventilation/perfusion (VA/Q) inequalities, and (iv) diffusion limitations. Given the difficult nature of performing truly mechanistic studies in exercising humans, the EIH literature has been slow to evolve. Respiratory factors limiting maximal exercise and EIH have previously been reviewed elsewhere [35, 45, 47, 92, 177], however recent 76 investigations have forced scientists to reconsider the factors responsible for EIH. In addition, recent studies have sought to examine other athletic populations in relation to EIH, in particular women and older athletes. Insights from animal physiology have also provided information on EIH. Therefore, the purpose of this review is to provide a contemporary review of EIH and the mechanism(s) thought to be responsible. 2. RELEVANCE Reductions of PaC>2 and SaC>2 during exercise are of interest to the exercise physiologist because they indicate that the pulmonary system may be a limiting factor to maximal oxygen uptake (VC^max) and exercise performance. Reductions in SaC»2 from resting values have negative effects on V02max [67, 96, 123, 132, 174, 216]. To date, only one study has examined the level of arterial desaturation at which maximal performance capacity is impaired [121]. Subjects performed 3 five-min cycle performance tests under normoxia, mild hypoxemia, or moderate hypoxemia. Mean SaC>2 values were 96, 90, and 87% respectively. Mild hypoxemia elicited a detrimental effect but did not reach statistical significance. Moderate hypoxemia caused a significant decline in performance. As was correctly pointed out by the authors, a linear trend was observed between decreasing levels of SaCh and diminished work, and in terms of athletic performance, statistical significance holds little importance. The factors, which provide a limit to VC^max, have been a source of debate for many years. The traditional view is that oxygen delivery to working muscle represents the primary limiting factor to exercise VO2 and performance and that the oxygen content of arterial blood is adequate to meet all exercise-imposed metabolic demands [9]. The reader is directed to reviews of cardiovascular [202], and peripheral limitations [228] to VC^max. It has been postulated that in some elite aerobic athletes the limit to VC^max, and possibly performance, is the respiratory 77 system [45, 47]. Accordingly, the first step in the supply of oxygen to working muscle becomes the "weak link". Elite athletes undergo training adaptations in skeletal muscle and the cardiovascular system, which eventually surpasses the capability of the pulmonary system. Dempsey speculates further that this occurs because of the pulmonary system's inability to adapt despite many years of aerobic training. If this supposition holds true then the pulmonary system does in fact pose a constraint to maximal exercise performance because of the inability to match the metabolic requirements of the athlete. 3. MECHANISMS OF EXERCISE INDUCED HYPOXEMIA 3.1 VENO-ARTERIAL SHUNT A veno-arterial shunt is an anatomical phenomenon that allows mixture of venous and arterial blood causing a decrease in Pa02. Approximately 50% of the [A-a]D02 in resting and exercising humans can be explained by veno-arterial shunts [8, 69, 240]. In subjects with EIH and a widened [A-a]D02 during maximal exercise, breathing hyperoxic gas (24-26% O2) causes Pa02 to approach normal levels [48, 176]. If veno-arterial shunts were the cause of EIH breathing such an inspirate would have little effect on Pa02 because of the venous and arterial blood mixture. Based on these findings, veno-arterial shunts have been excluded as an important determinant of EIH. 3.2 HYPOVENTILATION Minute ventilation (VE) increases during progressive exercise via elevations in tidal volume (VT) and respiratory frequency (/b). At low levels of exercise intensity both VT and fb increase, while at higher work loads increases in VE are due principally to /b while VT remains constant. VE increases with exercise intensity raising alveolar PO2 (PAO2) to provide a sufficient pressure gradient for oxygen diffusion. Arterial carbon dioxide tension (PaCCh) is also maintained through increased VE where alveolar PC02 (PACO2) is reduced, facilitating C02 diffusion from arterial blood. Inadequate ventilation, or hypoventilation, during exercise would lead to an inadequate PAO2, decreased driving gradient from alveoli to blood, and elevated PaCC^. Hypoventilation is clinically characterized by the retention of CO2 (PaC02 > 40 mmHg). It has been suggested that hypoventilation can occur during exercise despite the presence of stimuli to increase ventilation such as elevated PaCC«2, temperature, blood catecholamines, metabolic acidosis or a decrease in PaC>2 [45, 48]. Indeed, hypoventilation has been implicated as a causative factor of EIH [38, 45, 47, 48, 162]. Athletes who developed EIH showed minimal compensatory hyperventilation during exercise (PaCCh was only 1-4 mmHg below resting values) and those who hyperventilated the least showed the greatest hypoxemia [48]. However, given that retention of CO2 is the cardinal marker of hypoventilation and athletes who exhibit EIH have not retained C02, hypoventilation has been ruled out as an important factor in EIH [176,178]. Alternatively, it may be that hypoventilation during exercise is relative and permits a necessary trade-off. Conceivably, arterial blood gas homeostasis is sacrificed to minimize the work of breathing. Dempsey has describe the situation where if the trained male runner at a V02 of 5 1-min"1 were to achieve the same pressures of O2 and CO2 as in the untrained individual exercising at a VO2 of only 3 1-min"1, he would have to sustain a ventilation in excess of 200 1-min"1 , whereas the untrained individual would need only achieve ~ 120 1-min"1 ventilation [45]. Perhaps respiratory muscle pressure development in proportion to, but not out of proportion to, increases VCO2, and respiratory muscle fatigue is spared at the expense of arterial blood gas homeostasis [45]. The 79 development of EIH is not limited to the human athlete. Significant arterial hypoxemia has been reported in horses [20-22, 156, 231]. The mechanism for EIH in the horse remains equally elusive but is possibly related to hypoventilation. Horses have been shown to have retain CO2 with PaC02 values 5-10 mmHg in excess of resting values during heavy exercise [21]. A critical review of the literature suggests that relative alveolar hypoventilation warrants reexamination and it can not be excluded as was previously concluded [176, 178]. Hypoventilation during exercise would likely be caused by one or a combination of the following (i) respiratory muscle fatigue, (ii) inadequate drive to breathe, or (iii) mechanical limitation. Respiratory muscle fatigue is defined as the inability of the respiratory muscles (RM) to continue generating a given pleural pressure. RM fatigue , if sufficient in magnitude, may be of physiologic significance to the regulation of ventilation [46]. RM fatigue has been successfully demonstrated following voluntary hyperpnea [15] and marathon running [126]. Bye et al. [35] asked if the fatigue of RM during exercise is a coincidental phenomenon or does it actually set limits to exercise capacity that could be improved if the ventilatory muscles improved? This was addressed by Fairbarn and co-workers [56] who showed 4 weeks of RM training increased RM endurance but had no effect on maximal cycling performance in highly-trained cyclists. The available data is convincing that RM endurance improves with specific RM training [28, 29, 57, 146]. However, the effect of improved RM endurance on whole body exercise and VO2 is debatable. The two studies of Boutellier [28,29] are unique because they show large increases in VChmax of 38% and 50% in non-athletes and athletes respectively. Methodological differences among studies may be reflected in these results, however the large improvements in VC^max seem incredible and these studies have yet to be replicated. If the proportion of total VO2 going to the RM is high, the amount of O2 available to the locomotor muscles could be reduced. Therefore a stage could be reached when the VO2 during exercise is insufficient to meet the needs of both the RM and the other exercising muscles. As a result either the exercising muscles or the RM or both would go into oxygen debt and exercise tolerance would be limited [35]. At high levels of exercise the percentage of total blood flow to the RM is 14-16% [82]. Thus the RM are in direct competition with skeletal muscle for oxygen and cardiac output. Dempsey et al. [46] have predicted that RM, if placed under sufficient load during heavy exercise, receive adequate blood flow at the expense of limb locomotor muscles under conditions where total Q is at or near capacity. It appears that at V02max, high demand for respiratory muscle blood flow causes locomotor muscle vasoconstriction and compromises limb blood flow [78, 86]. Heavy exercise places substantial demands on the RM to produce and sustain high levels of ventilatory work. While it seems that RM receive adequate blood supply during maximal exercise, the question remains: do respiratory muscles fatigue during strenuous exercise? RM, in particular the diaphragm, can be considered highly resistant to fatigue. The natural recruitment of motor units in the diaphragm optimizes its fatigue resistance (i.e., type I fibers followed by type Ha then type lib) [46]. The diaphragm, as the primary muscle of inspiration, is required to be fatigue resistant. In humans RM fatigue has been investigated using alterations in pulmonary function, maximal voluntary ventilation, maximal mouth, pleural, or transdiaphragmatic pressure development (Pdi) [34, 41, 98, 126]. Volitional measurements of ventilatory performance after exercise have indicated the presence of RM fatigue [126, 128, 233]. However caution must be take when interpreting these 81 results. Test conditions in "field" studies are difficult to control and may not be sufficiently objective or independent of whole-body fatigue [46]. More objectively, non-volitional measures can be used to evaluate diaphragm fatigue. Bilateral phrenic nerve stimulation (BPNS) has shown that the human diaphragm can become fatigued [12, 13, 15]. BPNS involves stimulation of the phrenic nerves bilaterally behind the sternocleidomastoid muscles in the neck [10, 11]. Using BPNS, diaphragm fatigue can be divided into high and low frequency fatigue [10, 148]. High-frequency fatigue is characterized by an inability to maintain a given Pdi at high rates of phrenic nerve stimulation (50-100 Hz), whereas low-frequency fatigue is characterized by failure to maintain adequate pressures at low rates of stimulation (1-20 Hz) [46]. Exercise-induced low frequency diaphragm fatigue is seen in normal individuals [12, 13, 102, 125]. Most recently BPNS has been used at low and high frequencies to determine that both low and high frequency fatigue is present following near maximal exercise (95% VC^max) [14]. What are the consequences of low/high frequency fatigue of the diaphragm? It is unknown if diaphragm fatigue affects VE during exercise. The relationship between diaphragm fatigue, VE, and EIH remains to be determined. While speculative, it is possible that if diaphragm fatigue occurs during strenuous exercise, ventilation could be compromised resulting in relative alveolar hypoventilation and EIH. Pulmonary ventilation is tightly coupled to metabolic demand and is of paramount importance to the maintenance of blood gas homeostasis. The control of ventilation during exercise is a complex and not yet fully understood process. The exactness of pulmonary ventilation is seen during heavy work where the rate and depth of breathing are adjusted quickly and precisely to meet metabolic demands. The stimuli, or drive to breathe, are mediated in large part by peripheral chemoreceptors. Peripheral chemoreceptors are sensitive to changes in PO2, 82 PCO2, temperature, pH, and potassium concentrations during exercise [51, 241]. Endurance performance and chemosensitivity have been linked [131], where endurance athletes have blunted responses to hypoxia and hypercapnia compared to other athletes and sedentary controls [36, 131, 208]. A blunted hypoxic ventilatory response (HVR) and/or hypercapnic ventilatory response (HCVR), results in an inappropriate VE and a lowered arterial oxygenation. An attractive hypothesis put forth was that athletes who exhibit EIH may have diminished peripheral chemosensitivity resulting in inadequate VE and consequently EIH [91]. However, resting HVR was not significantly correlated with maximal VE (r = 0.08), VE/VO2 (r = 0.1), or arterial desaturation (r = 0.06), indicating that ventilation was adequate in subjects with EIH. More recently, contrasting data demonstrate that the role of chemosensitivity in EIH is of importance [81]. HVR and HCVR at rest were significantly lower in individuals with EIH. HVR was significantly related to both VE/V02 (r = 0.43) and VE/VC02 (r = 0.61). In another attempt to examine this relationship significant positive correlations were reported between SaC>2 and PET02 (r = 0.72), PET02 and VE/V02 (r = 0.91), Sa02 and VE/V02 (r = 0.74), and HCVR at rest and Sa02 (r = 0.45) [144]. It is worth noting that few subjects met the criteria for EIH; only 3 of 31 subjects had an SaC>2 < 92, while 28 were > 92. A significant relationship (r = 0.698) was also calculated between SaC>2 and VE/VO2 during maximal exercise [226]. The discrepancies between these studies and variability among correlation coefficients are difficult to reconcile. The indirect nature of the data is problematic, but more importantly it must be reemphasized that statistically significant correlations do not imply causation [151]. Regardless, what may be of more consequence to understanding EIH is the relationship between HVR and HCVR to EIH during exercise rather than at rest. Cooper et al. found that highly trained cyclists who develop EIH have been shown to have a lower HCVR at rest and during exercise at 25%, 83 50%, and 66% VChmax than do athletes without EIH [43]. These introductory data suggest a role for altered ventilatory control in the development of EIH. In summary, the relationship between chemosensitivity and EIH remains debatable from the available data and warrants further attention. A mechanical limitation of the chest wall and lung structures may constrain ventilation during strenuous exercise. In the untrained, expiratory or inspiratory flow limitation is minimal during maximal exercise, end-expiratory lung volumes remain well below resting values and VT operates along the steepest portion of the pressure/volume relationship [46]. Typically, maximal /b, VT, and VE during exercise are determined by lung structures and an adequate reserve exists. Given the theory that the morphology of the lung does not change with exercise training [45], it is reasonable to suggest that those capable of extremely high VC^max and VEmax, as a result of exercise training, may exceed the functional limits of the chest wall and lung. Peak expiratory flow rates have been measured during exercise that approach and exceed maximal expiratory flow measured during maximal expiratory flow-volume (MEFV) curve [103]. During normoxic helium breathing (He-02) VE increases and the respiratory system becomes "unloaded" resulting in augmented hyperventilation during exercise [48]. It was hypothesized that He-02 breathing increases the MEFV loop and allows for a larger mechanical reserve and a subsequent increase in VE. If hyperventilation with He-02 breathing reduces EIH then hypoventilation can be considered an important component of EIH. During a treadmill run of 3-4 min at 75-95% V02max subjects breathed either room air or 21% O2 balance helium [48]. He-02 breathing caused hyperventilation, hypocapnia, increased PETO2 (4-11 mmHg), and caused Pa02 to increase 5-15 mm Hg in four of five runners compared to breathing room air. The effects were quickly reversed by the resumption of room-air breathing. This suggests that 84 the mechanics of the respiratory system set VE at a lower level at the cost of arterial oxygenation. Dissimilar results have been reported in subjects breathing a similar He-02 mixture during a graded exercise test to exhaustion [32]. Maximal ventilation was increased significantly but Sa02 values did not differ between He-02 and room air breathing. The conflict between these two studies may be related to differences in duration, mode, and intensity of exercise (3-4 min running at 75-95% V02max vs. incremental cycle test to exhaustion). The potential importance of a mechanical limitation is demonstrated when He-02 breathing was shown to prolong endurance time and reduce VO2 during heavy work [1], suggesting that high levels of ventilatory work may impact endurance capacity. Locomotion and breathing are not independent during exercise. Entrainment, or synchronization between locomotor activities and breathing has been described during running, cycling, kayaking, and rowing [23, 66, 119, 138, 139, 157, 221]. Because both exercise and respiration rely on cyclical movements impacting on the thoracic complex, to circumvent any mechanical constraints developing as a result of limb motion, breathing must be made to fit the locomotory cycle [30]. Entrainment is not as clearly described in humans as it has been in other species such as the dog and the horse (for review see [46]). In humans, the degree of entrainment is strongly influenced by the type of exercise. This is reflected in the high variability of entrainment across modes of exercise. It remains possible that at high levels of exercise, ventilation and locomotion are tightly coupled and that ventilation is partially determined by locomotion. To date, only one study has sought to examine the relationship between EIH and entrainment [138]. This preliminary study (n = 5), showed that elite kayakers who involuntarily select a breathing frequency-to-stroke rate of 1:1 had consistently lower Sa02 values (Sa02 = 89%) at V02max than do those who select a 1:2 ratio (Sa02 = 91%). It is tempting to speculate 85 further on the relationship between entrainment, hypoventilation, and EIH but the lack of data makes even this impossible. 3.3 VENTILATION-PERFUSION INEQUALITY The bases of the lung are better perfused while the apices are better ventilated. Consequently, the alveoli at the apex of the lung have less blood flow but are well ventilated, while the base is well perfused but less well ventilated. Regardless, in normal healthy lungs VA/Q is reasonably well matched and allows for adequate pulmonary gas exchange at rest and during exercise. Dispersion of VA/Q occurs when ventilation to an area of the lung is compromised and blood passing by does not participate in gas exchange. A mismatching of VA/Q will lower Pa02 as blood will be inadequately oxygenated. This concept has been related to EIH where abnormalities of VA/Q are seen during heavy exercise [74, 93, 204, 223 230], and prolonged exercise [89]. VA/Q mismatch, as determined by the Multiple Inert Gas Elimination Technique (MIGET), increases with exercise intensity to a V02 -3.5 1-min"1, beyond this the ratio does not worsen further while [A-a]D02 continues to widen [74, 93]. It seems likely that VA/Q inequality contributes significantly to EIH, in fact it has been calculated that VA/Q may contribute > 60% to the [A-a]D02 [93]. In a unique investigation, Pederson et al. [159] measured PaC»2 and SaC>2 in highly trained cyclists during upright and supine cycling. A more homogenous VA/Q distribution during supine cycling would be reflected in a favorable environment for gas exchange compared to upright cycling. No differences between positions were seen in PaC>2 and SaC^. VC^max was lower in the supine position, but altered body position (and presumably VA/Q, although not measured) did not affect arterial O2 desaturation. This suggests a minimal role for VA/Q mismatch on PaC^ and 86 Sa02- While these results are intriguing, the lack of a VA/Q measure makes evaluation of their physiological importance difficult. While several studies have clearly shown that VA/Q inequality occurs during exercise the cause remains speculative but may be related to non-uniform pulmonary vasoconstriction, reduced gas mixing in the large airways or the development of interstitial pulmonary edema [203]. The favored hypothesis in the literature for VA/Q inequality is the formation of interstitial pulmonary edema [89, 106]. The etiology of pulmonary edema during exercise is discussed below. 3.4 REDUCED PULMONARY TRANSIT TIME During exercise there are several mechanisms which help to maintain adequate pulmonary gas exchange. Pulmonary capillary blood volume (Vc) increases through a 3-fold recruitment of capillaries above resting values. The rise in Vc accommodates exercise related increases in cardiac output and provides adequate transit time of red blood cells for diffusion equilibrium to occur. In some athletes Q can reach over 40 1-min"1 [50]. In these elite athletes, Q may continue to increase beyond the point at which Vc has reached its morphological limits; consequently pulmonary transit time (PTT) would be reduced [45]. Under resting conditions red blood cells remain in the pulmonary capillary for ~ 0.75 s [105], while during intense exercise PTT has been estimated to be reduced to ~ 0.25 s [235]. In agreement with Dempsey's model [45], this represents a significant diffusion disequilibrium for 02 at the end of the pulmonary capillary. Experimental data examining PTT during exercise have been sparse. It has been observed that Vc does not plateau, nor does mean PTT fall below 0.46 s despite further increases 87 in exercise intensity and that no relationship exists between mean PTT and a widened [A-a]D02 [234]. Oppositely, PTT has been significantly correlated with [A-a]D02 (r = - 0.59) [88]. In this study, estimated mean transit time during maximal exercise was 0.39-0.41 s. This would be sufficient for diffusion disequilibrium to occur [45]. The difference between these two studies has been ascribed to exercise intensity [88]; where subjects exercised at 88% V02max [234] and > 90% V02max [88]. It is difficult to discern between these two workloads, however subjects exercising at near-maximal intensities would be more likely to have a substantial reduction in PTT. Despite the available theory and data decreased PTT can not be solely responsible for EIH. Athletes capable of high aerobic work (i.e., V02max > 65 ml-kg"1-min"1) would have comparable cardiac output during maximal exercise. Yet, only 50% of these athletes develop EIH. Based on the theoretical model [45], and the observation that PTT decreases during heavy work [88, 234], it seems likely that a shortened PTT contributes to EIH in athletes capable of high Q. Further study is required to determine the magnitude of its importance. 3.5 PULMONARY EDEMA One of the principal factors which limits gas exchange through the alveolar-capillary membrane is the distance between the membrane and the red blood cell. Any increase in this distance hinders gas equilibrium from occurring. Athletes with EIH may possibly enlarge this distance through the formation of interstitial pulmonary edema [33, 129, 185, 204, 230]. The mechanism responsible for the accumulation of extravascular water remains to be determined but is likely caused by increases in capillary hydrostatic pressure, capillary permeability, capillary surface area, or a lymphatic insufficiency [235]. The pulmonary blood-gas barrier is required to be extremely thin for gas exchange but also immensely strong because the capillary wall stresses become very high during exercise. Stress-failure of the blood-gas barrier would cause high-permeability pulmonary edema or hemorrhage [236]. At a capillary transmural pressure of 40 mmHg ultrastructural changes are seen using a rabbit model [225]. Changes include disruption of the capillary endothelial layer, alveolar epithelial layer and in some cases all layers of the wall. In humans mean pulmonary artery pressure (PPA) and capillary wedge pressure (PWEDGE) can reach values ~ 40 and 27 mmHg respectively [189, 230]. Increases in PPA with exercise in humans have been consistently reported [70, 101, 188, 222]. Pulmonary capillary pressure must be deduced from the above mentioned pressures. Unfortunately, accurate measures of PPA and PWEDGE in athletes with high Q and EIH are few. Measures of these pressures are required to confirm existing theories on capillary stress failure during exercise. Nonetheless, the pressures observed by Wagner et al. [230] are within the range where stress failure of pulmonary capillaries have been seen in animals [225, 239]. Stress failure of the pulmonary capillaries at these elevated pressures may cause leakage of fluid and transient pulmonary edema. Accumulation of interstitial fluid is usually removed by lymph flow, but may be compromised during exercise [42]. Another possibility is that the distention of lung capillaries during exercise, via increased blood volume, could increase permeability and promote fluid shifts [225]. Using sheep, it has been shown that 15 min after prolonged heavy exercise (40-60 min) that pulmonary lymph flow is significantly higher than at rest [153]. Possibly, the lymph system is unable to clear the interstitial space during exercise, resulting in pulmonary edema. This hypothesis remains to be tested. 