Anna Bredberg

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.

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.

Exhaled particles as markers of small airway inflammation in subjects with asthma.

Exhaled breath contains suspended particles of respiratory tract lining fluid from the small airways. The particles are formed when closed airways open during inhalation. We have developed a method called Particles in Exhaled air (PExA®) to measure and sample these particles in the exhaled aerosol. Here, we use the PExA® method to study the effects of birch pollen exposure on the small airways of individuals with asthma and birch pollen allergy. We hypothesized that birch pollen‐induced inflammation could change the concentrations of surfactant protein A and albumin in the respiratory tract lining fluid of the small airways and influence the amount of exhaled particles. The amount of exhaled particles was reduced after birch pollen exposure in subjects with asthma and birch pollen allergy, but no significant effect on the concentrations of surfactant protein A and albumin in exhaled particles was found. The reduction in the number of exhaled particles may be due to inflammation in the small airways, which would reduce their diameter and potentially reduce the number of small airways that open and close during inhalation and exhalation.

Analysis of manganese and iron in exhaled endogenous particles

Background: many full-time welders experience some sort of respiratory disorder e.g., asthma, bronchitis and metal fume fever. Thus, welding aerosols are thought to cause airway inflammation. There is a need for markers of welding aerosols in exposure assessments, and as most welding aerosols contain manganese and iron, these metals may possibly be used as an indicator. We have previously developed a novel non-invasive technique to collect endogenous particles in exhaled air (PEx). This study is designed to (i) develop a method for analysis of manganese and iron in PEx and (ii) investigate whether the manganese and/or iron content of PEx changes after exposure to welding aerosols. Methods: nine individuals were experimentally exposed to welding fumes. PEx was collected at three time points for each individual; before, after and 24 hours after exposure. Analyses of PEx samples were performed using Inductively Coupled Plasma Mass Spectrometry (ICP-MS). Results: four out of nine individuals showed an increase in manganese and iron levels after exposure to welding aerosols. The mean manganese and iron concentration increased from, <LOD to 82–84 pg L−1 (range from 0 to LOD for values <LOD) and 20–86 to 2600 pg L−1 of exhaled air respectively. Conclusions: an ICP-MS method for analysis of manganese and iron in PEx has been developed. The method could easily be expanded to include other trace metals of interest, such as cadmium, nickel or chromium. This first attempt to evaluate PEx as a tool for exposure assessments of airborne metals indicates that the method has potential.

Bronchodilatation increases number of particles in exhaled air in subjects with asthma

Background Particles in exhaled air (PEx) are derived from the small airways and are formed during airway closure and re-opening. They mainly contain surfactant; both phospholipid and protein composition in PEx resemble that of BAL. Measurements of surfactant protein A in PEx from 100 l exhaled air were shown to be highly reproducible, making the PEx a promising tool in the monitoring of asthma. The number of exhaled particles varies substantially, mainly among subjects, but also within subjects. To enable a correct interpretation of the results using PEx it is crucial to examine how airway constriction affects the number of exhaled particles. Aim To examine the effect of bronchodilatation on exhaled PEx concentration. Method 16 subjects with pollen-asthma and 14 healthy non-atopic subjects (all non-smokers) were examined before and after bronchodilation during the pollen season and outside the pollen-season. PEx, spirometry, blood-samples and answers to a questionnaire were obtained. The subjects performed a breathing maneuvers allowing for airway closure and re-opening and PEx concentrations in about 60 l of exhaled air were measured with an in-house developed instrument based on particle impaction.