Ann-Charlotte Almstrand

Experimental exposure to wood smoke: effects on airway inflammation and oxidative stress

Background: Particulate air pollution affects cardiovascular and pulmonary disease and mortality. A main hypothesis about the mechanisms involved is that particles induce inflammation in lower airways, systemic inflammation and oxidative stress. Objectives: To examine whether short-term exposure to wood smoke in healthy subjects affects markers of pulmonary inflammation and oxidative stress. Methods: 13 subjects were exposed first to clean air and then to wood smoke in a chamber during 4-hour sessions, 1 week apart. The mass concentrations of fine particles at wood smoke exposure were 240–280 μg/m3, and number concentrations were 95 000–180 000/cm3, about half of the particles being ultrafine (<100 nm). Blood and breath samples were taken before and at various intervals after exposure to wood smoke and clean air and examined for exhaled nitric oxide and Clara cell protein in serum and urine, and malondialdehyde in exhaled breath condensate. Results: Exposure to wood smoke increased alveolar nitric oxide 3 hours post-exposure while malondialdehyde levels in breath condensate were higher both immediately after and 20 hours after exposure. Serum Clara cell protein was increased 20 hours after exposure. Conclusions: Wood smoke at levels that can be found in smoky indoor environments caused an inflammatory response and signs of increased oxidative stress in the respiratory tract, especially in the lower airways.

Exhaled breath malondialdehyde as a marker of effect of exposure to air pollution in children with asthma

BACKGROUND Assessment of the adverse effects of oxidative stress related to air pollution is limited by the lack of biological markers of dose to the lungs. OBJECTIVE We evaluated the use of exhaled breath condensate (EBC) malondialdehyde as a biomarker of exposure to traffic-related pollution in children with asthma as part of a panel study in Mexico City. METHODS Standard spirometry and collection of EBC and nasal lavage were performed. Environmental monitoring sites were located within 5 km of the childrens homes and schools. Data were analyzed by using generalized estimating equations. RESULTS A total of 480 samples of malondialdehyde were obtained from 107 patients with asthma, with a median level of 18.7 (interquartile range [IQR], 12.4-28.7) nmol. Ambient particulates less than 2.5 microg/m(3) and ozone levels on the day of sampling were significantly associated with higher malondialdehyde levels. A 14.2-microg/m(3) (IQR) increase in 8-hour moving average particulates less than 2.5 microg/m(3) in size was associated with a 1.12-nmol increase in malondialdehyde and a 15.9-ppb (IQR) increase in 8-hour moving average ozone with a 1.16-nmol increase in malondialdehyde. Malondialdehyde levels were inversely associated with forced vital capacity and FEV(1) and positively associated with IL-8 levels in nasal lavage. CONCLUSION Exhaled breath condensate malondialdehyde was related to both air pollution exposure and changes in lung function and inflammatory markers.

Airway Monitoring by Collection and Mass Spectrometric Analysis of Exhaled Particles

We describe a new method for simultaneously collecting particles in exhaled air for subsequent chemical analysis and measuring their size distribution. After forced exhalation, particles were counted and collected in spots on silicon wafers with a cascade impactor. Several phospholipids were identified by time-of-flight secondary ion mass spectrometric analysis of the collected spots, suggesting that the particles originated from the lower airways. The amount of particles collected in ten exhalations was sufficient for characterizing the phospholipid composition. The feasibility of the technique in respiratory research is demonstrated by analysis of the phospholipid composition of exhaled particles from healthy controls, patients with asthma, and patients with cystic fibrosis. We believe this technology will be useful for monitoring patients with respiratory disease and has a high potential to detect new biomarkers in exhaled air.

