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8-isoprostane levels in exhaled breath condensate of pregnant women compared to non-pregnant women; is there a baseline ...

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Title:
8-isoprostane levels in exhaled breath condensate of pregnant women compared to non-pregnant women; is there a baseline difference?
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Book
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English
Creator:
Szollas, Rosemary
Publisher:
University of South Florida
Place of Publication:
Tampa, Fla
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Subjects / Keywords:
Inflammatory markers
Asthma
Pregnancy
Preeclampsia
Oxidative stress
Dissertations, Academic -- Public Health -- Masters -- USF
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bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

Notes

Abstract:
ABSTRACT: This study investigated whether or not there was an overall difference in the pulmonary oxidative marker, 8-isoprostane (PGF2-a), found in exhaled breath condensate, during pregnancy versus the non-pregnant state. The utility of this information was important secondary to the effect of maternal asthma on pregnancy and outcome, as it has been demonstrated in past studies that overall pregnant females with asthma have been shown to have an increased risk of exacerbation requiring medical intervention.The primary goal of this study sought to determine if there is a difference in PGF2a levels in the exhaled breath condensate of pregnant versus non-pregnant females. In order to achieve this goal a cross-sectional study was performed consisting of two groups and were compared to one another. A group of 16 healthy, non-pregnant females aged 18-35 years old was compared to a group of 6 healthy, pregnant females in their third trimester of pregnancy. Both groups exhaled breath^ condensate was collected and 8-isoprostane levels determined and compared to each other. Both groups compared did not report a history of environmental allergies, asthma, and smoking. The non-pregnant group showed a mean 8-isoprostane level of 11.513pg/ml (C.I. 8.7633- 14.263). The pregnant group showed a mean 8-isoprostane level of 17.34 pg/ml (C.I. -4.209-38.889).Although a crude observable difference between the means of the two groups was determined, this pilot study did not show a statistically significant difference between the means of the pregnant versus non-pregnant group when they were statistically compared. This finding is primarily due to the small sample size of both groups. A power calculation determined that each group would require 25 participants in order to establish a statistically significant difference in the 8-isoprostane levels in exhaled breath condensate.The implication is that a larger scale study is needed in order to conclusively determine if there is^ a statistically significant difference between the exhaled breath condensate 8-isoprostane levels in pregnant versus non-pregnant females.
Thesis:
Thesis (M.A.)--University of South Florida, 2006.
Bibliography:
Includes bibliographical references.
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System requirements: World Wide Web browser and PDF reader.
System Details:
Mode of access: World Wide Web.
Statement of Responsibility:
by Rosemary Szollas.
General Note:
Title from PDF of title page.
General Note:
Document formatted into pages; contains 49 pages.

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aleph - 001796788
oclc - 156852979
usfldc doi - E14-SFE0001606
usfldc handle - e14.1606
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8-isoprostane levels in exhaled breath condensate of pregnant women compared to non-pregnant women; is there a baseline difference?
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ABSTRACT: This study investigated whether or not there was an overall difference in the pulmonary oxidative marker, 8-isoprostane (PGF2-a), found in exhaled breath condensate, during pregnancy versus the non-pregnant state. The utility of this information was important secondary to the effect of maternal asthma on pregnancy and outcome, as it has been demonstrated in past studies that overall pregnant females with asthma have been shown to have an increased risk of exacerbation requiring medical intervention.The primary goal of this study sought to determine if there is a difference in PGF2a levels in the exhaled breath condensate of pregnant versus non-pregnant females. In order to achieve this goal a cross-sectional study was performed consisting of two groups and were compared to one another. A group of 16 healthy, non-pregnant females aged 18-35 years old was compared to a group of 6 healthy, pregnant females in their third trimester of pregnancy. Both groups exhaled breath^ condensate was collected and 8-isoprostane levels determined and compared to each other. Both groups compared did not report a history of environmental allergies, asthma, and smoking. The non-pregnant group showed a mean 8-isoprostane level of 11.513pg/ml (C.I. 8.7633- 14.263). The pregnant group showed a mean 8-isoprostane level of 17.34 pg/ml (C.I. -4.209-38.889).Although a crude observable difference between the means of the two groups was determined, this pilot study did not show a statistically significant difference between the means of the pregnant versus non-pregnant group when they were statistically compared. This finding is primarily due to the small sample size of both groups. A power calculation determined that each group would require 25 participants in order to establish a statistically significant difference in the 8-isoprostane levels in exhaled breath condensate.The implication is that a larger scale study is needed in order to conclusively determine if there is^ a statistically significant difference between the exhaled breath condensate 8-isoprostane levels in pregnant versus non-pregnant females.
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PAGE 1

8-Isoprostane Levels in Exha led Breath Condensate of Preg nant Women Compared to Non-Pregnant Women; Is Th ere a Baseline Difference? by Rosemary Szollas, M.D. A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Public Health Department of Occupational Medicine College of Public Health University of South Florida Major Professor: Stuart M. Brooks, M.D. Robert Haight, M.D. Thomas Truncale, M.D. Date of Approval: April 5, 2006 Keywords: inflammatory markers, asthma, pr egnancy, preeclampsia, oxidative stress Copyright 2006, Rosemary Szollas, M.D.

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Dedication I would like to thank Dr. St uart Brooks for his guid ance through the residency program and through this research project. I would also like to thank Karen Olson for all of her help and support. Throughout my time in the residency with her I have made a wonderful friend. I would like to thank Robert Haight for hi s help with th e statistical calculations, and advice on paper thickness. Last but not least I would like to thank Melinda Tyler for her kindness and patience. It has been a pleasure for me to work with these individuals throughout my time in the occupational medicine residency program. I could not have met my academic and professional goals wi thout their help.

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i Table of Contents List of Tables iii List of Figures iv Abstract v Introduction 1 Isoprostanes 1 Pregnancy 3 Exhaled Breath Condensate 6 Research Question 8 Hypothesis 8 Goals and Objectives 9 Utility and Significance 9 Materials and Methods 10 Study Design 10 Facilities and Equipment 10 Participant Recruitment 11 Study Subjects and Restrictions 12 Study Qu estionnaire and Eligibility 12 Physical Examination 13 Exhaled Breath Condensate Collection and Storage 13

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ii Data Collection and 8-isoprostane Analysis 13 Acquisition of Spirometry 17 Results 20 Study Population Characteristics 20 8-Isoprostane Measurements 21 Biostatistical Da ta for 8-Isoprostane Measurements 26 Conclusions 29 References 31 Appendices 33 Appendix A: Questionnaire Form 35 Appendix B: Adult Univers ity of South Florida Informed Consent Document 37

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iii List of Tables Table 1. Effects of isoprostane s (8-iso-prostaglandin (PG)F2 ) in asthma 5 Table 2. Advantages and limitations of collection and analyzing exhaled breath condensates 8 Table 3. Demographic characteristics of subjects both pregnant and non-pregnant 21 Table 4. 8-isoprostane concentrations in pregnant and non-pregnant females 22 Table 5. Standard concentrations (known ) and actual absorbance concentrations Detected 24 Table 6. Standard concentrations (known ) and actual absorbance concentrations detected using standard curve w ithout known concentrations 500pg/ml, 250 pg/ml and 125 pg/ml 25 Table 7. Spirometric data for all subjects tested including pregnant and n on-pregnant females 25 Table 8. Statistical analysis of mean 8-Is oprostane values of 20 subjects tested 26 Table 9. T-tests used to analyze difference between means of the two groups 27 Table 10. Variables involved in power calculations 27

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iv List of Figures Figure 1. Lipid Pero xidation Pathways 2 Figure 2. Schematic representation of an EBC collection apparatus 6 Figure 3. Cayman Chemical 8isoprostane kit precision curve 16 Figure 4. 8-isoprostane Concentrations Taken from Samples in Pregnant Females 23 Figure 5. 8-Isoprostane Concentrations in Samp les Taken from Non-pregnant 24