89 Mechanical stress failure of the blood-gas barrier during exercise in humans has yet to be proven. However, from the available data on pulmonary pressures and current stress-failure theory it seems possible that pulmonary edema can occur with exercise. Even a mild degree of pulmonary edema could pose an important limit to gas exchange. Coupling decreased PTT and pulmonary edema would impact on diffusion equilibrium. In addition, development of pulmonary edema may compromise VA/Q relationships. Anecdotal reports following heavy exercise have reported pulmonary edema [137]. Indeed, much of the evidence for pulmonary edema is indirect and circumstantial. No techniques have yet shown directly the development of a small degree of edema in man. However, exercise has been shown to cause perivascular cuffing in the pig [203]. Pigs exercised for 6-7 min at the highest speed they could sustain. Animals that exercised had a higher percentage of pulmonary arteries with perivascular edema than did non-exercised controls (33.8% vs. 20.0%). These direct data show that edema can occur during short-term heavy exercise in an animal model. Unfortunately, indications of extravascular lung water accumulation in exercising humans have been indirect and inconsistent. Trans-thoracic electrical impedance (TEI) has been used in an attempt to quantify the accumulation of extravascular water [33]. TEI was decreased for 30 min following exercise and was related to the accumulation lung water. However, training status was not reported nor was a measure of arterial oxygenation. The physiological importance of these findings remains unclear because the measurement of TEI is non-specific and can not explain the exact source of hindered impedance. The decrease may have been due to elevated intravascular volume and not interstitial edema. More recent results [186] demonstrate the opposite where TEI was increased following 90 maximal exercise. These conflicting studies, and the non-specific nature of TEI highlight the limitation of using TEI as a tool to evaluate pulmonary edema. Decreases in the diffusing capacity of the lung (DL) post-exercise have been identified as indicators of pulmonary edema [33, 129, 185, 204]. However, data from these studies have been conflicting and it has been unclear if DL changes are exclusive to elite athletic populations. Sheel et al. [212] determined that endurance trained, moderately trained and untrained subjects all experience the same degree of change in DL following exercise. The change in DL was due primarily to a decrease in Vc rather than pulmonary edema. The observed decrease in Vc post-exercise is supported by the work from several studies [75-77,140]. The mechanism for decreased Vc is unclear, however the fact that it occurs even after mild exercise makes pulmonary edema seem an unlikely explanation [76]. While pulmonary edema may occur during exercise post-exercise measures of DL are not a reflection of the processes occurring during exercise. Using chest radiography, pulmonary edema was not detectable [65]. However, the group of subjects were un-trained therefore EIH nor the development of pulmonary edema would not have been expected. Using well trained triathletes (VC^max = 68 ml-kg"1-min"1) following a triathlon race computerized tomographies (CT) did reveal the possibility of mild edema [39]. CT lung density and mass increased 19% and 21% respectively following the race. Despite this, it is not clear to what degree increased CT density reflects the presence of pulmonary edema and extravascular water rather than an increase in intravascular volume. These results remain indirect and inconclusive. In addition, visual analysis of CT scans did not reveal an obvious image of acute pulmonary edema. In a recent study McKenzie et al. [141] examined changes in extravascular water (EW) using a magnetic resonance scanner pre- and post-45 min of maximum 91 sustainable cycling. EW increased significantly by 9.4% implying that transient interstitial edema did in fact occur. Unfortunately, no measure of arterial oxygenation (pulse oximeter or blood gases) were made during exercise. Given the above mentioned studies it would seem that pulmonary edema may occur [39, 141]. Alternatively, since neither of these studies measured arterial oxygenation it could be concluded that the mild edema observed is not related to decrements of PaC>2 or SaC>2. The answer to this question awaits further study. Normal volunteers who performed moderate or heavy exercise over a period of 2-7 h have shown significantly increased concentrations of protein in bronchoalveolar lavage fluid compared to non-exercising controls [54]. Increases in red blood cells protein in bronchoalveolar lavage fluid have been reported following a 7-min cycling race situation compared to sedentary controls [94]. Although no measure of VO2 was made it was assumed that exercise effort was maximal as there was a cash prize for the fastest time. Unfortunately, no measure of arterial oxygenation was made. These results suggest that the integrity of the blood-gas barrier may be impaired during exercise. Consistent with this theme is the observation that sustained submaximal exercise does not alter the integrity of the lung blood-gas barrier in elite athletes [95]. While the evidence for exercise-induced pulmonary hemorrhage in humans is lacking, this phenomenon occurs frequently in thoroughbred racehorses. Evidence of stress failure of pulmonary capillaries has been described in horses, including disruptions of the capillary endothelial and alveolar epithelial layers, red blood cells in the alveolar wall interstitium, protein-containing fluid and red blood cells in the alveolar spaces, interstitial edema [238]. The authors surmised that the ultrastructural changes were consistent with those changes previously seen in rabbit lungs at high capillary transmural pressure. It becomes attractive to extrapolate the above 92 mentioned studies back to the human athlete although adequate measures of pulmonary pressures are currently lacking and do not allow for such a comparison. The pulmonary endothelium is exposed to the entire cardiac output and is possibly vulnerable to injury. Strenuous exercise can induce an acute inflammatory response marked by leukocytosis and neutrophil activation and release of inflammatory mediators. Inflammatory mediators such as eicosanoids, reactive oxygen species, cytokines all could negatively affect pulmonary vasomotor tone or membrane permeability. A significant correlation (r = 0.8) between the change in plasma histamine and the decrease in Pa02 in young and masters athletes compared to controls [7, 149]. Nitric oxide (NO) derived from the pulmonary vascular endothelium may play a role in the regulation of pulmonary vascular tone. Endogenously produced NO has been detected in the exhaled air (VNO) of humans [72] and is altered during exercise [130, 167]. VNO increases to significantly higher levels in highly trained aerobic athletes compared to controls [130]. The role VNO may have in the development of EIH is unknown. Based on these preliminary data the role of inflammatory mediators and NO in EIH remains to be well defined. However, given that the pulmonary microvascular blood flow is know to increase during exercise these are areas that warrant further investigation. In summary, evidence for pulmonary edema in humans remains unclear. Indications of extravascular lung water accumulation in humans have been indirect and inconsistent. Stress failure of the blood-gas barrier is an attractive theory, which awaits sufficient experimental evidence. 93 4. GENDER To date, all investigations except one [80] that have examined the incidence, relevance, or mechanisms of EIH have utilized male subjects. From this one descriptive study it appears that women do indeed develop EIH, and at a lower VC^max compared to matched males. Males with a VOimax within 15% of predicted normal (40-50 ml-kg"1-min"1) do not demonstrate EIH, while 40%o of women with a V02max within 15% normal (35-50 ml-kg^-min"1) experienced EIH [80]. The observation of gender-dependent EIH suggests that women are more susceptible to EIH than men. Gender differences were addressed by McClaran et al. [136] in a companion paper where the same women were observed to have significant flow limitations due to their smaller lung volumes and lower maximal flow rates (i.e., mechanical limitation). Possibly, relative hypoventilation was responsible for EIH in these women. Utilizing the same group of women, the effects of prior exercise on EIH was examined [217]. EIH was lessened, not enhanced by prior exercise. This argues for a functionally based cause for EIH that is only present during exercise rather than a persistent mechanism (i.e., pulmonary edema). These observations have recently been corroborated by McKenzie et al. [140] in a group of well-trained male cyclists. Given the paucity of data examining EIH in women, it is difficult to draw substantial conclusions. However, the pattern of EIH appears to be different between men and women. The incidence of EIH in women needs to be well established before useful gender comparisons can be made. Mechanism(s) responsible for EIH may be different between men and women and gender comparisons may help to elucidate the causative factors of EIH. 94 5. AGE The development of EIH has been documented in older athletes [149, 150, 179]. Few studies have been completed on this group of athletes, however significant reductions of Pa02 and Sa02 are seen at VC^max. EIH may occur more frequently in older athletes than young athletes. Seventy percent of older athletes have been shown to experience EIH [134]. Prefaut et al. [179] described a different pattern of EIH between older and younger athletes. For the same absolute work load, the drop in Pa02 was greater in older athletes for training conditions less rigorous than in young athletes (i.e., training intensity lower in older athletes). The pattern of widening [A-a]DC>2 was similar between young and old athletes, but was greater in magnitude the older group at a given %VC>2max. It is known that the normal aging process reduces lung elastic recoil and increases the stiffness of the chest wall. This can result in reduced vital capacity and expiratory flow rates, thus the MEFV loop is reduced. Whether or not the older athlete is mechanically constrained during exercise, resulting in EIH, remains to be investigated adequately. 6. SUMMARY Exercise-induced hypoxemia negatively affects VC^max and exercise performance capacity in highly trained male endurance athletes. EIH also affects women and older athletes. EIH is likely a multifactorial phenomenon related to diffusion limitations, inappropriate exercise ventilation, and inequality of VA/Q. The relative contribution of each postulated cause remains debatable. Further study is required to completely examine the role of hypoventilation and the complex ventilatory control mechanisms involved. Stress-failure of the pulmonary capillary and the development of pulmonary edema can not yet be confirmed or refuted from the available 95 data. Although not yet described in the literature, the mechanism(s) for EIH are possibly varied between individuals. Single-subject design studies may possibly reveal this. To borrow the words of Dr. J. A. Dempsey "...given the long list of our still untested speculations, we could use a bit more data" [45]. 96 APPENDIX B REVIEW OF LITERATURE: EXHALED NITRIC OXIDE 1. INTRODUCTION Endogenously produced nitric oxide (NO) has been detected in the exhaled air of humans [72] and the amount of exhaled NO is altered during exercise [130, 167]. It is now well established that NO is produced in a variety of both human and animal cells with numerous biological functions. NO is believed to exert regulatory functions in the circulatory, pulmonary, nervous and immune systems. Expiratory NO concentrations (CNO) and the production rate of NO (VNO) have been examined during physical exercise. It is believed that the formation of NO may play a role in the normal pulmonary response to exercise. This is a contemporary issue of relevance to exercise scientists. However, the effects and relevance of NO to exercise are not yet completely understood. This review summarizes the current literature of the physiology of NO, expiratory NO, and the relationship between exhaled NO and exercise. 2. PHYSIOLOGY OF NITRIC OXIDE NO is produced from one of the nitrogen atoms of the N-guanidino terminals of the common amino acid L-arginine and molecular oxygen. In addition to NO, citrulline is a product of this reaction. The L-form of arginine is specific to the reaction, as a number of analogs and the D-enantiomer of arginine are inactive. Production of NO occurs via a metabolic pathway requiring an isoform of the enzyme NO synthase (NOS), calcium (Ca2+), nicotinamide adenine dinucleotide phosphate, tetrahydrobiopterin, flavin adenine dinucleotide, flavin mononucleotide, and calmodulin depending on the cellular location and isoform of NOS [85]. Three primary isoforms of NOS exist; (1) constitutive form, dependent on Ca2+ and calmodulin found in neural 97 tissue (nNOS), (2) Ca2+/calmodulin dependent constitutive enzyme found in endothelial cells (eNOS), and (3) Ca2+ independent form isolated from macrophages and vascular smooth muscle cells following induction with specific cytokines (iNOS). Despite extensive research, the specific mechanism for the NOS oxidation of L-arginine to NO remains unclear. It is known that the more active forms of NOS are the constitutive forms (nNOS and eNOS). Activation of iNOS requires gene transcription, therefore increased NO production occurs several hours after exposure to stimuli as compared to the constitutive forms which can be activated pharmacologically or physically within seconds or minutes. The production of NO is believed to cause relaxation of smooth muscle cells via a signal transduction system. NO activates guanylate cyclase (GC), the enzyme which stimulates the conversion of 3', 5'-cyclic guanosine triphosphate (GTP) to 3', 5'-cyclic guanosine monophosphate (cGMP). Two major subtypes of GC exist; particulate guanylate cyclase (pGC) and the more active soluble guanylate cyclase (sGC). The presence of a cGMP-dependent protein kinase also appears obligatory for the activation of cGMP [85]. The NO-cGMP signal transduction system causes relaxation of smooth muscle by decreasing the concentration of free calcium (Ca2+) in smooth muscle cytosol. Three mechanisms have been proposed to account for the smooth muscle relaxant properties of cGMP; (1) cGMP may stimulate Ca2+ extrusion by activation of the Ca2+-ATPase on the plasma membrane [171, 211]; (2) cGMP may facilitate uptake of Ca2+ into the endoplasmic reticulum by activation of a Ca2+-ATPase [184,227]; (3) Na+-Ca2+ exchange [63]. Nitric oxide-relaxation of smooth muscle is an important physiological process, yet there is no consensus on the mechanism of decreased Ca2+. It is possible that a combination of the aforementioned factors leads to a lowering of Ca2+. 