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
1.0
2.0
CHAPTER 1: Thesis overview
1.1
Background
2
1.2
Strategy
4
1.3
Aims and objectives
7
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
3.0
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
Chapter 3: General Methodology
3.1
Incremental cycling protocol
60
3.2
Respiratory measurements
60
3.3
Heart rate (HR)
61
3.4
Arterial oxygen saturation (SaO2)
61
3.5
Near Infrared Spectroscopy (NIRS)
61
3.6
Blood sampling
62
3.7
Ozone-based chemiluminescence:
63
Detection of plasma NO metabolites
4.0
3.8
sICAM-1, sVCAM-1 and 3-NT
65
3.9
Ascorbate free radical (A•)
66
3.10
Haemoglobin (Hb)
66
3.11
Haematocrit (Hcrt)
67
3.12
Blood gas
67
3.13
IH machine
68
Study 1: The impact of acute hypoxia on cerebral and muscle oxygenation
and systemic molecular biomarkers of vascular function during exercise
4.1
Introduction
70
4.2
Research aims and objectives
72
4.3
Working hypothesis
72
4.4
Methodology
73
4.5
Statistical analysis
76
4.6
Results
76
4.7
Discussion
87
4.8
Conclusions
100
5.0
Study 2:The effect of intermittent hypoxia on cerebral and muscle
oxygenation and molecular biomarkers of vascular function
during exercise in hypoxia
6.0
5.1
Introduction
103
5.2
Research aims and objectives
104
5.3
Working hypothesis
104
5.4
Methodology
105
5.5
Statistical analysis
108
5.6
Results
109
5.7
Discussion
120
5.8
Conclusions
130
General discussion and conclusions
6.1
Overview
132
6.2
Study 1: The impact of acute hypoxia on cerebral and muscle
133
oxygenation and systemic molecular biomarkers of vascular
function during exercise
6.3
Study 2: Intermittent hypoxia: implications for cerebral and
136
muscle oxygenation and molecular biomarkers of vascular function
6.4
References
Future research
138
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 V 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
V 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 bloodborne 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) V 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 V 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 sVCAM1) were measured before and immediately after maximal exercise. The key observations
from this study were: 1) a tendency for IH to reduce submaximal V O2 and increase
V 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 V O2peak. IH may be used to improve
exercise economy and V 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 V O2max at various
altitudes compared to sea-level. Aerobically trained subjects ( V O2max ≥ 63
ml.kgˉ1.minˉ1) are represented by the dashed regression line, untrained
individuals ( V 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
V O2max
Maximal oxygen consumption
MAP
Mean arterial blood pressure
MCAV
Middle cerebral artery velocity
Qm
Muscle blood flow with muscle
V 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)
V 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
V 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 ( V 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 V 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 V 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.
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
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
Summary of key research
findings and future directions
Figure 1.1 – A schematic overview of thesis
Literature review
Sections 2.18 – 2.22
Intermittent hypoxia
and vascular function
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 V 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 O 2 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 V 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 V O2 when CaO2 is
reduced during submaximal exercise (Bourdillon et al. 2009; Calbet 2000; GonzalezAlonso 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
V 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 V 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 V O2max in
hypoxia is related to the reduction in O2 supply (Richardson et al. 1999).
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 βadrenergic 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.
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 V 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 V 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 (GonzalezAlonso 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 V 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).
2.3 Aerobic exercise performance in acute hypoxia.
The decrease in V 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 V 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 V 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 V 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 V 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).
Figure 2.3 – Relationship between aerobic capacity and reduction in V O2max at various
altitudes compared to sea-level. Aerobically trained subjects ( V O2max ≥ 63 ml.kgˉ1.minˉ1)
are represented by the dashed regression line, untrained individuals ( V 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).
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.
2.5 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 O 2dependant 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).
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 BeerLambert 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].
∆C =
∆ODλ
єλ · L · DPF
(3)
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 V 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 V 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
V 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 V O2 across a range of
exercise intensities. The profile can be explained mechanistically by the relationship
between muscle blood flow (Qm) with muscle V O2 ( V 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 V O2m and early attainment of the plateau region in the
HHb profile. The plateau indicates the kinetics of V O2m and Qm are linear and may be
interpreted as evidence of maximal O2 extraction and V O2max and therefore raises the
possibility that incremental exercise performance is driven by the HHb profile.
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 V 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 O2sensitive 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).
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 ischemiareperfusion (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).
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.
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 SNitrosothiols (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 NOdependant 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).
→
RSNO + NO2•
NO+ + O2
→
N2O3 (+ RSH) →
HNO2 + H+
→
NO+ + H2O
(2)
RSH + NO+ →
RSNO + H+
(3)
(1)
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 V O2 (Larsen et al. 2007) and V 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).
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).
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 NOHb 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 3NT 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% V 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 V 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 V 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).
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 min after reoxygenation (Yu et al.
1998). The rapid kinetics of HIF-1α ensures adaptation to IH can occur after only a few
exposures and increasing vascular NO storage in rats (Manukhina et al. 2000) and HVR
in humans (Foster et al. 2005).
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).
Peng et al. (2006) showed the elevated carotid body output to acute and chronic IH in
wild type (WT) mice was completely absent in HIF-1α deficient mice. Compared to WT
mice, basal HIF-1α protein expression in cortical tissue was 50% lower in HIF-1α
deficient mice. There was also no change in HIF-1α expression following 10 days IH in
HIF-1α deficient mice whereas expression was significantly enhanced in WT mice
exposed to IH. ROS generation is a critical signaling event for HIF-1α expression since
the increased ROS levels were observed only in WT mice whereas antioxidant treatment
prior to IH prevented HIF-1α activation and cardio-respiratory changes in WT mice
(Peng et al. 2006). A reduction in the threshold sensitivity for HIF-1 activation was
documented during adaptation to IH (Liu et al. 2005). This would appear to be an
important factor that drives the cross-adaptive benefits of IH and modulated through an
enhanced sensitivity of ROS signaling for HIF-1α expression and downstream release of
protective factors such as EPO (Liu et al 2005) and HSP70 (Yeh et al. 2010).
Subsequently, a prolonged activation of HIF-1α after IH was attributed to the reduction in
I-R injury in rat kidneys by attenuating the oxidative stress response and cell apoptosis
(Yang et al. 2009) whilst administration of a free radical scavenger abolishes the
protective effects of hypoxic preconditioning (Rauca et al. 2000). Furthermore, two
hours after hypoxic preconditioning in mice with a genetic predisposition to over-express
SOD1 showed an elevated HIF-1α activation threshold during the 15 hours severe
hypoxic exposure that followed which downregulated HIF-1α expression and impaired
neuroprotective effects to injury (Liu et al 2005). These results suggest ROS signaling
plays a central role in hypoxic preconditioning by down-regulating the activation
threshold sensitivity for HIF-1α expression during future stressful events.
Similarly in humans ROS generation is a critical signaling event for HIF-1α transcription
in vivo. During an acute 12 hour continuous hypoxic exposure, Pialoux et al. (2009d)
showed an increased HIF-1α expression within 1-2 hours before returning to baseline.
These changes correlated positively with blood bourne markers of oxidative stress and
HIF-1 target genes erythropoietin (EPO) and vascular endothelial growth factor (VEGF)
which followed the same kinetic pattern. The increased HVR following 18 days of living
at moderate altitude and training at sea-level in elite athletes was also related by an
increased sensitivity and up-regulation of HIF-1α expression which coincided with
increased oxidative stress levels (Pialoux et al. 2009a).
Treatment of OSA with
antioxidant supplementation mitigated the respiratory changes associated with the
condition such as progressive augmentation and ventilatory long term facilitation (Lee et
al. 2009). It is possible antioxidant supplementation may be detrimental to individuals
adapting to IH although this remains to be shown in healthy, human subjects.
NO donors can suppress HIF-1 activation and HIF-1α stabilisation in cell culture systems
under hypoxic conditions (Liu et al. 1998b; Sogawa et al. 1998) through an up-regulation
of O2-dependant prolyl hydroxylase enzymes (PHD) and in particular PHD-2 (BerchnerPfannschmidt et al. 2007).
Since these enzymes are involved in the O2-dependant
degradation of HIF-1α, a reduction in PHD-2 activity would allow HIF-1α stabilization
for target gene expression in hypoxia (Berchner-Pfannschmidt et al. 2007). Therefore the
sensitivity for HIF-1 pathway activation following IH can be up-regulated by increased
ROS generation or down-regulated by NO production.
2.20
Adaptive mechanisms 2: ROS and NO regulation
The magnitude of ROS and NO generation determines whether the adaptive response has
beneficial or adverse effects on vascular homeostasis and functionality (Manukhina et al
2006; Serebrovskaya 2002; Neubauer et al. 2001). A summary of studies examining IH
with direct implications on NO-ROS regulation is presented in Table 2.1. An optimal IH
regime and time course for the protective response has yet to be determined. If the insult
occurs too soon after IH could allow insufficient time for cells to recover and increase
susceptibility to oxidative injury.
If the recovery period is too long, the threshold
sensitivity for HIF-1α activation returns to normal and window of opportunity for
protection is lost. Protection can last from 60 minutes (Bertuglia 2008) and up to to 24
hours after IH (Ryou et al. 2008; Beguin et al 2005; Zong et al 2004; Ding et al. 2005)
although one report showed protection against myocardial ischemia in rats persisted for 5
weeks after IH consisting of 7,000m 8hours/day, 5 days/week for 7 weeks (Neckar et al.
2004).
A similar NO-ROS mediated response has been identified for ischemic
preconditioning where an early (< 3 hours) and late (1-4 days) phase of resistance against
ischemic injury are reported (Bolli 2000). It is possible that a similar time frame exists
after IH. Although, Cai et al. (2003) showed the protective mechanism activated by
hypoxia was present at 24 hours but not 30 minutes after IH. Since HVR remains
elevated for days after IH and is fully restored within 1-2 weeks (Foster et al. 2005;
Katayama et al. 2001 & 2005), it is possible the protective effects decays concurrently
with changes in respiratory chemosensitivity and presumably ROS-HIF-1α activation
sensitivity.
IH has the advantage of exposing individuals to more severe hypoxic episodes whilst
avoiding some of the detrimental effects associated with prolonged exposures such as
pulmonary and cerebral oedema and other symptoms associated with acute mountain
sickness (Bartsch et al. 2004). The molecular processes driving adaptation appear to
differ between the two types of hypoxic exposure. Peng & Prabhakar (2004) showed that
hypoxic sensitivity of isolated carotid bodies from rats was enhanced after 10 days IH
(15s of 5% O2 + 5 min of 21% O2, 9 episodes/hour, 8 hour/day) but not after 10 days
continuous hypoxic exposure (4 hours per day at 0.4 atm).
Carotid body
chemosensitivity was attenuated in rats pretreated with a potent superoxide scavenger
prior to administration of IH implying that the primary mechanism driving adaptation is
mediated by the number of reoxygenation phases and associated oxidative stress rather
than the absolute duration of each hypoxic episode. These findings are supported by
others that show IH is a more potent preconditioning stimulus for eliciting tissue
protection against organ I-R injury than continuous hypoxia (Beguin et al. 2005; Milano
et al. 2002). However in healthy humans, 10 days breathing continuous (FIO2 = 12%, 30
min/day) versus IH exposure (6x5 min episodes of 12% O2 followed by 5 min normoxia)
had similar effects on HVR and cerebrovascular responsiveness to progressive isocapnic
hypoxia (Foster et al. 2005).
Manukhina et al. (2006) previously described a central role for NO during adaptation to
IH. An improved regulation of cardiovascular homeostasis after IH was related to a more
effective synthesis and storage of NO thus preventing NO deficiency or by limiting NO
overproduction
during
inflammatory
reactions
which
together
maintain
NO
bioavailability at ‘optimal’ levels. The enhancement of NO regulation can stimulate other
protective mechanisms such as improved hypoxic sensing, HSP/antioxidant activities and
more efficient mitochondrial O2 utilisation making the adaptive response more robust and
increasing overall resistance to injury. However severe IH can lead to systemic and
pulmonary hypertension, increased susceptibility inflammation, impaired NO production,
ROS overproduction and haemodynamic dysfunction at rest and during exercise in
humans (Foster et al. 2009; Wang et al. 2007a & b; Neubauer et al. 2001). Foster et al
(2005) reported a blunted cerebral oxygenation response during progressive isocapnic
hypoxic exposure after 10 sessions of IH in healthy human subjects.
An impaired
hypoxic vasodilation response was also reported in rats after severe IH and was explained
mechanistically by a ROS-induced inactivation of NO production (Phillips et al 2004). In
a second study by Phillips et al. (2006) the impaired myogenic activation and
noradrenaline-induced vasoconstrictor responsiveness of skeletal muscle resistance
arteries was restored by treatment with a superoxide scavenger administered in the
drinking water throughout the hypoxic intervention. In humans, short (Foster et al. 2009;
Pialoux et al. 2009c) and long duration IH (Pialoux et al. 2009d) increased HVR and
conincided with increased oxidative stress, elevations in MAP, reduction in vascular NO
metabolite bioavailability and impaired cerebrovascular response to hypoxia. These
studies highlight excessive generation of ROS can lead to the progression of vascular
complications arising from IH.
Antioxidant supplementation restored endothelial
function assessed by FMD (Grebe et al 2006) and alleviated the respiratory changes
shown in OSA patients (Lee et al. 2009) highlighting ROS-NO generation and interplay
as an important molecular signalling mechanism driving acute and chronic responses to
IH in health and disease.
2.21 IH and endothelial activation
Expression of CAMs is elevated in OSA patients (Dyugovskaya et al. 2002; Ohga et al.
