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Gordon, Robert L.,
A comparison of exhaled breath nitric oxide between old and young individuals
h [electronic resource] /
by Robert L. Gordon, Jr. M.D.
[Tampa, Fla.] :
University of South Florida,
Includes bibliographical references.
Text (Electronic thesis) in PDF format.
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ABSTRACT: BACKGROUND: Older individuals suffer from higher rates of pulmonary infections than younger individuals. In addition, older individuals have increased morbidity and mortality due to pulmonary infections when compared to younger individuals. The physiological and immunological reasons for these aforementioned differences are not clear. Recently, non-invasive markers of the lung's physiologic and immunologic status have been recognized. This study employs one of these non-invasive markers, exhaled nitric oxide, in an attempt to determine how the airways may change with age, predisposing older individuals to pulmonary diseases and poorer outcomes as compared to younger individuals. METHODS: Exhaled nitric oxide measurements were obtained from a group of 25 older subjects (61 to 79 years old, median 72 years old) and a group of 23 younger subjects (21 to 30 years old, median 24 years old) that were non-smokers with no history of pulmonary disease, no recent respiratory infections, and no history of environmental allergies. A focused history and physical exam along with spirometry were used to confirm the normal pulmonary status of each subject. Exhaled nitric oxide was measured following the American Thoracic Society recommendations using the Sievers Nitric Oxide Analyzer 280i. The exhaled nitric oxide values for the old and young groups were compared using the Wilcoxon Rank Sum test. RESULTS: For the older subjects, the median exhaled NO concentration was 36.9 ppb. For the younger subjects, the median exhaled NO concentration was 18.7 ppb. These exhaled NO concentrations are significantly different (p = 0.0011). CONCLUSIONS: The exhaled NO concentrations are significantly higher in older individuals than in younger individuals. The reasons for this difference along with the significance are unclear and further studies will be necessary to further evaluate these issues.
Adviser: Stuart M. Brooks
nitric oxide synthase.
noninvasive markers of airway inflammation.
x Public Health
t USF Electronic Theses and Dissertations.
A Comparison of Exhaled Breath Nit ric Oxide Between Old and Young Individuals by Robert L. Gordon, Jr., M.D. A thesis submitted in partial fulfillment of the requirement s for the degree of Master of Science in Public Health Department of Environmental and Occupational Health College of Public Health University of South Florida Major Professor: Stuart M. Brooks, M.D. Robert R. Haight, M.D., M.S.P.H. Philip P. Roets, Sc.D. Date of Approval: March 30, 2004 Keywords: chemiluminescence, nitric oxide synthase, aging, noninvasive markers of airway infl ammation, pulmonary infection Copyright 2004 Robert L. Gordon, Jr., M.D.
Dedication I would like to dedicate this work to my wife, Kim, who has encouraged me to strive for excellence.
Acknowledgements I would like to thank Dr. Robert Ha ight for the countless hours of assistance he provided to me during this project. I would also like to thank Dr. Stuart Brooks for providing me encouragem ent and inspiration while I worked on this project.
i Table of Contents List of Tables iii List of Figures iv Abstract v Introduction 1 Background 1 Airway Nitric Oxide 3 Biochemistry 3 Cellular and Anatomic Sources of Nitric Oxide Synthases 4 Function of Endogenous Nitric Oxide within the Lower 5 Respiratory Tract Clinical Significance of Nitric Oxide 7 Measurement of Nitric Oxide 9 Materials and Methods 12 Study Design 12 Measurement of Exhaled Nitric Oxide 13 Spirometry 16 Measurement of Exhaled Breat h Condensate pH 16 Statistical Analysis 18 Results 19 Baseline Characteristics of t he Study Populations 19 Spirometric Measurements 19 Exhaled Breath Condensate pH Measurements 21 Exhaled Nitric Oxide Measurements 21 Discussion 22 Conclusion 27 References 28
ii Appendices 34 Appendix A: Exhaled Breath Condens ate and Exhaled Breath 35 Nitric Oxide Study Questionnaire Appendix B: Age Distrib utions within Each Age Category 37 Appendix C: Age Category and Gender Distributions for Entire 39 Study Population Appendix D: Distributions of Characteristics and Measurements by 41 Age Category Appendix E: Analysis of NO and pH by Age Category 68
iii List of Tables Table 1. Factors Which Influence Exhaled Nitric Oxide 10 Table 2. Baseline Characteristics of Study Populations 20 Table 3. Spirometric Measurements of Study Populations 21 Table 4. Exhaled Breath Nitric Oxide and Exhaled Breath 21 Condensate Deaerated pH
iv List of Figures Figure 1. The Chemical Reactions Involved in the Chemiluminescence 11 Method of Measuring Exhaled Nitric Oxide Figure 2. Sievers 280i Nitric Oxide Analyzer with Mouthpiece 15 Figure 3. Performance of Exhaled Nit ric Oxide Maneuver 15 Figure 4. pHTube Used to Collect Br eath Condensate (Cooling Sleeve 17 Not Attached) Figure 5. Measurement of pH of Br eath Condensate While Undergoing 18 Deaeration with Argon Gas
v A Comparison of Exhaled Breath Nitri c Oxide Between Older and Younger Individuals Robert L. Gordon, Jr., M.D. ABSTRACT BACKGROUND: Older individuals suffer from higher rates of pulmonary infections than younger individuals. In addition, older individuals have increased morbidity and mortality due to pulmonary in fections when compared to younger individuals. The physiological an d immunological reasons for these aforementioned differences are not clear. Recently, non-invasive markers of the lungÂ’s physiologic and immunologic status have been recognized. This study employs one of these non-invasive markers, exhaled nitric oxide, in an attempt to determine how the airways may change with age, predisposing older individuals to pulmonary diseases and poorer outcomes as compared to younger individuals. METHODS: Exhaled nitric oxide measur ements were obtained from a group of 25 older subjects (61 to 79 years old, median 72 y ears old) and a group of 23 younger subjects (21 to 30 years old, median 24 y ears old) that were nonsmokers with no history of pulmonary diseas e, no recent respiratory infections, and no history of environmental allergies. A focused history and physical exam along with spirometry were used to conf irm the normal pulmonary status of each subject. Exhaled nitric oxide was meas ured following the Am erican Thoracic
vi Society recommendations using the Si evers Nitric Oxide Analyzer 280i. The exhaled nitric oxide values for the ol d and young groups were compared using the Wilcoxon Rank Sum test. RESULTS: For the older subjects, t he median exhaled NO concentration was 36.9 ppb. For the younger subjects, the median exhaled NO concentration was 18.7 ppb. These exhaled NO concentra tions are significantly different (p = 0.0011). CONCLUSIONS: The exhaled NO concentrati ons are significantly higher in older individuals than in younger individuals. T he reasons for this difference along with the significance are unclear and further studies will be necessary to further evaluate these issues.
