Oemed-2011-100255 1.7

OEM Online First, published on July 5, 2012 as 10.1136/oemed-2011-100255
Association between exhaled breath condensatenitrate + nitrite levels with ambient coarse particleexposure in subjects with airways disease Sarah Manney,1 C M Meddings,2 R M Harrison,2,12 A H Mansur,1 A Karakatsani,3A Analitis,4 Klea Katsouyanni,4 D Perifanou,4 I G Kavouras,5 N Kotronarou,5J J de Hartog,6 J Pekkanen,7,8 K Ha ¨meri,9 Harry ten Brink,10 Gerard Hoek,6 Objectives Studies of individual inflammatory responses to exposure to air pollution are few but are important in defining the most sensitive markers in better < Short-term increases in particulate matter air understanding pathophysiological pathways in the lung.
pollution have been associated with respiratory The goal of this study was to assess whether exposure to airborne particles is associated with oxidative stress in < Oxidative stress and inflammation may form part of the mechanism of the cardiorespiratory Methods The authors assessed exposure to particulate effects of particulate matter air pollution, but the matter air pollution in four European cities in relation to direct evidence in epidemiological studies is levels of nitrite plus nitrate (NOx) in exhaled breath condensate (EBC) measurements in 133 subjects with < We evaluated the association between particle asthma or chronic obstructive pulmonary disease using number, fine and coarse particle mass and EBC an EBC capture method developed for field use. In each subject, three measurements were collected. Exposure measurements included particles smaller than 10 mm chronic obstructive pulmonary disease in four (PM10), smaller than 2.5 mm (PM2.5) and particle number counts at a central site, outdoors near the subject’s < The coarse particle concentration at a central site was significantly associated with increased Results There were positive and significant relationships nitrate and nitrite concentrations in EBC. No between EBC NOx and coarse particles at the central associations with other particle metrics were sampling sites (increase of 20.4% (95% CI 6.1% to 36.6%) per 10 mg/m3 increase of coarse particles of the < Our findings add to evidence that coarse previous day) but not between EBC NOx and other particles have health effects that air pollution particle measures. Associations tended to be stronger in subjects not taking steroid medication.
Conclusions An association was found betweenexposure to ambient coarse particles at central sites andEBC NOx, a marker of oxidative stress. The lack ofassociation between PM measures more indicative of multiple locations and times is logistically chal- personal exposures (particularly indoor exposure) means lenging. Assessment of airway inflammation is now interpretation should be cautious. However, EBC NOx relatively easy using non-invasive means such asinduced sputum or exhaled breath condensate may prove to be a marker of PM-induced oxidative stressin epidemiological studies.
(EBC),6 although neither has been assessed inepidemiological studies. Of the two, EBC collectionis theoretically easier to undertake in field studiesand allows measurement of pH, ions and small molecules such as glutathione, nitrate and nitrite Exposure to air pollution is associated with deaths (markers of oxidative stress) as well as larger mole- from and hospital admissions for chronic respiratory cules putatively involved in the inflammatory and cardiac disease.1e4 The most likely mechanism for these effects is the induction by inhaled particles, However, the laboratory-based EBC apparatus does particularly in the PM2.5 fraction (particles smaller not lend itself to epidemiological field studies, while than 2.5 mm), of lung inflammation involving a simpler method, the R-tube, is limited by the need oxidative stress.5 However, direct evidence for for a freezer to keep the sleeve cold.7 EBC NOx (total changes in inflammatory state within the lung in nitrate + nitrite) is suggested as a reliable marker epidemiological studies is scarce as measurement of due to its stability and close correlation with other oxidative stress in the lung in many individuals at Manney S, Meddings CM, Harrison RM, et al. Occup Environ Med (2012). doi:10.1136/oemed-2011-100255 Copyright Article author (or their employer) 2012. Produced by BMJ Publishing Group Ltd under licence.
The RUPIOH (Relationship between Ultrafine and fine Partic- home environment. Subjects were non-current smokers living in ulate matter in Indoor and Outdoor air and respiratory Health) a household without a smoker. There was no criterion for years study is an EU-funded study designed to explore the distribution since quitting smoking. Past smokers quitted smoking on average of various particle metrics both indoors and outdoors11e13 in 19.3 years before recruitment. Two subjects quitted in the year conjunction with clinical parameters in patients with asthma or before recruitment. Details of treatment were obtained for each chronic obstructive pulmonary disease (COPD). It also aimed to subject, in particular dose of inhaled corticosteroid (ICS).
