Fractional exhaled nitric oxide levels in relation to work‐related respiratory burden and sensitization to wheat flour and multigrain in bakers

Abstract Background Work‐related lower airway symptoms (WR‐LAS), rhinitis (WRR), and asthma (WRA) are very common among bakers, due to airborne exposure to wheat flour and multigrain. Limited data is available regarding fractional exhaled nitric oxide (FeNO) in bakers in relation to respiratory burden and occupational sensitization in a real‐life situation. Objective To analyze FeNO levels in relation to WRR, WR‐LAS, and WRA with regard to allergic sensitization to occupational allergen in bakers. Methods Cross‐sectional, observational study of 174 bakers employed in traditional small bakeries in the Verona District. Subjects did FeNO measurements, spirometry, methacholine challenge, and skin prick test to common inhalant aeroallergens and bakeries occupational allergens. Results FeNO levels were higher in subjects sensitized to occupational allergens compared with bakers not sensitized to occupational allergens (22.8 ppb (18.9, 27.6) vs. 12.0 ppb (9.9, 14.5), p < 0.05). FeNO levels were higher in bakers with WRR and occupational sensitization (25.4 (20.6, 31.3)) than in bakers with WRR without occupational sensitization compared and bakers without respiratory burden (13.4 (9.6, 18.6) and 11.9 (9.8, 14.5), both p < 0.001). Similar findings were found for WR‐LAS with regard to the same categories (31.2 (24.1, 40.4) vs 13.3 (11.4, 15.6) and 15.3 (8.5, 27.5), p < 0.001 and p = 0.005). Bakers with WRA, with or without occupational allergic sensitization, had higher levels of FeNO than bakers without respiratory burden (both p ≤ 0.001). These findings were consistent after adjustments for gender, age, height, weight, smoking, and sensitization to common inhalant aeroallergens and lung function. Conclusions WRR and lower airway symptoms in bakers sensitized to occupational allergens relate to increased FeNO. Our study suggests that FeNO is associated with work‐related allergic inflammation in occupational sensitized bakers, but future studies are needed to assess how FeNO should be integrated in the diagnostic work‐up of occupational disease in bakers.

future studies are needed to assess how FeNO should be integrated in the diagnostic work-up of occupational disease in bakers.

K E Y W O R D S
allergic sensitization, bakery, fractional exhaled nitric oxide, lower airway symptoms, occupational exposure, work-related asthma, work-related rhinitis 1 | BACKGROUND A high prevalence of work-related allergic asthma in bakers is well known. 1 Occupational rhinitis is even two to four times more common. 2,3 Moreover, both diseases frequently coexist. 4 This relationship is due to airborne exposure in bakeries to wheat flour and consequent sensitization that results in onset of allergic symptoms at work. 5 Small family-run bakeries are generally characterized by poor automation, so production processes are often carried out manually (i.e., weighing, dumping, mixing bagged ingredients, and cleaning). To produce a large variety of bread, it is necessary to use many types of flour with additives or multigrain, a blend of several cereal flours, seed flours, and enzymes. Therefore, the probability of sensitizing and developing allergic respiratory symptoms when exposed both to flour and multigrain might increase with increasing exposure. 6 Fractional exhaled nitric oxide (FeNO) is a marker of type 2 inflammation. 7 FeNO reflects the production of nitric oxide (NO) in the respiratory epithelium by activation of inducible NO synthetase as response to different triggers, as for example, allergen exposure. 8 FeNO is useful as an aid in diagnostic work-up and the follow-up of patients with asthma. 9 FeNO appears to find a role in occupational asthma with recent studies suggesting use of FeNO both in relation to work exposures and standardized inhalation allergen challenges. An increase of 20 ppb in FeNO in relation to occupational exposure could be an additional diagnostic tool in the workup of asthma and help in establishing the diagnosis of occupational asthma in about 20% of the cases with suspected occupational asthma, according to a recent study. 10 Increase of FeNO in relation to specific inhalation challenge with occupational allergens could be combined with lung function information and provided to be useful in the diagnostic workup of occupational asthma. 11 A study among bakers, farmers and healthcare workers showed that a significant increase in FeNO occurred 24 h after a specific inhalation test. 12 However, allergen inhalation challenges are performed to little extent, especially in bakers sensitized to several occupational allergens, and therefore it is of interest to study if FeNO measurements in real life reflect the degree of occupational sensitization and exposure.
Studies on the relationship between FeNO and respiratory symptoms/asthma in bakers are scant. In a population of apprentice bakers, an increase in FeNO is related to the onset of bronchial hyperresponsiveness (BHR) regardless of atopy, suggesting that the measurement of FeNO in workers exposed to agents is capable to identify workers at risk of occupational asthma. 13 Few information is available on the prevalence and coexistence of work-related nasal and asthma-like disorders in bakers using enzymes and/or multigrain and their effects on FeNO. In supermarket bakers with allergic respiratory symptoms increased FeNO values were detected in sensitized to cereals and α-amylase and wheat immunoglobulin E (IgE) levels accounted for most of the variability in FeNO. 14 FeNO is well-studied in relation to common inhalant aeroallergen sensitization, and higher levels have been reported especially with regard to allergic sensitization to perennial allergens. 15 However, limited data is available regarding FeNO related to IgE sensitization towards occupational allergens due to many different factors that influence the levels of FeNO.
The aim of the present study was to analyze FeNO levels in bakers in relation to respiratory symptoms and sensitization to occupational allergens in bakeries.

