Redox regulation of vascular NO bioavailability during ...
Redox regulation of vascular NO bioavailability during hypoxia:
Implications for oxygen transport and exercise performance
A thesis submitted for the degree of Doctor of Philosophy
awarded by the University of Glamorgan
by
John Woodside
Faculty of Health, Sport and Science
University of Glamorgan
September 2010
Contents: ii
Acknowledgements: v
Abstract: vi
List of tables: viii
List of figures: ix
Definition of terms: xii
Conferences and publications xv
1. CHAPTER 1: Thesis overview
1. Background 2
2. Strategy 4
3. Aims and objectives 7
2. CHAPTER 2: Literature review
2.1 The oxygen transport cascade 9
2.2 Cerebral circulation 12
2.3 Aerobic exercise performance in acute hypoxia 15
2.4 Origins of fatigue 17
2.5 Near-infrared spectroscopy (NIRS) 20
2.6 Skeletal muscle oxygenation and exercise 22
2.7 Cerebral oxygenation and exercise 25
2.8 Technical considerations and limitations of NIRS 27
2.9 Nitric oxide (NO) 29
2.10 Fate of vascular NO 31
2.11 S-Nitrosothiols (RSNO) 31
2.12 Nitrate (NO3•) 33
2.13 Nitrite (NO2•) 34
2.14 Reapportionment of NO metabolites 36
2.15 Oxidative inactivation, vascular dysfunction and nitrotyrosine 37
2.16 NO and exercise 38
2.17 Cell adhesion molecules (CAM) 40
2.18 Intermittent hypoxia (IH) 42
2.19 Adaptive mechanisms 1: Genetic modulation 43
2.20 Adaptive mechanisms 2: ROS and NO regulation 46
2.21 IH and endothelial activation 48
2.22 IH and exercise performance 52
2.23 Summary 56
2.24 Specific aims and working hypotheses 57
3. Chapter 3: General Methodology
1. Incremental cycling protocol 60
2. Respiratory measurements 60
3. Heart rate (HR) 61
4. Arterial oxygen saturation (SaO2) 61
5. Near Infrared Spectroscopy (NIRS) 61
6. Blood sampling 62
7. Ozone-based chemiluminescence: 63
Detection of plasma NO metabolites
8. sICAM-1, sVCAM-1 and 3-NT 65
9. Ascorbate free radical (A•) 66
10. Haemoglobin (Hb) 66
11. Haematocrit (Hcrt) 67
12. Blood gas 67
13. IH machine 68
4. Study 1: The impact of acute hypoxia on cerebral and muscle oxygenation and systemic molecular biomarkers of vascular function during exercise
1. Introduction 70
2. Research aims and objectives 72
3. Working hypothesis 72
4. Methodology 73
5. Statistical analysis 76
6. Results 76
7. Discussion 87
8. Conclusions 100
5. Study 2:The effect of intermittent hypoxia on cerebral and muscle oxygenation and molecular biomarkers of vascular function
during exercise in hypoxia
1. Introduction 103
2. Research aims and objectives 104
3. Working hypothesis 104
4. Methodology 105
5. Statistical analysis 108
6. Results 109
7. Discussion 120
8. Conclusions 130
6. General discussion and conclusions
1. Overview 132
2. Study 1: The impact of acute hypoxia on cerebral and muscle 133
oxygenation and systemic molecular biomarkers of vascular
function during exercise
3. Study 2: Intermittent hypoxia: implications for cerebral and 136
muscle oxygenation and molecular biomarkers of vascular function
4. Future research 138
References 140
Acknowledgements
My sincere thanks go to the following people:
To the subjects for their time and energy during each study,
To Dr. Phillip James and Dr. Jane McEneny for their help and expertise in running the biochemical analysis,
To my friends, family and girlfriend Jo for their support,
…. and of course to Professor Damian Bailey for his expertise and supervision in putting this thesis together. I now look forward to pursuing my own research career with the knowledge of having Damian as my mentor and collaborator.
Abstract
The reduction in [pic]O2peak at altitude is well documented. Maximal exercise in hypoxia is accelerated through a reduction in O2 supply with contributions from central and peripheral origins of fatigue. Changes in cerebral and muscle oxygenation have not been well characterised during incremental exercise in hypoxia. It is possible attainment of [pic]O2peak is driven by the oxygenation profile of these tissues whilst changes in molecular biomarkers of endothelial function could provide some insight into the mechanisms driving systemic and regional O2 delivery and vascular hypoxic sensing capabilities.
The first study of this thesis examined the impact of acute hypoxia (FIO2 = 0.12) on the cerebral and muscle oxygenation response to incremental cycling exercise using NIRS (n = 14; age: 23 ± 5yr; height: 1.80 ± 0.07m; weight: 84 ± 8kg). The profiles were characterised at equivalent relative and absolute exercise intensities and molecular blood-borne markers of O2 sensing and function were measured before and immediately after maximal exercise for changes in oxidative stress (A• and 3-NT), NO metabolites (NOx, NO3•, NO2• and RSNO) and cell adhesion molecules (sICAM-1 and sVCAM-1). The key observations from this study were: 1) [pic]O2peak decreased by 22% and the magnitude of cerebral and muscle deoxygenation (↓O2Hb and ↑HHb) was greater in hypoxia, 2) the slope for the relative HHb response was similar between conditions whereas there was an accelerated slope across the absolute workloads in hypoxia implying cycling performance was driven by a premature attainment of maximal O2 extraction capacity of the muscle, 3) there was no evidence suggesting cerebral O2 metabolism was impaired in hypoxia however since SaO2 was 78 ± 4% at PPO it is possible the reduction in systemic O2 delivery could have influenced central fatigue, 4) there was a tendency for a rightward shift in the cerebral THb profile in hypoxia and although muscle THb peaked at 80% PPO in both trials, the response also tended to be lower in hypoxia, 5) there was no change in oxidative stress markers and NOx after exercise, 6) RSNO increased and NO2• decreased after maximal exercise. The decline in NO2• was attenuated in hypoxia possibly due to a blunted NO2•-HHb-NO pathway and may explain the systemic hypoperfusion response, 7) The increase in sICAM-1 and sVCAM-1 after exercise was augmented in normoxia, 8) Only when normoxia and hypoxia data was pooled was there a correlation between sVCAM-1 pre-post exercise and [pic]O2peak.
Intermittent hypoxia (IH) may be used to improve the efficiency of exercise training and as a pre-acclimatisation strategy prior to high altitude ascent. The purpose of the second study was to evaluate the efficacy of a 10 day IH regime consisting of 9x 5 min daily exposures of 9.5% O2 breathing followed by equal periods of normoxia on submaximal and maximal cardiorespiratory responses to exercise in hypoxia. Additionally, cerebral and muscle oxygenation was monitored throughout incremental cycling to exhaustion and changes in NO metabolites (NO3•, NO2• and RSNO) and CAMs (sICAM-1 and sVCAM-1) were measured before and immediately after maximal exercise. The key observations from this study were: 1) a tendency for IH to reduce submaximal [pic]O2 and increase [pic]O2peak in hypoxia, 2) IH increased the muscle THb response to exercise due an increased intercept for both the muscle O2Hb and HHb in the absence of any change in slope, 4) cerebral oxygenation increased (↑O2Hb) at rest and during exercise, 4) the reduction in nitrite was attenuated in the IH group whilst resting sICAM-1 decreased and the pre-post maximal exercise increase in sICAM-1 was augmented after IH.
It is concluded that exercise performance in acute hypoxia is driven by the magnitude of hypoxaemia and an accelerated rate of cerebral and muscle deoxygenation. Molecular biomarkers of endothelial function in particular, NO2• and CAMs, are also influenced by hypoxia and may contribute to the reduction in [pic]O2peak. IH may be used to improve exercise economy and [pic]O2peak in hypoxia by improving cerebral and muscle oxygenation in the absence of any change in central O2 delivery. It is possible a recalibration of mechanisms that affect NO bioactivation could have enhanced vascular hypoxic sensitivity, O2 delivery and adaptation within brain and muscle tissue which ultimately translated to an improved hypoxic exercise performance. These results give motivation for athletes and mountaineers to incorporate an IH strategy prior to athletic performance at altitude.
List of Tables
Table 2.1 Examples of IH studies and summary of key research findings with implications for vascular function and NO/ROS balance
Table 2.2 Summary of investigations examining the effects of IH and exercise responses at sea-level or in acute hypoxia
Table 3.1 Volume and gas concentration measurements taken from IH machine
Table 4.1 Subject characteristics
Table 4.2 Average cardiorespiratory responses to exercise in hypoxia and normoxia
Table 4.3 Slopes and intercept for muscle oxygenation
Table 5.1 Subject characteristics
Table 5.2 The effect of IH on cardiorespiratory variables
Table 5.3 Slope and intercept for NIRS muscle oxygenation measurements
List of figures
Figure 1.1 Schematic overview of thesis
Figure 2.1 The O2 cascade at rest and during maximal exercise (Richardson et al. 1995 & 2006). Inset shows impact of hyperventilation on ODC during maximal exercise in normoxia (white triangles) and hypoxia FIO2 = 0.105, black triangle) (Calbet et al. 2003).
Figure 2.2 Changes in cerebral O2 delivery (SaO2 x MCAV) measured at different altitudes (( 150m; ♦ 3,610m; ● 4,750m; ▲ 5,260m) and exercise intensities. Measurements were taken within 36 hours of arrival at each altitude (Imray et al. 2005)
Figure 2.3 Relationship between aerobic capacity and reduction in [pic]O2max at various altitudes compared to sea-level. Aerobically trained subjects ([pic]O2max ≥ 63 ml.kgˉ1.minˉ1) are represented by the dashed regression line, untrained individuals ([pic]O2max ≤ 51 ml.kgˉ1.minˉ1) by the dotted line and mean by the solid line (Fulco et al. 1998).
Figure 2.4 Schematic diagram illustrating the effect of decreased convective O2 transport on exercise performance and influence from central and peripheral origins of fatigue (Amann and Calbet 2008)
Figure 2.5 Representation of NIR light transported through tissue (ƒtis) and blood (ƒbl) in the muscle and by volume fractions of arterioles (ωart), capillaries (ωcap) and venules (ωven). The light penetrates tissue in a banana shape for tissue absorbency and oxygenation evaluation.
Figure 2.6 Sigmoidal HHb profile during incremental exercise (Ferreira et al. 2007)
Figure 2.7 Patterns of cerebral (top) and muscle (bottom) oxygenation (O2Hb, HHb and THb) during incremental cycling exercise in normoxia (Rupp and Perrey 2008).
Figure 2.8 Synthesis of NO from L-arginine (Manukhina et al. 2006)
Figure 2.9 NO synthesis in endothelium for vasodilation and implications for vascular NO storage for delivery of vasodilation and vascular O2 sensing and oxygenation state of the RBC (O2Hb or HHb) (Dejam et al. 2004)
Figure 2.10 The reaction between HHb and nitrite yielding NO and metHb (Hb-Fe(III) is optimal when Hb is 50% saturated (eqn.1). The reaction between O2Hb and NO to form nitrate dominate when O2Hb levels are greater than HHb (eqn.2) (Gladwin et al. 2005).
Figure 2.11 O2-dependant nitrite reductase (low O2) and oxidase activities of Hb (high O2). In the oxidase reaction nitrate is the primary product and intermediate formation of nitrogen dioxide (NO2). Nitrite reductase activities are associated with formation of NO-Hb products, HbNO and SNOHb (Gladwin et al 2004).
Figure 2.12 Effect of hypoxia (left, Steiner et al. 2002) and antioxidant administration (right, Wood et al. 1999a) on leukocyte adherence. Leukocyte count was measured as the number of cells that remained stationary for longer than 30sec during video analysis
Figure 2.13 Regulation of the HIF-1α subunit by O2 through hydroxylation. There are three hydroxylation sites: two prolyl residues in the O2-dependent destruction domain (ODDD) and an asparaginyl residue in the C terminal transactivation domain (CTAD). In the presence of O2 these are hydroxylated by prolyl hydroxylase (PHD) and FIH (factor inhibiting HIF) enzymes, respectively. The prolyl hydroxylation allows capture by von Hipple-Lindau protein (VHL), leading to ubiquitylation and destruction, and the asparaginyl hydroxylation blocks transactivator recruitment; (Maxwell 2005)
Figure 2.14 Changes in the number of adherent leukocytes during as period of breathing room air; 0-10min, hypoxia (10% O2); 20-20min and during the recovery period; 20-30 min in acclimatised and non-acclimatised rats (Wood et al 1999b)
Figure 2.15 Working hypotheses for study 1
Figure 2.16 Working hypothesis for study 2
Figure 3.1 Example of raw chemiluminescence signal observed for plasma sample
Figure 3.2 A typical EPR spectra for the A• detected in human venous blood
Figure 4.1 Working hypotheses for study 1
Figure 4.2 Schematic representation of experimental design. Cerebral and muscle oxygenation measurements were taken at baseline, rest and throughout incremental exercise (IE) in normoxia (21% O2) and hypoxia (12% O2).
Figure 4.3 The effect of hypoxia on heart rate during incremental exercise
Figure 4.4 The effect of hypoxia on SaO2 during incremental exercise
Figure 4.5 Muscle oxygenation profile at rest and during exercise
Figure 4.6 Cerebral oxygenation profile at rest and during exercise
Figure 4.7 Oxidative stress markers before and after maximal exercise
Figure 4.8 NO metabolites before and after maximal exercise
Figure 4.9 sVCAM-1 and sICAM-1 before and after maximal exercise
Figure 4.10 Blood pH before and after maximal exercise
Figure 4.11 Correlation between VO2peak and pooled (pre-post) exercise sVCAM-1
Figure 4.12 Correlation between baseline nitrite and change pre-post exercise
Figure 5.1 Working hypothesis for study 2
Figure 5.2 Experimental design for study 2. Subjects performed 2x incremental exercise (IE) tests in hypoxia (FIO2 = 0.12) before and 3 days after 2x 5 day blocks of intermittent hypoxia (IH) or control (IN). NIRS assessment for cerebral and muscle oxygenation was measured throughout IE and blood samples (B) were taken before and immediately after IE.
Figure 5.3 Average daily resting SaO2 after each IH or IN exposure.
Figure 5.4 Effect of IH on maximal and submaximal VO2
Figure 5.5 Effect of IH on SaO2 during incremental exercise
Figure 5.6 Effect of IH on heart rate during incremental exercise
Figure 5.7 Cerebral oxygenation before and after IH or IN
Figure 5.8 Muscle oxygenation before and after IH or IN
Figure 5.9 NOx at rest and after maximal hypoxic exercise
Figure 5.10 Nitrate before and after maximal hypoxic exercise.
Figure 5.11 Nitrite at rest and after maximal hypoxic exercise
Figure 5.12 RSNO at rest and after maximal hypoxic exercise
Figure 5.13 sVCAM-1 at rest and after maximal hypoxic exercise
Figure 5.14 sICAM-1 before and after maximal hypoxic exercise
Figure 5.15 Blood pH at rest and after maximal hypoxic exercise
Definition of terms
ATP Adenosine triphosphate
VA/Q Alveolar ventilation/ perfusion ratio
CaO2 Arterial oxygen content
SaO2 Arterial oxygen saturation
A• Ascorbate free radical
Q Cardiac output
CAM Cell adhesion molecules
CNS Central nervous system
CBF Cerebral blood flow
CMR Cerebral metabolic ratio
CAD Coronary artery disease
cGMP Cyclic guanosine monophosphate
HHb Deoxyhaemoglobin
DPF Differential path factor
N2O3 Dinitrogen trioxide
DPG Diphosphoglycerate
eNOS Endothelial nitric oxide synthase
EPO Erythropoietin
EIH Exercise induced hypoxaemia
FMD Flow mediated dilatation
FBF Forearm blood flow
Hct Haematocrit
Hb Haemoglobin
HR Heart rate
HIF Hypoxic inducible factor
HVR Hypoxic ventilatory response
iNOS Inducible nitric oxide synthase
FIO2 Inspiratory oxygen fraction
PIO2 Inspiratory oxygen pressure
IH Intermittent hypoxia
I-R Ischemia-reperfusion
LBF Leg blood flow
LDL Low density lipoproteins
[pic]O2max Maximal oxygen consumption
MAP Mean arterial blood pressure
MCAV Middle cerebral artery velocity
Qm Muscle blood flow with muscle
[pic]O2m Muscle oxygen consumption
Mb Myoglobin
NIRS Near-infrared spectroscopy
NO3• Nitrate
NO Nitric oxide
NOA Nitric Oxide chemiluminescence analyser
NOS Nitric oxide synthase
NO2• Nitrite
3-NT 3-Nitrotyrosine
OSA Obstructive sleep apnoea
O2 Oxygen
ODC Oxygen dissociation curve
O2Hb Oxyhaemoglobin
O3 Ozone
PCO2 Partial pressure of carbon dioxide (in mmHg)
PaCO2 Partial pressure of carbon dioxide in arterial blood (in mmHg)
PO2 Partial pressure of oxygen (in mmHg)
PAO2 Partial pressure of oxygen in the alveoli (in mmHg)
(PAO2-PaO2) Partial pressure of oxygen in the alveoli minus arterial blood (in mmHg)
PaO2 Partial pressure of oxygen in the arterial blood (in mmHg)
[pic]O2oeak Peak oxygen consumption
PPO Peak power output
PBMC Peripheral blood mononuclear cells
ONOO• Peroxynitrite
PHD Prolyl hydroxylase enzymes
ROS Reactive oxygen species
RBC Red blood cell
O2• Superoxide anion
SOD Superoxide dismutase
AlbS-NO S-nitrosoalbumin
GSNO S-nitrosogluthione
SNO-Hb S-nitrosohaemoglobin
RSNO S-nitrosothiols
sICAM-1 soluble intracellular cell adhesion molecule-1
sVCAM-1 soluble vascular cell adhesion molecule-1
THb Total Haemoglobin
VEGF Vascular endothelial growth factor
[pic]E Ventilation rate
WT Wild type
XO Xanthane oxidase
Conferences and publications
Conferences
• Intermittent hypoxia: Implications of redox regulation and systemic oxygen delivery. Birmingham Medical Research Expeditionary Society conference 2006. (Oral)
• Implications of acute exercise and inspiratory hypoxia for systemic NO bioavailability. Hypoxia Symposium 2009. Lake Louise, Canada (Poster)
• Exercise and hypoxia: Implications for cerebral and muscle oxygenation and systemic NO bioavailability. Birmingham Medical Research Expeditionary Society conference 2009. (Oral)
Publication in preparation
• The impact of acute hypoxia on cerebral and muscle oxygenation and systemic molecular biomarkers of vascular function during exercise.
▪ Intended journal: Journal of Applied Physiology
• Implications for intermittent hypoxia on cerebral and muscle oxygenation and molecular biomarkers of vascular function during exercise in hypoxia.
▪ Intended journal: Journal of Applied Physiology
Chapter 1
Thesis overview
1.1 Background
A critical component of endurance performance is the ability to extract oxygen (O2) from the atmosphere in the lungs and the integrity of the cardiovascular system to transport O2 in the blood, mostly as oxyhaemoglobin (O2Hb), through the peripheral circulation until delivering O2 to the contracting muscle for aerobic metabolism. The intention is to ensure optimal transfer of O2 to the mitochondria for ATP synthesis and therefore any breakdown along the O2 transport cascade will reflect the efficiency of O2 delivery and present as a limitation to the individuals aerobic capacity. This was highlighted by Richardson et al. (1999) during maximal knee extension exercise in normoxia, hypoxia and hyperoxia who concluded maximal oxygen consumption ([pic]O2max) in healthy humans was proportionally related to the decrease in O2 supply rather than through impaired mitochondrial metabolic rate. It is also important that the brain maintains adequate O2 supply to preserve optimal functionality. A decrease cerebral O2 delivery has been attributed to the decline in aerobic performance at high altitude and independently of a significant muscle fatigue which implies cerebral oxygenation can directly inhibit central motor output to the muscle (Amann et al. 2007; Imray et al. 2005; Noakes et al. 2001; Rasmussen et al. 2010). It is likely O2-dependant central fatigue functions as a protective mechanism preserving normal cerebral homeostasis by preventing further reductions in cerebral capillary O2 levels and could explain why the decline in [pic]O2max is greater than predicted for the given reduction in O2 supply at altitudes greater than 4,000m (Fulco et al. 1998). Therefore the exercise performance limitation in hypoxia can be attributed to central and peripheral origins of fatigue.
Near-infrared spectroscopy (NIRS) provides a non-invasive assessment of cerebral and muscle oxygenation. However the oxygenation profiles of these tissues have not been well defined during incremental exercise performance in hypoxia. Since performance in hypoxia appears to be driven by the associated increases in metabolic stress for a given absolute workload due to the decrease in [pic]O2max, an examination of the oxygenation profiles at equivalent absolute and relative exercise intensities would give some insight into the kinetics of microvascular O2 exchange in the brain and muscle during incremental exercise. Subudhi et al. (2007) reported a critical reduction in cerebral oxygenation during incremental exercise in 12% O2 conditions in the workloads preceding exhaustion in well trained subjects who resided at moderate altitude and with recent experience of exercise at high altitude. Furthermore, Ferreira et al. (2007b) suggested incremental exercise performance is driven by the muscle O2 extraction profile evaluated by the NIRS deoxyhaemoglobin (HHb) signal. Similar studies have yet to be replicated during exercise in hypoxia in untrained, unacclimatised sea-level residents.
The inherent ability for the cardiovascular system to respond to low O2 levels ensures O2 delivery matches its demand. The production of nitric oxide (NO) and bioavailability of its more stable vascular metabolites particularly nitrite (NO2•) are key regulators of endothelial function, vasodilation and O2 sensing during hypoxic exposure and exercise (Cosby et al. 2003; Gladwin et al. 2000; Maher et al. 2008). These studies show that an effective and efficient NO-induced vasodilation requires the complex interaction and activation of several signaling pathways related to oxygenation status, the redox biochemical environment and exposure to inflammatory stimuli. Although NO regulation is an important factor in O2 delivery during exercise, it is possible that intense exercise can perturb vascular homeostasis by inhibiting NO release and could present a limitation to exercise performance. Plasma nitrite levels decreased after maximal exercise in normoxia (Larsen et al. 2010) however other reports have shown an increase in nitrite after maximal exercise and was a key determinant of aerobic capacity in healthy subjects (Rassaf et al. 2007) and in patients with vascular disease (Allen et al. 2009). The discrepancy between these studies warrants further examination whilst it is unknown how (systemic) nitrite concentrations are affected by maximal exercise in hypoxia which in turn could have haemodynamic and O2 delivery implications.
Intermittent hypoxia (IH) is initiated by episodic hypoxia-normoxia breathing and is characterised by a swinging of arterial O2 saturations (SaO2). The repeated activation of hypoxic sensitive pathways together with periods of reoxygenation leads to the production of NO and reactive O2 species (ROS) which are important molecular signaling factors driving adaptation to IH (Bailey et al. 2001; Manukhina et al. 2006; Peng et al. 2003; Peng and Prabhakar 2004). Furthermore with each hypoxic episode cardiovascular, cerebrovascular and ventilatory hypoxic sensitivity is enhanced (Ainslie et al. 2008; Foster et al. 2005; Katayama et al. 2001). Consequently IH is often employed by athletes to enhance the efficiency of exercise training and mountaineers to accelerate the acclimatisation process during a high altitude ascent although there is some controversy regarding the efficacy of IH for aerobic performance (Levine 2002). The severity, intermittence and duration of each hypoxic episode is critical to adaptation and will determine if there is sufficient biochemical stimulus to initiate a protective effect or when there is a chronic overproduction of ROS, that might be detrimental to endothelial function (Neubauer 2001; Wang et al. 2007). An improved systemic O2 delivery, antioxidant/prooxidant regulation and a more efficient vascular NO storage and release during exercise would provide strong motivation for individuals to incorporate an IH strategy into their pre-competition preparation. However there is limited research examining the effect of IH on NO regulation, cerebral and muscle oxygenation and maximal aerobic exercise performance in hypoxia.
1.2 Strategy
The literature review is essentially divided into three formal sections. The relevance of these sections to each experimental chapter is presented in figure 1.1.
• Section 1 is a general overview of factors that affect O2 delivery to the muscle and brain during exercise with implications for aerobic performance in normoxia and hypoxia. NIRS theory, its limitations and a summary of previous research findings during exercise in normoxia and acute hypoxia are summarised in this review section.
• Section 2 describes the regulation of vascular endothelail NO production and metabolite bioavailability; plasma nitrite (NO2•), nitrate (NO3•) and S-nitrosothiols (RSNO). The production of 3-nitrotyrosine (3-NT) and release of cell adhesion molecules (CAM) as biomarkers of endothelial function are also presented. Study one then examined the impact of acute hypoxia on cerebral and muscle oxygenation and systemic molecular biomarkers of vascular function during exercise.
• Section 3 descirbes the practical implications of IH. This section is split broadly into the effects of IH on 1) molecular mechanisms driving adaption with a particular focus on NO-ROS balance when determining if the adaptive response has protective or detrimental effects, and 2) a summary of previous research findings on the effects of IH on sea-level and hypoxic exercise. Study two examined the effect of intermittent hypoxia on cerebral and muscle oxygenation and molecular biomarkers of vascular function during exercise in hypoxia.
[pic]
Figure 1.1 – A schematic overview of thesis
1.3 - Aims and objectives
Study 1: The impact of acute hypoxia on cerebral and muscle oxygenation and systemic molecular biomarkers of vascular function during exercise
Aim 1 -To characterise changes in muscle and cerebral oxygenation during incremental exercise to exhaustion in normoxia and hypoxia. Specifically, the oxygenation profiles of O2Hb, HHb and THb of the vastus lateralis muscle and prefrontal cortex region of the brain will be evaluated at equivalent absolute and relative exercise intensities.
Aim 2 - To examine the effect of maximal exercise in hypoxia on molecular blood-borne markers of vascular O2 sensing and endothelial function. Specifically biomarkers of oxidative stress, NO metabolite bioavailability and CAM will be measured before and immediately after maximal exercise.
Aim 3 - To establish if the reduction in aerobic capacity in hypoxia is related to the change in blood borne markers of vascular function after maximal exercise.
Study 2: Implications for intermittent hypoxia on cerebral and muscle oxygenation and molecular biomarkers of vascular function during exercise in hypoxia
Aim 4 - To evaluate the effect of IH on exercise performance in hypoxia.
Aim 5 - To examine if the effects of IH on the cerebral and muscle oxygenation during exercise in hypoxia.
Aim 6 - To determine if adaptation is driven by enhanced NO metabolite bioavailability and its vascular protective effects. Subsequently it is possible that the increased CAM release after maximal exercise will be attenuated after IH.
Chapter 2
Literature review
2.1 The oxygen transport cascade
The ability for cells and tissue to survive and function optimally depends critically on O2 availability for aerobic metabolism. Hypoxia is a physiological condition characterised by diminished O2 availability and can be a direct consequence of clinical conditions such as chronic obstructive pulmonary disease, the environment and exercise. A deficiency of O2 in the atmosphere as experienced at high altitude results in inadequate O2 supply to the muscle and adversely affects aerobic exercise performance (Richardson et al. 1999). During submaximal exercise it is possible for skeletal muscle to function with reduced arterial O2 content (CaO2) by compensatory increases in cardiac output, leg blood flow, O2 extraction (Lundby et al. 2006; Roach et al. 1999) and anaerobic energy contributions as characterised by a leftward shift in the blood lactate curve (Friedmann et al. 2005; Ozcelik and Kelestimur 2004). The decrease in [pic]O2max is therefore associated with exhaustion of these compensatory responses and accelerated accumulation of muscle fatigue products. The decrease in PO2 from air to the mitochondria is known as the O2 transport cascade (figure 2.2). The pathway determines the efficiency of O2 delivery as it moves along the physiological O2 gradient from the atmosphere, through the lungs and peripheral circulation until it is transported to the mitochondria for ATP synthesis. The magnitude of each step along the cascade will vary according to the level of inspiratory hypoxia, exercise intensity and the individuals’ ability to activate the compensatory mechanisms which counterbalance the reduction in CaO2. Significant impairment in the transfer of O2 at any one point along the cascade can have adverse effects downstream and will be reflected by the reduction in aerobic performance. The four key events which determine the efficiency of the O2 transport cascade are described below.
1) A high pulmonary ventilation rate is needed to maintain alveolar PO2 levels (PAO2) which is particularly important in hypoxia for improving the efficiency of O2 diffusion from the alveoli to the pulmonary circulation. During acute and chronic hypoxic exposure an enhanced sensitivity of carotid body chemoreceptors to low O2 levels increases ventilation rate at rest and during exercise increasing SaO2 although there is considerable individual variability in this response (Benoit et al. 1995; Katayama et al. 2001; Zhang and Robbins 2000). Insufficient ventilation impairs pulmonary gas exchange during exercise (Holmberg and Calbet 2007) and the decline in aerobic capacity at altitude has been correlated with a reduced exercise hyperventilation response (Gavin et al. 1998). Increased ventilation also decreases arterial PCO2 (PaCO2) elevating blood pH and causes a leftward shift in the O2 dissociation curve (ODC) which can facilitate O2 uploading to haemoglobin (Hb) in the lungs during exercise in hypoxia (Calbet et al. 2003) (figure 2.2). An augmented hypoxic ventilatory response (HVR) at rest has been consistently reported after chronic altitude exposure (Hupperets et al. 2004) and IH (Foster et al. 2005; Katayama et al. 2001 & 2005) and may translate to an increased exercise ventilatory response (Katayama et al. 2001; Townsend et al. 2002 & 2005) although not all studies show this in normoxia (Foster et al. 2006) and hypoxia (Beidleman et al. 2003).
2) The efficiency of pulmonary gas O2 exchange in the lung is driven by the PO2 gradient between alveoli and pulmonary capillary blood. The efficiency of the process is reflected by the (PAO2-PaO2) difference where PaO2 is the partial pressure of O2 in arterial blood and values of 0-5mmHg are expected in healthy individuals and increases during exercise (Calbet et al. 2003a). The (PAO2-PaO2) difference can increase due to an alveolar ventilation/perfusion (VA/Q) inequality and shunting of blood (Vogiatzis et al. 2008). Subsequently in hypoxia the PO2 pressure gradient is small limiting O2 diffusion to lung capillaries and at 5,300m ~40% of the reduction in CaO2 was explained by the reduced inspiratory O2 fraction and diffusion limitation (FIO2) (Calbet et al. 2003a). Additionally, during exercise above 3,000m the reduced transit time for RBC through pulmonary capillaries does not allow for a complete gas equilibration between alveoli and capillary blood and the (PAO2-PaO2) difference increases linearly with cardiac output (Calbet et al. 2008). Pulmonary hypertension and increased interstitial edema can also impair diffusion capacity at rest and during exercise in hypoxia (Dehnert et al. 2005). The impairments in pulmonary gas exchange may be improved after altitude acclimatisation due to increased HVR (Calbet et al. 2003a), enhanced recruitment of pulmonary capillaries thus increasing the mean transit time for O2 diffusion (Capen and Wagner, Jr. 1982) and elevated diphosphoglycerate (DPG) levels in red blood cells (RBC) by causing a rightward shift in the ODC thus for a given PO2 more O2 binds with Hb to form O2Hb (Calbet et al. 2003; Wagner et al. 2007). However, although O2 delivery increased by ~50% after altitude acclimatisation [pic]O2max improved by only 13% because most of the extra O2 available is distributed to tissues other than exercising muscle (Calbet et al. 2003a).
3) Compensatory increases in cardiac output (Q), vasodilation, capillary recruitment, O2 extraction and vascular conductance maintains O2 delivery and [pic]O2 when CaO2 is reduced during submaximal exercise (Bourdillon et al. 2009; Calbet 2000; Gonzalez-Alonso et al. 2001; Lundby et al. 2006). These compensatory mechanisms become exhausted as exercise intensity increases and an accelerated accumulation of muscle fatigue products increases proportionally with the decline in O2 supply (Richardson et al. 1999) whilst at altitudes above 4,000m the reduction in maximal heart rate (HR), cardiac output and leg blood flow results in even more significant impairments in O2 delivery and [pic]O2max (Calbet, et al. 2003; Lundby et al. 2006; Mollard et al. 2007).
4) The PO2 gradient between the muscle capillary and mitochondria determines the O2 extraction capacity of the muscle although this is unaffected by O2 supply and is maintained at ~90% and ~80% during maximal cycling (Lundby et al. 2006) and knee extension exercise respectively (Richardson et al. 1999). Sympathetic vasoconstriction facilitates O2 transfer by matching O2 delivery with demand and redistributing blood to the most active muscle regions since increasing NO bioavailability (Larsen et al. 2010) and ATP infusions (Lundby et al. 2008) decrease O2 extraction and [pic]O2max at sea-level and altitude. Intracellular O2 levels of the muscle are calculated from measurement of Mb desaturation using H-NMR spectroscopy. At rest this has been estimated at ~34mmHg in normoxia and ~23mmHg in 10% O2 and decreases dramatically during exercise where levels of 2-5mmHg in normoxia and hypoxia have been reported (figure 2.1) (Richardson et al. 1995; 1999; 2001 & 2006). The magnitude of Mb desaturation does not reflect exercise intensity since the response tends to plateau at workloads greater than 50% of maximal work rate (Richardson et al. 2001) and there is little benefit gained from further reductions in intracellular muscle oxygenation and the decrease in [pic]O2max in hypoxia is related to the reduction in O2 supply (Richardson et al. 1999).
[pic]
Figure 2.1 - The O2 cascade at rest and during maximal exercise (Richardson et al. 1995 & 2006). Inset shows impact of hyperventilation on ODC during maximal exercise in normoxia (white triangles) and hypoxia FIO2 = 0.105, black triangle)(Calbet et al. 2003b).