assess the relationship between exposures for a range of particlemetrics and EBC NOx, a marker of oxidative stress. The main findings from the exposure measurements imply that while Written informed consent was obtained from each subject. The measurements at a central site reflect temporal changes in particle study was approved by local medical ethics committees in all metrics outside specific homes, they may characterise indoor centres before the start of the fieldwork.
exposure to ambient particles less well for ultrafine particles thanfor fine particle mass.11e14 No exposure metric was associated with any index of lung function.13 This does not exclude the Airborne concentrations of particles smaller than 10 mm (PM10), possibility of significant inflammatory change in the airways due smaller than 2.5 mm (PM2.5), coarse particles (PMcoarse) and total to pollutant exposure. Which particle metric is best associated PNCs were measured at a central monitoring site for each centre with a specific health or physiological end point has been a matter during the entire approximately 18 months study period and of much discussion, although the fine fraction seems to be where both outside and inside each subject’s home. The central urban toxicity most resides5 and there is some evidence that particle background site measurements were continuous for the duration numbers (a surrogate of particle surface area) may be a better of the project and were selected to avoid local sources of air pollution. The measurements in and near the subjects’ homes In the RUPIOH study, a validated simple method was used for were performed for the week of observation for each individual.
collecting EBC in the field to determine whether glutathione and Each individual was visited three times in that week. Harvard NOx levels in EBC were associated with exposure to particles impactors were used for sampling of PM10 and PM2.5 (set at a both indoors and outdoors.16 A second goal was to evaluate the flow rate of 10 litres/min) as integrated 24-h averages from noon feasibility of using these markers of exposure to oxidant stress in to noon using timers. Noon to noon samples were taken to increase efficiency of obtaining seven daily samples given limitedequipment. The coarse fraction was taken as the difference between PM10 and PM2.5. For indoor sampling, silent pumps specifically designed for home measurements were used. TSI The study was conducted from October 2002 to March 2004 in model 3022A Condensation Particle Counters (TSI Incorporated, four European cities: Helsinki (Finland), Athens (Greece), Shoreview, Minnesota, USA) were used to obtain total PNCs Amsterdam (the Netherlands) and Birmingham (UK). The full for all particles >7 nm in diameter. Continuous measure- methodology has been described elsewhere.11e14 During the ments (PNC) were averaged to the same noon to noon time whole study period, a reference site at an urban background intervals.11 12 14 There were 11%, 14% and 10% observations location in each city was used to monitor particle mass con- with missing exposure measurements for the indoor, outdoor centration and particle number concentration (PNC). At various and central site, respectively. Traffic count data obtained from locations (urban and suburban) covering the participating cities, the municipality were used to classify a home as a major road homes of subjects on streets of both low and high traffic density with either asthma or COPD were selected for the study. Airpollution monitoring was done for 1 week in these homes both indoors (living room) and outdoors (garden or balcony). During EBC samples were collected using a validated system developed in this week, respiratory health was characterised by spirometry, Birmingham.16 This comprised two TeflonÒ tubes of 7.9 mm a symptom diary and EBC collection. EBC was collected three diameter (Du Pont, Wilmington, Delaware, USA) submerged in times during that week in each subject. This paper focuses on a container of ice/ice packs of 8 cubic litre capacity. Subjects were collection of EBC and levels in EBC of NOx and glutathione in asked to breathe continuously through a mouthpiece via a two- way non-rebreathing valve (Intersurgical LtdÓ Berkshire, England)attached to the tubes while wearing a nose clip for 15 min. At the end of the sampling period, samples were tipped from the end of Subjects were aged 35 years or older, had a doctor diagnosis of the tubes into sample tubes, which were kept on dry ice until chronic respiratory disease (asthma, COPD, as defined by Global being returned to the local laboratory where they were kept at Initiative for Asthma17 or Global Initiative for Chronic Obstruc- À708C for later central testing in Birmingham as a single batch. All tive Lung Disease18 criteria) and had experienced respiratory collections were made under direct supervision. This method symptoms in the past 12 months. Those patients who had not produces an average volume of 1.5 ml of condensate in laboratory received a definite diagnosis of asthma or COPD (especially in the studies.16 Saliva contamination is potentially a problem. We Netherlands) were classified as non-specific chronic lung disease.19 addressed this by discarding those that were clearly contaminated Because of difficulties to recruit a sufficient number of subjects on visual inspection (frothy, opaque appearance with more viscous (especially) in the Netherlands, some subjects without lung disease were included. Patients with severe disease, defined as useof bronchodilating reliever medications more than three times a day, nebulised bronchodilators or long-term oxygen treatment, were excluded. Preference was given to non-working subjects to Total glutathione was determined by use of a validated assay kit eliminate potential confounding by occupational exposures and to (Trevigen IncÒ, Gaithersburg, Maryland, USA).16 20 This used an obtain a closer approximation of personal exposures in the indoor enzymatic recycling method, employing glutathione reductase, Manney S, Meddings CM, Harrison RM, et al. Occup Environ Med (2012). doi:10.1136/oemed-2011-100255 for the quantification of total glutathione. Measurement of ab- data for all cities combined, as the number of observations per sorbance at 405 nm was completed using a spectrophotometric city was limited. Our main model adjusted for city, outdoor plate reader (Labsystems Multiskan MS, Thermo Fisher Scientific temperature and season. An indicator variable for city was IncÓ, Waltham, Massachusetts, USA). The standard limit of included in the regression model to adjust for potential detection of the glutathione assay was >3.12 picomoles/50 ml.
