Thesis for the Degree of Doctor of Philosophy, Östersund 2015
Cardiovascular, hematological and dietary means to cope
with environmentally induced hypoxia in humans
Harald K. Engan
Main supervisor:
Professor Erika Schagatay
Co-supervisor:
Professor Hans-Christer Holmberg
Department of Health Sciences, Östersund, Mid Sweden University, Sweden
Mid Sweden University, SE-831 25 Östersund, Sweden
ISSN 1652-893X
Mid Sweden University Doctoral Thesis 213
ISBN 978-91-88025-01-2
i
Akademisk avhandling som med tillstånd av Mittuniversitetet i Östersund
framläggs till offentlig granskning för avläggande av filosofie doktorsexamen i
Hälsovetenskap med inriktning Idrottsvetenskap den 24 april 2015, klockan 13.00 i
sal F234, Mittuniversitetet Östersund. Seminariet kommer att hållas på norska.
Cardiovascular, hematological and dietary means to cope with
environmentally induced hypoxia in humans
Harald K. Engan
© Harald K.Engan, 2015
Department of Health Sciences, Faculty of Human Sciences
Mid Sweden University, SE-831 25 Östersund
Sweden
Telephone:
+46 (0)771-975 000
Printed by Mid Sweden University, Sundsvall, Sweden, 2015. Cover photograps by
Harald.K Engan (Mt. Everest, Nepal) and Erika Schagatay (freediver,Egypt)
ii
Cardiovascular, hematological and dietary means to cope with
environmentally induced hypoxia in humans
Harald K. Engan
Department of Health Sciences
Mid Sweden University, SE-831 25 Östersund, Sweden
ISSN 1652-893X, Mid Sweden University Doctoral Thesis 213; ISBN 978-91-8802501-2
ABSTRACT
Environmentally induced hypoxia triggers a variety of physiological responses in
the human body that aim to maintain homeostasis. During breath-holding the
cardiovascular diving response reduces oxygen consumption by inducing
bradycardia as well as redistributing blood flow to organs most sensitive to
hypoxia, most notably the heart and the brain. In addition, spleen contraction
temporarily increases the oxygen carrying capacity by releasing stored red blood
cells from the splenic reservoir. There is great individual variability in the
cardiovascular diving response and the spleen contraction, and the extent to which
the responses can be modified by training, hypoxic exposure and other factors such
as hypercapnia have not been clearly defined. The mechanisms related to the
initiation of spleen contraction are also unclear.
Recent investigations show that dietary nitrate may provide another means to cope
with hypoxia. Through metabolism to nitric oxide it improves efficiency of muscle
work and even reduces resting metabolic rate – both advantageous in hypoxic
situations. It is not known, however, if dietary nitrate supplementation can improve
performance and reduce oxygen consumption during breath-holding.
iii
This thesis general aim is to increase our understanding of the human ability to
cope with naturally hypoxia. The more specific goals are to determine: if two
weeks of breath-hold training will modify the cardiovascular diving response and
alter the extent of splenic contraction caused by breath-holding (Paper I); if longterm exposure to hypoxia at high altitude alters the extent of splenic contraction in
response to breath-holding and exercise (Paper II); if hypercapnia influences the
spleen related increase in blood hemoglobin concentration (Hb) during breathholding (Paper III); and if the duration of breath-holding is increased via reduced
oxygen usage by acute dietary nitrate supplementation (Paper IV).
The studies contained within this thesis resulted in several important findings. The
cardiovascular diving response was augmented after two weeks of intense breathhold training, but there was no effect on the blood-boosting spleen contraction
(Paper I). Long-term high altitude exposure did however enhance spleen
contraction, suggesting acclimatization to hypoxia had occured (Paper II). The
enhanced spleen contraction after high altitude exposure may be due to the longlasting hypoxic stimulus that develops in such environments. During breathholding, a high level of carbon dioxide is also present, and may be another
important factor modifying the spleen contraction (Paper III). Dietary nitrate
supplementation prolonged breath-hold duration and seemed to reduce metabolic
rate during breath-holding by a mechanism unrelated to the cardiovascular diving
response (Paper IV).
This thesis shows that humans can employ several effective physiological and
dietary mechanisms to improve their coping with low-oxygen situations. Adequate
stimuli and sufficient exposure (training) are however required for the
physiological mechanisms to be optimally employed, and the major contribution
from this thesis was to increase our understanding of how to enhance these effects.
iv
Keywords:
hypoxia,
spleen
contraction,
hemoglobin,
dietary
nitrate,
azzlimatization, breath holding
SAMMANDRAG PÅ SVENSKA
Omgivningsinducerad hypoxi initierar en rad fysiologiska reaktioner hos
människan som syftar till att upprätthålla homeostasis i kroppen. Vid andhållning
reducerar den kardiovaskulära dykresponsen syreförbrukningen genom att sänka
hjärtfrekvensen och omfördela blodet till de organ som är mest känsliga för hypoxi,
såsom hjärta och hjärna. Dessutom ger kontraktion av mjälten en tillfällig ökning
av den syrebärande förmågan genom frisättning av lagrade röda blodkroppar från
mjältens blodreservoar. Det finns en stor individuell variation i omfattningen av
både den kardiovaskulära dykresponsen och mjälteskontraktionen, och det är oklart
i vilken utsträckning dessa kan modifieras med träning, hypoxiexponering och
andra faktorer såsom hyperkapni.
Nyligen genomförda studier har visat att nitrat i kosten kan ge andra förutsättningar
för människan att hantera hypoxi på. Genom metabolismen omvandlas nitrat till
kväveoxid som leder till effektivare muskelarbete och sänkt metabolism, vilket
skulle kunna vara fördelaktigt vid hypoxiska sitationer. Det är dock inte klarlagt
om nitrattillskott kan öka prestationsförmågan och minska syreförbrukningen vid
anhållning.
Det övergripande syftet med denna avhandling är att öka förståelsen för
människans förmåga att hantera naturlig hypoxi. Mer specifikt är målen att
undersöka: om två veckors anhållningsträning modifierar den kardiovaskulära
dykresponsen och påverkar mjältens kontraktionsförmåga (Artikel I); om långtids
v
exponering för hypoxi på hög höjd påverkar mjältens förmåga att kontrahera vid
anhållning ock fysisk arbete (Artikel II ); om hyperkapni påverkar den mjältes
relaterade ökningen av hemoglobin koncentrationene (Hb) efter anhållning (Artikel
III): och om tillskott av nitrat via kosten leder till en ökning i anhållningstiden
genom minskad syreförbrukning(Artikel IV).
Studierna i denna avhandling ledde till flera viktiga upptäckter. Den
kardiovaskulära dykresponsen ökade efter två veckor av anhållningsträning, men
träningen
hade
ingen
effekt
på
mjälteskontraktionen
(Artikel
I).
Långtidsexponering för hög höjd ökade dock mjältens kontraktionsförmåga, vilket
indikerar att en acklimatisering till hypoxi skett (Artikel II). Ökningen av mjältens
förmåga att kontrahera kan dels ha orsakats av långvarig hypoxi under
höjdexponeringen. Under anhållning är nivå av koldioxid högt, och det kan vara en
annan viktig faktor som modifierar mjälteskontraktionen. (Artikel III). Nitrat via
kosten ökade anhållningstiden och minskade syreåtgången genom mekanismer inte
orsakade av den kardiovaskulära dykresponsen (Artikel IV).
Avhandlingen visar att människan kan rekrytera flera effektiva fysiologiska och
dietbaserade mekanismer för att bättre hantera situationer med låg syrenivå.
Adekvat stimuli och tillräcklig exponering (träning) är dock nödvändigt för att de
fysiologiska reaktionerna ska kunna utnyttjas optimalt, och de viktigaste fynden i
denna avhandlingen ökar vår förståelse för hur detta åstadkoms via träning och
diet.
Nyckelord: hypoxi, mjälteskontraksion, hemoglobin, dietärt nitrat, acklimatisering,
apné
vi
SAMMENDRAG PÅ NORSK
Under hypoksi p.g.a lavt oksygennivå i miljøet iverksetter mennesket en rekke
fysiologiske responser som forsøker å opprettholde homeostasen i kroppen. Når
mennesket holder pusten, reduserer den kardiovaskulære dykkeresponsen
oksygenforbruket ved å senke hjertefrekvensen, samtidig som blodet redistribueres
til organer som er mest sensitive til hypoxi, slik som hjertet og hjernen. I tillegg
fører kontraksjon av milten, som fungerer som et blodreservoar, til en midlertidig
økning i tilgangen til oksygen ved å frigjøre lagrede røde blodceller. De
individuelle variasjonene i graden av den kardiovasculære dykkeresponsen og
miltkontrasjonen er store, og det er ikke klarlagt i hvilket omfang disse responsene
kan modifiseres med trening, hypoksieksponering og andre faktorer slik som
hyperkapni.
Nylige undersøkelser har vist at diettbasert nitrattilskudd representerer en annen
måte å håndtere hypoksi på for mennesket. Gjennom metabolisering til
nitrogenoksid fører nitratsupplementering til et mer effektivt muskelarbeid og
reduksjon av metabolismen, noe som kan være fordelaktig under situasjoner med
hypoksi. På en annen side er det ikke klarlagt om diettbasert nitratsupplementering
kan øke prestasjonene og redusere oksygenforbruket under ”breath-holding”.
Den overgripende hensikten med denne avhandlingen er å øke forståelsen av
menneskets evne til å håndtere naturlig hypoksi. Mer spesifikt er målet å
undersøke: om to uker ”breath-hold” trening modifiserer den kardiovaskulære
dykkeresponsen og endrer miltens kontraksjonsevne (Artikkel I); om langtids
hypoksieksponering på stor høyde endrer miltens kontraksjonevne under ”breathholding” og fysisk arbeid (Artikkel II); om hyperkapni påvirker den miltrelaterte
økningen i hemoglobin konsentrasjon (Hb) etter ”breath-holding” (Artikkel III): og
vii
om tilskudd av diettbasert nitrat fører til en økning i ”breath-hold” tiden (Artikkel
IV).
Studiene i denne avhandlingen førte til flere viktige funn. Den kardiovaskulære
dykkeresponsen økte etter to uker med ”breath-hold” trening, men treningen hadde
ingen effekt på miltkontraksjonen (Artikkel I). Langtids høydeeksponering førte
derimot til en økning i miltens evne til å kontrahere, noe som indikerer en
akklimatisering til hypoksi (Artikkel II). Økningen i miltens evne til kontraksjon
kan skyldes langvarig hypoksistimuli ved høydeeksponering. Under ”breathholding” er nivået av karbondioksid høyt, og det kan representere en annen viktig
faktor
som
modifiserer
miltenkontraksjonen
(Artikkel
III).
Diettbasert
nitratsupplementering økte ”breath-holding” varigheten og virket til å redusere
forbruket av oksygen med mekanismer som ikke skyldes den kardiovaskulære
dykkeresponsen (Artikkel IV).
Avhandlingen viser at mennesket kan benytte seg av flere effektive fysiologiske og
diettbaserte mekanismer for å håndtere situasjoner med lavt oksygennivå bedre.
Det virker nødvendig med både hensiktsmessige stimuli og tilstrekkelig
eksponering (trening) for at de fysiologiske responsene skal kunne utnyttes
optimalt, og de viktigste funnene i denne avhandlingen øker vår forståelse for
hvordan dette påvirkes med trening og diett.
Nøkkelord:
hypoksi,
miltkontraksjon,
akklimatisering, apne
viii
hemoglobin,
diettbasert
nitrat,
ABBREVATIONS
HR-heart rate
MAP-mean arterial pressure
NO- Nitric Oxide
NO2- -Nitrite
NO3- -Nitrate
O2-Oxygen
CO2-Carbon dioxide
IMPORTANT TERMS
Hypobaric hypoxia
The environmental state caused by a reduction in partial pressure of O2 due
to a reduction in ambient barometric pressure
Normobaric hypoxia
The environmental state caused by a reduction in partial pressure of O2 due
to a reduction in O2 fraction (e.g. % O2 content) of air under constant
barometric pressure
Normoxia
The environmental state where the partial pressure of O2 is equal to that
occuring at sea level when PO2 is 160 mmHg (21% O2 content)
Systemic hypoxia
The physiological state caused by a reduction in O2 levels in the blood that
is not due to a disruption of blood flow
Asphyxia
The combined effects of hypoxia, hypercapnia and acidosis resulting from
abnormal breathing and when the supply of O2 to the lungs is insufficient.
ix
Hypoxemia
The physiological state of inadequate level of O2 in arterial blood of
various causes, which may limit the supply of O2 required for aerobic
tissue metabolism. In humans, it is also defined as a condition where
arterial oxygen tension (PAO2) is below normal (normal PAO2 = 80-100
mmHg).
Anoxia
The physiological state of complete deprivation of O2 to the tissues.
Acclimatization
A physiological adjustment to a change in the environment allowing an
individual to maintain performance in this specific environmental
condition.
Adaptation
The evolutionarily process whereby an organism becomes better able to
survive and reproduce in its habitat by natural selection, thus affecting the
genetic properties of a group of organisms across generations.
Metabolic rate
The amount of energy liberated or expended in a given unit of time of an
animal. Basal metabolic rate is the minimal rate of energy expenditure of
endothermic animals at rest.
x
TABLE OF CONTENTS
ABSTRACT ...................................................................................................................... III
SAMMANDRAG PÅ SVENSKA .................................................................................. V
SAMMENDRAG PÅ NORSK ....................................................................................... VII
ABBREVATIONS ............................................................................................................. IX
IMPORTANT TERMS ..................................................................................................... IX
TABLE OF CONTENTS .................................................................................................. XI
LIST OF PAPERS ......................................................................................................... XIII
1.
INTRODUCTION ........................................................................................................1
1.1.
THE EVOLUTION OF PHYSIOLOGICAL REGULATION OF OXYGEN...............................1
1.2.
THE PHYSIOLOGICAL SETTING OF ENVIRONMENTALLY INDUCED HYPOXIA .............4
1.3.
EXPOSURE TO ENVIRONMENTALLY INDUCED HYPOXIA ...........................................6
1.3.1.
Hypobaric hypoxia .........................................................................................7
1.3.2.
Hypoxia induced by breath-holding ...............................................................7
1.4.
CARDIOVASCULAR, SPLENIC AND DIETARY REGULATIONS DURING HYPOXIA........10
1.4.1.
Regulation of heart function .........................................................................11
1.4.2.
Characteristics and initiation of the cardiovascular diving response ..........12
1.4.3.
Physiological function of the cardiovascular diving response ..................... 15
1.5.
ROLE OF DIETARY NITRATE SUPPLEMENTATION DURING BREATH-HOLDING .........18
1.6.
TRANSITORY STORAGE OF ERYTHROCYTES IN THE SPLEEN ...................................22
1.6.1.
Contraction of the spleen in some mammals ................................................ 22
1.6.2.
The human spleen ......................................................................................... 23
1.6.3.
Circulatory functions of the spleen in humans ............................................. 25
1.6.4.
Mediation and control of the spleen contraction ..........................................25
1.6.5.
Spleen contraction during exercise .............................................................. 27
1.6.6.
Spleen contraction during breath-holding and hypoxia ............................... 27
2.
AIMS OF THE THESIS ............................................................................................ 30
xi
3.
METHODS ................................................................................................................. 31
3.1.
SUBJECTS .............................................................................................................. 31
3.2.
ARTERIAL O2 SATURATION AND CARDIOVASCULAR MEASUREMENTS ................... 32
3.2.1.
Arterial O2 saturation and heart rate ........................................................... 32
3.2.2.
Blood pressure measurements ......................................................................33
3.3.
RESPIRATORY MOVEMENT MEASUREMENTS.......................................................... 35
3.4.
RESPIRATORY GAS MEASUREMENTS......................................................................35
3.5.
