USF Libraries
USF Digital Collections

Exhaled breath nitric oxide

MISSING IMAGE

Material Information

Title:
Exhaled breath nitric oxide is there a baseline difference due to ethnicity
Physical Description:
Book
Language:
English
Creator:
Patel, Sunita I
Publisher:
University of South Florida
Place of Publication:
Tampa, Fla.
Publication Date:

Subjects

Subjects / Keywords:
African descent
Caucasian
Male
Non-parametric
Generalizability
Dissertations, Academic -- Public Health -- Masters -- USF   ( lcsh )
Genre:
government publication (state, provincial, terriorial, dependent)   ( marcgt )
bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

Notes

Summary:
ABSTRACT: The air that humans exhale contains various chemical markers whose levels have been associated with various respiratory disorders. Therefore, measurement of these markers offers a potential method of examining airway disease status. Furthermore, exhaled breath offers the advantage of being easy to collect and non-invasive. Hence, these exhaled breath markers are potentially of significant clinical use in examining airways. Therefore, examination of exhaled breath has become the subject of intense study. Current research is targeting the development of methods and parameters for looking at these markers. The goal of this cross-sectional pilot study was to consider the variability in the measurement of these exhaled breath markers between members of different ethnic populations. Specifically, measurements of the exhaled breath marker Nitric Oxide (NO) were compared between two ethnic groups (Caucasian men versus men of African descent).Ten healthy men in each group were studied to examine whether baseline NO measurements differed between them. In this study, a cross-sectional design was used. The study sample consisted of young, healthy men with no history of environmental allergies, asthma, or lung diseases and no significant smoking history. A total of twenty-five men volunteered for the study, including fourteen men of Caucasian descent and eleven men of African descent. Because four men were excluded and one withdrew, ten men in each ethnic group were included in the final analysis. The source population from which the sample was drawn included students and workers. All participants were residing in Florida at the time of study. Ideally, the target population for this study was young, healthy, working men.Large inter-measurement variation was seen between the participants of each ethnic group.This was hypothesized to be attributed to a tri-modal distribution due to the existence of 3 populations of subjects: (1) asymptomatic with normal airways, where NO levels were under 30 parts per billion (ppb); (2) asymptomatic with airway pathology, where NO levels were over 30 ppb; and (3) asymptomatic just before the onset of an upper respiratory tract infection, where NO levels were over 60 ppb. This pilot study did not find statistically significant evidence that there is a difference in the baseline exhaled breath NO measurements between the two ethnic groups studied. Nonetheless, in participants with NO levels under 30 ppb the mean of the African group was found to be 7.6 ppb lower than the mean of the Caucasian group when attempts were made to exclude individuals with underlying airway pathology or imminent upper respiratory tract infection.
Thesis:
Thesis (M.S.P.H.)--University of South Florida, 2005.
Bibliography:
Includes bibliographical references.
System Details:
System requirements: World Wide Web browser and PDF reader.
System Details:
Mode of access: World Wide Web.
Statement of Responsibility:
by Sunita I Patel.
General Note:
Title from PDF of title page.
General Note:
Document formatted into pages; contains 106 pages.

Record Information

Source Institution:
University of South Florida Library
Holding Location:
University of South Florida
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
aleph - 001680965
oclc - 62458856
usfldc doi - E14-SFE0001156
usfldc handle - e14.1156
System ID:
SFS0025477:00001


This item is only available as the following downloads:


Full Text
xml version 1.0 encoding UTF-8 standalone no
record xmlns http:www.loc.govMARC21slim xmlns:xsi http:www.w3.org2001XMLSchema-instance xsi:schemaLocation http:www.loc.govstandardsmarcxmlschemaMARC21slim.xsd
leader nam Ka
controlfield tag 001 001680965
003 fts
005 20060215071036.0
006 m||||e|||d||||||||
007 cr mnu|||uuuuu
008 051205s2005 flu sbm s000 0 eng d
datafield ind1 8 ind2 024
subfield code a E14-SFE0001156
035
(OCoLC)62458856
SFE0001156
040
FHM
c FHM
049
FHMM
090
RA425 (Online)
1 100
Patel, Sunita I.
0 245
Exhaled breath nitric oxide
h [electronic resource] :
b is there a baseline difference due to ethnicity /
by Sunita I Patel.
260
[Tampa, Fla.] :
University of South Florida,
2005.
502
Thesis (M.S.P.H.)--University of South Florida, 2005.
504
Includes bibliographical references.
516
Text (Electronic thesis) in PDF format.
538
System requirements: World Wide Web browser and PDF reader.
Mode of access: World Wide Web.
500
Title from PDF of title page.
Document formatted into pages; contains 106 pages.
520
ABSTRACT: The air that humans exhale contains various chemical markers whose levels have been associated with various respiratory disorders. Therefore, measurement of these markers offers a potential method of examining airway disease status. Furthermore, exhaled breath offers the advantage of being easy to collect and non-invasive. Hence, these exhaled breath markers are potentially of significant clinical use in examining airways. Therefore, examination of exhaled breath has become the subject of intense study. Current research is targeting the development of methods and parameters for looking at these markers. The goal of this cross-sectional pilot study was to consider the variability in the measurement of these exhaled breath markers between members of different ethnic populations. Specifically, measurements of the exhaled breath marker Nitric Oxide (NO) were compared between two ethnic groups (Caucasian men versus men of African descent).Ten healthy men in each group were studied to examine whether baseline NO measurements differed between them. In this study, a cross-sectional design was used. The study sample consisted of young, healthy men with no history of environmental allergies, asthma, or lung diseases and no significant smoking history. A total of twenty-five men volunteered for the study, including fourteen men of Caucasian descent and eleven men of African descent. Because four men were excluded and one withdrew, ten men in each ethnic group were included in the final analysis. The source population from which the sample was drawn included students and workers. All participants were residing in Florida at the time of study. Ideally, the target population for this study was young, healthy, working men.Large inter-measurement variation was seen between the participants of each ethnic group.This was hypothesized to be attributed to a tri-modal distribution due to the existence of 3 populations of subjects: (1) asymptomatic with normal airways, where NO levels were under 30 parts per billion (ppb); (2) asymptomatic with airway pathology, where NO levels were over 30 ppb; and (3) asymptomatic just before the onset of an upper respiratory tract infection, where NO levels were over 60 ppb. This pilot study did not find statistically significant evidence that there is a difference in the baseline exhaled breath NO measurements between the two ethnic groups studied. Nonetheless, in participants with NO levels under 30 ppb the mean of the African group was found to be 7.6 ppb lower than the mean of the Caucasian group when attempts were made to exclude individuals with underlying airway pathology or imminent upper respiratory tract infection.
590
Adviser: Stuart Brooks, MD.
653
African descent.
Caucasian.
Male.
Non-parametric.
Generalizability.
690
Dissertations, Academic
z USF
x Public Health
Masters.
773
t USF Electronic Theses and Dissertations.
4 856
u http://digital.lib.usf.edu/?e14.1156



PAGE 1

Exhaled Breath Nitric Oxide: Is Ther e A Baseline Difference Due To Ethnicity? by Sunita I. Patel, M.D. A thesis submitted in partial fullfillment of the requirements for the degree of Master of Science in Public Health Department of Environmenta l And Occupational Health College of Public Health University of South Florida Major Professor: Stuart Brooks, M.D. Rony Francois, M.D., M.S.P.H., Ph.D. Robert Haight, M.D., M.S.P.H. Thomas Truncale, M.D., M.S.P.H. Date of Approval April 15, 2005 Keywords: African descent, Caucasian, male, non-parametric, generalizability Copyright 2005 Sunita Indravadan Patel, M.D.

PAGE 2

Acknowledgments I would like to thank Stuart Brooks, M.D. ; Rony Francois, M.D., M.S.P.H., Ph.D; Robert Haight, M.D., M.S.P.H.; James McCluskey, M.D., M.S.P.H., Ph.D(c); and Thomas Truncale, M.D., M.S.P.H., each of whom provided me with valuable input, support, and encouragement in completing this project. I would also like to thank Wei Wang, M. Sc., Ph.D, to whom I am especia lly grateful for his assistance with the statistical analyses perf ormed in this project.

PAGE 3

i Table of Contents List of Tables iii List of Figures iv Abstract v Introduction 1 Methods of Studying Airway Disease 1 Nitric Oxide in Exhaled Breath 2 Race and Ethnicity Considerations 5 Epidemiologic Considerations 6 Research Question 7 Hypothesis 8 Goals and Objectives 9 Significance and Utility of Research 10 Materials and Methods 11 Overall Study Design 11 Facilities and Equipment 11 Participant Recruitment 12 Study Subjects and Restrictions 12 Study Questionnaire and Eligibility 13 Subject Safety Considerations 14 Physical Examination 16 Acquisition of Spirometry 17 Measurement of Exhaled Breath Nitric Oxide 19

PAGE 4

ii Data Collection and Analysis 23 Study Results 25 Exclusions 25 Questionnaire Collection 26 Medical Information 30 Spirometry Measurements 33 Exhaled Breath Nitric Oxide Measurements 34 Discussion 42 Findings 42 Implications 44 Future Directions 45 Study Limitations 45 Conclusion 50 References 51 Appendices 54 Appendix A: Initial Medical and Ethnicity Questionnaires 55 Appendix B: Exit Questionnaire 63 Appendix C: Recruitment Flyer 64 Appendix D: Adult Informed Consent Form 66 Appendix E: Power Analysis 88 Appendix F: SAS Program Used 91

PAGE 5

iii List of Tables Table 1. Factors That Affect NO Meas urements in Healthy Subjects 3 Table 2. Number of Subjects in Each Ethnic Group 25 Table 3. Sociodemographic Characteristics 28 Table 4. Pearson-Product Co rrelation Coefficient 30 Table 5. Medical Information 31 Table 6. Statistical Analysis of Average NO Levels for all 20 Participants 34 Table 7. Wilcoxon Scores (Rank Su ms) for Variable Average NO 36 Classified by Variable Ethnicity Table 8. Statistical Analysis of Average NO Levels When Excluding 37 Extreme Values Table 9. Wilcoxon Scores (Rank Su ms) for Variable Average NO 38 Classified by Variable Ethnic ity When Extreme Values Are Excluded Table 10 Statistical Analysis of Average NO Levels When Excluding 40 Levels over 30 ppb. Table 11. t-Test of Average NO Le vels When Excluding Average NO 40 Values Over 30 Table 12. Calculating The Po wer Of The Study 41

PAGE 6

iv List of Figures Figure 1. Nitric Oxide Synthesis 4 Figure 2. Genetic Induction of NOS 4 Figure 3. Nitric Oxide Flow Rate Versus Time 19 Figure 4. Flowmeter Design 20 Figure 5. Effect of Inhalation of Air Thr ough the Flowmeter Versus Inhalation 20 of Ambient Air Figure 6. Participant Distribution By Age 26 Figure 7. Average NO Level Of Each Subj ect By Ethnicity 34

PAGE 7

v Exhaled Breath Nitric Oxide: Is There A Baseline Difference Due To Ethnicity? Sunita I. Patel, M.D. ABSTRACT The air that humans exhale contains various chemical markers whose levels have been associated with various respiratory di sorders. Therefore, measurement of these markers offers a potential method of examin ing airway disease status. Furthermore, exhaled breath offers the advantage of be ing easy to collect an d non-invasive. Hence, these exhaled breath markers are potentially of significant clinical use in examining airways. Therefore, examination of exhaled breath has become the subject of intense study. Current research is ta rgeting the development of methods and parameters for looking at these markers. The goal of this cross-secti onal pilot study was to consid er the variability in the measurement of these exhaled breath marker s between members of different ethnic populations. Specifically, measurements of th e exhaled breath marker Nitric Oxide (NO) were compared between two ethnic groups (Caucasian men versus men of African descent). Ten healthy men in each group were studied to examine whether baseline NO measurements differed between them.

PAGE 8

vi In this study, a cross-sectional design was used. The study sample consisted of young, healthy men with no history of environmen tal allergies, asthma or lung diseases and no significant smoking history. A total of twenty-five men volunteered for the study, including fourteen men of Caucasian descen t and eleven men of African descent. Because four men were excluded and one w ithdrew, ten men in each ethnic group were included in the final analysis. The source population from which the samp le was drawn included students and workers. All participants were residing in Fl orida at the time of st udy. Ideally, the target population for this study was young, healthy, working men. Large inter-measurement variation was seen between the participants of each ethnic group. This was hypothesized to be attributed to a tri-modal distribution due to the existence of 3 populations of subjects: (1) asymptomatic with normal airways, where NO levels were under 30 parts per billion (ppb); (2) asymptomatic with airway pathology, where NO levels were over 30 ppb; and (3) as ymptomatic just before the onset of an upper respiratory tract in fection, where NO levels were over 60 ppb. This pilot study did not find statistically significant evidence that there is a difference in the baseline exhaled breath NO measurements between the two ethnic groups studied. Nonetheless, in participants with NO levels under 30 ppb the mean of the African group was found to be 7.6 ppb lower than the mean of the Caucasian group when attempts were made to exclude indivi duals with underlying airway pathology or imminent upper respirat ory tract infection. In order to find statistical significance in the results, a power analysis using the standard deviation of 7.7 ppb that was found in th is study indicates that at least thirty-two

PAGE 9

vii eligible participants with NO levels u nder 30 ppb would be required. Only 13 such participants were examined in this study. Thus, at least fift y eligible participants would be required to find significant results. The implication is that even though stat istical significance was not achieved, the crude mean averages differed between the tw o groups in participants with NO levels under 30 ppb. This implies that a larger-sca le well-designed study is warranted before NO is used in clinical settings in th e diagnosis and monitoring of patients.