1 Introduction Background Respiratory infections, particula rly pneumonia, are known to cause significant morbidity and mortality in the elderly population.1 2 The incidence of pneumonia has been shown to increase wit h age with incidences of 91.6/100,000 for individuals younger than 45 years of age, 277.2/100,000 for individuals aged 45 to 64 years, and 1012.3/100,000 for indi viduals older than 65 years in one study.3 Factors that are known to predispos e the elderly to pneumonia are vast and include: age greater than 65 years, underlying comorbid illnesses such as chronic obstructive pulmonary disease, diabetes mellitus, congestive heart failure, malignancy, poor nutrition, instituti onalization, recent hospitalization, and smoking.1, 4 It is evident, as individuals increa se in age, they acquire more of the aforementioned risk factors bringing into q uestion whether increasing age itself is an independent risk factor fo r developing pneumonia. Aging in itself has been shown to lead to physiological and immunological changes that may predispose the aged population to pneumonia and poorer outcomes once contracted. The physi ological changes include: impaired mucociliary transport, enhanced oropharyng eal colonization, increased aspiration potential, decreased ability to expec torate, decreased respiratory muscle
2 endurance, and decreased forced expiratory volume in one second (FEV1).1, 4 Although not fully elucidated, the i mmune system has been shown to undergo alterations as individuals age such that there is impaired immune surveillance, decreased ability to mount an appropriate immune response, and inappropriate immune responses.5 Exhaled respiratory markers have been recognized that correlate with infection and inflammation.6 These include nitric oxide (NO), airway pH, eicosanoids, hydrogen peroxide, hy drocarbons, carbon monoxide, and NOrelated products.6 NO has been studied extensively since Gu stafsson recognized that it was present in exhaled breath.7 As will be discussed later, NO has many immunological and physiological roles withi n the human. In particular, NO has been found to be positively correlated with inflammation and infection within the lower respiratory tract.6, 8-10 This study was conducted to determine if there is a difference in endogenous NO concentrations derived from the lower respiratory airways between younger and older individuals in an attempt to see how the airways may change with age, predisposing the older population to pulmonary infections and increased morbidity and mortality with pulm onary infections. Other studies have not shown a difference between these tw o groups, however, those studies have generally included less people, part icularly older individuals.11-13
3 Airway Nitric Oxide Biochemistry NO is a free-radical which is form ed enzymatically and non-enzymatically. NO itself is very reac tive and undergoes a multitud e of reactions with other molecules within the human respirator y tract to produce other compounds with varying functions. NO may be formed when L-argini ne is oxidized producing NO and Lcitrulline. This reaction is catalyzed by a group of three enzymes known as nitric oxide synthases (NOSs) with the assistanc e of various co-factors. NOSs are divided into constitutive and inducible types There are two types of constitutive NOS (cNOS) which are known as Type I or neuronal NOS (nNOS) and Type III or endothelial NOS (eNOS). These enzymes are dependent on calcium and calmodulin for activation. There is one ty pe of inducible NOS known as Type II or inducible NOS (iNOS) which is not dependent on calcium and calmodulin for activation. NO is also produced non-enzymatically wi thin the airways. One method is via nitrite (NO2 -) protonation to form nitrous acid (HNO2) which then releases NO gas when an acidic environment is present such as during an asthma exacerbation.14 Another source of NO is via the release of NO from S-nitrosothiols within the airway tissues.6, 15 The nitrosothiols are originally produced by NO reacting with thiol groups (RSH) to produce the nitrosothiols
4 (RS-NO). The nitrosothiols are exce llent carriers of NO and provide it stabilization to act at dist ant sites from production.16 Nitric oxide is highly reactive with other molecules besides the thiols. When NO combines with the superox ide anion, peroxynitrite (OONO-) is formed.16 This molecule is a pro-oxidant and is known to be toxic to many molecules such as nucleic acids, lipids, and proteins.16, 17 Also, it is known to be very toxic to microbes.6, 17 In addition, NO reacts with O2 to form nitrite and nitrate in aqueous solutions by reactions catalyzed by transition metals such as iron.9, 17 NO also reacts with the hem e moiety of hemoglobin to form methemoglobin.17 Cellular and Anatomic Sources of the Nitric Oxide Synthases All three of the nitric oxide syn thases have been identified in airway mucosa.18 Neuronal NOS has been found in neur ons, bronchial epithelial cells, and skeletal muscles.9, 19 Endothelial NOS has been localized to platelets, endothelial cells, endocardial cells, bronchi al epithelial cells, and myocardial cells.9, 20 Inducible NOS has been demonstrated in the epithelial linings of the airways, macrophages, neutrophils, eosinophils, and fibroblasts.9, 17 As has been described above, the cNOSs are calciu m/calmodulin dependent for activation whereas iNOS is not dependent on calcium/ calmodulin for activation. Inducible NOS is induced indirectly by inflammato ry cytokines such as interferon gamma (IFN), interleukin (IL) Â– 1 tumor necrosis factor Â– alpha (TNF), and others. The inflammatory cytokines induce the production of transcription factors such
5 and nuclear factorkappa B (NFB) and signal transducers and activators of transcription-1 (STAT-1) which in turn lead to the transcription of the iNOS gene sequence.21-24 It is of note that iNOS produces up to 1,000 times more NO than cNOS and that cellular production continues for hours after the stimulus.22 In addition, iNOs has now been show n to be expressed at low levels constitutively in airway epithelium.25 As mentioned earlier, all three of the enzymes are produced within the airways. However the upper airways need to be distinguished from the lower airways with regards to synthase activity and NO levels. The upper airways, which include the paranasal sinuses and nose, have been shown to produce several fold higher levels of NO than the lower airways.22, 26, 27 The type of synthase in these areas is genotypically similar to iNOS of the lower airways.26 However, it is expressed at rela tively high levels constitutively.26, 28 The lower airways are capable of producing NO via a ll three types of synthases. For years there was great debate over where the majo rity of the exhaled NO was derived from within the lower airwa ys. It has now been determi ned that the majority of the exhaled NO is derived from the airways proximal to the alveoli.29 Function of Endogenous Nitric Oxide wit hin the Lower Respiratory Tract Nitric oxide is a mediator, either directly or via other NO containing reaction products for the nonadr energic, noncholinergic (N ANC) neural inhibitory impulses from nerves innervati ng the respiratory tract.30 Neuronal NOS is the synthase which is involved in the producti on of NO for this neural function. The
6 NANC neural pathways are the only bronch odilatory pathways in the airways of humans.31 It has been postulated that the br onchodilatory effect mediated by the NANC neural impulses is important in bal ancing cholinergic bronchoconstriction rather than causing signific ant independent bronchodilatation.32 The bronchodilatory effect mediated by NO or NO containing products is via stimulation of the intracellular enzym e guanylate cyclase which catalyzes the conversion of guanylate triphosphate (GTP) to cyclic guanylate 3Â’5Â’ monophosphate (cGMP) which ultimately l eads to relaxation of the smooth muscles within the airways.9 NO and NO containing products are involved in vasodilatation in the pulmonary and bronchial circulations.33, 34 Endothelial NOS is the synthase which is involved in the production of NO for this vasodilatory function at baseline. The vasodilatation is m ediated in a similar fashion as the bronchodilatation. When present in abnormally elevated amount s, NO is a medi ator that is involved in the inflammatory process in multiple ways. Inducible NOS is the synthase responsible for production of NO in very large quantities which contribute to an inflammatory response. NO, due to its vasodilatory properties, causes exaggerated vasodilatation al ong with plasma exudation carrying inflammatory cytokines and cells to specific sites within the lung to cause or perpetuate an inflammatory response.9 NO, through an intermediary, peroxyn itrite, may lead to significant inflammation within the airways through its oxidizing effects on tissues within the
