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Non-invasive method <strong>of</strong> measuring airway inflammation: exhaled nitric oxide<br />
Dr Catherine Ann BYRI\IES<br />
FRACB MBChB (New Zeatand)<br />
Graduate certificate in crinicat Education (New south wales)<br />
<strong>The</strong> <strong>University</strong> <strong>of</strong> <strong>Auckland</strong><br />
Department <strong>of</strong> Paediatrics<br />
Faculty <strong>of</strong> Medical & Health Sciences<br />
Private Bag92ol9<br />
<strong>Auckland</strong> 1142<br />
New Zealand<br />
Ph: &93737 599extn 89770<br />
Fax: @93737 486<br />
Email: c.byrnes@auckland.ac.nz<br />
Senior Lecturer & Honorary Consultant<br />
Paediatric Respiratory Medicine
UNIVERSITV OF AUCKI'AND<br />
Abstract<br />
- - oEC 2008<br />
Backeround<br />
UBRARY<br />
Nitric oxide (NO) was well known to be a component <strong>of</strong> air pollution, <strong>of</strong>ten in the form <strong>of</strong><br />
nitrogen dioxide (NOt. However its importance in biological systems altered dramatically<br />
with the discovery in 1987 that it was the 'endothelial-derived relaxing factor'. Since then<br />
there has been an explosion <strong>of</strong> research on NO demonstrating that this gaseous molecule was<br />
a widespread physiological mediator and was simultaneously recognised as a vital component<br />
<strong>of</strong> immune function contributing to macrophage-mediated cytotoxicity. NO was therefore a<br />
key molecule in modulating inflammation, including airway inflammation.<br />
<strong>The</strong> aim <strong>of</strong> this thesis was:<br />
l. To adapt a NO chemiluminscence analyser from measuring ainvay pollution to measuring<br />
exhaled air in human subjects.<br />
2. To measure NO levels in exhaled air in adult subjects.<br />
3. To evaluate whether altering measurement parameters altered the levels <strong>of</strong> NO obtained.<br />
4. To adapt this technique from adults to measure exhaled NO in children.<br />
5. To compare levels <strong>of</strong> NO from healthy children to groups <strong>of</strong> asthmatic children on either<br />
bronchodilator therapy only, or on regular inhaled corticosteroids.<br />
6. To compare the levels <strong>of</strong> NO in a pilot group <strong>of</strong> asthmatic children before and after<br />
commencement <strong>of</strong> inhaled corticosteroids.<br />
Methods<br />
A Dasibi Environmental Corporation Model 2t07 chemiluminescence analyser was adapted<br />
specifically requiring a reduction in response time, which was achieved by modification <strong>of</strong> the<br />
circuitry and re-routing <strong>of</strong> the analogue signal directly to a chart recorder, achieving a<br />
reduction <strong>of</strong> the response time by 807o. Addition <strong>of</strong> a number <strong>of</strong> analysers allowed the<br />
measurement <strong>of</strong> exhaled NO, carbon dioxide (COz), mouth pressure and flow for each<br />
exhalation from total lung capacity. Twenty adult subjects (in total) were then studied looking<br />
at direct (NO, CO2, mouth pressure) versus t-piece (with the addition <strong>of</strong> flow) measurements<br />
making five exhalations from total lung capacity, at 3-minute intervals (direct/t-piece/direct or<br />
t-piece/direct/t-piece in series). <strong>The</strong> area <strong>of</strong> NO under the curve versus the peak <strong>of</strong> the NO<br />
trace was compared and the exhalation pattern <strong>of</strong> NO versus COz was compared.<br />
Measurement conditions were altered to evaluate the effect <strong>of</strong> individual parameters on the<br />
exhaled NO result. This included separately assessing different expiratory flows, different<br />
expiratory mouth pressures, the effect <strong>of</strong> a high versus a low background NO level and the<br />
tl
effect <strong>of</strong> drinking water (<strong>of</strong> varying temperatures) prior to exhalation. Healthy control<br />
children were then enrolled to the study from a local school (Park Walk primary School) and<br />
compared with asthmatic children enrolled from outpatient clinics at the Royal Brompton<br />
Hospital. <strong>The</strong> asthmatic children were further divided into those on bronchodilator treatment<br />
only and those on regular inhaled corticosteroid therapy. NO was also measured before and<br />
two weeks after commencing inhaled corticosteroid therapy in previously steroid-naive<br />
asthmatics.<br />
Results<br />
It was possible to modify a chemiluminescence analyser to enable measurement <strong>of</strong> exhaled<br />
No' In 12 healthy subjects (mean age 32 years, 6 males) peak direct NO levels were g4.g<br />
parts per billion (ppb) (standard error <strong>of</strong> the mean (SEM) l4.0ppb), significantly higher than<br />
4l'2ppb (SEM 10.8ppb) measured via the t-piece system. <strong>The</strong> exhaled NO rose to an early<br />
peak and plateau while the COz levels continued to rise to peak late in the exhalation. <strong>The</strong><br />
mean times to peak NO levels were32.2 seconds (s) (direct) and 23.1s (t-piece), which was<br />
significantly different from the mean times to peak CO2levels at 50.5s (direct) and 51.4s (t-<br />
piece, p
<strong>The</strong>re was no significant difference between boys and girls for either the direct or the t-piece<br />
recordings. In comparison with normal children, 15 asthmatic children on bronchodilator<br />
therapy only had much higher levels <strong>of</strong> exhaled NO at 126.1ppb via the direct system (SD<br />
77.lppb, p4.001) and 109.5ppb via the t-piece system (SD l06.8ppb, p
esults suggested that the methods <strong>of</strong> measuring exhaled NO required standardization and that<br />
it could potentially be a non-invasive measure <strong>of</strong> airway inflammation to follow - particularly<br />
in children with asthma who were conmencing inhaled steroid treatment.<br />
V
This is Dedicated:<br />
Dedication<br />
To the strong women in my family from my great, great grandmother<br />
VI<br />
to my sister Angela who always believes in me.
United Kingdom<br />
Acknowledgements<br />
Pr<strong>of</strong>essor Andrew Bush invited me to a research position at the Royal Brompton Hospital<br />
and has been supportive from day one for both my research career and clinical training. His<br />
own approach I seek to emulate with his excellent clinical acumen, approach to children and<br />
their families. In addition, he maintains a huge research output and when working with him in<br />
this capacity his attention to detail and editing ability is superb. I, like most <strong>of</strong> the paediatric<br />
respiratory fraternity, count Andy, and his wife sue Bush, as friends.<br />
Pr<strong>of</strong>essor Peter Barnes from the then National Heart & Lung Institute contributed valuable<br />
advice and supervised other research projects that I conducted. He rather bravely allowed me<br />
to join his laboratory as one <strong>of</strong> only three 'medics' among the 25 scientists from whom I<br />
learnt about statistics, information technology and attention to detail.<br />
Dr Seinka Dinarevic assisted with the studies on the children and provided a link to the local<br />
school from where the children were recruited.<br />
Park Walk Primary School, Iondon who were approachable regarding the research and<br />
interested in assisting the study. <strong>The</strong> children enrolled from the school and from the<br />
Brompton Hospital clinics were terrific -<br />
humorous, enthusiastic, willing to help and could<br />
always be depended upon to question some factor about the testing or the research that I had<br />
not covered with them. <strong>The</strong>y loved all the switches even more than I did.<br />
Caroline Busst was the biomedical engineer who provided the main assistance with regard to<br />
modifying the analyser as we went through and assisted with troubleshooting whenever that<br />
was necessary. She brought completely different knowledge to mine to this project from an<br />
engineering and particularly electrical engineering capacity, which complimented the clinical<br />
and practical knowledge that I was able to <strong>of</strong>fer.<br />
I would like acknowledge the great 'Fellows' that I worked with at the Royal Brompton<br />
Hospital. We were embarking on clinical and research careers and it was great to be part <strong>of</strong><br />
the group and they remain friends to this day; Paul Munyard, Lara Shekerdemian, Jane<br />
Davies, Clare Hogg, Kate Brown and Adam Jaffe.<br />
vll
New Zealand<br />
<strong>The</strong> Paediatric Department at the Faculty <strong>of</strong> Health and Medical Sciences, <strong>University</strong> <strong>of</strong><br />
<strong>Auckland</strong> all contributed particularly in the final days <strong>of</strong> submission assisting with pro<strong>of</strong>-<br />
reading and formatting.<br />
A very special thank you to Jan Tate the CF nurse specialist and friend who has always been<br />
very supportive and made my daily working life better, particularly at times <strong>of</strong> clinical<br />
overload. She also helped take the photos used in this thesis -<br />
late into the night.<br />
Pr<strong>of</strong>essor Innes Asher has always been supportive <strong>of</strong> myself in the clinical, research and<br />
teaching arenas and has helped greatly with her capacity, despite the many hats she currently<br />
wears, in <strong>of</strong>fering advice and editing comments.<br />
Dr Elizabeth Bdwards who was my first research fellow, the first Ph.D student that I<br />
supervised which she completed before me and now both colleague and friend as well as<br />
fellow netball enthusiast.<br />
Merrin Harger, project manager <strong>of</strong> subsequent research with whom I shared an <strong>of</strong>fice. She<br />
was amusing every day and left me one <strong>of</strong> her inspirational artworks when she left to embark<br />
on a new career as an artist (that we all were so talented).<br />
Mirjana Jaksic, always generous with her own clinical time, I thank for supporting me in<br />
particular by picking up the occasional extra clinic, so that I could catch up.<br />
vill
Personal<br />
My family has always been completely supportive in everything that I do and my parents,<br />
Daphne Mary Pemberton Byrnes and Brian Liston Dominic Byrnes, were thrilled to see<br />
me start at medical school all those many years ago, although, sadly, were not alive to see me<br />
graduate. And my special Aunt, Veronica Commins, who has supported my sister and myself<br />
through our careers and also, sadly, died before seeing this dedication to her years <strong>of</strong><br />
generosity. My brothers, sisters and their families all <strong>of</strong>fer their special supports - I feel lucky<br />
to be part <strong>of</strong> a large whanau, and I hope they will be pleased to see me emerge from my study.<br />
It is a fttre person who believes in you so wholeheartedly, even at times when you yourself<br />
have misgivings and I would like to thank my sister, Angela Platt-Byrnes, for ringing me<br />
every Saturday and every Sunday to ensure that I was ... and to encourage me to be ... sitting<br />
at my desk working on this thesis.<br />
And to my own support network: Dr Sue Armstrong-Wahlers, Dr Michael Wahlers and<br />
Trudi Fava.<br />
IX
Contents<br />
Abstract ........tr<br />
Dedication .................... VI<br />
Acknowledgements.... ...................VtI<br />
Contents .......X<br />
List <strong>of</strong> Tables ..............XV<br />
List <strong>of</strong> Figures ........... XVI<br />
List <strong>of</strong> Abbreviatons.............. ....XVI[<br />
Chapter l: <strong>The</strong> burden <strong>of</strong> respiratory disease in New 7-ealand.,.. ..................... 1<br />
1.1 Introduction............... ....... I<br />
I.2 <strong>The</strong> burden <strong>of</strong> paediatric respiratory disease in New 7naland.... .............2<br />
1.3 <strong>The</strong> burden <strong>of</strong> asthma disease in New 7naland..... ................. 5<br />
I.4 <strong>The</strong> diagnosis <strong>of</strong> asthma -<br />
Page<br />
the quandaries in children ...........7<br />
1.4.1 <strong>The</strong> asthma diagnosis in national and international guidelines. .......7<br />
1.4.2 What is meant by 'wheeze'?.............. ..........7<br />
1.4.3 Where does cough fit in? ......... l0<br />
1.4.4 Investigations ............. .............. 12<br />
1.5 Treatment and concerns............... ......,,.....,.....,..21<br />
1.5.1 Possible adverse effects <strong>of</strong> asthma treatment in children ..............21<br />
L.5.2 A marker for inflammation would be useful... .............23<br />
1.6 Asthma as an inflammatory disease.............. .....24<br />
1.6.1 What has been learnt from autopsy studies? ................24<br />
1.6.2 Studies <strong>of</strong> inflammation using bronchoscopy and biopsy .............25<br />
1.6.3 Studies <strong>of</strong> inflammation using bronchoalveolar lavage............... ....................29<br />
L.6.4 Inflammatory markers in induced sputum ...................31<br />
1.6.4 (i) Studies in adult subjects..... ................31<br />
1.6.4 (ii) Induced sputum in children ................34<br />
1.6.4 (iii) Technical aspects <strong>of</strong> bronchoscopy, bronchial biopsy, bronchoalveolar<br />
lavage and/or induced sputum...... ...... 36<br />
1.6.4 (iv) Safety aspects <strong>of</strong> bronchoscopy, bronchial biopsy, bronchoalveolar lavage<br />
and/or induced sputum ..... 38<br />
1.6.5 Studies <strong>of</strong> inflammatory markers in blood and urine.. ................... 41<br />
1.6.6 Comparison <strong>of</strong> inflammation results between the different samples.. .............44<br />
1.7 Chapter summary ...........45<br />
Chapter 2: Nitric oxide: pollutant to mediator .............49<br />
2.I Introduction............... .....49<br />
2.2 Pollution, nitrogen oxides and disease ...............49<br />
2.2.L Concerns regarding pollution... ..................49<br />
2.2.2 Disease and pollution............. .................... 5l<br />
2.2.3 Nitrogen oxides in pollution .... 56<br />
2.3 <strong>The</strong> discovery <strong>of</strong> nitric oxide in biological systems ............ 57<br />
2.3.1 Vascular control .....57<br />
2.3.2 Immune function ....60<br />
2.3.3 Nitric oxide -<br />
a common pathway .............61<br />
2.3.4 Nitric oxide in physiological roles .............62<br />
2.3.4 (i) Cardiovascular.......... .......62<br />
2.3.4 (ii) Nervous system - central and peripheral............. ..................64<br />
2.3.4 (iii) Host defence.............. ....... 66<br />
2.4 Chapter summary ...........68<br />
Chapter 3: <strong>The</strong> synthesis, reactivity and control <strong>of</strong> nitric oxide........ .............. 70
3.1 Introduction............... .....70<br />
3.2 Properties <strong>of</strong> nitric oxide ..................70<br />
3.3 Reactions <strong>of</strong> nitric oxide......... ..........73<br />
3.3.1 Reactive nitrogen species and superoxide reactions.............. ,........73<br />
3.3.2 Reactions with transition metars ind metalroproteins ....................75<br />
3.3.3 Nitrogen thiols and amines .......76<br />
3.3.4 Reaction with amino acids......... ..........,......77<br />
3.3.5 Reaction with lipids. ..........,......77<br />
3.3.6 Reactions with genes ................7g<br />
3.3.7 Making sense <strong>of</strong> the reactions ....................7g<br />
3.4 Nitric oxide synthase isoenzymes ............... .......7g<br />
3.4.1 Constitutive nitric oxide synthases .............g1<br />
3.4.2 Inducible nitric oxide synthase.... ...............g2<br />
3.4.3 Control <strong>of</strong> the nitric oxide synthase isoen2ymes................ ............g3<br />
3.4.3 (i) <strong>The</strong> constitutive forms ......g3<br />
3.4.3 (ii) <strong>The</strong> inducible form ...........85<br />
3.4.4 Nicotinamide adenosine di-nucleotide phosphate oxidase and inducible nitric<br />
oxide synthase ........g7<br />
3'4'5 Drug! & other agents that affect the isoenzymes and nitric oxide production. gg<br />
3.4.5 (i) Drugs ..............88<br />
3.4.5 (ii) Nitric oxide synthase inhibitors.. ........g9<br />
3.5 Chapter summary... .........91<br />
Chapter 4: Methods to measure nitric oxide ................g2<br />
4.1 Introduction............... .....g2<br />
4.2 L-arginine, L-citrulline and cyclic guanosine monophosphate................................93<br />
4.3 Methaemoglobin....... ......g3<br />
4.4 Nitrite and nitrate. ...........g4<br />
4.5 Nitric oxide........ .............95<br />
4.6 Chemiluminescence... .....g7<br />
4.6.1 Calibration ............103<br />
4.6.2 Safety and toxicity ..................104<br />
_ 4.7 Chapter summary... .......10g<br />
Chapter 5: Methodological assessment <strong>of</strong> the chemiluminescence ana1yser................... l l0<br />
5.1 Introduction ............... ... I l0<br />
5.2, <strong>The</strong> equipment and personnel.. ....... I l0<br />
5.2.I Chemiluminescence Analyser ,Model 2107, ............110<br />
5.2.2 Capnograph, pressure transducer, flow meter and chart recorder.................. I I I<br />
5.2.3 Personnel .............. I 15<br />
5.3 Direct versus reseryoir measurement .............. ................... I 16<br />
5.4 Assessments <strong>of</strong> the analysers... .......117<br />
5.5 Other measurements............ ...........120<br />
_ 5.6 Chapter summary... .......121<br />
Chapter 6: Methodological studies <strong>of</strong> exhaled nitric oxide in healthy adult subjects: direct<br />
versus t-piece testing ..............122<br />
6.1 Introduction............... ...lLz<br />
6.2 Nitric oxide and the nitric oxide synthases in the lung .......... ..............122<br />
6.3 <strong>The</strong> need for methodological experiments ............... ..........125<br />
6.4 <strong>The</strong> aims <strong>of</strong> the exhaled nitric oxide methodological experiments ...... 130<br />
6.5 Setting up for the experiments........... ...............131<br />
6.5.1 Set-up and calibrations......... ....................131<br />
6.5.2 <strong>The</strong> exhalation protocol.............. .............. 136<br />
6.5.3 Ethics and Consent................ ...................13g<br />
XI
6.5.4 <strong>The</strong> subjects............... ............. 138<br />
6.5.5 Statistical analysis .................. 138<br />
6.6 Methodological experiment one... ..139<br />
6.6.1 Hypotheses................ ............. 139<br />
6.6.2 Aims .,139<br />
6.6.3 Procedure ............. 139<br />
6.6.4 Results..... ............. 140<br />
6.6.4 (i) Direct versus t-piece nitric oxide and carbon dioxide measurement..... 140<br />
6.6.4 (ii) Peak nitric oxide versus area under the nitric oxide curye ..l4<br />
6.6.4 (iii) Comparison <strong>of</strong> exhaled nitric oxide and carbon dioxide ..... 146<br />
6.7 Discussion: <strong>The</strong> origin <strong>of</strong> the exhaled nitric oxide ............ 148<br />
Chapter 7: Methodological studies <strong>of</strong> exhaled nitric oxide in healthy adult subjects:<br />
investigating test parameters .. 153<br />
7.1 Introduction............... ... 153<br />
7.2 Ethics, subjects, calibrations and statistical analysis .............. ............. 154<br />
7.3 Methodologicalexperimenttwo-effect<strong>of</strong> expiratoryflow...... .......... 155<br />
7.3.1 Hypothesis ............ 155<br />
7.3.2 Aim.......... ............. 155<br />
7.3.3 Procedure ............. 155<br />
7.3.4 Results..... ............. 156<br />
7 .4 Methodological experiment three - effect <strong>of</strong> pressure ....... 158<br />
7.4.1 Hypotheses................ ............. 158<br />
7.4.2 Aim.......... ............. 158<br />
7.4.3 Procedure ............. 158<br />
7.4.4 Results..... ............. 160<br />
7.5 Methodological experiment four - effect <strong>of</strong> ambient nitric oxide ........ 163<br />
7.5.1 Hypothesis ............ 163<br />
7.5.2 Aims ... 163<br />
7.5.3 Procedure ............. 163<br />
7.5.4 Results..... ............. 164<br />
7.6 Methodological experiment five - effect <strong>of</strong> water consumption........................... 168<br />
7.6.1 Hypothesis ............ 168<br />
7.6.2 Aim.......... ............. 168<br />
7.6.3 Procedure ............. 168<br />
7.6.4 Results..... ............. 169<br />
7.7 Discussion: which measurement factors alter nitric oxide levels? ...... 170<br />
Chapter 8: Exhaled nitric oxide in healthy and asthmatic children ............... 178<br />
8.1 Introduction............... ... 178<br />
8.2 Ethics, consent, statistics and subjects................ ............... 179<br />
8.3 Methodology <strong>of</strong> exhaled nitric oxide measurement in healthy chi1dren................ 180<br />
8.3.1 Hypotheses................ ............. 180<br />
8.3.2 Aims ... 180<br />
8.3.3 Protocol ................ 180<br />
8.3.4 Results..... ............. 182<br />
8.4 Discussion: exhaled nitric oxide results in healthy children.... ............ 186<br />
8.5 Methodology <strong>of</strong> exhaled nitric oxide measurement in asthmatic children ............ 191<br />
8.5.1 Background............... ............. 191<br />
8.5.2 <strong>The</strong> asthmatic subjects ...........I92<br />
8.5.3 Protocol ................ 193<br />
8.5.4 Results..... ............. 193<br />
8.6 Discussion: exhaled nitric oxide in asthmatic children.. .... 196<br />
Chapter 9: Exhaled and nasal NO to today........ ........202<br />
xll
9.1 Introduction............... ...202<br />
9.2 Technical factors affecting resurts across research groups....... ............202<br />
9.3 Standardisation ............ ...................204<br />
9.3.1 Single breath online measurement.............. ................205<br />
9.3.1 (i) F1ow......... .....206<br />
9.3.1 (ii) Mouth pressure ...............211<br />
9.3.1 (iii) Nasal clips and breath_holding............ ...............212<br />
9.3.1 (iv) <strong>The</strong> recorded measurement......... ......213<br />
9.3.1 (v) <strong>The</strong> effect <strong>of</strong> ambient nitric oxide ....213<br />
9.4 Online spontaneous or tidal breathing measurement.............. ..............214<br />
9.5 OffJine measurement.............. .......216<br />
9.6 Nasal nitric oxide measurement.............. .........220<br />
9.7 Physiological alterations that may affect measurement.............. .........221<br />
9.7.1 Size and gender....... ................221<br />
9.7.2 Age .......... ,............222<br />
9.7.3 Circadian rhythm. ...,...............223<br />
9.7.4 Menstrual cycle and pregnancy ................223<br />
9.7.5 Food and beverages ................224<br />
9.7 .6 Summary <strong>of</strong> physiological factors that could alter nitric oxide levels ....,......224<br />
9.8 Nitric oxide levels in asthma and atopy ...........224<br />
9.8.1 Does nitric oxide correlate with other asthmatic inflammatory markers?......22s<br />
9.8.2 Does nitric oxide correlate with lung function and bronchial hyperresponsiveness?<br />
....................226<br />
9.8.3 Can nitric oxide be used for diagnosis <strong>of</strong> asthma?.............. .........227<br />
9.8.4 Is nitric oxide associated with symptoms and severity <strong>of</strong> asthma? ................22g<br />
9.8.5 can nitric oxide predict deterioration in asthma control? ....,.......23t<br />
9.8.6 What happens to nitric oxide during an acute asthma attack .......233<br />
9.8.7 What is the effect <strong>of</strong> atopy alone on nitric oxide?....... .................233<br />
9.8.8 Can nitric oxide be an outcome measure for assessing treatment? ................235<br />
9.8.8 (i) Corticosteroids............. ...235<br />
9.8.8 (ii) Anti-leukotriene receptor antagonists .................237<br />
9.8.8 (iii) Long acting p2 agonisrs............... .....23g<br />
9.8.8 (iv) Nedocromil sodium .......,23g<br />
9.8.8 (v) <strong>The</strong>ophylline.............. .....23g<br />
9.8.8 (vi) Novel medications................ ............23g<br />
9.8.9 Summary <strong>of</strong> nitric oxide in asthma and atopy.. ..........240<br />
9.9 Nitric oxide levels in primary ciliary dyskinesia................... ...............241<br />
9.10 Nitric oxide levels in cystic fibrosis.................. .................242<br />
9.ll Nitric oxide levels in bronchiectasis......... ........245<br />
9.12 Nitric oxide levels in upper respiratory tract infections.......... .............245<br />
9.13 Nitric oxide levels in chronic obstructive pulmonary disease (copD).... ..............246<br />
9.14 Nitric oxide levels in smokers ........247<br />
9.15 Nitric oxide levels in interstitial lung diseases ...................24g<br />
9.16 Nitric oxide levels in exercise ........250<br />
9.17 Nitric oxide measurements in infants ...............252<br />
9.17.1 Methodology.............. .............252<br />
9.17.2 lrvels <strong>of</strong> nitric oxide in infants with different respiratory diseases ...............256<br />
9.17 .3 Prenatal and maternal effects on nitric oxide levet........:......... ...257<br />
9.17.4 Effects <strong>of</strong> treatment on nitric oxide levels........ ..........257<br />
9.17.5 Summary <strong>of</strong> nitric oxide findings in infants.. .............257<br />
_ 9.18 Chapter surnmary... .......25g<br />
Chapter l0: Reflections ............262<br />
xill
XIV
List <strong>of</strong> Tables<br />
Table 1.1: Alternative diagnoses in children with whee2e............... ........................g<br />
lable 3.1: Properties <strong>of</strong> nitric oxide .................71<br />
Table 3.2: Reactions with nitric oxide ..............72<br />
Table 3.3: <strong>The</strong> characteristics <strong>of</strong> the nitric oxide synthase isoen2ymes................................... g1<br />
Table 4.1: <strong>The</strong> companies providing chemiluminescence analysers adaptable for nitric oxide<br />
measurement in 1995. ...................101<br />
Table 5.1: <strong>The</strong> delay and response time <strong>of</strong> the NO, COz, mouth pressure and flow meter<br />
analysers used.......... ...llg<br />
fu9l" 6.1: <strong>The</strong> published results <strong>of</strong> exhaled nitric oxide in adults.... ...................127<br />
full" 6.2: Compete results for an individual subjecr .......143<br />
Table 6.3 NO and COz results from single exhalitions in twelve adult subjects............. ......143<br />
Table 7.1: NO, COz and duration <strong>of</strong> each exhalation at different expiratory flows...............157<br />
Table 7.2: Comparison <strong>of</strong> the first and repeated last set <strong>of</strong> exhalation, uf the same expiratory<br />
flow""""" ...................16r<br />
Table 7.3: NO, COz and duration <strong>of</strong> each exhalation at different expiratory mouth pressures<br />
.....,....,,,.,.., |62<br />
Table 7.4: Comparison <strong>of</strong> the first and repeated last set <strong>of</strong> exhalations performed at the same<br />
SPt]afory mouth pressure ............162<br />
Table 7.5: Exhaled NO results with high and low background NO concentrations...............l6g<br />
Table 7.6: Exhaled COz results with high and low background NO concentrations .............16g<br />
Table 8.1: Coefficients <strong>of</strong> variation <strong>of</strong> peak NO, peak COz and mouth pressure measurements<br />
made by both the direct and rpiece systems, and <strong>of</strong> flow made by the rpiece system......... lg5<br />
Table 9.1: <strong>The</strong> recommended standard for single breath online Nb meaiur.-"nt ...............206<br />
Table 9.2: <strong>The</strong> recommended standards for single breath and tidal breathing <strong>of</strong>f-line NO<br />
measurement.............. ..,,,...,,,,.....,.21g<br />
Table 9.3: <strong>The</strong> recommended standard for nasal No measurement...... ...............220<br />
XV<br />
Page
List <strong>of</strong> Figures<br />
Figure l.l: Top l0 causes <strong>of</strong> avoidable admissions, 0-24 years, 1999.......... .......... 3<br />
Figure 2.1: Diagram <strong>of</strong> blood vessels in cross section showing the endothelial layer. ...........59<br />
Figure 3.1: <strong>The</strong> molecular structure <strong>of</strong> NO....... ................. 70<br />
Figure 3.2: <strong>The</strong> reaction to generate nitric oxide ...............71<br />
Figure 3.3: <strong>The</strong> nitric oxide synthase reaction showing the generation <strong>of</strong> the constituent atoms<br />
<strong>of</strong> nitric oxide........ ....... 80<br />
Figure 3.4: <strong>The</strong> chemical structures <strong>of</strong> the nitric oxide synthase inhibitors ..........90<br />
Figure 4.1: Chemical reaction between nitric oxide and ozone. .......... 98<br />
Figure 4.2:Diagram <strong>of</strong> the chemiluminscence analyser.... .................. 99<br />
Figure 4.3: Relationship between chemiluminscence in millivolts to nitric oxide concentration<br />
in picomo1es.............. ................... 100<br />
Figure 4.4: Reaction equations used to release nitric oxide from other nitrogen compounds 103<br />
Figure 5.1: Schematic diagram <strong>of</strong> how the analysers were placed .... Llz<br />
Figure 5.2: An example <strong>of</strong> tracing from the testing....... .. 113<br />
Figure 5.3: Photographs <strong>of</strong> one <strong>of</strong> the children performing the exhalation........................... I 16<br />
Figure 6.1: Recording <strong>of</strong> the calibrations for NO, CO2 and pressure ana1ysers.................... 133<br />
Figure 6.2: Recording <strong>of</strong> the calibration <strong>of</strong> the flow rotameter and pneumotachograph....... 135<br />
Figure 6.3: Schematic diagrams <strong>of</strong> the two different types <strong>of</strong> connections: direct and t-piece<br />
measurements............. .................. 137<br />
Figure 6.4: Example <strong>of</strong> the chart recording rolls for the direct versus t-piece measurements.<br />
140<br />
Figure 6.5: Example <strong>of</strong> recording on one subject -<br />
Page<br />
direct and t-piece measurements .......... 141<br />
Figure 6.6: Mean exhaled NO levels measured by direct and t-piece systems......................I42<br />
Figure 6.7: Example <strong>of</strong> a recording for the two systems............... .... L42<br />
Figure 6.8: Mean exhaled COz levels measured by direct and t-piece systems..... ................ I44<br />
Figure 6.9: Comparisons <strong>of</strong> the peak NO with area under the curve... ................ 145<br />
Figure 6.10a: Time to peak NO and peak CO2 measured by the direct system..................... 146<br />
Figure 6.10b: Time to peak NO and peak CO2 measured by the t-piece system...................147<br />
Figure 6.1Ia: COz levels at peak NO and at peak CO2 measured by the direct system ........147<br />
Figure 6.1Ib: CO2 levels at peak NO and at peak CO2 measured by the t-piece system ...... 148<br />
Figure 7.1: Exhaled NO results with increasing expiratory flows .....I57<br />
Figure 7.2:Example <strong>of</strong> the tracing in one subject at two different expiratory flows ............ 158<br />
Figure 7.3: <strong>The</strong> effect <strong>of</strong> pressure on the calibration gas measurement ......... ..... 160<br />
Figure 7.4: Exhaled NO results at increasing expiratory mouth pressure ........... 161<br />
Figure 7.5: Measurement <strong>of</strong> exhaled NO incorporating the reservoir system ..... 165<br />
Figure 7.6: Photographs <strong>of</strong> the changing ambient NO concentration recordings.................. 166<br />
Figure 7.7: Recording from one subject showing NO results with exhalations from low and<br />
high background NO.... ................ 167<br />
Figure 7.8: <strong>The</strong> effect <strong>of</strong> high and low pressure on the calibration gas measurement........... 167<br />
Figure 7 .9: <strong>The</strong> effect <strong>of</strong> consuming water on the subsequent exhaled NO levels measured 170<br />
Figure 7.10: Hyperbolic relationship between exhaled NO and sampling flow rate.............172<br />
Figure 7.11: Exhaled NO determined at single breath plateau concentrations at increasing<br />
expiratory flows and at increasing expiratory mouth pressures... ......173<br />
Figure 8.1: Peak exhaled NO results in healthy children.... ............... 183<br />
Figure 8.2a: Comparison <strong>of</strong> peak exhaled NO in boys and girls measured by the direct system.<br />
184<br />
Figure 8.2b: Comparison <strong>of</strong> peak exhaled NO in boys and girls measured by the t-piece<br />
system......<br />
................... 184<br />
XVI
Fig-ure 8.3a: Mean peak exhaled No levels in control and asthmatic children measured direct<br />
to the analysers ...........1g4<br />
Figure 8.3b: Mean peak exhaled No levels in control and asthmatic children measured via the<br />
t-piece sampling system ...............195<br />
Figure 8.4a: <strong>The</strong> "fl:91<strong>of</strong> commencing inhaled corticosteroid therapy on peak exhaled NO<br />
levels in asthmatic children measured via the direct method .............196<br />
Figure 8.4b: <strong>The</strong> tfl.9t. <strong>of</strong> commencing inhaled corticosteroid therapy on peak exhaled NO<br />
levels in asthmatic children measured via the t-piece sampling sysrem....... ........196<br />
Figure 9.1: A two-compartment model <strong>of</strong> NO eichange._............... ...................20g<br />
Figure 9.2:Plateau nasal NO increasing with ug"............ ..................223<br />
ligure 9.3: Diagram <strong>of</strong> the measurement <strong>of</strong> lung function and NO in an infant................. ...253<br />
:i::::::::..Y:ill:. publications with a focui on nirric oxide research per year re80_2006<br />
,,.....,........262<br />
XVII
List <strong>of</strong> Abbreviations<br />
ABPA allergic bronchopulmonary dysplasia<br />
ATS American Thoracic Society<br />
ATP adenosine triphosphate<br />
BAL bronchoalveolar lavage<br />
BH4 tetrahydrobiopterin<br />
BTS British Thoracic Society<br />
C3, C5 complement factor 3, complement factor 5<br />
cAMP cyclic adenosine 3',5'monophosphate<br />
CATI, CAT2, CAT2B, CAT 3<br />
cationic amino acid proteins<br />
CB chronic bronchitis<br />
CD3+ cell marker for T lymPhocyte<br />
CD4+ cell marker for T helper lymphocyte<br />
CD8+ cell marker for T cytoxic lymphocyte<br />
CF cystic fibrosis<br />
cGMP cyclic guanosine monophosphate<br />
cNOS constituent nitric oxide synthase<br />
CNS central nervous system<br />
Co cobalt<br />
CO carbon monoxide<br />
COz carbon dioxide<br />
COPD chronic obstructive pulmonary disease<br />
CSF central spinal fluid<br />
Cu copper<br />
DMSO dimethyl sulfoxide<br />
DNA deoxyribonucleic acid<br />
DTT dithiothreitol<br />
ECMO extracorporeal membrane oxygenation<br />
ECP eosinophil cationic protein<br />
ECRHS European Community Respiratory Health Survey<br />
EDRF endothelial derived relaxing factor<br />
XVIII
ELISA Enzyme-linked immunosorbent assav<br />
eNOS endothelial nitric oxide synthase<br />
EPNIEPX eosinophilic neuraminadase<br />
EPO eosinophilic peroxidise<br />
ERS European Respiratory Society<br />
ERSTF European Respiratory Society Task Force<br />
FAD flavin adenosine dinucleotide<br />
Fe iron<br />
FBFzs-tsn forced expiratory flow at 25 to 75 percent <strong>of</strong> forced vital capacity<br />
FEVr forced expiratory volume in 1 second<br />
FEVo.s forced expiratory volume in half a second<br />
FMN flavin mononucleotide<br />
FVC forced vital capacity<br />
GINA Global Initiative for Asthma<br />
GM-csF Granulocyte macrophage - colony stimulating factor<br />
GTP guanosine S-triphosphate<br />
Hzo<br />
HzS hydrogen sulphide<br />
HNO2 nitrous acid<br />
HRCT high resolution computerised tomography<br />
ICAMI intracellular adhesion molecule I<br />
IFNI interferon gamma<br />
IgA immunoglobulin A<br />
IgG immunoglobulin G<br />
IgE immunoglobulin E<br />
IHCS inhaled corticosteroids<br />
nl, L2, IL3, nA, IL5, IL6, ILg, ILg, ILl0, ILI l, I[-l2, ILl3, Ll7<br />
interleukin l, interleukin 2, interreukin 3, interleukin 4, interleukin 5,<br />
interleukin 6, interleukin 8 interleukin 9, interleukin 10, interleukin<br />
I l, interleukin 12, interleukin 13, interleukin l7<br />
iNOS inducible nitric oxide synthase<br />
ISAAC Inbrnational Study <strong>of</strong> Asthma and Allergies in Childhood<br />
Kb kilobases<br />
Umin litres per minute<br />
LABA long acting 92 agonisr<br />
xrx
LADA N*N* dimethyl L-arginine<br />
LFA1 lympocyte function-associated antigen I<br />
L-NAME N* arginine methyl ester<br />
L-NMMA N* monomethyl L-arginine<br />
L-NOARG N*-nitroarginine<br />
LNIO N- imino-ethyl-ornithine<br />
LPC lysophosphotidylcholine<br />
LPS lipopolysaccharide<br />
mC4, LTD4, WM leukotriene C4,leukotriene D4,leukotriene Bl<br />
MBP major basic protein<br />
mg/ml milligrams per millilitre<br />
mls/min millilitres per minute<br />
Mn manganese<br />
mRNA messenger ribonucleic acid<br />
Nz nitrogen<br />
NzO nitrous oxide ("laughing gas")<br />
NzOs dihydrogen trioxide<br />
NzOl dihydrogen tetraoxide<br />
NADPH nicotinamide adenosine di-nucleotide phosphate<br />
NANC non adrenergic, non cholinergic (nerves)<br />
NF-KB nuclear factor-kappa B<br />
NHANES 1 National Health and Nutrition Examination Survey I<br />
nl-/min nanolitres per minute<br />
nNOS neuronal nitric oxide synthase<br />
NO nitric oxide<br />
NO + nitrosoium cation<br />
NO- nitroxyl anion<br />
NOz nitrogen dioxide<br />
NOz- nitrite<br />
No:- nitrate<br />
NO2Tyr 3-nitrotyrosine<br />
NOS nitric oxide synthase/s<br />
NOx nitrogen oxides (usually NO, NOz and NO:)<br />
NZPAG New Zealand Paediatric Asthma Guidelines<br />
Oz oxygen
O2-<br />
Or<br />
oH-<br />
oNoo-<br />
ONOOH<br />
PaOz<br />
PCD<br />
PCzo<br />
PDzo<br />
PEF<br />
Pmo<br />
ppb<br />
ppm<br />
PMIO<br />
redox<br />
ROC<br />
RS<br />
RSV<br />
SD<br />
SEM<br />
SGC<br />
SIGN<br />
SLE<br />
Soz<br />
TB<br />
rGFp<br />
Tnl<br />
Ts2<br />
Ts3<br />
TNFa<br />
TI.[FP<br />
type I (nNOS)<br />
type II (iNOS)<br />
superoxide anion<br />
ozone<br />
hydroxyl anion<br />
peroxynitrite<br />
peroxy nitrous acid<br />
pulmonary artery oxygen<br />
primary ciliary dyskinesia<br />
provocation concentration producing20vo fall in the forced expiratory<br />
volume in one second<br />
provocation dose producing 20vo fall in the forced expiratory volume<br />
in one second<br />
peak expiratory flow<br />
mouth pressure<br />
parts per billion<br />
parts per million<br />
particulate matter with a diameter <strong>of</strong> less than 10pm<br />
reduction oxidative reactions<br />
recei ver-operator curve<br />
sulphur thios<br />
Respiratory Syncytial Virus<br />
standard deviation<br />
standard error <strong>of</strong> the mean<br />
soluble guanylate cyclase<br />
Scottish Intercollegiate Guidelines Network<br />
systemic lupus erythematosis<br />
sulphur dioxide<br />
tuberculosis<br />
transforming growth factor beta<br />
T helper lymphocyte cell type I<br />
T helper lymphocyte cell type 2<br />
T helper lymphocyte cell type 3<br />
tumour necrosis factor alpha<br />
tumour necrosis factor beta<br />
Type I neuronal nitric oxide synthase<br />
Type II induced nitric oxide synthase<br />
XXI
t}ryem(eNoS)Typeltrendothelialnifticoxidesynthase<br />
LrK<br />
pgnn<br />
LJMCEF<br />
USA<br />
W light<br />
V/Q<br />
7^<br />
United Kingdom<br />
micrograms Pre millilitre<br />
UnitedNations International Children's Fund<br />
United States <strong>of</strong> America<br />
ultraviolet light<br />
ventilation and perfusion ratio<br />
zinc<br />
xxll
1.1<br />
chapter 1: <strong>The</strong> burden <strong>of</strong> respiratory disease in New znaland<br />
Introduction<br />
This opening chapter will set the scene as to why I felt that this area <strong>of</strong> research was <strong>of</strong><br />
importance and <strong>of</strong> interest. <strong>The</strong> burden <strong>of</strong> respiratory disease in New Tnaland is high, and<br />
clearly apparent to anyone who trains or works here in the field <strong>of</strong> medicine. It was an<br />
obvious problem as I came through the <strong>Auckland</strong> School <strong>of</strong> Medicine, it was obvious when I<br />
chose a rural setting to do my first house year, and it remained obvious when I commenced<br />
training in paediatrics. <strong>The</strong> burden appeared to be borne disproportionately by children, and<br />
by children <strong>of</strong> two specific community groups - Maori and Pacific Island. In the l9g0s and<br />
1990s, New Zealand was leading the world in both the incidence <strong>of</strong> asthma and, more<br />
appallingly, asthma mortality. Although the diagnosis <strong>of</strong> asthma in children was not always<br />
clear, we embarked on anti-inflammatory drug treatment, possibly for years. While asthma<br />
was thought to be a reversible disease, it has since transpired that an element <strong>of</strong> irreversibility<br />
could develop which has been termed 'airway remodelling'.<br />
This thesis commenced ten years ago and is based on work I conducted while employed as a<br />
fellow and senior registrar and lecturer at the Royal Brompton Hospital in london. My<br />
interest, then as now' was in paediatric respiratory disease. I was concerned that we seemed to<br />
be making treatment decisions regarding the use <strong>of</strong> potential toxic anti-inflammatory agents,<br />
specifically the use <strong>of</strong> steroids, without measuring any inflammatory parameter. Rather, we<br />
were using history, examination and other surrogate measures such as x-rays and lung<br />
function (where possible and where children were old enough) to determine correct diagnosis<br />
and response to treatment. So for the individual child; was the diagnosis correct? Was the<br />
child on too much or unnecessary medication? - a treatment with potentially significant side<br />
effects, such as adrenal suppression and growth failure. Or was the child on too little<br />
medication? - suffering symptoms daily with the possible development <strong>of</strong> irreversible<br />
disease, or even death. I saw many children in the clinic that were in both categories; on ..too<br />
much" steroid therapy for too long incorrectly, on "too little" with unnecessary morbidity, and<br />
so very few seemed'Just right". I planned and conducted the research outlined in this thesis,<br />
which I saw as a unique opportunity to investigate a possible solution to this quandary. <strong>The</strong><br />
research presented is thus now historical. It has already contributed to medical literature,<br />
including the formulation by the European Respiratory Society and American Thoracic<br />
Society <strong>of</strong> standardized procedures for the measurement <strong>of</strong> exhaled nitric oxide (NO), the<br />
marker I chose to explore to indicate airway inflammation. I have continued to research this
area, some <strong>of</strong> it has been expanded from the work presented here, and in these experiments I<br />
dotted the 'i's, crossed the't's and was hands on adapting the individual machinery. It remains<br />
eligible to present and I have lived with it so long that I am pleased to do so. This thesis<br />
presents the research leading to the experiments that I undertook and describes the<br />
development <strong>of</strong> NO as a measure <strong>of</strong> inflammation during this time and subsequently to the<br />
end 2006.<br />
In this chapter I review the burden <strong>of</strong> respiratory disease in paediatrics in New Zealand. This<br />
review was part <strong>of</strong> a monograph edited by myself and Pr<strong>of</strong>essor Innes Asher, and in which I<br />
also wrote two chapters. This will be followed by a brief overview <strong>of</strong> asthma, the difficulty <strong>of</strong><br />
accurate diagnosis and the background to it being designated an inflammatory disease. <strong>The</strong><br />
following chapter will present how NO was considered a pollutant, then became recognised as<br />
a vital biological agent, and then known as an inflammatory marker. In Chapter 3, the<br />
characteristics <strong>of</strong> NO will be examined as these make it a difficult gas to measure. Chapter 4<br />
will discuss the possible options for measuring NO and in Chapter 5 I will describe how a<br />
machine that measured airway pollution became adapted to measure exhaled air. In Chapters<br />
6 and 7 the feasibility <strong>of</strong> measuring NO in exhaled air in adults was assessed; particularly<br />
exploring what factors affected the NO readings. <strong>The</strong>se then describe the standardized method<br />
I developed which was used as presented in Chapter 8 to enable exhaled NO in healthy<br />
children to be measured and in asthmatic children on bronchodilator or steroid treatment, as<br />
well as a smaller goup that was commencing steroid therapy. <strong>The</strong> penultimate chapter,<br />
Chapter 9 will be devoted to reviewing the literature regarding NO to the present day, and<br />
where this investigation is now best utilized. <strong>The</strong> final chapter, Chapter 10, will be a brief<br />
commentary what I have learnt through the process <strong>of</strong> this research and writing.<br />
I hope you enjoy this story.<br />
1.2<br />
<strong>The</strong> burden <strong>of</strong> paediatric respiratory disease in New Zealand<br />
<strong>The</strong> 'Trying to Catch Our Breath' monograph (Anonymous 2006) published last year was an<br />
initiative by twelve leaders from the New Zealand paediatric health community, supported by<br />
the Asthma and Respiratory Foundation <strong>of</strong> New 7*aland (Te Taumatua Huango, Mate Ha o<br />
Aotearoa), to document and present the darming data that existed on the incidence,<br />
prevalence and the severity <strong>of</strong> respiratory disease in children. It was presented to the Right<br />
Honourable Mr Peter Hodgsen, Minister for Health in April 2006 by the Asthma and<br />
Respiratory Foundation <strong>of</strong> New 7*aland (Te Taumatua Huango, Mate Ha o Aotearoa). <strong>The</strong><br />
title <strong>of</strong> the document was deliberately ambiguous as the group also feel breathless in trying to<br />
2
deal with the sheer numbers <strong>of</strong> children requiring assessment and treatment with these<br />
disorders. <strong>The</strong> rates <strong>of</strong> most respiratory conditions are higher than comparable westernized<br />
countries and the severity <strong>of</strong> these conditions more significant. Much <strong>of</strong> this is preventable. In<br />
1999, '<strong>The</strong> Top Ten Report' (Graham, lrversha et al. 2001) detailed the top ten issues<br />
affecting the health and well-being <strong>of</strong> children and young people in <strong>Auckland</strong> and Waikato,<br />
including a review <strong>of</strong> the top ten causes <strong>of</strong> potentially avoidable hospital admissions in New<br />
Zealanders agedO-24 years (see Figure Ll).<br />
Figure 1.1: Top 10 causes <strong>of</strong> avoidabre admissions, 0-24 years, 1g9g<br />
Urlnary Infecilon<br />
Convulsions<br />
ENT Infections<br />
Gastroenteritis<br />
bronchlolltls<br />
Pneumonla<br />
]ake1 from the.'T-rying to catch our Breath monograph (Anonymous 2006) but adapted from ,<strong>The</strong><br />
Igp_f"! Report' (Graham, Leversha et at. 2001).<br />
ENT = Ear, nose and throat<br />
over half <strong>of</strong> these potentially avoidable admissions were respiratory or ear, nose and throat<br />
conditions' Disease is exacerbated by the proportion <strong>of</strong> children living in poverty (which is<br />
defined as living in a household with an income below 6ovo <strong>of</strong>the median family income net<br />
<strong>of</strong> housing costs). This has increased from l6vo in r987/r98g to 2gEo in 20ffi12001 (Ministry<br />
<strong>of</strong> Social Development2N2).Indeed New Zealand has one <strong>of</strong> the worst rates <strong>of</strong> child poverty<br />
in rich countries (UNICEF 2005). <strong>The</strong> burden <strong>of</strong> disease particularly impacts on certain ethnic<br />
groups within New Zealand. Maori children currently make up 25vo <strong>of</strong> all children living in<br />
New Zealand and, as a collective group, Pacific peoples make up 6.5vo <strong>of</strong> the New Zealand<br />
population' However both <strong>of</strong> these groups have significantly higher proportions <strong>of</strong> children<br />
aged less than fourteen yeius <strong>of</strong> age - 35vo <strong>of</strong> Maori children (Te puni Kokiri 2oo2) and 3gvo<br />
<strong>of</strong> Pacific children (Statistics New Zealand 2002) compared to 20vo <strong>of</strong> total New Zealanders.
In addition, both <strong>of</strong> these groups are disadvantaged with regard to disposable income,<br />
adequate housing, educational opportunities, unemployment rates and access to healthcare<br />
(Ministry <strong>of</strong> Health 2002; Statistics New Zealand2002; Asher 2006).<br />
A review <strong>of</strong> the statistics for the different paediatric respiratory conditions follows. Nationally<br />
the admission rate for children < one year <strong>of</strong> age for lower respiratory tract infection,<br />
predominantly bronchiolitis and pneumonia, is L02.61t000 but up to 176.5/1000 in certain<br />
regions (Graham, Irversha et al. 2001). <strong>The</strong> rates <strong>of</strong> admission for bronchiolitis for children <<br />
one year <strong>of</strong> age have increased from 26.611000 children in 1988 to 58.U1000 in 1998, an<br />
increase <strong>of</strong> ll87o (Vogel, knnon et al.2003). In addition, this increase in admission rates is<br />
accompanied by more severe disease in those admitted in comparison to the previous decade<br />
and in comparison to paediatric admissions <strong>of</strong> bronchiolitis in other developed countries. Of<br />
the children admitted; 59Vo required oxygen, 2lVo required nasogastric fluids, 22Vo<br />
intravenous fluids, 8Vo werc admitted with apnoea and 3.LVo reeuired ventilation (Vogel,<br />
lrnnon et al. 2003). Pneumonia continues to be a worldwide problem with acute lower<br />
respiratory tract infections being an important cause <strong>of</strong> mortality in children < five years <strong>of</strong><br />
age and remains one <strong>of</strong> the leading causes <strong>of</strong> disability-adjusted life years lost worldwide.<br />
This largely reflects children in developing countries where it is estimated that 4.3 million<br />
children aged < five years die annually from lower respiratory tract infections (Garenne,<br />
Ronsmans et al. t992). <strong>The</strong> incidence tends to be higher in children < five years <strong>of</strong> age at 34-<br />
40 cases per 1000 compared to any other age group except possibly for adults > 75 years<br />
(Mclntosh 2002). A recent study conducted in New 7-ealand ascertained that the national<br />
pneumonia admission rate for children 0-14 years <strong>of</strong> age was 4.0/1000 based on the discharge<br />
diagnoses for pneumonia in 1998 to 1999 (Milne,Irnnon et al. 2003). <strong>The</strong>se admissions were<br />
skewed toward the younger age group. This equated to t,534 admissions per 100,000 for<br />
children aged < two years, 562 admissions per 100,000 for children aged 2-4 yeus and 170<br />
admissions for children aged 5-9 years. Extrapolation <strong>of</strong> this data amounts to approximately<br />
3000 paediatric hospital admissions for pneumonia in New Tx,aland per year. Furthermore,<br />
from 1988 to 1995 there was an annual increase <strong>of</strong> SVo in the hospital admission rate. This<br />
increase was not due to admitting children with less severe disease; indeed the disease<br />
severity also seemed to be on the rise (Ministry <strong>of</strong> Health 1998; Grant 2006). Even pertussis,<br />
which is largely preventable by immunisation, continues to be a significant problem. Reported<br />
coverage <strong>of</strong> pertussis immunisation shows that only 60Vo <strong>of</strong> children are fully immunised,<br />
with only 42Vo <strong>of</strong> Maori and 45Vo <strong>of</strong> Pacific children completing the recommended
immunisation schedule (Ministry <strong>of</strong> Health 1992; Grant 2000; Grant, pati et al.211l;Turner<br />
2006).<br />
Bronchiectasis has been decreasing in most developed countries but this does not appear to be<br />
the case in New Zealand.In studies that I led, subsequent to the research presented in this<br />
thesis, a prospective investigation was conducted utilising the 'New Zealand paediatric<br />
surveillance Unit', covering 947o <strong>of</strong> paediatricians nationally. This requested notification and<br />
subsequent data on any new diagnosis <strong>of</strong> bronchiectasis made in children < 15 years through<br />
2001 and 2002 (Twiss, Metcalfe et al. 2005). <strong>The</strong> incidence from this study put bronchiectasis<br />
at 3'7/100,000/year, which is seven times greater than the only other comparable national<br />
study done in Finnish children (Sayajakangas, Keistinen et al. 1998). This equates to l/1700<br />
live born children being diagnosed with bronchiectasis before the age <strong>of</strong> 15 years. If the<br />
incidence rate was to remain static and all these children survived to 15 years <strong>of</strong> age, this<br />
would equate to a prevalence <strong>of</strong> 1/3000 children overall. Again a disparity is seen with the<br />
incidence found to be three times higher in Maori children (l/1700) and twelve times higher<br />
in Pacific Island children (1/650) than those with European ethnicity. As well as being<br />
common, the disease was severe as demonstrated in both the <strong>Auckland</strong> study @dwards, Asher<br />
et al. 2003) and the National study (Twiss, Metcalfe et al. 2005) with g3-93 Vo havingbilateral<br />
disease, and 6l-64%o having three or more <strong>of</strong> the six lobes <strong>of</strong> the lung involved. In addition<br />
the incidence <strong>of</strong> tuberculosis (TB) has decreased for adults in New 7*aland,over the recent<br />
years' but disturbingly there has been no such reduction in paediatric rates as demonstrated by<br />
our review <strong>of</strong> paediatric TB cases through the 1990s (Howie, Voss et al. 2005). Again<br />
significant ethnic disparities occur in these rates; in children < 15 years, pacific children<br />
account for lTVo <strong>of</strong> all cases <strong>of</strong> TB and Maori children for l5vo compared to Europea n at 3Vo.<br />
Unsurprisingly, this condition is also associated with socioeconomic deprivation (Ministry <strong>of</strong><br />
Health 2002).<br />
<strong>The</strong>se reports all confirm the extent <strong>of</strong> paediatric respiratory disease in New Zealand,and the<br />
fact that they do not appear to be on the wane despite the improving statistics seen in other<br />
developed and comparable countries.<br />
1.3<br />
Asthma is one <strong>of</strong> the most common chronic diseases in the world with an estimated 300<br />
million people affected and the number <strong>of</strong> disability-adjusted life years lost estimated at<br />
approximately 15 million per year (GINA 2O04; GINA zOO5). Asthma has also had a<br />
demonstrated increase in prevalence in the 1980s and 1990s as evidenced by a number <strong>of</strong><br />
5
studies conducted throughout the United Kingdom, Australia and New 7-ealand, (Robertson,<br />
Heycock et al. l99l; Peat, van den Berg et al. 1994; Rona, Chinn et al. 1995; Mitchell and<br />
Asher 1997; Asher and Grant 2006). In the 'Global Burden <strong>of</strong> Asthma' report (GINA 2004)<br />
the proportion <strong>of</strong> the population said to have 'clinical' asthma in New Zealand is given as<br />
l1.l%o and in England (the country I did the research reported in this thesis) is given as<br />
l5.3%o. <strong>The</strong> case fatality rate for asthma is 4.6/100,000 asthmatics in New Zealand and<br />
3.211N,000 asthmatics in England. In the 2006 ranking for asthma mortality from one<br />
(highest) to 68 (lowest) New Zealand appears at 17 and England at 26. <strong>The</strong> International<br />
Study <strong>of</strong> Asthma and Allergies in Childhood (ISAAC), (Weiland, Bjorksten et al.2004) and<br />
the European Community Respiratory Health Survey (ECRHS), (Anonymous 1996) asked<br />
about self reported wheezing in the previous twelve months as one <strong>of</strong> their core questions,<br />
having demonstrated this had good specificity and sensitivity for diagnosis <strong>of</strong> asthma. In 13-<br />
14 year old children, England and New Tnaland were ranked six and seven out <strong>of</strong> 84 countries<br />
where the listing was from one being the highest percentage <strong>of</strong> positive responses to 84 being<br />
lowest percentage <strong>of</strong> positive responses (Anonymous 1998). Furthermore, New Zealand<br />
ranked second when the symptom prevalence <strong>of</strong> wheeze in 13-14 year olds was determined<br />
by responses to a video questionnaire in 44 countries (Anonymous 1998).<br />
In addition to community prevalence, asthma is a major cause <strong>of</strong> hospital admissions for<br />
children, particularly noticeable in younger age groups. Admission rates had been increasing<br />
through the 1970s, 1980s and early 1990s (Anderson, Bailey et al. 1980; Jackson and Mitchell<br />
1983; Mitchell 1985; Hyndman, Williams et al.1994) with possible stabilisation or reduction<br />
in the most recent years (Kemp and Pearce t997; British Thoracic Society 20OI; Akinbami<br />
and Schoendorf 2002). <strong>The</strong>re were two major epidemics <strong>of</strong> asthma mortality and New<br />
7-ealand featured highly in both. <strong>The</strong> first mortality increase was observed in several countries<br />
through the 1960s and included Australia, England, Norway, Scotland, Wales and New<br />
7*,aland. <strong>The</strong> second epidemic was seen in New 7*,aland alone when mortality rates reached<br />
4.1/100,000, which appeared linked to the use <strong>of</strong> the relatively non-selective B-2 agonist<br />
'Fenoterol'. A 'Fenoterol' case control study was undertaken which looked at ll7 patients<br />
between 5-45 years dyrng <strong>of</strong> asthma between 1981 and 1983. Each index case was matched<br />
with four controls and the use <strong>of</strong> 'Fenoterol' gave a relative risk <strong>of</strong> death <strong>of</strong> 1.59, slightly<br />
higher in females at 1.65 compared to males at l.M. However it was greater in the patients<br />
who were less than 20 years <strong>of</strong> age with an odds ratio <strong>of</strong> 2.08 (Crane, Pearce et al. 1989).<br />
Fenoterol was subsequently withdrawn from the drug tariff and, coinciding with the greatly<br />
reduced use after 1990, there was a significant reduction in the number <strong>of</strong> deaths (Crane,
Pearce et al. 1995). Despite the stabilization in the last few years and the reduction in previous<br />
high mortality, asthma remains a prevalent paediatric disease (Kercsmar 2006; Weinberger<br />
and Abu-Hasan 2006).<br />
t.4<br />
L4.I <strong>The</strong> asthma diagnosis in national and international guidelines<br />
Prior to the difficulties regarding treatment, particularly in children, the first hurdle is making<br />
a correct diagnosis <strong>of</strong> asthma which cornmences by having an appropriate definition. <strong>The</strong><br />
2005 'British Guideline on the Management <strong>of</strong> Asthma' from the British Thoracic Society &<br />
Scottish Intercollegiate Guidelines Network (SIGN) guideline states; ,.<strong>The</strong> diagnosis <strong>of</strong><br />
asthma is a clinical one; there is no confirmatory diagnostic blood test, radiographic or<br />
histopathological investigation. In some people a diagnosis can be corroborated by suggestive<br />
changes in lung function tests" (SIGN 2005). <strong>The</strong> problem <strong>of</strong> accurate diagnosis appears to<br />
have existed for many decades. "<strong>The</strong> clinical diagnosis <strong>of</strong> asthma is not always simple and the<br />
absence <strong>of</strong> an agreed definition <strong>of</strong> the disease is a problem with many descriptions existing,,<br />
appeared in 1959 from a conference on 'terminology' (Anonymous 1959). <strong>The</strong> difficulties <strong>of</strong><br />
diagnosis and differential diagnoses are apparent in the paediatric section <strong>of</strong> the SIGN<br />
guideline and in two paediatric asthma guidelines published in 2005; 'pocket Guide for<br />
Asthma Management and Prevention in Children' from the 'Global Initiative for Asthma,<br />
(GINA) (GINA 2005), and the 'Management <strong>of</strong> Asthma in Children aged I -<br />
15 years' from<br />
the Paediatric Society <strong>of</strong> New 7*aland (NZPAG) (Paediatric Society <strong>of</strong> New Tnaland2005).<br />
Ultimately these publications agree that it is a clinical diagnosis based on a history <strong>of</strong><br />
appropriate symptoms <strong>of</strong> wheeze, which varies over time, improving either spontaneously or<br />
as a result <strong>of</strong> treatment and with wheezeheard, during times <strong>of</strong> exacerbation. However all<br />
indicate this should occur in the absence <strong>of</strong> other features that could suggest congenital,<br />
suppurative or atypical respiratory disease, or is not responsive to asthma treatment.<br />
L4.2 Wat is meant by 'wheeze'?<br />
Even here there are at least two difficulties. Firstly, what is meant by 'wheeze' and secondly,<br />
that not all wheeze is asthma. Parental reporting <strong>of</strong> wheezing does not always coincide with<br />
what doctors mean by wheezing (Cane, Ranganathan et al. 2000;Cane and McKenzie Z0(lI).<br />
Of parents presenting with infants with a problem <strong>of</strong> 'noisy breathing', 59Vo used the term<br />
'wheeze' to describe their concern. However, after being shown video clips illustrating<br />
wheezing and other airway noises, only 36Vo continued to describe their infant as wheezing.
Video clips <strong>of</strong> infants with a variety <strong>of</strong> breathing noises led to 30Vo <strong>of</strong> respondents using other<br />
words to describe wheezing and30Vo using the term 'wheeze' incorrectly @lphick, Sherlock<br />
et al. 2001).<br />
In addition, all that wheezes is not asthma (see Table 1.1). Up to 40Eo <strong>of</strong> infants who have<br />
been hospitalised with bronchiolitis may have subsequent wheezing episodes, usually in<br />
association with viral infections, up to five years <strong>of</strong> age (Martinez, Wright et al. 1995) and<br />
approximately LUVo continue to wheeze after age five years (Noble, Murray et al. 1997;<br />
Sigurs, Bjarnason et al. 2000; Hall 2001). In the Tucson study <strong>of</strong> over 1200 children, wheeze<br />
was reported in almost 50Vo at some time between birth and six years (Martinez, Wright et al.<br />
1995). Similar studies from Europe suggest l5-327o <strong>of</strong> children had wheezing in the first five<br />
years <strong>of</strong> life (Strachan 1985; Park, Golding et al. 1986). Sixty to 80Vo <strong>of</strong> infants who start<br />
wheezing in their first two years <strong>of</strong> life do not go on to have asthma. This group is found to<br />
have diminished airway function present prior to the onset <strong>of</strong> wheeze, and wheeze <strong>of</strong>ten<br />
ceases around the age <strong>of</strong> 3-5 years (Morgan and Martinez L992; Holberg, Wright et al. 1993;<br />
Wilson 1994; Martinez, Wright et al. 1995; Clough, Keeping et al. 1999) which confirms the<br />
findings <strong>of</strong> a much earlier study @oesen 1953). However, a minority <strong>of</strong> wheezy infants will<br />
develop later asthma (Wilson 1994; Martinez, Wright et al. 1995; Cochran 1998). <strong>The</strong>se<br />
children continued to have wheezy episodes, developed an increased total serum<br />
immunoglobulin E (IgE) bV age 9 months, developed sensitivity to a panel <strong>of</strong> aeroallergens by<br />
age 6 years, and had significantly lower lung function by age 6 years (Martinez 2002).In<br />
prospective follow up studies from childhood to adolescence, up to 80Vo <strong>of</strong> asthmatic children<br />
were reported to lose their symptoms during puberty (Balfour-Lynn 1985; Peat, Salome et al.<br />
1989; Nicolai, Illi et al. 1998). Thus asthma may then remit in later childhood or may<br />
continue throughout childhood and into adult life (von Mutius 2001; Weinberger 200.3).<br />
Table 1.1: Alternative diagnoses in children with wheeze<br />
Angio-oedemia<br />
Aspiration<br />
syndromes<br />
including foreign bodies Ainray compression bronchogenic or pulmonary<br />
cyst, lymph nodes, tumour<br />
(carcinoid, lymphoma),<br />
vascular ring or sling<br />
q1-antiprotease<br />
deficiency<br />
(older child)<br />
Bacterial tracheitis respiratory syncytial virus,<br />
metapneumovirus,<br />
parainfluenza, influenza,<br />
adenovirus<br />
Anaphylactic<br />
reactions<br />
Angioedema<br />
Bronchiolitis Aspiration including foreign bodies
Inhalational irritants<br />
pertussis, mycoplasma,<br />
tuberculosis, any other<br />
bacterial infection<br />
smoke, drugs<br />
syndromes<br />
Bronchiolitis -<br />
recurrent<br />
Bronchiolitis<br />
obliterans<br />
Cardiac conditions<br />
Chronic lung<br />
disease <strong>of</strong><br />
prsmaturity<br />
Chronic suppurative<br />
disease<br />
Congenital<br />
abnormality<br />
Eosinophilic<br />
bronchitis<br />
Gastrooeosphageal<br />
reflux<br />
Genetic disorders<br />
lmmune<br />
deficiencies<br />
Inhalational irritants<br />
Malacic syndromes<br />
Post-infectious<br />
Sarcoidosis<br />
Tuberculosis<br />
Vasculitic disorders<br />
Vocal cord<br />
dysfunction<br />
respiratory syncytial virus,<br />
metapneumovirus,<br />
parainfluenza, influenza,<br />
adenovirus<br />
congestive heail failure,<br />
vascular compression (ring or<br />
sling)<br />
bronchiectasis, chronic<br />
bronchitis<br />
anatomic lesion:<br />
cystic malformations, foregut<br />
malformations, granulation<br />
tissue, tracheoesophageal<br />
fistula<br />
+/- aspiration<br />
cystic fibrosis, primary ciliary<br />
dyskinesia<br />
smoke, glue, drugs<br />
tracheomalacia,<br />
bronchomalacia<br />
adenovirus<br />
pertussis<br />
mycoplasma<br />
Wegeners's granulomastosis,<br />
other<br />
Despite these two confounding issues, wheeze remains a key element in the diagnostic criteria<br />
cited for asthma in all the guidelines (Paediatric Society <strong>of</strong> New Zealand 2005), (GINA<br />
2005), (SIGN 2005) and confirmed by surveyed clinician opinion (Werk, Steinbach et al.<br />
2000). <strong>The</strong>re is no standard way <strong>of</strong> estimating the frequency or severity <strong>of</strong> episodes <strong>of</strong> wheeze<br />
to identify that which is purely consistent with asthma and classical diagnosis usually relies<br />
on associated symptoms and findings (GINA 2005). In addition, the performance <strong>of</strong> a p-2<br />
agonist treatment trial for acute wheeze are described in these guidelines and also in two<br />
recent bronchiolitis guidelines, 'Wheeze and Chest Infection in Infants under One year' from
the Paediatric Society <strong>of</strong> New 7*aland which I co-chaired (Byrnes, Vogel et al. 2005) and a<br />
report from the Agency for Healthcare Research and Quality, from the United States<br />
Department <strong>of</strong> Health and Human Services (Viswanathan, King et al. 2003).<br />
1.4.3 Where does coughfit in?<br />
<strong>The</strong> use <strong>of</strong> cough as a diagnostic symptom is another area that is not straightforward. When<br />
asked what clinical factors were associated with establishing an initial diagnosis <strong>of</strong> asthma,<br />
96Vo <strong>of</strong> the responding clinicians thought the requirement was recurrent wheeze, 90Vo<br />
symptomatic improvement with a bronchodilator, and 89Vo recunent cough, but there was<br />
disagreement over what combinations <strong>of</strong> these factors were important (Werk, Steinbach et al.<br />
2000). "Troublesome cough is a characteristic <strong>of</strong> asthma" (Li, I-ex et al. 2003) and "cough is<br />
as frequent a symptom as is wheezing" (Weinberger and Abu-Hasan 2006) has been reported.<br />
Controversy has focused on whether 'cough variant asthma' (sometimes called 'cough<br />
equivalent asthma') truly exists, with researchers both for and against.<br />
It is thought that asthma could occur without wheezing if the obstruction involves the small<br />
airways predominantly, and then coughing or shortness <strong>of</strong> breath may be the only complaint<br />
(Kercsmar 2006).In a follow-up study <strong>of</strong> I25 preschool children with recurrent cough but no<br />
other symptoms at the time <strong>of</strong> enrolment, 56Vo <strong>of</strong> them were symptom free at follow-up while<br />
36.8Vo continued to have recurrent cough in the absence <strong>of</strong> a 'cold', and7 .2Vo had developed<br />
recurrent episodes <strong>of</strong> wheeze @rooke, Lambert et al. 1998). One hundred children with a<br />
mean age <strong>of</strong> 5.7 years with chronic cough were followed for three years. Of the 75 deemed as<br />
having 'cough variant asthma', 28 (377o) went on to develop more 'classical' asthma<br />
symptoms (Todokoro, Mochizuki et al.2N3). Bronchial hyper-reactivity, nocturnal cough<br />
and developing cough at an earlier age may each be predictors <strong>of</strong> those who go on to get more<br />
typical asthmatic symptoms (Brooke, Lambert et al. 1998), (Todokoro, Mochizuki et al.<br />
2003).It has been suggested in adults that those with cough who go on to develop classical<br />
asthma have a threshold for airway hyper-responsiveness somewhere between asthmatic and<br />
normal subjects (Koh, Jeong et al. 1999; Mochizuki, Arakawa et al. 2005) and 30Vo <strong>of</strong> those<br />
with cough and bronchial hyper-responsiveness will go on to develop asthmatic symptoms<br />
(Fujimura, Nishizawa et al. 2005). Eosinophilic inflammation (eosinophils, eosinophil<br />
cationic protein (ECP), interleukin 5 (IL5)) has been demonstrated in some patients with<br />
persistent cough; a pattern <strong>of</strong> inflammation similar to asthmatics seen in adults (Lee, Cho et<br />
al. 200| De Diego, Martinez et al. 2005) and in children (Yoo, Koh et al.2004). Basement<br />
membrane thickening, a pathological feature <strong>of</strong> asthma, was demonstrated in 'classical'<br />
l0
asthmatics at 8.6 microns, in 'cough variant asthmatic' subjects at7.l microns and both were<br />
significantly different than healthy controls at 5.0 microns (Niimi, Matsumoto et al. 2000).<br />
Others are less enthusiastic for this diagnostic category -<br />
given that cough is a very corlmon<br />
symptom. A diagnosis <strong>of</strong> cough variant asthma could result in a significant increased number<br />
<strong>of</strong> children being treated' "Isolated chronic cough in children is rarely asthma, and the term<br />
'cough-variant-asthma' should not be used" (Chang, Landau et al. 2006). Wheezing reported<br />
by children had a very good discriminating ability for asthma whereas cough less so in over<br />
1600 school children aged 8-12 years (Yu, Wong et aI.2004). In asthmatic children who had<br />
cough as a predominant symptom, the cough occurred early and heralded the onset <strong>of</strong> an<br />
exacerbation. Asthma scores did relate to cough scores, although neither cough receptor<br />
sensitivity nor cough scores related to any specific inflammatory marker (Chang, Harrhy et al.<br />
2002). In 1245 children 6-12 years <strong>of</strong> age comparing symptoms <strong>of</strong> persistent cough with<br />
symptoms <strong>of</strong> wheeze, the former had less demonstrable atopy and less morbidity. <strong>The</strong> authors<br />
concluded that while 'cough variant asthma' was not shown, there remained a significant<br />
number <strong>of</strong> children with persistent cough diagnosed and treated as having asthma @aniran,<br />
Peat et al. 1999). So in the presence <strong>of</strong> persistent cough, if history, examination, chest x-ray<br />
and spirometry are normal, the cough could be regarded as non-specific and the child<br />
regarded as in the category <strong>of</strong> 'normal', as opposed to heralding a morbid or pre-morbid<br />
condition (Chang and Powell 1998; Bush 2002).<br />
Finally, treatment trials for cough using inhaled corticosteroids (IHCS) (Dicpinigaitis 2006;<br />
Matsumoto, Niimi et aL.2006) and a leukotriene receptor antagonist (Spector and Tan 2004)<br />
showed an improvement in symptoms. However the natural history <strong>of</strong> post-viral cough is that<br />
most will have resolved in 3-4 weeks and only l\Vo will continue for more than 25 days, with<br />
gradual resolution. Improvement could therefore have erroneously been attributed to<br />
treatment. <strong>The</strong> Cochrane Systematic Review <strong>of</strong> the efficacy <strong>of</strong> IHCS for non-specific cough<br />
in children > two years <strong>of</strong> age evaluated two trials enrolling a total <strong>of</strong> 123 children. <strong>The</strong> first<br />
trial using beclomethasone dipropionate 4o0ltglday (metered dose inhaler via a spacer) found<br />
no difference from placebo in reducing cough frequency measured objectively or scored<br />
subjectively. <strong>The</strong> second trial using fluticasone propionate 2mglday for three days followed<br />
by I mg/day for 1l days (metered dose inhaler via a spacer) showed a significant<br />
improvement in nocturnal cough frequency after two weeks, but also with a significant<br />
(though smaller) improvement with placebo (Tomerak, McGlashan et al. 2005). <strong>The</strong>re was no<br />
difference between the use <strong>of</strong> inhaled salbutamol and placebo (Tomerak, Vyas et al. 2005) or<br />
ll
inhaled anti-cholinergic agents and placebo (Chang, McKean et al.20O4) in children with<br />
persistent non-specifi c cough.<br />
1.4.4 Investigations<br />
<strong>The</strong>re are few investigations that are specific to asthma in adults and children. <strong>The</strong>re are no<br />
diagnostic chest x-ray findings; the film may be normal, or there may be hyperinflation,<br />
interstitial infiltrates or atelectasis with mucus plugging (Arthur 2000; Kercsmar 2006).<br />
Hyperinflation, the most common pattern associated with asthma, can also be seen in<br />
bronchiolitis and cystic fibrosis (CF) (Arthur 2000). Similarly, studies have shown that chest<br />
x-rays are not useful in determining the difference between bronchiolitis and pneumonia<br />
(Khamapirad and Glezen 1987; Davies, Wang et al. 1996; Byrnes, Vogel et al. 2005). In a<br />
survey <strong>of</strong> school age children referred to a respiratory clinic,30Vo had prior diagnoses <strong>of</strong><br />
pneumonia on chest xray appearances but presented with symptoms identical to those<br />
subsequently associated with their asthma diagnoses (Castro-Rodriguez, Holberg et al. 1999;<br />
Mahabee-Gittens, Bachman et al. 1999).<br />
Both total IgE and skin prick testing to common allergens have been used as a method <strong>of</strong><br />
detecting atopy, which overlaps the asthma syndrome. IgE levels appear to have been<br />
increasing over the last decades coinciding with the increase in asthma (Burrows, Martinez et<br />
al. 1989; Burney, Malmberg et al. 1997). Higher levels <strong>of</strong> IgE have been associated with<br />
increased atopy and an increased incidence <strong>of</strong> wheeze (Call, Smith et al. L992; Heymann,<br />
Carper et al. 2O04). An association has been demonstrated between the level <strong>of</strong> IgE and<br />
airway hyper-responsiveness as measured by bronchial challenges (Muranaka, Suzuki et al.<br />
1974; Burney, Britton et al. 1987). In a long term infant follow up study, the mean total serum<br />
IgE at nine months <strong>of</strong> age was significantly higher among those who became persistent<br />
wheezers compared to those who did not wheeze. Within the group <strong>of</strong> eady wheezers, those<br />
who were wheeze free by ages I I and 16 years had total IgE levels similar to those who never<br />
wheezed (Martinez 2002). However, the findings are not always consistent. Total IgE levels<br />
were found to be higher in some cohorts <strong>of</strong> children than others despite similar asthma<br />
prevalence, and the total IgE was higher in countries where house dust mite was the dominant<br />
source <strong>of</strong> allergen @latts-Mills and Heyman 2006). <strong>The</strong> ECHRS study involving 37 centres in<br />
16 countries demonstrated variation in the prevalence <strong>of</strong> response to at least one specific IgE<br />
which ranged from 60Vo in Albacete (Spain) to 45Vo in Christchurch (New 7*aland) and was<br />
not always consistent with asthma prevalence. <strong>The</strong> study also showed significant variations<br />
between countries for total IgE and for prevalence <strong>of</strong> any specific IgE. At all ages women had<br />
t2
a 26Vo lower total serum IgE than men, which is surprising when asthma tends to be more<br />
prevalent in women in adulthood. <strong>The</strong>re was a lack <strong>of</strong> association between the prevalence <strong>of</strong><br />
atopy (defined as a positive response to any <strong>of</strong> the four specific allergens used in skin prick<br />
testing in all centres) and the geometric mean <strong>of</strong> total serum IgE. It is probable that the total<br />
and specific IgE are influenced differently and while raised IgE is associated both with atopy<br />
and with asthma, the relationship is not clear (Burney, Malmberg et al. 1997). Furthermore,<br />
IgE antibody production is T cell dependent, and the immune response to allergens can also<br />
include other immunoglobulins such as immunoglobulin Ga (IgG+) and immunoglobulin A<br />
(IgA) which may give further variations (Platts-Mills and Heyman 2006).It is true to say that<br />
if a child is thought to have bad asthma, but is non-atopic, then the search for an alternative<br />
diagnosis should be undertaken immediately.<br />
Pulmonary function tests have been one <strong>of</strong> the mainstays <strong>of</strong> assessing respiratory disease in<br />
older children and adults. A measurement in forced expiratory volume in I second (FEVI)<br />
alone can miss small airway obstruction in patients and the forced expiratory flow rate over<br />
25-75Vo <strong>of</strong> forced vital capacity (FEF25 -tsw) may well be a more sensitive indicator,<br />
particularly in children and/or in mild disease; however, the measurement may be very<br />
variable. Subjects can have a reduction in this parameter <strong>of</strong> more than two standard deviations<br />
below predicted norms even with no clinical symptoms (Kercsmar 2006). <strong>The</strong>re is a<br />
significant overlap with measurements between healthy children and those with episodes <strong>of</strong><br />
wheeze (if not current), and the diagnostic value <strong>of</strong> baseline lung function tests is generally<br />
thought to be poor (Dundas and McKenzie 2006). <strong>The</strong> guidelines suggest a test <strong>of</strong><br />
bronchodilator responsiveness for asthma either clinically and/or utilising improvement in<br />
lung function (usually FEVr) by an arbitrary amount, sometimes 157o. Despite this, the SIGN<br />
guideline states "a definitive diagnosis <strong>of</strong> asthma can be difficult to obtain in young children.<br />
It is <strong>of</strong>ten not possible to measure ainvay function in order to confirm the presence <strong>of</strong> variable<br />
airway obstruction" (SIGN 2005).<br />
<strong>The</strong> other option for possible diagnosis and monitoring <strong>of</strong> asthma is to use peak expiratory<br />
flow (PEF). This is defined as the largest expiratory flow achieved with a maximal forced<br />
effort from a position <strong>of</strong> maximum inspiration sustained for longer than l0 milliseconds<br />
(Wright and McKerrow 1959). PEF reflects a range <strong>of</strong> physiological characteristics including<br />
lung elastic recoil, large airway calibre and lung volume (Ruffin Z0O4). <strong>The</strong> original .peak<br />
Flow Meter' was pioneered by Martin Wright in 1959 (Wright and McKenow 1959) and is<br />
now supplanted by newer models but these have retained the advantages <strong>of</strong> the original in<br />
being low cost and easily portable. In the late 1970s the use <strong>of</strong> PEF recordings was first<br />
13
ecommended to monitor asthma at home (Seaton 1978). This became part <strong>of</strong> an international<br />
consensus document in 1992 (Irnfant rgg2). <strong>The</strong> pEF technique was standardised in an<br />
American Thoracic society paper in 1994 (American Thoracic Society and Association.<br />
lgg4).By 1998 all the published asthma guidelines included PEF monitoring as part <strong>of</strong> an<br />
individualised management strategy (woolcock, Rubinfeld et al' 1989; National Heart Blood<br />
and Lung Institute 1991; British Thoracic Society 1993; GINA 1995; Jain, Kavuru et al'<br />
1998). <strong>The</strong> concept was to provide an objective measurement <strong>of</strong> airflow obstruction'<br />
particularly for the significant proportion <strong>of</strong> patients who otherwise had difficulty in<br />
recognising their asthma severity (Rubinfeld and Pain L976;Burdon, Juniper et al' 1982; Sly'<br />
Landau et al. 1985; Kendrick, Higgs et al. 1993; Fishwick and Beasley 1996)'<br />
However, there are issues with using pEF as this predominately reflects alteration in large<br />
airway calibre which is different to FEVr that reflects changes in calibre <strong>of</strong> both large and<br />
medium sized airways (Osmanliev, Bowley et al' t982; Robinson' Chaudhary et al' 1984;<br />
Dolyniuk and Fahey 1986; Gregg 2000). PEF is effort dependant so is susceptible to both<br />
respiratory muscle strength and patient motivation (Tzelepis, pavleas et al. 2005). while the<br />
reproducibility <strong>of</strong> pEF has been reported to be good with a coefficient <strong>of</strong> variation in well<br />
trained subjects <strong>of</strong> between 5 to I4Vo, still more than Sovo <strong>of</strong> asthmatic patients show a 107o<br />
difference and 33vo <strong>of</strong> patients show more than a 20vo difference between the percent<br />
predicted value <strong>of</strong> FEVr and PEF (Kelly and Gibson 1988; Paggiaro' Moscato et al' 1997)' In<br />
addition, the standard deviation for PEF readings is consistently greater than that <strong>of</strong> FEVr' In<br />
another study, the coefficient <strong>of</strong> pEF repeatability was 107o for healthy adults but rTvo for<br />
healthy children, LTVo inasthmatic adults and2SVo in asthmatic children @nright, shenill et<br />
al. 1995). This poorer repeatability in asthmatic subjects has been confirmed by other studies<br />
(Meijer, Postma et al. 1996; Timonen, Nielsen et al' IggT; Holcr<strong>of</strong>t' Eisen et al' 2003)' In<br />
addition, significant differences in variability were also described between encouraged PEF<br />
readings done in a laboratory and those done at home (Reddel' Ware et al' 1998; Gannon'<br />
Belcher et al. 1999).<br />
<strong>The</strong> two methods <strong>of</strong> using pEF to detect or monitor asthma was either by determining daily<br />
PEF variability or by using the comparison <strong>of</strong> the current PEF to a subject's best PEF (Jain'<br />
Kavuru et al. l99g). pEF variability is associated with acute asthma exacerbations @ellia,<br />
Cibella et al. 1985; Beasley, Cushley et al. 1989; Jindal, Aggarwal et al' 2002)' Cross<br />
sectional studies have demonstrated significant (albeit weak) correlations between PEF<br />
variability and overall severity <strong>of</strong> inflammation, symptoms scores, FEVr and bronchial hyper-<br />
responsiveness (Higgins, Britton et al. lgg2;Brand, Duiverman et al' 1997; Douma' Kerstjens<br />
L4
et al. 2000; Jindal, Aggarwal et al. 2002) with similar (but weaker) conelations found in<br />
longitudinal studies (Brand, Duiverman et al. lggg: Kamps and Brand 2001). However, the<br />
degree <strong>of</strong> variability considered 'abnormal' is difficult to determine (Jindal, Aggarwal et al.<br />
2002) with a difference <strong>of</strong> 20Vo between repeated PEF recording suggested as necessary to<br />
identify asthma (Hetzel l98l; Jamison and McKinley 1993; National Asthma Education and<br />
Prevention Program 1997). Studies have shown no clear demarcation between normal<br />
individuals and asthmatics (Higgins, Britton et al. 1989; Parameswaran, Belda et al. lggg),<br />
with the greatest overlap between groups occurring in children (Albertini, politano et al. l9g9;<br />
Goldberg, Springer et al. 2001). PEF variability <strong>of</strong> 20Vo gave a sensitivity <strong>of</strong> 5l.77o,<br />
specificity <strong>of</strong> 82.4Vo, positive predictive value <strong>of</strong> 13.97o and a negative predictive value <strong>of</strong><br />
95.5Vo for detecting asthma in one study (Goldberg, Springer et al. 2001). In a second study<br />
20vo variability gave a sensitivity <strong>of</strong> 36vo, specificity 90Vo, positive predictive value <strong>of</strong> 16.47o<br />
(Kunzli, Stutz et al. 1999) while this cut <strong>of</strong>f missed three quarters <strong>of</strong> the asthmatic patients in<br />
a third study (Goldstein, Yeza et al. 2001). <strong>The</strong> relationship between the magnitude <strong>of</strong> pEF<br />
variability and response to bronchodilators is poor on or <strong>of</strong>f IHCS therapy (Connolly 1979;<br />
Kerstjens, Brand et al. 1994; Douma, Kerstjens et al. 2000; Iwasaki, Kubota et al. 2000) and<br />
the relationship between PEF measurement when on long acting p2 agonist treatment is<br />
complex depending on the timing <strong>of</strong> the last dose (Reddel, Jenkins et al. 1999; Reddel, Ware<br />
et al. 1999). Also both daily peak flow levels and mean peak flow may fall so that the<br />
variability may not change (Reddel, Jenkins et al. 1999). In addition, there appears to be a<br />
circadian rhythm for PEF in all individuals (Hetzel and Clark 1980; Hetzel lggl) bur<br />
exaggerated in patients with asthma (Turner-Warwick 1977; Connolly 1979; Bagg and<br />
Hughes 1980; Hetzel and Clark 1980).<br />
<strong>The</strong> other option recommended is to use a percentage <strong>of</strong> maximum pEF value for an<br />
individual to discern asthma. A reduction from best PEF <strong>of</strong> 16.5To Bave asensitivity <strong>of</strong> SlVo<br />
and a specificity <strong>of</strong> 98.7Vo, while a reduction <strong>of</strong> 2o%o gave a sensitivity <strong>of</strong> only 40Vo and<br />
specificity <strong>of</strong> 99.3Vo (Aggarwal, Gupta et aL.2002). In children, percent predicted pEF only<br />
weakly correlated symptoms or airway responsiveness (Kolbe, Richards et al. 1996; Brand,<br />
Duiverman et al. 1997; den Otter, Reijnen et al. 1997). This also assumes the patients will<br />
know their best PEF measurement but only 297o <strong>of</strong> 104 subjects were able to report this<br />
@iner, Brenner et al. 2001). This group also showed that over subsequent days, 45Vo <strong>of</strong><br />
patients had a measured PEF greater than their reported personal best. Thus if calculated on<br />
the reported figure both over-treatment in as man y as 30Vo <strong>of</strong> clinically stable patients<br />
(Douma, Kerstjens et al. 1998) and inappropriate emergency department discharges based on<br />
15
improvement to 70Vo PEF would have occurred (Diner, Brenner et al. 2001). Predicted PEF<br />
values has also been shown to be alter with age, sex, race, height and smoking making advice<br />
based on "predicted values" difficult (Gregg and Nunn 1973; Nunn and Gregg 1989; Higgins,<br />
Britton et al. 1992; Higgins, Britton et al. 1993; Boezen, Schouten et al. 1994; Boezen,<br />
Schouten et al. 1995; Bellia, Cuttitta et al. 1997; Jain, Kavuru et al. 1998; Bellia, Catalano et<br />
al. 2004).In addition, significant variation in predicted PEF values has been demonstrated<br />
even within the same populations (Jindal, Aggarwal et al.2002).<br />
<strong>The</strong> number <strong>of</strong> PEF measurements also contributes to accuracy and studies have used between<br />
two and twelve readings (Gannon, Newton et al. 1998; Jindal, Aggarwal et al.2N2). Taking<br />
twelve measurements daily as the gold standard, using four <strong>of</strong> these detected only 60-807o <strong>of</strong><br />
the variability and using two measurements detected only 20-45Vo <strong>of</strong> the variability<br />
@'Alonzo, Steinijans et al. 1995). Another study showed that four, three and two daily<br />
measurements explained 90-95Vo,70-82Vo and 55Vo <strong>of</strong> the diurnal variability respectively<br />
(Gupta, Aggarwal et al. 2000). Patients also need to repeat the measurements several times on<br />
each occasion as, on review <strong>of</strong> 5,809 test sessions, it was the third manoeuvre that most<br />
frequently (40Vo <strong>of</strong> the time) gave the highest reading (Gannon, Belcher et al. 1999).<br />
Practically, it would be difficult to request patients to do anything beyond two readings per<br />
day over several days, let alone weeks or months. With a circadian rhythm playing a part,<br />
ideally but also difficult would be to request the PEFs be done regularly at specific times <strong>of</strong><br />
the day. Finally, the age at which the child becomes able to use a peak flow effectively is also<br />
variable (Clough 1996).<br />
Overall the recommendation is not to use PEF for diagnosis, but international guidelines<br />
continue to recommend PEF monitoring in the assessment <strong>of</strong> asthma, although more recently<br />
for selected groups; those with severe asthma, poor perceivers, as a short term monitoring<br />
procedure for exacerbation or to assess the adjustment <strong>of</strong> the medication dose (Reddel 2006).<br />
PEF monitoring did reduce symptoms and improve other parameters such as days lost from<br />
work, physician consultations and hospital visits in some studies (Ignacio-Garcia and<br />
Gonzalez-Santos 1995; Cowie, Revitt et al. 1997). However, others showed no major changes<br />
in outcomes in adults (Jones, Mullee et al. 1995; Turner, Taylor et al. 1998; Adams, Boath et<br />
al.200l; Tierney, Roesner et aL.2004) or children with asthma (Mortimer, Fallot et al. 2003).<br />
In children, it was difficult to perform (Gorelick, Stevens et al.2004) and was not useful in<br />
dictating management during acute asthma (Wensley and Silverman 2OM). A Cochrane<br />
review in 2004 concluded there was not enough data even combining seven trials (921 adults<br />
and 46 children) to show that personalised self management plans with or without PEF<br />
16
monitoring improved asthma outcomes (Toelle and Ram 2004). A further Cochrane review<br />
(Bhogal, Tnmek et al. 2006) in 2006 assessed four trials with 355 children comparing<br />
symptom based written action asthma plans to PEF monitoring and found no differences in<br />
the rate <strong>of</strong> exacerbation, oral steroid courses, admissions, symptoms, lung function, school<br />
absenteeism or quality <strong>of</strong> life (Charlton, Charlton et al. 1990; Yoos, Kitzman et a:.2002;I*ta,<br />
Schlie et al. 2004; Wensley and Silverman 2004). Symptom monitoring was actually<br />
preferred over peak flow monitoring by the children. <strong>The</strong> final conclusion from this analysis<br />
was "symptom based written action plans are superior to peak flow written action plans for<br />
preventing acute care visits." (Bhogal, Tnmek et aL.2006). <strong>The</strong> daily use <strong>of</strong> pEF in a large<br />
prospective study was not perceived to be useful by most families and therefore unlikely to be<br />
adhered to by many (McMullen, yoos et aI.2002).<br />
Electronic recording spirometers were compared to hand written diaries in 6l subjects aware<br />
<strong>of</strong> the recording. Adherence to PEF monitoring over 72 weeks gradually declined fromg67o<br />
to 89Vo with l3vo <strong>of</strong> participants ultimately withdrawn because <strong>of</strong> poor adherence (Reddel,<br />
Toelle et al.2002). When participants were unaware <strong>of</strong> electronic recording and completed a<br />
pen and paper diary over 3 month periods, 64Vo and,44vo adherence to monitoring was noted<br />
(Chowienczyk, Parkin et al. 1994; Verschelden, Cartier et al. 1996). Two groups <strong>of</strong> children<br />
reported 96.6Vo and 94.8Vo PEF monitoring compliance while the simultaneously monitoring<br />
electronic device recorded compliance at 73.4Vo and 80.9Vo. Compliance decreased<br />
significantly from week one to week four such that overall the compliance was less than 50Vo<br />
in l2.5%o <strong>of</strong> the children and 50-75Vo in 20Vo <strong>of</strong> the children. Invention <strong>of</strong> some pEF<br />
measurements occurred and increased threefold during the study time (Kamps, Roorda et al.<br />
2001). This adds to the difficulty <strong>of</strong> giving action points, especially if figures are made up<br />
and./or it is not perceived as useful.<br />
In addition to these difficulties, differences have also been demonstrated when using different<br />
PEF techniques. <strong>The</strong>se include fast or slow exhalation (Richards 1993; Tzelepis, Zakynthinos<br />
et al. 1997), a breath-hold at total lung capacity (Matsumoto, Walker et al. 1996), if the<br />
position <strong>of</strong> the instrument is changed in relation to the mouth (Nolan, Tolley et al. 1999) or<br />
with posture differences (Bongers and O'Driscoll 2006; Lin, parthasarathy et al. 2006).<br />
Increased air trapping also reduced the ability <strong>of</strong> a normal PEF to predict a normal FEVI or<br />
FEF25-75E" from 83 to 53Vo potentially falsely reassuring an individual <strong>of</strong> having better<br />
pulmonary function than is correct @id, yandell et al. 2000).<br />
t7
Furthermore, investigators have also found a poor agreement between the brands <strong>of</strong> peak flow<br />
metres when tested at sea level (Jackson 1995), at altitude (Gardner, Crapo et al. 1992) and in<br />
the clinic (Gregg 1991; Quanjer t992; Burge 1993; Dahlqvist, Eisen et al. 1993; Gregg 1998;<br />
Nolan, Tolley et al. 1999) even after the technical requirements for meters were established<br />
by the National Heart, Lung and Blood Institute in 1991 (National Heart Lung and Blood<br />
Institute 1991). Differences have also been seen between the values obtained from new or old<br />
meters <strong>of</strong> the same type (Douma, van der Mark et al. 1997; Nazir, Razaq et al. 2005).<br />
Finally there is also a report <strong>of</strong> adverse reactions to peak flow monitoring with two patients<br />
having a herniation <strong>of</strong> abdominal content and three having depression or neurotic<br />
preoccupation with their PEF values which the authors calculated as a side effect incidence <strong>of</strong><br />
1.1 cases per thousand patients in the study population (Schoch, Nierh<strong>of</strong>f et al. 1998). <strong>The</strong>re<br />
has been a report about the potential <strong>of</strong> fungal contamination <strong>of</strong> peak flow devices (Ayres,<br />
Whitehead et al. 1989). So PEF monitoring is also not the answer for accurate diagnosis and<br />
may not <strong>of</strong>fer significant advantage for long term monitoring in most asthmatic patients over<br />
symptom assessment.<br />
Airway challenges (both direct and indirect) have also been used to make an asthma<br />
diagnosis, although this happens less <strong>of</strong>ten in paediatric field outside <strong>of</strong> the research arena.<br />
Airway direct challenges have been used widely and are well standardised most commonly<br />
using methacholine or histamine. <strong>The</strong>se act as direct stimuli on the effector cells,<br />
predominantly airway smooth muscle, but also on mucus glands and airway microvasculature,<br />
causing airflow limitation (Adelroth, Hargreave et al. 1986; Woolcock, Anderson et al. 1991).<br />
<strong>The</strong> responses follow a continuous distribution within the population, which leads to<br />
difficulty when determining a normal, versus an asthmatic response @ehaut, Rachiele et al.<br />
l9S3). This has been arbitrarily defined (currently set at
sensitive and to have good negative predictive value in getting a response in asthmatics who<br />
have recent or current symptoms. This comes at the cost <strong>of</strong> being less specific and having less<br />
positive predictive value or being useful for population screening for asthma (Cockcr<strong>of</strong>t,<br />
Murdock et al. 1992). As part <strong>of</strong> the ECRHS, bronchial hyper-reactivity using standardised<br />
methacholine challenges was tested in over 13,000 adult subjects in 35 centres from 16<br />
countries showing considerable variation in incidence (Chinn, Burney et al. 1997). In some<br />
this mirrored their known rates <strong>of</strong> asthma disease, such as being high in New Zealand, Great<br />
Britain, Australia and the USA, and low in Iceland and Switzerland. <strong>The</strong>re was less <strong>of</strong> an<br />
obvious connection in others, for example showing high rates <strong>of</strong> hyper-reactivity in Denmark<br />
which has far lower documented asthma prevalence than, for example, Italy, Spain or Sweden<br />
which all had low reactivity rates. In an early study <strong>of</strong> 307 adults, bronchial reactivity was<br />
noted in 3Vo <strong>of</strong> normal subjects, l00vo <strong>of</strong> symptomatic asthmatics, 69Vo <strong>of</strong> currently<br />
asymptomatic asthmatics (Cockcr<strong>of</strong>t, Killian et al. 1977). In other studies, the proportion <strong>of</strong><br />
individuals with bronchial hyper-responsiveness was only about 40-60Vo <strong>of</strong> those reporting<br />
current wheeze (Backer, Dirksen et al. 1991; Backer, Groth et al. l99l; Backer and Ulrik<br />
1992; GINA 2005).<br />
As well as some 'normals' responding in what is deemed the asthmatic range (Cockcr<strong>of</strong>t,<br />
Killian et al. 1977; Cockcr<strong>of</strong>t, Murdock et al. 1992). the direct airway challenges are also non-<br />
specific, as subjects with other respiratory diseases also react, most notably cough (Brooke,<br />
Lambert et al. 1998), chronic obstructive pulmonary disease (COPD) and/or chronic<br />
bronchitis (CB) (Ramsdell, Nachtwey et al. 1982; Ramsdale, Roberts et al. 1985; Calverley,<br />
Burge et al. 2003).<br />
Direct airway challenges in children are limited to older age groups. In over 2000 children<br />
aged 7-10 years tested with a cumulative dose <strong>of</strong> 3.9;rmol <strong>of</strong> histamine, only 5BVo <strong>of</strong> the<br />
children with asthma and current symptoms responded. While 52Vo <strong>of</strong> those diagnosed with<br />
asthma responded overall, 53Vo <strong>of</strong> subjects demonstrating bronchial hyperactivity had no<br />
asthma diagnosis (Pattemore, Asher et al. 1990). In three regions with similar asthma<br />
admissions (<strong>Auckland</strong>, inland New South Wales and coastal New South Wales) between 700<br />
to over l'000 children were tested at each centre and while two groups had similar rates <strong>of</strong><br />
bronchial hyper-reactivity to methacholine challenges, the third had a much lower rate (Asher,<br />
Pattemore et al. 1988). <strong>The</strong> hyper-reactivity to direct airway challenge has a similar sensitivity<br />
and specificity pr<strong>of</strong>ile for asthma diagnosis as questionnaire data (Asher, pattemore et al.<br />
1988; Backer, Dirksen et al. I99l; Bakke, Baste et al. l99l; Backer and LJlrik 1992; Joos,<br />
O'Connor et al. 2003). In one <strong>of</strong> these studies, the level <strong>of</strong> bronchial hyper-reactivity was a<br />
l9
poorer predictor <strong>of</strong> a diagnosis <strong>of</strong> asthma than responses to the question 'has your child had<br />
wheezing in the past 12 months' on written questionnaires @attemore, Asher et al. 1990). In<br />
495 children aged 7-16 years, the subjects with asthma represented a subgroup within the<br />
responsive end when inhaling increasing concentrations <strong>of</strong> histamine rather than the asthma<br />
and the 'normals' having separate distribution peaks. Selecting a low dose <strong>of</strong> 2.4mglrnl-r as<br />
the cut <strong>of</strong>f point gave a specificity <strong>of</strong> 98Vo for asthma, but a positive predictive value <strong>of</strong> only<br />
