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Non-invasive method of measuring airway inflammation: exhaled nitric oxide
Dr Catherine Ann BYRI\IES
FRACB MBChB (New Zeatand)
Graduate certificate in crinicat Education (New south wales)
The University of Auckland
Department of Paediatrics
Faculty of Medical & Health Sciences
Private Bag92ol9
Auckland 1142
New Zealand
Ph: &93737 599extn 89770
Fax: @93737 486
Email: c.byrnes@auckland.ac.nz
Senior Lecturer & Honorary Consultant
Paediatric Respiratory Medicine

UNIVERSITV OF AUCKI'AND
Abstract
- - oEC 2008
Backeround
UBRARY
Nitric oxide (NO) was well known to be a component of air pollution, often in the form of
nitrogen dioxide (NOt. However its importance in biological systems altered dramatically
with the discovery in 1987 that it was the 'endothelial-derived relaxing factor'. Since then
there has been an explosion of research on NO demonstrating that this gaseous molecule was
a widespread physiological mediator and was simultaneously recognised as a vital component
of immune function contributing to macrophage-mediated cytotoxicity. NO was therefore a
key molecule in modulating inflammation, including airway inflammation.
The aim of this thesis was:
l. To adapt a NO chemiluminscence analyser from measuring ainvay pollution to measuring
exhaled air in human subjects.
2. To measure NO levels in exhaled air in adult subjects.
3. To evaluate whether altering measurement parameters altered the levels of NO obtained.
4. To adapt this technique from adults to measure exhaled NO in children.
5. To compare levels of NO from healthy children to groups of asthmatic children on either
bronchodilator therapy only, or on regular inhaled corticosteroids.
6. To compare the levels of NO in a pilot group of asthmatic children before and after
commencement of inhaled corticosteroids.
Methods
A Dasibi Environmental Corporation Model 2t07 chemiluminescence analyser was adapted
specifically requiring a reduction in response time, which was achieved by modification of the
circuitry and re-routing of the analogue signal directly to a chart recorder, achieving a
reduction of the response time by 807o. Addition of a number of analysers allowed the
measurement of exhaled NO, carbon dioxide (COz), mouth pressure and flow for each
exhalation from total lung capacity. Twenty adult subjects (in total) were then studied looking
at direct (NO, CO2, mouth pressure) versus t-piece (with the addition of flow) measurements
making five exhalations from total lung capacity, at 3-minute intervals (direct/t-piece/direct or
t-piece/direct/t-piece in series). The area of NO under the curve versus the peak of the NO
trace was compared and the exhalation pattern of NO versus COz was compared.
Measurement conditions were altered to evaluate the effect of individual parameters on the
exhaled NO result. This included separately assessing different expiratory flows, different
expiratory mouth pressures, the effect of a high versus a low background NO level and the
tl

effect of drinking water (of varying temperatures) prior to exhalation. Healthy control
children were then enrolled to the study from a local school (Park Walk primary School) and
compared with asthmatic children enrolled from outpatient clinics at the Royal Brompton
Hospital. The asthmatic children were further divided into those on bronchodilator treatment
only and those on regular inhaled corticosteroid therapy. NO was also measured before and
two weeks after commencing inhaled corticosteroid therapy in previously steroid-naive
asthmatics.
Results
It was possible to modify a chemiluminescence analyser to enable measurement of exhaled
No' In 12 healthy subjects (mean age 32 years, 6 males) peak direct NO levels were g4.g
parts per billion (ppb) (standard error of the mean (SEM) l4.0ppb), significantly higher than
4l'2ppb (SEM 10.8ppb) measured via the t-piece system. The exhaled NO rose to an early
peak and plateau while the COz levels continued to rise to peak late in the exhalation. The
mean times to peak NO levels were32.2 seconds (s) (direct) and 23.1s (t-piece), which was
significantly different from the mean times to peak CO2levels at 50.5s (direct) and 51.4s (t-
piece, p

There was no significant difference between boys and girls for either the direct or the t-piece
recordings. In comparison with normal children, 15 asthmatic children on bronchodilator
therapy only had much higher levels of exhaled NO at 126.1ppb via the direct system (SD
77.lppb, p4.001) and 109.5ppb via the t-piece system (SD l06.8ppb, p

esults suggested that the methods of measuring exhaled NO required standardization and that
it could potentially be a non-invasive measure of airway inflammation to follow - particularly
in children with asthma who were conmencing inhaled steroid treatment.
V

This is Dedicated:
Dedication
To the strong women in my family from my great, great grandmother
VI
to my sister Angela who always believes in me.

United Kingdom
Acknowledgements
Professor Andrew Bush invited me to a research position at the Royal Brompton Hospital
and has been supportive from day one for both my research career and clinical training. His
own approach I seek to emulate with his excellent clinical acumen, approach to children and
their families. In addition, he maintains a huge research output and when working with him in
this capacity his attention to detail and editing ability is superb. I, like most of the paediatric
respiratory fraternity, count Andy, and his wife sue Bush, as friends.
Professor Peter Barnes from the then National Heart & Lung Institute contributed valuable
advice and supervised other research projects that I conducted. He rather bravely allowed me
to join his laboratory as one of only three 'medics' among the 25 scientists from whom I
learnt about statistics, information technology and attention to detail.
Dr Seinka Dinarevic assisted with the studies on the children and provided a link to the local
school from where the children were recruited.
Park Walk Primary School, Iondon who were approachable regarding the research and
interested in assisting the study. The children enrolled from the school and from the
Brompton Hospital clinics were terrific -
humorous, enthusiastic, willing to help and could
always be depended upon to question some factor about the testing or the research that I had
not covered with them. They loved all the switches even more than I did.
Caroline Busst was the biomedical engineer who provided the main assistance with regard to
modifying the analyser as we went through and assisted with troubleshooting whenever that
was necessary. She brought completely different knowledge to mine to this project from an
engineering and particularly electrical engineering capacity, which complimented the clinical
and practical knowledge that I was able to offer.
I would like acknowledge the great 'Fellows' that I worked with at the Royal Brompton
Hospital. We were embarking on clinical and research careers and it was great to be part of
the group and they remain friends to this day; Paul Munyard, Lara Shekerdemian, Jane
Davies, Clare Hogg, Kate Brown and Adam Jaffe.
vll

New Zealand
The Paediatric Department at the Faculty of Health and Medical Sciences, University of
Auckland all contributed particularly in the final days of submission assisting with proof-
reading and formatting.
A very special thank you to Jan Tate the CF nurse specialist and friend who has always been
very supportive and made my daily working life better, particularly at times of clinical
overload. She also helped take the photos used in this thesis -
late into the night.
Professor Innes Asher has always been supportive of myself in the clinical, research and
teaching arenas and has helped greatly with her capacity, despite the many hats she currently
wears, in offering advice and editing comments.
Dr Elizabeth Bdwards who was my first research fellow, the first Ph.D student that I
supervised which she completed before me and now both colleague and friend as well as
fellow netball enthusiast.
Merrin Harger, project manager of subsequent research with whom I shared an office. She
was amusing every day and left me one of her inspirational artworks when she left to embark
on a new career as an artist (that we all were so talented).
Mirjana Jaksic, always generous with her own clinical time, I thank for supporting me in
particular by picking up the occasional extra clinic, so that I could catch up.
vill

Personal
My family has always been completely supportive in everything that I do and my parents,
Daphne Mary Pemberton Byrnes and Brian Liston Dominic Byrnes, were thrilled to see
me start at medical school all those many years ago, although, sadly, were not alive to see me
graduate. And my special Aunt, Veronica Commins, who has supported my sister and myself
through our careers and also, sadly, died before seeing this dedication to her years of
generosity. My brothers, sisters and their families all offer their special supports - I feel lucky
to be part of a large whanau, and I hope they will be pleased to see me emerge from my study.
It is a fttre person who believes in you so wholeheartedly, even at times when you yourself
have misgivings and I would like to thank my sister, Angela Platt-Byrnes, for ringing me
every Saturday and every Sunday to ensure that I was ... and to encourage me to be ... sitting
at my desk working on this thesis.
And to my own support network: Dr Sue Armstrong-Wahlers, Dr Michael Wahlers and
Trudi Fava.
IX

Contents
Abstract ........tr
Dedication .................... VI
Acknowledgements.... ...................VtI
Contents .......X
List of Tables ..............XV
List of Figures ........... XVI
List of Abbreviatons.............. ....XVI[
Chapter l: The burden of respiratory disease in New 7-ealand.,.. ..................... 1
1.1 Introduction............... ....... I
I.2 The burden of paediatric respiratory disease in New 7naland.... .............2
1.3 The burden of asthma disease in New 7naland..... ................. 5
I.4 The diagnosis of asthma -
Page
the quandaries in children ...........7
1.4.1 The asthma diagnosis in national and international guidelines. .......7
1.4.2 What is meant by 'wheeze'?.............. ..........7
1.4.3 Where does cough fit in? ......... l0
1.4.4 Investigations ............. .............. 12
1.5 Treatment and concerns............... ......,,.....,.....,..21
1.5.1 Possible adverse effects of asthma treatment in children ..............21
L.5.2 A marker for inflammation would be useful... .............23
1.6 Asthma as an inflammatory disease.............. .....24
1.6.1 What has been learnt from autopsy studies? ................24
1.6.2 Studies of inflammation using bronchoscopy and biopsy .............25
1.6.3 Studies of inflammation using bronchoalveolar lavage............... ....................29
L.6.4 Inflammatory markers in induced sputum ...................31
1.6.4 (i) Studies in adult subjects..... ................31
1.6.4 (ii) Induced sputum in children ................34
1.6.4 (iii) Technical aspects of bronchoscopy, bronchial biopsy, bronchoalveolar
lavage and/or induced sputum...... ...... 36
1.6.4 (iv) Safety aspects of bronchoscopy, bronchial biopsy, bronchoalveolar lavage
and/or induced sputum ..... 38
1.6.5 Studies of inflammatory markers in blood and urine.. ................... 41
1.6.6 Comparison of inflammation results between the different samples.. .............44
1.7 Chapter summary ...........45
Chapter 2: Nitric oxide: pollutant to mediator .............49
2.I Introduction............... .....49
2.2 Pollution, nitrogen oxides and disease ...............49
2.2.L Concerns regarding pollution... ..................49
2.2.2 Disease and pollution............. .................... 5l
2.2.3 Nitrogen oxides in pollution .... 56
2.3 The discovery of nitric oxide in biological systems ............ 57
2.3.1 Vascular control .....57
2.3.2 Immune function ....60
2.3.3 Nitric oxide -
a common pathway .............61
2.3.4 Nitric oxide in physiological roles .............62
2.3.4 (i) Cardiovascular.......... .......62
2.3.4 (ii) Nervous system - central and peripheral............. ..................64
2.3.4 (iii) Host defence.............. ....... 66
2.4 Chapter summary ...........68
Chapter 3: The synthesis, reactivity and control of nitric oxide........ .............. 70

3.1 Introduction............... .....70
3.2 Properties of nitric oxide ..................70
3.3 Reactions of nitric oxide......... ..........73
3.3.1 Reactive nitrogen species and superoxide reactions.............. ,........73
3.3.2 Reactions with transition metars ind metalroproteins ....................75
3.3.3 Nitrogen thiols and amines .......76
3.3.4 Reaction with amino acids......... ..........,......77
3.3.5 Reaction with lipids. ..........,......77
3.3.6 Reactions with genes ................7g
3.3.7 Making sense of the reactions ....................7g
3.4 Nitric oxide synthase isoenzymes ............... .......7g
3.4.1 Constitutive nitric oxide synthases .............g1
3.4.2 Inducible nitric oxide synthase.... ...............g2
3.4.3 Control of the nitric oxide synthase isoen2ymes................ ............g3
3.4.3 (i) The constitutive forms ......g3
3.4.3 (ii) The inducible form ...........85
3.4.4 Nicotinamide adenosine di-nucleotide phosphate oxidase and inducible nitric
oxide synthase ........g7
3'4'5 Drug! & other agents that affect the isoenzymes and nitric oxide production. gg
3.4.5 (i) Drugs ..............88
3.4.5 (ii) Nitric oxide synthase inhibitors.. ........g9
3.5 Chapter summary... .........91
Chapter 4: Methods to measure nitric oxide ................g2
4.1 Introduction............... .....g2
4.2 L-arginine, L-citrulline and cyclic guanosine monophosphate................................93
4.3 Methaemoglobin....... ......g3
4.4 Nitrite and nitrate. ...........g4
4.5 Nitric oxide........ .............95
4.6 Chemiluminescence... .....g7
4.6.1 Calibration ............103
4.6.2 Safety and toxicity ..................104
_ 4.7 Chapter summary... .......10g
Chapter 5: Methodological assessment of the chemiluminescence ana1yser................... l l0
5.1 Introduction ............... ... I l0
5.2, The equipment and personnel.. ....... I l0
5.2.I Chemiluminescence Analyser ,Model 2107, ............110
5.2.2 Capnograph, pressure transducer, flow meter and chart recorder.................. I I I
5.2.3 Personnel .............. I 15
5.3 Direct versus reseryoir measurement .............. ................... I 16
5.4 Assessments of the analysers... .......117
5.5 Other measurements............ ...........120
_ 5.6 Chapter summary... .......121
Chapter 6: Methodological studies of exhaled nitric oxide in healthy adult subjects: direct
versus t-piece testing ..............122
6.1 Introduction............... ...lLz
6.2 Nitric oxide and the nitric oxide synthases in the lung .......... ..............122
6.3 The need for methodological experiments ............... ..........125
6.4 The aims of the exhaled nitric oxide methodological experiments ...... 130
6.5 Setting up for the experiments........... ...............131
6.5.1 Set-up and calibrations......... ....................131
6.5.2 The exhalation protocol.............. .............. 136
6.5.3 Ethics and Consent................ ...................13g
XI

6.5.4 The subjects............... ............. 138
6.5.5 Statistical analysis .................. 138
6.6 Methodological experiment one... ..139
6.6.1 Hypotheses................ ............. 139
6.6.2 Aims .,139
6.6.3 Procedure ............. 139
6.6.4 Results..... ............. 140
6.6.4 (i) Direct versus t-piece nitric oxide and carbon dioxide measurement..... 140
6.6.4 (ii) Peak nitric oxide versus area under the nitric oxide curye ..l4
6.6.4 (iii) Comparison of exhaled nitric oxide and carbon dioxide ..... 146
6.7 Discussion: The origin of the exhaled nitric oxide ............ 148
Chapter 7: Methodological studies of exhaled nitric oxide in healthy adult subjects:
investigating test parameters .. 153
7.1 Introduction............... ... 153
7.2 Ethics, subjects, calibrations and statistical analysis .............. ............. 154
7.3 Methodologicalexperimenttwo-effectof expiratoryflow...... .......... 155
7.3.1 Hypothesis ............ 155
7.3.2 Aim.......... ............. 155
7.3.3 Procedure ............. 155
7.3.4 Results..... ............. 156
7 .4 Methodological experiment three - effect of pressure ....... 158
7.4.1 Hypotheses................ ............. 158
7.4.2 Aim.......... ............. 158
7.4.3 Procedure ............. 158
7.4.4 Results..... ............. 160
7.5 Methodological experiment four - effect of ambient nitric oxide ........ 163
7.5.1 Hypothesis ............ 163
7.5.2 Aims ... 163
7.5.3 Procedure ............. 163
7.5.4 Results..... ............. 164
7.6 Methodological experiment five - effect of water consumption........................... 168
7.6.1 Hypothesis ............ 168
7.6.2 Aim.......... ............. 168
7.6.3 Procedure ............. 168
7.6.4 Results..... ............. 169
7.7 Discussion: which measurement factors alter nitric oxide levels? ...... 170
Chapter 8: Exhaled nitric oxide in healthy and asthmatic children ............... 178
8.1 Introduction............... ... 178
8.2 Ethics, consent, statistics and subjects................ ............... 179
8.3 Methodology of exhaled nitric oxide measurement in healthy chi1dren................ 180
8.3.1 Hypotheses................ ............. 180
8.3.2 Aims ... 180
8.3.3 Protocol ................ 180
8.3.4 Results..... ............. 182
8.4 Discussion: exhaled nitric oxide results in healthy children.... ............ 186
8.5 Methodology of exhaled nitric oxide measurement in asthmatic children ............ 191
8.5.1 Background............... ............. 191
8.5.2 The asthmatic subjects ...........I92
8.5.3 Protocol ................ 193
8.5.4 Results..... ............. 193
8.6 Discussion: exhaled nitric oxide in asthmatic children.. .... 196
Chapter 9: Exhaled and nasal NO to today........ ........202
xll

9.1 Introduction............... ...202
9.2 Technical factors affecting resurts across research groups....... ............202
9.3 Standardisation ............ ...................204
9.3.1 Single breath online measurement.............. ................205
9.3.1 (i) F1ow......... .....206
9.3.1 (ii) Mouth pressure ...............211
9.3.1 (iii) Nasal clips and breath_holding............ ...............212
9.3.1 (iv) The recorded measurement......... ......213
9.3.1 (v) The effect of ambient nitric oxide ....213
9.4 Online spontaneous or tidal breathing measurement.............. ..............214
9.5 OffJine measurement.............. .......216
9.6 Nasal nitric oxide measurement.............. .........220
9.7 Physiological alterations that may affect measurement.............. .........221
9.7.1 Size and gender....... ................221
9.7.2 Age .......... ,............222
9.7.3 Circadian rhythm. ...,...............223
9.7.4 Menstrual cycle and pregnancy ................223
9.7.5 Food and beverages ................224
9.7 .6 Summary of physiological factors that could alter nitric oxide levels ....,......224
9.8 Nitric oxide levels in asthma and atopy ...........224
9.8.1 Does nitric oxide correlate with other asthmatic inflammatory markers?......22s
9.8.2 Does nitric oxide correlate with lung function and bronchial hyperresponsiveness?
....................226
9.8.3 Can nitric oxide be used for diagnosis of asthma?.............. .........227
9.8.4 Is nitric oxide associated with symptoms and severity of asthma? ................22g
9.8.5 can nitric oxide predict deterioration in asthma control? ....,.......23t
9.8.6 What happens to nitric oxide during an acute asthma attack .......233
9.8.7 What is the effect of atopy alone on nitric oxide?....... .................233
9.8.8 Can nitric oxide be an outcome measure for assessing treatment? ................235
9.8.8 (i) Corticosteroids............. ...235
9.8.8 (ii) Anti-leukotriene receptor antagonists .................237
9.8.8 (iii) Long acting p2 agonisrs............... .....23g
9.8.8 (iv) Nedocromil sodium .......,23g
9.8.8 (v) Theophylline.............. .....23g
9.8.8 (vi) Novel medications................ ............23g
9.8.9 Summary of nitric oxide in asthma and atopy.. ..........240
9.9 Nitric oxide levels in primary ciliary dyskinesia................... ...............241
9.10 Nitric oxide levels in cystic fibrosis.................. .................242
9.ll Nitric oxide levels in bronchiectasis......... ........245
9.12 Nitric oxide levels in upper respiratory tract infections.......... .............245
9.13 Nitric oxide levels in chronic obstructive pulmonary disease (copD).... ..............246
9.14 Nitric oxide levels in smokers ........247
9.15 Nitric oxide levels in interstitial lung diseases ...................24g
9.16 Nitric oxide levels in exercise ........250
9.17 Nitric oxide measurements in infants ...............252
9.17.1 Methodology.............. .............252
9.17.2 lrvels of nitric oxide in infants with different respiratory diseases ...............256
9.17 .3 Prenatal and maternal effects on nitric oxide levet........:......... ...257
9.17.4 Effects of treatment on nitric oxide levels........ ..........257
9.17.5 Summary of nitric oxide findings in infants.. .............257
_ 9.18 Chapter surnmary... .......25g
Chapter l0: Reflections ............262
xill

XIV

List of Tables
Table 1.1: Alternative diagnoses in children with whee2e............... ........................g
lable 3.1: Properties of nitric oxide .................71
Table 3.2: Reactions with nitric oxide ..............72
Table 3.3: The characteristics of the nitric oxide synthase isoen2ymes................................... g1
Table 4.1: The companies providing chemiluminescence analysers adaptable for nitric oxide
measurement in 1995. ...................101
Table 5.1: The delay and response time of the NO, COz, mouth pressure and flow meter
analysers used.......... ...llg
fu9l" 6.1: The published results of exhaled nitric oxide in adults.... ...................127
full" 6.2: Compete results for an individual subjecr .......143
Table 6.3 NO and COz results from single exhalitions in twelve adult subjects............. ......143
Table 7.1: NO, COz and duration of each exhalation at different expiratory flows...............157
Table 7.2: Comparison of the first and repeated last set of exhalation, uf the same expiratory
flow""""" ...................16r
Table 7.3: NO, COz and duration of each exhalation at different expiratory mouth pressures
.....,....,,,.,.., |62
Table 7.4: Comparison of the first and repeated last set of exhalations performed at the same
SPt]afory mouth pressure ............162
Table 7.5: Exhaled NO results with high and low background NO concentrations...............l6g
Table 7.6: Exhaled COz results with high and low background NO concentrations .............16g
Table 8.1: Coefficients of variation of peak NO, peak COz and mouth pressure measurements
made by both the direct and rpiece systems, and of flow made by the rpiece system......... lg5
Table 9.1: The recommended standard for single breath online Nb meaiur.-"nt ...............206
Table 9.2: The recommended standards for single breath and tidal breathing off-line NO
measurement.............. ..,,,...,,,,.....,.21g
Table 9.3: The recommended standard for nasal No measurement...... ...............220
XV
Page

List of Figures
Figure l.l: Top l0 causes of avoidable admissions, 0-24 years, 1999.......... .......... 3
Figure 2.1: Diagram of blood vessels in cross section showing the endothelial layer. ...........59
Figure 3.1: The molecular structure of NO....... ................. 70
Figure 3.2: The reaction to generate nitric oxide ...............71
Figure 3.3: The nitric oxide synthase reaction showing the generation of the constituent atoms
of nitric oxide........ ....... 80
Figure 3.4: The chemical structures of the nitric oxide synthase inhibitors ..........90
Figure 4.1: Chemical reaction between nitric oxide and ozone. .......... 98
Figure 4.2:Diagram of the chemiluminscence analyser.... .................. 99
Figure 4.3: Relationship between chemiluminscence in millivolts to nitric oxide concentration
in picomo1es.............. ................... 100
Figure 4.4: Reaction equations used to release nitric oxide from other nitrogen compounds 103
Figure 5.1: Schematic diagram of how the analysers were placed .... Llz
Figure 5.2: An example of tracing from the testing....... .. 113
Figure 5.3: Photographs of one of the children performing the exhalation........................... I 16
Figure 6.1: Recording of the calibrations for NO, CO2 and pressure ana1ysers.................... 133
Figure 6.2: Recording of the calibration of the flow rotameter and pneumotachograph....... 135
Figure 6.3: Schematic diagrams of the two different types of connections: direct and t-piece
measurements............. .................. 137
Figure 6.4: Example of the chart recording rolls for the direct versus t-piece measurements.
140
Figure 6.5: Example of recording on one subject -
Page
direct and t-piece measurements .......... 141
Figure 6.6: Mean exhaled NO levels measured by direct and t-piece systems......................I42
Figure 6.7: Example of a recording for the two systems............... .... L42
Figure 6.8: Mean exhaled COz levels measured by direct and t-piece systems..... ................ I44
Figure 6.9: Comparisons of the peak NO with area under the curve... ................ 145
Figure 6.10a: Time to peak NO and peak CO2 measured by the direct system..................... 146
Figure 6.10b: Time to peak NO and peak CO2 measured by the t-piece system...................147
Figure 6.1Ia: COz levels at peak NO and at peak CO2 measured by the direct system ........147
Figure 6.1Ib: CO2 levels at peak NO and at peak CO2 measured by the t-piece system ...... 148
Figure 7.1: Exhaled NO results with increasing expiratory flows .....I57
Figure 7.2:Example of the tracing in one subject at two different expiratory flows ............ 158
Figure 7.3: The effect of pressure on the calibration gas measurement ......... ..... 160
Figure 7.4: Exhaled NO results at increasing expiratory mouth pressure ........... 161
Figure 7.5: Measurement of exhaled NO incorporating the reservoir system ..... 165
Figure 7.6: Photographs of the changing ambient NO concentration recordings.................. 166
Figure 7.7: Recording from one subject showing NO results with exhalations from low and
high background NO.... ................ 167
Figure 7.8: The effect of high and low pressure on the calibration gas measurement........... 167
Figure 7 .9: The effect of consuming water on the subsequent exhaled NO levels measured 170
Figure 7.10: Hyperbolic relationship between exhaled NO and sampling flow rate.............172
Figure 7.11: Exhaled NO determined at single breath plateau concentrations at increasing
expiratory flows and at increasing expiratory mouth pressures... ......173
Figure 8.1: Peak exhaled NO results in healthy children.... ............... 183
Figure 8.2a: Comparison of peak exhaled NO in boys and girls measured by the direct system.
184
Figure 8.2b: Comparison of peak exhaled NO in boys and girls measured by the t-piece
system......
................... 184
XVI

Fig-ure 8.3a: Mean peak exhaled No levels in control and asthmatic children measured direct
to the analysers ...........1g4
Figure 8.3b: Mean peak exhaled No levels in control and asthmatic children measured via the
t-piece sampling system ...............195
Figure 8.4a: The "fl:91of commencing inhaled corticosteroid therapy on peak exhaled NO
levels in asthmatic children measured via the direct method .............196
Figure 8.4b: The tfl.9t. of commencing inhaled corticosteroid therapy on peak exhaled NO
levels in asthmatic children measured via the t-piece sampling sysrem....... ........196
Figure 9.1: A two-compartment model of NO eichange._............... ...................20g
Figure 9.2:Plateau nasal NO increasing with ug"............ ..................223
ligure 9.3: Diagram of the measurement of lung function and NO in an infant................. ...253
:i::::::::..Y:ill:. publications with a focui on nirric oxide research per year re80_2006
,,.....,........262
XVII

List of Abbreviations
ABPA allergic bronchopulmonary dysplasia
ATS American Thoracic Society
ATP adenosine triphosphate
BAL bronchoalveolar lavage
BH4 tetrahydrobiopterin
BTS British Thoracic Society
C3, C5 complement factor 3, complement factor 5
cAMP cyclic adenosine 3',5'monophosphate
CATI, CAT2, CAT2B, CAT 3
cationic amino acid proteins
CB chronic bronchitis
CD3+ cell marker for T lymPhocyte
CD4+ cell marker for T helper lymphocyte
CD8+ cell marker for T cytoxic lymphocyte
CF cystic fibrosis
cGMP cyclic guanosine monophosphate
cNOS constituent nitric oxide synthase
CNS central nervous system
Co cobalt
CO carbon monoxide
COz carbon dioxide
COPD chronic obstructive pulmonary disease
CSF central spinal fluid
Cu copper
DMSO dimethyl sulfoxide
DNA deoxyribonucleic acid
DTT dithiothreitol
ECMO extracorporeal membrane oxygenation
ECP eosinophil cationic protein
ECRHS European Community Respiratory Health Survey
EDRF endothelial derived relaxing factor
XVIII

ELISA Enzyme-linked immunosorbent assav
eNOS endothelial nitric oxide synthase
EPNIEPX eosinophilic neuraminadase
EPO eosinophilic peroxidise
ERS European Respiratory Society
ERSTF European Respiratory Society Task Force
FAD flavin adenosine dinucleotide
Fe iron
FBFzs-tsn forced expiratory flow at 25 to 75 percent of forced vital capacity
FEVr forced expiratory volume in 1 second
FEVo.s forced expiratory volume in half a second
FMN flavin mononucleotide
FVC forced vital capacity
GINA Global Initiative for Asthma
GM-csF Granulocyte macrophage - colony stimulating factor
GTP guanosine S-triphosphate
Hzo
HzS hydrogen sulphide
HNO2 nitrous acid
HRCT high resolution computerised tomography
ICAMI intracellular adhesion molecule I
IFNI interferon gamma
IgA immunoglobulin A
IgG immunoglobulin G
IgE immunoglobulin E
IHCS inhaled corticosteroids
nl, L2, IL3, nA, IL5, IL6, ILg, ILg, ILl0, ILI l, I[-l2, ILl3, Ll7
interleukin l, interleukin 2, interreukin 3, interleukin 4, interleukin 5,
interleukin 6, interleukin 8 interleukin 9, interleukin 10, interleukin
I l, interleukin 12, interleukin 13, interleukin l7
iNOS inducible nitric oxide synthase
ISAAC Inbrnational Study of Asthma and Allergies in Childhood
Kb kilobases
Umin litres per minute
LABA long acting 92 agonisr
xrx

LADA N*N* dimethyl L-arginine
LFA1 lympocyte function-associated antigen I
L-NAME N* arginine methyl ester
L-NMMA N* monomethyl L-arginine
L-NOARG N*-nitroarginine
LNIO N- imino-ethyl-ornithine
LPC lysophosphotidylcholine
LPS lipopolysaccharide
mC4, LTD4, WM leukotriene C4,leukotriene D4,leukotriene Bl
MBP major basic protein
mg/ml milligrams per millilitre
mls/min millilitres per minute
Mn manganese
mRNA messenger ribonucleic acid
Nz nitrogen
NzO nitrous oxide ("laughing gas")
NzOs dihydrogen trioxide
NzOl dihydrogen tetraoxide
NADPH nicotinamide adenosine di-nucleotide phosphate
NANC non adrenergic, non cholinergic (nerves)
NF-KB nuclear factor-kappa B
NHANES 1 National Health and Nutrition Examination Survey I
nl-/min nanolitres per minute
nNOS neuronal nitric oxide synthase
NO nitric oxide
NO + nitrosoium cation
NO- nitroxyl anion
NOz nitrogen dioxide
NOz- nitrite
No:- nitrate
NO2Tyr 3-nitrotyrosine
NOS nitric oxide synthase/s
NOx nitrogen oxides (usually NO, NOz and NO:)
NZPAG New Zealand Paediatric Asthma Guidelines
Oz oxygen

O2-
Or
oH-
oNoo-
ONOOH
PaOz
PCD
PCzo
PDzo
PEF
Pmo
ppb
ppm
PMIO
redox
ROC
RS
RSV
SD
SEM
SGC
SIGN
SLE
Soz
TB
rGFp
Tnl
Ts2
Ts3
TNFa
TI.[FP
type I (nNOS)
type II (iNOS)
superoxide anion
ozone
hydroxyl anion
peroxynitrite
peroxy nitrous acid
pulmonary artery oxygen
primary ciliary dyskinesia
provocation concentration producing20vo fall in the forced expiratory
volume in one second
provocation dose producing 20vo fall in the forced expiratory volume
in one second
peak expiratory flow
mouth pressure
parts per billion
parts per million
particulate matter with a diameter of less than 10pm
reduction oxidative reactions
recei ver-operator curve
sulphur thios
Respiratory Syncytial Virus
standard deviation
standard error of the mean
soluble guanylate cyclase
Scottish Intercollegiate Guidelines Network
systemic lupus erythematosis
sulphur dioxide
tuberculosis
transforming growth factor beta
T helper lymphocyte cell type I
T helper lymphocyte cell type 2
T helper lymphocyte cell type 3
tumour necrosis factor alpha
tumour necrosis factor beta
Type I neuronal nitric oxide synthase
Type II induced nitric oxide synthase
XXI

t}ryem(eNoS)Typeltrendothelialnifticoxidesynthase
LrK
pgnn
LJMCEF
USA
W light
V/Q
7^
United Kingdom
micrograms Pre millilitre
UnitedNations International Children's Fund
United States of America
ultraviolet light
ventilation and perfusion ratio
zinc
xxll

1.1
chapter 1: The burden of respiratory disease in New znaland
Introduction
This opening chapter will set the scene as to why I felt that this area of research was of
importance and of interest. The burden of respiratory disease in New Tnaland is high, and
clearly apparent to anyone who trains or works here in the field of medicine. It was an
obvious problem as I came through the Auckland School of Medicine, it was obvious when I
chose a rural setting to do my first house year, and it remained obvious when I commenced
training in paediatrics. The burden appeared to be borne disproportionately by children, and
by children of two specific community groups - Maori and Pacific Island. In the l9g0s and
1990s, New Zealand was leading the world in both the incidence of asthma and, more
appallingly, asthma mortality. Although the diagnosis of asthma in children was not always
clear, we embarked on anti-inflammatory drug treatment, possibly for years. While asthma
was thought to be a reversible disease, it has since transpired that an element of irreversibility
could develop which has been termed 'airway remodelling'.
This thesis commenced ten years ago and is based on work I conducted while employed as a
fellow and senior registrar and lecturer at the Royal Brompton Hospital in london. My
interest, then as now' was in paediatric respiratory disease. I was concerned that we seemed to
be making treatment decisions regarding the use of potential toxic anti-inflammatory agents,
specifically the use of steroids, without measuring any inflammatory parameter. Rather, we
were using history, examination and other surrogate measures such as x-rays and lung
function (where possible and where children were old enough) to determine correct diagnosis
and response to treatment. So for the individual child; was the diagnosis correct? Was the
child on too much or unnecessary medication? - a treatment with potentially significant side
effects, such as adrenal suppression and growth failure. Or was the child on too little
medication? - suffering symptoms daily with the possible development of irreversible
disease, or even death. I saw many children in the clinic that were in both categories; on ..too
much" steroid therapy for too long incorrectly, on "too little" with unnecessary morbidity, and
so very few seemed'Just right". I planned and conducted the research outlined in this thesis,
which I saw as a unique opportunity to investigate a possible solution to this quandary. The
research presented is thus now historical. It has already contributed to medical literature,
including the formulation by the European Respiratory Society and American Thoracic
Society of standardized procedures for the measurement of exhaled nitric oxide (NO), the
marker I chose to explore to indicate airway inflammation. I have continued to research this

area, some of it has been expanded from the work presented here, and in these experiments I
dotted the 'i's, crossed the't's and was hands on adapting the individual machinery. It remains
eligible to present and I have lived with it so long that I am pleased to do so. This thesis
presents the research leading to the experiments that I undertook and describes the
development of NO as a measure of inflammation during this time and subsequently to the
end 2006.
In this chapter I review the burden of respiratory disease in paediatrics in New Zealand. This
review was part of a monograph edited by myself and Professor Innes Asher, and in which I
also wrote two chapters. This will be followed by a brief overview of asthma, the difficulty of
accurate diagnosis and the background to it being designated an inflammatory disease. The
following chapter will present how NO was considered a pollutant, then became recognised as
a vital biological agent, and then known as an inflammatory marker. In Chapter 3, the
characteristics of NO will be examined as these make it a difficult gas to measure. Chapter 4
will discuss the possible options for measuring NO and in Chapter 5 I will describe how a
machine that measured airway pollution became adapted to measure exhaled air. In Chapters
6 and 7 the feasibility of measuring NO in exhaled air in adults was assessed; particularly
exploring what factors affected the NO readings. These then describe the standardized method
I developed which was used as presented in Chapter 8 to enable exhaled NO in healthy
children to be measured and in asthmatic children on bronchodilator or steroid treatment, as
well as a smaller goup that was commencing steroid therapy. The penultimate chapter,
Chapter 9 will be devoted to reviewing the literature regarding NO to the present day, and
where this investigation is now best utilized. The final chapter, Chapter 10, will be a brief
commentary what I have learnt through the process of this research and writing.
I hope you enjoy this story.
1.2
The burden of paediatric respiratory disease in New Zealand
The 'Trying to Catch Our Breath' monograph (Anonymous 2006) published last year was an
initiative by twelve leaders from the New Zealand paediatric health community, supported by
the Asthma and Respiratory Foundation of New 7*aland (Te Taumatua Huango, Mate Ha o
Aotearoa), to document and present the darming data that existed on the incidence,
prevalence and the severity of respiratory disease in children. It was presented to the Right
Honourable Mr Peter Hodgsen, Minister for Health in April 2006 by the Asthma and
Respiratory Foundation of New 7*aland (Te Taumatua Huango, Mate Ha o Aotearoa). The
title of the document was deliberately ambiguous as the group also feel breathless in trying to
2

deal with the sheer numbers of children requiring assessment and treatment with these
disorders. The rates of most respiratory conditions are higher than comparable westernized
countries and the severity of these conditions more significant. Much of this is preventable. In
1999, 'The Top Ten Report' (Graham, lrversha et al. 2001) detailed the top ten issues
affecting the health and well-being of children and young people in Auckland and Waikato,
including a review of the top ten causes of potentially avoidable hospital admissions in New
Zealanders agedO-24 years (see Figure Ll).
Figure 1.1: Top 10 causes of avoidabre admissions, 0-24 years, 1g9g
Urlnary Infecilon
Convulsions
ENT Infections
Gastroenteritis
bronchlolltls
Pneumonla
]ake1 from the.'T-rying to catch our Breath monograph (Anonymous 2006) but adapted from ,The
Igp_f"! Report' (Graham, Leversha et at. 2001).
ENT = Ear, nose and throat
over half of these potentially avoidable admissions were respiratory or ear, nose and throat
conditions' Disease is exacerbated by the proportion of children living in poverty (which is
defined as living in a household with an income below 6ovo ofthe median family income net
of housing costs). This has increased from l6vo in r987/r98g to 2gEo in 20ffi12001 (Ministry
of Social Development2N2).Indeed New Zealand has one of the worst rates of child poverty
in rich countries (UNICEF 2005). The burden of disease particularly impacts on certain ethnic
groups within New Zealand. Maori children currently make up 25vo of all children living in
New Zealand and, as a collective group, Pacific peoples make up 6.5vo of the New Zealand
population' However both of these groups have significantly higher proportions of children
aged less than fourteen yeius of age - 35vo of Maori children (Te puni Kokiri 2oo2) and 3gvo
of Pacific children (Statistics New Zealand 2002) compared to 20vo of total New Zealanders.

In addition, both of these groups are disadvantaged with regard to disposable income,
adequate housing, educational opportunities, unemployment rates and access to healthcare
(Ministry of Health 2002; Statistics New Zealand2002; Asher 2006).
A review of the statistics for the different paediatric respiratory conditions follows. Nationally
the admission rate for children < one year of age for lower respiratory tract infection,
predominantly bronchiolitis and pneumonia, is L02.61t000 but up to 176.5/1000 in certain
regions (Graham, Irversha et al. 2001). The rates of admission for bronchiolitis for children <
one year of age have increased from 26.611000 children in 1988 to 58.U1000 in 1998, an
increase of ll87o (Vogel, knnon et al.2003). In addition, this increase in admission rates is
accompanied by more severe disease in those admitted in comparison to the previous decade
and in comparison to paediatric admissions of bronchiolitis in other developed countries. Of
the children admitted; 59Vo required oxygen, 2lVo required nasogastric fluids, 22Vo
intravenous fluids, 8Vo werc admitted with apnoea and 3.LVo reeuired ventilation (Vogel,
lrnnon et al. 2003). Pneumonia continues to be a worldwide problem with acute lower
respiratory tract infections being an important cause of mortality in children < five years of
age and remains one of the leading causes of disability-adjusted life years lost worldwide.
This largely reflects children in developing countries where it is estimated that 4.3 million
children aged < five years die annually from lower respiratory tract infections (Garenne,
Ronsmans et al. t992). The incidence tends to be higher in children < five years of age at 34-
40 cases per 1000 compared to any other age group except possibly for adults > 75 years
(Mclntosh 2002). A recent study conducted in New 7-ealand ascertained that the national
pneumonia admission rate for children 0-14 years of age was 4.0/1000 based on the discharge
diagnoses for pneumonia in 1998 to 1999 (Milne,Irnnon et al. 2003). These admissions were
skewed toward the younger age group. This equated to t,534 admissions per 100,000 for
children aged < two years, 562 admissions per 100,000 for children aged 2-4 yeus and 170
admissions for children aged 5-9 years. Extrapolation of this data amounts to approximately
3000 paediatric hospital admissions for pneumonia in New Tx,aland per year. Furthermore,
from 1988 to 1995 there was an annual increase of SVo in the hospital admission rate. This
increase was not due to admitting children with less severe disease; indeed the disease
severity also seemed to be on the rise (Ministry of Health 1998; Grant 2006). Even pertussis,
which is largely preventable by immunisation, continues to be a significant problem. Reported
coverage of pertussis immunisation shows that only 60Vo of children are fully immunised,
with only 42Vo of Maori and 45Vo of Pacific children completing the recommended

immunisation schedule (Ministry of Health 1992; Grant 2000; Grant, pati et al.211l;Turner
2006).
Bronchiectasis has been decreasing in most developed countries but this does not appear to be
the case in New Zealand.In studies that I led, subsequent to the research presented in this
thesis, a prospective investigation was conducted utilising the 'New Zealand paediatric
surveillance Unit', covering 947o of paediatricians nationally. This requested notification and
subsequent data on any new diagnosis of bronchiectasis made in children < 15 years through
2001 and 2002 (Twiss, Metcalfe et al. 2005). The incidence from this study put bronchiectasis
at 3'7/100,000/year, which is seven times greater than the only other comparable national
study done in Finnish children (Sayajakangas, Keistinen et al. 1998). This equates to l/1700
live born children being diagnosed with bronchiectasis before the age of 15 years. If the
incidence rate was to remain static and all these children survived to 15 years of age, this
would equate to a prevalence of 1/3000 children overall. Again a disparity is seen with the
incidence found to be three times higher in Maori children (l/1700) and twelve times higher
in Pacific Island children (1/650) than those with European ethnicity. As well as being
common, the disease was severe as demonstrated in both the Auckland study @dwards, Asher
et al. 2003) and the National study (Twiss, Metcalfe et al. 2005) with g3-93 Vo havingbilateral
disease, and 6l-64%o having three or more of the six lobes of the lung involved. In addition
the incidence of tuberculosis (TB) has decreased for adults in New 7*aland,over the recent
years' but disturbingly there has been no such reduction in paediatric rates as demonstrated by
our review of paediatric TB cases through the 1990s (Howie, Voss et al. 2005). Again
significant ethnic disparities occur in these rates; in children < 15 years, pacific children
account for lTVo of all cases of TB and Maori children for l5vo compared to Europea n at 3Vo.
Unsurprisingly, this condition is also associated with socioeconomic deprivation (Ministry of
Health 2002).
These reports all confirm the extent of paediatric respiratory disease in New Zealand,and the
fact that they do not appear to be on the wane despite the improving statistics seen in other
developed and comparable countries.
1.3
Asthma is one of the most common chronic diseases in the world with an estimated 300
million people affected and the number of disability-adjusted life years lost estimated at
approximately 15 million per year (GINA 2O04; GINA zOO5). Asthma has also had a
demonstrated increase in prevalence in the 1980s and 1990s as evidenced by a number of
5

studies conducted throughout the United Kingdom, Australia and New 7-ealand, (Robertson,
Heycock et al. l99l; Peat, van den Berg et al. 1994; Rona, Chinn et al. 1995; Mitchell and
Asher 1997; Asher and Grant 2006). In the 'Global Burden of Asthma' report (GINA 2004)
the proportion of the population said to have 'clinical' asthma in New Zealand is given as
l1.l%o and in England (the country I did the research reported in this thesis) is given as
l5.3%o. The case fatality rate for asthma is 4.6/100,000 asthmatics in New Zealand and
3.211N,000 asthmatics in England. In the 2006 ranking for asthma mortality from one
(highest) to 68 (lowest) New Zealand appears at 17 and England at 26. The International
Study of Asthma and Allergies in Childhood (ISAAC), (Weiland, Bjorksten et al.2004) and
the European Community Respiratory Health Survey (ECRHS), (Anonymous 1996) asked
about self reported wheezing in the previous twelve months as one of their core questions,
having demonstrated this had good specificity and sensitivity for diagnosis of asthma. In 13-
14 year old children, England and New Tnaland were ranked six and seven out of 84 countries
where the listing was from one being the highest percentage of positive responses to 84 being
lowest percentage of positive responses (Anonymous 1998). Furthermore, New Zealand
ranked second when the symptom prevalence of wheeze in 13-14 year olds was determined
by responses to a video questionnaire in 44 countries (Anonymous 1998).
In addition to community prevalence, asthma is a major cause of hospital admissions for
children, particularly noticeable in younger age groups. Admission rates had been increasing
through the 1970s, 1980s and early 1990s (Anderson, Bailey et al. 1980; Jackson and Mitchell
1983; Mitchell 1985; Hyndman, Williams et al.1994) with possible stabilisation or reduction
in the most recent years (Kemp and Pearce t997; British Thoracic Society 20OI; Akinbami
and Schoendorf 2002). There were two major epidemics of asthma mortality and New
7-ealand featured highly in both. The first mortality increase was observed in several countries
through the 1960s and included Australia, England, Norway, Scotland, Wales and New
7*,aland. The second epidemic was seen in New 7*,aland alone when mortality rates reached
4.1/100,000, which appeared linked to the use of the relatively non-selective B-2 agonist
'Fenoterol'. A 'Fenoterol' case control study was undertaken which looked at ll7 patients
between 5-45 years dyrng of asthma between 1981 and 1983. Each index case was matched
with four controls and the use of 'Fenoterol' gave a relative risk of death of 1.59, slightly
higher in females at 1.65 compared to males at l.M. However it was greater in the patients
who were less than 20 years of age with an odds ratio of 2.08 (Crane, Pearce et al. 1989).
Fenoterol was subsequently withdrawn from the drug tariff and, coinciding with the greatly
reduced use after 1990, there was a significant reduction in the number of deaths (Crane,

Pearce et al. 1995). Despite the stabilization in the last few years and the reduction in previous
high mortality, asthma remains a prevalent paediatric disease (Kercsmar 2006; Weinberger
and Abu-Hasan 2006).
t.4
L4.I The asthma diagnosis in national and international guidelines
Prior to the difficulties regarding treatment, particularly in children, the first hurdle is making
a correct diagnosis of asthma which cornmences by having an appropriate definition. The
2005 'British Guideline on the Management of Asthma' from the British Thoracic Society &
Scottish Intercollegiate Guidelines Network (SIGN) guideline states; ,.The diagnosis of
asthma is a clinical one; there is no confirmatory diagnostic blood test, radiographic or
histopathological investigation. In some people a diagnosis can be corroborated by suggestive
changes in lung function tests" (SIGN 2005). The problem of accurate diagnosis appears to
have existed for many decades. "The clinical diagnosis of asthma is not always simple and the
absence of an agreed definition of the disease is a problem with many descriptions existing,,
appeared in 1959 from a conference on 'terminology' (Anonymous 1959). The difficulties of
diagnosis and differential diagnoses are apparent in the paediatric section of the SIGN
guideline and in two paediatric asthma guidelines published in 2005; 'pocket Guide for
Asthma Management and Prevention in Children' from the 'Global Initiative for Asthma,
(GINA) (GINA 2005), and the 'Management of Asthma in Children aged I -
15 years' from
the Paediatric Society of New 7*aland (NZPAG) (Paediatric Society of New Tnaland2005).
Ultimately these publications agree that it is a clinical diagnosis based on a history of
appropriate symptoms of wheeze, which varies over time, improving either spontaneously or
as a result of treatment and with wheezeheard, during times of exacerbation. However all
indicate this should occur in the absence of other features that could suggest congenital,
suppurative or atypical respiratory disease, or is not responsive to asthma treatment.
L4.2 Wat is meant by 'wheeze'?
Even here there are at least two difficulties. Firstly, what is meant by 'wheeze' and secondly,
that not all wheeze is asthma. Parental reporting of wheezing does not always coincide with
what doctors mean by wheezing (Cane, Ranganathan et al. 2000;Cane and McKenzie Z0(lI).
Of parents presenting with infants with a problem of 'noisy breathing', 59Vo used the term
'wheeze' to describe their concern. However, after being shown video clips illustrating
wheezing and other airway noises, only 36Vo continued to describe their infant as wheezing.

Video clips of infants with a variety of breathing noises led to 30Vo of respondents using other
words to describe wheezing and30Vo using the term 'wheeze' incorrectly @lphick, Sherlock
et al. 2001).
In addition, all that wheezes is not asthma (see Table 1.1). Up to 40Eo of infants who have
been hospitalised with bronchiolitis may have subsequent wheezing episodes, usually in
association with viral infections, up to five years of age (Martinez, Wright et al. 1995) and
approximately LUVo continue to wheeze after age five years (Noble, Murray et al. 1997;
Sigurs, Bjarnason et al. 2000; Hall 2001). In the Tucson study of over 1200 children, wheeze
was reported in almost 50Vo at some time between birth and six years (Martinez, Wright et al.
1995). Similar studies from Europe suggest l5-327o of children had wheezing in the first five
years of life (Strachan 1985; Park, Golding et al. 1986). Sixty to 80Vo of infants who start
wheezing in their first two years of life do not go on to have asthma. This group is found to
have diminished airway function present prior to the onset of wheeze, and wheeze often
ceases around the age of 3-5 years (Morgan and Martinez L992; Holberg, Wright et al. 1993;
Wilson 1994; Martinez, Wright et al. 1995; Clough, Keeping et al. 1999) which confirms the
findings of a much earlier study @oesen 1953). However, a minority of wheezy infants will
develop later asthma (Wilson 1994; Martinez, Wright et al. 1995; Cochran 1998). These
children continued to have wheezy episodes, developed an increased total serum
immunoglobulin E (IgE) bV age 9 months, developed sensitivity to a panel of aeroallergens by
age 6 years, and had significantly lower lung function by age 6 years (Martinez 2002).In
prospective follow up studies from childhood to adolescence, up to 80Vo of asthmatic children
were reported to lose their symptoms during puberty (Balfour-Lynn 1985; Peat, Salome et al.
1989; Nicolai, Illi et al. 1998). Thus asthma may then remit in later childhood or may
continue throughout childhood and into adult life (von Mutius 2001; Weinberger 200.3).
Table 1.1: Alternative diagnoses in children with wheeze
Angio-oedemia
Aspiration
syndromes
including foreign bodies Ainray compression bronchogenic or pulmonary
cyst, lymph nodes, tumour
(carcinoid, lymphoma),
vascular ring or sling
q1-antiprotease
deficiency
(older child)
Bacterial tracheitis respiratory syncytial virus,
metapneumovirus,
parainfluenza, influenza,
adenovirus
Anaphylactic
reactions
Angioedema
Bronchiolitis Aspiration including foreign bodies

Inhalational irritants
pertussis, mycoplasma,
tuberculosis, any other
bacterial infection
smoke, drugs
syndromes
Bronchiolitis -
recurrent
Bronchiolitis
obliterans
Cardiac conditions
Chronic lung
disease of
prsmaturity
Chronic suppurative
disease
Congenital
abnormality
Eosinophilic
bronchitis
Gastrooeosphageal
reflux
Genetic disorders
lmmune
deficiencies
Inhalational irritants
Malacic syndromes
Post-infectious
Sarcoidosis
Tuberculosis
Vasculitic disorders
Vocal cord
dysfunction
respiratory syncytial virus,
metapneumovirus,
parainfluenza, influenza,
adenovirus
congestive heail failure,
vascular compression (ring or
sling)
bronchiectasis, chronic
bronchitis
anatomic lesion:
cystic malformations, foregut
malformations, granulation
tissue, tracheoesophageal
fistula
+/- aspiration
cystic fibrosis, primary ciliary
dyskinesia
smoke, glue, drugs
tracheomalacia,
bronchomalacia
adenovirus
pertussis
mycoplasma
Wegeners's granulomastosis,
other
Despite these two confounding issues, wheeze remains a key element in the diagnostic criteria
cited for asthma in all the guidelines (Paediatric Society of New Zealand 2005), (GINA
2005), (SIGN 2005) and confirmed by surveyed clinician opinion (Werk, Steinbach et al.
2000). There is no standard way of estimating the frequency or severity of episodes of wheeze
to identify that which is purely consistent with asthma and classical diagnosis usually relies
on associated symptoms and findings (GINA 2005). In addition, the performance of a p-2
agonist treatment trial for acute wheeze are described in these guidelines and also in two
recent bronchiolitis guidelines, 'Wheeze and Chest Infection in Infants under One year' from

the Paediatric Society of New 7*aland which I co-chaired (Byrnes, Vogel et al. 2005) and a
report from the Agency for Healthcare Research and Quality, from the United States
Department of Health and Human Services (Viswanathan, King et al. 2003).
1.4.3 Where does coughfit in?
The use of cough as a diagnostic symptom is another area that is not straightforward. When
asked what clinical factors were associated with establishing an initial diagnosis of asthma,
96Vo of the responding clinicians thought the requirement was recurrent wheeze, 90Vo
symptomatic improvement with a bronchodilator, and 89Vo recunent cough, but there was
disagreement over what combinations of these factors were important (Werk, Steinbach et al.
2000). "Troublesome cough is a characteristic of asthma" (Li, I-ex et al. 2003) and "cough is
as frequent a symptom as is wheezing" (Weinberger and Abu-Hasan 2006) has been reported.
Controversy has focused on whether 'cough variant asthma' (sometimes called 'cough
equivalent asthma') truly exists, with researchers both for and against.
It is thought that asthma could occur without wheezing if the obstruction involves the small
airways predominantly, and then coughing or shortness of breath may be the only complaint
(Kercsmar 2006).In a follow-up study of I25 preschool children with recurrent cough but no
other symptoms at the time of enrolment, 56Vo of them were symptom free at follow-up while
36.8Vo continued to have recurrent cough in the absence of a 'cold', and7 .2Vo had developed
recurrent episodes of wheeze @rooke, Lambert et al. 1998). One hundred children with a
mean age of 5.7 years with chronic cough were followed for three years. Of the 75 deemed as
having 'cough variant asthma', 28 (377o) went on to develop more 'classical' asthma
symptoms (Todokoro, Mochizuki et al.2N3). Bronchial hyper-reactivity, nocturnal cough
and developing cough at an earlier age may each be predictors of those who go on to get more
typical asthmatic symptoms (Brooke, Lambert et al. 1998), (Todokoro, Mochizuki et al.
2003).It has been suggested in adults that those with cough who go on to develop classical
asthma have a threshold for airway hyper-responsiveness somewhere between asthmatic and
normal subjects (Koh, Jeong et al. 1999; Mochizuki, Arakawa et al. 2005) and 30Vo of those
with cough and bronchial hyper-responsiveness will go on to develop asthmatic symptoms
(Fujimura, Nishizawa et al. 2005). Eosinophilic inflammation (eosinophils, eosinophil
cationic protein (ECP), interleukin 5 (IL5)) has been demonstrated in some patients with
persistent cough; a pattern of inflammation similar to asthmatics seen in adults (Lee, Cho et
al. 200| De Diego, Martinez et al. 2005) and in children (Yoo, Koh et al.2004). Basement
membrane thickening, a pathological feature of asthma, was demonstrated in 'classical'
l0

asthmatics at 8.6 microns, in 'cough variant asthmatic' subjects at7.l microns and both were
significantly different than healthy controls at 5.0 microns (Niimi, Matsumoto et al. 2000).
Others are less enthusiastic for this diagnostic category -
given that cough is a very corlmon
symptom. A diagnosis of cough variant asthma could result in a significant increased number
of children being treated' "Isolated chronic cough in children is rarely asthma, and the term
'cough-variant-asthma' should not be used" (Chang, Landau et al. 2006). Wheezing reported
by children had a very good discriminating ability for asthma whereas cough less so in over
1600 school children aged 8-12 years (Yu, Wong et aI.2004). In asthmatic children who had
cough as a predominant symptom, the cough occurred early and heralded the onset of an
exacerbation. Asthma scores did relate to cough scores, although neither cough receptor
sensitivity nor cough scores related to any specific inflammatory marker (Chang, Harrhy et al.
2002). In 1245 children 6-12 years of age comparing symptoms of persistent cough with
symptoms of wheeze, the former had less demonstrable atopy and less morbidity. The authors
concluded that while 'cough variant asthma' was not shown, there remained a significant
number of children with persistent cough diagnosed and treated as having asthma @aniran,
Peat et al. 1999). So in the presence of persistent cough, if history, examination, chest x-ray
and spirometry are normal, the cough could be regarded as non-specific and the child
regarded as in the category of 'normal', as opposed to heralding a morbid or pre-morbid
condition (Chang and Powell 1998; Bush 2002).
Finally, treatment trials for cough using inhaled corticosteroids (IHCS) (Dicpinigaitis 2006;
Matsumoto, Niimi et aL.2006) and a leukotriene receptor antagonist (Spector and Tan 2004)
showed an improvement in symptoms. However the natural history of post-viral cough is that
most will have resolved in 3-4 weeks and only l\Vo will continue for more than 25 days, with
gradual resolution. Improvement could therefore have erroneously been attributed to
treatment. The Cochrane Systematic Review of the efficacy of IHCS for non-specific cough
in children > two years of age evaluated two trials enrolling a total of 123 children. The first
trial using beclomethasone dipropionate 4o0ltglday (metered dose inhaler via a spacer) found
no difference from placebo in reducing cough frequency measured objectively or scored
subjectively. The second trial using fluticasone propionate 2mglday for three days followed
by I mg/day for 1l days (metered dose inhaler via a spacer) showed a significant
improvement in nocturnal cough frequency after two weeks, but also with a significant
(though smaller) improvement with placebo (Tomerak, McGlashan et al. 2005). There was no
difference between the use of inhaled salbutamol and placebo (Tomerak, Vyas et al. 2005) or
ll

inhaled anti-cholinergic agents and placebo (Chang, McKean et al.20O4) in children with
persistent non-specifi c cough.
1.4.4 Investigations
There are few investigations that are specific to asthma in adults and children. There are no
diagnostic chest x-ray findings; the film may be normal, or there may be hyperinflation,
interstitial infiltrates or atelectasis with mucus plugging (Arthur 2000; Kercsmar 2006).
Hyperinflation, the most common pattern associated with asthma, can also be seen in
bronchiolitis and cystic fibrosis (CF) (Arthur 2000). Similarly, studies have shown that chest
x-rays are not useful in determining the difference between bronchiolitis and pneumonia
(Khamapirad and Glezen 1987; Davies, Wang et al. 1996; Byrnes, Vogel et al. 2005). In a
survey of school age children referred to a respiratory clinic,30Vo had prior diagnoses of
pneumonia on chest xray appearances but presented with symptoms identical to those
subsequently associated with their asthma diagnoses (Castro-Rodriguez, Holberg et al. 1999;
Mahabee-Gittens, Bachman et al. 1999).
Both total IgE and skin prick testing to common allergens have been used as a method of
detecting atopy, which overlaps the asthma syndrome. IgE levels appear to have been
increasing over the last decades coinciding with the increase in asthma (Burrows, Martinez et
al. 1989; Burney, Malmberg et al. 1997). Higher levels of IgE have been associated with
increased atopy and an increased incidence of wheeze (Call, Smith et al. L992; Heymann,
Carper et al. 2O04). An association has been demonstrated between the level of IgE and
airway hyper-responsiveness as measured by bronchial challenges (Muranaka, Suzuki et al.
1974; Burney, Britton et al. 1987). In a long term infant follow up study, the mean total serum
IgE at nine months of age was significantly higher among those who became persistent
wheezers compared to those who did not wheeze. Within the group of eady wheezers, those
who were wheeze free by ages I I and 16 years had total IgE levels similar to those who never
wheezed (Martinez 2002). However, the findings are not always consistent. Total IgE levels
were found to be higher in some cohorts of children than others despite similar asthma
prevalence, and the total IgE was higher in countries where house dust mite was the dominant
source of allergen @latts-Mills and Heyman 2006). The ECHRS study involving 37 centres in
16 countries demonstrated variation in the prevalence of response to at least one specific IgE
which ranged from 60Vo in Albacete (Spain) to 45Vo in Christchurch (New 7*aland) and was
not always consistent with asthma prevalence. The study also showed significant variations
between countries for total IgE and for prevalence of any specific IgE. At all ages women had
t2

a 26Vo lower total serum IgE than men, which is surprising when asthma tends to be more
prevalent in women in adulthood. There was a lack of association between the prevalence of
atopy (defined as a positive response to any of the four specific allergens used in skin prick
testing in all centres) and the geometric mean of total serum IgE. It is probable that the total
and specific IgE are influenced differently and while raised IgE is associated both with atopy
and with asthma, the relationship is not clear (Burney, Malmberg et al. 1997). Furthermore,
IgE antibody production is T cell dependent, and the immune response to allergens can also
include other immunoglobulins such as immunoglobulin Ga (IgG+) and immunoglobulin A
(IgA) which may give further variations (Platts-Mills and Heyman 2006).It is true to say that
if a child is thought to have bad asthma, but is non-atopic, then the search for an alternative
diagnosis should be undertaken immediately.
Pulmonary function tests have been one of the mainstays of assessing respiratory disease in
older children and adults. A measurement in forced expiratory volume in I second (FEVI)
alone can miss small airway obstruction in patients and the forced expiratory flow rate over
25-75Vo of forced vital capacity (FEF25 -tsw) may well be a more sensitive indicator,
particularly in children and/or in mild disease; however, the measurement may be very
variable. Subjects can have a reduction in this parameter of more than two standard deviations
below predicted norms even with no clinical symptoms (Kercsmar 2006). There is a
significant overlap with measurements between healthy children and those with episodes of
wheeze (if not current), and the diagnostic value of baseline lung function tests is generally
thought to be poor (Dundas and McKenzie 2006). The guidelines suggest a test of
bronchodilator responsiveness for asthma either clinically and/or utilising improvement in
lung function (usually FEVr) by an arbitrary amount, sometimes 157o. Despite this, the SIGN
guideline states "a definitive diagnosis of asthma can be difficult to obtain in young children.
It is often not possible to measure ainvay function in order to confirm the presence of variable
airway obstruction" (SIGN 2005).
The other option for possible diagnosis and monitoring of asthma is to use peak expiratory
flow (PEF). This is defined as the largest expiratory flow achieved with a maximal forced
effort from a position of maximum inspiration sustained for longer than l0 milliseconds
(Wright and McKerrow 1959). PEF reflects a range of physiological characteristics including
lung elastic recoil, large airway calibre and lung volume (Ruffin Z0O4). The original .peak
Flow Meter' was pioneered by Martin Wright in 1959 (Wright and McKenow 1959) and is
now supplanted by newer models but these have retained the advantages of the original in
being low cost and easily portable. In the late 1970s the use of PEF recordings was first
13

ecommended to monitor asthma at home (Seaton 1978). This became part of an international
consensus document in 1992 (Irnfant rgg2). The pEF technique was standardised in an
American Thoracic society paper in 1994 (American Thoracic Society and Association.
lgg4).By 1998 all the published asthma guidelines included PEF monitoring as part of an
individualised management strategy (woolcock, Rubinfeld et al' 1989; National Heart Blood
and Lung Institute 1991; British Thoracic Society 1993; GINA 1995; Jain, Kavuru et al'
1998). The concept was to provide an objective measurement of airflow obstruction'
particularly for the significant proportion of patients who otherwise had difficulty in
recognising their asthma severity (Rubinfeld and Pain L976;Burdon, Juniper et al' 1982; Sly'
Landau et al. 1985; Kendrick, Higgs et al. 1993; Fishwick and Beasley 1996)'
However, there are issues with using pEF as this predominately reflects alteration in large
airway calibre which is different to FEVr that reflects changes in calibre of both large and
medium sized airways (Osmanliev, Bowley et al' t982; Robinson' Chaudhary et al' 1984;
Dolyniuk and Fahey 1986; Gregg 2000). PEF is effort dependant so is susceptible to both
respiratory muscle strength and patient motivation (Tzelepis, pavleas et al. 2005). while the
reproducibility of pEF has been reported to be good with a coefficient of variation in well
trained subjects of between 5 to I4Vo, still more than Sovo of asthmatic patients show a 107o
difference and 33vo of patients show more than a 20vo difference between the percent
predicted value of FEVr and PEF (Kelly and Gibson 1988; Paggiaro' Moscato et al' 1997)' In
addition, the standard deviation for PEF readings is consistently greater than that of FEVr' In
another study, the coefficient of pEF repeatability was 107o for healthy adults but rTvo for
healthy children, LTVo inasthmatic adults and2SVo in asthmatic children @nright, shenill et
al. 1995). This poorer repeatability in asthmatic subjects has been confirmed by other studies
(Meijer, Postma et al. 1996; Timonen, Nielsen et al' IggT; Holcroft' Eisen et al' 2003)' In
addition, significant differences in variability were also described between encouraged PEF
readings done in a laboratory and those done at home (Reddel' Ware et al' 1998; Gannon'
Belcher et al. 1999).
The two methods of using pEF to detect or monitor asthma was either by determining daily
PEF variability or by using the comparison of the current PEF to a subject's best PEF (Jain'
Kavuru et al. l99g). pEF variability is associated with acute asthma exacerbations @ellia,
Cibella et al. 1985; Beasley, Cushley et al. 1989; Jindal, Aggarwal et al' 2002)' Cross
sectional studies have demonstrated significant (albeit weak) correlations between PEF
variability and overall severity of inflammation, symptoms scores, FEVr and bronchial hyper-
responsiveness (Higgins, Britton et al. lgg2;Brand, Duiverman et al' 1997; Douma' Kerstjens
L4

et al. 2000; Jindal, Aggarwal et al. 2002) with similar (but weaker) conelations found in
longitudinal studies (Brand, Duiverman et al. lggg: Kamps and Brand 2001). However, the
degree of variability considered 'abnormal' is difficult to determine (Jindal, Aggarwal et al.
2002) with a difference of 20Vo between repeated PEF recording suggested as necessary to
identify asthma (Hetzel l98l; Jamison and McKinley 1993; National Asthma Education and
Prevention Program 1997). Studies have shown no clear demarcation between normal
individuals and asthmatics (Higgins, Britton et al. 1989; Parameswaran, Belda et al. lggg),
with the greatest overlap between groups occurring in children (Albertini, politano et al. l9g9;
Goldberg, Springer et al. 2001). PEF variability of 20Vo gave a sensitivity of 5l.77o,
specificity of 82.4Vo, positive predictive value of 13.97o and a negative predictive value of
95.5Vo for detecting asthma in one study (Goldberg, Springer et al. 2001). In a second study
20vo variability gave a sensitivity of 36vo, specificity 90Vo, positive predictive value of 16.47o
(Kunzli, Stutz et al. 1999) while this cut off missed three quarters of the asthmatic patients in
a third study (Goldstein, Yeza et al. 2001). The relationship between the magnitude of pEF
variability and response to bronchodilators is poor on or off IHCS therapy (Connolly 1979;
Kerstjens, Brand et al. 1994; Douma, Kerstjens et al. 2000; Iwasaki, Kubota et al. 2000) and
the relationship between PEF measurement when on long acting p2 agonist treatment is
complex depending on the timing of the last dose (Reddel, Jenkins et al. 1999; Reddel, Ware
et al. 1999). Also both daily peak flow levels and mean peak flow may fall so that the
variability may not change (Reddel, Jenkins et al. 1999). In addition, there appears to be a
circadian rhythm for PEF in all individuals (Hetzel and Clark 1980; Hetzel lggl) bur
exaggerated in patients with asthma (Turner-Warwick 1977; Connolly 1979; Bagg and
Hughes 1980; Hetzel and Clark 1980).
The other option recommended is to use a percentage of maximum pEF value for an
individual to discern asthma. A reduction from best PEF of 16.5To Bave asensitivity of SlVo
and a specificity of 98.7Vo, while a reduction of 2o%o gave a sensitivity of only 40Vo and
specificity of 99.3Vo (Aggarwal, Gupta et aL.2002). In children, percent predicted pEF only
weakly correlated symptoms or airway responsiveness (Kolbe, Richards et al. 1996; Brand,
Duiverman et al. 1997; den Otter, Reijnen et al. 1997). This also assumes the patients will
know their best PEF measurement but only 297o of 104 subjects were able to report this
@iner, Brenner et al. 2001). This group also showed that over subsequent days, 45Vo of
patients had a measured PEF greater than their reported personal best. Thus if calculated on
the reported figure both over-treatment in as man y as 30Vo of clinically stable patients
(Douma, Kerstjens et al. 1998) and inappropriate emergency department discharges based on
15

improvement to 70Vo PEF would have occurred (Diner, Brenner et al. 2001). Predicted PEF
values has also been shown to be alter with age, sex, race, height and smoking making advice
based on "predicted values" difficult (Gregg and Nunn 1973; Nunn and Gregg 1989; Higgins,
Britton et al. 1992; Higgins, Britton et al. 1993; Boezen, Schouten et al. 1994; Boezen,
Schouten et al. 1995; Bellia, Cuttitta et al. 1997; Jain, Kavuru et al. 1998; Bellia, Catalano et
al. 2004).In addition, significant variation in predicted PEF values has been demonstrated
even within the same populations (Jindal, Aggarwal et al.2002).
The number of PEF measurements also contributes to accuracy and studies have used between
two and twelve readings (Gannon, Newton et al. 1998; Jindal, Aggarwal et al.2N2). Taking
twelve measurements daily as the gold standard, using four of these detected only 60-807o of
the variability and using two measurements detected only 20-45Vo of the variability
@'Alonzo, Steinijans et al. 1995). Another study showed that four, three and two daily
measurements explained 90-95Vo,70-82Vo and 55Vo of the diurnal variability respectively
(Gupta, Aggarwal et al. 2000). Patients also need to repeat the measurements several times on
each occasion as, on review of 5,809 test sessions, it was the third manoeuvre that most
frequently (40Vo of the time) gave the highest reading (Gannon, Belcher et al. 1999).
Practically, it would be difficult to request patients to do anything beyond two readings per
day over several days, let alone weeks or months. With a circadian rhythm playing a part,
ideally but also difficult would be to request the PEFs be done regularly at specific times of
the day. Finally, the age at which the child becomes able to use a peak flow effectively is also
variable (Clough 1996).
Overall the recommendation is not to use PEF for diagnosis, but international guidelines
continue to recommend PEF monitoring in the assessment of asthma, although more recently
for selected groups; those with severe asthma, poor perceivers, as a short term monitoring
procedure for exacerbation or to assess the adjustment of the medication dose (Reddel 2006).
PEF monitoring did reduce symptoms and improve other parameters such as days lost from
work, physician consultations and hospital visits in some studies (Ignacio-Garcia and
Gonzalez-Santos 1995; Cowie, Revitt et al. 1997). However, others showed no major changes
in outcomes in adults (Jones, Mullee et al. 1995; Turner, Taylor et al. 1998; Adams, Boath et
al.200l; Tierney, Roesner et aL.2004) or children with asthma (Mortimer, Fallot et al. 2003).
In children, it was difficult to perform (Gorelick, Stevens et al.2004) and was not useful in
dictating management during acute asthma (Wensley and Silverman 2OM). A Cochrane
review in 2004 concluded there was not enough data even combining seven trials (921 adults
and 46 children) to show that personalised self management plans with or without PEF
16

monitoring improved asthma outcomes (Toelle and Ram 2004). A further Cochrane review
(Bhogal, Tnmek et al. 2006) in 2006 assessed four trials with 355 children comparing
symptom based written action asthma plans to PEF monitoring and found no differences in
the rate of exacerbation, oral steroid courses, admissions, symptoms, lung function, school
absenteeism or quality of life (Charlton, Charlton et al. 1990; Yoos, Kitzman et a:.2002;I*ta,
Schlie et al. 2004; Wensley and Silverman 2004). Symptom monitoring was actually
preferred over peak flow monitoring by the children. The final conclusion from this analysis
was "symptom based written action plans are superior to peak flow written action plans for
preventing acute care visits." (Bhogal, Tnmek et aL.2006). The daily use of pEF in a large
prospective study was not perceived to be useful by most families and therefore unlikely to be
adhered to by many (McMullen, yoos et aI.2002).
Electronic recording spirometers were compared to hand written diaries in 6l subjects aware
of the recording. Adherence to PEF monitoring over 72 weeks gradually declined fromg67o
to 89Vo with l3vo of participants ultimately withdrawn because of poor adherence (Reddel,
Toelle et al.2002). When participants were unaware of electronic recording and completed a
pen and paper diary over 3 month periods, 64Vo and,44vo adherence to monitoring was noted
(Chowienczyk, Parkin et al. 1994; Verschelden, Cartier et al. 1996). Two groups of children
reported 96.6Vo and 94.8Vo PEF monitoring compliance while the simultaneously monitoring
electronic device recorded compliance at 73.4Vo and 80.9Vo. Compliance decreased
significantly from week one to week four such that overall the compliance was less than 50Vo
in l2.5%o of the children and 50-75Vo in 20Vo of the children. Invention of some pEF
measurements occurred and increased threefold during the study time (Kamps, Roorda et al.
2001). This adds to the difficulty of giving action points, especially if figures are made up
and./or it is not perceived as useful.
In addition to these difficulties, differences have also been demonstrated when using different
PEF techniques. These include fast or slow exhalation (Richards 1993; Tzelepis, Zakynthinos
et al. 1997), a breath-hold at total lung capacity (Matsumoto, Walker et al. 1996), if the
position of the instrument is changed in relation to the mouth (Nolan, Tolley et al. 1999) or
with posture differences (Bongers and O'Driscoll 2006; Lin, parthasarathy et al. 2006).
Increased air trapping also reduced the ability of a normal PEF to predict a normal FEVI or
FEF25-75E" from 83 to 53Vo potentially falsely reassuring an individual of having better
pulmonary function than is correct @id, yandell et al. 2000).
t7

Furthermore, investigators have also found a poor agreement between the brands of peak flow
metres when tested at sea level (Jackson 1995), at altitude (Gardner, Crapo et al. 1992) and in
the clinic (Gregg 1991; Quanjer t992; Burge 1993; Dahlqvist, Eisen et al. 1993; Gregg 1998;
Nolan, Tolley et al. 1999) even after the technical requirements for meters were established
by the National Heart, Lung and Blood Institute in 1991 (National Heart Lung and Blood
Institute 1991). Differences have also been seen between the values obtained from new or old
meters of the same type (Douma, van der Mark et al. 1997; Nazir, Razaq et al. 2005).
Finally there is also a report of adverse reactions to peak flow monitoring with two patients
having a herniation of abdominal content and three having depression or neurotic
preoccupation with their PEF values which the authors calculated as a side effect incidence of
1.1 cases per thousand patients in the study population (Schoch, Nierhoff et al. 1998). There
has been a report about the potential of fungal contamination of peak flow devices (Ayres,
Whitehead et al. 1989). So PEF monitoring is also not the answer for accurate diagnosis and
may not offer significant advantage for long term monitoring in most asthmatic patients over
symptom assessment.
Airway challenges (both direct and indirect) have also been used to make an asthma
diagnosis, although this happens less often in paediatric field outside of the research arena.
Airway direct challenges have been used widely and are well standardised most commonly
using methacholine or histamine. These act as direct stimuli on the effector cells,
predominantly airway smooth muscle, but also on mucus glands and airway microvasculature,
causing airflow limitation (Adelroth, Hargreave et al. 1986; Woolcock, Anderson et al. 1991).
The responses follow a continuous distribution within the population, which leads to
difficulty when determining a normal, versus an asthmatic response @ehaut, Rachiele et al.
l9S3). This has been arbitrarily defined (currently set at

sensitive and to have good negative predictive value in getting a response in asthmatics who
have recent or current symptoms. This comes at the cost of being less specific and having less
positive predictive value or being useful for population screening for asthma (Cockcroft,
Murdock et al. 1992). As part of the ECRHS, bronchial hyper-reactivity using standardised
methacholine challenges was tested in over 13,000 adult subjects in 35 centres from 16
countries showing considerable variation in incidence (Chinn, Burney et al. 1997). In some
this mirrored their known rates of asthma disease, such as being high in New Zealand, Great
Britain, Australia and the USA, and low in Iceland and Switzerland. There was less of an
obvious connection in others, for example showing high rates of hyper-reactivity in Denmark
which has far lower documented asthma prevalence than, for example, Italy, Spain or Sweden
which all had low reactivity rates. In an early study of 307 adults, bronchial reactivity was
noted in 3Vo of normal subjects, l00vo of symptomatic asthmatics, 69Vo of currently
asymptomatic asthmatics (Cockcroft, Killian et al. 1977). In other studies, the proportion of
individuals with bronchial hyper-responsiveness was only about 40-60Vo of those reporting
current wheeze (Backer, Dirksen et al. 1991; Backer, Groth et al. l99l; Backer and Ulrik
1992; GINA 2005).
As well as some 'normals' responding in what is deemed the asthmatic range (Cockcroft,
Killian et al. 1977; Cockcroft, Murdock et al. 1992). the direct airway challenges are also non-
specific, as subjects with other respiratory diseases also react, most notably cough (Brooke,
Lambert et al. 1998), chronic obstructive pulmonary disease (COPD) and/or chronic
bronchitis (CB) (Ramsdell, Nachtwey et al. 1982; Ramsdale, Roberts et al. 1985; Calverley,
Burge et al. 2003).
Direct airway challenges in children are limited to older age groups. In over 2000 children
aged 7-10 years tested with a cumulative dose of 3.9;rmol of histamine, only 5BVo of the
children with asthma and current symptoms responded. While 52Vo of those diagnosed with
asthma responded overall, 53Vo of subjects demonstrating bronchial hyperactivity had no
asthma diagnosis (Pattemore, Asher et al. 1990). In three regions with similar asthma
admissions (Auckland, inland New South Wales and coastal New South Wales) between 700
to over l'000 children were tested at each centre and while two groups had similar rates of
bronchial hyper-reactivity to methacholine challenges, the third had a much lower rate (Asher,
Pattemore et al. 1988). The hyper-reactivity to direct airway challenge has a similar sensitivity
and specificity profile for asthma diagnosis as questionnaire data (Asher, pattemore et al.
1988; Backer, Dirksen et al. I99l; Bakke, Baste et al. l99l; Backer and LJlrik 1992; Joos,
O'Connor et al. 2003). In one of these studies, the level of bronchial hyper-reactivity was a
l9

poorer predictor of a diagnosis of asthma than responses to the question 'has your child had
wheezing in the past 12 months' on written questionnaires @attemore, Asher et al. 1990). In
495 children aged 7-16 years, the subjects with asthma represented a subgroup within the
responsive end when inhaling increasing concentrations of histamine rather than the asthma
and the 'normals' having separate distribution peaks. Selecting a low dose of 2.4mglrnl-r as
the cut off point gave a specificity of 98Vo for asthma, but a positive predictive value of only
6OVo and a sensitivity of only 57Vo (Backer, Groth et al. l99l).
Using indirect challenges is a more recent phenomenon from the late 1980s and eady 1990s
(Manning, Watson et al. 1993; Joos, O'Connor et al. 2003). These are termed indirect as the
triggers act on cells, which then release mediators or cytokines to cause a secondary
bronchoconstriction and airflow limitation. They include physical stimuli such as exercise,
osmotic stimuli such as hypertonic saline or mannitol, or pharmacological stimuli such as
adenosine or bradykinin. These challenges are thought to be a better reflection of the active
airway inflammation as observed in asthma (Joos, O'Connor et al. 2003). Also IHCS reduce
responsiveness to these indirect challenges, as in asthma, while in contrast they only have a
small effect on histamine and methacholine challenges. The indirect challenges have
demonstrated less sensitivity but more specificity in differentiating asthmatics from 'normals'
(Vasar, Braback et al. 1996; Godfrey, Springer et al. 1999; Joos, O'Connor et al. 2003). In
addition in children, an exercise challenge has also been shown to be better at distinguishing
asthma from other chronic airway disorders such as CF, bronchiolitis obliterans, ciliary
dyskinesia and bronchiectasis (Avital, Springer et al. 1995; Godfrey, Springer et al. 1999).
There is a statistical, albeit weak, correlation between the methods; however there are
individuals who have positive exercise challenges and negative histamine or methacholine
challenges. A hypertonic saline challenge in adults has been shown to identify exercise
induced asthma (Belcher, I-ee et al. 1989; Boulet, Turcotte et al. 1989; Smith and Anderson
1990; Brannan, Koskela et al. 1998). The possibility of having exercise induced asthma and
not responding to hypertonic saline or mannitol challenges occurs, but only in those with very
mild asthma @rannan, Koskela et al. 1998). In children, those with a history of current
wheeze were seven times more likely to have a positive response to hypertonic saline than
asymptomatic children (Riedler, Reade et al. 1994). However the use of osmotic challenges to
date have been seen more frequently as a treatment to improve mucociliary clearance and
lung function, predominantly in children with CF (Eng, Morton et al. 1996; Rodwell and
Anderson 1996; Robinson, Daviskas et al. 1999).
20

