Exhaled Nitric Oxide and Breath Condensate pH in Asthmatic Reactions Induced by Isocyanates

Original Research ASTHMA

Exhaled Nitric Oxide and Breath Condensate pH in Asthmatic Reactions Induced by Isocyanates Silvia Ferrazzoni, PhD; Maria Cristina Scarpa, BS; Gabriella Guarnieri, MD; Massimo Corradi, MD; Antonio Mutti, MD; and Piero Maestrelli, MD

Background: We investigated the usefulness of measurements of fractional exhaled nitric oxide (FeNO) and pH of exhaled breath condensate (EBC) for monitoring airway response after specific inhalation challenges with isocyanates in sensitized subjects. Methods: Lung function (FEV1), FeNO, and pH in argon-deaerated EBC were measured before and at intervals up to 30 days after a specific inhalation challenge in 15 subjects with isocyanate asthma, in 24 not sensitized control subjects exposed to isocyanates, and in 3 nonasthmatic subjects with rhinitis induced by isocyanate. Induced sputum was collected before and 24 h after isocyanate exposure. Results: Isocyanate-induced asthmatic reactions were associated with a rise in sputum eosinophil levels at 24 h (p < 0.01), and an increase in FeNO at 24 h (p < 0.05) and 48 h (p < 0.005), whereas FeNO level did not vary with isocyanate exposure in subjects with rhinitis and in control subjects. FeNO changes at 24 h positively correlated with corresponding sputum eosinophil changes (␳ ⴝ 0.66, p < 0.001). A rise in pH was observed in the afternoon samples of EBC, irrespective of the occurrence of isocyanate-induced asthmatic reactions. Conclusions: We demonstrated that isocyanate-induced asthmatic reactions are associated with a consistent delayed increase in FeNO but not with the acidification of EBC. (CHEST 2009; 136:155–162) Abbreviations: EBC ⫽ exhaled breath condensate; FeNO ⫽ fractional exhaled nitric oxide; IQR ⫽ interquartile range; NO ⫽ nitric oxide; ppb ⫽ parts per billion; SIC ⫽ specific inhalation challenge

the mechanisms involved in isocyanateA lthough induced asthma remain unclear, this common type of occupational asthma shares many functional

From the Department of Environmental Medicine and Public Health (Drs. Ferrazzoni, Guarnieri, Maestrelli, and Ms. Scarpa), University of Padova, Padova, Italy; and the Department of Clinical Medicine, Nephrology, and Health Sciences (Drs. Corradi and Mutti), University of Parma, Parma, Italy. This research was supported by the Italian Ministry of University and Research (PRIN 2005); by the University of Padova; and by Associazione Ricerca Cura Asma, Padova, Italy. The authors have reported to the ACCP that no significant conflicts of interest exist with any companies/organizations whose products or services may be discussed in this article. Manuscript received October 1, 2008; accepted January 16, 2009. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (www.chestjournal. org/site/misc/reprints.xhtml). Correspondence to: Piero Maestrelli, MD, Dipartimento di Medicina Ambientale e Sanita` Pubblica, Universita` degli Studi di Padova, via Giustiniani 2, 35128 Padova, Italy; e-mail: [email protected] DOI: 10.1378/chest.08-2338 www.chestjournal.org

and morphologic features with allergic asthma.1 Because specific IgE antibodies are present in a minority of patients with asthma induced by isocyanates, the “gold standard” for diagnoses of isocyanateinduced asthma is the specific inhalation challenge (SIC) in the laboratory. Studies2,3 performed during SIC have allowed a better characterization of isocyanate-induced asthmatic reaction. Using induced sputum, the most consistent finding4,5 is an association between asthmatic reaction induced by occupational agents, including isocyanates, and influx of eosinophils into the airways between 6 and 24 h after SIC. This finding has been reproduced6 in natural settings, demonstrating that subjects with occupational asthma exhibit sputum eosinophilia after exposure to the offending agent at work. These observations provided the rationale7 for using eosinophils in induced sputum as an additional tool to improve the specificity of peak expiratory flow monCHEST / 136 / 1 / JULY, 2009