PAGE 10

1 INTRODUCTION Methods of Studying Airway Disease Airway inflammation has been seen in a variety of pulmonary disorders, including asthma, chronic obstructive pulmona ry disease, and bronchiectasis. Current methods to evaluate for disease or inflammation include invasive procedures such as bronchoscopy, bronchial biopsy, and bronchoalveolar lavage Although it is not invasive, sputum induction can involve discomfort to patients. With this in mind, an alternate method of examining airway disease that is non-invasive and produces less discomfort to patients has been actively sought by researchers. Examination of exhaled breath markers offers a possible alternative method. When people exhale, they breathe out warm, hu midified air. This exhaled air contains gases, water vapor, and various volatile substa nces that come from the airways and the fluid that lines the airways. Several of these dissolved substa nces have been shown to be markers of airway disease or inflammation. Si nce this exhaled air is easily obtained in a non-invasive manner with little discomfort to patients, it shows promise as a method for examining the degree of airway disease and/or monitoring patient response to treatment. However, measurements of markers in exha led breath can vary based on the parameters used to study them. For example, the equipment used, the exhalation duration, the method of acquiring samples, and the expiratory flow rate can all affect measured values.

PAGE 11

2 Nitric Oxide in Exhaled Breath One of the exhaled breath markers currently being investigated is nitric oxide (NO). NO has been found to be elevated in various disease states, such as asthma and COPD during exacerbations. Other factors have also been found to affect NO levels. (See Table 1) [11] For example, a recent study performed by Doctors Robert Haight and Robert Gordon comparing nitric oxide levels in old versus young people found increased exhaled breath NO levels in older indivi duals (median = 36.9 ppb) when compared to young people (median=18.7 ppb). [17] Genetic variation may also play a role in the amount of NO that is exhaled. An understanding of possible sources of genetic variation may be obtained by examining the mechanism of NO synthesis. (See Figur es 1 and 2) Endogenous NO is primarily synthesized from arginine via the enzyme Nitric Oxide Synthase (NOS). (Kharitanov, 2001, p. 1694) [8] There are 3 different forms of NOS: (1) T ype INeuronal NOS, (2) Type IIInducible NOS, and (3) Type IIIEndothelial NOS. T ypes I and III are activated by small rises in intracellular calcium 2 o to cell activation. (Kharitanov, 2001, p. 1694) [8] Type II has a much greater level of activ ity; is independent of calci um concentration ; may be induced by inflammatory cytokines, endot oxin, and viral infec tions; and may show increased expression in inflammatory diseases. (Kharitanov, 2001, p. 1694) [8]

PAGE 12

3 Table 1Factors That Affect NO Measurements in Healthy Subjects INCREASED NO LEVELS DECREASED NO LEVELS Pharmacologic Papaverin NOS inhibitors L-arginine Oxymetazoline Sodium nitroprusside ACE inhibitors (enalapril) Physiologic and procedural Arginine ingestion Repeated spirometry nitrite/nitrate-enriched food Acute & transient after forced exhalation Physical exercise Sputum induction Menstrual cycle Reduced body temperature Environmental, Occupational Air pollution (NO, ozone) Water vapour, CO 2 nitrous oxide, heptane Occupational hazards: 100% inspired O 2 Fluoride, dust Moderate altitude Rubber latex Ozone, chlorine dioxide Formaldehyde (domestic) exposure Electromagnetic field generated by cellular phone (nasal NO) Habitual/ Infections URTI Smoking Alcohol ingestion Definition of abbreviations: URTI = upper respiratory tract infection, ACE = angiotensin-converting enzyme. Modified from Kharitanov SA and Barnes PJ Exhaled Markers of Pulmonary Disease. Am J Respir Crit Care Med 2001; 163: 1693-1722. [8]

PAGE 13

Figure 1. Nitric Oxide Synthesis (Enzyme) (Amino Acid) Modified from Kharitanov SA and Barnes PJ. Exhaled Markers of Pulmonary Disease. Am J Respir Crit Care Med 2001; 163: 1693-1722. [8] Figure 2. Genetic Induction of NOS Inflammatory Mediators Nucleus Muscle Modified from Kharitanov SA and Barnes PJ. Exhaled Markers of Pulmonary Disease. Am J Respir Crit Care Med 2001; 163: 1693-1722. [8] 4

PAGE 14

5 Genetic polymorphisms of all three isofor ms of NOS have been detected. Associations have been found between polymor phisms in the NOS1 gene and asthma in Caucasian populations In pati ents with mild asthma, there is a significant association between the length of the AAT repeat polym orphism in intron 20 of the NOS1 gene and exhaled NO levels. (Kharitanov, 2001, p. 1694) [8] These findings leave open the possibility that genetic varia tions among different ethnic grou ps could result in variations in baseline NO levels betw een different ethnic groups. It has been assumed that baseline NO le vels are the same am ong different ethnic groups. No prior studies have directly examined whether a difference may exist between the different ethnic groups. This study will examine whether ethnicity could be a factor that affects NO levels. Race and Ethnicity Considerations A race is defined as any population that differs from other populations of its species in the frequency of one or more genes. (Bryant, 2004) It is an arbitrary classification based on group. In comparison, an ethnic group is defined as a group of people who share a similar cultural and regi onal origin, hold common norms and beliefs, and form part of a larger population, interacting with people from other segments of society. Ethnic groups (1) share fundamental cultural and norms that distinguish them from other groups, (2) communicate and intera ct together, reaffi rming their ethnic identity, and (3) are recognized by members a nd other groups as distinct. (Bryant, 2004) [15] These qualities can create variations among pe ople. (See Next Section on Epidemiologic Considerations)

PAGE 15

6 In conducting a study, the most valid me thod of ethnic assessment is selfidentification. (Bryant, 2004) [ 15] In keeping with this, to assign ethnicity, participants of this study will be asked to identify their own ethnicity as a strict criterion. It must be recognized that historical patterns of mi gration, intermarriage, and distribution of genetic characte ristics suggest that there ar e no pure races. (Bryant, 2004) [15] As an example, a person who may be identified as Caucasian may have one parent who is Caucasian while another parent is Hispanic. To gain insight into the possibility of mixed ethnicity, pa rticipants will also be asked to identify the birthplace of their parents and whether they identify the ethnicity of two gene rations of ancestors differently from themselves. Epidemiologic Considerations A primary consideration in any research study is internal va lidity. Internal validity is defined as the extent to which the results of a study are correct for the particular group being studied. Internal validity is a prerequisite for external validity. (Sanchez-Anguiano, 2004) Once findings have b een made through research with care to preserve internal validity, a general epidem iologic challenge then becomes to provide information that can be applied to populati ons outside the specific one studied. [22] External validity is defined as the exte nt to which the results of a study apply to people who were not in the study. (Sanchez-Anguiano, 2004) Many times, in an attempt to preserve internal validity, exte rnal validity may become compromised. A potential cause of loss of external va lidity is ethnic differences. [22]

PAGE 16

7 In 1999 and 2005, the American Thoracic Society promulgated recommended practices and instruments for obtaining and measuring NO in exhaled breath. Ethnic considerations were not included in these recommendations. [3], [19] Ethnic differences have sometimes been found to result in variations in parameter measurements based on different factors su ch as socio-economic status (eg. access to health care), environmental factors, and ge netic variations. For instance, pulmonary spirometry test results require adjustments in people of African descent in order to portray a more accurate clinical representa tion when measuring their pulmonary function. [18] This suggests the possi bility that other pulmonary monitoring parameters may require similar adjustments based on ethnic differences. These adjustments results from generalized differences in variab les such as those listed above. Specifically, in this study, measurement of exhaled breath levels of NO were compared between two dominant ethnic groups in American society: people of nonHispanic Caucasian descent and people of Af rican descent. In performing this study, the goal was to begin to provide an examinati on of the generalizabil ity of exhaled breath measurements to various populations. Research Question Are Baseline Measurement Values of Airway Markers of Inflammation In Exhaled Breath Generalizable To Different Ethnic Populations?

PAGE 17

8 Hypothesis To study this, measurements of NO leve ls in exhaled breath were compared between people of Caucasian descent and pe ople of African descent. The null hypothesis was there is no difference in baseline measurem ents of exhaled breath nitric oxide levels between people of Caucasian descent and pe ople of African descent. The alternate hypothesis was that baseline measurements of markers of inflammation in exhaled breath varied among different ethnic populations. As measurement protocols are developed, such variations, if they exist, should be considered to improve accuracy in detecti ng and monitoring methods of airway disease states.

PAGE 18

9 Goals and Objectives The specific objective of the proposed st udy was to examine whether a difference may exist in baseline clinical measuremen ts of exhaled breath NO levels between young, healthy men in the two ethnic groups. The goals were: [1] to begin to provide an examination of the generalizability of measur ements of exhaled breath markers to various populations, which may serve as the basis for a larger and more in-depth study [2] to examine whether baseline NO measurements are appropriately representative of individuals from different et hnic backgrounds, [3] to examin e whether adjustment factors based on ethnicity need to be considered as protocols for measuring markers in exhaled breath become advanced for potential use in clin ical settings to improve clinical accuracy in detection and monitoring of lung diseases.

PAGE 19

10 Significance and Utility of Research The study was unique since it looked at a hypot hesis that had not been previously examined in measurements of exhaled breath. The examination provided insight into the possible applicability of measurements of markers in e xhaled breath to others groups in the general population. In performing the st udy, insight was provided to researchers who are actively developing methods and parameters to use markers in exhaled breath for the detection of pulmonary disease or for monitoring treatment.

PAGE 20

11 Materials and Methods Overall Study Design In this pilot study, the variable studied was ethni city and the outcome of interest was levels of the marker nitric oxi de in exhaled breath. A cross-sectional design was utilized where individual level data was obtained simultaneously. Ten men were compared in each of two ethnic groups. The men in the first group were of African descent, whereas the men in the second group were of Caucasian descent. In this examinati on, only healthy men aged 18-45 were studied. Health was defined as having no history of allergies, asthma, lung diseases, and no significant smoking history (defined as less than one-half pack year history with no smoking in the past 2 years). Also, since ce rtain medications have been found to affect NO levels, those participants taking any of the medications listed in Table 1 were excluded. Finally, men with severe heart disease or coronary vessel disease were excluded as a safety precaution. Facilities and Equipment The participants were seen at the College of Public Health at the University of South Florida in the Breath Laboratory (MHH Room 323). The records were maintained in a secured cabinet in this room. The key to access the laboratory was distributed by the University of South Florida only to authori zed personnel (obtained th rough the College of Public Health). The necessary equipment di scussed above that was needed to perform this project was available in this room.

PAGE 21

12 Participant Recruitment Healthy subjects were asked to volunteer fo r the study through one or two visits at the Breath Laboratory at the College of Public Health (MHH Room 323), an airconditioned, comfortable room. They were as ked to sit in the same comfortable chair. Subjects who expressed a willingness to vol unteer were given information about the study procedure, the risks, the benefits, and the alternatives to the procedure. They were then asked to demonstrate an understanding of all components discussed. They were encouraged to ask questions at any time. If they still agreed to proceed, they were asked to sign an informed consent. They were monitored by a physician throughout the visit via direct observation. Because performance of spirometry maneuvers immediately before NO measurement affects the NO levels obtained, subjects were given the option of presenting for either one or two visits base d on their own convenience. Those subjects who chose to present for one visit were asked to perform NO maneuvers before spirometry was obtained. Those subject who completed the study over two visits performed the spirometry maneuvers on the first visit and NO maneuvers on the second visit. For one hour pr ior to the visit where NO levels we re obtained, subjects were asked not to eat or drink anything and to refrai n from strenuous exercise as recommended in both the 1999 and 2005 American Thoracic Society (ATS) criteria. [2], [19] Study Subjects and Restrictions Only normal, healthy people aged 18-45 years old who denied any history of environmental allergies, asthma, or other lung di seases were invited to participate. Also, they must not have suffered from any recent upper or lower respiratory tract infections.