systematic differences in EBC NOx between cities. Outdoor Following repeatability experiments and further assay develop- temperature on the day of EBC collection was included as a ment in our own laboratory, a limit of detection of >1.56 pico- potential confounder. Outdoor temperature data were collected moles/50 ml was established. Samples were thoroughly defrosted from the nearest station of the respective meteorological offices.
and mixed using a vortex mixer prior to analysis.
We additionally adjusted for season, defined as the four seasonscorresponding to the meteorological seasons, for example, December, January and February were defined as winter. To Nitrate and nitrite combined is referred to here as NOx as in account for repeated measurements within subjects, a random previous studies,21 but this is not to be confused with NOx in ambient air (NO2 plus NO). EBC NOx was determined as We additionally investigated a model having city-specific a coloured azo-dye product of the Griess reaction, using a spectro- terms for season and temperature, by including interaction photometric plate reader (Labsystems Multiskan MS) at 540 nm.
terms of city and these variables. Instead of indicator variables The concentration of total NOx is indirectly measured by deter- for season, we also included trend terms (linear, quadratic, mining both nitrate and nitrite levels in the sample. The standard cubic). Finally, we performed an analysis adding temperature limit of detection of the NOx assay was >3.12 mmol/l; following squared to the model to accommodate potential non-linear repeatability experiments and further assay development in our own laboratory established a limit of detection of >1.56 mmol/l.
EBC NOx data were transformed using a log10 function due to Samples were thoroughly defrosted and mixed using a vortex deviations from the normality assumption. We assessed the mixer prior to analysis. Samples below the detection limit (DL) effects of same day (lag 0, from yesterday noon to today noon) were set to one half of that value (eg, 1.56 mmol/l).
and previous days’ (lag 1e2 days) air pollution. Effect estimateswere calculated for an increase of 10 mg/m3 PM10, 10 mg/m3 PM2.5, 10 000 particles per$cm3 PNC and 10 mg/m3 coarse particles.
Respiratory symptoms, medication use and time spent outdoors We assessed effect modification by disease classification, inhaled were recorded three times daily in a diary for the study week.
steroid usage, sex, age, season, city, time spent outside the home ICS dose was expressed as the daily dose in microgram equiva- (continuous) and traffic near the home (major road yes/no) using lents of beclometasone, taking fluticasone as twice as potent as an interaction term of the exposure variable and the effect modi- beclometasone and budesonide as equipotent. ICS dose data fier. Analysis of the air pollution effects was done using mixed- were split into steroid naive (0 mg), low-dose (1e999 mg) and effects models (PROC MIXED) in SAS (V.9.2; SAS Institute Inc.).
high-dose (>999 mg) groups, using the median as the cut-off Analysis of the association between subject average EBC NOx and point to define high and low dose. Exposure to indoor sources of subject characteristics (eg, disease status) was performed with pollution (eg, cooking, smoking) during sampling was obtained from a timeeactivity diary kept by participants during the 1-weekmeasurement period with a 30 min resolution. Lung function was measured three times daily using a home spirometer.13 Subject characteristicsEBC was collected in 147 individuals. Data from 14 subjects were excluded from analysis as samples were contaminated with Associations between air pollution exposure and EBC NOx were saliva. One hundred and thirty-three individuals remained for assessed by linear regression. All analyses were performed with EBC analysis, of which 111 subjects with more than one valid Patient characteristics: baseline data for all subjects and for individual cities Data presented are mean 6 SD, with minimum and maximum in parentheses unless otherwise indicated.
