Published ahead of print on August 21, 2008, doi:10.1164/rccm.200711-1731OC
© 2008 American Thoracic Society doi: 10.1164/rccm.200711-1731OC
Biomarkers of Airway Acidity and Oxidative Stress in Exhaled Breath Condensate from Grain Workers1 Experimental Medicine Program, 2 School of Environmental Health, and 3 Division of Respiratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada Correspondence and requests for reprints should be addressed to Ron Do, M.Sc., McGill University Health Centre Research Institute, Royal Victoria Hospital, H7.39, 687 Pine Avenue West, Montreal, Quebec H3A 1A1, Canada. E-mail: ron.do{at}mail.mcgill.ca
Rationale: Grain workers report adverse respiratory symptoms due to exposures to grain dust and endotoxin. Studies have shown that biomarkers in exhaled breath condensate (EBC) vary with the severity of airway inflammation. Objectives: The purpose of the study was to evaluate biomarkers of airway acidity (pH and ammonium [NH4+]) and oxidative stress (8-isoprostane) in the EBC of grain workers. Methods: A total of 75 workers from 5 terminal elevators participated. In addition to EBC sampling, exposure monitoring for inhalable grain dust and endotoxin was performed; spirometry, allergy testing, and a respiratory questionnaire derived from that of the American Thoracic Society were administered. Measurements and Main Results: Dust and endotoxin levels ranged from 0.010 to 13 mg/m3 (median, 1.0) and 8.1 to 11,000 endotoxin units/m3 (median, 610) respectively. EBC pH values varied from 4.3 to 8.2 (median, 7.9); NH4+ values from 22 to 2,400 µM (median, 420); and 8-isoprostane values from 1.3 to 45 pg/ml (median, 11). Univariate and multivariable analyses revealed a consistent effect of cumulative smoking and obesity with decreased pH and NH4+, and intensity of grain dust and endotoxin with increased 8-isoprostane. Duration of work on the test day was associated with decreased pH and NH4+, whereas duration of employment in the industry was associated with decreased 8-isoprostane. Conclusions: Chronic exposures are associated with airway acidity, whereas acute exposures are more closely associated with oxidative stress. These results suggest that the collection of EBC may contribute to predicting the pathological state of the airways of workers exposed to acute and chronic factors.
Key Words: exhaled breath condensate • biomarkers • airway acidity • oxidative stress • grain dust and endotoxin
Workers in grain storage and transfer industries have been shown to have both acute and chronic respiratory impairment related to airborne exposure to components of grain dust (1, 2) and associated endotoxin (3). Adverse health outcomes have included asthma (4), acute cross-shift reductions in airflow rates (2), and chronic airflow obstruction. Acute and chronic airflow obstruction are dose related to both the level of grain dust (1, 4, 5) and endotoxin (3, 6). Epidemiological investigation of specific risk factors for airflow obstruction among workers in the grain industry is hampered by logistical challenges associated with conducting studies at the worksite. Traditional approaches to measuring pathological changes in the airways, such as sputum induction, bronchoscopy, and nasal lavage, have limited applicability in occupational settings due to high degrees of invasiveness, specialized equipment and facilities, time commitments, and associated risks. The collection of exhaled breath condensate (EBC) from workers may be a useful addition in occupational field studies to sample the epithelial fluid of the airways (7) because the procedure is safe and simple to perform. Fluctuations in the acid–base equilibrium (pH/ammonium [NH4+]) of EBC can provide a quantitative measure of airway acidity in asthma (8–10), chronic obstructive pulmonary disease (9, 11), and other respiratory disorders (12, 13). The regulation of airway pH is maintained by the production and release of acids and bases, such as ammonia in various buffer systems of the airways. Excessive or uncontrolled acidification of the airways can be caused by disturbance of the airway pH homeostatic regulatory system (14, 15), where acid stress can lead to adverse respiratory effects, such as epithelial sloughing and mucous plugging (16). Oxidative stress, which is caused by an imbalance between the level of oxidants and antioxidants in the airway epithelium, has also been linked to inflammation (17, 18). Elevated biomarkers of oxidative stress have been measured in the EBC of individuals with asthma (19–21) and chronic obstructive pulmonary disease (22). In human airway epithelial cells, organic dust exposure (swine barn) causes airway acidification by the secretion of excess protons (23). In addition, endotoxin induces lipid peroxidation, resulting in the formation of reactive oxidant species (24) and airway hyperreactivity (25). Only a few studies have investigated the use of EBC to measure adverse changes in the airways of workers in occupational settings (7, 26, 27). Our study was conducted in 2003 as part of an ongoing project performed by our research team involving a cross-section of workers employed at five terminal grain elevators in the port of Vancouver, Canada. Our objective was to evaluate the relationship, among grain elevator workers, between EBC biomarkers of airway acidity (pH and NH4+) and oxidative stress (8-isoprostane) on the one hand, and personal characteristics and work exposures on the other. Some of the results of this study have been previously reported in abstract form (28, 29).
