Exhaled Nitric Oxide during Academic Examination Stress in Students with Asthma

Online Data Supplement Exhaled Nitric Oxide during Academic Examination Stress in Students with Asthma Thomas Ritz 1, Ana F. Trueba 1,2, Jiayan Liu 3, Richard J. Auchus 3,4, and David Rosenfield 1 1 Department of Psychology, Southern Methodist University, Dallas, TX, USA 2 Department of Psychology, Universidad San Francisco de Quito, Quito, Ecuador 3 Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor, MI 4 Department of Internal Medicine, UT Southwestern Medical Center, Dallas, TX, USA

Expanded Methods for Selected Sections Measures FeNO was measured using a hand-held electrochemical analyzer (NIOXmino, Aerocrine, Solna, Sweden; E1) following standard recommendations(e2,e3)3,26). Participants were instructed to avoid exercise or heavier physical activity in the two hours before the assessments and food or drinks except for water should be avoided for one hour before assessments. Temporal stability of exhaled nitric oxide measurements has been demonstrated in prior research (E4,E5). A handheld spirometer (CareFusion/JaegerAM2) was used to obtain forced expiratory flow in the 1 st second (FEV 1 ) and peak expiratory flow (PEF) as the best of three valid forced expiratory maneuvers. These assessments were scheduled at the end of the session to avoid influences on FeNO (E6). Percent of predicted FEV 1 and PEF were determined with reference to standard norm populations(e7). Saliva was collected with Salivettes (Sarstedt, Inc., Newton, NC) containing sterile cotton plugs, which participants were instructed to place in the mouth for two minutes. Samples were subsequently stored at 80 Celsius. Salivary cortisol was determined using a Coat-A-Count cortisol kit TKCO2 (Siemens Medical Solutions Diagnostics, Los Angeles, CA) with a detection limit of 0.03 pg/ml with serial dilution of the lowest calibrator standard. Participants filled in the 20-item Positive Affect Negative Affect Schedule (PANAS-NA; E8) for current mood. The 10 items of the negative affect scale were scored. The 14-item Hospital Anxiety and Depression Scale (HADS; E9) was used for measuring anxious and E2

depressive mood in the past week with 7 items each. The 10-item Perceived Stress Scale (PSS; E10) measured feelings of stress, lack of control, and being overwhelmed in the past 4 weeks. Additionally, the 44-item Wisconsin Upper Respiratory Symptom Survey 44 (WURSS; E11) was used to control for respiratory infection symptom, the 32-item symptom severity scale was analyzed. Participants with asthma also completed the 5-item Asthma Control Test (ACT; E12). Data Analysis Basic biometric and demographic variables were compared by t-test or 2 -test. Multilevel models (MLMs) were used to analyze the longitudinal data because MLM is an intent-to-treat analysis that retains all participants regardless of missing data or dropout, thereby maximizing power and generalizability (n=5 asthmatic, n=3 controls did not complete the second exam stress assessment, the small number precluded meaningful statistical comparison between completers and non-completers on early exam stress levels and negative affect). The growth curve of the outcomes over time were modeled as quadratic. However, quadratic effects were dropped from final models if they were nonsignificant to arrive at the most parsimonious model and to minimize Type II error(e13, E14). Gender, age, and cold symptoms were used as covariates in all initial models, but cold symptoms were not significant in any model, so they were dropped from all final analyses. Because the raw scores for FeNO and cortisol were skewed (skewness>2.0), log transformation was used to reduce skewness (post-transformed scores skewness<1.0). All references to FeNO and cortisol in the Results section refer to these log-transformed values. E3

After calculating the growth curves for each outcome measure over the three time points, we investigated whether the changes over time were moderated by baseline levels of stress (PSS) and/or depressive symptoms (HADS-D). To make sure that results for each were not due to their relation with the other, both were included as simultaneous moderators of the growth curve of each outcome. To assess moderation, interactions were formed between the moderators and all of the growth curve parameters (intercept, linear slope, quadratic slope). Again, nonsignificant interactions were dropped and final models were then calculated. We examined also whether within-subjects changes in cortisol were related to withinsubjects changes in FeNO and negative affect over time. Following a recommended procedure (E15) we converted the raw scores for each assessment of cortisol, FeNO, and negative affect into deviation scores. These deviation scores represent the difference between the score at each time point and the person s average score over all three time points, as shown in the following formula: Ydev ij = Y ij - Ymean i where Ydev ij is the deviation score on an outcome variable for individual i at assessment j, Y ij is the raw outcome score for individual i at assessment j, and Ymean i is the average score on outcome Y for individual i over the three assessments. These deviation scores were then used in the analyses examining the relations between within-subjects change in cortisol and withinsubjects change in FeNO and negative affect. In particular, we added the deviations scores for cortisol to the growth curve model for FeNO and for negative affect, to determine the relation between deviations in cortisol and deviations in outcome, over and above any relation that might be due to their coincident change over time. E4