FEV1 ¼ forced expiratory volume in one second, comparing measured versus predicted, see reference.13A, asthma; C, COPD; FVC, forced vital capacity; ICS, inhaled corticosteroid; NS, non-specific lung disease; SN, inhaled steroid naive.
Manney S, Meddings CM, Harrison RM, et al. Occup Environ Med (2012). doi:10.1136/oemed-2011-100255 Mean particle levels indoors, outside the homes and at the central sites for the four centres separately and combined 36.3613.9 13.468.8 18002620242 28.0617.3 59.1636.0 31.7624.4 17254613341 25.8612.7 58.7625.5 34.4621.9 25971613980 8.566.1 17347620282 23.7618.0 34.8619.6 11.465.0 8.666.7 13536617234 19.0616.3 32.1627.1 13.4615.6 18906615596 18.1615.2 32.2624.4 14.0615.6 1985269726 Data are given as mean 6 SD. Mass metrics are in microgram per cubic metre. PNC ¼ particle number count given as number of particles/cubic centimetre air. PM10 and PM2.5 are particlessmaller than 10 mm and 2.5 mm, respectively. PMcoarse is particles between 2.5 and 10 mm.
EBC measurement (below) were included in the final data analysis (table 1). Sixty-three subjects were labelled as asthma From the 133 subjects, we had 310 valid NOx analyses available.
and 24 as COPD. Twenty-four subjects were labelled as non- EBC NOx was above the DL in 88.4% of samples tested (273/ specific chronic lung disease or were without chronic respiratory 310). Values below the DL were set to 0.78 mmol/l (0.5 3 DL).
disease (25 from the Dutch centre). The 22 excluded subjects did We had three valid observations per subject for 68 subjects, two not differ from the 111 subjects in distribution over the cities valid observations per subject for 43 subjects and one valid and disease status. For the main analysis, the three groups were observation per person for 20 subjects and two subjects with no valid observations. These 22 observations were excluded as wecannot analyse within subject temporal variability in relation to temporal variability of air pollution. The between-days coeffi- Data for each subject’s home monitoring results and the central cient of variation of the assay was 9% for concentrations near site information pertain to their individual study week. Full information on the particle measures and their spatial distribu- The mean value for the whole group (all cities) was 9.5 mmol/l tion have been published elsewhere,11 12 14 but the mean results (table 3). EBC NOx levels were statistically significantly for each metric are summarised in table 2. PM concentrations different between the four cities, with the highest levels found differed significantly between the cities, with the highest in Athens and the lowest level in Amsterdam and Helsinki. For concentrations observed in Athens and the lowest in Helsinki.
asthma and COPD (all cities), the mean levels were 9.6 and Concentrations also differed between central site, residential 11.2 mmol/l, respectively. Differences in EBC NOx between outdoor and indoor locations. Within each location, significant disease status were, however, statistically non-significant. In temporal variability was present. There were low correlations a regression model with both city and disease status, only city between indoor and outdoor/central site measurements for was a significant predictor of EBC NOx. We could not identify any significant predictor of baseline EBC, except city. Age, sex,disease, medication and smoking status (formerenever), pack- years smoked, traffic near the home, educational status, lung function were not associated with EBC NOX and did not Glutathione was only detectable in those samples where explain the higher values in Athens.
contamination with saliva was known due to the visual appear-ance of the sample so these data were unusable for assessment of lower respiratory tract glutathione content. Our assay was not Associations between air pollutant levels and EBC NOx are sensitive enough to detect the low glutathione levels reported in shown in table 4. PNC and PM2.5 concentrations were not a study performed after our analyses were carried out.22 related to EBC NOx for any of the evaluated locations and lags(0, 1, 2 days). The coarse particle concentrations measured at thecentral site and to some extent the home outdoor location wasrelated to EBC NOx. The most significant association was foundwith a 1-day lag for central sites and 2-day lag for the homeoutdoor measurements.
Exhaled breath condensate NOx levels (in micromoles/litre) for all cities and stratified by city and disease status An analysis limited to subjects with asthma and COPD showed very similar associations as in the main analysis, for example, for previous day (lag 1) coarse particles at the central site, the effect estimate was 13.8% (À0.3 to 29.9%). Interaction analyses showed no differences in the effect estimates for coarse particles between the three disease categories (table 5). There was a tendency towards more significant associations between coarse particle levels and EBC NOx in subjects that did not take inhaled steroid medication in particular in those with asthma (table 5). However, the differences between subgroups were not statistically signifi- cant. There were also no significant differences in effect estimates Levels of NOx that were below the detection limit (DL) were set to 0.5 3 the DL. Thus, all between city, season and above and below the median (3 h) values samples are included in the analysis.