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itoring in diagnosing occupational asthma. Induced sputum is minimally invasive, and some study subjects are unable to produce adequate samples. In addition, induction of sputum is time consuming, and processing the samples is laborious and needs to be performed soon after collection.8 Therefore, the search for additional noninvasive markers of airway inflammation is relevant. The use of fractional exhaled nitric oxide (FeNO) for assessing airway inflammation in asthma is totally noninvasive, quick, and relatively simple to perform.9 Evaluation of FeNO was equivalent to sputum eosinophils in diagnosing asthma, and management of asthma according to FeNO levels was effective in reducing the number of maintenance doses of inhaled corticosteroids.10,11 Some occupational studies12–19 have investigated the role of FeNO in assessing occupational asthma but with inconsistent results. Measurement of pH in exhaled breath condensate (EBC) is considered a robust variable in determining the degree of acidification of EBC in patients with various inflammatory lung diseases, including asthma. Acidification of the airways was demonstrated in acute exacerbation of asthma20 as well as after milder worsening of asthma due to short-term exposure to traffic pollution.21 Because asthmatic reactions induced by laboratory exposure to occupational agents in sensitized subjects resemble a short-term worsening of the disease, an acidification of the airways may occur in this circumstance. To our knowledge, no studies using EBC pH have been performed in occupational asthma. We hypothesized that asthmatic reactions induced by isocyanates are associated with an increase of FeNO and with an acidification of EBC. The aim of this study was to determine the time course of changes in FeNO and EBC pH after SIC with isocyanates in sensitized subjects. Materials and Methods Subjects Forty-two consecutive subjects referred to our center for suspected occupational asthma, who performed SIC with isocyanates, were included in the study. The subjects were allocated to the isocyanate asthma group on the basis of positive responses to SIC (ie, an early asthmatic reaction, a late asthmatic reaction, or both). Fifteen subjects with occupational asthma due to isocyanates (toluene diisocyanate, methylene-diisocianate, or 1,6hexamethylene diisocyanate) and 24 subjects with workplace exposure to isocyanates but with negative response to SIC were examined. In addition, three subjects who exhibited rhinitis induced by isocyanates but not bronchoconstriction were studied as a control for sensitization to isocyanate without asthma. All subjects had been free from respiratory tract infections within the preceding 2 months. No subjects received inhaled or oral glucocorticosteroids within 4 weeks or long-acting bronchodilators