An independent laboratory study was conducted previously to assess technical factors associated with biomarkers measured in the present study (30). In that study, six EBC samples were collected from each of five current smokers and five never-smokers over 1 month. The current field study was nested in a larger cross-sectional survey of employees in five terminal grain elevators in Vancouver. Workers were chosen randomly from within specified job titles. The selected job titles represented the complete range of anticipated exposure levels to particulate matter and endotoxin (see the online supplement for details). All participants gave informed, written consent, and confidentiality of personal identifiers was ensured. EBC collection was performed using the R-Tube (Respiratory Research, Inc, Charlottesville, VA). It proved unworkable to require workers to refrain from eating or drinking throughout the workday. Hence, a 0.3-µm filter provided by Respiratory Research, Inc. was attached between the mouth valve and collecting tube to minimize contamination of food and drink. Filter effects were assessed in four repeated EBC samples taken from two individuals with and without a filter (n = 16). No significant effect was found for all biomarker measurements (P > 0.05; data not shown). EBC sample storage and pH/NH4+ measurements were performed as described by Do and colleagues (30). EBC collection was performed for 15 minutes at a tidal breathing rate, and samples were subjected to argon deaeration at 350 µl/minute for 10 minutes. The 8-isoprostane content of EBC samples was determined with an enzyme-linked immunoassay according to manufacturer's instructions (Cayman Chemical, Ann Arbor, MI) (see the online supplement). Full-shift (6 h minimum) personal exposure monitoring for dust and endotoxin was performed on the same day as EBC collection. Respiratory health assessment, lung function, and atopy tests were performed within 2 weeks of EBC collection (see the online supplement). Although exposure monitoring was conducted for the complete shift (as required for the full study), EBC collection was performed at random times throughout the day. For data analysis purposes, exposure concentrations were adjusted to take into consideration the duration of work on the testing day before EBC collection by multiplying the concentration value by the proportion of the full shift before EBC testing.
Data were analyzed with SAS version 9.1 (SAS Institute, Cary, NC) and statistical graphs were produced with R (R project, Vienna, Austria). In the laboratory study, the intraindividual variability was assessed with the coefficient of variation (CV) (see the online supplement). In the field study, normal probability plots were used to assess normality of the variables. In order to minimize the effect of outliers and to achieve univariate normality, variables were transformed (see the online supplement). In univariate analyses, Student's t test (Satterthwaite if unequal variances) or analysis of variance was used to compare categorical distributions; simple linear regression was used to compare continuous variables. Multivariable analyses were performed with regression modeling for EBC marker parameters with marginally significant (P
The intraindividual variability of pH, NH4+, and 8-isoprostane (over a 1-mo period) was assessed in an independent laboratory study. Results for pH and NH4+ have been reported previously (30). Briefly, the CV was larger in current smokers than in never-smokers for pH and NH4+ (intraindividual CV for never-smokers: 2.2% for pH, 10% for NH4+; current smokers: 12% for pH, 13% for NH4+). The CVs for 8-isoprostane measurements were identical between current smokers (32%) and never-smokers (32%).