*Differences statistically significant (p <0.0001), t-test on slope linear regression analysis.
yDifferences not significantly different, t-test on slope linear regression analysis.
yes/no) and temperature on the day before the test.
Manney S, Meddings CM, Harrison RM, et al. Occup Environ Med (2012). doi:10.1136/oemed-2011-100255 Associations between air pollution concentrations on the same day and two previous days and EBC NOx for all cities combined Associations are expressed as the percentage change of NOx in EBC for increments of 10 mg/m3 for PM10, PM2.5 and PMcoarse; 10 000 p/cc for PNC. All models adjusted for city, season andtemperature on day of the test. Lag 0 is the effect of air pollution for the 24-h noonenoon period ending on the day of EBC collection.
#p<0.10; *p<0.05.
EBC, exhaled breath condensate.
field has promise as a tool for assessing the impact of air Effect estimates were similar when city-specific temperature pollution exposure in epidemiological studies, particularly and season effects were included. The estimate for coarse particle (central site, lag 1) was 19.4% (95% CI 4.6 to 36.3) and18.9% (95% CI 4.0 to 35.9) when season and temperature were specified as city-specific, respectively. Model fit (Akaike’s We have shown that, when considering the whole group, Information criterion) was worse for models with city-specific regardless of centre or diagnosis, PMcoarse at the central site was terms. Models that included cubic trend terms instead of the the strongest predictor for EBC NOx. The effect was shown season indicators also showed similar effects. For coarse parti- across all cities, which strengthens the belief that this is cles, central site lag 1, the effect estimate was 18.8% (95% CI a consistent rather than a chance finding. The consistency across the three evaluated lags also suggests a non-chance finding. Theassociation was further robust to various adjustments. If this is truly a causal association, it might be expected that there would This study has shown an association between measured expo- be an association between EBC NOx and more personal PM sure to ambient coarse particles at central sites and EBC NOx, exposure measures, such as indoor PM or PM levels outside each a marker of oxidative stress. This was seen consistently across all home (whatever the size fraction). We did find a significant four cities. There was no effect associated with PM2.5, but association with home outdoor coarse particles, though not effects from PM10 fell between those seen with the coarse and stronger than with central site PM. We did not find any asso- PM2.5 fractions. As we have already shown no association of ciation with coarse particles in indoor air, where people spend lung function with any measure of PM in this study,13 this a large fraction of their time. This suggests that the observed suggests that EBC NOx may reflect an effect of PM exposure association with central site coarse particles is either a chance before such effects become marked enough to change airflow.
finding or that there is something different about the coarse This study also shows that measurement of EBC NOx in the particles at the central site either in terms of toxicity or that the Associations between coarse particles at the central sites of same day and two previous days and EBC NOx in subgroups defined by disease classification and inhaled steroid use for all cities combined Disease classification (NS¼non-specific lung disease) and inhaled steroid use (naive¼no inhaled steroid). Associations are expressed as the percentage change of NOx in EBC for increments of10 mg/m3. All models adjusted for city, season and temperature on day of the test. Lag 0 is effect of air pollution of the 24-h noonenoon period ending on the day of EBC collection.
#p<0.10; *p<0.05.
EBC, exhaled breath condensate.
Manney S, Meddings CM, Harrison RM, et al. Occup Environ Med (2012). doi:10.1136/oemed-2011-100255 mix centrally reflects better overall exposure for these individ- baseline questionnaire (smoking status, pack-years smoked, uals. Various studies have documented differences in chemical educational level, age, sex), disease status, medication use and composition between particles generated indoors and outdoor traffic density near the home did not explain the difference (Brunekreef et al23 and references therein). This emphasises the need to collect information on the chemical composition andsurface characteristics of indoor and outdoor coarse particles. It is likely that particles measured indoors were to an appreciable The RUPIOH study is the largest field-based study of its kind to extent from indoor sources, though we did not formally separate employ EBC as a method to assess oxidative stress in the context particles from indoor and outdoor origin. Central site outdoor of indoor and outdoor air pollution, involving 133 subjects in measurements may also better reflect total individual exposure four European cities with repeated measures of EBC over time.