Thus determining the presence or absence of bronchial hyper-responsiveness to a trigger at a
certain concentration is not necessarily diagnostic of asthma. The direct challenges lack
specificity, while the indirect challenges lack sensitivity. Both challenge types are required to
be done in the laboratory and while they are achievable with children, there is both a safety
component and a significant time commitment to be considered. As repeated measures to
monitor response to treatment or progression of asthma, they are less than satisfactory for
these reasons.
In summary while the diagnosis of asthma may seem straightforward in some who fit the
'classical' asthma profile, there are many difficulties to be faced when testing for asthma. In
addition, the investigations are more suited for asthma determination in adults than in
children. A diagnosis of asthma in children remains difficult, when in this group getting the
correct diagnosis is important if the usual asthma treatment is to be recommended.
1.5
Treatment and concerns
1.5.1 Possible adverse fficts of asthmatreatment in children
Much is known about the underlying pathology of asthma, and this will be discussed in depth
in the next section of this chapter. This has resulted in all the guidelines (GINA 2005;
Paediatric Society of New Zealand. 20O5; SIGN 2005) recommending anti-inflammatory
treatment; with IHCS at step two as the first preventer and the introduction of oral
corticosteroids at steps four or five with unresponsive and difficult asthma, and for acute
treatment. The balance is between the correct diagnosis, appropriate management and to
prevent irreversible airway thickening on the one hand, against the development of side
effects with treatment on the other.
The adverse effects as listed in the guidelines for IHCS include; adrenal cortical insufficiency,
growth failure, dysphonia and oral candidiasis. The adverse effects listed for oral
corticosteroids is somewhat lengthier; adrenal insufficiency which may cause devastating
hypoglycaemia, adrenal suppression, growth failure, hypertension, diabetes mellitus,
reduction in bone mineral density, skin atrophy, straie, poor healing, immunosuppression and
cataracts. Doses of IHCS of greater than 400p gtday of beclomethasone dipropionate or
equivalent in children have been associated with side effects (Calpin, Macarthur et al. 1997:
Sharek and Bergman 2000). There have been reports of IHCS affecting short term growth
(Agertoft and Pedersen 1997), intermediate term growth over months to a year (Tinkelman,
Reed et al. 1993; Doull, Freezer et al. 1995; Simons 1997; Verbeme, Frost et al. 1997;Allen,
2l

Bronsky et al. 1998), and a long-term effect over a period of years (Anonymous 2000).
However, observational studies have suggested that despite long term IHCS in moderate
doses, final adult height is not affected (Agertoft and Pedersen 2000; Peters 2006). While all
guidelines recommend regular monitoring of the child's height, isolated growth failure is not
a reliable indicator of adrenal suppression @unlop, Carson et al. 2004).
On higher doses (800pg/day of beclomethasone dipropionate or equivalent) of IHCS or oral
steroids, blood pressure should be monitored, bone density assessed and cataracts should be
screened for, if being used for greater than a few months. Children may be more at risk of
developing cataracts and glaucoma @enfro and Snow 1992; Nootheti and Bielory 2006),
though one study disagreed (Simons, Persaud et al. 1993). IHCS at the recorrmended levels,
and in some studies even high doses of fluticasone, had minimal effects on bone mineral
density (Hopp, Degan et al. 1995; Kelly, Clee et al. 1995; Agertoft and Pedersen 1998;
Griffiths, Sim et al.2004). However others found that after years of treatment, asthmatics had
lower total body bone mineral density than controls (Boot, de Jongste et al. 1997; Allen,
Thong et al. 2000) with a negative relationship between this density and the total cumulative
doses of IHCS used (Wong, Walsh et al. 2000). Oral candidiasis has been found in up to 5Vo
of adult patients with asthma with positive mouth cultures up to 25Vo and dysphonia is also
said to occur in up to 58Vo of patients at some time (Barnes, Pedersen et al. 1998).
The issues regarding side effects of these medications should also be considered in two other
populations. Firstly, healthy subjects (for example if the diagnosis is not secure) may have
greater systemic side effects to IHCS and oral steroids than asthmatics (Brindley, Falcoz et al.
2000; Brutsche, Brutsche et al. 2000; Falcoz, Oliver et al. 2000; Mackie, McDowall et al.
2000). Secondly, in the last years, treatment with IHCS has been assessed in younger
children; those with preschool and infant wheeze and/or bronchiolitis. Most of the studies in
the preschool group have tried to target those who are more likely to develop asthma over
time - with either recurrent wheezy episodes, personal history of atopy, a family history of
asthma or a positive asthma predictive index (Castro-Rodriguez, Holberg et al. 2000;
Guilbert, Morgan et aI.2006). These have enrolled between 31 and 625 children aged six to
47 months have received doses from 40pglkg to 1000ug via nebuliser or 300-800pg/day via
metered dose inhaler with a spacer and face mask for between 6 and 26 weeks. Improvements
were seen in symptom free days (Bisgaard, Gillies et al. 1999; Roorda, Mezei et al. 2001;
Teper, Colom et al.20[.4; Guilbert, Morgan et al.2N6), in lung function parameters @ao and
McKenzie 2OO2; Teper, Colom et al.20O4), in bronchial hyper-reactivity (Stick, Burton et al.
1995) and in use of additional 'rescue' medication @isgaard, Gillies et al. 1999; Teper,
22

Colom et al.20O4; Guilbert, Morgan et aL.2006). Side effects noted were growth velocity
(Guilbert, Morgan et al. 2006), adrenal suppression, increased cough and hoarseness, and
cataract formation (Bisgaard, Allen et al. 2004). An earlier Cochrane review in 2000 had
suggested that episodic high dose IHCS was partially effective. However, they concluded that
"there is no current evidence to favour maintenance low dose IHCS in the prevention and
management of episodic mild viral wheeze of childhood" (McKean and Ducharme). In
addition, more recent Cochrane reviews do not support the use of IHCS in acute bronchiolitis
or used to prevent wheezing post acute illness in 2006 (Blom, Ermers et al.) or the use of oral
steroids for bronchiolitis in 2004 (Patel, Platt et al.). So while at the current time steroids are
not recommended in this age group, the increased use exposes many more children to
corticosteroid therapy, often before a definitive diagnosis of classic asthma is able to be made
(de Blic and Scheinmann 2000).
In fact it is interesting to note within the guidelines, the significant difference in the level of
evidence available for the different age groups. For example in the pharmacological
management chapter of the SIGN guidelines (SIGN 2005), the graded evidence for the
recommendations is presented in age brackets (>12 years and adults, 5-12 years and < 5
years). Across these age groups the evidence frequently drops from '1++' (strong evidence
with high quality meta-analyses and systematic reviews of randomised controlled trials) down
to '4' (expert opinion) in the youngest age group.
1.5.2 A marker for inflammation would be usefuI
There is a need to minimize side effects, while preventing morbidity (and mortality). This is
important for better individual health, and is also important financially - reducing work and
school days lost. Other financial burdens can be experienced by a family with a child with
uncontrolled asthma as revealed in this quote taken from the 'Trying to Catch Our Breath,
monograph (Anonymous 2006) :
"I visited a family who had almost no furniture in the house. They had taken their child
to the doctor one evening in the previous week for an asthma attack. They spent $80 for
the visit and the medications. This was their entire food budget for the following week.
They were eating white bread and butter." (Claire Richards, Asthma Nurse Educator,
Porirua Asthma Service, New Zealand)
In 2005, the GINA guideline (GINA 2005) suggested to "titrate the dose of inhaled
corticosteroid to the lowest dose at which effective control of asthma is maintained". The
23

SIGN guideline (SIGN 2005) stated "the smallest dose of inhaled steroids compatible with
maintaining disease control should be used. At higher doses add on agents for example long
acting p-2 agonists should be actively considered". In the NzpAG @aediatric
Society of New
7*,aland2005) again the recommendation is to "titrate the dose of inhaled corticosteroids to
the lowest dose at which effective control of asthma is maintained". The difficulty here is how
to measure what the appropriate low dose is and whether control has been achieved.
Ultimately, treating the pathological changes rather than being guided by symptoms alone
might well be useful. In the next section, I will present that data suggesting that asthma is an
to measure, non invasive, marker of airway
inflammatory disease, and whY an easY
inflammation might be of great value'
1.6
During the last decades there has been a gradual move from the concept of asthma as a
disease of bronchoconstriction to that of an inflammatory disorder, and from the concept of
complete reversibility to accepting that some irreversible airway damage can occur' The
current definition of asthma proposed by the GINA committee is "Asthma is a chronic
inflammatory disorder of the airway in which many cells play a role, in particular mast cells'
eosinophils, and T lymphocytes" (GINA 2005). In the following sections, I will briefly
summarise findings from autopsy studies, then biopsy and lavage' induced sputum' blood and
urine samples in asthmatic patients compared to normal subjects. while a full description of
the research that has resulted in these two major premise alterations is beyond the scope of
this thesis, the purpose of the next sections are two-fold; to background the concept of asthma
as an inflammatory disease and to show that many of these samples are difficult to obtain.
This makes them more appropriate for single or pre and post intervention determinations
rather than repeated longitudinal or population assessments. I have also concentrated on what
was known at the time of my research commencing but have included how this is viewed to
date. NO will be discussed in the next chapter'
1.6.1 What has been leamtfrom autopsy studies?
The hint that asthma was an inflammatory disease occurred early. In the late 19ft century
Charcot-I-eyden crystals (crystalline structures shaped as double nalrow pyramids)'
curschmanns spirals (coiled fibrils of mucus) and eosinophilia in sputum were recognised
during severe exacerbations of asthma (ossler rg92). This was followed early and mid last
century by autopsy studies of adults dying from asthma (Huber and Koessler 1922; Bullen
1952;Dunnill 1960) and much later by autopsy studies in children (cutz, Irvison et al' 1978)'
24

These described gross pathological changes with diffuse mucus plugging of the airways,
sloughing of the epithelial lining, an increase in airway smooth muscle and oedema of the
peribronchial tissues. Light microscopy showed mucus plugs, goblet cell hyperplasia and
apparent thickening of bronchial basement membranes with increased smooth muscle. There
was an infiltration of inflammatory cells with a marked increase in eosinophils. The later
paediatric study (Cutz, Levison et al. 1978) also examined the specimens under electron
microscopy. This again demonstrated mucus plugs, which consisted of epithelial, macrophage
and eosinophil cells and cell fragments. A thickened basement membrane appeared to be
composed of collagen deposits and the submucosa was infiltrated by a mixture of
inflammatory cells, predominantly eosinophils. Recent autopsy studies have compared
pathology specimens between patients with fatal asthma, non-fatal asthma and controls
(Koshino, Teshima et al. 1993; Kepley, McFeeley et al. 2001; Chen, Samson et a1.2004).
Significant smooth muscle shortening, increased submucosal gland area, increased mucus
plugging and an increase in basophils was seen in the cases of fatal asthma compared to the
other groups. These contributed to airway wall thickening with smooth muscle hypertrophy
and increased collagen along the basement membrane. Compared to ten controls, 18 adults
who had died of asthma had proteoglycans prominent in the extra cellular matrix creating
most of the basement membrane thickening seen, with differing types of proteoglycans
expressed in the two groups (de Medeiros Matsushita, da silva et al. 2005).
1.6.2 Studies of inflammation using bronchoscopy and biopsy
The use of bronchoscopy was developed initially as a therapeutic measure by Dr Chevalier
Jackson in 1904 (Reynolds 1987), and later became a way of obtaining airway samples. The
flexible fibreoptic bronchoscope was introduced into medical centres in the early 1970s
(Ikeda 1970) with appropriate guidelines (Smiddy, Ruth et al. l97l; Sackner, Wanner er al.
1972)' which were later modified to include standards for bronchoalveolar lavage (BAL)
(Bernstein, Boushey et al. 1985; Reynolds 1987; Turner-Warwick and Haslam l9g7) and
detailed indications and protocols for the procedure were subsequently published (Klech and
Pohl 1989; Kelch and Hutter 1990; Bleecker, McFadden et al. l99Z: Rennard, Aalbers et al.
1998; Haslam and Baughman 1999; Reynolds 2000). The development of paediatric
bronchoscopy commenced a decade later (Wood and Sherman 1980; Wood l9g5) when
construction of smaller bronchoscopes became possible and these entered clinical practice in
the mid 1990s (Perez and Wood 1994; Wood 1996; Rennard, Aalbers er al. 1998; ERS 2000;
Wood 20[l; Midulla, de Blic et aL.2003). Thus these technical advances allowed biopsy and
BAL studies in 'living' patients to be undertaken.
25

In brief, the human airway can be divided into three layers; the inner wall (epithelium,
basement membrane and the submucosa), the outer wall (loose connective tissue), and the
smooth muscle layer (Bergeron and Boulet 2006). All layers have shown some alterations in
obstructive respiratory disease compared to normal lung. with regard to the presence of
inflammation, biopsy studies have shown increases in eosinophils, eosinophilic proteins, T
lymphocytes (Foresi, Bertorelli et al. 1990; Poston, chanez et al. 1992: Laitinen, Laitinen et
al. 1gg6; carrou, cooke et al. lggT) and certain cytokines (or their messenger ribonucleic
acid (mRNA)) such as interleukin 2 (n-2), interleukin 3 (IL3), IL5, granulocyte macrophage
colony stimulating factor (GM-CSF), tumor necrosis factor alpha (TNF0) and rantes when
compared to both normal subjects and patients with other respiratory diseases (Azzawi'
Bradley et al. 1990; Ackerman, Marini er al. 1994; Humbert, Durham et al' 1996; Powell'
Humbert et al. 1996; Vrugt, Wilson et al. lggg). The presence of inflammation was confirmed
even in mild asthma @easley,
Burgess et al. 1993; Laitinen, Laitinen et al' 1996;
Grootendorst, Sont et al. t997; Laitinen, Karjalainen et al. 2000; ward, Reid et al' 2005) and
in patients during disease remission (Foresi, Bertorelli et al. 1990). A positive correlation with
each of these parameters to the severity of asthma has been demonstrated by some studies
(Beasley, Burgess et al. 1993; Carroll, cooke et al. 1997; Vrugt, wilson et al' 1999; Amin'
Ludviksdottir et al. 2000; Barnes, Burke et al. 2000; Benayoun, Druilhe et al' 2003)' In
addition, some markers more commonly associated with infective diseases, such as
interleukin g (ILg) and eotaxin, have also been demonstrated to be positively conelated with
asthma severity (Lamkhioued, Renzi et al. 1997; Pepe, Foley et al' 2005)' However' the
correlations of these markers with severity have not been unanimous @enayoun,
Druilhe et
al.2oI3;shahana, Bjornsson et al. 2005) and overall no unique inflammatory profile has been
demonstrated for all asthmatic patients (Kavuru, Dweik et al. 1999)'
In addition to the increase in inflammation demonstrated in asthma, there have also been
increases noted in the airway wall components. These include airway smooth muscle' mucus
glands, and submucosal and extra-cellular matrix tissue which have also been related to
symptom severity and bronchial hyper-responsiveness
(Saetta, Di Stefano et al' 1991; Chetta'
Foresi et al. 1997;Benayoun, Druilhe et al. 2003; chen, samson et al'20o4; Pepe' Foley et al'
2005). The presence of high numbers of fibroblasts has been the most consistent feature
demonstrated to relate to severity of disease and poor response to treatment (wenzel'
Schwartz et ar. lggg; Boulet, Turcotte et al. 2000). In the last decade, these findings have
taken on new importance as to their relevance to possible remodelling of the airway resulting
in irreversible changes occurring (Jeffery, Laitinen et al' 2000; wilson and Bamford 2001;
26

Black and Johnson 2002; Jeffery 2004; Kariyawasam and Robinson 2005: ward and walters
2005). This airway remodelling in asthma has been found in both large and small airways
(Saetta, Di Stefano et al. l99l; James, Maxwell et al. 2OO2) and the changes are the same
regardless of the asthma being atopic, occupational or intrinsic (Homer and Elias 2000). The
inner wall components include loss of epithelial integrity (Naylor 1962; Laitinen, Heino et al.
1985; Jeffery, Wardlaw et al. 1989) and hyperplasia of the globlet cells in the sub-epithelium
(Aikawa, Shimura et al. 1992: Ordonez, Khashayar et al. 2001). While rhickening of the
basement membrane was also described in the original autopsy and other studies above, it
now appears that the true basement membrane (the 'lamina rare' and 'lamina densa') is not
grossly altered (Roche, Beasley et al. 1989; Homer and Elias 2000) with the actual fibril
content of the membrane found to be similar in asthmatic and control groups (Saglani,
Molyneux et al. 2006). It is the area that sits immediately below the true basement membrane,
'the lamina reticularis' that is increased and this has been termed 'subepithelial fibrosis' or
'reticular or sub-basement membrane thickening' (Boulet, Laviolette et al. 1997;Elias, Zhu et
al. 1999; Beckett and Howarth 2OO3). Here, enhanced collagen deposition has been
demonstrated with subtypes I, III and IV, fibronectin, tenascin, Iumican and biglycan @oche,
Beasley et al. 1989; Laitinen, Laitinen et al. 1996; Laitinen, Altraja et al. 1997;Wilson and Li
1997 Huang, Olivenstein et al. t999; Benayoun, Druilhe et al. 2003; Karjalainen, Lindqvist
et al. 2O03). Myofibroblasts are specialized cells that have features of both myocytes and
fibroblasts and are sources of interstitial collagens so are likely contributing to this deposition
(Homer and Elias 2000). Myofibroblast number has been demonstrated to correlate directly to
the thickness of this layer (Brewster, Howarth et al. 1990; Hoshino, Nakamura et al. l99g).
However, it is the increased thickness of the smooth muscle layer that contributes most to the
overall thickness of the airway. This has variably been described as due to hypertrophy and/or
hyperplasia (Hossain 1973; Ebina, Takahashi et al. 1993; Cho, Seo et al. 1996;James lggT).
The smooth muscle contraction has been shown to particularly narrow the airway where the
entire lumen is surrounded as in the smaller bronchi prior to respiratory bronchioles. This is
consistent with computer modeling of ainvay function, showing that the smaller airways are
the site of the greatest increase in airway resistance in asthma (Hogg lggT). Finally, changes
have also been described in the airway vasculature with an increase in both size and number
of vessels and this angiogenesis also contributes to airway oedema seen (Li and Wilson 1997;
Tanaka, Yamada et al.20O3). Given the variable nature of the findings it is difficult to show
the exact nature of the progression, and this is the subject of ongoing research (Boulet 2000).
27

The ability to biopsy has led to studies of the early (Gonzalez,Diaz et al. 1987; Georas, Liu et
al. 1992) and late asthma reactions (Robertson, Kerigan et al. 1974; Crimi, Chiaramondia et
al. 1991) and a comparison of both (Metzger, Richerson et al. 1986; Metzger, Richerson et al.
1986; Aalbers, Kauffman et al. 1993) in response to deliberate allergy provocation. These
demonstrated an influx of neutrophils at 6-8 hours followed by an influx of eosinophils at24-
48 hours which continued more than a week but seemed to resolve by day 16. An increase in
the cellular expression of the mRNA of IL5 and GM-CSF occurred at 18 to 48 hours
(Ohnishi, Sur et al. 1993; Tang, Rolland et al. 1997) with an increase in total proteins and
ainvay permeability also in the late phase (Taylor, Hill et al.1996; Teeter and Bleecker 1996).
In assessing response to treatment in asthmatic patients treated with moderate to high doses of
IHCS (400mcg to l600mcdday beclomethasone equivalent) for between two weeks and up 5-
l0 years, most studies have reported a decrease in T-lymphocytes, eosinophils, and mast cell
numbers in the airway wall (Lundgren, Soderberg et al. 1988; Burke, Power et al. 1992;
Booth, Richmond et al. 1995; Djukanovic, Homeyard et al. 1997; Olivieri, Chetta et al. L997i
Barnes, Burke et al. 2000). As well, decreased dendritic cell number @urke, Power et al.
1992; Trigg, Manolitsas et al. 1994; Bentley, Hamid et al. 1996) and reduced inflammatory
cell activation measured as less gmnule secretory production and less cell membrane
activation markers have also been noted @entley, Hamid et al. 1996; Olivieri, Chetta et al.
1997; Barnes, Burke et al. 2000; Ward, Reid et al. 2005). The expression of mRNA for nA,
IL5 and GM-CSF within cells also decreased with steroid treatment (Sousa, Poston et al.
1993; Bentley, Hamid et al. 1996). Interestingly, the effects have been more marked in the
biopsy samples than the corresponding lavage studies (Booth, Richmond et al. 1995;
Thompson, Teschler et al. 1996: Djukanovic, Homeyard et al. 1997; Olivieri, Chetta et al.
1997). The effect of steroids on the thickness of the epithelium and basement membrane have
been less conclusive; some studies showing improvement (Olivieri, Chetta et al. 1997; Ward
and Walters 2005) that has not been demonstrated in others even after years of treatment
(Lundgren, Soderberg et al. 1988; Wenzel, Szefler et al. 1997; Wenzel, Schwartz et al. 1999;
Barnes, Burke et al. 2000).
Paediatric studies have been more recent, in keeping with the later technical bronchoscopy
development and the additional concerns regarding safety in this group. These have shown
similar findings to the adult asthma studies with eosinophilic and lymphocytic inflammation,
and an increase in the same cytokines (interleukin 4 (IL+), IL5, rantes) (de Blic, Tillie-
Irblond et al. 2OO4; Payne, Qiu et al. 2O04; Pohunek, Warner et al. 2005). The findings
associated with airway remodeling (smooth muscle hypertrophy and reticular basement
28

membrane thickening) were present early in a study in children with an age range down to 1.2
years (Pohunek, Warner et al. 2005) and did not show differences between those with few
symptoms compared to those with persistent symptoms or 'difficult to control' asthma
(cokugras, Akcakaya et al. 2001; Jenkins, cool et aL.2003; payne, Rogers et al.29613; de
Blic, Tillie-kblond et al. 2004; Fedorov, Wilson et al. 2005). In addition, the findings were
not associated with age, length of symptoms, lung function or bronchial reactivity (payne,
Rogers et al. 2003). Bronchial wall thickening on high resolution computerized tomography
(HRCT) scan was shown to correlate with eosinophils numbers, ECp and sub-basement
membrane thickening (de Blic, Tillie-Irblond et al. 2005).
1.6.3 studies of inflammation using bronchoalveolar lavage
In adults the standard bronchoscope wedges between the 4th and 6ft order bronchus
(Hunninghake, Gadek et al. 1979) typically localising a lung zone with 106 alveoli or a
volume of l65ml at total lung capacity and 45ml at residual volume. In the 1960s, two studies
initially started using catheters to do small volume bronchopulmonary lavage for assessment
of disease @nley, Swenson et al. 1967; pratt, Finley et al. 1969). standard guidelines for
clinical, diagnostic and research practices have been developed for use in adults (Klech and
Pohl 1989; American Thoracic Society 1990; Rennard, Aalbers et al. 1998; Reynolds 2000),
and more recently for use in children (ERS 2000; Midulla, de Blic et al. 2003). Lavage studies
have confirmed many of the biopsy study findings in asthmatics when compared to normal
subjects. Increases are seen in eosinophils, T-lymphocytes, eosinophilic proteins and
cytokines such as n-4,L5, rantes and chemo-attractant factors with excellent recent reviews
detailing these findings (Barrios, Kheradmand et aL.2006; Graham 2006: Tulic and Hamid
2006). An antigenic or exercise challenge resulted in an observed increase in eosinophils
(Kirby, Hargreave et al. 1987; Wardlaw, Dunnette et al. 1988; Bousquet, Van Vyve et al.
1993; Krug, Tschernig et al. 2001), T cell lymphocytes (Virchow, Walker et al. 1995;
Schuster, Tschernig et al. 2000), mast cells (Kirby, Hargreave et al. 1987; Bousquet, Van
Vyve et al. 1993; Olsson, Rak et al. 2000), basophils and neutrophils (Kirby, Hargreave et al.
1987; Mattoli, Mattoso et al. 1991; Bousquet, Van Vyve et al. 1993), complement factors
C3A and C5 (Krug, Erpenbeck et al. 2001) and eosinophilic proteins (Wardlaw, Dunnette et
al. 1988).
Many more proteins can be measured in lavage fluid and this can demonstrat e the effect of
cells' even if the latter are not as visible. For example, mast cells account for up to 20Vo of the
inflammatory cells and basophils are also prominent in biopsy studies, but both are only a
29

small proportion of cells recovered by BAL @unnill 1960; Foresi, Bertorelli et al. 1990;
Koshino, Teshima et al. 1993; Macfarlane, Kon et al. 2000). Provocation studies have shown
the early asthma response is associated with increased histamine, tryptase, prostaglandinD2,
prostaglandin F2, thromboxane and leukotrienes which are released by these two cell types
(Murray, Tonnel et al. 1986; Wenzel, Fowler et al. 1988; Liu, Hubbard et al. l99l; Chilton,
Averill et al. 1996; Kavuru, Dweik et al. 1999). Adhesion molecules such as intracellular
adhesion molecule I (ICAM l) and lymphocyte function-associated antigen I (IJA 1) were
also increased (Williams, Johnson et al. 1992). The cytokines associated with an allergic
profile - IL3, IIA,ILI and GM-CSF -
and their receptors in cell surfaces were measured in
high amounts (Virchow, Walker et al. 1995; Chung and Barnes 1999; Hamid, Tulic et al.
2003). Increases have also been seen in IL9 and ILl3 which have been associated with mucus
hypersecretion and IgE regulation (Louahed, Toda et al. 2000; Shimbara, Christodoulopoulos
et al. 2000; louahed, Zhou et al. 2001) as well as increased interleukin 11 (Lll) and
interleukin 17 (lLl7) that have pro-fibrotic activity @inarsson, Geba et al. 1995; Minshall,
Chakir et al. 2000; Molet, Hamid et al. 2001). On the other hand, decreased interleukin 10
(L10), an anti-inflammatory cytokine, was seen during acute asthma and this returned to
levels similar to controls with recovery (Borish, Aarons et al. 1996). Chemokines have been
demonstrated as present and rising with airway challenges including eotaxin, monocyte
chemoattractant protein and rantes as well as TGFp which has been noted to have a role in the
subepithelial fibrosis (Hamid, Tulic et al. 2003). With specific challenges, such as using
ragweed antigen, specific IgE and IgA have also been shown to be increased in
bronchoalveolar lavage fluid @eebles, Hamilton et al. 2001). Similar to the findings from the
biopsy studies, with treatment of either IHCS or oral corticosteroids, while there was a
reduction of eosinophil, mast and epithelial cell numbers in lavage fluid, the eosinophilic
proteins did not reduce and there was not a direct correlation between the inflammatory cell
decrease and improvement in symptoms or bronchial hyper-responsiveness @uddridge, Ward
et al. L993; Booth, Richmond et al. 1995; Djukanovic, Homeyard et al.1997; Olivieri, Chetta
et al. t997).
The paediatric literature in this area has been more recent but has largely been in agreement
with the findings in adults. The increase in eosinophilic inflammation and eosinophil proteins
were seen (Barbato, Panizzolo et al. 2001; Just, Fournier et al.2002; Najafi, Demanet et al.
2003), as well as increased neutrophils which were associated with persistent symptoms (Just,
Fournier et al. 2OO2), 'difficult to treat' asthma (de Blic, Tillie-Irblond et al. 2004; Payne,
Qiu et al. 2OO4) and status asthmaticus (Lamblin, Gosset et al. 1998; Tonnel, Gosset et al.
30

2001). In paediatrics, BAL has also been used to compare inflammatory profiles with other
diseases that present with wheeze. An increase in eosinophil cell and proteins was seen in a
group of asthmatics compared to control and bronchiolitis groups (Kim, Chung et al. 2000;
Marguet, Dean et al.2C0l; Kim, Kim et al.2003; Kim, Kim et al. 2005). However, ILg levels
also correlated with the eosinophil level in the asthmatic group (Kim, Kim et al. 2003) and the
number of neutrophils appeared to reflect severity of asthma (Marguet, Dean et al. 2001). A
recent summary of these studies into airway inflammatory profiles in paediatric asthma has
been published (Warner 2003).
1.6.4 Inflammatory markers in induced sputum
1.6.4 (i) Studies in adult subjects
Non-isotonic ultrasonically produced aerosols were first used more than fifty years ago to
examine bronchial reactivity (Cheney and Butler lgT}).Currently, nebulised hypertonic saline
is used for three main reasons. Firstly, it can act as an airway challenge to evaluate hyper-
responsiveness. Secondly, it can be utilised as a treatment to aid expectoration of the
thickened sputum produced in some respiratory diseases, particularly CF @ng, Morton et al.
1996; Wark, McDonald et al. 2005; Donaldson, Bennett et al. 2006: Elkins, Robinson et al.
2006). Thirdly, it has been used to induce sputum for microbiological assessment (for
example in CF and TB) and/or for the harvest of cells and cytokines for research. The
technique consists of inhaling saline at 3-7Vo concentration generated by an ultrasonic
nebuliser, which is necessary to give a sufficient output for saline aerosol generation, for l0 to
30 minutes (Belda, Hussack et al. 2001). Patients or research subjects are requested to cough
and try to expectorate sputum into a container every 3-5 minutes (paggraro, Chanez et al.
2002). Dithiothreitol O.lVo is used to break down mucus suphylhydryl bonds and to disperse
cells. The samples are then centrifuged with slides made of the cellular component and
stained to identify cell viability, total cell counts, the percentage of squamous cells and
differential percentage or numbers of the other cell types. The sputum supernatant following a
cytospin preparation is then used for measurement of soluble mediators. The mechanism by
which hypertonic saline causes sputum production is uncertain, but thought to be a
combination of the osmotic movement of water into airways, the induction of cough and the
increased rate of mucociliary clearance (Umeno, McDonald et al. 1990; Holz, Kips et al.
2000; Paggiaro, Chanez et aL.2002). This technique was first described in the early 1990s to
obtain sputum samples from asthmatic and normal adult subjects (Fahy, Liu et al. lgg3). A
task force of European Respiratory Society members developed the ..Standardised
3l

methodology of sputum induction and processing" @jukanovic, Sterk et al. 2002) which has
a detailed report on sputum induction @aggiaro, Chanez et al.2002), methods of processing
@fthimiadis, Spanevello et al. 20f.2) and measurement of fluid-phase mediators (Kelly,
Keatings et aI,2002) concentrating on techniques and findings in asthma and COPD subjects.
Pre-treatment with p2 agonists as appropriate and lung function and/or oxygen saturation
monitoring is recommended throughout the procedure @aggiaro, Chanez et al.2002).
The eady studies confirmed and enlarged on the inflammatory profiles seen in asthmatics
compared to normal subjects as had been described in the biopsy and lavage studies above.
An eosinophilic dominant inflammation was seen with increases in total cell numbers, as well
as individual eosinophil and neutrophil counts, eosinophil derived proteins [ECP, eosinophil
derived neurotoxin (EDNI and major basic protein (MBP)1, (Pin, Gibson et al. 1992; Fahy,
Liu et al. 1993; Foresi, I-eone et al. 1997; Louis, Shute et al. 1997; Bacci, Cianchetti et al.
1998; Park, Whang et al. 1998; Rosi, Ronchi et al. 2000) the allergic profile interleukins (IL3,
nA, L5, Ll0) and certain proteins such as tryptase, eosinophilic chemo-attractant factors,
and adhesion molecules (Shoji, Kanazawa et al. 1998; Hamzaoui, Brahim et al. 2000).
Correlations between these sputum inflammatory cells and markers and clinical parameters
have been investigated. The strongest associations appear to be during an acute asthma
exacerbation with increases in eosinophils, activated eosinophils and ECP (Jang and Choi
2000; Jang, Choi et al. 2000), basophils and mast cells @in, Freitag et al. 1992; Gauvreau,
I*e et al. 2000). During times of increased symptoms, eosinophil number, ECP, IL5 and
albumin were all found to independently contribute to abnormalities of FEVr and FVC in
asthmatics (Fujimoto, Kubo et al. 1997; Bacci, Cianchetti et al. 1998; Shoji, Kanazawa et al.
1998; Grebski, Wu et al. 1999; Woodruff, Khashayar et al. 2001; Bartoli, Bacci et al.2N4).
Severe asthma was predicted by high eosinophil numbers, ECP and albumin concentrations,
though there was no such gradation in mild to moderate asthmatics (Fujimoto, Kubo et al.
1997; Bartoli, Bacci et aL.2ffi4). The eosinophil counts gave a sensitivity of 68.3Vo, and a
specificity of 55.3Vo for moderate to severe asthma compared to mild, and was of a higher
diagnostic value for asthma than sputum ECP or methacholine challenge (Park, Whang et al.
1998; Rosi, Ronchi et al. 2000).
I 'Eosinophil protein X' and 'eosinophil derived neurotoxin' is the same protein but referred to by these two
different names throughout studies, and denoted as eitler 'EPX' or 'EDN'. I have elected to refer to it as
'eosinophil derived neurotoxin' and 'EDN'. Eosinophil cationic protein, eosinophil derived neurotoxin, major
basic protein and eosinophil peroxidase are all separate proteins.
32

The correlations are weaker when looking at asthmatics during times of stability
(Grootendorst, Sont et al. 1997; Ronchi, Piragino et al. lggT). No relationship was
demonstrated with steroid dose or lung function test results in over 100 stable asthmatics and
40 normal subjects in one community sample of adults (Iouis, Sele et a\.2002). Eosinophils
were frequently within the normal range and not a useful diagnostic tool in a population
divided into 'normal subjects', 'asthmatics', 'wheeze but no asthma' and 'industrial exposure
to irritants' (I-emiere, Walker et al. 2001). Eosinophils, ECp, MBp, EDN and IL5 correlated
with methacholine challenge and FEVr in adult asthmatics (Pizzichini ,Pizzichiniet al. 1996).
However, the eosinophil numbers only accounted for 167o of the variance of the methacholine
challenge across one group, while the TNFo levels correlated more closely (Obase, Shimoda
et al. 2001). Other studies showed no correlation between eosinophilic inflammatory
parameters and lung function (Hashimoto, Minoguchi et al. 1999; Rosi and Scano 2000),
methacholine (Rosi and Scano 2000) or histamine challenges (Iredale, Wanklyn et al. 1994;
Hashimoto, Minoguchi et al. 1999). Eosinophils, leukotriene Ea (LTE+), ECp and rantes were
all increased in poorly controlled asthmatics rather than being consistently high in severe
asthmatics, for example if patients were well controlled (Romagnoli, Vachier et al. 2002).In
addition, they were high only with symptoms even in long-term corticosteroid dependent
patients (Tarodo de la Fuente, Romagnoli et al. 1999). Considerable heterogeneity in sputum
counts were seen in over 250 adult asthmatics and normal subjects with eosinophils of all
amounts (Green, Brightling et al.2OO2).
Many studies have used sputum to determine the best predictor of response to steroids. When
commencing longer term IHCS treatment in placebo controlled trials, sputum eosinophils
correlated to the response in a number of studies (van Rensen, Straathof et al. 1999; Aldridge,
Hancox et al. 2002: Deykin, Lazarus et al. 2005; Brightling 2006) and was a better marker
than ECP levels in either sputum or blood (van Rensen, Straathof et al. 1999; Rosi, Ronchi et
al. 2000; Aldridge, Hancox et al. 2002: Deykin, Lazarus et al.2o05), baseline lung function
@eykin, Lazarus et al. 2005) or methacholine challenge results (van Rensen, Straathof et al.
1999; Deykin, Lazarus et al. 2005). Acutely, sputum eosinophils and ECp fell with oral or
intravenous steroid treatment (claman, Boushey et al.lgg4; Rosi, Lanini et al. 2002) with the
eosinophil count giving a positive predictive value of 68Vo, sensitivity of 54Vo and specificity
of 76Vo for an increase in FEVr >l5%o with steroid therapy (Little, Chalmers et al. 2000).
Eosinophils, neutrophils, ECP, IL5 and fibrinogen all increased when treatment was reduced
but the eosinophil count was consistently the most accurate, across a number of studies, to
predict when treatment reduction would lead to symptom development (Pizzichini, pizzichini
33

et al. 1999; Giannini, Di Franco et al. 2000; Jatakanon, Lim et al. 2000; Green, Brightling et
al. 2002). The results seen across all the studies suggest that the inflammation measured in
this way reflects current asthma control rather than grading chronicity or severity per se.
As indicated in the biopsy and lavage studies above, increased neutrophil counts may also be
a marker for poorly responsive disease. In severe asthmatics, there were more neutrophils and
less eosinophils compared to mild asthmatics (Loh, Kanabar et al. 2005) and neutrophilic
inflammation was dominant in the minority of subjects from over 250 adult asthmatics who
demonstrated less corticosteroid response (Green, Brightling et al. 2002). Both eosinophilic
inflammation and neutrophilic inflammation were found to independently contribute to
abnormalities of FEVr in 205 adult asthmatics (Woodrufl Khashayar et d. 2001). Treating
the neutrophil count resulted in better control than monitoring lung function or symptoms
(Chlumsky, Striz et al.20O6). Neutrophils were also increased acutely, making up more than
757o of the sputum cells in 10 of 18 adult subjects during acute asthma while eosinophils
made up more thanT1Vo of the cells in only three subjects (Fahy, Kim et al. 1995).
1.6.4 (ii) Induced sputum in children
Sputum induction in children developed more recently and therefore in some of the studies
from the 2000s it has been combined with NO measurement. As this will be covered in depth
in later parts of the thesis, I will only discuss the cell and cytokine inflammatory markers
from induced sputum in this section. The technique has been used for getting bacterial, viral,
fungal and tuberculosis cultures to determine infective aetiology (Merrick, Sepkowitz et al.
L997; Utsunomiya, Ahmed et al. 1998; Ordonez, Henig et al. 2003; kiso, Mudido et al. 2005;
Ratjen 2W6). Using induced sputum to assess inflammatory disease was first described in
1980 when presence of an eosinophilia was used to diagnose co-existent asthma in children
with CF was investigated (Sly and Hutchison 1980). In 1995 sputum and nasal smears taken
from I 11 young children presenting acutely with wheeze showed increased eosinophils,
neutrophils and basophils in those deemed 'asthmatic' compared to those who were wheezy
but unresponsive to asthma treatment (Twaddell, Gibson et al. 1996). Increased eosinophils at
3.8Vo and epithelial cells at tl.S%o were seen in 16 children with uncontrolled asthma
compared to 15 with controlled asthma at 2.5Vo and lO.SVo and 72 healthy non-asthmatic
children at0.3Vo and l.5Vo respectively (Cai, Carty et al. 1998). Mast cells, prominent in adult
asthma studies, were found in only 4 of the 42 asthmatic children (Cai, Carty et al. 1998). An
increase in eosinophil percentage and ECP was seen in 50 asthmatic children compared to
fifteen children with chronic bronchitis (CB) and 25 healthy children (Yazicioglu, Ones et al.
34

1999).In 146 children with asthma and 37 controls, the percentage of eosinophils increased
from a mean of l.S%o in children who had infrequent asthma to2.3Vo with frequent asthma
and to 3.8Vo if they had persisting symptoms, compared to controls at lTo. Similarly, ECp
Ievels increased from l l3ng/ml in infrequent asthma to 220nglml in frequent asthma and
37Snglml in the persistent asthmatics compared to 139ng/ml in the control goup. While there
were significant differences between the asthmatic groups, no difference was demonstrated
between the 'infrequent exacerbation' and 'control' children (Gibson, Simpson et al. 2003).
However, in 27 healthy and 60 asthmatic children, no correlation was seen with any marker of
airway inflammation and asthma severity as measured by lung function (Wilson, Bridge et al.
2000). In addition, in 58 asthmatic children, it was the methacholine challenge rather than the
sputum eosinophil counts or ECP that related to the presence of recent symptoms (Wilson,
James et al. 2001). No correlation was demonstrated between sputum eosinophils and FEVI or
sputum ECP in 25 asthmatic children (piacentini, Bodini et al. 1999). Further, no correlations
were demonstrated in 32 children with stable asthma between ambulatory cough frequency,
sputum inflammatory parameters or FEVI (Li, Irx et al. 2003). In 40 children with difficult
asthma, only nine children had abnormal sputum cytology; six had a predominant sputum
eosinophilia while three had a predominant neutrophilia (Irx, Payne et al. 2005).
Within one hour of arriving in the emergency department with acute asthma, eight children in
one study and 38 in a second took less time to produce an induced sputum sample than when
done during a time of stability. The sputum showed higher numbers of total cells, eosinophils,
neutrophils, basophils and mast cells compared to macrophages which was the dominant cell
during the recuperative phase when the sampling was repeated two weeks later with symptom
resolution (Twaddell, Gibson et al. 1996; Norzila, Fakes et al. 2000). The second study also
demonstrated reductions in ECP, myeloperoxidase, IL5 and IL8 by the recovery sample. The
primary cell of inflammation was either eosinophil or neutrophil or showed a co-dominant
pattern (Norzila, Fakes et al. 2000). Following a treatment with single nebulised
glucocorticoid dose, there was a reduction in eosinophil percentage in 30 asthmatic children
but ECP, IL5, GM-CSF and albumin levels, as well as FEV1, remained unchanged (Oh,I*e et
al.1999).
A number of studies have looked at the inflammation in induced sputum before and after
commencing IHCS treatment for more chronic asthma symptoms. In 14 children reduced
sputum and serum eosinophils and ECP levels were seen after two weeks of IHCS treatment
(Sorva, Metso et al. 1997). And in 60 children, there was reduction in sputum eosinophils
35

over a six month treatment prograrnme, with no colTesponding reduction in those on disodium
cromoglycate (Rytila, Pelkonen et al. 2004). In the 'Childhood Asthma Management
Program' (CAMP) study, IHCS treated patients had a lower sputum percentage of eosinophils
at 0.2Vo versus 0.87o than those treated with nedocromil sodium or placebo. In repeated
sputum inductions during reduction of medication, sputum eosinophil counts predicted an
exacerbation (Covar, Spahn et al. 2004).
1.6.4 (iii) Technical aspects of bronchoscopy, bronchial biopsy, bronchoalveolar lavage
and/or induced sputum
Despite the increasing ability to sample from the lung with the use of bronchoscopy, airway
biopsy, BAL and induced sputum, there remain questions over the success, reproducibility
and the safety of these techniques. To start with, there is significant variability both within
and between biopsy samples even when taken from one individual @ichmond, Booth et al.
1996; Sont, Willems et al. 1997; Jeffery, Laitinen et al. 2000). The parameters from biopsy
measurements are less reproducible than physiological tests, with even diurnal variation in
number and function of inflammatory cells @oulter, Burke et al. 2000). Biopsy specimens are
also likely to be taken from quite different sites, from both proximal and distal airways, which
may not be comparable @alzar, Wenzel et al. 2N2; Boulet 2N2; James and Carroll 2002).
BAL variability was investigated by doing 180 procedures in 2O clinically stable but
symptomatic asthmatic adults. The standard deviation for total cell counts was 78 X 103
cells/ml and for macrophages was 16%o,lymphocytes was l3%o and eosinophils 17o (Ward,
Gardiner et al. 1995; Ruffin 1996). The lavage profile seen in asthma, has also been seen in
allergic rhinitis (Gutierrez, Prieto et al. 1998; Alvarez, Olaguibel et al. 2000; Polosa, Ciamarra
et al. 2000; Boulay and Boulet 200.2; Beeh, Beier et al. 2O03; Wilson, Duong et al. 2005;
Bonay, Neukirch et al. 2006; Hara, Fujimura et al. 2006) and in subjects with atopic
dermatitis (Kyllonen, Malmberg et al. 2006).
The initial success rate of hypertonic saline in obtaining samples in adults was 757o (Pin,
Gibson et al. 1992; Foresi, Irone et al. L997; Hashimoto, Minoguchi et al. 1999) but
improved to 83Vo to 93Vo with refinement of the procedure (Vlachos-Mayer, Irigh et al.
2000; Bartoli, Bacci et al.2OO4). The success of sputum induction in children has been given
as 60 to 74Vo in controls (Wilson N M 2000) and 6IVo to 94Vo in asthmatics (Norzila, Fakes et
al. 2000; Wilson, Bridge et al. 2000; Li, I-ex et al. 2003; Covar, Spahn et al.2004; Rytila,
Pelkonen et al. 2OO4; I-ex, Payne et al. 2005). It is more successful in acute than chronic
asthma (Norzila, Fakes et al. 2000; Rytila, Pelkonen et al.2004) and more successful in the
36

older children, particularly those > 12 years of age (Norzila, Fakes et al. 2000; Covar, Spahn
et aL.2004; Rytila, Pelkonen et aL.2004; kx, Payne et al. 2005). In the CAMp study, 90 out
of 117 children (75Vo) with mild to moderate asthma across eight centres provided an
adequate sputum sample for analysis (Covar, Spahn et al.20O4). The total cell counts were
the only similar result when induced sputum samples were examined on two separate days,
two weeks apart in the same 37 subjects (Thomas, Yates et al. lggg). Considerable
heterogeneity was also documented, particularly in eosinophils, in samples from over 250
adult asthmatics and normal subjects (Green, Brightling et a\.2002). Some of the variability
seen between groups may be due to differing techniques. A range of 0.9 to lT%o concentration
of saline has been used, with the measurement being of either the whole expectorated sample
or a sample separated from saliva or by picking out mucus plugs. The plugs were thought to
provide more cells with better cell viability (Gershman, Wong et al. 1996; pjzzichini,
Pizzichini et al. 1996; Spanevello, Beghe et al. 1998) while selected sputum had higher
concentrations of soluble markers such as ECP (Gershman, Wong et al. 1996: Spanevello,
Beghe et al. 1998). Using 0-5 minutes of hypertonic saline gave more neutrophils, while using
a longer period of 10-15 minutes gave higher concentrations of eosinophils ,nA and,Il5 with
lymphocytes, macrophages and epithelial cells remaining unchanged (Taha, Hamid et al.
2004)- A comparison of induced and spontaneous sputum samples showed an increased
number and more viable cells in the former process (Pizzichini ,Pizzichiniet al. 1996).
In addition, the induction procedure itself may alter findings. Consecutive sputum samples
during one sputum induction displayed an increasing neutrophil gradient (Holz, Jorres et al.
1998; Nightingale, Rogers et al. 1998; Richter, Holz et al. 1999 Belda, Hussack et al. 2001)
which occurred within six hours and lasted for 24 (Holz, Jorres et al. 1998; Nightingale,
Rogers et al. 1998) with decreasing mucin and increasing concentrations of surfactant
(Gershman, Liu et al. 1999). This effect was observed with both high and low output
nebulisers (Berlyne, Irmiere et al. 1998). Repeated sputum induction over days also resulted
in increased neutrophils with decreasing macrophage and eosinophil numbers (Belcher,
Murdoch et al. 1988; Holz, Richter et al. 1998) and the effect of hypertonic saline on nasal
mucosa resulted in increased histamine and leukotriene release (Silber, Proud et al. lggg).
The exact cause of this inflammatory response is unknown but the additional fluid,
hypertonicity, frequent coughing, or possibly bacterial contamination could all be
contributory (Holz, Richter et al. 1998; Nightingale, Rogers et al. 1998; Sacco, Fregonese et
al. 2000).
37

There are other studies suggesting sample repeatability was acceptable, with correlation
coefficients between O.7-O.87 for macrophage, lymphocyte, neutrophil and eosinophil counts
in asthmatic, rhinitis, CF and normal subjects (Pin, Gibson et al. 1992; in 't Veen, de Gouw et
al. 1996; Spanevello, Migliori et al. 1997; Bacci, Cianchetti et ^1. 2002; Beier, Beeh et al.
2004; Smountas, Lands et al. 2004).The repeatability of the soluble markers albumin,
fibrinogen, IL8 and ECP were also thought to be acceptable in adult subjects with mild to
moderate asthma (in't Veen, de Gouw et al. 1996).
Performing all of these procedures requires highly experienced staff, particularly with
children, also recognising the need for general anaesthesia for bronchoscopy, as well as highly
qualified laboratory technicians (see Section L.6.4 (iii) below regarding safety). The
procedures are time-consuming which limits their use in clinical practice. For sputum
induction, it is estimated that should a patient produce a suitable sample within 10 minutes of
starting the procedure (and it can take up to 30 minutes) in a well practiced laboratory, the
total time required for processing through to a result is 100 minutes and the sample requires
processing within 2 hours of collection (Holz, Kips et al. 2000). The time spent to make the
procedure 'child friendly' is likely to be longer. The CAMP researchers across eight centres
commented "this procedure still remains a research tool in asthma because of its requirements
for technical expertise" (Covar, Spahn et al. 2OO4).
1.6.a (iv) Safety aspects of bronchoscopy, bronchial biopsy, bronchoalveolar lavage and/or
induced sputum
In 159 asthmatic adults undergoing 273 bronchoscopies in six studies, there were 34 adverse
event episodes which included bronchospasm, pleuritic chest pain, shortness of breath, fever,
fluJike symptoms and haemoptysis @lston, Whittaker et al.2004). Another safety review
reported that after six to eight biopsies taken per procedure in 57 patients; 40Vo had cough,
l2Vo cough and bronchospasm and3.5Vo required additional rescue medication (Iapanainen,
Lindqvist et al.2N2). Arterial saturation decreased in 50 asthmatic patients from a mean of
977o Io a mean of 92Vo and from 98Vo to 93Vo in 25 normal subjects during bronchoscopy and
biopsy, without correlation to the pre-operative lung functions tests (Van Vyve, Chanez et al.
1992). Bacteraemia is also described with fibreoptic bronchoscopy, with 26 of 200
consecutive patients having positive blood cultures after the procedure despite the majority of
patients having no prior respiratory illness (Yigla, Oren et al. 1999). A prospective analysis of
the financial cost of complications from flexible bronchoscopy over a 30 month period
revealed of the 1,009 bronchoscopies performed in 660 adults as an outpatient procedure,
38

complications occulred in SVo necessitating hospital admission in 0.5Vo, resulting in an
additional cost of US $6,996 to treat complications but with an additional US $34,500 in
those requiring hospitalisation (Colt and Matsuo 2001).
The largest study looking at side effects with flexible bronchoscopies in children, was a
retrospective analysis of 2,836 procedures over 2l years, which concluded that it was a safe
procedure. However 2l had life threatening hypoxaemia, l7 had laryngospasm or
bronchospasm and 4Vo had nasopharyngeal bleeding (Nussbaum 2002). A large prospective
study of 1,328 procedures (excluding intensive care examinations) recorded at least one
complication in 6.9Vo of cases, of which 5.2Vo were mild and l3To were major with one
pneumothorax (de Blic, Marchac et al. 2002).In 170 children with respiratory symptoms
having bronchoscopy and lavage plus at least three endobronchial biopsies, fluctuations of
oxygen saturation and end tidal carbon dioxide (CO, were seen in all patients but only one
had a prolonged desaturation episode. In addition, minor bleeding was demonstrated at the
site of the biopsies but no other side effects occurred (Salva, Theroux et al.2N3). Of 3g
asthmatic children undergoing bronchoscopy, one had desaturation, two had fever and in four
their asthma worsened over the following week. Of 35 non-asthmatic children undergoing
bronchoscopy for another reason (not 'normals'), there were there were 17 adverse events
which were predominantly laryngospasm and apnoea (Payne, McKenzie et al. 2001). In 66
children < 5 years of age a comparison was made between having bronchoalveolar lavage
alone and the addition of endobronchial biopsies with approximately half the group in each.
Complications occurred in 24Vo and, lSVo respectively including cough, desaturation, apnoea
and laryngospasm (Saglani, Payne et al. 2OO3). In 42 CF children and 39 with other
indications for bronchoscopy, the complication rate was l3.37o and, l7.97o respectively
(Molina-Teran, Hilliard et al. 2006).
So despite the progress in knowledge from the biopsy and lavage studies - they are still
invasive, require bronchoscopy, and sedation in adults and often general anaesthesia in
children. It is therefore particularly difficult to justify use of these techniques in early age
groups and/or repeatedly which is where the most knowledge regarding progression of disease
is likely to come. Discussions about safety in the literature has presented both pro and con
arguments (Connett 2000; Larsen and Holt 2000; Shields and Riedler 2000) ending with one
commentator suggesting "because of the invasive nature of the investigation there arc few
conditions for which repeat sampling can be justified" (Connett 2000). The debate has
continued more recently with a series of letters discussing the appropriateness of biopsy in
particular as a research tool in children (Mallory 2006;Bush and Davies zo07).
39

So is induced sputum in adults and children safe? Particularly those with asthma? In 34
asthmatic and normal subjects, a mean fall in FEVr of 5.3Vo was seen, but with a maximum of
20Vo (Pin, Gibson et al. 1992). Pre-treatment with a 9z agonist did not prevent
bronchoconstriction in all the asthmatic subjects, especially in those with a baseline low FEVr
(Wong and Fahy t997). Such pre{reatment in mild asthmatics resulted in an unchanged
FEV1, but with a reduction observed in the normal subjects (Thomas, Yates et al. 1999). One
group also documented that an overuse of F, agonists the day before sputum induction
reduced the protective effect of the pre-medication (Pizzichini, Pizzichini et al. 1997). While
samples were successfully obtained even in subjects with 'difficult to control' asthma, severe
bronchoconstriction occurred in22Vo with development of symptoms and a reduction in FEVr
of > l1%o (ten Brinke, de Lange et al. 2001). Another study noted that the procedure had to be
stopped because of side effects in l2Vo overall and in 17-l8%o of those with severe asthma (de
la Fuente, Romagnoli et al. 1998). When performed as part of their routine clinical visit, the
procedure was well tolerated in over 300 asthmatics, even among those with more severe
respiratory obstruction with an FEVr down to 60Vo predicted or one litre. However in patients
with a baseline FEVr between 4OVo and 59Vo, there was a mean 8Vo reduction in FEVr and in
patients with a baseline FEVr of < 40Vo predicted, 6Vo fellby 2OVo (Vlachos-Mayer, Leigh et
al. 2000). A decrease in arterial oxygen saturations of 6Vo in asthmatic patients, 5.3Vo in
smokers and 67o in healthy subjects has been reported suggesting that patients needed
monitoring during this procedure (Castagnaro, Chetta et al. 1999).
Reviewing the safety in children; in 53 sputum induction alone was tolerated in 98Vo, with
94Vo completing the procedure,92To providing an adequate sample but 4Vo experienced a
>l1vo fall in FEVr (Jones, Hankin et al. 2001). In 182 children who had combined sputum
induction and bronchial provocation testing using hypertonic saline, the procedure was
completed by 90Vo with a distressing cough in l3%o and mucosal irritation with wheeze in IVo
and fewer, 70Vo, successful samples obtained. So while the two investigations can be
combined, this was less well tolerated and resulted in less sampling success (Jones, Hankin et
al. 2001). In 40 children with a baseline FEVr > 65Vo predicted, seven (l8vo) had symptoms
of shortness of breath and wheezing and of these three (8Vo) had a significant fall in FEVr of
greater than 2O7o despite pre-treatment with a bronchodilator (Lex, Payne et al. 2005).In the
CAMP study, nine (87o) of 117 children with mild to moderate asthma developed significant
bronchospasm requiring treatment (Covar, Spahn et aI.2004).
40

1.6.5 Studies of inflammatory markers in blood and uine
Inflammatory markers have also been studied in blood and urine and I am going to briefly
review what has been found in this area. Blood and urine samples are relatively easy to obtain
when compared to the general anaesthesia or sedation required for bronchoscopy, lavage and
biopsy, are more consistently obtainable than induced sputum and less likely to cause side
effects than these other procedures. However blood testing is still seen as invasive by children
and most adults. Also, these compartments are removed from the direct area of interest and
reflect systemic findings, which may or may not be related to the lung.
As with results from sampling the lung, monitoring of inflammatory markers in blood have
shown an increase in eosinophils, ECP and EDN levels in children and adults with asthma
compared to control groups. In some studies these correlated with asthma severity or current
symptomatology (Krisdansson, Shimizu et al. 1994; Parra, Prieto et al. 1996; Rao, Frederick
et al. 1996; Remes, Korppi et al. 1998; Yazicioglu, Ones et al. 1999; Stelmach, Gorski et al.
2002), and although other studies also found higher levels, no such correlations were found
(Pizzichini,Pizzichini et al. 1997; Bacci, Cianchetti et al. 1998; Niimi and Matsumoto 1999;
Stelmach, Jerzynska et al. 2001; Stelmach, Jerzynska et al. 2001; Aldridge, Hancox et al.
2002; Reichenbach, Jarisch et al. 2002) or even mixed results were obtained (Currie, Syme_
Grant et al. 2o03). In adults, a large cross sectional study of almost 7000 subjects found
physician diagnosed asthma was significantly associated with serum eosinophil count
(Schwartz and Weiss 1993). Serum eosinophil and ECP levels during acute asthma were
corelated with more severe exacerbations and less bronchodilator responsiveness in 48 adults
(Lee, Ire et al. 1997). Plasma total IgE, plasma specific IgE, blood eosinophil percentage and
exhaled NO were thought to account for 55.5Vo of the variance seen in bronchial hyper-
reactivity (I-eung, Wong et al. 2005). Urinary levels of EDN did increase at time of asthma
exacerbation (Cottin, Deviller et al. l99g).
In children, ECP and eosinophilic peroxidase (EPO) in both serum and urine showed reduced
gradation in levels from asthmatics with current symptoms to symptom free asthmatic
children and to healthy children (Lonnkvist, Hellman et al. 2001). Both urinary EDN and
serum ECP were significantly higher in children with atopic asthma than control subjects
(Kristjansson, Strannegard et al. 1996). In both studies levels decreased with IHCS treatment
(Kristjansson, Strannegard et al. 1996;Lonnkvist, Hellman et al. 2001) and in one increased if
treatment was reduced (Lonnkvist, Hellman et al. 2001). Urinary EDN levels were increased
in 80 asthmatic children compared to 24 controls, and were higher in symptomatic than
4l

asymptomatic patients independent of the presence of atopy or the treatment modality.
Urinary EDN levels in the asthmatics correlated to lung function, and in the subgroup of 15
children re-examined two months after commencement of treatment, the reduction of EDN
also correlated with the changes in lung function (Lugosi, Halmerbauer et al. 1997). While no
correlation was found between urinary EDN values and lung function in two other studies of
155 and 39 children, there was a similar reduction of EDN three months after commencing
IHCS treatment (Krisdansson, Strannegard et al. 1996; Labbe, Aublet-Cuvelier et al. 2001).
Circadian variations of the eosinophil proteins in urine samples have been demonstrated with
high early morning and nocturnal peaks compared to lower levels in the afternoon in both
asthmatic and non-atopic, non-asthmatic groups of children (Storm van's Gravesande, Mattes
et al. 1999; Wolthers and Heuck 2003).
A more explored area using these samples has been in measuring the levels of the leukotriene
goup of proteins, particularly in urine samples. These proteins were shown to increase early
in adult asthmatic groups following an airway challenge (Westcott, Smith et al. 1991; Kumlin,
Dahlen et al. 1992; O'Sullivan, Roquet et al. 1998; Bancalari, Conti et al. L999) with peak
excretion occurring at two hours (Kumlin and Dahlen 2000; Mai, Bottcher et al. 2005). The
results were more variable when assessing whether levels remained elevated during the late
asthmatic response - not seen in one study (Manning, Rokach et al. 1990) but documented in
two others (O'Sullivan, Roquet et al. 1998; Bancalari, Conti et al. 1999). LT& was high in
184 adults presenting with moderate to severe acute asthma and reduced two weeks later in
the 146 followed up during recovery (Green, Malice et al. 2OO4). Compared to control
subjects, there was an increase in the LTEI level in asthmatics with nocturnal asthma
exacerbations but not in asthmatics without, although the numbers in each group were less
than ten (Bellia, Bonanno et al. 1996). Patients with asthma did excrete more LT& over a24
hour period than normal subjects, again with less than ten subjects in each group (Asano,
Lilly et al. 1995). In 40 severe asthmatics, 25 mild to moderate asthmatics and 20 non-
asthmatic controls, there was a gradation LTE4 levels despite treatment with high dose IHCS
in the severe group (Vachier, Kumlin et al. 2003). LTE4 in urine was higher in 35 atopic
asthmatic than32 non-atopic children with RSV bronchiolitis and 23 controls (Oh, Shin et al.
200s).
Other studies have been less positive about the correlations between inflammatory markers
measured in blood and urine samples and asthmatic disease. In adults, no correlation between
clinical status, functional status and serum ECP was seen during five months in asthmatics
either admitted for acute asthma or who were seasonally sensitised to birch and tree pollens
42

and had intermittent symptoms (de Blay, Purohit et al. 1998). The combination of serum ECp
with peak expiratory flow did not translate into a useful combination to determine IHCS
adjustment and was less useful than symptomatology alone or combined with other lung
function parameters (Lowhagen, Wever et aL.2002). Also, the blood inflammatory markers
did not reflect health related quality of life scores in subjects with mild asthma (Ehrs,
Sundblad et al. 2006).
In children, serum ECP levels followed during acute exacerbation and recovery of asthma in
11 asthmatics did not show a uniform pattern of increasing then decreasing values over this
period (Niggemann, Ertel et al. 1996). In 34 children with moderate asthma, none of the
markers measured reflected asthma activity, including serum eosinophils, serum ECp, serum
EDN, urinary EDN or urinary histamine levels (Hoekstra, Grol et al. 1998). Serial
measurements of ECP and EDN in urine samples taken monthly did not provide additional
information in monitoring childhood asthma or contribute to practical management in a
prospective study that followed children over a six month period measuring diary
symptomatology and peak flow (wojnarowski, Roithner et al. 1999).
In addition, the markers when raised were not found to be specific for asthma. Considerable
overlap was seen in blood eosinophil and ECP levels in asthmatics and controls, with no
difference in the mean levels measured in adults with asthma or with allergic rhinitis or in
those who had both (Sin, Terzioglu et al. 1998). While physician diagnosed bronchitis was
more significantly associated with neutrophil count, it was also associated with eosinophil
count and, as well, chronic sputum production was also associated with both cells (Schwartz
and Weiss 1993).In children, serum ECP and EDN was higher in the 36 asthmatic than 166
healthy children but higher rates than controls were also seen in those with allergic
rhinoconjunctivitis, atopic dermatitis and/or allergic skin sensiti zation. An elevated ECp gave
an odds ratio of 2.3 for asthma, but 2.9 for atopic dermatitis. Similarly, an elevated EDN gave
an odds ration of 2.61for asthma, but 5.23 for allergic rhinoconjunctivitis (Remes, Korppi et
al. 1998). Considerable overlap existed in another study comparing serum ECp levels in 2l
children with asthma and/or atopic dermatitis (Kristjansson, Shimizu et al. lgg4). In 207
children aged 24-41 months and 76 aged 0-23 months in whom repeated samples were
obtained in a large longitudinal childhood asthma study, the serum ECp was shown to be
influenced by a number of factors including age, the presence of active eczema and the
presence of maternal smoking in a dose dependent fashion (Lodrup Carlsen, Halvorsen et al.
1998). ln 72 infants with recurrent wheezing, there was no correlation between serum ECp
and the development of asthma and similarly no correlation between serum ECp and
43

onchial hyper-responsiveness (Reichenbach, Jarisch et al. 2002). Neither eosinophil
numbers nor ECP levels could predict the intensity of response to bronchial inflammatory
reaction in 46 house dust mite allergic children (Vila-Indurain, Munoz-l-apez et d. 1999).
Negative findings also occurred in studies into the leukotrienes. While there were significant
associations between lung function parameters and urinary LTEa, it did not distinguish
between groups of 49 mild and 3l moderate to severe asthmatics (Severien, Attlich et al.
2000). A single urine sample did not predict bronchial hyper-responsiveness or degree of
airflow obstruction in 41 asthmatics (Smith, Hawksworth et al. 1992). There was no increase
in LTCa, LTD4 or LTE4 in urine after an exercise challenge in adults (Taylor, Wellings et al.
1992). The measurement of urinary EDN in children with either acute or chronic asthma,
lower or upper respiratory tract infections or controls did not show significant differences
between these groups (Oymar and Bjerknes 2001).
So in all age groups blood and urine inflammatory parameters have failed to show consistent
discrimination between asthma and other allergic diseases, and in children between asthma
and other respiratory diseases, and also failed to determine asthma severity. The cells and
proteins in these systemic samples may not necessarily indicate what is happening in the
lungs as they are being measured from more distant sites. In addition, the measurement made
from blood samples by the time these reach the laboratory is made up of both the circulating
ECP and what is released from the eosinophils after the blood has been drawn into the tube
(Venge 1995). While these are easier samples to obtain with the benefit of fewer side effects
than sampling nonproductive respiratory secretions directly, blood testing is still perceived as
an invasive procedure and urine samples can be tiresome to obtain, particularly in children.
1.6.6 Comparison of intlammation results between the different samples
Comparisons have been made between the levels of inflammatory parameters obtained
through the different tissue or fluid samples. The cellular composition of induced sputum
correlates well with bronchoalveolar lavage and wash, but to a lesser extent with bronchial
biopsies (Fahy, Wong et al. 1995; Maestrelli, Saetta et al. 1995; Grootendorst, Sont et al.
1997; Keatings, Evans et al. 1997; Pizzichini, Pizzichini et al. 1998). For example, mast cells
usually make up only a small proportion of cells recovered by lavage but are as much as 20Vo
of the inflammatory cells in biopsy studies (Dunnill 1960; Foresi, Bertorelli et al. 1990).
Likewise there are more basophils in the biopsy specimens than in the lavage specimens
(Koshino, Teshima et al. 1993; Macfarlane, Kon et al. 2000). On the other hand as induced
sputum is rich in neutrophils and eosinophils but poor in lymphocytes, the origin of the
u

sample is thought to be from the larger airways (Keatings, Evans et al. 1997; Nocker, Out et
al. 2000). Induced sputum eosinophilic measures did predict exacerbations better than
bronchoalveolar lavage samples (Nocker, Out et al. 2000). While correlations were identified
between blood eosinophils and serum ECP, and sputum eosinophils and sputum ECp, in a
number of studies it was the sputum ECP that best correlated to bronchial obstruction, and the
sputum ECP and eosinophils that best correlated to methacholine challenge in asthmatics
(Sorva, Metso et al. 1997; Grebski, Wu et al. 1999; Piacentini, Bodini et al. 1999). Sputum
eosinophils and ECP were related to the symptom scores and FEV1, while no relationship was
demonstrated between blood eosinophils and symptom scores (Bacci, Cianchetti et al. 1998).
Finally, the measurement of ECP was easier to do from induced sputum than from blood or
lavage fluid, required less technical input and time and had better correlation to disease
activity (Barck, Lundahl et al. 2005). In a comparison between blood and urine samples, one
study showed a better correlation between lung function and the inflammatory parameters
measured in the urine samples (Labbe, Aublet-cuvelier et al. 2001).
t.7
Chapter summary
So in this introductory outline; the burden of paediatric respiratory disease in New Zealand is
great and asthma in both adults and children is a major cause of morbidity, stimulating my
interest in conducting research in this area. The diagnosis of asthma, particularly in children,
can be difficult with no single diagnostic or prognostic marker. Asthma continues to be a
clinical diagnosis based on history of symptoms, few examination findings and response to
treatment, with possible confirmation from lung function testing in older children. However
the mainstay of treatment is anti-inflammatory medication, which is associated with
significant side effects such as adrenal cortical insufficiency, growth failure, dysphonia and
oral candidiasis for IHCS use, and adrenal insufficiency which may cause devastating
hypoglycaemia, adrenal suppression, growth failure, hypertension, diabetes mellitus,
reduction in bone mineral density, skin atrophy, straie, poor healing, immunosuppression and
cataracts for oral corticosteroid use (GINA 2002; GINA 2005). We do not routinely measure a
marker for inflammation, but rather base decisions and treatment regimes on surrogare
markers such as symptoms, lung function, chest xray appearances, bronchial reactivity - to
determine successful outcomes.
The theory that asthma is an inflammatory disease was first indicated by autopsy studies in
early and mid last century. With the development of bronchoscopy, biopsy studies became
possible and these consolidated the pathogenesis of asthma as an inflammatory disease. This
45

led to research which looked for less invasive methods of assessing the inflammatory
component of asthma and bronchoalveolar lavage and this led to the development of the
ability to induce sputum using nebulised hypertonic saline. These samples allowed increasing
measurement not only of the cellular component, but also of the proteins that formed the
asthmatic inflammation. These measurements could be done repeatedly so parameters were
followed longitudinally to determine what happened during acute exacerbations of asthma, in
response to treatment and over longer periods of time during chronic asthma. Studies were
also undertaken using blood and urine samples. In the main there has been a recognition that
the majority of asthma and other allergic diseases have an increased allergic inflammatory
profile with an eosiniphilic dominant pattern, raised levels of eosinophilic proteins @CP,
EPO, EDN and MBP), and cytokines (IL3, nA,n-5, rantes). While over the last decades this
was thought to be an imbalance between T lymphocyte classes, between the T helper type 2
(Ts2) and T helper type I (Tnl), (Colavita, Reinach et al. 2000; Holgate, Davies et al. 2000;
Busse and Irmanske 2001; Peters 2003), very recently it has been thought there may be
upregulation of both classes with a third type, the T lymphocyte regulatory cells playing a
pivotal role (Lazarus, Raby et al.2004; Goldman 2007). Certainly some asthma is known to
have a neutrophil dominant or neutrophil present pattern, with the markers more commonly
associated with infective conditions. This is seen more often in severe persistent asthma
(Wenzel, Szefler et al. 1997i Jatakanon, Uasuf et al. 1999), associated with a poor response to
treatment (McDougall and Helms 2006; Wenzel 2006), has been demonstrated in some acute
exacerbations (Martin, Cicutto et al. 1991; Fahy, Kim et al. 1995; Ordonez, Shaughnessy et
al. 2000), and in sudden fatal asthma (Sur, Crotty et al. 1993). While the eosinophils and
neutrophils themselves and the proteins they release have shown the best association with
asthma exacerbations and response to treatment - neither have been completely consistent. In
addition, obtaining the appropriate samples can be difficult.
So, while contributing enonnously to our understanding of asthma, the use of bronchoscopy,
biopsy and BAL, induced sputum, blood and urine testing all have their disadvantages,
particularly when assessing disease in children. The first three options remain invasive,
require anaesthesia in children and are associated with risks and side effects, especially in
children with respiratory disease, and therefore cannot be done frequently. They also require
specialised personnel in terms of theatre and bronchoscopy staff. Induced sputum results in
less satisfactory sample success in children than in adults, although successful samples can be
obtained in up to 757o of control children. It also can be associated with side effects, as
nebulised hypertonic saline is also used as an airway challenge. This test also requires
46

experienced staff to undertake the procedure, and laboratory staff to process samples within a
short time frame. It also represents a significant time commitment - even in the most
experienced hands and a sample taken in the shortest time, the minimum time to from start to
getting a result for one sample is estimated to be 100 minutes (Holz, Kips et al. 2000). Both
blood and urine sampling have been found to be less useful, probably because the site of
measurement is distant from the site of interest, the lung. In addition, apart from obtaining
urine samples, the other sampling procedures all require a hospital or clinical setting.
These studies started to come about at the same time as my own research commenced. In view
of all these difficulties a simpler, direct, non-invasive technique for measuring airway
inflammation was sought, that could be achieved easily by children, and could be repeated
regularly with time. Because measurement of lung function is a common procedure in
children and does not involve collecting specimens and there is no anaesthesia or needles, the
result is instant -
a test analogous to this would seem ideal.
In 1987 came the announcement that a gaseous molecule, NO, was responsible for acting as
both a widespread physiological mediator and was also involved in host defense (Hibbs,
Taintor et al. 1987; Ignarro, Buga et al. 1987 Khan and Furchgott 1987). This resulted in an
explosion of research in the early 1990s identifying both NO and the enzymes that produced it
- the nitric oxide synthases (NOS) in all biological systems. NO was shown to play a major
role in host defense and inflammation within the lung as in other systems. Toward the end of
1995,I also went to a one day seminar on NO at the Royal Society of Medicine in I-ondon to
hear Professor Salvador Moncada, one of the major early researchers into NO in the
cardiovascular system, talk. I was struck by the importance of this one tiny molecule, and the
unfolding story with more to come. This seemed like a great opportunity to further explore the
possibility of NO as a potential marker for airway inflammation; however there were a
number of hurdles to overcome. Measuring NO was going to be difficult given its gaseous,
short lived and highly reactive nature. As well there was the difficulty of measuring levels in
exhaled air from human subjects, especially in children.
This research was conducted at the Royal Brompton Hospital in London from the mid 1990s.
On returning to New Zealand, it was acceptable to submit this research over time as a thesis.
Some of the findings within this thesis are now well-known, but were unknown, new and
exciting when first demonstrated. The findings contributed to the knowledge as to what
technical aspects were important in the measurement of NO, to the standardising of protocols
for measurement, and to what were important capabilities to be developed for the newer NO
47

analyser machines. This thesis presents one researcher negotiating research in one area of
medicine -
which will be presented how it unfolded at the time and will be reviewed with the
up to date NO literature in the penultimate chapter.
In the chapter to follow, I will review what was known about NO and nitrogen dioxide (NOz)
in pollution where it was recognised to be a major component, and its effects at the population
level on respiratory disease. The study of pollution was where the machines were first
developed in order to analyse levels. The chapter will then describe the recognition of NO as a
physiological mediator and its key role in inflammation. Chapter 3 will review the actions and
interactions of NO as well as the NOS enzymes that produce this molecule in vivo. Chapter 4
will elaborate on the methods of measuring this short lived, reactive molecule. Chapter 5 will
discuss how the chemiluminescence analyser, the method of choice for the subsequent
experiments, could be modified to measure NO in exhaled air in humans using an analyser
developed for NO measurement in the environment. Chapter 6 is the corlmencement of the
research in normal, control adult subjects looking at the measurement of exhaled NO through
two different methods, either directly into the NO analyser which also allowed the
measurement of mouth pressure and COz, and through a t-piece system which enabled the
measurement of flow in addition to the other parameters. Chapter 7 describes the research
undertaken to see what technical aspects of measurement altered the NO levels obtained.
Chapter 8 describes the research as I measured exhaled NO in normal and then asthmatic
children. It briefly reviews the effect of age, atopy, the presence of pets or the presence of
smoking in the home on the NO obtained in a group of healthy pre-pubertal children. There is
a comparison of results from this control group to a group of asthmatics on bronchodilator
therapy only and a group of asthmatics who were on chronic inhaled corticosteroid therapy. A
small number of steroid naive asthmatics were measured before and after commencing
inhaled corticosteroids. Chapter 9 summarises what was learned about measurement of
exhaled NO from these studies and how that has contributed to the literature. This is followed
by a further review of the literature to the current time and the place of NO measurement as it
currently stands. Finally chapter l0 will present the final thoughts with regard to this research
project and 'where to from here'.
NB: Some of the work presented in this opening chapter formed the monograph for the
Asthma and Respiratory Foundation of New Zealand; (Anonymous 2006) in which I
contibuted two chapters and edited in conjunction with Professor Innes Asher. This was
presented to The Honorable Mr Peter Hodgeson, Minister of Health April 2006, and is
av ailabl e on tw o w eb s it e s : www. asthmanz. c o. nz o r www. p ae diat ric s. o r 8. nz.
48

2.1 Introduction
Chapter 2: Nitric oxide: pollutant to mediator
There are two lines of research, from quite disparate areas, that came together to make the
studies described in Chapters 6, 7 and, 8 possible. The first is the measurement of air
pollution, which has been increasingly important due to recognition that different pollutants
cause or contribute to human disease, including nitrogen oxides; and the second is the
increasing recognition of the importance of nitric oxide (NO) in biological systems in the last
25 years. I will examine the developments in both of these areas that led to the
commencement of the research described in this thesis.
2.2 Pollution. nitrogen oxides and disease
2.2.1 Concerns regarding pollution
There is now no doubt that episodes of air pollution have resulted in excess deaths. Concerns
with regard to pollution and its effects on health date back to early last century, although the
detrimental effects of poor air quality and poor housing have been recognised for far longer.
Indoor air pollution can be traced to some 200,000 years ago, when humans first moved to
colder climates necessitating the construction of shelters and use of indoor fires for cooking,
warmth and light. These mostly 'inside' fires likely resulted in an excess exposure to levels of
pollution as evidenced by the carbon soot found in prehistoric caves (Brims and Chauhan
2005). Severe outdoor air pollution episodes have been documented since the early 17ft
century but these became more frequent and more severe throughout the 19ft century. The
most definitive and well documented episodes were the great smogs that occurred in London,
United Kingdom, in 1948, 1952 and 1962, with the most severe occurring in December 1952
(Brims and Chauhan 2005). The smoke concentration at this time was 50 times above the
average level, and this rise in pollution was followed by an equally sharp rise in mortality
(Ministry of Health, UK 1954). An estimated human death toll of 4,000 occurred during this
period which was three times the expected mortality for that time of year. Most deaths were
among infants, the elderly and those with chronic respiratory disease (Anderson, ponce de
Leon et al. 1996).In addition, emergency hospital admissions tripled for respiratory disease,
and doubled for cardiovascular disease over the same period (Schwartz l99l). These led to
the Clean Air Act in 1956 and 1968 in the United Kingdom (UK), with further modifications
to the Act made in 1993.
49