3. EXHALED NITRIC OXIDE Exhaled endogenous NO was first documented in the expired gas of rabbits, guinea pigs and humans using chemiluminescence [72]. The presence of NO in human breath has been confirmed using gas chromatography-mass spectrometry [124]. Indeed, numerous studies have detected NO in the air of normal, healthy humans (Table 6) and in many different animal species [206]. Reported human values of expired NO are variable, ranging from 2 to 80 ppb. Discrepancy between studies can be attributed to three methodological concerns. Firstly, gas collection has been done using a variety of exhalation maneuvers; direct exhalation, breath-holding, use of nose clip, use of a face mask, fixed expiratory flow rate, direct sampling vs. mixing chamber, and positive end expiratory pressures [71]. Exhaled NO concentrations are sensitive to expiratory flow rates where NO can rise 11-fold as flow increases [214]. To date, there is no standardized measurement technique, however the single-breath measurement is gaining acceptance [86]. Given the range of NO values reported in the literature, it important to be cognizant of the measurement technique used when interpreting and comparing results. Secondly, mixing of nasal and lower airway air gases can significantly elevate NO concentrations. The upper airways, especially the nose, contribute the largest amount of NO to exhaled air [113, 193, 205]. The proportion of exhaled NO derived from the lower airways ranges from 90% [205] to 50% [167]. There is considerable debate surrounding the proportion of NO from lower vs. upper airway, yet it appears reasonable to conclude that some portion of exhaled NO is of a lower airway origin. Lastly, NO is highly reactive with oxygen and various synthetic materials. Studies that do not account for this can have erroneous NO values depending on the reactivity or absorbency of the equipment to NO. 99 4. EXHALED NITRIC OXIDE DURING EXERCISE Exercise alters nitric oxide concentration (CNO) and production rate (VNO). Only two investigations have reported no change in CNO during exercise [19, 100], most existing data points to a decrease in CNO during exercise (Table 6). CNO in the nasal passages is predominant in resting humans with the lower airways contributing a smaller fraction [113, 193, 205]. The contribution of the nasal passages to exhaled NO has recently been observed to decrease during exercise [127, 167, 192], while the contribution of lower airways increases [167]. Nasal levels of NO appear to drop rapidly in response to exercise. Nasal cavity NO levels dropped 47% after only one minute of exercise compared with rest with a maximal reduction of 76% during heavy work [127]. The mechanisms underlying the reduction in CNO can only be speculated on at this stage. At first inspection, it would seem that CNO is decreased during exercise due to increased ventilation. However, it has been calculated that the decreased nasal CNO cannot be explained merely by dilution [127]. It is possible that during exercise, a shift occurs where a greater percentage of total CNO comes from a lower airway source rather than the nasal cavity [167]. The shift may be reflective of a change from nasal to oral breathing at the onset of exercise explaining the immediate decrease in CNO [127]. Mucosal blood flow is reduced during exercise in both the sinus [57] and the nose [154]. Regional differences in NO production during exercise likely exist. An increase in endothelial production could, in fact, explain the higher amounts of NO found in orally exhaled air during exercise since NO derived from the pulmonary endothelium may enter the airway lumen via the alveoli. The production rate of NO (VNO) is the product of expired NO concentration and VE (VNO = NO x VE). VNO increases with physical work (Table 6). There is considerable variability 100 reported in the rate of exercise induced increased VNO. One difficulty in interpreting the data is a lack of consistency in units. Another source of confusion is that the effects of physical conditioning on VNO during rest and exercise have not been adequately examined. It is widely accepted that physical conditioning affects the cardiovascular response to exercise. Training-induced alterations to cardiac output (Q), pulmonary blood flow, and ventilation (VE) may influence the production of NO. Basal CNO has been found to be identical between high aerobic capacity athletes and non-athletes [40, 130] and higher in one subject described as an athlete [19]. During exercise, it has been observed that athletes maintain a higher CNO than do moderately trained and sedentary individuals at a given exercise intensity [130]. In disagreement with these data, no difference in CNO was found between trained men, sedentary women, and sedentary men at rest or during cycle exercise. [40] The dissimilar results may be related to the different levels of physical conditioning and maximal aerobic capacities of the respective athletic groups; 4.4 1-min"1 [40] and 5.6 1-min'1 [130]. These two groups would likely have different VE, Q, and pulmonary blood flow, thus they would have different rates of NO production. To date, these are the only data available addressing this issue, leaving the question of the effects of physical conditioning on basal and exercising CNO unresolved. Regardless, VNO increases in both athletic and non-athletic populations during dynamic exercise. As mentioned, the rate of increase has been reported to be different between athletes and non-athletes [130] and the same [40]. This disunity is possibly related to the method of reporting data (Table 6). When VNO is expressed per unit of body mass (pmol-kg"1-min"1), differences are seen between athletes and non-athletes [130]. However, when VNO is expressed in absolute terms (nmol-min"1), no differences between groups are observed [40]. In a preliminary study of unpublished observations, Phillips et al. [167] describe a statistically 101 significant correlation between exhaled NO output and body surface area (r = 0.59). Based on this finding NO output values were expressed per square meter of body surface area. These results require further study to be confirmed or refuted. Given the sparse data and inconsistency between studies, it is difficult to conclude what role physical conditioning and body surface area plays in the determination of VNO. A growing body of evidence suggests that exercise training increases basal nitric oxide production in the systemic vasculature [5, 68, 108, 115, 210, 213, 232]. A lowering of blood pressure and heart rate, and an increase in coronary blood flow and capillary density of skeletal muscle characterize chronic exercise training. The decrease in blood pressure may be mediated by an augmented basal production of NO and its subsequent vasodilatory effects [172]. The significance of this may be to serve as a protective effect in disease states such as atherosclerosis, cardiac failure and perhaps hypertenstion [108, 115], It is reasonable to suggest that an upregulation of NO production in the systemic vasculature caused by training could be mirrored in the pulmonary vasculature. This hypothesis remains to be adequately tested. 5. NITRIC OXIDE PRODUCTION DURING EXERCISE It is widely reported that NO is detected in the exhaled air of humans. However, the origin and stimuli of exhaled NO are poorly understood. A better understanding of where and how exhaled NO originates is necessary to determine its significance. There is contradictory evidence to suggest that NO is primarily released from the upper airways, lower airways or from both areas. Schedin et al. [205], using patients with tracheostomies and a group of anesthetized healthy women, determined that the lower airways and lung are one source of NO, but the quantity is lower compared to the upper airways (10% lower vs. 90% upper). These important 102 data have been corroborated by several groups suggesting that the major contributor to exhaled NO at rest is the nasal space with a lesser addition from the lower airways [4, 37, 113, 205]. The cellular source of NO from the upper respiratory tract may be airway epithelial cells in addition to a nasal source [211]. In contrast to upper airway data, ~ 50% of NO in mixed expired air has been found to originate from a lower airway source when air from the lung was partitioned from that of the nasopharynx by balloon occlusion [167]. In support of a lower airway origin, fractional alveolar and expired concentration of NO and CO2 have been measured and it has been predicted that exhaled NO was derived from a similar region of the lung as CO2 [27]. Conversely, NO levels have been shown to rise to an early peak and plateau, while CO2 levels continue to rise and peak later during exhalation [37]. It is difficult to reconcile the disparity between these two studies. A lower airway origin of exhaled NO has also been documented in both normal [111, 166] and asthmatic individuals [111]. The cellular source of NO in the lower respiratory tract remains uncertain, however, studies with isolated perfused pig lung preparation suggests an alveolar surface origin, rather than the pulmonary circulation, [44] and may be derived from endothelial NO synthase expressed in the alveolar walls of normal lungs [117]. Based on the available data, it can be concluded that mixed expired air contains both a fraction of lower and upper airway derived NO. More NO is likely derived from the upper airways and nasal passages than the lower airways at rest, yet the respective proportions remain uncertain. During exercise, lower airway sources of NO may become more pronounced. Given that the site of NO production is debatable, the stimulus for production remains equally obscure. However, two schools of thought have emerged in the literature; increased pulmonary blood flow [19, 100, 130] and increased ventilation [166, 167, 169, 219]. In the 103 peripheral vasculature, an increase in shear stress leads to enhanced endothelial NO release [163]. Shear stress on the blood vessel wall arises from the frictional force caused by blood flow, although other physical stimuli such as pulsatility and changes in transmural pressure may be important [170, 201]. It is recognized that an acute increase in blood flow will lead to an increase in NO production and a relaxation of underlying smooth muscle. It is believed that shear stress causes deformation of endothelial cells and initiates the NO-cGMP signal transduction system causing changes in smooth muscle tone [142, 242]. However, the shear stress-vasodilation response within the pulmonary architecture is not as clearly defined as in the peripheral vasculature. Much evidence for a shear stress/pulmonary endothelium source of NO in expired air is indirect; statistically significant positive correlations are seen between VNO and heart rate, VO2, VCO2, VE, and cardiac index [40, 100, 130, 167, 169]. While these data provide some insight, it is difficult to extrapolate from correlative evidence as to the causative factors involved with increased VNO during exercise. Several studies have attempted to manipulate the variables that could alter VNO. TO determine the effects of altered pulmonary blood flow, Pogliaghi et al. [169] measured VNO with head-out water immersion (T pulmonary blood flow) or increased gravity (I pulmonary blood flow) at rest and during cycling. No differences were observed between conditions at similar levels of ventilation and it was concluded that altering pulmonary vascular endothelial stress does not alter lung output of NO. Despite the uniqueness of these data, they remain difficult to interpret as no pulmonary measures were made and it is uncertain to what extent a shear stress response occurred. Other studies have attempted to manipulate pulmonary blood flow with dobutamine infusion [167]. Dobutamine infusion did not increase NO production as compared to exercise. However, caution must be taken for ruling out a vascular 104 origin of NO, as the level of exercise was only moderate. Possibly, an effect of increased blood flow on increased VNO output would be evident at a higher exercise intensity. The most physiological method of increasing cardiac output and skeletal and coronary blood flow is through exercise. Moreover, it was also observed that mean VNO was increased during dobutamine infusion compared with rest control VNO. Interestingly, patients with heart disease have decreased basal production of expired NO [220]. While not conclusive, these data imply that impaired cardiac function reduces the shear stress response of pulmonary endothelial cells and is reflected in basal NO levels. The stimulus for NO production during rest or exercise will likely need to be resolved using an animal model. Indeed, evidence from an isolated pig lung preparation indicates that the pulmonary vascular endothelium likely contributes to the NO found in exhaled air [44]. From these data, it can be concluded that the lung itself can synthesize adequate NO to account for CNO seen in exhaled air. However, it is not clear if the cells within the lung are in fact the source of exhaled NO in an intact animal model or human. Presently, a non-endothelial source of NO can not be completely ruled out. Voluntary increases in ventilation without exercise have been used in an attempt to determine the site of release of exhaled NO [19, 100, 166, 167]. By increasing voluntary ventilation VNO increases. Stretching of the lung parenchyma or airway epithelia by positive end expiratory pressure increases VNO in rabbits [165, 219]. It is therefore possible that changing ventilation alters VNO. The observations of lung distention effects on exhaled NO suggest the possibility of stretch receptors coupled to NO formation within the lung. Further study is required to fully examine this hypothesis. 