within 48 h prior to the study. Each subject provided written informed consent, and the local ethics committee approved the study. Study Design Subjects were examined on 5 consecutive days, and then 7 and 30 days after SIC with isocyanate. On day 1, clinical and occupational histories were collected, skin-prick tests with common allergens and pulmonary function tests were performed, and baseline sputum was induced. On day 2, a single-blind sham inhalation challenge with air was performed. On day 3, the subjects underwent SIC with the appropriate isocyanate (ie, toluene diisocyanate, methylene-diisocianate, or 1,6-hexamethylene diisocyanate). The type of isocyanate to test was chosen according to each subject’s occupational exposure and the history. FEV1 and FeNO were monitored for 7 h after sham and isocyanate exposures. EBC was collected before and 7 h after sham and isocyanate exposures. On days 4 and 5 (ie, 24 and 48 h after exposure to isocyanate), FEV1 and FeNO were measured and EBC collected. After exposure, sputum was induced 24 h and 7 days after SIC. FEV1 and FeNO were assessed again 7 and 30 days after SIC. The subjects did not return to work until the end of the study. Inhalation Challenges FVC and FEV1 were measured by a dry spirometer (PFT Horizon, model 922; SensorMedics; Milan, Italy).4 Airway responsiveness to methacholine aerosol was assessed as previously described,2 and results were expressed as the cumulative provocative dose causing a 20% fall in FEV1 (in milligrams of methacholine). SICs with isocyanates were performed as described previously.4 Briefly, subjects were exposed to the isocyanate vapor (mean [⫾ SE], concentration, 10 ⫾ 3 parts per billion [ppb]) for a maximum of 30 min in an exposure chamber. The concentration of isocyanate was continuously monitored (Single Point Monitor; Zellweger Analytics Inc; Lincolnshire, IL). Because the threshold limit value suggested by the American Conference of Governmental Industrial Hygienists22 for short-term workplace exposure is 20 ppb, we can reasonably exclude that a mean laboratory exposure to 10 ⫾ 3 ppb isocyanate during the inhalation challenge induces unspecific irritant effects. An asthmatic reaction was considered to occur when FEV1 decreased by at least 20% from baseline within 1 h (early) or ⱖ 2 h (late) after exposure to isocyanates. The diagnosis of rhinitis induced by isocyanates was made when sneezing, nasal itching, rhinorrhea, and blocked nose occurred within 1 h from active exposure and not after sham exposure. FeNO Measurement FeNO was measured with a chemiluminescence analyzer (NIOX Nitric Oxide Monitoring System, version 2.0; Aerocrine AB; Solna, Sweden) prior to spirometric measurements according to recommendations from the American Thoracic Society.9 Individual FeNO was calculated as the mean of three correct exhalations.23 Measurement of pH in EBC EBC was collected with the apparatus described by Goldoni et al24 during tidal breathing for 15 min into the condenser kept at a temperature of ⫺55°C. The subjects had not eaten or drunk at least 1 h before the collection. Argon gas was bubbled in 200-␮L

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EBC aliquots for 3 min to remove air. Then, pH was measured using a pH meter (pH300; Hanna Instruments; Padova, Italy) with an accuracy of ⫾ 0.01, as previously described.25 In preliminary experiments, we found that pH increases bubbling of argon gas up to 2 min; deaeration for 4, 8, and 10 min did not induce further changes in pH. Therefore, we established that the bubbling of argon gas for 3 min is sufficient for the optimal deaeration of 200-␮L EBC samples. Amylase was measured using an enzymatic colorimetric test (lower detection limit, 3U/L) [IFCC; Roche Diagnostic Modular Analytics; Milan, Italy] to assess salivary contamination. Samples containing amylase were discarded. Sputum Induction and Analysis The induction of sputum was performed as previously described.4 Briefly, FEV1 was measured before and 15 min after salbutamol inhalation (400 ␮g). Next, the subject inhaled a hypertonic saline solution (3 to 4%) using an ultrasonic nebulizer (DeVilbiss Ultra Neb; DeVilbiss; Somerset, PA) for 5-min periods up to 20 min. The sputum sample plugs were selected from saliva. Each sample was weighted and incubated with a 1% dithiothreitol solution for 10 min. The cell suspension was cytocentrifuged (Cytospin 3; ThermoShandon, Ltd; Cheshire, UK), and two slides were fixed and stained by a rapid laboratory stain (Diff-Quick; Medion Diagnostics GmbH; Dudingen, Switzerland) for differential cell counts. Statistical Analysis Differences among the study groups were analyzed with the Mann-Whitney test or ␹2 test for continuous and categorical variables, respectively. Correlation coefficients were calculated using the Spearman rank correlation. Changes of different parameters after bronchial challenges were analyzed with the Wilcoxon signed rank test for sputum cells and EBC pH, and the Student t test for paired data for FEV1 and FeNO, separately in SIC-positive and SIC-negative groups. The Bonferroni adjustment for multiple comparisons was applied. The analysis was performed using a statistical software package (Statview, version 5.0.1; SAS Institute Inc; Cary, NC). A p value of ⬍ 0.05 was considered significant.