The pollution of the 'great smog of London' and contemporaneously elsewhere was to do
with the burning of coal and generation of 'black smoke' (Ministry of Health 1954). Smog
has become the common name given to the "soup" produced from photochemical reactions in
the atmosphere. Because of the role that heat and sunlight play in its production, the highest
levels of smog are recorded on hot, sunny days. Summer smog is composed mainly of ground
level ozone and particulate matter. Because ozone is not produced at high levels in cold
weather, winter smog is composed mainly of particulate matter and sulphur dioxide (World
Health Organisation 2003).
However with the increase in mechanisation and industrialization, particularly with increasing
motor vehicle use, the relative contribution of pollutants from the different sources has
changed over the last few decades. There has been a reduction from industrial sources and
domestic heating, and an increase from car exhaust emissions. The combustion of these fossil
fuels produces carbon monoxide, nitric oxides, benzene, sulphur dioxide, and particulate
matter. In addition, diesel engines, which are also on the rise, emit much higher emissions of
the gases and 100 times more particles compared with gasoline engines equipped with modern
exhaust treatment systems @iedl and Diaz-Sanchez 2005).
Reflecting the increasing pollution problem, 172 individual compounds and 17 compound
classes as hazardous air pollutants of concern in the USA in 1990 (Clean Air Act 1990). Of
these, the compounds that have been associated with respiratory and/or cardiovascular disease
are particulate matter with a diameter of less than l0pm (PM10), ozone, nitric oxides and
nitrogen dioxide (NOz) in particular, sulphur dioxide (SOz), carbon monoxide (CO), lead and
volatile organic compounds (Anonymous 1995; Anonymous 1996).
This association between large specific pollutant episodes and increased disease and/or deaths
is well documented for these early episodes. It has subsequently been more difficult to tease
out whether less, but more continuous, pollutant exposure had similar health effects and has
resulted in support both for (Bates 2000) and against (Gamble 1998) and necessitated large
committee reviews of the topic (Anonymous 1995; Anonymous 1996). The suspicion that
there was an adverse effect arose because of the noted increasing rate of allergic disease
occurring in parallel and geographically coincident with the increased industrialisation. Even
in 1873 when the first report of environmental exposure causing disease was documented
(Charles Harrison Blackley), that 'hay fever' or 'hay asthma' was caused by grass pollen, it
was even then observed to be more common in urban than in rural settings (Brims and
Chauhan 2005). The difficulties in confirming the effects of a lower pollution dose over a
50

longer period of time were manifold. Firstly, there were many differing pollutants available to
measure. Secondly, there was likely to be an interaction with pollutants and other
environmental and/or infective triggers for disease. Thirdly, there was uncertainty that an
increase in disease rates could occur at levels that were below World Health Organisation
(WHO) recommendations for safety of the time (World Health Organisation 2c413; World
Health Organisation working group 2003).
Analysis of the London data from 1958 to 1972 rcveals a pattern that the daily mortality was
indeed associated with pollution levels on the previous day. This was especially so for
particulate matter, SOz and acidic aerosol pollution (Mazumdar, Schimmel et al. l9g2:
Schwartz and Marcus 1990), but it was difficult to determine whether a single pollutant was
responsible and authors concluded it may be the interactions between the three that were
important (Ito, Thurston et al. 1993). A further large study analysed air pollution and daily
mortality in London between 1982 and 1992 as part of an across Europe project. The
strongest association was found with ozone recorded the same day, and particulates recorded
the previous day with small but positive effects also seen with NOz and SOz on all causes of
death (excluding accidents), cardiovascular mortality and respiratory mortality (Anderson,
Ponce de kon et al. 1996). Even relatively low levels of air pollution have been shown to be
associated with increased health problems and with day to day variations in mortality
(Schwartz t993). From the 1960s, through three decades of London winters, there was a
highly significant association between mortality and levels of particulate matter and/or SOz
comparing levels and mortality (Schwartz and Marcus 1990). In the USA, an association
between levels of air pollution and overall mortality was also demonstrated (Ostro 1993;
Schwartz 1994), as well as between particulate matter (PMlg) and an observed increase of
respiratory disease within the levels usually measured in urban areas @ockery and pope
1994).
2.2.2 Disease and pollution
So what has been discovered in terms of disease? Some subgroups of population appear more
sensitive to the effects of air pollution; these include young children, the elderly and people
with pre-existing chronic cardiac and respiratory disease such as chronic obstructive
pulmonary disease (COPD) and asthma (Anonymous 1995; Anonymous 1996). For example,
the analyses of fine particulate air pollution and mortality in nine counties across California
showed that the particulate level was related in an increase in overall mortality, as well as for
deaths out of hospital, respiratory disease, cardiovascular disease, diabetes and deaths in
51

adults greater than 65 years (Ostro, Broadwin et al. 2006). Increased rates of myocardial
infarction, dysrhythmias, increased blood pressure, heart failure and hospitalisation for
cardiac events have been associated with higher air pollution levels (Pope, Verrier et al. 1999;
Peters, Liu et al. 2000; Linn and Gong 200I; Peters, Dockery et al. 2001; Pope, Burnett et al.
2W4\.
The 'Swiss Study on Air Pollution and Lung Diseases in Adults' involving over nine
thousand subjects showed positive conelations of levels of particulate matter, ozone and NOz
with chronic cough, chronic sputum production, and shortness of breath during the day,
during the night and on exertion, but not with other respiratory symptoms (Zemp, Elsasser et
al. 1999). This study also showed reduced lung function parameters for FVC, FEVr and
FEFzs-zs which negatively correlated with the same three pollutants (Ackermann-Liebrich,
Iruenberger et al. 1997, Schindler, 2001 #652). A major study in six cities in the USA
demonstrated a three fold increase in chronic cough with increased exposure to particulate
pollution @ockery and Pope 1994). Throughout Califomia from 1973 to 1987 site and season
specific particulates were monitored and showed correlations between the development of
symptoms of airway obstructive disease, increased severity of obstructive disease, chronic
productive cough and increased asthma (Abbey, Hwang et al. 1995). Stronger associations
were seen with those who were also occupationally exposed to dusts and fumes. Increased
symptoms of cough, bronchitis, asthma, and COPD were associated with increased air
pollution (Schwartz t993; Souza, Saldiva et al. 1998). Particulate matter was found to be
associated with respiratory diagnoses made by a physician and chronic bronchitis in a study
involving 53 urban areas throughout the United States with graduated levels of pollution
(Schwartz 1993).In Australia, ozone levels, particulate matter and the concentration of SOz
had strong associations with emergency department review of respiratory illnesses overall,
and for asthma exacerbations in twelve hospitals, though it proved difficult to detect out
differences between the pollutants (Atkinson, Anderson et al. 1999). Particulate matter levels
were also positively associated with hospital admissions for asthma and for COPD @rbas and
Hyndman 2005).
Histopathologic changes have been shown in lung tissue samples of individuals who died due
to violent causes depending on their pollutant exposure history. With the increased pollution,
there was increased presence of inflammatory reaction, wall thickening, ild secretory
hyperplasia. There were also increased carbon deposits along the regional lymphatic drainage
(Souza, Saldiva et al. 1998).
52

Studies on traffic exposure, (including detailed studies on occupational exposure to increased
vehicular emissions (Raaschou-Nielsen, Nielsen et al. 1995; Zagury,6 Moullec et al. 2000;
Roegner, Sieber et al. 2002; Seshagiri 2003; Lai, Liou et al. 2005) and occupational NO
exposure (Azan, Williams et al. 1996; Markhorst, I-eenhoven et al. 1996; Olin, Ljungkvist et
al. 1999; Phillips, Hall et al. 1999 Qureshi, Shah et al. 20O3; Maniscalco, Grieco et al.
2004)), have also demonstrated that there is a consistent effect of long term exposure to car
traffic on non-specific respiratory symptoms (Abbey, Hwang et al. 1995) and lung function
(Ackermann-Liebrich, kuenberger et al. 1997). Proximity to traffic exposure was a risk
factor for wheezing, asthma severity and prevalence (Nitta, Sato et al. 1993; Oosterlee,
Drijver et al. 1996, Brunekreef, 1997 #640).In three Japanese cross sectional questionnaire
studies in five thousand women participants, the estimated odds ratio for chronic cough,
chronic sputum production, chronic wheeze and chest infections with sputum were increased
the closer the subjects lived to roadways with heavy traffic (Nitta, Sato et al. 1993). Air
pollution appears more likely to exacerbate existing asthma rather than generate new cases,
although it is associated with reduced lung function in healthy children (Barnes 1994; Segala
1999). There are fewer studies that have looked at the effects of air pollution on upper airway
disease rates, but general practitioner consultations due to upper airway symptoms increase on
days of higher concentrations of particulate matter and SOz (Gordian, Ozkaynak et al. 1996;
Hernandez-Garduno, Perez-Neria et al. 1997;Hajat, Anderson et al.2oo2).
Only a few authors have looked specifically at the effects of air pollution on children, where
both outdoor air pollution and indoor air quality have been implicated as causal factors for
respiratory diseases and respiratory symptoms (Nicolai 1999). An increase in mortality in
children during severe episodes of air pollution has been shown and this includes an increase
in the general rate of mortality, an increase in respiratory mortality and an increase in peri-
neonatal mortality (Bates 1995; Anderson, Ponce de kon et al. 1996). As well as mortality,
an increase in respiratory morbidity has been shown. The respiratory illnesses sensitive to
increased pollutants in children include acute bronchitis, cough, asthma and pneumonia.
Similarly to adults, this is most often attributable to an increase in particulate matter (Aunan
1996). In the 'Six Cities Study of Air Pollution and Health' reported rates of chronic cough,
bronchitis and chest illness were positively associated with all measures of particulate
pollution and positively, though less strongly, associated with SOz and NOz @ockery,
Speizer et al. 1989). Particulate matter in air pollution has been shown to triple the prevalence
of chronic cough, nocturnal cough and bronchitis in a cross sectional study of over 4,000
Swiss children aged six to 15 years from ten different communities with the highest pMl0
53

levels compared to those children living in less exposed areas (Braun-Fahrlander, Vuille et al.
1997). In a prospective, observational study over one year and comparing 1025 children
presenting with wheezy episodes with other admissions as a control group, the day to day
variations in the concentration of ozone were associated with an increased incidence in acute
wheezy episodes @uchdahl, Parker et al. 1996). Children living along a busy street were
found to have a higher prevalence for most respiratory symptoms than children living along a
quiet street. This was most significant for medication use and for episodes of wheeze
(Oosterlee, Drijver et al. 1996). Children admitted with asthma were more likely to live in an
area of high traffic flow compared to admissions for non-respiratory reasons or a community
sample of children with a significantly linear trend observed for traffic flow for children
living less than 500 metres from a main road @dwards, Walters et al. 1994). This study also
showed that children were more likely to be admitted for non-respiratory reasons compared to
the community sample of children if they lived within 200 metres of a main road. Similarly
results for atopy and road traffic have also been demonstrated with, for example, higher rates
of allergic sensitization found in children playing more than one hour per day near major
traffic thoroughfares (Brunekreef, Janssen et al. 1997).
Some studies have incorporated lung function testing to look at the effects of air pollution.
The lungs develop throughout childhood, with peak lung function occurring between 20 and
25 years, followed by a plateau of ten years before beginning to decline. A deficit in growth
caused by air pollution effects, for example, would most likely translate into a reduced
baseline function that would be carried throughout life (Gilliland, Gauderman et al. 2ffi2;
Gauderman 2006). Air pollution data from monitoring stations in twelve California
communities demonstrated that a greater proportion of young adults with and FEVr less than
80Vo predrcted lived in the areas of higher carbon and PM10 (Gauderman, Vora et al.2007).
An inverse dose dependent relationship was found between the carbon content of airway
macrophages in induced sputum and FEVr, FVC and FEFzs-zs in healthy children living in an
area with a variation of PMl0, primarily due to emissions from road traffic (Kulkarni, Pierse
et al. 2O06). Reviewing the age goup six years to 24 years of age, FVC, FEVr and PEF all
showed statistically significant negative correlations with the annual concentrations of total
suspended particles, NOz and ozone (Schwartz 1989). The relationships still persisted when
children with pre-existing respiratory illnesses and smokers were included. Furthermore in a
longitudinal three year study, children exposed to high PMl0 levels during the summer had a
reduced growth of FEVr and FEFzs-zs (Horak, Studnicka et al. 2002). A reduction in lung
function with higher levels of ozone was also demonstrated in children from three schools in
54

Mexico (Castillejos, Gold et al. 1992), with atopic status being an additional risk factor
(Jorres, Nowak et al. 1996). In children with moderate to severe asthma attending surnmer
camps over three years, air pollution levels (predominantly ozone) were associated with acute
asthma exacerbations, chest symptoms and a reduction in lung function (Thurston, Lippmann
et al.1997).
Increased absences in school and kindergarten or playschool attendances have also been
associated with an increase in particulate matter -
which was more pronounced in six to nine
year olds than older age groups (Pope, Schwartz et al. lgg2: Ransom and pope lgg2). There
has also been an association with rises in levels of pollutants on prolonging episodes of
infection (Bates 1995). Increased levels of pollutants have also been associated with an
increased risk of developing upper respiratory tract infections and symptoms in children
(Jaakkola, Paunio et al. 1991, Ostro, 1999 #657), particularly with high concentrations of
SOz, and moderate levels of NOz and particulate matter (von Mutius, Sherrill et al. 1995).
Clearly, it would be helpful to confirm these findings using more specific experiments to
determine a dose response and the mechanism of damage which causes disease. This has been
done in a few animal models which have demonstrated the development of infectious and
allergic lung disease with increased severity when exposure to air pollutants (ozone, NO2,
SOz). These pollutants increased damage in infection and inflammation in mouse and guinea
pig populations predominantly but also in a dog and a monkey model (Gilmour 1995;
Miyabara, Takano et al. 1998; Ng, Kokot et al. 1998; Selgrade 200O; Whitekus, Li et al.
2002). While it is more problematic to confirm a dose response in humans, it has been
possible to do nasal challenges with allergen, with or without the addition of diesel exhaust
particles, showing exaggerated responses in the presence of the particles @iaz-Sanchez, Tsien
et al. 1996; Diaz-Sanchez, Jyrala et al. 2000; Bastain, Gilliland et al, 2003; Gilliland, Li et al.
2004).It is now thought that oxidant stress is the mechanism that underlies the toxic effect of
most forms of air pollution (Nel, Diaz-Sanchez et al. 2001; Kelly 2003; Kelly and Sandstrom
2004; Gauderman 2006). The lung is actually well equipped to deal with oxidative stress as
the lung lining fluid is rich in enzymatic and low molecular weight non-enzymatic
antioxidants. In addition, there are intracellular defences with glutathione based enzymes that
use a variety of the products of oxidative stress as substrates and therefore prevent their build-
up. However individuals have differing genotypes of this enzqe family, which appear to
make them more or less susceptible to damage from air pollutants, including active or passive
smoke inhalation (Miyabara, Yanagisawa et al. 1998; Mudway and Kelly 2000; Gilliland,
Rappaport et aL.2002; Bastain, Gilliland et aI.2003; Gilliland, Li et al. 2004).
55

More recently 'Air Quality Indexes' were developed to better inform the public about air
quality or the degree of pollution on any given day. These are composite measures of
common air pollutants for which there are demonstrable adverse effects on health and the
environment. At the current time these include CO, NOz, SO2, ground level ozone, suspended
particulate matter (sometimes expressed as a coefficient of haze) and total reduced sulphur
compounds. The Index is reported on a scale of 0 to 100 (or more) with scales of 'very good"
air quality being 0-15 and 'very poor' near the 100 mark (Hilternann, Stolk et al. 1998;
Abelsohn, Stieb et al.2002).
2.2.3 Nitrogen oxides in pollution
NO is a potentially toxic molecule; and both NO and its interrelated forms were discovered as
components in pollution. Space shuttle and jet plane exhaust, lightening and smog are all
known to have a high proportion of NO (Nathan 1992). Nitrogen oxides were detected in the
evaluation of air pollution from the Titan tr firings @iamond and Johnson 1965). NO forms
part of the origin of the shuttle glow which was observed in 1983, and was carefully examined
because of concern that it would interfere with space-based spectroscopy. It was found to be
the reaction between NO and 02 forming an excited form of NO2 which released light as it
desorbed (Viereck, Murad et al. 1991). Increases in air traffic have resulted in increases in
nitrogen oxides in the troposphere, again causing concern about the effects in atmospheric
chemistry (Johnson, Henshaw et al. 1992). There exists an array of compounds that includes
NO, nitrosoium cation (NO +), nitroxyl anion (NO-), peroxynitrite (ONOO-) and hydroxyl
anion (OH-). Most of these are inherently unstable compounds and interact with other
elements particularly ozone and oxygen, so the main compound measurable in pollution from
this class of gases is the stable gas NO2. The ambient NOz levels in London vary between25-
58ppb (24 hour average), European air levels are 78ppb (24 hour average) and the level in the
USA is 53ppb (one year average) (Nicolai 1999).
Many epidemiological studies of outdoor pollution have found associations between NO2
exposure and disease, even at levels below the current WHO guidelines. The 'Air Pollution on
Health: European Approach' studies incorporated data from 15 cities with a total population
of greater than 25 million. A rise in NOz over the one hour safety maximum of 50 pglm3 was
associated with a 2.6Vo increase in asthma admissions and a l.3%o increase in daily all cause
mortality (Touloumi, Katsouyanni et al. 1997). In Toronto, from the total number of
premature deaths related to air pollution, NOz was assessed to be responsible for almost 407o,
and, of the hospital admissions, 60Vo was also thought to be attributable to NOz levels, with
56

the other main pollutants studied being CO and SOz (Abelsohn, Stieb et al.2O02). Three
studies have looked at the effect of ambient NO2 concentrations on respiratory illnesses in
children. Firstly, a significant association between the frequency of croup and the daily
measured level of NOz for the peak periods (September and March) of disease rate was found
in five German communities (Schwartz, Spix et al. l99l). Secondly, the risk of admission to
hospital with wheezing bronchitis was significantly related to outdoor NO2 exposure in girls
(although not boys) in Stockholm, however the presence of a gas stove in the home was also a
significant risk factor (Pershagen, Rylander et al. 1995). A third study showed worsened
virus-induced asthma in children following exposure to higher NO2 levels (Chauhan, Krishna
et al. 1998).
However examination of the potential harmful effects of the pollutant NO has been
investigated down to the individual cigarette. NO exists in an English cigarette ',at a level of
1,000 parts per billion with the level of a French cigarette being one million parts per billion"
(Borland and Higenbottam 1987). NO corrodes metals and interacts with plastics. There are
particular problems with the development of even more dangerous combinations as listed
above in the presence of higher concentrations of oxygen (see Chapter 3 and Chapter 4
regarding toxicity).
So by the time this research began, the nitrogen oxides were known to contribute to pollution,
to contribute to the excess of cardiovascular and respiratory disease and mortality seen with
increased air pollution and studies were well underway using analysers to measure daily
levels for air quality recordings and to ascertain their continued association with disease.
2.3 The discovery of nitric oxide in biological systems
2.3.1 Vascular control
It is increasingly rare to discover something completely novel. The discovery of NO and its
importance in biological systems, in particular the opening up of a new concept of the use of
gases as biological mediators, was truly new. Initially the exact nature of NO as a mediator
was not recognised and it was labelled as 'Endothelial Derived Relaxing Factor' (EDRF).
In 1916 Mitchell observed that oxides of nitrogen were produced in mammals showing more
nitrogen in urine than consumed (Mitchell, A. et al. 1916). In l92g Tannenbaum confirmed
that mammals produced nitrogen oxide by showing production occurring in the intestine
(Mitchell 1928; Tannenbaum, Fett et al. 1928). In the 1970s research was undertaken on
categorising biological 'amines' such as adrenaline, noradrenaline, histamine and
57

acetylcholine. These were known to contribute to both cell to cell and nerve cell to terminal
tissue interactions. One of the main investigators in this area, Professor R. F. Furchgott, was
studying the mechanism of contraction and relaxation of isolated of blood vessels. Using
identical chemicals, the studies initially yielded conflicting results. Laboratory investigations
subsequently revealed that the results were dependent on which technician conducted the
experiment. One technician found that acetylcholine always relaxed the vessels and another
found that it always contracted the vessels. Investigating further, Furchgott discovered that the
reason for this anomaly was the presence or absence of the endothelial monolayer, as one
technician meticulously kept the endothelium intact and the other always removed the
endothelium in preparation. The presence or absence of this endothelium resulted in quite
different reactions of the blood vessel to the applied mediator (Furchgott, Jothianandan et al.
1984). This also explained a somewhat surprising finding which had been made many years
earlier in his research career. He found that acetylcholine (a well known vasodilator in
animals and in perfused organ studies) elicited contraction rather than relaxation on an aortic
strip. However, the preparation technique unknowingly stripped the specimen of endothelial
cells @urchgott and Bhadrakom 1953). It was this that led to the coining of the term for the
substance responsible as the 'EDRF' @urchgott andZawadzki 1980).
Advances in this area occurred with the development of perfusion-bioassay procedures which
allowed more direct studies on the properties of EDRF than the previous experiments which
had been carried out in organ chambers. Intact endothelial cells were held upstrearn, a section
of blood vessel wall stripped of endothelium was held downstream so substances could be
perfused in turn to see whether each test substance inhibited EDRF by interfering with its
synthesis, its release or inactivated EDRF after release. This approach was also used to assess
the rate of decay of EDRF by altering the transit time of the perfusate (Furchgott 1998).
Initially this experiment resulted in confusion, as estimations of the EDRF half life were
estimated from four to 50 seconds. However later this was able to be explained by the
differences in the superoxide anion (O2-) concentration in the perfusion fluid as this rapidly
inactivates EDRF (see Chapter 3.2'Reactions of nitric oxide'), (Gryglewski, Palmer et al.
1986; Rubanyi and Vanhoutte 1986). These perfusion bioassay studies also allowed
demonstration of the release of EDRF from vessels with increased shear stress, usually
secondary to increased flow thereby suggesting that EDRF had a continuous physiological
role in the control of regional blood flow and regulation of blood pressure.
58

Figure 2.1: Diagram of blood vessels in cross section showing the endothelial layer
ExtltNll ElAtllC Xlti'l!8ANE
IMETNAI. ElAtIK
^itilEt^ltE
ARTERY
IUNICA IMNtrA
:NOOTHEUUM
crtcut.At ,$utcttt
tAt cttts
vlsEts 0F ArytNlrn
AOVIN'|I|a
Taken from 'Access Excellence'- The Heart and the Circulatory System by Roger E phillips Jr., from
the National Health Museum, "The Site for Health, Teachers and-Learners', t-+tl K st, Suite 1300,
Washington DC 2000s.
These and other researchers continued pursuing the identity of this substance with the
evolving characteristics becoming increasingly similar to a compound of nitrogen, resulting in
experiments comparing 'EDRF' with nitrogen oxides. Furchgott initially announced that
EDRF could actually be the inorganic gas 'NO' at a conference in July 1986 (Khan and
Furchgott 1987)' This same conclusion was proposed at the same conference by Ignarro as a
result of his experiments on bovine pulmonary arteries (Ignarro, Buga et al. l9g7; Ignarro,
Byrns et al. 1988). Similarly in 1987, Palmer, Fenige and Moncada showed that vascular
endothelial cells generated NO and that the amounts generated accounted for the biological
activity of the named EDRF mediator (Moncada, Palmer et al. 1988). This was a novel
suggestion as - until this time -
mediators.
gases had not been thought capable of acting as biological
It had been recognised that endothelium dependant relaxation of blood vessels was associated
with increased cyclic guanosine 3', 5' -monophosphate (cGMp) levels in vascular muscle.
This was demonstrated earlier in a number of studies using rat aorta (Rapoport and Murad
1983), rabbit aorta (Diamond and Chu 1983, Furchgott, 1983 #415), bovine coronary arteries
(Holzmann 1982) or bovine pulmonary arteries (Ignarro, Burke et al. 1984). Rises in cGMp
are produced by activation of soluble guanylate cyclase (sGC), a magnesium sensitive
hexodimer protein which contains two heme molecules. Under basal conditions, the guanylate
59

cyclase does not produce significant levels of NO, but the relaxant factor, EDRF, was thought
to alter the binding in so much that it increased the activation of the enzyme by 400 fold.
However it is now known that it is nitrosylation of the guanylate cyclase that results in high
levels of cGMP which both prevents entry and promotes movement out of the cell of calcium
resulting in vasodilatation. The Moncada research goup then identified that the substrate for
NO synthesis was L-arginine and also went on to synthesise an inhibitor of NO formation by
producing a false competitive inhibitor of L-arginine; N* monomethyl L-arginine (L-
NMMA), (Gardiner, Compton et al. 1990). It was by using this inhibitor that further
clarification of the role of NO was possible.
2.3.2 Immunefunction
At the same time that EDRF was being identified as NO, other research groups were looking
at NO in a different biological system -
that of host immune defence.
Macrophages were known to be able to inhibit tumour growth and induce tumour cell death
(Nathan 1992). In 1987 (Hibbs, Vavrin et al. 1987) it was suggested that the macrophage
cytotoxicity might in part be due to the ability of the macrophages to synthesise NO, nitrite
and nitrates from L-arginine. At the tissue level, NO was known specifically to be toxic to
cells. A number of bacteria were also known to be able to generate reactive species of nitro-
oxygen suspected as occasionally causing lung disease. However the main interest in these
bacteria was not in the medical arena but centred on the importance of bacteria using these
pathways causing meat to degenerate. Organisms that had demonstrated the production of
nitro-oxygen compounds included Achromobacter cycloclastes, Alcaligenes faecalds, and
Bacillus halodenitrificans (Godden, Turley et al. 1991).
The suggestion that the use of NO in this way could occur within living animals came from
the demonstration that germ free rats excreted more nitrate than they ingested (Green,
Tannenbaum et al. 1981). This was also found in humans in a stable state but further
investigations showed that the amounts increased dramatically during intercurrent infection
(Green, Ruiz de Luzuriaga et al. l98l). Other researchers confirmed this by injecting rats and
mice with pro-inflammatory or infectious agents and showing that there was an increase in the
amount of nitrates that were excreted (Wagner, Young et al. 1983, Stuehr, 1985 #580). In part
this was described by using macrophages from these rats in vitro and showing that these cells
could produce nitrite and nitrate by oxidizing L-arginine (Iyengar, Stuehr et al. 1987) to yield
citrulline and a compound with the ability to react with amines and generate nitrosamines
(Miwa, Stuehr et al. 1987). It was thought that a compound with such ability had to be an
60

oxide of nitrogen which was more reactive than nitrite or nitrate. It was also shown that
macrophages used an oxidative form of injury associated with iron loss and involving
inhibition of iron-sulphur enzymes to inflict damage on tumour cells and fungi. L-arginine
was then identified as the necessary metabolite in the extracellular medium to sustain this
form of macrophage-mediated cytotoxicity (Hibbs 1991). Substituting L-arginine analogs
blocked the cytotoxicity in a stereospecific manner and blocked the production of nitrite by
activated macrophages. Nitrite or nitrate substances alone could not replicate the cytotoxicity
of the nitrite-producing macrophage except when in an acidic pH - a condition coincidentally
in which nitrite can generate NO (Hibbs, Vavrin et al, 1987; Stuehr, Gross et al. l9g9). NO
(rather than other nitro-oxygen species) specifically mimicked the pattern of macrophage-
mediated cytotoxicity and scavengers of NO blocked the cytostatic effect of macrophages.
Funher investigation showed that this cytostatic effect used by the macrophages was also
operational against yeasts, helminths, protozoa and mycobacteria (Nathan and Hibbs 1991).
2.3.3 Nitric oxide -
a common pathway
These two separate areas of investigation into immune function and vasomotor control came
together in 1989, when Stuler showed that the compound secreted by activated alveolar
macrophages with L-Arginine availability, demonstrated the same bioactivity as that
described for EDRF and coincided with the chemical reactivity profile of NO (Stuehr, Gross
et al. 1989).
When these two major discoveries came together, it suggested NO synthesis from L-arginine
might in fact be a widespread pathway for the regulation of cell function and communication.
NO has the lowest molecular weight of any known bioactive mediator that cells produce. The
molecule's chemical reactivity means that its half life is short and interaction specificity is
minimal. Therefore, it was surprising that such a simple, fleeting, indiscriminating reactant
could convey enough information in a regulatory manner to help control vital mechanisms
such as vascular tone and neurotransmission (see below Section 2.3.4).It was also surprising
that a mediator involved in homeostasis, was also involved in host defence to destroy micro-
organisms and tumour cells (Nathan 1992). Following the demonstration that both
endothelium cells and macrophages were able to synthesis this new mediator, there was an
explosion of research demonstrating that many other cells were able to produce NO. This
molecule was shown to have physiological roles in many neuronal tissues; including the
cerebellum, the cerebral cortex and the hypothalamus, as well as in ganglion cells of the
autonomic nervous system (Garthwaite, Charles et al. 1988; Bredt and Snyder 1990;
6t

Garthwaite l99l), and the non adrenergic, non cholinergic (NANC) peripheral nerves (Bredt,
Hwang et al. 1990; Rand 1992).
NO was subsequently found, with likely mediator roles, in the renal cortex epithelial cells,
adrenal glands, gut, genital system and has also been found in amniotic fluid (Nathan 1992).
Thus NO was realised to be an important gas used in biological systems in many species
including humans, and within humans NO was seen in a wide variety of tissues as a gaseous
mediator. The type of roles it fulfils in three of these key systems (cardiovascular,
neurological and immunity) will be discussed briefly below, while the role of NO in the lung
is discussed in more depth in Chapter 4,preparatory to measuring levels from the airway.
2.3.4 Nitic oxide in physiological roles
2.3.4(i) Cardiovascular
NO is produced by nitric oxide synthases (NOS) which generate NO from the semi-essential
amino acid L-arginine and release picomolar amounts of NO in response to receptor
stimulation (see Chapter 3 and 4 for detail on chemical reactions and pathways). In this way
NO is responsible for the basal vasomotor tone and essential for the regulation of blood flow
and blood pressure. NO dependent vasodilator tone is in part maintained through the release
of NO in response to physical activation of endothelium cells by stimuli such pulsatile flow
and shear stress. This role was in part determined by the studies described above before NO
was so-named, and in part been determined subsequently by the use of the non-functioning
competitive inhibitor of this enzyme, L-NMMA. A potent vasoconstrictor, L-NMMA
constricts vascular beds and produces a hypertensive response in animals with high potency
@lsner, Muntze et al. 1992) and has been shown to cause vasoconstriction in the forearm
circulation in humans (Gardiner, Compton et al. 1990). As documented below, NO is also
released by non-adrenergic non-cholinergic (NANC) nerve terminals and this may also
contribute to the regulation of blood flow and pressure. The success of compounds used in
angina and hypertension such as nitro glycerine and sodium nitroprusside are now known to
be because these compounds imitate endogenous nitro-vasodilator compounds by relaxing
blood vessels @oel, Godber et al. 2000).
NO also inhibits platelet aggregation and adhesion. This occurs with the increase in cGMP
activity within platelets and subsequent phosphorylation of proteins that regulate platelet
activation (Radomski, Palmer et al. 1987; Radomski, Palmer et al. 1987). It can also be
generated either by the endothelium or the platelets themselves generating NO to act as a
62

negative feedback mechanism to inhibit platelet activation (Mehta, Chen et al. 1995). NO is
also involved in the reaction of leukocytes with vessels walls, again inhibiting activation. NO
inhibits vascular smooth muscle cell hyperplasia which may reduce the development of
pulmonary hypertension (Papapetropoulos, Garcia-Cardena et al. 1997; Ziche, parenti et al.
1997). Endothelial dependant relaxation demonstrated in human vascular tissue is greater in
the arteries than the veins, suggesting that arteries may generate more NO. This relaxation is
inhibited in a number of patient groups with pulmonary vascular disease. The response is
decreased in atherosclerotic coronary arteries compared with normal coronary arteries
(Egashira, Suzuki et al. 1995), in children and adult patients with hypercholesterolemia
(Quyyumi, Mulcahy et al. 1997), and in patients with essential hypertension (panza,Garcia et
al. 1995). It is also Iower in the vessels of diabetic animals (Sobrevia, Nadal et al. 1996) and
in pulmonary arteries obtained from patients undergoing heart lung transplantation @usting
and Macdonald 1995). NO dysfunction has also been noted as contributing to hean failure
(Hanssen, Brunini et al. 1998; Sharma, Coats et al. 2000), and to the pathogenesis of sickle
cell disease @nwonwu, Xu et al. 1990; Waugh, Daeschner et al. 2001). Finally, it has been
shown when solutions of L-arginine (substrate for NO generation) are exposed to cigarette
smoke, there is a depletion of L-arginine and the formation of cyanomethyl-L-arginine and
this is likely to act as a competitive inhibitor and has been shown to have an inhibitory effect
on NOS. This may contribute to the vascular compromise seen in long term smokers (Wong
2000).
NO is also associated with significant pathology within the vascular system in regards to
sepsis (Vallance and Moncada 1993).In endotoxin shock in animals, the generation of NO is
directly related to the degree of systemic hypotension. This is also seen as a characteristic of
septic shock in humans and it is also suggested as being responsible for the hypotension
induced by chemo/radiation therapy seen in patients with cancer who undergo such treatment.
Endotoxin also induces NOS production and activity in venous smooth muscle cells and
increased NO here may play a role in the hypotension seen with endotoxaemia. Similarly
endotoxin induces NOS production in the myocardium, which may contribute to the dilated
cardiomyopathy development. There have been trials using inhibitors of No to reverse the
hypotension which has been induced in animal models by lipopolysaccharide or endotoxin via
TNF0. However the level of inhibition of NOS is crucial for outcome, since high doses cause
severe vasoconstriction and end organ damage in multiple organs and thus causes rapid death.
With titration being the key factor and difficult to control in experiments to date without
63

significant complication, this treatment has not yet been able to become part of standard
clinical management strategies (Petros, Lamb et al. 1994; Grover, Zaccardelli et al. 1999).
2.3.4 (ii) Nervous system - central and peripheral
NO has become accepted as a non-endogenous messenger, signal mediator and non classical
neurotransmitter with NO synthases (see Chapter 3.4 'Nitric oxide synthase isoenzymes')
detected in varying amounts in all areas of both animal and human brain @redt and Snyder
1990). NO synthases are highly similar to NADPH diaphosphorase which is found in about
2Vo of the neurons in the cerebral cortex @astian and Hibbs 1994), and the two enzymes are
closely colocalised in both the brain and the peripheral nervous system. Glutamate is the
main excitory neurotransmitter and an interaction between this and NO was demonstrated
early (Garthwaite, Charles et al. 1988). After specific receptor stimulation, NO is released
from a postsynaptic source to act at a presynaptic region on one or more neurons in any
direction. This results in an increase in glutamate and a stable increase in synaptic
transmission, a phenomenon known as long-term potentiation @liss and Collingndge 1993),
which is linked to memory formation (ODell, Hawkins et al. 1991). Experiments in animals
have shown that inhibiting NO synthesis by analogue competitive inhibitors impairs learning
behaviour (Son, Hawkins et al. 1996). NO has also been shown to have a role in feeding
behaviour, nocioception and olfaction (Bagetta, Iannone et al. 1993). An increasing body of
work has continued to demonstrate the importance of NO in both short and long term memory
(Susswein, Katzoff et al. 2004). In addition, the NO produced by the perivascular nerves of
the cerebral arteries directly modulates vascular control (Gonzalez, Barroso et al. 1997). NO
may also have a role in modulating pain (Moore, Babbedge et al. 1993).
At low concentrations, NO plays a role in vasodilatation and neurotransmission, however at
higher concentrations it can be neurotoxic. It also may be that NO can possess either
neurodestructive or neuroprotective properties depending on its oxidation reduction status
(see Chapter 3 'Reactions with nitric oxide'). Microglial cells, which are of monocyte-
macrophage derivation, can express the form of NOS which gives very high levels of NO (see
Chapter 3: inducible NOS) within the central nervous system. These cells are implicated in
the pathogenesis of neurodegenerative disease with excess generation of NO and high
glutamate levels acting via receptors shown to mediate cell death in focal ischemia, Krabbe's
disease, Huntington's disease and Alzheimer's disease (Choi 1988; Meldrum and Garthwaite
1990; Snyder L993; Vodovotz, Lucia et al. 1996; Dawson and Dawson 1998; Akiyama,
Barger et al. 2000). For example, NOS inhibitors blocked ischemic damage following middle
64

cerebral artery ligation in several animal models (Dawson, Dawson et al. 1992; Yun, Dawson
et al. 1997) and the NOS knock out mice had smaller infarcts after cerebral ischaemia
(Iadecola 1997). The neurons which actually have high levels of both the NOS and the
NADPH enzymes appear resistant to a variety of toxic insults including Huntington's disease,
Alzheimer's disease and vascular stroke (Ferrante, Kowall et al. 1985; Koh, peters et al.
1986). These neurons are also rich in manganese super oxide dismutase, and if this
contributes to the neuroprotection, it may be similar to the reason for the variation of the half
life initially given in the cardiovascular experiments reported above -
i.e. that it depends on
the extent of super oxide anion availability. Excess NO levels have also been implicated in
demyelinating conditions such as multiple sclerosis (Parkinson, Mitrovic et al. t997) and
periventricular leukomalacia (Koprowski , Zheng et al. 1993). There is evidence showing that
the production of NO is significantly raised within the multiple sclerosis lesions, but also in
the cerebral spinal fluid, blood, and urine of these patients (Lin, Lin et al. 1993). In addition,
the lack of ability to generate NO is important, such as seen in patients with Duchenne
Muscular Dystrophy in whom skeletal muscle lack expression of the neuronal NOS
(Brenman, Chao et al. 1995).
As mentioned, NO is also found in peripheral nerves where it contributes to sensory
transmission and is a transmitter and/or modulator in non-adrenergic non-cholinergic (NANC)
neryes, although there is considerable variation depending on both experimental conditions
and the different species in which the experiments are carried out. The NANC nerves cause
vasodilatation and relaxation of animal and human tracheal muscle via NO release, and this is
not inhibited by NOS inhibitors (Stuart-Smith, Bynoe et al. 1994: Baba, yoshida et al. 199g;
Sipahi, Ercan et al. 1998). In human airway specimens, the NANC NO mediator relaxation is
more important in the distal airways (Belvisi, Stretton et al. 1992: Ellis and Undem lgg2).
This response is reduced in recipient transplanted human bronchial tissue and in specimens
from patients with CF (Stretton, Mak et al. 1990).
In the gastrointestinal tract, NO seems to mediate relaxation including dilatation of the
stomach, the sigmoid colon and the internal anal sphincter in humans. Selectively blocking
the NANC mediated relaxation of the gastrointestinal tract in mice resulted in marked
stomach enlargement and inner circular gut muscle layer hypertrophy, thought to be
compensatory for the inability of the pyloric sphincter to relax (Huang, Dawson et al. 1993;
Bredt 1999). Histochemical studies of biopsy specimens from infants with hypertrophic
pyloric stenosis and biopsy studies from adults with cardiac aplasia seem to confirm this with
reduced NOS enzyme demonstrated (Vanderwinden, Mailleux et al. 1992). Also the gene
65

locus for the enzyme is a susceptibility locus for infantile pyloric stenosis (Chung, Curtis et
al. 1996).
NO also has many roles in the reproductive system (Rosselli, Keller et al. 1998), an example
being a reduction of NO being associated with pre-eclampsia and premature delivery. NO
functions as the NANC transmitter which leads to relaxation of the corpus cavernosum and
thus the development of penile erections with its effect able to be blocked in animal studies
(Burnett, Lowenstein et al. 1992). A local reduction in NO is associated with decreased
bladder capacity and hyperactivity (Burnett, Calvin et al. 1997) with knock-out mice
displaying hypertrophied urinary bladders and loss of neurally mediated relaxation of urethral
and bladder muscle, which provides a model for voiding disorders in humans. Studies in
animals show that there is increased expression of NOS during pregnancy with increased
urinary excretion of nitrite and nitrate (Conrad, Joffe et al. 1993; Conrad, Vill et al. 1993).
The vasodilatation and decrease in blood pressure during pregnancy is likely to be also due to
increased NO secretion with uterine NO synthesis preventing myometrial contraction.
2.3.4 (iii) Host defence
That NO was used in host defence was discovered in one of the original areas of research that
delineated the importance of NO. The use of NO may have originated in evolution as a first
line defence against invading micro-organisms (Hibbs 1991; Bogdan 2001). Resistance to
tumour cells and cancer development was shown to be enhanced in a non-specific way by
bacterial products (Nathan t992), Activated macrophages were demonstrated to synthesise
nitrite and nitrate, with generation dependant on L-arginine and inhibited by analogues of L-
arginine (Hibbs, Vavrin et al. 1987). The generation of NO is now a known feature of many
immune cells (dendritic cells, NK cells, mast cells, monocytes, macrophages, microglial cells,
Kupffer cells, eosinophils and neutrophils) as well as other cells involved in the immune
reaction (such as endothelial cells, epithelial cells, vascular smooth muscle cells, fibroblasts,
keratinocytes, chondrocytes, hepatocytes, mesangial cells and Schwann cells), (Bogdan
2001). The NO produced in these cells comes from the inducible form of NOS having the
ability to generate much larger amounts of NO than the other enzyme types; the local
concentration of NO synthesis is an important determinant of cytotoxicity. NO is a ubiquitous
pathogen-killing agent and it is truly a generalist with a non specific response to infection. It
can be toxic to many kinds of pathogen including viruses, bacteria and parasites (both
intracellular and extracellular) as well as some metazoan parasites (James 1995; Bogdan
2O0L; Colasanti, Gradoni et al. 2002). NO mediated killing of bacteria is thought to have been
66

developed as a two step process. Firstly, interferon garnma (IFy) and TNFa activate
macrophages and promote NOS synthesis of NO. The definitive second step is the respiratory
burst during phagocytosis. It could be an adaptive host defence mechanism in humans. For
example, in African children a mutation in the promoter gene seems to be associated with
increased NO production which appears to provide significant protection against malaria
(Hobbs, Udhayakumar et al. 2002).
NO also regulates lymphocye function and may have a role in inhibiting subsets of T helper
cells. However most of the normal host cells are susceptible to necrosis or apoptosis from the
inhibition of mitochondrial enzymes and DNA damage caused by this molecule, particularly
if the cells producing NO are over stimulated. NO is not restricted to a single defined receptor
but can act widely. It is present in both acute and chronic inflammation. In these areas NO is
likely to have a number of roles ranging from enhanced vasodilatation, the formation of the
oedema, modulation of sensory nerve endings and increased leukocyte activity that possibly
induces higher NO levels to be produced as a reaction to organisms and therefore causes
increased tissue cytotoxicity. No in respiratory inflammation will be elaborated on in
Chapters 3,4 and 5.
There are many other examples. Increased NO production was noted in inflammatory disease
(Boughton-Smith, Evans et al. 1993; Tran, Visser et al. 1993; Alican and Kubes 1996;
Lrvine, Pettei et al. 1998), particularly in ulcerative colitis (Lundberg, Hellstrom et al. tgg4)
though less convincingly for Crohns disease (Rachmilewitz, Stamler et al. 1995). This has
been confirmed in animal models with Macaque monkeys (Ribbon s, Zhang et al. 1995),
guinea pigs (Miller, Thompson et al. 1995) and rats (Kankuri, Vaali et al. 2001). Increased
urinary nitrite is higher at times of disease exacerbation in humans (Goggins, Shah et al.
2001). Inhibitors have been shown to ameliorate induced chronic ileitis in animal models.
There are also increased nitrite concentrations in plasma and synovial fluid in patients with
rheumatoid arthritis, osteoarthritis, (Stefanovic-Racic, Stadler et al. 1993; Amin, Di Cesare et
al' 1995; St Clair, Wilkinson et al. 1996: Stichtenoth and Frolich l99g), type I diabetes
@izirik, Flodstrom et al. 1996), giant cell arteritis, systemic lupus erythematosis (SLE),
spondolarthopathy and Sjogrens syndrome (Belmont, Irvartovsky et al. 1997; Strand 1997:
Strand, kone et al. 1998). In SLE, for example, serum nitrite levels correlated with level of
antibodies to double stranded deoxyribonucleic acid @NA) and to symptom scores although
the highest correlation was between serum nitrite and renal disease. Biopsies in non-lesional
skin showed there were increases NOS expression during periods of active SLE (Belmont,
Levartovsky et al. 1997).In animal models of arthritis, a competitive NOS inhibitor blocked
67

NO synthesis and in so doing blocked paw swelling and histopathological changes in the joint
(Stefanovic-Racic, Meyers et al. 1994).
Sepsis is a special picture. Sepsis begins with the exposure to an infectious agent which
induces a cascade of events that initially tries to compensate for the problem with tachycardia,
peripheral vasoconstriction, fever (usually) and increased polymorphonuclear cells. This is
followed by progressive vasodilatation with a high cardiac output and decreased vascular
resistance resulting in hypovolaemic shock and decreased venous return to the heart. This can
progress to a state which is resistant to vasopressor agents and is often combined with a pro-
coagulant state. Increased levels of serum nitrates were found to be correlated with the degree
of systemic vasodilatation (Ochoa, Udekwu et al. 1991; Yoshizumi, Perrella et al. 1993). In
animal studies, NOS inhibitors were used to treat induced sepsis but this was far from
universally successful. The studies did not show improved haemodynamic parameters such as
mean arterial pressure, but there was a further derangement of local blood flow @ooke,
Meyer et al. 1995) resulting in worsened renal (Schwartz, Mendonca et al. 1997) and liver
impairment (Gundersen, Corso et al. 1997) and worsened inflammation generally (Aaron,
Valenza et al. 1998). The timing and dosage were found to be critical, as their use
preventatively or early in shock resulted in worse outcomes (Cohen, Huberfeld et al. 1996;
Strand, Leone et al. 1998). In one study of 12 patients with severe sepsis and hypotension,
low doses of L-NMMA (NOS inhibitor) did result in an increase in pulmonary vascular
resistance but also led to a decrease in cardiac output causing concern that this would result in
poorer tissue perfusion @etros, Lamb et al. 1994). In a further pilot study involving 32
patients with septic shock, the infusion of L-NMMA also resulted in an increase in vascular
tone and a decrease in cardiac index within the first hour of therapy. The infusion continued
for up to eight hours and mean arterial pressure was sustained with a 6O-8OVo reduction of
noradrenaline use (Grover, Zaccardelli et al. 1999). However a much larger multi-centre trial
was then undertaken enrolling 797 patients with septic shock allocated to receive the NOS
inhibitor or placebo for up to 7 days or 14 days in addition to standard therapy. It was
terminated early because of a trend to higher mortality in the treated group by day 28 from
multiple organ failure (Serrano, Casas et al.2004).
2.4
Chapter summary
The idea for doing research into NO in the lung in human adult subjects; in healthy subjects
and in those with respiratory disease, in particular asthma, came at a time when the areas of
environmental pollution and mediator research were rapidly developing. The first area
68

included the expanding knowledge and measurement of airway pollution and the connection
between higher or longer duration of daily exposure and increased cardiovascular and
respiratory mortality and morbidity. Through the 1990s increasing effort was made to clarify
the effects of the different pollutant components. Particulate matter, especially respirable
particles at less than l0 microns in diameter, and ozone have been consistently associated
with excess disease and mortality. However the nitrogen oxides have also had associations
with increased respiratory disease (bronchitis, COPD, pneumonia and asthma) and for more
episodes of wheeze with increased symptoms and lower lung function in adults and children
with asthma.
The second area led from the startling discovery of the gas NO as a ubiquitous, biological
messenger acting as a physiological mediator, a neurotransmitter and a mechanism of host
defence. Since this was realised, research into NO in every biological system flourished.
However there were a number of hurdles to overcome in measuring this molecule. Measuring
NO was going to be difficult given its gaseous, short lived and highly reactive nature, and
there were also the difficulties of measuring levels in exhaled air from human subjects.
In 1992 NO was named Science Magazine's "Molecule of the Year". In 1998 Robert F.
Furchgott, Louis J. Ignano and Ferid Murad were winners of the 1998 Nobel prize in
Medicine for their research in identifying firstly EDRF, and then renaming and identifying
NO as the mediator in vascular epithelium.
The following chapter will discuss the synthesis and control of NO production in detail, the
interactions of NO with other chemical and biological compounds, and the synthesis and
control of the nitric oxide synthase enzymes that produce it. This knowledge is preparatory to
discussing how to measure No concentrations in the respiratory system.
69

3.1
Chapter 3: The synthesis, reactivity and control of nitric oxide
Introduction
The previous chapter detailed the two areas where nitrogen compounds were being
increasingly recognised for their importance; air pollution and biological systems. In air
pollution the nitrogen oxides were associated with excess mortality, in particular respiratory
and cardiovascular morbidity; and in the biological systems, NO was found to be a
widespread fundamental messenger. In this chapter, I will detail how NO is produced and
cover its highly reactive and interactive properties. I will discuss the nitric oxide synthase
(NOS) enzymes and how they are controlled in the physiological roles and the host defence
roles of NO. The properties of NO needed to be understood when preparing to design
experiments to measure it in vivo.
3.2 Properties of nitric oxide
Figure 3.1: The molecular structure of NO
ao OO
oN=O..
NO is a clear, colourless gas. It is a free radical with an unpaired electron (. N=O, abbreviated
NO, see Figure 3.1) and therefore it is highly reactive with a half life of seconds and readily
combines with other free radicals (Beckman and Crow 1993). However it does not self react,
possibly because its bond length is intermediate between double and triple bond lengths
(Braker and Mossman 1975). Its small size and the fact that it is a relatively non-polar
molecule means that it moves readily through hydrophobic lipid membranes (Shaw and
Vosper 1977 Malinski, Taha et al. 1993; Liu, Miller et al. 1998). Its reactivity means that it
binds quickly with transition metals such as iron, copper, cobalt, or manganese that are central
ions to many cytochromes and oxidases, and in the case of iron, haemoglobulin. NO can be an
oxidant or a reducing agent depending on the 'redox' environment. NO is soluble in water up
to 2 millimoles per litre at 200C in one atmosphere, but has a high partition coefficient so it
usually exists as a gas. NO reacts rapidly with oxygen in air producing nitrogen dioxide (NOz)
(Vallance and Collier 1994). NO is very soluble in lipid and water and is therefore fully
diffusible in the environment of the cell (Archer 1993; Henry, Lepoivre et al. 1993).
70

Table 3.1: Properties of nitric oxide
Molecular weight 30.006 (1 mol= 0.030006 kg)
N...O bond distance 1.1508 angstroms
Partition coefficient approximately 20
Saturated NO solution approximately 3mM
Oxidation products Nitrite, nitrate
Dissociation t lze Hb-NO approximately 3 hours
Absolute density 101.325 kPa at 25oC
Solubility in H2O at OoC
Solubility in HzO at 2OoC
Solubility in H2O at 6OoC
7.34 ml/100m1
4.6 m/100m1
2.37 m/100m1
This table is modified from Archer S. Measurement of nitric oxide in biological models FASEB Journal.
1 993; 7(2): 349-60 (Archer 1 99s).
NO is generated by the stereospecific enzyme NOS which acts on L-arginine cleaving the
terminal guanidino nitrogen to produce NO and L-citrulline. This is accomplished via five
separate steps and requires 3 co-factors [tetrahydrobiopterin (BH4) and flavoproteins; flavin
adenosine dinucleotide (FAD), flavin mononucleotide (FMN)], and 2 co-substrates [oxygen
and nicotinamide adenosine dinucleotide phosphate (NADPH)I in the presence of calmodulin
and calcium (Kwon, Nathan et al. 1990; Stuehr, Kwon et al. 1991; Barnes and Belvisi 1993:
Knowles and Moncada 1994). L-arginine is a semi-essential non-aromatic amino acid and is
present in nuts (especially brazils and almonds), shellfish, and meat (bacon, beef and game)
(Vallance and Collier 1994). The necessary elements are demonstrated in figure 3.2.
Figure 3.2: The reaction to generate nitric oxide
t{Tglnlna-...+
l+ttrullino
The formation of No by No synthase involves the conversion of L-arginine to L-citrulline with several
co-facto.rs including the flavones and the tetrahydrobiopterin. There are a number of known
competitive
-nitrite,
inhibitors for the NO synthase enzym-es. NCj itselt can oxidiseO to nitrate or
perioxyitrite.
7l

BHa = tetrahyrobiopterin, FAD = flavin adenosine dinucleotideA FMN = flavin mononucleotide, H2O =
water, L-NAME= = No-nitroarginine methyl ester, L-NMMA = N"-mono-methyl-L-arginine, L-NOARG =
No-nitroarginine NADPH = nicotinamide adenosine nucleotide phosphate, NO = nitric oxide, NO2- =
nitrite, NOi = nitrate NO synthase = nitric oxide synthase, Oe = oXYgell, ONOO'= peroxynitrate,
Taken from Barnes PJ, Belvisi MG. Nitric oxide and lung disease. Thorax 1993; 48: 1034-1043
(Barnes and Belvisi 1993).
Table 3.2: Reactions with nitric oxide
Once NO is formed, there are a number of possible pathways of reactions(Nathan 1992; Stamler,
Jaraki et al. 1992; Stamler, Simon et al. 1992; Henry, Lepoivre et al. 1993; Anggard 1994; Gaston,
Drazen et al. 1994; Vallance and Collier 1994; Eiserich, Patel et al. 1998):
1. Redox reactions in the presence of Oz:
(a) To create reactive nitrogen species
(b) To create superoxides:
+ nitrate (NOz-)
2. (a) Reactions with transition metals: cobalt (Co)
--+ nitrate (NOg-)
+ nitrous acid (HNO2)
+ nitrogen dioxide (NO2)
+ perioxynitrite ONOO-
+ p€roxlnitrous acid (ONOOH)
(both strong oxidising agents)
copper (Cu)
iron (Fe)
manganese (Mn)
zinc (Zn)
(b) Reactions with haem and nonhaem metalloproteins:
ascorbate oxidase
catalase
ceruloplasmin
cytochrome c
haemoglobin
lipo-oxgenase
myoglobin
succinate dehydrogenase
tyronase
3. (a) Interactions with nitrogen thiols and amines:
-+ nitrosothiols
+ nitrosamines
--+ sulphur thiols
(b) lnteractions with sulphur:
4. Reactions with amino acid
5. Reactions with liPids
'These compounds are more often designated as Nw than NG and the former is used in the text but the latter was
used in this diagram.
72

3.3 Reactions of nitric oxide
The reaction of NO at any given time is dependent on a number of factors; the rate of reaction
and therefore the concentrations of the species with which NO reacts the fastest, the
concentration of NO, the reduction-oxidation (redox) environment in which it is found and
therefore the presence and concentration of Oz, the availability of other interacting
compounds and the pH (see Table 3.2). As well as local reactions, it is now believed that the
interaction between NO and thiols and amines may be a way of stabilising NO in a bioactive
form, potentially facilitating NO transport in tissue (Gaston, Drazen et al. 1994). I will review
the different pathways briefly below.
3.3.1 Reactive nitrogen species and superoxide reactions
High levels of NO exposure can cause significant damage. It can damage all classes of
macromolecules including DNA. It can destroy mitochondrial enzymes, prevent DNA
synthesis, and inhibit protein synthesis (Hibbs, Taintor et al. 1988; Curran, Ferrari et al. l99l;
Kwon, Stuehr et al. 1991; Stadler, Billiar et al. 1991; Lancaster 1992; Irpoivre, Flaman et al.
r9e2).
However it has become increasingly obvious that reactions with NO in the presence of Oz
forming NO derived reactive nitrogen species are equally important in mediating toxic injury.
These compounds include nitrite (Noz-), nitrate (No:-), No2, nitrous acid (HNoz) or
dinitrogen trioxide (NzOa). It also interacts with Oz to produce superoxides such as
perioxynitrite (ONOO-) and peroxynitrous acid (ONOOH), both strong oxidising agents.
These can continue to react with all classes of biomolecules including lipids, DNA, thiols,
amino acids and metals leading to the two mechanisms of damage which are oxidation and
nitration @iserich, Patel et al. 1998).
NOz is produced by the reaction of NO with Oz and oxidation of NOz to NOr. 6,
myeloperoxidase (Ignarro, Fukuto et al. 1993). This reaction will therefore occur in areas of
high macrophage numbers and high 02 concentration and therefore is commonly seen in the
Iung. The exposure of human plasma to NOz causes rapid loss of ascorbate, uric acid, c-
tocopherol, bilirubin, and protein thiols as well as increasing lipid peroxidation (Halliwell, Hu
et al. 1992). Exposure of lung lining to NOz leads to loss of ascorbic acid, uric acid. and
glutathione (Postlethwait, Langford et al. 1995: Kelly and Terley lggT). Under acidic
conditions NO2 becomes protonated to form HNO2. At least two biological compartments
experience pH low enough to allow HNO2 formation in vivo; the stomach with a pH 2.5-4.5
73

(Knowles, McWeeny et al. 1974) and within neutrophil phagocytes with a pH 3.0-6.5 (Cech
and Lehrer 1984). This makes the production of this acid possible in high inflammatory
conditions and after dietary intake of certain nutriments such as smoked and cured foods.
High levels of both NO and NOz during inflammation in the presence of the inducible
nitrogen synthase enzyme (see below) forms dinitrogen trioxde (NzOs). Both NOz- and NOt-
are moderately stable and can be released on tissue contact (Moro, Darley-Usmar et al. t994;
White, Moellering et al. 1997). NO can undergo electron oxidation and reduction reactions
generating nitrosonium cation (NO+) and nitroxyl anion (NO-) and these can participate in
numerous other redox reactions. NO can also regulate apoptotic signals through formation of
reactive NO species (Tamir, Irwis et al. 1993; Kim, Talanian et al. 1997; Nakano, Terato et
aI.2003).
Perioxynitrite anion (ONOO) and peroxynitrous acid (ONOOII) are oxidants which can also
react with numerous targets. The formation requires NO and Oz- anion produced by many cell
types - neutrophils, macrophages, smooth muscle cells endothelial cells and fibroblasts
(Ischiropoulos, Zhu et al. 1992; Kooy and Royall 1994; Boota, Zar et al. 1996; Thom, Xu et
al. 1997). They interact with glucose, fructose, and mannitol (White, Moellering et al. 1997;
Skinner, White et al. 1998), and DNA bases. They hydroxylate aromatic amino acids (such as
tyrosine, tryptophan, phenylalanine), and oxidise thiols and lipids @eckman, Ischiropoulos et
al. t992; Alvarez, Rubbo et al. 1996; Beckman 1996). Perioxynitrite can directly inhibit
oxidation reactions by chain terminating lipid peroxyl and alkoxyl radicals or through
regulation of cell signalling pathways that lead to induction of antioxidant enzymes (@ubbo,
Radi et al. L994; ODonnell, Chumley et al. 1997; Moellering, McAndrew et al. 1998). It can
nitrate both free and protein bound tyrosine residues to give a 3-nitrotyrosine compound
found in high levels in many inflammatory conditions (see below) (van der Vliet, O'Neill et
al. 1994; Alvarez, Rubbo et al. 1996). Addition of ONOO to red blood cells results in
methaemoglobin formation. ONOO also reacts with iron-sulphur enzymes and can lead to
inactivation (Castro, Rodriguez et al. 1994; Bouton, Hirling et al. 1997).
While responsible for many toxic reactions - it is possible that this also provides a mechanism
for removal as the ONOO compound isomerises into NOs- and NOz- at neutral pH (Irwis,
Tamir et al. 1995; Munzel, Sayegh et al. 1995; Pfeiffer, Gorren et al. 1997). It is also possible
that the ONOO- reaction with COz which then degrades which may also be a protective
mechanism (Lymar and Hurst 1996; Uppu and Pryor 1996).
74

3.3.2 Reactions with transition metals and metalloproteins
The biological chemistry of NO is strongly mediated through reaction with transition metals.
The reaction is with catalytic metal centres such as iron (Fe) in both haem and nonhaem
proteins (Tsai 1994). The high affinity of NO for the ferrous iron (Fe2+ in the reduced state) in
haemoglobin leads to a key interaction (Sharma, Traylor et al. 1987; Eich, Li et al. 1996). In
vivo, this reaction of NO and oxyhaemoglobin to form methaemoglobin and nitrate (NOl)
represents the major pathway for scavenging the endogenous NO production and is a
significant route of NO removal (Lancaster, Langrehr et al. lgg2). While this reaction is
rapid, in fact it would be too rapid to allow NO to interact with the subendothelial layer in the
vascular compartment (Lancaster, Langrehr et al. 1992). However, it is now known that when
the haemoglobin is in erythrocytes, the reaction with NO is limited by diffusion into the cell
and the half life is increased (Liu, Miller et al. 1998). Studies have shown that 70Vo ofthe NO
in humans is recovered as NO3- excreted through the urine (Westfelt, Benthin et al. 1995).
Oxyhaemoglobin can also directly oxidase NOz and NO3 forming methaemoglobin.
NO reaction with other Fe containing proteins can lead to either activation or inactivation
depending on the protein. For example, the reaction with soluble guanylyl cyclase (sGC) leads
to a conformational change which results in up to a 300 times increase in activation of the
enzyme. Activation of sCG results in the formation of cyclic quanosine monophosphate
(cGMP) from guanosine 5-triphosphate (GTp) (Ignarro, Degnan et al. l9g2) and accounts for
major signal transduction activity of NO. It is likely to be this mechanism that is the primary
one mediating vessel relaxation and the inhibition of platelet aggregation (Moncada and
Higgs l99l),In contrast, formation of a nitrosyl complex with cytochrome c oxidase leads to
reversible inhibition which is competitive with 02 binding @iserich, Patel et al. 1998).
NO also interacts with other metals such as zinc and copper. Formation of nitrosyl
compounds with co-ordinating cysteine residues results in release of zinc (Kroncke, Fehsel et
al. 1994). Formation of nitrosyl compounds with copper also occurs, and is particularly
important in copper containing enzymes such as the cytochrome c oxidase (Radi 1996). It is
through this type of interaction with metal ion containing proteins (metalloproteins) that NO
effects the enzymes of mitochondrial respiration and this is also a mechanism of the
macrophage mediated defence and involves, for example, cytochrome c oxidase and catalases
(Cleeter, Cooper et al. 1994; Torres, Darley-Usmar et al. 1995). Part of this is an NO and Oz
competition at the cytochrome c oxidase binding site for 02 and therefore NO inhibition is
75

more potent at low oxygen tensions (Brown and Cooper 1994). Again the most dramatic
effects will occur in high levels of NO seen in inflammation.
There are also regulatory proteins in which the interaction of NO can modulate translation of
mRNA sequences which contain Fe responsive elements such as ferritin and transferrin
receptors @antopoulos, Weiss et al. 1996). In this way NO can also interact to regulate Fe
levels in cells.
3.3.3 Nitrogen thiols and amines
S-nitrosothiols (S-nitrosoglutathione, S-nitrosoalbumin, S-nitrosohaemoglobin as examples)
are stable molecules that can potentially transfer NO+ to other thiols or transport and release
NO in distant areas and in other tissues (Stamler, Simon et al. 1992; Jia, Bonaventura et al.
1996; Funai, Davidson et al. 1997). This provides another mechanism by which NO regulates
protein function. It has long been demonstrated that nitrosothiol compounds are generated
during the curing process of meat with the interaction of nitrite @mi-Miwa, Okitani et al.
1976). Exactly how the NO is released from these compounds and when is unclear; however
these compounds have been suggested to account for some of the NO biological actions
(Stamler 1994). For example, S-nitrosation has been shown to confer vasodilatory properties
on other specific proteins. On the tissue type plasminogen activator enzyme, which dissolves
fibrin in blood clots, s-nitrosation confers vasodilatory and anti-platelet functions (Stamler,
Simon et al. 1992). The effect of shear stress to increase NO may also operate via s-
nitrosation of plasma proteins. The vasodilatation effects of organic nitrates used in treatment
such as nitroglycerin may also occur through the intermediate formation of these compounds
(Fung, Chong et al. 1988). They are therefore potential therapeutic agents for pharmacological
NO delivery @utler, Flitney et al. 1995; Stamler 1995; Upchurch, Welch et al. 1996). S-
nitrosothiols have been detected in the nervous system where they may function as
neurotransmitters and use in animal models mimic NANC actions @arbier and Irfebvre
1994; Liu, Gillespie et al. 1994; Slivka, Chuttani et al. 1994). Several reports have identified
proteins whose activity changes upon S-nitrosation in vitro (Stamler 1994; Stamler, Toone et
al.1997).
These compounds can also allow NO to inhibit cytokine dependent signalling in vascular
endothelial and smooth muscle cells. Both s-nitrosoglutathione and sodium nitroprusside
prevent activation of the transcription factor NF-KB and expression of vascular cell adhesion
molecule-l. Nitrosothiols can also regulate calcium transits and again can alter signal
76

transduction pathways in this manner by altering ion homeostasis (Stoyanovsky, Murphy et
al.1997; Xu, Eu et al. 1998).
3.3.4 Reaction with amino acids
The importance of the interaction of NO species with amino acids is exemplified by the
reaction with tyrosine to form 3-nitrotyrosine. This reaction can severely compromise
function of proteins with tyrosine residues in the active site, disrupting structure and activity
@iserich, Patel et al. 1998). This reaction was first described in 1990 (Ohshima, Friesen et al.
1990) and the level of 3-nitrotyrosine is now commonly used diagnostic marker of NO
derived oxidants in both human disease states and animal models (Schmidt, Hofmann et al.
1996). An "overwhelming body of evidence has amassed in the last several years revealing
that NO2Tyr (3-nitrotyrosine) is a reliable footprint of reactive nitrogen species production
that spatially and temporally parallels tissue and cellular injury" @iserich, Patel et al. 1998).
Although a direct relationship between 3-nitrotyrosine formation and pathological outcomes
remains poorly characterised, it is dramatically elevated in diverse diseases such as
atherosclerosis, sepsis, acute and chronic lung disease, neurodegenerative diseases,
inflammatory bowel disease, chronic organ rejection, myocardial inflammation and tobacco
smoking (Ischiropoulos, Zhu et al. 1992; Eiserich, Patel et al. 1998). The compound can be
incorporated into tuberculin which disrupts the cell cytoskeleton @iserich, I{ristova et al.
1998) and can attenuate adrenoreceptor agonists in vivo (Kooy and Royall 1994). The
formation of 3-nitrotyrosine is dependent on increased NO production and frequently
associated with activated phagocytes (neutrophils, monocytes, eosinophils, macrophages). NO
can also interact with amino acid radicals in proteins @iserich, Cross et al. 1996).
3.3.5 Reaction with lipids.
The reactive nitrogen species (NO, NO2, ONOO) also interacts with unsaturated lipids which
leads to either initiation of oxidation, altering the rates and products of lipid oxidation
products or inhibition (see below). For example, the unsaturated free fatty acids arachidonic
acid and linoleate are substrates for the enzymatic synthesis of bioactive medicators such as
prostaglandins and leukotrienes through the activities of lipo-oxygenases, cyclo-oxygenases
and cyochrome Pa56. These play a role in regulation of blood pressure, platelet aggregation,
and bronchosconstriction. Free unsaturated lipids and those esterified to phospholipids are a
significant component to biomembranes pulmonary surfactant and plasma lipoproteins.
Uncontrolled oxidation as can be caused by NO and related species can lead to changes in
integrity and fluidity of biomembranes. The interaction can also lead to the formation of other
77

toxic products such as isoprostanes and reactive aldehydes @iserich, Patel et al. 1998). Lipid
peroxidation is a characteristic feature of nearly all inflammatory diseases (Kuhn, Belkner et
al. 1994; Quinlan, Lamb et al. 1996; Kuhn, Heydeck et al. 1997; Li, Maher et al. 1997).
On the other hand, NO can also serve as a protective factor. There are also lipid derived
radicals both free and membrane bound which contribute to inflammatory conditions by
inducing membrane damage again resulting in alteration of fluidity and integrity, as well as
the production of eicosanoids and eicosonoid-like isomers which possess potent bioactivity.
Direct reaction of NO with radical lipids results in inhibition of lipid oxidation and in this way
may prevent lipid oxidation induced tissue damage (Rubbo, Radi et al. 1994; Gutierrez,
Nieves et al. 1996; Wink, Cook et al. 1996; ODonnell, Chumley et al. 1997).
3.3.6 Reactions with genes
Transcriptional regulation of several genes by NO have been reported. Interestingly, these are
in general anti-inflammatory (Naruse, Shimizu et al. 1994; Pilz, Suhasini et al. 1995);
examples include regulation of adhesion molecules, the haem metabolising enzyme,
haemoxygenase (Foresti, Clark et al.1997) and glutathione synthesis (Moellering, McAndrew
et al. 1998).
3.3.7 Making sense of the reactions
In essence, the reactions that are important in biological systems depend on the amount of NO
and where the NO is formed. The short half life and reactivity means NO is likely to act as a
local messenger molecule, transferring messages within and between individual cells. In
biological systems, when small amounts are formed as a mediator for physiological processes,
the NO preferentially binds to haem, hence in the blood stream most NO is quickly bound to
haemoglobin. From here it rapidly decomposes to yield predominantly nitrate (NOl-) and
some nitrite (NOz') and the compounds are eliminated in the urine with a half life of five to
eight hours. NO will also interact with other heme containing proteins such as enz).mes
particularly sGC which results in a rapid increase in activity and production of cGMP. Excess
NO is mopped up by other nitrosation reactions, for example nitrosothiols and these are now
known to be active in their own right, and a way of stabilising NO in a bioactive form and
potentially facilitating NO transport in tissue.
When released in much larger amounts, it is likely that NO more often reacts with other
elements such as metals, for example copper, iron and zinc proteins releasing free Cu*, Fe*
and Zn** and generating Oz and highly toxic hydroxyl radicals leading to effect massive
78

oxidative injury. High levels of NO disrupt DNA and cause both genotoxic and cytotoxic
damage. If there are changes in the pH, in the thiol content or in the redox state, this can result
in increased damaging and carcinogenic potential. Of interest is the reaction of NO with
amino acids such as tyrosine, which in itself is now used as a marker in several inflammatory
diseases.
Finally, there is now discussion that the original EDRF concept is made up of the actions of
NO and the actions of some of the post NO production of other compounds, in particular the
s-nitrosothiols (Myers, Minor et al. 1990; Vanin l99l; Jia, Bonaventura et al. 1996; Liu,
Miller et al. 1998).
3.4
Nitric oxide synthase isoenzymes
The nitric oxide synthases are large complex proteins that unusually contain both oxidative
and reductive domains. They have homology with cytochrome P+so which is also unique
(Bredt, Hwang et al. l99l; Vallance and Moncada 1994). They are synthases rather than
synthetases as they do not use adenosine triphosphate (ATP) in their reactions (Knowles and
Moncada 1994). NOS needs the substrate (L-arginine), the co-substrates (O2 and NADpI{)
and the co-factors (8H4, FAD, FMN) (see Chapter 3.2 above). The reaction is a five electron
mono-oxygenate oxidation pathway which involves two separate mono-oxygenation steps in
sequence (see Figure 3.2, Knowles, 1994 #712). In step one N*-hydroxyarginine is an
intermediate species formed (Stuehr, Kwon et al. l99l) in the first reaction by aquiring one
02 molecule and one NADPH molecule (Kwon, Nathan et al. 1990) and the presence of BII+
(Kwon, Nathan et al. 1989; Tayeh and Marletta 1989). The second step is the oxidation of N*-
hydroxyarginine to form citrulline and *NO. Flavin coenzymes are involved in the transfer of
electrons to form a reduced oxygen species (Stuehr, Cho et al. 1991; Stuehr, Fasehun et al.
1991). There are consensus binding sites for FAD, FMN and NADPH located in the carboxyl
terminal portion of the NOS protein, and also a consensus binding site for calmodulin. The
other co-factor BFIa binds to NO on a 1:1 stoichiometry basis and has a redox role in enzyme
activity (Hevel and Marletta 1992; Knowles and Moncada 1994). All three isoenzymes are
phosphorylated (Nathan and Xie 1994).
79

Figure 3.3: The nitric oxide synthase reaction showing the generation of the constituent atoms of nitric
oxide
*}^rilfl*t
En,
o.E
l(NADPtl + H'l
I NADT
'tT H
.ArA./"WNH'
tNl
o
The boxed 'O' and 'N' atoms show the constituent atoms of NO - see text for abbreviations. Taken
from Knowles RG, Moncada S. Nitric oxide synthases in mammals. Biochemical Journal 1994; 298
(2):249-255 (Knowles and Moncada 1994).
There are two forms of NOS; the constitutive form (cNOS) and the inducible form (iNOS).
The constitutive form is in turn made up of two types -
the endothelial NOS (eNOS) and the
neuronal NOS (nNOS), although there is only one form of the inducible NOS. They have also
been classified as type I (nNOS), type tI (iNOS) and type III (eNOS) which was the order in
which they were first purified with the first isolation of their DNA (Bredt, Hwang et al. 1990;
Bredt and Snyder 1990; Janssens, Simouchi et al. 1992; I-ammers, Barnes et al. 1992;
Lowenstein and Snyder 1992; Lyons, Orloff et al. 1992; Xie, Cho et al. 1992; Forstermann,
Closs et al. 1994). The homology between the isoforms in humans is between 5l-587o with
approximately 5LVo between nNOS and iNOS, and 54Vo homology between eNOS and iNOS.
(Nathan L992; Chartrain, Geller et al. 1994; Forstermann, Closs et al. 1994; Marsden, Heng et
al. 1994; Nathan and Xie 1994). Across species, the amino acid sequences for each isoform is
well conserved at greater than gOVo for the constitutive forms and greater than 807o for the
inducible forms (Forstermann, Closs et al. 1994). They predominantly operate in the areas for
80

which they are named; eNOS in vascular cells, nNOS in the CNS and peripheral nerves, and
iNOS within the immune cells, although these divisions are not so strict as first thought and
some cells therefore can produce two different types of the enzyme (Nathan 1992). The
enzymes are coded for on differing chromosomes; chromosome 7 for eNOS (Zl-22 kb,26
exons), chromosome l2for nNOS (150kb, 29 exons) and chromosome 17 foriNOS (37kb,26
exons) (Marsden, Heng et al. 1993). The characteristics of the NOS enzymes are listed in
Table 3.3 (Byrnes, Bush et al. 1996).
Table 3.3: The characteristics of the nitric oxide synthase isoenzymes
Enzyme
nNOS
Constitutive/
inducible
constitutive
Type NO
oroduclion
picomoles
Galcium/
calmodulin
dependent
Ghromosome
location
7: 26 exons
span
21 kb
eNOS constitutive ill picomoles dependent 12i 28 exons
span
>100 kb
iNOS inducible tl nanomoles independsnt 17: 26 exons
span
37 kb
a CNS' central nervous system; PNS, peripheral nervous system; |NANC, inhibitory noradrenergic
noncholinergic
b TNF, tumor necrosis factor; lL, interleukin.
This table is taken from Byrnes CA, Bush A, Shinebourne EA "Measuring expiratory nitric oxide in
humans" Methods in Enzymology 1996; 26g:45g-413 (Byrnes, Bush et d. rbsoi.
3.4.1 Constitutive nitric oxide wnthases
Activation of the two constitutive isoenzymes (nNOS and eNOS) depends on the levels of
calmodulin and calcium. The NOS enzyme lies dormant until an increase in cellular calcium
in the presence calmodulin occurs. A calcium concentration of 200 to 400 nanomoles allows
half the maximum activity of the enzyme. This results in a sustained release of NO over
several minutes, with picomole amounts of NO released, and this acts locally. The enzymes
are stimulated by a number of mediators, depending on where they are situated, and these
include bradykinin, acetylcholine, calcium ionophore, histamine, leukotriene, platelet
activating factor (serotonin/thrombin) and exercise. In this way these enzymes participate in
maintaining physiological balance within systems as discussed in the previous Chapter 2.3.4.
The nNOS was the first of the isoenzymes to be purified and cloned (Bredt, Hwang et al.
l99l; Schmidt, Pollock et al. 1991). There is wide expression and high activity of nNOS
isoenzyme in the brain and throughout the peripheral nervous system in the NANC nerves,
81
Gells" in which
enzvme is found
cNS: especially
cerebellum
PNS: gut,
bladder,
reproductive
organs
iNANC neruac
endothelial cells,
mast c€lls,
platelets,
smooth muscle
cells. neutroohils
macrophages,
neutrophils,
airway epithelial
cells,
fibroblasts, mast
cells
Enzyme.
stimulationo
acetylcholine,
bra)kinin,
Ca'* ionophore
histamins,
leukotrieng,
PAF
serotonin,
thrombin,
shear stress
ilpoporysacnand
e,
y-interferon,
TNFq,
TNFp, tll, tL2,
leptochoic acid,
picolinic acid

providing NO as a neurotransmitter (Forstermann, Closs et al. 1994). However it has also
been found in the spinal cord @un, Dun et al. 1992), in sympathetic ganglia and adrenal
glands @un, Dun et al. 1993; Sheng, Gagne et al. 1993), in the epithelial cells of the lung,
uterus and stomach (Schmidt, Gagne et al.1992), in pancreatic islet cells (Schmidt, Warner et
al. 1992) and in human skeletal muscle (Forstennann, Closs et al. 1994). While in the main
nNOS NO production functions mostly in neurotransmission, it is also utilised to maintain
muscle tone, for example, in the gastrointestinal tract and skeletal muscle. These enzymes are
involved in homeostasis throughout the body from memory, behaviour, circadian rhythms, to
the gastroenterology tract, renal function and reproductive activity. It is likely that this
isoenzyme form is the largest proportion of constitutive NOS in humans (Forstermann, Closs
et al. 1994; Knowles and Moncada 1994). The eNOS isoenzyme within arteries give a basal
level of NO production continuously, which maintains the vascular blood flow and blood
pressure. This isoenzyme is largely responsible for the NO which inhibits platelet aggregation
and platelet adhesion.
3.4.2 Inducible nitric oxide svnthase
The main method of activation, the amount of NO produced and the regulation of this form of
the enzyme is very different from the constitutive forms. The iNOS isoform is regulated at
transcriptional level with activation requiring both a primary and a secondary signal before
the mRNA for the enzyme is produced. The priming agent is interferon gamma (IFNI) or an
interferon inducing agent like bacterial lipopolysaccharide (LPS) (Weinberg, Chapman et al.
1978; Stuehr and Marletta 1985; Drapier, Wietzerbin et al. 1988; Sherman, loro et al. 1991;
Munoz-Fernandez, Fernandez et al. 1992). The category of factors which can act as the
secondary agent continues to be enlarged by every new study. These include interleukin 1
(n-l), IL2, TNFo, tumor necorsis factor p (TNFp), muramyl dipeptide, lipoteichoic acid,
picolinic acid (metabolite of l-tryptophan), ozone, cAMP elevating agents, ultravoilet light,
ozone and trauma (Ding, Nathan et al. 1988; Drapier and Hibbs 1988; Lorsbach, Murphy et
al. 1993; Bastian and Hibbs 1994; Nathan and Xie 1994).In addition, a number of viral and
antimicrobial products from mycobacteium tuberculosis, salmonella typhimuium and
protozoan parasites have also been found to stimulate this form of the enzyme (MacMicking,
North et ^1. 1997; Shoda, Kegerreis et al. 2001; Thoma-Uszynski, Stenger et al. 2001). Some
food components are also capable of inducing NO and may be the mechanism of action of
some food carcinogens, og soybean trypsin inhibitor, beta amylase (Nathan 1997). For
example, nitrites and nitrates are important antimicrobial, flavouring and colouring agents in
meat and fish products (Chow and Hong 2W2).
82