105 6. SIGNIFICANCE OF EXHALED NITRIC OXIDE Endogenously produced pulmonary NO has been found to have several physiological functions. Possible roles include VA/Q matching, maintenance of low pulmonary vascular resistance, and airway relaxation. The idea that an optimal VA/Q ratio may be regulated by NO is supported by data showing hypoxia in anesthetized animals upon administration of NOS inhibitors [163]. The importance of NO is evident when inspired NO induces pulmonary vasodilation and improves VA/Q relationships [60, 168, 182, 197]. Endogenous NO was recently found to be critical to the maintenance of a low pulmonary vascular resistance in eNOS knockout mice who developed pulmonary hypertension [55]. It is thought that NO is continuously released to maintain pulmonary arterial tone, systemic pressure, and cardiac output [218]. Patients with heart disease have reduced NO levels, perhaps explaining why they exhibit an increased vasoconstriction of their pulmonary blood vessels [220]. It would seem that endogenously produced NO is necessary for maintenance of VA/Q matching and a low pulmonary vascular resistance, the extent to which expired concentrations of exhaled NO reflect this process requires clarification. NO has also been detected in asthmatic patients and is elevated in comparison to non asthmatics [4, 111, 112]. The increase in exhaled NO in asthmatics likely reflects increased activity of iNOS that may be induced by inflammatory cytokines [73]. Exhaled NO may reflect airway inflammation in asthma, and may be used as means of monitoring inflammatory events [18]. During exercise Q increases in proportion to exercise intensity and the pulmonary circulation is able to accommodate the increase with little rise in pulmonary pressure. This is due to the distension and recruitment of the pulmonary capillaries. It has been traditionally thought 106 that this process is passive in nature where the pulmonary circulation is fully recruited. It is only possible to speculate at this stage, however it seems likely that NO may aid in mediating the VA/Q response during exercise. Further research is required to determine the relationship between exercise, NO, pulmonary pressures and VA/Q relationships. Patients with chronic obstructive pulmonary disease (COPD) who inhaled 40 parts per million of NO during submaximal exercise improved VA/Q relationships and Pa02 compared to breathing room air [196, 197]. Interestingly, some highly trained athletes experience hypoxemia during heavy exercise [48, 91]. Approximately 2/3 of their hypoxemia can be explained by a VA/Q mismatch [93]. The possible role of NO in this phenomenon has yet to be examined. 7. SUMMARY Nitric oxide is present in varying concentrations in the exhaled air of exercising humans. The concentration decreases with exercise intensity, while the production rate of NO increases. With exercise, the lower airways increase their production rate while nasal production decreases. NO may be partly responsible for the normal pulmonary response to exercise and exhaled NO may reflect this process. At the present time, there is considerable debate in the nitric oxide literature. Further research is warranted to determine the role of NO in the pulmonary response to exercise in normal, athletic, and clinical populations. 107 TABLE 6. Published values for exhaled nitric oxide during rest and exercise. Author Exercise Subjects Basal CNO Exercise CNO Basal VNO Exercise VNO Persson [166] cycling submaxial NR n= 12 male ? female ? 10 ppb 6 ppb 10 nl/min 30 nl/min Bauer [19] cycling submaximal NR n = 4 male 12.9 ppb 14.3 ppb 72.1 pmol/min/kg 135.6 pmol/min/kg Iwamoto [100] running maximal S n = 8 2 female 6 male 26.3 ppb 26 ppb 12.3 nmol/min NR Matsumoto [135] cycling maximal NR n = 5 male 10 ppb 6 ppb 121 nl/min 398 nl/min Trolin[224] cycling submaximal NR n = 8 4 female 4 male 11.9 ng/1 5.31 ng/1 11 lng/min 209 ng/min Maroun [130] cycling moderate S I A male n = 8/group 12 ppb 11 12 5 ppb 5 9 85 pM/kg/min 86 85 110 pM/kg/min 110 450 Phillips [167] cycling supine submaximal NR n = 8 male ? female ? NR NR 53 nl/min/m2 160 nl/min/m2 Chirpaz-Oddou [40] cycling maximal S male, n = 7 S female, n = 7 A male, n = 8 14.8 ppb 16.2 15.9 9 ppb 10.5 10.5 5 nmol/min 5 6 18 nmol/min 20 37 Pogliaghi [169] cycling maximal S n= 10 male 16.3 ppb 6.1 ppb 137.7 ppb/min 544.7 ppb/min Definition of abbreviations: NR = training status not reported; CNO = nitric oxide concentration; VNO = exhaled nitric oxide output; S = sedentary; I = intermediate; A = athlete; NR = not reported. 108 APPENDIX C REVIEW OF LITERATURE: INHALED NITRIC OXIDE DURING EXERCISE 1. INTRODUCTION Until recently, nitric oxide (NO) was regarded as a toxic gas responsible for a portion of the morbidity related to air pollution [158]. Currently NO is recognized as a major endogenous mediator of multiple physiological processes, and the use of inhaled NO is prevalent in anesthesia and critical care. Although many findings are preliminary, inhaled NO has found a place in clinical medicine. The magnitude and importance of its use awaits the results of further study. This review is not intended to be a comprehensive resource of inhaled NO literature, rather it briefly summarizes the physiology of inhaled NO, the clinical effects of inhaled NO, and describes the available literature examining inhaled NO during exercise. 2. PHYSIOLOGY OF INHALED NITRIC OXIDE NO was reported in 1987 to be an important endothelium-derived relaxing factor [99, 155]. Furchgott and Zawadzki [62] first demonstrated that endothelial cells must be present for acetylcholine to induce relaxation of isolated rabbit aorta. Subsequent to these studies, NO has been identified as an important biological second messenger where NO is found endogenously or is provided exogenously during therapy. The mechanisms of action are thought to be the same. Inhalation of NO is believed to cause relaxation of smooth muscle cells via a signal transduction system. NO activates guanylate cyclase (GC), the enzyme which stimulates the conversion of 3', 5'-cyclic guanosine triphosphate (GTP) to 3', 5'-cyclic guanosine monophosphate (cGMP). Two major subtypes of GC exist; particulate guanylate cyclase (pGC) and the more active soluble guanylate cyclase (sGC). The presence of a cGMP-dependent 109 protein kinase also appears obligatory for the activation of cGMP [85]. The NO-cGMP signal transduction system causes relaxation of smooth muscle by decreasing the concentration of free Ca in smooth muscle cytosol. Three mechanisms have been proposed to account for the smooth muscle relaxant properties of cGMP; (i) cGMP may stimulate Ca2+ extrusion by activation of the Ca2+-ATPase on the plasma membrane [171, 209], (ii) cGMP may facilitate uptake of Ca2+ into the endoplasmic reticulum by activation of a Ca2+-ATPase [184, 227], (iii) Na+-Ca2+ exchange [63]. NO-relaxation of smooth muscle is an important physiological process, yet there is no consensus on the mechanism responsible for the decrease in Ca2+. It is possible that a combination of the aforementioned factors leads to a lowering of Ca2+. Nonetheless, selective relaxation of pulmonary vascular smooth muscle has numerous clinical applications. 3. CLINICAL EFFECTS OF INHALED NITRIC OXIDE Short-term inhalation of gaseous NO causes selective vasodilation without systemic effects. When inhaled, NO diffuses across the alveoli to the pulmonary vasculature where it exerts a vasodilating effect. NO, in vivo, has a biological half-life between 111-130 ms, depending on the ambient oxygen concentration [110], and is quickly inactivated by hemoglobin to form methemoglobin and nitrate and nitrite ions [191]. The actual and potential uses of NO are summarized in Table 7. Indeed, NO inhalation (5-80 ppm) has been shown to have positive effects on arterial oxygenation in respiratory-compromised patients. Excellent reviews of these effects are described elsewhere in detail [24, 97, 144, 153]. A synopsis of the clinical effects of inhaled NO is presented below. Under normal conditions, the blood vessels surrounding underventilated areas of the lung will constrict resulting in a decreased perfusion of these areas and a preservation of VA/Q 110 matching. This occurrence, termed hypoxic vasoconstriction (HPV), serves to limit arterial hypoxemia but can lead to increased pulmonary artery pressure (PPA). In patients with chronic pulmonary hypertension [i.e., adult respiratory distress syndrome (ARDS)] the administration of intravenous vasodilators (i.e., nitroprusside, nitroglycerin) can have beneficial effects (I PPA) but these are typically at the expense of systemic hypotension and worsening hypoxemia [183]. Inhalation of NO in ARDS patients (2-20 ppm for 2-27 d) has been shown to decrease mean PPA and improve arterial oxygenation [25]. In such a case as ARDS, when hypoxia and increased PPA are chronic it is routinely observed that inhaled NO improves Pa02 and PPA. A decrease in pulmonary vascular resistance (PVR), via NO, may also serve to improve right ventricular function [58]. Using normal volunteers, Frostell et al. [59] examined the effects of inhaled NO during normoxia and acute hypoxia (Fi02 = 12%). Normoxic inhalation of NO (40 ppm) had no effect on pulmonary or systemic hemodynamics. Hypoxia induced mild pulmonary hypertension by increasing PPA (+5 mmHg) and PVR (+50 dynes-s_1-cm ~5) and inhalation of NO decreased PPA and PVR to control values without causing systemic vasodilation. Inhaled NO therapy may have also found a place in high-altitude medicine. Mountaineers who had previously documented HAPE ascended to 4559m (barometric pressure = 440 mmHg) [207]. Ten of 18 subjects exhibited radiographic evidence of HAPE. Inhalation of NO (40 ppm for 15 min) decreased systolic pulmonary-artery pressure (from 66 to 40 mmHg), improved Sa02 (from 67 to 73%) and Pa02 (from 36 to 41 mmHg), and lessened the [A-a]D02 (from 15 to 11 mmHg). While it seems clear that inhaled NO can improve oxygenation in some patient populations, this effect should not be considered to be a general response but is dependent upon the pathophysiology of the disease/condition. Ill Hopkins et al. [90], utilizing anesthetized dogs, examined how inhalation of NO (80 ppm) can alter pulmonary gas exchange under conditions of: (i) normal lungs, (ii) shunt, and (iii) VA/Q inequality. In dogs with VA/Q inequality, NO variably affected matching which was improved in some dogs and worsened in others. These data are in agreement with other reports of NO inhalation in patients with COPD where Pa02 values have been shown to increase slightly [3], have no effect [145], and decrease with VA/Q inequality [17]. VA/Q inequality is the most common gas exchange abnormality and marked intrapulmonary shunt is absent in COPD [229]. Critical analysis of COPD-NO inhalation studies reveals that grouped data may not be indicative of the NO-inhalation response. Based upon the above mentioned dog data [90], and the variable responses in gas exchange [3, 17, 145], it can be concluded that areas of low VA/Q matching contribute to the variable clinical effects of inhaled NO. Hopkins et al. [90], surmised that inhaled NO improved gas exchange when the normal areas of the lung increased blood flow, but worsened it when both the normal areas and the areas of low VA/Q ratio had a fall in PVR. This would likely explain why the response of NO inhalation is variable in COPD patients, and emphasizes the need to understand the pathophysiology of the lung disease/condition. While the effects of NO inhalation are widely described in the clinical literature, few data exist concerning the response of normal individuals. It is useful to succinctly recount the existing data here. Brett et al. [31], recently characterized the response of the pulmonary circulation in normal individuals to inhaled NO (n = 8; NO = 0, 20, and 40 ppm). No significant changes were observed for Q or DLCO implying that the normal pulmonary vascular bed is not amenable to vasodilation by inhaled NO. This is in agreement with normal lungs in dogs where NO did not alter Pa02 or VA/Q matching [90]. 