BLOOD VARIABLE MEASUREMENTS .......................................................................36
3.6.
SPLEEN VOLUME MEASUREMENTS ........................................................................36
3.7.
EXPERIMENTAL PROTOCOLS.................................................................................. 38
3.7.1
Voluntary breath-holding ............................................................................. 38
3.7.2.
Dietary nitrate supplementation ...................................................................39
3.8.
DATA ANALYSIS AND STATISTICS ..........................................................................40
3.9.
ETHICS .................................................................................................................. 41
4.
MAIN RESULTS ......................................................................................................42
5.
DISCUSSION ............................................................................................................. 47
5.1.
THE HEART RATE RESPONSE AFTER BREATH-HOLD TRAINING (PAPER I) ............... 48
5.2.
DIETARY NITRATE SUPPLEMENTATION DURING STATIC BREATH-HOLDING (PAPER
IV)
51
5.3.
ENHANCEMENT OF SPLEEN CONTRACTION BY HYPERCAPNIA (PAPER III) ............. 56
5.4. ACCLIMATIZATION POTENTIAL OF THE SPLEENS CONTRACTILITY .............................. 60
5.4.1.
Training effects ............................................................................................. 60
5.4.2.
Effects of long-term altitude exposure .......................................................... 61
5.5.
IMPLICATIONS AND FUTURE DIRECTIONS OF WORK ............................................... 62
5.5.1.
Diving response related studies....................................................................62
5.5.2.
Spleen related studies ................................................................................... 62
5.5.3.
Dietary nitrate related studies ......................................................................63
6.
CONCLUSIONS ........................................................................................................64
7.
ACKNOWLEDGEMENTS ....................................................................................... 66
8.
REFERENCES ........................................................................................................... 68
xii
LIST OF PAPERS
This thesis is mainly based on the following four papers, herein referred to
by their Roman numerals:
Paper I
Engan H, Richardson MX, Lodin-Sundström A, van Beekvelt M,
Schagatay E. (2013). Effects of two weeks of daily apnea training on diving
response, spleen contraction, and erythropoiesis in novel subjects. Scandinavian
Journal of Medicine & Science in Sports 23 (3): 340-348.
Paper II
Engan HK, Lodin-Sundström A, Schagatay F, Schagatay E. (2014).
The effect of climbing Mount Everest on spleen contraction and increase in
hemoglobin concentration during breath-holding and exercise. High Altitude
Medicine & Biology 15(1): 52-57.
Paper III
Richardson MX, Engan, HK, Lodin-Sundström A, Schagatay E.
(2012). Effect of hypercapnia on spleen-related haemoglobin increase during
apnea. Diving and Hyperbaric Medicine 42 (1): 4-9.
Paper IV
Engan HK, Jones, AM, Ehrenberg F, Schagatay, E. (2012). Acute
dietary nitrate supplementation improves dry static apnea performance. Respiratory
Physiology & Neurobiology182 (2-3):53-59.
The published papers are reproduced with kind permission of the publisher.
xiii
1.
INTRODUCTION
1.1. The evolution of physiological regulation of oxygen
Oxygen (O2) is both a vital gas and, with sufficient partial pressure, a lethal toxin
whose presence in the atmosphere and surface layer of the ocean began to rise 2.4
billion years ago (Gaillard, Scaillet et al. 2011). It is required for aerobic
respiration, and subsequently energy production via oxidative phosphorylation - a
prerequisite for complex organisms to sustain higher metabolic activity (Maina
2002; Pisani, Cotton et al. 2007).1
Harnessing energy from oxidative phosphorylation comes with a cost, however.
Cumulative cellular O2 stress may cause cellular damage and inevitability
senescence and death (Imlay 2013). Evolutionarily, the conflict between the need
for effective energy production and minimizing cellular O2 stress likely led to an
assortment of gas-exchange structures, ranging from simple air-blood interfaces in
for example the surface or skin in animals such as the adult bullfrog (Rana
catesbeiana) (Burggren and West 1982), to the complex mammalian lung working
in conjunction with the cardiovascular system (Hsia, Schmitz et al. 2013). These
systems evolved to deliver enough O2 and eliminate enough carbon dioxide (CO2)
in order for the animals to allow high enough activity to overcome specific
environmental and predatory pressures (both as predator and prey), while at the
same time limiting the energy cost of breathing as well as the oxidative stress
1
The origin of aerobic life forms is thought to be from an endosymbiotic process in which
ancestral bacteria were ingested by eukaryotic cells and evolved into modern mitochondria
(Pissani, Cotton et al. 2007). The ancestral bacteria were likely facultative anaerobes that
possessed O2-based electron transport chain as a pathway for detoxifying O 2. However,
later as a part of an organelle, the electron transport chain was exploited for aerobic energy
production to serve the host cell (Hsia, Schmitz et al. 2013).
1
within cells and organelles (Hsia, Schmitz et al. 2013). A sequential, step-wise
reduction of O2 tension from ambient air to that in the mitochondria is a
fundamental feature of the respiratory-cardiovascular systems, and helps to
maintain the balance between uptake and distribution and cellular protection.
Metalloproteins such as hemoglobin (Hb) and myoglobin have evolved to fulfill
the need of controlled storage, transport and release of O2 and other gases
(Waldron, Rutherford et al. 2009), although the Antarctic icefishes are exceptional
examples of vertebrates without Hb (Sidell and O'Brien 2006). Nevertheless, in
most vertebrates these proteins play crucial roles during the stepwise reduction of
O2 tension from the atmosphere through consecutive resistances across the
pulmonary, cardiac, macrovascular and microvascular systems to the cell and
mitochondria (Hsia, Schmitz et al. 2013). In the working human leg muscle, the
mitochondrial O2 tension at half of the maximal metabolic rate has been estimated
at approximately 0.02 and 0.2 mmHg (Wittenberg and Wittenberg 1989) which is,
interestingly, in the range of the ancient atmospheric level 2 billion years ago
(Gaillard, Scaillet et al. 2011). Increasing the O2 tension above these levels reduces
mitochondrial activity (Sagone 1985).
Parts of the intertwined mammalian respiratory, hematological and cardiovascular
systems investigated in this thesis have likely evolved in pre-mammalian life forms
under the variations of atmospheric O2 content throughout history to finely regulate
O2 partial pressure, ensuring delivery during various environmental challenges.
These could include periods of relative hypoxia. Simultaneously there was a need
for protecting the cells against oxidative damage (Hsia, Schmitz et al. 2013). The
responses of these systems can be found across phylogeny in for example
mammals such as seals and pigs, but also in reptiles and fish (Davis, Polasek et al.
2004).
One situation with limited O2 availability in air breathers is during breath-hold
diving. The possibility to harvest from the protein-rich environment in the sea
2
likely represent a selection pressure for diving characteristic (Uhen 2007). These
resulting adaptations are most notably found in semi-aquatic mammals such as
seals and fully aquatic whales (Kooyman, Castellini et al. 1981). Although they
have evolved from different non aquatic ancestors, co-evolution has brought about
similar solutions, involving means to store excess oxygen, lower metabolic
demands, and thereby refrain from breathing during extended periods (Berta,
Sumich et al. 2005; Uhen 2007).
As shown in this thesis, these characteristics are also found, although to a lesser
extent, in terrestrial mammals such as humans, allowing activity in both terrestrial
and aquatic ecological niches. The response characteristics related to breath-hold
diving are found to some extent in all vertebrates examined and include
bradycardia, tissue hypoperfusion and hypometabolism of hypoperfused tissues
(Hochachka, Gunga et al. 1998; Hochachka 2000). These traits presumably
evolved primarily through negative selection, that is any mutations affecting them
will not survive (Hochachka, Gunga et al. 1998). On the other hand, physiological
traits such as large blood volume, erythrocyte mass and likely spleen volume are
more malleable and correlate with more prolonged diving behaviors. It is therefore
suggested that these traits have evolved mainly through positive selection to enable
prolonged diving and high aerobic endurance capacity (Hochachka, Gunga et al.
1998; Hochachka 2000).
This thesis does not include detailed aspects of mammalian or human evolution to
various O2 tensions in the environment, although some comparison with our closest
counterparts in the animal kingdom is elucidated upon, e.g. diving mammals. This
thesis focuses on the present range of cardiovascular, hematological and dietary
strategies to cope with hypoxic challenge from natural environments and includes
voluntary human behaviors such as breath-holding diving. The results may
however reveal evolutionary steps occurring during more recent human evolution
(Schagatay 2011). The temporal dimensions of these strategies are important
3
during environmental and behavioral challenges. These can typically be divided
into three categories, of which the first two will be discussed in this thesis: 1) acute
effects, 2) acclimatization and 3) adaptation (Hochachka, Gunga et al. 1998). In
general, when exposed to environmental factors (oxygen, temperature, light, water,
food, parasite/pathogens) special sensing structures transfer their information to
physiological and biochemical systems. These systems may respond almost acutely
following the environmental challenge and aim to reestablish homeostasis.
Moreover, responses requiring hours or days to achieve a new steady state are
referred to as acclimatization. Acute responses and acclimatization are possible
only within a given individual. All parts of this cascade (from stimulus reception to
acclimatization) can change in group´s genetic makeup across generations and is
defined as “phylogenetic adaptation” (Hochachka, Gunga et al. 1998).
1.2. The physiological setting of environmentally induced
hypoxia
Nature sets the environmental conditions that can sustain life. The challenges of
such conditions stimulate evolutionary forces, setting the stage for development of
complex life forms. Higher organisms have evolved to maintain certain critical
body characteristics such as temperature, pH and blood glucose within tight limits.
This homeostasis (from Greek ‘hómoios’, "similar", and ‘stásis’, "standing still”),
a concept formulated by Claude Bernard in 1865 (Cooper 2008), can be maintained
even in environmental variation, albeit not without limit.
The unassisted human can only live in an environmental with a relatively O2-rich
atmosphere. Deliberate or accidental exposure to O2-poor environments can
potentially disturb our homeostatic regulation and progressively disrupt cellular
biochemical processes, resulting in physiological dysfunction (Essop 2007). The
term hypoxia is used to describe when there is an inadequate supply of O2 to
certain tissues (tissue hypoxia) or the entire body (systemic hypoxia). Although
4
there is no generally accepted definition of tissue hypoxia, this state is commonly
considered to exist when the supply of O2 is inadequate to meet the O2 demands of
cellular metabolic processes (Saito, Nishimura et al. 2002).
There are several subtypes of hypoxia, based on etiology and tissue localization
(Pierson 2000) summarized in table 1.
Table 1. Types of hypoxia based on etiology and tissue localization
Types of hypoxia
Etiology and/or tissue localization
Anemic hypoxia
Decreased concentration of functional
hemoglobin or reduced number of red
blood cells (anemia, hemorrhage)
Ischemic hypoxia
Inadequate perfusion of blood
Affinity hypoxia
Reduced ability of hemoglobin to release
O2
Stagnant hypoxia
Intravascular
impairment
stasis
of
venous
caused
outflow
by
or
decreased arterial inflow
Hypoxic hypoxia
Defective O2 perfusion in the lung
caused by abnormal pulmonary function,
airway obstruction and/or a low tension
of O2
5
1.3. Exposure to environmentally induced hypoxia
Allowing a reduction in tissue oxygenation may be beneficial for an organism
because it allows for certain advantageous activities to be conducted despite
inhospitable environments for related species. Weddell seals for example, may
perform long exploratory dives under the Antarctic ice sheet and can obtain food
from great depths (Kooyman, Kerem et al. 1973) (Figure 1). The present thesis is
concerned with this kind of hypoxia, which may be self-induced at least in humans
for example at high altitude or during breath-hold diving. This will involve altering
internal levels of O2 and allow a certain level of hypoxemia to develop. The
conditions in which (voluntary) hypoxia occurs will also often induce changes in
carbon dioxide (CO2) and hydrogen ions (H+), for example at altitude where
hypocapnia and alkalosis result from an associated increase in pulmonary
ventilation (Elsner 1989).
Figure 1. The Weddell seal (Leptonychotes weddellii), one of the most accomplished
mammalian diver species capable of > 80 minutes dives and diving depths to > 600 m, has
evolved efficient cardiovascular and hematological mechanisms allowing for long hunting
expeditions and underwater exploration (Kooyman 1966; Zapol, Hill et al. 1989) Credit: ©
Andrea Leone Dreamsteam.com
6
1.3.1.
Hypobaric hypoxia
The reduction in partial pressure of O2 at altitude stimulates physiological
responses and acclimatization in humans to maintain adequate tissue oxygenation.
Stimulation of peripheral arterial chemoreceptors leads to more rapid ventilation,
and enhanced sympathetic activity increases cardiac output, pulmonary vascular
resistance and mean pulmonary arterial pressure (West 1982). Hemoglobin (Hb)
concentration in the blood is also elevated during the first hours at higher altitude
as a result of water diuresis. Moreover, hypoxia increases renal secretion of erythropoietin
(EPO), augmenting peripheral O2 delivery by raising the number of erythrocytes,
although this process takes several days (Peacock 1998). High altitude Tibetans
have been shown to have >10-fold-higher circulating concentrations of bioactive
nitric oxide (NO) products and double the forearm blood flow measured at high
altitude compared to low-altitude residents measured at low altitude (Erzurum,
Ghosh et al. 2007). At least in native highlanders, this suggests that NO has an
important role in regulating vascular function at high altitude.
1.3.2.
Hypoxia induced by breath-holding
Breath-holding or the voluntary arrest of breathing, by etymology differs from the
more general concept of apnea, which simply refers to cessation of breathing. The
interruption of the unconscious pattern of breathing that characterizes voluntary
breath-holding most likely does not involve control of the central respiratory
rhythm (Foster and Sheel 2005), but rather suppression of central respiratory drive
by voluntarily “holding” the chest at fixed lung volume (Parkes 2006). Human
breath-holding occurs for example during competitive free diving (Schagatay
2011), synchronized swimming (Rodriguez-Zamora, Iglesias et al. 2012) and
7
harvest diving (Schagatay, Lodin-Sundstrom et al. 2011), as well as in infants
while swimming spontaneously (Goksor, Rosengren et al. 2002).
During a breath-hold, gas transport from the surrounding is halted and O2 for
metabolism must be obtained from the limited stores in the lungs, blood and tissues
(Schagatay 2009). Prolonged breath-holds may produce significant hypoxemia,
with insufficient oxygenation to sustain aerobic processes (Scholander, Hammel et
al. 1962; Ferretti 2001; Andersson, Liner et al. 2002; Muth, Radermacher et al.
2003; Andersson and Evaggelidis 2009; Marabotti, Piaggi et al. 2013).2
In humans, the O2 saturation level in the arterial blood (SaO2) can be measured
conveniently and non-invasively and is often used as an indication of the severity
of hypoxemia (Spyer 1981; Findley, Ries et al. 1983). The relationship between
the partial pressure of oxygen in the alveoli (PAO2) and in the arteries (PaO2) is
shown to be constant during breath-holding (Hong, Lin et al. 1971) implying that
the level of O2 measured in the arteries during breath-hold reflects the level in the
lung. SaO2 in healthy humans breathing at sea-level is normally 97- 99 %, but
trained breath-hold divers can reduce this value to less than 50 % during breathholding; further reductions may induce unconsciousness (Findley, Ries et al. 1983).
Upon breath-holding after a maximal inhalation of normal air not preceded by
hyperventilation, the end-tidal partial pressure of O2 is shown to fall from its
normal level of approximately 100 mm Hg to about 62 mm Hg, while the end-tidal
partial pressure of CO2 rises simultaneously from approximately 40 mm Hg to
2
Although work described here could be generalized to diving conditions, the present
studies utilize a breath-holding model at sea level conditions, whereas with diving to depth,
as predicted by Boyle`s and Dalton`s law, the change of ambient pressure during descent
and ascent affects the partial pressure of the respiratory gases. While hyperoxia and
pronounced hypercapnia could occur during the initial phase of deep diving, pronounced
hypoxia may be expected upon ascending (Muth,C. M.,P. Radermacher, et al. 2003).