2.2 Cerebral circulation
In contrast to muscle tissue, capillary recruitment is not possible in the cerebral circulation and O2 delivery can be estimated by global changes in cerebral blood flow (CBF) measured non-invasively by Doppler ultrasound as velocity changes in the middle cerebral artery (MCAV) (Ainslie and Poulin 2004; Subudhi et al. 2008) or by NIRS as a change in the O2Hb signal (Foster et al. 2005; Subudhi et al. 2007). The changes in CBF is regulated by the complex and dynamic interplay between several factors such as cerebral autoregulation, sympathetic nerve activation, vasodilation, cardiac output, CO2 and metabolism which together form an important protective feature in the brain (Ide and Secher 2000; Ogoh and Ainslie 2009; Rasmussen et al. 2006). A change in PaCO2 is regarded as the most sensitive influence of CBF at rest, during exercise and resting hypoxia exposure (Ainslie and Poulin 2004; Rasmussen et al. 2007). Cerebral autoregulation maintains a constant supply of blood to the brain within a wide range of MAP (between 60 and 150mmHg) to protect it from swelling and over-perfusion (LASSEN 1959). The response is enhanced at rest by hypocapnia and attenuated by hypercapnia (Paulson et al. 1990) and therefore its effectiveness is modified by individual hypoxic chemosensitivities and sympathetic activation (Ainslie & Poulin 2004). An impaired cerebral autoregulation response was observed during prolonged resting altitude exposure above 3,440m (Jansen et al. 2000; Jansen et al. 2007) by mechanisms which may be related to increased systemic free radical production (Bailey et al. 2009b).
CBF increases during low-moderate intensity exercise due to increased blood pressure, cardiac output and PaCO2 but when the workload exceeds ventilatory threshold the decline in PaCO2 and loss of cerebral autoregulatory control results in a reduction in CBF and O2 delivery (Imray et al. 2005; Ogoh et al. 2005; Subudhi et al. 2008). Although MCAV can be maintained at sea-level values during submaximal exercise in hypoxia through increased neurogenic activity and sympathoexcitation overrides the hypocapnic induced vasoconstriction lowering of CBF (Ainslie et al. 2007), the decline in maximal cardiac output and perfusion pressure further aggravates cerebral deoxygenation during maximal hypoxic exercise (Imray et al. 2005). This was evident during exercise with β-adrenergic blockade (Ide et al. 2000; Seifert et al. 2009). Local vasodilator factors can also influence CBF such as ATP (Gonzalez-Alonso et al. 2004) and NO bioavailability (Peebles et al. 2008; White et al. 1998 &. 2000) although NO blockade had no effect on CBF during hypoxic and hypercapnia exposure in humans (Ide et al. 2007). It was suggested that during resting poikilocapnic hypoxic exposure transient increases in cerebral vasodilation factors was responsible for balancing the hyperventilation induced lowering of PaCO2 and associated vasoconstriction resulting in relatively little change in CBF (Ainslie and Poulin 2004). An exercise induced haemoconcentration partially compensates the reduction in CBF increasing CaO2 by 5-10% whilst cerebral aerobic metabolism can be maintained by elevated O2 extraction and anaerobic energy provision as indicated by an increased lactate turnover (Gonzalez-Alonso et al. 2004; Ide et al. 2000). The cerebral O2 delivery profile follows the same pattern in hypoxia compared to sea-level during incremental exercise although the overall magnitude of oxygenation is less in hypoxia (Figure 2.3). The reduction may be partly restored by altitude acclimatisation and elevations in CBF, CaO2 and Hb concentrations.
[pic]
Figure 2.2 Changes in cerebral O2 delivery (SaO2 x MCAV) measured at different altitudes ((, 150m; ♦, 3,610m; ●, 4,750m; ▲, 5,260m) and exercise intensities. Measurements were taken within 36 hours of arrival at each altitude (Imray et al. 2005).
Cerebral metabolism is supported almost exclusively by carbohydrate metabolism and changes in global cerebral metabolism can be estimated at rest and during exercise by arterial-jugular venous differences for O2, glucose and lactate. Whole brain [pic]O2 was estimated to increase from ~60 ml.minˉ¹ at rest to 90 ml.minˉ¹ during maximal cycling exercise (Dalsgaard et al. 2004; Gonzalez-Alonso et al. 2004; Ide et al. 2000) although these values do not reflect the regional changes that can occur. The cerebral metabolic ratio (CMR) provides a global index for alterations in cerebral metabolism based on a typical ‘normal’ cerebral O2 to carbohydrate uptake ratio of six to one whereby a CMR of six indicates carbohydrate oxidation matches its uptake. Increased brain activity is associated with a reduction in the CMR since carbohydrate uptake increases disproportionately with O2. Although during submaximal exercise CMR remains relatively stable reflecting an overall ability for cerebral O2 and substrate delivery to match its demand, the decline in CBF during intense exercise increases the net uptake of O2, glucose and lactate of which more than 50% is not oxidised and CMR values as low as 1.7 during maximal rowing exercise (Volianitis et al. 2008) and ~3 during cycling exercise have been reported (Dalsgaard and Secher 2007; Gonzalez-Alonso et al. 2004; Ide et al. 2000). Cerebral O2 extraction is measured by Cerebral [pic]O2 ÷ O2 delivery (Oja et al. 1999) which increases from (36% at rest to an estimated limit of 50% at exhaustion and fatigue was associated with enhanced rather than impaired uptake of O2 and substrates even with reductions in cerebral O2 delivery caused by heat stress (Gonzalez-Alonso et al 2004). Similarly, Volianitis et al. (2008) showed the decrease in CMR during maximal rowing exercise was unaffected by changes in CaO2 by breathing a hyperoxic, normoxic and hypoxic gas (FIO2 = 0.17). However because the arterial-jugular venous difference for lactate was attenuated and the O2-glucose index was 20% higher during the hypoxia trial suggests a change in the cerebral metabolic substrate preference.
The lack of capillary recruitment in the cerebral circulation becomes critical when metabolism rises by increasing the diffusion distance for O2. The greater O2 reserve across the brain may act as a protective mechanism by allowing the possibility for [pic]O2 to increase when CBF declines although further reductions in O2 delivery may restrict large increases in cerebral O2 extraction because an even lower PO2 gradient in hypoxia will be too low to allow sufficient diffusion to the mitochondria (Kayser 2003). An impaired cerebral metabolism may be more likely to occur when exercise is performed in more severe hypoxia where cerebral oxygenation and central fatigue can directly influence aerobic performance (Amann et al. 2007; Subudhi et al. 2007). A 15% or greater decline in cerebral O2 delivery by reducing CBF through hyperventilation induced cerebral vasoconstriction or by breathing 10% O2 was attributed to decreased maximal handgrip strength, increased cerebral lactate release and fatigue was related to O2-dependant central fatigue (Rasmussen et al. 2006).
3. Aerobic exercise performance in acute hypoxia.
The decrease in [pic]O2max in hypoxia is mostly due to the reduction in PIO2 (or FIO2) which serves to widen the (PAO2-PaO2) diffusion gradient and causing significant impairment to O2 delivery during exercise (Calbet et al. 2003; Fulco et al. 1998; Woorons et al. 2005). Altitudes as low as 580m elicited a 4% greater decline in SaO2 during maximal exercise compared to sea-level in endurance athletes resulting in a 7% reduction in [pic]O2peak and highlights the sensitivity of the O2 supply limitation to aerobic performance in the elite (Gore et al. 1997) and it was suggested for every 1% drop in SaO2 below 92% there is a 1% drop in [pic]O2max (Powers et al. 1989). Maximal aerobic performance over a range of hypoxic exposures was reflected by a proportional reduction in SaO2 and driven by the individual properties of the ODC (Ferretti et al. 1997). Each individual responds differently to hypoxia which can alter the ‘critical threshold altitude’ initiating the decrement in [pic]O2max whereby factors such as gender and fitness level of the individual can have a strong influence on the response (Harms et al. 2000; Mollard et al. 2007; Woorons et al. 2007). The decline in [pic]O2max at sea-level compared to various altitudes in well trained and sedentary individuals is presented in figure 2.3. At altitudes greater than 4,000m the decrement in performance is greater than predicted for a given reduction in O2 supply suggesting factors other than CaO2 such as brain oxygenation, size of active muscle mass and central delivery limitations also play a key role (Amann et al. 2007; Benoit et al. 2003; Fulco et al. 1998; Roach et al. 1999).
[pic]
Figure 2.3 – Relationship between aerobic capacity and reduction in [pic]O2max at various altitudes compared to sea-level. Aerobically trained subjects ([pic]O2max ≥ 63 ml.kgˉ1.minˉ1) are represented by the dashed regression line, untrained individuals ([pic]O2max ≤ 51 ml.kgˉ1.minˉ1) by the dotted line and mean by the solid line (Fulco et al. 1998).
2.4 Origins of fatigue
Fatigue reflects a multi-factorial response whereby the end product is an impaired inability to activate the neuromuscular system. Amann and Calbet (2008) defined fatigue as: ‘Acute impairments of performance during exercise as reflected by the failure to generate a given force or power output which is attributed to mechanisms susceptible to alterations in convective O2 transport.’ Fatigue can have ‘central’ and ‘peripheral’ components switching, albeit not exclusively, from one that is predominately peripheral in origin to one of central origin and existing with or without sensory feedback from the muscle (figure 2.4) and mediated by the severity of hypoxaemia (Amann et al. 2007).
[pic]
Figure 2.4 – Schematic diagram illustrating the effect of decreased convective O2 transport on exercise performance and influence from central and peripheral origins of fatigue (Amann and Calbet 2008).
Peripheral fatigue is generally associated with failure to excite the neuromuscular system through impaired uptake and release of Ca2+ in the sarcoplasmic reticulum (Duhamel et al. 2004), inhibition of Na+-K+-ATPase activity (Sandiford et al. 2005) or via an accumulation of muscle metabolites such as hydrogen ions and phosphates which can inhibit excitation-contraction coupling (Haseler et al. 1999). Similar changes in peripheral fatigue mechanisms have been observed at maximal exercise in normoxic and hypoxic conditions despite a marked reduction in performance in hypoxia, therefore the decline in performance is a direct result of an accelerated accumulation of these fatigue products. The reduction in hypoxic performance may be through an enhanced sensitivity activation of group III and IV muscle afferents which relay sensory inhibitory feedback from the muscle to the CNS (Amann et al. 2007). The central nervous system (CNS) is highly sensitive to reductions in O2 levels and blockade of group III and IV muscle afferents did not affect exercise time to exhaustion in extreme hypoxia (Kjaer et al. 1999). However acute and chronic hypoxia can blunt the responsiveness of type III/IV fibers to fatiguing stimuli leaving an inability to detect metabolic changes in the muscle in rats (Arbogast et al. 2000; Dousset et al. 2001; Dousset et al. 2003). The ineffectiveness of sensorimotor control may explain the reduced endurance time to fatigue reported in subjects acclimated to high altitude by mechanisms and could involve the inhibitory effect of NO on discharge rate of group III-IV muscle afferents (Arbogast et al. 2002) and/or NO induced depression of muscle energetics (Murrant and Reid 2001).
Amann and colleagues (Amann et al. 2006 & 2007; Romer et al. 2007) showed a dominant effect on aerobic performance by peripheral fatigue assessed by changes in potentiated quadriceps twitch force before and after constant load cycling to exhaustion in conditions ranging from hyperoxia to moderate hypoxia (FIO2 = 0.30 – 0.12). However the influence of O2-sensitive central fatigue became dominant in severe hypoxia (FIO2 = 0.10) since potentiated quadriceps twitch force was one third less at the point of exhaustion compared to normoxia whilst HR, blood lactate and rating of perceived exertion were also lower during this trial. Rapidly switching the breathing circuit to a hyperoxic gas mixture at the point of task failure had no effect on prolonging exercise in normoxia and moderate hypoxia whereas exercise duration was markedly enhanced in severe hypoxia. Similar findings were observed by Subudhi et al. (2008) during incremental cycling exercise at 4,300m conditions using the same gas switch model. It was suggested that increasing muscle oxygenation at the point of exhaustion in normoxia and moderate hypoxia had limited effect on reversing peripheral fatigue after a ‘critical threshold’ is attained. Whereas in severe hypoxia, enhanced cerebral oxygenation and rapidly reversibility of O2-sensitive central fatigue and prolonged exercise performance. Subsequently, Amann et al. (2007) proposed a critical SaO2 threshold of 70-75% where central motor performance deteriorates as a direct consequence of CNS hypoxia.
The precise mechanisms regulating O2-sensitive central fatigue is unclear. There is no evidence showing cerebral O2 extraction and carbohydrate metabolism is impaired in moderate hypoxia although this has not yet been examined during whole body exercise in severe hypoxia. Due to the rapid reversibility of central fatigue the most likely explanation could be related to an altered release of CNS neurotransmitters such as dopamine, noradrenaline, and serotonin due to their association with the sensation of fatigue, motivation and arousal (Amann and Kayser 2009). These O2-sensitive neurotransmitters can cause dysfunction within the basal ganglia and prefrontal cortex therefore affecting limbic-motor interaction and impairing cortical activation and motor unit recruitment during exercise (Chaudhuri and Behan 2000). The O2-neurotransmitter theory and link with central fatigue is so far speculative and evidence from chronic fatigue syndrome patients using pharmacological manipulation such as 5-HT receptor antagonists or other nutritional based treatment therapies have so far been unsuccessful in enhancing functionality (Brouwers et al. 2002; The et al. 2007; The et al. 2010). Such interventions have yet to be implemented during exercise in severe hypoxia as a means of improving exercise performance.
5. Near-infrared spectroscopy (NIRS)
NIRS provides non-invasive assessment of changes in cerebral and skeletal muscle oxygenation based on the relative transparency of human tissue for NIR radiation in the 650-900nm region. The spectrophotometer generates the NIR radiation from an optode which penetrates the skin, subcutaneous fat and underlying vascular tissue which is either scattered or absorbed by a second optode positioned parallel to the original light source (figure 2.5). By selecting specific wavelengths of NIR radiation and knowledge of O2-dependant light absorption properties of Hb, the main component of the erythrocyte in blood, and myoglobin (Mb) which is present in the skeletal muscle, it is possible to track concentration changes over time on their oxygenation status and defined as (O2Hb+Mb) and (HHb+Mb). Since Hb and Mb share identical optical characteristics, NIRS is unable to distinguish between the two species making it impossible to dissociate between intra to extra vascular O2 exchange and therefore the measurement reflects an overall change in tissue oxygantion. The relative signal contribution of Hb and Mb to the overall signal has been estimated to be 65% and 35% respectively although the Mb contribution may increase to 80% in some individuals and are also likely to change during exercise due to blood volume shifts and increased membrane permeability (Lai et al. 2009).
[pic]
Figure 2.5 Representation of NIR light transported through tissue (ƒtis) and blood (ƒbl) in the muscle and by volume fractions of arterioles (ωart), capillaries (ωcap) and venules (ωven). The light penetrates tissue in a banana shape for tissue absorbency and oxygenation evaluation
Tissue oxygenation is calculated using the Beer-Lambert law (1) which quantifies the attenuation of light passing through a non-scattered medium or the medium’s optical density (ODλ). Since biological tissue is considered a scattering medium, a correction factor known as the differential path-length factor (DPF) must be added to the Beer-Lambert equation (2) to account for light scattering (Delpy et al. 1987).
ODλ = log(Io/I) = єλ · C · L (1)
ODλ = log (Io/I ) = єλ · C · L · DPF + (ODλ)R (2)
ODλ is the optical density of the tissue, Io is incident light, I is emergent light, єλ is the specific extinction coefficient of the chromophore (mM-1·cm-1), [C] is the chromophore concentration ([Hb] + [Mb]), L is the distance between where light enters and leaves the medium (cm) and (ODλ)R represents the light losses due to scattering and absorption by other O2-independent chromophores in tissue. Since it is assumed (ODλ)R remains constant, the equation can be converted to changes in chromophore concentrations shown in equation 3 although this assumption may lead to an overestimation in oxygenation values (Ferreira et al. 2007a). This equation is valid for a medium with only one chromophore however the spectral extinction coefficients of various chromophores together with the solution of multiple sets of equations leads to an algorithm that converts the changes in optical absorbance to changes in [O2Hb+Mb] and [HHb+Mb].
[pic]
The underlying vascular bed is composed of 70-75% venous, 15-20% capillary and 10% arterial blood although the relative distribution across each blood compartment may change during exercise due to arteriolar dilation and capillary recruitment (Boushel et al. 2001; Lai et al. 2009). An increase in O2Hb and THb is associated with enhanced regional O2 delivery and blood volume respectively and since arterial blood is well-oxygenated, increased O2Hb are reflected by parallel changes in arterial blood flow when the rate of [pic]O2 remains constant (van Beekvelt et al. 2001). The O2Hb signal is sensitive to alterations in blood volume whereas the HHb signal is less sensitive and has been used as an estimate of tissue deoxygenation through increased O2 extraction (Ferreira et al. 2007b; Grassi et al. 1999; Grassi et al. 2003). Subsequently NIRS may be used to assess changes in microvascular O2 delivery and utilisation at rest and during exercise (Subudhi et al. 2007; Foster et al. 2005) and for evaluating vascular function in conjunction with other routine clinical cardiovascular examinations (van Beekvelt et al. 2002). Therefore previously studies have incorporated NIRS to assess the effectiveness of rehabilitation strategies on O2 reperfusion rate following vascular occlusion in patients with chronic heart failure (Gerovasili et al. 2009), as a monitoring tool to assess changes in brain and muscle oxygenation during exercise (Subudhi et al. 2007) and for evaluating the severity of peripheral arterial disease in Type 2 diabetics during exercise whereby a blunted exercise blood volume expansion response was interpreted as evidence of impaired vasodilation (Mohler, III et al. 2006; Vardi and Nini 2008).
2.6 Skeletal muscle oxygenation and exercise
Changes in muscle oxygenation have been assessed during submaximal (Chuang et al. 2002; DeLorey et al. 2004; Ferreira et al. 2005; Grassi et al. 2003; Jones et al. 2006) and during incremental exercise to exhaustion (Costes et al. 1996; Ferreira et al. 2007b; Grassi et al. 1999; Rupp and Perrey 2008). Muscle deoxygenation increases with workload because microvascular O2 demand exceeds its supply and is characterised by decreased O2Hb and increased HHb (figure 2.6 & 2.7). This makes it possible to evaluate the affect of various interventions on adaptation within the muscle such as exercise training (Costes et al. 2001; Neary et al. 2002), intermittent hypoxia (Marshall et al. 2008) and nutritional supplements such as bicarbonate (Nielsen et al. 2002). Additionally patients with CHF showed an accelerated muscle deoxygenation and steeper oxygenation slope during incremental exercise to exhaustion compared to control subjects (Belardinelli et al. 1995). Thus NIRS may be used to assess kinetic changes in muscle oxygenation during exercise.
An accelerated rise in muscle deoxygenation corresponded with workloads at lactate and ventilatory threshold during incremental exercise and therefore NIRS may be used to assess metabolic threshold points (Belardinelli et al. 1995b; Bhambhani et al. 1997; Grassi et al. 1999). The response was attributed to the onset of metabolic acidosis and a rightward shift in the ODC which facilitated O2 offloading from Hb and maintained [pic]O2 through increased extraction (Grassi et al 2003). Similar observations were shown during constant load exercise above lactate threshold where the progressive rise in pulmonary [pic]O2 was reciprocated by a similar elevation in muscle deoxygenation (Grassi et al 2003; Chuang et al 2002; Jones et al. 2006; Ferreira et al. 2005). These findings reflect an adaptive response in the muscle and regulated by the dynamic changes in microvascular blood flow and O2 availability. Ferreira et al. (2007b) reported that incremental exercise in normoxia is characterised by an S-shaped HHb profile (figure 2.6). The dynamic nature of the profile signifies the efficiency of microvascular O2 exchange and tight coupling between O2 delivery and extraction maintaining muscle [pic]O2 across a range of exercise intensities. The profile can be explained mechanistically by the relationship between muscle blood flow (Qm) with muscle [pic]O2 ([pic]O2m) where the key features are the slope and plateau region whereby a greater slope signifies less of a difference between the kinetics of Qm compared to [pic]O2m and early attainment of the plateau region in the HHb profile. The plateau indicates the kinetics of [pic]O2m and Qm are linear and may be interpreted as evidence of maximal O2 extraction and [pic]O2max and therefore raises the possibility that incremental exercise performance is driven by the HHb profile.
[pic]
Figure 2.6 Sigmoidal HHb profile during incremental exercise (Ferreira et al. 2007b)
Typically only venous blood is deoxygenated at rest and during exercise at sea-level, whereas in hypoxia arterial and venous blood is deoxygenated and therefore it is expected that hypoxia would further aggravate the muscle deoxygenation response to exercise. Subudhi et al. (2007 & 2008) showed in a group of untrained subjects and competitive cyclists that the magnitude of muscle deoxygenation at equivalent absolute and relative workloads in 12% O2 was greater than at sea-level although a similar oxygenation pattern was observed between trials. However not all reports support this notion despite significant reductions in SaO2 and may be related to factors such as the magnitude of hypoxia, exercise modality, aerobic condition of the subject and muscle group investigated. No change in muscle oxygenation compared to sea-level was observed when performing single leg knee-extension exercise in 12% O2 (DeLorey et al. 2004), ankle extension exercise in 11% O2 (Rupp and Perrey 2009) and steady state cycling in 14% O2 (Ainslie et al. 2007). Although muscle oxygenation measured on the vastus lateralis was maintained at similar values to normoxia when cycling in 16% O2, the magnitude of deoxygenation was greater in the lateral gastrocnemius highlighting regional differences in oxygenation across active muscle groups during exercise (Heubert et al. 2005). Incremental exercise at 1,000m, 2,500m and 4,000m had relatively no effect on muscle O2Hb and HHb in untrained individuals whereas trained athletes showed an augmented muscle deoxygenation response when exercise was performed at altitudes greater than 2,500m (Bourdillon et al. 2009b). These results suggest surpassing a critical threshold altitude is necessary for significant muscle deoxygenation to occur and the advantages of exercise training are lost during exercise in hypoxia thereby increasing the susceptibility for impaired aerobic performance in endurance athletes.
Endurance athletes with exercise induced hypoxaemia (EIH) showed an augmented muscle deoxygenation response compared to non-EIH athletes with identical [pic]O2max values (Legrand et al. 2005). The authors suggested the response was a compensatory mechanism aimed to counteract the reduction in O2 delivery through increased O2 extraction. Preventing EIH by breathing a hyperoxic gas mixture improved maximal rowing performance however there was relatively no change in muscle oxygenation and the improved performance was attributed to elevated cerebral oxygenation (Nielsen et al. 1999). By switching the breathing circuit to a hyperoxic gas at the point of exhaustion during incremental and constant load exercise in severe hypoxia partially restored muscle oxygenation prior to attaining a new plateau which represented a continuing balance between O2 delivery and consumption and exercise performance was improved (Sububhi et al. 2008; Amann et al. 2007). It was suggested the mechanisms for the improved performance were related to increased cerebral oxygenation and reversibility of O2-sensitive central fatigue factors although the affect of improved muscle oxygenation should not be ignored.
2.7 Cerebral oxygenation and exercise
Changes in cerebral oxygenation using NIRS are typically measured at the prefrontal cortex region of the forehead. These measurements correlate well with changes in calculated cerebral capillary oxygenation levels estimated from arterial and jugular venous blood saturations (Rasmussen et al. 2007; Kim et al. 2000). The prefrontal cortex region of the brain is associated with executive cognitive function and reduced oxygenation of this brain region has been associated with the decision to stop exercising (Subudhi et al. 2007). The prefrontal cortex also projects to other cortical regions more directly associated with central motor activation such as the pre-motor and motor cortices and comparison between prefrontal cortex oxygenation with these regions have yielded similar patterns of oxygenation during incremental exercise in normoxia and hypoxia (Subudi et al. 2009). Since the prefrontal region of the forehead allows for a more accessible and reproducible probe placement, NIRS studies tend to examine this area of the brain. Due to the different DPF values accounting for NIR light penetrating bone tissue and because capillary recruitment is not possible in the cerebral circulation makes it inappropriate to directly compare oxygenation changes between the muscle and brain.
A typical NIRS trace for cerebral O2Hb, HHb and THb during incremental cycling exercise is presented in figure 2.7. During low intensity exercise cerebral oxygenation remains relatively unchanged but increases as workload progresses due to elevations in cerebral metabolism, CBF, cardiac output, PaCO2, reduced cerebrovascular resistance and vasodilation (Ide et al. 1998; Imray et al. 2005; Rasmussen et al. Nybo 2006). Cerebral oxygenation then plateaus or decreases prior to maximal exercise (Bhambhani et al. 2007; Rupp & Perrey 2008; Subudhi et al. 2007 & 2008) although cerebral metabolism is maintained through increased O2 extraction as indicated by the continued rise in HHb. The decrease in cerebral oxygenation is attributed to increased cerebrovascular resistance (Imray et al. 2005) and/or hypocapnic-induced cerebral vasoconstriction by blunting CBF at workloads greater than ventilatory threshold (Bhambhini et al. 2007). Adaptation to IH also blunted the cerebral oxygenation response to exercise due to an elevated ventilation response through a reduced CBF and O2 delivery (Marshall et al. 2007). Arterial desaturation contributes to the decrease in cerebral oxygenation during intense exercise in normoxia and is improved when breathing a hyperoxic gas (Neilsen et al. 1999) whilst inspiratory hypoxia profoundly reduces cerebral oxygenation at rest (Foster et al. 2005) during exercise (Subudhi et al. 2007; Ainslie et al. 2007).
[pic]
Figure 2.7 Patterns of cerebral (top) and muscle (bottom) oxygenation (O2Hb, HHb and THb) during incremental cycling exercise in normoxia (Rupp and Perrey 2008).
2.8 Technical considerations and limitations of NIRS
The technical issues and limitations of using NIRS for monitoring cerebral and muscle tissue oxygenation have been previously documented (Ainslie et al 2007; Ferrari et al. 2004; Grassi et al. 2003; Neary 2004; Perrey 2008; Quaresima et al. 2003). The unknown intra- to extra-vascular volume shifts and redistribution cross the arterial, capillary and venous blood compartments in favour of the capillary volume fractions may limit interpretation during exercise. Slight variation may be introduced by changes in probe placement, adipose tissue thickness, Hb concentration as well as differences in muscle architecture and recruitment patterns during exercise. Using a constant path length, absorption and scattering coefficients to convert the NIRS signal to O2Hb and HHb concentrations limits the accuracy of the oxygenation value and Ferreira et al. (2007b) showed by assuming a constant scattering coefficient can lead to an overestimation of muscle NIRS signal during exercise.
It is not possible to distinguish the relative contribution of Hb and Mb to the overall NIRS signal (Lai et al. 2009; Nioka et al. 2006). Hb is considered to be about 10-fold greater than Mb in human blood although much of the signal could be derived from extra-vascular tissue increasing the relative contribution of Mb by up to 50% of the total signal (Nioka et al 2006). Lai et al. (2009) suggested 35 and 65% contribution of the signal is derived from Mb and Hb respectively. These values may change during exercise where the Hb contribution may increase due to the elevated blood volume and capillary recruitment. Chance et al. (1992) suggested the Mb contribution is no greater than 25% in human limbs however Tran et al. (1999) proposed the NIRS signal mainly measures Mb since the Mb desaturation kinetics measured by NMR was matched those observed by NIRS during exercise and pressure cuffing of the leg.
Investigations attempting to validate the NIRS signal during exercise through comparison with femoral venous blood O2 saturation have been contradictory. This is due to the multi-compartmental signal derived from NIRS and contaminating factors in venous blood O2 saturation such as drainage from non-exercising muscle. Esaki et al. (2005) reported a significant but weak correlation between the NIRS O2Hb signal and femoral venous O2 saturation during normoxic knee extension exercise ranging from 20-60% peak work rate whilst Mancini et al. (1994) observed similar results during handgrip exercise. Costes et al. (1996) also showed no correlation during exercise in normoxia however the relationship appeared to be stronger during steady state exercise in hypoxia.
Cerebral NIRS measurements show agreement with other brain imaging techniques such as PET scanning (Rostrup et al. 2002) and Doppler Ultrasound (Girth et al. 1997) and can reliably monitor changes in cerebral capillary O2 saturation estimated from arterial to jugular venous saturation differences at rest and during exercise in normoxia and hypoxia (Ide & Secher 2000;Rasmussen et al. 2007. Since 70-80% of blood in the cerebral circulation is venous (Madsen and Secher 1999; Schmidek et al. 1985) suggests small amounts of blood contained in capillaries and arteries form a significant part of the measurement. The penetration depths are limited to superficial layers 2–3 mm deep which is sufficient to illuminate cortical gray matter although deeper brain structures associated with motor, respiratory, cardiovascular, and temperature regulation such as the basal ganglia, brain stem, and cerebellum are inaccessible using NIRS. Discrete regions of the brain may respond differently during exercise. However Subudhi et al. (2008) showed the oxygenation profiles during incremental exercise in normoxia and hypoxia yielded similar patterns between the prefrontal, premotor and motor regions.
Despite these limitations, NIRS is generally accepted as an appropriate imaging tool for examining muscle and cerebral oxygenation at rest and during exercise.
2.9 Nitric oxide (NO)
Furchgott & Zawadzki (1980) were the first to identify the endothelium as a key regulator of smooth muscle relaxation. Their experiments showed that segments of aorta with intact endothelium relaxed in response to acetylcholine but constricted when the endothelium was removed. The molecule responsible for the relaxation was initially called endothelium-derived relaxing factor but was later renamed as NO by Ignarro et al. (1987). NO is now recognised as an important modulator of vascular homeostasis where its effective release and storage has important implications in health and disease (Dejam et al. 2004; Gladwin et al. 2004; Kleinbongard et al. 2006; Lauer et al. 2002). NO is produced through shear stress exerted on the endothelium or by the oxygenation status of the RBC and essentially functions as a vascular O2 sensor which becomes critical for vasodilation and O2 delivery in hypoxia and during exercise. NO can also exert cytoprotection, anti-inflammatory and antioxidant effects in models of ischemia-reperfusion (I-R) injury (Duranski et al. 2005; Webb et al. 2004) however, excessive NO production can contribute to I-R injury via peroxynitrite formation resulting in tissue dysfunction (Flogel et al. 1999; Schulz and Wambolt 1995).
NO is produced in the endothelium by the five-electron oxidation of the terminal guanidine group of L-arginine in a two-step reaction (figure 2.8). The reaction is catalysed by the enzyme eNOS in the presence of flavins and tetrahydrobiopterin as cofactors and NADPH and molecular O2. NO and L-citrulline is formed via the enzyme bound intermediate, N-hydroxyarginine (Lauer et al. 2002).
[pic]
Figure 2.8 Synthesis of NO from L-arginine (Manukhina et al. 2006).
NO released from the endothelium diffuses into vascular smooth muscle where it activates soluble guanylyl cyclase by binding to its heme group. This increases cyclic guanosine monophosphate (cGMP) which activates GMP-dependent kinases and downstream signaling that ultimately decreases intracellular calcium concentration and vasorelaxation. In conditions where NO production is impaired or bioavailability is reduced results in endothelial dysfunction and impaired vasodilation in models of forearm I-R injury in heathy humans (Loukogeorgkis et al. 2006) and is characteristic of various clinical conditions such as cardiovascular disease (Kleinbongard et al. 2006), sleep apnea (Atkeson et al. 2009) and diabetes (James et al. 2004). Experiments where NOS activity is pharmacologically inhibited document impaired blood flow and FMD response at rest and during exercise (Cosby et al. 2003; Jones et al. 2004; Radegran and Saltin 1999). Whereas exercise training (Goto et al. 2003; Vassalle et al. 2003), IH (Bertuglia 2008; Manukhina et al. 2006; Wang et al. 2007a) and NO donors (Duranski, et al. 2005; Maher et al. 2008; Maxwell et al. 2001; Webb et al. 2004) enhances vasodilation, tissue protection and exercise performance.
[pic]
Figure 2.9 NO synthesis in endothelium for vasodilation and implications for vascular NO storage for delivery of vasodilation and vascular O2 sensing and oxygenation state of the RBC (O2Hb or HHb) (Dejam et al. 2004)
2.10 Fate of vascular NO
Only ~5-20 nM is required to elicit vasodilation (Beckman and Koppenol 1996). The direct measurement of free NO in blood is extremely difficult due to its short half-life estimated to be ~1.8 msecs which may vary depending on the concentration of free and RBC bound Hb which are potent scavengers of free NO (Liu et al. 1998) and the distribution density of capillary blood vessels (Lancaster, Jr. 1994). Once NO is released from the endothelium and into blood vessel lumen can lead to the formation of more stable and readily detectable NO-metabolites: nitrate (NO3•), nitrite (NO2•) and S-Nitrosothiols (RSNO) (figure 2.9). NO also reacts with the superoxide anion (O2•) to produce the highly reactive and damaging oxidant, peroxynitrite (ONOO•). Since peroxynitrite decays in only a few seconds and modifies tyrosine residues forming nitrotyrosine (3-NT) make this a reliable marker for peroxynitrite generation and NO-dependant oxidative stress (Beckman & Koppenol 1996). Direct intra-arterial infusion or inhalation of NO gas elevated plasma concentrations of RSNO, nitrate and nitrite which led to peripheral blood vessel relaxation (Cannon, III et al. 2001; Cosby et al. 2003; Rassaf et al. 2002a & b). Thus by preserving NO bioavailability and preventing inactivation by Hb, molecular O2 or superoxide, the vascular ‘NO reservoir’ is then transported in the blood to target sites in downstream vasculature where they are enzymatically converted back to NO depending on local O2 conditions and metabolism. Each metabolite can be modulated by the activation and interaction of several signaling pathways. In particular, the sensitivity of the nitrite-HHb-NO pathway is of interest for eliciting vasodilation in regions where O2 supply is low and metabolism is elevated (Gladwin et al. 2004).
