Airway diffusing capacity of nitric oxide and steroid therapy in asthma

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contributions and thus may not be the optimal parameter to describe pulmonary NO exchange.
The potential for greater clinical insight is accompanied by the need for new and robust analytic approaches to characterize NO in the exhaled breath.Because NO is produced throughout the respiratory tract, factors like expiratory flow rate substantially influence the NO concentration in the exhaled breath (C exh ) (21,47,54).To account for this and other determinants of NO concentration, we and others have described NO exchange using a biologically relevant two-compartment model (airway and alveolar compartments) and a series of flowindependent NO exchange parameters (20,42,48,52).The flow-independent parameters potentially provide clinically relevant information about NO exchange.For example, the alveolar NO concentration is elevated in allergic alveolitis (alveolar inflammation), whereas airway wall NO flux is elevated in asthma (bronchial inflammation) (37).
Inflammation is characteristic of asthma and induces the expression of several steroid-sensitive enzymes, such as NO synthase (NOS) and glutaminase, which impact NO metabolism (3,23,43).Consequently, corticosteroids, which attenuate the inflammatory process, also reduce the concentration of NO in the exhaled breath (31,41).This feature of corticosteroid therapy may be useful in monitoring the inflammatory status of the airways, but, by reducing the concentration of NO in the exhaled breath to near normal, may mask steroid-independent alterations in airway NO physiology that are of potential clinical significance.
The airway diffusing capacity of NO (D aw,NO ) is the conductance for the transfer of NO between the airway wall and the gas stream (48,52,53).It depends on both the physical features of the airway wall (e.g., airway surface area or tissue thickness) and the rate of chemical consumption (4,53), both of which may be altered in asthma.Recently, Silkoff et al. (48) demonstrated that D aw,NO was elevated in asthma independent of steroid therapy by measuring multiple C NO,plat at small flow rates (Ͻ50 ml/s).However, values for the flow-independent NO exchange parameters may depend on the breathing maneuver and analytic technique utilized.Thus the goal of the present study was to apply our alternate breathing and analytic technique (20-s preexpiratory breath hold followed by a decreasing flow-rate maneuver) in asthma to confirm the results of Silkoff et.al. and potentially provide additional insight into the pathophysiology that marks chronic asthma.

Subjects.
Twenty-four healthy adults and 20 subjects with a clinical history of asthma (8 steroid naive and 12 steroid treated) participated in Address for reprint requests and other correspondence: S. C. George, Dept. of Chemical Engineering and Materials Science, 916 Engineering Tower, Univ. of California, Irvine, CA 92697-2575 (E-mail: scgeorge @uci.edu).
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this study.Inclusion criteria for the healthy subjects was a forced expiratory volume in 1 s (FEV 1)-to-forced vital capacity (FVC) ratio (FEV1/FVC) Ͼ0.80; exclusion criteria were a history of smoking at any time, heart disease, or lung disease.Inclusion criteria for the asthma group were a clinical history of reversible bronchoconstriction and a current FEV 1/FVC Ͻ0.75, regardless of the use of corticosteroids; exclusion criteria were a history of smoking at any time, heart disease, and lung disease other than asthma.We then subdivided the adults with a clinical history of asthma into two groups: 1) steroid naive and 2) steroid treated.In addition, each of the adult subjects with asthma also completed a previously validated asthma control questionnaire (see AP- PENDIX A) to assess clinical symptoms of asthma over the past 7 days (27,28).Subject characteristics are presented in Tables 1 and 2, including details of their clinical history.The Institutional Review Board at the University of California, Irvine approved the protocol, and written, informed consent was obtained from all subjects.
Experimental protocol.Each subject performed two types of exhalation maneuvers: one necessary to estimate the flow-independent NO exchange parameters, and the other according to the ATS guidelines (2).The first maneuver was five repetitions of a 20-s preexpiratory breath hold followed by a decreasing flow rate (from ϳ6 to ϳ1% of vital capacity per second) maneuver (53) to estimate several flowindependent NO exchange parameters.A positive pressure of Ͼ5 cmH 2O was maintained to prevent nasal contamination during the breath hold (2), and a Starling resistor (Hans Rudolph, Kansas City, MO) with a variable resistance was used to progressively decrease the flow rate during the exhalation.After breath hold, the exhalation valve was opened, allowing the patient to expire.A schematic of the experimental apparatus has been previously presented (53).The second maneuver was a vital capacity maneuver performed in triplicate to collect plateau NO concentration based on the ATS guidelines (2).We also included an exhalation flow rate of 250 ml/s (ATS guideline is 50 ml/s) consistent with the guidelines of the ERS (29).After measuring the indexes of NO exchange dynamics, general spirometry, such as FVC, and FEV 1 normalized by FVC (FEV1/FVC) were measured in all subjects (Vmax229; Sensormedics, Yorba Linda, CA) by using the best performance (see Table 1) from three consecutive maneuvers.