Field Study Participants: Demographic and Exposure Characteristics
Work and exposure characteristics for participants are shown in Table 2. Full-shift grain dust concentrations ranged from 0.010 to 13 mg/m3 (median, 0.99 mg/m3), with 10.7% of samples measuring over the American College of Governmental Industrial Hygienists threshold limit value of 4 mg/m3 (31). Full-shift endotoxin concentrations ranged from 8.1 to 11,000 endotoxin units (EU)/m3 (median, 610 EU/m3), with 88% of samples measuring over the recommended health-based exposure limit for endotoxin (50 EU/m3) (32). After adjustment for the duration of work before EBC collection, dust concentrations ranged from 0.0022 to 12 mg/m3 (median, 0.34 mg/m3), and endotoxin ranged from 1.3 to 4,500 EU/m3 (median, 270 EU/m3). Dust and endotoxin values were highly correlated (r = 0.68 for full-shift original values; r = 0.87 for log-transformed values [Figure 1], both P < 0.0001). No differences were found for adjusted dust or endotoxin between grain elevators (P > 0.05).
EBC Biomarker Measurements EBC pH values ranged from 4.3 to 8.2 (median, 7.9); NH4+ from 22 to 2,400 µM (median, 420) and 8-isoprostane values varied from 1.3 to 45 pg/ml (median, 11). The distribution of EBC pH was negatively skewed with the appearance of two modes: a minor mode from 4.5 to 5.5, and major mode from 7.5 to 8.5 (Figure 2A). The distributions of both 8-isoprostane and NH4+ were positively skewed (Figures 2B and 2C). EBC pH and NH4+ were highly correlated (r = 0.53; P < 0.0001), whereas little correlation was found between 8-isoprostane and either pH (r = 0.11; P = 0.3) or NH4+ (r = 0.13; P = 0.3) (Figure 3).
Factors Associated with EBC Biomarkers: Univariate Results In univariate analyses, both pH and NH4+ were associated with certain jobs (gallery/bintop/distributor/annex, P < 0.05), whereas pH, but not NH4+, was influenced by duration of work on the test day before EBC collection (P = 0.04). Decreased NH4+ was associated with increased duration of employment in the grain industry only (P = 0.03). A relationship between decreased pH and NH4+ with obesity was found (P = 0.02 and 0.05, respectively). Several smoking variables decreased pH and NH4+, including current smoking status (P = 0.07 for pH and P = 0.1 for NH4+), current smoking amount (packs/d now: P = 0.05 for pH and P = 0.06 for NH4+) and cumulative smoking amount (pack-years for current smokers: P = 0.02 for pH and marginal P = 0.07 for NH4+). Neither obesity nor current smoking were significantly associated with 8-isoprostane levels. 8-isoprostane was significantly elevated at two of the five grain terminal elevators. A trend was seen in jobs involving unloading and handling grain (i.e., grain cleaner, trackshed, gallery/distributor/bintop/annex, general laborer/sweeper) when compared with jobs inside offices (panel/quality control operators, supervisor, custodian) (P = 0.08). There was a positive association between 8-isoprostane and measured levels of adjusted grain dust (P = 0.05), but only a marginal association between 8-isoprostane and endotoxin (P = 0.09). Paradoxically, a negative association was found with duration of employment in the grain industry (P < 0.0001). There was no relationship seen between any of the EBC biomarkers and atopy, reported history of current or prior asthma, respiratory symptoms, or spirometry measures.
Multivariable Results
Results from the multivariable regression modeling are shown in Table 3 and Table E2. Graphical displays for statistically significant results are also shown in Figures E1–E7. The factors associated with decreased pH and NH4+ were similar, and included cumulative smoking amount (among current smokers), longer duration of work on the study day before EBC collection, and either obesity (associated with pH) or duration of employment in the industry (associated with NH4+). Factors associated with increased 8-isoprostane included intensity of both grain dust and endotoxin (all P
Interactions between Obesity, Smoking, and Occupational Exposures Interactions between obesity, current smoking status, and adjusted dust concentrations with the three biomarker measurements were investigated by inclusion of the main effects and interaction term in the model. No interactions were found with obesity (P > 0.05 for interaction). An interaction between current smoking status and grain dust exposure was found for pH (P = 0.05 for the interaction term). The association with smoking status was strongest in individuals with low dust exposure values (median of <0.34 mg/m3; P < 0.05) compared with those with high values (>0.34 mg/m3; P > 0.05).