2.11 S-Nitrosothiols (RSNO)
Rassaf et al. (2002a) suggested that NO gas administered intravenously exerts most of its systemic effects via its conversion to RSNO. Since only a small amount of NO is converted to RSNO its physiological significance is not entirely clear because of analytical limitations and the fact that it exists in small concentrations in human blood typically less than 50nM (Bailey et al. 2009a; Marley et al. 2000; Rassaf et al. 2002a). The formation of RSNO involves multiple pathways which lead to the NO-dependent S-nitrosation of thiol-containing proteins and peptides such as glutathione and albumin and producing RSNOs such as S-nitrosogluthione (GSNO) and S-nitrosoalbumin (AlbS-NO) (Hogg 2002; Stamler et al. 1992). RSNOs are subdivided into low and high molecular weight species which significantly contribute to the NO reservoir although the main form of RSNO in plasma is AlbS-NO (Giustarini et al. 2003). The precise route for RSNO generation depends on biochemical factors such as pH and oxygenation status (Hogg 2002;Jia et al. 1996). The majority of RSNO is synthesised from the reaction between thiol (RSH) and acidified nitrite (eqns 1 and 2). Alternatively, in neutral pH and well oxygenated solutions the reaction involves the autooxidation of NO and formation of dinitrogen trioxide (N2O3). This intermediate is a good nitrosating agent which then reacts with thiol to produce RSNO and nitrite (eqn 3). RSNO can also be produced using peroxynitrite as a nitrosating agent which is more likely to develop during inflammation reactions (Williams 1997) and can occur independently of O2 (Marley et al. 2001).
NO+ + O2 → N2O3 (+ RSH) → RSNO + NO2• (1)
HNO2 + H+ → NO+ + H2O (2)
→ RSH + NO+ → RSNO + H+ (3)
RSNOs regulate vascular tone, platelet aggregation and blood flow and increases in RSNO by as little as 30nM was shown to exert vasodilation in conduit and resistance arteries (Rassaf et al. 2002a). The regeneration of NO from RSNO requires metal ions such as copper (Williams 1997) and iron (Vanin et al. 1997) and reducing agents. In vitro evidence show xanthine oxidase decomposes RSNO via superoxide anion dependent and independent mechanisms and in the presence of O2 to form peroxynitrite resulting in tissue injury (Trujillo et al. 1998). Furthermore, the increased RSNO that followed I-R injury in rabbit liver was associated with inducible NOS (iNOS) expression, impaired blood flow and mitochondrial dysfunction during the late phase of reperfusion (Glantzounis et al. 2007). Antioxidant administration eliminated organ injury, inhibited iNOS activity and prevented the increase in RSNO implying RSNO is produced during inflammatory reactions. The protective role of RSNO was demonstrated by reducing I-R injury in skeletal muscle (Hallstrom et al. 2002) and myocardium in animals (Dworschak et al. 2004) by enhancing O2 delivery and consumption.
2.12 Nitrate (NO3•)
Nitrate forms the largest vascular storage pool for NO (Rassaf et al. 2002b). It is produced in the blood by the O2Hb-dependant reaction with nitrite producing nitrate and methaemoglobin (equation 4) before it is released back into plasma (figure 2.10).
NO2• + O2Hb → NO3• + MetHb (4)
Plasma nitrate levels are affected by exercise training possibly due to a chronically elevated eNOS activation (Vassalle et al. 2003; Wang 2005) and by diet via ingestion of nitrate containing foods such as beetroot (Larsen et al. 2010; Lundberg et al. 2008) and is profoundly reduced after 4 days of low nitrate diet (Wang et al. 1997). Nitrate is broken down by nitrate reductase activity of commensal bacteria in the oral cavity before entering the circulation when saliva is swallowed. Subsequently, increases in nitrate were prevented when subjects were instructed not to swallow after ingestion of a large bolus of nitrate (Lundberg and Govoni 2004) or by antibacterial mouthwash (Govoni et al. 2008) highlighting its salivary origin. Nitrate can be reduced back to NO by xanthane oxidase (XO) under hypoxic (Millar et al. 1998) and normoxic conditions (Jansson et al. 2008). However the high background concentrations and long half life of nitrate of ~6 hours raises questions regarding its physiological significance in the human blood and should not be relied upon as a marker of eNOS activity (Lauer et al. 2001). Lauer et al. (2001) showed no change in nitrate following acetylcholine induced elevations in forearm blood flow (FBF). Rather increases in nitrite were observed whilst NOS inhibition dose dependently reduced nitrite and FBF indicating only nitrite reflects acute changes in eNOS activity. Similar observations were reported during flow mediated dilation (FMD) of the brachial artery (Rassaf et al. 2006) and no change in nitrate was observed following an acute bout of maximal exercise (Rassaf et al. 2007). Therefore nitrate may be considered an inert vascular NO metabolite. Dietary supplementation of sodium nitrate was shown to reduce submaximal [pic]O2 (Larsen et al. 2007) and [pic]O2max although exercise time to exhaustion was increased (Larsen et al. 2010). The authors speculated this could be a result of improved mitochondrial efficiency although it is more likley mediated by improved nitrite bioavailability and blunted O2 extraction.
2.13 Nitrite (NO2•)
Nitrite is the primary oxidation product of NO in human blood (Dejam et al. 2004). Administration of NOS inhibitors decrease plasma nitrite by 80% resulting in elevated vascular resistance, reduced forearm blood flow and impaired endothelium dependant vasodilation highlighting the sensitivity of nitrite as a biomarker of eNOS activity (Kelm et al. 1999; Kleinbongard et al. 2003). Nitrite can also be presented into human circulation from dietary sources such as cured meat products (Lundberg & Govoni 2004) and beetroot (Larsen et al 2010). Its concentration in human plasma is subject to some individual variation (typically 100 to 500nM) depending on lifestyle factors such as regular exercise, diet and smoking (Kleinbongard et al. 2006; Tsuchiya et al 2002) and chronic altitude exposure where a 10-fold greater nitrite concentration was reported in high altitude Tibetan natives at 4,200m compared to low-altitude residents which resulted in more than double forearm blood flow at rest and during exercise (Erzurum et al. 2007).
Nitrite levels correlate positively with endothelial function evaluated by FMD in humans (Kleinbongard et al. 2006). Furthermore, Duranski et al. (2005) showed in mice intravenous infusion of sodium nitrite at doses as low as 1.2nmol (and optimal at 48nmol) reduced I-R injury of the heart and liver after 30-45 minutes occlusion by factors independent of eNOS and nitrite-HHb-NO bioactivation. The protective effects of nitrite have been reported in other models of I-R injury via modulation of hypoxic gene activation and other protective factors such as haemoxygenase-1, heat shock protein expression (Bryan et al. 2005; Gladwin et al. 2006; Raat et al. 2009; Webb et al. 2004) or mitochondrial complex-1 inhibition and mechanisms limiting cellular superoxide production (Shiva et al. 2007). Lang et al. (2007) gave inhaled NO gas to humans undergoing liver transplantation and showed a twofold increase in plasma nitrite which was associated with improved restoration of liver function and reduced injury. Prevention of nitrite depletion via dietary sources also reduced liver damage after IR injury and prevented leukocyte adhesion, vascular inflammation and endothelial dysfunction in mice (Bryan 2009; Raat et al. 2009; Stokes et al. 2009).
Nitrite mediated hypoxic-vasodilation ensures O2 delivery matches its demand in hypoxia at rest (Maher et al. 2008) and during exercise with and without NOS inhibition (Cosby et al. 2003; Gladwin et al. 2000). The mechanisms regulating nitrite mediated vasodilation depend on its reactivity with HHb in RBCs. Despite concentrations of nitrite being 5-fold greater in RBC compared to plasma, nitrite can enter the RBC by pH-mediated diffusion or via a sodium dependant phosphate transporters (Dejam et al. 2005; May et al. 2000). The nitrite reductase activity of HHb increases NO production in low pH and hypoxic conditions (Cosby et al. 2003; Nagababu et al. 2003 & 2006) in processes which may involve xanthine oxidoreductase (Webb et al. 2008). This relationship was first described by Doyle et al. (1981) who showed optimal conditions for the reaction between nitrite and HHb to form NO is when Hb O2 saturation is between 40-60% (figure 2.10). Alternatively, nitrite can induce vasodilation independently of nitrite reductase activities whereby NO or nitrite enters the RBC leading to formation of nitroso proteins and nitrosyl products and subsequently, SNO-Hb releases RSNO during Hb desaturation for vasodilation (Dalsgaard et al. 2007;Jia et al. 1996;Stamler et al. 1997).
[pic]
1) HHb + NO2• → Hb-Fe(III) + NO
2) O2Hb + NO → Hb-Fe(III) + NO3•
Figure 2.10 The reaction between HHb and nitrite yielding NO and metHb (Hb-Fe(III) is optimal when Hb is 50% saturated (eqn.1). The reaction between O2Hb and NO to form nitrate dominate when O2Hb levels are greater than HHb (eqn.2) (Gladwin et al. 2005).
2.14 Reapportionment of NO metabolites
The arterial-venous difference for nitrite across the forearm at rest increases during exercise suggesting the nitrite reductase activity of HHb increases along the physiological O2 gradient depending on metabolic and environmental conditions (Dejam et al. 2005; Gladwin et al. 2000). In contrast, RSNOs present a reversed response whereby its concentration increases along the physiological O2 gradient (Gladwin et al. 2000). RBC bound NO levels also increase from coronary artery to vein (Rogers et al. 2007) therefore rather than assuming a net loss or gain of NO metabolites, it is possible a reapportionment or re-compartmentalisation of NO metabolites maintain total vascular NO pool. Rogers et al. (2007) showed no change in the total NO pool across the pulmonary and coronary circulation whilst the O2-dependant decrease in plasma nitrite and protein NO was matched by increased Hb bound NO. Blockade of eNOS activity had little effect on these changes suggesting a source another other than the endothelium is responsible. The reapportionment was reversed across the pulmonary vasculature as a function of Hb O2 saturation signifying an important role for the pulmonary circulation in normalising NO metabolite distribution. Others have shown similar cross-pulmonary (McMahon et al. 2002) and peripheral vascular exchange of NO metabolites as a function of Hb O2 saturation via reductase or oxidase activities (Gladwin et al. 2004) (figure 2.11).
[pic]
Figure 2.11 O2-dependant nitrite reductase (low O2) and oxidase activities of Hb (high O2). In the oxidase reaction nitrate is the primary product and intermediate formation of nitrogen dioxide (NO2). Nitrite reductase activities are associated with formation of NO-Hb products, HbNO and SNOHb (Gladwin et al 2004).
2.15 Oxidative inactivation, vascular dysfunction and nitrotyrosine
Gryglewski et al. (1986) was the first to show the inhibitory effects of oxidative stress on NO production in cultured endothelial cells whereby NO synthesis was protected from breakdown by SOD and Cu2+ but was inactivated by Fe2+ and concluded superoxide generation contributed to the instability of NO release. The reaction between NO and the superoxide anion (O2•) results in the formation of the highly reactive peroxynitrite anion (Beckman & Koppenol 1996). Its production in various tissues reflect NO-mediated oxidative stress and initiate reactions with lipids, DNA, and proteins that range from subtle changes in cell signaling to extensive oxidative injury and cellular apoptosis (Pacher et al. 2007). Impaired NO mediated hypoxic vasodilation in skeletal muscle of hypertensive and diabetic rats was related to an elevated oxidative stress since treatment with superoxide scavengers restored flow mediated dilation and improved the arteriolar dilator response to pharmacological stimulation (Frisbee 2001; Frisbee and Stepp 2001). Similar observations were shown in obstructive sleep apnoea patients, a condition characterised by elevations in vascular oxidative stress and reduced NO availability whereby intravenous vitamin C administration restored the FMD response to levels comparable with healthy control subjects (Grebe et al. 2006).
3-NT is produced by the diffusion limited nitration of free or protein bound tyrosine with peroxynitrite. Its presence in biological tissue is decribed as evidence of peroxynitrite formation (Beckman & Koppenol 1996; Mohiuddin et al. 2006). Protein bound tyrosine attached to low density lipoproteins (LDL) are prime candidates for nitration amongst the plasma proteins (Khan et al. 1998). The reaction is catalysed by the metal centers of SOD although superoxide production can be dismutated by SOD activity thus reducing peroxynitrite and nitrotyrosine formation in LDL (Khan et al 1998). However, NO may out-compete SOD for superoxide when produced in high concentrations, favouring peroxynitrite formation and 3-NT levels rise when large fluxes of superoxide is produced more rapidly than it is dismutated such as during intense exercise (Fatouros et al. 2004). The oxidation of nitrite to nitrogen dioxide by MPO can also nitrogenate tyrosine in inflammatory conditions such as atherosclerosis and is also related to increased 3-NT formation (Mohiuddin et al. 2006; Pennathur et al. 2004). However the small concentration of nitrite and nitrogen dioxide in vivo is much lower than necessary to cause significant nitration in vitro suggesting peroxynitrite is the most likely source of 3-NT formation in human blood (Beckman & Koppenol 1996).
The detection of 3-NT in human plasma has been subject to technical concerns due to a lack of sensitivity and specificity from some detection methodologies such as HPLC and ELISA (Tsikas et al. 2002). At rest these have varied considerably ranging from undetectable to 120nM in healthy humans and increases in clinical populations associated with chronic inflammation such as diabetes (Ceriello et al. 2001; Shishehbor et al. 2003;Tsikas et al. 2002). The presence of nitrotyrosine in diabetic patients suggests a potential role of peroxynitrite formation in vascular dysfunction and is implicated as inflammatory mediators in coronary artery disease (CAD) and arteriosclerosis (Ceriello et al. 2001; Shishehbor et al. 2003). Bailey et al. (2009b) showed an increased net output of cerebral 3-NT levels following 9 hr passive exposure to hypoxia (FIO2 = 12.9%) in healthy male subjects which correlated with the development of acute mountain sickness and therefore could contribute to other cerebrovascular and cardiovascular complications. The elevated nitrotyrosine levels documented following an acute bout of maximal exercise in older men was reduced by endurance training whilst detraining abolished these exercise induced adaptations (Fatouros et al 2004). It is unknown if healthy, young subjects follow a similar pattern following an acute bout of exercise.
2.16 NO and exercise
The primary physiological stimulus for eNOS activation is shear stress, the frictional force on the blood vessel wall due to blood flow (Dimmeler et al. 1999; Rubanyi et al. 1986). The magnitude of the stimulus increases during exercise and NO may be a strong candidate for initiating the microcirculatory adjustments that occur during acute exercise and regulate vascular adaptation to chronic exercise (Maiorana et al. 2003; Tschakovsky and Joyner 2008). It should be recognised that other vasodilators such as ATP (Gonzalez-Alonso et al. 2002), constrictor factors e.g. endothelin-1 (Wray et al. 2007), sympathetic activation, (systemic or metabolic induced) hypoxia (Hansen et al. 2000) as well as from the direct action of the muscle pump itself (Sheriff 2003) contribute to muscle blood flow and may interact with NO as a function of exercise intensity. In patients with CAD, an acute bout of intense exercise caused significant elevations in oxidative stress and impaired FMD whereas moderate intensity exercise improved FMD (Farsidfar et al. 2008). Similar observations were shown following a 12 week exercise training intervention where moderate intensity training at 50% [pic]O2max enhanced NO dependant vasodilation (Goto et al. 2003). These effects were abolished by NOS inhibitors and high intensity exercise training at 75%VO2max which was associated with increased oxidative stress and impaired forearm blood flow response to acetylcholine. These studies suggest an intensity dependant effect on NO-induced vasodilation during exercise and modulated by the balence between NO and oxidative stress.
The effects of acute exercise on plasma NO metabolites have been contradictory. Gladwin et al. (2000) showed during handgrip exercise nitrite consumption was increased estimated by arterial-venous gradients across the active muscle. Others have shown an increase in systemic nitrite 10 minutes after maximal cycling where blood was taken from a (forearm) vein and was a good predictor of exercise capacity and discrimated the severity of cardiovascular disease (Allen et al. 2009; Rassaf et al. 2007). In contrast, Larsen et al. (2010) showed a decrease in nitrite in healthy subjects immediately after exercise which was explained mechanistically by a greater rate of nitrite consumption versus the rate of shear stress induced eNOS activation and NO oxidation back to nitrite. These discrepancies may be indicative of the kinetic nitrite response during and after maximal exercise where haemodynamic control is also likely to differ. Ingestion of large doses of nitrate profoundly increased plasma nitrite and nitrate compared to a low nitrate diet resulting in reduced submaximal (Larsen et al. 2007) and [pic]O2max although exercise time to exhaustion was significantly enhanced (Larsen et al. 2009). Similar observations were shown by direct ATP infusion during maximal cycling exercise at 4559m resulting in a 20% reduction in leg [pic]O2max compared to control (Lundby et al. 2008). Therefore it is likely some degree of muscle vasoconstriction is needed to match O2 delivery with demand during exercise.
NOS inhibition studies tend to show a blunted hyperaemic response during exercise resulting in impaired performance (Jones et al. 2004; Maiorana et al. 2003; Maxwell et al. 1998; Wilkerson et al. 2004). Since maximal HR is also reduced makes it possible the response was related to the decline in central delivery rather than microcirculatory factors. NOS inhibition studies should however be interpreted with caution because the effects of the drug appear to be masked by the hyperaemia induced dilution effect resulting in inadequate blocking levels. In particular, during exercise with large perfusion-to-muscle mass ratio there appears to be a lack of effect from NOS inhibition whereas smaller muscle groups tend to have a greater effect (Maiorana et al. 2003).
2.17 Cell adhesion molecules (CAM)
The main function of CAM is to reduce the speed of circulating leucocytes, drive the process of firm attachment and modulate trans-endothelial migration of immune cells to the inflamed region (Smith 1993). Expression of ICAM-1 and VCAM-1 on neutrophils and endothelial cells play a key role in this process and are reflected by their appearance in plasma following enzymatic cleavage or shedding (Leeuwenberg et al. 1992; Shiu et al. 2000). Subsequently sVCAM-1 and sICAM-1 are regarded as reliable markers of endothelial function and activation in healthy subjects (Holmlund et al. 2002), in clinical conditions such as sleep apnea (Ursavas et al. 2007) and type 2 diabetes mellitus (Singhania et al. 2008) or following an acute vascular insult and mechanical injury such as stroke (Frijns and Kappelle 2002) and intense exercise (Akimoto et al. 2002; Monchanin et al. 2007; Rehman et al. 1997; Silvestro et al. 2002) but remain relatively unaffected by moderate intensity exercise (Jilma et al. 1997; Simpson et al. 2006). However sICAM-1 may inhibit leukocyte adhesion by binding to CD11a/CD18 and peripheral blood mononuclear cells (PBMC) leaving less available for endothelial binding and therefore a reduced sICAM-1 and sVCAM-1 response may reflect a more efficient leukocyte binding (Mills et al. 2006; Ohno and Malik 1997).
Concentrations of sICAM-1 and sVCAM-1 are regulated by a variety of signaling factors and activation pathways. Exposure to pro-inflammatory mediators such as TNF-α, IL-1 and NF-kappa B stimulate endothelial cell surface expression and release of CAMs and regulates endothelial cell mortality via NO dependant pathways (Kevil et al. 2004; Sahnoun et al. 1998). Activation may also be modulated by catecholamine release since treatment with B-adrenergic antagonists attenuated the exercise induced increase in sICAM-1 in humans (Rehman et al. 1997). Endothelial cells exposed to shear stress and TNF-α showed an elevated ICAM-1 and reduced VCAM-1 expression suggesting differential roles of shear stress during cytokine induced endothelial cell activation (Chiu et al. 2004). Increased sVCAM-1 was associated with oxidative stress and vascular complications arising from type-2 diabetes (Singhania et al 2008). Administration with low doses of vitamin C showed a tendency to reduce basal concentrations of sICAM-1 (Witkowska 2005) and attenuated the increase in sICAM-1 following maximal exercise in subjects with intermittent claudication whilst preventing the acute impairment in FMD (Silvestro et al. 2002). Steiner et al. (2002) showed the extent of leucocyte adherence in mesenteric venules of rats was proportional to the rise in ROS while breathing 15, 10 and 7.5% O2 (figure 2.12). Although breathing 15% O2 increased ROS generation, there was no change in leucocyte adherence suggesting attainment of a threshold ROS value is required before initiating an inflammatory response. Administration of NO donors and antioxidants prevented leucocyte adherence during 10% O2 exposure and since there was no difference in shear rate between groups gives further evidence that the response is modulated by the balance between ROS and NO (figure 2.12, Wood et al. 1999a).
[pic][pic]
Figure 2.12 Effect of hypoxia (left, Steiner et al. 2002) and antioxidant administration (right, Wood et al. 1999a) on leukocyte adherence. Leukocyte count was measured as the number of cells that remained stationary for longer than 30sec during video analysis.
Previous studies show NOS inhibitors dose-dependently augment and NO donors inhibit leukocyte adherence and expression of ICAM-1 and VCAM-1 following an acute inflammatory stimulus and highlight the anti-inflammatory effects of NO (Berendji-Grun et al. 2001; Dal et al. 2006; De et al. 1995; Lindemann et al. 2000). Supplementation of nitrite in the drinking water of hypercholesterolemic mice inhibited leukocyte adhesion and emigration through the endothelium preventing arteriolar dysfunction and reducing vascular inflammation (Stokes et al. 2009). However the specific pathway which NO modulates expression of CAM and transmigration of neutrophils is unclear although the effects may be controlled through mechanisms involving cyclic GMP (Dal Secco et al. 2006). Also, increased peroxynitrite formation were observed in cultured endothelial cells in direct contact with neutrophils (Sohn et al. 2003) highlighting a role for peroxynitrite as a cell signaling molecule during inflammation reactions and direction for the acute immune response.
2.18 Intermittent hypoxia (IH)
IH is characterised by recurrent episodes of low O2 breathing interspersed with periods of normoxia (reoxygenation) where each hypoxic exposure ranges from a few seconds in duration to several minutes or hours and is characterised by a constant swinging of SaO2. IH can elicit beneficial or detrimental effects depending on the duration, intermittence and depth of hypoxia (Beguin et al. 2005; Neubauer 2001) initiating adaption at various levels along the O2 transport cascade by enhancing carotid body chemosensitivity, mitochondrial efficiency and cerebrovascular and cardiovascular hypoxic sensitivity (Foster et al. 2005; Ainslie et al. 2007; Katayma et al. 2001 & 2005). In addition, IH exerts tissue protection by functioning as a preconditioning stimulus against future ischemic or hypoxic stress (Bertuglia 2008; Zong et al. 2004). These cross-adaptive effects have led to the wide-spread application of IH into a variety of healthy and clinical settings (Serebrovskaya 2002). For example, competitive athletes and mountaineers incorporate IH into their training schedule when preparing for athletic performance at altitude and sea-level to enhance efficiency of training, O2 delivery and utilisation and to ultimately improve aerobic performance (Beidleman et al. 2003; Katayama et al. 2003; Wang et al. 2007a). However the effects of IH on aerobic and anaerobic exercise performance remain a contentious issue (Levine 2002). IH has also been used in a clinical setting for the treatment of conditions such as asthma, hypertension and diabetes through a more efficient and effective NO production and storage capacity and by reducing oxidative stress (Serebrovskaya 2002; Manukhina et al. 2006). The majority of current literature on IH has focused on animal research and there is a lack of studies examining the effect of IH on vascular function in humans. The remainder of this literature review will focus on the mechanisms driving adaptation with implications for ROS and NO, endothelial activation and exercise performance.
2.19 Adaptive mechanisms 1: Genetic modulation
Adaptation to hypoxia is complex and subject to considerable individual variability (Chapman et al. 1998). The response requires direct interplay between several signaling factors and central to this is requires activation of the O2-sensitive transcription factor, hypoxia inducible factor-1 (HIF-1) (Semenza et al. 1997). HIF-1 is a heterodimer composed of HIF-1α and HIF-1β subunits although it is expression of HIF-1α that is regulated by O2 availability in a dose-dependent manner and regulates the specific adaptations to hypoxia such as angiogenesis and erythropoiesis (Semenza et al. 1997; Wang et al. 1995; Maxwell 2005) (figure 2.13). Expression of HIF-1α decreases progressively during continuous hypoxia exposure (Chavez et al. 2000) and rapidly degrades in the presence of O2 with a half-life of 1.1, pedalling frequency < 70 rpm and heart rate within 10% age related maximum.
3.3 Heart rate
Heart rate was measured during the final 10 seconds of each workload and at peak power output (PPO) using a Polar heart rate monitor. Each subject was fitted with an adjustable chest strap and monitor with build-in electrodes and measurement of heart rate was displayed continuously throughout the test on a watch fixed to the handlebars of the cycle ergometer.
3.4 Arterial oxygen saturation (SaO2)
SaO2 was measured using finger pulse oximetry (Novametrix, Oxypleth 520A, UK). The light emitter was placed of the finger nail side and a detector on the opposite side of the finger. The light emitter passes red and infrared light though the finger and the ratio of light emittence and absorption determine the SaO2. During exercise trials, subjects were requested to rest their hand on top of the handlebars to avoid restricting blood flow to the finger during exercise. A reliable signal was confirmed by a plethysmograph waveform displayed on the pulse oximeter.
3.5 Near Infrared Spectroscopy (NIRS)
Prior to each exercise test, subjects were instrumented with two pairs of near-infrared (NIR) probes (Oxymon, Artinis, The Netherlands) for measurement of cerebral and muscle oxygenation. The NIR probes were placed over the belly of the right vastus lateralis and the left frontal cortex region of the forehead. The light emitter and detector probes were held in place by a plastic spacer with a fixed optode distance of ~4cm and ~5cm for the cerebral and muscle measurements respectively (Subudhi et al. 2007). This particular optode arrangement minimises the effect of skin blood flow known to contribute to the NIRS signal (Owen-Reece et al. 1996) and presents a penetration depth for cerebral tissue which is enough to illuminate the grey matter but not deeper brain structures. The spacer was fixed to the skin by double sided tape and elastic bandages were also used to prevent light and movement artifacts.
Changes in tissue oxygenation across time are calculated using the Beer-Lambert law which quantifies the attenuation of light passing through a non-scattered medium. When applied to biological tissues the modified Beer-Lambert equation which accounts for light scattering is as follows:
ODλ = log (Io/I) = єλ · с · Lo · DPF + ODλx
Where ODλ is the mediums optical density, Io is the incident light intensity, I is the transmitted light intensity, єλ is the extinction coefficient of the chromophore (mMˉ¹ · cmˉ¹), c is the concentration of the chromophore (mMˉ¹), Lo is the distance (cm) between the light entry and exit, and λ is the wavelength of the light used. The DPF is given by L/Lo, where L is the average distance travelled by each photon. ODλx accounts for attenuation due to scattering and absorption of other chromophores.
Optical densities of two continuous wavelengths of NIR light at 780nm (deoxygenated signal) and 850nm (oxygenated signal) and DPFs of 4.95 (Duncan et al. 1995) and 5.93 (van der Zee et al. 1992) for muscle and cerebral measurements respectively are used to calculate cerebral and muscle oxygenation respectively. It is not possible to get absolute measurement of oxygenation because the exact DPFs for brain and muscle tissue for each individual are not known. Subsequently, baseline measurements were set after 15 minutes semi-supine rest in normoxia and tissue oxygenation measurements were taken as 60 second averages at rest and at the end of each workload up until the point of volitional exhaustion. Each oxygenation value is expressed as micromolar changes from baseline (∆µM) in O2Hb and HHb and calculated oxygenation variable THb (O2Hb + HHb).
3.6 Blood sampling
All blood samples were taken from an antecubital vein after a 12 hour overnight fast. A blood pressure cuff was fixed to the distal region of the subjects’ bicep and cuff pressure was increased to 90mmHg. A prominent antecubital vein was sterilised and an 18 gauge cannula was inserted and connected to a 3-way sterile stopcock. Resting blood samples were taken after 20 minutes after cannulation and immediately after maximal exercise. An initial 3ml blood sample was disregarded before a 5ml syringe sample was taken for duplicate measures of blood gas, Hb and Hct. Subsequent blood samples were drawn into two 5ml ethylenediaminetetra-acetic acid (EDTA) vacutainers. After each blood sample was taken 5ml of physiological saline mixed with 0.1ml heparin was injected to keep the line patent. The EDTA vacutainer was centrifuged immediately for 10 minutes at 3,000 rpm in 4ºC. 1ml plasma was transferred immediately into 1.5ml plastic vials (Eppendorf, Germany), snap frozen in liquid nitrogen and stored in -80ºC before analysis.
3.7 Ozone-based chemiluminescence: measurement of plasma NO metabolites
All chemicals were purchased from Sigma except glacial acetic acid, HPLC grade and nitrite free water from Fisher Scientific. Using the appropriate reductive method, ozone-based chemiluminescence is a highly specific, reproducible and sensitive technique for detection of plasma NO metabolites; nitrite, nitrate and RSNO. The technique has been validated with other analytical techniques for NO metabolites such as HPLC (Dejam et al. 2005) with referenced limits of detection less than 1pmol (Yang et al. 2003). Plasma samples were injected into the gas purge vessel containing the reducing agent which can selectively reduce NO species to release NO gas into solution. Helium is purged through the mixture to force the NO gas out of solution and into the headspace where it is carried through a sodium hydroxide trap to the reaction chamber of the Sievers NO chemiluminescence analyser (NOA). The NOA generates ozone (O3) which reacts with NO gas yielding NO2* in an excited state. A photon is emitted as the excited electron returns to its ground state which is detected as chemiluminescence (hv):
NO + O3 → NO2* + O2
NO2* → NO2 + hv
The chemiluminescent reaction is rapid and takes place in the gas phase therefore the NOA is connected directly in line with where the NO gas is formed. The emitted light is detected and amplified by a photomultiplier tube to generate an electrical signal which is then recorded by data recording software. Quantification of the NO metabolite involves conversion of the NO amplified signal into an actual concentration using the standard curve.
The tri-iodide reducing reagent is the most extensively used reducing agent when measuring nitrite and RSNO in human blood (Rogers et al 2008; Yang et al. 2003). This is due to its powerful reductive capabilities and ability to reduce a number of NO metabolites. Samples need to be pre-treated to ensure specificity of signal for the NO metabolite of interest. The addition of acidic sulphanilamide eliminates nitrite from the sample by converting it into a diazonium cation which is undetectable by chemiluminescence and the remaining signal represents RSNO.
Tri-iodide (I3ֿ) is available in solution as free iodine (I ˉ) and iodide (I2). Free iodine reacts with nitrite producing stoichiometric amounts of NO and iodide side products.
I3 ˉ → I2 + I ˉ
2NO2ˉ + 2I ˉ + 4H+ → 2NO +I2 + 2H2O
I3ֿ also reduces RSNO, releasing free iodine, thiyl radicals which combine to form the disulfide RSSR and nitrosonium cation. By reaction with free iodine, nitrosonium cation is readily converted to NO gas resulting in NO gas from RSNO.
I3ˉ + 2RSNO → 3I ˉ + RSSR + 2NO
2NO + 2I ˉ → 2NO + I2
Freshly prepared tri-iodide solution was made each day. The 5ml tri-iodide solution was contained in the glass purge vessel containing 20µL of antifoam and heated to 50ºC in a thermostatically controlled water bath. Frozen plasma samples were thawed in a 37ºC water bath for 3 minutes. A 200µl plasma sample was injected directly through septum on sidearm of purge vessel for determination of nitrite + RSNO (figure 3.1). A second 270µl plasma sample was pre-treated with 30µl acidified sulphanilamide and incubated at room temperature in the dark for 15mins to eliminate nitrite from the sample. At the end of incubation period, 200µl of the sulphanilamide plasma was injected into the glass purge vessel for determination of plasma RSNO. The pre-treated sample was subtracted form the untreated sample to determine nitrite.
[pic]
Figure 3.1 – Example of raw chemiluminescence signal observed for plasma sample
Plasma nitrate is analysed using a vanadium chloride reducing agent. Thirty ml of vanadium chloride solution was contained in a glass purge vessel containing 20µL of antifoam which was heated to 85ºC in a thermostatically controlled water bath. Frozen plasma samples were thawed in a 37ºC water bath for 3 minutes. A 20µl plasma sample was injected directly through septum on sidearm of purge vessel for measurement of total NO (i.e. nitrate, nitrite and RSNO). The average of two consecutive measurements was taken as the definitive value. Nitrate was determined by subtracting nitrite + RSNO from the total NOx value. The inter- and intra-assay CV for this assay is 7 and 10%.
3.8 sICAM-1, sVCAM-1 and 3-NT
Coating and saturation - The 3-NT measurements were analysed using ready-to-use commercially available kits from Hycult Biotechnology with a minimum detection limit of 2nM (The Netherlands). Adhesion molecules were analysed by the ELISA method using commercially available kits from Eli-pair (Diaclone, Besancon, France). For each plate 100µL of capture antibody was added to each well of the microplate. The plate was covered with an adhesive sealer and incubated overnight in 4°C. After 2 washes with 400µL of PBS-Tween 0.05%, each well was blocked with 250µL of saturation buffer for 2 hours at room temperature. The plate was then emptied and left to dry for 24 hours.
Method - Standards and plasma samples were diluted in standard diluent buffer and 100µL was added to each well including blank cells for zero standard concentration. Standard and plasma samples were analysed in duplicate. The vial of biotinylated detection anti-ICAM-1, VCAM-1 and 3-NT was reconstituted with 0.55ml of PBS 0.1% azide. For each plate, 100µL of this solution was added into 5ml of biotinylated antibody diluent buffer and 50µL was distributed into each well. The antigen and biotinylated detection antibody were co-incubated for 1 hour at room temperature and were washed 3 times with 400µL of wash buffer.