because they characterise the whole area where the subject This method of EBC collection, developed for ease of use in the moves during the day including those particles that penetrate field, proved cheap and portable, suitable for use in a patient’s indoors. We did not find a difference in coarse particle associa- home, was easily adopted by different research groups and was tion with EBC NOx between subjects spending <3 h away from well accepted. However, 18% of the total number of samples home (the median) and those who spent more. However, we did were contaminated with saliva, which is unacceptably high, not have information on where subjects spent their time out of but could be reduced by greater vigilance in the field. EBC NOx home (indoors, outdoors). The consistency of the finding across was detected in 88.4% of all samples with a range of values all cities of the association with coarse particles and the indicating that it has potential as a marker of oxidative stress in apparent size gradation of effect by particle size at the central site may, however, lend support to this not being a chance Questions have been raised about the measurement of NOx nding, even though the potential mechanisms remain elusive.
species in EBC. The full effect of EBC sample contamination Another explanation for the lack of an indoor coarse particle with NOx species in the general laboratory environment (eg, effect is the smaller contrast in indoor coarse particles (table 2).
glassware) or the collection device (eg, silicon/TeflonÒ or glass If this is a true association, it coheres with the recognised inner surface) has yet to be established. The ATS/ERS taskforce association of the coarse fraction with health impacts24 and in on EBC published recommendations to minimise the potential this context is likely to reflect not just vehicle emissions. Data for such contamination, suggesting that all surfaces coming into from the UK25 suggest that the main components of coarse contact with EBC during collection or analysis should be rinsed particles are non-exhaust particles from road traffic (eg, brake thoroughly with distilled/deionised water, as ambient NO and tyre wear particles) sodium chloride and soil-derived parti- oxidises rapidly on surfaces increasing the chance of contami- cles The average sampling height was 5.4 m above the ground nation.32 33 In this study, particular care was taken to minimise surface, hence a resuspended road dust contribution is likely. It the likelihood of such contamination occurring, both during the has been suggested that the coarse fraction is associated more collection and analysis of EBC. All collection tubes and non- with respiratory24 than cardiac end points, and these data would rebreathing valves underwent a final rinse in distilled water prior to use, and only sterile items were used in the storage andanalysis of samples, for example, cryovials and pipette tips. With regard to the collection device, we selected TeflonÒ collection There are few epidemiological studies that have linked air tubes due to the materials known non-reactive properties, while pollution with markers of oxidative stress in EBC, most of them being made entirely of carbon and fluorine, negating the chance in children.26e31 Significant associations with various markers of NOx species leaching into the sample during collection. EBC such as malondialdehyde and pH have been reported, but no study evaluated NOx and coarse particles.
been shown to affect the concentration in any way, giving There are a wide variety of inflammatory mediators that may a greater flexibility for the storage of domiciliary samples.34 be measured to monitor oxidative stress in airways disease. EBC It is unlikely that different degrees of misclassification of NOx (total nitrate + nitrite) is suggested as a reliable marker due exposure have played a role in finding associations for specific to its stability and close correlation with other common markers particle metrics, as the lowest misclassification was found for of oxidative stress, such as hydrogen peroxide, which is more challenging to measure due to its rapid breakdown and 8- isoprostane.8 9 The measurement of more complex lipid- orprotein-based molecules such as eicosanoids or cytokines is more challenging due to the low concentrations of these markers in EBC These data suggest that EBC NOx may be useful as an indicator and the inconsistency in commercial assays. EBC NOx is a marker of pollutant-associated oxidative stress in an epidemiological of oxidative/nitrosative stress, which correlates moderately with setting. NOx was found in the majority of EBC samples, but exhaled NO.10 EBC NOx measurements have been employed as levels did not differ significantly between the asthma and COPD a more stable end product of NO metabolism in the airways.
subgroups. Coarse particle concentration at the central site was EBC NOx is an indicator of oxidative stress in the absence of the best predictor of EBC NOx, although the lack of association changes in lung function previously reported13 and that use of EBC with more personal measures of particle exposure (particularly the NOx should be considered in further population-based studies.
indoor exposure) makes interpretation difficult. EBC NOx shouldbe further investigated as a marker of oxidative stress in subjects with airways disease in response to air pollution exposure.
The higher levels of EBC NOx in the Athens subjects are difficultto explain. Higher long-term exposure or different particle Author affiliations1Department of Respiratory Medicine, Birmingham Heart of England NHS Trust, composition could be a factor, but an alternative explanation lies in differences in the selection of patients or other lifestyle 2Division of Environmental Health Risk Management, University of Birmingham, factors. An analysis including lifestyle factors available from the Manney S, Meddings CM, Harrison RM, et al. Occup Environ Med (2012). doi:10.1136/oemed-2011-100255 32nd Department of Pulmonary Medicine, “ATTIKON” University Hospital, Medical McCafferty JB, Bradshaw TA, Tate S, et al. Effects of breathing pattern and School, National and Kapodistrian University of Athens, Athens, Greece inspired air conditions on breath condensate volume, pH, nitrite, and protein 4Department of Hygiene, Epidemiology and Medical Statistics, Medical School, concentrations. Thorax 2004;59:694e8.