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v 8-Isoprostane Levels in Exha led Breath Condensate of Pr egnant Women Compared to Non-Pregnant Women; Is Th ere a Baseline Difference? Rosemary Szollas ABSTRACT This study investigated whether or not there was an overall difference in the pulmonary oxidative marker, 8-isoprostane (PGF2), found in exhaled breath condensate, during pregnancy versus the nonpregnant state. The utility of this information was important secondary to the e ffect of maternal asthma on pregnancy and outcome, as it has been demonstrated in past studies that overall pregnant females with asthma have been shown to have an incr eased risk of exacerbation requiring medical intervention. The primary goal of this study sought to determine if there is a difference in PGF2 levels in the exhaled breath condensate of pregnant versus non-pregnant females. In order to achieve this goal a cross-sec tional study was performed consisting of two groups and were compared to one another. A group of 16 healthy, non-pregnant female s aged 18-35 years old was compared to a group of 6 healthy, pregnant females in their third trimester of pregnancy. Both groups exhaled breath condensate was collected and 8-isoprostane levels determined and compared to each other. Both groups compared did not report a history of environmental allergies, asthma, and smoking. The non-pre gnant group showed a mean 8-isoprostane

PAGE 8

vi level of 11.513pg/ml (C.I. 8.763314.263). The pregnant group showed a mean 8isoprostane level of 17.34 pg/ ml (C.I. -4.209-38.889). Although a crude observable difference betw een the means of the two groups was determined, this pilot study di d not show a statistically sign ificant difference between the means of the pregnant versus non-pregnant gr oup when they were statistically compared. This finding is primarily due to the sm all sample size of both groups. A power calculation determined that each group would re quire 25 participants in order to establish a statistically significant difference in th e 8-isoprostane levels in exhaled breath condensate. The implication is that a larger scale st udy is needed in order to conclusively determine if there is a statistically significant difference between the exhaled breath condensate 8-isoprostane levels in pre gnant versus non-pregnant females.

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1 Introduction Isoprostanes Isoprostanes are derived from membrane lipids via peroxidation with compounds such as free radicals and reactive oxygen species1. This non-enzymatic production of isoprostanes catalyzed by free radicals was first described by Morrow and colleagues in 1990. It is distinctly different from the cl assical enyzmatically produced isoprostanes via cyclooxygenase (COX) enzymatic pathways (Figure 1). Since their discovery, the usefulness of these compounds as bio-markers in various disease states has been studied. In particular, 8-isoprostane or PGF2 as a marker of oxidative stress in patients with asthma has been studied by Montushi and colleagues in 1999.2 Measurement of PGF2 has been done by collection of exhaled br eath condensates (EBC). This method of collection and analyzing oxidati ve products in the lung allows for a simple non-invasive mean of examining the human lower respiratory tracts3. Elevated levels of 8-isoprostanes have shown to correlate with level of disease severity in various disease states. Just why this occurs is not fully understood. 8isoprostane produces a numb er of biological responses such as smooth muscle contraction, vasoconstricti on, and platelet activation1. These responses are thought to be a result of an isoprostane receptor binding and subsequent signaling pathway involving phospholipase and kinase enzymatic activity resulting in Ca ion channel activation1.Various other markers have been found in the breath and typically vary by

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2 disease state. In particular, PGF2 in the exhaled breath condensate, plasma, and serum has been found to be elevated in individuals with asthma. Two separate studies have shown PGF2 to be elevated in a group of asthma tic children compared to a group of non-pregnant children.4, 5 Figure 1. Mechanisms leading to lipid peroxidation in asthma.6

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3 Other studies have shown 8-isoprostane levels to be elevated in seve re respiratory failure in infants.7 Therefore various diseas e states exist where exhale d breath condensates have been showed to be elevated. Pregnancy Not surprisingly, given its biological re sponses, 8-isoprostane has also been studied in its relationship to a healthy state and various disease stat es during pregnancy. 8-isoprostane, as previously discusse d, produces smooth muscle contraction, vasoconstriction, and platelet activation1. Preeclampsia is defined as a pregnancydependent syndrome diagnosed on the basis of increased blood pressu re and proteinuria. Endothelial dysfunction is hallmark of preeclam psia and is central in explaining the main features of preeclampsia: hypertension, edem a, proteinuria, and ac tivated homeostasis8. For example, plasma concentrations of free 8-isoprostane has been found to be significantly greater in a st udy analyzing levels before delivery in patients with preeclampsia than in control subjects.8 It is thought that oxidative lipid derivatives are uteroplacental compounds that may cause dysfunctional maternal endothelium.8 Recently, a study reported an increased cont ent of lipid peroxides in preeclamptic decidual placental tissue in preeclamptic pa tients at delivery co mpared with controls8. In addition, another study by Moret ti and colleagues demonstrat ed increased markers of oxidative stress in preeclamptic women versus uncomplicated pregnancy and nonpregnant controls by measuring various volat ile organic compounds in the exhaled breath condensate of these women9.

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4 Although levels of 8-isoprostanes have been shown to be elevated in the exhaled breath condensate of individuals with asthma, no study to date has measured the levels of 8-isoprostane in the exhaled breath condensat e of pregnant females. Asthmatic females who become pregnant have been demonstrated to be more at risk of low birth weight neonates, pre-term delivery, and complications such as preeclampsia, especially in the absence of actively managed asthma tr eated with inhaled corticosteroids.10 These mechanisms responsible for changes in as thma with pregnancy, or alterations in pregnancy outcomes due to asthma have not been thoroughly explored. Asthma is a chronic inflammatory disord er of the airways involving variable airflow obstruction and increased airway res ponsiveness to a variety of stimuli. The airway mucosal inflammatory response in asth ma is characterized by increased vascular permeability with edema of airway walls, mucus hypersecretion with small airway plugging and infiltration with inflam matory cells, typically eosinophils.6 The role of oxidant and antioxidant in asthmatic airway inflammation is ill-defi ned. Isoprostanes are one of the groups of lipid pe roxidation products that contri bute to the pathophysiological changes seen in asthma.6 8-isoprostane, in particularl y, has been demonstrated to be elevated in certain individuals with vari ous disease states, including asthma. These responses may be partially the result of elevated levels of 8-isoprostane.2 As previously stated, 8-isoprostane is a known smooth mu scle constrictor, as well as vascular constrictor, it has also been demonstrat ed to cause airway hyperresponsiveness and airway obstruction, as well as plasma exudation.11 effects of 8-isoprostane in asthma can are listed in Table 1 below.

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5 Table 1. Effects of isoprostane s (8-iso-prostaglandin (PG)F2 ) in asthma6 In vitro /animal models: Human studies: Smooth muscle constriction 22, 23 8-iso-PGF2 in asthma (plasma 28; breath condensate 29) Airway hyperreponsiveness 24 Airway obstruction 25 8-iso-PGF2 with asthma severity (plasma 28; breath condensate 29) Plasma exudation 25 Vascular constriction 26, 27 8-iso-PGF2 with allergen challenge (urine and bronchoalveolar lavage 30) Given that mechanisms responsible for changes in asthma with pregnancy, or alterations in pregnancy outcomes due to asth ma have not been t horoughly explored, it seems prudent that more research in this are be done as to why this occurs. It is well known that pregnancy alone produces pulm onary changes including increased tidal volume leading to increased minute ventilat ion leading to an increased PAO2 and decreased PACO2 and PaCO2. However, whethe r or not an increase in oxidative stress in the lungs of the pregnant female o ccurs, also remains unknown. The effect of pregnancy on asthma consensus has remained the same for many years that one-third worsens, one-third remain the same and one -third improve. Regardless, those that worsen are at an increased risk of deliveri ng low birth weight neonates, pre-term delivery and complications including preeclampsia. In addition to the in creased risk of poor outcomes, pregnant females with asthma ha ve an increased risk of an exacerbation requiring medical attention which may also increase the risk of a poor outcome.10