6OVo and a sensitivity <strong>of</strong> only 57Vo (Backer, Groth et al. l99l).<br />
Using indirect challenges is a more recent phenomenon from the late 1980s and eady 1990s<br />
(Manning, Watson et al. 1993; Joos, O'Connor et al. 2003). <strong>The</strong>se are termed indirect as the<br />
triggers act on cells, which then release mediators or cytokines to cause a secondary<br />
bronchoconstriction and airflow limitation. <strong>The</strong>y include physical stimuli such as exercise,<br />
osmotic stimuli such as hypertonic saline or mannitol, or pharmacological stimuli such as<br />
adenosine or bradykinin. <strong>The</strong>se challenges are thought to be a better reflection <strong>of</strong> the active<br />
airway inflammation as observed in asthma (Joos, O'Connor et al. 2003). Also IHCS reduce<br />
responsiveness to these indirect challenges, as in asthma, while in contrast they only have a<br />
small effect on histamine and methacholine challenges. <strong>The</strong> indirect challenges have<br />
demonstrated less sensitivity but more specificity in differentiating asthmatics from 'normals'<br />
(Vasar, Braback et al. 1996; Godfrey, Springer et al. 1999; Joos, O'Connor et al. 2003). In<br />
addition in children, an exercise challenge has also been shown to be better at distinguishing<br />
asthma from other chronic airway disorders such as CF, bronchiolitis obliterans, ciliary<br />
dyskinesia and bronchiectasis (Avital, Springer et al. 1995; Godfrey, Springer et al. 1999).<br />
<strong>The</strong>re is a statistical, albeit weak, correlation between the methods; however there are<br />
individuals who have positive exercise challenges and negative histamine or methacholine<br />
challenges. A hypertonic saline challenge in adults has been shown to identify exercise<br />
induced asthma (Belcher, I-ee et al. 1989; Boulet, Turcotte et al. 1989; Smith and Anderson<br />
1990; Brannan, Koskela et al. 1998). <strong>The</strong> possibility <strong>of</strong> having exercise induced asthma and<br />
not responding to hypertonic saline or mannitol challenges occurs, but only in those with very<br />
mild asthma @rannan, Koskela et al. 1998). In children, those with a history <strong>of</strong> current<br />
wheeze were seven times more likely to have a positive response to hypertonic saline than<br />
asymptomatic children (Riedler, Reade et al. 1994). However the use <strong>of</strong> osmotic challenges to<br />
date have been seen more frequently as a treatment to improve mucociliary clearance and<br />
lung function, predominantly in children with CF (Eng, Morton et al. 1996; Rodwell and<br />
Anderson 1996; Robinson, Daviskas et al. 1999).<br />
20
Thus determining the presence or absence <strong>of</strong> bronchial hyper-responsiveness to a trigger at a<br />
certain concentration is not necessarily diagnostic <strong>of</strong> asthma. <strong>The</strong> direct challenges lack<br />
specificity, while the indirect challenges lack sensitivity. Both challenge types are required to<br />
be done in the laboratory and while they are achievable with children, there is both a safety<br />
component and a significant time commitment to be considered. As repeated measures to<br />
monitor response to treatment or progression <strong>of</strong> asthma, they are less than satisfactory for<br />
these reasons.<br />
In summary while the diagnosis <strong>of</strong> asthma may seem straightforward in some who fit the<br />
'classical' asthma pr<strong>of</strong>ile, there are many difficulties to be faced when testing for asthma. In<br />
addition, the investigations are more suited for asthma determination in adults than in<br />
children. A diagnosis <strong>of</strong> asthma in children remains difficult, when in this group getting the<br />
correct diagnosis is important if the usual asthma treatment is to be recommended.<br />
1.5<br />
Treatment and concerns<br />
1.5.1 Possible adverse fficts <strong>of</strong> asthmatreatment in children<br />
Much is known about the underlying pathology <strong>of</strong> asthma, and this will be discussed in depth<br />
in the next section <strong>of</strong> this chapter. This has resulted in all the guidelines (GINA 2005;<br />
Paediatric Society <strong>of</strong> New Zealand. 20O5; SIGN 2005) recommending anti-inflammatory<br />
treatment; with IHCS at step two as the first preventer and the introduction <strong>of</strong> oral<br />
corticosteroids at steps four or five with unresponsive and difficult asthma, and for acute<br />
treatment. <strong>The</strong> balance is between the correct diagnosis, appropriate management and to<br />
prevent irreversible airway thickening on the one hand, against the development <strong>of</strong> side<br />
effects with treatment on the other.<br />
<strong>The</strong> adverse effects as listed in the guidelines for IHCS include; adrenal cortical insufficiency,<br />
growth failure, dysphonia and oral candidiasis. <strong>The</strong> adverse effects listed for oral<br />
corticosteroids is somewhat lengthier; adrenal insufficiency which may cause devastating<br />
hypoglycaemia, adrenal suppression, growth failure, hypertension, diabetes mellitus,<br />
reduction in bone mineral density, skin atrophy, straie, poor healing, immunosuppression and<br />
cataracts. Doses <strong>of</strong> IHCS <strong>of</strong> greater than 400p gtday <strong>of</strong> beclomethasone dipropionate or<br />
equivalent in children have been associated with side effects (Calpin, Macarthur et al. 1997:<br />
Sharek and Bergman 2000). <strong>The</strong>re have been reports <strong>of</strong> IHCS affecting short term growth<br />
(Agert<strong>of</strong>t and Pedersen 1997), intermediate term growth over months to a year (Tinkelman,<br />
Reed et al. 1993; Doull, Freezer et al. 1995; Simons 1997; Verbeme, Frost et al. 1997;Allen,<br />
2l
Bronsky et al. 1998), and a long-term effect over a period <strong>of</strong> years (Anonymous 2000).<br />
However, observational studies have suggested that despite long term IHCS in moderate<br />
doses, final adult height is not affected (Agert<strong>of</strong>t and Pedersen 2000; Peters 2006). While all<br />
guidelines recommend regular monitoring <strong>of</strong> the child's height, isolated growth failure is not<br />
a reliable indicator <strong>of</strong> adrenal suppression @unlop, Carson et al. 2004).<br />
On higher doses (800pg/day <strong>of</strong> beclomethasone dipropionate or equivalent) <strong>of</strong> IHCS or oral<br />
steroids, blood pressure should be monitored, bone density assessed and cataracts should be<br />
screened for, if being used for greater than a few months. Children may be more at risk <strong>of</strong><br />
developing cataracts and glaucoma @enfro and Snow 1992; Nootheti and Bielory 2006),<br />
though one study disagreed (Simons, Persaud et al. 1993). IHCS at the recorrmended levels,<br />
and in some studies even high doses <strong>of</strong> fluticasone, had minimal effects on bone mineral<br />
density (Hopp, Degan et al. 1995; Kelly, Clee et al. 1995; Agert<strong>of</strong>t and Pedersen 1998;<br />
Griffiths, Sim et al.2004). However others found that after years <strong>of</strong> treatment, asthmatics had<br />
lower total body bone mineral density than controls (Boot, de Jongste et al. 1997; Allen,<br />
Thong et al. 2000) with a negative relationship between this density and the total cumulative<br />
doses <strong>of</strong> IHCS used (Wong, Walsh et al. 2000). Oral candidiasis has been found in up to 5Vo<br />
<strong>of</strong> adult patients with asthma with positive mouth cultures up to 25Vo and dysphonia is also<br />
said to occur in up to 58Vo <strong>of</strong> patients at some time (Barnes, Pedersen et al. 1998).<br />
<strong>The</strong> issues regarding side effects <strong>of</strong> these medications should also be considered in two other<br />
populations. Firstly, healthy subjects (for example if the diagnosis is not secure) may have<br />
greater systemic side effects to IHCS and oral steroids than asthmatics (Brindley, Falcoz et al.<br />
2000; Brutsche, Brutsche et al. 2000; Falcoz, Oliver et al. 2000; Mackie, McDowall et al.<br />
2000). Secondly, in the last years, treatment with IHCS has been assessed in younger<br />
children; those with preschool and infant wheeze and/or bronchiolitis. Most <strong>of</strong> the studies in<br />
the preschool group have tried to target those who are more likely to develop asthma over<br />
time - with either recurrent wheezy episodes, personal history <strong>of</strong> atopy, a family history <strong>of</strong><br />
asthma or a positive asthma predictive index (Castro-Rodriguez, Holberg et al. 2000;<br />
Guilbert, Morgan et aI.2006). <strong>The</strong>se have enrolled between 31 and 625 children aged six to<br />
47 months have received doses from 40pglkg to 1000ug via nebuliser or 300-800pg/day via<br />
metered dose inhaler with a spacer and face mask for between 6 and 26 weeks. Improvements<br />
were seen in symptom free days (Bisgaard, Gillies et al. 1999; Roorda, Mezei et al. 2001;<br />
Teper, Colom et al.20[.4; Guilbert, Morgan et al.2N6), in lung function parameters @ao and<br />
McKenzie 2OO2; Teper, Colom et al.20O4), in bronchial hyper-reactivity (Stick, Burton et al.<br />
1995) and in use <strong>of</strong> additional 'rescue' medication @isgaard, Gillies et al. 1999; Teper,<br />
22
Colom et al.20O4; Guilbert, Morgan et aL.2006). Side effects noted were growth velocity<br />
(Guilbert, Morgan et al. 2006), adrenal suppression, increased cough and hoarseness, and<br />
cataract formation (Bisgaard, Allen et al. 2004). An earlier Cochrane review in 2000 had<br />
suggested that episodic high dose IHCS was partially effective. However, they concluded that<br />
"there is no current evidence to favour maintenance low dose IHCS in the prevention and<br />
management <strong>of</strong> episodic mild viral wheeze <strong>of</strong> childhood" (McKean and Ducharme). In<br />
addition, more recent Cochrane reviews do not support the use <strong>of</strong> IHCS in acute bronchiolitis<br />
or used to prevent wheezing post acute illness in 2006 (Blom, Ermers et al.) or the use <strong>of</strong> oral<br />
steroids for bronchiolitis in 2004 (Patel, Platt et al.). So while at the current time steroids are<br />
not recommended in this age group, the increased use exposes many more children to<br />
corticosteroid therapy, <strong>of</strong>ten before a definitive diagnosis <strong>of</strong> classic asthma is able to be made<br />
(de Blic and Scheinmann 2000).<br />
In fact it is interesting to note within the guidelines, the significant difference in the level <strong>of</strong><br />
evidence available for the different age groups. For example in the pharmacological<br />
management chapter <strong>of</strong> the SIGN guidelines (SIGN 2005), the graded evidence for the<br />
recommendations is presented in age brackets (>12 years and adults, 5-12 years and < 5<br />
years). Across these age groups the evidence frequently drops from '1++' (strong evidence<br />
with high quality meta-analyses and systematic reviews <strong>of</strong> randomised controlled trials) down<br />
to '4' (expert opinion) in the youngest age group.<br />
1.5.2 A marker for inflammation would be usefuI<br />
<strong>The</strong>re is a need to minimize side effects, while preventing morbidity (and mortality). This is<br />
important for better individual health, and is also important financially - reducing work and<br />
school days lost. Other financial burdens can be experienced by a family with a child with<br />
uncontrolled asthma as revealed in this quote taken from the 'Trying to Catch Our Breath,<br />
monograph (Anonymous 2006) :<br />
"I visited a family who had almost no furniture in the house. <strong>The</strong>y had taken their child<br />
to the doctor one evening in the previous week for an asthma attack. <strong>The</strong>y spent $80 for<br />
the visit and the medications. This was their entire food budget for the following week.<br />
<strong>The</strong>y were eating white bread and butter." (Claire Richards, Asthma Nurse Educator,<br />
Porirua Asthma Service, New Zealand)<br />
In 2005, the GINA guideline (GINA 2005) suggested to "titrate the dose <strong>of</strong> inhaled<br />
corticosteroid to the lowest dose at which effective control <strong>of</strong> asthma is maintained". <strong>The</strong><br />
23
SIGN guideline (SIGN 2005) stated "the smallest dose <strong>of</strong> inhaled steroids compatible with<br />
maintaining disease control should be used. At higher doses add on agents for example long<br />
acting p-2 agonists should be actively considered". In the NzpAG @aediatric<br />
Society <strong>of</strong> New<br />
7*,aland2005) again the recommendation is to "titrate the dose <strong>of</strong> inhaled corticosteroids to<br />
the lowest dose at which effective control <strong>of</strong> asthma is maintained". <strong>The</strong> difficulty here is how<br />
to measure what the appropriate low dose is and whether control has been achieved.<br />
Ultimately, treating the pathological changes rather than being guided by symptoms alone<br />
might well be useful. In the next section, I will present that data suggesting that asthma is an<br />
to measure, non invasive, marker <strong>of</strong> airway<br />
inflammatory disease, and whY an easY<br />
inflammation might be <strong>of</strong> great value'<br />
1.6<br />
During the last decades there has been a gradual move from the concept <strong>of</strong> asthma as a<br />
disease <strong>of</strong> bronchoconstriction to that <strong>of</strong> an inflammatory disorder, and from the concept <strong>of</strong><br />
complete reversibility to accepting that some irreversible airway damage can occur' <strong>The</strong><br />
current definition <strong>of</strong> asthma proposed by the GINA committee is "Asthma is a chronic<br />
inflammatory disorder <strong>of</strong> the airway in which many cells play a role, in particular mast cells'<br />
eosinophils, and T lymphocytes" (GINA 2005). In the following sections, I will briefly<br />
summarise findings from autopsy studies, then biopsy and lavage' induced sputum' blood and<br />
urine samples in asthmatic patients compared to normal subjects. while a full description <strong>of</strong><br />
the research that has resulted in these two major premise alterations is beyond the scope <strong>of</strong><br />
this thesis, the purpose <strong>of</strong> the next sections are two-fold; to background the concept <strong>of</strong> asthma<br />
as an inflammatory disease and to show that many <strong>of</strong> these samples are difficult to obtain.<br />
This makes them more appropriate for single or pre and post intervention determinations<br />
rather than repeated longitudinal or population assessments. I have also concentrated on what<br />
was known at the time <strong>of</strong> my research commencing but have included how this is viewed to<br />
date. NO will be discussed in the next chapter'<br />
1.6.1 What has been leamtfrom autopsy studies?<br />
<strong>The</strong> hint that asthma was an inflammatory disease occurred early. In the late 19ft century<br />
Charcot-I-eyden crystals (crystalline structures shaped as double nalrow pyramids)'<br />
curschmanns spirals (coiled fibrils <strong>of</strong> mucus) and eosinophilia in sputum were recognised<br />
during severe exacerbations <strong>of</strong> asthma (ossler rg92). This was followed early and mid last<br />
century by autopsy studies <strong>of</strong> adults dying from asthma (Huber and Koessler 1922; Bullen<br />
1952;Dunnill 1960) and much later by autopsy studies in children (cutz, Irvison et al' 1978)'<br />
24
<strong>The</strong>se described gross pathological changes with diffuse mucus plugging <strong>of</strong> the airways,<br />
sloughing <strong>of</strong> the epithelial lining, an increase in airway smooth muscle and oedema <strong>of</strong> the<br />
peribronchial tissues. Light microscopy showed mucus plugs, goblet cell hyperplasia and<br />
apparent thickening <strong>of</strong> bronchial basement membranes with increased smooth muscle. <strong>The</strong>re<br />
was an infiltration <strong>of</strong> inflammatory cells with a marked increase in eosinophils. <strong>The</strong> later<br />
paediatric study (Cutz, Levison et al. 1978) also examined the specimens under electron<br />
microscopy. This again demonstrated mucus plugs, which consisted <strong>of</strong> epithelial, macrophage<br />
and eosinophil cells and cell fragments. A thickened basement membrane appeared to be<br />
composed <strong>of</strong> collagen deposits and the submucosa was infiltrated by a mixture <strong>of</strong><br />
inflammatory cells, predominantly eosinophils. Recent autopsy studies have compared<br />
pathology specimens between patients with fatal asthma, non-fatal asthma and controls<br />
(Koshino, Teshima et al. 1993; Kepley, McFeeley et al. 2001; Chen, Samson et a1.2004).<br />
Significant smooth muscle shortening, increased submucosal gland area, increased mucus<br />
plugging and an increase in basophils was seen in the cases <strong>of</strong> fatal asthma compared to the<br />
other groups. <strong>The</strong>se contributed to airway wall thickening with smooth muscle hypertrophy<br />
and increased collagen along the basement membrane. Compared to ten controls, 18 adults<br />
who had died <strong>of</strong> asthma had proteoglycans prominent in the extra cellular matrix creating<br />
most <strong>of</strong> the basement membrane thickening seen, with differing types <strong>of</strong> proteoglycans<br />
expressed in the two groups (de Medeiros Matsushita, da silva et al. 2005).<br />
1.6.2 Studies <strong>of</strong> inflammation using bronchoscopy and biopsy<br />
<strong>The</strong> use <strong>of</strong> bronchoscopy was developed initially as a therapeutic measure by Dr Chevalier<br />
Jackson in 1904 (Reynolds 1987), and later became a way <strong>of</strong> obtaining airway samples. <strong>The</strong><br />
flexible fibreoptic bronchoscope was introduced into medical centres in the early 1970s<br />
(Ikeda 1970) with appropriate guidelines (Smiddy, Ruth et al. l97l; Sackner, Wanner er al.<br />
1972)' which were later modified to include standards for bronchoalveolar lavage (BAL)<br />
(Bernstein, Boushey et al. 1985; Reynolds 1987; Turner-Warwick and Haslam l9g7) and<br />
detailed indications and protocols for the procedure were subsequently published (Klech and<br />
Pohl 1989; Kelch and Hutter 1990; Bleecker, McFadden et al. l99Z: Rennard, Aalbers et al.<br />
1998; Haslam and Baughman 1999; Reynolds 2000). <strong>The</strong> development <strong>of</strong> paediatric<br />
bronchoscopy commenced a decade later (Wood and Sherman 1980; Wood l9g5) when<br />
construction <strong>of</strong> smaller bronchoscopes became possible and these entered clinical practice in<br />
the mid 1990s (Perez and Wood 1994; Wood 1996; Rennard, Aalbers er al. 1998; ERS 2000;<br />
Wood 20[l; Midulla, de Blic et aL.2003). Thus these technical advances allowed biopsy and<br />
BAL studies in 'living' patients to be undertaken.<br />
25
In brief, the human airway can be divided into three layers; the inner wall (epithelium,<br />
basement membrane and the submucosa), the outer wall (loose connective tissue), and the<br />
smooth muscle layer (Bergeron and Boulet 2006). All layers have shown some alterations in<br />
obstructive respiratory disease compared to normal lung. with regard to the presence <strong>of</strong><br />
inflammation, biopsy studies have shown increases in eosinophils, eosinophilic proteins, T<br />
lymphocytes (Foresi, Bertorelli et al. 1990; Poston, chanez et al. 1992: Laitinen, Laitinen et<br />
al. 1gg6; carrou, cooke et al. lggT) and certain cytokines (or their messenger ribonucleic<br />
acid (mRNA)) such as interleukin 2 (n-2), interleukin 3 (IL3), IL5, granulocyte macrophage<br />
colony stimulating factor (GM-CSF), tumor necrosis factor alpha (TNF0) and rantes when<br />
compared to both normal subjects and patients with other respiratory diseases (Azzawi'<br />
Bradley et al. 1990; Ackerman, Marini er al. 1994; Humbert, Durham et al' 1996; Powell'<br />
Humbert et al. 1996; Vrugt, Wilson et al. lggg). <strong>The</strong> presence <strong>of</strong> inflammation was confirmed<br />
even in mild asthma @easley,<br />
Burgess et al. 1993; Laitinen, Laitinen et al' 1996;<br />
Grootendorst, Sont et al. t997; Laitinen, Karjalainen et al. 2000; ward, Reid et al' 2005) and<br />
in patients during disease remission (Foresi, Bertorelli et al. 1990). A positive correlation with<br />
each <strong>of</strong> these parameters to the severity <strong>of</strong> asthma has been demonstrated by some studies<br />
(Beasley, Burgess et al. 1993; Carroll, cooke et al. 1997; Vrugt, wilson et al' 1999; Amin'<br />
Ludviksdottir et al. 2000; Barnes, Burke et al. 2000; Benayoun, Druilhe et al' 2003)' In<br />
addition, some markers more commonly associated with infective diseases, such as<br />
interleukin g (ILg) and eotaxin, have also been demonstrated to be positively conelated with<br />
asthma severity (Lamkhioued, Renzi et al. 1997; Pepe, Foley et al' 2005)' However' the<br />
correlations <strong>of</strong> these markers with severity have not been unanimous @enayoun,<br />
Druilhe et<br />
al.2oI3;shahana, Bjornsson et al. 2005) and overall no unique inflammatory pr<strong>of</strong>ile has been<br />
demonstrated for all asthmatic patients (Kavuru, Dweik et al. 1999)'<br />
In addition to the increase in inflammation demonstrated in asthma, there have also been<br />
increases noted in the airway wall components. <strong>The</strong>se include airway smooth muscle' mucus<br />
glands, and submucosal and extra-cellular matrix tissue which have also been related to<br />
symptom severity and bronchial hyper-responsiveness<br />
(Saetta, Di Stefano et al' 1991; Chetta'<br />
Foresi et al. 1997;Benayoun, Druilhe et al. 2003; chen, samson et al'20o4; Pepe' Foley et al'<br />
2005). <strong>The</strong> presence <strong>of</strong> high numbers <strong>of</strong> fibroblasts has been the most consistent feature<br />
demonstrated to relate to severity <strong>of</strong> disease and poor response to treatment (wenzel'<br />
Schwartz et ar. lggg; Boulet, Turcotte et al. 2000). In the last decade, these findings have<br />
taken on new importance as to their relevance to possible remodelling <strong>of</strong> the airway resulting<br />
in irreversible changes occurring (Jeffery, Laitinen et al' 2000; wilson and Bamford 2001;<br />
26
Black and Johnson 2002; Jeffery 2004; Kariyawasam and Robinson 2005: ward and walters<br />
2005). This airway remodelling in asthma has been found in both large and small airways<br />
(Saetta, Di Stefano et al. l99l; James, Maxwell et al. 2OO2) and the changes are the same<br />
regardless <strong>of</strong> the asthma being atopic, occupational or intrinsic (Homer and Elias 2000). <strong>The</strong><br />
inner wall components include loss <strong>of</strong> epithelial integrity (Naylor 1962; Laitinen, Heino et al.<br />
1985; Jeffery, Wardlaw et al. 1989) and hyperplasia <strong>of</strong> the globlet cells in the sub-epithelium<br />
(Aikawa, Shimura et al. 1992: Ordonez, Khashayar et al. 2001). While rhickening <strong>of</strong> the<br />
basement membrane was also described in the original autopsy and other studies above, it<br />
now appears that the true basement membrane (the 'lamina rare' and 'lamina densa') is not<br />
grossly altered (Roche, Beasley et al. 1989; Homer and Elias 2000) with the actual fibril<br />
content <strong>of</strong> the membrane found to be similar in asthmatic and control groups (Saglani,<br />
Molyneux et al. 2006). It is the area that sits immediately below the true basement membrane,<br />
'the lamina reticularis' that is increased and this has been termed 'subepithelial fibrosis' or<br />
'reticular or sub-basement membrane thickening' (Boulet, Laviolette et al. 1997;Elias, Zhu et<br />
al. 1999; Beckett and Howarth 2OO3). Here, enhanced collagen deposition has been<br />
demonstrated with subtypes I, III and IV, fibronectin, tenascin, Iumican and biglycan @oche,<br />
Beasley et al. 1989; Laitinen, Laitinen et al. 1996; Laitinen, Altraja et al. 1997;Wilson and Li<br />
1997 Huang, Olivenstein et al. t999; Benayoun, Druilhe et al. 2003; Karjalainen, Lindqvist<br />
et al. 2O03). My<strong>of</strong>ibroblasts are specialized cells that have features <strong>of</strong> both myocytes and<br />
fibroblasts and are sources <strong>of</strong> interstitial collagens so are likely contributing to this deposition<br />
(Homer and Elias 2000). My<strong>of</strong>ibroblast number has been demonstrated to correlate directly to<br />
the thickness <strong>of</strong> this layer (Brewster, Howarth et al. 1990; Hoshino, Nakamura et al. l99g).<br />
However, it is the increased thickness <strong>of</strong> the smooth muscle layer that contributes most to the<br />
overall thickness <strong>of</strong> the airway. This has variably been described as due to hypertrophy and/or<br />
hyperplasia (Hossain 1973; Ebina, Takahashi et al. 1993; Cho, Seo et al. 1996;James lggT).<br />
<strong>The</strong> smooth muscle contraction has been shown to particularly narrow the airway where the<br />
entire lumen is surrounded as in the smaller bronchi prior to respiratory bronchioles. This is<br />
consistent with computer modeling <strong>of</strong> ainvay function, showing that the smaller airways are<br />
the site <strong>of</strong> the greatest increase in airway resistance in asthma (Hogg lggT). Finally, changes<br />
have also been described in the airway vasculature with an increase in both size and number<br />
<strong>of</strong> vessels and this angiogenesis also contributes to airway oedema seen (Li and Wilson 1997;<br />
Tanaka, Yamada et al.20O3). Given the variable nature <strong>of</strong> the findings it is difficult to show<br />
the exact nature <strong>of</strong> the progression, and this is the subject <strong>of</strong> ongoing research (Boulet 2000).<br />
27
<strong>The</strong> ability to biopsy has led to studies <strong>of</strong> the early (Gonzalez,Diaz et al. 1987; Georas, Liu et<br />
al. 1992) and late asthma reactions (Robertson, Kerigan et al. 1974; Crimi, Chiaramondia et<br />
al. 1991) and a comparison <strong>of</strong> both (Metzger, Richerson et al. 1986; Metzger, Richerson et al.<br />
1986; Aalbers, Kauffman et al. 1993) in response to deliberate allergy provocation. <strong>The</strong>se<br />
demonstrated an influx <strong>of</strong> neutrophils at 6-8 hours followed by an influx <strong>of</strong> eosinophils at24-<br />
48 hours which continued more than a week but seemed to resolve by day 16. An increase in<br />
the cellular expression <strong>of</strong> the mRNA <strong>of</strong> IL5 and GM-CSF occurred at 18 to 48 hours<br />
(Ohnishi, Sur et al. 1993; Tang, Rolland et al. 1997) with an increase in total proteins and<br />
ainvay permeability also in the late phase (Taylor, Hill et al.1996; Teeter and Bleecker 1996).<br />
In assessing response to treatment in asthmatic patients treated with moderate to high doses <strong>of</strong><br />
IHCS (400mcg to l600mcdday beclomethasone equivalent) for between two weeks and up 5-<br />
l0 years, most studies have reported a decrease in T-lymphocytes, eosinophils, and mast cell<br />
numbers in the airway wall (Lundgren, Soderberg et al. 1988; Burke, Power et al. 1992;<br />
Booth, Richmond et al. 1995; Djukanovic, Homeyard et al. 1997; Olivieri, Chetta et al. L997i<br />
Barnes, Burke et al. 2000). As well, decreased dendritic cell number @urke, Power et al.<br />
1992; Trigg, Manolitsas et al. 1994; Bentley, Hamid et al. 1996) and reduced inflammatory<br />
cell activation measured as less gmnule secretory production and less cell membrane<br />
activation markers have also been noted @entley, Hamid et al. 1996; Olivieri, Chetta et al.<br />
1997; Barnes, Burke et al. 2000; Ward, Reid et al. 2005). <strong>The</strong> expression <strong>of</strong> mRNA for nA,<br />
IL5 and GM-CSF within cells also decreased with steroid treatment (Sousa, Poston et al.<br />
1993; Bentley, Hamid et al. 1996). Interestingly, the effects have been more marked in the<br />
biopsy samples than the corresponding lavage studies (Booth, Richmond et al. 1995;<br />
Thompson, Teschler et al. 1996: Djukanovic, Homeyard et al. 1997; Olivieri, Chetta et al.<br />
1997). <strong>The</strong> effect <strong>of</strong> steroids on the thickness <strong>of</strong> the epithelium and basement membrane have<br />
been less conclusive; some studies showing improvement (Olivieri, Chetta et al. 1997; Ward<br />
and Walters 2005) that has not been demonstrated in others even after years <strong>of</strong> treatment<br />
(Lundgren, Soderberg et al. 1988; Wenzel, Szefler et al. 1997; Wenzel, Schwartz et al. 1999;<br />
Barnes, Burke et al. 2000).<br />
Paediatric studies have been more recent, in keeping with the later technical bronchoscopy<br />
development and the additional concerns regarding safety in this group. <strong>The</strong>se have shown<br />
similar findings to the adult asthma studies with eosinophilic and lymphocytic inflammation,<br />
and an increase in the same cytokines (interleukin 4 (IL+), IL5, rantes) (de Blic, Tillie-<br />
Irblond et al. 2OO4; Payne, Qiu et al. 2O04; Pohunek, Warner et al. 2005). <strong>The</strong> findings<br />
associated with airway remodeling (smooth muscle hypertrophy and reticular basement<br />
28
membrane thickening) were present early in a study in children with an age range down to 1.2<br />
years (Pohunek, Warner et al. 2005) and did not show differences between those with few<br />
symptoms compared to those with persistent symptoms or 'difficult to control' asthma<br />
(cokugras, Akcakaya et al. 2001; Jenkins, cool et aL.2003; payne, Rogers et al.29613; de<br />
Blic, Tillie-kblond et al. 2004; Fedorov, Wilson et al. 2005). In addition, the findings were<br />
not associated with age, length <strong>of</strong> symptoms, lung function or bronchial reactivity (payne,<br />
Rogers et al. 2003). Bronchial wall thickening on high resolution computerized tomography<br />
(HRCT) scan was shown to correlate with eosinophils numbers, ECp and sub-basement<br />
membrane thickening (de Blic, Tillie-Irblond et al. 2005).<br />
1.6.3 studies <strong>of</strong> inflammation using bronchoalveolar lavage<br />
In adults the standard bronchoscope wedges between the 4th and 6ft order bronchus<br />
(Hunninghake, Gadek et al. 1979) typically localising a lung zone with 106 alveoli or a<br />
volume <strong>of</strong> l65ml at total lung capacity and 45ml at residual volume. In the 1960s, two studies<br />
initially started using catheters to do small volume bronchopulmonary lavage for assessment<br />
<strong>of</strong> disease @nley, Swenson et al. 1967; pratt, Finley et al. 1969). standard guidelines for<br />
clinical, diagnostic and research practices have been developed for use in adults (Klech and<br />
Pohl 1989; American Thoracic Society 1990; Rennard, Aalbers et al. 1998; Reynolds 2000),<br />
and more recently for use in children (ERS 2000; Midulla, de Blic et al. 2003). Lavage studies<br />
have confirmed many <strong>of</strong> the biopsy study findings in asthmatics when compared to normal<br />
subjects. Increases are seen in eosinophils, T-lymphocytes, eosinophilic proteins and<br />
cytokines such as n-4,L5, rantes and chemo-attractant factors with excellent recent reviews<br />
detailing these findings (Barrios, Kheradmand et aL.2006; Graham 2006: Tulic and Hamid<br />
2006). An antigenic or exercise challenge resulted in an observed increase in eosinophils<br />
(Kirby, Hargreave et al. 1987; Wardlaw, Dunnette et al. 1988; Bousquet, Van Vyve et al.<br />
1993; Krug, Tschernig et al. 2001), T cell lymphocytes (Virchow, Walker et al. 1995;<br />
Schuster, Tschernig et al. 2000), mast cells (Kirby, Hargreave et al. 1987; Bousquet, Van<br />
Vyve et al. 1993; Olsson, Rak et al. 2000), basophils and neutrophils (Kirby, Hargreave et al.<br />
1987; Mattoli, Mattoso et al. 1991; Bousquet, Van Vyve et al. 1993), complement factors<br />
C3A and C5 (Krug, Erpenbeck et al. 2001) and eosinophilic proteins (Wardlaw, Dunnette et<br />
al. 1988).<br />
Many more proteins can be measured in lavage fluid and this can demonstrat e the effect <strong>of</strong><br />
cells' even if the latter are not as visible. For example, mast cells account for up to 20Vo <strong>of</strong> the<br />
inflammatory cells and basophils are also prominent in biopsy studies, but both are only a<br />
29
small proportion <strong>of</strong> cells recovered by BAL @unnill 1960; Foresi, Bertorelli et al. 1990;<br />
Koshino, Teshima et al. 1993; Macfarlane, Kon et al. 2000). Provocation studies have shown<br />
the early asthma response is associated with increased histamine, tryptase, prostaglandinD2,<br />
prostaglandin F2, thromboxane and leukotrienes which are released by these two cell types<br />
(Murray, Tonnel et al. 1986; Wenzel, Fowler et al. 1988; Liu, Hubbard et al. l99l; Chilton,<br />
Averill et al. 1996; Kavuru, Dweik et al. 1999). Adhesion molecules such as intracellular<br />
adhesion molecule I (ICAM l) and lymphocyte function-associated antigen I (IJA 1) were<br />
also increased (Williams, Johnson et al. 1992). <strong>The</strong> cytokines associated with an allergic<br />
pr<strong>of</strong>ile - IL3, IIA,ILI and GM-CSF -<br />
and their receptors in cell surfaces were measured in<br />
high amounts (Virchow, Walker et al. 1995; Chung and Barnes 1999; Hamid, Tulic et al.<br />
2003). Increases have also been seen in IL9 and ILl3 which have been associated with mucus<br />
hypersecretion and IgE regulation (Louahed, Toda et al. 2000; Shimbara, Christodoulopoulos<br />
et al. 2000; louahed, Zhou et al. 2001) as well as increased interleukin 11 (Lll) and<br />
interleukin 17 (lLl7) that have pro-fibrotic activity @inarsson, Geba et al. 1995; Minshall,<br />
Chakir et al. 2000; Molet, Hamid et al. 2001). On the other hand, decreased interleukin 10<br />
(L10), an anti-inflammatory cytokine, was seen during acute asthma and this returned to<br />
levels similar to controls with recovery (Borish, Aarons et al. 1996). Chemokines have been<br />
demonstrated as present and rising with airway challenges including eotaxin, monocyte<br />
chemoattractant protein and rantes as well as TGFp which has been noted to have a role in the<br />
subepithelial fibrosis (Hamid, Tulic et al. 2003). With specific challenges, such as using<br />
ragweed antigen, specific IgE and IgA have also been shown to be increased in<br />
bronchoalveolar lavage fluid @eebles, Hamilton et al. 2001). Similar to the findings from the<br />
biopsy studies, with treatment <strong>of</strong> either IHCS or oral corticosteroids, while there was a<br />
reduction <strong>of</strong> eosinophil, mast and epithelial cell numbers in lavage fluid, the eosinophilic<br />
proteins did not reduce and there was not a direct correlation between the inflammatory cell<br />
decrease and improvement in symptoms or bronchial hyper-responsiveness @uddridge, Ward<br />
et al. L993; Booth, Richmond et al. 1995; Djukanovic, Homeyard et al.1997; Olivieri, Chetta<br />
et al. t997).<br />
<strong>The</strong> paediatric literature in this area has been more recent but has largely been in agreement<br />
with the findings in adults. <strong>The</strong> increase in eosinophilic inflammation and eosinophil proteins<br />
were seen (Barbato, Panizzolo et al. 2001; Just, Fournier et al.2002; Najafi, Demanet et al.<br />
2003), as well as increased neutrophils which were associated with persistent symptoms (Just,<br />
Fournier et al. 2OO2), 'difficult to treat' asthma (de Blic, Tillie-Irblond et al. 2004; Payne,<br />
Qiu et al. 2OO4) and status asthmaticus (Lamblin, Gosset et al. 1998; Tonnel, Gosset et al.<br />
30
2001). In paediatrics, BAL has also been used to compare inflammatory pr<strong>of</strong>iles with other<br />
diseases that present with wheeze. An increase in eosinophil cell and proteins was seen in a<br />
group <strong>of</strong> asthmatics compared to control and bronchiolitis groups (Kim, Chung et al. 2000;<br />
Marguet, Dean et al.2C0l; Kim, Kim et al.2003; Kim, Kim et al. 2005). However, ILg levels<br />
also correlated with the eosinophil level in the asthmatic group (Kim, Kim et al. 2003) and the<br />
number <strong>of</strong> neutrophils appeared to reflect severity <strong>of</strong> asthma (Marguet, Dean et al. 2001). A<br />
recent summary <strong>of</strong> these studies into airway inflammatory pr<strong>of</strong>iles in paediatric asthma has<br />
been published (Warner 2003).<br />
1.6.4 Inflammatory markers in induced sputum<br />
1.6.4 (i) Studies in adult subjects<br />
Non-isotonic ultrasonically produced aerosols were first used more than fifty years ago to<br />
examine bronchial reactivity (Cheney and Butler lgT}).Currently, nebulised hypertonic saline<br />
is used for three main reasons. Firstly, it can act as an airway challenge to evaluate hyper-<br />
responsiveness. Secondly, it can be utilised as a treatment to aid expectoration <strong>of</strong> the<br />
thickened sputum produced in some respiratory diseases, particularly CF @ng, Morton et al.<br />
1996; Wark, McDonald et al. 2005; Donaldson, Bennett et al. 2006: Elkins, Robinson et al.<br />
2006). Thirdly, it has been used to induce sputum for microbiological assessment (for<br />
example in CF and TB) and/or for the harvest <strong>of</strong> cells and cytokines for research. <strong>The</strong><br />
technique consists <strong>of</strong> inhaling saline at 3-7Vo concentration generated by an ultrasonic<br />
nebuliser, which is necessary to give a sufficient output for saline aerosol generation, for l0 to<br />
30 minutes (Belda, Hussack et al. 2001). Patients or research subjects are requested to cough<br />
and try to expectorate sputum into a container every 3-5 minutes (paggraro, Chanez et al.<br />
2002). Dithiothreitol O.lVo is used to break down mucus suphylhydryl bonds and to disperse<br />
cells. <strong>The</strong> samples are then centrifuged with slides made <strong>of</strong> the cellular component and<br />
stained to identify cell viability, total cell counts, the percentage <strong>of</strong> squamous cells and<br />
differential percentage or numbers <strong>of</strong> the other cell types. <strong>The</strong> sputum supernatant following a<br />
cytospin preparation is then used for measurement <strong>of</strong> soluble mediators. <strong>The</strong> mechanism by<br />
which hypertonic saline causes sputum production is uncertain, but thought to be a<br />
combination <strong>of</strong> the osmotic movement <strong>of</strong> water into airways, the induction <strong>of</strong> cough and the<br />
increased rate <strong>of</strong> mucociliary clearance (Umeno, McDonald et al. 1990; Holz, Kips et al.<br />
2000; Paggiaro, Chanez et aL.2002). This technique was first described in the early 1990s to<br />
obtain sputum samples from asthmatic and normal adult subjects (Fahy, Liu et al. lgg3). A<br />
task force <strong>of</strong> European Respiratory Society members developed the ..Standardised<br />
3l
methodology <strong>of</strong> sputum induction and processing" @jukanovic, Sterk et al. 2002) which has<br />
a detailed report on sputum induction @aggiaro, Chanez et al.2002), methods <strong>of</strong> processing<br />
@fthimiadis, Spanevello et al. 20f.2) and measurement <strong>of</strong> fluid-phase mediators (Kelly,<br />
Keatings et aI,2002) concentrating on techniques and findings in asthma and COPD subjects.<br />
Pre-treatment with p2 agonists as appropriate and lung function and/or oxygen saturation<br />
monitoring is recommended throughout the procedure @aggiaro, Chanez et al.2002).<br />
<strong>The</strong> eady studies confirmed and enlarged on the inflammatory pr<strong>of</strong>iles seen in asthmatics<br />
compared to normal subjects as had been described in the biopsy and lavage studies above.<br />
An eosinophilic dominant inflammation was seen with increases in total cell numbers, as well<br />
as individual eosinophil and neutrophil counts, eosinophil derived proteins [ECP, eosinophil<br />
derived neurotoxin (EDNI and major basic protein (MBP)1, (Pin, Gibson et al. 1992; Fahy,<br />
Liu et al. 1993; Foresi, I-eone et al. 1997; Louis, Shute et al. 1997; Bacci, Cianchetti et al.<br />
1998; Park, Whang et al. 1998; Rosi, Ronchi et al. 2000) the allergic pr<strong>of</strong>ile interleukins (IL3,<br />
nA, L5, Ll0) and certain proteins such as tryptase, eosinophilic chemo-attractant factors,<br />
and adhesion molecules (Shoji, Kanazawa et al. 1998; Hamzaoui, Brahim et al. 2000).<br />
Correlations between these sputum inflammatory cells and markers and clinical parameters<br />
have been investigated. <strong>The</strong> strongest associations appear to be during an acute asthma<br />
exacerbation with increases in eosinophils, activated eosinophils and ECP (Jang and Choi<br />
2000; Jang, Choi et al. 2000), basophils and mast cells @in, Freitag et al. 1992; Gauvreau,<br />
I*e et al. 2000). During times <strong>of</strong> increased symptoms, eosinophil number, ECP, IL5 and<br />
albumin were all found to independently contribute to abnormalities <strong>of</strong> FEVr and FVC in<br />
asthmatics (Fujimoto, Kubo et al. 1997; Bacci, Cianchetti et al. 1998; Shoji, Kanazawa et al.<br />
1998; Grebski, Wu et al. 1999; Woodruff, Khashayar et al. 2001; Bartoli, Bacci et al.2N4).<br />
Severe asthma was predicted by high eosinophil numbers, ECP and albumin concentrations,<br />
though there was no such gradation in mild to moderate asthmatics (Fujimoto, Kubo et al.<br />
1997; Bartoli, Bacci et aL.2ffi4). <strong>The</strong> eosinophil counts gave a sensitivity <strong>of</strong> 68.3Vo, and a<br />
specificity <strong>of</strong> 55.3Vo for moderate to severe asthma compared to mild, and was <strong>of</strong> a higher<br />
diagnostic value for asthma than sputum ECP or methacholine challenge (Park, Whang et al.<br />
1998; Rosi, Ronchi et al. 2000).<br />
I 'Eosinophil protein X' and 'eosinophil derived neurotoxin' is the same protein but referred to by these two<br />
different names throughout studies, and denoted as eitler 'EPX' or 'EDN'. I have elected to refer to it as<br />
'eosinophil derived neurotoxin' and 'EDN'. Eosinophil cationic protein, eosinophil derived neurotoxin, major<br />
basic protein and eosinophil peroxidase are all separate proteins.<br />
32
<strong>The</strong> correlations are weaker when looking at asthmatics during times <strong>of</strong> stability<br />
(Grootendorst, Sont et al. 1997; Ronchi, Piragino et al. lggT). No relationship was<br />
demonstrated with steroid dose or lung function test results in over 100 stable asthmatics and<br />
40 normal subjects in one community sample <strong>of</strong> adults (Iouis, Sele et a\.2002). Eosinophils<br />
were frequently within the normal range and not a useful diagnostic tool in a population<br />
divided into 'normal subjects', 'asthmatics', 'wheeze but no asthma' and 'industrial exposure<br />
to irritants' (I-emiere, Walker et al. 2001). Eosinophils, ECp, MBp, EDN and IL5 correlated<br />
with methacholine challenge and FEVr in adult asthmatics (Pizzichini ,Pizzichiniet al. 1996).<br />
However, the eosinophil numbers only accounted for 167o <strong>of</strong> the variance <strong>of</strong> the methacholine<br />
challenge across one group, while the TNFo levels correlated more closely (Obase, Shimoda<br />
et al. 2001). Other studies showed no correlation between eosinophilic inflammatory<br />
parameters and lung function (Hashimoto, Minoguchi et al. 1999; Rosi and Scano 2000),<br />
methacholine (Rosi and Scano 2000) or histamine challenges (Iredale, Wanklyn et al. 1994;<br />
Hashimoto, Minoguchi et al. 1999). Eosinophils, leukotriene Ea (LTE+), ECp and rantes were<br />
all increased in poorly controlled asthmatics rather than being consistently high in severe<br />
asthmatics, for example if patients were well controlled (Romagnoli, Vachier et al. 2002).In<br />
addition, they were high only with symptoms even in long-term corticosteroid dependent<br />
patients (Tarodo de la Fuente, Romagnoli et al. 1999). Considerable heterogeneity in sputum<br />
counts were seen in over 250 adult asthmatics and normal subjects with eosinophils <strong>of</strong> all<br />
amounts (Green, Brightling et al.2OO2).<br />
Many studies have used sputum to determine the best predictor <strong>of</strong> response to steroids. When<br />
commencing longer term IHCS treatment in placebo controlled trials, sputum eosinophils<br />
correlated to the response in a number <strong>of</strong> studies (van Rensen, Straath<strong>of</strong> et al. 1999; Aldridge,<br />
Hancox et al. 2002: Deykin, Lazarus et al. 2005; Brightling 2006) and was a better marker<br />
than ECP levels in either sputum or blood (van Rensen, Straath<strong>of</strong> et al. 1999; Rosi, Ronchi et<br />
al. 2000; Aldridge, Hancox et al. 2002: Deykin, Lazarus et al.2o05), baseline lung function<br />
@eykin, Lazarus et al. 2005) or methacholine challenge results (van Rensen, Straath<strong>of</strong> et al.<br />
1999; Deykin, Lazarus et al. 2005). Acutely, sputum eosinophils and ECp fell with oral or<br />
intravenous steroid treatment (claman, Boushey et al.lgg4; Rosi, Lanini et al. 2002) with the<br />
eosinophil count giving a positive predictive value <strong>of</strong> 68Vo, sensitivity <strong>of</strong> 54Vo and specificity<br />
<strong>of</strong> 76Vo for an increase in FEVr >l5%o with steroid therapy (Little, Chalmers et al. 2000).<br />
Eosinophils, neutrophils, ECP, IL5 and fibrinogen all increased when treatment was reduced<br />
but the eosinophil count was consistently the most accurate, across a number <strong>of</strong> studies, to<br />
predict when treatment reduction would lead to symptom development (Pizzichini, pizzichini<br />
33
et al. 1999; Giannini, Di Franco et al. 2000; Jatakanon, Lim et al. 2000; Green, Brightling et<br />
al. 2002). <strong>The</strong> results seen across all the studies suggest that the inflammation measured in<br />
this way reflects current asthma control rather than grading chronicity or severity per se.<br />
As indicated in the biopsy and lavage studies above, increased neutrophil counts may also be<br />
a marker for poorly responsive disease. In severe asthmatics, there were more neutrophils and<br />
less eosinophils compared to mild asthmatics (Loh, Kanabar et al. 2005) and neutrophilic<br />
inflammation was dominant in the minority <strong>of</strong> subjects from over 250 adult asthmatics who<br />
demonstrated less corticosteroid response (Green, Brightling et al. 2002). Both eosinophilic<br />
inflammation and neutrophilic inflammation were found to independently contribute to<br />
abnormalities <strong>of</strong> FEVr in 205 adult asthmatics (Woodrufl Khashayar et d. 2001). Treating<br />
the neutrophil count resulted in better control than monitoring lung function or symptoms<br />
(Chlumsky, Striz et al.20O6). Neutrophils were also increased acutely, making up more than<br />
757o <strong>of</strong> the sputum cells in 10 <strong>of</strong> 18 adult subjects during acute asthma while eosinophils<br />
made up more thanT1Vo <strong>of</strong> the cells in only three subjects (Fahy, Kim et al. 1995).<br />
1.6.4 (ii) Induced sputum in children<br />
Sputum induction in children developed more recently and therefore in some <strong>of</strong> the studies<br />
from the 2000s it has been combined with NO measurement. As this will be covered in depth<br />
in later parts <strong>of</strong> the thesis, I will only discuss the cell and cytokine inflammatory markers<br />
from induced sputum in this section. <strong>The</strong> technique has been used for getting bacterial, viral,<br />
fungal and tuberculosis cultures to determine infective aetiology (Merrick, Sepkowitz et al.<br />
L997; Utsunomiya, Ahmed et al. 1998; Ordonez, Henig et al. 2003; kiso, Mudido et al. 2005;<br />
Ratjen 2W6). Using induced sputum to assess inflammatory disease was first described in<br />
1980 when presence <strong>of</strong> an eosinophilia was used to diagnose co-existent asthma in children<br />
with CF was investigated (Sly and Hutchison 1980). In 1995 sputum and nasal smears taken<br />
from I 11 young children presenting acutely with wheeze showed increased eosinophils,<br />
neutrophils and basophils in those deemed 'asthmatic' compared to those who were wheezy<br />
but unresponsive to asthma treatment (Twaddell, Gibson et al. 1996). Increased eosinophils at<br />
3.8Vo and epithelial cells at tl.S%o were seen in 16 children with uncontrolled asthma<br />
compared to 15 with controlled asthma at 2.5Vo and lO.SVo and 72 healthy non-asthmatic<br />
children at0.3Vo and l.5Vo respectively (Cai, Carty et al. 1998). Mast cells, prominent in adult<br />
asthma studies, were found in only 4 <strong>of</strong> the 42 asthmatic children (Cai, Carty et al. 1998). An<br />
increase in eosinophil percentage and ECP was seen in 50 asthmatic children compared to<br />
fifteen children with chronic bronchitis (CB) and 25 healthy children (Yazicioglu, Ones et al.<br />
34
1999).In 146 children with asthma and 37 controls, the percentage <strong>of</strong> eosinophils increased<br />
from a mean <strong>of</strong> l.S%o in children who had infrequent asthma to2.3Vo with frequent asthma<br />
and to 3.8Vo if they had persisting symptoms, compared to controls at lTo. Similarly, ECp<br />
Ievels increased from l l3ng/ml in infrequent asthma to 220nglml in frequent asthma and<br />
37Snglml in the persistent asthmatics compared to 139ng/ml in the control goup. While there<br />
were significant differences between the asthmatic groups, no difference was demonstrated<br />
between the 'infrequent exacerbation' and 'control' children (Gibson, Simpson et al. 2003).<br />
However, in 27 healthy and 60 asthmatic children, no correlation was seen with any marker <strong>of</strong><br />
airway inflammation and asthma severity as measured by lung function (Wilson, Bridge et al.<br />
2000). In addition, in 58 asthmatic children, it was the methacholine challenge rather than the<br />
sputum eosinophil counts or ECP that related to the presence <strong>of</strong> recent symptoms (Wilson,<br />
James et al. 2001). No correlation was demonstrated between sputum eosinophils and FEVI or<br />
sputum ECP in 25 asthmatic children (piacentini, Bodini et al. 1999). Further, no correlations<br />
were demonstrated in 32 children with stable asthma between ambulatory cough frequency,<br />
sputum inflammatory parameters or FEVI (Li, Irx et al. 2003). In 40 children with difficult<br />
asthma, only nine children had abnormal sputum cytology; six had a predominant sputum<br />
eosinophilia while three had a predominant neutrophilia (Irx, Payne et al. 2005).<br />
Within one hour <strong>of</strong> arriving in the emergency department with acute asthma, eight children in<br />
one study and 38 in a second took less time to produce an induced sputum sample than when<br />
done during a time <strong>of</strong> stability. <strong>The</strong> sputum showed higher numbers <strong>of</strong> total cells, eosinophils,<br />
neutrophils, basophils and mast cells compared to macrophages which was the dominant cell<br />
during the recuperative phase when the sampling was repeated two weeks later with symptom<br />
resolution (Twaddell, Gibson et al. 1996; Norzila, Fakes et al. 2000). <strong>The</strong> second study also<br />
demonstrated reductions in ECP, myeloperoxidase, IL5 and IL8 by the recovery sample. <strong>The</strong><br />
primary cell <strong>of</strong> inflammation was either eosinophil or neutrophil or showed a co-dominant<br />
pattern (Norzila, Fakes et al. 2000). Following a treatment with single nebulised<br />
glucocorticoid dose, there was a reduction in eosinophil percentage in 30 asthmatic children<br />
but ECP, IL5, GM-CSF and albumin levels, as well as FEV1, remained unchanged (Oh,I*e et<br />
al.1999).<br />
A number <strong>of</strong> studies have looked at the inflammation in induced sputum before and after<br />
commencing IHCS treatment for more chronic asthma symptoms. In 14 children reduced<br />
sputum and serum eosinophils and ECP levels were seen after two weeks <strong>of</strong> IHCS treatment<br />
(Sorva, Metso et al. 1997). And in 60 children, there was reduction in sputum eosinophils<br />
35
over a six month treatment prograrnme, with no colTesponding reduction in those on disodium<br />
cromoglycate (Rytila, Pelkonen et al. 2004). In the 'Childhood Asthma Management<br />
Program' (CAMP) study, IHCS treated patients had a lower sputum percentage <strong>of</strong> eosinophils<br />
at 0.2Vo versus 0.87o than those treated with nedocromil sodium or placebo. In repeated<br />
sputum inductions during reduction <strong>of</strong> medication, sputum eosinophil counts predicted an<br />
exacerbation (Covar, Spahn et al. 2004).<br />
1.6.4 (iii) Technical aspects <strong>of</strong> bronchoscopy, bronchial biopsy, bronchoalveolar lavage<br />
and/or induced sputum<br />
Despite the increasing ability to sample from the lung with the use <strong>of</strong> bronchoscopy, airway<br />
biopsy, BAL and induced sputum, there remain questions over the success, reproducibility<br />
and the safety <strong>of</strong> these techniques. To start with, there is significant variability both within<br />
and between biopsy samples even when taken from one individual @ichmond, Booth et al.<br />
1996; Sont, Willems et al. 1997; Jeffery, Laitinen et al. 2000). <strong>The</strong> parameters from biopsy<br />
measurements are less reproducible than physiological tests, with even diurnal variation in<br />
number and function <strong>of</strong> inflammatory cells @oulter, Burke et al. 2000). Biopsy specimens are<br />
also likely to be taken from quite different sites, from both proximal and distal airways, which<br />
may not be comparable @alzar, Wenzel et al. 2N2; Boulet 2N2; James and Carroll 2002).<br />
BAL variability was investigated by doing 180 procedures in 2O clinically stable but<br />
symptomatic asthmatic adults. <strong>The</strong> standard deviation for total cell counts was 78 X 103<br />
cells/ml and for macrophages was 16%o,lymphocytes was l3%o and eosinophils 17o (Ward,<br />
Gardiner et al. 1995; Ruffin 1996). <strong>The</strong> lavage pr<strong>of</strong>ile seen in asthma, has also been seen in<br />
allergic rhinitis (Gutierrez, Prieto et al. 1998; Alvarez, Olaguibel et al. 2000; Polosa, Ciamarra<br />
et al. 2000; Boulay and Boulet 200.2; Beeh, Beier et al. 2O03; Wilson, Duong et al. 2005;<br />
Bonay, Neukirch et al. 2006; Hara, Fujimura et al. 2006) and in subjects with atopic<br />
dermatitis (Kyllonen, Malmberg et al. 2006).<br />
<strong>The</strong> initial success rate <strong>of</strong> hypertonic saline in obtaining samples in adults was 757o (Pin,<br />
Gibson et al. 1992; Foresi, Irone et al. L997; Hashimoto, Minoguchi et al. 1999) but<br />
improved to 83Vo to 93Vo with refinement <strong>of</strong> the procedure (Vlachos-Mayer, Irigh et al.<br />
2000; Bartoli, Bacci et al.2OO4). <strong>The</strong> success <strong>of</strong> sputum induction in children has been given<br />
as 60 to 74Vo in controls (Wilson N M 2000) and 6IVo to 94Vo in asthmatics (Norzila, Fakes et<br />
al. 2000; Wilson, Bridge et al. 2000; Li, I-ex et al. 2003; Covar, Spahn et al.2004; Rytila,<br />
Pelkonen et al. 2OO4; I-ex, Payne et al. 2005). It is more successful in acute than chronic<br />
asthma (Norzila, Fakes et al. 2000; Rytila, Pelkonen et al.2004) and more successful in the<br />
36
older children, particularly those > 12 years <strong>of</strong> age (Norzila, Fakes et al. 2000; Covar, Spahn<br />
et aL.2004; Rytila, Pelkonen et aL.2004; kx, Payne et al. 2005). In the CAMp study, 90 out<br />
<strong>of</strong> 117 children (75Vo) with mild to moderate asthma across eight centres provided an<br />
adequate sputum sample for analysis (Covar, Spahn et al.20O4). <strong>The</strong> total cell counts were<br />
the only similar result when induced sputum samples were examined on two separate days,<br />
two weeks apart in the same 37 subjects (Thomas, Yates et al. lggg). Considerable<br />
heterogeneity was also documented, particularly in eosinophils, in samples from over 250<br />
adult asthmatics and normal subjects (Green, Brightling et a\.2002). Some <strong>of</strong> the variability<br />
seen between groups may be due to differing techniques. A range <strong>of</strong> 0.9 to lT%o concentration<br />
<strong>of</strong> saline has been used, with the measurement being <strong>of</strong> either the whole expectorated sample<br />
or a sample separated from saliva or by picking out mucus plugs. <strong>The</strong> plugs were thought to<br />
provide more cells with better cell viability (Gershman, Wong et al. 1996; pjzzichini,<br />
Pizzichini et al. 1996; Spanevello, Beghe et al. 1998) while selected sputum had higher<br />
concentrations <strong>of</strong> soluble markers such as ECP (Gershman, Wong et al. 1996: Spanevello,<br />
Beghe et al. 1998). Using 0-5 minutes <strong>of</strong> hypertonic saline gave more neutrophils, while using<br />
a longer period <strong>of</strong> 10-15 minutes gave higher concentrations <strong>of</strong> eosinophils ,nA and,Il5 with<br />
lymphocytes, macrophages and epithelial cells remaining unchanged (Taha, Hamid et al.<br />
2004)- A comparison <strong>of</strong> induced and spontaneous sputum samples showed an increased<br />
number and more viable cells in the former process (Pizzichini ,Pizzichiniet al. 1996).<br />
In addition, the induction procedure itself may alter findings. Consecutive sputum samples<br />
during one sputum induction displayed an increasing neutrophil gradient (Holz, Jorres et al.<br />
1998; Nightingale, Rogers et al. 1998; Richter, Holz et al. 1999 Belda, Hussack et al. 2001)<br />
which occurred within six hours and lasted for 24 (Holz, Jorres et al. 1998; Nightingale,<br />
Rogers et al. 1998) with decreasing mucin and increasing concentrations <strong>of</strong> surfactant<br />
(Gershman, Liu et al. 1999). This effect was observed with both high and low output<br />
nebulisers (Berlyne, Irmiere et al. 1998). Repeated sputum induction over days also resulted<br />
in increased neutrophils with decreasing macrophage and eosinophil numbers (Belcher,<br />
Murdoch et al. 1988; Holz, Richter et al. 1998) and the effect <strong>of</strong> hypertonic saline on nasal<br />
mucosa resulted in increased histamine and leukotriene release (Silber, Proud et al. lggg).<br />
<strong>The</strong> exact cause <strong>of</strong> this inflammatory response is unknown but the additional fluid,<br />
hypertonicity, frequent coughing, or possibly bacterial contamination could all be<br />
contributory (Holz, Richter et al. 1998; Nightingale, Rogers et al. 1998; Sacco, Fregonese et<br />
al. 2000).<br />
37
<strong>The</strong>re are other studies suggesting sample repeatability was acceptable, with correlation<br />
coefficients between O.7-O.87 for macrophage, lymphocyte, neutrophil and eosinophil counts<br />
in asthmatic, rhinitis, CF and normal subjects (Pin, Gibson et al. 1992; in 't Veen, de Gouw et<br />
al. 1996; Spanevello, Migliori et al. 1997; Bacci, Cianchetti et ^1. 2002; Beier, Beeh et al.<br />
2004; Smountas, Lands et al. 2004).<strong>The</strong> repeatability <strong>of</strong> the soluble markers albumin,<br />
fibrinogen, IL8 and ECP were also thought to be acceptable in adult subjects with mild to<br />
moderate asthma (in't Veen, de Gouw et al. 1996).<br />
Performing all <strong>of</strong> these procedures requires highly experienced staff, particularly with<br />
children, also recognising the need for general anaesthesia for bronchoscopy, as well as highly<br />
qualified laboratory technicians (see Section L.6.4 (iii) below regarding safety). <strong>The</strong><br />
procedures are time-consuming which limits their use in clinical practice. For sputum<br />
induction, it is estimated that should a patient produce a suitable sample within 10 minutes <strong>of</strong><br />
starting the procedure (and it can take up to 30 minutes) in a well practiced laboratory, the<br />
total time required for processing through to a result is 100 minutes and the sample requires<br />
processing within 2 hours <strong>of</strong> collection (Holz, Kips et al. 2000). <strong>The</strong> time spent to make the<br />
procedure 'child friendly' is likely to be longer. <strong>The</strong> CAMP researchers across eight centres<br />
commented "this procedure still remains a research tool in asthma because <strong>of</strong> its requirements<br />
for technical expertise" (Covar, Spahn et al. 2OO4).<br />
1.6.a (iv) Safety aspects <strong>of</strong> bronchoscopy, bronchial biopsy, bronchoalveolar lavage and/or<br />
induced sputum<br />
In 159 asthmatic adults undergoing 273 bronchoscopies in six studies, there were 34 adverse<br />
event episodes which included bronchospasm, pleuritic chest pain, shortness <strong>of</strong> breath, fever,<br />
fluJike symptoms and haemoptysis @lston, Whittaker et al.2004). Another safety review<br />
reported that after six to eight biopsies taken per procedure in 57 patients; 40Vo had cough,<br />
l2Vo cough and bronchospasm and3.5Vo required additional rescue medication (Iapanainen,<br />
Lindqvist et al.2N2). Arterial saturation decreased in 50 asthmatic patients from a mean <strong>of</strong><br />
977o Io a mean <strong>of</strong> 92Vo and from 98Vo to 93Vo in 25 normal subjects during bronchoscopy and<br />
biopsy, without correlation to the pre-operative lung functions tests (Van Vyve, Chanez et al.<br />
1992). Bacteraemia is also described with fibreoptic bronchoscopy, with 26 <strong>of</strong> 200<br />
consecutive patients having positive blood cultures after the procedure despite the majority <strong>of</strong><br />
patients having no prior respiratory illness (Yigla, Oren et al. 1999). A prospective analysis <strong>of</strong><br />
the financial cost <strong>of</strong> complications from flexible bronchoscopy over a 30 month period<br />
revealed <strong>of</strong> the 1,009 bronchoscopies performed in 660 adults as an outpatient procedure,<br />
38
complications occulred in SVo necessitating hospital admission in 0.5Vo, resulting in an<br />
additional cost <strong>of</strong> US $6,996 to treat complications but with an additional US $34,500 in<br />
those requiring hospitalisation (Colt and Matsuo 2001).<br />
<strong>The</strong> largest study looking at side effects with flexible bronchoscopies in children, was a<br />
retrospective analysis <strong>of</strong> 2,836 procedures over 2l years, which concluded that it was a safe<br />
procedure. However 2l had life threatening hypoxaemia, l7 had laryngospasm or<br />
bronchospasm and 4Vo had nasopharyngeal bleeding (Nussbaum 2002). A large prospective<br />
study <strong>of</strong> 1,328 procedures (excluding intensive care examinations) recorded at least one<br />
complication in 6.9Vo <strong>of</strong> cases, <strong>of</strong> which 5.2Vo were mild and l3To were major with one<br />
pneumothorax (de Blic, Marchac et al. 2002).In 170 children with respiratory symptoms<br />
having bronchoscopy and lavage plus at least three endobronchial biopsies, fluctuations <strong>of</strong><br />
oxygen saturation and end tidal carbon dioxide (CO, were seen in all patients but only one<br />
had a prolonged desaturation episode. In addition, minor bleeding was demonstrated at the<br />
site <strong>of</strong> the biopsies but no other side effects occurred (Salva, <strong>The</strong>roux et al.2N3). Of 3g<br />
asthmatic children undergoing bronchoscopy, one had desaturation, two had fever and in four<br />
their asthma worsened over the following week. Of 35 non-asthmatic children undergoing<br />
bronchoscopy for another reason (not 'normals'), there were there were 17 adverse events<br />
which were predominantly laryngospasm and apnoea (Payne, McKenzie et al. 2001). In 66<br />
children < 5 years <strong>of</strong> age a comparison was made between having bronchoalveolar lavage<br />
alone and the addition <strong>of</strong> endobronchial biopsies with approximately half the group in each.<br />
Complications occurred in 24Vo and, lSVo respectively including cough, desaturation, apnoea<br />
and laryngospasm (Saglani, Payne et al. 2OO3). In 42 CF children and 39 with other<br />
indications for bronchoscopy, the complication rate was l3.37o and, l7.97o respectively<br />
(Molina-Teran, Hilliard et al. 2006).<br />
So despite the progress in knowledge from the biopsy and lavage studies - they are still<br />
invasive, require bronchoscopy, and sedation in adults and <strong>of</strong>ten general anaesthesia in<br />
children. It is therefore particularly difficult to justify use <strong>of</strong> these techniques in early age<br />
groups and/or repeatedly which is where the most knowledge regarding progression <strong>of</strong> disease<br />
is likely to come. Discussions about safety in the literature has presented both pro and con<br />
arguments (Connett 2000; Larsen and Holt 2000; Shields and Riedler 2000) ending with one<br />
commentator suggesting "because <strong>of</strong> the invasive nature <strong>of</strong> the investigation there arc few<br />
conditions for which repeat sampling can be justified" (Connett 2000). <strong>The</strong> debate has<br />
continued more recently with a series <strong>of</strong> letters discussing the appropriateness <strong>of</strong> biopsy in<br />
particular as a research tool in children (Mallory 2006;Bush and Davies zo07).<br />
39
So is induced sputum in adults and children safe? Particularly those with asthma? In 34<br />
asthmatic and normal subjects, a mean fall in FEVr <strong>of</strong> 5.3Vo was seen, but with a maximum <strong>of</strong><br />
20Vo (Pin, Gibson et al. 1992). Pre-treatment with a 9z agonist did not prevent<br />
bronchoconstriction in all the asthmatic subjects, especially in those with a baseline low FEVr<br />
(Wong and Fahy t997). Such pre{reatment in mild asthmatics resulted in an unchanged<br />
FEV1, but with a reduction observed in the normal subjects (Thomas, Yates et al. 1999). One<br />
group also documented that an overuse <strong>of</strong> F, agonists the day before sputum induction<br />
reduced the protective effect <strong>of</strong> the pre-medication (Pizzichini, Pizzichini et al. 1997). While<br />
samples were successfully obtained even in subjects with 'difficult to control' asthma, severe<br />
bronchoconstriction occurred in22Vo with development <strong>of</strong> symptoms and a reduction in FEVr<br />
<strong>of</strong> > l1%o (ten Brinke, de Lange et al. 2001). Another study noted that the procedure had to be<br />
stopped because <strong>of</strong> side effects in l2Vo overall and in 17-l8%o <strong>of</strong> those with severe asthma (de<br />
la Fuente, Romagnoli et al. 1998). When performed as part <strong>of</strong> their routine clinical visit, the<br />
procedure was well tolerated in over 300 asthmatics, even among those with more severe<br />
respiratory obstruction with an FEVr down to 60Vo predicted or one litre. However in patients<br />
with a baseline FEVr between 4OVo and 59Vo, there was a mean 8Vo reduction in FEVr and in<br />
patients with a baseline FEVr <strong>of</strong> < 40Vo predicted, 6Vo fellby 2OVo (Vlachos-Mayer, Leigh et<br />
al. 2000). A decrease in arterial oxygen saturations <strong>of</strong> 6Vo in asthmatic patients, 5.3Vo in<br />
smokers and 67o in healthy subjects has been reported suggesting that patients needed<br />
monitoring during this procedure (Castagnaro, Chetta et al. 1999).<br />
Reviewing the safety in children; in 53 sputum induction alone was tolerated in 98Vo, with<br />
94Vo completing the procedure,92To providing an adequate sample but 4Vo experienced a<br />
>l1vo fall in FEVr (Jones, Hankin et al. 2001). In 182 children who had combined sputum<br />
induction and bronchial provocation testing using hypertonic saline, the procedure was<br />
completed by 90Vo with a distressing cough in l3%o and mucosal irritation with wheeze in IVo<br />
and fewer, 70Vo, successful samples obtained. So while the two investigations can be<br />
combined, this was less well tolerated and resulted in less sampling success (Jones, Hankin et<br />
al. 2001). In 40 children with a baseline FEVr > 65Vo predicted, seven (l8vo) had symptoms<br />
<strong>of</strong> shortness <strong>of</strong> breath and wheezing and <strong>of</strong> these three (8Vo) had a significant fall in FEVr <strong>of</strong><br />
greater than 2O7o despite pre-treatment with a bronchodilator (Lex, Payne et al. 2005).In the<br />
CAMP study, nine (87o) <strong>of</strong> 117 children with mild to moderate asthma developed significant<br />
bronchospasm requiring treatment (Covar, Spahn et aI.2004).<br />
40
1.6.5 Studies <strong>of</strong> inflammatory markers in blood and uine<br />
Inflammatory markers have also been studied in blood and urine and I am going to briefly<br />
review what has been found in this area. Blood and urine samples are relatively easy to obtain<br />
when compared to the general anaesthesia or sedation required for bronchoscopy, lavage and<br />
biopsy, are more consistently obtainable than induced sputum and less likely to cause side<br />
effects than these other procedures. However blood testing is still seen as invasive by children<br />
and most adults. Also, these compartments are removed from the direct area <strong>of</strong> interest and<br />
reflect systemic findings, which may or may not be related to the lung.<br />
As with results from sampling the lung, monitoring <strong>of</strong> inflammatory markers in blood have<br />
shown an increase in eosinophils, ECP and EDN levels in children and adults with asthma<br />
compared to control groups. In some studies these correlated with asthma severity or current<br />
symptomatology (Krisdansson, Shimizu et al. 1994; Parra, Prieto et al. 1996; Rao, Frederick<br />
et al. 1996; Remes, Korppi et al. 1998; Yazicioglu, Ones et al. 1999; Stelmach, Gorski et al.<br />
2002), and although other studies also found higher levels, no such correlations were found<br />
(Pizzichini,Pizzichini et al. 1997; Bacci, Cianchetti et al. 1998; Niimi and Matsumoto 1999;<br />
Stelmach, Jerzynska et al. 2001; Stelmach, Jerzynska et al. 2001; Aldridge, Hancox et al.<br />
2002; Reichenbach, Jarisch et al. 2002) or even mixed results were obtained (Currie, Syme_<br />
Grant et al. 2o03). In adults, a large cross sectional study <strong>of</strong> almost 7000 subjects found<br />
physician diagnosed asthma was significantly associated with serum eosinophil count<br />
(Schwartz and Weiss 1993). Serum eosinophil and ECP levels during acute asthma were<br />
corelated with more severe exacerbations and less bronchodilator responsiveness in 48 adults<br />
(Lee, Ire et al. 1997). Plasma total IgE, plasma specific IgE, blood eosinophil percentage and<br />
exhaled NO were thought to account for 55.5Vo <strong>of</strong> the variance seen in bronchial hyper-<br />
reactivity (I-eung, Wong et al. 2005). Urinary levels <strong>of</strong> EDN did increase at time <strong>of</strong> asthma<br />
exacerbation (Cottin, Deviller et al. l99g).<br />
In children, ECP and eosinophilic peroxidase (EPO) in both serum and urine showed reduced<br />
gradation in levels from asthmatics with current symptoms to symptom free asthmatic<br />
children and to healthy children (Lonnkvist, Hellman et al. 2001). Both urinary EDN and<br />
serum ECP were significantly higher in children with atopic asthma than control subjects<br />
(Kristjansson, Strannegard et al. 1996). In both studies levels decreased with IHCS treatment<br />
(Kristjansson, Strannegard et al. 1996;Lonnkvist, Hellman et al. 2001) and in one increased if<br />
treatment was reduced (Lonnkvist, Hellman et al. 2001). Urinary EDN levels were increased<br />
in 80 asthmatic children compared to 24 controls, and were higher in symptomatic than<br />
4l
asymptomatic patients independent <strong>of</strong> the presence <strong>of</strong> atopy or the treatment modality.<br />
Urinary EDN levels in the asthmatics correlated to lung function, and in the subgroup <strong>of</strong> 15<br />
children re-examined two months after commencement <strong>of</strong> treatment, the reduction <strong>of</strong> EDN<br />
also correlated with the changes in lung function (Lugosi, Halmerbauer et al. 1997). While no<br />
correlation was found between urinary EDN values and lung function in two other studies <strong>of</strong><br />
155 and 39 children, there was a similar reduction <strong>of</strong> EDN three months after commencing<br />
IHCS treatment (Krisdansson, Strannegard et al. 1996; Labbe, Aublet-Cuvelier et al. 2001).<br />
Circadian variations <strong>of</strong> the eosinophil proteins in urine samples have been demonstrated with<br />
high early morning and nocturnal peaks compared to lower levels in the afternoon in both<br />
asthmatic and non-atopic, non-asthmatic groups <strong>of</strong> children (Storm van's Gravesande, Mattes<br />
et al. 1999; Wolthers and Heuck 2003).<br />
A more explored area using these samples has been in measuring the levels <strong>of</strong> the leukotriene<br />
goup <strong>of</strong> proteins, particularly in urine samples. <strong>The</strong>se proteins were shown to increase early<br />
in adult asthmatic groups following an airway challenge (Westcott, Smith et al. 1991; Kumlin,<br />
Dahlen et al. 1992; O'Sullivan, Roquet et al. 1998; Bancalari, Conti et al. L999) with peak<br />
excretion occurring at two hours (Kumlin and Dahlen 2000; Mai, Bottcher et al. 2005). <strong>The</strong><br />
results were more variable when assessing whether levels remained elevated during the late<br />
asthmatic response - not seen in one study (Manning, Rokach et al. 1990) but documented in<br />
two others (O'Sullivan, Roquet et al. 1998; Bancalari, Conti et al. 1999). LT& was high in<br />
184 adults presenting with moderate to severe acute asthma and reduced two weeks later in<br />
the 146 followed up during recovery (Green, Malice et al. 2OO4). Compared to control<br />
subjects, there was an increase in the LTEI level in asthmatics with nocturnal asthma<br />
exacerbations but not in asthmatics without, although the numbers in each group were less<br />
than ten (Bellia, Bonanno et al. 1996). Patients with asthma did excrete more LT& over a24<br />
hour period than normal subjects, again with less than ten subjects in each group (Asano,<br />
Lilly et al. 1995). In 40 severe asthmatics, 25 mild to moderate asthmatics and 20 non-<br />
asthmatic controls, there was a gradation LTE4 levels despite treatment with high dose IHCS<br />
in the severe group (Vachier, Kumlin et al. 2003). LTE4 in urine was higher in 35 atopic<br />
asthmatic than32 non-atopic children with RSV bronchiolitis and 23 controls (Oh, Shin et al.<br />
200s).<br />
Other studies have been less positive about the correlations between inflammatory markers<br />
measured in blood and urine samples and asthmatic disease. In adults, no correlation between<br />
clinical status, functional status and serum ECP was seen during five months in asthmatics<br />
either admitted for acute asthma or who were seasonally sensitised to birch and tree pollens<br />
42
and had intermittent symptoms (de Blay, Purohit et al. 1998). <strong>The</strong> combination <strong>of</strong> serum ECp<br />
with peak expiratory flow did not translate into a useful combination to determine IHCS<br />
adjustment and was less useful than symptomatology alone or combined with other lung<br />
function parameters (Lowhagen, Wever et aL.2002). Also, the blood inflammatory markers<br />
did not reflect health related quality <strong>of</strong> life scores in subjects with mild asthma (Ehrs,<br />
Sundblad et al. 2006).<br />
In children, serum ECP levels followed during acute exacerbation and recovery <strong>of</strong> asthma in<br />
11 asthmatics did not show a uniform pattern <strong>of</strong> increasing then decreasing values over this<br />
period (Niggemann, Ertel et al. 1996). In 34 children with moderate asthma, none <strong>of</strong> the<br />
markers measured reflected asthma activity, including serum eosinophils, serum ECp, serum<br />
EDN, urinary EDN or urinary histamine levels (Hoekstra, Grol et al. 1998). Serial<br />
measurements <strong>of</strong> ECP and EDN in urine samples taken monthly did not provide additional<br />
information in monitoring childhood asthma or contribute to practical management in a<br />
prospective study that followed children over a six month period measuring diary<br />
symptomatology and peak flow (wojnarowski, Roithner et al. 1999).<br />
In addition, the markers when raised were not found to be specific for asthma. Considerable<br />
overlap was seen in blood eosinophil and ECP levels in asthmatics and controls, with no<br />
difference in the mean levels measured in adults with asthma or with allergic rhinitis or in<br />
those who had both (Sin, Terzioglu et al. 1998). While physician diagnosed bronchitis was<br />
more significantly associated with neutrophil count, it was also associated with eosinophil<br />
count and, as well, chronic sputum production was also associated with both cells (Schwartz<br />
and Weiss 1993).In children, serum ECP and EDN was higher in the 36 asthmatic than 166<br />
healthy children but higher rates than controls were also seen in those with allergic<br />
rhinoconjunctivitis, atopic dermatitis and/or allergic skin sensiti zation. An elevated ECp gave<br />
an odds ratio <strong>of</strong> 2.3 for asthma, but 2.9 for atopic dermatitis. Similarly, an elevated EDN gave<br />
an odds ration <strong>of</strong> 2.61for asthma, but 5.23 for allergic rhinoconjunctivitis (Remes, Korppi et<br />
al. 1998). Considerable overlap existed in another study comparing serum ECp levels in 2l<br />
children with asthma and/or atopic dermatitis (Kristjansson, Shimizu et al. lgg4). In 207<br />
children aged 24-41 months and 76 aged 0-23 months in whom repeated samples were<br />
obtained in a large longitudinal childhood asthma study, the serum ECp was shown to be<br />
influenced by a number <strong>of</strong> factors including age, the presence <strong>of</strong> active eczema and the<br />
presence <strong>of</strong> maternal smoking in a dose dependent fashion (Lodrup Carlsen, Halvorsen et al.<br />
1998). ln 72 infants with recurrent wheezing, there was no correlation between serum ECp<br />
and the development <strong>of</strong> asthma and similarly no correlation between serum ECp and<br />
43
onchial hyper-responsiveness (Reichenbach, Jarisch et al. 2002). Neither eosinophil<br />
numbers nor ECP levels could predict the intensity <strong>of</strong> response to bronchial inflammatory<br />
reaction in 46 house dust mite allergic children (Vila-Indurain, Munoz-l-apez et d. 1999).<br />
Negative findings also occurred in studies into the leukotrienes. While there were significant<br />
associations between lung function parameters and urinary LTEa, it did not distinguish<br />
between groups <strong>of</strong> 49 mild and 3l moderate to severe asthmatics (Severien, Attlich et al.<br />
2000). A single urine sample did not predict bronchial hyper-responsiveness or degree <strong>of</strong><br />
airflow obstruction in 41 asthmatics (Smith, Hawksworth et al. 1992). <strong>The</strong>re was no increase<br />
in LTCa, LTD4 or LTE4 in urine after an exercise challenge in adults (Taylor, Wellings et al.<br />
1992). <strong>The</strong> measurement <strong>of</strong> urinary EDN in children with either acute or chronic asthma,<br />
lower or upper respiratory tract infections or controls did not show significant differences<br />
between these groups (Oymar and Bjerknes 2001).<br />
So in all age groups blood and urine inflammatory parameters have failed to show consistent<br />
discrimination between asthma and other allergic diseases, and in children between asthma<br />
and other respiratory diseases, and also failed to determine asthma severity. <strong>The</strong> cells and<br />
proteins in these systemic samples may not necessarily indicate what is happening in the<br />
lungs as they are being measured from more distant sites. In addition, the measurement made<br />
from blood samples by the time these reach the laboratory is made up <strong>of</strong> both the circulating<br />
ECP and what is released from the eosinophils after the blood has been drawn into the tube<br />
(Venge 1995). While these are easier samples to obtain with the benefit <strong>of</strong> fewer side effects<br />
than sampling nonproductive respiratory secretions directly, blood testing is still perceived as<br />
an invasive procedure and urine samples can be tiresome to obtain, particularly in children.<br />
1.6.6 Comparison <strong>of</strong> intlammation results between the different samples<br />
Comparisons have been made between the levels <strong>of</strong> inflammatory parameters obtained<br />
through the different tissue or fluid samples. <strong>The</strong> cellular composition <strong>of</strong> induced sputum<br />
correlates well with bronchoalveolar lavage and wash, but to a lesser extent with bronchial<br />
biopsies (Fahy, Wong et al. 1995; Maestrelli, Saetta et al. 1995; Grootendorst, Sont et al.<br />
1997; Keatings, Evans et al. 1997; Pizzichini, Pizzichini et al. 1998). For example, mast cells<br />
usually make up only a small proportion <strong>of</strong> cells recovered by lavage but are as much as 20Vo<br />
<strong>of</strong> the inflammatory cells in biopsy studies (Dunnill 1960; Foresi, Bertorelli et al. 1990).<br />
Likewise there are more basophils in the biopsy specimens than in the lavage specimens<br />
(Koshino, Teshima et al. 1993; Macfarlane, Kon et al. 2000). On the other hand as induced<br />
sputum is rich in neutrophils and eosinophils but poor in lymphocytes, the origin <strong>of</strong> the<br />
u
sample is thought to be from the larger airways (Keatings, Evans et al. 1997; Nocker, Out et<br />
al. 2000). Induced sputum eosinophilic measures did predict exacerbations better than<br />
bronchoalveolar lavage samples (Nocker, Out et al. 2000). While correlations were identified<br />
between blood eosinophils and serum ECP, and sputum eosinophils and sputum ECp, in a<br />
number <strong>of</strong> studies it was the sputum ECP that best correlated to bronchial obstruction, and the<br />
sputum ECP and eosinophils that best correlated to methacholine challenge in asthmatics<br />
(Sorva, Metso et al. 1997; Grebski, Wu et al. 1999; Piacentini, Bodini et al. 1999). Sputum<br />
eosinophils and ECP were related to the symptom scores and FEV1, while no relationship was<br />
demonstrated between blood eosinophils and symptom scores (Bacci, Cianchetti et al. 1998).<br />
Finally, the measurement <strong>of</strong> ECP was easier to do from induced sputum than from blood or<br />
lavage fluid, required less technical input and time and had better correlation to disease<br />
activity (Barck, Lundahl et al. 2005). In a comparison between blood and urine samples, one<br />
study showed a better correlation between lung function and the inflammatory parameters<br />
measured in the urine samples (Labbe, Aublet-cuvelier et al. 2001).<br />
t.7<br />
Chapter summary<br />
So in this introductory outline; the burden <strong>of</strong> paediatric respiratory disease in New Zealand is<br />
great and asthma in both adults and children is a major cause <strong>of</strong> morbidity, stimulating my<br />
interest in conducting research in this area. <strong>The</strong> diagnosis <strong>of</strong> asthma, particularly in children,<br />
can be difficult with no single diagnostic or prognostic marker. Asthma continues to be a<br />
clinical diagnosis based on history <strong>of</strong> symptoms, few examination findings and response to<br />
treatment, with possible confirmation from lung function testing in older children. However<br />
the mainstay <strong>of</strong> treatment is anti-inflammatory medication, which is associated with<br />
significant side effects such as adrenal cortical insufficiency, growth failure, dysphonia and<br />
oral candidiasis for IHCS use, and adrenal insufficiency which may cause devastating<br />
hypoglycaemia, adrenal suppression, growth failure, hypertension, diabetes mellitus,<br />
reduction in bone mineral density, skin atrophy, straie, poor healing, immunosuppression and<br />
cataracts for oral corticosteroid use (GINA 2002; GINA 2005). We do not routinely measure a<br />
marker for inflammation, but rather base decisions and treatment regimes on surrogare<br />
markers such as symptoms, lung function, chest xray appearances, bronchial reactivity - to<br />
determine successful outcomes.<br />
<strong>The</strong> theory that asthma is an inflammatory disease was first indicated by autopsy studies in<br />
early and mid last century. With the development <strong>of</strong> bronchoscopy, biopsy studies became<br />
possible and these consolidated the pathogenesis <strong>of</strong> asthma as an inflammatory disease. This<br />
45
led to research which looked for less invasive methods <strong>of</strong> assessing the inflammatory<br />
component <strong>of</strong> asthma and bronchoalveolar lavage and this led to the development <strong>of</strong> the<br />
ability to induce sputum using nebulised hypertonic saline. <strong>The</strong>se samples allowed increasing<br />
measurement not only <strong>of</strong> the cellular component, but also <strong>of</strong> the proteins that formed the<br />
asthmatic inflammation. <strong>The</strong>se measurements could be done repeatedly so parameters were<br />
followed longitudinally to determine what happened during acute exacerbations <strong>of</strong> asthma, in<br />
response to treatment and over longer periods <strong>of</strong> time during chronic asthma. Studies were<br />
also undertaken using blood and urine samples. In the main there has been a recognition that<br />
the majority <strong>of</strong> asthma and other allergic diseases have an increased allergic inflammatory<br />
pr<strong>of</strong>ile with an eosiniphilic dominant pattern, raised levels <strong>of</strong> eosinophilic proteins @CP,<br />
EPO, EDN and MBP), and cytokines (IL3, nA,n-5, rantes). While over the last decades this<br />
was thought to be an imbalance between T lymphocyte classes, between the T helper type 2<br />
(Ts2) and T helper type I (Tnl), (Colavita, Reinach et al. 2000; Holgate, Davies et al. 2000;<br />
Busse and Irmanske 2001; Peters 2003), very recently it has been thought there may be<br />
upregulation <strong>of</strong> both classes with a third type, the T lymphocyte regulatory cells playing a<br />
pivotal role (Lazarus, Raby et al.2004; Goldman 2007). Certainly some asthma is known to<br />
have a neutrophil dominant or neutrophil present pattern, with the markers more commonly<br />
associated with infective conditions. This is seen more <strong>of</strong>ten in severe persistent asthma<br />
(Wenzel, Szefler et al. 1997i Jatakanon, Uasuf et al. 1999), associated with a poor response to<br />
treatment (McDougall and Helms 2006; Wenzel 2006), has been demonstrated in some acute<br />
exacerbations (Martin, Cicutto et al. 1991; Fahy, Kim et al. 1995; Ordonez, Shaughnessy et<br />
al. 2000), and in sudden fatal asthma (Sur, Crotty et al. 1993). While the eosinophils and<br />
neutrophils themselves and the proteins they release have shown the best association with<br />
asthma exacerbations and response to treatment - neither have been completely consistent. In<br />
addition, obtaining the appropriate samples can be difficult.<br />
So, while contributing enonnously to our understanding <strong>of</strong> asthma, the use <strong>of</strong> bronchoscopy,<br />
biopsy and BAL, induced sputum, blood and urine testing all have their disadvantages,<br />
particularly when assessing disease in children. <strong>The</strong> first three options remain invasive,<br />
require anaesthesia in children and are associated with risks and side effects, especially in<br />
children with respiratory disease, and therefore cannot be done frequently. <strong>The</strong>y also require<br />
specialised personnel in terms <strong>of</strong> theatre and bronchoscopy staff. Induced sputum results in<br />
less satisfactory sample success in children than in adults, although successful samples can be<br />
obtained in up to 757o <strong>of</strong> control children. It also can be associated with side effects, as<br />
nebulised hypertonic saline is also used as an airway challenge. This test also requires<br />
46
experienced staff to undertake the procedure, and laboratory staff to process samples within a<br />
short time frame. It also represents a significant time commitment - even in the most<br />
experienced hands and a sample taken in the shortest time, the minimum time to from start to<br />
getting a result for one sample is estimated to be 100 minutes (Holz, Kips et al. 2000). Both<br />
blood and urine sampling have been found to be less useful, probably because the site <strong>of</strong><br />
measurement is distant from the site <strong>of</strong> interest, the lung. In addition, apart from obtaining<br />
urine samples, the other sampling procedures all require a hospital or clinical setting.<br />
<strong>The</strong>se studies started to come about at the same time as my own research commenced. In view<br />
<strong>of</strong> all these difficulties a simpler, direct, non-invasive technique for measuring airway<br />
inflammation was sought, that could be achieved easily by children, and could be repeated<br />
regularly with time. Because measurement <strong>of</strong> lung function is a common procedure in<br />
children and does not involve collecting specimens and there is no anaesthesia or needles, the<br />
result is instant -<br />
a test analogous to this would seem ideal.<br />
In 1987 came the announcement that a gaseous molecule, NO, was responsible for acting as<br />
both a widespread physiological mediator and was also involved in host defense (Hibbs,<br />
Taintor et al. 1987; Ignarro, Buga et al. 1987 Khan and Furchgott 1987). This resulted in an<br />
explosion <strong>of</strong> research in the early 1990s identifying both NO and the enzymes that produced it<br />
- the nitric oxide synthases (NOS) in all biological systems. NO was shown to play a major<br />
role in host defense and inflammation within the lung as in other systems. Toward the end <strong>of</strong><br />
1995,I also went to a one day seminar on NO at the Royal Society <strong>of</strong> Medicine in I-ondon to<br />
hear Pr<strong>of</strong>essor Salvador Moncada, one <strong>of</strong> the major early researchers into NO in the<br />
cardiovascular system, talk. I was struck by the importance <strong>of</strong> this one tiny molecule, and the<br />
unfolding story with more to come. This seemed like a great opportunity to further explore the<br />
possibility <strong>of</strong> NO as a potential marker for airway inflammation; however there were a<br />
number <strong>of</strong> hurdles to overcome. Measuring NO was going to be difficult given its gaseous,<br />
short lived and highly reactive nature. As well there was the difficulty <strong>of</strong> measuring levels in<br />
exhaled air from human subjects, especially in children.<br />
This research was conducted at the Royal Brompton Hospital in London from the mid 1990s.<br />
On returning to New Zealand, it was acceptable to submit this research over time as a thesis.<br />
Some <strong>of</strong> the findings within this thesis are now well-known, but were unknown, new and<br />
exciting when first demonstrated. <strong>The</strong> findings contributed to the knowledge as to what<br />
technical aspects were important in the measurement <strong>of</strong> NO, to the standardising <strong>of</strong> protocols<br />
for measurement, and to what were important capabilities to be developed for the newer NO<br />
47
analyser machines. This thesis presents one researcher negotiating research in one area <strong>of</strong><br />
medicine -<br />
which will be presented how it unfolded at the time and will be reviewed with the<br />
up to date NO literature in the penultimate chapter.<br />
In the chapter to follow, I will review what was known about NO and nitrogen dioxide (NOz)<br />
in pollution where it was recognised to be a major component, and its effects at the population<br />
level on respiratory disease. <strong>The</strong> study <strong>of</strong> pollution was where the machines were first<br />
developed in order to analyse levels. <strong>The</strong> chapter will then describe the recognition <strong>of</strong> NO as a<br />
physiological mediator and its key role in inflammation. Chapter 3 will review the actions and<br />
interactions <strong>of</strong> NO as well as the NOS enzymes that produce this molecule in vivo. Chapter 4<br />
will elaborate on the methods <strong>of</strong> measuring this short lived, reactive molecule. Chapter 5 will<br />
discuss how the chemiluminescence analyser, the method <strong>of</strong> choice for the subsequent<br />
experiments, could be modified to measure NO in exhaled air in humans using an analyser<br />
developed for NO measurement in the environment. Chapter 6 is the corlmencement <strong>of</strong> the<br />
research in normal, control adult subjects looking at the measurement <strong>of</strong> exhaled NO through<br />
two different methods, either directly into the NO analyser which also allowed the<br />
measurement <strong>of</strong> mouth pressure and COz, and through a t-piece system which enabled the<br />
measurement <strong>of</strong> flow in addition to the other parameters. Chapter 7 describes the research<br />
undertaken to see what technical aspects <strong>of</strong> measurement altered the NO levels obtained.<br />
Chapter 8 describes the research as I measured exhaled NO in normal and then asthmatic<br />
children. It briefly reviews the effect <strong>of</strong> age, atopy, the presence <strong>of</strong> pets or the presence <strong>of</strong><br />
smoking in the home on the NO obtained in a group <strong>of</strong> healthy pre-pubertal children. <strong>The</strong>re is<br />
a comparison <strong>of</strong> results from this control group to a group <strong>of</strong> asthmatics on bronchodilator<br />
therapy only and a group <strong>of</strong> asthmatics who were on chronic inhaled corticosteroid therapy. A<br />
small number <strong>of</strong> steroid naive asthmatics were measured before and after commencing<br />
inhaled corticosteroids. Chapter 9 summarises what was learned about measurement <strong>of</strong><br />
exhaled NO from these studies and how that has contributed to the literature. This is followed<br />
by a further review <strong>of</strong> the literature to the current time and the place <strong>of</strong> NO measurement as it<br />
currently stands. Finally chapter l0 will present the final thoughts with regard to this research<br />
project and 'where to from here'.<br />
NB: Some <strong>of</strong> the work presented in this opening chapter formed the monograph for the<br />
Asthma and Respiratory Foundation <strong>of</strong> New Zealand; (Anonymous 2006) in which I<br />
contibuted two chapters and edited in conjunction with Pr<strong>of</strong>essor Innes Asher. This was<br />
presented to <strong>The</strong> Honorable Mr Peter Hodgeson, Minister <strong>of</strong> Health April 2006, and is<br />
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.<br />
48
2.1 Introduction<br />
Chapter 2: Nitric oxide: pollutant to mediator<br />
<strong>The</strong>re are two lines <strong>of</strong> research, from quite disparate areas, that came together to make the<br />
studies described in Chapters 6, 7 and, 8 possible. <strong>The</strong> first is the measurement <strong>of</strong> air<br />
pollution, which has been increasingly important due to recognition that different pollutants<br />
cause or contribute to human disease, including nitrogen oxides; and the second is the<br />
increasing recognition <strong>of</strong> the importance <strong>of</strong> nitric oxide (NO) in biological systems in the last<br />
25 years. I will examine the developments in both <strong>of</strong> these areas that led to the<br />
commencement <strong>of</strong> the research described in this thesis.<br />
2.2 Pollution. nitrogen oxides and disease<br />
2.2.1 Concerns regarding pollution<br />
<strong>The</strong>re is now no doubt that episodes <strong>of</strong> air pollution have resulted in excess deaths. Concerns<br />
with regard to pollution and its effects on health date back to early last century, although the<br />
detrimental effects <strong>of</strong> poor air quality and poor housing have been recognised for far longer.<br />
Indoor air pollution can be traced to some 200,000 years ago, when humans first moved to<br />
colder climates necessitating the construction <strong>of</strong> shelters and use <strong>of</strong> indoor fires for cooking,<br />
warmth and light. <strong>The</strong>se mostly 'inside' fires likely resulted in an excess exposure to levels <strong>of</strong><br />
pollution as evidenced by the carbon soot found in prehistoric caves (Brims and Chauhan<br />
2005). Severe outdoor air pollution episodes have been documented since the early 17ft<br />
century but these became more frequent and more severe throughout the 19ft century. <strong>The</strong><br />
most definitive and well documented episodes were the great smogs that occurred in London,<br />
United Kingdom, in 1948, 1952 and 1962, with the most severe occurring in December 1952<br />
(Brims and Chauhan 2005). <strong>The</strong> smoke concentration at this time was 50 times above the<br />
average level, and this rise in pollution was followed by an equally sharp rise in mortality<br />
(Ministry <strong>of</strong> Health, UK 1954). An estimated human death toll <strong>of</strong> 4,000 occurred during this<br />
period which was three times the expected mortality for that time <strong>of</strong> year. Most deaths were<br />
among infants, the elderly and those with chronic respiratory disease (Anderson, ponce de<br />
Leon et al. 1996).In addition, emergency hospital admissions tripled for respiratory disease,<br />
and doubled for cardiovascular disease over the same period (Schwartz l99l). <strong>The</strong>se led to<br />
the Clean Air Act in 1956 and 1968 in the United Kingdom (UK), with further modifications<br />
to the Act made in 1993.<br />
49
<strong>The</strong> pollution <strong>of</strong> the 'great smog <strong>of</strong> London' and contemporaneously elsewhere was to do<br />
with the burning <strong>of</strong> coal and generation <strong>of</strong> 'black smoke' (Ministry <strong>of</strong> Health 1954). Smog<br />
has become the common name given to the "soup" produced from photochemical reactions in<br />
the atmosphere. Because <strong>of</strong> the role that heat and sunlight play in its production, the highest<br />
levels <strong>of</strong> smog are recorded on hot, sunny days. Summer smog is composed mainly <strong>of</strong> ground<br />
level ozone and particulate matter. Because ozone is not produced at high levels in cold<br />
weather, winter smog is composed mainly <strong>of</strong> particulate matter and sulphur dioxide (World<br />
Health Organisation 2003).<br />
However with the increase in mechanisation and industrialization, particularly with increasing<br />
motor vehicle use, the relative contribution <strong>of</strong> pollutants from the different sources has<br />
changed over the last few decades. <strong>The</strong>re has been a reduction from industrial sources and<br />
domestic heating, and an increase from car exhaust emissions. <strong>The</strong> combustion <strong>of</strong> these fossil<br />
fuels produces carbon monoxide, nitric oxides, benzene, sulphur dioxide, and particulate<br />
matter. In addition, diesel engines, which are also on the rise, emit much higher emissions <strong>of</strong><br />
the gases and 100 times more particles compared with gasoline engines equipped with modern<br />
exhaust treatment systems @iedl and Diaz-Sanchez 2005).<br />
Reflecting the increasing pollution problem, 172 individual compounds and 17 compound<br />
classes as hazardous air pollutants <strong>of</strong> concern in the USA in 1990 (Clean Air Act 1990). Of<br />
these, the compounds that have been associated with respiratory and/or cardiovascular disease<br />
are particulate matter with a diameter <strong>of</strong> less than l0pm (PM10), ozone, nitric oxides and<br />
nitrogen dioxide (NOz) in particular, sulphur dioxide (SOz), carbon monoxide (CO), lead and<br />
volatile organic compounds (Anonymous 1995; Anonymous 1996).<br />
This association between large specific pollutant episodes and increased disease and/or deaths<br />