8
about 54 mm Hg (Lin, Lally et al. 1974). The same maximal inhalation breath-hold
results in a reduction of arterial partial pressure of O2 (PaO2) of approximately 10
mm Hg per minute (Ferris, Engel et al. 1946). According to Lin (1982), O2
disappears from lung at approximately a linear rate during the first 2 minutes of
breath-holding. After this period, the rate diminishes but continues nonetheless in
an attempt to support metabolism, but arterial O2 saturation eventually is affected
by lowered pulmonary PO2 in the lungs, and the level of O2 in the venous blood
progressively decreases (Lin 1982).
The O2 desaturation during breath-holding is determined by the O2 stores initially
available, primarily in the venous blood and lungs (Findley, Ries et al. 1983;
Fletcher, Costarangos et al. 1989; Sasse, Berry et al. 1996), and the rate of usage by
metabolic processes (Lindholm, Sundblad et al. 1999; Andersson, Liner et al.
2002). Inhalation of hyperoxic gas prior to breath-holding can prolong its duration
by extending the period until the lungs begin to become depleted of O2. Klocke and
and Rahn (1959) found that immediately prior inhalation of O2 extended breathholding time to as long as 14 minutes, with some subjects absorbing the entire
pulmonary vital capacity volume of O2. The elevation in metabolic rate during
steady state exercise training (at 167 kgm ∙ min-1) for example, results in
approximately 60 % more rapid depletion of O2 stores, thereby reducing the
duration of breath-holding to the same extent (Lin, Lally et al. 1974).
The continuous production of CO2 by metabolically active tissues and its lack of
clearance during breath-holding will lead to CO2 accumulation in the venous and
subsequently arterial blood, a state referred to as hypercapnia (Mithoefer 1959; Del
Castillo, Lopez-Herce et al. 2012). The partial pressure of CO2 in the arterial
blood increases even more due to pulmonary shrinkage as well as the Haldane
effect from oxygenation of Hb (Mithoefer 1959). The partial pressure of CO2
(PCO2) in venous blood is elevated to a lesser extent by the same Haldane effect as
well as buffering of CO2 by the tissues (Mithoefer 1959; Tyuma 1984).
9
CO2 transfer to the lungs falls progressively during breath-holding, and in
particularly extended breath-holding a retrograde diffusion of CO2 from its higher
level in the lungs into arterial blood may be noted (Hong, Lin et al. 1971). The
Haldane effect and an elevation of alveolar PCO2 caused by lung volume shrinkage
results in a faster increase of the alveolar-arterial PCO2 than mixed venous PCO2
(Lanphier and Rahn 1963).
Blood acidity also rises during breath-holding due to the dissolution of CO2 in
plasma into carbonic acid (Gooden 1994). Moreover, the hypoxic state may
enhance glycolysis, resulting in an elevated blood concentration of lactate
(Hochachka and Mommsen 1983; Rodríguez-Zamora, Engan et al. 2013). This
situation favors unloading of O2 from hemoglobin, which might lead to more rapid
depletion of O2 from this compartment as it moves to peripheral tissues. However,
this potential “loss” of O2 to the periphery is counteracted by a potent
vasoconstriction that virtually eliminates peripheral blood flow during breathholding (Lin 1982).
It should be noted that CO2 is the dominant factor for cessation of breath-holding in
non-divers, while in trained divers this is more dependent on the development of
hypoxia, as these individuals have a reduced ventilatory response to hypercapnia
(Davis, Graves et al. 1987) .
1.4. Cardiovascular, splenic and dietary regulations during
hypoxia
Respiratory organs in all air-breathing animals have likely evolved to ensure
effective gas exchange with the atmosphere, and transport of respiratory gases to
tissues should ideally match metabolic activity. This thesis focuses on a series of
responses developed to optimize this match and to prioritize O2 delivery to the
10
most vital functions, as well as to improve O2 efficiency. These mechanisms
include regulation of metabolic rate and distribution of blood flow from the
cardiovascular system, and a temporarily increase of
O2 carrying capacity
resulting from spleen contraction during hypoxia induced by, for example, breathholding in humans. It also includes regulation of metabolic activity during breathholding through the dietary nitrate-nitrite-nitric oxide pathway.
1.4.1.
Regulation of heart function
The cardiovascular system can maintain a sufficient blood supply to tissue under a
wide range of conditions, including breath-holding. The autonomic regulation of
the heart obtains input about arterial pressure via baroreceptors located mainly in
the carotid sinus and the aorta. I also receive input about the levels of O2 and CO2
in the blood via chemosensors primarily located in the carotid bodies and brain
(Spyer 1981).3
The cardiac pacemaker at the sinoatreal (SA) node of the heart receives
sympathetic innervation via the glosspharyngeal nerve and parasympthateic
innervation via the vagus nerve column (Langley 1898). The heart rate (HR), being
at a spontaneous level at around 100 beats per minute without influence (Rushmer,
Crystal et al. 1953), and cardiac contractility can be changed by vagal tonus and
sympathetic activation mediated via release of norephineprine from postganglionic
terminals to the SA node and cardiac muscle fibers (Heesch 1999). They can also
be changed by release of catecholamine from adrenal medulla (Heesch 1999).
Cardiac output, the product of HR and stroke volume (SV), and blood pressure, the
product of cardiac output and muscle tonus of the vessels of the circulatory system,
are key targets for autonomic adjustment during breath-holding.
3
A structural description of the human heart is beyond the scope of this thesis, but the
interested reader is referred to an extensive review by Rushmer, Crystal et al. (1954).
11
1.4.2.
Characteristics and initiation of the cardiovascular diving response
A collection of reflexes occurring during breath-holding and breath-hold diving in
humans and other air-breathing animals, especially divers, is referred to as the
cardiovascular diving response (Elsner and Gooden 1983; Gooden 1994). This
response is characterized by a lowering of HR (Landsberg 1975) and thereby
cardiac output (Heistad, Abbound et al. 1968; Liner 1994), as well as centralization
of blood due to peripheral vasoconstriction (Heistad, Abbound et al. 1968). During
breath-holding and breath-hold diving in humans HR often exhibits three
distinctive phases: 1) an increase (tachycardia) for 5-10 s; followed by 2) a decline
during which HR is inversely proportional to time elapsed (Jung and Stolle 1981),
and finally 3) a fully established and relatively stable bradycardia after
approximately 30 s of breath-holding (Lin 1982). Recently Costalat and colleagues
(2015) investigated the HR kinetics during prolonged static breath-holds in trained
breath-hold divers, and identified a breaking point preceding a second and final
drop in HR which was paralleled by a marked decrease in SpO2. The author named
it the “oxygen conserving breaking point” and suggested it could represent an
unique adaptive feature against hypoxic damages in the human bradycardia
(Costalat, Pichon et al. 2015).
The bradycardia is often expressed as the HR reduction from what it was during the
last few minutes preceding breath-holding. In certain diving mammals, such as the
Weddell seal, the reduction in cardiac output and elevation of total peripheral
resistance are so closely balanced during a dive that systemic arterial pressure
remains stable (Butler 1982). In humans, however, mean arterial blood pressure
(MAP) is increased during breath-holding (Ferrigno, Ferretti et al. 1997). It should
be noted that even though the diving response constitutes several reflexive
responses, the bradycardia correlates strongly with the peripheral vasoconstriction
(Schagatay and Andersson 1998), and is often used as an outcome variable for the
12
whole response, as is this case during research of diving mammals (Panneton
2013).
Two principal sensory inputs, cessation of breathing and the stimulation of facial
cold receptors, elicit the human cardiovascular diving response (Campbell, Gooden
et al. 1969; Dykes 1974; Angell-James, Elsner et al. 1981; Schuitema and Holm
1988) (Figure 2). It is the cessation of breathing (or breath-holding) that initiates
and sustains this response (Foster and Sheel 2005), but it is potentiated by cooling
the trigeminal facial cold receptors in the upper face in relation to the ambient air,
whereby greater temperature differences increase the potentiating (Schuitema and
Holm 1988; Schagatay and Holm 1996). The initiation of the response therefore
occurs before any hypoxia stimuli develop. Chemoreceptors found in the carotid
and aortic bodies may also regulate the cardiovascular system. For example,
stimulation resulting from decreased PO2 causes a reduction in HR and peripheral
vasoconstriction, but only during cessation of breathing (Gooden 1994). The effect
of PO2 on the magnitude of the cardiovascular diving response is most likely
mediated via the carotid body, whose primary function is to detect and respond to
hypoxemia (Donnelly 1997). Respiratory arrest in combination with stimulation of
the carotid bodies by arterial hypoxemia and hypercapnia causes a cardiovascular
response that is similar to the diving response, including bradycardia and peripheral
vasoconstriction (de Burgh Daly 1997). Interestingly, an experiment on immersed
harbor seals revealed that perfusing their carotid and aortic bodies with blood of
varying oxygen and carbon dioxide concentrations could concomitantly alter the
diving response in these animals (de Burgh Daly, Elsner et al. 1977). Changes in
PCO2 appear to have little influence on the control of the response in humans,
however (Lin, Shida et al. 1983; Gooden 1994).
In humans, initiation of bradycardia at the onset of breath-holding involves a
complex interaction between the respiratory center and the cardiac autonomic
centers in the central nervous system (Elsner and Gooden 1983; Gooden 1994). In
13
general, this bradycardia is produced by parasympathetic activity of the vagus
nerve (Elsner and Gooden 1983; Gooden 1994). The associated peripheral
vasoconstriction is the result of a simultaneous increase of sympathetic outflow to
peripheral vessels (Elsner and Gooden 1983) .
Cold stimulation of the face without breath-holding leads to bradycardia similar to
that which occurs during breath-holding alone, and combining the two stimuli
elicits twice the response (Elsner and Gooden 1983; Hurwitz and Furedy 1986;
Marsh, Askew et al. 1995). Since chilling of the forehead and eyes alone, but not of
other facial areas of similar size produces such bradycardia, the receptors involved
must be located primarily in the region of forehead and eyes, which is innervated
by the ophthalmic branch of the trigeminal nerve (Schuitema and Holm 1988;
Schagatay and Holm 1996).
Several other factors including exercise (Bergman, Campbell et al. 1972; Wein,
Andersson et al. 2007), pulmonary volume (Andersson and Schagatay 1998),
alterations in intrathoracic pressure (Angell-James and Daly 1978; Angell-James,
Elsner et al. 1981), age (Gooden 1994; Holm, Schagatay et al. 1998) and
psychological factors (e.g., anxiety) (Wolf 1994) have been shown to modify the
cardiovascular diving response.
14
Figure 2. Overview of the main factors eliciting and modifying the cardiovascular diving
response in humans. 1) Decreased nervous activity to the respiratory muscles initiates
breath-holding. 2) Cessation of cyclic stimulation of lung stretch receptors and stimulation of
facial cold receptors convey signals to the brainstem, which by 3) increasing sympathetic
stimulation to peripheral vessels initiates vasoconstriction. 4) At the same time bradycardia
is induced by increased parasympathetic activity,5) and possibly by decreased sympathetic
activity. 6) The bradycardia and the vasoconstriction may be modified by chemoreceptors
and baroreceptors during the course of the breath-hold when these stimuli develop. CIC
(cardioinhibitory centre), VMC (vasomotor centre) Figure adapted from Schagatay (1996).
1.4.3.
Physiological function of the cardiovascular diving response
The diving response has most likely evolved to maintain oxygenation of vital
organs when breathing is interrupted (Alboni, Alboni et al. 2011) and has been
15
observed to exist to some extrent in all vertebrates examined to date (Schreer and
Kovacs 1997; Panneton 2013). It is most well characterized in diving mammals
such as seals (Panneton 2013), which demonstrate a more rapid and pronounced
response than in humans (Foster and Sheel 2005). It is noteworthy that the
characteristic bradycardia, peripheral vasoconstriction and metabolic suppression
may occur not only in response to diving but to other hypoxic conditions as well,
such as during hibernation and during birth when uterine contractions temporarily
occlude placental vessels that supply the fetus with blood (Rial, Barbal et al. 2000).
The fact that many vertebrates, including reptilian species, develop bradycardia
when O2 supply is limited suggests that the response is evolutionarily ancient
(Hochachka 2000; Rial, Barbal et al. 2000; Taylor, Leite et al. 2010). Because of
its importance during diving (Elsner and Gooden 1983), its relatively early
evolutionary development (Scholander, Hammel et al. 1962), and its apparent
conservation among species (Hochachkta 1998), diving bradycardia has been
referred to as the “master switch of life”. Furthermore, the finding that fish develop
bradycardia when taken out of water supports the conclusion that hypoxia, rather
than immersion per se, is the key stimulus for initiating bradycardia (Rial, Barbal
et al. 2000). Nonetheless, the response is initiated prior to hypoxia in most diving
mammals (including humans) suggesting that it may have a pre-emptive function
(Elsner and Gooden 1983; Alboni, Alboni et al. 2011).
Indeed, a number of investigations in humans have concluded that the primary role
of the cardiovascular diving response is conservation of O2 (Andersson and
Schagatay 1998; Lindholm, Sundblad et al. 1999; Ferretti 2001; Andersson, Liner
et al. 2002). Earlier studies (Craig 1963; Hong, Lin et al. 1971) did not always
arrive at the same conclusion, however, perhaps because they included untrained
subjects with limited experience of breath-holding which could only manage short
duration breath-holds. More modern research showed that the cardiovascular
diving response potentially conserves O2 in several ways. The bradycardia reduces
16
O2 consumption of the myocardium since cardiac power output is roughly
proportional to the HR (Lin 1982). In addition, the reduced metabolic activity of
the heart is associated with a depressed ventricular contractile force (negative
inotropic effect), probably due to increased vagal activity and simultaneously
reduced sympathetic activity (Daly, Angell-James et al. 1979).
Conservation of O2 is also a result of reduced cardiac output and peripheral
vasoconstriction, since less blood flow to the lungs will limit pulmonary gas
exchange (Andersson, Biasoletto-Tjellstrom et al. 2008). Importantly, the
vasoconstriction of peripheral and visceral capillary beds redirects blood from the
gut, kidneys, liver and skeletal muscle to the organs most sensitive to asphyxia,
mainly the heart and brain (Butler 1982). The lower perfusion of the peripheral and
visceral beds forces the peripheral tissue to rely on local stores of O 2 and high
energy phosphates, and a shift to anaerobic metabolism (Ferrigno, Ferretti et al.
1997; Andersson, Liner et al. 2004). Although hypometabolism may also occur in
at least some underperfused mammalian organs during prolonged dives (Kooyman
and Ponganis 1998; Hurley and Costa 2001), the high metabolic demand limits
this effect in the brain, liver and heart (Wang, Ying et al. 2010).
In light of its “hard-wired” characteristics, the potential to augment the
cardiovascular diving response through training is contentious. In one study,
humans who trained breath-holding daily for 14 days exhibited a more pronounced
reduction in HR followed by attenuated arterial O2 desaturation (Schagatay, van
Kampen et al. 2000). Nonetheless, present knowledge about intermittent hypoxic
training effects on the time-course of HR reduction and potential implications for
O2 conservation is incomplete. Neither has the potential interaction of the diving
response with other responses that might allow prolonged diving, such as splenic
contraction, nor the spleen contractions own susceptibility to training, been
investigated.
17
1.5. Role of dietary nitrate supplementation during breathholding
While several studies have identified the metabolic restrictions imposed by the
cardiovascular diving response, two methods to voluntarily limit metabolic rate
have also been examined during breath-holding, namely fasting (Lindholm,
Conniff et al. 2007; Schagatay and Lodin-Sundstrom 2014) and meditation and
yoga relaxation techniques (Liner and Linnarsson 1994; Telles, Reddy et al. 2000).