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6 Since reducing asthmatic inflammation a nd preventing exacerbat ions contributes to improved outcomes in outcomes for mother and fetus it becomes apparent that studies in this area should be focused on understa nding changes in airway inflammation in pregnant females with asthma, which may lead to more effective treatment and target management of pregnant asthmatic females.10 Exhaled Breath Condensate Exhaled breath condensate co llection (EBC) is a noninvasive tool to collect breath in order to analyze its contents. The me thod involves an indivi dual breathing into a cooled chamber for approximately 15 minutes. The condensed breath is then analyzed for various substances.12 Figure 2. Schematic representati on of a EBC collection apparatus13 As previously discussed 8-isoprostane measurements in the exhaled breath condensate of pregnant females have not b een performed to date. This method of

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7 collection and analyzing oxidati ve products in the lung allows for a simple non-invasive mean of examining the human lower respiratory tracts3. The appeal of EBC lies in its ability to non-invasively collect a wide range of nonvolatile molecules from the respiratory tract.12 However despite its utility with respect to the noninvasive nature EBC portrays, there are drawbacks to analyzing breath in this manner. One of the major problems lie s in that of dilution which probably affects the concentration of most biomarkers analyzed with in EBC. In addition, ambient air pollutants may influence biomarkers. Natu rally EBC contains mostly water vapor, approximately >99.99%.12 Previous studies done in atte mpts to produce repeatability in measuring substances were unsuccessful and s howed to be variable between subjects and within repeated samples of the same subject.12 The collection chamber of the device and c ontainer that collects, cools, and stores the breath is composed either of glass, polystyrene or polypropylene. The composition the EBC may be altered by the adhesive pr operties of the material from which the container is made. This can be minimized by using a polypropylene container, as less adsorption occurs as compared to polystyrene.13 In addition, collection technique to mini mize saliva contamination is one of the most important concerns regard ing the collection procedure. Certain substances such as nitrite and hydrogen peroxide present in high quantities ca n contaminate sample data interpretation. This can be minimized by keeping the mouth dry during collection.13 Table 2 given below illustrates the va rious advantages and limitations of collection and analysis of exha led breath condensate collection.

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8 Table 2. Advantages and lim itations of collections and analysis of exhaled breath condensate13 Advantages 1. Simple, point-of-care intervention 2. Inclusive rather than in trusive (e.g., healthy childre n, mechanically ventilated neonates) 3. Domiciliary 4. Longitudinal sampling 5. Nonvolatile compounds associated with pulmonary pathophysiology 6. Amplified DNA and RNA from prokaryotic and eukaryotic cells 7. Pharmacokinetics/pharmacodynamics of drugs 8. Solute clearance Limitations 1. Lack of standard breath-sampling method 2. Not anatomic site specific 3. Lack of evidence for the origin of the aero sol particles (bronchi versus terminal airways) 4. Concentration artifact (due to evaporation of samples) 5. Feasibility and utility of biomarkers unrelated to oxidative stress not tested 6. Little information on biomarkers of interstitial lung disease Research Question Is there a baseline difference between 8-is oprostane levels in the exhaled breath condensate of pregnant women versus t hose levels in non-pregnant females? Hypothesis The null hypothesis states that there wa s no difference in the exhaled breath condensate oxidative marker PGF2 in pregnant females in their third trimester of pregnancy versus non-pregnant females. The alternative hypothesis st ates that there was a difference between the exhaled brea th condensate oxidative marker PGF2 in pregnant females in their third trimester of pregnancy versus non-pregnant females. The utility of the study may prove to provide a better unders tanding of oxidative stress in the pregnant

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9 state and may prove to provide a baseline measurement of PGF2 levels in the monitoring of airway, and other di sease states in pregnancy. Goals and Objectives The primary objective of this study serves to determine if there is a difference in PGF2 levels in the exhaled breath condensate of pregnant versus non-pregnant females. In addition, the following goals will be were 1) To assess the applicability of measuring baseline PGF2 levels in pregnant females versus non-pregnant females as a measure of oxidative stress 2) To begin to provide an examination of the generalizability of measurements of exhaled breath markers to pregnant populations, which may serve as the basis for a larger and more in-depth study 3) To examine whether baseline 8-isoprostane levels are appropriately representative of healthy pregnant females. Utility and Significance This study was unique in that it examined exhaled breath condensate levels in the breath of pregnant females which has not been previously performed. The utility of the study proved to provide a better understanding of oxidative st ress in the pregnant state and proved to provide a baseline measurement of PGF2 levels in the monitoring of airway and other disease states in pregnancy.

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10 Materials and Methods Study Design The study design was a cross-sectional de sign comparing two groups: a group of pregnant women ages 18 to 35 years in thei r third trimester and a group of non-pregnant women in the same age group. Exposure c onsisted of pregnancy and non-exposure consisted of non-pregnant. The outco me consisted of levels of PGF2 oxidative marker in exhaled breath condensate. Both groups were screened to exclude those with a recent history of upper respiratory infections or smoking and a ny history of environmental allergies or lung disease. In addition any individual in the pregnant group was excluded for any complication of pregnancy, including but not limited to gestational diabetes, hyperemesis gravidarum, preeclampsia, hype rtension, or any other complication of pregnancy. Inclusion criteria include female sex, within the age range of 18-35 years, nonpregnant state (including indi viduals that have previously been pregnant), and pregnant state – specifically within the third trim ester of pregnancy. Informed consent was obtained before any testing began. 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).

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11 Exhaled breath condensate samples were collected using Jaeger air condensing device (Jaeger, Wuerzburg, Germany 1999). The Jaeger instrument was located in the Breath Lab – MHH room 323. Samples were stored at approximately -70 degrees Celsius in The Breath Lab freezer. Once samples were ready to be analyzed they were removed from the freezer and de-thawed and analyzed using a commercially available enzyme linked immunoassay (EIA) kit, ordered from Caymen Chemical. The EIA kits were processed on a 96 well plate and analyzed using a spectrophotometer ( Quant Universal Microplate spectrophotometer,2004), wh ich is also available within the Breath Lab (MHH Room 323). All samples were used in their entirety. Spirometry was performed using the KoKo spirometry software and devices also located within the Breath Lab – Room M HH 323. Mouthpieces needed for performing the testing were disposable and locat ed with in the Breath Lab. Participant Recruitment Subjects were recruited to volunteer to pa rticipate in the study through a one time visit to The Breath Lab (Room MHH 323) ove r a range of months spanning from November 2005 to March 2006. Volunteers we re recruited thr ough primarily three mechanisms: 1) Advertisements in the form of a poster for non-pr egnant and pregnant females were displayed in various areas th roughout the University of South Florida Tampa campus and the Tampa General Hospit al Genesis OBGYN clinic (Appendix A,B) 2) A mass email targeting female students a nd staff in the University of South Florida health science centers was sent out twice 3) Researchers personally manned a booth located in the Tampa General Hospital Genesis OBGYN clinic to recruit in person.

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12 Researchers did not approach volunteers; rather potential su bjects voluntarily approached the booth. Study Subjects and Restrictions Healthy pregnant and non-pr egnant female subjects we re recruited who denied any history of environmental al lergies, asthma, or other lung diseases. Also, they must not have suffered from any recent upper or lo wer respiratory tract infections. 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 years, and no si gnificant second-hand smoke exposure. Subjects must refrain fr om strenuous exercise, food, or drink for one hour prior to the test. Pregnant subjects were restricted to the third trimester of pr egnancy. Previous history of pregnancy or deliver y did not exclude subjects. Gestational dates were taken in the medical history portion of the questionnaire form (Appendix A). Study Questionnaire and Eligibility In order to determine eligibility for potentia l healthy subjects, subjects were initially asked a series of questions. The questions were asked by telephone when the subject called in response to a recruitment flyer or email. If the potential subject denied any previous history of environmental allergy, de nied any recent URI in fections within the past two weeks, denied any chronic diseases and met the age criteria range the participant was scheduled for an appointment for testing with in The Breath Lab. Once the subject was met in the Breath Lab the Health Qu estionnaire was given to each subject. Responses were reviewed by re searcher and subject in a private cubicle.