Colour development – A 100µL solution of HRP-Strep with HRP-Streptavidin diluent buffer was added into each well and incubated for 20 minutes. Then 100µL of TMB was distributed and left to develop for 10 minutes in the dark. This reaction was stopped with 100µL of 1M H2SO4 and absorbance was read at 450nm with a reference filter set to 630nm. Concentrations of sICAM-1 and sICAM-1 were then calculated from the standard curve in ng/mL and the averages of the duplicate samples were taken as the definitive value. The intra and inter assay CVs were 4.7 and 11.8% respectively for sVCAM-1. The intra and inter assay CVs were 5.8 and 1.5% respectively for sICAM-1.
3.9 Ascorbate free radical (A•)
A• is regarded as a direct biomarker of oxidative stress (Buettner & Jurkiewicz, 1993; Bailey, 2004; Bailey et al. 2006). Exactly 1 ml of K-EDTA plasma was injected into a high-sensitivity multiple-bore sample cell (AquaX, Bruker Daltonics Inc., Billerica, MA, USA) housed within a TM110 cavity of an EPR spectrometer operating at X-band (9.87 GHz). Samples were analysed using a modulation frequency of 100 kHz, modulation amplitude of 0.65 gauss (G), microwave power of 10mW, receiver gain of 2×105, time constant of 41 ms, magnetic field centre of 3477 G and scan width of ± 50 G for three incremental scans. After identical baseline correction and filtering, each of the spectral peak-to-trough line heights was normalized relative to the square root of line width in G and the mean considered a measure of the relative concentration of A• following conformation of peak-to-trough line-width conformity and double integration on a random selection of samples. A typical EPR trace is presented in figure 3.2. The intra- and inter assay coefficients of variation were both 80% PPO) (Figure 4.5E). The HHb: workload slope greater during the hypoxia trial when expressed in absolute but not relative terms which suggests the attainment of maximal rate of deoxygenation (by O2 extraction) was driven by the relative metabolic stress. Visual inspection of the relative HHb profile revealed a tendency for a sigmoidal profile in normoxia and hyperbolic profile in hypoxia (Figure 4.5E). The difference in change in HHb from 60W to PPO (([HHb]60-PPO, Norm: 13.9 ± 4.5 ΔµM; Hyp 14.1 ± 3.2µM, P < 0.05) and from 30 to 100%PPO (([HHb]30-100%, Norm: 12.5 ± 3.9µM; Hyp: 13.1 ± 2.7µM, P > 0.05) was not different between trials further suggesting attainment of PPO could be driven by the O2 extraction response. Muscle THb increased with workload peaking at 80% PPO in both trials (figure 4.5 C & F). Peak THb tended to be higher in normoxia whereas during low intensity exercise THb was higher in hypoxia. The transition from rest to exercise (60W) resulted in a greater difference in hypoxia versus normoxia for HHb and THb (P < 0.05) whereas there was no significant effect for O2Hb.
Cerebral oxygenation - Due to technical issues one subject was excluded from the cerebral analysis. There was considerable individual variability for each cerebral NIRS measurement. In contrast to muscle, cerebral O2Hb increased during incremental exercise and tended to plateau at workloads >80% PPO (Figure 4.6 A & D). Cerebral HHb was greater throughout the hypoxia trail however after a period of relatively no change during the low-moderate intensity workloads, cerebral HHb increased at workloads >50% PPO in both tests (Figure 4.6E). Cerebral THb increased with workload (P < 0.05) however there was a tendency for a rightward shift in hypoxia and at PPO THb tended to be greater in normoxia (Figure 4.6F).
Table 4.3 - Muscle oxygenation slopes and intercept
| |∆O2Hb/∆WR |∆HHb/∆WR |∆THb/∆WR |∆HbDiff/∆WR |
| |Absolute |Relative |
|Age (year) |21 ± 1 |26 ± 6 |
|Height (m) |1.8 ± 0.7 |1.8 ± 0.6 |
|Mass (kg) |84 ± 7 |81 ± 6 |
|Hb (g/dL) |15.0 ± 1.0 |15.2 ± 0.6 |
|Hct (%) |45 ± 2 |47 ± 2 |
Experimental design
The experimental design for this study is presented in figure 5.2. Subjects were instructed to refrain from intense exercise and alcohol consumption for 48 h prior to each test and reported to the lab after a 12 hour overnight fast. A venous cannula was inserted into an antecubital vein (section 3.6) and NIRS probes were applied to the prefrontal cortex region of the forehead and belly of the right vastus lateralis muscle (section 3.5). Subjects performed 2 incremental cycling tests to volitional exhaustion before and 3 days after 2x 5 day blocks of IH or control (intermittent normoxia, IN). The incremental exercise test was conducted in an environmental chamber: temperature: ~20°C, relative humidity: ~50% and FIO2 = 0.12. Baseline measurements were taken in normoxia and NIRS assessment of cerebral and muscle oxygenation was measured continuously throughout a 10 minute resting stabilisation period in the chamber and during incremental exercise. Each cycle test adopted the same protocol (section 3.1). Determination of [pic]O2, [pic]CO2 and [pic]E was measured off-line using the Douglas bag method (section 3.2) during the final 60 seconds of the 60W workload and prior to volitional exhaustion. Heart rate and SaO2 were determined using short range telemetry system (section 3.3) and finger tip pulse oximeter (section 3.4). Fasted blood samples were taken at baseline and immediately after maximal exercise and analysed immediately for blood gas measurements, Hb and Hct or centrifuged at 3,000 rpm in 4°C. Plasma was aliquoted into 1.5 ml polypropylene tubes, snap frozen in liquid nitrogen and stored in -80°C until analysis for the following NO metabolites; nitrite nitrate and RSNO and CAM; sICAM-1 and sVCAM-1. Since blood samples were taken from a vein protruding a ‘non-active’ muscle, subjects were instructed to refrain from gripping the handle bars and the potential confounding influence of forearm ischemia.
[pic]
B B B B
Figure 5.2 - Experimental design for study 2. Subjects performed 2x incremental exercise (IE) tests in hypoxia (FIO2 = 0.12) before and 3 days after 2x 5 day blocks of intermittent hypoxia (IH) or control (IN). NIRS assessment for cerebral and muscle oxygenation was measured throughout IE and blood samples (B) were taken before and immediately after IE.
IH exposure
While in a seated position, subjects in the IH group breathed hypoxic air (FIO2 = 9.5% O2) through a hand held mask via an automated IH machine (section 3.13). Each exposure involved a 5 min cycle of hypoxic air breathing alternating with 5-min ambient air breathing for a total of 90-min. Each subject was exposed to 9 hypoxic-reoxygenation episodes per session for 10 sessions conducted over 2x 5 day blocks. SaO2 was measured at the end of each 5-min hypoxia exposure using finger tip pulse oximetry (section 3.4). This protocol was identical to the one utilised by Stuke et al. (2005) who reported a 20% improvement in exercise time to exhaustion in normoxia. Subjects were blinded to these measurements throughout each hypoxic exposure. The control group followed an identical protocol but breathed normoxic air (FIO2 = 0.21).
Cerebral and muscle oxygenation analysis
Cerebral and muscle oxygenation were expressed as relative changes from baseline (∆µM) set in normoxia while in an upright seated position. Changes in O2Hb, HHb and THb in both tissues were averaged over the final 60 seconds of the 10 minute rest period in hypoxia and during the final minute of each workload ranging from 60 to 180W and prior to volitional exhaustion. Throughout exercise, subjects were encouraged to refrain from any sudden movement of the head and upper body to avoid potential movement artifacts which are likely to impact on the signal for cerebral measurement.
Blood analysis
NO metabolites: nitrite, RSNO and nitrate
For a detailed description refer to methodology section 3.7.
sICAM-1 and sVCAM-1
For a detailed description refer to methodology section 3.8.
Hb and Hcrt
For a detailed description refer to methodology section 3.10 for Hb and 3.11 for Hct.
Blood gas
For a detailed description refer to methodology section 3.12.
5.5 Statistical analysis
Cerebral and muscle oxygenation changes were expressed as micromolar changes from resting baseline (∆µmol) and plotted against workload. Linear regression analysis was performed to determine the slope and intercept of the relationship between muscle oxygenation versus workload (∆µmol/W). The linear regression analysis was taken between each absolute workload ranging from 60 to 180W. Shapiro-Wilk W tests were employed to confirm distribution normality. Data were subsequently analysed with a three-way repeated measures analysis of variance [Group (IH vs. IN) x exercise intensity (rest-60-180W-PPO)] for each cerebral and muscle NIRS measurement or [Group (IH vs. IN) x State (rest vs. maximal exercise)] for blood measurements. Following a significant main effect and interaction, post-hoc Bonferroni-corrected paired samples t-tests were performed to locate differences. Significance was established at P < 0.05 and values presented as mean ± SD.
5.6 Results
Each subject completed all 10 IH sessions and average SaO2 response for each day is presented in figure 5.3. Some subjects reported initial discomfort and mild headache during the first few days of IH however these symptoms dissipated over the course of adaptation although there was no change in SaO2.
Figure 5.3 - Average daily resting SaO2 after each IH or IN exposure.
[pic]
Cardiorespiratory measurements
The [pic]O2 data are presented on figure 5.4. After IH, there was a tendency for [pic]O2peak to increase whilst [pic]O2 was lower during the 60W steady state workload although these changes were not significant. In the IH group, SaO2 increased slightly from 84 ± 4 to 87 ± 4% (P = 0.07) during the 10 minute rest period and during the 60W workload (figure 5.5) however there was no change in HR at rest or during exercise (figure 5.6). Average cardiorespiratory responses during the steady state 60W workload and at PPO are presented in Table 5.2. At PPO there was a 2.0% increase in [pic]CO2peak after IH (P < 0.05). During the 60W workload [pic]E/VO2 increased by 1.0 ± 0.9 L/min in the IH group (P < 0.01) and was related the decrease in [pic]O2 at 60W rather than augmented ventilatory response. There was no change in any cardiorespiratory measurements compared to baseline in the control group.
Figure 5.4 – Effect of IH on maximal and submaximal [pic]O2
[pic]
There was a main effect for intensity and time x intensity x group. After IH, there was a 3.5% decrease in [pic]O2 during the 60W workload (P = 0.06) and 2.3% increase in [pic]O2peak (0.07).
Figure 5.5 – Effect of IH on SaO2 during incremental exercise
[pic][pic]
After IH, SaO2 tended to be higher at rest and during the 60W steady state workload however as exercise progressed there was no difference from baseline.
Figure 5.6 – Effect of IH on heart rate during incremental exercise
[pic][pic]
There were no main effects for heart rate during incremental exercise after IH and IN.
|GROUP |Intermittent Hypoxia |Control |
|TIME |Before |After |Before |After |
|INTENSITY |60W |
|[pic]E, l/min | 31.7 ± 4.0 |
|[pic]E/VO2 |24.0 ± 1.9 |
|[pic]E/VCO2 |25.5 ± 1.8 |
|RER |0.94 ± 0.04 |
Table 5.2 – The effect of IH on cardiorespiratory measurements
* denotes different from before IH/IN (P < 0.05)
Cerebral oxygenation
Changes in cerebral oxygenation at rest and during incremental exercise are presented in figure 5.7. The IH group showed only a trend for O2Hb to increase at rest and during the high intensity workloads. There was no change in HHb however there was tendency for a leftward shift in the THb curve after IH. There was no change in any cerebral oxygenation variable in the control group.
Muscle oxygenation
Due to technical issues 2 subjects were excluded from the control group and results from 7 subjects were examined. Muscle oxygenation responses are presented in figure 5.8 and the results from the linear regression analysis for the slope and intercept values are presented in Table 5.3. There was no change in any resting muscle oxygenation variables in either group. Although there was no change in any slope measurements, the intercept was significantly greater for O2Hb and THb (P < 0.05) and tended be higher for HHb (P = 0.07) after IH. There was no change in any muscle oxygenation variables in the control group.
Table 5.3 - Slope and intercept for NIRS muscle oxygenation measurements
| |∆O2Hb/∆WR |∆HHb/∆WR |∆THb/∆WR |∆HbDiff/∆WR |
|Slope |Intercept |Slope |Intercept |Slope |Intercept |Slope |Intercept | |Before IH
After
IH |-0.05
±
0.02
-0.05
±
0.03 |-4.9
±
4.8
-2.0
±
6.7* |0.09
±
0.03
0.07
±
0.03 |2.8
±
4.0
7.2
±
6.6 |0.04
±
0.03
0.03
±
0.02 |-2.1
±
4.9
5.1
±
10.2* |-0.14
±
0.04
-0.12
±
0.05 |-7.8
±
7.4
-9.2
±
8.4 | |Before IN
After IN |-0.05
±
0.02
-0.04
±
0.03 |-6.5
±
6.4
-7.6
±
6.0 |0.09
±
0.02
0.09
±
0.02 |2.2
±
2.5
3.9
±
4.3 |0.04
±
0.02
0.05
±
0.02 |-4.6
±
5.6
-3.7
±
8.5 |-0.13
±
0.04
-0.13
±
0.05 |-8.7
±
-8.0
-11.6
±
6.0 | |
* Significantly different from baseline testing
Intermittent Hypoxia
[pic] [pic] [pic]
Intermittent Normoxia
[pic] [pic] [pic]
Figure 5.7 - Cerebral oxygenation before and after IH or IN
Intermittent Hypoxia
[pic] [pic] [pic]
Intermittent Normoxia
[pic] [pic] [pic]
Figure 5.8 - Muscle oxygenation before and after IH or IN
Figure 5.9 – NOx at rest and after maximal exercise
[pic]
Figure 5.9 shows the effect of IH and IN on NOx before and after maximal exercise in hypoxia. There were no significant main effects after IH other than a tendency for a time x intensity x group effect (P = 0.06). Resting NOx tended to increase after IH (P < 0.05) however there was no change after maximal exercise. There was no change in NOx at rest and after exercise in the control group.
Figure 5.10 – Nitrate before and after maximal exercise
[pic]
Figure 5.10 shows the effect of IH and IN on nitrate at rest and after maximal exercise in hypoxia. There were no significant main effects although there was a tendency for a time x intensity x group effect (P = 0.07) whereby resting nitrate increased after IH however there was no change after maximal exercise. There was no change in nitrate at rest and after exercise in the control group.
Figure 5.11 – Nitrite at rest and after maximal exercise
[pic]
Figure 5.11 shows the effect of IH and IN on nitrite at rest and after maximal exercise in hypoxia. There was no significant change in nitrite at rest and after maximal exercise in either group. However the decrease in nitrite pre-post maximal exercise was attenuated after IH (pre-post difference before IH: 75 ± 58 nM vs. after IH: 43 ± 86 nmol, P = 0.07).
Figure 5.12 – RSNO at rest and after maximal exercise
[pic]
Figure 5.12 shows the effect of IH on RSNO before and after maximal incremental exercise in hypoxia. RSNO tended to increase after IH however the effect was not significant (P > 0.05).
Figure 5.13 – sVCAM-1 at rest and after maximal exercise
[pic]
Figure 5.13 illustrates the effect of IH on sVCAM-1 at rest and after maximal exercise in hypoxia. At rest sVCAM-1 decreased after IH (P < 0.05). The increase in sVCAM-1 pre-post maximal exercise tended to be greater after IH (before IH: 77 ± 71 vs. after IH: 105 ± 33 pg/mL, P > 0.05). There was no change in the control group.
Figure 5.14 – sICAM-1 before and after maximal exercise
[pic]
Figure 5.14 shows the effect of IH on sICAM-1 at rest and after maximal exercise in hypoxia. There was nearly a main effect for time x intensity x group (P = 0.08). Resting sICAM-1 decreased after IH and the increase pre-post exercise difference was significantly greater after IH (pre-IH: 55 ± 45 versus post-IH: 90 ± 27 ng/mL, P < 0.05). There was no change in the control group.
Figure 5.15 – Blood pH at rest and after maximal exercise
[pic]
Figure 5.15 shows the effect of IH on blood pH before and after maximal exercise in hypoxia. Blood pH decreased after maximal exercise (P < 0.05) however the difference pre-post maximal exercise was unchanged after IH or IN.
5.7 Discussion
The purpose of this study was to examine the effect of 10 days IH on incremental exercise performance in hypoxia. Cerebral and muscle oxygenation was measured at rest and throughout exercise to evaluate adaptation at the microvascular level. Additionally, systemic molecular biomarkers of endothelial function were measured at rest and immediately after maximal exercise. The key findings from this study are as follows:
1) IH tended to improve exercise economy and [pic]O2peak in hypoxia. These results provide motivation for athletes and mountaineers to implement IH as part of their preparations for athletic performance at altitude.
2) IH increased the muscle THb response to exercise. Since both the slope for O2Hb and HHb increased suggests the overall balance between O2 delivery and uptake was unchanged after IH. Therefore we relate the muscle THb response to an enhanced vascular O2 sensitivity and vasodilation response rather than potential metabolic changes.
3) IH tended to increase cerebral O2Hb at rest and during exercise. Since arterial blood is mostly oxygenated the improved cerebral O2Hb response could be evidence of increased arterial blood flow in the absence of any changes in SaO2. Consequently the THb: workload curve showed a leftward shift. Therefore the improved aerobic performance after IH could have influences from peripheral and central fatigue mechanisms. Since cerebral HHb was unaffected by IH, if central fatigue was related to the improved [pic]O2peak the precise mechanism is likely to be regulated through an increased cerebral oxygenation per se rather than metabolic changes.
4) At rest, nitrate levels increased whilst the decrease in nitrite pre-post maximal exercise was attenuated after IH. Since our study was limited by low subject numbers, we tentatively speculate these results signify a recalibration of vascular O2 sensing mechanisms which could be driven a more effective eNOS production or more efficient utilisation of the nitrite-HHb-NO pathway during exercise.
5) Resting sICAM-1 decreased after IH. Additionally, the increase in sICAM-1 after maximal exercise was augmented by IH signifying an enhanced susceptibility to the systemic ‘stresses’ of exercise. Alternatively, the response could be related to an elevated THb response by increasing the total surface area for endothelial cell surface expression and release of sICAM-1.
Training protocol
There are various IH paradigms ranging in depth, duration and intermittence of hypoxia. The IH pattern will determine if there is sufficient biochemical stimulus to initiate a protective response or be detrimental to vascular function and hypoxic sensitivity (Beguin et al. 2005). We specifically chose a short duration IH protocol as opposed to a continuous hypoxic exposure because 4-8 weeks of continuous IH (12% O2, 1hr/d, 5 d/week) led to an overproduction of ROS, depleted antioxidant defense and subsequently endothelial dysfunction at rest and during exercise (Wang et al. 2007a & b). The effect of repeated, short duration IH on vascular responsiveness to hypoxia at rest and during exercise in healthy humans is less well known but may allow for exposure to a more severe hypoxic stimulus whist avoiding the potential detrimental effects of continuous hypoxia. The 10 day IH paradigm used in the present study was identical to that of Stuke et al (2005) who reported a 20% improvement in sea-level cycling time to exhaustion. Each subject was exposed to 90 x 5-minute IH episodes of 9.5% O2 equating to a total hypoxic exposure time of 450 minutes. This duration of hypoxia is comparable to previous IH investigations in human subjects who showed enhanced cardiovascular, ventilatory and cerebrovascular hypoxic sensitivity at rest and during submaximal exercise (Foster et al. 2005; Koehle et al. 2007; Ainslie et al. 2008).
It is possible the adaptive mechanisms reported in previous (short duration) IH studies differ from that shown in the present study because subjects were exposed to a higher FIO2 which may be sufficient to initiate a chemosensitive response however may be inadequate to initiate the molecular cascade of events driving (vascular) adaptation to more severe IH. These studies include breathing hypoxic exposures of 12% O2 (Foster et al. 2005; Koehle et al. 2007; Querido et al. 2009), clamping SaO2 to an individual target of 80% (Marshall et al. 2007) or by using a graded decline in FIO2 starting with 12% O2 before progressively lowering to 9% O2 over the course of adaptation (Julien et al. 2004). The decision to breathe 9.5% O2 with equal periods of normoxia evoked aggressive oscillations in SaO2 which dropped on average to 66%. We are confident our IH protocol was of sufficient depth and duration to switch activation between O2-sensitive (hypoxia) and inflammatory (reoxygenation) pathways and initiate a variety of adaptive mechanisms in muscle, brain and vascular tissue which translated to improved [pic]O2peak.
Our findings could be related to the fact exercise was conduced in hypoxia since previous studies have been less successful in improving aerobic performance at sea-level but by using a ‘less aggressive’ IH regime (Marshall et al. 2007; Julian et al. 2004; Foster et al. 2006). However a dose-dependant response was reported during adaptation to moderate versus severe continuous IH whereby severe IH elicited detrimental affects on vascular function via an overproduction of ROS whereas moderate IH had beneficial effects on vascular function through improved NO production although [pic]O2max increased in both groups (Wang et al. 2007a & b). The generation of ROS is considered to play a key role in the adaptive process during IH and Steiner et al. (2002) gave evidence showing the increase in ROS in the mesenteric venules of rats was inversely related to PO2 during exposure to hypoxia ranging from 15, 10 and 7.5 % O2. Since there was no change in leukocyte adherence in 15% O2 implies ROS must reach a threshold level prior to initiating activation of endothelial cells. Further research is required to evaluate the influence of hypoxic intensity during adaptation to short duration IH in humans where it is possible attainment of a threshold hypoxic stimulus is critical for adaptation. However prolonged activation of the same signaling pathway during continuous hypoxic exposure appears to have detrimental effects. The reoxygenation periods may have an important role in preventing an adverse response to IH although we recognise that repeated hypoxic exposures can be detrimental to vascular function in healthy humans (Foster et al. 2009; Pialoux et al 2009).
Due to the aggressive nature of our IH regime some subjects reported symptoms of acute mountain sickness such as mild headache however this effect dissipated within the first few days of adaptation. We believe from this anecdotal evidence that our intervention was successful in enhancing cerebral tolerance to severe IH in healthy subjects despite showing no obvious changes in SaO2. Our subjective observations could signify an improved cerebrovascular responsiveness to reduce potential brain swelling after each hypoxic exposure or alternatively IH could elicit structural and/or hypoxic sensitivity changes within the brain and specifically the blood brain barrier to preserve functionality over the course of adaptation. Future research is warranted examining the structural adaptations within the brain to IH using more comprehensive imaging techniques such as fMRI. In a recent review, Burtscher et al. (2008) suggested 1-4 hour daily exposure of ~4,000m for 1-5 weeks had the potential to reduce symptoms of AMS and improve exercise performance at high altitude. In the present study, a prolonged resting hypoxic exposure would have determined if there is neurological and neurocognitive benefits to be gained after IH in addition to the improvements in exercise performance.
Haematology
Traditionally IH is used to stimulate EPO release and red blood cell production (Levine & Stray-Gundersen 1997). In the current study, Hb and haematocrit values before and after maximal exercise was not different from baseline after IH. Others have documented similar results following 2-4 weeks IH (Julien et al. 2004; Marshall et al. 2007) however 9 continuous hypoxia exposures of ~5,000m for 90-180min/day increased RBC count, reticulocyte level and Hb concentration (Rodriguez et al. 1999 & 2000). Regulation of the transcription factor HIF-1α plays a key role in the expression and activation of numerous hypoxia related genes including EPO and rapidly degrades in the presence of O2 (Semenza et al. 1997). Our short 5 minute cycles of IH would have prevented a sustained activation of HIF-1α thus eliminating a potential erythropoeitic response.
Cardiorespiratory
Although SaO2 at rest and during the 60W steady state workload tended to be higher after IH, these findings were not related to an enhanced ventilatory response. This negative result was surprising since one of the more consistent observations from previous IH studies is an enhanced carotid body chemosensitivity and HVR (Ainslie et al. 2007; Foster et al. 2006). An elevation in HVR plateaued 3 days into a 7 day course of IH consisting of 12x 5-minute episodes of 12% O2 separated with 5 minutes normoxia (Koehle et al. 2007). Since our evaluation of IH was conducted 3 days after the final hypoxic exposure, our results could be related to the decay in HVR which can return to baseline 3-5 days after IH (Foster et al. 2005). Regardless, an increased HVR measured at rest does not always translate to increased ventilatory response to exercise (Foster et al. 2006) although an enhanced exercise ventilatory response in hypoxia (Katayama et al. 2001) and at sea-level (Townsend et al. 2005) after IH have been documented. The increased SaO2 in the present study could be related to an improved ventilation-perfusion matching (Lundby et al. 2006). It is possible IH increased SaO2 through improved capillary recruitment in the lungs thereby increasing the mean transit time for O2 diffusion (Capen & Wagner, Jr. 1982). Alternatively, an elevated DPG levels in red blood cells after altitude acclimatisation can cause a leftward shift in the ODC thus for a given PO2 less O2 binds with Hb forming O2Hb (Calbet et al. 2003; Wagner et al. 2007).
Exercise economy was shown to improve after IH as demonstrated by the reduction in [pic]O2 during the 60W workload. Similar findings have been reported after adaptation to longer duration IH during exercise at sea-level (Katayama et al 2003 & 2004; Gore et al. 2001; Saunders et al. 2004) and in hypoxia (Beidleman et al. 2008) and can be explained mechanistically by intrinsic changes within the mitochondria and increased O2 delivery. In the present study, the augmented SaO2 and THb response increased O2 delivery during the low intensity workloads. Elevated local and systemic vasodilators factors such as ATP and NO have been shown to decrease [pic]O2 during maximal (Lundby et al 2008; Larsen et al 2009) and submaximal exercise (Larsen et al 2005) therefore it is possible some degree of vasoconstriction is needed to match O2 delivery with demand during exercise. The increased THb response may have restricted leg [pic]O2 by impairing O2 extraction during submaximal exercise whereas during maximal exercise IH resulted in favourable effects on muscle energetic function and increased [pic]O2peak. Previous reports show no change in [pic]O2max at sea-level after IH where it may be less likely further increases in O2 delivery would enhance aerobic performance in healthy subjects (Foster et al. 2006; Julien et al. 2004; Marshall et al 2007). It has been suggested IH is more effective in improving exercise capacity in patient populations characterised by chronic hypoxemia such as coronary artery disease and chronic obstructive pulmonary disease and were attributed to increases in total Hb mass, lung diffusion capacity and more efficient ventilation (Burtsher et al. 2009). The present study extends these observations to an improved aerobic capacity in healthy human subjects with superimposed hypoxaemia however there was no change in any of the factors mentioned above at PPO. Rather the NIRS results tend to suggest the primary mechanisms driving the improved [pic]O2peak in hypoxia is more likely to be regulated by regional microcirculatory changes in the brain and muscle rather than factors related to improvements in central O2 delivery.
Muscle and cerebral oxygenation
At rest there was no change in muscle oxygenation in hypoxia after the intervention. In contrast, cerebral oxygenation increased (↑O2Hb) highlighting the sensitivity of the brain to respond acutely and chronically to hypoxia and could explain our subjective observation of neurological benefits over the course of adaptation. Our results could simply be related to the increased SaO2 and/or CBF may have increased since changes in O2Hb correlate with MCAVmean measured by transcranial Doppler ultrasound (Al-Rawi et al 2001). The present study contradicts previous reports that show impaired cerebrovascular O2 sensitivity in animals and humans after IH (Phillips et al. 2005; Foster et al. 2005 & 2007; Querido et al. 2008; Ainslie et al. 2007). These impairments were related to elevations in oxidative stress, blunted NO production and increased HVR and associated reductions in CBF due to hypocapnic-induced vasoconstriction. However the blunted cerebrovascular responsiveness to hypoxia normalises within days after IH (Foster et al. 2005) and on return to sea-level after altitude acclimatisation (Norcliffe et al. 2005).
Cerebral O2Hb also increased during intense exercise in the IH group but without any change in SaO2. Since the decline in nitrite pre-post maximal exercise was attenuated possibly due to increased eNOS activity, could be evidence of improved cerebral vasodilation and O2 delivery at rest and during exercise. A critical reduction in cerebral oxygenation is regarded as a limiting factor to exercise performance in hypoxia (Amann et al. 2007; Subudhi et al. 2007; Imray et al. 2005). Therefore the increased [pic]O2peak could be related to attenuation or desenitisation of O2-sensitive central fatigue factors thus preserving central motor output in the absence of any changes in cerebral metabolism since the cerebral HHb profile as an estimate of changes in tissue O2 extraction (Ferreira et al. 2007), did not change after IH. Two previous studies have examined the effects of IH on cerebral and muscle oxygenation during incremental cycling at sea-level (Marshall et al. 2007) and steady state exercise in 14% O2 (Ainslie et al. 2008). In contrast to the present study, both reported a decline in cerebral oxygenation after IH which the authors related to an augmented exercise hyperventilation and hypocapnic-vasoconstriction induced reduction in CBF. Since there was less of a decrease in muscle oxygenation compared to baseline, Marshall et al. (2007) concluded that the unchanged [pic]O2peak after IH in a group of athletes exhibiting EIH reflected a balance between peripheral and central adaptations. However it is unlikely small reductions in cerebral oxygenation would impair exercise performance in normoxia. In the present study, the increased intercept for the linear regression between muscle O2Hb and HHb versus workload without a change in slope is more likely to signify an elevated vasodilation response as a direct consequence of the metabolic changes that occur and may be a specific response to exercise in a hypoxic environment. This could in turn have translated to improve O2 delivery and utilisation during the high intensity workloads. Subsequently the increased [pic]O2peak in hypoxia after IH could have been regulated by both central and peripheral fatigue mechanisms.
NO and CAMs
It has been suggested NO plays a key role in driving vascular adaptation to IH (Manukhina et al. 2006). Subsequently the protective effects of IH were abolished by administration of NOS inhibitors and reproduced with NO donors (Malyshev et al. 1999). In the present study, nitrate and NOx concentrations increased at rest in healthy, human subjects. It is possible acute, transient increases in NO production with each hypoxic exposure was followed by its uptake by O2Hb during the reoxygenation phase and NO is converted to nitrate before being released back into plasma and prolonging its bioavailability. Our results do not support the possibility of a sustained NO production over the course of adaption since basal concentrations of nitrite which provide a reliable index of eNOS activity (Kleinbongard et al. 2003) remain unchanged. Vascular NO bioavailability was reported to be 10 fold greater among Tibetan residents at 4,200m compared to sea-level residents and resulted in more than doubling of forearm blood flow at rest and during exercise (Erzurum et al. 2007). These results highlight the importance of enhanced NO bioavailability during adaptation to hypoxia but also suggest the adaptive response can occur rapidly since the total hypoxic exposure time over the 10 day intervention was 450 minutes. There are contrasting reports on the effects of IH on NO storage in human and animals and can be attributed to the IH regime which determines if the adaptive response is detrimental or beneficial to endothelial function and NO bioavailability. Manukhina et al (1999) reported an increased NO storage in rats within the first few days of IH and increased as adaptation developed. An increased NOx was also reported by Wang et al. (2007) in healthy male subjects after 4 weeks severe (12% O2, 1h/day, 5 days/week) but not moderate (15% O2) IH. Whereas in OSA patients, a condition characterised by as many as 60 hypoxic-reoxygenation events per hour, NOx levels were reduced compared to control (Neubaurer 2001; Atkeson et al. 2009).
We are confident our IH regime elicited a protective response since the increase in nitrate at rest was mirrored by decreases in sVCAM-1 and sICAM-1. Concentrations of sICAM-1 have been related to the degree of endothelial activation and endothelial dysfunction in humans (Holmlund et al. 2002). The expression of ICAM-1 on endothelial cells following an acute inflammatory stimulus was also related to the level of NO present and degree of NOS activity (Berendji-Grun et al. 2001; Lindemann et al. 2000; Dal Secco et al. 2006). It is possible our results reflect a structural remodeling of blood vessels and downregulated basal (shear stress induced) vascular tone due to the increased NO bioavailability. Alternatively, low doses of vitamin C and E have been shown to decrease sICAM-1 (Witkowska et al. 2005) therefore our results could signify enhanced antioxidant status after IH.
Exercise - Two key observations from this study were the attenuated decline in nitrite and augmented CAM release pre-post maximal exercise. It is possible that both are related directly or indirectly to an enhanced vascular O2 sensitivity through increased eNOS activation whereby the resulting systemic hyperperfusion response increased the total surface area for shear stress induced expression and release of CAM. A similar (protective) preconditioned response after IH was reported in animals during subsequent exposure to more severe hypoxia (Liu et al. 2005; Kaminski et al. 2006) and in various models of ischemia-reperfusion injury (Zong et al. 2004; Cai et al. 2003; Beguin et al. 2005; Bertuglia 2008). It would appear the common mechanisms which regulate hypoxic preconditioning from these studies are related to reduced threshold sensitivity for HIF-1α activation which can enhance endothelial NO release and capillary perfusion, prevent the development of a NO-deficient condition and offer protection against NO over-production and oxidative stress. Since NO production increases proportionally with changes in nitrite and HHb concentrations (Cosby et al. 2003) raises the possibility that the adaptive response is more effective during exercise in hypoxia than in normoxia. The precise mechanism whereby IH attenuates the reduction in nitrite may also be regulated via direct communication between the RBC and endothelial cells (Buehler and Alayash 2004). A recalibration of RBC-NO hypoxic sensitivity following IH could modify the effectiveness and efficiency of the nitrite-HHb-NO pathway although this has yet to be investigated. Since the adaptive response persisted for 3 days after the final hypoxic exposure our findings compare to the late phase preconditioning shown during ischemic preconditioning (Bolli 2000). It is therefore likely the two preconditioning stimuli share common metabolic triggers and signaling pathways.