Airstream analysis.A chemiluminesence NO analyzer (NOA280, Sievers, Boulder, CO) was used to measure the C exh.The instrument was calibrated on a daily basis by using a certified NO gas (45 parts/million in N2; Sievers, Boulder, CO).The zero-point calibration was performed with a NO filter (Sievers) immediately before the collection of a profile.The flow rate and pressure signals were measured by using a pneumotachometer (RSS100, Hans Rudolph, Kansas City, MO).The pneumotachometer was calibrated daily and was set to provide the flow in units of STPD.
Data analysis and parameter estimation.Experimental singleexhalation profiles with the 20-s preexpiratory breath hold were characterized by the peak concentration in phases I and II (C NO,peak); the peak width (W50) in phases I and II, defined as the exhaled volume in which the NO concentration was Ͼ50% of CNO,peak; and the total volume of phases I and II (VI,II), defined as the point of zero slope (dCexh/dV ϭ 0, where V is volume) in the exhalation profile (53) (Fig. 1).The constant-flow-rate single exhalations were characterized by the C NO,plat, as previously described by the ATS and the ERS (2,29).
A previously described two-compartment model was used to estimate four flow-independent NO exchange parameters: 1) maximum flux of NO from the airways (JЈ aw,NO; pl/s); 2) Daw,NO [pl⅐s Ϫ1 ⅐parts per billion (ppb) Ϫ1 ]; 3) steady-state alveolar concentration (Calv,ss; ppb); and 4) mean airway tissue NO concentration (Caw,NO; ppb; equal to the ratio of JЈ aw,NO to Daw,NO).A simple schematic of the twocompartment model and flow-independent parameters is presented in Fig. 2, and a detailed description of the mathematical estimation of the parameters has been previously described (53).
The source of NO from the airways can be described by the instantaneous flux of NO from the airways (J aw,NO; pl/s).Jaw,NO depends on the flow-independent parameters and is expressed as a linear function of the airway gas-phase concentration (C air) by the following J aw,NO ϭ JЈ aw,NO Ϫ D aw,NO C air (1) or JЈ aw,NO is equal to the product Daw,NO ‫ء‬ Caw,NO (Eq.2).Conceptually, JЈ aw,NO approaches Jaw,NO as the product Daw,NO ‫ء‬ Cair approaches zero.Daw,NO is the conductance for mass transfer (transfer factor or airway diffusing capacity) of NO between the airway tissue and the gas phase.The alveolar region is characterized by Calv,ss, which is equivalent to the alveolar tissue concentration (25,52).tage that intersubject and interpopulation variations in flow rate can be accounted for by calculating C NO,plat at a precise desired flow rate (e.g., 50 ml/s).Statistics.To detect differences among the three groups of subjects, data were analyzed by using ANOVA and post hoc paired comparisons of treatment means.In those instances in which Levene's test rejected homogeneity of variance, tests for group differences relied on Welch's ANOVA or Satterthwaite's method to adjust the test to account for this problem.To detect significant relationships between the parameters that characterize NO exchange and either asthma symptoms or standard indexes of lung function (e.g., FEV 1), we utilized first-and second-order partial correlation coefficients, respectively.For example, to determine the relationship between NO parameters and lung function for all subjects, the second-order partial correlation coefficient factors out the effect of having asthma or being treated with steroids by subtracting the group mean from each individual score.As to the question of normality, in addition to screening variables for excessive skewness, all tests of group differences were rerun by using a log transformation of the dependent variables.Because the log transformation of each variable did not impact the results, all statistical tests were reported by using the untransformed data.Finally, a P value Ͻ0.05 was considered statistically significant, and all results were produced by using the GLM procedure of SAS.