is well documented for these early episodes. It has subsequently been more difficult to tease<br />
out whether less, but more continuous, pollutant exposure had similar health effects and has<br />
resulted in support both for (Bates 2000) and against (Gamble 1998) and necessitated large<br />
committee reviews <strong>of</strong> the topic (Anonymous 1995; Anonymous 1996). <strong>The</strong> suspicion that<br />
there was an adverse effect arose because <strong>of</strong> the noted increasing rate <strong>of</strong> allergic disease<br />
occurring in parallel and geographically coincident with the increased industrialisation. Even<br />
in 1873 when the first report <strong>of</strong> environmental exposure causing disease was documented<br />
(Charles Harrison Blackley), that 'hay fever' or 'hay asthma' was caused by grass pollen, it<br />
was even then observed to be more common in urban than in rural settings (Brims and<br />
Chauhan 2005). <strong>The</strong> difficulties in confirming the effects <strong>of</strong> a lower pollution dose over a<br />
50
longer period <strong>of</strong> time were manifold. Firstly, there were many differing pollutants available to<br />
measure. Secondly, there was likely to be an interaction with pollutants and other<br />
environmental and/or infective triggers for disease. Thirdly, there was uncertainty that an<br />
increase in disease rates could occur at levels that were below World Health Organisation<br />
(WHO) recommendations for safety <strong>of</strong> the time (World Health Organisation 2c413; World<br />
Health Organisation working group 2003).<br />
Analysis <strong>of</strong> the London data from 1958 to 1972 rcveals a pattern that the daily mortality was<br />
indeed associated with pollution levels on the previous day. This was especially so for<br />
particulate matter, SOz and acidic aerosol pollution (Mazumdar, Schimmel et al. l9g2:<br />
Schwartz and Marcus 1990), but it was difficult to determine whether a single pollutant was<br />
responsible and authors concluded it may be the interactions between the three that were<br />
important (Ito, Thurston et al. 1993). A further large study analysed air pollution and daily<br />
mortality in London between 1982 and 1992 as part <strong>of</strong> an across Europe project. <strong>The</strong><br />
strongest association was found with ozone recorded the same day, and particulates recorded<br />
the previous day with small but positive effects also seen with NOz and SOz on all causes <strong>of</strong><br />
death (excluding accidents), cardiovascular mortality and respiratory mortality (Anderson,<br />
Ponce de kon et al. 1996). Even relatively low levels <strong>of</strong> air pollution have been shown to be<br />
associated with increased health problems and with day to day variations in mortality<br />
(Schwartz t993). From the 1960s, through three decades <strong>of</strong> London winters, there was a<br />
highly significant association between mortality and levels <strong>of</strong> particulate matter and/or SOz<br />
comparing levels and mortality (Schwartz and Marcus 1990). In the USA, an association<br />
between levels <strong>of</strong> air pollution and overall mortality was also demonstrated (Ostro 1993;<br />
Schwartz 1994), as well as between particulate matter (PMlg) and an observed increase <strong>of</strong><br />
respiratory disease within the levels usually measured in urban areas @ockery and pope<br />
1994).<br />
2.2.2 Disease and pollution<br />
So what has been discovered in terms <strong>of</strong> disease? Some subgroups <strong>of</strong> population appear more<br />
sensitive to the effects <strong>of</strong> air pollution; these include young children, the elderly and people<br />
with pre-existing chronic cardiac and respiratory disease such as chronic obstructive<br />
pulmonary disease (COPD) and asthma (Anonymous 1995; Anonymous 1996). For example,<br />
the analyses <strong>of</strong> fine particulate air pollution and mortality in nine counties across California<br />
showed that the particulate level was related in an increase in overall mortality, as well as for<br />
deaths out <strong>of</strong> hospital, respiratory disease, cardiovascular disease, diabetes and deaths in<br />
51
adults greater than 65 years (Ostro, Broadwin et al. 2006). Increased rates <strong>of</strong> myocardial<br />
infarction, dysrhythmias, increased blood pressure, heart failure and hospitalisation for<br />
cardiac events have been associated with higher air pollution levels (Pope, Verrier et al. 1999;<br />
Peters, Liu et al. 2000; Linn and Gong 200I; Peters, Dockery et al. 2001; Pope, Burnett et al.<br />
2W4\.<br />
<strong>The</strong> 'Swiss Study on Air Pollution and Lung Diseases in Adults' involving over nine<br />
thousand subjects showed positive conelations <strong>of</strong> levels <strong>of</strong> particulate matter, ozone and NOz<br />
with chronic cough, chronic sputum production, and shortness <strong>of</strong> breath during the day,<br />
during the night and on exertion, but not with other respiratory symptoms (Zemp, Elsasser et<br />
al. 1999). This study also showed reduced lung function parameters for FVC, FEVr and<br />
FEFzs-zs which negatively correlated with the same three pollutants (Ackermann-Liebrich,<br />
Iruenberger et al. 1997, Schindler, 2001 #652). A major study in six cities in the USA<br />
demonstrated a three fold increase in chronic cough with increased exposure to particulate<br />
pollution @ockery and Pope 1994). Throughout Califomia from 1973 to 1987 site and season<br />
specific particulates were monitored and showed correlations between the development <strong>of</strong><br />
symptoms <strong>of</strong> airway obstructive disease, increased severity <strong>of</strong> obstructive disease, chronic<br />
productive cough and increased asthma (Abbey, Hwang et al. 1995). Stronger associations<br />
were seen with those who were also occupationally exposed to dusts and fumes. Increased<br />
symptoms <strong>of</strong> cough, bronchitis, asthma, and COPD were associated with increased air<br />
pollution (Schwartz t993; Souza, Saldiva et al. 1998). Particulate matter was found to be<br />
associated with respiratory diagnoses made by a physician and chronic bronchitis in a study<br />
involving 53 urban areas throughout the United States with graduated levels <strong>of</strong> pollution<br />
(Schwartz 1993).In Australia, ozone levels, particulate matter and the concentration <strong>of</strong> SOz<br />
had strong associations with emergency department review <strong>of</strong> respiratory illnesses overall,<br />
and for asthma exacerbations in twelve hospitals, though it proved difficult to detect out<br />
differences between the pollutants (Atkinson, Anderson et al. 1999). Particulate matter levels<br />
were also positively associated with hospital admissions for asthma and for COPD @rbas and<br />
Hyndman 2005).<br />
Histopathologic changes have been shown in lung tissue samples <strong>of</strong> individuals who died due<br />
to violent causes depending on their pollutant exposure history. With the increased pollution,<br />
there was increased presence <strong>of</strong> inflammatory reaction, wall thickening, ild secretory<br />
hyperplasia. <strong>The</strong>re were also increased carbon deposits along the regional lymphatic drainage<br />
(Souza, Saldiva et al. 1998).<br />
52
Studies on traffic exposure, (including detailed studies on occupational exposure to increased<br />
vehicular emissions (Raaschou-Nielsen, Nielsen et al. 1995; Zagury,6 Moullec et al. 2000;<br />
Roegner, Sieber et al. 2002; Seshagiri 2003; Lai, Liou et al. 2005) and occupational NO<br />
exposure (Azan, Williams et al. 1996; Markhorst, I-eenhoven et al. 1996; Olin, Ljungkvist et<br />
al. 1999; Phillips, Hall et al. 1999 Qureshi, Shah et al. 20O3; Maniscalco, Grieco et al.<br />
2004)), have also demonstrated that there is a consistent effect <strong>of</strong> long term exposure to car<br />
traffic on non-specific respiratory symptoms (Abbey, Hwang et al. 1995) and lung function<br />
(Ackermann-Liebrich, kuenberger et al. 1997). Proximity to traffic exposure was a risk<br />
factor for wheezing, asthma severity and prevalence (Nitta, Sato et al. 1993; Oosterlee,<br />
Drijver et al. 1996, Brunekreef, 1997 #640).In three Japanese cross sectional questionnaire<br />
studies in five thousand women participants, the estimated odds ratio for chronic cough,<br />
chronic sputum production, chronic wheeze and chest infections with sputum were increased<br />
the closer the subjects lived to roadways with heavy traffic (Nitta, Sato et al. 1993). Air<br />
pollution appears more likely to exacerbate existing asthma rather than generate new cases,<br />
although it is associated with reduced lung function in healthy children (Barnes 1994; Segala<br />
1999). <strong>The</strong>re are fewer studies that have looked at the effects <strong>of</strong> air pollution on upper airway<br />
disease rates, but general practitioner consultations due to upper airway symptoms increase on<br />
days <strong>of</strong> higher concentrations <strong>of</strong> particulate matter and SOz (Gordian, Ozkaynak et al. 1996;<br />
Hernandez-Garduno, Perez-Neria et al. 1997;Hajat, Anderson et al.2oo2).<br />
Only a few authors have looked specifically at the effects <strong>of</strong> air pollution on children, where<br />
both outdoor air pollution and indoor air quality have been implicated as causal factors for<br />
respiratory diseases and respiratory symptoms (Nicolai 1999). An increase in mortality in<br />
children during severe episodes <strong>of</strong> air pollution has been shown and this includes an increase<br />
in the general rate <strong>of</strong> mortality, an increase in respiratory mortality and an increase in peri-<br />
neonatal mortality (Bates 1995; Anderson, Ponce de kon et al. 1996). As well as mortality,<br />
an increase in respiratory morbidity has been shown. <strong>The</strong> respiratory illnesses sensitive to<br />
increased pollutants in children include acute bronchitis, cough, asthma and pneumonia.<br />
Similarly to adults, this is most <strong>of</strong>ten attributable to an increase in particulate matter (Aunan<br />
1996). In the 'Six Cities Study <strong>of</strong> Air Pollution and Health' reported rates <strong>of</strong> chronic cough,<br />
bronchitis and chest illness were positively associated with all measures <strong>of</strong> particulate<br />
pollution and positively, though less strongly, associated with SOz and NOz @ockery,<br />
Speizer et al. 1989). Particulate matter in air pollution has been shown to triple the prevalence<br />
<strong>of</strong> chronic cough, nocturnal cough and bronchitis in a cross sectional study <strong>of</strong> over 4,000<br />
Swiss children aged six to 15 years from ten different communities with the highest pMl0<br />
53
levels compared to those children living in less exposed areas (Braun-Fahrlander, Vuille et al.<br />
1997). In a prospective, observational study over one year and comparing 1025 children<br />
presenting with wheezy episodes with other admissions as a control group, the day to day<br />
variations in the concentration <strong>of</strong> ozone were associated with an increased incidence in acute<br />
wheezy episodes @uchdahl, Parker et al. 1996). Children living along a busy street were<br />
found to have a higher prevalence for most respiratory symptoms than children living along a<br />
quiet street. This was most significant for medication use and for episodes <strong>of</strong> wheeze<br />
(Oosterlee, Drijver et al. 1996). Children admitted with asthma were more likely to live in an<br />
area <strong>of</strong> high traffic flow compared to admissions for non-respiratory reasons or a community<br />
sample <strong>of</strong> children with a significantly linear trend observed for traffic flow for children<br />
living less than 500 metres from a main road @dwards, Walters et al. 1994). This study also<br />
showed that children were more likely to be admitted for non-respiratory reasons compared to<br />
the community sample <strong>of</strong> children if they lived within 200 metres <strong>of</strong> a main road. Similarly<br />
results for atopy and road traffic have also been demonstrated with, for example, higher rates<br />
<strong>of</strong> allergic sensitization found in children playing more than one hour per day near major<br />
traffic thoroughfares (Brunekreef, Janssen et al. 1997).<br />
Some studies have incorporated lung function testing to look at the effects <strong>of</strong> air pollution.<br />
<strong>The</strong> lungs develop throughout childhood, with peak lung function occurring between 20 and<br />
25 years, followed by a plateau <strong>of</strong> ten years before beginning to decline. A deficit in growth<br />
caused by air pollution effects, for example, would most likely translate into a reduced<br />
baseline function that would be carried throughout life (Gilliland, Gauderman et al. 2ffi2;<br />
Gauderman 2006). Air pollution data from monitoring stations in twelve California<br />
communities demonstrated that a greater proportion <strong>of</strong> young adults with and FEVr less than<br />
80Vo predrcted lived in the areas <strong>of</strong> higher carbon and PM10 (Gauderman, Vora et al.2007).<br />
An inverse dose dependent relationship was found between the carbon content <strong>of</strong> airway<br />
macrophages in induced sputum and FEVr, FVC and FEFzs-zs in healthy children living in an<br />
area with a variation <strong>of</strong> PMl0, primarily due to emissions from road traffic (Kulkarni, Pierse<br />
et al. 2O06). Reviewing the age goup six years to 24 years <strong>of</strong> age, FVC, FEVr and PEF all<br />
showed statistically significant negative correlations with the annual concentrations <strong>of</strong> total<br />
suspended particles, NOz and ozone (Schwartz 1989). <strong>The</strong> relationships still persisted when<br />
children with pre-existing respiratory illnesses and smokers were included. Furthermore in a<br />
longitudinal three year study, children exposed to high PMl0 levels during the summer had a<br />
reduced growth <strong>of</strong> FEVr and FEFzs-zs (Horak, Studnicka et al. 2002). A reduction in lung<br />
function with higher levels <strong>of</strong> ozone was also demonstrated in children from three schools in<br />
54
Mexico (Castillejos, Gold et al. 1992), with atopic status being an additional risk factor<br />
(Jorres, Nowak et al. 1996). In children with moderate to severe asthma attending surnmer<br />
camps over three years, air pollution levels (predominantly ozone) were associated with acute<br />
asthma exacerbations, chest symptoms and a reduction in lung function (Thurston, Lippmann<br />
et al.1997).<br />
Increased absences in school and kindergarten or playschool attendances have also been<br />
associated with an increase in particulate matter -<br />
which was more pronounced in six to nine<br />
year olds than older age groups (Pope, Schwartz et al. lgg2: Ransom and pope lgg2). <strong>The</strong>re<br />
has also been an association with rises in levels <strong>of</strong> pollutants on prolonging episodes <strong>of</strong><br />
infection (Bates 1995). Increased levels <strong>of</strong> pollutants have also been associated with an<br />
increased risk <strong>of</strong> developing upper respiratory tract infections and symptoms in children<br />
(Jaakkola, Paunio et al. 1991, Ostro, 1999 #657), particularly with high concentrations <strong>of</strong><br />
SOz, and moderate levels <strong>of</strong> NOz and particulate matter (von Mutius, Sherrill et al. 1995).<br />
Clearly, it would be helpful to confirm these findings using more specific experiments to<br />
determine a dose response and the mechanism <strong>of</strong> damage which causes disease. This has been<br />
done in a few animal models which have demonstrated the development <strong>of</strong> infectious and<br />
allergic lung disease with increased severity when exposure to air pollutants (ozone, NO2,<br />
SOz). <strong>The</strong>se pollutants increased damage in infection and inflammation in mouse and guinea<br />
pig populations predominantly but also in a dog and a monkey model (Gilmour 1995;<br />
Miyabara, Takano et al. 1998; Ng, Kokot et al. 1998; Selgrade 200O; Whitekus, Li et al.<br />
2002). While it is more problematic to confirm a dose response in humans, it has been<br />
possible to do nasal challenges with allergen, with or without the addition <strong>of</strong> diesel exhaust<br />
particles, showing exaggerated responses in the presence <strong>of</strong> the particles @iaz-Sanchez, Tsien<br />
et al. 1996; Diaz-Sanchez, Jyrala et al. 2000; Bastain, Gilliland et al, 2003; Gilliland, Li et al.<br />
2004).It is now thought that oxidant stress is the mechanism that underlies the toxic effect <strong>of</strong><br />
most forms <strong>of</strong> air pollution (Nel, Diaz-Sanchez et al. 2001; Kelly 2003; Kelly and Sandstrom<br />
2004; Gauderman 2006). <strong>The</strong> lung is actually well equipped to deal with oxidative stress as<br />
the lung lining fluid is rich in enzymatic and low molecular weight non-enzymatic<br />
antioxidants. In addition, there are intracellular defences with glutathione based enzymes that<br />
use a variety <strong>of</strong> the products <strong>of</strong> oxidative stress as substrates and therefore prevent their build-<br />
up. However individuals have differing genotypes <strong>of</strong> this enzqe family, which appear to<br />
make them more or less susceptible to damage from air pollutants, including active or passive<br />
smoke inhalation (Miyabara, Yanagisawa et al. 1998; Mudway and Kelly 2000; Gilliland,<br />
Rappaport et aL.2002; Bastain, Gilliland et aI.2003; Gilliland, Li et al. 2004).<br />
55
More recently 'Air Quality Indexes' were developed to better inform the public about air<br />
quality or the degree <strong>of</strong> pollution on any given day. <strong>The</strong>se are composite measures <strong>of</strong><br />
common air pollutants for which there are demonstrable adverse effects on health and the<br />
environment. At the current time these include CO, NOz, SO2, ground level ozone, suspended<br />
particulate matter (sometimes expressed as a coefficient <strong>of</strong> haze) and total reduced sulphur<br />
compounds. <strong>The</strong> Index is reported on a scale <strong>of</strong> 0 to 100 (or more) with scales <strong>of</strong> 'very good"<br />
air quality being 0-15 and 'very poor' near the 100 mark (Hilternann, Stolk et al. 1998;<br />
Abelsohn, Stieb et al.2002).<br />
2.2.3 Nitrogen oxides in pollution<br />
NO is a potentially toxic molecule; and both NO and its interrelated forms were discovered as<br />
components in pollution. Space shuttle and jet plane exhaust, lightening and smog are all<br />
known to have a high proportion <strong>of</strong> NO (Nathan 1992). Nitrogen oxides were detected in the<br />
evaluation <strong>of</strong> air pollution from the Titan tr firings @iamond and Johnson 1965). NO forms<br />
part <strong>of</strong> the origin <strong>of</strong> the shuttle glow which was observed in 1983, and was carefully examined<br />
because <strong>of</strong> concern that it would interfere with space-based spectroscopy. It was found to be<br />
the reaction between NO and 02 forming an excited form <strong>of</strong> NO2 which released light as it<br />
desorbed (Viereck, Murad et al. 1991). Increases in air traffic have resulted in increases in<br />
nitrogen oxides in the troposphere, again causing concern about the effects in atmospheric<br />
chemistry (Johnson, Henshaw et al. 1992). <strong>The</strong>re exists an array <strong>of</strong> compounds that includes<br />
NO, nitrosoium cation (NO +), nitroxyl anion (NO-), peroxynitrite (ONOO-) and hydroxyl<br />
anion (OH-). Most <strong>of</strong> these are inherently unstable compounds and interact with other<br />
elements particularly ozone and oxygen, so the main compound measurable in pollution from<br />
this class <strong>of</strong> gases is the stable gas NO2. <strong>The</strong> ambient NOz levels in London vary between25-<br />
58ppb (24 hour average), European air levels are 78ppb (24 hour average) and the level in the<br />
USA is 53ppb (one year average) (Nicolai 1999).<br />
Many epidemiological studies <strong>of</strong> outdoor pollution have found associations between NO2<br />
exposure and disease, even at levels below the current WHO guidelines. <strong>The</strong> 'Air Pollution on<br />
Health: European Approach' studies incorporated data from 15 cities with a total population<br />
<strong>of</strong> greater than 25 million. A rise in NOz over the one hour safety maximum <strong>of</strong> 50 pglm3 was<br />
associated with a 2.6Vo increase in asthma admissions and a l.3%o increase in daily all cause<br />
mortality (Touloumi, Katsouyanni et al. 1997). In Toronto, from the total number <strong>of</strong><br />
premature deaths related to air pollution, NOz was assessed to be responsible for almost 407o,<br />
and, <strong>of</strong> the hospital admissions, 60Vo was also thought to be attributable to NOz levels, with<br />
56
the other main pollutants studied being CO and SOz (Abelsohn, Stieb et al.2O02). Three<br />
studies have looked at the effect <strong>of</strong> ambient NO2 concentrations on respiratory illnesses in<br />
children. Firstly, a significant association between the frequency <strong>of</strong> croup and the daily<br />
measured level <strong>of</strong> NOz for the peak periods (September and March) <strong>of</strong> disease rate was found<br />
in five German communities (Schwartz, Spix et al. l99l). Secondly, the risk <strong>of</strong> admission to<br />
hospital with wheezing bronchitis was significantly related to outdoor NO2 exposure in girls<br />
(although not boys) in Stockholm, however the presence <strong>of</strong> a gas stove in the home was also a<br />
significant risk factor (Pershagen, Rylander et al. 1995). A third study showed worsened<br />
virus-induced asthma in children following exposure to higher NO2 levels (Chauhan, Krishna<br />
et al. 1998).<br />
However examination <strong>of</strong> the potential harmful effects <strong>of</strong> the pollutant NO has been<br />
investigated down to the individual cigarette. NO exists in an English cigarette ',at a level <strong>of</strong><br />
1,000 parts per billion with the level <strong>of</strong> a French cigarette being one million parts per billion"<br />
(Borland and Higenbottam 1987). NO corrodes metals and interacts with plastics. <strong>The</strong>re are<br />
particular problems with the development <strong>of</strong> even more dangerous combinations as listed<br />
above in the presence <strong>of</strong> higher concentrations <strong>of</strong> oxygen (see Chapter 3 and Chapter 4<br />
regarding toxicity).<br />
So by the time this research began, the nitrogen oxides were known to contribute to pollution,<br />
to contribute to the excess <strong>of</strong> cardiovascular and respiratory disease and mortality seen with<br />
increased air pollution and studies were well underway using analysers to measure daily<br />
levels for air quality recordings and to ascertain their continued association with disease.<br />
2.3 <strong>The</strong> discovery <strong>of</strong> nitric oxide in biological systems<br />
2.3.1 Vascular control<br />
It is increasingly rare to discover something completely novel. <strong>The</strong> discovery <strong>of</strong> NO and its<br />
importance in biological systems, in particular the opening up <strong>of</strong> a new concept <strong>of</strong> the use <strong>of</strong><br />
gases as biological mediators, was truly new. Initially the exact nature <strong>of</strong> NO as a mediator<br />
was not recognised and it was labelled as 'Endothelial Derived Relaxing Factor' (EDRF).<br />
In 1916 Mitchell observed that oxides <strong>of</strong> nitrogen were produced in mammals showing more<br />
nitrogen in urine than consumed (Mitchell, A. et al. 1916). In l92g Tannenbaum confirmed<br />
that mammals produced nitrogen oxide by showing production occurring in the intestine<br />
(Mitchell 1928; Tannenbaum, Fett et al. 1928). In the 1970s research was undertaken on<br />
categorising biological 'amines' such as adrenaline, noradrenaline, histamine and<br />
57
acetylcholine. <strong>The</strong>se were known to contribute to both cell to cell and nerve cell to terminal<br />
tissue interactions. One <strong>of</strong> the main investigators in this area, Pr<strong>of</strong>essor R. F. Furchgott, was<br />
studying the mechanism <strong>of</strong> contraction and relaxation <strong>of</strong> isolated <strong>of</strong> blood vessels. Using<br />
identical chemicals, the studies initially yielded conflicting results. Laboratory investigations<br />
subsequently revealed that the results were dependent on which technician conducted the<br />
experiment. One technician found that acetylcholine always relaxed the vessels and another<br />
found that it always contracted the vessels. Investigating further, Furchgott discovered that the<br />
reason for this anomaly was the presence or absence <strong>of</strong> the endothelial monolayer, as one<br />
technician meticulously kept the endothelium intact and the other always removed the<br />
endothelium in preparation. <strong>The</strong> presence or absence <strong>of</strong> this endothelium resulted in quite<br />
different reactions <strong>of</strong> the blood vessel to the applied mediator (Furchgott, Jothianandan et al.<br />
1984). This also explained a somewhat surprising finding which had been made many years<br />
earlier in his research career. He found that acetylcholine (a well known vasodilator in<br />
animals and in perfused organ studies) elicited contraction rather than relaxation on an aortic<br />
strip. However, the preparation technique unknowingly stripped the specimen <strong>of</strong> endothelial<br />
cells @urchgott and Bhadrakom 1953). It was this that led to the coining <strong>of</strong> the term for the<br />
substance responsible as the 'EDRF' @urchgott andZawadzki 1980).<br />
Advances in this area occurred with the development <strong>of</strong> perfusion-bioassay procedures which<br />
allowed more direct studies on the properties <strong>of</strong> EDRF than the previous experiments which<br />
had been carried out in organ chambers. Intact endothelial cells were held upstrearn, a section<br />
<strong>of</strong> blood vessel wall stripped <strong>of</strong> endothelium was held downstream so substances could be<br />
perfused in turn to see whether each test substance inhibited EDRF by interfering with its<br />
synthesis, its release or inactivated EDRF after release. This approach was also used to assess<br />
the rate <strong>of</strong> decay <strong>of</strong> EDRF by altering the transit time <strong>of</strong> the perfusate (Furchgott 1998).<br />
Initially this experiment resulted in confusion, as estimations <strong>of</strong> the EDRF half life were<br />
estimated from four to 50 seconds. However later this was able to be explained by the<br />
differences in the superoxide anion (O2-) concentration in the perfusion fluid as this rapidly<br />
inactivates EDRF (see Chapter 3.2'Reactions <strong>of</strong> nitric oxide'), (Gryglewski, Palmer et al.<br />
1986; Rubanyi and Vanhoutte 1986). <strong>The</strong>se perfusion bioassay studies also allowed<br />
demonstration <strong>of</strong> the release <strong>of</strong> EDRF from vessels with increased shear stress, usually<br />
secondary to increased flow thereby suggesting that EDRF had a continuous physiological<br />
role in the control <strong>of</strong> regional blood flow and regulation <strong>of</strong> blood pressure.<br />
58
Figure 2.1: Diagram <strong>of</strong> blood vessels in cross section showing the endothelial layer<br />
ExtltNll ElAtllC Xlti'l!8ANE<br />
IMETNAI. ElAtIK<br />
^itilEt^ltE<br />
ARTERY<br />
IUNICA IMNtrA<br />
:NOOTHEUUM<br />
crtcut.At ,$utcttt<br />
tAt cttts<br />
vlsEts 0F ArytNlrn<br />
AOVIN'|I|a<br />
Taken from 'Access Excellence'- <strong>The</strong> Heart and the Circulatory System by Roger E phillips Jr., from<br />
the National Health Museum, "<strong>The</strong> Site for Health, Teachers and-Learners', t-+tl K st, Suite 1300,<br />
Washington DC 2000s.<br />
<strong>The</strong>se and other researchers continued pursuing the identity <strong>of</strong> this substance with the<br />
evolving characteristics becoming increasingly similar to a compound <strong>of</strong> nitrogen, resulting in<br />
experiments comparing 'EDRF' with nitrogen oxides. Furchgott initially announced that<br />
EDRF could actually be the inorganic gas 'NO' at a conference in July 1986 (Khan and<br />
Furchgott 1987)' This same conclusion was proposed at the same conference by Ignarro as a<br />
result <strong>of</strong> his experiments on bovine pulmonary arteries (Ignarro, Buga et al. l9g7; Ignarro,<br />
Byrns et al. 1988). Similarly in 1987, Palmer, Fenige and Moncada showed that vascular<br />
endothelial cells generated NO and that the amounts generated accounted for the biological<br />
activity <strong>of</strong> the named EDRF mediator (Moncada, Palmer et al. 1988). This was a novel<br />
suggestion as - until this time -<br />
mediators.<br />
gases had not been thought capable <strong>of</strong> acting as biological<br />
It had been recognised that endothelium dependant relaxation <strong>of</strong> blood vessels was associated<br />
with increased cyclic guanosine 3', 5' -monophosphate (cGMp) levels in vascular muscle.<br />
This was demonstrated earlier in a number <strong>of</strong> studies using rat aorta (Rapoport and Murad<br />
1983), rabbit aorta (Diamond and Chu 1983, Furchgott, 1983 #415), bovine coronary arteries<br />
(Holzmann 1982) or bovine pulmonary arteries (Ignarro, Burke et al. 1984). Rises in cGMp<br />
are produced by activation <strong>of</strong> soluble guanylate cyclase (sGC), a magnesium sensitive<br />
hexodimer protein which contains two heme molecules. Under basal conditions, the guanylate<br />
59
cyclase does not produce significant levels <strong>of</strong> NO, but the relaxant factor, EDRF, was thought<br />
to alter the binding in so much that it increased the activation <strong>of</strong> the enzyme by 400 fold.<br />
However it is now known that it is nitrosylation <strong>of</strong> the guanylate cyclase that results in high<br />
levels <strong>of</strong> cGMP which both prevents entry and promotes movement out <strong>of</strong> the cell <strong>of</strong> calcium<br />
resulting in vasodilatation. <strong>The</strong> Moncada research goup then identified that the substrate for<br />
NO synthesis was L-arginine and also went on to synthesise an inhibitor <strong>of</strong> NO formation by<br />
producing a false competitive inhibitor <strong>of</strong> L-arginine; N* monomethyl L-arginine (L-<br />
NMMA), (Gardiner, Compton et al. 1990). It was by using this inhibitor that further<br />
clarification <strong>of</strong> the role <strong>of</strong> NO was possible.<br />
2.3.2 Immunefunction<br />
At the same time that EDRF was being identified as NO, other research groups were looking<br />
at NO in a different biological system -<br />
that <strong>of</strong> host immune defence.<br />
Macrophages were known to be able to inhibit tumour growth and induce tumour cell death<br />
(Nathan 1992). In 1987 (Hibbs, Vavrin et al. 1987) it was suggested that the macrophage<br />
cytotoxicity might in part be due to the ability <strong>of</strong> the macrophages to synthesise NO, nitrite<br />
and nitrates from L-arginine. At the tissue level, NO was known specifically to be toxic to<br />
cells. A number <strong>of</strong> bacteria were also known to be able to generate reactive species <strong>of</strong> nitro-<br />
oxygen suspected as occasionally causing lung disease. However the main interest in these<br />
bacteria was not in the medical arena but centred on the importance <strong>of</strong> bacteria using these<br />
pathways causing meat to degenerate. Organisms that had demonstrated the production <strong>of</strong><br />
nitro-oxygen compounds included Achromobacter cycloclastes, Alcaligenes faecalds, and<br />
Bacillus halodenitrificans (Godden, Turley et al. 1991).<br />
<strong>The</strong> suggestion that the use <strong>of</strong> NO in this way could occur within living animals came from<br />
the demonstration that germ free rats excreted more nitrate than they ingested (Green,<br />
Tannenbaum et al. 1981). This was also found in humans in a stable state but further<br />
investigations showed that the amounts increased dramatically during intercurrent infection<br />
(Green, Ruiz de Luzuriaga et al. l98l). Other researchers confirmed this by injecting rats and<br />
mice with pro-inflammatory or infectious agents and showing that there was an increase in the<br />
amount <strong>of</strong> nitrates that were excreted (Wagner, Young et al. 1983, Stuehr, 1985 #580). In part<br />
this was described by using macrophages from these rats in vitro and showing that these cells<br />
could produce nitrite and nitrate by oxidizing L-arginine (Iyengar, Stuehr et al. 1987) to yield<br />
citrulline and a compound with the ability to react with amines and generate nitrosamines<br />
(Miwa, Stuehr et al. 1987). It was thought that a compound with such ability had to be an<br />
60
oxide <strong>of</strong> nitrogen which was more reactive than nitrite or nitrate. It was also shown that<br />
macrophages used an oxidative form <strong>of</strong> injury associated with iron loss and involving<br />
inhibition <strong>of</strong> iron-sulphur enzymes to inflict damage on tumour cells and fungi. L-arginine<br />
was then identified as the necessary metabolite in the extracellular medium to sustain this<br />
form <strong>of</strong> macrophage-mediated cytotoxicity (Hibbs 1991). Substituting L-arginine analogs<br />
blocked the cytotoxicity in a stereospecific manner and blocked the production <strong>of</strong> nitrite by<br />
activated macrophages. Nitrite or nitrate substances alone could not replicate the cytotoxicity<br />
<strong>of</strong> the nitrite-producing macrophage except when in an acidic pH - a condition coincidentally<br />
in which nitrite can generate NO (Hibbs, Vavrin et al, 1987; Stuehr, Gross et al. l9g9). NO<br />
(rather than other nitro-oxygen species) specifically mimicked the pattern <strong>of</strong> macrophage-<br />
mediated cytotoxicity and scavengers <strong>of</strong> NO blocked the cytostatic effect <strong>of</strong> macrophages.<br />
Funher investigation showed that this cytostatic effect used by the macrophages was also<br />
operational against yeasts, helminths, protozoa and mycobacteria (Nathan and Hibbs 1991).<br />
2.3.3 Nitric oxide -<br />
a common pathway<br />
<strong>The</strong>se two separate areas <strong>of</strong> investigation into immune function and vasomotor control came<br />
together in 1989, when Stuler showed that the compound secreted by activated alveolar<br />
macrophages with L-Arginine availability, demonstrated the same bioactivity as that<br />
described for EDRF and coincided with the chemical reactivity pr<strong>of</strong>ile <strong>of</strong> NO (Stuehr, Gross<br />
et al. 1989).<br />
When these two major discoveries came together, it suggested NO synthesis from L-arginine<br />
might in fact be a widespread pathway for the regulation <strong>of</strong> cell function and communication.<br />
NO has the lowest molecular weight <strong>of</strong> any known bioactive mediator that cells produce. <strong>The</strong><br />
molecule's chemical reactivity means that its half life is short and interaction specificity is<br />
minimal. <strong>The</strong>refore, it was surprising that such a simple, fleeting, indiscriminating reactant<br />
could convey enough information in a regulatory manner to help control vital mechanisms<br />
such as vascular tone and neurotransmission (see below Section 2.3.4).It was also surprising<br />
that a mediator involved in homeostasis, was also involved in host defence to destroy micro-<br />
organisms and tumour cells (Nathan 1992). Following the demonstration that both<br />
endothelium cells and macrophages were able to synthesis this new mediator, there was an<br />
explosion <strong>of</strong> research demonstrating that many other cells were able to produce NO. This<br />
molecule was shown to have physiological roles in many neuronal tissues; including the<br />
cerebellum, the cerebral cortex and the hypothalamus, as well as in ganglion cells <strong>of</strong> the<br />
autonomic nervous system (Garthwaite, Charles et al. 1988; Bredt and Snyder 1990;<br />
6t
Garthwaite l99l), and the non adrenergic, non cholinergic (NANC) peripheral nerves (Bredt,<br />
Hwang et al. 1990; Rand 1992).<br />
NO was subsequently found, with likely mediator roles, in the renal cortex epithelial cells,<br />
adrenal glands, gut, genital system and has also been found in amniotic fluid (Nathan 1992).<br />
Thus NO was realised to be an important gas used in biological systems in many species<br />
including humans, and within humans NO was seen in a wide variety <strong>of</strong> tissues as a gaseous<br />
mediator. <strong>The</strong> type <strong>of</strong> roles it fulfils in three <strong>of</strong> these key systems (cardiovascular,<br />
neurological and immunity) will be discussed briefly below, while the role <strong>of</strong> NO in the lung<br />
is discussed in more depth in Chapter 4,preparatory to measuring levels from the airway.<br />
2.3.4 Nitic oxide in physiological roles<br />
2.3.4(i) Cardiovascular<br />
NO is produced by nitric oxide synthases (NOS) which generate NO from the semi-essential<br />
amino acid L-arginine and release picomolar amounts <strong>of</strong> NO in response to receptor<br />
stimulation (see Chapter 3 and 4 for detail on chemical reactions and pathways). In this way<br />
NO is responsible for the basal vasomotor tone and essential for the regulation <strong>of</strong> blood flow<br />
and blood pressure. NO dependent vasodilator tone is in part maintained through the release<br />
<strong>of</strong> NO in response to physical activation <strong>of</strong> endothelium cells by stimuli such pulsatile flow<br />
and shear stress. This role was in part determined by the studies described above before NO<br />
was so-named, and in part been determined subsequently by the use <strong>of</strong> the non-functioning<br />
competitive inhibitor <strong>of</strong> this enzyme, L-NMMA. A potent vasoconstrictor, L-NMMA<br />
constricts vascular beds and produces a hypertensive response in animals with high potency<br />
@lsner, Muntze et al. 1992) and has been shown to cause vasoconstriction in the forearm<br />
circulation in humans (Gardiner, Compton et al. 1990). As documented below, NO is also<br />
released by non-adrenergic non-cholinergic (NANC) nerve terminals and this may also<br />
contribute to the regulation <strong>of</strong> blood flow and pressure. <strong>The</strong> success <strong>of</strong> compounds used in<br />
angina and hypertension such as nitro glycerine and sodium nitroprusside are now known to<br />
be because these compounds imitate endogenous nitro-vasodilator compounds by relaxing<br />
blood vessels @oel, Godber et al. 2000).<br />
NO also inhibits platelet aggregation and adhesion. This occurs with the increase in cGMP<br />
activity within platelets and subsequent phosphorylation <strong>of</strong> proteins that regulate platelet<br />
activation (Radomski, Palmer et al. 1987; Radomski, Palmer et al. 1987). It can also be<br />
generated either by the endothelium or the platelets themselves generating NO to act as a<br />
62
negative feedback mechanism to inhibit platelet activation (Mehta, Chen et al. 1995). NO is<br />
also involved in the reaction <strong>of</strong> leukocytes with vessels walls, again inhibiting activation. NO<br />
inhibits vascular smooth muscle cell hyperplasia which may reduce the development <strong>of</strong><br />
pulmonary hypertension (Papapetropoulos, Garcia-Cardena et al. 1997; Ziche, parenti et al.<br />
1997). Endothelial dependant relaxation demonstrated in human vascular tissue is greater in<br />
the arteries than the veins, suggesting that arteries may generate more NO. This relaxation is<br />
inhibited in a number <strong>of</strong> patient groups with pulmonary vascular disease. <strong>The</strong> response is<br />
decreased in atherosclerotic coronary arteries compared with normal coronary arteries<br />
(Egashira, Suzuki et al. 1995), in children and adult patients with hypercholesterolemia<br />
(Quyyumi, Mulcahy et al. 1997), and in patients with essential hypertension (panza,Garcia et<br />
al. 1995). It is also Iower in the vessels <strong>of</strong> diabetic animals (Sobrevia, Nadal et al. 1996) and<br />
in pulmonary arteries obtained from patients undergoing heart lung transplantation @usting<br />
and Macdonald 1995). NO dysfunction has also been noted as contributing to hean failure<br />
(Hanssen, Brunini et al. 1998; Sharma, Coats et al. 2000), and to the pathogenesis <strong>of</strong> sickle<br />
cell disease @nwonwu, Xu et al. 1990; Waugh, Daeschner et al. 2001). Finally, it has been<br />
shown when solutions <strong>of</strong> L-arginine (substrate for NO generation) are exposed to cigarette<br />
smoke, there is a depletion <strong>of</strong> L-arginine and the formation <strong>of</strong> cyanomethyl-L-arginine and<br />
this is likely to act as a competitive inhibitor and has been shown to have an inhibitory effect<br />
on NOS. This may contribute to the vascular compromise seen in long term smokers (Wong<br />
2000).<br />
NO is also associated with significant pathology within the vascular system in regards to<br />
sepsis (Vallance and Moncada 1993).In endotoxin shock in animals, the generation <strong>of</strong> NO is<br />
directly related to the degree <strong>of</strong> systemic hypotension. This is also seen as a characteristic <strong>of</strong><br />
septic shock in humans and it is also suggested as being responsible for the hypotension<br />
induced by chemo/radiation therapy seen in patients with cancer who undergo such treatment.<br />
Endotoxin also induces NOS production and activity in venous smooth muscle cells and<br />
increased NO here may play a role in the hypotension seen with endotoxaemia. Similarly<br />
endotoxin induces NOS production in the myocardium, which may contribute to the dilated<br />
cardiomyopathy development. <strong>The</strong>re have been trials using inhibitors <strong>of</strong> No to reverse the<br />
hypotension which has been induced in animal models by lipopolysaccharide or endotoxin via<br />
TNF0. However the level <strong>of</strong> inhibition <strong>of</strong> NOS is crucial for outcome, since high doses cause<br />
severe vasoconstriction and end organ damage in multiple organs and thus causes rapid death.<br />
With titration being the key factor and difficult to control in experiments to date without<br />
63
significant complication, this treatment has not yet been able to become part <strong>of</strong> standard<br />
clinical management strategies (Petros, Lamb et al. 1994; Grover, Zaccardelli et al. 1999).<br />
2.3.4 (ii) Nervous system - central and peripheral<br />
NO has become accepted as a non-endogenous messenger, signal mediator and non classical<br />
neurotransmitter with NO synthases (see Chapter 3.4 'Nitric oxide synthase isoenzymes')<br />
detected in varying amounts in all areas <strong>of</strong> both animal and human brain @redt and Snyder<br />
1990). NO synthases are highly similar to NADPH diaphosphorase which is found in about<br />
2Vo <strong>of</strong> the neurons in the cerebral cortex @astian and Hibbs 1994), and the two enzymes are<br />
closely colocalised in both the brain and the peripheral nervous system. Glutamate is the<br />
main excitory neurotransmitter and an interaction between this and NO was demonstrated<br />
early (Garthwaite, Charles et al. 1988). After specific receptor stimulation, NO is released<br />
from a postsynaptic source to act at a presynaptic region on one or more neurons in any<br />
direction. This results in an increase in glutamate and a stable increase in synaptic<br />
transmission, a phenomenon known as long-term potentiation @liss and Collingndge 1993),<br />
which is linked to memory formation (ODell, Hawkins et al. 1991). Experiments in animals<br />
have shown that inhibiting NO synthesis by analogue competitive inhibitors impairs learning<br />
behaviour (Son, Hawkins et al. 1996). NO has also been shown to have a role in feeding<br />
behaviour, nocioception and olfaction (Bagetta, Iannone et al. 1993). An increasing body <strong>of</strong><br />
work has continued to demonstrate the importance <strong>of</strong> NO in both short and long term memory<br />
(Susswein, Katz<strong>of</strong>f et al. 2004). In addition, the NO produced by the perivascular nerves <strong>of</strong><br />
the cerebral arteries directly modulates vascular control (Gonzalez, Barroso et al. 1997). NO<br />
may also have a role in modulating pain (Moore, Babbedge et al. 1993).<br />
At low concentrations, NO plays a role in vasodilatation and neurotransmission, however at<br />
higher concentrations it can be neurotoxic. It also may be that NO can possess either<br />
neurodestructive or neuroprotective properties depending on its oxidation reduction status<br />
(see Chapter 3 'Reactions with nitric oxide'). Microglial cells, which are <strong>of</strong> monocyte-<br />
macrophage derivation, can express the form <strong>of</strong> NOS which gives very high levels <strong>of</strong> NO (see<br />
Chapter 3: inducible NOS) within the central nervous system. <strong>The</strong>se cells are implicated in<br />
the pathogenesis <strong>of</strong> neurodegenerative disease with excess generation <strong>of</strong> NO and high<br />
glutamate levels acting via receptors shown to mediate cell death in focal ischemia, Krabbe's<br />
disease, Huntington's disease and Alzheimer's disease (Choi 1988; Meldrum and Garthwaite<br />
1990; Snyder L993; Vodovotz, Lucia et al. 1996; Dawson and Dawson 1998; Akiyama,<br />
Barger et al. 2000). For example, NOS inhibitors blocked ischemic damage following middle<br />
64
cerebral artery ligation in several animal models (Dawson, Dawson et al. 1992; Yun, Dawson<br />
et al. 1997) and the NOS knock out mice had smaller infarcts after cerebral ischaemia<br />
(Iadecola 1997). <strong>The</strong> neurons which actually have high levels <strong>of</strong> both the NOS and the<br />
NADPH enzymes appear resistant to a variety <strong>of</strong> toxic insults including Huntington's disease,<br />
Alzheimer's disease and vascular stroke (Ferrante, Kowall et al. 1985; Koh, peters et al.<br />
1986). <strong>The</strong>se neurons are also rich in manganese super oxide dismutase, and if this<br />
contributes to the neuroprotection, it may be similar to the reason for the variation <strong>of</strong> the half<br />
life initially given in the cardiovascular experiments reported above -<br />
i.e. that it depends on<br />
the extent <strong>of</strong> super oxide anion availability. Excess NO levels have also been implicated in<br />
demyelinating conditions such as multiple sclerosis (Parkinson, Mitrovic et al. t997) and<br />
periventricular leukomalacia (Koprowski , Zheng et al. 1993). <strong>The</strong>re is evidence showing that<br />
the production <strong>of</strong> NO is significantly raised within the multiple sclerosis lesions, but also in<br />
the cerebral spinal fluid, blood, and urine <strong>of</strong> these patients (Lin, Lin et al. 1993). In addition,<br />
the lack <strong>of</strong> ability to generate NO is important, such as seen in patients with Duchenne<br />
Muscular Dystrophy in whom skeletal muscle lack expression <strong>of</strong> the neuronal NOS<br />
(Brenman, Chao et al. 1995).<br />
As mentioned, NO is also found in peripheral nerves where it contributes to sensory<br />
transmission and is a transmitter and/or modulator in non-adrenergic non-cholinergic (NANC)<br />
neryes, although there is considerable variation depending on both experimental conditions<br />
and the different species in which the experiments are carried out. <strong>The</strong> NANC nerves cause<br />
vasodilatation and relaxation <strong>of</strong> animal and human tracheal muscle via NO release, and this is<br />
not inhibited by NOS inhibitors (Stuart-Smith, Bynoe et al. 1994: Baba, yoshida et al. 199g;<br />
Sipahi, Ercan et al. 1998). In human airway specimens, the NANC NO mediator relaxation is<br />
more important in the distal airways (Belvisi, Stretton et al. 1992: Ellis and Undem lgg2).<br />
This response is reduced in recipient transplanted human bronchial tissue and in specimens<br />
from patients with CF (Stretton, Mak et al. 1990).<br />
In the gastrointestinal tract, NO seems to mediate relaxation including dilatation <strong>of</strong> the<br />
stomach, the sigmoid colon and the internal anal sphincter in humans. Selectively blocking<br />
the NANC mediated relaxation <strong>of</strong> the gastrointestinal tract in mice resulted in marked<br />
stomach enlargement and inner circular gut muscle layer hypertrophy, thought to be<br />
compensatory for the inability <strong>of</strong> the pyloric sphincter to relax (Huang, Dawson et al. 1993;<br />
Bredt 1999). Histochemical studies <strong>of</strong> biopsy specimens from infants with hypertrophic<br />
pyloric stenosis and biopsy studies from adults with cardiac aplasia seem to confirm this with<br />
reduced NOS enzyme demonstrated (Vanderwinden, Mailleux et al. 1992). Also the gene<br />
65
locus for the enzyme is a susceptibility locus for infantile pyloric stenosis (Chung, Curtis et<br />
al. 1996).<br />
NO also has many roles in the reproductive system (Rosselli, Keller et al. 1998), an example<br />
being a reduction <strong>of</strong> NO being associated with pre-eclampsia and premature delivery. NO<br />
functions as the NANC transmitter which leads to relaxation <strong>of</strong> the corpus cavernosum and<br />
thus the development <strong>of</strong> penile erections with its effect able to be blocked in animal studies<br />
(Burnett, Lowenstein et al. 1992). A local reduction in NO is associated with decreased<br />
bladder capacity and hyperactivity (Burnett, Calvin et al. 1997) with knock-out mice<br />
displaying hypertrophied urinary bladders and loss <strong>of</strong> neurally mediated relaxation <strong>of</strong> urethral<br />
and bladder muscle, which provides a model for voiding disorders in humans. Studies in<br />
animals show that there is increased expression <strong>of</strong> NOS during pregnancy with increased<br />
urinary excretion <strong>of</strong> nitrite and nitrate (Conrad, J<strong>of</strong>fe et al. 1993; Conrad, Vill et al. 1993).<br />
<strong>The</strong> vasodilatation and decrease in blood pressure during pregnancy is likely to be also due to<br />
increased NO secretion with uterine NO synthesis preventing myometrial contraction.<br />
2.3.4 (iii) Host defence<br />
That NO was used in host defence was discovered in one <strong>of</strong> the original areas <strong>of</strong> research that<br />
delineated the importance <strong>of</strong> NO. <strong>The</strong> use <strong>of</strong> NO may have originated in evolution as a first<br />
line defence against invading micro-organisms (Hibbs 1991; Bogdan 2001). Resistance to<br />
tumour cells and cancer development was shown to be enhanced in a non-specific way by<br />
bacterial products (Nathan t992), Activated macrophages were demonstrated to synthesise<br />
nitrite and nitrate, with generation dependant on L-arginine and inhibited by analogues <strong>of</strong> L-<br />
arginine (Hibbs, Vavrin et al. 1987). <strong>The</strong> generation <strong>of</strong> NO is now a known feature <strong>of</strong> many<br />
immune cells (dendritic cells, NK cells, mast cells, monocytes, macrophages, microglial cells,<br />
Kupffer cells, eosinophils and neutrophils) as well as other cells involved in the immune<br />
reaction (such as endothelial cells, epithelial cells, vascular smooth muscle cells, fibroblasts,<br />
keratinocytes, chondrocytes, hepatocytes, mesangial cells and Schwann cells), (Bogdan<br />
2001). <strong>The</strong> NO produced in these cells comes from the inducible form <strong>of</strong> NOS having the<br />
ability to generate much larger amounts <strong>of</strong> NO than the other enzyme types; the local<br />
concentration <strong>of</strong> NO synthesis is an important determinant <strong>of</strong> cytotoxicity. NO is a ubiquitous<br />
pathogen-killing agent and it is truly a generalist with a non specific response to infection. It<br />
can be toxic to many kinds <strong>of</strong> pathogen including viruses, bacteria and parasites (both<br />
intracellular and extracellular) as well as some metazoan parasites (James 1995; Bogdan<br />
2O0L; Colasanti, Gradoni et al. 2002). NO mediated killing <strong>of</strong> bacteria is thought to have been<br />
66
developed as a two step process. Firstly, interferon garnma (IFy) and TNFa activate<br />
macrophages and promote NOS synthesis <strong>of</strong> NO. <strong>The</strong> definitive second step is the respiratory<br />
burst during phagocytosis. It could be an adaptive host defence mechanism in humans. For<br />
example, in African children a mutation in the promoter gene seems to be associated with<br />
increased NO production which appears to provide significant protection against malaria<br />
(Hobbs, Udhayakumar et al. 2002).<br />
NO also regulates lymphocye function and may have a role in inhibiting subsets <strong>of</strong> T helper<br />
cells. However most <strong>of</strong> the normal host cells are susceptible to necrosis or apoptosis from the<br />
inhibition <strong>of</strong> mitochondrial enzymes and DNA damage caused by this molecule, particularly<br />
if the cells producing NO are over stimulated. NO is not restricted to a single defined receptor<br />
but can act widely. It is present in both acute and chronic inflammation. In these areas NO is<br />
likely to have a number <strong>of</strong> roles ranging from enhanced vasodilatation, the formation <strong>of</strong> the<br />
oedema, modulation <strong>of</strong> sensory nerve endings and increased leukocyte activity that possibly<br />
induces higher NO levels to be produced as a reaction to organisms and therefore causes<br />
increased tissue cytotoxicity. No in respiratory inflammation will be elaborated on in<br />
Chapters 3,4 and 5.<br />
<strong>The</strong>re are many other examples. Increased NO production was noted in inflammatory disease<br />
(Boughton-Smith, Evans et al. 1993; Tran, Visser et al. 1993; Alican and Kubes 1996;<br />
Lrvine, Pettei et al. 1998), particularly in ulcerative colitis (Lundberg, Hellstrom et al. tgg4)<br />
though less convincingly for Crohns disease (Rachmilewitz, Stamler et al. 1995). This has<br />
been confirmed in animal models with Macaque monkeys (Ribbon s, Zhang et al. 1995),<br />
guinea pigs (Miller, Thompson et al. 1995) and rats (Kankuri, Vaali et al. 2001). Increased<br />
urinary nitrite is higher at times <strong>of</strong> disease exacerbation in humans (Goggins, Shah et al.<br />
2001). Inhibitors have been shown to ameliorate induced chronic ileitis in animal models.<br />
<strong>The</strong>re are also increased nitrite concentrations in plasma and synovial fluid in patients with<br />
rheumatoid arthritis, osteoarthritis, (Stefanovic-Racic, Stadler et al. 1993; Amin, Di Cesare et<br />
al' 1995; St Clair, Wilkinson et al. 1996: Stichtenoth and Frolich l99g), type I diabetes<br />
@izirik, Flodstrom et al. 1996), giant cell arteritis, systemic lupus erythematosis (SLE),<br />
spondolarthopathy and Sjogrens syndrome (Belmont, Irvartovsky et al. 1997; Strand 1997:<br />
Strand, kone et al. 1998). In SLE, for example, serum nitrite levels correlated with level <strong>of</strong><br />
antibodies to double stranded deoxyribonucleic acid @NA) and to symptom scores although<br />
the highest correlation was between serum nitrite and renal disease. Biopsies in non-lesional<br />
skin showed there were increases NOS expression during periods <strong>of</strong> active SLE (Belmont,<br />
Levartovsky et al. 1997).In animal models <strong>of</strong> arthritis, a competitive NOS inhibitor blocked<br />
67
NO synthesis and in so doing blocked paw swelling and histopathological changes in the joint<br />
(Stefanovic-Racic, Meyers et al. 1994).<br />
Sepsis is a special picture. Sepsis begins with the exposure to an infectious agent which<br />
induces a cascade <strong>of</strong> events that initially tries to compensate for the problem with tachycardia,<br />
peripheral vasoconstriction, fever (usually) and increased polymorphonuclear cells. This is<br />
followed by progressive vasodilatation with a high cardiac output and decreased vascular<br />
resistance resulting in hypovolaemic shock and decreased venous return to the heart. This can<br />
progress to a state which is resistant to vasopressor agents and is <strong>of</strong>ten combined with a pro-<br />
coagulant state. Increased levels <strong>of</strong> serum nitrates were found to be correlated with the degree<br />
<strong>of</strong> systemic vasodilatation (Ochoa, Udekwu et al. 1991; Yoshizumi, Perrella et al. 1993). In<br />
animal studies, NOS inhibitors were used to treat induced sepsis but this was far from<br />
universally successful. <strong>The</strong> studies did not show improved haemodynamic parameters such as<br />
mean arterial pressure, but there was a further derangement <strong>of</strong> local blood flow @ooke,<br />
Meyer et al. 1995) resulting in worsened renal (Schwartz, Mendonca et al. 1997) and liver<br />
impairment (Gundersen, Corso et al. 1997) and worsened inflammation generally (Aaron,<br />
Valenza et al. 1998). <strong>The</strong> timing and dosage were found to be critical, as their use<br />
preventatively or early in shock resulted in worse outcomes (Cohen, Huberfeld et al. 1996;<br />
Strand, Leone et al. 1998). In one study <strong>of</strong> 12 patients with severe sepsis and hypotension,<br />
low doses <strong>of</strong> L-NMMA (NOS inhibitor) did result in an increase in pulmonary vascular<br />
resistance but also led to a decrease in cardiac output causing concern that this would result in<br />
poorer tissue perfusion @etros, Lamb et al. 1994). In a further pilot study involving 32<br />
patients with septic shock, the infusion <strong>of</strong> L-NMMA also resulted in an increase in vascular<br />
tone and a decrease in cardiac index within the first hour <strong>of</strong> therapy. <strong>The</strong> infusion continued<br />
for up to eight hours and mean arterial pressure was sustained with a 6O-8OVo reduction <strong>of</strong><br />
noradrenaline use (Grover, Zaccardelli et al. 1999). However a much larger multi-centre trial<br />
was then undertaken enrolling 797 patients with septic shock allocated to receive the NOS<br />
inhibitor or placebo for up to 7 days or 14 days in addition to standard therapy. It was<br />
terminated early because <strong>of</strong> a trend to higher mortality in the treated group by day 28 from<br />
multiple organ failure (Serrano, Casas et al.2004).<br />
2.4<br />
Chapter summary<br />
<strong>The</strong> idea for doing research into NO in the lung in human adult subjects; in healthy subjects<br />
and in those with respiratory disease, in particular asthma, came at a time when the areas <strong>of</strong><br />
environmental pollution and mediator research were rapidly developing. <strong>The</strong> first area<br />
68
included the expanding knowledge and measurement <strong>of</strong> airway pollution and the connection<br />
between higher or longer duration <strong>of</strong> daily exposure and increased cardiovascular and<br />
respiratory mortality and morbidity. Through the 1990s increasing effort was made to clarify<br />
the effects <strong>of</strong> the different pollutant components. Particulate matter, especially respirable<br />
particles at less than l0 microns in diameter, and ozone have been consistently associated<br />
with excess disease and mortality. However the nitrogen oxides have also had associations<br />
with increased respiratory disease (bronchitis, COPD, pneumonia and asthma) and for more<br />
episodes <strong>of</strong> wheeze with increased symptoms and lower lung function in adults and children<br />
with asthma.<br />
<strong>The</strong> second area led from the startling discovery <strong>of</strong> the gas NO as a ubiquitous, biological<br />
messenger acting as a physiological mediator, a neurotransmitter and a mechanism <strong>of</strong> host<br />
defence. Since this was realised, research into NO in every biological system flourished.<br />
However there were a number <strong>of</strong> hurdles to overcome in measuring this molecule. Measuring<br />
NO was going to be difficult given its gaseous, short lived and highly reactive nature, and<br />
there were also the difficulties <strong>of</strong> measuring levels in exhaled air from human subjects.<br />
In 1992 NO was named Science Magazine's "Molecule <strong>of</strong> the Year". In 1998 Robert F.<br />
Furchgott, Louis J. Ignano and Ferid Murad were winners <strong>of</strong> the 1998 Nobel prize in<br />
Medicine for their research in identifying firstly EDRF, and then renaming and identifying<br />
NO as the mediator in vascular epithelium.<br />
<strong>The</strong> following chapter will discuss the synthesis and control <strong>of</strong> NO production in detail, the<br />
interactions <strong>of</strong> NO with other chemical and biological compounds, and the synthesis and<br />
control <strong>of</strong> the nitric oxide synthase enzymes that produce it. This knowledge is preparatory to<br />
discussing how to measure No concentrations in the respiratory system.<br />
69
3.1<br />
Chapter 3: <strong>The</strong> synthesis, reactivity and control <strong>of</strong> nitric oxide<br />
Introduction<br />
<strong>The</strong> previous chapter detailed the two areas where nitrogen compounds were being<br />
increasingly recognised for their importance; air pollution and biological systems. In air<br />
pollution the nitrogen oxides were associated with excess mortality, in particular respiratory<br />
and cardiovascular morbidity; and in the biological systems, NO was found to be a<br />
widespread fundamental messenger. In this chapter, I will detail how NO is produced and<br />
cover its highly reactive and interactive properties. I will discuss the nitric oxide synthase<br />
(NOS) enzymes and how they are controlled in the physiological roles and the host defence<br />
roles <strong>of</strong> NO. <strong>The</strong> properties <strong>of</strong> NO needed to be understood when preparing to design<br />
experiments to measure it in vivo.<br />
3.2 Properties <strong>of</strong> nitric oxide<br />
Figure 3.1: <strong>The</strong> molecular structure <strong>of</strong> NO<br />
ao OO<br />
oN=O..<br />
NO is a clear, colourless gas. It is a free radical with an unpaired electron (. N=O, abbreviated<br />
NO, see Figure 3.1) and therefore it is highly reactive with a half life <strong>of</strong> seconds and readily<br />
combines with other free radicals (Beckman and Crow 1993). However it does not self react,<br />
possibly because its bond length is intermediate between double and triple bond lengths<br />
(Braker and Mossman 1975). Its small size and the fact that it is a relatively non-polar<br />
molecule means that it moves readily through hydrophobic lipid membranes (Shaw and<br />
Vosper 1977 Malinski, Taha et al. 1993; Liu, Miller et al. 1998). Its reactivity means that it<br />
binds quickly with transition metals such as iron, copper, cobalt, or manganese that are central<br />
ions to many cytochromes and oxidases, and in the case <strong>of</strong> iron, haemoglobulin. NO can be an<br />
oxidant or a reducing agent depending on the 'redox' environment. NO is soluble in water up<br />
to 2 millimoles per litre at 200C in one atmosphere, but has a high partition coefficient so it<br />
usually exists as a gas. NO reacts rapidly with oxygen in air producing nitrogen dioxide (NOz)<br />
(Vallance and Collier 1994). NO is very soluble in lipid and water and is therefore fully<br />
diffusible in the environment <strong>of</strong> the cell (Archer 1993; Henry, Lepoivre et al. 1993).<br />
70
Table 3.1: Properties <strong>of</strong> nitric oxide<br />
Molecular weight 30.006 (1 mol= 0.030006 kg)<br />
N...O bond distance 1.1508 angstroms<br />
Partition coefficient approximately 20<br />
Saturated NO solution approximately 3mM<br />
Oxidation products Nitrite, nitrate<br />
Dissociation t lze Hb-NO approximately 3 hours<br />
Absolute density 101.325 kPa at 25oC<br />
Solubility in H2O at OoC<br />
Solubility in HzO at 2OoC<br />
Solubility in H2O at 6OoC<br />
7.34 ml/100m1<br />
4.6 m/100m1<br />
2.37 m/100m1<br />
This table is modified from Archer S. Measurement <strong>of</strong> nitric oxide in biological models FASEB Journal.<br />
1 993; 7(2): 349-60 (Archer 1 99s).<br />
NO is generated by the stereospecific enzyme NOS which acts on L-arginine cleaving the<br />
terminal guanidino nitrogen to produce NO and L-citrulline. This is accomplished via five<br />
separate steps and requires 3 co-factors [tetrahydrobiopterin (BH4) and flavoproteins; flavin<br />
adenosine dinucleotide (FAD), flavin mononucleotide (FMN)], and 2 co-substrates [oxygen<br />
and nicotinamide adenosine dinucleotide phosphate (NADPH)I in the presence <strong>of</strong> calmodulin<br />
and calcium (Kwon, Nathan et al. 1990; Stuehr, Kwon et al. 1991; Barnes and Belvisi 1993:<br />
Knowles and Moncada 1994). L-arginine is a semi-essential non-aromatic amino acid and is<br />
present in nuts (especially brazils and almonds), shellfish, and meat (bacon, beef and game)<br />
(Vallance and Collier 1994). <strong>The</strong> necessary elements are demonstrated in figure 3.2.<br />
Figure 3.2: <strong>The</strong> reaction to generate nitric oxide<br />
t{Tglnlna-...+<br />
l+ttrullino<br />
<strong>The</strong> formation <strong>of</strong> No by No synthase involves the conversion <strong>of</strong> L-arginine to L-citrulline with several<br />
co-facto.rs including the flavones and the tetrahydrobiopterin. <strong>The</strong>re are a number <strong>of</strong> known<br />
competitive<br />
-nitrite,<br />
inhibitors for the NO synthase enzym-es. NCj itselt can oxidiseO to nitrate or<br />
perioxyitrite.<br />
7l
BHa = tetrahyrobiopterin, FAD = flavin adenosine dinucleotideA FMN = flavin mononucleotide, H2O =<br />
water, L-NAME= = No-nitroarginine methyl ester, L-NMMA = N"-mono-methyl-L-arginine, L-NOARG =<br />
No-nitroarginine NADPH = nicotinamide adenosine nucleotide phosphate, NO = nitric oxide, NO2- =<br />
nitrite, NOi = nitrate NO synthase = nitric oxide synthase, Oe = oXYgell, ONOO'= peroxynitrate,<br />
Taken from Barnes PJ, Belvisi MG. Nitric oxide and lung disease. Thorax 1993; 48: 1034-1043<br />
(Barnes and Belvisi 1993).<br />
Table 3.2: Reactions with nitric oxide<br />
Once NO is formed, there are a number <strong>of</strong> possible pathways <strong>of</strong> reactions(Nathan 1992; Stamler,<br />
Jaraki et al. 1992; Stamler, Simon et al. 1992; Henry, Lepoivre et al. 1993; Anggard 1994; Gaston,<br />
Drazen et al. 1994; Vallance and Collier 1994; Eiserich, Patel et al. 1998):<br />
1. Redox reactions in the presence <strong>of</strong> Oz:<br />
(a) To create reactive nitrogen species<br />
(b) To create superoxides:<br />
+ nitrate (NOz-)<br />
2. (a) Reactions with transition metals: cobalt (Co)<br />
--+ nitrate (NOg-)<br />
+ nitrous acid (HNO2)<br />
+ nitrogen dioxide (NO2)<br />
+ perioxynitrite ONOO-<br />
+ p€roxlnitrous acid (ONOOH)<br />
(both strong oxidising agents)<br />
copper (Cu)<br />
iron (Fe)<br />
manganese (Mn)<br />
zinc (Zn)<br />
(b) Reactions with haem and nonhaem metalloproteins:<br />
ascorbate oxidase<br />
catalase<br />
ceruloplasmin<br />
cytochrome c<br />
haemoglobin<br />
lipo-oxgenase<br />
myoglobin<br />
succinate dehydrogenase<br />
tyronase<br />
3. (a) Interactions with nitrogen thiols and amines:<br />
-+ nitrosothiols<br />
+ nitrosamines<br />
--+ sulphur thiols<br />
(b) lnteractions with sulphur:<br />
4. Reactions with amino acid<br />
5. Reactions with liPids<br />
'<strong>The</strong>se compounds are more <strong>of</strong>ten designated as Nw than NG and the former is used in the text but the latter was<br />
used in this diagram.<br />
72
3.3 Reactions <strong>of</strong> nitric oxide<br />
<strong>The</strong> reaction <strong>of</strong> NO at any given time is dependent on a number <strong>of</strong> factors; the rate <strong>of</strong> reaction<br />
and therefore the concentrations <strong>of</strong> the species with which NO reacts the fastest, the<br />
concentration <strong>of</strong> NO, the reduction-oxidation (redox) environment in which it is found and<br />
therefore the presence and concentration <strong>of</strong> Oz, the availability <strong>of</strong> other interacting<br />
compounds and the pH (see Table 3.2). As well as local reactions, it is now believed that the<br />
interaction between NO and thiols and amines may be a way <strong>of</strong> stabilising NO in a bioactive<br />
form, potentially facilitating NO transport in tissue (Gaston, Drazen et al. 1994). I will review<br />
the different pathways briefly below.<br />
3.3.1 Reactive nitrogen species and superoxide reactions<br />
High levels <strong>of</strong> NO exposure can cause significant damage. It can damage all classes <strong>of</strong><br />
macromolecules including DNA. It can destroy mitochondrial enzymes, prevent DNA<br />
synthesis, and inhibit protein synthesis (Hibbs, Taintor et al. 1988; Curran, Ferrari et al. l99l;<br />
Kwon, Stuehr et al. 1991; Stadler, Billiar et al. 1991; Lancaster 1992; Irpoivre, Flaman et al.<br />
r9e2).<br />
However it has become increasingly obvious that reactions with NO in the presence <strong>of</strong> Oz<br />
forming NO derived reactive nitrogen species are equally important in mediating toxic injury.<br />
<strong>The</strong>se compounds include nitrite (Noz-), nitrate (No:-), No2, nitrous acid (HNoz) or<br />
dinitrogen trioxide (NzOa). It also interacts with Oz to produce superoxides such as<br />
perioxynitrite (ONOO-) and peroxynitrous acid (ONOOH), both strong oxidising agents.<br />
<strong>The</strong>se can continue to react with all classes <strong>of</strong> biomolecules including lipids, DNA, thiols,<br />
amino acids and metals leading to the two mechanisms <strong>of</strong> damage which are oxidation and<br />
nitration @iserich, Patel et al. 1998).<br />
NOz is produced by the reaction <strong>of</strong> NO with Oz and oxidation <strong>of</strong> NOz to NOr. 6,<br />
myeloperoxidase (Ignarro, Fukuto et al. 1993). This reaction will therefore occur in areas <strong>of</strong><br />
high macrophage numbers and high 02 concentration and therefore is commonly seen in the<br />
Iung. <strong>The</strong> exposure <strong>of</strong> human plasma to NOz causes rapid loss <strong>of</strong> ascorbate, uric acid, c-<br />
tocopherol, bilirubin, and protein thiols as well as increasing lipid peroxidation (Halliwell, Hu<br />
et al. 1992). Exposure <strong>of</strong> lung lining to NOz leads to loss <strong>of</strong> ascorbic acid, uric acid. and<br />
glutathione (Postlethwait, Langford et al. 1995: Kelly and Terley lggT). Under acidic<br />
conditions NO2 becomes protonated to form HNO2. At least two biological compartments<br />
experience pH low enough to allow HNO2 formation in vivo; the stomach with a pH 2.5-4.5<br />
73
(Knowles, McWeeny et al. 1974) and within neutrophil phagocytes with a pH 3.0-6.5 (Cech<br />
and Lehrer 1984). This makes the production <strong>of</strong> this acid possible in high inflammatory<br />
conditions and after dietary intake <strong>of</strong> certain nutriments such as smoked and cured foods.<br />
High levels <strong>of</strong> both NO and NOz during inflammation in the presence <strong>of</strong> the inducible<br />
nitrogen synthase enzyme (see below) forms dinitrogen trioxde (NzOs). Both NOz- and NOt-<br />
are moderately stable and can be released on tissue contact (Moro, Darley-Usmar et al. t994;<br />
White, Moellering et al. 1997). NO can undergo electron oxidation and reduction reactions<br />
generating nitrosonium cation (NO+) and nitroxyl anion (NO-) and these can participate in<br />
numerous other redox reactions. NO can also regulate apoptotic signals through formation <strong>of</strong><br />
reactive NO species (Tamir, Irwis et al. 1993; Kim, Talanian et al. 1997; Nakano, Terato et<br />
aI.2003).<br />
Perioxynitrite anion (ONOO) and peroxynitrous acid (ONOOII) are oxidants which can also<br />
react with numerous targets. <strong>The</strong> formation requires NO and Oz- anion produced by many cell<br />
types - neutrophils, macrophages, smooth muscle cells endothelial cells and fibroblasts<br />
(Ischiropoulos, Zhu et al. 1992; Kooy and Royall 1994; Boota, Zar et al. 1996; Thom, Xu et<br />
al. 1997). <strong>The</strong>y interact with glucose, fructose, and mannitol (White, Moellering et al. 1997;<br />
Skinner, White et al. 1998), and DNA bases. <strong>The</strong>y hydroxylate aromatic amino acids (such as<br />
tyrosine, tryptophan, phenylalanine), and oxidise thiols and lipids @eckman, Ischiropoulos et<br />
al. t992; Alvarez, Rubbo et al. 1996; Beckman 1996). Perioxynitrite can directly inhibit<br />
oxidation reactions by chain terminating lipid peroxyl and alkoxyl radicals or through<br />
regulation <strong>of</strong> cell signalling pathways that lead to induction <strong>of</strong> antioxidant enzymes (@ubbo,<br />
Radi et al. L994; ODonnell, Chumley et al. 1997; Moellering, McAndrew et al. 1998). It can<br />
nitrate both free and protein bound tyrosine residues to give a 3-nitrotyrosine compound<br />
found in high levels in many inflammatory conditions (see below) (van der Vliet, O'Neill et<br />
al. 1994; Alvarez, Rubbo et al. 1996). Addition <strong>of</strong> ONOO to red blood cells results in<br />
methaemoglobin formation. ONOO also reacts with iron-sulphur enzymes and can lead to<br />
inactivation (Castro, Rodriguez et al. 1994; Bouton, Hirling et al. 1997).<br />
While responsible for many toxic reactions - it is possible that this also provides a mechanism<br />
for removal as the ONOO compound isomerises into NOs- and NOz- at neutral pH (Irwis,<br />
Tamir et al. 1995; Munzel, Sayegh et al. 1995; Pfeiffer, Gorren et al. 1997). It is also possible<br />
that the ONOO- reaction with COz which then degrades which may also be a protective<br />
mechanism (Lymar and Hurst 1996; Uppu and Pryor 1996).<br />
74
3.3.2 Reactions with transition metals and metalloproteins<br />
<strong>The</strong> biological chemistry <strong>of</strong> NO is strongly mediated through reaction with transition metals.<br />
<strong>The</strong> reaction is with catalytic metal centres such as iron (Fe) in both haem and nonhaem<br />
proteins (Tsai 1994). <strong>The</strong> high affinity <strong>of</strong> NO for the ferrous iron (Fe2+ in the reduced state) in<br />
haemoglobin leads to a key interaction (Sharma, Traylor et al. 1987; Eich, Li et al. 1996). In<br />
vivo, this reaction <strong>of</strong> NO and oxyhaemoglobin to form methaemoglobin and nitrate (NOl)<br />
represents the major pathway for scavenging the endogenous NO production and is a<br />
significant route <strong>of</strong> NO removal (Lancaster, Langrehr et al. lgg2). While this reaction is<br />
rapid, in fact it would be too rapid to allow NO to interact with the subendothelial layer in the<br />
vascular compartment (Lancaster, Langrehr et al. 1992). However, it is now known that when<br />
the haemoglobin is in erythrocytes, the reaction with NO is limited by diffusion into the cell<br />
and the half life is increased (Liu, Miller et al. 1998). Studies have shown that 70Vo <strong>of</strong>the NO<br />
in humans is recovered as NO3- excreted through the urine (Westfelt, Benthin et al. 1995).<br />
Oxyhaemoglobin can also directly oxidase NOz and NO3 forming methaemoglobin.<br />
NO reaction with other Fe containing proteins can lead to either activation or inactivation<br />
depending on the protein. For example, the reaction with soluble guanylyl cyclase (sGC) leads<br />
to a conformational change which results in up to a 300 times increase in activation <strong>of</strong> the<br />
enzyme. Activation <strong>of</strong> sCG results in the formation <strong>of</strong> cyclic quanosine monophosphate<br />
(cGMP) from guanosine 5-triphosphate (GTp) (Ignarro, Degnan et al. l9g2) and accounts for<br />
major signal transduction activity <strong>of</strong> NO. It is likely to be this mechanism that is the primary<br />
one mediating vessel relaxation and the inhibition <strong>of</strong> platelet aggregation (Moncada and<br />
Higgs l99l),In contrast, formation <strong>of</strong> a nitrosyl complex with cytochrome c oxidase leads to<br />
reversible inhibition which is competitive with 02 binding @iserich, Patel et al. 1998).<br />
NO also interacts with other metals such as zinc and copper. Formation <strong>of</strong> nitrosyl<br />
compounds with co-ordinating cysteine residues results in release <strong>of</strong> zinc (Kroncke, Fehsel et<br />
al. 1994). Formation <strong>of</strong> nitrosyl compounds with copper also occurs, and is particularly<br />
important in copper containing enzymes such as the cytochrome c oxidase (Radi 1996). It is<br />
through this type <strong>of</strong> interaction with metal ion containing proteins (metalloproteins) that NO<br />
effects the enzymes <strong>of</strong> mitochondrial respiration and this is also a mechanism <strong>of</strong> the<br />
macrophage mediated defence and involves, for example, cytochrome c oxidase and catalases<br />
(Cleeter, Cooper et al. 1994; Torres, Darley-Usmar et al. 1995). Part <strong>of</strong> this is an NO and Oz<br />
competition at the cytochrome c oxidase binding site for 02 and therefore NO inhibition is<br />
75
more potent at low oxygen tensions (Brown and Cooper 1994). Again the most dramatic<br />
effects will occur in high levels <strong>of</strong> NO seen in inflammation.<br />
<strong>The</strong>re are also regulatory proteins in which the interaction <strong>of</strong> NO can modulate translation <strong>of</strong><br />
mRNA sequences which contain Fe responsive elements such as ferritin and transferrin<br />
receptors @antopoulos, Weiss et al. 1996). In this way NO can also interact to regulate Fe<br />
levels in cells.<br />
3.3.3 Nitrogen thiols and amines<br />
S-nitrosothiols (S-nitrosoglutathione, S-nitrosoalbumin, S-nitrosohaemoglobin as examples)<br />
are stable molecules that can potentially transfer NO+ to other thiols or transport and release<br />
NO in distant areas and in other tissues (Stamler, Simon et al. 1992; Jia, Bonaventura et al.<br />
1996; Funai, Davidson et al. 1997). This provides another mechanism by which NO regulates<br />
protein function. It has long been demonstrated that nitrosothiol compounds are generated<br />
during the curing process <strong>of</strong> meat with the interaction <strong>of</strong> nitrite @mi-Miwa, Okitani et al.<br />
1976). Exactly how the NO is released from these compounds and when is unclear; however<br />
these compounds have been suggested to account for some <strong>of</strong> the NO biological actions<br />
(Stamler 1994). For example, S-nitrosation has been shown to confer vasodilatory properties<br />
on other specific proteins. On the tissue type plasminogen activator enzyme, which dissolves<br />
fibrin in blood clots, s-nitrosation confers vasodilatory and anti-platelet functions (Stamler,<br />
Simon et al. 1992). <strong>The</strong> effect <strong>of</strong> shear stress to increase NO may also operate via s-<br />
nitrosation <strong>of</strong> plasma proteins. <strong>The</strong> vasodilatation effects <strong>of</strong> organic nitrates used in treatment<br />
such as nitroglycerin may also occur through the intermediate formation <strong>of</strong> these compounds<br />
(Fung, Chong et al. 1988). <strong>The</strong>y are therefore potential therapeutic agents for pharmacological<br />
NO delivery @utler, Flitney et al. 1995; Stamler 1995; Upchurch, Welch et al. 1996). S-<br />
nitrosothiols have been detected in the nervous system where they may function as<br />
neurotransmitters and use in animal models mimic NANC actions @arbier and Irfebvre<br />
1994; Liu, Gillespie et al. 1994; Slivka, Chuttani et al. 1994). Several reports have identified<br />
proteins whose activity changes upon S-nitrosation in vitro (Stamler 1994; Stamler, Toone et<br />
al.1997).<br />
<strong>The</strong>se compounds can also allow NO to inhibit cytokine dependent signalling in vascular<br />
endothelial and smooth muscle cells. Both s-nitrosoglutathione and sodium nitroprusside<br />
prevent activation <strong>of</strong> the transcription factor NF-KB and expression <strong>of</strong> vascular cell adhesion<br />
molecule-l. Nitrosothiols can also regulate calcium transits and again can alter signal<br />
76
transduction pathways in this manner by altering ion homeostasis (Stoyanovsky, Murphy et<br />
al.1997; Xu, Eu et al. 1998).<br />
3.3.4 Reaction with amino acids<br />
<strong>The</strong> importance <strong>of</strong> the interaction <strong>of</strong> NO species with amino acids is exemplified by the<br />
reaction with tyrosine to form 3-nitrotyrosine. This reaction can severely compromise<br />
function <strong>of</strong> proteins with tyrosine residues in the active site, disrupting structure and activity<br />
@iserich, Patel et al. 1998). This reaction was first described in 1990 (Ohshima, Friesen et al.<br />
1990) and the level <strong>of</strong> 3-nitrotyrosine is now commonly used diagnostic marker <strong>of</strong> NO<br />
derived oxidants in both human disease states and animal models (Schmidt, H<strong>of</strong>mann et al.<br />
1996). An "overwhelming body <strong>of</strong> evidence has amassed in the last several years revealing<br />
that NO2Tyr (3-nitrotyrosine) is a reliable footprint <strong>of</strong> reactive nitrogen species production<br />
that spatially and temporally parallels tissue and cellular injury" @iserich, Patel et al. 1998).<br />
Although a direct relationship between 3-nitrotyrosine formation and pathological outcomes<br />
remains poorly characterised, it is dramatically elevated in diverse diseases such as<br />
atherosclerosis, sepsis, acute and chronic lung disease, neurodegenerative diseases,<br />
inflammatory bowel disease, chronic organ rejection, myocardial inflammation and tobacco<br />
smoking (Ischiropoulos, Zhu et al. 1992; Eiserich, Patel et al. 1998). <strong>The</strong> compound can be<br />
incorporated into tuberculin which disrupts the cell cytoskeleton @iserich, I{ristova et al.<br />
1998) and can attenuate adrenoreceptor agonists in vivo (Kooy and Royall 1994). <strong>The</strong><br />
formation <strong>of</strong> 3-nitrotyrosine is dependent on increased NO production and frequently<br />
associated with activated phagocytes (neutrophils, monocytes, eosinophils, macrophages). NO<br />
can also interact with amino acid radicals in proteins @iserich, Cross et al. 1996).<br />
3.3.5 Reaction with lipids.<br />
<strong>The</strong> reactive nitrogen species (NO, NO2, ONOO) also interacts with unsaturated lipids which<br />
leads to either initiation <strong>of</strong> oxidation, altering the rates and products <strong>of</strong> lipid oxidation<br />
products or inhibition (see below). For example, the unsaturated free fatty acids arachidonic<br />
acid and linoleate are substrates for the enzymatic synthesis <strong>of</strong> bioactive medicators such as<br />
prostaglandins and leukotrienes through the activities <strong>of</strong> lipo-oxygenases, cyclo-oxygenases<br />
and cyochrome Pa56. <strong>The</strong>se play a role in regulation <strong>of</strong> blood pressure, platelet aggregation,<br />
and bronchosconstriction. Free unsaturated lipids and those esterified to phospholipids are a<br />
significant component to biomembranes pulmonary surfactant and plasma lipoproteins.<br />
Uncontrolled oxidation as can be caused by NO and related species can lead to changes in<br />
integrity and fluidity <strong>of</strong> biomembranes. <strong>The</strong> interaction can also lead to the formation <strong>of</strong> other<br />
77
toxic products such as isoprostanes and reactive aldehydes @iserich, Patel et al. 1998). Lipid<br />
peroxidation is a characteristic feature <strong>of</strong> nearly all inflammatory diseases (Kuhn, Belkner et<br />
al. 1994; Quinlan, Lamb et al. 1996; Kuhn, Heydeck et al. 1997; Li, Maher et al. 1997).<br />
On the other hand, NO can also serve as a protective factor. <strong>The</strong>re are also lipid derived<br />
radicals both free and membrane bound which contribute to inflammatory conditions by<br />
inducing membrane damage again resulting in alteration <strong>of</strong> fluidity and integrity, as well as<br />
the production <strong>of</strong> eicosanoids and eicosonoid-like isomers which possess potent bioactivity.<br />
Direct reaction <strong>of</strong> NO with radical lipids results in inhibition <strong>of</strong> lipid oxidation and in this way<br />
may prevent lipid oxidation induced tissue damage (Rubbo, Radi et al. 1994; Gutierrez,<br />
Nieves et al. 1996; Wink, Cook et al. 1996; ODonnell, Chumley et al. 1997).<br />
3.3.6 Reactions with genes<br />
Transcriptional regulation <strong>of</strong> several genes by NO have been reported. Interestingly, these are<br />
in general anti-inflammatory (Naruse, Shimizu et al. 1994; Pilz, Suhasini et al. 1995);<br />
examples include regulation <strong>of</strong> adhesion molecules, the haem metabolising enzyme,<br />
haemoxygenase (Foresti, Clark et al.1997) and glutathione synthesis (Moellering, McAndrew<br />
et al. 1998).<br />
3.3.7 Making sense <strong>of</strong> the reactions<br />
In essence, the reactions that are important in biological systems depend on the amount <strong>of</strong> NO<br />
and where the NO is formed. <strong>The</strong> short half life and reactivity means NO is likely to act as a<br />
local messenger molecule, transferring messages within and between individual cells. In<br />
biological systems, when small amounts are formed as a mediator for physiological processes,<br />
the NO preferentially binds to haem, hence in the blood stream most NO is quickly bound to<br />
haemoglobin. From here it rapidly decomposes to yield predominantly nitrate (NOl-) and<br />
some nitrite (NOz') and the compounds are eliminated in the urine with a half life <strong>of</strong> five to<br />
eight hours. NO will also interact with other heme containing proteins such as enz).mes<br />
particularly sGC which results in a rapid increase in activity and production <strong>of</strong> cGMP. Excess<br />
NO is mopped up by other nitrosation reactions, for example nitrosothiols and these are now<br />
known to be active in their own right, and a way <strong>of</strong> stabilising NO in a bioactive form and<br />
potentially facilitating NO transport in tissue.<br />
When released in much larger amounts, it is likely that NO more <strong>of</strong>ten reacts with other<br />
elements such as metals, for example copper, iron and zinc proteins releasing free Cu*, Fe*<br />
and Zn** and generating Oz and highly toxic hydroxyl radicals leading to effect massive<br />
78
oxidative injury. High levels <strong>of</strong> NO disrupt DNA and cause both genotoxic and cytotoxic<br />
damage. If there are changes in the pH, in the thiol content or in the redox state, this can result<br />
in increased damaging and carcinogenic potential. Of interest is the reaction <strong>of</strong> NO with<br />
amino acids such as tyrosine, which in itself is now used as a marker in several inflammatory<br />
diseases.<br />
Finally, there is now discussion that the original EDRF concept is made up <strong>of</strong> the actions <strong>of</strong><br />
NO and the actions <strong>of</strong> some <strong>of</strong> the post NO production <strong>of</strong> other compounds, in particular the<br />
s-nitrosothiols (Myers, Minor et al. 1990; Vanin l99l; Jia, Bonaventura et al. 1996; Liu,<br />
Miller et al. 1998).<br />
3.4<br />
Nitric oxide synthase isoenzymes<br />
<strong>The</strong> nitric oxide synthases are large complex proteins that unusually contain both oxidative<br />
and reductive domains. <strong>The</strong>y have homology with cytochrome P+so which is also unique<br />
(Bredt, Hwang et al. l99l; Vallance and Moncada 1994). <strong>The</strong>y are synthases rather than<br />
synthetases as they do not use adenosine triphosphate (ATP) in their reactions (Knowles and<br />
Moncada 1994). NOS needs the substrate (L-arginine), the co-substrates (O2 and NADpI{)<br />
and the co-factors (8H4, FAD, FMN) (see Chapter 3.2 above). <strong>The</strong> reaction is a five electron<br />
mono-oxygenate oxidation pathway which involves two separate mono-oxygenation steps in<br />
sequence (see Figure 3.2, Knowles, 1994 #712). In step one N*-hydroxyarginine is an<br />
intermediate species formed (Stuehr, Kwon et al. l99l) in the first reaction by aquiring one<br />
02 molecule and one NADPH molecule (Kwon, Nathan et al. 1990) and the presence <strong>of</strong> BII+<br />
(Kwon, Nathan et al. 1989; Tayeh and Marletta 1989). <strong>The</strong> second step is the oxidation <strong>of</strong> N*-<br />
hydroxyarginine to form citrulline and *NO. Flavin coenzymes are involved in the transfer <strong>of</strong><br />
electrons to form a reduced oxygen species (Stuehr, Cho et al. 1991; Stuehr, Fasehun et al.<br />
1991). <strong>The</strong>re are consensus binding sites for FAD, FMN and NADPH located in the carboxyl<br />
terminal portion <strong>of</strong> the NOS protein, and also a consensus binding site for calmodulin. <strong>The</strong><br />
other co-factor BFIa binds to NO on a 1:1 stoichiometry basis and has a redox role in enzyme<br />
activity (Hevel and Marletta 1992; Knowles and Moncada 1994). All three isoenzymes are<br />
phosphorylated (Nathan and Xie 1994).<br />
79
Figure 3.3: <strong>The</strong> nitric oxide synthase reaction showing the generation <strong>of</strong> the constituent atoms <strong>of</strong> nitric<br />
oxide<br />
*}^rilfl*t<br />
En,<br />
o.E<br />
l(NADPtl + H'l<br />
I NADT<br />
'tT H<br />
.ArA./"WNH'<br />
tNl<br />
o<br />
<strong>The</strong> boxed 'O' and 'N' atoms show the constituent atoms <strong>of</strong> NO - see text for abbreviations. Taken<br />
from Knowles RG, Moncada S. Nitric oxide synthases in mammals. Biochemical Journal 1994; 298<br />
(2):249-255 (Knowles and Moncada 1994).<br />
<strong>The</strong>re are two forms <strong>of</strong> NOS; the constitutive form (cNOS) and the inducible form (iNOS).<br />
<strong>The</strong> constitutive form is in turn made up <strong>of</strong> two types -<br />
the endothelial NOS (eNOS) and the<br />
neuronal NOS (nNOS), although there is only one form <strong>of</strong> the inducible NOS. <strong>The</strong>y have also<br />
been classified as type I (nNOS), type tI (iNOS) and type III (eNOS) which was the order in<br />
which they were first purified with the first isolation <strong>of</strong> their DNA (Bredt, Hwang et al. 1990;<br />
Bredt and Snyder 1990; Janssens, Simouchi et al. 1992; I-ammers, Barnes et al. 1992;<br />
Lowenstein and Snyder 1992; Lyons, Orl<strong>of</strong>f et al. 1992; Xie, Cho et al. 1992; Forstermann,<br />
Closs et al. 1994). <strong>The</strong> homology between the is<strong>of</strong>orms in humans is between 5l-587o with<br />
approximately 5LVo between nNOS and iNOS, and 54Vo homology between eNOS and iNOS.<br />
(Nathan L992; Chartrain, Geller et al. 1994; Forstermann, Closs et al. 1994; Marsden, Heng et<br />
al. 1994; Nathan and Xie 1994). Across species, the amino acid sequences for each is<strong>of</strong>orm is<br />
well conserved at greater than gOVo for the constitutive forms and greater than 807o for the<br />
inducible forms (Forstermann, Closs et al. 1994). <strong>The</strong>y predominantly operate in the areas for<br />
80
which they are named; eNOS in vascular cells, nNOS in the CNS and peripheral nerves, and<br />
iNOS within the immune cells, although these divisions are not so strict as first thought and<br />
some cells therefore can produce two different types <strong>of</strong> the enzyme (Nathan 1992). <strong>The</strong><br />
enzymes are coded for on differing chromosomes; chromosome 7 for eNOS (Zl-22 kb,26<br />
exons), chromosome l2for nNOS (150kb, 29 exons) and chromosome 17 foriNOS (37kb,26<br />
exons) (Marsden, Heng et al. 1993). <strong>The</strong> characteristics <strong>of</strong> the NOS enzymes are listed in<br />
Table 3.3 (Byrnes, Bush et al. 1996).<br />
Table 3.3: <strong>The</strong> characteristics <strong>of</strong> the nitric oxide synthase isoenzymes<br />
Enzyme<br />
nNOS<br />
Constitutive/<br />
inducible<br />
constitutive<br />
Type NO<br />
oroduclion<br />
picomoles<br />
Galcium/<br />
calmodulin<br />
dependent<br />
Ghromosome<br />
location<br />
7: 26 exons<br />
span<br />
21 kb<br />
eNOS constitutive ill picomoles dependent 12i 28 exons<br />
span<br />
>100 kb<br />
iNOS inducible tl nanomoles independsnt 17: 26 exons<br />
span<br />
37 kb<br />
a CNS' central nervous system; PNS, peripheral nervous system; |NANC, inhibitory noradrenergic<br />
noncholinergic<br />
b TNF, tumor necrosis factor; lL, interleukin.<br />
This table is taken from Byrnes CA, Bush A, Shinebourne EA "Measuring expiratory nitric oxide in<br />
humans" Methods in Enzymology 1996; 26g:45g-413 (Byrnes, Bush et d. rbsoi.<br />
3.4.1 Constitutive nitric oxide wnthases<br />
Activation <strong>of</strong> the two constitutive isoenzymes (nNOS and eNOS) depends on the levels <strong>of</strong><br />
calmodulin and calcium. <strong>The</strong> NOS enzyme lies dormant until an increase in cellular calcium<br />
in the presence calmodulin occurs. A calcium concentration <strong>of</strong> 200 to 400 nanomoles allows<br />
half the maximum activity <strong>of</strong> the enzyme. This results in a sustained release <strong>of</strong> NO over<br />
several minutes, with picomole amounts <strong>of</strong> NO released, and this acts locally. <strong>The</strong> enzymes<br />
are stimulated by a number <strong>of</strong> mediators, depending on where they are situated, and these<br />
include bradykinin, acetylcholine, calcium ionophore, histamine, leukotriene, platelet<br />
activating factor (serotonin/thrombin) and exercise. In this way these enzymes participate in<br />
maintaining physiological balance within systems as discussed in the previous Chapter 2.3.4.<br />
<strong>The</strong> nNOS was the first <strong>of</strong> the isoenzymes to be purified and cloned (Bredt, Hwang et al.<br />
l99l; Schmidt, Pollock et al. 1991). <strong>The</strong>re is wide expression and high activity <strong>of</strong> nNOS<br />
isoenzyme in the brain and throughout the peripheral nervous system in the NANC nerves,<br />
81<br />
Gells" in which<br />
enzvme is found<br />
cNS: especially<br />
cerebellum<br />
PNS: gut,<br />
bladder,<br />
reproductive<br />
organs<br />
iNANC neruac<br />
endothelial cells,<br />
mast c€lls,<br />
platelets,<br />
smooth muscle<br />
cells. neutroohils<br />
macrophages,<br />
neutrophils,<br />
airway epithelial<br />
cells,<br />
fibroblasts, mast<br />
cells<br />
Enzyme.<br />
stimulationo<br />
acetylcholine,<br />
bra)kinin,<br />
Ca'* ionophore<br />
histamins,<br />
leukotrieng,<br />
PAF<br />
serotonin,<br />
thrombin,<br />
shear stress<br />
ilpoporysacnand<br />
e,<br />
y-interferon,<br />
TNFq,<br />
TNFp, tll, tL2,<br />
leptochoic acid,<br />
picolinic acid
providing NO as a neurotransmitter (Forstermann, Closs et al. 1994). However it has also<br />
been found in the spinal cord @un, Dun et al. 1992), in sympathetic ganglia and adrenal<br />
glands @un, Dun et al. 1993; Sheng, Gagne et al. 1993), in the epithelial cells <strong>of</strong> the lung,<br />
uterus and stomach (Schmidt, Gagne et al.1992), in pancreatic islet cells (Schmidt, Warner et<br />
al. 1992) and in human skeletal muscle (Forstennann, Closs et al. 1994). While in the main<br />
nNOS NO production functions mostly in neurotransmission, it is also utilised to maintain<br />
muscle tone, for example, in the gastrointestinal tract and skeletal muscle. <strong>The</strong>se enzymes are<br />
involved in homeostasis throughout the body from memory, behaviour, circadian rhythms, to<br />
the gastroenterology tract, renal function and reproductive activity. It is likely that this<br />
isoenzyme form is the largest proportion <strong>of</strong> constitutive NOS in humans (Forstermann, Closs<br />
et al. 1994; Knowles and Moncada 1994). <strong>The</strong> eNOS isoenzyme within arteries give a basal<br />
level <strong>of</strong> NO production continuously, which maintains the vascular blood flow and blood<br />
pressure. This isoenzyme is largely responsible for the NO which inhibits platelet aggregation<br />
and platelet adhesion.<br />
3.4.2 Inducible nitric oxide svnthase<br />
<strong>The</strong> main method <strong>of</strong> activation, the amount <strong>of</strong> NO produced and the regulation <strong>of</strong> this form <strong>of</strong><br />
the enzyme is very different from the constitutive forms. <strong>The</strong> iNOS is<strong>of</strong>orm is regulated at<br />
transcriptional level with activation requiring both a primary and a secondary signal before<br />
the mRNA for the enzyme is produced. <strong>The</strong> priming agent is interferon gamma (IFNI) or an<br />
interferon inducing agent like bacterial lipopolysaccharide (LPS) (Weinberg, Chapman et al.<br />
1978; Stuehr and Marletta 1985; Drapier, Wietzerbin et al. 1988; Sherman, loro et al. 1991;<br />
Munoz-Fernandez, Fernandez et al. 1992). <strong>The</strong> category <strong>of</strong> factors which can act as the<br />
secondary agent continues to be enlarged by every new study. <strong>The</strong>se include interleukin 1<br />
(n-l), IL2, TNFo, tumor necorsis factor p (TNFp), muramyl dipeptide, lipoteichoic acid,<br />
picolinic acid (metabolite <strong>of</strong> l-tryptophan), ozone, cAMP elevating agents, ultravoilet light,<br />
ozone and trauma (Ding, Nathan et al. 1988; Drapier and Hibbs 1988; Lorsbach, Murphy et<br />
al. 1993; Bastian and Hibbs 1994; Nathan and Xie 1994).In addition, a number <strong>of</strong> viral and<br />
antimicrobial products from mycobacteium tuberculosis, salmonella typhimuium and<br />
protozoan parasites have also been found to stimulate this form <strong>of</strong> the enzyme (MacMicking,<br />
North et ^1. 1997; Shoda, Kegerreis et al. 2001; Thoma-Uszynski, Stenger et al. 2001). Some<br />
food components are also capable <strong>of</strong> inducing NO and may be the mechanism <strong>of</strong> action <strong>of</strong><br />
some food carcinogens, og soybean trypsin inhibitor, beta amylase (Nathan 1997). For<br />
example, nitrites and nitrates are important antimicrobial, flavouring and colouring agents in<br />
meat and fish products (Chow and Hong 2W2).<br />
82
<strong>The</strong> induction requires gene transcription and mRNA production, and therefore occurs several<br />
hours after the exposure to the activating agents. However this also means that the production<br />
<strong>of</strong> NO can go on for days until the enzyme is broken down. Also important is that when<br />
activated, nanomole levels <strong>of</strong> NO are produced; ie 100 times that <strong>of</strong> the endothelial or<br />
neuronal isoenzyme production. NO exposure can damage all classes <strong>of</strong> macromolecules<br />
including DNA, it can destroy mitochondrial enzymes, inhibit protein synthesis, and cause<br />
apoptosis <strong>of</strong> cells (Hibbs, Taintor et al. 1988; Curran, Ferrari et al. 1991; Kwon, Stuehr et al.<br />
l99l; Stadler, Billiar et al. 1991; Lancaster, Langrehr et al. 1992: Lepoivre, Flaman et al.<br />
1992; Tamir, Irwis et al. 1993; Nakano, Terato et al. 2003). So the regulation <strong>of</strong> this enzqe<br />
is extremely important given its unique role in host defence but also ability to inflict<br />
significant host damage (Nathan lggT\.<br />
Under certain conditions such as in the absence L-arginine or if BFI+ is limited (or in the<br />
presence <strong>of</strong> adriamycin which may in part explain the toxicity <strong>of</strong> this drug), all the NOS<br />
enzymes, but more <strong>of</strong>ten seen with this inducible form <strong>of</strong> the enzyme, can reduce 02 via the<br />