Both methods aim to reduce metabolic rate already before breath-holding and
thereby improve breath-hold performance. Supplementation with dietary nitrate
(NO3-) may be a third, previously unexamined voluntary method, based on recent
discoveries of the ergogenic and metabolic effects of such supplementation in
exercising and resting humans.
The physiological effects of NO3- arise from its bioconversion to nitric oxide (NO),
an ubiquitous and evolutionary ancient signaling molecule (Olson, Donald et al.
2012) involved in regulation of several physiological responses including
mitochondrial respiration, mitochondrial biogenesis, calcium homeostasis, vascular
conductance and blood flow (Stamler and Meissner 2001; Dejam, Hunter et al.
2004; Cooper and Giulivi 2007; Larsen, Schiffer et al. 2011). Until recently it was
believed that NO3- and nitrite (NO2-) were merely byproducts of the pathway for
NO production, where NO could only be produced by oxidation of L-arginine,
requiring oxygen and nitric oxide synthase enzymes (NOS) for the reaction
(Gladwin, Schechter et al. ; Lundberg, Weitzberg et al. 2004). Only recently this
view has changed, with studies showing that humans and other mammals (Morris
2007; Racké and Warnken 2010), produce NO through both the L-arginine
pathway and the dietary nitrate-nitrite-nitric oxide (NO3-- NO2- -NO) pathway. The
best natural sources for NO3-
are celery, cress, chervil, lettuce, red beetroot,
18
spinach, and rocket (rucola), which typically contain over 250 mg nitrate per 100 g
fresh weight (Santamaria 2006; Hord, Tang et al. 2009; Jones 2014).
For a human at sea level with a normal dietary variation roughly half of plasma NO
is derived from L-arginine, and the other half from dietary NO3- (Zuckerbraun,
George et al. 2011). The two pathways differ fundamentally in their functional
capacity under hypoxic conditions. The L-arginine pathway involves the three
isoforms of nitric oxide synthases (NOS) - endothelial (eNOS), neuronal (nNOS)
and inducible (iNOS) - all of which oxidize L-arginine to L-citrulline and NO
utilizing O2 as a co-substrate (Stuehr 1999). Because of its dependency on O2,
hypoxia reduces the activity of this pathway (McQuillan, Leung et al. 1994;
Ostergaard, Stankevicius et al. 2007; Lundberg, Weitzberg et al. 2008; Lundberg
and Weitzberg 2009). During hypoxia both the production of eNOS (mRNA) (Ho,
Man et al. 2012), and its activity are reduced (Shaul, Wells et al. 1993;
Berkenbosch, Baribeau et al. 2000). Indeed, a reduction in the PO2 in the
endothelial tissue from 150 to 40 mmHg decreased NO production 52 % (Shaul,
Wells et al. 1993), and in both in vivo and in vitro trabecular smooth muscle
lowering PO2 leads to a gradual decrease in NO production (Kim, Vardi et al. 1993;
Robinson, Baumgardner et al. 2008).
The NO3-- NO2--NO pathway generates comparatively more NO when production
from L-arginine is attenuated during hypoxia (Giraldez, Panda et al. 1997;
Østergaard, Stankevicius et al. 2007; Sparacino-Watkins, Lai et al. 2012). Ingested
NO3- is rapidly absorbed by the gut and thereafter metabolized first to bioactive
NO2- and then to NO via the enterosalivary pathway (Benjamin, O'Driscoll et al.
1994; Lundberg and Govoni 2004) (Figure 3). This pathway involves active
absorption of NO3- by the salivary glands and its subsequent secretion into saliva,
where it is partially reduced to NO2- by facultative lingual bacteria. Then, in the
acidic gastric milieu some NO2- is converted to NO, some of which is absorbed into
the blood stream and converted back to NO2-. Thereafter, in the blood and
19
peripheral tissue NO2-- can
deoxyhemoglobin,
be converted into NO by proteins such as
deoxymyoglobin, neuroglobin, and cytochrome c oxidase
(Lundberg and Govoni 2004; Lundberg, Weitzberg et al. 2008).
Figure 3. The pathway taken by dietary nitrate, derived from consuming beetroot juice.
Systemically absorbed nitrate is concentrated 10-fold in the salivary glands and enters an
enterosalivary circulation where it is reduced to nitrite by bacterial nitrate reductases on the
dorsal surface of the tongue, and swallowed into the stomach providing a source of
systemically available nitrite/NO. Nitrite is transported in the arterial circulation to peripheral
vessels, where lower O2 tension favors the reduction of nitrite to NO, causing for example
vasodilatation and improved mitochondrial efficiency. Figure is adapted from Webb, Patel et
al. (2008).
20
Several recent studies suggest that dietary NO3- supplementation influences
physiological processes both at rest and during exercise in humans, whereby both
acute and long term NO3- supplementation may reduce the O2 cost and improve
tolerance and/or endurance performance in a variety of exercise modalities (Larsen,
Weitzberg et al. 2007; Bailey, Winyard et al. 2009; Vanhatalo, Bailey et al. 2010;
Lansley, Winyard et al. 2011; Breese, McNarry et al. 2013; Wylie, Mohr et al.
2013; Jones 2014; Kelly, Vanhatalo et al. 2014). Dietary NO3- supplementation, at
concentrations achievable through consumption of vegetables, furthermore appears
to result in more efficient energy production without increasing lactate
concentrations during submaximal exercise (Larsen, Weitzberg et al. 2007). As
submaximal O2 consumption has been considered intransigent to a variety of acute
exercise and pharmacological interventions, as well as to be essentially unaltered
by ageing or training (Jones, Wilkerson et al. 2003; Wilkerson, Berger et al. 2006;
Berger and Jones 2007; Bailey, Wilkerson et al. 2009), the prospect of its
improvement in normoxic conditions through normal dietary supplementation is
appealing. Furthermore, newer research has observed improved exercise tolerance,
oxidative function and arterial O2 status following NO3- supplementation in
hypoxia as well (Vanhatalo, Fulford et al. 2011; Kelly, Vanhatalo et al. 2014;
Casey, Treichler et al. 2015).
The ergogenic effect of reducing the O2 cost of exercise via NO3- is related to
enhanced muscle efficiency (Bailey, Fulford et al. 2010). Static breath-holding
places little demand on muscles, which would render the potential reduction of O2
consumption by NO3- supplementation somewhat redundant during this kind of
breath-hold. Research has, however, reported reductions in resting O2 uptake in
both healthy persons (Larsen, Schiffer et al. 2014) as well as patients with
peripheral arterial disease (Kenjale, Ham et al. 2011) through dietary NO3supplementation. In light of these findings, paper IV describes a study of voluntary
breath-holding performance and arterial O2 status in humans following NO3supplementation.
21
1.6. Transitory storage of erythrocytes in the spleen
In contrast to the metabolic restrictions associated with the diving response and the
potential effects of NO3- supplementation, the spleen may augment O2 availability
by mobilizing a transient blood pool during hypoxia. Many terrestrial and diving
mammals can mobilize large numbers of erythrocytes from the spleen and venous
pools during hypoxic challenge in order to improve oxygenation of metabolically
active tissue (Butler and Jones 1997). Such “autotransfusion”
improves O2-
carrying capacity and aerobic performance in highly active mammals such as the
fox, horse and grey dog, as well as the diving capacity of seals, among others
(Stewart and McKenzie 2002). Humans possess this function as well (Hurford,
Hong et al. 1990; Schagatay, Andersson et al. 2001).
1.6.1.
Contraction of the spleen in some mammals
The original observations on domestic animals, primarily dogs and cats, showed
the spleen’s varying volume and ability to rapidly discharge stored erythrocytes
(Schafer and Moore 1896; Barcroft, Khanna et al. 1932; Barcroft, Nisimaru et al.
1932). Further studies showed a return of blood volume to normal within minutes
after large infusions of saline, dextrose or whole blood in intact, but not in
splenectomized dogs (Roberts and Crandall 1933). Subsequent investigations on
animals with wide range of aerobic capacities, such as sheep (Turner and Hodgetts
1959), goats (Anderson and Rogers 1957), pigs (Hannon, Bossone et al. 1985) and
horses (Persson, Ekman et al. 1973), all documented significant elevation of
hematocrit during exercise, an effect that was eliminated via splenectomy. The
spleen was also noted to have an important role in restoring blood volume in
animals following conditions such as hemorrhage and shock (Guntheroth and
Mullins 1963; Carneiro and Donald 1977).
22
In marine mammals, splenic volume correlates well with the duration of the species
diving and foraging ability at sea (Kooyman, Castellini et al. 1981; Hochachka
1998). The deep diving Weddell seal stores approximately two thirds of its
erythrocytes in the spleen during rest, mobilizing these through splenic contraction
during diving and taking them up for storage again during rest (Qvist, Hill et al.
1986; Thornton, Spielman et al. 2001). Such reuptake by the spleen during nonhypoxic periods prevents excessive circulatory viscosity, which is important in
these animals with a very high hematocrit (Elsner and Meiselman 1995). Thus, the
conceivable physiological functions of spleen contraction is to help sustain tissue
oxygenation under physiologically stressful conditions and to minimize blood
viscosity during periods of rest.
1.6.2.
The human spleen
Originating from the mesodermal mesenchyme, the spleen is located in the upper
left abdomen beneath the diaphragm (Figure 4). It is surrounded by a smooth
external coat (the tunica serosa), while a fibroelastic internal coat (the tunica
albuginea) extends inward to form small fibrous bands, or trabecula, which
constitute the framework of the spleen (Takubo, Miyamoto et al. 1986). Within this
framework are two functionally different spaces: the red pulp and the white pulp.
The white pulp, its colour due to the large number of lymphocytes it contains,
performs important immunological functions. Interested readers are referred to
Bronte and Pittet (2013) for an extensive review of these functions. The red pulp,
colored by erythrocytes, represents one of the largest blood filters in the body
(Steiniger and Barth 2000).
23
Figure 4. Illustrations of the spleens location in the human body and the spleen with veins
and arteries. Credit: Rob 3000 Dreamstime.com-Spleen Photo
Blood enters the spleen via the splenic artery and then empties into the splenic
vein, which in turn drains into the superior mesenteric vein. Within the spleen,
arterial blood is localized into venous sinuses via filtering through fibrous slits by
striated
muscle
filaments
in
the
endothelial
lining
of
these
sinuses.
Immunohistochemical staining reveals contractile proteins within the walls of
arteries, veins, splenic capsule and trabeculae, as well as in the reticular cells of the
white pulp and sinus lining cells of the red pulp (Pinkus, Warhol et al. 1986).
Only about 75 % of the blood that enters the spleen is returned to the splenic vein,
likely due to filtration of plasma into the lymphatic system, which occurs in the red
pulp and results in an elevated hematocrit in the spleen’s stored contents (Isbister
1997). While old or deformed erythrocytes may be trapped and recycled in the
reticular meshwork here (Willekens, Roerdinkholder-Stoelwinder et al. 2003), the
remaining functional erythrocytes represent a reservoir of 200-250 mL of blood
24
(Bakovic, Valic et al. 2003) with more than twice the hematocrit of normal arterial
blood (MacDonald, Schmidt et al. 1991).
1.6.3.
Circulatory functions of the spleen in humans
Contraction of the human spleen was traditionally considered physiologically
insignificant, perhaps because splenectomy could be performed without any readily
perceived side-effects. However, since the documentation a of pronounced spleen
contraction after diving Korean Ama in 1990 (Hurford, Hong et al. 1990) and then
the observations of a reversible Hb elevation after apneas in non-diving volunteers,
but not in splenectomized subjects (Schagatay, Andersson et al. 2001; Bakovic,
Eterovic et al. 2005), a substantial number of studies have confirmed that the
human spleen contracts in a similar manner to that of several diving and running
mammals. Palada and collegues (2007) demonstrated that blood flow in the splenic
artery was unaffected during breath-holding, suggesting that splenic contraction
was active, rather than a passive collapse due to symphatetically mediated splenic
arterial constriction as was suggested previously (Allsop, Peters et al. 1992).
Furthermore the reversible elevations of Hb does not reflect hemoconcentration
due to extravasation of plasma, nor is it due to diuresis (Schagatay, Andersson et
al. 2001; Bakovic, Eterovic et al. 2005; Schagatay, Haughey et al. 2005).
1.6.4.
Mediation and control of the spleen contraction
Relatively little is known about the mechanism(s) that mediate spleen contraction
in humans, although numerous animal studies implicate the sympathetic adrenergic
system. All innervation of the rat, mouse, dog and human spleen is sympathetic,
and indeed the spleen is among the most densely adrenergically innervated organs
(Felten 2000). Several studies have showed that neurostimulation, epinephrine, and
norephineprine all cause alpha (α)-mediated contraction of the mammalian spleen
(Ayers, Davies et al. 1972; Davies, Powis et al. 1978; Ojiri, Noguchi et al. 1993;
25
Hurford, Hochachka et al. 1996; Kaufman, Siegel et al. 1998), while cholinergic
innervation of the spleen is limited (Reilly 1985).
Chemoreception might also stimulate splenic contraction via the adrenergic system.
The binding of α2-adrenergic receptors to their catecholamine agonist appears to
enable and even potentiate splenic contraction in rats following short-term hypoxia
(Kuwahira, Kamiya et al. 1999). Hypoxia has also been shown to cause a reduction
in splenic mass in dogs that can be reversed by normoxia (Kramer and Luft 1951).
In cats and dogs hypoxia appears to exert effects similar to those caused by electric
stimulation of the splenic nerve, resulting ultimately in emptying of its stored blood
(Donald and Aarhus 1974). Similarly, Hoka and colleagues (1989) found that
approximately 60 % of the pronounced reduction in splenic volume, caused by
severe hypoxia was due to sympathetic discharge by efferent nerves. During
hypoxic challenge hematocrit in the splenic vein during splenic contraction in dogs
is on average 80 % higher than in arterial blood and more highly oxygenated, with
a return to normal values upon restoration to normoxia. Such findings emphasize
the role of the spleen as a dynamic reservoir of well oxygenated blood in animals
(Kramer and Luft 1951).
In humans, hypoxia alone or in combination with breath-holding induces splenic
contraction, most potently in combination of both (Lodin-Sundstrom and
Schagatay 2010). One explanation for the stronger response during breath-holding
than during eupneic hypoxia could be the high partial pressure of carbon dioxide
(PaCO2) arising from breath-holding, although the possibility that PaCO2 itself can
initiate or modify splenic contraction has not yet been clarified. Therefore, a goal
of this thesis was to evaluate if there is a specific effect of PCO 2 on the splenic
response (Paper III).
26
1.6.5.
Spleen contraction during exercise
Both supine and upright exercise has been shown to decrease splenic erythrocyte
count (Sandler, Kronenberg et al. 1984). In one study using upright cycling it was
reduced to 34 % of resting counts (Laub, Hvid-Jacobsen et al. 1993) and in
another study by 56 % during submaximal and maximal position cycling (Stewart,
Warburton et al. 2003). Wolski (1999) documented a graded spleen volume
reduction from 338 mL to 283mL, 219mL and 143mL after 10, 20 and 30 min of
exercise, respectively; with no difference between untrained or aerobically trained
subjects.
1.6.6.
Spleen contraction during breath-holding and hypoxia
As with diving mammals, the human spleen thus has an ability to contract during
breath-holding, elevating Hb and Hct (Hurford, Hong et al. 1990; Schagatay,
Andersson et al. 2001; Bakovic, Eterovic et al. 2005) in a manner not attributable
to hemoconcentration (Schagatay, Andersson et al. 2001; Espersen, Frandsen et al.