RESULTS
FVC, FEV 1 , FEV 1 /FVC, and the clinical history of the subjects with asthma are presented in Tables 1 and 2, respectively.FEV 1 /FVC was more reproducible than FEV 1 alone.The mean maximum variability (defined as the difference between the maximum and minimum value normalized by the mean of the three repeated maneuvers) for FEV 1 /FVC was 5.8% (range 1.5-10.2%)and 2.9% (range 0-10.2%) for steroid-naive and steroid-treated asthma subjects, respectively.For FEV 1 alone, the mean maximum variability was slightly higher for each group: 8.9% (range 0.7-20.6%)and 5.2% (range 1.5-17.9%)for steroid-naive and steroid-treated asthma subjects, respectively.FEV 1 /FVC was significantly lower in both groups of subjects with asthma compared with healthy adults.However, there was no difference in FEV 1 /FVC or clinical symptoms (as assessed by the composite score on the Fig. 2. Schematic of 2-compartment model used to describe NO exchange dynamics.Cexh is the sum of two contributions, the alveolar region and the airway region, which depends on 3 flow-independent parameters: maximum total volumetric flux of NO from the airway wall (JЈ aw,NO; pl/s), diffusing capacity of NO in the airways (Daw,NO; pl⅐s Ϫ1 ⅐ppb Ϫ1 ), and steady-state alveolar concentration (Calv,ss; ppb).Jaw,NO is the total flux (pl/s) of NO between the tissue and gas phase in the airway and is an inverse function of the exhalation flow rate (V ˙E) and is the sum of two terms: JЈ aw,NO Ϫ Daw,NO ‫ء‬ Cair (airway gas phase concentration).If Daw,NO increases while JЈ aw,NO is held constant (note that this necessitates a decrease in the wall concentration, Caw,NO, as JЈ aw,NO is the product of Daw,NO ‫ء‬ Caw,NO), then Jaw,NO decreases (see text for details).If exhalation flow rate is held constant (i.e., 50 ml/s as suggested by the American Thoracic Society), then Cexh approaches a constant value in phase III of the exhalation profile and is equivalent to CNO,plat (NO plateau concentration in phase III).t, Time.In each panel, the control profile (solid line) is shown together with the exhaled profile when one of the flow-independent parameters is doubled (dashed line).A: the decreasing V ˙E is also shown on the y-axis.This informal sensitivity analysis demonstrates graphically which part of the profile is impacted by each parameter.It can be seen that each parameter uniquely impacts the exhaled profile and can thus be uniquely determined.Note that Daw,NO primarily impacts phases I and II, Calv,ss impacts primarily phase III, whereas JЈ aw,NO impacts all 3 phases.In addition, note that an increase in Daw,NO (while holding JЈ aw,NO and Calv,ss) decreases the NO concentration in phases I and II if JЈ aw,NO (Eq.2) and Calv,ss are held constant, but would increase the concentration in phases I and II if Caw,NO (Eq.2) and Calv,ss were held constant (Eq.2).In the former case, Caw,NO must be decreased to hold JЈ aw,NO constant (product of Daw,NO ‫ء‬ Caw,NO), whereas, in the later case, JЈ aw,NO would increase as Caw,NO is constant.asthma control questionnaire) between the two groups of subjects with asthma.
Of the 20 subjects with asthma, three of the steroid treated (subjects 2, 10, and 12) were not able to complete the 20-s breath hold, and thus we utilized a 10-s breath hold, which may increase the confidence interval of D aw,NO (44,53).To highlight differences among groups in exhaled concentrations, a composite exhalation profile for each group was attained (Fig. 4) by taking the mean exhaled concentration at equivalent exhaled volume intervals for each of the three groups.The three asthmatic subjects who were not able to complete the 20-s breath hold were excluded from the composite exhalation profile.Steroid-naive subjects with asthma had an increased concentration of NO in all phases of the exhalation profile compared with both steroid-treated subjects with asthma and healthy controls.Although the NO exhalation profile for steroid-treated subjects with asthma and healthy controls is similar (Fig. 4B), there are important differences that reflect alterations in the flow-independent NO parameters.Steroidtreated subjects with asthma have elevated NO in phase III that is reflected in a steeper phase III slope.This steeper slope reflects a greater airway wall flux (JЈ aw,NO ) as opposed to an elevated C alv,ss , which would cause a uniform increase in NO concentration over phase III (52,53).The elevated JЈ aw,NO would result in a much larger C NO,peak than actually observed, and this results in an elevated D aw,NO as described below.