flavin c<strong>of</strong>actors to O2'- (Xia, Dawson et al. 1996; Cosentino, Patton et al. 1998; Vasquez-<br />
Vivar, Kalyanaraman et al. l99S). <strong>The</strong> simultaneous generation <strong>of</strong> 02- and NO then results in<br />
producing ONOO- causing further cellular injury.<br />
3.4.3 Contol <strong>of</strong> the nitric oxide synthase isoen4tmes<br />
3.4.3 (i) <strong>The</strong> constitutive forms<br />
All <strong>of</strong> the enzymes can be regulated at pre-transcriptional, post transcriptional and post<br />
translational levels (Papapetropoulos, Rudic et al. 1999). However the main control factors<br />
appear to be different for each. <strong>The</strong> control <strong>of</strong> the constitutive enzymes is predominantly<br />
maintained by the need for the presence <strong>of</strong> substrate and c<strong>of</strong>actors, while the control <strong>of</strong> the<br />
inducible form is predominantly by the need for two signals for transcription.<br />
<strong>The</strong> constitutive enzymes require both the presence <strong>of</strong> calmodulin and a certain level <strong>of</strong><br />
calcium from 200 to 400 nmols to commence production, and if the calcium concentration<br />
falls below this level then production ceases. <strong>The</strong> substrate L-arginine is required and L-<br />
arginine exists within and outside the cell. A low substrate amount can occur as a result <strong>of</strong> the<br />
absolute availability <strong>of</strong> L-arginine, or the ability <strong>of</strong> the cell to take up L-arginine, or the ability<br />
<strong>of</strong> the cell to regenerate L-citrulline back to L-arginine to act as further substrate. Vascular<br />
cells, for example, are able to perform this regeneration with the use <strong>of</strong> an enzyme<br />
arginosuccinate synthetase. BFI+ is also required, and this is produced by another pathway.<br />
83
<strong>The</strong> enzyme <strong>of</strong> this second pathway - guanosine triphosphate cyclohydrolase I - is in turn<br />
affected by certain cytokines and inflammatory products. So while this operates as a control<br />
for the is<strong>of</strong>orms, in fact the limiting effect may be more important for the iNOS enzyme<br />
which is present in higher amounts, particularly in infection and inflammation when much<br />
greater levels <strong>of</strong> these cytokines are around and when the production <strong>of</strong> NO is higher (Stuehr<br />
1999; Werner-Felmayer, Golderer et al.2002). This will be discussed in more depth below in<br />
Section 3.4.3 (ii).<br />
<strong>The</strong> rates <strong>of</strong> formation <strong>of</strong> NO and L-citrulline are non linear for all isoenzyme forms which<br />
demonstrates there is negative feedback inhibition and this is also a mechanism to control<br />
overproduction (Rogers and Ignarro 19921, Assreuy, Cunha et al. 1993; Bastian and Hibbs<br />
r9e4).<br />
In addition, there are some differing control mechanisms operating for each <strong>of</strong> the constitutive<br />
isoenzymes. Firstly, the nNOS form can be regulated by alternative splicing to give multiple<br />
transcripts and molecular diversity. This means that the protein can be spliced in different<br />
places giving different molecular weights which possibly fulfil a differing role. <strong>The</strong>re are two<br />
major transcriptional clusters identified within the human nNOS gene, with one form lacking<br />
approximately 315 base pairs, or 105 amino acids and this smaller version is seen<br />
predominantly in the peripheral nervous system, renal tract and in the male reproductive tract<br />
(Papapetropoulos, Rudic et al. 1999).<br />
Secondly, regarding the eNOS form, it was noted that some physiological situations led to<br />
increased eNOS expression such as shear stress and exercise training (Nishida, Harrison et al.<br />
1992; Sessa, Ftitchard et al. 1994; Uematsu, Ohara et al. 1995), and situations <strong>of</strong> local<br />
hypoxia (Marsden, Schappert et al. 1992). While both iNOS and nNOS exist in soluble forms,<br />
eNOS exists in a particulate form (Nathan 1992). <strong>The</strong> importance <strong>of</strong> this is that the proper<br />
localisation <strong>of</strong> eNOS is a pre-requisite to enable it to interact with specific proteins that will<br />
allow full activity. Post translation, cNOS undergoes a process <strong>of</strong> acylation (Shaul, Smart et<br />
al. 1996) which appears necessary to anchor the enzyme to the membrane in the Golgi<br />
apparatus, and in the plasmalemmal vesicles (the caveolae). <strong>The</strong>re are some regulatory<br />
proteins that play a role in promoting this within the cells (C-protein coupled receptors,<br />
caveolin or Hsp90), that contribute to the correct localisation and that can also be individually<br />
influenced therefore contributing to more or less NO being produced. For example, Hsp90<br />
increases with increased histamine or fluid shear stress (Garcia-Cardena, Fan et al. 1998).<br />
Changes in phosphorylation <strong>of</strong> eNOS have been observed with shear stress (Corson, James et<br />
84
al. 1996; Eng, Morton et al. 1996; Fleming, Bauersachs et al. 1998), secondary to calcium<br />
mobilising agents (Michel, Li et al. 1993), tyrosine phosphatase inhibitors (Garcia-Cardena,<br />
Fan et al. 1996), and a number <strong>of</strong> protein kinases (Hirata, Kuroda et al. 1995; Chen,<br />
Mitchelhill et al. 1999). <strong>The</strong> phosphorylation <strong>of</strong> the enzyme makes it more sensitive to<br />
calcium necessary for the active pathway.<br />
<strong>The</strong> most potent activator is lysophosphotidylcholine (IlC), a major phospholipid found in<br />
oxidised low density lipoproteins and this allows up to an 11 fold increase in mRNA<br />
induction and eNOS production, although there is a varying discrepancy between the levels <strong>of</strong><br />
the mRNA and both the absolute protein levels and the levels <strong>of</strong> activity pointing to continued<br />
control later in the pathway (Papapetropoulos, Rudic et al. 1999). Interestingly, the content <strong>of</strong><br />
LPC in atherosclerotic vessels is higher than normal vessels. Statin-based cholesterol<br />
lowering drugs result in a modest increase in the eNOS mRNA levels by a post transcriptional<br />
mechanism involving stabilisation (Laufs and Liao 1998). Oestrogens are capable <strong>of</strong> modestly<br />
increasing eNOS expression (Wiener, Itokazu et al. 1995; Kleinert, Wallerath et al. 1998).<br />
<strong>The</strong> activity <strong>of</strong> the enzyme is reduced by the presence <strong>of</strong> TNFo which destabilises the eNOS<br />
mRNA and reduces the half life <strong>of</strong> the enzyme from 48 to 3 hours (Yoshizumi, Perrella et al.<br />
le93).<br />
3.4.3 (ii) <strong>The</strong> inducible form<br />
Control <strong>of</strong> the iNOS isoenzyme can also operate at pre-transcriptional, post transcriptional<br />
and post translational levels, and drugs also interact to affect enzyme generation. However,<br />
different to the constitutive forms, control, particularly <strong>of</strong> gross overproduction, is extremely<br />
important to host survival.<br />
Firstly, Iooking at the pre-transcriptional level; the activation <strong>of</strong> the iNOS promoter region is<br />
the main control for this isoenzyme. Most <strong>of</strong> the work looking at effects at a transcriptional<br />
level has been done in explanted mouse macrophages or macrophage cell lines (MacMicking,<br />
Xieet al. 1997; Papapetropoulos,Rudic etal. 1999).Thishasrevealedanumber<strong>of</strong> binding<br />
sequences including interferon gamma (INFI), TNFo, TNFp, interferon alpha (IFNa), NF-KB,<br />
gamma activated sites, interleukin 6 (IL6), activating protein sites and a basal transcription<br />
site. However the importance <strong>of</strong> only 2 <strong>of</strong> these has been clearly documented. <strong>The</strong> NF-rB site<br />
is important for iNOS induction (Nunokawa, Ishida et al. 1994) and LPS induction also<br />
operates through this site (Nathan 1992), and a cluster <strong>of</strong> four sites for IFNI are important for<br />
NOS transcription (Xie, Kashiwabara et al. 1994). <strong>The</strong> need for second signal as well as IFNy<br />
or an IFNy-like substance to activate iNOS is probably an important control to prevent<br />
85
inappropriate production from commencing (Lorsbach, Murphy et al. 1993). Another possible<br />
protective mechanism is, while INF1 induces iNOS, it also induces production <strong>of</strong> ILl0 an<br />
anti-inflammatory cytokine. Pre-treatment <strong>of</strong> macrophages with IL10 inhibits their ability to<br />
express iNOS or to produce NO, although once iNOS exists then it has no effect (Cunha,<br />
Moncada et al. 1992). IL,l added with ILl0 acts even more strongly in inhibiting the<br />
production <strong>of</strong> the enzyme (Oswald, Gazzinelli et al. 1992).<br />
Secondly, examining the post transcriptional control, certain cytokines can have either<br />
stabilising or destabilising effects on the mRNA for iNOS, thus greatly altering its half life.<br />
For example, IFNI stabilises the mRNA prolonging its action while transforming growth<br />
factor beta (TGFp) can destabilise the mRNA and reduce its transcription as well as<br />
accelerating its breakdown (Vodovotz, Bogdan et al. 1993; Imai, Hirata et al. 1994; Sirsjo,<br />
Soderkvist et al. 1994; Perrella, Patterson et al. 1996; MacMicking, Xie et al. 1997). While<br />
this inhibits the production <strong>of</strong> iNOS in this way, it does not effect cNOS production (Ding,<br />
Nathan et al. 1990).<br />
Thirdly, post translational control, as mentioned above, includes the availability <strong>of</strong> the<br />
substrate and c<strong>of</strong>actors. However the inducible form is independent from some <strong>of</strong> the<br />
regulations described for the constitutive forms. While all the isoenzymes require calmodulin<br />
as a c<strong>of</strong>actor, it is tightly bound to this form <strong>of</strong> the enzyme (Xie, Cho et al. 1992). In addition,<br />
the constitutive enzymes require calcium at levels <strong>of</strong> 200 nanomoles, whereas the inducible<br />
form can activate with calcium at much lower levels down to 39 nanomoles and notably the<br />
cellular basal level <strong>of</strong> calcium is nearer 70 to 100 nanomoles. This also explains why this<br />
form <strong>of</strong> the enzyme can keep producing NO when the constitutive enzymes have stopped<br />
production once the calcium falls below a certain level @astian and Hibbs 1994). A low<br />
amount <strong>of</strong> the substrate L-arginine acts as a control mechanism, there are three possibilities as<br />
mentioned above. <strong>The</strong>re may be an absolute low availability <strong>of</strong> L-arginine. <strong>The</strong> uptake <strong>of</strong><br />
arginine into the cell occurs through a sodium and pH dependent pathway, mediated by a<br />
family <strong>of</strong> cationic amino acid proteins (CATI, CATZ, CAT2B, CAT3), (Closs, Scheld et al.<br />
2000; Nicholson, Manner et al. 2001), but this can be up-regulated by the presence <strong>of</strong> LPS.<br />
External to the cell, the arginine levels are controlled by the enzyme arginase which degrades<br />
arginine to urea and ornithine (Gotoh and Mori L999; Munder, Eichmann et al. 1999;<br />
Rutschman, Lang et al. 2001). Macrophages contribute to this, possibly also developed as a<br />
protective mechanism, as they release an abundance <strong>of</strong> arginase. However macrophages and<br />
vascular smooth muscle cells can also regenerate arginine from citrulline and therefore re-<br />
utilize the citrulline amino acid. This is with the use <strong>of</strong> another enzyme arginosuccinate<br />
86
synthetase present in these cells, again upregulated by LPS and IFNy (Hattori, Campbell et al.<br />
1994; Nussler, Billiar et al. 1994; Nagasaki, Gotoh et al. 1996). <strong>The</strong> availability <strong>of</strong> c<strong>of</strong>actor<br />
BFIa may also affect iNOS activity (Stuehr 1999; Werner-Felmayer, Golderer et al. 2002').<br />
One <strong>of</strong> the key enzymes (guanosine triphosphate cyclohydroylase I, as mentioned above)<br />
which generates this c<strong>of</strong>actor is affected by the presence <strong>of</strong> certain cytokines with IFN1,<br />
TNFo, ILI and LPS increasing its activity, while the protective cytokines IL4, ILl0 and<br />
TGFP suppressing activity. IL8 also causes a concentration dependent inhibition <strong>of</strong> iNOS<br />
(McCall, Palmer et al.1992). Fibroblast growth factors inhibit NO synthesis and this may be a<br />
particular protective factor for the retina against damage (Goureau, Irpoivre et al. 1993).<br />
Finally, in keeping with the other isoenzyme forms, the rates <strong>of</strong> formation <strong>of</strong> NO and L-<br />
citrulline from iNOS are non linear which suggests there is negative feedback inhibition<br />
(Rogers and Ignarro 1992; Assreuy, Cunha et al. 1993; Connelly, Palacios-Callender et al.<br />
200r).<br />
Studies recently have suggested an "adaptive NO resistance" where some cells likely exposed<br />
to high levels <strong>of</strong> NO have an inducible NO resistance mechanism @emple 2004) and on<br />
subsequent exposure to high levels <strong>of</strong> NO the loss <strong>of</strong> cells reduces from 80 to20Vo. <strong>The</strong>se<br />
resistance mechanisms also operate against other free radicals (Kim, Bergonia et al. 1995;<br />
Bishop, Marquis et al. 1999).<br />
Some <strong>of</strong> the effects <strong>of</strong> iNOS have been clarified by the use <strong>of</strong> NOS knock out mice -<br />
demonstrating both beneficial and detrimental roles. Mice with no iNOS have altered immune<br />
responses and decreased survival to bacterial, viral and parasitic infection (MacMicking,<br />
Nathan et al. 1995; Wei, Charles et al. 1995). <strong>The</strong>y also have increased leukocyte adhesion to<br />
endothelium during toxaemia, poor wound repair and incomplete regeneration e.g. to liver<br />
biopsies and resections (Hickey, Sharkey et al. 1997; Rai, I-ee et al. 1998; Yamasaki,<br />
Edington et al. 1998). However they are also resistant to endotoxin induced mortality, end<br />
organ damage after haemorrhagic shock, or hypoxic injury, and develop less eosinophilia in<br />
allergic airways disease (Wei, Charles et al. 1995; Nathan 1997; Hierholzer, Harbrecht et al.<br />
1998; Ling, Gengaro et al. 1998).<br />
3.4.4 Nicotinamide adenosine di-nucleotide phosphate oxidase and inducible nitric oxide<br />
synthase<br />
<strong>The</strong>se are the two key enzymes involved in host defence. <strong>The</strong>re is 36Vo homology between<br />
them and both are present in macrophages, one <strong>of</strong> the key defence cells.<br />
87
Nicotinamide adenosine dinucleotide phosphate oxidase (NADPII) is involved in the<br />
respiratory burst generating Oz- as a result <strong>of</strong> phagocytic triggering (Segal 1989). <strong>The</strong> Oz-<br />
produced is a precursor <strong>of</strong> other reactive oxygen species such as hydrogen peroxide (HzOz)<br />
and hydroxyl radical (OH.). NADPH oxidase is produced primarily in<br />
polymorphonucleocytes, monocytes and macrophages - all expendable cells in host defence.<br />
It is membrane bound but has membrane and cytoplasmic subunits and is responsible for<br />
defence against extracellular pathogens and engulfed organisms accessible to phagocytic<br />
cells. Activation and priming occurs with INFI, PAF, GCSF, GMCSF, ILl, IL6, IL8, TNFa<br />
and substance P. Activation can also occur with complement factor CF5a, chemotactic<br />
factors, phorbol esters and fatty acids.<br />
In comparison, iNOS can be induced in virtually all nucleated somatic cells and is responsible<br />
for the defence against pathogens that survive and proliferate in the intracellular environment.<br />
It lies within the cytoplasm and kills pathogens that enter the cells (Nathan 1992). When it<br />
was realised that biosynthesis <strong>of</strong> NO was possible by the same cells, it meant that these cells<br />
were capable <strong>of</strong> the production <strong>of</strong> a range <strong>of</strong> reactive potentially toxic oxynitrogen species<br />
such as nitrosium (NO*), nitroxyl ions (NO-), NO2, and S-nitrosothiols (Stamler, Jaraki et al.<br />
1992). However, although macrophages can have both NADPH oxidase and iNOS present,<br />
O; and NO are not <strong>of</strong>ten simultaneously produced thus avoiding the formation <strong>of</strong><br />
perioxynitrite (ONOO-). <strong>The</strong>y appear to be independently regulated although INFI induces<br />
both enzyme systems. It may in part depend on the presence <strong>of</strong> L-arginine and the isomeric<br />
form in which it is found (Ding, Nathan et al. 1988; Martin and Edwards 1993; Bastian and<br />
Hibbs 1994).In isomerism the 'cis'form is when the two substituent groups are orientated in<br />
the same direction, while the 'trans'form is when the substituents are oriented in opposing<br />
directions. In the trans form, ONOO- undergoes homolytic cleavage to form highly reactive<br />
molecules HO* and NOz which can produce significant and irreversible damage to microbes,<br />
but also to host cells. However the cis form, ONOO- reilranges to form a non-toxic nitrate<br />
and it may be that the conditions for formation <strong>of</strong> these cis and trans isomers are what<br />
determine their formation in these cells.<br />
3.4.5 Drugs and other agents that affect the isoenrymes and nitrtc oxide production<br />
3.4.s (i) Drugs<br />
Corticosteroids inhibit the expression <strong>of</strong> inducible but not constitutive forms <strong>of</strong> NOS. This<br />
occurs at transcriptional level, with the ability to inhibit the induction <strong>of</strong> iNOS, but they are<br />
ineffective once the enzyme is synthesised. <strong>The</strong>re are no recognisable steroid responsive<br />
88
elements in the iNOS promoter region. Similarly the antifungal imidazoles drugs inhibit the<br />
induction <strong>of</strong> NOS. Methotrexate inhibits the synthesis <strong>of</strong> BFIa, therefore limiting the<br />
availability <strong>of</strong> this as a c<strong>of</strong>actor (Werner-Felmayer, Werner et al. 1990; Gross, Jaffe et al.<br />
1991).<br />
Diphenylene iodonium is an aromatic compound that binds to the flavoprotein c<strong>of</strong>actors, but<br />
is not a therapeutic option, because it was realised that very high doses would be required<br />
(Stuehr, Fasehun et al. 1991). Carbon monoxide binds to the haem in NOS and inhibits<br />
enzyme activity (White, Moellering et al. 1997). Compounds that bind calmodulin can affect<br />
cNOS but not iNOS (Bredt and Snyder 1990; Stuehr, Cho et al. l99l). Antioxidants protect<br />
NO.<br />
Calcium mobilising agents (Michel, Li et al. 1993) and tyrosine phosphatase inhibitors<br />
(Garcia-Cardena, Fan et al. 1996) both increase the rate <strong>of</strong> phosphorylation <strong>of</strong> eNOS resulting<br />
in increased sensitivity to calcium and increased production. Statin-based cholesterol lowering<br />
drugs result in a modest increase in the eNOS mRNA levels by a post transcriptional<br />
mechanism involving stabilisation (Laufs and Liao 1998). Oestrogens are capable <strong>of</strong> modestly<br />
increasing eNOS expression (Weiner, Knowles et al. lgg4: Kleinert, Wallerath et al. l99g).<br />
<strong>The</strong> angiotensin converting enzyme inhibitors inhibit the breakdown <strong>of</strong> bradykinin, and<br />
bradykinin stimulates particularly the endothelial cells to produce NO, so act to increase<br />
activity. Finally NO is the active moiety <strong>of</strong> the glyceryl trinitrate and other agents that have<br />
been trialled as anti-hypertensives and for bronchodilator properties.<br />
3.4.5 (ii) Nitric oxide synthase inhibitors<br />
<strong>The</strong> NOS inhibitors have been essential for evaluating the role <strong>of</strong> NO in physiological and<br />
pathophysiological processes, and have been used by infusion in both laboratory animals and<br />
humans to judge effects. L-arginine analogues have been created to act as competitive false<br />
substrates to inhibit NOS (see Chapter 2.3).<strong>The</strong>y have now been widely used to study the NO<br />
generating NOS pathways which has been helpful in delineating the effects <strong>of</strong> NO in a variety<br />
<strong>of</strong> systems (Hibbs, Taintor et al. 1987; Hibbs, Vavrin et al. 1987; Palmer, Rees et al. lggg).<br />
This was first accomplished by the modification <strong>of</strong> guanidino group <strong>of</strong> L-arginine to create a<br />
series <strong>of</strong> inhibitors. <strong>The</strong>se include L-NMMA (N* monomethyl L-arginine) which has<br />
substituted a methyl group, L-NAME (N* arginine methyl ester) which has substituted a nitro<br />
group and similar substitutions for the related compounds; L-NNA (N* - nitro - L-arginine),<br />
LADMA (N*N* dimethyl L-arginine), and L-MO (N* -<br />
iminoethyl-L-ornithine) (Knowles<br />
and Moncada 1992) (see Figure 3.3). <strong>The</strong>y are stereo specific, acting at the level <strong>of</strong> the<br />
89
enzyme and are competitively reversed by the normal substrate L-arginine, but not D-<br />
arginine. With prolonged incubation, the inhibitors appear to exhibit non-competitive<br />
properties (Marletta, Tayeh et al. 1990). Ebselen is a selenium containing antioxidant (2 -<br />
phenyl, 2-benzisoselenazol-3-(2h)-one) which is synthetic analog <strong>of</strong> glutathione peroxidase<br />
and is able to inhibit both iNOS and cNOS (Hibbs, Taintor et al. 1990).<br />
Figure 3.4: <strong>The</strong> chemical structures <strong>of</strong> the nitric oxide synthase inhibitors<br />
cooe ,,4,\,*"<br />
cooo<br />
'\r/t'<br />
,,A__^^(.*<br />
lW-Amino-u-arglnlnc<br />
tt'\*lt'<br />
,,4,,\'*" -.A,r"-f'<br />
'.)\".<br />
l-Arglnine<br />
r-NMMA<br />
\ t-NNA<br />
cooo<br />
t€anlvanrnc<br />
<strong>The</strong> structures <strong>of</strong> arginine and <strong>of</strong> the arginine analogues most frequently used as inhibitors <strong>of</strong> the NO<br />
synthases with the predominant ionic species at neutral pH shown.<br />
Taken from Knowles RG, Moncada S. Nitric oxide synthases in mammals. BiochemicalJournal 1994;<br />
298 (21:249-255 (Knowles and Moncada 1994).<br />
Some NOS inhibitors occur naturally such as aminoguanadine a competitive inhibitor, and<br />
two <strong>of</strong> the compounds discussed above (L-NMMA and L-NAME) also occur naturally in very<br />
small amounts, and so may also operate as a control at substrate level. Interestingly, the<br />
90
inhibitor compounds do not have equal responses with the 3 isoenzymes, for example gNOS<br />
is more sensitive to L-NAME and iNOS is more sensitive to L-NMMA. Aminoguanadine has<br />
been shown to have 10 to100 times the affinity for iNOS compared to cNOS. LNIO is a<br />
particularly potent inhibitor <strong>of</strong> the neutrophil form <strong>of</strong> iNOS suggesting subclasses <strong>of</strong> the<br />
enzymes. Selective inhibitors <strong>of</strong> cerebral NOS have also been described, possibly utilising the<br />
differing length transcriptions described for this iso-enzyme (Moore, Babbedge et al. 1993).<br />
3.5<br />
Chapter summarv<br />
NO is a free radical with an unpaired electron which is highly reactive with a half life <strong>of</strong><br />
seconds. It is produced by the NOS enzymes which exist as constitutive and inducible forms.<br />
<strong>The</strong>y require L-arginine as a substrate, plus co-substrates (NADPH and 02) and co-factors<br />
(BtI+ and flavoproteins). <strong>The</strong> neurological and endothelial constitutive forms generate<br />
continuous low levels <strong>of</strong> NO to maintain physiological processes and are largely controlled by<br />
calcium levels and available factors to be active. Activation <strong>of</strong> the inducible form requires a<br />
primary and a secondary signal before transcription occurs. However it then goes on to<br />
produce far greater amounts <strong>of</strong> NO for far longer than the other forms and is free from some<br />
<strong>of</strong> the controls <strong>of</strong> the other enzymes. It is this isoenzyme that is responsible for the host<br />
defence availability <strong>of</strong> this molecule.<br />
NO reacts to form a number <strong>of</strong> related compounds known as the reactive nitrogen species<br />
(nitrate, nitrite, nitrous oxide and nitrogen dioxide) and in the presence <strong>of</strong> higher<br />
concentrations <strong>of</strong> oxygen, it forms superoxides (perioxynitrite and perioxynitrous acid). <strong>The</strong>se<br />
compounds in turn are highly reactive and operate to kill intracellular and extracellular<br />
pathogens, but also to cause toxicity and destruction to the host cells and host DNA. NO also<br />
reacts with thiol compounds (S-nitrosoglutathione, S-nitrosoalbumin, S-nitrosohaemoglobin)<br />
and these have been increasingly recognised as a mechanism by which NO can be stabilised,<br />
transported and transferred to other compounds and other tissues. <strong>The</strong> compounds have also<br />
been demonstrated to have active roles regulating protein function and to have their own<br />
vasodilatory properties. A key interaction <strong>of</strong> NO is the reaction with metals in the centre <strong>of</strong><br />
metalloproteins, and this forms the main method <strong>of</strong> excretion <strong>of</strong> NO. NO reacts with the<br />
ferrous iron in haemoglobin to form methaemoglobin and nitrate, the latter <strong>of</strong> which is then<br />
excreted in the urine. NO and related compounds can in addition react with genes, amino<br />
acids and lipids. NO itself is a clear and colourless gas. Having examined the properties <strong>of</strong><br />
NO and the production pathway - the next chapter will discuss the technical options <strong>of</strong> how to<br />
measure NO levels.<br />
9l
4.1 Introduction<br />
Chapter 4: Methods to measure nitric oxide<br />
<strong>The</strong> previous chapter reviewed the production <strong>of</strong> NO, its reactions and interactions in vivo.<br />
This chapter will examine the available methods <strong>of</strong> measuring NO. <strong>The</strong>re were three issues<br />
that I believed needed to be addressed; firstly, how the production <strong>of</strong> this evanescent molecule<br />
could be measured as accurately as possible, secondly, which technique was the most<br />
appropriate for measuring the production <strong>of</strong> NO in the lung and thirdly, how could it be done<br />
non-invasively such that it could be used with children -<br />
so preferably no venepuncture, no<br />
biopsy or bronchoscopy and lavage required. A technique based on the way it was measured<br />
in airway pollution but adapted to exhaled air in a manner analogous to lung function was<br />
sought.<br />
From the previous chapter (see Chapter 3. 1 'Introduction'), it is known that NO is formed<br />
stereo-specifically from the guanidino nitrogen <strong>of</strong> L-arginine and oxygen. This is<br />
accomplished via five separate steps (see Figure 3.3) and requires c<strong>of</strong>actors<br />
(tetrahydrobiopterin and flavins FAD, FMN) and cosubstrates (O2, NADPH) as well as<br />
calmodulin and calcium.<br />
<strong>The</strong>refore the possible factors that could be measured to assess the activity <strong>of</strong> this pathway<br />
are:<br />
a<br />
o<br />
o<br />
o<br />
O<br />
o<br />
o<br />
<strong>The</strong> decrease in L-arginine<br />
<strong>The</strong> formation <strong>of</strong> L-citrulline<br />
<strong>The</strong> activity <strong>of</strong> cyclic guanosine monophosphate (cGMP)<br />
<strong>The</strong> formation <strong>of</strong> NO<br />
<strong>The</strong> formation <strong>of</strong> methaemoglobinuria<br />
<strong>The</strong> formation <strong>of</strong> nitrite (NO2')<br />
<strong>The</strong> formation <strong>of</strong> nitrate (NO3)<br />
<strong>The</strong>se, plus the measurement <strong>of</strong> co-factors and the other nitrogen compounds have all been<br />
explored. A detailed review <strong>of</strong> all methods to measure NO in all compounds and in all tissues<br />
is presented in "Methods in Nitric Oxide Research" edited by Martin Feelisch & Jonathan J<br />
Stamler, published by John Wiley & Sons Ltd, Baffins Lane, Chichester, West Sussex,<br />
England, 1996. It is immediately obvious that many <strong>of</strong> these compounds will be inappropriate<br />
to use for detection in lung exhalate. I will briefly review the options before reviewing in<br />
more depth the technique <strong>of</strong> NO measurement with the use <strong>of</strong> a chemiluminscence analyser.<br />
92
4.2<br />
<strong>The</strong> use <strong>of</strong> L-arginine as a substrate for NO production is a relatively specific reaction. This<br />
allows the production <strong>of</strong> NO to be correlated to the loss <strong>of</strong> L-arginine and the gain <strong>of</strong> L-<br />
citrulline, and it is possible to measure both <strong>of</strong> these in a laboratory setting (Senshu, Sato et al.<br />
1992). However the baseline concentrations <strong>of</strong> L-arginine need to be known, which is difficult<br />
in vivo. Also, while the formation <strong>of</strong> L-citrulline can be measured (Hecker, Sessa et al. 1990,<br />
Senshu, 1992 #898; Bredt and Schmidt 1996), this is made more difficult in vivo where<br />
certain cells such as vascular endothelial cells and macrophages (and likely others) are able to<br />
regenerate L-citrulline back to L-arginine at a variable rate to act as further substrate (see<br />
Chapter 3.4.3 'Control <strong>of</strong> the nitric oxide synthase isoenzymes'). This recycling clearly<br />
precludes the use <strong>of</strong> measurement <strong>of</strong> the levels <strong>of</strong> these compounds to accurately reflect the<br />
production <strong>of</strong> NO.<br />
<strong>The</strong> NOS enzymes stimulate soluble guanylate cylase (sGC) with subsequent accumulation <strong>of</strong><br />
cyclic guanosine monophosphate (cGMP). Again, it is possible to measure the levels <strong>of</strong> this<br />
protein, however, this requires access to, or the releasing <strong>of</strong>, the protein from its usual<br />
intracellular site. This is also non specific for NO as both sGC and sGMP operate in many<br />
cellular enzyme pathways (Schultz, Bohme et al. 1969; White and Aurbach 1969; Archer<br />
L993; Craven andIgnarro 1996).<br />
4.3<br />
Methaemoglobin<br />
NO can be measured by the transformation <strong>of</strong> reduced haemoglobin (Fe 2) as it is oxidised to<br />
methaemoglobin (Fe 3*; which can then measured by spectrophotometry (detection threshold<br />
= I nmol). <strong>The</strong> reaction is rapid and will be almost stoichometric under most experimental<br />
conditions, with the only interfering compound likely to be superoxide anion production<br />
(Sutton, Roberts et al. 1976). <strong>The</strong> advantages are that spectrophotometers are widely<br />
available, there is no need to acidify the sample and that methaemoglobulin remains relatively<br />
stable. Accumulation <strong>of</strong> oxyhaemoglobin is not a problem due to the much higher affinity <strong>of</strong><br />
haemoglobin for NO than Oz. <strong>The</strong> disadvantages are that it is more useful for biological<br />
samples other than exhalate, considerable expertise is required and the assay will detect other<br />
nitrosyl grcups in any given sample (Noack, Kubitzek et al. lgg2; Murphy and Noack 1994;<br />
Feelisch, Kubitzek et al. 1996).<br />
93
4.4 Nitrite and nitrate<br />
<strong>The</strong> measurement <strong>of</strong> NO in biological tissues has been difficult in the past because <strong>of</strong> the<br />
short halfJife, the small amounts produced, and its lability in the presence <strong>of</strong> Oz.<br />
By contrast nitrite and nitrate are stable metabolites whose presence in blood, urine and tissue<br />
in humans was demonstrated many years ago (Mitchell, A. et al. 1916; Mitchell 1928;<br />
Tannenbaum, Fett et al. 1928). <strong>The</strong> diazotization assay is the standard technique for<br />
measuring inorganic nitrite and was described more than 150 years ago by Greiss (Greiss<br />
1864; Greiss 1879). A two step assay is carried out with the reagents sulphanilic and N-(1-<br />
naphthyl) ethylenediamine first mixed and then incubated with a nitrite containing sample<br />
which interacts in a 1-l ratio generating a purple azo dye which can be monitored by<br />
spectrophotometry at a wave length <strong>of</strong> 546nm (Greiss 1864; Green, Wagner et al. L982). This<br />
remains the most common method <strong>of</strong> measuring nitrite and nitrate (Schmidt and Kelm 1996)<br />
in fluid such as blood and urine (Wishnok, Tannenbaum et al. 1993; Baylis and Vallance<br />
1998). Studies have suggested that it is circulating nitrite measurable in serum or plasma<br />
rather than nitrate that more reflects the NO synthesis in both humans and animals (Bode-<br />
Boger, Boger et al. 1999; Lauer, Preik et al. 2IN_I; Kleinbongard, Dejam et al. 2006).<br />
However, the excretion rate in urine <strong>of</strong> nitrate is a more non-invasive method to measure<br />
whole body NO synthesis and is particularly useful for obtaining baseline results and then<br />
assessing responses to physical and pharmacological treatment (Kanno, Hirata et al. 1992;<br />
Bode-Boger, Boger er al. 1994 Borgonio, Witte et al. 1999; Bode-Boger, Boger et d. 2000).<br />
<strong>The</strong>re are some difficulties in making these measurements in biological assays as NO<br />
decomposes to nitrite and nitrate at different rates depending on the ambient conditions and<br />
on the redox environment <strong>of</strong> the fluid being measured. As well, these two compounds are<br />
present at different concentrations in urine, serum and/or blood therefore requiring the ability<br />
to measure and calculate across a wide concentration range potentially from low nanomolar to<br />
high micromolar levels (Schmidt and Kelm 1996). Although adaptation to enable this has<br />
occurred as studies on the nitrogen compounds have progressed (Green, Wagner et al. 1982;<br />
Corras and Wakid 1990; Ohta, Araki et al. 1994; Tsikas 2005). However even a very recent<br />
comprehensive review <strong>of</strong> the topic states "Despite the chemical simplicity <strong>of</strong> nitrite and<br />
nitrate, accurate and interference-free quantification <strong>of</strong> nitrite and nitrate in biological fluids<br />
as indicators <strong>of</strong> NO synthesis may be difficult" (Tsikas 2005). <strong>The</strong> Greiss reaction gives an<br />
assay that is designed to measure single samples, although it can be modified to give online<br />
94
esults (Tracey, Linden et al. 1990), but may not be sensitive or specific enough to measure<br />
NO alone (Schmidt and Kelm t996),<br />
NO will rapidly convert to nitrite and nitrate in biological samples and by adding acids or<br />
reducing substances these can be transformed back to release the NO molecule (Verdon,<br />
Burton et al. 1995; Kelm, Dahmann et al. 1997). However acidification and/or reduction will<br />
release NO from all nitroso compounds, alkyl or inorganic nitrites, the nitrosamines and<br />
nitrosothiols. If the sample contains compounds other than nitrite and nitrate, then there may<br />
be overestimation <strong>of</strong> the NO that existed as a biologically active or inflammatory mediator.<br />
4.5 Nitric oxide<br />
It has become possible to measure NO directly down to the cellular level by using<br />
microelectrodes. This cornmenced with the modification <strong>of</strong> a miniature 02 electrode, and<br />
sealing it so that only low molecular weight gases could enter. By introducing a positive<br />
voltage, NO was oxidised on the surface <strong>of</strong> the electrode and could be measured over l-<br />
3pmol range (Shibuki 1990). <strong>The</strong> probe was approximately 2mm in diameter. Modification <strong>of</strong><br />
this then allowed a much smaller microsensor to be developed using a semi conductor<br />
polymeric porphyrin and a cationic exchanger on a sharpened carbon tip which reduced the<br />
size to 5 microns (Malinski and Taha 1992; Taha, Kiechle et al.1992) and could measure NO<br />
as an electrical current. This has allowed NO measurement in cardiac cells (Xian ,Zhang et al.<br />
2000; Kanai, Pearce et al. 2001; Katrlik and Zalesakova2002),brain and nerve cells (Shibuki<br />
1990; Taha, Kiechle et al. 1992: Malinski, Bailey et al. 1993; Kumar, Porterfield et al. 2001),<br />
osteoclasts (Silverton, Adebanjo et al. 1999), trachea and main bronchi from an animal model<br />
(Ricciardolo, Vergnani et al. 2000) and in suspensions <strong>of</strong> mitochondria and cells such as<br />
platelets, leukocytes and in cell cultures (Wadsworth, Stankevicius et aL.2006). <strong>The</strong> detection<br />
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<br />
low detection limit is also matched by high sensitivity and selectivity although it does also<br />
detect catecholamine activity (Tu, Xue et al. 2000). It has also been achieved as an online<br />
measurement so release <strong>of</strong> NO could be followed in activated macrophages, which correlated<br />
well with the nitrite concentration determined by the Greiss assay (Cserey and Gratzl 2001).<br />
However a recent review suggested that further methodological development <strong>of</strong> these<br />
microsensors was needed in order to avoid the influence <strong>of</strong> changes in temperature, pH, and<br />
oxygen on the measurements (Wadsworth, Stankevicius et al. 2006).<br />
My interest was to look at measurement in exhaled breath. A number <strong>of</strong> very early methods <strong>of</strong><br />
detection depended on colorimetric analysis but for exhalate these were too slow and too<br />
95
insensitive (Braker and Mossman 1975; Archer 1993). For example, the use <strong>of</strong> moist iodide<br />
paper to detect the oxidation products <strong>of</strong> NO in air has a sensitivity <strong>of</strong> 300ppm and has a<br />
response time <strong>of</strong> 5 minutes. When collecting samples to measure the NO concentration, the<br />
presence <strong>of</strong> oxygen will continue to degrade the sample. This means the sample needs to be<br />
measured quickly, oxygen contamination must be avoided (which is difficult in biological<br />
specimens), or the loss <strong>of</strong> NO with these reactions needs to be compensated for by converting<br />
the other compounds back to releasing NO just prior to measurement.<br />
It is possible to use collected expirate which can be bubbled through degassed water and the<br />
NO can be trapped by nitroso compounds or reduced haemoglobin to form stable adducts that<br />
can then be detected by electron paftrmagnetic resonance (detection threshold = I nmol).<br />
Electron paramagnetic resonance spectroscopy allows a specific assessment <strong>of</strong> molecules<br />
whose energy levels are altered in the presence <strong>of</strong> a magnetic field and this property is typical<br />
<strong>of</strong> molecules with an uneven number <strong>of</strong> electrons (Henry, Ducrocq et al. l99l; Henry,<br />
Irpoivre et al. L993; Singel and Lancaster 1996). Usually a continuous wave <strong>of</strong><br />
electromagnetic radiation in the microwave frequency is applied to the sample. When the<br />
frequency <strong>of</strong> this is equivalent to the energy difference <strong>of</strong> the electron spin energy levels,<br />
which is known as the 'resonance', the radiation is absorbed and the absorption leads to the<br />
generation <strong>of</strong> a signal which is measured. This technology has proved useful in detecting the<br />
movement <strong>of</strong> NO, for example determining the specific parentage <strong>of</strong> L-arginine to NO to<br />
methaemoglobin, determining the active site for NO or the binding site with other<br />
compounds. Interestingly, despite the unpaired electron, the NO molecule is only detectable in<br />
an excited state, while at a 'ground' state (unexcited state) the coupling <strong>of</strong> electron spin and<br />
the orbital angular momentum makes it resonant silent on the spectroscope. It is possible to<br />
identify signals at different frequencies which allow different molecules within compounds to<br />
be identified. This is unless a wide band <strong>of</strong> frequency is occupied as is seen with the iron in<br />
the haem proteins and from some <strong>of</strong> the other compounds used to trap NO such as metals or<br />
thiols. <strong>The</strong> compounds used for trapping NO in this way are also sensitive to pH. At acidic pH<br />
levels, the nitroso and nitrone compounds are unstable and can yield NO-type spectra even in<br />
the absence <strong>of</strong> NO (Anoyo and Kohno 1991). At alkaline pH there can be inhibition <strong>of</strong> the<br />
reaction between NO and haemoglobin (Feelisch, Kubitzek et al. 1996; Hakim, Sugimori et<br />
al. 1996). Finally, the equipment required to perform electron paramagnetic resonance was,<br />
and remains, expensive and considerable expertise is necessary (Archer 1993). In addition,<br />
more direct methods <strong>of</strong> measuring exhalation were becoming available.<br />
96
NO can be detected by standard gas chromatograph techniques @ai, Payne et al. 1987; Tsikas,<br />
Boger et al. 1994), although initially this was found to be much less sensitive than most <strong>of</strong><br />
other methods discussed here, partly because <strong>of</strong> the necessity for periodic manipulation and<br />
thus disturbance <strong>of</strong> the reaction mixture during the sample processing. It is now increasingly<br />
combined with scanning mass spectrophotometers where NO in air can be detected (Kelm,<br />
Feelisch et al. 1988). Sensitivity has been limited by the resolving power (13500) required to<br />
differentiate between ttN2 1m/z 30.00022) and NO (m1229.99799). euantification was further<br />
complicated by the vast difference in the concentration <strong>of</strong> these two gases, namely parts per<br />
thousand for l5N2 and parts per billion for NO. Specific experiments can be designed using the<br />
isotope <strong>of</strong> lsN as a specific label for NO. In the mass spectrometer, gases are admitted under<br />
very low pressures into an ion source where they are ionized in a high energy electron flux.<br />
<strong>The</strong> positive ions formed are accelerated in an electric field and then deflected in a magnetic<br />
field. <strong>The</strong> radius <strong>of</strong> the deflection is inversely proportional to the number <strong>of</strong> the particles. A<br />
multi-collector device for a number <strong>of</strong> particles can be used. It can also be used to measure<br />
the evolution <strong>of</strong> gas from a liquid sample. <strong>The</strong> reaction mixture is 'sparged' with an inert<br />
compound such as argon (sparging is to agitate by introducing a compressed gas), a plunger is<br />
lowered to the liquid surface and closed to the atmosphere. <strong>The</strong> reaction is then commenced<br />
with injections <strong>of</strong> reagents, inhibitors, enzymes or cells - and in the case <strong>of</strong> NO stripping,<br />
obtaining NO back from the compounds to which has bonded by acidification. <strong>The</strong> gas then<br />
diffuses into a vacuum line through a cold trap which removes water vapour, and the<br />
remaining gases canying NO and other compounds <strong>of</strong> interest are scanned and analysed at<br />
brief intervals (Payne, Le Gall et al. 1996). This has been used to study nitrogen metabolism,<br />
not only in biological fluids but also, for example, to assess bacterial activity in river water<br />
etc. However using labelled arginine, it has been used to measure NOS activity (Tsikas 2OO4)<br />
and very recently to assess whole body NO synthesis in healthy children after subjects had<br />
oral doses <strong>of</strong> the labelled substrate with the measurement <strong>of</strong> the subsequently collected urine<br />
(Forte, Ogborn et al. 2006). For all these measurements, isotope labelling is required, and<br />
therefore they are not so useful for repeated exhalation measurement under different<br />
conditions.<br />
4.6<br />
Chemiluminescence<br />
<strong>The</strong> method that held the most promise for measuring NO in direct gas samples such as<br />
exhaled air was chemiluminescence.<br />
97
A group <strong>of</strong> instruments have been developed to enable the measurement <strong>of</strong> certain compound<br />
concentrations within samples by using light reactions. <strong>The</strong>se include:<br />
o 'spectrophotometers', mentioned above, which measure how light is absorbed by the<br />
specimen with the light being generated by the meter itself.<br />
o 'Fluorometers' which measure light emitted by the specimen after excitation <strong>of</strong> the sample<br />
generated by the meter.<br />
o 'Luminometers' which measure light generated with no light or excitation input - this<br />
makes them comparatively simple compared to the other devices as they only require a<br />
reaction chamber, a light detector and a recorder (and for the measurement <strong>of</strong> NO, a<br />
photomultipler).<br />
<strong>The</strong> chemiluminscence analysers (see Figure 4.2) were originally developed to measure NO<br />
as an atmospheric pollutant as discussed in Chapter 2.2. <strong>The</strong> measurement is based on the<br />
observation that the reaction <strong>of</strong> NO with ozone produces light (see Figure 4.1).<br />
Figure 4.1: Chemical reaction between nitric oxide and ozone<br />
NO + Os + NO2' 1'. 9,<br />
NO"+NOz+hf<br />
(. = unstable electron)<br />
(hf = light)<br />
<strong>The</strong> interaction <strong>of</strong> ozone with gases such as NO, NOz, CO and SOz was described in the early<br />
1960s (AIHA 1966), although ozone reacts most readily with NO. In the excited state the<br />
electrons are unstable and they dissipate energy as they regain their original state. This light is<br />
sufficient to make chemiluminescence one <strong>of</strong> the most sensitive NO assays available. <strong>The</strong><br />
chemiluminescence reaction <strong>of</strong> NO and Or is very fast and has a low activation energy <strong>of</strong> 10.5<br />
joules (Johnston and Crosby 1954). At room temperature the rate constant <strong>of</strong> the reaction<br />
(both steps) is l0-7 I mol-r s-t lclyne, Thrush et al. 1964). This high speed means that the<br />
chemiluminscence assay is able to detect rapid changes in NO concentration and therefore can<br />
be adapted for on-line measurement. It was, for example, used early to monitor NO<br />
concentrations when this was delivered as a treatment trial in intensive care for pulmonary<br />
hypertension @epke-Zaba, Higenbottam et al. 1991; Kinsella, Neish et al. 1992; Roberts,<br />
Polaner et al. 1992: Gerlach, Rossaint et al. 1993). NOz reacts with ozone more slowly and<br />
the reaction requires higher activation energy so the chemiluminscence levels <strong>of</strong> NO are not<br />
affected by NO2, even if the latter is present in high concentrations (Johnston and Crosby<br />
I9l4;Fontijn, Sabadell et al. 1970).<br />
98
<strong>The</strong> first chemiluminscence analyser to measure NO was built in 1970 utilizing these<br />
principles <strong>of</strong> chemical reaction. A vacuum <strong>of</strong> reduced pressure (approximately negative l-15<br />
mmHg) is used to draw the gaseous sample into a reaction chamber through a valve where<br />
ozone is generated by electrical discharge. <strong>The</strong> vacuum is also necessary to evacuate gases<br />
that could potentially absorb energy from NO2., and to stabilise NO by removing 02, to<br />
prevent any 02 reacting with the NO to produce NOz which does not emit light (Hampl,<br />
Walters et al. 1996). <strong>The</strong> NO and 03 are then mixed in front <strong>of</strong> a photomultiplier tube<br />
sensitive to low levels <strong>of</strong> light at the red sensitive end <strong>of</strong> the spectrum (660 - 900nm). <strong>The</strong><br />
photons from the reaction strike a photosensitive surface and the impact releases electrons<br />
which are accelerated toward an electron sensitive surface (the first dynode) by an electric<br />
field. Each electron impact on this first dynode then causes emission <strong>of</strong> several electrons and<br />
these are accelerated to a second dynode. This step is repeated but the electrons are attracted<br />
to the terminal electrically charged element - the anode - and the resulting current is<br />
measured. This amplification achieved by the photomultiplier is necessary to measure the<br />
signal as the NO2'rosction emits a relatively weak red light (Hampl, Walters et al. 1996) and<br />
means that each electron emitted from the original photosensitive surface becomes a signal<br />
from millions <strong>of</strong> electrons at the anode (Turner 1985).<br />
Figure 4.2: Diagram <strong>of</strong> the chemiluminscence analyser<br />
AmlogdChl<br />
cocputor/rrconbr<br />
I Flowwrctcr<br />
i (o9ddlll)<br />
!<br />
Sampb<br />
<strong>The</strong> original group demonstrated that the light emitted was indeed directly proportional to the<br />
linear content <strong>of</strong> the specimen and I have included the graph <strong>of</strong> their results (see Figure 4.3).<br />
<strong>The</strong>y used increasing volumes from one to one hundred micro-litres <strong>of</strong> a commercially<br />
available gas in gas tight, Nz flushed syringes. <strong>The</strong> chemiluminscence signal was recorded as<br />
the peak height at an integration time <strong>of</strong> two seconds. This showed a linear relationship <strong>of</strong><br />
chemiluminscence measured in millivolts to NO concentration measured from zero to fifty<br />
picomoles.<br />
Figure 4.3: Relationship between chemiluminscsnce in millivolts to nitric oxide concentration in<br />
picomoles.<br />
m<br />
e*<br />
Eto<br />
plo<br />
tr*<br />
!-<br />
E-<br />
610<br />
dgnrl.l7.9'l{Onurr-tt<br />
0<br />
0tr0t6m?lgl$'|oa5<br />
t{Oernln[(pfllol)<br />
Taken from Archer S. Measurement <strong>of</strong> Nitric oxide in biological models. FASEB Journal 1993; 7(2):<br />
349-360 (Archer 1993).<br />
<strong>The</strong> background output <strong>of</strong> the photomultiplier is relatively stable, unlike the other light source<br />
meters which can have surges. <strong>The</strong> dark current (as it is known) can be affected by changes in<br />
temperature, light and alterations in voltage, and by very high levels <strong>of</strong> NO. Large<br />
disturbances in the dark current may take days to stabilise. For this reason most are equipped<br />
with a cooler as cooling the photomultiplier tube improved the signaUnoise ratio. Preferably<br />
the analyser should also be kept in a temperature controlled room and away from any other<br />
equipment that potentially generates heat.<br />
A red cut <strong>of</strong>f filter separates the reaction chamber and the photomultiplier to prevent the<br />
detection <strong>of</strong> light with wavelengths below that <strong>of</strong> the NO/O3 reaction. <strong>The</strong>se include blue or<br />
ultraviolet emission <strong>of</strong> alkenes and sulphur containing species such as the reaction <strong>of</strong><br />
hydrogen sulphide (HzS). <strong>The</strong>se reactions are unlikely to be a major problem in practice as the<br />
compounds are present on a greatly reduced scale compared to NO with far less or no<br />
biological activity, and they are not volatile. As the NO/OI reaction takes place in the gas<br />
phase, this is ideal for measurement <strong>of</strong> gaseous samples such as exhaled air. <strong>The</strong> resulting<br />
100
NO2 produced can be removed by a soda lime column and resulting Og can be removed using<br />
a charcoal column; or the exhaust can be immediately directed outside. I opted to use columns<br />
to 'scrub' the compounds from the exhaust air. <strong>The</strong> luminescence signal as measured by a<br />
photon counter and transmitted as an electrical signal and (in the early adapted machine which<br />
we used) this was then transmitted to a chart recorder. In the later versions <strong>of</strong> the machine the<br />
signal went to an analogue digital computer.<br />
Sensitivity: <strong>The</strong> detection threshold <strong>of</strong> NO is 20-50pmol. In aqueous solutions the<br />
chemiluminescence assay has been reported to detect as little as 10-13 M <strong>of</strong> NO<br />
(Zafrnou and McFarland 1980). <strong>The</strong> chemiluminescence/1.{O curye has been<br />
found to be linear between NO doses <strong>of</strong> 0-50pmol (Fontijn, Sabadell et al.<br />
1970) and 300-3000 pmol (Menon, Wolf et al. 1989; Brien, Mclaughlin et al.<br />
19e1).<br />
Specificity: Chemiluminescence is almost exclusively due to NO. As mentioned above,<br />
there are few other substances that react to produce light and these are either<br />
non-volatile or are not biologically important such as the production <strong>of</strong> HzS<br />
and alkenes. One author (Archer 1993) found that at very high levels a solvent<br />
used for many drugs (dimethyl sulfoxide - 'DMSO') could also cause a<br />
chemiluminescent signal. This is unlikely to be a problem in exhalate and<br />
pertains more to the use <strong>of</strong> high dose chemicals on explanted or cultured tissue<br />
(Mottu, Laurent et al. 2000). However this chemical is a known antioxidant<br />
and more recently has been employed as a treatment in certain inflammatory<br />
conditions, most particularly in interstitial cystitis (bladder inflammation), but<br />
also for some rheumatologic diseases (Santos, Figueira-Coelho et al. 2OO3i<br />
Chancellor and Yoshimura 2004; Parsons 2004).<br />
<strong>The</strong> NO chemiluminscence analysers available in 1995 were designed for measuring NO<br />
concentrations within 2-4000pbb and 40-400ppm range in a continuous ambient air sample.<br />
<strong>The</strong>y had been adapted for online recording and had a stabilised measurement capability as<br />
measured by drift without using an auto zero over 24 hours. In most this was cited at between<br />
zero and two ppb. <strong>The</strong>y operated in ambient temperatures <strong>of</strong> 5-400 C and humidity <strong>of</strong> 0-95Vo.<br />
<strong>The</strong> estimates <strong>of</strong> NO concentrations were decreased 10-15Vo at l00%o humidity, however NO<br />
pre-drying <strong>of</strong> the expirate was not thought to be necessary for most environments @gure 4.2<br />
does have a pre-drying unit added in the diagram). <strong>The</strong> original response time <strong>of</strong> these<br />
machine developed to measure airway pollution were long, too long for the purpose that we<br />
101
equired it. For example, there was a 90 second delay in the model we used (Model 207,<br />
Dasabi Corporation), but modification <strong>of</strong> the circuitry and the sending <strong>of</strong> the pre-computer<br />
analogue signal directly to the chart recorder (with assistance from the Biomedical<br />
Engineering Department at the Royal Brompton Hospital, London, United Kingdom: Carolyn<br />
Busst and Ron Sinclair) decreased the response time - a factor checked in the first<br />
experiments made to establish the analyser's capability (see Chapter 5). <strong>The</strong> option <strong>of</strong> a<br />
reader, chart recorder or an analogue digital computer may seem unusual now in the days <strong>of</strong><br />
easy, fast computer access, and the new analysers have these inbuilt for signal display. <strong>The</strong><br />
sampling flow <strong>of</strong> the NO analyser depends on the vacuum pump rate. <strong>The</strong> NO analysers listed<br />
above at the time that these experiments began sampled at fixed rates between 200 and 800<br />
mls/min. This too was modified for use on the first experiments using this machine (see<br />
Chapter 5). <strong>The</strong> newer purpose built analysers have a wider range <strong>of</strong> sampling flow options <strong>of</strong><br />
25, 50,100,250 and 5OOmls/min.<br />
Table 4.1: <strong>The</strong> companies providing chemiluminescence analysers adaptable for nitric oxide<br />
measurement in 1995<br />
ChemLab Instruments Ltd, Hornchurch, United Kingdom<br />
Columbia Scientific Industries Corporation, Austin, Texas,<br />
United States <strong>of</strong> America.<br />
Dasibi Environmental Corporation, Glendale, Galifornia,<br />
United States <strong>of</strong> America (Model2107)<br />
Eco Physics, Durnten, Switzerland (CLD 700)<br />
Lear-Seig ler/Mon itor Laboratories, En glewood, Colorado,<br />
United States <strong>of</strong> America.<br />
Seivers Instruments, Boulder, Colorado,<br />
United States <strong>of</strong> America. (NOA')<br />
<strong>The</strong>rmoelectron, Warrington, United Kingdom. (Model 42)<br />
In addition to detecting NO in gas samples, I will mention briefly that the technique can be<br />
used for liquid samples. At room temperature NO has a partition coefficient <strong>of</strong> 2O; that is in a<br />
sample containing both liquid and gas, there is 20 times more NO in the gas phase than<br />
dissolved in the liquid phase. So if a fluid with no air contact is then injected into a chamber,<br />
the NO quickly escapes into the gas phase and this can be aspirated into the<br />
chemiluminscence analyser. <strong>The</strong> rest <strong>of</strong> the NO can then be "stripped" from the solution by<br />
bubbling the solution with an inert gas under vacuum conditions usually at a rate <strong>of</strong> between<br />
8-10 mls/min driving it into the gas phase which can then be measured (Chung and Fung<br />
1990; Archer, Shultz et al. 1995; Hampl, Walters et al. 1996). However it is vital to avoid<br />
foam or bubbles getting into the reaction chamber as this can then coat the PMT and impair its<br />
102
function. This is especially important with biological specimens as proteinaceous material<br />
(such as albumin and blood) foams during stripping and therefore provides more difficulties.<br />
<strong>The</strong> chemiluminscence assay can also be used to measure the other intermediates and other<br />
end products <strong>of</strong> NO oxidation such as s-nitrosothiols, nitrite and nitrate. <strong>The</strong> compounds can<br />
be made to release the NO by reducing them with acid (usually hydrochloric acid, citric acid<br />
or glacial acetic acid is used). This gives the nitrosonium ion (NO) which then can react with<br />
anions (such as iodine which is commonly used) which then dissociates to give NO, water and<br />
iodine, with the No able to be measured in the chemiluminscence analyser.<br />
Figure 4.4: Reaction equations used to release nitric oxide from other nitrogen compounds<br />
NOz' + 2H+ -> NO* + H2O<br />
NO* + l'+ ONl<br />
2ONl +2NO+lz<br />
As can be seen, this is more difficult than the direct gas measurement and involves more<br />
steps. In addition, all the reagents must be replaced with every sample and extra time required<br />
for signal <strong>of</strong> the acid and iodine compounds alone. It is very temperature and (clearly) pH<br />
dependent. However, as mentioned above, when correctly performed it remains very sensitive<br />
to NO in small amounts (Zafiriou and McFarland 1980).<br />
4.6.1 Calibration<br />
Machine calibration had to be carried out regularly, and at the time a number <strong>of</strong> commercial<br />
gas companies <strong>of</strong>fered cylinders with graduated NO concentrations:<br />
o BOC Gases, Surrey Research Park, Guildford, Surrey, UK<br />
o Scott Specialty Gases, Troy, Michigan<br />
o Matheson, East Rutherford, New Jersey<br />
(Modified from our original published chapter (Byrnes, Bush et al. 1996)).<br />
<strong>The</strong>se were prepared by oxidation <strong>of</strong> ammonia at 5000C over platinum gauze or produced by<br />
passing an electric arc through the air (Braker and Mossman L975).Individual calibration<br />
samples <strong>of</strong> NO can be prepared chemically either by adding acids to sodium nitrite (NaNOz),<br />
or mixing NOz- and a denitrifying enzyme (Pai, Payne et al. 1987). A preparation <strong>of</strong> a<br />
saturated NO solution (NO = 3 mM) is needed. Double distilled cold water is bubbled with<br />
103
helium for 30 minutes to remove Oz. <strong>The</strong> water is then bubbled with pure NO (>99.0Vo1) for<br />
30 minutes in a glass sampling bulb. Samples can be aspirated through a rubber gas-tight<br />
syringe. Before aspiration Nz should be injected into the glass bulb to exclude Oz (Archer<br />
1993). Once a saturated NO solution has been made serial dilutions <strong>of</strong> the NO gas or solution<br />
can be made using deoxygenated HzO. Having reviewed the alternatives, the company that I<br />
used was BOC Gases (Suney Research Park, Guildford, Suney, United Kingdom) who were<br />
local to where the research was taking place and who regularly supplied gases to the Royal<br />
Brompton Hospital. Also they could provide NO in cylinders <strong>of</strong> appropriate concentrations<br />
suitable for calibration <strong>of</strong> the levels we intended to measure (see below).<br />
Whether personal or commercial preparations <strong>of</strong> NO are being made for calibration purposes<br />
it is imperative to avoid contamination with Oz which will reduce the NO in the sample. Zero<br />
calibration can be done with NO free certified compressed air. Alternatively, passing room air<br />
though the ozone generator <strong>of</strong> the chemiluminescence analyser, which converts all NOx to<br />
nitrogen dioxide and then passing the resultant gas through soda lime and activated charcoal<br />
to remove the NOz and ozone respectively can also produce NOx free air. <strong>The</strong> most useful<br />
calibrations gas concentrations are zero and concentrations within the usual levels measured,<br />
with the final calibration just above the highest levels likely encountered to avoid the need for<br />
linear extrapolation beyond the range. Not all the companies could provide appropriate<br />
concentrations or had them in stock. In normal subjects on the current analysers with the<br />
current measurement protocols, the range is 0-25ppb for oral exhaled NO and 250-1000ppb<br />
for nasal NO but the levels on the older machines were <strong>of</strong>ten higher (0-80ppb) for oral<br />
exhaled measurements. <strong>The</strong> reason for these differences will be come clear when reviewing<br />
the methodology experiments that I completed (see Chapter 5). I always had a zero and two<br />
NO cylinder concentrations for calibration for the experiments described in the work <strong>of</strong> this<br />
thesis. <strong>The</strong>y were either 27ppb and 103ppb and ll0ppb or 55ppb and 118ppb when the first<br />
cylinders were exhausted.<br />
4.6.2 Safety and toxicity<br />
In the final part in this chapter I want to review the toxicity <strong>of</strong> NO, and the two gases<br />
generated using the chemiluminescent technique -<br />
NOz and ozone. <strong>The</strong> reactions and effect <strong>of</strong><br />
NO at the cellular level has been covered in Chapter 3 sections 3.2 'Properties <strong>of</strong> nitric oxide'<br />
and 3.3 'Reactions <strong>of</strong> nitric oxide'. NO must be handled with care -<br />
the toxicity <strong>of</strong> NO is due<br />
to the gas itself and also the interactions that occur with oxygen when it forms a dimeric form<br />
<strong>of</strong> NOz - a reddish brown toxic gas @udvari, O'Neil et al. 1989) which in high 02<br />
r04
concentration can form the superoxide perioxynitrite (OONO). In part the effects <strong>of</strong> these<br />
gases have been covered when reviewing the noxious effects <strong>of</strong> air pollution with particular<br />
reference to the nitrogen oxides in Chapter 2.2. NO and, even more so, NOz are common air<br />
pollutants (Morrow 1984; Anonymous 1995; Anonymous 1996) with inspired NO<br />
concentrations from airway pollution ranging from 20-500ppb in non-polluted areas to<br />
1.5ppm or greater in polluted areas (Aranda and Pearl 2000). As previously mentioned,<br />
cigarette smoke contains NO concentrations in order <strong>of</strong> lOOppm (Norman and Keith 1965), or<br />
higher depending on the cigarette type (Borland and Higenbottam 1987) and cigarette smoke<br />
can inhibit NADPH and myeloperoxidase (Nguyen, Finkelstein et al. 2001). While this does<br />
not result in an acute respiratory event, it might cause an increase risk <strong>of</strong> mutations over time<br />
and thus it contributes to cigarette smoke carcinogenic potential.<br />
With the use <strong>of</strong> nitrous oxide (NzO, "laughing gas") effects were noted when N2O cylinders<br />
were contaminated with NO showing that high NO concentrations could give rise to acute<br />
pulmonary oedema and methamoglobinaemia (Anonymous 1967; Clutton-Brock 1967). High<br />
levels <strong>of</strong> NO were needed to get this response in experiments with animals. For example,<br />
exposure <strong>of</strong> lambs to 80ppm <strong>of</strong> NO for three hours in 2l%o oxygen did not result<br />
methaemoglobinaemia or any <strong>of</strong> the acute problems previously documented (Frostell, Fratacci<br />
et al. 1991). Exposure <strong>of</strong> dogs to 20,000ppm did result in this acute respiratory event with<br />
high morbidity and mortality (Shiel 1967). Inhalation <strong>of</strong> 50ppm <strong>of</strong> NO impairs the<br />
performance <strong>of</strong> learned tasks and prolongs brainstem evoked potential responses in rats,<br />
without significantly elevating methaemoglobin levels (Groll-Knapp, Haider et al. 1988). In<br />
healthy subjects inhaling lOOppm <strong>of</strong> NO for three hours, methaemoglobulin levels increased<br />
by I.77Vo <strong>of</strong> total haemoglobin to a peak at 45 minutes. Serum nitrogen oxides (NOz, NOI)<br />
increased from 36.7 to 124umol L-l at 172 minutes and then declined (Young, Sear et al.<br />
1996). Inhaled NO at 5ppm resulted in thickened alveolar membranes in rabbits (Hugod<br />
1979) and NO at 43ppm and NO2 at 3.6ppm narrowed the surfactant hysteresis, altered<br />
epithelium ultra-structure and caused interstitial atrophy (Hugod 1979). Intermittent NO<br />
exposure over a 9 week period in rats resulted in degeneration <strong>of</strong> interstitial cells and the<br />
interstitial matrix leading to an emphysematous-like destruction <strong>of</strong> alveolar septa (Mercer,<br />
Cosra et al. 1995).<br />
NOz is more toxic and has long been known to cause respiratory damage and fatality<br />
(Kooiker, Schuman et al. 1963: Wagner, 1965 #1030). It was early recognised as a concern in<br />
atmospheric pollution (Kennebeck, Wetherington et al. 1963). NOz has been shown to be<br />
taken up easily by lung lining fluid at a rate related to its speed <strong>of</strong> reaction to glutathione<br />
105
(Postlethwait and Bidani 1990; Postlethwait, I-angford et al. 1990). This is increased with<br />
alkalosis and hyperthermia @ostlethwait, Langford et al. 1991; Postlethwait and Bidani<br />
1994). A number <strong>of</strong> studies have been conducted to determine the acute effects <strong>of</strong> NOz<br />
exposure at levels seen in air pollution on airway lining (usually the nose), lavage fluid, lung<br />
function and airway reactivity in normal and asthmatic adult subjects. <strong>The</strong> exposures have<br />
ranged from two to six hours at levels from 200 to 800ppb, aild were to NOz alone or<br />
combined with either ozone or SOz @evalia, Rusznak et al. 1996; Devalia, Bayram et al.<br />
1997; Jenkins, Devalia et al. 1999). <strong>The</strong>se exposures resulted in disruption <strong>of</strong> the airway<br />
epithelia, resulting in an increase <strong>of</strong> inflammatory cells with eosinophil 'priming', and a<br />
release <strong>of</strong> inflammatory cytokines such as TNFo and IL8. Increased airway hyper-reactivity<br />
in normal (Utell, Frampton et al. l99l) as well as asthmatic subjects was also demonstrated<br />
@evalia, Rusznak et al. 1996; Devalia, Rusznak et al. 1996; Jenkins, Devalia et al. 1999).<br />
Cilia were also disrupted and, after exposure to NOz at 2ppm for 4 hours, ultra-structurally<br />
altered cilia with excess matrix and multiple ciliary ru(enomes were seen (Carson, Collier et<br />
al. L993). Intermittent NOz exposure over a period <strong>of</strong> weeks in rats also resulted in<br />
emphysematous like destruction <strong>of</strong> alveolar septa with reduction in the ventilatory surface<br />
@vans, Stephens et al. 1972; Freeman, Crane et al. 1972; Mercer, Costa et al. 1995). <strong>The</strong>re<br />
was also hyperplasia <strong>of</strong> the respiratory epithelium with squamous metaplasia (Maejima,<br />
Suzuki et al. 1992), disturbed surfactant (Muller, Barth et al. 1992) and aldehyde release<br />
(Robison, Forman et al. 1995). Prolonged inhalation <strong>of</strong> 2ppm NOz is associated with terminal<br />
bronchial epithelial hypertrophy and alveolar cell hyperplasia (Sherwin, Dibble et al. t972).<br />
Gas mixtures <strong>of</strong> NOz and NO, each in concentrations <strong>of</strong> less than lppm, have been shown to<br />
reduce peak expiratory flows and diffusion capacity in beagles (Bloch, kwis et al. 1972).ln<br />
studies using lower concentrations (similar to pollution values) the damage in the airway was<br />
related to duration <strong>of</strong> exposure (Maejima, Suzuki et al. 1992), but when exposed to high doses<br />
the dose was more damaging than the duration <strong>of</strong> exposure (Lehnert, Archuleta et al. 1994).<br />
NOz <strong>of</strong> l75ppm has been used in rats to create severe lung damage but avoiding death to<br />
develop an ARDS model for study (Meulenbelt, Dormans et al. 1992). NOz has been<br />
recognised as the product <strong>of</strong> grain fermentation responsible for the syndrome <strong>of</strong> pulmonary<br />
oedema and haemorrhagic bronchiolitis obliterans known as silo fillers disease (Ramirez and<br />
Dowell l97l; Horvath, doPico et al. 1978; Leavey, Dubin et d. 2004).<br />
<strong>The</strong> Occupational Safety and Health Administration guidelines (NIOSH 2005) recommend<br />
that the safety limit <strong>of</strong> mixed nitrogen oxides is below 25ppm. Inhalation at this concentration<br />
may cause immediate pulmonary irritation and higher doses may cause haemorrhagic<br />
r06
pulmonary oedema in days. <strong>The</strong> guideline states that inhaling NO at a dose <strong>of</strong> 25ppm for<br />
eight hours is the maximum safe dose and time limit. <strong>The</strong> level considered an immediate<br />
damage to life and health is estimated at 100 to 150ppm based on older toxicology studies<br />
(Carson, Rosenholtz et al. 1962; Sax 1975). For NOz the recommendation has been altered<br />
down from 5ppm to only lppm (in 1996) for eight hours as the exposure limit (NIOSH 2005).<br />
kvels <strong>of</strong> 10 to 20ppm have demonstrated to be a mild irritant (Patty 1963), levels <strong>of</strong> lOOppm<br />
are dangerous causing haemorrhagic pulmonary oedema (NRC 1985), and 150 to 200ppm<br />
fatal from haemorrhagic pulmonary oedema (Braker and Mossman 1975).<br />
Ozone is a colourless to blue gas with a pungent odour. Again the 'usual' exposure to ozone is<br />
through airway pollution as mentioned in Chapter 2.2, (Anonymous 1995; Anonymous 1996)<br />
where it is one <strong>of</strong> the most toxic elements reaching one hour mean ambient concentrations <strong>of</strong><br />
200-400uglm3 1Rnu, 1,966; Lippmann 1989; van Bree and Last lggT). Shorr term ozone<br />
exposure causes reduction in lung function, increased airway hyperactivity, increased airway<br />
inflammation, increased respiratory symptoms and hospital admissions (van Bree, Marra et al.<br />
1995; Nikasinovic, Momas et al. 2003). Exposure to long term elevated ozone levels is again<br />
associated with reduced lung function, increased respiratory symptoms, exacerbations <strong>of</strong><br />
asthma and airway cell and tissue changes (van Bree, Mara et al. 1995; Anderson, Ponce de<br />
I-eon et al. 1998). At the tissue level, ozone with its high reactivity is usually consumed as it<br />
passes through the first layer <strong>of</strong> tissue it contacts at the airway/air interface or lung/air<br />
interface (Pryor 1992; Pryor, Squadrito et al. 1995). Since the lung lining fluid is 0.1 to 20<br />
microns thick, in the thinnest parts it can react directly with cells and it has been shown, for<br />
example, to act with the type II macrophages disrupting surfactant activity. <strong>The</strong> majority <strong>of</strong><br />
the interface area has thicker lining fluid and ozone is transformed into other reactive species<br />
such as aldehyde and hydrogen peroxide, and these go on to cause the airway damage. This<br />
causes epithelial disruption and increased permeability with an increase in inflammatory cells.<br />
<strong>The</strong> disruption <strong>of</strong> cell membranes also results in a release <strong>of</strong> cytokine, cyclo-oxygenase and<br />
liopoxygenase products. In studies looking at toxicity, differences have been demonstrated<br />
between species with regards to dose and length <strong>of</strong> exposure that results in damage (Dormans,<br />
van Bree et al. 1999). Inhaled ozone is also used in animals to give a lung fibrosis model for<br />
study (Cross, Hesterberg et al. 1981; Yu, Song et al. 2001). In direct comparison <strong>of</strong> toxicity,<br />
ozone was found to cause four times as much damage to the airways as NO2 (Chang, Mercer<br />
et al. 1988) and be ten times more toxic to macrophages (Rietjens, Poelen et al. 1986).<br />
<strong>The</strong> Occupational Safety and Health administration guidelines recommended exposure limit<br />
for ozone is 0.lppm (0.2mglm3) (NIOSH 2005). <strong>The</strong> immediate danger to life and health level<br />
to7
is set at 5ppm (AIHA 1966) based on pulmonary oedema that developed in welders who had a<br />
severe acute exposure to an estimated 9ppm <strong>of</strong> ozone (plus other pollutants) (Giel, Kleinfeld<br />
et al. t957). Fifteen to 20ppm is lethal to animals in two hours, exposure to 50ppm for one<br />
hour "would likely prove fatal to humans" (King 1963).<br />
4.7 Chapter summary<br />
This chapter has reviewed the possible compounds and methods that could be used to<br />
determine the level <strong>of</strong> NO based around the known pathway <strong>of</strong> formation from L-arginine to<br />
L-citrulline and NO, and the nitrogen compounds. <strong>The</strong> chemiluminescence technique was the<br />
most appropriate for use in measuring exhaled NO in humans. This analysis is based on the<br />
reaction <strong>of</strong> NO with ozone which produces light, and the light intensity has been shown to be<br />
directly proportional to the concentration <strong>of</strong> NO in the sample. <strong>The</strong> chemiluminscence<br />
technique appeared to be both appropriately sensitive and specific for NO for this purpose.<br />
<strong>The</strong> chemiluminescent NO analysers available at that time had been developed to measure<br />
NO in air pollution. <strong>The</strong> job now was to assess their use and examine what technical<br />
alterations were required to enable measurement <strong>of</strong> NO in exhaled air in humans in a reliable<br />
and reproducible way. This was needed before discussion as to what these levels meant, and<br />
before a trial <strong>of</strong> measurement in children. Initial testing showed that the response time <strong>of</strong> the<br />
analyser was too slow for our purpose initially, but a rerouting <strong>of</strong> the signal to a chart recorder<br />
decreased the response time and meant that it was practical for use for the studies that follow.<br />
Finally in this chapter, I have reviewed the toxicity <strong>of</strong> NO, NOz and ozone. Although I was<br />
only going to be measuring NO in exhalate and therefore it was unlikely to reach high levels,<br />
I was using NO calibration gases. NOz and ozone are the two gases created by this technique<br />
so I wanted to also assess their toxicity, although I was planning to remove them immediately<br />
by having them 'scrubbed' with a soda lime column and a charcoal column respectively.<br />
<strong>The</strong> next chapter examines the chemiluminescent analyser Model 2107 @asibi<br />
Environmental Corporation, Glendale, California, USA) in more detail, and the<br />
commencement <strong>of</strong> the machine and subject methodology studies. What was known about<br />
exhaled NO levels at the time <strong>of</strong> cornmencement <strong>of</strong> these studies in 1995 will also be<br />
reviewed here.<br />
108
NB: <strong>The</strong> review presented in this chapter and the start <strong>of</strong> the machine methodology presented<br />
at the beginning <strong>of</strong> the next chapterformed the basis <strong>of</strong> the publication:<br />
Byrnes CA, Bush A and Shineboume EA "Methods to Measure Expiratory Nitric Ortde in<br />
People" in "A Volume <strong>of</strong> Methods in Enqmology" edited by Lester Packeter Academic press<br />
Inc. 1996.<br />
109
5.1<br />
Chapter 5: Methodological assessment <strong>of</strong> the chemiluminescence analyser<br />
Introduction<br />
<strong>The</strong> previous chapter reviewed the methods available to measure NO in biological systems<br />
and determined that the most appropriate method for measuring NO in exhaled air using a<br />
chemiluminescence analyser, originally developed to assess NO pollution levels. <strong>The</strong><br />
technical aspects <strong>of</strong> the chemiluminescence analysers were reviewed and the direct correlation<br />
between the intensity <strong>of</strong> light measured and the concentration <strong>of</strong> NO described. <strong>The</strong><br />
instrument that I ultimately used was the Dasibi Environmental Corporation 'Model 2107'.In<br />
this chapter the characteristics <strong>of</strong> this analyser will be reviewed, as well as the other<br />
equipment. Connections between a number <strong>of</strong> analysers were developed to enable<br />
simultaneous measurement <strong>of</strong> NO, COz, flow and pressure. I will also mention the other<br />
people involved in the work and the role each person undertook.<br />
5.2 <strong>The</strong> equipment and personnel<br />
5.2.1 Chemilurninescence Analyser'Model 2107'<br />
<strong>The</strong> chemiluminescence analyser Model 2107 (Dasibi Environmental Corporation, Glendale,<br />
California, USA; local supplier Quantitech Ltd, Unit 3 Wolverton Rd, Old Wolverton, Mlton<br />
Keynes, UK) was designed for measuring NO concentrations within a 2-4000pbb range in a<br />
continuous ambient air sample and operated in ambient temperatures <strong>of</strong> 5400 C and humidity<br />
<strong>of</strong> O-9SVo.It had been adapted for online recording with a sampling rate at 240 mls/min and<br />
had a highly stabilised measurement capability (drift without auto zero over 24 hours <strong>of</strong><br />
lppb). <strong>The</strong> original response time <strong>of</strong> this machine was long at 90 seconds, too long for the<br />
purpose for which it was now required. Modification <strong>of</strong> the circuitry and the sending <strong>of</strong> the<br />
pre-computer analogue signal directly to the chart recorder decreased the response time to 6.4<br />
seconds (see below). <strong>The</strong> estimates <strong>of</strong> NO concentrations were decreased by lo-l1Vo at IOOVo<br />
humidity; however NO pre-drying <strong>of</strong> the expirate was not thought to be necessary (this may<br />
have been considered differently if the experiments were conducted in areas <strong>of</strong> high humidity<br />
such as within New Zealand). I did use a water absorber when doing the reservoir set <strong>of</strong><br />
experiments only (see Section 6.4). This machine operated on the same principles as<br />
described in the last chapter. <strong>The</strong> gaseous specimen was drawn by the vacuum pump into a<br />
reaction chamber where the ozone was generated internally by an electrical discharger. <strong>The</strong><br />
NO and 03 wore mixed in front <strong>of</strong> a red-sensitive photomultiplier tube, the light emission <strong>of</strong><br />
this reaction was measured at 660 to 900nm in the red sensitive range, and the signal<br />
110
amplified and detected as a current. As mentioned in the previous chapter, the dark current <strong>of</strong><br />
the photomultiplier can be affected by changes in temperature and this model had an inbuilt<br />
cooler that kept the photomultiplier temperature at -200C to improve the signaUnoise ratio.<br />
<strong>The</strong> machine itself should ideally also be kept in a temperature controlled room (as was<br />
suggested in the machine manual). In our case it was placed in a small air conditioned room<br />
within the hospital away from any other equipment that potentially generated heat - though<br />
there was one window into the room which resulted in some temperature change. <strong>The</strong> NO<br />
analyser was re-serviced just prior to our experiments taking place.<br />
<strong>The</strong> NO analyser was calibrated with azero and two known NO gas concentrations before and<br />
after every subject during the methodological studies and between every two subjects when<br />
later studies in children were being undertaken. Two <strong>of</strong> the gases used were commercial<br />
preparations <strong>of</strong> NO made for calibration purposes by BOC Gases (BOC Gases, 10 Priestley<br />
Rd, Surrey Research Park, Guildford, Surrey, UK) and as the studies progressed included<br />
concentrations <strong>of</strong> 37ppb, 55ppb lO3ppb, l lOppb and I lSppb with the balance being nitrogen<br />
(Nz). Calibration gases at these low levels were extremely difficult to get at the<br />
commencement <strong>of</strong> these studies as these gas mixtures had no known use other than<br />
calibration, and this was not widely used at this time. <strong>The</strong> third gas was a zero calibration and<br />
this was prepared by passing room air through the ozone generator <strong>of</strong> the chemiluminescence<br />
analyser, which converted all NOx to NOz. This was then passed through a soda lime and<br />
activated charcoal column to remove the NOz and Ot respectively and then passed direct into<br />
the analyser. <strong>The</strong> resulting NOz and 03 from the experiment were removed in the same<br />
manner by passing through the soda lime column and the charcoal column. To minimise NO<br />
loss by absorption or interaction with other surfaces (see Section 5.4), teflon tubing (4mm<br />
extemal diameter) was used as the most inert substance to connect the machinery and to<br />
sample from the subject.<br />
5.2.2 Capnograph, pressure transducer, Jlow meter and chart recorder<br />
In addition to measuring NO itself, we wanted to be able to measure other parameters. At the<br />
time, the early publications available on exhaled NO from the lung, which will be reviewed in<br />
the next chapter (see Section 6.3), seemed to show a consistency in the trends found in NO<br />
results in different subject groups but the absolute levels <strong>of</strong> NO reported between the<br />
investigators were very different. This suggested that some <strong>of</strong> the measurement techniques<br />
were altering the NO results recorded. <strong>The</strong>re was also discussion in the literature about where<br />
the measured NO from exhalation was anatomically being generated. In view <strong>of</strong> this, we<br />
lll
wanted to compare the patterns <strong>of</strong> exhaled NO with COz, the latter being largely <strong>of</strong> alveolar<br />
origin. For this purpose, I elected to measure expiratory COz, flow and pressure in addition to<br />
the expiratory NO levels which meant developing a number <strong>of</strong> monitors connected in parallel<br />
that could operate simultaneously. <strong>The</strong> equipment used, as described below, was all<br />
equipment that was able to be 'seconded' from the adult lung function laboratory and put<br />
together by myself and one <strong>of</strong> the biomedical engineers (Ms Carolyn Busst, see below) and<br />
the ultimate format is demonstrated in Figure 5.1.<br />
Figure 5.1: Schematic diagram <strong>of</strong> how the analysers were placed<br />
This is a schematic diagram <strong>of</strong> the analysers and connections required to measure nitric oxide, carbon<br />
dioxide, mouth pressure and flow. <strong>The</strong> signals from each <strong>of</strong> the 4 machines went directly to, and were<br />
displayed on, a Linseis 4 channel pen chart recorder (Linseis Limited, Cambridge, United Kingdom)<br />
with each signal denoted by a different colour.<br />
<strong>The</strong> COz levels were measured with a Morgan Capnograph (PK Morgan, Kent, United<br />
Kingdom) which sampled at2O0 mls/min-I. Calibration <strong>of</strong> COz was done using ^zero and gas<br />
cylinder <strong>of</strong> known COz concentration at 5.9Vo and later 5.7Vo also provided by BOC gases<br />
(address given above). <strong>The</strong> mouth pressure was measured using a Medex Straingauge<br />
pressure transducer (Medex Medical Incorporated, Rossendale, United Kingdom) which was<br />
connected at right angles to the flow via a connector in the mouthpiece. This was then<br />
directed through a Galtec pressure amplifier (Galtec Limited, Isle <strong>of</strong> Skye, United Kingdom)<br />
which measured the pressure in mmHg and showed a linear and stable response. <strong>The</strong> flow<br />
was measured via a flow meter to a low flow pneumotachograph (CT Platon Limited, Jays<br />
t12
Close, Viables, Basingstoke, United Kingdom) and a GM electrospirometer (GM Instruments<br />
Limited, Kilwinning, United Kingdom) then gave an electrical output that could be sent to the<br />
chart recorder. <strong>The</strong> pneumotachograph was initially calibrated by passing flow from an air<br />
cylinder set at known flow rates (100, 250, 500, 750 and l0o0mls/min) and was linear over<br />
this range but then seemed to read slightly but consistently high after 1l0omls/min. In initial<br />
self trials (myself and Carolyn Busst) the response from human exhalation seemed unsteady<br />
or 'noisy'. This appeared to be the effect <strong>of</strong> the small but repeated voluntary alterations in<br />
expiratory flow by the subject (one <strong>of</strong> us) trying to maintain a flow without adequate<br />
feedback. It disappeared when the steady flow <strong>of</strong> compressed air was put through the<br />
rotameter and the pneumotachograph at designated flow rates. In view <strong>of</strong> this, we then added<br />
the rotameter as part <strong>of</strong> the routine exhalation. Subsequently, all the subjects were directed to<br />
watch the rotameter during exhalation and aim to maintain a set flow rate by keeping a<br />
'bobbin' at a set number on the upright scale. This improved the steady quality <strong>of</strong> the signal.<br />
<strong>The</strong> rotameter was a Platon glass tube c6 with stainless steel float 6D (Flowmeter Model<br />
GTV - CT Platon Ltd, address above).<br />
Figure 5.2: An example <strong>of</strong> tracing from the testing<br />
sec9-<br />
-?a.S<br />
<strong>The</strong> first thing to note is that the chart record is read from right to left which is how it was generated<br />
from the chart recorder. Note the <strong>of</strong>fset <strong>of</strong> the traces from the different analysers which were displayed<br />
n3<br />
- 4?o<br />
!4sr,J<br />
I<br />
+415<br />
i<br />
-g,2<br />
it,+<br />
j<br />
i56<br />
-48<br />
i l(-,t l ;4.o<br />
i-_<br />
ieo<br />
I<br />
I<br />
i 01.5<br />
i<br />
"45i<br />
'1- ?L5<br />
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ito<br />
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- lE'6<br />
-,t5.5<br />
-12,+<br />
- t,a<br />
+C.L<br />
it'l<br />
o'<br />
;rn<br />
; k''l - tf l3t<br />
No(ppb; ; o2%)<br />
1cm=22.5 1cm=o.9<br />
-o :o<br />
MP(mmHg)<br />
lcm=3. 1<br />
o
in different colours that became the standard: NO in green, COz in red, and mouth pressure in blue. As<br />
this is an example from the direct method <strong>of</strong> analysis, flow is not denoted. <strong>The</strong> measurements <strong>of</strong> each<br />
parameter were calculated from the calibration markings (see Chapter 6).<br />
As can be seen in Figure 5.2, the NO rose to a plateau which <strong>of</strong>ten had additional small<br />
deviations, the COz continued to rise to a peak throughout the exhalation, the mouth pressure<br />
(and flow with the t-piece measurements) were under voluntary control and therefore less<br />
exact plateaux markings were seen and the lines <strong>of</strong> measurement were drawn through the<br />
middle to determine that they had been appropriately close to requested levels. Note the <strong>of</strong>fset<br />
<strong>of</strong> the traces from the different analysers which were displayed in different colours that<br />
became the standard: NO in green, COz in red, and mouth pressure in blue. As this is an<br />
example form the direct method <strong>of</strong> analysis, flow is not denoted but when measured, as<br />
during the t-piece sampling technique, was displayed as a brown trace. <strong>The</strong> measurements <strong>of</strong><br />
each parameter were calculated from the calibration markings.<br />
We (Carolyn Busst and myself) checked the reported sampling rates <strong>of</strong> the NO and COz<br />
analysers. As mentioned, the sampling rate was determined by the vacuum pump within the<br />
NO analyser and had a fixed rate. Similarly, the sampling rate <strong>of</strong> the CO2 analyser was also<br />
determined by the vacuum pump which could apparently be adjusted by pressing the<br />
calibration button on the front <strong>of</strong> the analyser panel and the rate changed by pressing the up<br />
and down arrow keys. We left the sampling rate at the setting it was on when we received it<br />
from rhe Adult Physiology Laboratory (Royal Brompton Hospital). This was in the middle <strong>of</strong><br />
the range suggested by the manufacturers and was used for all assessments. <strong>The</strong> check <strong>of</strong> the<br />
machines' sampling was repeated a number <strong>of</strong> times prior to development <strong>of</strong> the protocol for<br />
the experiments and again between the methodological experiments conducted with adult<br />
subjects and commencement <strong>of</strong> the studies on paediatric subjects. We listed what was to<br />
become our standard procedure as the following steps:<br />
o Connect the outlet line from the mouthpiece t-piece connection to the lower port <strong>of</strong> the<br />
rotarmeter.<br />
o Turn the rotameter needle valve <strong>of</strong>f to ensure that the room air cannot be sampled via<br />
the rotameter.<br />
o Disconnect the pneumotachograph from the rotameter.<br />
o Run the paper to record zero sampling.<br />
o Connect the outlet side <strong>of</strong> the pneumotachograph to the inlet <strong>of</strong> the mouth piece.<br />
tt4
o<br />
o<br />
Ensure there is no leak in the connections and room air will now be drawn through the<br />
pneumotachograph by the NO analyser and capnograph thus measuring their sampling<br />
rates.<br />
Run the chart recorder to mark the level <strong>of</strong> the combined sampling rates.<br />
Label the chart record.<br />
Reconnect the pneumotachograph to the upper port <strong>of</strong> the rotameter.<br />
5.2.3 Personnel<br />
Adjustments to the original NO analyser circuitry and the sending <strong>of</strong> the pre-compurer<br />
analogue signal directly to the chart recorder were made with the assistance <strong>of</strong> the Biomedical<br />
Engineering Department, Royal Brompton Hospital (Mr Ron l-ogan-Sinclair [later <strong>of</strong> logan<br />
Research Ltd, Rochester, Kent, UK] and Ms Carolyn Busst). <strong>The</strong> connections and adaptation<br />
<strong>of</strong> the equipment to do all the measurements simultaneously and the experiments on assessing<br />
machine capability were done by myself and Ms Carolyn Busst. I designed and conducted the<br />
methodological experiments on the healthy adult subjects. I also designed the studies<br />
involving normal and asthmatic children as subjects. Following ethical approval from the<br />
Royal Brompton Hospital Ethics Committee, Dr Seinka Dinarevic and I gave presentations to<br />
the local primary school and, with individual signed parental or caregiver consent, we<br />
enrolled children the school. I also enrolled all the asthmatic subjects from the weekly<br />
paediatric asthma and/or the weekly paediatric general respiratory clinic at the Royal<br />
Brompton Hospital in which I was working. Dr Seinka Dinarevic helped transporting the<br />
children to and from the local school, and we collected the consent and the questionnaire data,<br />
did the clinical examination, measured height and weight, performed the lung function and<br />
exhaled NO testing on all the children. <strong>The</strong> manual calculation <strong>of</strong> the levels for each<br />
parameter from the chart recorder using the calibration measurements was completed by us<br />
both' I entered the data into a statistical analysis programme (Statistical Products and Services<br />
Solutions, SPSS Inc, Chicago, USA - package U 7.0), while Dr Seinka Dinarevic double<br />
checked the entries. We shared the drafting <strong>of</strong> the paediatric paper, while I wrote the other<br />
papers in conjunction with contributory editing and suggestions from the other named authors<br />
on each. I remained the key driver and contact for all this work. <strong>The</strong> same equipment was<br />
used for all the following experiments and the work was carried out in the Paediatric<br />
Department at the Royal Brompton Hospital, South Kensington, London, England3. <strong>The</strong> main<br />
supervisor for this work was Pr<strong>of</strong>essor Andrew Bush. In addition, Pr<strong>of</strong>essor Peter Barnes was<br />
eNow the 'Royal Brompton & Harefield National Health Trust, South Kensington, London NW3 6Np, U.K.<br />
115
an advisor for this work and main supervisor for other research done at the time (publications<br />
listed as Appendix 1), and Dr Elliot Shinebourne also acted as advisor, particularly in the<br />
early stages.<br />
5.3 Direct versus reservoir measurement<br />
<strong>The</strong> concept <strong>of</strong> the proposed methodological experiments was to have a subject exhale into<br />
teflon tubing connected to NO, CO2, flow and mouth pressure meters allowing measurement<br />
<strong>of</strong> each. It was established that all the equipment could come directly <strong>of</strong>f the mouth piece,<br />
with the exception <strong>of</strong> flow connector. This was due to the necessity <strong>of</strong> the addition <strong>of</strong> a<br />
rotameter to provide a visual feedback guide for the subjects regarding expiratory flow and<br />
added a level <strong>of</strong> complexity to the system. In view <strong>of</strong> this, I elected to do a series <strong>of</strong><br />
experiments in which one set <strong>of</strong> exhalations was connected directly to the NO analyser,<br />
pressure and COz monitors (known as the 'direct method') and a second set <strong>of</strong> exhalations<br />
where a t-piece system enabled the additional measurement <strong>of</strong> flow (known as the 't-piece<br />
method' or 't-piece sampling system'). A comparison <strong>of</strong> the two techniques is shown<br />
diagrammatically in the next chapter in Figure 6.5. Below is a photograph <strong>of</strong> one <strong>of</strong> the<br />
children performing the procedure with the analysers labelled in Figure 5.3. Unfortunately I<br />
did not take any photographs <strong>of</strong> the subjects during the methodological experiments.<br />
Figure 5.3: Photographs <strong>of</strong> one <strong>of</strong> the children performing the exhalation
<strong>The</strong> photographs are taken from behind (a) and from the right side (b) <strong>of</strong> one <strong>of</strong> the paediatric subjects<br />
'Jonathon'that was testing the procedure (used with permission) as he performs a single exhalation<br />
into the mouthpiece. I have labelled the connections and analysers as seen below (not all visible in<br />
both photographs):<br />
1. Mouth piece.<br />
2. Connector.<br />
3. NO analyser (Model2107, Dasibi Corporation).<br />
4. CO2 analyser (Morgan capnograph).<br />
5. Low flow pneumotachograph (CT Platon Limited).<br />
6. Mouth pressure analyser (Medex Straingauge pressure transducer).<br />
7. Galtec pressure amplifier (Galtec Limited).<br />
8. GM electrospirometer (GM Instruments Limited).<br />
9. Four channel pen chart recorder (Linseis Limited).<br />
10. Timing device.<br />
11. NO zero calibration column.<br />
12. Calibration gases (BOC gases Ltd).<br />
13. <strong>The</strong> f irst <strong>of</strong> the pages listing instructions on the wall.<br />
5.4 Assessments <strong>of</strong> the analysers<br />
<strong>The</strong> response time <strong>of</strong> each analyser had to be determined to enable direct comparisons. <strong>The</strong><br />
chart recorder recorded continuously at a paper speed <strong>of</strong> 50mm/second and was set so that as<br />
the recording came <strong>of</strong>f, the signals were read from right to left. An oscilloscope was<br />
connected to the system with a three way tap which on turning allowed the test gas to flow<br />
down at the apparatus at a rate comparable to the subjects' expiratory flow rate (500mls/min<br />
was chosen) starting at the subject mouthpiece. <strong>The</strong> output <strong>of</strong> the analyzers was displayed on<br />
the same oscilloscope. <strong>The</strong> measurement <strong>of</strong> the delay time and the response time was then<br />
measured with a stop watch.<br />
rt7
Delay time: the time between turning on the switch and the onset <strong>of</strong> the analyser<br />
signal<br />
Response time: the rise time that from onset <strong>of</strong> signal to 95Vo <strong>of</strong> plateau or a square wave<br />
<strong>The</strong> experiment was repeated 10 times in all with times recorded for each <strong>of</strong> the analysers.<br />
Results are presented in the Table 5.1.<br />
Table 5.1: <strong>The</strong> delay and response time <strong>of</strong> the NO, COz, mouth pressure and flow meter analysers<br />
used<br />
Analyser Delay time Response tlme<br />
NO analyser Model2107 5.9 seconds<br />
(SEM 0.8 s)<br />
CO2 Morgan capnograph 0.11 seconds<br />
(SEM 0.003 s)<br />
Medex Staingauge pressure<br />
transducer (mouth pressure)<br />
SEM = standard error <strong>of</strong> the mean, s = secondsr lrls = milliseconds<br />
This established that the delay time for the NO analyzer at 5.9 seconds (SEM 0.8s), and95Vo<br />
response time at 0.5 seconds (SEM 0.1s) showed the NO analyser responded much more<br />
slowly than the CO2 analyser at a delay time <strong>of</strong> 0.1 I seconds (SEM 0.003s) and 95Vo response<br />
time at 0.41 milliseconds (SEM 2.9ms). It has to be remembered that the delay time was<br />
influenced only by the time that it took for the gases, flow or pressure to get to the analysers<br />
and the time to cornmence registering the change. <strong>The</strong> response time should be a square wave<br />
for 'on/<strong>of</strong>f' phenomena like the pressure and the flow, but could be influenced by other<br />
parameters in the measurement <strong>of</strong> the gases. <strong>The</strong> pattern <strong>of</strong> excretion through the exhaled<br />
breath was unknown for NO, and was known to increase with the duration <strong>of</strong> exhalation for<br />
COz. So while the response time was measured with these two gases, it was the delay time<br />
that was the key factor. In essence it meant that when comparing the NO and COz recordings,<br />
an allowance needed to be made for the difference in time delay <strong>of</strong> the measurements from<br />