7 lungs.35 In addition, because of its toxic e ffect on microbes, it may serve as an innate defense mechanism within the lung.17 NO has also been shown to accentuate inflammation by modulating the immune system. It is believ ed that NO may selectivel y inhibit T-helper 1 (Th1) lymphocytes.36 This in turn may lead to decreased levels of IFNwhich is produced by the Th1 lymphocytes. As a result of decreased levels of IFN, Th2 cells proliferate liberati ng substantial amounts IL-5 which drive eosinophilic inflammation in conditions such as asthma.36-38 This effect is most prominent when elevated levels of NO are pr esent after induction of iNOS. NO has also been shown to mediate ciliary motion and stimulate airway secretions.39 In individuals with primary c iliary dyskinesia (PCD), exhaled NO values have been shown to be very low. This is significant as only PCD has such a characteristically low exhaled NO value.40 As can be appreciated from t he above discussion regarding NO production and function, NOÂ’s roles and t he roles of the synthases are quite complex. Due to its m any functions, NO may ther efore have beneficial or deleterious effects depending on the under lying situation within the lung. Clinical Significance of Nitric Oxide Nitric oxide is present in the airw ays of healthy individuals and individuals with various airway diseases. In addition all three of the synthases are produced in both healthy individuals and those with airway diseases.6, 25 The expression of iNOS in the lower airways of healthy indi viduals had long been debated but there
8 is now evidence that it is indeed express ed at a basal rate, ramping up when it is specifically induced by inflammatory cytokines involved in various airway diseases resulting in el evated exhaled NO levels.25 NO has been studied extensively in individuals with airway conditions as a potential marker for inflammation. Many believe that this marker will revolutionize the non-invasive monitoring of airway diseases. Exhaled NO has been studied most extens ively in asthmatics. Elevated levels of exhaled NO in asthmatics have been well documented in the literature.41, 42 This rise in NO is believed to be related to the increased expression of iNOS due to an inflammatory state via inflammatory cytokines and to the non-enzymatic production of NO fr om nitrite in the acidic bronchial environment of asthmatics.14, 43 This marker is not specific for asthma but may assist in differentiating healthy indivi duals from asthmatics when there is a question.44 Another potential use of NO in asthmatics is for the monitoring of corticosteroid and/or anti-leukotriene ther apies. Levels of NO have been shown to decline in asthmatics treated with corticosteroids (inhaled and oral) along with the leukotriene inhibitors.45-48 The reason for the decline associated with corticosteroids has been postulated to be due to decreased iNOS levels secondary to inhibition of iNOS tr anscription factors such as NFB and a reduction of inflammatory cytokines.45, 49 In addition, exhaled NO has been shown to increase before other markers of asthma exacerbation such as lung function, provocative concentration 20% (PC20), sputum eosinophilia, or asthma
9 symptoms.50 Due to this ability, it has been proposed that NO levels may be followed to determine the Â“lo ss of controlÂ” of asthma.50 Exhaled nitric oxide has also been s hown to be elevated in other disease states. Adrie et al. described elevated exha led nitric oxide levels in mechanically ventilated patients with pneumonia.10 In addition, individuals with unstable chronic obstructive disease, bronchiecta sis, and exposure to outdoor pollutants have also been shown to have elevated levels of exhaled NO.51, 52 Elevated exhaled nitric oxide levels have also been demonstrated in individuals given Larginine orally or via i nhalation, and during exercise.51-55 Table 1 summarizes the conditions in which exhaled NO may be increased. Exhaled nitric oxide has been shown to be decreased in various respiratory disease states and include: cystic fibrosis, PCD, and pulmonary hypertension.51 Decreased exhaled nitric oxide levels have also been demonstrated in those who are smokers. In addition, various drugs have been shown to reduce exhaled NO levels such as inhaled NOS inhibitors, oral or inhaled corticosteroids, anti-l eukotriene medications, and alcohol.45-48, 51, 56, 57 Table 1 summarizes the conditions in which exhaled NO may be decreased. Measurement of Nitric Oxide The most commonly used method for detecting NO in exhaled breath is via chemiluminescence. With this method, exhaled NO reacts with an excess of ozone (O3) within an exhaled breath NO anal yzer to produce nitrogen dioxide (NO2) with an electron in the excited state (NO2*). NO2* then transforms to the
10 ground state (NO2) while emitting electromagnetic radiation (chemiluminescence) ranging from 600-3,000 nm in wavelength. This chemiluminescence is detected by a photomultiplier tube and is converted in to an equivalent electrical display of NO concentration. With this me thod, NO can be detected down to a concentration of less than 1 part per bill ion (ppb). Refer to Figure 1 for the chemical reactions involved with the chemiluminescence method of measuring exhaled NO concentration. Table 1. Factors Which In fluence Exhaled Nitric Oxide6, 58 Increased NO Decreased NO Drugs: L-Arginine ingestion or inhalation, nitroprusside, ACE inhibitors Drugs: NOS inhibitors, oral or inhaled corticosteroids Respiratory tract infections Primary ciliary dyskinesis Sarcoidosis Pulmonary hypertension Asthma exacerbation Cystic Fibrosis Unstable COPD Smoking Bronchiectasis Alcohol ingestion Allergic/atopic state Forced exhalation Exercise Repeated spirometry Exposure to outdoor pollutants Sputum induction ACE = angiotensin converting enzyme; CO PD = chronic obstructive pulmonary disease; NOS=nitric oxide synthase
11 Figure 1. The Chemical Reactions Invo lved in the Chemiluminescence Method of Measuring Exhaled Nitric Oxide NO + 03 N02* + O2 NO2* NO2 + h There are two types of exhaled NO collection methods which may be used in order to analyze the breath within an ana lyzer. One method is called the Â“onlineÂ” method. This method involves an individual exhaling a single breath into a mouthpiece which provides expiratory pos itive pressure against exhalation to prevent contamination of t he sample by paranasal and nasal NO by closing the velopharyngeal aperture during the maneuver The mouthpiece is connected to the breath analyzer by tubing which immedi ately analyzes the breath for NO and provides a real-time read-out of the NO concentration. The other method, known as the Â“off-lineÂ” measurement, involves an individual breathing continuously into a specialized bag via a mouthpiece for a defi ned amount of time. After collection, the sample is then analyzed by the chemiluminescence machine. With this method the sample is analyzed at a later time. The American Thoracic Society (ATS) and the European Respiratory Society (ERS) have both published extens ive guidelines on the measurement of exhaled NO in an attempt to standardize the procedure.58, 59