Results Table 1 reports the characteristics of the subjects. Airway hyperresponsiveness was present in 13 subjects who were SIC positive and in 7 subjects who were SIC negative. Subjects who were SIC negative had longer intervals from last exposure to isocyanates at work than those who were SIC positive, but the difference was not statistically significant. The time course of changes in FEV1 after exposure to isocyanates in the SIC-positive and SICnegative groups is shown in Figure 1. Baseline FeNO was higher in the SIC-positive group than in the SIC-negative group (geometric mean, 62 ppb [SE, 1.3 ppb] vs 23 ppb [SE, 1.2 ppb], respectively; p ⬍ 0.01) and in the rhinitic group (geometric mean 28 ppb [SE, 1.6 ppb]). No significant changes in FeNO were observed after sham exposure in any groups. The time course of FeNO after isocyanates www.chestjournal.org

Table 1—Characteristics of the Study Subjects Characteristics Gender Male Female Age, yr Smoking status, No. Nonsmokers or ex-smokers Current smokers Atopy Duration of exposure, yr Duration of symptoms, yr Interval from last exposure to isocyanates,* d FEV1, % predicted Methacholine PD20,† mg Isocyanate tested, No. Toluene diisocyanate Methylenediisocianate 1,6-hexamethylene diisocyanate Type of asthmatic reaction, No. Early Late Dual

SIC-Negative Group

SIC-Positive Group

Rhinitis Group

15 9 39 ⫾ 2

8 7 42 ⫾ 2

2 1 37 ⫾ 7

20

12

2

4 16 7.9 ⫾ 1.4

3 3‡ 6.8 ⫾ 1.5

1 3 13.3 ⫾ 10.9

3.6 ⫾ 0.7

3.1 ⫾ 0.7

1.8 ⫾ 0.7

37 (242)

66 (106)

2 (15)

104 ⫾ 3 1.00 (1.15)

91 ⫾ 4 0.28 (1.41)‡

103 ⫾ 6 1.10 (1.65)

8

8

0

7

6

1

9

1

2

4 5 6

Values are given as No., or mean ⫾ SE, unless otherwise indicated. PD20 ⫽ provocative dose causing a 20% fall in FEV1. *Data are given as the median (IQR). †Data are given as the geometric mean (SE). ‡p ⬍ 0.005 vs SIC-negative group.

in the SIC-positive and SIC-negative groups is shown in Figure 2. In the SIC-positive group, FeNO decreased significantly from 30 min (geometric mean, 45 ppb [SE, 1.2 ppb]) to 2 h (geometric mean, 54 [SE, 1.2 ppb]) after isocyanate exposure. FeNO then increased progressively and reached its maximum levels between 24 h (geometric mean, 115 ppb [SE, 1.2 ppb]) and 48 h (geometric mean, 118 ppb [SE, 1.3 ppb]) after exposure. On day 7 after isocyanate challenge, FeNO was still higher than baseline, although the difference was not significant. In the SIC-negative and rhinitic groups, no significant changes in FeNO were observed (Fig 2). In the four subjects who exhibited a single early asthmatic reaction, FeNO changes showed a similar time course, with a maximum increase at 48 h in all subjects (baseline: geometric mean, 60 ppb [SE, 1.5 ppb]; 24 h: geometric mean, 70 ppb [SE, 1.5 ppb; 17% CHEST / 136 / 1 / JULY, 2009

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exposure

100

#

*

m in

80

h

ΔFEV1 (%)

120

60

#

*

*

#

#

# SIC-positive SIC-negative

40 20

30

da

ys

ys da 7

h

h 48

24

h

h 7

h

h

6

5

h

h

4

3

2

1

30

0

Time #p <0.005 C

*pC<0.05 vs baseline

Figure 1. Time course of FEV1 after SIC with isocyanates in sensitized (F, SIC positive; n ⫽ 15) and nonsensitized (f, SIC negative; n ⫽ 24) subjects. Data are presented as the mean ⫾ SE. pc ⫽ values corrected for multiple comparisons.