Expanded Results for Selected Sections Asthma-Specific Effects Individuals with asthma had higher initial levels of FeNO and tended to have greater reductions in FeNO (b=.12, t(110)=1.90, p=.061) over time than healthy controls. The average reduction of FeNO was -11. 5 ppb in asthma, compared to -1.2 ppb in controls. Because of the steeper FeNO decline in asthma, log-transformed values were not significantly different (b=-.22, t(105)=-1.78, p=.077) from healthy controls in late stress. Cortisol levels increased less from the low stress time point to late stress in asthma than in controls (b=.05, t(86)=2.26, p=.027). Negative affect increased more at the beginning of the high-stress period for controls than for asthmatics, leading to higher negative affect among controls at the early stress time point (b=3.32, t(103)=2.24,p=.017). But for both controls and asthmatics, negative affect was higher during both stress periods than during the low stress time (for controls: b=.7.57, t(106)=10.26, p<.001 for early stress vs. low stress and b=2.95, t(104)=4.04, p<.001 for late stress vs. low stress; for asthma: b=4.72, t(103)=4.71, p<.001 for early stress vs. low stress, and b=3.81, t(101)=3.82, p<.001 for late stress vs. low stress). For controls, momentary stress was higher at both early and late stress time points than at the low stress time point (b=1.08, t(100)=3.46, p=.001, and b=1.06, t(99)=3.15, p=.002), but did not differ between early stress and late stress. For asthmatics, momentary stress was not higher during the stress periods than during the low stress time point (b=.66, t(96)=1.54, p=.127, and b=.06, t(95)=0.13,p=.897 for the early and late stress time points). Neither FEV 1 nor PEF changes differed between groups, but individuals with asthma were marginally lower than controls on overall FEV 1 (b=3.87, t(107)=1.68, p=.097). E5

Dependency of Findings on Initial Depression and Perceived Stress Higher depressive symptoms at baseline led to a steeper decline in FeNO over time, regardless of group (b=.06, t(84)=-2.09,p=.039 for the depressive symptoms x Time interaction). Baseline PSS had no effect on controls (p=.872), but for asthmatics, higher PSS was associated with lower FeNO during non-stress times (b=-.25, t(92)=-2.49p=.015), while not being related to FeNO during the stressful exam period (ps>.374). Neither PSS nor depressive symptoms were significantly related to cortisol. Higher baseline PSS was associated with marginally faster rates of decline in FEV 1 over time, across groups (b=1.28,t(88)=1.94,p=.055), and higher baseline depressive symptoms were related to faster rates of decline in PEF over time, across groups (b=-1.99, t(64)=-2.27, p=.026).higher baseline PSS was associated with lower PEF across time and groups, b=4.85, t(83)=-2.26, p=.026, as well as higher average momentary stress levels across time for asthmatics (b=.78, t(86)=4.22,p<.001) but not for controls (b=.05, t(87)=0.35, p=.728). Finally, higher baseline depressive symptoms were related to higher reported negative affect across time and groups (b=1.98, t(88)=4.23,p<.001) and higher momentary stress level s across time and groups (b=.36, t(84)=3.15, p=.002). Effects of Seasons and Inhaled Corticosteroid Use in Asthma To examine the impact of seasons (spring versus fall), allergies, inhaled corticosteroids, or maintenance medication (including leukotriene modifiers) on FeNO, cortisol, negative affect, and spirometric lung function, we added these variables to the growth curve models. None of these variables affected any of the temporal trajectories. Inhaled corticosteroid use and allergies were related to overall higher FEV 1 (b=8.98, t(26)=2.67, p=.013, and b=5.51, E6

t(107)=2.49, p=.014, respectively) and marginally to lower cortisol (b=-.12, t(21)=-1.98,p=.061, and b=.06, t(102)=1.85, p=.067, respectively). Finally, allergies were also related to higher negative mood (b=3.07, t(106)=3.57, p=.001). None of the participants reported changes in medication between time points. E7

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