112 4. INHALED NITRIC OXIDE DURING EXERCISE Studies that have sought to examine the effects of inhaled NO during exercise have been few. Nonetheless, these investigations have provided valuable insight into the effects of NO therapy. It is well established that COPD patients may develop pulmonary hypertension when exercising [243]. Roger and colleagues [197], demonstrated that in COPD patients, inhaled NO (40 ppm) reduced pulmonary hypertension and improved VA/Q ratios during exercise. However, the effect of NO on Pa02 was different at rest than during exercise. During rest, Pa02 and VA/Q ratios were worsened while during exercise they both improved slightly. The authors concluded that during exercise the effect is likely explained by a preferential distribution of inhaled NO to well-ventilated alveolar units with faster time constants and normal VA/Q ratios. The results are in agreement with the previous discussion of the variability of NO inhalation at rest in COPD patients. Patients with left ventricular dysfunction and chronic heart failure were examined during exercise conditions of control and NO inhalation (30 ppm) [26]. VT and ventilatory equivalent for oxygen (VE/V02) were significantly reduced during exercise with NO inhalation compared to control. There was also a strong, but non-significant, trend for VE to be reduced (P = 0.051). The results of this study are difficult to interpret physiologically. It is unclear what mechanisms are responsible for alterations in ventilatory parameters. Hemodynamic measurements at rest were determined from a Swan-Ganz catheter placed in the pulmonary artery and Q was determined by the thermodilution technique. No statistical differences were observed for hemodynamic and gas exchange variables. It is also worth noting that the authors of this study utilized in excess of 20 paired t-tests (i.e., large probability for Type I error). In a separate investigation, Koelling et al. [118], examined patients undergoing cardiac transplantation evaluation (n = 14). Right heart catheterization was performed and Q was 113 determined by thermodilution. Exercise was performed under normal conditions and with NO (40 ppm). Patients with greater PPA, larger LV end-diastolic volumes, and lower RV ejection fractions were those benefited the most from NO. Reduction of resistance, via NO, within the pulmonary vessels likely leads to augmented maximum workload and VO2 at the anaerobic threshold. This effect was not observed during treadmill exercise in sheep [120]. With inhalation of NO (30 ppm), pulmonary vascular tone was unchanged at rest, while during exercise the usual reduction in PVR was not observed. The implication of this observation is that the pulmonary vasculature is already maximally dilated during exercise by flow-related pressure effects [120, 187]. To date, only one study has sought to investigate the effects of NO inhalation on pulmonary gas exchange during exercise in highly trained athletes [49]. Male endurance athletes (n = 9: V02max ~ 65 mlkg"1-rnin"1) performed a maximal cycle test to exhaustion while inhaling either (i) room air, or (ii) room air combined with 15 ppm NO. No differences between conditions were observed for V02max or maximal work. Inhalation of NO caused Pa02 to decrease at rest, 50%, 75%, and 100% V02max (1 9.5, 15.0, 14.6, and 5.1 mmHg). Interestingly, delta Pa02 continuously fell without NO (13.5%). With NO, after a preliminary drop, a stabilization of delta Pa02 was observed between 75 and 100% V02max. This trend was also reflected in [A-a]D02. Inhalation of NO also abolished histamine release between 75 and 100% V02max but EIH was not reversed. The physiological significance of these results remains to be determined. As concluded by Durand and co-workers [49], the negative effect of NO on gas exchange at rest was unexpected and continued during exercise; and these preliminary results should be interpreted with caution. 114 5. SUMMARY Nitric oxide is a potent and selective pulmonary vasodilator. In patients with lung disease, inhalation of NO can improve VA/Q matching, reduced pulmonary vascular resistance, and improve arterial oxygenation. These effects appear to continue during exercise. In normal individuals the effect of NO inhalation during exercise is not clearly established. From the available preliminary data (one study), it appears that athletes with exercise-induced hypoxemia have worsened gas exchange at rest with NO inhalation and a stabilization occurs as exercise progresses. TABLE 7. Actual and potential uses of inhaled nitric oxide. Adapted from Kacmarek [109]. Persistent pulmonary hypertension of the newborn Congenital cardiac anomalies Adult respiratory distress syndrome Chronic pulmonary hypertension Cardiac disease Pulmonary disease Asthma Cardiac surgery Lung transplantation Pulmonary embolism Acute myocardial infarction 116 APPENDIX D INDIVIDUAL DATA: EXHALED NITRIC OXIDE TABLE 8. Individual subject data for age, height, mass and sport. Definition of abbreviations: Tri. = triathlete, Road = road cyclist, Mtn. = off-road, mountain cyclist. SUBJECT AGE HEIGHT MASS SPORT (yr) (cm) (kg) 1 (MB) 30 179.6 75.1 Road 2(GA) 29 184.0 81.0 Tri. 3 (TD) 29 173.0 62.7 Mtn. 4(PH) 26 179.0 72.6 Tri. 5 (JM) 27 171.0 61.6 Road 6(KS) 22 172.5 67.3 Mtn. 7 (MM) 22 185.5 73.4 Tri. 8(OU) 24 180.0 62.7 Road 9(SD) 28 173.1 66.9 Road 10 (MM) 24 187.0 79.4 Tri. 11 (MK) 27 183.0 71.3 Road 12 (JJ) 25 182.7 73.7 Road 13 (MP) 27 175.9 68.2 Mtn. 14 (RR) 26 182.3 80.5 Mtn. 15 (MS) 20 181.0 73.2 Mtn. 16 (TB) 30 181.3 76.3 Road 17 (JB) 36 184.5 94.1 Road 18 (DM) 24 170.7 71.0 Mtn. 117 TABLE 9. Individual subject data for respiratory variables during maximal cycle ergometer exercise. Definition of abbreviations: VC^max = maximal oxygen consumption, VE = minute ventilation, RER = respiratory exchange ratio. SUBJECT VC^max (1-min'1) V02max (ml-kg'^min"1) VE (BTPS) (1-min1) RER (VC02/V02) 1 (MB) 5.18 72.96 179.42 1.15 2(GA) 5.47 66.95 199.47 1.22 3 (TD) 4.63 74.44 173.16 1.21 4(PH) 5.08 69.97 168.1 1.2 5 (JM) 3.9 63.31 154.92 1.13 6(KS) 4.22 62.7 188.37 1.17 7 (MM) 4.38 59.67 152.3 1.14 8(OU) 4.09 61.04 185.84 1.17 9(SD) 4.72 68.51 163.91 1.15 10 (MM) 4.61 58.06 178.76 1.19 11 (MK) 4.63 64.94 173.1 1.24 12 (JJ) 5.11 69.34 170.12 1.14 13 (MP) 4.92 72.14 169.77 1.18 14 (RR) 5.46 67.82 176.89 1.15 15 (MS) 5.5 75.14 173.84 1.14 16 (TB) 5.16 67.63 176.02 1.14 17 (JB) 6.42 68.2 203.69 1.06 18 (DM) 5.31 74.89 154.54 1.14 TABLE 10. Individual subject data for heart rate, saturation, and power during maximal cycle ergometer exercise. Definition of abbreviations: HRmax = maximal heart rate, Sa02 percent oxyhemoglobin saturation. SUBJECT Sa02 - Rest Sa02 - Lowest HRmax Power (%) (%) (beats-min"1) (watts) 1 (MB) 97.69 93.34 187 449 2(GA) 97.66 91.01 197 450 3 (TD) 98.14 92.32 193 458 4(PH) 97.66 93.65 198 439 5 (JM) 97.49 95.12 183 372 6(KS) 97.69 93.62 192 462 7 (MM) 98.14 95.12 182 444 8(OU) 98.14 93.85 184 472 9(SD) 98.66 94.64 180 469 10 (MM) 96.75 91.13 187 449 11 (MK) 98.82 94.56 187 462 12 (JJ) 98.61 88.57 192 456 13 (MP) 98.14 93.88 194 467 14 (RR) 98.02 90.11 189 484 15 (MS) 98.11 89.65 199 502 16 (TB) 98.08 93.91 197 478 17 (JB) 97.72 94.24 208 494 18 (DM) 97.47 91.41 182 456 TABLE 11. Individual subject data for concentration of nitric oxide (CNO) at rest and during incremental exercise (watts). CNO values are in ppm. SUBJECT Rest 100 200 250 300 350 400 450 1 (MB) 22.7 18.8 16.5 20.0 17.7 17.1 21.1 22.5 2(GA) 11.5 8.2 8.2 7.9 7.7 8.7 9.5 11.1 3 (TD) 10.4 10.6 10 11.8 11.7 13 14.1 14.3 4(PH) 4.6 3.1 1.8 2.1 2.0 1.5 2.1 2.3 5 (JM) 6.4 5.3 4.0 4.1 3.3 3.3 _ 6(KS) 6.4 2.8 2.3 3.4 5.2 3.7 3.3 7.9 7 (MM) 5.3 5.7 4.7 4.9 4.5 4.9 4.2 4.9 8(OU) 22.1 18.0 18.0 18.4 17.5 16.2 17.0 18.5 9(SD) 4.0 4.9 5.0 4.2 2.9 4.6 3.2 4.3 10 (MM) 6.7 4.9 3.9 4.1 3.6 4.1 4.9 6.6 11 (MK) 11.8 12.3 12.1 13.0 11.7 12.8 13.2 14.0 12 (JJ) 5.0 3.3 2.7 2.7 2.0 3.0 3.1 3.9 13 (MP) 2.5 2.6 2.0 3.3 2.5 2.6 2.0 2.5 14 (RR) 4.7 2.3 2.2 2.0 2.4 2.2 2.0 2.0 15 (MS) 3.0 2.1 1.5 2.1 5.1 3.4 4.9 2.3 16 (TB) 8.6 4.7 4.0 4.0 10.1 6.2 6.4 5.5 17 (JB) 3.5 3.2 3.4 2.3 8.5 3.0 5.1 3.4 18 (DM) 3.9 4.7 6.9 2.8 2.0 3.1 3.6 3.6 TABLE 12. Individual subject data for production rate of nitric oxide (VNO) at rest and during incremental exercise (watts). VNO values are in nmol-min"1. SUBJECT Rest 100 200 250 300 350 400 450 1 (MB) 7.6 21.1 28.8 31.0 53.5 59.1 112.1 134.1 2(GA) 4.3 13.2 19.5 21.7 26.2 43.4 57.5 67.8 3 (TD) 3.7 15.5 20.8 34.3 39.0 55.5 73.3 78.6 4(PH) 1.7 3.4 2.9 4.7 5.9 5.8 10.8 14.6 5(JM) 2.3 6.3 8.5 12.7 11.5 15.7 0.0 0.0 6(KS) 2.3 3.8 4.9 9.1 17.6 14.2 16.1 49.4 7 (MM) 2.0 6.1 9.5 13.0 14.0 17.3 20.4 24.8 8(OU) 8.4 27.4 35.3 48.7 53.4 59.8 80.2 104.6 9(SD) 2.4 7.2 8.0 10.6 9.1 15.9 14.5 23.4 10 (MM) 3.8 8.5 9.8 11.9 12.5 18.6 27.6 37.2 11 (MK) 6.4 15.4 22.8 26.8 33.4 45.1 55.3 80.5 12 (JJ) 2.0 5.8 6.4 8.0 6.5 12.1 16.5 22.1 13 (MP) 1.0 3.4 3.3 8.1 7.6 9.3 9.0 14.1 14 (RR) 2.0 4.0 5.0 5.7 8.2 8.7 9.5 11.8 15 (MS) 1.0 2.6 2.8 4.9 13.5 10.7 19.0 11.5 16 (TB) 3.3 5.5 7.6 10.2 30.3 21.0 28.2 31.4 17 (JB) 1.3 4.5 7.4 6.1 22.7 10.7 21.0 20.9 18 (DM) 2.2 6.1 12.5 5.6 5.5 10.7 16.2 18.2 121 TABLE 13. Concentration of nitric oxide (CNO) (ppm) in subjects with normal (NOS) and low (LOS) oxyhemoglobin saturation at rest and during incremental exercise. Values are means ± SE. * significantly different than Rest, P < 0.05. WORK NOS LOS (watts) ("=12) (/i = 6) Rest 9.0 ±1.9 5.8 ±1.2 100 7.7 ± 1.7 4.3 ±0.9 200 7.0 ±1.7 4.2 ±1.1 250 7.6 + 1.9 3.6 ±0.9 300 8.1 ± 1.6 3.8 ±0.9 350 7.4 ±1.6 4.0 ±1.0 400 8.3 ±2.0 4.7 ±1.1 450 9.1 ±2.1 4.9 ±1.4 TABLE 14. Production rate of nitric oxide (VNO) (nmol-min"1) in subjects with normal (NOS) and low (LOS) oxyhemoglobin saturation at rest and during incremental exercise. Values are means + SE. * significantly different than Rest, P < 0.05. WORK NOS LOS (watts) (n=12) (n = 6) Rest 3.5 ±0.5 2.6 ±0.7 100 10.0 ± 1.6 6.7 ±2.3 200 13.3 ±2.2 9.3 ±3.1 250 17.9 ±2.9 9.6 ±4.0 300 24.8 ±3.7 12.0 ±4.9 350 27.5 ±4.5 17.4 ±6.0 400 36.7 ± 7.2 24.4 ± 10.1 450 48.0 ±8.7 28.1 ± 12.1 122 APPENDIX E INDIVIDUAL DATA: INHALED NITRIC OXIDE TABLE 15. Individual subject data for age, height, mass, sport, level of competition, and amount of training per week. Definition of abbreviations: Tri. = triathlete, Road = road cyclist, Mtn = off-road, mountain cyclist. SUBJECT AGE HEIGHT MASS SPORT LEVEL OF TRAINING (yr) (cm) (kg) COMPETITION (h/wk) 1(RR) 27 183.4 80.4 Mtn. National 10-15 2(SF) 27 181.7 80 Mtn. National 10-15 3(AP) 27 195.3 71.7 Road Provincial 10-15 4(TD) 31 173.1 64.1 Mtn. National 10-15 5(NS) 24 180.0 78.7 Road National 10-15 6 (LB) 30 183.4 80.0 Tri. National 10-15 7(BJ) 35 173.0 68.5 Tri. Provincial 5-10 TABLE 16. Individual subject data for pulmonary function. Actual value with % predicted value below in parentheses. Definition of abbreviations: FVC = forced vital capacity, FEVi = forced expired volume in 1 second, FEF25-75% = forced expiratory flow at 25-75% of FVC, FEFmax = maximal forced expiratory flow rate, DLCO = diffusion capacity of the lung for carbon monoxide, VA = alveolar volume, DLCO/VA = diffusion capacity of the lung for carbon monoxide/alveolar volume. SUBJECT FVC FEVi FEF25-75% FEVi/FVC FEFmax DLCO VA DLCO/ (L) (L) (1-sec"1) (%) (1-sec"1) (L) VA 1(RR) 5.73 4.54 4.26 79.2 7.57 38.19 7.01 5.45 (93) (97) (83) (94) (73) (115) - -2(SF) 5.75 4.93 5.02 75.85 10.62 36.53 7.06 5.17 (100) (103) (100) (102) (104) (110) - -3(AP) 6.69 5.52 5.14 82.85 10.31 36.56 9.20 3.98 (97) (96) (88) (98) (90) (106) - -4(TD) 4.93 3.84 3.41 77.84 9.87 36.38 5.92 6.15 (131) (129) (114) (97) (126) (148) - -5(NS) 5.39 3.93 3.20 72.87 10.46 43.69 7.88 5.54 (95) (83) (80) (86) (104) (131) - -6 (LB) 6.32 5.42 5.31 85.77 10.51 40.31 9.96 4.05 (108) (112) (105) (102) (103) (123) - -7(BJ) 4.31 3.86 5.33 89.59 10.31 27.28 6.59 4.14 (90) (98) (127) (108) (114) (89) - -123 TABLE 17. Individual subject data for respiratory variables during normoxic (Fi02 = 21%) maximal cycle ergometer exercise. Definition of abbreviations: V02max = maximal oxygen consumption, VE = minute ventilation, RER = respiratory exchange ratio. SUBJECT V02max (1-min"1) V02max (ml-kg^-min"1) VE (BTPS) (1-min"1) RER (VC02/V02) 1(RR) 5.28 65.67 176.15 1.22 2(SF) 5.11 63.88 187.15 1.23 3(AP) 4.57 63.74 168.70 1.22 4(TD) 4.37 68.17 162.23 1.20 5 (NS) 5.21 65.45 169.60 1.16 6 (LB) 5.28 66.00 190.54 1.19 7(BJ) 4.38 63.92 177.11 1.22 TABLE 18. Individual subject data for heart rate, saturation, and power during normoxic (Fi02 = 21%) maximal cycle ergometer exercise. Definition of abbreviations: HRmax = maximal heart rate, Sa02 = percent oxyhemoglobin saturation. SUBJECT HRmax Sa02 - Rest Sa02 - Lowest Power (beats-min"1) (%) (%) (watts) 