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13 Any medications and health problems were re viewed by the researcher. If subjects met inclusion criteria they were invited to then move on to the informed consent documentation in order to participate in the study. Physical Examination All subjects were visually inspected for any cyanosis and clubbing. Also, auscultation of the heart and lungs was performed pr ior to any breathing tests and at the conclusion of each visit. In auscultation, a stethoscope is used to listen to both heart sounds and breath sounds. Auscultation is ro utinely and correctly done while disrobed, however, for our purposes assessment was done over outer garments. Pregnant females were examined identically to their non-pre gnant counterparts; of note, gestational age was not assessed on physical exam ination of pregnant females. Exhaled Breath Condensat e Collection and Storage Data Collection and 8-isoprostane Analysis Exhaled breath condensate samples were collected using Jaeger air condensing device (Jaeger, Wuerzburg, Germany 1999). The Jaeger instrument was located in the Breath Lab – MHH room 323. The Jaeger inst rumentation was turned on at least 15 minutes prior to EBC collection in order for adequate cooling time of the collection chamber to approximately -30 degrees Celsiu s. All valves and collection tubing was cleaned with cavicide solution and thoroughly dried prior to use. A disposable mouth piece and nose clip was given to each subj ect for collection. Apparatus was assembled and subjects were asked to sit in a chair during the fifteen minute collection time. Individual were asked to breathe tidally through their mouths, after a nose clip was

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14 applied, into the two-way non -re-breathable mouth piece co nnected to the collection chamber portion for a total of fifteen minutes. Subjects were asked to discard any excessive saliva produced into a tissue period ically. Reading mate rial was provided to each subject and timer was sent to 15 minutes with alarms at 10 and 5 minutes remaining. Subjects were notified that removal of their mouth from the tubing in order to cough, sneeze, etc was permitted. However, subjects were instructed that refraining from removing their mouth from the tubing was ideal in order to optimize sample collection. After fifteen minutes, collec tion chamber cups were unscrewed from the chamber and breath condensate was immediately transferre d to cryo safe storage tubes in a split sample using an analytic pipette. On aver age two ml of condensate was obtained and 1 ml stored in each tube. Samples were th en capped and placed in a -70 degree Celsius controlled freezer. Tubes were pre-labeled with subject identification numbers that identified samples with indi vidual subjects. Key sample ID sheets were stored in a laboratory workbook in Room MHH 323. Sample s were collected in this manner from November 2005 to March 2006. 8-isoprostane concentrations were measur ed in pregnant and non-pregnant subject samples using a commercially availabl e enzyme immunoassay (EIA) kit (Cayman Chemical Company, 8-isopros tane EIA kit model # 516351, Ann Arbor, MI). The 8isoprostane detection limit for the kit is 5 pg/ml. Normal Plasma 8-isoprostane levels for healthy subjects: 40-100 pg/ml (Cayman Chemical), Normal Urine 8-isoprostane levels for health subjects: 10-50 pg/ml (Cayma n Chemical), exhaled breath condensate measurements were not provi ded by Cayman Chemical. Exhaled Breath Condensate 8-

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15 isoprostane levels (two differe nt studies) demonstrated 8-is oprostane levels in a sample of 10 children (male) asthmatics levels were 9.4-29.5 pg/ml, same study sampled 10 healthy children (male) – levels were 2.1-3.0 (Baraldi, et.al., Thorax 2003). 8-isoprostane levels were demonstrated in a sample of 12 hea lthy children – avg. of 34.2+/-4.5 pg/ml, compared to a group of 12 as thmatic steroid nave children – avg of 56.4+/-7.7. (Baraldi, et.al., Chest 2003). Analyzing the 8-isoprostane kit was done via spectrophotometry ( Quant Universal Micro plate spectr ophotometer,2004). Absorbance readings of the EIA kit plates were taken according to the manuf acturer’s recommendation at 420 nm, 977 nm and 900 nm (the higher wavelengths were take n to negate background measurements). The EIA 8-isoprostane EIA kit detects 8-isoprostane starting from a concentration of 5 pg/ml. The intra and interassay precision for the kit is depicted below (Figure 3). Precision which is the component of accuracy th at is concerned with the consistency of the stability of results is demonstrated in Figure 3 as the degree to which the kit provides consistent results from one application to the next.

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16 Figure 3. Cayman chemical 8-isoprostane kit precision curve (Cayman Chemical, kit number 516351) The EIA 8-isoprostane laboratory analys is kit works via an Enzyme Linked Immunoassay Analysis (ELISA) method. It is based on the competition between 8isoprostane and 8-isoprostaneacetylcholinesterase (AChE) for a limited number of 8isoprostane-specific rabbit antiserum binding si tes. So, the concen tration of AChE is known, and the concentration of 8-isoprosta ne is unknown. The re action between bound AChE and the antiserum binding sites produces a yellow color which is absorbed at 412 nm of light via the spectrophotometer. Ther efore the amount of 8-isoprostane in each

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17 sample is inversely proportional to the am ount of bound 8-isoprostane AChE, which is in turn directly proportional to absorbance. (See below). Absorbance {Bound 8-Isoprostane Tracer} 1/{8-Isoprostane} In order to calculate concentrations from the absorbance values the following manner: 1. We average the absorban ce readings from the NSB wells. (NSB well = nonspecific binding that occurs even in the absence of antiserum – this artificially elevates total antiserum bound). 2. We average the absorbance reading from the B0 wells (B0 is the max tracer the antiserum can bind in the absence of free analyte). 3. We subtract NSB average from the B0 avg. This is the corrected B0 or the actual real maximum amount bound. 4. We calculate % B/B0 (%sample or standard bound /maximum bound) for all our sample wells. Once this is done we can use the standard curve to locate the %B/B0 and find the corresponding concentration on the xaxis. The standard curve was produced via a set of known concentrations of 8isoprostane provided in the EIA kit. Acquisition of Spirometry Spirometry, which is a common clini cal method employed in assessing lung function, was performed on each s ubject after collecting exhale d breath condensate. The Koko spirometer was used and calibrated us ing a standard 3-liter syringe to ambient temperature, humidity, and barometric pr essure at least once a day on days when participants were examined. The spirometer was additionally re-calibrated when at least

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18 six hours had elapsed since the prior calibration or at the discretion of the examiner. The raw FEV1 and FVC measurements obtained fo r each participant were automatically compared to their predicted normal values based on age, ethnicit y, weight, height, and non-smoking status in determining their percen t 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-vol ume 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 nose clip 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. 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 spirometric criteria.

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19 Adverse effects were unlikely 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 br ief in duration. All s ubjects 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 precauti ons 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.