1999; Ursavas et al. 2007). Similarly in rats, 60 recurrent obstructive apneas lasting 5
seconds for 3 hours triggered a significant rise in leukocyte-endothelial cell interactions
on the venular endothelium as demonstrated by increased leukocyte rolling flux and
number of rolling leukocytes (Nacher et al. 2007).
In vivo studies however yield
contradictory findings on the effect of IH on CAM expression and are likely to reflect
differences in experimental models and IH regime. Ichikawa et al. (1997) demonstrated
an increased ICAM-1 and P-selectin expression on HUVECs after one hour of hypoxia
followed by one hour reoxygenation. A downregulated ICAM-1 and VCAM-1 expression
in human endothelial cells was shown after exposure to sustained hypoxia however both
increased after 4 hours hypoxia followed by 16-28 hours reoxygenation (Willam et al.
1999). In contrast, Lattimore et al. (2005) showed no change after IH in isolated human
monocytes adhesion to endothelial cells and ICAM-1 and VCAM-1 expression.
Rat aortic endothelail cells preconditioned with 1 hour hypoxia prevented CAM
expression following subsequent exposure to anoxia-reoxygenation (Beauchamps et al.
(1999). The changes in leukocyte adherence evoked by IH may be mediated by NO since
the diminished vascular NO bioavailability in OSA patients is reversed after nCPAP
treatment (Ip et al. 2000). The increased leukocyte adherence in the cerebral circulation
4, 24 and 48 hours after IH (twelve 30-s periods of hypoxia every 5 min for 1 hour) in
wild type mice was elevated in eNOS knockout mice whereas there was no change in
adherence in nNOS knockouts (Altay et al. 2004). These results suggest an NO-isoform
dependant role whereby nNOS activity promotes leucocyte-endothelial adherence
whereas eNOS activity blunts the response and exerts neuroprotection. The effect of NO
and hypoxia on the cell adhesive response was characterised by Wood et al. (1999b).
Leukocyte adhesion was assessed in rat venules during an acute hypoxic (10% O2)
exposure before and after 3 weeks acclimatisation to 10% O2. The results showed that
before acclimatisation acute hypoxia promoted the leukocyte-endothelial adhesion and
was explained mechanistically by a reduction in NOS activity and depletion in NO levels
that was independent of changes in shear rate (figure 2.14). There was no leukocyte
adhesion in preacclimatised rats due to compensatory increases in NO formation and was
related to an increased contribution from iNOS whilst administration of an NO donor in
non-acclimatised rats also decreased the number of adherent leukocytes.
.
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).
Table 2.1 Examples of IH studies and summary of key research findings with implications for vascular function and NO/ROS balance.
Reference
IH protocol
Research summary and conclusions
Animal studies
Phillips et al. 2004
Philippi et al. 2010
Bertuglia 2008
Ryou et al. 2008
Beguin et al. 2005
Manukhina et al. 2000
Mallet et al. 2006
Asha et al. 2005
Ding et al. 2005
10% O2 for 1 min at 4-min intervals; 12
↓ endothelium dependant vasodilator response to Ach and hypoxia in skeletal muscle and cerebral
h/day for 14 d (rats)
resistance arteries, sensitivity unaffected by NO donors after IH,
10% O2 for 2 min norm: hyp cycles for
↓endothelium vasodilation after 2 weeks IH, 3 days insufficient to cause impairment, longer
3, 14, 28 or 56 d (rats)
exposures did not worsen the effect, evidence of ↑oxidative stress and eNOS expression
6 min cycles of 8:21% O2 breathing every 8
↓oxidative stress, ↑ NO-induced vasodilation and ↓NOx during I/R injury and Ach stimulation
h for 21 d (hamsters)
maintaining capillary perfusion, protective effects abolished by NOS inhibition
5–8 cycles of FIO2 9.5–10%, 5–10 min
suppressed myocardial NOS activity, nitrite release, eNOS content, and excessive NO formation
with 4 min norm, 20 d (dogs)
upon reperfusion without compromising reactive hyperemia
40 s 5 or 10% O2, 20 s norm for 30 min or 4
4 h 5% O2 ↑infarct size, 4 h 10% O2 ↓infarct size, ↔ of 30 min IH or 4 hr continuous hyp. Effect
h, or 4hr continuous 10% O2 (rat)
abolished by NOS inhibition and mitochondrial K ATP channel blockade
40 sessions at 5,000m for 10 min and ↑
Graded ↑ vascular NO storage, adaptation prevented impairment of endothelium dependant
to 5 hr exposures after 10 sessions (rat)
relaxation and protection against NO overproduction and/or deficiency
5–8 cycles of FIO2 9.5–10%, 5–10 min
IH cardioprotection evoked by β1-adrenergic activation, single IH session failed to protect ischemic
with 4 min norm, 20 d (dogs)
myocardium, protection incomplete after 10 sessions
Exposure to 5,700, 90 min/d for 9 d or
↑ antioxidant activity and oxidative stress in both IH groups, these size of these effects was related
6,300, 30 min/d for 15 d (rat)
to trained status, 6300 m appears a more effective stimulus for triggering metabolic adaptations
5,000m, 6h/d for 42 d (rat)
improved recovery of post-ischemic ventricular function and coronary blood flow, ↑NOx,
↑cardioprotection to I/R injury after IH abolished by iNOS inhibition
Li et al. 2007
30sec of 5% or 10% O2 followed by
10% O2 leads to hypercholesterolemia and lipid peroxidation in liver, the degree of metabolic
30sec norm for 28 d (mice)
impairment was dependent on the severity of the hypoxic stimulus
Human studies
Pialoux et al 2009a &
2 min cycles of hyp PETO2 = 45mmHg
↑ oxidative stress without compensatory ↑ in antioxidant activity, these mechanisms modulate the
c
with norm 6 hr/d for 4 d
↑HVR
Foster et al 2009
2 min cycles of hyp PETO2 = 45mmHg
↑ MAP, ↓ NOx, ↑cerebral vascular resistance and pressor response to acute hypoxia
with norm 6 hr/d for 4 d
Foster et al 2005
Wang et al 2007a
10 d of 6x 5min 12% O2 with norm
similar effects on ventilatory and cardiovascular response to acute progressive hypoxia and hindered
(SDIH) or 30 min continuous 12 O2
cerebral oxygenation from 2 types of IH, ↑ MAP only after SDIH
4 weeks, 15 or 12% O2, 1hr/d, 5d/week
Adaptation to 12% O2 (but not 15% O2) ↓blood anti-oxidative capacity, ↑lipid peroxidation, ↑NOx,
↑MAP response to exercise, ↓hyperaemic arterial blood flow, ↓venous compliance and ↓vascular
responsiveness to Ach. Both IH regimes ↑ventilatory threshold and ↑VO2max
Wang et al 2007b
8 weeks, 15 or 12% O2, 1hr/d, 5d/week
Both IH regimes ↓ eosinophil-platelet aggregation, ↓ IL-1β production, ↑anti-oxidant content and ↓
lipid peroxidation induced by severe exercise or acute hypoxia exposure. Adaptation to 12% O2↑ IL6 and ↑ IL-10 at rest, post exercise and during acute hypoxia exposure
Note: ↑= increase or improved, ↓ = decrease, ↔ = no change
2.22
IH and exercise performance
Athletes and mountaineers incorporate IH at rest or during exercise into their training
program to replicate some of the key features of altitude acclimatisation as a means of
enhancing systemic O2 delivery and utilisation with the ultimate goal of improving
exercise performance at sea-level or altitude (Bailey et al. 2001; Dufour et al. 2006;
Katayama et al. 2001 & 2003; Levine and Stray-Gundersen 1997).
Studies so far
investigating these hypoxic interventions are rather disjointed due to differences in
hypoxic administration (see table 2.3), methods for evaluating performance, aerobic
condition and health of the subjects (Burtscher et al. 2009) and individual variations in
response to hypoxia and the concept of ‘responders’ and ‘non-responders’ to altitude
training (Chapman et al. 1998).
Subsequently, the efficacy of IH on exercise
performance has often been the source of debate (Levine 2002). There is some evidence
suggesting submaximal and maximal performance can be improved due to improved
SaO2, exercise efficiency, increased RBC mass and improved buffering capacity. A
summary of previous research findings on the effect of IH specifically during short
duration resting exposures on sea-level and hypoxia performance is presented in Table
2.3.
There is more persuading evidence showing IH as an effective pre-acclimatisation
strategy by eliciting cardiovascular, cerebrovascular and ventilatory adaptation prior to
subsequent resting hypoxic exposure (Katayama et al. 2001; Foster et al. 2005; Ainslie et
al. 2007). However there are only a few studies examining the effects of IH on exercise
performance at altitude (Table 2.3). These investigations tend to show an improved SaO2
and exercise economy as well as enhanced ventilatory response to submaximal exercise.
Adaptation within the muscle and brain of sedentary subjects have also been reported in
untrained subjects (Ainslie et al. 2008) and in athletes exhibiting symptoms of EIH
(Marshall et al. 2007) where an augmented ventilatory response resulted in a blunted
cerebral oxygenation response whereas favourable effects may be shown in the muscle.
A more aggressive IH regime may be more effective strategy in delivering benefical
effects on exercise performance (Stuke et al. 2005) despite detrimental effects on vascular
function (Wang et al. 2007a). It is possible the beneficial effects on muscle energetic
function could over-ride the impairments in haemodynamic control and O2 delivery.
These effects have yet to be shown during maximal exercise in hypoxia where systemic
O2 delivery and regional utilisation in the brain and muscle is critical to aerobic
performance (Amann et al 2007; Subudhi et al. 2007).
Wang et al. (2007a) showed four weeks of moderate (FIO2 = 0.15, 1hr/day) and severe IH
(FIO2 = 0.12, 1hr/day) produced similar improvements in ventilatory threshold and
V O2max in healthy men at sea-level.
This was despite impairment in vascular
haemodynamics as assessed by the elevated exercising blood pressure, impaired resting
endothelial dependant vasodilation response (evaluated by reactive hyperaemia
stimulation and blood flow responsiveness to acetylcholine), increased NO metabolite
levels, reduced plasma antioxidant status and increased oxidative stress in the severe IH
group whereas no such effects were observed in the moderate IH group. A second study
by Wang et al. (2007b) showed 8 weeks of the same moderate and severe IH regime
increased V O2max and suppressed the extent of eosinophil-platelet aggregation, proinflammatory IL-1β secretion and the decline in antioxidant status and increased lipid
peroxidation induced by maximal exercise. However severe IH was also associated with
elevations in resting and post exercise plasma IL-6 and IL-10. These results suggest the
potential risk of cardiovascular complications derived from endothelial dysfunction and
pro-inflammatory stimulation is dependant on the severity of the IH regime at rest and
during exercise.
Table 2.2
Summary of investigations examining the effects of IH and exercise responses at sea-level or in acute hypoxia
Authors
IH protocol
Summary of key findings
15 days, 6 cycles of 6:4 min hyp-to-norm,
↔ aerobic and anaerobic performance
Sea-level performance
Tadibi et al. 2007
FIO2 = 0.10 to 0.11
Julian et al. 2004
4wk of 5:5min hyp-to-norm for 70 min,
↔ endurance performance and haematological variables
FIO2 = 0.12 wk1, 0.11 wk2, 0.10 wk 3 & 4
Katayama et al. 2004
3 wk, 3 days/wk, 90 min/day at 4,500m
↑TT performance, ↑ submaximal exercise efficiency, ↔ maximal
Katayama et al. 2003
& 14 days, 3hr/day at FIO2 = 0.12
cardiorespiratory and haematological variables
Stuke et al. 2005
10 days, 5:5min hyp-to-norm for 90 min
↑constant load cycling performance to exhaustion at 80% PPO
FIO2 = 0.09
Marshall et al 2007
Foster et al. 2006
10 days, 90 min/day, 7:3 min hyp-to-norm,
↔ aerobic performance, ↑SaO2 and V E at PPO, response reflected a balance
SaO2 at 80%
between cerebral (↓) and muscle (↑) oxygenation changes
10 exposures of 5:5min hyp-to-norm for 60
↔ aerobic performance or ventilatory response to exercise
min or 30min, FIO2 = 0.12
↑aerobic and anaerobic performance which remained for up to 10 days after IH
Bonetti et al. 2006
15 exposures of 3 or 5 min intervals hyp-
Bonetti et al. 2009
to-norm for 60 min, SaO2 = 90-76%
Burtscher et al. 2004
15 sessions, 3-5 hypoxic (14-10% O2)
↑O2 carrying capacity and V O2, V E, CaO2 and ↓ blood lactate at maximal exercise
exposures for 3-5min with 3-min norm
in elderly men with and without CAD. During submaximal exercise; ↓HR,
↓systolic BP, ↓blood lactate and ↓RPE
Rodriguez et al. 1999
9 days, 3-5hour/day, at 4,000-5,500m with
↑ incremental exercise performance and V Emax, rightward shift in blood lactate-
and without exercise during acclimation
velocity curve, ↑O2 carrying capacity, no added effect of exercise
Casas et al. 2000
17 days, 3-5hour/day, at 4,000-5,500m
↑ RBC count and [Hb], ↑ V O2max, rightward shift in blood lactate curve and
ventilatory threshold (↑anaerobic threshold), ↓HR,
Truijens et al. 2008
4,000-5,500m, 3hr/day, 5days/week for 4wk
Rodriguez et al. 2007
↔ Maximal aerobic performance and submaximal economy in well trained
athletes
Pre-acclimation studies and hypoxic exercise performance
Katayama et al. 2001
7 days, 4,500m for 1 hour/day
↑resting HVR and SaO2, VE and VE/O2 at 40 and 70% of VO2max at 4,500m, ↔VO2
but ↑SaO2 during max ex, adaptation persisted 1 wk after IH
Ainslie et al. 2008
Beidlemann et al. 2003
10-12 sessions, 5:5-min hyp-to-norm for 90
↑VE, ↑SaO2, ↓PCO2, ↓MCAv, ↑muscle and ↓cerebral oxygenation during steady
min, target SaO2 = 90-75%
state ex in 14% O2
4 h/day, 5 days/wk, 4,300 m for 3 wks (rest
↑SaO2, VO2max and time trial performance at 4,300m
or exercise)
Beidlemann et al. 2008
Beidleman et al. 2009
4 h/day, 5 days/wk, 4,300 m for 7 days (rest
↑TT performance, ↑SaO2, ↓HR, VO2 and ↓RPE at 40 and 70% of VO2max at
or exercise)
4,300m, No added affect of exercise,
3-h rest and exercise, PO2 of 90 mm Hg for
↔ TT performance or SaO2, HR, and RPE at 40 and 60% of VO2max at 4,300m
1wk
Katayama et al. 2007
7 days, 1hr/day at FIO2 = 0.15 or 0.12
↔ VE and SaO2 during max and submaximal exercise at 2,500m, ↑resting HVR
only in FIO2 = 0.12 group
Note: ↑= increase or improved, ↓ = decrease, ↔ = no change compared to baseline
2.24
Summary
Central and peripheral fatigue factors can play an important role in the reduction of
aerobic performance in hypoxia. It is possible the reduction in V O2max in hypoxia is
determined by the magnitude and profile of the cerebral and muscle oxygenation
response to incremental exercise. The aim of study 1 is to characterise the changes in
cerebral and muscle oxygenation using NIRS during incremental exercise and
establish if the rate of deoxygenation through a reducion O2 delivery and increased O2
extraction, and in turn the development of central and peripheral fatigue, is
accelerated in hypoxia. The initial physiological response to hypoxia leads to a
disruption of normal homeostasis where adaptation is complex and can involve
activation of multiple signalling pathways. In particular, the O2 sensing ability of
blood vessels in the microcirculation ensures adequate delivery of O2 to critical
regions
matches
its
demand.