SIC-positive SIC-negative SIC-rhinitis

160 140 120 exposure 100 80 60 40 * * 20 0

*

#

ys da 30

ys da 7

h 48

h 24

h

h 7

6

h 5

h 4

h 3

h

*

2

1

30

m in h

FeNO (ppb)

increase]; 48 h: geometric mean, 81 ppb [SE, 1.6 ppb; 35% increase]). However, the magnitude of late FeNO increase was less than that observed in subjects with late or dual reactions (baseline: geometric mean, 70 ppb [SE, 1.3 ppb]; 24 h: geometric mean, 138 ppb [SE, 1.2 ppb; 97% increase]; 48 h: geometric mean, 143 ppb [SE, 1.3 ppb; 104% increase]). The measurement of EBC pH was carried out in 19 subjects who were SIC negative and 10 subjects who were SIC positive. EBC pH increased significantly for both groups 7 h after sham exposure compared to baseline (Fig 3, a and b). The same pattern was observed on isocyanate exposure day, but the differences were not statistically significant (Fig 3, c and d). The median EBC pH of the samples collected in the morning at time 0 and 24 h after exposure did not differ (Fig 3). No significant changes in EBC pH were detected at subsequent time points after isocyanate exposure in any group of subjects (Table 2).

Time *pC<0.05

#p <0.005 C

vs baseline

Figure 2. Time course of FeNO after SIC with isocyanates in sensitized subjects (F, SIC positive; n ⫽ 15), nonsensitized subjects (f, SIC negative; n ⫽ 24), and sensitized subjects with rhinitic responses only (Œ, SIC rhinitis; n ⫽ 3). Data are presented as the geometric mean ⫾ SE. See the legend of Figure 1 for abbreviation not used in the text.

Adequate sputum samples at baseline and 24 h after exposure were obtained from 12 subjects who were SIC positive and 15 who were SIC negative. In the SIC-positive group, there was a significant increase in the percentage of sputum eosinophils at 24 h after exposure to isocyanates (median, 9.8%; interquartile range [IQR], 12.5) compared to baseline (median, 4.4%; IQR, 9.2) [Fig 4, a]. In the SIC-negative group, sputum eosinophils slightly decreased at 24 h after exposure to isocyanates (median, 0.9%; IQR, 2.1) compared to baseline sputum eosinophils (median, 1.5; IQR, 7.3). No significant changes in the percentage of sputum neutrophils were detected in either group (Fig 4, b). Adequate sputum samples at day 7 were obtained in 14 subjects who were SIC negative and in 6 subjects who were SIC positive. Eosinophil levels returned to baseline values by day 7 in the SIC-positive group (median, 3.8%; IQR, 9.3), whereas no changes over time were detected in the SIC-negative group (median 1.25%; IQR, 2.1). When the SIC-positive and SIC-negative groups were analyzed together, baseline and 24-h values of FeNO positively correlated with the corresponding percentages of sputum eosinophils (baseline: ␳ ⫽ 0.49, p ⬍ 0.01; 24 h: ␳ ⫽ 0.71, p ⬍ 0.001). In addition, the changes in FeNO at 24 and 48 h positively correlated with the sputum eosinophil changes at 24 h (24 h: ␳ ⫽ 0.66, p ⬍ 0.001; 48 h: ␳ ⫽ 0.66, p ⬍ 0.002). No significant relationships were detected between exhaled nitric oxide (NO) changes and the magnitude of late asthmatic reactions induced by isocyanate exposure. However, a strong positive correlation was detected between the 30-min fall in FEV1 during the early asthmatic response in the SIC-positive group and the corresponding decrease of FeNO (r ⫽ 0.76, p ⬍ 0.001).