PAGE 22

13 [2], [19] The study was restri cted to men only because there have been reports that the level of exhaled breath NO differs between me n and women. [16] Subjects must also have no significant smoking history as defined by less than one-half pack-year history of smoking, no smoking within the past 2 year s, and no significant second-hand smoke exposure. Subjects must refrain from st renuous exercise, food, or drink for one hour prior to the test at a minimum pe r 1999 and 2005 ATS Criteria [2], [19]. Study Questionnaire and Eligibility During each subjects first visit, a questionnaire was initially given that asked for medical and demographic information. For t hose who participated in a second visit, the subjects were asked a brief questionnaire on the second visit to ascertain the absence of changes during the interim between visits. Duri ng each visit, subjects were also given a brief exit questionnaire to help assess any possible adverse effects from the study visit itself. (See Appendices A and B) The medical history was elicited to de termine health, and the demographic questionnaire determined age, attained ethnic aff iliation, and asked for SES variables. (See Appendix A) In this study, participants were administered a demographic questionnaire to assess ethnic ity that specifically asked, How would you classify your ethnicity? They were also asked where they and their parents were born. This was done to assess migration patterns and differences between subjects within each ethnic group. They were asked if they would classify the ethnicity of anyone in two prior generations of their ancestors differently from themselv es. Anyone who classifi ed 50% or more of their ancestors differently from themselves would be disqualified. To consider socio-

PAGE 23

14 economic status differences between the tw o groups (which may act as a potential confounding factor), participants were also as ked for their income level and occupation. Subjects who fulfilled the criteria of age, ethnicity, and being healthy then underwent brief physical examination including auscultation. Those subjects found to be healthy after questionnaire, interview, and physical exam ination were invited to participate in the remainder of the study. To be eligible for the study, all participants needed to show normal spirometry. Those subjects who chose to participate in two visits performed spirometry on the first visit and the NO maneuvers on the second visit. Alternatively, those subjects who chose to participate durin g a single visit performed the NO maneuvers prior to spirometry. The reason for this is that perf ormance of spirometry maneuvers could affect NO levels. Subjects who chose to participat e in two visits were given a brief entry questionnaire on the second visit, and auscultation was performed again to ensure absence of change in health st atus during the interval between the first and second visits. Subject Safety Considerations Subject safety was given the utmost cons ideration in this st udy. Subjects were told they may discontinue at any time. The subjects were healthy individuals who agreed to participate in th e study. The entire study was non-invasive. Furthermore, physical examination and in itial medical screening questionnaires were reviewed directly by a physician prio r to any procedure be ing performed. Only subjects who had normal physical examination were allowed to continue in the study. Spirometry, which is commonly used in cl inical practice to a ssess lung function, was

PAGE 24

15 obtained as an eligibility cr iterion. To obtain spirometr y, subjects blew out through a disposable filter (to prevent infection) usi ng maximum effort after a maximal inhalation. Measurement of exhaled breath NO, which is cu rrently still a research tool, involved tidal breathing and normal respiration through a disposable plastic f ilter. Therefore, it was even safer to obtain than spirometric measurements. During each visit, subjects were also gi ven a brief exit questionnaire to help assess any possible adverse effects from the stud y visit itself. For t hose who participated in a second visit, the subjects were asked a brief questionnaire on the second visit to ascertain the absence of changes dur ing the interim between visits. Participation in this study did not affect the standard care subjects would receive from their personal physicians unless [1] a bnormal auscultation or spirometry was found, in which case the subject was advised to consult their personal physician, or [2] an adverse effect occurred. In the latter case, most events would only be temporary and subside within seconds or minutes after st opping the activity. Although rare, a subject could experience chest pain or other advers e event during spiromet ry or exhaled breath nitric oxide measurement. In that event, emergency equipm ent was available at all times during the study to assist the individual until appropriate em ergency personnel arrives. To ensure patient safety, all subjects we re directly observed by a physician who was ACLS-certified during the enti re visit. The sampling procedure was completely noninvasive. Since real-time measurements of exhaled breath were made, no samples were kept beyond their immediate measurement. For those individuals who participat ed in two visits, a questionnaire was administered at the start of the second visit to ensure the absence of new symptoms in the

PAGE 25

16 interval between visits. Also, these participants were verbally asked if there had been any changes in their health status or medications in the interim. Physical Examination All subjects were assessed for cyanos is and clubbing. Also, auscultation was performed prior to any breathing tests a nd at the conclusion of each visit. Cyanosis is a bluish discoloration of the skin resulting from an inadequate amount of oxygen in the blood. Cyanosis occurs when oxygen-depleted blood, which is bluish rather than red, circulates through the skin. Cyanosis can be caused by many types of severe lung or heart disease or certain malformations that produce low levels of oxygen in the blood. Clubbing is an enlargement of the tips of the fingers or toes and a loss of the angle where the nails emerge. Finger clubbing occurs when the am ount of soft tissue beneath the nailbeds increases. The reason this increase occurs is not clear, but clubbing seems to occur with some pulmonary disorders (lung can cer, lung abscess, bronchiectasis), but not with others (pneumonia, asthma, emphysema) Finger clubbing also occurs with some congenital heart diseases or, in some cases may be inherited and not indicate any disease. (Merck, 2005) [20] In auscultation, a stethoscope is used to listen to both heart sounds and breath sounds. Any subject demonstrating cyanosis, clubbing, or abnormal breath sounds was immediately disqualified from the study. Au scultation of the heart was also performed for the purposes of subject safety.

PAGE 26

17 Acquisition of Spirometry Spirometry, which is a common clini cal method employed in assessing lung function, was performed as part of the entry criteria into the study. The Koko spirometer was used and calibrated usi ng a standard 3-liter syringe to ambient temperature, humidity, and barometric pressure at least once a day on days when participants were examined. The spirometer was additionally re-calibrated when at least six hours had elapsed since the prior calibration or at the discretion of the examiner. The raw FEV1 and FVC measurements obtained for each participan t were automatically compared to their predicted normal values based on age, ethni city, weight, height, and non-smoking status in determining their percent of predicted values using parameters as set forth by Crapo et. al. The spirometry parameters examined on each study participant included FEV1, FVC, FEV1/FVC ratio, and the flow-volume loop. At least three spirometric measurements were obtained on each subjec t, with at least one flow-volume loop showing good effort. Proper technique was en sured by evaluating the flow-volume curve and continuance of the expiratory maneuver for at least six seconds according to ATS criteria. A noseclip was placed on the subjects noses during the study to prevent nasal breathing in order to obtain more accurate spir ometry results. Subjects were asked to forcibly exhale for at least six seconds through a disposable single-use filter after maximal inspiration, followed by anothe r maximal inhalation. The maneuver was demonstrated to them to help achieve consistency.

PAGE 27

18 Any subject with abnormal spirometry measurements was notified of this information and advised to consult a healthcar e provider and disqualifi ed from the study. All subjects with an FEV1/FVC ratio greater than 70% of predicted, FEV1 greater than 80% of predicted, FVC greater than 70% of predicted, and a normal appearing flowvolume loop were eligible for the study by spirom etric criteria. In t hose participants who did not meet all of these stri ct criteria, the overall clinical picture, including medical history and physical examination were assessed individually to determine study eligibility. When eligibility due to spirometry did not meet th ese strict criteria, Dr. Stuart Brooks (faculty advisor, Board Certified in Pulmonary Medicine) was consulted to determine whether the participant would be eligible for the study. Adverse effects were unlikey to occur during this phase of the study because subjects identified themselves as being in good health, the room was temperature controlled, and this phase of the study was brief in duration. All subject were seated during the spirometry. In the event that ad equate trials could not be obtained in the seated position as demonstrated by their flow-volume loops, subjects were asked to stand and instructed on safety precautions in the event of symptoms. Nonetheless, there was the slight possi bility of unusual symptoms such as lightheadedness, dizziness, chest pains, palp itations, or shortness of breath during the spirometry from overexertion in breathing. As a precaution, subjects were instructed to stop, be seated, and notify the physician examiner immediately if they experienced any symptoms at any time.

PAGE 28

19 Measurement of Exhaled Breath Nitric Oxide The Nitric Oxide Analyzer was calibrate d at least on a daily basis on days where subjects were being examined. It was calibra ted again when at leas t 6 hours had elapsed since the last calibrati on. Additional calibrations occurred at the judgment of the study examiner. NO analysis calibrations were ma de using zero NO air and air containing 45 ppm NO. The flow meter was calib rated with a 3-liter syringe. Subjects were asked to maximally inhale ambient air and then to immediately breathe out normally through a single-use disposab le filter mouthpiece attached to a flow meter at a constant flow rate of 50 mill iliters per second as recommended by the ATS. The mouthpiece also provided a specific amount of resistance during exhalation to allow for velopharyngeal closure to prevent contamination of the exhaled breath NO measurements with nasal nitric oxide. (See Figures 3 and 4) Because exhaled breath NO concentrations depended on the ra te of airflow, subjects exhale d at a constant airflow rate determined by the Nitric Oxide Analyzer (NOA) through biofeedback by real-time computer display of their own flow rates. The NO flow rate was equal to the product of NO concentration in the exhaled air multiplied by the airflow rate. [2], [19] The following ATS recommendations were followed. ATS recommended an exhalation duration of at least six seconds ( up to 30 seconds) to obtai n at least a 3-second plateau of the measured NO level. The plateau was defined as the first portion of the NO versus time profile where [the difference bewe en the start of the plateau and the end of the plateau varied by no more than] 10%. O nline electronic analysis of NO profiles allowed automatic identification of valid NO plateaus according to these criteria. Repeated reproducible exhalations were performed, resulting in three NO plateau

PAGE 29

values that agreed within 10% of the mean value. [The average NO level] was then calculated as the mean of these three values. At least 30 seconds of relaxed tidal breathing off the NO measurement circuit elapsed between exhalations to allow the subject to rest. (ATS, 2005, p. 916) (See Figure 5) [2] In certain cases, where automatic computer selection chose clearly incorrect plateau levels, the most appropriate 3-second plateau that met ATS criteria was selected for the calculation of the average NO level. Figure 3. Nitric Oxide Flow Rate Versus Time [2], [19] Plateau 1 Plateau 2 Plateau 3 Modified from the American Thoracic Society The mouthpiece apparatus was specially designed with an attached filter during inhalation to provide inhalation air at less than 5 ppb NO. Alternativelyas used in this studyambient air was inhaled, which caused an early peak in real-time NO levels. In this case, plateau levels of NO were equivalent to when NO-free air was inhaled. Figure 4. Flowmeter Design [2], [19] 20

PAGE 30

Modified From the American Thoracic Society p. .916 Air Place Mouth Here Figure 5. Effect of Inhalation of Air Through the Flowmeter Versus Inhalation of Ambient Air [2], [19] Modified From the American Thoracic Society, 2005, p.916 Early Peak Gradual Rise Inhale air with <5ppb NO Inhale Ambient Air Nitric oxide in exhaled breath was collected in a chamber in the Sievers 280i Nitric Oxide Analyzer (Boulder, CO) and measured according to recommended methods set forth by the American Thoracic Society in 1999 and 2005. The machinery contained 21

PAGE 31

an ozone generator that produced ozone in the chamber containing the exhaled breath. The ozone reacted with the NO within the chamber according to the following photochemical reactions: NO + O 3 NO 2 + O 2 NO 2 NO 2 + hv NO reacted with ozone to form nitrogen dioxide in an electronically excited state, which then emitted light in the red and near-infra-red region of the electromagnetic spectrum to return to the more stable state. This red light generated from the reaction was detected by a thermoelectrically cooled, red-sensitive photomultiplier tube within the machine. The Sievers 280i Nitric Oxide Analyzer (Boulder, CO), a high-sensitivity detector for measuring NO based on a gas phase chemiluminescent reaction between nitric oxide and ozone, was used in measuring NO levels. The Nitric Oxide Analyzer (NOA) had a sensitivity of 1 ppb NO and measured from 0.1 to 500 ppb nitric oxide within a timeframe of 500msec. (Sievers, 1995) [14] Since this limb of the study was not a maximal forced exhalation, subjects were even less likely to experience symptoms during this phase of the study. However, subjects were be given the same instructions as during the spirometry phase of the study. Data Collection and Analysis To appropriately assess the research question, a large number of eligible subjects would have been required to participate. Primarily because of the limited availability of subjects who were willing to participate and who met the criteria for entry into this study, 22

PAGE 32

23 it had been undertaken as a pi lot study. Therefore, the study was performed with the goal of examining at least twenty eligible subjec ts to provide insight into the question of whether a difference may exist between the two ethnic groups. A power calculation was performed which was derived using alpha= 0.05 and power=80% to show the level of difference in the means of the two groups that was needed for significance given a specific sample size. (See Appendix E) To do the power calculation, a recent study by Kharitanov et al. [9] which reported an av erage NO level of 17.8 with a standard deviation of 6.8 was used for the calculations. In any study where there are two groups being compared, th e participants are ideally randomly assigned to each group. However, random assignment is not possible when two distinct groups are being used such as men versus wome n or young versus old. That was the case in this study, where the tw o distinct groups were people of Caucasian descent versus people of African descent. The mean of each of the two groups in a study sample may be compared using a Students t-Test, a parametric analysis, when the population studied is normally distributed. When the population is not norma lly distributed, a non-parametric analysis must be used. In the case where two distin ct groups are being compared, non-parametric tests include the Wilcoxon Ranked-Sum Test or the Mann-Whitney U Test. Because the overall study set was not normally distribut ed (probably tri-modal), the Wilcoxon RankSum Test was used in the statistical analysis of this data. When those individuals with extremely high NO levels were excluded, the re maining data consisted of those subjects who were asymptomatic but either did or did not have underlying airway pathology. Therefore, a probable bi-modal distribution rema ined, also requiring calculation using the

PAGE 33

24 Wilcoxon Rank-Sum Test. When all participants with levels over 30 were also excluded, the data better resembled a normal distributi on for which a Students t-Test could be used. However, upon statistical analysis using SAS, the average NO values obtained between the two ethnic groups were found to c onsist of different variances. Hence, the Satterthwaite Test was a more appropriate test to use in this case than the Students tTest. (See Appendixces G and H ) Statistical calculations were performe d by inputting the data into Statistical Analysis Software (SAS), which calculated the data automatically. For this study, SAS was utilized to calculate de scriptive statistics such as the mean, median, variance, standard error of the mean, standard de viation, range, minimum and maximum values, skewness, and kurtosis both for each ethnic group and for all of the study subjects together. SAS was also used to calculate infe rential statistics: the Satterthwaite T-Test was completed for those subjects with average NO levels below 30. The Wilcoxon Rank-Sum Test was used for stat istical analysis of data that was not normally distributed. In a crude attempt to assess whether there might be a correlati on between SES and NO levels, a Pearson-Product Correlation Coefficient was calculated.