The
balance
between
ROS
and
NO
production/inactivation are key regulators of vascular function and O2 sensing during
exercise. There is limited research examining the effects of acute hypoxia on changes
in molecular biomarkers of vascular function which could influence haemodynamic
stability and aerobic performance and are examined in study 1.
Depending on the severity and intermittence of hypoxia, it is possible IH may have
beneficial or detrimental effects on vascular function in healthy humans.
The
increasing popularity of IH by athletes and mountaineers as a means of enhancing O2
delivery and aerobic performance requires further examination. There is no research
examining the effects of severe, short duration IH as a preconditioning stimulus to
enhance cerebral and muscle oxygenation and aerobic performance in hypoxia.
Reports have suggested that vascular adaptation to IH is driven by NO although there
is no evidence documenting the potential beneficial effects of IH on vascular
endothelial function and O2 sensing ability during maximal exercise in hypoxia.
Study 2 will examine the effects of 10 days of severe IH on cardiorespiratory response
to submaximal and maximal exercise and changes in systemic molecule biomarkers of
vascular function, cerebral and muscle oxygenation and ultimately aerobic
performance in hypoxia.
2.25 Specific aims and working hypotheses
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 bloodborne 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.
Exercise + Hypoxia
Systemic O2
delivery
Regional O2
delivery
Vascular
Function
SaO2
Cerebral
Oxygenation
Muscle
Oxygenation
(↑Central Fatigue) (↑Peripheral Fatigue)
V O2peak
Figure 2.15
Working hypotheses for study 1
Study 2 – The effect of 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 the increased CAM
release after maximal exercise will be attenuated after IH.
Intermittent Hypoxia
Exercise + Hypoxia
Systemic O2
delivery
Regional O2
delivery
Vascular
Function
SaO2
Cerebral
Oxygenation
Muscle
Oxygenation
(↓Central Fatigue) (↓Peripheral Fatigue)
V O2peak
Figure 2.16
Working hypotheses for study 2
Chapter 3
General Methodology
3.1 Incremental cycling protocol
All exercise tests were performed on the same stationary cycle ergometer. The
adjustable seat and handlebars was fixed to each individual’s preference and their feet
were secured to the pedals with straps. Each exercise test commenced with 4 minutes
steady state cycling at 60 W before rising 30 W every 2 minutes until volitional
exhaustion or when pedal frequency dropped below 70 rpm (Benoit et al. 1997). The
2 minute increment was chosen because it is long enough to attain a plateau in muscle
oxygenation at each workload below lactate threshold whilst minimising the effect of
the O2 slow component during the high intensity workloads (Cheung et al. 2002;
Grassi et al. 2003). Subjects were requested to maintain a pedal frequency of 80 rpm
throughout the test. Peak power output (PPO) is defined as the power output recorded
immediately before termination of incremental exercise and calculated as:
PPO = WLfull + [(t /120)*30]
Where WLfull is the value for the last workload completed in full and t is the time (in
seconds) over which the last (uncompleted) workload is maintained.
3.2 Respiratory measurements
Owing to the instability of on-line gas analysis systems in a hypoxic environment,
expired gas samples were analysed using the ‘gold standard’ Douglas bag method.
Quantification of V O2max assumes further increases in workload during incremental
exercise do not result in additional increases in V O2 as PPO approaches and is defined
by the ‘plateau’ region in the workload- V O2 profile. In the present study, aerobic
capacity will be referred to as V O2peak since it is not appropriate to assume attainment
of a plateau using one expired gas sample prior to termination of the test. Subjects
breathed through a two-way non re-breathing valve (Hans-Rudolph, Kansas, U.S.A)
and into a 150L Douglas bag via 1.5m Falconia tubing. Each expired gas sample was
taken beginning on inspiration with a sampling period of 60 seconds. Samples of
expired air were dried using calcium sulphate crystals (Drierite, U.S.A) and gas
fractions were analysed for 60 seconds using a paramagnetic O2 and infra-red CO2
analysers (Servomex 1400B4, Sussex, UK). A zero point was established for the O 2
and CO2 analysers using pure nitrogen and then calibrated using a specialised gas
mixture containing 16.9% O2, 5.04% CO2 and balanced N2 (BOC gases, Surrey, UK).
The volume of the Douglas bag was measured using a dry gas meter (Harvard,
Edenbridge, UK) at a flow rate of 70L/min. Measurement of O2 uptake ( V O2) and
CO2 production ( V CO2) corrected to STPD was computed using the Haldane
transformation equation (Wasserman et al. 1994).
Expired gas samples were
collected during the final minute of the 60 W steady state workload and prior to
volitional exhaustion for determination of V O2peak. Attainment of maximal exercise
was verified by the following criteria: RER > 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.
Chemiluminescence
signal (mV)
Untreated
NO2• + RSNO
Treated
RSNO
Time (seconds)
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 Xband (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
<5%.
Figure 3.2 – A typical EPR spectrum for the A• detected in human venous blood
3.10
Haemoglobin (Hb)
Whole blood haemoglobin concentration was measured using a dual wave photometer
(HemoCue B-Haemoglobin, Sheffield, UK).
A 10µl venous blood sample was
transferred immediately into a microcuvette and inserted into the photometer. Sodium
desoxycholate lyses the erythrocytes releasing the haemoglobin. The Hb is converted
to methemoglobin by sodium nitrite which combines with azide to form
azidemethemoglobin.
The absorbance is measured at two wavelengths (570 and
880nm) and Hb concentration is presented in g/dL.
Samples were analysed in
duplicate and the average of the two was taken as the definitive value.
3.11
Haematocrit (Hct)
A venous blood sample was transferred from the syringe and into a 75mm heparinised
capillary tube (Hawksley and Son Ltd, Sussex, UK). The capillary tube was sealed
and centrifuged for 4 minutes at 11,800 rpm. The packed erythrocytes were then
measured to the nearest 0.5% using a micro haematocrit reader (Hawksley and Son
Ltd, UK). Samples were analysed in duplicate and the average of the two was taken
as the definitive value.
3.12
Blood gas
A 5 ml sample of venous blood was drawn from the syringe and analysed PO2, PCO2
and pH in anaerobic conditions using a Rapid lab 248 blood gas analyser (Chiron
Diagnostics, Essex, UK). An automated calibration was conducted every hour and on
each testing day a quality control was analysed. Samples were analysed in duplicate
and the average of the two was taken as the definitive value.
3.13
IH machine
The IH machine (Inside out, New Zealand) operated by drawing ambient air through a
molecular sieve via a flow meter and into an outlet pipe.
The generator was
automated to produce hypoxic air in 5 minute cycles. Subjects could breathe the
hypoxic air through a hand held face mask connected to the outlet pipe. To calibrate
the system, a Douglas bag was connected to the outlet pipe via corrugated tubing and
gas samples were taken at 2 minute intervals and analysed for FIO2 and FICO2 using a
paramagnetic O2 and infra-red CO2 analyser (Servomex 1400B4, Sussex, UK). Flow
was measured using a dry gas meter (Harvard Ltd, Edenbridge, UK). The outlet gas
concentration was pre-set for 9% O2 as per protocol for the IH study and gas samples
were taken over 9 episodes of hypoxia. The results are presented in Table 3.1. On
average O2 was 9.5%, CO2 was 0.03% and flow was 53.5L/min.
Sample
1
2
3
4
5
6
7
8
9
O2
9.5
9.4
9.6
9.6
9.5
9.5
9.6
9.5
9.6
CO2
Flow
L/min
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.03
53.9
53.9
53.7
53.8
53.3
53.3
53.2
53.1
53.3
Table 3.1 Volume and gas concentration measurements taken from IH machine
Chapter 4
Study 1
The impact of acute hypoxia on cerebral and muscle oxygenation and systemic
molecular biomarkers of vascular function during exercise
4.1
Introduction
The dose dependent relationship between muscle O2 supply and V O2max in acute
hypoxia has been well documented (Richardson et al. 1999; Ferretti et al. 1997).
However, at altitudes above 4,000m the decline in V O2max is larger than expected for
the reduction in CaO2 (Roach et al. 1999).
A critical reduction in cerebral
oxygenation was observed prior to termination of incremental exercise in hypoxia
(Subudhi et al 2007). Subsequently, an accelerated development of ‘central’ and
‘peripheral’ fatigue mechanisms have been attributed to the impairment in hypoxic
exercise performance (Amann et al. 2006). An important protective feature of the
brain is its ability to maintain cerebral blood flow although during intense exercise the
hyperventilation induced hypocapnia and ensuing vasoconstriction decreases cerebral
blood flow and O2 delivery (Nybo and Rasmussen 2007). Increased O2 extraction
estimated by global arterial-venous differences maintains the required level of
cerebral aerobic metabolism in normoxia and moderate hypoxia (Ide et al. 2000;
Volianitis et al. 2008) although there are no studies to show if cerebral metabolism is
impaired during intense exercise in severe hypoxia where it has been proposed central
motor output may be impaired directly by O2 availability (Amann et al. 2006). It is
possible that like skeletal muscle maximal cerebral O2 extraction capacity is
unaffected during exercise in acute hypoxia (Lundby et al. 2006) and attainment of
maximal cerebral O2 extraction capacity may be achieved at a reduced workload.
NIRS presents an accurate and reliable non-invasive assessment of haemodynamic
changes and metabolism across muscle and brain tissue (Rasmussen et al. 2007;
Chance et al. 1992; Delpy et al. 1987). It is possible to profile the dynamic changes in
cerebral and muscle oxygenation at rest and during exercise to identify potential
impairments in microcirculatory O2 exchange and vascular function. Incremental
exercise testing is used extensively to monitor cardiovascular fitness however the
profiles for muscle and cerebral oxygenation are not well defined in hypoxia. Recent
reports show a sigmoidal muscle HHb profile during incremental exercise in
normoxia reflecting the non-linear relationship between microvascular muscle blood
flow and O2 uptake (Boone et al. 2009; Ferreira et al. 2007). A more linear profile
from the onset of incremental exercise and up to 75% PPO was followed by a
‘plateau’ phase at workloads exceeding 90% PPO in athletes residing at moderate
altitude (Subudhi et al. 2007). A similar pattern was observed in hypoxia but the
magnitude of deoxygenation was greater however not all studies show an aggravated
muscle deoxygenation response to exercise at altitudes up to 4,000m (Ainslie et al.
2007; Bourdillon et al. 2009; DeLorey et al. 2004). The discrepancy between studies
warrants further examination with direct comparison between the kinetic changes in
muscle and cerebral oxygenation during incremental exercise in hypoxia and
normoxia.
An effective and efficient NO production is critical for vascular homeostasis
regulating vascular tone in normoxia, hypoxia and during exercise (Maher et al. 2008;
Gladwin et al. 2000; Blitzer et al. 1996). NO is also known to function as a cell
signaling molecule exerting anti-inflammatory and anti-atherogenic properties within
the vessel wall thus downregulating endothelial CAM expression and transmigration
of leukocytes (Dal Secco et al. 2006; De Caterina et al. 1995). The vascular NO
metabolites: nitrate, nitrite and RSNO form an active NO reservoir that is transported
in the blood until re-release of NO depending on the local biochemical and
environmental conditions. These metabolites are essential modulators of vasodilation
through their effects on vascular hypoxic sensing (van Frassen et al. 2009) and tissue
protection (Duranski et al. 2005; Giustarini et al. 2003). The beneficial effects of
exercise on endothelial function have been reported in healthy and patient populations
(Di Francescomarino et al 2009; Rush et al 2005). Exercise induced elevations in
shear stress leads to a greater NO production from endothelial cells resulting in
vasodilation to increase the delivery of O2 and substrates to the muscle (Tschakovsky
and Joyner 2008; Koller & Kaley 1991). The effectiveness of the response appears to
be dose dependant on exercise intensity although it is not clear if the relationship is a
beneficial or detrimental one during high intensity exercise. Acute high intensity
exercise was shown to impair FMD in patients with coronary heart disease whereas
moderate intensity exercise improved the response (Farsidfar et al. 2008). In healthy
male subjects, moderate intensity exercise training enhanced NO-mediated
endothelial function however high intensity exercise was associated with increased
oxidative stress and impaired NO-dependant vasodilation (Goto et al. 2003; Bergholm
et al. 1999). In contrast, high intensity interval exercise improved FMD compared to
moderate intensity exercise in post-infarction heart failure patients (Wisloff et al.