The induction requires gene transcription and mRNA production, and therefore occurs several
hours after the exposure to the activating agents. However this also means that the production
of NO can go on for days until the enzyme is broken down. Also important is that when
activated, nanomole levels of NO are produced; ie 100 times that of the endothelial or
neuronal isoenzyme production. NO exposure can damage all classes of macromolecules
including DNA, it can destroy mitochondrial enzymes, inhibit protein synthesis, and cause
apoptosis of cells (Hibbs, Taintor et al. 1988; Curran, Ferrari et al. 1991; Kwon, Stuehr et al.
l99l; Stadler, Billiar et al. 1991; Lancaster, Langrehr et al. 1992: Lepoivre, Flaman et al.
1992; Tamir, Irwis et al. 1993; Nakano, Terato et al. 2003). So the regulation of this enzqe
is extremely important given its unique role in host defence but also ability to inflict
significant host damage (Nathan lggT\.
Under certain conditions such as in the absence L-arginine or if BFI+ is limited (or in the
presence of adriamycin which may in part explain the toxicity of this drug), all the NOS
enzymes, but more often seen with this inducible form of the enzyme, can reduce 02 via the
flavin cofactors to O2'- (Xia, Dawson et al. 1996; Cosentino, Patton et al. 1998; Vasquez-
Vivar, Kalyanaraman et al. l99S). The simultaneous generation of 02- and NO then results in
producing ONOO- causing further cellular injury.
3.4.3 Contol of the nitric oxide synthase isoen4tmes
3.4.3 (i) The constitutive forms
All of the enzymes can be regulated at pre-transcriptional, post transcriptional and post
translational levels (Papapetropoulos, Rudic et al. 1999). However the main control factors
appear to be different for each. The control of the constitutive enzymes is predominantly
maintained by the need for the presence of substrate and cofactors, while the control of the
inducible form is predominantly by the need for two signals for transcription.
The constitutive enzymes require both the presence of calmodulin and a certain level of
calcium from 200 to 400 nmols to commence production, and if the calcium concentration
falls below this level then production ceases. The substrate L-arginine is required and L-
arginine exists within and outside the cell. A low substrate amount can occur as a result of the
absolute availability of L-arginine, or the ability of the cell to take up L-arginine, or the ability
of the cell to regenerate L-citrulline back to L-arginine to act as further substrate. Vascular
cells, for example, are able to perform this regeneration with the use of an enzyme
arginosuccinate synthetase. BFI+ is also required, and this is produced by another pathway.
83

The enzyme of this second pathway - guanosine triphosphate cyclohydrolase I - is in turn
affected by certain cytokines and inflammatory products. So while this operates as a control
for the isoforms, in fact the limiting effect may be more important for the iNOS enzyme
which is present in higher amounts, particularly in infection and inflammation when much
greater levels of these cytokines are around and when the production of NO is higher (Stuehr
1999; Werner-Felmayer, Golderer et al.2002). This will be discussed in more depth below in
Section 3.4.3 (ii).
The rates of formation of NO and L-citrulline are non linear for all isoenzyme forms which
demonstrates there is negative feedback inhibition and this is also a mechanism to control
overproduction (Rogers and Ignarro 19921, Assreuy, Cunha et al. 1993; Bastian and Hibbs
r9e4).
In addition, there are some differing control mechanisms operating for each of the constitutive
isoenzymes. Firstly, the nNOS form can be regulated by alternative splicing to give multiple
transcripts and molecular diversity. This means that the protein can be spliced in different
places giving different molecular weights which possibly fulfil a differing role. There are two
major transcriptional clusters identified within the human nNOS gene, with one form lacking
approximately 315 base pairs, or 105 amino acids and this smaller version is seen
predominantly in the peripheral nervous system, renal tract and in the male reproductive tract
(Papapetropoulos, Rudic et al. 1999).
Secondly, regarding the eNOS form, it was noted that some physiological situations led to
increased eNOS expression such as shear stress and exercise training (Nishida, Harrison et al.
1992; Sessa, Ftitchard et al. 1994; Uematsu, Ohara et al. 1995), and situations of local
hypoxia (Marsden, Schappert et al. 1992). While both iNOS and nNOS exist in soluble forms,
eNOS exists in a particulate form (Nathan 1992). The importance of this is that the proper
localisation of eNOS is a pre-requisite to enable it to interact with specific proteins that will
allow full activity. Post translation, cNOS undergoes a process of acylation (Shaul, Smart et
al. 1996) which appears necessary to anchor the enzyme to the membrane in the Golgi
apparatus, and in the plasmalemmal vesicles (the caveolae). There are some regulatory
proteins that play a role in promoting this within the cells (C-protein coupled receptors,
caveolin or Hsp90), that contribute to the correct localisation and that can also be individually
influenced therefore contributing to more or less NO being produced. For example, Hsp90
increases with increased histamine or fluid shear stress (Garcia-Cardena, Fan et al. 1998).
Changes in phosphorylation of eNOS have been observed with shear stress (Corson, James et
84

al. 1996; Eng, Morton et al. 1996; Fleming, Bauersachs et al. 1998), secondary to calcium
mobilising agents (Michel, Li et al. 1993), tyrosine phosphatase inhibitors (Garcia-Cardena,
Fan et al. 1996), and a number of protein kinases (Hirata, Kuroda et al. 1995; Chen,
Mitchelhill et al. 1999). The phosphorylation of the enzyme makes it more sensitive to
calcium necessary for the active pathway.
The most potent activator is lysophosphotidylcholine (IlC), a major phospholipid found in
oxidised low density lipoproteins and this allows up to an 11 fold increase in mRNA
induction and eNOS production, although there is a varying discrepancy between the levels of
the mRNA and both the absolute protein levels and the levels of activity pointing to continued
control later in the pathway (Papapetropoulos, Rudic et al. 1999). Interestingly, the content of
LPC in atherosclerotic vessels is higher than normal vessels. Statin-based cholesterol
lowering drugs result in a modest increase in the eNOS mRNA levels by a post transcriptional
mechanism involving stabilisation (Laufs and Liao 1998). Oestrogens are capable of modestly
increasing eNOS expression (Wiener, Itokazu et al. 1995; Kleinert, Wallerath et al. 1998).
The activity of the enzyme is reduced by the presence of TNFo which destabilises the eNOS
mRNA and reduces the half life of the enzyme from 48 to 3 hours (Yoshizumi, Perrella et al.
le93).
3.4.3 (ii) The inducible form
Control of the iNOS isoenzyme can also operate at pre-transcriptional, post transcriptional
and post translational levels, and drugs also interact to affect enzyme generation. However,
different to the constitutive forms, control, particularly of gross overproduction, is extremely
important to host survival.
Firstly, Iooking at the pre-transcriptional level; the activation of the iNOS promoter region is
the main control for this isoenzyme. Most of the work looking at effects at a transcriptional
level has been done in explanted mouse macrophages or macrophage cell lines (MacMicking,
Xieet al. 1997; Papapetropoulos,Rudic etal. 1999).Thishasrevealedanumberof binding
sequences including interferon gamma (INFI), TNFo, TNFp, interferon alpha (IFNa), NF-KB,
gamma activated sites, interleukin 6 (IL6), activating protein sites and a basal transcription
site. However the importance of only 2 of these has been clearly documented. The NF-rB site
is important for iNOS induction (Nunokawa, Ishida et al. 1994) and LPS induction also
operates through this site (Nathan 1992), and a cluster of four sites for IFNI are important for
NOS transcription (Xie, Kashiwabara et al. 1994). The need for second signal as well as IFNy
or an IFNy-like substance to activate iNOS is probably an important control to prevent
85

inappropriate production from commencing (Lorsbach, Murphy et al. 1993). Another possible
protective mechanism is, while INF1 induces iNOS, it also induces production of ILl0 an
anti-inflammatory cytokine. Pre-treatment of macrophages with IL10 inhibits their ability to
express iNOS or to produce NO, although once iNOS exists then it has no effect (Cunha,
Moncada et al. 1992). IL,l added with ILl0 acts even more strongly in inhibiting the
production of the enzyme (Oswald, Gazzinelli et al. 1992).
Secondly, examining the post transcriptional control, certain cytokines can have either
stabilising or destabilising effects on the mRNA for iNOS, thus greatly altering its half life.
For example, IFNI stabilises the mRNA prolonging its action while transforming growth
factor beta (TGFp) can destabilise the mRNA and reduce its transcription as well as
accelerating its breakdown (Vodovotz, Bogdan et al. 1993; Imai, Hirata et al. 1994; Sirsjo,
Soderkvist et al. 1994; Perrella, Patterson et al. 1996; MacMicking, Xie et al. 1997). While
this inhibits the production of iNOS in this way, it does not effect cNOS production (Ding,
Nathan et al. 1990).
Thirdly, post translational control, as mentioned above, includes the availability of the
substrate and cofactors. However the inducible form is independent from some of the
regulations described for the constitutive forms. While all the isoenzymes require calmodulin
as a cofactor, it is tightly bound to this form of the enzyme (Xie, Cho et al. 1992). In addition,
the constitutive enzymes require calcium at levels of 200 nanomoles, whereas the inducible
form can activate with calcium at much lower levels down to 39 nanomoles and notably the
cellular basal level of calcium is nearer 70 to 100 nanomoles. This also explains why this
form of the enzyme can keep producing NO when the constitutive enzymes have stopped
production once the calcium falls below a certain level @astian and Hibbs 1994). A low
amount of the substrate L-arginine acts as a control mechanism, there are three possibilities as
mentioned above. There may be an absolute low availability of L-arginine. The uptake of
arginine into the cell occurs through a sodium and pH dependent pathway, mediated by a
family of cationic amino acid proteins (CATI, CATZ, CAT2B, CAT3), (Closs, Scheld et al.
2000; Nicholson, Manner et al. 2001), but this can be up-regulated by the presence of LPS.
External to the cell, the arginine levels are controlled by the enzyme arginase which degrades
arginine to urea and ornithine (Gotoh and Mori L999; Munder, Eichmann et al. 1999;
Rutschman, Lang et al. 2001). Macrophages contribute to this, possibly also developed as a
protective mechanism, as they release an abundance of arginase. However macrophages and
vascular smooth muscle cells can also regenerate arginine from citrulline and therefore re-
utilize the citrulline amino acid. This is with the use of another enzyme arginosuccinate
86

synthetase present in these cells, again upregulated by LPS and IFNy (Hattori, Campbell et al.
1994; Nussler, Billiar et al. 1994; Nagasaki, Gotoh et al. 1996). The availability of cofactor
BFIa may also affect iNOS activity (Stuehr 1999; Werner-Felmayer, Golderer et al. 2002').
One of the key enzymes (guanosine triphosphate cyclohydroylase I, as mentioned above)
which generates this cofactor is affected by the presence of certain cytokines with IFN1,
TNFo, ILI and LPS increasing its activity, while the protective cytokines IL4, ILl0 and
TGFP suppressing activity. IL8 also causes a concentration dependent inhibition of iNOS
(McCall, Palmer et al.1992). Fibroblast growth factors inhibit NO synthesis and this may be a
particular protective factor for the retina against damage (Goureau, Irpoivre et al. 1993).
Finally, in keeping with the other isoenzyme forms, the rates of formation of NO and L-
citrulline from iNOS are non linear which suggests there is negative feedback inhibition
(Rogers and Ignarro 1992; Assreuy, Cunha et al. 1993; Connelly, Palacios-Callender et al.
200r).
Studies recently have suggested an "adaptive NO resistance" where some cells likely exposed
to high levels of NO have an inducible NO resistance mechanism @emple 2004) and on
subsequent exposure to high levels of NO the loss of cells reduces from 80 to20Vo. These
resistance mechanisms also operate against other free radicals (Kim, Bergonia et al. 1995;
Bishop, Marquis et al. 1999).
Some of the effects of iNOS have been clarified by the use of NOS knock out mice -
demonstrating both beneficial and detrimental roles. Mice with no iNOS have altered immune
responses and decreased survival to bacterial, viral and parasitic infection (MacMicking,
Nathan et al. 1995; Wei, Charles et al. 1995). They also have increased leukocyte adhesion to
endothelium during toxaemia, poor wound repair and incomplete regeneration e.g. to liver
biopsies and resections (Hickey, Sharkey et al. 1997; Rai, I-ee et al. 1998; Yamasaki,
Edington et al. 1998). However they are also resistant to endotoxin induced mortality, end
organ damage after haemorrhagic shock, or hypoxic injury, and develop less eosinophilia in
allergic airways disease (Wei, Charles et al. 1995; Nathan 1997; Hierholzer, Harbrecht et al.
1998; Ling, Gengaro et al. 1998).
3.4.4 Nicotinamide adenosine di-nucleotide phosphate oxidase and inducible nitric oxide
synthase
These are the two key enzymes involved in host defence. There is 36Vo homology between
them and both are present in macrophages, one of the key defence cells.
87

Nicotinamide adenosine dinucleotide phosphate oxidase (NADPII) is involved in the
respiratory burst generating Oz- as a result of phagocytic triggering (Segal 1989). The Oz-
produced is a precursor of other reactive oxygen species such as hydrogen peroxide (HzOz)
and hydroxyl radical (OH.). NADPH oxidase is produced primarily in
polymorphonucleocytes, monocytes and macrophages - all expendable cells in host defence.
It is membrane bound but has membrane and cytoplasmic subunits and is responsible for
defence against extracellular pathogens and engulfed organisms accessible to phagocytic
cells. Activation and priming occurs with INFI, PAF, GCSF, GMCSF, ILl, IL6, IL8, TNFa
and substance P. Activation can also occur with complement factor CF5a, chemotactic
factors, phorbol esters and fatty acids.
In comparison, iNOS can be induced in virtually all nucleated somatic cells and is responsible
for the defence against pathogens that survive and proliferate in the intracellular environment.
It lies within the cytoplasm and kills pathogens that enter the cells (Nathan 1992). When it
was realised that biosynthesis of NO was possible by the same cells, it meant that these cells
were capable of the production of a range of reactive potentially toxic oxynitrogen species
such as nitrosium (NO*), nitroxyl ions (NO-), NO2, and S-nitrosothiols (Stamler, Jaraki et al.
1992). However, although macrophages can have both NADPH oxidase and iNOS present,
O; and NO are not often simultaneously produced thus avoiding the formation of
perioxynitrite (ONOO-). They appear to be independently regulated although INFI induces
both enzyme systems. It may in part depend on the presence of L-arginine and the isomeric
form in which it is found (Ding, Nathan et al. 1988; Martin and Edwards 1993; Bastian and
Hibbs 1994).In isomerism the 'cis'form is when the two substituent groups are orientated in
the same direction, while the 'trans'form is when the substituents are oriented in opposing
directions. In the trans form, ONOO- undergoes homolytic cleavage to form highly reactive
molecules HO* and NOz which can produce significant and irreversible damage to microbes,
but also to host cells. However the cis form, ONOO- reilranges to form a non-toxic nitrate
and it may be that the conditions for formation of these cis and trans isomers are what
determine their formation in these cells.
3.4.5 Drugs and other agents that affect the isoenrymes and nitrtc oxide production
3.4.s (i) Drugs
Corticosteroids inhibit the expression of inducible but not constitutive forms of NOS. This
occurs at transcriptional level, with the ability to inhibit the induction of iNOS, but they are
ineffective once the enzyme is synthesised. There are no recognisable steroid responsive
88

elements in the iNOS promoter region. Similarly the antifungal imidazoles drugs inhibit the
induction of NOS. Methotrexate inhibits the synthesis of BFIa, therefore limiting the
availability of this as a cofactor (Werner-Felmayer, Werner et al. 1990; Gross, Jaffe et al.
1991).
Diphenylene iodonium is an aromatic compound that binds to the flavoprotein cofactors, but
is not a therapeutic option, because it was realised that very high doses would be required
(Stuehr, Fasehun et al. 1991). Carbon monoxide binds to the haem in NOS and inhibits
enzyme activity (White, Moellering et al. 1997). Compounds that bind calmodulin can affect
cNOS but not iNOS (Bredt and Snyder 1990; Stuehr, Cho et al. l99l). Antioxidants protect
NO.
Calcium mobilising agents (Michel, Li et al. 1993) and tyrosine phosphatase inhibitors
(Garcia-Cardena, Fan et al. 1996) both increase the rate of phosphorylation of eNOS resulting
in increased sensitivity to calcium and increased production. Statin-based cholesterol lowering
drugs result in a modest increase in the eNOS mRNA levels by a post transcriptional
mechanism involving stabilisation (Laufs and Liao 1998). Oestrogens are capable of modestly
increasing eNOS expression (Weiner, Knowles et al. lgg4: Kleinert, Wallerath et al. l99g).
The angiotensin converting enzyme inhibitors inhibit the breakdown of bradykinin, and
bradykinin stimulates particularly the endothelial cells to produce NO, so act to increase
activity. Finally NO is the active moiety of the glyceryl trinitrate and other agents that have
been trialled as anti-hypertensives and for bronchodilator properties.
3.4.5 (ii) Nitric oxide synthase inhibitors
The NOS inhibitors have been essential for evaluating the role of NO in physiological and
pathophysiological processes, and have been used by infusion in both laboratory animals and
humans to judge effects. L-arginine analogues have been created to act as competitive false
substrates to inhibit NOS (see Chapter 2.3).They have now been widely used to study the NO
generating NOS pathways which has been helpful in delineating the effects of NO in a variety
of systems (Hibbs, Taintor et al. 1987; Hibbs, Vavrin et al. 1987; Palmer, Rees et al. lggg).
This was first accomplished by the modification of guanidino group of L-arginine to create a
series of inhibitors. These include L-NMMA (N* monomethyl L-arginine) which has
substituted a methyl group, L-NAME (N* arginine methyl ester) which has substituted a nitro
group and similar substitutions for the related compounds; L-NNA (N* - nitro - L-arginine),
LADMA (N*N* dimethyl L-arginine), and L-MO (N* -
iminoethyl-L-ornithine) (Knowles
and Moncada 1992) (see Figure 3.3). They are stereo specific, acting at the level of the
89

enzyme and are competitively reversed by the normal substrate L-arginine, but not D-
arginine. With prolonged incubation, the inhibitors appear to exhibit non-competitive
properties (Marletta, Tayeh et al. 1990). Ebselen is a selenium containing antioxidant (2 -
phenyl, 2-benzisoselenazol-3-(2h)-one) which is synthetic analog of glutathione peroxidase
and is able to inhibit both iNOS and cNOS (Hibbs, Taintor et al. 1990).
Figure 3.4: The chemical structures of the nitric oxide synthase inhibitors
cooe ,,4,\,*"
cooo
'\r/t'
,,A__^^(.*
lW-Amino-u-arglnlnc
tt'\*lt'
,,4,,\'*" -.A,r"-f'
'.)\".
l-Arglnine
r-NMMA
\ t-NNA
cooo
t€anlvanrnc
The structures of arginine and of the arginine analogues most frequently used as inhibitors of the NO
synthases with the predominant ionic species at neutral pH shown.
Taken from Knowles RG, Moncada S. Nitric oxide synthases in mammals. BiochemicalJournal 1994;
298 (21:249-255 (Knowles and Moncada 1994).
Some NOS inhibitors occur naturally such as aminoguanadine a competitive inhibitor, and
two of the compounds discussed above (L-NMMA and L-NAME) also occur naturally in very
small amounts, and so may also operate as a control at substrate level. Interestingly, the
90

inhibitor compounds do not have equal responses with the 3 isoenzymes, for example gNOS
is more sensitive to L-NAME and iNOS is more sensitive to L-NMMA. Aminoguanadine has
been shown to have 10 to100 times the affinity for iNOS compared to cNOS. LNIO is a
particularly potent inhibitor of the neutrophil form of iNOS suggesting subclasses of the
enzymes. Selective inhibitors of cerebral NOS have also been described, possibly utilising the
differing length transcriptions described for this iso-enzyme (Moore, Babbedge et al. 1993).
3.5
Chapter summarv
NO is a free radical with an unpaired electron which is highly reactive with a half life of
seconds. It is produced by the NOS enzymes which exist as constitutive and inducible forms.
They require L-arginine as a substrate, plus co-substrates (NADPH and 02) and co-factors
(BtI+ and flavoproteins). The neurological and endothelial constitutive forms generate
continuous low levels of NO to maintain physiological processes and are largely controlled by
calcium levels and available factors to be active. Activation of the inducible form requires a
primary and a secondary signal before transcription occurs. However it then goes on to
produce far greater amounts of NO for far longer than the other forms and is free from some
of the controls of the other enzymes. It is this isoenzyme that is responsible for the host
defence availability of this molecule.
NO reacts to form a number of related compounds known as the reactive nitrogen species
(nitrate, nitrite, nitrous oxide and nitrogen dioxide) and in the presence of higher
concentrations of oxygen, it forms superoxides (perioxynitrite and perioxynitrous acid). These
compounds in turn are highly reactive and operate to kill intracellular and extracellular
pathogens, but also to cause toxicity and destruction to the host cells and host DNA. NO also
reacts with thiol compounds (S-nitrosoglutathione, S-nitrosoalbumin, S-nitrosohaemoglobin)
and these have been increasingly recognised as a mechanism by which NO can be stabilised,
transported and transferred to other compounds and other tissues. The compounds have also
been demonstrated to have active roles regulating protein function and to have their own
vasodilatory properties. A key interaction of NO is the reaction with metals in the centre of
metalloproteins, and this forms the main method of excretion of NO. NO reacts with the
ferrous iron in haemoglobin to form methaemoglobin and nitrate, the latter of which is then
excreted in the urine. NO and related compounds can in addition react with genes, amino
acids and lipids. NO itself is a clear and colourless gas. Having examined the properties of
NO and the production pathway - the next chapter will discuss the technical options of how to
measure NO levels.
9l

4.1 Introduction
Chapter 4: Methods to measure nitric oxide
The previous chapter reviewed the production of NO, its reactions and interactions in vivo.
This chapter will examine the available methods of measuring NO. There were three issues
that I believed needed to be addressed; firstly, how the production of this evanescent molecule
could be measured as accurately as possible, secondly, which technique was the most
appropriate for measuring the production of NO in the lung and thirdly, how could it be done
non-invasively such that it could be used with children -
so preferably no venepuncture, no
biopsy or bronchoscopy and lavage required. A technique based on the way it was measured
in airway pollution but adapted to exhaled air in a manner analogous to lung function was
sought.
From the previous chapter (see Chapter 3. 1 'Introduction'), it is known that NO is formed
stereo-specifically from the guanidino nitrogen of L-arginine and oxygen. This is
accomplished via five separate steps (see Figure 3.3) and requires cofactors
(tetrahydrobiopterin and flavins FAD, FMN) and cosubstrates (O2, NADPH) as well as
calmodulin and calcium.
Therefore the possible factors that could be measured to assess the activity of this pathway
are:
a
o
o
o
O
o
o
The decrease in L-arginine
The formation of L-citrulline
The activity of cyclic guanosine monophosphate (cGMP)
The formation of NO
The formation of methaemoglobinuria
The formation of nitrite (NO2')
The formation of nitrate (NO3)
These, plus the measurement of co-factors and the other nitrogen compounds have all been
explored. A detailed review of all methods to measure NO in all compounds and in all tissues
is presented in "Methods in Nitric Oxide Research" edited by Martin Feelisch & Jonathan J
Stamler, published by John Wiley & Sons Ltd, Baffins Lane, Chichester, West Sussex,
England, 1996. It is immediately obvious that many of these compounds will be inappropriate
to use for detection in lung exhalate. I will briefly review the options before reviewing in
more depth the technique of NO measurement with the use of a chemiluminscence analyser.
92

4.2
The use of L-arginine as a substrate for NO production is a relatively specific reaction. This
allows the production of NO to be correlated to the loss of L-arginine and the gain of L-
citrulline, and it is possible to measure both of these in a laboratory setting (Senshu, Sato et al.
1992). However the baseline concentrations of L-arginine need to be known, which is difficult
in vivo. Also, while the formation of L-citrulline can be measured (Hecker, Sessa et al. 1990,
Senshu, 1992 #898; Bredt and Schmidt 1996), this is made more difficult in vivo where
certain cells such as vascular endothelial cells and macrophages (and likely others) are able to
regenerate L-citrulline back to L-arginine at a variable rate to act as further substrate (see
Chapter 3.4.3 'Control of the nitric oxide synthase isoenzymes'). This recycling clearly
precludes the use of measurement of the levels of these compounds to accurately reflect the
production of NO.
The NOS enzymes stimulate soluble guanylate cylase (sGC) with subsequent accumulation of
cyclic guanosine monophosphate (cGMP). Again, it is possible to measure the levels of this
protein, however, this requires access to, or the releasing of, the protein from its usual
intracellular site. This is also non specific for NO as both sGC and sGMP operate in many
cellular enzyme pathways (Schultz, Bohme et al. 1969; White and Aurbach 1969; Archer
L993; Craven andIgnarro 1996).
4.3
Methaemoglobin
NO can be measured by the transformation of reduced haemoglobin (Fe 2) as it is oxidised to
methaemoglobin (Fe 3*; which can then measured by spectrophotometry (detection threshold
= I nmol). The reaction is rapid and will be almost stoichometric under most experimental
conditions, with the only interfering compound likely to be superoxide anion production
(Sutton, Roberts et al. 1976). The advantages are that spectrophotometers are widely
available, there is no need to acidify the sample and that methaemoglobulin remains relatively
stable. Accumulation of oxyhaemoglobin is not a problem due to the much higher affinity of
haemoglobin for NO than Oz. The disadvantages are that it is more useful for biological
samples other than exhalate, considerable expertise is required and the assay will detect other
nitrosyl grcups in any given sample (Noack, Kubitzek et al. lgg2; Murphy and Noack 1994;
Feelisch, Kubitzek et al. 1996).
93

4.4 Nitrite and nitrate
The measurement of NO in biological tissues has been difficult in the past because of the
short halfJife, the small amounts produced, and its lability in the presence of Oz.
By contrast nitrite and nitrate are stable metabolites whose presence in blood, urine and tissue
in humans was demonstrated many years ago (Mitchell, A. et al. 1916; Mitchell 1928;
Tannenbaum, Fett et al. 1928). The diazotization assay is the standard technique for
measuring inorganic nitrite and was described more than 150 years ago by Greiss (Greiss
1864; Greiss 1879). A two step assay is carried out with the reagents sulphanilic and N-(1-
naphthyl) ethylenediamine first mixed and then incubated with a nitrite containing sample
which interacts in a 1-l ratio generating a purple azo dye which can be monitored by
spectrophotometry at a wave length of 546nm (Greiss 1864; Green, Wagner et al. L982). This
remains the most common method of measuring nitrite and nitrate (Schmidt and Kelm 1996)
in fluid such as blood and urine (Wishnok, Tannenbaum et al. 1993; Baylis and Vallance
1998). Studies have suggested that it is circulating nitrite measurable in serum or plasma
rather than nitrate that more reflects the NO synthesis in both humans and animals (Bode-
Boger, Boger et al. 1999; Lauer, Preik et al. 2IN_I; Kleinbongard, Dejam et al. 2006).
However, the excretion rate in urine of nitrate is a more non-invasive method to measure
whole body NO synthesis and is particularly useful for obtaining baseline results and then
assessing responses to physical and pharmacological treatment (Kanno, Hirata et al. 1992;
Bode-Boger, Boger er al. 1994 Borgonio, Witte et al. 1999; Bode-Boger, Boger et d. 2000).
There are some difficulties in making these measurements in biological assays as NO
decomposes to nitrite and nitrate at different rates depending on the ambient conditions and
on the redox environment of the fluid being measured. As well, these two compounds are
present at different concentrations in urine, serum and/or blood therefore requiring the ability
to measure and calculate across a wide concentration range potentially from low nanomolar to
high micromolar levels (Schmidt and Kelm 1996). Although adaptation to enable this has
occurred as studies on the nitrogen compounds have progressed (Green, Wagner et al. 1982;
Corras and Wakid 1990; Ohta, Araki et al. 1994; Tsikas 2005). However even a very recent
comprehensive review of the topic states "Despite the chemical simplicity of nitrite and
nitrate, accurate and interference-free quantification of nitrite and nitrate in biological fluids
as indicators of NO synthesis may be difficult" (Tsikas 2005). The Greiss reaction gives an
assay that is designed to measure single samples, although it can be modified to give online
94

esults (Tracey, Linden et al. 1990), but may not be sensitive or specific enough to measure
NO alone (Schmidt and Kelm t996),
NO will rapidly convert to nitrite and nitrate in biological samples and by adding acids or
reducing substances these can be transformed back to release the NO molecule (Verdon,
Burton et al. 1995; Kelm, Dahmann et al. 1997). However acidification and/or reduction will
release NO from all nitroso compounds, alkyl or inorganic nitrites, the nitrosamines and
nitrosothiols. If the sample contains compounds other than nitrite and nitrate, then there may
be overestimation of the NO that existed as a biologically active or inflammatory mediator.
4.5 Nitric oxide
It has become possible to measure NO directly down to the cellular level by using
microelectrodes. This cornmenced with the modification of a miniature 02 electrode, and
sealing it so that only low molecular weight gases could enter. By introducing a positive
voltage, NO was oxidised on the surface of the electrode and could be measured over l-
3pmol range (Shibuki 1990). The probe was approximately 2mm in diameter. Modification of
this then allowed a much smaller microsensor to be developed using a semi conductor
polymeric porphyrin and a cationic exchanger on a sharpened carbon tip which reduced the
size to 5 microns (Malinski and Taha 1992; Taha, Kiechle et al.1992) and could measure NO
as an electrical current. This has allowed NO measurement in cardiac cells (Xian ,Zhang et al.
2000; Kanai, Pearce et al. 2001; Katrlik and Zalesakova2002),brain and nerve cells (Shibuki
1990; Taha, Kiechle et al. 1992: Malinski, Bailey et al. 1993; Kumar, Porterfield et al. 2001),
osteoclasts (Silverton, Adebanjo et al. 1999), trachea and main bronchi from an animal model
(Ricciardolo, Vergnani et al. 2000) and in suspensions of mitochondria and cells such as
platelets, leukocytes and in cell cultures (Wadsworth, Stankevicius et aL.2006). The detection
rate has been as low as 10-20 Mol, with a linear response between 8.0 to 4.8 x 10-6 mol/L. This
low detection limit is also matched by high sensitivity and selectivity although it does also
detect catecholamine activity (Tu, Xue et al. 2000). It has also been achieved as an online
measurement so release of NO could be followed in activated macrophages, which correlated
well with the nitrite concentration determined by the Greiss assay (Cserey and Gratzl 2001).
However a recent review suggested that further methodological development of these
microsensors was needed in order to avoid the influence of changes in temperature, pH, and
oxygen on the measurements (Wadsworth, Stankevicius et al. 2006).
My interest was to look at measurement in exhaled breath. A number of very early methods of
detection depended on colorimetric analysis but for exhalate these were too slow and too
95

insensitive (Braker and Mossman 1975; Archer 1993). For example, the use of moist iodide
paper to detect the oxidation products of NO in air has a sensitivity of 300ppm and has a
response time of 5 minutes. When collecting samples to measure the NO concentration, the
presence of oxygen will continue to degrade the sample. This means the sample needs to be
measured quickly, oxygen contamination must be avoided (which is difficult in biological
specimens), or the loss of NO with these reactions needs to be compensated for by converting
the other compounds back to releasing NO just prior to measurement.
It is possible to use collected expirate which can be bubbled through degassed water and the
NO can be trapped by nitroso compounds or reduced haemoglobin to form stable adducts that
can then be detected by electron paftrmagnetic resonance (detection threshold = I nmol).
Electron paramagnetic resonance spectroscopy allows a specific assessment of molecules
whose energy levels are altered in the presence of a magnetic field and this property is typical
of molecules with an uneven number of electrons (Henry, Ducrocq et al. l99l; Henry,
Irpoivre et al. L993; Singel and Lancaster 1996). Usually a continuous wave of
electromagnetic radiation in the microwave frequency is applied to the sample. When the
frequency of this is equivalent to the energy difference of the electron spin energy levels,
which is known as the 'resonance', the radiation is absorbed and the absorption leads to the
generation of a signal which is measured. This technology has proved useful in detecting the
movement of NO, for example determining the specific parentage of L-arginine to NO to
methaemoglobin, determining the active site for NO or the binding site with other
compounds. Interestingly, despite the unpaired electron, the NO molecule is only detectable in
an excited state, while at a 'ground' state (unexcited state) the coupling of electron spin and
the orbital angular momentum makes it resonant silent on the spectroscope. It is possible to
identify signals at different frequencies which allow different molecules within compounds to
be identified. This is unless a wide band of frequency is occupied as is seen with the iron in
the haem proteins and from some of the other compounds used to trap NO such as metals or
thiols. The compounds used for trapping NO in this way are also sensitive to pH. At acidic pH
levels, the nitroso and nitrone compounds are unstable and can yield NO-type spectra even in
the absence of NO (Anoyo and Kohno 1991). At alkaline pH there can be inhibition of the
reaction between NO and haemoglobin (Feelisch, Kubitzek et al. 1996; Hakim, Sugimori et
al. 1996). Finally, the equipment required to perform electron paramagnetic resonance was,
and remains, expensive and considerable expertise is necessary (Archer 1993). In addition,
more direct methods of measuring exhalation were becoming available.
96

NO can be detected by standard gas chromatograph techniques @ai, Payne et al. 1987; Tsikas,
Boger et al. 1994), although initially this was found to be much less sensitive than most of
other methods discussed here, partly because of the necessity for periodic manipulation and
thus disturbance of the reaction mixture during the sample processing. It is now increasingly
combined with scanning mass spectrophotometers where NO in air can be detected (Kelm,
Feelisch et al. 1988). Sensitivity has been limited by the resolving power (13500) required to
differentiate between ttN2 1m/z 30.00022) and NO (m1229.99799). euantification was further
complicated by the vast difference in the concentration of these two gases, namely parts per
thousand for l5N2 and parts per billion for NO. Specific experiments can be designed using the
isotope of lsN as a specific label for NO. In the mass spectrometer, gases are admitted under
very low pressures into an ion source where they are ionized in a high energy electron flux.
The positive ions formed are accelerated in an electric field and then deflected in a magnetic
field. The radius of the deflection is inversely proportional to the number of the particles. A
multi-collector device for a number of particles can be used. It can also be used to measure
the evolution of gas from a liquid sample. The reaction mixture is 'sparged' with an inert
compound such as argon (sparging is to agitate by introducing a compressed gas), a plunger is
lowered to the liquid surface and closed to the atmosphere. The reaction is then commenced
with injections of reagents, inhibitors, enzymes or cells - and in the case of NO stripping,
obtaining NO back from the compounds to which has bonded by acidification. The gas then
diffuses into a vacuum line through a cold trap which removes water vapour, and the
remaining gases canying NO and other compounds of interest are scanned and analysed at
brief intervals (Payne, Le Gall et al. 1996). This has been used to study nitrogen metabolism,
not only in biological fluids but also, for example, to assess bacterial activity in river water
etc. However using labelled arginine, it has been used to measure NOS activity (Tsikas 2OO4)
and very recently to assess whole body NO synthesis in healthy children after subjects had
oral doses of the labelled substrate with the measurement of the subsequently collected urine
(Forte, Ogborn et al. 2006). For all these measurements, isotope labelling is required, and
therefore they are not so useful for repeated exhalation measurement under different
conditions.
4.6
Chemiluminescence
The method that held the most promise for measuring NO in direct gas samples such as
exhaled air was chemiluminescence.
97

A group of instruments have been developed to enable the measurement of certain compound
concentrations within samples by using light reactions. These include:
o 'spectrophotometers', mentioned above, which measure how light is absorbed by the
specimen with the light being generated by the meter itself.
o 'Fluorometers' which measure light emitted by the specimen after excitation of the sample
generated by the meter.
o 'Luminometers' which measure light generated with no light or excitation input - this
makes them comparatively simple compared to the other devices as they only require a
reaction chamber, a light detector and a recorder (and for the measurement of NO, a
photomultipler).
The chemiluminscence analysers (see Figure 4.2) were originally developed to measure NO
as an atmospheric pollutant as discussed in Chapter 2.2. The measurement is based on the
observation that the reaction of NO with ozone produces light (see Figure 4.1).
Figure 4.1: Chemical reaction between nitric oxide and ozone
NO + Os + NO2' 1'. 9,
NO"+NOz+hf
(. = unstable electron)
(hf = light)
The interaction of ozone with gases such as NO, NOz, CO and SOz was described in the early
1960s (AIHA 1966), although ozone reacts most readily with NO. In the excited state the
electrons are unstable and they dissipate energy as they regain their original state. This light is
sufficient to make chemiluminescence one of the most sensitive NO assays available. The
chemiluminescence reaction of NO and Or is very fast and has a low activation energy of 10.5
joules (Johnston and Crosby 1954). At room temperature the rate constant of the reaction
(both steps) is l0-7 I mol-r s-t lclyne, Thrush et al. 1964). This high speed means that the
chemiluminscence assay is able to detect rapid changes in NO concentration and therefore can
be adapted for on-line measurement. It was, for example, used early to monitor NO
concentrations when this was delivered as a treatment trial in intensive care for pulmonary
hypertension @epke-Zaba, Higenbottam et al. 1991; Kinsella, Neish et al. 1992; Roberts,
Polaner et al. 1992: Gerlach, Rossaint et al. 1993). NOz reacts with ozone more slowly and
the reaction requires higher activation energy so the chemiluminscence levels of NO are not
affected by NO2, even if the latter is present in high concentrations (Johnston and Crosby
I9l4;Fontijn, Sabadell et al. 1970).
98

The first chemiluminscence analyser to measure NO was built in 1970 utilizing these
principles of chemical reaction. A vacuum of reduced pressure (approximately negative l-15
mmHg) is used to draw the gaseous sample into a reaction chamber through a valve where
ozone is generated by electrical discharge. The vacuum is also necessary to evacuate gases
that could potentially absorb energy from NO2., and to stabilise NO by removing 02, to
prevent any 02 reacting with the NO to produce NOz which does not emit light (Hampl,
Walters et al. 1996). The NO and 03 are then mixed in front of a photomultiplier tube
sensitive to low levels of light at the red sensitive end of the spectrum (660 - 900nm). The
photons from the reaction strike a photosensitive surface and the impact releases electrons
which are accelerated toward an electron sensitive surface (the first dynode) by an electric
field. Each electron impact on this first dynode then causes emission of several electrons and
these are accelerated to a second dynode. This step is repeated but the electrons are attracted
to the terminal electrically charged element - the anode - and the resulting current is
measured. This amplification achieved by the photomultiplier is necessary to measure the
signal as the NO2'rosction emits a relatively weak red light (Hampl, Walters et al. 1996) and
means that each electron emitted from the original photosensitive surface becomes a signal
from millions of electrons at the anode (Turner 1985).
Figure 4.2: Diagram of the chemiluminscence analyser
AmlogdChl
cocputor/rrconbr
I Flowwrctcr
i (o9ddlll)
!
Sampb

The original group demonstrated that the light emitted was indeed directly proportional to the
linear content of the specimen and I have included the graph of their results (see Figure 4.3).
They used increasing volumes from one to one hundred micro-litres of a commercially
available gas in gas tight, Nz flushed syringes. The chemiluminscence signal was recorded as
the peak height at an integration time of two seconds. This showed a linear relationship of
chemiluminscence measured in millivolts to NO concentration measured from zero to fifty
picomoles.
Figure 4.3: Relationship between chemiluminscsnce in millivolts to nitric oxide concentration in
picomoles.
m
e*
Eto
plo
tr*
!-
E-
610
dgnrl.l7.9'l{Onurr-tt
0
0tr0t6m?lgl$'|oa5
t{Oernln[(pfllol)
Taken from Archer S. Measurement of Nitric oxide in biological models. FASEB Journal 1993; 7(2):
349-360 (Archer 1993).
The background output of the photomultiplier is relatively stable, unlike the other light source
meters which can have surges. The dark current (as it is known) can be affected by changes in
temperature, light and alterations in voltage, and by very high levels of NO. Large
disturbances in the dark current may take days to stabilise. For this reason most are equipped
with a cooler as cooling the photomultiplier tube improved the signaUnoise ratio. Preferably
the analyser should also be kept in a temperature controlled room and away from any other
equipment that potentially generates heat.
A red cut off filter separates the reaction chamber and the photomultiplier to prevent the
detection of light with wavelengths below that of the NO/O3 reaction. These include blue or
ultraviolet emission of alkenes and sulphur containing species such as the reaction of
hydrogen sulphide (HzS). These reactions are unlikely to be a major problem in practice as the
compounds are present on a greatly reduced scale compared to NO with far less or no
biological activity, and they are not volatile. As the NO/OI reaction takes place in the gas
phase, this is ideal for measurement of gaseous samples such as exhaled air. The resulting
100

NO2 produced can be removed by a soda lime column and resulting Og can be removed using
a charcoal column; or the exhaust can be immediately directed outside. I opted to use columns
to 'scrub' the compounds from the exhaust air. The luminescence signal as measured by a
photon counter and transmitted as an electrical signal and (in the early adapted machine which
we used) this was then transmitted to a chart recorder. In the later versions of the machine the
signal went to an analogue digital computer.
Sensitivity: The detection threshold of NO is 20-50pmol. In aqueous solutions the
chemiluminescence assay has been reported to detect as little as 10-13 M of NO
(Zafrnou and McFarland 1980). The chemiluminescence/1.{O curye has been
found to be linear between NO doses of 0-50pmol (Fontijn, Sabadell et al.
1970) and 300-3000 pmol (Menon, Wolf et al. 1989; Brien, Mclaughlin et al.
19e1).
Specificity: Chemiluminescence is almost exclusively due to NO. As mentioned above,
there are few other substances that react to produce light and these are either
non-volatile or are not biologically important such as the production of HzS
and alkenes. One author (Archer 1993) found that at very high levels a solvent
used for many drugs (dimethyl sulfoxide - 'DMSO') could also cause a
chemiluminescent signal. This is unlikely to be a problem in exhalate and
pertains more to the use of high dose chemicals on explanted or cultured tissue
(Mottu, Laurent et al. 2000). However this chemical is a known antioxidant
and more recently has been employed as a treatment in certain inflammatory
conditions, most particularly in interstitial cystitis (bladder inflammation), but
also for some rheumatologic diseases (Santos, Figueira-Coelho et al. 2OO3i
Chancellor and Yoshimura 2004; Parsons 2004).
The NO chemiluminscence analysers available in 1995 were designed for measuring NO
concentrations within 2-4000pbb and 40-400ppm range in a continuous ambient air sample.
They had been adapted for online recording and had a stabilised measurement capability as
measured by drift without using an auto zero over 24 hours. In most this was cited at between
zero and two ppb. They operated in ambient temperatures of 5-400 C and humidity of 0-95Vo.
The estimates of NO concentrations were decreased 10-15Vo at l00%o humidity, however NO
pre-drying of the expirate was not thought to be necessary for most environments @gure 4.2
does have a pre-drying unit added in the diagram). The original response time of these
machine developed to measure airway pollution were long, too long for the purpose that we
101

equired it. For example, there was a 90 second delay in the model we used (Model 207,
Dasabi Corporation), but modification of the circuitry and the sending of the pre-computer
analogue signal directly to the chart recorder (with assistance from the Biomedical
Engineering Department at the Royal Brompton Hospital, London, United Kingdom: Carolyn
Busst and Ron Sinclair) decreased the response time - a factor checked in the first
experiments made to establish the analyser's capability (see Chapter 5). The option of a
reader, chart recorder or an analogue digital computer may seem unusual now in the days of
easy, fast computer access, and the new analysers have these inbuilt for signal display. The
sampling flow of the NO analyser depends on the vacuum pump rate. The NO analysers listed
above at the time that these experiments began sampled at fixed rates between 200 and 800
mls/min. This too was modified for use on the first experiments using this machine (see
Chapter 5). The newer purpose built analysers have a wider range of sampling flow options of
25, 50,100,250 and 5OOmls/min.
Table 4.1: The companies providing chemiluminescence analysers adaptable for nitric oxide
measurement in 1995
ChemLab Instruments Ltd, Hornchurch, United Kingdom
Columbia Scientific Industries Corporation, Austin, Texas,
United States of America.
Dasibi Environmental Corporation, Glendale, Galifornia,
United States of America (Model2107)
Eco Physics, Durnten, Switzerland (CLD 700)
Lear-Seig ler/Mon itor Laboratories, En glewood, Colorado,
United States of America.
Seivers Instruments, Boulder, Colorado,
United States of America. (NOA')
Thermoelectron, Warrington, United Kingdom. (Model 42)
In addition to detecting NO in gas samples, I will mention briefly that the technique can be
used for liquid samples. At room temperature NO has a partition coefficient of 2O; that is in a
sample containing both liquid and gas, there is 20 times more NO in the gas phase than
dissolved in the liquid phase. So if a fluid with no air contact is then injected into a chamber,
the NO quickly escapes into the gas phase and this can be aspirated into the
chemiluminscence analyser. The rest of the NO can then be "stripped" from the solution by
bubbling the solution with an inert gas under vacuum conditions usually at a rate of between
8-10 mls/min driving it into the gas phase which can then be measured (Chung and Fung
1990; Archer, Shultz et al. 1995; Hampl, Walters et al. 1996). However it is vital to avoid
foam or bubbles getting into the reaction chamber as this can then coat the PMT and impair its
102

function. This is especially important with biological specimens as proteinaceous material
(such as albumin and blood) foams during stripping and therefore provides more difficulties.
The chemiluminscence assay can also be used to measure the other intermediates and other
end products of NO oxidation such as s-nitrosothiols, nitrite and nitrate. The compounds can
be made to release the NO by reducing them with acid (usually hydrochloric acid, citric acid
or glacial acetic acid is used). This gives the nitrosonium ion (NO) which then can react with
anions (such as iodine which is commonly used) which then dissociates to give NO, water and
iodine, with the No able to be measured in the chemiluminscence analyser.
Figure 4.4: Reaction equations used to release nitric oxide from other nitrogen compounds
NOz' + 2H+ -> NO* + H2O
NO* + l'+ ONl
2ONl +2NO+lz
As can be seen, this is more difficult than the direct gas measurement and involves more
steps. In addition, all the reagents must be replaced with every sample and extra time required
for signal of the acid and iodine compounds alone. It is very temperature and (clearly) pH
dependent. However, as mentioned above, when correctly performed it remains very sensitive
to NO in small amounts (Zafiriou and McFarland 1980).
4.6.1 Calibration
Machine calibration had to be carried out regularly, and at the time a number of commercial
gas companies offered cylinders with graduated NO concentrations:
o BOC Gases, Surrey Research Park, Guildford, Surrey, UK
o Scott Specialty Gases, Troy, Michigan
o Matheson, East Rutherford, New Jersey
(Modified from our original published chapter (Byrnes, Bush et al. 1996)).
These were prepared by oxidation of ammonia at 5000C over platinum gauze or produced by
passing an electric arc through the air (Braker and Mossman L975).Individual calibration
samples of NO can be prepared chemically either by adding acids to sodium nitrite (NaNOz),
or mixing NOz- and a denitrifying enzyme (Pai, Payne et al. 1987). A preparation of a
saturated NO solution (NO = 3 mM) is needed. Double distilled cold water is bubbled with
103

helium for 30 minutes to remove Oz. The water is then bubbled with pure NO (>99.0Vo1) for
30 minutes in a glass sampling bulb. Samples can be aspirated through a rubber gas-tight
syringe. Before aspiration Nz should be injected into the glass bulb to exclude Oz (Archer
1993). Once a saturated NO solution has been made serial dilutions of the NO gas or solution
can be made using deoxygenated HzO. Having reviewed the alternatives, the company that I
used was BOC Gases (Suney Research Park, Guildford, Suney, United Kingdom) who were
local to where the research was taking place and who regularly supplied gases to the Royal
Brompton Hospital. Also they could provide NO in cylinders of appropriate concentrations
suitable for calibration of the levels we intended to measure (see below).
Whether personal or commercial preparations of NO are being made for calibration purposes
it is imperative to avoid contamination with Oz which will reduce the NO in the sample. Zero
calibration can be done with NO free certified compressed air. Alternatively, passing room air
though the ozone generator of the chemiluminescence analyser, which converts all NOx to
nitrogen dioxide and then passing the resultant gas through soda lime and activated charcoal
to remove the NOz and ozone respectively can also produce NOx free air. The most useful
calibrations gas concentrations are zero and concentrations within the usual levels measured,
with the final calibration just above the highest levels likely encountered to avoid the need for
linear extrapolation beyond the range. Not all the companies could provide appropriate
concentrations or had them in stock. In normal subjects on the current analysers with the
current measurement protocols, the range is 0-25ppb for oral exhaled NO and 250-1000ppb
for nasal NO but the levels on the older machines were often higher (0-80ppb) for oral
exhaled measurements. The reason for these differences will be come clear when reviewing
the methodology experiments that I completed (see Chapter 5). I always had a zero and two
NO cylinder concentrations for calibration for the experiments described in the work of this
thesis. They were either 27ppb and 103ppb and ll0ppb or 55ppb and 118ppb when the first
cylinders were exhausted.
4.6.2 Safety and toxicity
In the final part in this chapter I want to review the toxicity of NO, and the two gases
generated using the chemiluminescent technique -
NOz and ozone. The reactions and effect of
NO at the cellular level has been covered in Chapter 3 sections 3.2 'Properties of nitric oxide'
and 3.3 'Reactions of nitric oxide'. NO must be handled with care -
the toxicity of NO is due
to the gas itself and also the interactions that occur with oxygen when it forms a dimeric form
of NOz - a reddish brown toxic gas @udvari, O'Neil et al. 1989) which in high 02
r04

concentration can form the superoxide perioxynitrite (OONO). In part the effects of these
gases have been covered when reviewing the noxious effects of air pollution with particular
reference to the nitrogen oxides in Chapter 2.2. NO and, even more so, NOz are common air
pollutants (Morrow 1984; Anonymous 1995; Anonymous 1996) with inspired NO
concentrations from airway pollution ranging from 20-500ppb in non-polluted areas to
1.5ppm or greater in polluted areas (Aranda and Pearl 2000). As previously mentioned,
cigarette smoke contains NO concentrations in order of lOOppm (Norman and Keith 1965), or
higher depending on the cigarette type (Borland and Higenbottam 1987) and cigarette smoke
can inhibit NADPH and myeloperoxidase (Nguyen, Finkelstein et al. 2001). While this does
not result in an acute respiratory event, it might cause an increase risk of mutations over time
and thus it contributes to cigarette smoke carcinogenic potential.
With the use of nitrous oxide (NzO, "laughing gas") effects were noted when N2O cylinders
were contaminated with NO showing that high NO concentrations could give rise to acute
pulmonary oedema and methamoglobinaemia (Anonymous 1967; Clutton-Brock 1967). High
levels of NO were needed to get this response in experiments with animals. For example,
exposure of lambs to 80ppm of NO for three hours in 2l%o oxygen did not result
methaemoglobinaemia or any of the acute problems previously documented (Frostell, Fratacci
et al. 1991). Exposure of dogs to 20,000ppm did result in this acute respiratory event with
high morbidity and mortality (Shiel 1967). Inhalation of 50ppm of NO impairs the
performance of learned tasks and prolongs brainstem evoked potential responses in rats,
without significantly elevating methaemoglobin levels (Groll-Knapp, Haider et al. 1988). In
healthy subjects inhaling lOOppm of NO for three hours, methaemoglobulin levels increased
by I.77Vo of total haemoglobin to a peak at 45 minutes. Serum nitrogen oxides (NOz, NOI)
increased from 36.7 to 124umol L-l at 172 minutes and then declined (Young, Sear et al.
1996). Inhaled NO at 5ppm resulted in thickened alveolar membranes in rabbits (Hugod
1979) and NO at 43ppm and NO2 at 3.6ppm narrowed the surfactant hysteresis, altered
epithelium ultra-structure and caused interstitial atrophy (Hugod 1979). Intermittent NO
exposure over a 9 week period in rats resulted in degeneration of interstitial cells and the
interstitial matrix leading to an emphysematous-like destruction of alveolar septa (Mercer,
Cosra et al. 1995).
NOz is more toxic and has long been known to cause respiratory damage and fatality
(Kooiker, Schuman et al. 1963: Wagner, 1965 #1030). It was early recognised as a concern in
atmospheric pollution (Kennebeck, Wetherington et al. 1963). NOz has been shown to be
taken up easily by lung lining fluid at a rate related to its speed of reaction to glutathione
105

(Postlethwait and Bidani 1990; Postlethwait, I-angford et al. 1990). This is increased with
alkalosis and hyperthermia @ostlethwait, Langford et al. 1991; Postlethwait and Bidani
1994). A number of studies have been conducted to determine the acute effects of NOz
exposure at levels seen in air pollution on airway lining (usually the nose), lavage fluid, lung
function and airway reactivity in normal and asthmatic adult subjects. The exposures have
ranged from two to six hours at levels from 200 to 800ppb, aild were to NOz alone or
combined with either ozone or SOz @evalia, Rusznak et al. 1996; Devalia, Bayram et al.
1997; Jenkins, Devalia et al. 1999). These exposures resulted in disruption of the airway
epithelia, resulting in an increase of inflammatory cells with eosinophil 'priming', and a
release of inflammatory cytokines such as TNFo and IL8. Increased airway hyper-reactivity
in normal (Utell, Frampton et al. l99l) as well as asthmatic subjects was also demonstrated
@evalia, Rusznak et al. 1996; Devalia, Rusznak et al. 1996; Jenkins, Devalia et al. 1999).
Cilia were also disrupted and, after exposure to NOz at 2ppm for 4 hours, ultra-structurally
altered cilia with excess matrix and multiple ciliary ru(enomes were seen (Carson, Collier et
al. L993). Intermittent NOz exposure over a period of weeks in rats also resulted in
emphysematous like destruction of alveolar septa with reduction in the ventilatory surface
@vans, Stephens et al. 1972; Freeman, Crane et al. 1972; Mercer, Costa et al. 1995). There
was also hyperplasia of the respiratory epithelium with squamous metaplasia (Maejima,
Suzuki et al. 1992), disturbed surfactant (Muller, Barth et al. 1992) and aldehyde release
(Robison, Forman et al. 1995). Prolonged inhalation of 2ppm NOz is associated with terminal
bronchial epithelial hypertrophy and alveolar cell hyperplasia (Sherwin, Dibble et al. t972).
Gas mixtures of NOz and NO, each in concentrations of less than lppm, have been shown to
reduce peak expiratory flows and diffusion capacity in beagles (Bloch, kwis et al. 1972).ln
studies using lower concentrations (similar to pollution values) the damage in the airway was
related to duration of exposure (Maejima, Suzuki et al. 1992), but when exposed to high doses
the dose was more damaging than the duration of exposure (Lehnert, Archuleta et al. 1994).
NOz of l75ppm has been used in rats to create severe lung damage but avoiding death to
develop an ARDS model for study (Meulenbelt, Dormans et al. 1992). NOz has been
recognised as the product of grain fermentation responsible for the syndrome of pulmonary
oedema and haemorrhagic bronchiolitis obliterans known as silo fillers disease (Ramirez and
Dowell l97l; Horvath, doPico et al. 1978; Leavey, Dubin et d. 2004).
The Occupational Safety and Health Administration guidelines (NIOSH 2005) recommend
that the safety limit of mixed nitrogen oxides is below 25ppm. Inhalation at this concentration
may cause immediate pulmonary irritation and higher doses may cause haemorrhagic
r06

pulmonary oedema in days. The guideline states that inhaling NO at a dose of 25ppm for
eight hours is the maximum safe dose and time limit. The level considered an immediate
damage to life and health is estimated at 100 to 150ppm based on older toxicology studies
(Carson, Rosenholtz et al. 1962; Sax 1975). For NOz the recommendation has been altered
down from 5ppm to only lppm (in 1996) for eight hours as the exposure limit (NIOSH 2005).
kvels of 10 to 20ppm have demonstrated to be a mild irritant (Patty 1963), levels of lOOppm
are dangerous causing haemorrhagic pulmonary oedema (NRC 1985), and 150 to 200ppm
fatal from haemorrhagic pulmonary oedema (Braker and Mossman 1975).
Ozone is a colourless to blue gas with a pungent odour. Again the 'usual' exposure to ozone is
through airway pollution as mentioned in Chapter 2.2, (Anonymous 1995; Anonymous 1996)
where it is one of the most toxic elements reaching one hour mean ambient concentrations of
200-400uglm3 1Rnu, 1,966; Lippmann 1989; van Bree and Last lggT). Shorr term ozone
exposure causes reduction in lung function, increased airway hyperactivity, increased airway
inflammation, increased respiratory symptoms and hospital admissions (van Bree, Marra et al.
1995; Nikasinovic, Momas et al. 2003). Exposure to long term elevated ozone levels is again
associated with reduced lung function, increased respiratory symptoms, exacerbations of
asthma and airway cell and tissue changes (van Bree, Mara et al. 1995; Anderson, Ponce de
I-eon et al. 1998). At the tissue level, ozone with its high reactivity is usually consumed as it
passes through the first layer of tissue it contacts at the airway/air interface or lung/air
interface (Pryor 1992; Pryor, Squadrito et al. 1995). Since the lung lining fluid is 0.1 to 20
microns thick, in the thinnest parts it can react directly with cells and it has been shown, for
example, to act with the type II macrophages disrupting surfactant activity. The majority of
the interface area has thicker lining fluid and ozone is transformed into other reactive species
such as aldehyde and hydrogen peroxide, and these go on to cause the airway damage. This
causes epithelial disruption and increased permeability with an increase in inflammatory cells.
The disruption of cell membranes also results in a release of cytokine, cyclo-oxygenase and
liopoxygenase products. In studies looking at toxicity, differences have been demonstrated
between species with regards to dose and length of exposure that results in damage (Dormans,
van Bree et al. 1999). Inhaled ozone is also used in animals to give a lung fibrosis model for
study (Cross, Hesterberg et al. 1981; Yu, Song et al. 2001). In direct comparison of toxicity,
ozone was found to cause four times as much damage to the airways as NO2 (Chang, Mercer
et al. 1988) and be ten times more toxic to macrophages (Rietjens, Poelen et al. 1986).
The Occupational Safety and Health administration guidelines recommended exposure limit
for ozone is 0.lppm (0.2mglm3) (NIOSH 2005). The immediate danger to life and health level
to7

is set at 5ppm (AIHA 1966) based on pulmonary oedema that developed in welders who had a
severe acute exposure to an estimated 9ppm of ozone (plus other pollutants) (Giel, Kleinfeld
et al. t957). Fifteen to 20ppm is lethal to animals in two hours, exposure to 50ppm for one
hour "would likely prove fatal to humans" (King 1963).
4.7 Chapter summary
This chapter has reviewed the possible compounds and methods that could be used to
determine the level of NO based around the known pathway of formation from L-arginine to
L-citrulline and NO, and the nitrogen compounds. The chemiluminescence technique was the
most appropriate for use in measuring exhaled NO in humans. This analysis is based on the
reaction of NO with ozone which produces light, and the light intensity has been shown to be
directly proportional to the concentration of NO in the sample. The chemiluminscence
technique appeared to be both appropriately sensitive and specific for NO for this purpose.
The chemiluminescent NO analysers available at that time had been developed to measure
NO in air pollution. The job now was to assess their use and examine what technical
alterations were required to enable measurement of NO in exhaled air in humans in a reliable
and reproducible way. This was needed before discussion as to what these levels meant, and
before a trial of measurement in children. Initial testing showed that the response time of the
analyser was too slow for our purpose initially, but a rerouting of the signal to a chart recorder
decreased the response time and meant that it was practical for use for the studies that follow.
Finally in this chapter, I have reviewed the toxicity of NO, NOz and ozone. Although I was
only going to be measuring NO in exhalate and therefore it was unlikely to reach high levels,
I was using NO calibration gases. NOz and ozone are the two gases created by this technique
so I wanted to also assess their toxicity, although I was planning to remove them immediately
by having them 'scrubbed' with a soda lime column and a charcoal column respectively.
The next chapter examines the chemiluminescent analyser Model 2107 @asibi
Environmental Corporation, Glendale, California, USA) in more detail, and the
commencement of the machine and subject methodology studies. What was known about
exhaled NO levels at the time of cornmencement of these studies in 1995 will also be
reviewed here.
108

NB: The review presented in this chapter and the start of the machine methodology presented
at the beginning of the next chapterformed the basis of the publication:
Byrnes CA, Bush A and Shineboume EA "Methods to Measure Expiratory Nitric Ortde in
People" in "A Volume of Methods in Enqmology" edited by Lester Packeter Academic press
Inc. 1996.
109

5.1
Chapter 5: Methodological assessment of the chemiluminescence analyser
Introduction
The previous chapter reviewed the methods available to measure NO in biological systems
and determined that the most appropriate method for measuring NO in exhaled air using a
chemiluminescence analyser, originally developed to assess NO pollution levels. The
technical aspects of the chemiluminescence analysers were reviewed and the direct correlation
between the intensity of light measured and the concentration of NO described. The
instrument that I ultimately used was the Dasibi Environmental Corporation 'Model 2107'.In
this chapter the characteristics of this analyser will be reviewed, as well as the other
equipment. Connections between a number of analysers were developed to enable
simultaneous measurement of NO, COz, flow and pressure. I will also mention the other
people involved in the work and the role each person undertook.
5.2 The equipment and personnel
5.2.1 Chemilurninescence Analyser'Model 2107'
The chemiluminescence analyser Model 2107 (Dasibi Environmental Corporation, Glendale,
California, USA; local supplier Quantitech Ltd, Unit 3 Wolverton Rd, Old Wolverton, Mlton
Keynes, UK) was designed for measuring NO concentrations within a 2-4000pbb range in a
continuous ambient air sample and operated in ambient temperatures of 5400 C and humidity
of O-9SVo.It had been adapted for online recording with a sampling rate at 240 mls/min and
had a highly stabilised measurement capability (drift without auto zero over 24 hours of
lppb). The original response time of this machine was long at 90 seconds, too long for the
purpose for which it was now required. Modification of the circuitry and the sending of the
pre-computer analogue signal directly to the chart recorder decreased the response time to 6.4
seconds (see below). The estimates of NO concentrations were decreased by lo-l1Vo at IOOVo
humidity; however NO pre-drying of the expirate was not thought to be necessary (this may
have been considered differently if the experiments were conducted in areas of high humidity
such as within New Zealand). I did use a water absorber when doing the reservoir set of
experiments only (see Section 6.4). This machine operated on the same principles as
described in the last chapter. The gaseous specimen was drawn by the vacuum pump into a
reaction chamber where the ozone was generated internally by an electrical discharger. The
NO and 03 wore mixed in front of a red-sensitive photomultiplier tube, the light emission of
this reaction was measured at 660 to 900nm in the red sensitive range, and the signal
110

amplified and detected as a current. As mentioned in the previous chapter, the dark current of
the photomultiplier can be affected by changes in temperature and this model had an inbuilt
cooler that kept the photomultiplier temperature at -200C to improve the signaUnoise ratio.
The machine itself should ideally also be kept in a temperature controlled room (as was
suggested in the machine manual). In our case it was placed in a small air conditioned room
within the hospital away from any other equipment that potentially generated heat - though
there was one window into the room which resulted in some temperature change. The NO
analyser was re-serviced just prior to our experiments taking place.
The NO analyser was calibrated with azero and two known NO gas concentrations before and
after every subject during the methodological studies and between every two subjects when
later studies in children were being undertaken. Two of the gases used were commercial
preparations of NO made for calibration purposes by BOC Gases (BOC Gases, 10 Priestley
Rd, Surrey Research Park, Guildford, Surrey, UK) and as the studies progressed included
concentrations of 37ppb, 55ppb lO3ppb, l lOppb and I lSppb with the balance being nitrogen
(Nz). Calibration gases at these low levels were extremely difficult to get at the
commencement of these studies as these gas mixtures had no known use other than
calibration, and this was not widely used at this time. The third gas was a zero calibration and
this was prepared by passing room air through the ozone generator of the chemiluminescence
analyser, which converted all NOx to NOz. This was then passed through a soda lime and
activated charcoal column to remove the NOz and Ot respectively and then passed direct into
the analyser. The resulting NOz and 03 from the experiment were removed in the same
manner by passing through the soda lime column and the charcoal column. To minimise NO
loss by absorption or interaction with other surfaces (see Section 5.4), teflon tubing (4mm
extemal diameter) was used as the most inert substance to connect the machinery and to
sample from the subject.
5.2.2 Capnograph, pressure transducer, Jlow meter and chart recorder
In addition to measuring NO itself, we wanted to be able to measure other parameters. At the
time, the early publications available on exhaled NO from the lung, which will be reviewed in
the next chapter (see Section 6.3), seemed to show a consistency in the trends found in NO
results in different subject groups but the absolute levels of NO reported between the
investigators were very different. This suggested that some of the measurement techniques
were altering the NO results recorded. There was also discussion in the literature about where
the measured NO from exhalation was anatomically being generated. In view of this, we
lll

wanted to compare the patterns of exhaled NO with COz, the latter being largely of alveolar
origin. For this purpose, I elected to measure expiratory COz, flow and pressure in addition to
the expiratory NO levels which meant developing a number of monitors connected in parallel
that could operate simultaneously. The equipment used, as described below, was all
equipment that was able to be 'seconded' from the adult lung function laboratory and put
together by myself and one of the biomedical engineers (Ms Carolyn Busst, see below) and
the ultimate format is demonstrated in Figure 5.1.
Figure 5.1: Schematic diagram of how the analysers were placed
This is a schematic diagram of the analysers and connections required to measure nitric oxide, carbon
dioxide, mouth pressure and flow. The signals from each of the 4 machines went directly to, and were
displayed on, a Linseis 4 channel pen chart recorder (Linseis Limited, Cambridge, United Kingdom)
with each signal denoted by a different colour.
The COz levels were measured with a Morgan Capnograph (PK Morgan, Kent, United
Kingdom) which sampled at2O0 mls/min-I. Calibration of COz was done using ^zero and gas
cylinder of known COz concentration at 5.9Vo and later 5.7Vo also provided by BOC gases
(address given above). The mouth pressure was measured using a Medex Straingauge
pressure transducer (Medex Medical Incorporated, Rossendale, United Kingdom) which was
connected at right angles to the flow via a connector in the mouthpiece. This was then
directed through a Galtec pressure amplifier (Galtec Limited, Isle of Skye, United Kingdom)
which measured the pressure in mmHg and showed a linear and stable response. The flow
was measured via a flow meter to a low flow pneumotachograph (CT Platon Limited, Jays
t12

Close, Viables, Basingstoke, United Kingdom) and a GM electrospirometer (GM Instruments
Limited, Kilwinning, United Kingdom) then gave an electrical output that could be sent to the
chart recorder. The pneumotachograph was initially calibrated by passing flow from an air
cylinder set at known flow rates (100, 250, 500, 750 and l0o0mls/min) and was linear over
this range but then seemed to read slightly but consistently high after 1l0omls/min. In initial
self trials (myself and Carolyn Busst) the response from human exhalation seemed unsteady
or 'noisy'. This appeared to be the effect of the small but repeated voluntary alterations in
expiratory flow by the subject (one of us) trying to maintain a flow without adequate
feedback. It disappeared when the steady flow of compressed air was put through the
rotameter and the pneumotachograph at designated flow rates. In view of this, we then added
the rotameter as part of the routine exhalation. Subsequently, all the subjects were directed to
watch the rotameter during exhalation and aim to maintain a set flow rate by keeping a
'bobbin' at a set number on the upright scale. This improved the steady quality of the signal.
The rotameter was a Platon glass tube c6 with stainless steel float 6D (Flowmeter Model
GTV - CT Platon Ltd, address above).
Figure 5.2: An example of tracing from the testing
sec9-
-?a.S
The first thing to note is that the chart record is read from right to left which is how it was generated
from the chart recorder. Note the offset of the traces from the different analysers which were displayed
n3
- 4?o
!4sr,J
I
+415
i
-g,2
it,+
j
i56
-48
i l(-,t l ;4.o
i-_
ieo
I
I
i 01.5
i
"45i
'1- ?L5
i
bo--
I
I
L7,Z
rL?
I
I
.4'S
-0.g
)
ito
lr
I
- lE'6
-,t5.5
-12,+
- t,a
+C.L
it'l
o'
;rn
; k''l - tf l3t
No(ppb; ; o2%)
1cm=22.5 1cm=o.9
-o :o
MP(mmHg)
lcm=3. 1
o

in different colours that became the standard: NO in green, COz in red, and mouth pressure in blue. As
this is an example from the direct method of analysis, flow is not denoted. The measurements of each
parameter were calculated from the calibration markings (see Chapter 6).
As can be seen in Figure 5.2, the NO rose to a plateau which often had additional small
deviations, the COz continued to rise to a peak throughout the exhalation, the mouth pressure
(and flow with the t-piece measurements) were under voluntary control and therefore less
exact plateaux markings were seen and the lines of measurement were drawn through the
middle to determine that they had been appropriately close to requested levels. Note the offset
of the traces from the different analysers which were displayed in different colours that
became the standard: NO in green, COz in red, and mouth pressure in blue. As this is an
example form the direct method of analysis, flow is not denoted but when measured, as
during the t-piece sampling technique, was displayed as a brown trace. The measurements of
each parameter were calculated from the calibration markings.
We (Carolyn Busst and myself) checked the reported sampling rates of the NO and COz
analysers. As mentioned, the sampling rate was determined by the vacuum pump within the
NO analyser and had a fixed rate. Similarly, the sampling rate of the CO2 analyser was also
determined by the vacuum pump which could apparently be adjusted by pressing the
calibration button on the front of the analyser panel and the rate changed by pressing the up
and down arrow keys. We left the sampling rate at the setting it was on when we received it
from rhe Adult Physiology Laboratory (Royal Brompton Hospital). This was in the middle of
the range suggested by the manufacturers and was used for all assessments. The check of the
machines' sampling was repeated a number of times prior to development of the protocol for
the experiments and again between the methodological experiments conducted with adult
subjects and commencement of the studies on paediatric subjects. We listed what was to
become our standard procedure as the following steps:
o Connect the outlet line from the mouthpiece t-piece connection to the lower port of the
rotarmeter.
o Turn the rotameter needle valve off to ensure that the room air cannot be sampled via
the rotameter.
o Disconnect the pneumotachograph from the rotameter.
o Run the paper to record zero sampling.
o Connect the outlet side of the pneumotachograph to the inlet of the mouth piece.
tt4

o
o
Ensure there is no leak in the connections and room air will now be drawn through the
pneumotachograph by the NO analyser and capnograph thus measuring their sampling
rates.
Run the chart recorder to mark the level of the combined sampling rates.
Label the chart record.
Reconnect the pneumotachograph to the upper port of the rotameter.
5.2.3 Personnel
Adjustments to the original NO analyser circuitry and the sending of the pre-compurer
analogue signal directly to the chart recorder were made with the assistance of the Biomedical
Engineering Department, Royal Brompton Hospital (Mr Ron l-ogan-Sinclair [later of logan
Research Ltd, Rochester, Kent, UK] and Ms Carolyn Busst). The connections and adaptation
of the equipment to do all the measurements simultaneously and the experiments on assessing
machine capability were done by myself and Ms Carolyn Busst. I designed and conducted the
methodological experiments on the healthy adult subjects. I also designed the studies
involving normal and asthmatic children as subjects. Following ethical approval from the
Royal Brompton Hospital Ethics Committee, Dr Seinka Dinarevic and I gave presentations to
the local primary school and, with individual signed parental or caregiver consent, we
enrolled children the school. I also enrolled all the asthmatic subjects from the weekly
paediatric asthma and/or the weekly paediatric general respiratory clinic at the Royal
Brompton Hospital in which I was working. Dr Seinka Dinarevic helped transporting the
children to and from the local school, and we collected the consent and the questionnaire data,
did the clinical examination, measured height and weight, performed the lung function and
exhaled NO testing on all the children. The manual calculation of the levels for each
parameter from the chart recorder using the calibration measurements was completed by us
both' I entered the data into a statistical analysis programme (Statistical Products and Services
Solutions, SPSS Inc, Chicago, USA - package U 7.0), while Dr Seinka Dinarevic double
checked the entries. We shared the drafting of the paediatric paper, while I wrote the other
papers in conjunction with contributory editing and suggestions from the other named authors
on each. I remained the key driver and contact for all this work. The same equipment was
used for all the following experiments and the work was carried out in the Paediatric
Department at the Royal Brompton Hospital, South Kensington, London, England3. The main
supervisor for this work was Professor Andrew Bush. In addition, Professor Peter Barnes was
eNow the 'Royal Brompton & Harefield National Health Trust, South Kensington, London NW3 6Np, U.K.
115

an advisor for this work and main supervisor for other research done at the time (publications
listed as Appendix 1), and Dr Elliot Shinebourne also acted as advisor, particularly in the
early stages.
5.3 Direct versus reservoir measurement
The concept of the proposed methodological experiments was to have a subject exhale into
teflon tubing connected to NO, CO2, flow and mouth pressure meters allowing measurement
of each. It was established that all the equipment could come directly off the mouth piece,
with the exception of flow connector. This was due to the necessity of the addition of a
rotameter to provide a visual feedback guide for the subjects regarding expiratory flow and
added a level of complexity to the system. In view of this, I elected to do a series of
experiments in which one set of exhalations was connected directly to the NO analyser,
pressure and COz monitors (known as the 'direct method') and a second set of exhalations
where a t-piece system enabled the additional measurement of flow (known as the 't-piece
method' or 't-piece sampling system'). A comparison of the two techniques is shown
diagrammatically in the next chapter in Figure 6.5. Below is a photograph of one of the
children performing the procedure with the analysers labelled in Figure 5.3. Unfortunately I
did not take any photographs of the subjects during the methodological experiments.
Figure 5.3: Photographs of one of the children performing the exhalation

The photographs are taken from behind (a) and from the right side (b) of one of the paediatric subjects
'Jonathon'that was testing the procedure (used with permission) as he performs a single exhalation
into the mouthpiece. I have labelled the connections and analysers as seen below (not all visible in
both photographs):
1. Mouth piece.
2. Connector.
3. NO analyser (Model2107, Dasibi Corporation).
4. CO2 analyser (Morgan capnograph).
5. Low flow pneumotachograph (CT Platon Limited).
6. Mouth pressure analyser (Medex Straingauge pressure transducer).
7. Galtec pressure amplifier (Galtec Limited).
8. GM electrospirometer (GM Instruments Limited).
9. Four channel pen chart recorder (Linseis Limited).
10. Timing device.
11. NO zero calibration column.
12. Calibration gases (BOC gases Ltd).
13. The f irst of the pages listing instructions on the wall.
5.4 Assessments of the analysers
The response time of each analyser had to be determined to enable direct comparisons. The
chart recorder recorded continuously at a paper speed of 50mm/second and was set so that as
the recording came off, the signals were read from right to left. An oscilloscope was
connected to the system with a three way tap which on turning allowed the test gas to flow
down at the apparatus at a rate comparable to the subjects' expiratory flow rate (500mls/min
was chosen) starting at the subject mouthpiece. The output of the analyzers was displayed on
the same oscilloscope. The measurement of the delay time and the response time was then
measured with a stop watch.
rt7

Delay time: the time between turning on the switch and the onset of the analyser
signal
Response time: the rise time that from onset of signal to 95Vo of plateau or a square wave
The experiment was repeated 10 times in all with times recorded for each of the analysers.
Results are presented in the Table 5.1.
Table 5.1: The delay and response time of the NO, COz, mouth pressure and flow meter analysers
used
Analyser Delay time Response tlme
NO analyser Model2107 5.9 seconds
(SEM 0.8 s)
CO2 Morgan capnograph 0.11 seconds
(SEM 0.003 s)
Medex Staingauge pressure
transducer (mouth pressure)
SEM = standard error of the mean, s = secondsr lrls = milliseconds
This established that the delay time for the NO analyzer at 5.9 seconds (SEM 0.8s), and95Vo
response time at 0.5 seconds (SEM 0.1s) showed the NO analyser responded much more
slowly than the CO2 analyser at a delay time of 0.1 I seconds (SEM 0.003s) and 95Vo response
time at 0.41 milliseconds (SEM 2.9ms). It has to be remembered that the delay time was
influenced only by the time that it took for the gases, flow or pressure to get to the analysers
and the time to cornmence registering the change. The response time should be a square wave
for 'on/off' phenomena like the pressure and the flow, but could be influenced by other
parameters in the measurement of the gases. The pattern of excretion through the exhaled
breath was unknown for NO, and was known to increase with the duration of exhalation for
COz. So while the response time was measured with these two gases, it was the delay time
that was the key factor. In essence it meant that when comparing the NO and COz recordings,
an allowance needed to be made for the difference in time delay of the measurements from
each machine and for the 2mm offset of the pens on the recorder.
Finally, confirmation of the detection of the appropriate gases was checked for the two
analysers. Cylinders of IOOVo Oz, 5.9Vo COz and 37ppb of NO were run through the NO
analyser and the CO2 analyser with only the NO gas detected and the others giving a zero
reading on the NO analyser machine and only the COz gas detected with the others giving a
zero reading on the CO2 analyser machine.
7.3 milliseconds
(SEM 0.2 ms)
Platon flow meter 40.2 milliseconds
(SEM O.8 ms)
118
Seconds
(SEM 0.1 s)
41 milliseconds
(SEM 2.9 ms)
No appreciable delay
No appreciable delay