2002; Bakovic, Eterovic et al. 2005). The response is less prominent than in diving
mammals, however, with reductions of 18-25 % observed after breath-holding in
humans resulting in a concurrent increase of Hb concentration typically in the
range of 3-6 % (Hurford, Hong et al. 1990; Schagatay, Andersson et al. 2001;
Bakovic, Valic et al. 2003; Schagatay, Haughey et al. 2005).
Three to five sessions of breath-holding typically maximizes the spleen contraction
(Schagatay, Haughey et al. 2005) although Bakovic and associates (2005) have
suggested a single breath-hold can elicit a full response. The potential influence of
the duration of breath-holding on the extent of contraction is controversial: there is
one report of pronounced splenic contraction after short breath-holding (Espersen,
Frandsen et al. 2002), while in another study this contraction increased with breathhold durations (Schagatay, Haughey et al. 2005). A biphasic contraction pattern has
been observed in humans, characterized by immediate splenic contraction followed
27
by gradual contraction during the course of the breath-hold (Lodin-Sundström,
Richardson et al. 2009). This suggests the involvement of different stimuli in
triggering the response during breath-holding, one of which is likely hypoxia.
Others are probably specific to breath-holding, however, as 20 min of normobaric
hypoxia has been shown to elicit less spleen contraction than 2-min of breathholding (Lodin-Sundstrom and Schagatay 2010).
Across series of breath-holds separated by 2-3min intervals, the spleen remains
contracted (Schagatay, Andersson et al. 2001; Bakovic, Valic et al. 2003;
Schagatay, Haughey et al. 2005). Upon removal of triggering stimuli the spleen
returns to its normal resting volume in about 10 minutes (Schagatay, Andersson et
al. 2001; Espersen, Frandsen et al. 2002; Bakovic, Eterovic et al. 2005; Schagatay,
Haughey et al. 2005).
The normal resting size of the spleen varies greatly between individuals
(Prassopoulos, Daskalogiannaki et al. 1997; Hosey, Mattacola et al. 2006), as does
its ability to contract (Schagatay et al. 2005). The initial observations of spleen
contraction in Ama divers (Figure 5) (Hurford, Hong et al. 1990) raised questions
as to whether this response is only prominent in experienced breath-holders and
whether it can be improved by training or environmental acclimatization. There is a
strong correlation between splenic volume and breath-hold diving capacity, with
spleens as large as up to 600 ml in the best breath-hold divers (Schagatay,
Richardson et al. 2012). The increase in Hb following breath-holding was greater
in breath-hold divers than untrained subjects or endurance athletes (Richardson, de
Bruijn et al. 2005). A significant spleen contraction resulted only in the divers
compared to control subjects (Prommer, Ehrmann et al. 2007). Whether these
differences are due to predisposition or training is unclear. The growth of accessory
spleens after surgical removal of the original organ (Voet, Afschrift et al. 1983)
demonstrates pronounced regenerative ability and therefore implies a potential for
growth e.g during acclimatization or training.
28
Figure 5. An Ama diver at work collecting abalone and turbo shells. The Ama have an over
2000 year old tradition of sea harvesting for e.g. sea-molluscs (Schagatay, Lodin-Sundstrom
et al. 2011). Photo: Erika Schagatay
A logical query for further examination is then how the spleen may respond to, and
affects the outcome of, exposure to altitude. Physiological reactions to decreased
PO2 are often dependent on the rate, as well as the degree of reduction. When onset
of hypoxia is rapid, the ventilation is increased and changes in cardiovascular
dynamics occur within minutes, including sympathetically augmentation of HR,
constriction of venous capacitance vessels and centralization of blood volume
(West 1982). During days and weeks at higher altitude, a hypoxia-mediated
increase of erythropoietin (EPO) production and release stimulates erythropoiesis,
thereby elevating the blood concentration of Hb (Gore, Sharpe et al. 2013).
However, “autotransfusion” from the spleen may enhance the delivery of
29
oxygenated blood to metabolically active tissues on a considerably shorter timescale.
The present thesis examines for the first time whether the resting volume of the
spleen and/or its ability to contract can be altered with repeated breath-hold
training (Paper I) or with long-term exposure to high altitude (Paper II).
2.
AIMS OF THE THESIS
The general aim of this thesis was to increase our understanding of the human
ability to cope with hypoxia by investigating cardiovascular and hematological
responses as well as the influence of dietary nitrate during hypoxic interventions.
Specific research questions addressed were:
Breath-hold training (I)
Does two weeks of breath-hold training modify the cardiac components of the
diving responses to breath-holding? Does such training alter the extent of splenic
contraction caused by breath-holding?
Long-term hypobaric hypoxic exposure (II)
Does long-term exposure to hypoxia during climbing at high altitude alter the
extent of splenic contraction in response to breath-holding and exercise?
Hypercapnia (III)
Does hypercapnia influence the spleen-related increase in circulating Hb following
breath-holding?
30
Acute dietary nitrate supplementation (IV)
Is the duration of breath-holding affected by acute dietary nitrate supplementation?
Are the oxygen costs and heart rate response during voluntary breath-holding
modified with dietary nitrate supplementation?
3.
METHODS
3.1. Subjects
Thirty-eight healthy subjects volunteered to take part in different studies in the
present thesis. The characteristics of the subjects are summarized in table 2.
Table 2. Subject characteristics in each study.
Study nr
Age
(years)
26 (5)
Body
mass (kg)
72 (9)
Height (m)
Study
population/characteristics
1.7 (0.1)
Study II:
n=8
35 (7)
71 (13)
1.7 (0.1)
Study III:
n=8
28 (7)
78 (19)
1.8 (0.1)
Study IV:
n=12
32 (7)
69 (10)
1.8 (0.1)
6
males,
all
subjects
Caucasian, limited lifetime
experience in breath-holding
with
no current breath-holding
activity
7 males,5 Sherpas and 3
Caucasian (1female) Trained
mountain climbers
4
males,
all
subjects
Caucasian, limited lifetime
experience in breath-holding,
with
no current breath-hold activity
9 males, all subjects
Caucasian, subjects trained
for breath-hold diving for at
least 2 h/week
Study I:
n=10
Mean (SD) values for all subjects. n= number of subjects.
31
3.2. Arterial O2 saturation and cardiovascular measurements
3.2.1.
Arterial O2 saturation and heart rate
There are several ways to measure the O2 level in the body depending on the site of
interest. Some well known methods to measure O2 level at various sites include
ergospirometry (respiratory gas analysis) for systemic O2 consumption (Hollmann
and Prinz 1997), arterial blood gas analysis to determine the level arterial partial
pressure of O2 (Syabbalo 1997), near-infrared spectroscopy (NIRS) for monitoring
of regional hemoglobin O2 saturation values (McCully and Hamaoka 2000) and
arterial O2 saturation (SaO2) to measures the percentage of Hb binding sites in the
arterial bloodstream occupied by O2 (Nitzan, Romem et al. 2014). In the works of
this thesis SaO2 was obtained by finger pulse oximetry (Paper I and II ;Biox 3700e,
Ohmeda, Madison, Wisconsin, USA, Paper III, IV; Medair Lifesense LS1-9R,
Nonin Medical Inc., Medair AB, Delsbo, Sweden). A pulse oximeter is an easily
administered, non-invasive tool that indirectly monitors arterial O2 saturation. It
utilizes an electronic processor and a pair of light-emitting diodes sending red light
(wavelength 660 nm) and infrared light (wavelength 940 nm) towards a photodiode
through a translucent body part, in this thesis the fingertip. The pulse oximeter
computes the percentage of Hb saturated with O2 (SpO2) by measuring differences
in red and infrared absorbance of fully oxygenated and deoxygenated arterial Hb
(Mendelson 1992).
At sea level normal SaO2 values are 97 % to 99 % in the healthy individuals.
Clinical hypoxemia is defined as SaO2 values at or lower than 90 %, which
according to the oxyhemoglobin curve corresponds to a PaO2 of 60 mmHg
depending on e.g the PaCO2 (Siggaard-Andersen and Siggaard-Andersen 1990). In
healthy subjects the differences between pulse oximetry SaO2 (SpO2) values and
32
SaO2 values obtained via analysis of arterial blood sample are considered to be
small and stable (Perkins, McAuley et al. 2003). Erroneous readings may occur as
a result of hypoperfusion, vasoconstriction or movement of the limb being
measured (Barker and Shah 1996; Bohnhorst, Peter et al. 2000; Gehring,
Hornberger et al. 2002; Shah, Ragaswamy et al. 2012), although such disturbances
are readily detectable.
Heart rate can be detected by a number of methods including palpation,
electrocardiography
(ECG),
photopletysmography,
oscillometry
and
phonocardiograph (Laukkanen and Virtanen 1998; Achten and Jeukendrup 2003).
In this thesis, HR was also measured by the pulse oximetry device, which detects
fluctuations of light due to the change in flow of arterial blood produced during the
cardiac cycle and thereby calculates HR, after automatically correcting for the
effects of other tissues (Mendelson 1992). For a more detailed description of this
method, the reader is referred to the individual papers (I, II, III, IV).
3.2.2.
Blood pressure measurements
The mean arterial pressure (MAP) describes the average arterial pressure during a
single cardiac cycle. Two main physiological components determining MAP are
cardiac output and systemic vascular resistance. This means that factors affecting
these two parameters including ventricular preload, venous compliance and blood
volume also will affect MAP (Daniels, Kimball et al. 1996). At normal or low heart
rates it can be estimated according to the following equation: MAP = DP + 1/3(SPDP) where SP and DP are systolic and diastolic pressure, respectively (Razminia,
Trivedi et al. 2004). MAP is considered to be the blood perfusion pressure at the
organ level, and it is expected that values below 60 mmHg are insufficient to
sustain adequate oxygenation of the tissue of vital organs (1984).
33
In paper I MAP was measured continuously from the finger via automated
sphygmonometer (Finapres 2300, Ohmedia, Madison, WI, USA). In this method a
cuff is wrapped around the finger, and the diameter of an artery under the cuff is
kept constant, or “clamped” at a certain set-point diameter in spite of changes in
arterial pressure during each heart beat. Changes in diameters are detected via
infrared photoplethysmograph built into the finger cuff and immediately adjusted
for by a pressure servo-controller system to prevent the diameter change. In this
way the device is able to compute the intra-arterial pressure during a cardiac cycle
by adjusting and tracking the finger cuff pressure (Bogert and van Lieshout 2005).
This device has shown good correlation with measurements of MAP obtained via
invasive (i.e intra radial catheter) methods during both rest and light exercise
(Raamat, Jagomagi et al. 2003). Reliability of finger sphygmomanometer
measurements can be influenced by room temperature, change of limb posture
(Netea, Lenders et al. 2003) and extreme peripheral vasoconstriction (Pickering
2002) in which case the detector elicits a warning signal. In study I the two former
aspects were held constant and should not have influenced measurements.
Blood pressure was measured in paper IV using an automated upper arm inflation
blood pressure monitor (Omron M41 automated blood pressure monitor, Omron
Healthcare, Europe).This device uses an empirically derived algorithm to calculate
pressure (Pickering 2002), but tends to underestimate the systolic pressure in
comparison to direct intra-arterial measurements. Reliability of automated cuff
system can be influenced by room temperature, positioning of the arm, isometric
exercise (muscle tension), talking, background noise and arterial inflexibility due to
old age (Pickering 2002). In paper IV these factors were held constant and
disturbances limited, and should therefore have not substantially influenced
measurement.
34
3.3. Respiratory movement measurements
In paper I, breathing movements were monitored using a lab-developed chest
bellows placed around the circumference of the thorax below the xiphoid process.
Breathing changes the chest circumference and in turn the tension on the bellows,
such that oscillations are amplified and converted to a digital signal for online
recording. Breathing movements were recorded during breath-holding to identify
the onset of involuntary breathing movements, often referred to as the
physiological breaking point of the breath-hold (Lin, Lally et al. 1974).
3.4. Respiratory gas measurements
In paper I and III inspired and expired percentages of O2 and CO2 were measured
via gas analyzer (Normocap Oxy, Datax-Ohmeda, Helsinki, Finland) utilizing
paramagnetic and spectrophotometric technologies for measurement of O2 and CO2
respectively. The paramagnetic sensor technology works on the principle that O2
molecules experience a force in a magnetic field that is proportional to the O2
partial pressure, which in turn gives rise to electrical signals that can be translated
into standard units. The spectrophotometric technology works on the principle that
CO2 absorbs infrared radiation. When a beam of infrared light passes across the gas
sample, the CO2 leads to a reduction in the amount of light falling to the sensor
which changes the voltage in a circuit and can be translated into standard units
(Soubani 2001; Jaffe 2008).
The CO2 level prior to breath-holding was measured to ensure that it was similar
between repeated breath-holds, and to detect any unwarranted hyperventilation. In
was also used to ensure that hyperventilation, when intended, occurred at the level
defined by the protocol (Paper III). Expired CO2 levels were used as indicators of
35
CO2 production during breath-holding. In paper III inspired and expired O2 levels
were measured to identify any potential hypoxic influence.
3.5. Blood variable measurements
The main hematological measurement in these studies contained in this thesis was
Hb (reported in g/L). The measures were obtained either via intravenous catheter in
the antecubital region of the arm (Venflon Pro, Beckton-Dickson AB, Helsingborg,
Sweden), or via capillary puncture of the finger. Intravenous samples were
analyzed for Hb and Hct via automated blood analysis unit (Micros 60 Analyzer,
ABX Diagnosis, Montpellier, France). Capillary samples were analyzed in
triplicate via Hb analyzer (B-Hemoglobin Photometer, Hemocue AB, Ängelholm,
Sweden). In paper I blood samples for analyses of reticulocytes were transported in
a timely manner to an accredited hospital laboratory in Östersund, Sweden and
analyzed via automated blood analysis unit (Cell –Din, ABBOTT, San Fransisco,
California, USA). For details of measurements of blood variables see papers I, II
and III.
3.6. Spleen volume measurements
Spleen size was measured in two studies (I and II) using triaxial measurements of
frozen images obtained via portable ultrasound apparatus ( Mindray DP-6600,
Shenzen Mindray Bio-Medical Electronics Co., LTd., Shenzhen, China). In this
method, a narrow beam of ultrasound is radiated from a transducer towards the
tissue to be imaged. This tissue reflects and scatters the beam, giving rise to echoes
that are detected by a transducer, recorded and displayed as an image (Hosey,
Mattacola et al. 2006). Ultrasoundography was performed by an experienced
technician. The spleen was visualized with the participant in the supine decubitus
position (Paper I) or sitting position (Paper II) with the ultrasound probe angled
36
intercostally from a posterolateral position. Measurements were taken in the
sagittal and transverse plans yielding three different diameters on the observed
spleen image: maximal length (L), maximal thickness (T) and maximal width (W).
After the ultrasound operator had manually detected the edges of the spleen at its
maximal outer dimensions using the displayed image, the device’s software
calculated the according diameters (Figure 6). These diameters were used for
calculation of spleen volume, using the following formula developed by S.
Pilström: L (WT-T2)/3. This formula is based on observed ultrasound images of the
shape of spleen, and expresses the difference between two ellipsoids divided by
two. The calculated volume using this formula was compared to that obtained by
Breiman and associates (Breiman, Beck et al. 1982) and Sonmez and associates
(Sonmez, Ozturk et al. 2007) using the following formula: length x thickness x
width x 0.523, showing that the two methods on average differed by less than 4 mL
or less than 1 % of the measured volume. A limitation of the method is that relies
on operator skills to discriminate the edges and might be affected by the spleens
shape and contour of the image (Hoefs, Wang et al. 1999).