each machine and for the 2mm <strong>of</strong>fset <strong>of</strong> the pens on the recorder.<br />
Finally, confirmation <strong>of</strong> the detection <strong>of</strong> the appropriate gases was checked for the two<br />
analysers. Cylinders <strong>of</strong> IOOVo Oz, 5.9Vo COz and 37ppb <strong>of</strong> NO were run through the NO<br />
analyser and the CO2 analyser with only the NO gas detected and the others giving a zero<br />
reading on the NO analyser machine and only the COz gas detected with the others giving a<br />
zero reading on the CO2 analyser machine.<br />
7.3 milliseconds<br />
(SEM 0.2 ms)<br />
Platon flow meter 40.2 milliseconds<br />
(SEM O.8 ms)<br />
118<br />
Seconds<br />
(SEM 0.1 s)<br />
41 milliseconds<br />
(SEM 2.9 ms)<br />
No appreciable delay<br />
No appreciable delay
When planning the protocol, it was obvious that the response time <strong>of</strong> the NO analyser limited<br />
the possible respiratory manoeuvres. I next considered online measurement <strong>of</strong> repeated single<br />
exhalations analogous to lung function testing versus exhaling into a reservoir that was then<br />
fed into the analysers. I briefly explored the use <strong>of</strong> a reservoir, as this had three theoretical<br />
advantages:<br />
o it would allow the measurement <strong>of</strong> a whole exhaled breath, whereas the online<br />
measurement which would control flow may mean that at low flows the subject would<br />
not reach complete exhalation before requiring to take another breath,<br />
o it would allow me as operator to put the collected sample through the machine at a<br />
constant flow instead <strong>of</strong> requiring the individual subject's voluntary but imperfect<br />
control,<br />
o performing tidal breathing over a set time or exhaling into a bag may have been easier<br />
when it came time to measure the children rather than relying on their being able to do<br />
controlled breaths into the analysers.<br />
At the time that I was commencing the experiments, there had been some studies into NO<br />
levels in exhaled air where a reservoir system had been used. <strong>The</strong> problems that had been<br />
detected and needed to be overcome involved No instability -<br />
a topic that has been touched<br />
on repeatedly. Firstly, although in O2-free and haemoglobulin-/ree solutions NO may be<br />
stable, in the presence <strong>of</strong> 02, NO levels rapidly decrease by 50Vo in 8-20 minutes. Secondly,<br />
NO is adsorbed by most plastics giving low and/or variable readings. NO also interacts with<br />
transition metals and with the sulftrydryl groups contained in rubber. Reservoir systems that<br />
had been used were polyvinyl chloride, rubber and fluoroethylene propylene' Preliminary<br />
studies in one group demonstrated that at levels <strong>of</strong> 5-30ppb there was no NO loss in the<br />
polyethylene reservoir at 6 and 12 hours post collection, although they commented that any<br />
loss may have been within the noise <strong>of</strong> the measurement that they were getting at that time <strong>of</strong><br />
10Zo (Schilling, Holzer et al. Igg4). Another group had found that using polyethylene tubing<br />
compared to Teflon tubing resulted in loss <strong>of</strong> NO presumably as it adsorbed to the former,<br />
particularly at higher (>40ppb) levels <strong>of</strong> NO (Kharitonov, logan-Sinclair et al. 1994). One<br />
group with a Douglas bag collection (an anaesthetic bag used to collect exhaled air, usually<br />
made <strong>of</strong> rubber) described no perceivable problems, but two other groups found them<br />
unreliable at higher NO concentrations (Kharitonov, logan-Sinclair et al. 1994)'<br />
Subsequently Mylar foil bags or balloons were used and seem to hold NO levels stable for at<br />
least 48 hours (Steerenberg, Nierkens et al. 2000). However, this research group also<br />
discarded the first part <strong>of</strong> exhalation to get lower respiratory tract samples and found that it<br />
119
led to an increased water content that decreased measured NO levels, though this could be<br />
minimised by exhaling through a water absorber into the reservoir bags (Steerenberg,<br />
Nierkens et al. 2000). NO online measurement correlated well with NO measured via a<br />
balloon technique (mylar foil) when measured immediately during times <strong>of</strong> low air pollution<br />
and high pollution days (van Amsterdam, Verlaan et al. 1999). Another group disagreed and<br />
showed that the online measurement was higher than the mylar bag reservoir results when<br />
both were measured immediately (Silk<strong>of</strong>f, Stevens et al. 1999), though the <strong>of</strong>fline<br />
measurements were similar to the end expiratory online measurements (Jobsis, Schellekens et<br />
al. 2001). NO concentration in the mylar bag was also shown to actually increase at24 and48<br />
hours after collection (Silk<strong>of</strong>f, Stevens et al. 1999). When the first European Respiratory<br />
Society Task Force (ERSTF) recommendations were published regarding the measurement <strong>of</strong><br />
exhaled air, they recommended the online technique <strong>of</strong> measurement (Kharitonov, Alving et<br />
al. 1997).In some initial testing we did, teflon and "wine cask bags" (which are mylar foil<br />
bags) did not seem to absorb NO, but a Douglas bag did seem to absorb NO and the material<br />
<strong>of</strong> the bag became sticky. <strong>The</strong> use <strong>of</strong> teflon or teflon coated tubing to connect the reservoir or<br />
the subject to the analysers appeared to minimise NO loss.<br />
In view <strong>of</strong> the purpose <strong>of</strong> the studies, the online technique seemed more appropriate as it<br />
allowed comparison <strong>of</strong> signals throughout the exhalation and allowed an alteration <strong>of</strong> the test<br />
conditions to investigate how this affected NO results. A slow controlled exhalation from total<br />
lung capacity appeared to be the most satisfactory exhalation for observing the results. This<br />
manoeuvre yielded obvious NO and CO2 traces and most people, including most children in<br />
the age group studied, could be easily trained to perform this correctly. Thus I could discard<br />
the reservoir system for most <strong>of</strong> the studies which I believe excluded another uncertain<br />
element from test measures.<br />
5.5 Other measurements<br />
For all the experiments utilising the healthy adult volunteers and normal and asthmatic<br />
children, a respiratory examination was carried out, and weight (minimal clothing, no shoes)<br />
and height were recorded on a digital Seca scale (Seca 770 medical scales, Seca Ltd, 4802<br />
Glenwood Rd, Brooklyn NYI1234, USA) and a Harpenden stadiometer (Harpenden Portable<br />
Stadiometer, Crosswell, Crymych, Pembrokeshire, SA41 3UF, UK) respectively. Lung<br />
function was measured according to the American Thoracic Society (ATS) criteria (American<br />
Thoracic Society and Association. 1994) on a compact vitalograph (Vitalograph Ltd, Maids<br />
t20
Moreton House, Buckingham, United Kingdom) using the Polgar predictive equation (Polgar<br />
and Promadh at.l97l) from a standing position for all subjects.<br />
5.6 Chapter summary<br />
This chapter has described the equipment, the sequence <strong>of</strong> analysers and the capabilities <strong>of</strong><br />
each that was used for testing in the studies presented in the following chapters (Chapters 6, 7<br />
and 8). Chapter 6 will begin with a brief review <strong>of</strong> what was known about NO in the lung, the<br />
early published articles in the measurement <strong>of</strong> exhaled NO and describe the first <strong>of</strong> five<br />
methodological experiments carried out in healthy adult volunteers'<br />
NB: <strong>The</strong> review and data presented in Chapters 4 and 5 formed<br />
the basis <strong>of</strong> the publication:<br />
Byrnes CA, Bush A and shinebourne EA "Methods to Measure Expiratory Nitric oxide in<br />
People" in "AVolume <strong>of</strong> Methods in Enrymology" edited by l*ster Packeter Academic Press<br />
Inc. 1996.<br />
t2l
Chapter 6: Methodological studies <strong>of</strong> exhaled nitric oxide in healthy adult subjects:<br />
6.1 Introduction<br />
direct versus t-piece testing<br />
So far, I have examined why a marker <strong>of</strong> airway inflammation that was easy to measure,<br />
particularly in terms <strong>of</strong> being non-invasive, would be useful. I have outlined the developing<br />
knowledge on NO as a physiological agent and inflammatory marker. <strong>The</strong> previous chapters<br />
then explored how NO could be measured by chemiluminscence and presented data on<br />
adapting an appropriate analyser with our own initial methodology testing. In this chapter, I<br />
will very briefly review NO in the lung, the literature on exhaled NO available to me at the<br />
beginning <strong>of</strong> my research, and present the methodology experiments themselves. <strong>The</strong> first<br />
experiment, which will be presented in detail in this chapter, was to assess the ability to<br />
measure NO from exhalation in adults and to compare NO, CO2, mouth pressure and flow<br />
traces using two methods; direct to analyser and side arm sampling systems. <strong>The</strong> following<br />
chapter will detail further experiments designed to examine a number <strong>of</strong> technical factors to<br />
assess whether they alter the levels <strong>of</strong> exhaled NO obtained. <strong>The</strong>se investigations were in<br />
response to the newly presented and published literature in this area where, although the<br />
conclusions were similar from different research groups regarding NO results in different<br />
populations, it remained curious that the absolute levels <strong>of</strong> NO being documented were very<br />
different. How the results from these experiments then fit into subsequent research, current<br />
literature and new discoveries are detailed in Chapter 9.<br />
6.2 Nitric oxide and the nitric oxide synthases in the lung<br />
From the literature presented in Chapter 2.2 'Nitric oxide in biological systems', and in<br />
Chapter 3.4 'Nitric oxide synthase isoenzymes', it was clear that NO and the NOS enzymes<br />
were widespread throughout human organ systems. In the lung, NO has four major roles -<br />
vasodilator, bronchodilator, neurotransmitter for the NANC neryes and as an immune and<br />
inflammatory mediator. By the time <strong>of</strong> this research all three is<strong>of</strong>orms <strong>of</strong> the enzyme had been<br />
detected in the human respiratory tract @arnes and Belvisi t993; Jorens, Vermeire et al.<br />
1993; Marsden, Heng et al. 1993; Moncada and Higgs 1993; Singh and Evans 1997).<br />
localisation <strong>of</strong> NOS in lung tissue was demonstrated by immunohistochemical labelling<br />
using both polyclonal and monoclonal NOS antibodies @redt, Hwang et al. 1991; Schmidt,<br />
Gagne et al. 1992; Hamid, Springall et al. 1993; Hattori, Kosuga et al. 1993; Kobzik, Bredt et<br />
al. 1993; Forstermann, Closs et al. 1994; Rengasamy, Xue et al. 1994; Kawai, Bloch et al.<br />
t995; Ambalavanan, Mariani et al. 1999). <strong>The</strong> detection <strong>of</strong> NOS within the respiratory tree<br />
r22
initially appeared somewhat site-specific. Early studies showed NOS presence in bronchial<br />
epithelial cells with little in the respiratory terminal bronchi, smooth muscle cells or alveolar<br />
sacs. However, this changed following stimulation <strong>of</strong> the iNOS enzyme, when there was some<br />
mild uniform labelling <strong>of</strong> epithelium in large cartilaginous airways with iNOS in normal<br />
trachea and main bronchi, but with much grcatet labelling in the more peripheral bronchi'<br />
This was first demonstrated when comparing normal and inflamed rat lung samples that had<br />
been stimulated with intra-tracheal lipopolysaccharide, and in human tissue from four airway<br />
and ten parenchymal specimens obtained from uninvolved areas <strong>of</strong> surgical tumour resections<br />
stimulated with TNF0. While the presence <strong>of</strong> the constitutive forms along the bronchi before<br />
the infective stimulation seemed appropriate given the eNOS and nNOS activity in<br />
physiological regulation, it was uncertain why iNOS should be seen there. <strong>The</strong> researchers<br />
suggested that it may be in response to continual low grade induction <strong>of</strong> iNOS activity in<br />
response to airborne toxins (Hamid, Springall et al. 1993; Kobzik, Bredt et al' 1993)'<br />
Turning from the presence <strong>of</strong> the enzymes to NO itself: when administered exogenously, NO<br />
gas was shown to relax tracheal and airway smooth muscle in vitro taken from different<br />
sources such as bovine trachea, guinea pig trachea and human airway smooth muscle (Jansen'<br />
Drazen et al. 1992). <strong>The</strong> gas also had the capacity to relax tissues that had previously been<br />
constricted with methacholine, histamine or leuktriene Da (Jansen, Drazen et al. 1992; Gaston,<br />
Drazen et al. Igg4). NOS inhibition resulted in exaggerated bronchosconstrictor responses in<br />
animal models (Ricciardolo, Mistretta et al. 1996; Nogami, Umeno et al' 1998; Boer'<br />
Duyvendak et al. 1999) and human specimens (Ricciardolo, Di Maria et al. 1997; Taylor,<br />
McGrath et al. 1998). <strong>The</strong>se responses suggested that NO had a role in modulating basal<br />
airway tone both directly and via the NANC nerves and, as discussed previously in Chapter 2,<br />
is now thought to be one <strong>of</strong> the main mediators for regional airflow and blood flow matching'<br />
<strong>The</strong> reaction products <strong>of</strong> NO such as s-nitrosoglutathione and s-nitrosalbumin also had<br />
bronchodilator effects (Jansen, Drazen et al. lgg2; Asano, Chee et al. 1994; Gaston, Drazen et<br />
al. 1994;Gaston, Drazen et al. 1994; Bannenberg, Xue et al. 1995) with lower levels noted in<br />
asthmatics (Gaston, Sears et al. 1998). In the lung, low concentrations <strong>of</strong> NO caused ainvay<br />
smooth relaxation during bronchospasm (Buga, Gold et al. 1989; Jansen, Drazen et al' 1992;<br />
Gaston, Drazen et al. Igg4). As seen systemicallY, the NO production from the eNOS<br />
isoenzyme was found to maintain vasomotor tone (Stamler, Osborne et al. 1993)' Pulmonary<br />
arterial endothelial cells released NO in response to acetylcholine and bradykinin agonists<br />
which relaxed pulmonary arterial, venous and lymphatic smooth muscle (Crawley, Liu et al'<br />
1990; Dinh-Xuan, Higenbottam et al. 1991). Release <strong>of</strong> NO from endothelial cells in the<br />
123
pulmonary circulation appeared to counteract hypoxic vasoconstriction responses to<br />
catacholamines and prostaglandin F2c, with NO release decreased in chronic hypoxia<br />
(Crawley, Liu et al. 1990). In vivo the NANC nerves were shown to cause vasodilatation and<br />
relaxation <strong>of</strong> animal and human tracheal muscle using NO as the mediator and these were not<br />
inhibited by NOS inhibitors (Stuart-Smith, Bynoe et al. 1994; Baba, Yoshida et al. 1998;<br />
Sipahi, Ercan et al. 1998). However, human airway specimens suggested this NO mediator<br />
relaxation via the NANC neryes was more important in the response <strong>of</strong> distal rather than<br />
proximal airways (Ellis and Undem 1992).In addition, the NO-dependent NANC dilatation <strong>of</strong><br />
human bronchi was reduced in transplanted tissue and in specimens from patients with CF<br />
@elvisi, Barnes et al. 1995; Belvisi, Ward et al. 1995). Nitrosothiols <strong>of</strong> low molecular weight,<br />
such as S-nitrosoglutathione, and NOz were also detected in alveolar fluid from normal<br />
individuals at concentrations sufficient to suggest regulation <strong>of</strong> basal airway tone @arnes<br />
1993; Gaston, Reilly et al. 1993).<br />
<strong>The</strong> NOS enzymes were considered to have a role in mucociliary clearance, and even now<br />
this remains uncertain as the precise regulation <strong>of</strong> ciliary beat frequency is still unclear.<br />
Similar to NOS activity, ciliary beat frequency is increased by a rise in intracellular calcium<br />
with possible intracellular mechanisms involving cAMP, calmodulin, and inositol 1,4,5-<br />
triphosphate. Increases <strong>of</strong> NO have been detected when ciliary beat increases, although no<br />
consistent correlation has been shown between NO levels and cilia activity (Wanner, Salathe<br />
et al. 1996; Dirksen 1998; Yeates 1998; Uzlaner and Priel 1999). Ciliated cells have been<br />
shown to have increased NO production and increased beat frequency after treatment with L<br />
arginine (Li, Shirakami et al. 2000) and L- NMMA caused a 4OVo decrease in ciliary beat<br />
frequency lasting 60 minutes which was reversed with L-arginine administration (Jain,<br />
Rubinstein et al. 1993).<br />
Finally NOS activation in macrophages, neutrophils and mast cells exerts bactericidal and<br />
fungicidal effects primarily through the oxygen superoxide formation <strong>of</strong> the NO products<br />
such as perioxynitrite as covered in Chapters 2.3.4 and 3.4.2. <strong>The</strong>se are important in host<br />
defence but in excess can have similady detrimental effects on the normal cells. Higher<br />
concentrations <strong>of</strong> NO in combination with oxidant generation leads to inflammation and<br />
oedema (Beckman, Beckman et al. 1990; Mulligan, Hevel et al. 1991; Mulligan, Warren et al.<br />
L992) and these potent oxidants deplete glutathione, uric acid and ascorbate in lung lining<br />
fluid (Williams, Rhoades et al. I97l; Kelly, Shah et al. t997). Exposure <strong>of</strong> alveolar type II<br />
cells particularly to perioxynitrite leads to pr<strong>of</strong>ound inhibition <strong>of</strong> surfactant synthesis (Gaston,<br />
124
Drazen et al. 1994), and the reactions can form potentially carcinogenic nitrosamines such a<br />
NzOg and NzO+ (Miwa, Stuehr et al. 1987).<br />
6.3 <strong>The</strong> need for methodological experiments<br />
In a key development, NO was then detected in exhaled air (Gustafsson, Irone et al. 1991;<br />
Archer 1993;[rone, Gustafsson et al. 1994). In 1991 Gustafsson and I-eone first showed that<br />
NO could be detected in the exhaled air <strong>of</strong> tracheostomized animals and normal humans<br />
(Gustafsson, Irone et al. 1991). Exhaled No was then measured in single and tidal breathing<br />
in eight adult subjects giving levels between 8.3 to 20.3ppb (Borland, Cox et al' 1993)'<br />
Exhaled NO was measured at a mean peak <strong>of</strong> 3.25ppb in ten subjects with normal breathing,<br />
increasing to 100.25ppb with a sixty second breath hold prior to exhalation, and decreasing to<br />
4.?5ppb with hyperventilation. During exercise, the mean peak levels in five subjects dropped<br />
from gppb to 5-6ppb with 50 and 100 watt exercise on an ergometer cycle, although the total<br />
NO excretion (NO concentration times number <strong>of</strong> breaths) increased (Persson, Wiklund et al'<br />
1993). It was shown to be increased in normal subjects during a time <strong>of</strong> upper respiratory tract<br />
infection to a mean peak <strong>of</strong> 315ppb during symptoms and reducing to a mean peak <strong>of</strong> 87ppb<br />
three weeks later during recovery (Kharitonov, Yates et al. 1995). NO was shown in three<br />
studies at this time to be lower in chronic smokers, with Persson et al measuring a mean in<br />
mixed exhaled air <strong>of</strong> 3.9ppb in 6 smokers compared to their 20 normal subjects at 8.4 ppb<br />
(persson, Zetterstrom et al. 1994). Kharitonov et al reporting a lower mean peak <strong>of</strong> 42ppb in<br />
4l smokers compared to 88ppb in 73 nonsmokers in single exhalations (Kharitonov, Robbins<br />
et al. 1995). Schilling et al documenting a mean <strong>of</strong> l6ppb in 12 smoking females versus<br />
2lppb in 2l non-smoking females, and a mean <strong>of</strong> 15ppb in 24 smoking males versus l9ppb in<br />
24 non-smoking males by using a reservoir method to collect tidal breathing (Schilling'<br />
Holzer et al. 1994). In this paper Schilling et al also demonstrated a reduction <strong>of</strong> exhaled NO<br />
in ten hypertensive patients to a mean <strong>of</strong> 13.7ppb (Schilling, Holzer et al. 1994). One study<br />
reported varying levels <strong>of</strong> exhaled NO throughout the menstrual cycle in seven women from a<br />
peak exhaled No at 150ppb midcycle to a low <strong>of</strong> 59ppb during menstruation (Kharitonov'<br />
logan-Sinclair et al. L994). As by far the greatest amounts <strong>of</strong> NO in vitro were shown to be<br />
produced from iNOS stimulation, (Nathan 1992; Barnes and Belvisi 1993), it had been<br />
hypothesised to be a measure <strong>of</strong> airway inflammation (Barnes t993; Barnes and Kharitonov<br />
1996). This seemed to be confirmed when higher levels were found in animal asthma models<br />
when an exacerbation was induced (Persson and Gustafsson 1993; Endo, Uchido et al. 1995)'<br />
and then in adult asthmatic subjects compared to normal subjects in a number <strong>of</strong> early studies'<br />
Initially Kharitinov et al found levels <strong>of</strong> 283ppb compared to controls at 80.2ppb, Persson et<br />
r25
al found levels <strong>of</strong> 10.3ppb compared to controls at 8.4ppb, Massaro et al found levels <strong>of</strong><br />
13.2ppb compared to controls at 4.7ppb, and Robbins et al found levels <strong>of</strong> 174ppb compared<br />
to controls <strong>of</strong> 105.5ppb (Kharitonov, Yates et al. 1994; Persson, Zetterstrom et al. 1994;<br />
Massaro, Mehta et al. 1996; Robbins, Floreani et al. 1996). Alving et al demonstrated no<br />
overlap between their control group with a range <strong>of</strong> 5-16ppb and their asthmatic group who<br />
had a range measured at 2I-3Ippb (Alving, Weitzberg et al. 1993). Massaro demonstrated<br />
that the NO levels were even higher during a period <strong>of</strong> acute asthma in seven patients<br />
requiring emergency department treatment with a reduction <strong>of</strong> NO beginning by 48 hours<br />
(Massaro, Gaston et al. 1995) and Kharitinov et al showed a reduction over three weeks in<br />
eleven asthmatic patients who commenced on IHCS therapy. However, as can be seen above,<br />
the absolute mean concentrations <strong>of</strong> exhaled NO obtained by these separate workers in similar<br />
patient groups and normal subjects using similar techniques appeared so disparate as to be<br />
confusing.<br />
In addition, the regional source <strong>of</strong> NO within the respiratory tract was debated. Initially<br />
Borland et al suggested that the NO measured in exhaled air from a single full exhalation and<br />
during tidal breathing was <strong>of</strong> alveolar origin like COz (Borland, Cox et al. 1993). Persson et al<br />
then suggested that NO was formed preferentially in the small airways such as the terminal<br />
and respiratory bronchioles (Persson, Wiklund et al. 1993). By comparing the concentrations<br />
<strong>of</strong> NO exhaled during tidal breathing through either nose or mouth, Alving et al suggested<br />
that the major contribution came from the nasal space with a minor addition from the lower<br />
airways (Alving, Weitzberg et al. 1993). Lundberg et al demonstrated a decreasing<br />
concentration <strong>of</strong> exhaled NO when sampling progressively down the respiratory tract at the<br />
nose, mouth and, in four tracheotomised patients, below the vocal cords (Lundberg, Farkas-<br />
Szallasi et al. 1995).<br />
Another goup also looked at exhaled concentrations <strong>of</strong> NO in five adults with tracheostomy<br />
during spontaneous breathing where oral NO concentrations were 9 - 25.7ppb while via the<br />
tracheostomy this dropped to l.l - 6.5ppb. Eleven patients admitted for minor abdominal<br />
surgery had similar decreases in NO levels measured under anaesthesia from nasal (mean<br />
54ppb), oral (mean l3ppb) and following intubation (mean 1.3ppb, which was close to the<br />
lower detection limit <strong>of</strong> their analyser). Seven patients intubated and ventilated in intensive<br />
care with multiple diagnoses all had NO concentration levels close to the detection limit <strong>of</strong> the<br />
analyser (range <strong>of</strong> 0.0 - 1.3ppb) (Schedin, Frostell et al. 1995). By isolating the nasal passages<br />
from the rest <strong>of</strong> the respiratory tract with voluntary closure <strong>of</strong> the s<strong>of</strong>t palate, Kimberly et al<br />
showed the release <strong>of</strong> NO in the nasal passages was approximately seven times greater than<br />
126
the rest <strong>of</strong> the respiratory tracr (Kimberly, Nejadnik et al. 1996). Higher levels still were<br />
demonstrated in the paranasal sinuses with an age-dependent increase in keeping with sinus<br />
pneumatisation (Kimberly, Nejadnik et al. 1996). It was postulated that the sinus production<br />
<strong>of</strong> NO diffusing into the nasal spaces gave the high levels <strong>of</strong> NO then measured in the nasal<br />
cavity. Gerlach et al measured NO concentrations in the nasopharynx and trachea showing<br />
higher levels during inspiration than expiration (Gerlach, Rossaint et al. 1993). This led to the<br />
suggestion that there may be absorption <strong>of</strong> NO by the lower respiratory tract (giving a concept<br />
<strong>of</strong> auto-inhalation). However they could not exclude an exhaled component coming from<br />
pulmonary synthesis <strong>of</strong> NO.<br />
<strong>The</strong> variation in NO levels obtained made it difficult to comparc results presented by different<br />
groups (see Table 6.1).<br />
Table 6.1: <strong>The</strong> published results <strong>of</strong> exhaled nitric oxide in adults<br />
Authors and PaPer Subject numbers Method <strong>of</strong><br />
measurement<br />
Borland et al, 1993<br />
(Borland, Cox et al.<br />
1 ss3)<br />
Schedin et al, 1995<br />
(Schedin, Frostellet<br />
al. 1995)<br />
Gerlach et al 1994<br />
(Gerlach, Rossaint et<br />
al. 1994)<br />
Kimberly et al, 1996<br />
(Kimberly, Nejadnik et<br />
al. 1996)<br />
I controls<br />
5 controls<br />
11 conlrols<br />
10 controls<br />
10 smokers<br />
5 controls<br />
8 controls<br />
single exhalation<br />
tidal breathing<br />
mouth breathing<br />
tracheostomy breathing<br />
tidal breathing via nose<br />
tidalbreathing via<br />
mouth<br />
following intubation<br />
Soontaneous<br />
breathing:<br />
trachea<br />
controlled ventilation<br />
trachea<br />
controlled ventilation<br />
Bronchoscooe<br />
sampling:<br />
at epiglottis, nose<br />
breathing<br />
at epiglottis, mouth<br />
breathing<br />
in trachea, nose<br />
breathing<br />
in trachea, mouth<br />
breathing<br />
Tidal breathino over 2<br />
minutes:<br />
during nasalbreathing<br />
during oral breathing<br />
Sinole exhalations:<br />
exhalation<br />
after 30s breath-hold<br />
r27<br />
Results in controls Results In<br />
dieease<br />
8.1ppb<br />
14.7ppb<br />
16ppb<br />
4.6ppb<br />
64ppb<br />
1 Sppb<br />
0.3ppb<br />
89ppb (insP)<br />
42ppb (ep)<br />
2ppb (insp)<br />
63ppb (exP)<br />
72ppb (insp)<br />
31ppb (exp)<br />
2ppb (insp)<br />
49ppb (exp)<br />
22ppb<br />
9ppb<br />
18ppb<br />
9ppb
Authors and Pap€r Sublect numbers Method <strong>of</strong><br />
measurement<br />
Jilma et al, 1996<br />
(Jilma, Kastner et al.<br />
1996)<br />
Persson et al, 1993<br />
(Persson, Wiklund et<br />
al. 1993)<br />
Sato et al, 1996<br />
(Sato, Sakamaki et al.<br />
1996)<br />
Morris et al, 1996<br />
(Morris, Sooranna et<br />
al. 1996)<br />
Kharitonov et al, 1994<br />
(Kharitonov, Logansinclair<br />
et al. 1994)<br />
Trolin et al, 1994<br />
(Trolin, Anden et al.<br />
1 994)<br />
Robbins et al, 1996<br />
(Robbins, Floreaniet<br />
al. 1996)<br />
lwamoto et al, 1994<br />
(lwamoto, Pendergast<br />
et al. 1994)<br />
Martin el al, 1996<br />
(Martin, Bryden et al.<br />
1 996)<br />
Alving et al 1993<br />
(Alving, Weitzberg et<br />
al. 1993)<br />
22male controls<br />
21 female controls<br />
10 controls<br />
5 controls<br />
single exhalations 34ppb<br />
tidal breathing<br />
following brealh-hold <strong>of</strong><br />
60s<br />
during hyperventilation<br />
pre-exercise<br />
exercise at 50 and 100<br />
watts<br />
16 controls reservoir collection<br />
no breath-hold<br />
breath-hold for 10<br />
seconds<br />
breath-hold for 60<br />
seconds<br />
5 female controls single exhalations daily<br />
for one month<br />
40 male controls<br />
19 female controls<br />
7 females through a<br />
menstrual cycle<br />
27 controls<br />
8 with exercise<br />
91 controls<br />
18 asthmatics<br />
23 smokers<br />
I controls with<br />
exercise<br />
18 controls<br />
32 allergic rhinitis<br />
18 conlrols<br />
32 allergic rhinitis<br />
18 controls<br />
32 allergic rhinitis<br />
12 controls<br />
8 asthmatics<br />
single e;fialations<br />
gg<br />
single exhalation<br />
total output over one<br />
minute<br />
single exhalation<br />
total output<br />
single exhalation<br />
reservoir collection<br />
single exhalation<br />
reservoir collection<br />
single exhalation<br />
reservoir collection<br />
single exhalation at rest<br />
one minute collection:<br />
at rest<br />
exercise<br />
hyperventilation<br />
single exhalation<br />
following breath-hold <strong>of</strong><br />
10s<br />
following breath-hold <strong>of</strong><br />
:::,,,,<br />
Tidalbreathing:<br />
nasal inhalation<br />
oralinhalation<br />
with URTI<br />
oral inhalation<br />
128<br />
Results In controls Results ln<br />
dlsease<br />
20ppb<br />
3.2Sppb<br />
100.25ppb<br />
4.75ppb<br />
9ppb<br />
5-6ppb<br />
(numbers from graph)<br />
't2ppb<br />
14ppb<br />
1Sppb<br />
51 ng g-1<br />
No varialion across<br />
the month<br />
75ppb<br />
70ppb<br />
midcycle 150ppb<br />
menstruation 59ppb<br />
8.6ppb<br />
90.9ppb<br />
2.8ppb<br />
171.2ppb<br />
105.5ppb<br />
14.Sppb<br />
26.3ppb<br />
9.Sppb<br />
graphs for individuals<br />
4.8ppb<br />
1 1.1ppb<br />
15.6ppb<br />
32.1ppb<br />
23ppb<br />
9ppb<br />
32ppb<br />
'l74.2ppb<br />
27.2ppb<br />
39.6ppb<br />
7.3ppb<br />
16.3ppb<br />
34.Oppb<br />
62.Oppb<br />
1 lppb
Authors and PaPer Sublect numbers Method <strong>of</strong><br />
meagurement<br />
Persson et al, 1994<br />
(Persson, Zetterstrom<br />
et al. 1994)<br />
Kharitonov et al, 1994<br />
(Kharitonov, Yates et<br />
al. 1994)<br />
Kharitonov et al, 1995<br />
(Kharitonov, Yates et<br />
al. 1995)<br />
Massaro et al, 1996<br />
(Massaro, Mehta et<br />
al. 1996)<br />
Massaro et al 1995<br />
(Massaro, Gaston et<br />
al. 1995)<br />
Kharitonov et al, 1995<br />
(Kharitonov, Robbins<br />
et al. 1995)<br />
schilling et al, 1994<br />
(Schilling, Holzer et<br />
al. 1994)<br />
Kharitonov et al, 1996<br />
(Kharitonov, Yates et<br />
al. 1996)<br />
Kharitonov et al, 1995<br />
(Kharitonov, Yates et<br />
al. 1995)<br />
Kharitonov et al, 1995<br />
(Kharitonov, Wells et<br />
al. 1995)<br />
20 controls<br />
23 asthmatics<br />
6 smokers<br />
67 controls<br />
61 asthmatics<br />
52 asthmatics on<br />
tHcs<br />
25 asthmatics:<br />
single responders<br />
baseline<br />
single responders<br />
27 hours<br />
dual responders<br />
baseline<br />
dual responders 27<br />
hours<br />
5 controls<br />
5 asthmatics<br />
90 controls<br />
43 asthmatics<br />
73 controls<br />
41 smokers<br />
21 female controls<br />
12 female smokers<br />
24 male controls<br />
24 male smokers<br />
10 with<br />
hypertension<br />
11 asthmatics<br />
baseline<br />
following IHCS<br />
18 (URTI)<br />
during recovery<br />
79 (controls)<br />
20 bronchiectasis<br />
19 bronchiectasis<br />
on IHCS<br />
mixed exhaled air<br />
,t at aa<br />
single exhalation<br />
single exhalation<br />
pre and post allergen<br />
challenge<br />
mixed expired air<br />
at carina with<br />
bronchoscoPe<br />
mixed expired air<br />
at carina with<br />
bronchoscoPe<br />
mixed air<br />
single exhalation<br />
reservoir collection<br />
single exhalation<br />
single exhalation<br />
single exhalation<br />
Resulte In contlols Rasults In<br />
dlsease<br />
8.4ppb<br />
80.2ppb<br />
4.7ppb<br />
7.0ppb<br />
6.2ppb<br />
88ppb<br />
21 ppb<br />
1 9ppb<br />
89ppb<br />
10.3ppb<br />
3.9ppb<br />
283ppb<br />
101ppb<br />
214ppb<br />
233ppb<br />
258ppb<br />
264ppb<br />
13.2ppb<br />
40.Sppb<br />
13.9ppb<br />
42ppb<br />
16ppb<br />
1 5ppb<br />
13.7ppb<br />
203ppb<br />
120ppb<br />
31 Sppb<br />
88.3ppb<br />
285ppb<br />
88ppb<br />
This table lists the published results <strong>of</strong> exhaled NO in adults by the beginning <strong>of</strong> 1997 showing the<br />
wide range <strong>of</strong> techniques and even with app-arentty similar. techniquei the wide range <strong>of</strong> results<br />
reported. <strong>The</strong> mean refufts <strong>of</strong> the exhaleo No ior eac'h subiect group are presented here. I have listed<br />
129
only those measured by mouth or lower respiratory tract rather than nasal or sinus measurement<br />
which will be discussed in a later chapter.<br />
(exp = expiratory, IHCS = inhaled corticosteroids, insp = inspiratory, rg = nanogram, ppb = parts per<br />
billion, s = seconds, single responders = edrly asthmatic response only, late responders = both early<br />
and late asthmatic response, URTI = upper respiratory tract infection).<br />
6.4 <strong>The</strong> aims <strong>of</strong> the exhaled nitric oxide methodological experiments<br />
<strong>The</strong> following methodological experiments were therefore designed and conducted in adult<br />
subjects to try and answer two main questions:<br />
1. Were NO and CO2 produced from the same region within the lung?<br />
2. Which technical factors during measurement led to changes in the NO results and<br />
therefore would require standardisation in future research in this area?<br />
<strong>The</strong> aims <strong>of</strong> the methodological investigations were:<br />
L. To determine whether exhaled NO could be measured by the chemiluminscence analyser<br />
now modified from its original purpose <strong>of</strong> measuring NO in air pollution.<br />
2. To compare the exhalation pattern <strong>of</strong> NO versus COz from a series <strong>of</strong> single exhalations.<br />
3. To compare the results <strong>of</strong> NO using the peak level versus the area under the curve <strong>of</strong> the<br />
recording trace.<br />
4. To compare the results <strong>of</strong> NO and COz between two methods <strong>of</strong> measurement; either<br />
direct exhalation to the NO analyser (which also allowed measurement <strong>of</strong> CO2, and mouth<br />
pressure) or exhalation via a t-piece system (which measured these parameters with the<br />
additional measurement <strong>of</strong> expiratory flow).<br />
5. To assess the reproducibility <strong>of</strong> the NO and COz measurements.<br />
6. To investigate whether altering the expiratory flow changed the exhaled NO levels<br />
measured.<br />
7. To assess whether pressure had any effect on the exhaled NO levels by two methods:<br />
(a) Delivering the calibration gas at different pressures to the NO analyser.<br />
(b) Altering the expiratory mouth pressure voluntarily by adult subjects.<br />
8. To investigate whether ambient NO levels affected exhaled NO levels.<br />
9. To investigate whether water consumption immediately before exhalation changed the NO<br />
results obtained (this was added in response to a chance finding).<br />
<strong>The</strong> experiments and results from aims one to five listed above will be detailed in this chapter.<br />
<strong>The</strong> experiments and results conducted to answer aims six to nine where a specific single<br />
condition was varied to assess the effect on the exhaled NO levels obtained are detailed in<br />
Chapter 7.<br />
130
6.5 Setting up for the experiments<br />
In the previous chapter, the capabilites and set-up <strong>of</strong> the analysers was described. In this<br />
section, I will outline how the analysers were calibrated prior to each subject in the<br />
methodological experiments and the basic procedure to serve as an introduction to all the<br />
subsequent experiments on adult volunteer subjects. Changes in this exact procedure and<br />
results <strong>of</strong> each set <strong>of</strong> studies will be outlined as separate experiments. I will present and then<br />
discuss the initial findings after the first investigations which compared the two sampling<br />
techniques with NO, CO2, expiratory mouth pressure, and (in the t-piece system) expiratory<br />
flow results.<br />
6.5.1 Set-up and calibrations<br />
<strong>The</strong>se instructions were set out by Carolyn Busst and myself after many individual tests <strong>of</strong> the<br />
machinery and then posted next to the NO analyser to follow each time I did these studies to<br />
ensure a standard procedure. I have re-presented them here as they were written in the present<br />
tense and with numerical representation <strong>of</strong> the recorded numbers for times and frequency or<br />
repetitions, but have added in italics some additional notes that came to be part <strong>of</strong> the set-up<br />
as I progressed through and became more experienced. <strong>The</strong>re was no change to the nature <strong>of</strong><br />
the investigations, but made it easier to standardise the machines and have the set-up running<br />
efficiently. <strong>The</strong> purpose <strong>of</strong> these procedures was two-fold; to calibrate the analysers, in<br />
particular the NO and COz analysers, and to mark the levels on the chart recorder to enable<br />
calculation <strong>of</strong> the study parameters.<br />
One hour before the study:<br />
1. Check that the NO analyser and the vacuum pump are switched on and that the ozone<br />
generator in the analyser is switched on (toggle switch left hand side, front panel). [NB: I<br />
found the analyser and vacuutn more stable and took less time to reach a stable level if I<br />
let it run continuously Monday to Friday or over weekends as well if experiments were<br />
being underraken. This is in keeping with how it would have been used oiginally as a<br />
continuous monitor <strong>of</strong> air NO pollution.l<br />
2. Switch on the capnograph (switch located at the back <strong>of</strong> the machine).<br />
g. Switch on the electrospirometer (switch located at the back <strong>of</strong> the machine).<br />
4. Check that the calibration gas cylinders and air cylinders are not empty'<br />
S. Check the paper in the chart recorder. If in doubt regarding having sufficient to record the<br />
whole next subject experiment, replace with a new roll. Keep the old ones for test runs.<br />
r3l
6. Check that the pens work. Fill with ink if necessary, careful not to overfill as this can lead<br />
to smudges on the traces.<br />
7. Check that the mouthpiece is clean and that the sample tubing is free from water. If it is<br />
not, flush the tubing with compressed air from the cylinder.<br />
8. Do not switch on the pressure equipment as it is battery powered and this will just waste<br />
the battery.<br />
Calibration just before the study:<br />
On chart recorder note:<br />
o Date.<br />
o Study name.<br />
o Subject name.<br />
o Parameters and voltage scales [these remained unchanged and would only have mattered<br />
if there was a log scale change in the concentrations <strong>of</strong> the gases, pressure orflow that we<br />
were measuring, see NO calibration belowl.<br />
o Paper speed.<br />
o Barometric pressure.<br />
o Temperature.<br />
o <strong>The</strong> background ambient NO level.<br />
<strong>The</strong> procedures for calibrating the analysers:<br />
Calibration <strong>of</strong> the NO analyser:<br />
o Make sure the rotameter valve is closed.<br />
o Tnro calibration: connect NO sampling probe to the black, cylindrical NO charcoal scrub<br />
unit.<br />
o NO concentration calibration one: connect NO sampling probe to the calibration gas 'one'<br />
nozzle - turn on the cylinder and record the level noting on the chart record.<br />
o NO concentration calibration two: connect NO sampling probe to the calibration gas 'two'<br />
nozzle - turn on the cylinder and record the level noting on the chart record.<br />
o Turn <strong>of</strong>f the cylinders.<br />
o Connect the sample port to the mouth-piece.<br />
o Run the chart recorder to record both NO and COz baseline ambienUroom air values and<br />
note results on the chart record.<br />
132
Note that ordcring o1'ncw NO gases cylinclcrs takcs 2 months t() sort out ancl arrive stl whct-l ttr<br />
a quarter 1-ull cortrtttcncc tl-tc order tor tl-tc ncxt cylinclcr'<br />
NB: Tlie calibration gascs usecl clr-rring thc tirnc ol'thc str,rclies wcrc 37 ppb. l03ppb' ll0ppb'<br />
55ppb, ancl llSppl-, F-or an cxarrplc <strong>of</strong> calitrration on the chart rccorcl scc Figure 6.1.<br />
Figure 6.1: Recording <strong>of</strong> the calibrations for NO, CO2 and pressure analysers<br />
Pr6s3urc cqlibralion in 2 mmHg stepg<br />
<strong>The</strong> charl recording is read f rom right to left and the green tracing shows NO, the red tracing CO2 wtth<br />
the zero. ambient and calibrations for each. <strong>The</strong> blue tracing is for mouth pressure and this is shown<br />
as calibration in sets <strong>of</strong> 2mmHq.<br />
c'o. latibraion s.sz<br />
:I .l<br />
-t r: -<br />
Cal it-rration o1' thc capnograph:<br />
. Make surc thc r()lantctcr valvc is closccl.<br />
o Prcss the CAL button on the front o1'thc pancl. <strong>The</strong> punrp clctcrtltitlcs the sarupling ratc ol'<br />
thc CO: plobc. (Thc santpling rate can bc changcd by pressing thc Lrp and down ilrrow<br />
| 11<br />
Details<br />
recorded<br />
:<br />
r<br />
ZarONO I .-r --- r--
keys but it is currently in the middle <strong>of</strong> the range suggested by the manufacturers, and<br />
used for all the early assessments).<br />
o Tnro calibration: connect the COz sampling probe to the NO/i.{z tubing (NO calibration<br />
gas) and press the CAL button to accept the zero calibration - mark on chart record - turn<br />
<strong>of</strong>f the gas.<br />
o COz calibration gas one -<br />
connect the COz sampling probe to the COz calibration gas<br />
nozzle used and press CAL button to accept calibration - mark on the chart recorder - turn<br />
<strong>of</strong>f the gas.<br />
o Barometric calibrations: Press the CAL button until the barometric pressure is displayed -<br />
press the up arrow to set the barometric pressure - the COz bobbin will fall for 3 seconds<br />
and then rise back up again and the current barometric pressure will be displayed - make a<br />
note on chart record.<br />
o Connect the sample port to the mouthpiece.<br />
o Press display on the front panel to obtain the end-tidal COz display.<br />
o Run the chart recorder to record both NO and COz baseline values noting the ambient<br />
levels (more important for the NO recordings) (see Figure 6.1).<br />
Calibration <strong>of</strong> the flow rotameter:<br />
o Tnro calibration: with the air supply turned <strong>of</strong>f and the rotameter needle valve turned <strong>of</strong>f -<br />
connect the air supply tubing to the bottom inlet <strong>of</strong> the rotameter - turn the air supply on -<br />
run the chart recorder and label the trace.<br />
o Calibration levels: open the valve until the rotameter bobbin moves and calibrate to<br />
l50mls/min, 22lmlslmin, 325mls/min, 40Omls/min, 500mls/min, 600mls/min,<br />
700mls/min, 800mls/min, 900mls/min, 1000mls/min at each level run the chart recorder<br />
each time and note the calibration on the chart record - beyond this range the readings<br />
become less stable when repeated over time and therefore less likely to be accurate.<br />
o Leave the rotameter valve open - turn <strong>of</strong>f the air supply, the bobbin will fall to zero<br />
releasing the pressure in the air line (see Figure 6.2).<br />
134
Figure 6.2: Recording <strong>of</strong> the calibration <strong>of</strong> the flow rotameter and pneumotachograph<br />
<strong>The</strong> chart recording is read from right to left and the brown tracing shows the levels <strong>of</strong> flow calibrations<br />
in mls/min. <strong>The</strong> calibrations are worked out on the sheet.<br />
Mouth pressure.<br />
o Open the rotameter needle valve.<br />
o Ensure all connections are tight.<br />
o Switch on the pressure generator (small black sliding switch, right hand side).<br />
o Tero the display with the small black screw on the left hand side front panel <strong>of</strong> the<br />
pressure generator.<br />
o Z.ero calibration: connect the line from the pressure generator to the test point <strong>of</strong> the<br />
Medex pressure transducer and ensure that it is secure - zero the pressure generator using<br />
the wheel in the centre <strong>of</strong> the generator - zero the pressure monitor gauge making sure the<br />
needle is on the zero <strong>of</strong> the scale -<br />
record the zero level on the chart record.<br />
135
Calibration pressures: the test port <strong>of</strong> the pressure transducer is on the opposite side <strong>of</strong> the<br />
transducer to the mouthpiece - to mimic an increase in mouthpiece pressure it is necessary<br />
to create an equivalent negative pressure at the test port -<br />
move the wheel in the direction<br />
indicated by the vacuum alrow to the right <strong>of</strong> the wheel - a negative figure is noted on the<br />
generator display -<br />
increase the vacuum by increments <strong>of</strong> 2mmHg from 0-20 mmHg and<br />
run the chart paper at each level and mark on the chart record.<br />
o Ensure that when calibrating the trace does not drift down which suggests a leak and in<br />
this case recheck the joints.<br />
o At the end, disconnect the generator and switch it <strong>of</strong>f to save the batteries and ensure the<br />
needle on the pressure monitor is on zero (see Figure 6.1).<br />
For the testing the chart speed can be set 20mm/min or 5Omm/min - the latter should be used<br />
for the subjects.<strong>The</strong> scaling factor on the CO2 channel on the pen recorder should be set to l0<br />
volt full scale deflection (i.e. set the the toggle switch to V and the pointer on the votage knob<br />
pointing to l0 on the V scale), and that <strong>of</strong> the NO channel to 500mV. <strong>The</strong> scale <strong>of</strong> the NO<br />
channel will have to be changed if the calibration gases contain more than 490 ppb NO /nor<br />
required during this timel.<br />
At the end <strong>of</strong> the study:<br />
o Repeat the calibrations in reverse order immediately the study has finished.<br />
r Check that the gas cylinders are <strong>of</strong>f.<br />
o If no further studies on the day -<br />
turn everything <strong>of</strong>f except the NO analyser.<br />
o If there is a gap between studies turn <strong>of</strong>f the pressure monitor only to conserye the battery.<br />
r <strong>The</strong> ozone generator can be switched <strong>of</strong>f overnight but it should be switched on again an<br />
hour before the study to stabilise.<br />
o <strong>The</strong> NO analyser can be switched <strong>of</strong>f if it is not going to be used for long periods - switch<br />
<strong>of</strong>f the ozone generator I't, then the NO analyser and finally the vacuum pump.<br />
6.5.2 <strong>The</strong> exhalation protocol<br />
Standard protocol for exhalation is described here and forms the basis for all the subsequent<br />
experiments - any deviation in order to assess different factors is documented in each<br />
experiment.<br />
136
Direct to analysers method:<br />
o <strong>The</strong> subject sits at rest for a minimum <strong>of</strong> 5 minutes.<br />
. Each subject inhales to total lung capacity then performs a slow vital capacity manoeuvre to<br />
residual volume over 30-60 seconds.<br />
. 5 exhalations are made consecutively at 3-minute intervals.<br />
o Nose clips are worn 5 seconds before the exhalation, and taken <strong>of</strong>f between measurements.<br />
. Measurements <strong>of</strong> NO, mouth pressure and COz are made.<br />
. Mouth pressure is standardised to 4 mmHg by a fixed restriction.<br />
. <strong>The</strong> sampling rate <strong>of</strong> the combined analysers was 44Omls/min.<br />
T-piece sampling system to analysers method:<br />
o <strong>The</strong> subject sits at rest for a minimum <strong>of</strong> 5 minutes.<br />
. Each subject inhales to total lung capacity then performs a slow vital capacity manoeuvre to<br />
residual volume over 30-60 seconds.<br />
o 5 exhalations are made consecutively at 3-minute intervals.<br />
o Nose clips are worn 5 seconds before the exhalation, and taken <strong>of</strong>f between measurements.<br />
o Measurements <strong>of</strong> NO, mouth pressure and CO2, and flow are made.<br />
. Mouth pressure is standardised to 4 mmHg by a fixed restriction.<br />
. Flow in the t-piece was standardised by having the subject exhale at a constant rate <strong>of</strong><br />
221mllmin. <strong>The</strong> subjects controlled their expiratory flow rates voluntarily observing the<br />
rotameter bobbin level <strong>of</strong> the pneumotachograph for visual feedback.<br />
o <strong>The</strong> sampling rate <strong>of</strong> the combined analysers including the flow was 665mls/min<br />
Figure 6.3: Schematic diagrams <strong>of</strong> the two different types <strong>of</strong> connections: direct and t-piece<br />
measurements<br />
Direct:<br />
O<br />
0<br />
t37
T-piece:<br />
tO<br />
<strong>The</strong> first figure shows the connections from the mouthpiece in the direct system to the NO analyser,<br />
the CO2 analyser, the pressure analyser and the chart recorder. <strong>The</strong> second figure shows the t-piece<br />
connection allowing additional measurement <strong>of</strong> flow.<br />
6.5.3 Ethics and Consent<br />
<strong>The</strong> studies received prospective approval by the Royal Brompton Hospital Ethics Committee<br />
and informed consent was obtained from each <strong>of</strong> the volunteer subjects.<br />
6.5.4 <strong>The</strong> subjects<br />
In total, twenty healthy volunteers (mean age 35.9 years, range 18-52 years, 9 males) took<br />
part in these methodological studies. Following consent, each subject completed a<br />
questionnaire (see Appendix 2). <strong>The</strong> subjects were non-atopic with no history <strong>of</strong> respiratory<br />
or cardiac disease and taking no medication. All had normal spirometry (Compact<br />
Vitalograph, Vitalograph Ltd, Buckingham, United Kingdom). Two female subjects involved<br />
in the variable flow and variable pressure experiments smoked two cigarettes per day; the rest<br />
were nonsmokers.<br />
6.5.5 Statistical analvsis<br />
<strong>The</strong> results were analysed using the Statistical Products and Services Solutions, (Package U<br />
7.0, SPSS Inc, Chicago, USA). This employs the Student's t-test for matched pairs with mean,<br />
138<br />
E<br />
CL<br />
(U<br />
o)<br />
o<br />
-c<br />
o(0<br />
o<br />
E<br />
J<br />
o<br />
c<br />
ct
standard error <strong>of</strong> the mean (SEM) and ranges given. A p-value <strong>of</strong> less than 0'05 was<br />
considered significant.<br />
6.6<br />
o<br />
a<br />
o<br />
Methodological exPeriment one<br />
Direct versus t-piece NO measurement<br />
Peak NO versus area under the NO curve<br />
Exhalation patterns <strong>of</strong> NO versus COz<br />
6.6.1 Hypotheses<br />
l. <strong>The</strong> levels <strong>of</strong> NO measured in exhaled air are dependent on the techniques <strong>of</strong><br />
measurement.<br />
Z. <strong>The</strong>re will be good correlation between the peak NO levels and the area under<br />
recording curve <strong>of</strong> the NO measurement.<br />
3. Curves for NO and COz in a single exhalation should be identical if they<br />
predominantly being produced in the same department within the lung.<br />
6.6.2 Aims<br />
1. To compare exhaled NO levels measured by two different techniques'<br />
Z. To compare results <strong>of</strong> NO measured as peak level with area under the curve.<br />
3. To compare the peak levels and curves <strong>of</strong> NO and COz in a single exhalation.<br />
6.6.3 Procedure<br />
Each subject abstained from food and drink for four hours prior to the experiment. <strong>The</strong><br />
experiments were made if the ambient level <strong>of</strong> No was less than 10ppb, and all the testing<br />
was done with inhalation from ambient room air. <strong>The</strong> procedures as described in the previous<br />
section for starting and calibrating the analysers were made before and after each subject. <strong>The</strong><br />
exact procedure followed is as described above in Section 6.5.2. Twelve subjects were<br />
enrolled and each subject continued until five sets <strong>of</strong> measurable exhalations were made for<br />
each set <strong>of</strong> conditions with the first set being determined randomly by tossing a coin (heads -<br />
direct, tails -<br />
t-piece). This was followed after a five minute break by measuring exhalation<br />
under the other set <strong>of</strong> conditions. Again, after a five minute break, the first conditions were<br />
measured again. Expiratory mouth pressure was requested to be voluntarily kept at 4mmHg<br />
by the subjects and was measured on the chart recorder. During the exhalations into the t-<br />
piece system, the subjects were also requested to maintain a set expiratory flow by watching<br />
139<br />
the
the rotameter. Tracings <strong>of</strong> each parameter (see in previous chapter Figure 5.2; NO in green,<br />
COz in red, pressure in blue, flow in brown) were recorded on the chart recorder and each<br />
then calculated using the calibration recordings. <strong>The</strong> NO and the COz peak levels on the<br />
recording was measured. <strong>The</strong> peak NO levels were then compared with the area under the<br />
curye. <strong>The</strong> latter was measured by drawing a baseline along the recording congruent with the<br />
zero reading. <strong>The</strong> baseline and the curved NO record were then traced out and the area within<br />
the margins calculated. Each result was the mean <strong>of</strong> five exhalations. <strong>The</strong> comparisons<br />
presented between the direct and the t-piece measurements are the first measurements <strong>of</strong> each.<br />
<strong>The</strong> final repeated set was compared against the first set to assess whether there were<br />
differences over this time.<br />
6.6.4 Results<br />
Figure 6.4: Example <strong>of</strong> the chart recording rolls for the direct versus t-piece measurements.<br />
6.6.4 (i) Direct versus t-piece nitric oxide and carbon dioxide measurement<br />
Fifteen measurements were performed successfully on each <strong>of</strong> the twelve subjects (six male,<br />
six female) with their lung function as percent predicted for gender, age and height by the<br />
Polgar predictive equation (Polgar and Promadhat l97l) having an overall mean FVC <strong>of</strong><br />
IOTVo (range 96-I2OVo) and FEV1 <strong>of</strong> IO2Vo (range 93Vo-lI5Vo). <strong>The</strong> direct mean peak<br />
r40
concenrration <strong>of</strong> exhaled NO was 84.8ppb (SEM 14.0, range 25.I - 189.0). <strong>The</strong>re was greater<br />
variation shown in the female subjects with a mean <strong>of</strong> 92.4ppb (SEM 36.9,26.2 - 189.0) than<br />
the male subjecrs with a mean <strong>of</strong> 71.7ppb (SEM 28.7, range 25.1 - 141.0). <strong>The</strong> peak exhaled<br />
NO levels measured through the t-piece system were lower in all the subjects with a total<br />
subject mean <strong>of</strong> 41.2ppb (SEM 10.8, range 10.00 - 101.6). <strong>The</strong> mean in males was 33.2ppb<br />
(SEM 8.4) versus females <strong>of</strong> 50.7ppb (SEM 12.3) (see Figure 6.6). <strong>The</strong> data for one<br />
individual as the change occurred from t-piece to direct measurement is shown in Figure 6.7.<br />
Note again that the chart recording is read from right to left so the exhalation commences at<br />
the right hand side <strong>of</strong> each set <strong>of</strong> tracings. <strong>The</strong> scales were added for convenience <strong>of</strong> viewing<br />
this single tracing as the scales were worked out on each graph depending on the checks with<br />
the calibration gases. <strong>The</strong> main point depicted is the change with higher NO levels being<br />
measured in the direct rather than the t-piece system.<br />
Figure 6.5: Example <strong>of</strong> recording on one subject - direct and t-piece measurements<br />
Note the chart is read from right to left, NO is recorded in green, COz is recorded in red, mouth<br />
pressure is recorded in blue and flow (seen only in the t-piece measurements) is recorded in brown'<br />
<strong>The</strong> exhaled NO levels increase when the tiace moves from t-piece to direct recordings. <strong>The</strong><br />
calculations made for each exhalation can also be seen on the chart.<br />
t4l
Figure 6.6: Mean exhaled NO levels measured by direct and t-piece systems<br />
180<br />
160<br />
140<br />
E 120<br />
EL<br />
tr<br />
= 100<br />
o<br />
g80<br />
o<br />
;60<br />
g<br />
E40<br />
x<br />
ul<br />
20<br />
0<br />
T-plece<br />
Comparisons <strong>of</strong> mean exhaled peak NO by the direct and t-piece system (n=12, each mean <strong>of</strong> 5<br />
exhalations, SEM = standard error <strong>of</strong> the mean).<br />
Figure 6.7: Example <strong>of</strong> a recording for the two systems<br />
200<br />
180<br />
160<br />
1.10<br />
-o 120<br />
CL<br />
Sroo<br />
z' Bo<br />
60<br />
40<br />
20<br />
0<br />
ED<br />
-<br />
Ere<br />
e9<br />
a<br />
g6<br />
Eg<br />
3<br />
Eo<br />
=<br />
Direcl Toiece<br />
ffi<br />
876543210<br />
Time min<br />
Rrll<br />
cq<br />
NO<br />
6<br />
5<br />
4<br />
is<br />
35'<br />
C)<br />
2<br />
This is an example <strong>of</strong> the raw data from one individual showing the NO and COz and mouth pressure<br />
(P,*) with the appropriate scales added along the '/ axes. <strong>The</strong> main point to note is the difference<br />
seen in the exhaled NO concentration between the two methods; higher in the direct versus the tpiece<br />
system <strong>of</strong> measurement.<br />
t42<br />
t<br />
0
Table 6.2 shows all the results on one subject to demonstrate that each <strong>of</strong> the testing<br />
parameters are measured five times with the first set <strong>of</strong> conditions repeated. <strong>The</strong> parameters <strong>of</strong><br />
pressure and flow, although standardised voluntarily, were also measured to ensure they were<br />
adequately controlled.<br />
Table 6.2: Compete results for an individualsubject<br />
NO In<br />
pPb<br />
COzas<br />
% total<br />
9a8e3<br />
I have also included Table 6.3 with the mean results and ranges from all the subjects for<br />
exhaled NO and COz, each the mean <strong>of</strong> 5 readings per subject. <strong>The</strong> coefficient <strong>of</strong> variation for<br />
individuals was LTVo for the direct method and llVo for the t-piece method. <strong>The</strong> background<br />
level <strong>of</strong> NO ranged from 1 to llppb; this did not alter the conclusions so was not taken into<br />
account in the calculations.<br />
Mouth<br />
pressure<br />
In mmHg<br />
Tlme in<br />
secondg<br />
Time<br />
NO<br />
peak<br />
Time<br />
COz<br />
peak<br />
COz at<br />
NO peak<br />
NO at<br />
COz<br />
peak<br />
Area<br />
under the<br />
culve<br />
Direct 1 41 6.2 4.O 58.3 32.4 50.4 5.4 37.7 6.43<br />
Direct 2 45 5.9 4.O 59.4 32.4 54.0 5.3 42.2 7.71<br />
Direct 3 50 6.3 4.2 64.8 27.6 60.3 4.9 46.6 8.93<br />
Direct 4 48 6.4 4.2 64.8 34.8 56.6 5.7 44.4 8.77<br />
Direct 5 52 6.4 4.0 66.0 24.0 60.5 5.7 46.6 9.8<br />
T-piece 1 10 6.8 4.0 60.6 6.0 48.0 3.7 8.9 1.27<br />
T-piece 2 10 6.4 4.2 51.6 8.4 63.6 4.5 11.1 1.6<br />
T-piece 3 10 6.6 4.1 58.3 12.O 63.5 4.9 13.1 1.2<br />
T-piece 4 11 6.5 4.2 60.0 '12.0 58.8 4.9 8.8 1.5<br />
T-piece 5 11 6.7 4.0 60.6 21.6 49.1 5.9 11.6 1.7<br />
Table 6.3 NO and COz results from single exhalations in twelve adult subjects<br />
Sublect<br />
No.<br />
Gender Peak<br />
NO'<br />
Range<br />
NO<br />
Dlrect<br />
Peak<br />
COzb<br />
Range<br />
COz<br />
Ratlo<br />
NO/COz<br />
Peak<br />
NO<br />
Range<br />
NO<br />
t-plece<br />
P€ak<br />
COz<br />
Range<br />
COz<br />
Ratlo<br />
NO/COz<br />
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<br />
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<br />
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<br />
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<br />
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<br />
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<br />
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<br />
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<br />
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<br />
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<br />
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<br />
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<br />
t43
NO and COz results from single exhalations as the mean <strong>of</strong> five recordings in twelve adult subjects by<br />
direct or t-piece system measurements (m = mdle, f = female). (a. NO measured in parts per billion. b.<br />
CO2 measured as % <strong>of</strong> totalgas expired).<br />
<strong>The</strong>re was no significant difference between peerk COz level by the direct measurements with<br />
a mean <strong>of</strong> 5.8l%o (range 4.0Vo to 6.9Vo) or t-piece measurements with a mean <strong>of</strong> 6.l9Vo (range<br />
4.4-7.2Vo, p = 0.66) or between male and female subjects (5.84Vo venius 5.75Vo direct, p =<br />
0.64, and 6.4l%o versus 6.03Vo t-piece, p = 0.44), (see Figure 6.8). <strong>The</strong>re was no difference in<br />
repeated last set <strong>of</strong> exhalations for either NO or COz whether it was a repetition <strong>of</strong> the direct<br />
or the t-piece measurements, although the coefficient <strong>of</strong> variation had improved suggesting<br />
that there may be some improvement with training.<br />
Figure 6.8: Mean exhaled COz levels measured by direct and t-piece systems<br />
Comparisons <strong>of</strong> mean exhaled peak CO2 by the direct and t-piece system (n--12, each mean <strong>of</strong> 5<br />
exhalations, SEM = standard error <strong>of</strong> the mean).<br />
6.6.4 (ii) Peak nitric oxide versus area under the nitric oxide curve<br />
<strong>The</strong> correlation coefficients comparing the results <strong>of</strong> peak NO level versus using area under<br />
the curve for these traces were 0.87 for the direct results and 0.81 for the t-piece results and<br />
are shown below in Figure 6.9.<br />
8<br />
7<br />
6<br />
o o<br />
$E<br />
E<br />
"ge d4<br />
6<br />
5<br />
E3<br />
* (\l<br />
92<br />
t-t<br />
1<br />
-<br />
Dlect<br />
t44<br />
-)<br />
T-plece
Figure 6.9: Comparisons <strong>of</strong> the peak No with area under the curve<br />
Figure 6.9a: Direct measurement.<br />
o<br />
Eru (,<br />
o<br />
=20<br />
s<br />
t'u a<br />
Ero<br />
Figure 6.9b: T-Piece measurement<br />
o<br />
14<br />
't2<br />
3,0<br />
(,<br />
o z8<br />
q,<br />
E<br />
o6<br />
E tg4<br />
2<br />
0<br />
50 100<br />
correlation <strong>of</strong> the peak NO levels with the measurements <strong>of</strong> area under the NO curve with a<br />
correlation coefficient ol 0.87 (direct) and 0'81 (t'piece)'a<br />
a <strong>The</strong> conelation here is slightly better than that given in the'Methods to Measure NO' article' This was an<br />
invited article and I incluJfi tfi" aut" completedit that time but continued with the study'<br />
r45<br />
150<br />
Peak level NO ln Parts Per Hlllon<br />
20 40 @ 80 100<br />
Peak level <strong>of</strong> NO In parts per bllllon<br />
200<br />
1n
6.6.4 (iii) Comparison <strong>of</strong> exhaled nitric oxide and carbon dioxide<br />
<strong>The</strong>re was a significant difference in the mean time to reach peak NO levels between the<br />
direct (mean 32.2 seconds) and t-piece method (mean 23.1 seconds, p ( 0.001) while there<br />
was no difference between the time to reach CO2 peaks between the direct (mean 50.5<br />
seconds) and t-piece methods (mean 51.4 seconds, p = 0.5). In addition, the time taken for the<br />
peak NO level to be reached and the time taken for the peak CO2 level to be reached were<br />
compared. In the direct measurements, the mean time to reach peak NO was 32.2 seconds<br />
(SEM 4.3), which was significantly less (p
Figure 6.10b: Time to peak NO and peak COz measured by the t'piece system<br />
80<br />
70<br />
a<br />
:60<br />
o<br />
E50 o<br />
.s 40<br />
.g 30<br />
F20<br />
10<br />
0<br />
Tlmeto<br />
NO peak<br />
Tlme to Duraton <strong>of</strong><br />
CO2 pak total exhalatbn<br />
Comparisons <strong>of</strong> the time in seconds taken to reach peak NO,.taken to reach peak COz with the total<br />
time for exhalation in measurements by the t-piece system (n=12, each mean <strong>of</strong> 5 exhalations)'<br />
I also marked the coz level at the time <strong>of</strong> No peak to compare the difference between the<br />
coz level at this time and at the time <strong>of</strong> co2 peak, which occurred later in the exhalation. For<br />
the direct measurements, the COz level measured at peak NO was 4.88Vo (SEM 0'14) which<br />
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<br />
88<br />
o<br />
!g'I<br />
ot<br />
E6<br />
F5 g4<br />
4<br />
t!<br />
E3<br />
E2<br />
{r<br />
8o GOrat NO peak PeakCO,<br />
:<br />
Comparisons <strong>of</strong> the COz conc€Dtrations in percent <strong>of</strong> total expired gases at the time <strong>of</strong> peak NO and<br />
at peak COz in measurements by the direct system (n=12, each mean <strong>of</strong> 5 exhalations).<br />
6.7 Discussion: <strong>The</strong> origin <strong>of</strong> the exhaled nitric oxide<br />
<strong>The</strong> study demonstrated that NO from a single exhalation could be measured by two methods;<br />
either by direct connection to the NO analyser or by using a t-piece side arm sampling system.<br />
However the results <strong>of</strong> NO were significantly different with an approximate halving <strong>of</strong> the<br />
peak levels from the direct to t-piece technique, while there was no difference seen in the COz<br />
results. <strong>The</strong>se methods gave similar individual coefficients <strong>of</strong> variation as other groups<br />
(Kharitonov, Yates et al. 1994; Monis, Caroll et al. 1995; Jilma, Kastner et al. 1996;<br />
Massaro, Mehta et al, 1996; Hogman, Stromberg et al. 1997). <strong>The</strong> NO results from the female<br />
subjects showed greater variability, and this has been noted previously as possibly being an<br />
effect <strong>of</strong> the menstrual cycle in one (Kharitonov, lngan-Sinclair et al. 1994) but not a second<br />
paper (Jilma, Kastner et al. 1996). Our subjects were chosen to minimise any other<br />
confounding factors most notably being non atopic, non-asthmatic and having normal lung<br />
function. As noted in the total group <strong>of</strong> subjects from which all the studies were done, there<br />
were two smokers (two cigarettes per day) which was not ideal but I believed controlling for<br />
atopy was more important when excluding subjects. As it happened, neither subject took part<br />
in this direct versus t-piece experiment but were involved in later experiments.<br />
Previous researchers had been unable to agree whether NO was <strong>of</strong> alveolar or airway origin<br />
(Alving, Weitzberg et al. 1993; Borland, Cox et al. 1993; Persson, Wiklund et al. 1993). It is<br />
known that the vast bulk <strong>of</strong> COz is <strong>of</strong> alveolar origin, having diffused across the<br />
alveolar:capillary membrane from the pulmonary circulation. If COz and NO were both <strong>of</strong><br />
alveolar origin, the time to reach their respective peaks and the ratio <strong>of</strong> peak NO to COz<br />
would remain the same even if measurement conditions were varied. I thus measured exhaled<br />
148
COz and NO using two different conditions. In the direct experiments the subject exhaled<br />
directly into the NO analyz er at M0 mls/min while in the indirect studies exhalation was into<br />
a t-piece system at a faster rate <strong>of</strong> 665 mUmin. <strong>The</strong> time to reach peak co2 level, and the level<br />
<strong>of</strong> the peak CO2 were the same in both manoeuvres. However the NO traces were very<br />
different. Firstly, the time to reach peak NO was shorter than the time to peak COz in both<br />
manoeuvre s (32.2 versus 50.5 seconds in direct measurements, and 23.1 versus 51.4 seconds<br />
in t-piece measurements). Secondly, altering the rate <strong>of</strong> exhalation altered the time to peak<br />
NO independently <strong>of</strong> the time to peak CO2. Thirdly, at peak NO levels the COz levels were<br />
below their maximal peak. Fourthly, the ratio <strong>of</strong> peak NO to peak CO2 differed between the<br />
two measurement methods. Thus although it is possible that a small amount <strong>of</strong> NO is <strong>of</strong><br />
alveolar origin, the bulk <strong>of</strong> NO must be being produced proximal to the alveolar membrane'<br />
In any physiological study, it is important to consider whether the choice <strong>of</strong> apparatus or the<br />
manoeuvre selected could have significantly affected the conclusions. Ideally, I would have<br />
used fast response time analysers, with identical delay times. Fast-response time analysers for<br />
NO had been used by others and are now available in the much later purpose-built machines<br />
(see Chapter 9). At this time, Persson et al had used a NO analyser with an integration time <strong>of</strong><br />
0.01 second to study the changes in NO at rest and during exercise. <strong>The</strong>y came to similar<br />
conclusions, that NO and coz were not from identical lung compartments, using a very<br />
different analyser and manoeuvre (Persson, wiklund et al. 1993). Our analyser would have<br />
been unable to detect rapid transient changes in NO. However, had these occurred, and they<br />
had not been described by other workers, they would have strengthened rather than detracted<br />
from these conclusions. Rapid transients have not been described for CO2, and their presence<br />
in the NO signal would have implied a different source, which is the main conclusion <strong>of</strong> this<br />
research and our PaPer.<br />
I considered the possibility that differing pressures exerted on the No analyser may have<br />
accounted for the difference in readings and I describe two experiments in the next chapter<br />
(see Sectio n 7 .4) specifically to answer this point (Byrnes, Dinarevic et al' 1997)' However' I<br />
was careful to standardise to a mouth pressure <strong>of</strong> 4mmHg during the expiratory manoeuvre to<br />
eliminate the possibility <strong>of</strong> this artefact. <strong>The</strong> pressure drop from mouth to analyser was<br />
measured for both direct and t-piece apparatus, and found to be essentially the same' <strong>The</strong> two<br />
manoeuvres used deliberately different expiratory flows to try to separate the COz and NO<br />
signals. However the sampling flow to the NO analyser remained constant throughout this<br />
study.<br />
149
I also considered whether the different response times <strong>of</strong> the analysers could have affected our<br />
conclusions. Ideally I would have liked equal analyser response times or a single machine<br />
(such as a mass spectrophotometer) to differentiate both gases simultaneously. <strong>The</strong> NO<br />
response was slower, but the NO peaked first. Had the NO response time been the same as<br />
that <strong>of</strong> the COz analyser, the NO peak would have been even earlier, thus again the analyser<br />
differences would serve to obscure, not enhance our conclusions.<br />
We elected to study a single slow exhalation for the purposes <strong>of</strong> these studies, accepting it<br />
may not be physiological or necessarily the best method for clinical practice. However, it was<br />
designed to supply an answer to the question posed. <strong>The</strong> possibility exists that the results were<br />
affected by back diffusion <strong>of</strong> alveolar NO into the alveolar capillary blood, and by nasal<br />
contamination <strong>of</strong> the exhalate. During a prolonged expiration, if alveolar epithelium were the<br />
source <strong>of</strong> NO, then back diffusion would allow some to be carried away combined with<br />
haemoglobin. However, although this could affect the height <strong>of</strong> the NO peak, it is<br />
inconceivable that it could affect the time to reach peak, and thus my conclusions are<br />
unaffected by this concern. Furthermore, the total time <strong>of</strong> expiration was very similar in the<br />
two different manoeuvres, suggesting that, were this mechanism to be operative, it would<br />
apply equally between the two tests.<br />
Higher levels <strong>of</strong> NO are produced by the paranasal and nasal spaces when compared to oral<br />
exhalation (Alving, Weitzberg et al. 1993; Lundberg, Rinder et al. 1994; Lundberg, Weitzberg<br />
et al. L994; Kimberly, Nejadnik et al. 1996) and these sites contribute a major proportion <strong>of</strong><br />
the NO concentration measured by oral exhalation. Indeed the paranasal sinuses also<br />
contribute a major proportion <strong>of</strong> the NO concentration measured by nasal exhalation<br />
(Lundberg, Rinder et al. 1994; Lundberg, Farkas-Szallasi et al. 1995). However if all <strong>of</strong> the<br />
NO came from the nasopharyngeal region then the pattern <strong>of</strong> the exhaled NO trace would be<br />
expected to both peak and fall <strong>of</strong>f early rather than give the continued plateau seen in this<br />
experiment. NO had been measured in exhaled air <strong>of</strong> tracheostomised rabbits (measured by<br />
chemilumnescence), guinea pigs (measured by Quattro mass spectrometry) (Gustafsson,<br />
Leone et al. 1991) and in humans (measured by chemilunescence) (Lundberg, Weitzberg et al.<br />
1994).I attempted to exclude nasal contamination by asking the subject to wear a noseclip,<br />
and to exhale against a moderate resistance (ammHg). One group had used nasal suction<br />
combined with an (amplitude unstated) expiratory resistance (Silk<strong>of</strong>fl Kesten et al. 1995) and<br />
another had excluded nasal NO contamination by voluntary closure <strong>of</strong> the s<strong>of</strong>t palate<br />
(Kimberly, Nejadnik et al. 1996) but these methods were more difficult and by no means<br />
universally used. Whereas I cannot exclude a degree <strong>of</strong> nasal contamination, I believe that our<br />
150
technique will have excluded most contamination, and that in any case the degfee<br />
contamination will have been identical because <strong>of</strong> the very careful standardisation<br />
experimental conditions including expiratory mouth pressure. This study was not designed to<br />
differentiate nasal and bronchial sources <strong>of</strong> NO.<br />
I elected to analyse the peak NO rather than the area under the curve. <strong>The</strong> two appear to be<br />
closely correlated (Kharitonov, Yates et al. tgg4; Byrnes, Bush et al. 1996) and had good<br />
correlation coefficients greater than 0.8 for both techniques in this study. I aimed to determine<br />
the sources <strong>of</strong> the two gases, not the total amounts produced, which we would expect to be<br />
independent <strong>of</strong> the experimental conditions. Thus the different behaviour <strong>of</strong> the peak signals<br />
as the experimental conditions change gives more useful information than the area under the<br />
curve.<br />
It might be argued that if airways <strong>of</strong> volume 200mls contribute a peak NO at 85 ppb, a total <strong>of</strong><br />
10-10 mols, and the alveolar expirate (four litres) a plateau <strong>of</strong> 60, a total <strong>of</strong> 10-8 mols, then<br />
most NO comes from the alveoli. This assumes that subjects exhaled to residual volume,<br />
which is not the case. Furthermore, if this model was correct, peak COz (definitely alveolar)<br />
and peak NO should move in the same direction as experimental conditions vary. <strong>The</strong> reverse<br />
was seen. In eight subjects peak No fell using the t-piece while coz rosei in the other four<br />
peak NO fell and peak CO2 also decreased. It is impossible to account for this on a model <strong>of</strong><br />
major alveolar production <strong>of</strong> NO. <strong>The</strong> model that best accounts for our data is continuous<br />
airway production <strong>of</strong> No, diluted variably in a flow-dependent manner by No free (or low<br />
concentration NO) alveolar gas, analogous with a gaseous phase dye dilution method. This<br />
data cannot exclude that alveolar gas contains small amounts <strong>of</strong> NO.<br />
This study also demonstrated that the conditions <strong>of</strong> measurement critically affect No levels<br />
obtained. Thus both the time to reach the No peak and the peak level <strong>of</strong> No reached were<br />
quite different in the two methods used in this study although the patterns <strong>of</strong> the traces were<br />
similar. I considered whether the difference in results could have been artifactual due to minor<br />
differences in the apparatus used in the two techniques. Teflon tubing which does not absorb<br />
NO connected the mouth piece to the NO analyzer.In the t-piece system there was a two and<br />
a half centimetre extension <strong>of</strong> plastic tubing leading from the mouth piece to the actual t-piece<br />
with the teflon tubing then leading to the NO analyzer <strong>of</strong>f one arm and further tubing <strong>of</strong>f the<br />
second arm passing directly to the pneumotachograph for flow analysis. This is unlikely to<br />
account for the magnitude <strong>of</strong> differences obtained. <strong>The</strong> other alteration was that moving from<br />
151<br />
<strong>of</strong><br />
<strong>of</strong>
the direct to the indirect method led to an increase in flow rate from 440mUmin to 665<br />
mUmin. This may have been enough to account for the different results.<br />
Clearly, at the time <strong>of</strong> this research, there were major and unexplained differences between<br />
results from different investigators as documented in Table 6.1. <strong>The</strong>se results showed that the<br />
levels <strong>of</strong> NO measured in exhalate were critically dependent on measurement conditions. This<br />
may be related to expired flow rates but other factors may be important. It has been suggested<br />
that orally-exhaled NO concentrations correlate with the inhaled ambient concentrations <strong>of</strong><br />
NO @otsch, Demirakca et al. 1996), although we found in a subsequent experiment a high<br />
ambient NO rendered exhaled NO levels difficult to interpret @yrnes, Dinarevic et al. 1997).<br />
Our current study was only undertaken if the ambient NO levels were low -<br />
while we aimed<br />
for less than l0ppb, on one occasion by the end <strong>of</strong> the session the ambient NO had drifted up<br />
to llppb hence the range given from l-llppb in the results. As mentioned the greater NO<br />
levels and wider variations seen in females may be due to possible effects <strong>of</strong> the menstrual<br />
cycle (Kharitonov, Iogan-Sinclair et al. 1994) and this was not controlled for in the present<br />
study. However, as the measurements were done sequentially on each subject on the same<br />
day, conclusions as to the origin <strong>of</strong> NO from this study should not be affected.<br />
As increasing research on NO in exhaled air was being undertaken in subjects in health and<br />
different respiratory and vascular diseases at the time, it was increasingly important to<br />
understand where and at what level NO was being generated so that abnormal results could be<br />
properly interpreted.<br />
NB: <strong>The</strong> results <strong>of</strong> this researchformed the basis <strong>of</strong> the following publications:<br />
. Byrnes CA, Dinareyic S, Bzsst CA, Bush A, Shinebournes EA. Is nitric oxide produced at<br />
airway or alveolar level? European Respiratory Joumal 1997 10 (5) 1021-1025<br />
. Byrnes CA, Bush A and Shineboume EA "Methods to Measure Expiratory Nitric Oxide in<br />
People" in "A Volume <strong>of</strong> Methods in Enrymology" edited by Lcster Packeter Academic<br />
Press Inc. 1996.<br />
t52
Chapter 7: Methodological studies <strong>of</strong> exhaled nitric oxide in healthy adult subjects:<br />
7.t Introduction<br />
investigating test Parameters<br />
<strong>The</strong> previous chapter described the commencement <strong>of</strong> the methodological studies into exhaled<br />
NO in adult volunteers. <strong>The</strong> first investigation was the measurement and comparison <strong>of</strong> NO<br />
and COz in single exhalations by two methods; direct to analyser and via t-piece sampling<br />
systems. In both methods the results and patterns <strong>of</strong> NO and COz could be determined, as well<br />
as expiratory mouth pressure measurement and, in the t-piece system, expiratory flow could<br />
also be recorded. This was an attempt to answer the question as to whether NO and COz came<br />
from the same areas within the lung. This experiment also demonstrated a significant<br />
difference in the peak exhaled NO levels with an almost halving <strong>of</strong> the NO result when<br />
moving from the direct to t-piece measurements with no corresponding reduction seen in the<br />
peak CO2 levels. <strong>The</strong> main change between these two techniques appeared to be an increase<br />
in the expiratory flow which was required for the t-piece sampling. I have already noted in the<br />
previous chapter (Section 6.4) the different levels <strong>of</strong> NO reported by different researchers<br />
despite investigating similar population groups (healthy controls, asthmatics, smokers) and<br />
summarised these in Table 6.1. I thought it was vital to understand which parameters when<br />
altered gave different exhaled NO results. This was needed to be able to recommend<br />
standardised procedures so that better interpretation <strong>of</strong> results would be possible both between<br />
different research groups and in comparisons <strong>of</strong> results in health and disease.<br />
As mentioned in Section 6.4, the aims in designing the methodological experiments were to<br />
answer two main questions Posed:<br />
l. Were NO and CO2 produced from the same regions within the lung?<br />
2. Which technical factors during measurements led to changes in the exhaled NO results?<br />
<strong>The</strong> results from the direct versus t-piece analysis suggested that the exhalation patterns seen<br />
were different between the gases. While it was known that COz was <strong>of</strong> alveolar origin, the<br />
pattern <strong>of</strong> NO exhalation suggested that it came from a more proximal source such as the<br />
airways. So I felt that we had answered the first question. <strong>The</strong> experiments detailed<br />
throughout this chapter were to answer the second question' <strong>The</strong> parameters chosen to<br />
examine, by changing each one alone and maintaining an otherwise standardised procedure,<br />
were:<br />
o <strong>The</strong> effect <strong>of</strong> expiratory flow<br />
suggested by discrePancies in<br />
- an effect <strong>of</strong> flow altering the level <strong>of</strong> NO had been<br />
the published literature summarised in Section 6.3 '<strong>The</strong><br />
153
need for methodological experiments' and from the first methodological experiment<br />
conducted and described in the last chapter.<br />
<strong>The</strong> effect <strong>of</strong> pressure -<br />
to be assessed both by applying a calibration gas under pressure<br />
to the NO analysers and by looking at the effect <strong>of</strong> expiratory mouth pressure in NO<br />
results.<br />
<strong>The</strong> effect <strong>of</strong> the ambient NO when high - this was not always standardised and<br />
sometimes not reported in early literature and could have been a source <strong>of</strong> error in results<br />
presented by different groups.<br />
<strong>The</strong> effect <strong>of</strong> water consumption - this was due to a chance finding during one<br />
experiment, where a person drank some water during a series <strong>of</strong> test exhalations and the<br />
NO results dropped significantly for the next readings. I considered that asking both<br />
healthy and, more particularly, subjects with any respiratory disease to inspire and exhale<br />
fully may well lead to coughing in some for which a corlmon response is to give a glass<br />
<strong>of</strong> water. If this then resulted in decreasing the levels <strong>of</strong> subsequent NO measurements,<br />
this could easily skew results.<br />
<strong>The</strong> peak exhaled NO was shown to have a good correlation with the area under the NO curve<br />
in the previous experiment, so I elected to proceed with the peak measurements as they were a<br />
more straightforward measurement, without the possibility <strong>of</strong> introducing error when tracing<br />
around a curve, drawing a line for zero along the lower edge <strong>of</strong> the recording and then<br />
calculating the area within these boundaries. I will summarise the subjects, ethics and<br />
statistics again at the beginning <strong>of</strong> this chapter pertaining to all the investigations in the adult<br />
subjects. For each experiment I will present the hypothesis, aim, procedure and results. I will<br />
discuss the results and how they contribute to findings in the literature <strong>of</strong> all four experiments<br />
at the end <strong>of</strong> the chapter. As the direct and t-piece experiment was deemed 'methodological<br />
experiment one', these are labelled methodological experiments two to five.<br />
7.2<br />
Ethics. subjects. calibrations and statistical analysis<br />
<strong>The</strong> studies received prospective approval by the Royal Brompton Hospital Ethics Committee<br />
and informed consent was obtained from each <strong>of</strong> the volunteer subjects.<br />
In total, twenty healthy volunteers (mean age 35.9 years, range 18-52 years, 9 males) took<br />
part in these methodological studies. Following consent, each subject completed a<br />
questionnaire (see Appendix l). <strong>The</strong>y were non-atopic with no history <strong>of</strong> respiratory or<br />
cardiac disease and taking no medication. All had normal spirometry (Compact Vitalograph,<br />
Vitalograph Limited, Buckingham, United Kingdom) and results are presented as percent<br />
r54
predicted or age, sex and height as defined by the Polgar reference equation (see results in<br />
Section 6.6.4). Two female subjects involved in experiments two and three smoked two<br />
cigarettes per day; the rest were nonsmokers.<br />
<strong>The</strong> set-up <strong>of</strong> the analysers and the calibrations before and after each subject doing each set <strong>of</strong><br />
exhalations for the individual experiments was carried out in the same manner as described in<br />
Section 6.5.1 .Set up and calibrations' and Section 6.5.2 '<strong>The</strong> exhalation protocol' <strong>of</strong> the<br />
previous chapter. Any specific differences from these procedures are documented under the<br />
separate experiments when outlined below. <strong>The</strong> procedures were written out prior to<br />
performing them, I have therefore re-presented them here using the present tense and<br />
numerical documentation as in the original instructions that I devised'<br />
<strong>The</strong> results were analysed using the Statistical Products and Services Solutions @ackage 7'0,<br />
SPSS Inc, Chicago, USA). This employs Students t-test for matched pairs' An analysis <strong>of</strong><br />
variance for repeated measures was used to determine the effects <strong>of</strong> flow, mouth pressure, NO<br />
background and pre/post water consumption using the subjects as a blocking variable'<br />
Differences are represented by mean, standard error <strong>of</strong> the mean (SEM) or 95Vo confidence<br />
intervals (SD) where appropriate. <strong>The</strong> correlation coefficient for NO levels at the increasing<br />
expiratory flows was calculated for each individual subject and is given for the group as a<br />
whole.<br />
7.3<br />
7.3.1 Hypothesis<br />
1. <strong>The</strong> levels <strong>of</strong> NO measured in exhaled air will alter with changes in the expiratory flow.<br />
7.3,2 Aim<br />
1. To compare exhaled NO levels measured at different expiratory flows'<br />
7.3.3 Procedure<br />
Ten adult volunteers (six females) were enrolled. All measurements were made by the t-piece<br />
system. <strong>The</strong> subjects performed five exhalations at each <strong>of</strong> four expiratory flows: 25Omls/min'<br />
500mls/min, 75omls/min, and 1lo0mls/min with a final set <strong>of</strong> measurements at the first flow<br />
to rule out an order effect. Switching the machinery on in advance and calibration between<br />
and after each subject occurred as discussed in the previous chapter (Section 6.5'l)'<br />
155
<strong>The</strong> exhalations were carried out in the standard manner:<br />
o <strong>The</strong> subject abstains from food and drink for 4 hours prior to the experiment.<br />
o <strong>The</strong> subject sits at rest for at least 5 minutes.<br />
r <strong>The</strong> chart recorder is started 30 seconds prior to each exhalation to document baseline pre-<br />
exhalation measurements.<br />
o <strong>The</strong> subject puts on nasal clips 5 seconds prior to each exhalation and exhales by mouth,<br />
and removes the clips between exhalations.<br />
o <strong>The</strong> subject inhales to total lung capacity then performs a slow vital capacity manoeuvre<br />
to residual volume over 30-60 seconds.<br />
o <strong>The</strong> exhalations occur 3 minutes apart.<br />
o <strong>The</strong> subject exhales at a constant mouth pressure <strong>of</strong> 4mmHg voluntarily controlled by the<br />
subject observing the pressure gauge and keeping it at level '10' on the scale.<br />
o <strong>The</strong> subject exhales at a constant flow for 5 exhalations at each <strong>of</strong> 4 expiratory flows. <strong>The</strong><br />
flow is voluntarily controlled by the subject keeping the rotameter at specific levels:<br />
Flow Rotameter level<br />
250 mls/min 1.5<br />
500 mls/min 3.0<br />
750 mls/min 4.5<br />
1100 mls/min 6.0<br />
o <strong>The</strong> flow rate at which participants started, either 25Omls/min and increasing<br />
consecutively or 1l00mls/min and decreasing consecutively, was made by tossing a coin.<br />
o <strong>The</strong> subject continues until 5 exhalations at each flow level adequate for measurement are<br />
made.<br />
o A final set <strong>of</strong> measurements at the first flow was made.<br />
o <strong>The</strong> subject was breathing ambient air when the natural background level <strong>of</strong> NO was less<br />
than 10ppb.<br />
7.3.4 Results<br />
<strong>The</strong> mean peak concentrations <strong>of</strong> NO at the four expiratory flow settings were 79.0ppb (SEM<br />
15.5) at 250mls/min, 67.8ppb (SEM 13.5) at 500mls/min, 59.lppb (SEM ll.2) at 75Omls/min<br />
and 54.1ppb (SEM 10.7) at ll00mls/min (see Table 7.1). <strong>The</strong>re were strong linear relations<br />
between the NO concentrations obtained at the four flow levels for each individual subject<br />
with a correlation coefficient <strong>of</strong> 0.85 (see Figure 7.1). <strong>The</strong> analysis <strong>of</strong> variance showed a<br />
highly significant difference across the flow rates (p
individual subjects (see Figure 7 .2 for an example <strong>of</strong> the recording on one subject)' <strong>The</strong> mean<br />
decrease in exhaled NO was 35ppb (957o C| 25.7,43.4) from the expiratory flow rate <strong>of</strong><br />
25omls/min to ll0omls/min. <strong>The</strong>re were no significant differences in the mean CO2 levels,<br />
mean mouth pressure (controlled) or mean duration <strong>of</strong> exhalation in the measurements made<br />
at the differing flow rates. <strong>The</strong>re was no difference between the first baseline and repeated<br />
baseline <strong>of</strong> NO when the expiratory flow was the same (p-0.9 95Vo CI -16'9, 17'0), excluding<br />
an order effect (see Table 7.2).<br />
Table 7.1: NO, COz and duration <strong>of</strong> each exhalation at different expiratory flows<br />
NO is measured as peak NO in parts per billion, COz is measured as peak aq the percentage <strong>of</strong><br />
QOz<br />
total gases, duration is measured in seconds. Lach result is the mean <strong>of</strong> five exhalations for every<br />
subject. a. Duratn = duration <strong>of</strong> exhalation.<br />
Figure 7.1: Exhaled NO results with increasing expiratory flows<br />
160<br />
og rao<br />
.E 120<br />
$ 1oo<br />
o80<br />
=60 *40<br />
t<br />
,i 20<br />
0<br />
250 mls/mln 500 mlr/mln 750 mle/mln 1100 mlr/mln<br />
Sublect Gender Age NO COz Durtn' NO COz Durtn NO COz Durtn NO COr Durtn<br />
1 Female 32 23 4.7 57 17 4.5 59 23 4.5 55 4.5 50<br />
2 Male 33 102 7.2 52 89 7.0 55.0 73 6.3 55 TI 6.2 51<br />
3 Male 37 78 6.0 56 68 6.1 56 60 5.9 62 51 5.9 62<br />
4 Male 49 85 6.4 66 70 5.7 59 79 5.8 62 70 6.0 61<br />
c Female 43 31 6.2 55 23 6.2 54 19 5.8 54 17 6.0 54<br />
6 Female 32 147 6.2 63 115 6.1 64 100 6.2 68 92 6.1 63<br />
7 Female 37 83 5.7 50 86 5.8 52 82 5.9 47 76 6.0 44<br />
8 Female 35 143 6.4 71 127 6,2 7',| 95 5.2 67 85 6.2 69<br />
9 Male 43 67 6.7 84 60 6.9 72.1 43 2.0 71.0 38 7.0 71<br />
10 Female 36 30 6.1 58 24 6.0 57 18 5.9 55 15 5.8 50<br />
L<br />
__ F.-f<br />
2g 500 7s0<br />
Explratory flows In mls/mln<br />
Mean peak exhaled No levels at different expiratory flows for ten individuals (six females). Each point<br />
is the mean <strong>of</strong> five exhalations.<br />
t57<br />
------<br />
a<br />
11(n
Figure 7.2: Example <strong>of</strong> the tracing in one subject at two different expiratory flows<br />
7 .4 Methodological experiment three -<br />
7.4.1 Hypotheses<br />
effect <strong>of</strong> pressure<br />
l. <strong>The</strong> levels <strong>of</strong> NO will alter with different pressure.<br />
2. <strong>The</strong> levels <strong>of</strong> NO measured in exhaled air will be different with changes in expiratory<br />
mouth pressure.<br />
7.4.2 Aim<br />
1. To compare NO levels measured using a calibration gas delivered to the NO analyser at<br />
different pressures.<br />
2. To compare NO levels measured at different expiratory mouth pressures.<br />
7.4.3 Procedure<br />
(i) A calibration gas <strong>of</strong> 110ppb was applied to the NO analyser as measured via the direct<br />
system and was delivered to the analyser at pressures from 4 to 4OmmHg in increasing<br />
increments <strong>of</strong> 4mmHg determined by the pressure gauge, and the NO level recorded.<br />
158
(ii) Ten adult volunteers (eight females) were enrolled. All measurements were made by<br />
the t-piece sampling system. <strong>The</strong> subjects performed five exhalations at each <strong>of</strong> four<br />
expiratory mouth pressures <strong>of</strong> 4mmHg, 8mmHg, l2mmHg and l6mmHg with a final<br />
set <strong>of</strong> measurements at the first mouth pressure to rule out an order effect. Switching<br />
the machinery on in advance and calibration between and after each subject occurred<br />
as discussed in the previous chapter (Section 6'5'1)'<br />
<strong>The</strong> exhalations were carried out in the standard manner:<br />
o <strong>The</strong> subject abstains from food and drink for 4 hours prior to the experiment'<br />
o <strong>The</strong> subject sits at rest for at least 5 minutes.<br />
r <strong>The</strong> chart recorder is started 30 seconds prior to each exhalation to document baseline pre-<br />
exhalation measurements.<br />
o <strong>The</strong> subject puts on nasal clips 5 seconds prior to each exhalation and exhales by mouth,<br />
and removes the clips between exhalations'<br />
o <strong>The</strong> subject inhales to total lung capacity then performs a slow vital capacity manoeuvre<br />
to residual volume over 30-60 seconds.<br />
o <strong>The</strong> exhalations occur 3 minutes apart.<br />
o <strong>The</strong> subject exhales at a constant flow <strong>of</strong> 5O0mls/min voluntarily controlled by the subject<br />
observing the rotameter and keeping it at level '3' on the scale.<br />
o <strong>The</strong> subject exhales at a constant mouth pressure for 5 exhalations at each <strong>of</strong> 4 different<br />
pressures which are voluntarily controlled by the subject keeping the pressure gauge at<br />
specific levels:<br />
Mouth Pressure<br />
4 mmHg<br />
8 mmHg<br />
12 mmHg<br />
16 mmHg<br />
Pressure Gauge Level<br />
o <strong>The</strong> initial pressure <strong>of</strong> either 4mmHg and increasing consecutively or 16mmHg and<br />
decreasing consecutively was made by tossing a coin'<br />
o <strong>The</strong> subject continues until 5 exhalations at each pressure level adequate for measurement<br />
are made.<br />
o A final set <strong>of</strong> measurements at the first pressure is made'<br />
o <strong>The</strong> testing is done at a time when the ambient NO level is less than 10ppb.<br />
159<br />
10<br />
20<br />
30<br />
40
7.4.4 Results<br />
<strong>The</strong>re was an increase in the peak NO signal <strong>of</strong> 5.6ppb seen across the range from 4mmHg to<br />
4OmmHg <strong>of</strong> pressure when the calibration gas was applied to the NO analyser in increments<br />
<strong>of</strong> 4mmHg, with the mean and standard deviation shown for each level in Figure 7.3.<br />
Figure 7.3: <strong>The</strong> etfect <strong>of</strong> pressure on the calibration gas measurement<br />
1{O<br />
Euo<br />
; 100<br />
t<br />
€&<br />
CL<br />
.E 60<br />
og 640<br />
o<br />
En<br />
81210?fJ2428323640<br />
pros$Jra ol oallbratori gas dellvery In mmHg<br />
<strong>The</strong> mean and standard deviation <strong>of</strong> the NO levels <strong>of</strong> a known calibration gas with a concentration <strong>of</strong><br />
110ppb delivered to the NO analyser at incremental pressures with an increase <strong>of</strong> 5.6ppb<br />
demonstrated over this range.<br />
<strong>The</strong> mean peak concentrations <strong>of</strong> NO obtained at the four mouth pressure settings were<br />
61.0ppb (SEM 15.1) at 4mmHg,55.2ppb (SEM 12.3) at 8mmHg, 47.3ppb (SEM 8.4) at<br />
l2mmHg and 4O.lppb (SEM 7.2) at l6mmHg (see Table 7.2).<strong>The</strong>re was no significant<br />
difference in the exhaled NO levels obtained at each mouth pressure. Figure 7.4 shows that in<br />
the majority <strong>of</strong> patients NO levels did not change. However in two subjects who found the<br />
higher pressures difficult to sustain (see reduced duration <strong>of</strong> exhalation in the Table 7.3 for<br />
these two subjects at this higher pressure) there was a drop in NO as mouth pressure<br />
increased. <strong>The</strong>re was no difference between the first baseline and repeated final baseline <strong>of</strong><br />
NO when the mouth pressure was the same (p = 0.M 95Vo CI -16.7, 15.6) thus excluding an<br />
order effect (see Table 7 .4). <strong>The</strong>re was no significant difference between the flows measured<br />
at the different mouth pressure readings. <strong>The</strong> peak expired CO2 at different mouth pressure<br />
levels tended to decrease. <strong>The</strong> mean peak values were 5.907o (SEM 0.83) at 4mmHg, 5.83Vo<br />
160
(sEM 0.82) at 8mmHg, 5.67o7o (SEM 0.86) at l2mmHg and then 5.53Vo (SEM 0.95) at<br />
l6mmHg. <strong>The</strong>re was also a tendency for the duration <strong>of</strong> exhalation to decrease, from 49'9<br />
seconds (SEM 4.9) at4mmHg, 49.6 seconds (SEM 5.6) at 8mmHg, 44'2 seconds (SEM 6'3)<br />
at l2mmHg to 40.9 seconds (SEM 5.5) at 16mmHg'<br />
Figure 7.4: Exhaled No results at increasing expiratory mouth pressure<br />
160<br />
ll<br />
& 140<br />
.E tzo<br />
E 100<br />
o80<br />
=60 *40 s<br />
fi200<br />
Mean peak exhaled NO levels at different expiratory mouth pressures for ten individuals (eight<br />