When planning the protocol, it was obvious that the response time of the NO analyser limited
the possible respiratory manoeuvres. I next considered online measurement of repeated single
exhalations analogous to lung function testing versus exhaling into a reservoir that was then
fed into the analysers. I briefly explored the use of a reservoir, as this had three theoretical
advantages:
o it would allow the measurement of a whole exhaled breath, whereas the online
measurement which would control flow may mean that at low flows the subject would
not reach complete exhalation before requiring to take another breath,
o it would allow me as operator to put the collected sample through the machine at a
constant flow instead of requiring the individual subject's voluntary but imperfect
control,
o performing tidal breathing over a set time or exhaling into a bag may have been easier
when it came time to measure the children rather than relying on their being able to do
controlled breaths into the analysers.
At the time that I was commencing the experiments, there had been some studies into NO
levels in exhaled air where a reservoir system had been used. The problems that had been
detected and needed to be overcome involved No instability -
a topic that has been touched
on repeatedly. Firstly, although in O2-free and haemoglobulin-/ree solutions NO may be
stable, in the presence of 02, NO levels rapidly decrease by 50Vo in 8-20 minutes. Secondly,
NO is adsorbed by most plastics giving low and/or variable readings. NO also interacts with
transition metals and with the sulftrydryl groups contained in rubber. Reservoir systems that
had been used were polyvinyl chloride, rubber and fluoroethylene propylene' Preliminary
studies in one group demonstrated that at levels of 5-30ppb there was no NO loss in the
polyethylene reservoir at 6 and 12 hours post collection, although they commented that any
loss may have been within the noise of the measurement that they were getting at that time of
10Zo (Schilling, Holzer et al. Igg4). Another group had found that using polyethylene tubing
compared to Teflon tubing resulted in loss of NO presumably as it adsorbed to the former,
particularly at higher (>40ppb) levels of NO (Kharitonov, logan-Sinclair et al. 1994). One
group with a Douglas bag collection (an anaesthetic bag used to collect exhaled air, usually
made of rubber) described no perceivable problems, but two other groups found them
unreliable at higher NO concentrations (Kharitonov, logan-Sinclair et al. 1994)'
Subsequently Mylar foil bags or balloons were used and seem to hold NO levels stable for at
least 48 hours (Steerenberg, Nierkens et al. 2000). However, this research group also
discarded the first part of exhalation to get lower respiratory tract samples and found that it
119

led to an increased water content that decreased measured NO levels, though this could be
minimised by exhaling through a water absorber into the reservoir bags (Steerenberg,
Nierkens et al. 2000). NO online measurement correlated well with NO measured via a
balloon technique (mylar foil) when measured immediately during times of low air pollution
and high pollution days (van Amsterdam, Verlaan et al. 1999). Another group disagreed and
showed that the online measurement was higher than the mylar bag reservoir results when
both were measured immediately (Silkoff, Stevens et al. 1999), though the offline
measurements were similar to the end expiratory online measurements (Jobsis, Schellekens et
al. 2001). NO concentration in the mylar bag was also shown to actually increase at24 and48
hours after collection (Silkoff, Stevens et al. 1999). When the first European Respiratory
Society Task Force (ERSTF) recommendations were published regarding the measurement of
exhaled air, they recommended the online technique of measurement (Kharitonov, Alving et
al. 1997).In some initial testing we did, teflon and "wine cask bags" (which are mylar foil
bags) did not seem to absorb NO, but a Douglas bag did seem to absorb NO and the material
of the bag became sticky. The use of teflon or teflon coated tubing to connect the reservoir or
the subject to the analysers appeared to minimise NO loss.
In view of the purpose of the studies, the online technique seemed more appropriate as it
allowed comparison of signals throughout the exhalation and allowed an alteration of the test
conditions to investigate how this affected NO results. A slow controlled exhalation from total
lung capacity appeared to be the most satisfactory exhalation for observing the results. This
manoeuvre yielded obvious NO and CO2 traces and most people, including most children in
the age group studied, could be easily trained to perform this correctly. Thus I could discard
the reservoir system for most of the studies which I believe excluded another uncertain
element from test measures.
5.5 Other measurements
For all the experiments utilising the healthy adult volunteers and normal and asthmatic
children, a respiratory examination was carried out, and weight (minimal clothing, no shoes)
and height were recorded on a digital Seca scale (Seca 770 medical scales, Seca Ltd, 4802
Glenwood Rd, Brooklyn NYI1234, USA) and a Harpenden stadiometer (Harpenden Portable
Stadiometer, Crosswell, Crymych, Pembrokeshire, SA41 3UF, UK) respectively. Lung
function was measured according to the American Thoracic Society (ATS) criteria (American
Thoracic Society and Association. 1994) on a compact vitalograph (Vitalograph Ltd, Maids
t20

Moreton House, Buckingham, United Kingdom) using the Polgar predictive equation (Polgar
and Promadh at.l97l) from a standing position for all subjects.
5.6 Chapter summary
This chapter has described the equipment, the sequence of analysers and the capabilities of
each that was used for testing in the studies presented in the following chapters (Chapters 6, 7
and 8). Chapter 6 will begin with a brief review of what was known about NO in the lung, the
early published articles in the measurement of exhaled NO and describe the first of five
methodological experiments carried out in healthy adult volunteers'
NB: The review and data presented in Chapters 4 and 5 formed
the basis of the publication:
Byrnes CA, Bush A and shinebourne EA "Methods to Measure Expiratory Nitric oxide in
People" in "AVolume of Methods in Enrymology" edited by l*ster Packeter Academic Press
Inc. 1996.
t2l

Chapter 6: Methodological studies of exhaled nitric oxide in healthy adult subjects:
6.1 Introduction
direct versus t-piece testing
So far, I have examined why a marker of airway inflammation that was easy to measure,
particularly in terms of being non-invasive, would be useful. I have outlined the developing
knowledge on NO as a physiological agent and inflammatory marker. The previous chapters
then explored how NO could be measured by chemiluminscence and presented data on
adapting an appropriate analyser with our own initial methodology testing. In this chapter, I
will very briefly review NO in the lung, the literature on exhaled NO available to me at the
beginning of my research, and present the methodology experiments themselves. The first
experiment, which will be presented in detail in this chapter, was to assess the ability to
measure NO from exhalation in adults and to compare NO, CO2, mouth pressure and flow
traces using two methods; direct to analyser and side arm sampling systems. The following
chapter will detail further experiments designed to examine a number of technical factors to
assess whether they alter the levels of exhaled NO obtained. These investigations were in
response to the newly presented and published literature in this area where, although the
conclusions were similar from different research groups regarding NO results in different
populations, it remained curious that the absolute levels of NO being documented were very
different. How the results from these experiments then fit into subsequent research, current
literature and new discoveries are detailed in Chapter 9.
6.2 Nitric oxide and the nitric oxide synthases in the lung
From the literature presented in Chapter 2.2 'Nitric oxide in biological systems', and in
Chapter 3.4 'Nitric oxide synthase isoenzymes', it was clear that NO and the NOS enzymes
were widespread throughout human organ systems. In the lung, NO has four major roles -
vasodilator, bronchodilator, neurotransmitter for the NANC neryes and as an immune and
inflammatory mediator. By the time of this research all three isoforms of the enzyme had been
detected in the human respiratory tract @arnes and Belvisi t993; Jorens, Vermeire et al.
1993; Marsden, Heng et al. 1993; Moncada and Higgs 1993; Singh and Evans 1997).
localisation of NOS in lung tissue was demonstrated by immunohistochemical labelling
using both polyclonal and monoclonal NOS antibodies @redt, Hwang et al. 1991; Schmidt,
Gagne et al. 1992; Hamid, Springall et al. 1993; Hattori, Kosuga et al. 1993; Kobzik, Bredt et
al. 1993; Forstermann, Closs et al. 1994; Rengasamy, Xue et al. 1994; Kawai, Bloch et al.
t995; Ambalavanan, Mariani et al. 1999). The detection of NOS within the respiratory tree
r22

initially appeared somewhat site-specific. Early studies showed NOS presence in bronchial
epithelial cells with little in the respiratory terminal bronchi, smooth muscle cells or alveolar
sacs. However, this changed following stimulation of the iNOS enzyme, when there was some
mild uniform labelling of epithelium in large cartilaginous airways with iNOS in normal
trachea and main bronchi, but with much grcatet labelling in the more peripheral bronchi'
This was first demonstrated when comparing normal and inflamed rat lung samples that had
been stimulated with intra-tracheal lipopolysaccharide, and in human tissue from four airway
and ten parenchymal specimens obtained from uninvolved areas of surgical tumour resections
stimulated with TNF0. While the presence of the constitutive forms along the bronchi before
the infective stimulation seemed appropriate given the eNOS and nNOS activity in
physiological regulation, it was uncertain why iNOS should be seen there. The researchers
suggested that it may be in response to continual low grade induction of iNOS activity in
response to airborne toxins (Hamid, Springall et al. 1993; Kobzik, Bredt et al' 1993)'
Turning from the presence of the enzymes to NO itself: when administered exogenously, NO
gas was shown to relax tracheal and airway smooth muscle in vitro taken from different
sources such as bovine trachea, guinea pig trachea and human airway smooth muscle (Jansen'
Drazen et al. 1992). The gas also had the capacity to relax tissues that had previously been
constricted with methacholine, histamine or leuktriene Da (Jansen, Drazen et al. 1992; Gaston,
Drazen et al. Igg4). NOS inhibition resulted in exaggerated bronchosconstrictor responses in
animal models (Ricciardolo, Mistretta et al. 1996; Nogami, Umeno et al' 1998; Boer'
Duyvendak et al. 1999) and human specimens (Ricciardolo, Di Maria et al. 1997; Taylor,
McGrath et al. 1998). These responses suggested that NO had a role in modulating basal
airway tone both directly and via the NANC nerves and, as discussed previously in Chapter 2,
is now thought to be one of the main mediators for regional airflow and blood flow matching'
The reaction products of NO such as s-nitrosoglutathione and s-nitrosalbumin also had
bronchodilator effects (Jansen, Drazen et al. lgg2; Asano, Chee et al. 1994; Gaston, Drazen et
al. 1994;Gaston, Drazen et al. 1994; Bannenberg, Xue et al. 1995) with lower levels noted in
asthmatics (Gaston, Sears et al. 1998). In the lung, low concentrations of NO caused ainvay
smooth relaxation during bronchospasm (Buga, Gold et al. 1989; Jansen, Drazen et al' 1992;
Gaston, Drazen et al. Igg4). As seen systemicallY, the NO production from the eNOS
isoenzyme was found to maintain vasomotor tone (Stamler, Osborne et al. 1993)' Pulmonary
arterial endothelial cells released NO in response to acetylcholine and bradykinin agonists
which relaxed pulmonary arterial, venous and lymphatic smooth muscle (Crawley, Liu et al'
1990; Dinh-Xuan, Higenbottam et al. 1991). Release of NO from endothelial cells in the
123

pulmonary circulation appeared to counteract hypoxic vasoconstriction responses to
catacholamines and prostaglandin F2c, with NO release decreased in chronic hypoxia
(Crawley, Liu et al. 1990). In vivo the NANC nerves were shown to cause vasodilatation and
relaxation of animal and human tracheal muscle using NO as the mediator and these were not
inhibited by NOS inhibitors (Stuart-Smith, Bynoe et al. 1994; Baba, Yoshida et al. 1998;
Sipahi, Ercan et al. 1998). However, human airway specimens suggested this NO mediator
relaxation via the NANC neryes was more important in the response of distal rather than
proximal airways (Ellis and Undem 1992).In addition, the NO-dependent NANC dilatation of
human bronchi was reduced in transplanted tissue and in specimens from patients with CF
@elvisi, Barnes et al. 1995; Belvisi, Ward et al. 1995). Nitrosothiols of low molecular weight,
such as S-nitrosoglutathione, and NOz were also detected in alveolar fluid from normal
individuals at concentrations sufficient to suggest regulation of basal airway tone @arnes
1993; Gaston, Reilly et al. 1993).
The NOS enzymes were considered to have a role in mucociliary clearance, and even now
this remains uncertain as the precise regulation of ciliary beat frequency is still unclear.
Similar to NOS activity, ciliary beat frequency is increased by a rise in intracellular calcium
with possible intracellular mechanisms involving cAMP, calmodulin, and inositol 1,4,5-
triphosphate. Increases of NO have been detected when ciliary beat increases, although no
consistent correlation has been shown between NO levels and cilia activity (Wanner, Salathe
et al. 1996; Dirksen 1998; Yeates 1998; Uzlaner and Priel 1999). Ciliated cells have been
shown to have increased NO production and increased beat frequency after treatment with L
arginine (Li, Shirakami et al. 2000) and L- NMMA caused a 4OVo decrease in ciliary beat
frequency lasting 60 minutes which was reversed with L-arginine administration (Jain,
Rubinstein et al. 1993).
Finally NOS activation in macrophages, neutrophils and mast cells exerts bactericidal and
fungicidal effects primarily through the oxygen superoxide formation of the NO products
such as perioxynitrite as covered in Chapters 2.3.4 and 3.4.2. These are important in host
defence but in excess can have similady detrimental effects on the normal cells. Higher
concentrations of NO in combination with oxidant generation leads to inflammation and
oedema (Beckman, Beckman et al. 1990; Mulligan, Hevel et al. 1991; Mulligan, Warren et al.
L992) and these potent oxidants deplete glutathione, uric acid and ascorbate in lung lining
fluid (Williams, Rhoades et al. I97l; Kelly, Shah et al. t997). Exposure of alveolar type II
cells particularly to perioxynitrite leads to profound inhibition of surfactant synthesis (Gaston,
124

Drazen et al. 1994), and the reactions can form potentially carcinogenic nitrosamines such a
NzOg and NzO+ (Miwa, Stuehr et al. 1987).
6.3 The need for methodological experiments
In a key development, NO was then detected in exhaled air (Gustafsson, Irone et al. 1991;
Archer 1993;[rone, Gustafsson et al. 1994). In 1991 Gustafsson and I-eone first showed that
NO could be detected in the exhaled air of tracheostomized animals and normal humans
(Gustafsson, Irone et al. 1991). Exhaled No was then measured in single and tidal breathing
in eight adult subjects giving levels between 8.3 to 20.3ppb (Borland, Cox et al' 1993)'
Exhaled NO was measured at a mean peak of 3.25ppb in ten subjects with normal breathing,
increasing to 100.25ppb with a sixty second breath hold prior to exhalation, and decreasing to
4.?5ppb with hyperventilation. During exercise, the mean peak levels in five subjects dropped
from gppb to 5-6ppb with 50 and 100 watt exercise on an ergometer cycle, although the total
NO excretion (NO concentration times number of breaths) increased (Persson, Wiklund et al'
1993). It was shown to be increased in normal subjects during a time of upper respiratory tract
infection to a mean peak of 315ppb during symptoms and reducing to a mean peak of 87ppb
three weeks later during recovery (Kharitonov, Yates et al. 1995). NO was shown in three
studies at this time to be lower in chronic smokers, with Persson et al measuring a mean in
mixed exhaled air of 3.9ppb in 6 smokers compared to their 20 normal subjects at 8.4 ppb
(persson, Zetterstrom et al. 1994). Kharitonov et al reporting a lower mean peak of 42ppb in
4l smokers compared to 88ppb in 73 nonsmokers in single exhalations (Kharitonov, Robbins
et al. 1995). Schilling et al documenting a mean of l6ppb in 12 smoking females versus
2lppb in 2l non-smoking females, and a mean of 15ppb in 24 smoking males versus l9ppb in
24 non-smoking males by using a reservoir method to collect tidal breathing (Schilling'
Holzer et al. 1994). In this paper Schilling et al also demonstrated a reduction of exhaled NO
in ten hypertensive patients to a mean of 13.7ppb (Schilling, Holzer et al. 1994). One study
reported varying levels of exhaled NO throughout the menstrual cycle in seven women from a
peak exhaled No at 150ppb midcycle to a low of 59ppb during menstruation (Kharitonov'
logan-Sinclair et al. L994). As by far the greatest amounts of NO in vitro were shown to be
produced from iNOS stimulation, (Nathan 1992; Barnes and Belvisi 1993), it had been
hypothesised to be a measure of airway inflammation (Barnes t993; Barnes and Kharitonov
1996). This seemed to be confirmed when higher levels were found in animal asthma models
when an exacerbation was induced (Persson and Gustafsson 1993; Endo, Uchido et al. 1995)'
and then in adult asthmatic subjects compared to normal subjects in a number of early studies'
Initially Kharitinov et al found levels of 283ppb compared to controls at 80.2ppb, Persson et
r25

al found levels of 10.3ppb compared to controls at 8.4ppb, Massaro et al found levels of
13.2ppb compared to controls at 4.7ppb, and Robbins et al found levels of 174ppb compared
to controls of 105.5ppb (Kharitonov, Yates et al. 1994; Persson, Zetterstrom et al. 1994;
Massaro, Mehta et al. 1996; Robbins, Floreani et al. 1996). Alving et al demonstrated no
overlap between their control group with a range of 5-16ppb and their asthmatic group who
had a range measured at 2I-3Ippb (Alving, Weitzberg et al. 1993). Massaro demonstrated
that the NO levels were even higher during a period of acute asthma in seven patients
requiring emergency department treatment with a reduction of NO beginning by 48 hours
(Massaro, Gaston et al. 1995) and Kharitinov et al showed a reduction over three weeks in
eleven asthmatic patients who commenced on IHCS therapy. However, as can be seen above,
the absolute mean concentrations of exhaled NO obtained by these separate workers in similar
patient groups and normal subjects using similar techniques appeared so disparate as to be
confusing.
In addition, the regional source of NO within the respiratory tract was debated. Initially
Borland et al suggested that the NO measured in exhaled air from a single full exhalation and
during tidal breathing was of alveolar origin like COz (Borland, Cox et al. 1993). Persson et al
then suggested that NO was formed preferentially in the small airways such as the terminal
and respiratory bronchioles (Persson, Wiklund et al. 1993). By comparing the concentrations
of NO exhaled during tidal breathing through either nose or mouth, Alving et al suggested
that the major contribution came from the nasal space with a minor addition from the lower
airways (Alving, Weitzberg et al. 1993). Lundberg et al demonstrated a decreasing
concentration of exhaled NO when sampling progressively down the respiratory tract at the
nose, mouth and, in four tracheotomised patients, below the vocal cords (Lundberg, Farkas-
Szallasi et al. 1995).
Another goup also looked at exhaled concentrations of NO in five adults with tracheostomy
during spontaneous breathing where oral NO concentrations were 9 - 25.7ppb while via the
tracheostomy this dropped to l.l - 6.5ppb. Eleven patients admitted for minor abdominal
surgery had similar decreases in NO levels measured under anaesthesia from nasal (mean
54ppb), oral (mean l3ppb) and following intubation (mean 1.3ppb, which was close to the
lower detection limit of their analyser). Seven patients intubated and ventilated in intensive
care with multiple diagnoses all had NO concentration levels close to the detection limit of the
analyser (range of 0.0 - 1.3ppb) (Schedin, Frostell et al. 1995). By isolating the nasal passages
from the rest of the respiratory tract with voluntary closure of the soft palate, Kimberly et al
showed the release of NO in the nasal passages was approximately seven times greater than
126

the rest of the respiratory tracr (Kimberly, Nejadnik et al. 1996). Higher levels still were
demonstrated in the paranasal sinuses with an age-dependent increase in keeping with sinus
pneumatisation (Kimberly, Nejadnik et al. 1996). It was postulated that the sinus production
of NO diffusing into the nasal spaces gave the high levels of NO then measured in the nasal
cavity. Gerlach et al measured NO concentrations in the nasopharynx and trachea showing
higher levels during inspiration than expiration (Gerlach, Rossaint et al. 1993). This led to the
suggestion that there may be absorption of NO by the lower respiratory tract (giving a concept
of auto-inhalation). However they could not exclude an exhaled component coming from
pulmonary synthesis of NO.
The variation in NO levels obtained made it difficult to comparc results presented by different
groups (see Table 6.1).
Table 6.1: The published results of exhaled nitric oxide in adults
Authors and PaPer Subject numbers Method of
measurement
Borland et al, 1993
(Borland, Cox et al.
1 ss3)
Schedin et al, 1995
(Schedin, Frostellet
al. 1995)
Gerlach et al 1994
(Gerlach, Rossaint et
al. 1994)
Kimberly et al, 1996
(Kimberly, Nejadnik et
al. 1996)
I controls
5 controls
11 conlrols
10 controls
10 smokers
5 controls
8 controls
single exhalation
tidal breathing
mouth breathing
tracheostomy breathing
tidal breathing via nose
tidalbreathing via
mouth
following intubation
Soontaneous
breathing:
trachea
controlled ventilation
trachea
controlled ventilation
Bronchoscooe
sampling:
at epiglottis, nose
breathing
at epiglottis, mouth
breathing
in trachea, nose
breathing
in trachea, mouth
breathing
Tidal breathino over 2
minutes:
during nasalbreathing
during oral breathing
Sinole exhalations:
exhalation
after 30s breath-hold
r27
Results in controls Results In
dieease
8.1ppb
14.7ppb
16ppb
4.6ppb
64ppb
1 Sppb
0.3ppb
89ppb (insP)
42ppb (ep)
2ppb (insp)
63ppb (exP)
72ppb (insp)
31ppb (exp)
2ppb (insp)
49ppb (exp)
22ppb
9ppb
18ppb
9ppb

Authors and Pap€r Sublect numbers Method of
measurement
Jilma et al, 1996
(Jilma, Kastner et al.
1996)
Persson et al, 1993
(Persson, Wiklund et
al. 1993)
Sato et al, 1996
(Sato, Sakamaki et al.
1996)
Morris et al, 1996
(Morris, Sooranna et
al. 1996)
Kharitonov et al, 1994
(Kharitonov, Logansinclair
et al. 1994)
Trolin et al, 1994
(Trolin, Anden et al.
1 994)
Robbins et al, 1996
(Robbins, Floreaniet
al. 1996)
lwamoto et al, 1994
(lwamoto, Pendergast
et al. 1994)
Martin el al, 1996
(Martin, Bryden et al.
1 996)
Alving et al 1993
(Alving, Weitzberg et
al. 1993)
22male controls
21 female controls
10 controls
5 controls
single exhalations 34ppb
tidal breathing
following brealh-hold of
60s
during hyperventilation
pre-exercise
exercise at 50 and 100
watts
16 controls reservoir collection
no breath-hold
breath-hold for 10
seconds
breath-hold for 60
seconds
5 female controls single exhalations daily
for one month
40 male controls
19 female controls
7 females through a
menstrual cycle
27 controls
8 with exercise
91 controls
18 asthmatics
23 smokers
I controls with
exercise
18 controls
32 allergic rhinitis
18 conlrols
32 allergic rhinitis
18 controls
32 allergic rhinitis
12 controls
8 asthmatics
single e;fialations
gg
single exhalation
total output over one
minute
single exhalation
total output
single exhalation
reservoir collection
single exhalation
reservoir collection
single exhalation
reservoir collection
single exhalation at rest
one minute collection:
at rest
exercise
hyperventilation
single exhalation
following breath-hold of
10s
following breath-hold of
:::,,,,
Tidalbreathing:
nasal inhalation
oralinhalation
with URTI
oral inhalation
128
Results In controls Results ln
dlsease
20ppb
3.2Sppb
100.25ppb
4.75ppb
9ppb
5-6ppb
(numbers from graph)
't2ppb
14ppb
1Sppb
51 ng g-1
No varialion across
the month
75ppb
70ppb
midcycle 150ppb
menstruation 59ppb
8.6ppb
90.9ppb
2.8ppb
171.2ppb
105.5ppb
14.Sppb
26.3ppb
9.Sppb
graphs for individuals
4.8ppb
1 1.1ppb
15.6ppb
32.1ppb
23ppb
9ppb
32ppb
'l74.2ppb
27.2ppb
39.6ppb
7.3ppb
16.3ppb
34.Oppb
62.Oppb
1 lppb

Authors and PaPer Sublect numbers Method of
meagurement
Persson et al, 1994
(Persson, Zetterstrom
et al. 1994)
Kharitonov et al, 1994
(Kharitonov, Yates et
al. 1994)
Kharitonov et al, 1995
(Kharitonov, Yates et
al. 1995)
Massaro et al, 1996
(Massaro, Mehta et
al. 1996)
Massaro et al 1995
(Massaro, Gaston et
al. 1995)
Kharitonov et al, 1995
(Kharitonov, Robbins
et al. 1995)
schilling et al, 1994
(Schilling, Holzer et
al. 1994)
Kharitonov et al, 1996
(Kharitonov, Yates et
al. 1996)
Kharitonov et al, 1995
(Kharitonov, Yates et
al. 1995)
Kharitonov et al, 1995
(Kharitonov, Wells et
al. 1995)
20 controls
23 asthmatics
6 smokers
67 controls
61 asthmatics
52 asthmatics on
tHcs
25 asthmatics:
single responders
baseline
single responders
27 hours
dual responders
baseline
dual responders 27
hours
5 controls
5 asthmatics
90 controls
43 asthmatics
73 controls
41 smokers
21 female controls
12 female smokers
24 male controls
24 male smokers
10 with
hypertension
11 asthmatics
baseline
following IHCS
18 (URTI)
during recovery
79 (controls)
20 bronchiectasis
19 bronchiectasis
on IHCS
mixed exhaled air
,t at aa
single exhalation
single exhalation
pre and post allergen
challenge
mixed expired air
at carina with
bronchoscoPe
mixed expired air
at carina with
bronchoscoPe
mixed air
single exhalation
reservoir collection
single exhalation
single exhalation
single exhalation
Resulte In contlols Rasults In
dlsease
8.4ppb
80.2ppb
4.7ppb
7.0ppb
6.2ppb
88ppb
21 ppb
1 9ppb
89ppb
10.3ppb
3.9ppb
283ppb
101ppb
214ppb
233ppb
258ppb
264ppb
13.2ppb
40.Sppb
13.9ppb
42ppb
16ppb
1 5ppb
13.7ppb
203ppb
120ppb
31 Sppb
88.3ppb
285ppb
88ppb
This table lists the published results of exhaled NO in adults by the beginning of 1997 showing the
wide range of techniques and even with app-arentty similar. techniquei the wide range of results
reported. The mean refufts of the exhaleo No ior eac'h subiect group are presented here. I have listed
129

only those measured by mouth or lower respiratory tract rather than nasal or sinus measurement
which will be discussed in a later chapter.
(exp = expiratory, IHCS = inhaled corticosteroids, insp = inspiratory, rg = nanogram, ppb = parts per
billion, s = seconds, single responders = edrly asthmatic response only, late responders = both early
and late asthmatic response, URTI = upper respiratory tract infection).
6.4 The aims of the exhaled nitric oxide methodological experiments
The following methodological experiments were therefore designed and conducted in adult
subjects to try and answer two main questions:
1. Were NO and CO2 produced from the same region within the lung?
2. Which technical factors during measurement led to changes in the NO results and
therefore would require standardisation in future research in this area?
The aims of the methodological investigations were:
L. To determine whether exhaled NO could be measured by the chemiluminscence analyser
now modified from its original purpose of measuring NO in air pollution.
2. To compare the exhalation pattern of NO versus COz from a series of single exhalations.
3. To compare the results of NO using the peak level versus the area under the curve of the
recording trace.
4. To compare the results of NO and COz between two methods of measurement; either
direct exhalation to the NO analyser (which also allowed measurement of CO2, and mouth
pressure) or exhalation via a t-piece system (which measured these parameters with the
additional measurement of expiratory flow).
5. To assess the reproducibility of the NO and COz measurements.
6. To investigate whether altering the expiratory flow changed the exhaled NO levels
measured.
7. To assess whether pressure had any effect on the exhaled NO levels by two methods:
(a) Delivering the calibration gas at different pressures to the NO analyser.
(b) Altering the expiratory mouth pressure voluntarily by adult subjects.
8. To investigate whether ambient NO levels affected exhaled NO levels.
9. To investigate whether water consumption immediately before exhalation changed the NO
results obtained (this was added in response to a chance finding).
The experiments and results from aims one to five listed above will be detailed in this chapter.
The experiments and results conducted to answer aims six to nine where a specific single
condition was varied to assess the effect on the exhaled NO levels obtained are detailed in
Chapter 7.
130

6.5 Setting up for the experiments
In the previous chapter, the capabilites and set-up of the analysers was described. In this
section, I will outline how the analysers were calibrated prior to each subject in the
methodological experiments and the basic procedure to serve as an introduction to all the
subsequent experiments on adult volunteer subjects. Changes in this exact procedure and
results of each set of studies will be outlined as separate experiments. I will present and then
discuss the initial findings after the first investigations which compared the two sampling
techniques with NO, CO2, expiratory mouth pressure, and (in the t-piece system) expiratory
flow results.
6.5.1 Set-up and calibrations
These instructions were set out by Carolyn Busst and myself after many individual tests of the
machinery and then posted next to the NO analyser to follow each time I did these studies to
ensure a standard procedure. I have re-presented them here as they were written in the present
tense and with numerical representation of the recorded numbers for times and frequency or
repetitions, but have added in italics some additional notes that came to be part of the set-up
as I progressed through and became more experienced. There was no change to the nature of
the investigations, but made it easier to standardise the machines and have the set-up running
efficiently. The purpose of these procedures was two-fold; to calibrate the analysers, in
particular the NO and COz analysers, and to mark the levels on the chart recorder to enable
calculation of the study parameters.
One hour before the study:
1. Check that the NO analyser and the vacuum pump are switched on and that the ozone
generator in the analyser is switched on (toggle switch left hand side, front panel). [NB: I
found the analyser and vacuutn more stable and took less time to reach a stable level if I
let it run continuously Monday to Friday or over weekends as well if experiments were
being underraken. This is in keeping with how it would have been used oiginally as a
continuous monitor of air NO pollution.l
2. Switch on the capnograph (switch located at the back of the machine).
g. Switch on the electrospirometer (switch located at the back of the machine).
4. Check that the calibration gas cylinders and air cylinders are not empty'
S. Check the paper in the chart recorder. If in doubt regarding having sufficient to record the
whole next subject experiment, replace with a new roll. Keep the old ones for test runs.
r3l

6. Check that the pens work. Fill with ink if necessary, careful not to overfill as this can lead
to smudges on the traces.
7. Check that the mouthpiece is clean and that the sample tubing is free from water. If it is
not, flush the tubing with compressed air from the cylinder.
8. Do not switch on the pressure equipment as it is battery powered and this will just waste
the battery.
Calibration just before the study:
On chart recorder note:
o Date.
o Study name.
o Subject name.
o Parameters and voltage scales [these remained unchanged and would only have mattered
if there was a log scale change in the concentrations of the gases, pressure orflow that we
were measuring, see NO calibration belowl.
o Paper speed.
o Barometric pressure.
o Temperature.
o The background ambient NO level.
The procedures for calibrating the analysers:
Calibration of the NO analyser:
o Make sure the rotameter valve is closed.
o Tnro calibration: connect NO sampling probe to the black, cylindrical NO charcoal scrub
unit.
o NO concentration calibration one: connect NO sampling probe to the calibration gas 'one'
nozzle - turn on the cylinder and record the level noting on the chart record.
o NO concentration calibration two: connect NO sampling probe to the calibration gas 'two'
nozzle - turn on the cylinder and record the level noting on the chart record.
o Turn off the cylinders.
o Connect the sample port to the mouth-piece.
o Run the chart recorder to record both NO and COz baseline ambienUroom air values and
note results on the chart record.
132

Note that ordcring o1'ncw NO gases cylinclcrs takcs 2 months t() sort out ancl arrive stl whct-l ttr
a quarter 1-ull cortrtttcncc tl-tc order tor tl-tc ncxt cylinclcr'
NB: Tlie calibration gascs usecl clr-rring thc tirnc ol'thc str,rclies wcrc 37 ppb. l03ppb' ll0ppb'
55ppb, ancl llSppl-, F-or an cxarrplc of calitrration on the chart rccorcl scc Figure 6.1.
Figure 6.1: Recording of the calibrations for NO, CO2 and pressure analysers
Pr6s3urc cqlibralion in 2 mmHg stepg
The charl recording is read f rom right to left and the green tracing shows NO, the red tracing CO2 wtth
the zero. ambient and calibrations for each. The blue tracing is for mouth pressure and this is shown
as calibration in sets of 2mmHq.
c'o. latibraion s.sz
:I .l
-t r: -
Cal it-rration o1' thc capnograph:
. Make surc thc r()lantctcr valvc is closccl.
o Prcss the CAL button on the front o1'thc pancl. The punrp clctcrtltitlcs the sarupling ratc ol'
thc CO: plobc. (Thc santpling rate can bc changcd by pressing thc Lrp and down ilrrow
| 11
Details
recorded
:
r
ZarONO I .-r --- r--

keys but it is currently in the middle of the range suggested by the manufacturers, and
used for all the early assessments).
o Tnro calibration: connect the COz sampling probe to the NO/i.{z tubing (NO calibration
gas) and press the CAL button to accept the zero calibration - mark on chart record - turn
off the gas.
o COz calibration gas one -
connect the COz sampling probe to the COz calibration gas
nozzle used and press CAL button to accept calibration - mark on the chart recorder - turn
off the gas.
o Barometric calibrations: Press the CAL button until the barometric pressure is displayed -
press the up arrow to set the barometric pressure - the COz bobbin will fall for 3 seconds
and then rise back up again and the current barometric pressure will be displayed - make a
note on chart record.
o Connect the sample port to the mouthpiece.
o Press display on the front panel to obtain the end-tidal COz display.
o Run the chart recorder to record both NO and COz baseline values noting the ambient
levels (more important for the NO recordings) (see Figure 6.1).
Calibration of the flow rotameter:
o Tnro calibration: with the air supply turned off and the rotameter needle valve turned off -
connect the air supply tubing to the bottom inlet of the rotameter - turn the air supply on -
run the chart recorder and label the trace.
o Calibration levels: open the valve until the rotameter bobbin moves and calibrate to
l50mls/min, 22lmlslmin, 325mls/min, 40Omls/min, 500mls/min, 600mls/min,
700mls/min, 800mls/min, 900mls/min, 1000mls/min at each level run the chart recorder
each time and note the calibration on the chart record - beyond this range the readings
become less stable when repeated over time and therefore less likely to be accurate.
o Leave the rotameter valve open - turn off the air supply, the bobbin will fall to zero
releasing the pressure in the air line (see Figure 6.2).
134

Figure 6.2: Recording of the calibration of the flow rotameter and pneumotachograph
The chart recording is read from right to left and the brown tracing shows the levels of flow calibrations
in mls/min. The calibrations are worked out on the sheet.
Mouth pressure.
o Open the rotameter needle valve.
o Ensure all connections are tight.
o Switch on the pressure generator (small black sliding switch, right hand side).
o Tero the display with the small black screw on the left hand side front panel of the
pressure generator.
o Z.ero calibration: connect the line from the pressure generator to the test point of the
Medex pressure transducer and ensure that it is secure - zero the pressure generator using
the wheel in the centre of the generator - zero the pressure monitor gauge making sure the
needle is on the zero of the scale -
record the zero level on the chart record.
135

Calibration pressures: the test port of the pressure transducer is on the opposite side of the
transducer to the mouthpiece - to mimic an increase in mouthpiece pressure it is necessary
to create an equivalent negative pressure at the test port -
move the wheel in the direction
indicated by the vacuum alrow to the right of the wheel - a negative figure is noted on the
generator display -
increase the vacuum by increments of 2mmHg from 0-20 mmHg and
run the chart paper at each level and mark on the chart record.
o Ensure that when calibrating the trace does not drift down which suggests a leak and in
this case recheck the joints.
o At the end, disconnect the generator and switch it off to save the batteries and ensure the
needle on the pressure monitor is on zero (see Figure 6.1).
For the testing the chart speed can be set 20mm/min or 5Omm/min - the latter should be used
for the subjects.The scaling factor on the CO2 channel on the pen recorder should be set to l0
volt full scale deflection (i.e. set the the toggle switch to V and the pointer on the votage knob
pointing to l0 on the V scale), and that of the NO channel to 500mV. The scale of the NO
channel will have to be changed if the calibration gases contain more than 490 ppb NO /nor
required during this timel.
At the end of the study:
o Repeat the calibrations in reverse order immediately the study has finished.
r Check that the gas cylinders are off.
o If no further studies on the day -
turn everything off except the NO analyser.
o If there is a gap between studies turn off the pressure monitor only to conserye the battery.
r The ozone generator can be switched off overnight but it should be switched on again an
hour before the study to stabilise.
o The NO analyser can be switched off if it is not going to be used for long periods - switch
off the ozone generator I't, then the NO analyser and finally the vacuum pump.
6.5.2 The exhalation protocol
Standard protocol for exhalation is described here and forms the basis for all the subsequent
experiments - any deviation in order to assess different factors is documented in each
experiment.
136

Direct to analysers method:
o The subject sits at rest for a minimum of 5 minutes.
. Each subject inhales to total lung capacity then performs a slow vital capacity manoeuvre to
residual volume over 30-60 seconds.
. 5 exhalations are made consecutively at 3-minute intervals.
o Nose clips are worn 5 seconds before the exhalation, and taken off between measurements.
. Measurements of NO, mouth pressure and COz are made.
. Mouth pressure is standardised to 4 mmHg by a fixed restriction.
. The sampling rate of the combined analysers was 44Omls/min.
T-piece sampling system to analysers method:
o The subject sits at rest for a minimum of 5 minutes.
. Each subject inhales to total lung capacity then performs a slow vital capacity manoeuvre to
residual volume over 30-60 seconds.
o 5 exhalations are made consecutively at 3-minute intervals.
o Nose clips are worn 5 seconds before the exhalation, and taken off between measurements.
o Measurements of NO, mouth pressure and CO2, and flow are made.
. Mouth pressure is standardised to 4 mmHg by a fixed restriction.
. Flow in the t-piece was standardised by having the subject exhale at a constant rate of
221mllmin. The subjects controlled their expiratory flow rates voluntarily observing the
rotameter bobbin level of the pneumotachograph for visual feedback.
o The sampling rate of the combined analysers including the flow was 665mls/min
Figure 6.3: Schematic diagrams of the two different types of connections: direct and t-piece
measurements
Direct:
O
0
t37

T-piece:
tO
The first figure shows the connections from the mouthpiece in the direct system to the NO analyser,
the CO2 analyser, the pressure analyser and the chart recorder. The second figure shows the t-piece
connection allowing additional measurement of flow.
6.5.3 Ethics and Consent
The studies received prospective approval by the Royal Brompton Hospital Ethics Committee
and informed consent was obtained from each of the volunteer subjects.
6.5.4 The subjects
In total, twenty healthy volunteers (mean age 35.9 years, range 18-52 years, 9 males) took
part in these methodological studies. Following consent, each subject completed a
questionnaire (see Appendix 2). The subjects were non-atopic with no history of respiratory
or cardiac disease and taking no medication. All had normal spirometry (Compact
Vitalograph, Vitalograph Ltd, Buckingham, United Kingdom). Two female subjects involved
in the variable flow and variable pressure experiments smoked two cigarettes per day; the rest
were nonsmokers.
6.5.5 Statistical analvsis
The results were analysed using the Statistical Products and Services Solutions, (Package U
7.0, SPSS Inc, Chicago, USA). This employs the Student's t-test for matched pairs with mean,
138
E
CL
(U
o)
o
-c
o(0
o
E
J
o
c
ct

standard error of the mean (SEM) and ranges given. A p-value of less than 0'05 was
considered significant.
6.6
o
a
o
Methodological exPeriment one
Direct versus t-piece NO measurement
Peak NO versus area under the NO curve
Exhalation patterns of NO versus COz
6.6.1 Hypotheses
l. The levels of NO measured in exhaled air are dependent on the techniques of
measurement.
Z. There will be good correlation between the peak NO levels and the area under
recording curve of the NO measurement.
3. Curves for NO and COz in a single exhalation should be identical if they
predominantly being produced in the same department within the lung.
6.6.2 Aims
1. To compare exhaled NO levels measured by two different techniques'
Z. To compare results of NO measured as peak level with area under the curve.
3. To compare the peak levels and curves of NO and COz in a single exhalation.
6.6.3 Procedure
Each subject abstained from food and drink for four hours prior to the experiment. The
experiments were made if the ambient level of No was less than 10ppb, and all the testing
was done with inhalation from ambient room air. The procedures as described in the previous
section for starting and calibrating the analysers were made before and after each subject. The
exact procedure followed is as described above in Section 6.5.2. Twelve subjects were
enrolled and each subject continued until five sets of measurable exhalations were made for
each set of conditions with the first set being determined randomly by tossing a coin (heads -
direct, tails -
t-piece). This was followed after a five minute break by measuring exhalation
under the other set of conditions. Again, after a five minute break, the first conditions were
measured again. Expiratory mouth pressure was requested to be voluntarily kept at 4mmHg
by the subjects and was measured on the chart recorder. During the exhalations into the t-
piece system, the subjects were also requested to maintain a set expiratory flow by watching
139
the

the rotameter. Tracings of each parameter (see in previous chapter Figure 5.2; NO in green,
COz in red, pressure in blue, flow in brown) were recorded on the chart recorder and each
then calculated using the calibration recordings. The NO and the COz peak levels on the
recording was measured. The peak NO levels were then compared with the area under the
curye. The latter was measured by drawing a baseline along the recording congruent with the
zero reading. The baseline and the curved NO record were then traced out and the area within
the margins calculated. Each result was the mean of five exhalations. The comparisons
presented between the direct and the t-piece measurements are the first measurements of each.
The final repeated set was compared against the first set to assess whether there were
differences over this time.
6.6.4 Results
Figure 6.4: Example of the chart recording rolls for the direct versus t-piece measurements.
6.6.4 (i) Direct versus t-piece nitric oxide and carbon dioxide measurement
Fifteen measurements were performed successfully on each of the twelve subjects (six male,
six female) with their lung function as percent predicted for gender, age and height by the
Polgar predictive equation (Polgar and Promadhat l97l) having an overall mean FVC of
IOTVo (range 96-I2OVo) and FEV1 of IO2Vo (range 93Vo-lI5Vo). The direct mean peak
r40

concenrration of exhaled NO was 84.8ppb (SEM 14.0, range 25.I - 189.0). There was greater
variation shown in the female subjects with a mean of 92.4ppb (SEM 36.9,26.2 - 189.0) than
the male subjecrs with a mean of 71.7ppb (SEM 28.7, range 25.1 - 141.0). The peak exhaled
NO levels measured through the t-piece system were lower in all the subjects with a total
subject mean of 41.2ppb (SEM 10.8, range 10.00 - 101.6). The mean in males was 33.2ppb
(SEM 8.4) versus females of 50.7ppb (SEM 12.3) (see Figure 6.6). The data for one
individual as the change occurred from t-piece to direct measurement is shown in Figure 6.7.
Note again that the chart recording is read from right to left so the exhalation commences at
the right hand side of each set of tracings. The scales were added for convenience of viewing
this single tracing as the scales were worked out on each graph depending on the checks with
the calibration gases. The main point depicted is the change with higher NO levels being
measured in the direct rather than the t-piece system.
Figure 6.5: Example of recording on one subject - direct and t-piece measurements
Note the chart is read from right to left, NO is recorded in green, COz is recorded in red, mouth
pressure is recorded in blue and flow (seen only in the t-piece measurements) is recorded in brown'
The exhaled NO levels increase when the tiace moves from t-piece to direct recordings. The
calculations made for each exhalation can also be seen on the chart.
t4l

Figure 6.6: Mean exhaled NO levels measured by direct and t-piece systems
180
160
140
E 120
EL
tr
= 100
o
g80
o
;60
g
E40
x
ul
20
0
T-plece
Comparisons of mean exhaled peak NO by the direct and t-piece system (n=12, each mean of 5
exhalations, SEM = standard error of the mean).
Figure 6.7: Example of a recording for the two systems
200
180
160
1.10
-o 120
CL
Sroo
z' Bo
60
40
20
0
ED
-
Ere
e9
a
g6
Eg
3
Eo
=
Direcl Toiece
ffi
876543210
Time min
Rrll
cq
NO
6
5
4
is
35'
C)
2
This is an example of the raw data from one individual showing the NO and COz and mouth pressure
(P,*) with the appropriate scales added along the '/ axes. The main point to note is the difference
seen in the exhaled NO concentration between the two methods; higher in the direct versus the tpiece
system of measurement.
t42
t
0

Table 6.2 shows all the results on one subject to demonstrate that each of the testing
parameters are measured five times with the first set of conditions repeated. The parameters of
pressure and flow, although standardised voluntarily, were also measured to ensure they were
adequately controlled.
Table 6.2: Compete results for an individualsubject
NO In
pPb
COzas
% total
9a8e3
I have also included Table 6.3 with the mean results and ranges from all the subjects for
exhaled NO and COz, each the mean of 5 readings per subject. The coefficient of variation for
individuals was LTVo for the direct method and llVo for the t-piece method. The background
level of NO ranged from 1 to llppb; this did not alter the conclusions so was not taken into
account in the calculations.
Mouth
pressure
In mmHg
Tlme in
secondg
Time
NO
peak
Time
COz
peak
COz at
NO peak
NO at
COz
peak
Area
under the
culve
Direct 1 41 6.2 4.O 58.3 32.4 50.4 5.4 37.7 6.43
Direct 2 45 5.9 4.O 59.4 32.4 54.0 5.3 42.2 7.71
Direct 3 50 6.3 4.2 64.8 27.6 60.3 4.9 46.6 8.93
Direct 4 48 6.4 4.2 64.8 34.8 56.6 5.7 44.4 8.77
Direct 5 52 6.4 4.0 66.0 24.0 60.5 5.7 46.6 9.8
T-piece 1 10 6.8 4.0 60.6 6.0 48.0 3.7 8.9 1.27
T-piece 2 10 6.4 4.2 51.6 8.4 63.6 4.5 11.1 1.6
T-piece 3 10 6.6 4.1 58.3 12.O 63.5 4.9 13.1 1.2
T-piece 4 11 6.5 4.2 60.0 '12.0 58.8 4.9 8.8 1.5
T-piece 5 11 6.7 4.0 60.6 21.6 49.1 5.9 11.6 1.7
Table 6.3 NO and COz results from single exhalations in twelve adult subjects
Sublect
No.
Gender Peak
NO'
Range
NO
Dlrect
Peak
COzb
Range
COz
Ratlo
NO/COz
Peak
NO
Range
NO
t-plece
P€ak
COz
Range
COz
Ratlo
NO/COz
1 m 29.4 25-33 5.6 5.2-6.1 5.3 12.2 12-13 6.7 6.3-6.9 1.8
2 m 43.6 41-45 5.8 5.7-5.9 7.5 35.2 34-38 6.2 6.0-6.3 5.7
3 m 45.1 42-59 4.4 4.0-4.6 10.3 26.9 18-39 5.3 5.0-5.4 5.1
4 m 63.6 59-68 6.5 6.3-6.8 9.8 14.2 14-15 7.1 6.9-7.2 2.0
5 m 118.6 103-151 6.0 5.8-6.1 19.8 66.2 24-71 6.5 6.4-6.6 10.2
6 m 129.9 125-141 6.8 6.6-6.9 19.1 30.8 29-3'f! 7.2 7.1-7.3 4.3
7 I 47.2 41-52 6.3 5.9-6.4 7.5 10.4 10-1 1 6.7 6.5-6.8 1.6
I t 50.8 26-'110 5.2 5.2-5.5 9.4 30.3 29-32 5.4 5.3-5.6 5.6
I t 52.6 32-72 5.1 4.1-5.6 10.3 40.8 34-56 4.9 4.4-5.2 8.5
10 I 111 7l-160 5.6 5.4-5.6 19.8 92.7 83-102 5.3 5.2-5.3 1.7
11 t 123.9 26-147 6.8 6.7-6.9 18.5 72.7 69-74 6.7 6.5-6.9 10.7
12 I 166.8 143-189 5.5 5.2-5.8 30.3 50.8 46-56 6.2 6.0-6.3 8.2
t43

NO and COz results from single exhalations as the mean of five recordings in twelve adult subjects by
direct or t-piece system measurements (m = mdle, f = female). (a. NO measured in parts per billion. b.
CO2 measured as % of totalgas expired).
There was no significant difference between peerk COz level by the direct measurements with
a mean of 5.8l%o (range 4.0Vo to 6.9Vo) or t-piece measurements with a mean of 6.l9Vo (range
4.4-7.2Vo, p = 0.66) or between male and female subjects (5.84Vo venius 5.75Vo direct, p =
0.64, and 6.4l%o versus 6.03Vo t-piece, p = 0.44), (see Figure 6.8). There was no difference in
repeated last set of exhalations for either NO or COz whether it was a repetition of the direct
or the t-piece measurements, although the coefficient of variation had improved suggesting
that there may be some improvement with training.
Figure 6.8: Mean exhaled COz levels measured by direct and t-piece systems
Comparisons of mean exhaled peak CO2 by the direct and t-piece system (n--12, each mean of 5
exhalations, SEM = standard error of the mean).
6.6.4 (ii) Peak nitric oxide versus area under the nitric oxide curve
The correlation coefficients comparing the results of peak NO level versus using area under
the curve for these traces were 0.87 for the direct results and 0.81 for the t-piece results and
are shown below in Figure 6.9.
8
7
6
o o
$E
E
"ge d4
6
5
E3
* (\l
92
t-t
1
-
Dlect
t44
-)
T-plece

Figure 6.9: Comparisons of the peak No with area under the curve
Figure 6.9a: Direct measurement.
o
Eru (,
o
=20
s
t'u a
Ero
Figure 6.9b: T-Piece measurement
o
14
't2
3,0
(,
o z8
q,
E
o6
E tg4
2
0
50 100
correlation of the peak NO levels with the measurements of area under the NO curve with a
correlation coefficient ol 0.87 (direct) and 0'81 (t'piece)'a
a The conelation here is slightly better than that given in the'Methods to Measure NO' article' This was an
invited article and I incluJfi tfi" aut" completedit that time but continued with the study'
r45
150
Peak level NO ln Parts Per Hlllon
20 40 @ 80 100
Peak level of NO In parts per bllllon
200
1n

6.6.4 (iii) Comparison of exhaled nitric oxide and carbon dioxide
There was a significant difference in the mean time to reach peak NO levels between the
direct (mean 32.2 seconds) and t-piece method (mean 23.1 seconds, p ( 0.001) while there
was no difference between the time to reach CO2 peaks between the direct (mean 50.5
seconds) and t-piece methods (mean 51.4 seconds, p = 0.5). In addition, the time taken for the
peak NO level to be reached and the time taken for the peak CO2 level to be reached were
compared. In the direct measurements, the mean time to reach peak NO was 32.2 seconds
(SEM 4.3), which was significantly less (p

Figure 6.10b: Time to peak NO and peak COz measured by the t'piece system
80
70
a
:60
o
E50 o
.s 40
.g 30
F20
10
0
Tlmeto
NO peak
Tlme to Duraton of
CO2 pak total exhalatbn
Comparisons of the time in seconds taken to reach peak NO,.taken to reach peak COz with the total
time for exhalation in measurements by the t-piece system (n=12, each mean of 5 exhalations)'
I also marked the coz level at the time of No peak to compare the difference between the
coz level at this time and at the time of co2 peak, which occurred later in the exhalation. For
the direct measurements, the COz level measured at peak NO was 4.88Vo (SEM 0'14) which
was significantly lower than the peak CO2 at 5.8lVo (SEM 0'21,p

Figure 6.11b: COz levels at peak NO and at peak CO2 measured by the t-piece system
88
o
!g'I
ot
E6
F5 g4
4
t!
E3
E2
{r
8o GOrat NO peak PeakCO,
:
Comparisons of the COz conc€Dtrations in percent of total expired gases at the time of peak NO and
at peak COz in measurements by the direct system (n=12, each mean of 5 exhalations).
6.7 Discussion: The origin of the exhaled nitric oxide
The study demonstrated that NO from a single exhalation could be measured by two methods;
either by direct connection to the NO analyser or by using a t-piece side arm sampling system.
However the results of NO were significantly different with an approximate halving of the
peak levels from the direct to t-piece technique, while there was no difference seen in the COz
results. These methods gave similar individual coefficients of variation as other groups
(Kharitonov, Yates et al. 1994; Monis, Caroll et al. 1995; Jilma, Kastner et al. 1996;
Massaro, Mehta et al, 1996; Hogman, Stromberg et al. 1997). The NO results from the female
subjects showed greater variability, and this has been noted previously as possibly being an
effect of the menstrual cycle in one (Kharitonov, lngan-Sinclair et al. 1994) but not a second
paper (Jilma, Kastner et al. 1996). Our subjects were chosen to minimise any other
confounding factors most notably being non atopic, non-asthmatic and having normal lung
function. As noted in the total group of subjects from which all the studies were done, there
were two smokers (two cigarettes per day) which was not ideal but I believed controlling for
atopy was more important when excluding subjects. As it happened, neither subject took part
in this direct versus t-piece experiment but were involved in later experiments.
Previous researchers had been unable to agree whether NO was of alveolar or airway origin
(Alving, Weitzberg et al. 1993; Borland, Cox et al. 1993; Persson, Wiklund et al. 1993). It is
known that the vast bulk of COz is of alveolar origin, having diffused across the
alveolar:capillary membrane from the pulmonary circulation. If COz and NO were both of
alveolar origin, the time to reach their respective peaks and the ratio of peak NO to COz
would remain the same even if measurement conditions were varied. I thus measured exhaled
148

COz and NO using two different conditions. In the direct experiments the subject exhaled
directly into the NO analyz er at M0 mls/min while in the indirect studies exhalation was into
a t-piece system at a faster rate of 665 mUmin. The time to reach peak co2 level, and the level
of the peak CO2 were the same in both manoeuvres. However the NO traces were very
different. Firstly, the time to reach peak NO was shorter than the time to peak COz in both
manoeuvre s (32.2 versus 50.5 seconds in direct measurements, and 23.1 versus 51.4 seconds
in t-piece measurements). Secondly, altering the rate of exhalation altered the time to peak
NO independently of the time to peak CO2. Thirdly, at peak NO levels the COz levels were
below their maximal peak. Fourthly, the ratio of peak NO to peak CO2 differed between the
two measurement methods. Thus although it is possible that a small amount of NO is of
alveolar origin, the bulk of NO must be being produced proximal to the alveolar membrane'
In any physiological study, it is important to consider whether the choice of apparatus or the
manoeuvre selected could have significantly affected the conclusions. Ideally, I would have
used fast response time analysers, with identical delay times. Fast-response time analysers for
NO had been used by others and are now available in the much later purpose-built machines
(see Chapter 9). At this time, Persson et al had used a NO analyser with an integration time of
0.01 second to study the changes in NO at rest and during exercise. They came to similar
conclusions, that NO and coz were not from identical lung compartments, using a very
different analyser and manoeuvre (Persson, wiklund et al. 1993). Our analyser would have
been unable to detect rapid transient changes in NO. However, had these occurred, and they
had not been described by other workers, they would have strengthened rather than detracted
from these conclusions. Rapid transients have not been described for CO2, and their presence
in the NO signal would have implied a different source, which is the main conclusion of this
research and our PaPer.
I considered the possibility that differing pressures exerted on the No analyser may have
accounted for the difference in readings and I describe two experiments in the next chapter
(see Sectio n 7 .4) specifically to answer this point (Byrnes, Dinarevic et al' 1997)' However' I
was careful to standardise to a mouth pressure of 4mmHg during the expiratory manoeuvre to
eliminate the possibility of this artefact. The pressure drop from mouth to analyser was
measured for both direct and t-piece apparatus, and found to be essentially the same' The two
manoeuvres used deliberately different expiratory flows to try to separate the COz and NO
signals. However the sampling flow to the NO analyser remained constant throughout this
study.
149

I also considered whether the different response times of the analysers could have affected our
conclusions. Ideally I would have liked equal analyser response times or a single machine
(such as a mass spectrophotometer) to differentiate both gases simultaneously. The NO
response was slower, but the NO peaked first. Had the NO response time been the same as
that of the COz analyser, the NO peak would have been even earlier, thus again the analyser
differences would serve to obscure, not enhance our conclusions.
We elected to study a single slow exhalation for the purposes of these studies, accepting it
may not be physiological or necessarily the best method for clinical practice. However, it was
designed to supply an answer to the question posed. The possibility exists that the results were
affected by back diffusion of alveolar NO into the alveolar capillary blood, and by nasal
contamination of the exhalate. During a prolonged expiration, if alveolar epithelium were the
source of NO, then back diffusion would allow some to be carried away combined with
haemoglobin. However, although this could affect the height of the NO peak, it is
inconceivable that it could affect the time to reach peak, and thus my conclusions are
unaffected by this concern. Furthermore, the total time of expiration was very similar in the
two different manoeuvres, suggesting that, were this mechanism to be operative, it would
apply equally between the two tests.
Higher levels of NO are produced by the paranasal and nasal spaces when compared to oral
exhalation (Alving, Weitzberg et al. 1993; Lundberg, Rinder et al. 1994; Lundberg, Weitzberg
et al. L994; Kimberly, Nejadnik et al. 1996) and these sites contribute a major proportion of
the NO concentration measured by oral exhalation. Indeed the paranasal sinuses also
contribute a major proportion of the NO concentration measured by nasal exhalation
(Lundberg, Rinder et al. 1994; Lundberg, Farkas-Szallasi et al. 1995). However if all of the
NO came from the nasopharyngeal region then the pattern of the exhaled NO trace would be
expected to both peak and fall off early rather than give the continued plateau seen in this
experiment. NO had been measured in exhaled air of tracheostomised rabbits (measured by
chemilumnescence), guinea pigs (measured by Quattro mass spectrometry) (Gustafsson,
Leone et al. 1991) and in humans (measured by chemilunescence) (Lundberg, Weitzberg et al.
1994).I attempted to exclude nasal contamination by asking the subject to wear a noseclip,
and to exhale against a moderate resistance (ammHg). One group had used nasal suction
combined with an (amplitude unstated) expiratory resistance (Silkoffl Kesten et al. 1995) and
another had excluded nasal NO contamination by voluntary closure of the soft palate
(Kimberly, Nejadnik et al. 1996) but these methods were more difficult and by no means
universally used. Whereas I cannot exclude a degree of nasal contamination, I believe that our
150

technique will have excluded most contamination, and that in any case the degfee
contamination will have been identical because of the very careful standardisation
experimental conditions including expiratory mouth pressure. This study was not designed to
differentiate nasal and bronchial sources of NO.
I elected to analyse the peak NO rather than the area under the curve. The two appear to be
closely correlated (Kharitonov, Yates et al. tgg4; Byrnes, Bush et al. 1996) and had good
correlation coefficients greater than 0.8 for both techniques in this study. I aimed to determine
the sources of the two gases, not the total amounts produced, which we would expect to be
independent of the experimental conditions. Thus the different behaviour of the peak signals
as the experimental conditions change gives more useful information than the area under the
curve.
It might be argued that if airways of volume 200mls contribute a peak NO at 85 ppb, a total of
10-10 mols, and the alveolar expirate (four litres) a plateau of 60, a total of 10-8 mols, then
most NO comes from the alveoli. This assumes that subjects exhaled to residual volume,
which is not the case. Furthermore, if this model was correct, peak COz (definitely alveolar)
and peak NO should move in the same direction as experimental conditions vary. The reverse
was seen. In eight subjects peak No fell using the t-piece while coz rosei in the other four
peak NO fell and peak CO2 also decreased. It is impossible to account for this on a model of
major alveolar production of NO. The model that best accounts for our data is continuous
airway production of No, diluted variably in a flow-dependent manner by No free (or low
concentration NO) alveolar gas, analogous with a gaseous phase dye dilution method. This
data cannot exclude that alveolar gas contains small amounts of NO.
This study also demonstrated that the conditions of measurement critically affect No levels
obtained. Thus both the time to reach the No peak and the peak level of No reached were
quite different in the two methods used in this study although the patterns of the traces were
similar. I considered whether the difference in results could have been artifactual due to minor
differences in the apparatus used in the two techniques. Teflon tubing which does not absorb
NO connected the mouth piece to the NO analyzer.In the t-piece system there was a two and
a half centimetre extension of plastic tubing leading from the mouth piece to the actual t-piece
with the teflon tubing then leading to the NO analyzer off one arm and further tubing off the
second arm passing directly to the pneumotachograph for flow analysis. This is unlikely to
account for the magnitude of differences obtained. The other alteration was that moving from
151
of
of

the direct to the indirect method led to an increase in flow rate from 440mUmin to 665
mUmin. This may have been enough to account for the different results.
Clearly, at the time of this research, there were major and unexplained differences between
results from different investigators as documented in Table 6.1. These results showed that the
levels of NO measured in exhalate were critically dependent on measurement conditions. This
may be related to expired flow rates but other factors may be important. It has been suggested
that orally-exhaled NO concentrations correlate with the inhaled ambient concentrations of
NO @otsch, Demirakca et al. 1996), although we found in a subsequent experiment a high
ambient NO rendered exhaled NO levels difficult to interpret @yrnes, Dinarevic et al. 1997).
Our current study was only undertaken if the ambient NO levels were low -
while we aimed
for less than l0ppb, on one occasion by the end of the session the ambient NO had drifted up
to llppb hence the range given from l-llppb in the results. As mentioned the greater NO
levels and wider variations seen in females may be due to possible effects of the menstrual
cycle (Kharitonov, Iogan-Sinclair et al. 1994) and this was not controlled for in the present
study. However, as the measurements were done sequentially on each subject on the same
day, conclusions as to the origin of NO from this study should not be affected.
As increasing research on NO in exhaled air was being undertaken in subjects in health and
different respiratory and vascular diseases at the time, it was increasingly important to
understand where and at what level NO was being generated so that abnormal results could be
properly interpreted.
NB: The results of this researchformed the basis of the following publications:
. Byrnes CA, Dinareyic S, Bzsst CA, Bush A, Shinebournes EA. Is nitric oxide produced at
airway or alveolar level? European Respiratory Joumal 1997 10 (5) 1021-1025
. Byrnes CA, Bush A and Shineboume EA "Methods to Measure Expiratory Nitric Oxide in
People" in "A Volume of Methods in Enrymology" edited by Lcster Packeter Academic
Press Inc. 1996.
t52

Chapter 7: Methodological studies of exhaled nitric oxide in healthy adult subjects:
7.t Introduction
investigating test Parameters
The previous chapter described the commencement of the methodological studies into exhaled
NO in adult volunteers. The first investigation was the measurement and comparison of NO
and COz in single exhalations by two methods; direct to analyser and via t-piece sampling
systems. In both methods the results and patterns of NO and COz could be determined, as well
as expiratory mouth pressure measurement and, in the t-piece system, expiratory flow could
also be recorded. This was an attempt to answer the question as to whether NO and COz came
from the same areas within the lung. This experiment also demonstrated a significant
difference in the peak exhaled NO levels with an almost halving of the NO result when
moving from the direct to t-piece measurements with no corresponding reduction seen in the
peak CO2 levels. The main change between these two techniques appeared to be an increase
in the expiratory flow which was required for the t-piece sampling. I have already noted in the
previous chapter (Section 6.4) the different levels of NO reported by different researchers
despite investigating similar population groups (healthy controls, asthmatics, smokers) and
summarised these in Table 6.1. I thought it was vital to understand which parameters when
altered gave different exhaled NO results. This was needed to be able to recommend
standardised procedures so that better interpretation of results would be possible both between
different research groups and in comparisons of results in health and disease.
As mentioned in Section 6.4, the aims in designing the methodological experiments were to
answer two main questions Posed:
l. Were NO and CO2 produced from the same regions within the lung?
2. Which technical factors during measurements led to changes in the exhaled NO results?
The results from the direct versus t-piece analysis suggested that the exhalation patterns seen
were different between the gases. While it was known that COz was of alveolar origin, the
pattern of NO exhalation suggested that it came from a more proximal source such as the
airways. So I felt that we had answered the first question. The experiments detailed
throughout this chapter were to answer the second question' The parameters chosen to
examine, by changing each one alone and maintaining an otherwise standardised procedure,
were:
o The effect of expiratory flow
suggested by discrePancies in
- an effect of flow altering the level of NO had been
the published literature summarised in Section 6.3 'The
153

need for methodological experiments' and from the first methodological experiment
conducted and described in the last chapter.
The effect of pressure -
to be assessed both by applying a calibration gas under pressure
to the NO analysers and by looking at the effect of expiratory mouth pressure in NO
results.
The effect of the ambient NO when high - this was not always standardised and
sometimes not reported in early literature and could have been a source of error in results
presented by different groups.
The effect of water consumption - this was due to a chance finding during one
experiment, where a person drank some water during a series of test exhalations and the
NO results dropped significantly for the next readings. I considered that asking both
healthy and, more particularly, subjects with any respiratory disease to inspire and exhale
fully may well lead to coughing in some for which a corlmon response is to give a glass
of water. If this then resulted in decreasing the levels of subsequent NO measurements,
this could easily skew results.
The peak exhaled NO was shown to have a good correlation with the area under the NO curve
in the previous experiment, so I elected to proceed with the peak measurements as they were a
more straightforward measurement, without the possibility of introducing error when tracing
around a curve, drawing a line for zero along the lower edge of the recording and then
calculating the area within these boundaries. I will summarise the subjects, ethics and
statistics again at the beginning of this chapter pertaining to all the investigations in the adult
subjects. For each experiment I will present the hypothesis, aim, procedure and results. I will
discuss the results and how they contribute to findings in the literature of all four experiments
at the end of the chapter. As the direct and t-piece experiment was deemed 'methodological
experiment one', these are labelled methodological experiments two to five.
7.2
Ethics. subjects. calibrations and statistical analysis
The studies received prospective approval by the Royal Brompton Hospital Ethics Committee
and informed consent was obtained from each of the volunteer subjects.
In total, twenty healthy volunteers (mean age 35.9 years, range 18-52 years, 9 males) took
part in these methodological studies. Following consent, each subject completed a
questionnaire (see Appendix l). They were non-atopic with no history of respiratory or
cardiac disease and taking no medication. All had normal spirometry (Compact Vitalograph,
Vitalograph Limited, Buckingham, United Kingdom) and results are presented as percent
r54

predicted or age, sex and height as defined by the Polgar reference equation (see results in
Section 6.6.4). Two female subjects involved in experiments two and three smoked two
cigarettes per day; the rest were nonsmokers.
The set-up of the analysers and the calibrations before and after each subject doing each set of
exhalations for the individual experiments was carried out in the same manner as described in
Section 6.5.1 .Set up and calibrations' and Section 6.5.2 'The exhalation protocol' of the
previous chapter. Any specific differences from these procedures are documented under the
separate experiments when outlined below. The procedures were written out prior to
performing them, I have therefore re-presented them here using the present tense and
numerical documentation as in the original instructions that I devised'
The results were analysed using the Statistical Products and Services Solutions @ackage 7'0,
SPSS Inc, Chicago, USA). This employs Students t-test for matched pairs' An analysis of
variance for repeated measures was used to determine the effects of flow, mouth pressure, NO
background and pre/post water consumption using the subjects as a blocking variable'
Differences are represented by mean, standard error of the mean (SEM) or 95Vo confidence
intervals (SD) where appropriate. The correlation coefficient for NO levels at the increasing
expiratory flows was calculated for each individual subject and is given for the group as a
whole.
7.3
7.3.1 Hypothesis
1. The levels of NO measured in exhaled air will alter with changes in the expiratory flow.
7.3,2 Aim
1. To compare exhaled NO levels measured at different expiratory flows'
7.3.3 Procedure
Ten adult volunteers (six females) were enrolled. All measurements were made by the t-piece
system. The subjects performed five exhalations at each of four expiratory flows: 25Omls/min'
500mls/min, 75omls/min, and 1lo0mls/min with a final set of measurements at the first flow
to rule out an order effect. Switching the machinery on in advance and calibration between
and after each subject occurred as discussed in the previous chapter (Section 6.5'l)'
155

The exhalations were carried out in the standard manner:
o The subject abstains from food and drink for 4 hours prior to the experiment.
o The subject sits at rest for at least 5 minutes.
r The chart recorder is started 30 seconds prior to each exhalation to document baseline pre-
exhalation measurements.
o The subject puts on nasal clips 5 seconds prior to each exhalation and exhales by mouth,
and removes the clips between exhalations.
o The subject inhales to total lung capacity then performs a slow vital capacity manoeuvre
to residual volume over 30-60 seconds.
o The exhalations occur 3 minutes apart.
o The subject exhales at a constant mouth pressure of 4mmHg voluntarily controlled by the
subject observing the pressure gauge and keeping it at level '10' on the scale.
o The subject exhales at a constant flow for 5 exhalations at each of 4 expiratory flows. The
flow is voluntarily controlled by the subject keeping the rotameter at specific levels:
Flow Rotameter level
250 mls/min 1.5
500 mls/min 3.0
750 mls/min 4.5
1100 mls/min 6.0
o The flow rate at which participants started, either 25Omls/min and increasing
consecutively or 1l00mls/min and decreasing consecutively, was made by tossing a coin.
o The subject continues until 5 exhalations at each flow level adequate for measurement are
made.
o A final set of measurements at the first flow was made.
o The subject was breathing ambient air when the natural background level of NO was less
than 10ppb.
7.3.4 Results
The mean peak concentrations of NO at the four expiratory flow settings were 79.0ppb (SEM
15.5) at 250mls/min, 67.8ppb (SEM 13.5) at 500mls/min, 59.lppb (SEM ll.2) at 75Omls/min
and 54.1ppb (SEM 10.7) at ll00mls/min (see Table 7.1). There were strong linear relations
between the NO concentrations obtained at the four flow levels for each individual subject
with a correlation coefficient of 0.85 (see Figure 7.1). The analysis of variance showed a
highly significant difference across the flow rates (p

individual subjects (see Figure 7 .2 for an example of the recording on one subject)' The mean
decrease in exhaled NO was 35ppb (957o C| 25.7,43.4) from the expiratory flow rate of
25omls/min to ll0omls/min. There were no significant differences in the mean CO2 levels,
mean mouth pressure (controlled) or mean duration of exhalation in the measurements made
at the differing flow rates. There was no difference between the first baseline and repeated
baseline of NO when the expiratory flow was the same (p-0.9 95Vo CI -16'9, 17'0), excluding
an order effect (see Table 7.2).
Table 7.1: NO, COz and duration of each exhalation at different expiratory flows
NO is measured as peak NO in parts per billion, COz is measured as peak aq the percentage of
QOz
total gases, duration is measured in seconds. Lach result is the mean of five exhalations for every
subject. a. Duratn = duration of exhalation.
Figure 7.1: Exhaled NO results with increasing expiratory flows
160
og rao
.E 120
$ 1oo
o80
=60 *40
t
,i 20
0
250 mls/mln 500 mlr/mln 750 mle/mln 1100 mlr/mln
Sublect Gender Age NO COz Durtn' NO COz Durtn NO COz Durtn NO COr Durtn
1 Female 32 23 4.7 57 17 4.5 59 23 4.5 55 4.5 50
2 Male 33 102 7.2 52 89 7.0 55.0 73 6.3 55 TI 6.2 51
3 Male 37 78 6.0 56 68 6.1 56 60 5.9 62 51 5.9 62
4 Male 49 85 6.4 66 70 5.7 59 79 5.8 62 70 6.0 61
c Female 43 31 6.2 55 23 6.2 54 19 5.8 54 17 6.0 54
6 Female 32 147 6.2 63 115 6.1 64 100 6.2 68 92 6.1 63
7 Female 37 83 5.7 50 86 5.8 52 82 5.9 47 76 6.0 44
8 Female 35 143 6.4 71 127 6,2 7',| 95 5.2 67 85 6.2 69
9 Male 43 67 6.7 84 60 6.9 72.1 43 2.0 71.0 38 7.0 71
10 Female 36 30 6.1 58 24 6.0 57 18 5.9 55 15 5.8 50
L
__ F.-f
2g 500 7s0
Explratory flows In mls/mln
Mean peak exhaled No levels at different expiratory flows for ten individuals (six females). Each point
is the mean of five exhalations.
t57
------
a
11(n

Figure 7.2: Example of the tracing in one subject at two different expiratory flows
7 .4 Methodological experiment three -
7.4.1 Hypotheses
effect of pressure
l. The levels of NO will alter with different pressure.
2. The levels of NO measured in exhaled air will be different with changes in expiratory
mouth pressure.
7.4.2 Aim
1. To compare NO levels measured using a calibration gas delivered to the NO analyser at
different pressures.
2. To compare NO levels measured at different expiratory mouth pressures.
7.4.3 Procedure
(i) A calibration gas of 110ppb was applied to the NO analyser as measured via the direct
system and was delivered to the analyser at pressures from 4 to 4OmmHg in increasing
increments of 4mmHg determined by the pressure gauge, and the NO level recorded.
158

(ii) Ten adult volunteers (eight females) were enrolled. All measurements were made by
the t-piece sampling system. The subjects performed five exhalations at each of four
expiratory mouth pressures of 4mmHg, 8mmHg, l2mmHg and l6mmHg with a final
set of measurements at the first mouth pressure to rule out an order effect. Switching
the machinery on in advance and calibration between and after each subject occurred
as discussed in the previous chapter (Section 6'5'1)'
The exhalations were carried out in the standard manner:
o The subject abstains from food and drink for 4 hours prior to the experiment'
o The subject sits at rest for at least 5 minutes.
r The chart recorder is started 30 seconds prior to each exhalation to document baseline pre-
exhalation measurements.
o The subject puts on nasal clips 5 seconds prior to each exhalation and exhales by mouth,
and removes the clips between exhalations'
o The subject inhales to total lung capacity then performs a slow vital capacity manoeuvre
to residual volume over 30-60 seconds.
o The exhalations occur 3 minutes apart.
o The subject exhales at a constant flow of 5O0mls/min voluntarily controlled by the subject
observing the rotameter and keeping it at level '3' on the scale.
o The subject exhales at a constant mouth pressure for 5 exhalations at each of 4 different
pressures which are voluntarily controlled by the subject keeping the pressure gauge at
specific levels:
Mouth Pressure
4 mmHg
8 mmHg
12 mmHg
16 mmHg
Pressure Gauge Level
o The initial pressure of either 4mmHg and increasing consecutively or 16mmHg and
decreasing consecutively was made by tossing a coin'
o The subject continues until 5 exhalations at each pressure level adequate for measurement
are made.
o A final set of measurements at the first pressure is made'
o The testing is done at a time when the ambient NO level is less than 10ppb.
159
10
20
30
40

7.4.4 Results
There was an increase in the peak NO signal of 5.6ppb seen across the range from 4mmHg to
4OmmHg of pressure when the calibration gas was applied to the NO analyser in increments
of 4mmHg, with the mean and standard deviation shown for each level in Figure 7.3.
Figure 7.3: The etfect of pressure on the calibration gas measurement
1{O
Euo
; 100
t
€&
CL
.E 60
og 640
o
En
81210?fJ2428323640
pros$Jra ol oallbratori gas dellvery In mmHg
The mean and standard deviation of the NO levels of a known calibration gas with a concentration of
110ppb delivered to the NO analyser at incremental pressures with an increase of 5.6ppb
demonstrated over this range.
The mean peak concentrations of NO obtained at the four mouth pressure settings were
61.0ppb (SEM 15.1) at 4mmHg,55.2ppb (SEM 12.3) at 8mmHg, 47.3ppb (SEM 8.4) at
l2mmHg and 4O.lppb (SEM 7.2) at l6mmHg (see Table 7.2).There was no significant
difference in the exhaled NO levels obtained at each mouth pressure. Figure 7.4 shows that in
the majority of patients NO levels did not change. However in two subjects who found the
higher pressures difficult to sustain (see reduced duration of exhalation in the Table 7.3 for
these two subjects at this higher pressure) there was a drop in NO as mouth pressure
increased. There was no difference between the first baseline and repeated final baseline of
NO when the mouth pressure was the same (p = 0.M 95Vo CI -16.7, 15.6) thus excluding an
order effect (see Table 7 .4). There was no significant difference between the flows measured
at the different mouth pressure readings. The peak expired CO2 at different mouth pressure
levels tended to decrease. The mean peak values were 5.907o (SEM 0.83) at 4mmHg, 5.83Vo
160

(sEM 0.82) at 8mmHg, 5.67o7o (SEM 0.86) at l2mmHg and then 5.53Vo (SEM 0.95) at
l6mmHg. There was also a tendency for the duration of exhalation to decrease, from 49'9
seconds (SEM 4.9) at4mmHg, 49.6 seconds (SEM 5.6) at 8mmHg, 44'2 seconds (SEM 6'3)
at l2mmHg to 40.9 seconds (SEM 5.5) at 16mmHg'
Figure 7.4: Exhaled No results at increasing expiratory mouth pressure
160
ll
& 140
.E tzo
E 100
o80
=60 *40 s
fi200
Mean peak exhaled NO levels at different expiratory mouth pressures for ten individuals (eight
females). Each point is the mean of five exhalations'
TableZ.2:Comparison of the first and repeated last set of exhalations at the same expiratory flow
Sublect Gender Age Expiratory
flow
48tz
Explratory mouth Pl€ssure in mmHg
Flrst Set of Exhalations
Nolcorlorttn'
NO is measured as peak No in parts per billion, coz is measured as peak coz as the percentage of
ioLt gir"r, duration is measured in seconds. Lach resutt is the mean of five exhalations for every
subject. a. Duratn = duration of exhalation.
161
Last
NO
iet of Exhalatlons
Co, Durtn
I
1 Female 32 250mls/min 23 4.7 57 28 4.5 51
3 Male 37 250mls/min 78 6.0 56 80 5.9 58
5 Female 43 250mls/min 31 6.2 55 28 5.4 57
6 Female 32 250mls/min 147 6.2 63 145 6.3 64
7 Female 37 250mls/min 83 5.7 50 111 5.8 47
I Female 35 250mls/min 't43 6.4 7',! 167 6.1 66
10 Female 36 250mls/min 30 6.1 58 30 5.7 54
2 Male 33 1100mls/min 77 6.2 51 75 6.7 55
4 Male 49 1100mls/min 70 6.0 61 65 5.8 60
I Male 43 1100mls/min 38 7.0 71 34 6.8 74