In the present study, we selected two biomarkers of acid stress (pH and NH4+) and one biomarker of oxidative stress (8-isoprostane) to represent some of the pathological processes that occur in the airways. In a previous study (30), the reproducibility of the acid stress biomarkers was assessed among never-smokers and current smokers. The intraindividual variability for these biomarkers was small among never-smokers, suggesting that the biomarkers are reproducible, and that a single sample taken in a field study may be a reasonable reflection of airway status. The larger variability found among current smokers (particularly for pH) suggests that the EBC assay can be used to distinguish the inflammatory effects of smoking on the airways. No difference in the variability was seen between never-smokers and current smokers for 8-isoprostane, indicating that current smoking status may not play a large role in influencing the reproducibility of this biomarker. In the field study, a unique bimodal distribution was found for EBC pH. The minor mode consisted mostly of low pH values (Figure 2A), which may have arisen from intrinsic airway acidification due to respiratory disease, such as asthma (8), gastroesophageal reflux (33), or acidic particle inhalation from the environment (14, 34). Paget-Brown and colleagues (35) report that only 6.4% of samples in their normal, healthy reference data set had pH values of 7.3 or lower. In comparison, our study had a much higher proportion of samples with low pH (25.3%), which could be explained by their exposure to proinflammatory agents (i.e., grain dust and endotoxin). We showed that EBC pH and NH4+ were highly correlated with each other, confirming that NH3 plays a role as a basic buffer in pH homeostasis in the airways (36). We have also shown that neither pH nor NH4+ are correlated with 8-isoprostane, suggesting that airway acidity and oxidative stress events are distinct components of airway pathophysiology. Chronic factors, such as smoking and obesity, were more closely associated with decreased pH and NH4+, whereas acute exposures, such as intensity of grain dust and endotoxin exposure, were closely associated with increased 8-isoprostane. In addition, other occupational factors, such as duration of work on the study day, influenced decreased pH and NH4+. The directions of the associations are consistent with what we expected from previous literature in which pathological changes in the airways coincide with decreased pH and NH4+ for acid stress events and with increased 8-isoprostane for oxidative stress events. An exception to this expectation is the association of decreased 8-isoprostane with duration of employment in the industry. The discrepancy may be due to the healthy worker effect, where workers leave the industry or transfer to lower-exposure jobs after a certain period of time because they can no longer tolerate the high exposures to the lung irritant (37). The effect of smoking on EBC pH/NH4+ in the low-exposure group only may indicate that smoking-related acid stress events in workers may mediate transfers to lower exposure jobs. Furthermore, the negative association of duration of employment and 8-isoprostane suggests that the selective forces of the healthy worker effect are strongest in those workers who are susceptible to oxidative stress events. A possible scenario might involve some workers having strong acute oxidative stress responses to high concentrations of dust or endotoxin, resulting in those workers choosing to leave the industry after a relatively short period of time. On the other hand, the expected associations of increased duration of employment in the industry (in univariate analyses only) and cumulative smoking with decreased NH4+ suggests that chronic exposures may also trigger subtle adverse effects via secretion of acids that can accumulate slowly over time.