2009) and healthy humans with endothelial dysfunction induced by postprandial
lipemia (Tyldum et al. 2009).
Nitrite is considered a reliable biomarker of eNOS activity and changes in its plasma
concentrations discriminates the severity of endothelial dysfunction in patients with
cardiovascular disease (Kleinbongard et al. 2003 & 2006) and predicts aerobic
performance in normoxia (Rassaf et al. 2007). Since hypoxia (McQuillan et al. 1994)
and oxidative stress (Steiner et al. 2002) inhibits eNOS activity, the NO2•-HHb-NO
pathway could play a more pivotal role in the delivery of O2 during exercise
particularly in hypoxia. Only a few studies have examined the effects of nitrite during
exercise and there appears to be some discrepancy in the literature. Some show
increased plasma nitrite after maximal exercise (Allen et al. 2009; Rassaf et al. 2007)
whereas others report a decrease (Larsen et al. 2009).
The impact of maximal
exercise on systemic molecular biomarkers of endothelial function requires further
examination with implications for aerobic performance and changes in systemic and
regional oxygenation during exercise in normoxia and acute hypoxia.
4.2
Research aims and objectives
1) To characterise changes in muscle and cerebral oxygenation during
incremental exercise to exhaustion in normoxia and acute 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.
2) To examine the impact of acute hypoxia on molecular blood-borne markers of
vascular O2 sensing and function after maximal exercise.
Specifically
biomarkers of oxidative stress, NO metabolite bioavailability and CAM will
be measured before and immediately after maximal exercise.
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.
4.3
Working hypothesis
“Hypoxia will aggravate the cerebral and muscle deoxygenation response to
exercise and accelerate the deoxygenation profile of both tissues which will
ultimately determine aerobic performance. Exercise in hypoxia will also lead to a
greater increase in oxidative stress levels compared to normoxia resulting in further
reductions in NO bioavailability and increased CAM response.”
Exercise + Hypoxia
Endothelial
function
Regional
Oxygenation
SaO2
ROS
NO
Cerebral Oxygenation
3-NT
CAM
Muscle Oxygenation
V O2peak
Increase or
decrease
compared to normoxia
Figure 4.1 Working hypotheses for study 1
4.4
Methodology
Subjects
Fourteen apparently healthy male volunteers were recruited in the present
investigation. Subject characteristics are presented in Table 4.1. Subjects were sealevel residents and had not spent any time at altitude in the 6 months prior to
participating in the study. All were non-smokers, physically active and had no prior
history of disease. Subjects were free of any nutritional supplements such as oral antioxidants and anti-inflammatorys. Each subject was given a full verbal and written
explanation of the experimental procedures and completed one familiarisation trial in
normoxia. The study was approved by the University of Glamorgan Human Ethics
committee and each subject provided written informed consent prior to participation.
Table 4.1 - Subject characteristics
Age (yr)
Stature (m)
Body mass (kg)
Hct (%)
Hb (g/dL)
23 ± 5
1.80 ± 0.07
84 ± 8
46 ± 2
15.0 ± 0.7
Experimental design
Each subject performed 2 experimental incremental cycle tests to volitional
exhaustion in an environmental chamber (Design and environmental, UK) in
normoxia (FIO2 ~ 0.21) and hypoxia conditions (FIO2 ~ 0.12).
The study was
conducted using single blinded, randomised, cross-over design with a minimum of 7
days separating each trial (figure 4.2). Subjects were instructed to refrain from intense
exercise and alcohol consumption for 48 hours and reported to the lab between 8.30
and 9.30am after a 12 hour overnight fast. Subjects were instrumented with cerebral
and muscle NIR probes in a room adjacent to the environmental chamber (section 3.5)
and cannulated in a prominent antecubital vein (section 3.6). Bloods samples were
taken before and immediately after maximal exercise and were centrifuged
immediately at 3,000rpm in 4°C prior to being snap frozen in liquid nitrogen and
stored in -80°C until future analysis.
Baseline
21% O2
Baseline
12% O2
NIRS
Bloods
Figure 4.2 Schematic representation of experimental design. Cerebral and muscle
oxygenation measurements were taken throughout exercise in normoxia and hypoxia.
Blood samples were taken before and immediately after exercise.
Incremental cycling test protocol
Subjects entered the environmental chamber and remained in an upright seated
position for a 10 minutes stabilisation period where NIRS, HR and SaO2
measurements were taken. An incremental cycling test to volitional exhaustion was
then conducted (section 3.1).
Determination of submaximal (steady-state 60W
workload) and peak V O2, V CO2 and V E was measured using the Douglas bag method
(section 3.2). Expired gas samples were measured during the final minute of the 60W
workload with one further sample taken during the final minute of the test prior to
volitional exhaustion. Heart rate was determined using a short range telemetry system
(section 3.3) and SaO2 measured by fingertip pulse oximetry placed on a left index
finger (sections 3.4). HR and SaO2 were measured continuously throughout exercise
and recorded at the end of each workload and at the point of volitional exhaustion.
Cerebral and muscle oxygenation measurement
The NIR probes were positioned over the left prefrontal cortex region of the forehead
and on the belly of the vastus lateralis muscle. Baseline measurements were set in an
upright seated position in normoxia and cerebral and muscle oxygenation were
expressed as relative changes from baseline. After entering the environmental
chamber, NIRS measurements were obtained after the 10 minute rest period and at the
end of each workload until termination of the test. Changes in cerebral and muscle
O2Hb, HHb and THb were averaged over the final 60 seconds of each workload and
at the point of 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 oxygenation.
Blood analysis
Plasma NO metabolites
Nitrate, Nitrite and RSNO was measured by ozone based chemiluminescence method
using a tri-iodide reagent. For a detailed description refer to methodology section 3.7.
Cell adhesion molecules
sVCAM-1 and sICAM-1 was analysed by ELISA method. For a detailed description
refer to methodology section 3.8.
3-Nitrotyrosine
3-NT was measured by ELISA method. For a detailed description refer to
methodology section 3.8.
Ascorbate free radical (A•)
A• was measured by EPR method. For a detailed description refer to methodology
section 3.9.
Hb and Hct
For a detailed description refer to methodology section 3.10 and 3.11.
Blood gas variables
For a detailed description refer to methodology section 3.12.
4.5
Statistical analysis
Cerebral and muscle oxygenation changes were expressed as micromolar changes
from resting baseline (∆µmol) and plotted against absolute (W) and relative (%PPO)
workloads. Linear regression analysis was performed to determine the slope of the
relationship between muscle oxygenation versus workload (∆µmol/W). The slope
measurements were taken between absolute workloads ranging from 60 to 180W and
relative workloads from 30 to 80% PPO. Shapiro-Wilk W tests were employed to
confirm distribution normality. Data were subsequently analysed with a two-way
repeated measures ANOVA [inspirate (normoxia vs. hypoxia) x exercise intensity].
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. Values are presented as mean ± SD. A Pearson Correlation
Coefficient was used to identify potential relationships between selected biochemical
variables and V O2peak.
4.6
Results
All fourteen subjects completed both exercise trials however due to technical issues
one subject was excluded from the NIRS analysis. Cardiorespiratory data is presented
in Table 2. Compared to normoxia, there was a 16% and 22% decrease in PPO (Norm
= 280 ± 29 W, Hyp = 235 ± 21 W, P < 0.05) and V O2peak respectively during the
hypoxia trial.
Therefore, exercise from 60 to 180W corresponded to relative
workload changes of 22 ± 2 to 65 ± 7% PPO in normoxia which increased from 26 ±
3 to 77 ± 8% PPO in hypoxia. Changes in SaO2, and HR during incremental exercise
are presented in figures 4.3 and 4.4 respectively. There were profound reductions in
SvO2 (Norm: 58.4 ± 18.2% vs. Hyp: 31.5 ± 15.5%, P < 0.05) and PvO2 (Norm: 38.2 ±
10.7 vs. Hyp: 23.2 ± 6.9mmHg, P < 0.05) at PPO during the hypoxia trial (P < 0.05).
Table 4.2 – Average cardiorespiratory responses to exercise in hypoxia and normoxia
Inspirate:
Normoxia
Hypoxia
Intensity:
60W
Peak
60W
Peak
V E, L/min
26.5 ± 3.04
136.5 ± 17.1*
32.2 ± 3.4†
133.1 ± 10.9*
Main effect for Intensity (P < 0.05); Interaction effect (P < 0.05)
V O2, L/min
1.27 ± 0.09
3.88 ± 0.29*
1.28 ± 0.08
3.01 ± 0.19†*
Main effects for Intensity and Inspirate (P < 0.05); Interaction effect (P < 0.05)
V E/ V O2
20.9 ± 2.6
35.2 ± 3.2*
25.2 ± 2.0
44.2 ± 2.6†*
Main effects for Intensity and Inspirate (P < 0.05); Interaction effect (P < 0.05)
V E/ V CO2
23.8 ± 2.5
31.9 ± 2.7*
26.5 ± 2.2
36.5 ± 2.4†*
Main effects for Intensity and Inspirate (P < 0.05); Interaction effect (P < 0.05)
Values are means ± SD. *different vs. 60W for given Inspirate; †different (P < 0.05) vs.
Normoxia for given Intensity.
Figure 4.3 – Effect of hypoxia on heart rate during incremental exercise
210
†
†
†
180
Heart rate (b.p.m)
†
†
150
†
Normoxia
120
90
Hypoxia
†
60
30
Rest
60
90
120
150
180
PPO
Workload (W)
† indicates
different from hypoxia vs. normoxia for given workload (P < 0.05).
Heart rate was greater at rest and for a given submaximal workload in hypoxia (P <
0.05). However at PPO heart rate was greater in normoxia (P < 0.05). There were
main effects for intensity and inspirate (P < 0.05) and an interaction effect (P < 0.05).
The transition from rest to exercise (60W) was also greater in hypoxia (P < 0.05).
Figure 4.4 – The effect of hypoxia on SaO2 during incremental exercise
110
†
†
†
†
†
†
†
90
Normoxia
Hypoxia
(%)
SaO2 (%)
100
80
70
60
Rest
60
90
120
150
180
PPO
Workload (W)
†different (P < 0.05) vs. Normoxia for given intensity.
SaO2 was lower at rest and throughout incremental exercise in hypoxia (P < 0.05). The
transition from rest to exercise (60W) was also greater in hypoxia (P < 0.05) highlighting
a diffusion limitation during exercise in hypoxia although there was little change in SaO2
as each workload progressed.
Muscle and cerebral oxygenation
Muscle and cerebral oxygenation changes during each relative (30-100% PPO) and
absolute workloads (rest-60-180W-PPO) in normoxia and hypoxia are presented in
figure 4.5 and figure 4.6 respectively. The results for the linear regression analysis for
muscle oxygenation slopes and intercepts are presented in Table 4.3.
Muscle oxygenation – In normoxia, muscle O2Hb remained unchanged during lowmoderate intensity exercise before decreasing during the high intensity workloads. In
hypoxia muscle O2Hb decreased from the onset of exercise (figure 4.5 A & D). The
linear regression slope and intercept for muscle O2Hb were greater when expressed as
absolute and relative exercise intensities in hypoxia. Muscle HHb increased from the
onset of exercise although the rate of increase tended to lessen as PPO was
approached (workloads >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
Slope
Norm
Hyp
Intercept
Norm
Hyp
∆HHb/∆WR
∆THb/∆WR
∆HbDiff/∆WR
Absolute
Relative
Absolute
Relative
Absolute
Relative
Absolute
Relative
-0.012
±
0.037
-0.038
±
0.090
0.071
±
0.024
0.211
±
0.075
0.063
±
0.027
0.172
±
0.066
-0.080
±
0.053
-0.249
±
0.153
-0.054
±
0.027†
-0.116
±
0.042†
0.092
±
0.025†
0.217
±
0.052
0.042
±
0.018†
0.101
±
0.039†
-0.143
±
0.045†
-0.332
±
0.088†
-3.8
±
4.6
-3.2
±
5.1
-3.9
±
3.1
-4.6
±
4.0
-7.6
±
4.3
-7.8
±
4.7
0.1
±
6.6
1.3
±
7.6
-7.3
±
3.8†
-7.6
±
3.6†
3.1
±
3.8†
3.1
±
4.1†
-4.2
±
4.9†
-4.5
±
4.8
-10.6
±
5.7†
-10.7
±
6.0†
† denotes significantly different from normoxia (P < 0.05).