Table 7.3: NO, COz and duration of each exhalation at ditferent expiratory mouth pressures
Sublect Gender Ago NO COz Durtn
I
4 mmHg 8 mmHg 12 mmHg 16 mmHg
NO COz Durtn NO GOz Durtn NO COz Durtn
1 Female 52 142 6.8 51 112 6.6 44 75 6.8 44 67 6.5 40
2 Female 36 46 5.8 51 & 6.0 54 40 5.6 32 36 5.1 32
3 Female 36 38 4.8 48 42 4.7 51 47 4.5 49 42 4.2 46
4 Female 30 11 4.6 29 11 4.5 33 11 4.1 27 4 4.3 29
5 Male ,f3 s9 6.8 81 56 6.8 80 56 6.9 85 52 6.8 n
6 Female 32 107 6.4 62 101 6.2 61 81 5.8 47 57 5.6 39
7 Female 43 34 6.1 46 27 5.9 & 24 5.7 36 21 5.7 35
I Female 36 51 6.0 32 53 6.0 34 44 6.1 u 42 6.0 29
9 Male 4{r 56 5.9 54 53 5.9 53 56 5.7 44 51 5.8 41
10 Female 32 62 5.1 42 58 4.9 41 55 4.6 44 53 4.3 43
NO is measured as peak NO in parts per billion, COz is measured as peak COz as the percentage of
total gases, duration is measured in seconds. Each result is the mean of five exhalations for every
subject. a. Duratn = duration of exhalation.
Table 7.4: Comparison of the first and repeated last set of exhalations performed at the same
expiratory mouth pressure
Sublect Gender Age Explratory
pressure
First Set of Exhalations Last Set of Exhalations
NO COz Durtnt NO GOz Durtn
2 Female 36 4mmHg 46 5.8 51 78 6.1 il
3 Female 36 4mmHg 38 4.8 48 42 4.6 54
5 Male 43 4mmHg 59 6.8 81 59 6.9 81
6 Female 32 4mmHg 107 6.4 62 110 6.5 63
8 Female 36 4mmHg 51 6.0 32 65 6.0 35
10 Female 32 4mmHg 62 5.1 42 53 5 45
1 Female 52 16mmHg 142 6.8 51 106 6.5 48
4 Female 30 16mmHg 4 4.3 29 7 4.3 20
7 Female 43 16mmHg 21 5.7 35 25 5.4 30
9 Male 43 16mmHg 51 5.8 41 53 5.7 39
NO is measured as peak NO in parts per billion, COz is measured as peak COz as the percentage of
total gases, duration is measured in seconds. Each result is the mean of five exhalations for every
subject. a. Duratn = duration of exhalation.
r62

7.5
7.5.1 Hypothesis
1. Exhaled NO measurements will be affected by the ambient NO level'
7.5.2 Aims
l. To compare NO levels measured at a time of high and low ambient NO concentrations
with the high level being background and low level provided by breathing from a
reservoir system containing a low NO concentration'
7.5.3 Procedure
Three adult volunteers (all females) were enrolled and all measurements were made by the t-
piece sampling sYstem.
The first set of exhalations was carried out in the standard manner:
r The subject abstains from food and drink for 4 hours prior to the experiment'
o The subject sits at rest for at least 5 minutes'
o The chart recorder is started 30 seconds prior to each exhalation to document baseline pre
exhalation measurements.
o The subject puts on nasal clips 5 seconds prior to each exhalation and exhales by mouth,
and removes the clips between exhalations'
o The subject inhales to total lung capacity then performs a slow vital capacity manoeuvre
to residual volume over 30-60 seconds.
o The exhalations occur 3 minutes apart.
o The subject exhales at a constant mouth pressure of 4mmHg voluntarily controlled by the
subject observing the pressure gauge and keeping it at level '10' on the scale'
o The subject exhales at a constant flow at 5OOmls/min voluntarily controlled by the subject
observing the rotameter, keeping it at a level at '3' on the scale'
o At the end of each exhalation the connections are reversed to allow the subject and the
analysers to continue to sample from the low NO reservoir'
o The subject was breathing ambient air when the natural background level of NO was high
- above the NO concentration normally measured on exhalation.
o The subject continues until 5 exhalations adequate for measurement are made'
163

The second set of exhalations involved a reservoir system (see Figure 7.5):
r The ambient level is checked to make sure it is high - greater than the levels of known
exhaled NO results of the subjects, ambient NO > 75ppb.
o A reservoir bag (made of aluminium) is filled from a tank of NO free air (measured NO
level 0-2ppb).
o The reservoir bag is switched on-line to the analysers and the NO level in the bag is
checked to be < 5ppb.
o The subject breathes for 5 minutes from the reservoir bag. The subject exhales through a
one-way valve to avoid reservoir contamination.
o The analysers sampled from the reservoir system between exhalations.
o For the measured exhalation the connections were turned so that the subject exhaled to the
analysers.
o The subject exhales at a constant mouth pressure of 4mmHg voluntarily controlled by the
subject observing the pressure gauge and keeping it at level '10' on the scale.
o The subject exhales at a constant flow at 5O0mls/min voluntarily controlled by the subject
observing the rotameter, keeping it at '3' on the scale.
o At the end of each exhalation the connections were reversed to allow the subject and the
analysers to continue to sample from the low NO reservoir.
o This means none of the analysers are exposed to the high ambient level of NO during this
set of 5 exhalations.
The third set of exhalations was carried out in the standard manner:
r The measurements made were a repeat of the first set detailed above.
o Between exhalations the subject was breathing as normal in the known high ambient NO
level.
o Between exhalations the analysers were sampling as usual from the known high ambient
NO level.
Note: in this experiment the measurement used was NO plateaux rather than NO peak levels,
as the NO dropped from high level during the first and third set therefore a peak was not
obvious, while a plateau was more easily established.
7.5.4 Results
The ambient NO concentration was variable, although usually did not change quickly but
drifted up and down over hours. To demonstrate the appearance of the changing background
r64

NO level, I have enclosed photographs (see Figure 7 .6 of two of the recordings made from the
NO analyser over a 24 hour period with a slow paper speed.
Figure 7.5: Measurement of exhaled NO incorporating the reservoir system
tO
Diagrammatic representation of the reservoir system allowing both machines and subject to sample
froli low NO/NOiree air; NO, CO2, expiratory mbuth pressure and expiratory flow are all measured.
165

Figure 7.6: Photographs of the changing ambient NO concentration recordings
The ambient air NO concentration ranged from 91-200ppb during these experiments with a
mean background NO concentration of 134.5ppb (SEM 11.9). The background concentration
of the reservoir system was lppb (range 0-2ppb, SEM 0.2). Three subjects completed the
experiment (mean age 37 years, all female). The mean plateau NO concentrations during the
first set of exhalations with the high background NO was 94.9ppb (SEM 3.4ppb, range 84-
l0lppb). The mean plateau exhaled NO concentration in the set of exhalations during which
both the machine and subject were sampling from the NO-free reservoir system was 123.lppb
(range 103-l64ppb, SEM I9.4). The mean plateau NO concentrations during the last set of
exhalations with the high background NO was 99.9ppb (SEM 20.5ppb, range 53-15lppb).
Figure 7.7 shows a typical trace for one subject showing the rise to NO plateau during an
exhalation made from the reservoir system and the fall to an NO plateau during an exhalation
made from the high ambient NO. In the direct method, the NO level measured from the
reservoir system was 41ppb (95Vo CI I9.9,62.7) greater than the measurements made before
and after the reservoir system was used (see Figure 7.8). There were no significant differences
r66

in the NO plateaux seen before and after the set of exhalations done with the reservoir (p=0'2,
7 ,95Vo CI -10.9, 35.8, see Table 7.5). No difference was seen in the COz or duration between
the three sets of exhalations (see Table 7.6).
Figure 7.7: Recording from one subject showing NO results with exhalations from low and high
background NO
Ri3. to llo l.yal ttotl I low losarvoit llo Gonccntration
F.ll to IO l.v.l ftom I llgi ar|bi.nt XO Gooc.ntr.tlon
Figure 7.8: The effect of high and low pressure on the calibration gas measurement
Comparison of plateau exhaled NO levels following inhalation of high (ambient) labelled as 'pre' and
,post; or low (reservoir) labelled as 'reservoir' N-O concentrations. Each point is a mean of five
exhalations.
.o EL
o.
.g
o
g
o
z E-96sxul
Reservoir Post
r67

Table 7.5: Exhaled NO results with high and low background NO concentrations
Sublects Amblent NO Pre (hlgh NO) mean
(range)
One
(SD)
Two
(cAB)
Three
(cMB)
200.1 ppb 1 19.6ppb
(105.8-135.7ppb)
136.1ppb 94.9ppb
(84.0-100.8ppb)
91.4ppb 30.8ppb
(26.9-35.2ppb)
Reservolr (low NO)
mean (range)
202.4ppb
(154.1-225.4ppb)
134.0ppb
(102.9-163.8ppb)
32.9ppb
(20.3-38.6ppb)
Post (hlgh NO)
mean (range)
162.4ppb
(98.9-2a6.1ppb)
100.0ppb
(52.5-1s1.2ppb)
15.8ppb
(16.2-22.3ppb)
NB: The 'high' and 'bW NO note in the column headings refers to the ambient NO concentration
during each set of exhalations measured, with each result the mean of five exhalations. NO is
measured in parts per billion.
Table 7.6: Exhaled COz results with high and low background NO concentrations
Sublects Pre (hlsh NO)
mean (range)
One
(SD)
Two
(cAB)
Three
CMB
NB: The 'high' and 'low' NO note in the column headings refers to the ambient NO concentration
during each set of exhalations measured, with each result the mean of five exhalations. COz is
measured as percentage of totalgases and exhalation duration measured in seconds.
7.6 Methodological experiment five -
7.6.1 Hypothesis
effect of water consumption
l. Exhaled NO measurements will be affected by the consumption of water.
7.6.2 Aim
1. To compare NO levels measured by standard procedure before and after a set of testing in
which water was consumed immediately before each exhalation.
7.6.3 Procedure
Ten adult volunteers were enrolled (eight females) and all measurements were made by the t-
piece sampling system.
co25.2% (5.0-5.5)
Duration 52.2 secs
(45-60 seconds)
COzS.7To (5.6'5.8)
Duration 66.0 secs
(63-72 seconds)
COzS.5"/o (5.3'5.9)
Duration 55.2 secs
(48-63 seconds)
The first set of exhalations was carried out in the standard manner:
o The subject abstains from food and drink for 4 hours prior to the experiment.
168
Reservolr (low NO)
mean (range)
co2 5.1% (4.9'5.2)
Duration 54.0 secs
(36-63 seconds)
COz5.7o/" (5.6'5.8)
Duration 65.4 secs
(57-69 secs)
coz 5.6% (5.4-5.7)
Duration 58.2 secs
(54-69 seconds)
Post (hlgh NO)
mean (range)
co25.1"h (5.0-5.2)
Duration 51.6 secs
(39-63 seconds)
coz5.8To (5.7-5.9)
Duration 56.4 secs
(42-63 seconds)
COz5.5o/o (5.4-5.6)
Duration 57.0 secs
(54-60 seconds)

. The subject sits at rest for at least 5 minutes'
o The chart recorder is started 30 seconds prior to each exhalation to document baseline pre
exhalation measurements.
o The subject puts on nasal clips 5 seconds prior to each exhalation and exhales by mouth,
and removes the clips between exhalations'
o The subject inhales to total lung capacity then performs a slow vital capacity manoeuvre
to residual volume over 30-60 seconds'
o The exhalations occur 3 minutes apart.
o The subject exhales at a constant mouth pressure of 4mmHg voluntarily controlled by the
subject observing the pressure gauge and keeping it at '10' on the scale'
o The subject exhales at a constant flow at 500mls/min voluntarily controlled by the subject
observing the rotameter, keeping it at '3' on the scale'
o The subject was breathing ambient air when the natural background level of NO was less
than lOPPb.
o The subject continues until 5 exhalations adequate for measurement are made'
For the second set of exhalations:
o The procedure followed the standard set above'
o The subject consumed 60mls of water 20 seconds to 5 seconds prior to the next
exhalation. The water temperature was hot in five subjects and cold for five subjects'
The third set of exhalations was carried out in the standard manner detailed above as for the
first set of exhalations.
7.6.4 Results
The mean peak NO concentrations in the 10 subjects was 93'7ppb (SEM 20'8)' This
decreased by 23ppb (957o CI I2.g, 33.I) to 70.8ppb (sEM 16'5, p-0'002) during the
measuremenrs made with the subjects drinking 60mls of water before each exhalation' There
was then a significant increase of l7ppb (95Vo CI -9.7, -24'l) to 87'8ppb (SEM 18'7' p -
0.001, see Figure ?.9) in the subsequent recordings made in the standard way' The
consumption of water reduced the levels on No obtained regardless of the water temperature'
In the testing with cold water the mean NO went from 76.9ppb to a mean of 57.3ppb, and
then returned to a mean of 72.3ppb in the final set of exhalations. With hot water the mean
NO of 110.5ppb dropped to 84ppb and then returned to 102.8ppb. There was no difference in
r69

the NO concentrations taken pre and post the water experiment (p=0.095). There was
difference in the COz or duration of expiration across the three sets of exhalations made.
Figure 7.9: The etfect of consuming water on the subsequent exhaled NO levels measured
.o 2fi
e
Eo 't
a lso
o = 100
E Ps0
x
ttJ
0
Comparison of peak exhaled NO levels with the exhalation performed in a standard manner pre and
post a set of exhalations done where the consumption of either hot (in red) or cold (in black) took
place just prior to each exhalation. Each point is a mean of five exhalations.
7.7 Discussion: which measurement factors alter nitric oxide levels?
The methodological experiments described in Chapter 6 and 7 demonstrated that the
measurement of NO in exhaled air was feasible. A number of cross-sectional studies looking
at exhaled NO concentrations in different subject groups had been reported. The findings
within each research team had been consistent, but the absolute levels of NO reported by the
investigators in similar subject $oups were very different. All these investigators used NO
chemiluminescence analysers which, although developed by different companies, had similar
sensitivities and they stated that regular calibration was being performed. The study groups
were similar. For example, the results in healthy control subjects through the 1990s were
reported with means of 3.25ppb, 4.7Sppb and 100.25ppb under different conditions @ersson,
Wiklund et al. 1993), 4.7ppb (Massaro, Mehta et al. 1996),6.2ppb (Massaro, Gaston et al.
1995), 8.lppb (Borland, Cox et al. 1993), 8.4ppb @ersson, Zetterstrom et al. 1994), 8.6ppb
(Trolin, Anden et al. 1994), 9ppb (Alving, Weitzberg et al. 1993), 11.lppb (Martin, Bryden et
al. 1996), l6ppb (Schedin, Frostell et al. 1995), l9ppb and 2lppb (Schilling, Holzer et al.
1994),22ppb (Kimberly, Nejadnik et al. 1996), 26.3ppb (Iwamoto, Pendergast et al. 1994),
34ppb (Jilma, Kastner et al. 1996),42ppb (Gerlach, Rossaint et al. 1994), 51 NO g-r 6Morris,
Sooranna et al. 1996), 70ppb and 75ppb (Kharitonov, Logan-Sinclair et al. 1994), 80.2ppb
(Kharitonov, Yates et al. 1994), 88ppb (Kharitonov, Robbins et al. 1995), 89ppb (Kharitonov,
Wells et al. 1995) and l05.5ppb (Robbins, Floreani et al. 1996). Similar ranges in NO results
t70

etween these researchers were documented in the literature for other subject groups such as
those with asthma, bronchiectasis, hypertension and smokers. The measurement of NO in
these studies did vary between mean peak exhaled NO, mean plateau exhaled NO and NO
measured during tidal breathing. This led me to the suspicion that the technique of
measurement critically affected the No levels obtained. In the first methodological study in
twelve healthy adults (Byrnes, Dinarevic et al. 1997), I demonstrated that the concentration of
NO obtained could be halved depending on whether the subject was breathing directly into
the No analyser (mean 84.Sppb) or breathing through a t-piece system (mean 41.2ppb)' The
main difference between these two methods was a change in flow from 440 mls/min to 665
mls/min. The methodology experiments detailed in this chapter were therefore designed to
determine what factors altered the concentration of exhaled No obtained.
In the second methodological study, changing the expiratory flow rate significantly altered the
mean concentration of NO obtained. The higher the expiratory flow' the lower the
concentrations recorded, with a mean decrease of 35ppb when moving from 25omls/min to
1100mls/min in ten subjects (see Figure 7.1). Most of the literature at the time in the cross
sectional studies had usually mentioned the samplin g rate of the machine only, or measured
tidal volumes over a L-2 minute time periods with none specifically noting a standard
expiratory flow. Alving et al reported that if the airflow was increased in 12 healthy controls
from2umin-l to 5 Umin-l a slight increase in the levels of No was noted -
the data were not
shown and the increase was noted to be insignificant. This proved a different finding from
most others (Alving 1993). Imada et al showed a hyperbolic relationship between the NO
concentration and the sampling flow rate (see Figure 7'10) with a marked reduction of
exhaled No when increasing flow in one subject (Imada 1996).
t7r

Figure 7.10: Hyperbolic relationship between exhaled NO and sampling flow rate
800
e 800
5'
e 400
200
0
305*(x-0.07)
r=0.995, p

Figure 7.1 1: Exhaled No determined at single breath plateau concentrations at increasing expiratory
flows and at increasing expiratory mouth pressures
E r.o
Ci 15.0
2 ; 10.0
6
E s.o
ril o.o
r 1.5
o
'".
-E t.o
O'1""'O"'O"''
I
15
l0
5
0
1.5
?? I T I
+-f "'1""?"'1'.0
II--
o
U
x
o 0.5
z 051015n
Arway presswo, cmHP
T t "la-...+..o]..{
T '.--.1
, J{""'t"'i
fr^
5o 10 150 200 250 s
grytmryflownF, mLrl
Taken from Hogman M, Stromberg s, schedin Frostell c, Hedenstierna G, Gustafsson LE' Nitric oxide
from the human r"rpit'"tow tract-efficiently quantified by standarized single bl99!h measurements'
n.i" iny"iologica Sclndinavia 1997; 159: i+S'Sae (Hogman, Stromberg et al' 1997)'
In the pressure experiment, there were no significant differences between the mean exhaled
NO concentrations at 4mmHg, 8mmHg, l2mmHg and 16mmHg' In Figure 7 '3 it can be seen
that most of the ten subjects appeared to have flat traces across the sets of exhalation but the
top two and the bottom one subjects' traces do appear to have a reduction in No with the
increased pressure settings. There was a trend towards differences between the peak COz
levels reached being 5.907o at 4mmHg, 5.837o at 8mmHg, 5'83Vo at l2mmHg and 5'53Vo
reached at 16mmHg. This is likely to be accounted for by there also being differences in the
duration of exhalation with 49.9s at 4mmHg, 49.6s at 8mmHg ,44.2s at l2mmHg and 40'9s at
16mmHg. The differences probably reflect the difficulty for subjects of maintaining
exhalation against a strong resistance at constant flow to generate the higher mouth pressures'
subjects found it tiring to maintain this pressure and found it difficult to keep their lips tight
around the mouthpiece so as not to allow any air to escape when exhaling' When designing
the experiment in the preliminary trials both myself and Carolyn Busst tried a range of
expiratory mouth pressures up to 2OmmHg, and we quickly dropped this last high pressure as
being extremely difficult to exhale against. Two other studies published at the same time
showed no difference in the NO with differing pressures. In five subjects there was no
difference between an expiratory pressure of 6mmHg and 2ommHg (Silkoff 1997 ' Marked
flow dependence). And in six subjects at expiratory pressures over 0 to 20cmHzO there was
no difference in exhaled NO (see Figure 7.9 above, Hogman)' It would appear that mouth
173

pressure over the likely range encountered (such as the lower three pressure settings that I
tested) has no relevant effects on the measurements in most individuals. Conespondingly
there was no difference in NO, CO2, flow or duration of exhalation across these readings.
However as two, possibly three, individuals did show an effect, this again suggested the need
also to standardise mouth pressure for exhaled NO measurements.
The background NO concentration is variable and occasionally can be extremely high. In
many of the published studies, the ambient NO level at the time of testing was not stated and I
felt this may be another reason for error in the results obtained between and within groups.
This could occur particularly if testing was done on different days pre and post an intervention
with a subject group when the background levels may differ over time. In three subjects, I
showed that there were large differences in the mean exhaled NO concentration obtained
depending on the concentration of the NO being inhaled. In this experiment, the exhaled NO
measured was higher when inhaling low reservoir NO than when inhaling high ambient
levels. Three studies documented inhalation from NO-free air (Alving, Weitzberg et al. 1993;
Schilling, Holzer et al. 1994; Steerenberg, Nierkens et al. 2000) with one having noted that
the ambient air contained high and variable amounts of NO concentration (6-192ppb) leading
them to prepare and recommend an NO free reservoir for inhalation (Schilling, Holzer et al.
L994). Another study mentioned that the ambient NO air was recorded and the absolute zero
was adjusted just before each measurement by flushing the NO analyser with NO free
certified air (Kharitonov, Logan-sinclair et al. L994). A number of studies recorded levels of
ambient air at 0-20ppb (Persson, Wiklund et al. 1993), 5-20ppb (Kharitonov, Yates et al.
Lgg6), 0-38ppb (Kharitonov, Yates et al. 1994; Jilma, Kastner et al. 1996; Kimberly,
Nejadnik et al. 1996) and 0-68ppb (Kharitonov, Robbins et al. 1995) but did not seem to
affect the readings and therefore were not taken into account. One goup had specifically
requested two volunteers to inhale an NO concentration of 800ppb, hold their breath for
fifteen seconds and then exhale into the NO analyser. As they documented no change in the
NO concentration before and after inhalation, they concluded that the inspired NO must
disappear from the respiratory tract within that time (Kharitonov, Yates et al. 1994).
While the effect of ambient NO was either not specifically tested or was not published at this
time by any of these other groups, some researchers did investigate whether inhalation via the
nose or the mouth made a difference to the exhaled NO levels measured. This was an area of
variability, now known to be the degree of contamination of exhaled NO by nasal and sinus
production. The measured NO levels have been found to be greatest in the paranasal sinuses
(Lundberg, Farkas-Szallasi et al. 1995) with an age-dependent increase in keeping with sinus
174

pneumatisation. A progressive decrease in NO concentration had been found when sampling
progressively down the respiratory tract from nasal passages (Alving, Weitzberg et al' 1993;
Kimberly, Nejadnik et al. 1996), oral cavity (Gerlach, Rossaint et al. 1994), and below the
vocal cords (Lundberg, Weitzberg et al. 1994). The slow expiration time, particularly in the
direct measurements, did raise the possibility that I was merely recording inspired air,
contaminated by NO produced from the nose and sinuses, and exhaled unchanged' Schedin et
al showed the difference in exhaled NO in tidal breathing when the inhalation was by nose
giving a mean of 64ppb and by mouth giving a mean of 13ppb (Schedin, Frostell et al' 1995)'
Similarly when single exhalations were measured, nasal inhalation gave exhaled oral results
of 32ppb and22ppbwhile inhalation by mouth gave exhaled oral results of gppb and 9ppb in
two other studies (Alving l993;Kimberly 1996). However using a faster (t-piece) method in
our own study did not reveal an abrupt discontinuity suggestive of emptying unchanged dead
space prior to measuring true exhaled NO. The other area of difference between groups that
was studied by others at this time, which I did not exatnine, was the effect breath-holding had
on the subsequent exhaled NO. Using collection into a reservoir system, Sato et al showed
that the NO levels increased in proportion to the duration of exhalation and to duration of a
pre-exhalation breath-hold in 16 controls (Sato, Sakamaki et al. 1996)' In 18 controls Martin
et al reported the increase of exhaled No from no breath-hold at ll.lppb to l5'6ppb and
32.lppb in exhalations following ten and 60 second breath-holds respectively (Martin, Bryden
et al. 1996). The findings were simil ar in 32 subjects with allergic rhinitis from a baseline
exhaled NO at 16.2ppb to 34ppb following a ten second breath-hold and 62ppb following a
sixty second breath-hold (Martin, Bryden et al. 1996). Kimberly et al in eight controls showed
an increase in exhaled No from Tppb to 23ppb with a 30 second breath-hold (Kimberly'
Nejadnik et al. 1996). persson et al recorded the most dramatic increase from 3.25ppb with
tidal breathing to 100.25ppb with a breath-hold of 60 seconds (Persson, Wiklund et al' 1993)'
Kharitinov et al determined that a breath-hold for 20 seconds gave an initial peak of NO but
end expiration values were similar to non breath-hold results (Kharitonov, Chung et al' 1996)'
However, I believed that the finding with the ambient NO in this chapter suggested that
measurements of exhaled NO must be done on days with low ambient backgound NO' or by
using an NO-free circuit such as described.
The final experiment may seem an unusual test to have performed, but was based on a chance
finding that following drinking a glass of water, the NO levels on the next exhalations
dropped dramatically. The reason I thought that this may be significant was that when doing
lung function or full inspiratory/expiratory manoeuvres, a corrmon side effect, particularly in
175

subjects with respiratory disease, is to cough and an equally common response is to give the
person water to drink. This may be important if the levels of NO were then reduced. Exhaled
NO measured in the standard way did decrease significantly from 93.7ppb to 70.8ppb when
60mls of water was drunk between 20 and five seconds before the exhalation, and was not
different whether that water temperature was hot or cold. Many studies did not comment on
consumption of food or drink prior to the testing. One group reported asking the subjects not
to consume caffeine within two hours before the testing procedure (Kharitonov, O'Connor et
al. 1995; Kharitonov, Yates et al. 1996). Another goup did mention having a water absorber
installed in the proximal expiratory port in collections into a reservoir system (Schilling,
Holzer et al. 1994). The mechanism of the fall in the exhaled NO that I documented is
unknown. One group reported in two papers that the gastrointestinal tract had higher levels of
NO present which could be measured when 'belching' occurred (Lundberg, Weitzberg et al.
1994; Herulf, Ljung et al. 1998). The levels cited were far greater than those measured during
exhalation -
so it is possible that the gastro-intestinal tract may contribute to the exhaled NO
levels and was reduced by drinking water. Another possibility is that of quenching of the NO
gas. One study showed that prolonged sampling of wet gas resulted in an increased time for
the initial calibration to reach stable values from five to 20 minutes. They also demonstrated a
reduction in exhaled levels with increased water vapour and concluded that a decrease of NO
readings in water-saturated samples may have contributed to variation across studies (van der
Mark, Kort et al.1997).
In conclusion, several factors critically affect the measured mean peak concentration of
exhaled NO. An increase in expiratory flow rate resulted in a decreased concentration of NO.
Although there was no difference at increasing expiratory mouth pressures for most of the
subjects, individuals showed decreased exhaled NO concentrations and across all subjects
there was a decreased duration of exhalation and COz measurement. A high inspired
background NO made the exhaled NO concentrations difficult to interpret, and drinking water
just prior to measurement decreased the exhaled NO obtained. These findings confirmed that
the measurement of exhaled NO concentrations in humans should be performed in a standard
manner for the levels to have any meaning and to enable different teams of investigators to
compare results. These findings may have accounted for some of the discrepant results
reported in the literature. Since these results were published there has been a considerable
increase in the body of literature on exhaled NO with further studies in all these areas and
these will be covered to the present day in Chapter 9.
176

One aim of investigating the parameters that altered the levels of exhaled NO in these
methodological studies was to enable standardisations for future studies. In the next chapter, I
will describe the exhaled No research that I did with healthy and asthmatic children. The
results presented here in chapters 6 and 7 did make up some of the literature reviewed for the
first American Thoracic society and European Respiratory society recommendations to
standardise the methods of measuring exhaled NO'
The work in this chapter formed the basis of the publication:
. Byrnes CA, Dinarevjc g Bnsst CA, Bush A, Shinebournes EA. Is nitric oxide produced at
airway or alveolar level? European Respiratory Journal 1997 I0 (5) 1021-1025
In addition Mr Ron Logan-Sinclair went on to d.evelop the LR2000 series of NO analysers
using some of the ideas generatedfrom this research (I'agan Research Ltd, Unit 82, Spectrum
Business Centre, Anthony's Way, Rochester, Kent, United Kingdom), while Carolyn Busst
went on to run a ski lodge in Switzerland!
177

8.1
Chapter 8: Exhaled nitric oxide in healthy and asthmatic children
Introduction
The protocol was now established and I felt we would be able to measure NO in a
reproducible manner in children by standardising all of the parameters discovered to alter NO
readings. Initially I wanted to measure exhaled NO in healthy children: that is children with
no respiratory diagnosis or any acute or chronic respiratory symptoms. This was to confirm
that the technique was possible in children and to confirm some of the findings from the adult
studies presented in the previous chapters. Included was a comparison of the results from the
direct versus t-piece sampling systems that incorporated a change in flow. I then wanted to
compare the exhaled NO levels from these healthy 'control' children to those from asthmatic
children in two categories; children on bronchodilator therapy only versus those on regular
IHCS. Finally I wanted to measure the exhaled NO pre and post IHCS cornmencement in any
child who was on bronchodilator therapy only but who clinically required the introduction of
a preventer for asthma control.
At the time of corlmencement of this research there had been very little published on exhaled
or nasal NO in children. There had been a series of studies looking at the levels of NO from
nasal and sinus passages. High concentrations of NO were found when aspirating air from the
sinuses of five patients undergoing surgery at 9.1 parts per million (note that most of the
exhaled NO reported is measured in parts per billign) (Lundberg, Farkas-Szallasi et al. 1995).
An age-dependent increase in mean nasal NO plateau concentrations was also demonstrated
in 49 subjects with an age range of 0-62 years, thought to be consistent with the development
and pneumatisation of the paranasal sinuses (diagram included in the next chapter as Figure
9.2) (Lundberg, Farkas-Szallasi et al. 1995). The NO levels sampled directly from one nostril
and in exhaled air were also significantly less in four children with Kartageners syndrome at
4ppb when compared to 20 healthy children at22lppb (Lundberg, Weitzberg et al. 1994).
The following experiments ran from 1995-1997. At the time that we published these findings
in 1996 and through 1997, a number of research groups also published results of their
investigations into exhaled and nasal NO in children. In the main these were comparisons
between control subjects and those with respiratory diseases. In the next section, I will present
the subjects, protocols and results from the studies we conducted in healthy children and
discuss the findings comparing with these other results from the literature. In the second part
of the chapter I will present the literature available from studies in groups of adult asthmatics
as again these were published ahead of any results in children. The procedures followed were
178

the same for the children with asthma as those undertaken in the healthy children. I will
present the results and discuss the findings comparing our results with those then published
looking at similar groups of children.
8.2
Prospective ethical approval for the study was given by the Royal Brompton Hospital Ethics
Committee (Royal Brompton Hospital, Sydney St, London SW3 6NP, United Kingdom; now
Royal Brompton and Harefield National Health Trust)'
The subjects were recruited from a local school ("Park walk Primary school"' South
Kensington, London) within walking distance of the hospital. Following our initial approach
and discussion with the school board, the principal and the teachers of the children in the age
groups that we were hoping to enrol, a pamphlet about the study and a questionnaire were sent
home with childre n in 2classes aged 9-11 years to their parent/s or caregiver/s (Appendix 2)'
The school was familiar with the hospital and had been involved in previous (completely
separate) research. Two of the class teachers had participated in an "Asthma at school"
education programme run by myself and the respiratory nurse specialist (Ms Lizzie Webber)
at the Royal Brompton Hospital which ran for three years and was open to teachers in primary
schools throughout London.
Each child particiPated if:
o He/she had returned a signed consent and a questionnaire filled out by their parent/s'
o He/she was willing to be part of the study'
o He/she had permission of the class teacher to attend the hospital for an hour'
o He/she had no recorded respiratory, cardiac or other major chronic disease' (The
exception was children with asthma who were later recruited into the second part of
this studY).
o He/she had no upper or lower respiratory tract infection for at least four weeks'
The children were walked by the investigators from the school to the hospital, completed the
study and returned to the school always having been in our care' The children attended the
hospital in pairs from 1030 hours to 1200 hours and were returned in time for lunch break'
The aim was to enrol at least 30 paediatric subjects'
The results were analysed using the Statistical Products and services solutions, (Package u
7.0, SPSS Inc, Chicago, uSA). This employs Students t-test for matched pairs with mean'
179

standard error of the mean (SEM), standard deviation (SD) and ranges given where
appropriate. In the goup of asthmatic children that were measured before and after the
commencement of IHCS, medians are give. A p-value of less than 0.05 was considered
significant.
8.3 Methodology of exhaled nitric oxide measurement in healthy children
8.3.1 Hypotheses
1. NO can be measured in exhaled air in children.
2. NO levels will be different in the two methods of measurement; via the direct and the t-
piece sampling systems.
3. NO levels will be higher in children with current or past personal history of atopy such as
allergic rhinitis and/or enzema (excluding asthma in the first part of the study).
4. NO levels will be higher in children with a family history of atopy (allergic rhinitis and/or
eczema and/or asthma) in first degree relatives.
5. NO levels will be higher in children who may be expected to have environmental reasons
for airway inflammation such as environmental smoke or presence of funy peUs within
the household.
8.3.2 Aims
l-. To measure exhaled NO in healthy children by two methods; via the direct and the t-piece
sampling systems.
2. To compare the NO levels of children with and without a personal history of atopy
(having excluded asthma).
3 . To compare the NO levels of children with and without a family history of atopy in first
degree relatives.
4. To compare the NO levels of children with and without the presence of a household
smoker.
5 . To compare the NO levels of children with and without the presence of a household funy
pet.
8.3.3 Protocol
The questionnaire is added as Appendix 3. Questions were asked regarding information on the
following:
180

Child's personal medical hlstory
Current or past history
of respiratory illness
Current or past history of asthma
Current or past history of atoPY
(eczema, allergic rhinitis)
Current medications
Family and environmental history
Fami[ history of atoPY
presence of smokers in the household
Presence of Pets (and tYPe)
in the household
The child had abstained from food and drink for two hours prior to the experiment'
Height was measured on a Harpenden stadiometer (Harpenden Portable Stadiometer'
Crosswell, Crymych, Pembrokeshire, UK) and weight was measured on a digital Seca scale
(Seca 770 medical scales, Seca Ltd, 4802 Glenwood Rd, Brooklyn NYl1234)' Lung function
was measured via spirometry and in accordance to the ATS criteria (American Thoracic
Society and Association lgg4) and the best of three reproducible flow volume loops was
recorded using a Compact Vitalograph (Vitalograph Limited, Buckingham, United Kingdom)'
The results are presented as percent predicted for age, sex and height as defined by the Polgar
reference equation. All measurements were conducted by myself (predominantly taking care
of the NO readings) or Dr Senka Dinarevic (predominantly taking care of the lung function
testing). All results were then worked out by using the calibrations on the chart recording
system by both and entered into the SPSS database by myself and checked by Dr Senka
Dinarevic.
The experiments were made if the ambient level of NO was less than 10ppb and all inhalation
was done from ambient air. The procedures as described in the previous section (Section 6.5)
for setting up and calibrating all equipment were made before and after every two subjects'
The procedure listed below is re-presented exactly as it was designed and placed on the wall
adjacent to the analysers. It is therefore in the present tense and with numerical rather than
written designation of numbers.
Direct to analYsers method:
o The child sits at rest for at least 5 minutes
r Each child inhales to total lung capacity then performs a slow vital capacity manoeuvre to
residual volume or for as long as possible
o 5 exhalations are made consecutively at 3-minute intervals
. Nose clips are worn 5 seconds before the exhalation, and taken off between measurements
. Measurements of NO, mouth pressure and COz are made
o Mouth pressure is standardised to 4 mmHg by a fixed restriction
o The combined analysers sampled at 44Omls/min
181

T-piece sampling system to analysers method:
o The child sits at rest for at least 5 minutes
o Each child inhales to total lung capacity then performs a slow vital capacity manoeuvre to
residual volume or for as long as possible
o 5 exhalations are made consecutively at 3 minute intervals
. Nose clips are worn 5 seconds before the exhalation, and taken off between measurements
o Measurements of NO, mouth pressure and CO2, and, in addition, flow are made
o Mouth pressure is standardised to 4 mmHg by a fixed restriction
r Flow in the t-piece is standardised by having the subject exhale at a constant rate of
225mllmin. The subjects control their expiratory flow rates voluntarily using feedback by
observing the signal from the pneumotachograph
r The combined analysers plus flow then sampled at 665mls/min
The children did one set of five exhalations under each condition. The subjects were
randomised by tossing a coin (heads - direct, tails -
t-piece) to do either direct or t-piece first,
followed by a five minute break then the other set of conditions. The children continued until
five sets of measurable exhalations were made or it was obvious that the procedure was
difficult for the individual child.
The recordings were made in the same way as in the adult study with exhaled NO traced on
the chart recording system in green, exhaled COz in red, expiratory mouth pressure in blue
and flow in the t-piece sampling system in brown. A photograph of a child practising the
procedure is seen in Figure 5.4. The peak NO levels and area under the curve of the NO trace
were measured and compared. The mean mouth pressure was kept at 4mmHg in the direct and
t-piece sampling, and the mean flow in the t-piece conditions kept at 225mllmin in addition to
the flow required for the other analysers. Each result presented is the mean of five
exhalations.
8.3.4 Results
We recruited 39 healthy children (23 girls, 16 boys) mean age9.9 years (range 9-11 years)
from the local school. They had no current cardiac or respiratory disease, no upper respiratory
tract infection for the preceding four weeks and were on no oral medications. Their lung
function showed a mean FVC of 99Vo predrcted (SD 11.3), FEVr of 95Vo predicted, (SD 9.0)
and FEFzs-tsEoof 887o predicted for age gender and height (SD 12.8). There was no correlation
with exhaled NO results and lung function parameters. The correlation coefficient values for
the comparison of peak NO levels to the results obtained by measuring area under the curve
of the NO signal was 0.75 for the direct measurements and 0.34 for the t-piece measurements
t82

(see Figure 8.1). The following assessments used peak exhaled NO levels in keeping with our
work in adults.
The mean level of NO measured by direct exhalation into the analyser was 49.6 ppb' (SD
37.8, range lt.s-lgl.2ppb). The mean level of No measured via the t-piece system was29'7
ppb, (SD 2'7.1, range 5.I-t4l.2ppb). The reduction of the exhaled NO peak seen between the
two systems with the higher flow required for the t-piece measurement was consistent in all
subjects and consistent with the results in the adult experiments (see Figure 8'1)'
Figure 8.1: Peak exhaled NO results in healthy children
.o CL
o.
.E
o
.9
o zE.e6gxUJ
150
100
comparison of the peak exhaled No results in the direct to analyser and the t-piece sampling system
in 39 healthy children with each data point being a mean of five exhalations'
There was no significant difference between the direct mean NO levels in boys at 43'lppb
(SD 40.5, range ll.5-lg7.2ppb) and girls with mean NO of 55.2 ppb (SD 35'4, runge 17 '7-
124.gppb, p=0.43). There was also no significant difference between the t-piece mean NO
levels in boys at 25.6ppb (SD 2g.z,range 7.3-L4l.2ppb) and girls with mean NO 33'8ppb (SD
25.l,range 5'l-g4'3 PPb, p=0'11)' (see Figure 8'2a and 8'2b)'
183

Figure 8.2a: Comparison of peak exhaled NO in boys and girls measured by the direct system
o 200
ft reo
.s 160
p rco
g t2o
! roo
880
E60
fi40 20
0
No difference was seen when comparing the peak exhaled NO results between 23 girls and 16
boys aged 9-11 years in the direct to analyser sampling system with each data point being a
mean of five exhalations.
Figure 8.2b: Comparison of peak exhaled NO in boys and girls measured by the t-piece system
200
o 180
B reo
E reo
o a 120
o 100
=80 fi60
fi40
20
0
A
t
I
Boys Glrls
No difference was seen when comparing the peak exhaled NO results between 23 girls and 16
boys aged 9-11 years in the t-piece sampling system with each data point being a mean of five
exhalations.
There was no significant difference between the mean COz levels by direct and t-piece
measurements. The mean COz was 5.4Vo (SD 0.66, range 3.8-6.2Vo) direct compared to 5.52Vo
(SD 0.66, range 3.9-6.3Vo) via t-piece measurements (p=0.44). Duration of the exhalation had
184
A
A
a
I
T

a mean value of 32.9 seconds for the direct method (SD 1 !.4, range 15'8-65'8s) and a mean
value of 28.6 seconds for the t-piece method (SD 7.8, range 14'8-48s)' The mouthpiece was
standardised at 4.OmmHg in both techniques and the flow to 225mlslmin in the t-piece
method by voluntary control using visual feedback to the children. There was still some
variation around this depending on how successful the children were at maintaining the
pressure and flow as requested during a slow exhalation from total lung capacity which is
shown in Table 8.1.
Table 8.1: Coefficients of variation of peak NO, peak COzand mouth pressure measurements made
by both the direct and t-piece systems, and of flow made by the t-piece system
Direct method:
Peak NO in PPb
CQ2"/" totalgases
T-piece method:
Peak NO in pPb
CO2"/o totalgases
Mouth pressure in
Flow in mls/min
comparisons were made within this group of children of other factors that could result in
ainvay inflammation which may give higher results of exhaled No. Regarding a personal
history of atopy; the question sent home to the parents was:
Has he/she ever had eczema?
Has he/she ever had hay fever?
25.9
0.44
22.4
29.9
0.44
12.5
14
Yes/l'{o
Yes/No
Does he/she have eczemalha.yfever now? YesA'{o
2.6-67.4
0.41-0.81
4.5-57.0
0.5-65
0.41-0.81
2.9-36.1
1.8-30.3
There were eleven children that had a personal past or current history of allergic rhinitis
compared to 28 children who did not. At the time of testing none had current nasal symptoms
but three did have current eczema and were using appropriate topical creams including
hydrocortisone cream. There was no significant difference in the exhaled No levels in the
atopic children at 34.5ppb (sD 11.8) compared to non-atopic children at 45'6ppb (sD 38'5)
via the direct method or via the t-piece method measured at29'6ppb (SD 24) versus 24'8ppb
(SD 26.2) respectively. We also looked at whether family history of atopy had any effect on
the exhaled No levels in the children. The question sent home to the families was:
185

if yes who?
if yes who?
if yes who?
Any first-degree relative (parent or sibling) who was stated to have at least one of the
conditions listed was counted as a positive and any more distant relative or no family member
having any of the conditions listed were counted as a negative. Of the 39 children, 18 had a
positive family history of atopy compared to 2l that did not and there were no differences
seen in the direct exhaled NO levels at 40.6ppb (SD 26.7) versus 44.0ppb (SD 38.9), or in the
t-piece levels 25.3ppb (SD 21.4) versus 27.0ppb (SD 29.0).
The possibility of smoking causing airway inflammation and affecting the results of exhaled
NO was explored. The question sent home to the parents was: "Does anyone in the family
smoke? Yes or No" When the questionnaires were checked through with the children it was
determined whether the family smoker lived in the household. There were no differences seen
in the 19 children who lived with one smoker in the household compared to the 20 children
from non-smoking households at 37.lppb (SD17.4) versus 49.0ppb (SD 45.8) via direct and
22.2ppb (SD 20.4) versus 31.1ppb (SD 30.3) via the t-piece system. The children were not
asked about active smoking.
The possibility of a reaction to pet dander causing airway inflammation and affecting the
results of exhaled NO was explored. The question sent home to the parents was: "Do you
have any pets? If yes please name type". When the questionnaires were checked through with
the children the type of pet was checked again. Cats, dogs and any furry animal living within
the house were counted as positive, no matter the number involved. Fish and birds were
counted as a negative, as were not having pets. There were no differences in the peak exhaled
NO between those thirteen children who had a household furry pet (or pets) and those 26 that
did not at 39.3ppb (SD 29.2) versus 43.9ppb (SD 35.7) via direct and 23.5ppb (SD 23.2)
versus 27.6ppb (SD 26.7) via the t-piece system. There was no attempt made to look at
possible dose-response for any of these questionnaire replies.
8.4
Does anyone else in the family have any of the following:
Asthma
E*zema
Allergic Rhinitis
YesA.[o
YesA.[o
Yes/I.[o
Discussion: exhaled nitric oxide results in healthy children
This study confirmed that it was possible to measure exhaled NO levels in children aged
between nine and eleven years by using these two different techniques. As in the adult studies
there was a consistent difference between the NO levels obtained with the direct and the t-
186

piece methods. The main difference between these techniques is an increased flow of
Z2lmlstmin, and the adult technical studies revealed that with increasing flow there was a
decreasing level of exhaled NO obtained. In this case, an increase in flow of 52Vo led to a
reduction of exhaled NO of 407o.
The study was not designed to look at age-related differences in exhaled NO, as had been
suggested by Lundberg et al as occurring with possible development and pneumatisation of
the sinuses (Lundberg, Farkas-Szallasi et al. 1995). The children studied were in a nalrow age
range to try and standardise the results and to limit other extraneous effects. The age was
chosen as those likely to be able to do lung function tests and therefore complete the exhaled
No testing successfully. However the results in these 'normal' children were significantly
lower than the results obtained from the twelve healthy adults studied initially except for the
comparison of t-piece measurement in males. The children had mean exhaled No level
4g.6ppb compared to 84.8ppb in adults via direct measurement and mean NO 29'7ppb
compared to 41.2ppb in t-piece measurement. The boys had a mean exhaled NO of 43'lppb
(direct) and 25.6ppb (t-piece) compared to 71.7ppb (direct) and 33'2ppb (t-piece) of male
adults. The girls had a mean exhaled NO of 55.2ppb (direct) and 33.8ppb (t-piece) compared
to 92.4ppb (direct) and 50.7ppb (t-piece) of female adults. The differences werc more
significant for the direct than the t-piece technique. There was no difference within this close
two year age band, though it was noticeable that the nine year old children found it more
difficult to control expiration within the prescribed limits than the older children did' This
niurow and young age bracket was also chosen to try and enrol children that were prepubertal
following suggestions that exhaled No may vary through the menstrual cycle (Kharitonov'
Logan-sinclair et al. 1gg4). This ruled out a confounding factor if it was later discovered that
pubertal change had effects on exhaled No in either male or females, although, in fact' this
remains unknown. In both the children and the adults there was no significant difference in
exhaled NO between the genders.
There were significant differences between the duration of exhalation with the mean being
32.9 seconds for the children and 56.2 seconds for the adults in the direct measurements, and
2g.6 seconds for the children and 53.5 seconds for the adults in the t-piece measurements'
Three children had long exhalations of greater than 50 seconds for all of their direct method
exhalations and longer than 40 seconds for all the t-piece method exhalations and all had the
higher levels of peak coz measured. Interestingly, these three had swimming training and
were on representative teams either at the school and/or swim clubs'
r87

There are limitations with regard to the questionnaire data. The questionnaire was developed
as one of convenience and piloted on ten families in the paediatric general respiratory and
asthma clinics to ensure that it was easy to understand and easy to fill out. Two modifications
were made as a result of this - the description of wheeze was added, and the words for
describing amount of cough were changed. Park Walk School is an English-speaking school
but did have families of many nationalities so English was not necessarily the first language
of all the parents who were filling out the questionnaires and signing the consent form. The
questionnaire did not go through a validation process, save our own initial pilot. We did not
interact with the parents of the children directly. The questionnaires were distributed by the
teachers through the two classrooms and returned to the teacher from those happy to
participate. We did not have any data on the children who were given the questionnaire and
did not return it or did not gain consent for the study. I therefore cannot compare the
demographics of those who participated and those who did not to see if there were any major
differences between them. Forty-six children attended the hospital for the study from a
possible fifty-six in two classes. The classes were not stable through the study period with
additional movement of six children moving into and out of the classes because of moving
within the school and or two children from one family leaving the school altogether. Two
children that attended the hospital were ruled out because of inability to do lung function
successfully, although interestingly both were able to complete the exhaled NO tests. Five
children had asthma, four on bronchodilator therapy only and one on regular IHCS therapy
who briefly attended my asthma clinic -
these children were measured but not documented in
this segment of the study. The teachers ruled out seven children either because they had
special educational needs or because of misbehaviour - I do not know whether their families
had or had not consented. This gave us a response of 73Vo overall or 63Vo successful 'normal'
subject results.
The questionnaire responses were checked with the children on the day that they were brought
to the hospital to study. However it was obvious that the children were confident on some of
the responses and less confident on others. They tended to be confident on name, age,
nationality, current conditions such as asthma, hayfever or eczema, current medications, the
presence of family pets or presence of household smokers. They were less confident, and
there was more variability among the children about the degree of confidence, on answers
regarding recent 'colds', past history of conditions and family history of conditions, and for
these we had to rely predominantly on the written responses. We had documented nationality
but did not look at differences between them because of low numbers. The six nationalities
188

were loosely grouped as British, Indian, Arabic, African, European and South American with
one to fifteen children in each group.
we prospectively elected to enter into the database a positive or negative response to the
questions and not categorise answers any further. We therefore did not investigate possible
dose-response relationships for atopy (personal or family), passive smoking or presence of
pets. For example, we did not try to quantify the numbers of cigarettes smoked by the one or
more household smokers and look at the exhaled NO levels in this group. Likewise we did not
quantify the number of atopic responses within the family and see if there was any correlation
between atopy severity and exhaled No. This was for a number of reasons' Firstly, as
mentioned we were relying on questionnaire data which could be variably accurate' Secondly,
in the straight .positive and negative' categorisation of responses there were no significant
differences that I thought should be further investigated in this setting. Thirdly, the numbers
with a positive response to one of these areas ranged from eleven to nineteen so the numbers
were becoming small. Finally, it would have been difficult with these small numbers to
quantify one response in exclusion of other responses. At the time, our sample of 39 children
was the largest study in this area of research but the lack of findings of these factors that
might cause some degree of airway inflammation may be secondary to lack of numbers
causing a type two statistical error.
Despite the questionnaire and normal lung function, we had one boy who was an outlier with
direct peak No ot l97.2ppb and t-piece peak No of l4l.2ppb which was 73ppb and 43ppb
higher than the next highest result. On history, he occasionally wheezed with viral infections
and, following parental permission had 2OVo fall in FEV1 at2 mglml histamine on challenge'
It is possible he had mild asthma or was on the extreme of the normal range of ainuay
reactivity. He was a European boy with no personal or family history, from a non-pet owning'
non-smoking household.
By time of publication of this study, other investigators during the same period had also
begun to examine the measurement of NO in children, both in normal subjects and those who
with a range of respiratory diseases. In the main, the control children studied had no
respiratory disease, were on no medications and usually were recorded to have no upper
respiratory tract infection for between two and six weeks prior to the studies' Unfortunately'
as mentioned in the previous chapters, with all the early research each individual research
group developed their own techniques and thus every group utilised a different method of
measurement. As with the adult data, the absolute results in the healthy children differed
189

significantly between research groups, partly because of different techniques utilised. I-anz et
al reported a mean exhaled NO of 14ppb (+/- 2ppb) in seven children with single exhalations
from total lung capacity into a reservoir bag that was then fed through the NO analyser (lanz,
Irung et al. 1997). They also showed that there was no difference in the NO levels when re-
measured five days later (Lanz, I-eung et al. 1997). Using a similar method of slow exhalation
into a reservoir and then put through the analyser, Nelson et al reported a much lower mean
exhaled NO of 5.05ppb (+/- 0.4ppb) in 2I children (Nelson, Sears et al. 1997). Baraldi et al
showed that NO reached a steady plateau when measured during tidal breathing after one to
two minutes and reported mean levels of 5.4ppb in 16 children (Baraldi, Azzolin et ^1. 1997).
Lundberg et al also measured plateau oral exhaled NO levels with tidal breathing and reached
a similar figure of 4.8ppb (SD 1.1) in 19 children (Lundberg, Nordvall et al. 1996). Balfour-
Lynn et al measured NO from single exhalations taking the point of NO when the end tidal
COz reached a plateau in an attempt to compare alveolar levels for both oral and nasal
measurement. The 57 control children had a mean oral exhaled NO of 4.8 ppb (range l.l to
23ppb), and mean nasal NO of 1024 ppb (range 158 - 2502ppb) @alfour-Lynn, Laverty et al.
1996). Two studies measured controls across wide age ranges that incorporated an unknown
number of children. Dotch et al measured exhaled NO from single exhalations although the
response time of the analyser was not fast enough at 25 seconds for a 9OVo response rate to
measure one breath, so the subject was required to exhale four to five times into the analyser
via mouth inhalation and with nose clips in place. In 68 controls aged four to 34 years the
mean NO result was 3.0 ppb (+l-2.Sppb) by these single exhalations, with a minute ventilation
concentration of NO of 25 ppb (+l- 27 ppb) and direct nasal NO level of 96 ppb (+l- 47 ppb)
@otsch, Demirakca et al. 1996).
In another study, Grasemann measured NO by single exhalations to an analyser that had a 10
second response time. The mean exhaled NO level in 30 control subjects aged six to 37 years
was 9.1 ppb (+/- 3.6ppb). Lundberg et al measured the plateau NO signal by continuous
sampling from oral exhalation or nasal exhalation or by direct nasal measurement in 19
children. The mean oral level was 4.8ppb (SD 1.1), the mean nasal level was 2lppb (SD 9.1)
and the mean directly measured nasal level was 239ppb (SD 20) (Lundberg, Nordvall et al.
1996). Most of these researchers therefore reported absolute levels of exhaled NO that were
less than the results that I have reported from my studies. However I had elected to use single
exhalations measured immediately by the analysers, while these others used tidal breathing or
plateau measurements with or without a reservoir technique over several breaths.
190

In comparison to the early literature in the adults, the research groups studying children
appeared to more often consider the effect of ambient NO. Our experiments were only
undertaken if the ambient NO was less than 10ppb, as I had already shown in the previous
methodology experiment in adults (Section ?.6) that the ambient NO did affect the exhaled
NO levels. Similarly, Grasemann et al commented that "In preliminary experiments we
observed that the higher NO concentrations in ambient air were associated with increased
exhaled NO concentrations." (Grasemann, Michler et al. 1997). Ambient NO in this paper
was reported at between two and22 ppb in one paper which authors thought unnecessary to
take into account, but the exhaled levels in this paper were 1.1 to 23pb in the control children
which was exactly this ambient range (Balfour-Lynn, Laverty et al' 1996)' One group noted
that when measuring the exhaled levels in their control group it did not change when
measured in ambient or NO-free air, thus all their subsequent experiments were caried out
inhaling room air. However at the time their ambient NO level was reported as 3'95ppb +/-
0.98ppb and their NO free m at 3.97 +f 1.14ppb so there was no difference in the inhaled
levels and this ambient level may have changed over time (Nelson, Sears et al.1997} Finally
one group stipulated that they did all their measurements with the children inhaling No-free
air (Baraldi, Azzolin et al. 1997),
we found no correlation between exhaled No levels and gender or lung function parameters.
Nelson et al also found that there was no correlation between NO and height, weight, gender
or FEVr in either the control or asthmatic children (Nelson, Sears et al. 1997). Balfour-Lynn
et al found no correlation between exhaled NO and FEVr or FVC across control and CF
groups of children (Balfour-Lynn, Laverty et al' L996)'
In our study, we have documented that exhaled NO could be measured in children, reporting
normal values obtained with these techniques. As the continuation of this research, I then
moved on to investigate the exhaled NO levels in asthmatic children'
8.5
8.5./ Background
presence of higher levels of exhaled No in asthma were first indicated in two animal models
where exhaled NO significantly increased following induction of an asthma exacerbation
using ova-albumin sensitised guinea pigs and rabbits (Persson and Gustafsson 1993; Endo'
uchido et al. 1995). There followed a number of publications in adult subjects showing that
exhaled No levels appeared to be raised in patients with asthma when compared to healthy
191

controls. Kharitinov et al reported levels of 283ppb in 6l asthmatics compared to 67 controls
at 80.2ppb when measured with single exhalations (Kharitonov, Yates et al. 1994). Persson et
al measuring mixed exhaled air found levels of 10.3ppb in 23 asthmatics compared to 20
controls at 8.4ppb (Persson, Zetterstrom et al. 1994). Massaro et al using a similar technique
found levels of l3.2ppb in five asthmatics compared to five controls at 4.7ppb (Massaro,
Mehta et al. 1996). Robbins et al measured single exhalations and a reservoir collection in 18
asthmatics and 91 controls, reporting significant differences between the groups with both
techniques, of l74ppb compared to l05.5ppb and 27.2ppb compared to 14.5ppb @obbins,
Floreani et al. 1996). Alving et al demonstrated no overlap between their control group with a
range of 5-l6ppb and their asthmatic group who had a range measured at 2l-3lppb (Alving,
Weitzberg et al. 1993). Martin et al also found significant differences between 18 control
subjects and 32 patients with allergic rhinitis when measured by single exhalation with
ll.lppb compared to l6.3ppb, following a ten second breath-hold at 15.6ppb compared to
34.0ppb and following a 60 second breath-hold at 32.lppb and 62ppb (Martin, Bryden et al.
1996). Massaro demonstrated that the NO levels were even higher at 13.9ppb during a period
of acute asthma in seven patients requiring emergency department treatment. A reduction of
NO began after 48 hours which soon became indistinguishable from control NO levels at
6.2ppb (Massaro, Gaston et al. 1995). Kharitinov et al showed a reduction from 203ppb to
120ppb over three weeks in eleven asthmatic patients and an increase on their mean FEVr as
percent predicted from92Vo to 99Vo after commencing 800pgs budesonide dipropionate twice
per day, with no corresponding change when the same patients were randomised to placebo
(Kharitonov, Yates et al. 1996). As by far the greatest amounts of NO in vitro were shown to
be produced from iNOS stimulation, these high levels in asthma were hypothesised by these
groups to be a measure of airway inflammation (Barnes 1993; Barnes and Kharitonov 1996).
Following measurement of exhaled NO in healthy children, I was then interested to assess
whether these same findings as had been documented in adults could be found in children
with asthma.
8.5.2 The asthmatic subjects
The children were recruited from the paediatric respiratory or asthma outpatient clinics at the
Royal Brompton Hospital where I was seeing patients. Five children were also enrolled from
the Park Walk Primary School (four on bronchodilators only and one on regular IHCS therapy
who was subsequently also seen in the asthma clinic). They were presented with the same
questionnaire as used for the control children. Following informed consent from their parent/s
or caregiver/s, the children were enrolled if:
t92

o There was a signed consent form and a questionnaire filled out by their parent/s'
o The child was willing to be part of the study'
o The child had a doctor diagnosis of asthma'
o The child was on either bronchodilator therapy only or bronchodilator therapy and
long term IHCS therapy. No child studied was on any other asthma medications (such
as long acting p2 agonist therapy, theophylline or oral steroids). some of the children
were on topical treatments for eczema'
o There had been no change in their asthma treatment in the last six weeks'
o There was no current or recent upper or lower respiratory tract infection for at least
four weeks.
o The child had recorded no other respiratory, cardiac or other major chronic disease'
The ethical consent obtained for the study and the statistical analysis have been documented
previously in Section 8.2.
8.5.3 Protocol
The protocols for the asthmatic subjects were identical to those used the control subjects and
are listed in Section g.3.3 above. The two techniques of measurement were used: the direct to
analysers method and the t-piece sampling method' The children did one set of five
exhalations under each condition and continued until five sets of measurable exhalations were
made, or it became obvious that either the procedure was too difficult or it was exacerbating
their asthma. As the children were enrolled from either the morning clinics or the school (as
previously organised) all the children were measured at the same time during the day - late
morning between 1000 hours and 1300 hours'
8.5.4 Results
In total 31 asthmatic children were recruited for the studies of exhaled NO in asthma: fifteen
were on bronchodilator treatment only and 16 on regular IHCS therapy. The children with
asthma on bronchodilator treatment only had an FVC of 92Vo (SD 14.5) and FEVr of 78Vo
(sD 10.4) percent predicted by the Polgar reference equation @olgar and Promadh^t I97I)'
The children with asthma on regular IHCS had a mean FVC of 987o (SD 18.5) and FEVr of
g6qo (SD 17.5). The mean peak No level measured by the direct method in asthmatic children
on bronchodilator treatment only was 126.1ppb (SD 77.1, range L4.4-36l.lppb) which was
significantly higher than compared to the healthy children (mean 49'6ppb, SD 37'4' range
n.s-lgl.2ppb see previous results, p

direct method in asthmatic children on regular IHCS therapy was 48.7ppb (SD 43.3, range
8.6-106.4ppb) which was significantly lower than the asthmatic children on bronchodilator
treatment only (p

Figure g.3b: Mean peak exhaled No levels in control and asthmatic children measured via the t-piece
sampling system
lt 150
g o.
.g
:/ 100
a-
a
':r
-i i !
!!!
normals
asthmatics
bronchodilhlor
therapy only
mean
and SEM
aslhmatics
regular lnhaled
cortioostsroids
Mean peak exhaled NO levels in healthy children (n=39), asthmatics on bronchodilator therapy only
(n=15) and asthmatic children on regular IHCS therapy (n=16) measured by the t'piece sampling
system. Note the Y scale is discontinuous to accommodate the outliers'
There were no differences in the CO2 levels, mouth pressures, and durations of expiration
between the different groups or between the two methods within each group as noted in Table
8.2.
Six asthmatic children on bronchodilator treatment only but deemed clinically to require the
introduction of IHCS therapy to improve their asthma control were recruited for the
longitudinal study. Prior to commencing steroids the median exhaled NO was 1245ppb
(range 67.6-330.6ppb). Following treatment for two weeks on either budesonide diproprionate
4001t"gtwice per day in five subjects or budesonide diproprionate 2}}ltgtwice per day in one
subject all delivered via a Turbohaler@, NO fell to a median level of 48.6ppb (range 36'8-
153.6ppb). This reflected a decrease in all the children with only one subject now having a
different result from that observed in normal children (see Figures 8.4a and 8'4b)'
195

Figure 8.4a: The eftect of commencing inhaled corticosteroid therapy on peak exhaled NO levels
asthmatic children measured via the direct method
3s0
fl soo
CL
.s zso
o
B zoo
o z 150
E
fi roo
E
,i 50
0
Median = 124.5ppb
-
Pre Steroid NO
Post Steroid NO
E
Median = 48.6ppb
Figure 8.4b: The etfect of commencing inhaled corticosteroid therapy on peak exhaled NO levels in
asthmatic children measured via the t-piece sampling system
8.6
300
250
o
o.
o.
.c 200
o
g 150
o
zE
100
6
-c
x
ur 50
Discussion: exhaled nitric oxide in asthmatic children
Median = 41.3 ppb
-
Asthma is a chronic disease of airway inflammation that is treated with anti-inflammatory
drugs. However in the routine clinical setting we measure lung function rather than any
inflammatory parameters. The gold standard of assessment of airway inflammation is
bronchoscopy and bronchial biopsy as discussed in Chapter 1. Research then began to suggest
that NO could be measured in exhaled air and may be related to airway inflammation in adult
asthmatics (Alving, Weitzberg et al. L993; Kharitonov, Yates et al. 1994; Persson,
196

Zetterstrom et al. 1994; Kharitonov, Yates et al. 1995; Massaro, Gaston et al' 1995;
Kharitonov, Chung et al. 1996; Martin, Bryden et al. 1996; Massaro, Mehta et al' 1996;
Robbins, Floreani et al. 1996; Kharitonov, Rajakulasingam et al. 1997). In the experiments
above we set out to assess whether the pattern of NO excretion was similar in children as
determined in adults. In 39 children with no known respiratory problems it was possible to
measure exhaled No with mean levels of 49ppb (direct) and 29.7ppb (t-piece). I have also
shown that there was a significant increase in mean NO concentrations to t26.7ppb (direct)
and to l09.5ppb (t-piece) in asthmatic children receiving bronchodilator treatment only' There
was a significant decrease in children on regular IHCS therapy with mean NO levels of
48.7ppb (direct) and of 42.5ppb (t-piece). There was no difference between the exhaled No
levels of the healthy children and the stable asthmatics on regular IHCS therapy. There was
no significant difference in the mean age of the children in the three groups, although the age
range of the children recruited from the outpatient clinics was greater than those in the control
group. So while the effect of puberty and age was controlled for the healthy children' it is
possible that this may have affected results in the other two groups (Lundberg, Farkas-Szallasi
et al. 1995; Franklin, Taplin et al. 1999). The lowest age of a child that we successfully
studied was six years. All of the testing was completed in the mornings between 1000 hours
and 1300 hours so we could disregard any concerns regarding circadian rhythm effects
(Mattes, Storm van's Gravesande et al.2oo2). The pattern of No exhalation appeared to be
the same as the patterns seen in the adult subjects, and the pattern was the same in asthmatic
children as in healthy children, although the levels seen in children were lower' Similar to the
results from the adult subjects in the methodological experiments' we saw a reduction of the
NO levels between the direct and t-piece method of sampling in all the groups of children'
However the magnitude of the reduction was different. There is an increase from 440mls/min
in the direct sampling technique to 665mls/min in the t-piece sampling method, an increased
flow of 517o. This led to a SOVo reduction of the No levels in the adult experiment
(methodological experiment one described in Chapter 6), a 407o percent reduction in the
healthy control children, a 24Vo reduction in the asthmatic children on bronchodilator
treatment only and an 8Vo reduction in the asthmatic children on IHCS therapy' The variation
seen when measuring children was greater than that in adults, and there was a wide range of
exhaled No levels within all three paediatric groups. I have previously discussed one boy who
was an outlier in the group of healthy children. one subject recorded as an asthmatic on
bronchodilator treatment only had mean exhaled levels of 14'4ppb via the direct method
which was 53ppb lower than the next value, and 13.4ppb via the t-piece method which was
l7ppb lower than the next value. She had not had any episodes of asthma for two years and
r97

had last used her p2 agonist inhaler ten months previously. While there had been an increase
noted of exhaled NO levels with upper respiratory tract infections (Kharitonov, Yates et al.
1995), none of the children tested had had a respiratory tract infection, an exacerbation of
asthma within four weeks and any changes in medication for six weeks. Interpretation of
single NO results, however, must be made with caution. In a small group of six children
measured before and two weeks after starting ICHS, the exhaled NO levels dropped
significantly from a median of l24.5ppb to 48.6ppb and in all but one subject had returned to
the normal range. This suggested that the exhaled NO could be a useful monitoring tool for
airway inflammation in asthma.
A number of groups also published in the area of exhaled NO in paediatric controls, asthma
and cystic fibrosis through 1996 and 1997. I-anz et al looked at three groups of children:
seven asthmatics, six atopic but non-asthmatic children and seven controls with no asthma all
aged between eight to eighteen years with exhaled NO at 52ppb (+/- 5ppb), l6ppb (+/- 2ppb)
and l4ppb (+l-zppb) respectively. The exhaled NO in the asthmatic group decreased to l4ppb
(+/- lppb) when treated with oral steroids for 14 days (Lanz, Leung et al.1997). The other two
groups were re-measured five days later and showed no difference in their exhaled NO
readings. By a different method, Nelson et al measured expired NO concentrations via a slow
exhalation into a reservoir system, which was then sealed and subsequently run through the
NO analyser. Twenty one control children aged five to eighteen years had a mean NO of 5.05
ppb which was significantly lower than thirteen asthmatic children with a mean exhaled NO
at 16.3pp. This decreased linearly as airflow obstruction improved with corlmencement of
systemic steroid therapy of prednisone Z-4mglkglday. However, even when five of the
asthmatic patients had normalisation of their lung function, the exhaled NO was still higher
than the control children at 13.5ppb (Nelson, Sears et al. 1997). This goup speculated that
"NO assays may prove to be a more sensitive measure of childhood asthma than spirometrS/."
Baraldi et al measured sixteen children aged six to thirteen years during tidal breathing with
children inhaling NO free air with all measurements. NO reached a steady plateau during oral
breathing after one to two minutes. The mean level in the normal children was 5.4ppb (+/-
0.appb) significantly lower than sixteen children measured during an acute asthmatic attack at
31.3ppb (+l- 4.2ppb)and although these levels decreased with a five day course of oral
steroids (prednisone 1 mg/kg per day), the mean levels remained significantly higher than
controls at 16.5ppb (+l- 2.3ppb) (Baraldi, Azzolinet al.1997).
Two studies looked at NO levels in children with allergic rhinitis and/or sinusitis. In 36
asthmatic children all taking IHCS, there was no difference with direct nasal sampling of NO
198

levels between the asthmatics with allergic rhinitis at 252ppb (SD 20) compared to those
wirhout rhinitis at 256ppb (SD 26) (Lundberg, Nordvall et al. 1gg6). In sixteen children with
acute maxillary sinusitis the mean nasal concentration was 70 ppb (+l S.7ppb) increased to
220ppb (+/- 15ppb) after oral antibiotic therapy. In comparison nine children with upper
respiratory tract infections but not thought to have sinusitis had mean nasal NO levels of
249ppb (+t- 32ppb), which did not change after oral antibiotic treatment (Baraldi, Azzolin et
al. t997).
In studies comparing children with asthma and children with CF, Lundberg et al also
compared the plateau oral exhaled NO levels demonstrating no difference between 19 control
children and eight children with cF at a mean of 4.8ppb (sD 1.2) and 5'8ppb (SD 0'8)
respectively. However there was a significant increase in the 36 children with asthma to
l3.gppb (SD 2.5). These higher levels were seen in the asthmatic children despite their taking
a range of medication with twelve on low dose IHCS (defined as 0-100pgs budesonide or
equivalent per day), 16 on moderate doses of IHCS (defined as 200-400pgs budesonide or
equivalent per day) and eight on high doses of IHCS (defined as 600-800pgs budesonide or
equivalent per day). I note there appear to be gaps in these ranges i.e. 100-200pgs and 400-
600pgs which are not discussed but it is likely that there was no child on treatment in these
ranges which would have required odd dosing regimes. With nasal exhalation sampling, there
was no difference between the control group or this asthmatic group (recalling that all were on
IHCS therapy) at 21ppb (SD 9.1) versus 27ppb (SD 2.6), although a possible trend was noted
of a decreasing NO when on the higher steroid doses. There was also no difference in these
two groups when having direct nasal sampling (via a nasal olive) with the controls measured
at 239ppb (SD 20) and the collective asthmatic group measured at 254ppb (SD 17)' However
in both the nasal measurements those that were able to perform the technique from the group
of children with cF showed significantly lower levels with their nasal breathing giving levels
between 9-l5ppb and their nasal direct sampling giving levels between 40-105ppb (Lundberg'
Nordvall et al. 1996). In 68 controls, 90 asthmatics and 67 subjects with CF with a collective
age range from four to 34 years, Dotsch et al used a slowly responding analyser measuring a
series of single exhalations and showed a correlation between exhaled NO levels and the
ambient NO concentration. They then reported results between the three groups studied only
in the subjects that were measured during days of zero ambient No concentration (Dotsch,
Demirakca et al. 1996). The mean level of exhaled NO in 30 asthmatics aged four to fourteen
years was 8.0ppb (+/- 6.lppb), significantly higher than in 37 controls aged four to 34 years at
3.0ppb (+l 2.5ppb). Twenty three patients with CF aged five to 32 years tended to have a
t99

higher level of exhaled NO at 4.9ppb (+l-2.6ppb) than the controls but this did not reach
significance. They also found no conelation between NO and the lung function in any goup
@otsch, Demirakca et al. 1996). In another study, Grasemann et al measured NO by single
exhalations to an analyser that had a 10 second response time across a wide age range of
subjects (Grasemann, Michler et al.1997). The mean exhaled NO level in 30 control subjects
aged six to 37 years was 9.1 ppb (+/- 3.6ppb) which was significantly lower in 27 subjects
with CF aged six to 40 years at a time of disease stability at 5.9ppb (+l- 2.6ppb). This goup
commented that : "In preliminary experiments we observed that the higher NO concentrations
in ambient air were associated with increased exhaled NO concentrations." In view of this,
they went on to calculate the difference between the NO levels taking into account this
ambient NO concentration and still found a significant difference with the control group
having levels of 5.0ppb (+f3.lppb) compared to the CF group at 1.5ppb (+/- l.2ppb)
(Grasemann, Michler et al. 1997). Finally Balfour-Lynn et al measured plateau exhaled NO at
the time of COz plateau by placing a plastic nose piece just inside one nostril which was then
connected to the Teflon tubing of the NO analyser. They showed a significant difference
between the nasal measurements from 57 control children at l024ppb (95Vo CI896-1l52ppb)
when compared to children with CF; in thirteen on IHCS at 522ppb (95Vo CI313-730ppb) or
50 not on IHCS at 460ppb (95Vo C[399-520ppb). There was no difference in the exhaled NO
levels in these two groups and the use of IHCS did not affect the exhaled oral levels of NO in
the CF group with the results being means of 4.8ppb (95Vo Cl3.8-5.8ppb) in control children,
4.7ppb (957o Cl4.0-5.3ppb) in those with CF not on steroids and 3.6ppb (957o Cl2.5-4.8ppb)
in CF children on steroids. There was also no significant difference in the NO levels in the CF
goup between those 26 colonised with Pseudomonas aeruginosa compared to the 24 non-
colonised. There did appear to be a difference with a lower level in those 31 colonised with
Staphyloccocal aureus at a mean of 4ppb (95Vo CI3.44.6ppb) and those 19 which did not
have this organism at 5.8ppb (95Vo CI 4.4-7.2ppb). As there were 63 children in total there
must have been some considerable overlap between having Staphylococcal aureus and/or
Pseudomonas aeruginosa, plus the correct identification of the true presence of lower
respiratory tract pathogens in this age group with CF is known to be difficult so the overlaps
within these groups may have obscured findings (Balfour-Lynn, Laverty et al. 1996).
This completed my work in this area. I had demonstrated some key findings of factors that
affected exhaled NO measurements and would require standardisation for procedures in the
future. These were expiratory flow, expiratory mouth pressure, the need for low ambient NO
levels or to inhale from a reservoir of NO free air, and the need to prevent water consumption
200

during the testing procedure. I had demonstrated that it was possible to measure exhaled NO
in children, both healthy controls and asthmatics. In fact in some of the control children, they
found the exhaled NO measurements an easier task than completing lung function testing to
ATS criteria standards. I had demonstrated significantly higher levels of exhaled NO was
found in children with asthma on bronchodilator therapy only when compared to healthy
children and asthmatics on regular IHCS therapy, with no difference between the latter two
groups. Finally I had also demonstrated in a small group of asthmatic children the exhaled NO
levels reduced significantly following two weeks of IHCS treatment' These findings
contributed to the evolving literature to suggest that NO was a marker for ainvay
inflammation in asthma. I was invited with other research groups working in this area to
present this data to a European Society Task Force (ESTF) meeting in Stockholm in
September 1996 as a satellite working party to the European Respiratory Society Annual
Conference where the first discussion regarding standardisation procedures took place. This
later became the basis for the first publication setting out the best standard practices based on
the data available (Kharitonov, Alving et al. 1997)'
In the following chapter I will review how research in exhaled No and nasal No
measurement has progressed. I will review the findings on factors that altered No results in
both healthy groups and those with respiratory diseases. I will show how the standardisation
procedure for testing has continued to evolve to the present day. I will then review the
findings in the literature across adults, children and infants with a variety of conditions. I will
end with an opinion as to the best use of exhaled and nasal NO testing is at the current time'
The work in this chapterformed the basis of the publications:
o Dinarevic S, Byrnes CA, Bush A, Shinebourne EA' Measurement of expired nitric
oxide levels in child.ren. Pediatric Pulmonology 1996; 22: 396-401
. Byrnes CA, Dinarevic s, shineboume EA, Barnes PJ, Bush A' ExhAled No
measurements in normal and asthmatic child,ren. Pediatric Pulmonology 1997; 24:
312-318
20t

9.1 Introduction
Chapter 9: Exhaled and nasal NO to today
Having already presented my own research and preliminary discussion around the findings,
this chapter reviews where the research on exhaled NO had reached by 2000 and describes the
continued progression in this field to 2006. I will begin with a review of the official
statements from two international respiratory societies - the European Respiratory Society
(ERS) and American Thoracic Society (ATS) - both of which aimed to standardise the
techniques of NO measurement. I will present what is now known regarding NO in health and
in respiratory diseases in adults, children and infants. In the final paragraph I will give my
opinion as to where I believe this information is most useful.
9.2 Technical factors affecting results across research groups
By the end of the 1990s, exhaled and nasal NO had been measured in many groups; in normal
subjects (both adult and paediatric populations), as well as in those with asthma, non-
asthmatic atopy, chronic obstructive pulmonary disease (COPD), chronic bronchitis, cystic
fibrosis (CF), ciliary disorders, bronchiectasis and interstitial lung disease. As alluded to in
previous chapters, some findings remained consistent across the research groups. The best
examples of consistency in exhaled oral NO results were the high levels measured in steroid
naive asthmatics compared to normal controls which reduced with steroids, and the lower NO
levels in asthmatics already on inhaled or oral corticosteroids. For nasal NO measurements,
the most consistent findings were the low levels seen in patients with primary ciliary
dyskinesia and CF. However, other results were not consistent across research groups and
even when conclusions were similar, the absolute levels of exhaled NO were very different. It
was this discrepancy that led to the commencement of the methodological studies discussed in
the previous chapters. There were many reasons for the differences in the absolute levels that
were reported. I will list here what I believe constituted the main sources of variation.
1. Modification of analysers - the early analysers had been modified from machines
originally developed to measure NO in pollution using the chemiluminscence reaction,
similar to the machine that I adapted. The analysers used at that time included:
o Aerocrine AB, PO Box L024, Solna, Sweden.
o CLA 510s, Horiba, Kyoto, Japan.
o CLD 700AL, Eco Physics, AG, Basal or Duerten, Switzerland.
202

a
o
Model 2107, Dasibi Environmental corporation, Glendale, california, united states
of America.
Model 42, Thermoelectron, Warrington, United Kingdom'
Sievers Model 280A, GE Analytical Instruments, Boulder, Colorado' United States of
America.
These had differing response times ranging from 5-25 seconds while the subsequent
purpose built models from 2000 improved this to 0.02-0.3 seconds. The above models all
had differing sampling flows which ranged from 50-500mls/s (machine sampling rate)'
and different sensitivities to NO ranging from 0'3 to 5ppb'
2. Different methods of sampling - the three commonest techniques used for sampling the
exhalation were:
o Direct to the machine.
o Using a t-piece or side arm sampling'
o From a reseryoir of stored exhalations'
3. Variation of reservoir and tubing materials -
the type of material used to collect the
sample also varied which may have resulted in some absorption or adsorption of No as
discussed in Chapter 4. This is particularly important in reservoir studies with the use of
bags and tubing made of polyethylene, tedlar, mylar, teflon coated, metal and 'Douglas'
bags (a type of anaesthetic bag made of rubber)'
4. Method of cleaning and/or filter use - there was also the possibility of interference from
hygiene and infection control procedures. This isn't discussed in the early papers' At the
time, as currently, standard infection control procedures were recommended with regard
to doing routine spirometry (American Thoracic Society and Association' 1994)' It is
likely most researchers were following a similar type of infection control when measuring
No and this may have resulted in interference - either with use of inline filters and/or
chemicals for cleaning. Data presented in one abstract showed that the NO concentration
was greatly augmented by alcohol containing disinfectant (Meijer, Kerstjens et al' 1996)
at a time when the use of chlorhexidine (also an alcohol containing disinfectant) was
common in usual lung function laboratory hygiene practices' One study showed no
differences between a disposable mouth piece (sievers@) and a mouth piece containing a
filter (FIEpA) (Irme, Kasahara et a.^.2002), but there has been little research in this area'
5. Different exhalation techniques - subjects were asked to perform exhalation differently:
203