Discussion We demonstrated a consistent delayed increase of FeNO after an asthmatic reaction induced by isocyanates. The exposure to isocyanate did not affect FeNO concentration in nonsensitized subjects, even if they had airway hyperresponsiveness. In contrast, isocyanate-induced asthmatic reactions were not associated with the acidification of EBCs. The study examined the full time course of changes in levels of functional and inflammatory markers after SIC. During early bronchoconstriction, FeNO decreased, and the changes at 30 min after exposure to isocyanate were strongly correlated with the corresponding degree of reduction in FEV1. A chemical reaction of inhaled isocyanates with NO or its precursors in the airways might have occurred, but this explanation for the early decrease of FeNO

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Figure 3. Individual values of pH in EBC in the SIC-positive group (n ⫽ 10) and the SIC-negative (n ⫽ 19). a: before, 7 h, and 24 h after exposure to air in the SIC-negative group. b: before, 7 h, and 24 h after exposure to air in the SIC-positive group. c: before, 7 h, and 24 h after exposure to isocyanates in the SIC-negative group. d: before, 7 h, and 24 h after exposure to isocyanates in the SIC-positive group. n.s. ⫽ not significant; post ⫽ after SIC.

would implicate a different behavior of the chemicals in the SIC-positive and SIC-negative groups. In fact, in the SIC-negative group, no changes in FeNO were detected after isocyanate inhalation. A number of studies26 have shown that repeated spirometry can reduce FeNO levels. However, the sequence of forced expiratory maneuvers necessary to monitor an early reaction was similar in the SIC-positive and SIC-negative groups. The rapid onset of FeNO decrease and the correlation with the FEV1 fall suggest a mechanical effect

Table 2—Time Course of pH in EBC After Specific Inhalation Challenge With Isocyanates in the Three Study Groups

Group SIC-negative (n ⫽ 19) SIC-positive (n ⫽ 10) Rhinitis (n ⫽ 3)

After Exposure

Before Exposure

7h

24 h

48 h

7.79 (0.33)

7.92 (0.21)

7.60 (0.37)

7.73 (0.32)

7.79 (0.32)

7.96 (0.57)

7.72 (0.55)

7.73 (0.84)

7.85 (0.22)

8.20 (0.08)

8.40 (0.71)

8.00 (0.52)

Values are expressed as the median (IQR). www.chestjournal.org

of bronchoconstriction on NO output. It has been suggested27,28 that the occlusion of small airways during bronchoconstriction may trap the NO produced there, leading to a reduction in exhaled NO concentrations. In addition, because FeNO is measured by a constant mouth flow, the reduction in the volume of conducting airways during bronchoconstriction will lead to an increase of luminal airflow compared to a not-constricted condition. A shorter residence time in the airways will decrease the amount of NO loaded to the bolus of expirate, resulting in lower FeNO levels.23,29 This explanation is consistent with the observations27,28,30 that airway constriction induced by histamine or methacholine challenge is associated briefly with a reduction of FeNO. For the same reason, an increase of FeNO during late bronchoconstriction might have been underestimated. Indeed, significant changes of FeNO were detected at 24 h when FEV1 had returned to baseline values. It is well established that the course of airway inflammation does not necessarily parallel bronchoconstriction. The time course of FeNO changes, which we observed in patients with isocyanate-induced asthma, is similar to that detected in patients with atopic asthma after SIC CHEST / 136 / 1 / JULY, 2009

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eosinophils (%)

a

p=0.01 100 90 80 70 60 50 40 30 20 10 0

p=0.041

pre

24 h post

SIC-positive

neutrophils (%)

b

pre

24 h post

SIC-negative

n.s. 100 90 80 70 60 50 40 30 20 10 0

n.s.

pre 24 h post SIC-positive

pre 24 h post SIC-negative

Figure 4. Individual percentages of sputum cells before and 24 h after exposure to isocyanates in the SIC-positive group (n ⫽ 12) and the SIC-negative group (n ⫽ 15). a: sputum eosinophils; b: sputum neutrophils. pre ⫽ before SIC. See the legend of Figure 3 for abbreviation not used in the text.