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20 Results Study Population Characteristics The study population consiste d of 22 subjects total; a total of 17 non-pregnant females and 5 pregnant females. Pregnancy dates in terms of weeks ranged from 25-32 weeks. The age of the population ranged from eighteen to thirty five years with a median age of twenty-three years. Fourteen of the twenty-two subjects’ occupations were students, one a teacher, two waitresses, one c oordinator, one scientis t, and one lab tech. None of the subjects were smokers, and one reported a past smoking history of 2 packyear history. Sixteen of the twenty-two subjects reported no pa st medical history problems. Nine of the twenty-two s ubjects reported no use of over the counter medications, vitamins or supplements. Date of onset of last menstrual cycle was asked and recorded for non-pregnant subjects and ranged from 1-28 days with a mean of 14. Symptoms of cough, phlegm production, itchy or runny nose, throat irrita tion, and chest tightness or pain was asked on a four point scale zero = none, one = a little, two = moderate three = very much. One subject reported throat irrita tion and rated it as a little. Two subjects repo rted cough and both rated it as a little. Five subjects reported phlegm production one reported it as moderate, four reported it as a little. Four subjects re ported an itchy or runny nose and three reported it as a little, one reported it as moderate. One subject reported chest

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21 tightness or pain and rated it as a little. Tabl e 3 lists population characte ristics, as well as Appendix I. Table 3. Demographic characteristic s of subjects both pregnant and non pregnant Subjec t Occupatio n Preg nant Weeks Menses Smokin g PMH Meds Age A1 Student No 14 0 Hernia 30 A2 Student No 7 0 Insomni a OCP/Ben /Flex 22 A3 Student No 21 0 OCP/MV I 23 A4 Student No 21 0 25 A5 No 14 0 20 A6 Student No 21 0 OCP 23 A7 Teacher No 28 0 21 A8 Student No 5 0 Food allergy 20 A9 Student No 10 0 24 A10 Sales No 7 0 Childhd asthma OCP/Mot rin 20 A11 Student No 21 0 29 A12 Student No 14 0 28 A13 Student No 21 0 OCP/Mot rin 20 A14 Waitress No 7 0 Celebrex 18 A15 Coordinato r No 14 0 Motrin 28 A16 Student No 1 0 Migrain e h/a Axert 25 A17 Student No 14 0 OCP 24 B18 Lab Tech Yes 30 0 0 35 B19 Scientist Yes 31 0 2 Effexor/ MVI 33 B20 Waitress Yes 32 0 0 Gestati on. DM Glyb/MV I 26 B21 Student Yes 25 0 0 MVI/ Tylen 20 B22 Student Yes 25 0 0 MVI 28

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22 *OCP = oral contraceptive pill, Ben = Benadr yl, Flex = Flexeril, MVI = multivitamin 8-Isoprostane Measurements The data obtained from analyzing exha led breath condensate samples from study subjects using the 8-isoprostane EIA kit (C ayman Chemical) are given below in graph and tabular forms. The standard curve used to calculate 8-isoprostane concentrations is given in Appendix K and L. Appendix K sta ndard curve shows values prior to removing concentrations 500 pg/ml and 250 pg/. Remova l of these concentrations as shown in standard curve showed no significant change in concentration calculations for study samples. 8-isoprostane concentrations obt ained from standard curve were used for biostatistical manipulations. Table 4. 8-isoprostane concentrations in pregnant and non-pregnant females. Sample ID #1 – CONC (PG/ML) #2CONC (PG/ML) #3 – CONC (PG/ML) AVG SD A1 4.182 7.298 16.689 9.389 6.511 A2 16.925 14.207 16.498 15.877 1.462 A3 8.803 4.374 7.555 6.911 2.284 A4 18.469 8.700 13.759 13.642 4.886 A5 A6 5.372 10.199 7.538 7.703 2.417 A7 7.311 8.553 5.979 7.281 1.288 A8 8.048 10.690 11.368 10.035 1.754 A9 24.335 16.402 15.625 18.788 4.820 A10 24.421 11.708 13.945 16.692 6.787 A11 8.048 11.368 10.621 10.012 1.742 A12 9.837 6.188 12.744 9.590 3.285 A13 16.901 18.010 11.116 15.342 3.702 A14 11.533 5.770 9.616 8.973 2.935 A15 11.389 8.120 7.967 9.159 1.933 A16 38.277 14.207 13.684 22.056 14.050 B1 12.971 10.655 5.562 9.730 3.790 B2 13.502 21.182 10.881 15.188 5.354 B3 10.805 4.642 6.313 7.253 3.187 B4 52.832 42.412 48.122 47.789 5.218 B5 8.349 4.954 6.651 2.401 A = NON-PREGNANT, Mean = 11.51333, B = PREGNANT, Mean = 16.64

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23 Concentrations were calculated as a f unction of the light absorbance of the 8isoprostane AChE tracer which is indirectly proportional to the concentration of 8isoprostane. Given below in figures 4 and 5 are pregnant and nonpregnant concentrations obtain from subject exhaled breath condensate samples. Figure 4. 8-isoprostane con centrations taken from samples in pregnant females 8-Isoprostane Levels in Pregnant Females0 10 20 30 40 50 60 12345 Subject Number8-isoprostane concentrations ( p g /ml )

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24 Figure 5. 8-isoprostane concentrations in samples taken from non-pregnant females 8-Isoprostane levels in non-pregnant females (pg/ml)0 5 10 15 20 25 13579111315 subject number8-isoprostane concentration (pg /ml ) Table 5 depicts standard curve valu es used for concentrati on calculations. These values were obtained by using known 8-isoprostane concentra tion standards given in the kit. Table 5. Standard concentrations (known) a nd actual absorbance concentrations detected STD # Known Conc (pg/ml) Calc conc #1 (pg/ml) Calc Conc #2 (pg/ml) Calc conc#3 (pg/ml) AVg SD 1 500.000 2 250.000 207.790 164.255 166.470 179.505 24.521 3 125.000 94.526 86.222 141.009 107.253 29.528 4 62.5000 70.312 82.303 90.090 80.902 9.963 5 31.300 26.648 28.106 34.964 29.906 4.441 6 15.600 17.383 11.143 20.294 16.273 4.675 7 7.800 9.304 6.860 4.870 7.011 2.221 8 3.900 4.516 5.228 4.872 .504

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25 Table 6. Standard concentrations (known) a nd actual absorbance concentrations detected using standard curve without known con centrations 500pg/ml, 250 pg/ml and 125 pg/ml STD # Known Conc (pg/ml) Calc conc #1 (pg/ml) Calc Conc #2 (pg/ml) Calc conc#3 (pg/ml) AVg SD 1 500.000 2 250.000 3 125.000 4 62.5000 57.886 57.886 5 31.300 27.485 28.807 34.683 30.325 3.832 6 15.600 18.450 11.753 21.408 17.204 4.947 7 7.800 9.700 6.940 4.693 7.111 2.508 8 3.900 4.296 5.096 4.969 .565 Utilizing the standard curve data shown in table 6 above to obtain sample data concentrations for subjects di d not statistically alter calcul ated sample concentrations. The statistical calculations fo r 8-isoprostane concentrations of non-pregnant and pregnant subjects are given below in Table 8. Table 7. Spirometric data for all subjects tested including pregna nt and non-pregnant females Subject Number FEV1/FVC A1 A2 93% A3 A4 111% A5 90% A6 98% A7 77% A8 84% A9 91% A10 88% A11 A12 94% A13 97% A14 87% A15 96% A16 99% A17 B1 B2 91% B3 88% B4 94% B5 92%

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26 No subjects were excluded based on sp irometric measurements. The above values do not classify any of the subjects as having an obstructive respiratory component. Biostatistical Data for 8Isoprostane Measurements Table 8. Statistical analysis of mean 8-is oprostane values of 20 subjects tested Statistical Measure Non-Pregnant Pregnant N 15 5 Mean 11.513 17.34 Median 9.6 9.7 Mode 10.0 Standard Deviation 4.966 17.355 Skewness 0.841 2.039 Coeff Variation 43.131 100.08 Sum Weights 15 5 Sum Observations 172.7 86.7 Variance 24.659 301.193 Kurtosis -.2966 4.225 Std Error Mean 1.282 7.761 Lower CL of the mean 8.7633 -4.209 Upper CL of the mean 14.263 38.889 Lower CL of Std Dev 3.6356 10.398 Upper CL of Std Dev 7.8317 49.87

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27 The data given above show a difference between the means of the two groups. The standard deviation of each group is larg e and demonstrates a largely skewed set of data in each of the two sets. In addition th e Lower CL of the means and upper CL for the means of each group overlap. Performing a T-test on the data in order to determine if the difference between the means of the two groups was significant the following data was obtained as shown in Table 9 below. Table 9. T-tests used to analyze difference between means of the two groups Variable Method Variances DF T value Pr > t 8-iso Pooled Equal 18 -1.22 0.2397 8-iso Satterthwaite Unequal 4.22 -0.74 0.4980 The statistical analysis (Satterthwaite Te st for normally distributed data with different variances) failed to show a differen ce between the two means of the data groups as the p-value was greater than 0.05. The power of the study was calcu lated and shown to be .518. Table 10. Variables involv ed in power calculations 1 N1=15 2 N2=5 Critical Value Power 11.513 17.34 4.9659 17.3550.05 4.84434 .518 Because = 1 – Power, beta (or the probabi lity of making a type II error) was calculated to be e qual to 48.2%.