For the oral measurements options included:
o Single exhaled breath.
o Tidal breathing.
o Exhalation subsequent to a breath-hold with the suggested breath-hold ranging from 5
to 30 seconds.
o With or without the use of nose clips.
o Subject sitting or standing.
For the nasal measurements options included:
o Single exhalation via the nose.
o Sampling while tidal breathing.
o Direct sampling by nasal olive.
6. Different ranges of expiratory flows - differing flows were utilized ranging from 10-
1O0mls/min.
7. Arbitrary measures of expiratory mouth pressure -
either no specific expiratory pressure
was set, or it was not reported. In those that did standardise expiratory mouth pressure, the
pressures ranged from 5-20cmHzO.
8. Differences in recording and reporting of the results - the result reported was a different
part of the recorded NO signal, including:
o Peak.
o Plateau.
o Area under the curve.
o The NO measurement at the peak COz level.
Given the multitude of permutations, it was not surprising that different levels and at times
different results in similar populations of subjects were being reported in the literature.
9.3 Standardisation
In view of these different levels reported at that time, a European Respiratory Society Task
Force (ERSTF) was assembled to review techniques and a meeting organised in Stockholm,
September 1996, a meeting I attended. This was the first attempt to standardise measurements
for future NO research. This meeting resulted in an "Exhaled and Nasal Nitric Oxide
Measurements; Recommendations" (Kharitonov, Alving et al. 1997). Two years later, a task
204

force from the ATS with many of the researchers from the ERSTF meeting combined to
produce ..Recommendations of Standardised Procedures for the Online and Off-line
Measurement of Exhaled Lower Respiratory Nitric oxide and Nasal Nitric oxide in Adults
and childre n - lggg,,, which was adopted in July of that year as an official statement of the
ATS (American Thoracic Society and Association. 1999). Following this, there have been
two further documents written regarding standardisation of No measurement (Baraldi' de
Jongste et a:.2002; American Thoracic Society and European Respiratory Society 2005)' The
established ERSTF went on to publish "Measurement of Exhaled Nitric oxide in Children'
ZO0I- (Baraldi, de Jongste et al.2OO2). Finally a joint statement was prepared by the ATS and
ERS, which was adopted by both societies in 2O04 and subsequently published in 2005' This
was the ..ATS/ERS Recommendations for Standardized Procedures for the Online and Off-
line Measurement of Exhaled Lower Respiratory Nitric oxide and Nasal Nitric oxide 2w5"
(American Thoracic Society and European Respiratory Society 2005)'
In this section I will review the current 'standardisation' documents and discuss some of the
studies that led to these recommendations and the developments from 1997 to 2005' I will
critique my research to identify if the procedures I developed were correct and if they
contributed to the variability seen between research groups in light of subsequent findings'
The papers published from the research described in the previous chapters have been cited in
these documents.
There are a number of separate acceptable procedures to measure NO in adults and children -
each now having a standard method of measurement. These are:
1. Single breath online measurement (see Table 9'1)'
2. online measurement of exhaled No during spontaneous or tidal breathing (see Table 9'2)'
3. Offline measurement via a reservoir collection system (for delayed analysis)'
4. Nasal NO measurements (see Table 9.3).
Following this, I will review the more recent research on measuring exhaled NO in infants
using single breath or tidal breathing techniques'
9.3.1 Single breath online rneasurement
The current technique recommended for single breath online measurement for adults and
children (> 6 years of age) who are able to achieve a single exhalation is presented in Table
9.1.
205

Table 9.1: The recommended standard for single breath online NO measurement
Single breath online measurement for adults and children:
o the subject should be seated comfortably with the mouth piece at the proper height and
position.
o tho subject, particularly a child, should breathe quietly for five minutes to acclimatise.
. a nose clip should nof be used.
o the use of NO free air (containing less than Sppb) for inhalation is preferable.
o tho subject inhales to total lung capacity.
o tho exhalation takes place immediately after inhalation with no breath-hold.
o the subject exhales at a constant flow of S0mls/s and the expiratory flow is maintained at a
constant level.'
o the expiratory pressure should be maintained between 5 and 20cmHzO.
. visual biofeedback should be available to assist subjects in controlling their exhalation rate and
pressure.
. a peak and then a plateau should be seen in the recording.
. an NO plateau of greater than three seconds identified during an exhalation of six seconds in
adults and a plateau of two seconds identified during an exhalation of four seconds in children is
required.
o the plateau levelshould be used as the recorded measurement.
. the final result reported should be the mean of three readings within 10% variability or two within
5% variability.
. separate exhalations should be done at greater than 30 second intervals.
l. Note: fhis is the standard rwommendation unless using ditferent expintory flows to enable musurement of NO trwt
difterent lung compttments, see section bolow: 9.3.1.(i) Flw*
e.3.1 (i) Flow
Early studies recornmended a slow and prolonged exhalation between 10 and l5Umin (16-
25mls/s) for a duration of 5-30 seconds (Kharitonov, Alving et al. L997). However in
subsequent statements, a flow rate of 0.05Us (SOmls/s) was been the main recommendation.
Based on our own and other studies, it has been repeatedly demonstrated that exhaled NO is
markedly flow dependent with a reduction in concentration when the exhalation rate increases
(Alving, Weitzberg et al. 1993; Imada, Iwamoto et al. 1996; Kharitonov, Chung et al. 1996;
Lundberg, Weitzberg et al. 1996; Byrnes, Dinarevic etal.1997; Byrnes, Dinarevic etal.1997;
Byrnes, Dinarevic et al. 1997; Hogman, Stromberg et al. L997; Silkoff, McClean et al. 1997).
The 50mls/s flow has been shown in subsequent studies to be acceptable and reproducible in
adults (Silkofl McClean et ^1. 1997; Deykin, Massaro et al.2OO2; Kharitonov, Gonio et al.
2003), in adolescents (Kissoon, Duckworth et al. 2000) and in children (Baraldi, Azzolin et al.
1999; Kissoon, Duckworth et al. 2002; Pedroletti, Zetterquist et al. 2002; Pijnenburg,
Lissenberg et al.20O2; Shin, Rose-Gottron et al. 2OO2; Kharitonov, Gonio et al. 2003). From
a practical standpoint, fast exhalations in children result in a rapid decline in lung volume and
206

difficulty in sustaining exhalations long enough for the NO values to plateau (Kissoon'
Duckworth et al. 1999). Conection of expiratory flow for lung size has been debated given
the widely different lung volumes throughout early childhood, but this was not shown to
reduce exhaled NO variability (Kroesbergen, Jobsis et al' 1999)' There is no current
recommendation for correcting the expiratory flow for either weight or height in children or
adults.
Compartment modeling has further demonstrated the importance of the expiratory flow used -
with the flow determining where the exhaled No measured is being produced within the lung'
The exchange dynamics of NO is different from the other previously well studied gases such
as oz and coz because of differences in physical and biochemical properties' Firstly, No is
highly reactive. Secondly, it is actively produced basally in response to various stimuli'
Thirdly, it binds avidly to haemoglobin with different kinetics. I have alluded to these factors
previously in Chapter 6 when comparing the differing NO and COz exhalation pattern' where
the latter is known to be predominantly produced in the alveolar compartment' We used this
finding to suggest that NO was coming from a different part of the lung to COz'
One research group (Tsoukias and George 1998) used a two compartment model to describe
the pulmonary exchange dynamics of No, and to try and understand the results of the No
levels being presented in the literature (see figure 9.1 below)' Tsoukias' research group
depicted the first compartment as a rigid or non-expansile compartment representing the
conducting zoneithe airways from the trachea through to generation 17 as defined by Weibel
(weibel 1963). The second compartment was described as a flexible or expansile
compartment representing the respiratory bronchioles to the alveolar region' Both
compartments were surrounded by a layer of tissue representing the bronchial mucosa in the
airway compartment and the alveolar membrane in the alveolar compartment' The next layer
was blood which represented the bronchial circulation and the pulmonary circulation in the
airway and al veolar compartments respectivel y'
Using this model, the group then derived a series of mathematical equations to describe each
of the possible pathways for NO. This included NO production in the tissue, transfer of NO
from tissue to blood (and therefore loss of NO) and transfer of NO from tissue to alveolar or
airway space. Equations were also derived for the behaviour of No in intra-thoracic air'
assuming a well mixed compartment and accounting for convective flow of No in and out of
the compartments in inspiratory and expiratory manoeuvres, and for diffusion back into
tissue. The blood layers were considered an infinite sink for NO. All the parameters were
207

taken from the available literature including the production rate of NO in the tissue layer.
Using these equations they could then explain the pattern of NO seen in single exhalations
under different conditions including with and without breath holds and at different expiratory
flows. Overall, the model worked well and appeared to mirror actual results from the human
studies, although the predictions of the initial peaks seen with single exhalations were of
variable accuracy. Generally the model underestimated the peak in single exhalations and
overestimated the peak after breath-holds. The researchers stated in their paper that "Initial
reports have suggested that ambient levels of NO do not affect the exhalation profile." I also
could not ascertain if they took into account any upper airway contamination or whether, like
a later group (Silkoff, Sylvester et al. 2000), they assumed closed vellum. These could be
potential sources of error.
Figure 9.1: A two-compartment model of NO exchange
x
€ €
(J
E
o
nr.v(t)
Car,v(r)
o''f*
:I
v;,c;
Dead space
ALVeoli
Ftg. l. Schematlc of Z-compartment model for nltric oxtde (NO)
pulmonary exchange. Flrst comPartment rePresents relatively nonexpanslle
conducting airways; second compartment represents exPansile
alveoli. Each compartment ls adJacent to a layer of tissue that is
capable of producing and consumlng NO. Exterior to tissue is a layer
of-blood that represents bronchlal or.pulmonarv clrculation and
seryes as an infinlte slnk for NO. Ve and V1, exPlratory and
Insplratory flow, respectively; Ce and Cr, exPiratory and inspiratory
concentration, respectlvely; Ca1 and C61v, airway and alveolar concentratlon,
respectivCly; Var and Vap, airway and alveolar volume,
respectively; Jrrg,ar, and Jr:g,atv, total flux of NO from tlssue to alr and
from alveolar tlssue, respectively; t, time; V volume.
A two-compartment model of NO exchange dynamics using equations to derive the measurement of
NO from different lung compartments depending on the expiratory flow.
Taken from Tsoukias NM, George SC. A fwo-compartment model of pulmonary nitric oxide exchange
dynamics. Joumalof Applied Physiology 1998;85(2): 653-666 (Tsoukias and George 1998).
208

These predictions built on work from a previous group who had also derived equations to
model NO interactions in the lung (Hyde, Geigel et al. 1997). Their model was able to predict
NO results from the lower airway using a one compartment model but was unable to account
for the upper airway contamination. A later group also used a two compartment model and
reported that a breath hold of 10 to 20 seconds meant that the concentration of NO was
equilibrated - "alveolar airway" production of NO now equaled the amount of NO diffusing
out of the airways (pietropaoli, perillo et al. 1999). The subsequent exhalation could then give
the alveolar content of No. Finally, using similarly derived equations, another group re-
analysed their earlier data, from which they had concluded that NO levels correlated with
expiratory flow (Silkoff, Sylvester et al. 2000). They determined that a even closer
relationship existed between the exhaled No concentration (defined as the quantity of No
exhaled per unit time) and the expiratory flow. They felt this allowed a better estimation of
the quantity of NO diffusing into the exhaled gas and therefore better reflected tissue NO
concentration.
In essence, I believe these groups were making the point that studies were presenting No
results in a far too simplistic manner. NO was being presented as a single result from a single
compartment when in reality the NO concentration varied in different parts of the airway
system. In addition, the levels were always interpreted as if the NO was solely the result of
NO production, while in reality the NO reading was a combination of production' diffusion
(dictated by the rate of removal by capillary blood throughout the lung) and ventilation' The
importance of the modeling described above was to define specifically that different
expiratory rates would allow evaluation of different lung compartments. This could be critical
in appropriately discerning inflammation in different diseases; for example the conducting
airways in the airway disease of asthma or the alveolar compartment in alveolar diseases such
as in interstitial lung disease. This theory was tested by measuring 'fractionated' NO in a
cross section of subjects using three exhalation flows at 100, 1?5 and 370m1/s in 40 newly
diagnosed, steroid naive asthmatics, 17 patients with allergic alveolitis and 57 healthy
controls (Lehrimaki, Kankaanranta et al. 2001). The asthmatic patients did indeed have higher
bronchial NO output at 25nLJs compared to those with alveolitis or healthy controls which
were both measured at 0.7n[./s. On the other hand, the subjects with alveolitis had higher
alveolar NO concentrations at 4.1ppb than the asthmatic or healthy subjects which were both
measured at l.lppb. Thus there was no difference in No between those with asthma and
healthy controls when the alveolar compartment was measured (I-ehtimaki, Kankaanranta et
al. 2001). Finally they demonstrated that the concentration of NO in the bronchial
209

compartment correlated with the serum level of the EPX and bronchial hyper-responsiveness
in those with asthma, while the alveolar NO concentration in patients with alveolitis
correlated inversely with the pulmonary diffusing capacity.
A number of studies have now been conducted assessing NO in this manner. These have
demonstrated a higher exhaled NO in the conducting airways in asthmatics compared with
controls, with no mean difference of NO measured in alveolar compartment in otherwise
stable asthmatics (Hogman, Holmkvist et al. 2002; I-ehtimaki, Kankaanranta et al. 20fr2;
Mahut, Delacourt et ^l.2OO4; Shin, Rose-Gottron et al. 2004; Lehtimaki, Kankaanranta et al.
2005). The reduction of NO in response to steroid therapy also occurs in the airway
compartment (Silkoff, Sylvester et al. 2000; Irhtimaki, Kankaanranta et al. 2OOl; Shin, Rose-
Gottron et al.2004). In a cross sectional study of proximal and distal sampling in children, it
was the proximal NO output that correlated better with the FEVr (Mahut, Delacourt et al.
20M). However, as no theory is perfect, both high airway and high alveolar NO has been
demonstrated on highly symptomatic asthmatics or those with ongoing nocturnal symptoms
(Mahut, Delacourt et al.2004; Shin, Rose-Gottron et al.2004; I€htimaki, Kankaanranta et al.
2005). Also, high airway levels of NO were also seen in subjects with allergic rhinitis despite
having no airway symptoms (Hogman, Holmkvist et al.2002). Iow ainvay levels were seen
in smokers and patients with COPD, though the alveolar levels in NO were increased in the
COPD patients (Hogman, Holmkvist et al. 2002). Airway exhaled NO levels were no
different between CF and healthy control adolescents, but lower levels were seen in the
alveolar compartment and tissue concentration in the healthy goup (Shin, Rose-Gottron et al.
2N2).Increased rates of alveolar NO have been described in allergic alveolitis (khtimaki,
Kankaanranta et al. 2001) and scleroderma (Girgis, Gugnani et al. 2002) with negative
correlations between NO levels and the diffusion factor demonstrated in both.
The nomenclature used has also required adaptation as discussed in the 2002 and 2005
standardisation documents (Baraldi, de Jongste et al. 2OO2; American Thoracic Society and
European Respiratory Society 2005). The previous recommendations were to express exhaled
NO or nasal NO in terms of ppb (equivalent to nIJL of exhaled air). The standard single
exhalation remains at 50mls/s, now suggested to be denoted as F4o0.05. NO output
representing the rate of exhaled NO is denoted by Voo and calculated from the product of NO
concentration in nL/L and expiratory flow rate in Umin in the following equation:
Vno (nUmln) = NO (nUL) x alrflow rate (Umln)
2ro

The ATS guideline states "terms such as NO release, NO excretion, NO secretion and NO
production are to be discouraged when referring to Voo" (American Thoracic Society
European Respiratory Society 2005).
A constant expiratory flow is required with any exhalation measured and has been
reconrmended following the findings from early studies. This is most easily achieved with
appropriate biofeedback using a gauge or computer display for subjects to maintain expiratory
flow within specified limits. All newer machines as exemplified by the companies that took
part in the 2005 recommendations (Aerocrine, Eco Physics, Eco Medics, Ionics Instruments
and Ekips Technologies) have these available. However there are other options which include
the use of dynamic resisters (Kharitonov, Gonio et al. 2003), operator controlled flow
(Baraldi, Scollo et al. 2000), starling resisters (Hogman, Stromberg et ^l' 1997; Tsoukias'
Tannous et al. 1998) and server controlled devices (Silkoff, Bates et al. 2004)' These
techniques have usually been tried in young children but can also be useful in other subjects
that have difficulties controlling their exhalation such as those with neuromuscular disease' In
children aged 4-g years, 507o could not perform the single breath online technique adequately'
However, the addition of a dynamic flow resister allowed exhalation with a variable mouth
pressure while maintaining constant expiratory flow and resulted in only 77o of the children
unable to perform the manoeuvre (Baraldi, scollo et al. 2000).
9.3.1 (ii) Mouth Pressure
An expiratory mouth pressure of between five and 2}cttttLzO is currently recommended' This
degree of resistance is needed for velum closure of the soft palate to prevent nasal
contamination of the exhaled NO and has been validated by nasal COz measurements (Silkoff'
McClean et al. L997) and nasal argon insufflation (Kharitonov and Barnes 1997)' Many
studies have confirmed higher sinus and nasal levels when comparing sinus' nasal' exhaled
and/or lower airway sampling (Alving, weitzberg et al. 1993; Lundberg, Rinder et al' 1994;
Lundberg, weitzberg et al. 1994; Schedin, Frostell et al. 1995; Dillon, Hampl et al' 1996;
Imada, Iwamoto et al. 1996; Kimberly, Nejadnik et al. 1996). The other options described to
prevent nasal contamination has been continuous nasal aspiration (Silkoff, Kesten et al' 1995)
or to inflate a balloon in the posterior nasal pharynx to separate the two compartments
(Schedin, Frostell et al. 1995; Kimberly, Nejadnik et al. 1996). Clearly, exhaling against an
appropriate mouth pressure is the easiest option and has been most widely used'
Unlike the effects of flow on NO, the effects of varying mouth pressure has not demonstrated
consistent findings in all studies. Two studies showed no difference in plateau NO
2lr
and

measurement with variable expiratory mouth pressure (Hogman, Stromberg et al. 1997;
Silkoff, McClean et al. t997), but another showed increasing exhaled NO concentrations with
increasing expiratory pressure from two to ten cmHzO (Kondo, Haniuda et al. 2003). Our own
findings suggested the opposite with a reduction of exhaled NO with increasing mouth
pressure in two of ten subjects (Byrnes, Dinarevic et al. 1997). However, as noted in the 2005
recommendations, a pressure above 20cmHzo should be avoided as it may be "uncomfortable
for patients or subjects to maintain". Indeed, I think this may have contributed to the fact that
there was a falling off of the exhaled NO in our study in two subjects. Having tried it
repeatedly myself, while pressures of 4mmHg (5.4cmHzO) and 8mmHg (10.9cmHzO) were
easy to maintain, l2mmHg (16.3cmHzO) became a little more difficult but l6mmHg
(21.8crnHzO), the highest expiratory pressure sampled in our study, was very tiring to exhale
against, and 2OmmHg Q7.2cn*l2O) almost impossible to maintain and therefore was not
used. One needs to recall that in the earlier studies a far longer exhalation time was also
required with the older modified analysers.
9.3.1 (iii) Nasal clips and breath-holding
While the early studies varied as to whether or not nose clips were used, the current
recommendation is not to wear them when doing exhaled NO in any of the methods of
measurement as this may actually increase the risk of having nasal and sinus contamination of
the oropharyngeal and exhaled sample (American Thoracic Society and European Respiratory
Society 2005). The previous standards suggested that a nose clip may be worn to prevent
inhaled air to contaminate the exhaled sample (see below) (Baraldi, de Jongste et al.2002).
In addition, the inhalation is now recommended as being through the mouth with higher initial
exhaled NO demonstrated following an inhaled breath through the nose when compared to
inhalation through the mouth @hillips, Giraud et al. 1996; Robbins, Floreani et al. 1996).Any
degree of breath-holding also results in NO accumulation in the nasal and oropharyngeal
spaces giving higher exhaled levels @ersson, Wiklund et al. 1993; Lundberg, Weitzberg et al.
L994; Kharitonov, Chung et al. 1996; Kimberly, Nejadnik et al. 1996; Massaro, Mehta et al.
1996; Sato, Sakamaki et al. 1996). Breath-holding was shown to increase NO levels in a time
dependent manner in atopic and healthy subjects (Martin, Bryden et al. 1996). Breath-holding
appears to affect the peak more than the plateaux levels in healthy and asthmatic subjects
(Shinkai, Suzuki et al.2O02). The effect of breath-holding also depended on the ambient NO;
when the ambient NO was greater than 10ppb, exhaled NO was decreased whereas if the
212

ambient No was less than 10ppb the exhaled No was increased after a 10 second breath-hold
(Jobsis, Schellekens et al. 2001).
9.3.1 (iv) The recorded measurement
The NO recorded in the literature has been peak, plateau, area under the curve or NO plateau
signal at COz peak. The current recommendation is for plateau levels to be reported' unless
calculating NO output. This requires a plateau of three seconds following exhalation of at
least six seconds for participants older than 12 years of age' or two seconds following
exhalation for at least four seconds for children less than 12 years of age' The peak
measurement has shown more variability as influenced by the degfee of dead space' ambient
NO and inspiration taken via nose or mouth. An expiratory flow of 50mls/s ensures an
acceptable time to plateau, an acceptable rate of decline in lung volumes and is appropriate
for children at less than 12 years of age, as well as those with vital capacities less than one
litre (Pfaff and Morgan l994;Canady, Platts-Mills et al. 1999; Franklin, Taplin et al' 1999;
Kissoon, Duckworth et al. 1999).
9.3.1 (v) The effect of ambient nitric oxide
Recommendations since 199? have been to record the ambient NO as this fluctuates
considerably. A note from one of the authors (A Sovijarvi, Helsinki University Central
Hospital, Finland) in this document states; the ambient NO was noted to vary between I and
600ppb during winter months in their studies (Kharitonov, Alving et al. 1997). Initially it was
suggested that measurements should only be performed with an ambient NO less than 40ppb'
with improved standardisation and more sensitive and rapidly responding analysers this is
now too high. The 2005 recommendation is that the ambient NO be less than 5ppb and/or that
subjects inhale NO free air while being tested. The newer NO analysers including the smaller'
more transportable units such as the MINO@ (Aerocrine AB, Solna, Sweden) or z-700 No
meter @asibi Environmental Corporation, Glendale, California, United States of America)
have a scrubbing facility within the inhalation limb. This may be more important for reservoir
collections during tidal breathing where an increased amount of ambient NO is exhaled and
thus collected. In our experiments we elected to measure only when the NO in ambient air
was less than 10ppb. However on many days this precluded measurement which delayed the
experiments. Unlike other researchers, we found exhalation from a high NO concentration
compared to exhalation from a low NO concentration resulted in a drop in the overall NO
level as discussed in Chapter 7. On review now, it may have been because we were waiting
for a plateau to develop with a drop from the high ambient NO reading and it took some time
2r3

to settle. We may have been measuring the latter part of the exhalation in error and this
potentially may give lower levels than the peak or the true plateau. Thus, we may have been
comparing the plateau in the exhalation from the low ambient NO with the last part of the
exhalation from the high ambient NO. In two studies NO was significantly higher when
measured in a total of 215 children including healthy controls, non-asthmatic atopics and
asthmatics at times of high ambient NO (>10ppb, mean l9ppb) and at times of low ambient
NO (10ppb was the cut-off used) and
measured NO increased if the ambient NO was lower (NOclOppb) (Jobsis, Schellekens et al.
2001). Nasal NO in healthy children correlated with ambient NO and, in those children
greater than 12 years, it was the only factor correlating with the measured expiratory levels
(Struben, Wieringa et al. 2005). More recently there has been a study suggesting that even
levels of 5-10ppb ambient NO may have an effect on the exhaled results, with no difference
seen at less than 5ppb, upholding the most recent recommendations (Franklin, Turner et al.
2004).
Other studies have examined effects of pollution on exhaled NO. Ambient NO levels, as well
as ambient CO levels, were positively correlated with the exhaled NO results in 16 non-
smoking healthy subjects (Van Amsterdam, Verlaan et al. 1999). Mean air pollution was also
correlated with the exhaled NO levels in 16 healthy subjects measured on two separate days
(van Amsterdam, Verlaan et al. 1999) and 18 subjects measured on four separate days
(Steerenberg, Snelder et al. t999) at times of differing pollutant levels. At times of high
ambient NO, an increased risk of developing respiratory symptoms described as "sore throat,
runny nose, having a cold or being sick at home in the following week" was also shown in 68
children (Fischer, Steerenberg et al.2N2).
9.4
Online spontaneous or tidal breathing measurement
A single exhaled breath with online measurement remains the measurement of choice.
However, in some studies 50Vo of children aged 4-8 years of age @araldi, Scollo et al. 2000),
l\Vo of children aged 9-16 years (Baraldi, Scollo et al. 2000) and 30Vo of children aged 4-16
years of age (Jobsis, Schellekens et al. L999) could not pedonn this technique appropriately.
Online measurement during tidal breathing, usually as a mean of three breaths, has been
explored as an option for younger children and infants. One study measured exhalation
following quiet breathing with the operator controlling the expiratory flow by varying
214

expiratory resistance. A flexible rubber tube 9cm long with a diameter of 0'9cm and an end
resistor of 2mm was placed in the circuit. The operator could bend the flexible device during
exhalation to provide resistance and allow manual control of the expiratory flow by direct
checking on the monitor without requiring active cooperation from the child' of the 115
children enrolled, lr0 (g3vo)were able to perform this manoeuvre compared to 73 (63Vo)who
were able to perform the active single exhaled breath (Baraldi, Scollo et al. 2000)' In another
study, NO was measured online in 67 children during controlled tidal breathing (767o wete
aged2-5 years) with flow measured by a pneumotachograph and displayed on the computer
screen with a negligible delay of 0.1 second allowing the operator to target the exhaled flow
within preset limits of 0.4 to 0.6Us by continuously adapting the outlet resistance. of the nine
children (l3zo) who failed the measurement; seven had asthma, two had episodic wheeze'
four were aged two, three were aged three and two were aged five' The visualisation of the
online measurements allowed breath to breath profiles to be scrutinised to ensure "a stable'
calm and reproducible breathing pattern" (Buchvald and Bisgaard 2001)'
Tidal breathing results in lower NO levels than single exhalations and this is exaggerated at
higher No readings (Silkoff, Mcclean et al. 1997; Rutgers, Meijer et al. 1998; Franklin,
Turner et a;.2004t. While a reasonable correlation has been found between the two (Silkoff'
Stevens et al. 1999; Kissoon, Duckworth et al. 2000; Franklin, Turner et al' 2N4), individual
agreement can be poor (Rutgers, Meijer et al. 1998; Franklin, Turner et al.200,4)' The lower
results could be explained by increased inhaled air dilution from a low NO source and
variable flow during tidal breathing, and the possibility of breath-holding and nasal leakage in
single breaths (Hyde, Geigel et al. 1997; Silkoff, Mcclean et al. 1997; Rutgers, Meijer et al'
l99g). This can be improved by controlling expiratory flow during tidal breathing -
but when
only passive co-operation is available and considerable operator experience appears necessary
to manipulate the resistance correctly (Baraldi, Scollo et al. 2000; Buchvald and Bisgaard
2001). I would also be concerned that the rubber tubing itself, in one method described, may
result in some NO loss following reaction with the sulphur hydryl groups' This online
spontaneous breathing method still "requires passive cooperation as the child needs to breath
slowly and regularly through a tight fitted mask, close the mouth around a mouth piece which
is often the limiting factor, as well as being able to tolerate breathing against resistance"
(Buchvald and Bisgaard 2001). Despite this, results have shown the same patterns as seen
with the single online measurements in the cross-sectional population studies (Baraldi'
Azzolin et al. 1997; Baraldi, Azzolin et al. 1998; Baraldi, Dario et al. 1999). Use of this in
infants is discussed below in Section 9.16.
2t5

9.5 Off-line measurement
NO determinations can be measured from samples collected into a reservoir system and later
fed through a cheminiluminescence analyzer. Similar to early online experiments, while the
absolute levels of NO measured by reservoir collection varied between investigators, the
findings across disease entities remained consistent (Alving, Weitzberg et al. 1993; Persson,
Wiklund et al. 1993; Kharitonov, Yates et al. L994; Kharitonov, Yates et al. 1995; Massaro,
Gaston et al. 1995; Deykin, Halpern et al. 1998).
A number of standardisation issues arose with this technique. Firstly, the concern regarding
NO reactivity and that inert collection bags would be required. Other factors that could affect
stability included temperature, light exposure, damage to bags, leaks and pressure changes.
Mylar or tedlar bags were early reported as the most stable (Kharitonov, Alving et al. 1997)
with NO levels consistent up to 12 hours post collection (Massaro, Gaston et al. 1995;
Massaro, Mehta et al. 1996; Canady, Platts-Mills et al. 1999; Jobsis, Schellekens et al. 2001).
Some investigators reported continued stability for 24-48 hours @aredi, Loukides et al. 1998;
Djupesland, Qian et al. 2001), while others found an increase in the NO concentration
(Silkoffl Stevens et al. 1999). NO collected in a reservoir containing silica gel was stable for
24 hours @aredi, Ioukides et al. 1998). One $oup
used a tube collection system showing
better online and off-line agreement than bag collections for both exhaled and nasal levels
@jupesland, Qian et al. 2001). Reservoir stability also depends on temperature. NO results
appeared to be stable for nine hours at 4o to 37oC but increased between nine and 48 hours in
samples with initially low concentrations. It was not stable at higher temperatures and the use
of a drying agent did not improve the stability (Bodini, Pijnenburg et al. 2003). One study
evaluated 185 breaths in bags that were re-used 10-20 times flushing three times with NO free
air between uses and stored at 22oC. Over a four day period, the samples with low NO
increased in a linear fashion, and those with high NO from asthmatic subjects showed a
gradual decrease (Silkoff, Stevens et al. L999; Kharitonov, Gonio et al. 2OO3). Higher
temperatures made the changes more rapid and lower temperatures made it less likely to
occur. The size of the balloon was not thought to be critical but was recommended to be
similar to, or larger than, the subjects' vital capacity (Linn, Avila et al. 2004).
Secondly, uncertainty existed as to whether the collection should be a single exhaled breath,
several 'single exhaled breaths' or tidal breathing. The reproducibility with single breaths
appeared to be fair or good with intra-subject coefficients of variation down to 5Vo (Gaston,
216

Drazen et al. 1994; Nelson, Sears et al. 1997; Canady, Platts-Mills et al' 1999; Jobsis'
Schellekens et al. 2001).
Thirdly, there was debate as to whether the whole exhaled breath should be collected. Early
experiments discarded the first part of exhalation to reduce contamination by inhaled ambient
NO, dead space and nasopharyngeal space with a good correlation demonstrated between the
remaining exhalate and single online exhalation (Borland, Cox et al.1993; Massaro, Mehta et
al. 1996). Similarly, studies that discarded the first tidal volume when measuring during tidal
breathing also achieved a closer approximation to online values (Paredi, Loukides et al' 1998;
Jobsis, Raatgeep et al. 2001). The uncertainty was in deciding the absolute amount to discard
and how to get consistency across subjects. While 150 to 200mls were used in the adult
studies even greater uncertainty existed in the correct amount to discard in paediatric studies'
The 'discard' was achieved employing spring loaded or manually activated valves' or low
compliance reservoirs placed in series with the main collection vessel. Subsequently
collecting the whole exhalation was shown to provide identical sensitivity and specificity as
single breath online recordings, despite the fact that the absolute NO levels were not identical
(Silkoffl Stevens et al. 1999; Djupesland, Qian et al. 2001; Jobsis, Raatgeep et al' 2001;
Deykin, Massaro et a:.2002). The standardisation has therefore altered accordingly between
lggT and2005 from discarding the first o.7sLexhaled (or 0.5L if the vital capacity was less
than 2L) @uropean Respiratory Society 1993; Kharitonov, Alving et al. 1997) to collecting
the entire breath (American Thoracic Society and Association. L999;Baraldi, de Jongste et al'
2002).
Similar across all methods, higher flow rates result in decreased concentration of NO
recovered from the exhaled reservoir sample (Hogman, Stromberg et al' t997; Silkofl
McClean et al. 1997). Despite this, subject group differences can be seen at expiratory rates
between 50 and 500mls/s as long as the flow remains identical for all subjects in any
comparison (Jobsis, Raatgeep et al. 2001; Deykin, Massaro et al' 2002)' With collection of
tidal breathing, using standard expiratory flow reduces variability and improves comparison
with online results (Kissoon, Duckworth et al. 2000). This can be controlled by the subject or
by the operator (Massaro, Mehta et al. 1996; Baraldi, Azzolin et al. 1997i Baraldi, Carra et al'
t999; Baraldi, Dario et al. 1999; Jobsis, Schellekens et al. 2001). The tasldorce ultimately
proposed recommendations for 50mls/s exhalation for both offline and online collections as
showing good correlation in the paediatric arena (Baraldi, de Jongste et al.2002)' It is still
possible to sample different compartments using a different flow even with this technique
2r7

(Hyde, Geigel et al. 1997; Tsoukias and George 1998; Tsoukias, Tannous et al. 1998; Silkoff,
Sylvester et al. 2000).
One of the reasons that higher levels were thought to be obtained using either single or tidal
breathing into a reservoir was because of an open soft palate and upper nasopharyngeal
contamination (Sharma, Traylor et al. 1987; Schilling, Holzer et al. 1994; Kimberly, Nejadnik
et al. 1996; Kharitonov and Barnes 1997). A single exhalation against even low resistance
into the reservoir gave much lower levels (Massaro, Gaston et al. 1995). A resistor is now
included in most reservoir bags with a recorlmendation of standard expiratory flow and
standard expiratory pressure of 5cmHzO for both single breath (Massaro, Gaston et al. 1995;
Deykin, Halpern et al. 1998) and tidal breathing reservoir collections (Canady, Platts-Mills et
al. 1999; Jobsis, Schellekens et al. 2001). A dynamic flow restrictor is possible to aid the
operator to standardise the exhaled breath with less able subjects by altering the resistance to
give a standard flow during the exhalation @ijnenburg, Lissenberg et al. 2002), although
again I imagine this must introduce an element of complexity.
From 1999, inhaling via the mouth prior to exhalation into the reservoir bag was added to the
protocol. By 2005 this recommendation had been revised to include the inhalation of NO free
gas or via an NO scrubbing filter in the inspiratory limb. High concentrations of NO in the
inspired air of greater than 20ppb significantly increased the offline exhaled NO
measurements (Gustafsson, Irone et al. l99l; Zayasu, Sekizawa et al. 1997). Again wearing
a nose clip and breath holding are not recommended for similar reasons to the single breath
online measurements (see section 9.3.1 (ii)) (Jobsis, Schellekens et al. 2OOt). Following these
guidelines, subjects tested on multiple days had a variation between 1 and 5ppb, which is
similar to that found in immediate online measurements, although in this study the collection
was delayed for more than three seconds to discard the dead space (Borland, Cox et al. 1993;
Massaro, Mehta et al. 1996). For the standard method to measure off-line collections as either
single exhaled breaths or as tidal breathing collections see Table 9.2.
2r8

Table 9.2: The recommended standards for single breath and tidal breathing otf-line NO measurement
The current recommended technique for single exhalation collection into a reservoir is:
r inhale through the mouth from NO free air (

9.6 Nasal nitric oxide measurement
For completion, I am going to briefly cover nasal NO although I did not measure this
parameter as part of my research. Presentation of standard techniques for this measurement
commenced in 1999 (American Thoracic Society and Association. 1999), with minor
amendments in 2002 and 2005 (Baraldi, de Jongste et al.2N2; American Thoracic Society
and European Respiratory Society 2005). The NO concentration is far greater in the nose than
the lower respiratory tract and is measured in parts per million as opposed to parts per billion
(Alving, Weitzberg et al. 1993; Gerlach, Rossaint et al. 1994; Lundberg, Farkas-Szallasi et al.
1995). NO concentration is even higher in the para-nasal sinuses (Lundberg, Rinder et al.
1994; Lundberg, Farkas-Szallasi et al. 1995; Haight, Qian et al. 2000).
Table 9.3: The recommended standard for nasal NO measurement
The current recommended technique for measurement of nasal NO is:
o tho subject is seated
o two nasalolives are placed with a central lumen in the nares.
o these must be of sufficient size to occlude the nostril.
o d sdlTrple is aspirated continuously at a constant rate from one narus.
o gas is entrained via the other narus giving a trans-nasal flow in series.
o a target sampling airflow of 0.25 to O.3Umin is recommended.
. oropharyngeal and lower airway contamination is prevented by one of the methods
suggested (see paragraph below).
. mean NO values are calculated for a stable plateau of greater than ten seconds or five
breaths (Silkotf, Chatkin et al. 1999; Ratjen, Kavuk et al. 2000).
o the higher flow rate (compared to exhaled oral sampling) at 0.25-3Umin allows a steady
plateau levelof NO concentration in subjects within 20-30 seconds.
This trans-nasal flow reflects the most cornmon method of measurement. An alternative is to
have continual aspiration of both nares; one to discard and one to measure. Velum closure can
be achieved in a number of ways; the most common is to slowly exhale orally against a
resistance similar to the exhaled NO recommendation (Arnal, Didier et al. 1997; Kharitonov,
Rajakulasingam et al. 1997; Silkofl McClean et al. 1997; Dubois, Douglas et al. 1998;
Silkoff, Chatkin et al. 1999). Other options that have also been effective are purse-lip
breathing via the mouth (Rodenstein and Stanescu 1983), breath-holding with the velum
closed (Kimberly, Nejadnik et al. 1996) or voluntary elevation of the soft palate by a trained
subject (Giraud, Nejadnik et al. 1998). Of note for nasal NO measurements, healthy adults
have significantly better repeatability than healthy children (Kharitonov, Walker et al. 2005).
220

9.7
In this next section, I review physiological variables that may alter NO results, recognistng
there are conflicting findings reported in most parameters.
9.7.1 Size and gender
Firstly, there have been a number of investigations across animal species, and the reasons I
refer to this is with regarding the possible effect of size, and to describe an extraordinary
study done in elephants! Exhaled No results have been similar in rabbits, guinea pigs and rats
(Gustafsson, Ifone et al. 1991; Stewart, Yalenza et al' 1995) but lower levels found in
Antarctica seals (Stanek 1995). one heroic study measured direct exhalation with a one litre
syringe and continuous online measurement in four elephants, finding no difference between
these and human subjects. They also showed no difference of NO with height of the elephants
which ranged from 240cm to 260cm (although how significant a 20cm difference in height is
at these levels remains questionable), or weight which ranged from 2800kg to 4000kg
(Lewandowski, Busch et al. 1996).
Secondly, with regard to gender, differences have been found in some studies (Tsang, kung
et a;.2002; van der Ire, van den Bosch et a\.2002. Olivieri, Talamini et al.2006) but not in
other adult (Jilma, Kastner et al. 1996; Morris, Sooranna et al. 1996; Bartley, Fergusson et al'
1999; Kharitonov, Gonio et al. 2003) or in paediatric studies (Baraldi, Dario et al' 1999)'
Measurements in over 100 (van der lfe, van den Bosch et al. 2002) and over 200 healthy
adults (olivieri, Talamini et al. 2006) found normal exhaled No values to be significantly
higher in men. One study showed these higher levels correlated with height and weight but
not with age (Tsang, Ip et al. 2001). In another study, when corrected for body weight' men
were calculated to exhale 50Vo moreNO than women (Morris, Sooranna et al' 1996)' Exhaled
NO measured in nUmin/m2 correlated with body surface area in one (Phillips, Giraud et al'
1996) but not a second study (Jilma, Kastner et al' 1996)'
paediatric studies have also looked for gender differences. In almost 1000 13-14 year olds,
higher levels of exhaled NO were independently related to male gender, as well as to the
presence of wheeze and rhinoconjunctivitis (Nordvall, Janson et al. 2005)' In 258 children'
exhaled No did not correlate with height, weight, body mass index or body surface area but
again demonstrated a higher result in boys (wong, Liu et al. 2005). In over 100 healthy
children using both online and offline analyses, body area, age and FEFzs-zs were significant
predictors of exhaled No (Kissoon, Duckworth et al. 2o02). In children aged 4-6 years
22r

measured over two 24 hour periods, there was a gender difference (again higher in boys than
girls) in the first assessment, but not in a second assessment (Napier and Turner 2005).
However other paediatric studies have not confirmed these associations; showing no
correlation observed between NO and height or spirometric data (Baraldi, Azzolin et al.
1999), and no correlation between nasal NO and body mass index (Struben, Wieringa et al.
2005). At the current time the reference ranges for 'normal' are given as one range, with no
gender correction.
9.7.2 Age
Early in the research literature, it was suggested that NO increased with age correlating to
pneumatisation of sinuses (see Figure 9.2) (Lundberg, Farkas-Szallasi et al. 1995). In over
650 healthy children aged4-17 years in three studies, exhaled NO significantly increased with
age in both off-line and online analyses (Franklin, Taplin et al. 1999; Kissoon, Duckworth et
al.2[f]l2; Buchvald, Baraldi et al. 2005). Nasal NO in 340 healthy children aged 6-17 years
also increased with age which would appear appropriate if it does relate to sinus development
(Struben, Wieringa et al. 2005). An increase of exhaled NO was confirmed in a younger age
goup of 2-5 year olds using tidal breathing (Avital, Uwyyed et al. 2003). One study also
demonstrated an increase between 1 and 24 hours of age in 13 healthy newborn infants
(Schedin, Norman et al. 1996). However, again findings have been inconsistent with no
correlation between age and exhaled NO seen in some groups (Baraldi, Azzolin et al. 1999;
Bartley, Fergusson et al. 1999). Despite the findings above, the current practice is to use one
range for normal values with no correction for the age of the subjects. It was however one of
our reasons for choosing a tight age bracket when measuring the exhaled NO in children in
the methodology study (see Chapter 8).
222

Figure 9.2: Plateau nasalNO increasing with age
E
o.
o,
o Eo
g go('
Eot,
o z6(to2
0.1
0.01
0.001
10 20 30 40
Agc (Years)
50 60
The plateau nasal NO levels increased with age in 49 subjects, suggested by the authors to correlate
with pneumatisation of the sinuses.
Taken from Lundberg JON, Farkas-szallasi T, Weitzberg E, Rinder J, Lidholm J' Angaard A' Hokfelt T'
Lundberg JM, Alving K. High nitric oxide proOuction.ii human paranasal sinuses' Nature Medicine
i99a; t (i): gzo'gz4 (Lundberg, Farkas-szallasi et al' 1995)'
9.7.3 Circadian rhYthm.
One study in children found the lowest exhaled NO and urinary EPX levels occurred at 7pm
and the highest at Tamin 20 asthmatics compared to six healthy children (Mattes' Storm van's
Gravesande et a:.2002). Otherwise no circadian differences have been demonstrated in adult
subjects with exhaled No (Kharitonov, Gonio et al. 2003) or nasal No measurements
(Bartley, Fergusson et al. Iggg). No was higher at night in asthmatics with nocturnal
symptoms compared to asthmatics without nocturnal symptoms and higher at all times
compared to healthy controls, but no circadian variation in any group was demonstrated (ten
Hacken, van der Vaart et al. 1998; Georges, Bartelson et al' 1999; Palm' Graf et al' 2000)' In
children aged 4-6 years, exhaled NO was reproducible though a 24 hour period (Napier and
Turner 2005) and in six children measured at different times on six consecutive days, no
circadian rhythm was found (Latzin, Beck et al'2002)'
9.7.4 Menstrual cycle and pregnancy
There are few studies to review here. No significant changes were found with repeated
measurements over weeks in healthy controls (Jilma, Kastner et al' 1996; Morris' Sooranna et
al. 1996), while one study did seem to demonstrate high exhaled NO levels in 40 asthmatic
223

women who coincidently experienced worsening of sputum scores, eosinophils and lung
function pre-menstrually (Oguzulgen, Turktas et al.2002). No difference has been shown in
exhaled NO during pregnancy between 10 and 42 weeks (Morris, Caroll et al. 1995).
9.7.5 Food and beverages
In our own study, we found that drinking water prior to exhalation transiently reduced NO
levels @yrnes, Dinarevic et al. 1997). Caffeine also decreased exhaled NO in one paper
(Bruce, Yates et at.2002) but not in another (Taylor, Smith et al.2004). Alcohol ingestion
reduced exhaled NO in both asthmatic and healthy subjects (Persson, Cederqvist et al. 1994;
Yates, Kharitonov et al. 1996) with a small decrease in exhaled NO observed up to 34 hours
after drinking (Jones, Fransson et al. 2005). Smoking also significantly reduces exhaled NO
and will this be discussed below with the studies on COPD subjects (see Section 9.15). The
reduced NO levels subsequent to both cigarette smoking and alcohol consumption was
suggested to be secondary to down regulation of iNOS (Steerenberg and van Amsterdam
20M).In contrast increased exhaled NO has been found after the ingestion of nitrite or nitrate
containing foods (such as lettuce) with a maximum effect occurring two hours after ingestion
(Zetterquist, Pedroletti et al. 1999; Olin, Aldenbratt et al. 2001). The gastric lumen has been
noted to have high levels of NO, therefore contamination with gastric air results in very high
exhaled NO (Lundberg, Weitzberg et al.1994).
9.7.6 Summary of physiologicalfactors that could alter nitric oxide levels
In conclusion, exhaled NO is likely to be higher in males than females, and relates to age
through childhood but not adulthood, possibly in relation to pneumatisation of the sinuses. It
is reduced with current smoking, recent ingestion of water, alcohol or caffeine and increased
with accidental gut contamination or after nitrogen-compound rich meals. There may be
differences throughout the 24 hour day in asthmatic subjects with nocturnal asthma, and
through the menstrual cycle in those who experience premenstrual exacerbations of asthma.
However, relationships with height, weight, surface area, menstrual cycle in non-asthmatic
women, or a circadian rhythm in non-asthmatic individuals has not been demonstrated.
Despite some of the findings above, there is only one range of 'normal' values given for the
results of measurement for oral or nasal results.
9.8 Nitric oxide levels in asthma and atopy
Since commencement of the studies on exhaled NO, more research has been conducted in
asthmatic populations than any other group; in particular assessing the use of NO for
224

screening, diagnostic and monitoring purposes. Initially cross sectional studies were carried
out with comparisons to the traditional inflammatory and clinical markers used in asthma (see
Chapter 1). Research then graduated to the use of longitudinal studies to assess the predictive
value of NO for presence, severity and to denote exacerbations of asthma' Longitudinal
studies have also been used to assess treatment effects in standard and new therapies' The
relationships between NO and asthmatic inflammatory markers, lung function testing'
bronchodilator responsiveness and airway challenges have all been studied. I have grouped
the studies presented here under headings of the hypotheses that the studies were devised to
address.
g.g.l Does nitric oxide correlate with other asthmatic inflammatory markers?
Exhaled NO was found to correlate with sputum eosinophil numbers in asthmatics and
healthy controls in adults (Jatakanon, Lim et al. 1998; Berlyne, Parameswaran et al' 2000;
Tsujino, Nishimura et al. 2000; Reid, Johns et al. 2003; silkoff, Ifnt et al' 2005; Fujimoto'
Yamaguchi et al. 2006; Zietkowski, Bodzenta-Lukaszyk et al' 2006) and in children
(Piacentini, Bodini et al. L999; Little, Chalmers et al. 2000; Warke, Fitch et al' 2002; Sacco'
sale et al. 2003; Mahut, Delclaux et a:.2004: Thomas, Gibson et al. 2005; Li, Tsang et al'
2006). while these have been demonstrated in asthmatics whether on or off a range of
medications, the correlations are strongest for steroid naive patients. These findings have been
consistent across the studies that used variable expiratory flows, and were confirmed in one
study that compared results in these subject groups across a range of flows (Beny, shaw et al'
2005). A positive correlation has also been demonstrated between exhaled No and serum
eosinophil cell counts in adults and children (Tsujino, Nishimura et al' 2000; Reid' Johns et
al.Z}o3:Silvestri, sabatini et al. 2003; Strunk, Szefler et al. 2003; Irhtimaki' Kankaanranta
et al. 2005; Zietkowski, Bodzenta-Lukaszyk et al' 2006). Correlations have been described
between NO and sputum ECP levels (Piacentini, Bodini et al. 1999; Warke, Fitch et al' 2002;
Thomas, Gibson et al. 2005), as well as No and lerum ECP levels (Aziz, wilson et al' 2000;
Dal Negro, Micheletto et al. 2003; Strunk, Szefler et al' 2003; Mahut, Delclaux et al' 200,4i
Zietkowski, Bodzenta-Lukaszyk et al. 2006). Perhaps unsurprisingly, in studies that compared
both, it was sputum rather than serum markers that had stronger correlations with exhaled NO
levels. A positive correlation was also shown with the leukotriene group of inflammatory
markers - leukotriene%,leukotriene B4 and 8-isoprostane (Mondino, Ciabattoni et al' 2W4)'
and a negative correlation with the anti-inflammatory cytokines IL-4 and IL-13 (Shome'
Starnes et al. 2006).
225

While these cross-sectional and the longitudinal studies described below (see Section 9.8.5)
have confirmed these findings, it has not been universal. For example, in 58 asthmatic
children no association between NO and eosinophils or ECP from sputum or serum was
shown (Wilson, James et al. 2001). Other studies did not demonstrate a relation between one
specific parameter such as NO with serum eosinophils (Turktas, Oguzulgen et al. 2003), with
serum ECP (del Giudice, Brunese et al. 2004) or with sputum leukotriene levels (Strunk,
Szefler et al. 2003), although they confirmed other associations. Iooking at these studies,
there is no apparent difference in methodology compared to others with positive findings,
although they are more often heterogeneous groups of asthmatics on a wide range of
medications, with smaller numbers of subjects and tended to be 'doctor diagnosed' asthmatics
rather than those diagnosed with specific testing when in clinic which was also conducting the
research.
Direct comparisons of NO with bronchial biopsy results have also been studied. One goup
found a correlation between exhaled NO and each of the eosinophil, lymphocyte and mast cell
components (Silkoffl lrnt et al. 2005), while another study found that NO was only related to
the total number of inflammatory cells present (Turktas, Oguzulgen et al. 2003). In 31
children with difficult asthma, there was a relationship between exhaled NO and the
eosinophil score in seven of 2l children for whom biopsies and NO were obtained. The
strongest relationship was an exhaled NO >7ppb and raised eosinophils at baseline in children
who had persistent symptoms despite high dose prednisolone for two weeks (Payne,
McKenzie et al. 2001). Exhaled NO was also shown to correlate with the degree of airway re-
modeling on biopsy samples in a group of 28 children (Mahut, Delclaux et al.2004) and the
degree of bronchial wall thickening seen on a CT scan in nine asthmatic children (Ketai,
Harkins et al. 2005).
9.8.2 Does nitric oxide correlate with lung function and bronchial hyper-responsiveness?
Comparisons of exhaled NO with lung function parameters have produced variable results.
While many have shown a significant negative correlation, particularly to FEV1, in adults (de
Gouw, Hendriks et al. 1998; Ho, Wood et al. 2000; Dal Negro, Micheletto et al. 2003;
Nogami, Shoji et al. 2003) and children (Colon-Semidey, Marshik et al. 2000; Piacentini,
Bodini et al. 2000; Spallarossa, Battistini et al. 2001; Beck-Ripp, Griese et al. 2002;
Malmberg, Pelkonen et al. 2003; del Giudice, Brunese et al. 2004; Saito, Inoue et al. 2004)
others were not able to demonstrate this (al-Ali, Eames et al. 1998; Silkoff, McClean et al.
1998; Piacentini, Bodini et al. 1999; Ho, Wood et al. 2000; Silvestri, Spallarossa et al. 2000;
226

Turktas, oguzulgen et a:.2003; Mappa, cardinale et al. 2005; Spergel, Fogg et al' 2005;
Thomas, Gibson et al. 2005; Zietkowski, Bodzenta-Lukaszyk et al. 2006). Again some of
these studies described correlations with certain parameters only such as FEF25-75 (Mahut'
Delacourt et al. 2004; Battaglia, den Hertog et al. 2005; Irhtimaki, Kankaanranta et al' 2005)'
FEVr/FVC ratio (Strunk, Szefler et al. 2003), residual volume (Mappa' Cardinale et al' 2005)'
or peak flow lability (al-Ali, Eames et al. 1998; Lim, Jatakanon et al' 2000)' In other studies
the correlations were only significant in certain groups such as atopic, rather than non-atopic'
asthmatics (Franklin, Stick et al. 2004)'
Positive correlations have been demonstrated between NO levels and airway reactivity as
measured by methacholine challenges (Jatakanon, Lim et al' 1998; Henriksen' Lingaas-
Holmen et al. 2000; wilson, Dempsey et al. 2001; Prieto, Gutierrez et al' 2002; Prieto'
Gutierrez et al. 2002; Buchvald, Eiberg et al. 2003; Langley, Goldthorpe et al' 2oo3;
Malmberg, Pelkonen et al. 2003; Nogami, Shoji et al. 2003; Berkman' Avital et al' 2005;
Ehrs, Sundblad et al. 2006), histamine challenges (al-Ali, Eames et al' 1998; de Gouw'
Hendriks et al. 1998; Dupont, Rochette et al. 1998; Dupont, Demedts et al' 2003; Zietkowski'
Bodzenta-Lukaszyk et al. 2006), or adenosine S-monophospate challenges (de Gouw'
Hendriks et al. 1998; Aziz,wilson et al. 2000; Prieto, Gutierrez et al' 2002; Prieto' uixera et
aI.2002;Prieto, Bruno et al. 2003; Berkman, Avital et al' 2005)' Again some studies found
correlations only in certain groups of subjects such as atopic asthmatics (Franklin' Stick et al'
2004) or those on IHCS (Reid, Johns et al. 2003) rather than all asthmatic and control
subjects.
g.8.3 Can nitric oxide be usedfor diagnosis of asthma?
Studies have assessed whether measurement of exhaled NO can be used to diagnose asthma'
either to screen a general population or to screen new clinic referrals' One group evaluated 47
consecutive adult patients referred with symptoms suggestive of asthma' The individual
sensitivities for each of the conventional tests (p.* flow, spirometry, serum eosinophils,
bronchodilator responsiveness and airway challenge) were up to 47Vo; much lower than for
exhaled No at ggvo andsputum eosinophili a at 86Vo. The results for conventional tests were
not improved when using a trial of oral steroid as a diagnostic test. The researchers concluded
that exhaled No and induced sputum analysis, particularly when combined, were superior to
the other conventional approaches in diagnosing asthma (smith, cowan et al' 2004)' The
same group looked at the predictive accuracy of exhaled NO to identify steroid
responsiveness to IHCS for four weeks in 52 adults presenting with undiagnosed respiratory
227

symptoms. Steroid response was significantly greater in those with the higher exhaled NO
levels (> a7ppb) independent of the original diagnostic label (asthma, COPD or chronic
bronchitis) (Smith, Cowan et al. 2005). A similar study evaluated 95 patients presenting with
respiratory symptoms in which 40 were subsequently diagnosed as asthmatic. A baseline
value of NO at >7ppb best differentiated between asthmatics and non-asthmatics with a
sensitivity of 82.5Vo and a specificity of 88.9Vo. The generated receiver-operated curves
(ROC) gave a value for exhaled NO of 0.89, similar to both methacholine and adenosine-S
monophosphate challenges, better than lung function or exercise testing (Berkman, Avital et
al. 2005). In 50 consecutive subjects being screened for exercise induced bronchospasm,
exhaled NO at

exhaled NO was the best predictor, when compared to lung function testing and
bronchodilator response, to discriminate those who subsequently developed asthma with the
sensitivity of g6vo and a specificity of g27o at a cut off level chosen 1.5 standard deviations
above the average for controls (Malmberg2004)'
g.8.4 Is nitic oxid.e associatedwith symptoms and severiry of asthma?
positive correlations with exhaled No have been shown with bronchodilator use and
symptom scores (Lanz,Irung et al. 1997; Tsujino, Nishimura et al. 2000; Roberts, Hurley et
a:.2004;warke, Mairs et a,..2004: Pijnenburg, Bakker et al. 2005; spergel, Fogg et al' 2005;
prasad, Langford et al. 2006), cough (Li, Irx et al. 2003) and bronchodilator responsiveness
(Colon-Semidey, Marshik et al. 2000; Little, Chalmers et al' 2000; Dupont' Demedts et al'
2xl3;Malmberg, Pelkonen et al. 2003; Silvestri, sabatini et al. 2003; Pijnenburg, Bakker et
al. 2005; Fujimoto, Yamaguchi et al. 2006; Zietkowski, Bodzenta-Lukaszyk et al' 2006)' In
many of the studies, the No levels and the sputum eosinophil levels had stronger correlations
to symptoms than other parameters measured. Again the findings were not universal' For
example, in some studies no correlation was seen between NO and bronchial hyper-
responsiveness (al-Ali, Eames et al. 1998; Silkoff, Mcclean et al. 1998; Chan-Yeung' obata
et al. 1999; van Rensen, Straathof et al. t999;Ho, Wood et al. 2000; Silvestri, Spallarossa et
al. 2000; del Giudice, Brunese et a:.2004:Thomas, Gibson et al. 2005; Jentzsch, le Bourgeois
et al. 2006), NO and symptoms (al-Ali, Eames et al. 1998; Griese, Koch et al' 2000; Sippel'
Holden et al. 2000) or NO and medication use (al-Ali, Eames et al. 1998; Sippel, Holden et al'
2000). In addition, no relationship was determined in 77 adult asthmatics between exhaled
NO and quality of life (Ehrs, Sundblad et al' 2006)'
There are a number of possible explanations for the inconsistencies. Firstly, the correlation
with severity is difficult depending on how 'severity' is defined' we know that exhaled No is
higher in steroid naive asthmatics, so those on increased amounts of treatment may have
reduced levels of No but their requirement for higher IHCS doses or additional medications
to control symptoms deems them more 'severe'. Several studies using either the GINA
guidelines or the ATS asthma guidelines to categorize asthmatic patients into gradations of
severity have been unable to show a relationship to exhaled No levels in adults (stirling'
Kharitonov et al. 1998; Chan-Yeung, obata et al. Iggg; Lim, Jatakanon et al' 2000; sippel'
Holden et al. 2000) or in children (Griese, Koch et al. 2000)' In one study of 30 children
divided into mild, moderate and severe groups' the exhaled NO did appear to correlate with
asthma severity (Delgado-Corcoran' Kissoon et al' 2004), and those on oral steroids with
229

'difficult asthma' also had higher exhaled NO suggesting ongoing inflammation (Stirling,
Kharitonov et al. 1998; Payne, Adcock et al. 2001). Secondly, studies in asthma have
commonly considered it as a single diagnostic entity when we increasingly have come to
consider it as a more heterogeneous disease. Differing factors within the asthma 'syndrome'
have been emphasized such as atopic versus non-atopic (or allergic versus intrinsic), and,
more recently, defined by the cell most prominent in the airway inflammation; eosinophilic
versus neutrophilic versus both or neither. In the cross sectional studies, most have consider
asthmatics as a single group without the nicety of these divisions. Thirdly, other factors may
influence asthma development, such as genetics. One goup studied the genotype of the NOS
enzyme in adult asthmatics and COPD patients, and showed that if there were high numbers
of "AAT" sequences (greater than 12 repeats) in the specific region of "intron 20" then much
lower levels of exhaled NO were obtained (Wechsler, Grasemann et al. 2000). Fourthly, we
are comparing exhaled NO as a predictor against other single measurements such as sputum
eosinophils or FEVr when it is unlikely that these are all isolated parameters. In a study of 92
children with 59Vo receiving IHCS, factor analysis selected four factors explaining 55.5Vo of
the total variance. These factors were exhaled NO, plasma total IgE, plasma specific IgE and
peripheral blood eosinophil percentage. The researchers suggested that these pararneters
operate as a group and should not be viewed as having non-overlapping characteristics
(I-eung, Wong et al. 2005).
Since cross sectional studies have not demonstrated a good correlation between asthma
severity and NO, perhaps it is more appropriate to look at NO levels in controlled versus
uncontrolled asthmatics. In 32 adults about to commence on a comparison of treatments,
baseline exhaled NO was higher in those presenting with symptoms (Gratziou, Rovina et al.
2001). In 45 asthmatic children higher NO levels were obtained in those that were recently
symptomatic, although all had levels above normals (Mahut, Delacourt et at.2004), and in 14
of 22 atopic asthmatic children with current symptoms the exhaled NO was two standard
deviations above the normal control group (Silvestri, Spallarossa et al. 1999). In studies
looking at NO levels in a total of 625 asthmatic patients with allergy as a trigger, the NO
levels were higher in those recently exposed (Artlich, Busch et al. 1999; Simpson, Custovic et
al. 1999; Langley, Goldthorpe et ^1. 2OO3), in those with perennial exposure compared to
seasonal allergies (Olin, Alving et al. 2004) and in those sensitised with common indoor
allergens such as dust mite, cat and dog.
Is this pertinent to clinical practice? In the following studies, the physicians attending the
children were blinded to the exhaled NO readings when making their concurrent assessment
230

and treatment decisions. Exhaled NO correctly predicted 21 asthmatic children deemed to be
,uncontrolled, versus 3l who were 'well controlled' (Meyts, proesmans et al. 2003). Fifteen
asthmatic children followed over 3-4 visits showed that exhaled No was significantly
correlated with the degree of control @elgado-corcoran'
Kissoon et al' 2oo4)' In 74
asthmatics and 31 healthy control children, the exhaled No level when raised above normal
(designated as > l3ppb in this study) had a sensitivity of 0'67 and a specificity of 0'65 to
predicted the requirement for a step-up in therapy (Griese, Koch et al' 2000)'
g.g.5 can nitric oxide predict deterioration in asthma control?
The studies above looked at concurrent exhaled No and symptoms' In one study' cited above'
the cross sectional analysis of 100 asthmatic patients from 7 to 80 years of age showed that
exhaled NO correlated better with markers of control such as lung function and sputum
eosinophils rather than markers of severity such as history of respiratory failure, healthcare
use, fixed air flow obstruction or an asthma severity score (Sippel, Holden et al' 2000)'
However, it would be a more useful tool if it could predict loss of control when following
asthmatic individuals' longitudinally'
In 22 moderate to severe asthmatics seen in a routine clinic visit, those who subsequently had
went on to have an exacerbation within two weeks had significantly higher mean exhaled No
(2gppb versus l3ppb) compared to those who remained stable (Harkins, Fiato et al' 2004)'
Irvels of exhaled No were followed prospectively in 44 asthmatics on daily IHCS and
LABA therapy over l8 months, withzzhaving one or more exacerbations' while the baseline
FEVr remained the best predictor, the Roc curve for exhaled No to predict exacerbation was
0.71. An exhaled NO of 28ppb gave a sensitivity of 0.59, a specificity of 0'82' a positive
predictive value of o.77 and a negative predictive value of 0'87 for having increased
symptoms. So, an exhaled NO >28ppb gave a relative risk of 3'4 for an exacerbation'
although authors concluded that combining exhaled NO and FEVr was the most useful (Gelb'
Flynn Taylor et al. 2006).ln 44 sensitized children prospectively followed though one grass
pollen season, NO was best associated with the mean pollen count the week before the No
measurement and was significantly associated with asthma control (Roberts' Hurley et al'
2004).In 105 asthmatic and healthy children, exhaled No did not depict asthma severity but
correlated with changes in asthma therapy. Irvels >l3ppb, had a sensitivity of 0'67 and a
specificity of 0.65 to predict step-up in therapy (Griese, Koch et al' 2000)' In 2L asthmatic
children with seasonal allergic asthma compared to 21 healthy children that were followed
throughout a grass pollen season, their No levels were higher, increased through the pollen
23r

season and then reduced down to baseline. However, their exhaled NO did not necessarily
correlate with FEV1 @araldi, Carra et al. 1999). Exhaled NO did relate to recent symptoms
versus no symptoms when dividing 133 children aged 5-14 years attending a hospital clinic
into 'uncontrolled' and 'controlled' asthma groups. The researchers also felt that NO
predicted subgroups as to whether IHCS or medication was increased, decreased or remained
unchanged (Warke, Mairs et al. 2004).
There have also been studies where exhaled NO was measured prospectively while IHCS
doses were reduced. After three months of good control on moderate doses of IHCS (500-
1000pg/day beclomethasone), 37 asthmatics had their individual doses halved for twelve
weeks. Parameters which detected the ten that developed an exacerbation were; a baseline
exhaled NO level > 20ppb, or a baseline exhaled NO level > l5ppb plus a bronchoconstriction
response to the adenosine S-monophosphate challenge, or a rapid increase in exhaled NO over
the first weeks of the assessments (Prieto, Bruno et al. 2003). In 78 asthmatic adults, IHCS
treatment was withdrawn over six weeks with 60 subjects developing symptoms. Both the
baseline measurement of exhaled NO and an increase of 60Vo over the baseline reading had
positive predictive values between 80-90Vo for an exacerbation and was similar to the
predictive values using sputum eosinophils or hypertonic saline airway responsiveness (Jones,
Kittelson et al. 2001). Similarly, nine of 40 children with a mean age of 12.2 years relapsed 2-
4 weeks after withdrawal of steroids. The exhaled NO was higher at a mean of 35.3ppb versus
15.7ppb at two weeks and 40.8ppb versus l5.9ppb at four weeks. A value of 49ppb at four
weeks after steroid discontinuation had the best sensitivity at TLVo and specificity at 93Vo for
an asthma relapse @ijnenburg, Hoftruis et al. 2005). One study was less conclusive.
Seventeen moderate asthmatic adults had their daily dose of IHCS halved at 20 day intervals
until loss of control or replacement with placebo. Of the parameters which were measured
every ten days, the sputum eosinophil percentage altered 20 days ahead of deterioration while
changes in exhaled NO, FEVr and methacholine PC26 were only observed once symptoms had
already developed @elda, Parameswaran et al. 2006).
Studies have also compared changes in exhaled NO with traditional clinical and lung function
assessments to dictate changes in treatment.ln 97 adult asthmatics, future treatment decisions
regarding steroid adjustment were assigned randomly to be based either on exhaled NO levels
q! on an algorithm based on the conventional GINA 2OO2 guideline (GINA 2002). The
optimal IHCS dose per individual was determined in the first phase and patients were then
followed for L2 months. The mean final doses of inhaled fluticasone used were 3701tglday for
the 46 patients in the exhaled NO group and 64Lltglday for the 48 patients in the guideline
232

group. Rates of exacerbation were 0.49 episodes/patient/year in the exhaled No managed
group and 0.90 in the guideline group, representin g a 45.6Vo exacerbation reduction in the No
group, although this did not reach significance. There were no significant differences in
pulmonary function changes, sputum eosinophils or the use of oral prednisone (Smith' Cowan
et al. 2005). Similar to the adult study, children on IHCS were allocated to groups where
treatment decisions were made by symptomatology in 46 or with the addition of exhaled No
in 39. Over the next year, those monitored using the NO readings had improved airway hyper-
responsiveness from 2.5 to 1.1 doubling dose, lesS Severe exacerbations at eight versus 18',
and a trend to improved FEVr (Pijnenburg, Bakker et al. 2005). These studies suggest that No
can successfully be used longitudinally for individual patients to predict exacerbations and to
aid management deci sions re gardin g treatment'
9.8.6 Wat happens to nitric oxide during an acute asthma attack?
Most studies have examined NO levels during chronic asthma disease over time' So what
happens to NO levels in the acute phase? Studies have used allergen challenges to simulate an
acute asthmatic exacerbation. It appears that even when the No is similar in the asthmatic and
control groups at baseline, No increases in the asthmatic groups during the late asthmatic
reaction (Obata, Dittrick et al. 1999; Paredi, Irckie et al' 1999; Khatri' Hammel et al' 2003;
Ihre, Gyllfors et al. 2006). It was found to increase three fold from baseline 24 hours post
challenge (Khatri, Hammel et al. 2003). Repeated low dose allergen inhalation in 8 mild
asthmatic subjects demonstrated an increased exhaled NO despite no change seen in asthma
symptoms or lung function (Ihre, Gyllfors et al. 2006)' These findings suggest that in acute
asthma an increased production requiring synthesis of the NOS enzymes occulred and
therefore some time was needed to appreciate the full insult (Khatri, Hammel et al' 2003)'
This may also explain why the use of No to dictate treatment acutely has been less successful'
one study used the exhaled No levels to dictate the degree of treatment given to adult
patients presenting to the emergency department with an asthma exacerbation' The study was
discontinued after the first 53 enrolled subjects as those in whom the management offered was
decided on their exhaled NO levels did less well when followed up than those in whom the
management was decided by the more traditional measures of clinical scores or spirometry
(Gill, Walker et al. 2005).
g.8.7 What is the effect of atopy alone on nitric oxide?
In assessing the validity of exhaled No to assist in screening, diagnosis and longitudinal
monitoring of asthma control, the effect of atopy alone must be taken into account' A number
233

of cross sectional studies with a total of 949 subjects have shown that there is a higher mean
exhaled NO which decreases through the groups of atopic asthmatics, non-atopic asthmatics,
atopic subjects and non-atopic controls. However, considerable overlap between groups exist
so that a single cut off may not discern exactly into which group an individual belongs (Frank,
Adisesh et al. 1998; Barreto, Villa et al. 2001; Henriksen, Holmen et al. 200L; Prieto,
Gutierrez et al.2002; Jouaville, Annesi-Maesano et al. 2003; Cardinale, de Benedictis et al.
2005; Prasad, Langford et al. 2006). In these studies, asthmatics divided into those who were
atopic and non-atopic showed those with atopy had higher levels of exhaled NO. In 28
asthmatics on IHCS treatment, NO levels were positively correlated with total IgE and
positive skin prick testing more than bronchial hyper-reactivity, lung function or asthma
severity (Ho, Wood et al. 2000).In92 steroid naive asthmatics, the NO was high in those that
were atopic with no correlation to any other parameters (Silvestri, Sabatini et al. 2W3).In2l3
children, exhaled NO was higher in children with atopy alone compared to those with non-
atopic asthma and was correlated to the wheal size of skin prick tests (Barreto, Villa et al.
2001). In 53 steroid naive asthmatic and 96 non-asthmatic children who had skin prick testing
to 12 common allergens, the exhaled NO was higher in the asthmatic versus non-asthmatic
individuals but was also higher in atopic (including those with allergic rhinitis alone) versus
non-atopic children (Jouaville, Annesi-Maesano et al. 2003). In 53 subjects, no significant
difference was found between non-atopic asthmatics and controls (Ludviksdottir, Janson et al.
1999). Similarly of 140 subjects, llVo of those with asthma but not deemed allergic had NO
levels within the normal range (Zietkowski, Bodzenta-Lukaszyk et al. 2006).
Looking at the effect of atopy alone, 38 subjects with allergic rhinitis but not asthma had a
raised exhaled NO @rieto, Gutierrez et al.2002) and in 64 atopic Pacific Island subjects, NO
levels correlated with house dust mite wheal reaction and clinical atopy (Moody, Fergusson et
al. 2000). More studies have been conducted evaluating exhaled NO with atopic status in
children - mostly within community school cohorts. In 356 children from an unselected
population, exhaled NO was two fold higher in atopic individuals (noted as having positive
skin prick tests and a high IgE) with high eosinophil counts compared to atopic individuals
with low eosinophil counts. However the exhaled NO in both these groups was higher than
the non-atopic subjects regardless of the eosinophil count, suggesting it was a link with atopy
not just eosinophilic inflammation (Barreto, Villa et al. 2005). Exhaled NO related to both
eosinophil levels and atopy but not necessarily to asthma and/or wheeze in the last 12 months
in 155 children (Franklin, Turner et al. 2003). NO also correlated with skin prick testing and
sputum eosinophils but not airway responsiveness in 107 children (Thomas, Gibson et al.
234

2005). Exhaled NO showed strongly positive correlations to non-specific IgE and mite
specific IgE with weaker positive correlations to specific IgE to cat and cedar in 278 children
(Saito, Inoue et al. 2OO4). Finally in 374 school children high exhaled NO levels were
associated with pet sensitisation in atopic children, and respiratory infections and home
window pane condensation in non-atopic children rather than other factors (Janson, Kalm-
Stephens et al. 2005). In a hospital clinic setting in 45 children, there was a correlation
between eczema and exhaled NO but not asthma, allergic rhinitis, food allergY, the use of
inhaled corticosteroids, anti leukotriene therapy, antihistamine therapy or lung function
(Jentzsch, le Bourgeois et al. 2006).
Thus atopy alone is an important confounding factor when using exhaled NO to assess
asthmatic and healthy individuals.
9.8.8 Can nitric oxide be an outcome measure for assessing treatment?
9.8.8 (i) Corticosteroids
Similar to my own study, there have now been many publications confirming that levels of
exhaled NO is high in asthmatics on bronchodilator therapy only and is reduced in asthmatics
on steroid therapy often to the equivalent levels found in healthy controls using adult (Alving,
Weitzberg et al. 1993; Kharitonov, Yates et al. L994; Persson, Zetterstrom et al. 1994;
Kharitonov, Chung et al. 1996; Massaro, Mehta et al. 1996; al-Ali, Eames et al. 1998;
Stirling, Kharitonov et al. 1998; Berlyne, Parameswaran et al. 2000; Lim, Jatakanon et al.
2000; Tsujino, Nishimura et al. 2000; Reid, Johns et al. 2003; Shin, Rose-Gottron et al.2004)
and paediatric subjects (Byrnes, Dinarevic et al. L997; Nelson, Sears et al. 1997; Silvestri,
Spallarossa et al. 1999; Colon-Semidey, Marshik et al. 2000; Little, Chalmers et al. 2000;
Piacentini, Bodini et al. 2000; Silvestri, Spallarossa et al. 2000; Visser, de Wit et al. 2000;
Avital, Uwyyed et al. 2001). Many of these studies also determined that the raised exhaled
NO levels in asthmatics reduced when they were given inhaled or oral steroids from five days
to several weeks in adults (Silkoff, McClean et al. 1998; Jatakanon, Kharitonov et al. 1999;
Lim, Jatakanon et al. 1999; van Rensen, Straathof et al. 1999; Wilson and Lipworth 2000;
Silkoff, McClean et al. 2001; Jones, Herbison et al.20O2: Kharitonov, Donnelly et al. 2002;
Currie, Syme-Grant et al. 2003; Dal Negro, Micheletto et al. 2003; Tnidler, Kleerup et al.
2004; Brightling, Green et al. 2005; Silkoff 2005; Smith, Cowan et al. 2005; Zietkowski,
Bodzenta-Lukaszyk et al. 2006) and children (Byrnes, Dinarevic et al. 1997i Artlich, Busch et
al.1999;Lanz,I-eung et al. 1999; Colon-Semidey, Marshik et al. 2000; Piacentini, Bodini et
al. 2000; Spallarossa, Battistini et al. 2001; Beck-Ripp, Griese et al. 2002; Mondino,
235

Ciabattoni et al. 2004; Petersen, Agertoft et al. 2004; Cardinale, de Benedictis et al. 2005;
Prasad, Langford et al. 2006;Zeiger, Szefler et al. 2006). In addition to commencing IHCS in
steroid naive patients, some studies also showed a dose response reduction of exhaled NO
(Srirling, Kharitonov et al. 1998; Colon-Semidey, Marshik et al. 2000; Piacentini, Bodini et
al. 2000; Silkofl McClean et al. 2001; Jones, Herbison et al.2002; Kharitonov, Donnelly et
al. 2002). Two studies demonstrated a plateau in the reduction of exhaled NO in asthmatic
children at 400pgs beclomethasone or equivalent (Jatakanon, Kharitonov et al. 1999; Wilson
and Lipworth 2000) but was not seen between 50 and 100pgs hydrofluoroalkane-
beclomethasone (Petersen, Agertoft et al. 20M). One study showed that at a lower dose of
200pgs beclomethasone there was a longer time required for the reduction of exhaled NO to
the lowest levels measured compared to commencing at double this dose @al Negro,
Micheletto et al.2003), although another study found no additional reduction in exhaled NO
after ten days of treatment even with low doses (Spallarossa, Battistini et al. 2001). The
reduction of NO levels has even been seen with single nebulised steroid doses (Baraldi,
Azzolin et al. 1997;Lanz,Irung et al. 1999; Tsai, Lee et al. 2001). In addition, these findings
have been confirmed using tidal breathing techniques (Visser, de Wit et al. 2000) and
reservoir samples @araldi, Dario et al. 1999; Colon-Semidey, Marshik et al. 2000). Many of
these individual papers have been already been discussed in more detail in Chapters 6,7 and
8. Only one cross sectional hospital based study in 392 adult patients with a range of asthma
severity showed no difference in the exhaled NO between those on or not on IHCS, although
the researchers did show a relationship between exhaled NO and airway reactivity (measured
by methacholine challenge) and atopic status (Langley, Goldthorpe et al. 2003).
In addition to using NO as an outcome measure to follow asthmatics treated with
conventional inhaled steroids (considered here to be fluticasone, budesonide and
beclomethasone), NO has also been used to assess whether novel steroids have a similar
effect. Significant improvements in exhaled NO levels and induced sputum eosinophil counts
were seen in three separate studies of ciclosonide -
one using it alone (Wilson, Duong et al.
2006), one comparing it to budesonide (Kanniess, Richter et al. 2001) and one comparing it to
fluticasone (ke, Fardon et al. 2005). Four week treatment periods in 25 children with the
newer beclomethasone derivative, hydrofluoroalcane-beclomethasone diproprionate at doses
of 50pg and l00pg daily also resulted in reducing exhaled NO, associated with improvements
in FEVr and exercise challenges @etersen, Agertoft et al.2004).
236