Mean (ϮSD) C NO,peak for steroid-naive, steroid-treated, and healthy subjects was 192 Ϯ 127, 82 Ϯ 42, and 67 Ϯ 29 ppb, respectively.C NO,peak for steroid-naive subjects with asthma was statistically larger than that for the other two groups.W 50 for steroid-naive, steroid-treated, and healthy subjects was 189 Ϯ 60, 171 Ϯ 49, and 190 Ϯ 51 ml, respectively, and was not different among groups.V I,II for steroid-naive, steroidtreated, and healthy subjects was 657 Ϯ 98, 604 Ϯ 127, and 668 Ϯ 142 ml, respectively, and was also not different among the groups.
As shown in Fig. 5, JЈ aw,NO and D aw,NO are elevated in steroid-naive subjects with asthma relative to healthy controls.The use of corticosteroids does not impact D aw,NO , but is associated with a significantly lower JЈ aw,NO and C aw,NO that are equivalent to those in healthy adults.C alv,ss is not different among the three groups.
The experimental values of C NO,plat at the target flow rates of 50 and 250 ml/s, respectively, are presented in Table 3 (for healthy adults and subjects with asthma) along with the modelpredicted C NO,plat (Eq.3, C NO,plat * ) at exhalation flow rates of exactly 50 and 250 ml/s.C NO,plat * and C NO,plat were not statistically different from each other, with the exception of the steroid-naive group of asthmatic subjects at 250 ml/s (see Table 3).Statistical differences between the groups did not depend on the choice of C NO,plat or C NO,plat * .Thus, to control for small variations in the exhalation flow rate between groups (e.g., mean exhalation flow rate at the target of 50 ml/s was 62 ml/s and 55 ml/s for steroid-naive and steroid-treated groups, respectively), statistical differences between groups are presented by using C NO,plat * (Fig. 6).C NO,plat * was 13.0 Ϯ 5.97 and 5.17 Ϯ 2.97 ppb for healthy adults, 53.9 Ϯ 33.0 and 16.1 Ϯ 9.46 ppb for steroid-naive adults with asthma, and 23.2 Ϯ 14.3 and 7.76 Ϯ 5.34 ppb for steroid-treated adults with asthma at flow rates of 50 and 250 ml/s, respectively.C NO,plat * at 50 ml/s is significantly higher for both groups of subjects with asthma compared with healthy controls (Fig. 6), whereas only the steroid-naive subjects with asthma have a higher C NO,plat * at 250 ml/s.D aw,NO was inversely correlated with both FEV 1 (%predicted) and FVC (%predicted) (Fig. 7, A and B).In contrast, C aw,NO was positively correlated with FVC (%predicted).JЈ aw,NO and C alv,ss were not correlated with any lung function indexes.C NO,plat * at either V ˙E was not correlated with indexes of lung function, but C NO,plat was inversely correlated with FEV 1 /FVC (%predicted) (Fig. 8).The asthma control questionnaire composite score was not correlated with any of the NO exchange parameters.

DISCUSSION
In the present study, we estimate flow-independent NO exchange parameters with a single exhalation breathing technique and plateau C exh by following ATS and ERS guidelines in a group of subjects with a low FEV 1 (FEV 1 /FVC Ͻ 0.75) and a clinical history of asthma.We found that the use of corticosteroids was associated with a decrease in C NO,plat at flow rates of 50 and 250 ml/s, as well as a decrease in the flow-independent parameters that reflect airway tissue concentration (JЈ aw,NO and C aw,NO , respectively).In contrast, D aw,NO Fig. 4. A: composite experimental NO exhalation profiles are presented for the 20-s breath hold followed by a decreasing flow rate maneuver for steroid-naive (SN) asthma subjects (thick line) and healthy adults (HA) (thin line).B: composite experimental NO exhalation profiles are presented for the 20-s breath hold followed by a decreasing flow rate maneuver for steroid-treated (ST) asthma subjects (thick line) and HA (thin line).Values are means Ϯ SD.