12 Materials and Methods Study Design Individuals for two groups, a y ounger group and an older group, were recruited to participate in this study wh ich was performed in the Breath Lab at the University of South Florida in Tampa, Florida. Informed consent was obtained from each of the study parti cipants prior to the conducti on of the study after the research protocol along with the ri sks and benefits of the study were fully explained. All personal information and data gathered in this study were done per the guidelines of the Health Insu rance Portability and Accountability Act (HIPAA). The Institutional Review Boar d at the University of South Florida approved the study protocol used for this particular study prior to its conduction. The study subjects consisted of an ol der group of 25 individuals (61 to 79 years old, median 72 years old) and a y ounger group of 23 indi viduals (21 to 30 years old, median 24 years old) that were non-smokers with no history of lung disease, no recent respiratory infe ctions, and no history of environmental allergies. A focused history using a questionnaire (Appendix A) and a physical exam were performed prior to beginning t he testing to assess for any symptoms or signs of respiratory ill ness or impairment. None of the research participants had any symptoms or signs of respiratory illness or impa irment that would have
13 excluded them from this st udy. In addition, the subjec ts abstained from eating or drinking except for water for 6 hours prior to the study. Next, exhaled NO was assessed. A fter the exhaled NO measurement, spirometry was performed to further c onfirm normal respir atory status. Spirometry was performed after the NO measurement as it has been show to affect the exhaled NO concentrati on if done prior to the exhaled NO assessment.58 After the spirometry, exhaled breath condensate (EBC) was obtained to assess the airway pH. T he methods for obtai ning exhaled NO, spirometric, and EBC meas urements will be described in detail later in the materials and methods section. Measurement of Ex haled Nitric Oxide Exhaled NO measurements were performed in accordance with the protocol described by the ATS.58 The on-line measurements of exhaled NO were made using a chemiluminescence analyzer (Sievers, NOAÂ™ 280i; Boulder, CO) with a response time of 67 msec which is sensitive to NO over a range of approximately 0.5 ppb to 500,000 ppb. Stan dardization of t he analyzer with the zero air filter (Sievers; Boulder, CO) containing KMnO4 and activated carbon and the calibration NO gas of 45 parts per m illion (ppm) (Sievers; Boulder, CO) were performed each day prior to use per t he manufacturerÂ’s recommendations. The chemiluminescence analyzer was connect ed to a personal computer that contained the NO analysis software (Sie vers, NO AnalysisÂ™ Software Version
14 3.2; Boulder, CO) which pr ovided a graphical display of exhaled flow rate and exhaled NO concentration dur ing the analysis process. Prior to the subject performing th e test, the procedur e was demonstrated by the examiner multiple times. Next, the subject was seated in a comfortable manner such that he could view the atta ched computer monitor which displayed the real-time measurements of NO and fl ow rate so that biofeedback would be provided to assist in the acquisition of acceptable measurem ents. The subject then inhaled through their mouth via t he provided mouthpiece to total lung capacity (TLC). The inspired air was filt ered of room NO by a cartridge attached to the distal end of the mout hpiece. Once reaching TLC, the subject exhaled in a continuous manner against resistance wh ile maintaining the target ATS recommended exhalation flow rate of 50 ml /sec until a stable NO plateau of at least 3 seconds was obtained.58 After each maneuver, t he subject was given a 30 second break before performing the test again.58 Repeated exhalations were performed until three or more exhaled NO values were obtained that met ATS criteria and were within 5% of t he mean to assure reproducibility.58 The resultant mean of these values was then taken to be the exhaled NO c oncentration for that subject. Figure 2 depicts t he NO analyzer and mouthpiec e. Figure 3 depicts the maneuver required for exhaled NO measurement.
15 Figure 2: Sievers 280i Nitric Oxide Analyzer with Mouthpiece Figure 3: Performance of Exhaled Nitric Oxide Maneuver Mouthpiece with attached flowmeter NO filtering cartridge
16 Spirometry Spirometry was performed on each of the subjects using the PuritanBennett, Renaissance II Spirometer (Carlsbad, CA) per ATS protocol.60 Forced expiratory volume in one second (FEV1) and forced expiratory volume in 6 seconds (FEV6), a surrogate for forced vital capacity (FVC), were measured with the spirometer.61 In addition, the spir ometer calculated the FEV1/FEV6 ratio. HankinsonÂ’s spirometric reference val ues, which were incorporated into the spirometer, were used to determine if the subjectÂ’s spirometric values were within normal limits.62 In addition, the subjectsÂ’ FEV1/FEV6 ratio had to be equal to or greater than 70% in the younger group and equal to or greater than 65% in the older group to be considered normal for this study. Measurement of Exhal ed Breath Condensate pH Exhaled breath condensate collect ion and deaeration of the condensate were performed as has been described in the literature by using the RTubeÂ™ Exhaled Breath Condensate System (Respira tory Research, Inc; Charlottesville, VA) with the specialized pHTubeÂ™.63 In this procedure, the subject breathed normally into the specialized plastic pHTubeÂ™ which was surrounded by a cooling sleeve for 15 minutes while weari ng nose clips. Due to the surrounding cooling sleeve, the breath vapor wa s condensed into a breath condensate (liquid). After obtaining the condensat e, the sample was deaerated with argon gas by utilizing the Exhaled Breath C ondensate System Â“standard plungerÂ” to remove the carbon dioxide from the sample while monitoring the pH to assess for
17 stabilization of the pH using a pH me ter (Denver Instrument Company, Model 250 pH/Ion/Conductivity Meter; Arvada, CO). Once the pH stabilized, that value was taken to be the pH of the condensate for that subject. In addition, each of the condensate samples were tested spec trophotometrically (Thermo Spectronic, GenesysÂ™ 2 Spectrophotometer; Rochester, NY) for amylase to assure that there was no salivary contam ination of the exhaled br eath condensate using a specialized kit which assayed for -amylase quantitatively in a kinetic fashion (Pointe Scientific, Inc., Liquid Amylase Re agent Set; Lincoln Park, MI). Figure 4 depicts the specialized tube used for colle ction of condensate. Figure 5 depicts the process of measuring pH while the condensate sample is being deaerated with argon gas. Figure 4: pHTube Used to Collect Breath Condensate (Cooling Sleeve Not Attached)
18 Figure 5: Measurement of pH of Breath Condensate While Undergoing Deaeration with Argon Gas Statistical Analysis The statistical software package JMP IN Version 5.1 (SAS Institute, Inc.; Cary, NC ) was utilized to perform the statisti cal analysis. The exhaled NO values and the exhaled breath condensate pH valu es were compared for the younger and older groups using the Wilcoxon Rank Sum test. The Wilcoxon Rank Sum test was used to analyze the data because the frequency distributions of the data were found not to be normally distribut ed when plotted. The Holm method was then applied to limit the chances of Ty pe I error that may have been introduced due to multiple testing.64
19 Results Baseline Characteristics of the Study Populations Twenty-three younger subjects were recruited with a median age of 24 years and a range of 21 to 30 years. F ourteen (61%) were females and 9 (39%) were males. One individual in the younger group had ulcerative colitis. The rest did not have any medical conditions. Twent y-five older subjects were recruited with a median age of 72 years and a range of 61 to 79 years. Eleven (44%) were females and 14 (56%) were males. Health problems included: hypertension, coronary artery disease, hyperlipidemia, hypothyroidism, gastroesophageal reflux disease, and cancer. Refer to Table 2 for a complete description of baseline characteristics of the study populations. In addition, none of the individuals in either group had smoked within the past ten years, had been diagnosed with or had symptoms of allergies (allergic rhin itis or sinusitis), had been diagnosed with any lung disease including asthma, had a re spiratory tract infection recently, or used anti-histamines recently. Spirometric Measurements Spirometric measurements ar e summarized in Table 3.