There is evidence showing severe IH can impair NO-dependant vasodilation and compromise vascular protection in rats (Phillips et al. 2004) and human subjects (Wang et al. 2007). The interaction between the NO-ROS pathways as adaption develops will determine if adaptation elicits a protective response or in circumstances where there is a chronic overproduction of ROS could be detrimental to endothelial function. For example, Beguin et al. (2005) reported enhanced protection against myocardial I-R injury 24 hours after IH consisting of 40 seconds breathing 10% O2 followed by 20 seconds of normoxia for 4 hours. In contrast, identical cycles of 5% O2 breathing for 4 hours increased susceptibility to myocardial injury whilst 30 minutes of IH or 4 hours continuous 10% O2 breathing did not modify infarct size implying a critical threshold stimulus is required prior to initiation of an adaptive response. Furthermore, Bertuglia (2008) demonstrated IH consisting of 6 min breathing 8% O2 followed by 6 min normoxia every 8 hours for 21 days greatly reduced oxidative stress and stimulated NO-vasodilation in the microcirculation of the hamster cheek pouch during I/R injury thus maintaining capillary perfusion whereas an NO inhibitor aggravated capillary perfusion and I/R damage. Further studies in humans are required to validate our observations.
There are several potential mechanisms that can explain the augmented increase in sICAM-1 after IH. Since nitrite concentrations were better preserved after IH it could have been expected for the CAM response to be downregulated and our results would therefore contradict the potential protective effects of elevated nitrite levels (Webb et al. 2004; Duranski et al. 2005). However, endogenously released NO during inflammation negatively effects the leukocyte-endothelial interaction (Kubes et al. 1991; Dal Secco et al. 2003) whilst sICAM-1 can inhibit adhesion by binding to CD11a/CD18 and PBMC leaving less available for endothelial binding (Mill et al 2006; Ohno et al 1997). There is also evidence showing hypoxic acclimatisation reverses the rapid increase in leukocyte adherence in rat microvasculature during subsequent acute hypoxic exposure (Wood et al. 1999). The response is mediated by increased NO production since a specific iNOS inhibitor partially restored the response. Therefore it is possible the increased circulating CAMs signify weaker leukocyte-endothelial interactions after IH. Our results also comply with previous findings in OSA patients that show an increased susceptibility to oxidant stress and vascular injury (Neubaurer 2001; Atkeson et al. 2009). Alternatively the systemic hyperperfusion response effectively elevated the total surface area for shear stress induced expression and release of sICAM-1. Increases in endothelium dependant vasodilation induced by 2 hours intra-arterial infusion of acetycholine and nitroglycerine correlated with increased plasma concentrations of sVCAM-1 and VEGF (Schmidt-Lucke et al 2003) supporting this hypothesis. Since the increased Hb and haematocrit levels before and after maximal exercise did not change after IH, our findings cannot be explained by increased blood viscosity.
5.8 Conclusions
In the present study, IH consisting of 2x 5 day blocks breathing 9.5% O2 for 5 minutes followed by equal periods of normoxia for 90 minutes was successful in increasing cerebral oxygenation at rest and during exercise in hypoxia. These findings add support to our subjective observation of neurological benefits over the course of adaptation and raise the possibility that IH could be an effective strategy in preventing AMS during prolonged altitude exposure. The main aim of this investigation was to examine the effects of IH on submaximal and maximal response to exercise in hypoxia. Cardiorespiratory, cerebral and muscle oxygenation responses were monitored during incremental exercise and molecular blood-borne markers of vascular function were measured at rest and immediately after maximal exercise. Despite showing an increased SaO2 during steady state exercise after IH, there was surprisingly no change in the exercise chemsensitivity since ventilation rate was unchanged during steady state and maximal exercise. The decrease in [pic]O2 during the 60W workload signifies improved exercise economy and together with the increase in [pic]O2peak in the IH group gives motivation for athletes and mountaineers to implement a similar IH regime prior to athletic performance at altitude. Our findings could signify a recalibration of factors regulating vascular O2 sensitivity and adaptations within the brain and muscle which translated to improved O2 delivery and exercise performance in hypoxia through an attenuated development of peripheral and central fatigue. The precise mechanisms regulating adaptation are unclear however are likely to involve a reduced threshold sensitivity for HIF-1α activation, a more effective regulation of the nitrite-HHb-NO pathway and/or increased eNOS activity. The augmented elevation in sICAM-1 after maximal exercise could signify a weakened leukocyte-endothelial interaction directly through increased endogenous NO production, increased susceptibility to oxidative stress or may be related to the enhanced THb response to exercise.
Chapter 6
General discussion and conclusions
6.1 Overview
This final chapter will provide a summary of each research aim and reflect on the key observations from the two experimental studies together with recommendations for future research. The overall theme of this thesis focused on systemic and regional changes in O2 delivery and metabolism and the vascular response to exercise in hypoxia. There is limited research examining the effect of acute hypoxia on the kinetic changes in cerebral and muscle oxygenation during incremental exercise. Furthermore, the impact of acute hypoxia on the regulation of systemic blood-borne molecular biomarkers of endothelial function and O2 sensing during exercise is not well defined in healthy human subjects. Study 1 (Chapter 4) investigated regional changes in cerebral and muscle oxygenation during incremental exercise in normoxia and hypoxia using NIRS. It has been suggested that aerobic exercise performance is impaired in hypoxia by accelerating the development of central and peripheral fatigue (Amann et al. 2007). Therefore the oxygenation profiles were characterised at equivalent absolute and relative workloads to determine if the slope of the profile and in turn the rate of deoxygenation was accelerated in hypoxia. Since vascular function is adversely affected in hypoxia by mechanisms which appear to be related to the regulation of NO metabolites and oxidative stress (Bailey et al. 2008 & 2009), changes in systemic blood borne markers of oxidative/nitrative stress (A• and 3-NT), NO bioavailability (nitrate, nitrite and RSNO) and endothelial activation (sICAM-1 and sVCAM-1) were measured before and immediately after maximal exercise.
IH has been used as a pre-acclimatisation strategy prior to acute hypoxia exposure at rest and during exercise (Ainslie et al. 2008; Katayama et al. 2001; Beidleman et al. 2008). Although some have suggested IH can adversely affect cerebrovascular function in humans (Foster et al. 2005), there is good evidence showing O2 delivery is improved after IH and the molecular pathways driving vascular adaptation are regulated by the balance between NO and ROS formation (Manukhina et al. 2006). Study 2 (Chapter 5) sought to determine if 10 days of IH enhanced cerebral and muscle oxygenation during incremental exercise in hypoxia and in turn aerobic performance. This study examined if these potential changes are driven through enhanced activation/sensitivity of NO generating pathways by increasing systemic NO bioavailability during maximal exercise.
6.2 – Study 1: The impact of acute hypoxia on cerebral and muscle oxygenation and systemic molecular biomarkers of vascular function during exercise
▪ Aim 1 - To characterise changes in muscle and cerebral oxygenation during incremental exercise to exhaustion in normoxia and hypoxia. Specifically, the oxygenation profiles of O2Hb, HHb and THb of the vastus lateralis muscle and prefrontal cortex region of the brain will be evaluated at equivalent absolute and relative exercise intensities.
The magnitude of cerebral and muscle deoxygenation was greater at rest and throughout incremental exercise in hypoxia and is consistent with previous reports (Subudhi et al 2007). Therefore it is likely O2-dependant central and peripheral fatigue factors contributed to the 22% reduction in [pic]O2peak in hypoxia. Although it is not possible to differentiate the NIRS signal to arterial, capillary and venous blood, we attribute the deoxygenation response to a ventilation/perfusion inequality and reduction in O2 delivery. The transition from rest to exercise contributed significantly to the decline in tissue oxygenation and signifies an even greater diffusion limitation when exercise and hypoxia are combined. There was considerable inter-subject variability in the magnitude of deoxygenation during exercise and can be attributed to the heterogeneity of human tissue in addition to different muscle recruitment and activation patterns. We acknowledge there was considerable individual SaO2 variation during rest and exercise in hypoxia although there was no indication from our data to suggest individual differences in muscle and cerebral oxygenation are accounted for solely by the variability in SaO2.
A change in the HHb signal is regarded as an estimate of tissue deoxygenation by O2 extraction and was shown to be an important factor driving incremental exercise performance in normoxia (Ferreira et al. 2007). Attainment of [pic]O2peak was preceded by a reduced rate or plateau region in the HHb profile which could be interpreted as evidence of the maximal O2 extraction capacity of muscle and brain during maximal exercise. The slope for the relative muscle HHb response was similar between trials however there was a greater slope across the absolute workloads in hypoxia which suggests PPO was driven by an accelerated rate of deoxygenation from the onset of exercise in hypoxia. A visual inspection of the muscle HHb profile revealed a tendency for a sigmoidal pattern in normoxia supporting observations from others (Ferreira et al. 2007; Boone et al. 2009), whereas there was a hyperbolic pattern in hypoxia.
The relative cerebral HHb profile followed a near identical pattern in both trials where there was an accelerated increase at workloads greater than 50% PPO. This suggests cerebral metabolism increases as a function of the relative metabolic stress in hypoxia presumably as a consequence of a reduction in CBF (Bhambhani et al. 2007). There was no suggestion that cerebral metabolism was impaired during the hypoxia trial therefore if central fatigue influenced performance it is most likely due the direct inhibitory effect of a reduction in cerebral O2 delivery (Amann et al. 2006). Together these findings suggest that the cerebral and muscle HHb profile are related to relative changes in metabolic stress. It is possible that attainment of [pic]O2peak was driven by an earlier onset of peripheral and/or central fatigue and maximal O2 extraction capacity of muscle and brain tissue.
▪ Aim 2 - To examine the effects of maximal exercise in hypoxia on molecular blood-borne markers of vascular O2 sensing and function. Specifically biomarkers of oxidative stress, NO metabolite bioavailability and CAM will be measured before and immediately after maximal exercise.
The total NO metabolite pool was unaffected by maximal exercise. However nitrite decreased at PPO in both tests suggesting the rate of nitrite consumption or inactivation of eNOS occurs at a greater rate than it is produced by NO oxidation. The decline in nitrite was attenuated in hypoxia and could be explained by a blunted activation of the nitrite-HHb-NO pathway which functions optimally when PO2 pressures range from 20-40mmHg and Hb saturations of 40-60% (Gladwin et al. 2003). At PPO SvO2 was 31% in hypoxia versus 58% in normoxia therefore it is likely nitrite reductase activity of HHb could have been impaired by the severity of hypoxaemia. Although there was no change in the oxidative stress markers, we do not rule out the possibility of an oxidative inactivation that could have influenced the partial depletion of nitrite since previous studies from our lab show an elevated muscle and systemic free radical output during exercise using identical analytical techniques (Bailey et al. 2001, 2003 & 2007; Davison et al. 2006). Since nitrite is an essential mediator of vascular function and O2 sensing in hypoxia and during exercise (van Faassen et al. 2009), this mechanism is a prime candidate for explaining the systemic hypoperfusion response shown during exercise in hypoxia whereby there tended to be a rightward shift in the cerebral THb curve and the muscle THb response which peaked at approximately 80% PPO tended to be lower in hypoxia. The elevation in RSNO after maximal exercise is less well understood but could signify a partial reapportionment of vascular NO metabolites. This response may be initiated by the systemic stress of maximal exercise since previous studies indicate a role for RSNO in regulating haemodynamic stability, O2 delivery and utilisation during I-R injury of skeletal muscle (Hallstrom et al. 2002). There was a significant increase in both sICAM-1 and sVCAM-1 after maximal exercise. The increase tended to mirror the changes in nitrite since the magnitude of CAM release was also greater in normoxia. It is possible the release of the CAMs was blunted as a direct consequence of the reduction in total surface area for endothelial shear stress induced activation or could reflect a more efficient leukocyte-endothelial interaction in hypoxia.
▪ Aim 3 -To establish if the reduction in aerobic capacity in hypoxia is related to the change in blood-borne markers of vascular function after maximal exercise.
Moderate relationships were observed between the change in pre-post exercise difference in nitrite and CAMs with [pic]O2peak when the normoxia and hypoxia tests were combined. This would imply that the degree of exercise induced endothelial function/dysfunction and integrity of the vascular system to delivery O2 to highly active tissues is an important factor driving aerobic performance. Our study is in agreement with two previous reports in healthy subjects (Rassaf et al. 2007) and those presenting with low cardiovascular risk factors up to fully diagnosed peripheral arterial disease (Allen et al 2009) after maximal exercise in normoxia. This is further evident in studies that show chronic exercise training increases muscle blood flow and aerobic capacity in conjunction with enhancements in NO-dependant vasodilation and endothelial function (Goto et al. 2003; Maiorana et al. 2003).
6.3 – Study 2: Intermittent hypoxia: implications for cerebral and muscle oxygenation and molecular biomarkers of vascular function
▪ Aim 4 - To evaluate the effect of IH on exercise performance in hypoxia.
Although the implications of IH on sea-level performance have been well documented, the effect of IH on aerobic performance in hypoxia has received little attention. The second study showed that 10 sessions of IH consisting of 5 minutes breathing 9.5% O2 interspersed with 5 minutes of normoxia for 90 minutes tended to improve submaximal exercise economy and [pic]O2peak in 12% O2. Our IH protocol utilised a more severe IH exposure compared to others that showed no change in [pic]O2max at sea-level (Julian et al. 2004; Foster et al. 2006; Marshall et al. 2007). We employed an identical protocol to that of Stuke et al. (2005) who reported a dramatic increase in sea-level exercise time to exhaustion. It is possible that a more severe IH exposure is required to exert adaptation in vascular, muscle and the brain which ultimately resulted in improving [pic]O2peak. Our findings are consistent with others examining exercise performance at sea-level in healthy subjects (Wang et al. 2007) and patients with coronary artery disease (Burtscher et al. 2004) and in hypoxia (Beidleman et al. 2003) and relate the performance improvements in hypoxia to an attenuated development of central and peripheral fatigue.
▪ Aim 5 - To examine the effect of IH on the cerebral and muscle oxygenation response to exercise in hypoxia.
We are the first to examine the effects of IH on changes in cerebral and muscle oxygenation during incremental exercise in hypoxia. Despite some initial concerns that IH could impair vascular function, our results give evidence of an enhanced cerebral and muscle oxygenation response to exercise as well as neurological benefits over the course of adaptation. Therefore results from this study suggest the primary mechanism driving the improved [pic]O2peak in hypoxia is related to microvascular changes in brain and muscle tissue rather than central O2 delivery factors since SaO2 and HR at PPO was unaffected by IH. It is possible the increased blood volume response throughout submaximal exercise could reduce leg [pic]O2 by increasing the O2 diffusion gradient from capillary to mitochondria. Lundby et al. (2008) suggested some vasoconstriction is needed to match O2 delivery with demand and it is possible the increased THb response presumably due to enhanced vasodilation resulted in a lowering of steady state [pic]O2 whereas during maximal exercise a greater THb could have resulted in favourable effects on muscle energetics and [pic]O2peak by increasing O2 delivery. Subsequently, the increased intercept in the linear regression between muscle O2Hb and HHb versus workload in the absence of a change in slope could be interpreted as evidence of an elevated vascular O2 sensitivity and vasodilation as a direct consequent of the increased metabolic activity.
Although there was no change in muscle oxygenation during the initial 10 minute rest period, cerebral oxygenation was improved highlighting the sensitivity of brain tissue to respond to hypoxia and provide some physiological rationale into our subjective observation of neurological benefits. Cerebral oxygenation was also enhanced during the high intensity workloads in the absence of any significant changes in SaO2, therefore the increased [pic]O2peak could be related to an attenuation or desensitisation of central fatigue factors but in the absence of potential changes in cerebral aerobic metabolism since the HHb profile was unaffected by IH.
▪ Aim 6 - To determine if adaptation is driven by an enhanced NO metabolite bioavailability and its vascular protective effects. Subsequently the increased CAM release after maximal exercise will be attenuated after IH.
After IH the increased NOx at rest was mirrored by a reduction in sICAM-1. Increased NOx levels was also reported by Wang et al. (2007) in healthy male subjects after 4 weeks continuous IH resulting in improved VO2max at sea-level. Together this would imply NOx levels may have important implications in prolonging NO storage during adaptation to IH, reducing endothelial activation and improving aerobic performance. The decline in nitrite pre-post maximal exercise was attenuated after IH. It is possible that IH initiated a recalibration of vascular O2 sensitivity through mechanisms that involve a reduced threshold sensitivity for HIF-1α activation and driven either by a more effective eNOS production or utilisation of the nitrite-HHb-NO pathway.
There are a few potential mechanisms that can explain the augmented sICAM-1 response to maximal exercise after IH. It is possible that the severity of the IH intervention could have increased susceptibility for exercise mediated oxidative and/or increased vasodilation induced endothelial expression and release of CAM. Secondly, since there was an increase in the THb response during exercise after IH, the elevation in total surface area for flow mediated endothelial activation could have facilitated a greater expression and release of sICAM-1 into the systemic circulation. Alternatively, since endogenously released NO during inflammation can negatively effect the leukocyte-endothelial binding (Kubes et al. 1991; Dal Secco et al. 2003), it is possible a more effective nitrite-HHb-NO bioactivation or enhanced eNOS activity after IH initiated a weaker leukocyte-endothelial interaction through increased NO production.
6.4 –Future research
As demonstrated throughout the literature review and experimental chapters, much work is still required in the area of exercise, O2 sensing and vascular function. The following information presents recommendations for future research as directed from this thesis and may contribute to our understanding of vascular physiology.
Preconditioning - There is good research comparing adaptation to continuous and intermittent hypoxia on vascular function in animals with direct implications for ROS generation and NO production. However there is a need for this research to be replicated in human subjects by examining adaptation to IH of different frequency, depth and durations which will allow us to identify the most effective method for administering IH prior to exercise or exposure to other systemic/local stress. Furthermore, since there is an early (within hours) and late phase (>24hours) preconditioned response to ischemic preconditioning (Bolli 2002), the time course for optimal effects of hypoxic preconditioning remains to be determined where it is likely the two stimuli share a common metabolic trigger and molecular adaptive pathway. A comparison with other preconditioning methods such as ischemic preconditioning on the vascular response to exercise should be considered for investigation.
NO bioavailability and exercise – A study examining the kinetic changes in nitrite and CAM with increasing workload would determine if the decrease in nitrite and increase in CAMs after maximal exercise was the result of a dose-dependant relationship with exercise intensity or if there the change was initiated by a threshold metabolic stimulus. In addition to the plasma-NO metabolites, an examination of changes in RBC-NO species would establish if there is a net loss in NO bioavailability or reapportionment across vascular NO compartments. These studies can be replicated across a range of vascular phenotypes such as in endurance trained subjects whose vascular function is closer to its ‘optimal’ limit compared to those with cardiovascular complications such as diabetes and endothelial dysfunction associated with ageing. Since we proposed oxidative inactivation as a potential mechanism for the decline in nitrite immediately after maximal exercise, a study investigating the effects of antioxidant supplementation would determine if this explanation is valid.
Cerebral and muscle oxygenation – Further research is required to validate NIRS as a monitoring tool in sport science research. This will allow practitioners to evaluate more effectively interventions aimed at improving microvascular O2 delivery and metabolism for athletic performance and can include other pre-acclimation strategies such as sleeping in hypoxic tents, exercising training in hypoxia or nutritional interventions for example, nitrite and acetazolamide supplementation. The mechanisms regulating O2-sensitive central fatigue are still unclear and future research focusing on treatment therapies which can reduce or eliminate its deleterious effects would have direct implications for exercise performance at altitude. Ingestion of branched chain amino acids or tryptophan have been suggested to reduce central fatigue during exercise in the heat and in patients with chronic fatigue syndrome by mechanisms involving 5-HT (Georgiades et al. 2003; Blomstrand 2001). Similar studies have yet to be conducted in hypoxia. The effect of graded levels of hypoxia on cerebral and muscle oxygenation would determine if there is a threshold level of hypoxia which elicits a more profound decrease in oxygenation. It is possible a similar S-shaped relationship between the level of hypoxia and SaO2 at maximal exercise exists where the decline in [pic]O2peak is accelerated due to the properties of the ODC (Ferretti et al. 1997) and could coincide with an even greater reduction in cerebral and muscle oxygenation.
References
Ainslie, P.N., Barach, A., Murrell, C., Hamlin, M., Hellemans, J., & Ogoh, S. 2007. Alterations in cerebral autoregulation and cerebral blood flow velocity during acute hypoxia: rest and exercise. Am.J.Physiol Heart Circ.Physiol, 292, (2) 976-983
Ainslie, P.N., Hamlin, M., Hellemans, J., Rasmussen, P., & Ogoh, S. 2008. Cerebral hypoperfusion during hypoxic exercise following two different hypoxic exposures: independence from changes in dynamic autoregulation and reactivity. Am.J.Physiol Regul.p Physiol, 295, (5) 1613-1622
Ainslie, P.N. & Poulin, M.J. 2004a. Respiratory, cerebrovascular and pressor responses to acute hypoxia: dependency on PET(CO2). Adv.Exp.Med.Biol., 551, 243-249
Ainslie, P.N. & Poulin, M.J. 2004b. Ventilatory, cerebrovascular, and cardiovascular interactions in acute hypoxia: regulation by carbon dioxide. J.Appl.Physiol, 97, (1) 149-159
Akimoto, T., Furudate, M., Saitoh, M., Sugiura, K., Waku, T., Akama, T., & Kono, I. 2002. Increased plasma concentrations of intercellular adhesion molecule-1 after strenuous exercise associated with muscle damage. Eur.J.Appl.Physiol, 86, (3) 185-190
Al-Rawi, P.G., Smielewski, P., & Kirkpatrick, P.J. 2001. Evaluation of a near-infrared spectrometer (NIRO 300) for the detection of intracranial oxygenation changes in the adult head. Stroke, 32, (11) 2492-2500
Allen, J.D., Miller, E.M., Schwark, E., Robbins, J.L., Duscha, B.D., & Annex, B.H. 2009. Plasma nitrite response and arterial reactivity differentiate vascular health and performance. Nitric.Oxide., 20, (4) 231-237
Allen, J.D., Cobb, F.R., & Gow, A.J. 2005. Regional and whole-body markers of nitric oxide production following hyperemic stimuli. Free Radic.Biol.Med., 38, (9) 1164-1169
Altay, T., Gonzales, E.R., Park, T.S., & Gidday, J.M. 2004. Cerebrovascular inflammation after brief episodic hypoxia: modulation by neuronal and endothelial nitric oxide synthase. J.Appl.Physiol, 96, (3) 1223-1230
Amann, M., Eldridge, M.W., Lovering, A.T., Stickland, M.K., Pegelow, D.F., & Dempsey, J.A. 2006. Arterial oxygenation influences central motor output and exercise performance via effects on peripheral locomotor muscle fatigue in humans. J.Physiol, 575, (Pt 3) 937-952
Amann, M. & Kayser, B. 2009. Nervous system function during exercise in hypoxia
2. High Alt.Med.Biol., 10, (2) 149-164
Amann, M., Romer, L.M., Subudhi, A.W., Pegelow, D.F., & Dempsey, J.A. 2007. Severity of arterial hypoxaemia affects the relative contributions of peripheral muscle fatigue to exercise performance in healthy humans. J.Physiol, 581, (Pt 1) 389-403
Amann, M. & Calbet, J.A. 2008. Convective oxygen transport and fatigue. J.Appl.Physiol, 104, (3) 861-870
Arbogast, S., Darques, J.L., & Jammes, Y. 2002. Interactions between endogenous nitric oxide and hypoxemia in activation of group IV muscle afferents. Muscle Nerve, 26, (2) 194-200
Arbogast, S., Vassilakopoulos, T., Darques, J.L., Duvauchelle, J.B., & Jammes, Y. 2000. Influence of oxygen supply on activation of group IV muscle afferents after low-frequency muscle stimulation. Muscle Nerve, 23, (8) 1187-1193
Arnould, T., Michiels, C., & Remacle, J. 1993. Increased PMN adherence on endothelial cells after hypoxia: involvement of PAF, CD18/CD11b, and ICAM-1. Am.J.Physiol, 264, (5 Pt 1) 1102-1110
Asha, D.S., Subramanyam, M.V., Vani, R., & Jeevaratnam, K. 2005. Adaptations of the antioxidant system in erythrocytes of trained adult rats: impact of intermittent hypobaric-hypoxia at two altitudes. Comp Biochem.Physiol C.Toxicol.Pharmacol., 140, (1) 59-67
Atkeson, A., Yeh, S.Y., Malhotra, A., & Jelic, S. 2009. Endothelial function in obstructive sleep apnea. Prog.Cardiovasc.Dis., 51, (5) 351-362
Bailey, D.M., Davies, B., & Young, I.S. 2001. Intermittent hypoxic training: implications for lipid peroxidation induced by acute normoxic exercise in active men. Clin.Sci.(Lond), 101, (5) 465-475
Bailey, D.M., Evans, K.A., James, P.E., McEneny, J., Young, I.S., Fall, L., Gutowski, M., Kewley, E., McCord, J.M., Moller, K., & Ainslie, P.N. 2009a. Altered free radical metabolism in acute mountain sickness: implications for dynamic cerebral autoregulation and blood-brain barrier function. J.Physiol, 587, (Pt 1) 73-85
Bailey, D.M., Taudorf, S., Berg, R.M., Lundby, C., McEneny, J., Young, I.S., Evans, K.A., James, P.E., Shore, A., Hullin, D.A., McCord, J.M., Pedersen, B.K., & Moller, K. 2009b. Increased cerebral output of free radicals during hypoxia: implications for acute mountain sickness? Am.J.Physiol Regul.p Physiol, 297, (5) 1283-1292
Bailey, D.M., Roukens, R., Knauth, M., Kallenberg, K., Christ, S., Mohr, A., Genius, J., Storch-Hagenlocher, B., Meisel, F., McEneny, J., Young, I.S., Steiner, T., Hess, K., & Bartsch, P. 2006. Free radical-mediated damage to barrier function is not associated with altered brain morphology in high-altitude headache. J.Cereb.Blood Flow Metab, 26, (1) 99-111
Bailey, D.M., Young, I.S., McEneny, J., Lawrenson, L., Kim, J., Barden, J., & Richardson, R.S. 2004. Regulation of free radical outflow from an isolated muscle bed in exercising humans. Am.J.Physiol Heart Circ.Physiol, 287, (4) 1689-1699
Bartsch, P., Bailey, D.M., Berger, M.M., Knauth, M., & Baumgartner, R.W. 2004. Acute mountain sickness: controversies and advances. High Alt.Med.Biol., 5, (2) 110-124
Beauchamp, P., Richard, V., Tamion, F., Lallemand, F., Lebreton, J.P., Vaudry, H., Daveau, M., & Thuillez, C. 1999. Protective effects of preconditioning in cultured rat endothelial cells: effects on neutrophil adhesion and expression of ICAM-1 after anoxia and reoxygenation. Circulation, 100, (5) 541-546
Beckman, J.S. & Koppenol, W.H. 1996. Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and ugly. Am.J.Physiol, 271, (5 Pt 1) 1424-1437
Beguin, P.C., Joyeux-Faure, M., Godin-Ribuot, D., Levy, P., & Ribuot, C. 2005. Acute intermittent hypoxia improves rat myocardium tolerance to ischemia. J.Appl.Physiol, 99, (3) 1064-1069
Beidleman, B.A., Muza, S.R., Fulco, C.S., Cymerman, A., Ditzler, D., Stulz, D., Staab, J.E., Skrinar, G.S., Lewis, S.F., & Sawka, M.N. 2004. Intermittent altitude exposures reduce acute mountain sickness at 4300 m. Clin.Sci.(Lond), 106, (3) 321-328
Beidleman, B.A., Muza, S.R., Fulco, C.S., Cymerman, A., Ditzler, D.T., Stulz, D., Staab, J.E., Robinson, S.R., Skrinar, G.S., Lewis, S.F., & Sawka, M.N. 2003. Intermittent altitude exposures improve muscular performance at 4,300 m. J.Appl.Physiol, 95, (5) 1824-1832
Beidleman, B.A., Muza, S.R., Fulco, C.S., Cymerman, A., Sawka, M.N., Lewis, S.F., & Skrinar, G.S. 2008. Seven intermittent exposures to altitude improves exercise performance at 4300 m. Med.Sci.Sports Exerc., 40, (1) 141-148
Beidleman, B.A., Muza, S.R., Fulco, C.S., Jones, J.E., Lammi, E., Staab, J.E., & Cymerman, A. 2009. Intermittent hypoxic exposure does not improve endurance performance at altitude. Med.Sci.Sports Exerc., 41, (6) 1317-1325
Belardinelli, R. 1998. Muscle oxygenation kinetics measured by near-infrared spectroscopy during recovery from exercise in chronic heart failure. G.Ital.Cardiol., 28, (8) 866-872
Belardinelli, R., Georgiou, D., & Barstow, T.J. 1995a. Near infrared spectroscopy and changes in skeletal muscle oxygenation during incremental exercise in chronic heart failure: a comparison with healthy subjects. G.Ital.Cardiol., 25, (6) 715-724
Benoit, H., Busso, T., Castells, J., Denis, C., & Geyssant, A. 1995. Influence of hypoxic ventilatory response on arterial O2 saturation during maximal exercise in acute hypoxia. Eur.J.Appl.Physiol Occup.Physiol, 72, (1-2) 101-105
Benoit, H., Busso, T., Castells, J., Geyssant, A., & Denis, C. 2003. Decrease in peak heart rate with acute hypoxia in relation to sea level VO(2max). Eur.J.Appl.Physiol, 90, (5-6) 514-519
Berchner-Pfannschmidt, U., Yamac, H., Trinidad, B., & Fandrey, J. 2007. Nitric oxide modulates oxygen sensing by hypoxia-inducible factor 1-dependent induction of prolyl hydroxylase 2. J.Biol.Chem., 282, (3) 1788-1796
Berendji-Grun, D., Kolb-Bachofen, V., & Kroncke, K.D. 2001. Nitric oxide inhibits endothelial IL-1[beta]-induced ICAM-1 gene expression at the transcriptional level decreasing Sp1 and AP-1 activity. Mol.Med., 7, (11) 748-754
Bertuglia, S. 2008. Intermittent hypoxia modulates nitric oxide-dependent vasodilation and capillary perfusion during ischemia-reperfusion-induced damage. Am.J.Physiol Heart Circ.Physiol, 294, (4) 1914-1922
Bergholm, R., Makimattila, S., Valkonen, M., Liu, M.L., Lahdenpera, S., Taskinen, M.R., Sovijarvi, A., Malmberg, P., & Yki-Jarvinen, H. 1999. Intense physical training decreases circulating antioxidants and endothelium-dependent vasodilatation in vivo. Atherosclerosis, 145, (2) 341-349
Bhambhani, Y., Malik, R., & Mookerjee, S. 2007. Cerebral oxygenation declines at exercise intensities above the respiratory compensation threshold. Respir.Physiol Neurobiol., 156, (2) 196-202
Blitzer, M.L., Loh, E., Roddy, M.A., Stamler, J.S., & Creager, M.A. 1996. Endothelium-derived nitric oxide regulates systemic and pulmonary vascular resistance during acute hypoxia in humans. J.Am.Coll.Cardiol., 28, (3) 591-596
Bhambhani, Y.N., Buckley, S.M., & Susaki, T. 1997. Detection of ventilatory threshold using near infrared spectroscopy in men and women. Med.Sci.Sports Exerc., 29, (3) 402-409
Bolli, R. 2000. The late phase of preconditioning67. Circ.Res., 87, (11) 972-983
Bonetti, D.L., Hopkins, W.G., & Kilding, A.E. 2006. High-intensity kayak performance after adaptation to intermittent hypoxia. Int.J.Sports Physiol Perform., 1, (3) 246-260
Bonetti, D.L., Hopkins, W.G., Lowe, T.E., Boussana, A., & Kilding, A.E. 2009. Cycling performance following adaptation to two protocols of acutely intermittent hypoxia
1. Int.J.Sports Physiol Perform., 4, (1) 68-83
Boone, J., Koppo, K., Barstow, T.J., & Bouckaert, J. 2009. Pattern of deoxy[Hb+Mb] during ramp cycle exercise: influence of aerobic fitness status. Eur.J.Appl.Physiol, 105, (6) 851-859
Bourdillon, N., Mollard, P., Letournel, M., Beaudry, M., & Richalet, J.P. 2009. Interaction between hypoxia and training on NIRS signal during exercise: contribution of a mathematical model. Respir.Physiol Neurobiol., 169, (1) 50-61
Bourdillon, N., Mollard, P., Letournel, M., Beaudry, M., & Richalet, J.P. 2009b. Non-invasive evaluation of the capillary recruitment in the human muscle during exercise in hypoxia. Respir.Physiol Neurobiol., 165, (2-3) 237-244
Boushel, R., Langberg, H., Olesen, J., Gonzales-Alonzo, J., Bulow, J., & Kjaer, M. 2001. Monitoring tissue oxygen availability with near infrared spectroscopy (NIRS) in health and disease. Scand.J.Med.Sci.Sports, 11, (4) 213-222
Brevetti, L.S., Paek, R., Brady, S.E., Hoffman, J.I., Sarkar, R., & Messina, L.M. 2001. Exercise-induced hyperemia unmasks regional blood flow deficit in experimental hindlimb ischemia. J.Surg.Res., 98, (1) 21-26
Brouwers, F.M., Van Der, W.S., Bleijenberg, G., Van Der, Z.L., & van der Meer, J.W. 2002. The effect of a polynutrient supplement on fatigue and physical activity of patients with chronic fatigue syndrome: a double-blind randomized controlled trial. QJM., 95, (10) 677-683
Bryan, N.S. 2009. Cardioprotective actions of nitrite therapy and dietary considerations
7. Front Biosci., 14, 4793-4808
Bryan, N.S., Fernandez, B.O., Bauer, S.M., Garcia-Saura, M.F., Milsom, A.B., Rassaf, T., Maloney, R.E., Bharti, A., Rodriguez, J., & Feelisch, M. 2005. Nitrite is a signaling molecule and regulator of gene expression in mammalian tissues. Nat.Chem.Biol., 1, (5) 290-297
Buehler, P.W. & Alayash, A.I. 2004. Oxygen sensing in the circulation: "cross talk" between red blood cells and the vasculature. Antioxid.Redox.Signal., 6, (6) 1000-1010
Buettner, G.R. & Jurkiewicz, B.A. 1993. Ascorbate free radical as a marker of oxidative stress: an EPR study. Free Radic.Biol.Med., 14, (1) 49-55
Burtscher, M., Gatterer, H., Szubski, C., Pierantozzi, E., & Faulhaber, M. 2009. Effects of interval hypoxia on exercise tolerance: special focus on patients with CAD or COPD. Sleep Breath.