Effects on airways of short-term exposure to two kinds of wood smoke in a chamber study of healthy humans

Introduction: Air pollution causes respiratory symptoms and pulmonary disease. Airway inflammation may be involved in the mechanism also for cardiovascular disease. Wood smoke is a significant contributor to air pollution, with complex and varying composition. We examined airway effects of two kinds of wood smoke in a chamber study. Materials and Methods: Thirteen subjects were exposed to filtered air and to wood smoke from the start-up phase and the burn-out phase of the wood-burning cycle. Levels of PM2.5 were 295 µg/m3 and 146 µg/m3, number concentrations 140 000/cm3 and 100 000/cm3. Biomarkers in blood, breath and urine were measured before and on several occasions after exposure. Effects of wood smoke exposure were assessed adjusting for results with filtered air. Results: After exposure to wood smoke from the start-up, but not the burn-out session, Clara cell protein 16 (CC16) increased in serum after 4 hours, and in urine the next morning. CC16 showed a clear diurnal variation. Fraction of exhaled nitric oxide (FENO) increased after wood smoke exposure from the burn-out phase, but partly due to a decrease after exposure to filtered air. No other airway markers increased. Conclusions: The results indicate that relatively low levels of wood smoke exposure induce effects on airways. Effects on airway epithelial permeability was shown for the start-up phase of wood burning, while FENO increased after the burn-out session. CC16 seems to be a sensitive marker of effects of air pollution both in serum and urine, but its function and the significance need to be clarified.

TOF-SIMS analysis of exhaled particles from patients with asthma and healthy controls

Particles in exhaled air (PEx) may reflect the composition of respiratory tract lining fluid (RTLF); thus, there is a need to assess their potential as sources of biomarkers for respiratory diseases. In the present study, we compared PEx from patients with asthma and controls using time-of-flight–secondary ion mass spectrometry (TOF-SIMS) and multivariate analysis. Particles were collected using an instrument developed in-house. 15 nonsmoking subjects with physician-diagnosed asthma and 11 nonsmoking healthy controls performed 10 consecutive forced exhalations into the instrument. Particle concentrations were recorded and samples of particles collected on silicon plates were analysed by TOF-SIMS. Subjects with asthma exhaled significantly lower numbers of particles than controls (p=0.03) and the ratio of unsaturated to saturated phospholipids was significantly lower in samples from subjects with asthma (0.25 versus 0.35; p=0.036). Orthogonal partial least squares-discriminant analysis models showed good separation between both positive and negative spectra. Molecular ions from phosphatidylcholine and phosphatidylglycerol, and protein fragments were found to discriminate the groups. We conclude that analysis of PEx is a promising method to examine the composition of RTLF. In the present explorative study, we could discriminate between subjects with asthma and healthy controls based on TOF-SIMS spectra from PEx.

Exhaled Endogenous Particles Contain Lung Proteins

BACKGROUND We recently developed a novel, noninvasive method for sampling nonvolatile material from the distal airways. The method is based on the collection of endogenous particles in exhaled air (PEx). The aim of this study was to characterize the protein composition of PEx and to verify that the origin of PEx is respiratory tract lining fluid (RTLF). METHOD Healthy individuals exhaled into the sampling device, which collected PEx onto a silicon plate inside a 3-stage impactor. After their extraction from the plates, PEx proteins were separated by SDS-PAGE and then analyzed by LC-MS. Proteins were identified by searching the International Protein Index human database with the Mascot search engine. RESULTS Analysis of the pooled samples identified 124 proteins. A comparison of the identified PEx proteins with published bronchoalveolar lavage (BAL) proteomic data showed a high degree of overlap, with 103 (83%) of the PEx proteins having previously been detected in BAL. The relative abundances of the proteins were estimated according to the Mascot exponentially modified protein abundance index protocol and were in agreement with the expected protein composition of RTLF. No amylase was detected, indicating the absence of saliva protein contamination with our sampling technique. CONCLUSIONS Our data strongly support that PEx originate from RTLF and reflect the composition of undiluted RTLF.

Surfactant protein A and albumin in particles in exhaled air

In this study we test the hypothesis that endogenous particles in exhaled air (PEx), non-invasively sampled from lower airways, are well suited for the analysis of respiratory tract lining fluid (RTLF) proteins, i.e., surfactant protein A (SP-A) and albumin. Ten healthy volunteers were included in the study and participated in two sampling sessions. Blood, exhaled breath condensate (EBC) and PEx were collected at each session. 100 L of breath were collected for each exhaled sample. Serum and exhaled samples were analyzed for SP-A using an in-house ELISA. Albumin was analyzed in exhaled samples using a commercial ELISA kit. SP-A detection rates were 100%, 21%, and 89% for PEx, EBC and serum, respectively. Albumin was detected in PEx, but not in EBC. SP-A measurements in PEx showed good repeatability with an intra-individual coefficient of variation of 13%. Both SP-A and albumin showed significant correlation to mass of PEx (r(s) = 0.93, p < 0.001 and r(s) = 0.86, p = 0.003, respectively). Sampling and analysis of PEx is a valid non-invasive method to monitor RTLF proteins sampled from the lower respiratory tract, as demonstrated here by example of SP-A and albumin analysis.