1(RR) 187 96.84 89.82 487 2(SF) 189 98.14 90.61 485 3(AP) 183 97.66 90.89 460 4(TD) 190 97.66 90.92 417 5(NS) 192 97.66 88.28 466 6 (LB) 194 97.68 90.92 452 7(BJ) 183 98.73 90.21 377 TABLE 19. Individual subject data for respiratory variables during hypoxic (Fi02 = 14%) maximal cycle ergometer exercise. Definition of abbreviations: V02max = maximal oxygen consumption, VE = minute ventilation, RER = respiratory exchange ratio. SUBJECT V02max V02max VE (BTPS) RER (1-min"1) (ml-kg"'-min"1) (1-min'1) (VC02/V02) 1(RR) 3.91 47.74 165.60 1.15 2(SF) 4.66 57.96 188.67 1.14 3(AP) 4.10 56.26 166.63 1.10 4(TD) 3.78 59.06 156.34 1.14 5 (NS) 4.05 51.46 164.87 1.21 6 (LB) 5.18 64.45 159.32 0.98 7(BJ) 4.06 59.27 178.82 1.13 124 TABLE 20. Individual subject data for heart rate, saturation, and power during hypoxic (Fi02 = 14%) maximal cycle ergometer exercise. Definition of abbreviations: HRmax = maximal heart rate, SaC<2 = percent oxyhemoglobin saturation. SUBJECT HRmax (beats-min"1) Sa02 - Rest (%) Sa02 - Lowest (%) Power (watts) 1(RR) 178 95.70 78.86 431 2(SF) 183 97.17 74.02 400 3(AP) 177 96.55 76.27 363 4(TD) 186 97.66 77.28 351 5 (NS) 176 96.68 98.07 371 6 (LB) 181 98.14 82.81 374 7(BJ) 178 97.17 73.11 310 TABLE 21 to TABLE 46 Individucal subject data for blood gas and cardiorespiratory measurements during rest and at each min of 5-min cycle ergometry tests under conditons of normoxia (N), normoxia + 20 ppm nitric oxide (N/NO), hypoxia (H), and hypoxia + 20 ppm nitric oxide (H/NO). Definition of abbreviations: Pa02 = arterial pressure of O2, [A-a]D02 = alveolar-arterial difference for O2, Sa02 = oxyhemoglobin saturation, PaC02 = arterial pressure of CO2, HCO3" = bicarbonate, VO2 = oxygen consumption; VE = minute ventilation; RER = respiratory exchange ratio; HR = heart rate. TABLE 21. Individual subject data for SUBJECT 1 (RR) during five-minute exercise under condition H. TIME VARIABLE REST 1 min 2 min 3 min 4 min 5 min pH 7.42 7.39 7.34 7.33 7.31 7.30 Pa02 (mm Hg) 59 51 50 49 48 48 Sa02(%) 90.9 85.4 83.4 82.4 80.7 79.7 [A-a]D02 (mm Hg) 5.1 9.7 15.2 17.6 21.4 20.9 PaC02 (mm Hg) 41 39 36 34 32 33 HCO3"1 (mrnoM"1) 27 24 19 18 16 16 Base Excess (mmolT1) 2.6 -0.7 -5.1 -6.5 -8.4 -8.9 V02 (lmin"1) 0.27 2.99 2.88 2.94 2.95 3.12 V02(mlkg"1-min"1) 3.86 37.24 35.87 36.61 36.74 38.85 VE (1-min"1) 10.0 105.56 127.56 124.0 133.06 139.26 RER (VC02/V02) .91 1.04 1.10 1.08 1.12 1.14 HR (beats-min"1) 89 167 167 171 173 174 Power (watts) 0 375 315 320 315 315 TABLE 22. Individual subject data for SUBJECT 1(RR) during five-minute exercise under condition H/NO. TIME VARIABLE REST 1 min 2 min 3 min 4 min 5 min pH 7.41 7.37 7.32 7.29 7.27 7.25 Pa02(mmHg) 63 46 46 44 43 44 Sa02(%) 97.3 81.1 77.9 74.7 73.1 72.5 [A-a]D02 (mm Hg) 0.4 7.1 14.0 15.3 16.7 16.6 PaC02(mmHg) 43 41 41 39 36 35 HCO3"1 (mmoll"1) 27 24 21 18 16 15 Base Excess (mmolT1) 2.7 -0.9 -4.7 -7.6 -9.5 -11.0 V02 (1-min"1) 0.86 3.84 3.78 4.21 4.03 4.16 V02(ml-kg"'-min"1) 10.71 47.82 47.07 52.43 50.19 51.81 VE (1-min"1) 28.59 100.66 109.19 118.67 112.47 117.87 RER (VC02/V02) 1.12 1.02 1.23 1.12 1.08 1.04 HR (beats-min"1) 70 158 163 164 166 166 Power (watts) 0 375 330 300 300 300 126 TABLE 23. Individual subject data for SUBJECT 2 (SF) during five-minute exercise under condition N. TIME VARIABLE REST 1 min 2 min 3 min 4 min 5 min pH 7.42 — ~ 7.24 ~ — Pa02 (mm Hg) 105 ~ ~ 83 ~ — Sa02 (%) 98.0 — — 94.5 — --[A-a]D02 (mm Hg) 0.6 — ~ 29.6 — — PaC02(mmHg) 37 ~ — 35 ~ ~ HCO3"1 (mmol-1"1) 24 — ~ 15 ~ — Base Excess (mmolT1) 0.3 — ~ -11.5 — ~ V02 (1-min"1) 0.70 3.59 5.18 5.23 5.39 5.50 V02 (ml-kg'-min"1) 8.70 44.60 68.32 68.70 68.70 71.68 VE (1-min"1) 17.48 74.83 155.93 170.62 175.57 180.07 RER (VC02/V02) 0.74 0.71 1.00 0.95 1.01 0.94 HR (beats-min"1) 105 158 173 181 185 190 Power (watts) 0 445 390 420 420 420 TABLE 24. Individual subject data for SUBJECT 2 (SF) during five-minute exercise under condition N/NO. TIME VARIABLE REST 1 min 2 min 3 min 4 min 5 min pH 7.41 7.39 7.32 7.26 7.20 7.17 Pa02(mmHg) 105 78 80 76 73 74 Sa02(%) 98.0 96.0 95.0 94.0 92.0 92.0 [A-a]D02 (mm Hg) 1.9 30.1 28.5 35.8 41.4 43.6 PaC02 (mm Hg) 36 38 39 37 35 31 HCO3"1 (mmol-1"1) 24 23 20 17 15 13 Base Excess (mmol-1"1) 0.3 -1.4 -4.6 -8.7 -10.8 -13.3 V02 (1-min"1) 0.75 3.57 4.78 5.07 5.10 5.12 V02 (ml-kg^-min"1) 8.45 52.67 69.32 74.03 77.71 77.64 VE (1-min"1) 16.85 88.83 142.65 169.63 169.59 177.97 RER (VC02/V02) 0.82 0.91 0.95 0.99 1.01 0.98 HR (beats-min"1) 106 165 178 184 187 190 Power (watts) 0 445 445 450 400 450 127 TABLE 25. Individual subject data for SUBJECT 2 (SF) during five-minute exercise under condition H. TIME VARIABLE REST 1 min 2 min 3 min 4 min 5 min pH 7.44 7.41 7.35 — 7.29 --Pa02 (mm Hg) 69 42 40 ~ 39 --Sa02(%) 94.0 78.0 74.0 — 69.0 — [A-a]D02 (mm Hg) 0.0 20.9 29.7 ~ 31.8 — PaC02(mmHg) 36 33 32 — 31 ~ HCO3"1 (mmol-f1) 24 23 20 ~ 15 — Base Excess (mmolT1) 1.5 -2.7 -6.3 ~ -10.2 ~ V02 (1-min"1) 0.79 3.11 4.23 4.32 4.76 4.92 V02(ml-kg"1-min"1) 9.81 38.63 53.66 55.65 59.13 59.25 VE (1-min"1) 15.6 107.83 141.70 159.65 185.34 181.01 RER (VC02/V02) 0.87 0.89 1.09 1.11 1.10 1.10 HR (beatsmin1) 79 158 177 177 182 182 Power (watts) 0 350 375 400 380 360 TABLE 26. Individual subject data for SUBJECT 2 (SF) during five-minute exercise under condition H/NO. TIME VARIABLE REST 1 min 2 min 3 min 4 min 5 min pH 7.43 7.45 7.40 7.36 7.32 7.26 Pa02 (mm Hg) 73 42 43 42 42 42 Sa02(%) 95.0 80.0 79.0 77.0 74.0 71.0 [A-a]D02(mmHg) 0.2 20.1 21.7 24.5 24.1 25.2 PaC02(mmHg) 38 36 35 32 31 30 HCO3"1 (mmoir1) 25 21 18 ~ 15 — Base Excess (mmolT1) 1.19 2.2 -2.0 -5.5 -8.4 -12.2 V02 (1-min"1) 0.39 4.11 4.52 4.53 4.50 4.22 V02 (ml-kg'-min"1) 4.84 51.06 56.15 56.27 56.02 51.18 VE (1-min"1) 21.15 136.64 166.43 169.14 161.25 158.07 RER (VC02/V02) 0.89 0.96 1.01 0.97 0.92 0.92 HR (beats-min"1) 116 175 179 184 184 186 Power (watts) 0 350 360 300 280 290 TABLE 27. Individual subject data for SUBJECT 3 (AP) during five-minute exercise under condition N. TIME VARIABLE REST 1 min 2 min 3 min 4 min 5 min pH 7.38 7.38 7.32 7.27 7.22 7.17 Pa02 (mm Hg) 91 94 92 88 94 93 Sa02(%) 96.8 97.7 96.4 95.6 95.8 95.2 [A-a]D02 (mm Hg) 5.2 5.7 12.3 17.4 15.8 17.9 PaC02(mmHg) 44 41 40 39 35 34 HCO3"1 (mmol-1"1) 26 24 21 18 14 13 Base Excess (mmolT1) 0.6 -0.1 -4.9 -8.4 -12.7 -15.7 V02 (1-min"1) 0.23 2.9 3.52 3.41 3.34 3.33 V02(ml-kg"1-min'1) 10.60 49.09 47.56 48.95 46.95 46.58 VE (1-min"1) 14.03 114.62 133.71 133.71 133.23 133.67 RER (VC02/V02) 0.83 0.70 0.74 0.78 0.80 0.81 HR (beats-min"1) 91 168 176 179 180 181 Power (watts) 0 410 410 390 390 380 TABLE 28. Individual subject data for SUBJECT 3 (AP) during five-minute exercise condition N/NO. TIME VARIABLE REST 1 min 2 min 3 min 4 min 5 min pH 7.41 7.39 7.32 7.26 7.20 7.17 Pa02 (mmHg) 105 94 93 90 83 84 Sa02(%) 97.29 97.1 96.5 95.8 94.0 93.6 [A-a]D02 (mm Hg) 1.4 3.4 10.2 17.6 25.7 25.8 PaC02 (mm Hg) 40 43 41 37 36 35 HCO3"1 (mmol-1"1) 25 26 21 17 14 13 Base Excess (mmol-1"1) 1.4 1.3 -4.6 -9.5 -12.9 -15.4 V02 (1-min"1) 1.00 4.17 3.88 4.70 4.87 5.19 V02(ml-kg"1-min"1) 13.95 58.16 54.11 65.55 67.92 73.92 VE (1-min"1) 17.34 105.92 133.92 133.54 134.58 133.57 RER (VC02/V02) 0.83 0.84 1.03 0.98 0.92 0.88 HR (beats-min"1) 80 165 174 177 178 179 Power (watts) 0 410 400 380 370 350 TABLE 29. Individual subject data for SUBJECT 3 (AP) during five-minute exercise under condition H. TIME VARIABLE REST 1 min 2 min 3 min 4 min 5 min pH 7.39 7.41 — 7.33 7.28 7.24 Pa02 (mm Hg) 68 53 ~ 48 47 47 Sa02(%) 93.3 87.7 — 81.5 78.6 76.4 [A-a]D02(mm Hg) 1.1 14.1 — 20.3 20.8 20.4 PaC02 (mm Hg) 43 37 — 31 30 29 HCO3"1 (mmoir1) 26 23 — 17 14 12 Base Excess (mmolT1) 1.4 -0.2 ~ -7.8 -10.9 -13.6 V02 (1-min"1) 0.67 3.49 3.60 3.62 3.68 4.00 V02 (ml-kg^-min"1) 9.34 48.68 50.21 50.49 51.32 55.79 VE (1-min"1) 16.63 130.93 132.31 130.59 131.88 131.29 RER (VC02/V02) 1.15 1.21 1.05 1.03 0.97 0.92 HR (beats-min"1) 98 164 170 174 176 178 Power (watts) 0 330 330 310 310 310 TABLE 30. Individual subject data for SUBJECT 3 (AP) during five-minute exercise condition H/NO. TIME VARIABLE REST 1 min 2 min 3 min 4 min 5 min pH 7.40 7.43 7.37 7.31 7.27 7.23 Pa02 (mm Hg) 54 47 46 45 45 45 Sa02(%) 88.1 84.4 80.5 77.7 75.9 73.9 [A-a]D02 (mm Hg) 3.5 17.0 20.1 22.0 22.3 20.4 PaC02(mm Hg) 40 36 33 31 30 30 HCO3"1 (mmoll"1) 25 24 19 16 14 13 Base Excess (mmolT1) 0.9 0.5 -5.1 -8.8 -11.5 -13.6 V02 (1-min"1) 0.46 4.04 4.21 4.25 4.29 4.42 V02 (ml-kg^-min"1) 6.42 56.35 58.72 59.27 59.83 61.65 VE (1-min"1) 12.50 125.50 141.15 141.17 146.07 150.83 RER (VC02/V02) 0.97 1.05 1.02 0.98 0.92 0.89 HR (beats-min"1) 102 164 171 173 176 177 Power (watts) 0 330 330 300 280 280 TABLE 31. Individual subject data for SUBJECT 4 (TD) during five-minute exercise under condition N. TIME VARIABLE REST 1 min 2 min 3 min 4 min 5 min pH 7.40 7.39 7.33 7.25 7.32 7.19 Pa02(mmHg) 109 95 85 81 81 77 Sa02(%) 98.0 97.2 95 94.4 93.9 92.6 [A-a]D02 (mm Hg) 0.3 18.5 30.3 33.3 33.6 37.8 PaC02 (mm Hg) 39 35 34 34 32 31 HCO3"1 (mmol-r1) 24 21 18 15 13 12 Base Excess (mmol-r1) 0.0 -2.9 -7.0 -11.1 -13.4 -15 V02 (1-min"1) 0.64 4.41 4.55 4.79 4.79 4.83 V02 (ml-kg'-min"1) 9.98 68.80 70.98 74.73 74.73 75.35 VE (1-min"1) 23.74 153.90 154.36 155.28 155.09 155.97 RER (VC02/V02) 0.91 1.01 1.04 1.00 0.94 0.91 HR (beats-min"1) 99 174 178 178 178 177 Power (watts) 0 380 330 320 290 285 TABLE 32. Individual subject data for SUBJECT 4 (TD) during five-minute exercise condition N/NO. TIME VARIABLE REST 1 min 2 min 3 min 4 min 5 min pH 7.41 7.37 7.28 7.25 7.23 7.21 Pa02 (mmHg) 95 85 77 77 75 74 Sa02(%) 97.4 96.3 94 93.5 92.7 91.9 [A-a]D02(mmHg) 13.7 28.0 34.2 34.3 36.5 38.3 PaC02(mm Hg) 36 32 34 33 31 30 HCO3"1 (mmol-r1) 23 19 16 15 13 12 Base Excess (mmolT1) -0.7 -4.8 -9.5 -11.1 -13.5 -14.7 V02 (1-min"1) 0.8 4.46 4.84 4.97 5.24 5.20 V02(ml-kg"1-min'1) 12.35 68.83 74.68 76.70 80.86 80.25 VE (1-min"1) 19.0 152.27 157.66 156.63 156.86 156.72 RER (VC02/V02) 0.90 0.90 0.91 0.88 0.82 0.81 HR (beats-min"1) 100 164 175 176 177 177 Power (watts) 0 360 300 280 280 280 TABLE 33. Individual subject data for SUBJECT 4 (TD) during five-minute exercise under condition H. TIME VARIABLE REST 1 min 2 min 3 min 4 min 5 min pH 7.44 7.41 7.31 7.31 7.27 7.25 Pa02 (mm Hg) 64 45 42 42 42 42 Sa02(%) 93.5 81.7 74.2 74.2 71.5 71.1 [A-a]D02 (mm Hg) 1.4 23.9 26.6 26.6 27.1 26.7 PaC02(mm Hg) 32 31 31 31 29 28 HCO3"1 (mmol-r1) 22 20 15 15 14 13 Base Excess (mmol-1"1) -0.7 -3.1 -9.4 -9.4 -11.8 -13.0 V02 (1-min"1) 0.31 3.57 3.60 3.60 3.59 3.71 V02(ml-kg"1-min"1) 4.78 55.09 55.56 55.56 55.40 57.25 VE (1-min"1) 15.73 136.38 133.43 133.43 135.04 139.13 RER (VC02/V02) 0.95 1.04 1.03 1.03 0.97 0.92 HR (beats-min"1) 102 171 175 175 175 175 Power (watts) 0 289 230 230 210 200 TABLE 34. Individual subject data for SUBJECT 4 (TD) during five-minute exercise condition H/NO. TIME VARIABLE REST 1 min 2 min 3 min 4 min 5 min pH 7.42 7.40 7.35 7.30 7.26 7.24 Pa02 (mm Hg) 62 46 43 42 42 42 Sa02(%) 92.2 82.8 76.8 73.9 71.4 69.5 [A-a]D02(mmHg) 0.1 23.1 25.4 25.3 25.7 25.9 PaC02(mmHg) 36 31 31 31 29 28 HCO3"1 (mmol-1"1) 23 19 17 15 13 12 Base Excess (mmol-1"1) -0.2 -4.1 -6.8 -9.7 -12.5 -14.0 V02 (1-min"1) 0.31 3.59 3.82 3.90 3.80 3.90 V02 (ml-kg'-min"1) 4.78 55.40 58.95 60.19 58.64 60.19 VE (1-min"1) 16.68 140.83 143.95 144.13 143.84 142.34 RER (VC02/V02) 0.97 1.05 1.02 0.98 0.92 0.89 HR (beats-min"1) 98 171 175 175 177 179 Power (watts) 0 310 239 230 210 200 TABLE 35. Individual subject data for SUBJECT 5 (NS) during five-minute exercise under condition N. TIME VARIABLE REST 1 min 2 min 3 min 4 min 5 min pH 7.40 7.35 7.28 7.22 7.18 7.17 Pa02 (mm Hg) 106 85 80 74 73 72 Sa02(%) 99.0 97.2 95.2 93.8 92.4 91.1 [A-a]D02 (mm Hg) 6.8 25.2 28.3 27.0 31.9 34.0 PaC02 (mm Hg) 35 40 41 41 40 38 HCO3"1 (mmol-1"1) 21 21 19 16 15 13 Base Excess (mmol-1"1) -31 -4.1 -7.3 -10.9 -13.2 -14.4 V02 (lmin"1) 0.27 4.08 4.34 4.49 4.59 4.67 V02 (ml-kg^-min"1) 3.39 51.26 54.52 56.53 57.66 58.67 VE (1-min"1) 10.39 132.87 146.56 154.39 155.85 166.12 RER (VC02/V02) 1.07 1.12 1.21 1.24 1.24 1.21 HR (beats-min'1) 104 172 175 177 178 182 Power (watts) 0 400 380 350 350 350 TABLE 36. Individual subject data for SUBJECT 5 (NS) during five-minute exercise condition N/NO. TIME VARIABLE REST 1 min 2 min 3 min 4 min 5 min pH 7.41 7.39 7.31 7.26 7.22 7.19 Pa02 (mmHg) 96 74 72 68 67 63 Sa02(%) 98.3 95.8 94.3 92.5 91.0 89.7 [A-a]D02 (mm Hg) 21.4 42.6 42.2 46.8 48.6 55.5 PaC02(mmHg) 33 35 40 40 39 35 HCO3"1 (mmoll'1) 21 21 20 18 16 13 Base Excess (mmol-1"1) -3.2 -3.5 -6.0 -8.7 -11.6 -14.2 V02 (1-min"1) 0.69 4.79 4.90 5.09 5.02 5.00 V02 (ml-kg'-min"1) 8.67 60.18 61.56 63.94 63.07 62.81 VE (1-min"1) 14.81 114.38 142.20 156.18 156.77 158.73 RER (VC02/V02) 0.81 0.89 1.03 1.09 1.11 1.10 HR (beats-min"1) 106 171 178 181 185 186 Power (watts) 0 385 369 350 340 330 TABLE 37. Individual subject data for SUBJECT 5 (NS) during five-minute exercise under condition H. TIME VARIABLE REST 1 min 2 min 3 min 4 min 5 min pH 7.42 7.39 7.33 7.29 7.22 7.17 Pa02 (mm Hg) 62 42 40 37 40 42 Sa02(%) 94.2 80.7 75.8 71.4 69.8 69.2 [A-a]D02 (mm Hg) 0.3 18.4 25.0 33.3 32.6 30.6 PaC02(mmHg) 37 35 35 31 29 29 HCO3"1 (mrnoH"1) 24 20 18 14 12 11 Base Excess (mmolT1) -0.1 -3.7 -6.8 -11.0 -14.2 -16.4 V02 (1-min"1) 0.54 3.70 4.02 3.96 4.04 4.12 V02(ml-kg"1-min"1) 6.78 46.48 50.50 49.75 50.75 51.76 VE (1-min"1) 20.05 135.87 149.18 152.89 164.10 165.15 RER (VC02/V02) 