PAGE 34

25 Study Results Exclusions In response to recruitment strategies, approximately 15 to 20 men besides those who actually participated expressed an intere st in being part of the study. These men were excluded on initial contac t based on failure to meet all of the entry criteria. The primary reasons included not meeting the ag e criteria, a history of significant smoking, environmental allergies, or asthma. Failure in proper communication was dem onstrated by five men who presented to the lab for testing but did not m eet the inclusion criteria. They were excluded early in the informed consent process. One Caucas ian was excluded due to a history of environmental allergies. Another Caucasian male was excluded for current upper respiratory tract infection. He was invited back into the study but chose to withdraw. Two men of African descent were excluded due to a significant smoking history. A fifth person was excluded early in the questionnair e process after discovering that he was Hispanic. No one was excluded due to abnormal auscultation. All of the men who underwent auscultation had normal cardiac examin ations with S1 and S2 heart sounds with regular rate and rhythm and no murmurs, rubs, or gallops. Lung exams were clear to auscultation bilatera lly with no audible wheezes, rales, or rhonchi. No cyanosis or clubbing was noted in the upper extremities.

PAGE 35

26 All of the men who completed the spir ometry maneuvers had FEV1/FVC ratio values above 70%. After complete particip ation, two Caucasian men were excluded for markedly prolonged expiratory phases on thei r flow-volume loops despite having FEV1, FVC, and FEV1/FVC ratio values that were above the cutoffs for exclusion from the study. Interestingly, both of these men were Caucasian and had the best FEV1/FVC ratios between 80-85%, whereas the men who were included had best FEV1/FVC ratios between 90-110%. One of these two men wa s found to have an average NO level under 10 parts per billion (ppb), while the othe r had an NO level between 40-50 ppb. A third Caucasian male who completed the study wa s subsequently eliminated because he reported taking oral steroids. His NO level was between 10-15 ppb. One participant of African descent who completed the study and de nied any history of allergies but reported significant runny/ irritated nasal passages compounded with poor spirometry results was eliminated due to both spirometry and possible concurrent URI or newonset of allergies. His NO level was 54.5 ppb. Questionnaire Collection The following tables demonstrate the results of the demographic information that was collected during the study. TABLE 2. Number of Subject s in Each Ethnic Group Ethnicity Frequency Percent African 10 50.00 Caucasian 10 50.00

PAGE 36

Distribution By Age Age (yrs) 19 20 21 22 23 24 31 33 34 36 39 40 African 0 1 0 1 2 3 1 0 1 1 0 0 Ethnicity Caucasian 1 0 2 1 1 0 0 1 0 1 1 2 Figure 6. Participant Distribution By Age 192021222324313334363940 African 00.511.522.53 Number of ParticipantsAge (in years)Distribution By Age African Caucasian 27

PAGE 37

28 Table 3. Sociodemographic Characteristics Ethnicity African Caucasian Birthplace US 6 10 Africa 3 0 Jamaica 1 0 Different Ancestry By Ethnicity? No 8 9 YesParents Born in Haiti 1 0 Yes-1/4 Hispanic 0 1 Yes-Less than 1/4 Various Mixed 1 0 Current Occupation Para-Professional (Occupation Code=1) 1 2 Student (Occupation Code=1) 6 3 Professional (Occupation Code=2) 3 5 Annual Salary $0-$20,000 6 4 $20,000-$50,000 3 4 $50,000-$100,000 0 1 Over $100,000 1 1

PAGE 38

29 Two variables for socio-economic status (SES) were elicited by the demographic questionnaire. These included current inco me and current occupation. Because there may be an association between lower SES and asthma, and nitric oxide levels are higher in people with asthma, a Pearson-Product Moment Correlation was performed to determine if lower SES is correlated with a higher nitric oxide level. [21] To do this, the raw data needed to be conve rted into numerical values to plug into the equation for the Pearson-Product Moment Correlation. Accordingly, the subject responses were classified into three groups for occupation: Student, Para-Professional, or Professional. Students and Para-Professi onals each received a score of one point whereas professionals received a score of tw o points. Salary information, which was elicited at $10,000 intervals, wa s rated at one point for a salary of <$20,000; two points for $20,000-$50,000; three points for $50,000-$100,000, and four points for >$100,000. The scores from the employment data and the salary data were then multiplied together for each individual to arrive at a score repr esenting their socio-economic status. These values were used in the statistical analysis ag ainst nitric oxide levels to better visualize the descriptive data already obtained.

PAGE 39

30 Table 4. Pearson-Product Correlation Coefficient Average NO3 Variables: [1]Salary Code [2]Occupation Code, [3] SES Code Product Variable N Mean Std Dev Sum Minimum Maximum Average NO 20 33.55500 23.47091 671.10000 8.90000 94.40000 Salary Code 20 1.75000 0.96655 35.00000 1.00000 4.00000 Occupation Code 20 1.40000 0.50262 28.00000 1.00000 2.00000 SES Code Product 20 2.80000 2.35305 56.00000 1.00000 8.00000 Pearson Correlation Coefficients, N = 20 Prob > |r| under H0: Rho=0 SalaryCode OccupationCode SESCodeProduct r p-value -0.05156 0.8291 -0.21656 0.3591 -0.12463 0.6006 When the raw data was considered, a hi gher NO level was weakly inversely correlated with SES. However, because the p-valu e was greater than 0.05, the test was not statisrically significant. Medical Information Following are [1] the medical problem s that were reported by participants on the medical questionnaire, [2]the findings on the physical exam, and [3]the adverse events reported by participants upon completion of the study.

PAGE 40

31 Table 5. Medical Information Ethnicity African Caucasian Prior Respiratory Tract Ailments None 10 8 Reports Bronchitis >5 years ago 0 1 Once told he had child-h ood asthma but never took medications for this; he ha s never had any symptoms 0 1 Current Medications None 7 7 Diabetes medication 1 0 Glucovance, Enalapril 1 0 Maxzide, ASA 1 0 Topical Steroids 0 1 Tramadol 0 1 Doxycycline for acne 0 1 Inhalation Exposures Denied 10 7 Firefighter x 5yrs 0 1 Roomate smokes, but not in common areas 0 2

PAGE 41

32 Ethnicity Other Medical Condition s Reported By Subjects African Caucasian None 6 3 Acne 0 1 Atopic Dermatitis 0 1 Back Pain; Anxiety 0 1 Diabetes Mellitus; Palpitations from Coffee 1 0 HTN 1 0 HTN; Diabetes 1 0 Irregular Heart Beat 0 1 Irregular Heart Beat when eating pepperoni 0 1 Musculoskeletal Complaints 1 0 Prior subdural hematoma 0 1 groin hernia age 4 repaired 0 1 Ethnicity Physical Exam African Caucasian WNL 9 9 Obesity 1 1

PAGE 42

33 Ethnicity Adverse Events African Caucasian None 9 9 None/ got URI that night 1 0 Raspy Voice and tiredness several h ours later; sick the next day 0 1 Spirometry Measurements Of the twenty-four participants on whom spirometry was performed, three Caucasians were excluded: two for having a prolonged expiratory phase on spirometry, and one for taking oral steroids. A fourth male of African descent was excluded for low FEV1 and FVC values; he also reporting ir ritated nasal passages with rhinorrhea. The following participants were entered in to the study after approval was received from Dr. Brooks. One morbidly obese ma n (>300 pounds) of African descent who had low FEV1 and FVC values had a normal fl ow-volume loop and normal FEV1/FVC ratio. His low FEV1 and FVC values were attributed to his increased gi rth pushing against the diaphragm, creating limitations in total lung capacity. One man of African descent who was 79 inches tall had normally appeari ng flow-volume loops and normal FEV1/FVC ratios but low individual FEV1 and FVC readings. Because spirometry is less accurate at the extremes of height, he was included in the study. Two men w ith normally appearing flow-volume loops, normal FEV1/FVC ratios, and with FEV1 and FVC trials very close

PAGE 43

to 80% were kept in the study after discussion with Dr. Brooks (some degree of operator error was involved in each of these two cases). Exhaled Breath Nitric Oxide Measurements 010203040506070809010013579Average NO Level (ppb ) African Descent Caucasian Descent13579 Caucasian Descent The data obtained during this study are shown in the figure below: Figure 7. Average NO Level Of Each Subject By Ethnicity (Note: Each + Or x Represents One Participant) Visualization of the overall results in Figure 7 show that the data obtained was not normally distributed for either group. Inspection of the distribution of values for African descent reveals that the data may have contained three or four modes. The statistical analysis of the data is presented in Table 6. Table 6. Statistical Analysis of Average NO Levels for all 20 Participants Overall African Caucasian N 20 10 10 Sum Observations 671.1 356.6 314.5 34

PAGE 44

35 Table 6. Statistical Analysis of Aver age NO Levels for all 20 Participants Median 28.80000 29.20000 27.40000 Mean 33.555 35.66 31.45 Lower 95% Confidence Level for Mean 22.5702756 14.1217452 20.2160745 Upper 95% Confidence Level for Mean 44.5397244 57.1982548 42.6839255 Std Deviation 23.4709109 30.1084041 15.703945 Variance 550.883658 906.516 246.613889 Std Error Mean 5.24825522 9.52111338 4.96602345 Skewness 1.5873052 1.2238969 2.61550195 Kurtosis 1.94120303 0.45564928 7.43705885 Range 85.50000 85.50000 52.90000 Minimum 8.9 8.9 21.0 Maximum 94.4 94.4 73.9 The above data showed that the mean of the African group was slightly higher than the mean of the Caucasian group. The medians differed by only 1.8 ppb. The African group had a greater variance wher eas the Caucasian group showed less variance in data with significantly greater kurtosis (peakedness of the curve).

PAGE 45

36 Table 7. Wilcoxon Scores (Rank Sums) for Variable Average NO Classified by Variable Ethnicity Ethnicity N Sum of Scores Expected Under H0 Std Dev Under H0 Mean Score African 10 104.50 105.0 13.223782 10.450 Caucasian 10 105.50 105.0 13.223782 10.550 Average scores were used for ties. Wilcoxon Two-Sample Test Statistic 104.5000 Normal Approximation Z 0.0000 Two-Sided Pr > |Z| 1.0000 t Approximation Two-Sided Pr > |Z| 1.0000 Z includes a continuity correction of 0.5. To show a significant result, the twotailed z-score calculated by the WilcoxonRank Sum analysis would need to be at le ast 1.96 (alpha=0.05%). Thus non-parametric statistical analysis using Wilcoxon-Rank Sum failed to reject the null hypothesis that there is no difference between the means of the two ethnic groups. When the extreme values above fifty were removed from the data set, the following non-normally distributed results were obtained:

PAGE 46

37 Table 8. Statistical Analysis of Averag e NO Levels When Excluding Extreme Values Overall African Caucasian N 17 8 9 Sum Observations 420.8 180.2 240.6 Median 25.90000 20.85000 25.90000 Mean 24.7529412 22.525 26.7333333 Lower 95% CL for Mean 19.8197848 11.6675971 22.7272157 Upper 95% CL for Mean 29.6860976 33.3824029 30.7394510 Std Deviation 9.59473278 12.986999 5.21176554 Variance 92.0588971 168.662143 27.1625 Std Error Mean 2.32706451 4.59159753 1.73725518 Skewness -0.2552595 0.25915577 0.5170626 Kurtosis -0.8535283 -2.0829279 -1.1213304 Range 30.70000 30.70000 14.50000 Minimum 8.9 8.9 21.0 Maximum 39.6 39.6 35.5

PAGE 47

38 Again, the above data showed a significan tly different variance between the two groups. This time, however, the mean of th e African group is somewhat lower than the mean of the Caucasian group. The median s differed between the two groups by 5.05 ppb. Table 9. Wilcoxon Scores (Rank Sums) for Variable Average NO Classified by Variable Ethnicity When Extreme Values Are Excluded Ethnicity N Sum of Scores Expected Under H0 Std Dev Under H0 Mean Score African 8 65.50 72.0 10.385935 8.187500 Caucasian 9 87.50 81.0 10.385935 9.722222 Average scores were used for ties. Wilcoxon Two-Sample Test Statistic 65.5000 Normal Approximation Z -0.5777 One-Sided Pr < Z 0.2817 Two-Sided Pr > |Z| 0.5635 t Approximation One-Sided Pr < Z 0.2858 Two-Sided Pr > |Z| 0.5715 Z includes a continuity correction of 0.5.

PAGE 48

39 Non-parametric analysis by the Wilc oxon Rank-Sum test failed to find a statistical difference between the means of the two ethnic groups since the two-sided pvalue is greater than 0.05. It must be kept in mind that this was a p ilot study. Because of the small sample size, there was less reliability in the statistical analysis. A significantly larger sample size would have been needed to determine whether or not a truly significant difference exists. When the data was re-calculated using only those values below 30 in a crude attempt to exclude those asymptomatic part icipants with underly ing airway pathology, the following data was obtained: Table 10. Statistical Analysis of Average NO Levels When Excluding Levels over 30 ppb Overall African Caucasian N 13 6 7 Sum Observations 274.7 102.2 172.5 Median 22.50000 12.60000 22.90000 Mean 21.1307692 17.0333333 24.6428571 Lower 95% CL for Mean 16.4543913 7.0101150 21.3635183 Upper 95% CL for Mean 25.8071471 27.0565517 27.9221960 Std Deviation 7.73858133 9.55105579 3.54582249 Variance 59.885641 91.2226667 12.5728571 Std Error Mean 2.14629629 3.8992022 1.34019493

PAGE 49

40 Table 10. Statistical Analysis of Average NO Levels When Excluding Levels over 30 ppb Skewness -0.4877608 0.8640237 0.61145295 Kurtosis -1.2966622 -1.8466342 -1.6296301 Range 20.80000 20.80000 8.70000 Minimum 8.9000000 8.9 21.0 Maximum 29.7000000 29.7 29.7 Table 11. t-Test of Average NO Levels When Excluding Average NO Values Over 30 Variable Method Variances DF t Value Pr > |t| Average_NO Pooled Equal 11 -1.97 0.0748 Average_NO Satterthwaite Unequal 6.18 -1.85 0.1131 This time, the means of the two groups differed by 7.6 ppb, whereas the medians differed by 10.3 ppb. Although the two groups still had different variances, the difference in the variances between the two ethnic groups was not as great as be fore. Again, the statistical analysis (Satterthwaite Test for normally dist ributed data with diffe rent variances) failed to show a difference between the two mean s of the ethnic groups as the p-value was greater than 0.05. However, the mean of th e African group had dropped even more than the decline in the mean of the Caucasian group so that there was an even greater difference between the means of the tw o groups in this small pilot study.