ABSOLUTE WORKLOADS
Muscle O2 Hb (∆µM)
5
†
†
†
†
†
-5
-10
-15
-20
-25
-30
20
†
15
10
10
†
25
†
0
†
†
†
60
90
120 150 180 Peak
Workload (W)
5
0
-5
rest 60
B
8
6
4
2
0
-2
-4
-6
-8
-10
†
-10
rest
A
†
30
Muscle THb (∆µM)
†
Muscle H Hb (∆µM)
10
90
120 150 180 Peak
Workload (W)
rest 60
C
90
120 150 180 Peak
Workload (W)
RELATIVE WORKLOADS
30
-10
-15
†
†
-20
-25
†
†
†
†
†
25
20
15
10
5
0
†
30
40
50 60 70 80 90 100
Workload (%PPO)
E
30 40 50 60 70 80 90 100
Workload (%PPO)
Hypoxia
Figure 4.5 - Muscle oxygenation profile at rest and during exercise
†different (P < 0.05) vs. Normoxia for given intensity
8
4
0
-4
-8
-5
-30
D
12
Muscle THb (∆µM)
-5
Muscle H Hb (∆µM)
Muscle O2 Hb (∆µM)
0
Normoxia
30 40 50 60 70 80 90 100
F
Workload (%PPO)
30
15
25
10
5
0
-5
25
†
†
20
15
†
†
†
†
†
10
5
Cerebral THb (∆µM)
20
Cerebral HHb (∆µM)
Cerebral O2 Hb (∆µM)
ABSOLUTE WORKLOADS
20
15
10
5
-10
0
0
-15
-5
-5
A
rest
60
90
120
150
180
Peak
Workload (W)
rest
B
60
90
120
150
180 Peak
Workload (W)
C
rest
60
90 120 150 180 Peak
Workload (W)
30
15
25
10
5
0
-5
-10
20
15
10
5
0
-5
-15
D
Cerebral THb (∆µM)
20
Cerebral HHb (∆µM)
Cerebral O2 Hb (∆µM)
RELATIVE WORKLOADS
30
40
50
60
70
80
90 100
Workload (%PPO)
E
30 40 50 60 70 80 90 100
Workload (%PPO)
Hypoxia
Figure 4.6 - Cerebral oxygenation profile at rest and during exercise
†different (P < 0.05) vs. Normoxia for given intensity
Normoxia
30
25
20
15
10
5
0
-5
F
30 40 50 60 70 80 90 100
Workload (%PPO)
Figure 4.7 –Oxidative stress biomarkers before and after exercise
3000
200
2500
1500
120
(nM)
3-NT (nM)
2000
(SQRT)
A. (SQRT)
160
80
1000
40
500
A
0
Normoxia
Hypoxia
Before
B
0
Normoxia
Hypoxia
After
Oxidative stress - Figure 4.7 illustrates the effect of maximal exercise in normoxia
and hypoxia on blood markers of oxidative stress; A• (A) and 3-NT (B). There was
no main effect for condition and state (P > 0.05) for either metabolite. There was a
tendency for 3-NT to increase after maximal exercise however there was considerable
individual variability in the response and the difference was not significant (P > 0.05).
60
50
50
Nitrate (µM)
60
40
30
(µM)
NOx (µM)
Figure 4.8 – NO metabolites before and after exercise
40
30
20
20
10
10
0
A
0
Normoxia
Hypoxia
B
Normoxia
NOx
Hypoxia
Nitrate
120
900
100
C
RSNO (nM)
600
450
(nM)
Nitrite (nM)
750
80
60
300
40
150
20
0
0
Hypoxia
Normoxia
Nitrite
D
Normoxia
Hypoxia
RSNO
Before
After
NO metabolites - Figure 4.8 illustrates changes in NO metabolites before and after
maximal exercise in normoxia and hypoxia (A-NOx, B-nitrate, C-nitrite and DRSNO). There were no main effects for workload or inspirate for NOx and nitrate (P
> 0.05). Changes in nitrite showed a main effect for intensity (P < 0.05) but not
inspirate. There was nearly a significant interaction effect for nitrite (P = 0.07) where
the decrease after maximal exercise was greater in normoxia (138 ± 93 nM) compared
to hypoxia (78 ± 92 nM) (P = 0.07). There was an effect size for changes in RSNO for
intensity (P < 0.05) but not condition.
900
750
750
600
450
300
150
450
300
150
0
A
600
(ng/ml)
sICAM-1 (ng/ml)
900
0
B
Hypoxia
Normoxia
Before
Normoxia
Hypoxia
After
CAMs - Figure 4.9 presents changes in sVCAM-1 (A) and sICAM-1 (B) before and
after maximal exercise in normoxia and hypoxia.
There were main effects for
intensity and interaction (P < 0.05) but not inspirate for both sVCAM-1 and sICAM1. Maximal exercise increased sVCAM-1 by 139 ± 41ng/ml in normoxia and 104 ±
49ng/ml in hypoxia. This increase in sVCAM-1 was greater in normoxia (P < 0.05).
The 97 ± 39 ng/ml increase in sICAM-1 after maximal exercise in normoxia was also
greater than the 77 ± 28 ng/ml increase shown in hypoxia (P < 0.05).
Figure 4.10 – Blood pH before and after maximal exercise
7.450
7.400
Before
After
7.350
7.300
H
pH
sVCAM-1 (ng/ml)
Figure 4.9 – sVCAM-1 and sICAM-1 before and after maximal exercise
7.250
7.200
7.150
7.100
Normoxia
Hypoxia
Figure 4.10 demonstrates changes in blood pH before and after maximal exercise in
normoxia and hypoxia. There was an effect size for intensity and interaction and a
main effect for inspirate (P = 0.08). The decline in blood pH in normoxia (-0.162 ±
0.041AU) was greater than the reduction in hypoxia (-0.148 ± 0.066AU) (P < 0.05).
Correlation analysis
Performance - There was no significant correlation observed between changes in
selected biochemical variables (before-after maximal exercise) with V O2peak in
normoxia (nitrite; r2 = -0.22, P > 0.05, sVCAM-1; r2 = 0.29, P > 0.05 and sICAM-1; r2
= 0.24, P > 0.05) and hypoxia (nitrite; r2 = -0.07, P > 0.05, sVCAM-1; r2 = 0.04, P >
0.05 and sICAM-1; r2 = 0.03, P > 0.05). However when normoxia and hypoxia data
were pooled we observed a significant correlation between V O2peak and sVCAM-1
pre-post maximal exercise (r = 0.39, P < 0.05, figure 5.7) whereas the correlation
between V O2peak and nitrite (r2 = -0.36, P = 0.06) and sICAM-1 (r2 = 0.34, P = 0.07)
was approaching significance.
In normoxia, there was a significant correlation between pooled (before and after
exercise) nitrite and PvO2 (r2 = -0.51, P < 0.05). No significant correlations were
observed between pooled before and after exercise biochemical variables (nitrite,
sICAM-1 sVCAM-1) in normoxia. In hypoxia a correlation between pooled before
and after exercise nitrite and sVCAM-1 (r2 = 0.51, P < 0.05) and between sVCAM-1
and SvO2 (r2 = -0.45, p < 0.05) and PvO2 (r2 = 0.44, P < 0.05). Further significant
correlations were observed between sICAM-1 and SvO2 (r2 = -0.45, P < 0.05) and
PvO2 (r2 = -0.44, P < 0.05).
There was a significant correlation when pooled
normoxic and hypoxia data for resting baseline nitrite with the magnitude of decrease
after maximal exercise (r2 = -0.46, P < 0.05, Figure 5.8). This would imply baseline
vascular endothelial measurements are predictors of their bioactivation response to
exercise stress. No significant correlations were observed between basal levels of
sICAM-1 and sVCAM-1 and their subsequent change after maximal exercise.
Figure 4.11 – Correlation between V O2peak versus (pre-post) exercise sVCAM-1
250
r2 = 0.39
P < 0.05
150
100
(ng/ml)
∆sVCAM-1 (ng/ml)
200
50
0
1
2
3
4
5
-50
V O2peak (L/min)
Figure 4.12 – Correlation between baseline nitrite and change pre-post exercise
100
0
200
400
600
800
1000
-100
r2 = -0.46
P < 0.05
-200
(nM)
∆ Nitrite (nM)
0
-300
-400
Baseline nitrite (nM)
4.7
Discussion
The aim of this investigation was to characterise changes in cerebral and muscle
oxygenation during incremental exercise in normoxia and hypoxia. Additionally, the
independent effects of maximal exercise and acute hypoxia on molecular biomarkers
of endothelial function were examined. In summary, the results showed:
1) Despite some individual variability the magnitude of cerebral and muscle
deoxygenation (↓O2Hb and ↑HHb) was greater at rest and throughout incremental
exercise in hypoxia. At rest, the cerebral oxygenation response tended to be more
sensitive to changes in hypoxia than muscle and is consistent with previous reports
(Ainslie et al 2007).
2) The muscle O2 extraction response was estimated through changes in the HHb
signal and has been suggested to be an important factor driving incremental exercise
performance in normoxia (Ferreira et al 2007). In the present study, the rise in HHb
with workload was accelerated in hypoxia and associated increases in relative
metabolic stress. Since the slope for the relative HHb response was similar between
trials whereas the slope was greater across the absolute workloads in hypoxia implies
exercise performance was driven by an early attainment of the ‘plateau’ region in the
HHb profile and O2 extraction capacity of the muscle. A similar observation was
shown for cerebral HHb response although there was no suggestion that cerebral
metabolism was impaired during the hypoxia trial. If central fatigue contributed to
exercise performance in hypoxia, it is more likely that the reduction in cerebral O2
availability or delivery resulted in the impairment in central motor output rather than a
limitation in cerebral aerobic metabolism.
3) During the high intensity workloads in hypoxia there was a tendency for a reduced
and rightward shift in the cerebral THb profile and reduced peak muscle THb
response.
The systemic hypoperfusion response could be related to a blunted
vasodilation or endothelial dysfunction. This finding is consistent with previous
reports in trained cyclists during incremental exercise in hypoxia (Subudhi et al 2007)
and in patients with diabetes with related impairments in haemodynamic
responsiveness to exercise (Mohler et al. 2006).
4) There was no change in plasma biomarkers of oxidative stress (3-NT and A•) after
maximal exercise. This finding contradicts previous observations from our lab using
identical analytical techniques in healthy human subjects during hypoxia exposure at
rest (Bailey et al. 2009) and during exercise (Davison et al. 2006).
5) The total NO metabolite pool was unaffected by maximal exercise. However
nitrite decreased suggesting the rate of nitrite consumption (or oxidative inactivation?)
occurs at a greater rate than it is produced via eNOS bioactivation. The attenuated
decline of nitrite in hypoxia could be the result of a blunted nitrite reductase activity
of HHb evoked by the severity of hypoxaemia. This could in turn be responsible for
the systemic hypoperfusion response. In contrast, RSNO was elevated after maximal
exercise and could signify a partial reapportionment of active vascular NO
metabolites and mediated primarily through the systemic stress of exercise.
6) sICAM-1 and sVCAM-1 increased after maximal exercise. The response mirrored
the changes shown for nitrite since the magnitude of rise was greater in normoxia.
Subsequently pooled pre and post exercise data showed a correlation between nitrite
and sVCAM-1 in hypoxia.
7) No relationships were observed between molecular biomarkers of endothelial
function with V O2peak when the normoxia and hypoxia tests were examined
independently. However when normoxia and hypoxia data were pooled there were
correlations between selected blood markers pre-post exercise and V O2peak.
Muscle oxygenation
In the present study, muscle oxygenation was measured during incremental exercise
in normoxia and acute hypoxia and the dynamic response was characterised in
relation to relative and absolute workload profiles. Typically only venous blood is
deoxygenated in normoxia however since arterial and venous blood compartments are
deoxygenated in hypoxia our results showing exercise in hypoxia aggravated a greater
muscle deoxygenation response would have been expected. Previous studies have
yielded inconsistent findings where some have shown an elevated muscle
deoxygenation during hypoxic exercise compared to normoxia (Subudhi et al. 2007;
Costes et al. 1996) whereas others have reported no difference when performing
single leg extension exercise in 12% O2 (DeLorey et al. 2004), ankle extension
exercise in 11% O2 (Rupp and Perry 2009) and cycling exercise in 14% O2 (Ainslie et
al. 2007). Furthermore, Bourdillion et al. (2009) showed that compared to sea-level
exercise at altitudes up to 4,000m had no effect on muscle oxygenation in untrained
male subjects, however there was a greater deoxygenation response at altitudes above
2,500m in trained athletes which the authors related to a greater participation of
arterial blood to the NIRS signal. Regional changes across distinct muscle groups can
also be affected differently by exercise in hypoxia. Heubert et al. (2004) showed no
difference between normoxia and 16% O2 in vastus lateralis oxygenation during
steady state cycling exercise whereas there was a greater deoxygenation response in
the lateral gastrocnemius muscle in hypoxia.
There was some inter-subject variability in the magnitude of the NIRS signal making
it impossible to compare between subjects. This can be attributed to the heterogeneity
of human tissue and individual differences in subcutaneous fat, blood and skeletal
muscle composition in addition to differences in muscle recruitment and activation
patterns during exercise. We acknowledge the considerable individual SaO2 variation
to resting exposure and exercise in hypoxia (Benoit et al. 1995) although there was no
indication from our data to suggest individual differences in muscle oxygenation are
accounted for by the variability in SaO2. Despite these differences, a common muscle
oxygenation pattern was observed between individuals irrespective of condition.
The 22% reduction of V O2peak in hypoxia signifies an inability to maintain the
required ATP production levels via aerobic mechanisms. The ventilation/perfusion
mismatch and reduction in O2 supply is largely responsible for the reduction in
V O2peak through an earlier onset of lactate threshold and accelerated accumulation of
peripheral fatigue by-products such as hydrogen ions and inorganic phosphates
(Hogan et al. 1999).