| Population
A total of 229 traditional bakeries in Verona area were invited, through Bakers Labour Organization of Verona, to participate in a cross-sectional questionnaire-based survey finalized to a preventive medicine project. This resulted in 211 bakeries (92%) that accepted the invitation and 727 bakery employees who responded the questionnaire. 16 All responding bakers were offered to participate in a clinical visit. The present study is a cross-sectional, observational study based on 174 bakery workers that accepted to participate in the clinical visit. The study was approved by the local Ethics Committee (Prog. no. 1827).

| Clinical visit
The clinical interview included a questionnaire based on the European Community Respiratory Health Survey. 17 Smoking status was questionnaire-assessed, and we classified the subjects as nonand current smokers. Additional questions concerning the onset of work-related nasal and respiratory symptoms triggered by exposure to wheat flour and/or multigrain during the work shift and improved when away from work were included, according to literature. [18][19][20][21] The clinical visit was performed in the late morning on the same day that the subject has worked in the bakery.

| Exhaled NO
FeNO was measured at 50 ml/s flow using a Chemiluminescence Analyzer (CLD88; Ecomedics). FeNO was measured in accordance with international guidelines, and hence before spirometry. 9 The pulmonary technician performing both FeNO and pulmonary function testing was blinded to the answers from the questionnaire.

| Pulmonary function testing
Each patient underwent baseline spirometry (SensorMedics V-Max 22) in accordance with the ATS/ERS guidelines. In subjects without airway obstruction, methacholine challenge (MB3 Dosimeter; Mefar) was performed according to guidelines. 22 The European Coal and Steel reference values have been used. 23 The methacholine challenge was stopped when a cumulative dose of 2 mg of methacholine was reached or when the forced expiratory value at 1 s (FEV 1 ) had fallen by 20% or more below the best baseline FEV 1 following diluent inhalation (PD 20 FEV 1 ). The test was considered as positive for a provocative dose of methacholine (PD 20 FEV 1 ) ≤1 mg. 24 We have used the definition proposed by ERS for lower limit of normal for FEV 1 /FVC (88% of predicted for men and 89% of predicted for women). 25  were also tested. Histamine phosphate (10 mg/ml) and normal saline were used as positive and negative controls, respectively. Positive SPT was defined as a weal diameter ≥3 mm. Allergic sensitization to common inhalant aeroallergens was defined if at least one positive SPT was found to any of the common inhalant aeroallergens included.

| Case classification
The following respiratory symptoms: cough, wheezing, chest tightness, and shortness of breath as well as nasal symptoms (sneezy, runny, or blocked nose in absence of a cold) triggered by exposure to wheat flour and/or multigrain were assessed to be work related if worsening during the work shift and improving when away from work.
WRR: presence of work-related nasal symptoms with or without sensitization to any occupational allergens.
WR-LAS: presence of work-related lower airway symptoms with or without sensitization to any occupational allergens.
WRA: presence of work-related asthma-like symptoms and airway obstruction and/or positivity to the nonspecific bronchoprovocation test (PD 20 FEV 1 < 1000 mcg) with or without sensitization to any occupational allergens. A total of five subjects could not be classified due to missing needed information.
A p < 0.05 was considered as statistically significant.
Unpaired t-test of log-transformed FeNO levels was used for comparisons between different groups. FeNO levels are presented as geometric mean (95% confidence interval).
Multiple logistic regression models were used to study the relation between work-related rhinitis (WRR), work-related lower airway symptoms (WR-LAS), and work-related asthma (WRA) with and without occupational sensitization in relation to FeNO after adjustments for gender, age, height, weight, smoking, and allergic sensitization to common inhalant aeroallergens. Further adjustment for FEV 1

| RESULTS
A total of 174 bakery workers working in bakery for a median of 11 years (interquartile range: 5-21 years) have been included in the present study. The subjects' characteristics are given in Table 2.