PAGE 50

41 Using the and data obtained from the normally-distributed participants in this study with average NO levels under 30 ppb, the pow er of the analysis of was calculated to be 36.5%. Table 12. Calculating The Power Of The Study 1 N 1 =6 2 N 2 =7 NCP Critical Value Power 17.0333 24.6429 7.73858 0.05 3.12391 4.84434 0.36483 Because = 1 Power, beta (or the probability of making a type II error) was calculated to be equal to 63.5%.

PAGE 51

42 Discussion Findings To reiterate, the question of interest has been whether a difference exists in the average NO levels between two ethnic groups: Ca ucasians and people of African descent. First, information on demogra phics, socio-economic status, and medical information was obtained and described. The de scriptions were summarized as: [1] There was more age variability in the Caucasian group when compared to the African group; [2] all Caucasians were born in the United States, whereas four of the people in the African group were born outside the Un ited States; [3] two men in the African group and one man in the Caucasian group report ha ving different ancestry; [4] five Caucasians worked in professional occupations, whereas only three of the African men worked in professional capacities; [5] most participants re ported annual incomes under $50,000. Next, in a crude attempt to examine whether a correlation may exist between socio-economic status (SES) and average NO levels, individuals were given numerical assignments for their stated occupation and income responses, which were then multiplied together and used to calculate a Pearson Product Correlation Coefficient (r). The analysis resulted in a weakly negative r-value, indicating that there may be a weak inverse relationship between SES and NO levels However, as expected with the small sample size studied, the analysis did not yi eld a statistically significant result. The average NO levels automatically determined by the NOA for each of the participants were then plotted to visua lize the variability in NOA-de termined average NO levels

PAGE 52

43 obtained. (See Figure 7) Insp ection of this figure pointed to a non-normal di stribution of the data with possible tri-modal distribut ion. One mode centered around extreme NOAdetermined average NO values over 60 ppb. A second mode included individuals with normal airways. A possible third mode could include asymptomatic individuals with underlying airway pathology with NO levels between 30-40 ppb. Therefore, the average NO level data was analyzed in 3 ways: [1] nonparametric analysis of the average NO leve ls for all 20 eligible participants, [2] nonparametric analysis of the average NO leve ls excluding participants with extreme NO levels over 70 ppb, and [3] parametric anal ysis of the average NO levels excluding participants with averag e NO levels over 30 ppb. In this pilot study, none of the statistical analyses showed significance. Nonetheless, when attempts were made to exclude individuals with average NO levels over 30 ppb so that the data becomes normally distributed, the mean and median of the average NO levels for the African group was 7.6 ppb and 10.3 ppb lower than the mean and median for the Caucasian group, respectively. This indicated th e possibility that a statistically significant differe nce could be found in this an alysis group when a larger sample size is studied. The power and of the study for the group of participants with NO levels under 30 ppb were calculated to be 36.5% and 63.5%, respectively. Thus, there is a high likelihood of committing a Type II error in this group. Therefore, in participants with NO levels under 30 ppb, the high Type II error rate indicates a high probability of missing a true difference between the two means if one really exists. In order to find statistical significance in the results, a power analysis was performed. It indicated that at least thirty-two eligible participants with NO levels under

PAGE 53

44 30 would be required. (See appendix E). At le ast sixty eligible participants would be required to obtain a study sample of si xteen in each group. The number of willing participants needed would be substantial given the strict criteria of the study where anyone with a known history of smoking, enviro nmental allergies, asthma, or other lung diseases was excluded. Even then, those pa rticipants who were asymptomatic but who demonstrated either [1]abnormalities after sp irometric testing (for example, prolonged expiratory phase), or [2]high NO levels possible indicative of underlying airway pathology or an imminent upper respiratory tract infection would also need to be excluded. Of the three participants who were excluded due to clinically abnormal spirometry, two had high NO levels, whereas one had a low NO level. Thus, the presence of abnormal spirometry did not absolutely predict a high NO level in this tiny number of excluded participan ts for spirometry criteria. As may be expected, one participant taking oral steroi ds had a low NO level. Implications The implication gleaned from this stu dy was that statistical significance was not achieved due to the small sample size. However, the crude mean of the average NO levels in the normally-distributed male pa rticipants (with average NO levels under 30 ppb) of African descent was found to be 7.6 ppb lower than that for their Caucasian counterparts. This implies that a larger well-designed study ma y find a statistically significant difference and provides a basi s for performing a more extensive study including both men and women and examining varying ethnic backgrounds (for example,

PAGE 54

45 Alaskan native, Pacific islander, etc.) in addition to those studied here prior to the use of NO in clinical settings to in the diagnosis or monitoring of patients. Future Directions After investigators of a concurrent ca psaicin study menti oned that certain asymptomatic participants in their study with extremely high NO levels became ill with an upper respiratory tract infection within the next several days of testing, three participants with NO levels over sixty were as ked to notify the inves tigator of this study if they became ill within the next week. Two of the three study participants reported the onset of an upper respiratory tract infection with in the next two days. The third participant had not yet made contact with th e investigator. This generated the hypothesis that NO levels may be extremely high in norma l individuals up to a few days prior to the onset of an upper respiratory tract infection. Because the current study was not designed to investigate this hypothesis, this conclusion would need to be further investigated in a properly designed and controlled study to ex amine whether or not this is the case. Other hypotheses generated by this pilot study include a possible weak inverse correlation between SES and average NO level and a possible tri-modal distribution of NO levels. These would also need to be i nvestigated in larger, well-controlled studies designed to specifically examine these questions. Study Limitations As a pilot study with only 20 eligible part icipants, the entire study was of limited value. Nonetheless, the goal here had been to provide a cu rsory look at the question of

PAGE 55

46 whether a difference may exist in baseline nitr ic oxide levels in members of different ethnicities. Limitations of the study coul d effect on its validity. In this study, calculation of the sample size was based on the mean and standard deviation as measured in a prior study. [22] However, it was unknown whether th ese were the true population values for these parameters. The values measured for mean and standard de viation of average NO levels in this study varied significantly from the prior values. This could affect the accuracy of the measurement. Errors due to chance may have occurred due to small sampling size. The stronger the association is in na ture, the easier it is to find with a small sample size. The closer the true association is to the null value, the larger sample size you need to find it to be statistically significant. (Sanchez-Anguiano, 2004, unit 6) [22] Therefore the results of this study showed that it was highly unlikely that there was a strong association between exhaled breath NO levels and ethnicity. When the sample is too small, a study is more likely to miss an association, increasing the likelihood of a Type II error. (Sanchez-Ang uiano, 2004, unit 6) [22] A Type II error may have been committed in this study where a weaker association could still be present and would not be detected by this study. Subjects were asked to participate based on the eligibility criteria of no history of allergies, asthma, or lung diseases and no si gnificant smoking history. Participants with NO values over 30 may have had imminent upper respiratory tract infection or underlying airway pathology despite being asym ptomatic. With this in mind, the data was also calculated when the participants with levels over 30 ppb were excluded. In

PAGE 56

47 doing this, the sample size was reduced even more, further increas ing the likelihood of error due to chance. Another way the results of the study may have been limited was through bias. There were numerous biases that may have occurr ed during the study. An example included prevalence-incidence bias, where participants who were mild or silent cases were missed by the entry criteria and allowed to participate. [22] Thus, they were misclassified. Other biases may have in cluded observer bias because no blinding occurred during the study. Self-selection bias was present since vol unteers were used. [22] Also, a susceptibility bias was present since all subjects were students or workers, creating a healthy worker effect. Thus, the study could not be generalized to the general population. [22] Unknown confounders may also have affected the study. In this study, one participant reported taking en alapril on a regular basis. The investigator had inadvertently overlooked that this medication has been found to raise exhaled breath NO levels (See Table 1). However, because the participants average NO level was below 10 ppb, his NO level off the medication would have been even lower than the measured value. In that case, the difference betw een the means of the two ethnic groups would have increased slightly, giving more power to the study. In essence, the effect of the oversight was in the direction closer to th e null hypothesis. Because the power of the study was so low, it did not dramatically impact the results of the study. Methods of controlling for confounders included restriction, randomization, and matching. [22] Multiple restrictions were us ed in the study design to attempt to control for confounders. Although randomization is a powerful method of controlling for

PAGE 57

48 confounders, it could not be used in this st udy because two intact groupsCaucasians and Africanswere being compared. Matching woul d also have been beneficial. However, it would have been difficult and tedious. On ce a factor had been matched, it could not be studied as a risk factor for disease. Becau se the matching would have been by factors of socio-economic status, their relationship to NO levels could not have been examined if participants were matched based on these f actors. (Sanchez-Anguia no, 2004, unit 8) [22] [epi unit 8 p. 12] In performing statistical an alyses where two distinct groups were compared, three underlying assumptions were made: (1)Assu mption of Normality, (2)Assumption of Homogeneity of Variance, and (3)A ssumption of Independence. [23] Statistical analyses are most robust to violations of the Normality Assumption. Still, this was addressed by using a n on-parametric Wilcoxon Ranked-Sum Test for analysis of data that was not normally distributed. [23] Statistical analyses are moderately robust to the Homogeneity of Variance Assumption. In this study, the two groups ha d different variances, so this assumption was violated. However, performance of a Sa tterthwaite analysis attempted to account for violations of homogeneity of variance in the group with NO levels under 30 ppb. Nonetheless, the Satterthwaite analysis is less reliable for small sample sizes. [23] Statistical analyses are the least robust to the Assumption of Independence, the most serious of the violations. In this st udy, care was taken not to allow people who were genetically related to each other participat e in the study. Everyone participating in the study had some association to the University of South Florida (USF). Two participants, one Caucasian male and one African ma le, were significant others of USF

PAGE 58

49 student/employees. Otherwise, everyone in the study was directly a student or employee of USF.

PAGE 59

50 Conclusion Given the small sample size (due to difficulty in subject recruitment), the limited population that the study could be generalized to, and the various other limitations listed above, this pilot study is limited in its value. Despite this, a raw difference between the means and medians of the average NO levels for the two ethnic groups was noted when only those participants with average NO levels under 30 ppb we re included. Therefore, differences in baseline NO levels between members of different ethnic groups requires further study prior to the inst itution of NO measurements in c linical settings for patient care.

PAGE 60

51 References 1. Townsend, MC. American College of O ccupational and Environmental Medicine. ACOEM position paper: spirometry in th e occupational settin g. Retrieved from www.acoem.com/position on 04/14/05. 2.American Thoracic Society-Official Stat ement. Recommendation for standardized procedure for the online and offline measurem ent of exhaled lower respiratory nitric oxide and nasal nitric oxide in adults and children-1999. Am J Respir Crit Care Med 1999; 160:2104-2117. 3.American Thoracic Society. L ung function testing: selectio n of reference values and interpretative strategies. Am J Re spir Crit Care Med 1991; 144:1202-1218. 4.Baraldi E, Ghiro L, et al. Safety and success of exhaled breath condensate collection in asthma. Arch Dis Child 2003; 88:358-360. 5.Gaston B. Breath condensate analysis: Perhaps worth studying after all. Am J Respir Crit Care Med 2003; 167:292-293. 6.Griese M, Latzin P, and Beck J. A noninva sive method to collect nasally exhaled air condensate in humans of all ages. Eu r J Clin Investigation 2001; 31:915-920. 7.Hunt J. Exhaled breath condensate: An evol ving tool for non-i nvasive evaluation of lung disease. J Allergy Clin Immunol 2002; 110: 28-34. 8.Kharitanov SA and Barnes PJ. Exhaled Ma rkers of Pulmonary Disease. Am J Respir Crit Care Med 2001; 163: 1693-1722.