Although maximal muscle O2 extraction capacity remains
unaffected by hypoxia (Lundby et al. 2006; Richardson et al. 1999). The HHb signal
reflects an imbalance between O2 delivery and utilisation and can be used as a
surrogate measure of tissue deoxygenation through increased O2 extraction (Ferreira
et al. 2007; DeLory et al. 2003).
Although absolute changes in fractional O2
extraction values are unobtainable using NIRS, it has been shown that during maximal
exercise fractional O2 extraction remains at ~90% in low altitude natives during
maximal cycling exercise at sea-level and following acute and chronic hypoxia
exposure (Lundby et al. 2006). Our study assessed if an altered muscle HHb profile
presents a performance limitation during incremental exercise in hypoxia. In both
trials, attainment of V O2peak was preceded by a reduced rate of increase in the HHb
signal as workload approached PPO which has been interpreted as evidence of
maximal muscle O2 extraction and V O2max (Subudhi et al 2007; Esaki et al 2005).
Since the magnitude of the HHb response after the onset of exercise to PPO
([HHb60-PPO] and [HHb30-100%]) was similar between trials suggests the reduction in
V O2peak was driven by an upward shift in the HHb profile thus for a given change in
workload, the change in HHb was greater in hypoxia.
The increase in relative
metabolic stress was critical to hypoxic performance and premature attainment of
maximal muscle O2 extraction capacity whereas enhanced O2 supply can result in
slower HHb kinetics (Jones et al. 2006). In particular, the transition from rest to
exercise contributed significantly to the response highlighting an augmented
pulmonary diffusion limitation when exercise and hypoxia are combined.
A visual inspection of the HHb profile revealed a tendency for a sigmoidal pattern in
normoxia supporting observations from others (Ferreira et al. 2007; Boone et al
2009). Ferreira et al. (2007) suggested the sigmoidal pattern represents the non-linear
relationship between muscle blood flow (and O2 delivery) and O2 uptake whereby
during the low-moderate intensity workloads muscle blood flow increased at a faster
rate than it is taken up which then slows progressively as exercise intensity
approaches PPO. Our results presented a hyperbolic pattern in hypoxia due to the
increased O2 extraction rate from the onset of exercise until PPO.
A precise
mechanistic explanation for these findings is unclear due to the unknown
contributions of Hb and Mb to the overall NIRS signal. However an accelerated
myoglobin desaturation at 50-60% maximum work rate was reported by Richardson et
al. 2001 and could explain the accelerated rise in HHb at near identical workloads in
the present study. Further studies are needed to elucidate the relative contribution of
Mb and Hb to the overall NIRS signal during incremental cycling exercise in
normoxia and hypoxia. Although O2Hb and HHb exist in equilibrium, the different
profiles relate to the sensitivity of O2Hb signal to changes in THb whereas the HHb
remains largely unaffected by volumetric changes (Grassi et al. 2003). During low
intensity exercise in normoxia, V O2 is maintained through adequate intracellular pO2
levels without widening the RBC to mitochondria PO2 gradient (Richardson et al.
1999 & 2001). The initiating event of increased O2 extraction rate in normoxia
appears when intracellular or capillary PO2 levels, the driving force for muscle O2
diffusion, reaches its critical minimum value typically at anaerobic threshold or 50%
PPO (Richardson et al 2001; Stringer et al. 1994; Grassi et al. 2003 & 1999; Chaung
et al. 2002). The event causes a rightward shift the ODC due to the Bohr Effect
facilitating O2 release from O2Hb and subsequent uptake by mitochondria.
The elevation in muscle THb during exercise can be caused by a combination of
factors such as increased cardiac output and redistribution of blood to the active
muscle region, capillary recruitment as well as local vasodilator factors resulting in an
increased volumetric fraction of the capillary blood to the NIRS signal (Lai et al.
2009). Mortensen et al. (2005) showed a leveling off or decline in muscle blood flow
during incremental exercise in normoxia at workloads >80% PPO and could explain
why THb peaked at equivalent workloads in the present study. The ∆THb/∆WL slope
was reduced in hypoxia due to a hyper-perfusion response during low intensity
workloads whereas during the high intensity exercise THb tended to be lower
compared to normoxia. The enhanced THb response during low intensity exercise
could represent a compensatory mechanism that counteracts the reduction in CaO2
through activation of local O2 sensitive vasodilator factors and therefore increasing
leg blood flow and O2 delivery. The decreased maximal THb in hypoxia could be
related to a reduction in central perfusion pressure by a lowering of maximal HR and
cardiac output (Lundby et al 2006). Alternatively, a blunted vasodilation could have
impaired blood volume expansion through inactivation of O2-sensitive vasodilator
factors such as NO (Shibasaki et al. 2008) and ATP (Lundby et al 2008; GonzalezAlonso et al 2001) due to the increased oxidative stress associated with hypoxic
exercise (Bailey et al. 2001; Davison et al. 2006) and secondary to potential
impairments in endothelial function. Alternatively a greater decline in PaCO2 and
associated vasoconstriction could also have blunted the THb response during exercise
in hypoxia (Chin et al. 2007).
Exercise induced hypoxia causes a metabolic
attenuation of muscle sympathetic vasoconstriction (Hansen et al. 2000) however
since
sympathetic
vasoconstriction
facilitates
O2
transfer
in
the
muscle
microcirculation during exercise in hypoxia (Lundby et al. 2008) the reduction in THb
could favour O2 uptake by matching O2 delivery with demand.
Cerebral oxygenation
SaO2 dropped to 96% and 78% at the point of volitional exhaustion in 21 and 12% O2
conditions respectively. There was considerable individual variability in the reduction
of SaO2 at PPO in hypoxia ranging from 71 to 84%. Central motor activation can be
directly affected by reductions in O2 delivery whereby an estimated 6-7mmHg decline
in cerebral mitochondrial pO2 or 15% decrease in cerebral O2 delivery was associated
with impaired maximal handgrip performance (Rasmussen et al. 2007). In contrast
muscle functionality is maintained despite similar reductions in O2 availability
highlighting a reduced tolerance of cerebral tissue to hypoxia. This is due to a lack of
capillary recruitment in the cerebral circulation which increases the diffusion distance
for O2 (Secher et al. 2008). The critical threshold for hypoxia where central motor
activation is impaired may be reduced further by prolonged dynamic exercise and
when recruiting a larger muscle mass such as that experienced during cycling
exercise. Amann et al. (2006) showed that compared to normoxia and moderate
hypoxia (15% O2), exhaustion during constant load cycling in 10% O2 occurred in the
absence of significant peripheral quadriceps fatigue estimated by the change in force
output pre- versus post-exercise response to supra-maximal magnetic femoral nerve
stimulation. However exercise time to exhaustion was prolonged only in 10% O2 after
switching the inspiratory circuit to a hyperoxic gas mixture at the point of task failure.
Similar observations were shown during incremental cycling exercise in 4,300m
conditions despite slight improvements in muscle oxygenation (Subudhi et al. 2008).
The authors related their findings to the critical but irreversible effects of peripheral
fatigue whereas the improved performance in severe hypoxia was related to the rapid
reversibility of O2-sensitive central fatigue. A threshold SaO2 of ~75% was proposed
where fatigue can switch, albeit not exclusively, from one which is predominantly
peripheral in origin to one of central origin (Amann et al. 2007). Since this value falls
within the SaO2 range of the present study it is likely O2-sensitive central fatigue
would have impaired incremental exercise performance in hypoxia.
The precise mechanisms mediating O2-senisitive central fatigue is not fully
understood. The CNS receives directly and indirectly inhibitory stimuli that can
impair central motor activation and resonate from inhibitory group III and IV muscle
afferents. These can be triggered by the accelerated accumulation and stimulatory
affects of muscle fatigue metabolites in hypoxia however blockade of group III and
IV muscle afferents did not affect exercise time to exhaustion in extreme hypoxia
(Kjaer et al. 1999).
Rather it has been suggested that hypoxia can blunt the
responsiveness of type III/IV fibres to fatiguing stimuli leaving an inability to detect
metabolic changes in the muscle during acute and chronic hypoxia (Arogast et al.
2000; Dousset et al. 2001 & 2003). Other factors impairing motor neuron excitability
have been proposed such as IL-6 release or altered ion channel function through direct
activation
of
O2-sensitive
brain
neurotransmitters
for
example
dopamine,
noradrenaline and serotonin which are all associated with feelings of tiredness,
motivation and lethargy (Secher et al. 2008). The rapid reversibility of central fatigue
when cerebral oxygenation is restored lends support the neurotransmitter theory.
One of the main objectives of this investigation was to characterise the cerebral
oxygenation response to incremental exercise in hypoxia and normoxia. It is unlikely
that cerebral deoxygenation limited exercise performance in normoxia since
theoretically subjects would have not been able to start exercising in hypoxia at the
equivalent level of deoxygenation shown at PPO in normoxia. In contrast to other
reports (Subudhi et al. 2007; Thomas and Stephane 2008), we did not observe a
‘critical’ reduction in cerebral O2Hb prior to exhaustion. Our data showed a plateau
in O2Hb at workloads >90%PPO with near identical patterns between conditions.
After an initial plateau phase we observed an increase in cerebral HHb at workloads
exceeding 60-70% PPO in both tests. The response signifies a compensatory increase
in O2 extraction to sustain cerebral aerobic metabolism when CBF and O2 delivery
becomes blunted at workloads greater than ventilatory threshold and associated
hypocapnic induced vasoconstriction (Bhambhani et al. 2007; Gonzalez-Alonso et al.
2004; Subudhi et al. 2008). Thus cerebral O2 extraction increased when exercise
intensity matches the associated metabolic stress irrespective of condition and HHb
tended to plateau as PPO was approached. Therefore there is no clear evidence to
suggest brain O2 extraction capacity was impaired in hypoxia however further
research is required to confirm this. A cerebral O2 extraction fraction of 50% has
been documented during maximal exercise and fatigue was associated with enhanced
rather than impaired cerebral uptake of O2 in normoxia (Gonzalez-Alonso et al 2004)
and moderate hypoxia (Volianitis et al 2008). The reduction in CBF prevents overperfusion in the brain and the greater O2 reserve across cerebral circulation compared
to the 90% extraction fraction in muscle creates an important protective mechanism
preserving cerebral homeostasis and function when O2 supply is compromised.
It has been suggested there is a ‘steal’ of blood from the cerebral circulation in order
to meet the metabolic demand of the muscle/lung/heart (Nielsen et al. 1999). Our
results showed cerebral THb increased continuously throughout incremental exercise
therefore changes in THb is not entirely related to PaCO2 and subsequent
vasoconstriction after ventilatory threshold is reached although there was a tendency
for a rightward shift in the cerebral THb profile in hypoxia. Similar observations
were reported by Subudhi et al (2007) at workloads greater than 75% PPO. Changes
in PaCO2 is regarded as the most sensitive influence of cerebral blood flow during
normoxic exercise and hypoxia exposure at rest (Ainslie and Poulin 2004; Rasmussen
et al. 2006). However despite a marked reduction in PETCO2 during steady state
cycling in hypoxia, compensatory increases in neurogenic activity and/or
sympathoexcitation maintained the elevation in cerebral blood flow and THb
compared to normoxia (Ainslie et al. 2007). It is unclear if these mechanisms persist
during intense exercise particularly since maximal heart rate and cardiac output is
reduced in hypoxia.
Subsequently the drop in perfusion pressure and increased
cerebrovascular resistance during intense exercise in hypoxia could adversely affect
the rise in cerebral THb (Imray et al. 2005; Seifert et al. 2009; Ide et al. 1998). It also
possible the rightward shift in the cerebral THb profile in hypoxia in addition to the
reduction in peak muscle THb could result from the severity of hypoxia and/or
oxidative inactivation of vasodilator factors such as NO. This is described in more
detail in the NO metabolite part of the discussion section.
Oxidative stress
EPR provides a direct measure of oxidative stress in human blood (Buettner &
Jurkiewicz 1993; Bailey et al. 2004). In the present study there was no change in
ascorbate free radical production after maximal exercise. Previously, increased
ascorbate free radical generation was promoted by maximal exercise in normoxia
using this detection technique (Davison et al. 2002; Bailey et al 2003). Combined
exercise and hypoxia resulted in enhanced free radical output (Davison et al. 2006;
Bailey et al. 2003 & 2004) whilst the elevation was attenuated by regular hypoxic
compared to normoxic exercise training (Bailey et al. 2001). These results suggest
hypoxic exercise is a more effective stimulus for eliciting free radical production and
is a necessity for promoting molecular adaptation although it is unclear why we
showed no change after normoxic and hypoxic exercise.
A compensatory response to increased superoxide anions generation during exercise
is an elevated superoxide dismutase activity (Lawson et al. 1997). SOD activity is
also known to catalyze the reaction between NO and superoxide anion to produce the
highly potent oxidant peroxynitrite before giving rise to nitrated species such as 3-NT
making it a reliable marker of NO-dependant oxidative stress (Beckman & Koppenol
1996; Pacher et al. 2007). We observed considerable individual variability in basal 3NT concentrations using an ELISA detection method with levels ranging from
undetectable up to 330nM although the values were consistent across each individual.
There was a tendency for 3-NT to increase after maximal exercise. This observation
is supported by others during incremental treadmill exercise to exhaustion using a
high sensitivity HPLC method with a detection limit of 0.2µM (Faturous et al. 2004).