Figure 6. Ultrasound image of the human spleen. Diagonals with maximal length(1) and
thickness(2) are marked as dotted lines. Photo: Angelica Lodin-Sundström
37
3.7. Experimental protocols
3.7.1
Voluntary breath-holding
Voluntary breath-holding was the intervention used to induce hypoxia in all
studies. Successive breath-holds with less than 10 minutes in between have been
shown to increase breath-hold duration (Heath and Irwin 1968; Hentsch and Ulmer
1984), which in turn has been causally linked to increased
spleen
contraction(Schagatay, Andersson et al. 2001). This model has been used in many
breath hold-associated investigations of the cardiovascular diving response and
human spleen contraction (Schagatay and Holm 1996; Schagatay 2009), and it was
deemed appropriate to employ the same protocol in the present studies to ensure
compatibility with previous works.
All studies followed essentially the same breath-holding protocol, with the
exception of that in paper II, where ergometer cycle exercise was added, in quiet,
controlled environments. On experimental test days, study participants were
instructed to refrain from tobacco, caffeine containing beverages, exercise and
breath-holding outside the experimental protocol. Full meals were to be avoided at
least 2 hours prior to testing and all eating was to be avoided one hour prior to
testing. Before interventions a detailed instruction of procedures were given orally
and in writing after which study participants confirmed their informed consent with
a signature. All necessary measurement equipment probes were then placed on the
participants. Thereafter study participants rested in a prone position (except for the
study presented in paper II) to minimize metabolic activity (Rubini, Paoli et al.
2012) and allow blood mixing and stabilization of the transcapillary fluid exchange
(Lundvall and Bjerkhoel 1985; Hinghofer-Szalkay and Moser 1986). In paper II a
sitting position was maintained prior to breath-holding, as this was the posture that
was to be used during the remainder of the protocol involving cycling exercise.
38
Two or three successive breath-holds spaced by at least two minutes of rest were
performed by the study participants in all studies, with breath-hold duration as a
measured variable. All breath-holds were preceded by a 2 minute countdown
period. A nose clip was applied 30 s before the breath-holds, as well as a mask or a
mouthpiece was placed for inspiration of pre breath-hold gas mixture or for
measurements for inspired or expired gases (study I, III, IV). Study participants
began each breath-hold following a 10 s verbal countdown from the experimenter,
and according to instruction exhaled fully and then inhaled deeply but not
maximally before ceasing to breathe. These instructions have previously been
shown to result in approximately 85 % of vital capacity (Schagatay 2009) which is
optimal for non-immersion breath holds as it avoids the risk of overfilling the lungs
and subsequent risk for syncope due to impeded venous return (Potkin, Cheng et al.
2007; Schagatay 2009). In the study reported in paper III pre breath-hold
hyperventilation was required, and the study participants were given verbal
feedback regarding rate and depth of breathing by the experimenter. Other than
this, the breath-hold was conducted in the same manner as in non- hyperventilation
trials. Time cues during breath-holds were not used, but in the study reported in
paper III participants were verbally instructed to terminate the submaximal breathholds on cue. Additionally, verbal cues were given in the study reported in paper II
to denote the start and termination of cycling exercise. Just prior to resumption of
breathing the experimenter provided study participants with a mask or a
mouthpiece through which the participants were instructed to expire at the end of
their breath-hold.
3.7.2.
Dietary nitrate supplementation
Inorganic NO3- can be supplemented through processed NO3- -rich vegetable
products (Butler and Feelisch 2008). In paper IV a concentrate of organic beetroot
juice was used as a donor of inorganic NO3-. The 70 ml beetroot juice servings
contained 5.0 mmol NO3- while the 70 ml NO3- depleted beetroot juice servings
39
used as a placebo contained 0.003 mmol NO3- (Lansley, Winyard et al. 2011).In
the placebo servings, NO3- was removed via ion-exchanging resin, while the color,
taste, smell and texture remained indistinguishable from the non-placebo servings
(Lansley, Winyard et al. 2011). The experimental intervention in paper IV was
designed to be 2.5 hours after dietary NO3- ingestion. While plasma NO2- was not
measured, it has been shown that concentration of this substance in the plasma is
maximal 2-3 h following ingestion (Webb, Patel et al. 2008), and similar protocols
have been adopted in several studies (Webb, Patel et al. 2008; Wylie, Kelly et al.
2013; Bentley, Gray et al. 2014).
3.8. Data analysis and statistics
3.8.1.
Data analysis
All experiments in this thesis were controlled crossover trails, whereby each study
participant served as their own control. Absolute values of the respiratory,
cardiovascular, splenic and hematological variables, as well as the relative changes
during breath-hold and exercise, were analyzed. A randomized, double-blind
placebo controlled design was used in the study presented in paper IV, a design
considered to be the “Gold Standard” in intervention-based studies (Misra 2012).
3.8.2.
Statistical analysis
The main statistical outcome was the mean difference of the variable of interest
between treatments or from control, whereby intra-individual comparisons were
utilized. A period of 30 s or 60 s prior to each breath-hold was established as a
reference period for the subsequent breath-hold. This reference period was then
used to analyze changes occurring during the following breath-hold, as well as
changes among the breath-holds performed in a series. The chosen statistical test
for each study varied depending on the analysis required, including the following:
Student`s t-test, one-way analysis of variance (ANOVA), and two-way ANOVA
40
repeated measures (ANOVA RM). A Bonferroni correction for the Student`s t-test
was used for multiple comparisons when applicable. Both absolute values using the
units and scales of the variable of interests, and relative values as percentage
change from reference or control values were presented. In all studies statistical
significance was accepted at P<0.05 and non-significant trends were denoted for
P< 0.1.
In the study presented in paper III all variables were log transformed before
analysis to reduce non-uniformity of error. Excel templates were used for the
calculations, purpose-designed for analyses using physiological data (Available
from: Internet Society for Sport Science. http://www.sportsci.org/resource/stats/).
3.9. Ethics
Experiments were conducted in accordance with the Declaration of Helsinki
regarding research involving human participants, and had been ethically approved
by the human research ethics board of Umeå University. The study presented in
paper II was additionally approved by the Nepali Health Research council (NHRC)
as data collection occurred in that country. Prior to the experiments subjects were
thoroughly informed of the procedures, measurements and possible risks involved,
after which they gave their written informed consent. Subjects were aware they
could abort the protocol at any time, but no subject did so.
41
4.
MAIN RESULTS
The individual papers:
Paper I:
Effects of two weeks of daily apnea training on diving response, spleen
contraction, and erythropoiesis in novel subjects
In this study, which aimed to investigate the effects of breath-hold training on
diving response, spleen contraction and erythropoiesis, 10 untrained participants
followed a breath-hold training program consisting of 10 maximal breath-holds
each day for two weeks. Study participants were tested before and after the training
period in a protocol consisting of 3 maximal breath-holds spaced by 2 minutes
pauses, as well as an additional breath-hold protocol conducted after the training
period in which the participants repeated the same breath-hold durations as they
performed in the test series prior to the training period. Following the training
period, maximal breath-hold duration had increased by 44 s (28 %; P<0.01).
Diving bradycardia started 3 s earlier and was more pronounced after the training
period. Spleen contraction was, however, similar in all tests. The SaO2 nadir after
the breath-holds of the same duration was 84 % pre-training and 89 % post-training
(P<0.05), while it was 72 % (P<0.05) after maximal breath-holds post-training
(Figure 7). Baseline Hb remained unchanged after the training period, while the
reticulocyte count had increased by 15 % (P<0.05). It was concluded that the
higher SaO2 after repeating breath-holds of similar duration after training was
mainly due to the more pronounced diving response, as no enhancement of spleen
contraction or Hb was observed.
42
Figure 7. Percent change in arterial hemoglobin oxygen saturation (SaO2) from baseline
values during the third breath-hold in all three tests. During “mimicposttest” the duration of
the breath-holds in the posttest was the same as in the pretest, while during the”
maxposstest” the duration of the breath-holds in the posttest was maximal. Values are
means (SD) for 9 subjects. *P<0.05 .
Paper II
The Effect of Climbing Mount Everest on Spleen Contraction and Increase in
Hemoglobin Concentration During Breath-holding and Exercise
In this study, the effects of long-term altitude exposure on spleen contraction
during breath-holding and exercise were investigated after a 45-day Mt. Everest
(8848 m) expedition. Eight climbers performed the following protocol to evoke
spleen contraction before and after the successful climb: 5 min ambient air
respiration at 1370 m during rest, 20 min O2 respiration, 20 min ambient air
respiration at 1370 m, three maximal-effort breath-holds spaced by 2 min, 10 min
ambient air respiration, 5 min of cycling at 100 W, and finally 10 min ambient air
43
respiration. Ultrasonic imaging was used to determine spleen volume. Mean (SD)
baseline spleen volume was unchanged at 213 (101) ml before and 206 (52) ml
after the expedition. Before the expedition, spleen volume was 176 (80) ml after
three breath-holds (NS), while after the expedition three breath-holds resulted in a
spleen volume of 122 (36) ml (P = 0.01). The pre-expedition spleen volume
following 5 minutes of cycling was 186 (89) ml compared with 112 (389) ml after
the expedition (P = 0.03; Figure 8). Breath-hold duration and cardiovascular
responses to breath-holding were unchanged after the expedition. It was concluded
that spleen contraction was enhanced after long-term climbing at altitude.
Figure 8. Mean percent (SD) change in spleen volume from baseline immediately
following each breath-hold (BH1, BH2, BH3), exercise, and recovery pre- and postaltitude. * denotes significance at p < 0.05 in spleen volume reduction from
baseline and between tests while + denotes trend at p < 0.1 for difference of
spleen volume between tests (n=8).
44
Paper III
Effect of hypercapnia on spleen-related haemoglobin increase during apnea
This study investigated the influence of hypercapnia on spleen-related increase in
Hb concentration. Eight non-divers performed three separate series of breath-holds
after inspiring different CO2 levels mixed in O2. Each series consisted of 3 breathholds spaced by 2 min pauses, with individually fixed breath-hold durations: one
with pre-breathing of 5% CO2 in O2 (‘Hypercapnia’); one with pre-breathing of
100% O2 (‘Normocapnia’); and one with hyperventilation of 100% O2
(‘Hypocapnia’). In a fourth trial, study participants breathed 5 % CO2 in O2 for the
same duration as their breath-holds (‘Eupneic hypercapnia’). Hb concentration
increased by 4 % after breath-holds in the ‘Hypercapnia’ trial (P = 0.002) and by 3
% in the ‘Normocapnia’ trial (P = 0.011), while the ‘Hypocapnia’ and ‘Eupneic
hypercapnia’ trials showed no changes. The period without involuntary breathing
movements, known to depend on the CO2 level, was longest in the ‘Hypocapnia’
trial and shortest in the ‘Hypercapnia’ trial. No differences in the cardiovascular
diving response were observed between trials and no arterial O2 desaturation
occurred in any trial.
Spleen size was measured in a subgroup of three subjects using ultrasonic imaging.
A decrease in spleen size was evident in the hypercapnic trial (-33 %), whereas in
the hypocapnia trial spleen size increased (30 %), while only minor changes
occurred in the other trials. It was concluded that there may be a dose-response
effect of CO2 on splenic emptying during breath-holds in the absence of hypoxia.
45
Paper IV
Acute dietary nitrate supplementation improves dry static apnea performance
This study investigated the effects of acute dietary nitrate supplementation on
breath-hold duration. Twelve well-trained breath-hold divers ingested 70 ml of
nitrate rich beetroot juice (BR) containing 5.0 mmol of nitrate and 70 ml of
placebo beetroot juice (PL) containing
0.003 mmol of nitrate on separate
occasions in a weighted order. At 2.5 hours after ingestion study participants
performed 2 sub-maximal breath-holds of 2 min duration spaced by 2 min pauses,
followed by a final maximal breath-hold. After ingestion of nitrate rich beetroot
juice, maximal breath-hold duration increased by 11 % compared to placebo (PL:
250± 58 vs. BR: 278 ± 64 s; P = 0.04). The mean nadir for SaO2 after the two submaximal breath-holds was 97.2 ± 1.6% in PL and 98.5 ± 0.9% in BR (P =
0.03;Figure 9). Relative to PL, BR reduced resting mean arterial pressure by 2 %
(PL: 86 ± 7 vs. BR: 84 ± 6 mmHg; P = 0.04). The reduction in HR from baseline
due to the diving response did not differ between PL and BR. It was concluded that
acute dietary nitrate supplementation increased breath-hold duration by reducing
metabolic cost due to effects unrelated to the diving response.
46
Figure 9. Mean (SD) percent change in nadir arterial hemoglobin oxygen saturation (SaO2)
from the two sub-maximal breath-holds and maximal breath-holds in placebo and nitrate
conditions.*P≤0.05. Note that the time to breath-hold termination was extended following
nitrate supplementation in maximal trails (n=12).
5.
DISCUSSION
The results presented in this thesis show that humans employ various physiological
responses when faced with hypoxic challenge from the environment, and that some
of these responses can be altered through training. These responses include
conservation of metabolism via reflex bradycardia, redistribution of blood flow,
and expulsion of erythrocytes from the spleen into the circulation. There is also
behavioral acclimatization that can be employed, including ingestion of exogenous
dietary sources of nitrate (NO3-) to more efficiently use the available O2. One
discovery was that the HR response during breath-holding occurred earlier and was
more pronounced following a training period. The first finding is novel whereas the
latter finding is in accordance with previous studies of Schagatay, van Kampen et
al (2000), and this effect is likely a key factor in the observed improvements in
performance seen in apnea athletes that trained regularly. Another novel finding
47
was that the spleen response is enhanced by prolonged periods at high altitude,
which involves exercise and long-term chronic hypoxic exposure. This effect was
not seen during a shorter period of breath-hold training, suggesting that long-term
hypoxic exposure with exercise is a more powerful stimulus for the splenic
response than intermittent hypoxia from breath-holding. Finally, the dose-response
effect of CO2 on spleen contraction during breath-holding without changes in
oxyhemoglobin saturation levels suggests that CO2 may also trigger the response.
5.1. The heart rate response after breath-hold training (Paper I)
The greater magnitude of bradycardia during breath-holding following training was
particularly notable in the last phase of the breath-hold. The earlier occuring and
augmented bradycardia in combination with a diminished arterial O2 desaturation
during breath-holding suggest that the diving bradycardia was more O2 conserving
following training.
This is strengthened by the fact that neither spleen contraction nor baseline Hb
concentration increased after training, a finding not previously studied. Earlier
studies showed that the diving response is important for O2 conservation in humans
(Schagatay and Andersson 1998; Lindholm, Sundblad et al. 1999; Andersson,
Liner et al. 2002). The study described in paper I in this thesis confirms Schagatay
and colleagues (2000) observations of a more pronounced cardiovascular diving
response and improved O2 conservation during breath-holding after two weeks of
breath-hold training. However, none of the earlier studies included measurements
of spleen contraction. The spleen contraction could have been a confounding factor
responsible for attenuating arterial deoxygenation during breath-holding, thereby
leading to incorrect conclusions about the effects of diving bradycardia in humans.
In the present study both of these responses were measured in parallel, and this
48
study showed no augmentation of spleen contraction during breath-holding after
the training period, while HR reduction developed faster and was more
pronounced.
A number of investigations in animals have revealed a greater variability in diving
bradycardia during dives in non-captivity compared to those performed in forced
submersion, with a stronger, more definite response occurring in the latter
(Kooyman and Campbell 1972; Williams, Kooyman et al. 1991; Ponganis,
Kooyman et al. 1997). The results of voluntary breath-hold investigations may
therefore not be directly reflective of the maximum response possible, which is
likely reserved for situations involving acute asphyxia. Should the human diving
response be modified by higher cortical influence, as some field studies have
revealed in marine mammals that adjust their diving response depending on
situational circumstances (Kooyman 1989; Butler and Jones 1997; McCulloch
2012), it would be plausible that part of the faster and more pronounced HR
reduction represents a learned response to a goal-oriented behavior in which the
individual regulates their responses to maximize breath-hold duration.