PAGE 61

52 9.Kharitanov SA, Gonio F, Kelly C, Meah S, Barnes PJ. Reproducibility of Exhaled Nitric Oxide Measurements in Healthy and Asthmatic Adults and Children. Eur Respir J 2003; 21: 433-438. 10.Montuschi P, Barnes, PJ. Analysis of exhaled breath condensate for monitoring airway inflammation. Trends in Pharmacological Sciences 2002; 23:232-237. 11.Montuschi P CM, Ciabattoni G, et al. 8-isop rostane as a biomarker of oxidative stress in interstitial lung disease. Am J Respir Crit Care Med 1998; 158:1524-1527. 12.Mutlu GM GK, Robbins RA, Danziger LH,. Collection and analysis of exhaled breath condensate in humans. Am J Resp ir Crit Care Med 2001; 164:731-737. 13.Paredi P, Kharitanov SA, and Barnes PJ. Analysis of expired air for oxidation products. Am J Respir Crit Care Med 2002; 166: 531-537. 14.Sievers Nitric Oxide Analyzer NOATM 280i Operation and Maintenance Manual, 1995. 15. Bryant C, Social Behavior al Sciences Lecture Presentation, University of South Florida: UNIT 6; week of February 9-15, 2004. 16. Tsang KW, Ip SK, et al. Exhaled Nitric Oxide: The Effects of Age, Gender, and Body Size. Lung 2001; 179: 83-91. 17. Gordon R. Haight R. A Comparison of Exhaled Breath Nitric Oxide Between Old and Young Individuals. Masters Thesis: University of South Florida: Spring, 2004. Retrieved from http://purl.fcla.edu.proxy.usf.edu/fcla/etd/SFE0000346 on 04/14/05. 18. ACOEM position paper on spirometry 19. ATS/ERS Recommendations for Standardized Procedures for the Online and Offline Measurement of Exhaled Lower Respiratory Nitric Oxide and Nasal Nitric Oxide, 2005 Am J Respir Crit Care Med Vol 171. pp 912, 2005

PAGE 62

53 20. http://www.merck.com/mmhe/sec04/ch039/ch039b.html retrieved 04/14/05. 21. Mielck A, Reitmeir P, and Wjst M. Severity of childhood as thma by socioeconomic status. International Journal of Epidemiology, Vol 25, 388-393. 22. Sanchez-Anguiano A. Epid emiology Lecture Notes(EPB 6000), University of South Florida: Summer, 2004. 23. Blair, Clifford. Biostatistics I Lecture No tes (EPB 6050) University of South Florida: Fall, 2003.

PAGE 63

54 Appendices

PAGE 64

55 Appendix A: Initial Medical and Ethnicity Questionnaires Subject number:_______ Exhaled Breath Study Questionnaire Todays Date _______________________________________ Gender: Male Female (circle one) What is your date of birth? ______________________ How old are you? _____________________________ years old. Where were you born? ____________________________________________ How would you classify your ethnicity? (Please check one of the following): _______Caucasian _______African American _______OtherPlease explain: ________________________________________ Are any of your biological parents or grandpa rents from a differen t ethnicity than your stated ethnicity above? YES NO (circle one) Where was your mother born? _____________________________________ Where was your father born? _______________________________________

PAGE 65

56 What is your current occupation? ________Student ________OtherPlease Specify__________________________________________ _____________________________________________________________________ What is your current annual salary ? Please check one of the following: (Please note that this information is used solely to further subdivide the subject population. Like all of the other personal information obtained for this study, this information will be kept strictly confidential.) ________$0 to $10,000 per year ________$60,000 to $70,000 per year ________$10,000 to $20,000 per year ________$70,000 to $80,000 per year ________$20,000 to $30,000 per year ________$80,000 to $90,000 per year ________$30,000 to $40,000 per year ________$90,000 to $100,000 per year ________$40,000 to $50,000 per year ________More than $100,000 per year

PAGE 66

57 ________$50,000 to $60,000 per year Otherplease explain: _______________________________________________ Do you currently have, or have you ever had any of the following conditions listed below? (Please circle YES or NO) YES NO CHEST PAIN, PALPITATIONS, IRREGULAR HEART BEAT, OR HEART DISEASE? YES NO HIGH BLOOD PRESSURE? YES NO ASTHMA, BRONCHITIS, EMPHYSEMA, OR OTHER LUNG OR BREATHING DISORDERS? YES NO DIFFICULT OR HEAVY BREATHING YES NO A LARGE AMOUNT OF PHLEGM PRODUCTION YES NO PREGNANCY? IF YOU HAVE CIRCLED YE S TO ANY OF THE ABOVE QUESTIONS, PLEASE EXPLAIN: ___________________________________________________________

PAGE 67

58 ___________________________________________________________ Do you have any health problems or past me dical history of health problems that you have seen a physician for? Please list them below. 1._________________________________________________________ 2. ________________________________________________________ 3. ________________________________________________________ 4. ________________________________________________________ 5.________________________________________________________ Are you taking any medications? If so pl ease list them below. (Including over the counter medications) 1. ________________________________________________________ 2. ________________________________________________________ 3. ________________________________________________________

PAGE 68

59 4. ________________________________________________________ 5. ________________________________________________________ 6. ________________________________________________________ 7. ________________________________________________________ At this point in time, to what de gree do you note the following symptoms? (a) Phlegm production: ___NONE ___VERY LITTLE ___MODERATE AMOUNT ___VERY MUCH (b) Runny or irritated nose or nasal passages: ___NONE ___VERY LITTLE ___MODERATE AMOUNT ___VERY MUCH (c) Throat irritation or burning sensation: ___NONE ___VERY LITTLE ___MODERATE AMOUNT ___VERY MUCH (d) Sensation of a weight or tightness of the chest: ___NONE ___VERY LITTLE ___MODERATE AMOUNT ___VERY MUCH

PAGE 69

60 (e) Feeling of chest pa in, burning, or tightness: ___NONE ___VERY LITTLE ___MODERATE AMOUNT ___VERY MUCH If you have ever smoked, please answer the following: How many packs per day did you smoke? ________________________ For how many years did you smoke? ____________________________ When did you stop smoking? __________________________________ On what date were you last ill? _____________________________________ What illness did you have? ________________________________________ Please circle YES or NO to the following questions: Are you exposed to second hand smoke at home or at work?

PAGE 70

61 YES or NO Were you or are you exposed to any gases, dusts, or fumes at your job? YES or NO If so, please explain: ____________________________________________ _____________________________________________________________________ Do you ever wheeze or become short of breath? YES or NO This is the end of this questionnaire. Thank you for taking the time to fill it out. Special thanks to Dr. Gwyn Crump for allowing the use of his questionn aire in this study.

PAGE 71

62 Second Visit Initial Study Questionaire SUBJECT NUMBER: ________________________________________ Are you currently experiencing any of the following symptoms? (Please Circle YES or NO) (1) Difficult or heavy breathing? YES NO (2) Feeling of chest pain, tightness, pressure, or burning? YES NO (3) Any new symptoms that were not present during your first visit here? YES NO (4) Any other unusual symptoms? YES NO

PAGE 72

63 Appendix B: Exit Questionnaire Post-Study Questionaire SUBJECT NUMBER: ________________________________________ Are you currently experiencing any of the following symptoms? (Please Circle YES or NO) (1) Difficult or heavy breathing? YES NO (2) Feeling of chest pain, tightness, pressure, or burning? YES NO (3) Any new symptoms that were not present when you arrived here today? YES NO (4) Any other unusual symptoms? YES NO Special Thanks To Dr. Gwyn Crump for develo ping the majority of this questionnaire!

PAGE 73

Appendix C: Recruitment Flyer YYoouu AArree IInnvviitteedd TToo PPaarrttiicciippaattee!! WWhhaatt?? A $20.00 total honorarium will be given to each volunteer who completes the study for valuable time spent participating. This study only makes measurements of the normal air breathed out by subjects. The tests will measure lung function & a constituent of the air people breathe out called Nitric Oxide. This research study is non-invasive, meaning you will not be given any drugs or subjected to invasive procedures such as needlesticks as part of the study. WWhhoo?? HHeeaalltthhyy mmeenn with normal lungs who have nneevveerr ssmmookkeedd and have nnoo aalllleerrggiieess .. Candidates must be 1188--4455 yyrrss oolldd & either CCaauuccaassiiaann or ppeeooppllee ooff AAffrriiccaann ddeesscceenntt .. WWhhyy?? The study is designed to examine whether a certain marker found in the air we normally breathe out is different in normal young, healthy people of African descent as compared to Caucasians. Your participation in this study may help to determine whether 64

PAGE 74

adjustments should be made in measurement of this marker in people of African descent to help more accurately portray when disease is or is not occurring. Hopefully, the results will benefit science. WWhheenn?? The study will take about two hours to complete. You may choose to participate either: During two visits which will last for approximately one hour each, or During a single visit which will last approximately two hours. WWhheerree?? USF College of Public Health Call (813) 391-9385 IIff yyoouu mmaayy bbee iinntteerreesstteedd oorr hhaavvee aannyy qquueessttiioonnss,, pplleeaassee ccaallll ((881133)) 339911--99338855 oorr ee-mmaaiill uuss aatt ssppaatteell55@@hhsscc..uussff..eedduu 65

PAGE 75

66 Appendix D: Adult Informed Consent Form Informed Consent for an Adult University of South Florida Information for People Being Asked to Take Part in Research Studies IRB Study # 103323 Doctors and researchers at University of South Florida (USF) study diseases and other health problems people have. We try to find better ways to help treat these health problems. To do this, we need the help of people who agree to take part in a research study. Title of Research Study: Exhaled Breath Nitric Oxide: Is There a Baseline Difference Due to Ethnicity? Persons in Charge of Study: Sunita I. Patel, MD; Stuart M. Brooks, MD Where the study will be done: University of South Florida, College of Public Health, MHH Room 323 Who is paying for the study: Sunshine Education Research Center Should you take part in this study? This form tells you about this research study. You can decide if you want to take part in it. You do not have to take part. Reading this form should help you decide if you want

PAGE 76

67 to take part in the study. If, at any time, you have any questions feel free to ask the person explaining this study to you. Before you decide: Read this form. Talk about this study with the study doctor or the person explaining the study. You can have someone with you when you talk about the study. Find out what the study is about. This form explains: The purpose of this research study. What will happen during this study and what you will need to do. The potential benefits of being in this study, if any. The risks of having problems because you are in this study. The answers to any questions you might have. You can ask questions: You may have questions this form does not answer. If you do, ask the study doctor as you go along. You dont have to guess at things you dont understand. Ask the people doing the study to explain things in a way you can understand. After you read this form, you can: Take your time to think about the inform ation that has been provided to you. Have a friend or family member read the form. Talk it over with your regular doctor.

PAGE 77

68 Its up to you: If you choose to be in the study, then you can sign the form. If you do not want to take part in this study, you do not sign the form. Why is this research being done? The purpose of this study is to find out if there is a difference between Africans and Caucasians in the amount of a certain substa nce in the air we breat he out called nitric oxide. This research could eventually lead to development of more accurate baseline measurements of this and other markers in exhaled breath in African s or individuals of other ethnicities, such as Asians, Alaskan Natives, Hispanics, etc. The air we breathe out is currently the subj ect of intense researc h. There are certain substances in this air whose levels are affected by specific diseases associated with the lungs. In this study, one of these substances in the breath is called nitric oxide. Your breath will be measured by having you breath out into a single-use, clean, disposable tube that has never been used before. Each tube is discarded after you have used it. Two ethnic groups are being examined in this st udy because, in the past, ethnic differences have sometimes been found to affect certain clinical measurements. Why are you being asked to take part? You must be a healthy pers on of a certain ethnicity and age group to be eligible for the study. You will not be eligible if you have had any recent upper or lower respiratory tract diseases, heart or blood vessel disease, or hern ias. You may have had a recent respiratory tract disease if you have had any recent feve r, chills, coughing, shortness of breath or difficulty breathing, nasal congestion, runny nose, sinus congestion or headache, colored phlegm or (green, yellow, brown, black, or red), crackling or whistling sounds heard

PAGE 78

69 when you breathe, diagnosis by a doctor of nose/sinus/lung infections, or recent treatment with antibiotics for any of these symptoms. You may not be eligible if you have any other diseases or health problems. We are asking you to take part in this study because you are a healthy young Caucasian or African adult between 18-45 year of age We want to find out if there is a difference in the exhaled breath measurements between healthy Caucasians and Africans. Your regular medical treatment will not actually be part of the research study. In fact you will not be eligible for the study if you suffer from any significant heart or lung disease or other significant medical problems. How long will you be asked to stay in the study? You will be asked to spend a total of about 2 hours in this study. You may choose to complete this study on two one -hour visits at least twelve hours apart. On each visit, this hour is used to complete the screening questionnaires, perform a limited non-invasive physical examina tion, and for breathing tests. Alternatively, you may choose to complete the study in a si ngle 2-hour visit. If you select this option, the same procedures w ill be performed, but in a different order. How often will you need to come for study visits? A study visit is one you have with the study doctor. This visit is different than the visits you make with your regular docto r. You will need to come for up to two study visits in all. At these study visits, the doctor will obtain a questionnaire both before and after testing, perform a brief physical exam consisting prim arily of listening to your heart and lungs using a stethoscope, and perform the breathing tests.

PAGE 79

70 How many other people will take part? About 50 people will take part in this study at USF. Will the medical treatment you get from your re gular doctor change if you take part in this study? The kind of treatment you now get from your regular doctor will not change because you take part in this study. You will keep seeing your regular doctor. Your regular doctor will give you the same kind of treatment you would get anyway, whether you take part in the study or not. If you need it, you can Use other medicines prescribed th at will help your disease. Get any surgery you need. You will need to talk to the study doctor about any surgery you have planned. Talk to your regular doctor about the treatments you may need. If you have an emergency, you can get emergency care. Other procedures can be used for meas uring airway disease. These include bronchoalveolar lavage/ bronchi al biopsy (which are invasive), or sputum induction (which can be less comfortable). These procedures, which can be ordered by your regular doctor if needed clinically, will not be performed here. What other choices do you have if you deci de not to take part in this study? An alternative of being part of this research study is to not participate in this study. There will be no changes in your life. You will con tinue with your regular life style and are instructed to visit your pers onal physician for any respirat ory or non-respiratory problem

PAGE 80

71 you were being followed for before the study. The same is true if you decide to participate in the study. If you decide you do not want to take part in this study that is okay. There are other choices such as chest X-ray/CT, bronchoalveo lar lavage, biopsy, or sputum induction that are considered to be the current standard of clini cal care for diagnosi ng airway disease. How do you get started? You must be of a certain ethnicity and age group to be eligible for the study. You must also be healthy to participat e in this study. Volunteers must be between ages 18 and 45. If you decide to participate in this study, you will be required to review this informed consent, discuss the study and your possibl e participation with the study doctor and decide whether you are interested in participating. If you decide to take part in this study, you will need to sign this consent form. This informed consent must be signed before any study-related test or pro cedure can be done. After signing this informed consent, screening tests will be comple ted to help determine if you meet the requirements to be in the study. You will be asked to fill out questionnaires, undergo a limited physical examination and perform a limited breathing test called spirometry. Screening tests are tests done to see if you are able to be in the study. In order to qualify, you must record a normal health history. You must record a negative respiratory questionnaire response to treatment for any l ung, nasal, or sinus di seases. You must not be suffering from any major cardiovascular condition. There are different tests performed. These te sts are considered non-invasive and do not involve drawing blood or inserting t ubes into your throat or nose.