National and Kapodistrian University of Athens, Athens, Greece Hunt J, Byrns RE, Ignarro LJ, et al. Condensed expirate nitrite as a home marker for 5National Observatory Athens, Institute for Environmental Research and Sustainable acute asthma. Lancet 1995;346:1235e6.
Ganas K, Loukides S, Papatheodorou G, et al. Total nitrite/nitrate in expired breath 6Institute for Risk Assessment Sciences, University of Utrecht, Utrecht, The condensate of patients with asthma. Respir Med 2001;95:649e54.
¨bsis Q, et al. Exhaled nitric oxide and biomarkers in exhaled breath condensate indicate the presence, severity and control of childhood Department of Environmental Health, National Institute for Health and Welfare, ´rot-Kornobis N, Hulo S, Edme´ JL, et al. Analysis of nitrogen oxides (NOx) in the Institute of Public Health and Clinical Nutrition, University of Eastern Finland, Kuopio, exhaled breath condensate (EBC) of subjects with asthma as a complement to exhaled nitric oxide (FeNO) measurements: a cross-sectional study. BMC Res Notes 9Department of Physics, University of Helsinki, Helsinki, Finland 10Energy Research Center of the Netherlands, Business Unit ECN Clean Fossil Fuels, Hoek G, Kos G, Harrison R, et al. Indoor-outdoor relationships of particle number and mass in four European cities. Atmos Environ 2008;42:156e69.
11Institute of Occupational and Environmental Medicine, University of Birmingham, Puustinen A, Hameri K, Pekkanen J, et al. Spatial variation of particle number and mass over four European cities. Atmos Environ 2007;41:6622e36.
12Department of Environmental Sciences/Center of Excellence in Environmental de Hartog JJ, Ayres JG, Karakatsani A, et al. Indoor and outdoor fine and ultrafine Studies, King Abdulaziz University, Jeddah, Saudi Arabia particles in relation to lung function in asthma / COPD patients in four European cities.
Occup Environ Med 2010;67:2e10.
Lianou M, Chalbot MC, Kotronarou A, et al. Dependence of outdoor particulate mass Acknowledgements The project ‘Relationship between Ultrafine and fine and number concentrations on residential and traffic features in urban areas. J Air Particulate matter in Indoor and Outdoor air and respiratory Health’ was funded by the EU ENVIRONMENT and CLIMATE Research Programme, contract QLRT-2001- Donaldson K, Stone V. Current hypotheses on the mechanisms of toxicity of 00452. The project was coordinated by the Institute of Risk Assessment Sciences, ultrafine particles. Ann Ist Super Sanita 2003;39:405e10.
with additional funding from the Municipal Health Service Amsterdam. The Manney S. Studies of Markers of Inflammation and Oxidative Stress in a Newly contribution of following persons to the fieldwork of the project is gratefully Developed Exhaled Breath Condensate Collecting System. Thesis (PhD). University of acknowledged: Niilo Kalakoski, Jyrki Martikainen, Arto Puustinen, Marko Vallius (Helsinki), Ino Vei, Evangelos Akylas, Dimitrios Papagiannis, Antonios Foutougios, GINA Guidelines for Asthma. http://www.ginasthma.com/ (accessed 31 Jul 2009).
Spyros Lykoudis, Elena Arvanitaki, Vana Athanasiadi, Maria Lianou (Athens), Kees GOLD guidelines for COPD. http://www.goldcopd.com/ (accessed 31 Jul 2009).
Meliefste, Hans Jongsma, Marjan Tewis, Nicolette van der Heijden-de Hartog, Jack ¨ter GH, de Monchy JG, et al. The Dutch hypothesis (chronic non- van Gommeren, Gerard Kos, Piet Jongejan, Joop van Wijnen (Amsterdam), Steve specific lung disease) revisited. Eur Respir J 1991;4:479e89.
Baker MA, Cerniglia GJ, Zaman A. Microtiter plate assay for the measurement ofglutathione and glutathione disulfide in large numbers of biological samples. Anal Contributors SM, AHM and JGA were responsible for development of the exhaled breath condensate methodology, including the laboratory analysis of the samples. SM Liu J, Sandrini A, Thurston MC, et al. Nitric oxide and exhaled breath nitrite/nitrates and JGA provided the first draft text of the paper. RMH, KK, NK, JP, HtB, KH, JGA and in chronic obstructive pulmonary disease patients. Respiration 2007;74:617e23.