| FeNO in relation to anthropometric variables
FeNO was not significantly related to either height, weight, or BMI.

| FeNO in relation to lung function and BHR
A weak relation of increased FeNO with lower FEV 1 (% predicted) was found in all subjects (r = −0.16, p = 0.04). Increased FeNO was also related with lower FEV 1 /FVC ratio (r = −0.31, p < 0.001) and subjects with low FEV 1 /FVC ratio had higher levels than subjects

| FeNO in relation to sensitization to common inhalant aeroallergens
FeNO was increased in subjects sensitized to common inhalant aer-

| FeNO in relation to occupational sensitization
FeNO was elevated in subjects sensitized to all occupational allergens, compared with nonsensitized subjects, in univariate analyses, with exception for amylase (Table 3). Being sensitized to any occupational allergen was also related to higher levels of FeNO (Table 3) and being sensitized to only one occupational allergen or at least two occupational allergens resulted in increased levels of FeNO compared with subjects nonsensitized to occupational allergens:

| FeNO in relation to symptoms upon exposure
FeNO was higher in subjects presenting wheeze, chest tightness or shortness of breath upon exposure, compared with subjects without the respective symptom in univariate analyses (Table 4). Similarly, nasal symptoms upon exposure related with higher FeNO levels (   Abbreviations: CI, confidence interval; FeNO, fractional exhaled nitric oxide.

| FeNO in relation to airway symptoms and sensitization by occupational allergens
T A B L E 3 FeNO levels (geometric mean (95% CI)) with regard to sensitization to respective occupational allergen (univariate analyses)

| FeNO in relation to years working in bakery
No relation between FeNO and years working in bakery was found (p = 0.48).

| FeNO in relation to asthma, rhinitis, and sensitization to occupational allergens
Subjects with WRA, disregarding occupational allergic sensitization status had higher levels of FeNO than subjects without asthma and rhinitis ( Table 1). The results from the univariate analyses are presented also as FeNO percentual increase, having as reference the group without WRR and WRA, in Table 1 These results were consistent after adjusting for potential confounders, such as gender, age, height, weight, smoking, and common inhalant aeroallergen sensitization ( Table 1). The results were consistent for further adjustments for FEV 1 /FVC (data not shown).

| DISCUSSION
FeNO levels were significantly higher in bakers sensitized to any occupational allergens than bakers not sensitized to bakery allergens.
A novel finding was the dose-response effect on FeNO with larger exposure. 27 We could report a dose-response relation between FeNO and allergic sensitization to occupational allergens, in line with results regarding sensitization to multiple common inhalant aeroallergens. 15 In line with thus, by using the "in vivo" SPT model, the association of two different allergens can increase the magnitude of the wheal skin response, in comparison with single allergen. 28  and with occupational sensitization related to increased FeNO might be due to the fact that these subjects already developed airways obstruction or had BHR as this was part of our asthma definition. In line with this, we found significant inverse relationship among FeNO levels and airway obstruction, evaluated as a lower FEV 1 /FVC ratio or positive BHR, in agreement with literature. 34 However, it has to be highlighted that the large majority of cases with asthma were seen in subjects sensitized to occupational allergens. The small size of the group with asthma without occupational sensitization warrants some caution in interpreting the results in that group.
Work-related cough is not associated with increased levels of

| CONCLUSION
Our study confirms the association of FeNO with work-related respiratory symptoms and disease in bakers sensitized and exposed to occupational allergens. Bakers sensitized to occupational allergens OLIVIERI ET AL. had increased FeNO levels if WRR was present and further increased levels of FeNO if work-related asthma was present. Future studies need to focus on the diagnostic work-up of WRR and asthma and which cut-offs should be used in the assessment of occupational respiratory disease in bakers.