Burtscher, M., Pachinger, O., Ehrenbourg, I., Mitterbauer, G., Faulhaber, M., Puhringer, R., & Tkatchouk, E. 2004. Intermittent hypoxia increases exercise tolerance in elderly men with and without coronary artery disease. Int.J.Cardiol., 96, (2) 247-254
Cai, Z., Manalo, D.J., Wei, G., Rodriguez, E.R., Fox-Talbot, K., Lu, H., Zweier, J.L., & Semenza, G.L. 2003. Hearts from rodents exposed to intermittent hypoxia or erythropoietin are protected against ischemia-reperfusion injury. Circulation, 108, (1) 79-85
Calbet, J.A. 2000. Oxygen tension and content in the regulation of limb blood flow. Acta Physiol Scand., 168, (4) 465-472
Calbet, J.A., Boushel, R., Radegran, G., Sondergaard, H., Wagner, P.D., & Saltin, B. 2003a. Determinants of maximal oxygen uptake in severe acute hypoxia. Am.J.Physiol Regul.p Physiol, 284, (2) 291-303
Calbet, J.A., Boushel, R., Radegran, G., Sondergaard, H., Wagner, P.D., & Saltin, B. 2003b. Why is VO2 max after altitude acclimatization still reduced despite normalization of arterial O2 content? Am.J.Physiol Regul.p Physiol, 284, (2) R304-R316
Calbet, J.A., Robach, P., Lundby, C., & Boushel, R. 2008. Is pulmonary gas exchange during exercise in hypoxia impaired with the increase of cardiac output? Appl.Physiol Nutr.Metab, 33, (3) 593-600
Cannon, R.O., III, Schechter, A.N., Panza, J.A., Ognibene, F.P., Pease-Fye, M.E., Waclawiw, M.A., Shelhamer, J.H., & Gladwin, M.T. 2001. Effects of inhaled nitric oxide on regional blood flow are consistent with intravascular nitric oxide delivery. J.Clin.Invest, 108, (2) 279-287
Capen, R.L. & Wagner, W.W., Jr. 1982. Intrapulmonary blood flow redistribution during hypoxia increases gas exchange surface area. J.Appl.Physiol, 52, (6) 1575-1580
Casas, M., Casas, H., Pages, T., Rama, R., Ricart, A., Ventura, J.L., Ibanez, J., Rodriguez, F.A., & Viscor, G. 2000. Intermittent hypobaric hypoxia induces altitude acclimation and improves the lactate threshold. Aviat.Space Environ.Med., 71, (2) 125-130
Ceriello, A., Mercuri, F., Quagliaro, L., Assaloni, R., Motz, E., Tonutti, L., & Taboga, C. 2001. Detection of nitrotyrosine in the diabetic plasma: evidence of oxidative stress. Diabetologia, 44, (7) 834-838
Chance, B., Wang, N.G., Maris, M., Nioka, S., & Sevick, E. 1992. Quantitation of tissue optical characteristics and hemoglobin desaturation by time- and frequency-resolved multi-wavelength spectrophotometry. Adv.Exp.Med.Biol., 317, 297-304
Chapman, R.F., Stray-Gundersen, J., & Levine, B.D. 1998. Individual variation in response to altitude training. J.Appl.Physiol, 85, (4) 1448-1456
Chaudhuri, A. & Behan, P.O. 2000. Fatigue and basal ganglia. J.Neurol.Sci., 179, (S 1-2) 34-42 available from: PM:11054483
Chavez, J.C., Agani, F., Pichiule, P., & LaManna, J.C. 2000. Expression of hypoxia-inducible factor-1alpha in the brain of rats during chronic hypoxia. J.Appl.Physiol, 89, (5) 1937-1942
Chin, L.M., Leigh, R.J., Heigenhauser, G.J., Rossiter, H.B., Paterson, D.H., & Kowalchuk, J.M. 2007. Hyperventilation-induced hypocapnic alkalosis slows the adaptation of pulmonary O2 uptake during the transition to moderate-intensity exercise. J.Physiol, 583, (Pt 1) 351-364
Chiu, J.J., Lee, P.L., Chen, C.N., Lee, C.I., Chang, S.F., Chen, L.J., Lien, S.C., Ko, Y.C., Usami, S., & Chien, S. 2004. Shear stress increases ICAM-1 and decreases VCAM-1 and E-selectin expressions induced by tumor necrosis factor-[alpha] in endothelial cells. Arterioscler.Thromb.Vasc.Biol., 24, (1) 73-79
Chuang, M.L., Ting, H., Otsuka, T., Sun, X.G., Chiu, F.Y., Hansen, J.E., & Wasserman, K. 2002. Muscle deoxygenation as related to work rate. Med.Sci.Sports Exerc., 34, (10) 1614-1623
Cosby, K., Partovi, K.S., Crawford, J.H., Patel, R.P., Reiter, C.D., Martyr, S., Yang, B.K., Waclawiw, M.A., Zalos, G., Xu, X., Huang, K.T., Shields, H., Kim-Shapiro, D.B., Schechter, A.N., Cannon, R.O., III, & Gladwin, M.T. 2003. Nitrite reduction to nitric oxide by deoxyhemoglobin vasodilates the human circulation. Nat.Med., 9, (12) 1498-1505
Costes, F., Barthelemy, J.C., Feasson, L., Busso, T., Geyssant, A., & Denis, C. 1996. Comparison of muscle near-infrared spectroscopy and femoral blood gases during steady-state exercise in humans. J.Appl.Physiol, 80, (4) 1345-1350
Costes, F., Prieur, F., Feasson, L., Geyssant, A., Barthelemy, J.C., & Denis, C. 2001. Influence of training on NIRS muscle oxygen saturation during submaximal exercise. Med.Sci.Sports Exerc., 33, (9) 1484-1489
Crawford, J.H., Isbell, T.S., Huang, Z., Shiva, S., Chacko, B.K., Schechter, A.N., rley-Usmar, V.M., Kerby, J.D., Lang, J.D., Jr., Kraus, D., Ho, C., Gladwin, M.T., & Patel, R.P. 2006. Hypoxia, red blood cells, and nitrite regulate NO-dependent hypoxic vasodilation. Blood, 107, (2) 566-574
Dal, S.D., Moreira, A.P., Freitas, A., Silva, J.S., Rossi, M.A., Ferreira, S.H., & Cunha, F.Q. 2006. Nitric oxide inhibits neutrophil migration by a mechanism dependent on ICAM-1: role of soluble guanylate cyclase. Nitric.Oxide., 15, (1) 77-86
Dalsgaard, M.K., Ogoh, S., Dawson, E.A., Yoshiga, C.C., Quistorff, B., & Secher, N.H. 2004. Cerebral carbohydrate cost of physical exertion in humans. Am.J.Physiol Regul.p Physiol, 287, (3)534-540
Dalsgaard, M.K. & Secher, N.H. 2007. The brain at work: a cerebral metabolic manifestation of central fatigue? J.Neurosci.Res., 85, (15) 3334-3339
Dalsgaard, T., Simonsen, U., & Fago, A. 2007. Nitrite-dependent vasodilation is facilitated by hypoxia and is independent of known NO-generating nitrite reductase activities. Am.J.Physiol Heart Circ.Physiol, 292, (6) 3072-3078
Davison, G.W., George, L., Jackson, S.K., Young, I.S., Davies, B., Bailey, D.M., Peters, J.R., & Ashton, T. 2002. Exercise, free radicals, and lipid peroxidation in type 1 diabetes mellitus. Free Radic.Biol.Med., 33, (11) 1543-1551
Davison, G.W., Morgan, R.M., Hiscock, N., Garcia, J.M., Grace, F., Boisseau, N., Davies, B., Castell, L., McEneny, J., Young, I.S., Hullin, D., Ashton, T., & Bailey, D.M. 2006. Manipulation of systemic oxygen flux by acute exercise and normobaric hypoxia: implications for reactive oxygen species generation. Clin.Sci.(Lond), 110, (1) 133-141
De, C.R., Libby, P., Peng, H.B., Thannickal, V.J., Rajavashisth, T.B., Gimbrone, M.A., Jr., Shin, W.S., & Liao, J.K. 1995. Nitric oxide decreases cytokine-induced endothelial activation. Nitric oxide selectively reduces endothelial expression of adhesion molecules and proinflammatory cytokines. J.Clin.Invest, 96, (1) 60-68
Dehnert, C., Grunig, E., Mereles, D., von, L.N., & Bartsch, P. 2005. Identification of individuals susceptible to high-altitude pulmonary oedema at low altitude. Eur.Respir.J., 25, (3) 545-551
Dejam, A., Hunter, C.J., Pelletier, M.M., Hsu, L.L., Machado, R.F., Shiva, S., Power, G.G., Kelm, M., Gladwin, M.T., & Schechter, A.N. 2005. Erythrocytes are the major intravascular storage sites of nitrite in human blood. Blood, 106, (2) 734-739
Dejam, A., Hunter, C.J., Schechter, A.N., & Gladwin, M.T. 2004a. Emerging role of nitrite in human biology. Blood Cells Mol.Dis., 32, (3) 423-429
DeLorey, D.S., Kowalchuk, J.M., & Paterson, D.H. 2004a. Effects of prior heavy-intensity exercise on pulmonary O2 uptake and muscle deoxygenation kinetics in young and older adult humans. J.Appl.Physiol, 97, (3) 998-1005
DeLorey, D.S., Shaw, C.N., Shoemaker, J.K., Kowalchuk, J.M., & Paterson, D.H. 2004c. The effect of hypoxia on pulmonary O2 uptake, leg blood flow and muscle deoxygenation during single-leg knee-extension exercise. Exp.Physiol, 89, (3) 293-302
Delpy, D.T., Cope, M.C., Cady, E.B., Wyatt, J.S., Hamilton, P.A., Hope, P.L., Wray, S., & Reynolds, E.O. 1987. Cerebral monitoring in newborn infants by magnetic resonance and near infrared spectroscopy. Scand.J.Clin.Lab Invest Suppl, 188, 9-17
Di, F.S., Sciartilli, A., Di, V., V, Di, B.A., & Gallina, S. 2009. The effect of physical exercise on endothelial function. Sports Med., 39, (10) 797-812
Dimmeler, S., Hermann, C., Galle, J., & Zeiher, A.M. 1999. Upregulation of superoxide dismutase and nitric oxide synthase mediates the apoptosis-suppressive effects of shear stress on endothelial cells. Arterioscler.Thromb.Vasc.Biol., 19, (3) 656-664
Ding, H.L., Zhu, H.F., Dong, J.W., Zhu, W.Z., Yang, W.W., Yang, H.T., & Zhou, Z.N. 2005. Inducible nitric oxide synthase contributes to intermittent hypoxia against ischemia/reperfusion injury. Acta Pharmacol.Sin., 26, (3) 315-322
Dousset, E., Decherchi, P., Grelot, L., & Jammes, Y. 2001. Effects of chronic hypoxemia on the afferent nerve activities from skeletal muscle. Am.J.Respir.Crit Care Med., 164, (8 Pt 1) 1476-1480
Dousset, E., Decherchi, P., Grelot, L., & Jammes, Y. 2003. Comparison between the effects of chronic and acute hypoxemia on muscle afferent activities from the tibialis anterior muscle. Exp.Brain Res., 148, (3) 320-327
Doyle, M.P., Pickering, R.A., DeWeert, T.M., Hoekstra, J.W., & Pater, D. 1981. Kinetics and mechanism of the oxidation of human deoxyhemoglobin by nitrites. J.Biol.Chem., 256, (23) 12393-12398
Dufour, S.P., Ponsot, E., Zoll, J., Doutreleau, S., Lonsdorfer-Wolf, E., Geny, B., Lampert, E., Fluck, M., Hoppeler, H., Billat, V., Mettauer, B., Richard, R., & Lonsdorfer, J. 2006. Exercise training in normobaric hypoxia in endurance runners. I. Improvement in aerobic performance capacity. J.Appl.Physiol, 100, (4) 1238-1248
Duhamel, T.A., Green, H.J., Sandiford, S.D., Perco, J.G., & Ouyang, J. 2004. Effects of progressive exercise and hypoxia on human muscle sarcoplasmic reticulum function. J.Appl.Physiol, 97, (1) 188-196
Duncan, A., Meek, J.H., Clemence, M., Elwell, C.E., Tyszczuk, L., Cope, M., & Delpy, D.T. 1995. Optical pathlength measurements on adult head, calf and forearm and the head of the newborn infant using phase resolved optical spectroscopy. Phys.Med.Biol., 40, (2) 295-304
Duranski, M.R., Greer, J.J., Dejam, A., Jaganmohan, S., Hogg, N., Langston, W., Patel, R.P., Yet, S.F., Wang, X., Kevil, C.G., Gladwin, M.T., & Lefer, D.J. 2005. Cytoprotective effects of nitrite during in vivo ischemia-reperfusion of the heart and liver. J.Clin.Invest, 115, (5) 1232-1240
Dworschak, M., Franz, M., Hallstrom, S., Semsroth, S., Gasser, H., Haisjackl, M., Podesser, B.K., & Malinski, T. 2004. S-nitroso human serum albumin improves oxygen metabolism during reperfusion after severe myocardial ischemia. Pharmacology, 72, (2) 106-112
Dyugovskaya, L., Lavie, P., & Lavie, L. 2002. Increased adhesion molecules expression and production of reactive oxygen species in leukocytes of sleep apnea patients. Am.J.Respir.Crit Care Med., 165, (7) 934-939
Erzurum, S.C., Ghosh, S., Janocha, A.J., Xu, W., Bauer, S., Bryan, N.S., Tejero, J., Hemann, C., Hille, R., Stuehr, D.J., Feelisch, M., & Beall, C.M. 2007. Higher blood flow and circulating NO products offset high-altitude hypoxia among Tibetans. Proc.Natl.Acad.Sci.U.S.A, 104, (45) 17593-17598
Esaki, K., Hamaoka, T., Radegran, G., Boushel, R., Hansen, J., Katsumura, T., Haga, S., & Mizuno, M. 2005. Association between regional quadriceps oxygenation and blood oxygen saturation during normoxic one-legged dynamic knee extension. Eur.J.Appl.Physiol, 95, (4) 361-370
Fadel, P.J., Keller, D.M., Watanabe, H., Raven, P.B., & Thomas, G.D. 2004. Noninvasive assessment of sympathetic vasoconstriction in human and rodent skeletal muscle using near-infrared spectroscopy and Doppler ultrasound. J.Appl.Physiol, 96, (4) 1323-1330
Farsidfar, F., Kasikcioglu, E., Oflaz, H., Kasikcioglu, D., Meric, M., & Umman, S. 2008. Effects of different intensities of acute exercise on flow-mediated dilatation in patients with coronary heart disease. Int.J.Cardiol., 124, (3) 372-374
Fatouros, I.G., Jamurtas, A.Z., Villiotou, V., Pouliopoulou, S., Fotinakis, P., Taxildaris, K., & Deliconstantinos, G. 2004. Oxidative stress responses in older men during endurance training and detraining. Med.Sci.Sports Exerc., 36, (12) 2065-2072
Ferrari, M., Mottola, L., & Quaresima, V. 2004. Principles, techniques, and limitations of near infrared spectroscopy. Can.J.Appl.Physiol, 29, (4) 463-487
Ferreira, L.F., Hueber, D.M., & Barstow, T.J. 2007a. Effects of assuming constant optical scattering on measurements of muscle oxygenation by near-infrared spectroscopy during exercise. J.Appl.Physiol, 102, (1) 358-367
Ferreira, L.F., Koga, S., & Barstow, T.J. 2007b. Dynamics of noninvasively estimated microvascular O2 extraction during ramp exercise. J.Appl.Physiol, 103, (6) 1999-2004
Ferreira, L.F., Townsend, D.K., Lutjemeier, B.J., & Barstow, T.J. 2005. Muscle capillary blood flow kinetics estimated from pulmonary O2 uptake and near-infrared spectroscopy. J.Appl.Physiol, 98, (5) 1820-1828
Ferretti, G., Moia, C., Thomet, J.M., & Kayser, B. 1997. The decrease of maximal oxygen consumption during hypoxia in man: a mirror image of the oxygen equilibrium curve J.Physiol, 498 ( Pt 1), 231-237
Flogel, U., Decking, U.K., Godecke, A., & Schrader, J. 1999. Contribution of NO to ischemia-reperfusion injury in the saline-perfused heart: a study in endothelial NO synthase knockout mice. J.Mol.Cell Cardiol., 31, (4) 827-836
Foster, G.E., Brugniaux, J.V., Pialoux, V., Duggan, C.T., Hanly, P.J., Ahmed, S.B., & Poulin, M.J. 2009. Cardiovascular and cerebrovascular responses to acute hypoxia following exposure to intermittent hypoxia in healthy humans. J.Physiol, 587, (Pt 13) 3287-3299
Foster, G.E., McKenzie, D.C., Milsom, W.K., & Sheel, A.W. 2005. Effects of two protocols of intermittent hypoxia on human ventilatory, cardiovascular and cerebral responses to hypoxia. J.Physiol, 567, (Pt 2) 689-699
Foster, G.E., McKenzie, D.C., & Sheel, A.W. 2006. Effects of enhanced human chemosensitivity on ventilatory responses to exercise. Exp.Physiol, 91, (1) 221-228
Friedmann, B., Frese, F., Menold, E., & Bartsch, P. 2005. Individual variation in the reduction of heart rate and performance at lactate thresholds in acute normobaric hypoxia. Int.J.Sports Med., 26, (7) 531-536
Frijns, C.J. & Kappelle, L.J. 2002. Inflammatory cell adhesion molecules in ischemic cerebrovascular disease. Stroke, 33, (8) 2115-2122
Frisbee, J.C. 2001. Impaired dilation of skeletal muscle microvessels to reduced oxygen tension in diabetic obese Zucker rats. Am.J.Physiol Heart Circ.Physiol, 281, (4) 1568-1574
Frisbee, J.C. & Stepp, D.W. 2001. Impaired NO-dependent dilation of skeletal muscle arterioles in hypertensive diabetic obese Zucker rats. Am.J.Physiol Heart Circ.Physiol, 281, (3) 1304-1311
Fulco, C.S., Rock, P.B., & Cymerman, A. 1998. Maximal and submaximal exercise performance at altitude. Aviat.Space Environ.Med., 69, (8) 793-801
Furchgott, R.F. & Zawadzki, J.V. 1980. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature, 288, (5789) 373-376
Gavin, T.P., Derchak, P.A., & Stager, J.M. 1998. Ventilation's role in the decline in VO2max and SaO2 in acute hypoxic exercise. Med.Sci.Sports Exerc., 30, (2) 195-199
Gerovasili, V., Drakos, S., Kravari, M., Malliaras, K., Karatzanos, E., Dimopoulos, S., Tasoulis, A., nastasiou-Nana, M., Roussos, C., & Nanas, S. 2009. Physical exercise improves the peripheral microcirculation of patients with chronic heart failure. J.Cardiopulm.Rehabil.Prev., 29, (6) 385-391
Giustarini, D., Milzani, A., Colombo, R., le-Donne, I., & Rossi, R. 2003. Nitric oxide and S-nitrosothiols in human blood. Clin.Chim.Acta, 330, (1-2) 85-98
Gladwin, M.T., Crawford, J.H., & Patel, R.P. 2004. The biochemistry of nitric oxide, nitrite, and hemoglobin: role in blood flow regulation. Free Radic.Biol.Med., 36, (6) 707-717
Gladwin, M.T., Raat, N.J., Shiva, S., Dezfulian, C., Hogg, N., Kim-Shapiro, D.B., & Patel, R.P. 2006. Nitrite as a vascular endocrine nitric oxide reservoir that contributes to hypoxic signaling, cytoprotection, and vasodilation. Am.J.Physiol Heart Circ.Physiol, 291, (5) 2026-2035
Gladwin, M.T., Schechter, A.N., Kim-Shapiro, D.B., Patel, R.P., Hogg, N., Shiva, S., Cannon, R.O., III, Kelm, M., Wink, D.A., Espey, M.G., Oldfield, E.H., Pluta, R.M., Freeman, B.A., Lancaster, J.R., Jr., Feelisch, M., & Lundberg, J.O. 2005. The emerging biology of the nitrite anion. Nat.Chem.Biol., 1, (6) 308-314
Gladwin, M.T., Shelhamer, J.H., Schechter, A.N., Pease-Fye, M.E., Waclawiw, M.A., Panza, J.A., Ognibene, F.P., & Cannon, R.O., III 2000a. Role of circulating nitrite and S-nitrosohemoglobin in the regulation of regional blood flow in humans. Proc.Natl.Acad.Sci.U.S.A, 97, (21) 11482-11487
Gladwin, M.T., Shelhamer, J.H., Schechter, A.N., Pease-Fye, M.E., Waclawiw, M.A., Panza, J.A., Ognibene, F.P., & Cannon, R.O., III 2000b. Role of circulating nitrite and S-nitrosohemoglobin in the regulation of regional blood flow in humans. Proc.Natl.Acad.Sci.U.S.A, 97, (21) 11482-11487
Glantzounis, G.K., Rocks, S.A., Sheth, H., Knight, I., Salacinski, H.J., Davidson, B.R., Winyard, P.G., & Seifalian, A.M. 2007. Formation and role of plasma S-nitrosothiols in liver ischemia-reperfusion injury. Free Radic.Biol.Med., 42, (6) 882-892
Gonzalez-Alonso, J., Dalsgaard, M.K., Osada, T., Volianitis, S., Dawson, E.A., Yoshiga, C.C., & Secher, N.H. 2004. Brain and central haemodynamics and oxygenation during maximal exercise in humans. J.Physiol, 557, (Pt 1) 331-342
Gonzalez-Alonso, J., Olsen, D.B., & Saltin, B. 2002. Erythrocyte and the regulation of human skeletal muscle blood flow and oxygen delivery: role of circulating ATP. Circ.Res., 91, (11) 1046-1055
Gonzalez-Alonso, J., Richardson, R.S., & Saltin, B. 2001. Exercising skeletal muscle blood flow in humans responds to reduction in arterial oxyhaemoglobin, but not to altered free oxygen. J.Physiol, 530, (Pt 2) 331-341
Gore, C.J., Little, S.C., Hahn, A.G., Scroop, G.C., Norton, K.I., Bourdon, P.C., Woolford, S.M., Buckley, J.D., Stanef, T., Campbell, D.P., Watson, D.B., & Emonson, D.L. 1997. Reduced performance of male and female athletes at 580 m altitude. Eur.J.Appl.Physiol Occup.Physiol, 75, (2) 136-143
Gore, C.J., Hahn, A.G., Aughey, R.J., Martin, D.T., Ashenden, M.J., Clark, S.A., Garnham, A.P., Roberts, A.D., Slater, G.J., & McKenna, M.J. 2001. Live high:train low increases muscle buffer capacity and submaximal cycling efficiency. Acta Physiol Scand., 173, (3) 275-286
Goto, C., Higashi, Y., Kimura, M., Noma, K., Hara, K., Nakagawa, K., Kawamura, M., Chayama, K., Yoshizumi, M., & Nara, I. 2003. Effect of different intensities of exercise on endothelium-dependent vasodilation in humans: role of endothelium-dependent nitric oxide and oxidative stress. Circulation, 108, (5) 530-535
Govoni, M., Jansson, E.A., Weitzberg, E., & Lundberg, J.O. 2008. The increase in plasma nitrite after a dietary nitrate load is markedly attenuated by an antibacterial mouthwash. Nitric.Oxide., 19, (4) 333-337
Grassi, B., Pogliaghi, S., Rampichini, S., Quaresima, V., Ferrari, M., Marconi, C., & Cerretelli, P. 2003. Muscle oxygenation and pulmonary gas exchange kinetics during cycling exercise on-transitions in humans. J.Appl.Physiol, 95, (1) 149-158
Grassi, B., Quaresima, V., Marconi, C., Ferrari, M., & Cerretelli, P. 1999. Blood lactate accumulation and muscle deoxygenation during incremental exercise. J.Appl.Physiol, 87, (1) 348-355
Grebe, M., Eisele, H.J., Weissmann, N., Schaefer, C., Tillmanns, H., Seeger, W., & Schulz, R. 2006. Antioxidant vitamin C improves endothelial function in obstructive sleep apnea. Am.J.Respir.Crit Care Med., 173, (8) 897-901
Gryglewski, R.J., Palmer, R.M., & Moncada, S. 1986. Superoxide anion is involved in the breakdown of endothelium-derived vascular relaxing factor. Nature, 320, (6061) 454-456
Hallstrom, S., Gasser, H., Neumayer, C., Fugl, A., Nanobashvili, J., Jakubowski, A., Huk, I., Schlag, G., & Malinski, T. 2002. S-nitroso human serum albumin treatment reduces ischemia/reperfusion injury in skeletal muscle via nitric oxide release. Circulation, 105, (25) 3032-3038
Hansen, J., Sander, M., Hald, C.F., Victor, R.G., & Thomas, G.D. 2000. Metabolic modulation of sympathetic vasoconstriction in human skeletal muscle: role of tissue hypoxia. J.Physiol, 527 Pt 2, 387-396
Harms, C.A., McClaran, S.R., Nickele, G.A., Pegelow, D.F., Nelson, W.B., & Dempsey, J.A. 2000. Effect of exercise-induced arterial O2 desaturation on VO2max in women. Med.Sci.Sports Exerc., 32, (6) 1101-1108
Haseler, L.J., Hogan, M.C., & Richardson, R.S. 1999. Skeletal muscle phosphocreatine recovery in exercise-trained humans is dependent on O2 availability. J.Appl.Physiol, 86, (6) 2013-2018
Heubert, R.A., Quaresima, V., Laffite, L.P., Koralsztein, J.P., & Billat, V.L. 2005. Acute moderate hypoxia affects the oxygen desaturation and the performance but not the oxygen uptake response. Int.J.Sports Med., 26, (7) 542-551
Hogg, N. 2002. The biochemistry and physiology of S-nitrosothiols. Annu.Rev.Pharmacol.Toxicol., 42, 585-600
Holmberg, H.C. & Calbet, J.A. 2007. Insufficient ventilation as a cause of impaired pulmonary gas exchange during submaximal exercise. Respir.Physiol Neurobiol., 157, (2-3) 348-359
Holmlund, A., Hulthe, J., Millgard, J., Sarabi, M., Kahan, T., & Lind, L. 2002. Soluble intercellular adhesion molecule-1 is related to endothelial vasodilatory function in healthy individuals. Atherosclerosis, 165, (2) 271-276
Hupperets, M.D., Hopkins, S.R., Pronk, M.G., Tiemessen, I.J., Garcia, N., Wagner, P.D., & Powell, F.L. 2004. Increased hypoxic ventilatory response during 8 weeks at 3800 m altitude. Respir.Physiol Neurobiol., 142, (2-3) 145-152
Ichikawa, H., Flores, S., Kvietys, P.R., Wolf, R.E., Yoshikawa, T., Granger, D.N., & Aw, T.Y. 1997. Molecular mechanisms of anoxia/reoxygenation-induced neutrophil adherence to cultured endothelial cells. Circ.Res., 81, (6) 922-931
Ide, K., Boushel, R., Sorensen, H.M., Fernandes, A., Cai, Y., Pott, F., & Secher, N.H. 2000a. Middle cerebral artery blood velocity during exercise with beta-1 adrenergic and unilateral stellate ganglion blockade in humans. Acta Physiol Scand., 170, (1) 33-38
Ide, K., Pott, F., Van Lieshout, J.J., & Secher, N.H. 1998. Middle cerebral artery blood velocity depends on cardiac output during exercise with a large muscle mass. Acta Physiol Scand., 162, (1) 13-20
Ide, K., Schmalbruch, I.K., Quistorff, B., Horn, A., & Secher, N.H. 2000b. Lactate, glucose and O2 uptake in human brain during recovery from maximal exercise. J.Physiol, 522 Pt 1, 159-164
Ide, K. & Secher, N.H. 2000. Cerebral blood flow and metabolism during exercise. Prog.Neurobiol., 61, (4) 397-414
Ide, K., Worthley, M., Anderson, T., & Poulin, M.J. 2007. Effects of the nitric oxide synthase inhibitor L-NMMA on cerebrovascular and cardiovascular responses to hypoxia and hypercapnia in humans. J.Physiol, 584, (Pt 1) 321-332
Ignarro, L.J., Buga, G.M., Wood, K.S., Byrns, R.E., & Chaudhuri, G. 1987. Endothelium-derived relaxing factor produced and released from artery and vein is nitric oxide. Proc.Natl.Acad.Sci.U.S.A, 84, (24) 9265-9269
Imray, C.H., Myers, S.D., Pattinson, K.T., Bradwell, A.R., Chan, C.W., Harris, S., Collins, P., & Wright, A.D. 2005. Effect of exercise on cerebral perfusion in humans at high altitude. J.Appl.Physiol, 99, (2) 699-706
Ip, M.S., Lam, B., Chan, L.Y., Zheng, L., Tsang, K.W., Fung, P.C., & Lam, W.K. 2000. Circulating nitric oxide is suppressed in obstructive sleep apnea and is reversed by nasal continuous positive airway pressure. Am.J.Respir.Crit Care Med., 162, (6) 2166-2171
Isbell, T.S., Gladwin, M.T., & Patel, R.P. 2007. Hemoglobin oxygen fractional saturation regulates nitrite-dependent vasodilation of aortic ring bioassays. Am.J.Physiol Heart Circ.Physiol, 293, (4) 2565-2572
James, P.E., Lang, D., Tufnell-Barret, T., Milsom, A.B., & Frenneaux, M.P. 2004. Vasorelaxation by red blood cells and impairment in diabetes: reduced nitric oxide and oxygen delivery by glycated hemoglobin. Circ.Res., 94, (7) 976-983
Jansen, G.F., Krins, A., Basnyat, B., Bosch, A., & Odoom, J.A. 2000. Cerebral autoregulation in subjects adapted and not adapted to high altitude. Stroke, 31, (10) 2314-2318
Jansen, G.F., Krins, A., Basnyat, B., Odoom, J.A., & Ince, C. 2007. Role of the altitude level on cerebral autoregulation in residents at high altitude. J.Appl.Physiol, 103, (2) 518-523
Jansson, E.A., Huang, L., Malkey, R., Govoni, M., Nihlen, C., Olsson, A., Stensdotter, M., Petersson, J., Holm, L., Weitzberg, E., & Lundberg, J.O. 2008. A mammalian functional nitrate reductase that regulates nitrite and nitric oxide homeostasis. Nat.Chem.Biol., 4, (7) 411-417
Jia, L., Bonaventura, C., Bonaventura, J., & Stamler, J.S. 1996. S-nitrosohaemoglobin: a dynamic activity of blood involved in vascular control Nature, 380, (6571) 221-226
Jilma, B., Eichler, H.G., Stohlawetz, P., Dirnberger, E., Kapiotis, S., Wagner, O.F., Schutz, W., & Krejcy, K. 1997. Effects of exercise on circulating vascular adhesion molecules in healthy men. Immunobiology, 197, (5) 505-512
Jones, A.M., Berger, N.J., Wilkerson, D.P., & Roberts, C.L. 2006. Effects of "priming" exercise on pulmonary O2 uptake and muscle deoxygenation kinetics during heavy-intensity cycle exercise in the supine and upright positions. J.Appl.Physiol, 101, (5) 1432-1441
Jones, A.M., Wilkerson, D.P., & Campbell, I.T. 2004. Nitric oxide synthase inhibition with L-NAME reduces maximal oxygen uptake but not gas exchange threshold during incremental cycle exercise in man. J.Physiol, 560, (Pt 1) 329-338