The present study also suggests that the cardiovascular diving response and the
spleen contraction are separate responses. The opposite was proposed in early
studies (Schagatay, Andersson et al. 2001; Espersen, Frandsen et al. 2002),
although more recent studies have revealed that the initiation and temporal and
functional aspects of the two responses are different (Schagatay, Haughey et al.
2005; Schagatay, Andersson et al. 2007; Lodin-Sundstrom and Schagatay 2010).
The cardiovascular diving response is reflexively initiated by breath-holding, and
facial chilling – the latter a response involving stimulation of trigeminal coldreceptors (Schuitema and Holm 1988). However, the spleen contraction is not
improved by facial chilling and has a differnt time pattern for its development,
suggesting different etiology of the responses (Schagatay, Andersson et al. 2007).
49
The exact mechanism by which the more pronounced bradycardia was achieved
after breath-hold training cannot be determined from the present study. Since only
dry breath-holds were performed and ambient temperature was kept constant,
stimuli arising from temperature-sensitive trigeminal face receptors can be
eliminated. Other mechanisms, however, that alter control systems of the diving
response including stimuli input, functions of central parasympathic and vasomotor
centers, excitatory neural connections and changes of signal transference to and in
the heart itself are likely candidates. Investigations of endurance athletes have
revealed a lower resting HR after long term training due to increased
parasympthateic tone (Alom, Bhuiyan et al. 2011), and remodeling of the sinoatrial
node has also been documented after exercise training (Boyett, D'Souza et al.
2013). Potentiation of the diving response by face immersion in humans is related
to increased vagal activity (Hayashi, Ishihara et al. 1997), and changes in this
neural pathway could be involved in an increase of the response after training.
Animal studies have revealed that sustained stimulation of the carotid bodies by
hypoxic blood may result in bradycardia and vasoconstriction (Donnelly 1997).
While hypercapnia, and in particular hypoxia, are important chemoreceptor stimuli
for maintenance of the diving response in humans as well (Lin, Shida et al. 1983) ,
it is less feasible that such contributions increase the response as O2 desaturation
was attenuated following the training period. Whether long-term intermittent
hypoxia could potentiate chemoreceptor input needs to be further investigated.
In humans, the general HR reduction during breath-holding ranges from 15- 40 %
of resting HR although a small proportion of humans can develop bradycardia
below 20 beats/minute (Alboni, Alboni et al. 2011). The magnitude of HR
reduction after two weeks of breath-hold training in the present study is in line with
that described by Schagatay et al. (2000). Terblanche and colleagues (2004)
showed that the diving response in individual humans is reproducible over many
years, suggesting relatively strong inherent response characteristics. Genetic
50
variability of the diving response has indeed been investigated, and such a
component suggested, in rats (Fahlman, Bostrom et al. 2011). Thus, the response is
likely to some extent be inherent and to another part a response to training.
5.2. Dietary nitrate supplementation during static breathholding (Paper IV)
Another novel finding in this thesis was that dietary NO3- supplementation
increased breath-hold duration, a discovery which implies that the NO3- -NO2- -NO
pathway can modify metabolic function in resting and hypoxic tissues. Although
the exact mechanism responsible for this effect cannot be determined from the
present study, the higher arterial O2 saturation seen in the in high [NO3- ] trial
supports reduced metabolic rate as a candidate mechanism.
Much of the current knowledge related to the ergogenic effects of dietary NO3supplementation derives from exercise studies, where the effect is linked to
increased muscle efficiency (Jones 2014). Reduced O2 consumption after NO3supplementation seems to be a consistent finding during submaximal
exercise(Larsen, Weitzberg et al. 2007; Bailey, Winyard et al. 2009; Bailey,
Fulford et al. 2010; Vanhatalo, Bailey et al. 2010; Kenjale, Ham et al. 2011;
Lansley, Winyard et al. 2011; Larsen, Schiffer et al. 2011), while the reduction is
slightly more inconsistent during maximal exercise, with only some studies
observing decreased O2 consumption (Larsen, Weitzberg et al. 2010; Bescos,
Rodriguez et al. 2011; Lansley, Winyard et al. 2011) and others showing no effect
(Larsen, Weitzberg et al. 2007; Bailey, Winyard et al. 2009).
It should be emphasized that the results of the previous exercise studies, involving
a high degree of muscle work, cannot be directly converted to resting hypoxic
conditions for example at static breath-holding where voluntary muscle work is
minimal. Potential hemodynamic changes after dietary NO3- supplementation, such
as increased peripheral blood flow and improved vascular conductance (Webb,
51
Patel et al. 2008; Lidder and Webb 2013), may yield different effects given the
dissimilar role of the circulatory system between exercise and resting breathholding; during exercise the increased O2 requirement of the active skeletal muscle
is met by increased cardiac output, greater muscle perfusion and increased O 2
extraction (Bangsbo 2000; Korthuis 2011), but during static breath-holding, muscle
blood flow and O2 consumption is limited due to a redistribution of the cardiac
output to supply mainly the brain and the heart (Schagatay 2009). Hypothetically,
an
increased
peripheral
blood
flow
could
potentially
counteract
the
vasoconstriction component of the diving response whereas it would be beneficial
during exercise.
A similarity between exercise studies and paper IV, however, was the unchanged
HR following NO3- ingestion suggesting no alteration of cardiac function. These
findings were confirmed in a recent experiment in our lab showing that the more
pronounced HR reduction during face-immersion breath-holds vs. dry breath-holds
was similar after NO3- ingestion as compared to placebo (unpublished data).
Instead, the effects of NO3- supplementation could lie in the periphery during
breath-holding, a situation also found during exercise as indicated by a lower
difference in O2 content in the arterial and venous blood during exercise following
NO3- ingestion (Kenjale, Ham et al. 2011).
The increased breath-hold duration after NO3- supplementation described in the
study in paper IV could be due to a reduction of metabolic rate. This is an
interpretation of the indirect evidence seen in the sub-maximal breath-holds trials
with NO3- supplementation, where arterial O2 saturation was higher than in the
trails without supplementation. Previous research may support this interpretation.
A recent study showed a tendency for increased (7.8 %) voluntary dry breath-hold
duration following NO3- supplementation in healthy females (Collofello, Moskalik
et al. 2014). Reduced metabolic perturbation and increased exercise tolerance in
hypoxic conditions after dietary NO3- supplementation has been observed
52
(Vanhatalo, Fulford et al. 2011), as has a strong tendency for reduced O2
consumption at rest after dietary supplementation in patients suffering from
peripheral arterial disease Kenjale et al. (2011). A recent crossover-study also
showed that dietary supplementation of 0.1 mmol NO3-/kg/day over 3-4 days
resulted in 4.2 % reduction in resting metabolic rate in healthy subjects compared
to the placebo administration (Larsen, Schiffer et al. 2014). The observations in the
present study and that described by Collefello and collegues (2014) are, however,
in contrast to a study by Schiffer and collegues (2013), who showed no
improvement in breath-hold duration and arterial O2 saturation during static breathholding following NO3- supplementation. These authors speculated that NO3supplementation reduced the efficiency of the diving response via NO-mediated
vasodilatation in the microcirculation and enhanced peripheral blood perfusion.
However, there are several methodological differences between the two studies that
might explain the diverging results. First, Schiffer and colleagues (2013) examined
three days of NO3- ingestion in comparison to the acute dose of beetroot juice in the
present study and also the study by Collefello and colleagues (2014). Next, the
subjects in Schiffer and colleagues (2013) study were able to hold their breath
down to an arterial O2 saturation of 57-63 %. In comparison, the subjects in our
study had an average arterial O2 saturation of approximately 75 % at the
termination of breath-hold. This indicates that the subject in the present study were
less well-trained, which was also the case in the study by Collefello and colleagues
(2014). Previous research has demonstrated that the circulatory and ergogenic
effects of dietary NO3- supplementation are less prominent in well trained subjects
compared to less trained subjects (Jones 2014). It is unknown if the same
differences occur regarding breath-hold training status, but the possibility seems
likely.
A basis for any physiological effect of NO3- supplementation (during breathholding) is that the activity and ingestion imposes a change in NO metabolism. It is
established that during normoxic conditions some NO is synthesized by the O2
53
dependent nitric oxide synthases (NOS), and that the oxidation of L-arginine to NO
and L-citrulline by endothelial NOS (eNOS) is progressively slowed during
hypoxia (McQuillan, Leung et al. 1994; Abu-Soud, Ichimori et al. 2000;
Ostergaard, Stankevicius et al. 2007; Ho, Man et al. 2012). Whether the hypoxia
during breath-holding could temporarily reduce the production of NO from Larginine to an extent that influences physiological function is unknown. More
certain, though, is that the NO3- -NO2- -NO pathway is potentiated under hypoxic
and acidic conditions (Lundberg and Weitzberg 2010). The study described in
paper IV showed effects already after 2 min of breath-holding when O2 saturation
was still high (97-98%), when significant hypoxia-induced conversion of NO2- to
NO would not be expected (Lundberg, Weitzberg et al. 2008). Further research
should be conducted to determine if the peripheral vasoconstriction during breathholding creates a suitable hypoxic milieu to facilitate the NO3- -NO2- -NO pathway,
or if other mechanisms are responsible.
The current basis of knowledge suggest that NO3- supplementation could promote
metabolic restrictions through several molecular mechanism, which is not
surprising given the ubiquity and multifarious roles of NO in physiological
regulation. Increased muscular efficiency following NO3- ingestion appears to be
coupled to effects of NO on the sarcoplasmatic reticulum calsium ATPase or the
actin-myosin ATPase causing a lower ATP cost of force production during
exercise (Ishii, Sunami et al. 1998; Bailey, Fulford et al. 2010; Evangelista, Rao et
al. 2010). The tendency towards slower arterial O2 desaturation during maximal
duration dynamic breath-holding during cycle ergometer exercise following dietary
NO3- supplementation (Schiffer, Larsen et al. 2013), and that ingestion of beetroot
juice resulted in a reduced arterial O2 desaturation during dynamic breath-holding
in experienced breath-hold divers (Patrician and Schagatay 2014), may result from
such increased muscular efficiency. Other mechanisms may be at work during
exposure to hypoxia while resting, such as improved mitochondrial efficiency. This
feasibly occurs through reduced proton leak across the inner mitochondrial
54
membrane caused by a downregulation of two mitochondrial proteins (3 (UCP-3)
and adenine nucleotide translocase (ANT)), as suggested by Larsen and colleagues
(2014). Interestingly, this investigation was accompanied with an in vitro
experiment revealing that NO2- infusion to myogenic stem cells from the human
skeletal muscle reduced the basal myotube O2 consumption to
60 % of untreated
cells (Larsen, Schiffer et al. 2014), supporting the earlier observations of improved
mitochondrial efficiency following NO3- ingestion (Larsen, Schiffer et al. 2011).
Larsen and colleagues (2011) showed that the expression of ANT following
nitrate supplementation was reduced, and that NO3- supplementation increased the
mitochondrial phosphate/O2 (P/O) ratio (the amount of O2 consumed per ATP
produced), which was closely correlated with the reduction in whole-body O2
consumption during submaximal cycling. Future research regarding the effects of
dietary NO3- prior to breath-holding should include measurements of both arterial
PO2 and local tissue O2 consumption, for example with near infrared spectroscopy
(NIRS) as further support for these potential mechanisms.
A final candidate mechanism worth considering is increased peripheral blood
perfusion following dietary NO3- supplementation, previously observed in studies
in rats (Ferguson, Hirai et al. 2013) and humans (Thomas, Liu et al. 2001).
Vanhatalo et al. (2010) also reported that muscle oxidative function in hypoxia was
restored to that measured in normoxia following NO3- supplementation, and
suggested that this could be related to improved perfusion of peripheral loci in the
muscle that were relatively more hypoxic. Following the results from these studies,
the longer breath-hold duration following NO3- supplementation could reflect a
more homogenous delivery of O2 within the tissue thereby reducing CO2 and H+
production. Future research should address whether the peripheral vasoconstriction
occurring during breath-hold may be counteracted by NO-donor vasodilatation.
55
5.3. Enhancement of spleen contraction by hypercapnia (Paper
III)
Earlier investigations suggested that the initiation and modification of spleen
contraction most likely involves some kind of chemoreception and hypoxia is a
known trigger (Kramer and Luft 1951; Hoka, Bosnjak et al. 1989; Kuwahira,
Kamiya et al. 1999; Richardson, de Bruijn et al. 2009). The study described in
paper III was the first to find a role for hypercapnia in the triggering of spleen
contraction, even in the absence of hypoxia. This occurred only during breathholding, however. Based on this finding, it is likely that spleen contraction has not
evolved mainly to buffer the CO2 in blood. Assuming that the CO2 concentration
used in the present study (5 % with the remaining 95 % O2) was sufficient to
trigger chemoreceptive activity, it is worthwhile to consider the structures and
mechanisms responsible for this observation.
Hypoxia increases the discharge of the carotid bodies which in turn send signals
via efferent pathways to the central nervous system (brain respiratory center in
medulla oblongata) in which they are integrated and processed to yield functional
responses such as increased breathing when at extreme levels (O'Regan and
Majcherczyk 1982). The temporal characteristics of the response/signal transfer are
designed for immediate adjustments even for modest changes in PaO2, but not for
breathing which is mostly regulated by the level of CO2 (Nattie 1999). As cellular
responses to hypoxia need more time to be initiated (Prabhakar 2006), it is
reasonable that breath-hold induced changes in PaO2 are recognized by carotid
body receptors and serve as a signal for homeostatic regulations. With respect to
detection of PaCO2, central chemoreceptors located on the ventrolateral medullary
surface are able to register changes in PaCO2 indirectly through changes in pH in
cerebral spinal fluid due to the reaction of CO2 with water (H2O) to form carbonic
acid (H2CO3). In addition, the peripheral chemoreceptors both in the carotid artery
and in the aortic arch react to changes in PaCO2, although the aortic receptors are
56
less sensitive than the carotid receptors to changes in PaCO2 and pH (O'Regan and
Majcherczyk 1982).
For the spleen to contract following chemoreceptive stimulus, an effector signal
must be transmitted. In most mammals, the splenic nerve is composed of 98 %
sympathetic nerve fibers that terminates into the spleen (Klein, Wilson et al. 1982),
suggestion an effector might plausible be neuronally-derived catecholamine release
into the spleen. It is, however, possible that nervous inputs to the spleen could be
exerted directly from the central nervous system without any chemical-sensitive
neuronal mechanism involved, such as excitatory neurons projecting from the
ventrolateral medulla to the spleen (Beluli and Weaver 1991). Observations of
immediate spleen contraction upon breath-holding, with corresponding increase in
HR and decrease in MAP, also support a neutrally-mediated contraction by
unloading of baroreceptors (Palada, Eterovic et al. 2007; Bakovic, Pivac et al.
2013). Rapid spleen contraction has also been observed following infusion by lowdose epinephrine, with concomitant decreases in blood pressure and increases in
muscle sympathetic nerve activity which lends further support to direct central
nervous system control of the spleen (Bakovic, Pivac et al. 2013).
Most likely is both chemoreception as well as direct neural innervation involved in
splenic contraction. Under conditions involving breath-holding, the major
contraction is found to correspond with the degree of hypoxia (Richardson, Lodin
et al. 2008; Richardson, de Bruijn et al. 2009). With respect to changes in PaCO2
following breath-holding, these are reflected in changes in extracellular pH, and
can be manifested/reacted upon within seconds (Ahmad and Loeschcke 1982).