NO EXCHANGE PARAMETERS IN ASTHMA
J Appl Physiol • VOL 96 • JANUARY 2004 • www.jap.org was elevated in both groups of asthmatic subjects and was independent of the use of corticosteroids.These findings are in good agreement with previously published data by Silkoff et al. (48), despite using a different breathing maneuver and analytic technique to estimate the flow-independent NO exchange parameters.In addition, we found that D aw,NO is inversely correlated with both FEV 1 and FVC (%predicted), independent of the presence of asthma and steroid use.Thus we confirm that D aw,NO may reflect physiological changes in the lungs that impact lung function independent of the use of corticosteroids.
Because the initial reports that FE NO in asthma was elevated (1,30), subsequent studies have focused on exploring the correlation between C exh and other inflammatory markers (i.e., eosinophils), clinical interventions such as corticosteroids, and standard indexes of lung function (i.e., FEV 1 /FVC).Corticosteroid treatment significantly decreases C NO,plat in subjects with asthma (31,40,41), and the dose of steroid is inversely related to C NO,plat (32).In addition, an increase in C NO,plat has recently been shown to be equally effective as sputum eosinophils and airway hyperresponsiveness to hypertonic saline as a predictor for loss of asthma control (26).However, the present study, as well as that of Silkoff et al. (48), demonstrates the presence of steroid-independent factors (i.e., D aw,NO ) that can also contribute to the elevated levels of NO in the exhaled breath of asthmatic subjects.
We are also now aware of disease states in which exhaled concentration of NO is in the normal range only because abnormalities in the flow-independent determinants of NO concentration balance each other.For example, in scleroderma, the alveolar concentration of NO is elevated, whereas the airway wall flux of NO is reduced (15).In cystic fibrosis, the D aw,NO (transfer factor) is elevated, but the airway wall concentration is reduced, leading to an C exh that is similar to that of healthy controls (45).
Silkoff et al. (48) first reported that D aw,NO is fourfold higher in subjects with asthma and that this increase is independent of steroid treatment, whereas JЈ aw,NO decreases.Lehtimaki et al. (38) then demonstrated that steroid treatment reduces JЈ aw,NO in newly diagnosed asthma subjects (previously steroid naive) by utilizing multiple constant-flow rate maneuvers (52,54).Most recently, Hogman et al. (22) also recently demonstrated that D aw,NO is increased 1.5-fold in a group of atopic asthmatic subjects.Although we utilized a different breathing maneuver and technique to estimate the flow-independent NO parameters, our results are consistent with previously reported trends (22,38,48) and also demonstrates that D aw,NO is inversely correlated with FEV 1 and FVC (%predicted) and C aw,NO is positively correlated with FVC.The positive correlation of C aw,NO with FVC is likely due to the fact that it is inversely related to D aw,NO (i.e., C aw,NO ϭ JЈ aw,NO /D aw,NO ).Of note is the fact that Silkoff et al. (48) reported that values of JЈ aw,NO , D aw,NO , and C aw,NO after steroid use in asthmatic subjects were all positively correlated with FEV 1 /FVC (%predicted).These important differences may be due to differences in study design and the technique used to estimate the flow-independent NO parameters.Nonetheless, future studies will need to continue to  investigate the relationship between NO flow-independent parameters and lung function.
C exh necessarily reflects both the chemical and physical properties of the airway wall and alveoli, as well as the endogenous production rate from NOS isoforms in the airway and alveoli.Our ability to estimate the flow-independent NO parameters, which depend on these properties from the exhaled concentration signal, can be illustrated by using the composite exhalation profile (Fig. 4A).Our laboratory (53) has previously demonstrated that only phases I and II are sensitive to changes in D aw,NO (if D aw,NO increases, less NO is exhaled in phases I and II), only phase III is sensitive to C alv,ss (if C alv,ss increases, there is a uniform increase across exhaled volume in phase III), and all three phases are sensitive to JЈ aw,NO (if JЈ aw,NO increases, there is more NO exhaled in all phases, and the impact on phase III is a steeper slope) (see Fig. 3).Thus the observed changes in the composite profile of each group are consistent with our reported values of the flow-independent parameters.For example, steroid-treated subjects with asthma have a steeper slope in phase III and a higher concentration (necessitating a larger JЈ aw,NO ), yet a similar amount of NO in phases I and II (necessitating a larger D aw,NO to balance the increased JЈ aw,NO ).Of note is the fact that, among the parameters characterizing phases I and II of the exhalation profile, only C NO,peak differs among the groups (W 50 and V I,II are not different among the three groups).This is consistent with altered NO production and transport in the airway wall during the breath hold, but also suggests that the volume accumulating NO during the breath hold and subsequently eliminated during exhalation is similar among the three groups.