20 Table 2. Baseline Characteri stics of Study Populations Age Category Characteristic Old (n =25) Young (n = 23) Gender Female (11) 44% Male (14) 56% Female (14) 61% Male (9) 39% Median age 72 years old 24 years old Health problems Hypertension (12) 48% CAD (3) 12% Hyperlipidemia (6) 24% Hypothyroidism (2) 8% GERD (2) 8% Cancer (2) 8% None (5) 20% None (22) 96% Ulcerative colitis (1) 4% Medications NSAIDs/ ASA (10) 40% Vitamins and minerals (12) 48% Statins (6) 24% ACE inhibitors (5) 20% PPIs (2) 8% Synthroid (2) 8% None (1) 4% Birth control pill (6) 26% Imuran (1) 4% Antibiotic (1) 4% Vitamins and minerals (1) 4% None (15) 65% History of tobacco use (>10 years ago) Yes (16) 64% No (9) 36% Yes (2) 9% No (21) 91% Occupation Retired (19) 76% Nurse (1) 4% Professor (3) 12% Restaurant manager (1) 4% Adm. assistant (4) 17% Nurse (3) 13% Student (16) 70% Exposure to secondary smoke Yes (3) 12% No (22) 88% Yes (1) 4% No (22) 96% Past exposures Firefighter (1) 4% Coal dust, coke oven (1) 4% None (23) 92% Chlorine gas (1) 4% None (22) 96% CAD = coronary artery disease; GE RD = gastroesophageal reflux disease; NSAIDs/ASA = nonsteroidal anti-inflamma tory drugs / aspirin; PPIs = proton pump inhibitors; Adm. assistant = administrative assistant.
21 Table 3. Spirometric Measur ements of Study Populations Age Category Measurement Old Young Median FEV1 (percentage of normal) 94% 95% Median FEV6 (percentage of normal) 95% 92% Median FEV1/ FEV6 78% 87% FEV1 = forced expiratory volume in 1 second; FEV6 = forced expiratory volume in 6 seconds Exhaled Breath Condens ate pH Measurements The median deaerated pHs for each of the groups are depicted in Table 4. The range of pH for the older group was 5. 26 to 8.12. The range for the pH for the younger group was 6.44 to 8.12. The median pHs of the older age group and the younger age group we re both 7.72. Exhaled Nitric Oxide Measurements The median exhaled nitric oxide conc entrations for each of the groups are depicted in Table 4. The range for exhal ed NO for the older group was 6.0 ppb to 103 ppb. The range for exhaled for NO for the younger group was 6.4 ppb to 214 ppb. The median exhaled NO concent rations of the older and younger age groups were 36.9 ppb and 18.7 ppb, respectively. Table 4. Exhaled Breath Nitric Ox ide and Exhaled Breath Condensate Deaerated pH Measurement Median for Old Median for Young P Value Deaerated pH 7.72 7.72 0.959 Nitric Oxide (ppb) 36.9 18.7 0.0011 ppb=parts per billion
22 Discussion Exhaled NO has been shown to be elevated in lower respiratory conditions in which inflammation is present such as asthma and pneumonia. Two mechanisms are thought to be responsib le for the increase in NO. One is the up-regulation of iNOS due to inflamma tory cytokines and the other is due to an increased release of NO from molecu lar intermediates due to an acidic airway.14, 43 Despite the uncertainty with r egards to the mechanism of NO elaboration from the lower ai rways, exhaled NO has the potential for assisting in the diagnosis and prognosis of airway dise ases and monitoring their treatments. The current study compared NO concentrations between a younger group of 23 individuals (median age of 24 years) and an older group of 25 individuals (median age of 72 years) that were non-smokers wit h no history of pulmonary disease, no recent respiratory infe ctions, and no history of environmental allergies. This study was conducted to determine if there is a difference in endogenous NO concentrations derived from the lower respiratory airways between younger and older individuals in an attempt to see how the airways may change with age, predisposing the older population to pulmonary infections and increased morbidity and mortalit y with pulmonary infections. This study showed that exhaled NO concentrations of the older and younger age groups were 36.9 ppb and 18.7 ppb, respectively. This represented a significant statistical differ ence between the two groups with a value of
23 0.0011 per the Wilcoxon Rank Sum test. Th is statistical significance held true when adjusted for multiple testing with the Holm method. In addition, there was no significant difference between the exhaled breath condensate pHs between the two groups with each hav ing a median of 7.72. This is the first study to show a significant difference in exhaled NO between a younger and an older population. Previous studies have not shown a difference. Ekroos et al. f ound no correlation between age and NO.11 However, the age range was 21-48 years and the majority of the subjects were in their twenties and thirties. Their study di d not really address a possible difference between a young population and an aged population due to its limited age sampling. Tsang et al. studied a gr oup of 121 healthy adults with an age range of 20-79 years and did not find a rela tionship between age and NO levels.13 On evaluation of their data, t here were noted to be several high NO outliers in the younger age ranges that may have significant ly affected their data. In addition, Tsang et al. did not divide their sample into two distinct groups in order to analyze for a difference as was done in this study using the Wilcoxon Rank Sum test. Instead, they looked for a correlati on using regression. With their method, a linear relationship would have been nece ssary to reveal a difference as opposed to the method used presently which would pick up any difference between the two distinct age groups. Ther efore, the difference in the findings between the current study and the study of Tsang et al. may be due to the use of different statistical methods. It is also possible that the difference may be related to the different population samples as the current study wa s conducted in the
24 United States of America and the study of Tsang et al. was conducted in China. In an abstract by Duncan et al., no corre lation between age and exhaled NO was found in a group of 59 healthy individuals.12 However, this study had a limited age range with the oldest category reaching a peak of 60 years. Also, there were only two individuals in the oldest age category of Â“51-60. Â” In addition, the Â“offlineÂ” technique was used which differed from the Â“on-lineÂ” tec hnique used in the present study. The mechanisms responsible for the el evated NO concentrations in the older population are not clear at present. This study showed a difference in exhaled NO concentrations between the age groups despite similar pHs between the groups. Therefore, this is more compatible with a NOS-mediated increase rather than an airway pH-mediated incr ease in NO. If indeed the elevated NO concentration is due to NOS, what is the underlying reason for the increased NOS activity and what type of NOS is responsible? It has been proposed that aged individuals have a baseline airway inflammation despite being clinically healthy.12, 65, 66 Bronchoalveolar lavage examination has supported th is hypothesis by showing that older individuals have elevated lymphocytes with an elevat ed CD4+/CD8+ T cell ratio, elevated IgM, IgA, and IgG levels, elevated IL-6 and IL-8, and elevated neutrophils.5 It is therefore possible that a low-grade inflammation may provide the necessary constituents such as inflammatory cytoki nes to induce the expression of iNOS in bronchial epithelial cells, macrophages, and neutrophils leading to the elevated NO levels in the older population.