Results from Faturous et al. (2004) showed basal 3-NT levels of ~5µM before
increasing to ~8µM after exhaustive exercise which differs considerably from the
values shown in the present study. The detection of 3-NT using ELISA has been
previously validated (Khan et al. 1998) and the lowest detection limit of our kit was
2nM. Others have reported basal 3-NT levels of 2nM using GC/MS/MS detection
technique (Tsikas et al. 2002) which represents the lowest detection limit from our
ELISA kit. Previous studies from our lab have found increase cerebral net output of
3-NT during prolonged hypoxic exposure using the ELISA detection technique
(Bailey et al. 2009). Therefore we are confident the measurement reflects an actual
change in nitrative stress in the present study. The ELISA technique may lack the
sensitivity and specificity of the GC/MS/MS method thus validation of our findings
using this technique is recommended.
NO metabolites
The total NOx storage pool remained unchanged afetr maximal exercise. Increases in
NOx have been documented after exercise (Allen et al. 2005; Yang et al. 2007).
Since NOx consists predominantly of nitrate and this metabolite is regarded as inert
during forearm reactive hyperaemia (Lauer et al. 2001) it is unlikely to exert any
regulatory function in the microcirculation during intense exercise (Rassaf et al 2007;
Allen et al 2009). Nitrate represents a stable end product of chronic eNOS activity via
the reaction between nitrite or NO and O2Hb before it redistributed back into the
plasma (Kelm 1999). A novel conversion pathway of nitrate back to nitrite or NO
under physiological conditions has yet to be determined and it is possible the large
background dosages of nitrate could mask any potential favorable effects.
Exercise is generally associated with elevations in shear stress induced eNOS
activation for NO production (Tschakovsky and Joyner 2008; Koller & Kaley 1991).
Plasma nitrite is the primary oxidative NO metabolite in human blood of which ~7090% is derived from eNOS activity (Kleinbongard et al. 2003 & 2006).
The
decreased nitrite after maximal exercise implies its rate of removal occurs at a greater
rate than it is produced. As an essential mediator of vascular function and O2 sensing
(van Faassen et al. 2009), the nitrite-HHb-NO pathway may become critical during
maximal exercise in order to maintain blood flow and O2 delivery when the
endothelium may otherwise become damaged or dysfunctional due to mechanical
injury and eNOS derived NO production may be impaired through oxidative
inactivation and associated metabolic stress.
In particular, the nitrite reductase
activity of deoxyhaemoglobin becomes a potent source of NO release when O2
availability becomes limited as a function of exercise or inspiratory hypoxia (Maher et
al. 2008; Cosby et al. 2003). Thus there are a few mechanistic explanations which
could be responsible for the decrease in nitrite although is likely to involve the
combination of several regulating factors.
Gladwin et al. (2000) classically
demonstrated nitrite (consumption) becomes the major substrate source for NO
generation during forearm exercise in normoxia and the local biochemical
environment determined nitrite-NO induced vasodilation. Optimal conditions for
nitrite reductase activity as a NO generator range from PO2 pressures of 20-40mmHg
and hemoglobin saturations of 40-60% (Gladwin et al. 2004; Isbell et al. 2007;
Crawford et al. 2006). At maximal exercise SvO2 was 31% in hypoxia versus 58% in
normoxia in the present study therefore it is likely nitrite reductase activity of HHb or
its uptake by the erythrocyte could have been impaired by the severity of hypoxaemia
and independently of the effects exercise. This would support our observations of a
systemic hypo-perfusion response during exercise in hypoxia. It is unlikely that
enhanced eNOS activation would increase nitrite production during the hypoxia trial
since O2 is an essential cofactor for its bioactivation (McQuillan et al. 1994).
Furthermore, hypoxia can independently lead to free radical generation which would
further aggravate the oxidative inactivation of eNOS (Steiner et al. 2002). Although
there was no evidence of oxidative stress after exercise in the present study, there is
evidence showing a regulatory role for ROS on microvascular O2 sensing and blood
flow control (Wolin et al. 2005).
Although the concentration of nitrite in RBCs is 5 times greater than plasma (Dejam
et al. 2004), under stressful conditions nitrite may be actively removed from the
plasma by the erythrocyte through a sodium-dependant phosphate transporter (May et
al. 2000). Enzymatic activity of this pathway increases with temperature, hypoxia and
acidic conditions and in the presence of high levels of superoxide dismutase therefore
intense exercise presents an ideal biochemical environment for nitrite plasma-RBC
transfer. A partial replenishment of nitrite from the RBC is unlikely to explain the
attenuated decline in hypoxia since the anion exchanger regulating nitrite transport
across the erythrocyte membrane inhibits its export under low O2 tensions (Vitturi et
al 2009).
However RSNO export from the erythrocyte may be enhanced by
deoxyhaemoglobin (Cosby et al 2003) although the precise role of RSNO is unclear
due to its small concentrations in human blood. RSNO concentrations rise during
inflammation reactions leading to the formation of peroxynitrite (Trujillo et al 1998;
Glantzounis et al. 2007) and could explain the increase in RSNO and 3-NT after
maximal exercise.
It is also possible exercise could have initiated a partial
reapportionment between NO species across the vascular NO storage compartments in
order to preserve the total NO pool. An increase in Hb bound NO was matched by
decreases in plasma nitrite along the physiological O2 gradient in the pulmonary and
coronary circulation (Rogers et al. 2007). Therefore Hb may function as a nitrite
buffer until local conditions signal for NO release via RBC-NO intermediates
(Stamler et al. 1996). Future studies examining changes in NO species across plasma
and RBC compartments during exercise are recommended.
We are confident the decrease in nitrite is representative of the changes induced by
lower limb exercise although we refer to the changes as ‘systemic’ because blood
samples were obtained from a forearm vein.
Subsequently, our subjects were
instructed not to grip onto the handle bars of the cycle ergometer to avoid the potential
confounding influence of forearm ischemia. The present study contrasts two other
reports during maximal exercise.
Recently, Allen et al. (2009) documented an
increase in nitrite after maximal exercise in subjects with low cardiovascular risk
factors. This compared to the decreased nitrite observed in those with fully diagnosed
peripheral arterial disease and the change in nitrite after exercise was related to the
degree of endothelial dysfunction. Similarly, Rassaf et al. (2007) showed an increase
in nitrite after maximal exercise in healthy male subjects and was predictive of
aerobic capacity. The present study showed no relationship between nitrite levels and
V O2peak in normoxia and hypoxia however when pooled normoxic and hypoxic data
was presented there was a moderate correlation. The differences between the present
study and the aforementioned could be related to the timing of the post-exercise blood
sample where samples were taken 10 minutes after termination of maximal exercise.
This in turn could be reflective of a post-exercise hyperemic response where
haemodynamic control is likely to differ from that immediately after the test. Larsen
et al. (2009) also reported a decrease in nitrite one-minute after maximal exercise was
performed before returning to pre-exercise values within 30 minutes of rest. The
decrease was interpreted as a greater rate of nitrite consumption compared to the rate
of NO oxidation back to nitrite. Future studies examining the kinetic nitrite response
during and after exercise at different intensities would determine if the response is
driven by a dose dependant relationship.
A constant infusion of small doses of nitrite would realise the physiological
significance of a partially depleted reserve during exercise. Nitrite infusion studies
have shown increase in cerebral blood flow, reduced blood pressure (Rifkind et al.
2007), decreased organ I-R injury in rats (Duranski et al. 2005; Webb et al. 2002) and
increased forearm arterial blood flow by up to 50% in humans during hypoxic
exposure but not in normoxia (Pinder et al. 2008). Dietary supplementation of nitrate
profoundly increased plasma nitrite levels during incremental exercise leading to a
reduction in submaximal (Larsen et al. 2007) and V O2max although exercise time to
exhaustion tended to improve (Larsen et al. 2009). Similar observations were shown
with direct ATP infusion by lowering muscle O2 extraction (Lundby et al 2008) which
could suggest during maximal exercise some vasoconstriction or reduced vasodilation
is needed to match O2 delivery with demand. However the reduction in nitrite may be
negligible in terms of its vasodilation effects since only small (~5-20nM) amounts of
NO is required to elicit vasodilation (Beckman & Koppenol 1996; Bellamy et al
2000).
Cell adhesion molecules
In the present study, maximal exercise provoked an increase in sICAM-1 and
sVCAM-1. The main function of CAM is to reduce the speed of circulating
leukocytes, drive the process of firm attachment and support the trans-endothelial
migration of immune cells to the inflamed region (Frijns & Kappelle 2002). Surface
expression of CAMs on leukocytes and endothelial cells are released into the blood
by enzymatic cleavage or shedding (Brevetti et al. 2001) and it is assumed that
circulating sVCAM-1 and sICAM-1 reflect their expression on vascular cells
(Leeuwenberg et al. 1992) and degree of endothelial damage and activation
(Holmlund et al. 2002). There is good evidence showing CAM expression and release
into peripheral circulation increases after intense exercise (Monchanin et al.
2006;Akimoto et al. 2002;Shepard 2003) before returning to baseline after 1 hour
recovery (Rehman et al. 1997). Expression of ICAM-1 and VCAM-1 is mediated by
a series of signaling factors affected by exercise including shear stress (Morigi et al
1995), sympathetic activity (Rehman et al. 1997), pro-inflammatory mediators such as
TNF-α (Chiu et al. 2004), increased oxidative stress (Silvestro et al. 2002) and NO
bioavailability (Dal Secco et al. 2006).
However the magnitude of endothelial
activation appears dependent on exercise intensity since there was no change in blood
lymphocyte expression of adhesion/activation molecules following moderate intensity
exercise (Simpson et al. 2006).
Our finding that the CAM response was attenuated in hypoxia was unexpected. Since
there was a systemic hypoperfusion response during exercise in hypoxia, it is possible
the reduction in blood volume could have limited the total surface area for shear stress
induced endothelial activation and CAM release. Alternatively, in vivo studies show
a reduction in PO2 causes an increase in leukocyte adherence (Arnould et al. 1993;
Shreeniwas et al. 1992). It is therefore possible the attenuated increase in circulating
CAMs could reflect a more efficient leukocyte binding in hypoxia. The magnitude of
leukocyte adherence in hypoxia is related to several factors such as the severity of
hypoxia, attainment of a threshold level of oxidative stress, balance between ROS
generation and NO availability and is independent of changes in shear stress induced
activation (Wood et al. 1999a; Steiner et al. 2002). All of these factors could have
influenced CAM release during the hypoxia trial.
The CAM response reflected a mirror image of the nitrite response and there was a
moderate correlation between pooled pre and post exercise sVCAM-1 and nitrite data
in hypoxia.
Decreased nitrite levels is associated with endothelial dysfunction
(Kleinbongard et al. 2006) whereas increased levels have been shown to exert
protective effects in models of ischemia/reperfusion injury (Duranski et al. 2005;
Webb et al. 2004). NO can directly inhibit cytokine induced endothelial activation,
leukocyte adherence and expression of ICAM-1 and VCAM-1 in a concentration
dependant manner (Berendji-Grun et al. 2001; Lindemann et al. 2000; De Caterina et
al. 1995).
Subsequently, administration of a NO donor attenuated the hypoxia
induced leukocyte adherence in rat mesenteric venules in non-acclimatised but not in
acclimatised rats (Wood et al 1999b). The impairment in FMD and increase in
sICAM-1 after maximal exercise was abolished by vitamin C administration in
patients with intermittent claudication giving further evidence of direct interplay
between endothelial activation, NO availability and oxidative stress (Silvestro et al.
2002).
Increasing nitrite availability through dietary intervention in hyper-
cholesterolemic mice inhibited leukocyte adhesion and emigration (Stokes et al.
2009). Whether a similar intervention is as effective in reducing the CAM response to
maximal exercise is yet to be determined.
4.8
Conclusions
In summary, our results showed the magnitude of cerebral and muscle deoxygenation
was greater at rest and throughout incremental cycling exercise in hypoxia. The
oxygenation profile in both tissues was related to changes in the relative metabolic
stress. Our results suggest attainment of V O2peak was driven by the pattern of the
muscle O2 extraction response and slope of the linear regression analysis between the
muscle HHb versus workload. It is unlikely the magnitude cerebral deoxygenation
affected performance in normoxia however may have limited incremental exercise
performance in hypoxia. The HHb profile from cerebral and muscle gave some
insight into the metabolic changes that occur during exercise. Both showed a reduced
rate of increase or tendancy for a plateau in HHb in the workloads preceding exercise
exhaustion indicating attainment of maximal O2 extraction capacity and an
accelerated increase in the HHb response can account for the decrease in V O2peak in
hypoxia. It is possible at PPO the decline in SaO2 in hypoxia influenced exercise
performance directly by O2-sensitive central fatigue and associated impairment in
central motor output.
We observed a systemic hypo-perfusion response (↓THb) in both tissues during high
intensity exercise in hypoxia. The present study also sought to determine the effects
of maximal exercise on molecular blood-borne markers of vascular endothelial
function and O2 sensing. The reduction in nitrite implies the mechanisms which lead
to a reduction in its bioavailability i.e. consumption through nitrite-HHb-NO
bioactivation and/or oxidative inactivation outbalance its production by eNOS
activity. It is possible the hypo-perfusion response can be explained by the ‘steal’
phenomenon, a reduced PCO2 and/or blunted activation of the nitrite-HHb-NO
pathway due to the severity of hypoxaemia since the decline in nitrite after maximal
exercise was attenuated in hypoxia. The reduction in nitrite after maximal exercise
was mirrored by the increased in CAM. The precise mechanistic interpretation for
this is unclear but could signify the anti-inflammatory effects of enhanced vascular
nitrite availability or could be due to the direct effects of hypoxia and more effective
leukocyte-endothelial interaction. The decline in nitrite and increased RSNO after
maximal exercise appears to be driven by the systemic stress of exercise or signifies a
partial reapportionment between NO species across the vascular NO storage
compartments. We showed no relationship between any blood metabolite and aerobic
performance in normoxia or hypoxia however when normoxia and hypoxia data were
pooled we reported a moderate relationship between selected biomarkers of vascular
function and V O2peak. An examination of interventions to enhance O2 delivery and
utilisation and improve NO regulation during exercise in hypoxia such as adaptation
to intermittent hypoxia is recommended.