females). Each point is the mean <strong>of</strong> five exhalations'<br />
TableZ.2:Comparison <strong>of</strong> the first and repeated last set <strong>of</strong> exhalations at the same expiratory flow<br />
Sublect Gender Age Expiratory<br />
flow<br />
48tz<br />
Explratory mouth Pl€ssure in mmHg<br />
Flrst Set <strong>of</strong> Exhalations<br />
Nolcorlorttn'<br />
NO is measured as peak No in parts per billion, coz is measured as peak coz as the percentage <strong>of</strong><br />
ioLt gir"r, duration is measured in seconds. Lach resutt is the mean <strong>of</strong> five exhalations for every<br />
subject. a. Duratn = duration <strong>of</strong> exhalation.<br />
161<br />
Last<br />
NO<br />
iet <strong>of</strong> Exhalatlons<br />
Co, Durtn<br />
I<br />
1 Female 32 250mls/min 23 4.7 57 28 4.5 51<br />
3 Male 37 250mls/min 78 6.0 56 80 5.9 58<br />
5 Female 43 250mls/min 31 6.2 55 28 5.4 57<br />
6 Female 32 250mls/min 147 6.2 63 145 6.3 64<br />
7 Female 37 250mls/min 83 5.7 50 111 5.8 47<br />
I Female 35 250mls/min 't43 6.4 7',! 167 6.1 66<br />
10 Female 36 250mls/min 30 6.1 58 30 5.7 54<br />
2 Male 33 1100mls/min 77 6.2 51 75 6.7 55<br />
4 Male 49 1100mls/min 70 6.0 61 65 5.8 60<br />
I Male 43 1100mls/min 38 7.0 71 34 6.8 74
Table 7.3: NO, COz and duration <strong>of</strong> each exhalation at ditferent expiratory mouth pressures<br />
Sublect Gender Ago NO COz Durtn<br />
I<br />
4 mmHg 8 mmHg 12 mmHg 16 mmHg<br />
NO COz Durtn NO GOz Durtn NO COz Durtn<br />
1 Female 52 142 6.8 51 112 6.6 44 75 6.8 44 67 6.5 40<br />
2 Female 36 46 5.8 51 & 6.0 54 40 5.6 32 36 5.1 32<br />
3 Female 36 38 4.8 48 42 4.7 51 47 4.5 49 42 4.2 46<br />
4 Female 30 11 4.6 29 11 4.5 33 11 4.1 27 4 4.3 29<br />
5 Male ,f3 s9 6.8 81 56 6.8 80 56 6.9 85 52 6.8 n<br />
6 Female 32 107 6.4 62 101 6.2 61 81 5.8 47 57 5.6 39<br />
7 Female 43 34 6.1 46 27 5.9 & 24 5.7 36 21 5.7 35<br />
I Female 36 51 6.0 32 53 6.0 34 44 6.1 u 42 6.0 29<br />
9 Male 4{r 56 5.9 54 53 5.9 53 56 5.7 44 51 5.8 41<br />
10 Female 32 62 5.1 42 58 4.9 41 55 4.6 44 53 4.3 43<br />
NO is measured as peak NO in parts per billion, COz is measured as peak COz as the percentage <strong>of</strong><br />
total gases, duration is measured in seconds. Each result is the mean <strong>of</strong> five exhalations for every<br />
subject. a. Duratn = duration <strong>of</strong> exhalation.<br />
Table 7.4: Comparison <strong>of</strong> the first and repeated last set <strong>of</strong> exhalations performed at the same<br />
expiratory mouth pressure<br />
Sublect Gender Age Explratory<br />
pressure<br />
First Set <strong>of</strong> Exhalations Last Set <strong>of</strong> Exhalations<br />
NO COz Durtnt NO GOz Durtn<br />
2 Female 36 4mmHg 46 5.8 51 78 6.1 il<br />
3 Female 36 4mmHg 38 4.8 48 42 4.6 54<br />
5 Male 43 4mmHg 59 6.8 81 59 6.9 81<br />
6 Female 32 4mmHg 107 6.4 62 110 6.5 63<br />
8 Female 36 4mmHg 51 6.0 32 65 6.0 35<br />
10 Female 32 4mmHg 62 5.1 42 53 5 45<br />
1 Female 52 16mmHg 142 6.8 51 106 6.5 48<br />
4 Female 30 16mmHg 4 4.3 29 7 4.3 20<br />
7 Female 43 16mmHg 21 5.7 35 25 5.4 30<br />
9 Male 43 16mmHg 51 5.8 41 53 5.7 39<br />
NO is measured as peak NO in parts per billion, COz is measured as peak COz as the percentage <strong>of</strong><br />
total gases, duration is measured in seconds. Each result is the mean <strong>of</strong> five exhalations for every<br />
subject. a. Duratn = duration <strong>of</strong> exhalation.<br />
r62
7.5<br />
7.5.1 Hypothesis<br />
1. Exhaled NO measurements will be affected by the ambient NO level'<br />
7.5.2 Aims<br />
l. To compare NO levels measured at a time <strong>of</strong> high and low ambient NO concentrations<br />
with the high level being background and low level provided by breathing from a<br />
reservoir system containing a low NO concentration'<br />
7.5.3 Procedure<br />
Three adult volunteers (all females) were enrolled and all measurements were made by the t-<br />
piece sampling sYstem.<br />
<strong>The</strong> first set <strong>of</strong> exhalations was carried out in the standard manner:<br />
r <strong>The</strong> subject abstains from food and drink for 4 hours prior to the experiment'<br />
o <strong>The</strong> subject sits at rest for at least 5 minutes'<br />
o <strong>The</strong> chart recorder is started 30 seconds prior to each exhalation to document baseline pre<br />
exhalation measurements.<br />
o <strong>The</strong> subject puts on nasal clips 5 seconds prior to each exhalation and exhales by mouth,<br />
and removes the clips between exhalations'<br />
o <strong>The</strong> subject inhales to total lung capacity then performs a slow vital capacity manoeuvre<br />
to residual volume over 30-60 seconds.<br />
o <strong>The</strong> exhalations occur 3 minutes apart.<br />
o <strong>The</strong> subject exhales at a constant mouth pressure <strong>of</strong> 4mmHg voluntarily controlled by the<br />
subject observing the pressure gauge and keeping it at level '10' on the scale'<br />
o <strong>The</strong> subject exhales at a constant flow at 5OOmls/min voluntarily controlled by the subject<br />
observing the rotameter, keeping it at a level at '3' on the scale'<br />
o At the end <strong>of</strong> each exhalation the connections are reversed to allow the subject and the<br />
analysers to continue to sample from the low NO reservoir'<br />
o <strong>The</strong> subject was breathing ambient air when the natural background level <strong>of</strong> NO was high<br />
- above the NO concentration normally measured on exhalation.<br />
o <strong>The</strong> subject continues until 5 exhalations adequate for measurement are made'<br />
163
<strong>The</strong> second set <strong>of</strong> exhalations involved a reservoir system (see Figure 7.5):<br />
r <strong>The</strong> ambient level is checked to make sure it is high - greater than the levels <strong>of</strong> known<br />
exhaled NO results <strong>of</strong> the subjects, ambient NO > 75ppb.<br />
o A reservoir bag (made <strong>of</strong> aluminium) is filled from a tank <strong>of</strong> NO free air (measured NO<br />
level 0-2ppb).<br />
o <strong>The</strong> reservoir bag is switched on-line to the analysers and the NO level in the bag is<br />
checked to be < 5ppb.<br />
o <strong>The</strong> subject breathes for 5 minutes from the reservoir bag. <strong>The</strong> subject exhales through a<br />
one-way valve to avoid reservoir contamination.<br />
o <strong>The</strong> analysers sampled from the reservoir system between exhalations.<br />
o For the measured exhalation the connections were turned so that the subject exhaled to the<br />
analysers.<br />
o <strong>The</strong> subject exhales at a constant mouth pressure <strong>of</strong> 4mmHg voluntarily controlled by the<br />
subject observing the pressure gauge and keeping it at level '10' on the scale.<br />
o <strong>The</strong> subject exhales at a constant flow at 5O0mls/min voluntarily controlled by the subject<br />
observing the rotameter, keeping it at '3' on the scale.<br />
o At the end <strong>of</strong> each exhalation the connections were reversed to allow the subject and the<br />
analysers to continue to sample from the low NO reservoir.<br />
o This means none <strong>of</strong> the analysers are exposed to the high ambient level <strong>of</strong> NO during this<br />
set <strong>of</strong> 5 exhalations.<br />
<strong>The</strong> third set <strong>of</strong> exhalations was carried out in the standard manner:<br />
r <strong>The</strong> measurements made were a repeat <strong>of</strong> the first set detailed above.<br />
o Between exhalations the subject was breathing as normal in the known high ambient NO<br />
level.<br />
o Between exhalations the analysers were sampling as usual from the known high ambient<br />
NO level.<br />
Note: in this experiment the measurement used was NO plateaux rather than NO peak levels,<br />
as the NO dropped from high level during the first and third set therefore a peak was not<br />
obvious, while a plateau was more easily established.<br />
7.5.4 Results<br />
<strong>The</strong> ambient NO concentration was variable, although usually did not change quickly but<br />
drifted up and down over hours. To demonstrate the appearance <strong>of</strong> the changing background<br />
r64
NO level, I have enclosed photographs (see Figure 7 .6 <strong>of</strong> two <strong>of</strong> the recordings made from the<br />
NO analyser over a 24 hour period with a slow paper speed.<br />
Figure 7.5: Measurement <strong>of</strong> exhaled NO incorporating the reservoir system<br />
tO<br />
Diagrammatic representation <strong>of</strong> the reservoir system allowing both machines and subject to sample<br />
froli low NO/NOiree air; NO, CO2, expiratory mbuth pressure and expiratory flow are all measured.<br />
165
Figure 7.6: Photographs <strong>of</strong> the changing ambient NO concentration recordings<br />
<strong>The</strong> ambient air NO concentration ranged from 91-200ppb during these experiments with a<br />
mean background NO concentration <strong>of</strong> 134.5ppb (SEM 11.9). <strong>The</strong> background concentration<br />
<strong>of</strong> the reservoir system was lppb (range 0-2ppb, SEM 0.2). Three subjects completed the<br />
experiment (mean age 37 years, all female). <strong>The</strong> mean plateau NO concentrations during the<br />
first set <strong>of</strong> exhalations with the high background NO was 94.9ppb (SEM 3.4ppb, range 84-<br />
l0lppb). <strong>The</strong> mean plateau exhaled NO concentration in the set <strong>of</strong> exhalations during which<br />
both the machine and subject were sampling from the NO-free reservoir system was 123.lppb<br />
(range 103-l64ppb, SEM I9.4). <strong>The</strong> mean plateau NO concentrations during the last set <strong>of</strong><br />
exhalations with the high background NO was 99.9ppb (SEM 20.5ppb, range 53-15lppb).<br />
Figure 7.7 shows a typical trace for one subject showing the rise to NO plateau during an<br />
exhalation made from the reservoir system and the fall to an NO plateau during an exhalation<br />
made from the high ambient NO. In the direct method, the NO level measured from the<br />
reservoir system was 41ppb (95Vo CI I9.9,62.7) greater than the measurements made before<br />
and after the reservoir system was used (see Figure 7.8). <strong>The</strong>re were no significant differences<br />
r66
in the NO plateaux seen before and after the set <strong>of</strong> exhalations done with the reservoir (p=0'2,<br />
7 ,95Vo CI -10.9, 35.8, see Table 7.5). No difference was seen in the COz or duration between<br />
the three sets <strong>of</strong> exhalations (see Table 7.6).<br />
Figure 7.7: Recording from one subject showing NO results with exhalations from low and high<br />
background NO<br />
Ri3. to llo l.yal ttotl I low losarvoit llo Gonccntration<br />
F.ll to IO l.v.l ftom I llgi ar|bi.nt XO Gooc.ntr.tlon<br />
Figure 7.8: <strong>The</strong> effect <strong>of</strong> high and low pressure on the calibration gas measurement<br />
Comparison <strong>of</strong> plateau exhaled NO levels following inhalation <strong>of</strong> high (ambient) labelled as 'pre' and<br />
,post; or low (reservoir) labelled as 'reservoir' N-O concentrations. Each point is a mean <strong>of</strong> five<br />
exhalations.<br />
.o EL<br />
o.<br />
.g<br />
o<br />
g<br />
o<br />
z E-96sxul<br />
Reservoir Post<br />
r67
Table 7.5: Exhaled NO results with high and low background NO concentrations<br />
Sublects Amblent NO Pre (hlgh NO) mean<br />
(range)<br />
One<br />
(SD)<br />
Two<br />
(cAB)<br />
Three<br />
(cMB)<br />
200.1 ppb 1 19.6ppb<br />
(105.8-135.7ppb)<br />
136.1ppb 94.9ppb<br />
(84.0-100.8ppb)<br />
91.4ppb 30.8ppb<br />
(26.9-35.2ppb)<br />
Reservolr (low NO)<br />
mean (range)<br />
202.4ppb<br />
(154.1-225.4ppb)<br />
134.0ppb<br />
(102.9-163.8ppb)<br />
32.9ppb<br />
(20.3-38.6ppb)<br />
Post (hlgh NO)<br />
mean (range)<br />
162.4ppb<br />
(98.9-2a6.1ppb)<br />
100.0ppb<br />
(52.5-1s1.2ppb)<br />
15.8ppb<br />
(16.2-22.3ppb)<br />
NB: <strong>The</strong> 'high' and 'bW NO note in the column headings refers to the ambient NO concentration<br />
during each set <strong>of</strong> exhalations measured, with each result the mean <strong>of</strong> five exhalations. NO is<br />
measured in parts per billion.<br />
Table 7.6: Exhaled COz results with high and low background NO concentrations<br />
Sublects Pre (hlsh NO)<br />
mean (range)<br />
One<br />
(SD)<br />
Two<br />
(cAB)<br />
Three<br />
CMB<br />
NB: <strong>The</strong> 'high' and 'low' NO note in the column headings refers to the ambient NO concentration<br />
during each set <strong>of</strong> exhalations measured, with each result the mean <strong>of</strong> five exhalations. COz is<br />
measured as percentage <strong>of</strong> totalgases and exhalation duration measured in seconds.<br />
7.6 Methodological experiment five -<br />
7.6.1 Hypothesis<br />
effect <strong>of</strong> water consumption<br />
l. Exhaled NO measurements will be affected by the consumption <strong>of</strong> water.<br />
7.6.2 Aim<br />
1. To compare NO levels measured by standard procedure before and after a set <strong>of</strong> testing in<br />
which water was consumed immediately before each exhalation.<br />
7.6.3 Procedure<br />
Ten adult volunteers were enrolled (eight females) and all measurements were made by the t-<br />
piece sampling system.<br />
co25.2% (5.0-5.5)<br />
Duration 52.2 secs<br />
(45-60 seconds)<br />
COzS.7To (5.6'5.8)<br />
Duration 66.0 secs<br />
(63-72 seconds)<br />
COzS.5"/o (5.3'5.9)<br />
Duration 55.2 secs<br />
(48-63 seconds)<br />
<strong>The</strong> first set <strong>of</strong> exhalations was carried out in the standard manner:<br />
o <strong>The</strong> subject abstains from food and drink for 4 hours prior to the experiment.<br />
168<br />
Reservolr (low NO)<br />
mean (range)<br />
co2 5.1% (4.9'5.2)<br />
Duration 54.0 secs<br />
(36-63 seconds)<br />
COz5.7o/" (5.6'5.8)<br />
Duration 65.4 secs<br />
(57-69 secs)<br />
coz 5.6% (5.4-5.7)<br />
Duration 58.2 secs<br />
(54-69 seconds)<br />
Post (hlgh NO)<br />
mean (range)<br />
co25.1"h (5.0-5.2)<br />
Duration 51.6 secs<br />
(39-63 seconds)<br />
coz5.8To (5.7-5.9)<br />
Duration 56.4 secs<br />
(42-63 seconds)<br />
COz5.5o/o (5.4-5.6)<br />
Duration 57.0 secs<br />
(54-60 seconds)
. <strong>The</strong> subject sits at rest for at least 5 minutes'<br />
o <strong>The</strong> chart recorder is started 30 seconds prior to each exhalation to document baseline pre<br />
exhalation measurements.<br />
o <strong>The</strong> subject puts on nasal clips 5 seconds prior to each exhalation and exhales by mouth,<br />
and removes the clips between exhalations'<br />
o <strong>The</strong> subject inhales to total lung capacity then performs a slow vital capacity manoeuvre<br />
to residual volume over 30-60 seconds'<br />
o <strong>The</strong> exhalations occur 3 minutes apart.<br />
o <strong>The</strong> subject exhales at a constant mouth pressure <strong>of</strong> 4mmHg voluntarily controlled by the<br />
subject observing the pressure gauge and keeping it at '10' on the scale'<br />
o <strong>The</strong> subject exhales at a constant flow at 500mls/min voluntarily controlled by the subject<br />
observing the rotameter, keeping it at '3' on the scale'<br />
o <strong>The</strong> subject was breathing ambient air when the natural background level <strong>of</strong> NO was less<br />
than lOPPb.<br />
o <strong>The</strong> subject continues until 5 exhalations adequate for measurement are made'<br />
For the second set <strong>of</strong> exhalations:<br />
o <strong>The</strong> procedure followed the standard set above'<br />
o <strong>The</strong> subject consumed 60mls <strong>of</strong> water 20 seconds to 5 seconds prior to the next<br />
exhalation. <strong>The</strong> water temperature was hot in five subjects and cold for five subjects'<br />
<strong>The</strong> third set <strong>of</strong> exhalations was carried out in the standard manner detailed above as for the<br />
first set <strong>of</strong> exhalations.<br />
7.6.4 Results<br />
<strong>The</strong> mean peak NO concentrations in the 10 subjects was 93'7ppb (SEM 20'8)' This<br />
decreased by 23ppb (957o CI I2.g, 33.I) to 70.8ppb (sEM 16'5, p-0'002) during the<br />
measuremenrs made with the subjects drinking 60mls <strong>of</strong> water before each exhalation' <strong>The</strong>re<br />
was then a significant increase <strong>of</strong> l7ppb (95Vo CI -9.7, -24'l) to 87'8ppb (SEM 18'7' p -<br />
0.001, see Figure ?.9) in the subsequent recordings made in the standard way' <strong>The</strong><br />
consumption <strong>of</strong> water reduced the levels on No obtained regardless <strong>of</strong> the water temperature'<br />
In the testing with cold water the mean NO went from 76.9ppb to a mean <strong>of</strong> 57.3ppb, and<br />
then returned to a mean <strong>of</strong> 72.3ppb in the final set <strong>of</strong> exhalations. With hot water the mean<br />
NO <strong>of</strong> 110.5ppb dropped to 84ppb and then returned to 102.8ppb. <strong>The</strong>re was no difference in<br />
r69
the NO concentrations taken pre and post the water experiment (p=0.095). <strong>The</strong>re was<br />
difference in the COz or duration <strong>of</strong> expiration across the three sets <strong>of</strong> exhalations made.<br />
Figure 7.9: <strong>The</strong> etfect <strong>of</strong> consuming water on the subsequent exhaled NO levels measured<br />
.o 2fi<br />
e<br />
Eo 't<br />
a lso<br />
o = 100<br />
E Ps0<br />
x<br />
ttJ<br />
0<br />
Comparison <strong>of</strong> peak exhaled NO levels with the exhalation performed in a standard manner pre and<br />
post a set <strong>of</strong> exhalations done where the consumption <strong>of</strong> either hot (in red) or cold (in black) took<br />
place just prior to each exhalation. Each point is a mean <strong>of</strong> five exhalations.<br />
7.7 Discussion: which measurement factors alter nitric oxide levels?<br />
<strong>The</strong> methodological experiments described in Chapter 6 and 7 demonstrated that the<br />
measurement <strong>of</strong> NO in exhaled air was feasible. A number <strong>of</strong> cross-sectional studies looking<br />
at exhaled NO concentrations in different subject groups had been reported. <strong>The</strong> findings<br />
within each research team had been consistent, but the absolute levels <strong>of</strong> NO reported by the<br />
investigators in similar subject $oups were very different. All these investigators used NO<br />
chemiluminescence analysers which, although developed by different companies, had similar<br />
sensitivities and they stated that regular calibration was being performed. <strong>The</strong> study groups<br />
were similar. For example, the results in healthy control subjects through the 1990s were<br />
reported with means <strong>of</strong> 3.25ppb, 4.7Sppb and 100.25ppb under different conditions @ersson,<br />
Wiklund et al. 1993), 4.7ppb (Massaro, Mehta et al. 1996),6.2ppb (Massaro, Gaston et al.<br />
1995), 8.lppb (Borland, Cox et al. 1993), 8.4ppb @ersson, Zetterstrom et al. 1994), 8.6ppb<br />
(Trolin, Anden et al. 1994), 9ppb (Alving, Weitzberg et al. 1993), 11.lppb (Martin, Bryden et<br />
al. 1996), l6ppb (Schedin, Frostell et al. 1995), l9ppb and 2lppb (Schilling, Holzer et al.<br />
1994),22ppb (Kimberly, Nejadnik et al. 1996), 26.3ppb (Iwamoto, Pendergast et al. 1994),<br />
34ppb (Jilma, Kastner et al. 1996),42ppb (Gerlach, Rossaint et al. 1994), 51 NO g-r 6Morris,<br />
Sooranna et al. 1996), 70ppb and 75ppb (Kharitonov, Logan-Sinclair et al. 1994), 80.2ppb<br />
(Kharitonov, Yates et al. 1994), 88ppb (Kharitonov, Robbins et al. 1995), 89ppb (Kharitonov,<br />
Wells et al. 1995) and l05.5ppb (Robbins, Floreani et al. 1996). Similar ranges in NO results<br />
t70
etween these researchers were documented in the literature for other subject groups such as<br />
those with asthma, bronchiectasis, hypertension and smokers. <strong>The</strong> measurement <strong>of</strong> NO in<br />
these studies did vary between mean peak exhaled NO, mean plateau exhaled NO and NO<br />
measured during tidal breathing. This led me to the suspicion that the technique <strong>of</strong><br />
measurement critically affected the No levels obtained. In the first methodological study in<br />
twelve healthy adults (Byrnes, Dinarevic et al. 1997), I demonstrated that the concentration <strong>of</strong><br />
NO obtained could be halved depending on whether the subject was breathing directly into<br />
the No analyser (mean 84.Sppb) or breathing through a t-piece system (mean 41.2ppb)' <strong>The</strong><br />
main difference between these two methods was a change in flow from 440 mls/min to 665<br />
mls/min. <strong>The</strong> methodology experiments detailed in this chapter were therefore designed to<br />
determine what factors altered the concentration <strong>of</strong> exhaled No obtained.<br />
In the second methodological study, changing the expiratory flow rate significantly altered the<br />
mean concentration <strong>of</strong> NO obtained. <strong>The</strong> higher the expiratory flow' the lower the<br />
concentrations recorded, with a mean decrease <strong>of</strong> 35ppb when moving from 25omls/min to<br />
1100mls/min in ten subjects (see Figure 7.1). Most <strong>of</strong> the literature at the time in the cross<br />
sectional studies had usually mentioned the samplin g rate <strong>of</strong> the machine only, or measured<br />
tidal volumes over a L-2 minute time periods with none specifically noting a standard<br />
expiratory flow. Alving et al reported that if the airflow was increased in 12 healthy controls<br />
from2umin-l to 5 Umin-l a slight increase in the levels <strong>of</strong> No was noted -<br />
the data were not<br />
shown and the increase was noted to be insignificant. This proved a different finding from<br />
most others (Alving 1993). Imada et al showed a hyperbolic relationship between the NO<br />
concentration and the sampling flow rate (see Figure 7'10) with a marked reduction <strong>of</strong><br />
exhaled No when increasing flow in one subject (Imada 1996).<br />
t7r
Figure 7.10: Hyperbolic relationship between exhaled NO and sampling flow rate<br />
800<br />
e 800<br />
5'<br />
e 400<br />
200<br />
0<br />
305*(x-0.07)<br />
r=0.995, p
Figure 7.1 1: Exhaled No determined at single breath plateau concentrations at increasing expiratory<br />
flows and at increasing expiratory mouth pressures<br />
E r.o<br />
Ci 15.0<br />
2 ; 10.0<br />
6<br />
E s.o<br />
ril o.o<br />
r 1.5<br />
o<br />
'".<br />
-E t.o<br />
O'1""'O"'O"''<br />
I<br />
15<br />
l0<br />
5<br />
0<br />
1.5<br />
?? I T I<br />
+-f "'1""?"'1'.0<br />
II--<br />
o<br />
U<br />
x<br />
o 0.5<br />
z 051015n<br />
Arway presswo, cmHP<br />
T t "la-...+..o]..{<br />
T '.--.1<br />
, J{""'t"'i<br />
fr^<br />
5o 10 150 200 250 s<br />
grytmryflownF, mLrl<br />
Taken from Hogman M, Stromberg s, schedin Frostell c, Hedenstierna G, Gustafsson LE' Nitric oxide<br />
from the human r"rpit'"tow tract-efficiently quantified by standarized single bl99!h measurements'<br />
n.i" iny"iologica Sclndinavia 1997; 159: i+S'Sae (Hogman, Stromberg et al' 1997)'<br />
In the pressure experiment, there were no significant differences between the mean exhaled<br />
NO concentrations at 4mmHg, 8mmHg, l2mmHg and 16mmHg' In Figure 7 '3 it can be seen<br />
that most <strong>of</strong> the ten subjects appeared to have flat traces across the sets <strong>of</strong> exhalation but the<br />
top two and the bottom one subjects' traces do appear to have a reduction in No with the<br />
increased pressure settings. <strong>The</strong>re was a trend towards differences between the peak COz<br />
levels reached being 5.907o at 4mmHg, 5.837o at 8mmHg, 5'83Vo at l2mmHg and 5'53Vo<br />
reached at 16mmHg. This is likely to be accounted for by there also being differences in the<br />
duration <strong>of</strong> exhalation with 49.9s at 4mmHg, 49.6s at 8mmHg ,44.2s at l2mmHg and 40'9s at<br />
16mmHg. <strong>The</strong> differences probably reflect the difficulty for subjects <strong>of</strong> maintaining<br />
exhalation against a strong resistance at constant flow to generate the higher mouth pressures'<br />
subjects found it tiring to maintain this pressure and found it difficult to keep their lips tight<br />
around the mouthpiece so as not to allow any air to escape when exhaling' When designing<br />
the experiment in the preliminary trials both myself and Carolyn Busst tried a range <strong>of</strong><br />
expiratory mouth pressures up to 2OmmHg, and we quickly dropped this last high pressure as<br />
being extremely difficult to exhale against. Two other studies published at the same time<br />
showed no difference in the NO with differing pressures. In five subjects there was no<br />
difference between an expiratory pressure <strong>of</strong> 6mmHg and 2ommHg (Silk<strong>of</strong>f 1997 ' Marked<br />
flow dependence). And in six subjects at expiratory pressures over 0 to 20cmHzO there was<br />
no difference in exhaled NO (see Figure 7.9 above, Hogman)' It would appear that mouth<br />
173
pressure over the likely range encountered (such as the lower three pressure settings that I<br />
tested) has no relevant effects on the measurements in most individuals. Conespondingly<br />
there was no difference in NO, CO2, flow or duration <strong>of</strong> exhalation across these readings.<br />
However as two, possibly three, individuals did show an effect, this again suggested the need<br />
also to standardise mouth pressure for exhaled NO measurements.<br />
<strong>The</strong> background NO concentration is variable and occasionally can be extremely high. In<br />
many <strong>of</strong> the published studies, the ambient NO level at the time <strong>of</strong> testing was not stated and I<br />
felt this may be another reason for error in the results obtained between and within groups.<br />
This could occur particularly if testing was done on different days pre and post an intervention<br />
with a subject group when the background levels may differ over time. In three subjects, I<br />
showed that there were large differences in the mean exhaled NO concentration obtained<br />
depending on the concentration <strong>of</strong> the NO being inhaled. In this experiment, the exhaled NO<br />
measured was higher when inhaling low reservoir NO than when inhaling high ambient<br />
levels. Three studies documented inhalation from NO-free air (Alving, Weitzberg et al. 1993;<br />
Schilling, Holzer et al. 1994; Steerenberg, Nierkens et al. 2000) with one having noted that<br />
the ambient air contained high and variable amounts <strong>of</strong> NO concentration (6-192ppb) leading<br />
them to prepare and recommend an NO free reservoir for inhalation (Schilling, Holzer et al.<br />
L994). Another study mentioned that the ambient NO air was recorded and the absolute zero<br />
was adjusted just before each measurement by flushing the NO analyser with NO free<br />
certified air (Kharitonov, Logan-sinclair et al. L994). A number <strong>of</strong> studies recorded levels <strong>of</strong><br />
ambient air at 0-20ppb (Persson, Wiklund et al. 1993), 5-20ppb (Kharitonov, Yates et al.<br />
Lgg6), 0-38ppb (Kharitonov, Yates et al. 1994; Jilma, Kastner et al. 1996; Kimberly,<br />
Nejadnik et al. 1996) and 0-68ppb (Kharitonov, Robbins et al. 1995) but did not seem to<br />
affect the readings and therefore were not taken into account. One goup had specifically<br />
requested two volunteers to inhale an NO concentration <strong>of</strong> 800ppb, hold their breath for<br />
fifteen seconds and then exhale into the NO analyser. As they documented no change in the<br />
NO concentration before and after inhalation, they concluded that the inspired NO must<br />
disappear from the respiratory tract within that time (Kharitonov, Yates et al. 1994).<br />
While the effect <strong>of</strong> ambient NO was either not specifically tested or was not published at this<br />
time by any <strong>of</strong> these other groups, some researchers did investigate whether inhalation via the<br />
nose or the mouth made a difference to the exhaled NO levels measured. This was an area <strong>of</strong><br />
variability, now known to be the degree <strong>of</strong> contamination <strong>of</strong> exhaled NO by nasal and sinus<br />
production. <strong>The</strong> measured NO levels have been found to be greatest in the paranasal sinuses<br />
(Lundberg, Farkas-Szallasi et al. 1995) with an age-dependent increase in keeping with sinus<br />
174
pneumatisation. A progressive decrease in NO concentration had been found when sampling<br />
progressively down the respiratory tract from nasal passages (Alving, Weitzberg et al' 1993;<br />
Kimberly, Nejadnik et al. 1996), oral cavity (Gerlach, Rossaint et al. 1994), and below the<br />
vocal cords (Lundberg, Weitzberg et al. 1994). <strong>The</strong> slow expiration time, particularly in the<br />
direct measurements, did raise the possibility that I was merely recording inspired air,<br />
contaminated by NO produced from the nose and sinuses, and exhaled unchanged' Schedin et<br />
al showed the difference in exhaled NO in tidal breathing when the inhalation was by nose<br />
giving a mean <strong>of</strong> 64ppb and by mouth giving a mean <strong>of</strong> 13ppb (Schedin, Frostell et al' 1995)'<br />
Similarly when single exhalations were measured, nasal inhalation gave exhaled oral results<br />
<strong>of</strong> 32ppb and22ppbwhile inhalation by mouth gave exhaled oral results <strong>of</strong> gppb and 9ppb in<br />
two other studies (Alving l993;Kimberly 1996). However using a faster (t-piece) method in<br />
our own study did not reveal an abrupt discontinuity suggestive <strong>of</strong> emptying unchanged dead<br />
space prior to measuring true exhaled NO. <strong>The</strong> other area <strong>of</strong> difference between groups that<br />
was studied by others at this time, which I did not exatnine, was the effect breath-holding had<br />
on the subsequent exhaled NO. Using collection into a reservoir system, Sato et al showed<br />
that the NO levels increased in proportion to the duration <strong>of</strong> exhalation and to duration <strong>of</strong> a<br />
pre-exhalation breath-hold in 16 controls (Sato, Sakamaki et al. 1996)' In 18 controls Martin<br />
et al reported the increase <strong>of</strong> exhaled No from no breath-hold at ll.lppb to l5'6ppb and<br />
32.lppb in exhalations following ten and 60 second breath-holds respectively (Martin, Bryden<br />
et al. 1996). <strong>The</strong> findings were simil ar in 32 subjects with allergic rhinitis from a baseline<br />
exhaled NO at 16.2ppb to 34ppb following a ten second breath-hold and 62ppb following a<br />
sixty second breath-hold (Martin, Bryden et al. 1996). Kimberly et al in eight controls showed<br />
an increase in exhaled No from Tppb to 23ppb with a 30 second breath-hold (Kimberly'<br />
Nejadnik et al. 1996). persson et al recorded the most dramatic increase from 3.25ppb with<br />
tidal breathing to 100.25ppb with a breath-hold <strong>of</strong> 60 seconds (Persson, Wiklund et al' 1993)'<br />
Kharitinov et al determined that a breath-hold for 20 seconds gave an initial peak <strong>of</strong> NO but<br />
end expiration values were similar to non breath-hold results (Kharitonov, Chung et al' 1996)'<br />
However, I believed that the finding with the ambient NO in this chapter suggested that<br />
measurements <strong>of</strong> exhaled NO must be done on days with low ambient backgound NO' or by<br />
using an NO-free circuit such as described.<br />
<strong>The</strong> final experiment may seem an unusual test to have performed, but was based on a chance<br />
finding that following drinking a glass <strong>of</strong> water, the NO levels on the next exhalations<br />
dropped dramatically. <strong>The</strong> reason I thought that this may be significant was that when doing<br />
lung function or full inspiratory/expiratory manoeuvres, a corrmon side effect, particularly in<br />
175
subjects with respiratory disease, is to cough and an equally common response is to give the<br />
person water to drink. This may be important if the levels <strong>of</strong> NO were then reduced. Exhaled<br />
NO measured in the standard way did decrease significantly from 93.7ppb to 70.8ppb when<br />
60mls <strong>of</strong> water was drunk between 20 and five seconds before the exhalation, and was not<br />
different whether that water temperature was hot or cold. Many studies did not comment on<br />
consumption <strong>of</strong> food or drink prior to the testing. One group reported asking the subjects not<br />
to consume caffeine within two hours before the testing procedure (Kharitonov, O'Connor et<br />
al. 1995; Kharitonov, Yates et al. 1996). Another goup did mention having a water absorber<br />
installed in the proximal expiratory port in collections into a reservoir system (Schilling,<br />
Holzer et al. 1994). <strong>The</strong> mechanism <strong>of</strong> the fall in the exhaled NO that I documented is<br />
unknown. One group reported in two papers that the gastrointestinal tract had higher levels <strong>of</strong><br />
NO present which could be measured when 'belching' occurred (Lundberg, Weitzberg et al.<br />
1994; Herulf, Ljung et al. 1998). <strong>The</strong> levels cited were far greater than those measured during<br />
exhalation -<br />
so it is possible that the gastro-intestinal tract may contribute to the exhaled NO<br />
levels and was reduced by drinking water. Another possibility is that <strong>of</strong> quenching <strong>of</strong> the NO<br />
gas. One study showed that prolonged sampling <strong>of</strong> wet gas resulted in an increased time for<br />
the initial calibration to reach stable values from five to 20 minutes. <strong>The</strong>y also demonstrated a<br />
reduction in exhaled levels with increased water vapour and concluded that a decrease <strong>of</strong> NO<br />
readings in water-saturated samples may have contributed to variation across studies (van der<br />
Mark, Kort et al.1997).<br />
In conclusion, several factors critically affect the measured mean peak concentration <strong>of</strong><br />
exhaled NO. An increase in expiratory flow rate resulted in a decreased concentration <strong>of</strong> NO.<br />
Although there was no difference at increasing expiratory mouth pressures for most <strong>of</strong> the<br />
subjects, individuals showed decreased exhaled NO concentrations and across all subjects<br />
there was a decreased duration <strong>of</strong> exhalation and COz measurement. A high inspired<br />
background NO made the exhaled NO concentrations difficult to interpret, and drinking water<br />
just prior to measurement decreased the exhaled NO obtained. <strong>The</strong>se findings confirmed that<br />
the measurement <strong>of</strong> exhaled NO concentrations in humans should be performed in a standard<br />
manner for the levels to have any meaning and to enable different teams <strong>of</strong> investigators to<br />
compare results. <strong>The</strong>se findings may have accounted for some <strong>of</strong> the discrepant results<br />
reported in the literature. Since these results were published there has been a considerable<br />
increase in the body <strong>of</strong> literature on exhaled NO with further studies in all these areas and<br />
these will be covered to the present day in Chapter 9.<br />
176
One aim <strong>of</strong> investigating the parameters that altered the levels <strong>of</strong> exhaled NO in these<br />
methodological studies was to enable standardisations for future studies. In the next chapter, I<br />
will describe the exhaled No research that I did with healthy and asthmatic children. <strong>The</strong><br />
results presented here in chapters 6 and 7 did make up some <strong>of</strong> the literature reviewed for the<br />
first American Thoracic society and European Respiratory society recommendations to<br />
standardise the methods <strong>of</strong> measuring exhaled NO'<br />
<strong>The</strong> work in this chapter formed the basis <strong>of</strong> the publication:<br />
. Byrnes CA, Dinarevjc g Bnsst CA, Bush A, Shinebournes EA. Is nitric oxide produced at<br />
airway or alveolar level? European Respiratory Journal 1997 I0 (5) 1021-1025<br />
In addition Mr Ron Logan-Sinclair went on to d.evelop the LR2000 series <strong>of</strong> NO analysers<br />
using some <strong>of</strong> the ideas generatedfrom this research (I'agan Research Ltd, Unit 82, Spectrum<br />
Business Centre, Anthony's Way, Rochester, Kent, United Kingdom), while Carolyn Busst<br />
went on to run a ski lodge in Switzerland!<br />
177
8.1<br />
Chapter 8: Exhaled nitric oxide in healthy and asthmatic children<br />
Introduction<br />
<strong>The</strong> protocol was now established and I felt we would be able to measure NO in a<br />
reproducible manner in children by standardising all <strong>of</strong> the parameters discovered to alter NO<br />
readings. Initially I wanted to measure exhaled NO in healthy children: that is children with<br />
no respiratory diagnosis or any acute or chronic respiratory symptoms. This was to confirm<br />
that the technique was possible in children and to confirm some <strong>of</strong> the findings from the adult<br />
studies presented in the previous chapters. Included was a comparison <strong>of</strong> the results from the<br />
direct versus t-piece sampling systems that incorporated a change in flow. I then wanted to<br />
compare the exhaled NO levels from these healthy 'control' children to those from asthmatic<br />
children in two categories; children on bronchodilator therapy only versus those on regular<br />
IHCS. Finally I wanted to measure the exhaled NO pre and post IHCS cornmencement in any<br />
child who was on bronchodilator therapy only but who clinically required the introduction <strong>of</strong><br />
a preventer for asthma control.<br />
At the time <strong>of</strong> corlmencement <strong>of</strong> this research there had been very little published on exhaled<br />
or nasal NO in children. <strong>The</strong>re had been a series <strong>of</strong> studies looking at the levels <strong>of</strong> NO from<br />
nasal and sinus passages. High concentrations <strong>of</strong> NO were found when aspirating air from the<br />
sinuses <strong>of</strong> five patients undergoing surgery at 9.1 parts per million (note that most <strong>of</strong> the<br />
exhaled NO reported is measured in parts per billign) (Lundberg, Farkas-Szallasi et al. 1995).<br />
An age-dependent increase in mean nasal NO plateau concentrations was also demonstrated<br />
in 49 subjects with an age range <strong>of</strong> 0-62 years, thought to be consistent with the development<br />
and pneumatisation <strong>of</strong> the paranasal sinuses (diagram included in the next chapter as Figure<br />
9.2) (Lundberg, Farkas-Szallasi et al. 1995). <strong>The</strong> NO levels sampled directly from one nostril<br />
and in exhaled air were also significantly less in four children with Kartageners syndrome at<br />
4ppb when compared to 20 healthy children at22lppb (Lundberg, Weitzberg et al. 1994).<br />
<strong>The</strong> following experiments ran from 1995-1997. At the time that we published these findings<br />
in 1996 and through 1997, a number <strong>of</strong> research groups also published results <strong>of</strong> their<br />
investigations into exhaled and nasal NO in children. In the main these were comparisons<br />
between control subjects and those with respiratory diseases. In the next section, I will present<br />
the subjects, protocols and results from the studies we conducted in healthy children and<br />
discuss the findings comparing with these other results from the literature. In the second part<br />
<strong>of</strong> the chapter I will present the literature available from studies in groups <strong>of</strong> adult asthmatics<br />
as again these were published ahead <strong>of</strong> any results in children. <strong>The</strong> procedures followed were<br />
178
the same for the children with asthma as those undertaken in the healthy children. I will<br />
present the results and discuss the findings comparing our results with those then published<br />
looking at similar groups <strong>of</strong> children.<br />
8.2<br />
Prospective ethical approval for the study was given by the Royal Brompton Hospital Ethics<br />
Committee (Royal Brompton Hospital, Sydney St, London SW3 6NP, United Kingdom; now<br />
Royal Brompton and Harefield National Health Trust)'<br />
<strong>The</strong> subjects were recruited from a local school ("Park walk Primary school"' South<br />
Kensington, London) within walking distance <strong>of</strong> the hospital. Following our initial approach<br />
and discussion with the school board, the principal and the teachers <strong>of</strong> the children in the age<br />
groups that we were hoping to enrol, a pamphlet about the study and a questionnaire were sent<br />
home with childre n in 2classes aged 9-11 years to their parent/s or caregiver/s (Appendix 2)'<br />
<strong>The</strong> school was familiar with the hospital and had been involved in previous (completely<br />
separate) research. Two <strong>of</strong> the class teachers had participated in an "Asthma at school"<br />
education programme run by myself and the respiratory nurse specialist (Ms Lizzie Webber)<br />
at the Royal Brompton Hospital which ran for three years and was open to teachers in primary<br />
schools throughout London.<br />
Each child particiPated if:<br />
o He/she had returned a signed consent and a questionnaire filled out by their parent/s'<br />
o He/she was willing to be part <strong>of</strong> the study'<br />
o He/she had permission <strong>of</strong> the class teacher to attend the hospital for an hour'<br />
o He/she had no recorded respiratory, cardiac or other major chronic disease' (<strong>The</strong><br />
exception was children with asthma who were later recruited into the second part <strong>of</strong><br />
this studY).<br />
o He/she had no upper or lower respiratory tract infection for at least four weeks'<br />
<strong>The</strong> children were walked by the investigators from the school to the hospital, completed the<br />
study and returned to the school always having been in our care' <strong>The</strong> children attended the<br />
hospital in pairs from 1030 hours to 1200 hours and were returned in time for lunch break'<br />
<strong>The</strong> aim was to enrol at least 30 paediatric subjects'<br />
<strong>The</strong> results were analysed using the Statistical Products and services solutions, (Package u<br />
7.0, SPSS Inc, Chicago, uSA). This employs Students t-test for matched pairs with mean'<br />
179
standard error <strong>of</strong> the mean (SEM), standard deviation (SD) and ranges given where<br />
appropriate. In the goup <strong>of</strong> asthmatic children that were measured before and after the<br />
commencement <strong>of</strong> IHCS, medians are give. A p-value <strong>of</strong> less than 0.05 was considered<br />
significant.<br />
8.3 Methodology <strong>of</strong> exhaled nitric oxide measurement in healthy children<br />
8.3.1 Hypotheses<br />
1. NO can be measured in exhaled air in children.<br />
2. NO levels will be different in the two methods <strong>of</strong> measurement; via the direct and the t-<br />
piece sampling systems.<br />
3. NO levels will be higher in children with current or past personal history <strong>of</strong> atopy such as<br />
allergic rhinitis and/or enzema (excluding asthma in the first part <strong>of</strong> the study).<br />
4. NO levels will be higher in children with a family history <strong>of</strong> atopy (allergic rhinitis and/or<br />
eczema and/or asthma) in first degree relatives.<br />
5. NO levels will be higher in children who may be expected to have environmental reasons<br />
for airway inflammation such as environmental smoke or presence <strong>of</strong> funy peUs within<br />
the household.<br />
8.3.2 Aims<br />
l-. To measure exhaled NO in healthy children by two methods; via the direct and the t-piece<br />
sampling systems.<br />
2. To compare the NO levels <strong>of</strong> children with and without a personal history <strong>of</strong> atopy<br />
(having excluded asthma).<br />
3 . To compare the NO levels <strong>of</strong> children with and without a family history <strong>of</strong> atopy in first<br />
degree relatives.<br />
4. To compare the NO levels <strong>of</strong> children with and without the presence <strong>of</strong> a household<br />
smoker.<br />
5 . To compare the NO levels <strong>of</strong> children with and without the presence <strong>of</strong> a household funy<br />
pet.<br />
8.3.3 Protocol<br />
<strong>The</strong> questionnaire is added as Appendix 3. Questions were asked regarding information on the<br />
following:<br />
180
Child's personal medical hlstory<br />
Current or past history<br />
<strong>of</strong> respiratory illness<br />
Current or past history <strong>of</strong> asthma<br />
Current or past history <strong>of</strong> atoPY<br />
(eczema, allergic rhinitis)<br />
Current medications<br />
Family and environmental history<br />
Fami[ history <strong>of</strong> atoPY<br />
presence <strong>of</strong> smokers in the household<br />
Presence <strong>of</strong> Pets (and tYPe)<br />
in the household<br />
<strong>The</strong> child had abstained from food and drink for two hours prior to the experiment'<br />
Height was measured on a Harpenden stadiometer (Harpenden Portable Stadiometer'<br />
Crosswell, Crymych, Pembrokeshire, UK) and weight was measured on a digital Seca scale<br />
(Seca 770 medical scales, Seca Ltd, 4802 Glenwood Rd, Brooklyn NYl1234)' Lung function<br />
was measured via spirometry and in accordance to the ATS criteria (American Thoracic<br />
Society and Association lgg4) and the best <strong>of</strong> three reproducible flow volume loops was<br />
recorded using a Compact Vitalograph (Vitalograph Limited, Buckingham, United Kingdom)'<br />
<strong>The</strong> results are presented as percent predicted for age, sex and height as defined by the Polgar<br />
reference equation. All measurements were conducted by myself (predominantly taking care<br />
<strong>of</strong> the NO readings) or Dr Senka Dinarevic (predominantly taking care <strong>of</strong> the lung function<br />
testing). All results were then worked out by using the calibrations on the chart recording<br />
system by both and entered into the SPSS database by myself and checked by Dr Senka<br />
Dinarevic.<br />
<strong>The</strong> experiments were made if the ambient level <strong>of</strong> NO was less than 10ppb and all inhalation<br />
was done from ambient air. <strong>The</strong> procedures as described in the previous section (Section 6.5)<br />
for setting up and calibrating all equipment were made before and after every two subjects'<br />
<strong>The</strong> procedure listed below is re-presented exactly as it was designed and placed on the wall<br />
adjacent to the analysers. It is therefore in the present tense and with numerical rather than<br />
written designation <strong>of</strong> numbers.<br />
Direct to analYsers method:<br />
o <strong>The</strong> child sits at rest for at least 5 minutes<br />
r Each child inhales to total lung capacity then performs a slow vital capacity manoeuvre to<br />
residual volume or for as long as possible<br />
o 5 exhalations are made consecutively at 3-minute intervals<br />
. Nose clips are worn 5 seconds before the exhalation, and taken <strong>of</strong>f between measurements<br />
. Measurements <strong>of</strong> NO, mouth pressure and COz are made<br />
o Mouth pressure is standardised to 4 mmHg by a fixed restriction<br />
o <strong>The</strong> combined analysers sampled at 44Omls/min<br />
181
T-piece sampling system to analysers method:<br />
o <strong>The</strong> child sits at rest for at least 5 minutes<br />
o Each child inhales to total lung capacity then performs a slow vital capacity manoeuvre to<br />
residual volume or for as long as possible<br />
o 5 exhalations are made consecutively at 3 minute intervals<br />
. Nose clips are worn 5 seconds before the exhalation, and taken <strong>of</strong>f between measurements<br />
o Measurements <strong>of</strong> NO, mouth pressure and CO2, and, in addition, flow are made<br />
o Mouth pressure is standardised to 4 mmHg by a fixed restriction<br />
r Flow in the t-piece is standardised by having the subject exhale at a constant rate <strong>of</strong><br />
225mllmin. <strong>The</strong> subjects control their expiratory flow rates voluntarily using feedback by<br />
observing the signal from the pneumotachograph<br />
r <strong>The</strong> combined analysers plus flow then sampled at 665mls/min<br />
<strong>The</strong> children did one set <strong>of</strong> five exhalations under each condition. <strong>The</strong> subjects were<br />
randomised by tossing a coin (heads - direct, tails -<br />
t-piece) to do either direct or t-piece first,<br />
followed by a five minute break then the other set <strong>of</strong> conditions. <strong>The</strong> children continued until<br />
five sets <strong>of</strong> measurable exhalations were made or it was obvious that the procedure was<br />
difficult for the individual child.<br />
<strong>The</strong> recordings were made in the same way as in the adult study with exhaled NO traced on<br />
the chart recording system in green, exhaled COz in red, expiratory mouth pressure in blue<br />
and flow in the t-piece sampling system in brown. A photograph <strong>of</strong> a child practising the<br />
procedure is seen in Figure 5.4. <strong>The</strong> peak NO levels and area under the curve <strong>of</strong> the NO trace<br />
were measured and compared. <strong>The</strong> mean mouth pressure was kept at 4mmHg in the direct and<br />
t-piece sampling, and the mean flow in the t-piece conditions kept at 225mllmin in addition to<br />
the flow required for the other analysers. Each result presented is the mean <strong>of</strong> five<br />
exhalations.<br />
8.3.4 Results<br />
We recruited 39 healthy children (23 girls, 16 boys) mean age9.9 years (range 9-11 years)<br />
from the local school. <strong>The</strong>y had no current cardiac or respiratory disease, no upper respiratory<br />
tract infection for the preceding four weeks and were on no oral medications. <strong>The</strong>ir lung<br />
function showed a mean FVC <strong>of</strong> 99Vo predrcted (SD 11.3), FEVr <strong>of</strong> 95Vo predicted, (SD 9.0)<br />
and FEFzs-tsEo<strong>of</strong> 887o predicted for age gender and height (SD 12.8). <strong>The</strong>re was no correlation<br />
with exhaled NO results and lung function parameters. <strong>The</strong> correlation coefficient values for<br />
the comparison <strong>of</strong> peak NO levels to the results obtained by measuring area under the curve<br />
<strong>of</strong> the NO signal was 0.75 for the direct measurements and 0.34 for the t-piece measurements<br />
t82
(see Figure 8.1). <strong>The</strong> following assessments used peak exhaled NO levels in keeping with our<br />
work in adults.<br />
<strong>The</strong> mean level <strong>of</strong> NO measured by direct exhalation into the analyser was 49.6 ppb' (SD<br />
37.8, range lt.s-lgl.2ppb). <strong>The</strong> mean level <strong>of</strong> No measured via the t-piece system was29'7<br />
ppb, (SD 2'7.1, range 5.I-t4l.2ppb). <strong>The</strong> reduction <strong>of</strong> the exhaled NO peak seen between the<br />
two systems with the higher flow required for the t-piece measurement was consistent in all<br />
subjects and consistent with the results in the adult experiments (see Figure 8'1)'<br />
Figure 8.1: Peak exhaled NO results in healthy children<br />
.o CL<br />
o.<br />
.E<br />
o<br />
.9<br />
o zE.e6gxUJ<br />
150<br />
100<br />
comparison <strong>of</strong> the peak exhaled No results in the direct to analyser and the t-piece sampling system<br />
in 39 healthy children with each data point being a mean <strong>of</strong> five exhalations'<br />
<strong>The</strong>re was no significant difference between the direct mean NO levels in boys at 43'lppb<br />
(SD 40.5, range ll.5-lg7.2ppb) and girls with mean NO <strong>of</strong> 55.2 ppb (SD 35'4, runge 17 '7-<br />
124.gppb, p=0.43). <strong>The</strong>re was also no significant difference between the t-piece mean NO<br />
levels in boys at 25.6ppb (SD 2g.z,range 7.3-L4l.2ppb) and girls with mean NO 33'8ppb (SD<br />
25.l,range 5'l-g4'3 PPb, p=0'11)' (see Figure 8'2a and 8'2b)'<br />
183
Figure 8.2a: Comparison <strong>of</strong> peak exhaled NO in boys and girls measured by the direct system<br />
o 200<br />
ft reo<br />
.s 160<br />
p rco<br />
g t2o<br />
! roo<br />
880<br />
E60<br />
fi40 20<br />
0<br />
No difference was seen when comparing the peak exhaled NO results between 23 girls and 16<br />
boys aged 9-11 years in the direct to analyser sampling system with each data point being a<br />
mean <strong>of</strong> five exhalations.<br />
Figure 8.2b: Comparison <strong>of</strong> peak exhaled NO in boys and girls measured by the t-piece system<br />
200<br />
o 180<br />
B reo<br />
E reo<br />
o a 120<br />
o 100<br />
=80 fi60<br />
fi40<br />
20<br />
0<br />
A<br />
t<br />
I<br />
Boys Glrls<br />
No difference was seen when comparing the peak exhaled NO results between 23 girls and 16<br />
boys aged 9-11 years in the t-piece sampling system with each data point being a mean <strong>of</strong> five<br />
exhalations.<br />
<strong>The</strong>re was no significant difference between the mean COz levels by direct and t-piece<br />
measurements. <strong>The</strong> mean COz was 5.4Vo (SD 0.66, range 3.8-6.2Vo) direct compared to 5.52Vo<br />
(SD 0.66, range 3.9-6.3Vo) via t-piece measurements (p=0.44). Duration <strong>of</strong> the exhalation had<br />
184<br />
A<br />
A<br />
a<br />
I<br />
T
a mean value <strong>of</strong> 32.9 seconds for the direct method (SD 1 !.4, range 15'8-65'8s) and a mean<br />
value <strong>of</strong> 28.6 seconds for the t-piece method (SD 7.8, range 14'8-48s)' <strong>The</strong> mouthpiece was<br />
standardised at 4.OmmHg in both techniques and the flow to 225mlslmin in the t-piece<br />
method by voluntary control using visual feedback to the children. <strong>The</strong>re was still some<br />
variation around this depending on how successful the children were at maintaining the<br />
pressure and flow as requested during a slow exhalation from total lung capacity which is<br />
shown in Table 8.1.<br />
Table 8.1: Coefficients <strong>of</strong> variation <strong>of</strong> peak NO, peak COzand mouth pressure measurements made<br />
by both the direct and t-piece systems, and <strong>of</strong> flow made by the t-piece system<br />
Direct method:<br />
Peak NO in PPb<br />
CQ2"/" totalgases<br />
T-piece method:<br />
Peak NO in pPb<br />
CO2"/o totalgases<br />
Mouth pressure in<br />
Flow in mls/min<br />
comparisons were made within this group <strong>of</strong> children <strong>of</strong> other factors that could result in<br />
ainvay inflammation which may give higher results <strong>of</strong> exhaled No. Regarding a personal<br />
history <strong>of</strong> atopy; the question sent home to the parents was:<br />
Has he/she ever had eczema?<br />
Has he/she ever had hay fever?<br />
25.9<br />
0.44<br />
22.4<br />
29.9<br />
0.44<br />
12.5<br />
14<br />
Yes/l'{o<br />
Yes/No<br />
Does he/she have eczemalha.yfever now? YesA'{o<br />
2.6-67.4<br />
0.41-0.81<br />
4.5-57.0<br />
0.5-65<br />
0.41-0.81<br />
2.9-36.1<br />
1.8-30.3<br />
<strong>The</strong>re were eleven children that had a personal past or current history <strong>of</strong> allergic rhinitis<br />
compared to 28 children who did not. At the time <strong>of</strong> testing none had current nasal symptoms<br />
but three did have current eczema and were using appropriate topical creams including<br />
hydrocortisone cream. <strong>The</strong>re was no significant difference in the exhaled No levels in the<br />
atopic children at 34.5ppb (sD 11.8) compared to non-atopic children at 45'6ppb (sD 38'5)<br />
via the direct method or via the t-piece method measured at29'6ppb (SD 24) versus 24'8ppb<br />
(SD 26.2) respectively. We also looked at whether family history <strong>of</strong> atopy had any effect on<br />
the exhaled No levels in the children. <strong>The</strong> question sent home to the families was:<br />
185
if yes who?<br />
if yes who?<br />
if yes who?<br />
Any first-degree relative (parent or sibling) who was stated to have at least one <strong>of</strong> the<br />
conditions listed was counted as a positive and any more distant relative or no family member<br />
having any <strong>of</strong> the conditions listed were counted as a negative. Of the 39 children, 18 had a<br />
positive family history <strong>of</strong> atopy compared to 2l that did not and there were no differences<br />
seen in the direct exhaled NO levels at 40.6ppb (SD 26.7) versus 44.0ppb (SD 38.9), or in the<br />
t-piece levels 25.3ppb (SD 21.4) versus 27.0ppb (SD 29.0).<br />
<strong>The</strong> possibility <strong>of</strong> smoking causing airway inflammation and affecting the results <strong>of</strong> exhaled<br />
NO was explored. <strong>The</strong> question sent home to the parents was: "Does anyone in the family<br />
smoke? Yes or No" When the questionnaires were checked through with the children it was<br />
determined whether the family smoker lived in the household. <strong>The</strong>re were no differences seen<br />
in the 19 children who lived with one smoker in the household compared to the 20 children<br />
from non-smoking households at 37.lppb (SD17.4) versus 49.0ppb (SD 45.8) via direct and<br />
22.2ppb (SD 20.4) versus 31.1ppb (SD 30.3) via the t-piece system. <strong>The</strong> children were not<br />
asked about active smoking.<br />
<strong>The</strong> possibility <strong>of</strong> a reaction to pet dander causing airway inflammation and affecting the<br />
results <strong>of</strong> exhaled NO was explored. <strong>The</strong> question sent home to the parents was: "Do you<br />
have any pets? If yes please name type". When the questionnaires were checked through with<br />
the children the type <strong>of</strong> pet was checked again. Cats, dogs and any furry animal living within<br />
the house were counted as positive, no matter the number involved. Fish and birds were<br />
counted as a negative, as were not having pets. <strong>The</strong>re were no differences in the peak exhaled<br />
NO between those thirteen children who had a household furry pet (or pets) and those 26 that<br />
did not at 39.3ppb (SD 29.2) versus 43.9ppb (SD 35.7) via direct and 23.5ppb (SD 23.2)<br />
versus 27.6ppb (SD 26.7) via the t-piece system. <strong>The</strong>re was no attempt made to look at<br />
possible dose-response for any <strong>of</strong> these questionnaire replies.<br />
8.4<br />
Does anyone else in the family have any <strong>of</strong> the following:<br />
Asthma<br />
E*zema<br />
Allergic Rhinitis<br />
YesA.[o<br />
YesA.[o<br />
Yes/I.[o<br />
Discussion: exhaled nitric oxide results in healthy children<br />
This study confirmed that it was possible to measure exhaled NO levels in children aged<br />
between nine and eleven years by using these two different techniques. As in the adult studies<br />
there was a consistent difference between the NO levels obtained with the direct and the t-<br />
186
piece methods. <strong>The</strong> main difference between these techniques is an increased flow <strong>of</strong><br />
Z2lmlstmin, and the adult technical studies revealed that with increasing flow there was a<br />
decreasing level <strong>of</strong> exhaled NO obtained. In this case, an increase in flow <strong>of</strong> 52Vo led to a<br />
reduction <strong>of</strong> exhaled NO <strong>of</strong> 407o.<br />
<strong>The</strong> study was not designed to look at age-related differences in exhaled NO, as had been<br />
suggested by Lundberg et al as occurring with possible development and pneumatisation <strong>of</strong><br />
the sinuses (Lundberg, Farkas-Szallasi et al. 1995). <strong>The</strong> children studied were in a nalrow age<br />
range to try and standardise the results and to limit other extraneous effects. <strong>The</strong> age was<br />
chosen as those likely to be able to do lung function tests and therefore complete the exhaled<br />
No testing successfully. However the results in these 'normal' children were significantly<br />
lower than the results obtained from the twelve healthy adults studied initially except for the<br />
comparison <strong>of</strong> t-piece measurement in males. <strong>The</strong> children had mean exhaled No level<br />
4g.6ppb compared to 84.8ppb in adults via direct measurement and mean NO 29'7ppb<br />
compared to 41.2ppb in t-piece measurement. <strong>The</strong> boys had a mean exhaled NO <strong>of</strong> 43'lppb<br />
(direct) and 25.6ppb (t-piece) compared to 71.7ppb (direct) and 33'2ppb (t-piece) <strong>of</strong> male<br />
adults. <strong>The</strong> girls had a mean exhaled NO <strong>of</strong> 55.2ppb (direct) and 33.8ppb (t-piece) compared<br />
to 92.4ppb (direct) and 50.7ppb (t-piece) <strong>of</strong> female adults. <strong>The</strong> differences werc more<br />
significant for the direct than the t-piece technique. <strong>The</strong>re was no difference within this close<br />
two year age band, though it was noticeable that the nine year old children found it more<br />
difficult to control expiration within the prescribed limits than the older children did' This<br />
niurow and young age bracket was also chosen to try and enrol children that were prepubertal<br />
following suggestions that exhaled No may vary through the menstrual cycle (Kharitonov'<br />
Logan-sinclair et al. 1gg4). This ruled out a confounding factor if it was later discovered that<br />
pubertal change had effects on exhaled No in either male or females, although, in fact' this<br />
remains unknown. In both the children and the adults there was no significant difference in<br />
exhaled NO between the genders.<br />
<strong>The</strong>re were significant differences between the duration <strong>of</strong> exhalation with the mean being<br />
32.9 seconds for the children and 56.2 seconds for the adults in the direct measurements, and<br />
2g.6 seconds for the children and 53.5 seconds for the adults in the t-piece measurements'<br />
Three children had long exhalations <strong>of</strong> greater than 50 seconds for all <strong>of</strong> their direct method<br />
exhalations and longer than 40 seconds for all the t-piece method exhalations and all had the<br />
higher levels <strong>of</strong> peak coz measured. Interestingly, these three had swimming training and<br />
were on representative teams either at the school and/or swim clubs'<br />
r87
<strong>The</strong>re are limitations with regard to the questionnaire data. <strong>The</strong> questionnaire was developed<br />
as one <strong>of</strong> convenience and piloted on ten families in the paediatric general respiratory and<br />
asthma clinics to ensure that it was easy to understand and easy to fill out. Two modifications<br />
were made as a result <strong>of</strong> this - the description <strong>of</strong> wheeze was added, and the words for<br />
describing amount <strong>of</strong> cough were changed. Park Walk School is an English-speaking school<br />
but did have families <strong>of</strong> many nationalities so English was not necessarily the first language<br />
<strong>of</strong> all the parents who were filling out the questionnaires and signing the consent form. <strong>The</strong><br />
questionnaire did not go through a validation process, save our own initial pilot. We did not<br />
interact with the parents <strong>of</strong> the children directly. <strong>The</strong> questionnaires were distributed by the<br />
teachers through the two classrooms and returned to the teacher from those happy to<br />
participate. We did not have any data on the children who were given the questionnaire and<br />
did not return it or did not gain consent for the study. I therefore cannot compare the<br />
demographics <strong>of</strong> those who participated and those who did not to see if there were any major<br />
differences between them. Forty-six children attended the hospital for the study from a<br />
possible fifty-six in two classes. <strong>The</strong> classes were not stable through the study period with<br />
additional movement <strong>of</strong> six children moving into and out <strong>of</strong> the classes because <strong>of</strong> moving<br />
within the school and or two children from one family leaving the school altogether. Two<br />
children that attended the hospital were ruled out because <strong>of</strong> inability to do lung function<br />
successfully, although interestingly both were able to complete the exhaled NO tests. Five<br />
children had asthma, four on bronchodilator therapy only and one on regular IHCS therapy<br />
who briefly attended my asthma clinic -<br />
these children were measured but not documented in<br />
this segment <strong>of</strong> the study. <strong>The</strong> teachers ruled out seven children either because they had<br />
special educational needs or because <strong>of</strong> misbehaviour - I do not know whether their families<br />
had or had not consented. This gave us a response <strong>of</strong> 73Vo overall or 63Vo successful 'normal'<br />
subject results.<br />
<strong>The</strong> questionnaire responses were checked with the children on the day that they were brought<br />
to the hospital to study. However it was obvious that the children were confident on some <strong>of</strong><br />
the responses and less confident on others. <strong>The</strong>y tended to be confident on name, age,<br />
nationality, current conditions such as asthma, hayfever or eczema, current medications, the<br />
presence <strong>of</strong> family pets or presence <strong>of</strong> household smokers. <strong>The</strong>y were less confident, and<br />
there was more variability among the children about the degree <strong>of</strong> confidence, on answers<br />
regarding recent 'colds', past history <strong>of</strong> conditions and family history <strong>of</strong> conditions, and for<br />
these we had to rely predominantly on the written responses. We had documented nationality<br />
but did not look at differences between them because <strong>of</strong> low numbers. <strong>The</strong> six nationalities<br />
188
were loosely grouped as British, Indian, Arabic, African, European and South American with<br />
one to fifteen children in each group.<br />
we prospectively elected to enter into the database a positive or negative response to the<br />
questions and not categorise answers any further. We therefore did not investigate possible<br />
dose-response relationships for atopy (personal or family), passive smoking or presence <strong>of</strong><br />
pets. For example, we did not try to quantify the numbers <strong>of</strong> cigarettes smoked by the one or<br />
more household smokers and look at the exhaled NO levels in this group. Likewise we did not<br />
quantify the number <strong>of</strong> atopic responses within the family and see if there was any correlation<br />
between atopy severity and exhaled No. This was for a number <strong>of</strong> reasons' Firstly, as<br />
mentioned we were relying on questionnaire data which could be variably accurate' Secondly,<br />
in the straight .positive and negative' categorisation <strong>of</strong> responses there were no significant<br />
differences that I thought should be further investigated in this setting. Thirdly, the numbers<br />
with a positive response to one <strong>of</strong> these areas ranged from eleven to nineteen so the numbers<br />
were becoming small. Finally, it would have been difficult with these small numbers to<br />
quantify one response in exclusion <strong>of</strong> other responses. At the time, our sample <strong>of</strong> 39 children<br />
was the largest study in this area <strong>of</strong> research but the lack <strong>of</strong> findings <strong>of</strong> these factors that<br />
might cause some degree <strong>of</strong> airway inflammation may be secondary to lack <strong>of</strong> numbers<br />
causing a type two statistical error.<br />
Despite the questionnaire and normal lung function, we had one boy who was an outlier with<br />
direct peak No ot l97.2ppb and t-piece peak No <strong>of</strong> l4l.2ppb which was 73ppb and 43ppb<br />
higher than the next highest result. On history, he occasionally wheezed with viral infections<br />
and, following parental permission had 2OVo fall in FEV1 at2 mglml histamine on challenge'<br />
It is possible he had mild asthma or was on the extreme <strong>of</strong> the normal range <strong>of</strong> ainuay<br />
reactivity. He was a European boy with no personal or family history, from a non-pet owning'<br />
non-smoking household.<br />
By time <strong>of</strong> publication <strong>of</strong> this study, other investigators during the same period had also<br />
begun to examine the measurement <strong>of</strong> NO in children, both in normal subjects and those who<br />
with a range <strong>of</strong> respiratory diseases. In the main, the control children studied had no<br />
respiratory disease, were on no medications and usually were recorded to have no upper<br />
respiratory tract infection for between two and six weeks prior to the studies' Unfortunately'<br />
as mentioned in the previous chapters, with all the early research each individual research<br />
group developed their own techniques and thus every group utilised a different method <strong>of</strong><br />
measurement. As with the adult data, the absolute results in the healthy children differed<br />
189
significantly between research groups, partly because <strong>of</strong> different techniques utilised. I-anz et<br />
al reported a mean exhaled NO <strong>of</strong> 14ppb (+/- 2ppb) in seven children with single exhalations<br />
from total lung capacity into a reservoir bag that was then fed through the NO analyser (lanz,<br />
Irung et al. 1997). <strong>The</strong>y also showed that there was no difference in the NO levels when re-<br />
measured five days later (Lanz, I-eung et al. 1997). Using a similar method <strong>of</strong> slow exhalation<br />
into a reservoir and then put through the analyser, Nelson et al reported a much lower mean<br />
exhaled NO <strong>of</strong> 5.05ppb (+/- 0.4ppb) in 2I children (Nelson, Sears et al. 1997). Baraldi et al<br />
showed that NO reached a steady plateau when measured during tidal breathing after one to<br />
two minutes and reported mean levels <strong>of</strong> 5.4ppb in 16 children (Baraldi, Azzolin et ^1. 1997).<br />
Lundberg et al also measured plateau oral exhaled NO levels with tidal breathing and reached<br />
a similar figure <strong>of</strong> 4.8ppb (SD 1.1) in 19 children (Lundberg, Nordvall et al. 1996). Balfour-<br />
Lynn et al measured NO from single exhalations taking the point <strong>of</strong> NO when the end tidal<br />
COz reached a plateau in an attempt to compare alveolar levels for both oral and nasal<br />
measurement. <strong>The</strong> 57 control children had a mean oral exhaled NO <strong>of</strong> 4.8 ppb (range l.l to<br />
23ppb), and mean nasal NO <strong>of</strong> 1024 ppb (range 158 - 2502ppb) @alfour-Lynn, Laverty et al.<br />
1996). Two studies measured controls across wide age ranges that incorporated an unknown<br />
number <strong>of</strong> children. Dotch et al measured exhaled NO from single exhalations although the<br />
response time <strong>of</strong> the analyser was not fast enough at 25 seconds for a 9OVo response rate to<br />
measure one breath, so the subject was required to exhale four to five times into the analyser<br />
via mouth inhalation and with nose clips in place. In 68 controls aged four to 34 years the<br />
mean NO result was 3.0 ppb (+l-2.Sppb) by these single exhalations, with a minute ventilation<br />
concentration <strong>of</strong> NO <strong>of</strong> 25 ppb (+l- 27 ppb) and direct nasal NO level <strong>of</strong> 96 ppb (+l- 47 ppb)<br />
@otsch, Demirakca et al. 1996).<br />
In another study, Grasemann measured NO by single exhalations to an analyser that had a 10<br />
second response time. <strong>The</strong> mean exhaled NO level in 30 control subjects aged six to 37 years<br />
was 9.1 ppb (+/- 3.6ppb). Lundberg et al measured the plateau NO signal by continuous<br />
sampling from oral exhalation or nasal exhalation or by direct nasal measurement in 19<br />
children. <strong>The</strong> mean oral level was 4.8ppb (SD 1.1), the mean nasal level was 2lppb (SD 9.1)<br />
and the mean directly measured nasal level was 239ppb (SD 20) (Lundberg, Nordvall et al.<br />
1996). Most <strong>of</strong> these researchers therefore reported absolute levels <strong>of</strong> exhaled NO that were<br />
less than the results that I have reported from my studies. However I had elected to use single<br />
exhalations measured immediately by the analysers, while these others used tidal breathing or<br />
plateau measurements with or without a reservoir technique over several breaths.<br />
190
In comparison to the early literature in the adults, the research groups studying children<br />
appeared to more <strong>of</strong>ten consider the effect <strong>of</strong> ambient NO. Our experiments were only<br />
undertaken if the ambient NO was less than 10ppb, as I had already shown in the previous<br />
methodology experiment in adults (Section ?.6) that the ambient NO did affect the exhaled<br />
NO levels. Similarly, Grasemann et al commented that "In preliminary experiments we<br />
observed that the higher NO concentrations in ambient air were associated with increased<br />
exhaled NO concentrations." (Grasemann, Michler et al. 1997). Ambient NO in this paper<br />
was reported at between two and22 ppb in one paper which authors thought unnecessary to<br />
take into account, but the exhaled levels in this paper were 1.1 to 23pb in the control children<br />
which was exactly this ambient range (Balfour-Lynn, Laverty et al' 1996)' One group noted<br />
that when measuring the exhaled levels in their control group it did not change when<br />
measured in ambient or NO-free air, thus all their subsequent experiments were caried out<br />
inhaling room air. However at the time their ambient NO level was reported as 3'95ppb +/-<br />
0.98ppb and their NO free m at 3.97 +f 1.14ppb so there was no difference in the inhaled<br />
levels and this ambient level may have changed over time (Nelson, Sears et al.1997} Finally<br />
one group stipulated that they did all their measurements with the children inhaling No-free<br />
air (Baraldi, Azzolin et al. 1997),<br />
we found no correlation between exhaled No levels and gender or lung function parameters.<br />
Nelson et al also found that there was no correlation between NO and height, weight, gender<br />
or FEVr in either the control or asthmatic children (Nelson, Sears et al. 1997). Balfour-Lynn<br />
et al found no correlation between exhaled NO and FEVr or FVC across control and CF<br />
groups <strong>of</strong> children (Balfour-Lynn, Laverty et al' L996)'<br />
In our study, we have documented that exhaled NO could be measured in children, reporting<br />
normal values obtained with these techniques. As the continuation <strong>of</strong> this research, I then<br />
moved on to investigate the exhaled NO levels in asthmatic children'<br />
8.5<br />
8.5./ Background<br />
presence <strong>of</strong> higher levels <strong>of</strong> exhaled No in asthma were first indicated in two animal models<br />
where exhaled NO significantly increased following induction <strong>of</strong> an asthma exacerbation<br />
using ova-albumin sensitised guinea pigs and rabbits (Persson and Gustafsson 1993; Endo'<br />
uchido et al. 1995). <strong>The</strong>re followed a number <strong>of</strong> publications in adult subjects showing that<br />
exhaled No levels appeared to be raised in patients with asthma when compared to healthy<br />
191
controls. Kharitinov et al reported levels <strong>of</strong> 283ppb in 6l asthmatics compared to 67 controls<br />
at 80.2ppb when measured with single exhalations (Kharitonov, Yates et al. 1994). Persson et<br />
al measuring mixed exhaled air found levels <strong>of</strong> 10.3ppb in 23 asthmatics compared to 20<br />
controls at 8.4ppb (Persson, Zetterstrom et al. 1994). Massaro et al using a similar technique<br />
found levels <strong>of</strong> l3.2ppb in five asthmatics compared to five controls at 4.7ppb (Massaro,<br />
Mehta et al. 1996). Robbins et al measured single exhalations and a reservoir collection in 18<br />
asthmatics and 91 controls, reporting significant differences between the groups with both<br />
techniques, <strong>of</strong> l74ppb compared to l05.5ppb and 27.2ppb compared to 14.5ppb @obbins,<br />
Floreani et al. 1996). Alving et al demonstrated no overlap between their control group with a<br />
range <strong>of</strong> 5-l6ppb and their asthmatic group who had a range measured at 2l-3lppb (Alving,<br />
Weitzberg et al. 1993). Martin et al also found significant differences between 18 control<br />
subjects and 32 patients with allergic rhinitis when measured by single exhalation with<br />
ll.lppb compared to l6.3ppb, following a ten second breath-hold at 15.6ppb compared to<br />
34.0ppb and following a 60 second breath-hold at 32.lppb and 62ppb (Martin, Bryden et al.<br />
1996). Massaro demonstrated that the NO levels were even higher at 13.9ppb during a period<br />
<strong>of</strong> acute asthma in seven patients requiring emergency department treatment. A reduction <strong>of</strong><br />
NO began after 48 hours which soon became indistinguishable from control NO levels at<br />
6.2ppb (Massaro, Gaston et al. 1995). Kharitinov et al showed a reduction from 203ppb to<br />
120ppb over three weeks in eleven asthmatic patients and an increase on their mean FEVr as<br />
percent predicted from92Vo to 99Vo after commencing 800pgs budesonide dipropionate twice<br />
per day, with no corresponding change when the same patients were randomised to placebo<br />
(Kharitonov, Yates et al. 1996). As by far the greatest amounts <strong>of</strong> NO in vitro were shown to<br />
be produced from iNOS stimulation, these high levels in asthma were hypothesised by these<br />
groups to be a measure <strong>of</strong> airway inflammation (Barnes 1993; Barnes and Kharitonov 1996).<br />
Following measurement <strong>of</strong> exhaled NO in healthy children, I was then interested to assess<br />
whether these same findings as had been documented in adults could be found in children<br />
with asthma.<br />
8.5.2 <strong>The</strong> asthmatic subjects<br />
<strong>The</strong> children were recruited from the paediatric respiratory or asthma outpatient clinics at the<br />
Royal Brompton Hospital where I was seeing patients. Five children were also enrolled from<br />
the Park Walk Primary School (four on bronchodilators only and one on regular IHCS therapy<br />
who was subsequently also seen in the asthma clinic). <strong>The</strong>y were presented with the same<br />
questionnaire as used for the control children. Following informed consent from their parent/s<br />
or caregiver/s, the children were enrolled if:<br />
t92
o <strong>The</strong>re was a signed consent form and a questionnaire filled out by their parent/s'<br />
o <strong>The</strong> child was willing to be part <strong>of</strong> the study'<br />
o <strong>The</strong> child had a doctor diagnosis <strong>of</strong> asthma'<br />
o <strong>The</strong> child was on either bronchodilator therapy only or bronchodilator therapy and<br />
long term IHCS therapy. No child studied was on any other asthma medications (such<br />
as long acting p2 agonist therapy, theophylline or oral steroids). some <strong>of</strong> the children<br />
were on topical treatments for eczema'<br />
o <strong>The</strong>re had been no change in their asthma treatment in the last six weeks'<br />
o <strong>The</strong>re was no current or recent upper or lower respiratory tract infection for at least<br />
four weeks.<br />
o <strong>The</strong> child had recorded no other respiratory, cardiac or other major chronic disease'<br />
<strong>The</strong> ethical consent obtained for the study and the statistical analysis have been documented<br />
previously in Section 8.2.<br />
8.5.3 Protocol<br />
<strong>The</strong> protocols for the asthmatic subjects were identical to those used the control subjects and<br />
are listed in Section g.3.3 above. <strong>The</strong> two techniques <strong>of</strong> measurement were used: the direct to<br />
analysers method and the t-piece sampling method' <strong>The</strong> children did one set <strong>of</strong> five<br />
exhalations under each condition and continued until five sets <strong>of</strong> measurable exhalations were<br />
made, or it became obvious that either the procedure was too difficult or it was exacerbating<br />
their asthma. As the children were enrolled from either the morning clinics or the school (as<br />
previously organised) all the children were measured at the same time during the day - late<br />
morning between 1000 hours and 1300 hours'<br />
8.5.4 Results<br />
In total 31 asthmatic children were recruited for the studies <strong>of</strong> exhaled NO in asthma: fifteen<br />
were on bronchodilator treatment only and 16 on regular IHCS therapy. <strong>The</strong> children with<br />
asthma on bronchodilator treatment only had an FVC <strong>of</strong> 92Vo (SD 14.5) and FEVr <strong>of</strong> 78Vo<br />
(sD 10.4) percent predicted by the Polgar reference equation @olgar and Promadh^t I97I)'<br />
<strong>The</strong> children with asthma on regular IHCS had a mean FVC <strong>of</strong> 987o (SD 18.5) and FEVr <strong>of</strong><br />
g6qo (SD 17.5). <strong>The</strong> mean peak No level measured by the direct method in asthmatic children<br />
on bronchodilator treatment only was 126.1ppb (SD 77.1, range L4.4-36l.lppb) which was<br />
significantly higher than compared to the healthy children (mean 49'6ppb, SD 37'4' range<br />
n.s-lgl.2ppb see previous results, p
direct method in asthmatic children on regular IHCS therapy was 48.7ppb (SD 43.3, range<br />
8.6-106.4ppb) which was significantly lower than the asthmatic children on bronchodilator<br />
treatment only (p
Figure g.3b: Mean peak exhaled No levels in control and asthmatic children measured via the t-piece<br />
sampling system<br />
lt 150<br />
g o.<br />
.g<br />
:/ 100<br />
a-<br />
a<br />
':r<br />
-i i !<br />
!!!<br />
normals<br />
asthmatics<br />
bronchodilhlor<br />
therapy only<br />
mean<br />
and SEM<br />
aslhmatics<br />
regular lnhaled<br />
cortioostsroids<br />
Mean peak exhaled NO levels in healthy children (n=39), asthmatics on bronchodilator therapy only<br />
(n=15) and asthmatic children on regular IHCS therapy (n=16) measured by the t'piece sampling<br />
system. Note the Y scale is discontinuous to accommodate the outliers'<br />
<strong>The</strong>re were no differences in the CO2 levels, mouth pressures, and durations <strong>of</strong> expiration<br />
between the different groups or between the two methods within each group as noted in Table<br />
8.2.<br />
Six asthmatic children on bronchodilator treatment only but deemed clinically to require the<br />
introduction <strong>of</strong> IHCS therapy to improve their asthma control were recruited for the<br />
longitudinal study. Prior to commencing steroids the median exhaled NO was 1245ppb<br />
(range 67.6-330.6ppb). Following treatment for two weeks on either budesonide diproprionate<br />
4001t"gtwice per day in five subjects or budesonide diproprionate 2}}ltgtwice per day in one<br />
subject all delivered via a Turbohaler@, NO fell to a median level <strong>of</strong> 48.6ppb (range 36'8-<br />
153.6ppb). This reflected a decrease in all the children with only one subject now having a<br />
different result from that observed in normal children (see Figures 8.4a and 8'4b)'<br />
195
Figure 8.4a: <strong>The</strong> eftect <strong>of</strong> commencing inhaled corticosteroid therapy on peak exhaled NO levels<br />
asthmatic children measured via the direct method<br />
3s0<br />
fl soo<br />
CL<br />
.s zso<br />
o<br />
B zoo<br />
o z 150<br />
E<br />
fi roo<br />
E<br />
,i 50<br />
0<br />
Median = 124.5ppb<br />
-<br />
Pre Steroid NO<br />
Post Steroid NO<br />
E<br />
Median = 48.6ppb<br />
Figure 8.4b: <strong>The</strong> etfect <strong>of</strong> commencing inhaled corticosteroid therapy on peak exhaled NO levels in<br />
asthmatic children measured via the t-piece sampling system<br />
8.6<br />
300<br />
250<br />
o<br />
o.<br />
o.<br />
.c 200<br />
o<br />
g 150<br />
o<br />
zE<br />
100<br />
6<br />
-c<br />
x<br />
ur 50<br />
Discussion: exhaled nitric oxide in asthmatic children<br />
Median = 41.3 ppb<br />
-<br />
Asthma is a chronic disease <strong>of</strong> airway inflammation that is treated with anti-inflammatory<br />
drugs. However in the routine clinical setting we measure lung function rather than any<br />
inflammatory parameters. <strong>The</strong> gold standard <strong>of</strong> assessment <strong>of</strong> airway inflammation is<br />
bronchoscopy and bronchial biopsy as discussed in Chapter 1. Research then began to suggest<br />
that NO could be measured in exhaled air and may be related to airway inflammation in adult<br />
asthmatics (Alving, Weitzberg et al. L993; Kharitonov, Yates et al. 1994; Persson,<br />
196
Zetterstrom et al. 1994; Kharitonov, Yates et al. 1995; Massaro, Gaston et al' 1995;<br />
Kharitonov, Chung et al. 1996; Martin, Bryden et al. 1996; Massaro, Mehta et al' 1996;<br />
Robbins, Floreani et al. 1996; Kharitonov, Rajakulasingam et al. 1997). In the experiments<br />
above we set out to assess whether the pattern <strong>of</strong> NO excretion was similar in children as<br />
determined in adults. In 39 children with no known respiratory problems it was possible to<br />
measure exhaled No with mean levels <strong>of</strong> 49ppb (direct) and 29.7ppb (t-piece). I have also<br />
shown that there was a significant increase in mean NO concentrations to t26.7ppb (direct)<br />
and to l09.5ppb (t-piece) in asthmatic children receiving bronchodilator treatment only' <strong>The</strong>re<br />
was a significant decrease in children on regular IHCS therapy with mean NO levels <strong>of</strong><br />
48.7ppb (direct) and <strong>of</strong> 42.5ppb (t-piece). <strong>The</strong>re was no difference between the exhaled No<br />
levels <strong>of</strong> the healthy children and the stable asthmatics on regular IHCS therapy. <strong>The</strong>re was<br />
no significant difference in the mean age <strong>of</strong> the children in the three groups, although the age<br />
range <strong>of</strong> the children recruited from the outpatient clinics was greater than those in the control<br />
group. So while the effect <strong>of</strong> puberty and age was controlled for the healthy children' it is<br />
possible that this may have affected results in the other two groups (Lundberg, Farkas-Szallasi<br />
et al. 1995; Franklin, Taplin et al. 1999). <strong>The</strong> lowest age <strong>of</strong> a child that we successfully<br />
studied was six years. All <strong>of</strong> the testing was completed in the mornings between 1000 hours<br />
and 1300 hours so we could disregard any concerns regarding circadian rhythm effects<br />
(Mattes, Storm van's Gravesande et al.2oo2). <strong>The</strong> pattern <strong>of</strong> No exhalation appeared to be<br />
the same as the patterns seen in the adult subjects, and the pattern was the same in asthmatic<br />
children as in healthy children, although the levels seen in children were lower' Similar to the<br />
results from the adult subjects in the methodological experiments' we saw a reduction <strong>of</strong> the<br />
NO levels between the direct and t-piece method <strong>of</strong> sampling in all the groups <strong>of</strong> children'<br />
However the magnitude <strong>of</strong> the reduction was different. <strong>The</strong>re is an increase from 440mls/min<br />
in the direct sampling technique to 665mls/min in the t-piece sampling method, an increased<br />
flow <strong>of</strong> 517o. This led to a SOVo reduction <strong>of</strong> the No levels in the adult experiment<br />
(methodological experiment one described in Chapter 6), a 407o percent reduction in the<br />
healthy control children, a 24Vo reduction in the asthmatic children on bronchodilator<br />
treatment only and an 8Vo reduction in the asthmatic children on IHCS therapy' <strong>The</strong> variation<br />
seen when measuring children was greater than that in adults, and there was a wide range <strong>of</strong><br />
exhaled No levels within all three paediatric groups. I have previously discussed one boy who<br />
was an outlier in the group <strong>of</strong> healthy children. one subject recorded as an asthmatic on<br />
bronchodilator treatment only had mean exhaled levels <strong>of</strong> 14'4ppb via the direct method<br />
which was 53ppb lower than the next value, and 13.4ppb via the t-piece method which was<br />
l7ppb lower than the next value. She had not had any episodes <strong>of</strong> asthma for two years and<br />
r97
had last used her p2 agonist inhaler ten months previously. While there had been an increase<br />
noted <strong>of</strong> exhaled NO levels with upper respiratory tract infections (Kharitonov, Yates et al.<br />
1995), none <strong>of</strong> the children tested had had a respiratory tract infection, an exacerbation <strong>of</strong><br />
asthma within four weeks and any changes in medication for six weeks. Interpretation <strong>of</strong><br />
single NO results, however, must be made with caution. In a small group <strong>of</strong> six children<br />
measured before and two weeks after starting ICHS, the exhaled NO levels dropped<br />
significantly from a median <strong>of</strong> l24.5ppb to 48.6ppb and in all but one subject had returned to<br />
the normal range. This suggested that the exhaled NO could be a useful monitoring tool for<br />
airway inflammation in asthma.<br />
A number <strong>of</strong> groups also published in the area <strong>of</strong> exhaled NO in paediatric controls, asthma<br />
and cystic fibrosis through 1996 and 1997. I-anz et al looked at three groups <strong>of</strong> children:<br />
seven asthmatics, six atopic but non-asthmatic children and seven controls with no asthma all<br />
aged between eight to eighteen years with exhaled NO at 52ppb (+/- 5ppb), l6ppb (+/- 2ppb)<br />
and l4ppb (+l-zppb) respectively. <strong>The</strong> exhaled NO in the asthmatic group decreased to l4ppb<br />
(+/- lppb) when treated with oral steroids for 14 days (Lanz, Leung et al.1997). <strong>The</strong> other two<br />
groups were re-measured five days later and showed no difference in their exhaled NO<br />
readings. By a different method, Nelson et al measured expired NO concentrations via a slow<br />
exhalation into a reservoir system, which was then sealed and subsequently run through the<br />
NO analyser. Twenty one control children aged five to eighteen years had a mean NO <strong>of</strong> 5.05<br />
ppb which was significantly lower than thirteen asthmatic children with a mean exhaled NO<br />
at 16.3pp. This decreased linearly as airflow obstruction improved with corlmencement <strong>of</strong><br />
systemic steroid therapy <strong>of</strong> prednisone Z-4mglkglday. However, even when five <strong>of</strong> the<br />
asthmatic patients had normalisation <strong>of</strong> their lung function, the exhaled NO was still higher<br />
than the control children at 13.5ppb (Nelson, Sears et al. 1997). This goup speculated that<br />
"NO assays may prove to be a more sensitive measure <strong>of</strong> childhood asthma than spirometrS/."<br />
Baraldi et al measured sixteen children aged six to thirteen years during tidal breathing with<br />
children inhaling NO free air with all measurements. NO reached a steady plateau during oral<br />
breathing after one to two minutes. <strong>The</strong> mean level in the normal children was 5.4ppb (+/-<br />
0.appb) significantly lower than sixteen children measured during an acute asthmatic attack at<br />
31.3ppb (+l- 4.2ppb)and although these levels decreased with a five day course <strong>of</strong> oral<br />
steroids (prednisone 1 mg/kg per day), the mean levels remained significantly higher than<br />
controls at 16.5ppb (+l- 2.3ppb) (Baraldi, Azzolinet al.1997).<br />
Two studies looked at NO levels in children with allergic rhinitis and/or sinusitis. In 36<br />
asthmatic children all taking IHCS, there was no difference with direct nasal sampling <strong>of</strong> NO<br />
198
levels between the asthmatics with allergic rhinitis at 252ppb (SD 20) compared to those<br />
wirhout rhinitis at 256ppb (SD 26) (Lundberg, Nordvall et al. 1gg6). In sixteen children with<br />
acute maxillary sinusitis the mean nasal concentration was 70 ppb (+l S.7ppb) increased to<br />
220ppb (+/- 15ppb) after oral antibiotic therapy. In comparison nine children with upper<br />
respiratory tract infections but not thought to have sinusitis had mean nasal NO levels <strong>of</strong><br />
249ppb (+t- 32ppb), which did not change after oral antibiotic treatment (Baraldi, Azzolin et<br />
al. t997).<br />
In studies comparing children with asthma and children with CF, Lundberg et al also<br />
compared the plateau oral exhaled NO levels demonstrating no difference between 19 control<br />
children and eight children with cF at a mean <strong>of</strong> 4.8ppb (sD 1.2) and 5'8ppb (SD 0'8)<br />
respectively. However there was a significant increase in the 36 children with asthma to<br />
l3.gppb (SD 2.5). <strong>The</strong>se higher levels were seen in the asthmatic children despite their taking<br />
a range <strong>of</strong> medication with twelve on low dose IHCS (defined as 0-100pgs budesonide or<br />
equivalent per day), 16 on moderate doses <strong>of</strong> IHCS (defined as 200-400pgs budesonide or<br />
equivalent per day) and eight on high doses <strong>of</strong> IHCS (defined as 600-800pgs budesonide or<br />
equivalent per day). I note there appear to be gaps in these ranges i.e. 100-200pgs and 400-<br />
600pgs which are not discussed but it is likely that there was no child on treatment in these<br />
ranges which would have required odd dosing regimes. With nasal exhalation sampling, there<br />
was no difference between the control group or this asthmatic group (recalling that all were on<br />
IHCS therapy) at 21ppb (SD 9.1) versus 27ppb (SD 2.6), although a possible trend was noted<br />
<strong>of</strong> a decreasing NO when on the higher steroid doses. <strong>The</strong>re was also no difference in these<br />
two groups when having direct nasal sampling (via a nasal olive) with the controls measured<br />
at 239ppb (SD 20) and the collective asthmatic group measured at 254ppb (SD 17)' However<br />
in both the nasal measurements those that were able to perform the technique from the group<br />
<strong>of</strong> children with cF showed significantly lower levels with their nasal breathing giving levels<br />
between 9-l5ppb and their nasal direct sampling giving levels between 40-105ppb (Lundberg'<br />
Nordvall et al. 1996). In 68 controls, 90 asthmatics and 67 subjects with CF with a collective<br />
age range from four to 34 years, Dotsch et al used a slowly responding analyser measuring a<br />
series <strong>of</strong> single exhalations and showed a correlation between exhaled NO levels and the<br />
ambient NO concentration. <strong>The</strong>y then reported results between the three groups studied only<br />
in the subjects that were measured during days <strong>of</strong> zero ambient No concentration (Dotsch,<br />
Demirakca et al. 1996). <strong>The</strong> mean level <strong>of</strong> exhaled NO in 30 asthmatics aged four to fourteen<br />
years was 8.0ppb (+/- 6.lppb), significantly higher than in 37 controls aged four to 34 years at<br />
3.0ppb (+l 2.5ppb). Twenty three patients with CF aged five to 32 years tended to have a<br />
t99
higher level <strong>of</strong> exhaled NO at 4.9ppb (+l-2.6ppb) than the controls but this did not reach<br />
significance. <strong>The</strong>y also found no conelation between NO and the lung function in any goup<br />
@otsch, Demirakca et al. 1996). In another study, Grasemann et al measured NO by single<br />
exhalations to an analyser that had a 10 second response time across a wide age range <strong>of</strong><br />
subjects (Grasemann, Michler et al.1997). <strong>The</strong> mean exhaled NO level in 30 control subjects<br />
aged six to 37 years was 9.1 ppb (+/- 3.6ppb) which was significantly lower in 27 subjects<br />
with CF aged six to 40 years at a time <strong>of</strong> disease stability at 5.9ppb (+l- 2.6ppb). This goup<br />
commented that : "In preliminary experiments we observed that the higher NO concentrations<br />
in ambient air were associated with increased exhaled NO concentrations." In view <strong>of</strong> this,<br />
they went on to calculate the difference between the NO levels taking into account this<br />
ambient NO concentration and still found a significant difference with the control group<br />
having levels <strong>of</strong> 5.0ppb (+f3.lppb) compared to the CF group at 1.5ppb (+/- l.2ppb)<br />
(Grasemann, Michler et al. 1997). Finally Balfour-Lynn et al measured plateau exhaled NO at<br />
the time <strong>of</strong> COz plateau by placing a plastic nose piece just inside one nostril which was then<br />
connected to the Teflon tubing <strong>of</strong> the NO analyser. <strong>The</strong>y showed a significant difference<br />
between the nasal measurements from 57 control children at l024ppb (95Vo CI896-1l52ppb)<br />
when compared to children with CF; in thirteen on IHCS at 522ppb (95Vo CI313-730ppb) or<br />
50 not on IHCS at 460ppb (95Vo C[399-520ppb). <strong>The</strong>re was no difference in the exhaled NO<br />
levels in these two groups and the use <strong>of</strong> IHCS did not affect the exhaled oral levels <strong>of</strong> NO in<br />
the CF group with the results being means <strong>of</strong> 4.8ppb (95Vo Cl3.8-5.8ppb) in control children,<br />
4.7ppb (957o Cl4.0-5.3ppb) in those with CF not on steroids and 3.6ppb (957o Cl2.5-4.8ppb)<br />
in CF children on steroids. <strong>The</strong>re was also no significant difference in the NO levels in the CF<br />
goup between those 26 colonised with Pseudomonas aeruginosa compared to the 24 non-<br />
colonised. <strong>The</strong>re did appear to be a difference with a lower level in those 31 colonised with<br />
Staphyloccocal aureus at a mean <strong>of</strong> 4ppb (95Vo CI3.44.6ppb) and those 19 which did not<br />
have this organism at 5.8ppb (95Vo CI 4.4-7.2ppb). As there were 63 children in total there<br />
must have been some considerable overlap between having Staphylococcal aureus and/or<br />
Pseudomonas aeruginosa, plus the correct identification <strong>of</strong> the true presence <strong>of</strong> lower<br />
respiratory tract pathogens in this age group with CF is known to be difficult so the overlaps<br />
within these groups may have obscured findings (Balfour-Lynn, Laverty et al. 1996).<br />
This completed my work in this area. I had demonstrated some key findings <strong>of</strong> factors that<br />
affected exhaled NO measurements and would require standardisation for procedures in the<br />
future. <strong>The</strong>se were expiratory flow, expiratory mouth pressure, the need for low ambient NO<br />
levels or to inhale from a reservoir <strong>of</strong> NO free air, and the need to prevent water consumption<br />
200
during the testing procedure. I had demonstrated that it was possible to measure exhaled NO<br />
in children, both healthy controls and asthmatics. In fact in some <strong>of</strong> the control children, they<br />
found the exhaled NO measurements an easier task than completing lung function testing to<br />
ATS criteria standards. I had demonstrated significantly higher levels <strong>of</strong> exhaled NO was<br />
found in children with asthma on bronchodilator therapy only when compared to healthy<br />
children and asthmatics on regular IHCS therapy, with no difference between the latter two<br />
groups. Finally I had also demonstrated in a small group <strong>of</strong> asthmatic children the exhaled NO<br />
levels reduced significantly following two weeks <strong>of</strong> IHCS treatment' <strong>The</strong>se findings<br />