Our laboratory has previously reported analytic expressions for the flow-independent parameters that approximate the functional dependence on the surface area emitting NO [A i , where i is either airways (aw) or alveoli (alv); cm 2 ], solubility [partition coefficient ( t:air )], molecular diffusion [molecular diffusivity (D t,NO ); cm 2 /s], chemical consumption (lumped first-order rate reaction constant k; s Ϫ1 ), thickness of the tissue layer (L t,i ; cm), and chemical production [airway (S aw,NO ) and alveolar (S alv,NO ) production rate per unit volume; ml NO⅐s Ϫ1 ⅐cm Ϫ3 ] (45, 52).The analytic expressions are summarized in APPENDIX B and provide a level of quantitative insight into the mechanism of the observed changes in the flowindependent parameters.D aw,NO is independent of S aw,NO ; is a positive function of total surface area of airway space (A aw ), t:air , D t,NO , and k; and is an inverse function of the thickness of the airway tissue layer (L t,aw ) (Eq.B2 in APPENDIX B).Thus the increase in D aw,NO may be due to alterations in any of these parameters.The airway wall in asthma is generally considered to be thicker than in healthy controls due to remodeling processes, such as subepithelial fibrosis and increased mucous production (56).The thicker airway wall would tend to increase the diffusion distance for NO, and the mucus tends to be more viscous, which would decrease the "ease" at which NO can diffuse (i.e., decrease D t,NO ) (4).Both of these observations would decrease D aw,NO and contrast with our experimental observation, as well as that of Silkoff et al. (48), of an elevated D aw,NO .Enhanced chemical consumption, primarily with superoxide (9), can increase D aw,NO due to an increase in the radial concentration gradient (4,45).However, D aw,NO remains elevated after steroid treatment, which has been reported to suppress superoxide release (10).
An increase in A aw is a plausible mechanism for the increase in D aw,NO .Silkoff et al. (48) postulated that extension of the NO-producing nonadrenergic noncholinergic nerves from the large airways into the small airways may increase the A i , which is supported both directly and indirectly by several studies (6, 7, 16, 17, 39, 55).Expression of inducible NOS (iNOS) in the airways of subjects with asthma has been demonstrated (12,36,50), which could potentially increase A aw ; however, this possible mechanism would likely be sensitive to corticosteroid therapy, which is not the observation.
JЈ aw,NO has a similar functional dependence on the physical and chemical parameters of the airways (A aw , D t,NO , and L t,aw ) (see APPENDIX B) as D aw,NO .However, in contrast to D aw,NO , JЈ aw,NO is inversely related to k and is a positive function of an additional parameter, S aw,NO .An increase in S aw,NO by an increase in neuronal NOS expression from nonadrenergic noncholinergic nerves (6,7,16,17,39,55) or prokaryotic denitrification (13) may increase the exhaled concentration of NO and thus contribute to the observed increase in JЈ aw,NO for both steroid-naive and steroid-treated subjects with asthma.Other enzymatic and nonenzymatic chemical events in the airways, such as increased iNOS expression in the epithelium (18), nitrite reduction to NO at lower pH (23,24,35), and Snitrosoglutathione catabolism (5,11,14,49), could also increase S aw,NO and contribute to the increase in JЈ aw,NO for steroid-naive subjects with asthma.
Steroid treatment dramatically decreases the C exh (see Fig. 5), which corresponds to observed decreases in JЈ aw,NO and C aw,NO as well as C NO,plat at both 50 and 250 ml/s flow rates.As previously discussed, steroid therapy decreases superoxide production, which would correspond to a reduced consumption rate and an increase in JЈ aw,NO , which is not observed.The decrease in JЈ aw,NO in steroid-treated subjects with asthma may be related to 1) the reduced iNOS activity in the epithelial and inflammatory cells in the airways (12,34,36,50,57); 2) reduced nitrite to NO reduction due to normalized airway pH (23,24,35); 3) decreased prokaryotic colonization (13); and 4) inhibition of arginase upregulation (33).The decrease in C aw,NO (a ratio of JЈ aw,NO over D aw,NO ) for steroid-treated subjects with asthma is due to the decrease in JЈ aw,NO , whereas D aw,NO is not changed.