PAGE 81

72 We will do these screening tests: 1. Questionnaires: You will be asked to fill out questionnaires about your health, ethnicity, and socioeconomic status. They w ill include medical questionnaires. A poststudy symptom questionnaire will also be given at the end of each visit. 2. Physical Examination: Listening wi th a stethoscope to your chest and abdomen. 3. Spirometry: This is a common, frequently used and accepted medical test for evaluating how your lungs are working. A bnormal spirometry may indicate that you have a lung disease such as emphysema, chr onic bronchititis, asthma, and other diseases. The test involves taking in a deep breath and th en blowing out into a tube as hard and as fast as you can. The spirometer measures th e amount and speed with which you forcibly exhale your deep breath. You will be notified if your spirometry test results are abnormal and advised to see your personal physician. The test results will come back right away. At the end of each of these screening tests, you and the research team will decide whet her or not you should be in the study. What will you need to do to get ready for this study? You will need to refrain from strenuous phys ical exercise (such as running, jogging, working out with exercise equipment, partic ipating in sports, or doing any heavy physical labor), eating, or drinking for a mini mum of one hour prior to the visit. You must be of a certain ethnicity and age group to be eligible for the study. You must also be healthy to participat e in this study. Volunteers must be between ages 18 and 45. If you decide to participate in this study, you will be required to review this informed

PAGE 82

73 consent, discuss the study and your possibl e participation with the study doctor and decide whether you are interested in participating. This informed consent must be signed before any study-related test or procedure can be done. After signing this informed consent, medical tests will be completed to help determine if you meet the requirements to be in the study. You will be asked to fill out a questionnaire, undergo a physical examination and perform a breathing test. In order to qualify, you must record a negative respiratory questionnaire res ponse to treatment for respirat ory diseases, including asthma, emphysema, chronic bronchitis, sinusitis and in terstitial lung disease. You must not be suffering from any major car diovascular condition. There are different tests performed. These te sts are considered non-invasive and do not involve drawing blood or inserting t ubes into your throat or nose. What will happen during this study? Only normal, healthy, non-smoking subjects without second hand smoke exposure or history of heart/lung/other dise ases ages 18-45 years old will be invited to participate. You will be asked to verify that they have not had any signs of recent upper or lower respiratory tract infections. This would include any recent fever, chills, coughing, shortness of breath or difficulty breathing, nasal congestion, runny nose, sinus congestion or headache, colored phlegm or (green, yellow, brown, black, or red), crackling or whistling sounds heard when you breathe, di agnosis by a doctor of nose/sinus/lung infections, or recent treatment with antibiotics for any of these symptoms. At a minimum of 1 hour prior to each visit, su bjects will be instructed not to eat or drink or participate in strenuous exercise. If you decide to participat e in two one-hour visits:

PAGE 83

74 On the first visit, you will be asked to volunteer for the study. If you volunteer, you will be given information about the study pro cedure, the risks, the benefits, and the alternatives to the procedur e. Then you will be asked if you have any questions and demonstrate an understanding of everything di scussed. If you still agree to proceed, you will be asked to sign an informed consent. You will be taken to the Breath Laboratory at the College of Public Health (MHH Room 323)an air-conditioned, comfortable room, and sit in a comfortable chair. You will be asked to complete a symptom questionnaire, medical history form to determine health, and a short questionnaire to determine age and ethnicity. Those who fulfill the criteria of age, ethnicity, and being healthy will be asked to participate in the study. You will then undergo brief physical examination including auscultation. You will be directly watched by a physician througho ut the remainder of the study via direct observation. Limited spirometry (including FEV1, FVC, a nd FEV1/FVC ratio), which is part of a common method done in hospitals and clinics to check lung f unction, will be done to help verify that your breathing is normal. A loose-fitting device resemb ling a clothespin will be placed on the nose during the study to obtain better spirometry results. You will be asked to forcibly exhale into a disposable single-use tube using maximum effort after maximal inhalation. Anyone who has abnormal spirometry measurements will be notified of this information and advised to c onsult a healthcare provi der and disqualified for the remainder of the study. Adverse effects are extremely uncommon duri ng this phase of the study because subjects are young/ healthy individuals, the room is temperature controlled, you will be seated

PAGE 84

75 during the spirometry, and this phase of the st udy is brief in duration. Nonetheless, there is the remote possibility of unusual symptoms such as lightheadedness, dizziness, chest pains, palpitations, or shortness of breath during the spirometry from overexertion in breathing. As a precaution, you will be inst ructed to notify the physician examiner immediately if you experience any symptoms at any time. Following this, auscultation will be performed again and you will be gi ven another brief symptom questionnaire to make sure you have no adverse effects from spirometry. On the second visit, which will occur at l east 12 hours after the first visit, you will be asked if there have been any changes in your he alth status since the first visit. You will be given the same safety and autonomy instructions as during the spirometry phase of the study. After physical exam (primarily auscul tation), you will be asked to rinse out your mouth with tap water to prevent contamination of the sample. Next, the actual study exhaled breat h testing, which is strictly pa rt of the research, will be performed to measure the amount of nitric oxi de in the breath. You will be asked to breathe out through a disposable plastic mouthpie ce connected to a filter at a constant rate of flow after a maximal inspiration (which is where you take as deep a breath in as you can to fill your lungs with as much air as possible). Following this, the investigator will again perform a brief physical examination usin g a stethoscope to list en to your heart and lungs and you will be given another brief symptom questionnaire to make sure you have no adverse effects from the second visit. Some study visits may be longer or shorter than one hour depending primarily on both the equipment and subject variability in breathing. At your last visit, you will be asked to verify the absence of symptoms prior to leaving.

PAGE 85

76 If you decide to participate in a single two-hour visit: The same procedures described above for two one-hour visits will be performed. However, The study exhaled breath testing, which is strictly part of th e research, will be completed prior to spirometry. Also, examin ation with a stethoscope will be performed at the beginning of the visit, after the study exhaled breath testing, and at the end of the visit. We will study two groups of people: People in one group will be Caucasian. Pe ople in the other group will be of African background. Sometimes people of different et hnicities may exhale different levels of markers that we can measure in the breath. We want to examine whether levels of the marker nitric oxide are different between Caucasia ns and Africans at baseline. Right now, we do not know for sure if the meas urement of nitric oxide in your breath will accurately measure lung disease. We are doing this study to find out if the measurement of nitric oxide in your breath is different based on your ethnicity. If the measurement is not accurate, you may s till have airway or lung disease that is undetected by the study. You should consult your own physician regardless of the study results if you have any concerns or experience any symptoms at any time. During this study, here is what you will need to do: Subjects will be instructed to refrain from eating, drinking, or stre nuous exercise for at least one hour prio r to the visit. Will you be paid for taking part in this study?

PAGE 86

77 We will pay you for the time you volunteer while being in this study. You will receive a total of twenty dollars for completion of the study. If you select to participate in two one-hour visits, you will receive five dollars for completing the first visit and fifteen do llars for completing the second visit. If you select to participate in a single two-hour visit, you will receive twenty dollars if you complete the entire study. What will it cost you to take part in this study? The study will pay the costs of limited physical exam (primarily auscultation), limited spirometry, and the experimental nitric oxide measurement. You will not have to pay the fees for tests in this study that are not a part of regular medical care for your disease/condition. Those subjects that do not have a parking permit for on-campus parking will have to pay for any parking costs incurred as required fr om the University of South Florida by taking part in this study. You will have to pay for your regular care or an y other costs. Your insurance plan should cover your regular costs. Your insurance plan will not have to pay for study costs of limited physical exam, limited spirometry, or exhaled breath nitric oxide measurement. What are the potential benefits if you take part in this study? We dont know if you will get any health benefits by taking part in this study. We do not know if nitric oxide measurement will help in diagnosing airway or lung diseases. That is why we are doing this study. This research study should help us learn whether nitric oxide measurement will help in diagnosing airway or lung diseases.

PAGE 87

78 Since a medical questionnaire, limited physical exam, and limited spirometry will be performed, overt abnormalities detected by your examiner will be relayed to you. You will be instructed to consult your re gular doctor regarding these findings. No matter what, we will learn more about exha led breath nitric oxide levels in Africans compared to Caucasians. We will learn more about whether differences do or do not exist. What we learn may help others who are actively studying nitric oxide levels in exhaled breath for potential use in clinical settings. What are the risks if you take part in this study? The treatment might not help. Right now we do not know for sure if the measurements are consistent. Because of this, you will have to consult your regular physicia n if you are experiencing any shortness of breath, other symptoms, or have any concer ns about disease st atus. If you do, your condition/disease may get worse. There may be adverse effects. You may have problems because of the procedure used in this study. These problems are called adverse effects. Some adverse effects are just a bother. Others could harm you. There may be some adverse effects that we dont know about yet. Here are the known adverse effects th at could happen with this study: This study poses minimal risk to you. Spirom etry is a standard, routine physical test commonly performed in hospitals and some clin ics. It is a noninvasive and relatively safe procedure and involves the requirement fo r maximal expiratory effort. Thus, there is a minimal risk that you may breathe too hard during the forced e xpiratory maneuvers. This could result in: [1]hyperventilation, wh ere too much carbon dioxide is blown off

PAGE 88

79 in a short period of time or [2]excessive physical exertion. Imme diate effects you could experience from hyperventilation or excessive physical exertion may include lightheadedness, dizziness, chest pain or pressu re, rapid heart beat, or shortness of breath. These usually resolve upon discontinuing th e procedure. You are instructed to immediately notify the examiner if any symptoms result. A determination by the examiner of whether or not to continue w ith the study will be made on an individual basis. At any time, both you and the examiner have the right to stop the study for any reason. If you were to con tinue despite these symptoms loss of consciousness or cardiopulmonary arrest could re sult in rare cases, requiring emergency treatment and/or hospitalization. In such rare cases permanent effects could result. Patients with history of any t ype of hernia are excluded fr om the study since they could potentially aggravate the hernia using maximal expiratory effort. The procedure for collecting exhaled breath nitric oxide is very similar to the procedure for spirometry, except that the subject is as ked to breathe out at a slow regular rate instead of the forced maximal exhalation requi red in spirometry. Thus, it poses even less risk and discomfort to th e subject than spirometry. Private medical information will be collected during the study to find out your health status. The health informati on is being collected only as a guide for participation in the study. The questionnaires used in the study must remain confidential. To maintain your confidentiality, the questionnaires and recorded data from the study will be kept in a locked file cabinet. Access to the material will only be made to the investigators of the study. Also, as an additional measure to en sure confidentiality, each person will be given a subject number. This will be the only id entifier listed on the quest ionnaires to link the

PAGE 89

80 confidential information to the subject. The ma ster list linking subj ects to their subject numbers will also be kept in th e locked cabinet, but separately. During the study, the safety of the normal subjects will be maintained by: [1] the presence of a physician at all times of testin g; [2] careful direct physician observation and monitoring of subjects during testing, and [3] instruction of subjects to immediately discontinue the exercise and notify the physician should they experience any shortness of breath, chest pain or pressure, dizziness, lightheadedness, or other unusual symptoms. No medications will be given during the study, but emergency equipment will be present, and a physician will be in atte ndance for every study patient. If you have any of these or any other problems, notify your study doctor immediately. If these side effects bother or worry you, or if you have other problems, call your study doctor at (813) 974-7545. If you have an em ergency, go to the closest emergency room or clinic for treatment. You may also have problems from the medi cal treatment you would usually get. We may need to stop your treatment. If we find that the breathing tests are causing adverse events, we will stop the procedure. Early stopping criteria include: 1. For individual subjects: The following criteria will be used to discontinue participation in the study: [1] You wish to withdraw from the study. [2] You do not meet the criteria for inclusion into the study. [3] You demonstrate abnormalities on spirometry, physical examination, or monitoring through the study.

PAGE 90

81 [4] You complain of chest pain or pressure, palpitations, or tachypnea. [5] If you experience other symptoms, they will be assessed by a doc tor and assisted in any way needed. A decision will be made whet her you may continue to participate in the study provided that your safety has not been co mpromised. In this case, the decision to continue would require mu tual willingness on your part to continue the study. [6] The safety of the researcher is compromised (for example, a hostile subject). 2. For the study: The study will be terminated if thehealth or safety of the volunteers or examiners becomes jeopardized by an unforeseen event. Is there any risk to your unborn childr en if you take part in this study? Pregnant women will not be allowed to par ticipate in the study. There are no adverse effects for men with partne rs of childbearing age. If you are a woman You are excluded from the study if you are or may be pregnant or breastfeeding due to the study protocol, since the study results may be affected. If you are pregnant and you inadvertently take part in this study, the risk of adverse effects is still minimal, especially if you are in the first trimeste r of pregnancy. Nonetheless, it could be that your unborn children may have problems now or in the future. Tell one of the study doctors right away if: You are pregnant. You get pregnant. You are breastfeeding.