GH contributed to the design of the RUPIOH study and interpretation of the results.
Corradi M, Folesani G, Andreoli R, et al. Aldehydes and glutathione in exhaled breath CMM, AK, DP, IGK and JJdH contributed to the conduct and design of the local condensate of children with asthma exacerbation. Am J Respir Crit Care Med studies. AA, SM and GH contributed to the statistical analysis. All authors commented on the draft text and agree with the text. Primary responsibility for the study is with Brunekreef B, Janssen NA, de Hartog JJ, et al. Personal, indoor, and outdoor exposures to PM2.5 and its components for groups of cardiovascular patients inAmsterdam and Helsinki. Res Rep Health Eff Inst 2005;127:1e70; discussion 71e9.
Funding European Union (grant number QLRT-2001-00452).
Brunekreef B, Forsberg B. Epidemiological evidence of effects of coarse airborneparticles on health. Eur Respir J 2005;26:309 Thorpe A, Harrison RM. Sources and properties of non-exhaust particulate matter from road traffic: a review. Sci Total Environ 2008;400:270e82.
Laumbach RJ, Kipen HM. Acute effects of motor vehicle traffic-related air pollution Ethics approval Ethics approval was provided by Medical Ethical Committees in the exposures on measures of oxidative stress in human airways. Ann N Y Acad Sci Liu L, Poon R, Chen L, et al. Acute effects of air pollution on pulmonary function, Provenance and peer review Not commissioned; externally peer reviewed.
airway inflammation, and oxidative stress in asthmatic children. Environ Health Data sharing statement All data were available to all authors. The final data analysis files were prepared and shared by SM, JGA and GH.
Barraza-Villarreal A, Sunyer J, Hernandez-Cadena L, et al. Air pollution, airwayinflammation, and lung function in a cohort study of Mexico City schoolchildren.
Environ Health Perspect 2008;116:832e8.
Epton MJ, Dawson RD, Brooks WM, et al. The effect of ambient air pollution onrespiratory health of school children: a panel study. Environ Health 2008;7:16.
Romieu I, Barraza-Villarreal A, Escamilla-Nun Analitis A, Katsouyanni K, Dimakopoulou K, et al. Short-term effects of ambient malondialdehyde as a marker of effect of exposure to air pollution in children with particles on cardiovascular and respiratory mortality. Epidemiology 2006;17:230e3.
asthma. J Allergy Clin Immunol 2008;121:903e9.
Pope CA III, Burnett RT, Thun MJ, et al. Lung cancer, cardiopulmonary Trenga CA, Sullivan JH, Schildcrout JS, et al. Effect of particulate air pollution on mortality, and long-term exposure to fine particulate air pollution. JAMA lung function in adult and pediatric subjects in a Seattle panel study. Chest Atkinson RW, Anderson HR, Sunyer J, et al. Acute effects of particulate air pollution Moncada S, Palmer RM, Higgs EA. Biosynthesis of nitric oxide from L-arginine. A on respiratory admissions: results from APHEA 2 project. air pollution and health: pathway for the regulation of cell function and communication. Biochem Pharmacol a European approach. Am J Respir Crit Care Med 2001;164:1860e6.
Le Tertre A, Medina S, Samoli E, et al. Short term effects of particulate air pollution Horvath I, Hunt J, Barnes PJ; ATS/ERS Task Force on Exhaled Breath Condensate.
on cardiovascular disease in eight European cities: a quantitative summary. J Epi Exhaled breath condensate: methodological recommendations and unsolved questions. Eur Respir J 2005;26:523e48.
Ayres JG, Borm P, Cassee F, et al. Evaluating the toxicity of airborne particulate Hoffmeyer F, Harth V, Merget R, et al. Exhaled breath condensate analysis: matter and nanoparticles by measuring oxidative stress potential - a workshop report evaluation of a methodological setting for epidemiological field studies. J Physiol and consensus statement. Inhal Toxicol 2008;20:75e99.
Manney S, Meddings CM, Harrison RM, et al. Occup Environ Med (2012). doi:10.1136/oemed-2011-100255 Association between exhaled breath
condensate nitrate + nitrite levels with
ambient coarse particle exposure in
subjects with airways disease
Sarah Manney, C M Meddings, R M Harrison, et al.
Occup Environ Med published online July 5, 2012doi: 10.1136/oemed-2011-100255 Updated information and services can be found at: References
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