Contrasting effects of hypoxia and hypercapnia on sympathetic activity in humans
exist (Somers, Mark et al. 1989; Tamisier, Nieto et al. 2004), although some
studies are more conclusive in that sympathoexcitation is greater with hypoxia than
hypercapnia (Cutler, Swift et al. 1985; O'Donnell, Schwartz et al. 1996;
Leuenberger, Hogeman et al. 2007). Combined, hypoxia and hypercapnia may
57
cross-influence and trigger a communal symphatoexitatory response (Lahiri and
DeLaney 1975; Morgan, Crabtree et al. 1995), but it could be that the spleen
becomes fully contracted by a certain amount of either stimulus and no further
contraction can be obtained with additional stimulus load. That said, Richardson
and colleagues (2009) showed that spleen related increases in Hb was achieved in
hypercapnia without hypoxia to a similar extend as pure hypoxia.
It is not known which mechanisms are responsible for the enhanced spleen
contraction after altitude exposure as described by the study in paper II.
Intermittent hypoxic exposure has been shown to augment sympathetic nervous
activity both in short-term exposure (Morgan, Crabtree et al. 1995; Xie, Skatrud et
al. 2001) and longer-term exposure (Hui, Striet et al. 2003; Gilmartin, Tamisier et
al. 2008). The latter may, however, also inhibit sympathetic nervous activity given
enough time (Hui, Striet et al. 2003; Tamisier, Hunt et al. 2007). The more
pronounced spleen contraction after breath-holding after long-term altitude
dwelling could be related to elevated sympathetic drive. Earlier work on healthy
subjects residing at altitude has however showed a decreased baseline spleen
volume when returning to low altitude (Sonmez, Ozturk et al. 2007) which in
contrast to the study presented in paper II and in subjects residing at moderate
altitude (up to 5200 m) for 6 weeks (unpublished data). The reduced spleen size in
Sonmez and colleagues (2007) study could be due to a sympathetic derived tonic
contraction of the spleen, although no data of the splenic contractile response or
details of its measurement conditions were provided.
Other output mechanisms to the spleen cannot be ruled out to be responsible for the
improved spleen contraction. It is shown that catecholamines are released into the
circulation following hypoxia (Prabhakar, Kumar et al. 2012) and freediving
(Chmura, Kawczynski et al. 2014), and it is generally accepted that humoral
stimulation via adrenoreceptors (α1, α2, β1 and β2) located in the splenic capsule
and parenchyma can mediate spleen contraction (Ayers, Davies et al. 1972; Stewart
58
and McKenzie 2002). More specifically, stimulation of α-adrenoreceptors has been
shown to cause spleen contraction while stimulation of β-adrenoreceptors to cause
spleen relaxation (Olsson, Kutti et al. 1976; Kutti, Freden et al. 1977; Freden, Vilen
et al. 1979). For example in hooded and harp seals, α -adrenoreceptor activation
with epinephrine resulted in forceful spleen contraction within 1–3 min of
administration while stimulation of β -receptors and cholinergic receptors did not
cause capsular contraction (Hance, Robin et al. 1982). At least in seals, the rapid
contraction of the spleen suggests a neural origin of the initial signal upon
submersion, while the contraction may be sustained during dive by catecholamine
released from the adrenal gland. Interestingly, the human spleen contraction during
breath-holding has been shown to be of a biphasic nature in which the first
contraction occur immediately upon initiation of the breath-hold (Palada, Eterovic
et al. 2007), while the second contractile phase occur minutes into the breath-hold
(Lodin-Sundström, Richardson et al. 2009). The first rapid contraction may
represent a sympathetically derived response similar to that found after injection of
low doses of epinephrine (Bakovic, Pivac et al. 2013), while the second contractile
phase could be linked to chemoreceptor input stimuli resulting in a release of
systemic catecholamine similar to that recorded during chronic intermittent
hypoxia (Prabhakar, Kumar et al. 2012).
Results from animal studies reveal that hypoxia-evoked adrenal catecholamine
secretion is neurogenic, requiring activation of the sympathetic nervous system
(Seidler and Slotkin 1986; Yokotani, Okada et al. 2002), whereas the adrenal
medulla is relatively insensitive to direct effects of acute hypoxia (Thompson,
Jackson et al. 1997; Keating, Rychkov et al. 2001). Interestingly, Lin and
colleagues (1983) hypothesized that hypercapnia may enhance sympathoadrenal
catecholamine release. However, the isolated effect of increased plasma
catecholamine on spleen contraction during breath-holding is yet to be investigated
in humans.
59
5.4. Acclimatization potential of the spleens contractility
(Paper I and II)
5.4.1.
Training effects
The novel finding that long-term altitude exposure increased spleen contraction at
exercise and breath-holding suggests that the utilization of splenic blood storage
can be altered given an adequate stimulus over time. Such possibilities were
already discussed following observations of greater spleen-related increases in Hb
concentration during breath-holding in trained breath-hold divers than in elite
skiers and untrained individuals (Richardsson and others, 2005). Additionally,
large spleen volumes were found in elite freedivers (Schagatay 2009), augmented
spleen contraction was shown in breath-hold trained individuals (Bakovic et al
2003, Prommer and others, 2007) and breath-holding capacity was found to
correlate well with spleen volume (Schagatay, Richardson et al. 2012). The
increased spleen contraction could feasibly be due to predisposion, breath-hold
training, or a combination of both. While the study described in paper (I) showed
no increase in the magnitude of the spleen contraction after breath-hold training the
training dose or duration may have been insufficient. It also remains uncertain if
prolonged breath-holding training represents an adequate stimulus leading to
changes in spleen contractility, in comparison to altitude simulation that elicits a
more powerful contraction (Lodin-Sundström and Schagatay 2010). The study in
paper I did, however, find significant levels of spleen contraction even in
individuals with no experience in breath-holding, as have previous studies
(Schagatay, Andersson et al. 2001; Espersen, Frandsen et al. 2002; Baković, Valic
et al. 2003), indicating the spleen contraction is likely a “hard wired” trait that is
activated upon physiological stress, similar to in terrestrial mammals and airbreathing divers (Stewart and McKenzie 2002).
60
5.4.2.
Effects of long-term altitude exposure
In contrast to two weeks of breath-hold training, long-term altitude exposure
resulted in increased spleen contraction, representing a novel finding that may
provide new insight into the acclimatization processes when entering a low O2
environment. Earlier reports of spleen contraction upon exposure to normobaric
hypoxia (Lodin and Schagatay, 2010), and that the spleen contraction follows a
graded patter according to the level of hypoxia when subjects are progressing to
higher altitude (Lodin-Sundström, Söderberg et al. 2014), support the present
study`s findings.
Spleen contraction may benefit the individual before altitude-induced polycythemia
develops, which can take days to weeks (Gore, Sharpe et al. 2013). Increased
spleen contraction may also be beneficial for acclimatized individuals when
additional mobilization of available O2 resources are needed, such as during hard
work at high altitude. When not contracted, the spleen may also serves to reduce
blood viscosity (Stewart and McKenzie 2002) which can be beneficial during high
altitude polycythemia.
The similar physiological mechanisms among mammals for tolerating endurance
exercise and hypoxia have long been known (Hochachkta 1998). Dogs, being
superb endurance runners, are known for their significant splenic contributions
which improves maximal O2 consumption (VO2max) during exercise (Longhurst,
Musch et al. 1986). Interestingly they are able to acclimatize within minutes to
altitude allowing them, “to chase their prey up and down a mountain with minimal
changes in hypoxic acclimatization during the exertion”(Dane, Hsia et al. 2006) .
Though humans, comparatively speaking, are less impressive endurance athletes,
the human spleen volume decreases progressively with increasing altitude
61
(Richardson and Schagatay 2007). The alternating hypoxic strain with exercise
achieved during prolonged stays at high altitude may be a potent stimulus for
spleen contraction. Whether such responses could contribute to individual
differences in susceptibility to altitude sickness is presently unknown.
5.5. Implications and future directions of work
5.5.1.
Diving response related studies
Current knowledge of the training effects of the human diving response are
restricted to observations after two studies of two weeks training duration, and it
would be interesting to investigate the effects of longer periods of breath-hold
training on the response. Group comparisons between different groups of breathhold divers and untrained subjects indeed suggest training effects (Schagatay and
Andersson 1998; Schagatay, Richardson et al. 2012).
5.5.2.
Spleen related studies
It is most likely worthwhile to investigate longer periods than 2 weeks of breathhold training on spleen contraction. The most obvious application of an enhanced
spleen contraction would be in breath-hold activities such as freediving,
competitive apnea and synchronized swimming. Sperlich and colleagues (2014)
found no improvement in performance after a 4km cycling time trail after
“priming” with breath-hold induced spleen contraction vs. a trail without breathhold spleen “priming”. However, it might be that the spleen contracted during the
warmup before the cycling trail as indicated by the very small spleen volumes
before start and the small difference in volume after the trail. Research attention
could be directed towards possible effects for athletic performance in endurance
sports of relative short duration where improved early phase VO2 kinetics could be
of importance.
62
One study showed that spleen contraction was enhanced after physical activity in
patients with severe chronic obstructive pulmonary disease (COPD) as compared to
patients with mild COPD, suggesting that the contraction is related to the severity
of the disease and possibly degree of hypoxia (Stenfors, Hubinette et al. 2009). It
would be intriguing to further investigate the physiological role of spleen
contraction in pathological conditions where O2 availability is limited. Similarly
intriguing would be determination of the characteristics of the altitude exposure
necessary to enhanced spleen contraction, for example exposure duration, hypoxic
dosage and work rates.
In humans, spleen volume changes have been measured using ultrasound,
computerized tomography scanning (CT) and radionucleotide imaging (Hoefs,
Wang et al. 1999; Espersen, Frandsen et al. 2002; Schagatay 2009). Although these
methods prove effective in measuring spleen size reductions, limitations can be
imposed by these methods in determining organ contours and variations in shape.
Future studies aiming to address exact volume changes of the spleen would do well
to employ magnetic resonance imaging (MRI), which can produce multiple images
of great detail in fraction of seconds and also provide information of regional blood
flow. One study has indeed already done this (Thornton, Spielman et al. 2001)
where MRI was used to determine spleen volume changes in northern elephant seal
pups, and today it represents the best option available for accurate volume
determination in laboratory settings. This technology, being detailed and
temporally accurate, could also be used to investigate if there is any contribution
from other potential blood storage pools - perhaps the liver being the most likely
candidate.
5.5.3.
Dietary nitrate related studies
The study described in paper IV, indicated a reduction of metabolic rate in static
breath-hold after dietary NO3- supplementation. It would therefore be of interest to
63
elucidate the potential of NO3- administration in modulating metabolic rate and
aspects of regional blood flow in different models of tissue-specific and global
hypoxia. Reduction of hypoxic load by virtue of low cost dietary means is certainly
of relevance for several pathological conditions such as peripheral artery disease,
COPD, placenta insufficiency, reperfusion injuries as well as in environmental
conditions involving altitude exposure. To gain a better understanding of the
mechanisms of NO3- supplementation, future studies could benefit from involving
measurements of full body O2 consumption, mitochondrial respiration and
hemodynamic variables. In the context of breath-hold induced hypoxia, it would be
interesting to include measurements of the near-infrared spectroscopy (NIRS)
method, the latter to gain insight into local blood flow and tissue oxygenation.
6.
CONCLUSIONS
General conclusions
Humans have evolved several distinctive physiological responses that can
ameliorate, or at least delay the onset of potentially detrimental effects during
situations that do not allow sufficient gas exchange with the environment, such as
during breath-holding and travel at high altitude. This thesis shows that these
responses can be augmented in humans through sufficient exposure to stimuli over
time, and in some cases through voluntary training, thereby enhancing the capacity
to sustain hypoxic stress. In the former case, long-term high altitude climbing
enhanced spleen contraction and thereby increased the utilization of intrinsic O2
storage capacity. In the latter case, recurrent breath-hold training enhanced the O2
conserving cardiovascular diving response, while the blood boosting spleen
contraction was not affected. This thesis further demonstrates that CO2 is an
important stimulus for spleen contraction, something which often accompanies
hypoxic stimuli and may help initiate responses. Additionally, dietary nitrate,
acting as a nitric oxide donor, seemed to reduce the metabolic rate by a mechanism
64
that is separate from the cardiovascular diving response. In summary, this thesis
reveals that the human species possess several malleable responses when faced
with hypoxia that can allow activity in challenging environments despite the
constant need for O2 to maintain energy production via the aerobic metabolic
pathways.
Specific conclusions
Paper I
The metabolic rate during breath-holding was reduced following two weeks of
intermittent exposure to hypoxia via breath-hold training, as a result of a more
pronounced diving response that also had an earlier onset. The contraction of the
spleen, and subsequent expulsion of its concentrated blood stores, was unaffected
by the training.
Paper II
Long-term exposure to high altitude increased splenic contraction, probably due to
a general acclimatization to chronic hypoxia as well as due to transient periods of
more acute hypoxia while performing physical work at altitude.
Paper III
Increased levels of CO2 inspired prior to breath-holding appear to trigger splenic
contraction during the breath-hold in a dose-dependent manner, despite the absence
of hypoxia. Eupneic hypercapnia did not have the same effect.
Paper IV
Acute dietary nitrate supplementation may prolong breath-holding by reducing
metabolic activity.
65
Funding
The work in this thesis was supported by funding from:
Swedish National Centre for Research in Sports (Centrum för idrottsforsking, CIF)
for all studies and Swedish National Winter Sports Centre for study II.
7.
ACKNOWLEDGEMENTS
I feel greatly indebted to many people, friends and colleagues for not only making
this thesis possible, but most important for making it such a fantastic and fun
experience! The Environmental Physiology Group (EPG) at Mid Sweden
University has become a cornerstone for personal and intellectual development
during the last years, and also a platform to explore spectacular environments and
cultures worldwide. Albert Einstein once expressed a concern; “It is a miracle that
curiosity survives formal education”. I think the learning atmosphere in the EPG
proves the opposite can be true also in formal education. Here curiosity thrives. I
want to thank everybody in the group for making it such a pleasant group to work
in. I particularly want to thank the following:
My supervisor, Professor Erika Schagatay and head of the EPG for being positive
and enthusiastic about everything, for opening my eyes for how interesting and fun
science can be -and how and where it can be done(!), for encouraging scientific
discussions and for expanding my knowledge in mammalian physiology. Thank
you for these years, and the trust and flexibility given. It has been a great pleasure
and privilege to work under your supervision.
66
My co-supervisor Professor H-C Holmberg for generous support, including
manuscript and thesis revision.
Fellow PhD-Student Angelica Lodin-Sundström, my collaborator and friend
through the whole PhD-period. Thank you for your kindness and support, all great
work together, for all exiting travels together in Nepal and Egypt, and for your
ability to work and plan systematically-making every logistic challenge running
smoothly.
Dear friends and colleagues Alexander Patrician, Dr. Matt Richardson, Helana
Haughey Vigetun and Dr. Lara Rodrigues Zamora and other co-workers across
these years for your positive spirit and unselfish assistance during field and lab
work as well as manuscript preparation.
I`m also indebted to all my collaborators and friends at LHL-Klinikkene Røros,
Norway, which in a flexible manner have made it possible to combine the PhD
work while also working in interesting projects with you. Similarly, I appreciate
what I have learned from former teachers and fellow students at primary school,
high school and university in order for me to develop my curiosity and the skills
needed to work systematically.
I also want to express my special thanks to Chirring Dorje Sherpa, Ngawang Tashi
Sherpa, Furtemba Sherpa, Mingma Tensing Sherpa and other of your colleagues at
Rolwaling Excursions and Karma Sherpa for your hospitality, kindness and
assistance during our works in the Himalaya; and not at least to our subject
volunteers who where kind to participate in the studies. Without your efforts this
thesis would not have been possible.
A special thanks also to the Engan and Solhaug families for practical and moral
support, and last but not least to my dear wife Berit Rein Solhaug, the one person
67
who have always supported me in all facets of life, kept my moral up when faced
with obstacles and to “endured” the numerous “trans-Scandinavian” trips and
“away from home” periods.
8.
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