PAGE 91

82 If you are a woman If you take part in this study, you must use a good birth control method, like oral contraceptives or condoms. What if you get sick or hurt while you are in the study? If you need emergency care: Go to your nearest hospital or emergency room right away. Call 911 for help. It is important that you tell the doc tors at the hospital or em ergency room that you are participating in a research study. If possible, take a copy of this consent form with you when you go. You should know that the USF does not provide emergency care. Call the study doctors as soon as you can. They will need to know that you are hurt or ill. Call Dr. Sunita Patel, MD or Dr. Stuart Brooks, MD at (813) 974-7545. If it is NOT an emergency, and you get hurt or sick while you are ta king part in this study: Go to your regular doctor. It is important that you tell your regular doctor that you are participating in a research study. If possible, take a copy of this consent form with you when you go. The USF Medical Clinics may not be able to give the kind of help you need. You may need to get help somewhere else. If you are harmed while taking part in the study: The state of Florida enjoys what is called "sovereign immunity." This means that you usually cannot sue the state of Florida. However, the state has waived sovereign immunity (agreed to be sued) in certain situations. One of those situ ations is if a state employee, such as your study doctor or other USF employee, is negligent in doing his or

PAGE 92

83 her job in a way that harms you during the study. The money that you might recover from the state of Florida is limited in amount. You can also call the USF Self Insurance Pr ograms (SIP) at 1-813974-8008 if you think: You were harmed because you took part in this study. Someone from the study did something wrong that caused you harm, or didnt do something they should have done. Ask the SIP to look into what happened. What will we do to keep your study records private? Federal law says we must keep your study reco rds private. Private medical information will be collected during the study to ascertain the health status of the subjects. The health information is being collected only as a para meter for participation in the study. It is obtained after discussion with the subjects that the study is only open to healthy subjects. Thus, the questionnaires in the study must rema in confidential. We will keep the records of this study private by keeping them in a lock ed file cabinet. Access to the material will only be made to the investigators of the study. Also, as an additional measure to ensure confidentiality, each subject will be given a subject number. This will be the only identifier listed on the questionnaires to link the confidential information to the subject. The master list linking subjects to their subjec t numbers will also be kept in the locked cabinet, but separately. However, certain people may need to see your study records. By law, anyone who looks at your records must keep them completely confidential. The only people who will be allowed to see these records are:

PAGE 93

84 The medical staff who are taking care of you. Certain government and university people who need to know more about the study. For example, individuals who provide oversight on this study may need to look at your records. These include the University of S outh Florida Institutiona l Review Board (IRB) and the staff that work for the IRB. Othe r individuals who work for USF that provide other kinds of oversight may al so need to look at your records. Other individuals who may look at your records include: the Florida Department of Health, people from the Food and Drug Administration (FDA), the Unit ed States Department of Health and Human Services, and the United St ates Department of Labor. This is done to make sure that we are doing the study in the right way. Th ey also need to make sure that we are protecting your rights and your safety.) People at the company who paid for this st udy at the Sunshine Education and Research Center (funded by the National Institute of Occupational Safety and Health, a governmental department under the United States Department of La bor may look at the study records and pertinent porti ons of your medical records to make sure the study is done in the right way. We may publish what we find out from this st udy. If we do, we will not let anyone know your name. We will not publish anything else that would let people know who you are. What happens if you decide not to take part in this study? You should only take part in this st udy if you want to take part. If you decide not to take part: You will not be in trouble or lose any rights you normally have. You will still have the same health care benefits.

PAGE 94

85 You can still get your regular trea tments from your regular doctor. What if you join the study and decide you want to stop later on? You can decide after signing this informed consent document that you no longer want to take part in this study. If you decide you want to stop taki ng part in the study, tell the study staff as soon as you can. If you deci de to stop, you can continue getting care from your regular doctor. Are there reasons we might take you out of the study later on? Even if you want to stay in the study, there may be reasons we will need to take you out of it. You may be taken out of this study if: We find out it is not safe for you to stay in the study. For example, your health may worsen. Or we may find out that the devi ce used to measure spirometry or exhaled breath nitric oxide might harm you. Then you may be taken out of the study. You are not coming for your st udy visits when scheduled. You can get the answers to your questions. If you have any questions about this st udy, call Dr. Sunita Patel at (813) 974-7545. If you have questions about your rights as a person who is taking part in a study, call the Division of Research Compliance of the Univ ersity of South Flor ida at (813) 974-9343. Signatures for Consent to Take Part in this Research Study It is up to you to decide whether you want to take part in this study. If you want to take part, please read the statements below and sign the form if the statements are true. I freely give my consent to take part in this study. I understand that this I am agreeing to take part in research. I have received a copy of this consent form to take with me.

PAGE 95

I choose to participate in the study as: (Please select only one option) Two visits which will last approximately one hour per visit. A single visit which will last approximately two hours. ____________________________________________ ___________ Signature of Person Taking Part in Study Date ____________________________________________ Printed Name of Person Taking Part in Study ____________________________________________ ___________ Signature of Witness Date ____________________________________________ Printed Name of Witness Statement of Person Obtaining Informed Consent I have carefully explained to the person taking part in the study what he or she can expect. I hereby certify that when this person signs this form, to the best of my knowledge, he or she understands: What the study is about. What needs to be done. What the potential benefits might be. 86

PAGE 96

87 What the known risks might be. I also certify that he or she does not have any problems that could make it hard to understand what it means to take part in this study. This person speaks the language that was used to explain this study. This person reads well enough to understand this form or, if not, this person is able to hear and understand when the form is read to him or her. This person does not have a medical problem that makes it hard to understand what is being explained and can, therefore, give informed consent. This person is not taking drugs that make it hard to unders tand what is being explained and can, therefore, give informed consent. ____________________________________________ ___________ Signature of Person Obtaining Informed Consent Date __________________________________________________ Printed Name of Person Obtaining Informed Consent ____________________________________________ ___________ Signature of Witness Date ____________________________________________ Printed Name of Witness]

PAGE 97

88 Appendix E: Power Analysis Power Calculation: Using power= 80% and = 0.05, the following formula was derived: a = 2.8 2 2 /n where a = level of difference re quired for significance, = standard deviation of the population, n = sample size. sigma 6.8 7.7 10 1 26.927 30.49 39.598 2 19.040 21.56 28.000 3 15.546 17.604 22.862 4 13.463 15.245 19.799 5 12.042 13.636 17.709 6 10.993 12.448 16.166 7 10.177 11.524 14.967 8 9.520 10.78 14.000 9 8.976 10.163 13.199 10 8.515 9.642 12.522

PAGE 98

89 11 8.119 9.193 11.939 12 7.773 8.802 11.431 13 7.468 8.457 10.983 14 7.196 8.149 10.583 15 6.952 7.873 10.224 16 6.732 7.623 9.899 17 6.531 7.395 9.604 18 6.347 7.187 9.333 19 6.177 6.995 9.084 20 6.021 6.818 8.854 30 4.916 5.567 7.230 40 4.257 4.821 6.261 50 3.808 4.312 5.600 60 3.476 3.936 5.112 70 3.218 3.644 4.733 80 3.010 3.409 4.427 90 2.838 3.214 4.174 100 2.693 3.049 3.960

PAGE 99

123456789101112131415161718192030405060708090100 = 0.0005.00010.00015.00020.00025.00030.00035.00040.000Difference Between the Means of the 2 GroupsNumber of Participants in Each GroupPower Analysis =6.8 =7.7 =10 90

PAGE 100

91 Appendix F: SAS Program Used *Read an Excel spreadsheet and import it in to SAS as filename=hospital when correcting for the confounders; PROC IMPORT DATAFILE = 'F:\thesis_excel_data_fbile.xls' OUT = hospital; PROC PRINT DATA = hospital; TITLE 'RawData'; RUN; *Create a new SAS file=sorted th at sorts the data by Ethnicity; PROC SORT DATA = hospital OUT = sorted; BY Ethnicity Average_NO; PROC PRINT DATA = sorted; RUN; *Create a new SAS file=ranges that sorts the data by Average_NO; PROC SORT DATA = hospital OUT = ranges; BY Average_NO; PROC PRINT DATA = ranges; RUN; *Initiate export of th e data to HTML file; ODS HTML BODY = "F:\sorted"; CONTENTS = "F:\sorted"; PAGE = "F:\sorted";

PAGE 101

92 FRAME = "F:\sorted"; *Creates a report of total number of subjects in Each Ethnic Group; PROC FREQ DATA = sorted; TABLES Ethnicity; TITLE 'Number of Subjects in Each Ethnic Group'; RUN; *Creates a report of Age by ethnicity; PROC TABULATE DATA = sorted; CLASS Age Ethnicity; TABLE Age, Ethnicity; TITLE 'Age By Ethnicity'; RUN; *Creates a report of birthplace by ethnicity; PROC TABULATE DATA = sorted; CLASS Birthplace Ethnicity; TABLE Birthplace, Ethnicity; TITLE 'Birthplace By Ethnicity'; RUN; *Creates a report of AncestralDifference by ethnicity; PROC TABULATE DATA = sorted; CLASS AncestralDifference Ethnicity; TABLE AncestralDifference, Ethnicity; TITLE 'Different Ancestry By Ethnicity';

PAGE 102

93 RUN; *Creates a report of CurrentOccupation by ethnicity; PROC TABULATE DATA = sorted; CLASS CurrentOccupation Ethnicity; TABLE CurrentOccupation, Ethnicity; TITLE 'Current Occupation By Ethnicity'; RUN; *Creates a report of AnnualSalary by ethnicity; PROC TABULATE DATA = sorted; CLASS AnnualSalary Ethnicity; TABLE AnnualSalary, Ethnicity; TITLE 'Annual Salary By Ethnicity'; RUN; *Creates a report of RespiratoryTractConditions by ethnicity; PROC TABULATE DATA = sorted; CLASS RespiratoryTractConditions Ethnicity; TABLE RespiratoryTractConditions, Ethnicity; TITLE 'Respiratory Tract Conditions By Ethnicity'; RUN; *Creates a report of Inhalation Exposures by ethnicity; PROC TABULATE DATA = sorted; CLASS InhalationExposures Ethnicity; TABLE InhalationExposures, Ethnicity;

PAGE 103

94 TITLE 'Inhalation Exposures By Ethnicity'; RUN; *Creates a report of OtherMed icalConditions by ethnicity; PROC TABULATE DATA = sorted; CLASS OtherMedicalConditions Ethnicity; TABLE OtherMedicalConditions, Ethnicity; TITLE 'OtherMedicalConditions By Ethnicity'; RUN; *Creates a report of CurrentMedications by ethnicity; PROC TABULATE DATA = sorted; CLASS CurrentMedications Ethnicity; TABLE CurrentMedications, Ethnicity; TITLE 'CurrentMedications By Ethnicity'; RUN; *Creates a report of Phys icalExam by ethnicity; PROC TABULATE DATA = sorted; CLASS PhysicalExam Ethnicity; TABLE PhysicalExam, Ethnicity; TITLE 'Physical Exam By Ethnicity'; RUN; *Creates a report of Adve rseEvents by ethnicity; PROC TABULATE DATA = sorted; CLASS AdverseEvents Ethnicity;

PAGE 104

95 TABLE AdverseEvents, Ethnicity; TITLE 'Adverse Events By Ethnicity'; RUN; *Create a statistical summary of Av erage_NO values overall ADDED ALPHA; PROC UNIVARIATE DATA = sort ed mu0=33.5 ALPHA= .05; VAR Average_NO; TITLE 'Statistical Summary Overall'; RUN; *Create a summary of NO de scriptive statistics overal l REMOVED DUPLICATES and ADDED CONFIDENCE LIMITS; PROC MEANS DATA = sorted MIN MAX ALPHA=.05 CLM; VAR Average_NO; TITLE 'Means*Summary Overall'; RUN; *Create a statistical summary of Average_NO values by ethnicity ADDED ALPHA; PROC UNIVARIATE DATA = sort ed mu0=33.5 ALPHA= .05; VAR Average_NO; BY Ethnicity; TITLE 'Statistical Summary By Ethnicity'; RUN; *Create a summary of NO descriptive statistics REM OVED DUPLICATES and ADDED CONFIDENCE LIMITS; PROC MEANS DATA = sorted MIN MAX ALPHA=.05 CLM;

PAGE 105

96 VAR Average_NO; BY Ethnicity; TITLE 'Means*Summary By Ethnicity'; RUN; *Create a GRAPH of Average_NO versus ethnicity ADDED ALPHA; PROC PLOT DATA = sorted; PLOT Average_NO Ethnicity; TITLE 'Statistical Summary'; RUN; *Correlate SES data to NO levels; PROC CORR DATA = sorted; VAR SalaryCode OccupationCode SESCodeProduct; WITH Average_NO; TITLE 'Correlate SES Data to NO Levels'; RUN; *Perform a students T-Test on the normally distributed values under 30; proc ttest data=sorted; var average_no; class ethnicity; RUN; *Calculate Wilcoxon Ranked-Sum; proc npar1way data = sorted wilcoxon; class ethnicity;

PAGE 106

97 var Average_NO; run; data twosample; Mu1=17.0333333; Mu2=24.6428571; StDev=7.73858133; N1=6; N2=7; Alpha=0.05; NCP = (Mu2-Mu1)**2/((StDev**2)* (1/N1 + 1/N2)); CriticalValue = FINV(1-Alpha, 1, N1+N2-2, 0); Power = SDF(F, Critical Value, 1, N1+N2-2, NCP); proc print data=twosample; run; *Closes the HTML function; ODS HTML CLOSE;