25 Another potential reason for the elevat ed NO concentrations in the older population may be due to an immune system dysregulation without inflammation leading to inappropriate NOS ( iNOS or cNOS) expression. It is also possible that NO concentrations are elevated at baseline in the aged in an attempt to compensate for an impaired immune system as NO has been shown to be toxic to microorganisms and therefore may be of assistance to the immune system. This baseline com pensatory increase could potentially be mediated by elevated cNOS or iNOS expression. Lastly, the increase in NO concentrations in older individuals may be secondary to an incompetent soft palate allowing for contamination of exhaled lower airway NO with upper airway NO despite performing the maneuver properly. The significance of the elevated NO concentrations in the aged population is not clear. Whether the elevated le vels further support the hypothesis of baseline airway inflammation in the aged remains to be determined. If the elevated NO levels are due to an i mmune system dysregulation or a compensatory mechanism to assists an impaired immune system then NO levels may be useful in identifying those with immune systems which are not completely competent to fight infect ion. If this is indeed the case, an elevated NO concentration may serve as a marker for a dysregulated or impaired immune system which would predispose older indivi duals to pulmonary infections such as pneumonia with poorer outcomes. Finally, it is possible that the difference in NO concentration between the older and younger groups is not clinically significant.
26 Additional investigations are nece ssary to elucidate the mechanisms and significances of elevated NO levels in the aged. A study which employed immunohistochemical methods to determi ne the differential expression of the NOSs between the age groups w ould elucidate which NOS is responsible for the elevated NO concentrations in the aged. Another study of value would be to create a prospective study in which basel ine serial measurements of NO would be taken in an older population while they are free of respiratory disease. The group would then be followed for the inciden ce of pulmonary infections along with the morbidity and mortality secondary to pulmonary infections in order to determine if a relationship exists between baseline elevated NO levels and pulmonary infections in the aged. An additi onal study that would be interesting would be to determine the effect of bas eline inflammation in the aged on NO levels. This could be accomplished by performing bronchoscopic biopsy evaluations for inflammation in younger and older individuals and correlating the levels of inflammation with the exhaled NO concentrations. Another potentially valuable study would be to perform bronc hoalveolar lavages or induced sputum studies on older and younger individuals to assess the milieu of cells and mediators and their relationships with exhaled NO concentrations.
27 Conclusion This study evaluated exhaled NO levels between younger and older populations to gain further insight into possible differences between the airways of these two groups. It was found that ol der individuals have si gnificantly higher NO concentrations than younger indivi duals despite each group having similar pHs. The reasons for this differenc e along with the relationship to pulmonary infections are unclear and further studies will be necessary to further evaluate these issues.
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35 Appendix A: Exhaled Breath Condensate and Exha led Breath Nitric Oxide Study Questionnaire Subject number:_______ Exhaled Breath Condensate and exhaled Breath Nitric Oxide Study Questionnaire TodayÂ’s Date _______________________________________ Gender: Male Female (circle one) 1) How old are you? ______________years 2) Do you have any health problems that you see a physician for? Please list them below. 1._________________________________________________________ 2. ________________________________________________________ 3. ________________________________________________________ 4. ________________________________________________________ 5.________________________________________________________ 3) Are you taking any medications? If so please list them below. (Including over the counter medications) 1._________________________________________________________ 2. ________________________________________________________ 3. ________________________________________________________
36 Appendix A (Continued) 4. ________________________________________________________ 5.________________________________________________________ 4) If you have ever smoked, answer the following. How many packs per day did you smoke? ________________________ For how many years did you smoke? ____________________________ When did you stop smoking? __________________________________ 5) On what date were you last ill? _________________________________________ 6) What illness did you have? ____________________________________________ 7) What is your occupation? _____________________________________________ 8) Are you exposed to second hand smoke at home or at work? Please circle Â“yesÂ” or Â“noÂ”. YES or NO 9) Were you or are you exposed to any gases, dusts, or fumes at your job? YES or NO If so, please explain: ____________________________________________
37 Appendix B: Age Distributions within Each Age Category Category: Old Distribution: Age 60 65 70 75 80 Quantiles 100.0% maximum 79.000 99.5% 79.000 97.5% 79.000 90.0% 77.500 75.0% quartile 76.000 50.0% median 71.500 25.0% quartile 67.000 10.0% 63.000 2.5% 61.000 0.5% 61.000 0.0% minimum 61.000 Moments Mean 71.291667 Std Dev 5.3363329 Std Err Mean 1.0892744 upper 95% Mean 73.545002 lower 95% Mean 69.038331 N 24