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28 Conclusions To reiterate, the question of interest ha s been whether a difference exists in the average 8-isoprostane levels between two groups: Pregnant Females and Non-Pregnant Females aged 18-35 years. When analyzing the demographic charac teristics of the two groups it became evident that two subjects in the non-pregna nt group could be excluded. One subject reported a history of childhood asthma; another re ported a history of f ood allergies. Both of these subjects were excluded from data calculations. Subjec t A10 self reported a history of childhood asthma; her 8-isoprostane concentr ation was 16.692 pg/ml. The subject A8 self reported a hi story of food allergy; her 8isoprostane concentration was 10.035. All other data collected from subjects was used for calculations. The means of the non-pregnant and pregnant groups was 11.513 and 17.34 respectively. There is an obvious differen ce between these two means, but is this difference statistically significan t? According to the t-test performed on the two means there was not a statistical difference found. The null hypothesis stated that there was no difference in the exhaled breath c ondensate oxidative marker PGF2 in pregnant females in their third trimester of pregnancy vers us non-pregnant females. The alternative hypothesis stated that there was a difference between the exhaled breath condensate oxidative marker PGF2 in pregnant females in their third trimester of pregnancy versus non-pregnant females. Based on our t-test we must fail to reject the null hypothesis. As to why a difference between the two groups was not found could primarily be due to the sample size of the study. Power is largely affected by samp le size, that is, if

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29 the sample study size is small the study w ill have insufficient power to detect real associations. Since Power = 1the smaller the power the larg er the beta (probablility of not finding an association in a sample wh en one exists in the population). The standard deviations of both the pre gnant group and non-pregnant group were large; 4.966 and 17.355 respectively. This could be in part due to the distribution of 8isoprostanes in females, both pregnant and non-pr egnant is large in nature. However, we cannot statistically confirm this as our sample size is not large enough for us to conclude that we have statistically found a difference between the two means. As previously discussed, Staff, et.al. reported an increased content of lipid peroxides in preeclamptic deci dual placental tissue in preeclam ptic patients at delivery compared with controls.8 Another study by Moretti and colleagues demonstrated increased markers of oxidative stress in preeclamptic women versus uncomplicated pregnancy and non-pregnant controls by meas uring various volatile organic compounds in the exhaled breath condensate of these women9 This study analyzed non-pregnant, healthy pregnant and preeclamptic pregnant fe males. We sought out to determine if a difference exists between baseline levels of 8-isoprostanes between a group of healthy pregnant females and non-pregna nt females. This difference between the means of the two data sets was shown and is an observed difference, however is not a statistically significant difference. The implication that the crude means of the two groups indicated an observable difference but not a statistically significant di fference provides a basis for the need of future studies. Future studies would need to address the nece ssity of larger sample sizes

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30 in each group. This serves as a challenge as participant recruitment of pregnant females in their 3rd trimester was low in this study.

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31 References 1. Janssen LJ. Isoprostanes: an overvi ew and putative roles in pulmonary pathophysiology. Am J Physiol Lung Cell Mol Physiol 2001;280(6):L1067-82. 2. Montuschi P, Corradi M, Ciabattoni G, Nightingale J, Kharitonov SA, Barnes PJ. Increased 8-Isoprostane, a Marker of Oxidativ e Stress, in Exhaled Condensate of Asthma Patients. Am J Respir Cr it Care Med 1999;160(1):216-20. 3. Van Hoydonck PGA, Wuyts WA, Vana udenaerde BM, Schouten EG, Dupont LJ, Temme EHM. Quantitative analysis of 8-isopr ostane and hydrogen peroxide in exhaled breath condensate. Eur Re spir J 2004;23(2):189-92. 4. Baraldi E, Carraro S, Alinovi R, et al Cysteinyl leukotrienes and 8-isoprostane in exhaled breath condensate of children with asthma exacerbations. Thorax 2003;58(6):505-9. 5. Baraldi E, Ghiro L, Piovan V, et al. Increased Exhaled 8-Isoprostane in Childhood Asthma. Chest 2003;124(1):25-31. 6. Wood LG, Gibson PG, Garg ML. Biomar kers of lipid peroxidation, airway inflammation and asthma. Eur Respir J 2003;21(1):177-86. 7. Goil S, Truog WE, Barnes C, Norberg M, Rezaiekhaligh M, Thibeault D. Eight-epiPGF2[alpha]: A possible marker of lipid peroxidation in term infants with severe pulmonary disease. The Journal of Pediatrics 1998;132(2):349-51. 8. Staff AC, Halvorsen B, Ranheim T, He nriksen T. Elevated level of free 8-isoprostaglandin F2[alpha] in the decidua basa lis of women with preeclampsia. American Journal of Obstetrics and Gynecology 1999;181(5):1211-5. 9. Moretti M, Phillips M, Abouzeid A, Ca taneo RN, Greenberg J. Increased breath markers of oxidative stress in normal pregna ncy and in preeclampsia. American Journal of Obstetrics and Gynecology 2004;190(5):1184-90. 10. Murphy VE, Gibson PG, Smith R, C lifton VL. Asthma during pregnancy: mechanisms and treatment implications. Eur Respir J 2005;25(4):731-50. 11. Held H-D, Uhlig S. Mechanisms of Endotoxin-Induced Airway and Pulmonary Vascular Hyperreactivity in Mice. Am J Respir Crit Care Med 2000;162(4):1547-52.

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32 12. Holz O. Catching breath: monitoring ai rway inflammation using exhaled breath condensate. Eur Respir J 2005;26(3):371-2. 13. Mutlu GM, Garey KW, Robbins RA, Danz iger LH, Rubinstein I. Collection and Analysis of Exhaled Breath Condensate in Humans. Am J Respir Crit Care Med 2001;164(5):731-7.

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33 Appendices

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34 Appendix A: Questionnaire Form Health Questionnaire What is your date of birth?___________________________ Your current age?____________________________ What is your current occupation?_________________________________ Have you ever been pregnant? YES NO G___P___A___ Are you currently pregnant? YES NO EDC_________ When was your last menstrual period?___________________________________ Do you have any medical complications relate d to your pregnancy such as diabetes, hypertension, morning sickness, etc? YES NO If yes, what conditions?__________________________________________________ Do you smoke? YES NO Are you exposed to second hand smoke? YES NO Have you ever smoked? YES NO How many packs per day did you smoke?___________________ For how many years did you smoke?_______________________

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35 Appendix A (Continued) When did you quit smoking? _______________________________________________ Do you have any medical problems? YES NO Please list them here. _________________________________________________________________ __________________________________________________________________ __________________________________________________________________ Have you ever had any of the following conditions? Chest pain, palpitations, irregu lar heart beat, or heart disease? YES NO High blood pressure? YES NO Asthma, bronchitis, emphysema, or other lung disorder? YES NO Do you see your health care provider on a routin e basis for any medical condition? YES NO If so, what?______________________________________________________________ Do you take any prescription or ov er-the-counter medications? YES NO If so, what? ________________________________________________________________ ________________________________________________________________ __________________________________________________________________ When is the last time you remember being ill?_______________ What illness did you have?___________________________________________ To what degree do you have the following symptoms at this time? Cough: NONE A LITTLE MODERATE VERY MUCH

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36 Appendix A (Continued) Phlegm production: NONE A LITTLE MODERATE VERY MUCH Itchy or runny nose: NONE A LITTLE MODERATE VERY MUCH Throat irritation: NONE A LITTLE MODERATE VERY MUCH Chest tightness or pain: NONE A LITTLE MODERATE VERY MUCH Have you been exposed to any gases, dusts, or fume s at home or on the job? YES NO If so, please explain: ________________________________________________________________________ ________________________________________________________________________ _______________________________________________________________________ Post Study Questionnaire Did you have any concerns about the way the in formed consent process was handled? YES NO Did you have any problems with the Health Questionnaire? YES NO Did you have any discomfort or concerns af ter the physical exam? YES NO Did you have any discomfort or concerns after the nitric oxide test? YES NO Did you have any discomfort or concerns after completing the spirometry? YES NO Please elaborate. Use the back of this page is necessary.