In summary, we have estimated both flow-independent NO exchange parameters and plateau C exh following ATS guidelines, in subjects with low FEV 1 /FVC and a clinical history of asthma.D aw,NO is elevated independent of corticosteroid use, whereas JЈ aw,NO , C aw,NO , and C NO,plat (at both 50 and 250 ml/s) are all reduced by the use of steroids.In addition, D aw,NO is inversely correlated with pulmonary function, independent of the presence of asthma and steroid use.In agreement with Silkoff et al. (48), we conclude that D aw,NO may reflect changes in the lungs that impact function and that are not impacted by steroid therapy and thus may provide clinical information not available from C exh alone.6) On average, during the past week, how many puffs of shortacting bronchodilator (e.g., Ventolin) have you used each day?
Each question is answered by the subject on a scale of 0-6, representing the absence of symptoms (score of 0) to severe symptoms (score of 6).The composite score is then the mean of the six scores.Thus a higher composite score reflects more asthmatic symptoms.The questionnaire has been shown to have improved discriminative and evaluative measurement properties than an asthma control diary (27).

Fig. 3 Fig. 1 .
Fig. 1.Definition of CNO,peak, W50, and VI,II are presented by a schematic of a representative exhalation nitric oxide (NO) profile using the single-breath technique with a preexpiratory breath hold and a decreasing exhalation flow rate.CNO,peak is the maximum concentration of NO in phases I and II; W50 is the width of the phase I and II peak calculated by taking the volume (V) at which the exhaled concentration (Cexh) is Ͼ50% of CNO,peak; and VI,II is the volume of phases I and II.The distinction between phases I and II and phase III is the point of zero slope in the exhalation profile, as previously described (53).ppb, Parts per billion.

Fig. 3 .
Fig.3.The 2-compartment model prediction of the exhaled NO profile is shown for the single-exhalation maneuver with a 20-s preexpiratory breath hold.Representative values for lung volumes of a healthy adult have been used, and the "control" values for the flow-independent parameters are as follows: Daw,NO ϭ 5 pl⅐s Ϫ1 ⅐ppb Ϫ1 (A); JЈ aw,NO ϭ 750 pl⅐s Ϫ1 (B); Calv,ss ϭ 3 ppb (C).In each panel, the control profile (solid line) is shown together with the exhaled profile when one of the flow-independent parameters is doubled (dashed line).A: the decreasing V ˙E is also shown on the y-axis.This informal sensitivity analysis demonstrates graphically which part of the profile is impacted by each parameter.It can be seen that each parameter uniquely impacts the exhaled profile and can thus be uniquely determined.Note that Daw,NO primarily impacts phases I and II, Calv,ss impacts primarily phase III, whereas JЈ aw,NO impacts all 3 phases.In addition, note that an increase in Daw,NO (while holding JЈ aw,NO and Calv,ss) decreases the NO concentration in phases I and II if JЈ aw,NO (Eq.2) and Calv,ss are held constant, but would increase the concentration in phases I and II if Caw,NO (Eq.2) and Calv,ss were held constant (Eq.2).In the former case, Caw,NO must be decreased to hold JЈ aw,NO constant (product of Daw,NO ‫ء‬ Caw,NO), whereas, in the later case, JЈ aw,NO would increase as Caw,NO is constant.

Fig. 7 .
Fig. 7. Second-order partial correlation analysis demonstrates a significant inverse relationship between Daw,NO and forced expiratory volume in 1 s (FEV1; %predicted) (A), Daw,NO and forced vital capacity (FVC; %predicted) (B), and a positive relationship between Caw,NO and FVC (%predicted) (C) in a total of 44 subjects.⌬, Difference between the individual score of each subject and the group mean value to which each subject belongs.ϩ, HA (n ϭ 24); E, ST (n ϭ 12); F, SN (n ϭ 8).

Fig. 6 .
Fig. 6.Individual and population mean (solid bar) values of the plateau exhaled concentration for nitric oxide as predicted by the model (C NO,plat * ; Eq. 3) using the flow-independent parameters for each subject.A: exhalation flow rate of exactly 50 ml/s; B: exhalation flow rate of exactly 250 ml/s.F, SN; E, ST; {, HA.Statistically different from *HA and # SN subjects with asthma: P Ͻ 0.05.

Table 1 .
Physical characteristics of subjects

Table 3 .
Model-predicted and experimental C NO,plat of subjects