38 Appendix B (Continued) Category: Young Distribution: Age 17.5 20 22.5 25 27.5 30 32.5 Quantiles 100.0% maximum 30.000 99.5% 30.000 97.5% 30.000 90.0% 27.600 75.0% quartile 26.000 50.0% median 24.000 25.0% quartile 21.000 10.0% 19.400 2.5% 18.000 0.5% 18.000 0.0% minimum 18.000 Moments Mean 23.608696 Std Dev 2.9808347 Std Err Mean 0.621547 upper 95% Mean 24.897705 lower 95% Mean 22.319686 N 23
39 Appendix C: Age Cat egory and Gender Distributions for Entire Study Population Age Category Distribution fo r Entire Study Population old young old young Frequencies Level Count Prob old 25 0.52083 young 23 0.47917 Total 48 1.00000 2 Levels
40 Appendix C (Continued) Gender Distribution for En tire Study Population female male female male Frequencies Level Count Prob female 25 0.52083 male 23 0.47917 Total 48 1.00000 2 Levels
41 Appendix D: Distributions of Characteristics and Meas urements by Age Category Age Category: Old FEV1 70 80 90 100 110 120 130 Quantiles 100.0% maximum 122.00 99.5% 122.00 97.5% 122.00 90.0% 116.00 75.0% quartile 101.50 50.0% median 94.00 25.0% quartile 87.50 10.0% 76.60 2.5% 73.00 0.5% 73.00 0.0% minimum 73.00 Moments Mean 94.48 Std Dev 12.467023 Std Err Mean 2.4934046 upper 95% Mean 99.626134 lower 95% Mean 89.333866 N 25
42 Appendix D (C ontinued) FEV6 70 80 90 100 110 120 130 Quantiles 100.0% maximum 123.00 99.5% 123.00 97.5% 123.00 90.0% 109.40 75.0% quartile 103.50 50.0% median 95.00 25.0% quartile 87.50 10.0% 77.60 2.5% 77.00 0.5% 77.00 0.0% minimum 77.00 Moments Mean 95.04 Std Dev 11.570364 Std Err Mean 2.3140729 upper 95% Mean 99.816012 lower 95% Mean 90.263988 N 25
43 Appendix D (C ontinued) FEV1/FEV6 65 70 75 80 85 90 95 Quantiles 100.0% maximum 91.000 99.5% 91.000 97.5% 91.000 90.0% 84.400 75.0% quartile 81.000 50.0% median 78.000 25.0% quartile 74.500 10.0% 70.000 2.5% 69.000 0.5% 69.000 0.0% minimum 69.000 Moments Mean 77.96 Std Dev 5.2399109 Std Err Mean 1.0479822 upper 95% Mean 80.122929 lower 95% Mean 75.797071 N 25
44 Appendix D (C ontinued) Gender female male female male Frequencies Level Count Prob female 11 0.44000 male 14 0.56000 Total 25 1.00000 2 Levels Health Problems breastca cad, bph cad, htn, hyperlipid gerd htn htn, AR, MR, hypothyroid htn, hyperlip htn, hyperlip, afib, bladder ca htn, mvp htn,gout, hypothyroid hyperlipid none oa osteopen platelet disorder breastca cad, bph cad, htn, hyperlipid gerd htn htn, AR, MR, hypothyroid htn, hyperlip htn, hyperlip, afib, bladder ca htn, mvp htn,gout, hypothyroid hyperlipid none oa osteopen platelet disorder
45 Appendix D (C ontinued) Frequencies Level CountProb breastca 10.04000 cad, bph 10.04000 cad, htn, hyperlipid 20.08000 gerd 20.08000 htn 40.16000 htn, AR, MR, hypothyroid 10.04000 htn, hyperlip 20.08000 htn, hyperlip, afb, bladder ca 10.04000 htn, mvp 10.04000 htn,gout, hypothyroid 10.04000 hyperlipid 10.04000 none 50.20000 oa 10.04000 osteopen 10.04000 platelet disorder 10.04000 Total 251.00000 15 Levels Medications acupril, glucosamine, chondroitin asa, naprosyn, hytrin bppill, asa, ca, vitD, vitE centrum, triamterene, ca, vitd, fosamax ditropan, maxide evista, vitD feldene, oscal, vitD, premarin fosimax, glucosamine/choldroitin, folate, vit D, ca lipitor, cardura, alelite, plendal, vioxx lipitor,toprol, altace, plavix, asa, ocuvit, mvi, vitc, vite, vi mvi, vite mvi, vite, asa nexium, imipramine, actinal, premarin nifedipine, vitc, vite, asa, zocor none norvasc, asa, mvi, vitC, vitE pcn, prempro premerin, protonix prinivil, diazide, lipitor rythmol, zocor, dig, plendil, trerazosin synthroid, lisinopril, allopurinol vasotec, dynacirc, lasix verapamil, maxide, synthroid verapamil, toprol, lipitor, asa,naproxen, viagra voltaran, lipitor, folate, ca, vitD, MVI
46 Appendix D (C ontinued) Frequencies Level Count Prob acupril, glucosamine, chondroitin 1 0.04000 asa, naprosyn, hytrin 1 0.04000 bppill, asa, ca, vitD, vitE 1 0.04000 centrum, triamterene, ca, vitd, fosamax 1 0.04000 ditropan, maxide 1 0.04000 evista, vitD 1 0.04000 feldene, oscal, vitD, premarin 1 0.04000 fosimax, glucosamine/ choldroitin, folate, vit D, ca 1 0.04000 Lipitor, cardura, alelite, plendal, vioxx 1 0.04000 lipitor,toprol, altace, plavix, asa, ocuvit, mvi, vitc, vite, vi 1 0.04000 mvi, vite 1 0.04000 mvi, vite, asa 1 0.04000 nexium, imipramine, actinal, premarin 1 0.04000 nifedipine, vitc, vite, asa, zocor 1 0.04000 none 1 0.04000 norvasc, asa, mvi, vitC, vitE 1 0.04000 pcn, prempro 1 0.04000 premerin, protonix 1 0.04000 prinivil, diazide, lipitor 1 0.04000 rythmol, zocor, dig, plendil, trerazosin 1 0.04000 synthroid, lisinopril, allopurinol 1 0.04000 vasotec, dynacirc, lasix 1 0.04000 verapamil, maxide, synthroid 1 0.04000 verapamil, toprol, lipitor, asa,naproxen, viagra 1 0.04000 voltaran, lipitor, folate, ca, vitD, MVI 1 0.04000 Total 25 1.00000 25 Levels Smoking History no yes no yes
47 Appendix D (C ontinued) Frequencies Level Count Prob no 9 0.36000 yes 16 0.64000 Total 25 1.00000 2 Levels Packs Smoked When Smoker 0.5cigpd 0.5ppd 1ppd 2cigpd 2pipes 2ppd 3ppd <1ppd na 0.5cigpd 0.5ppd 1ppd 2cigpd 2pipes 2ppd 3ppd <1ppd na Frequencies Level Count Prob 1 0.04000 0.5cigpd 1 0.04000 0.5ppd 2 0.08000 1ppd 5 0.20000 2cigpd 2 0.08000 2pipes 1 0.04000 2ppd 1 0.04000 3ppd 1 0.04000 <1ppd 2 0.08000 na 9 0.36000 Total 25 1.00000 10 Levels
48 Appendix D (C ontinued) Years Smoked na na Frequencies Level Count Prob na 9 1.00000 Total 9 1.00000 1 Levels Last Ill unknown unknown
49 Appendix D (C ontinued) Frequencies Level Count Prob unknown 16 1.00000 Total 16 1.00000 1 Levels Illness When Last Ill na uri na uri Frequencies Level Count Prob na 16 0.64000 uri 9 0.36000 Total 25 1.00000 2 Levels
50 Appendix D (C ontinued) Occupation nretired nurse prof reseraunt retired transcrip nretired nurse prof reseraunt retired transcrip Frequencies Level Count Prob nretired 1 0.04000 nurse 1 0.04000 prof 3 0.12000 reseraunt 1 0.04000 retired 18 0.72000 transcrip 1 0.04000 Total 25 1.00000 6 Levels