with Dermatophagoides pteronyssinus31 and is in agreement with the observation32 that eosinophils in BAL fluid peaked 42 h after allergen challenge. The shorter time course of sputum eosinophil increase compared with that of BAL fluid eosinophils and FeNO could be due to different compartments of the respiratory tract sampled by the different techniques, that is, more proximal airways using induced sputum than using BAL fluid or exhaled NO. Inconsistent results obtained by previous studies12–14,17,18 that analyzed FeNO after SIC with occupational agents may have been due to the insufficient duration of monitoring. The last measurement of FeNO was obtained 20 to 24 h after challenge, whereas successive time points were not considered. Although none of the subjects who were SIC positive had returned to work, the FeNO level 7 days after challenge was still higher than at baseline. It is unlikely that the elevated FeNO level at 7 days is due to environmental exposure to allergens because only three of the subjects who were SIC positive were atopic. It has been demonstrated33 that human mononuclear cells take up and accumulate isocyanate-albumin conjugates up to 3 days in vitro; therefore, it is conceivable that an inflammatory response is maintained over time after SIC in vivo.

This prolonged FeNO increase means that SIC should be performed at a sufficient duration from the last exposure to isocyanates to avoid the rise in FeNO being blunted by an already elevated FeNO level at baseline. This observation might explain why patients with high basal FeNO levels in the study by Piipari et al17 did not show a significant elevation of FeNO level despite a bronchoconstriction induced by occupational agents. Our data confirm that FeNO level reflects the degree of airway eosinophilia in asthma12,34 because of a positive correlation between sputum eosinophil levels and FeNO levels before and after SIC, and a significant relationship between the changes in levels of both markers after SIC. Corticosteroids inhibit inducible NO synthase and induce falls in FeNO35; therefore, we were careful to examine subjects who had not received corticosteroids for at least 4 weeks prior to the study. This reason may partially explain why our results were more consistent than those of studies12–13,18 that included patients on corticosteroid treatment at the time of the test. Two of our subjects had to be treated with a single dose of 25 mg of oral prednisone at 7 h to reverse the late asthmatic reaction. Exhaled NO levels rose in both subjects at 24 and 48 h, as did the percentage of sputum eosinophils at 24 h. However, we cannot exclude that increases in FeNO and eosinophil levels would have been greater if the subjects had not been treated with corticosteroids. It has been suggested36 that the measurement of EBC pH can be used as a marker for acute worsening of childhood asthma. The mechanism leading to low airway pH in patients with asthma is still undetermined, and whether this phenomenon actually occurs in lower airways has been challenged.37 We had no evidence that isocyanate-induced asthma is associated with the acidification of EBC. This finding might be due to differences in the pathophysiology of isocyanate asthma, or, more likely, it reflects the differences between natural asthma exacerbation and asthmatic reactions induced in laboratory. Airway acidification leading to the conversion of nitrite to NO gas cannot therefore explain the increase in FeNO levels detected after isocyanate-induced asthmatic reactions.36 However, higher EBC pH at 7 h after exposure of SIC might have attenuated the FeNO rise, favoring the reaction of airway NO with nitrite.38 EBC samples collected in the afternoon (7 h after exposure) exhibited a higher pH compared to morning values (ie, baseline, 24 h, and 48 h), suggesting a circadian rhythm of EBC pH. A small effect of inhaled isocyanates on EBC pH might be hidden by a circadian rhythm and explain the less significant increase in EBC pH after active exposure. When repeated daily measurements of EBC pH are then performed, appropriate controls are needed for

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a reliable interpretation of data. An optimal study design would randomize the order of sham vs isocyanate experimental exposures. However, this design was not practicable. The unpredictable duration of the effects of active exposure would have required intervals of some weeks between the two challenges with the need to keep the subjects off work in order to avoid the interference of workplace exposure.

Conclusions Our results suggest that FeNO is a useful measurement in the evaluation of patients with occupational asthma, particularly when the causitive agent is a low-molecular-weight compound, and the assessment of airway response on specific exposure is necessary for a diagnosis because conventional immunologic tests are not applicable to demonstrate sensitization. The analysis of the time course of FeNO changes after SIC in the laboratory provides the necessary information for an appropriate use of this tool in a natural setting, such as the workplace. ACKNOWLEDGMENT: The authors thank Giovanna Fulgeri for secretarial assistance and Dr. Roberta Venturini and Luigi Zedda for technical assistance.

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