Effects of breath holding at low and high lung volumes on amount of exhaled particles

Exhaled breath contains particles originating from the respiratory tract lining fluid. The particles are thought to be generated during inhalation, by reopening of airways closed in the preceding expiration. The aim here was to explore processes that control exhaled particle concentrations. The results show that 5 and 10s breath holding at residual volume increased the median concentration of particles in exhaled air by 63% and 110%, respectively, averaged over 10 subjects. An increasing number of closed airways, developing on a timescale of seconds explains this behaviour. Breath holds of 5, 10 and 20s at total lung capacity decreased the concentration to 63%, 45% and 28% respectively, of the directly exhaled concentration. The decrease in particle concentration after breath holding at total lung capacity is caused by gravitational settling in the alveoli and associated bronchioles. The geometry employed here when modelling the deposition is however not satisfactory and ways of improving the description are discussed.

Surfactant Protein A in Exhaled Endogenous Particles Is Decreased in Chronic Obstructive Pulmonary Disease (COPD) Patients: A Pilot Study

Background Exhaled, endogenous particles are formed from the epithelial lining fluid in small airways, where surfactant protein A (SP-A) plays an important role in pulmonary host defense. Based on the knowledge that chronic obstructive pulmonary disease (COPD) starts in the small airway epithelium, we hypothesized that chronic inflammation modulates peripheral exhaled particle SP-A and albumin levels. The main objective of this explorative study was to compare the SP-A and albumin contents in exhaled particles from patients with COPD and healthy subjects and to determine exhaled particle number concentrations. Methods Patients with stable COPD ranging from moderate to very severe (n = 13), and healthy non-smoking subjects (n = 12) were studied. Subjects performed repeated breath maneuvers allowing for airway closure and re-opening, and exhaled particles were optically counted and collected on a membrane using the novel PExA® instrument setup. Immunoassays were used to quantify SP-A and albumin. Results COPD patients exhibited significantly lower SP-A mass content of the exhaled particles (2.7 vs. 3.9 weight percent, p = 0.036) and lower particle number concentration (p<0.0001) than healthy subjects. Albumin mass contents were similar for both groups. Conclusions Decreased levels of SP-A may lead to impaired host defense functions of surfactant in the airways, contributing to increased susceptibility to COPD exacerbations. SP-A in exhaled particles from small airways may represent a promising non-invasive biomarker of disease in COPD patients.

Comparison of exhaled endogenous particles from smokers and non-smokers using multivariate analysis.

Background: Smoking, along with many respiratory diseases, has been shown to induce airway inflammation and alter the composition of the respiratory tract lining fluid (RTLF). We have previously shown that the phospholipid and protein composition of particles in exhaled air (PEx) reflects that of RTLF. In this study, we hypothesized that the composition of PEx differs between smokers and non-smokers, reflecting inflammation in the airways. Objective: It was the aim of this study to identify differences in the phospholipid composition of PEx from smokers and non-smokers. Methods: PEx from 12 smokers and 13 non-smokers was collected using a system developed in-house. PEx was analysed using time-of-flight secondary ion mass spectrometry, and the mass spectral data were evaluated using multivariate analysis. Orthogonal partial least squares (OPLS) was used to relate smoking status, lung function and pack years to the chemical composition of RTLF. The discriminating ions identified by OPLS were then used as explanatory variables in traditional regression analysis. Results: There was a clear discrimination between smokers and non-smokers according to the chemical composition, where phospholipids from smokers were protonated and sodiated to a larger extent. Poor lung function showed a strong association with higher response from all molecular phosphatidylcholine species in the samples. Furthermore, the accumulated amount of tobacco consumed was associated with variations in mass spectra, indicating a dose-response relationship. Conclusion: The chemical composition of PEx differs between smokers and non-smokers, reflecting differences in the RTLF. The results from this study may suggest that the composition of RTLF is affected by smoking and may be of importance for lung function.