Julian, C.G., Gore, C.J., Wilber, R.L., Daniels, J.T., Fredericson, M., Stray-Gundersen, J., Hahn, A.G., Parisotto, R., & Levine, B.D. 2004. Intermittent normobaric hypoxia does not alter performance or erythropoietic markers in highly trained distance runners. J.Appl.Physiol, 96, (5) 1800-1807
Kalliokoski, K.K., Oikonen, V., Takala, T.O., Sipila, H., Knuuti, J., & Nuutila, P. 2001. Enhanced oxygen extraction and reduced flow heterogeneity in exercising muscle in endurance-trained men. Am.J.Physiol Endocrinol.Metab, 280, (6) 1015-1021
Kaminski, A., Kasch, C., Zhang, L., Kumar, S., Sponholz, C., Choi, Y.H., Ma, N., Liebold, A., Ladilov, Y., Steinhoff, G., & Stamm, C. 2007. Endothelial nitric oxide synthase mediates protective effects of hypoxic preconditioning in lungs. Respir.Physiol Neurobiol., 155, (3) 280-285
Katayama, K., Matsuo, H., Ishida, K., Mori, S., & Miyamura, M. 2003. Intermittent hypoxia improves endurance performance and submaximal exercise efficiency. High Alt.Med.Biol., 4, (3) 291-304
Katayama, K., Sato, K., Hotta, N., Ishida, K., Iwasaki, K., & Miyamura, M. 2007. Intermittent hypoxia does not increase exercise ventilation at simulated moderate altitude. Int.J.Sports Med., 28, (6) 480-487
Katayama, K., Sato, K., Matsuo, H., Hotta, N., Sun, Z., Ishida, K., Iwasaki, K., & Miyamura, M. 2005. Changes in ventilatory responses to hypercapnia and hypoxia after intermittent hypoxia in humans. Respir.Physiol Neurobiol., 146, (1) 55-65
Katayama, K., Sato, K., Matsuo, H., Ishida, K., Iwasaki, K., & Miyamura, M. 2004. Effect of intermittent hypoxia on oxygen uptake during submaximal exercise in endurance athletes. Eur.J.Appl.Physiol, 92, (1-2) 75-83
Katayama, K., Sato, Y., Morotome, Y., Shima, N., Ishida, K., Mori, S., & Miyamura, M. 2001a. Intermittent hypoxia increases ventilation and Sa(O2) during hypoxic exercise and hypoxic chemosensitivity. J.Appl.Physiol, 90, (4) 1431-1440
Katayama, K., Shima, N., Sato, Y., Qiu, J.C., Ishida, K., Mori, S., & Miyamura, M. 2001c. Effect of intermittent hypoxia on cardiovascular adaptations and response to progressive hypoxia in humans. High Alt.Med.Biol., 2, (4) 501-508
Kayser, B. 2003. Exercise starts and ends in the brain. Eur.J.Appl.Physiol, 90, (3-4) 411-419
Kelm, M., Preik-Steinhoff, H., Preik, M., & Strauer, B.E. 1999. Serum nitrite sensitively reflects endothelial NO formation in human forearm vasculature: evidence for biochemical assessment of the endothelial L-arginine-NO pathway. Cardiovasc.Res., 41, (3) 765-772
Kevil, C.G., Pruitt, H., Kavanagh, T.J., Wilkerson, J., Farin, F., Moellering, D., rley-Usmar, V.M., Bullard, D.C., & Patel, R.P. 2004. Regulation of endothelial glutathione by ICAM-1: implications for inflammation. FASEB J., 18, (11) 1321-1323
Khan, J., Brennand, D.M., Bradley, N., Gao, B., Bruckdorfer, R., & Jacobs, M. 1998. 3-Nitrotyrosine in the proteins of human plasma determined by an ELISA method. Biochem.J., 330 ( Pt 2), 795-801
Kim, M. B., Ward, D. S., Cartwright, C. R., Kolano, J., Chlebowski, S., & Henson, L. C. 2000, "Estimation of jugular venous O2 saturation from cerebral oximetry or arterial O2 saturation during isocapnic hypoxia", J.Clin.put., vol. 16, no. 3, pp. 191-199
Kjaer, M., Hanel, B., Worm, L., Perko, G., Lewis, S.F., Sahlin, K., Galbo, H., & Secher, N.H. 1999. Cardiovascular and neuroendocrine responses to exercise in hypoxia during impaired neural feedback from muscle. Am.J.Physiol, 277, (1 Pt 2) 76-85
Kleinbongard, P., Dejam, A., Lauer, T., Jax, T., Kerber, S., Gharini, P., Balzer, J., Zotz, R.B., Scharf, R.E., Willers, R., Schechter, A.N., Feelisch, M., & Kelm, M. 2006a. Plasma nitrite concentrations reflect the degree of endothelial dysfunction in humans. Free Radic.Biol.Med., 40, (2) 295-302
Kleinbongard, P., Dejam, A., Lauer, T., Rassaf, T., Schindler, A., Picker, O., Scheeren, T., Godecke, A., Schrader, J., Schulz, R., Heusch, G., Schaub, G.A., Bryan, N.S., Feelisch, M., & Kelm, M. 2003. Plasma nitrite reflects constitutive nitric oxide synthase activity in mammals. Free Radic.Biol.Med., 35, (7) 790-796
Koehle, M.S., Sheel, A.W., Milsom, W.K., & McKenzie, D.C. 2007. Two patterns of daily hypoxic exposure and their effects on measures of chemosensitivity in humans. J.Appl.Physiol, 103, (6) 1973-1978
Koller, A. & Kaley, G. 1991. Endothelial regulation of wall shear stress and blood flow in skeletal muscle microcirculation. Am.J.Physiol, 260, (3 Pt 2) 862-868
Kubes, P., Suzuki, M., & Granger, D.N. 1991. Nitric oxide: an endogenous modulator of leukocyte adhesion. Proc.Natl.Acad.Sci.U.S.A, 88, (11) 4651-4655
Lai, N., Zhou, H., Saidel, G.M., Wolf, M., McCully, K., Gladden, L.B., & Cabrera, M.E. 2009. Modeling oxygenation in venous blood and skeletal muscle in response to exercise using near-infrared spectroscopy. J.Appl.Physiol, 106, (6) 1858-1874
Lancaster, J.R., Jr. 1994. Simulation of the diffusion and reaction of endogenously produced nitric oxide. Proc.Natl.Acad.Sci.U.S.A, 91, (17) 8137-8141
Lang, J.D., Jr., Teng, X., Chumley, P., Crawford, J.H., Isbell, T.S., Chacko, B.K., Liu, Y., Jhala, N., Crowe, D.R., Smith, A.B., Cross, R.C., Frenette, L., Kelley, E.E., Wilhite, D.W., Hall, C.R., Page, G.P., Fallon, M.B., Bynon, J.S., Eckhoff, D.E., & Patel, R.P. 2007. Inhaled NO accelerates restoration of liver function in adults following orthotopic liver transplantation. J.Clin.Invest, 117, (9) 2583-2591
Larsen, F.J., Weitzberg, E., Lundberg, J.O., & Ekblom, B. 2007. Effects of dietary nitrate on oxygen cost during exercise. Acta Physiol (Oxf), 191, (1) 59-66
Larsen, F.J., Weitzberg, E., Lundberg, J.O., & Ekblom, B. 2010. Dietary nitrate reduces maximal oxygen consumption while maintaining work performance in maximal exercise. Free Radic.Biol.Med., 48, (2) 342-347
LASSEN, N.A. 1959. Cerebral blood flow and oxygen consumption in man. Physiol Rev., 39, (2) 183-238
Lattimore, J.D., Wilcox, I., Nakhla, S., Langenfeld, M., Jessup, W., & Celermajer, D.S. 2005. Repetitive hypoxia increases lipid loading in human macrophages-a potentially atherogenic effect. Atherosclerosis, 179, (2) 255-259
Lauer, T., Kleinbongard, P., & Kelm, M. 2002. Indexes of NO bioavailability in human blood. News Physiol Sci., 17, 251-255
Lauer, T., Preik, M., Rassaf, T., Strauer, B.E., Deussen, A., Feelisch, M., & Kelm, M. 2001. Plasma nitrite rather than nitrate reflects regional endothelial nitric oxide synthase activity but lacks intrinsic vasodilator action. Proc.Natl.Acad.Sci.U.S.A, 98, (22) 12814-12819
Lawson, D.L., Chen, L., & Mehta, J.L. 1997. Effects of exercise-induced oxidative stress on nitric oxide release and antioxidant activity. Am.J.Cardiol., 80, (12) 1640-1642
Lee, D.S., Badr, M.S., & Mateika, J.H. 2009. Progressive augmentation and ventilatory long-term facilitation are enhanced in sleep apnoea patients and are mitigated by antioxidant administration. J.Physiol, 587, (Pt 22) 5451-5467
Leeuwenberg, J.F., Smeets, E.F., Neefjes, J.J., Shaffer, M.A., Cinek, T., Jeunhomme, T.M., Ahern, T.J., & Buurman, W.A. 1992. E-selectin and intercellular adhesion molecule-1 are released by activated human endothelial cells in vitro. Immunology, 77, (4) 543-549
Legrand, R., Ahmaidi, S., Moalla, W., Chocquet, D., Marles, A., Prieur, F., & Mucci, P. 2005. O2 arterial desaturation in endurance athletes increases muscle deoxygenation. Med.Sci.Sports Exerc., 37, (5) 782-788
Loukogeorgakis, S.P., Panagiotidou, A.T., Yellon, D.M., Deanfield, J.E., & MacAllister, R.J. 2006. Postconditioning protects against endothelial ischemia-reperfusion injury in the human forearm. Circulation, 113, (7) 1015-1019
Levine, B.D. 2002. Intermittent hypoxic training: fact and fancy. High Alt.Med.Biol., 3, (2) 177-193
Levine, B.D. & Stray-Gundersen, J. 1997. "Living high-training low": effect of moderate-altitude acclimatization with low-altitude training on performance. J.Appl.Physiol, 83, (1) 102-112
Li, J., Savransky, V., Nanayakkara, A., Smith, P.L., O'Donnell, C.P., & Polotsky, V.Y. 2007. Hyperlipidemia and lipid peroxidation are dependent on the severity of chronic intermittent hypoxia. J.Appl.Physiol, 102, (2) 557-563
Lindemann, S., Sharafi, M., Spiecker, M., Buerke, M., Fisch, A., Grosser, T., Veit, K., Gierer, C., Ibe, W., Meyer, J., & Darius, H. 2000. NO reduces PMN adhesion to human vascular endothelial cells due to downregulation of ICAM-1 mRNA and surface expression. Thromb.Res., 97, (3) 113-123
Liu, J., Narasimhan, P., Yu, F., & Chan, P.H. 2005. Neuroprotection by hypoxic preconditioning involves oxidative stress-mediated expression of hypoxia-inducible factor and erythropoietin. Stroke, 36, (6) 1264-1269
Liu, X., Miller, M.J., Joshi, M.S., Sadowska-Krowicka, H., Clark, D.A., & Lancaster, J.R., Jr. 1998a. Diffusion-limited reaction of free nitric oxide with erythrocytes. J.Biol.Chem., 273, (30) 18709-18713
Liu, Y., Christou, H., Morita, T., Laughner, E., Semenza, G.L., & Kourembanas, S. 1998b. Carbon monoxide and nitric oxide suppress the hypoxic induction of vascular endothelial growth factor gene via the 5' enhancer. J.Biol.Chem., 273, (24) 15257-15262
Lundberg, J.O. & Govoni, M. 2004. Inorganic nitrate is a possible source for systemic generation of nitric oxide. Free Radic.Biol.Med., 37, (3) 395-400
Lundberg, J.O., Weitzberg, E., & Gladwin, M.T. 2008. The nitrate-nitrite-nitric oxide pathway in physiology and therapeutics. Nat.Rev.Drug Discov., 7, (2) 156-167
Lundby, C., Boushel, R., Robach, P., Moller, K., Saltin, B., & Calbet, J.A. 2008. During hypoxic exercise some vasoconstriction is needed to match O2 delivery with O2 demand at the microcirculatory level. J.Physiol, 586, (1) 123-130
Lundby, C., Sander, M., van, H.G., Saltin, B., & Calbet, J.A. 2006a. Maximal exercise and muscle oxygen extraction in acclimatizing lowlanders and high altitude natives. J.Physiol, 573, (Pt 2) 535-547
Madsen, P.L. & Secher, N.H. 1999. Near-infrared oximetry of the brain. Prog.Neurobiol., 58, (6) 541-560
Maher, A.R., Milsom, A.B., Gunaruwan, P., Abozguia, K., Ahmed, I., Weaver, R.A., Thomas, P., Ashrafian, H., Born, G.V., James, P.E., & Frenneaux, M.P. 2008. Hypoxic modulation of exogenous nitrite-induced vasodilation in humans. Circulation, 117, (5) 670-677
Maiorana, A., O'Driscoll, G., Taylor, R., & Green, D. 2003. Exercise and the nitric oxide vasodilator system. Sports Med., 33, (14) 1013-1035
Mallet, R.T., Ryou, M.G., Williams, A.G., Jr., Howard, L., & Downey, H.F. 2006. Beta1-Adrenergic receptor antagonism abrogates cardioprotective effects of intermittent hypoxia. Basic Res.Cardiol., 101, (5) 436-446
Malyshev, I.Y., Zenina, T.A., Golubeva, L.Y., Saltykova, V.A., Manukhina, E.B., Mikoyan, V.D., Kubrina, L.N., & Vanin, A.F. 1999. NO-dependent mechanisms of adaptation to hypoxia. Nitric.Oxide., 3, (2) 105-113
Mancini, D.M., Bolinger, L., Li, H., Kendrick, K., Chance, B., & Wilson, J.R. 1994. Validation of near-infrared spectroscopy in humans. J.Appl.Physiol, 77, (6) 2740-2747
Manukhina, E.B., Downey, H.F., & Mallet, R.T. 2006a. Role of nitric oxide in cardiovascular adaptation to intermittent hypoxia. Exp.Biol.Med.(Maywood.), 231, (4) 343-365
Manukhina, E.B., Mashina, S.Y., Smirin, B.V., Lyamina, N.P., Senchikhin, V.N., Vanin, A.F., & Malyshev, I.Y. 2000. Role of nitric oxide in adaptation to hypoxia and adaptive defense. Physiol Res., 49, (1) 89-97
Marley, R., Feelisch, M., Holt, S., & Moore, K. 2000. A chemiluminescense-based assay for S-nitrosoalbumin and other plasma S-nitrosothiols. Free Radic.Res., 32, (1) 1-9
Marley, R., Patel, R.P., Orie, N., Ceaser, E., rley-Usmar, V., & Moore, K. 2001. Formation of nanomolar concentrations of S-nitroso-albumin in human plasma by nitric oxide. Free Radic.Biol.Med., 31, (5) 688-696
Marshall, H.C., Hamlin, M.J., Hellemans, J., Murrell, C., Beattie, N., Hellemans, I., Perry, T., Burns, A., & Ainslie, P.N. 2008. Effects of intermittent hypoxia on SaO(2), cerebral and muscle oxygenation during maximal exercise in athletes with exercise-induced hypoxemia. Eur.J.Appl.Physiol, 104, (2) 383-393
Maxwell, A.J., Ho, H.V., Le, C.Q., Lin, P.S., Bernstein, D., & Cooke, J.P. 2001a. L-arginine enhances aerobic exercise capacity in association with augmented nitric oxide production. J.Appl.Physiol, 90, (3) 933-938
Maxwell, A.J., Schauble, E., Bernstein, D., & Cooke, J.P. 1998. Limb blood flow during exercise is dependent on nitric oxide. Circulation, 98, (4) 369-374
Maxwell, P.H. 2005. Hypoxia-inducible factor as a physiological regulator. Exp.Physiol, 90, (6) 791-797
May, J.M., Qu, Z.C., Xia, L., & Cobb, C.E. 2000. Nitrite uptake and metabolism and oxidant stress in human erythrocytes. Am.J.Physiol Cell Physiol, 279, (6) 1946-1954
McMahon, T.J., Moon, R.E., Luschinger, B.P., Carraway, M.S., Stone, A.E., Stolp, B.W., Gow, A.J., Pawloski, J.R., Watke, P., Singel, D.J., Piantadosi, C.A., & Stamler, J.S. 2002. Nitric oxide in the human respiratory cycle. Nat.Med., 8, (7) 711-717
McQuillan, L.P., Leung, G.K., Marsden, P.A., Kostyk, S.K., & Kourembanas, S. 1994. Hypoxia inhibits expression of eNOS via transcriptional and posttranscriptional mechanisms. Am.J.Physiol, 267, (5 Pt 2) 1921-1927
Milano, G., Corno, A.F., Lippa, S., von Segesser, L.K., & Samaja, M. 2002. Chronic and intermittent hypoxia induce different degrees of myocardial tolerance to hypoxia-induced dysfunction. Exp.Biol.Med.(Maywood.), 227, (6) 389-397
Millar, T.M., Stevens, C.R., Benjamin, N., Eisenthal, R., Harrison, R., & Blake, D.R. 1998. Xanthine oxidoreductase catalyses the reduction of nitrates and nitrite to nitric oxide under hypoxic conditions. FEBS Lett., 427, (2) 225-228
Mills, P.J., Hong, S., Redwine, L., Carter, S.M., Chiu, A., Ziegler, M.G., Dimsdale, J.E., & Maisel, A.S. 2006. Physical fitness attenuates leukocyte-endothelial adhesion in response to acute exercise. J.Appl.Physiol, 101, (3) 785-788
Mohiuddin, I., Chai, H., Lin, P.H., Lumsden, A.B., Yao, Q., & Chen, C. 2006. Nitrotyrosine and chlorotyrosine: clinical significance and biological functions in the vascular system. J.Surg.Res., 133, (2) 143-149
Mohler, E.R., III, Lech, G., Supple, G.E., Wang, H., & Chance, B. 2006. Impaired exercise-induced blood volume in type 2 diabetes with or without peripheral arterial disease measured by continuous-wave near-infrared spectroscopy. Diabetes Care, 29, (8) 1856-1859
Mollard, P., Woorons, X., Letournel, M., Lamberto, C., Favret, F., Pichon, A., Beaudry, M., & Richalet, J.P. 2007. Determinants of maximal oxygen uptake in moderate acute hypoxia in endurance athletes. Eur.J.Appl.Physiol, 100, (6) 663-673
Mollard, P., Woorons, X., ntoine-Jonville, S., Jutand, L., Richalet, J.P., Favret, F., & Pichon, A. 2008. 'Oxygen uptake efficiency slope' in trained and untrained subjects exposed to hypoxia. Respir.Physiol Neurobiol., 161, (2) 167-173
Monchanin, G., Serpero, L.D., Connes, P., Tripette, J., Wouassi, D., Bezin, L., Francina, A., Ngongang, J., de la, P.M., Massarelli, R., Gozal, D., Thiriet, P., & Martin, C. 2007. Effects of progressive and maximal exercise on plasma levels of adhesion molecules in athletes with sickle cell trait with or without alpha-thalassemia. J.Appl.Physiol, 102, (1) 169-173
Morigi, M., Zoja, C., Figliuzzi, M., Foppolo, M., Micheletti, G., Bontempelli, M., Saronni, M., Remuzzi, G., & Remuzzi, A. 1995. Fluid shear stress modulates surface expression of adhesion molecules by endothelial cells. Blood, 85, (7) 1696-1703
Mortensen, S.P., Dawson, E.A., Yoshiga, C.C., Dalsgaard, M.K., Damsgaard, R., Secher, N.H., & Gonzalez-Alonso, J. 2005. Limitations to systemic and locomotor limb muscle oxygen delivery and uptake during maximal exercise in humans. J.Physiol, 566, (Pt 1) 273-285
Murrant, C.L. & Reid, M.B. 2001. Detection of reactive oxygen and reactive nitrogen species in skeletal muscle. Microsc.Res.Tech., 55, (4) 236-248
Nacher, M., Serrano-Mollar, A., Farre, R., Panes, J., Segui, J., & Montserrat, J.M. 2007. Recurrent obstructive apneas trigger early systemic inflammation in a rat model of sleep apnea. Respir.Physiol Neurobiol., 155, (1) 93-96
Nagababu, E., Ramasamy, S., Abernethy, D.R., & Rifkind, J.M. 2003. Active nitric oxide produced in the red cell under hypoxic conditions by deoxyhemoglobin-mediated nitrite reduction. J.Biol.Chem., 278, (47) 46349-46356
Nagababu, E., Ramasamy, S., & Rifkind, J.M. 2006. S-nitrosohemoglobin: a mechanism for its formation in conjunction with nitrite reduction by deoxyhemoglobin. Nitric.Oxide., 15, (1) 20-29
Neary, J.P. 2004. Application of near infrared spectroscopy to exercise sports science
1. Can.J.Appl.Physiol, 29, (4) 488-503
Neary, J.P., McKenzie, D.C., & Bhambhani, Y.N. 2002. Effects of short-term endurance training on muscle deoxygenation trends using NIRS. Med.Sci.Sports Exerc., 34, (11) 1725-1732
Neckar, J., Ostadal, B., & Kolar, F. 2004. Myocardial infarct size-limiting effect of chronic hypoxia persists for five weeks of normoxic recovery. Physiol Res., 53, (6) 621-628
Neubauer, J.A. 2001a. Invited review: Physiological and pathophysiological responses to intermittent hypoxia. J.Appl.Physiol, 90, (4) 1593-1599
Nielsen, H.B., Boushel, R., Madsen, P., & Secher, N.H. 1999. Cerebral desaturation during exercise reversed by O2 supplementation. Am.J.Physiol, 277, (3 Pt 2) 1045-1052
Nielsen, H.B., Bredmose, P.P., Stromstad, M., Volianitis, S., Quistorff, B., & Secher, N.H. 2002. Bicarbonate attenuates arterial desaturation during maximal exercise in humans. J.Appl.Physiol, 93, (2) 724-731
Nioka, S., Wang, D.J., Im, J., Hamaoka, T., Wang, Z.J., Leigh, J.S., & Chance, B. 2006. Simulation of Mb/Hb in NIRS and oxygen gradient in the human and canine skeletal muscles using H-NMR and NIRS. Adv.Exp.Med.Biol., 578, 223-228
Noakes, T.D., Peltonen, J.E., & Rusko, H.K. 2001. Evidence that a central governor regulates exercise performance during acute hypoxia and hyperoxia. J.Exp.Biol., 204, (Pt
Norcliffe, L.J., Rivera-Ch, M., Claydon, V.E., Moore, J.P., Leon-Velarde, F., Appenzeller, O., & Hainsworth, R. 2005. Cerebrovascular responses to hypoxia and hypocapnia in high-altitude dwellers. J.Physiol, 566, (Pt 1) 287-294
Nybo, L. & Rasmussen, P. 2007. Inadequate cerebral oxygen delivery and central fatigue during strenuous exercise. Exerc.Sport Sci.Rev., 35, (3) 110-11818) 3225-3234
Ogoh, S. & Ainslie, P.N. 2009. Cerebral blood flow during exercise: mechanisms of regulation. J.Appl.Physiol, 107, (5) 1370-1380
Ogoh, S., Dalsgaard, M.K., Yoshiga, C.C., Dawson, E.A., Keller, D.M., Raven, P.B., & Secher, N.H. 2005. Dynamic cerebral autoregulation during exhaustive exercise in humans. Am.J.Physiol Heart Circ.Physiol, 288, (3) 1461-1467
Ohga, E., Nagase, T., Tomita, T., Teramoto, S., Matsuse, T., Katayama, H., & Ouchi, Y. 1999. Increased levels of circulating ICAM-1, VCAM-1, and L-selectin in obstructive sleep apnea syndrome. J.Appl.Physiol, 87, (1) 10-14
Ohno, S. & Malik, A.B. 1997. Polymorphonuclear leucocyte (PMN) inhibitory factor prevents PMN-dependent endothelial cell injury by an anti-adhesive mechanism. J.Cell Physiol, 171, (2) 212-216
Oja, J.M., Gillen, J.S., Kauppinen, R.A., Kraut, M., & van Zijl, P.C. 1999. Determination of oxygen extraction ratios by magnetic resonance imaging. J.Cereb.Blood Flow Metab, 19, (12) 1289-1295
Owen-Reece, H., Elwell, C.E., Wyatt, J.S., & Delpy, D.T. 1996. The effect of scalp ischaemia on measurement of cerebral blood volume by near-infrared spectroscopy. Physiol Meas., 17, (4) 279-286
Ozcelik, O. & Kelestimur, H. 2004. Effects of acute hypoxia on the determination of anaerobic threshold using the heart rate-work rate relationships during incremental exercise tests. Physiol Res., 53, (1) 45-51
Pacher, P., Beckman, J.S., & Liaudet, L. 2007. Nitric oxide and peroxynitrite in health and disease. Physiol Rev., 87, (1) 315-424
Padilla, D.J., McDonough, P., Behnke, B.J., Kano, Y., Hageman, K.S., Musch, T.I., & Poole, D.C. 2007. Effects of Type II diabetes on muscle microvascular oxygen pressures
1. Respir.Physiol Neurobiol., 156, (2) 187-195
Paulson, O.B., Strandgaard, S., & Edvinsson, L. 1990. Cerebral autoregulation. Cerebrovasc.Brain Metab Rev., 2, (2) 161-192
Peebles, K.C., Richards, A.M., Celi, L., McGrattan, K., Murrell, C.J., & Ainslie, P.N. 2008. Human cerebral arteriovenous vasoactive exchange during alterations in arterial blood gases. J.Appl.Physiol, 105, (4) 1060-1068
Peng, Y., Yuan, G., Overholt, J.L., Kumar, G.K., & Prabhakar, N.R. 2003. Systemic and cellular responses to intermittent hypoxia: evidence for oxidative stress and mitochondrial dysfunction. Adv.Exp.Med.Biol., 536, 559-564
Peng, Y.J. & Prabhakar, N.R. 2004. Effect of two paradigms of chronic intermittent hypoxia on carotid body sensory activity. J.Appl.Physiol, 96, (3) 1236-1242
Peng, Y.J., Yuan, G., Ramakrishnan, D., Sharma, S.D., Bosch-Marce, M., Kumar, G.K., Semenza, G.L., & Prabhakar, N.R. 2006. Heterozygous HIF-1alpha deficiency impairs carotid body-mediated systemic responses and reactive oxygen species generation in mice exposed to intermittent hypoxia. J.Physiol, 577, (Pt 2) 705-716
Pennathur, S., Bergt, C., Shao, B., Byun, J., Kassim, S.Y., Singh, P., Green, P.S., McDonald, T.O., Brunzell, J., Chait, A., Oram, J.F., O'Brien, K., Geary, R.L., & Heinecke, J.W. 2004. Human atherosclerotic intima and blood of patients with established coronary artery disease contain high density lipoprotein damaged by reactive nitrogen species. J.Biol.Chem., 279, (41) 42977-42983
Perrey, S. 2008. Non-invasive NIR spectroscopy of human brain function during exercise
2. Methods, 45, (4) 289-299
Philippi, N.R., Bird, C.E., Marcus, N.J., Olson, E.B., Chesler, N.C., & Morgan, B.J. 2010. Time course of intermittent hypoxia-induced impairments in resistance artery structure and function. Respir.Physiol Neurobiol., 170, (2) 157-163
Phillips, S.A., Olson, E.B., Lombard, J.H., & Morgan, B.J. 2006. Chronic intermittent hypoxia alters NE reactivity and mechanics of skeletal muscle resistance arteries. J.Appl.Physiol, 100, (4) 1117-1123
Phillips, S.A., Olson, E.B., Morgan, B.J., & Lombard, J.H. 2004. Chronic intermittent hypoxia impairs endothelium-dependent dilation in rat cerebral and skeletal muscle resistance arteries. Am.J.Physiol Heart Circ.Physiol, 286, (1) 388-393
Pialoux, V., Brugniaux, J.V., Fellmann, N., Richalet, J.P., Robach, P., Schmitt, L., Coudert, J., & Mounier, R. 2009a. Oxidative stress and HIF-1 alpha modulate hypoxic ventilatory responses after hypoxic training on athletes. Respir.Physiol Neurobiol., 167, (2) 217-220
Pialoux, V., Brugniaux, J.V., Rock, E., Mazur, A., Schmitt, L., Richalet, J.P., Robach, P., Clottes, E., Coudert, J., Fellmann, N., & Mounier, R. 2009b. Antioxidant status of elite athletes remains impaired 2 weeks after a simulated altitude training camp. Eur.J.Nutr.
Pialoux, V., Hanly, P.J., Foster, G.E., Brugniaux, J.V., Beaudin, A.E., Hartmann, S.E., Pun, M., Duggan, C.T., & Poulin, M.J. 2009c. Effects of exposure to intermittent hypoxia on oxidative stress and acute hypoxic ventilatory response in humans. Am.J.Respir.Crit Care Med., 180, (10) 1002-1009
Pialoux, V., Mounier, R., Brown, A.D., Steinback, C.D., Rawling, J.M., & Poulin, M.J. 2009d. Relationship between oxidative stress and HIF-1 alpha mRNA during sustained hypoxia in humans. Free Radic.Biol.Med., 46, (2) 321-326
Powers, S.K., Lawler, J., Dempsey, J.A., Dodd, S., & Landry, G. 1989. Effects of incomplete pulmonary gas exchange on VO2 max. J.Appl.Physiol, 66, (6) 2491-2495
Prabhakar, N.R., Peng, Y.J., Overholt, J.L., & Kumar, G.K. 2004. Detection of oxygen sensing during intermittent hypoxia. Methods Enzymol., 381, 107-120
Quaresima, V., Lepanto, R., & Ferrari, M. 2003. The use of near infrared spectroscopy in sports medicine. J.Sports Med.Phys.Fitness, 43, (1) 1-13
Querido, J.S., Rupert, J.L., McKenzie, D.C., & Sheel, A.W. 2009. Effects of intermittent hypoxia on the cerebrovascular responses to submaximal exercise in humans. Eur.J.Appl.Physiol, 105, (3) 403-409
Raat, N.J., Noguchi, A.C., Liu, V.B., Raghavachari, N., Liu, D., Xu, X., Shiva, S., Munson, P.J., & Gladwin, M.T. 2009a. Dietary nitrate and nitrite modulate blood and organ nitrite and the cellular ischemic stress response. Free Radic.Biol.Med., 47, (5) 510-517
Radegran, G. & Saltin, B. 1999. Nitric oxide in the regulation of vasomotor tone in human skeletal muscle. Am.J.Physiol, 276, (6 Pt 2) 1951-1960
Rasmussen, P., Dawson, E.A., Nybo, L., van Lieshout, J.J., Secher, N.H., & Gjedde, A. 2007. Capillary-oxygenation-level-dependent near-infrared spectrometry in frontal lobe of humans. J.Cereb.Blood Flow Metab, 27, (5) 1082-1093
Rasmussen, P., Nielsen, J., Overgaard, M., Krogh-Madsen, R., Gjedde, A., Secher, N.H., & Petersen, N.C. 2010. Reduced muscle activation during exercise related to brain oxygenation and metabolism in humans. J.Physiol, 588, (Pt 11) 1985-1995
Rasmussen, P., Plomgaard, P., Krogh-Madsen, R., Kim, Y.S., Van Lieshout, J.J., Secher, N.H., & Quistorff, B. 2006a. MCA Vmean and the arterial lactate-to-pyruvate ratio correlate during rhythmic handgrip. J.Appl.Physiol, 101, (5) 1406-1411
Rasmussen, P., Stie, H., Nielsen, B., & Nybo, L. 2006b. Enhanced cerebral CO2 reactivity during strenuous exercise in man. Eur.J.Appl.Physiol, 96, (3) 299-304
Rasmussen, P., Dawson, E. A., Nybo, L., Van Lieshout, J. J., Secher, N. H., & Gjedde, A. 2007, "Capillary-oxygenation-level-dependent near-infrared spectrometry in frontal lobe of humans", J.Cereb.Blood Flow Metab, vol. 27, no. 5, pp. 1082-1093.