contributed to the evolving literature to suggest that NO was a marker for ainvay<br />
inflammation in asthma. I was invited with other research groups working in this area to<br />
present this data to a European Society Task Force (ESTF) meeting in Stockholm in<br />
September 1996 as a satellite working party to the European Respiratory Society Annual<br />
Conference where the first discussion regarding standardisation procedures took place. This<br />
later became the basis for the first publication setting out the best standard practices based on<br />
the data available (Kharitonov, Alving et al. 1997)'<br />
In the following chapter I will review how research in exhaled No and nasal No<br />
measurement has progressed. I will review the findings on factors that altered No results in<br />
both healthy groups and those with respiratory diseases. I will show how the standardisation<br />
procedure for testing has continued to evolve to the present day. I will then review the<br />
findings in the literature across adults, children and infants with a variety <strong>of</strong> conditions. I will<br />
end with an opinion as to the best use <strong>of</strong> exhaled and nasal NO testing is at the current time'<br />
<strong>The</strong> work in this chapterformed the basis <strong>of</strong> the publications:<br />
o Dinarevic S, Byrnes CA, Bush A, Shinebourne EA' Measurement <strong>of</strong> expired nitric<br />
oxide levels in child.ren. Pediatric Pulmonology 1996; 22: 396-401<br />
. Byrnes CA, Dinarevic s, shineboume EA, Barnes PJ, Bush A' ExhAled No<br />
measurements in normal and asthmatic child,ren. Pediatric Pulmonology 1997; 24:<br />
312-318<br />
20t
9.1 Introduction<br />
Chapter 9: Exhaled and nasal NO to today<br />
Having already presented my own research and preliminary discussion around the findings,<br />
this chapter reviews where the research on exhaled NO had reached by 2000 and describes the<br />
continued progression in this field to 2006. I will begin with a review <strong>of</strong> the <strong>of</strong>ficial<br />
statements from two international respiratory societies - the European Respiratory Society<br />
(ERS) and American Thoracic Society (ATS) - both <strong>of</strong> which aimed to standardise the<br />
techniques <strong>of</strong> NO measurement. I will present what is now known regarding NO in health and<br />
in respiratory diseases in adults, children and infants. In the final paragraph I will give my<br />
opinion as to where I believe this information is most useful.<br />
9.2 Technical factors affecting results across research groups<br />
By the end <strong>of</strong> the 1990s, exhaled and nasal NO had been measured in many groups; in normal<br />
subjects (both adult and paediatric populations), as well as in those with asthma, non-<br />
asthmatic atopy, chronic obstructive pulmonary disease (COPD), chronic bronchitis, cystic<br />
fibrosis (CF), ciliary disorders, bronchiectasis and interstitial lung disease. As alluded to in<br />
previous chapters, some findings remained consistent across the research groups. <strong>The</strong> best<br />
examples <strong>of</strong> consistency in exhaled oral NO results were the high levels measured in steroid<br />
naive asthmatics compared to normal controls which reduced with steroids, and the lower NO<br />
levels in asthmatics already on inhaled or oral corticosteroids. For nasal NO measurements,<br />
the most consistent findings were the low levels seen in patients with primary ciliary<br />
dyskinesia and CF. However, other results were not consistent across research groups and<br />
even when conclusions were similar, the absolute levels <strong>of</strong> exhaled NO were very different. It<br />
was this discrepancy that led to the commencement <strong>of</strong> the methodological studies discussed in<br />
the previous chapters. <strong>The</strong>re were many reasons for the differences in the absolute levels that<br />
were reported. I will list here what I believe constituted the main sources <strong>of</strong> variation.<br />
1. Modification <strong>of</strong> analysers - the early analysers had been modified from machines<br />
originally developed to measure NO in pollution using the chemiluminscence reaction,<br />
similar to the machine that I adapted. <strong>The</strong> analysers used at that time included:<br />
o Aerocrine AB, PO Box L024, Solna, Sweden.<br />
o CLA 510s, Horiba, Kyoto, Japan.<br />
o CLD 700AL, Eco Physics, AG, Basal or Duerten, Switzerland.<br />
202
a<br />
o<br />
Model 2107, Dasibi Environmental corporation, Glendale, california, united states<br />
<strong>of</strong> America.<br />
Model 42, <strong>The</strong>rmoelectron, Warrington, United Kingdom'<br />
Sievers Model 280A, GE Analytical Instruments, Boulder, Colorado' United States <strong>of</strong><br />
America.<br />
<strong>The</strong>se had differing response times ranging from 5-25 seconds while the subsequent<br />
purpose built models from 2000 improved this to 0.02-0.3 seconds. <strong>The</strong> above models all<br />
had differing sampling flows which ranged from 50-500mls/s (machine sampling rate)'<br />
and different sensitivities to NO ranging from 0'3 to 5ppb'<br />
2. Different methods <strong>of</strong> sampling - the three commonest techniques used for sampling the<br />
exhalation were:<br />
o Direct to the machine.<br />
o Using a t-piece or side arm sampling'<br />
o From a reseryoir <strong>of</strong> stored exhalations'<br />
3. Variation <strong>of</strong> reservoir and tubing materials -<br />
the type <strong>of</strong> material used to collect the<br />
sample also varied which may have resulted in some absorption or adsorption <strong>of</strong> No as<br />
discussed in Chapter 4. This is particularly important in reservoir studies with the use <strong>of</strong><br />
bags and tubing made <strong>of</strong> polyethylene, tedlar, mylar, teflon coated, metal and 'Douglas'<br />
bags (a type <strong>of</strong> anaesthetic bag made <strong>of</strong> rubber)'<br />
4. Method <strong>of</strong> cleaning and/or filter use - there was also the possibility <strong>of</strong> interference from<br />
hygiene and infection control procedures. This isn't discussed in the early papers' At the<br />
time, as currently, standard infection control procedures were recommended with regard<br />
to doing routine spirometry (American Thoracic Society and Association' 1994)' It is<br />
likely most researchers were following a similar type <strong>of</strong> infection control when measuring<br />
No and this may have resulted in interference - either with use <strong>of</strong> inline filters and/or<br />
chemicals for cleaning. Data presented in one abstract showed that the NO concentration<br />
was greatly augmented by alcohol containing disinfectant (Meijer, Kerstjens et al' 1996)<br />
at a time when the use <strong>of</strong> chlorhexidine (also an alcohol containing disinfectant) was<br />
common in usual lung function laboratory hygiene practices' One study showed no<br />
differences between a disposable mouth piece (sievers@) and a mouth piece containing a<br />
filter (FIEpA) (Irme, Kasahara et a.^.2002), but there has been little research in this area'<br />
5. Different exhalation techniques - subjects were asked to perform exhalation differently:<br />
203
For the oral measurements options included:<br />
o Single exhaled breath.<br />
o Tidal breathing.<br />
o Exhalation subsequent to a breath-hold with the suggested breath-hold ranging from 5<br />
to 30 seconds.<br />
o With or without the use <strong>of</strong> nose clips.<br />
o Subject sitting or standing.<br />
For the nasal measurements options included:<br />
o Single exhalation via the nose.<br />
o Sampling while tidal breathing.<br />
o Direct sampling by nasal olive.<br />
6. Different ranges <strong>of</strong> expiratory flows - differing flows were utilized ranging from 10-<br />
1O0mls/min.<br />
7. Arbitrary measures <strong>of</strong> expiratory mouth pressure -<br />
either no specific expiratory pressure<br />
was set, or it was not reported. In those that did standardise expiratory mouth pressure, the<br />
pressures ranged from 5-20cmHzO.<br />
8. Differences in recording and reporting <strong>of</strong> the results - the result reported was a different<br />
part <strong>of</strong> the recorded NO signal, including:<br />
o Peak.<br />
o Plateau.<br />
o Area under the curve.<br />
o <strong>The</strong> NO measurement at the peak COz level.<br />
Given the multitude <strong>of</strong> permutations, it was not surprising that different levels and at times<br />
different results in similar populations <strong>of</strong> subjects were being reported in the literature.<br />
9.3 Standardisation<br />
In view <strong>of</strong> these different levels reported at that time, a European Respiratory Society Task<br />
Force (ERSTF) was assembled to review techniques and a meeting organised in Stockholm,<br />
September 1996, a meeting I attended. This was the first attempt to standardise measurements<br />
for future NO research. This meeting resulted in an "Exhaled and Nasal Nitric Oxide<br />
Measurements; Recommendations" (Kharitonov, Alving et al. 1997). Two years later, a task<br />
204
force from the ATS with many <strong>of</strong> the researchers from the ERSTF meeting combined to<br />
produce ..Recommendations <strong>of</strong> Standardised Procedures for the Online and Off-line<br />
Measurement <strong>of</strong> Exhaled Lower Respiratory Nitric oxide and Nasal Nitric oxide in Adults<br />
and childre n - lggg,,, which was adopted in July <strong>of</strong> that year as an <strong>of</strong>ficial statement <strong>of</strong> the<br />
ATS (American Thoracic Society and Association. 1999). Following this, there have been<br />
two further documents written regarding standardisation <strong>of</strong> No measurement (Baraldi' de<br />
Jongste et a:.2002; American Thoracic Society and European Respiratory Society 2005)' <strong>The</strong><br />
established ERSTF went on to publish "Measurement <strong>of</strong> Exhaled Nitric oxide in Children'<br />
ZO0I- (Baraldi, de Jongste et al.2OO2). Finally a joint statement was prepared by the ATS and<br />
ERS, which was adopted by both societies in 2O04 and subsequently published in 2005' This<br />
was the ..ATS/ERS Recommendations for Standardized Procedures for the Online and Off-<br />
line Measurement <strong>of</strong> Exhaled Lower Respiratory Nitric oxide and Nasal Nitric oxide 2w5"<br />
(American Thoracic Society and European Respiratory Society 2005)'<br />
In this section I will review the current 'standardisation' documents and discuss some <strong>of</strong> the<br />
studies that led to these recommendations and the developments from 1997 to 2005' I will<br />
critique my research to identify if the procedures I developed were correct and if they<br />
contributed to the variability seen between research groups in light <strong>of</strong> subsequent findings'<br />
<strong>The</strong> papers published from the research described in the previous chapters have been cited in<br />
these documents.<br />
<strong>The</strong>re are a number <strong>of</strong> separate acceptable procedures to measure NO in adults and children -<br />
each now having a standard method <strong>of</strong> measurement. <strong>The</strong>se are:<br />
1. Single breath online measurement (see Table 9'1)'<br />
2. online measurement <strong>of</strong> exhaled No during spontaneous or tidal breathing (see Table 9'2)'<br />
3. Offline measurement via a reservoir collection system (for delayed analysis)'<br />
4. Nasal NO measurements (see Table 9.3).<br />
Following this, I will review the more recent research on measuring exhaled NO in infants<br />
using single breath or tidal breathing techniques'<br />
9.3.1 Single breath online rneasurement<br />
<strong>The</strong> current technique recommended for single breath online measurement for adults and<br />
children (> 6 years <strong>of</strong> age) who are able to achieve a single exhalation is presented in Table<br />
9.1.<br />
205
Table 9.1: <strong>The</strong> recommended standard for single breath online NO measurement<br />
Single breath online measurement for adults and children:<br />
o the subject should be seated comfortably with the mouth piece at the proper height and<br />
position.<br />
o tho subject, particularly a child, should breathe quietly for five minutes to acclimatise.<br />
. a nose clip should n<strong>of</strong> be used.<br />
o the use <strong>of</strong> NO free air (containing less than Sppb) for inhalation is preferable.<br />
o tho subject inhales to total lung capacity.<br />
o tho exhalation takes place immediately after inhalation with no breath-hold.<br />
o the subject exhales at a constant flow <strong>of</strong> S0mls/s and the expiratory flow is maintained at a<br />
constant level.'<br />
o the expiratory pressure should be maintained between 5 and 20cmHzO.<br />
. visual bi<strong>of</strong>eedback should be available to assist subjects in controlling their exhalation rate and<br />
pressure.<br />
. a peak and then a plateau should be seen in the recording.<br />
. an NO plateau <strong>of</strong> greater than three seconds identified during an exhalation <strong>of</strong> six seconds in<br />
adults and a plateau <strong>of</strong> two seconds identified during an exhalation <strong>of</strong> four seconds in children is<br />
required.<br />
o the plateau levelshould be used as the recorded measurement.<br />
. the final result reported should be the mean <strong>of</strong> three readings within 10% variability or two within<br />
5% variability.<br />
. separate exhalations should be done at greater than 30 second intervals.<br />
l. Note: fhis is the standard rwommendation unless using ditferent expintory flows to enable musurement <strong>of</strong> NO trwt<br />
difterent lung compttments, see section bolow: 9.3.1.(i) Flw*<br />
e.3.1 (i) Flow<br />
Early studies recornmended a slow and prolonged exhalation between 10 and l5Umin (16-<br />
25mls/s) for a duration <strong>of</strong> 5-30 seconds (Kharitonov, Alving et al. L997). However in<br />
subsequent statements, a flow rate <strong>of</strong> 0.05Us (SOmls/s) was been the main recommendation.<br />
Based on our own and other studies, it has been repeatedly demonstrated that exhaled NO is<br />
markedly flow dependent with a reduction in concentration when the exhalation rate increases<br />
(Alving, Weitzberg et al. 1993; Imada, Iwamoto et al. 1996; Kharitonov, Chung et al. 1996;<br />
Lundberg, Weitzberg et al. 1996; Byrnes, Dinarevic etal.1997; Byrnes, Dinarevic etal.1997;<br />
Byrnes, Dinarevic et al. 1997; Hogman, Stromberg et al. L997; Silk<strong>of</strong>f, McClean et al. 1997).<br />
<strong>The</strong> 50mls/s flow has been shown in subsequent studies to be acceptable and reproducible in<br />
adults (Silk<strong>of</strong>l McClean et ^1. 1997; Deykin, Massaro et al.2OO2; Kharitonov, Gonio et al.<br />
2003), in adolescents (Kissoon, Duckworth et al. 2000) and in children (Baraldi, Azzolin et al.<br />
1999; Kissoon, Duckworth et al. 2002; Pedroletti, Zetterquist et al. 2002; Pijnenburg,<br />
Lissenberg et al.20O2; Shin, Rose-Gottron et al. 2OO2; Kharitonov, Gonio et al. 2003). From<br />
a practical standpoint, fast exhalations in children result in a rapid decline in lung volume and<br />
206
difficulty in sustaining exhalations long enough for the NO values to plateau (Kissoon'<br />
Duckworth et al. 1999). Conection <strong>of</strong> expiratory flow for lung size has been debated given<br />
the widely different lung volumes throughout early childhood, but this was not shown to<br />
reduce exhaled NO variability (Kroesbergen, Jobsis et al' 1999)' <strong>The</strong>re is no current<br />
recommendation for correcting the expiratory flow for either weight or height in children or<br />
adults.<br />
Compartment modeling has further demonstrated the importance <strong>of</strong> the expiratory flow used -<br />
with the flow determining where the exhaled No measured is being produced within the lung'<br />
<strong>The</strong> exchange dynamics <strong>of</strong> NO is different from the other previously well studied gases such<br />
as oz and coz because <strong>of</strong> differences in physical and biochemical properties' Firstly, No is<br />
highly reactive. Secondly, it is actively produced basally in response to various stimuli'<br />
Thirdly, it binds avidly to haemoglobin with different kinetics. I have alluded to these factors<br />
previously in Chapter 6 when comparing the differing NO and COz exhalation pattern' where<br />
the latter is known to be predominantly produced in the alveolar compartment' We used this<br />
finding to suggest that NO was coming from a different part <strong>of</strong> the lung to COz'<br />
One research group (Tsoukias and George 1998) used a two compartment model to describe<br />
the pulmonary exchange dynamics <strong>of</strong> No, and to try and understand the results <strong>of</strong> the No<br />
levels being presented in the literature (see figure 9.1 below)' Tsoukias' research group<br />
depicted the first compartment as a rigid or non-expansile compartment representing the<br />
conducting zoneithe airways from the trachea through to generation 17 as defined by Weibel<br />
(weibel 1963). <strong>The</strong> second compartment was described as a flexible or expansile<br />
compartment representing the respiratory bronchioles to the alveolar region' Both<br />
compartments were surrounded by a layer <strong>of</strong> tissue representing the bronchial mucosa in the<br />
airway compartment and the alveolar membrane in the alveolar compartment' <strong>The</strong> next layer<br />
was blood which represented the bronchial circulation and the pulmonary circulation in the<br />
airway and al veolar compartments respectivel y'<br />
Using this model, the group then derived a series <strong>of</strong> mathematical equations to describe each<br />
<strong>of</strong> the possible pathways for NO. This included NO production in the tissue, transfer <strong>of</strong> NO<br />
from tissue to blood (and therefore loss <strong>of</strong> NO) and transfer <strong>of</strong> NO from tissue to alveolar or<br />
airway space. Equations were also derived for the behaviour <strong>of</strong> No in intra-thoracic air'<br />
assuming a well mixed compartment and accounting for convective flow <strong>of</strong> No in and out <strong>of</strong><br />
the compartments in inspiratory and expiratory manoeuvres, and for diffusion back into<br />
tissue. <strong>The</strong> blood layers were considered an infinite sink for NO. All the parameters were<br />
207
taken from the available literature including the production rate <strong>of</strong> NO in the tissue layer.<br />
Using these equations they could then explain the pattern <strong>of</strong> NO seen in single exhalations<br />
under different conditions including with and without breath holds and at different expiratory<br />
flows. Overall, the model worked well and appeared to mirror actual results from the human<br />
studies, although the predictions <strong>of</strong> the initial peaks seen with single exhalations were <strong>of</strong><br />
variable accuracy. Generally the model underestimated the peak in single exhalations and<br />
overestimated the peak after breath-holds. <strong>The</strong> researchers stated in their paper that "Initial<br />
reports have suggested that ambient levels <strong>of</strong> NO do not affect the exhalation pr<strong>of</strong>ile." I also<br />
could not ascertain if they took into account any upper airway contamination or whether, like<br />
a later group (Silk<strong>of</strong>f, Sylvester et al. 2000), they assumed closed vellum. <strong>The</strong>se could be<br />
potential sources <strong>of</strong> error.<br />
Figure 9.1: A two-compartment model <strong>of</strong> NO exchange<br />
x<br />
€ €<br />
(J<br />
E<br />
o<br />
nr.v(t)<br />
Car,v(r)<br />
o''f*<br />
:I<br />
v;,c;<br />
Dead space<br />
ALVeoli<br />
Ftg. l. Schematlc <strong>of</strong> Z-compartment model for nltric oxtde (NO)<br />
pulmonary exchange. Flrst comPartment rePresents relatively nonexpanslle<br />
conducting airways; second compartment represents exPansile<br />
alveoli. Each compartment ls adJacent to a layer <strong>of</strong> tissue that is<br />
capable <strong>of</strong> producing and consumlng NO. Exterior to tissue is a layer<br />
<strong>of</strong>-blood that represents bronchlal or.pulmonarv clrculation and<br />
seryes as an infinlte slnk for NO. Ve and V1, exPlratory and<br />
Insplratory flow, respectively; Ce and Cr, exPiratory and inspiratory<br />
concentration, respectlvely; Ca1 and C61v, airway and alveolar concentratlon,<br />
respectivCly; Var and Vap, airway and alveolar volume,<br />
respectively; Jrrg,ar, and Jr:g,atv, total flux <strong>of</strong> NO from tlssue to alr and<br />
from alveolar tlssue, respectively; t, time; V volume.<br />
A two-compartment model <strong>of</strong> NO exchange dynamics using equations to derive the measurement <strong>of</strong><br />
NO from different lung compartments depending on the expiratory flow.<br />
Taken from Tsoukias NM, George SC. A fwo-compartment model <strong>of</strong> pulmonary nitric oxide exchange<br />
dynamics. Joumal<strong>of</strong> Applied Physiology 1998;85(2): 653-666 (Tsoukias and George 1998).<br />
208
<strong>The</strong>se predictions built on work from a previous group who had also derived equations to<br />
model NO interactions in the lung (Hyde, Geigel et al. 1997). <strong>The</strong>ir model was able to predict<br />
NO results from the lower airway using a one compartment model but was unable to account<br />
for the upper airway contamination. A later group also used a two compartment model and<br />
reported that a breath hold <strong>of</strong> 10 to 20 seconds meant that the concentration <strong>of</strong> NO was<br />
equilibrated - "alveolar airway" production <strong>of</strong> NO now equaled the amount <strong>of</strong> NO diffusing<br />
out <strong>of</strong> the airways (pietropaoli, perillo et al. 1999). <strong>The</strong> subsequent exhalation could then give<br />
the alveolar content <strong>of</strong> No. Finally, using similarly derived equations, another group re-<br />
analysed their earlier data, from which they had concluded that NO levels correlated with<br />
expiratory flow (Silk<strong>of</strong>f, Sylvester et al. 2000). <strong>The</strong>y determined that a even closer<br />
relationship existed between the exhaled No concentration (defined as the quantity <strong>of</strong> No<br />
exhaled per unit time) and the expiratory flow. <strong>The</strong>y felt this allowed a better estimation <strong>of</strong><br />
the quantity <strong>of</strong> NO diffusing into the exhaled gas and therefore better reflected tissue NO<br />
concentration.<br />
In essence, I believe these groups were making the point that studies were presenting No<br />
results in a far too simplistic manner. NO was being presented as a single result from a single<br />
compartment when in reality the NO concentration varied in different parts <strong>of</strong> the airway<br />
system. In addition, the levels were always interpreted as if the NO was solely the result <strong>of</strong><br />
NO production, while in reality the NO reading was a combination <strong>of</strong> production' diffusion<br />
(dictated by the rate <strong>of</strong> removal by capillary blood throughout the lung) and ventilation' <strong>The</strong><br />
importance <strong>of</strong> the modeling described above was to define specifically that different<br />
expiratory rates would allow evaluation <strong>of</strong> different lung compartments. This could be critical<br />
in appropriately discerning inflammation in different diseases; for example the conducting<br />
airways in the airway disease <strong>of</strong> asthma or the alveolar compartment in alveolar diseases such<br />
as in interstitial lung disease. This theory was tested by measuring 'fractionated' NO in a<br />
cross section <strong>of</strong> subjects using three exhalation flows at 100, 1?5 and 370m1/s in 40 newly<br />
diagnosed, steroid naive asthmatics, 17 patients with allergic alveolitis and 57 healthy<br />
controls (Lehrimaki, Kankaanranta et al. 2001). <strong>The</strong> asthmatic patients did indeed have higher<br />
bronchial NO output at 25nLJs compared to those with alveolitis or healthy controls which<br />
were both measured at 0.7n[./s. On the other hand, the subjects with alveolitis had higher<br />
alveolar NO concentrations at 4.1ppb than the asthmatic or healthy subjects which were both<br />
measured at l.lppb. Thus there was no difference in No between those with asthma and<br />
healthy controls when the alveolar compartment was measured (I-ehtimaki, Kankaanranta et<br />
al. 2001). Finally they demonstrated that the concentration <strong>of</strong> NO in the bronchial<br />
209
compartment correlated with the serum level <strong>of</strong> the EPX and bronchial hyper-responsiveness<br />
in those with asthma, while the alveolar NO concentration in patients with alveolitis<br />
correlated inversely with the pulmonary diffusing capacity.<br />
A number <strong>of</strong> studies have now been conducted assessing NO in this manner. <strong>The</strong>se have<br />
demonstrated a higher exhaled NO in the conducting airways in asthmatics compared with<br />
controls, with no mean difference <strong>of</strong> NO measured in alveolar compartment in otherwise<br />
stable asthmatics (Hogman, Holmkvist et al. 2002; I-ehtimaki, Kankaanranta et al. 20fr2;<br />
Mahut, Delacourt et ^l.2OO4; Shin, Rose-Gottron et al. 2004; Lehtimaki, Kankaanranta et al.<br />
2005). <strong>The</strong> reduction <strong>of</strong> NO in response to steroid therapy also occurs in the airway<br />
compartment (Silk<strong>of</strong>f, Sylvester et al. 2000; Irhtimaki, Kankaanranta et al. 2OOl; Shin, Rose-<br />
Gottron et al.2004). In a cross sectional study <strong>of</strong> proximal and distal sampling in children, it<br />
was the proximal NO output that correlated better with the FEVr (Mahut, Delacourt et al.<br />
20M). However, as no theory is perfect, both high airway and high alveolar NO has been<br />
demonstrated on highly symptomatic asthmatics or those with ongoing nocturnal symptoms<br />
(Mahut, Delacourt et al.2004; Shin, Rose-Gottron et al.2004; I€htimaki, Kankaanranta et al.<br />
2005). Also, high airway levels <strong>of</strong> NO were also seen in subjects with allergic rhinitis despite<br />
having no airway symptoms (Hogman, Holmkvist et al.2002). Iow ainvay levels were seen<br />
in smokers and patients with COPD, though the alveolar levels in NO were increased in the<br />
COPD patients (Hogman, Holmkvist et al. 2002). Airway exhaled NO levels were no<br />
different between CF and healthy control adolescents, but lower levels were seen in the<br />
alveolar compartment and tissue concentration in the healthy goup (Shin, Rose-Gottron et al.<br />
2N2).Increased rates <strong>of</strong> alveolar NO have been described in allergic alveolitis (khtimaki,<br />
Kankaanranta et al. 2001) and scleroderma (Girgis, Gugnani et al. 2002) with negative<br />
correlations between NO levels and the diffusion factor demonstrated in both.<br />
<strong>The</strong> nomenclature used has also required adaptation as discussed in the 2002 and 2005<br />
standardisation documents (Baraldi, de Jongste et al. 2OO2; American Thoracic Society and<br />
European Respiratory Society 2005). <strong>The</strong> previous recommendations were to express exhaled<br />
NO or nasal NO in terms <strong>of</strong> ppb (equivalent to nIJL <strong>of</strong> exhaled air). <strong>The</strong> standard single<br />
exhalation remains at 50mls/s, now suggested to be denoted as F4o0.05. NO output<br />
representing the rate <strong>of</strong> exhaled NO is denoted by Voo and calculated from the product <strong>of</strong> NO<br />
concentration in nL/L and expiratory flow rate in Umin in the following equation:<br />
Vno (nUmln) = NO (nUL) x alrflow rate (Umln)<br />
2ro
<strong>The</strong> ATS guideline states "terms such as NO release, NO excretion, NO secretion and NO<br />
production are to be discouraged when referring to Voo" (American Thoracic Society<br />
European Respiratory Society 2005).<br />
A constant expiratory flow is required with any exhalation measured and has been<br />
reconrmended following the findings from early studies. This is most easily achieved with<br />
appropriate bi<strong>of</strong>eedback using a gauge or computer display for subjects to maintain expiratory<br />
flow within specified limits. All newer machines as exemplified by the companies that took<br />
part in the 2005 recommendations (Aerocrine, Eco Physics, Eco Medics, Ionics Instruments<br />
and Ekips Technologies) have these available. However there are other options which include<br />
the use <strong>of</strong> dynamic resisters (Kharitonov, Gonio et al. 2003), operator controlled flow<br />
(Baraldi, Scollo et al. 2000), starling resisters (Hogman, Stromberg et ^l' 1997; Tsoukias'<br />
Tannous et al. 1998) and server controlled devices (Silk<strong>of</strong>f, Bates et al. 2004)' <strong>The</strong>se<br />
techniques have usually been tried in young children but can also be useful in other subjects<br />
that have difficulties controlling their exhalation such as those with neuromuscular disease' In<br />
children aged 4-g years, 507o could not perform the single breath online technique adequately'<br />
However, the addition <strong>of</strong> a dynamic flow resister allowed exhalation with a variable mouth<br />
pressure while maintaining constant expiratory flow and resulted in only 77o <strong>of</strong> the children<br />
unable to perform the manoeuvre (Baraldi, scollo et al. 2000).<br />
9.3.1 (ii) Mouth Pressure<br />
An expiratory mouth pressure <strong>of</strong> between five and 2}cttttLzO is currently recommended' This<br />
degree <strong>of</strong> resistance is needed for velum closure <strong>of</strong> the s<strong>of</strong>t palate to prevent nasal<br />
contamination <strong>of</strong> the exhaled NO and has been validated by nasal COz measurements (Silk<strong>of</strong>f'<br />
McClean et al. L997) and nasal argon insufflation (Kharitonov and Barnes 1997)' Many<br />
studies have confirmed higher sinus and nasal levels when comparing sinus' nasal' exhaled<br />
and/or lower airway sampling (Alving, weitzberg et al. 1993; Lundberg, Rinder et al' 1994;<br />
Lundberg, weitzberg et al. 1994; Schedin, Frostell et al. 1995; Dillon, Hampl et al' 1996;<br />
Imada, Iwamoto et al. 1996; Kimberly, Nejadnik et al. 1996). <strong>The</strong> other options described to<br />
prevent nasal contamination has been continuous nasal aspiration (Silk<strong>of</strong>f, Kesten et al' 1995)<br />
or to inflate a balloon in the posterior nasal pharynx to separate the two compartments<br />
(Schedin, Frostell et al. 1995; Kimberly, Nejadnik et al. 1996). Clearly, exhaling against an<br />
appropriate mouth pressure is the easiest option and has been most widely used'<br />
Unlike the effects <strong>of</strong> flow on NO, the effects <strong>of</strong> varying mouth pressure has not demonstrated<br />
consistent findings in all studies. Two studies showed no difference in plateau NO<br />
2lr<br />
and
measurement with variable expiratory mouth pressure (Hogman, Stromberg et al. 1997;<br />
Silk<strong>of</strong>f, McClean et al. t997), but another showed increasing exhaled NO concentrations with<br />
increasing expiratory pressure from two to ten cmHzO (Kondo, Haniuda et al. 2003). Our own<br />
findings suggested the opposite with a reduction <strong>of</strong> exhaled NO with increasing mouth<br />
pressure in two <strong>of</strong> ten subjects (Byrnes, Dinarevic et al. 1997). However, as noted in the 2005<br />
recommendations, a pressure above 20cmHzo should be avoided as it may be "uncomfortable<br />
for patients or subjects to maintain". Indeed, I think this may have contributed to the fact that<br />
there was a falling <strong>of</strong>f <strong>of</strong> the exhaled NO in our study in two subjects. Having tried it<br />
repeatedly myself, while pressures <strong>of</strong> 4mmHg (5.4cmHzO) and 8mmHg (10.9cmHzO) were<br />
easy to maintain, l2mmHg (16.3cmHzO) became a little more difficult but l6mmHg<br />
(21.8crnHzO), the highest expiratory pressure sampled in our study, was very tiring to exhale<br />
against, and 2OmmHg Q7.2cn*l2O) almost impossible to maintain and therefore was not<br />
used. One needs to recall that in the earlier studies a far longer exhalation time was also<br />
required with the older modified analysers.<br />
9.3.1 (iii) Nasal clips and breath-holding<br />
While the early studies varied as to whether or not nose clips were used, the current<br />
recommendation is not to wear them when doing exhaled NO in any <strong>of</strong> the methods <strong>of</strong><br />
measurement as this may actually increase the risk <strong>of</strong> having nasal and sinus contamination <strong>of</strong><br />
the oropharyngeal and exhaled sample (American Thoracic Society and European Respiratory<br />
Society 2005). <strong>The</strong> previous standards suggested that a nose clip may be worn to prevent<br />
inhaled air to contaminate the exhaled sample (see below) (Baraldi, de Jongste et al.2002).<br />
In addition, the inhalation is now recommended as being through the mouth with higher initial<br />
exhaled NO demonstrated following an inhaled breath through the nose when compared to<br />
inhalation through the mouth @hillips, Giraud et al. 1996; Robbins, Floreani et al. 1996).Any<br />
degree <strong>of</strong> breath-holding also results in NO accumulation in the nasal and oropharyngeal<br />
spaces giving higher exhaled levels @ersson, Wiklund et al. 1993; Lundberg, Weitzberg et al.<br />
L994; Kharitonov, Chung et al. 1996; Kimberly, Nejadnik et al. 1996; Massaro, Mehta et al.<br />
1996; Sato, Sakamaki et al. 1996). Breath-holding was shown to increase NO levels in a time<br />
dependent manner in atopic and healthy subjects (Martin, Bryden et al. 1996). Breath-holding<br />
appears to affect the peak more than the plateaux levels in healthy and asthmatic subjects<br />
(Shinkai, Suzuki et al.2O02). <strong>The</strong> effect <strong>of</strong> breath-holding also depended on the ambient NO;<br />
when the ambient NO was greater than 10ppb, exhaled NO was decreased whereas if the<br />
212
ambient No was less than 10ppb the exhaled No was increased after a 10 second breath-hold<br />
(Jobsis, Schellekens et al. 2001).<br />
9.3.1 (iv) <strong>The</strong> recorded measurement<br />
<strong>The</strong> NO recorded in the literature has been peak, plateau, area under the curve or NO plateau<br />
signal at COz peak. <strong>The</strong> current recommendation is for plateau levels to be reported' unless<br />
calculating NO output. This requires a plateau <strong>of</strong> three seconds following exhalation <strong>of</strong> at<br />
least six seconds for participants older than 12 years <strong>of</strong> age' or two seconds following<br />
exhalation for at least four seconds for children less than 12 years <strong>of</strong> age' <strong>The</strong> peak<br />
measurement has shown more variability as influenced by the degfee <strong>of</strong> dead space' ambient<br />
NO and inspiration taken via nose or mouth. An expiratory flow <strong>of</strong> 50mls/s ensures an<br />
acceptable time to plateau, an acceptable rate <strong>of</strong> decline in lung volumes and is appropriate<br />
for children at less than 12 years <strong>of</strong> age, as well as those with vital capacities less than one<br />
litre (Pfaff and Morgan l994;Canady, Platts-Mills et al. 1999; Franklin, Taplin et al' 1999;<br />
Kissoon, Duckworth et al. 1999).<br />
9.3.1 (v) <strong>The</strong> effect <strong>of</strong> ambient nitric oxide<br />
Recommendations since 199? have been to record the ambient NO as this fluctuates<br />
considerably. A note from one <strong>of</strong> the authors (A Sovijarvi, Helsinki <strong>University</strong> Central<br />
Hospital, Finland) in this document states; the ambient NO was noted to vary between I and<br />
600ppb during winter months in their studies (Kharitonov, Alving et al. 1997). Initially it was<br />
suggested that measurements should only be performed with an ambient NO less than 40ppb'<br />
with improved standardisation and more sensitive and rapidly responding analysers this is<br />
now too high. <strong>The</strong> 2005 recommendation is that the ambient NO be less than 5ppb and/or that<br />
subjects inhale NO free air while being tested. <strong>The</strong> newer NO analysers including the smaller'<br />
more transportable units such as the MINO@ (Aerocrine AB, Solna, Sweden) or z-700 No<br />
meter @asibi Environmental Corporation, Glendale, California, United States <strong>of</strong> America)<br />
have a scrubbing facility within the inhalation limb. This may be more important for reservoir<br />
collections during tidal breathing where an increased amount <strong>of</strong> ambient NO is exhaled and<br />
thus collected. In our experiments we elected to measure only when the NO in ambient air<br />
was less than 10ppb. However on many days this precluded measurement which delayed the<br />
experiments. Unlike other researchers, we found exhalation from a high NO concentration<br />
compared to exhalation from a low NO concentration resulted in a drop in the overall NO<br />
level as discussed in Chapter 7. On review now, it may have been because we were waiting<br />
for a plateau to develop with a drop from the high ambient NO reading and it took some time<br />
2r3
to settle. We may have been measuring the latter part <strong>of</strong> the exhalation in error and this<br />
potentially may give lower levels than the peak or the true plateau. Thus, we may have been<br />
comparing the plateau in the exhalation from the low ambient NO with the last part <strong>of</strong> the<br />
exhalation from the high ambient NO. In two studies NO was significantly higher when<br />
measured in a total <strong>of</strong> 215 children including healthy controls, non-asthmatic atopics and<br />
asthmatics at times <strong>of</strong> high ambient NO (>10ppb, mean l9ppb) and at times <strong>of</strong> low ambient<br />
NO (10ppb was the cut-<strong>of</strong>f used) and<br />
measured NO increased if the ambient NO was lower (NOclOppb) (Jobsis, Schellekens et al.<br />
2001). Nasal NO in healthy children correlated with ambient NO and, in those children<br />
greater than 12 years, it was the only factor correlating with the measured expiratory levels<br />
(Struben, Wieringa et al. 2005). More recently there has been a study suggesting that even<br />
levels <strong>of</strong> 5-10ppb ambient NO may have an effect on the exhaled results, with no difference<br />
seen at less than 5ppb, upholding the most recent recommendations (Franklin, Turner et al.<br />
2004).<br />
Other studies have examined effects <strong>of</strong> pollution on exhaled NO. Ambient NO levels, as well<br />
as ambient CO levels, were positively correlated with the exhaled NO results in 16 non-<br />
smoking healthy subjects (Van Amsterdam, Verlaan et al. 1999). Mean air pollution was also<br />
correlated with the exhaled NO levels in 16 healthy subjects measured on two separate days<br />
(van Amsterdam, Verlaan et al. 1999) and 18 subjects measured on four separate days<br />
(Steerenberg, Snelder et al. t999) at times <strong>of</strong> differing pollutant levels. At times <strong>of</strong> high<br />
ambient NO, an increased risk <strong>of</strong> developing respiratory symptoms described as "sore throat,<br />
runny nose, having a cold or being sick at home in the following week" was also shown in 68<br />
children (Fischer, Steerenberg et al.2N2).<br />
9.4<br />
Online spontaneous or tidal breathing measurement<br />
A single exhaled breath with online measurement remains the measurement <strong>of</strong> choice.<br />
However, in some studies 50Vo <strong>of</strong> children aged 4-8 years <strong>of</strong> age @araldi, Scollo et al. 2000),<br />
l\Vo <strong>of</strong> children aged 9-16 years (Baraldi, Scollo et al. 2000) and 30Vo <strong>of</strong> children aged 4-16<br />
years <strong>of</strong> age (Jobsis, Schellekens et al. L999) could not pedonn this technique appropriately.<br />
Online measurement during tidal breathing, usually as a mean <strong>of</strong> three breaths, has been<br />
explored as an option for younger children and infants. One study measured exhalation<br />
following quiet breathing with the operator controlling the expiratory flow by varying<br />
214
expiratory resistance. A flexible rubber tube 9cm long with a diameter <strong>of</strong> 0'9cm and an end<br />
resistor <strong>of</strong> 2mm was placed in the circuit. <strong>The</strong> operator could bend the flexible device during<br />
exhalation to provide resistance and allow manual control <strong>of</strong> the expiratory flow by direct<br />
checking on the monitor without requiring active cooperation from the child' <strong>of</strong> the 115<br />
children enrolled, lr0 (g3vo)were able to perform this manoeuvre compared to 73 (63Vo)who<br />
were able to perform the active single exhaled breath (Baraldi, Scollo et al. 2000)' In another<br />
study, NO was measured online in 67 children during controlled tidal breathing (767o wete<br />
aged2-5 years) with flow measured by a pneumotachograph and displayed on the computer<br />
screen with a negligible delay <strong>of</strong> 0.1 second allowing the operator to target the exhaled flow<br />
within preset limits <strong>of</strong> 0.4 to 0.6Us by continuously adapting the outlet resistance. <strong>of</strong> the nine<br />
children (l3zo) who failed the measurement; seven had asthma, two had episodic wheeze'<br />
four were aged two, three were aged three and two were aged five' <strong>The</strong> visualisation <strong>of</strong> the<br />
online measurements allowed breath to breath pr<strong>of</strong>iles to be scrutinised to ensure "a stable'<br />
calm and reproducible breathing pattern" (Buchvald and Bisgaard 2001)'<br />
Tidal breathing results in lower NO levels than single exhalations and this is exaggerated at<br />
higher No readings (Silk<strong>of</strong>f, Mcclean et al. 1997; Rutgers, Meijer et al. 1998; Franklin,<br />
Turner et a;.2004t. While a reasonable correlation has been found between the two (Silk<strong>of</strong>f'<br />
Stevens et al. 1999; Kissoon, Duckworth et al. 2000; Franklin, Turner et al' 2N4), individual<br />
agreement can be poor (Rutgers, Meijer et al. 1998; Franklin, Turner et al.200,4)' <strong>The</strong> lower<br />
results could be explained by increased inhaled air dilution from a low NO source and<br />
variable flow during tidal breathing, and the possibility <strong>of</strong> breath-holding and nasal leakage in<br />
single breaths (Hyde, Geigel et al. 1997; Silk<strong>of</strong>f, Mcclean et al. 1997; Rutgers, Meijer et al'<br />
l99g). This can be improved by controlling expiratory flow during tidal breathing -<br />
but when<br />
only passive co-operation is available and considerable operator experience appears necessary<br />
to manipulate the resistance correctly (Baraldi, Scollo et al. 2000; Buchvald and Bisgaard<br />
2001). I would also be concerned that the rubber tubing itself, in one method described, may<br />
result in some NO loss following reaction with the sulphur hydryl groups' This online<br />
spontaneous breathing method still "requires passive cooperation as the child needs to breath<br />
slowly and regularly through a tight fitted mask, close the mouth around a mouth piece which<br />
is <strong>of</strong>ten the limiting factor, as well as being able to tolerate breathing against resistance"<br />
(Buchvald and Bisgaard 2001). Despite this, results have shown the same patterns as seen<br />
with the single online measurements in the cross-sectional population studies (Baraldi'<br />
Azzolin et al. 1997; Baraldi, Azzolin et al. 1998; Baraldi, Dario et al. 1999). Use <strong>of</strong> this in<br />
infants is discussed below in Section 9.16.<br />
2t5
9.5 Off-line measurement<br />
NO determinations can be measured from samples collected into a reservoir system and later<br />
fed through a cheminiluminescence analyzer. Similar to early online experiments, while the<br />
absolute levels <strong>of</strong> NO measured by reservoir collection varied between investigators, the<br />
findings across disease entities remained consistent (Alving, Weitzberg et al. 1993; Persson,<br />
Wiklund et al. 1993; Kharitonov, Yates et al. L994; Kharitonov, Yates et al. 1995; Massaro,<br />
Gaston et al. 1995; Deykin, Halpern et al. 1998).<br />
A number <strong>of</strong> standardisation issues arose with this technique. Firstly, the concern regarding<br />
NO reactivity and that inert collection bags would be required. Other factors that could affect<br />
stability included temperature, light exposure, damage to bags, leaks and pressure changes.<br />
Mylar or tedlar bags were early reported as the most stable (Kharitonov, Alving et al. 1997)<br />
with NO levels consistent up to 12 hours post collection (Massaro, Gaston et al. 1995;<br />
Massaro, Mehta et al. 1996; Canady, Platts-Mills et al. 1999; Jobsis, Schellekens et al. 2001).<br />
Some investigators reported continued stability for 24-48 hours @aredi, Loukides et al. 1998;<br />
Djupesland, Qian et al. 2001), while others found an increase in the NO concentration<br />
(Silk<strong>of</strong>fl Stevens et al. 1999). NO collected in a reservoir containing silica gel was stable for<br />
24 hours @aredi, Ioukides et al. 1998). One $oup<br />
used a tube collection system showing<br />
better online and <strong>of</strong>f-line agreement than bag collections for both exhaled and nasal levels<br />
@jupesland, Qian et al. 2001). Reservoir stability also depends on temperature. NO results<br />
appeared to be stable for nine hours at 4o to 37oC but increased between nine and 48 hours in<br />
samples with initially low concentrations. It was not stable at higher temperatures and the use<br />
<strong>of</strong> a drying agent did not improve the stability (Bodini, Pijnenburg et al. 2003). One study<br />
evaluated 185 breaths in bags that were re-used 10-20 times flushing three times with NO free<br />
air between uses and stored at 22oC. Over a four day period, the samples with low NO<br />
increased in a linear fashion, and those with high NO from asthmatic subjects showed a<br />
gradual decrease (Silk<strong>of</strong>f, Stevens et al. L999; Kharitonov, Gonio et al. 2OO3). Higher<br />
temperatures made the changes more rapid and lower temperatures made it less likely to<br />
occur. <strong>The</strong> size <strong>of</strong> the balloon was not thought to be critical but was recommended to be<br />
similar to, or larger than, the subjects' vital capacity (Linn, Avila et al. 2004).<br />
Secondly, uncertainty existed as to whether the collection should be a single exhaled breath,<br />
several 'single exhaled breaths' or tidal breathing. <strong>The</strong> reproducibility with single breaths<br />
appeared to be fair or good with intra-subject coefficients <strong>of</strong> variation down to 5Vo (Gaston,<br />
216
Drazen et al. 1994; Nelson, Sears et al. 1997; Canady, Platts-Mills et al' 1999; Jobsis'<br />
Schellekens et al. 2001).<br />
Thirdly, there was debate as to whether the whole exhaled breath should be collected. Early<br />
experiments discarded the first part <strong>of</strong> exhalation to reduce contamination by inhaled ambient<br />
NO, dead space and nasopharyngeal space with a good correlation demonstrated between the<br />
remaining exhalate and single online exhalation (Borland, Cox et al.1993; Massaro, Mehta et<br />
al. 1996). Similarly, studies that discarded the first tidal volume when measuring during tidal<br />
breathing also achieved a closer approximation to online values (Paredi, Loukides et al' 1998;<br />
Jobsis, Raatgeep et al. 2001). <strong>The</strong> uncertainty was in deciding the absolute amount to discard<br />
and how to get consistency across subjects. While 150 to 200mls were used in the adult<br />
studies even greater uncertainty existed in the correct amount to discard in paediatric studies'<br />
<strong>The</strong> 'discard' was achieved employing spring loaded or manually activated valves' or low<br />
compliance reservoirs placed in series with the main collection vessel. Subsequently<br />
collecting the whole exhalation was shown to provide identical sensitivity and specificity as<br />
single breath online recordings, despite the fact that the absolute NO levels were not identical<br />
(Silk<strong>of</strong>fl Stevens et al. 1999; Djupesland, Qian et al. 2001; Jobsis, Raatgeep et al' 2001;<br />
Deykin, Massaro et a:.2002). <strong>The</strong> standardisation has therefore altered accordingly between<br />
lggT and2005 from discarding the first o.7sLexhaled (or 0.5L if the vital capacity was less<br />
than 2L) @uropean Respiratory Society 1993; Kharitonov, Alving et al. 1997) to collecting<br />
the entire breath (American Thoracic Society and Association. L999;Baraldi, de Jongste et al'<br />
2002).<br />
Similar across all methods, higher flow rates result in decreased concentration <strong>of</strong> NO<br />
recovered from the exhaled reservoir sample (Hogman, Stromberg et al' t997; Silk<strong>of</strong>l<br />
McClean et al. 1997). Despite this, subject group differences can be seen at expiratory rates<br />
between 50 and 500mls/s as long as the flow remains identical for all subjects in any<br />
comparison (Jobsis, Raatgeep et al. 2001; Deykin, Massaro et al' 2002)' With collection <strong>of</strong><br />
tidal breathing, using standard expiratory flow reduces variability and improves comparison<br />
with online results (Kissoon, Duckworth et al. 2000). This can be controlled by the subject or<br />
by the operator (Massaro, Mehta et al. 1996; Baraldi, Azzolin et al. 1997i Baraldi, Carra et al'<br />
t999; Baraldi, Dario et al. 1999; Jobsis, Schellekens et al. 2001). <strong>The</strong> tasldorce ultimately<br />
proposed recommendations for 50mls/s exhalation for both <strong>of</strong>fline and online collections as<br />
showing good correlation in the paediatric arena (Baraldi, de Jongste et al.2002)' It is still<br />
possible to sample different compartments using a different flow even with this technique<br />
2r7
(Hyde, Geigel et al. 1997; Tsoukias and George 1998; Tsoukias, Tannous et al. 1998; Silk<strong>of</strong>f,<br />
Sylvester et al. 2000).<br />
One <strong>of</strong> the reasons that higher levels were thought to be obtained using either single or tidal<br />
breathing into a reservoir was because <strong>of</strong> an open s<strong>of</strong>t palate and upper nasopharyngeal<br />
contamination (Sharma, Traylor et al. 1987; Schilling, Holzer et al. 1994; Kimberly, Nejadnik<br />
et al. 1996; Kharitonov and Barnes 1997). A single exhalation against even low resistance<br />
into the reservoir gave much lower levels (Massaro, Gaston et al. 1995). A resistor is now<br />
included in most reservoir bags with a recorlmendation <strong>of</strong> standard expiratory flow and<br />
standard expiratory pressure <strong>of</strong> 5cmHzO for both single breath (Massaro, Gaston et al. 1995;<br />
Deykin, Halpern et al. 1998) and tidal breathing reservoir collections (Canady, Platts-Mills et<br />
al. 1999; Jobsis, Schellekens et al. 2001). A dynamic flow restrictor is possible to aid the<br />
operator to standardise the exhaled breath with less able subjects by altering the resistance to<br />
give a standard flow during the exhalation @ijnenburg, Lissenberg et al. 2002), although<br />
again I imagine this must introduce an element <strong>of</strong> complexity.<br />
From 1999, inhaling via the mouth prior to exhalation into the reservoir bag was added to the<br />
protocol. By 2005 this recommendation had been revised to include the inhalation <strong>of</strong> NO free<br />
gas or via an NO scrubbing filter in the inspiratory limb. High concentrations <strong>of</strong> NO in the<br />
inspired air <strong>of</strong> greater than 20ppb significantly increased the <strong>of</strong>fline exhaled NO<br />
measurements (Gustafsson, Irone et al. l99l; Zayasu, Sekizawa et al. 1997). Again wearing<br />
a nose clip and breath holding are not recommended for similar reasons to the single breath<br />
online measurements (see section 9.3.1 (ii)) (Jobsis, Schellekens et al. 2OOt). Following these<br />
guidelines, subjects tested on multiple days had a variation between 1 and 5ppb, which is<br />
similar to that found in immediate online measurements, although in this study the collection<br />
was delayed for more than three seconds to discard the dead space (Borland, Cox et al. 1993;<br />
Massaro, Mehta et al. 1996). For the standard method to measure <strong>of</strong>f-line collections as either<br />
single exhaled breaths or as tidal breathing collections see Table 9.2.<br />
2r8
Table 9.2: <strong>The</strong> recommended standards for single breath and tidal breathing otf-line NO measurement<br />
<strong>The</strong> current recommended technique for single exhalation collection into a reservoir is:<br />
r inhale through the mouth from NO free air (
9.6 Nasal nitric oxide measurement<br />
For completion, I am going to briefly cover nasal NO although I did not measure this<br />
parameter as part <strong>of</strong> my research. Presentation <strong>of</strong> standard techniques for this measurement<br />
commenced in 1999 (American Thoracic Society and Association. 1999), with minor<br />
amendments in 2002 and 2005 (Baraldi, de Jongste et al.2N2; American Thoracic Society<br />
and European Respiratory Society 2005). <strong>The</strong> NO concentration is far greater in the nose than<br />
the lower respiratory tract and is measured in parts per million as opposed to parts per billion<br />
(Alving, Weitzberg et al. 1993; Gerlach, Rossaint et al. 1994; Lundberg, Farkas-Szallasi et al.<br />
1995). NO concentration is even higher in the para-nasal sinuses (Lundberg, Rinder et al.<br />
1994; Lundberg, Farkas-Szallasi et al. 1995; Haight, Qian et al. 2000).<br />
Table 9.3: <strong>The</strong> recommended standard for nasal NO measurement<br />
<strong>The</strong> current recommended technique for measurement <strong>of</strong> nasal NO is:<br />
o tho subject is seated<br />
o two nasalolives are placed with a central lumen in the nares.<br />
o these must be <strong>of</strong> sufficient size to occlude the nostril.<br />
o d sdlTrple is aspirated continuously at a constant rate from one narus.<br />
o gas is entrained via the other narus giving a trans-nasal flow in series.<br />
o a target sampling airflow <strong>of</strong> 0.25 to O.3Umin is recommended.<br />
. oropharyngeal and lower airway contamination is prevented by one <strong>of</strong> the methods<br />
suggested (see paragraph below).<br />
. mean NO values are calculated for a stable plateau <strong>of</strong> greater than ten seconds or five<br />
breaths (Silkotf, Chatkin et al. 1999; Ratjen, Kavuk et al. 2000).<br />
o the higher flow rate (compared to exhaled oral sampling) at 0.25-3Umin allows a steady<br />
plateau level<strong>of</strong> NO concentration in subjects within 20-30 seconds.<br />
This trans-nasal flow reflects the most cornmon method <strong>of</strong> measurement. An alternative is to<br />
have continual aspiration <strong>of</strong> both nares; one to discard and one to measure. Velum closure can<br />
be achieved in a number <strong>of</strong> ways; the most common is to slowly exhale orally against a<br />
resistance similar to the exhaled NO recommendation (Arnal, Didier et al. 1997; Kharitonov,<br />
Rajakulasingam et al. 1997; Silk<strong>of</strong>l McClean et al. 1997; Dubois, Douglas et al. 1998;<br />
Silk<strong>of</strong>f, Chatkin et al. 1999). Other options that have also been effective are purse-lip<br />
breathing via the mouth (Rodenstein and Stanescu 1983), breath-holding with the velum<br />
closed (Kimberly, Nejadnik et al. 1996) or voluntary elevation <strong>of</strong> the s<strong>of</strong>t palate by a trained<br />
subject (Giraud, Nejadnik et al. 1998). Of note for nasal NO measurements, healthy adults<br />
have significantly better repeatability than healthy children (Kharitonov, Walker et al. 2005).<br />
220
9.7<br />
In this next section, I review physiological variables that may alter NO results, recognistng<br />
there are conflicting findings reported in most parameters.<br />
9.7.1 Size and gender<br />
Firstly, there have been a number <strong>of</strong> investigations across animal species, and the reasons I<br />
refer to this is with regarding the possible effect <strong>of</strong> size, and to describe an extraordinary<br />
study done in elephants! Exhaled No results have been similar in rabbits, guinea pigs and rats<br />
(Gustafsson, Ifone et al. 1991; Stewart, Yalenza et al' 1995) but lower levels found in<br />
Antarctica seals (Stanek 1995). one heroic study measured direct exhalation with a one litre<br />
syringe and continuous online measurement in four elephants, finding no difference between<br />
these and human subjects. <strong>The</strong>y also showed no difference <strong>of</strong> NO with height <strong>of</strong> the elephants<br />
which ranged from 240cm to 260cm (although how significant a 20cm difference in height is<br />
at these levels remains questionable), or weight which ranged from 2800kg to 4000kg<br />
(Lewandowski, Busch et al. 1996).<br />
Secondly, with regard to gender, differences have been found in some studies (Tsang, kung<br />
et a;.2002; van der Ire, van den Bosch et a\.2002. Olivieri, Talamini et al.2006) but not in<br />
other adult (Jilma, Kastner et al. 1996; Morris, Sooranna et al. 1996; Bartley, Fergusson et al'<br />
1999; Kharitonov, Gonio et al. 2003) or in paediatric studies (Baraldi, Dario et al' 1999)'<br />
Measurements in over 100 (van der lfe, van den Bosch et al. 2002) and over 200 healthy<br />
adults (olivieri, Talamini et al. 2006) found normal exhaled No values to be significantly<br />
higher in men. One study showed these higher levels correlated with height and weight but<br />
not with age (Tsang, Ip et al. 2001). In another study, when corrected for body weight' men<br />
were calculated to exhale 50Vo moreNO than women (Morris, Sooranna et al' 1996)' Exhaled<br />
NO measured in nUmin/m2 correlated with body surface area in one (Phillips, Giraud et al'<br />
1996) but not a second study (Jilma, Kastner et al' 1996)'<br />
paediatric studies have also looked for gender differences. In almost 1000 13-14 year olds,<br />
higher levels <strong>of</strong> exhaled NO were independently related to male gender, as well as to the<br />
presence <strong>of</strong> wheeze and rhinoconjunctivitis (Nordvall, Janson et al. 2005)' In 258 children'<br />
exhaled No did not correlate with height, weight, body mass index or body surface area but<br />
again demonstrated a higher result in boys (wong, Liu et al. 2005). In over 100 healthy<br />
children using both online and <strong>of</strong>fline analyses, body area, age and FEFzs-zs were significant<br />
predictors <strong>of</strong> exhaled No (Kissoon, Duckworth et al. 2o02). In children aged 4-6 years<br />
22r
measured over two 24 hour periods, there was a gender difference (again higher in boys than<br />
girls) in the first assessment, but not in a second assessment (Napier and Turner 2005).<br />
However other paediatric studies have not confirmed these associations; showing no<br />
correlation observed between NO and height or spirometric data (Baraldi, Azzolin et al.<br />
1999), and no correlation between nasal NO and body mass index (Struben, Wieringa et al.<br />
2005). At the current time the reference ranges for 'normal' are given as one range, with no<br />
gender correction.<br />
9.7.2 Age<br />
Early in the research literature, it was suggested that NO increased with age correlating to<br />
pneumatisation <strong>of</strong> sinuses (see Figure 9.2) (Lundberg, Farkas-Szallasi et al. 1995). In over<br />
650 healthy children aged4-17 years in three studies, exhaled NO significantly increased with<br />
age in both <strong>of</strong>f-line and online analyses (Franklin, Taplin et al. 1999; Kissoon, Duckworth et<br />
al.2[f]l2; Buchvald, Baraldi et al. 2005). Nasal NO in 340 healthy children aged 6-17 years<br />
also increased with age which would appear appropriate if it does relate to sinus development<br />
(Struben, Wieringa et al. 2005). An increase <strong>of</strong> exhaled NO was confirmed in a younger age<br />
goup <strong>of</strong> 2-5 year olds using tidal breathing (Avital, Uwyyed et al. 2003). One study also<br />
demonstrated an increase between 1 and 24 hours <strong>of</strong> age in 13 healthy newborn infants<br />
(Schedin, Norman et al. 1996). However, again findings have been inconsistent with no<br />
correlation between age and exhaled NO seen in some groups (Baraldi, Azzolin et al. 1999;<br />
Bartley, Fergusson et al. 1999). Despite the findings above, the current practice is to use one<br />
range for normal values with no correction for the age <strong>of</strong> the subjects. It was however one <strong>of</strong><br />
our reasons for choosing a tight age bracket when measuring the exhaled NO in children in<br />
the methodology study (see Chapter 8).<br />
222
Figure 9.2: Plateau nasalNO increasing with age<br />
E<br />
o.<br />
o,<br />
o Eo<br />
g go('<br />
Eot,<br />
o z6(to2<br />
0.1<br />
0.01<br />
0.001<br />
10 20 30 40<br />
Agc (Years)<br />
50 60<br />
<strong>The</strong> plateau nasal NO levels increased with age in 49 subjects, suggested by the authors to correlate<br />
with pneumatisation <strong>of</strong> the sinuses.<br />
Taken from Lundberg JON, Farkas-szallasi T, Weitzberg E, Rinder J, Lidholm J' Angaard A' Hokfelt T'<br />
Lundberg JM, Alving K. High nitric oxide proOuction.ii human paranasal sinuses' Nature Medicine<br />
i99a; t (i): gzo'gz4 (Lundberg, Farkas-szallasi et al' 1995)'<br />
9.7.3 Circadian rhYthm.<br />
One study in children found the lowest exhaled NO and urinary EPX levels occurred at 7pm<br />
and the highest at Tamin 20 asthmatics compared to six healthy children (Mattes' Storm van's<br />
Gravesande et a:.2002). Otherwise no circadian differences have been demonstrated in adult<br />
subjects with exhaled No (Kharitonov, Gonio et al. 2003) or nasal No measurements<br />
(Bartley, Fergusson et al. Iggg). No was higher at night in asthmatics with nocturnal<br />
symptoms compared to asthmatics without nocturnal symptoms and higher at all times<br />
compared to healthy controls, but no circadian variation in any group was demonstrated (ten<br />
Hacken, van der Vaart et al. 1998; Georges, Bartelson et al' 1999; Palm' Graf et al' 2000)' In<br />
children aged 4-6 years, exhaled NO was reproducible though a 24 hour period (Napier and<br />
Turner 2005) and in six children measured at different times on six consecutive days, no<br />
circadian rhythm was found (Latzin, Beck et al'2002)'<br />
9.7.4 Menstrual cycle and pregnancy<br />
<strong>The</strong>re are few studies to review here. No significant changes were found with repeated<br />
measurements over weeks in healthy controls (Jilma, Kastner et al' 1996; Morris' Sooranna et<br />
al. 1996), while one study did seem to demonstrate high exhaled NO levels in 40 asthmatic<br />
223
women who coincidently experienced worsening <strong>of</strong> sputum scores, eosinophils and lung<br />
function pre-menstrually (Oguzulgen, Turktas et al.2002). No difference has been shown in<br />
exhaled NO during pregnancy between 10 and 42 weeks (Morris, Caroll et al. 1995).<br />
9.7.5 Food and beverages<br />
In our own study, we found that drinking water prior to exhalation transiently reduced NO<br />
levels @yrnes, Dinarevic et al. 1997). Caffeine also decreased exhaled NO in one paper<br />
(Bruce, Yates et at.2002) but not in another (Taylor, Smith et al.2004). Alcohol ingestion<br />
reduced exhaled NO in both asthmatic and healthy subjects (Persson, Cederqvist et al. 1994;<br />
Yates, Kharitonov et al. 1996) with a small decrease in exhaled NO observed up to 34 hours<br />
after drinking (Jones, Fransson et al. 2005). Smoking also significantly reduces exhaled NO<br />
and will this be discussed below with the studies on COPD subjects (see Section 9.15). <strong>The</strong><br />
reduced NO levels subsequent to both cigarette smoking and alcohol consumption was<br />
suggested to be secondary to down regulation <strong>of</strong> iNOS (Steerenberg and van Amsterdam<br />
20M).In contrast increased exhaled NO has been found after the ingestion <strong>of</strong> nitrite or nitrate<br />
containing foods (such as lettuce) with a maximum effect occurring two hours after ingestion<br />
(Zetterquist, Pedroletti et al. 1999; Olin, Aldenbratt et al. 2001). <strong>The</strong> gastric lumen has been<br />
noted to have high levels <strong>of</strong> NO, therefore contamination with gastric air results in very high<br />
exhaled NO (Lundberg, Weitzberg et al.1994).<br />
9.7.6 Summary <strong>of</strong> physiologicalfactors that could alter nitric oxide levels<br />
In conclusion, exhaled NO is likely to be higher in males than females, and relates to age<br />
through childhood but not adulthood, possibly in relation to pneumatisation <strong>of</strong> the sinuses. It<br />
is reduced with current smoking, recent ingestion <strong>of</strong> water, alcohol or caffeine and increased<br />
with accidental gut contamination or after nitrogen-compound rich meals. <strong>The</strong>re may be<br />
differences throughout the 24 hour day in asthmatic subjects with nocturnal asthma, and<br />
through the menstrual cycle in those who experience premenstrual exacerbations <strong>of</strong> asthma.<br />
However, relationships with height, weight, surface area, menstrual cycle in non-asthmatic<br />
women, or a circadian rhythm in non-asthmatic individuals has not been demonstrated.<br />
Despite some <strong>of</strong> the findings above, there is only one range <strong>of</strong> 'normal' values given for the<br />
results <strong>of</strong> measurement for oral or nasal results.<br />
9.8 Nitric oxide levels in asthma and atopy<br />
Since commencement <strong>of</strong> the studies on exhaled NO, more research has been conducted in<br />
asthmatic populations than any other group; in particular assessing the use <strong>of</strong> NO for<br />
224
screening, diagnostic and monitoring purposes. Initially cross sectional studies were carried<br />
out with comparisons to the traditional inflammatory and clinical markers used in asthma (see<br />
Chapter 1). Research then graduated to the use <strong>of</strong> longitudinal studies to assess the predictive<br />
value <strong>of</strong> NO for presence, severity and to denote exacerbations <strong>of</strong> asthma' Longitudinal<br />
studies have also been used to assess treatment effects in standard and new therapies' <strong>The</strong><br />
relationships between NO and asthmatic inflammatory markers, lung function testing'<br />
bronchodilator responsiveness and airway challenges have all been studied. I have grouped<br />
the studies presented here under headings <strong>of</strong> the hypotheses that the studies were devised to<br />
address.<br />
g.g.l Does nitric oxide correlate with other asthmatic inflammatory markers?<br />
Exhaled NO was found to correlate with sputum eosinophil numbers in asthmatics and<br />
healthy controls in adults (Jatakanon, Lim et al. 1998; Berlyne, Parameswaran et al' 2000;<br />
Tsujino, Nishimura et al. 2000; Reid, Johns et al. 2003; silk<strong>of</strong>f, Ifnt et al' 2005; Fujimoto'<br />
Yamaguchi et al. 2006; Zietkowski, Bodzenta-Lukaszyk et al' 2006) and in children<br />
(Piacentini, Bodini et al. L999; Little, Chalmers et al. 2000; Warke, Fitch et al' 2002; Sacco'<br />
sale et al. 2003; Mahut, Delclaux et a:.2004: Thomas, Gibson et al. 2005; Li, Tsang et al'<br />
2006). while these have been demonstrated in asthmatics whether on or <strong>of</strong>f a range <strong>of</strong><br />
medications, the correlations are strongest for steroid naive patients. <strong>The</strong>se findings have been<br />
consistent across the studies that used variable expiratory flows, and were confirmed in one<br />
study that compared results in these subject groups across a range <strong>of</strong> flows (Beny, shaw et al'<br />
2005). A positive correlation has also been demonstrated between exhaled No and serum<br />
eosinophil cell counts in adults and children (Tsujino, Nishimura et al' 2000; Reid' Johns et<br />
al.Z}o3:Silvestri, sabatini et al. 2003; Strunk, Szefler et al. 2003; Irhtimaki' Kankaanranta<br />
et al. 2005; Zietkowski, Bodzenta-Lukaszyk et al' 2006). Correlations have been described<br />
between NO and sputum ECP levels (Piacentini, Bodini et al. 1999; Warke, Fitch et al' 2002;<br />
Thomas, Gibson et al. 2005), as well as No and lerum ECP levels (Aziz, wilson et al' 2000;<br />
Dal Negro, Micheletto et al. 2003; Strunk, Szefler et al' 2003; Mahut, Delclaux et al' 200,4i<br />
Zietkowski, Bodzenta-Lukaszyk et al. 2006). Perhaps unsurprisingly, in studies that compared<br />
both, it was sputum rather than serum markers that had stronger correlations with exhaled NO<br />
levels. A positive correlation was also shown with the leukotriene group <strong>of</strong> inflammatory<br />
markers - leukotriene%,leukotriene B4 and 8-isoprostane (Mondino, Ciabattoni et al' 2W4)'<br />
and a negative correlation with the anti-inflammatory cytokines IL-4 and IL-13 (Shome'<br />
Starnes et al. 2006).<br />
225
While these cross-sectional and the longitudinal studies described below (see Section 9.8.5)<br />
have confirmed these findings, it has not been universal. For example, in 58 asthmatic<br />
children no association between NO and eosinophils or ECP from sputum or serum was<br />
shown (Wilson, James et al. 2001). Other studies did not demonstrate a relation between one<br />
specific parameter such as NO with serum eosinophils (Turktas, Oguzulgen et al. 2003), with<br />
serum ECP (del Giudice, Brunese et al. 2004) or with sputum leukotriene levels (Strunk,<br />
Szefler et al. 2003), although they confirmed other associations. Iooking at these studies,<br />
there is no apparent difference in methodology compared to others with positive findings,<br />
although they are more <strong>of</strong>ten heterogeneous groups <strong>of</strong> asthmatics on a wide range <strong>of</strong><br />
medications, with smaller numbers <strong>of</strong> subjects and tended to be 'doctor diagnosed' asthmatics<br />
rather than those diagnosed with specific testing when in clinic which was also conducting the<br />
research.<br />
Direct comparisons <strong>of</strong> NO with bronchial biopsy results have also been studied. One goup<br />
found a correlation between exhaled NO and each <strong>of</strong> the eosinophil, lymphocyte and mast cell<br />
components (Silk<strong>of</strong>fl lrnt et al. 2005), while another study found that NO was only related to<br />
the total number <strong>of</strong> inflammatory cells present (Turktas, Oguzulgen et al. 2003). In 31<br />
children with difficult asthma, there was a relationship between exhaled NO and the<br />
eosinophil score in seven <strong>of</strong> 2l children for whom biopsies and NO were obtained. <strong>The</strong><br />
strongest relationship was an exhaled NO >7ppb and raised eosinophils at baseline in children<br />
who had persistent symptoms despite high dose prednisolone for two weeks (Payne,<br />
McKenzie et al. 2001). Exhaled NO was also shown to correlate with the degree <strong>of</strong> airway re-<br />
modeling on biopsy samples in a group <strong>of</strong> 28 children (Mahut, Delclaux et al.2004) and the<br />
degree <strong>of</strong> bronchial wall thickening seen on a CT scan in nine asthmatic children (Ketai,<br />
Harkins et al. 2005).<br />
9.8.2 Does nitric oxide correlate with lung function and bronchial hyper-responsiveness?<br />
Comparisons <strong>of</strong> exhaled NO with lung function parameters have produced variable results.<br />
While many have shown a significant negative correlation, particularly to FEV1, in adults (de<br />
Gouw, Hendriks et al. 1998; Ho, Wood et al. 2000; Dal Negro, Micheletto et al. 2003;<br />
Nogami, Shoji et al. 2003) and children (Colon-Semidey, Marshik et al. 2000; Piacentini,<br />
Bodini et al. 2000; Spallarossa, Battistini et al. 2001; Beck-Ripp, Griese et al. 2002;<br />
Malmberg, Pelkonen et al. 2003; del Giudice, Brunese et al. 2004; Saito, Inoue et al. 2004)<br />
others were not able to demonstrate this (al-Ali, Eames et al. 1998; Silk<strong>of</strong>f, McClean et al.<br />
1998; Piacentini, Bodini et al. 1999; Ho, Wood et al. 2000; Silvestri, Spallarossa et al. 2000;<br />
226
Turktas, oguzulgen et a:.2003; Mappa, cardinale et al. 2005; Spergel, Fogg et al' 2005;<br />
Thomas, Gibson et al. 2005; Zietkowski, Bodzenta-Lukaszyk et al. 2006). Again some <strong>of</strong><br />
these studies described correlations with certain parameters only such as FEF25-75 (Mahut'<br />
Delacourt et al. 2004; Battaglia, den Hertog et al. 2005; Irhtimaki, Kankaanranta et al' 2005)'<br />
FEVr/FVC ratio (Strunk, Szefler et al. 2003), residual volume (Mappa' Cardinale et al' 2005)'<br />
or peak flow lability (al-Ali, Eames et al. 1998; Lim, Jatakanon et al' 2000)' In other studies<br />
the correlations were only significant in certain groups such as atopic, rather than non-atopic'<br />
asthmatics (Franklin, Stick et al. 2004)'<br />
Positive correlations have been demonstrated between NO levels and airway reactivity as<br />
measured by methacholine challenges (Jatakanon, Lim et al' 1998; Henriksen' Lingaas-<br />
Holmen et al. 2000; wilson, Dempsey et al. 2001; Prieto, Gutierrez et al' 2002; Prieto'<br />
Gutierrez et al. 2002; Buchvald, Eiberg et al. 2003; Langley, Goldthorpe et al' 2oo3;<br />
Malmberg, Pelkonen et al. 2003; Nogami, Shoji et al. 2003; Berkman' Avital et al' 2005;<br />
Ehrs, Sundblad et al. 2006), histamine challenges (al-Ali, Eames et al' 1998; de Gouw'<br />
Hendriks et al. 1998; Dupont, Rochette et al. 1998; Dupont, Demedts et al' 2003; Zietkowski'<br />
Bodzenta-Lukaszyk et al. 2006), or adenosine S-monophospate challenges (de Gouw'<br />
Hendriks et al. 1998; Aziz,wilson et al. 2000; Prieto, Gutierrez et al' 2002; Prieto' uixera et<br />
aI.2002;Prieto, Bruno et al. 2003; Berkman, Avital et al' 2005)' Again some studies found<br />
correlations only in certain groups <strong>of</strong> subjects such as atopic asthmatics (Franklin' Stick et al'<br />
2004) or those on IHCS (Reid, Johns et al. 2003) rather than all asthmatic and control<br />
subjects.<br />
g.8.3 Can nitric oxide be usedfor diagnosis <strong>of</strong> asthma?<br />
Studies have assessed whether measurement <strong>of</strong> exhaled NO can be used to diagnose asthma'<br />
either to screen a general population or to screen new clinic referrals' One group evaluated 47<br />
consecutive adult patients referred with symptoms suggestive <strong>of</strong> asthma' <strong>The</strong> individual<br />
sensitivities for each <strong>of</strong> the conventional tests (p.* flow, spirometry, serum eosinophils,<br />
bronchodilator responsiveness and airway challenge) were up to 47Vo; much lower than for<br />
exhaled No at ggvo andsputum eosinophili a at 86Vo. <strong>The</strong> results for conventional tests were<br />
not improved when using a trial <strong>of</strong> oral steroid as a diagnostic test. <strong>The</strong> researchers concluded<br />
that exhaled No and induced sputum analysis, particularly when combined, were superior to<br />
the other conventional approaches in diagnosing asthma (smith, cowan et al' 2004)' <strong>The</strong><br />
same group looked at the predictive accuracy <strong>of</strong> exhaled NO to identify steroid<br />
responsiveness to IHCS for four weeks in 52 adults presenting with undiagnosed respiratory<br />
227
symptoms. Steroid response was significantly greater in those with the higher exhaled NO<br />
levels (> a7ppb) independent <strong>of</strong> the original diagnostic label (asthma, COPD or chronic<br />
bronchitis) (Smith, Cowan et al. 2005). A similar study evaluated 95 patients presenting with<br />
respiratory symptoms in which 40 were subsequently diagnosed as asthmatic. A baseline<br />
value <strong>of</strong> NO at >7ppb best differentiated between asthmatics and non-asthmatics with a<br />
sensitivity <strong>of</strong> 82.5Vo and a specificity <strong>of</strong> 88.9Vo. <strong>The</strong> generated receiver-operated curves<br />
(ROC) gave a value for exhaled NO <strong>of</strong> 0.89, similar to both methacholine and adenosine-S<br />
monophosphate challenges, better than lung function or exercise testing (Berkman, Avital et<br />
al. 2005). In 50 consecutive subjects being screened for exercise induced bronchospasm,<br />
exhaled NO at
exhaled NO was the best predictor, when compared to lung function testing and<br />
bronchodilator response, to discriminate those who subsequently developed asthma with the<br />
sensitivity <strong>of</strong> g6vo and a specificity <strong>of</strong> g27o at a cut <strong>of</strong>f level chosen 1.5 standard deviations<br />
above the average for controls (Malmberg2004)'<br />
g.8.4 Is nitic oxid.e associatedwith symptoms and severiry <strong>of</strong> asthma?<br />
positive correlations with exhaled No have been shown with bronchodilator use and<br />
symptom scores (Lanz,Irung et al. 1997; Tsujino, Nishimura et al. 2000; Roberts, Hurley et<br />
a:.2004;warke, Mairs et a,..2004: Pijnenburg, Bakker et al. 2005; spergel, Fogg et al' 2005;<br />
prasad, Langford et al. 2006), cough (Li, Irx et al. 2003) and bronchodilator responsiveness<br />
(Colon-Semidey, Marshik et al. 2000; Little, Chalmers et al' 2000; Dupont' Demedts et al'<br />
2xl3;Malmberg, Pelkonen et al. 2003; Silvestri, sabatini et al. 2003; Pijnenburg, Bakker et<br />
al. 2005; Fujimoto, Yamaguchi et al. 2006; Zietkowski, Bodzenta-Lukaszyk et al' 2006)' In<br />
many <strong>of</strong> the studies, the No levels and the sputum eosinophil levels had stronger correlations<br />
to symptoms than other parameters measured. Again the findings were not universal' For<br />
example, in some studies no correlation was seen between NO and bronchial hyper-<br />
responsiveness (al-Ali, Eames et al. 1998; Silk<strong>of</strong>f, Mcclean et al. 1998; Chan-Yeung' obata<br />
et al. 1999; van Rensen, Straath<strong>of</strong> et al. t999;Ho, Wood et al. 2000; Silvestri, Spallarossa et<br />
al. 2000; del Giudice, Brunese et a:.2004:Thomas, Gibson et al. 2005; Jentzsch, le Bourgeois<br />
et al. 2006), NO and symptoms (al-Ali, Eames et al. 1998; Griese, Koch et al' 2000; Sippel'<br />
Holden et al. 2000) or NO and medication use (al-Ali, Eames et al. 1998; Sippel, Holden et al'<br />
2000). In addition, no relationship was determined in 77 adult asthmatics between exhaled<br />
NO and quality <strong>of</strong> life (Ehrs, Sundblad et al' 2006)'<br />
<strong>The</strong>re are a number <strong>of</strong> possible explanations for the inconsistencies. Firstly, the correlation<br />
with severity is difficult depending on how 'severity' is defined' we know that exhaled No is<br />
higher in steroid naive asthmatics, so those on increased amounts <strong>of</strong> treatment may have<br />
reduced levels <strong>of</strong> No but their requirement for higher IHCS doses or additional medications<br />
to control symptoms deems them more 'severe'. Several studies using either the GINA<br />
guidelines or the ATS asthma guidelines to categorize asthmatic patients into gradations <strong>of</strong><br />
severity have been unable to show a relationship to exhaled No levels in adults (stirling'<br />
Kharitonov et al. 1998; Chan-Yeung, obata et al. Iggg; Lim, Jatakanon et al' 2000; sippel'<br />
Holden et al. 2000) or in children (Griese, Koch et al. 2000)' In one study <strong>of</strong> 30 children<br />
divided into mild, moderate and severe groups' the exhaled NO did appear to correlate with<br />
asthma severity (Delgado-Corcoran' Kissoon et al' 2004), and those on oral steroids with<br />
229
'difficult asthma' also had higher exhaled NO suggesting ongoing inflammation (Stirling,<br />
Kharitonov et al. 1998; Payne, Adcock et al. 2001). Secondly, studies in asthma have<br />
commonly considered it as a single diagnostic entity when we increasingly have come to<br />
consider it as a more heterogeneous disease. Differing factors within the asthma 'syndrome'<br />
have been emphasized such as atopic versus non-atopic (or allergic versus intrinsic), and,<br />
more recently, defined by the cell most prominent in the airway inflammation; eosinophilic<br />
versus neutrophilic versus both or neither. In the cross sectional studies, most have consider<br />
asthmatics as a single group without the nicety <strong>of</strong> these divisions. Thirdly, other factors may<br />
influence asthma development, such as genetics. One goup studied the genotype <strong>of</strong> the NOS<br />
enzyme in adult asthmatics and COPD patients, and showed that if there were high numbers<br />
<strong>of</strong> "AAT" sequences (greater than 12 repeats) in the specific region <strong>of</strong> "intron 20" then much<br />
lower levels <strong>of</strong> exhaled NO were obtained (Wechsler, Grasemann et al. 2000). Fourthly, we<br />
are comparing exhaled NO as a predictor against other single measurements such as sputum<br />
eosinophils or FEVr when it is unlikely that these are all isolated parameters. In a study <strong>of</strong> 92<br />
children with 59Vo receiving IHCS, factor analysis selected four factors explaining 55.5Vo <strong>of</strong><br />
the total variance. <strong>The</strong>se factors were exhaled NO, plasma total IgE, plasma specific IgE and<br />
peripheral blood eosinophil percentage. <strong>The</strong> researchers suggested that these pararneters<br />
operate as a group and should not be viewed as having non-overlapping characteristics<br />
(I-eung, Wong et al. 2005).<br />
Since cross sectional studies have not demonstrated a good correlation between asthma<br />
severity and NO, perhaps it is more appropriate to look at NO levels in controlled versus<br />
uncontrolled asthmatics. In 32 adults about to commence on a comparison <strong>of</strong> treatments,<br />
baseline exhaled NO was higher in those presenting with symptoms (Gratziou, Rovina et al.<br />
2001). In 45 asthmatic children higher NO levels were obtained in those that were recently<br />
symptomatic, although all had levels above normals (Mahut, Delacourt et at.2004), and in 14<br />
<strong>of</strong> 22 atopic asthmatic children with current symptoms the exhaled NO was two standard<br />
deviations above the normal control group (Silvestri, Spallarossa et al. 1999). In studies<br />
looking at NO levels in a total <strong>of</strong> 625 asthmatic patients with allergy as a trigger, the NO<br />
levels were higher in those recently exposed (Artlich, Busch et al. 1999; Simpson, Custovic et<br />
al. 1999; Langley, Goldthorpe et ^1. 2OO3), in those with perennial exposure compared to<br />
seasonal allergies (Olin, Alving et al. 2004) and in those sensitised with common indoor<br />
allergens such as dust mite, cat and dog.<br />
Is this pertinent to clinical practice? In the following studies, the physicians attending the<br />
children were blinded to the exhaled NO readings when making their concurrent assessment<br />
230
and treatment decisions. Exhaled NO correctly predicted 21 asthmatic children deemed to be<br />
,uncontrolled, versus 3l who were 'well controlled' (Meyts, proesmans et al. 2003). Fifteen<br />
asthmatic children followed over 3-4 visits showed that exhaled No was significantly<br />
correlated with the degree <strong>of</strong> control @elgado-corcoran'<br />
Kissoon et al' 2oo4)' In 74<br />
asthmatics and 31 healthy control children, the exhaled No level when raised above normal<br />
(designated as > l3ppb in this study) had a sensitivity <strong>of</strong> 0'67 and a specificity <strong>of</strong> 0'65 to<br />
predicted the requirement for a step-up in therapy (Griese, Koch et al' 2000)'<br />
g.g.5 can nitric oxide predict deterioration in asthma control?<br />
<strong>The</strong> studies above looked at concurrent exhaled No and symptoms' In one study' cited above'<br />
the cross sectional analysis <strong>of</strong> 100 asthmatic patients from 7 to 80 years <strong>of</strong> age showed that<br />
exhaled NO correlated better with markers <strong>of</strong> control such as lung function and sputum<br />
eosinophils rather than markers <strong>of</strong> severity such as history <strong>of</strong> respiratory failure, healthcare<br />
use, fixed air flow obstruction or an asthma severity score (Sippel, Holden et al' 2000)'<br />
However, it would be a more useful tool if it could predict loss <strong>of</strong> control when following<br />
asthmatic individuals' longitudinally'<br />
In 22 moderate to severe asthmatics seen in a routine clinic visit, those who subsequently had<br />
went on to have an exacerbation within two weeks had significantly higher mean exhaled No<br />
(2gppb versus l3ppb) compared to those who remained stable (Harkins, Fiato et al' 2004)'<br />
Irvels <strong>of</strong> exhaled No were followed prospectively in 44 asthmatics on daily IHCS and<br />
LABA therapy over l8 months, withzzhaving one or more exacerbations' while the baseline<br />
FEVr remained the best predictor, the Roc curve for exhaled No to predict exacerbation was<br />
0.71. An exhaled NO <strong>of</strong> 28ppb gave a sensitivity <strong>of</strong> 0.59, a specificity <strong>of</strong> 0'82' a positive<br />
predictive value <strong>of</strong> o.77 and a negative predictive value <strong>of</strong> 0'87 for having increased<br />
symptoms. So, an exhaled NO >28ppb gave a relative risk <strong>of</strong> 3'4 for an exacerbation'<br />
although authors concluded that combining exhaled NO and FEVr was the most useful (Gelb'<br />
Flynn Taylor et al. 2006).ln 44 sensitized children prospectively followed though one grass<br />
pollen season, NO was best associated with the mean pollen count the week before the No<br />
measurement and was significantly associated with asthma control (Roberts' Hurley et al'<br />
2004).In 105 asthmatic and healthy children, exhaled No did not depict asthma severity but<br />
correlated with changes in asthma therapy. Irvels >l3ppb, had a sensitivity <strong>of</strong> 0'67 and a<br />
specificity <strong>of</strong> 0.65 to predict step-up in therapy (Griese, Koch et al' 2000)' In 2L asthmatic<br />
children with seasonal allergic asthma compared to 21 healthy children that were followed<br />
throughout a grass pollen season, their No levels were higher, increased through the pollen<br />
23r
season and then reduced down to baseline. However, their exhaled NO did not necessarily<br />
correlate with FEV1 @araldi, Carra et al. 1999). Exhaled NO did relate to recent symptoms<br />
versus no symptoms when dividing 133 children aged 5-14 years attending a hospital clinic<br />
into 'uncontrolled' and 'controlled' asthma groups. <strong>The</strong> researchers also felt that NO<br />
predicted subgroups as to whether IHCS or medication was increased, decreased or remained<br />
unchanged (Warke, Mairs et al. 2004).<br />
<strong>The</strong>re have also been studies where exhaled NO was measured prospectively while IHCS<br />
doses were reduced. After three months <strong>of</strong> good control on moderate doses <strong>of</strong> IHCS (500-<br />
1000pg/day beclomethasone), 37 asthmatics had their individual doses halved for twelve<br />
weeks. Parameters which detected the ten that developed an exacerbation were; a baseline<br />
exhaled NO level > 20ppb, or a baseline exhaled NO level > l5ppb plus a bronchoconstriction<br />
response to the adenosine S-monophosphate challenge, or a rapid increase in exhaled NO over<br />
the first weeks <strong>of</strong> the assessments (Prieto, Bruno et al. 2003). In 78 asthmatic adults, IHCS<br />
treatment was withdrawn over six weeks with 60 subjects developing symptoms. Both the<br />
baseline measurement <strong>of</strong> exhaled NO and an increase <strong>of</strong> 60Vo over the baseline reading had<br />
positive predictive values between 80-90Vo for an exacerbation and was similar to the<br />
predictive values using sputum eosinophils or hypertonic saline airway responsiveness (Jones,<br />
Kittelson et al. 2001). Similarly, nine <strong>of</strong> 40 children with a mean age <strong>of</strong> 12.2 years relapsed 2-<br />
4 weeks after withdrawal <strong>of</strong> steroids. <strong>The</strong> exhaled NO was higher at a mean <strong>of</strong> 35.3ppb versus<br />
15.7ppb at two weeks and 40.8ppb versus l5.9ppb at four weeks. A value <strong>of</strong> 49ppb at four<br />
weeks after steroid discontinuation had the best sensitivity at TLVo and specificity at 93Vo for<br />
an asthma relapse @ijnenburg, H<strong>of</strong>truis et al. 2005). One study was less conclusive.<br />
Seventeen moderate asthmatic adults had their daily dose <strong>of</strong> IHCS halved at 20 day intervals<br />
until loss <strong>of</strong> control or replacement with placebo. Of the parameters which were measured<br />
every ten days, the sputum eosinophil percentage altered 20 days ahead <strong>of</strong> deterioration while<br />
changes in exhaled NO, FEVr and methacholine PC26 were only observed once symptoms had<br />
already developed @elda, Parameswaran et al. 2006).<br />
Studies have also compared changes in exhaled NO with traditional clinical and lung function<br />
assessments to dictate changes in treatment.ln 97 adult asthmatics, future treatment decisions<br />
regarding steroid adjustment were assigned randomly to be based either on exhaled NO levels<br />
q! on an algorithm based on the conventional GINA 2OO2 guideline (GINA 2002). <strong>The</strong><br />
optimal IHCS dose per individual was determined in the first phase and patients were then<br />
followed for L2 months. <strong>The</strong> mean final doses <strong>of</strong> inhaled fluticasone used were 3701tglday for<br />
the 46 patients in the exhaled NO group and 64Lltglday for the 48 patients in the guideline<br />
232
group. Rates <strong>of</strong> exacerbation were 0.49 episodes/patient/year in the exhaled No managed<br />
group and 0.90 in the guideline group, representin g a 45.6Vo exacerbation reduction in the No<br />
group, although this did not reach significance. <strong>The</strong>re were no significant differences in<br />
pulmonary function changes, sputum eosinophils or the use <strong>of</strong> oral prednisone (Smith' Cowan<br />
et al. 2005). Similar to the adult study, children on IHCS were allocated to groups where<br />
treatment decisions were made by symptomatology in 46 or with the addition <strong>of</strong> exhaled No<br />
in 39. Over the next year, those monitored using the NO readings had improved airway hyper-<br />
responsiveness from 2.5 to 1.1 doubling dose, lesS Severe exacerbations at eight versus 18',<br />
and a trend to improved FEVr (Pijnenburg, Bakker et al. 2005). <strong>The</strong>se studies suggest that No<br />
can successfully be used longitudinally for individual patients to predict exacerbations and to<br />
aid management deci sions re gardin g treatment'<br />
9.8.6 Wat happens to nitric oxide during an acute asthma attack?<br />
Most studies have examined NO levels during chronic asthma disease over time' So what<br />
happens to NO levels in the acute phase? Studies have used allergen challenges to simulate an<br />
acute asthmatic exacerbation. It appears that even when the No is similar in the asthmatic and<br />
control groups at baseline, No increases in the asthmatic groups during the late asthmatic<br />
reaction (Obata, Dittrick et al. 1999; Paredi, Irckie et al' 1999; Khatri' Hammel et al' 2003;<br />
Ihre, Gyllfors et al. 2006). It was found to increase three fold from baseline 24 hours post<br />
challenge (Khatri, Hammel et al. 2003). Repeated low dose allergen inhalation in 8 mild<br />
asthmatic subjects demonstrated an increased exhaled NO despite no change seen in asthma<br />
symptoms or lung function (Ihre, Gyllfors et al. 2006)' <strong>The</strong>se findings suggest that in acute<br />
asthma an increased production requiring synthesis <strong>of</strong> the NOS enzymes occulred and<br />
therefore some time was needed to appreciate the full insult (Khatri, Hammel et al' 2003)'<br />
This may also explain why the use <strong>of</strong> No to dictate treatment acutely has been less successful'<br />
one study used the exhaled No levels to dictate the degree <strong>of</strong> treatment given to adult<br />
patients presenting to the emergency department with an asthma exacerbation' <strong>The</strong> study was<br />
discontinued after the first 53 enrolled subjects as those in whom the management <strong>of</strong>fered was<br />
decided on their exhaled NO levels did less well when followed up than those in whom the<br />
management was decided by the more traditional measures <strong>of</strong> clinical scores or spirometry<br />
(Gill, Walker et al. 2005).<br />
g.8.7 What is the effect <strong>of</strong> atopy alone on nitric oxide?<br />
In assessing the validity <strong>of</strong> exhaled No to assist in screening, diagnosis and longitudinal<br />
monitoring <strong>of</strong> asthma control, the effect <strong>of</strong> atopy alone must be taken into account' A number<br />
233
<strong>of</strong> cross sectional studies with a total <strong>of</strong> 949 subjects have shown that there is a higher mean<br />
exhaled NO which decreases through the groups <strong>of</strong> atopic asthmatics, non-atopic asthmatics,<br />
atopic subjects and non-atopic controls. However, considerable overlap between groups exist<br />
so that a single cut <strong>of</strong>f may not discern exactly into which group an individual belongs (Frank,<br />
Adisesh et al. 1998; Barreto, Villa et al. 2001; Henriksen, Holmen et al. 200L; Prieto,<br />
Gutierrez et al.2002; Jouaville, Annesi-Maesano et al. 2003; Cardinale, de Benedictis et al.<br />
2005; Prasad, Langford et al. 2006). In these studies, asthmatics divided into those who were<br />
atopic and non-atopic showed those with atopy had higher levels <strong>of</strong> exhaled NO. In 28<br />
asthmatics on IHCS treatment, NO levels were positively correlated with total IgE and<br />
positive skin prick testing more than bronchial hyper-reactivity, lung function or asthma<br />
severity (Ho, Wood et al. 2000).In92 steroid naive asthmatics, the NO was high in those that<br />
were atopic with no correlation to any other parameters (Silvestri, Sabatini et al. 2W3).In2l3<br />
children, exhaled NO was higher in children with atopy alone compared to those with non-<br />
atopic asthma and was correlated to the wheal size <strong>of</strong> skin prick tests (Barreto, Villa et al.<br />
2001). In 53 steroid naive asthmatic and 96 non-asthmatic children who had skin prick testing<br />
to 12 common allergens, the exhaled NO was higher in the asthmatic versus non-asthmatic<br />
individuals but was also higher in atopic (including those with allergic rhinitis alone) versus<br />
non-atopic children (Jouaville, Annesi-Maesano et al. 2003). In 53 subjects, no significant<br />
difference was found between non-atopic asthmatics and controls (Ludviksdottir, Janson et al.<br />
1999). Similarly <strong>of</strong> 140 subjects, llVo <strong>of</strong> those with asthma but not deemed allergic had NO<br />
levels within the normal range (Zietkowski, Bodzenta-Lukaszyk et al. 2006).<br />
Looking at the effect <strong>of</strong> atopy alone, 38 subjects with allergic rhinitis but not asthma had a<br />
raised exhaled NO @rieto, Gutierrez et al.2002) and in 64 atopic Pacific Island subjects, NO<br />
levels correlated with house dust mite wheal reaction and clinical atopy (Moody, Fergusson et<br />
al. 2000). More studies have been conducted evaluating exhaled NO with atopic status in<br />
children - mostly within community school cohorts. In 356 children from an unselected<br />
population, exhaled NO was two fold higher in atopic individuals (noted as having positive<br />
skin prick tests and a high IgE) with high eosinophil counts compared to atopic individuals<br />
with low eosinophil counts. However the exhaled NO in both these groups was higher than<br />
the non-atopic subjects regardless <strong>of</strong> the eosinophil count, suggesting it was a link with atopy<br />
not just eosinophilic inflammation (Barreto, Villa et al. 2005). Exhaled NO related to both<br />
eosinophil levels and atopy but not necessarily to asthma and/or wheeze in the last 12 months<br />
in 155 children (Franklin, Turner et al. 2003). NO also correlated with skin prick testing and<br />
sputum eosinophils but not airway responsiveness in 107 children (Thomas, Gibson et al.<br />
234
2005). Exhaled NO showed strongly positive correlations to non-specific IgE and mite<br />
specific IgE with weaker positive correlations to specific IgE to cat and cedar in 278 children<br />
(Saito, Inoue et al. 2OO4). Finally in 374 school children high exhaled NO levels were<br />
associated with pet sensitisation in atopic children, and respiratory infections and home<br />
window pane condensation in non-atopic children rather than other factors (Janson, Kalm-<br />
Stephens et al. 2005). In a hospital clinic setting in 45 children, there was a correlation<br />
between eczema and exhaled NO but not asthma, allergic rhinitis, food allergY, the use <strong>of</strong><br />
inhaled corticosteroids, anti leukotriene therapy, antihistamine therapy or lung function<br />
(Jentzsch, le Bourgeois et al. 2006).<br />
Thus atopy alone is an important confounding factor when using exhaled NO to assess<br />
asthmatic and healthy individuals.<br />
9.8.8 Can nitric oxide be an outcome measure for assessing treatment?<br />
9.8.8 (i) Corticosteroids<br />
Similar to my own study, there have now been many publications confirming that levels <strong>of</strong><br />
exhaled NO is high in asthmatics on bronchodilator therapy only and is reduced in asthmatics<br />
on steroid therapy <strong>of</strong>ten to the equivalent levels found in healthy controls using adult (Alving,<br />
Weitzberg et al. 1993; Kharitonov, Yates et al. L994; Persson, Zetterstrom et al. 1994;<br />
Kharitonov, Chung et al. 1996; Massaro, Mehta et al. 1996; al-Ali, Eames et al. 1998;<br />
Stirling, Kharitonov et al. 1998; Berlyne, Parameswaran et al. 2000; Lim, Jatakanon et al.<br />
2000; Tsujino, Nishimura et al. 2000; Reid, Johns et al. 2003; Shin, Rose-Gottron et al.2004)<br />
and paediatric subjects (Byrnes, Dinarevic et al. L997; Nelson, Sears et al. 1997; Silvestri,<br />
Spallarossa et al. 1999; Colon-Semidey, Marshik et al. 2000; Little, Chalmers et al. 2000;<br />
Piacentini, Bodini et al. 2000; Silvestri, Spallarossa et al. 2000; Visser, de Wit et al. 2000;<br />
Avital, Uwyyed et al. 2001). Many <strong>of</strong> these studies also determined that the raised exhaled<br />
NO levels in asthmatics reduced when they were given inhaled or oral steroids from five days<br />
to several weeks in adults (Silk<strong>of</strong>f, McClean et al. 1998; Jatakanon, Kharitonov et al. 1999;<br />
Lim, Jatakanon et al. 1999; van Rensen, Straath<strong>of</strong> et al. 1999; Wilson and Lipworth 2000;<br />
Silk<strong>of</strong>f, McClean et al. 2001; Jones, Herbison et al.20O2: Kharitonov, Donnelly et al. 2002;<br />
Currie, Syme-Grant et al. 2003; Dal Negro, Micheletto et al. 2003; Tnidler, Kleerup et al.<br />
2004; Brightling, Green et al. 2005; Silk<strong>of</strong>f 2005; Smith, Cowan et al. 2005; Zietkowski,<br />
Bodzenta-Lukaszyk et al. 2006) and children (Byrnes, Dinarevic et al. 1997i Artlich, Busch et<br />
al.1999;Lanz,I-eung et al. 1999; Colon-Semidey, Marshik et al. 2000; Piacentini, Bodini et<br />
al. 2000; Spallarossa, Battistini et al. 2001; Beck-Ripp, Griese et al. 2002; Mondino,<br />
235
Ciabattoni et al. 2004; Petersen, Agert<strong>of</strong>t et al. 2004; Cardinale, de Benedictis et al. 2005;<br />
Prasad, Langford et al. 2006;Zeiger, Szefler et al. 2006). In addition to commencing IHCS in<br />
steroid naive patients, some studies also showed a dose response reduction <strong>of</strong> exhaled NO<br />
(Srirling, Kharitonov et al. 1998; Colon-Semidey, Marshik et al. 2000; Piacentini, Bodini et<br />
al. 2000; Silk<strong>of</strong>l McClean et al. 2001; Jones, Herbison et al.2002; Kharitonov, Donnelly et<br />
al. 2002). Two studies demonstrated a plateau in the reduction <strong>of</strong> exhaled NO in asthmatic<br />