51 Appendix D (C ontinued) Second Hand Smoke no yes no yes Frequencies Level Count Prob no 22 0.88000 yes 3 0.12000 Total 25 1.00000 2 Levels Exposures no yes no yes
52 Appendix D (C ontinued) Frequencies Level Count Prob no 22 0.88000 yes 3 0.12000 Total 25 1.00000 2 Levels Explanation of Exposures asbestoscourths coaldust, cokeoven fire fighter na asbestoscourths coaldust, cokeoven fire fighter na Frequencies Level CountProb asbestoscourths 10.04000 coaldust, cokeoven 10.04000 fire fighter 10.04000 na 220.88000 Total 251.00000 4 Levels
53 Appendix D (C ontinued) NO Concentrations 0 50 100 150 200 250 Quantiles 100.0% maximum 214.00 99.5% 214.00 97.5% 214.00 90.0% 90.70 75.0% quartile 56.60 50.0% median 36.90 25.0% quartile 26.30 10.0% 14.44 2.5% 6.00 0.5% 6.00 0.0% minimum 6.00 Moments Mean 47.772 Std Dev 41.525038 Std Err Mean 8.3050076 upper 95% Mean 64.912693 lower 95% Mean 30.631307 N 25
54 Appendix D (C ontinued) pH Deaerated 5 5.5 6 6.5 7 7.5 8 8.5 Quantiles 100.0% maximum 8.1200 99.5% 8.1200 97.5% 8.1200 90.0% 8.0400 75.0% quartile 7.9700 50.0% median 7.7200 25.0% quartile 7.1750 10.0% 6.5280 2.5% 5.2600 0.5% 5.2600 0.0% minimum 5.2600 Moments Mean 7.4996 Std Dev 0.6650343 Std Err Mean 0.1330069 upper 95% Mean 7.7741127 lower 95% Mean 7.2250873 N 25
55 Appendix D (C ontinued) Age Category: Young FEV1 70 80 90 100 110 120 130 Quantiles 100.0% maximum 123.00 99.5% 123.00 97.5% 123.00 90.0% 112.40 75.0% quartile 100.75 50.0% median 95.00 25.0% quartile 87.50 10.0% 81.00 2.5% 80.00 0.5% 80.00 0.0% minimum 80.00 Moments Mean 95.590909 Std Dev 10.795321 Std Err Mean 2.3015702 upper 95% Mean 100.37729 lower 95% Mean 90.804532 N 22
56 Appendix D (C ontinued) FEV6 70 75 80 85 90 95 100 105 110 115 Quantiles 100.0% maximum 111.00 99.5% 111.00 97.5% 111.00 90.0% 105.00 75.0% quartile 103.25 50.0% median 92.50 25.0% quartile 88.00 10.0% 83.60 2.5% 74.00 0.5% 74.00 0.0% minimum 74.00 Moments Mean 94.681818 Std Dev 9.0679255 Std Err Mean 1.9332882 upper 95% Mean 98.702311 lower 95% Mean 90.661325 N 22
57 Appendix D (C ontinued) FEV1/FEV6 70 75 80 85 90 95 100 Quantiles 100.0% maximum 98.000 99.5% 98.000 97.5% 98.000 90.0% 95.400 75.0% quartile 91.000 50.0% median 87.000 25.0% quartile 81.750 10.0% 77.200 2.5% 74.000 0.5% 74.000 0.0% minimum 74.000 Moments Mean 86.545455 Std Dev 6.2467307 Std Err Mean 1.3318075 upper 95% Mean 89.3151 lower 95% Mean 83.775809 N 22
58 Appendix D (C ontinued) Gender female male female male Frequencies Level Count Prob female 14 0.60870 male 9 0.39130 Total 23 1.00000 2 Levels Health Problems none ulcerativecolitis none ulcerativecolitis
59 Appendix D (C ontinued) Frequencies Level CountProb none 220.95652 ulcerativecolitis 10.04348 Total 231.00000 2 Levels Medications bcp doxy, bcp imuran none weightlosssulp bcp doxy, bcp imuran none weightlosssulp Frequencies Level CountProb bcp 50.21739 doxy, bcp 10.04348 imuran 10.04348 none 150.65217 weightlosssulp 10.04348 Total 231.00000 5 Levels
60 Appendix D (C ontinued) Smoking History no yes no yes Frequencies Level Count Prob no 21 0.91304 yes 2 0.08696 Total 23 1.00000 2 Levels Packs Smoked When Smoker 2cigpd 3cigpd na 2cigpd 3cigpd na
61 Appendix D (C ontinued) Frequencies Level Count Prob 2cigpd 1 0.04348 3cigpd 1 0.04348 na 21 0.91304 Total 23 1.00000 3 Levels Years Smoked na na Frequencies Level Count Prob na 21 1.00000 Total 21 1.00000 1 Levels
62 Appendix D (C ontinued) Last Ill unknown unknown Frequencies Level Count Prob unknown 16 1.00000 Total 16 1.00000 1 Levels Illness When Last Ill bronchitis gastro na uri bronchitis gastro na uri
63 Appendix D (C ontinued) Frequencies Level Count Prob bronchitis 1 0.04348 gastro 1 0.04348 na 16 0.69565 uri 5 0.21739 Total 23 1.00000 4 Levels Occupation ce-director facman manager nurse secret student ce-director facman manager nurse secret student Frequencies Level Count Prob ce-director 1 0.04348 facman 1 0.04348 manager 1 0.04348 nurse 3 0.13043 secret 1 0.04348 student 16 0.69565 Total 23 1.00000 6 Levels
64 Appendix D (C ontinued) Second Hand Smoke 2permo no yes 2permo no yes Frequencies Level Count Prob 2permo 1 0.04348 no 21 0.91304 yes 1 0.04348 Total 23 1.00000 3 Levels Exposures no yes no yes
65 Appendix D (C ontinued) Frequencies Level Count Prob no 22 0.95652 yes 1 0.04348 Total 23 1.00000 2 Levels Explanation of Exposures Cl2twoyrago na Cl2twoyrago na Frequencies Level Count Prob Cl2twoyrago 1 0.04348 na 22 0.95652 Total 23 1.00000 2 Levels
66 Appendix D (C ontinued) NO Concentations 0 25 50 75 100 125 Quantiles 100.0% maximum 107.00 99.5% 107.00 97.5% 107.00 90.0% 63.60 75.0% quartile 27.00 50.0% median 18.70 25.0% quartile 12.70 10.0% 9.82 2.5% 6.40 0.5% 6.40 0.0% minimum 6.40 Moments Mean 25.221739 Std Dev 23.23606 Std Err Mean 4.8450535 upper 95% Mean 35.269765 lower 95% Mean 15.173713 N 23
67 Appendix D (C ontinued) pH Deaerated 6.5 7 7.5 8 Quantiles 100.0% maximum 8.1200 99.5% 8.1200 97.5% 8.1200 90.0% 8.0640 75.0% quartile 7.9100 50.0% median 7.7200 25.0% quartile 7.3000 10.0% 6.9540 2.5% 6.4400 0.5% 6.4400 0.0% minimum 6.4400 Moments Mean 7.5917391 Std Dev 0.4261845 Std Err Mean 0.0888656 upper 95% Mean 7.7760351 lower 95% Mean 7.4074431 N 23
68 Appendix E: Analysis of NO and pH by Age Category Oneway Analysis of NO By Age Category NO 0 50 100 150 200 old young agecat Wilcoxon / Kruskal-Wallis Tests (Rank Sums) Level Count Score SumScore Mean(Mean-Mean0)/Std0 old 25 77130.84003.261 young 23 40517.6087-3.261 2-Sample Test, Normal Approximation S Z Prob>|Z| 405 -3.26083 0.00111-way Test, ChiSquare Approximation ChiSquare DF Prob>ChiSq 10.7004 1 0.0011
69 Appendix E (Continued) Oneway Analysis of pH Dearate By Age Category phdearate 5 5.5 6 6.5 7 7.5 8 8.5 old young agecat Wilcoxon / Kruskal-Wallis Tests (Rank Sums) Level Count Score SumScore Mean(Mean-Mean0)/Std0 old 25 615.524.62000.052 young 23 560.524.3696-0.052 2-Sample Test, Normal Approximation S Z Prob>|Z| 560.5 -0.05161 0.95881-way Test, ChiSquare Approximation ChiSquare DF Prob>ChiSq 0.0038 1 0.9506