9.8.8 (ii) Anti-leukotriene receptor antagonists
In this class of drugs, the two predominantly studied in conjunction with NO have been
montelukast and pranlukast. Firstly, NO has been monitored when these have been used as an
addition to regular treatment. An open label six week trial of oral montelukast in 20 adult
asthmatics already on IHCS resulted in significant improvements for levels of exhaled NO,
coinciding with an improvement in exercise tolerance (Berkman, Avital et al. 200.3).
Similarly, in 20 stable asthmatics, a crossover trial showed that montelukast, as opposed to
placebo, reduced exhaled NO as well as reducing peak flow variability and symptom score
over a two week treatment period (Sandrini, Ferreira et al. 2003). When montelucast was
added to treatment of 22 adults with mild to moderate asthma using fluticasone 2501t9 and
salmeterol 50pg twice a day, montelukast l0mg a day, compared to placebo, reduced levels of
exhaled NO and blood eosinophilia (Currie, lre et al. 2003). In 35 children treated with IHCS
in whom the exhaled NO was greater than 20ppb, those that had montelukast added to their
treatment had a significant reduction of exhaled NO which increased back to baseline level
after withdrawal of the drug (Ghiro, Zanconato et al. 2002). While the reduction of NO
coincided with some clinical improvements, there was no improvement in others such as
bronchial responsiveness (Berkman, Avital et aL.2003), FEVr (Ghiro, Zanconato et al.2002;
Currie, Ire et al. 2OO3; Sandrini, Ferreira et al. 2003) or in measured exhaled HzOz (Sandrini,
Ferreira et al.2OO3). Only one study conducted in 25 children did not show a difference in
exhaled NO with the addition of montelukast to IHCS treatment (Strauch, Moske et al. 2003).
Pranlukast added to a six week reduction of inhaled budesonide did prevent asthma
deterioration compared to placebo, but did not show a difference in levels of exhaled NO,
although NO levels in the placebo group increased (Tamaoki, Kondo etal.1997).
Secondly, NO has also been used as an outcome measure when these medication have been
used alone. In steroid naive children (twelve to 2l subjects in each study), treatment with
montelukast (5 or l0mgs per day) for between two and eight weeks resulted in a reduction in
exhaled NO in two studies (Bratton, Lanz et al. 1999;l-ee,Lu et al. 2005) but not in a third
(Spahn, Covar et al. 20O6) with the individual researchers also showing improvements in
FEV', (Bratton, Lanz et al. 1999), salbutamol use (Bratton,Lanz et al. 1999), residual volume
(Spahn, Covar et ^l.2006) and serum ECP levels (Spahn, Covar et al.2OO6). They did not
demonstrate changes in symptom scores (Spahn, Covar et al. 2006) and the exhaled NO
returned to pre treatment levels two weeks after withdrawal of the medication (Ire, Lai et al.
2005). In younger steroid narve children aged ten months to five years (54 in total) with an
early diagnosis of asthma, significant improvements were seen in levels of exhaled NO,
237

FEV9.5, symptom score and airways resistance but not bronchodilator responsiveness after
four weeks treatment with 4mg daily of montelukast (Straub, Minocchieri et al. 2005; Straub,
Moeller et al. 2005). In a study of twelve asthmatic children where montelukast reduced
levels of exhaled NO, the four individuals that were heterogeneous at the gene locus of the
leukotriene C4 synthase had a far better response than those who were homozygotes (Whelan,
Blake et al. 2003).
Thirdly, NO has been measured as one factor in comparison studies between montelukast and
IHCS which have all resulted in a greater reduction of levels of NO in the IHCS arm
(Kanniess, Richter et^1.2N2; Peroni, Bodini et aI.2005;Zniger, Szefleret aI.2006). This
coincided with improvements of hyperreactivity (Kanniess, Richter et aL.2002; Peroni, Bodini
et al. 2005), FEVr (Kanniess, Richter et al. 2002; Tniger, Szefler et al. 2006), sputum
eosinophils (Kanniess, Richter et al.2002), asthma control questionnaire and salbutamol use
(7*ige4 Szefler et al. 2006) with both treatments, again greater in the IHCS groups. However,
the other antagonist pranlukast at 450mg per day did not change levels of exhaled NO in 30
adults compared to IHCS but did show improvement in all other parameters measured
(Yamauchi, Tanifuji et al. 20Ol). Comparisons between this drug class and the long actingp2
agonists (LABAs) are described below.
9.S.8 (iii) Long acting p2 agonists
Exhaled NO has been used as an outcome measure when looking at the addition of long
acting p2 agonists (LABAs) to IHCS. Neither eformoterol nor salmeterol have shown any
effect on the exhaled NO when used alone in comparison to either IHCS therapy alone
(Priero, Gutierrez et al.2OO2) or to the combination of IHCS and LABA (Aziz, Wilson et al.
2000; Currie, Syme-Grant et al. 2N3), and no difference where they have been added to
IHCS (Yates, Kharitonov et al. 1997; ke, Jackson et al. 2003). This is despite improvements
in other parameters such as FEV1, adenosine S-monophosphate challenges, sputum and blood
eosinophil counts and serum ECP (Aziz. Wilson et al. 2000; Prieto, Gutierrez et aL 2402;
Currie, Syme-Grant et al. 2003; I-ee, Jackson et al. 2003).
In comparisons between montelukast and LABAs, neither of two studies showed an effect on
the level of exhaled NO (Aziz, Wilson et al. 2000; Wilson, Dempsey et al. 2001). This is
interesting given that in most of the montelukast studies, this medication did result in a
reduction in exhaled NO. The studies here really just confirm that the LABAs do not have
anti -infl ammatory properties.
238

9.8.8 (iv) Nedocromil sodium
In two paediatric studies, this medication had no effect on levels of exhaled NO when
compared to IHCS in 32 sensitised patients followed through a pollen season (Gratziou,
Rovina et al. 2001) or in ll8 four to six year olds studied as part of the childhood asthma
management program (CAMP) studies (Covar, Szefler et al. 2003).
9.8.8 (v) Theophylline
Despite theophylline being a previously commonly used drug, there has only been one study
looking at its effect on levels of exhaled NO. In a double blind crossover study 15 patients
with mild asthma on theophylline of 250mg twice per day had a reduction in eosinophil
counts in sputum, BAL, and biopsy samples but no change in levels of exhaled NO and no
improvements in lung function or bronchial hyper-responsiveness (Lim, Tomita et al. 2001).
9.8.8 (vi) Novel medications
L-NAME -
Nebulised pretreatment with this non-selective NOS inhibitor reduced levels of
exhaled NO from baseline in 22 asthmatics with either an early asthmatic response only or
both an early and late asthmatic response to allergen challengebyTT andTIVo respectively.
Despite this result in NO, there was no change on the magnitude of the other acute responses
in either group (Taylor, McGrath et al. 1998).
L-Arginine -
L-arginine (the substrate for the NOS enzymes) was nebulised as single doses of
0,759,1.5g and 39 to healthy subjects and asthmatics with 6-7 in each group. This increased
levels of exhaled NO in a dose dependent fashion at 60 minutes showing a negative
correlation to the fall in FEVr (Sapienza, Kharitonov et al. 1998). Nebulisation of 2.59 of L-
arginine and D-arginine on separate days to eight steroid naive asthmatics both resulted in
bronchoconstriction. There was an initial proportional decline of NO then, with resolution of
the constriction,the NO increased so by three hours it was higher than baseline (Chambers and
Ayres 2001). It is hard to know whether L-arginine is acting as a substrate in these studies or
just as an airway irritant.
Omalizumab -
This humanised monoclonal antibody to Ig E was used in a steroid reduction
study in adults with allergic asthma over one year. In the 18 that received active medication
compared to the l l receiving placebo, the variability of the exhaled NO was significantly less
and remained similar to the levels obtained when the children were on higher doses of IHCS
prior to the reduction phase (Silkoff, Romero et al. 2004).
239

Indomethacin - This medication alters the cyclo-oxygenase products of the arachidonic acid
pathway which play a role in bronchoconstriction and airway inflammation. Indomethacin
50mg daily or placebo was given to 38 asthmatics on beclomethasone > 1500pg daily for six
weeks while the IHCS was reduced to half at week two and to a third at week four. While
levels of exhaled NO increased in both groups, the effects were less pronounced in the
indomethacin group and corresponded to fewer exacerbations (Tamaoki, Nakata et al. 2000).
PGE2|PGF2a -
These prostaglandins also provide negative feedback in the cyclo-oxygenase
pathway and a trial of inhaled PGE2 and PGF2a significantly reduced levels of exhaled NO in
both normal and asthmatic subjects. This was not associated with any alteration of lung
function in the normal subjects, but PGE2 caused an increase in FEVr in the asthmatic group
(Kharitonov, Sapienza et al. 1998).
9.8.9 Summnry of nitric oxide in asthma and atopy
How do we make sense of all this information regarding NO and asthmalatopy"! NO does
correlate with other clinical and inflammatory parameters of asthma -
most particularly in the
steroid narve asthmatic and most particularly with sensitivity and specificity seen with
eosinophil counts. The utilization of NO to contribute to diagnosis seems hopeful. It
performed as well as eosinophil counts in induced sputum (Smith, Cowan et al.200/.) and
methacholine or adenosine S-monophosphate challenges (Berkman, Avital et al. 2005).
However this again is best used in steroid naive patients and the effect of atopy alone in
elevating NO levels must be taken into account. It is easier to show NO differences in groups
of subjects such 'asthma', 'atopy', 'other respiratory disease' or 'controls', rather than being
able to place one individual into the correct category based on the exhaled NO reading. NO
does appear to determine current or recent symptoms and is more likely to be useful in the
individual when followed longitudindly for loss of control or response to treatment. Studies
using NO to guide treatment have shown beneficial effects. However, again eosinophils were
more sensitive and of the most benefit when these two parameters are combined @rightling,
Green et al. 2005; Pijnenburg, Bakker et al. 2005; Smith, Cowan et al. 2005; Belda,
Parameswaran et al. 2006). In medications that have anti-inflammatory effects such as the
different IHCS, leukotriene receptor antagonists, indomethacin and prostaglandins, it appears
the result is to decrease NO levels. NO remains unchanged in those without such properties as
in the LABAs. In these studies, when NO did show a reduction, this often coincided with
other clinical benefits.
240

However, the major advantage of using measurements exhaled NO, particularly over sputum
eosinophil counts, blood tests or airway challenges is obvious -
particulady in children. It is a
non-invasive test, analogous to lung function testing to which patients are very familiar' It is
also likely to be available in more clinics and can be performed more quickly and more
cheaply with immediate results.
g.g Nitric oxide levels in primar.v ciliary dyskinesia
This is an area where NO measurement was found early to be of particular value. Initially No
was found to be absent in nasal measurements in four children with Kartagener's syndrome
(Lundberg, Weitzberg et al. 1994). This led to the evaluation of 21 children with primary
ciliary dyskinesia (PCD) in whom both nasal and oral NO was found to be significantly lower
when compared to 60 healthy children. While there was some overlap with regard to
individual lower airway results, only one child was an outlier from each group in the nasal
NO ranges (Karadag, James et al. 1999). This has been confirmed in a recent study where the
impairment of NO output was less pronounced in lower than the upper nasal respiratory tract
after sampling at several sites in 17 PCD and 28 healthy subjects (Matrut, Escudier et al.
2006). In another large cross-sectional study involving 102 subjects, the concentration of
exhaled NO was significantly lower in both PCD and CF patients than bronchiectasis and
healthy subjects. The nasal NO, however, was markedly reduced only in the PCD subjects
(Horvath, Loukides et al. 2003). This change appears to occur early in life as seen in two
infants with pCD aged four and six months having significantly lower levels of NO compared
to five healthy controls (Baraldi, Pasquale et a|.2004). These low NO levels found are despite
levels of nitrite, nitrate and S-nitrosothiol when measured in exhaled breath condensate being
no different from normals (Csoma, Bush et al. 2003)'
Use of NO as a screening tool for this condition has been investigated. From the study above
reviewing children already diagnosed with PCD, the authors determined a cut off of 250ppb
for nasal NO as having both a positive and a negative predictive value of 0.95. This correctly
detected 20 of 21 subjects as having PCD and conectly detected 19 of 20 as not having PCD
(Karadag, James et al. Lggg). Measuring 31 children with PCD, 2l with non-CF
bronchiectasis, 17 with CF, 35 with asthma and 53 healthy controls, a nasal NO of 250ppb
gave a sensitivity of 977o and a specificity of gOVo for a diagnosis of PCD (Narang, Ersu et al'
2OOZ). Of 34 children referred for investigation for possible PCD to a tertiary clinic, the 17
that proved positive on biopsy again had significantly lower level of nasal NO. This group
determined a nasal NO of 105ppb gave a specificity of 88Vo and a positive predictor value of
24r

89Vo for PCD, and above that excluded PCD with 1007o certainty (Corbelli, Bringolf-Isler et
al. 2004). Reviews now recommend the use of nasal NO to screen for PCD, usually with the
250ppb cut-off (Silkoff 2004; Stehling, Roll et al. 2006).
Nebulised L-arginine given to ten patients with PCD and ten healthy individuals increased
ciliary beat frequency (Inukides, Kharitonov et al. 1998). L-arginine given intravenously
resulted in an increase of nasal and exhaled NO in both PCD and CF subjects, although the
mean concentrations were still significantly lower than normals (Grasemann, Gartig et al.
1999). Neither study showed that the increase in NO translated into other clinical effects or
improvements.
The mechanism behind the low nasal NO in PCD is not yet determined. In other diseases
where low levels are obtained such as CF @alfour-Lynn, Laverty et al. 1996; Dotsch,
Demirakca et al. 1996; Lundberg, Nordvall et al. 1996), diffuse panbronchiolitis (Nakano, Ide
et al. 2000), paranasal sinus inflammatory disease (Arnal, Flores et al. 1999), chronic sinusitis
(Lindberg, Cervin et al. 1997; Deja, Busch et al. 2003) (described in sections below), it is
thought to be secondary to thickened secretions and/or obstruction of the ostea preventing NO
diffusion. However, in clinically stable PCD patients with low NO, their ostea were noted to
be open (Wodehouse, Kharitonov et al. 2003) and sinuses clear (Amal, Flores et al. 1999).
NO has been implicated in control of ciliary function. In our own study (conducted after the
data presented in this thesis), we found no correlation between exhaled NO levels and ciliary
beat frequency in 135 children of European, Maori and Pacific ethnicities. However, we did
find an increase in the percentage of ciliary structural defects, three times that reported in
previous control groups (Edwards, Douglas et al. 2005). Endothelial NOS is located at the
basal micro-tubular membrane in ciliated epithelium (Xue, Botkin et al. 1996) with a
suggestion that NO acts as an intermediate messenger @uner and Lindberg 1999) and TNFc
or ILI can up-regulate ciliary motility seeming to act through NO via induction of NOS (Jain,
Rubinstein et al. 1993). Further research is needed in this area for an adequate explanation of
this phenomenon of low NO levels in this condition.
9.10 Nitric oxide levels in cystic fibrosis
Studies of NO in subjects with cystic fibrosis (CF) have resulted in conflicting data.
Contributing factors are likely to be the different techniques used for measurement when these
studies began in the late 1990s, as well as the variation in age of the patients, co-morbidities
and treatments, not always specified. Cross-sectional studies in children have shown no
difference in levels of oral exhaled NO across CF, asthmatics on IHCS and healthy groups,
242

ut those with CF did have significantly lower nasal NO levels (Balfour-Lynn, Laverty et al'
1996;Dotsch, Demirakca et al. 1996; Lundberg, Nordvall et al. 1996). However, three studies
in adult patients with CF showed lower NO levels from both the oral and nasal sampling
(Grasemann, Michler et al. 1997; Jones, Hegab et al. 2000; Thomas, Kharitonov et al' 2000)'
There has been no relationship demonstrated between the oral and nasal NO concentrations,
although one study demonstrated a positive correlation between ambient and measured NO
levels in all subject groups @otsch, Demirakca et al. 1996). In infants' one study showed
reduced levels of exhaled NO in five infants with CF compared to 1l healthy infant controls
at a mean age of 48.6 days (Elphick, Demoncheaux et al. 2001), but a more recent study
showed no difference in exhaled NO levels in 18 infants with CF and 23 healthy infants at a
mean age of 64.9 weeks (Franklin, Hall et al. 2006). Franklin's study also demonstrated an
inverse relationship between age and exhaled No levels in the CF but not the healthy infant
group (Franklin, Hall et al. 2006).
Studies of correlations of NO level to lung function have also been variable in the cF
population. No correlation was demonstrated with levels of either nasal or oral NO in three
studies with 135 subjects @otsch, Demirakca et al. 1996: Thomas, Kharitonov et al' 2000;
Jaffe, slade et al. 2003), while another two studies showed a weak but statistically significant
positive relationship with FEVr in 63 subjects (Grasemann, Michler et al. t997; Ho, Innes et
al. lggg). It should be noted that this is a different response than seen in asthmatics as the cF
patients with better lung function appeared to have higher exhaled NO levels. No association
was observed between the CF genotype and NO values (Thomas, Kharitonov et al' 2000;
Texereau, Fajac et al. 2005) but an inverse correlation between No and the transepithelial
baseline potential difference measured has been described (Texereau, Fajac et al. 2005)'
Whether infection and type of organism has had an effect on the exhaled No levels has also
been examined. There was no difference in nasal or exhaled NO levels in children chronically
infected with staphylococcal aureus (22 infected versus 11 non-infected) (Balfour-Lynn,
Laverty et al. 1996). Nasal NO was significantly lower in L7 of 33 who were chronically
infected with pseudomonas aeruginosa in one study (Balfour-Lynn, Laverty et al. 1996) but
not confirmed in two others with 49 of the 68 subjects who had been chronically infected
(Thomas, Kharitonov et al. 2000; Jaffe, Slade et al. 2003). Exhaled NO was lower in nine
patients considered high risk of developing allergic bronchopulmonary aspergillosis (ABPA)
compared to a low risk group (Lim, Chambers et al.2003) but, as the high risk patients were
on corticosteroids, this may reflect steroid use as opposed to ABPA risk per se'
243

This low NO occurs despite higher than normal levels of NO metabolites of nitrite and nitrate
(Francoeur and Denis 1995; Grasemann, Ioannidis et al. 1998; Jones, Hegab et al. 2000) and
nitrotyrosine (Jones, Hegab et al. 2000) which have been demonstrated in sputum and saliva
samples. These metabolite levels have been correlated with sputum IL8 (Francoeur and Denis
1995) and lung function (Grasemann, Ioannidis et al. 1998). No correlation was seen when
levels of exhaled NO were compared with markers of inflammation obtained from
bronchoalveolar lavage in 18 CF infants (Franklin, Hall et al. 2006). Immunostaining studies
of iNOS showed an inverse correlation to neutrophil counts and IL8 levels suggesting the
iNOS expression decreases in the CF children as ainvay inflammation increases (Wooldridge,
Deutsch et al.2004). Similarly, in tracheobronchial brushings from 40 children with CF, the
expression of mRNA for iNOS was found to be lower than seen in two healthy children,
which the authors suggested may have consequences for local antimicrobial defense (Moeller,
Horak et al. 2006).
Regarding NO as a possible outcome measure for treatment, no change in levels were seen
from the onset of an acute infective exacerbation in eight patients followed for seven days
during treatment (Ho, Innes et al. 1998). In contrast, increased exhaled NO levels, associated
with a significant improvement in lung function, was seen in fourteen children after a course
of IV antibiotics, although the NO level did not alter in six of the children that had chronic
pseudomonas infection (Jaffe, Slade et al. 2003). In six children that were commenced on
dornase alpha (Rh DNAse, 'pulmozyme'), five had exhaled NO changes in parallel to the
changes of pulmonary function tests, with no change in those receiving placebo (Grasemann,
Lax et al. 2004). With single inhalations of L-arginine, there were increased levels of NO,
FEVr and oxygen saturation (Grasemann, Kurtz et al. 2006). Oral L-arginine compared to
placebo showed that a single dose of 200mgkg also resulted in increasing levels of exhaled
NO which continued for six weeks with continued supplementation but there was no change
of lung function (Grasemann, Grasemann et al. 2005). Another study administered oral L-
arginine daily for four weeks getting an increase in plasma L-arginine levels but there was no
change in either exhaled NO levels or FEVI @verard and Donnelly 2005).
On the face of it, it is hard to believe that the level of NO in such an inflammatory condition is
low, especially when the NO metabolites are higher than that found in asthmatic and control
groups. There are a number of possible explanations:
o Mucosal swelling with thickened mucus may result in mechanical obstruction of the
sinus ostea which could impede the flow of NO into the nasal cavity. Consistent with
this is that almost all patients with CF develop nasal and sinus disease.
244

Some of these studies, particularly the early studies, may have included subjects on
inhaled or oral corticosteroids and not differentiated them within the study groups.
NO may degrade within the mucus giving low levels reaching the airway lumen for
measurement.
Pseudomonas aeruginosa produces a pigment pyocinen which in vitro has been shown
to inactivate NO (Wanen,I-oi et al. 1990).
While the true reason is likely to be a combination of these factors, the obstruction to
diffusion of NO into the airway and trapping of NO within the mucus, probably plays the
major role. The low nasal NO level seems a consistent finding in this population, and in adults
oral NO also appears to be low, with a possible correlation between higher lung function and
higher NO levels.
9.11 Nitric oxide levels in bronchiectasis
An initial paper suggested both elevated levels exhaled NO and a correlation between this and
the chest CT scan score in 20 patients with bronchiectasis, but neither were seen in another 19
on IHCS treatment (Kharitonov, Wells et al. 1995). Subsequent papers have not confirmed
these findings in 16 (Ho, Innes et al. 1998), 3l (Horvath, Loukides et al' 2003) and 109
(Tsang, Irung et a\.2}}2)patients with bronchiectasis measured at a time of disease stability'
Those with pseudomonas aeruginosa infection (25 of 109) did have significantly lower levels
of exhaled NO although their sputum NO concentration was the same' There was no
correlation between the levels of exhaled No and FEVI, FVC or the number of bronchiectatic
lobes involved (Tsang, lrung et al. 2o02). similar to findings in cF, it is surprising not to
find elevated NO in such an inflammatory and infective disease. The mucus layer may well be
a barrier to detection of the production of NO, and in an inflamed environment the presence of
reactive oxygen species may result in interaction with this highly reactive molecule resulting
in effective removal of NO to form other compounds, such as perioxynitrite. The use of NO
here is likely to be as a screen in bronchiectasis - if low nasal No levels are obtained, then CF
and PCD testing must be undertaken.
g.l2 Nitric oxide levels in upper resoiratory tract infections
The effect that upper respiratory tract infections (URTI) have on exhaled NO levels has also
been studied. In 18 non-asthmatic adults during an URTI, increased exhaled NO levels to
315ppb were noted, decreasing to 87ppb during recovery at two to three weeks which became
similar to aged matched healthy controls (Kharitonov, Yates et al. 1995). When 79 COPD
245

patients were followed through one winter, there were increases in levels of exhaled NO when
they had a 'cold', 'a sore throat', or 'dyspnoea combined with a cold' compared to times of
stability (Bhowmik, Seemungal et al. 2005). Two studies actually infected adult subjects with
respiratory viruses. In the first, seven subjects infected with human rhinovirus 16 had
significant increases in NO levels by days two and three compared to seven placebo
'infections' (de Gouw, Grunberg et al. 1998). In the second, six subjects infected with the
same virus had increases in both nasal and lower airway NO levels with positive correlations
to the severity of infection as measured by organism count, inflammatory markers from nasal
lavages and nasal scrapings, and day four symptom scores (Sanders, Proud et al.20O4).
However in 16 children with acute maxillary sinusitis, the NO nasal measurements dropped
and then increased following antibiotic treatment over a two week period @araldi, Azzolin et
al. 1997). This suggests that NO does increase with upper respiratory tract infections, but may
be obstructed from measurement if there is significant sinus obstruction.
9.13 Nitric oxide levels in chronic obstructive pulmonary disease (COPD)
In cross-sectional studies of chronic obstructive pulmonary disease (COPD) subjects, levels of
exhaled NO has been similar to, or higher than, healthy controls though not to levels seen in
steroid narve asthmatics (Clini, Bianchi et al. 1998; Kanazawa, Shoji et al. 1998; Rutgers,
Meijer et al. 1998; Corradi, Majori et al. 1999; Zietkowski, Kucharewicz et al. 2005). This has
been confirmed using single breath (Clini, Bianchi et al. 1998; Kanazawa, Shoji et al. 1998;
Maziak, Loukides et al. 1998; Rutgers, Meijer et al. 1998; Zietkowski, Kucharewicz et al.
2005), tidal breathing @utgers, Meijer et al. 1998) and reservoir techniques (Corradi, Majori
et al. 1999). A significant negative correlation has been shown with FEVr at times of disease
stability (Clini, Bianchi et al. 1998; Maziak, l.oukides et al. 1998; Ansarin, Chatkin et al.
2001). A correlation has also been found between baseline FEV1 and the change in the level
of exhaled NO following an eight week rehabilitation exercise program in mild, moderate and
severe COPD patients but not those with cor pulmonale (Clini, Bianchi et al. 2000; Clini,
Bianchi et al. 2001; Clini, Bianchi et al.2002). With regard to other physiological parameters,
exhaled NO was found to be inversely correlated with oxygen saturation and diffusion
transfer factor @r-CO) (Ansarin, Chatkin et al. 2001) and positively correlated with the
residual lung volume, total lung capacity (Ansarin, Chatkin et al. 2001), and degree of airflow
obstruction (Clini, Bianchi et al. 1998). These all suggest that the worse the disease is, as
determined by these parameters, the higher the NO level obtained (Ansarin, Chatkin et al.
2001). More recently, multiple flows to assess the different airway compartments have been
employed in this population (see Section 9.3.1 (i)). In all studies to date NO was found to be
246

high in the alveolar compartment which correlated with severity and progression regardless of
the patient's smoking habit or current treatment (Tsoukias and George 1998; Kharitonov and
Barnes 2004; Brindicci, Ito et al. 2005). As well, No metabolites of nitrite and nitrate have
been higher in induced sputum in COPD patients compared to normals (Kanazawa, Shoji et
al. 199g). Lung biopsies from patients with severe COPD had higher iNOS seen than those
that had more mild disease and normal controls (Maestrelli, Paska et al. 2003).
COpD patients having frequent exacerbations had higher levels of exhaled NO than stable
current smokers and ex-smokers with COPD or chronic bronchitis (Agusti, Villaverde et al.
1999; Silkoff, Martin et al. 2001). Similarly during admission, the level of exhaled NO was
high and remained elevated at discharge despite intravenous steroid therapy, but when the
patients were once again clinically stable it had reduced to levels similar to control subjects
(Agusti, Villaverde et al. lggg). The NO levels correlated to the drop in FEV1 during
exacerbation in one study (Silkoff, Martin et al. 2001), but did not relate to FEVr or other
variables such as FVC, lung volumes, diffusing capacity or pulmonary gas exchange at the
time of discharge in another study (Agusti, Villaverde et al. 1999). Assessing the effect of
pollution; an association between exhaled NO and particulate matter when measured in a
cross section of the population was significantly greater in subjects who had a doctor
diagnosis of coPD (Adamkiewicz, Ebelt et al. 2004). Exhaled No levels measured
seasonally and during exacerbations in 79 COPD outpatients showed higher levels during
winter months, as well as being higher during the 68 exacerbations seen in 38 of these
patients. The degree of elevation, however, was noted to be lower in those more severely
affected (Bhowmik, Seemungal et al. 2005). Elevated No levels decreased significantly in 47
patients commencing IHCS therapy but there were no changes in FEVr (Zietkowski'
Kucharewi cz et a:.2005). Two studies showed higher exhaled No levels that positively
correlated with post-bronchodilator FEVr suggesting NO may be a marker for inflammation'
responsive to bronchodilator treatment similar to asthmatics, although these COPD patients
were not known to be atopic (Papi, Romagnoli et al. 2000; Zietkowski, Kucharewicz et al'
2005). Both exhaled No and co levels were low in 19 individuals with alpha-l antitrypsin
deficiency compared to both controls and coPD patients (Machado, Stoller et al' 2002)'
9.14 Nitric oxide levels in smokers
NO is known to be contained within cigarettes as previously discussed (see Section 2'2'3)'
Exact concentrations between brands differ, but cigarettes produced in France and the United
States exceeded the NO content of those produced in the UK up to 3-5 fold with no other
247

differences noted based on filter, design, tar, nicotine or CO yield. This research was done in
the 1980s and the relationship to what is made and sold now here in New Zealand has not
been studied. Many studies have demonstrated that both peak and plateau NO is lower in
smokers than non-smokers who are otherwise 'healthy' with normal lung function (Persson,
Zetterstrom et al. 1994; Schilling, Holzer et al. 1994; Kharitonov, Robbins et al. 1995;
Robbins, Millatmal et al. 1997; Verleden, Dupont et al. 1999; Balint, Donnelly et al. 2001;
Mazzone, Cusa et al. 2001; Hogman, Holmkvist et ^1.200.2; Horvath, Donnelly et al.2O04).
In mild asthmatics, smokers also had a lower level of exhaled NO when compared to non-
smokers (Persson, Zetterstrom et al. 1994; Verleden, Dupont et al. 1999; Horvath, Donnelly et
al. 2ffi4; McSharry, McKay et al. 2005). Nasal NO measurements in 'healthy' normal and
asthmatic smokers compared with their non-smoking counterparts were also reduced
(Robbins, Millatmal et al. 1997; Olin, Hellgren et al. 1998). This reduction of NO was
correlated with 'pack years' in current (McSharry, McKay et al. 2005) and ex-smokers
(Corradi, Majori et al. 1999). Two studies also found significant reductions in levels of
exhaled NO and NO output in a total of ll8 healthy infants exposed to pre-natal cigarette
smoke compared to the non-exposed (Hall, Reinmann et al.2OO2; Frey, Kuehni et al. 2OO4).
The effects of a single cigarette have also been studied. A transient reduction in peak exhaled
NO occurs immediately (Kharitonov, Robbins et al. 1995) and at 50 minutes with a co-
incident rise in HzOz (Horvath, Donnelly et al. 2OO4). However another study found an
increase level of NO at one and ten minutes (Chambers, Tunnicliffe et al. 1998) while a fourth
found no change in the levels of exhaled NO, S-nitrosothiol or nitrotyrosine at 30 and 90
minutes, although nitrite and nitrate levels were significantly increased (Balint, Donnelly et al.
2001). A decrease in plasma nitrate and nitrite concentrations were seen after smoking a
single 'real' cigarette compared to a sham cigarette (Tsuchiya, Asada et al.2002). Passive
exposure and active smoking in usual non-smokers resulted in a reduction in the level of
exhaled NO when measured every 15 minutes to one hour (Yates, Breen et al. 2001).
Recently a murine model showed that cigarette smoke caused a reduction of NO by
decreasing iNOS mRNA transcription but did not alter the iNOS mRNA half life once already
developed (Hoyt, Robbins et al.20O3). It may be that the NO in the cigarettes operates as a
negative feedback mechanism, resulting in reduction of activity of the NOS enzymes.
The effect of quitting smoking on the subsequent levels of NO has also been examined. In 20
smokers, nine that were able to refrain from smoking for four weeks resulted in increased
levels of exhaled NO, which was no longer significantly lower than controls (Hogman,
Holmkvist et al.20O2). Fourteen smokers had an increase in their exhaled NO compared to
248

their baseline No when they gave up for one week. In the ten who were able to continue, their
level of exhaled NO had increased to normal non-smoking levels when re-measured at eight
weeks, while in those that were unable to sustain quitting, the No had dropped to the previous
low levels (Hogman, Holmkvist et al. 2OO2). This effect of smoking is unfortunate as it
renders the measurement of No unhelpful, particularly in the asthmatic population where it
could be a helpful longitudinal indicator of airway inflammation and treatment effects.
9.15 Nitric oxide levels in interstitial lung diseases
Interstitial lung diseases have been particularly helpful in proving the value of using differing
flows to measure NO from different areas within the lung (see Section 9.3.1 (i))' In 20
patienrs with scleroderma including five with additional pulmonary hypertension (Girgis,
Gugnani et a:.2002) and in 24 patients with scleroderma including twelve with and twelve
without clinical or radiological evidence of lung disease (Moodley and Lalloo 2001)'
significant differences in exhaled NO levels from control subjects were only seen when the
alveolar rather than airway concentrations were measured. There was also a negative
correlation demonstrated between the alveolar concentration of NO and the diffusion
coefficient in the parient groups (Moodley and Lalloo 2001; Girgis, Gugnani et al' 2002)' In
active fibrosing alveolitis, an alveolar NO increase was associated with increasing
lymphocyte cell counts and an alveolar decrease was associated with corticosteroid treatment
(paredi, Kharitonov et al. 1999). This is in contrast to studies using one standard expiratory
flow for measurement, where no difference was found in 34 patients with systemic sclerosis
(Sud, Khullar et al. 2000), or in 52 patients with sarcoidosis (Wilsher, Fergusson et al' 2005)'
One study did find higher exhaled NO levels using the standard flow in 47 patients with
systemic sclerosis, particularly those with interstitial lung disease, and demonstrated an
inverse correlation with pulmonary artery pressure but no correlation to age, disease duration,
current therapy or the type of disease be it limited or diffuse (Rolla, Colagrande et al' 2000)'
One early paper also measured exhaled NO continuously in expired air at baseline and during
an exercise test and calculated the output of NO, which was low in six subjects with
pulmonary fibrosis compared to normal subjects. The authors suggested that that this patient
group may fail to increase NO output by failing to recruit the capillary bed during exercise
(Riley, Porszasz et al. lgg|). Nasal NO was 887o lower than normals in diffuse
panbronchiolitis, a pulmonary disease of unknown origin confined usually to individuals of
Japanese, Korean or chinese descent (Nakano, Ide et al. 2000). All current and future work in
this area of interstitial lung disease should use the ability to measure NO in the alveolar
compartment for meaningful results.
249

9.L6 Nitric oxide levels in exercise
The effects of exercise on levels of exhaled NO in healthy, athletic adults, and those with
respiratory and/or cardiac disease has been investigated, with a few studies also done in
children. In healthy adults, the measurement of exhaled NO with exercise (treadmill or
bicycle) showed a decrease when measuring the absolute levels in single breaths, but there
was a two to three fold increase in NO output taking into account the increased ventilatory
minute volume @ersson, Wiklund et al. 1993; Iwamoto, Pendergast et al. 1994; Trolin, Anden
et al. 1994; Phillips, Giraud et al. 1996; Yasuda, Itoh et al. L997). The increased output more
closely related to increased ventilation than increased blood flow @hillips, Giraud et al. 1996)
and significantly correlated with CO2 production (Iwamoto, Pendergast et al. 1994). Two
studies assessed the contribution from nasal or oral exhalations showing that most of the NO
coming from the lower airways (Lundberg, Rinder et al. 1997; Yasuda, Itoh et al. 1997) and
from the airway rather than alveolar compartment @ersson, Wiklund et al. 1993). The nasal
cavity NO reduced by 47Vo after one minute and 76Vo after five minutes of exercise
(Lundberg, Rinder et al. 1997).
There have been many studies done in elite athletes. When compared to those with lower
levels of fitness, the athletes had a significantly more linear increase in NO output (Maroun,
Mehta et al. 1995; Chirpaz-Oddou, Favre-Juvin et al. L997; Sheel, Edwards et al. 2000;
Verges, Flore et al. 2005). This output correlated well with oxygen consumption, COz
production, minute ventilation and heart rate (Chirpaz-Oddou, Favre-Juvin et al. 1997).In one
study, athletes that developed exercise induced hypoxemia had lower levels of NO compared
to those who did not seen (Kippelen, Caillaud et al. 2N2), but this was not confirmed in
another (Sheel, Edwards et al. 2000). Exercise in cold challenge conditions resulted in lower
levels of exhaled NO output being achieved and a slower time to recovery with lower FEVr
(Therminarias, Flore et al. 1998; Pendergast, Krasney et al. 1999). These results suggested
that physical conditioning increases expiratory NO output during exercise and this may be due
to an increased vascular and/or epithelial production of NO. The enhanced vascular NO
production may be the result of increased share stress or of an up regulation of the endothelial
NOS gene expression (Maroun, Mehta et al. 1995).
Can NO predict development of exercise induced bronchospasm? Higher baseline levels were
significantly associated with bronchoconstriction and histamine responsiveness in atopic
rather than non-atopic steroid naive healthy conscripts (Rouhos, Ekroos et al. 2005). In 50
consecutive subjects, a ROC curve yielded a value of 0.636 when comparing those who did
250

and did not develop exercise-induced bronchospasm. A result of

another study of 111 school children with asthma, exercise induced bronchospasm could be
excluded with a probability of 90Vo in those with baseline NO levels

Figure 9.3: Diagram of the measurement of lung function and No in an infant
Syringe
Pneumotach
Pump
Resistancs
Blow-off at 20 crn H2O
chemiluminescence analyser
Taken from wildhaber JH, Hail GL, Stick sM. Measurements of exhaled nitric oxide with the single'
breath technique and positive expiratory ;;;il6 il infants..American Journal of Respiratory and
Criticat Care Medicine 'iggg; 159:74-78 (Wildhaber, Hallet al. 1999)'
using this technique there was successful measurement of FEVo.s in 27 of 30 sedated infants
aged 3-24 months and successful measurement of NO in all. The end tidal COz level at the
nose was not changed during expiration, indicating that the velum was closed during the
manoeuvre (wildhaber, Hall et al. 1999). Studies have now been carried out using variations
on this technique reporting 95To+ success in sedated or quiet infants (Buchvald and Bisgaard
Zxl:;Martinez, Weist et al. 2003; Franklin, Turner et al' 2004; Straub' Moeller et al' 2005)'
The other method is by using tidal breathing where NO can be measured online or by
reservoir collection. Again the infant is in a supine position asleep following sedation' with
direct online measurement recorded during 20 seconds of quiet breathing (Franklin, Turner et
a:.2004), or using a mean of five or ten consecutive breaths (Hall' Reinmann et al' 20/0.2;
colnaghi, condo et al. 2003; Iripala, williams et al.2004). For offline measurements' tidal
breathing can be collected into a gas sampling balloon which can then be attached to the
sampler inlet of the NO analyser -
collecting over a set time period such as 15-45 seconds
@inakar, Craff et al. 2006), or during a set number of breaths (most use three or five)
(Anlich, Busch et al. 1998; Baraldi, Dario et al. 1999: Ratjen, Kavuk et al' 2000; Artlich'
Jonsson et al. 2001; Franklin, Turner et al. 2o04; Franklin, Turner et al' 2004; Moeller'
Franklin er al. 2004; Gabriele, Nieuwhof et al. 2006). Results have been reported as peak,
average levels and the avengecalculated from the area under the curve during expiration'
253

Almost all studies used sedation, with only a couple stating that they had not (Artlich, Busch
et al. 1998; Hall, Reinmann et al.200.2) and there was one study that had infants and young
children awake and in their parents' lap @araldi, Dario et al. 1999). One group investigated
whether sedation itself made a difference to the level of exhaled NO comparing results of 29
infants prior and subsequent to administering 60-100mg/kg of chloral hydrate. The mean
exhaled NO of 2l.4ppb when awake dropped significantly to l4.6ppb when asleep, associated
with an intra-subject co-efficient of variation of 20Vo when awake versus l0%o while sedated.
The authors suggested this may be explained by variation in expiratory flow, contamination
with ambient NO, breath-holding and crying, and often difficulty maintaining an appropriate
seal of the mask around an agitated infant. They were ultimately successful in getting readings
from29 of 39 infants when they were awake (but only 1l were 'co-operative'), compared to
achieving measurements in all the infants when sedated (Franklin, Turner et al. 2004).
Another study in 2O healthy non-sedated infants showed the intra-subject variability from
different phases of the exhalation ranged from 9.3 to l5.l%o with the inter-subject variation
much greater, ranging from 34.6 to 44.67o (Hall, Reinmann et al.2(N2).
Infants were shown to have a similar pattern of levels of exhaled NO to that seen in older
children and adults; rising to a peak with a rapid plateau. A linear relationship between NO
output and plateau was also demonstrated (Martinez, Weist et al. 2003). One study
investigated whether there was a difference between nasal and oral NO levels in 23 infants.
Most studies use a mask covering both mouth and nose and it is likely, given that infants tend
to preferentially breathe through their nose, that nasal NO may contribute significantly to
overall readings. The nasal tidal expiratory breaths were measured by continuing to place a
mask over both mouth and nose but ensuring that the mouth was firmly shut (not stated how)
and the oral tidal breaths were collected by blocking the nose with the rim of the mask. This
resulted in no significant difference between nose and mouth levels, which is different from
all other ages (Franklin, Turner et al.2N4). It may be that the method of exclusion of either
the oral or nasal exhalate was inadequate and/or that there was no effect from the presence of
sinuses in this age group to add to the usual higher nasal measurements.
There have been comparisons between the single breath and tidal breathing NO results.In 32
infants with recurrent wheeze, 16 with recurrent cough and 23 healthy infants a moderate
correlation of 0.6 was demonstrated between the two methods but with poor agreement
(Franklin, Turner et al.2004). This goup also noted that the single breath technique appeared
to be more sensitive to detect differences between infants with and without respiratory
symptoms (Franklin, Turner et aI.2004). In another study of 20 infants, the tidal exhaled NO
254

washout characteristics were noted to vary from breath to breath (Hall, Reinmann et al.2002)'
This group then divided the exhalation to assess whether one part of the expiratory signal
exhibited less intra-subject variability and therefore could potentially be the most
discriminatory. Firstly, based on the exhalation pattern of COz, they looked at the NO levels
which corresponded to phase one (from the convective airways), phase two (progressive
washout of the airways with alveolar gas) and phase three (alveolar compartment when
exhaled COz is high). Secondly, they divided the tidal volume into four quarters of exhalation
time. The most variable periods were phase one, and the first and fourth quarters'
Correspondingly, the second and third quarter time periods had about the same variability as
phase two, which was the best, followed by phase three. The group concluded that in the
absence of COz measurement as a guide, the reported result should come from the second and
third quarter of the exhalation (Hall, Reinmann et al' 2002)'
Similar to studies in other age groups, flow dependency of exhaled NO levels have been
demonstrated in infants by both methods. This was demonstrated in the original single breath
study comparing a second flow of 100mls/s as well as the standard of 5omls/s in three
subjects showing a significant difference in NO results (Wildhaber, Hall et al' 1999)'
Similarly, low to higher NO levels were obtained at expiratory flows of 50, 25 and l5mls/s in
five full term healthy infants (Martinez, Weist et al. 2003). Breath-by-breath analysis for tidal
breathing also showed that higher expiratory flow rates were associated with lower exhaled
NO levels (Franklin, Turner et a:.2004). In a large prospective healthy birth cohort of 98
infants the tidal breathing parameters of flow, breathing rate and expiratory time, measured at
age one month, all led to varying exhaled NO results. This group also came to the conclusion
that the third expiratory quartile of exhaled NO during tidal breathing was the most
reproducible (Frey, Kuehni et al.2004). In a direct comparison in 7l infants, the peak flow
was 128mls/s during tidal breathing and llmls/s during single breath measurements
(Franklin, Turner et al. 2004). This may also explain why single breath results versus tidal
breathing results usually have higher absolute exhaled No levels.
Other parameters have been seen to affect NO levels in infancy. Measurements in twelve
infants showed that ambient NO concentrations as low as 5-10 ppb compared to the inhalation
of NO free air resulted in an increase exhaled level (Franklin, Turner et al. 2004).In the 98
infants at one month, a gender difference, with boys having higher levels at 17 '7ppb than girls
at 14.6ppb, was noted. This remained significant after adjusting for weight and minute
ventilation (Frey, Kuehni et al.2004). In thirteen preterm infants the exhaled NO was almost
absent and gradually increased over the first 48 hours, while in eleven term infants there was a
255

peak exhaled NO production in the first hours of life (Colnaghi, Condo et al. 2003). In a series
of measurements of three premature infants while the absolute values were considerably
lower than older children, when corrected for the body weight it appeared comparable
(Artlich, Busch et al. 1998).
9.17.2 l*vels of nitic oxide in infants with diffirent respiratory diseases
Levels of exhaled NO were higher in wheezy infants compared with healthy controls (Baraldi,
Dario et al. 1999; Wildhaber, Hall et al. 1999; Franklin, Turner et al. 2004), as well as
compared to infants with a current URTI (Baraldi, Dario et al. 1999) and compared to infants
with cough (Franklin, Turner et aI.2ffi4). Having eczema also resulted in higher exhaled NO
levels in a group 88 infants in one study (Franklin, Turner et al.2O04) and 43 in the second
study @inakar, Craff et al.2006). One study in six infants with URTI found higher levels of
NO than healthy infants @araldi, Dario et al. 1999). However, two others found lower levels;
one measuing I7 infants with an URTI by nasal and oral and end tidal oral measurements
@atjen, Kavuk et al. 2000) and the second measuring 24 infants presenting with rhinorrhoea
with or without cough but not wheeze (Franklin, Turner et al. 2005). In eight of the infants
who had rhinorrhoea, repeated tests showed an increase from 7.5ppb to 34.1ppb when they
were symptom free (Franklin, Turner et d. 2005). Recently a large cross sectional study of
218 infants, including a number with different respiratory diseases and healthy controls, had
NO measured between 4.6 and 25.2 months of age. Higher levels were seen in 74 infants with
recurrent wheeze at l8.6ppb compared to 100 controls at l0.4ppb and 20 infants with
bronchopulmonary dysplasia at similar levels to controls with a mean of ll.8ppb, while 20
infants with CF had significantly lower levels at 5.9ppb (Gabriele, Nieuwhof et al. 2006).
Different results were found in infants with chronic lung disease in another study (I*ipala,
Williams et al.2004). Ten infants born at a median gestational age of 26 weeks and who had
developed chronic lung disease had higher peak nasal and face mask NO levels than ten term
infants and ten premature infants born at a median gestational age of 32 weeks without
chronic lung disease. Those with chronic lung disease were not receiving diuretics, inhaled or
systemic corticosteroids and bronchodilators or antibiotics at the time of measurement. All
infants were measured at a post conceptual age between 36 and 45 weeks. This meant the
infants with chronic lung disease were studied at an older post natal age at 85 days compared
to two days for the term grcup and32 days for the non-chronic lung disease premature goup.
There was, however, a wide range of NO levels in each group (Iripala, Williams et al. 2004).
256

g.17.3 Prenatal and maternal fficts on nitric oxide levels
In one already previously mentioned study which measured 98 infants at age one month'
exhaled NO levels were no different between infants from mothers with either asthma, other
atopic diseases or without atopic disease (Frey, Kuehni et aL.2004). These results differ from
another study where a family history of atopy gave a mean NO level of 41.lppb if it was both
parents, 24.2ppb if it was either parent and was lowest at 10'8ppb if it was neither parent
(wildhaber, Hall et al. 1999). What did have an effect in the Frey study was maternal
smoking or coffee consumption during pregnancy, both resulting in significantly decreasing
exhaled NO levels (Frey, Kuehni et al. 2004). Similarly' seven infants exposed to prenatal
cigarette smoke had lower exhaled NO than thirteen unexposed infants when measured
between 25 and58 days of age (Hall, Reinmann etal.2002).
g.17.4 Efficts of teatment on nitric oxide levels
Few studies have looked at levels of NO in response to treatment in this age group' Thirty one
infants aged 6-19 months with recurrent wheeze and raised exhaled NO determined by off-
line tidal breathing were treated with inhaled fluticasone 50pg twice per day or placebo for
four weeks. There was substantial reduction in the level of exhaled NO from 35ppb to
16.5ppb with only a small increase in FEV0.5 and no difference in the symptom scores
between groups (Moeller, Franklin et al.20O4). Similarly in 15 infants with recurrent wheeze
and high levels of exhaled No, this reduced by 527o after five days of prednisone becoming
no different to No levels of the control children (Baraldi, Dario et al. 1999).In 24 children
aged 10-26months with wheeze, allergy and a positive family history of asthma, montelukast
4mg per day for four weeks resulted in a significant decrease in levels of exhaled No from
29.8ppb to 19.0ppb and this did correspond to improvements in FEV0.5 and symptom scores
with no changes in the placebo group (Straub, Moeller et al. 2005). Finally in eight ventilated
infants with a mean gestational age of 25.8 weeks and post natal age of 55 days' exhaled NO
levels reduced from a mean of 6.5ppb to 4.2ppb co-inciding with a reduction 62 to 45Eo in
supplemental oxygen requirements with three days of dexamethasone treatment (Williams'
Bhat et al.20o4).
g.17.5 Summary of nitric oxide ftndings in infants
In summary, exhaled NO can be measured in infants by both single and tidal breathing
methods incorporating online and reservoir techniques. The advantages of the single breath
technique are, being able to use an expiratory pressure to standardise the desired expiratory
257

flow and to minimize nasal contamination. The tidal breathing method is less invasive and a
simpler collection method but it is not always possible to control flow or nasal NO
contamination. The most reproducible recordings have come from the second and third
exhalation phases, and with use of sedation in the infants. The results so far have
demonstrated a difference with gender, as suggested in some studies of older age groups, and
confirmed the effect of expiratory flow on NO. Antenatal effects include a decrease in infants
of smoking or caffeine ingesting mothers, though the effect of maternal (and patemal) atopy
has been either nil or resulted in an increased levels of NO. Higher levels of NO were
demonstrated in wheezy infants although the relation between this and those who develop
asthma versus those who have viral induced wheeze of infancy which is likely to disappear
between three and six years is not yet determined. The effect of a viral upper respiratory tract
infection is less consistent on NO levels. As with infant lung function, the measurement of
NO in this age group remains the premise of research groups and is not widely used in the
clinical arena.
9.18 Chapter summarv
Following early experiments throughout the 1990s, it was recognised that standardisation of
the method of measuring NO was required to allow better interpretation of results and
comparison between research groups. These began with a meeting in Stockholm in 1996 and
from 1997 to 2005 four documents were generated involving the ERS and the ATS detailing
standard procedures and then updating and refining these procedures as more data became
available (Kharitonov, Alving et al. 1997; American Thoracic Society and Association. 1999;
Baraldi, de Jongste et al.20O2l American Thoracic Society and European Respiratory Society
2005). What became increasingly apparent from very early in the research was the importance
of getting consistent and reproducible results. The most important factors to control were
expiratory flow and nasal contamination. Standard practices for single, tidal breathing,
reservoir collections and nasal measurements have been developed for both adults and
children. In addition, the use of different expiratory flows to assess NO from different lung
compartments has also been explored. This is important when wanting to demonstrate
inflammation in certain compartments in certain diseases, for example the airway
compartment in asthma and the alveolar compartment in interstitial lung diseases. In the latter
type of diseases, this technique should always be employed in the future to enable meaningful
results to be obtained.
258

Physiological variables also affect the results. Exhaled NO levels have been shown to be
higher in males than females, are reduced with cunent Smoking, recent ingestion of water'
alcohol or caffeine and increased with accidental gut contamination or after nitrogen-
compound rich meals. Levels of NO also increase with age throughout childhood' but not
adulthood, possibly in relation to pneumatisation of the sinuses. Relationships with height,
weight, surface area, menstrual cycle in non-asthmatic women' or a circadian rhythm has not
been demonstrated. However, there remains only one range of 'normal' values given for oral
or nasal NO.
By far the most work in exhaled and nasal NO has been undertaken in atopic and/or asthmatic
subjects with comparison to healthy controls. NO appears to correlate well with the
inflammatory cells, lung function and bronchial hyper-responsiveness in steroid naive
asthmatics and less well in those on IHCS or oral steroids. It does not correlate well with
.severity, of asthma as the NO result here is attenuated by treatment, and it is the requirement
for the higher levels of treatment with possible breakthrough symptoms that determine the
severity category in asthma guidelines(Anonymous 1997 GINA 20O2; GINA 2005; SIGN
2005). There is the potential for No measurements to contribute to diagnosis, management
when measured longitudinally in individuals (on any medication), and to wafii of loss of
Control. NO measurement can also be used as an outcome measure for ant-inflammatory
agents used in asthma treatment. However there are some cautions to be made. Firstly, it does
not appear useful to guide acute asthma treatment. Secondly, measurement of sputum
eosinophils appear as, or more, accurate than NO itself, and the changes in the eosinophil
counts may be a more accurate predictor of loss of control and a better target to tailor
treatment. It is increasingly apparent that, like most parameters we measure' it is likely to be
the pattern from a number of investigations along with an individual's history that best
describes their clinical disease rather than a single marker' The effect of atopy alone on
exhaled NO levels needs to be kept in mind with overlapping results between asthmatic and
atopic subjects. Unfortunately, smoking renders the test inaccurate and unhelpful' In addition'
the presence of high NO levels may be a marker for respiratory diseases that are steroid
sensitive no matter what the underlying disease is labelled.
In exercise, NO drops during single breath measurements, but the NO output is increased
when taking into account the increased rate of breathing. An inability to increase the NO
output during exercise was associated with more severe cardiac disease and, in elite athletes'
worse performance suggesting that the increased NO output may depend on the ability to
259

ecruit blood vessels in the lung. Studies have also suggested that a normal NO reading at the
beginning of exercise predicted that no exercise-induced bronchospasm occurred.
Low nasal NO appears to be helpful in screening for PCD and possibly CF. It is particularly
important to measure this in children with established bronchiectasis and detecting a low NO
level early in assessment for recurrent respiratory illnesses would be additionally beneficial
and would indicate that these diseases must be investigated and excluded. The low NO levels
seen in PCD remain unexplained, while those in CF and other conditions such as sinusitis
would appear to be secondary to sinus obstruction. Oral NO levels are also low in PCD, but
the nasal NO measurements (using a cut point of 250ppb) have minimal overlap with normal
children. Oral NO levels become low in CF with age and disease progression, probably with
entrapment of NO within thick mucus and therefore likely to interact with other compounds,
so the NO does not reach the air to be exhaled. NO levels are increased in acute infections in
both normal subjects and those with COPD, and increase during times of higher pollution and
also associated with respiratory symptoms. However 'smoking' remains a confounding factor
here -
again rendering the measurement of NO as useless. In interstitial lung disease, levels of
NO co-relate with severity, in particular that assessed by the diffusion co-efficient, but only
when measured in the alveolar compartment. The measurement of NO has been undertaken in
infants and here while the parameters are still being worked through, it appears that the results
are already low in those with PCD and CF, and high in children with wheeze, though it is still
to be clarified whether this will result in detecting asthma versus infant viral associated
wheeze, and whether it matters, as it may just detect disease that will respond to steroids.
The advantages of using NO measurement rather than (or in addition to) other parameters is
obvious. It is much easier to perform, especially in children, than lung function testing or
induced sputum for eosinophils or any of the blood measurements such as total IgE. In
addition it is quicker, with immediate results, and potentially can be done away off hospital
site. While it still needs an understanding of the test and qualified personnel, it can be taught
and undertaken more easily than the other investigations.
So where do I see it as cunently being MOST useful:
l. Nasal (and oral) measurement as a screen for PCD. If nasal levels are low - ensure
investigation for PCD and CF are carried out. While we do have a newborn screening
for CF here in New Zealand,87o of children are not detected.
2. As confirmation of asthma in steroid natve patients - when NO levels should be high.
260

3. As a marker to follow when commencing treatment, in essence IHCS in steroid naive
asthmatics (in non-smokers).
4. As a marker to follow longitudinally, particularly in poorly controlled asthmatics
contributing to assessing disease control, and response to changes in treatment (in
non-smokers).
5. It may become a method to assess the need for an exercise test in patients presenting
with a complaint of possible exercise induced bronchospasm - if No levels are normal
then an exercise test may not be required and investigations should proceed down
another avenue (in non-smokers).
The final, and brief, chapter will review where the research that I undertook contributed to
both the clarity and confusion of early knowledge in this area. I will review what I learnt from
the process of doing this research, writing up this thesis, and how this information has
ultimately altered my research and clinical practice'
26r

Chapter 10: Reflections
The outcome of any serious research can only be to make two questions grow where
only one grew before. (Torstein Veblin 1857-1929)
By the time I had finished the research presented in this thesis, I felt that in attempting to
answer a few questions - about the exact nature of exhaled NO and what made a difference to
the levels obtained - I now had far more questions than when I began. It is hard to
comprehend the rapid explosion regarding NO from the first discovery of its physiological
role in 1987. Now the vital roles it occupies as a widespread mediator to maintain
homeostasis and in host defence seems to be a long-known fact. The Medline searches reveal
the rapid growth of research in this area in the 1990s and 2000s with studies conducted
involving every organ system in every conceivable subject population (see Figure 10.1).
When I started with the investigations presented in these chapters, very little was known about
its role. Having acknowledged this, the measurement of exhaled NO is still not part of routine
clinical practice and the exact use of this technology and its place in clinical medicine
continues to be investigated today. In the thesis I have not touched on the measures of gases
in exhaled breath condensate. This is an even more recent rapidly expanding area of research
worthy of its own chapters by those involved in investigating its use.
Figure 10.1: Medline publications with a focus on nitric oxide research per year 1980-2006
16000
14000
o
5 12ooo
.g - loooo
.=6
E g. Sooo
o.L
E t 6000
L
.g 4ooo
5 I
2oqr
z
0 ,-alIl
1980 1983 1986 1989 1992 199s 1998 2001 2004
When I first cornmenced research, it was as a means to an end. By the end of the 1990s, I
knew that I wanted to train as a paediatric respiratory specialist given the burden of disease
seen in children in the UK where I was currently working, and, perhaps more particularly, at
I
Years 1980-2006
262

home in New 7*aland.In order to do that I aimed to get a senior registrar / clinical lecturer
post at a centre of excellence which for me, working in London at time, was the Royal
Brompton Hospital. I decided that research would definitely be a pre-requisite to this
appointment and so successfully obtained a research fellow job at the same hospital'
However, having started down a research path, I can no longer envisage not having research
as a major part of my role. Research involves the development of different skills to those
required in clinical medicine. At the time, I enjoyed considering in depth and reading widely
around one aspect of medicine. But, when I was asked by one scientist whether I used
,positive, or .negative' pipetting when I did enzyme-linked immunosorbent assays (ELISAS)
studies, I knew that I was in a different world. It teaches you attention to detail' the need to be
very methodical, the need to be sceptical and the need to question every factor' It also allows
an opportunity to work with people who are experts in different areas. From the scientists I
learnt patience, information technology skills, statistics and careful laboratory bench-work' I
also appreciated the very different knowledge and skills that Carolyn Busst as a medical and
electrical engineer brought to the research. The topics of research and critical reviews were
not previously emphasised but now occupy a central theme in current medical school training'
and I consider this important in developing questioning practitioners'
Of course, now I would do everything very differently. One problem with research' as the
quotation above suggests, is that no matter what topic you start with, it can expand
exponentially. If commencing this research now, I would restrict the work to investigating
exhaled NO and the long-actingp2 agonist ('salmeterol') trial that I was also conducting
during this time. In addition,I was undertaking clinical training with on-call commitment in a
paediatric cardiorespiratory intensive care setting' I would not' for example' have also
embarked on learning induced sputum techniques and ELISAs for processing IL8 and TNFg'
This latter research ultimately suffered for which I feel responsible. From the beginning I
found the children, particularly healthy controls, did not like the use of hypertonic saline for
sputum induction. This needed much greater attention and a full time researcher on this alone
to determine a satisfactory technique. However, by this time I had established the machines'
connections and protocols and was running with the exhaled NO research, and I was enjoying
it. Also, in comparison, the adult respiratory service had four research fellows dedicated to
investigating these areas of medicine which was a great deal more realistic. On the other hand,
I would now also make the most of any overlappin g area of research to combine
investigations. For example, at the time I was the principal investigator in a cross-over trial
involving two dosage regimes of salmeterol compared to one dosage regime of salbutamol
263

and I enrolled 52 asthmatic children. At the time there was a question as to whether the long
acting p2 agonist therapy had any anti-inflammatory effects. I was well placed to answer this
if I had measured the exhaled NO in these children, and/or connected them with an 'induced
sputum' researcher.
My experience in research has, I hopr, assisted me in the role of supervisor of other
researchers. I have learnt from my supervisors and from my own successes and failures. I
ensure regular contact no matter what is happening, and I limit the focus of the research. Early
enthusiasm often encourages taking on too big a project and then feeling ovenvhelmed or that
justice is not being done to every possible investigative avenue. I also returned to a one in two
on-call roster providing paediatric respiratory cover regionally and nationally. Again my 'take
home' message quite literally is to be certain to factor in time for writing. I believe important
contributors to success in research are attention to detail, regular review, maintaining focus,
and a realistic appreciation of the time required.
Since returning to New 7*aland my own clinical and research focus has shifted to the area I
am concerned which is a main burden of respiratory disease in New 7*,aland children -
infection and in particular the development of chronic suppurative lung disease resulting in
early morbidity and ultimately early adult mortality. My next endeavours are to continue to
develop a good management programme and research into aetiology, appropriate intervention
and strategies for prevention in this area of lung disease. While I also steadfastly remain a
clinician, I could not stop doing research now. I thank the Royal Brompton Hospital and Dr
Andy Bush in particular for opening up this path for me.
2&

Appendix I : Publications
Published manuscripts related to this thesis:
Appendices
o ,.Trying to catch our breath" The burden of preventable breathing diseases in children and
young people. Editors Innes Asher and Cass Byrnes. Asthma and Respiratory Foundation
of New 7-ealand. Te Taumatua Huango, Mate Ha o Aoteroa' www'asthmanz'co'nz and
www.paedi atrics.org.nz.
C A Byrnes: Chapter 9: The Burden of Bronchiolitis in New Zealand (40-45)'
c A Byrnes: Chapter I 1: The Burden of Bronchiectasis in New T.r,aland (51-55)'
o Byrnes cA, Dinarevic s, shinebourne EA, Barnes PJ, Bush A. I-evels Of nitric oxide in
exhaled air in normal and asthmatic children. Pediatric Pulmonolo gy 1997 24 (5) 312-318'
o Byrnes CA, Dinarevic S, Busst CA, Shinebourne EA, Bush A' Effect of measurement
conditions on measured levels of peak exhaled nitric oxide. Thorax 1997 52 (8) 697-701'
o Byrnes cA, Dinarevic S, Busst cA, Bush A, Shinebournes EA. Is nitric oxide produced
at airway or alveolar level? European Respiratory Journal 1997 l0 (5) 1021-1025'
o Dinarevic s, Byrnes cA, Shinebourne EA, Bush A. Measurement of exhaled nitric oxide
in children. Pediatric Pulmonolo gy 1996; 22: 396-40L'
o Byrnes CA, Bush A and shinebourne EA "Methods to Measure Expiratory Nitric oxide
in People" in "A Volume of Methods in Enzymology" edited by I-ester packeter
Academic Press Inc. 1996.
Publications based on research that has progressed from this thesis:
o phD thesis: .Nitric oxide and ciliary beat in normal and bronchiectatic children'' Dr
Elizabeth Edwards awarded 2004 (2001-2004)'
Prinicipal supervisor: Dr Cass Byrnes'
o Edwards EA, Douglas C, Broome S, Kolbe J, Jensen CG, Dewar A, Bush A' Byrnes CA'
Nitric oxide levels and ciliary beat frequency in indigenous New Zealand children'
Pediatric Pulmonolo gy 2005 ; 39: 238-246'
Presentations and published abstracts from this research and related to this research:
o Edwards EA, Douglas c, Broome s, Ferguson w, Kolbe J, Byrnes cA' Exhaled nitric
oxide levels in children with non-cystic fibrosis bronchiectasis in New Tnaland' European
Respiratory Meeting & European Respiratory Journal 2002;38: 338s'
265

Edwards EA, Douglas C, Broome S, Jensen C, Byrnes CA. Exhaled nitric oxide (NO) and
ciliary beat frequency (CBF) in normal and bronchiectatic New Zealand Children.
American Thoracic Meeting & American Journal of Respiratory & Critical Care Medicine
200r;163 (5): 4854.
Edwards EA, Douglas C, Broome S, Jensen C, Byrnes CA. Exhaled nitric oxide (NO) and
ciliary beat frequency (CBF) in normal and bronchiectatic New Zealand Children. New
TnalandPaediatric Society Meeting 2000. Young investigators award.
Edwards EA, Douglas C, Broome S, Jensen C, Byrnes CA. Exhaled nitric oxide (NO) and
ciliary beat frequency (CBF) in normal and bronchiectatic children. New 7*aland
Thoracic Society 2000. Young investigators award.
Measurements of exhaled nitric oxide in children - Australiasian Paediatric Respiratory
Group 1999 -
invited presentation.
Byrnes. CA. Exhaled nitric oxide. National New Zealand Respiratory Meeting 1998 -
invited presentation.
Byrnes CA, Dinarevic S, Shinbourne EA, Bush A. Exhaled nitric oxide in normal and
asthmatic children - British Paediatric Society - 1996.
Other Publications:
Byrnes C. Non cystic fibrosis bronchiectasis. Paediatric Respiratory Reviews 2A06;7
suppl 1: 5255-5257.
Twiss J, Stewart AW, Byrnes CA. Longitudinal pulmonary function of childhood
bronchiectasis and comparison with cystic fibrosis. Thorax 2006;61(5): 414 - 418.
Thomson JM, Wesley A, Byrnes CA, Nixon GM. Pulse intravenous methylprednisolone
for resistant allergic bronchopulmonary aspergillosis in cystic fibrosis. Pediatric
Pulmonolo gy 2006; 4l(2): 164-17 0.
"Accreditation of Paediatric Respiratory Function Assessment Services" Dr C A Byrnes -
by invitation Thoracic Society of Australia and New Zealand and Australasian Paediatric
Respiratory Group. www.thoracic. org. au March 200,6.
'Wheeze and Chest infections in children less than one year of age' - Co-chairs Dr Cass
Byrnes, Dr Alison Vogel. Evidence based guideline funded by a Ministry of Health
initiative. Guidelines endorsed by the Paediatric Society of New 7*aland, New Zealand
Guidelines Group, the Royal New Zealand College of General Practitioners and the Royal
Australasian College of Physicians. www.paediatrics.org.nz. April 2005.
266

o Howie S, Voss L, Baker M, Calder L, Grimwood K, Byrnes C. Tuberculosis in New
Tnaland, 1992-200I: a resurgence. Archives of Disease in Childhood 2005; 90: II57-
1161.
r Gillies J, Brown J, Byrnes C, Farell A, Graham D. PHARMAC and ventolin in New
Tlaland. New Zealand Medical Journal August 2005; Ll8:122O.
o Twiss J, Metcalfe R, Edwards E, Byrnes C. New Zealand national incidence of
bronchiectasis "too high" for a developed country. Archives of Disease in Childhood
2005;90(7): 737-740.
o Twiss J, Byrnes CA, Johnson R, Holland D. Nebulised gentamicin - suitable for
childhood bronchiectasis. International Journal of Pharmaceutics 2OO5;295: ll3-119
o Edwards EA, Douglas C, Broome S, Kolbe J, Jensen CG, Dewar A, Bush A, Byrnes CA.
Nitric oxide levels and ciliary beat frequency in indigenous New Zealand children.
Pediatric Pulmonolo gy 2005; 39: 238-246.
r Edwards EA, Metcalfe R, Milne DG, Thompson J, Byrnes CA. Retrospective review of
children presenting with non cystic fibrosis bronchiectasis: HRCT features and clinical
rel ationships. Pedi atric Pulmonolo gy 20O3 ; 36: 87 -93.
o Edwards EA, Asher MI, Byrnes CA. Paediatric bronchiectasis in the 2l't century:
experience of a tertiary childrens hospital in New T.rualand. Journal of Paediatric and Child
Health 2003; 39: 1 I L-117 .
o Vogel A, knnon D, Broadbent R, Byrnes CA, Grimwood K, Mildenhall L, Richardson
V, Rowley S. Palvimuzab prophylaxis of respiratory syncytial virus infection in high risk
infants. Journal of Paediatric and Child Health 2OO2;38: 550-554.
o Edwards EA, Butler SA, Byrnes CA. Paediatric non cystic fibrosis bronchiectasis -
recognition and management. New Ethicals Journal 2OO2; November: 58-65 (invited
article).
o Massie J, Poplawski N, Wilcken B, Goldblatt J, Byrnes C, Robertson C. Intron-8
polythymidine sequence in Australasian individuals with CF mutations RllTH and
Rll7C. European Respiratory Journal 2001 L7 1195-1200.
o Byrnes CA, Shrewsbury S, Barnes PJ, Bush A. Salmeterol in Paediatric Asthma. Thorax
2000; 55:780-784.
o Edwards E, Byrnes CA. Humidification difficulties in two tracheostomised children,
Journal of Anaesthetic and Intensive Care 1999; 27:656-658.
267

o Cass Byrnes & Nicky Holt. "Drugs used in Cystic Fibrosis". Medicines for Children.
British Paediatric Formulatory I't Edition, published by the Royal College of Paediatrics
and Child Health 1999.
Other presentations and published abstracts:
International Meetings:
o Byrnes CA. Pearls: Non-Cystic Fibrosis Bronchiectasis - 7fr International Congress on
Pediatric Pulmonology, Montreal, Canada July 2006 - invited presentation.
o Byrnes CA. Lung function accreditation guidelines - Australasian Paediatric Respiratory
Group Annual Scientific Meeting 2004 - invited presentation.
o Byrnes CA. Lung function and lung function accreditation guidelines - Australasian
Paediatric Respiratory Group Annual Scientific Meeting 2W3 - invited presentation.
o Bynes CA. Bronchiectasis - Australasian Paediatric Respiratory Group Annual Scientific
Meeting 2OO2 -
invited presentation.
. Byrnes CA, Barnes PJ, Bush A. Salmeterol trial in asthmatic children - American
Thoracic Society Annual Scientific Meeting 1999.
National Meetings:
o Byrnes CA. Australia and New 7*,aland Cystic Fibrosis Bronchoalveolar Lavage -
National Respiratory Meeting 2W7 - invited presentation.
o Byrnes CA. Paediatric workshop - Standards of Care in Cystic Fibrosis in New 7*aland -
National Respiratory Meeting 2007- invited presentation.
o Byrnes CA. Paediatric cases workshop - National Respiratory Meeting L999-2W6 -
invited presentation.
o Cheney JL, Carlin J, Cooper P, Byrnes C, Grimwood K, Armstrong D, Martin J, Dakin C,
Robertson C, Whitehead B, Francis P, Vidmar S, Wainwright CE. Adverse events
associated with flexible bronchoscopy and bronchoalveolar lavage in young children with
cystic fibrosis. North American Cystic Fibrosis Conference 2006.
r Wainwright CE, Carlin J, Cooper P, Byrnes C, Martin J, Grimwood K,, Armstrong D,
Francis P, Dakin C, Whitehead B, Carter R, Vidmar S, Cheney J, Tiddens H, Fink M,
Robertson C. Australasian Cystic Fibrosis BAL study analysis. North American Cystic
Fibrosis Conference 200,6.
o Byrnes CA. Pseudomonas aeruginosa -
management of ltt infection in children with
cystic fibrosis - National Respiratory Meeting 2003 - invited presentation.
268

o Byrnes CA. Cystic Fibrosis Bronchoalveolar Lavage Study -
National Cystic Fibrosis
Seminar February 2003 - invited presentation.
o Howie S, Voss L, Grimwood K, Byrnes CA. Tuberculosis disease in New 7*aland
Children. American Thoracic Meeting & American Journal of Respiratory & Critical Care
Medicine 2003;163 (5) :4854.
o Twiss J, Byrnes CA. Paediatric bronchiectasis: longitudinal lung function and comparison
with cystic fibrosis. New Zealand Paediatric Society Annual Conference 2003. Highly
commended award.
o Twiss J, Byrnes CA. Longitudinal lung function. Thoracic Society of Australia & New
Zealand2003.
o Wainwright C, Cooper P, Carlin J, James M, Armstrong D, Robertson I, Cooper D,
Whitehead B, Byrnes C, Francis P, Robertson C. Early infection with pseudomonas
aeruginosa can be effectively cleared in young children with cystic fibrosis. Australian
Cystic Fibrosis Conference 2003.
o Byrnes CA. Asthma Medications - New Tnaland Paediatric Update 2002 - invited
presentation.
r Howie S, Voss L, Grimwood K, Byrnes CA. Tuberculosis disease in New Tr,aland
Children. New Zraland Paediatric Society Annual Conference 2002.
o Wainwright C, Carlin J, Cooper P, Byrnes C, Whitehead B, Martin J, Cooper D,
Armstong D, Robertson C. Early infection with pseudomonas can be cleared in young
children with cystic fibrosis - North American Cystic Fibrosis Meeting 2002 Pediatic
Pulmonology Supplement 24: 357 .
o Vogel AM. Moodabe K, Aish H, Campanella S, Byrnes CA. Implementation of guidelines
for the management of cough and wheeze in the I't year of life in general practice. New
Zealand Paedi atric Society Annual Meeting 200L.
o Jaksic M, Asher MI, Byrnes CA. Streptokinase in the Management of Empyema.
American Thoracic Meeting & American Journal of Respiratory & Critical Care Medicine
2001; 163 (5): A854.
e Edwards EA, Asher MI, Metcalfe R, Byrnes CA. Non-cystic bronchiectasis - correlation
of high resolution CT scan and chest x-ray scores with clinical evaluation. American
Thoracic Meeting & American Journal of Respiratory & Critical Care Medicine 2001; 163
(5): A854.
o Edwards EA. Byrnes CA. Temperature control is vital for the measurement of cilia beat
frequency when establishing a hospital protocol. European Respiratory Meeting &
European Respiratory Journal 2001;18: 300s.
269

o Byrnes CA. Paediatric fibreoptic bronchoscopy - New Tnaland Paediatric Society Annual
Meeting 2001.
o Byrnes CA. Screening for respiratory ciliary dysfunction - New 7*,aland Paediatric
Society Annual Meeting 200I.
r Edwards EA, Metcalfe R, Asher MI, Byrnes CA. Bronchiectasis - Correlation of high
resolution CT scan and chest x-ray scores with clinical evaluation in 52 children. New
Tnaland Paedi atri c S oci ety Ann u al Conference 2000.
o Edwards EA, Douglas C, Broome S, Jensen C, Byrnes CA. Exhaled nitric oxide (NO) and
ciliary beat frequency (CBF) in normal and bronchiectatic children. New Zealand
Thoracic Society 2000.
o Edwards EA, Metcalfe R, Asher MI, Byrnes CA. Bronchiectasis - Correlation of high
resolution CT scan and chest x-ray scores with clinical evaluation in 52 children. New
7*aland Paediatric Society Annual Conference 2000.
o Byrnes CA. Home oxygen in children - National Respiratory Meeting 2000 - invited
presentation.
o Byrnes CA. Infection control in children with cystic fibrosis - National NZ Respiratory
Meeting 2000 - invited presentation.
o Byrnes CA. Obstructive sleep apnea - New 7*aland Paediatric Update 2000 - invited
presentation.
o Byrnes CA. Management of pleural effusions - New 7-ealand Paediatric Update 2000 -
invited presentation.
o Byrnes CA, Barnes PJ, Bush A. Salmeterol trial in asthmatic children - American
Thoracic Society Annual Scientific Meeting 1999.
Abstracts and poster presentations:
o McNamara DG, Byrnes CA, Rutland MD. Development of a technique to measure
mucociliary clearance in children with tracheostomies. Thoracic Society of Australia &
New Zealand Annual Scientific Meeting, May 2005.
o McMorran B, James A, Armstrong D, Cooper P, Carlin J, Martin J, Robertson I, Cooper
D, Whitehead B, Byrnes C, Francis P, Robertson C, Wainwright C, Wainwright B.
Proteomic comparisons in cystic fibrosis lung lavage fluid using ProteinChip@
technology. North American Annual Cystic Fibrosis Conference, October 2W3.
r Butler SA, Twiss J, Byrnes CA. Bronchiectasis outreach - a new initiative in New
7*aland. Fifth National Paediatric Physiotherapy Conference, Australasia, 2003.
270

Edwards EA, Dewar A, Jensen C, Bush A, Byrnes CA. Ciliary abnormalities in New
7*alandchildren with bronchiectasis. Cilia, Mucus and Mucociliary Group Meeting' USA
2002.
Edwards EA, Byrnes CA. Temperature control is vital for the measurement of cilia beat
frequency when establishing a hospital protocol. European Respiratory Meeting &
European Respiratory Journal 2001; 18: 300s'
Edwards EA, Asher MI, Metcalfe R, Byrnes cA. Non-cystic bronchiectasis - correlation
of high resolution CT scan and chest x-ray scores with clinical evaluation' American
Thoracic Meeting & American Journal of Respiratory & Critical Care Medicine 2001; 163
(5): A854.
Jaksic M, Asher MI, Byrnes cA. Streptokinase in the Management of Empyema'
American Thoracic Meeting & American Journal of Respiratory & Critical Care Medicine
2001; 163 (5): A854.
Current PhD suPervision:
o .Bronchiectasis in children and young people in New Tnaland'' Dr Jacob Twiss'
o .Effects of humidification on the airway in children with tracheostomies.' Dr David
McNamara.
27r

Appendix 2: Questionnaire for enrolling the children
9C Ar.gr13r
Itt JursrtRs ltrLt w nREllr@ .Irf aIRIcr @r:rDElfcE.
trttrE of ciltd.
Drte of bl,rth of child.
ltacloaall,Cy... ,.......o. r.......
w/drRl'
IIas he/she lrad a col.d or chest infection in tfte lasc month? tE8./TO
Ilas fie./she evet iad astlrCIa? tt lraercltc .tlr tlrrt dtrgraroct.. W9|TO
Does Jre./slre have asthna non? YE$/XO
Ilas ie./slra ever wheezed?(a& r r.trjrtllng touact oa Draetlrtng out) W9/tW
ooes ielslre cough. . . lrgvER. . . sodBgr.tE$. . . om8F. . . coilSmilrur.
Has he./she ever been prescribed 9entolin or BricanyJ?
If lcrrxbcn?.. ...,
Itas he/she ever had eczena?
ffas helshe ersr had ftay fcver?
Docs he/shc have eczena/hayfevet no*?
Oocs he./s[e have any other cfiest cotplalnt? prc.lsa n rrc......
Does anyone eJse jn the .faniy harre any of tie followittg:
aschnd Y$S/XO if yes uho?,, a . . , ,
ecsenr VES/NO lt yes who?.......
iayfever ws/No tf yes nha?.
tlrrr glvr r.Ltl'orrtlp tc tb cl.tll
Do you have any pets? Jf pr plcrsc n nc cJar.
Does anyone in the family snoke?.
wslTo
YE9/TO
t:Es/fro
t]3g/xO
rc8/NO
Note: the spelling error noted in the heading of these sheets was only noted after we had delivered
most of the questionnaires to the participating school - in view of that and as this how most of the
collected forms remain - | have left it in.
272

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