Rassaf, T., Heiss, C., Hendgen-Cotta, U., Balzer, J., Matern, S., Kleinbongard, P., Lee, A., Lauer, T., & Kelm, M. 2006. Plasma nitrite reserve and endothelial function in the human forearm circulation. Free Radic.Biol.Med., 41, (2) 295-301
Rassaf, T., Kleinbongard, P., Preik, M., Dejam, A., Gharini, P., Lauer, T., Erckenbrecht, J., Duschin, A., Schulz, R., Heusch, G., Feelisch, M., & Kelm, M. 2002a. Plasma nitrosothiols contribute to the systemic vasodilator effects of intravenously applied NO: experimental and clinical Study on the fate of NO in human blood. Circ.Res., 91, (6) 470-477
Rassaf, T., Lauer, T., Heiss, C., Balzer, J., Mangold, S., Leyendecker, T., Rottler, J., Drexhage, C., Meyer, C., & Kelm, M. 2007. Nitric oxide synthase-derived plasma nitrite predicts exercise capacity. Br.J.Sports Med., 41, (10) 669-673
Rassaf, T., Preik, M., Kleinbongard, P., Lauer, T., Heiss, C., Strauer, B.E., Feelisch, M., & Kelm, M. 2002b. Evidence for in vivo transport of bioactive nitric oxide in human plasma. J.Clin.Invest, 109, (9) 1241-1248
Rauca, C., Zerbe, R., Jantze, H., & Krug, M. 2000. The importance of free hydroxyl radicals to hypoxia preconditioning. Brain Res., 868, (1) 147-149
Rehman, J., Mills, P.J., Carter, S.M., Chou, J., Thomas, J., & Maisel, A.S. 1997. Dynamic exercise leads to an increase in circulating ICAM-1: further evidence for adrenergic modulation of cell adhesion. Brain Behav.Immun., 11, (4) 343-351
Richardson, R.S., Duteil, S., Wary, C., Wray, D.W., Hoff, J., & Carlier, P.G. 2006. Human skeletal muscle intracellular oxygenation: the impact of ambient oxygen availability. J.Physiol, 571, (Pt 2) 415-424
Richardson, R.S., Grassi, B., Gavin, T.P., Haseler, L.J., Tagore, K., Roca, J., & Wagner, P.D. 1999a. Evidence of O2 supply-dependent VO2 max in the exercise-trained human quadriceps. J.Appl.Physiol, 86, (3) 1048-1053
Richardson, R.S., Leigh, J.S., Wagner, P.D., & Noyszewski, E.A. 1999b. Cellular PO2 as a determinant of maximal mitochondrial O(2) consumption in trained human skeletal muscle. J.Appl.Physiol, 87, (1) 325-331
Richardson, R.S., Newcomer, S.C., & Noyszewski, E.A. 2001. Skeletal muscle intracellular PO(2) assessed by myoglobin desaturation: response to graded exercise. J.Appl.Physiol, 91, (6) 2679-2685
Richardson, R.S., Noyszewski, E.A., Kendrick, K.F., Leigh, J.S., & Wagner, P.D. 1995a. Myoglobin O2 desaturation during exercise. Evidence of limited O2 transport. J.Clin.Invest, 96, (4) 1916-1926
Rifkind, J.M., Nagababu, E., Barbiro-Michaely, E., Ramasamy, S., Pluta, R.M., & Mayevsky, A. 2007. Nitrite infusion increases cerebral blood flow and decreases mean arterial blood pressure in rats: a role for red cell NO. Nitric.Oxide., 16, (4) 448-456
Roach, R.C., Koskolou, M.D., Calbet, J.A., & Saltin, B. 1999. Arterial O2 content and tension in regulation of cardiac output and leg blood flow during exercise in humans. Am.J.Physiol, 276, (2 Pt 2) 438-445
Rodriguez, F.A., Casas, H., Casas, M., Pages, T., Rama, R., Ricart, A., Ventura, J.L., Ibanez, J., & Viscor, G. 1999. Intermittent hypobaric hypoxia stimulates erythropoiesis and improves aerobic capacity. Med.Sci.Sports Exerc., 31, (2) 264-268
Rodriguez, F.A., Truijens, M.J., Townsend, N.E., Stray-Gundersen, J., Gore, C.J., & Levine, B.D. 2007. Performance of runners and swimmers after four weeks of intermittent hypobaric hypoxic exposure plus sea level training. J.Appl.Physiol, 103, (5) 1523-1535
Rogers, S.C., Khalatbari, A., Datta, B.N., Ellery, S., Paul, V., Frenneaux, M.P., & James, P.E. 2007. NO metabolite flux across the human coronary circulation. Cardiovasc.Res., 75, (2) 434-441
Romer, L.M., Haverkamp, H.C., Amann, M., Lovering, A.T., Pegelow, D.F., & Dempsey, J.A. 2007. Effect of acute severe hypoxia on peripheral fatigue and endurance capacity in healthy humans. Am.J.Physiol Regul.p Physiol, 292, (1) 598-606
Rostrup, E., Law, I., Pott, F., Ide, K., & Knudsen, G.M. 2002. Cerebral hemodynamics measured with simultaneous PET and near-infrared spectroscopy in humans. Brain Res., 954, (2) 183-193
Rubanyi, G.M., Romero, J.C., & Vanhoutte, P.M. 1986. Flow-induced release of endothelium-derived relaxing factor. Am.J.Physiol, 250, (6 Pt 2) 1145-1149
Rupp, T. & Perrey, S. 2008. Prefrontal cortex oxygenation and neuromuscular responses to exhaustive exercise. Eur.J.Appl.Physiol, 102, (2) 153-163
Rupp, T. & Perrey, S. 2009. Effect of severe hypoxia on prefrontal cortex and muscle oxygenation responses at rest and during exhaustive exercise. Adv.Exp.Med.Biol., 645, 329-334
Rush, J.W., Denniss, S.G., & Graham, D.A. 2005. Vascular nitric oxide and oxidative stress: determinants of endothelial adaptations to cardiovascular disease and to physical activity. Can.J.Appl.Physiol, 30, (4) 442-474
Ryan, S. & McNicholas, W.T. 2008. Intermittent hypoxia and activation of inflammatory molecular pathways in OSAS. Arch.Physiol Biochem., 114, (4) 261-266
Ryou, M.G., Sun, J., Oguayo, K.N., Manukhina, E.B., Downey, H.F., & Mallet, R.T. 2008. Hypoxic conditioning suppresses nitric oxide production upon myocardial reperfusion. Exp.Biol.Med.(Maywood.), 233, (6) 766-774
Sahnoun, Z., Jamoussi, K., & Zeghal, K.M. 1998. [Free radicals and antioxidants: physiology, human pathology and therapeutic aspects (part II)]. Therapie, 53, (4) 315-339 ]
Sandiford, S.D., Green, H.J., Duhamel, T.A., Schertzer, J.D., Perco, J.D., & Ouyang, J. 2005. Muscle Na-K-pump and fatigue responses to progressive exercise in normoxia and hypoxia. Am.J.Physiol Regul.p Physiol, 289, (2) 441-449
Saunders, P.U., Telford, R.D., Pyne, D.B., Cunningham, R.B., Gore, C.J., Hahn, A.G., & Hawley, J.A. 2004. Improved running economy in elite runners after 20 days of simulated moderate-altitude exposure. J.Appl.Physiol, 96, (3) 931-937
Schmidek, H.H., Auer, L.M., & Kapp, J.P. 1985. The cerebral venous system. Neurosurgery, 17, (4) 663-678
Schmidt-Lucke, C., Reinhold, D., Ansorge, S., Klein, H.U., & Schmidt-Lucke, J.A. 2003. Changes of plasma concentrations of soluble vascular cell adhesion molecule-1 and vascular endothelial growth factor after increased perfusion of lower extremities in humans. Endothelium, 10, (3) 159-165
Schulz, R. & Wambolt, R. 1995. Inhibition of nitric oxide synthesis protects the isolated working rabbit heart from ischaemia-reperfusion injury. Cardiovasc.Res., 30, (3) 432-439
Secher, N.H., Seifert, T., & Van Lieshout, J.J. 2008. Cerebral blood flow and metabolism during exercise: implications for fatigue. J.Appl.Physiol, 104, (1) 306-314
Seifert, T., Rasmussen, P., Secher, N.H., & Nielsen, H.B. 2009. Cerebral oxygenation decreases during exercise in humans with beta-adrenergic blockade. Acta Physiol (Oxf), 196, (3) 295-302
Semenza, G.L., Agani, F., Booth, G., Forsythe, J., Iyer, N., Jiang, B.H., Leung, S., Roe, R., Wiener, C., & Yu, A. 1997. Structural and functional analysis of hypoxia-inducible factor 1. Kidney Int., 51, (2) 553-555
Serebrovskaya, T.V. 2002. Intermittent hypoxia research in the former soviet union and the commonwealth of independent States: history and review of the concept and selected applications. High Alt.Med.Biol., 3, (2) 205-221
Sheriff, D.D. 2003. Muscle pump function during locomotion: mechanical coupling of stride frequency and muscle blood flow. Am.J.Physiol Heart Circ.Physiol, 284, (6) 2185-2191
Shibasaki, M., Low, D.A., Davis, S.L., & Crandall, C.G. 2008. Nitric oxide inhibits cutaneous vasoconstriction to exogenous norepinephrine. J.Appl.Physiol, 105, (5) 1504-1508
Shishehbor, M.H., Aviles, R.J., Brennan, M.L., Fu, X., Goormastic, M., Pearce, G.L., Gokce, N., Keaney, J.F., Jr., Penn, M.S., Sprecher, D.L., Vita, J.A., & Hazen, S.L. 2003. Association of nitrotyrosine levels with cardiovascular disease and modulation by statin therapy. JAMA, 289, (13) 1675-1680
Shiu, Y.T., Udden, M.M., & McIntire, L.V. 2000. Perfusion with sickle erythrocytes up-regulates ICAM-1 and VCAM-1 gene expression in cultured human endothelial cells
11. Blood, 95, (10) 3232-3241
Shiva, S., Sack, M.N., Greer, J.J., Duranski, M., Ringwood, L.A., Burwell, L., Wang, X., MacArthur, P.H., Shoja, A., Raghavachari, N., Calvert, J.W., Brookes, P.S., Lefer, D.J., & Gladwin, M.T. 2007. Nitrite augments tolerance to ischemia/reperfusion injury via the modulation of mitochondrial electron transfer. J.Exp.Med., 204, (9) 2089-2102
Shreeniwas, R., Koga, S., Karakurum, M., Pinsky, D., Kaiser, E., Brett, J., Wolitzky, B.A., Norton, C., Plocinski, J., Benjamin, W., & . 1992. Hypoxia-mediated induction of endothelial cell interleukin-1 alpha. An autocrine mechanism promoting expression of leukocyte adhesion molecules on the vessel surface. J.Clin.Invest, 90, (6) 2333-2339
Silvestro, A., Scopacasa, F., Oliva, G., de, C.T., Iuliano, L., & Brevetti, G. 2002. Vitamin C prevents endothelial dysfunction induced by acute exercise in patients with intermittent claudication. Atherosclerosis, 165, (2) 277-283
Simpson, R.J., Florida-James, G.D., Whyte, G.P., & Guy, K. 2006. The effects of intensive, moderate and downhill treadmill running on human blood lymphocytes expressing the adhesion/activation molecules CD54 (ICAM-1), CD18 (beta2 integrin) and CD53. Eur.J.Appl.Physiol, 97, (1) 109-121
Singhania, N., Puri, D., Madhu, S.V., & Sharma, S.B. 2008. Assessment of oxidative stress and endothelial dysfunction in Asian Indians with type 2 diabetes mellitus with and without macroangiopathy. QJM., 101, (6) 449-455
Smith, C.W. 1993. Endothelial adhesion molecules and their role in inflammation
19. Can.J.Physiol Pharmacol., 71, (1) 76-87
Sogawa, K., Numayama-Tsuruta, K., Ema, M., Abe, M., Abe, H., & Fujii-Kuriyama, Y. 1998. Inhibition of hypoxia-inducible factor 1 activity by nitric oxide donors in hypoxia. Proc.Natl.Acad.Sci.U.S.A, 95, (13) 7368-7373
Sohn, H.Y., Krotz, F., Zahler, S., Gloe, T., Keller, M., Theisen, K., Schiele, T.M., Klauss, V., & Pohl, U. 2003. Crucial role of local peroxynitrite formation in neutrophil-induced endothelial cell activation. Cardiovasc.Res., 57, (3) 804-815
Stamler, J.S., Jaraki, O., Osborne, J., Simon, D.I., Keaney, J., Vita, J., Singel, D., Valeri, C.R., & Loscalzo, J. 1992. Nitric oxide circulates in mammalian plasma primarily as an S-nitroso adduct of serum albumin. Proc.Natl.Acad.Sci.U.S.A, 89, (16) 7674-7677
Stamler, J.S., Jia, L., Eu, J.P., McMahon, T.J., Demchenko, I.T., Bonaventura, J., Gernert, K., & Piantadosi, C.A. 1997. Blood flow regulation by S-nitrosohemoglobin in the physiological oxygen gradient. Science, 276, (5321) 2034-2037
Steiner, D.R., Gonzalez, N.C., & Wood, J.G. 2002. Interaction between reactive oxygen species and nitric oxide in the microvascular response to systemic hypoxia. J.Appl.Physiol, 93, (4) 1411-1418
Stokes, K.Y., Dugas, T.R., Tang, Y., Garg, H., Guidry, E., & Bryan, N.S. 2009. Dietary nitrite prevents hypercholesterolemic microvascular inflammation and reverses endothelial dysfunction. Am.J.Physiol Heart Circ.Physiol, 296, (5) 1281-1288
Stringer, W., Wasserman, K., Casaburi, R., Porszasz, J., Maehara, K., & French, W. 1994. Lactic acidosis as a facilitator of oxyhemoglobin dissociation during exercise. J.Appl.Physiol, 76, (4) 1462-1467
Subudhi, A.W., Dimmen, A.C., & Roach, R.C. 2007. Effects of acute hypoxia on cerebral and muscle oxygenation during incremental exercise. J.Appl.Physiol, 103, (1) 177-183
Subudhi, A.W., Lorenz, M.C., Fulco, C.S., & Roach, R.C. 2008. Cerebrovascular responses to incremental exercise during hypobaric hypoxia: effect of oxygenation on maximal performance. Am.J.Physiol Heart Circ.Physiol, 294, (1) 164-171
Subudhi, A. W., Miramon, B. R., Granger, M. E., & Roach, R. C. 2009, "Frontal and motor cortex oxygenation during maximal exercise in normoxia and hypoxia", J.Appl.Physiol, vol. 106, no. 4, pp. 1153-1158
Suzuki, Y.J., Jain, V., Park, A.M., & Day, R.M. 2006. Oxidative stress and oxidant signaling in obstructive sleep apnea and associated cardiovascular diseases. Free Radic.Biol.Med., 40, (10) 1683-1692
Svatikova, A., Wolk, R., Wang, H.H., Otto, M.E., Bybee, K.A., Singh, R.J., & Somers, V.K. 2004. Circulating free nitrotyrosine in obstructive sleep apnea. Am.J.Physiol Regul.p Physiol, 287, (2) 284-287
Tadibi, V., Dehnert, C., Menold, E., & Bartsch, P. 2007. Unchanged anaerobic and aerobic performance after short-term intermittent hypoxia. Med.Sci.Sports Exerc., 39, (5) 858-864
The, G.K., Bleijenberg, G., Buitelaar, J.K., & van der Meer, J.W. 2010. The effect of ondansetron, a 5-HT3 receptor antagonist, in chronic fatigue syndrome: a randomized controlled trial. J.Clin.Psychiatry, 71, (5) 528-533
The, G.K., Bleijenberg, G., & van der Meer, J.W. 2007. The effect of acclydine in chronic fatigue syndrome: a randomized controlled trial. PLoS.Clin.Trials, 2, (5) e19
Townsend, N.E., Gore, C.J., Hahn, A.G., Aughey, R.J., Clark, S.A., Kinsman, T.A., McKenna, M.J., Hawley, J.A., & Chow, C.M. 2005. Hypoxic ventilatory response is correlated with increased submaximal exercise ventilation after live high, train low. Eur.J.Appl.Physiol, 94, (1-2) 207-215
Townsend, N.E., Gore, C.J., Hahn, A.G., McKenna, M.J., Aughey, R.J., Clark, S.A., Kinsman, T., Hawley, J.A., & Chow, C.M. 2002. Living high-training low increases hypoxic ventilatory response of well-trained endurance athletes. J.Appl.Physiol, 93, (4) 1498-1505
Tran, T.K., Sailasuta, N., Kreutzer, U., Hurd, R., Chung, Y., Mole, P., Kuno, S., & Jue, T. 1999. Comparative analysis of NMR and NIRS measurements of intracellular PO2 in human skeletal muscle. Am.J.Physiol, 276, (6 Pt 2) 1682-1690
Truijens, M.J., Rodriguez, F.A., Townsend, N.E., Stray-Gundersen, J., Gore, C.J., & Levine, B.D. 2008. The effect of intermittent hypobaric hypoxic exposure and sea level training on submaximal economy in well-trained swimmers and runners. J.Appl.Physiol, 104, (2) 328-337
Trujillo, M., Alvarez, M.N., Peluffo, G., Freeman, B.A., & Radi, R. 1998. Xanthine oxidase-mediated decomposition of S-nitrosothiols. J.Biol.Chem., 273, (14) 7828-7834
Tschakovsky, M.E. & Joyner, M.J. 2008. Nitric oxide and muscle blood flow in exercise. Appl.Physiol Nutr.Metab, 33, (1) 151-161
Tsikas, D., Schwedhelm, E., & Frolich, J.C. 2002. Methodological considerations on the detection of 3-nitrotyrosine in the cardiovascular system. Circ.Res., 90, (6) 70
Tsuchiya, M., Asada, A., Kasahara, E., Sato, E.F., Shindo, M., & Inoue, M. 2002. Smoking a single cigarette rapidly reduces combined concentrations of nitrate and nitrite and concentrations of antioxidants in plasma. Circulation, 105, (10) 1155-1157
Tyldum, G.A., Schjerve, I.E., Tjonna, A.E., Kirkeby-Garstad, I., Stolen, T.O., Richardson, R.S., & Wisloff, U. 2009. Endothelial dysfunction induced by post-prandial lipemia: complete protection afforded by high-intensity aerobic interval exercise. J.Am.Coll.Cardiol., 53, (2) 200-206
Ursavas, A., Karadag, M., Rodoplu, E., Yilmaztepe, A., Oral, H.B., & Gozu, R.O. 2007. Circulating ICAM-1 and VCAM-1 levels in patients with obstructive sleep apnea syndrome. Respiration, 74, (5) 525-532
van Beekvelt, M.C., Colier, W.N., Wevers, R.A., & van Engelen, B.G. 2001. Performance of near-infrared spectroscopy in measuring local O(2) consumption and blood flow in skeletal muscle. J.Appl.Physiol, 90, (2) 511-519
van der Zee, P., Cope, M., Arridge, S.R., Essenpreis, M., Potter, L.A., Edwards, A.D., Wyatt, J.S., McCormick, D.C., Roth, S.C., Reynolds, E.O., & . 1992. Experimentally measured optical pathlengths for the adult head, calf and forearm and the head of the newborn infant as a function of inter optode spacing. Adv.Exp.Med.Biol., 316, 143-153
van Faassen, E.E., Bahrami, S., Feelisch, M., Hogg, N., Kelm, M., Kim-Shapiro, D.B., Kozlov, A.V., Li, H., Lundberg, J.O., Mason, R., Nohl, H., Rassaf, T., Samouilov, A., Slama-Schwok, A., Shiva, S., Vanin, A.F., Weitzberg, E., Zweier, J., & Gladwin, M.T. 2009. Nitrite as regulator of hypoxic signaling in mammalian physiology. Med.Res.Rev., 29, (5) 683-741
Vanin, A.F., Malenkova, I.V., & Serezhenkov, V.A. 1997. Iron catalyzes both decomposition and synthesis of S-nitrosothiols: optical and electron paramagnetic resonance studies. Nitric.Oxide., 1, (3) 191-203
Vardi, M. & Nini, A. 2008. Near-infrared spectroscopy for evaluation of peripheral vascular disease. A systematic review of literature. Eur.J.Vasc.Endovasc.Surg., 35, (1) 68-74
Vassalle, C., Lubrano, V., Domenici, C., & L'Abbate, A. 2003. Influence of chronic aerobic exercise on microcirculatory flow and nitric oxide in humans. Int.J.Sports Med., 24, (1) 30-35
Vitturi, D.A., Teng, X., Toledo, J.C., Matalon, S., Lancaster, J.R., Jr., & Patel, R.P. 2009. Regulation of nitrite transport in red blood cells by hemoglobin oxygen fractional saturation. Am.J.Physiol Heart Circ.Physiol, 296, (5) 1398-1407
Vogiatzis, I., Zakynthinos, S., Boushel, R., Athanasopoulos, D., Guenette, J.A., Wagner, H., Roussos, C., & Wagner, P.D. 2008. The contribution of intrapulmonary shunts to the alveolar-to-arterial oxygen difference during exercise is very small. J.Physiol, 586, (9) 2381-2391
Volianitis, S., Fabricius-Bjerre, A., Overgaard, A., Stromstad, M., Bjarrum, M., Carlson, C., Petersen, N.T., Rasmussen, P., Secher, N.H., & Nielsen, H.B. 2008. The cerebral metabolic ratio is not affected by oxygen availability during maximal exercise in humans. J.Physiol, 586, (1) 107-112
Wagner, P.D., Wagner, H.E., Groves, B.M., Cymerman, A., & Houston, C.S. 2007. Hemoglobin P(50) during a simulated ascent of Mt. Everest, Operation Everest II. High Alt.Med.Biol., 8, (1) 32-42 available from: PM:17394415
Wang, J., Brown, M.A., Tam, S.H., Chan, M.C., & Whitworth, J.A. 1997. Effects of diet on measurement of nitric oxide metabolites. Clin.Exp.Pharmacol.Physiol, 24, (6) 418-420
Wang, J.S. 2005. Effects of exercise training and detraining on cutaneous microvascular function in man: the regulatory role of endothelium-dependent dilation in skin vasculature. Eur.J.Appl.Physiol, 93, (4) 429-434
Wang, J.S., Chen, L.Y., Fu, L.L., Chen, M.L., & Wong, M.K. 2007a. Effects of moderate and severe intermittent hypoxia on vascular endothelial function and haemodynamic control in sedentary men. Eur.J.Appl.Physiol, 100, (2) 127-135
Wang, J.S., Lin, H.Y., Cheng, M.L., & Wong, M.K. 2007b. Chronic intermittent hypoxia modulates eosinophil- and neutrophil-platelet aggregation and inflammatory cytokine secretion caused by strenuous exercise in men. J.Appl.Physiol, 103, (1) 305-314
Wasserman, K. 1994. Coupling of external to cellular respiration during exercise: the wisdom of the body revisited. Am.J.Physiol, 266, (4 Pt 1) 519-539
Webb, A., Bond, R., McLean, P., Uppal, R., Benjamin, N., & Ahluwalia, A. 2004. Reduction of nitrite to nitric oxide during ischemia protects against myocardial ischemia-reperfusion damage. Proc.Natl.Acad.Sci.U.S.A, 101, (37) 13683-13688
Webb, A.J., Milsom, A.B., Rathod, K.S., Chu, W.L., Qureshi, S., Lovell, M.J., Lecomte, F.M., Perrett, D., Raimondo, C., Khoshbin, E., Ahmed, Z., Uppal, R., Benjamin, N., Hobbs, A.J., & Ahluwalia, A. 2008. Mechanisms underlying erythrocyte and endothelial nitrite reduction to nitric oxide in hypoxia: role for xanthine oxidoreductase and endothelial nitric oxide synthase. Circ.Res., 103, (9) 957-964
White, R.P., Deane, C., Hindley, C., Bloomfield, P.M., Cunningham, V.J., Vallance, P., Brooks, D.J., & Markus, H.S. 2000. The effect of the nitric oxide donor glyceryl trinitrate on global and regional cerebral blood flow in man. J.Neurol.Sci., 178, (1) 23-28
White, R.P., Deane, C., Vallance, P., & Markus, H.S. 1998. Nitric oxide synthase inhibition in humans reduces cerebral blood flow but not the hyperemic response to hypercapnia. Stroke, 29, (2) 467-472
Wilkerson, D.P., Campbell, I.T., & Jones, A.M. 2004. Influence of nitric oxide synthase inhibition on pulmonary O2 uptake kinetics during supra-maximal exercise in humans. J.Physiol, 561, (Pt 2) 623-635
Willam, C., Schindler, R., Frei, U., & Eckardt, K.U. 1999. Increases in oxygen tension stimulate expression of ICAM-1 and VCAM-1 on human endothelial cells. Am.J.Physiol, 276, (6 Pt 2) 2044-2052
Williams, D.L. 1997. Nitrosating agents: is peroxynitrite a likely candidate? Nitric.Oxide., 1, (6) 522-527
Wisloff, U., Ellingsen, O., & Kemi, O.J. 2009. High-intensity interval training to maximize cardiac benefits of exercise training? Exerc.Sport Sci.Rev., 37, (3) 139-146
Witkowska, A.M. 2005. Soluble ICAM-1: a marker of vascular inflammation and lifestyle. Cytokine, 31, (2) 127-134
Wolin, M.S., Ahmad, M., & Gupte, S.A. 2005. Oxidant and redox signaling in vascular oxygen sensing mechanisms: basic concepts, current controversies, and potential importance of cytosolic NADPH. Am.J.Physiol Lung Cell Mol.Physiol, 289, (2) 159-173
Wood, J.G., Johnson, J.S., Mattioli, L.F., & Gonzalez, N.C. 1999a. Systemic hypoxia promotes leukocyte-endothelial adherence via reactive oxidant generation. J.Appl.Physiol, 87, (5) 1734-1740
Wood, J.G., Mattioli, L.F., & Gonzalez, N.C. 1999b. Hypoxia causes leukocyte adherence to mesenteric venules in nonacclimatized, but not in acclimatized, rats. J.Appl.Physiol, 87, (3) 873-881
Woorons, X., Mollard, P., Lamberto, C., Letournel, M., & Richalet, J.P. 2005. Effect of acute hypoxia on maximal exercise in trained and sedentary women. Med.Sci.Sports Exerc., 37, (1) 147-154
Woorons, X., Mollard, P., Pichon, A., Lamberto, C., Duvallet, A., & Richalet, J.P. 2007. Moderate exercise in hypoxia induces a greater arterial desaturation in trained than untrained men. Scand.J.Med.Sci.Sports, 17, (4) 431-436
Wray, D.W., Nishiyama, S.K., Donato, A.J., Sander, M., Wagner, P.D., & Richardson, R.S. 2007. Endothelin-1-mediated vasoconstriction at rest and during dynamic exercise in healthy humans. Am.J.Physiol Heart Circ.Physiol, 293, (4) 2550-2556
Yang, Z., Wang, J.M., Chen, L., Luo, C.F., Tang, A.L., & Tao, J. 2007. Acute exercise-induced nitric oxide production contributes to upregulation of circulating endothelial progenitor cells in healthy subjects. J.Hum.Hypertens., 21, (6) 452-460
Yang, B.K., Vivas, E.X., Reiter, C.D., & Gladwin, M.T. 2003. Methodologies for the sensitive and specific measurement of S-nitrosothiols, iron-nitrosyls, and nitrite in biological samples. Free Radic.Res., 37, (1) 1-10
Yang, C.C., Lin, L.C., Wu, M.S., Chien, C.T., & Lai, M.K. 2009. Repetitive hypoxic preconditioning attenuates renal ischemia/reperfusion induced oxidative injury via upregulating HIF-1 alpha-dependent bcl-2 signaling. Transplantation, 88, (11) 1251-1260
Yeh, C.H., Hsu, S.P., Yang, C.C., Chien, C.T., & Wang, N.P. 2010. Hypoxic preconditioning reinforces HIF-alpha-dependent HSP70 signaling to reduce ischemic renal failure-induced renal tubular apoptosis and autophagy. Life Sci., 86, (3-4) 115-123
Yu, A.Y., Frid, M.G., Shimoda, L.A., Wiener, C.M., Stenmark, K., & Semenza, G.L. 1998. Temporal, spatial, and oxygen-regulated expression of hypoxia-inducible factor-1 in the lung. Am.J.Physiol, 275, (4 Pt 1) 818-826
Yuan, G., Adhikary, G., McCormick, A.A., Holcroft, J.J., Kumar, G.K., & Prabhakar, N.R. 2004. Role of oxidative stress in intermittent hypoxia-induced immediate early gene activation in rat PC12 cells. J.Physiol, 557, (Pt 3) 773-783
Zhang, S. & Robbins, P.A. 2000. Methodological and physiological variability within the ventilatory response to hypoxia in humans. J.Appl.Physiol, 88, (5) 1924-1932
Zong, P., Setty, S., Sun, W., Martinez, R., Tune, J.D., Ehrenburg, I.V., Tkatchouk, E.N., Mallet, R.T., & Downey, H.F. 2004. Intermittent hypoxic training protects canine myocardium from infarction. Exp.Biol.Med.(Maywood.), 229, (8) 806-812
-----------------------
General Introduction
Literature review Sections 2.1 – 2.8
Muscle and brain O2 delivery and NIRS
Literature review Sections 2.9 – 2.17
Redox regulation of vascular function
Literature review Sections 2.18 – 2.22
Intermittent hypoxia and vascular function
Summary of key research findings and future directions
Study 1
The impact of acute hypoxia on cerebral and muscle oxygenation and systemic molecular biomarkers of vascular function during exercise
Study 2
The effect of intermittent hypoxia on cerebral and muscle oxygenation and molecular biomarkers of vascular function during exercise in hypoxia
(3)
"OD»
T» · L · DPF
"C =
Exercise + Hypoxia
Muscle
Oxygenation
Cerebral
Oxygenation
[pic]O2peak
Systemic O2 delivery
SaO2
Regional O2 delivery
Vascular∆ODλ
єλ · L · DPF
∆C =
Exercise + Hypoxia
Muscle
Oxygenation
Cerebral
Oxygenation
[pic]O2peak
Systemic O2 delivery
SaO2
Regional O2 delivery
Vascular
Function
(↑Central Fatigue)
(↑Peripheral Fatigue)
Intermittent Hypoxia
Exercise + Hypoxia
(↓Peripheral Fatigue)
Systemic O2 delivery
[pic]O2peak
Muscle
Oxygenation
Vascular
Function
Cerebral
Oxygenation
SaO2
Regional O2 delivery
(↓Central Fatigue)
Untreated
NO2• + RSNO
Treated
RSNO
Time (seconds)
Chemiluminescence signal (mV)
Endothelial function
Regional Oxygenation
Increase or decrease compared to normoxia
30
60
90
120
150
180
210
Rest
60
90
120
150
180
PPO
Workload (W)
Heart rate (b.p.m)
Normoxia
Hypoxia
†
†
†
†
†
†
†
60
70
80
90
100
110
Rest
60
90
120
150
180
PPO
Workload (W)
SaO2 (%)
Normoxia
Hypoxia
†
†
†
†
†
†
†
Muscle THb (∆µM)
Muscle O2 Hb (∆µM)
Muscle H Hb (∆µM)
ABSOLUTE WORKLOADS
C
B
A
Workload (W)
Workload (W)
Workload (W)
RELATIVE WORKLOADS
Muscle THb (∆µM)
Muscle H Hb (∆µM)
Muscle O2 Hb (∆µM)
Workload (%PPO)
Workload (%PPO)
Workload (%PPO)
F
E
D
Normoxia
Hypoxia
A
B
C
Cerebral THb (∆µM)
Cerebral HHb (∆µM)
Cerebral O2 Hb (∆µM)
Workload (W)
Workload (W)
Workload (W)
ABSOLUTE WORKLOADS
RELATIVE WORKLOADS
F
E
D
Workload (%PPO)
Workload (%PPO)
Workload (%PPO)
Cerebral THb (∆µM)
Cerebral HHb (∆µM)
Cerebral O2 Hb (∆µM)
Normoxia
Hypoxia
Before
After
0
40
80
120
160
200
Normoxia
Hypoxia
3-NT (nM)
B
0
500
1000
1500
2000
2500
3000
Normoxia
Hypoxia
A. (SQRT)
A
RSNO
Nitrite
Nitrate
NOx
0
10
20
30
40
50
60
Normoxia
Hypoxia
NOx (µM)
A
0
20
40
60
80
100
120
Normoxia
Hypoxia
RSNO (nM)
D
0
10
20
30
40
50
60
Normoxia
Hypoxia
Nitrate (µM)
B
0
150
300
450
600
750
900
Normoxia
Hypoxia
Nitrite (nM)
C
After
Before
After
Before
0
150
300
450
600
750
900
Normoxia
Hypoxia
sVCAM-1 (ng/ml)
A
0
150
300
450
600
750
900
Normoxia
Hypoxia
sICAM-1 (ng/ml)
B
After
Before
7.100
7.150
7.200
7.250
7.300
7.350
7.400
7.450
Normoxia
Hypoxia
pH
r2 = 0.39
P < 0.05
-50
0
50
100
150
200
250
1
2
3
4
5
[pic]O2peak (L/min)
∆sVCAM-1 (ng/ml)
r2 = -0.46
P < 0.05
-400
-300
-200
-100
0
100
0
200
400
600
800
1000
Baseline nitrite (nM)
∆ Nitrite (nM)
Regional Oxygenation
Vascular function
Increase or decrease compared to normoxia
Endothelial function
Regional Oxygenation
Increase or decrease compared to control group
Normoxia
Hypoxia
50
60
70
80
90
100
110
1
2
3
4
5
6
7
8
9
10
Days of IH
SaO2 (%)
Post IH/IN
Pre IH/IN
VO2 (L/min)
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
60W
PPO
60W
PPO
IH
IN
70
75
80
85
90
95
Rest
60
90
120
150
180
Max
Workload (W)
SaO2 (%)
Pre IH
Post IH
A
Rest
60
90
120
150
180
Max
Workload (W)
Pre-IN
Post-IN
B
B
0
50
100
150
200
250
Rest
60
90
120
150
180
Max
Workload (W)
Heart rate (b.p.m)
Pre IH
Post IH
A
Rest
60
90
120
150
180
Max
Workload (W)
Pre IN
Post IN
A
-15
-10
-5
0
5
10
15
20
25
Rest
60
90
120
150
180
Peak
O2 Hb (∆µM)
0
5
10
15
20
25
30
35
Rest
60
90
120
150
180
Peak
HHb (∆µM)
B
-10
0
10
20
30
40
50
60
Rest
60
90
120
150
180
Peak
THb (∆µM)
C
-15
-10
-5
0
5
10
15
20
25
Rest
60
90
120
150
180
Peak
O2 Hb (∆µM)
D
0
5
10
15
20
25
30
35
Rest
60
90
120
150
180
Peak
HHb (∆µM)
E
-10
0
10
20
30
40
50
60
Rest
60
90
120
150
180
Peak
THb (∆µM)
F
-25
-20
-15
-10
-5
0
5
Rest
60
90
120
150
180
Peak
O2Hb (∆µM)
-5
0
5
10
15
20
25
30
35
Rest
60
90
120
150
180
Peak
HHb (∆µM)
-10
-5
0
5
10
15
20
Rest
60
90
120
150
180
Peak
THb (∆µM)
-25
-20
-15
-10
-5
0
5
Rest
60
90
120
150
180
Peak
O2Hb (∆µM)
-5
0
5
10
15
20
25
30
35
Rest
60
90
120
150
180
Peak
HHb (∆µ M)
-10
-5
0
5
10
15
20
Rest
60
90
120
150
180
Peak
THb (∆µ M)
Rest
0
10
20
30
40
50
60
70
Before
After
Before
After
Intermittent Hypoxia
Control
NOx (µM)
Maximal exercise
0
10
20
30
40
50
60
70
Before
After
Before
After
Intermittent Hypoxia
Control
Nitrate (µM)
Rest
Maximal exercise
0
100
200
300
400
500
600
700
800
900
Before
After
Before
After
Intermittent Hypoxia
Control
Nitrite (nM)
Rest
Maximal exercise
0
20
40
60
80
100
120
140
Before
After
Before
After
IH
Control
RSNO (nM)
Rest
Maximal exercise
0
200
400
600
800
1000
Before
After
Before
After
Intermittent Hypoxia
Control
sVCAM-1 (ng/mL)
Rest
Maximal exercise
0
200
400
600
800
Before
After
Before
After
Intermittent Hypoxia
Control
sICAM-1 (pg/mL)
Rest
Maximal exercise
7.05
7.10
7.15
7.20
7.25
7.30
7.35
7.40
7.45
Before
After
Before
After
Intermittent Hypoxia
Control
Blood pH
Rest
Maximal exercise
................
................
In order to avoid copyright disputes, this page is only a partial summary.
To fulfill the demand for quickly locating and searching documents.
It is intelligent file search solution for home and business.