Other explanations for the negative effect of duration of employment on 8-isoprostane may involve increased amplification of specific antioxidant host-defense systems in response to chronic occupational exposures (38). Acute exposure to grain dust and endotoxin can initially trigger oxidative stress in the airways, but this response may decrease over time as the airways adapt to the constant exposure. Attenuation of proinflammatory responses in the airways over time have already been shown in mice, where endotoxin-sensitive mice that had been preexposed to LPS for 4 days, and then exposed to LPS or sterile corn dust extract on the fifth day, had significantly lower concentrations of inflammatory agents (i.e., neutrophils and tumor necrosis factor- The associations of obesity with pH/NH4+ were interesting given that there was little evidence that supports the role of obesity in airway acidity. The effects of obesity on airway inflammation may originate from the adipose tissue itself, where obese individuals tend to have persistent low levels of systemic inflammation due to an increase in inflammatory mediators (40). In particular, nitric oxide (NO) synthase, an enzyme that catalyzes NO synthesis during inflammation, has been reported to be expressed in adipose tissue (41), and metabolites of NO have been shown to mediate airway acidity (8, 15). Recently, increased exhaled NO and decreased EBC pH has been reported in obese individuals (42, 43). Another contributing factor of obesity may involve its role in gastroesophageal reflux (44–46). Reflux can influence airway acidity through the aspiration of gastric contents into the esophagus and mouth, where the acidic droplets can be inhaled and/or exhaled (33). This would be similar to inhaling acid fogs, air pollution, or workplace exposures (14), and hence lead to adverse respiratory health effects. Recently, EBC pH has been shown to be more acidic in individuals with asthma with gastroesophageal reflux disease compared with asthma control subjects (47). It has been well established that tobacco smoking can induce the activation of inflammatory processes in the lung (48). The association of smoking and EBC pH/NH4+ in this study confirms results from a previous study from our group on an effect of acute and cumulative smoking amount with decreased EBC pH/NH4+ (30). Similar to the effects of obesity on airway acidity, the smoking results could be explained by NO metabolites in the airways (49, 50) or gastroesophageal reflux episodes (51). Other studies have also reported decreased EBC pH in smokers with asthma compared with nonsmokers with asthma (52, 53). These results suggest that exhaled acid stress biomarkers may accurately predict airway responses to chronic tobacco smoke inhalation. The present study shows that measures of occupational exposure known to be linked to airway inflammation were associated with the EBC biomarkers. Goldoni and colleagues (7) reported a dose–response relationship over a work shift with cobalt exposure and malondialdehyde, a marker of oxidative stress, in the EBC of metal workers. Similarly, Caglieri and colleagues (27) showed that not only do EBC malondialdehyde and H2O2 (another marker of oxidative stress) increase over a work shift in chrome-plating workers, but the measurements decrease significantly from the end of the work week to the beginning. This indicates a potential role for examining within-person changes in EBC 8-isoprostane (e.g., before and after controlled allergen challenge) in future research. A limitation of the study was that the study population was relatively small (n = 75). In addition, it would have been preferable to compare results with other traditional tests that measure airway inflammation, such as bronchoalveolar lavage or sputum induction. However, these tests are not feasible in occupational field studies. A strength of this study is that it was nested in a larger cross-sectional surveillance study, making a detailed, comprehensive workup of health history and work/exposure characteristics available for all of the study subjects. This enabled us to link this information with EBC biomarker measurements taken from samples that were collected on the same day when the personal exposure monitoring was performed. To date, this is one of the most comprehensive EBC studies in this regard. In summary, this article reports several new findings relating smoking, obesity, and occupational exposures to airway acidity and oxidative stress events in grain workers. Results from this study suggest that the collection of EBC may be a useful tool in occupational field studies to monitor the biological effects of exposure to proinflammatory agents on the airways of workers.
The authors thank Barbara Karlen and Yat Chow, as well as the research staff of the grain study for their help with field sampling and data collection. Gratitude is expressed to Tim Ma and Tom Barnjak for their assistance with the ammonium and 8-isoprostane analyses.
Supported by a research operating grant from the Canadian Institutes for Health Research and by fellowship support from WorkSafeBC. This article has an online supplement, which is accessible from the issue's table of contents at www.atsjournals.org Originally Published in Press as DOI: 10.1164/rccm.200711-1731OC on August 28, 2008 Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Received in original form November 25, 2007; accepted in final form August 21, 2008
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ABSTRACT
AT A GLANCE COMMENTARY
0.1) variables from univariate analyses offered using a manual, step-wise procedure. A priori variables based on theory or previous studies were tested (see the online supplement). 
) when compared with similar mice that had been preexposed to saline (