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37 Appendix B: Adult University of Sout h Florida Informed Consent Document Informed Consent for an Adult University of South Florida Information for People Being Asked to Take Part in Research Studies IRB Study #103718c Doctors and researchers at Un iversity of South Florida (U SF) 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 pe ople who agree to take part in a research study. Title of Research Study: 8-isoprostane levels in exhaled breath condensate of pregnant women compared to non-pregnant women, is there a baseline difference? Person in Charge of Study: Rosemary Szollas, M.D. and Stuart Brooks, M.D.; Karen Olson, M.D. 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 Resource 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. Readi ng this form should help you decide if you want 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 docto r 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.

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38 Appendix B (Continued) You don’t have to guess at things yo u don’t 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. It’s 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: The purpose of this study is to find out if th ere is an increased le vel of 8-isoprostane in the exhaled breath condensate of pregna nt women. 8-isoprostane is a chemical made in the body naturally. The amount of 8-isoprostane also increases in the lungs in some diseases of the lung. This study will help find out if pregnancy causes an increase in the production of 8-isoprostane in the lungs. This study will help find out if 8-isoprosta ne levels increase during pregnancy and help in understanding of how pregnancy affects asthma. To measure 8-isoprostane in the breath we need to collect your exhaled breath. Breathing normally into a mouth size tube will do this. The tube is cooled to a very low temperature thereby changing the gas you ex hale into a liquid form. The liquid is a suitable way to collect your br eath and store it for analyzing. Why are you being as ked to take part? We are asking you to take part in this st udy because you are a woma n of child bearing age who is pregnant or who is not pregna nt. We need to compare these two groups. In order to participate in this study, you n eed to be between 18 – 35 years of age, a non-smoker with no history of asthma, envir onmental allergies, chronic illness, or recent respiratory infection (i n the past month). If you are pregnant, you need to be at 28 weeks gestation or more (as determined by your personal hea lth care provider) when you come to the Breath Lab for the study. How long will you be asked to stay in the study? You will be asked to spend a bout three hours in this study. The sample collected from your breath will be stored for a maximum of three months, the

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39 Appendix B (Continued) analyzing of your sample will take about tw o hours, you will not be required to be present for this portion of the study. One hour includes the time it takes to complete the informed consent process and fill out a health questionnaire. You may choose to take more time to complete the informed consent. A brief physical examination, collection of exhaled breath condensate and spirometry will take about an hour. 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 doctor. You will need to come for one study visits in all. How many other people will take part? About fifty people will take part in this study at USF. Will the medical treatment you get from your regular 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. The only way that it could potentially change is if we discover a medical condition which was not previously di agnosed and your doctor chooses to treat you for it. You will keep seeing your regular doctor. Your regular doctor will give you the same kind of treatment you would get anyway, whet her you take part in the study or not. This study includes only hea lthy women whose only condition requiring treatment should be pregnancy. If you require any other treatme nt, please do not hesitate to get it. We need you to inform us of any treatment in the month before tes ting or in the week following testing, other than routine prenatal ca re, because it may affe ct or ability to use your results in this study.

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40 Appendix B (Continued) What other choices do you have if you decide not to take part in this study? If you decide you do not want to take part in this study, that is okay. We are performing some simple diagnostic tests. These are probably not necessary for your medical care if you are bei ng included in this study. If you wanted or needed them done, your pers onal health care pr ovider could perform a physical examination and order spirometr y. If you wanted to know the level of 8isoprostane in your breath, you would only be able to have it measured in a study such as this one. It is an experimental study which is not generally available. How do you get started? If you decide to take part in this study, you will need to sign this consent form. Then, we will do some screening tests. Scr eening tests are tests done to see if you are able to be in the study. Scr eening tests are different from the actual study procedures. The definition of screening is the application of a test to detect a potential disease or condition in a person who has no known signs of that disease or condi tion. The screening tests are done, therefore, prio r to the actual study procedures to make sure the researchers identify the correct population of indi viduals to include into the study. We will do these screening tests: 1. The health questionnaire will be used to confirm whether you meet the criteria for being involved in this study. In order to be included you need to be a non-smoker be 18 – 35 years of age have no environmental allergies such as hay fever have no chronic illnesses which require routine monitoring or treatment, especially asthma or other pulmonary diseases be twenty-eight or more weeks pregnant when you are scheduled to be tested OR be non-pregnant, having had a normal me nstrual period in the month prior to testing 2. The brief physical examination will include listening to your hear t and lungs. In order to be included you need to have

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41 Appendix B (Continued) clear lung sounds without wheezing, rales, or rhonchi (abnormal lung sounds) a regular heart beat without a ny murmurs, rubs, or gallops There is not a separate visit to complete the screening tests. The results will be available once the collected samples are analyzed. Then you and the research team will decide whether or not you continue in the study. What will you need to do to get ready for this study? For one hour prior to your scheduled appoint ment at the Breath Lab, you will need to refrain from eating, drinking, or performing any strenuous exercise such as running, weight lifting, or heavy physical labor. What will happen during this study? You will need to complete five things to complete this study: 1. This informed consent document 2. A health questionnaire 3. A brief physical exam where we listen to your heart and lungs 4. Spirometry 5. The collection of exhaled breath condensate If you have not completed the informed consent before you come to the Breath Lab, we will complete it at the beginning of your visit. If you agree to partic ipate in the study and sign the informed consent, we will proceed. The health questionnaire is a short assessm ent of your health hi story and any current symptoms you may be experienci ng. It will be used to collect demographic ( a group of characteristics defining huma n populations) information about you such as your age and medical information about your heal th history and recent symptoms. After you complete the health questionnaire, th e principal investigator (PI) will listen to your heart and lungs. She needs to see if you have wheezing or rhonchi (abnormal breath sounds) or an irregular heart beat or murmur. Any of thes e findings will probably result in your withdrawal from the study. Next, we will measure how fast you can blow the air out of your lungs and how much air you can blow out in a test called spirometry. This test is used routinely to monitor people of all ages who have lung disease. We are us ing it in our study to be certain that you do not have any type of undiagnosed lung disease which would affect our results. Spirometry takes about as much effort as blowing up a big balloon and is the most difficult test to perform in this study. Once again, we need to have three acceptable

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42 Appendix B (Continued) measurements to complete this test. We will not repeat this test more than eight times even if we do not get three acceptable tracings. Most people are able to complete this test in five attempts or less. You can take your time; you can take a break; or you can stop at any time. Finally, your exhaled breath will be collected and cooled in order to condense (turn from a gas to a liquid form) your br eath into a liquid for collecti on and storage. In order to collect your breath condensate we will ask you to breathe as you normally would through a disposable tube. We will ask you to do this for a maximum of fifteen minutes, in order to collect an adequate sample amount. If you need to break to cough, sneeze, or blow your nose, or for any other reason you will be asked to remove your mouth from the tube and resume breathing through the tube when co mfortable once again. We can collect at your pace if necessary. The time limit is not concrete as it is a recommended time period for collection by the manufacturer of the colle ction device, however if adequate sample size is obtained prior to fi fteen minutes you may stop if you would like to do so. After collection of exhaled breath condensate, we will ask you if you have any questions about the tests, any discomfort at all, or any other concerns All of these responses will be recorded on your h ealth questionnaire. Will you be paid for taking part in this study? Subjects will be offered twenty dollars upon completion of exhaled breath condensate collection and for their time sp ent participating in the study. If for any reason a subject is unable to complete the study, the following payment schedule will be used: 1. Completion of exhaled breat h condensate collection $20 We are not able to reimburse you for additi onal costs you incur such as parking fees, bus or taxi fare, childcare costs, or time away from work. What will it cost you to take part in this study? It will not cost you anything to be part of the study. You will have to pay for your regular car e or any other costs. Your insurance plan should cover your regular costs. Your insurance plan will not have to pay for any study costs. What are the potential benefits if you take part in this study? We don’t anticipate that you w ill get any health benefits from taking part in this study. We are performing diagnostic (used to identify disease or conditions) tests only; not providing any treatment.