1.04 1.08 1.15 1.14 1.15 1.14 HR (beats-min"1) 106 169 172 174 177 177 Power (watts) 0 320 297 285 285 285 TABLE 38. Individual subject data for SUBJECT 5 (NS) during five-minute exercise condition H/NO. TIME VARIABLE REST 1 min 2 min 3 min 4 min 5 min pH 7.41 7.40 7.34 7.29 7.24 7.19 Pa02(mmHg) 67 42 38 37 38 39 Sa02(%) 95.5 81.4 75.1 70.9 63.2 66.9 [A-a]D02(mmHg) 0.6 25.3 30.0 31.7 32.7 32.4 PaC02(mmHg) 38 34 35 34 32 31 HCO3"1 (mmol-1"1) 23 21 18 16 14 11 Base Excess (mmolT1) -0.9 -3.3 -6.6 -9.5 -12.5 -15.4 V02 (1-min"1) 0.62 3.61 3.65 4.10 3.98 4.05 V02 (ml-kg'-min"1) 7.79 45.35 45.85 51.51 50.00 50.88 VE (1-min"1) 38.57 136.70 132.02 147.25 149.22 160.32 RER (VC02/V02) 1.07 1.09 1.14 1.12 1.09 1.09 HR (beats-min"1) 106 169 173 175 177 180 Power (watts) 0 320 295 295 275 275 TABLE 39. Individual subject data for SUBJECT 6 (LB) during five-minute exercise under condition N. TIME VARIABLE REST 1 min 2 min 3 min 4 min 5 min pH 7.41 7.42 7.38 7.35 7.33 7.33 Pa02(mmHg) 98 95 95 94 93 92 Sa02(%) 98.0 98.0 97.2 97.2 96.8 97.0 [A-a]D02(mmHg) ~ — 22.1 24.5 28.7 35.8 PaC02(mmHg) 40 37 35 34 31 23 HCO3"1 (mmol-r1) 25 23 20 18 16 12 Base Excess (mmolT1) 0.4 -0.8 -3.9 -6.3 -8.3 -12.0 V02 (1-min1) — — 4.45 4.55 4.50 4.57 V02 (ml-kg^-min"1) ~ ~ 58.14 56.38 55.76 56.63 VE (1-min"1) — ~ 155.39 161.23 156.98 161.21 RER (VC02/V02) — ~ 1.16 1.19 1.22 1.16 HR (beats-min"1) 71 167 171 174 195 178 Power (watts) 0 370 320 340 310 310 TABLE 40. Individual subject data for SUBJECT 6 (LB) during five-minute exercise condition N/NO. TIME VARIABLE REST 1 min 2 min 3 min 4 min 5 min pH 7.39 7.42 ~ — 7.38 7.38 Pa02(mmHg) 98 97 — ~ 95 93 Sa02(%) 97.6 98.0 ~ — 98.2 98.5 [A-a]D02 (mm Hg) -- ~ —— ~ 17.5 20.4 PaC02 (mm Hg) 45 40 — — 36 36 HCO3"1 (mmol-r1) 27 26 — — 21 20 Base Excess (mmolT1) 1.2 1.1 — — -3.3 -4.0 V02 (1-min"1) ~ ~ ~ 4.82 5.24 5.48 V02(ml-kg"1-min"1) ~ — ~ 59.73 64.93 67.91 VE (1-min"1) 7.29 141.80 162.31 162.95 172.39 175.95 RER (VC02/V02) — ~ ~ 1.03 1.01 1.04 HR (beats-min"1) 75 163 166 168 172 178 Power (watts) 0 350 310 310 310 310 TABLE 41. Individual subject data for SUBJECT 6 (LB) during five-minute exercise under condition H. TIME VARIABLE REST 1 min 2 min 3 min 4 min 5 min pH 7.40 7.45 7.45 7.41 7.39 7.38 Pa02 (mm Hg) 95 93 70 53 52 49 Sa02(%) 94.8 90.8 88.7 88.0 86.2 84.2 [A-a]D02 (mm Hg) 0.7 0.2 0.1 15.4 16.4 18.8 PaC02 (mm Hg) 40 37 36 32 31 31 HCO3"1 (mmol-r1) 24 25 22 19 18 17 Base Excess (mmolT1) -0.4 -1.3 -1.6 -5.0 -5.8 -7.1 V02 (1-min"1) 0.34 3.71 3.85 4.54 4.59 4.65 V02 (ml-kg^-min"1) 4.21 45.97 52.28 56.26 56.88 57.62 VE (1-min"1) 8.05 150.53 150.53 170.46 181.63 178.62 RER (VC02/V02) 0.82 0.94 1.15 1.06 1.02 1.00 HR (beats-min"1) 71 167 167 171 174 176 Power (watts) 0 340 290 280 280 280 TABLE 42. Individual subject data for SUBJECT 6 (LB) during five-minute exercise condition H/NO. TIME VARIABLE REST 1 min 2 min 3 min 4 min 5 min pH 7.36 7.41 7.41 7.38 7.34 7.33 Pa02 (mm Hg) 71 50 47 48 44 44 Sa02(%) 94.8 86.6 83.1 83.3 79.0 78.2 [A-a]D02 (mm Hg) 0.2 21.9 27.1 26.7 31.8 31.0 PaC02(mmHg) 25 26 25 25 23 23 HCO3"1 (mmoir1) 15 16 15 15 12 12 Base Excess (mmolT1) -7.2 -6.3 -8.3 -10.0 -11.3 -12.1 V02 (1-min"1) 0.40 4.27 4.78 4.88 4.97 4.92 V02 (ml-kg'-min"1) 4.96 52.91 59.23 60.47 61.59 60.97 VE (1-min"1) 15.73 156.78 187.71 182.10 178.07 180.97 RER (VC02/V02) 0.81 0.96 1.01 1.04 1.00 0.96 HR (beats-min"1) 69 161 167 169 170 174 Power (watts) 0 340 330 280 263 263 TABLE 43. Individual subject data for SUBJECT 7 (BJ) during five-minute exercise under condition N. TIME VARIABLE REST 1 min 2 min 3 min 4 min 5 min pH 7.39 7.34 7.29 7.22 7.20 7.17 Pa02 (mm Hg) 114 97 95 90 87 87 Sa02(%) 98.1 97.0 96.0 95.4 94.7 94.2 [A-a]D02 (mm Hg) 0.9 16.7 22.4 29.3 34.2 34.8 PaC02 (mm Hg) 38 38 36 34 31 30 HCO3"1 (mmol-1"1) 23 21 18 14 12 11 Base Excess (mmol-1"1) -0.9 -4.3 -8.1 -12.7 -14.9 -16.7 V02 (1-min"1) 0.38 3.53 3.78 3.73 3.58 3.59 V02 (ml-kg'-min"1) 5.54 51.46 55.10 55.37 52.19 52.33 VE (1-min"1) 14.69 125.12 154.54 166.37 169.34 163.04 RER (VC02/V02) 0.95 1.11 1.20 1.21 1.17 1.15 HR (beats-min"1) 65 163 172 175 175 174 Power (watts) 0 370 370 340 310 310 TABLE 44. Individual subject data for SUBJECT 7 (BJ) during five-minute exercise under condition N/NO. TIME VARIABLE REST 1 min 2 min 3 min 4 min 5 min pH 7.39 7.39 7.34 7.31 7.28 7.26 Pa02 (mmHg) 93 86 86 83 79 80 Sa02(%) 97.1 96.6 96.1 95.5 94.4 94.4 [A-a]D02 (mm Hg) 18.1 27.4 33.1 38.4 42.6 42.9 PaC02(mmHg) 33 31 31 30 29 27 HCO3"1 (mmoll"1) 20 19 17 15 13 12 Base Excess (mmol-1"1) -3.2 -4.4 -7.4 -9.6 -11.6 -13.1 V02 (1-min"1) 0.29 2.64 3.04 3.47 3.45 3.72 V02 (ml-kg'-min"1) 10.64 38.05 44.17 48.25 50.00 54.23 VE (1-min"1) 9.12 113.11 138.59 152.85 157.53 158.29 RER (VC02/V02) 0.85 0.85 1.06 1.13 1.09 1.06 HR (beats-min"1) 89 162 167 173 175 178 Power (watts) 0 350 310 310 310 310 TABLE 45. Individual subject data for SUBJECT 7 (BJ) during five-minute exercise under condition H. TIME VARIABLE REST 1 min 2 min 3 min 4 min 5 min pH 7.40 7.43 7.36 7.32 7.28 7.24 Pa02 (mm Hg) 75 46 41 40 39 40 Sa02(%) 95.2 83.6 75.6 71.7 68.5 67.3 [A-a]D02 (mm Hg) 5.2 23.7 29.1 29.9 34.2 36.2 PaC02(mmHg) 34 32 32 31 29 26 HCO3"1 (mmol-r1) 21 21 21 18 16 13 Base Excess (mmolT1) -2.1 -1.9 -5.6 -8.5 -11.6 -14.6 V02 (1-min"1) 0.73 2.61 3.03 3.31 3.43 3.72 V02 (ml-kg^-min"1) 4.21 45.97 38.05 50.58 50.29 54.23 VE (1-min"1) 12.52 119.26 134.60 159.69 161.08 168.10 RER (VC02/V02) 0.85 1.11 1.13 1.12 1.15 1.17 HR (beats-min"1) 71 167 167 171 174 176 Power (watts) 0 340 260 250 240 240 TABLE 46. Individual subject data for SUBJECT 7 (BJ) during five-minute exercise under condition H/NO. TIME VARIABLE REST 1 min 2 min 3 min 4 min 5 min pH 7.43 7.43 7.38 7.32 7.27 7.24 Pa02 (mm Hg) 60 41 40 40 40 40 Sa02(%) 91.9 78.5 74.5 71.6 68.7 66.6 [A-a]D02(mm Hg) 4.7 28.7 31.9 31.8 35.0 35.6 PaC02(mmHg) 31 30 30 29 27 26 HCO3"1 (mmol-r1) 20 20 17 15 12 11 Base Excess (mmolT1) -2.0 -2.8 -5.8 -9.1 -12.8 -14.6 V02 (1-min"1) 0.29 2.60 3.12 3.47 3.33 3.50 V02 (ml-kg^-min"1) 4.23 37.90 45.48 50.58 48.54 51.02 VE (1-min"1) 15.19 142.37 168.01 178.50 184.66 178.98 RER (VC02/V02) 0.89 1.03 1.13 1.08 1.15 1.13 HR (beats-min"1) 69 161 167 169 170 174 Power (watts) 0 280 265 245 235 230 V 138 APPENDIX F GROUP DATA: INHALED NITRIC OXIDE TABLE 47. Oxygen consumption (VO2,1-min"1) during rest and at each min of 5-min cycle ergometry tests under conditions of normoxia (N), normoxia + 20 ppm nitric oxide (N/NO), hypoxia (H), and hypoxia + 20 ppm nitric oxide (H/NO). Values are means (± SD). REST 1 MIN 2 MIN 3 MIN 4 MIN 5 MIN N 0.44 (0.21) 3.70 (0.58) 4.30 (0.59) 4.37 (0.68) 4.37 (0.77) 4.42 (0.81) N/NO 0.70 (0.26) 3.93 (0.85) 4.29 (0.81) 4.69 (0.61) 4.82 (0.69) 4.95 (0.62) H 0.52 (0.22) 3.31 (0.42) 3.60 (0.50) 3.76 (0.56) 3.86 (0.64) 4.03 (0.61) H/NO 0.48 (0.20) 3.72 (0.56) 3.98 (0.56) 4.19 (0.45) 4.13 (0.52) 4.17 (0.44) TABLE 48. Oxygen consumption (VO2, ml-kg^-min"1) during rest and at each min of 5-min cycle ergometry tests under conditions of normoxia (N), normoxia + 20 ppm nitric oxide (N/NO), hypoxia (H), and hypoxia + 20 ppm nitric oxide (H/NO). Values are means (± SD). REST 1 MIN 2 MIN 3 MIN 4 MIN 5 MIN N 7.64 (3.08) 53.04 (9.23) 58.67 (9.14) 60.05 (9.54) 59.33 (10.44) 60.21 (11.17) N/NO 10.81 (2.37) 55.58 (11.39) 60.77 (12.12) 64.70 (10.27) 67.42 (11.09) 69.37 (9.77) H 6.53 (2.55) 45.48 (6.42) 50.13 (6.21) 53.05 (3.06) 53.96 (3.69) 55.98 (2.68) H/NO 5.50 (1.34 49.83 (7.03) 54.06 (6.60) 56.38 (4.40) 55.77 (5.37) 55.93 (5.51) 139 TABLE 49. Minute ventilation (VE, lmin"1) during rest and at each min of 5-min cycle ergometry tests under conditions of normoxia (N), normoxia + 20 ppm nitric oxide (N/NO), hypoxia (H), and hypoxia + 20 ppm nitric oxide (H/NO). Values are means (± SD). REST 1 MIN 2 MIN 3 MIN 4 MIN 5 MIN N 16.07 (4.98) 120.27 (29.20) 150.08 (8.73) 156.93 (12.98) 157.68 (14.59) 160.01 (15.23) N/NO 14.07 (4.77) 119.39 (23.51) 146.22 (11.21) 155.30 (12.21) 157.86 (13.37) 160.21 (16.07) H 14.10 (4.15) 126.62 (16.44) 138.62 (8.74) 147.24 (17.74) 156.02 (22.94) (157.51) 20.52 H/NO 21.22 (9.29) 134.21 (17.48) 149.78 (26.20) 153.71 (23.33) 153.65 (24.10) 155.63 (21.76) TABLE 50. Respiratory exchange ratio (RER, VCO2/VO2) during rest and at each min of 5-min cycle ergometry tests under conditions of normoxia (N), normoxia + 20 ppm nitric oxide (N/NO), hypoxia (H), and hypoxia + 20 ppm nitric oxide (H/NO). Values are means (± SD). REST 1 MIN 2 MIN 3 MIN 4 MIN 5 MIN N 0.87 (0.16) 0.93 (0.21) 1.06 (0.18) 1.06 (0.18) 1.06 (0.18) 1.03 (0.16) N/NO 0.84 (0.04) 0.88 (0.03) 1.00 (0.06) 1.02 (0.09) 0.99 (0.11) 0.98 (0.11) H 0.94 (0.12) 1.04 (0.11) 1.12 (0.04) 1.09 (0.04) 1.08 (0.07) 1.06 (0.08) H/NO 0.95 (0.11) 1.01 (0.06) 1.07 (0.09) 1.05 (0.08) 1.02 (0.09) 0.99 (0.09) 140 TABLE 51. Heart rate (beats-min"1) during rest and at each min of 5-min cycle ergometry tests under conditions of normoxia (N), normoxia + 20 ppm nitric oxide (N/NO), hypoxia (H), and hypoxia + 20 ppm nitric oxide (H/NO). Values are means (± SD). REST 1 MIN 2 MIN 3 MIN 4 MIN 5 MIN N 89 167 174 177 179 180 (17) (6) (3) (3) (4) (6) N/NO 93 165 173 177 179 181 (13) (3) (5) (6) (6) (5) H 89 166 171 174 176 177 (14) (4) (4) (2) (3) (3) H/NO 92 166 171 173 175 177 (18) (6) (5) (6) (6) (6) TABLE 52. Arterial pressure of O2 (Pa02, mmHg) during rest and at each min of 5-min cycle ergometry tests under conditions of normoxia (N), normoxia + 20 ppm nitric oxide (N/NO), hypoxia (H), and hypoxia + 20 ppm nitric oxide (H/NO). Values are means (± SD). REST 1 MIN 2 MIN 3 MIN 4 MIN 5 MIN N 103.8 (8.2) 93.2 (4.7) 89.4 (6.7) 85.0 (7.2) 85.6 (8.8) 84.2 (9.3) N/NO 98.7 (5.2) 85.7 (8.9) 81.6 (8.1) 78.8 (8.2) 78.7 (9.7) 78.0 (10.2) H 66.7 (5.4) 47.4 (4.9) 44.3 (5.3) 44.2 (5.2) 43.3 (4.2) 44.3 (3.4) H/NO 64.3 (6.6) 44.9 (3.3) 43.3 (3-4) 42.6 (3-6) 42.0 (2.4) 42.3 (2.2) 141 TABLE 53. Alveolar-arterial difference for O2 ([A-a]D02, mmHg) during rest and at each min of 5-min cycle ergometry tests under conditions of normoxia (N), normoxia + 20 ppm nitric oxide (N/NO), hypoxia (H), and hypoxia + 20 ppm nitric oxide (H/NO). Values are means (± SD). REST 1 MIN 2 MIN 3 MIN 4 MIN 5 MIN N 2.8 (3.0) 16.5 (8.1) 23.1 (7.0) 26.9 (5.5) 28.9 (7.6) 32.0 (8.1) N/NO 11.3 (9.2) 26.3 (14.2) 29.6 (11.9) 34.6 (10.7) 35.4 (11.6) 37.7 (12.8) H 2.0 (2.2) 15.8 (8.6) 21.0 (11.5) 23.9 (7.2) 26.3 (6.9) 25.6 (6.8) H/NO 1.4 (1.9) 20.5 (7.0) 24.3 (6.2) 25.3 (5.7) 26.9 (6.6) 26.7 (6.8) TABLE 54. Oxyhemoglobin saturation (Sa02, %) during rest and at each min of 5-min cycle ergometry tests under conditions of normoxia (N), normoxia + 20 ppm nitric oxide (N/NO), hypoxia (H), and hypoxia + 20 ppm nitric oxide (H/NO). Values are means (± SD). REST 1 MIN 2 MIN 3 MIN 4 MIN 5 MIN N 98.0 (0.7) 97.4 (0.4) 96.3 (0.4) 95.2 (0.7) 94.7 (1.3) 94.0 (1.8) N/NO 97.7 (0.3) 96.6 (0.6) 95.2 (1.3) 94.3 (1.2) 93.7 (1.0) 93.4 (1.0) H 93.7 (1.4) 84.0 (4.3) 79.0 (4.9) 78.2 (4.5) 74.9 (5.6) 74.7 (4.3) H/NO 93.5 (4.0) 82.1 (1.9) 78.1 (1.6) 75.6 (1.8) 72.2 (1.9) 71.2 (1.9) 142 TABLE 55. Arterial pressure of CO2 (PaCCh, mmHg) during rest and at each min of 5-min cycle ergometry tests under conditions of normoxia (N), normoxia + 20 ppm nitric oxide (N/NO), hypoxia (H), and hypoxia + 20 ppm nitric oxide (H/NO). Values are means (± SD). REST 1 MIN 2 MIN 3 MIN 4 MIN 5 MIN N 38.8 (3.1) 38.2 (2.4) 37.2 (3.1) 36.2 (3.1) 33.8 (3.8) 31.2 (5.5) N/NO 37.2 (4.6) 36.5 (4.7) 37.0 (4.3) 35.4 (3.9) 34.3 (3.7) 32.3 (3.6) H 37.6 (4.0) 34.9 (3.0) 33.7 (2.3) 31.7 (1.2) 30.1 (1.2) 29.3 (2.4) H/NO 35.9 (6.0) 33.4 (4.9) 32.9 (5.0) 31.6 (4.3) 29.7 (4.1) 29.0 (3.8) TABLE 56. Values for pH during rest and at each min of 5-min cycle ergometry tests under conditions of normoxia (N), normoxia + 20 ppm nitric oxide (N/NO), hypoxia (H), and hypoxia + 20 ppm nitric oxide (H/NO). Values are means (± SD). REST 1 MIN 2 MIN 3 MIN 4 MIN 5 MIN N 7.40 (0.01) 7.38 (0.03) 7.32 (0.04) 7.26 (0.05) 7.23 (0.06) 7.21 (0.07) N/NO 7.41 (0.01) 7.39 (0.02) 7.32 (0.02) 7.27 (0.02) 7.26 (0.06) 7.24 (0.08) H 7.42 (0.02) 7.41 (0.02) 7.36 (0.04) 7.33 (0.04) 7.29 (0.05) 7.26 (0.07) H/NO 7.41 (0.02) 7.41 (0.03) 7.37 (0.03) 7.32 (0.04) 7.28 (0.04) 7.25 (0.04) 143 TABLE 57. (HCO3", mmolT1) during rest and at each min of 5-min cycle ergometry tests under conditions of normoxia (N), normoxia + 20 ppm nitric oxide (N/NO), hypoxia (H), and hypoxia + 20 ppm nitric oxide (H/NO). Values are means (± SD). REST 1 MIN 2 MIN 3 MIN 4 MIN 5 MIN N 23.8 (1.7) 22.0 (1.4) 19.2 (1.3) 16.0 (1.7) 14.0 (1.6) 12.2 (0.8) N/NO 23.3 (2.6) 22.3 (3.2) 18.8 (2.2) 16.4 (1.3) 15.3 (3.0) 13.8 (3.1) H 24.1 (2.1) 22.0 (2.0) 19.2 (1.9) 16.8 (1.9) 15.0 (1.9) 13.7 (2.3) H/NO 22.6 (4.0) 21.3 (3.3) 18.4 (2.4) 15.9 (1.8) 13.9 (1.7) 12.4 (1.4) TABLE 58. Base excess (mmolT1) during rest and at each min of 5-min cycle ergometry tests under conditions of normoxia (N), normoxia + 20 ppm nitric oxide (N/NO), hypoxia (H), and hypoxia + 20 ppm nitric oxide (H/NO). Values are means (± SD). REST 1 MIN 2 MIN 3 MIN 4 MIN 5 MIN N -0.5 (1.4) -2.4 (1.9) -6.2 (1.8) -10.2 (2.4) -12.5 (2.5) -14.8 (1.8) N/NO -0.7 (2.1) -2.0 (2.7) -6.4 (2.1) -9.5 (1.0) -10.6 (3.7) -12.5 (4.2) H 0.3 (1.6) -1.9 (1.3) -5.4 (2.0) -8.0 (2.1) -10.4 (2.7) -12.2 (3.5) H/NO -0.7 (3.3) -1.4 (2.2) -5.6 (2.0) -8.6 (1.6) -11.2 (1.7) -13.3 (1.6) 

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