Chapter 5
Implications for intermittent hypoxia on cerebral and muscle oxygenation and
molecular biomarkers of vascular function during exercise in hypoxia
5.1
Introduction
Intermittent hypoxia is used by athletes to enhance the efficiency and tolerance to
exercise training, in mountaineers as a pre-acclimatisation strategy prior to high
altitude ascent and for the prevention and treatment of various clinical conditions
(Serebrovskaya 2002; Manukhina et al. 2006; Benardi 2001). IH has been shown to
enhance ventilatory chemosensitivity and cerebrovascular responsiveness at rest
(Foster et al. 2005) and during exercise (Katayama et al. 2001) in hypoxia, reduce the
incidence of acute mountain sickness (Beidleman et al. 2004) and improve resistance
to ischemic injury through NO-dependant mechanisms (Kaminski et al. 2007).
Additionally IH at rest can improve aerobic performance in normoxia and hypoxia by
a variety of mechanisms that enhance O2 transport capacity and utilisation such as
increased red cell mass (Levine et al. 1997), increased SaO2 (Beidleman et al. 2008)
and pulmonary ventilation (Wang et al. 2007) as well as induce adaptation at the
muscle level through enhanced exercise efficiency (Katayama et al. 2004) and
buffering capacity (Gore et al. 2001).
However the effects of IH on exercise
performance remains a controversial area of research (Levine 2002).
Short duration IH consists of repeated exposures of breathing low O2 interspersed
with equal periods of normoxia breathing. Each exposure can range from 9 to 12%
O2 and lasts for 3-7 minutes in duration (Marshall et al. 2007; Bernardi 2001;
Burtscher et al. 2004). The adaptive pathway can differ depending on the pattern of
hypoxia-reoxygenation and compared to continuous hypoxic exposure, IH has the
advantage of exposing subjects to a more intense hypoxic stimulus.
Vascular
adaptation to IH appears to be driven through a more effective release and storage of
NO (Manukhina et al 2006). The cyclic activation of O2 sensitive pathways leads to
repeated production of NO and ROS.
These are important signaling molecules
driving adaptation to IH since the addition of a potent scavenger of superoxide ions
inhibited the O2 sensing ability of carotid bodies in rats (Peng & Prabhakar 2004)
whilst NO inhibition prevented the protective effects of hypoxic preconditioning
(Beguin et al. 2005; Bertuglia 2008). The severity, duration and intermittence of the
hypoxic exposure is critical and will determine if there is sufficient biochemical
stimulus to elicit an adaptive response or be detrimental to vascular function (Steiner
et al 2002; Beguin et al 2005). A chronic overproduction of ROS can lead to a
preferential activation of inflammatory over adaptive pathways and development of
endothelial dysfunction in healthy humans (Wang et al. 2007) and is characterised
within the pathology of sleep apnoea patients (Ryan and McNicholas 2008).
It is possible IH could lead to a recalibration of O2 sensing mechanisms by a more
efficient and effective regulation of ROS and activation of eNOS and/or the nitriteHHb-NO pathway during exercise. In our previous study we related the decrement in
V O2peak in hypoxia to the magnitude of decrease in cerebral and muscle oxygenation
and an accelerated HHb kinetics. This could have been driven by the severity of
hypoxaemia, a blunted activation/sensitivity of the nitrite-HHb-NO pathway or
inactivation of eNOS activity resulting in a tendency for a systemic hypoperfusion
response. An improved endothelial function and integrity of the vascular system to
deliver O2 to the brain and muscle during exercise could ultimately improve aerobic
performance in hypoxia through a combination of central and peripheral fatigue
mechanisms.
5.2
Research aims and objectives
There are currently no studies examining the effect of IH on incremental exercise
performance in hypoxia. The main aims of this investigation are:
1) To evaluate the effect of IH regime on exercise performance in hypoxia.
2) To examine the effect of IH on the cerebral and muscle oxygenation response to
exercise.
3) 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
5.3 Working hypothesis
“Intermittent hypoxia will enhance cerebral and muscle oxygenation during
incremental exercise in hypoxia.
This will be due to an improved systemic
(SaO2) and regional O2 delivery. Endothelial function will also be improved by
IH estimated though increased nitrite bioavailability at maximal exercise and as
a direct consequence the rise in CAM will be also be attenuated. This will lead to
an attenuated development of central and peripheral fatigue and ultimately
result in an increased V O2peak in hypoxia.”
Intermittent Hypoxia
Exercise + Hypoxia
Endothelial
function
Regional
Oxygenation
SaO2
NO
Cerebral oxygenation
CAM
Muscle oxygenation
V O2peak
Increase or
decrease
compared to control group
Figure 5.1 - Working hypothesis for study 2
5.4
Methodology
Subjects
Eighteen apparently healthy male volunteers participated in this study and were
randomly assigned to one of 2 groups; IH (n = 9) or control (n = 9). Subject
characteristics are presented in Table 5.1. All experimental procedures and protocols
were approved by the Human Ethics committee of the University of Glamorgan.
Subjects provided written informed consent prior to participating in the investigation.
All subjects were sea-level residents and had not spent any time at altitude in the 6
months prior to the study. All subjects were non-smokers, physically active with no
known physician diagnosed illnesses prior to participation.
Each subject was
requested to abstain from any antioxidant or anti-inflammatory supplements and
medication over the course of the study.
The requirements of the study were
explained in full. Each subject made a preliminary visit to the laboratory which
served as a familiarisation session to laboratory equipment and experimental
procedures and a full simulated experimental exercise trial was conducted.
Table 5.1 - Subject characteristics
IH
Control
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
45 ± 2
47 ± 2
Hct (%)
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 V O2, V CO2 and V E was measured offline 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.
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 hypoxicreoxygenation 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.
110
SaO2 (%)
100
Hypoxia
90
Normoxia
80
70
60
50
1
2
3
4
5
6
7
8
9
10
Days of IH
Cardiorespiratory measurements
The V O2 data are presented on figure 5.4. After IH, there was a tendency for V O2peak
to increase whilst V 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
V CO2peak after IH (P < 0.05). During the 60W workload V E/VO2 increased by 1.0 ±
0.9 L/min in the IH group (P < 0.01) and was related the decrease in V 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 V O2
3.5
VO2 (L/min)
3.0
2.5
Pre IH/IN
2.0
Post IH/IN
1.5
1.0
0.5
0.0
60W
PPO
60W
IH
PPO
IN
There was a main effect for intensity and time x intensity x group. After IH, there was
a 3.5% decrease in V O2 during the 60W workload (P = 0.06) and 2.3% increase in
V O2peak (0.07).
Figure 5.5 – Effect of IH on SaO2 during incremental exercise
95
85
80
(%)
SaO2 (%)
90
75
70
Rest
60
90
120
150
180
Max
Rest
Workload (W)
A
60
90
120
150
180
Max
Workload (W)
B
Pre IH
Post IH
Pre-IN
Post-IN
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
250
150
(b.p.m)
Heart rate (b.p.m)
200
100
50
0
Rest
A
60
90
120
150
180
Max
Rest
90
120
150
180
Max
Workload (W)
Workload (W)
Pre IH
60
Post IH
B
Pre IN
Post IN
There were no main effects for heart rate during incremental exercise after IH and IN.
Intermittent Hypoxia
GROUP
Before
TIME
Control
After
Before
After
INTENSITY
60W
Max
60W
Max
60W
Max
60W
Max
V CO2, l/min
1.24 ± 0.09
3.57 ± 0.18
1.20 ± 0.11
3.64 ± 0.16*
1.21 ± 0.08
3.61 ± 0.25
1.19 ± 0.10
3.57 ± 0.24
Main effect for intensity and time x intensity x group
V E, l/min
31.7 ± 4.0
134.7 ± 13.5
31.5 ± 3.7
137.1 ± 11.3
32.1 ± 3.4
133.9 ± 12.8
31.5 ± 3.1
135.7 ± 9.1
25.0 ± 1.9*
45.2 ± 2.9
25.2 ± 2.5
44.8 ± 3.4
24.9 ± 1.9
45.5 ± 3.5
26.3 ± 2.2
37.7 ± 2.1
26.5 ± 2.8
37.2 ± 3.3
26.6 ± 2.0
38.1 ± 3.3
0.95 ± 0.04
1.20 ± 0.03
0.95 ± 0.03
1.21 ± 0.04
0.94 ± 0.05
1.20 ± 0.04
Main effect for intensity
V E/VO2
24.0 ± 1.9
45.4 ± 4.8
Main effect for intensity
V E/VCO2
25.5 ± 1.8
37.7 ± 2.9
Main effect for intensity
RER
0.94 ± 0.04
1.20 ± 0.04
Main effect for intensity
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
-0.05
-4.9
0.09
2.8
0.04
-2.1
-0.14
-7.8
IH
±
±
±
±
±
±
±
±
0.02
4.8
0.03
4.0
0.03
4.9
0.04
7.4
After
-0.05
-2.0
0.07
7.2
0.03
5.1
-0.12
-9.2
IH
±
±
±
±
±
±
±
±
0.03
6.7*
0.03
6.6
0.02
10.2*
0.05
8.4
Before
-0.05
-6.5
0.09
2.2
0.04
-4.6
-0.13
-8.7
IN
±
±
±
±
±
±
±
±
0.02
6.4
0.02
2.5
0.02
5.6
0.04
-8.0
After
-0.04
-7.6
0.09
3.9
0.05
-3.7
-0.13
-11.6
IN
±
±
±
±
±
±
±
±
0.03
6.0
0.02
4.3
0.02
8.5
0.05
6.0
* Significantly different from baseline testing
Intermittent Hypoxia
25
35
60
30
50
25
40
15
10
5
0
20
15
-5
-10
-15
A
THb (∆µM)
HHb (∆µM)
O2 Hb (∆µM)
20
90
120 150
180 Peak
20
10
10
5
0
-10
0
Rest 60
30
Rest 60
B
90
120 150
180 Peak
C
Rest 60
90
120 150
180 Peak
Rest 60
90
120 150 180 Peak
Intermittent Normoxia
60
20
30
50
15
25
40
HHb (∆µM)
O2 Hb (∆µM)
10
5
0
THb (∆µM)
35
25
20
15
30
20
10
10
-10
5
0
-15
0
-10
-5
D
Rest 60
90
120
150
180 Peak
Figure 5.7 - Cerebral oxygenation before
E
Rest 60
and after
90
120
150
180 Peak
IH or IN
F
Intermittent Hypoxia
20
-5
-10
30
15
25
10
THb (∆µM)
HHb (∆µM)
O2Hb (∆µM)
0
20
(∆µM)
5
15
10
-15
5
-20
5
0
(∆µM)
35
-5
0
-10
-5
-25
Rest
60
90
120
150
180
Rest
Peak
60
90
120
150
180
Rest
60
90
Rest
60
90
Peak
120
150
180
Peak
Intermittent Normoxia
20
35
15
30
-10
-15
25
THb (∆µ M)
HHb (∆µ M)
-5
(∆µM)
O2Hb (∆µM)
0
20
15
10
5
-20
0
-25
-5
Rest 60
90
120
150
180 Peak
Figure 5.8 - Muscle oxygenation before
10
5
0
M)
5
-5
-10
Rest 60
and after
90
120
150
IH or IN
180 Peak
120
150
180
Peak
Figure 5.9 – NOx at rest and after maximal exercise
70
60
NOx (µM)
50
40
Rest
30
Maximal exercise
20
10
0
Before
After
Before
Intermittent Hypoxia
After
Control
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
70
Nitrate (µM)
60
50
40
Rest
30
Maximal exercise
20
10
0
Before
After
Intermittent Hypoxia
Before
After
Control
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
900
800
600
500
Rest
400
Maximal exercise
(nM)
Nitrite (nM)
700
300
200
100
0
Before
After
Intermittent Hypoxia
Before
After
Control
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
140
120
RSNO (nM)
100
80
Rest
60
Maximal exercise
40
20
0
Before
After
IH
Before
After
Control
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
1000
sVCAM-1 (ng/mL)
800
600
Rest
Maximal exercise
400
200
0
Before
After
Before
Intermittent Hypoxia
After
Control
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
sICAM-1 (pg/mL)
800
600
Rest
400
Maximal exercise
200
0
Before
After
Intermittent Hypoxia
Before
After
Control
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
7.45
7.40
Blood pH
7.35
7.30
Rest
7.25
Maximal exercise
7.20
7.15
7.10
7.05
Before
After
Intermittent Hypoxia
Before
After
Control
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 V 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
V 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 F IO2 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 V 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 V 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
V 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 V 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 V O2 by impairing O2
extraction during submaximal exercise whereas during maximal exercise IH resulted in
favourable effects on muscle energetic function and increased V O2peak. Previous reports
show no change in V 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
V 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 V 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 V 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
V 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 overproduction 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 NOvasodilation 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 (SchmidtLucke 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 V O2 during the 60W workload signifies improved
exercise economy and together with the increase in V 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 V 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 V 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 V 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 2040mmHg 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 IR 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 V 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 V O2peak in 12% O2. Our IH protocol utilised a more severe IH
exposure compared to others that showed no change in V 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 V 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 V 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 V 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 V O2 whereas during
maximal exercise a greater THb could have resulted in favourable effects on muscle
energetics and V 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 O 2
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 V 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 leukocyteendothelial 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 V 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.
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