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43 Appendix B (Continued) You may potentially benefit by learning more about your health as a result of your physical examination and te sting during this study. You may enjoy being part of a st udy and seeing how it is conducted. No matter what, we will learn more about changes in the lungs during pregnancy. What we learn may guide further re search about asthma and pregnancy. What are the risks if you take part in this study? There is very little risk that you incur by being part of this study. You may experience some disc omfort during the collecti on of the exhaled breath condensate or during the spirom etry. Spirometry, in particular, requires you to blow out as hard and fast as you can. This is something like blowing up a big, stiff balloon. You could potentially get an infection in the Breath Lab. This is probably less likely than getting an infection at the grocery st ore. Currently, all subj ects being tested are healthy subjects. Everyone gets a new, di sposable mouthpiece to use. Anything that gets contaminated is cleaned according to lab protocol. Should you experience any adverse symptoms or event, you will be under the direct supervision of a physician at all time s during the study. We have emergency equipment available and will call for emergency personnel (911) if needed. Is there any risk to your unborn children if you take part in this study? This study does not involve any treatm ent which could affect you unborn child. Two of the diagnostic studies which are be ing performed, physical examination and spirometry, are routinely performed in pre gnant women who have asthma without any adverse outcome. The experimental diagnostic study, collecti on and analyzing levels of exhaled breath 8-isoprostane, does not expose you to any phys ical, chemical, or biological hazard which could potentially harm you or your unbor n child. It is simply a measurement of a common constituent in exhaled breath. What if you get sick or hurt while you are in the study? If you need emergency care: Go to your nearest hospital or em ergency room right away. Call 911 for help. You should know that 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 Rosemary Szollas, M. D., or Stuart Brooks, M.D. at (813) 974-

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44 Appendix B (Continued) 7545. If it is NOT an emergency, and you get hurt or sick while you are taking part in this study: Go to your regular doctor. Please inform the study doctors. They will need to know if you are hurt or ill. Call Rosemary Szollas, M.D. or St uart Brooks, M.D. at (813) 974-7545. 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 situat ions. 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 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 Insuran ce Programs (SIP) at 1-813-974-8008 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 didn’t do something they should have done. Ask the SIP to look into what happened. Recruitment for this project has been conduc ted at a Tampa General clinical facility therefore participation in this research project will make you subject to the following: ADULT TAMPA GENERAL INJURY STATEMENT In the event you suffer an injury or illness as a result of participating in this research study please be aware that immediate, short-term medical treatment for the injuries or illness will be available to you from Tampa General Hospital. The cost of the medical treatment will be billed to you to the extent not covered by your insurance company or government program or study sponsor No other compensation will be offered. You are not giving up any legal rights by signing this form. If you believe you have experienced a reaction to the study medication or have been injured as a resu lt of research procedures performed at Tampa General Hospital, pl ease contact the Department of Risk Management at (813) 844-7666.

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45 Appendix B (Continued) What will we do to keep yo ur study records private? Federal law says we must keep your study records private. We have made prior arrangements to do this. To maintain subject confiden tiality, each subject will be given a random three digit subject number which will be recorded on the informed consent document and compiled in a master list of all subjects. This number will be the only identifier listed on the health questionnaire, the data collec tion form, the appointment list, spirometry report, and sample storage tubes. Wit hout the informed consent documents or the master list, it will not be possible to link any confidential information to the subject. The informed consent document and a master list of subjects will be kept in a locked file cabinet in the Breath Lab or in th e Occupational Medicine Residency Office. Access to this material will only be avai lable the investigators in the study. Questionnaires and recorded data from the st udy will be kept in a locked file cabinet in the Breath Lab or in the Occupational Me dicine Residency office separate from the informed consent documents and the master list whenever they are not in direct possession of the investigators. These doc uments will not contain any information which could, by itself, link them to you. 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: 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 oversigh t 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 Florid a Department of Health, people from the Food and Drug Administration (FDA) and the De partment of Health and Human Services (DHHS) (This is done to make sure that we are doing the study in the right way. They also need to make sure that we are protecting your rights and your safety.) 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.

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46 Appendix B (Continued) 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 inform ed consent document that you no longer want to take part in this study. If you decide you want to stop taking part in the study, tell the study staff as soon as you can. You can stop at any time without any adverse outcome. If you decide to stop, you can continue ge tting 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, ther e 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 fo r you to stay in the study. You experience a change in your health which is inconsiste nt with the study criteria. You miss your appointment at the Breath La b twice without call ing ahead of time to reschedule. You can get the answers to your questions. If you have any questions about this study, ca ll Rosemary Szollas, M.D. at (813) 9747545. If you have questions about your rights as a person who is taking pa rt in a study, call the Division of Research Compliance of the Univ ersity of South Flor ida at (813) 974-9343.

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47 Appendix B (Continued) 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. ____________________________________________ ___________ Signature of Person Taking Part in Study Date ____________________________________________ Printed Name of Person Taking Part in Study Optional Witness ____________________________________________ ___________ Signature of Witness Date ____________________________________________ Printed Name of Witness Statement of Person Obtaining Informed Consent I have carefully explained to the person taki ng 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. 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

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48 Appendix B (Continued) 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 understand what is being explained and can, therefore, give informed consent. ____________________________________________ ___________ Signature of Person Obtaining Informed Consent Date __________________________________________________ Printed Name of Person Obtaining Informed Consent Optional Witness ____________________________________________ ___________ Signature of Witness Date ____________________________________________ Printed Name of Witness Addendum to the Consent Consent to take and store additional samples. We are asking you to allow us to take and store additional samples of your exhaled breath for use in the future. These samples may be used for a variety of purposes. This means we will take some of your exhale d breath and cool it in order to collect the liquid form of your breath. We will then fr eeze this small amount of liquid (about half of a teaspoon) at below freezing temperatures in order to stabilize the compounds within your breath. Once we are capable of analyzing your sample within the breath lab we will de-thaw the sample and utilize it in a chemical assay kit that determines the concentration of 8-isoprostane within your br eath. Your entire sample colle cted will be used for this technique and we will not discard any of the sample. When you sign this below, you are agreeing to let us store and use your exhaled breath condensate for future research studies. We may use these samples to help us: Determine if 8-isoprostane is elevated in the exhaled breath condensate of pregnant women. Help us with understanding how pregnancy affects asthma. ____ I give my consent to provide my e xhaled breath condensate for that purpose.

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49 Appendix B (Continued) ____ I do not give my consent to my exhaled breath condensate for that purpose. ____________________________________________ ___________ Signature of Person Taking Part in Study Date ____________________________________________ Printed Name of Person Taking Part in Study Optional Witness ____________________________________________ ___________ Signature of Witness Date ____________________________________________ Printed Name of Witness