children at 400pgs beclomethasone or equivalent (Jatakanon, Kharitonov et al. 1999; Wilson<br />
and Lipworth 2000) but was not seen between 50 and 100pgs hydr<strong>of</strong>luoroalkane-<br />
beclomethasone (Petersen, Agert<strong>of</strong>t et al. 20M). One study showed that at a lower dose <strong>of</strong><br />
200pgs beclomethasone there was a longer time required for the reduction <strong>of</strong> exhaled NO to<br />
the lowest levels measured compared to commencing at double this dose @al Negro,<br />
Micheletto et al.2003), although another study found no additional reduction in exhaled NO<br />
after ten days <strong>of</strong> treatment even with low doses (Spallarossa, Battistini et al. 2001). <strong>The</strong><br />
reduction <strong>of</strong> NO levels has even been seen with single nebulised steroid doses (Baraldi,<br />
Azzolin et al. 1997;Lanz,Irung et al. 1999; Tsai, Lee et al. 2001). In addition, these findings<br />
have been confirmed using tidal breathing techniques (Visser, de Wit et al. 2000) and<br />
reservoir samples @araldi, Dario et al. 1999; Colon-Semidey, Marshik et al. 2000). Many <strong>of</strong><br />
these individual papers have been already been discussed in more detail in Chapters 6,7 and<br />
8. Only one cross sectional hospital based study in 392 adult patients with a range <strong>of</strong> asthma<br />
severity showed no difference in the exhaled NO between those on or not on IHCS, although<br />
the researchers did show a relationship between exhaled NO and airway reactivity (measured<br />
by methacholine challenge) and atopic status (Langley, Goldthorpe et al. 2003).<br />
In addition to using NO as an outcome measure to follow asthmatics treated with<br />
conventional inhaled steroids (considered here to be fluticasone, budesonide and<br />
beclomethasone), NO has also been used to assess whether novel steroids have a similar<br />
effect. Significant improvements in exhaled NO levels and induced sputum eosinophil counts<br />
were seen in three separate studies <strong>of</strong> ciclosonide -<br />
one using it alone (Wilson, Duong et al.<br />
2006), one comparing it to budesonide (Kanniess, Richter et al. 2001) and one comparing it to<br />
fluticasone (ke, Fardon et al. 2005). Four week treatment periods in 25 children with the<br />
newer beclomethasone derivative, hydr<strong>of</strong>luoroalcane-beclomethasone diproprionate at doses<br />
<strong>of</strong> 50pg and l00pg daily also resulted in reducing exhaled NO, associated with improvements<br />
in FEVr and exercise challenges @etersen, Agert<strong>of</strong>t et al.2004).<br />
236
9.8.8 (ii) Anti-leukotriene receptor antagonists<br />
In this class <strong>of</strong> drugs, the two predominantly studied in conjunction with NO have been<br />
montelukast and pranlukast. Firstly, NO has been monitored when these have been used as an<br />
addition to regular treatment. An open label six week trial <strong>of</strong> oral montelukast in 20 adult<br />
asthmatics already on IHCS resulted in significant improvements for levels <strong>of</strong> exhaled NO,<br />
coinciding with an improvement in exercise tolerance (Berkman, Avital et al. 200.3).<br />
Similarly, in 20 stable asthmatics, a crossover trial showed that montelukast, as opposed to<br />
placebo, reduced exhaled NO as well as reducing peak flow variability and symptom score<br />
over a two week treatment period (Sandrini, Ferreira et al. 2003). When montelucast was<br />
added to treatment <strong>of</strong> 22 adults with mild to moderate asthma using fluticasone 2501t9 and<br />
salmeterol 50pg twice a day, montelukast l0mg a day, compared to placebo, reduced levels <strong>of</strong><br />
exhaled NO and blood eosinophilia (Currie, lre et al. 2003). In 35 children treated with IHCS<br />
in whom the exhaled NO was greater than 20ppb, those that had montelukast added to their<br />
treatment had a significant reduction <strong>of</strong> exhaled NO which increased back to baseline level<br />
after withdrawal <strong>of</strong> the drug (Ghiro, Zanconato et al. 2002). While the reduction <strong>of</strong> NO<br />
coincided with some clinical improvements, there was no improvement in others such as<br />
bronchial responsiveness (Berkman, Avital et aL.2003), FEVr (Ghiro, Zanconato et al.2002;<br />
Currie, Ire et al. 2OO3; Sandrini, Ferreira et al. 2003) or in measured exhaled HzOz (Sandrini,<br />
Ferreira et al.2OO3). Only one study conducted in 25 children did not show a difference in<br />
exhaled NO with the addition <strong>of</strong> montelukast to IHCS treatment (Strauch, Moske et al. 2003).<br />
Pranlukast added to a six week reduction <strong>of</strong> inhaled budesonide did prevent asthma<br />
deterioration compared to placebo, but did not show a difference in levels <strong>of</strong> exhaled NO,<br />
although NO levels in the placebo group increased (Tamaoki, Kondo etal.1997).<br />
Secondly, NO has also been used as an outcome measure when these medication have been<br />
used alone. In steroid naive children (twelve to 2l subjects in each study), treatment with<br />
montelukast (5 or l0mgs per day) for between two and eight weeks resulted in a reduction in<br />
exhaled NO in two studies (Bratton, Lanz et al. 1999;l-ee,Lu et al. 2005) but not in a third<br />
(Spahn, Covar et al. 20O6) with the individual researchers also showing improvements in<br />
FEV', (Bratton, Lanz et al. 1999), salbutamol use (Bratton,Lanz et al. 1999), residual volume<br />
(Spahn, Covar et ^l.2006) and serum ECP levels (Spahn, Covar et al.2OO6). <strong>The</strong>y did not<br />
demonstrate changes in symptom scores (Spahn, Covar et al. 2006) and the exhaled NO<br />
returned to pre treatment levels two weeks after withdrawal <strong>of</strong> the medication (Ire, Lai et al.<br />
2005). In younger steroid narve children aged ten months to five years (54 in total) with an<br />
early diagnosis <strong>of</strong> asthma, significant improvements were seen in levels <strong>of</strong> exhaled NO,<br />
237
FEV9.5, symptom score and airways resistance but not bronchodilator responsiveness after<br />
four weeks treatment with 4mg daily <strong>of</strong> montelukast (Straub, Minocchieri et al. 2005; Straub,<br />
Moeller et al. 2005). In a study <strong>of</strong> twelve asthmatic children where montelukast reduced<br />
levels <strong>of</strong> exhaled NO, the four individuals that were heterogeneous at the gene locus <strong>of</strong> the<br />
leukotriene C4 synthase had a far better response than those who were homozygotes (Whelan,<br />
Blake et al. 2003).<br />
Thirdly, NO has been measured as one factor in comparison studies between montelukast and<br />
IHCS which have all resulted in a greater reduction <strong>of</strong> levels <strong>of</strong> NO in the IHCS arm<br />
(Kanniess, Richter et^1.2N2; Peroni, Bodini et aI.2005;Zniger, Szefleret aI.2006). This<br />
coincided with improvements <strong>of</strong> hyperreactivity (Kanniess, Richter et aL.2002; Peroni, Bodini<br />
et al. 2005), FEVr (Kanniess, Richter et al. 2002; Tniger, Szefler et al. 2006), sputum<br />
eosinophils (Kanniess, Richter et al.2002), asthma control questionnaire and salbutamol use<br />
(7*ige4 Szefler et al. 2006) with both treatments, again greater in the IHCS groups. However,<br />
the other antagonist pranlukast at 450mg per day did not change levels <strong>of</strong> exhaled NO in 30<br />
adults compared to IHCS but did show improvement in all other parameters measured<br />
(Yamauchi, Tanifuji et al. 20Ol). Comparisons between this drug class and the long actingp2<br />
agonists (LABAs) are described below.<br />
9.S.8 (iii) Long acting p2 agonists<br />
Exhaled NO has been used as an outcome measure when looking at the addition <strong>of</strong> long<br />
acting p2 agonists (LABAs) to IHCS. Neither eformoterol nor salmeterol have shown any<br />
effect on the exhaled NO when used alone in comparison to either IHCS therapy alone<br />
(Priero, Gutierrez et al.2OO2) or to the combination <strong>of</strong> IHCS and LABA (Aziz, Wilson et al.<br />
2000; Currie, Syme-Grant et al. 2N3), and no difference where they have been added to<br />
IHCS (Yates, Kharitonov et al. 1997; ke, Jackson et al. 2003). This is despite improvements<br />
in other parameters such as FEV1, adenosine S-monophosphate challenges, sputum and blood<br />
eosinophil counts and serum ECP (Aziz. Wilson et al. 2000; Prieto, Gutierrez et aL 2402;<br />
Currie, Syme-Grant et al. 2003; I-ee, Jackson et al. 2003).<br />
In comparisons between montelukast and LABAs, neither <strong>of</strong> two studies showed an effect on<br />
the level <strong>of</strong> exhaled NO (Aziz, Wilson et al. 2000; Wilson, Dempsey et al. 2001). This is<br />
interesting given that in most <strong>of</strong> the montelukast studies, this medication did result in a<br />
reduction in exhaled NO. <strong>The</strong> studies here really just confirm that the LABAs do not have<br />
anti -infl ammatory properties.<br />
238
9.8.8 (iv) Nedocromil sodium<br />
In two paediatric studies, this medication had no effect on levels <strong>of</strong> exhaled NO when<br />
compared to IHCS in 32 sensitised patients followed through a pollen season (Gratziou,<br />
Rovina et al. 2001) or in ll8 four to six year olds studied as part <strong>of</strong> the childhood asthma<br />
management program (CAMP) studies (Covar, Szefler et al. 2003).<br />
9.8.8 (v) <strong>The</strong>ophylline<br />
Despite theophylline being a previously commonly used drug, there has only been one study<br />
looking at its effect on levels <strong>of</strong> exhaled NO. In a double blind crossover study 15 patients<br />
with mild asthma on theophylline <strong>of</strong> 250mg twice per day had a reduction in eosinophil<br />
counts in sputum, BAL, and biopsy samples but no change in levels <strong>of</strong> exhaled NO and no<br />
improvements in lung function or bronchial hyper-responsiveness (Lim, Tomita et al. 2001).<br />
9.8.8 (vi) Novel medications<br />
L-NAME -<br />
Nebulised pretreatment with this non-selective NOS inhibitor reduced levels <strong>of</strong><br />
exhaled NO from baseline in 22 asthmatics with either an early asthmatic response only or<br />
both an early and late asthmatic response to allergen challengebyTT andTIVo respectively.<br />
Despite this result in NO, there was no change on the magnitude <strong>of</strong> the other acute responses<br />
in either group (Taylor, McGrath et al. 1998).<br />
L-Arginine -<br />
L-arginine (the substrate for the NOS enzymes) was nebulised as single doses <strong>of</strong><br />
0,759,1.5g and 39 to healthy subjects and asthmatics with 6-7 in each group. This increased<br />
levels <strong>of</strong> exhaled NO in a dose dependent fashion at 60 minutes showing a negative<br />
correlation to the fall in FEVr (Sapienza, Kharitonov et al. 1998). Nebulisation <strong>of</strong> 2.59 <strong>of</strong> L-<br />
arginine and D-arginine on separate days to eight steroid naive asthmatics both resulted in<br />
bronchoconstriction. <strong>The</strong>re was an initial proportional decline <strong>of</strong> NO then, with resolution <strong>of</strong><br />
the constriction,the NO increased so by three hours it was higher than baseline (Chambers and<br />
Ayres 2001). It is hard to know whether L-arginine is acting as a substrate in these studies or<br />
just as an airway irritant.<br />
Omalizumab -<br />
This humanised monoclonal antibody to Ig E was used in a steroid reduction<br />
study in adults with allergic asthma over one year. In the 18 that received active medication<br />
compared to the l l receiving placebo, the variability <strong>of</strong> the exhaled NO was significantly less<br />
and remained similar to the levels obtained when the children were on higher doses <strong>of</strong> IHCS<br />
prior to the reduction phase (Silk<strong>of</strong>f, Romero et al. 2004).<br />
239
Indomethacin - This medication alters the cyclo-oxygenase products <strong>of</strong> the arachidonic acid<br />
pathway which play a role in bronchoconstriction and airway inflammation. Indomethacin<br />
50mg daily or placebo was given to 38 asthmatics on beclomethasone > 1500pg daily for six<br />
weeks while the IHCS was reduced to half at week two and to a third at week four. While<br />
levels <strong>of</strong> exhaled NO increased in both groups, the effects were less pronounced in the<br />
indomethacin group and corresponded to fewer exacerbations (Tamaoki, Nakata et al. 2000).<br />
PGE2|PGF2a -<br />
<strong>The</strong>se prostaglandins also provide negative feedback in the cyclo-oxygenase<br />
pathway and a trial <strong>of</strong> inhaled PGE2 and PGF2a significantly reduced levels <strong>of</strong> exhaled NO in<br />
both normal and asthmatic subjects. This was not associated with any alteration <strong>of</strong> lung<br />
function in the normal subjects, but PGE2 caused an increase in FEVr in the asthmatic group<br />
(Kharitonov, Sapienza et al. 1998).<br />
9.8.9 Summnry <strong>of</strong> nitric oxide in asthma and atopy<br />
How do we make sense <strong>of</strong> all this information regarding NO and asthmalatopy"! NO does<br />
correlate with other clinical and inflammatory parameters <strong>of</strong> asthma -<br />
most particularly in the<br />
steroid narve asthmatic and most particularly with sensitivity and specificity seen with<br />
eosinophil counts. <strong>The</strong> utilization <strong>of</strong> NO to contribute to diagnosis seems hopeful. It<br />
performed as well as eosinophil counts in induced sputum (Smith, Cowan et al.200/.) and<br />
methacholine or adenosine S-monophosphate challenges (Berkman, Avital et al. 2005).<br />
However this again is best used in steroid naive patients and the effect <strong>of</strong> atopy alone in<br />
elevating NO levels must be taken into account. It is easier to show NO differences in groups<br />
<strong>of</strong> subjects such 'asthma', 'atopy', 'other respiratory disease' or 'controls', rather than being<br />
able to place one individual into the correct category based on the exhaled NO reading. NO<br />
does appear to determine current or recent symptoms and is more likely to be useful in the<br />
individual when followed longitudindly for loss <strong>of</strong> control or response to treatment. Studies<br />
using NO to guide treatment have shown beneficial effects. However, again eosinophils were<br />
more sensitive and <strong>of</strong> the most benefit when these two parameters are combined @rightling,<br />
Green et al. 2005; Pijnenburg, Bakker et al. 2005; Smith, Cowan et al. 2005; Belda,<br />
Parameswaran et al. 2006). In medications that have anti-inflammatory effects such as the<br />
different IHCS, leukotriene receptor antagonists, indomethacin and prostaglandins, it appears<br />
the result is to decrease NO levels. NO remains unchanged in those without such properties as<br />
in the LABAs. In these studies, when NO did show a reduction, this <strong>of</strong>ten coincided with<br />
other clinical benefits.<br />
240
However, the major advantage <strong>of</strong> using measurements exhaled NO, particularly over sputum<br />
eosinophil counts, blood tests or airway challenges is obvious -<br />
particulady in children. It is a<br />
non-invasive test, analogous to lung function testing to which patients are very familiar' It is<br />
also likely to be available in more clinics and can be performed more quickly and more<br />
cheaply with immediate results.<br />
g.g Nitric oxide levels in primar.v ciliary dyskinesia<br />
This is an area where NO measurement was found early to be <strong>of</strong> particular value. Initially No<br />
was found to be absent in nasal measurements in four children with Kartagener's syndrome<br />
(Lundberg, Weitzberg et al. 1994). This led to the evaluation <strong>of</strong> 21 children with primary<br />
ciliary dyskinesia (PCD) in whom both nasal and oral NO was found to be significantly lower<br />
when compared to 60 healthy children. While there was some overlap with regard to<br />
individual lower airway results, only one child was an outlier from each group in the nasal<br />
NO ranges (Karadag, James et al. 1999). This has been confirmed in a recent study where the<br />
impairment <strong>of</strong> NO output was less pronounced in lower than the upper nasal respiratory tract<br />
after sampling at several sites in 17 PCD and 28 healthy subjects (Matrut, Escudier et al.<br />
2006). In another large cross-sectional study involving 102 subjects, the concentration <strong>of</strong><br />
exhaled NO was significantly lower in both PCD and CF patients than bronchiectasis and<br />
healthy subjects. <strong>The</strong> nasal NO, however, was markedly reduced only in the PCD subjects<br />
(Horvath, Loukides et al. 2003). This change appears to occur early in life as seen in two<br />
infants with pCD aged four and six months having significantly lower levels <strong>of</strong> NO compared<br />
to five healthy controls (Baraldi, Pasquale et a|.2004). <strong>The</strong>se low NO levels found are despite<br />
levels <strong>of</strong> nitrite, nitrate and S-nitrosothiol when measured in exhaled breath condensate being<br />
no different from normals (Csoma, Bush et al. 2003)'<br />
Use <strong>of</strong> NO as a screening tool for this condition has been investigated. From the study above<br />
reviewing children already diagnosed with PCD, the authors determined a cut <strong>of</strong>f <strong>of</strong> 250ppb<br />
for nasal NO as having both a positive and a negative predictive value <strong>of</strong> 0.95. This correctly<br />
detected 20 <strong>of</strong> 21 subjects as having PCD and conectly detected 19 <strong>of</strong> 20 as not having PCD<br />
(Karadag, James et al. Lggg). Measuring 31 children with PCD, 2l with non-CF<br />
bronchiectasis, 17 with CF, 35 with asthma and 53 healthy controls, a nasal NO <strong>of</strong> 250ppb<br />
gave a sensitivity <strong>of</strong> 977o and a specificity <strong>of</strong> gOVo for a diagnosis <strong>of</strong> PCD (Narang, Ersu et al'<br />
2OOZ). Of 34 children referred for investigation for possible PCD to a tertiary clinic, the 17<br />
that proved positive on biopsy again had significantly lower level <strong>of</strong> nasal NO. This group<br />
determined a nasal NO <strong>of</strong> 105ppb gave a specificity <strong>of</strong> 88Vo and a positive predictor value <strong>of</strong><br />
24r
89Vo for PCD, and above that excluded PCD with 1007o certainty (Corbelli, Bringolf-Isler et<br />
al. 2004). Reviews now recommend the use <strong>of</strong> nasal NO to screen for PCD, usually with the<br />
250ppb cut-<strong>of</strong>f (Silk<strong>of</strong>f 2004; Stehling, Roll et al. 2006).<br />
Nebulised L-arginine given to ten patients with PCD and ten healthy individuals increased<br />
ciliary beat frequency (Inukides, Kharitonov et al. 1998). L-arginine given intravenously<br />
resulted in an increase <strong>of</strong> nasal and exhaled NO in both PCD and CF subjects, although the<br />
mean concentrations were still significantly lower than normals (Grasemann, Gartig et al.<br />
1999). Neither study showed that the increase in NO translated into other clinical effects or<br />
improvements.<br />
<strong>The</strong> mechanism behind the low nasal NO in PCD is not yet determined. In other diseases<br />
where low levels are obtained such as CF @alfour-Lynn, Laverty et al. 1996; Dotsch,<br />
Demirakca et al. 1996; Lundberg, Nordvall et al. 1996), diffuse panbronchiolitis (Nakano, Ide<br />
et al. 2000), paranasal sinus inflammatory disease (Arnal, Flores et al. 1999), chronic sinusitis<br />
(Lindberg, Cervin et al. 1997; Deja, Busch et al. 2003) (described in sections below), it is<br />
thought to be secondary to thickened secretions and/or obstruction <strong>of</strong> the ostea preventing NO<br />
diffusion. However, in clinically stable PCD patients with low NO, their ostea were noted to<br />
be open (Wodehouse, Kharitonov et al. 2003) and sinuses clear (Amal, Flores et al. 1999).<br />
NO has been implicated in control <strong>of</strong> ciliary function. In our own study (conducted after the<br />
data presented in this thesis), we found no correlation between exhaled NO levels and ciliary<br />
beat frequency in 135 children <strong>of</strong> European, Maori and Pacific ethnicities. However, we did<br />
find an increase in the percentage <strong>of</strong> ciliary structural defects, three times that reported in<br />
previous control groups (Edwards, Douglas et al. 2005). Endothelial NOS is located at the<br />
basal micro-tubular membrane in ciliated epithelium (Xue, Botkin et al. 1996) with a<br />
suggestion that NO acts as an intermediate messenger @uner and Lindberg 1999) and TNFc<br />
or ILI can up-regulate ciliary motility seeming to act through NO via induction <strong>of</strong> NOS (Jain,<br />
Rubinstein et al. 1993). Further research is needed in this area for an adequate explanation <strong>of</strong><br />
this phenomenon <strong>of</strong> low NO levels in this condition.<br />
9.10 Nitric oxide levels in cystic fibrosis<br />
Studies <strong>of</strong> NO in subjects with cystic fibrosis (CF) have resulted in conflicting data.<br />
Contributing factors are likely to be the different techniques used for measurement when these<br />
studies began in the late 1990s, as well as the variation in age <strong>of</strong> the patients, co-morbidities<br />
and treatments, not always specified. Cross-sectional studies in children have shown no<br />
difference in levels <strong>of</strong> oral exhaled NO across CF, asthmatics on IHCS and healthy groups,<br />
242
ut those with CF did have significantly lower nasal NO levels (Balfour-Lynn, Laverty et al'<br />
1996;Dotsch, Demirakca et al. 1996; Lundberg, Nordvall et al. 1996). However, three studies<br />
in adult patients with CF showed lower NO levels from both the oral and nasal sampling<br />
(Grasemann, Michler et al. 1997; Jones, Hegab et al. 2000; Thomas, Kharitonov et al' 2000)'<br />
<strong>The</strong>re has been no relationship demonstrated between the oral and nasal NO concentrations,<br />
although one study demonstrated a positive correlation between ambient and measured NO<br />
levels in all subject groups @otsch, Demirakca et al. 1996). In infants' one study showed<br />
reduced levels <strong>of</strong> exhaled NO in five infants with CF compared to 1l healthy infant controls<br />
at a mean age <strong>of</strong> 48.6 days (Elphick, Demoncheaux et al. 2001), but a more recent study<br />
showed no difference in exhaled NO levels in 18 infants with CF and 23 healthy infants at a<br />
mean age <strong>of</strong> 64.9 weeks (Franklin, Hall et al. 2006). Franklin's study also demonstrated an<br />
inverse relationship between age and exhaled No levels in the CF but not the healthy infant<br />
group (Franklin, Hall et al. 2006).<br />
Studies <strong>of</strong> correlations <strong>of</strong> NO level to lung function have also been variable in the cF<br />
population. No correlation was demonstrated with levels <strong>of</strong> either nasal or oral NO in three<br />
studies with 135 subjects @otsch, Demirakca et al. 1996: Thomas, Kharitonov et al' 2000;<br />
Jaffe, slade et al. 2003), while another two studies showed a weak but statistically significant<br />
positive relationship with FEVr in 63 subjects (Grasemann, Michler et al. t997; Ho, Innes et<br />
al. lggg). It should be noted that this is a different response than seen in asthmatics as the cF<br />
patients with better lung function appeared to have higher exhaled NO levels. No association<br />
was observed between the CF genotype and NO values (Thomas, Kharitonov et al' 2000;<br />
Texereau, Fajac et al. 2005) but an inverse correlation between No and the transepithelial<br />
baseline potential difference measured has been described (Texereau, Fajac et al. 2005)'<br />
Whether infection and type <strong>of</strong> organism has had an effect on the exhaled No levels has also<br />
been examined. <strong>The</strong>re was no difference in nasal or exhaled NO levels in children chronically<br />
infected with staphylococcal aureus (22 infected versus 11 non-infected) (Balfour-Lynn,<br />
Laverty et al. 1996). Nasal NO was significantly lower in L7 <strong>of</strong> 33 who were chronically<br />
infected with pseudomonas aeruginosa in one study (Balfour-Lynn, Laverty et al. 1996) but<br />
not confirmed in two others with 49 <strong>of</strong> the 68 subjects who had been chronically infected<br />
(Thomas, Kharitonov et al. 2000; Jaffe, Slade et al. 2003). Exhaled NO was lower in nine<br />
patients considered high risk <strong>of</strong> developing allergic bronchopulmonary aspergillosis (ABPA)<br />
compared to a low risk group (Lim, Chambers et al.2003) but, as the high risk patients were<br />
on corticosteroids, this may reflect steroid use as opposed to ABPA risk per se'<br />
243
This low NO occurs despite higher than normal levels <strong>of</strong> NO metabolites <strong>of</strong> nitrite and nitrate<br />
(Francoeur and Denis 1995; Grasemann, Ioannidis et al. 1998; Jones, Hegab et al. 2000) and<br />
nitrotyrosine (Jones, Hegab et al. 2000) which have been demonstrated in sputum and saliva<br />
samples. <strong>The</strong>se metabolite levels have been correlated with sputum IL8 (Francoeur and Denis<br />
1995) and lung function (Grasemann, Ioannidis et al. 1998). No correlation was seen when<br />
levels <strong>of</strong> exhaled NO were compared with markers <strong>of</strong> inflammation obtained from<br />
bronchoalveolar lavage in 18 CF infants (Franklin, Hall et al. 2006). Immunostaining studies<br />
<strong>of</strong> iNOS showed an inverse correlation to neutrophil counts and IL8 levels suggesting the<br />
iNOS expression decreases in the CF children as ainvay inflammation increases (Wooldridge,<br />
Deutsch et al.2004). Similarly, in tracheobronchial brushings from 40 children with CF, the<br />
expression <strong>of</strong> mRNA for iNOS was found to be lower than seen in two healthy children,<br />
which the authors suggested may have consequences for local antimicrobial defense (Moeller,<br />
Horak et al. 2006).<br />
Regarding NO as a possible outcome measure for treatment, no change in levels were seen<br />
from the onset <strong>of</strong> an acute infective exacerbation in eight patients followed for seven days<br />
during treatment (Ho, Innes et al. 1998). In contrast, increased exhaled NO levels, associated<br />
with a significant improvement in lung function, was seen in fourteen children after a course<br />
<strong>of</strong> IV antibiotics, although the NO level did not alter in six <strong>of</strong> the children that had chronic<br />
pseudomonas infection (Jaffe, Slade et al. 2003). In six children that were commenced on<br />
dornase alpha (Rh DNAse, 'pulmozyme'), five had exhaled NO changes in parallel to the<br />
changes <strong>of</strong> pulmonary function tests, with no change in those receiving placebo (Grasemann,<br />
Lax et al. 2004). With single inhalations <strong>of</strong> L-arginine, there were increased levels <strong>of</strong> NO,<br />
FEVr and oxygen saturation (Grasemann, Kurtz et al. 2006). Oral L-arginine compared to<br />
placebo showed that a single dose <strong>of</strong> 200mgkg also resulted in increasing levels <strong>of</strong> exhaled<br />
NO which continued for six weeks with continued supplementation but there was no change<br />
<strong>of</strong> lung function (Grasemann, Grasemann et al. 2005). Another study administered oral L-<br />
arginine daily for four weeks getting an increase in plasma L-arginine levels but there was no<br />
change in either exhaled NO levels or FEVI @verard and Donnelly 2005).<br />
On the face <strong>of</strong> it, it is hard to believe that the level <strong>of</strong> NO in such an inflammatory condition is<br />
low, especially when the NO metabolites are higher than that found in asthmatic and control<br />
groups. <strong>The</strong>re are a number <strong>of</strong> possible explanations:<br />
o Mucosal swelling with thickened mucus may result in mechanical obstruction <strong>of</strong> the<br />
sinus ostea which could impede the flow <strong>of</strong> NO into the nasal cavity. Consistent with<br />
this is that almost all patients with CF develop nasal and sinus disease.<br />
244
Some <strong>of</strong> these studies, particularly the early studies, may have included subjects on<br />
inhaled or oral corticosteroids and not differentiated them within the study groups.<br />
NO may degrade within the mucus giving low levels reaching the airway lumen for<br />
measurement.<br />
Pseudomonas aeruginosa produces a pigment pyocinen which in vitro has been shown<br />
to inactivate NO (Wanen,I-oi et al. 1990).<br />
While the true reason is likely to be a combination <strong>of</strong> these factors, the obstruction to<br />
diffusion <strong>of</strong> NO into the airway and trapping <strong>of</strong> NO within the mucus, probably plays the<br />
major role. <strong>The</strong> low nasal NO level seems a consistent finding in this population, and in adults<br />
oral NO also appears to be low, with a possible correlation between higher lung function and<br />
higher NO levels.<br />
9.11 Nitric oxide levels in bronchiectasis<br />
An initial paper suggested both elevated levels exhaled NO and a correlation between this and<br />
the chest CT scan score in 20 patients with bronchiectasis, but neither were seen in another 19<br />
on IHCS treatment (Kharitonov, Wells et al. 1995). Subsequent papers have not confirmed<br />
these findings in 16 (Ho, Innes et al. 1998), 3l (Horvath, Loukides et al' 2003) and 109<br />
(Tsang, Irung et a\.2}}2)patients with bronchiectasis measured at a time <strong>of</strong> disease stability'<br />
Those with pseudomonas aeruginosa infection (25 <strong>of</strong> 109) did have significantly lower levels<br />
<strong>of</strong> exhaled NO although their sputum NO concentration was the same' <strong>The</strong>re was no<br />
correlation between the levels <strong>of</strong> exhaled No and FEVI, FVC or the number <strong>of</strong> bronchiectatic<br />
lobes involved (Tsang, lrung et al. 2o02). similar to findings in cF, it is surprising not to<br />
find elevated NO in such an inflammatory and infective disease. <strong>The</strong> mucus layer may well be<br />
a barrier to detection <strong>of</strong> the production <strong>of</strong> NO, and in an inflamed environment the presence <strong>of</strong><br />
reactive oxygen species may result in interaction with this highly reactive molecule resulting<br />
in effective removal <strong>of</strong> NO to form other compounds, such as perioxynitrite. <strong>The</strong> use <strong>of</strong> NO<br />
here is likely to be as a screen in bronchiectasis - if low nasal No levels are obtained, then CF<br />
and PCD testing must be undertaken.<br />
g.l2 Nitric oxide levels in upper resoiratory tract infections<br />
<strong>The</strong> effect that upper respiratory tract infections (URTI) have on exhaled NO levels has also<br />
been studied. In 18 non-asthmatic adults during an URTI, increased exhaled NO levels to<br />
315ppb were noted, decreasing to 87ppb during recovery at two to three weeks which became<br />
similar to aged matched healthy controls (Kharitonov, Yates et al. 1995). When 79 COPD<br />
245
patients were followed through one winter, there were increases in levels <strong>of</strong> exhaled NO when<br />
they had a 'cold', 'a sore throat', or 'dyspnoea combined with a cold' compared to times <strong>of</strong><br />
stability (Bhowmik, Seemungal et al. 2005). Two studies actually infected adult subjects with<br />
respiratory viruses. In the first, seven subjects infected with human rhinovirus 16 had<br />
significant increases in NO levels by days two and three compared to seven placebo<br />
'infections' (de Gouw, Grunberg et al. 1998). In the second, six subjects infected with the<br />
same virus had increases in both nasal and lower airway NO levels with positive correlations<br />
to the severity <strong>of</strong> infection as measured by organism count, inflammatory markers from nasal<br />
lavages and nasal scrapings, and day four symptom scores (Sanders, Proud et al.20O4).<br />
However in 16 children with acute maxillary sinusitis, the NO nasal measurements dropped<br />
and then increased following antibiotic treatment over a two week period @araldi, Azzolin et<br />
al. 1997). This suggests that NO does increase with upper respiratory tract infections, but may<br />
be obstructed from measurement if there is significant sinus obstruction.<br />
9.13 Nitric oxide levels in chronic obstructive pulmonary disease (COPD)<br />
In cross-sectional studies <strong>of</strong> chronic obstructive pulmonary disease (COPD) subjects, levels <strong>of</strong><br />
exhaled NO has been similar to, or higher than, healthy controls though not to levels seen in<br />
steroid narve asthmatics (Clini, Bianchi et al. 1998; Kanazawa, Shoji et al. 1998; Rutgers,<br />
Meijer et al. 1998; Corradi, Majori et al. 1999; Zietkowski, Kucharewicz et al. 2005). This has<br />
been confirmed using single breath (Clini, Bianchi et al. 1998; Kanazawa, Shoji et al. 1998;<br />
Maziak, Loukides et al. 1998; Rutgers, Meijer et al. 1998; Zietkowski, Kucharewicz et al.<br />
2005), tidal breathing @utgers, Meijer et al. 1998) and reservoir techniques (Corradi, Majori<br />
et al. 1999). A significant negative correlation has been shown with FEVr at times <strong>of</strong> disease<br />
stability (Clini, Bianchi et al. 1998; Maziak, l.oukides et al. 1998; Ansarin, Chatkin et al.<br />
2001). A correlation has also been found between baseline FEV1 and the change in the level<br />
<strong>of</strong> exhaled NO following an eight week rehabilitation exercise program in mild, moderate and<br />
severe COPD patients but not those with cor pulmonale (Clini, Bianchi et al. 2000; Clini,<br />
Bianchi et al. 2001; Clini, Bianchi et al.2002). With regard to other physiological parameters,<br />
exhaled NO was found to be inversely correlated with oxygen saturation and diffusion<br />
transfer factor @r-CO) (Ansarin, Chatkin et al. 2001) and positively correlated with the<br />
residual lung volume, total lung capacity (Ansarin, Chatkin et al. 2001), and degree <strong>of</strong> airflow<br />
obstruction (Clini, Bianchi et al. 1998). <strong>The</strong>se all suggest that the worse the disease is, as<br />
determined by these parameters, the higher the NO level obtained (Ansarin, Chatkin et al.<br />
2001). More recently, multiple flows to assess the different airway compartments have been<br />
employed in this population (see Section 9.3.1 (i)). In all studies to date NO was found to be<br />
246
high in the alveolar compartment which correlated with severity and progression regardless <strong>of</strong><br />
the patient's smoking habit or current treatment (Tsoukias and George 1998; Kharitonov and<br />
Barnes 2004; Brindicci, Ito et al. 2005). As well, No metabolites <strong>of</strong> nitrite and nitrate have<br />
been higher in induced sputum in COPD patients compared to normals (Kanazawa, Shoji et<br />
al. 199g). Lung biopsies from patients with severe COPD had higher iNOS seen than those<br />
that had more mild disease and normal controls (Maestrelli, Paska et al. 2003).<br />
COpD patients having frequent exacerbations had higher levels <strong>of</strong> exhaled NO than stable<br />
current smokers and ex-smokers with COPD or chronic bronchitis (Agusti, Villaverde et al.<br />
1999; Silk<strong>of</strong>f, Martin et al. 2001). Similarly during admission, the level <strong>of</strong> exhaled NO was<br />
high and remained elevated at discharge despite intravenous steroid therapy, but when the<br />
patients were once again clinically stable it had reduced to levels similar to control subjects<br />
(Agusti, Villaverde et al. lggg). <strong>The</strong> NO levels correlated to the drop in FEV1 during<br />
exacerbation in one study (Silk<strong>of</strong>f, Martin et al. 2001), but did not relate to FEVr or other<br />
variables such as FVC, lung volumes, diffusing capacity or pulmonary gas exchange at the<br />
time <strong>of</strong> discharge in another study (Agusti, Villaverde et al. 1999). Assessing the effect <strong>of</strong><br />
pollution; an association between exhaled NO and particulate matter when measured in a<br />
cross section <strong>of</strong> the population was significantly greater in subjects who had a doctor<br />
diagnosis <strong>of</strong> coPD (Adamkiewicz, Ebelt et al. 2004). Exhaled No levels measured<br />
seasonally and during exacerbations in 79 COPD outpatients showed higher levels during<br />
winter months, as well as being higher during the 68 exacerbations seen in 38 <strong>of</strong> these<br />
patients. <strong>The</strong> degree <strong>of</strong> elevation, however, was noted to be lower in those more severely<br />
affected (Bhowmik, Seemungal et al. 2005). Elevated No levels decreased significantly in 47<br />
patients commencing IHCS therapy but there were no changes in FEVr (Zietkowski'<br />
Kucharewi cz et a:.2005). Two studies showed higher exhaled No levels that positively<br />
correlated with post-bronchodilator FEVr suggesting NO may be a marker for inflammation'<br />
responsive to bronchodilator treatment similar to asthmatics, although these COPD patients<br />
were not known to be atopic (Papi, Romagnoli et al. 2000; Zietkowski, Kucharewicz et al'<br />
2005). Both exhaled No and co levels were low in 19 individuals with alpha-l antitrypsin<br />
deficiency compared to both controls and coPD patients (Machado, Stoller et al' 2002)'<br />
9.14 Nitric oxide levels in smokers<br />
NO is known to be contained within cigarettes as previously discussed (see Section 2'2'3)'<br />
Exact concentrations between brands differ, but cigarettes produced in France and the United<br />
States exceeded the NO content <strong>of</strong> those produced in the UK up to 3-5 fold with no other<br />
247
differences noted based on filter, design, tar, nicotine or CO yield. This research was done in<br />
the 1980s and the relationship to what is made and sold now here in New Zealand has not<br />
been studied. Many studies have demonstrated that both peak and plateau NO is lower in<br />
smokers than non-smokers who are otherwise 'healthy' with normal lung function (Persson,<br />
Zetterstrom et al. 1994; Schilling, Holzer et al. 1994; Kharitonov, Robbins et al. 1995;<br />
Robbins, Millatmal et al. 1997; Verleden, Dupont et al. 1999; Balint, Donnelly et al. 2001;<br />
Mazzone, Cusa et al. 2001; Hogman, Holmkvist et ^1.200.2; Horvath, Donnelly et al.2O04).<br />
In mild asthmatics, smokers also had a lower level <strong>of</strong> exhaled NO when compared to non-<br />
smokers (Persson, Zetterstrom et al. 1994; Verleden, Dupont et al. 1999; Horvath, Donnelly et<br />
al. 2ffi4; McSharry, McKay et al. 2005). Nasal NO measurements in 'healthy' normal and<br />
asthmatic smokers compared with their non-smoking counterparts were also reduced<br />
(Robbins, Millatmal et al. 1997; Olin, Hellgren et al. 1998). This reduction <strong>of</strong> NO was<br />
correlated with 'pack years' in current (McSharry, McKay et al. 2005) and ex-smokers<br />
(Corradi, Majori et al. 1999). Two studies also found significant reductions in levels <strong>of</strong><br />
exhaled NO and NO output in a total <strong>of</strong> ll8 healthy infants exposed to pre-natal cigarette<br />
smoke compared to the non-exposed (Hall, Reinmann et al.2OO2; Frey, Kuehni et al. 2OO4).<br />
<strong>The</strong> effects <strong>of</strong> a single cigarette have also been studied. A transient reduction in peak exhaled<br />
NO occurs immediately (Kharitonov, Robbins et al. 1995) and at 50 minutes with a co-<br />
incident rise in HzOz (Horvath, Donnelly et al. 2OO4). However another study found an<br />
increase level <strong>of</strong> NO at one and ten minutes (Chambers, Tunnicliffe et al. 1998) while a fourth<br />
found no change in the levels <strong>of</strong> exhaled NO, S-nitrosothiol or nitrotyrosine at 30 and 90<br />
minutes, although nitrite and nitrate levels were significantly increased (Balint, Donnelly et al.<br />
2001). A decrease in plasma nitrate and nitrite concentrations were seen after smoking a<br />
single 'real' cigarette compared to a sham cigarette (Tsuchiya, Asada et al.2002). Passive<br />
exposure and active smoking in usual non-smokers resulted in a reduction in the level <strong>of</strong><br />
exhaled NO when measured every 15 minutes to one hour (Yates, Breen et al. 2001).<br />
Recently a murine model showed that cigarette smoke caused a reduction <strong>of</strong> NO by<br />
decreasing iNOS mRNA transcription but did not alter the iNOS mRNA half life once already<br />
developed (Hoyt, Robbins et al.20O3). It may be that the NO in the cigarettes operates as a<br />
negative feedback mechanism, resulting in reduction <strong>of</strong> activity <strong>of</strong> the NOS enzymes.<br />
<strong>The</strong> effect <strong>of</strong> quitting smoking on the subsequent levels <strong>of</strong> NO has also been examined. In 20<br />
smokers, nine that were able to refrain from smoking for four weeks resulted in increased<br />
levels <strong>of</strong> exhaled NO, which was no longer significantly lower than controls (Hogman,<br />
Holmkvist et al.20O2). Fourteen smokers had an increase in their exhaled NO compared to<br />
248
their baseline No when they gave up for one week. In the ten who were able to continue, their<br />
level <strong>of</strong> exhaled NO had increased to normal non-smoking levels when re-measured at eight<br />
weeks, while in those that were unable to sustain quitting, the No had dropped to the previous<br />
low levels (Hogman, Holmkvist et al. 2OO2). This effect <strong>of</strong> smoking is unfortunate as it<br />
renders the measurement <strong>of</strong> No unhelpful, particularly in the asthmatic population where it<br />
could be a helpful longitudinal indicator <strong>of</strong> airway inflammation and treatment effects.<br />
9.15 Nitric oxide levels in interstitial lung diseases<br />
Interstitial lung diseases have been particularly helpful in proving the value <strong>of</strong> using differing<br />
flows to measure NO from different areas within the lung (see Section 9.3.1 (i))' In 20<br />
patienrs with scleroderma including five with additional pulmonary hypertension (Girgis,<br />
Gugnani et a:.2002) and in 24 patients with scleroderma including twelve with and twelve<br />
without clinical or radiological evidence <strong>of</strong> lung disease (Moodley and Lalloo 2001)'<br />
significant differences in exhaled NO levels from control subjects were only seen when the<br />
alveolar rather than airway concentrations were measured. <strong>The</strong>re was also a negative<br />
correlation demonstrated between the alveolar concentration <strong>of</strong> NO and the diffusion<br />
coefficient in the parient groups (Moodley and Lalloo 2001; Girgis, Gugnani et al' 2002)' In<br />
active fibrosing alveolitis, an alveolar NO increase was associated with increasing<br />
lymphocyte cell counts and an alveolar decrease was associated with corticosteroid treatment<br />
(paredi, Kharitonov et al. 1999). This is in contrast to studies using one standard expiratory<br />
flow for measurement, where no difference was found in 34 patients with systemic sclerosis<br />
(Sud, Khullar et al. 2000), or in 52 patients with sarcoidosis (Wilsher, Fergusson et al' 2005)'<br />
One study did find higher exhaled NO levels using the standard flow in 47 patients with<br />
systemic sclerosis, particularly those with interstitial lung disease, and demonstrated an<br />
inverse correlation with pulmonary artery pressure but no correlation to age, disease duration,<br />
current therapy or the type <strong>of</strong> disease be it limited or diffuse (Rolla, Colagrande et al' 2000)'<br />
One early paper also measured exhaled NO continuously in expired air at baseline and during<br />
an exercise test and calculated the output <strong>of</strong> NO, which was low in six subjects with<br />
pulmonary fibrosis compared to normal subjects. <strong>The</strong> authors suggested that that this patient<br />
group may fail to increase NO output by failing to recruit the capillary bed during exercise<br />
(Riley, Porszasz et al. lgg|). Nasal NO was 887o lower than normals in diffuse<br />
panbronchiolitis, a pulmonary disease <strong>of</strong> unknown origin confined usually to individuals <strong>of</strong><br />
Japanese, Korean or chinese descent (Nakano, Ide et al. 2000). All current and future work in<br />
this area <strong>of</strong> interstitial lung disease should use the ability to measure NO in the alveolar<br />
compartment for meaningful results.<br />
249
9.L6 Nitric oxide levels in exercise<br />
<strong>The</strong> effects <strong>of</strong> exercise on levels <strong>of</strong> exhaled NO in healthy, athletic adults, and those with<br />
respiratory and/or cardiac disease has been investigated, with a few studies also done in<br />
children. In healthy adults, the measurement <strong>of</strong> exhaled NO with exercise (treadmill or<br />
bicycle) showed a decrease when measuring the absolute levels in single breaths, but there<br />
was a two to three fold increase in NO output taking into account the increased ventilatory<br />
minute volume @ersson, Wiklund et al. 1993; Iwamoto, Pendergast et al. 1994; Trolin, Anden<br />
et al. 1994; Phillips, Giraud et al. 1996; Yasuda, Itoh et al. L997). <strong>The</strong> increased output more<br />
closely related to increased ventilation than increased blood flow @hillips, Giraud et al. 1996)<br />
and significantly correlated with CO2 production (Iwamoto, Pendergast et al. 1994). Two<br />
studies assessed the contribution from nasal or oral exhalations showing that most <strong>of</strong> the NO<br />
coming from the lower airways (Lundberg, Rinder et al. 1997; Yasuda, Itoh et al. 1997) and<br />
from the airway rather than alveolar compartment @ersson, Wiklund et al. 1993). <strong>The</strong> nasal<br />
cavity NO reduced by 47Vo after one minute and 76Vo after five minutes <strong>of</strong> exercise<br />
(Lundberg, Rinder et al. 1997).<br />
<strong>The</strong>re have been many studies done in elite athletes. When compared to those with lower<br />
levels <strong>of</strong> fitness, the athletes had a significantly more linear increase in NO output (Maroun,<br />
Mehta et al. 1995; Chirpaz-Oddou, Favre-Juvin et al. L997; Sheel, Edwards et al. 2000;<br />
Verges, Flore et al. 2005). This output correlated well with oxygen consumption, COz<br />
production, minute ventilation and heart rate (Chirpaz-Oddou, Favre-Juvin et al. 1997).In one<br />
study, athletes that developed exercise induced hypoxemia had lower levels <strong>of</strong> NO compared<br />
to those who did not seen (Kippelen, Caillaud et al. 2N2), but this was not confirmed in<br />
another (Sheel, Edwards et al. 2000). Exercise in cold challenge conditions resulted in lower<br />
levels <strong>of</strong> exhaled NO output being achieved and a slower time to recovery with lower FEVr<br />
(<strong>The</strong>rminarias, Flore et al. 1998; Pendergast, Krasney et al. 1999). <strong>The</strong>se results suggested<br />
that physical conditioning increases expiratory NO output during exercise and this may be due<br />
to an increased vascular and/or epithelial production <strong>of</strong> NO. <strong>The</strong> enhanced vascular NO<br />
production may be the result <strong>of</strong> increased share stress or <strong>of</strong> an up regulation <strong>of</strong> the endothelial<br />
NOS gene expression (Maroun, Mehta et al. 1995).<br />
Can NO predict development <strong>of</strong> exercise induced bronchospasm? Higher baseline levels were<br />
significantly associated with bronchoconstriction and histamine responsiveness in atopic<br />
rather than non-atopic steroid naive healthy conscripts (Rouhos, Ekroos et al. 2005). In 50<br />
consecutive subjects, a ROC curve yielded a value <strong>of</strong> 0.636 when comparing those who did<br />
250
and did not develop exercise-induced bronchospasm. A result <strong>of</strong>
another study <strong>of</strong> 111 school children with asthma, exercise induced bronchospasm could be<br />
excluded with a probability <strong>of</strong> 90Vo in those with baseline NO levels
Figure 9.3: Diagram <strong>of</strong> the measurement <strong>of</strong> lung function and No in an infant<br />
Syringe<br />
Pneumotach<br />
Pump<br />
Resistancs<br />
Blow-<strong>of</strong>f at 20 crn H2O<br />
chemiluminescence analyser<br />
Taken from wildhaber JH, Hail GL, Stick sM. Measurements <strong>of</strong> exhaled nitric oxide with the single'<br />
breath technique and positive expiratory ;;;il6 il infants..American Journal <strong>of</strong> Respiratory and<br />
Criticat Care Medicine 'iggg; 159:74-78 (Wildhaber, Hallet al. 1999)'<br />
using this technique there was successful measurement <strong>of</strong> FEVo.s in 27 <strong>of</strong> 30 sedated infants<br />
aged 3-24 months and successful measurement <strong>of</strong> NO in all. <strong>The</strong> end tidal COz level at the<br />
nose was not changed during expiration, indicating that the velum was closed during the<br />
manoeuvre (wildhaber, Hall et al. 1999). Studies have now been carried out using variations<br />
on this technique reporting 95To+ success in sedated or quiet infants (Buchvald and Bisgaard<br />
Zxl:;Martinez, Weist et al. 2003; Franklin, Turner et al' 2004; Straub' Moeller et al' 2005)'<br />
<strong>The</strong> other method is by using tidal breathing where NO can be measured online or by<br />
reservoir collection. Again the infant is in a supine position asleep following sedation' with<br />
direct online measurement recorded during 20 seconds <strong>of</strong> quiet breathing (Franklin, Turner et<br />
a:.2004), or using a mean <strong>of</strong> five or ten consecutive breaths (Hall' Reinmann et al' 20/0.2;<br />
colnaghi, condo et al. 2003; Iripala, williams et al.2004). For <strong>of</strong>fline measurements' tidal<br />
breathing can be collected into a gas sampling balloon which can then be attached to the<br />
sampler inlet <strong>of</strong> the NO analyser -<br />
collecting over a set time period such as 15-45 seconds<br />
@inakar, Craff et al. 2006), or during a set number <strong>of</strong> breaths (most use three or five)<br />
(Anlich, Busch et al. 1998; Baraldi, Dario et al. 1999: Ratjen, Kavuk et al' 2000; Artlich'<br />
Jonsson et al. 2001; Franklin, Turner et al. 2o04; Franklin, Turner et al' 2004; Moeller'<br />
Franklin er al. 2004; Gabriele, Nieuwh<strong>of</strong> et al. 2006). Results have been reported as peak,<br />
average levels and the avengecalculated from the area under the curve during expiration'<br />
253
Almost all studies used sedation, with only a couple stating that they had not (Artlich, Busch<br />
et al. 1998; Hall, Reinmann et al.200.2) and there was one study that had infants and young<br />
children awake and in their parents' lap @araldi, Dario et al. 1999). One group investigated<br />
whether sedation itself made a difference to the level <strong>of</strong> exhaled NO comparing results <strong>of</strong> 29<br />
infants prior and subsequent to administering 60-100mg/kg <strong>of</strong> chloral hydrate. <strong>The</strong> mean<br />
exhaled NO <strong>of</strong> 2l.4ppb when awake dropped significantly to l4.6ppb when asleep, associated<br />
with an intra-subject co-efficient <strong>of</strong> variation <strong>of</strong> 20Vo when awake versus l0%o while sedated.<br />
<strong>The</strong> authors suggested this may be explained by variation in expiratory flow, contamination<br />
with ambient NO, breath-holding and crying, and <strong>of</strong>ten difficulty maintaining an appropriate<br />
seal <strong>of</strong> the mask around an agitated infant. <strong>The</strong>y were ultimately successful in getting readings<br />
from29 <strong>of</strong> 39 infants when they were awake (but only 1l were 'co-operative'), compared to<br />
achieving measurements in all the infants when sedated (Franklin, Turner et al. 2004).<br />
Another study in 2O healthy non-sedated infants showed the intra-subject variability from<br />
different phases <strong>of</strong> the exhalation ranged from 9.3 to l5.l%o with the inter-subject variation<br />
much greater, ranging from 34.6 to 44.67o (Hall, Reinmann et al.2(N2).<br />
Infants were shown to have a similar pattern <strong>of</strong> levels <strong>of</strong> exhaled NO to that seen in older<br />
children and adults; rising to a peak with a rapid plateau. A linear relationship between NO<br />
output and plateau was also demonstrated (Martinez, Weist et al. 2003). One study<br />
investigated whether there was a difference between nasal and oral NO levels in 23 infants.<br />
Most studies use a mask covering both mouth and nose and it is likely, given that infants tend<br />
to preferentially breathe through their nose, that nasal NO may contribute significantly to<br />
overall readings. <strong>The</strong> nasal tidal expiratory breaths were measured by continuing to place a<br />
mask over both mouth and nose but ensuring that the mouth was firmly shut (not stated how)<br />
and the oral tidal breaths were collected by blocking the nose with the rim <strong>of</strong> the mask. This<br />
resulted in no significant difference between nose and mouth levels, which is different from<br />
all other ages (Franklin, Turner et al.2N4). It may be that the method <strong>of</strong> exclusion <strong>of</strong> either<br />
the oral or nasal exhalate was inadequate and/or that there was no effect from the presence <strong>of</strong><br />
sinuses in this age group to add to the usual higher nasal measurements.<br />
<strong>The</strong>re have been comparisons between the single breath and tidal breathing NO results.In 32<br />
infants with recurrent wheeze, 16 with recurrent cough and 23 healthy infants a moderate<br />
correlation <strong>of</strong> 0.6 was demonstrated between the two methods but with poor agreement<br />
(Franklin, Turner et al.2004). This goup also noted that the single breath technique appeared<br />
to be more sensitive to detect differences between infants with and without respiratory<br />
symptoms (Franklin, Turner et aI.2004). In another study <strong>of</strong> 20 infants, the tidal exhaled NO<br />
254
washout characteristics were noted to vary from breath to breath (Hall, Reinmann et al.2002)'<br />
This group then divided the exhalation to assess whether one part <strong>of</strong> the expiratory signal<br />
exhibited less intra-subject variability and therefore could potentially be the most<br />
discriminatory. Firstly, based on the exhalation pattern <strong>of</strong> COz, they looked at the NO levels<br />
which corresponded to phase one (from the convective airways), phase two (progressive<br />
washout <strong>of</strong> the airways with alveolar gas) and phase three (alveolar compartment when<br />
exhaled COz is high). Secondly, they divided the tidal volume into four quarters <strong>of</strong> exhalation<br />
time. <strong>The</strong> most variable periods were phase one, and the first and fourth quarters'<br />
Correspondingly, the second and third quarter time periods had about the same variability as<br />
phase two, which was the best, followed by phase three. <strong>The</strong> group concluded that in the<br />
absence <strong>of</strong> COz measurement as a guide, the reported result should come from the second and<br />
third quarter <strong>of</strong> the exhalation (Hall, Reinmann et al' 2002)'<br />
Similar to studies in other age groups, flow dependency <strong>of</strong> exhaled NO levels have been<br />
demonstrated in infants by both methods. This was demonstrated in the original single breath<br />
study comparing a second flow <strong>of</strong> 100mls/s as well as the standard <strong>of</strong> 5omls/s in three<br />
subjects showing a significant difference in NO results (Wildhaber, Hall et al' 1999)'<br />
Similarly, low to higher NO levels were obtained at expiratory flows <strong>of</strong> 50, 25 and l5mls/s in<br />
five full term healthy infants (Martinez, Weist et al. 2003). Breath-by-breath analysis for tidal<br />
breathing also showed that higher expiratory flow rates were associated with lower exhaled<br />
NO levels (Franklin, Turner et a:.2004). In a large prospective healthy birth cohort <strong>of</strong> 98<br />
infants the tidal breathing parameters <strong>of</strong> flow, breathing rate and expiratory time, measured at<br />
age one month, all led to varying exhaled NO results. This group also came to the conclusion<br />
that the third expiratory quartile <strong>of</strong> exhaled NO during tidal breathing was the most<br />
reproducible (Frey, Kuehni et al.2004). In a direct comparison in 7l infants, the peak flow<br />
was 128mls/s during tidal breathing and llmls/s during single breath measurements<br />
(Franklin, Turner et al. 2004). This may also explain why single breath results versus tidal<br />
breathing results usually have higher absolute exhaled No levels.<br />
Other parameters have been seen to affect NO levels in infancy. Measurements in twelve<br />
infants showed that ambient NO concentrations as low as 5-10 ppb compared to the inhalation<br />
<strong>of</strong> NO free air resulted in an increase exhaled level (Franklin, Turner et al. 2004).In the 98<br />
infants at one month, a gender difference, with boys having higher levels at 17 '7ppb than girls<br />
at 14.6ppb, was noted. This remained significant after adjusting for weight and minute<br />
ventilation (Frey, Kuehni et al.2004). In thirteen preterm infants the exhaled NO was almost<br />
absent and gradually increased over the first 48 hours, while in eleven term infants there was a<br />
255
peak exhaled NO production in the first hours <strong>of</strong> life (Colnaghi, Condo et al. 2003). In a series<br />
<strong>of</strong> measurements <strong>of</strong> three premature infants while the absolute values were considerably<br />
lower than older children, when corrected for the body weight it appeared comparable<br />
(Artlich, Busch et al. 1998).<br />
9.17.2 l*vels <strong>of</strong> nitic oxide in infants with diffirent respiratory diseases<br />
Levels <strong>of</strong> exhaled NO were higher in wheezy infants compared with healthy controls (Baraldi,<br />
Dario et al. 1999; Wildhaber, Hall et al. 1999; Franklin, Turner et al. 2004), as well as<br />
compared to infants with a current URTI (Baraldi, Dario et al. 1999) and compared to infants<br />
with cough (Franklin, Turner et aI.2ffi4). Having eczema also resulted in higher exhaled NO<br />
levels in a group 88 infants in one study (Franklin, Turner et al.2O04) and 43 in the second<br />
study @inakar, Craff et al.2006). One study in six infants with URTI found higher levels <strong>of</strong><br />
NO than healthy infants @araldi, Dario et al. 1999). However, two others found lower levels;<br />
one measuing I7 infants with an URTI by nasal and oral and end tidal oral measurements<br />
@atjen, Kavuk et al. 2000) and the second measuring 24 infants presenting with rhinorrhoea<br />
with or without cough but not wheeze (Franklin, Turner et al. 2005). In eight <strong>of</strong> the infants<br />
who had rhinorrhoea, repeated tests showed an increase from 7.5ppb to 34.1ppb when they<br />
were symptom free (Franklin, Turner et d. 2005). Recently a large cross sectional study <strong>of</strong><br />
218 infants, including a number with different respiratory diseases and healthy controls, had<br />
NO measured between 4.6 and 25.2 months <strong>of</strong> age. Higher levels were seen in 74 infants with<br />
recurrent wheeze at l8.6ppb compared to 100 controls at l0.4ppb and 20 infants with<br />
bronchopulmonary dysplasia at similar levels to controls with a mean <strong>of</strong> ll.8ppb, while 20<br />
infants with CF had significantly lower levels at 5.9ppb (Gabriele, Nieuwh<strong>of</strong> et al. 2006).<br />
Different results were found in infants with chronic lung disease in another study (I*ipala,<br />
Williams et al.2004). Ten infants born at a median gestational age <strong>of</strong> 26 weeks and who had<br />
developed chronic lung disease had higher peak nasal and face mask NO levels than ten term<br />
infants and ten premature infants born at a median gestational age <strong>of</strong> 32 weeks without<br />
chronic lung disease. Those with chronic lung disease were not receiving diuretics, inhaled or<br />
systemic corticosteroids and bronchodilators or antibiotics at the time <strong>of</strong> measurement. All<br />
infants were measured at a post conceptual age between 36 and 45 weeks. This meant the<br />
infants with chronic lung disease were studied at an older post natal age at 85 days compared<br />
to two days for the term grcup and32 days for the non-chronic lung disease premature goup.<br />
<strong>The</strong>re was, however, a wide range <strong>of</strong> NO levels in each group (Iripala, Williams et al. 2004).<br />
256
g.17.3 Prenatal and maternal fficts on nitric oxide levels<br />
In one already previously mentioned study which measured 98 infants at age one month'<br />
exhaled NO levels were no different between infants from mothers with either asthma, other<br />
atopic diseases or without atopic disease (Frey, Kuehni et aL.2004). <strong>The</strong>se results differ from<br />
another study where a family history <strong>of</strong> atopy gave a mean NO level <strong>of</strong> 41.lppb if it was both<br />
parents, 24.2ppb if it was either parent and was lowest at 10'8ppb if it was neither parent<br />
(wildhaber, Hall et al. 1999). What did have an effect in the Frey study was maternal<br />
smoking or c<strong>of</strong>fee consumption during pregnancy, both resulting in significantly decreasing<br />
exhaled NO levels (Frey, Kuehni et al. 2004). Similarly' seven infants exposed to prenatal<br />
cigarette smoke had lower exhaled NO than thirteen unexposed infants when measured<br />
between 25 and58 days <strong>of</strong> age (Hall, Reinmann etal.2002).<br />
g.17.4 Efficts <strong>of</strong> teatment on nitric oxide levels<br />
Few studies have looked at levels <strong>of</strong> NO in response to treatment in this age group' Thirty one<br />
infants aged 6-19 months with recurrent wheeze and raised exhaled NO determined by <strong>of</strong>f-<br />
line tidal breathing were treated with inhaled fluticasone 50pg twice per day or placebo for<br />
four weeks. <strong>The</strong>re was substantial reduction in the level <strong>of</strong> exhaled NO from 35ppb to<br />
16.5ppb with only a small increase in FEV0.5 and no difference in the symptom scores<br />
between groups (Moeller, Franklin et al.20O4). Similarly in 15 infants with recurrent wheeze<br />
and high levels <strong>of</strong> exhaled No, this reduced by 527o after five days <strong>of</strong> prednisone becoming<br />
no different to No levels <strong>of</strong> the control children (Baraldi, Dario et al. 1999).In 24 children<br />
aged 10-26months with wheeze, allergy and a positive family history <strong>of</strong> asthma, montelukast<br />
4mg per day for four weeks resulted in a significant decrease in levels <strong>of</strong> exhaled No from<br />
29.8ppb to 19.0ppb and this did correspond to improvements in FEV0.5 and symptom scores<br />
with no changes in the placebo group (Straub, Moeller et al. 2005). Finally in eight ventilated<br />
infants with a mean gestational age <strong>of</strong> 25.8 weeks and post natal age <strong>of</strong> 55 days' exhaled NO<br />
levels reduced from a mean <strong>of</strong> 6.5ppb to 4.2ppb co-inciding with a reduction 62 to 45Eo in<br />
supplemental oxygen requirements with three days <strong>of</strong> dexamethasone treatment (Williams'<br />
Bhat et al.20o4).<br />
g.17.5 Summary <strong>of</strong> nitric oxide ftndings in infants<br />
In summary, exhaled NO can be measured in infants by both single and tidal breathing<br />
methods incorporating online and reservoir techniques. <strong>The</strong> advantages <strong>of</strong> the single breath<br />
technique are, being able to use an expiratory pressure to standardise the desired expiratory<br />
257
flow and to minimize nasal contamination. <strong>The</strong> tidal breathing method is less invasive and a<br />
simpler collection method but it is not always possible to control flow or nasal NO<br />
contamination. <strong>The</strong> most reproducible recordings have come from the second and third<br />
exhalation phases, and with use <strong>of</strong> sedation in the infants. <strong>The</strong> results so far have<br />
demonstrated a difference with gender, as suggested in some studies <strong>of</strong> older age groups, and<br />
confirmed the effect <strong>of</strong> expiratory flow on NO. Antenatal effects include a decrease in infants<br />
<strong>of</strong> smoking or caffeine ingesting mothers, though the effect <strong>of</strong> maternal (and patemal) atopy<br />
has been either nil or resulted in an increased levels <strong>of</strong> NO. Higher levels <strong>of</strong> NO were<br />
demonstrated in wheezy infants although the relation between this and those who develop<br />
asthma versus those who have viral induced wheeze <strong>of</strong> infancy which is likely to disappear<br />
between three and six years is not yet determined. <strong>The</strong> effect <strong>of</strong> a viral upper respiratory tract<br />
infection is less consistent on NO levels. As with infant lung function, the measurement <strong>of</strong><br />
NO in this age group remains the premise <strong>of</strong> research groups and is not widely used in the<br />
clinical arena.<br />
9.18 Chapter summarv<br />
Following early experiments throughout the 1990s, it was recognised that standardisation <strong>of</strong><br />
the method <strong>of</strong> measuring NO was required to allow better interpretation <strong>of</strong> results and<br />
comparison between research groups. <strong>The</strong>se began with a meeting in Stockholm in 1996 and<br />
from 1997 to 2005 four documents were generated involving the ERS and the ATS detailing<br />
standard procedures and then updating and refining these procedures as more data became<br />
available (Kharitonov, Alving et al. 1997; American Thoracic Society and Association. 1999;<br />
Baraldi, de Jongste et al.20O2l American Thoracic Society and European Respiratory Society<br />
2005). What became increasingly apparent from very early in the research was the importance<br />
<strong>of</strong> getting consistent and reproducible results. <strong>The</strong> most important factors to control were<br />
expiratory flow and nasal contamination. Standard practices for single, tidal breathing,<br />
reservoir collections and nasal measurements have been developed for both adults and<br />
children. In addition, the use <strong>of</strong> different expiratory flows to assess NO from different lung<br />
compartments has also been explored. This is important when wanting to demonstrate<br />
inflammation in certain compartments in certain diseases, for example the airway<br />
compartment in asthma and the alveolar compartment in interstitial lung diseases. In the latter<br />
type <strong>of</strong> diseases, this technique should always be employed in the future to enable meaningful<br />
results to be obtained.<br />
258
Physiological variables also affect the results. Exhaled NO levels have been shown to be<br />
higher in males than females, are reduced with cunent Smoking, recent ingestion <strong>of</strong> water'<br />
alcohol or caffeine and increased with accidental gut contamination or after nitrogen-<br />
compound rich meals. Levels <strong>of</strong> NO also increase with age throughout childhood' but not<br />
adulthood, possibly in relation to pneumatisation <strong>of</strong> the sinuses. Relationships with height,<br />
weight, surface area, menstrual cycle in non-asthmatic women' or a circadian rhythm has not<br />
been demonstrated. However, there remains only one range <strong>of</strong> 'normal' values given for oral<br />
or nasal NO.<br />
By far the most work in exhaled and nasal NO has been undertaken in atopic and/or asthmatic<br />
subjects with comparison to healthy controls. NO appears to correlate well with the<br />
inflammatory cells, lung function and bronchial hyper-responsiveness in steroid naive<br />
asthmatics and less well in those on IHCS or oral steroids. It does not correlate well with<br />
.severity, <strong>of</strong> asthma as the NO result here is attenuated by treatment, and it is the requirement<br />
for the higher levels <strong>of</strong> treatment with possible breakthrough symptoms that determine the<br />
severity category in asthma guidelines(Anonymous 1997 GINA 20O2; GINA 2005; SIGN<br />
2005). <strong>The</strong>re is the potential for No measurements to contribute to diagnosis, management<br />
when measured longitudinally in individuals (on any medication), and to wafii <strong>of</strong> loss <strong>of</strong><br />
Control. NO measurement can also be used as an outcome measure for ant-inflammatory<br />
agents used in asthma treatment. However there are some cautions to be made. Firstly, it does<br />
not appear useful to guide acute asthma treatment. Secondly, measurement <strong>of</strong> sputum<br />
eosinophils appear as, or more, accurate than NO itself, and the changes in the eosinophil<br />
counts may be a more accurate predictor <strong>of</strong> loss <strong>of</strong> control and a better target to tailor<br />
treatment. It is increasingly apparent that, like most parameters we measure' it is likely to be<br />
the pattern from a number <strong>of</strong> investigations along with an individual's history that best<br />
describes their clinical disease rather than a single marker' <strong>The</strong> effect <strong>of</strong> atopy alone on<br />
exhaled NO levels needs to be kept in mind with overlapping results between asthmatic and<br />
atopic subjects. Unfortunately, smoking renders the test inaccurate and unhelpful' In addition'<br />
the presence <strong>of</strong> high NO levels may be a marker for respiratory diseases that are steroid<br />
sensitive no matter what the underlying disease is labelled.<br />
In exercise, NO drops during single breath measurements, but the NO output is increased<br />
when taking into account the increased rate <strong>of</strong> breathing. An inability to increase the NO<br />
output during exercise was associated with more severe cardiac disease and, in elite athletes'<br />
worse performance suggesting that the increased NO output may depend on the ability to<br />
259
ecruit blood vessels in the lung. Studies have also suggested that a normal NO reading at the<br />
beginning <strong>of</strong> exercise predicted that no exercise-induced bronchospasm occurred.<br />
Low nasal NO appears to be helpful in screening for PCD and possibly CF. It is particularly<br />
important to measure this in children with established bronchiectasis and detecting a low NO<br />
level early in assessment for recurrent respiratory illnesses would be additionally beneficial<br />
and would indicate that these diseases must be investigated and excluded. <strong>The</strong> low NO levels<br />
seen in PCD remain unexplained, while those in CF and other conditions such as sinusitis<br />
would appear to be secondary to sinus obstruction. Oral NO levels are also low in PCD, but<br />
the nasal NO measurements (using a cut point <strong>of</strong> 250ppb) have minimal overlap with normal<br />
children. Oral NO levels become low in CF with age and disease progression, probably with<br />
entrapment <strong>of</strong> NO within thick mucus and therefore likely to interact with other compounds,<br />
so the NO does not reach the air to be exhaled. NO levels are increased in acute infections in<br />
both normal subjects and those with COPD, and increase during times <strong>of</strong> higher pollution and<br />
also associated with respiratory symptoms. However 'smoking' remains a confounding factor<br />
here -<br />
again rendering the measurement <strong>of</strong> NO as useless. In interstitial lung disease, levels <strong>of</strong><br />
NO co-relate with severity, in particular that assessed by the diffusion co-efficient, but only<br />
when measured in the alveolar compartment. <strong>The</strong> measurement <strong>of</strong> NO has been undertaken in<br />
infants and here while the parameters are still being worked through, it appears that the results<br />
are already low in those with PCD and CF, and high in children with wheeze, though it is still<br />
to be clarified whether this will result in detecting asthma versus infant viral associated<br />
wheeze, and whether it matters, as it may just detect disease that will respond to steroids.<br />
<strong>The</strong> advantages <strong>of</strong> using NO measurement rather than (or in addition to) other parameters is<br />
obvious. It is much easier to perform, especially in children, than lung function testing or<br />
induced sputum for eosinophils or any <strong>of</strong> the blood measurements such as total IgE. In<br />
addition it is quicker, with immediate results, and potentially can be done away <strong>of</strong>f hospital<br />
site. While it still needs an understanding <strong>of</strong> the test and qualified personnel, it can be taught<br />
and undertaken more easily than the other investigations.<br />
So where do I see it as cunently being MOST useful:<br />
l. Nasal (and oral) measurement as a screen for PCD. If nasal levels are low - ensure<br />
investigation for PCD and CF are carried out. While we do have a newborn screening<br />
for CF here in New Zealand,87o <strong>of</strong> children are not detected.<br />
2. As confirmation <strong>of</strong> asthma in steroid natve patients - when NO levels should be high.<br />
260
3. As a marker to follow when commencing treatment, in essence IHCS in steroid naive<br />
asthmatics (in non-smokers).<br />
4. As a marker to follow longitudinally, particularly in poorly controlled asthmatics<br />
contributing to assessing disease control, and response to changes in treatment (in<br />
non-smokers).<br />
5. It may become a method to assess the need for an exercise test in patients presenting<br />
with a complaint <strong>of</strong> possible exercise induced bronchospasm - if No levels are normal<br />
then an exercise test may not be required and investigations should proceed down<br />
another avenue (in non-smokers).<br />
<strong>The</strong> final, and brief, chapter will review where the research that I undertook contributed to<br />
both the clarity and confusion <strong>of</strong> early knowledge in this area. I will review what I learnt from<br />
the process <strong>of</strong> doing this research, writing up this thesis, and how this information has<br />
ultimately altered my research and clinical practice'<br />
26r
Chapter 10: Reflections<br />
<strong>The</strong> outcome <strong>of</strong> any serious research can only be to make two questions grow where<br />
only one grew before. (Torstein Veblin 1857-1929)<br />
By the time I had finished the research presented in this thesis, I felt that in attempting to<br />
answer a few questions - about the exact nature <strong>of</strong> exhaled NO and what made a difference to<br />
the levels obtained - I now had far more questions than when I began. It is hard to<br />
comprehend the rapid explosion regarding NO from the first discovery <strong>of</strong> its physiological<br />
role in 1987. Now the vital roles it occupies as a widespread mediator to maintain<br />
homeostasis and in host defence seems to be a long-known fact. <strong>The</strong> Medline searches reveal<br />
the rapid growth <strong>of</strong> research in this area in the 1990s and 2000s with studies conducted<br />
involving every organ system in every conceivable subject population (see Figure 10.1).<br />
When I started with the investigations presented in these chapters, very little was known about<br />
its role. Having acknowledged this, the measurement <strong>of</strong> exhaled NO is still not part <strong>of</strong> routine<br />
clinical practice and the exact use <strong>of</strong> this technology and its place in clinical medicine<br />
continues to be investigated today. In the thesis I have not touched on the measures <strong>of</strong> gases<br />
in exhaled breath condensate. This is an even more recent rapidly expanding area <strong>of</strong> research<br />
worthy <strong>of</strong> its own chapters by those involved in investigating its use.<br />
Figure 10.1: Medline publications with a focus on nitric oxide research per year 1980-2006<br />
16000<br />
14000<br />
o<br />
5 12ooo<br />
.g - loooo<br />
.=6<br />
E g. Sooo<br />
o.L<br />
E t 6000<br />
L<br />
.g 4ooo<br />
5 I<br />
2oqr<br />
z<br />
0 ,-alIl<br />
1980 1983 1986 1989 1992 199s 1998 2001 2004<br />
When I first cornmenced research, it was as a means to an end. By the end <strong>of</strong> the 1990s, I<br />
knew that I wanted to train as a paediatric respiratory specialist given the burden <strong>of</strong> disease<br />
seen in children in the UK where I was currently working, and, perhaps more particularly, at<br />
I<br />
Years 1980-2006<br />
262
home in New 7*aland.In order to do that I aimed to get a senior registrar / clinical lecturer<br />
post at a centre <strong>of</strong> excellence which for me, working in London at time, was the Royal<br />
Brompton Hospital. I decided that research would definitely be a pre-requisite to this<br />
appointment and so successfully obtained a research fellow job at the same hospital'<br />
However, having started down a research path, I can no longer envisage not having research<br />
as a major part <strong>of</strong> my role. Research involves the development <strong>of</strong> different skills to those<br />
required in clinical medicine. At the time, I enjoyed considering in depth and reading widely<br />
around one aspect <strong>of</strong> medicine. But, when I was asked by one scientist whether I used<br />
,positive, or .negative' pipetting when I did enzyme-linked immunosorbent assays (ELISAS)<br />
studies, I knew that I was in a different world. It teaches you attention to detail' the need to be<br />
very methodical, the need to be sceptical and the need to question every factor' It also allows<br />
an opportunity to work with people who are experts in different areas. From the scientists I<br />
learnt patience, information technology skills, statistics and careful laboratory bench-work' I<br />
also appreciated the very different knowledge and skills that Carolyn Busst as a medical and<br />
electrical engineer brought to the research. <strong>The</strong> topics <strong>of</strong> research and critical reviews were<br />
not previously emphasised but now occupy a central theme in current medical school training'<br />
and I consider this important in developing questioning practitioners'<br />
Of course, now I would do everything very differently. One problem with research' as the<br />
quotation above suggests, is that no matter what topic you start with, it can expand<br />
exponentially. If commencing this research now, I would restrict the work to investigating<br />
exhaled NO and the long-actingp2 agonist ('salmeterol') trial that I was also conducting<br />
during this time. In addition,I was undertaking clinical training with on-call commitment in a<br />
paediatric cardiorespiratory intensive care setting' I would not' for example' have also<br />
embarked on learning induced sputum techniques and ELISAs for processing IL8 and TNFg'<br />
This latter research ultimately suffered for which I feel responsible. From the beginning I<br />
found the children, particularly healthy controls, did not like the use <strong>of</strong> hypertonic saline for<br />
sputum induction. This needed much greater attention and a full time researcher on this alone<br />
to determine a satisfactory technique. However, by this time I had established the machines'<br />
connections and protocols and was running with the exhaled NO research, and I was enjoying<br />
it. Also, in comparison, the adult respiratory service had four research fellows dedicated to<br />
investigating these areas <strong>of</strong> medicine which was a great deal more realistic. On the other hand,<br />
I would now also make the most <strong>of</strong> any overlappin g area <strong>of</strong> research to combine<br />
investigations. For example, at the time I was the principal investigator in a cross-over trial<br />
involving two dosage regimes <strong>of</strong> salmeterol compared to one dosage regime <strong>of</strong> salbutamol<br />
263
and I enrolled 52 asthmatic children. At the time there was a question as to whether the long<br />
acting p2 agonist therapy had any anti-inflammatory effects. I was well placed to answer this<br />
if I had measured the exhaled NO in these children, and/or connected them with an 'induced<br />
sputum' researcher.<br />
My experience in research has, I hopr, assisted me in the role <strong>of</strong> supervisor <strong>of</strong> other<br />
researchers. I have learnt from my supervisors and from my own successes and failures. I<br />
ensure regular contact no matter what is happening, and I limit the focus <strong>of</strong> the research. Early<br />
enthusiasm <strong>of</strong>ten encourages taking on too big a project and then feeling ovenvhelmed or that<br />
justice is not being done to every possible investigative avenue. I also returned to a one in two<br />
on-call roster providing paediatric respiratory cover regionally and nationally. Again my 'take<br />
home' message quite literally is to be certain to factor in time for writing. I believe important<br />
contributors to success in research are attention to detail, regular review, maintaining focus,<br />
and a realistic appreciation <strong>of</strong> the time required.<br />
Since returning to New 7*aland my own clinical and research focus has shifted to the area I<br />
am concerned which is a main burden <strong>of</strong> respiratory disease in New 7*,aland children -<br />
infection and in particular the development <strong>of</strong> chronic suppurative lung disease resulting in<br />
early morbidity and ultimately early adult mortality. My next endeavours are to continue to<br />
develop a good management programme and research into aetiology, appropriate intervention<br />
and strategies for prevention in this area <strong>of</strong> lung disease. While I also steadfastly remain a<br />
clinician, I could not stop doing research now. I thank the Royal Brompton Hospital and Dr<br />
Andy Bush in particular for opening up this path for me.<br />
2&
Appendix I : Publications<br />
Published manuscripts related to this thesis:<br />
Appendices<br />
o ,.Trying to catch our breath" <strong>The</strong> burden <strong>of</strong> preventable breathing diseases in children and<br />
young people. Editors Innes Asher and Cass Byrnes. Asthma and Respiratory Foundation<br />
<strong>of</strong> New 7-ealand. Te Taumatua Huango, Mate Ha o Aoteroa' www'asthmanz'co'nz and<br />
www.paedi atrics.org.nz.<br />
C A Byrnes: Chapter 9: <strong>The</strong> Burden <strong>of</strong> Bronchiolitis in New Zealand (40-45)'<br />
c A Byrnes: Chapter I 1: <strong>The</strong> Burden <strong>of</strong> Bronchiectasis in New T.r,aland (51-55)'<br />
o Byrnes cA, Dinarevic s, shinebourne EA, Barnes PJ, Bush A. I-evels Of nitric oxide in<br />
exhaled air in normal and asthmatic children. Pediatric Pulmonolo gy 1997 24 (5) 312-318'<br />
o Byrnes CA, Dinarevic S, Busst CA, Shinebourne EA, Bush A' Effect <strong>of</strong> measurement<br />
conditions on measured levels <strong>of</strong> peak exhaled nitric oxide. Thorax 1997 52 (8) 697-701'<br />
o Byrnes cA, Dinarevic S, Busst cA, Bush A, Shinebournes EA. Is nitric oxide produced<br />
at airway or alveolar level? European Respiratory Journal 1997 l0 (5) 1021-1025'<br />
o Dinarevic s, Byrnes cA, Shinebourne EA, Bush A. Measurement <strong>of</strong> exhaled nitric oxide<br />
in children. Pediatric Pulmonolo gy 1996; 22: 396-40L'<br />
o Byrnes CA, Bush A and shinebourne EA "Methods to Measure Expiratory Nitric oxide<br />
in People" in "A Volume <strong>of</strong> Methods in Enzymology" edited by I-ester packeter<br />
Academic Press Inc. 1996.<br />
Publications based on research that has progressed from this thesis:<br />
o phD thesis: .Nitric oxide and ciliary beat in normal and bronchiectatic children'' Dr<br />
Elizabeth Edwards awarded 2004 (2001-2004)'<br />
Prinicipal supervisor: Dr Cass Byrnes'<br />
o Edwards EA, Douglas C, Broome S, Kolbe J, Jensen CG, Dewar A, Bush A' Byrnes CA'<br />
Nitric oxide levels and ciliary beat frequency in indigenous New Zealand children'<br />
Pediatric Pulmonolo gy 2005 ; 39: 238-246'<br />
Presentations and published abstracts from this research and related to this research:<br />
o Edwards EA, Douglas c, Broome s, Ferguson w, Kolbe J, Byrnes cA' Exhaled nitric<br />
oxide levels in children with non-cystic fibrosis bronchiectasis in New Tnaland' European<br />
Respiratory Meeting & European Respiratory Journal 2002;38: 338s'<br />
265
Edwards EA, Douglas C, Broome S, Jensen C, Byrnes CA. Exhaled nitric oxide (NO) and<br />
ciliary beat frequency (CBF) in normal and bronchiectatic New Zealand Children.<br />
American Thoracic Meeting & American Journal <strong>of</strong> Respiratory & Critical Care Medicine<br />
200r;163 (5): 4854.<br />
Edwards EA, Douglas C, Broome S, Jensen C, Byrnes CA. Exhaled nitric oxide (NO) and<br />
ciliary beat frequency (CBF) in normal and bronchiectatic New Zealand Children. New<br />
TnalandPaediatric Society Meeting 2000. Young investigators award.<br />
Edwards EA, Douglas C, Broome S, Jensen C, Byrnes CA. Exhaled nitric oxide (NO) and<br />
ciliary beat frequency (CBF) in normal and bronchiectatic children. New 7*aland<br />
Thoracic Society 2000. Young investigators award.<br />
Measurements <strong>of</strong> exhaled nitric oxide in children - Australiasian Paediatric Respiratory<br />
Group 1999 -<br />
invited presentation.<br />
Byrnes. CA. Exhaled nitric oxide. National New Zealand Respiratory Meeting 1998 -<br />
invited presentation.<br />
Byrnes CA, Dinarevic S, Shinbourne EA, Bush A. Exhaled nitric oxide in normal and<br />
asthmatic children - British Paediatric Society - 1996.<br />
Other Publications:<br />
Byrnes C. Non cystic fibrosis bronchiectasis. Paediatric Respiratory Reviews 2A06;7<br />
suppl 1: 5255-5257.<br />
Twiss J, Stewart AW, Byrnes CA. Longitudinal pulmonary function <strong>of</strong> childhood<br />
bronchiectasis and comparison with cystic fibrosis. Thorax 2006;61(5): 414 - 418.<br />
Thomson JM, Wesley A, Byrnes CA, Nixon GM. Pulse intravenous methylprednisolone<br />
for resistant allergic bronchopulmonary aspergillosis in cystic fibrosis. Pediatric<br />
Pulmonolo gy 2006; 4l(2): 164-17 0.<br />
"Accreditation <strong>of</strong> Paediatric Respiratory Function Assessment Services" Dr C A Byrnes -<br />
by invitation Thoracic Society <strong>of</strong> Australia and New Zealand and Australasian Paediatric<br />
Respiratory Group. www.thoracic. org. au March 200,6.<br />
'Wheeze and Chest infections in children less than one year <strong>of</strong> age' - Co-chairs Dr Cass<br />
Byrnes, Dr Alison Vogel. Evidence based guideline funded by a Ministry <strong>of</strong> Health<br />
initiative. Guidelines endorsed by the Paediatric Society <strong>of</strong> New 7*aland, New Zealand<br />
Guidelines Group, the Royal New Zealand College <strong>of</strong> General Practitioners and the Royal<br />
Australasian College <strong>of</strong> Physicians. www.paediatrics.org.nz. April 2005.<br />
266
o Howie S, Voss L, Baker M, Calder L, Grimwood K, Byrnes C. Tuberculosis in New<br />
Tnaland, 1992-200I: a resurgence. Archives <strong>of</strong> Disease in Childhood 2005; 90: II57-<br />
1161.<br />
r Gillies J, Brown J, Byrnes C, Farell A, Graham D. PHARMAC and ventolin in New<br />
Tlaland. New Zealand Medical Journal August 2005; Ll8:122O.<br />
o Twiss J, Metcalfe R, Edwards E, Byrnes C. New Zealand national incidence <strong>of</strong><br />
bronchiectasis "too high" for a developed country. Archives <strong>of</strong> Disease in Childhood<br />
2005;90(7): 737-740.<br />
o Twiss J, Byrnes CA, Johnson R, Holland D. Nebulised gentamicin - suitable for<br />
childhood bronchiectasis. International Journal <strong>of</strong> Pharmaceutics 2OO5;295: ll3-119<br />
o Edwards EA, Douglas C, Broome S, Kolbe J, Jensen CG, Dewar A, Bush A, Byrnes CA.<br />
Nitric oxide levels and ciliary beat frequency in indigenous New Zealand children.<br />
Pediatric Pulmonolo gy 2005; 39: 238-246.<br />
r Edwards EA, Metcalfe R, Milne DG, Thompson J, Byrnes CA. Retrospective review <strong>of</strong><br />
children presenting with non cystic fibrosis bronchiectasis: HRCT features and clinical<br />
rel ationships. Pedi atric Pulmonolo gy 20O3 ; 36: 87 -93.<br />
o Edwards EA, Asher MI, Byrnes CA. Paediatric bronchiectasis in the 2l't century:<br />
experience <strong>of</strong> a tertiary childrens hospital in New T.rualand. Journal <strong>of</strong> Paediatric and Child<br />
Health 2003; 39: 1 I L-117 .<br />
o Vogel A, knnon D, Broadbent R, Byrnes CA, Grimwood K, Mildenhall L, Richardson<br />
V, Rowley S. Palvimuzab prophylaxis <strong>of</strong> respiratory syncytial virus infection in high risk<br />
infants. Journal <strong>of</strong> Paediatric and Child Health 2OO2;38: 550-554.<br />
o Edwards EA, Butler SA, Byrnes CA. Paediatric non cystic fibrosis bronchiectasis -<br />
recognition and management. New Ethicals Journal 2OO2; November: 58-65 (invited<br />
article).<br />
o Massie J, Poplawski N, Wilcken B, Goldblatt J, Byrnes C, Robertson C. Intron-8<br />
polythymidine sequence in Australasian individuals with CF mutations RllTH and<br />
Rll7C. European Respiratory Journal 2001 L7 1195-1200.<br />
o Byrnes CA, Shrewsbury S, Barnes PJ, Bush A. Salmeterol in Paediatric Asthma. Thorax<br />
2000; 55:780-784.<br />
o Edwards E, Byrnes CA. Humidification difficulties in two tracheostomised children,<br />
Journal <strong>of</strong> Anaesthetic and Intensive Care 1999; 27:656-658.<br />
267
o Cass Byrnes & Nicky Holt. "Drugs used in Cystic Fibrosis". Medicines for Children.<br />
British Paediatric Formulatory I't Edition, published by the Royal College <strong>of</strong> Paediatrics<br />
and Child Health 1999.<br />
Other presentations and published abstracts:<br />
International Meetings:<br />
o Byrnes CA. Pearls: Non-Cystic Fibrosis Bronchiectasis - 7fr International Congress on<br />
Pediatric Pulmonology, Montreal, Canada July 2006 - invited presentation.<br />
o Byrnes CA. Lung function accreditation guidelines - Australasian Paediatric Respiratory<br />
Group Annual Scientific Meeting 2004 - invited presentation.<br />
o Byrnes CA. Lung function and lung function accreditation guidelines - Australasian<br />
Paediatric Respiratory Group Annual Scientific Meeting 2W3 - invited presentation.<br />
o Bynes CA. Bronchiectasis - Australasian Paediatric Respiratory Group Annual Scientific<br />
Meeting 2OO2 -<br />
invited presentation.<br />
. Byrnes CA, Barnes PJ, Bush A. Salmeterol trial in asthmatic children - American<br />
Thoracic Society Annual Scientific Meeting 1999.<br />
National Meetings:<br />
o Byrnes CA. Australia and New 7*,aland Cystic Fibrosis Bronchoalveolar Lavage -<br />
National Respiratory Meeting 2W7 - invited presentation.<br />
o Byrnes CA. Paediatric workshop - Standards <strong>of</strong> Care in Cystic Fibrosis in New 7*aland -<br />
National Respiratory Meeting 2007- invited presentation.<br />
o Byrnes CA. Paediatric cases workshop - National Respiratory Meeting L999-2W6 -<br />
invited presentation.<br />
o Cheney JL, Carlin J, Cooper P, Byrnes C, Grimwood K, Armstrong D, Martin J, Dakin C,<br />
Robertson C, Whitehead B, Francis P, Vidmar S, Wainwright CE. Adverse events<br />
associated with flexible bronchoscopy and bronchoalveolar lavage in young children with<br />
cystic fibrosis. North American Cystic Fibrosis Conference 2006.<br />
r Wainwright CE, Carlin J, Cooper P, Byrnes C, Martin J, Grimwood K,, Armstrong D,<br />
Francis P, Dakin C, Whitehead B, Carter R, Vidmar S, Cheney J, Tiddens H, Fink M,<br />
Robertson C. Australasian Cystic Fibrosis BAL study analysis. North American Cystic<br />
Fibrosis Conference 200,6.<br />
o Byrnes CA. Pseudomonas aeruginosa -<br />
management <strong>of</strong> ltt infection in children with<br />
cystic fibrosis - National Respiratory Meeting 2003 - invited presentation.<br />
268
o Byrnes CA. Cystic Fibrosis Bronchoalveolar Lavage Study -<br />
National Cystic Fibrosis<br />
Seminar February 2003 - invited presentation.<br />
o Howie S, Voss L, Grimwood K, Byrnes CA. Tuberculosis disease in New 7*aland<br />
Children. American Thoracic Meeting & American Journal <strong>of</strong> Respiratory & Critical Care<br />
Medicine 2003;163 (5) :4854.<br />
o Twiss J, Byrnes CA. Paediatric bronchiectasis: longitudinal lung function and comparison<br />
with cystic fibrosis. New Zealand Paediatric Society Annual Conference 2003. Highly<br />
commended award.<br />
o Twiss J, Byrnes CA. Longitudinal lung function. Thoracic Society <strong>of</strong> Australia & New<br />
Zealand2003.<br />
o Wainwright C, Cooper P, Carlin J, James M, Armstrong D, Robertson I, Cooper D,<br />
Whitehead B, Byrnes C, Francis P, Robertson C. Early infection with pseudomonas<br />
aeruginosa can be effectively cleared in young children with cystic fibrosis. Australian<br />
Cystic Fibrosis Conference 2003.<br />
o Byrnes CA. Asthma Medications - New Tnaland Paediatric Update 2002 - invited<br />
presentation.<br />
r Howie S, Voss L, Grimwood K, Byrnes CA. Tuberculosis disease in New Tr,aland<br />
Children. New Zraland Paediatric Society Annual Conference 2002.<br />
o Wainwright C, Carlin J, Cooper P, Byrnes C, Whitehead B, Martin J, Cooper D,<br />
Armstong D, Robertson C. Early infection with pseudomonas can be cleared in young<br />
children with cystic fibrosis - North American Cystic Fibrosis Meeting 2002 Pediatic<br />
Pulmonology Supplement 24: 357 .<br />
o Vogel AM. Moodabe K, Aish H, Campanella S, Byrnes CA. Implementation <strong>of</strong> guidelines<br />
for the management <strong>of</strong> cough and wheeze in the I't year <strong>of</strong> life in general practice. New<br />
Zealand Paedi atric Society Annual Meeting 200L.<br />
o Jaksic M, Asher MI, Byrnes CA. Streptokinase in the Management <strong>of</strong> Empyema.<br />
American Thoracic Meeting & American Journal <strong>of</strong> Respiratory & Critical Care Medicine<br />
2001; 163 (5): A854.<br />
e Edwards EA, Asher MI, Metcalfe R, Byrnes CA. Non-cystic bronchiectasis - correlation<br />
<strong>of</strong> high resolution CT scan and chest x-ray scores with clinical evaluation. American<br />
Thoracic Meeting & American Journal <strong>of</strong> Respiratory & Critical Care Medicine 2001; 163<br />
(5): A854.<br />
o Edwards EA. Byrnes CA. Temperature control is vital for the measurement <strong>of</strong> cilia beat<br />
frequency when establishing a hospital protocol. European Respiratory Meeting &<br />
European Respiratory Journal 2001;18: 300s.<br />
269
o Byrnes CA. Paediatric fibreoptic bronchoscopy - New Tnaland Paediatric Society Annual<br />
Meeting 2001.<br />
o Byrnes CA. Screening for respiratory ciliary dysfunction - New 7*,aland Paediatric<br />
Society Annual Meeting 200I.<br />
r Edwards EA, Metcalfe R, Asher MI, Byrnes CA. Bronchiectasis - Correlation <strong>of</strong> high<br />
resolution CT scan and chest x-ray scores with clinical evaluation in 52 children. New<br />
Tnaland Paedi atri c S oci ety Ann u al Conference 2000.<br />
o Edwards EA, Douglas C, Broome S, Jensen C, Byrnes CA. Exhaled nitric oxide (NO) and<br />
ciliary beat frequency (CBF) in normal and bronchiectatic children. New Zealand<br />
Thoracic Society 2000.<br />
o Edwards EA, Metcalfe R, Asher MI, Byrnes CA. Bronchiectasis - Correlation <strong>of</strong> high<br />
resolution CT scan and chest x-ray scores with clinical evaluation in 52 children. New<br />
7*aland Paediatric Society Annual Conference 2000.<br />
o Byrnes CA. Home oxygen in children - National Respiratory Meeting 2000 - invited<br />
presentation.<br />
o Byrnes CA. Infection control in children with cystic fibrosis - National NZ Respiratory<br />
Meeting 2000 - invited presentation.<br />
o Byrnes CA. Obstructive sleep apnea - New 7*aland Paediatric Update 2000 - invited<br />
presentation.<br />
o Byrnes CA. Management <strong>of</strong> pleural effusions - New 7-ealand Paediatric Update 2000 -<br />
invited presentation.<br />
o Byrnes CA, Barnes PJ, Bush A. Salmeterol trial in asthmatic children - American<br />
Thoracic Society Annual Scientific Meeting 1999.<br />
Abstracts and poster presentations:<br />
o McNamara DG, Byrnes CA, Rutland MD. Development <strong>of</strong> a technique to measure<br />
mucociliary clearance in children with tracheostomies. Thoracic Society <strong>of</strong> Australia &<br />
New Zealand Annual Scientific Meeting, May 2005.<br />
o McMorran B, James A, Armstrong D, Cooper P, Carlin J, Martin J, Robertson I, Cooper<br />
D, Whitehead B, Byrnes C, Francis P, Robertson C, Wainwright C, Wainwright B.<br />
Proteomic comparisons in cystic fibrosis lung lavage fluid using ProteinChip@<br />
technology. North American Annual Cystic Fibrosis Conference, October 2W3.<br />
r Butler SA, Twiss J, Byrnes CA. Bronchiectasis outreach - a new initiative in New<br />
7*aland. Fifth National Paediatric Physiotherapy Conference, Australasia, 2003.<br />
270
Edwards EA, Dewar A, Jensen C, Bush A, Byrnes CA. Ciliary abnormalities in New<br />
7*alandchildren with bronchiectasis. Cilia, Mucus and Mucociliary Group Meeting' USA<br />
2002.<br />
Edwards EA, Byrnes CA. Temperature control is vital for the measurement <strong>of</strong> cilia beat<br />
frequency when establishing a hospital protocol. European Respiratory Meeting &<br />
European Respiratory Journal 2001; 18: 300s'<br />
Edwards EA, Asher MI, Metcalfe R, Byrnes cA. Non-cystic bronchiectasis - correlation<br />
<strong>of</strong> high resolution CT scan and chest x-ray scores with clinical evaluation' American<br />
Thoracic Meeting & American Journal <strong>of</strong> Respiratory & Critical Care Medicine 2001; 163<br />
(5): A854.<br />
Jaksic M, Asher MI, Byrnes cA. Streptokinase in the Management <strong>of</strong> Empyema'<br />
American Thoracic Meeting & American Journal <strong>of</strong> Respiratory & Critical Care Medicine<br />
2001; 163 (5): A854.<br />
Current PhD suPervision:<br />
o .Bronchiectasis in children and young people in New Tnaland'' Dr Jacob Twiss'<br />
o .Effects <strong>of</strong> humidification on the airway in children with tracheostomies.' Dr David<br />
McNamara.<br />
27r
Appendix 2: Questionnaire for enrolling the children<br />
9C Ar.gr13r<br />
Itt JursrtRs ltrLt w nREllr@ .Irf aIRIcr @r:rDElfcE.<br />
trttrE <strong>of</strong> ciltd.<br />
Drte <strong>of</strong> bl,rth <strong>of</strong> child.<br />
ltacloaall,Cy... ,.......o. r.......<br />
w/drRl'<br />
IIas he/she lrad a col.d or chest infection in tfte lasc month? tE8./TO<br />
Ilas fie./she evet iad astlrCIa? tt lraercltc .tlr tlrrt dtrgraroct.. W9|TO<br />
Does Jre./slre have asthna non? YE$/XO<br />
Ilas ie./slra ever wheezed?(a& r r.trjrtllng touact oa Draetlrtng out) W9/tW<br />
ooes ielslre cough. . . lrgvER. . . sodBgr.tE$. . . om8F. . . coilSmilrur.<br />
Has he./she ever been prescribed 9entolin or BricanyJ?<br />
If lcrrxbcn?.. ...,<br />
Itas he/she ever had eczena?<br />
ffas helshe ersr had ftay fcver?<br />
Docs he/shc have eczena/hayfevet no*?<br />
Oocs he./s[e have any other cfiest cotplalnt? prc.lsa n rrc......<br />
Does anyone eJse jn the .faniy harre any <strong>of</strong> tie followittg:<br />
aschnd Y$S/XO if yes uho?,, a . . , ,<br />
ecsenr VES/NO lt yes who?.......<br />
iayfever ws/No tf yes nha?.<br />
tlrrr glvr r.Ltl'orrtlp tc tb cl.tll<br />
Do you have any pets? Jf pr plcrsc n nc cJar.<br />
Does anyone in the family snoke?.<br />
wslTo<br />
YE9/TO<br />
t:Es/fro<br />
t]3g/xO<br />
rc8/NO<br />
Note: the spelling error noted in the heading <strong>of</strong> these sheets was only noted after we had delivered<br />
most <strong>of</strong> the questionnaires to the participating school - in view <strong>of</strong> that and as this how most <strong>of</strong> the<br />
collected forms remain - | have left it in.<br />
272
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Bancalari, L., I. Conti, et al. (1999). 'E.tty tt-"^e in urinary leukotriel" E4 (LTB[) is<br />
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Allergy 54(12): 127 8-85.<br />
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